key: cord-311115-nimxnf6s authors: bednarska, k.; hallmann-szelińska, e.; kondratiuk, k.; brydak, l. b. title: antigenic drift of a/h3n2/virus and circulation of influenza-like viruses during the 2014/2015 influenza season in poland date: 2016-03-09 journal: respiratory contagion doi: 10.1007/5584_2016_216 sha: doc_id: 311115 cord_uid: nimxnf6s morbidity rates of influenza could be greatly reduced due to vaccination. however, the virus is able to evolve through genetic mutations, which is why vaccines with updated composition are necessary every season. their effectiveness depends on whether there is a good antigenic match between circulating viruses and vaccine strains. in poland, the 2014/2015 influenza epidemic started in week 5 (january/february) of 2015 and continued until week 17 (april) of 2015. the influenza activity was moderate with the highest incidence of influence-like illness at week 10/2015 (march). during that season, antigenic drift of influenza virus a/h3n2/ occurred causing higher rates of a/h3n2/ infections. among the 2416 tested specimens, 22.6 % of influenza cases were positive for a/h3n2/, while a/h1n1/pdm09 constituted 14.6 % cases. influenza a viruses were detected in co-circulation with influenza b viruses; the latter amounted to 34.1 % of all influenza detections. other detected causes of influenza-like illness consisted of respiratory syncytial virus (rsv), being predominant, and, sporadically, human coronavirus, parainfluenza 1–3, rhinovirus, and adenovirus. despite low vaccine effectiveness of solely one component, a/h3n2/, the vaccine could mitigate or shorten the length of influenza infection and reduce the number of severe outcomes and mortality. thus, vaccination against influenza remains the most effective way to prevent illness and possibly fatal outcomes. influenza reappears every season and is caused by circulating influenza type a and type b viruses. these viruses evolve through genetic mutations and resulting antigenic changes, what allows them to evade host immunity. influenza vaccines have to be updated periodically in order to be effective in the following season. hence, the constant need for seasonal vaccination. as it takes approximately 6 months to produce influenza vaccines, recommendations need to be done in advance. the world health organization (who) convenes annual meetings in february and september each year to establish the composition of influenza vaccine for the forthcoming season in the northern and southern hemispheres, respectively (chambers et al. 2015; who 2014a; webster et al. 2013 the 2014/2015 influenza epidemic season in poland started in week 5 (january/ february) of 2015 and continued until week 17 (april) of 2015. the influenza activity was moderate with the highest incidence of influenza-like illness (ili) at week 10/2015 (march) (fig. 1) . during this season, 2416 specimens were tested for influenza and influenza-like viruses, of which 21.2 % (n ¼ 513) were positive for influenza. in detail, there were, 34.1 % (n ¼ 175) influenza type b, 14.6 % (75) influenza a/h1n1/pdm09, 22.6 % (n ¼ 116) influenza a/h3n2/, and 28.7 % (n ¼ 147) non-subtyped influenza type a. there was observed a co-circulation of influenza type a and b viruses, with influenza a being the most prevalent one (fig. 2) . infections caused by influenza-like viruses accounted for 11.2 % (n ¼ 270) of tested specimens. respiratory syncytial virus (rsv) was the other predominant virus, constituting 87.0 % (n ¼ 235) of all ili cases, followed by sporadic cases of human coronavirus (n ¼ 10, 3.7 %), parainfluenza virus-1 (n ¼ 8, 3.0 %), rhinovirus (n ¼ 6, 2.2 %), adenovirus (n ¼ 5, 1.9 %), and parainfluenza viruses 2 and 3 (n ¼ 3, 1.1 % each (fig. 3) ). the highest percentage of influenza and ili confirmed cases was noted in the 0-4 years -group (46.4 %) , followed by the 45-64 years (12.3 %) and 26-44 years age-groups (11.6 %). the lowest morbidity was observed in the 15-25 years (4.9 %) and 10-14 years age-groups (5.4 %) (fig. 4) . the verification of specimen testing by vses demonstrated a complete agreement with the results obtained at the national influenza center (table 1) . in the epidemic seasons 2013/2014 and 2014/ 2015 in poland, the number of specimens received from primary care physicians was similar, although the percentage of influenza and influenza-like infection confirmations was 10 % higher in the latter season. the 2014/2015 morbidity in children aged 0-4 years was twice as much as it had been in the former epidemic season. the vast majority of infections in this group of children was induced by influenza-like viruses (67.2 %). in both seasons, in the case of influenza-like viruses, the predominant virus was rsv. infections caused by other respiratory viruses occurred sporadically. influenza cases were caused mainly by influenza type a (83.2 %). the proportion of influenza b was significantly higher in the 2014/2015 season than that in the previous season where it amounted to 1.2 % of all influenza confirmations (bednarska et al. 2015) . while comparing data from poland and europe, it is apparent that the proportion of circulating viruses of influenza type a and b remains similar. in europe and the whole world alike, the dominant strain was a/h3n2/, but it co-circulated with influenza a/h1n1/pdm09. the predominance of the a/h3n2/ strain also was noted in poland, even taking into account the fact that not all the specimens tested within the sentinel and non-sentinel influenza surveillance were subtyped (broberg et al. 2015; ecdc/who 2015; who 2015; rolfes et al. 2014 ). in the 2014/2015 season, antigenic drift of the subtype a/h3n2/ decreased the effectiveness of vaccine against influenza. yet vaccination remains the most effective and the cheapest method of influenza prevention (broberg et al. 2015) . since 1968, the advisory committee on immunization practices (acip) recommends trivalent vaccines against influenza. they contain two subtypes of influenza virus a and one lineage of influenza virus type b. it should be emphasized that people, particularly those who receive vaccination every season, substantially enhance their immunological memory. evaluation of the activity of influenza and influenza-like viruses in the epidemic season on behalf of the who european region and the european influenza surveillance network (2015) start of the 2014/15 influenza season in europe: drifted influenza a (h3n2) viruses circulate as dominant subtype identification of hemagglutinin residues responsible for h3n2 antigenic drift during the 2014-2015 influenza season from european centre for disease prevention and control. circulation of drifted influenza a (h3n2) viruses in the eu/eea from european centre for disease prevention and control (ecdc)/world health organization regional office for europe (who/europe) report prepared for the who annual consultation on the composition of influenza vaccine for the northern hemisphere report prepared for the who annual consultation on the composition of influenza vaccine for the northern hemisphere update: influenza activity -united states textbook of influenza recommended composition of influenza virus vaccines for use in the 2014-2015 northern hemisphere influenza season available from http:// gamapserver.who.int/gareports/default.aspx? acknowledgements this work was funded in parts by research projects 2011/01/b/nz7/06188 and niph-nih's subject 5/em.1. the authors would like to acknowledge physicians and employees of vses participating in sen-tinel program for their input into the influenza surveillance in poland. the authors declare no conflicts of interest in relation to this article. key: cord-296469-h0ma163u authors: gellin, bruce g.; qadri, firdausi title: preparing for the unpredictable: the continuing need for pandemic influenza preparedness date: 2016-10-26 journal: vaccine doi: 10.1016/j.vaccine.2016.09.023 sha: doc_id: 296469 cord_uid: h0ma163u nan preparing for the unpredictable: the continuing need for pandemic influenza preparedness ebola, zika, middle east respiratory syndrome (mers), chikungunya, severe acute respiratory syndrome (sars). the cadence of emerging infectious diseases that travel, transmit and stand ready to spark a public health emergency of international concern seems to be accelerating. these infectious diseases cause significant human suffering, can overwhelm unprepared health care systems, and significantly disrupt societies and economies. it is estimated that a severe pandemic similar to the 1918 ''spanish flu" could nowadays cost as much as 5% of global gross domestic product; the global cost of even a moderately severe pandemic could be as much as us$ 570 billion, or 0.7% of global income [1] . given the health, social and economic impact of infectious diseases with pandemic potential, there has been much attention to these microbial threats and, not surprisingly, the first question is always, ''is there a vaccine?" but too often we find that the vaccines we need most are not available when we need them most. a number of efforts are focused on better anticipating the vaccines that we may need [2] [3] [4] [5] [6] and a global emergency fund has been proposed to support accelerated vaccine development [7] . the current headlines about zika virus have replaced last year's headlines about ebola. . .and next year's headlines will undoubtedly feature something new. imperfect crystal balls fail to guide us with certainty. but looming in the background is the ever present threat of influenza, the only microbe that causes regular widespread epidemics around the world. not so exotic and all too familiar -casually referred to as the ''flu" -influenza is the virologist's trojan horse, as it readily and rapidly mutates (drift), has the ability to swap genetic elements from influenza viruses with origins in other species (shift) and, as an rna virus lacks repair and self-correcting mechanisms that are the basis of its genetic instability and makes it both dangerous and unpredictable. a reassortment leading to an extremely virulent and readily transmissible pandemic virus is inevitable. of the many things that need to be in place to prepare for and respond to the next influenza pandemic, vaccines -together with the capacity to mount a timely global vaccination effort -are paramount. a pandemic can only be brought under control through a shift in the population from susceptibility to immunity. but as we learned in the 2009 influenza pandemic, although our response time has improved, a significant shift in approach is needed if an effective vaccine is to be in place before the next pandemic emerges. with that goal in mind, the science of influenza and of vaccine development needs to be looked at with fresh eyes [8] . the holy grail is an influenza vaccine that provides a broader and more durable immune response than the natural infection and that can provide population immunity to pandemic influenza viruses before they emerge. a number of efforts are now focused on developing such a ''universal" influenza vaccine; however, the scientific challenges involved are enormous [9, 10] . until such a universal influenza vaccine becomes available, global influenza vaccine production capacity needs to be ready to respond when the next pandemic emerges. vaccines obviously need to be safe and effective, but they must also be available and affordable. while vaccine supply is clearly essential for a large-scale vaccination programme, the logistics and supporting policies needed to conduct effective and efficient vaccination campaigns in a range of community settings also need to be addressed and the needs of high-risk and vulnerable populations and of low-and middle-income countries accommodated. ultimately, the success of a vaccination programme will hinge not only on the technical aspects of its implementation, but on the demand for it among the population; this underscores the need to understand the factors that promote vaccine acceptance [11, 12] . within this broad framework, and with a focus on the role of vaccines in mitigating the impact of both seasonal and pandemic influenza, who established in 2006 the global action plan for influenza vaccines (gap) as ''a comprehensive strategy to reduce the present global shortage of influenza vaccines for seasonal epidemics and pandemic influenza in all countries of the world" [13] . the papers in this special issue of vaccine, provide a foretaste of the in-depth review to come in november 2016, when who will host the third and final consultation on gap as the programme, as it exists today, comes to close. the background papers prepared for this consultation are available on the who website (http:// www.who.int/influenza_vaccines_plan/news/gap3_nov16/en/). while this event is advertised as a programme review, in reality as an examination of the progress achieved during the decade, it should also serve to direct the global community on the path forward for the work that remains to achieve global pandemic influenza vaccine and vaccination preparedness. this review, combined with the current rethinking of global preparedness for emerging threats, the review of the pandemic influenza preparedness (pip) framework [14] , and who's restructuring of its approach to global health emergencies the global action plan for influenza vaccines was structured around three broad objectives that underpin pandemic influenza vaccine and vaccination preparedness: -evidence-based increase in seasonal influenza vaccine use; -increase in influenza vaccine production capacity and regulatory capacity; -research and development for improved influenza vaccines. the articles contained in this issue not only relate to the gap objectives but also reflect the principles and goals of pip and highlight the synergy of these two efforts. pip's pandemic preparedness goal of increasing the access to vaccines and other pandemicrelated supplies by developing countries (and the critical importance of improving the sharing of influenza viruses with human pandemic potential toward that end), will ultimately depend on influenza vaccine production capacity in place when the next pandemic occurs. these articles present a variety of perspectives on the system that will develop, produce, regulate, distribute and evaluate pandemic vaccines and mass vaccination programmes. they have been selected to represent some of the many elements of the system that will be necessary for a pandemic vaccine response -including clinical trials of new vaccines, new vaccine production platforms, national regulatory systems, implementation of policy recommendations for vaccine use, and assessment of the sustainability of the global influenza vaccine manufacturing capacity that gap has supported. progress has already been significant and measurable. one clear example is that global production of seasonal influenza vaccine increased from less than 500 million doses in 2006 to nearly 1.5 billion doses in 2015 [16, 17] . yet, there is a pressing need for additional burden of influenza illness studies to provide the essential data that will be looked to by policymakers to assess the role of vaccines in national influenza control strategies. it is the implementation of these policies that will determine demand. without a concomitant increase in global demand for seasonal influenza vaccine, the capacity that will produce the world's pandemic vaccines that gap has stimulated cannot be sustained [18] . at the end of the day there is the expectation that the efforts and investments made to prepare for a pandemic will result in the timely and equitable availability of a vaccine that is safe and effective, and that can be used in vaccination campaigns before the majority of the global population is exposed to the virus. however, a continuing dedicated effort is needed to ensure that all the elements of the system that will allow this to happen are in place before we need them. because we don't know when that day is, the global community must continue with speed and focus. and, as who develops a new structure to respond for the call to be prepared for all hazards, in its complex organizational chart pandemic influenza preparedness must remain front and center. the inclusive cost of pandemic influenza risk (working paper 22137) an r&d blueprint for action to prevent epidemics. accelerating r&d and saving lives. geneva: world health organization report from the world health organization's product development for vaccines advisory committee (pdvac) meeting antibiotic resistance threats in the united states the coalition for epidemic preparedness innovations (cepi) establishing a global vaccine-development fund (perspective) the compelling need for game-changing influenza vaccines edu/compelling-need-game-changing-influenza-vaccines> advances in the development of influenza virus vaccines induction of unnatural immunity: prospects for a broadly protective universal influenza vaccine (commentary) global vaccine action plan 2011-2020. geneva: world health organization sage working group on vaccine hesitancy. how to deal with vaccine hesitancy? global action plan for influenza vaccines. geneva: world health organization pandemic influenza preparedness (pip) framework. geneva: world health organization melbourne: peter doherty institute for infection and immunity global action plan for influenza vaccines a literature review to identify factors that determine policies for influenza vaccination key: cord-354877-n5du3bqt authors: vasoo, shawn; stevens, jane; singh, kamaljit title: rapid antigen tests for diagnosis of pandemic (swine) influenza a/h1n1 date: 2009-10-01 journal: clin infect dis doi: 10.1086/644743 sha: doc_id: 354877 cord_uid: n5du3bqt we found that the sensitivities of 3 rapid influenza antigen tests for pandemic influenza a/h1n1 virus were low to moderate: bd directigen ez flu a+b test (becton dickinson), 46.7%; binaxnow influenza a&b (inverness medical), 38.3%; and quickvue influenza a+b test (quidel), 53.3%. a patient with influenza-like illness who has a negative rapid antigen test result should undergo further testing using reverse-transcription polymerase chain reaction. influenza viruses and can cause similar symptoms, diagnosing influenza on the basis of clinical presentation alone is difficult, with reported sensitivity ranging from 38% [4] to 79% [5] . rapid influenza antigen tests (point-of-care tests) might prove useful, because they have a fast turnaround time (10-15 min) and require minimal training to perform. however, there are few data on the diagnostic accuracy of rapid influenza antigen tests for pandemic influenza a/h1n1 virus [6] . soon after the onset of the pandemic influenza a/h1n1 outbreak, we received reports of poor performance of rapid influenza antigen tests. we report our findings of the clinical accuracy of 3 commonly used rapid influenza tests for diagnosis of pandemic a/h1n1 influenza. methods. during the period from 1 may 2009 through 2 june 2009, a convenience sample of 84 positive, nonduplicate nasopharyngeal specimens were tested using 3 different rapid antigen test kits: bd directigen ez flu a+b test (becton dickinson), binaxnow influenza a&b (inverness medical), and quickvue influenza a+b test (quidel). testing was performed in accordance with the manufacturer's instructions, and each test result was interpreted by at least 2 of the study authors. nasopharyngeal specimens were collected from patients presenting with influenza-like illness using a sterile polyester nasopharyngeal swab and were transported to the microbiology laboratory in m4rt viral transport medium (remel). only specimens that were positive for respiratory viruses using the luminex xtag rvp (luminex) were included. the luminex xtag rvp is a multiplex rt-pcr assay that allows for the simultaneous detection of 17 respiratory virus types/subtypes, including metapneumovirus, coronaviruses, influenza a virus subtypes h1 and h3, influenza b virus, parainfluenza virus types 1-4, respiratory syncytial virus, adenovirus, and rhinovirus. all specimens were refrigerated, and rapid antigen tests were performed р48 h after specimen receipt. specimens included 60 nasopharyngeal samples positive for pandemic influenza a/h1n1 virus, with results confirmed using the cdc's rt-pcr assay for pandemic influenza a/h1n1 virus. control specimens included 24 nasopharyngeal specimens positive for other respiratory viruses, as follows: adenovirus ( ), coro-n p 1 navirus ( ), concurrent coronavirus and rhinovirus ( n p 3 n p ), metapneumovirus ( ), rhinovirus ( ), concurrent 1 n p 4 n p 4 rhinovirus and parainfluenza virus ( ), parainfluenza virus n p 1 ( ), and respiratory syncytial virus ( ). n p 7 n p 3 we calculated the sensitivity and specificity for each rapid antigen test. we determined the prevalence of pandemic a/ h1n1 influenza over the study period; this was used to calculate the positive predictive value (ppv) and negative predictive value (npv). patient charts were reviewed, if available, for potential factors that may have been associated with rapid antigen test result; these included patient age, duration of symptoms before presentation, inpatient or emergency department versus outpatient status, and the median number of rt-pcr fluorescence intensity (mfi) units. data analysis was performed using spss, version 16.0 (spss). the mann-whitney u test was used for comparison of continuous variables, and x 2 and mcnemar tests were used for categorical independent and paired variables, respectively. p values !.05 were considered to be statistically significant. this study was approved by the institutional review board of rush university medical center (chicago, il). results. the majority of patients with pandemic a/h1n1 influenza were children and young adults; the median age was 12.5 years (range, 7 months to 58 years). a total of 34 patients (57%) were assessed in the emergency department, and 26 patients (43%) were seen in physicians' offices. twenty-two (65%) of 34 patients evaluated in the emergency department were subsequently hospitalized. information for the duration of illness before presentation was available for 38 of 60 patients. the majority of patients (76%) presented р3 days after developing symptoms influenza-like illness (mean interval, 2.8 days; range, 1-9 days). control patients had a median age of 7 years (range, 4 months to 66 years); 14 control patients (58%) presented to the emergency department, and 10 (42%) were outpatients. the analytic performance of the 3 rapid antigen tests, compared with that of the luminex xtag rvp are presented in table 1 . the overall respective sensitivity and specificity of the bd directigen ez flu a+b test were 46.7% (28 of 60 specimens) and 100%; for the binaxnow influenza a&b test, 38.3% (23 of 60 specimens) and 100%; and for the quickvue influenza a+b test, 53.3% (32 of 60 specimens) and 100%. the quick-vue influenza a+b test was significantly more sensitive than the binaxnow influenza a&b test ( ), but there was no sig-p ! .01 nificant difference between the other rapid antigen tests. during the study period, the overall prevalence of pandemic a/h1n1 virus was 17.9% (95% confidence interval, 8.24%-27.6%) among all specimens submitted for respiratory virus rt-pcr testing. on the basis of this prevalence, the calculated ppvs were 100% for all 3 tests, and the calculated npvs were 89.6%, 88.2%, and 90.8% for bd directigen ez flu a+b test, binaxnow influenza a&b, and quickvue influenza a+b test, respectively. patient age and duration of symptoms prior to collection of the respiratory specimen did not correlate significantly with the results of rapid antigen tests ( ). a higher mfi unit with rt-pcr (which p 1 .05 correlates with higher viral load) was significantly associated with positive results for all rapid antigen tests ( ) (table 2) . p ! .01 discussion. rapid antigen tests are commonly used in many hospital laboratories, emergency departments, and doctors' offices. most influenza rapid antigen tests are lateral flow chromatographic immunoassays in which patient samples are applied to a chromatographic strip that contains monoclonal antibodies against influenza a and b viruses. on-site diagnosis of influenza by point-of-care tests has been shown to limit antibiotic prescriptions, use of blood cultures, and chest radiography and ultimately to reduce patient costs [7] . however, rapid antigen tests have demonstrated a wide range of sensitivities for detection of seasonal influenza viruses, ranging from 69%-96% for the bd directigen ez flu a+b test [8, 9] , 64.9%-73% for the binaxnow influenza a&b test [8, 10] , and 19.7%-85% for the quickvue influenza a+b test [11, 12] . we found that the 3 rapid antigen tests had low to modest sensitivities for diagnosis of pandemic influenza a/h1n1, although all tests demonstrated excellent specificity, with no cross-reactivity against other cocirculating respiratory viruses. possible reasons for the low sensitivities in our study include the fact that the sample collection technique was not standardized and that specimens were refrigerated and only tested after completion of rt-pcr, which may have led to antigen degradation. however, we think that this is unlikely, because our findings are consistent with those of a similar study by ginocchio et al [6] in which rapid antigen tests had a sensitivity of only 17.8% for pandemic influenza a/h1n1 virus, compared with the luminex xtag rvp. an alternative explanation may be related to differences in test sensitivity for detection of pandemic influenza a/h1n1 virus, compared with seasonal influenza viruses. in a study that used titered pandemic influenza a/h1n1 virus grown in cell cultures, rapid antigen tests required 1-1.5-log higher viral loads than did human seasonal virus for a positive result [13] . other limitations of our study include the fact that we employed a known convenience sample of positive specimens. because of the widespread interest regarding the clinical performance of rapid antigen tests for detection of pandemic influenza a/h1n1 virus, we used a convenience sample of positive specimens to allow for a quick yet relatively accurate assessment of these tests. in addition, all specimens were decoded, and both investigators performing the tests were blinded to the rt-pcr results. finally, because the design of our study did not permit an accurate determination of the npv and ppv, we calculated these values on the basis of the prevalence of novel influenza h1n1 during the study period. these values would clearly vary according to the prevalence of disease, and the ppv would be highest during the peak of an outbreak, with increased numbers of false-positive results when disease prevalence is low (table 1). despite modest sensitivities, rapid influenza antigen testing could prove to be useful because of the tests' speed, portability, and ease of performance. clinicians should be aware that patients presenting with an influenza-like illness and a negative rapid antigen test result should undergo further laboratory testing. however, a positive rapid antigen test result would allow presumptive diagnosis of novel influenza h1n1 virus infection and should lead to timely institution of infection control measures, treatment, and prophylaxis. finally, as we approach the 2009-2010 winter season, it is desirable for manufacturers to develop sensitive rapid antigen kits that can also differentiate seasonal influenza a viruses from pandemic influenza a/h1n1 virus in view of their different antiviral susceptibilities. novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans world health organization. influenza a(h1n1)-update 46 chicago department of public health. health alert, swine influenza update 19 accuracy of clinical diagnosis of influenza in outpatient children clinical signs and symptoms predicting influenza infection evaluation of multiple test methods for the detection of the novel 2009 influenza a (h1n1) during the new york city outbreak impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized prospective controlled trial performance of six influenza rapid tests in detecting human influenza in clinical specimens evaluation of the directigen flua+b test for rapid diagnosis of influenza virus type a and b infections comparison of the binax now flu a enzyme immunochromatographic assay and r-mix shell vial culture for the 2003-2004 influenza season evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods low sensitivity of rapid diagnostic test for influenza performance of influenza rapid point-of-care tests in the detection of swine lineage a (h1n1) influenza viruses potential conflicts of interest. k.s. is on the speaker panel for wyeth. s.v. and j.s.: no conflicts. key: cord-340611-7ftnttm0 authors: gensheimer, k. f title: challenges and opportunities in pandemic influenza planning: lessons learned from recent infectious disease preparedness and response efforts date: 2004-06-30 journal: international congress series doi: 10.1016/j.ics.2004.01.021 sha: doc_id: 340611 cord_uid: 7ftnttm0 abstract the impact of the next pandemic influenza is likely to be far greater, by orders of magnitude, than most bioterrorism (bt) scenarios. a written pandemic emergency plan and establishment of emergency management teams are critical to mounting a coordinated and effective response to what will be a catastrophic event. members of these teams should include public health, medical, emergency response and public safety officials, organized at each local, state and federal level. the tragic events of september 11, 2001 and the subsequent anthrax attacks have substantially increased funding and support for bioterrorism planning in the united states. thus, public health officials have an unprecedented opportunity to strengthen current systems' planning efforts by promoting dual use bioterrorism/pandemic influenza plans. combining lessons learned from the 2001 terrorist incidents, recent preevent smallpox vaccine programs and the history of past influenza pandemics, more effective strategies can be developed. for example, enhanced influenza surveillance systems can provide data that will not only provide early identification of a novel influenza strain, but will provide more timely recognition of other outbreaks of infectious diseases, including public health threats that may initially present as an influenza-like illness (ili). in recent years, we have witnessed emerging and reemerging infectious disease threats that have presented us with challenges similar to those posed by an influenza pandemic. such events highlight the need for advance planning to ensure an optimal response to a health emergency that is certain to be unpredictable, complex, rapidly evolving and accompanied by considerable public alarm. while advance warning for a terrorist attack is unlikely, the warning already exists for a possible new influenza strain, as evidenced by the recent cases of h5n1 in hong kong and the rapid global spread of cases of severe acute respiratory syndrome. influenza is transmitted readily from person to person, and because a novel influenza virus, by definition, is one to which the general population has little to no immunity, an influenza virus with pandemic potential has the potential to cause substantial morbidity, mortality, social disruption, and widespread panic. crosby's [1] book, ''america's forgotten pandemic: influenza 1918'' notes that more americans died of pandemic influenza than of war-related causalities throughout the entire history of this nation. despite the extent of morbidity experienced, little attention was focused on this catastrophic heath event. in contrast, the tragic events of september 11, coupled with the use of bacillus anthracis as a bioterrorist weapon of mass destruction, received considerable attention from the media, the public and the political leadership of this country. one of the greatest revelations in the aftermath of these unprecedented events was the realization that public health is a bona fide first responder. in the current era of concern for bioterrorism (bt) disaster-type preparedness, the public health community needs to acknowledge the leadership position it has achieved through these recent events and to accept yet another challenge of thinking broadly and creatively to address the many needs posed by a catastrophic infectious disease disasterbe it an influenza pandemic, a bioterrorism event or an emerging/reemerging infectious disease threat that is yet to be identified. planning for these events can no longer be postponed as advance planning and building of public health infrastructure can make a significant difference in our response. national efforts to prepare for the next influenza pandemic require support and collaboration from multiple partners at the state, local and federal level. establishing relationships with the medical community, law enforcement and public health agencies are not only critical in responding to a potential catastrophic event, but will enhance ongoing everyday work. the recent anthrax events demonstrated that public health's unfamiliarity with the emergency response system's incident command structure impeded investigative efforts. cross-department planning will facilitate a more effective response to pandemic influenza, strengthen ties between public health and emergency response sectors and complement other planning efforts for not only pandemic influenza but for other emergencies including acts of terrorism. a written pandemic emergency plan and an established emergency management team, which includes public health, medical, emergency response and public safety officials, are needed to provide effective leadership, coordination and an effective response to the next influenza pandemic. the planning and public health infrastructure needed to effectively address a bioterrorist event and an influenza pandemic overlap considerably. one such area of overlap is surveillance. global and domestic laboratory and disease surveillance must be strengthened to increase the likelihood of early detection and tracking of pandemic influenza or a bioterrorist event. improvements in state public health laboratory capacity through support of the laboratory response network (lrn) has enhanced rapid testing for influenza. because many potential bioterrorist agents initially cause symptoms that resemble an influenza-like illness (ili), it is critical for every state to have rule-out influenza testing capabilities available on a year-round basis. timely reporting of outbreaks and surveillance for influenza-like illness (ili) are directly relevant to tracking the progression and intensity of influenza activity and may provide an early indication of a bioterrorist event. continued support for the early aberration reporting system (e-ars) and other innovative surveillance strategies will benefit the public during seasonal influenza epidemics, an influenza pandmeic, and any other catastrophic disease event. another critical component of any catastrophic infectious disease plan and response is communication. a key lesson learned from the anthrax attacks was that the public demands up-to-date information on an ongoing basis throughout the emergency. factual information presented by trusted public health officials can assist in minimizing fear and hysteria. most health crises are similar to the recent anthrax attacks, where only a few cases are ultimately diagnosed, but the bulk of the populace seeks information on a rapidly unfolding scenario. demand for factual information will only be heightened for a highly contagious disease entity such as pandemic influenza. data generated as a result of a robust surveillance system can assist public health efforts in minimizing hysteria and preventing the dissemination of misinformation regarding the evolving pandemic: has the novel virus arrived; geographical areas of the country most severely affected; whether disease activity is increasing or decreasing and groups most severely affected. despite the many similarities between pandemic influenza and planning for other catastrophic infectious disease events, including an act of bioterrorism, critical differences do exist. unlike the anthrax events of 2001 where there was no forewarning, surveillance should provide days to months of warning for a pandemic, while the pandemic itself will last for several months or years. there will be no ''unaffected'' areas, as the pandemic influenza virus will be present virtually simultaneously in all parts of the country. mutual aid from either the federal government or other regions of the country will be unlikely, as all public health, medical and emergency resources will be dedicated to the disaster at hand locally. support from the federal government will be limited in such a scenario; despite the fact, such assistance usually comes during other states of emergency. resource deficiencies will exist for inpatient/outpatient medical services, biologic products, and key personnel. absenteeism among essential first-line medical and emergency workers will impact services rendered, as no one will be immune to infections from the novel pandemic influenza virus. vaccine will be the primary prevention tool, assuming that vaccine will be able to be developed in a timeframe that will be useful. promoting adult immunization programs, including increasing the use of influenza and pneumococcal vaccine during interpandemic years will also strengthen the public health response. the tragic events of september 11 and the subsequent anthrax attacks and other recent threats posed by severe acute respiratory syndrome and monkey pox have created considerable demand on the medical and public health communities nationwide. as a result, unprecedented resources for enhancing our public health preparedness and response infrastructure at all levels of government have been recently provided to all states by congressional appropriations in the form of bioterrorism cooperative agreements administered by the centers for disease control and prevention (cdc). the request for proposals explicitly note that planning moneys may be used ''. . . to upgrade state and local public health jurisdictions preparedness for and response to bioterrorism, other outbreaks of infectious disease, and other public health threats and emergencies. . .'' [2] . although these funds will be critical for strengthening this country's preparedness and response to bioterrorism, there exists substantial overlap between the public health infrastructure needed to address bioterrorism-related events and other potential public health threats including pandemic influenza. hence, the current climate presents an opportune time to engage in pandemic preparedness planning. taking advantage of the current funding opportunities will not only optimize our response to such a catastrophe, but will help to limit the total burden of disease in terms of morbidity and mortality, economic loss and social disruption caused by an influenza pandemic. by reflecting upon the lessons learned from the 1918 influenza pandemic and recent emerging infectious disease catastrophic events, the public health and medical community can work together to develop an effective preparedness and response plan to strengthen our national readiness to respond to an influenza pandemic as well as to strengthen the health system on which the plan depends. the state and local guidelines developed by the state and federal working group [3] address the various essential components of an infectious disease catastrophic response: surveillance, communication, emergency preparedness, recommendations for distribution of limited biological products, and infection control/medical management. many lessons were learned through the events of september 11 and the aftermath of the attacks using anthrax through the postal service. public health must assume a leadership position in planning effectively and utilizing newly generated resources to optimally prepare for the next public health catastrophe. like the emerging diseases and recurring disease that have occurred over the past several years, we need to think of bioterrorism and the threat posed by influenza pandemic as an emerging and recurring threat, which will probably continue into the foreseeable future. limiting our planning efforts narrowly focused on bioterrorism will be a lost opportunity. in the highly interconnected and readily traversed 'global village' of our time, one nations' problem soon becomes every nation's problem. . . [4] america's forgotten pandemic: the influenza of 1918 notice of cooperative agreement award: guidance for fiscal year 2002 supplemental funds for public health preparedness and response for bio-terrorism (announcement number 99051-emergency supplemental) pandemic influenza: a planning guide for state and local officials (2.1) national academy of sciences, institute of medicine, microbial threats to health: emergence, detection and response we wish to acknowledge the members of the ad hoc influenza pandemic conference planning and steering committee for their continuing dedication and contributions to pandemic planning: lynnette brammer, ron burger, zygmunt dembek, kristine ehresmann, john iskander, deva joseph, donna lazorik, ann moen, mack sewell and gregory wallace. key: cord-336915-dbu93ufh authors: aloizos, stavros; aravosita, paraskevi; mystakelli, christina; kanna, efthymia; gourgiotis, stavros title: h1n1 influenza viral infection in a postpartum young woman causes respiratory failure: what the care providers ought to know? date: 2012-10-23 journal: case rep pulmonol doi: 10.1155/2012/419528 sha: doc_id: 336915 cord_uid: dbu93ufh pregnant and postpartum women are considered a population at increased risk of hospitalization of h1n1 infection. we report the case of a young postpartum woman, who developed evidence of respiratory failure reaching the point of requiring intubation due to an h1n1 influenza virus infection two days after a caesarean delivery. we emphasize the diagnosis, management, and the outcome focusing on the question “what the care providers, including obstetric health care workers, ought to know?” diagnostic and management strategy for pregnant or postpartum women with novel influenza a (h1n1) viral infection and increased awareness amongst patients and health care professionals may result in improved survival. since 2009, the infection with the novel h1n1 influenza virus, popularly termed "swine flu," prompted the world health organization (who) to state that during pregnancy both mother and baby are at increased risk when infected with either pandemic or seasonal influenza and that pregnant women should be vaccinated [1] . a reported systematic literature review found that pregnancy was associated with increased risk of hospital and intensive care unit (icu) admission and death, while pregnant women who received delayed treatment with neuraminidase inhibitors or who had additional risk factors were more likely to develop severe disease and preterm births [2] . we report the case of a young postpartum caucasian woman, with no preexisting illness, presenting with respiratory manifestations of h1n1 influenza virus infection two days after caesarean delivery of a healthy newborn. the patient developed evidence of respiratory failure reaching the point of requiring intubation and a long time intensive care management. a 30-year-old postpartum woman reported cough, shortness of breath, myalgia, and fever until 38.3 • c. the patient had an uneventful cesarean delivery two days ago. she had been in good health throughout her pregnancy and had not traveled abroad or been exposed to anyone with confirmed or probable seasonal or novel influenza. however, she reported a 2-day history of rhinorrhea, cough, and temperature until 37.4 • c, before her delivery. she denied nausea, vomiting, diarrhea, or abdominal pain. she had no chronic medical problems, did not smoke or use illicit drugs, and rarely drank alcohol. she also was hesitant to receive both the h1n1 and influenza vaccines during her pregnancy. initial vital signs included bp: 110/55 mmhg, hr: 114 beats/minute, and spo 2 : 95% in room air, while she was tachypneic at 20 breaths/min. arterial blood gas analysis demonstrated ph: 7.44, pao 2 : 90 mmhg, and paco 2 : 32 mmhg. results of the initial laboratory analysis included wbc: 12,430 cells/μl, neutrophils: 88%, lymphocytes: 6%, crp: 10.9 mg/dl, sgot: 48 u/l, alkp: 139 u/l, and γ-gt: 201 u/l. her physical examination was notable for decreased breath sounds, rhonchi, and wheezing in bilateral lung fields. the initial chest radiograph revealed bilateral alveolar infiltrates and a dense area of consolidation with a small pleural reaction in the left hilar region ( figure 1 ). due to the fact that community-acquired pneumonia was suspected, a broad-spectrum antibiotic coverage was initiated, with the support of oxygen therapy and bronchodilators. at the same time, urine sample was sent for possible detection of pneumoococcus and legionella antigens. a rapid influenza diagnostic antigen test (ridt) was negative. during the first 24 hours after her admission in the icu, the patient's respiratory status continued to deteriorate. an arterial blood gas revealed ph: 7.41, pco 2 : 23 mmhg, and po 2 : 63 mmhg, while receiving 100% fio 2 via nonrebreather mask. there was also a characteristic radiological burden on chest radiograph with diffuse alveolar opacities extending to the left upper lung fields, but in the right middle and lower lobe ( figure 2 ). the decision was made to intubate the patient for impending respiratory failure. subsequent real-time reverse transcription-polymerase chain reaction (rt-pcr) analysis of a nasal swab specimen for influenza subtyping confirmed a diagnosis of novel influenza a (h1n1) infection. she was started on oseltamivir and broad-spectrum antibiotics for during her hospitalization, the patient remained hemodynamically stable with intensive care management without major complications or phenomena of other organs' failure. however, she sometimes showed worsening of her ventilation due to partial atelectasis. to resolve the problem of obstructive events, we performed three urgently bronchoscopies. in addition, acinetobacter baumannii was isolated from the bronchial secretions (ventilator-associated pneumonia; vap) and a new intravenous antibiotic scheme was administrated based on her bronchial cultures susceptibility. the patient was weaned from mechanical ventilation on day 15 and transferred to the obstetrical floor the following days. she continued to recover uneventfully and was discharged with her healthy newborn on day 20. the 4-month followup with chest ct continued to be abnormal with areas of pulmonary fibrosis while spirometry findings had features of restrictive syndrome. the risks of morbidity and mortality from seasonal and pandemic influenza h1n1 are now known to be greater in pregnant than in nonpregnant and postpartum women, especially pregnant women in the 3rd trimester [3] . in one study, the authors estimated that the relative risk of hospitalization, admission to icu, and death was 5.2, 6.5, and 1.4, respectively, for pregnant women [4] . gestational age is associated with higher risk of developing critical infection; the risk increases with the weeks of gestation while women in the 2nd or 3rd trimester of pregnancy have a higher rate of developing critical infection [5] . it may be related to specific immune suppression, decreased resistance, and physiological changes in pregnancy. the patients may present with symptoms for influenzalike illnesses (fever, malaise, cough, sore throat, rhinorrhea, headache, myalgia, vomiting, and diarrhea) and they may demonstrate significant abnormalities on complete blood counts and chest radiographs [6] . the median time from onset of symptoms to hospitalization ranges from 2 to 6 days. ridt detects influenza viral nucleoprotein antigen and is used as screening diagnostic tool. this test often provides results in 30 minutes or less; however, it has low sensitivity (10% to 70% for h1n1, while a negative test does not rule out influenza) and cannot distinguish between virus subtypes; so a specific diagnosis of influenza a (h1n1) cannot be established [7] . rt-pcr assays are the most sensitive (86%-100%) and specific tests available, but generally require 48 to 96 hours to process [7] . confirmatory tests include rt-pcr and viral cultures on properly collected upper respiratory tract specimens. although the isolation of h1n1 virus by culture is the "gold standard" diagnostic test, the procedure typically is too slow to guide clinical management [8] . early admission to the icu for respiratory support may be required. treatment of severe influenza a (h1n1) is primarily supportive, although a role for antiviral medications exists. currently, the cdc recommends treatment of all hospitalized patients with suspected, probable, or confirmed a (h1n1) or seasonal influenza with either oseltamivir or zanamivir [9] . treatment with oseltamivir should commence as soon as possible and antiviral treatment should be provided even if started later than 48 h. the recommended duration of therapy is 5 days, although a longer course can be considered in severe cases. also of note, new mothers should be considered as high risk and treated as such until 2 weeks postpartum. about 1% to 10% of patients with clinical illness due to the novel infection have required hospitalization and the overall case fatality ratio has been estimated as <0.5%. [10] rapidly progressive respiratory failure is relatively common and about 10% to 30% of hospitalized patients have required icu admission [10] . in the present case, we initially tried to stabilize the patient's hypoxemia using noninvasive positive pressure ventilation (nppv). there was no hemodynamic instability and no multiorgan, thus, there were no contraindications to application of nppv. unfortunately, we observed no spectacular results; nppv temporarily improved oxygenation and reduced the work of breathing, but did not alter the course of the disease. on the other hand, nppv is a procedure in which there is possibly increased risk of respiratory pathogen transmission [11] . our patient, in a few hours, required invasive mechanical ventilation (imv) support. imv, with a lung-protective ventilation strategy, is recommended as the appropriate approach for managing patients with pandemic a (h1n1) infection complicated by respiratory failure [10] . low-dose systemic steroids may be considered for patients with refractory septic shock [10] . however, early use of corticosteroids might prolong viral replication in severe acute respiratory syndrome (sars), while during the sars period in the icu, the use of systemic steroids is associated with an increase rate of mrsa, stenotrophomonas, and candida species acquisition, and ventilator-associated pneumonia [12] . in figure 4 , a diagnostic and treatment strategy of pregnant and postpartum women with influenza a (h1n1) virus infection is encompassed. specifically, risk factors for severe disease include pregnant women in their 2nd or 3rd trimester of pregnancy during the influenza season and women at any stage of pregnancy with certain chronic medical conditions. patients should be referred for icu assessment if fio 2 of >0.5 or oxygen at a rate of <10 l/min is required to maintain the spo 2 at 92%. all hospitalized patients with suspected influenza should be tested. nasal swabs with nasal secretions or nasopharyngeal aspirates or swabs are appropriate specimens for detecting human influenza a. rt-pcr or viral culture should be mainly performed as confirmatory testing. patients with illnesses compatible with pandemic a (h1n1) virus infection but with negative ridt results should be treated empirically based on the level of clinical suspicion, underlying medical conditions, severity of illness, and risk for complications. inactivated influenza vaccine can be safely and effectively administered during the 2nd and 3rd trimester. no study has demonstrated an increased risk of either maternal complications or adverse fetal outcomes or adverse events among children born to women who received inactivated influenza vaccine during pregnancy [13] . finally, consideration of the health of the fetus is an additional concern. clinicians should attempt to maintain a minimum pao 2 of 70 mm hg to ensure adequate fetal oxygenation. the fetus is better supported in uterus up to 32-34 weeks of gestational age, if the mother can maintain adequate hemodynamics, oxygenation, and ventilation. in conclusion, this case of h1n1 infection in relatively normal postpartum woman illustrates the increased risk of life threatening complications in this group. thus, increased awareness amongst patients and health care professionals and a higher uptake of prevention strategies may result in improved survival in future epidemics. it is critical that all health care providers use proper preventive measures to avoid infection and appropriately manage those who are affected. recommendations for ph1n1 vaccine in pregnancy 2009 pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature 2009 h1n1 influenza a and pregnancy outcomes in quantifying the risk of pandemic influenza in pregnancy and indigenous people in australia in 2009 h1n1 2009 influenza virus infection during pregnancy in the usa the resurgence of swine-origin influenza a (h1n1) a 29-year-old female at 33 weeks' gestation with respiratory failure novel influenza a (h1n1) viral infection in late pregnancy: report of a case updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the clinical management of pandemic 2009 influenza a(h1n1) infection effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients increase in methicillin-resistant staphylococcus aureus acquisition rate and change in pathogen pattern associated with an outbreak of severe acute respiratory syndrome safety of influenza vaccination during pregnancy key: cord-337721-who0xdyz authors: faggion, heloisa zimmerman; leotte, jaqueline; trombetta, hygor; pereira, luciane aparecida; lapinski, bruna amaral; nogueira, meri bordignon; vidal, luine rosele; almeida, bernardo machado; petterle, ricardo rasmussen; raboni, sonia mara title: influenza sentinel surveillance and severe acute respiratory infection in a reference hospital in southern brazil date: 2019-12-20 journal: revista da sociedade brasileira de medicina tropical doi: 10.1590/0037-8682-0498-2017 sha: doc_id: 337721 cord_uid: who0xdyz introduction: we report the results of the active surveillance of influenza infections in hospitalized patients and the evaluation of the seasonality and correlation with temperature and rainfall data. methods: during the 2-year study period, 775 patients were tested for 15 respiratory viruses (rvs). results: most of the 57% of (n=444) virus-positive samples were human rhinovirus and respiratory syncytial virus. however, 10.4% (n=46) were influenza virus (80% flua; 20% flub). age and sari were significantly associated with influenza. flub circulation was higher is 2013. conclusions: in the post-epidemic period, influenza remains an important cause of hospitalization in sari patients. influenza viruses cause seasonal infections and are associated with morbidity and mortality. the recommended treatment for influenza is neuraminidase inhibitors, and vaccination might be the best choice for public health prevention, considering the complications among high-risk patients 1,2 . the peak timing of influenza-like illness (ili) varies seasonally, and the characterization of the regional dispersion of this virus are important for introducing preventive measures. the many influenza a subtypes are determined by the presence of specific hemagglutinin and neuraminidase, and some subtypes are associated with pandemic episodes 1 . influenza b is classified into two genetic and antigenically distinct lineages, victoria and yamagata 1 . although not a potential pandemic virus, influenza b is often the cause of outbreaks in humans and is characterized by a clinical pattern of intermediate severity. however, fatalities among influenza b cases can occur, especially in the pediatric population 3 . influenza a subtypes h1n1pdm09 and h3n2 and both influenza b lineages have been detected in brazil during influenza seasons 4 . the clinical manifestations of influenza virus infection include fever, headache, myalgia, cough, and sore throat. the signs of severity include oxygen saturation <95%, dyspnea, and respiratory discomfort. the presence of severe disease sets the diagnosis of severe acute respiratory illness (sari), which also requires notification in the brazilian health system 5 . emerging influenza viruses include a/h7n9, a/h3n2(v), and a/h5n1 subtypes 6 . surveillance for these emerging viruses is important and includes both active and passive surveillance systems. the goal of these systems is the rapid and early identification of potentially epidemic strains to prevent their spread. surveillance systems are designed to detect triggers with possible hazards to public health to provide early warning and establish measures to minimize the risks of global effects 7 . this study reported the findings of 2 consecutive years of influenza infection active surveillance and respiratory virus (rv) investigation conducted in hospitalized patients, evaluated influenza seasonality in this region, correlated virus circulation with temperature and rainfall data, analyzed the epidemiological and clinical impacts, and compared the severity of influenza infections to those of other respiratory virus (orv) infections. this cross-sectional study was approved by the institutional ethics review board and performed at hospital de clínicas/ universidade federal do paraná (hc/ufpr), southern brazil. overall, 775 patients were selected from two databases. (i) the first group included 321 hospitalized individuals who underwent laboratory testing for rvs in 2012 (127/321; 40%) and 2013 (194/321; 60%). medical charts of the selected group were reviewed, focusing on epidemiology, clinical manifestations, outcomes, laboratory findings, and sari diagnosis criteria. (ii) the second group included another 454 cases of hospitalized patients in hc/ufpr that were previously notified as sari by the epidemiology hospital staff during 2012 and 2013. epidemiological and clinical data for this group were electronically retrieved from the sari notification system. sari was defined as influenza-like illness along with signs of severity (dyspnea, oxygen saturation <95%, or respiratory discomfort) 5 . meteorological data for curitiba-paraná (brazil), including monthly average temperature (°c) and rainfall (mm 3 ) from january 2012 to december 2013, were obtained from the sistema meteorológico do paraná (simepar) database. curitiba is located in southern brazil, at latitude 25.5°s, and has a temperate climate. rvs were detected by multiplex reverse transcription polymerase chain reaction (rt-pcr) using the seeplex® rv15 ace detection kit (seegene inc, korea, according to the manufacturer's protocol. this kit enables the simultaneous detection of multiple rvs: human adenovirus, human metapneumovirus, parainfluenza viruses (piv-1, piv-2, piv-3, and piv-4), influenza a (flua), influenza b (flub), respiratory syncytial virus (rsv-a, rsv-b), human rhinovirus type a and b (hrv a/b), human enterovirus, and human bocavirus, and human coronavirus type 229e/nl63 (alfa-coronaviruses) and oc43/hku1 (beta-coronaviruses). influenza a subtyping was performed using real-time rt-pcr (rtrt-pcr) according to the centers for disease control and prevention protocol 8 . influenza b-positive samples were tested to identify lineages using a single one-step rtrt-pcr, as previously reported 9 . data were analyzed using r (r core team, 2018), version 3.3.4. parametric and non-parametric tests were used to assess differences between continuous variables with normal and asymmetric distributions, respectively. fisher's exact and pearson's chi-square tests were used to assess differences between categorical variables, as appropriate. covariates were examined in univariate analysis to determine their association with influenza and other rv infections. those with p<0.05 were included in the multivariate analysis. using a stepwise conditional procedure, multivariate logistic regression models were conducted to identify independent predictors considering the presence of influenza or other rv infection as the endpoint. to verify the quality of the fit (goodness-of-fit), we used the hosmer-lemeshow test and qq-plot graph; both showed a good fit of the proposed model to the data. all p-values were two-tailed and p<0.05 was considered statistically significant. overall, 444 (58.8%) patients had virus-positive samples. as seen in table 1 , influenza virus was detected in 46 samples (10.4%), including 37 (80%) with influenza a and nine (20%) with influenza b. the most commonly detected virus was hrv (162 cases). viral co-detection occurred in 127 cases (29% of positive samples); among the influenza virus infections, we found eight co-detections, mainly with hrv. compared to orvs, there was a lower rate of co-detection among influenza cases (17%). whether viral co-infections may enhance pathogenicity during infection requires further investigation. overall, 83% (31/37) of influenza a samples were subtyped; of these, 24 (65%) were a/h1n1pdm09 and seven (20%) were seasonal a/h3. lineage differentiation of the influenza b-positive revealed that all were victoria-like (figure 1 ). both influenza and orv were predominant in children aged <2 years. this represented 35% (16/46) of the influenza cases and 73% (290/398) of orv cases. the median age differed significantly between influenza and orv cases, at 5.4 and 0.7 years, respectively ( table 1) . influenza infection affected more patients aged 14-60 years, a population that remains susceptible to the virus, as the public health system provides the vaccine to children (<5 years), elderly (>60 years), health workers, and patients with comorbidities. in addition, the likelihood of influenza infection increased proportionally with age. the vaccination status data had a large amount of missing information due to the retrospective nature of this study. of 46 patients with influenza, only 26% had records of receiving the influenza vaccine in the last year. these findings emphasize the need to expand immunization coverage in high-risk patients. most patients infected had not received the vaccine or those who presented a low immune response 10 . unlike in immediate post-pandemic years, a reduction in antiviral use in patients with influenza infection was observed. among all cases of influenza in this study, only 35% of the influenza-infected patients showed more cases of fever (93%), cough (100%), and myalgia than those in patients infected by orvs ( table 2) . even though influenza infections comprised only 10% of the total cases of rv detected in this study, these viruses represented 44% of all cases with myalgia. furthermore, influenza infections tended to cause more severe disease, with more cases of sari; however, this finding should be confirmed by prospective studies with higher numbers of patients. of the 775 total cases in this study, 70% (535/775) were diagnosed with sari and 57% (444/775) were virus-positive, 8% of which were positive for influenza ( table 2 ). adjusted analysis showed that patients who were older, with sari, and with myalgia had increased chances of positive results for influenza. therefore, older patients with sari diagnosis should be treated as having influenza infections until the laboratory investigation is completed. chest x-ray was performed in 55% (201/444) of the positive patients, 80% of which showed some abnormality. significant alterations such as interstitial pneumonia (36% and 32%, p=0.69), pulmonary consolidation (25% and 27%, p=1.00) and mixed patterns (25% and 5%, p<0.05) were reported in patients infected with influenza and orvs, respectively. most studies neglected the analysis of x-ray patterns in respiratory tract infections caused by influenza, which showed statistical relevance in this analysis. in 18 influenza-infected patients, the radiographic findings were either consolidation or mixed patterns, none of which had simultaneous bacterial detection. these results emphasize that severe tissue damage is found in hospitalized patients with pneumonia caused by influenza. comparison of the epidemiological and clinical characteristics reported in this study between patients with influenza virus a and b infections revealed no statistically significant results differences, although the low prevalence of influenza b virus may have impaired this assessment. studies that aimed to compare the clinical presentation of influenza patients across virus types and subtypes/lineages have reported divergent results; however, in general, despite differences in age distribution, the clinical illnesses produced by the different influenza virus types and subtypes are indistinguishable 11 . in both years, influenza cases were more common in june and july. the study results showed an association between influenza rates and decreased temperature (figure 2) . however, unlike previous findings, no association was observed between influenza circulation and rainfall during the study period 12 . studies on influenza seasonality in brazil have shown distinct patterns of viral circulation; in the northeast region, influenza circulates in the first 4 months of the year, overlapping with a period of higher humidity in that area 13, 14 . these different circulation profiles due to the climatic conditions of each region impact the vaccine effectiveness, which in tropical regions has been carried out in a period subsequent to viral circulation. the 10.4% influenza positivity among samples found is similar to that in previous reports 7, 12, 14 ; however, the present study tested a larger number of influenza a virus samples for subtypes. in brazil, there was a non-homogeneous distribution of influenza a subtypes. laboratory surveillance data in the southern region showed a higher frequency of influenza a/h1n1pdm than a/h3n2, at 61% and 38%, respectively. in the northeastern region, which has different climatic conditions, there was circulation of influenza a/h3n2 in the first 16 epidemiological weeks (ew) and then an increased detection of h1n1pdm between the 20 th. and 30 th ew, but with a lower intensity than occurred in the southeastern and southern regions 4 . these findings highlight the need to subtype influenza samples from distinct regions to improve the surveillance system. (13) 3/46 (7) 17/46 (37) among influenza b cases, 78% occurred in 2013 and reintroduction of the victoria lineage was observed. in 2013, 89% of the influenza b cases in the region were antigenically categorized as the b/brisbane/60/2008, victoria lineage 4 . in both years, the predominant circulating lineage was victoria-like, a variant different from that selected for vaccine composition (a/h1n1pdm and a/h3n2, a virus similar to the b/wisconsin/1/2010, yamagata lineage 10 ), highlighting the importance of updated information on circulating viruses to determine the vaccine composition. these findings emphasize the importance of public vaccination and studies on circulating virus strains. although the difference was not statistically significant, the rate of severe cases among influenza b (5/9 cases, 55%) was higher than previously reported 3 . more recent reports have also shown similar findings, with incidences of severe disease in influenza b of up to 50% 15 . this finding underscores the relevance of influenza b infection, which has usually been associated with mild disease this study had some limitations. the retrospective data collection may have contributed to the loss of some clinical and laboratory information. the parameters with higher rates of missing data were the vaccination status and the radiographic patterns. a prospective study involving a wider period of time would improve the analysis, allowing evaluation of the seasonality of influenza b cases. in conclusion, influenza infections were associated with seasonal and severe disease, occurred more commonly in older patients, and usually, in a non-immunized population. the influenza treatment rates should be increased and treatment should be initiated earlier, especially in critically ill patients. an increased frequency of influenza b infections occurred in 2013, probably due to a mismatch between the vaccine and the circulating lineage. strengthening surveillance systems within institutions is important to rapidly identify the circulation of pathogens that present a risk to public health. global update on the susceptibility of human influenza viruses to neuraminidase inhibitors the burden of influenza b : a structured literature review influenza: monitoramento até a semana epidemiológica 52 de protocolo de manejo clínico de síndrome respiratória aguda grave -srag warning signals from the volatile world of influenza viruses surveillance for emerging respiratory viruses who -world health organization. cdc protocol of realtime rtpcr for influenza a (h1n1) differentiation of influenza b virus lineages yamagata and victoria by real-time pcr brazilian health ministry. national immunization program clinical characteristics are similar across type a and b influenza virus infections sentinel surveillance of influenza and other respiratory viruses, brazil seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china influenza season in réunion dominated by influenza b virus circulation associated with numerous cases of severe disease the authors declare that there is no conflict of interest. key: cord-316217-ynh8d853 authors: yoshihara, keisuke; le, minh nhat; toizumi, michiko; nguyen, hien anh; vo, hien minh; odagiri, takato; fujisaki, seiichiro; ariyoshi, koya; moriuchi, hiroyuki; hashizume, masahiro; dang, duc anh; yoshida, lay‐myint title: influenza b associated paediatric acute respiratory infection hospitalization in central vietnam date: 2019-02-28 journal: influenza other respir viruses doi: 10.1111/irv.12626 sha: doc_id: 316217 cord_uid: ynh8d853 background: influenza b is one of the major etiologies for acute respiratory infections (ari) among children worldwide; however, its clinical‐epidemiological information is limited. we aimed to investigate the hospitalization incidence and clinical‐epidemiological characteristics of influenza b‐associated paediatric aris in central vietnam. methods: we collected clinical‐epidemiological information and nasopharyngeal swabs from ari children hospitalized at khanh hoa general hospital, nha trang, vietnam from february 2007 through june 2013. nasopharyngeal samples were screened for 13 respiratory viruses using multiplex‐pcrs. influenza b‐confirmed cases were genotyped by haemagglutinin gene sequencing. we analyzed the clinical‐epidemiological characteristics of influenza b lineages (victoria/yamagata) and who groups. results: in the pre‐a/h1n1pdm09 period, influenza b‐associated ari hospitalization incidence among children under five was low, ranging between 14.7 and 80.7 per 100 000 population. the incidence increased to between 51.4 and 330 in the post‐a/h1n1pdm09. influenza b ari cases were slightly older with milder symptoms. both victoria and yamagata lineages were detected before the a/h1n1pdm09 outbreak; however, victoria lineage became predominant in 2010‐2013 (84% victoria vs 16% yamagata). victoria and yamagata lineages did not differ in demographic and clinical characteristics. in victoria lineage, group1 ari cases were clinically more severe compared to group5, presenting a greater proportion of wheeze, tachypnea, and lower respiratory tract infection. conclusions: the current results highlight the increased incidence of influenza b‐related ari hospitalization among children in central vietnam in the post‐a/h1n1pdm09 era. furthermore, the difference in clinical severity between victoria lineage group1 and 5 implies the importance of influenza b genetic variation on clinical presentation. influenza viruses belong to the family orthomyxoviridae, which possess a segmented negative-stranded rna genome. 1,2 among three influenza types, namely, type-a, b and c, influenza a and b often cause seasonal acute respiratory infections (ari) epidemics and impose a high socioeconomic burden, particularly among children and the elderly population. 3, 4 in temperate climate regions, peaks of influenza-associated aris may appear in early autumn through winter, 5 whereas year-round circulation with no apparent seasonal trend may be seen in tropical climate regions. [6] [7] [8] [9] influenza b differs from type-a in terms of genetic composition and its ability to infect only humans and seals. 10, 11 two surface glycoproteins, haemagglutinin (ha) and neuraminidase (na), play pivotal roles during pathogenesis. 12 unlike influenza a, influenza b does not possess multiple subtypes. instead, influenza b obtains its genetic variation mainly through genetic drift, including nucleotide substitutions, insertions, and deletions, which explains the slower molecular evolutionary rate and smaller capacity to cause seasonal ari outbreaks compared to influenza a. 13, 14 genetic reassortment of influenza b genetic components was previously documented 15 ; however, its frequency is not as high as that of influenza a due to its limited animal reservoir. despite the clinical importance of influenza b among ari cases, majority of previous literature focused on influenza a. therefore, clinical and molecular epidemiological information on influenza b is relatively limited worldwide. the first influenza b strain b/lee/40 was isolated in 1940. 16 in the 1970s, influenza b diverged into two genetically and antigenically distinct lineages, victoria-like (b/victoria/2/87) and yamagata-like (b/yamagata/16/88) lineages. 17, 18 since then, the two lineages have been co-circulating in many regions of the world. 19 previous studies from cambodia, china, india, and taiwan have reported the increase of influenza b-associated ari cases in the following seasons after the emergence of the pandemic a/ h1n1pdm09 strain. 4, 17, 18, 20, 21 furthermore, some studies presented the circulation of victoria lineage as a dominant type in the post-a/h1n1pdm09 period, 17, 18, [20] [21] [22] [23] whereas others showed cocirculation of both lineages. 3, 4 regardless of a few epidemiological studies on influenza b lineage-specific clinical presentation, 21, 23 the clinical aspect of two genetically distinct lineages has not been clearly understood up to date. in this study, we investigated the incidence and clinicalepidemiological characteristics of paediatric hospitalized influenza b ari cases in vietnam. population-based prospective paediatric ari surveillance in khanh hoa province, nha trang, vietnam was established in february 2007. 24 all children from 16 communes admitted to the paediatric ward of khanh hoa general hospital (khgh), which is the only hospital in the region, due to ari symptoms (cough and/or difficulty breathing) from february 2007 to june 2013 were enrolled in the current study. nha trang city in south central region of vietnam has a hot and dry season or tropical climate with a short rainy or wet season from september to december. the detailed characterization of the target population was described in the previous study. 24 written informed consent from the parents or guardians of all the enrolled ari children were obtained. nasopharyngeal (np) swab, clinical-epidemiological information, chest radiograph, and laboratory test data were collected from each enrollee. currently, vaccination against influenza type a and b is not included in the nationwide immunization program in vietnam. its availability in our study site is limited and not commonly used among children. lower respiratory tract infection (lrti) was defined based on the modified world health organization (who) integrated management of childhood illnesses (imci) algorithms. 25 the presence of tachypnea (respiratory rate >60/min for children ≦1 month, >50/min for 2-11 months and >40/min for 12-59 months of age) was categorized as mild lrtis. furthermore, children with a general danger sign (the situation in which children were either unable to drink, under convulsion, or lethargy), chest-wall indrawing, or stridor were categorized as severe lrtis. radiologically-confirmed pneumonia was defined as the presence of either substantial alveolar consolidation or pleural effusion in chest x-ray result following the standardized interpretation method established by who vaccine trial investigators group. 26 ari cases with abnormal shadow but neither substantial alveolar consolidation nor pleural effusion were categorized into chest x-ray abnormality or other lower respiratory infections. 26 adenovirus, and bocavirus as previously described. 24 for the cur confirmed that there was statistical evidence of an increase in influenza b-associated paediatric ari hospitalization in "post-a/ with regard to the demographic and clinical characterization of overall paediatric ari hospitalization cases enrolled in the current study (n = 4,429), 2,602 were male (58.8%), and the median age (in months) was 16.6 (iqr: 8.6-27.3). other demographic information, including socioeconomic status, medical history, as well as detailed clinical information, was summarized in table 2 . the demographic and clinical characteristics between influenza b (n = 133) and non-influenza b ari groups (n = 4296) were compared ( table 2 ). the male proportion was higher in the non-influenza b ari group (50.4%, influenza b vs 59.0%, non-influenza b, p = 0.046). regarding the median age (in month), the influenza b ari group was older (22.9, influenza b vs 16.5, non-influenza b, p < 0.001), and the greater proportion of ari children were 3 years or older in the influenza b ari group. furthermore, family smoking was more frequent in the influenza b ari group (p = 0.003). with respect to the clinical information, the respiratory rate (per min) was faster in the non-influenza b ari group (32.0, influenza b vs 33.0, non-influenza b, p = 0.012) ( table 2) . furthermore, ari children with wheeze (p = 0.005) and breathing difficulty (p = 0.022) were more common in the non-influenza b ari group. the proportion of paediatric ari hospitalizations with chest x-ray abnormal findings was also greater in the non-influenza b ari group ( furthermore, in the comparison between influenza a and b, influenza b-associated ari hospitalizations were slightly older with different age distribution pattern (p = 0.002) (table s2) . parental smoking was more common in influenza b ari group (p = 0.011). regarding the clinical information, influenza a group was associated with higher body temperature (p = 0.003) as well as frequent presence of chest x-ray abnormality (p < 0.001). (table s3) . on the contrary, the overall number of yamagata lineage-related ari hospitalizations was limited throughout the investigation period in both pre-and post-a/ h1n1pdm09 periods. in the demographic characteristics comparison between influenza b table s3 . firstly, demographic information among who groups (1, 4, and 5) of victoria lineage was compared (table 4 ). there were no significant differences in sex distribution and median age among the three groups. daycare attendance was more common in group 5 com(table s4) . with regard to demographic characterization, no statistically significant differences were detected between groups 2 and 3. both group 2-and group 3-associated ari hospitalizations were commonly observed among children younger than 2 years. the results of a comparison in clinical manifestations did not reveal any significant differences. interestingly, we also observed unusual split of rsv seasonal peak in 2010 as we previously described. 37 further studies would be necessary to understand the effect of a/h1n1pdm09 on respiratory virus circulation trend. among all the paediatric ari hospitalization cases enrolled in the current study, 133 were influenza b positive ( table 2 ). the median age in influenza b ari group was older than non-influenza b aris, and influenza b group presented the higher proportion of family smoking, which is known to be one of the major risk factors for influenza morbidity and mortality. 38 we also have to take the lineage-specific sero-epidemiological information into account to gain a better understanding of influenza b lineage circulation pattern. a study from taiwan suggested switching of dominantly circulating lineage may occur every 2-3 years. 18 in the future study, we will continue monitoring the lineage-specific circulation trend of influenza b strains in order to investigate the lineagespecific viral transmissibility based on the viral genetic variation. regarding the demographic information of influenza b lineages, median age was slightly older in victoria lineage (table 3) , which contradicted the reports from china and slovenia that presented victoria lineage aris were younger than yamagata lineage. 21, 45 the difference in age of infection has been controversial due to the differences in inclusion criteria among studies. furthermore, clinical aspect of influenza b lineages is poorly understood. in the current study, the clinical data did not present a significant difference between lineages (table 3) , which was in line with previous reports from china, france, serbia, and slovenia. 41, [45] [46] [47] [48] although the body temperature was slightly higher in yamagata lineage aris, it was probably due to the difference in age distribution. in our study, the hospitalization duration did not differ between lineages, which con male sex (%) 41 we would like to express our sincere gratitude to the medical doctors, nurses, and laboratory technicians at khanh hoa general hospital and staffs in khanh hoa health service for their kind support on clinical sample collection and ari clinical data management. we would like to express our special appreciation for administrative staff and professors in leading program at nagasaki university, graduate school of biomedical sciences for their kind support during the research. this study was conducted in part at the joint usage/research center on tropical disease, institute of tropical medicine, nagasaki university, japan. the authors have declared that no competing interests exist. lay-myint yoshida https://orcid.org/0000-0002-7567-3248 evolutionary characteristics of influenza b virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism the gene structure and replication of influenza virus influenza b lineage circulation and hospitalization rates in a subtropical city surveillance and molecular characterization of human influenza b viruses during 2006-2010 revealed co-circulation of 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increase influenza virus pathogenicity in macaques the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis multipathogen infections in hospitalized children with acute respiratory infections dual respiratory virus infections dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis insights into the interaction between influenza virus and pneumococcus key: cord-302713-h3aoag4y authors: jauréguiberry, stéphane; boutolleau, david; grandsire, eric; kofman, tomek; deback, claire; aït‐arkoub, zaïna; bricaire, françois; agut, henri; caumes, eric title: clinical and microbiological evaluation of travel‐associated respiratory tract infections in travelers returning from countries affected by pandemic a(h1n1) 2009 influenza date: 2011-12-08 journal: j travel med doi: 10.1111/j.1708-8305.2011.00570.x sha: doc_id: 302713 cord_uid: h3aoag4y background: although acute respiratory tract infections (rti) have been recognized as a significant cause of illness in returning travelers, few studies have specifically evaluated the etiologies of rti in this population. methods: this prospective investigation evaluated travelers returning from countries with endemic influenza a(h1n1) 2009, and who were seen in our department at the onset of the outbreak (april–july 2009). patients were included if they presented with signs of rti that occurred during travel or less than 7 days after return from overseas travel. patients were evaluated for microbial agents with respifinder plus assay, and throat culture according to clinical presentation. results: a total of 113 travelers (m/f ratio 1.2:1; mean age 39 y) were included. they were mainly tourists (n = 50; 44.2%) mostly returning from north america (n = 65; 58%) and mexico (n = 21; 18.5%). the median duration of travel was 23 days (range 2–540 d). the median lag time between return and onset of illness was 0.2 days (range 10 d prior to 7 d after). the main clinical presentation of rti was influenza‐like illness (n = 76; 67.3%). among the 99 microbiologically evaluated patients, a pathogen was found by polymerase chain reaction (pcr) or throat culture in 65 patients (65.6%). the main etiological agents were influenza a(h1n1) 2009 (18%), influenza viruses (14%), and rhinovirus (20%). a univariate analysis was unable to show variables associated with influenza a(h1n1) 2009, whereas rhinorrhea was associated with viruses other than influenza (p = 0.04). conclusion: despite the a(h1n1) 2009 influenza pandemic, rhinovirus and other influenza viruses were also frequent causes of rti in overseas travelers. real‐time reverse transcription‐pcr and nasopharyngeal swab cultures are useful diagnostic tools for evaluating travelers with rti. stéphane jauréguiberry, md, * david boutolleau, phd, † ‡ eric grandsire, md, † ‡ tomek kofman, md, * claire deback, phd, † ‡ zaïna aït-arkoub, ‡ franç ois bricaire, md, * henri agut, md, phd, † ‡ and eric caumes, md * † r espiratory tract infections (rtis) are a significant cause of health problems, accounting for 7%-11% of consultations in returning travelers. 1, 2 the prevalence of rti is invariably higher in travelers presenting with fever, as rtis account for 14%-24% of the etiologies of fever. 2 -4 however, the spectrum of microbial agents causing rti in travelers has been investigated in only limited circumstances or selected populations. influenza is recognized as a significant cause of fever and rti infections in travelers. an australian study found that influenza was responsible for 5% of the 56 rtis diagnosed in 232 returning travelers and immigrants/refugees presenting with fever. 3 seroconversion for influenza virus was confirmed in 12% of 211 febrile swiss travelers compared with 2.8% for all swiss travelers surveyed; the incidence was estimated to be around one influenza-associated event per 100 person-months abroad. 5 however, a high number of rtis remain unexplained, mostly owing to a lack of evaluation and the rapid, spontaneous recovery of patients. at the end of april 2009, a new influenza virus a(h1n1) outbreak was identified in mexico and spread rapidly to north america then to europe and the rest of the world through international travelers. 6, 7 the rapid progression of the disease led the who to declare a phase 6 pandemic on june 11, 2009. 8 during the first months of the outbreak in france, travelers were given particular attention and those with presumed signs of influenza were advised to immediately consult dedicated infectious disease units until july 17, 2009. 9 this gave us an opportunity to evaluate the microbiological etiologies of rti in travelers during the first months of the new influenza virus a(h1n1) 2009 outbreak (april-july 2009). although cell culture is the ''gold standard'' for the detection of respiratory viruses, it is impractical for general use in travelers, so, we evaluated the use of a multiplex polymerase chain reaction (pcr) assay in this setting. in a consecutive manner, adult (>17 y old) patients, returning from abroad and who consulted within our department in paris, from april 27, 2009 to july 17, 2009 were enrolled in the study. no informed consent was required because clinical management was as per routine pandemic protocol. patients were included if they presented with signs suggestive of rti that had occurred during travel or <7 days after their return from countries endemic for influenza virus a(h1n1) 2009. rtis were classified as upper rti [tonsillitis, otitis, sinusitis, laryngitis, or influenza-like illness (ili)] and lower rti (bronchitis, lobar pneumonia, or diffuse pneumonia). ili was defined as the presence of the following signs: temperature >37.5 • c with respiratory (eg, cough, sore throat, rhinorrhea) and/or constitutional symptoms (eg, headache, myalgia, arthralgia, fatigue, chills) according to previously established criteria for respiratory illnesses. 10 ili and bronchitis were clinically diagnosed. lobar pneumonia was diagnosed on chest x-ray. endemic countries were those which declared outbreaks of new influenza virus a(h1n1) in their territories according to weekly published who bulletins. following admission, patients were isolated either in hospital or at home. the following epidemiologic data were collected: demographic findings (age and sex), travel history (destination and duration), and purpose of travel (tourism, business, or immigrants visiting friends and relatives). travel destination was classified according to the country visited. the time between return and symptom onset was also recorded. the following signs and symptoms were assessed: temperature, sore throat, rhinorrhea, cough, dyspnea, headache, myalgia, arthralgia, fatigue, chills, gastrointestinal signs (eg, diarrhea, vomiting), urinary tract symptoms, and cutaneous symptoms. the following biological data were recorded: serum creatinine, liver function tests, blood cell count, platelets count, and c-reactive protein. the different presentations of rti were classified according to clinical signs and the results of chest x-ray performed when pneumonia was clinically suspected. pneumococcal pneumonia was presumed if the patient presented with typical clinical signs, a compatible chest x-ray, and a favorable outcome with amoxicillin. no diagnostic confirmation, such as urinary pneumococcal or legionella pneumophila 1 antigen was performed. nasopharyngeal specimens were collected by trained nurses upon admission. at the virology laboratory, the first step of the diagnostic evaluation was to identify influenza a(h1n1) 2009 virus infection by means of real-time reverse transcription-pcr (rt-pcr), as previously described 11 to assess whether or not the patient should remain isolated. in addition, blood cultures were performed in cases with fever and those patients with tonsillitis received a pharyngeal swab for streptococcal evaluation. the second step of the etiologic diagnosis entailed an investigation for other respiratory viruses and intracellular bacteria potentially associated with rti. the following viral and bacterial assays were performed to detect a broad spectrum of microorganisms. respifinder plus (pathofinder, maastricht, the netherlands), a multiplex pcr assay 12 , is able to detect 15 viruses and 4 bacteria in a single reaction: influenza a virus (infa), influenza b virus (infb), influenza a (h5n1) virus (infa h5n1), respiratory syncytial virus (rsv; types a and b), parainfluenza virus (piv; types 1-4), human metapneumovirus (hmpv), rhinovirus, coronavirus (types oc43, 229e, nl63), adenovirus, chlamydophila pneumoniae, mycoplasma pneumoniae, l. pneumophila, and bordetella pertussis. furthermore, human bocavirus (hbov) dna was detected using the bocavirus r-gene kit (argène, varilhes, france), and enterovirus rna was evaluated following the method previously described. 13 all assays were performed using the remaining nasopharyngeal specimen frozen at −80 • c in the virology laboratory. variables were collected using microsoft excel 2002 software (microsoft windows xp professional, microsoft corp., redmond, wa, usa). the relative frequency of the diagnoses and their association with biological and clinical findings were analyzed. the statistical significance of differences in dichotomous variables was determined using chi-square tests with the fisher two-tailed exact test. all variables correlated in a univariate analysis with influenza were included in a stepwise backward regression model (significance level for exclusion was p ≥ 0.25) to identify predictors of the disease. statistical analyses were performed by spss statistical software 17.0 (spss inc., chicago, il, usa). a total of 113 travelers with signs of rti were included. the m/f ratio was 1.2:1, and the mean age was 39 years old. the reason for travel was mainly tourism (n = 50; 44.2%) to the united states (n = 59; 52.2%), canada (n = 6; 5.3%), and mexico (n = 21; 18.5%). the median duration of travel was 23 days (range 2-540 d). the median lag time between symptoms onset and return was 0.2 days (10 d before return to 7 d after) ( table 1) . the most common symptoms were fever, sore throat, and cough, found in more than 65% of the 113 patients ( this study provides a prospective and solid evaluation of etiological causes of rti in a population of returning travelers with rti regardless of intensity. the unusual situation surrounding the h1n1 pandemic allowed us to access a general population, accustomed to mild rti symptoms for which they do not usually consult. this was illustrated in a study of 779 american travelers visiting developing countries where 75 patients (10%) presented symptoms of rti after return but only 22 (3%) sought medical consultation for rti. 14 in france, at the beginning of the flu pandemic, travelers with any sign of rti were advised to promptly consult a clinician. 9 therefore, we were able to test most, if not all, our patients with rti, providing an accurate evaluation of the spectrum of respiratory pathogens that may target travelers. the age distribution in our study (>60% of our cases are more than 30 y old) is consistent with that found in a japanese study during the same outbreak. indeed the median age of confirmed cases of influenza a(h1n1) 2009 in japanese travelers (ie, 25 y old) was older than the median age of influenza confirmed cases who did not travel (ie, 15 y old). 15 older adults tend to travel more often than younger and therefore are perhaps more at risk of contracting respiratory disease. the clinical spectrum of rti in travelers is broad. in the geosentinel study in which rti was diagnosed in 1719 returning travelers (7.8% of all returning travelers), the main clinical presentations of rti were ''nonspecified'' upper rti (diagnosed in 47% of the patients), bronchitis (20%), pneumonia (13%), pharyngitis (13%), and ili (5%). 16 in an italian series of 540 hospitalized patients with a history of travel and fever, rti was diagnosed in 40 patients (7% of the febrile patients) and the most common rtis were pneumonia (35%) and tuberculosis (15%), whereas ili was found in 2.5% of the patients. 17 in contrast to previously reported literature and as an illustration of the inclusion bias discussed above, most (67%) of our patients had ili, a situation that does not routinely lead to a consultation. the spectrum of microbial agents causing rti had been previously described and include numerous viruses (eg, influenza, parainfluenza, respiratory syncitial virus, metapneumovirus, adenovirus, rhinovirus, and coronavirus) as well as some bacteria (eg, streptococcus sp., m. pneumoniae, l. pneumophila). 18 in the subset of our 99 patients evaluated with rt-pcr and a throat swab, an infectious agent was found in 65.6%. this is much higher than that observed in many other studies performed in travelers or during influenza season. in a series of 500 hajj pilgrims presenting with upper rti, 54 (10%) had a positive viral throat culture. 19 of these 54 positive cultures, 27 (50%) were due to influenza b, 7 (12%) due to rsv, 4 (7%) due to parainfluenza, and 3 (5%) due to influenza a. 19 in another study of 255 iranian pilgrims with rti, 83 (32%) had a viral pathogen isolated by throat culture. 20 of these 83 positive throat cultures, influenza was diagnosed in 25 (9.8%), followed by parainfluenza in 19 (7.4%), rhinovirus in 15 (5.9%), adenovirus in 14 (5.4%), enterovirus in 5 (2%), and rsv in 4 (1.6%); coinfection with two viruses was observed in one patient (0.4%). 20 of 67 german travelers that fulfilled the who case definition of suspected or probable severe acute respiratory syndrome (sars) during the 2003 outbreak, influenza and pivs accounted for 14.2 and 15.5% of the viral etiologies by rt-pcr, whereas 56.8% of the cases remain unexplained. 21 therefore, the viruses isolated in travelers include viruses other than infa and infb. in a study performed at san francisco university medical center during the influenza season, a viral agent was identified (through shell vial assay and pcr) in 103 (39%) of the patients with rti. 22 lastly, among 420 patients with ili recruited over 3 years in sao paulo (brazil), rt-pcr were performed on nasal washes and 61.8% were positive for respiratory viruses. 23 therefore, rt-pcr leads to an etiological diagnosis of rti in about two thirds of the cases. although this study took place during the early months of the influenza a(h1n1) 2009 outbreak, this strain of influenza virus was isolated only in 18% of the microbiological evaluated cases. we found that ili was mainly because of influenza (30%) but other viruses (37%) such as rhinovirus (22%) were also involved. this supports previous data in brazil where ili was reported in 240 of 420 patients (57.1%), with influenza and rhinovirus accounting for 30.9 and 19.6% of the ili etiologies, respectively. 23 otherwise viruses identified during passed flu epidemics were also diverse as reported in other studies. 22, 24 we were unable to identify risk factors for infection with influenza virus a(h1n1) in our patients with rti (data not shown), probably because of the limited number of cases evaluated during the inclusion period (april-july). thus, it was not possible to confirm the three factors (travel to the northern hemisphere during the period of december through february, visiting friends or relatives, and trip duration of >30 d) that had previously been shown to be associated with ili in travelers. 16 there are several limitations to this study. first, this is a monocentric study but at the onset of the outbreak there were only three centers available for such patients in paris, of which one cared for infants and adolescents only. second, the method used for diagnosing rti in this study could be improved. we chose a multiplex ligation-dependent probe amplification technology for diagnosing rti in our travelers. compared to cell culture, the ''gold standard'' for the detection of respiratory viruses, the sensitivity and specificity of this technology is satisfactory for clinical practice. depending on the pathogen, sensitivity varies from 90% to 99% and specificity is 100% for this device. 12 nevertheless, adequate performance and lack of interference from other analytes should be checked by other investigations. 25 moreover sampling requires good handling practice by the nurse to avoid carryover contamination and false negative results. nasal swabs need to be pushed deeply into the nasal cavity to obtain a good quality sample. furthermore, additional studies are needed to fully elucidate their ideal clinical application and performance characteristics. 26 third, a subset of patients did not undergo pcr evaluation because of various reasons such as technical issues on assays on weekends or nights. fourth, bronchoalveolar lavage was not performed due to lack of severity or treatment failure in case of pneumonia. finally it was impossible to have a denominator (ie number of air travelers) during this period. therefore incidence rate could not be assessed. these study findings demonstrate that, even at the onset of the influenza a(h1n1), rhinovirus and other influenza viruses were common. therefore, these viral infections should always be considered in the diagnosis of rti in returning travelers. systematic research of pathogens by rt-pcr and culture of nasopharyngeal swab lead to almost 70% diagnoses and could therefore be considered for use in travelers with rti. spectrum of disease and relation to place of exposure among ill returned travelers illnesses in travelers returning from the tropics: a prospective study of 622 patients fever in returned travelers: review of hospital admissions for a 3-year period fever in returned travelers: results from the geosentinel surveillance network 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authors thank alice perignon, marylin lecso for the management of patients and samples, and amy whereat, medical english consultant for proof reading the manuscript. the authors state they have no conflicts of interest to declare. key: cord-298776-tjw45t3f authors: al awaidi, salah; abusrewil, suleiman; abuhasan, muslim; akcay, meral; aksakal, fatma n.b.; bashir, uzma; elahmer, omar; esteghamati, abdoulreza; gahwagi, milad; mirza, yusuf k.; grasso, cindy; kassianos, george; khris, moulud; mardani, masoud; maltezou, helena; nourlil, jalal; oumzil, hicham; osterhaus, ab; picot, valentina; pehlivan, tamer; saadatian-elahi, mitra; tali, i̇lham; tarraf, hesham; ugur, baris; zaraket, hassan title: influenza vaccination situation in middle-east and north africa countries: report of the 7th mena influenza stakeholders network (mena-isn) date: 2018-08-17 journal: j infect public health doi: 10.1016/j.jiph.2018.07.003 sha: doc_id: 298776 cord_uid: tjw45t3f background: the middle east and north africa (mena) region faces a dual challenge with regard to influenza infection due to severe zoonotic influenza outbreaks episodes and the circulation of northern hemisphere human influenza viruses among pilgrims. methods: the mena influenza stakeholder network (mena-isn) was set-up with the aim of increasing seasonal influenza vaccination coverage by (i) enhancing evidence-based exchanges, and (ii) increasing awareness on the safety and benefits of seasonal vaccination. during the 7th mena-isn meeting, representatives from 8 countries presented their influenza surveillance, vaccination coverage and actions achieved and provided a list of country objectives for the upcoming 3 years. results: mena-isn countries share the goal to reduce influenza related morbidity and mortality. participants admitted that lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy are the major barrier to attaining higher influenza vaccination coverage in their countries. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. participants drew a list of strategic interventions to bridge gaps in the knowledge of influenza burden in this region. conclusions: participating countries concluded that despite an increase in vaccine uptake observed during the last few years, influenza vaccination coverage remains relatively low. priority areas should be identified and action plans tailored to each country situation set-up to investigate the best way to move forward. results: mena-isn countries share the goal to reduce influenza related morbidity and mortality. participants admitted that lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy are the major barrier to attaining higher influenza vaccination coverage in their countries. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. worldwide, influenza affects 10-20% of the population and causes more than 1 million deaths annually [1] . this vaccinepreventable infectious disease belongs to the list of the world health organization (who) recommended vaccines, yet the vaccine uptake is in general low in both developed and developing countries [2] . the finding of the who flu-net database showed that dynamic of influenza epidemics in the large majority of the middle east and north africa (mena) countries was in line with the northern hemisphere, with the largest peak observed between january and march [3] . annual vaccination campaigns in the mena region target primarily specific high-risk groups i.e. pregnant women; individuals >6 months with underlying chronic diseases, elderly, residents of long-term care facilities, children aged 6-59 months and health care providers. the mena region faces a dual challenge with regard to influenza infection. first, the region tackled severe zoonotic influenza outbreaks episodes with lethal cases during the last years [4] [5] [6] [7] [8] . second, the northern hemisphere human influenza viruses circulate in the region and are the most frequently detected respiratory viruses among pilgrims [9] , increasing the risk for further spread of the disease. in addition, there is a risk of human infection from middle east respiratory syndrome coronavirus (mers-cov). indeed, since 2012, outbreaks of mers-cov and very few sporadic travelrelated cases were recorded in saudi arabia [10] and returning haji [11] , respectively. although epidemiological analysis does not support human to human transmission, overcrowding, mass gathering and travel increase the fear about potential mers-cov international dissemination. it is very well known that diabetics have a significantly increased risk of flu-related hospitalization and death [12] . with approximately 37 million adults aged 20-79 living with diabetes, the mena region has the highest global prevalence of diabetes in the adult population [13] . this point emphasizes on the commitment to increase influenza vaccination coverage in this region. the few available published literatures on influenza vaccination coverage in mena report low coverage in this region [1, [14] [15] [16] . aligned with the objective of the who global influenza vaccine action plan to increase influenza awareness, the middle east and north africa influenza stakeholder network (mena-isn) has been initiated in 2014. mena-isn is a network of regional experts with the global mission to increase seasonal influenza vaccination coverage by (i) enhancing evidence-based exchanges with national and international actors, and (ii) increasing awareness on the safety and benefits of seasonal vaccination. mena-isn support the who initiative in building laboratory capacity and surveillance in the region and urge the governments to give high priority to the establishment and continued support for influenza surveillance systems; identify the needs of countries for establishing or improving existing surveillance networks; disseminate surveillance and disease burden data through publications and develop actions to increase vaccination coverage rates in health care professionals (hcps), pregnant women, people at risk, elderly and children. the 7th mena-isn meeting was organized by foundation mérieux on 9-10 september, 2017 in riga, latvia. a total of 25 participants from 8 countries (egypt, iran, lebanon, libya, morocco, oman, pakistan and saudi arabia) attended the meeting. in addition to mena-isn country representatives, experts from the who global influenza program, the college of general practitioners (uk), the hannover veterinary university (germany), and hellenic centre for disease prevention and control (greece) were also present. each country representative summarized their current situation of influenza surveillance, influenza vaccination coverage and actions achieved, and provided a list of country objectives for the upcoming 3 years. a panel discussion on the second day allowed the participants to discuss how to strengthen the network and its visibility and how to reinforce between country research and publications. herein, we report a summary of the country situation and actions to move forward. vaccination coverage in egypt remains low [17] . however, the ministry of health (moh) makes all efforts to monitor influenza and make public recommendations to the benefit of influenza vaccination particularly in high risk groups or in face of epidemics. egypt is one of the countries that have experienced a large epizootic of highly pathogenic avian influenza in poultry caused by the influenza a (h5n1) virus [18] . an integrated national plan for avian and pandemic influenza was developed in response to the rapid spread of avian influenza in this country. national influenza centers equipped with tests such as virus isolation, polymerase chain reaction (pcr), serology and sequencing exist and are currently functioning. the surveillance of severe acute respiratory infection (sari) is enhanced and there is also a significant increase in the available number of influenza vaccines doses. furthermore, several social mobilization and advocacy campaigns have been recently conducted. progress achieved so far and the main mid-and long-term objectives of the country are detailed in tables 1 and 2 respectively. influenza surveillance system has been set-up in 2004. the analysis of influenza a/h1n1pdm09 and a/h3n2 viruses collected in iran during the 2014-2015 provided evidence of co-circulation of several influenza a virus strains [19] . of the 200 influenza typing studies specimens, 80 were influenza a-positive, including 44 a/h1n1pdm09 and 36 a/h3n2, while 18 were influenza b-positive [19] . analysis of the a/h3n2 viruses showed a genetic drift from the vaccine strain a/texas/50/2012 with 5 mutations [19] . serological study among poultry workers from fars province of iran showed that exposure to avian h9n2 viruses had occurred in this population [7] . influenza surveillance system has been established since 2004 and an increased number of publications of influenza in peer-reviewed journals are among the most key actions achieved. progress achieved and country objectives for the upcoming 3 years are listed in tables 1 and 2 respectively. influenza surveillance has continued through the 2016-17 influenza season, during which nasopharyngeal swabs from 518 influenza-like illness (ili) cases were analyzed. the results showed circulation of both influenza b yamagata and victoria lineage viruses in addition to influenza a/h3n2 and a/h1n1pmd09, emphasizing the importance of introducing the quadrivalents influenza vaccine. among the study population, only 28% were vaccinated (unpublished data). analysis of flu vaccination uptake overtime showed relatively high vaccination coverage (40%-60%) until 2011 with a peak during the 2009 pandemic and decreased subsequently to reach 28% in 2016-17 (unpublished data). however, these figures mostly reflect the vaccination coverage of the population served by one major hospital in beirut. it was noted that further studies to include more representative population from throughout the country are needed to accurately reflect the vaccination coverage in the country. based on vaccine dose distribution, vaccination coverage in lebanon is estimated at 6%. increased flu vaccine coverage and increased laboratory capacity to isolate and detect influenza are the main successes that have been so far achieved. although vaccine uptake is improving, there is still a long way to reach optimal vaccination coverage in high-risk individuals. the search for advocacy continues and includes representatives from academic, public and the government. the main achievements and objectives are listed in tables 1 and 2 respectively. the number of available doses of influenza vaccine has increased by more than 5 folds since 2012. also, good progress has been achieved in surveillance, social mobilization and advocacy. progress achieved and the main objectives are listed in tables 1 and 2 respectively. influenza is one of the moh priorities in morocco. sentinel surveillance exists since 1995. however, influenza burden is not well understood. currently, 378 health centers in all districts and 80 private physicians in 9 cities are involved in the ili surveillance system. overall, 8 regional laboratories are in charge of detection and identification of influenza strains by reverse transcriptase pcr (rt-pcr.) the national institute of hygiene (rabat) and the pasteur institute (casablanca) have the capacity to perform rt-pcr, sequencing, virus isolation, and antiviral susceptibility screening. of the overall 178 samples analyzed in casablanca during the 2016-17 influenza season 95.5% belonged to type a(h3n2) and 4.54% were victoria b lineage. the main objectives for the upcoming years are listed in table 2 . oman sari surveillance has been launched at sentinel sites since 2008 and the central public health laboratory is recognized by the who as national influenza center (nic) since march 2009. in 2017, 648 patients were enrolled in sari surveillance. the highest numbers of sari cases were recorded among children 0-2 years old followed by those 2-4 years of age. the overall mortality rate due to influenza was 9.3% but no death was reported among pregnant women. influenza strains a (h1n1)pdm09, a(h3n2), and b viruses were detected during this season. influenza vaccine is recommended for hcps working in critical and non-critical units, administrative agents and educational institution staff. pre-vaccination awareness emails sent to hcps to inform them about the availability of the vaccine and the beginning of the vaccination campaign at the hospital. in addition, the awareness campaign includes presentations, person-person communication, pamphlet, banner, etc. vaccinators visit the wards to offer the vaccine. electronic and manual records are maintained at the institutional, governorate and national level. regarding pregnant women, influenza vaccination is integrated into the antenatal program and administered during antenatal visits. pre-vaccination counselling is also provided. furthermore, influenza advocacy campaigns provide awareness to the family and pregnant woman. the main progress achieved and future objectives are detailed in tables 1 and 2 respectively. currently, influenza is not considered as a health priority in pakistan. there is a distinct seasonality with peak activity levels observed in most regions during the winter season. available data indicates that there is a sizeable burden of influenza. however, influenza related morbidity and mortality estimates are not well known. in the key achievements include the implementation of sentinel influenza surveillance networks and the reinforcement of social mobilization and advocacy (table 1) . country specific vaccination priorities must be determined for policy recommendations. the main objectives are listed in table 2 . historically, vaccination coverage has been than 2% saudi arabia but higher rates are seen among hcps, pregnant women, elderly, haji and patients with chronic diseases. influenza surveillance system has been recently set-up in hospitals and public health centres in six regions. in order to increase vaccination coverage, a 3-phase strategic project (3p) has been launched in 2014 with the aim of reaching 30% vaccination coverage among at-risk population in 5 years scope. several public awareness campaigns have also been conducted and the moh is funding flu vaccination. the main achievements and future objectives are detailed in tables 1 and 2 respectively. the panel discussion focused on exploring ways to increase influenza vaccination coverage in the participating countries and involving the moh in the network. lack of knowledge about influenza, its consequences in terms of morbidity, mortality and economy is the major barrier to attaining higher influenza vaccination coverage in the mena-isn countries. as stressed by the participating countries, under awareness about influenza could itself be related to communication gaps. tailored communication messages to the community to promote influenza vaccination and to increase awareness on influenza infection have been put in place in several countries. this includes advertising panels in the streets, lay public activities in shopping malls and airports, bulk messages, social media campaigns, etc., but more needs to be done. besides lack of knowledge, other factors such as misbeliefs and personal experiences could lead to low vaccine uptake. participating countries concluded that a better understanding of the factors underlying hesitancy and acceptability of the influenza vaccine is needed to devise evidence-based communication campaigns aimed at increasing vaccination coverage. the adaptation of who-sage available tools is each country has been suggested as an appropriate means to determine the underlying determinants of vaccine hesitancy in each country. the cost of the vaccine is another key barrier that could contribute to low vaccination coverage. country representatives concluded that depending on the economic situation, countries should primarily focus on increasing vaccination coverage among known high-risk groups (diabetics, pregnant women, people with underlying chronic diseases, etc.). some countries believed that even within the high-risk groups, a priority list should be drawn according to influenza burden in each group and each country. from country presentations, it was evidenced that the use of private health care facilities is more usual than the public facilities. nevertheless, a lack of communication between these two sectors was noted. strategic communication by mena-isn and the use of uniform terms of references for local stakeholders could help in enhancing the involvement of moh and the private sector thereby improving the situation for vaccine implementation. as reflected by individual country presentations, all mena-isn countries share the goal to reduce influenza related morbidity and mortality. vaccination coverage has slightly increased in all participating countries, yet more work should be done to reach the optimal vaccination coverage. the main challenges to increase vaccine coverage were: under awareness about influenza, financial and political issues, and limited collaboration between private and public health sectors. country representatives agreed on the need to design studies aimed at accurately estimating vaccination coverage among different populations and risk groups and assessing the impact of vaccination on hospitalization, mortality and herd immunity. this can be presented to the moh to develop evidence-based policies or policy updates. the data can be also communicated with the public to increase their awareness and willingness for influenza vaccination. pharmacies can play a fundamental role as they are easily accessible, but policies in some countries prevent pharmacies from administrating vaccines. in parallel, region specific information about vaccine efficacy, economic impacts of influenza (absenteeism, hospitalization, etc.) should be provided in order to provide more confidence about influenza vaccine. incentives for general practitioners (gps) could also be useful in mena-isn countries and should be considered [17] . participating countries recommended the following steps to move forward • develop action plans tailored to each country situation by focusing on four main areas: (i) epidemiological and virological surveillance; (ii) vaccination; (iii) communication/awareness and (iv) advocacy • expand the influenza stakeholder network to involve decisionmakers including moh, gps, patients, industry, etc. • reinforce local influenza network and advisory groups • set-up research agenda to generate country and region-specific data • identify priority areas to investigate the best way to move forward • promote and increase collaborative research among the mena-isn countries • encourage publication and sharing of existing data • set-up a public mena-isn website to increase its visibility at national and international levels • improve/reinforce the link between mena-isn and who-emro pandemic influenza preparedness framework. the meeting was made possible thanks to fondation mérieux and unrestricted grants from sanofi pasteur. seasonal influenza vaccination coverage rates in 10 countries in africa seasonal influenza vaccine dose distribution in 157 countries epidemiology of seasonal influenza in the middle east and north africa regions, 2010-2016: circulating influenza a and b viruses and spatial timing of epidemics pathogenicity of highly pathogenic avian influenza virus h5n1 in naturally infected poultry in egypt cumulative number of confirmed human cases for avian influenza a(h5n1) reported to who influenza research in the eastern mediterranean region: the current state and the way forward serological evidence of h9n2 avian influenza virus exposure 7th mena-isn study group avian influenza outbreak in poultry in the lebanon and transmission to neighbouring farmers and swine cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during 2012-2015. influenza other respir viruses occurrence of the middle east respiratory syndrome coronavirus (mers-cov) across the gulf corporation council countries: four years update middle east respiratory syndrome coronavirus (mers-cov) summary and literature update-as of 11 benefits of flu vaccination for persons with diabetes mellitus: a review economic development and diabetes prevalence in mena countries: egypt and saudi arabia comparison global role and burden of influenza in pediatric respiratory hospitalizations, 1982-2012: a systematic analysis influenza vaccination in turkey: prevalence of risk groups, current vaccination status, factors influencing vaccine uptake and steps taken to increase vaccination rate influenza a and b infection in children in urban slum seasonal influenza vaccine dose distribution in 195 countries (2004-2013): little progress in estimated global vaccination coverage microevolution of highly pathogenic avian influenza a(h5n1) viruses isolated from humans molecular characterization and phylogenetic analysis of human influenza a viruses isolated in iran during the 2014-2015 season mg, it, km, ma, bu, and tp are employee of sanofi pasteur. others authors do not have any conflict of interest to declare. key: cord-331148-40gvay7i authors: hsieh, yu-chia; tsao, kuo-chien; huang, ching-tai; chang, kuang-yi; huang, yhu-chering; gong, yu-nong title: clinical characteristics of patients with laboratory-confirmed influenza a(h1n1)pdm09 during the 2013/2014 and 2015/2016 clade 6b/6b.1/6b.2-predominant outbreaks date: 2018-10-23 journal: sci rep doi: 10.1038/s41598-018-34077-4 sha: doc_id: 331148 cord_uid: 40gvay7i a novel pandemic influenza a(h1n1)pdm09 virus emerged in 2009 globally, and it continues to circulate in humans. the national influenza surveillance network in taiwan identified five a(h1n1)pdm09-predominant seasons, representing the 2009/2010, 2010/2011, 2012/2013, 2013/2014, and 2015/2016 outbreaks from 2009 to 2016. independently, a retrospective cohort study (which enrolled 639 infected patients during the five seasons) was conducted at chang gung memorial hospital to explore the risk factors associated with influenza a(h1n1)pdm09-related complications. a phylogenetic analysis of hemagglutinin (ha) sequences showed that the circulating a(h1n1)pdm09 virus belonged to clades 1, 2, and 8 in 2009/2010; clades 3, 4, 5, and 7 in 2010/2011; clades 7 and 6c in 2012/2013; clades 6b in 2013/2014; and 6b/6b.1/6b.2 in 2015/2016. compared to individuals infected in non-6b/6b.1/6b.2 seasons (2009/2010, 2010/2011, and 2012/2013), those infected in 6b/6b.1/6b.2 seasons (2013/2014 and 2015/2016) were at higher risk for influenza-related complications (adjusted odds ratio [aor]: 1.6, 95% confidence interval [ci]: 1.0–2.8), pneumonia (aor: 1.78, 95% ci: 1.04–3.04), mechanical ventilation (aor: 2.6, 95% ci: 1.2–5.6), and acute respiratory distress syndrome (aor: 5.5, 95% ci: 1.9–15.9). for the increased severity of infection during the influenza a(h1n1)pdm09 clade 6b/6b.1/6b.2 seasons, aspects related to the antigenic change of a(h1n1)pdm09 virus, immune response of the host, and environmental factors required further investigation. scientific reports | (2018) 8:15636 | doi: 10 .1038/s41598-018-34077-4 number of severe cases and outcomes in the groups at risk and healthy young adults; these events were associated with a(h1n1)pdm09 clade 6b.1 infection 5, 6 . since 1999, the taiwan centers for disease control (cdc) has established a nationwide surveillance system requiring contract virologic laboratories to perform continuous virologic surveillance for respiratory viruses, particularly influenza and enteroviruses; this system was established after an epidemic of enterovirus 71 in 1998 7 . the long-term national influenza surveillance network described the epidemiologic pattern of circulating viruses, and it has successfully identified the outbreaks of severe acute respiratory syndrome (sars)-associated coronavirus and adenovirus 8, 9 . moreover, it also identified the novel h7n9 and h6n1 influenza viruses 10 were significantly different between each season (table 1) . a phylogenetic analysis of hemagglutinin (ha) sequences recovered in these epidemics, along with geographically diverse global influenza a(h1n1) pdm09 viral sequences, has revealed that the sequences are members of clades 1, seasons versus non-clades 6b/6b.1/6b.2 seasons, the patients were classified into two groups. the median (interquartile range, iqr) age of patients in the 6b/6b.1/6b.2 season was older than that in the non 6b/6b.1/6b.2 seasons ( table 2 ). the number of infected individuals aged 50-64 years was higher in 6b/6b.1/6b.2 seasons than that in the non-clade 6b/6b.1/6b.2 seasons ( table 2 ). the rate of underlying conditions; complications, including pneumonia and acute respiratory distress syndrome (ards); icu admission; respiratory failure with mechanical ventilation; 30-day mortality; and in-hospital mortality in 6b/6b.1/6b.2 seasons were significantly higher than that in non-clade 6b/6b.1/6b.2 seasons ( table 3 ). the rate of underlying conditions; complications, such as ards; icu admission; and respiratory failure with mechanical ventilation in 6b/6b.1/6b.2 seasons was significantly higher than that in non-clade 6b/6b.1/6b.2 seasons (table 3) . the results of the logistic regression analysis on the risk factors associated with influenza a(h1n1)pdm09-related complications and pneumonia are shown in table 4 , and respiratory failure with mechanical ventilation and ards are also presented in table 5 . in the univariate analysis, 6b/6b.1/6b.2 season, age (50-64 years), onset to presentation, underlying conditions, obesity, smoking, alcoholism, and antiviral therapy were significant risk factors of complications, pneumonia, mechanical ventilation, and ards (tables 4 and 5 ). in the multivariate logistic regression analysis, 6b/6b.1/6b.2 season, age (50-64 years and ≥65 years), underlying conditions, and antiviral therapy were significant independent risk factors of complications, pneumonia, and mechanical ventilation (tables 4 and 5). only 6b/6b.1/6b.2 season and obesity were considered as significant independent risk factors of ards ( table 5 ). the effect of 6b/6b.1/6b.2 season on the total number of influenza-related complications was not significant in children aged ≤5 years. however, it was significantly stronger among individuals aged ≥6 years (table s1 ). among the hospitalized patients with laboratory-confirmed influenza a(h1n1)pdm09 infection, male patients and those with underlying conditions were significantly at risk for 30-day mortality (overall death within the first 30 days after hospital admission) and all-cause in-hospital mortality (overall death during hospital admission) as assessed using the multivariable cox proportional hazard model ( table 6 ). the same analysis showed that season was not associated with an increased risk for 30-day and all-cause in-hospital mortality (table 6 ). the during the 2013/2014 season, an unusually high hospitalization rate in adults aged 50-64 years was observed in the united states and mexico 3, 16 . the increased morbidity in middle-aged adults during the 2013/2014 season had been attributed to the low vaccination rate in this age group 16 . however, that hypothesis cannot explain the unusual number of severe cases because the vaccination rate had already been low during the previous years 16, 17 . an interesting study has shown that up to 42% of middle-aged adults born between 1965 and 1979, who had been exposed to seasonal h1n1 viruses circulating in 1977, had reduced serologic reactivity with the 2013/2014 a(h1n1)pdm09; notably, the 2013/2014 virus harbors the distinctive k163q ha antigenic mutation 18 . in the cohort of individuals born between 1965 and 1979, ha-specific antibodies with activity against a(h1n1)pdm09 must have been produced and shaped by exposure to prior-season h1n1 viruses (the so called "original antigenic sin") 19 . nonetheless, the ha-specific antibodies in this cohort failed to recognize the 2013/2014 a(h1n1) 25 . compared to the a/california/7/2009 vaccine virus, viruses of clade 6b harbor d97n, k163q, s185t, k283e, and a256t substitutions in ha1. viruses of subclade 6b.1 harbor further amino acid substitutions s84n, s162n, and i216t and 6b.2 and carry amino acid substitutions v152t and v173i 20 . more extensive studies must be conducted to identify the potential antigenic differences between clade 6b and subclade 6b.1/6b.2; such studies are expected to improve our understanding of how a(h1n1)pdm09 evolved (and continues to evolve) and how it affects and interacts with the human immune system. in the 2017/2018 season, the who has selected a new vaccine virus, which is the a/michigan/45/2015 (h1n1)pdm09-like virus (a member of the 6b.1 subclade), as the influenza vaccine virus component for the northern hemisphere. the national influenza surveillance network coordinated by the taiwan cdc was established more than 10 years ago. policies favoring government funding for vaccines and antiviral agents have been consistent during the subsequent intervals. between 2009 and 2015, government-funded vaccines have been administered primarily to those aged 6 months to 12 years, elderly individuals aged ≥65 years, healthcare workers, and individuals with underlying diseases. individuals aged 13-64 years were not included in the government-funded vaccination program. elementary school children aged 7-12 years had the highest influenza vaccination rate, with coverage reaching 60-70% annually 11 . the present study is limited by its observational nature and the incorporation of a retrospective investigation. a potential bias may exist due to the exclusion of all cases with a(h1n1)pdm09 infection for 7 years. nonetheless, no change was observed in terms of admission or management procedures during these outbreaks. the surveillance and reporting system in taiwan has long been established. taken together, the increased frequency of complications in 2013/2014 and 2015/2016 is unlikely due to detection bias. in addition, the major drawback of this study was the lack of documentation about the history of influenza vaccination in the records used to generate this study. however, a study by taiwan cdc has reported that 95% of patients with complications in the 2015/2016 season had not received the influenza vaccine 26 . the vaccine coverage rate in non-elderly adults and elderly individuals would have been low during each of the outbreaks, particularly during the first wave, given that no a(h1n1)pdm09 vaccine was available in 2009/2010. thus, the increased severity of influenza during the 2013/2014 and 2015/2016 seasons is unlikely to reflect a decreased rate of vaccination. the study has shown that taiwan experienced the greatest burden of influenza-related complications due to a(h1n1)pdm09 clades 6b/6b.1/6b.2 in the sixth year of its circulation. the reasons for the increased impact of influenza-related complications remain uncertain. aspects related to the antigenic change of a(h1n1)pdm09 virus, immune response of the host, and environmental factors required further investigation. this report shows the importance of influenza disease surveillance and requires that the influenza a(h1n1)pdm09 virus should always be considered. national influenza surveillance network. the network consists of eight regional commissioned laboratories located in the northern (n = 3), central (n = 2), southern (n = 2), and eastern (n = 1) parts of taiwan. these laboratories have steadily collected more than 8000 respiratory specimens for surveillance per year, including more than 1000 influenza virus specimens annually, all of which are sent to the taiwan cdc for the monitoring of influenza viral activity. the taiwan influenza express, a weekly online influenza surveillance report, has been published by the taiwan cdc from july to may of each year since 2005 (http://www.cdc.gov.tw/english/ submenu.aspx?treeid = 00ed75d6c887bb27&nowtreeid = 8f1e239d0fd8877a) 10, 27 . this report includes the total number of respiratory specimens; isolate number of influenza a(h1n1), influenza a(h3n2), and influenza b; and case number of laboratory-confirmed influenza cases in intensive care units (icus), a class of events that is considered a category 4 nationally notifiable disease. however, data on weeks 19-37 are not available annually. a confirmed case involved a patient who had acute influenza-like illness (temperature ≥ 38 °c with either cough or sore throat) and nasopharyngeal/throat or bronchoalveolar lavage samples harboring influenza a(h1n1)pdm09 virus as detected using real-time (rt) reverse-transcription polymerase chain reaction (pcr) assay or via viral culture 11, 28 . for the purposes of the present study, each season was defined as extending from july of the same year to may of the following year. the annual population figures provided by the department of household registration affairs of the interior ministry were used for the calculation of the incidence of laboratory-confirmed influenza a(h1n1)pdm09 cases in the icu. , a 4000-bed, university-affiliated teaching hospital that is located in northern taiwan and provides both primary and tertiary care. in addition, cgmh is one of the 8 regional commissioned laboratories of the taiwan cdc. patients who had acute influenza-like illness (temperature ≥ 38 °c with either cough or sore throat) and had influenza a(h1n1)pdm09 virus as detected using rt-pcr assay or via viral culture using respiratory specimens were included in the study. patients whose data are not available were excluded. the institutional review board of cgmht approved the study, and it was carried out in accordance with the relevant guidelines and regulations. informed consent was waived due to the study's retrospective nature. all medical records of the enrolled patients were reviewed. demographic characteristics, underlying medical conditions, clinical course, antiviral treatment (oseltamivir or zanamivir), mechanical ventilation, admission to an icu, and death were recorded using a structured questionnaire. body mass index (bmi), a measure of obesity, was calculated for patients whose height and weight data were available. obesity was defined as follows: 1) body weight ≥ 95th percentile in children < 2 years of age; 2) bmi ≥ 25 kg/m 2 in patients aged between 2 and 18 years; and 3) bmi > 28 kg/m 2 (chinese criteria) in patients > 18 years 29 . medical conditions associated with a high risk for influenza complications were defined based on those listed by the united states advisory committee on immunization practices 30 . patients with confirmed pneumonia on radiography, acute respiratory distress syndrome (ards), acute onset of cardiovascular, neurologic condition, respiratory failure with mechanical ventilation; those who were admitted in the icu; and those who died were considered to have influenza-related complications. pneumonia on radiography was diagnosed based on the presence of a consolidation, infiltrate, or opacity 31 . ards was defined according to the standard criteria 32 . the primary study outcome was the occurrence of (any) influenza-related complications. the secondary study outcomes were pneumonia, mechanical ventilation, ards, 30-day mortality, and in-hospital mortality. genetic characterization of the virus. a total of 82 isolated influenza a(h1n1)pdm09 virus were randomly selected for the analysis of viral hemagglutinin (ha) and neuraminidase (na) genes across the five seasons. the rna was extracted using the qiaamp viral rna mini kit (qigen, germany) according to the manufacturer's instructions. rt-pcr and primer pairs used for sequencing ha and na genes were performed, as previously described 33 . sanger sequencing of the viral ha and na genes was performed to establish clade designation and to detect differences in amino acid 33 . the obtained amplicons were assembled into a full-length 1,701-bp span for ha and 1410-bp for na using dnastar lasergene (dnastar, madison, wi). newly reported sequences in this study were deposited at the genbank database under the accession numbers shown in fig. s1 for ha and na genes. the evolution history was inferred by the maximum likelihood method based on the hasegawa-kishino-yano model 34 . the percentages of replicate trees (1,000 replicates) are shown next to the branches in which the associated taxa clustered together in the bootstrap test. phylogenetic analysis in this study was conducted using mega7 35 . statistical analysis. continuous variables were presented as medians and interquartile ranges (iqrs); categorical variables were presented as numbers and percentages. all analyses were performed using the statistical package for the social sciences software package version 22.0 (spss inc., chicago, il, the usa). the incidence rate ratio (irr) was generated using poisson regression with 95% confidence intervals to compare the rates of laboratory-confirmed influenza a(h1n1)pdm09 cases in the icu per 100,000 populations across different seasons; 95% confidence intervals for which the upper and lower bounds did not include 1 were considered as statistically significant. differences in categorical variables were compared using the chi-square test or a fisher's exact test. continuous variables were compared using the kruskal-wallis one-way analysis of variance test. multivariate logistic regression analysis and multivariate cox proportional hazards model were used for outcome analysis. the variables included sex, season, age group, onset to presentation, underlying condition, obesity, smoking, alcoholism, and antiviral therapy. variables with a p value < 0.1 in the univariate analysis were included in the multivariate model. the hosmer-lemeshow goodness-of-fit test was performed to assess the overall fit of the model. all statistical operations were two-tailed. p values ≤ 0.05 were considered statistically significant. world now at the start of 2009 influenza pandemic report prepared for the who annual consultation on the composition of influenza vaccine for the southern hemisphere influenza activity -united states, 2013-14 season and composition of the 2014-15 influenza vaccines report prepared for the who annual consultation on the composition of influenza vaccine for the southern hemisphere risk assessment of the 2015-2016 influenza season in thewho european region, week 40/2015 to week 04/2016 a major 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of influenza virus in taiwan risk factors for severe illness with 2009 pandemic influenza a(h1n1) virus infection in china prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip), 2009. mmwr recommendations and reports standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination amino acids transitioning of 2009 h1n1pdm in taiwan from dating of the human-ape splitting by a molecular clock of mitochondrial dna molecular evolutionary genetics analysis version 7.0 for bigger datasets this work was supported by a grant from national science council, taiwan, and three grants (grant cmrpg3f1871 and cmrpg3f1591 to yc hsieh, most 106-2320-b-182a-013-my3,nmrpg3g6071 to kc tsao) from the chang gung memorial hospital. y.c.h., c.t.h., y.c.h., and t.y.l. designed the study. k.c.t. and y.n.g. conducted the virologic characterization. y.c.h. and k.y.c. performed the statistical analysis. h.y.l. collected data. y.c.h. wrote the first draft of the manuscript, and all authors contributed to the final draft. all authors contributed to data interpretation and critically reviewed the manuscript. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-34077-4. the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-341364-dle938bt authors: thompson, catherine; zambon, maria title: influenza, respiratory syncytial virus and sars date: 2009-11-26 journal: medicine (abingdon) doi: 10.1016/j.mpmed.2009.10.003 sha: doc_id: 341364 cord_uid: dle938bt acute lower respiratory tract infections (lrtis) are a major worldwide health problem, particularly in childhood. about 30–50% of acute lrtis are viral in origin; of these, influenza and respiratory syncytial virus are associated with the greatest disease burden in humans. many different influenza a viruses occur naturally in animal reservoirs, and present a constant threat of zoonotic infections and global pandemics. the pandemic (h1n1) influenza virus that emerged in humans in 2009 contained a unique combination of genes originating in swine and the global human population was highly susceptible to the novel strain. the emergence of the severe acute respiratory syndrome coronavirus in 2003, and the ensuing worldwide epidemic, highlights the fact that respiratory viral infections in humans may originate in animals. preventative measures for influenza include annual vaccination and treatment with antiviral drugs such as the neuraminidase inhibitors oseltamivir and zanamivir. subtype-dependent resistance to antivirals can develop and should be closely monitored. influenza viruses are small (80e120 nm diameter), contain rna and are enveloped. there are three types e a, b, and c. type a is further classified according to the properties of the surface proteins haemagglutinin and neuraminidase. all a subtypes are found in aquatic birds, which are the natural reservoir ( figure 1) ; only a few subtypes circulate in humans and other mammals. type b and c influenza have only one subtype and are restricted to humans. influenza viruses circulating in humans (a h1n1, h3n2, b and c) cause respiratory tract disease. influenza a is generally considered to be clinically more severe than influenza b; influenza c causes only a mild illness confined to the upper respiratory tract. circulation of influenza a and b viruses in humans ( figure 2 ) causes unpredictable seasonal epidemics of disease in temperate climates, with excess population morbidity and mortality, usually occurring between october and march in the northern hemisphere and lasting about 6e8 weeks. 1 in the uk, 5000e10,000 deaths are associated with influenza a and b epidemics every year, and more than 20,000 in severe years. widespread pandemics of severe disease occur less frequently, occurring on at least three occasions in the last century (1918, 1957, 1968) , and have been associated with high mortality. in influenza a and b illness in humans ranges from subclinical or mild upper respiratory tract symptoms to more severe illness including laryngotracheitis and pneumonia or, less commonly, death from respiratory system failure. the most common presenting symptoms are cough, high temperature, joint pain and general malaise ( figure 3 ). the rapid onset and short incubation period (about 48 h) are characteristic, though incubation can last up to 4 days. individuals at greatest risk of complications are those with pre-existing cardiac and respiratory disease, the elderly, and those with impaired immune systems ( table 1 ). the severity of the illness reflects preexisting host immunity and the prevailing virus strain. protective immunity to influenza is conferred by antibodies. the ability of influenza to cause re-infections is related to the genetic mutability of the virus. in every replication round, mutant viruses are generated, some of which have a growth advantage because they can partially evade host immune responses. variants capable of causing epidemics in susceptible populations emerge by a process termed 'antigenic drift'. new influenza a drift variants arise every 2e3 years; influenza b drift variants arise every 4e5 years. the segmented nature of the influenza genome allows reassortment of segments when a single host is infected with more than one virus. this occurs regularly in aquatic birds, in which almost all combinations of influenza a virus segments can be detected, but is less common in mammals. it was previously thought that, for a novel subtype of influenza to arise in humans, reassortment of two virus subtypes had to occur in a mammal that could then transmit to humans. the pig was considered a suitable 'intermediate host', because both avian and human viruses, which differ slightly in their surface receptor requirements, can replicate in pigs. however, following transmission of h5n1 directly from birds to humans on 18 occasions in hong kong in 1997, and further zoonotic infections involving several different subtypes of influenza (table 2) , it is evident that cross-species barriers to transmission may be less stringent than was thought. nevertheless, the requirements for adaptation of avian viruses to mammals are poorly understood. cross-species transmission of novel subtypes into susceptible human populations (antigenic shift) are thought to be the source of pandemics of influenza. influenza pandemics occur when a novel influenza virus, against which there is little or no pre-existing immunity, emerges and spreads in the human population. in april 2009 a novel influenza virus originating from swine was identified in humans in mexico and the united states. 2 individual cases were rapidly detected in other countries and following the onset of community transmission the world health organization (who) declared the start of the 2009 influenza pandemic. the pandemic (h1n1) 2009 virus has a unique combination of genes obtained from swine viruses from north america and eurasia. substantial antigenic differences between the seasonal h1n1 virus that has circulated incidence (%) figure 3 groups at increased risk of complications of influenza c chronic respiratory disease c chronic heart disease c chronic renal failure c diabetes c immunosuppression from disease/treatment c asplenia/splenic dysfunction c frail elderly with co-morbidity since 1977 and the pandemic h1n1 virus meant the human population was highly susceptible to the new virus and it quickly became the dominant influenza strain circulating worldwide. it caused an unusual peak of influenza activity in many northern hemisphere countries, including the uk, during the summer months when few influenza detections are usually made ( figure 4 ). in the southern hemisphere during the normal winter influenza season, the pandemic strain rapidly became dominant. disease caused by the pandemic (h1n1) 2009 influenza virus is of moderate severity. presentations range from mild, selflimiting illness, with typical symptoms of fever, cough and sore throat, to severe disease requiring hospitalization. younger people appear to be more commonly affected, particularly for severe or fatal disease. this is in stark contrast to seasonal flu which generally has a greater impact on the over 65 years age group. severe respiratory failure has been seen in a small proportion of cases and certain underlying medical conditions may present an increased risk of developing severe and fatal disease ( table 2 ). an increased risk for complications during pregnancy has also been documented. 3 the presence of a large, mobile animal reservoir of influenza a virus suggests that eradication of this agent will be impossible. control strategies focus on limiting the opportunities for crossspecies transmission of novel subtypes; for example: housing domestic poultry in shelters to avoid contact with overflying migratory birds eliminating/reducing live bird markets housing aquatic birds and domestic poultry separately slaughtering domestic flocks infected with highly pathogenic influenza a viruses. these measures may achieve some success in preventing zoonotic transmission of influenza a to humans, but have little impact on its annual cycle. unprecedented levels of h5 circulating in domestic poultry in south east asia and elsewhere present a high risk for emergence of a novel pandemic influenza a strain. antibodies against haemagglutinin (and, to a limited extent, neuraminidase) can prevent disease caused by the same strain of virus. this is the basis of vaccination for influenza. currently, most vaccines used worldwide are subunit vaccines. however, bird-to-human transmission place the high variability of influenza virus means that antibodies to one strain confer only limited protection against drift variants. thus, influenza vaccines are given annually before the influenza season to boost pre-existing immunity, and the composition of the vaccine is updated regularly. in developed countries, the benefit of immunization has led to expansion of age-related vaccination policies. in the uk in 2000, vaccination was introduced for all individuals over 65 years of age, irrespective of preexisting illness. vaccination rates vary considerably between countries. there is increasing interest in vaccination of children. 4 live attenuated influenza vaccines are particularly suitable, because they induce broader, long-lasting immunity. child vaccination may also help to prevent transmission in the community in general. development of candidate pandemic vaccines and extensive testing of protocols as part of pandemic preparedness measures meant that vaccines for pandemic (h1n1) 2009 virus could be rapidly developed following the emergence of the novel virus. despite the preventive efficacy of vaccination, the need for treatment of severe influenza remains. amantadine (or the related compound rimantadine) was, until recently, the only anti-influenza drug available. it selectively targets a viral protein (m2) and inhibits viral replication, but its use has been limited in the last 30 years, partly because of side effects (dizziness, confusion) that particularly affect the elderly, and also because drug-resistant mutants arise frequently and can be readily transmitted. neuraminidase inhibitors are a more recently developed, novel class of anti-influenza compounds (table 3) . 5 they act on viral neuraminidase, prevent release of virus particles from infected cells, and are likely to be most efficacious when given early in illness. since 2000, the uk national institute for clinical excellence has recommended that neuraminidase inhibitors may be used for treatment and prophylaxis, with certain restrictions. the neuraminidase inhibitor drugs have been used for treatment of pandemic (h1n1) influenza in community and hospital settings. oseltamivir was widely used in the uk for treatment of confirmed cases and prophylaxis of close contacts during the early stages of the pandemic. as community transmission increased in the uk during july 2009, oseltamivir was prescribed to people diagnosed with influenza through the national pandemic flu service telephone health helpline (figure 4 ). neuraminidase inhibitors are frequently used for treatment of critically ill patients with pandemic influenza. during 2007 resistance to the neuraminidase inhibitor drug oseltamivir was detected in seasonal h1n1 influenza virus. 6 the resistant strain was found globally in people who had not been treated with the drug. clinical presentation did not appear to differ from infection with sensitive viruses, but circulation of the resistant strain had an impact on antiviral management of patients. the mutation conferring resistance was the well characterized h275y change in the viral neuraminidase gene, which confers resistance to oseltamivir while sensitivity to zanamivir remains. widespread community circulation of resistant virus was unexpected as it was previously shown that viruses with these mutations were at a disadvantage for replication and transmission. further concern was also due to the fact that the neuraminidase inhibitors, and oseltamivir in particular, are the drugs of choice for clinical use against seasonal influenza and zoonotic h5n1 infections and were stockpiled by governments as a pandemic preparedness measure. the pandemic (h1n1) influenza virus that subsequently emerged in april 2009 was sensitive to the neuraminidase inhibitor drugs and oseltamivir was widely used for treatment and prophylaxis. sporadic detections of neuraminidase inhibitor resistant pandemic virus have been made in treated patients, often in immunocompromised people who can shed virus for a prolonged period. rsv ( figure 5 ) is a negative-sense, non-segmented, enveloped rna virus of 100e300 nm diameter. it is best known as a cause of bronchiolitis in infants, but can cause respiratory tract infection in all age groups. upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis) range in severity from subclinical infection to pneumonia and death. more than 60% of children have been infected with rsv by their first birthday and more than 80% by 2 years of age; thereafter, individuals are infected approximately every 3 years. rsv infections in adults are probably under-recognized, and the severity of rsv infection in the elderly may be much underestimated as a cause of pneumonia. about 5% of elderly individuals are thought to become infected with rsv every year. transmission of rsv is primarily through large aerosol droplet or secretions, causing widespread nosocomial infection. outbreaks in adult and paediatric facilities are difficult to control. rsv infection in immunocompromised individuals is severe and lifethreatening; mortality may be 50e70% in adult bone marrow transplant recipients, in whom viral shedding may be prolonged. pathophysiology rsv infects the respiratory epithelium, leading to increased goblet cell production of mucus. dying infected ciliated epithelial cells combine with mucus to form plugs that block the airways; the consequences are most severe in very small babies with narrow airways. this leads to the characteristic signs and symptoms of atelectasis and the clinical syndrome of bronchiolitis. in rsv bronchitis and pneumonia, the peribronchiolar and interstitial infiltrate is characteristically lymphocytic. the severity of rsv infection is related to age. in young infants, the illness is seldom asymptomatic and lasts for 1e3 weeks. early signs of infection may include difficulty in feeding, nasal congestion, cough and otitis media compatible with urti. fever is often but not invariably present in rsv infection. abnormal breath sounds, tachypnoea and hypoxaemia suggest lower respiratory tract involvement. bronchiolitis and pneumonia are the two primary manifestations of progression to lrti; they may be difficult to distinguish and can occur simultaneously. the clinical features of bronchiolitis are wheezing and hyperaeration, and these are characteristic of infants with rsv infection. in the usa, rsv is estimated to cause 4500 deaths per year in children under 2 years of age. the risk of hospitalization in otherwise healthy under-2s is 0.5e2%, and 10e20% of children admitted to hospital require mechanical ventilation. these rates are higher in the first 6 months, and may be higher still in children with underlying acquired or congenital cardiopulmonary disease. older children and adults with rsv infection or re-infection usually have a milder or asymptomatic respiratory infection with a lower likelihood of lrti. adults with rsv-associated respiratory tract infections may experience prolonged symptoms. disease in the elderly may be particularly severe; up to 50% develop pneumonia. rsv infection is often diagnosed on the basis of the clinical features. the certainty of the diagnosis is increased when rsv is known to be circulating in a seasonal epidemic. chest radiography findings are non-specific and commonly include hyperaeration and peribronchial thickening, with areas of consolidation and interstitial infiltrates in patients with rsv pneumonia. there is a range of respiratory findings in immunocompromised adults, including pleural effusions. laboratory diagnosis depends on detection of viral antigen in respiratory secretions by immunofluorescence, rapid antigen tests or culture of the virus. serological tests are of little help in diagnosis of rsv infection, because they rely on the use of paired acute and convalescent sera. premature and very young infants are more likely to suffer acute apnoeic episodes and require assisted ventilation. bronchiolitis and pneumonia are the major complications of rsv disease in young children. children with congenital heart disease or chronic lung disease, and immunocompromised children and adults, are also at risk of severe disease. pneumonia is the major complication in adults and the elderly. estimates of the incidence range from 10% in nursing homes to 55% in a hospital in-patient population; estimated mortalities in the elderly are 3e5% in the former and 10e20% in the latter. rsv infection has very high mortality (50e70%) in severely immunocompromised individuals. supportive care is the mainstay of management of rsv disease in infancy. maintenance of oxygenation, hydration and nutrition is essential in hospitalized patients, and ventilatory support may be necessary in severe cases. a trial of bronchodilators may be beneficial. controlled trials of corticosteroids and vitamin a supplementation have not proved efficacy in infant rsv disease. there are two subtypes of rsv e a and b. the surface glycoproteins f and g are the major antigens of the virus to which neutralizing antibodies are directed. protective immunity to rsv is complex. antibodies generated during natural infection are not necessarily protective. a high proportion of primary rsv infections occur before 6 months of age, when maternal antibody levels are highest. overall, current (controversial) data suggest that neutralizing antibody to rsv is beneficial. neutralizing antibody titres in human sera correlate inversely with the likelihood of hospitalization as a result of rsv infection, and neutralizing antibody titre correlates with a reduced risk of re-infection. immunoglobulin infusions with high neutralizing titres of antibody to rsv (rsvig, table 4 ) have been used to treat rsv illness in normal-risk and high-risk infants. in normal infants, there is little evidence to justify rsvig for treatment of rsv infection. however, prophylaxis may be useful in high-risk infants (e.g. those with bronchopulmonary dysplasia). assessment of risk is important, because intravenous rsvig is contraindicated in congenital cyanotic heart disease. recombinant humanized rsv monoclonal antibodies are now available for intramuscular treatment and prophylaxis of rsv. early clinical data suggest that this preparation of passive antibodies may have wider application, with fewer limitations than rsvig; however, it is extremely expensive. few antiviral drugs are available for rsv. ribavirin is a guanosine analogue that has been used widely, though its precise mode of action is uncertain and many clinical studies show conflicting results. several studies have raised further doubts about the clinical effectiveness of ribavirin in infants and children at risk of severe rsv disease, and in ventilated children. in most centres, its use is now restricted to the immunocompromised or severely ill. several rsv vaccines (live attenuated, subunit and recombinant) are undergoing clinical trials. development of safe vaccines has been impeded by poor understanding of the factors governing immune protection in different age groups, 7 and early vaccination attempts in which more severe disease was seen in vaccinees. it is likely that there will be significant progress in future and that the types of vaccines suitable for different age groups may differ. peak virus shedding is 7e12 days after the onset of illness ( figure 6 ). the virus is found in various body fluids. infectious virus has not been recovered later than 21 days after illness onset. neutralizing antibody is detected from about 10 days post-onset. risk factors for sars cov infection are: close contact with civet cats/racoon dogs eating/preparing civet meat laboratory work with sars cov contact with a known case of sars. presentation is with fever and respiratory illness with cough and shortness of breath, progressing to acute respiratory distress syndrome and death in 10% of cases. fatalities increase significantly over the age of 40 years; mortality is up to 40% in the over-50s. onset of illness is 2e10 days post-infection, with a mean of about 5e6 days. about 60% of patients suffer later gastrointestinal symptoms of diarrhoea and vomiting. specific control measures have not yet been developed, though work is in progress on antiviral drugs and suitable vaccines. during the 2003 epidemic, various non-specific therapeutic measures were used with variable success: corticosteroids antimicrobials to prevent secondary bacterial infection positive-pressure ventilatory support anti-inflammatory drugs infusion of antisera from convalescent patients. the epidemic was controlled mainly through public health measures such as contact-tracing and quarantining. this policy was effective because there is little evidence of transmission before symptom onset, so infected individuals are easily identified. a use of intravenous respiratory syncytial virus immunoglobulin c consider for prophylaxis in infants < 2 years receiving oxygen therapy for bronchopulmonary dysplasia c infants born < 32 weeks with bronchopulmonary dysplasia are likely to benefit from 6e12 months' prophylaxis c infants born > 32 weeks with bronchopulmonary dysplasia may not benefit c should not be used in cyanotic congenital heart disease c not evaluated in paediatric or adult immunocompromised patients c main emphasis in nosocomial outbreaks should be infection control; efficacy is improved in such settings c initiate treatment before onset of respiratory syncytial virus season c defer live virus vaccines (e.g. mmr) until last dose the contribution of influenza to combined acute respiratory infections, hospital admissions and deaths in winter novel swine-origin influenza a virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans h1n1 2009 influenza virus infection during pregnancy in the usa the japanese experience with vaccinating school children against influenza neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir the potential impact of neuraminidase inhibitor resistant influenza age related differences in humoral immune response to respiratory syncytial virus infection in adults newly discovered coronavirus as the primary cause of severe acute respiratory syndrome key: cord-328525-80xk3gln authors: baier, claas; linderkamp, christin; beilken, andreas; thol, felicitas; heuser, michael; ebadi, ella; ganzenmueller, tina; heim, albert; bange, franz-christoph title: influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology date: 2018-09-24 journal: gms hyg infect control doi: 10.3205/dgkh000314 sha: doc_id: 328525 cord_uid: 80xk3gln introduction: respiratory syncytial virus (rsv) and influenza virus infections are a significant healthcare risk for immunocompromised patients. in addition to community onset, nosocomial acquisition and transmission may also occur. detection of asymptomatic shedders (e.g., patients in the incubation period or immunosuppressed long term shedders) facilitates control of nosocomial transmission. methods: to strengthen the existing infection control concept, a pcr-based screening for rsv and influenza virus was implemented for all patients lacking respiratory symptoms (asymptomatic patients) who were hospitalized on an adult and a pediatric hemato-oncological ward. laboratory results of this screening were analyzed retrospectively. results: 665 respiratory specimens were obtained for screening from 251 patients (26% were 18 years and younger) from december 2016 to april 2017. in 23 patients without respiratory symptoms, either influenza virus or rsv infection was found, resulting in a detection rate of about 9%. in 6 patients, the infection was presumably detected during the incubation period, because an increase of viral load was observed in subsequent specimens. positive screening results facilitated timely implementation of adequate infection control precautions. nosocomial clusters of rsv or influenza were not detected during the screening period on the two wards. conclusion: the seasonal screening program expanded our existing infection control concept in terms of patients lacking respiratory symptoms who shed influenza virus or rsv. it enabled us to identify 23 rsv or influenza infections in patients lacking respiratory symptoms in a 4-month period and thus to rapidly take isolation precautions. influenza virus (family orthomyxoviridae) and respiratory syncytial virus (rsv; family pneumoviridae) are both single-strand rna viruses and cause upper and lower respiratory tract infections in children and adults [1] , [2] , [3] , [4] . influenza virus and rsv are spread by droplet and contact transmission. therefore, nosocomial acquisition may occur due to exposure to another infected patient, visitor or healthcare workers (hcws), as well as by contact with contaminated, inanimate surfaces. immunocompromised patients with underlying hemato-oncological disorders or cancer are at risk for increased mortality, especially in the case of lower respiratory tract infection due to these viruses [5] , [6] , [7] . both community and nosocomial acquisition of influenza and rsv are possible in this patient group. nosocomial outbreaks in oncologic care facilities have been described [8] , [9] in spite of multiple infection control measures to prevent nosocomial transmission. these measures include stringent hand hygiene, patient isolation, and use of barrier precautions such as surgical masks or vaccination (influenza) [5] , [10] , [11] , [12] . during a localized nosocomial cluster with rsv at our clinic in the winter season 2015/2016, we introduced a rt-pcr-based screening of asymptomatic patients for rsv and influenza virus which contributed to the successful control of the rsv cluster [13] . to strengthen our existing infection control measures in hematology and oncology, we subsequently implemented a systematic influenza and rsv screening of patients lacking respiratory symptoms on a pediatric and an adult hemato-oncological ward as a prophylactic infection control measure in the following winter (2016/2017). this screening intended to identify patients shedding rsv or influenza virus without signs of disease and -in case of a positive screening test -to establish infection control measures similar to those used for typical symptomatic (e.g. cough, sneezing, fever, respiratory distress) patients with rsv or influenza. in this paper, we describe the laboratory results of this prophylactic screening program for asymptomatic patients (season 2016/2017) and discuss its value for infection control in hematology and oncology. the screening program for rsv and influenza was implemented on two wards located in the department of pediatric hematology and oncology and in the department of hematology, hemostasis, oncology and stem cell transplantation at hannover medical school. these departments are tertiary referral centers for children and adults, respectively, with hematologic and solid neoplasia. the pediatric ward contains 5 single (one-bed) and 5 twobed rooms. the adult ward has 4 single (one-bed) rooms, 6 two-bed rooms, and 3 four-bed rooms. all single rooms on the pediatric ward and 3 single rooms on the adult ward have an anteroom and high-efficiency particulate air filtration with the air flow directed to the hallway. single rooms are usually reserved for patients with severe immunosuppression or palliative settings. the wards are serviced by permanent healthcare workers (hcws) and housekeeping staff. parents are allowed to stay overnight with their children on the pediatric ward. during their stay on the ward, parents are permitted to wear street clothes. however, a regular change of their clothing is recommended. the pcr-based seasonal screening for rsv and influenza started in the second half of december 2016 and ended at the beginning of april 2017. the start and finish of the screening period were set jointly by the infection control staff, virologists and the clinicians involved based on available epidemiological data. on the adult ward, the screening was performed upon admission (admission screening) and once weekly (prevalence screening). on the pediatric ward, the pcr-based screening was performed once weekly. respiratory material was obtained from all patients lacking respiratory symptoms (asymptomatic patients). prophylactic prevalence screening was continued as long as patients stayed on the wards. patients with any symptoms of respiratory disease, e.g., cough, sneeze, fever or oxygen need (symptomatic patients), received a full diagnostic virology panel including influenza virus and rsv, and other respiratory viruses such as human metapneumovirus, adenovirus, parainfluenza virus, coronavirus and rhino-/enterovirus. in order to guarantee high adherence to the screening program, repeated audits and feedback talks took place. specimens were collected as a combined nasopharyngeal swab or pharyngeal lavage (oral wash) with sterile saline and were processed at the institute of virology. for realtime rt-pcr, rna was extracted using a qiaamp viral rna mini kit in a qiacube according to the manufacturer's instruction (qiagen, hilden, germany). cdna synthesis, amplification and detection of nucleic acid were performed in an applied biosystems ® 7500 real-time pcr system (life technologies, carlsbad, california, usa) using a commercially available one-step real-time rt-pcr kit (rsv/hmpv r-gene ® pcr kit, biomérieux, nürtingen, germany) according to the manufacturer's instructions, and an influenza a/b multiplex real time rt-pcr [14] . the influenza rt-pcr differentiates between types a and b. for semi-quantitative comparison of viral loads with follow-up specimens, the cycle threshold (ct) value of the real time pcr was used. a decrease of the ct by ≥4 values (usually equivalent to a more than 10-fold increase of viral load) was considered as a significant increase of viral load. infection control interventions for asymptomatic patients with rsv or influenza virus, detected by the screening infection control recommendations for asymptomatically infected patients with rsv and influenza virus were the same as for symptomatic patients. they were labeled in an electronic alert system, and it was recommended that the patients each be placed in a single (one-bed) room. each positive pcr test was electronically communicated to the infection control staff. contact and droplet precautions (surgical mask, gown, gloves) were mandatory for hcws and all visitors entering the room of affected patients. the patients were restricted to their single (onebed) room and were trained in hand hygiene. if they had to leave the room, they wore a surgical mask. two negative rt-pcr results at a minimum 2-day interval were required to stop all isolation precautions. hcws were strongly encouraged to let themselves be vaccinated against influenza before and during the winter season 2016/17. the vaccination rate among hcws for influenza was about 60% on the pediatric ward and about 30% percent on the adult ward. from december 2016 to april 2017, 251 asymptomatic patients were screened. 66 (26%) of these patients were aged 18 or younger and were screened on the pediatric ward. of the remaining 185 adult patients, 11 (5.9%) received swabs on the emergency ward, whereas 174 patients were screened exclusively on the adult hematooncological ward. overall, 665 screening samples were obtained, 23% of which (n=152) were from pediatric patients and 77% from adults (n=513). the mean screening sample number per patient was 2.3 for pediatric patients and 2.8 for adults. the majority (98%) of the screening samples were nasopharyngeal swabs (n=654). 11 samples were pharyngeal washes and were exclusively obtained from adult patients. altogether, 23 asymptomatic patients tested positive either for influenza or rsv in a screening sample, i.e., a combined detection rate of 9%. 11 patients (10 adults and one child) tested positive for influenza type a. 12 patients (5 adults and 7 children) were tested positive for rsv. co-infections with influenza virus and rsv were not detected. an overview is shown in table 1 . interestingly, in 6 patients (5 influenza virus patients and 1 rsv patient), a significant increase of the viral load in samples was observed following the positive screening specimen ( table 2) . course of viral load in 6 patients who showed an increase in viral load (presumed detection during the incubation period). the 6 patients showed an increase in viral load during their clinical course and were therefore presumably detected during the incubation period. 13 of the 23 (57%) asymptomatic patients were in twoand four-bed rooms at the time the screening test was reported positive (4 positive for rsv, 9 positive for influenza virus). 6 of these patients in shared rooms were immediately moved to separate single rooms, following the infection control recommendations. 2 patients with influenza were kept together, as they occupied the same 2-bed room when they simultaneously tested positive. 2 patients with rsv and 1 patient with influenza stayed in the shared rooms, and 2 patients had already been discharged when the test result became available. 10 of the 23 asymptomatic patients were already located in single (one-bed) rooms (for reasons other than infection control) and remained in this room after virus detection. for all patients identified (whether located in private or shared rooms after positive test results were available), barrier precautions (droplet and contact) were implemented as described above (see "materials and methods" section). on both wards, all patients presenting with symptoms of acute respiratory infections were routinely tested with a broad panel for respiratory viruses (including rsv and influenza). in these patients with symptoms of acute respiratory infection, we found 1 influenza case and 1 rsv case on the pediatric ward and 1 influenza case and 3 rsv cases on the adult ward from december 2016 to april 2017. prevalence of other respiratory viruses was low in this season on these two wards. 1 parainfluenza case, 6 human metapneumovirus cases and 4 rhinovirus cases were found on the pediatric ward. on the adult ward 1 parainfluenza case was detected. clusters of nosocomial transmission of rsv or influenza were not observed during the screening program period. respiratory tract infections caused by rsv and influenza virus are a significant healthcare risk for hospitalized and non-hospitalized patients with underlying hemato-oncological disease. during the winter season, acquisition of these viruses typically occurs in the community, and patients may be admitted with symptoms of infection or infection to a hospital. nosocomial acquisition due to transmission occurs as well [15] , [16] , [17] , [18] . recom-mendations and suggestions for controlling the spread of influenza and rsv in healthcare settings in general and especially in hemato-oncological patients exist (e.g. [5] , [10] ), but should be adapted to each hospital's specific needs. to extend our existing standard infection control measures, we introduced a prophylactic screening program for asymptomatic patients targeting rsv and influenza on an adult and a pediatric hemato-oncological ward. the screening was intended to detect patients that shed the virus without having signs or symptoms of disease. shedding my occur before the onset of disease (incubation period), during an asymptomatic clinical course, or following disease as a result of prolonged viral shedding, particularly in the immunocompromised host [19] . these groups of asymptomatic patients, who often remain unnoticed, can contribute to nosocomial spread. of the 23 asymptomatic shedders, we detected an increased viral load in 6 patients in follow-up specimens (see table 2 ). it is likely that these patients were in the incubation period. this group of patients is of particular interest for infection control strategies, since with increasing viral load, the risk of transmission also rises. we identified 23 of a total of 251 asymptomatic patients with an rsv or influenza virus infection (detection rate of about 9%).this means that about 11 asymptomatic patients needed to be screened to detect one shedder of either rsv or influenza virus. other studies found a similar prevalence in asymptomatic individuals for influenza (8.3%) and rsv (4.4%) using rt-pcr [20] , [21] . campbell et al. examined samples from asymptomatic and symptomatic patients prior to hematopoietic stemcell transplantation (hsct) in a study to evaluate the clinical outcomes associated with respiratory virus detection before hsct [22] . during a 5-year period, 308 asymptomatic adult and pediatric patients were screened, but only one patient with rsv and none with influenza virus were identified. interestingly, this study found other viruses (e.g., human rhinovirus or coronavirus) in 23 patients. these respiratory viruses were not included in our screening program, as they rarely cause severe lower respiratory tract disease. 13 out of 23 asymptomatic patients who tested positive for rsv or influenza virus in our study occupied shared patient rooms at the time of virus detection. the majority were moved to single rooms (one-bed rooms) or cohorting was used. droplet and contact precautions were used for all affected patients. isolation and barrier precautions regarding virus shedders is important, as transmission to roommates has been described [16] . the transfer to single (one-bed) rooms was challenging in some cases, due to the lack of single-room availability. nonetheless, because of the high mortality rate of rsv and influenza infection in hemato-oncological patients, isolation and barrier precautions were enforced. with these measures, we aimed to lower the risk of patient-to-patient transmission by reducing the exposure of contact patients. the usage of droplet and contact precautions (such as surgical mask and gown) was also intended to reduce exposure of hcws. all hcws, whether vaccinated against influenza or not, wore masks when they performed care in patients with rsv or influenza virus. a further benefit of early detection of influenza virus is the possibility of early antiviral therapy for infected patients and prophylaxis with antiviral agents for contact patients [5] . an additional effect of the screening program and the associated audits and feedback talks was the heightened awareness among hcws and visitors for respiratory viral infections in this highly susceptible, immunocompromised patient population. during the screening period, no nosocomial cluster of influenza virus or rsv was observed on the two wards. our investigation has limitations. first, it relied on virological laboratory data analyzed retrospectively. further information, such as the clinical course and outcome of the patients identified by screening, was not accessible for this study. we therefore could not correlate the laboratory results with the clinical course of the patients. viral loads were estimated semi-quantitatively by comparing ct values of real-time pcr between positive screening and follow-up specimens. however, this approach has been established for respiratory specimens by previous studies on influenza and various other pathogens [23] , [24] , [25] , [26] . multifaceted approaches are necessary to help to control the nosocomial spread of rsv and influenza in healthcare settings. infection control is based on standard precautions (e.g., compliance with hand hygiene, cough etiquette, etc.) together with rsv and influenza pathogenspecific precautionary measures (e.g. droplet precautions, isolation). in special high-risk settings such as hematology and oncology units, additional measures can be useful to lower the risk of hospital acquisition of rsv and influenza. therefore, we implemented a prophylactic rt-pcrbased rsv and influenza screening program targeting asymptomatic patients. this seasonal screening program enabled us to identify 23 patients who lacked symptoms for respiratory disease, but were infected with either rsv or influenza during a period of about 4 months. for these patients the same infection control practices were implemented as for symptomatic patients with rsv and influ-enza infection. in our view, this screening program proved useful for identifying asymptomatically infected patients with viral shedding, thus reducing the risk of transmission and potential nosocomial clusters of rsv and influenza virus on hemato-oncological wards. nevertheless, future studies on larger cohorts, including the analyses of clinical data, are necessary to further validate the use of a seasonal screening program as suggested here. competing interests respiratory syncytial virus -a comprehensive review systematic review of respiratory viral pathogens identified in adults with community-acquired pneumonia in europe community acquired respiratory virus infections in cancer patients -guideline on diagnosis and management by the infectious diseases working party of the german society for haematology and medical oncology respiratory viruses in transplant recipients: more than just a cold. clinical syndromes and infection prevention principles influenza virus infections in patients with malignancies --characteristics and outcome of the season 2014/15. a survey conducted by the infectious diseases working party (agiho) of the german society of haematology and medical oncology (dgho) consecutive yearly outbreaks of respiratory syncytial virus in a haemato-oncology ward and efficacy of infection control measures nosocomial outbreak of the pandemic influenza a (h1n1) 2009 in critical hematologic patients during seasonal influenza 2010-2011: detection of oseltamivir resistant variant viruses what transmission precautions best control influenza spread in a hospital? fourth european conference on infections in leukaemia (ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, and coronavirus effect of two-step hygiene management on the prevention of nosocomial influenza in a season with high influenza activity molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease diagnostic approach for the differentiation of the pandemic influenza a(h1n1)v virus from recent human influenza viruses by real-time pcr combining highresolution contact data with virological data to investigate influenza transmission in a tertiary care hospital active surveillance for influenza reduces but does not eliminate hospital exposure to patients with influenza laboratory-based surveillance of hospital-acquired respiratory virus infection in a tertiary care hospital practical prevention of nosocomial influenza transmission, "a hierarchical control" issue the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts influenza virus prevalence in asymptomatic and symptomatic subjects during pandemic and postpandemic periods respiratory syncytial virus evaluation among asymptomatic and symptomatic subjects in a university hospital in sao paulo, brazil in the period of 2009-2013. influenza other respi viruses clinical outcomes associated with respiratory virus detection before allogeneic hematopoietic stem cell transplant comparison of simplexa hsv 1 & 2 pcr with culture, immunofluorescence, and laboratorydeveloped taqman pcr for detection of herpes simplex virus in swab specimens a comparison of nasopharyngeal and oropharyngeal swabbing for the detection of influenza virus by real-time pcr assessment of the quantitative ability of advansure tb/ntm real-time pcr in respiratory specimens by comparison with phenotypic methods comparison of 2 real-time pcr assays for diagnosis of pneumocystis jirovecii pneumonia in human immunodeficiency virus (hiv) and non-hiv immunocompromised patients influenza and respiratory syncytial virus screening for the detection of asymptomatically infected patients in hematology and oncology the authors declare that they have no competing interests. we obtained ethical approval for this study from the ethics committee of the hannover medical school (number 3672-2017). this research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. we acknowledge the technical laboratory staff for the processing of screening specimens. all authors contributed to the manuscript according to the icmje (international committee of medical journal editors) recommendations: ah and cb were responsible for data acquisition. all authors were involved in analysis and interpretation of the data. tg and ah supervised laboratory diagnostics. cb and fcb prepared the manuscript. cb organized the drafting process. cl, ab, mh and ft implemented and supervised the screening program on the wards. all authors critically revised the manuscript and are accountable for accuracy and correctness. all authors have read and agreed to the final draft before submission. key: cord-354690-ywb9krdp authors: barr, margo; raphael, beverley; taylor, melanie; stevens, garry; jorm, louisa; giffin, michael; lujic, sanja title: pandemic influenza in australia: using telephone surveys to measure perceptions of threat and willingness to comply date: 2008-09-15 journal: bmc infect dis doi: 10.1186/1471-2334-8-117 sha: doc_id: 354690 cord_uid: ywb9krdp background: baseline data is necessary for monitoring how a population perceives the threat of pandemic influenza, and perceives how it would behave in the event of pandemic influenza. our aim was to develop a module of questions for use in telephone health surveys on perceptions of threat of pandemic influenza, and on preparedness to comply with specific public health behaviours in the event of pandemic influenza. methods: a module of questions was developed and field tested on 192 adults using the new south wales department of health's in-house computer assisted telephone interviewing (cati) facility. the questions were then modified and re field tested on 202 adults. the module was then incorporated into the new south wales population health survey in the first quarter of 2007. a representative sample of 2,081 adults completed the module. their responses were weighted against the state population. results: the reliability of the questions was acceptable with kappa ranging between 0.25 and 0.51. overall 14.9% of the state population thought pandemic influenza was very or extremely likely to occur; 45.5% were very or extremely concerned that they or their family would be affected by pandemic influenza if it occurred; and 23.8% had made some level of change to the way they live their life because of the possibility of pandemic influenza. in the event of pandemic influenza, the majority of the population were willing to: be vaccinated (75.4%), be isolated (70.2%), and wear a face mask (59.9%). people with higher levels of threat perception are significantly more likely to be willing to comply with specific public health behaviours. conclusion: while only 14.9% of the state population thought pandemic influenza was very or extremely likely to occur, a significantly higher proportion were concerned for self and family should a pandemic actually occur. the baseline data collected in this survey will be useful for monitoring changes over time in the population's perceptions of threat, and preparedness to comply with specific public health behaviours. if an outbreak of pandemic influenza should occur, it is essential that public health authorities are prepared to act. while resources have been prepared to educate the population about the nature of a threat and planned government actions, [1] it is necessary to understand the potential response of a population. most of the existing information about a population's response to the threat of pandemics comes from research on outbreaks of the sars coronavirus, most notably in hong kong, singapore, and canada, [2] [3] [4] [5] and on studies of risk perception and anticipated behaviours in a potential pandemic in humans from the avian influenza virus (especially the h5n1 subtype). [6] [7] [8] [9] to date, australia has been relatively unaffected by sars or h5n1; however, some of australia's neighbours have experienced limited outbreaks: for example, sars in hong kong and singapore; and h5n1 in indonesia and hong kong and china. globally, the threat of a pandemic of h5n1 is high. a key component of a population's response is the perception of risk or threat. research shows that in a sars outbreak willingness to comply with risk-reducing behaviours is linked to the perceived immediacy and seriousness of the threat. [2, 3, 5] three risk perception studies on potential avian influenza outbreaks were conducted in 2005. in the first study, lau et al. surveyed residents of hong kong on a potential outbreak of h5n1. [7] their study focussed on protective behaviours and likely compliance with them; however, the researchers also asked respondents about the perceived threat of h5n1 and the likelihood of it occurring within the next 12 months. it was found that 33% of respondents felt the chance of an outbreak was high or very high. lau's study also asked respondents how worried they would be about oneself or a family member contracting the virus in the event of a local outbreak; 54% said they would be very worried. in the second study, de zwart et al. compared the risk perceptions of european and asian respondents to the threat of avian influenza, [8] and measured self-efficacy beliefs to assess the likely compliance with protective health measures. overall the study found that 45% of respondents thought they were likely or very likely to become infected should an outbreak of avian influenza occur. this figure varied from 32% (denmark and singapore) to 61% (poland and spain). the researchers took a composite measure of risk perception and found that higher scores were observed in europe rather than asia. they found higher risk perceptions in females and older respondents; while lower self-efficacy beliefs in europe suggested that adherence to protective measures would be lower in europe. in the third study, di giuseppe et al. surveyed the knowledge and attitudes of an italian population to avian influenza. [9] they found that around 19% of respondents had a high risk perception and felt very much at risk of contracting avian influenza. in this study lower socioeconomic status and lower education levels were associated with higher risk perception, and those with a higher risk perception were more likely to comply with hygiene practices to avoid the spread of disease. our aim was to develop a module of questions for use in telephone health surveys on perceptions of threat of pandemic influenza, and on preparedness to comply with specific public health behaviours in the event of pandemic influenza. [7] [8] [9] as such, our literature search identified no relevant studies on response to pandemic influenza specifically, although other studies have been published on general threat perception and compliance with protective behaviours in the context of infectious diseases or other emergencies. the primary reference was a study by canadian researchers on anticipated public response to terrorism. [10] questions on the threat likelihood, effect on family, and behavioural compliance, were adapted with permission by subject matter experts and survey methodologists. each proposed question was considered for clarity, ease of administration, and possible biases. a set of 6 questions was developed for field-testing (table 1) , as well as an additional open question: "do you have any comments you would like to make on any of the questions or any other issues?" the pandemic influenza questions were field tested for test-retest reliability using the protocol of the new south wales health survey program. [11] the questions were then modified based on the results from the field testing and were re field tested. for both field tests the target sample was 200 persons living in the state aged 16 years and over stratified by geographical region. this sample size ensures that a kappa of 0.6 (good or excellent) is able to be detected at a significance level of 5% and a power of 80% when compared to a kappa of 0.4 or less (fair or poor) for response frequencies greater than 20%. [11] additional context added before question to provide better context. likelihood of being affected was changed to concern about being affected, to tap a sense of vulnerability rather than probability. responses altered to reflect concern and increase to 5 options. households were contacted using random digit dialling. one person aged 16 years and over from each household was randomly selected for field testing. trained interviewers conducted the interviews. up to 7 calls were made to establish initial contact with a household, and at least 5 calls were made to contact a selected respondent. when the respondent completed the first field test, an appointment was made for a retest at least a week later but within 3 weeks of the initial field test. if a respondent was unable to be contacted during this 2 week window they were deemed to be unavailable and their initial field test was deleted. [11] test-retest reliability and validity were estimated by cohen's kappa statistic for binary variables, and weighted kappa with cicchetti-allison weights for ordinal variables. unbalanced tables were corrected using the method described by crewson. [11] since erroneously low values of kappa can arise from skewed data, per cent agreement was also presented for categorical variables, calculated as the proportion of respondents in the same category at test and retest. responses for don't know and refused are also reviewed. [11] data manipulation and analysis were conducted using sas version 9.2. [11] the survey the new south wales population health survey is a continuous telephone survey of the health of the state population using the in-house cati facility of the new south wales department of health. [11] only residential phone numbers were used in the sample, as residential phone coverage in australia still remains high, [12] and results from persons who only have mobile phones has been shown to be comparable in the united states. [13, 14] the pandemic influenza module was administered as part of the survey between 22 january and 31 march 2007. the pandemic influenza questions were submitted to a lead ethics committee for approval prior to use. the survey also includes other modules on health behaviours, health status (including psychological distress, using the kessler k10 measure, and self-rated health status), and access to health services, as well as the demographics of respondents and households. the target population for the survey is all state residents living in households with private telephones. up to 7 calls were made to establish initial contact with a household, and 5 calls were made in order to contact a selected respondent. response categories were dichotomised into indicators of interest and don't knows and refused were removed. for the hypothetical questions -that is, likelihood of pandemic influenza, likelihood that family or self affected, willingness to comply with vaccination, isolation or wearing a face mask -the responses of extremely likely and very likely were combined into the indicator of interest. for the non-hypothetical question "changed way live because of the possibility of an influenza pandemic" responses a little, moderately, very much and extremely were combined into the indicator of interest: that is, changed life. the survey data were weighted to adjust for probability of selection and for differing non-response rates among males and females and different age groups. [15] data were manipulated and analysed using sas version 9.2. [11] the surveyfreq procedure in sas was used to analyse the data and calculate point estimates and 95 per cent confidence intervals for the prevalence estimates. for pairwise comparisons of subgroup estimates, the p-value for a two-tailed test was calculated using the normal distribution probability function probnorm in sas, assuming approximate normal distribution of each individual subgroup estimates with the estimated standard errors, and approximate normal distribution for the estimated difference. in total, 192 residents aged 16 years and over completed the first field test and 202 residents completed the second field test. estimates of test-retest reliability for the first and second field tests are shown in second field test. kappa values for the indicators derived from the questions ranged between 0.25 and 0.51 in the second field test. there were low don't know response rates (0-3.9%) and no respondent refused to answer any question. in response to the open question "do you have any comments you would like to make on any of the questions or any other issues?": 79% made positive comments about the questions, 48.7% found the question wording easy to understand and answer, and 29.9% found the subject matter relevant and interesting. of the respondents who had difficulty answering the questions, the main issues were: the questions were too vague (7.1%), response options were not descriptive enough (7.1%), or the topic area was difficult (6.5%). table 3 shows the responses to each question, including don't know and refused. the percentage of don't know or refused responses was low. table 4 shows the indicators for pandemic influenza likely, concern for self and family, and changed life by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. overall 14.9% of the population thought pandemic influenza was very or extremely likely, 45.5% were very or extremely concerned that they or their family would be affected by pandemic influenza, and 23.8% had made some (small to extreme) level of change to the way they live their life because of the possibility of pandemic influenza. when the indicators for pandemic influenza likelihood, concern for self and family and changed life were combined, as shown in figure 1 , the greatest proportion of the population (41.3%) thought pandemic influenza was unlikely to occur, would not be concerned for themselves or their family, and had not changed the way they lived their life because of the possibility of pandemic influenza. a quarter of the population (25.1%) thought pandemic influenza was unlikely to occur and had not made any changes to their lives, but would be concerned for themselves and their family in the event of pandemic influenza. table 4 also shows the combined indicators pandemic influenza likely and concern for self and family as well as pandemic influenza likely and concern for self and family and changed life by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. table 5 shows the indicators willing to receive vaccination, isolate themselves, or wear a face mask by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. overall, the majority of the population would be willing to receive vaccination (75.4%), willing to be isolated (70.2%), and willing to wear a mask (59.9%), if pandemic influenza were to occur. when the indicators for willing to receive vaccination, isolate themselves, and wear a face mask were combined, as shown in figure 2 , 48.3% reported being willing to receive vaccination, isolate themselves, and wear a face mask if pandemic influenza were to occur; 13.7% would not be willing to receive vaccination, isolate themselves and wear a face mask; 13.1% would be willing to receive vaccination, isolate themselves but not wear a face mask; and 10.4% would be willing to receive vaccination and wear a face mask but not isolate themselves. table 5 also shows the combined indicator for willing to receive vaccination, isolate themselves, and wear a face mask by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. table 6 shows the indicators for willing to receive vaccination, isolate themselves, or wear a face mask as well as complying with all the specific public health behaviours: that is, willing to receive vaccination, isolate themselves, and wear a face in people who think a pandemic influenza is very or extremely likely, and who are also very or extremely concerned for themselves and their family. this study shows it is possible to construct a small set of questions about threat perception for a general population, which can be used for health surveillance. field testing identifies improvements that can be made to the questions and the response structure, and highlights the population's interest in surveys of this nature. kappa values for the indicators ranged from 0.25-0.51, which is acceptable for hypothetical questions. the items had low don't know response rates (0-3.9%); no respondents refused to answer any of the questions; and the majority of respondents made positive comments about the questions. those reporting the highest levels of threat perception are older people, those with fair or poor self-rated health status, no formal qualifications, low household incomes, and those living in rural areas. perhaps surprisingly, there were no differences noted between the perceptions of men and women, or between those persons with or without children. overall, the majority of the population has taken no action, at this point, to change the way they live their life because of the possibility of pandemic influenza. the only two subgroups reporting moderate changes are those born overseas and those who speak a language other than english in the home. although direct comparisons with other studies are difficult to make, these findings suggest that the threat perceptions of the new south wales population are similar to those reported by residents of hong kong, even though australia has not been exposed directly to sars or h5n1. willingness to comply with specific public health behaviours is generally high (60-75%), with willingness to be vaccinated greater than being willing to be isolated, which in turn is greater than being willing to wear a face mask. there is clearly a lower level of willingness to comply with wearing a face mask, especially in younger people, those living in urban areas, and those who speak a language other than english in the home. current findings on compliance with protective behaviours are comparable with findings from studies conducted in hong kong in relation to anticipated sars and h5n1. [4, 7] a study about sars in hong kong indicates that those with higher risk perception and moderate levels of anxiety were more likely to take comprehensive precautionary measures against infection, and younger less educated males were least likely to adopt preventative measures. [3] our data suggest that younger people are less likely to comply with protective behaviours, while a higher level of formal education (a university degree or equivalent) is associated with higher willingness to comply with all protective behaviours, but especially wearing a face mask. a study of this nature has a number of limitations. first, people are being asked about a hypothetical event of which they have no experience. however, comparisons [4] and reported mask wearing rising from 11% in the early stages to 94% in the later stages of the outbreak. clearly data in that study support the increased likelihood of protective behaviours being adopted with increased risk perception; and, in our study, those with higher levels of threat perception were significantly more likely to be willing to comply with specific public health behaviours. prevalence estimates and 95% confidence intervals for response combinations to the three questions on health protection behaviours for pandemic influenza willing to be vaccinated, isolated, and wear a face mask willing to be vaccinated and isolated, but not willing to wear a face mask willing to be vaccinated and wear a face mask, but not willing to be isolated willing to be vaccinated, but not willing to be isolated or wear a face mask willing to be isolated and to wear a face mask, but not willing to be vaccinated willing to be isolated, but not willing to be vaccinated or to wear a face mask willing to wear a face mask, but not wiling to be vaccinated or isolated not willing to be vaccinated, isolated, or wear a face mask % our data indicate that while most respondents are very or extremely willing to perform a behaviour; the remaining respondents are expressing varying, but lower, degrees of willingness to perform these behaviours, with 21-31% indicating they would be moderately or a little willing, and 3-8% indicating they would be not at all willing to perform these behaviours. however, evidence such as data indicating very high levels of compliance with quarantine and minimal requirement for enforceable quarantine orders during sars in canada suggests that, in the event of a serious and immediate threat, the majority of those who are indecisive would shift their position and comply. [19] it is likely, however, that even with such a compliance 'shift' the relative compliance of sub groups within the population noted in our study will be upheld; as these patterns of compliance have been supported consistently by studies of actual protective behaviours. [3, 4] this study of the response of the new south wales population to the threat of pandemic influenza is part of a broader study of perceptions and behaviours around adverse events, including terrorism and global warming. as post-disaster studies generally report a lack of baseline data as a major handicap to understanding the trajectory for psychosocial recovery, [17, 18] our study takes the first steps in establishing baseline for data vital for emergency planning, against which impact and recovery can be monitored. australian health management plan for pandemic influenza canberra: australian government department of health and ageing infectious disease and risk: lessons from sars london: the nuffield trust the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong monitoring community psychological responses to the sars epidemic in hong kong: from day 10 to day 62 risk perception and compliance with quarantine during the sars outbreak avian influenza risk perception: hong kong anticipated and current preventative behaviours in response to an anticipated human-to-human h5n1 epidemic in hong kong chinese general population notes: level of statistical significance: * p < 0 â�¡ population level frequencies do not agree with table 3 as don't know/ refused responses were excluded from this analysis. ⧠for pairwise comparison testing in subgroups with more than two categories comparisons are made between each subgroup prevalence and the overall population prevalence. ï� psychological distress was measured using the k10. values range from 10-50, with 'high' psychological distress considered as being â�¥ 22 avian influenza risk perception a survey of knowledge, attitudes and practices towards avian influenza in an adult population of italy national public survey of perceived cbrn terrorism threat and preparedness. in university of ottawa in partnership with health canada and the canadian food inspection agency ottawa: institute of population health nsw population health survey: description of methods australian bureau of statistics: population survey monitor. catalogue no. 4103.0 canberra: abs estimation issues in dual frame sample of cell and landline numbers surveying households on cell phones: results and lessons. paper presented at the annual conference of the american association for public opinion research nsw population health survey: review of weighting procedures australian bureau of statistics: census of population and housing effects of fear and anger on perceived risks of terrorism: a national field experiment comparison of post-disaster psychiatric disorders after terrorist bombings in nairobi and oklahoma city public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto this study was funded by emergency management australia and the new south wales department of health. the following staff of the centre for epidemiology and research, new south wales department of health, assisted with the study: matthew gorringe, question development and data collection; raymond ferguson, sas programming and infrastructure; frances garden, comparing weighted survey data against census data. the authors declare that they have no competing interests. the authors contributed equally to this work.publish with bio med central and every scientist can read your work free of charge the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/8/117/pre pub key: cord-307918-8y89p11a authors: onyango, clayton o.; njeru, regina; kazungu, sidi; achilla, rachel; bulimo, wallace; welch, stephen r.; cane, patricia a.; gunson, rory n.; hammitt, laura l.; scott, j. anthony g.; berkley, james a.; nokes, d. james title: influenza surveillance among children with pneumonia admitted to a district hospital in coastal kenya, 2007–2010 date: 2012-12-15 journal: j infect dis doi: 10.1093/infdis/jis536 sha: doc_id: 307918 cord_uid: 8y89p11a background. influenza data gaps in sub-saharan africa include incidence, case fatality, seasonal patterns, and associations with prevalent disorders. methods. nasopharyngeal samples from children aged <12 years who were admitted to kilifi district hospital during 2007–2010 with severe or very severe pneumonia and resided in the local demographic surveillance system were screened for influenza a, b, and c viruses by molecular methods. outpatient children provided comparative data. results. of 2002 admissions, influenza a virus infection was diagnosed in 3.5% (71), influenza b virus infection, in 0.9% (19); and influenza c virus infection, in 0.8% (11 of 1404 tested). four patients with influenza died. among outpatients, 13 of 331 (3.9%) with acute respiratory infection and 1 of 196 without acute respiratory infection were influenza positive. the annual incidence of severe or very severe pneumonia, of influenza (any type), and of influenza a, was 1321, 60, and 43 cases per 100 000 <5 years of age, respectively. peak occurrence was in quarters 3–4 each year, and approximately 50% of cases involved infants: temporal association with bacteremia was absent. hypoxia was more frequent among pneumonia cases involving influenza (odds ratio, 1.78; 95% confidence interval, 1.04–1.96). influenza a virus subtypes were seasonal h3n2 (57%), seasonal h1n1 (12%), and 2009 pandemic h1n1 (7%). conclusions. the burden of influenza was small during 2007–2010 in this pediatric hospital in kenya. influenza a virus subtype h3n2 predominated, and 2009 pandemic influenza a virus subtype h1n1 had little impact. the coast of kenya, approximately 60 km north of mombasa. the district comprises a largely rural population of subsistence farmers and has an equatorial climate, with rain predominantly falling during april-july and october-december. kdh is the principal hospital facility for the population of the khdss. further details of the study area and respiratory disease surveillance at kdh and local clinics can be found in previous reports (2) (3) (4) 13) . nasopharyngeal wash or aspirate specimens were collected from eligible children aged 1 day to 12 years from january 2007 through december 2010 and were either stored in viral transport medium at −80°c (2007-2009) prior to molecular screening or screened prior to freezing in raw form. inpatients were eligible if they were admitted to the hospital with cough or difficulty breathing and either lower-chest-wall indrawing (defined as severe pneumonia) or 1 or more of the following: cyanosis, prostration, unconsciousness, or an oxygen saturation level <90% (a modification of the world health organization criteria for very severe pneumonia). children who were not residing in the khdss at admission, were admitted in extremis, were admitted for elective surgery, or received a diagnosis of neonatal tetanus were excluded. the following clinical and laboratory features obtained on admission or that relate to discharge outcome were compared between influenza-positive and influenza-negative children: duration of hospitalization >14 days, very severe pneumonia, wheezing, hypoxia (oxygen saturation level <90%, by fingertip pulse oximetry), circulatory shock (capillary refill time ≥3 seconds), severe anemia (hemoglobin level <5 g/dl), prematurity, congenital heart disease, positivity for hiv antibody (by 2 rapid tests), severe underweight (weight for age z score ≤3), slide positivity for plasmodium species, bacteremia, concurrent viral infection diagnosis, and death before discharge [2] . outpatient recruits were a convenience sample of children aged <13 years, enrolled for broad comparison with hospitaladmitted patients, who presented with either no signs of acute respiratory infection (non-ari) or signs of upper respiratory tract infection (urti) [2, 5] . individuals with urti had 1 or more of the following: cough, difficulty breathing, nasal discharge, runny or blocked nose, or sore throat. written informed consent was obtained from the parent or guardian of subjects. this study was approved by the kenyan national ethical review committee and the university of warwick biomedical research ethics subcommittee. diagnostic real-time reverse-transcription polymerase chain reaction (rt-pcr) rna was extracted from 140 µl of nasopharyngeal samples, using the qiagen viral rna miniprep kit (qiagen, united kingdom), or from 200 µl of nasopharyngeal samples, using the total nucleic acid extraction kit (roche applied science, germany) with a magna pure lc32 automated nucleic acid extractor, following the manufacturer's instructions. diagnostic screening for viral targets was performed using real-time rt-pcr. for specimens from kdh inpatients in 2007 and from outpatients in 2007-2008, reactions were tested by multiplex real-time rt-pcr, using fret hybridization probes as described by lassaunière et al [6] , with primers and probe targeting ns1 of influenza a virus and nucleoprotein of influenza b virus. all other samples were screened using the taqman qiagen quantifast multiplex method on the abi 7500 platform described by hammitt et al, targeting the matrix protein for influenza a and c viruses and targeting ns for influenza b virus [3] . concurrent targets in both rt-pcr assays included respiratory syncytial virus (rsv), adenovirus, rhinovirus, parainfluenza virus (piv) 1-3, human metapneumovirus, coronavirus (cov 229e, nl69, and oc43), and, differentially by assay, coronavirus hong kong [6] and piv4 [3] . an aliquot of each of the influenza a virus-positive specimens was shipped to the national influenza center (nic) in nairobi for subtyping. samples were subjected to rna extraction using the qiaamp viral rna isolation kit (qiagen). detection was performed using invitrogen superscript iii platinum one-step quantitative kit with primers and probes targeting seasonal influenza a virus h1n1 (a[h1n1]), a (h1n1)pdm09, and influenza a virus subtype h3n2 (a [h3n2]). statistical analysis was undertaken using stata, version 11.0 (statacorp, college station, tx) and microsoft office excel 2003 (microsoft, redmond, wa). the incidence of influenza among inpatients for age group i, i(i), per 100 000 population per year was estimated on the basis of the equation i(i) = [c (i)/n(i)p(i)].100,100, where c(i) is the average number of children per year admitted who were resident in the khdss in age group i, n(i) the midsurvey khdss population in age group i, and p(i) is the proportion of eligible children tested for influenza (ie, we assumed that children who were not tested would have had the same prevalence of influenza as those who were tested and scaled the incidence accordingly). for pneumonia incidence estimates, p(i) is set to 1. the kdhss population on 1 january 2009 was estimated to be 9451 individuals aged <1 year, 45 644 aged <5 years, and 108 708 aged <13 years. the population size and incidence estimation procedures have been described elsewhere [4] . the incidence estimation for the population proximal to the hospital was undertaken using cases involving children aged <5 years admitted from administrative sublocations within a 5km radius of the hospital and the corresponding midpoint population estimate from the khdss (12 339 as of 1 january 2009). the wilcoxon rank sum test was used to compare median ages; the χ 2 or fisher exact test was used to compare proportions, as appropriate; the score test ( procedure tabodds) was used to assess the trend in prevalence, by age; and the poisson probability distribution was used to assess whether observed cases of influenza in specified quarters of the year exceeded the expected number of cases. spearman rank correlation was used to test for a temporal association between monthly or quarterly numbers of influenza cases or influenza a virus infections and the number of cases of bacteremia or streptococcus pneumoniae infection. the analysis was undertaken with cases of influenza and bacteremia temporally in phase or between 1 and 4 months time step out of phase. the association between positivity for any influenza type and laboratory or clinical features on admission was assessed using logistic regression, adjusted for age group, to obtain odds ratios (ors) and 95% confidence intervals (95% cis). over the 4-year period, there were 2429 admissions to kdh involving individuals who were eligible for the study (57% were boys; median age, 9 months [interquartile range {iqr}, 3-22 months]). a total of 503 (21%) had very severe pneumonia (50% were boys; median age, 10 months [iqr, 2-34 months]; 55% were infants), and the in-hospital case-fatality rate was 6.5%. of the eligible inpatients, 2002 (82%) were tested for influenza (57% were boys; median age, 9 months [iqr, 3-21 months]; 58% were infants), and this percentage did not differ among those aged <1 year, 1-4 years, and ≥5 years (p = .333); 387 (19%) had very severe pneumonia (52% were boys; median age, 10 months [iqr, 2-34 months]; 53% were infants). stratified by severity, 84% of eligible inpatients with severe pneumonia were tested, compared with 77% of eligible inpatients with very severe pneumonia (p = .001). the case-fatality rate among inpatients who were untested was significantly higher than among those who were tested ( ; 37% were infants). compared with the median age of inpatients, the median ages of outpatients with non-ari (p = .004) or urti (p ≤ .0001) were higher. the prevalence of influenza virus of any type was 4.9% (99 of 2002 cases) among inpatients; 4.7% (76 of 1615) had severe pneumonia, and 5.9% (23 of 387) had very severe pneumonia (p = .299). among outpatients, the prevalence of influenza virus of any type was 3.9% (13 of 331) among those with urti and 0.5% (1 of 196) among those with non-ari. data stratified by virus type are presented in table 1 . influenza a virus was the most prevalent type among outpatients with pneumonia (3.5%) and outpatients with urti (3.3%). these proportions were unaltered by restricting the analysis to children <5 years of age. among outpatients classified as having the seasonal patterns of influenza a and b virus infection are shown in figure 1 . over the 4-year period, influenza a and b virus infection showed a significantly higher occurrence among inpatients during quarters 3 and 4, relative to the average for all quarters (22.5 cases expected vs 37.5 cases observed; p = .003). there was very little influenza activity in 2010. the majority of influenza b virus infections occurred in the fourth quarter of 2009. most influenza cases occurred after the main period of rainfall (april-july) and before peak temperatures (first quarter; figure 1 ). cases of bacteremia, and specifically s. pneumoniae infection, by quarter, are shown in figure 1 . no statistically significant correlation between influenza cases (or influenza a virus infections) and occurrence of bacteremia (or s. pneumoniae infection) was identified, either concurrently or delayed (p > .05). of figure 1a) , with around 50% of cases in infants (46% had influenza a virus infection, 47% had influenza b virus infection, and 66% had influenza c virus infection; p = .632). the proportion of inpatients with pneumonia who were found to be influenza positive showed a trend for increase with increasing age (supplementary figure 1b) . in the case of influenza a virus infection, the trend was significant: 1.7% of children aged 0-2 months had influenza a virus infection, compared with >5% of children aged ≥24 months (p = .005). analysis of the association between a diagnosis of infection with any influenza virus and components of a set of severity features yielded an increased odds of hypoxia among children with influenza, compared with those without influenza (ageadjusted or, 1.78; 95% ci, 1.04-1.96). no other severity feature was significantly associated with influenza (supplementary table 1 ). there were 4 deaths (age range, 7-32 months) among the 99 inpatients with influenza; one was infected with influenza a virus, 2 were infected with influenza b virus, and 1 was infected with influenza c virus. three were positive for hiv antibody, severely malnourished (weight-for-age z score, ≤4), and had a discharge diagnosis including immunosuppression, and 1 inpatient (who was infected with influenza a virus) had chronic heart disease. our study identified all 3 influenza viruses in circulation in this rural coastal kenya location among patients hospitalized with severe or very severe pneumonia and among outpatients with urti. however, the prevalence of infection with any influenza virus was low among inpatients with severe or very severe pneumonia (4.9%) and among outpatients with urti (3.9%). influenza a virus predominated, with identification in 3.5% of inpatients and 3.3% of outpatients with urti. correspondingly, relative to the incidence of severe or very severe pneumonia among hospitalized children aged <5 years (1323 cases per 100 000 per year), the incidences of influenza (64 cases per 100 000 per year) and influenza a virus infection (46 cases per 100 000 per year) were low. there was near absence of influenza in the convenience sample of children without signs of respiratory illness. our inpatient data are consistent with the results of a recent review of data on seasonal influenza from 15 published studies in sub-saharan africa [1] , which reported a median prevalence of 6.0% among hospitalized pediatric patients, with a range of 0%-16%. in the same review, the median prevalence of influenza among outpatients with ari was higher than we found, at 10% (range, 1%-25%; 11 studies), but comparisons should be cautioned because of a number of methodological differences. exploration of a range of severity features and concurrent illnesses revealed hypoxia to be more commonly associated with influenza among hospitalized children. calculation of the incidence of influenza-associated severe disease on the basis of hospital admission data is likely to underestimate the true burden in the community, as a result of the relationship between healthcare access and distance from the hospital. this is supported in the analysis, where it was shown that the incidence of influenza-associated admissions among children with severe or very severe pneumonia was about 70% greater in the population proximal to the hospital. we have previously shown a similar distance decay for severe rotavirus diarrhea [7] and severe rsv-associated pneumonia [4] and pneumonia and meningitis [8] . furthermore, a previous study of rsv among infants and young children in the hdss revealed that roughly 4 in 5 children identified with severe pneumonia in the outpatient setting were not admitted to the local hospital [9] . notwithstanding this underestimation, it is clear that the incidence of influenza-associated hospital admissions is significantly lower than that associated with either rhinovirus or rsv infection. while the etiology of rhinovirus as the causative agent of lower respiratory tract disease may be in question [10] , this is not the case for rsv, which is known to be a major cause of infant and childhood lower bronchiolitis and pneumonia in sub-saharan africa and globally [11] . while rsv is invariably among the most prevalent viruses in children admitted with lower respiratory tract illness, it is not always dominant over influenza [1] . in kenya, contemporary data on the relative prevalence of respiratory viruses among pneumonia-related admissions to the hospital are largely absent. further data are clearly needed in kenya to gauge the relative burden of disease due to respiratory viruses and thereby help support future health policy planning. in terms of seasonality, there was increased occurrence in the third and fourth quarters of each year, most notably for influenza a virus infection, except in 2010. these periods are characteristically times of lower rainfall levels (referred to locally as "second rains"), intermediate temperatures, and relative lower humidity. during the study period, a(h1n1)pdm09 entered kenya [12] , and cases of a(h1n1)pdm09 infection were identified in kdh from late 2010. it is possible that the introduction of a(h1n1)pdm09 disrupted the normal pattern of a(h3n2) activity in 2010; only a(h1n1)pdm09 was observed in the latter quarters of 2010. continued surveillance will reveal whether a (h1n1)pdm09 has any long-term effect on the circulation patterns of other influenza subtypes. however, in general, the contribution of a(h1n1)pdm09 to the burden of hospitalization-associated pneumonia was minimal in this setting. the possibility exists that the burden of influenza was underestimated because of associated, but delayed, invasive bacterial disease. however, we identified no evidence for an increased number of admissions in which bacteria (or s. pneumoniae, in particular) were detected in blood cultures during the quarter following peak occurrences of influenza. during the a(h1n1)pdm09 infection pandemic, antiviral therapy (oseltamivir) was prescribed to children admitted to kdh with severe acute respiratory illness on a presumptive basis (ie, prior to laboratory confirmation of influenza.) this would not have altered the pattern of observation of influenza described in this study, because nasopharyngeal specimens were collected prior to treatment with the antiviral. within the surrounding community, a(h1n1)pdm09 vaccination was undertaken in 2010 but was limited to target groups, including healthcare workers, pregnant women, and patients with chronic disease, totaling 2203 subjects (kenya ministry of health, personal communication). this number and the age group of subjects receiving vaccine would not have altered the pattern of a(h1n1) infection occurrence described in this study. half of the influenza cases occurred in infants, and the proportion of cases rapidly declined with age into older age groups. however, we noted that within the age group that we studied, the prevalence of influenza increased with age, suggesting that relative to other causes of cases of severe or very severe pneumonia associated with admission, those caused by influenza decline less rapidly with age. a limitation of the present study is the failure to recruit and test samples from approximately 20% of eligible children. the reasons for this have been previously described [2, 4] and were primarily due to parental refusal and, to a lesser extent, to discharge or death before sampling. failure to recruit and test was more common for critically ill children and for those who died while in the hospital. this almost certainly led to an underestimation of severity associated with influenza. a further limitation of the study is that results are based only on nasopharyngeal samples, and we now have definitive evidence that an oropharyngeal swab specimen provides added diagnostic value for detecting influenza in our setting (22% increased detection; 95% ci, 9-42), compared with nasopharyngeal specimens alone [3] . the low prevalence of influenza viruses in this study limits the power of the analysis to identify associations between virus presence and specific clinical features or coinfections. our study involved sampling of 2000 children; a definitive investigation of clinical associations will require a considerably larger sample size or a location with a markedly higher incidence of influenza a virus infection. we report very low prevalence of influenza in outpatient children without signs of acute respiratory infection, suggesting that influenza virus is rarely the cause of asymptomatic infection, and the data also suggest influenza is the cause of only 4% of urti cases. further interpretation of these data with regard to the association between influenza and severe disease is unwarranted since outpatient sampling was not contemporaneous throughout the period of surveillance of hospitalized patients, and collection was not frequency matched by age and location within the khdss. we therefore await the complete results of a larger and better-designed case-control study, which is ongoing. in conclusion, although the incidence of influenza was underestimated in this study, it is clear that influenza contributes only a small proportion of the total burden of hospitalizationassociated severe and very severe pneumonia among children in this rural coastal kenya setting. influenza a virus is the dominant influenza virus causing pediatric severe and very severe pneumonia. a seasonal signature for influenza was evident, but no temporal association was identified with invasive bacterial disease. although a(h1n1)pdm09 infection was observed, its contribution to disease was not substantial. hypoxia was more frequently identified among patients with influenza, and immunosuppression, severe malnutrition, or chronic heart disease were identified in all of the 4 influenza-associated deaths. given the low influenza prevalence, larger studies are required to investigate associations between influenza and disease severity or prevalent conditions, such as malaria, hiv infection, or malnutrition. additional comparative studies on viral diagnoses in severe pneumonia hospital admissions are warranted elsewhere in kenya. such data may be informative to the kenya ministry of health in their assessment of the role for influenza antivirals and vaccination in kenya. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org/). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the author. seasonal influenza epidemiology in sub-saharan africa: a systematic review viral etiology of severe pneumonia among kenyan infants and children added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection incidence and severity of respiratory syncytial virus pneumonia in rural kenyan children identified through hospital surveillance increased detection of respiratory viruses in paediatric outpatients with acute respiratory illness by real-time polymerase chain reaction using nasopharyngeal flocked swabs a novel multiplex real-time rt-pcr assay with fret hybridization probes for the detection and quantitation of 13 respiratory viruses incidence and clinical characteristics of group a rotavirus infections among children admitted to hospital in kilifi sensitivity of hospital-based surveillance for severe disease: a geographic information system analysis of access to care in kilifi district respiratory syncytial virus infection and disease in infants and young children observed from birth in kilifi district viral respiratory infections in hospitalized and community control children in alaska global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis introduction and transmission of 2009 pandemic influenza a (h1n1) virus-kenya specimen collection for the diagnosis of pediatric pneumonia influenza surveillance of kenyan children with pneumonia acknowledgments. we thank the clinical and laboratory staff, for their hard work in collection and processing of the specimens; mwanajuma ngama, for the coordination of patient recruitment and sample collecpotential conflicts of interest. key: cord-325141-x3txhjkr authors: grech, victor; gauci, charmaine; agius, steve title: vaccine hesitancy among maltese healthcare workers toward influenza and novel covid-19 vaccination date: 2020-10-01 journal: early hum dev doi: 10.1016/j.earlhumdev.2020.105213 sha: doc_id: 325141 cord_uid: x3txhjkr introduction vaccine hesitancy is a chronic public health threat. this study was carried out to ascertain maltese healthcare workers’ hesitancy to a novel covid-19 vaccine and correlate this with influenza vaccine uptake. methods a short, anonymous questionnaire was sent out to all of malta’s government sector healthcare workers via the service’s standard email services (11-19/09/2020). a total of 9,681 questionnaires were posted electronically, with 10.4% response. results the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly. doctors had the highest baseline uptake and highest likely influenza vaccine uptake next winter. the likely/undecided/unlikely to take a covid-19 vaccine were 52/22/26% respectively. males were likelier to take the vaccine. doctors were the occupation with the highest projected vaccine uptake. likelihood of taking covid-19 vaccine was directly related to the likelihood of influenza vaccination. concerns raised were related to insufficient knowledge about such a novel vaccine, especially unknown long term side effects. discussion the increased uptake of influenza vaccine is probably due to increased awareness of respiratory viral illness. doctors may have higher vaccine uptakes due to greater awareness and knowledge of vaccine safety. the proportions of who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid-19 are similar to rates reported in other countries. the higher male inclination to take the vaccine may be due the innate male propensity for perceived risk taking. shared covid-19 with influenza vaccine hesitancy implies an innate degree of vaccine reluctance/hesitancy and not merely reluctance based on novel vaccine knowledge gap. vaccine hesitancy is a chronic public health threat. this study was carried out to ascertain maltese healthcare workers' hesitancy to a novel covid-19 vaccine and correlate this with influenza vaccine uptake. a short, anonymous questionnaire was sent out to all of malta's government sector healthcare workers via the service's standard email services (11) (12) (13) (14) (15) (16) (17) (18) (19) /09/2020). a total of 9,681 questionnaires were posted electronically, with 10.4% response. the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly. doctors had the highest baseline uptake and highest likely influenza vaccine uptake next winter. the likely/undecided/unlikely to take a covid-19 vaccine were 52/22/26% respectively. males were likelier to take the vaccine. doctors were the occupation with the highest projected vaccine uptake. likelihood of taking covid-19 vaccine was directly related to the likelihood of influenza vaccination. concerns raised were related to insufficient knowledge about such a novel vaccine, especially unknown long term side effects. the increased uptake of influenza vaccine is probably due to increased awareness of respiratory viral illness. doctors may have higher vaccine uptakes due to greater awareness and knowledge of vaccine safety. the proportions of who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid-19 are similar to rates reported in other countries. the higher male inclination to take the vaccine may be due the innate male propensity for perceived risk taking. shared covid-19 with influenza vaccine hesitancy implies an innate degree of vaccine reluctance/hesitancy and not merely reluctance based on novel vaccine knowledge gap. hospital-acquired influenza has a high mortality, with an estimated median of 16% that rises up to 60% in high risk groups (e.g. transplant recipients and intensive care patients). (1, 2) healthcare workers who carry the influenza virus have been frequently identified as sources of hospital-acquired outbreaks.(3) annual influenza vaccination is strongly recommended for all healthcare workers,(4) but vaccination rates remain poor,(5) despite models that show that a significant proportion of hospital-acquired burden of disease is vaccine preventable. (6) in summer 2020, the world remains in thrall to pandemic covid-19, a respiratory virus with transmission characteristics similar to influenza. for this reason, vaccine development has accelerated at an unprecedented pace.(7) once a vaccine becomes available, it will be crucial to vaccinate healthcare workers so as to minimise nosocomial infections. it has been announced that in malta, frontliners (including all healthcare workers) will be given first priority for the first batch of vaccines that are anticipated to arrive in malta in december 2020. (8) an earlier study this year that surveyed maltese healthcare workers with regard to influenza vaccination showed that the proportion of workers who did not take the vaccine last year but who are likely to take the vaccine this winter halved from 41% to 21%. doctors had the highest baseline uptake (23% refused vaccination in 2019) and the highest likely uptake next winter (6% likely to refuse vaccination in 2020). (9) this study was carried out in order to ascertain the degree of vaccine hesitancy in maltese healthcare workers vis-à-vis a putative novel covid-19 vaccine later this year, and correlate this with influenza vaccination uptake. a short, anonymous questionnaire was sent out to all of malta's government sector healthcare workers via the service's standard email services. the period for which the questionnaire was open was from 11/09/2020 to 16/09/2020. the questionnaire was hosted via google forms and exported to bespoke excel spreadsheets for analysis. the questionnaire was sent to all healthcare workers in the main hospital (mater dei hospital), district primary care health centres, st. vincent de paul long term care facility, mount carmel mental health hospital, karin grech rehabilitation hospital and miscellaneous other smaller facilities. it commenced with the following introduction: malta has been fortunate to have the early allocation of a covid-19 vaccine later this year. the vaccine is licensed and approved and will have passed through phase 3 trials. priority will be given to front liners and to the vulnerable, followed later by the rest of the population. this is totally anonymous and a very short, public health survey for healthcare workers, please fill completely. the questions, formatted in tick boxes, covered sex, occupation (medical, nursing, allied profession and other, with the latter including support staff such as in administration, ward clerks, cleaners, etc.), place of work (as above), age bracket, whether the influenza vaccine was taken last winter and whether it would be taken this coming winter (yes/no). the following text was inserted in the questionnaire followed by several questions on a likert scale of 1-5. phase i, small groups of people receive the trial vaccine. in phase ii, the vaccine is given to people who have characteristics (such as age and physical health) similar to those for whom the vaccine is intended. in phase iii, the vaccine is given to thousands of people and checked for efficacy and safety. the covid vaccine that will arrive in malta will have gone through these phases and will be approved and licensed. based on this information, how likely are you to take the covid-19 vaccine? i am concerned as i don't know enough about the vaccine i am concerned about the short term side effects (e.g. fever etc) i am concerned about possible long term side effects i am concerned because i don't think the vaccine will be effective i am against vaccines in general for the first question in the list above, it was assumed that scores 1 and 2 were "unlikely", 4 and 5 were "likely" and a score of 3 was regard as undecided. for the likert questions following the first, all were allowed to tick vaccines whatever their likelihood of taking the vaccine. chi tests and chi tests for trend were used except for one two by two table with small values wherein a fischer exact test was used. a p value ≤0.05 was taken to represent a statistically significant result. a total of 9,681 questionnaires were posted electronically, with 1002 (10.4%) responses (table 1) . the proportion of maltese healthcare workers who will take the influenza vaccine increased significantly across the board when compared to last year irrespective of sex, workplace or occupation (table 2) . doctors had the highest baseline uptake and the highest likely influenza vaccine uptake next winter (table 2) . with regard to a covid-19 vaccine, approximately half of respondents were likely to take the vaccine and a quarter each were undecided or unlikely to take the vaccine. males were likelier to take the vaccine than females (chi=13.2, p=0.0003table 3). doctors were also the likeliest group to take the covid-19 vaccine and when compared against all others this was a highly significant difference (chi=21.8, p<0.0001table 3). an analysis by age showed that there was a significant increase in the likely uptake of the influenza vaccine at all ages (first two columns of table 4 with statistical analysis in next two columns). the covid-19 likelihood uptake pattern was as described above except for the over 65 age group as none of these fell in the "unlikely to take" category. the proportion of those likelier to take the covid-19 vaccine was directly related to the likelihood of their taking the influenza vaccine (table 5: chi=246.2, p<0.0001). covid-19 vaccine concerns are shown in table 6. the issues raised were only very slightly related to vaccine avoidance in general but more related to insufficient knowledge about such a vaccine and any potential side effects especially those in the long term. the increased proportion of maltese healthcare workers who plan to take the influenza vaccine this year when compared to last winter is probably due to increased awareness of respiratory viral illnesses in general in the wake of the covid-19 pandemic. interestingly, it is the medical profession who had the highest baseline influenza vaccine uptake and the highest likely influenza vaccine uptake next winter and this may be due to greater awareness and knowledge of vaccine safety. the same applies for this profession with regard to the covid-19 vaccine. the proportions of those who are likely/undecided/unlikely (half, quarter, quarter respectively) to take a covid-19 are similar to rates reported in other countries.(10) the higher male inclination to take the vaccine may be due to a combination of factors which could include the innate male propensity for perceived risk taking in the face of a novel vaccine. (11) the higher likely uptake of a covid-19 vaccine in the oldest age group is unsurprising as this is the most vulnerable group and therefore most likely, in their own selfinterest, to take this vaccine. vaccine hesitancy for covid-19 was similar to that for influenza implying an innate degree of vaccine reluctance/hesitancy and not merely a reluctance based on the concerns discussed below.(10) however, the concerns are, to some extent, valid. there are various types of vaccines in development and these include not only traditional vaccines but also next generation vaccines. (7) non-vaccination and vaccine hesitancy our findings are unsurprising as the availability of a vaccine does not automatically equate to 100% aggregate uptake. for example, an h1n1 influenza vaccine in 2009 had a population uptake of 0.4-59% across 22 countries. (12) the low acceptance and uptake of a safe vaccine for a high risk infection is well known and has been dubbed the "pandemic public health paradox".(13) this is a strong contributor to vaccine hesitancy and is a tragic public health outcome as vaccines only protect if a sufficient proportion of the population is vaccinated. (14) non-vaccination has been quite extensively studied and table 7 shows some of the commonest reasons for non-vaccination.(15) one specific example specifically related to this topic is the aforementioned 2009 h1n1 influenza vaccine which was initially claimed to have had associated mortality using the vaccine adverse event reporting system(vaers) system which was eventually disproved, but not before undermining public confidence in this important vaccine. (16) in 2019, the world health organization named vaccine hesitancy as one of the top ten threats to global health. (17) the reasons for hesitancy are varied and some common vaccine myths and their scientific rebuttals are summarised in table 8. (17) clearly, the reasons for non-vaccination are complex but misconceptions pertaining to safety predominate. trends in hesitancy are overall not promising with a recent study showing that vaccine confidence in europe is low compared to other regions of the world, such as africa (strongly agreeing with vaccine safety range 19% in lithuania to 66% in finland). a drop in j o u r n a l p r e -p r o o f journal pre-proof confidence trend was linked to political instability and religious extremism, with rogue leaders sometimes promoting natural, unproven and ineffective alternatives to vaccines.(18) a representative sample of circa 1000 adults in the us questioned from 16-20 april 2020 with regard to a putative covid-19 vaccine replied: 57.6% intended to be vaccinated, 31.6% were uncertain and 10.8% did not intend to be vaccinated. factors independently associated with vaccine hesitancy ("no"/"not sure") included younger age, black race, lower educational attainment, and not having received the influenza vaccine in the prior year. reasons specified for vaccine hesitancy included vaccine-specific concerns, a need for more information, antivaccine attitudes or beliefs, and a lack of trust.(10) overcoming hesitancy who advises a pre-emptive pro-vaccination strategy that psychologically impacts populations so as to maximize uptake when vaccines become available. (19) in the case of covid-19, national vaccination strategies must be in place in advance of vaccine availability so as to have a plan for population prioritisation for vaccination and to reduce the incidence of fear/concern vis-à-vis vaccination. (20) a crucial part of the latter aspect is the countering of fake news and misinformation that already percolates (especially via social media) in this regard. (20) suggested key guidelines/milestones are shown in table 9.(20) segmentation of target populations is vital and consists of the identification of groups who share similar beliefs/attitudes/behavioral patterns. this goes beyond easily pigeonholed fields such demographic/epidemiological data and greatly enables public heath planners to shape intervention/s to specific segment/s.(21) hesitancy already exists among healthcare workers with regard to ordinary vaccines, such as seasonal influenza vaccination. (22) (23) (24) it is anticipated that the next challenge will be vaccination for covid-19 when this desperately-awaited vaccine becomes available. indeed, questionnaires in this regard already reveal novel covid-19 vaccine hesitancy among healthcare workers. (25, 26) hesitancy is fuelled by social media, conspiracy theories and fake news, a topic about which entire volumes have been written. (27, 28) public health and healthcare worker employers must do their best to ensure that the proportion of vaccinated workers is as close to totality as possible. clinicians, legislators and even ethicists are increasingly cognisant of this aspect of healthcare, and are progressively mandating seasonal influenza vaccination for healthcare workers in some countries. this is not being envisaged for malta. the society for healthcare epidemiology has recommended that annual influenza vaccination should be a condition of employment for healthcare workers,(29) a stance endorsed almost universally by professional bodies.(5) indeed, ethicists have averred that: "given the mounting evidence for the efficacy of influenza vaccination in infection control […] the provision of health care by non-vaccinated health care workers is not merely suboptimal health care, but it is also at variance with generally accepted principles of health care ethics." (5) j o u r n a l p r e -p r o o f journal pre-proof this is because medical ethics upholds the dual principles of beneficence and nonmaleficence. the former infers the promotion of patients' well-being and the latter is primum non nocere. therefore "practicing without vaccination is maleficent because it falls below the standard of medical care".(5) it has in fact been shown that influenza vaccination of healthcare workers reduces influenza morbidity and mortality in influenza-vulnerable populations. (30) (31) (32) (33) the commonest reason for healthcare worker vaccination hesitancy is insufficient knowledge about its safety profile and irrational apprehension and it has been shown that improved information about the vaccine improves voluntary vaccine uptake. (34) our study partially supports this contention in that doctors were more likely to take the influenza vaccine, both last year and with even greater likelihood next winter, and this may be due to greater knowledge in this group of healthcare workers than in the other groups i.e. allied health professionals, nurses and others. healthcare workers and their institutions are professionally responsible for the care and wellbeing of their patients following evidence-based practices.(5) this study may have been biased by the announcement that astrazeneca's vaccine trial was voluntarily paused as for a standard review process that was triggered by a "single event of an unexplained illness that occurred in the uk phase iii trial", a routine action "to allow an independent committee to review the safety data". (35) vaccine passport several countries have suggested the introduction of covid-19 "immunity passports" following infection with the disease but this poses extensive scientific, practical, equitable, and legal challenges. (36) on the other hand, the introduction of a vaccination passport or certificate to vaccinees could be used as additional incentive to take the vaccine by exempting holders from physical restrictions, social distancing etc. (36) conclusions healthcare workers should be informed about and encouraged to take influenza vaccination. the introduction of a covid-19 vaccination "passport" may also be considered especially if it provides added benefit/s to the vaccinee. j o u r n a l p r e -p r o o f j o u r n a l p r e -p r o o f 'the unconcerned' -those who consider immunization a low priority and see no real perceived risk of vaccine-preventable diseases. 'the poorly reached' -those who have limited or difficult access to services, related to social exclusion, poverty and, in the case of more integrated and affluent populations, factors related to convenience. 'the active resisters' -those for whom personal, cultural, or religious beliefs discourage them from vaccinating. j o u r n a l p r e -p r o o f key concepts too many vaccines too soon. the number of immunologic components in vaccines have declined over time. the current 14 vaccines on the united states schedule contain 200 immunologic proteins in total, the smallpox vaccine contained 160. too many vaccines can "overwhelm" the immune system. epidemiologic data and biologic data show that cumulative increases in the number of vaccines have no effect on immune function. mmr vaccine causes autism. original study making this claim contained 12 children, the paper was subsequently retracted due to evidence of misrepresented data. multiple large scale studies, including a study of half a million children have shown no association between receipt of mmr and risk of autism. hpv vaccine increases risk of autoimmune disease. more than 270 million doses of hpv vaccine have been administered. repeated well-designed studies show no association between hpv and ai disease. influenza vaccine given in early pregnancy increases risk of miscarriage. a study of 2762 women showed no association between influenza vaccine and spontaneous abortion. j o u r n a l p r e -p r o o f influenza in the acute hospital setting influenza control in acute care hospitals a nosocomial outbreak of influenza during a period without influenza epidemic activity immunization of health-care personnel: recommendations of the advisory committee on immunization practices (acip) mmwr morb mortal wkly rep mandatory influenza vaccination for health care workers as the new standard of care: a matter of patient safety and nonmaleficent practice modeling the effects of influenza vaccination of health care workers in hospital departments developing a vaccine for covid-19 first delivery of potential covid-19 vaccine expected in december. times of malta influenza vaccination survey in maltese healthcare workers in the covid-19 era. malta medical school gazette attitudes toward a potential sars-cov-2 vaccine: a survey of u.s. adults age and gender differences in risk-taking behaviour as an explanation for high incidence of motor vehicle crashes as a driver in young males influenza a(h1n1)pdm09 vaccination policies and coverage in europe pandemic public health paradox media attention, risk perception and public reactions in 5 european countries when a covid-19 vaccine is ready, will we all be ready for it? under-vaccinated groups in europe and their beliefs, attitudes and reasons for non-vaccination; two systematic reviews risk of fatal adverse events after h1n1 influenza vaccine: limitations of passive surveillance data vaccine safety: myths and misinformation mapping global trends in vaccine confidence and investigating barriers to vaccine uptake: a large-scale retrospective temporal modelling study world health organization. the guide to tailoring immunization programs. world health organisation key guidelines in developing a pre-emptive covid-19 vaccination uptake promotion strategy measles still spreads in europe: who is responsible for the failure to vaccinate vaccine hesitancy and self-vaccination behaviors among nurses in southeastern france the continuum of influenza vaccine hesitancy among nursing professionals in hong kong knowledge and attitude towards vaccination among healthcare workers: a multicenter cross-sectional study in a southern italian region. vaccines (basel) vaccine hesitancy: the next challenge in the fight against covid-19 mistrust in biomedical research and vaccine hesitancy: the forefront challenge in the battle against covid-19 in italy social media and vaccine hesitancy: new updates for the era of covid-19 and globalized infectious diseases fake news and post-truth pronouncements in general and in early human development revised shea position paper: influenza vaccination of healthcare personnel influenza vaccination of health care workers in long-term-care hospitals reduces the mortality of elderly patients effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care: a randomised controlled trial effectiveness of an influenza vaccine programme for care home staff to prevent death, morbidity, and health service use among residents: cluster randomised controlled trial effect of influenza vaccination of nursing home staff on mortality of residents: a cluster-randomized trial influenza vaccination acceptance and refusal rates among health care personnel covid-19: oxford researchers halt vaccine trial while adverse reaction is investigated covid-19 immunity passports and vaccination certificates: scientific, equitable, and legal challenges the authors have no conflict of interest to declare. key: cord-342796-f7n8sxbu authors: stowe, j.; tessier, e.; zhao, h.; guy, r.; muller-pebody, b.; zambon, m.; andrews, n.; ramsay, m.; lopez bernal, j. title: interactions between sars-cov-2 and influenza and the impact of coinfection on disease severity: a test negative design date: 2020-09-18 journal: nan doi: 10.1101/2020.09.18.20189647 sha: doc_id: 342796 cord_uid: f7n8sxbu background: the potential impact of covid-19 alongside influenza on morbidity, mortality and health service capacity is a major concern as the northern hemisphere winter approaches. this study investigates the interaction between influenza and covid-19 during the latter part of the 2019-20 influenza season in england. methods: individuals tested for influenza and sars-cov-2 were extracted from national surveillance systems between 20/01/2020 and 25/04/2020. to estimate influenza infection on the risk of sars-cov-2 infection, univariable and multivariable analyses on the odds of sars-cov-2 in those who tested positive for influenza compared to those who tested negative for influenza. to assess whether a coinfection was associated with severe sars-cov-2 outcome, univariable and multivariable analyses on the odds of death adjusted for age, sex, ethnicity, comorbidity and coinfection status. findings: the risk of testing positive for sars-cov-2 was 68% lower among influenza positive cases, suggesting possible pathogenic competition between the two viruses. patients with a coinfection had a risk of death of 5.92 (95% ci, 3.21-10.91) times greater than among those with neither influenza nor sars-cov-2 suggesting possible synergistic effects in coinfected individuals. the odds of ventilator use or death and icu admission or death was greatest among coinfection patients showing strong evidence of an interaction effect compared to sars-cov-2/influenza acting independently. interpretation: cocirculation of these viruses could have a significant impact on morbidity, mortality and health service demand. testing for influenza alongside sars-cov-2 and maximising influenza vaccine uptake should be prioritised to mitigate these risks. it is likely that both sars-cov-2 and seasonal respiratory pathogens, most notably influenza, will be co-circulating as the northern hemisphere 2020/21 winter approaches. the potential impact of covid-19 alongside influenza on morbidity, mortality and health service capacity is a major concern, however, currently little is understood about the interaction between these two respiratory viruses 1,2 . there is existing evidence of pathogenic competition between respiratory viruses, including between influenza and seasonal coronaviruses [3] [4] [5] . this could be through immune-mediated interference resulting in some viruses to diminish during the peak of another virus, a phenomenon that has been recognised for many decades 3, 6, 7 . one study reported that influenza vaccination was associated with an increased risk of seasonal coronavirus 5 . to date there is some evidence of ectopic interaction between the sars-cov-2 protein and host proteins 8 , however there is no information on the pathogenic interaction between sars-cov-2 and influenza and the epidemiological impact of such interaction is unknown. if individuals are coinfected with both sars-cov-2 and influenza, this could lead to more severe disease outcomes. since the beginning of the sars-cov-2 pandemic, a number of case reports of sars-cov-2 and influenza coinfection with severe outcomes have been published 1, [9] [10] [11] [12] [13] . however, there is a propensity for case reports to highlight more severe cases and there has been no systematic analysis of disease outcomes in coinfected patients compared to non-coinfected controls. in the uk the 2019-2020 influenza season peaked early with activity declining significantly from january 2020 14 . the season saw lower activity with influenza a(h3n2) as the predominant strain 14 . the first sars-cov-2 infection occurred in late january 2020 arising from an imported case, and the distribution of sars-cov-2 rose with sustained community transmission from early march in the uk peaking on 7 april 2020 with 4,493 cases and on 21 april the total number of daily sars-cov-2 deaths peaked at 1,172 15 . as such there was only a limited period of overlap between influenza circulation and sars-cov-2 circulation. in this study, we explore the interaction between influenza and sars-cov-2 during the latter stages of the 2019-2020 influenza season in england. the aims of the study are two-fold; firstly, to assess whether infection with influenza is associated with a reduced risk of sars-cov-2 infection and secondly to assess whether coinfection with influenza is associated with a more severe sars-cov-2 outcome such as death, being admitted to hospital, admitted to icu or requiring ventilatory support. the sgss (second generation surveillance system) and datamart were used to obtain all influenza positive cases between 01/01/2020 and 02/06/2020 16, 17 . for the analyses data was restricted to the time period between 20/01/2020 up to 25/04/2020, when the first sars-cov-2 and influenza coinfection occurred and the last influenza sample was reported in datamart. individuals tested for influenza who had a negative result in datamart were also extracted. both groups were matched to sars-cov-2 test results (positive and negative) in sgss as of 02/06/2020. a coinfection was defined as positive for both influenza and sars-cov-2 within 7 days of each sample date. database coordinated by nhs digital that allows the tracing of information against personal demographics, and the date of death was extracted 18 . deaths from 6 days before to 28 days after the test result were included. test results were also linked to the secondary uses service (sus) dataset and the hospital episode statistics dataset, which contain information on all admitted patient care, outpatient and a&e attendances at nhs hospitals in england 19 20 . these datasets were used to identify patients in an intensive care unit (icu) and that required the use of a ventilator within 14 days before to 28 days after the earliest test sample date were as outcome variables as well as ethnicity 21 and comorbidities as covariates. comorbidities were identified using the international classification of diseases 10 th revision (icd-10) codes and grouped into the following categories: asplenia or dysfunction of the spleen, asthma, chronic heart disease, chronic kidney disease, chronic liver disease, chronic neurological disorders, chronic respiratory disease (excluding asthma), dementia including alzheimer's, diabetes, malignancies affecting the immune system, obesity, other neoplasms, rheumatological diseases, and transplantations and conditions affecting the immune system. for the comorbidities linkage, data was restricted to inpatient and outpatient hospital episodes in the last 5 years. effect of influenza infection on the risk of sars-cov-2 infection: the total number of positive and negative sars-cov-2 and influenza test results from weeks 1 to 17, 2020 were assessed. percent positivity was calculated for individuals with a sars-cov-2 and influenza coinfection and individuals with no influenza infection by dividing the number of individuals with sars-cov-2 positive results by the total number of individuals tested and multiplied by 100. additionally, the total number individuals with a sars-cov-2 and influenza coinfection were assessed by influenza type. to estimate the effect of recent influenza infection on the risk of sars-cov-2 infection, univariable and multivariable analyses on the odds of sars-cov-2 in those who tested positive for influenza compared to those who tested negative for influenza were conducted adjusting for age, sex, ethnicity, region, comorbidity and sample week. finally, to determine the influence of unmeasured confounding such as occupation, the analysis was stratified by age into children (under 19 years), working age adults and older adults (>65). severity and risk of death among individuals with a coinfection: the mortality rate among individuals with a sars-cov-2 and influenza coinfection and those with sars-cov-2 infection who tested negative for influenza was calculated by dividing the number of deaths by the total number of individuals tested by age group. to assess whether having a coinfection was associated with death, univariable and multivariable analyses on the odds of death adjusted for age, sex, ethnicity, comorbidity (0 or 1+) and coinfection status (flu negative/ sars-cov-2 negative; flu negative/ sars-cov-2 positive; flu positive/ sars-cov-2 negative; flu positive/ sars-cov-2 positive) was assessed. this analysis was repeated with a composite outcome of ventilator use or death use and a composite outcome of icu admission or death. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint a total of 19,256 individuals were tested for both influenza and sars-cov-2 between 20/01/2020 and 25/04/2020, when the last positive influenza test was detected in datamart. in total, 58 individuals had a sars-cov-2 and influenza coinfection, 992 had a positive influenza result and were negative for sars-cov-2, 4,443 had a positive sars-cov-2 result and were negative for influenza and the remaining 13,763 were negative for both sars-cov-2 and influenza during this period ( figure 1 ). of the 58 patients with a sars-cov-2 and influenza co-infection 32 (55.2%) cases were ages 70 years and older. of the 58 cases with a sars-cov-2 and influenza coinfection, 31 individuals had influenza type a (unsubtyped), 16 had influenza type b, 8 had influenza h1n1, one had influenza type a&b and two cases had unknown influenza type. week 12 had the highest reported sars-cov-2 and influenza coinfections (20 individuals, table 1 ). a total of 13,451 (70%) individuals linked to a hospital admission record in sus between 01/12/2020 and 24/08/2020 of which 12,253 individuals had an associated record in the 14 days before and up to 28 days after the earliest sars-cov-2 or influenza test date. a total 1,666 (6%) of individuals had an icu admission and 890 (5%) were ventilated (supplementary figure 1) . of the 19,256 cases, 2,469 (12.8%) died of which 25/58 (43.1%) of the sars-cov-2 and influenza coinfected cases died. sars-cov-2 positivity among influenza positive cases was generally lower than sars-cov-2 positivity among influenza test negatives ( table 1 ). the highest sars-cov-2 positivity rate for both influenza positive and negative cases was in week 14 (66.7% and 44.8%, respectively). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. overall 43.1% of cases with coinfection died compared to 26 .9% of those who tested positive only for sars-cov-2 (table 3) . age specific mortality rates were higher among older people with a sars-cov-2 and influenza coinfection (table 3) . for individuals with influenza only, the overall mortality rate was 48/992=4.8% and for those negative for both, the mortality rate was 1,203/13,763=8.7%. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint than those with only covid-19 where the odds of death was 2.61 time greater compared to no sars-cov-2 or influenza (table 4 ). for those only positive for influenza there was a slightly decreased mortality risk (or 0.64 (95% ci 0.47-0.89)). to formally test the interaction between influenza and sars-cov-2 the same model was fitted but with separate terms for influenza, sars-cov-2 and the interaction of influenza and sars-cov-2, this gave a significant interaction effect (p=<0.001) of an additional 3.60 odds of death (95% ci 1.83-7.11) compared to that expected if influenza and sars-cov-2 acted independently. when combining ventilator use or death into a composite variable, the odds was 6.43 times greater among individuals with coinfection (95% ci 3.61-11.47). the icu admission or death composite had an odds 6.33 times greater among individuals with coinfection (95% ci 3.57-11.23) ( table 4 ). a test for interaction for both the ventilator composite and icu composite gave a significant effect (p=<0.001) with additional 3.38 odds of coinfection (95% ci 1.81-6.34) and 3.39 odds of coinfection(95% ci 1.83-6.29), respectively compared to that expected if influenza and sars-cov-2 acted independently. we found that influenza infection was associated with a lower risk of sars-cov-2 infection, suggesting that there may be pathogenic competition between these two viruses. we also found strong evidence that coinfection with influenza and sars-cov-2 was associated with an increased risk of death or severe disease and that this appears to be beyond the additive effect of the two viruses acting independently. the risk of testing positive for sars-cov-2 was 68% lower among influenza positive cases. this is consistent with recent descriptive evidence from new york where <3% of those testing positive for . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint sars-cov-2 had coinfection with influenza whereas 13% of those testing negative for sars-cov-2 were influenza positive 22 . it is also consistent with existing evidence on the interaction between influenza and seasonal coronavirus and rhinovirus [3] [4] [5] 23 . there are biologically plausible mechanisms for such an effect, including stimulation of non-specific immune responses by the first infectious agent, such as the induction of a refractory state in bystander cells as a result of the antiviral effect of interferon induced as part of an innate immune response to an rna viral infection. our findings cannot distinguish between a reduced risk of sars-cov-2 among those first infected with influenza or vice versa. a recent study has suggests that sars-cov-2 has a lower growth rate than influenza and is suppressed if the infections start simultaneously, however, if an influenza infection were to occur after sars-cov-2 infection, a coinfection would be detected 24 . our findings would not support the relaxation of preventative measures against influenza, including vaccination, given the risk of morbidity and mortality from influenza 5,25,26 as well as our finding of adverse outcomes associated with influenza and sars-cov-2 coinfection. furthermore, results from brazil indicated a significantly lower odds of needing intensive care treatment, invasive respiratory support and death among patients with sars-cov-2 that received the inactivated trivalent influenza vaccine 27 . the international council on adult immunization highlights in their roadmap that influenza, pneumococcal and herpes zoster vaccines programmes are more urgent than ever before 28 . as a further potential implication on influenza vaccination, if there is a competitive effect between influenza and sars-cov-2, this effect may also be seen with live attenuated influenza vaccination (laiv) which if offered to children in england and could in turn have a role in outbreak management. further research on the pathology of influenza and sars-cov-2 coinfection such as the order of infection and the effect of timing of influenza infection on the risk of acquiring sars-cov-2 infection, as well as any effect of laiv is required. the results from this study indicate that the risk of death was nearly six times greater among individuals with a sars-cov-2 and influenza coinfection than those with neither influenza nor sars-cov-2 and that this effect is significantly higher than the risk associated with sars-cov-2 infection alone. similarly, the combined outcomes of ventilator use or death and icu admission or death gave similar results. these findings suggest a possible synergistic effect between sars-cov-2 and influenza once an individual is coinfected. the high mortality rate is consistent with case reports of severe outcomes in coinfected patients 12, 13, 29 . conversely, some case series have not seen increased severity with influenza and sars-cov-2 co-infection, where the outcomes have been similar to cases with sars-cov-2 only 30, 31 . synergistic effects have previously been reported between influenza and other respiratory viruses, for example by facilitating cell to cell spread 32 . these findings also emphasise the importance of influenza vaccination in at risk groups and early administration of antivirals where coinfection is identified or suspected. this also adds further weight to the need for effective vaccines against influenza, in particular among the elderly among whom vaccine effectiveness tends to be lower and among whom most coinfections were seen. this has been an area of development in recent years with the introduction of high dose and adjuvanted vaccines 33 . studies of other respiratory viral infections have not indicated adverse outcomes from coinfection, for example, a study assessing sars-cov and metapneumovirus in hong kong that showed that there was no significant difference in the outcomes, including deaths between those with a sars-cov and metapneumovirus coinfection versus sars-cov alone 34 . it is important to note, that these are case studies of hospitalised individuals and the comparisons do not adjust for potential confounders. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint to our knowledge, our study is the first epidemiological study that uses national level data on both positive and negative sars-cov-2 and influenza cases. by extracting all cases with a sars-cov-2 and influenza test result, and linking the data to hes we were able to assess the effects of sars-cov-2 and influenza co-infections compared to single infection and negative test results while controlling for variables such as ethnicity, comorbidities, sex and age which are known factors for sars-cov-2 morbidity [35] [36] [37] . furthermore, the test negative design controls for the propensity for more severe cases to be tested for other respiratory viruses. most of the sars-cov-2 tests were collected when the government policy was to test individuals on admission to hospital with lower respiratory tract infections and healthcare workers 38 . therefore, the majority of sars-cov-2 cases were individuals with moderate to severe symptoms and mild cases are likely to be missed. additionally, influenza test results collected from datamart are only collected from sentinel laboratories. however, the test negative controlled design means that none of the study arms were biased towards more severe outcomes as all were tested for both diseases. additionally, in our study the majority of cases with a sars-cov-2 coinfection had influenza subtype a. due to small numbers it was not possible to determine whether the risk of sars-cov-2 coinfection and severity of disease varied by influenza subtype. while in the 2019-2020 influenza season, the majority of influenza subtype a cases were h3n2, towards the end of the season there was a shift towards h1n1, which is consistent with our finding of more h1n1 cases among those coinfections that were subtyped 14 . the impact severity of influenza and sars-cov-2 coinfection by different subtypes should be further considered in the upcoming influenza season. furthermore, the influenza vaccination status of the patients was not available therefore we could not adjust for vaccination status of the patients in the model. while our findings provide evidence of pathogenic competition between influenza and sars-cov-2, a significant number of coinfections occur and they appear to be associated with higher mortality rates. further investigation is needed in order to understand the potential mechanisms for any synergistic interaction. cocirculation of these two viruses could have a significant impact on morbidity, mortality and health service demand. as the 2020-2021 northern hemisphere influenza season approaches, it is important that a high index of suspicion for coinfection is maintained. testing strategies should include influenza and other respiratory viruses as well as sars-cov-2 and measures should be adopted to prevent coinfection including maximising uptake of influenza vaccination, particularly in groups at higher risk of both diseases. authors jlb, et, js, na developed the study protocol. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september 18, 2020. . https://doi.org/10.1101/2020.09.18.20189647 doi: medrxiv preprint role of the funding source: the study was undertaken by authors at public health england as part of the routine functions of surveillance and control of communicable diseases. public health england, national infection service, immunisation and countermeasures division has provided vaccine manufacturers with post-marketing surveillance reports, which the marketing authorisation holders are required to submit to the uk licensing authority in compliance with their risk management strategy. a cost recovery charge is made for these reports. are co-infections with covid-19 and influenza low or underreported? an observational study examining current published literature including three new unpublished cases co-infections in people with covid-19: a systematic review and meta-analysis virus-virus interactions impact the population dynamics of influenza and the common cold increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine influenza vaccination and respiratory virus interference among department of defense personnel during the 2017-2018 influenza season viral interference and interferon in vitro growth profiles of respiratory syncytial virus in the presence of influenza virus covid-19, cilia, and smell co-infection with sars-cov-2 and influenza a virus high prevalence of sars-cov-2 and influenza a virus (h1n1) co-infection in dead patients in northeastern iran co-infection of influenza b virus and sars-cov-2: a case report from taiwan the epidemiology and clinical characteristics of co-infection of sars-cov-2 and influenza viruses in patients during covid-19 outbreak co-infection with covid-19 and influenza a virus in two died patients with acute respiratory syndrome surveillance of influenza and other respiratory viruses in the uk winter 2019 to 2020. 2020. 15. public health england. coronavirus (covid-19) in the uk a new laboratory-based surveillance system (respiratory datamart system) for influenza and other respiratory viruses in england: results and experience from laboratory reporting to public health england a guide for diagnostic laboratories data-tools-and-services/data-services/hospital-episode-statistics. 20. nhs digital. secondary uses service (sus) co-infection in sars-cov-2 infected patients: where are influenza virus and rhinovirus/enterovirus? interference between outbreaks of respiratory viruses sars-cov-2 coinfections: could influenza and the common cold be beneficial modeling the impact of mass influenza vaccination and public health interventions on covid-19 epidemics with limited detection capability influenza vaccination in the covid-19 era inactivated trivalent influenza vaccine is associated with lower mortality among covid-19 patients in brazil a global agenda for older adult immunization in the covid-19 era: a roadmap for action clinical characteristics of critically ill patients co-infected with sars-cov-2 and the influenza virus in wuhan, china. international journal of infectious diseases : ijid : official publication of the international society for coinfection with sars-cov-2 and influenza a virus a case series of patients coinfected with influenza and covid-19 enhanced growth of influenza a virus by coinfection with human parainfluenza virus type 2 end of season influenza vaccine effectiveness in adults and children in the united kingdom in 2017/18 co-circulation of human metapneumovirus and sars-associated coronavirus during a major nosocomial sars outbreak in hong kong asian and minority ethnic groups in england are at increased risk of death from covid-19: indirect standardisation of nhs mortality data intensive care national audit & research centre. icnarc report on covid-19 in critical care 08 preexisting comorbidities predicting severe covid-19 in older adults in the uk biobank community cohort key: cord-328979-xfze12ah authors: monto, arnold s; malosh, ryan e; evans, richard; lauring, adam s; gordon, aubree; thompson, mark g; fry, alicia m; flannery, brendan; ohmit, suzanne e; petrie, joshua g; martin, emily t title: data resource profile: household influenza vaccine evaluation (hive) study date: 2019-04-30 journal: int j epidemiol doi: 10.1093/ije/dyz086 sha: doc_id: 328979 cord_uid: xfze12ah nan acute respiratory infections (ari) are a major cause of morbidity worldwide. 1 some of the earliest studies to describe the epidemiology of these infections were household cohort studies. [2] [3] [4] [5] [6] [7] the largest of these were conducted in seattle, washington and tecumseh, michigan in the 1960s and 1970s 6, 7 and they provided information on the relative frequency, seasonality and symptomatic characteristics of ari shortly after many respiratory viruses were first identified. 7 given that the role of household structure in seasonal incidence and transmission of respiratory viruses was the primary objective, an individual's longitudinal history of infection was not a major focus. indeed, during the first phase of the tecumseh study of respiratory illness, households were maintained on report for only 1 year, and then gradually replaced so that the entire community could be represented over time. 8 these historical studies relied on cell culture for virus identification, a method that requires specimens to be processed quickly and is considerably less sensitive than current molecular methods. 9 for this reason, the tecumseh study and others relied extensively on serodiagnosis of influenza infection using twice yearly blood specimens. 10 it was not possible to time infections in those who only were serologically positive, limiting the ability to do robust analyses of transmission patterns. 11 with current molecular techniques, it has become much easier to identify a broad range of agents of respiratory infection. this allows documentation of infection, co-infection and subsequent re-infection. collecting specimens within a short time from the onset of symptoms still maximizes the likelihood of accurate and timely identification of viruses associated with a respiratory illness for studies of transmission and vaccine effectiveness. collection of regular blood specimens continues to be valuable as well, for both virus identification and for analysis of serologic correlates of protection. the household influenza vaccine evaluation (hive) study is an ongoing prospective household cohort study that began in 2010. the hive study was based on the original tecumseh study of respiratory illness 6 with several key modifications to illness surveillance and to laboratory methods for identification of respiratory viruses. while respiratory virus infections in general could be studied, the primary objective was to estimate the effectiveness of influenza vaccines using a cohort design for comparison with studies using the testnegative design (tnd). under the tnd, specimens are collected from participants who meet a respiratory illness case definition, and vaccination frequency is compared between those who test positive for influenza and those who test negative. 12 the tnd is focused on those individuals with illnesses severe enough to seek care, which reduces bias due to differential care-seeking behaviour but misses milder presentations of respiratory virus illnesses. [13] [14] [15] the use of a prospective cohort design has allowed inclusion of these mild illnesses in evaluations of vaccine effectiveness. here we report on the first phase of the hive study, conducted from 2010 through 2016. who is included? from 2010-2011 through 2013-2014, eligible households were those who lived within 30 miles of the study clinic in ann arbor mi, and who had at least four individuals who received primary care from the university of michigan health system (umhs) and at least two children <18 years old; in 2014-2015 the eligibility criteria were changed to allow three-person households. we recruited or re-recruited households each summer or autumn. previously participating households were invited to re-enroll provided that they continued to meet the eligibility criteria. we supplemented this recruitment with direct mail or email invitations to households in the source population. in total, we have collected data and specimens with active ari surveillance for 2850 individuals from 413 households ( table 1 ). the majority of these individuals were children <18 years of age, 73% were white (non-hispanic), and 51% were female. the study cohort size has ranged from 943 to 1441 participants in each year (213 to 340 households), and 1822 (64%) individuals have been followed longitudinally over multiple years. how often have they been followed up? active surveillance for ari meeting a case definition was conducted seasonally (october-may) from 2010-2014. beginning in october 2014, year round surveillance was initiated. hive study participants were contacted weekly by email or phone to ascertain new ari meeting the study case definition. for participants 3 years of age, the case definition consisted of two or more of the following symptoms: cough, fever/feverishness, nasal congestion, sore throat, body aches, chills, headache. for participants <3 years of age, the case definition consisted of two or more of the following symptoms: cough, fever/feverishness, nasal congestion/runny nose, trouble breathing, fussiness/ irritability, decreased appetite. each household received a calendar at enrollment to assist with recording illness onset dates. when illnesses were reported, the household was contacted by study staff to schedule a clinic visit for sample collection. at illness visits for ari meeting the case definition, trained research staff collected nasal and throat swabs (or nasal swabs only for participants <3 years of age) for detection of influenza and other respiratory viruses. since october 2014, an additional, self-collected or parentcollected specimen was also collected in the home on the day of symptom onset. beginning in december 2011, all household members 13 years of age were asked to contribute a blood specimen at the initial enrollment visit and at scheduled visits twice annually thereafter. each year, autumn blood specimens (pre-season) were collected after the majority of study participants had received influenza vaccine and prior to the start of the influenza season (beginning in november). spring (post-season) specimens were collected after local influenza circulation ended each year (may-june). at the annual enrollment visits, individual participants were queried about household and demographic characteristics and advisory committee on immunization practices (acip)-defined high-risk conditions. a detailed clinical history for these participants is extracted from the umhs electronic medical record (emr), or from their regular primary care provider if they are not part of the health system. influenza vaccination information, including date, lot number, vaccine type, dose and manufacturer, was confirmed using multiple sources. information from the michigan care improvement registry (a state-maintained repository of vaccination records that requires reporting of all childhood vaccines in michigan) was requested for all study participants. provider-based vaccine records were obtained directly from the umhs emr or through medical record requests for individuals with outside-system care providers. these records were supplemented by vaccine diary cards that were filled out by participants at the time of vaccination. there has historically been a high level of vaccine uptake in the hive study population, ranging from 59-69% (table 2) . respiratory specimens collected at illness visits were tested by real-time reverse transcriptase polymerase chain reaction (rt-pcr) for laboratory confirmation of influenza, using primers and probes from the centers for disease control and prevention. influenza subtype was determined for influenza a specimens and lineage was determined for influenza b specimens. from 2010 through 2016, a total of 581 influenza infections were identified. specimens are also tested by pcr for non-influenza respiratory viruses including respiratory syncytial virus, human metapneumovirus, parainfluenza, coronavirus and rhinovirus. symptoms present since illness onset were recorded and participants completed a follow-up survey reporting illness outcomes (e.g. duration of symptoms, care seeking behaviour). blood specimens were centrifuged, and the serum was separated and stored in 1 ml aliquots at -20 c until processing in serologic assays. collected sera were tested in parallel using hemagglutinin inhibition (hai) assays. hai assays use inactivated influenza vaccine subunit material (sanofi-pasteur) from relevant a/h3n2, a/h1n1 and b vaccine strains. anti-neuraminidase antibody (nai) was measured by an enzyme-linked lectin assay (ella) using inactivated, reassortant viral targets containing contemporary neuraminidase segments and a mismatched h6 hemagglutinin ha. 16 antibody titres measured from blood specimens collected in the autumn have been analysed as both pre-season titres for the coming influenza season as well as post-vaccination titres for those individuals who were vaccinated prior to collection. similarly, antibody titres measured from blood specimens collected in the spring have served as post-season titres as well as prevaccination titres in analyses for the following study year. a full description of data collected throughout study follow-up can be found in table 3 and the timeline for key study activities can be found in figure 1 . the original aims of this study were to estimate influenza vaccine effectiveness annually and to compare those estimates with those from tnd studies conducted in outpatient clinics during the same seasons. with additional funding, we have expanded on these original aims by collecting blood specimens for studies of antibody-mediated households 328 213 321 232 340 227 participants 1441 943 1426 1049 1431 996 influenza-positive individuals 125 32 111 50 202 38 influenza-positive specimens 130 32 117 52 210 40 strain a c 86 23 69 48 166 30 h1n1pdm09 27 1 3 47 0 28 h3n2 59 22 66 1 immunity, extending ari surveillance year-round, and incorporating laboratory testing for other respiratory viruses. statistical analyses to estimate vaccine effectiveness were performed annually. adjusted hazard ratios (ahr) were estimated by cox proportional hazard models with robust variance using a sandwich estimator to account for household clustering. adjusted models included age, high-risk health status and vaccination status. vaccination status was modelled as a time-varying covariate, with subjects considered vaccinated 14 days after vaccine receipt. vaccine effectiveness was calculated as 100 x . vaccine effectiveness (ve) has varied markedly by year and age group. in 2010-2011 and 2012-2013, for example, we observed ve against any influenza infection of approximately 30% in two of three age groups. ve was lowest among adults (18 years) in 2010-2011, however, young children (6 months to 8 years) had the lowest ve in 2012-2013. 17, 18 in 2013-2014, when influenza a/h1n1 predominated, we observed ve against community acquired influenza a/h1n1 infection of 54% (95% confidence interval -4 to 80). 19 during the 2014-2015 season, we observed no effect of vaccine on the risk of infection with antigenically drifted influenza a/h3n2, but point estimates against b yamagata were near 50%. the estimated ve against b yamagata appeared to be primarily driven by high effectiveness in young children. 20 in general our annual estimates of influenza ve have been consistent with those from the us flu ve network. [21] [22] [23] [24] [25] [26] during the 2010-2011 influenza season, we found that those who had been vaccinated in the previous and current year were less protected than those who received the vaccine in the current year only. 17 this was the first time in the recent era that such a reduction was documented, thus continuing historical debates on whether repeat vaccination resulted in reduced protection. [27] [28] [29] [30] these original findings of a repeat vaccination effect prompted a furthering of the hypothesis that antibody response to vaccination was decreased after multiple annual administration of vaccines of similar antigenic make-up. in order to evaluate this hypothesis, we began collecting blood specimens in december 2011. in the 2012-13 season, the predominant circulating influenza virus was a(h3n2). relative to those unvaccinated in both years, ve was higher in those vaccinated in the current year, lower in those vaccinated in the current and previous year, and lowest in those vaccinated in the previous year only. with the addition of blood collection in the cohort, we found that hai titres correlated with the observed ve for the type a viruses. lower post-vaccination titres were found among those vaccinated in both years relative to those vaccinated only in the current season. a similar difference in post-vaccination titres was not observed for influenza b in 2012-2013. importantly, the effects of prior season vaccination on ve and serologic susceptibility to infection have not been observed in all years. during the 2013-2014 season, a year in which influenza a(h1n1)pdm09 predominated, we found similar levels of effectiveness among those vaccinated in both the current and previous seasons compared with those vaccinated only in the current season. 19 consistent with this finding, hai and nai titres against influenza a(h1n1)pdm09 were similar comparing those vaccinated in both the current and prior season to those vaccinated only in the current season. antibody titres measured in twice annual blood specimens have enabled additional evaluations of the relationship between vaccination and antibody-mediated protection. the fact that participants were observed over time allowed us to demonstrate that in this highly vaccinated cohort, prevaccination antibody titres were generally high and a 4fold rise in titre following vaccination was less frequent than expected. 19, 20 finally, measuring antibody titres both against viruses similar to those that circulated locally and against those in the vaccine, we observed increased susceptibility among those with high vaccine-strain-specific antibody titres but low circulating-strain-specific antibody titres. 31 interrupting transmission is a major goal of influenza prevention strategies and could be key to controlling the burden of disease during a pandemic. the household may be an effective place to accomplish this goal due to the high proportion of transmission estimated to occur in this setting. we have used standard epidemiologic methods to estimate serial intervals by virus type and to identify household and individual level characteristics associated with secondary infection risk. 32 these methods remain susceptible to misclassification of a transmission event, as infections are generally linked retrospectively based on the time between onset of each illness and viral type and subtype despite continued risk of infection from the community. we have attempted to address this potential for misclassification in two ways. first, it is now possible using next-generation sequencing to differentiate the two types of transmission on the basis of genetic similarity as we have demonstrated with influenza a viruses. 33 second, we have adapted individual-based transmission models to account for risk of infection from both the community and the household and to allow for chains of transmission. 34 the hive study from the start has identified viruses other than influenza, and, for one season, bacterial agents associated with respiratory illnesses. 35 surveillance initially extended through much of the respiratory season and has been conducted year-round since october 2014; thus it has been possible to determine seasonality of these respiratory viruses. 36 in addition, the detection of multiple respiratory viruses has allowed us to describe the frequency of coinfection in different age groups. 36 the hive study has recently received continued core funding and is now recruiting existing and new households into a second phase of the cohort. a commitment to 5 years of follow-up with year-round surveillance for ari is now required of those entering the study. blood specimen collection, which in the past was limited to those 13 years of age, is now extended to younger children and includes collection of peripheral blood mononuclear cells (pbmcs) for enrollment / re-engagement blood collecæ�on ari surveillance figure 1 . timeline of study enrollment, acute respiratory illness (ari) surveillance and blood collection activities. blood collection for antibody studies began in the autumn of 2011. ari surveillance has been carried out continuously on a year-round basis since the autumn of 2014; ari surveillance was limited to the typical influenza season prior to that. studies of cell-mediated immunity. in addition, targeted recruitment strategies are now focused on children <36 months old and their households in an effort to study infections and immune response in early life. the main strength of the hive study is the longitudinal, prospective study design with intensive data collection. active surveillance for symptomatic illness with collection of respiratory specimens for laboratory-confirmation of influenza and other viruses allows for calculations of the incidence and determinants of infection over time, and builds on previous cohorts in the region that defined illness based on serology alone. using multiple sources, including participant medical records, to document influenza vaccination status and regular serologic sampling are major strengths of the cohort. the number of participants that have been prospectively followed for multiple years has enabled multi-year studies of immune response to infection and vaccination, studies that are increasingly important for understanding how individual influenza immunity evolves over time. 37 the illness sampling strategy requires participant symptom reporting. it is therefore important to note that subclinical infections are not captured by illness sampling, as this would require routine swabs in symptom-free individuals. however, the availability of end-of-season serologic specimens allows for analysis for uncaptured influenza virus infections in unvaccinated individuals. the intensive data collection increases the burden on study participants. it is therefore necessary to build relationships with the community to ensure enrollment and long-term participation. as a result the study is resource intensive and the study population is limited in terms of both sample size and generalizability. in particular the source population from which participants is drawn is limited geographically to households who are able to travel to the university of michigan study site for enrollment visits, blood draws and illness specimen collection. the hive study population consists of suburban residents and largely reflects the high education level and vaccination uptake in our region. one approach to increase sample size and generalizability would be to establish multi-site cohorts, presenting logistical considerations that might be challenging to unify under a common protocol. indeed, other longitudinal cohorts of respiratory illness have used varied recruitment strategies or follow-up methods tailored to their unique communities. 38, 39 our history of involvement in this community, stretching back to the 1960s, has given us the opportunity to optimize the cohort for longterm success. the investigators regularly collaborate with others to address key questions in respiratory virus epidemiology and immune correlates of protection. proposals for future collaborations using hive study specimens and data can be submitted to the study investigators for consideration. the hive study was supported by the centers for disease control and prevention (u01 ip000170, u01 ip000474) and the national institute of allergy and infectious diseases (r01 ai097150, r56 ai097150). conflict of interest: e.t.m has received grant support from merck and pfizer for work unrelated to this report. a.s.m. has received consultancy fees from sanofi, seqirus and novavax for work unrelated to this report. a.s.l. has received consultancy fees from sanofi for work unrelated to this report. all other authors declare no conflict of interest. â�¢ we collected longitudinal data on demographics, health history, influenza vaccination status and ari incidence. we collected specimens to detect occurrence by rt-pcr of influenza and other respiratory viruses and serum to determine antibody titres to hemagglutinin and neuraminidase. specimens are archived. â�¢ investigators interested in learning more about the hive study as well as data and specimen availability are welcome to email the study investigators. estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the global burden of disease study acute minor respiratory diseases prevailing in a group of families residing in baltimore, maryland, 1928-1930. prevalence, distribution and clinical description of observed cases a study of illness in a group of cleveland families i. plan of study and certain general observations acute upper respiratory infections in families illness in the home: a study of 25, 000 illnesses in a group of cleveland families the tecumseh study of respiratory illness: i. plan of study and observations on syndromes of acute respiratory disease the seattle virus watch. ii. objectives, study population and its observation, data processing and summary of illnesses acute respiratory illness in an american community. the tecumseh study rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 the tecumseh study of respiratory illness. ix. occurence of influenza in the community tecumseh study of illness. xiii. influenza infection and disease, 1976-1981 the testnegative design: validity, accuracy and precision of vaccine efficacy estimates compared to the gold standard of randomised placebocontrolled clinical trials the case testnegative design for studies of the effectiveness of influenza vaccine the test-negative design for estimating influenza vaccine effectiveness theoretical basis of the test-negative study design for assessment of influenza vaccine effectiveness an optimized enzymelinked lectin assay to measure influenza a virus neuraminidase inhibition antibody titers in human sera influenza vaccine effectiveness in the community and the household influenza vaccine effectiveness in households with children during the 2012-2013 season: assessments of prior vaccination and serologic susceptibility substantial influenza vaccine effectiveness in households with children during the 2013-2014 influenza season, when 2009 pandemic influenza a(h1n1) virus predominated the household influenza vaccine effectiveness study: lack of antibody response and protection following receipt of 2014-2015 influenza vaccine effectiveness of seasonal influenza vaccines in the united states during a season with circulation of all three vaccine strains influenza vaccine effectiveness in the 2011-2012 season: protection against each circulating virus and the effect of prior vaccination on estimates influenza vaccine effectiveness in the united states during 2012-2013: variable protection by age and virus type influenza vaccine effectiveness against 2009 pandemic influenza a(h1n1) virus differed by vaccine type during 2013-2014 in the united states influenza vaccine effectiveness in the united states by vaccine type influenza vaccine effectiveness in the united states during the 2015-2016 season the doctrine of original antigenic sin: separating good from evil assessment of inactivated influenza-a vaccine after three outbreaks of influenza a at christ's hospital efficacy of repeated annual immunization with inactivated influenza virus vaccines over a five year period variable efficacy of repeated annual influenza vaccination antibodies against the current influenza a(h1n1) vaccine strain do not protect some individuals from infection with contemporary circulating influenza a(h1n1) virus strains influenza transmission in a cohort of households with children stochastic processes constrain the within and between host evolution of influenza virus application of an individual-based transmission hazard model for estimation of influenza vaccine effectiveness in a household cohort co-colonization by streptococcus pneumoniae and staphylococcus aureus in the throat during acute respiratory illnesses frequency of acute respiratory illnesses and circulation of respiratory viruses in households with children over 3 surveillance seasons a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases mosaic: mobile surveillance for acute respiratory infections and influenzalike illness in the community community surveillance of respiratory viruses among families in the utah better identification of germs-longitudinal viral epidemiology (big-love) study the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. hive study research staff: barbara aaron; amy p. callear; rachel truscon; emileigh johnson; caroline k. cheng; anne kaniclides; natalie williams; casey martens. key: cord-296935-y77c4ro4 authors: couch, robert b.; atmar, robert l.; franco, luis m.; quarles, john m.; niño, diane; wells, janet m.; arden, nancy; cheung, sheree; belmont, john w. title: prior infections with seasonal influenza a/h1n1 virus reduced the illness severity and epidemic intensity of pandemic h1n1 influenza in healthy adults date: 2011-11-10 journal: clinical infectious diseases doi: 10.1093/cid/cir809 sha: doc_id: 296935 cord_uid: y77c4ro4 background. a new influenza a/h1n1 (ph1n1) virus emerged in april 2009, proceeded to spread worldwide, and was designated as an influenza pandemic. a/h1n1 viruses had circulated in 1918–1957 and 1977–2009 and were in the annual vaccine during 1977–2009. methods. serum antibody to the ph1n1 and seasonal a/h1n1 viruses was measured in 579 healthy adults at enrollment (fall 2009) and after surveillance for illness (spring 2010). subjects reporting with moderate to severe acute respiratory illness had illness and virus quantitation for 1 week; evaluations for missed illnesses were conducted over holiday periods and at the spring 2010 visit. results. after excluding 66 subjects who received ph1n1 vaccine, 513 remained. seventy-seven had reported with moderate to severe illnesses; 31 were infected with ph1n1 virus, and 30 with a rhinovirus. determining etiology from clinical findings was not possible, but fever and prominent myalgias favored influenza and prominent rhinorrhea favored rhinovirus. tests of fall and spring antibody indicated ph1n1 infection of 23% had occurred, with the rate decreasing with increasing anti-ph1n1 antibody; a similar pattern was seen for influenza-associated illness. a reducing frequency of ph1n1 infections was also seen with increasing antibody to the recent seasonal a/h1n1 virus (a/brisbane/59/07). preexisting antibody to ph1n1 virus, responses to a single vaccine dose, a low infection-to-illness ratio, and a short duration of illness and virus shedding among those with influenza indicated presence of considerable preexisting immunity to ph1n1 in the population. conclusions. the 2009 a/h1n1 epidemic among healthy adults was relatively mild, most likely because of immunity from prior infections with a/h1n1 viruses. shown to be a triple reassortant virus containing genes from swine, human, and avian influenza a viruses [3] . the virus spread rapidly throughout north america and worldwide, causing the world health organization (who) to declare the spread as pandemic influenza [4] . public health authorities mobilized for monitoring and control as the virus proceeded to cause epidemic influenza in the southern and northern hemispheres during the spring and summer of 2009. pandemic h1n1 influenza (ph1n1) peaked in the united states in october 2009, with minimal activity during the usual winter period of influenza [5] . retrospective estimates of the medical impact for the united states for the period april 2009 to april 2010 are 60.8 million cases, 274 304 hospitalizations, and 12 469 deaths, less than some seasonal epidemics but substantial in children [6] . the hemagglutinin (ha) of the ph1n1 virus is a swine virus ha similar to the ha of viruses isolated from swine in north america in recent years and related to influenza a/h1n1 viruses that have circulated in swine since first detected in 1930 [7] . antigenically related influenza a/h1n1 viruses circulated in humans from 1918 to 1956 and reappeared in 1977 as the ''russian flu'' [8] . at that time, persons $25 years of age were ''primed'' for h1 antigens from prior a/h1n1 virus infections; they experienced very little clinical influenza during the 1977-1978 a/h1n1 virus epidemic [9] . because a/h1n1 viruses had caused infections and influenza as well as having been included in the annual influenza vaccine since 1977, it seemed likely that large numbers of people would have a degree of immunity to the ph1n1 virus, with increasing susceptibility likely to increase with decreasing age. however, no specific age could be designated for full susceptibility as was possible in 1977. it seemed likely that young adults would have a high degree of susceptibility. a serological survey for specific antibody conducted by the centers for disease control and prevention had indicated very little ph1n1 antibody in them [10] . to assess the clinical and epidemiological impact of ph1n1 infections and to identify immunologic factors correlating with infections and illnesses, we conducted a prospective study of influenza in a young adult population. here we describe the infections and illnesses in this population with the ph1n1 virus, assess the role of prior seasonal h1n1 infections in occurrences of infection and illness with the ph1n1 virus, and discuss the similarity of the ph1n1 experience with that of the ''russian'' h1n1 influenza that emerged in 1977. the study was conducted at texas a&m university, college station. healthy persons ages 18 to 49 at the college and in the community were invited to enroll to be followed for acute respiratory illness (ari) through the influenza season. the protocol and informed consent were approved by the baylor college of medicine and texas a&m university institutional review boards before the study began. after subjects provided consent, a medical history was taken to ensure good health, and baseline specimens were obtained. surveillance for influenza began during the september 2009 enrollment period because ph1n1 as a cause of influenza was identified in the population during enrollment. subjects were given thermometers and instructions to call within 48 hours of onset for any ari. except for 4 days of the thanksgiving holiday period and 4 weeks of the christmas holiday period, a coordinator and physician were available every day to see patients. those persons presenting within 48 hours of onset with a new ari with fever or that caused them to miss school, work, or social activities were enrolled for evaluation. specimens were obtained and medical care was provided, including the antiviral zanamivir if indicated. ill persons were seen 2, 4, and 6 days later for repeat evaluation, specimen collections, and medical care and 21 days later for collection of convalescent specimens. for illnesses occurring during the christmas holiday, subjects obtained an oral temperature and completed a symptom and medication diary for 7 days; a physician reviewed the diary and blood was obtained on return to college station about 21 days after the illness onset. surveillance for influenza was terminated after 5.5 months; all subjects were asked to return for specimen collection and to provide a medical and ari history. the criteria for illness enrollment are those categorizing an illness as moderate or severe (table 1) . a study physician obtained an oral temperature, completed a symptom survey, and performed a respiratory system examination at each illness visit. each symptom or physical finding was graded as mild, moderate, or severe using a 1-3 scale. a retrospective survey for subjects with moderate to severe ari who had not reported to the study site was conducted at the final visit. based on symptom complexes, a reported ari was classified by a study physician as probable influenza, possible influenza, or not influenza before testing of fall and spring sera for evidence of ph1n1 infection. serum specimens obtained at enrollment, acute and convalescent visits for illnesses, and the terminal visit were tested simultaneously using hemagglutination-inhibition (hai) antibody tests following previously described methods [11, 12] . virus antigens were a locally obtained ph1n1 virus (a/baylor/09) and the most recently prevalent seasonal a/h1n1 virus (a/brisbane/59/07). a combined 8-ml nasal wash and throat swab specimen was collected at each illness visit. specimens were tested for all respiratory viruses in tissue cultures; influenza-positive specimens were titered in 96-well plates for quantity of virus. for quantitation, plates were incubated for 5 days and endpoints were determined by hemagglutination. all specimens were also tested by reverse-transcriptase polymerase chain reaction (rt-pcr) for respiratory viruses including influenza a, ph1n1 influenza, influenza b, picornavirus/rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses, coronaviruses, and adenoviruses, as described elsewhere [13] [14] [15] [16] [17] [18] [19] . rxc contingency and v 2 for trend tests were used for frequency comparisons and the mann-whitney u test and kruskal-wallis test for comparisons of means. when the study began, texas a&m university had 48 702 students and college station had a population of approximately 95 000. during september 2009, 615 healthy adults were enrolled in our study; 578 (95%) were between the ages of 18 and 30; 362 (59%) were male and 253 (41%) were female. three hundred ninety-four (64%) were white, 158 (26%) asian, 69 (11%) hispanic, 14 (2%) black, and the remainder multiracial or not reported. spring 2010 follow-up information and specimens were obtained from 579 (94%) subjects. sixty-six subjects had obtained vaccination with the 2009 ph1n1 vaccine and 50 of the 66 (76%) had developed a significant antibody response between fall and spring. none of the 66 reported ill with a ph1n1 infection. thirty-eight subjects had received only seasonal inactivated vaccine and 3 (7.9%) reported ill with a ph1n1 infection. the ph1n1 vaccinees were excluded; the final population for analysis was 513. the number of persons reporting to the university health center with an ari by week for the year after classes commenced is shown in figure 1 . all health center visits are coded; codes used for possible influenza were international classification of diseases, ninth revision codes that correlate with proven influenza [20] . also shown is the number of specimens that tested positive for influenza a in rapid diagnostic tests at the clinic or in rt-pcr tests from subjects reporting an ari. as shown, the ph1n1 epidemic was ongoing among students during the enrollment period (weeks 37-39); no other influenza virus was detected during surveillance. the epidemic peaked in a 4-week period in late september and early october (weeks 37-40); illnesses caused by ph1n1 virus continued at a low level throughout the remaining surveillance period. seventy-six subjects with 77 illnesses reported to the study site with a moderate or severe ari during the surveillance period. sixty-three (82%) of these illnesses yielded a virus; 24 were ph1n1 influenza virus, 22 rhinovirus, 7 both ph1n1 and ) median tissue culture infective dose (tcid 50 )/ml of specimen. the mean titer was only 80 tcid 50 2 days later and ,10 at the day 5 and day 7 visits. eighteen of these subjects were given zanamivir as treatment (day 1 or day 2 of illness) and 11 were not (physician decision). mean titers at presentation were higher for those treated (10 5.3 vs 10 4.0 ), but the means were not significantly different (mann-whitney u test). titers were low at subsequent visits and did not differ; similarly, the means between day 1 and day 3 for those treated and not treated did not differ. a comparison of illnesses among subjects infected with ph1n1 influenza, a rhinovirus, or both is shown in table 1 . at presentation, fever ($100°f) was more common among subjects with influenza (rxc contingency, p , .001). for mean symptom severity scores, myalgia scores were greater for influenza; rhinorrhea scores were greater for rhinovirus infections (p 5 .01 and ,.01, respectively; mann-whitney u test). myalgias and rhinorrhea were also different among the 3 groups (p 5 .02 and p 5 .01, respectively, kruskal-wallis test). rhinorrhea persisted as a major symptom for rhinovirus-infected subjects for the 7 days of observation. occurrences of ph1n1 influenza infections and illnesses in relation to serum hai antibody titer at enrollment are shown in table 2 . one hundred twenty-two (23.0%) subjects exhibited a significant antibody response to ph1n1 between enrollment (september 2009) and spring follow-up (march 2010). one hundred eighty-eight (37%) had serum antibody ($1:8) to ph1n1 virus at enrollment. there was an inverse correlation between baseline serum antibody titer to ph1n1 virus and occurrence of ph1n1 infection during the surveillance period (v 2 for trend, p , .001). thirty-one symptomatic influenza infections among enrolled subjects were detected, 30 by virus and serologic tests and 1 by serologic tests only. the apparent inverse correlation for infection and illness among those enrolled with ari was not significant. forty-five retrospectively identified aris were considered moderate to severe; ph1n1 infection was detected in 14 of 19 (74%) subjects with probable influenza, 4 of 9 (44%) subjectswith possible influenza, and 2 of 17 (12%) subjects not designated to have clinical influenza (the clinical diagnosis for the 2 was a severe cold). when the retrospectively identified moderate to severe aris with a significant antibody response were included, the inverse correlation between baseline titer and frequency of ph1n1 infection and illness was significant (v 2 for trend, p 5 .01). two studies of seasonal inactivated influenza vaccine indicating the antigenic relationship of seasonal h1n1 and ph1n1 viruses had been conducted in the population in the year preceding the 2009-2010 study, 1 among males only (september 2008) and the other (march 2009) in a mixed population similar to that of the present study. in the 2008 study, 60% had seasonal h1n1 antibody ($1:8) and 71% (confidence interval [ci], 63%-79%) developed a response to the seasonal vaccine virus; 17% had ph1n1 antibody and 19% (ci, 13%-27%) developed a response to ph1n1 virus. for the spring 2009 study, 70% had seasonal antibody and 62% (ci, 54%-70%) developed antibody to seasonal virus; 24% had ph1n1 antibody and 17% (ci, 12%-24%) developed a response to ph1n1 virus. forty-four of 48 antibody responses (92%) to ph1n1 were in subjects with prevaccination titers of ,1:8. the frequency of ph1n1 infections among participants in relation to their serum antibody titer to the recent seasonal h1n1 virus decreased with increasing baseline seasonal antibody titer (table 3 ; v 2 for trend, p 5 .02). a 44% reduction in occurrence of infection with ph1n1 influenza virus was noted among those with seasonal antibody at baseline. a reduction in frequencies of subjects with moderate to severe illness among those infected was not statistically significant (data not shown). this prospective study of 2009 pandemic influenza a infections and illnesses in a university community detected an infection frequency of 23%, with a moderate to severe ari frequency of 9.6%. the epidemic peaked in late september-early october 2009, but infections occurred for months thereafter. most participants were 18-30 years of age, and yet 37% had serum antibody to the ph1n1 virus at enrollment. a similar population at the same site 6 months earlier, preceding the spread of ph1n1 in the united states, exhibited an antibody frequency of 24%, suggesting that about 13% of the population was infected with the ph1n1 virus during the interval. there had been reports of outbreaks of ph1n1 influenza during that period in the united states and in texas. serum antibody responses to ph1n1 virus among young adults in the same population who were given seasonal inactivated vaccine containing a/brisbane/ 59/07 (h1n1) virus before the appearance of ph1n1 suggests that seasonal vaccine might contribute cross-reacting antibody and priming to ph1n1 virus. however, only 10% of the study population reported prior seasonal vaccination. consequently, prior a/h1n1 infections in combination with some ph1n1 infections preceding enrollment and not vaccinations will have induced the high frequency of preexisting ph1n1 antibody at enrollment. although rapid worldwide spread suggested high transmissibility, the ph1n1 viruses failed to become dominant as the cause of ari in our population during the period of maximal occurrence. rhinoviruses, known to be common causes of ari in college populations in early fall, were as prominent as ph1n1 viruses as a cause of illness, and some subjects were infected with both simultaneously [21, 22] . comparison of influenza and rhinovirus illnesses indicated that presentation with fever and prominent myalgias increased the likelihood that influenza virus infection induced the illness, while prominence of rhinorrhea increased the likelihood of a rhinovirus infection. however, it was not possible to make a designation of etiology based on clinical findings only. resistance to 2009 h1n1 influenza infection and illness in our study conformed to the well-documented inverse correlation with increasing preexposure serum anti-ha antibody. the significance of an antigenic relationship between h1n1 viruses was shown in a similar inverse correlation with the titer of antibody to the most recently preceding seasonal a/h1n1 virus. mean titers in our hai tests for those .25 years of age in 1977 and those 18-40 years in spring 2009 were both 1:6; titers $1:40 were detected in 20% of persons in our 1977 tests and $1:32 in 8% in 2009 tests [23] . despite the relatively low antibody titers in 1977, substantial immunity to a/h1n1 illness was seen in adults. the present study also found evidence of a substantial degree of immunity despite the fact that about two-thirds of the population lacked detectable serum hai antibody to the ph1n1 virus [9] . presence of heterologous immunity in the absence of detectable serum hai antibody to the epidemic virus has been shown previously [24] . additional evidence of preexisting immunity to ph1n1 virus in our study was indicated by the relatively low illness-to-infection ratio, the short duration of fever, the rapid disappearance of virus in respiratory secretions, and the antibody responses to a single dose of the 2009 h1n1 vaccine. the fact that immunity to ph1n1 is conveyed by prior seasonal a/h1n1 infection has been reported for animal model infections and suggested for humans [25] [26] [27] . three new and distinct introductions of influenza a/h1n1 viruses that caused widespread influenza occurred in 1918, 1977, and 2009 [1, 2, 8, 28] . in each instance, the viruses displayed a high capability for transmissibility and infectivity. the new strain of influenza a/h1n1, identified in the soviet union in 1977 (a/ussr/77 [h1n1]), proceeded to spread worldwide, with high infection rates among susceptibles that were comparable to those for the a/h2n2 and a/h3n2 pandemics of 1957 and 1968, respectively [29, 30] . since then, antigenic variants of the 1977 h1n1 virus have continued as causes of human infections and illnesses. in the spring of 2009, ph1n1 virus emerged and spread rapidly worldwide. thus, the a/h1n1 influenza viruses that emerged in 1918, 1977, and 2009 demonstrated a high capacity for transmissibility among humans. the 1918 h1n1 viruses caused high frequencies of severe influenza, a pattern also seen for the a/h2n2 and a/h3n2 virus pandemics [28] [29] [30] [31] [32] . however, this was not clear for the 1977 a/h1n1 viruses; although high ratios of symptomatic to asymptomatic infections were reported in some outbreaks, other reports described low illness rates among susceptible groups [9, 29, 33, 34] . in 1977, antibody prevalence, vaccine responses, and subsequent surveillance data indicated a high level of immunity among those .25 years of age despite relatively low levels of antibody; they were likely exposed to a/h1n1 viruses before the viruses disappeared in 1957 [9, 35, 36] . the 2009 a/h1n1 infections caused concern for a pandemic with high frequencies of severe disease that would cause high hospitalization and death rates. this concept did not adequately consider the high level of preexisting experience with a/h1n1 viruses in human populations. based on prior experience with a/h1n1 viruses in 1976-1977, a considerable degree of immunity was expected for ph1n1 viruses among those .55 years of age and against the swine ha among those .85 years [35, 36] . there was uncertainty as to which age groups would be fully susceptible to the 2009 virus, with accompanying high illness rates, because circulation of a/h1n1 viruses had occurred over the 32 years since they were reintroduced in 1977. this study has indicated that a high level of immunity existed among adults 18 years and older despite relatively low levels of antibody. the 2009 pandemic influenza experience indicated that the full susceptibility with high illness rates among healthy persons was only in children [37, 38] . overall, the human experience with influenza a/h1n1 viruses over the 93-year interval since 1918 has considerably increased our knowledge of influenza and influenza immunity. severe respiratory disease concurrent with the circulation of h1n1 influenza novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans triple-reassortant swine influenza a (h1) in humans in the united states new influenza a (h1n1) virus: global epidemiological situation epidemiology of 2009 pandemic influenza a (h1n1) in the united states estimating the burden of 2009 pandemic influenza a (h1n1) in the united states antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans global surveillance of influenza age distribution of patients with medically-attended illnesses caused by sequential variants of influenza a/h1n1: comparison to age-specific infection rates cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus diagnostic procedures for viral, rickettsial and chlamydial infections high doses of purified influenza a virus hemagglutinin significantly augment serum and nasal secretion antibody responses in healthy young adults comparison of real-time pcr assays with fluorescent-antibody assays for diagnosis of respiratory virus infections in children development of a real-time rt-pcr assay for detection and quantitation of parainfluenza virus 3 real-time pcr with an internal control for detection of all known human adenovirus serotypes design and performance of the cdc real-time reverse transcriptase pcr swine flu panel for detection of 2009 a(h1n1) pandemic influenza virus respiratory viral infections in patients with chronic obstructive pulmonary disease ldh concentration in nasal wash fluid as a biochemical predictor of bronchiolitis severity human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays code-based syndromic surveillance for influenzalike illness by international classification of diseases, ninth revision virologic studies of acute respiratory disease in young adults. iv. virus isolations during four years of surveillance acute respiratory illnesses in university (1962-1966), military and industrial (1962-1963) populations reactogenicity, immunogenicity and antibody persistence in adults given inactivated influenza virus vaccines an outbreak of an influenza type a variant in a closed population: the effect of homologous and heterologous antibody on illness and infection impact of prior seasonal influenza vaccination and infection on pandemic a(h1n1) influenza virus replication in ferrets multiple infections with seasonal influenza a virus induce crossprotective immunity against a(h1n1) pandemic influenza virus in a ferret model protective efficacy of seasonal influenza vaccination against seasonal and pandemic influenza virus infection during 2009 in hong kong the epidemiology of influenza epidemiological and clinical aspects of influenza epidemiology of the hong kong/68 variant of influenza a2 in britain morbidity and mortality characteristics of asian strain influenza hong kong influenza: the epidemiologic features of a high school family study analyzed and compared with a similar study during the 1957 asian influenza epidemic outbreak of influenza a/ussr/77 at marquette university communitywide surveillance of influenza after outbreaks due to h3n2 (a/victoria/75 and a/texas/77) and h1n1 (a/ussr/77) influenza viruses session v. summary of clinical studies. summary of clinical trials of influenza virus vaccines in adults summary of clinical trials of inactivated influenza vaccine-1978 incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study the infection attack rate and severity of 2009 pandemic h1n1 influenza in hong kong acknowledgments. we thank dr martha dannenbaum, scott draper, richard darnell, and other staff of the beutel health center at texas a&m university for their strong support in facilitating this research study. we thank the staff of the viral pathogens research unit laboratory for their high-quality effort and i. darlene kirk, ccrp, for aid in coordinating the study and preparing the manuscript.disclaimer. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does mention of trade names, commercial products, or organizations imply endorsement by the us government.financial support. this work was supported by the national institute of allergy and infectious diseases (public health service contract no1-ai-30039).potential conflicts of interest. all authors: no reported conflicts. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-352984-mzv9t7ex authors: jackson-lee, angela; barr, neil g.; randall, glen e. title: mandating influenza vaccinations for health care workers: analysing opportunities for policy change using kingdon’s agenda setting framework date: 2016-09-29 journal: bmc health serv res doi: 10.1186/s12913-016-1772-0 sha: doc_id: 352984 cord_uid: mzv9t7ex background: the consequences of annual influenza outbreaks are often underestimated by the general public. influenza poses a serious public health threat around the world, particularly for the most vulnerable populations. fortunately, vaccination can mitigate the negative effects of this common infectious disease. although inoculating frontline health care workers (hcws) helps minimize disease transmission, some hcws continue to resist participating in voluntary immunization programs. a potential solution to this problem is government-mandated vaccination for hcws; however, in practice, there are substantial barriers to the adoption of such policies. the purpose of this paper is to identify the likelihood of adopting a policy for mandatory immunization of hcws in ontario based on a historical review of barriers to the agenda setting process. methods: documents from secondary data sources were analysed using kingdon’s agenda setting framework of three converging streams leading to windows of opportunity for possible policy adoption. results: the problems, politics, and policies streams of kingdon’s framework have converged and diverged repeatedly over an extended period (policy windows have opened and closed several times). in each instance, a technically feasible solution was available. however, despite the evidence supporting the value of hcw immunization, alignment of the three agenda setting streams occurred for very short periods of time, during which, opposition lobby groups reacted, making the proposed solution less politically acceptable. conclusions: prior to the adoption of any new policies, issues must reach a government’s decision agenda. based on kingdon’s agenda setting framework, this only occurs when there is alignment of the problems, politics, and policies streams. understanding this process makes it easier to predict the likelihood of a policy being adopted, and ultimately implemented. such learning may be applied to policy issues in other jurisdictions. in the case of mandatory influenza vaccinations for hcws in ontario, it seems highly unlikely that a new policy will be adopted until perception of the problem’s importance is sufficient to overcome the political opposition to implementing a solution and thus, create a window of opportunity that is open long enough to support change. the consequences of annual influenza outbreaks are often underestimated by the general public. this circumstance may be due in part to a large proportion of healthy young adults experiencing only minor symptoms [1] , which has contributed to a lack of public awareness and complacency regarding the severity of its effects on vulnerable populations such as the elderly and individuals that are immunocompromised. despite this misconception, influenza poses a serious public health threat around the world. the world health organization (who) estimates that annual influenza epidemics cause 3 to 5 million cases of severe illness worldwide, along with 250,000 to 500,000 annual deaths [2] . these epidemics are also associated with overwhelmed clinics and hospitals, many of which need to account for increased staff absenteeism and productivity losses due to illness of health care workers (hcws) [2] . the impact of annual influenza epidemics on some of the most susceptible populations, such those living with chronic conditions, is even more striking. global metaanalyses of risk factors for severe disease from pandemic influenza found that 31 % of patients hospitalized had at least one other chronic medical condition, as did 52 % of those admitted to intensive care units and 62 % of fatal cases [3] . in the united states, individuals who were 65 or older consistently accounted for approximately 90 % of all influenza related deaths between 1976 and 2007 [3] . fortunately, vaccination can mitigate the negative effects of this common infectious disease. during the influenza seasons of 2010-2012, the centers for disease control and prevention found that influenza vaccination helped reduce children's risk of influenza-related intensive care unit admissions by 74 % [4] . the use of vaccinations has also been associated with a 77 % reduction in influenzarelated hospitalization among adults aged 50 or over [5] . in addition, rates of influenza infections were found to be lower in vaccinated hcws versus unvaccinated hcws [6] and other healthy adults [7] . however, while vaccination may reduce the risk of influenza in hcws, a recent systematic review reported the reduced risk to be less than 50 % as compared to hcws who are not vaccinated; which suggests the need for additional solutions beyond hcw vaccinations [6] . furthermore, recent reviews highlight the lack of clear evidence to support the benefit of vaccinating hcws [8] to prevent the spread of influenza to elderly [1] . within high-income countries, influenza vaccination is readily accessible and many jurisdictions have implemented publicly funded programs to cover the cost [9] . however, public vaccination uptake has been variable [10, 11] , and perhaps more importantly, some hcws continue to resist participation in vaccination programs. for example, in canada, voluntary uptake of the seasonal influenza vaccine by hcws remains below the 90 % recommended level [12] and varies substantially across health care organizations [13] . other studies have reported that in some locations more than 50 % of physicians also fail to take advantage of the annual influenza vaccination [13] . inadequate levels of hcw immunization can place the vulnerable populations they care for at greater risk of health complications [14] . a potential solution to this problem is governmentmandated inoculation for hcws. however, in practice, there are substantial barriers to the adoption of such policies. for instance, the attitudes of hcws in the united states toward whether to be vaccinated against influenza or not remains divided, prompting a backlash against suggestions of any mandatory policies [15] . surveys have revealed multiple reasons why hcws disapprove of mandated vaccination. these include beliefs that the decision to be vaccinated is personal, fears of side effects, and concern that influenza vaccines are ineffective [15] . there have also been legal obstacles to mandating vaccination as worker unions and professional associations have argued that such policies violate individual rights and freedoms [16] . specifically, in canada, it has been argued that mandated immunization violates one's right to refuse unwanted medical treatment under section 7 of the canadian charter of rights and freedoms [16] . new public policies are rarely adopted based solely on supportive evidence, public opinion, or interest group lobbying; rather, they typically require a confluence of events that is difficult to predict or orchestrate. through the use of relevant frameworks, theories, or models, policy analysis can provide a greater understanding of the various processes involved and, in turn, enhance the likelihood of recognizing and seizing opportunities for new policies to be adopted [17] . in this vein, kingdon's [18] three process streams framework helps to explain how three elements or "streams"-problems, politics, and policiesaffect policy agenda setting dynamics. kingdon argues that, while there is some interplay among the streams, they are largely independent of one another [18] . this framework can be used to explain how issues may make their way onto the government's decision agenda, which is a key step in the policy adoption process. the purpose of this paper is to identify the likelihood of adopting a policy for mandatory immunization of hcws in ontario based on a historical review of barriers to the agenda setting process. background information regarding the effectiveness and uptake of influenza vaccinations was gathered via narrative review, which included academic and newspaper articles, as well as government reports. subsequently, these materials were analysed using kingdon's agenda setting framework of three converging streams leading to windows of opportunity for possible policy adoption. no permissions to analyse the data used in this study were necessary or granted. the authors independently categorized historical events as falling primarily into the problems, politics, or policies stream (see table 1 ). categories are based on kingdon's description as noted below. any discrepancies in classification were resolved through discussion. in kingdon's framework, the problems stream involves the identification of a particular social problem that has gained public or decision-maker attention and cannot be easily ignored. these problems may come to light through awareness of a change in an indicator, such as an increased infection rate, or a focusing event/crisis, such as the severe acute respiratory syndrome (sars) outbreak in 2003. in the politics stream, the governmental agenda is formulated and the list of issues or problems to be given attention is prioritized. this stream is impacted by political events (such as changes in public opinion/national mood), and organized forces (such as a change in government due to elections). in the policies stream, experts analyse the various problems, and suggest technically feasible and politically acceptable solutions to them. generally, the three streams flow independently of one another; however, a time-limited window of opportunity may open when these streams converge (as politically acceptable solutions to prioritized problems are identified). at this point of convergence, issues are most likely to reach the government's decision agenda-where policy problems and their proposed solutions are under active discussion by government decision-makers-and thus, most likely to result in the adoption of a new policy. policy windows can also close rapidly. this tends to occur when: problems, politics, and policies are not adequately linked together; initiatives that are implemented lead to either failure or success; indicators of severity lessen; other critical items push the issue off the agenda; there has been movement through an "issue attention cycle" (i.e., a gradual decline of interest in the condition). using kingdon's agenda setting framework (three process streams that lead to windows of opportunity when they converge) the objective of this paper is to analyse the likelihood of government adopting a mandatory vaccination policy for hcws in ontario. ontario endured through a difficult 1999-2000 influenza season. hospital emergency departments experienced serious overcrowding [19] , which resulted in less access to health services for patients seeking critical care. this unfavourable set of circumstances was a focusing event/crisis (problems stream) that resulted in heightened media attention and highlighted the need for government action (politics stream) [19] . during this period, an effective influenza vaccine that had been used as part of a public program in other jurisdictions was available, suggesting that mandatory vaccination was a technically viable and politically acceptable solution (policies stream). in july 2000 it was announced that ontario would be the first province in the country to offer free influenza vaccinations to all citizens for the upcoming season to relieve the aforementioned pressure on emergency departments [19] . ontario's universal influenza immunization program (uiip) is offered annually through the ministry of health and long-term care (mohltc), with the goal of reducing the number of influenza cases and the associated negative impact on the health care system. all individuals who live, work, or attend school in the province, and are 6 months of age or older, are covered by the program [20] . in recent years, uiip has increased the accessibility of vaccinations by making them available at a variety of locations such as employer-sponsored clinics and pharmacies [20] . by immunizing a critical mass of the general population, as well as hcws, it was expected that there would be a dramatic reduction to the death toll as well as the social and economic costs associated with annual influenza outbreaks. studies have shown that since introducing uiip in ontario, the number of reported cases of influenza has [22] . despite broad public acceptance and substantial participation in the voluntary immunization program, pockets of hcw resistance persisted (politics stream), and outbreaks in long-term care facilities and hospitals continued to occur, resulting in preventable illness and death [23] . as a consequence, ontario's mohltc attempted to promote voluntary vaccination of hcws by requiring health care facilities to report vaccination rates [24] . by winter 2002, uiip was in its second year and the issue of mandatory vaccination of hcws was beginning to become controversial and disputed in the courts [25] . when a health care system in hamilton instituted a mandatory vaccination policy during outbreaks, or work suspension for non-compliance, 15 staff members refused inoculation and were removed from duty without pay. this led to arbitration where the union presented its case as forced medical treatment. the arbitrator agreed with the union position citing that the policy was not supported by regulatory or statutory authority. in the decision, the arbitration board declared a violation of section 7 of the charter of rights and freedoms, which assures security of the person. mandatory influenza vaccination, the arbitrator decided, was a forced medical act and therefore, contravened the charter (st. peter's health system v. cupe local 778, 2002) [25] . around the same time, a paramedic working in ontario was making a constitutional challenge against the addition of annual influenza immunization to the ontario ambulance act (north bay hospital v. cupe local 139, 2003) [25] . his challenge also argued that mandatory immunization violated section 7 of the charter. pressure from the unions resulted in an amendment to the ambulance act in 2002, replacing mandatory vaccination with a requirement for influenza education. these cases/ political events represented a coordinated force in opposition to the mandatory vaccination initiative (politics stream). as a result, the problems, politics and policies streams were no longer aligned, closing the window of opportunity for policy change. after the sars outbreak, significant changes came to the public health system in canada, bolstering pandemic preparedness and the ability to monitor emerging disease [26] . when an arbitration board faced a union challenging mandatory influenza vaccination, this time in british columbia, the arbitrator sided with the employer noting a requirement for general immunization in the union collective agreement. in this case the employee had choices: vaccination, anti-viral medication, or to be off work without pay. the arbitrator ruled that the choices, which were not dealt with in the st. peter`s decision, meant there was no violation of the charter under section 7. an important note in the decision summary was the rationale that preventing the spread of influenza to vulnerable populations is clear and was agreed upon by both the employer and the union, which allowed for a realignment of the policies stream with problems and politics streams. may 2009 saw the first wave of the h1n1 pandemic influenza strain. by july 2009, the public health agency of canada reported it still did not have a plan in place to manage hcws who refused the seasonal influenza vaccine [27] . the seasonal vaccine did not include protection from the novel h1n1 strain and thus, worldwide production of a vaccine was implemented. stories of production delays, low vaccine supplies, and rationing caused long lines at vaccination clinics and resulted in public anxiety [28] . even after this pandemic experience, uptake of the vaccine by hcws the following season remained low [23] . the h1n1 pandemic could have resulted in the opening of a new policy window, but with the relatively mild nature of the h1n1 strain, the mandatory vaccination issue did not gain adequate or sustained public attention (politics stream), closing another window of opportunity. a long-awaited economic appraisal of ontario's uiip was published in 2010 [21] . based on a measure of qualityadjusted life years, the study concluded that uiip was cost-effective and might be considered for adoption in other provinces and countries with similar populations, health care models, and influenza rates. this evidence bolstered ontario's influenza vaccination program and may have served as a catalyst to support further expansion of the program to include mandatory hcw vaccination, however, it was not sufficient to make mandatory vaccination a politically acceptable option. in other jurisdictions, most notably the us, consideration of mandatory vaccination for hcws was gaining momentum. a large us health care employer had published their successful program requiring influenza immunization for employment [14] . in canada, the province of british columbia initiated a task force in 2011 to investigate and recommend plans for mandatory immunization programs [29] . the task force visited nine us sites with programs in place before making recommendations in 2012 to the provincial government. pressure on the ontario government to act intensified as both the evidence and prominent organizations supported mandatory hcw vaccination. for instance, the influenza surveillance protocol for ontario hospitals was revised and included the national advisory committee on immunization statement that "… refusal of health care workers who have direct patient contact to be immunized against influenza implies failure in their duty of care to their patients [30] ." the association of medical microbiology and infectious disease also presented a position paper supporting mandatory immunization acknowledging that all efforts at voluntary programs had failed [19] . public health ontario, the canadian medical association journal, and the american academy of pediatrics joined in the call for mandatory immunization [31] . in july 2012, the british columbia government announced a new policy that would require either influenza immunization or the wearing of a surgical mask for all hcws during influenza season when in direct patient contact. by october 2012 the health sciences association of british columbia union had filed a grievance citing privacy issues relating to the freedom of information and privacy act (fipa) and violations of the human rights code, the charter of rights and freedoms, and their collective union agreement [29] . facing an election the following spring, the incumbent government did not repeal the policy but announced that it would wait for the outcome of the arbitration hearing and that workers who did not comply in the 2012-13 season would not face disciplinary action [32] . the government revised and softened the policy in july before going to arbitration [29] . the decision was released in october and the arbitrator had sided with the employer. it was determined the policy aligned with fipa and there were no violations to the union agreement, the charter of rights and freedoms or the human rights code. this court ruling provided the ontario government, and health care organizations, with greater assurances that movement in the direction of mandatory vaccination for hcws could be a viable policy option subject to hcws being given some limited alternative to vaccination. despite this, the ontario government did not step forward with a consistent policy for the province. in an effort to fill this vacuum, numerous high profile ontario hospitals announced plans to implement a mandatory "vaccinate or mask" policy that would require hcws who chose not to get vaccinated to wear a mask during the influenza season [33] . even though the canadian nurses' association supported mandatory influenza vaccinations for hcws, the ontario nurses' association filed a grievance in opposition to the "vaccinate or mask" policy adopted by several ontario hospitals. following several weeks of testimony, the arbitrator found that the policy was unreasonable (policies stream) [34] . concerns identified included that wearing a mask revealed personal health information about the hcws decision not to vaccinate, masks were not effective, and the policy was designed to coerce hcws [34] . this ruling leaves ontario hospitals with no clear policy direction for the foreseeable future. by analysing the progress of mandatory hcw immunization through the lens of kingdon's framework, the separate streams of problems, politics, and policies can be seen to converge and diverge repeatedly over an extended period (policy windows have opened and closed several times). since the implementation of uiip in ontario, there have been several potential opportunities to pursue a mandatory hcw vaccination policy. in each instance a technically feasible solution was available (policies stream). however, despite the policy's importanceprotecting the public-the problem's prominence (problems stream) and the political environment (politics stream) only aligned with the policies stream for a very short period of time. during these periods there was inadequate support for the issue to remain on the government's decision agenda long enough to result in the adoption of a new policy. in part, this seems to have been due to other problems gaining prominence and displacing the issue of mandatory vaccination of hcws from a priority position. in addition, each time this issue gained prominence, opposition lobby groups reacted, making the proposed solution less politically acceptable. although the call for mandatory hcw vaccination has withstood some legal challenges-including to the charter of rights and freedoms, the freedom of information and privacy act, and the human rights code-there remains small but committed groups who continue to oppose such a policy. as a result, it seems highly unlikely that a mandatory hcw vaccination policy will be adopted by the ontario government until perception of the problem's importance is sufficient to overcome the political opposition to implementing a solution. abbreviations fipa: freedom of information and privacy act; hcws: health care workers; sars: severe acute respiratory syndrome; uiip: universal influenza immunization program; who: world health organization acknowledgments not applicable. not applicable. the datasets during and/or analysed during the current study available from the corresponding author on reasonable request. authors' contributions aj was responsible for the conception and design, data collection, analysis of data, interpretation of data and drafting of manuscript. nb was responsible for revisions to design, analysis of data, interpretation of data and revisions to influenza vaccination for healthcare workers who care for people aged 60 or older living in long-term care 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lawyers health agency won't force flu shots on health-care workers the calm face at the centre of canada's immunization campaign in re the matter of an arbitration under the b.c. labour relations code on: public health agency of canada flu shot should be mandatory, journal says health-care workers cheer softened stand on flu shots re: healthcare worker influenza vaccination policy -vaccine required date and vaccine required period union says ontario nurses can't be forced to wear masks in flu season manuscript. gr was responsible for revisions to design, analysis of data, interpretation of data and revisions to manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable. submit your next manuscript to biomed central and we will help you at every step: key: cord-313693-qmkrn7pr authors: wong, bonnie c. k.; lee, nelson; li, yuguo; chan, paul k. s.; qiu, hong; luo, zhiwen; lai, raymond w. m.; ngai, karry l. k.; hui, david s. c.; choi, k. w.; yu, ignatius t. s. title: possible role of aerosol transmission in a hospital outbreak of influenza date: 2010-11-15 journal: clin infect dis doi: 10.1086/656743 sha: doc_id: 313693 cord_uid: qmkrn7pr background. we examined the role of aerosol transmission of influenza in an acute ward setting. methods. we investigated a seasonal influenza a outbreak that occurred in our general medical ward (with open bay ward layout) in 2008. clinical and epidemiological information was collected in real time during the outbreak. spatiotemporal analysis was performed to estimate the infection risk among patients. airflow measurements were conducted, and concentrations of hypothetical virus-laden aerosols at different ward locations were estimated using computational fluid dynamics modeling. results. nine inpatients were infected with an identical strain of influenza a/h3n2 virus. with reference to the index patient's location, the attack rate was 20.0% and 22.2% in the “same” and “adjacent” bays, respectively, but 0% in the “distant” bay (p=.04). temporally, the risk of being infected was highest on the day when noninvasive ventilation was used in the index patient; multivariate logistic regression revealed an odds ratio of 14.9 (95% confidence interval, 1.7–131.3; p=.015). a simultaneous, directional indoor airflow blown from the “same” bay toward the “adjacent” bay was found; it was inadvertently created by an unopposed air jet from a separate air purifier placed next to the index patient's bed. computational fluid dynamics modeling revealed that the dispersal pattern of aerosols originated from the index patient coincided with the bed locations of affected patients. conclusions. our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. source and engineering controls, such as avoiding aerosol generation and improving ventilation design, may warrant consideration to prevent nosocomial outbreaks. nosocomial transmission of influenza is frequently reported [1] [2] [3] . it typically occurs during seasonal peaks, and may involve almost all types of healthcare facilities [1, 2] . its consequences are considerable: it may result in significant disease and even fatality among hospitalized patients, because these patients often are older and/or have multiple comorbidities [1] [2] [3] . health care professionals are frequently involved, and the affected hospital units may require temporary closure with service suspension [2] [3] [4] [5] [6] . lack of preexisting immunity toward the recently emerged pandemic influenza h1n1 virus and its potential to cause serious disease in both young and older adults have further raised the importance of hospital infection control [7] [8] [9] . however, how influenza is transmitted in the health care setting and what control measures are effective have remained largely unclear [2, [8] [9] [10] [11] [12] . under natural conditions, influenza virus is transmitted predominantly via droplets and direct contact [13] . thus, adequate spacing, hand washing, and droplet precautions, including the use of face masks, are likely effective in preventing transmission [8, 9, 11] . however, in indoor health care settings, because of the special clinical and environmental conditions, aerosol transmission of diseases might become possible, as described for other viral and bacterial infections [1, 3, [13] [14] [15] [16] [17] . emerging evidence suggests that influenza infection can also be transmitted via the aerosol route, as shown in animal models and in experimental studies involving human subjects [3, 13, 18, 19] . in this study, we report an influenza outbreak that occurred in an acute medical ward. epidemiological, airflow and computational fluid dynamics analyses were performed. the possible role of aerosol transmission of influenza was examined. the implications of the results on hospital infection control strategies will be discussed. at prince of wales hospital (pwh; hong kong), an outbreak of influenza a occurred in an adult general medical ward in april 2008. in hong kong, peak influenza activities occur in both spring (january-april) and summer (july-september) [20] . pwh is a 1350-bed acute care general hospital operated by the hospital authority of hong kong that serves an urban population of 1.5 million. each year, 1200 adult cases of confirmed influenza are being treated at pwh [5, 21, 22] . a major nosocomial outbreak of severe acute respiratory syndrome (sars) occurred at pwh in 2003 [15, 23] ; since then, all patients hospitalized with acute febrile respiratory illnesses are put on droplet precautions; if influenza is confirmed, the patients will be isolated or cohorted in designated wards [21, 22] . as a hospital policy, all health care personnel working in medical wards are required to wear surgical face masks, and an on-duty nursing officer is responsible for monitoring compliance [5] . outbreak investigation. nosocomial outbreaks of infectious diseases, once identified, would be investigated by the outbreak management team (which consisted of physicians, microbiologists, infection control practitioners, nurses, and hospital epidemiologists) as part of the management protocol [23] . affected cases would be reviewed daily, and clinicoepidemiological information collected real time as the outbreak evolved. these data were studied and discussed in daily meetings throughout the whole outbreak period. control measures, including ward closure, patient isolation, and use of antiviral prophylaxis, would then be recommended. virological investigations for influenza have been described elsewhere [21, 22] . in brief, nasopharyngeal aspirate specimens were collected from symptomatic individuals and subjected to immunofluorescence assay and virus culture for diagnosis [21, 22] . in addition, for all confirmed cases, viral rna was directly extracted for sequencing of the whole length of the hemaggultinin gene, as described elsewhere [24] ; the nucleotide sequences in individual cases were then compared. finally, a hemagglutination inhibition assay was performed (using the virus isolate obtained from the index patient as antigen source) to detect antibody rise in the paired serum samples collected from affected individuals [25] . spatiotemporal analysis of epidemiological data. the epidemic curve for the outbreak was produced. the floor plan of the affected wards was studied; in pwh, the design of general medical wards followed an open bay ward layout [15] . for the analysis, all patients who had ever stayed with the index patient during the period of his presence in the ward (from admission to transfer or isolation) and all health care workers (hcws) who had worked in the same ward during the period were included. attack rates of influenza among inpatients were calculated each day on the basis of their locating "bay" [15] . univariate relationships between risk of acquiring influenza infection and spatiotemporal variables were analyzed using the x 2 test or the fisher exact test. variables with a p value !.1 were entered into a multivariate (stepwise) logistic regression model to identify independent factors associated with infection. odds ratio (ors) and 95% confidence intervals (cis) were reported for explanatory variables. in all analyses, a p value of !.05 was considered to indicate statistical significance. all probabilities were 2-tailed. statistical analysis was performed using pasw statistics software, version 17.0 (spss). airflow measurements and fluid dynamics analyses. information on the ward's ventilation systems was collected during the outbreak investigation. these included the location and size of air supply diffusers, return grills, and the highefficiency particulate absorbing (hepa) air purifier units that were present in each ward bay. airflow rate through each supply diffuser, return grill, air purifier, and exhaust fan was measured in detail, as described elsewhere [15] . dispersion of the hypothetical virus-laden aerosols, originated from the index patient's bed through the entire ward, was analyzed by computational fluid dynamics (cfd) method. the hypothetical virus-laden aerosols were modeled as gaseous and passive tracers, which have been shown to be able to model well the dispersion of droplet nuclei that were less than 5-10 microns in diameter. the commercial cfd software, fluent, version 6.2, was used [15, 26] . the predicted concentration fields were compared with the locations of affected patients found in the outbreak. the 'outbreak management team' was being alerted of a possible outbreak in a general medical ward on 4 april 2008, when 7 inpatients were found to have developed fever and respiratory symptoms. the ward was immediately closed to new admissions, and transfer to other hospitals or institutes was suspended; sick patients were isolated. for all hcws and the remaining patients, strict droplet precautions were implemented, and the individuals were required to wear surgical masks at all times. hand hygiene was reinforced. postexposure prophylaxis with oseltamivir was offered, and both groups were monitored for development of symptoms. discharged patients were put under continuous home medical surveillance for 1 week. the ward returned to normal function on 12 april, when the outbreak was declared over. an epidemiological investigation was started on 4 april. at the end of the outbreak, a total of 9 inpatients (patients a-i) were found to have symptoms that fulfilled the case definition of influenza-like illness (figures 1 and 2) . no visitor was known to be affected. the symptom onset dates of the first (index) figure 1 . epidemic curve of the influenza outbreak. patients were shown according to their symptom onset date (fever or new respiratory symptoms); the order does not necessarily reflect the order in which they acquired infection. the arrow indicates the time when the index patient (patient a) commenced bi-level positive airway pressure (bipap) ventilation support. prior to that, he was receiving supplemental oxygen therapy via nasal cannula. the bipap ventilation lasted for 116 h; he was subsequently transferred to the intensive care unit. patient i started to receive oseltamivir prophylaxis on 4 april (the ward was closed and sick patients were isolated); however, he soon became unwell and developed fever on 10 april, despite receipt of prophylaxis. staff 1 and 2 had symptoms; however, the results of serological tests for recent influenza infection were negative (table 1 ). case and the last case were 27 march and 10 april, respectively. all 9 inpatients tested positive for influenza a by immunofluorescence assay and culture of nasopharyngeal aspirate specimens. all isolates belonged to the h3n2 virus subtype (influenza a/brisbane/10/2007) and were 100% identical, as determined by nucleotide sequence analysis (table 1) . nasopharyngeal aspiration was not performed for the 2 hcws who had reported symptoms; however, serological test results did not suggest recent influenza infection ( table 1) . the index patient (patient a) ( figure 1 ) was a 68-year-old man who had underlying chronic obstructive pulmonary disease. he was admitted on 27 march with unresolved pulmonary shadows, despite having completed a course of antibiotic treatment, and increased dyspnea, which initially required suppleall secondary cases were detected to have fever and/or new onset of respiratory symptoms. once influenza was confirmed, the patients started to receive oseltamivir treatment (75 mg twice per day for 5 days) within 24 h after symptom onset. the clinical courses of 7 patients remained uncomplicated, and the patients were subsequently discharged. the remaining patient (patient c) (figure 1 ) was a 73-year-old man who had originally been admitted for pseudomonas pneumonia that had complicated advanced bronchiectasis; although afebrile, he developed lower respiratory tract complication after influenza infection and died of progressive respiratory failure. spatiotemporal analysis. the outbreak ward was a male general medical ward consisting of 30 beds arranged in 3 major bays (2 adjacent rear bays b and c separated by a ∼2-m wide corridor and 1 front bay a) (figure 2 ). the distance between adjacent beds was ∼1 m. the index patient's bed was located at the wall end of bay c, right next to a hepa air purifier. in total, 59 patients and 29 hcws had stayed or worked on the ward during the period from 27 march to 1 april. the overall attack rate among patients was 13.6% (8 of 59 subjects). we found that patients who had stayed in the "adjacent" bay b (attack rate, 22.2%) were affected to a degree similar to those who stayed in the "same" bay c with the index patient (attack rate, 20.0%); however, no patients in the front, "distant" bay a or side room were affected (attack rate, 0%; p p .041, by the fisher exact test). when analyzed according to date, presence in rear bays was associated with attack rates of 15.8%, 15.8%, 26.3%, and 26.3% on the dates 27-30 march, respectively. the risk of being infected was highest on 31 march (30.4%; p p .005) and 1 april (30.0%; p p .016), which coincided with the time of bipap ventilation use in the index patient. in a final multivariate logistic regression model, staying in the rear bays on 31 march was independently associated with a higher risk of acquiring influenza (or, 14.9; 95% ci, 1.7-131.3; p p .015). the attack rate among hcws was not analyzed in detail, because only 2 were reported to have had symptoms, and neither had laboratory-confirmed infection. they reported close contact with patients staying at all 3 bays while performing their routine duties. airflow measurements and analysis. air conditioning in the outbreak ward was provided by fan coil unit systems with a 4-way diffuser at the ceiling level in each of the 3 bays. the return air grills were located at the ceiling of the corridor. a hepa air purifier was placed at the wall end of each bay ( figures 2 and 3) ; it functioned by drawing in air from a lower level, and after filtration, injecting the air back into the ward at an upper level. during the outbreak, the fan setting of the hepa air purifiers was found to set to low in bays a and b and to medium in bay c inadvertently, with injection velocity measuring 1.47 m/s, 1.44 m/s, and 1.90 m/s respectively. airflow measurements revealed that, under this situation, there was an imbalance in the air supply to and return from different bays, and the net flow toward the corridor was 70-100 l/s ( figure 3 ). the injecting air velocities from the hepa air purifiers were substantially higher than that of the diffusers and thus dominated the overall airflow pattern. because of the higher air injection velocity from the air purifier in bay c compared with that in the adjacent bay b, air from bay c was expected to be "pushed" into the corridor and toward bay b (figure 4 ). cfd simulations were performed, and the distributions of hypothetical virus-laden aerosols originated from the index patient are shown in figure 5 . the normalized concentration of the hypothetical virus-laden aerosols was found to be the highest in bay c ("same"), followed by bay b ("adjacent"), and the lowest in bay a ("distant"). the estimated spatial distribution figure 5 . the spatial distribution of normalized concentration of hypothetical virus-laden aerosols (modeled as gaseous tracer) in the outbreak ward at a height of 1.1 m. the flow rates used in this model were those described in figure 3 . all high-efficiency particulate absorbing (hepa) filters were assumed to function with 100% filtration of the modeled droplet nuclei. the 3 hepa air purifiers are shown as black boxes, the 4 diffusers are shown by a square with an x, and the 4 returns are shown as a small rectangular filled box. affected patients are represented by white ovals (the index patient is marked as a red oval). was found to correspond to the locations of affected patients in the outbreak. large droplets would not have accounted for such distribution because of their fast deposition onto surfaces [27, 28] . we report a nosocomial outbreak of seasonal influenza in an acute ward setting. it was temporally related to the use of an aerosol-generating device in the index patient. this had occurred together with an imbalanced indoor airflow; and the spatial distribution of cases was found to follow the directional airflow and coincided with a cfd-estimated aerosol dispersal pattern. our findings suggest a possible role of aerosol transmission in this outbreak. we have previously reported aerosol transmission of sarsassociated coronavirus in a similar ward setting [15] . in that study, cfd modeling demonstrated a close relationship between concentration of virus-laden aerosols and the risk of acquiring infection in various ward locations. we studied this outbreak using similar approach, and our findings provide further evidence to support the hypothesis that, under suitable clinical and environmental conditions, aerosol transmission of influenza virus can occur [2, 3] . in this outbreak, we postulate that infectious aerosols were generated continuously through the use of noninvasive ventilation for the index patient (which projected for at least 1 m sagittally to bed end) [29] [30] [31] for 16 h and these aerosols were blown toward the adjacent bay by an imbalanced indoor airflow created by an air purifier's outflow jet, which was located at patient's bed end. we believe that droplets and contact routes of transmission cannot entirely explain the outbreak because (1) the epidemic curve suggested a point-source mode of infection, instead of successive propagation (especially for the aggregation of symptom onset on 2 and 3 april); (2) the index patient and most other inpatients were immobile during their illnesses and, therefore, direct contacts should have been minimal; (3) patients 12 m away were affected; and (4) locations of affected patients coincided with dispersal pattern of aerosols and the directional airflow. in this outbreak, patients in the "adjacent" and "same" bay were similarly affected, but the "distant" bay was spared. a more random distribution of infected cases would be expected if hcws had acted as vectors, because they were responsible for the care of patients in all bays (duties were assigned on the basis of function and not location) [15] . similarly, close patient-patient contact, visits to a contaminated common area, and transmission through a wandering, sick visitor are unlikely explanations in our setting [15] . finally, high air velocities (110 m/s), which can carry even large droplets beyond 2 m, were not found in our ward setting, and they would have been deposited too fast onto surfaces to account for the observed dispersal pattern [27, 28] . we hold the view that the predominant modes of influenza transmission are via droplets and direct contact [2, 3, 8, 13] . however, accumulating evidences suggest that the aerosol route may have a contributing role [3, 13, 18, 19] . in animal models, influenza virus (eg, h3n2 or h1n1) has been shown to transmit efficiently through air, whereas fomite or contact spread is relatively inefficient [13, 18, 19, [32] [33] [34] . in clinical studies, virus-laden particles less than 5-6 mm (ie, within the respirable aerosol fraction) have been detected in exhaled breaths of patients with influenza and in the air sampled from an acute healthcare setting during seasonal peak [19, 35, 36] . in contrast to natural coughing or sneezing, artificially generated respiratory particles are often much smaller in size ( less than 5-6 mm), can penetrate more readily into the lower respiratory tract, and can cause infection with a smaller dose [3, 13, 18, 19] . it has been shown that certain clinical procedures (eg, endotracheal intubation, cardiopulmonary resuscitation, noninvasive ventilation, and receipt of high-flow oxygen) can generate a large amount of respiratory aerosols [8, 9, 13, 16, 18, [29] [30] [31] , and transmission of respiratory infection related to some of these procedures-despite implementation of droplet precautions-has been reported [37] [38] [39] . since the outbreak, we have used a hierarchy of control measures to prevent influenza transmission in our hospital, such as administrative and source controls, engineering controls, and use of personal protective equipment [8, 9] . the policy of isolating or cohorting patients with suspected or confirmed influenza is reinforced; application of an aerosol-generating procedure is allowed only in adequately ventilated single rooms before influenza can be excluded; all patients with respiratory infections are required to wear face masks, which are freely provided, until symptoms subside; air-conditioning units and their settings are regularly checked to avoid airflow imbalance; and hcws are advised to use n-95 respirators, face shields, gloves, and gowns while performing aerosol-generating procedures and to receive annual influenza vaccines [2, 5, 8, 9] . we have adopted these measures on all medical wards, because there might be similar unsuspected or "invisible" patients with influenza acting as infection sources. we did not encounter another influenza outbreak in open wards during the subsequent 24 month period, which included the first wave of the influenza h1n1 pandemic [7, 20] . although each institute's infrastructure may be different, our findings suggest that the strategies of source and engineering controls might be important considerations to prevent nosocomial influenza transmission. our study is limited by its descriptive nature. we could not analyze the impact of influenza vaccination on the size of outbreak, because such information was unavailable from many patients; however, the general vaccine uptake rate in our community was reported to be very low [40, 41] . also, we could not entirely eliminate the role of sick hcws in transmitting infection, albeit no case of influenza was eventually confirmed among them. however, because all hcws were required to wear surgical face mask during work and to report any influ-enza-like illnesses through a daily reporting system (the presence of which would immediately exempt them from duty), the chance of hcws cross-infecting patients should be rather small [5] . furthermore, the spatial distribution of cases could not be easily explained by hcw-to-patient or patient-to-patient transmission, as discussed above [15] . our findings indicate the need to evaluate the infection risks of aerosol-generating procedures or devices, especially when applied to the disease state [8, 9, 29, 30, 37] ; the effectiveness of various source and administrative control strategies [2, 3, 8, 9, 39] ; the ventilation systems in different healthcare settings; and the impact of airflow and humidity on nosocomial influenza transmission with an architectural aerodynamics approach [13, 17, 42, 43] . in conclusion, our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. source and engineering controls, such as avoiding aerosol generation and improving ward ventilation design, may warrant consideration to prevent nosocomial outbreaks. hospital-acquired influenza: a synthesis using the outbreak reports and intervention studies of nosocomial infection (orion) statement influenza in the acute hospital setting transmission of influenza: implications for control in health care settings disruption of services in an internal medicine unit due to a nosocomial influenza outbreak preventing healthcare workers from acquiring influenza a nosocomial outbreak of influenza during a period without influenza epidemic activity world health organization. pandemic (h1n1) infection prevention and 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virus transmission is dependent on relative humidity and temperature factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises we thank mr. li liu and ms caroline xiaolei gao for their participation in the field measurement in the outbreak ward.potential conflicts of interest. all authors: no conflicts. key: cord-327819-7p05jk1h authors: trampuz, andrej; prabhu, rajesh m.; smith, thomas f.; baddour, larry m. title: avian influenza: a new pandemic threat? date: 2004-04-30 journal: mayo clinic proceedings doi: 10.4065/79.4.523 sha: doc_id: 327819 cord_uid: 7p05jk1h in december 2003, the largest outbreak of highly pathogenic avian influenza h5n1 occurred among poultry in 8 asian countries. a limited number of human h5n1 infections have been reported from vietnam and thailand, with a mortality rate approaching 70%. deaths have occurred in otherwise healthy young individuals, which is reminiscent of the 1918 spanish influenza pandemic. the main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. notably, sore throat, conjunctivitis, and coryza were absent. the h5n1 strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors, which can be used for treatment and prophylaxis. the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. the threat of a future pandemic from avian influenza is real. adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. this article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. in december 2003, the largest outbreak of highly pathogenic avian influenza h5n1 occurred among poultry in 8 asian countries. a limited number of human h5n1 infections have been reported from vietnam and thailand, with a mortality rate approaching 70%. deaths have occurred in otherwise healthy young individuals, which is reminiscent of the 1918 spanish influenza pandemic. the main presenting features were fever, pneumonitis, lymphopenia, and diarrhea. notably, sore throat, conjunctivitis, and coryza were absent. the h5n1 strains are resistant to amantadine and rimantadine but are susceptible to neu-raminidase inhibitors, which can be used for treatment and prophylaxis. the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. the threat of a future pandemic from avian influenza is real. adequate surveillance, development of vaccines, outbreak preparedness, and pandemic influenza planning are important. this article summarizes the current knowledge on avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. s ince mid-december 2003, 8 asian countries-cambodia, china, indonesia, japan, laos, south korea, thailand, and vietnam-have reported outbreaks of highly pathogenic avian influenza caused by the h5n1 strain among poultry. this is the largest outbreak of avian influenza in poultry ever described. most of these countries experienced outbreaks of avian influenza for the first time. during the past 3 months, more than 100 million domestic poultry have died, or they have been culled to contain the epidemic and prevent the potential transmission to humans. despite the widespread infection of avian influenza viruses among poultry in asia, reported infection in humans has been rare to date. as of , only vietnam and thailand have reported influenza h5n1 infection in humans. thirty-two human cases were laboratory confirmed, resulting in 22 deaths, a mortality rate approaching 70%. 1 however, most of the affected countries have limited capacity for surveillance of human disease. in addition, poultry production contributes substantially to the economies of the affected countries, resulting in considerable political pressure to minimize the extent and seriousness of the epidemic. therefore, the reported human cases of avian influenza may be underestimated. historically, this avian epidemic zoonosis is unprecedented in its scale, geographical distribution, economic losses, challenge for control, and potential public health consequences. 2 the potential development of a human influenza pandemic similar to that of 1918 due to spanish influenza is a major concern. two of the 3 key criteria that characterized the pandemic of 1918-1919 have already been fulfilled in the current epidemic: (1) the ability of the virus to infect humans resulting in high mortality and (2) a global immunologically naive human population. 3 the third criterion, efficient human-to-human transmission, has thus far not been observed. 4 the adaptation that would result in human-to-human transmission might involve changes in the receptor properties or improved viral replication efficiency. this adaptation might be achieved by mutation of an avian virus genome or by mixing segments of an avian virus with segments from a virus already adapted to humans (genetic reassortment), leading to the emergence of a new influenza subtype with pandemic potential. 5 both events may occur easily. first, influenza viruses mutate frequently, potentially allowing them to change the host receptor specificity from avian to human. 6 second, the segmented viral genome allows the exchange of genes between viruses if they infect the same host cell, which serves as the "mixing vessel." 7 in the current h5n1 outbreak in asia, no genetic reassortment among avian and human influenza viruses has been found; all genes are of avian origin. 4, 8 however, the widespread epidemic of avian influenza in domestic birds increases the likelihood for mutational events and genetic reassortment. in view of the high mortality observed in the limited number of infections in humans, the prospect of a severe pandemic is of considerable importance. 9 we review current knowledge about avian influenza, including the virology, epidemiology, diagnosis, and management of this emerging disease. for personal use. mass reproduce only with permission from mayo clinic proceedings. influenza viruses type a, b, and c belong to the family of orthomyxoviridae. these enveloped viruses contain a segmented single-stranded rna genome. a key difference among influenza virus types is their host range. 10 type a viruses have been isolated from a wide range of species, including humans, pigs, horses, seals, ferrets, mink, whales, and birds. types b and c viruses infect humans predominantly but have also been isolated from seals and pigs, respectively. 7 influenza a viruses can be divided into subtypes based on surface glycoproteins, hemagglutinin and neuraminidase. to date, 15 different hemagglutinin (h1-h15) and 9 neuraminidase (n1-n9) proteins have been identified. 10 hemagglutinin facilitates entry of the virus into host cells through its attachment to sialic acid on epithelial cell receptors, promotes membrane fusion, and elicits protective neutralizing antibody response. hemagglutinin is the crucial component of influenza vaccines. neuraminidase has enzyme activity, which cleaves sialic acid on virion proteins, facilitating the release of progeny virions from infected cells. it is an important target for antiviral agents. 11 although all influenza a virus subtypes have been found in birds, only 3 different hemagglutinin (h1, h2, or h3) and 2 different neuraminidase (n1 or n2) proteins have circulated widely in humans. the antigenic characteristics of influenza virus change gradually by accumulation of point mutations (antigenic drift) or profoundly by genetic reassortment (antigenic shift) in the genes encoding primarily hemagglutinin and neuraminidase. during antigenic drift, mutations occur because of the lack of the proofreading activity of viral rna polymerases. as a consequence, new antigenic variants emerge constantly, allowing the virus to evade immune recognition and giving rise to annual epidemics. the antigenic drift of influenza viruses requires the replacement of influenza strains used in the vaccine every several years. 12 during antigenic shift, new strains appear to which most humans have no immunity and that have the potential to cause severe global outbreaks of human influenza. these pandemic strains derive from nonhuman viruses by interspecies transmission of the whole virus or by genetic reassortment between avian and human viruses that have infected a single cell. pigs have receptors for both avian and human influenza viruses and have been considered an intermediate host for the reassortment of influenza viruses. reemergence of a previously circulating virus to which most of the population is immunologically naive is another mechanism by which pandemic strains can occur. 13 the nomenclature of influenza viruses includes the type of virus (a, b, or c), host of origin (excluding humans), geographical site of origin, strain number, and year of isolation, followed in parentheses by the antigenic description of the hemagglutinin and neuraminidase glycoproteins, eg, a/chicken/hong kong/258/97 (h5n1). avian influenza viruses do not replicate efficiently in humans, which suggested initially that direct avian-to-human transmission would not occur. the hemagglutinins of avian strains bind preferentially to host receptors terminating in an α(2,3)-linked sialic acid, whereas human strains bind preferentially to receptors terminating in an α(2,6)linkage. the predominance of these receptors in different tissues partly reflects the tropism of influenza in different species. 5 receptor specificity and inability of efficient replication in vivo were believed to provide a barrier against human infection by avian influenza virus. high doses of avian influenza virus strains were required for replication in volunteers. the first human infections by avian h5n1 virus in hong kong in 1997 showed that receptor specificity was not a definitive host restriction factor. 14 in this epidemic, an intermediate host was not necessary for transmission of avian strains to humans. all birds are thought to be susceptible to infection with avian influenza viruses. wild waterfowl and migrating bird populations provide an extensive natural reservoir for influenza a viruses ( figure 1 ). 15 the virus multiplies in the intestines of these birds, particularly in wild ducks, and they can carry the virus without developing signs of infection, indicating an optimal level of viral adaptation in these hosts. therefore, influenza is not an eradicable disease, and prevention and control of outbreaks are the only realistic goals. 16 unlike mammalian influenza virus strains, avian viruses have shown little antigenic variability during the past decades. the high level of genetic conservation suggests that avian viruses have reached an evolutionary stage in which antigenic changes provide no selective advantage. infected birds excrete large amounts of virus in respiratory secretions, saliva, and feces. in the northern hemisphere, the prevalence of avian influenza peaks between late summer and early winter when the birds leave their breeding grounds and migrate; up to 30% of these birds may excrete virus. 15 water sources that are contaminated by wild bird droppings and used by domestic poultry are a common and efficient means of viral spread. migratory birds are capable of flying long distances, and those migrating longitudinally appear to play a key role in viral transmission. 7 figure 1 . wild aquatic birds are the main reservoir for influenza a viruses, from which viruses can be transmitted to other hosts such as horses, pigs, poultry, whales, seals, and humans. as indicated by arrows, humans can also be infected by pigs and poultry. the fowl plague was first described in 1878 as a disease affecting chickens in italy. the causative agent was isolated from a chicken in 1902, which was the first identified influenza virus (the human influenza virus was not identified until 1933). domestic poultry flocks are highly vulnerable to avian influenza, particularly birds that are raised outdoors. once introduced into domestic flocks, the virus can spread among farms via contaminated inanimate objects, contributing to the rapid evolution of epidemics. avian influenza in birds is characterized by a wide spectrum of symptoms, ranging from a mild illness to a highly contagious, severe, and rapidly fatal disease with a mortality rate approaching 100%. the latter syndrome was formerly known as fowl plague; it is now termed highly pathogenic avian influenza and is characterized by coughing, sneezing, excessive lacrimation, cyanosis of the unfeathered skin, edema of the head, ruffled feathers, diarrhea, nervous system disorders, or sudden death without clinical signs. 15 in the past, most outbreaks of highly pathogenic avian influenza in poultry have been due to subtypes h5 or h7. epidemics of avian influenza among poultry have produced serious economic consequences. 7 in addition to a highly pathogenic avian influenza, viruses of low pathogenicity resulting in mild respiratory symptoms and reduced egg production can cause substantial economic losses. in 1995, 178 turkey farms in minnesota were affected by influenza virus h9n2, resulting in an economic loss of approximately $6 million. 15 moreover, avian influenza viruses of low pathogenicity can mutate into highly pathogenic viruses after circulating for several months. during the 1999-2001 epidemic in italy, the h7n1 virus mutated within 9 months to a highly pathogenic form, and more than 13 million birds died or were culled. 1 since 1997, outbreaks of highly pathogenic avian influenza have increased in frequency and severity, and the number of novel strains suggests that the next influenza pandemic is imminent. 9 in 2004, outbreaks of highly pathogenic avian influenza occurred among chickens at farms in delaware, pennsylvania, texas, and maryland. the diseased and exposed birds were killed, and a quarantine zone was implemented around the affected farms. on february 20, 2004, h5n1 infection was confirmed in 2 dead domestic cats and in a tiger in a zoo in thailand. to date, these animals have not been considered susceptible to influenza viruses. this raises concern about the need for surveillance in animals other than birds. 1 since the late 1970s, live poultry markets have been considered the source of human influenza viruses. these permanent live animal markets (wet markets) are embedded strongly in the food culture of asian countries. close human contact with live animals provides an ideal environment for the zoonotic transfer and evolution of infectious disease agents. 17 the most important control measure for containing the outbreak in hong kong in 1997 was rapid destruction of the entire poultry population, proper disposal of carcasses, and rigorous disinfection of farms. to reduce the risk of reemergence of avian influenza, all aquatic birds were removed from retail markets, including ducks, geese, and quails (source of the h5n1 strain). monthly "clean days" were introduced when all markets are emptied and cleaned simultaneously. new regulations mandate quarantine of poultry in designated farms in mainland china for 5 days and testing for h5 infection before importation to hong kong. 18 other control measures include continuous surveillance of influenza virus strains in humans and in birds, careful protection of cullers through appropriate personal protective equipment, restrictions on the movement of live poultry, and use of the human influenza vaccine to reduce the risk of coinfection in poultry workers and cullers. 4 a global outbreak of influenza usually occurs when a new influenza virus emerges, spreads, and causes disease worldwide. 13 descriptions of widespread and serious epidemics of respiratory disease suggestive of influenza have for personal use. mass reproduce only with permission from mayo clinic proceedings. *many people died within the first few days after infection, nearly half of whom were healthy young adults. †virus is still circulating. ‡illness occurred almost exclusively among persons younger than 20 years. this pandemic did not increase mortality. no. of deaths been recorded for centuries. hippocrates in ancient greece described the first case of influenza-like illness. in the 20th century, 4 human influenza pandemics emerged with intervals of 9 to 39 years (table 1 ). 14 the spanish influenza of 1918-1919 (h1n1) was the most devastating pandemic in recorded human history, resulting in 25 to 50 million deaths worldwide; nearly half of those who died were healthy young adults. this figure is at least double the number of soldiers killed on the battlefields of europe during world war i. other less catastrophic pandemics occurred in 1957 (asian influenza), 1968 (hong kong influenza), and 1977 (russian influenza). the asian (h2n2) and hong kong (h3n2) pandemic strains were generated by reassortment between human and avian viral genes. the russian influenza virus (h1n1) circulated in the 1950s and reemerged in 1977. the illness occurred almost exclusively among persons younger than 20 years. the relatively low mortality rate associated with this pandemic can be attributed to the immunity of older individuals who had antibodies from their previous exposure to nearly identical viruses. 7 to date, human infection with avian influenza viruses has been confirmed on several occasions ( table 2 ). in 1997, the first documented direct transmission of an avian influenza virus to humans occurred in hong kong, when an h5n1 strain caused a severe respiratory disease in 18 previously healthy young adults, 6 of whom died. 3 the outbreak in hong kong was controlled by the destruction of the entire poultry population; within 3 days, more than 1.5 million chickens were killed. in 1999, avian influenza h9n2 viruses were isolated for the first time in humans; 2 children were hospitalized with uncomplicated upper respiratory tract infection in hong kong, and both recovered completely. 19 in may 2001 and in february and april 2002, the poultry stocks in hong kong were again destroyed when the highly pathogenic h5n1 virus reemerged in flocks. however, no more human cases of h5n1 influenza were identified until february 2003, when 2 cases were confirmed in 2 hong kong residents after travel to china, 1 of whom died. 19 in february 2003, a large outbreak of h7n7 avian influenza occurred in poultry farms in the netherlands. 20 the virus caused mild illness in several farm workers, but a veterinarian who had visited an affected farm died of pneumonia. the virus isolated from the fatal case displayed 14 amino acid substitutions, which possibly contributed to the increased disease severity. all farm workers received mandatory influenza vaccination and prophylaxis with oseltamivir. 21 the h7n7 poultry outbreak subsequently spread to germany and belgium but was eventually controlled by destruction of more than 30 million domestic poultry. in february 2004, another outbreak of h7n7 avian influenza occurred on a poultry farm in british columbia, canada, and resulted in at least 5 humans with infection, mainly conjunctivitis. 1 recently, the crystal structure of the hemagglutinin from the 1918 virus was determined, explaining why this virus was able to spread efficiently in the human population despite retainment of the avian receptor-binding site. 6 epidemiological studies suggest that most human h5n1 infections resulted from contact with infected birds or surfaces contaminated with their excretions. however, evidence of limited human-to-human transmission of avian influenza viruses has occurred ( table 2) . a case-control study of 15 patients hospitalized for influenza virus h5n1 disease in 1997 was conducted in hong kong. 22 exposure to live poultry in the week before the onset of illness was significantly associated with h5n1 disease, whereas traveling, eating, or preparing poultry products and recent exposure to persons with respiratory illness showed no significant association. in a cohort study including 3 hospitals in hong kong at which patients infected with h5n1 strains had been admitted, a significantly higher seropositivity rate for personal use. mass reproduce only with permission from mayo clinic proceedings. for h5n1 was found among exposed health care workers (3.7%) than among nonexposed ones (0.7%), providing epidemiological evidence of transmission from infected patients to health care workers. 23 in the netherlands in 2003, 3 family members of farm workers were infected during the h7n7 outbreak, demonstrating the ability of human-to-human transmission. 20 the present epidemic of highly pathogenic avian influenza virus in asia is historically unprecedented and extremely challenging. 4 between december 5 and 11 in 2003, sudden death occurred in 19,000 chickens at a farm near seoul, south korea; this was later confirmed as being caused by the highly pathogenic avian influenza virus h5n1. on january 8, 2004 , authorities in vietnam also reported several outbreaks of avian influenza h5n1 at farms in the southern provinces. human cases of h5n1 avian influenza presenting as severe respiratory illness occurred, with a mortality rate of more than 70%. on january 11, 2004, the world health organization alerted the global outbreak alert and response network to support health authorities in the epidemiological investigation and containment of human cases. these h5n1 viruses are substantially different from the h5n1 viruses in outbreaks in hong kong in 1997 and 2003, indicating that the virus has mutated. on january 23, 2004, authorities in thailand reported an outbreak of highly pathogenic avian influenza among poultry, with laboratory-confirmed cases of h5n1 infection in humans. 24 japan, the european union, the united states, and other major export markets banned import of poultry products from the affected asian countries. preliminary clinical data from 10 confirmed human cases of h5n1 avian influenza from the current epidemic in vietnam were summarized recently. 25 the mean age of the patients was 13.7 years (range, 5-24 years); none had any clinically important preexisting medical conditions. of the 10 patients, 9 had a history of direct contact with poultry (chicken or ducks), with a median time before onset of illness of 3 days (range, 2-4 days). the main clinical features were fever, shortness of breath, cough, and diarrhea. notably, sore throat, coryza, and conjunctivitis were absent. all patients presented with pronounced lymphopenia (mean lymphocyte count, 0.7 × 10 9 /l) and important chest radiographic abnormalities including diffuse, multifocal, or patchy infiltrates or segmental or lobular consolidation. eight of the 10 patients (80%) died after a mean of 10 days of illness. the clinical features, together with liver dysfunction, renal failure, coagulopathy, and pancytopenia seen in the 1997 hong kong h5n1 outbreak, are similar to those seen in patients with severe acute respiratory syndrome. similarly, human infection with avian influenza h5n1 reported in 1997 in hong kong presented as an influenzalike illness with pneumonia. 26 reactive hemophagocytic syndrome was the most characteristic pathologic finding and probably contributed to the lymphopenia, liver dysfunction, and abnormal clotting profiles that were observed among these patients. 27 gastrointestinal manifestations, renal failure unrelated to rhabdomyolysis, and pancytopenia were unusually prominent. factors associated with severe disease included older age, delay in hospitalization, lower respiratory tract involvement, and leukopenia or lymphopenia at admission. the preliminary findings suggest that cytokine dysfunction contributes to the severity of for personal use. mass reproduce only with permission from mayo clinic proceedings. testing is indicated for hospitalized patients with radiographically confirmed pneumonia, acute respiratory distress syndrome, or other severe respiratory illness for which an alternate diagnosis has not been established and history of travel to a country with documented h5n1 avian influenza within 10 days of symptom onset testing should be considered on a case-by-case basis in consultation with state and local health departments for hospitalized or ambulatory patients with temperature >38°c (>100.4°f) and one or more of the following: cough, sore throat, shortness of breath and history of contact with domestic poultry (eg, visited a poultry farm, household raising poultry, or bird market) or a known or suspected human case of influenza h5n1 in an h5n1-affected country within 10 days of symptom onset h5n1 disease. 19 there is no evidence of h5n1 virus replication outside the respiratory tract. viral replication seems to trigger a burst of cytokine production that may ultimately result in multiorgan failure. all patients who present to a health care setting with fever and respiratory symptoms should be questioned regarding their recent travel and exposure history. 8 in patients at risk for avian influenza h5n1, laboratory testing is indicated ( table 3 ). the optimal specimen for influenza virus detection is a nasopharyngeal aspirate obtained within 3 days of the onset of symptoms; however, nasopharyngeal swabs and other specimens can be used. the h5n1 influenza virus can be detected by rapid antigen tests, virus culture, and reverse transcriptase-polymerase chain reaction (rt-pcr). at least 6 rapid antigen detection assays are commercially available and provide results in 15 to 30 minutes but are not as sensitive or specific as virus culture or rt-pcr. 10 virus isolation in cell cultures enables further antigenic and genetic characterization, drug susceptibility testing, and vaccine preparation. however, cultures take 2 to 10 days and must be performed under biosafety level 3+ laboratory conditions. conventional and real-time pcr assays detect diverse influenza type a viruses, including the avian h5n1 strains. 28, 29 polymerase chain reaction primers are directed to the conserved matrix gene of the virus, and therefore this assay would be expected to detect recombinant avian strains of the virus. of emphasis, avian influenza virus may be recombinant strains of the virus and may not contain the essential genes for replication in laboratory cultures. 14 rapid antigen detection and rt-pcr can be performed with standard biosafety level 2 laboratory conditions in a class ii biological safety cabinet. serologic diagnosis of influenza is based on the detection of a 4-fold or greater increase in specific antibody titer in paired serum samples; the first should be collected as soon as possible after onset of illness and the second, 10 to 14 days later. this limits the usefulness of serology in the diagnosis and treatment of acute illness. the current h5n1 strains are resistant to amantadine and rimantadine but are susceptible to neuraminidase inhibitors (oseltamivir and zanamivir). these drugs are effective for prophylaxis and treatment of influenza a virus infection, including the avian influenza h5n1. however, no efficacy trials have been performed due to the small number of human cases. because a potential vaccine may be in short supply, antiviral drugs may play an important role in reducing the severity and spread of infection during the first stages of a pandemic. 32 for prophylaxis, oseltamivir should be administered to individuals exposed to h5n1 avian influenza within 48 hours. therefore, a global influenza strategy in the 21st century calls for stockpiling antiviral drugs and drafting plans for rapid distribution. 9 in geographic areas affected by avian influenza, individuals should avoid contact with poultry, and they should perform thorough and frequent hand hygiene using soap and water or alcohol-based hand rubs. the virus is killed by heat (56°c for 3 hours, 60°c for 30 minutes, or 70°c for 1 minute) and common disinfectants such as alcohol, bleach, formalin, or iodine compounds. generally, 5% bleach solution is appropriate for dealing with biohazardous spillage. influenza virus can survive in feces for several months. it can survive in water for up to 4 days at 22°c, for more than 30 days at 0°c, and indefinitely in frozen material. 1 therefore, poultry, including the eggs, should be cooked thoroughly. recently, the centers for disease control and prevention issued guidelines for airline flight crews and persons meeting passengers arriving from areas with avian influenza. 30 protection of persons involved in outbreak eradication activities includes strict adherence to hand hygiene practices and the use of appropriate personal protective equipment (gloves, disposable clothing, shoe covers, safety goggles, and particulate respirators). 30 during handling of human specimens, formation of aerosols and droplets should be minimized. isolation precautions identical to those recommended for severe acute respiratory syndrome should be implemented for all hospitalized patients diagnosed as having or being evaluated for avian influenza h5n1 (table 4 ). 31 these precautions should be continued for 14 days after for personal use. mass reproduce only with permission from mayo clinic proceedings. the current inactivated trivalent human influenza vaccine provides no protection against the h5 and h7 avian influenza strains. however, an important control measure is providing the seasonal human influenza vaccine to people at risk for avian influenza to reduce the risk of coinfection with avian and human influenza viruses simultaneously and to decrease the possibility of reassortment. the resultant hybrid virus could be highly transmissible among humans. a prototype virus and candidate vaccines have been developed for protection against the h5n1 virus strain that infected 2 people and resulted in 1 death in hong kong in 2003. 2 however, the avian subtypes are rapidly lethal to chicken embryos, and therefore the traditional chicken embryo method is not applicable. alternative means of producing h5 and h7 vaccines were exploited, including the plasmid-based reverse genetic technology. 9 a vaccine strain can be created by merging selected hemagglutinin and neuraminidase genes from the target virus with a laboratory virus. the resulting virus is recognized by the human immune system and causes a protective immune response but no disease. however, such vaccines have yet to be studied in clinical trials, and safety testing must be completed. the occurrence of avian influenza h5n1 in humans is another reminder of our vulnerability to an emerging pandemic. several measures can help to minimize the global public health risk. an immediate priority is to halt further spread of epidemics in poultry populations that would reduce the opportunities for human exposure to the virus. clinicians should be cognizant of human influenza h5n1 infection among patients with the appropriate epidemiological exposure, so that patients can be identified quickly and managed appropriately and health care workers can be protected. the reemergence of h5n1 influenza in humans emphasizes the need to develop a vaccine against this virus. for personal use. mass reproduce only with permission from mayo clinic proceedings. available at: www.who.int/csr/disease/avian_influenza/en/. accessibility verified development of a vaccine effective against avian influenza h5n1 infection in humans human influenza a h5n1 virus related to a highly pathogenic avian influenza virus world health organization the pathogenesis of influenza in humans the structure and receptorbinding properties of the 1918 influenza hemagglutinin pandemic threat posed by avian influenza a viruses outbreaks of avian influenza a (h5n1) in asia and interim recommendations for evaluation and reporting of suspected cases-united states are we ready for pandemic influenza? influenza virus neuraminidase inhibitors realities and enigmas of human viral influenza: pathogenesis, epidemiology and control pandemic influenza: its origin and control influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission animal influenza virus surveillance influenza: an emerging disease wet markets-a continuing source of severe acute respiratory syndrome and influenza? outbreak of avian influenza a(h5n1) virus infection in hong kong in 1997 human infection with influenza h9n2 avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands case-control study of risk factors for avian influenza a (h5n1) disease, hong kong risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 cases of influenza a (h5n1)-thailand world health organization international avian influenza investigative team. avian influenza a (h5n1) in 10 patients in vietnam clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus pathology of fatal human infection associated with avian influenza a h5n1 virus detection of influenza a viruses from different species by pcr amplification of conserved sequences in the matrix gene multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses available at: www.cdc.gov/flu/avian/index.htm. accessibility verified sars: epidemiology, clinical presentation, management, and infection control measures prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) a. strict hand hygiene before and after all patient contact b. use of gloves and gown for all patient contact c. use of eye protection when within 3 feet (1 m) of the patient d. use of shoe covers when entering the isolation room e. placement of the patient in an airborne isolation room key: cord-309635-1tgovkr7 authors: wu, nicholas c.; wilson, ian a. title: structural biology of influenza hemagglutinin: an amaranthine adventure date: 2020-09-22 journal: viruses doi: 10.3390/v12091053 sha: doc_id: 309635 cord_uid: 1tgovkr7 hemagglutinin (ha) glycoprotein is an important focus of influenza research due to its role in antigenic drift and shift, as well as its receptor binding and membrane fusion functions, which are indispensable for viral entry. over the past four decades, x-ray crystallography has greatly facilitated our understanding of ha receptor binding, membrane fusion, and antigenicity. the recent advances in cryo-em have further deepened our comprehension of ha biology. since influenza ha constantly evolves in natural circulating strains, there are always new questions to be answered. the incessant accumulation of knowledge on the structural biology of ha over several decades has also facilitated the design and development of novel therapeutics and vaccines. this review describes the current status of the field of ha structural biology, how we got here, and what the next steps might be. four types of influenza virus, a, b, c, and d, are known. influenza a and b viruses can cause severe symptoms and mortality in the human population, whereas influenza c virus only manifests itself in mild disease and influenza d virus does not circulate in humans. a major difference between influenza a and b viruses is that influenza b virus is almost exclusively observed in humans, whereas influenza a virus has a diverse and extensive reservoir in aquatic birds that occasionally spills over to humans directly or via domestic animals, such as pigs, as new pandemics or emerging viruses [1] . as a result, influenza a viruses receive much more attention than other influenza types even though influenza a and b both co-circulate in the human population as seasonal viruses. influenza a virus can be further divided into subtypes based on the antigenicity of the surface glycoproteins hemagglutinin (ha) and neuraminidase (na), with 18 known subtypes of ha (h1-h18) and 11 subtypes of na (n1-n11). similar to influenza a virus, influenza b virus also has two surface glycoproteins ha and na, which diverged into two lineages, victoria and yamagata, during the 1980s [2] . in contrast, influenza c and d viruses only have one surface glycoprotein hemagglutinin-esterase fusion (hef) [3] that encompasses both ha and na activities. four known influenza a pandemics have been documented in human history, namely 1918 spanish flu (h1n1), 1957 asian flu (h2n2), 1968 hong kong flu (h3n2), and 2009 swine flu (h1n1), although others undoubtedly have occurred prior to these [4] . occasionally, other influenza a subtypes, such as h5n1, h5n6, h6n1, h7n7, h7n9, h9n2, and h10n8, also infect back in the early 1940s, george hirst reported the ability of influenza virus to agglutinate chicken red blood cells (rbcs) [10] and attributed this to adsorption of the ha onto the rbcs [11] . in the late 1940s, an enzyme from vibrio cholerae was discovered with the ability to prevent influenza virus from agglutinating red blood cells [12, 13] . subsequent identification of the enzymatic product revealed sialic acid as the receptor of influenza virus [14] . however, the location and molecular characteristics of the rbs were unclear until the first ha structure was determined in 1981 [9] . the rbs was identified partly due to its sequence conservation, structural resemblance to the wheat-germ agglutinin sialic acid-binding site [15] , and from mutations that affect receptor specificity [16] . the first structure of ha in complex with sialic acid in 1988 confirmed the location of the rbs and sialic acid as the host receptor of influenza virus [17] . the rbs of influenza a ha is composed of four structural elements, 130-loop, 150-loop, 190 -helix, and 220-loop, which are named after their positions on the primary amino acid sequence. similarly, rbs of influenza b ha is composed of the 140-loop, 190-helix, and 240-loop, which are structurally equivalent to the 130-loop, 150-loop, and 190-helix receptor specificity can also continue to evolve when seasonal viruses circulate in the human population, due to natural mutations that are likely a response to immune selection pressure. this phenomenon has recently been reported in human h3n2 viruses, which have evolved a preference for long, branched sialylated glycans with extended poly-n-acetyl-lactosamine (poly-lacnac) [ . in fact, evolutionary contingency, which describes sequence variants that were previously fit but then become unfit and extinct, as well as evolutionary entrenchment, which describes sequence variants that were previously unfit and then become fit and emerge, are common in the ha rbs of human h3n2 viruses [48] . as seasonal influenza viruses continue to evolve in the human population, it will be fascinating to observe how the receptor-binding mode is able to change (or not) in the future, which would allow the h3n2 virus to continue its over 50 years of sustained circulation in the human population. interestingly, bat influenza a viruses h17n10 and h18n11 do not utilize sialylated glycans as receptors [50, 51] . crystal structures of the ha from h17n10 and h18n11 viruses indicate that their rbs is highly acidic, which would electrostatically repulse sialic acid and hence would have substantially different biochemical properties from the other ha subtypes (i.e., h1-h16) even although the overall architecture of the rbs is roughly similar [50, 51] . recent studies have revealed that major histocompatibility complex class ii (mhc-ii) human leukocyte antigen dr isotype (hla-dr) can act as a receptor for bat influenza a viruses [52, 53] . however, a structure of the complex between bat influenza ha and hla-dr has not been reported. therefore, the receptor-binding mechanism of bat influenza ha remains elusive. influenza viruses have also been discovered in species as diverse as eel, toad, and hagfish using a meta-transcriptomic approach [54] . nonetheless, the has from these influenza viruses have not been functionally characterized and their receptors are currently unknown. additional influenza virus subtypes as well as types will likely be discovered in the future, and it will be to interesting to see whether other host receptors are employed. after attaching to the host receptor, endocytosis transports the influenza viral particle to the endosome, where the ph becomes acidic. the acidic ph triggers viral-host membrane fusion that is mediated by conformational rearrangements in the ha. the prerequisite for such conformational rearrangements is proteolytic processing of the ha. ha is translated as a single polypeptide chain ha0, which is then cleaved by host proteases into the ha1 and ha2 subunits. the membrane fusion machinery is encoded mainly by ha2, while ha1 is entirely responsible for receptor binding, as outlined in the previous section. the overall structure of uncleaved ha0 is almost identical to the cleaved ha [55] . the cleavage site on ha0 is presented as a surface loop on the ha stem, which is proximal to the viral membrane compared to the ha head. the amino acid sequence at the cleavage site is a well-characterized pathogenic factor [56] . while most influenza a strains carry a monobasic cleavage site, some highly pathogenic avian influenza a strains carry a polybasic cleavage site that can be processed by ubiquitously expressed furin. upon cleavage, the c-terminus of ha1 remains solvent exposed, whereas the n-terminal of ha2, which represents the hydrophobic fusion peptide, inserts into a buried cavity that is composed of ionizable residues including ha1 his17, as well as ha2 asp109, asp112, and lys117 [55] . this metastable conformation is then poised for low ph-induced structural rearrangements to accomplish viral-host membrane fusion. in fact, the ability of ha to undergo ph-dependent structural rearrangement has been known since the early 1980s based on circular dichroism, electron microscopy, and sedimentation analyses [57] . subsequent analyses demonstrated that, after conformational changes, ha is susceptible to trypsin digestion, where ha1 residues 28 to 328 (globular domain) are released, while ha1 residues 1 to 27 remain covalently linked through a disulfide bond to the intact ha2 subunit [57, 58] . a crystal structure of this trypsin-digested product containing the intact ha2 subunit, which represents the post-fusion conformation of ha, was reported in the mid-1990s [59] . the post-fusion conformation of ha2 features substantial rearrangements of helices and connecting segments to form a 100 å-long α-helix in each protomer, which assemble as a three-stranded coiled coil at trimer interface ( figure 2 ). in addition, the hydrophobic fusion peptide relocates to the top of the helix ready for membrane insertion. [60] , and state 5 (post-fusion conformation, pdb 1qu1) are shown [61] . of note, after fusion peptide is released from state 2, the fusion peptide becomes disordered [60] . in state 3, the membrane proximal region (yellow) is also disordered [60] . different components in the ha2 that are involved in structural rearrangements between pre-and post-fusion structures are in different colors. structures of intermediates during the ha fusion process have been probed by low-resolution cryo-electron microscopy (cryo-em) [62, 63] , as well as x-ray crystallography [64] . nonetheless, a more complete picture of ha fusion intermediate structures was described only recently [60] , by taking advantage of advances in high-resolution cryo-em [65] . specifically, after incubation of ha at low ph for different times (10 s, 20 s, 60 s, and 30 min), ha conformational changes were examined by cryo-em [60] (figure 2 ). three-dimensional (3d) classification and reconstruction at different time points revealed three sequential intermediate forms of ha, including one with a 150 å-long three-stranded α-helix coiled coil [60] . however, it is still unclear how the lipid bilayer membranes from the virus and host are fused together because most structural studies use the ha ectodomain and membranes are often excluded. the feasibility of structurally characterizing full-length ha, which includes the transmembrane region, has also recently been demonstrated by cryo-em [66] . therefore, future studies should be able to explore the conformational changes during influenza virus-host membrane fusion in the context of full-length ha and in the presence of a membrane. based on analysis of the first ha structure [9] with known natural antigenic variants and laboratory escape mutants at the time [67] [68] [69] [70] [71] [72] [73] [74] , four major antigenic sites (a-d) in the h3 ha were identified and reported in a back-to-back paper with the ha structure in 1981 [9, 75] . in the 1980s, a fifth antigenic site (e) was also identified [76, 77] . similarly, five major antigenic sites, namely sa, sb, ca1, ca2, and cb, were identified in h1 ha during the early 1980s [78, 79] . all of the major antigenic sites in h1 and h3 has as well as influenza b ha [80, 81] are located in the ha1 globular head domain and their immunodominance can change over natural evolution ( figure 3a,b) . however, the first structure of an antibody (hc19) in complex with ha was not reported until 1995 [82] . antibody hc19 targets the rbs, which explains its neutralization activity. however, hc19 also recognizes rbs-proximal regions, which are highly variable across strains. as a result, escape mutants to hc19 could be readily identified. consistently, subsequent studies demonstrated that major antigenic drift in seasonal influenza viruses is mostly driven by mutations within or near the rbs [83, 84] . it is therefore not surprising that some of the mutations that arise during natural evolution of human influenza virus can alter both ha antigenicity and receptor binding [47, 48, 85, 86] . furthermore, egg-based seasonal influenza vaccines often carry egg-adaptive mutations in the ha rbs that allow the vaccine strain to bind to α2-3 linkage sialylated glycans on the chorioallantoic membrane but can also alter the antigenicity of ha, thereby decreasing vaccine effectiveness [49, [87] [88] [89] . for example, one of the egg-adaptive mutations t160k would abolish an n-glycosylation site at n158 and appears to contribute to the poor seasonal influenza vaccine effectiveness in the 2016-2017 influenza season. in fact, accumulation of n-glycosylation in the ha1 globular head domain plays an important role in the antigenic drift of seasonal influenza virus [76, 90, 91] . a recent study has shown that n-glycosylation sites are added to has of seasonal influenza virus at discrete 5-to-7-year intervals, with an upper limit of~6 and~8 glycans in the ha1 globular head domains of h1n1 and h3n2, respectively [92] . the glycan form, occupancy, and heterogeneity at each n-glycosylation site on ha can be probed by mass spectrometry [93, 94] . moreover, some of the n-glycans on ha can be observed by x-ray crystallography and cryo-em [9, 64] . as compared to the variable globular head domain in ha1, the stem domain in ha2 is much more conserved. it had long been thought that neutralizing antibodies (nabs) do not target the stem domain until the discovery of a mouse ha stem antibody c179 in 1993 [97] . nevertheless, this observation was largely unappreciated and stem antibodies were not found in humans until the late 2000s [98] [99] [100] (figure 3c ). in the subsequent decade, many stem neutralizing and protective antibodies have been isolated and structurally characterized. unlike neutralizing antibodies to the ha head, which generally block receptor binding, stem antibodies typically protect by interfering with the fusion machinery [101] [102] [103] . due to high sequence conservation of the stem domain, stem antibodies usually exhibit higher breadth (i.e., broadly neutralizing antibodies, bnabs) and interact with a greater range of influenza subtypes and strains compared to nabs to the ha head. recurring molecular features are observed in stem abs. for example, the ighv1-69 antibody heavy-chain germline gene is commonly used by the immune system for generation of stem antibodies due to the presence of a germline-encoded ify motif, which can engage three highly conserved hydrophobic pockets in the ha stem region [98, [100] [101] [102] [104] [105] [106] . in addition, ighd3-9, one of the heavy-chain diversity genes that encodes for an important part of complementarity determining region 3 of the heavy chain (cdr h3), is also utilized in many stem antibodies. the ighd3-9 gene encodes an lxyfxwl motif that makes favorable interactions with four hydrophobic pockets in the ha stem. however, the breadth of some ha stem antibodies is often restricted to group 1 has (h1, h2, h5, h6, h8, h9, h11, h12 , h13, and h16), since a conserved n-glycan at ha1 residue 38 in most of the group 2 has (h3, h4, h7, h10, h14, and h15) can sterically hinder accessibility to the ha stem epitope [102, 107] . a few select ighv1-69-encoded stem antibodies can manage to navigate around the n-glycan at ha1 residue 38 to achieve cross-group neutralization [101, 104] . some group 2-specific abs bind to an epitope that is in the lower part of the stem domain and closer to the viral membrane ( figure 3c ) and hence can avoid a clash with the n-glycan at ha1 residue 38 [108, 109] . more recently, ighv6-1 was found to be a germline gene that is often utilized in cross-group stem abs [110] [111] [112] [113] . interestingly, the ancestral precursors of ighv6-1-encoded cross-group stem abs can be either group 1-or group 2-specific, depending on the cdr h3 sequences and conformations [113] . there is an accumulation of glycosylation sites during human h3n2 evolution. while many antigenic sites have now been masked by glycans (yellow), antigenic site b (blue) remains exposed due to its proximity to the rbs, making it immunodominant in recent human h3n2 strains [95, 96] . (c) broadly neutralizing epitopes that have been identified in the past decade are shown. (d) a recently identified trimeric interface epitope is illustrated. over the past decade, several cross-group bnabs that target ha rbs have also been discovered and characterized [114] [115] [116] [117] [118] [119] [120] (figure 3c ). while the reactivity of some rbs bnabs is mostly limited to a particular subtype [121] [122] [123] [124] [125] [126] , they are still considered as broadly neutralizing in the sense of covering most if not all strains within a subtype (e.g., 5j8 [121] and ch65 [124] to h1 ha, as well as f045-092 [115] and 019-10117-3c06 [120] to h3 ha). such antibodies could be very useful in protecting against antigenic drift in seasonal viruses, for example. in addition, subtype-specific bnabs that target the vestigial esterase subdomain [127, 128] , "lateral patch" epitope on ha1 [129] , and the junction between the ectodomain and membrane anchor have also been identified [66] . in 2019, an h7-specific bnab was shown to target an epitope that partly involves the ha protomer-protomer interface in ha1 [130] . such a finding demonstrated that an antibody epitope does not need to be completely solvent exposed in the prefusion conformation. in the same year, three other papers have reported an epitope that is almost exclusively in the ha protomer-protomer interface in ha1 [131] [132] [133] (figure 3d ). some interface-targeting antibodies can cross-react with all influenza a subtypes and confer in vivo protection despite the lack of neutralization activity [131, 132] . therefore, it is now quite clear that "breathing" of the ha trimer [134] can allow antibodies to access cryptic epitopes that are transiently exposed and were not originally thought to be accessible in the ha prefusion conformation. during the early 1990s, structural-based computational screening of around 55,000 small molecules resulted in the identification of benzoquinones and hydroquinones as ha fusion inhibitors [135] . one of the compounds, tert-butyl hydroquinone (tbhq), had its binding mode to ha reported in 2008 [136] . tbhq binds to a hydrophobic pocket in an interface region between ha monomers, which in turn stabilizes the ha prefusion conformation and prevents the conformational changes required for membrane fusion. interestingly, arbidol, which was developed as a general antiviral medication in russia especially for respiratory diseases during the late 1980s [137] , was more recently shown to inhibit ha-mediated membrane fusion by stabilizing the prefusion conformation [138, 139] . in 2017, the structure of arbidol in complex with ha revealed that the arbidol binds to a similar location in the ha stem as tbhq, but its binding site is much larger and more complex [140] . structure-based optimization of arbidol resulted in a compound with its affinity improved by two to three orders of magnitude, although it manifested low stability [141] . thus, the stem region on ha that is targeted by both arbidol and tbhq represents a promising target for future influenza antiviral development. over the past decade, structural characterization of bnabs to the ha have stimulated antiviral design, ranging from small protein binders [142] [143] [144] [145] to peptides [146] to small molecules [147, 148] . in addition, the discovery and characterization of bnabs to ha have reignited aspirations and novel approaches towards a more universal influenza vaccine [149] . while universal influenza vaccine design has largely been focused on the stem domain [150] [151] [152] [153] [154] , our recent study demonstrated the need to consider the potential for escape mutations to stem bnabs, which can more rapidly emerge in the h3 subtype compared to other subtypes, such as h1n1 [155] . indeed, some escape mutations have already been observed in low frequency in naturally circulating strains. to escape stem bnabs, mutations can either decrease binding of stem bnabs or enhance ha fusion ability [156] . similarly, escape mutants to rbs bnabs can be even more readily isolated [157, 158] . notwithstanding, studies in zika virus, ebola virus, hbv, and sars-cov-2 have shown that use of a well-designed antibody cocktail can minimize the emergence of escape mutants [159] [160] [161] [162] . thus, a universal influenza vaccine may need to induce a polyclonal response that targets both the rbs and stem domain to prevent or mitigate against escape. the advantage of simultaneously targeting rbs and the stem domain has been demonstrated by a multidomain antibody composed of four physically linked camelid single-domain antibodies-three of which target the stem domain and one the rbs [163] . this multidomain antibody is able confer "universal" in vivo protection against both influenza a and b viruses [163] . furthermore, the recent development of a "mosaic" nanoparticle that co-displays has from multiple subtypes provides possibilities to induce such a polyclonal bnab response [164] . our understanding of ha biology has advanced relentlessly every year since the first ha structure was reported in 1981. however, new unknowns in the structural biology of ha emerge as influenza viruses continues to evolve, new subtypes are found, and new zoonotic viruses enter the human population. for example, accumulation of natural mutations in the ha rbs has revealed unexpected changes in the receptor-binding modes during h3n2 evolution and motivated greater understanding of how the sialic acid receptor can continue to engage to an ever-changing binding site. in addition, discovery of the initial human bnabs to the ha stem has inspired the discovery of new epitopes in the ha targeted by different families of bnabs. the elucidation of how bnabs target neutralizing epitopes on the ha has further galvanized efforts to design a variety of different classes of therapeutic candidates against the ha [142] [143] [144] 146, 147] . such therapeutics could prevent influenza entry and infection compared to ameliorating infection as for drugs like tamiflu and relenza [165] . recent advances in cryo-em have greatly complemented x-ray crystallography and enhanced the ability to investigate full-length ha embedded in micelles or membranes [60, 66] . thus, many of the new as well as perennial unanswered questions can now begin to be addressed. in addition, the neuraminidase (na) is also undergoing its own reincarnation from the initial antibody work in the 1960s [166] and first structures in the 1980s [167] [168] [169] . na has been a neglected target on influenza virus [170] but is now undergoing a renaissance for vaccine design [8] . structural biology of ha, as well as na, will therefore remain a key component of influenza research until influenza virus ceases to be a global health concern, which is not yet on the horizon. we have experienced the wrath of the sars-cov-2 pandemic in 2020 and do not want to also experience an influenza pandemic like 1918 h1n1. thus, effective utilization of the available and emerging structural information on 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we thank the two anonymous reviewers for providing insightful comments. the authors declare no conflict of interest. key: cord-343050-1pfqgvie authors: huang, qiu sue; turner, nikki; baker, michael g; williamson, deborah a; wong, conroy; webby, richard; widdowson, marc-alain title: southern hemisphere influenza and vaccine effectiveness research and surveillance date: 2015-06-09 journal: influenza other respir viruses doi: 10.1111/irv.12315 sha: doc_id: 343050 cord_uid: 1pfqgvie the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012–2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012–2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. the 2009 influenza a(h1n1)pdm09 pandemic highlighted the need for improved scientific knowledge to support better pandemic preparedness and seasonal influenza control. the southern hemisphere influenza and vaccine effectiveness research and surveillance (shivers) project, a 5-year (2012-2016) multiagency and multidisciplinary collaboration, aimed to measure disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory infectious diseases of public health importance. two active, prospective, population-based surveillance systems were established for monitoring influenza and other respiratory pathogens among those hospitalized patients with acute respiratory illness and those enrolled patients seeking consultations at sentinel general practices. in 2015, a sero-epidemiological study will use a sample of patients from the same practices. these data will provide a full picture of the disease burden and risk factors from asymptomatic infections to severe hospitalized disease and deaths and related economic burden. the results during the first 2 years (2012-2013) provided scientific evidence to (a) support a change to nz's vaccination policy for young children due to high influenza hospitalizations in these children; (b) contribute to the revision of the world health organization's case definition for severe acute respiratory illness for global influenza surveillance; and (c) contribute in part to vaccine strain selection using vaccine effectiveness assessment in the prevention of influenza-related consultations and hospitalizations. in summary, shivers provides valuable international platforms for supporting seasonal influenza control and pandemic preparedness, and responding to other emerging/endemic respiratory-related infections. keywords disease burden, epidemiology, immunology, influenza, risk factors, vaccine effectiveness. the 2009 influenza a(h1n1)pdm09 pandemic provided a test of global preparedness to assess the epidemiology of a pandemic and to respond appropriately and rapidly. the world was illprepared to respond to a severe influenza pandemic or to any similarly global, sustained and threatening public health emergency. 1 one fundamental constraint highlighted during the pandemic was the limited understanding of the epidemiology and severity of the pandemic which in turn hampered international efforts to mount an appropriate response. 2 rapid assessment of the epidemiologic, virologic and clinic features of a pandemic is essential to provide critical information to decision-makers on how to minimize morbidity and mortality, and mitigate potential economic and societal disruption. soon after the pandemic virus emerged in april 2009 in mexico and spread globally, public health leaders, anxious to understand the full breadth of influenza epidemiology, turned their attention to countries in southern temperate areas with an upcoming influenza season. this demonstrated the absence of an established real-time system in the southern hemisphere to provide more complete surveillance of an influenza pandemic. also, such a system would help monitor the epidemiology of new strains of seasonal influenza and the effectiveness of vaccination, both for the southern hemisphere and for upcoming northern hemisphere seasons. in december 2010, the us centers for disease control and prevention (us-cdc) made a funding opportunity announcement for a temperate southern hemisphere site to conduct research on the disease burden, epidemiology, and prevention of influenza and other respiratory diseases of public health importance. new zealand (nz) is a temperate southern hemisphere country with a population of 4á4 million people. the influenza season mainly occurs from june to september. [3] [4] [5] [6] nz's predominantly public-funded healthcare system with associated integrated health information systems is a strong asset in conducting population-based research. all new zealanders are assigned a unique health identifier allowing tracking of healthcare utilization over time and confidential record linkage to multiple databases including hospitalization and surveillance data. additionally, patients are registered with primary care providers who maintain highly computerized records with detailed demography, immunization status and clinical information. the nz population is well characterized in terms of demographic structure, particularly by ethnicity and socio-economic status. indigenous maori and pacific peoples (collectively about 20% of the population) appear particularly vulnerable to influenza and other respiratory infections. 3, 7 in in this article, we describe the objectives and study designs of shivers. we also describe lessons learned from the first 2 years and planned future studies as well as international applications. the overarching aim of shivers is to comprehensively investigate the disease burden, epidemiology, aetiology, risk factors, immunology, effectiveness of vaccination and other prevention strategies for influenza and other respiratory viral diseases of public health importance. the project consists of 9 objectives as detailed in table 1 . they can be divided into five main streams: influenza disease burden data are essential to allocate limited health resources, assist influenza vaccination policy development and improve vaccine uptake, particularly for subpopulations at risk. however, the evidence to support valid and precise estimates of influenza disease burden globally remains weak with low quality, partly due to the short duration of studies and the heterogeneity of study settings and methods (statistical modelling, active versus passive case findings, virological versus clinical detection). [8] [9] [10] [11] [12] [13] [14] in addition, there is scarce information on sero-epidemiologic investigation of seasonal influenza at a population level. serology can detect both symptomatic and asymptomatic infections, thus estimating the true incidence of influenza infection. this parameter cannot be determined by either disease surveillance programmes or detection of virologically confirmed cases as they would vastly underestimate influenza incidence and overestimate severity. [15] [16] [17] [18] shivers allows calculation of rates of infection and different clinical presentations in the same population at the same time for an accurate picture of the relative severity of influenza infection in the population and vulnerable subpopulations at four levels: (a) severe hospitalized disease; (b) moderate disease requiring a general practice visit; (c) mild disease not requiring a general practice visit; (d) incidence of infection (symptomatic and asymptomatic). aetiology shivers provides an integrated platform for the study of respiratory diseases caused by influenza and other common and emerging respiratory pathogens. the aetiological component allows us to (1) monitor antigenic drift of seasonal influenza viruses, contributing to who's annual vaccine strain selection; (2) support pandemic preparedness including surveillance for new subtypes of influenza a viruses (e.g. a(h7n9)); (3) monitor common non-influenza respiratory pathogens to understand their impact on the disease and epidemiology; and (4) provide early detection for emerging respiratory viruses (e.g. mers-cov). there is increasing evidence in the literature for the importance of polymicrobial infections. however, there remain gaps in our understanding of respiratory virus codetection and whether this represents co-infection and affects clinical disease manifestations and severity. there are contradictory reports with some suggesting that co-infections increase the severity of respiratory disease, 19-23 while others have found either no association [24] [25] [26] [27] [28] or that co-infections may actually be protective. 29 additionally, bacterial coinfections associated with cases of influenza are a leading cause of severe morbidity and mortality: bacterial coinfections complicated nearly all influenza deaths in the 1918 pandemic and up to 34% of the 2009 a(h1n1)pdm09 infections managed in intensive care units worldwide. 30, 31 shivers will help our understanding of the potential role of pathogen co-detection in patient outcome, severity, aetiology, demography and underlying risk conditions. influenza vaccine strain selection requires annual consultations and frequent updates to match the antigenic drift of the circulating viral strains, and ample evidence indicates that influenza vaccine effectiveness (ve) varies not only by virus type (subtype) but also from year to year. 32, 33 robust and timely vaccine effectiveness estimates are important to measure the public health benefit of seasonal influenza control strategies, pandemic preparedness and vaccine strain selection. 34 many ve estimates derive from observational studies with existing data collecting systems which often have multiple limitations and biases, and there are international calls for more rigorous ve studies. [34] [35] [36] [37] [38] [39] shivers is providing robust and timely estimations of the protective effect of seasonal influenza vaccine in the prevention of hospitalizations and general practice consultations for laboratory-confirmed influenza using case test-negative control methods. 40, 41 immune response an individual's immune response to influenza infection can vary depending on many factors (e.g. age, underlying conditions and ethnicity). clinical observation during the 2009 pandemic indicated that the unexpected low morbidity and mortality rates in the elderly were in part due to their crossreactive immunity. 3, 42, 43 there are knowledge gaps regarding each component of adaptive immune responses in determining an individual's risk of acquiring influenza virus infection and the severity of the resulting disease: antihemagglutinin (ha) antibodies, antineuraminidase (na) antibodies, isotypes of responding antibodies, influenza-specific cd4+, cd8+ t cells, surface markers and key cytokines. 44, 45 additionally, there are scarce data on the correlation of cellular immunologic and neuraminidase targeted antibody responses in individuals with serologically (anti-ha antibodies) defined influenza infection. 46, 47 by interconnecting the epidemiological and immune studies in severe and moderate disease cases and high-risk subgroups (e.g. pacific and maori ethnic groups) and individuals with serologically defined influenza infection, shivers will facilitate our understanding of host immune responses that determine an individual's risk of acquiring influenza infection or developing severe disease. identification and quantification of risk factors for influenza infection and poor outcomes (hospitalization, icu treatment, death) provides evidence to inform decisions on targeted pharmaceutical (vaccinations, antivirals), healthcare (e.g. improved treatment of comorbidities) and non-pharmaceutical (e.g. exposure to infections) interventions to reduce the risk of seasonal and pandemic influenza. elderly people have a significantly higher risk of influenza-associated death compared with non-elderly people. 48 additionally, the 2009 pandemic in nz revealed that the risk of hospitalization and death was markedly higher for maori and pacific people, and those from the most deprived socioeconomic groups. 3, 42, 49 however, it is not clear whether these sociodemographic factors are independent risk factors for influenza. furthermore, some chronic health conditions (high body mass index, asthma and pregnancy) have been shown to increase the risk of having a poor outcome from influenza infection. [50] [51] [52] [53] in nz, household crowding has been identified as a risk factor for transmission of meningococcal disease, 54 rheumatic fever 55 and tuberculosis 56 and may also be contributing to higher rates of influenza for some populations. the household setting (crowding, housing conditions) may influence transmission of influenza, but these effects remain poorly understood. 57-59 shivers will provide a multifaceted understanding of influenza risk that considers organism, host and environmental factors and opportunities for intervention. this comprehensive and quantitative approach will include detailed consideration of the independent contributions of host ethnicity, socioeconomic position, chronic illness status, obesity, household environment exposures and infecting virus. study sites shivers study sites are located within two district health boards of the auckland region of nz: adhb and cmdhb ( figure 1 ). this is a predominantly urban population of 906 000 people, with a wide spectrum of socio-economic, cultural, ethnic and demographic groups broadly similar to the new zealand population. 60 we established two surveillance platforms (hospital and sentinel general practice) in the two dhbs: four publicly funded hospitals serve the secondary healthcare needs for all residents of the two dhbs: auckland city hospital and the associated starship children's hospital (adhb), and middlemore hospital and the associated kidz first children's hospital (cmdhb). in 2012, we began active, prospective, continuous, population-based surveillance for influenza and other respiratory pathogens among persons residing in the two dhbs hospitalized for respiratory disease (figure 2 ). research nurses reviewed daily records of all overnight acutely admitted inpatients to identify any inpatient with a suspected acute respiratory illness (ari). they interviewed these patients by applying the world health organization (who) interim sari case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 7 days, and requiring inpatient hospitalization'. 61 since 2013, the who final sari case definition has been used with onset changed from '7 days' to '10 days'. the patients were differentiated into sari cases (those who met the sari case definition) and non-sari patients (those with ari who did not meet the sari case definition). a case report form captured information on demography, history of presenting illness, comorbidities, influenza vaccination history, disease outcome and risk factors. 60 if a patient met the sari case definition, a respiratory specimen (nasopharyngeal swab or aspirate) was collected and tested simultaneously for influenza and other respiratory viruses by real-time reverse transcription (rt) polymerase chain reaction (pcr) techniques: influenza virus, respiratory syncytial virus (rsv), rhinovirus, parainfluenza virus types 1-3, adenovirus and human metapneumovirus ( figure 3) . 60 a systematic sample of about 50% of non-sari patients were also interviewed and provided a respiratory sample, in addition to those from whom a specimen was collected for clinical purposes. some sari cases and non-sari patients were also tested for clinical purposes for a range of respiratory bacteria (e.g. streptococcus pneumonia, in 2013, we began active, prospective, population-based surveillance for influenza and other respiratory pathogens among persons enrolled in sentinel general practices who seek medical consultations (figure 4 ). eighteen sentinel general practices situated within adhb and cmdhb were recruited. these practices have a combined total of 103 752 enrolled patients, covering approximately 12% of the adhb and cmdhb population. the participating general practitioners (gp) and practice nurses (pn) assessed all consultationseeking patients. if a patient met the influenza-like illness (ili) case definition: 'an acute respiratory illness with a history of fever or measured fever of ≥38°c, and cough, and onset within the past 10 days, and requiring consultation in that general practice', a respiratory specimen (nasopharyngeal swab or throat swab) was collected to test for influenza and other respiratory viruses by real-time rt-pcr ( figure 3 ). gp/pn documented the components of the case definition that were present and recorded patients who met the ili case definition in an advanced electronic form designed for the practice management system (pms). patient information already captured in the pms was automatically retrieved, including demography, comorbidities, vaccination history and regular medication list. further data were captured by interviewing ili patients regarding influenza vaccination obtained elsewhere, pregnancy and a clinician's judgement of obesity. to more fully understand the epidemiology of influenza, these two platforms will be leveraged for other studies: (a) sero-epidemiological study: to obtain rates of mild influenza illness that do not trigger gp visits as well as symptomatic/asymptomatic infections, the enrolled patients in those sentinel general practices will be used to randomly select a cohort of persons (stratified by age and ethnicity) and followed through one influenza season. the serologic surveys will measure preand post-season antibodies to circulating influenza strains using relevant vaccine reference strains as antigens; (b) immunology study: a subset of samples from severe, moderate influenza cases, related risk groups and individuals with serologically defined influenza infection are selected for the study of humoral and cellular immune responses ( figure 3) ; and (c) remaining studies: the combined laboratory testing results and metadata collected from these platforms are used to study vaccine effectiveness, interaction between respiratory pathogens, respiratory mortality, risk factors and economic burden and vaccine cost-effectiveness. our innovative study design interconnecting multiple objectives, in addition to exploiting nz's unique healthcare structure, will maximize efficiency and study power. the two surveillance platforms provided specimens and data to serve the nine objectives of shivers. figure 5 shows how each of the objectives is linked to each other and maps data and specimen flows between them. since its conception in 2006, sari surveillance has become a recognized international standard for monitoring hospitalized severe respiratory disease related to influenza and other pathogens. 61 occurred over this time. [65] [66] [67] it was designed to monitor trends in severe influenza disease and to best capture the majority of influenza respiratory disease to estimate the burden of influenza-associated respiratory hospitalizations, and risk factors for severe disease. 63 the initial sari case definition included the symptom onset of acute respiratory illness within 7 days. 61 the shivers results in 2012 showed that a small proportion (7%) of influenza cases had specimens collected 8-9 days after the symptom onset. these specimens only consisted of a small proportion (9%) of total specimens; thus, the cost of testing was minimal (manuscript in preparation). this result was shared with the who global influenza programme. it contributed to a change in the final who sari case definition, with onset shifting from 'within 7 days' to 'within 10 days'. 63 burden and epidemiology shivers allows the estimation of influenza disease burden and risk factors at various levels of severity. firstly, the disease burden of severe influenza is estimated from the hospital surveillance platform. it measured population-based incidence for sari-associated influenza hospitalizations including icu admissions and in-hospital deaths as it provided reliable numerators and denominators, thus without a need for additional healthcare utilization surveys. [68] [69] [70] [71] our first-year findings (30 april 2012 to 28 april 2013) showed that the sari-associated influenza hospitalization rate was substantial with the overall adjusted annual incidence of 54/ 100 000 persons (manuscript in preparation). this rate was similar to us data on influenza-associated hospitalizations during 1979-2001, with an average annual incidence of 36á8/ 100 000. 12 the very young (0-4 years) and elderly (≥65 years) had the highest sari-associated influenza hospitalization rates, consistent with trends identified in international literature, particularly those from developed countries with temperate climates. 10, [72] [73] [74] [75] a high rate of influenza-related hospitalizations and low vaccine uptake (6%) in young children (6 months to 4 years) from shivers led the nz government to change vaccination policy by extending free influenza vaccination to those in this age group who have been hospitalized or have a history of significant respiratory illness. 76 sari surveillance is likely to underestimate the true burden of severe influenza resulting in hospitalization. some patients will present with non-respiratory symptoms or respiratory disease that does not meet the sari case definition, or stay briefly in emergency department. 63, 66 shivers has begun to address this gap; a pilot study in 2012 testing persons with respiratory disease who did not meet the sari case definition showed that a small proportion (6%) of non-sari patients were positive for influenza viruses, compared with 18% of sari cases (manuscript in preparation). future work to expand the case definition to all acute hospital admissions in a sample of very young children will further expand our knowledge of the burden of influenza in this important group potentially protected by maternal immunization. additionally, sari and associated influenza cases will be linked to the hospital discharge data to determine the accuracy and validity of the discharge data by determining proportions of the principal discharge diagnosis code categories that are sari and influenza cases. this will help inform modelling studies of icd-coded data and help provide some validation of these with laboratory-confirmed data. sari surveillance is also likely to grossly undercount the actual number of influenza-associated deaths because only a minority of influenza-related sari deaths are correctly diagnosed, tested and recorded as such. additional influenza deaths resulting from secondary bacterial infections and exacerbation of pre-existing chronic conditions and atypical clinical presentations are not captured. 77 this limitation presents a challenge in accurately measuring influenzarelated mortality. future work on statistical modelling may allow for indirect estimation of 'excess' mortality attributable to influenza in those broad categories such as pneumonia, respiratory or circulatory deaths during influenza seasons. 78 secondly, the disease burden of moderate influenza is estimated using data from shivers ili surveillance. our findings from the 2013 season (29 april to 29 september) showed that the ili-associated influenza consultation rate was about 14 times higher than sari-associated influenza hospitalization rate (manuscript in preparation). additionally, ili-associated influenza consultations and sari-associated influenza hospitalizations showed contrasting socio-demographic patterns: higher rates of ili-associated influenza consultations were shown in preschoolers (aged 1-4 years), school-age children and adults (<65 years), those of asian ethnicity and those from least deprived socio-economic status (ses) groups. this was a different picture from sari surveillance where sari-associated influenza hospitalizations were more frequent in the very young (under 1 year), the elderly, m aori and pacific peoples and those from most deprived ses groups. 79 these results provided insights into the interplay between healthcare access opportunities and related health-seeking behaviours and the differential effect of the predominant strains on various age groups. thirdly, the disease burden of mild influenza not requiring medical consultation (e.g. school/work-related absenteeism) and influenza infection (symptomatic/asymptomatic) will be estimated from the shivers serosurvey. additionally, we will also conduct severity assessment using true numbers of infections as the denominator to calculate case/fatality and case/hospitalization ratios. furthermore, the data on influenza disease burden will allow us to estimate direct medical costs and indirect societal costs (e.g. loss of productivity, loss of earning and loss of life) for the study population and subpopulations. 80, 81 aetiology preliminary results in 2013 identified an under-recognized burden of non-influenza respiratory viruses, particularly rsv and rhinoviruses, in sari and ili cases in nz as we have never had active population-based study on these viruses previously although substantial burden of rsv and rhinovirus has been described elsewhere. 82, 83 ili-associated consultations and sari-associated hospitalizations for rsv and rhinovirus show different socio-demographic patterns (age, ethnicity and ses) from that of influenza. for example, both ili-and sari-associated rsv incidences were similarhigh rates for very young (<1 year and 1-4 years) followed by elderly (≥65 years). this presented a very different agespecific incidence profile from that of influenza (indicated above). this result, together with subsequent multiyear surveillance data, may provide insights on differential effects of various respiratory viruses on the age distribution of the host and disease severity. shivers surveillance platforms provided a systematic opportunity to estimate ve for the prevention of general practice visits and hospitalizations for rt-pcr-confirmed influenza from the same population in the same influenza season. 40, 41 a case test-negative control design is used to estimate annual propensity-adjusted vaccine effectiveness in both hospital and community settings. the data in 2013 showed moderate effectiveness of influenza vaccine against medically attended and hospitalized influenza in nz with 56% (95% ci 34,70) against influenza presenting to general practice and 52% (95% ci 32,66) protection against laboratory-confirmed influenza hospitalization. 41 immune responses shivers surveillance platforms also provided sera and whole-blood samples during acute and convalescent phases of infection to study humoral and cellular immune responses from a subset of severe (n = 39) and moderate (n = 29) influenza cases in 2013. with these samples, and using a combination of serological and immunological assays, we were able to (a) estimate the relative contribution of early adaptive and cellular immune responses to disease severity; (b) identify differences in the immune profiles between these diseases groups; and (c) identify immunological correlates of disease severity in subpopulations. data acquired so far indicate that sari cases may experience a more robust immunologic response during infection (i.e. greater increases in ha-and na-specific antibody titres as well as magnitude of t-cell response). the ability to parse out immunological differences between severe and moderate influenza cases in this pilot cohort highlights the value of adding the active immunology study to the shivers platforms. as nz has a well-characterized socio-demographic distribution (age, sex, ethnicity, deprivation) from population census data, socio-demographic risk factors can be characterized quite easily. shivers will use the results obtained from hospital and sentinel general practice surveillance to disentangle the effect of ethnic and socio-economic gradients. for other more specific risk factors (e.g. host factors such as comorbidities, and environmental factors such as housing conditions), there are limited data available on their distribution in the population. consequently, it is difficult to assess the importance of the risk factor data collected. there are several comparison/control groups such as hospital-based control populations without respiratory illness, serosurvey participants as a control group and sari/ili testnegative controls. these controls could be compared with sari/ili cases to estimate the importance of specific risk factors for influenza infection and related severe/moderate diseases including socio-economic, underlying medical conditions, health intervention, health service utilization, and environmental and behavioural factors. we have established active, prospective, population-based surveillance systems for a wide range of respiratory disease presentations and designed a portfolio of influenza studies based on these platforms. the shivers results during the first 2 years (2012-2013) provided scientific evidence to support change to nz's vaccination policy for young children due to high sari-associated influenza hospitalizations in these children; contribute to the finalization of the world health organization's sari case definition for global influenza surveillance; and contribute in part to vaccine strain selection with vaccine effectiveness assessment in the prevention of general practice visits and hospitalizations for laboratoryconfirmed influenza. in the next 3 years (2014-2016), this project will continue to help us to understand (1) the burden of influenza infection including symptomatic/asymptomatic infection and mild disease not requiring medical consultation; (2) influenza risk that considers organism, host and environmental factors; (3) the impact of viral-viral and viral-bacteria co-infections on clinical disease and severity; and (4) the nature of responding adaptive immune responses in determining individual's risk of acquiring influenza virus infection. over 5 years, we hope this project will shed more light on the burden of influenza and other respiratory pathogens in our study population and subgroups and estimate key epidemiologic parameters such as relative rates of infection, clinical disease, general practice visits and hospitalizations as well as risk factors for illness, effectiveness of vaccination, mechanisms of immunity and monitoring for new influenza viruses with pandemic potential such as a(h7n9) and other emerging viruses (e.g. mers-cov) and provide a framework for timely assessment of severity which is essential in an event of emergence of these pathogens. the platforms established here are relevant not only for new zealand policy, but also for the region and the world. it will provide robust systematic virologic, epidemiologic and vaccine effectiveness data on circulating pandemic or seasonal influenza viruses at a time when circulation in the northern hemisphere is low. this could provide valuable information on all emergent respiratory pathogens that have some winter seasonality. moreover, the data elements on a range of disease severities collected in the same population at the same time will generate epidemiologic parameters that maybe broadly generalizable and translatable to similar developed, southern and northern temperate countries worldwide. this will help enormously to better understand more basic surveillance data and to extrapolate those data in models to plan and predict influenza behaviour, generate burden estimates, model the impact of seasonal influenza vaccination to support more global use and better prepare for the next pandemic. in summary, shivers is expected to provide extensive data to guide improved methods for disease surveillance; improve clinical case management, early detection and optimization of laboratory diagnosis; inform vaccine strain selection and vaccine development; guide targeted vaccination strategies for population and subgroups; understand host immune responses and identify better immune diagnostic markers. korrapadu, louise optland, cecilia dela cruz. special thanks to it staff and sari surveillance participants. also, a special thanks to dr dean erdman from gastroenteritis and respiratory viruses laboratory branch, the u.s. centers for disease control and prevention who provided the real-time pcr assay for non-influenza respiratory viruses. support in kind is provided by the nz ministry of health. report of the review committee on the functioning of the international health regulations (2005) in relation to pandemic (h1n1) pandemic preparedness and response-lessons from the h1n1 influenza of 2009 surveillance for the 2009 pandemic influenza a (h1n1) virus and seasonal influenza viruses -new zealand influenza surveillance and immunisation in new zealand influenza surveillance in new zealand in 2005 pandemic influenza a(h1n1)v in new zealand: the experience from increasing incidence of serious infectious diseases and inequalities in new zealand: a national epidemiological study incidence of influenza in healthy adults and healthcare workers: a 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illness: systematic review and meta-analysis the second wave of 2009 pandemic influenza a(h1n1) in new zealand a novel risk factor for a novel virus: obesity and 2009 pandemic influenza a (h1n1) critical care services and 2009 h1n1 influenza in australia and new zealand risk factors and outcomes among children admitted to hospital with pandemic h1n1 influenza pandemic influenza a (h1n1) in pregnancy: a systematic review of the literature household crowding a major risk factor for epidemic meningococcal disease in auckland children acute rheumatic fever associated with household crowding in a developed country tuberculosis associated with household crowding in a developed country household transmission of the 2009 pandemic a/h1n1 influenza virus: elevated laboratory-confirmed secondary attack rates and evidence of asymptomatic infections comparative epidemiology of pandemic and seasonal influenza a in households impact of pollution, climate, and sociodemographic factors on spatiotemporal dynamics of seasonal respiratory viruses implementing hospitalbased surveillance for severe acute respiratory infections (sari) caused by influenza and other respiratory pathogens in new zealand interim global epidemiological surveillance standards for influenza generic protocol for influenza surveillance global epidemiological surveillance standards for influenza who regional office for europe guidance for influenza surveillance in humans validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in north india what are the most sensitive and specific sign and symptom combinations for influenza in patients hospitalized with acute respiratory illness? results from western kenya lessons from a one-year hospital-based surveillance of acute respiratory infections in influenza study in southern hemisphere ª 2015 the authors. influenza and other respiratory viruses published by berlin-comparing case definitions to monitor influenza incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh estimation of the national disease burden of influenza-associated severe acute respiratory illness in kenya and guatemala: a novel methodology the cost of influenza in thailand the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses the underrecognized burden of influenza in young children burden of seasonal influenza hospitalization in children adult hospitalizations for laboratory-positive influenza during the virologically confirmed populationbased burden of hospitalization caused by influenza a and b among children in hong kong high influenza hospitalisations lead to extension of free influenza vaccination to young children estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study influenza-associated primary care consultations and hospitalisations show contrasting socio-demorgraphic patterns medical and economic burden of influenza in the elderly population in central and eastern european countries influenza cost and cost-effectiveness studies globally-a review disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden the shivers investigation team (listed in an alphabetic order) the shivers project is funded by us cdc (1u01ip000480-01). the project is a 5-year research cooperative agreement between the authors declare that they have no competing interests. key: cord-340678-2e2s1gof authors: skowronski, danuta m; zou, macy; clarke, quinten; chambers, catharine; dickinson, james a; sabaiduc, suzana; olsha, romy; gubbay, jonathan b; drews, steven j; charest, hugues; winter, anne-luise; jassem, agatha; murti, michelle; krajden, mel; de serres, gaston title: influenza vaccine does not increase the risk of coronavirus or other non-influenza respiratory viruses: retrospective analysis from canada, 2010-11 to 2016-17 date: 2020-05-22 journal: clin infect dis doi: 10.1093/cid/ciaa626 sha: doc_id: 340678 cord_uid: 2e2s1gof influenza vaccine effectiveness against influenza and non-influenza respiratory viruses (nirv) was assessed by test-negative design using historic datasets of the community-based canadian sentinel practitioner surveillance network (spsn), spanning 2010-11 to 2016-17. vaccine significantly reduced the risk of influenza illness by >40% with no effect on coronaviruses or other nirv risk. influenza vaccine effectiveness (ve) is commonly estimated through the test-negative design (tnd), an observational method that compares the odds of vaccination among influenza test-positive cases to influenza test-negative controls through the odds ratio (or), with ve derived as (1-or)x100%. the core prerequisite for valid ve estimation by tnd is that vaccine has no effect on alternate etiologies of the same clinical syndrome included in the control group. comparison of per-protocol and tnd analyses of several large randomized-controlled trial (rct) datasets involving >6000 participants has verified this prerequisite for influenza ve estimation, with the or for influenza vaccine effect against non-influenza causes of influenza-like illness (ili) approximating 1.0 (ve approximating zero) [1] . if, however, influenza infection induces immunity that is cross-protective against noninfluenza respiratory viruses (nirv)(e.g. through non-specific innate immunity), then vaccination that effectively prevents influenza may indirectly result in greater nirv risk among vaccinated compared to unvaccinated individuals. cowling et.al. hypothesized such vaccine interference with infection-induced immunity to explain a significant four-fold increased nirv risk among 69 children randomized to receive the 2008-09 influenza vaccine compared to 46 children receiving placebo [2] . that small rct, however, included just 23 nirv cases and was under-powered to show ve against influenza, as required by the interference hypothesis [2] . conversely, in tnd analysis of six study seasons (2004[5, 6] . three other coronaviruses have been associated with more severe illness including sars-cov, mers-cov, and more recently sars-cov-2, the latter emerging in late 2019 and responsible for the ongoing pandemic of coronavirus disease 2019 (covid-19) [5, 6] . wolff's findings for seasonal coronaviruses, coincidentally published in january 2020, have triggered concern that influenza vaccination may detrimentally affect covid-19 risk [4] . here, we use historic datasets of the community-based canadian sentinel practitioner surveillance network (spsn) to assess the association between influenza vaccine and nirv risk, notably seasonal coronaviruses. we retrospectively applied tnd analysis to canadian spsn influenza ve study specimens collected during the 2010-11 to 2016-17 seasons[7], when specimens were tested for both influenza and nirv. specimens were included if collected november-april from consenting patients ≥1-year-old who presented within 7days of ili onset to a sentinel practitioner in the provinces of alberta, british columbia, ontario or quebec. ili was m a n u s c r i p t defined by fever and cough plus ≥1 of arthralgia, myalgia, prostration or sore throat. fever was not required for adults ≥65-years-old after 2010-11. specimens were tested for influenza and nirv at provincial public health reference laboratories by reverse-transcriptase-polymerase-chain-reaction (rrt-pcr) and/or commercial multiplex rt-pcr assays(supplementary_material_1). ontario panels did not include the hku1 coronavirus. during seasons for which ontario (2015-16) and alberta (2015-16/2016-17) did not perform multi-plex testing they were excluded from influenza and nirv analyses. participants who self-reported influenza vaccination≥2weeks before ili onset were considered vaccinated. participants with unknown timing or self-reporting vaccination<2weeks before ili onset were excluded; the latter were also explored as unvaccinated (per wolff) [4] . ors compared influenza vaccination rates among influenza and nirv test-positive cases relative to test-negative, pan-negative and nirv-positive controls. influenza test-positive specimens were excluded from nirv analyses. nirv cases were assessed in combination and separately grouped as coronaviruses, entero-/rhinoviruses(ev/rv), hmpv, parainfluenza, and rsv. coxsackie-/echovirus, adenovirus, and bocavirus estimates are not presented owing to limited detection but are included in combined nirv analyses. co-infections across nirv groupings were included among controls but not cases; in sensitivity analyses cases also included co-infections. all models adjusted for age, province, specimen-collection interval, calendar-time, and season; participants missing information for any of these covariates were excluded. comorbidity and sex were also assessed in sensitivity analyses but had no confounding effect. a c c e p t e d m a n u s c r i p t in this seven-season analysis by the canadian spsn, influenza vaccine was protective against medically-attended ili due to influenza viruses, significantly reducing the risk by >40%. conversely, influenza vaccine had no effect on non-influenza causes of ili, with the likelihood of vaccination among nirv cases relative to test-negative controls approaching unity. in particular, influenza vaccine did not affect seasonal coronavirus risk. our findings provide reassurance against the speculation that influenza vaccine may negatively affect covid-19 risk. addressing such speculation is important to maintain influenza vaccine coverage through the ongoing covid-19 pandemic. in assessing wolff's paper we identified a major methodological problem to account for his unexpected findings [4] . in combined nirv analysis, relative to pan-negative controls, wolff adjusted for age and excluded specimens that tested influenza-positive. in that analysis, shown in his table 3 , the or approached unity indicating no vaccine effect as expected. conversely, in unadjusted analysis of individual nirv outcomes (e.g. coronaviruses) wolff retained influenza test-positive specimens in nirv test-negative control groups, thereby violating the core prerequisite for valid tnd analysis. in the context of effective influenza vaccine, influenza cases would have lower likelihood of vaccination; as such, their inclusion would systematically reduce the proportion vaccinated in the control group and thereby inflate ors comparing vaccine exposure between nirv cases and controls. we illustrate the impact of this bias in supplementary_material_3, where we have re-analyzed wolff's data as well as our own, comparing influenza vaccine effect against nirv when influenza testpositive specimens are properly excluded (as per tnd prerequisite) or improperly included (as per wolff [4] ) within the control group. in both data sets and for all nirv, ors for influenza vaccination are biased higher when influenza cases are erroneously included in the control group. a c c e p t e d m a n u s c r i p t as for any observational design, random variation, bias and confounding may influence tnd findings. our seven-season analysis was based on substantial sample size, standardized ili testing indication, and multi-variate analysis to address those concerns; whereas, wolff relied upon a single season, general laboratory submissions, and univariate analysis, despite evidence in his dataset for confounding by age. the importance of adjustment for age and other potential confounders is reinforced by our analyses in which several unadjusted but no adjusted ors significantly differed from one(table_1;supplementary_table_s2a). vaccine status was self-reported in our study but recorded before specimen testing, minimizing differential misclassification. assays varied by province and season. two spsn provinces did not conduct nirv testing during 1-2 of the study seasons and hku1 was omitted from the coronavirus panel of one province all seasons. however, hku1 comprised a small proportion of coronavirus detections in other spsn provinces (15%;53/349) and findings were robust across nirv outcomes and in sensitivity analyses addressing variation in provincial contribution (not displayed). finally, although we did not find evidence for vaccine interference, population surveillance signals elsewhere suggesting cross-pathogen immunological interactions still warrant immuno-epidemiological investigation [3, 8, 9] . in conclusion, our findings provide reassurance that protective influenza vaccination does not negatively affect nirv risk, including coronaviruses. valid tnd estimates require that etiologies against which vaccine is effective are specifically excluded from the testnegative control group, and this applies also when exploring vaccine effects on non-vaccine target pathogens. these methodological insights have important implications for other tnd applications, including future evaluations of influenza vaccine effects against covid-19, and vice-versa when sars-cov-2 vaccines become available. a c c e p t e d m a n u s c r i p t the authors gratefully acknowledge the contribution of sentinel sites whose regular submission of specimens and data provide the basis of our analyses. we also wish to acknowledge those who provided administrative and coordination support and those who provided laboratory and technical support in each participating province. the views expressed herein do not necessarily represent the view of the public health agency of canada. funders had no role in data analysis or interpretation or in the decision to publish the test-negative design : validity, accuracy and precision of vaccine efficacy estimates compared to the gold standard of randomised placebo-controlled clinical trials increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness. clinical infectious diseases influenza vaccination and respiratory virus interference among department of defense personnel during the 2017-2018 influenza season. vaccine coronavirus infections in children including covid-19. an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection and association of antibody responses with severity of disease m a n u s c r i p t a c c e p t e d m a n u s c r i p t a c c e p t e d m a n u s c r i p t key: cord-336493-ggo9wsrm authors: huang, stephen s. h.; lin, zhen; banner, david; león, alberto j.; paquette, stéphane g.; rubin, barry; rubino, salvatore; guan, yi; kelvin, david j.; kelvin, alyson a. title: immunity toward h1n1 influenza hemagglutinin of historical and contemporary strains suggests protection and vaccine failure date: 2013-04-23 journal: sci rep doi: 10.1038/srep01698 sha: doc_id: 336493 cord_uid: ggo9wsrm evolution of h1n1 influenza a outbreaks of the past 100 years is interesting and significantly complex and details of h1n1 genetic drift remains unknown. here we investigated the clinical characteristics and immune cross-reactivity of significant historical h1n1 strains. we infected ferrets with h1n1 strains from 1943, 1947, 1977, 1986, 1999, and 2009 and showed each produced a unique clinical signature. we found significant cross-reactivity between viruses with similar ha sequences. interestingly, a/fortmonmouth/1/1947 antisera cross-reacted with a/ussr/90/1977 virus, thought to be a 1947 resurfaced virus. importantly, our immunological data that didn't show cross-reactivity can be extrapolated to failure of past h1n1 influenza vaccines, ie. 1947, 1986 and 2009. together, our results help to elucidate h1n1 immuno-genetic alterations that occurred in the past 100 years and immune responses caused by h1n1 evolution. this work will facilitate development of future influenza therapeutics and prophylactics such as influenza vaccines. t he major challenge in regard to influenza surveillance and management is the propensity of the influenza virus to mutate, altering its immunogenic properties thereby allowing it to evade immune recognition and cause disease. influenza a is classified according to the specific combination of its two surface molecules, hemagglutinin (ha) (h1 -h17) and neuraminidase (na) (n1 -n9) isotypes 1-3 and its diversity is attributed by two mechanisms: genetic mutation or by gene reassortment 4, 5 . typically genetic mutation is responsible for seasonal influenza outbreaks and the emergence of influenza pandemics is a consequence of gene reassortment among different strains and subtypes [4] [5] [6] . since the extensively documented influenza pandemic in 1918-20, there have been a total of five influenza pandemics that have resulted in millions of deaths worldwide, where the fatality rate has reached more than 2.5% as in the case of 1918 pandemic 5, 7 . in 1957, h2n2 surfaced and replaced h1n1 in the human population until 1977 when h1n1 resurfaced, leading to co-circulation of the 2 influenza subtypes. compared to h3n2 8 , the h1n1 subtype is believed to have a lower rate of antigenic drift which is associated with a smaller amount of mutations leading to amino acid changes 9 . importantly the evolutionary behaviours of h1n1 and h3n2 are divergent leading to a dynamic and ever changing influenza climate in the human population where one virus subtype typically dominates over the other 8, 9 . throughout the 100 year history of h1n1, the specific clinical parameters and immunogenic response to the genetic drift of h1n1 remains to be clarified. subsequent to the 1918 h1n1 pandemic were several h1n1 epidemics occurring from the 1920's to the late 1950's [9] [10] [11] which was followed by a 20 year disappearance and a re-emergence in the 1970's 12 . in 1947 a significant antigenic change transpired in the h1n1 virus creating strain distinct from previous 1943 viruses 13 . the new h1n1 virus termed ''a-prime'' was relatively mild although widespread (a pseudopandemic) and hypothesized to be a reassortant from two distinct h1n1 strains 9, 13, 14 . an unusual h1n1 virus emerged in 1951 that was associated with severe disease: also thought to be a reassortant virus with genes from novel viruses and older h1n1 segments from the 1940's 9 . succeeding a 20 year h1n1 disappearance, two h1n1 epidemics of interest came about, including the 1977 children's pandemic and a swine flu epidemic in 1976 that was feared to have pandemic potential and led to a massive public vaccination strategy 14 . the 1977 epidemic had limited infectivity to the immunologically naïve younger population (persons ,25 years of age) which is thought to be due to the similar circulating h1n1 viruses of the 1950's 14 . in 2009 a substantial change in the h1n1 virus occurred, unlike previous modifications, which allowed the virus to spread rapidly throughout the globe and prompted the who to declare a pandemic on 11 june 2009 15 . genomic analysis determined the virus was of swine origin and contained a triple reassortant of swine, human and avian influenza a genes 16 . unlike previous contemporary seasonal influenza outbreaks, the 2009 h1n1pdm had age-related disparities in the frequency and severity of infection where the older age groups were less susceptible to the disease 17, 18 . furthermore, these differences in age-related severity are hypothesized to be due to previous exposure to older h1n1 viruses with similar antigenic epitopes 17, 18 . after influenza exposure, the body generates antibodies against the specific influenza strain it has encountered. anti-ha production is often associated with immunity to the same or homologous influenza strains and some antibodies have virus neutralizing ability blocking viral entry to the host cell 1, 19 . importantly, previous exposure to influenza viruses influences how the body will respond to a subsequent influenza infection of the same or different genetic subtype 20 . to date, little is known about the immune and clinical response to h1n1 influenza viruses of the past 100 years and how the h1n1 subtype genetic drift and shift affected immune crossreactivity. previously nelson et al., conducted a large scale genetic analysis of 71 h1n1 sequences to determine the evolutionary history of this virus since 1918 9 . in our present study, we investigated the clinical characteristics and immune cross-reactivity of significant h1n1 influenza strains in the past 100 years in ferrets to determine the immunogenicity of important h1n1 viruses. we infected ferrets with historical h1n1 strains from 1943, 1947, 1977, 1986, 1999, and 2009 and monitored them for a 14-day time course to determine the clinical picture of each influenza strain infection and immune crossreactivity. our findings clarify the influenza clinical picture of historical h1n1 strains and the help to elucidate the antigenic alterations of h1n1 that occurred in the past 100 years. the work here will facilitate the understanding of the immune response toward h1n1 and with future work may aid the development of future influenza therapeutics and anti-viral treatments. ferrets infected with historical h1n1 strains show mild clinical symptoms. the ferret, mustela putorius furo, is a superlative animal model for respiratory infections and the influenza ferret infectome has recently been published [21] [22] [23] [24] [25] [26] [27] [28] [29] . ferrets show respiratory illness similar to humans and clinical features of disease are easily observed where fevers can persist days following infection of viruses such as 2009 h1n1pdm influenza 21, 23, 30 . as well as fever, nasal discharge and sneezing can also be observed in animals infected with influenza viruses 21 these influenza a viruses were chosen due to their emergence and influence in h1n1 genetic history (fig. 1 , strains used in this study are marked with an asterisks) as covered in the introduction. following infection, ferrets were monitored for body temperature, weight, inactivity level, sneezing and nasal discharge from each group were observed daily until day 14 post-infection (pi). infection by all strains produced an increase in temperature; the normal range for ferret temperature is indicated by the shaded area of each graph 23 ( supplementary fig. s1a ). the pandemic h1n1 strain ny/09 induced the greatest fever on day 2 to a temperature of 104% (of baseline). ncal/99 and marton/43 infection also caused a high temperature of 103% from baseline, which peaked on day 1 and day 2 pi, respectively ( table 1) . ussr/77 and taiwan/86 had moderate fevers and fm/47 had the smallest increase in temperature reaching only 101% above baseline ( table 1) . analysis of weight loss showed that animals infected with of all viruses except pandemic ny/09 were able to recover to original weight or greater following infection (supplementary fig. s1b and table 1 ). ny/09 infected ferrets had the most significant weight loss compared to normal weight fluctuations (shaded area) 23 which peaked at 91% of baseline weight on day 6 and day 7. ussr/77 and ncal/99 reached less than 95% and 95% of baseline weight, respectively, on day 2 pi ( table 1) . infection with taiwan/86 produced the smallest amount of weight loss and animals infected with fm/47 did not lose any weight at all ( table 1) . secondary clinical signs were also measured and analysed for all infections, including nasal discharge, sneezing, and inactivity level (supplementary table s1 ). ussr/77 infected ferrets had the highest amount of nasal discharge and ny/09 ferrets had the greatest amount of sneezing and lethargy. taken together, analysis of the complete clinical signs for each h1n1 strain infection suggested a unique clinical picture for each strain: ny/09 infection had the most severe with significant increase in temperature and an unrecovered weight loss compared to mildest strain, fm/47, which did not produce any weight loss and only a slight increase in temperature. analysis of ha immunogenicity in historical h1n1 strains. the ha protein, a homotrimeric protein that functions in viral entry into host cells 31 , and antibodies reactive toward ha have been associated with host resistance and a decrease in disease severity [32] [33] [34] . it has been shown that 60% of antibodies produced during an influenza infection are reactive toward the ha protein 35 . after determining the clinical signatures of ferrets infected with historical and contemporary h1n1 viruses, we went on to analyze the aspects of h1n1 ha immunogenicity. the hemagglutination inhibition (hi) assay, is a common assay used to determine the reactivity and/or amount of antibody to the ha protein produced during a viral infection and the cross-reactivity of antibodies raised toward these specific historically important h1n1 viruses has not been investigated. to determine the crossreactivity produced by infection among the historical and contemporary influenza strains, we used the harvested antisera from each ferret strain infection and perform hi assays with our panel of table s2) . interestingly, ferret anti-sera toward the marton/43 virus showed hi with its own virus and with the older pr/34 but not with its nearest chronological neighbour fm/47 (fig. 2a) . fm/47 and ussr/77 antisera recognized its respective virus and each other ( fig. 2b and c) . taiwan/86 and 2009 h1n1pdm (ny/09 or cal/09) antisera both only reacted with their respective viruses ( fig. 2d and h). ncal/99 antisera only showed hi with its own ncal/99 virus (fig. 2e) . unexpectedly, the si/06 and bris/07 antisera were able to inhibit the ncal/99 virus ( fig. 2f and g) . furthermore, si/06 antisera also cross-reacted with the bris/07 virus and the bris/07 antisera recognized the si/06 virus. taken together, these results showed antigenic cross-reactivity to infection among historical and contemporary h1n1 influenza strains. this data suggested similarities of the host immune response between marton table s4 ). importantly, no detection was observed for the tawain/ 86 virus and earlier viruses with the who h1n1 seasonal control. taken together, this work suggested that the who seasonal h1n1 antisera is only capable of detecting infection from recent seasonal h1n1 strains. we investigated the phylogenetic relationship and sequence homology of the ha gene among the h1n1 viruses used in this study and included other strains implicated in important historical h1n1 outbreaks. the temporal structure of phylogenetic tree was comprised of 3 groups separated by long braches (fig. 3a) . the 1918 pandemic strains and 2009 pandemic isolates each formed their own distinct cluster. the middle cluster contained 10 epidemic isolates. within this cluster, fm/47 was closer to ussr/77 than marton/43 which was more similar to pr/34. the 3 latest strains of seasonal h1n1, ncal/ 99, si/06 and bris/07, were positioned as a clade which was separated from the early stains by a long branch and clustal alignment of these three viruses ( supplementary fig. s2 ) showed several amino acid changes from ncal/99 and conserved between si/06 and bris/07: a3v, t99k, y111h, k157e, v182a, r224k, w268r, t283n, and n489d. we calculated the protein homology by using the most variable region of the ha gene (ha135-295) which covers most of the viral surface and sequence change in this area has a strong impact in immunogenicity 36 . we found that 94% and higher homology scores led to hi cross-reactivity in all the cases. on the other hand, crossreactivity with lower homologies of 91-93% may or may not take place (fig. 3b) . taken together, our results suggested an important relationship and cross-reactivity between fm/47 and ussr/77 as well as the ncal/99, si/06 and bris/07 group that can be related to the genetic ha sequence. further analysis of other influenza genes may also be important to the understanding of h1n1 evolution. the h1n1 influenza subtype is a significant viral agent affecting public health that has been responsible for 3 pandemics and several influenza epidemics globally. here we investigated the clinical response and subsequent immunogenicity following infection with historically and contemporarily relevant h1n1 strains in ferrets. importantly, we showed that infection with each strain produced a unique clinical signature which may be indicative of disease that affected humans. we also found significant cross-reactivity between viruses with similar ha aa sequences which can be extrapolated to protection or no protection following a subsequent infection with another h1n1 strain. these results are the first to compare the these results add to the understanding of how the immune system responds to an evolving virus. in the current study we analyzed the clinical parameters of evolutionary h1n1 strain infection in ferrets which has not been previously compared experimentally. our results of the evolutionary h1n1 strains showed that infection with the fm/47 resulted in the 14 . these records support our findings, where animals infected with fm/47 had the lowest percent nasal discharge, mildest fever and was the only virus infection which did not lead to weight loss. furthermore, ussr/77 infection produced a moderate disease comparable to the reports of a mild clinical disease 37 . our analysis of the clinical course of taiwan/86 infection in ferrets showed a moderate disease consisting of a mild biphasic temperature increase and minimal weight loss. these observations were in agreement with clinical disease reported during the 1986 naval base outbreak of influenza which there was no deaths but patients described fever, non-productive cough, sore throat and myalgia 38 . furthermore, in accordance with our ny/09 data which had the most significant temperature fluctuations and weight loss, analysis by shrestha and colleagues demonstrated that h1n1pdm was more severe than the previous circulating seasonal influenza viruses 39 . evolutionary relationships among historical influenza h1n1 strains were conducted in mega5 using the maximum likelihood method and 500 bootstrap replications (a). antibody cross-reactivity (determined by hi) among influenza strains is indicated by using the same colors. the numerical values displayed next to the branches indicate the percentage of bootstrap replicates in which the associated sequences clustered together. hemagglutinin protein sequence homology among several influenza strains (b). influenza strains were grouped according to the hi cross-reactivity observed in the serum from previously-infected ferrets (groups a through f). alignments of protein sequences were performed using the highly variable ha135-295 region, which belongs to the ha-rbd area. the influenza virus was first identified in 1932 and since then the epidemiology and phylogentic analysis of subsequent influenza epidemics and pandemics have been extensively investigated 9, 14, 40 . importantly, our results build on the previous investigations as we report the immunogenic evolution of the ha protein of significant pandemic and epidemic strains. the epidemic isolates examined by our phylogenetic analysis showed the 1918 and h1n1pdm 2009 strains to comprise each their own cluster and the seasonal strains ranging from 1934 to 2007 to cluster together. as expected by chronological analysis, our results showed marton/43 to cross-react with its predecessor pr/34 which was in agreement with the phylogenetic analysis that placed the ha of marton/43 close to pr/34 in the tree. extensive phylogenetic analysis preformed by nelson mi and colleagues suggested the 1947 strain to be a reassortant from two previous h1n1 viruses combining the pb1, na and m segements from a 1943 virus with the pb2, pa, ha, np and ns segments from an unknown h1n1 virus 9 . furthermore, they showed that the ha1 region of the 1947 ha was significantly different from the ha of the earlier 1940's strains 9 . in accordance with this analysis, our results showed that the fm/47 antisera did not inhibit hemagglutination with the earlier pr/34 and marton/43 viruses and the marton/ 43 antisera did not cross-react with the fm/47 virus. the inability for immunity induced by marton/43 virus infection to target the fm/47 virus compliments the influenza vaccine performance in the human population of the 1940's as well as previously published studies 41 . specifically, a related 1943 h1n1 virus strain (a/weiss/43), as well as a/pr/8/34 and b/lee/40 were used in the influenza vaccine preparation which was completely ineffective during the '47 influenza outbreak 14, 41 . although fm/47 antisera did not recognize the earlier h1n1 strains, it did cross-react with the ha molecule of the ussr/77 virus as did the ussr/77 antisera with the fm/47 virus. in support of these findings, the 1977 epidemic was thought to be the resurfacing of the fm/47 strain 9 . the ussr/77 outbreak was known as 'the children's pandemic' since only persons younger than 26 years old were affected 14 . during this time, older individuals were considered primed for the 1977 outbreak strain by previous infections from the 40's and 50's 37 which is supported by our immunological evidence. together, our results, the phylogenetic analysis and the epidemiological records suggest the triggering of a unique immune response following infection with marton/43 and fm/47 and a similar response from infection with the related strains fm/47 and ussr/77. in the fall of 1986 a significant outbreak of influenza occurred on a us naval base which was found to be caused by strains that had been circulating in asia since the spring of that year 38, [42] [43] [44] . taiwan/86 was isolated from this outbreak and genomic analysis of its ha have suggested that the taiwan/86 virus had evolved from viruses circulating in the early 1980's in hong kong 45 . furthermore, it has also been suggested that this virus was the product of a reassortment event that had occurred between two h1n1 viruses 9 . our results showed that the taiwan/86 strain shared lower homology to 99, 06 and 07 viruses seen on our phylogenetic tree. the immunogenic findings showed antisera produced from ferret infection with taiwan/86 was not able to inhibit hemagglutination with the other viruses on our virus panel, which compliments the literature describing the 1986 strain. the influenza vaccine in 1986 included the a/chile/1/83 influenza strain 38 . interestingly, during the 1986 us naval base outbreak, the soldiers who were vaccinated were poorly protected from infection with taiwan/1986 38 . furthermore, it was also shown that antibodies produced by vaccination with the a/chile/1/83 vaccine preparation were not able to provide hemagglutination inhibition toward the newly arisen taiwan/86 and taiwan/86-like viruses 44 . from this evidence, it was then recommended that the taiwan/86 strain be included in the subsequent influenza monovalent vaccine preparation 42, 44 . our findings together with the previous reports on the taiwan/86 outbreak suggest that the 1986 viruses were an antigenically unique group of strains that induced a specific immune response important when considering the evolution of h1n1 immunity. it can be argued that the h1n1 virus underwent minor genetic drift in the 10 year span from 1999 to 2009 which can be evidenced by the consistent inclusion of the ncal/99 in the influenza vaccine preparation 9, 46 . our genetic analysis showed the three contemporary h1n1 seasonal viruses, ncal/99, bris/07 and si/06, to be proximal to each other on the phylogenetic tree with a long separation from the previous taiwan/86 virus and in a distinct cluster from the 2009 h1n1 pandemic viruses. moreover, bris/07 and si/06 were closer to each other than they were to ncal/99. interestingly, the antisera generated by infection with ncal/99 did not cross-react with the other two contemporary seasonal h1n1 viruses although the si/06 and bris/07 antisera recognized the ncal/09 virus. these results suggested there to be an amino acid change in bris/07 and si/06 from ncal/99 that may have resulted in a new antigenic site that was not originally present in ncal/99 strain. to investigate this contention we generated a clustal alignment of these viruses which showed numerous amino acid changes between bris/07 and si/06 sequences with ncal/99. these results may indicate the gradual yet still present drift of the h1n1 virus in this time period. as well, the immunogenic evolution seen in our analysis may question the consistent usage of the ncal/99 virus in influenza vaccine preparations from 2000-2001 to 2006-2007 9,47 . the most significant emergent in h1n1 history since 1918 ensued in late spring 2009 and was declared a pandemic shortly after 15 . the ha from newly emerged h1n1 virus was identified to be similar to the ha of viruses isolated from north american swine which had recent and historical precedence 48 . this virus was designated 2009 h1n1 pandemic (h1n1pdm) and was shown to be a triple reassortant virus containing genes from swine, human and avian influenza a viruses 16 . in our phylogenetic analysis it was clearly evident that the triple reassortant viruses clustered far from the previously circulating seasonal h1n1 viruses in agreement with similar alignments. furthermore, hi assays performed using ferret antisera produced raised against the ny/09 h1n1pdm antisera was not able to recognize the ha of any other viruses. these results are supported by previous published findings that determined the cross-protection initiated following infection with h1n1pdm which was preceded by infection with contemporary seasonal h1n1 did not occur through an ha targeted antibody response 20 . interestingly, although an h1n1 component had been included in the seasonal influenza vaccine preparations since 1977 49 , antigen directed pre-existing immunity to 2009 h1n1pdm was only detected in the elderly suggesting the majority of the current human population was naïve to the this novel virus 50, 51 . cross-reactive antibodies found in the elderly population suggested that protective immunity was induced from exposure to an older h1n1 virus with antigenic similarities to 2009 h1n1pdm 25,50-52 . our study directly tested the ability of infection with older h1n1 strains from the years 1943 to 1947 to bind the ha molecule of the 2009 h1n1pdm virus. against current hypotheses, we found that the immune production of antisera from infection with viruses originating from the 1940's were not able to recognize the ha of the novel pandemic viruses. likewise, the h1n1pdm antisera did not detect the panel of viruses ranging from 1934 to 2007. recently, the effect of priming ferrets with historical h1n1 viruses on the immune response of secondary infection the h1n1pdm was described 53, 54 . these studies addressed distinct immunological questions as our study utilizing a different set of h1n1 viruses. these priming studies complimented our study as the historical h1n1 strains used were not the same as ours. the purpose of the previous studies was to determine protection from priming infection/infections whereas we queried immune detection, cross-reactivity and clinical analysis. importantly, a significant issue that remains is the affect of age on the outcome of infection/ www.nature.com/scientificreports scientific reports | 3 : 1698 | doi: 10.1038/srep01698 sequential infection with the historical and contemporary viruses. historical accounts mention clinical pathology among age groups but do not have a comprehensive analysis of the comparative severity 14, 38, 49, 55 . as well, it is not know how the aging immune system responds to the evolving influenza virus. although a protective response was seen in priming studies in adult animals, it is not know if protection of the same magnitude would remain during age related immunosenescence or if the clinical response in the aged would be similar as we have shown in this study. influenza a h1n1 and influenza a h3n2 have varying patterns of antigenic and genetic evolution where specifically h1n1 has a lower rate of acquired mutations 8, 9 . these evolutionary behaviours are divergent leading to a dynamic and ever changing influenza climate in the human population where one influenza a subtype typically dominates over the other. importantly, understanding the interplay between the h1n1 and h3n2 infection evolution remains a significant hurdle in influenza reasearch 8, 9 . nelson and colleagues investigated the genetic evolution of the h1n1 virus by analyzing the evolution of the whole virus as well as the individual viral segments from strains since 1918 9 . in this study the authors were able to identify instances where divergent clades co-circulated as well as events of intra-h1n1 subtype reassortment 9 . previously, smith and colleagues compared the antigenic and genetic evolution of the h3n2 virus from strains originating in 1968 to 2003 and found that antigenic and genetic evolution showed differing patterns 8 . antigenic evolution was punctuated where the strains formed clusters and genetic change was more even and gradual as time increased 8 . here we sought to elucidate the antigenic evolution of the h1n1 virus. as with the h3n2 virus 8 , we found the antigenic change did not vary evenly by year but instead corresponded to genetic changes in the ha molecule. these studies are significant for elucidating the intricacies of influenza evolution as the continuous change of the influenza virus is the greatest challenge to the management of disease and containment of further outbreaks. a better understanding of the evolution of the influenza virus and the interplay between the h3n2 and h1n1 strains will improve the design of prophylactics such as by guiding vaccine strain selection for yearly trivalent influenza vaccine or may even give insight to the development of a universal influenza vaccine. more work is needed to understand the true nature of influenza virus dynamics and evolution. animal models remain the most appropriate mode for the methodical scientific investigation of human influenza virus pathogenesis and the testing of influenza prophylactics and therapeutics. currently, the ferret is thought to be the most suitable animal model for respiratory influenza virus infection 56 since ferrets are physiologically susceptible to wild type influenza viruses due to a similar respiratory tract and are able to transmit the virus once infected. as mentioned previously, ferrets display similar clinical disease as humans following influenza infection which include fever, weight loss, and sneezing and these positive features have been well discussed previously 21, 24, 26, 30, 56 . as well, the ferret has been shown to have similar features as human in regard to ifn pathway function and is potential model for the study of human ifn-gamma signalling 24, 57 . although the ferret is an appropriate model, it is imperative to be mindful of the disparities between animal models and the nature human infection to have an accurate assessment of research findings especially from ferret-influenza studies. during experimental influenza infections in animal models, a specific and known amount of virus is administered to the animal in a controlled manor, usually by intranasal inoculation. modeling the infectious behaviour of a virus is more predictable when the infection route and dose are controlled. this is in contrast to natural infection where humans are infected with an unknown amount of virus in an unknown route, which must be considered when extrapolating results from animal model studies. most significantly, ferrets as well as other animal models used in influenza studies are typically 'specific pathogen free' (spf). in this case, the ferrets are determined to be influenza free prior to study initiation. unlike humans who have been previously exposed to various subtypes of influenza viruses, ferrets used in influenza studies are completely naïve to the virus. the nuances of the immune response subsequent to multiple influenza infections, such as the phenomenon of original antigenic sin 58 , has only started to be elucidated in the ferret model 53 and much work is still required to have a full understanding of the effect of sequential influenza infection on the specific immune response mounted. furthermore, since the influenza virus is subject to both genetic shift and genetic drift 35 , the clinical manifestations of sequential virus infections by a virus that undergone multiple genetic drifts would differ from a response of a virus that had undergone a significant genetic shift as that of the h1n1 2009 pandemic variant. influenza virus infection has been thought to plague the human population for hundreds of years, where symptoms of influenza can be traced back in the writings of the early greek cultures to 412 bc 40 . as many questions still exist in relation to evolution and emergence of influenza strains, we analyzed the immunological evolution of past and contemporary influenza viruses. our results shed light on the cross-protective immune response toward the ha molecule and suggest that unique immunity is induced dependent on the ha sequence. further work is needed to determine the evolutionary placement of the 1918 and 1934 virus immunogenicity, as well as the placement of newly emerging influenza strains. our results describing the antigenic evolution of the h1n1 influenza subtype together with previous work will add to the understanding of the transcendence of influenza viruses and have implications on the design of future influenza vaccines. ethics statement. all animal work was conducted in strict accordance with the canadian council of animal care (ccac) guidelines. the university health network (uhn) has certification with the animals for research act (permit number: #0045 and #0085 of the ontario ministry of agriculture, food and rural affairs) and follows nih guidelines (olaw #a5408-01). the animal use protocol was approved by the animal care committee (acc) of the uhn. infections and subsequent sample collection were performed under 5% isofluorane anesthesia in an effort to minimize suffering. infection and ferret monitoring. maintenance and monitoring of infected ferrets has been previously described 23 . briefly, male ferrets 4-6 months old were bred in an on-site spf ferret colony (university health network, toronto, on, canada). prior to infection, all ferrets were screened for influenza and shown to be seronegative by hi assay against circulating influenza a and b strains (2010-2011 who influenza reagent kit for identification of influenza isolates (who collaborating center for surveillance, epidemiology and control for influenza infection division)). the kits contain the circulating influenza strains for the particular year. prior to infection, ferrets were randomly selected and pair-housed in cages contained in bioclean portable laminar-flow clean-room enclosures (lab products, seaford, de) in the bsl-2 animal holding area. baseline body temperature and weight were measured on day 0 for each animal. temperatures were measured by using a subcutaneous implantable temperature transponder (biomedic data systems, inc., seaford, de). upon infection, ferrets were anesthetized and infected 1ml of virus preparation for each ferret (0.5ml in each nostril). ferrets were infected with marton/43 (n 5 12), fm/47 (n 5 10), ussr/77 (n 5 12), taiwan/86 (n 5 10), ncal/99 (n 5 14) , and ny/09 (n 5 12). all viruses were used at 10 5 eid 50 except for fm/47 which was infected at 10 6 eid 50 since it was reported to be a milder virus 14 . clinical signs (body temperature, body weight, level of activity, nasal discharge, and sneezing) were observed daily for 14 days pi. we examined animals at the same time each day for consistency. nasal discharge includes crusty nose, mucous, and transparent exudates/ fluids. the scores were calculated from the total animals displaying any nasal discharge symptom over the total number of animals. the sneezing scores were calculated from the total animals found sneezing over the total number of animals. scores were calculated daily for 14 days and only the peak values for each infection are www.nature.com/scientificreports scientific reports | 3 : 1698 | doi: 10.1038/srep01698 summarized. the inactivity scoring system is based on the reference reuman et al., 1989 59 to assess the inactivity level: 0, alert and playful; 0.5, alert but playful only when stimulated; 1, alert but not playful when stimulated; 2, neither alert nor playful when stimulated. a relative inactivity index was calculated as follows: s (day 1 to day 14) [score11] n /s (day 1 to day 14) n, where n equals the total number of observations. a value of 1 was added to each observation unit score so that a score of 0 could be divided by a denominator, resulting in an index value of 1.0 as the minimum value. determination of influenza specific antibody responses. influenza specific antibody responses from the uninfected or infected ferrets were measured by hi as previously described 23 . briefly, receptor destroying enzyme ([rde], accurate chemical & scientific corp., westbury, ny, usa) treated ferret anti-sera was serially diluted and hi titers were determined by the highest dilution that completely inhibited influenza hemagglutination (4hau) of turkey erythrocytes. comparative analysis of hemagglutinin sequences. phylogenetic analysis of influenza hemagglutinin dna sequences were conducted in mega5 60 using the maximum likelihood method based on the tamura-nei model 61 , 500 bootstrap repetitions were performed. to analyze the protein similarity of influenza hemagglutinin from different h1n1 strains, the aminoacid sequences from the protein region ha135-295 were aligned using clustalw2 62 . this region of the ha protein is part of the hemagglutinin receptor binding domain (ha-rbd), which is located in the external surface of the virus and it concentrates most of the antigenic potential of the ha protein 36 . amino acid ha genbank accession numbers for clustal alignment (supplementary fig. s2 ): a/newcaledonia/20/1999(h1n1) amino acid ha genbank 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viruses. mmwr 35 cdc recommendation of the immunization practices advisory committee monovalent influenza a(h1n1) vaccine sequence analysis of the haemagglutinin of a/taiwan/1/86, a new variant of human influenza a(h1n1) virus the evolution of human influenza viruses cdc update: influenza activity -united states and worldwide, 2006 -2007 and composition of the 2007 -2008 influenza vaccine antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans prior infections with seasonal influenza a/h1n1 virus reduced the illness severity and epidemic intensity of pandemic h1n1 influenza in healthy adults cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses sequential seasonal h1n1 influenza virus infections protect ferrets against novel 2009 h1n1 influenza the effect of priming with h1n1 influenza viruses of variable antigenic distance on challenge with 2009 pandemic h1n1 virus failure of influenza vaccine to prevent two successive outbreaks of influenza a h1n1 in a school community the ferret as a model organism to study influenza a virus infection cloning, expression and characterization of ferret cxcl10 understanding original antigenic sin in influenza with a dynamical system assessment of signs of influenza illness in the ferret model mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods estimation of the number of nucleotide substitutions in the control region of mitochondrial dna in humans and chimpanzees clustal w and clustal x version 2.0 we thank the li ka-shing foundation of canada, immune diagnostics & research, and shantou university medical college for the support of conducting this study. a/mexico/4108/2009 virus was obtained through the influenza reagent resource, influenza division, who collaborating center for surveillance, epidemiology and control of influenza key: cord-325197-j1uo8qmf authors: crimi, ettore; benincasa, giuditta; figueroa-marrero, neisaliz; galdiero, massimiliano; napoli, claudio title: epigenetic susceptibility to severe respiratory viral infections: pathogenic and therapeutic implications: a narrative review date: 2020-08-20 journal: br j anaesth doi: 10.1016/j.bja.2020.06.060 sha: doc_id: 325197 cord_uid: j1uo8qmf the emergence of highly pathogenic strains of influenza virus and coronavirus (cov) has been responsible for large epidemic and pandemic outbreaks characterised by severe pulmonary illness associated with high morbidity and mortality. one major challenge for critical care is to stratify and minimise the risk of multi-organ failure during the stay in the intensive care unit (icu). epigenetic-sensitive mechanisms, including deoxyribonucleic acid (dna) and ribonucleic acid (rna) methylation, histone modifications, and non-coding rnas may lead to perturbations of the host immune-related transcriptional programmes by regulating chromatin structure and gene expression patterns. viruses causing severe pulmonary illness can use epigenetic-regulated mechanisms during host–pathogen interaction to interfere with innate and adaptive immunity, adequacy of inflammatory response, and overall outcome of viral infections. for example, middle east respiratory syndrome-cov and h5n1 can affect host antigen presentation through dna methylation and histone modifications. the same mechanisms would presumably occur in patients with coronavirus disease 2019, in which tocilizumab may epigenetically reduce microvascular damage. targeting epigenetic pathways by immune modulators (e.g. tocilizumab) or repurposed drugs (e.g. statins) may provide novel therapeutic opportunities to control viral–host interaction during critical illness. in this article, we provide an update on epigenetic-sensitive mechanisms and repurposed drugs interfering with epigenetic pathways which may be clinically suitable for risk stratification and beneficial for treatment of patients affected by severe viral respiratory infections. influenza, and influenza a virus subtype h5n1 or avian influenza) and coronavirus (cov) (i.e. severe acute respiratory syndrome [sars]-cov, middle east respiratory syndrome [mers]-cov, and sars-cov-2) responsible for pandemic infections associated with high morbidity and mortality. 1e5 these viruses may cause a wide spectrum of respiratory manifestations associated with massive inflammatory cell infiltration and proinflammatory cytokine/chemokine release resulting in acute lung injury (ali); acute respiratory distress syndrome (ards); and, ultimately, death from multi-organ failure (mof). 6e8 molecular mechanisms regulating virusehost interactions can significantly affect the degree and adequacy of both immune and inflammatory responses influencing clinical outcomes. 9e11 for the critical care physicians, to identify and prevent the occurrence of this exuberant inflammatory response and to stratify the risk of mof in the intensive care unit (icu) are still major challenges. 12 epigenetics may influence host susceptibility to such viral infections. 13 deoxyribonucleic acid (dna) methylation, ribonucleic acid (rna) methylation, histone tail changes, and also non-coding rnas are heritable and acquired modifications able to alter gene expression at different levels without any changes in the primary dna sequence. epigenetic mechanisms, by regulating chromatin structure and gene expression patterns, modulate host immunity and inflammatory response. 14 in critical illness, such epigenetic modifications can promote the release of proinflammatory cytokines and activation of inflammatory cells, responsible for oxidative stress, endothelial dysfunction, apoptosis, and mof. 15 epigenetics can also regulate the interaction between host and multidrug-resistant bacteria. 16 the interaction of viruses with host cells can cause perturbations of transcriptional programmes involving such epigenetic mechanisms leading to viral shedding and inadequate immune response. 13 middle east respiratory syndrome-cov and h5n1 infections can antagonise the host immune response by modulating antigen presentation through dna methylation and histone modifications. 17e20 similar mechanisms would occur during coronavirus disease 2019 . 21, 22 indeed, advanced bioinformatic tools have predicted the possibility of using micrornas (mirnas) to inhibit infections caused by covid-19, sars-cov, and mers-cov by inhibiting the translation of viral proteins and viral replication. 21 as some epigenetic changes can be reversed by small agents, known as 'epidrugs', or alternatively, epigenetic pathways can be interfered by immune modulators, they might provide useful drug targets to ameliorate the clinical outcome during viral respiratory infections. 23, 24 ongoing trials will answer to this possible clinical application. the goal of the review was to provide an appropriate pathogenic scenario in which epigenetic-sensitive mechanisms and epidrugs may be clinically useful to stratify risk and treatment of patients in icu affected by severe viral respiratory infections. influenza viruses are enveloped, single-stranded rna viruses classified into three major serotypes: a, b, and c. influenza a viruses, the most extensively studied, are further classified based on the different subtypes of the two surface glycoproteins: haemagglutinin (h1eh18) and neuraminidase (n1en11) which facilitate virus binding to host respiratory epithelial cells via a sialic acid receptor and virions released from cells, respectively. 25 avian influenza a virus h5n1 26à28 and swine influenza a h1n1 29à34 infections have caused acute respiratory failure secondary to severe pneumonia and ards. the highly pathogenic avian influenza a virus h5n1, first described in 1996, caused severe pneumonia with high mortality (more than 60%), secondary to ards and mof. 26e28 risks factors for severe influenza a h1n1 infection included pregnancy, table 1 clinical and immunological features of viral respiratory infections by influenza viruses. 36e44 copd, chronic obstructive pulmonary disease; cr, conventional radiograph; ct, computed tomography; ggo, ground glass opacity; hf, heart failure; ifng, interferon g; il-1 ra, interleukin-1 receptor antagonist protein; ip-10, interferon-g-inducible protein-10; mcp-1, monocyte chemoattractant protein-1; mip 1-b, macrophage inflammatory protein 1-b; tlr-3, toll-like receptor type 3; tnf-a, tumour necrosis factor-alpha. obesity, asthma, and chronic obstructive pulmonary disease (copd). 29e34 mitigation of the pathogenicity of the pandemic virus was probably caused by the presence of a cross-reactive cell response against this virus, which was boosted by seasonal vaccination. 35 in table 1, we summarised clinical and immunological features of viral respiratory infections by influenza viruses. 36e44 coronaviruses coronaviruses, named after their crown-like structure, are enveloped, positive-sense rna viruses, containing the largest known genome amongst rna viruses. the cov genome encodes for 16 non-structural proteins, which form the viral replicase transcriptase complex, and four essential structural proteins, involved in the host immune response and virion assembly: the spike (s) protein, responsible for receptor binding and viral entry into the host cell; the membrane (m) and envelope (e) proteins, responsible for virus assembly and release; and the nucleocapsid (n) protein, important for rna synthesis and its final packaging into the viral particles. 45 the genome sequence of sars-cov-2 is about 79% identical to the sars-cov and 50% to the mers-cov. 46 these viruses cause severe respiratory infections (table 2) . 47e60 older adults with co-morbidities, especially cardiovascular diseases, diabetes, obesity, renal failure, and copd, are at higher risk of severe disease. 51, 56 icu admission for organ support occurs in about 20e30%, 5e36%, and 50e89% of patients infected with sars-cov, sars-cov-2, and mers-cov, respectively. 3,51e53,56 currently, global public health is facing the covid-19 pandemic as the third cov crisis in less than 20 yr (https://www.nih.gov/health-information/coronavirus). coronaviruses are capable of infecting several other organs, as demonstrated by the presence of sars-cov in circulating immune cells, neurones, intestinal mucosa, and epithelium of renal distal tubules. 61 coronavirusesehost interaction can influence susceptibility to cov infection and progression to severe disease. 3, 11 after entering the body via the respiratory system, a critical step for cell entry and infection is the binding of the envelope s glycoprotein to the epithelial cells through specific receptors. the s protein of the sars-cov and sars-cov-2 binds to the angiotensinconverting enzyme 2 (ace2) molecule present on cells through the receptor-binding domain, 62, 63 whilst mers-cov binds to the host cell protein dipeptidyl peptidase 4. 64 after binding to the host, the s protein needs to be activated by a cellular protease, the transmembrane protease serine 2, which cleaves s in two subunits liberating the fusion peptide that mediates the fusion of the viral envelope with the cellular membrane. differences in the structural and dynamic state of the receptor-binding domain 65 and s protein priming 66 between sars-cov and sars-cov-2 cause higher ace2-binding affinity of the sars-cov-2 and favour its evasion of the immune surveillance, 64 suggesting a potential explanation of the higher sars-cov-2 infectivity. 63 angiotensin-converting enzyme 2 can play a protective role in lung injury, 67 and its downregulation by sars-cov can contribute to progression to severe lung injury. 68 on the contrary, mers-cov regulates dipeptidyl peptidase 4 receptor in the lungs of smokers and copd, and this could explain their susceptibility to severe disease. 69 to survive in the host cells, the covs adopt multiple strategies to evade detection by the host immune system, allowing active virus replication. the virus can downregulate genes involved in the antigen presentation, such as retinoic-acidinducible gene and melanoma differentiation-associated protein 5, and interfere with intracellular signalling pathways through structural (proteins m and n) and non-structural proteins, so delaying interferon (ifn) expression. 3, 17, 70, 71 ultimately, a delayed but excessive reaction of the immune system with an uncontrolled expression of cytokines and chemokines (the so-called cytokine storm) associated with virus-induced cytopathic effects will result in lung epithelial and endothelial cell apoptosis and activation of the coagulation cascade, leading to vascular leakage; alveolar oedema; microvascular thrombosis; and, later, cell proliferation with pulmonary fibrosis. 3, 11, 47 cytokine storm in such patients is associated with more severe lung injury, icu admission, and worse outcome. 3, 51, 71 to date, no definitive treatment for viruses causing severe pulmonary illness can use three epigenetic-regulated ways during hostepathogen interaction: (i) they can affect host dna methylation signatures and mir-nas regulating a cassette of genes underlying innate and adaptive antiviral responses; (ii) they can encode for viral proteins that directly interact with the host modified histones; and (iii) they can manipulate the host mirna processing nuclear machinery to encode viral non-canonical mirna-like rna fragments (v-mirnas) regulating the viral life cycle and immune response. 73 here, we focus on epigenetic-sensitive mechanisms by which h5n1 and sars-cov-2 may affect susceptibility to pulmonary illness by interfering with both innate and adaptive immune responses in humans 74,75 ( fig. 1 and table 3 ). by combining multi-omics data, h5n1 antagonised the early host antiviral response by altering histone methylation at type i ifn-sensitive genes. 18 in detail, ns1 viral protein was associated with parallel increased h3k27me3 (repressive mark) and decreased h3k4me3 (active mark) levels favouring a heterochromatin state surrounding the smad9l, cfhr1, and ddx58 genes in human airway cells. 18 viral non-canonical mirna-like rna fragments induced cytokine storm and high mortality 76 ; however, v-mirna biogenesis and function remain to be clarified. 81 overall, the virus skill to produce functional mirnas can be also exploited to construct delivery systems of mirnas based on rna viruses as molecular vectors. 82 severe acute respiratory syndrome coronavirus 2 the high transmissibility and asymptomatic infection rates of sars-cov-2 may be caused by a more efficient virus replication and reduced ifn production in lung tissues. 83 as both sars-cov-2 and mers can reprogramme the host epigenome, we hypothesise a possible role for epigenetic drivers underlying susceptibility to covid-19. sawalha and colleagues 77 have proposed oxidativestress-induced epigenetic pathways linked to ace2 deregulation to increase susceptibility and severity of covid-19 in patients affected by systemic lupus erythematosus (sle). indeed, the ace2 gene cpg hypomethylation status characterising sle patients could be exacerbated upon sars-cov-2 infection leading to further ace2 protein overexpression in t cells, thus promoting viral infections and dissemination. 77 disease-related epigenetic perturbations might be hotspots favouring viral infection and provide risk biomarkers useful to stratify sensitiveness to infection and disease severity in patients more prone to disseminate sars-cov-2 infection. consistent with this, a bioinformatic analysis focusing on ace2 gene has supported the hypothesis for which dna methylation signatures are dependent on host cell type and gender and age, which are risk factors associated with increased susceptibility to covid-19 and poor prognosis. 84 patients affected by covid-19 are at higher risk of thromboembolic events and disseminated intravascular coagulation. 85 as evidence of neutrophil lung infiltration, barnes and colleagues 78 have emphasised the key role of netosis in contributing to organ damage and mortality of patients affected by covid-19. physiologically, netosis is a form of innate immunity, in which the neutrophil cell death is guided from histone h3 modifications and release of neutrophil extracellular traps (nets), complexes of dna fibres, histones, and proteins aimed to provide a scaffold for platelet adhesion and aggregation to entrap pathogens and avoid their diffusion. 86 moreover, nets can induce macrophages to secrete il1b to further sustain the signalling loop between macrophages and neutrophils, leading to progressive inflammatory damage. previously, netosis dysregulation was linked to thrombotic events, 86 ards, pulmonary inflammation, and extensive lung damage. 87 interestingly, there is evidence for which heparin can dismantle nets and prevent histone-induced platelet aggregation. this might represent the molecular basis for which heparin treatment reduces mortality in subjects affected by severe covid-19, which develop sepsis-induced coagulopathy. 88 thus, deoxyribonuclease i-mediated degradation of nets could provide a therapeutic avenue to suppress excess injury in patients severely affected by covid-19. 89 the epitranscriptome of sars-cov-2 was analaysed as a possible strategy to dissect the hidden layer of viral regulation. by using nanopore direct rna sequencing, almost 41 rna modification sites, mostly located in the aagaa motif on viral transcripts with shorter poly(a) tails, have been suggested. 90 as poly(a) tails play a relevant role in rna turnover and stability, the proposed modifications may represent one of the molecular mechanisms by which sars-cov-2 evades the host immune response and indices the cytokine storm. 90 besides, the authors did not identify what type of rna modifications occur at these sites and the mechanisms underlying covid-19 pathogenesis. 90 nowadays, the emergence of drug-resistant pathogens continuously increases, thus the discovery of novel drugs or the repositioning of already-approved drugs is needed. 16 epitherapy may provide further therapeutic opportunities to control viralehost interaction during critical illness. 91 in particular, the current emergence of covid-19 is guiding researchers towards the possible repurposing of food and drug administration (fda)-approved epidrugs, 92 including metformin and statins, which may be effective against the novel sars-cov-2 infection. here, we give an update on clinical evidence about the usefulness of novel and fda-approved drugs interfering with epigenetic pathways, which were applied to icu patients affected by highly pathogenic strains of influenza virus and cov, with a particular interest about the novel sars-cov-2 (table 4 ). in recent years, a few epidrugs have been introduced into clinic use (e.g. vorinostat and belinostat mainly to treat haematological malignancies), and a wide range of epigeneticbased drugs are undergoing trials, which will clarify whether pharmacological epigenetic modulation is of clinical interest ( table 5 ). the road from discovery to clinical approval requires long timelines; thus, the repurposing of old drugs interfering with epigenetic pathways is another goal. in this case, epigenetic effect is inevitably going to be 'off-target' in comparison with the drug action used in the first place. experimental evidence demonstrated that the effects of epidrugs were achieved at lower doses with prolonged exposure, whereas higher drug concentrations were detrimental. however, when ongoing trials will be completed, we will establish clinically if the dose used for epigenetic modification reversal would be the same as, less than, or more than that used for the original purpose. curcumin curcumin, belonging to the histone deacetylase inhibitor (hdaci) group, is a natural polyphenol extracted from turmeric with a wide range of molecular targets and drug activities, including anti-inflammatory properties. interestingly, after h1n1 infection, curcumin treatment downregulated the secretion of proinflammatory cytokines and expression of the nuclear factor kappa-light-chain enhancer of activated b cell (nf-kb) gene in human macrophages without affecting cell viability. 93 this evidence suggests that curcumin may confer protection against influenza a virus-induced ali by counteracting the cytokine signalling without damaging the immune system. 93 interestingly, curcumin and demetoxycurcumin have been indicated as possible inhibitors of covid-19 virus main protease, which plays a crucial function in controlling viral replication and transcription of sars-cov-2, suggesting a putative useful drugetarget interaction to be validated in clinical trials. 100 apabetalone an international consortium of scientists has identified 50 proteins as putative drug targets against covid-19. amongst these targets, bromodomain (brd) 2/4 would be relevant during interaction with the (e) envelope proteins of sars-cov-2 and viral reproduction. 94 by mimicking the histone structure, the (e) envelope proteins might potentially disrupt brdhistone complexes. brd proteins are epigenetic players that bind acetylated groups on histone proteins to aid in the recruitment of transcriptional machinery at promoter genes. apabetalone can directly inhibit bet2/4 proteinesars-cov-2 interaction and may downregulate the expression of ace2 receptors, which are exploited by the surface s glycoprotein to enter into human cells. 101 currently, apabetalone is not approved by fda, but has already shown clinical safety as demonstrated during the phase 3 trial (betonmace) focusing on secondary prevention of cardiovascular dysfunction in diabetics. 102 overall, this evidence suggests that apabetalone may potentially reduce viral infection and replication. in this way, resverlogix corporation (https://bit.ly/2cbaekb) invites collaborators for further research on apabetalone as a putative therapeutic strategy for covid-19. statins are hydroxymethylglutaryl-coenzyme a reductase inhibitors normalising lipid levels with pleiotropic epigeneticoriented effects by acting as hdaci. as statins also show anti-inflammatory effects, 103 they were thought to block the cytokine storm triggered by influenza viruses. 95 clinical evidence about the use of statins in the treatment of viral pneumonia is limited and provided mixed results. indeed, two trials reported that the anti-inflammatory effects of statins may reduce cardiovascular risk and mortality in icu patients affected by pneumonia. 109, 110 otherwise, the results of one randomised clinical trial did not support statin use in those patients with ventilator-associated pneumonia. 111 currently, there is no clinical or experimental evidence supporting the assertion that statins can improve clinical management of covid-19. as the rates of acute events and mortality associated with covid-19 infection are extremely high in patients with cardiovascular diseases (10.8%) and diabetics (7.3%), which generally use statins as primary or secondary prevention, these patients should continue the treatment when sars-cov-2 infection is suspected or diagnosed (https://www.acc.org/latest-in-cardiology/features/ /media/non-clinical/files-pdfs-excel-ms-word-etc/2020/ 02/s20028-acc-clinical-bulletin-coronavirus.pdf). statins are cleared by the liver metabolism and can increase the level of transaminases in cardio-hepatic patients 112 ; thus, strict evaluation and monitoring of statin therapy should be provided for covid-19 patients, which commonly show an elevation of the aminotransferases (aspartate transaminase and alanine aminotransferase), with occasional alkaline phosphatase and total bilirubin elevations underlying a high risk for hepatotoxicity. 113, 114 whether a de novo use of statin therapy may play a key role in preventing covid-19 complications remains to be elucidated. remarkably, experimental studies supported the hypothesis for which an early and high dose of statins might be a useful strategy for the treatment of mers-cov infections by directly affecting the tlremyd88enf-kb axis, which plays a pivotal role during cov infections. 96, 115, 116 statins are the most common fda-approved drugs classified as tlremyd88 antagonists; moreover, under normal conditions, statins did not strongly alter myd88 levels, whereas they maintain basal myd88 levels during stress and hypoxia. 96 this supports that statins might be protective for patients affected by covid-19. thus, the putative regulation of myd88 pathway via statins may be an attractive field of research to explore how to protect innate immune response against novel viral respiratory infections, including sars-cov-2. metformin, belonging to hdaci class, is the first-line antihyperglycaemic drug for type 2 diabetes (t2d) patients, which can indirectly reduce chronic inflammation by normalising glucose levels or directly impact on inflammatory pathways. recently, saenwongsa and colleagues 117 have demonstrated that after the trivalent inactivated influenza vaccine (tiv), both the igg antibody response and ifn-a expression were impaired in t2d patients treated with metformin via repression of rapamycin (mtor)-mediated pathway and impaired igg avidity index, leading to increased sensitiveness to h1n1 and h3n2 infection. this suggests that the tiv may not be suitable for t2d patients treated with metformin by emphasising the necessity of developing a more customised strategy for influenza prevention in high-risk groups. metformin may recover the influenza vaccine responses in t2d patients (treated and non-treated with metformin) by improving the bcell function via parallel downregulation of inflammation and upregulation of ampk phosphorylation (active form), a metabolic enzyme involved also in antibody responses. 97 understanding of the metformin effects on the immune system may guide the repurposing of this drug focused on therapeutic intervention on metabolism in inflammatory diseases. immunomodulators and antivirals: could they impact on epigenetic pathways underlying cardiovascular dysfunction in covid-19? the repurposing of both immunomodulators and antivirals, including tocilizumab (tcz), remdesevir, favipiravir, hydroxychloroquine, and chloroquine, could be a fast way to get effective treatments whilst a preventive vaccine will be available (https://www.who.int/emergencies/diseases/novelcoronavirus-2019). in the current covid-19 pandemic, tcz is one of the most promising repurposed drugs under clinical investigation for the treatment of severe hospitalised pneumonia patients. tocilizumab is a humanised monoclonal antibody that can counteract the cytokine storm by blocking the interleukin-6 (il-6) receptor signalling associated with a high risk of cardiovascular mortality. 118 immunomodulators can counteract the overactive inflammatory response, which seems to be the driver of increased disease severity. many old anti-inflammatory drugs are in clinical trials, such as sarilumab (nct04315298; phases 2 and 3) and tcz (nct04320615; phase 3). besides, the efficacy of antivirals inhibiting viral replication, such as favipiravir (nct04358549; phase 2) and remdesevir (nct04292730; phase 3), is being evaluated in clinical trials compared with standard of care. the impact of covid-19 on cardiovascular health is an urgent question for physicians. 119 as epigenetics plays an increasing role in cardiovascular diseases and inflammation, 102, 120 and also during sars-cov-2 infection, 77 we emphasised the need to clarify if these drugs could potentially impact the associated cardiac dysfunction modulating the severity of the disease. clinical evidence from rheumatoid arthritis patients demonstrated that tcz therapy can prevent cardiovascular dysfunction via two main epigenetic-sensitive mechanisms: (i) reduction of netosis; and (ii) upregulation of mirna-23, mirna-146, and mirna-223 serum levels. 98 overall, this suggests that tcz can improve the pro-atherosclerotic status by regulating dyslipidaemias, endothelial dysfunction, inflammation, and oxidative stress. 98 moreover, tcz-treated rheumatoid arthritis patients demonstrated a differential expression of 85 lncrnas in cd14 þ monocytes regulated by il-6 or tumour necrosis factor-alpha. 99 preliminary trials have suggested the usefulness of chloroquine or hydroxychloroquine repurposing in the treatment of covid-19, which is correlated with the ability of these antimalarial agents in interfering with the cellular-mediated viral endocytosis. 121 however, a randomised trial demonstrated that hydroxychloroquine failed in preventing symptomatic infection when taken within 4 days after exposure. 122 at epigenetic level, hydroxychloroquine can exert an inhibitory activity against the polycomb repressive complex 2 (prc2), responsible of switching chromatin towards a compacted state (inactive gene expression) in blood malignancies. 123 interestingly, influenza a viruses, mers-cov, and sars-cov are able to activate the prc2, which, in turn, increases the levels of h3k27me3 (repressive mark) at the promoters of targeted ifn-stimulated genes to counteract the host antiviral immune response. 124 this let us suggest that also sars-cov-2 might use the same mechanisms to inactive ifn-related pathways in infected cells; besides, since polycomb inhibitors have shown a general antiviral activity, 125 it might be useful to investigate whether hydroxychloroquine can directly impact on prc2 activity in covid-19. as reported, the clinical effect of i.v. remdesivir seems to be relatively modest; however, a randomised phase 3 clinical trial (nct04292899) did not find a significant difference in efficacy between 5-and 10-day courses of this drug. 126 until now, there is no direct evidence that antivirals, such as remdesevir and favipiravir, would impact epigenetic-sensitive ways during their mechanism of action. targeted epigenetic-sensitive molecular networks are temporally manipulated during virusehost interactions, providing further risk factors for viral shedding and inadequate immune response also in patients affected by covid-19, in which ace2 promoter hypomethylation may be one of the relevant drivers. during the current covid-19 pandemic, we need to understand why a part of the population becomes critically infected when exposed to low viral load, whilst other subjects are less responsive when exposed to high viral loads. as the influenza virus may also promote acute coronary syndromes (which can be reduced by vaccine), 127 the issue of coinfection (i.e. sars-cov-2 and influenza viruses) needs to be further explored both in terms of epigenetic-sensitive tandem events and in critical care. for better risk stratification, it would be needed to clarify the sars-cov-2 basic mechanisms of action and how these impact on the individual genetic/ epigenetic background and pre-existent cardio-metabolic diseases highly correlated with mortality rate, especially in the elderly. 128, 129 clinical treatment of mild forms of covid-19 should not be phobic from fever, which can promote an effective immune response and virus clearance from the body. 130 ongoing controlled clinical trials would clarify the repositioning of tcz, whereas curcumin, apabetalone, metformin, and statins could be an effective treatment to protect innate immune response against severe viral respiratory infections. in the era of network medicine, predictive analysis tools are playing a relevant role in addressing the covid-19 pandemic by providing maps of human proteins interacting with sars-cov-2 proteins 101 and a list of candidate repurpose drugs and potential drug combinations targeting 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hydroxychloroquine targeting sars-cov-2 using polycomb inhibitors as antiviral agents inhibitors of the histone methyltransferases ezh2/1 induce a potent antiviral state and suppress infection by diverse viral pathogens remdesivir for 5 or 10 days in patients with severe covid-19 influenza vaccination reduces cardiovascular events in patients with acute coronary syndrome cardiovascular involvement during covid-19 and clinical implications in elderly patients. a review can covid 2019 disease induce a specific cardiovascular damage or it exacerbates pre-existing cardiovascular diseases? covid-19: do not be phobic from fever network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 molecular networks in network medicine: development and applications this research was supported (in whole or in part) by hospital corporation of america (hca) or hca affiliated entity. the views expressed in this publication represent those of the authors and do not necessarily represent the official views of hca or any of its affiliated entities. design/implementation of the review: ec, gb, cn critical feedback: nf-m, mg writing of paper: all authors the authors declare that they have no conflicts of interest. key: cord-326160-mf0vh6iu authors: de wit, emmie; rasmussen, angela l.; feldmann, friederike; bushmaker, trenton; martellaro, cynthia; haddock, elaine; okumura, atsushi; proll, sean c.; chang, jean; gardner, don; katze, michael g.; munster, vincent j.; feldmann, heinz title: influenza virus a/anhui/1/2013 (h7n9) replicates efficiently in the upper and lower respiratory tracts of cynomolgus macaques date: 2014-08-12 journal: mbio doi: 10.1128/mbio.01331-14 sha: doc_id: 326160 cord_uid: mf0vh6iu in march 2013, three fatal human cases of infection with influenza a virus (h7n9) were reported in china. since then, human cases have been accumulating. given the public health importance of this virus, we performed a pathogenicity study of the h7n9 virus in the cynomolgus macaque model, focusing on clinical aspects of disease, radiographic, histological, and gene expression profile changes in the upper and lower respiratory tracts, and changes in systemic cytokine and chemokine profiles during infection. cynomolgus macaques developed transient, mild to severe disease with radiographic evidence of pulmonary infiltration. virus replicated in the upper as well as lower respiratory tract, with sustained replication in the upper respiratory tract until the end of the experiment at 6 days after inoculation. virus shedding occurred mainly via the throat. histopathological changes in the lungs were similar to those observed in humans, albeit less severe, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, pneumocyte hyperplasia, and fibroproliferative changes. analysis of gene expression profiles in lung lesions identified pathways involved in tissue damage during h7n9 infection as well as leads for development of therapeutics targeting host responses rather than virus replication. overall, h7n9 infection was not as severe in cynomolgus macaques as in humans, supporting the possible role of underlying medical complications in disease severity as discussed for human h7n9 infection (h. n. gao et al., n. engl. j. med. 368:2277–2285, 2013, doi:10.1056/nejmoa1305584). mitted, albeit inefficiently, via respiratory droplets or aerosols in the ferret model (10) (11) (12) (13) . in order to allow a better estimation of the effect of a potential h7n9 pandemic, we studied the a/anhui/ 1/2013 strain of this virus in the cynomolgus macaque model. this model was chosen because it most closely reflects the human physiology and the development of pneumonia, cytokine, and chemokine responses (14) , and the pattern of attachment of h7n9 virus to respiratory tissues of macaques was recently shown to be more similar to that in humans than that in other frequently used animal models (15) . upon inoculation with influenza virus a/anhui/ 1/2013, cynomolgus macaques developed transient, moderate disease with virus replication in the upper and lower respiratory tracts. the emerging h7n9 influenza virus was more pathogenic than seasonal influenza a virus and most isolates of the pandemic h1n1 virus but not as pathogenic as the 1918 spanish influenza virus or highly pathogenic avian influenza (hpai) h5n1 virus in cynomolgus macaques. year-old cynomolgus macaques (4 male and 4 female) were inoculated with 7 ϫ 10 6 50% tissue culture infectious doses (tcid 50 ) of a/anhui/1/2013 (h7n9) via a combination of ocular, oral, intranasal, and intratracheal inoculation as described previously (16) . animals started to show signs of disease at 1 day postinoculation (dpi). clinical disease peaked at 3 and 4 dpi (fig. 1a) and was moderate. six out of 8 animals showed obvious respiratory signs, such as increased respiration rates, abdominal breathing, and coughing (see table s1 in the supplemental material). nasal discharge and cough were noted in only one animal. hematologic and blood chemical analyses did not reveal any abnormalities during the course of infection (data not shown). radiographic changes in lungs of animals inoculated with a/anhui/1/2013. at 1, 2, 3, 4, and 6 dpi, ventral-dorsal and lateral chest x-rays were taken to monitor the development of pneumonia. radiographic changes in the lungs of inoculated animals were observed starting 2 and 3 dpi and were observed in all inoculated animals to various degrees (see table s2 in the supplemental material). the interstitial infiltration was observed first in the lower right lung lobe and, in individual animals, spread over time to the right middle, left lower, left middle, and right upper lung lobes and developed into severe diffuse interstitial infiltration ( fig. 2 ; also, see table s2 in the supplemental material). virus shedding in cynomolgus macaques inoculated with a/anhui/1/2013. clinical exams were performed at 0, 1, 2, 3, 4, and 6 dpi, and nasal, oropharyngeal, ocular, and rectal swabs were collected. swabs were initially analyzed for the presence of viral rna by real-time reverse transcription-pcr (rt-pcr). because of the large number of swabs in which viral rna was detected, all nasal, oropharyngeal, and ocular swabs were titrated on mdck cells; rectal swabs were not titrated, as only 3 rectal swabs were positive by pcr. oropharyngeal swabs were positive in virus titration by 1 dpi and remained positive in all animals until the end of the experiment at 6 dpi (fig. 1b) . not all nasal swabs were positive by virus titration; most of the virus shedding via the nose occurred in animals h7n9-7 and h7n9-8 (fig. 1b) . despite the ability of influenza a viruses of the h7 subtype to cause conjunctivitis, ocular swabs were only sporadically positive by virus titration: only the ocular swabs collected from h7n9-1 at 1, 2, and 3 dpi, the ocular swab collected from h7n9-4 at 1 dpi, and the ocular swab collected from h7n9-8 at 3 dpi were positive, with virus titers between 10 0.8 and 10 2.8 tcid 50 /ml (data not shown). during clinical exams, bronchalveolar lavages (bal) were performed, and the samples were analyzed for the presence of infectious virus. virus could be isolated from the bal fluid of all animals at 1 dpi; bal fluid remained positive throughout the experiment, with significant amounts of virus still detected at 6 dpi, ranging from 3.2 ϫ 10 2 tcid 50 /ml to 1.4 ϫ 10 4 tcid 50 /ml (fig. 1b) . gross lung pathology in cynomolgus macaques inoculated with a/anhui/1/2013. upon necropsy of 4 animals at 3 dpi, gross lesions were observed in the lungs of all animals. there was variation in the area of the lung affected between animals, but at least two lobes showed gross lesions in all animals, varying from 5% to 100% of tissue affected (fig. 1c ). in line with our observation on x-rays, the right lung lobes were more severely affected than the left lobes; this is likely a result of the intratracheal inoculation and the anatomy of the lung (17) . by 6 dpi, the area of the lung displaying gross lesions had increased, with 100% of all three right lung lobes being affected in 2 of 4 animals (fig. 1c) . virus titers in tissues of cynomolgus macaques inoculated with a/anhui/1/2013. for each animal, virus titers were determined in tissue samples collected from all 6 lung lobes and 2 lung lesions. in line with our x-ray and gross pathology observations, at 3 dpi virus could be detected in all three lobes of the right lung but not in all three lung lobes of the left lung in all animals (fig. 3a) . virus was present in the collected lung lesions, but unexpectedly, virus titers were not higher in lung lesions than in the collected lung lobe samples, indicating widespread virus replication throughout the lower respiratory tract. by 6 dpi, the virus titers in the lung lobes and lung lesions had decreased compared to those at 3 dpi, although this decrease was not statistically significant; the number of animals with positive virus titration also decreased (fig. 3a) . in other tissues of the respiratory tract, i.e., nasal turbinates, oropharynx, trachea, and right and left bronchus, virus titers were generally higher than in the lungs at 3 dpi and 6 dpi (fig. 3b) . interestingly, although virus titers in the lung lobes and lung lesions decreased between 3 and 6 dpi, virus titers in the other respiratory tract tissues did not. virus could also be detected in the tonsils and mediastinal lymph nodes of infected animals at 3 and 6 dpi (fig. 3b ). virus could be detected in the conjunctiva of only one animal at 3 and 6 dpi. the remaining tissues that were collected at 3 and 6 dpi (i.e., heart, liver, spleen, kidney, stomach, jejunum, ileum, transverse colon, and brain) were analyzed for the presence of viral rna by real-time rt-pcr. except for the liver, where vrna was detected in 6 out of 8 animals, vrna was detected sporadically in all tissues except in heart (see table s3 in the supplemental material). since cycle threshold values (c t ) in almost all of these samples were higher than the level at which virus titration is usually successful (c t ͻ 32), virus titration was not attempted on these samples. histopathology of respiratory tissues of cynomolgus macaques inoculated with a/anhui/1/2013. at 3 dpi, histopathology of the lungs was similar for all 4 animals necropsied and was characterized as mild to marked, acute, bronchointerstitial pneumonia. the pneumonia was characterized microscopically as mild to marked thickening of alveolar septa by fibrin, edema, neutrophils, and macrophages, and the alveoli contained small to large amounts of these same inflammatory components (fig. 3c ). mul-de wit et al. tifocally, hyaline membranes were observed. the lumens of terminal bronchioles frequently contained fibrin, edema, hyaline membranes, neutrophils, and macrophages, with multifocal necrosis and loss of lining epithelium. inflammation and necrosis of bronchial submucosal glands were frequently noted, with mild, subacute periglandular inflammation or more severe changes that ranged from neutrophils and macrophages within ductular lumens to necrosis of acinar and ductular epithelium, occasionally affecting the entire gland. larger bronchioles and bronchi were generally much less severely affected than terminal airways, with intact, viable lining epithelium and only occasional mild, neutrophilic luminal exudate. at 6 dpi, histopathology of the lungs was characterized as mild to marked, subacute to chronic, bronchointerstitial pneumonia. microscopic changes were again characterized by the presence of neutrophils, macrophages, fibrin, and edema within alveolar septa, alveoli, and terminal bronchioles (fig. 3d) . additionally, type ii pneumocyte hyperplasia was observed in extensive portions of each lung lobe, and large clumps of alveolar fibrin frequently engulfed neutrophils and macrophages and were lined and infiltrated by fibroblasts. immunohistochemistry (ihc) of sections of lung demonstrated low to moderate numbers of antigen-positive alveolar type i and type ii pneumocytes, macrophages, and epithelium lining bronchioles, bronchi, and bronchial submucosal glands (fig. 4) . the numbers of positively stained alveolar pneumocytes and bronchial and bronchiolar submucosal gland epithelial cells were similar at 3 and 6 dpi. the number of positively stained pulmonary macrophages was increased at 6 dpi compared with 3 dpi. positive staining in alveolar and submucosal macrophages was cytoplasmic, most likely indicating active phagocytosis of virus rather than replication of virus in these cells (fig. 4h ). antigenpositive macrophages were also consistently noted within mediastinal lymph nodes, with increased numbers of these cells being noted at day 6 dpi. low numbers of positively stained macrophages were noted in pharyngeal tonsils, oropharynges, tracheas, and extrapulmonary bronchi. there was more variation between individual animals in histopathological lesions in the remaining tissues. mild, subacute conjunctivitis was noted histologically in one of eight animals at 6 dpi, and low numbers of macrophages positive for influenza a virus antigen were present in the submucosa from that animal; the viral load in the eye swab collected from this animal (h7n9-7) at 6 dpi was still high (10 4 tcid 50 equivalents/ml), likely indicating active virus replication. influenza a virus-positive cells were noted in the epithelium of the nasal turbinates with minimal inflammation in 3 of 4 animals at 3 dpi ( fig. 5a and b ). although no mitable s2 in the supplemental material. in severe cases of infection at 3 dpi, lesions were characterized by edema, alveolar fibrin (black asterisk), and hyaline membrane formation (arrow). at 6 dpi, edema, organizing fibrin (white asterisk), and type ii pneumocyte hyperplasia (arrowheads) were observed. magnification (c and d), ϫ400. de wit et al. ( fig. 5e and f). minimal to moderate subacute inflammation was noted in at least one of the extrapulmonary bronchi from each animal, and in one animal there was an extensive area of marked inflammation with ulceration. upon ihc staining of the same sections of bronchi, few positively stained epithelial cells and/or macrophages were noted ( fig. 5g and h) . matching tissue sections were stained with hematoxylin and eosin (h&e) or studied by immunohistochemistry (ihc) using an anti-np monoclonal antibody (ihc; visible as red-brown staining). at 3 dpi, mild to marked thickening of alveolar septa by fibrin, edema, neutrophils, and macrophages was observed (a), and virus antigen was present in type i and type ii (black arrows) pneumocytes (b). inflammation and necrosis of bronchial submucosal glands were frequently noted (c), with virus antigen being present in submucosal gland (open arrowhead) and bronchial epithelium (white arrow). infection of the bronchial lining epithelium was more pronounced by 6 dpi (e and f). cytoplasmic staining of alveolar macrophages indicates active phagocytosis on 6 dpi (h; black arrowheads). (j) virus replication in bronchial submucosal gland epithelium on 6 dpi. magnification, ϫ400. inoculated with influenza virus a/anhui/1/2013. cynomolgus macaques were euthanized at 3 (a and b) and 6 (c to h) days postinoculation, and tissue was collected and stained with hematoxylin and eosin (h&e) or immunohistochemistry (ihc) using an anti-np monoclonal antibody (ihc; visible as redbrown staining). in the rostral nasal cavity (a and b) and nasopharynx (c and d), influenza virus a/anhui/1/2013 mainly replicated in the lining epithelium. in the trachea (e and f), cell debris (arrowhead) with immunopositive staining is visible in a submucosal gland. in the bronchus (g and h), bronchial lumen exudate is visible, with virus replication in the epithelium lining the bronchus. magnification, ϫ400. and vascular endothelial growth factor (vegf) levels. at 1 dpi, there was a statistically significant increase in levels of g-csf, il-1ra, il-6, il-10, il-15, mcp-1, and tnf-␣ (fig. 6) ; levels of ifn-␥, il-2, il-8, mip-1␣, mip-1␤, and vegf increased at that time point, but increases were not statistically significant ( fig. 6 ; also data not shown). by 6 dpi, all levels of cytokines and chemokines had returned to baseline. alterations in host gene expression upon infection with a/anhui/1/2013. to elucidate global host responses specifically associated with sites of virus-induced airway injury in influenza virus a/anhui/1/2013-infected macaques, we used microarrays to assess transcriptional profiles induced in lung lesions compared to the adjacent lung tissue. welch's t test (p ͻ 0.05; change ͼ 1.5-fold) identified 802 differentially expressed genes (deg) in the lesions compared to lung samples from the same animals at 3 dpi ( fig. 7a ; also, see table s4 in the supplemental material); these 802 genes were differentially expressed in all animals. of those, 429 were upregulated and 372 were downregulated in lesions compared to samples from the right lower lung lobe. using ingenuity pathway analysis (ipa), we assessed the functional significance of this molecular signature and constructed a network of critical molecules based on direct interactions in the ipa knowledge base (ipkb) (fig. 7b ). as expected, there are numerous transcripts upregulated from functional categories previously observed in cynomolgus macaque models of influenza infection (18) (19) (20) (21) , including known mediators of antiviral immunity, such as pattern recognition receptors and downstream signaling molecules (tlr4 and myd88), interferons and interferon-stimulated genes (ifnl3), interferon-regulatory factors (irf1, irf5, and jak3), and proinflammatory cytokines and inflammatory mediators (il-6, nlr family, nlrp3, tnfrsf1b, tnfrsf6b, and tnfsf8). we also identified a number of upregulated genes associated with leukocyte migration and differentiation (cxcl10, cxcl11, sele, il4r, il-18, and csf2ra). this suggests that molecules that recruit infiltrating effector leukocytes are increased at the site of lesions and is in line with the observed influx of neutrophils and macrophages observed microscopically in the lungs. moreover, hyaluronic acid synthase 2 (has2), a molecule known to be associated with lung injury, was strongly upregulated in the lung lesion samples. we also observed several functional categories of molecules that were downregulated in lesions relative to the adjacent lung tissue (fig. 7b) . these were associated predominantly with lipid metabolism and adipogenesis regulated by peroxisome proliferator-activated receptor alpha (ppar␣). we also observed downregulation of growth factors, particularly those related to insulin signaling and regulation of glucose levels (insr and igfbp5). by 6 dpi, 154 deg were identified in lung tissue versus lung lesions of animals euthanized at 6 dpi (see fig. s1 and table s4 in the supplemental material). due to substantial variability between animals, we were not able to determine any significantly enriched functional categories at this time point. next, the upstream analysis function in ipa was used to identify drugs predicted to act as significant upstream regulators of the key deg identified at 3 dpi. drugs that are predicted to inhibit pathological host responses were selected based on the activation z score. negative activation z scores are predicted to cause opposite or inhibitory effects on significant genes associated with pathology in the lesions. these findings were confirmed using connectivity map (cmap), a resource allowing comparison with transcriptional signatures from multiple cell types treated with a library of chemical and genetic perturbagens (22) , by determining if the connectivity score was also negative, indicating that the compound would have inhibitory effects on transcriptional signatures associated with lesion formation. we identified ten compounds in ipa (table 1) , four of which were perturbagens listed in cmap. we identified two compounds that met our criteria in ipa and cmap, rosiglitazone and simvastatin, predicted to have inhibitory effects on pathological host responses associated with lesions in influenza virus a/anhui/1/2013-infected animals ( table 1) . these molecules are both involved in regulating lipid biosynthesis and metabolism. rosiglitazone is an fda-approved diabetes drug that modulates ppar activity and insulin sensitivity and has been shown to affect rna virus replication (23) (24) (25) , virus-induced inflammation (26, 27) , and lung inflammation (28) (29) (30) . simvastatin is a fda-approved statin which lowers blood cholesterol by inhibiting 3-hydroxy-3-methyl-glutaryl coenzyme a (hmg-coa) reductase. it has been shown to reduce lung inflammation in mouse models of airway injury (31-34) and bacterial infections (35) (36) (37) . however, simvastatin was not shown to have a significant effect on influenza a virus pathogenesis in mice (38) (39) (40) . besides rosiglitazone and simvastatin, mainly immunomodulatory drugs were predicted to have inhibitory effects on pathological host responses to a/anhui/1/2013 infection. the ability of rosiglitazone to inhibit a/anhui/1/2013 replication was tested in mdck cells treated with different concentrations of this drug after infection with a/anhui/1/2013 at a multiplicity of infection of 0.001. twenty-four hours after addition of 100 m rosiglitazone, but not at lower concentrations, a statistically significant (p ϭ 0.0314; two-tailed unpaired t test) 14-fold reduction in virus titers in the supernatant of treated, infected cells compared to mock-treated cells was observed (see fig. s2a in the supplemental material); a small cytotoxic effect of the drug was also noticed at this time point in an mts [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium, inner salt] assay (see fig. s2b in the supplemental material). at 72 h after treatment, the inhibitory effect of rosiglitazone on a/anhui/1/2013 replication could no longer be detected (data not shown). china has shifted the focus away from hpai h5n1 to h7n9 as one of the main pandemic threats. although much attention has been drawn to the ability of this virus to be transmitted between ferrets, the pathogenicity of h7n9 influenza a virus in ferrets does not match the severity of disease observed in human cases (10) (11) (12) (13) . the physiology of the cynomolgus macaque lung is more similar to that of humans, and cytokine and chemokine responses to infection in macaques are similar to those in humans (14) . moreover, it was shown recently that the pattern of attachment of h7n9 influenza a virus to respiratory tissues of cynomolgus macaques is more similar to the attachment pattern in humans than that in other animal models frequently used for influenza a virus pathogenesis studies, including the ferret (15) . therefore, we studied the h7n9 infection of cynomolgus macaques in detail. in agreement with the observed abundant attachment of the h7n9 influenza a virus to the human upper and lower respiratory tracts (41) , and the replication of the h7n9 virus in ex vivo cultures of the human upper as well as lower respiratory tract (42) , h7n9 virus replicated well in the upper and lower respiratory tracts of cynomolgus macaques, as indicated by virus titers in nasal turbinates, oronasopharynges, tracheas, bronchi, and lung tissue samples, reflecting the previously described receptor distribution of influenza virus h7n9 in the macaque respiratory tract (15) . surprisingly, the virus titers in nasal turbinates, oronasopharynges, tracheas, and bronchi did not decrease between 3 and 6 dpi, unlike the virus titers in the lung samples, which did decrease. this extended virus replication in the upper respiratory tract could result in prolonged virus shedding and thus an increased risk of virus transmission. the histopathology of the lungs in fatal human h7n9 cases was similar to, albeit more severe than, that observed in cynomolgus macaques, with diffuse alveolar damage, infiltration of polymorphonuclear cells, formation of hyaline membranes, and, later after onset of symptoms, pneumocyte hyperplasia and fibroproliferative changes (4). however, clinically and histopathologically, h7n9 infection was not as severe in cynomolgus macaques as it has been described in humans, where h7n9 has a high case fatality rate. although viral characteristics could cause differences in disease severity between humans and macaques, another possible explanation for the discrepancy in disease severity could be the high percentage of human cases with underlying medical complications (3) . since some of the h7n9 influenza virus strains isolated from human cases have an r292k substitution in na that renders them partially resistant to treatment with neuraminidase inhibitors (12, (43) (44) (45) , it is important to identify drugs that either directly inhibit virus replication in the host or reduce the severity of disease and the level of lung injury after infection. by analyzing the gene expression profiles in lung lesions compared to adjacent infected, nonlesional lung tissue, we were able to predict drugs reported to act as upstream regulators of some of these genes that may thus play a role in the development of lung lesions during h7n9 infection. one of the predicted drugs, rosiglitazone, modestly reduced a similar data analysis resulted in the identification of a compound that reduced replication of the middle east respiratory syndrome coronavirus in vitro (46) ; however, the validity of this approach has not yet been tested in vivo. thus, this analysis of the gene expression profile hints at new avenues of treatment to explore in in vivo models, rather than revealing novel treatments directly applicable in the clinic. although it is difficult to compare studies investigating the pathogenicity of different influenza a viruses in the macaque model, because of different inoculation routes and doses used and because sampling schemes do not overlap between studies, the h7n9 infection in macaques in our study was similar to that described previously (12) . in both studies, animals developed transient clinical signs, with virus replication in the upper as well as lower respiratory tract and similar histopathological lesions in the lower respiratory tract. more importantly, h7n9 infection seemed to be clinically more severe than most infections with isolates of pandemic h1n1 (16); virus shedding from the throat was higher in h7n9-infected animals, and a larger area of the lung was affected with gross lesions (47) . inoculation of cynomolgus macaques with seasonal h3n2 influenza a virus resulted in infection with mild or even asymptomatic disease (48, 49) . cynomolgus macaques inoculated via the same route and with the same dose of 1918 spanish influenza virus developed lethal disease (50) , and hpai h5n1 influenza a virus inoculated into macaques at a 40-fold-lower dose caused more severe disease than h7n9 influenza a virus in this study (51) . thus, the emerging h7n9 influenza virus is more pathogenic than seasonal influenza a virus and most isolates of the pandemic h1n1 virus but not as pathogenic as the 1918 spanish influenza virus and hpai h5n1 virus in cynomolgus macaques. however, the pathogenicity of the h7n9 virus may decrease if the virus adapts further to solely using ␣2,6-linked sialic acids as the receptor for entry, as pandemic influenza viruses to date have done (52) (53) (54) (55) . exclusive attachment to ␣2,6-linked sialic acids would most likely result in a shift to replication mainly in the upper respiratory tract of humans, likely resulting in less severe disease, as has been described for the 2009 pandemic h1n1 virus (56) and upon adaptation of hpai h5n1 virus to efficient transmission via respiratory droplets or aerosols (57) . all animal experiments were approved by the institutional animal care and use committee of the rocky mountain laboratories and performed following the guidelines of the association for assessment and accreditation of laboratory animal care, international (aaalac), by certified staff in an aaalac-approved facility. cells. madin-darby canine kidney (mdck) cells were cultured in eagle's modified essential medium (emem) (gibco) supplemented with 10% fetal calf serum (fcs), 50 iu/ml penicillin, 50 g/ml streptomycin, 2 mm glutamine, 0.75 mg/ml sodium bicarbonate, and nonessential amino acids. virus. a/anhui/1/2013 (passage e2/e1) was obtained from the centers for disease control in atlanta, ga, and passaged once in mdck cells. animal study and sample collection. eight cynomolgus macaques (4 males, 4 females; age, 5 years; 4 to 6 kg) were inoculated with 7 ϫ 10 6 tcid 50 of a/anhui/1/2013 via a combination of intratracheal (4 ϫ 10 6 tcid 50 ; 4 ml), intranasal (1 ϫ 10 6 tcid 50 ; 500 l/nostril), oral (1 ϫ 10 6 tcid 50 ; 1 ml), and ocular (1 ϫ 10 6 tcid 50 ; 20 l/eye) routes. the animals were observed twice daily for clinical signs of disease and scored using a previously described clinical scoring system (58) . at 1, 2, 3, 4, and 6 days postinoculation, clinical exams were performed on anesthetized animals, and lateral x rays were taken and analyzed by a veterinarian. nasal, oral, urogenital, and rectal swabs were collected in 1 ml dulbecco's modified essential medium (dmem) with 50 u/ml penicillin and 50 g/ml streptomycin; bronchoalveolar lavages (bal) were performed using 10 ml sterile saline solution; blood was collected for hematology, blood chemistry analysis, and peripheral blood mononuclear cell (pbmc) isolation. the total white blood cell count, lymphocyte, platelet, reticulocyte, and red blood cell counts, hemoglobin and hematocrit values, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentrations were determined from edta-containing blood with the hemavet 950fsϩ hemoglobin analyzer (drew scientific). pbmc were isolated by centrifugation over a histopaque gradient (sigma) as per the manufacturer's recommendation. at 3 and 6 dpi, 4 macaques were euthanized, and samples of the conjunctivas, right and left eyes, nasal turbinates, tonsils, oronasopharynges, tracheas, right and left bronchi, all six lung lobes, lung lesions, mediastinal lymph nodes, hearts, livers, spleens, kidneys, stomachs, jejuna, ilea, transverse colons, olfactory bulbs, cerebella, brain stems, and bone marrow were collected. the percentage of the lung surface area affected by gross lung lesions was quantitated for each lung lobe, ventrally and dorsally, by a board-certified veterinary pathologist at the time of necropsy. histopathology and immunohistochemistry. histopathology and immunohistochemistry were performed on macaque tissues. after fixation for 7 days in 10% neutral buffered formalin and embedding in paraffin, tissue sections were stained with hematoxylin and eosin (h&e). to detect influenza a virus antigen, immunohistochemistry was performed using an anti-np monoclonal hb-65 antibody (evl, the netherlands) as a primary antibody. rna extraction. rna was extracted from swabs, bal fluid, and whole-blood samples using the qiaamp viral rna kit (qiagen) according to the manufacturer's instructions. tissues were stored at ϫ80°c until further processing; tissue samples (30 mg) were homogenized in rlt buffer, and rna was extracted using the rneasy kit (qiagen). quantitative real-time rt-pcr. a one-step real-time rt-pcr targeted at the matrix gene of influenza a virus was performed using the quantifast probe kit (qiagen) according to instructions of the manufacturer using the primers and probe described in reference 59. virus titrations. viruses were titrated by endpoint dilution in mdck cells. mdck cells were inoculated with tenfold serial dilutions of culture supernatants. one hour after inoculation, cells were washed with phosphate-buffered saline (pbs) and supplemented with infection medium (emem supplemented with 50 iu/ml penicillin, 50 g/ml streptomycin, 2 mm glutamine, 0.75 mg/ml sodium bicarbonate, nonessential amino acids, and 5 g/ml trypsin). three days after inoculation, the supernatants of infected cell cultures were tested for agglutination activity using turkey red blood cells as an indicator of infection of the cells. infectious titers were calculated from 5 replicates by the spearman-karber method (60) . serum cytokine and chemokine analysis. serum samples for analysis of cytokine/chemokine levels were inactivated with gamma radiation (5 megarads) according to standard operating procedures. concentrations of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, ifn-␥, il-1␤, il-1 receptor antagonist, il-2, il-4, il-5, il-6, il-8, il-10, il-12/23 (p40), il-13, il-15, il-17, mcp-1, mip-1␣, mip-1␤, soluble cd40 ligand (scd40l), transforming growth factor ␣, tnf-␣, vascular endothelial growth factor (vegf), and il-18 were measured on a bio-plex 200 instrument (bio-rad) using the nonhuman primate cytokine milliplex 23-plex map kit (millipore) according to the manufacturer's instructions. microarray data and functional analysis. rna was extracted using qiagen micro-rneasy spin columns per the manufacturer's protocol. low-yield samples were concentrated using the rna clean and concentrator kit (zymo research, irvine, ca). as multiple lesions were collected from each animal, equal masses of rna from these samples (3 or 4 samples per animal) were pooled to investigate comprehensive signatures from multiple pathological sites within the same individual. probe labeling was carried out using the agilent low-input processing protocol and hybridized to agilent rhesus macaque 8ϫ60k microarrays (agilent technologies, santa clara, ca) using the manufacturer's one-color analysis protocol. for comparisons of differentially expressed genes (deg) in infected lungs and lung lesions, raw array data were uploaded to genedata analyst 7.6 (genedata inc., san francisco, ca). data were normalized using the quantile normalization method, and the log 2 ratio expression was calculated relative to the mean probe values of the 4 right lower lung lobe samples per time point (lesion versus lung comparisons). statistically significant deg were identified using welch's t test (p ͻ 0.05; fold change, ն1.5). hierarchical clustering of deg was performed by the unweighted average method (unweighted pair group with arithmetic mean [upgma]) using spotfire decisionsite 9.1.1 (tibco, somerville, ma). analysis of functional enrichment was performed using ingenuity pathway analysis (ipa) software (ingenuity systems, redwood city, ca), and upstream drug efficacy predictions were made using both the upstream analysis function of ipa and connectivity map 02 (broad institute, cambridge, ma). drugs predicted to inhibit pathological host responses were selected based on the activation z score. negative activation z scores are predicted to cause opposite or inhibitory effects on significant genes associated with pathology in the lesions, and in this study we sought compounds with activation z scores less than ϫ2 and a p value less than 0.05. these findings were confirmed using connectivity map (cmap) (22) . cmap assigns enrichment scores ranging between ϫ1 and 1, and we accepted compounds with cmap enrichment scores less than ϫ0.1. these are compounds inducing transcriptional profiles that are negatively connected with the gene expression signature associated with lung pathology, confirming that these drugs may induce a transcriptional profile that is inhibitory to tissue damage and lesion formation. antiviral assay. confluent mdck cells in 24-well culture plates were infected in triplicate with influenza virus a/anhui/1/2013 at a multiplicity of infection of 0.001. after 1 h at 37°c, cells were washed once with pbs, and infection medium containing rosiglitazone (0 to 100 m) was added to the cells. cells were incubated for 24 h; supernatant was then harvested, stored at ϫ80°c for subsequent virus titration, and replaced with fresh infection medium containing rosiglitazone. supernatant was again collected at 72 h after infection and stored at ϫ80°c for subsequent virus titration. to determine a potential cytotoxic effect of rosiglitazone, mdck cells were simultaneously plated in 96-well culture plates and treated with rosiglitazone (0 to 100 m). after 24 h incubation, cytotoxicity was tested using the celltiter 96 aqueous one-solution cell proliferation assay (mts) (promega) according to the manufacturer's instructions. microarray data accession number. raw microarray data have been deposited in ncbi's gene expression omnibus database (gse48976) and are also available to the public at http://viromics.washington.edu. supplemental material for this article may be found at http://mbio.asm.org /lookup/suppl/doi:10.1128/mbio.01331-14/-/dcsupplemental. figure s1 , tif file, 0.1 mb. figure s2 , tif file, 0.1 mb. human infection with a novel avian-origin influenza a (h7n9) virus confirmed human cases of avian influenza a(h7n9) reported to who. world health organization clinical findings in 111 cases of influenza a (h7n9) virus infection clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h7n9) virus past, present, and possible future human infection with influenza virus a subtype h7 avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome genetic analysis of novel avian a(h7n9) influenza viruses isolated from patients in china the genesis and source of the h7n9 influenza viruses causing human infections in china genomic signature and protein sequence analysis of a novel influenza a (h7n9) virus that causes an outbreak in humans in china pathogenesis and transmission of avian influenza a (h7n9) virus in ferrets and mice limited airborne transmission of h7n9 influenza a virus between ferrets characterization of h7n9 influenza a 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role for the cell cycle in 2009 pandemic influenza virus-induced inflammation in macaque lungs the connectivity map: using gene-expression signatures to connect small molecules, genes, and disease inhibition of rotavirus infection in cultured cells by n-acetyl-cysteine, ppargamma agonists and nsaids inhibitory effect on hepatitis b virus in vitro by a peroxisome proliferator-activated receptor-gamma ligand, rosiglitazone hepatitis c virus ns5a protein increases hepatic lipid accumulation via induction of activation and expression of ppargamma pparalpha and ppargamma effectively protect against hiv-induced inflammatory responses in brain endothelial cells activation of peroxisome proliferator-activated receptor gamma (ppargamma) suppresses rho gtpases in human brain microvascular endothelial cells and inhibits adhesion and transendothelial migration of hiv-1 infected monocytes rosiglitazone, a peroxisome proliferatoractivated receptor-gamma agonist, attenuates acrolein-induced airway mucus hypersecretion in rats anti-inflammatory effects of thiazolidinediones in human airway smooth muscle cells modulation of airway remodeling and airway inflammation by peroxisome proliferator-activated receptor gamma in a murine model of toluene diisocyanate-induced asthma role of claudin-5 in the attenuation of murine acute lung injury by simvastatin statins decrease lung inflammation in mice by upregulating tetraspanin cd9 in macrophages simvastatin inhibits smoke-induced airway epithelial injury: implications for copd therapy differential effects of simvastatin on il-13-induced cytokine gene expression in primary mouse tracheal epithelial cells inhibitory effects of simvastatin on staphylococcus aureus lipoteichoic acid-induced inflammation in human alveolar macrophages impact of oral simvastatin therapy on acute lung injury in mice during pneumococcal pneumonia simvastatin is protective during staphylococcus aureus pneumonia simvastatin and oseltamivir combination therapy does not improve the effectiveness of oseltamivir alone following highly pathogenic avian h5n1 influenza virus infection in mice simvastatin treatment showed no prophylactic effect in influenza virusinfected mice effect of statin treatments on highly pathogenic avian influenza h5n1, seasonal and h1n1pdm09 virus infections in balb/c mice novel avian-origin influenza a (h7n9) virus attaches to epithelium in both upper and lower respiratory tract of humans tropism and innate host responses of a novel avian influenza a h7n9 virus: an analysis of ex-vivo and in-vitro cultures of the human respiratory tract association between adverse clinical outcome in human disease caused by novel influenza a h7n9 virus and sustained viral shedding and emergence of antiviral resistance r292k substitution and drug susceptibility of influenza a(h7n9) viruses resistance to neuraminidase inhibitors conferred by an r292k mutation in a human influenza virus h7n9 isolate can be masked by a mixed r/k viral population cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus pandemic 2009 h1n1 influenza virus causes diffuse alveolar damage in cynomolgus macaques induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines seasonal h3n2 influenza a virus fails to enhance staphylococcus aureus co-infection in a non-human primate respiratory tract infection model aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus pathogenesis of influenza a (h5n1) virus infection in a primate model receptor specificity in human, avian, and equine h2 and h3 influenza virus isolates early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice avian-type receptor-binding ability can increase influenza virus pathogenicity in macaques airborne transmission of influenza a/h5n1 virus between ferrets thoracic radiography as a refinement methodology for the study of h1n1 influenza in cynomologus macaques (macaca fascicularis) practical considerations for high-throughput influenza a virus surveillance studies of wild birds by use of molecular diagnostic tests beitrag zur kollektiven behandlung pharmakologischer reihenversuch [a contribution to the collective treatment of pharmacological experimental series key: cord-350593-bvmg7f15 authors: mcdonald, r.s.; sambol, a.r.; heimbuch, b.k.; brown, t.l.; hinrichs, s.h.; wander, j.d. title: proportional mouse model for aerosol infection by influenza date: 2012-08-21 journal: j appl microbiol doi: 10.1111/j.1365-2672.2012.05402.x sha: doc_id: 350593 cord_uid: bvmg7f15 aims: the aim of this study was to demonstrate a prototype tool for measuring infectivity of an aerosolized human pathogen – influenza a/pr/8/34 (h1n1) virus – using a small‐animal model in the controlled aerosol test system (cats). methods and results: intranasal inoculation of nonadapted h1n1 virus into c57bl, balb/c and cd‐1 mice caused infection in all three species. respiratory exposure of cd‐1 mice to the aerosolized virus at graduated doses was accomplished in a modified rodent exposure apparatus. weight change was recorded for 7 days postexposure, and viral populations in lung tissue homogenates were measured post mortem by dna amplification (qrt‐pcr), direct fluorescence and microscopic evaluation of cytopathic effect. plots of weight change and of pcr cycle threshold vs delivered dose were linear to threshold doses of ~40 tcid (50) and ~12 tcid (50), respectively. conclusions: mid (50) for inspired h1n1 aerosols in cd‐1 mice is between 12 and 40 tcid (50); proportionality to dose of weight loss and viral populations makes the cd‐1 mouse a useful model for measuring infectivity by inhalation. significance and impact of the study: in the cats, this mouse–virus model provides the first quantitative method to evaluate the ability of respiratory protective technologies to attenuate the infectivity of an inspired pathogenic aerosol. whereas aerosols and contact are accepted as modes of transmitting many disease-causing organisms, including legionella pneumophila, smallpox, severe acute respiratory syndrome (sars), coronaviruses, rhinovirus (fiegel et al. 2006 ) and tuberculosis (wells 1934; riley et al. 1959; mcclement and christianson 1980) , the role of bioaerosols as a transmission mechanism for influenza is less clearly understood (tellier 2006; tellier 2007a,b; lemieux and brankston, 2007; brankston et al. 2007; tang and li, 2007; lee 2007) . although a few publications have documented the transmissibility of influenza a through inhalation routes (tellier 2006 (tellier , 2009 , few studies to date have utilized a mouse model to investigate susceptibility to and pathogenicity of measured aerosol exposures. the lack of aerobiology studies results from several factors, including the need for specialized equipment to generate and monitor bioaerosols, the technical difficulty involved, inconsistency among reported techniques (lore et al. 2011 ) and the considerable cost of conducting this research (sherwood et al. 1988) . therefore, the most commonly described method of infecting mice with influenza virus is through the installation of fluid into the nasal passages (lu et al. 1999; govorkova et al. 2007; gillim-ross et al. 2008; chen et al. 2011) . for more than 75 years, laboratory mice have served as models for susceptibility to and pathogenesis of influenza disease (andrewes et al. 1934) . their low cost, small size, relative susceptibility to the virus and ease of handling make mice a favourable platform for studying influenza virus infections. the mouse is currently considered the primary model for the evaluation of influenza antiviral agents because it is a predictive indicator of the efficacy of such treatments in humans (sidwell and smee 2004) . the use of a well-characterized mouse model is especially important in studying the infectious pathways of new pandemic (pdm) subtypes of influenza a. indeed, the latest emergence of influenza has reignited interest in the use of mouse models (beigel and bray 2008) . although mathematical models have been used for decades (findeisen 1935; yeh et al. 1976 ; international commission on radiological protection (icrp) 1994; asgharian and anjilvel 1998; heyder 2004) to calculate particle deposition within the respiratory tract, such calculations of particle placement are able to rationalize but not to predict the resulting clinical effect. animal models allow closer approximation to a human response (schulman 1968; lowen et al. 2006; gustin et al. 2011) , and therefore, it is important to continue to further develop these models kawaoka 2012) . experimental inhalation exposure systems are an established tool and the subject of several reviews (drew and laskin 1973; macfarland 1983; cheng and moss 1995; jaeger et al. 2006; wong 2007) . the purpose of this study was to identify and evaluate a mouse model as a complement to a measured-dose bioaerosol delivery apparatus termed cats (controlled aerosol test system) for testing the clinical effectiveness of media used in respiratory protective equipment (rpe). this validation, which includes complementary data measured postmortem, renders the mice available to serve as a detector to evaluate the clinical significance of articles of rpe by directly measuring the change in infectivity the protective article causes. exposure to influenza virus often leads to a disease presenting as an acute and temporarily incapacitating infection of the upper respiratory tract that can be fatal. influenza illness is often associated with occurrences of annual or near-annual epidemics in temperate climate zones. within the last 100 years, influenza pandemics have occurred four times [1918 (h1n1), 1957 (h2n2), 1968 (h3n2) and 2009 (h1n1)] (oxford 2000) . pandemics are infrequent but often severe events because of the emergence of novel, unpredictable strains of influenza a virus caused by recombination of genetic material from two or more circulating virus subtypes. this antigenic shift can often lead to a new virus subtype with the ability to jump from one species into another, potentially naive species (e.g. avian influenza), and cause a large proportion of influenza-related deaths. a number of animal models have been studied to evaluate new vaccines and other approaches for preventing influenza-related disease (gubareva et al. 1998; ng et al. 2010) . green and kass (1964) conducted studies on the clearance of inhaled microbial aerosols from the murine respiratory tract. schulman and kilbourne (1963) studied mouse-to-mouse transmissibility of influenza virus. a factor complicating viral research in animal models is that a virus may be present in a host without causing disease. this may be due to restrictions such as the absence of appropriate receptors on certain cell types (e.g. tissue tropism) and the lack of intracellular processes required to generate infectious progeny viruses or induce cytolytic effects. differences in viral receptors have been documented for respiratory epithelial cells based on location in either the pharynx or peripheral lung (van riel et al. 2007 (van riel et al. , 2010 . in addition, either the organism or host cell may mount an immune response or generate intracellular molecules that disrupt the viral effects. therefore, differences may appear at either the cellular or tissue level or among susceptible hosts depending upon the method of infection, especially in regard to aerosol exposures (phalen et al. 2008) . this study examined these parameters related to efficient (near threshold) infection versus overwhelming infection of mice by exposure to aerosolized influenza virus. the cats is an apparatus that was designed, constructed and validated (stone 2010; stone et al., 2012) to deliver a precisely measured aerosol concentration, either directly or after passage through a filter medium, through a nose-only directed-flow inhalation exposure system (noies; ch technologies, westwood, nj, usa, jaeger et al. 2006) to individual mice (figs 1 and 2) . this low-flow, singlepass design consists of an aerosol generator, diffusion drier, charge neutralizer, filter holder, sampling points and noies unit (stone 2010; stone et al., 2012) . the cats generates a biological aerosol over a range of constant concentrations andafter conditioning and treatment, if any is applieddelivers the particles to the nose of a mouse constrained in a polycarbonate tube (cht-247; ch technologies) as a pure respiratory exposure. the main system components were connected using 0á5-inch (12á7-mm) stainless steel tubing with a minimum number of gradual bends. in operation, a single-jet collison nebulizer (bgi inc., waltham, ma, usa), regulated to 25-30 psi was used to atomize the viral suspensions. the aerosol, which acquires surface charges during atomization, passes through a 9á5-inch (23-cm) diffusion drier and then through a 2-mci 85 kr charge neutralizer (tsi inc., shoreview, mn, usa) to restore the 'normal' boltzmann equilibrium charge distribution. after passage through the filter holder and any filter medium mounted in it, the aerosol enters the 12-port noies, from which it exits the test system through the hepa-filtered exhaust. total flow rate through the system was regulated at the nebulizer to deliver 2á0 ± 0á1 l min à1 measured on exit by a mass flow metre (tsi model 4043e). the entire system was designed to fit inside a biological safety cabinet (baker company, sanford, me, usa; sg603-ats) for additional protection from generated aerosols. the unique feature of the system is an optional filter holder (triosyn corp, williston, vt, usa), which is capable of holding filter media samples 47 mm in diameter. smaller discs of filter media can be accommodated with the use of reducers. correlation of sampling ports stone et al. (2012 , stone 2010 ) demonstrated uniform distribution of aerosol to several ports of the cats. following transport and installation of the cats in an animal biosafety level 3 (abl3) facility at the university of nebraska medical center, the exposure system was retested to verify consistency of particle counts among all 12 ports, using tap water to generate test aerosol particles. from each of the 12 exposure ports, samples of particles delivered by a single-jet collison nebulizer were measured in triplicate using a scanning mobility particle sizer (smps) system (tsi inc., minneapolis, mn, usa). the aerosol particle size and concentration were determined using 6á0 l min à1 sheath air and 0á6 l min à1 sample air. data outputs from the smps were collected by the aerosol instrument manager ® software (ver. 8.1.0.0; tsi inc., minneapolis, mn, usa). influenza a/pr/8/34 (h1n1) virus was obtained from american type culture collection (rockville, md, usa) as frozen stock (atcc vr-1469). virus was propagated using cdc unit 15g.1 protocol (szretter et al. 2006) . titres were performed and calculated using the spearman-kärber method (armitage and allen 1950; finney 1964) . to assess the psd of aerosols containing influenza virus, samples were taken in triplicate from the sampling port located downstream from the cats, diluted with filtered air and routed to the smps. results indicated a single-mode, polydisperse aerosol in the size range 10-400 nm. three strains of female mice (c57bl, balb/c and cd-1) were purchased from charles river laboratories (portage facility, mi, usa). the mice were 6-8 weeks old and ranged in weight from 18 to 30 g. mice were randomly divided into groups assigned to specific exposure time points, and no more than five (all in a given exposure group) were housed per cage. animals were provided rodent chow (harlan teklad, usa) and water ad libitum and maintained on a 12-h light/dark cycle. all animal work was carried out in an abl3 facility following institutional and regulatory procedures. to minimize artefacts caused by stress during respiratory exposure sessions, mice were preconditioned daily during the week preceding their exposure sessions (nrc 2003 (nrc , 2011 ) by insertion into a mouse restraint device (ch247; ch technologies) for a period that did not exceed the maximum exposure time for that experiment. to select a suitable mouse strain for infection with the influenza a/pr/8/34 (h1n1) (not mouse-adapted) strain used in this study, two inbred (balb/c and c57bl) and one semi-outbred strain (cd-1) of 20-25 g female mice were tested for susceptibility to infection by the virus. individual base weights were determined prior to exposure, and all mice were weighed daily at a uniform, scheduled time throughout the study. the average weights from surviving exposed mice at day 7 were compared to the averages of control mice. all mice were euthanized by day 7 postinoculation. the inoculum, consisting of 30 ll of virus at a concentration of 4á74 9 10 7 median tissue culture infectious dose (tcid 50 ) ml à1 , was placed intranasally into each mouse. the dose was divided equally and placed dropletby-droplet by pipette into each naris of the anesthetized (ketamine/xylazine) mouse. following the same procedure, a 1 : 10 dilution of virus stock in 19 phosphatebuffered saline (pbs) medium was used to inoculate a second set of mice of the same three strains. in all, five mice per strain per dilution were used to determine susceptibility to the virus. three mice per strain were used as controls. each control mouse was intranasally inoculated with 30 ll total 1 9 pbs medium as previously described (jerrells et al. 2007) . in a preliminary study conducted to establish a baseline dose of virus capable of causing infection following aerosolization, the working stock of influenza virus was diluted 1 : 30 in endotoxin-free water (sigma, st. louis, mo, usa) and delivered into the collison nebulizer at 1á58 9 10 6 tcid 50 ml à1 . four sets of three cd-1 mice were emplaced in polycarbonate restraints, installed into the noies with the filter holder empty, and exposed to aerosolized virus at exposure times of 2, 6, 20 and 60 min. aliquots of the influenza working stock (4á74 9 10 7 tcid 50 ml à1 titre) were subsequently diluted to 1 : 300 and 1 : 1000 (v/v) in endotoxin-free water to prepare concentrations aerosolized during three successive mouse exposure series described below. the single-jet collison nebulizer was charged and allowed to run for 5 min to stabilize the system. the bypass valve directly upstream of the cats directed the aerosol through two hepa filters connected in series until exposure was initiated. nonanesthetized mice were carefully immobilized in the polycarbonate tubes so that the tip of the nose projected out of an opening in the front of the holder. the tubes were then inserted securely into a port on the cats. once the animals were emplaced, the test aerosol was directed through the system. vents inside the cavity of the cats directed an airstream containing the filtered aerosol at the nares of the mouse as her only source of breathing air. excess aerosol flow and exhaled air were continuously swept away to preclude inhalation of previously exhaled air. a spread of delivered doses (proportional to concentration, c, 9 time, t) around each dilution was achieved by varying the time of exposure. the bioaerosol dose received is calculated as follows: where vsf is the ratio of viable airborne counts, in tcid 50 ml à1 , to viable counts, in tcid 50 ml à1 , in the collison reservoir, and r a is the respiration rate per minute, and v a is the tidal volume in ml a (ml of air), respectively, of the cd-1 mouse. r a and v a are reported (fairchild 1972) to be 261 respirations/min and 0á16 ml a , respectively. thus, a mouse exposed for 2 min to a 300 : 1 dilution of a suspension containing 4á74 9 10 7 tcid 50 ml à1 of virus inhales a dose of d p ¼ 4á74 â 10 7 tcid 50 ml à1 â 1=300 â 9 â 10 à7 ml ml à1 a â 261 resp min à1 â 0á16 ml a resp à1 â 2 min ¼ 12 tcid 50 mice were exposed in groups for each preselected time (tables 2-4 ). at the end of the exposure period, the polycarbonate tubes holding the mice were removed, and the next group was inserted, until all mice for that series of experiments were exposed. when time points allowed, the mice were inserted in overlapping groups. unused ports were sealed with the supplier's standard plugs. all exposures were carried out within a biological safety cabinet. control mice for 1 : 30 (1á58 9 10 6 tcid 50 ml à1 ) and 1 : 300 (1á58 9 10 5 tcid 50 ml à1 ) exposure groups were placed in polycarbonate tubes during the testing equal to the maximum exposure time and exposed to aerosols generated from endotoxin-free water (sigma) containing no virus. for the 1 : 1000 (4á74 9 10 4 tcid 50 ) exposure group, two sets of controls were used. one mouse group was exposed as earlier, while a second group was exposed to uninfected allantoic fluid processed in the same manner as from influenza-infected eggs. mice were observed and weighed each of 7 days postexposure. severely distressed mice were euthanized after the day's weighing and, following the final weighing, all surviving mice were euthanized by administration of an overdose of ketamine/xylazine by intraperitoneal injection. a necropsy was conducted and selected portions of the lungs were selected for molecular, histological or virus culture assessment. lung tissues aseptically placed into 2á7 ml of cold bd universal virus transport medium (becton, dickinson and co., franklin lakes, nj, usa) were homogenized by hand using a closed ultra tissue grinder system (fisher scientific, pittsburgh, pa, usa) and then stored at à80°c. cell culture and molecular assays tcid 50 /cpe and dfa assays starting viral titres were quantified by cell culture endpoint-dilution assays performed using madin-darby canine kidney (mdck) cells and calculated using the spearman-kärber method in units of log 10 tcid 50 ml à1 . cell culture plates containing mdck cells were grown and maintained using standard cell culture techniques. presence of viable virus in homogenates of murine lung tissue was qualitatively assessed by two-concentration cell culture endpoint assays performed using mdck cells. cell culture plates containing mdck cells were grown and maintained using standard cell culture techniques. aliquots (1á0 ml) of lung homogenates were plated in serial 1 : 10 dilutions (in serum-free eagle's minimal essential medium (emem)) from 10 à1 to 10 à4 in quadruplicate on confluent cell monolayers. the samples remained in contact with the monolayer for a 1-h incubation before 1% bsa-serumfree emem with trypsin was added (bovine serum albumin). the plates were incubated for 5-6 days under 5% co 2 at 37°c prior to visualization under the microscope for cytopathic effect (cpe) or fluorescent-labelled antibody evaluation. test plates were read using a +/à system, in which + showed disruption of the monolayer and à showed that the monolayer remained confluent. direct fluorescent antibody assay (dfa) was used to qualitatively determine influenza infection of the mdck cell line using the d 3 ultra dfa respiratory virus screening and id kit (diagnostic hybrids inc., athens, oh, usa) per manufacturer's instructions. rna extraction and qrt-pcr ribonucleic acid (rna) was extracted from samples using the qiaamp ® minelute ® virus spin kit following the manufacturer's protocol (qiagen, valencia, ca, usa). rna amplification was performed using invitrogen's superscript iii platinum one-step quantitative real-time polymerase chain reaction (qrt-pcr) kit (invitrogen, grand island, ny, usa). the qrt-pcr assay was run on the roche lightcycler ® 480 real-time pcr system (roche diagnostics, indianapolis, in). assay conditions and reaction volumes were used from protocols previously described by the cdc (who 2009 ). the cycle threshold (ct) values from replicate runs were averaged for each time point and rounded to two decimal places. the recommended cut-off ct value of 30 was used as the criterion for infection. following fixation and routine processing, tissue sections were stained with hematoxylin and eosin and reviewed under standard light microscopy. compared to normal tissue, influenza-infected lungs showed lobular pneumonia with interbronchial inflammation. infected lungs also showed focal chronic inflammatory cell infiltration with a few neutrophils and some interstitial thickening. figure 3 shows the infiltrates in the infected tissue compared to uninfected tissue. after installation in the abl3 cabinet, a reverification of cats performance was conducted with water. the par-ticle counts at each port were averaged and again seen to be uniform within 10% (data not shown). a subsequent delivery of 100 mg l à1 sodium chloride in water showed number mean diameter (d 50,n ) = 74 nm and mass mean diameter (d 50,m ) = 208 nm over the range of particle diameters from 10 to 407 nm. figure 4 plots the coefficient of variation (cov) as a function of particle size at the 12 ports for the nacl aerosol measurements. groups of five mice were inoculated intranasally with one of two doses of virus and weighed daily for a week. per cent changes in average weights of exposed and control groups are indicated in table 1 . the nonmouse-adapted influenza virus produced obvious infection in all three strains of mice used. as no difference in gross infectivity was indicated by weight loss (table 1) , the less-expensive cd-1 mice were selected for further study. aerosol exposure (1á58 9 10 6 tcid 50 ml à1 ) two mice were used as unexposed controls. all of the mice survived to day 7, when they were euthanized and necropsied, and their lung tissue was examined by three different assays. at all four exposure time points, mouse lung tissues gave positive results from the qrt-pcr assay, for which a positive value was defined to be 31 ct. dfa and cpe assays on the lung tissue were also all positive ( table 2 ). there was a direct correlation between dose received (for convenience reckoned as ct values (product of concentration and exposure time)), with lower pcr ct values resulting from prolonged exposure. the mean ct value for control mice was 37, which was defined to be a negative response. values of 37 > ct > 31 were considered indeterminate. weight losses again showed proportionality to dose delivered. the results demonstrated that the influenza virus remained viable and capable of causing infection in cd-1 mice when aerosolized under test conditions. aerosol exposure (1á58 9 10 5 tcid 50 ml à1 ) because exposure to the 1 : 30 dilution of aerosolized virus resulted in massive but graduated infection of all tested mice, a greater dilution of the working stock was delivered in an effort to identify a threshold infective dose. the dilution was increased tenfold (to 1 : 300, resulting in delivery of a 1á58 9 10 5 tcid 50 ml à1 dispersion from the nebulizer and an overlap (as ct products) of the two smaller doses from the 1 : 30 dilutions), and the initial aerosol exposure sequence was repeated. three mice per time point were assayed by qrt-pcr. variation in ct values was observed in the 2-min exposure group, one mouse being clearly positive as reflected by ct values, and the other two mice falling within the indeterminate range (table 3 ). all mice in the 6-, 20-and 60-min time points were positive (<31 ct) with minimal variation in ct values. all control mice were negative (ct values ! 37). tcid 50 assays were performed on mice from group 1 at each time point. one mouse lung homogenate in group 1 that was indeterminate by qrt-pcr (36 ct) was negative by tcid 50 assay. all other lung homogenates tested positive by both methods. aerosol exposure (4á7 9 10 4 tcid 50 ml à1 ) although weight loss by the mice appeared to have reached baseline at the dose delivered in 6 min at 1 : 300 dilution, ct results from the aerosol challenge at 1 : 300 dilution show that all the mice exposed for 6 min or more received an infectious dosethat is, weight loss is an indicator of dose but ct measurements provide better sensitivity to detect an endpoint. in an effort to better define the threshold at which viral infection occurs, the stock suspension was further diluted to 4á7 9 10 4 tcid 50 ml à1 (1 : 1000), the exposure times were reduced, the number of mice per time group was increased to five, and two additional time points were added to increase the range and dose of aerosol exposure. results of this test are shown in table 4 . 4á74 9 10 7 tcid 50 ml à1 4á74 9 10 6 tcid 50 ml à1 controls sd, standard deviation. *one mouse died before day 7. table 2 results of three assays [pcr, direct fluorescent antibody assay (dfa) and cpe] from the homogenates of cd-1 murine lung tissue exposed to an aerosol generated from 1á58 9 10 6 tcid 50 ml à1 at the 3-min exposure time, no mice were positive for influenza virus as determined by ct value. in all four of the longer-exposure groups, a single mouse displayed a positive ct value. a trend may be suggested by the pattern of indeterminate values, but the quantitative ct values show an equally unconvincing opposite trend. all of the lung homogenates were tested by virus cell culture assay for tcid 50 and dfa. for homogenates whose ct value is <31, both tcid 50 and dfa were positive. it is accepted that influenza may be contracted through a variety of methods including large droplet and contact transmission. the only route of infection examined during this study was inhalation of droplet nuclei through nasal passageways. results should be expected to be different if other mucosal surfaces had been dosed with the same viral aerosols. this study sought only to determine the existence and scale of a measurable threshold aerosol infective dose in this animal model and to set parameters such as the mouse restraints in the experimentthat limit other exposure. although this study demonstrated infectivity of the aerosol, its residence time as dispersed fine particles was shorthundreds of milliseconds from nozzle to noseand viruses are known to spontaneously lose viability, so the importance of such bioaerosols as an environmental component remains uncertain. however, the experiment accurately simulates direct exposure to droplet nuclei generated by a cough, which can accordingly be concluded to be a mechanism for immediate transmission of this virus. the range of states resulting from influenza virus spans from asymptomatic infection to mild symptoms to pneumonia, which is often fatal. factors such as the strain of influenza virus that caused the illness, immune status of the host and/or age of the affected individual play an important role in recovery or progression of disease. this was evident in the 2009 outbreak of influenza with the influenza a virus h1n1 pandemic (pdm) strain, during which a disproportionately high percentage of morbidity occurred in children and young adults as well as in individuals with underlying conditions such as obesity and or diabetes (jhung et al. 2011) . influenza a (h1n1) virus selected for this study was chosen based on reports of its infectivity in mice (smee et al. 2008 )although it had not been mouse adaptedand its known hardiness in culture systems as a starting point for development of a mouse model for aerosolized influenza. our results showed a significant variability in morbidity and mortality among the mice exposed to aerosolized influenza virus. this appears to have been owing to individual susceptibility of the mice, because variability in the uniformity of the aerosol delivered was found not to be significant when each of the ports was analysed. in addition, significant differences were noted when mice were exposed over times ranging from 6 to 18 min with minimal or no morbidity when a low quantity of virus was sprayed over an extended period of time. our preferred explanation is that some of the mice were able to process and clear virus, while in others, it caused clinical infection and disease. additionally, the hardiness of immune response to influenza varies among the mice, resulting in different levels of susceptibility. for example, in table 3 at the lowest exposure time, one of the three mice was positive for infection, whereas in the lowest dose experiment (table 4) , one mouse was positive following only 6 min of exposure. as with many viruses, influenza produces a significant number of defective particles incapable of causing infection (huang 1973; pathak and nagy 2009) . this is further demonstrated by the wide variationranging from hundreds to thousandsin reported gene copy (total virions)-to-tcid 50 (infectious virion fraction) ratio (yang et al. 2011 ). sidorenko and reichl (2004) table 4 pcr ct values from homogenates of cd-1 murine lung tissue exposed to aerosol generated from a 4á74 9 10 4 tcid 50 ml à1 dilution of influenza virus over five different exposure times are indicated in parentheses: x = mouse death; pos = positive, ct < 31; ind = indeterminate, 37 > ct ! 31; and neg = negative, ct ! 37 level for influenza a (h1n1) to be 1 tcid 50 for using qrt-pcr and 0á1 tcid 50 using nested qrt-pcr. recognizing that a direct correlation may not always exist between a method that detects viable organisms and one based on viral genomes, our system showed excellent correlation between classical virology methods, morphology based on histological examination, clinical features and molecular quantification by qrt-pcr. influenza infection in mice has been monitored by several different parameters including mean time to death, lung weight and change in body weight (sidwell and smee 2000) . however, these indicators are difficult to interpret when the infectivity and challenge dose of the virus does not clinically manifest an illness (morbidity or mortality). therefore, we utilized qrt-pcr in comparison with tcid 50 to minimize the variability. virus replication in lung tissue is considered the most informative endpoint for efficacy studies because even modest changes in virus load can have a large impact on survivability (haga and horimoto 2010) . assays of postsacrifice tissue samples from the mice were uniformly positive in the 1 : 30 dilution series. in contrast with the data shown in fig. 4 , in the 1 : 300 dilution series only, the 2-min exposure (12 tcid 50 dose) group contained subjects that were not unequivocally positive for infection, both by ct (2 of 3) and by cpe (1 of 3). one-way anova and a two-tailed t-test did not find any statistical differences between the change in the mouse's weight and the pcr data; however, a more precise threshold value and statistical significance of this difference can be expected when the number of mice (n) in each exposure group is increased. likewise, the two sets of subjects that shared ct products (2 min 9 1 : 30 and 20 min 9 1 : 300, and 6 min 9 1 : 30 and 60 min 9 1 : 300) appear to show increased sensitivity with increasing aerosol concentration, which could be taken to imply that an acute exposure leads to greater infectivity than the same dose experienced more gradually. although this interpretation is intuitively reasonable, the volume of data supporting it is too limited to justify such a conclusion. owing to logistical constraints in the abl-3 facility, the psd was not measured during the exposures. however, stone et al. (2012; stone 2010 ) measured bioaerosol particles in the range 100~500 nm for a slightly smaller virus (ms2 coli phage) in the same apparatus. the absorbed dose was likely slightly smaller than the presented dose because deposition of inspired particles in this size range is incomplete and size dependent (stuart 1973; clay and clarke 1987; heyder 2004) . a plot of weight loss vs calculated inhaled dose (fig. 5 ) was fitted to a straight line, which intersects the mean weight change of the control group at approximately 40 tcid 50 . a third series of exposures was performed to a 1 : 1000 dilution, intended to improve definition of the threshold dose for weight loss; however, the results were equivocal, likely because the delivery was gradual enough that the mice developed an immune response that was able to manage the challenge, and/or the n of five was too small to average out what we presume to have been idiosyncrasies among the subjects. our results showed that qrt-pcr was more sensitive or that an excess of genome was present in comparison with the number of infectious virions as determined by tcid 50 and dfa assays. as the gold standard (schrauwen et al. 2011) for determining the moi has been tcid 50 and quantification of virus in mice exposed to influenza aerosols by qrt-pcr has not been previously reported, additional confirmatory studies were needed. we chose seven days as the terminal point for our study based on symptomatology in humans where virus production peaks approximately 48 h postinfection, and few virus particles are shed after day 6 (taubenberger and morens 2008). our results showed the delivered aerosol mid 50 to be at least 12 tcid 50 as determined by qrt-pcr ct value and significantly <40 tcid 50 as determined by obvious clinical response. however, the sample size must be expanded in the future to achieve greater resolution and statistical significance. in addition, future studies will utilize the influenza virus a (h1n1) pdm strain to determine variation in mid 50 between the two strains. the data and methods presented here contribute to a fundamental basis for refining studies of aerosol delivery of particles into animal models for study of a variety of clinical subjects, such as infectious doses and vaccine delivery. further work will be needed to more precisely define the median infective dose (mid 50 ) of the current influenza strain in the cd-1 mouse, and to better understand the effect of bioaerosol ageing and dose rate on infectivity. work presented herein validates aerosolization of one organism and delivery by a pure respiratory pathway into one murine host as a technique for assaying infectivity in the challenging bioaerosol. this work can serve as a starting point for a continuation of work using other microbial organisms and other animal hosts. the intended application of the aerosol influenza animal model described here is the assessment of the clinical effect of respiratory protection devices incorporating antimicrobial treatments. various approaches have been proposed to increase the effectiveness of respiratory filtering media including the addition of bioactive media. although materials such as silver nanoparticles (lala et al. 2007) , copper oxide (borkow et al. 2010) , iodinated compounds (heimbuch and wander 2006) and others (cecchini et al. 2004) have shown biocidal potential, only the iodine vector has been proposed (lee et al. 2009 ) to operate by a noncontact mechanism. additional studies will focus on evaluating such new technologies and, after replacement of the filter holder with a larger enclosure able to collect aerosols behind a filtering facepiece respirator (ffr) worn by an articulated headform, on quantifying the effect on protectivity of seal leakage and on optimizing the particle removal efficiency of ffrs and other rpe to maximize net protectivity. the susceptibility of mice to the viruses of human and swine influenza methods of estimating the ld 50 in 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influenza virus factors influencing the clearance of bacteria by the lung characterization of influenza a/hongkong/156/ 97 (h5n1) virus in a mouse model and protective effect of zanamivir on h5n1 infection in mice influenza virus aerosol exposure and analytical system for ferrets animal models to study influenza virus pathogenesis and control bioaerosol challenges to antimicrobial surface treatments: enchanced efficacy against ms2 coli phage of air filter media coated with polystyrene-4-methyltrimethylammonium triiodide deposition of inhaled in the human respiratory tract and consequences for regional targeting in respiratory drug delivery international commission on radiological protection (icrp). 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pathogenesis and immunity to influenza a (h5n1) viruses isolated from humans designs and operational characteristics of inhalation exposure equipment -a review clinical forms of tb prevention and treatment of influenza with hyperimmune bovine colostrum antibody guidelines for the care and use of mammals in neuroscience and behavioral research guide for the care and use of laboratory animals influenza a pandemics of the 20th century with special reference to 1918: virology, pathology and epidemiology defective interfering rnas: foes of viruses and friends of virologists nested real-time pcr assay has an increased sensitivity suitable for detection of viruses in aerosol studies the relevance of animal models for aerosol studies human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses aerial dissemination of pulmonary tuberculosis. a two-year study of contagion in a tuberculosis ward possible increased pathogenicity of pandemic (h1n1) 2009 influenza virus upon reassortment the use of an animal model to study transmission of influenza virus infection experimental transmission of influenza virus infection in mice. i. the period of transmissibility comparison of streptococcus zooepidemicus and influenza virus pathogenicity in mice by three pulmonary exposure routes structured model of influenza virus replication in mdck cells in vitro and in vivo assay systems for study of influenza virus inhibitors experimental disease models of influenza virus infections: recent developments treatment of influenza a (h1n1) virus infections in mice and ferrets with cyanovirin-n consistency and reproducibility of bioaerosol delivery for infectivity studies on mice design, construction and validation of a nose-only inhalation exposure system to measure infectivity of filtered bioaerosols in mice deposition of inhaled aerosols current protocols in microbiology; unit 15g.1 influenza: propagation, quantification, and storage reflection and reactiontransmission of influenza a in human beings review of aerosol transmission of influenza a virus questioning aerosol transmission of influenza: in response reflection and reaction-transmission of influenza a in human beings aerosol transmission of influenza a virus: a review of new studies on air-borne infection. study ii. droplets and droplet nuclei cdc protocol of realtime rtpcr for influenza a(h1n1) revision 2 inhalation exposure systems: design, methods, and operation concentrations and size distributions of airborne influenza a viruses measured indoors at a health centre, a day-care centre and on aeroplanes factors influencing the deposition of inhaled particles pilgrim's pride farms (live oak, fl) donated 100 embryonic eggs from stock-breeding hens, of which 80 were used for virus propagation and 20 served as controls. this work was supported in part by contract no. fa8650-07-c-5911, funded by the defense threat reduction agency. protocols for animal use were approved by the university of nebraska medical center institutional animal care and use committee and all active participants received all required training prior to the acquisition of mice. disclaimerpublication of this work does not indicate endorsement or approval of this work by the department of defence. key: cord-326614-cik3ino6 authors: corder, brigette n.; bullard, brianna l.; poland, gregory a.; weaver, eric a. title: a decade in review: a systematic review of universal influenza vaccines in clinical trials during the 2010 decade date: 2020-10-20 journal: viruses doi: 10.3390/v12101186 sha: doc_id: 326614 cord_uid: cik3ino6 on average, there are 3–5 million severe cases of influenza virus infections globally each year. seasonal influenza vaccines provide limited protection against divergent influenza strains. therefore, the development of a universal influenza vaccine is a top priority for the nih. here, we report a comprehensive summary of all universal influenza vaccines that were tested in clinical trials during the 2010–2019 decade. of the 1597 studies found, 69 eligible clinical trials, which investigated 27 vaccines, were included in this review. information from each trial was compiled for vaccine target, vaccine platform, adjuvant inclusion, clinical trial phase, and results. as we look forward, there are currently three vaccines in phase iii clinical trials which could provide significant improvement over seasonal influenza vaccines. this systematic review of universal influenza vaccine clinical trials during the 2010–2019 decade provides an update on the progress towards an improved influenza vaccine. globally, seasonal influenza virus epidemics are estimated to cause 3-5 million cases of severe infection and result in 290,000-650,000 deaths annually [1, 2] . mortality is increased in the elderly over 65 years, children under 5 years, and people in developing countries [3, 4] . in the united states alone, influenza virus infects between 9.2-35.6 million people each year, leading to 140,000-710,000 hospitalizations [5] . these annual influenza epidemics result in an estimated total economic loss of $87.1 billion each year due to direct medical costs and indirect costs such as projected lost earnings and loss of life [6] . while the disease burden for seasonal influenza epidemics is substantial, this is significantly increased during influenza pandemics. for example, it is estimated that 24% of the worldwide population was infected during the 2009 h1n1 swine influenza pandemic [7] . a substantial challenge in the development of an effective influenza vaccine is the significant viral population diversity. the current influenza vaccine can be either trivalent or quadrivalent. the trivalent vaccine contains a h1n1, h3n2, and an influenza b strain, with the quadrivalent vaccine including both yamagata and victoria influenza b lineage strains [2, 8] . the strains contained in the seasonal influenza vaccine are updated yearly to include those predicted to circulate in the upcoming influenza season. although the current influenza vaccine is effective at reducing morbidity and mortality due to seasonal influenza infections [9] , vaccine effectiveness estimates only range from 10 to 60% [8, 10] . the vaccine effectiveness is lowest when there is poor antigenic match to the circulating influenza strains [8, 11] . relevant information was extracted from the clinicaltrials.gov database including phase, vaccine target, vaccine platform, and results. if information was not available, relevant publications were analyzed. a summary of this data is reported in table 1 . all data were analyzed using graphpad prism 8.2 software. figures were designed in adobe illustrator 2020 (24.0.1). seasonal influenza vaccines provide limited protection and are updated annually to incorporate circulating strains. a vaccine which induces broad cross-protection against influenza remains a top priority for the national institute of health (nih). here we report a comprehensive review of universal influenza vaccine (uiv) clinical trials that were active between january 2010 and december 2019. in the last decade, 69 clinical trials investigating 27 vaccines were performed ( figure 1) . these trials include a variety of viral targets, vaccine platforms, and adjuvants to boost the immune response to vaccination. table 1 reports a chronological summary for each uiv clinical trial. the unique id for each trial is used for identification in subsequent figures. importantly, several uivs were tested in up to 9 clinical trials. since the trends may be skewed by these vaccines, we differentiate between vaccines and total clinical trials throughout this paper. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix 1 (m1), can induce strong t cell responses [122] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [123] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [124, 125] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure 2 ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. internal influenza proteins are attractive vaccine candidates since they are more conserved than the external glycoproteins [124] . this may result in broader cross-protection induced by the vaccine. one example is fp-01.1, a peptide-based vaccine which includes several cd4+ and cd8+ t-cell epitopes conjugated to a fluorocarbon chain. these epitopes are derived from internal influenza proteins including np, m1, polymerase basic 1 (pb1), and polymerase basic 2 (pb2). four trials were performed in the past decade utilizing 328 participants 18-74 years old. one phase i trial demonstrated that vaccination with fp-01.1 induced strong cellular responses in 75% of participants with a median response of 243 spot forming cells (sfc)/million peripheral blood mononuclear cells (pbmc) as measured using ifnγ elispot assay [65] . this cellular response was activated against several heterologous h1n1 and h3n2 strains indicating broad cross-reactivity [65] . another uiv targeting internal proteins is ovx836, which is a recombinant np vaccine. in preclinical trials, vaccinated mice were protected against three lethal influenza a virus (iav) challenges and induced stronger immunogenicity than wild-type np alone [118] . protection was further improved if mice were immunized with a combination of the seasonal inactivated vaccine and ovx836. in the past decade, 14 of the 27 vaccines in uiv clinical trials targeted external glycoproteins. although the ha protein has a high amount of diversity in the globular head, the ha stalk region is more conserved [125] . vaccines targeting the stalk region of ha have shown promise during development and are being investigated in several uiv clinical trials [109, 110, 112, 115, 116] . one such vaccine is ch8/1n1, h5/1n1, which utilizes a prime-boost immunization strategy to promote an viruses 2020, 12, 1186 7 of 22 immune response towards the ha stalk domain. the phase 1 trial included 65 individuals between 18 and 39 years of age. two viruses were modified with chimeric ha containing a homologous ha stalk and heterologous ha heads. these were administered as live attenuated or inactivated vaccines and boosted with a heterologous ha head vaccine 85 days later [109] . an oil-in-water adjuvant, as03, was included with the inactivated vaccine. after the prime vaccination, only the adjuvanted groups induced strong igg antibody responses. however, all groups demonstrated 2.2 to 5.6-fold increases in ha stalk specific igg antibodies after a heterologous boost. these h1 ha antibodies were cross reactive to h2, h9, and h18 ha, indicating broad cross-protection against group 1 ha [109]. influenza vaccines typically target specific viral antigens to maximize the immune response to vaccination. vaccination aims to induce a strong adaptive immune response which results in both t and b cell activation. these immune cells produce cytotoxic t cells and antibodies which can protect against future infection. vaccines targeting internal viral proteins, such as nucleoprotein (np) and matrix 1 (m1), can induce strong t cell responses [122] . viral surface (external) antigens, hemagglutinin (ha) and neuraminidase (na), are targeted by neutralizing antibodies [123] . traditionally, a robust antibody response has been the goal of influenza vaccination and has been the basis upon which vaccines have been tested and licensed [124, 125] . however, these antibodies provide limited protection against divergent influenza strains. since there are strengths for both internal and external strategies, many vaccines include multiple antigens to induce a strong humoral and cellular immune response. over the past decade, both internal and external influenza proteins were utilized in uiv clinical trials ( figure 2 ). other strategies which target whole virus or attenuated virus through gene deletion have also been investigated. however, recent vaccines have focused on external proteins, specifically ha. another vaccine utilized the full-length h5 ha protein in an oral recombinant adenovirus type 4 (ad4) vectored vaccine, ad4-h5-vtn. three clinical trials have enrolled 313 participants between 18 and 49 years of age to investigate this avian h5 influenza vaccine. three immunizations with ad4-h5-vtn resulted in low seroconversion, 11% for vaccinees and 7% for placebo [46] . participants were boosted with an inactivated h5n1 vaccine, which resulted in 100% seroconversion for vaccinees compared to 36% in the placebo group. vaccination with ad4-h5-vtn induced a significant t cell response after a single vaccination with a median 232 sfc/million pbmc. no serious adverse events were reported although vaccinees experienced higher rates of self-limited abdominal pain (16.8% vs. 2.4%), diarrhea (19.2% vs. 4.9%), and nasal congestion (32.8% vs. 14.6%) compared to the placebo. na was included in a dna vaccine, vgx-3400x [60] . the dna vaccine included plasmids containing na, ha, and m2e-np from h5n1 avian influenza. the vaccine was administered intramuscularly to over 200 participants 18-55 years of age during 4 clinical trials [59, [61] [62] [63] . no results have been posted to date. interestingly, na was only investigated in combination with other influenza proteins. furthermore, besides the vgx-3400x vaccine, na was only included in whole inactivated and vlp vaccine strategies. the na protein should be further investigated for its cross-protective potential against influenza [126] . antigens are presented to the immune system in different ways depending on the vaccine platform. most seasonal influenza vaccines utilize attenuated or inactivated wild-type viruses. these viruses display the external influenza proteins and stimulate strong antibody responses [127] . although this strategy has been utilized since 1945, it has consistently shown low efficacy for protection against mismatched influenza strains [10, 128] . therefore, a variety of vaccine platforms were investigated during the last decade to further improve influenza vaccination. although many vaccine platforms have been investigated, no single platform has thus far been demonstrated to show superior protection against influenza. a common platform for uiv clinical trials is viral vectors (29.4%), which utilize viral machinery to package, deliver, or display the vaccine antigen ( figure 3 ). viral vectors have been commonly used as molecular biology tools and are approved for several gene therapies [129] . one of these vaccines is mva-np+m1, which is a modified vaccinia virus ankara (mva) viral vector expressing the nucleoprotein (np) and matrix protein 1 (m1) genes from an h3n2 influenza strain [15] . in the past decade, nine clinical trials investigated mva-np+m1 enrolling over 7200 participants 18 years or older. results from these trials report an increase in t cell response to vaccination, which remained significant above baseline for 52 weeks in 50-59-year-old participants. however, the response was only significant for 12 weeks in subjects 60-69 years old and 3 weeks for participants over 70 years [15] . a subsequent trial using 6 healthy subjects reported no significant difference in t cell response 21 days post-vaccination [24] . the antibody response to vaccination was not reported. to further boost the immune response to mva-np+m1, a heterologous boost with a simian adenovirus viral vector chadox1-np+m1 was investigated [21] . in this study involving 72 participants, both the mva-np+m1 and chadox1-np+m1 vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses 5-fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day 14 after chadox1-np+m1 vaccination, but a decreased response by day 21 [83] . vaccinees were boosted with mva-np+m1, which again increased the t cell response; however, this response was not significant 21 days following the boost vaccination. a recent viral vectored vaccine is nasovax, an intranasal adenoviral vectored vaccine. though no results have been posted for this clinical trial, data presented at the 2019 world vaccine congress reported strong immunogenicity and protection [105] . indeed, vaccination with nasovax induced 100% seroconversion, which was maintained for over 1 year. the newest vaccine platform utilizes nanoparticles to deliver viral antigens [130] . one vaccine utilizing this method is val-506440 and val-339851, which are mrna ha from h10n8 and h7n9 influenza strains delivered in a lipid nanoparticle (lnp) [92] . two trials were performed utilizing 357 participants aged 18-64 years. for the h10n8 mrna vaccine, vaccination resulted in mild to moderate systemic adverse events including injection site pain (76.7-93.1% vs. 5.7-11.1%) and myalgia (47.8-70.9% vs. 2.9-3.7%) compared to the placebo. antibody responses were increased in a dose-dependent manner for the h10n8 lnp vaccine reaching 100% seroconversion at 100 µg compared to 5.7% for the placebo group. these levels remained seropositive (hai ≥ 10) for 6 months after immunization. the h7n9 lnp vaccine induced strong antibody titers for all vaccine doses with 96.3% seroconversion for the 25 µg dose group. participants vaccinated with the h7 vaccine displayed mild injection site pain compared to the placebo (43.3-80% vs. 5.6-13.9%). mva-np+m1 and chadox1-np+m1 vaccines were shown to boost t cell responses when administered individually or together. a heterologous boost, regardless of the order, increased t cell responses ~5-fold. another study investigating this heterologous strategy reported a significant increase in t cell responses at day 14 after chadox1-np+m1 vaccination, but a decreased response by day 21 [83] . vaccinees were boosted with mva-np+m1, which again increased the t cell response; however, this response was not significant 21 days following the boost vaccination. other nanoparticle vaccines include vrc-flunpf081-00-vp, which is a recombinant ha vaccine delivered in a ferritin nanoparticle, and vrc-flunpf099-00-vp, which is a ha stalk protein delivered in a ferritin nanoparticle. although neither trial has posted results, influenza ferritin nanoparticle vaccines have shown strong immunogenicity in mice and ferrets during preclinical trials [112] . another common vaccine platform utilizes recombinant protein of a viral antigen (26.5%). due to low immunogenicity, recombinant protein vaccines typically require the use of an adjuvant to enhance the immune response to vaccination [131, 132] . panblok is a recombinant ha protein administered with a novel stable emulsion adjuvant. four clinical trials were performed which enrolled 1264 participants 18-49 years old. in one adjuvant dose-dependent trial targeting h5 influenza, results demonstrated that all adjuvanted vaccines (3.8 µg, 7.5 µg, or 15 µg) increased seroconversion from 9% in the unadjuvanted group to 70% for participants who received an adjuvanted vaccine [76] . however, another trial targeting h7 influenza reported low seroconversion regardless of the adjuvant dose [78] . despite low antibody detection using hai assay, antibodies against h7 influenza were detected using elisa. passive transfer of these antibodies resulted in protection against lethal h7 challenge in mice. additionally, these antibodies were cross-reactive to h1, h4, h14, h3, h10, and h15, indicating broad immunity against both group 1 and group 2 influenza [78] . some vaccines deliver conserved immunogenic peptides of viral antigens. one such vaccine is flu-v, a peptide-based vaccine containing conserved epitopes from influenza a and b viruses and adjuvanted with montanide isa-51. over the past decade, 4 clinical trials were performed involving 408 participants between 18 and 60 years of age. vaccination with flu-v increased ifn-γ cellular responses 2-fold but did not induce antibody responses as expected [52] . in another study, seronegative males were vaccinated with flu-v and then challenged with h3n2 influenza virus [55] . participants vaccinated with flu-v showed reductions in viral load and symptoms as well as an 8-fold increase in ifn-γ cellular responses. nonstructural protein 1 (ns1) is an influenza protein that antagonizes the immune system by downregulating antiviral host proteins [133] . several attenuated virus vaccines in clinical trials have deleted this viral gene to improve the immune response to vaccination. both ghb11l1 and ghb16l2 are intranasal live attenuated viruses with the ns1 gene deleted, but neither clinical trial has published results from these studies. matrix protein 2 (m2) is an essential structural viral protein for influenza replication. the m2sr vaccine includes a virus that lacks the m2 protein resulting in non-infectious viral progeny, essentially a single-cycle virus [98] . all three m2sr vaccine trials are currently active. an ideal uiv will provide highly effective and long-lasting protection. this can be difficult to achieve when targeting internal proteins or using poorly immunogenic vaccine platforms. adjuvants are compounds that stimulate the immune system and improve vaccine efficacy [132] . this is commonly achieved by oil-in-water emulsions, which recruit immune cells to the site of vaccination [134] . another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [135] . new adjuvants continue to be discovered and explored, but few are licensed for use in the united states [136] . over the past decade, most adjuvants in uivs have been oil-in-water emulsions (39%) (figure 4 ). m-001 is a recombinant protein vaccine that contains common b and t cell epitopes from the ha, np, and m1 influenza proteins. seven trials were performed over the past decade, which enrolled 10,391 individuals over 18 years old. this vaccine was combined with an adjuvant, montanide isa 51vg, which increased igg titers 50-fold against the m-001 protein [137] . strong t cell responses to m-001 were shown for all groups regardless of adjuvant inclusion. a subsequent trial reported m-001 could be used as a stand-alone or priming vaccine for the seasonal influenza vaccine [31] . when compared to seasonal vaccination alone, participants primed with m-001 before seasonal vaccination showed elevated antibody responses for matched h1n1 (4-fold vs. 2.24-fold) and h3n2 (3.17-fold vs. 2.3-fold), but not influenza b (1.7-fold vs. 1.32-fold). additionally, m-001 vaccination increased both cd4+ and cd8+ t cell responses to h1n1, h3n2, and influenza b strains compared to baseline. another unpublished clinical trial reported that 70% of m-001-vaccinated participants had a 4-fold increase in hai titers compared to 41% for the control group [31] . this vaccine has moved into phase iii clinical trials and was scheduled for primary completion in may 2020. immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in 2 of 36 participants including erysipelas and gastroenteritis [99] . mild to moderate adverse events were recorded in 88.9% and 85.7% of vaccinated participants compared to 55.6% in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [135] . an example is vax125, which is a recombinant ha protein fused to the tlr5 ligand, flagellin. four clinical trials were performed using 911 participants over the age of 18. vaccine doses over 5 µg resulted iñ 8-fold elevated hai titers, 75% seroconversion, and 98% seroprotection rates for h1n1 influenza [40] . however, dose escalation over 8 µg and 12 µg was stopped due to serious adverse events [42] . vaccine doses ≥1.25 µg resulted in an average 19-fold increase in hai titer, 92% seroprotection, and 79% seroconversion against a matched h1n1 influenza strain [42] . viruses 2020, 12, x for peer review 12 of 23 immunose flu is an inactivated split vaccine with a novel lipid adjuvant, endocine. the immunogenicity of immunose flu was not reported, but vaccination resulted in serious adverse events in 2 of 36 participants including erysipelas and gastroenteritis [99] . mild to moderate adverse events were recorded in 88.9% and 85.7% of vaccinated participants compared to 55.6% in the saline placebo control group. another common group of adjuvants are toll-like receptor (tlr) agonists. these adjuvants bind and activate cellular host pathways, which leads to increased immune activation [135] . an example is vax125, which is a recombinant ha protein fused to the tlr5 ligand, flagellin. four clinical trials were performed using 911 participants over the age of 18. vaccine doses over 5 µ g resulted in ~8-fold elevated hai titers, 75% seroconversion, and 98% seroprotection rates for h1n1 influenza [40] . however, dose escalation over 8 µ g and 12 µ g was stopped due to serious adverse events [42] . vaccine doses ≥1.25 µ g resulted in an average 19-fold increase in hai titer, 92% seroprotection, and 79% seroconversion against a matched h1n1 influenza strain [42] . another tlr adjuvant is double-stranded rna (dsrna), which binds tlr3 and activates inflammatory pathways [138] . vxa-a1.1 utilizes this adjuvant by encoding dsrna and an h1n1 ha transgene in a recombinant adenovirus type 5 (ad5) vector. this oral vaccine has been studied in 4 clinical trials with 285 participants between 18 and 49 years of age. one trial reported increased antibody responses to matched h1n1 strains with an average of 7.7-fold increases in hai titers and 29-fold increases in microneutralization titers after vaccination [84] . vaccination resulted in mild side effects at similar rates to the placebo group. phase 2 clinical trial participants were immunized with vxa-a1.1 or the seasonal qiv vaccine and then challenged with an h1n1 influenza strain [87] . vaccination with vxa-a1.1 resulted in 48% protection compared to 38% with the seasonal vaccine. another tlr adjuvant is double-stranded rna (dsrna), which binds tlr3 and activates inflammatory pathways [138] . vxa-a1.1 utilizes this adjuvant by encoding dsrna and an h1n1 ha transgene in a recombinant adenovirus type 5 (ad5) vector. this oral vaccine has been studied in 4 clinical trials with 285 participants between 18 and 49 years of age. one trial reported increased antibody responses to matched h1n1 strains with an average of 7.7-fold increases in hai titers and 29-fold increases in microneutralization titers after vaccination [84] . vaccination resulted in mild side effects at similar rates to the placebo group. phase 2 clinical trial participants were immunized with vxa-a1.1 or the seasonal qiv vaccine and then challenged with an h1n1 influenza strain [87] . vaccination with vxa-a1.1 resulted in 48% protection compared to 38% with the seasonal vaccine. interestingly, although alum is one of the most commonly used fda-approved adjuvants, only one clinical trial in 2010 utilized this adjuvant [136] . hai-05 is a recombinant h5 ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [72] . this trial enrolled 100 individuals between 18 and 49 years of age and investigated the dose response of hai-05 with alum. interestingly, any combination of hai-05 (15, 45, and 90 µg) with alum resulted in minimal antibody titers while hai-05 alone (90 µg) induced the greatest antibody response (6.4-fold increase). this suggests the hai-05 induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [139] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [139] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure 5 ). over the past decade, only half of completed trials reported their findings ( figure 5e ). this delay is consistent regardless of clinical trial phase ( figure 5d ). one clinical trial in 2010 utilized this adjuvant [136] . hai-05 is a recombinant h5 ha protein vaccine that is produced in a plant-expression system, nicotiana benthamiana [72] . this trial enrolled 100 individuals between 18 and 49 years of age and investigated the dose response of hai-05 with alum. interestingly, any combination of hai-05 (15, 45 , and 90 µ g) with alum resulted in minimal antibody titers while hai-05 alone (90 µ g) induced the greatest antibody response (6.4-fold increase). this suggests the hai-05 induced low immunogenicity that was not improved by the addition of an adjuvant. in the us, new drugs and vaccines must complete four phases of clinical trials to be licensed and marketed for public use. phase i trials investigate the safety and dosage of the vaccine. typically, phase i trials have limited numbers of participants and do not assess efficacy due to low statistical power [139] . phase ii trials assess the dose response, efficacy, and side effects of the new vaccine. these trials include more study participants and can last longer than phase i trials. occasionally, phases i and ii can be combined into one clinical trial, phase i/ii. phase iii trials include a large sample size and assess participants for vaccine efficacy and adverse reactions. at this point, the new vaccine or drug may be approved for the market [139] . lastly, phase iv clinical trials involve post-marketing surveillance of the efficacy and safety of the new vaccine. importantly, not all clinical trial results are reported or published. it is common for results to be posted several years after the completion of a trial ( figure 5 ). over the past decade, only half of completed trials reported their findings ( figure 5e ). this delay is consistent regardless of clinical trial phase ( figure 5d ). as expected, most uiv clinical trials performed over the past decade were phase i trials (57.4%) ( figure 5 ). of the 27 vaccines, 11 have progressed past phase i (40.7%); however, only 3 vaccines (11%) have been tested in phase iii clinical trials. the first phase iii trial investigated inflexal v, a trivalent adjuvanted virus-like particle (vlp) vaccine [70] . this study included 205 children between 6 and 36 months and was completed in november 2010 [69] . all participants were immunized with a single full dose (0.5 ml) or with two doses (0.25 ml) of the inflexal v vaccine. results suggest that both vaccine groups demonstrated improved seroprotection and seroconversion rates. participants who received two 0.25 ml doses 4 weeks apart showed higher seroprotection rates for h1n1 (99.0), h3n2 (99.0), and influenza b (92.2). for h1n1 and h3n2, the two-dose regimen resulted in higher seroconversion and geometric mean titer (gmt) fold increases than the single-shot regimen. half of participants from each group experienced non-serious adverse events including pyrexia, malaise, rhinitis, cough, otitis media acute, as well as adverse events at the injection site including erythema, induration, pain, or hemorrhage. the second uiv tested in a phase iii clinical trial was m-001. this vaccine is a synthetic recombinant protein containing common linear influenza epitopes [31] . as discussed above, the adjuvanted m-001 vaccine has shown promising immunogenicity and the phase iii trial was scheduled for primary completion in may 2020 [31, 137] . the third vaccine tested in a phase iii clinical trial is nanoflu. this vaccine is a recombinant ha protein delivered in a nanoparticle with a saponin-based matrix-m adjuvant [107] . although results for the phase ii trial have not been posted, a press release from novavax stated that nanoflu induced superior hai antibody responses against homologous and drifted strains compared to the seasonal influenza vaccine. a phase iii clinical trial involving 2650 participants over 65 years of age was scheduled for primary completion in december 2019. this systematic review documents uivs that were tested in clinical trials from january 2010 to december 2019. although many papers have discussed strategies for uivs, few review papers address the translation of uiv strategies to clinical trials [140, 141] . this is the first systematic review of uivs in clinical trials. the definition of a "universal" influenza vaccine is highly debated [125, 141] . in 2018, the niaid announced that a uiv should (1) be at least 75% effective, (2) protect against group i and ii iav, (3) have durable protection that lasts at least 1 year, and (4) be suitable for all age groups [1] . since this standard was put forward towards the end of the decade, our definition of a uiv remains broader than the niaid requirements. here, we have defined a uiv as a vaccine that aims to induce better cross-protection than seasonal influenza vaccines. therefore, "supra-seasonal vaccines" which cover a large subset of influenza strains and vaccines against specific subtypes of influenza have been included in this analysis. the influenza diversity targeted by each vaccine varied. only 37% of universal vaccines were designed to protect against both influenza a and b viruses. other strategies focused on iav (22%) or a single subtype of iav (41%). importantly, no vaccines focused on influenza b virus (ibv) alone. furthermore, the current niaid requirements for a universal influenza vaccine do not require cross-protection against ibv. notably, the cdc reports that ibv is responsible for 72% of influenza cases reported for children and young adults each year [142] . overall, approximately 26% of annual influenza cases can be attributed to ibv [143] . the significant burden of ibv should be addressed in the design of universal influenza vaccines. some limitations to this review should be noted. first, information about clinical trials can be limited until the results are published. specifically, not all clinical trial summaries include information on vaccine design and mechanism. in these cases, previous publications and press releases for the vaccines were consulted. additionally, most results reported safety information and homologous vaccine efficacy, providing limited information on the cross-reactivity of each vaccine. second, we searched clinical trials registered through clinicaltrials.gov, which could potentially exclude some studies. there are other clinical trial databases such as eu clinical trials register, however, the clinicaltrials.gov database reports more accurate and updated information for clinical trials [144] . despite limited information, this review provides a comprehensive summary of the uivs tested in clinical trials. indeed, this is the first comprehensive review to also discuss efficacy and trends in vaccine development for influenza. the field of influenza vaccine development is ever progressing. this is reflected in new vaccine targets and platforms such as ha stalk and nanoparticles. researchers over the past decade have produced many promising influenza vaccines, each with strengths and limitations. the efficacy of a vaccine may induce strong protection against matched strains, but an effective uiv must induce strong cross-protection as well. this review identifies vaccines that report efficacy against matched strains alone. importantly, these vaccines may provide cross-protection if delivered in combination with vaccines targeting other influenza subtypes. however, this would require further research and investigation. influenza virus remains a major global pathogen despite the general widespread use of seasonal vaccines due to varying efficacy to drifted strains. a uiv remains a top priority for the nih and world health organization. this review provides an update on the progress towards a 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safety, reactogenicity and immunogenicity of the gamfluvac influenza and memory t cells: how to awake the force host immune response to influenza a virus infection nucleoprotein of influenza a virus is a major target of immunodominant cd8+ t-cell responses emerging influenza viruses and the prospect of a universal influenza virus vaccine influenza infection in humans induces broadly cross-reactive and protective neuraminidase-reactive antibodies comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults history and evolution of influenza control through vaccination: from the first monovalent vaccine to universal vaccines viral vectors in gene therapy nanoparticle vaccines against infectious diseases recombinant vaccines and the development of new vaccine strategies. braz correlates of adjuvanticity: a review on adjuvants in licensed vaccines functions of the influenza a virus ns1 protein in antiviral defense mechanisms of action of adjuvants toll-like receptor agonists: are they good adjuvants? adjuvants help vaccines work better safety and immunogenicity of multimeric-001-a novel universal influenza vaccine dsrna-activation of tlr3 and rlr signaling: gene induction-dependent and independent effects what are clinical trials and studies? available online the hurdles from bench to bedside in the realization and implementation of a universal influenza vaccine universal influenza virus vaccines and therapeutic antibodies impact of influenza b lineage-level mismatch between trivalent seasonal influenza vaccines and circulating viruses prevalence of clinical trial status discrepancies: a cross-sectional study of 10,492 trials registered on both clinicaltrials.gov and the european union clinical trials register key: cord-286368-kdwh4hgf authors: hui, david s.c.; lee, nelson; chan, paul k.s. title: a clinical approach to the threat of emerging influenza viruses in the asia‐pacific region date: 2017-07-05 journal: respirology doi: 10.1111/resp.13114 sha: doc_id: 286368 cord_uid: kdwh4hgf seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. patients with chronic co‐morbid illness, those at the extremes of age and pregnant women are at higher risks of complications requiring hospitalization, whereas young adults and obese individuals were also at increased risk during the a(h1n1) pandemic in 2009. avian influenza a(h5n1) and a(h7n9) viruses have continued to circulate widely in some poultry populations and infect humans sporadically since 1997 and 2013, respectively. the recent upsurge in human cases of a(h7n9) infections in mainland china is of great concern. sporadic human cases of avian a(h5n6), a(h10n8) and a(h6n1) have also emerged in recent years while there are also widespread poultry outbreaks due to a(h5n8) in many countries. observational studies have shown that treatment with a neuraminidase inhibitor (nai) for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when administered early in the course of illness. whether higher than standard doses of nai would provide greater antiviral effects in such patients will require further investigation. high‐dose systemic corticosteroids were associated with worse outcomes in patients with severe influenza. there is an urgent need for developing more effective antiviral therapies for treatment of influenza infections. severe acute respiratory infections such as avian influenza a(h5n1) 1 and a(h7n9) viruses 2 with pandemic potential have continued to circulate widely in some poultry populations and infect humans sporadically. infection caused by these viruses may result in severe disease and high case fatality rates because most humans have no background immunity to these viruses. sporadic human cases due to avian a(h5n6), 3 a(h10n8) 4 and a(h6n1) 5 have also emerged in recent years. the global circulation of oseltamivir-resistant seasonal influenza, the emergence of a(h1n1)pdm09 virus in 2009 followed by its continual circulation, 6 the rising number of a(h7n9) infections in humans 2 and the ongoing spread of a(h5n8) in recent months in the poultry populations in many countries in asia, africa, europe and middle east with pandemic potential 7 all point to an urgent need for developing more effective antiviral therapies to reduce morbidity and mortality. this article reviews the epidemiology, clinical, diagnostic and treatment aspects of these important and emerging influenza viruses that are posing threats to human health in the asia-pacific region. seasonal influenza is a major health problem. for instance, in hong kong, influenza a and b accounted for 39.4% and 10.2%, respectively, of hospital admissions confirmed to have viral infections. 8 the average annual incidence of hospitalization with laboratoryconfirmed influenza a infection was 107.7/10 000 and 18.3/10 000 among children aged <5 years and elderly ≥65 years, respectively. furthermore, influenza a was the most common (45.5%) virus detected from patients who died of respiratory viral infections. although influenza b is usually associated with a less degree of disease burden, its impact on human health is substantial. our previous study conducted in hong kong revealed that 24% of laboratory-confirmed influenza-associated hospital admissions were due to influenza b, and was much higher (41.9%) among older children and adolescents (aged 5-14 years). 9 this observation is similar to data in europe and north america. in the temperate region, influenza exhibits an annual seasonal peak in winter. however, the seasonality in tropical and subtropical regions is more variable. for instance, in hong kong, a subtropical city, most of the time, influenza exhibits two peaks during january-march and june-august, and occasionally, the two peaks merge without an obvious gap. 8, 10 such bimodal and variable seasonality of influenza is also observed in countries within the subtropical and tropical regions, and poses a challenge to implement annual influenza vaccination campaign as well as a question on whether the northern or southern hemisphere version of influenza vaccine would be more appropriate. a schematic diagram of the structure of influenza virion is shown in figure 1 . influenza represents one of the few examples of viruses with segmented genome. this, together with the widespread infection with different subtypes in various animal species, allows robust evolution through the process of 'reassortment'. the prerequisite for reassortment is a simultaneous infection with more than one strain of viruses in a host referred to as the 'mixing vessel' that allows replication and swabbing of gene segments between different strains of viruses. water birds are well-known mixing vessels, but the reassortants are more likely to have a tropism restricted to avian species. these new reassortants may pose problems in the land birds and poultry, and subsequently be of human concern. swine is another well-known mixing vessel. the unique property of swine is their susceptibility to both avian and human influenza viruses. thus, reassortants from swine are more likely to gain tropism for infection in humans. field observations made as early as 1990s have pointed out that influenza outbreaks that began with either pigs or people were then rapidly transferred to each other. 11 swine are likely to play the role of a mixing vessel in creating pandemic strains. although the 1918 pandemic strain was most probably an entirely avian-like virus, 12 the 1957 and 1968 pandemic strains were reassortants of human-and avian-origin viruses. 13 while the most recent pandemic, a(h1n1)pdm09, was not as severe as those that occurred before, there is no guarantee that the next pandemic will be as 'mild'. at present, a few subtypes (h5, h7 and h9) are of particular concern. cross-species infections, especially the recent strong wave of human a(h7n9) infection in mainland china underscores that the next pandemic could be imminent. 14, 15 countries should have pandemic preparedness in place, including antiviral stockpiling and pandemic vaccine preparations. these would be better achieved under joint multigovernment industrial partnership. human cases of the highly pathogenic avian influenza a(h5n1) were first detected in hong kong in 1997. 1 the number of affected countries increased between 2003 and 2008, with expansion from east and southeast asia, then to west asia and africa, with a sharp rise in egypt since november 2014. 16 as of 16 march 2017, there have been 453 deaths out of 858 human cases in 16 countries since 2003. 17 a canadian tourist died of a(h5n1)-related meningoencephalitis in alberta in january 2014 after visiting beijing in december 2013 without any obvious exposure to infected avian sources or environmental contamination. 18 a history of exposure to dead or sick poultry/wild birds occurs in over 60% of cases of human a(h5n1) infection. the incubation period for a(h5n1) infection ranges from 2 to 8 days but may be as long as 17 days. the clinical spectrum may range from asymptomatic infection, mild influenza-like illness to severe pneumonia, with multiorgan failure. 19 some of the a(h5n1) human cases have been linked to consumption of dishes made of raw, contaminated poultry blood. however, slaughter, defeathering, handling carcasses of infected poultry and preparing poultry for consumption, especially in household settings, appear to be important risk factors. 19, 20 among the fatal cases, the median duration from symptom onset to death was 9-10 days (range: 6-30 days). 20 viral rna was present in the blood of fatal a(h5n1) cases and this was associated with higher pharyngeal viral loads. nasopharyngeal swab samples, bronchoalveolar lavage and cerebrospinal fluid samples were positive in the canadian case for influenza a(h5n1) virus by various molecular testing methods, including sequencing. 18 in addition, lymphopenia and high chemokine/cytokine levels have been observed in severe a(h5n1) infection and these correlated with pharyngeal loads. 21 a new reassortant genotype of a(h5n1) containing the haemagglutinin (ha) and neuraminidase (na) genes from clade 1.1.2 and the internal genes from clade 2.3.2.1 was detected in 2013 and associated with the highest number of cases (n = 26) and deaths (n = 14) in cambodia. 22 complete viral genome analysis of the fatal case in canada has shown an ha gene of clade 2.3.2.1c and was a reassortant with an a(h9n2) subtype lineage polymerase basic 2 gene without mutations conferring resistance to adamantanes or na inhibitors (nai). 18 human-human transmission remains rare as a meta-analysis has shown that only 1-2% of more than 12 500 study participants from 20 studies exposed to patients with a(h5n1) infection had serological evidence for prior a(h5n1) infection. 23 delay in the delivery of appropriate treatment to patients with influenza a(h5n1) infection in indonesia was mainly related to delay in diagnosis rather than late presentation to healthcare settings. 24 age, country, per capita government health expenditure and delay from symptom onset to hospitalization were the risk factors for mortality related to a(h5n1) infection, emphasizing the importance of early diagnosis, treatment and supportive care. 25 a systematic review has shown that females were at higher risk of death (or: 1.75, 95% ci: 1.27-2.44) following a(h5n1) infection, whereas young age in particular <5 years was protective (or: 0.44, 95% ci: 0.25-0.79). 26 human infections with a novel avian influenza a (h7n9) virus were first reported in china in march 2013 in patients hospitalized with severe pneumonia. 27 there have been five seasonal epidemics in china since the virus was first discovered in 2013. the estimated risk of death (hospitalization fatality risk, hfr) among hospitalized cases of a(h7n9) infection in the second epidemic was 48% (95% credibility interval: 42-54%), which was slightly higher than the corresponding risk of 36% in the first wave. in the second epidemic, the hfr was estimated at 36% (95% ci: 28-45%) in patients aged <60 years but at 59% (95% ci: 51-67%) among those aged ≥60 years. 28 an upsurge in human a(h7n9) infections has been observed since november 2016 in mainland china. during this fifth epidemic wave, the number of human cases is higher than during the previous two waves during 2014-2015 and 2015-2016. the majority of recently reported human cases are associated with exposure to infected live poultry or contaminated environments, including wet markets where live poultry are sold. in addition, the human cases are more geographically widespread and cases are also reported from rural areas, unlike in previous epidemics (fig. 2) . 29, 30 the phylogenetic analysis on ha genes of isolates collected from humans suggested that the virus is evolving and with the more recent isolates clustered to a separate branch (supplementary figure s1 ). 31, 32 as of 5 april 2017, 1364 laboratory-confirmed cases of human infection with avian influenza a(h7n9) virus have been reported to world health organization (who). 33 human cases of a(h7n9) infection exported from the mainland of china have been confirmed in hong kong (n = 21), taiwan (n = 4), macau (n = 2), canada (n = 2) and malaysia (n = 1) in visitors who developed illness after returning from the mainland of china to their home cities. 29, 30, 33 the incubation period of human infection with the a(h7n9) virus ranges from 1 to 10 days, with an average of 5 days. the median time from poultry exposure to disease onset was 6 days, whereas the median time from illness onset to hospitalization, acute respiratory distress syndrome (ards) development, antiviral therapy and death were 4, 7, 6 and 21 days, respectively. 34 preexisting co-morbid conditions occurred in >60% of these cases. the prominent clinical features on admission were those of a severe influenza syndrome with fever, cough, fatigue and dyspnoea, whereas the most striking laboratory findings were marked lymphopenia and thrombocytopenia. elevated cytokine levels have been observed in patients and the excessive cytokine responses may contribute to the clinical severity of a(h7n9) infection. 35, 36 limited human to human transmission with at least two epidemiologically linked cases due to close household contact with the index patients has been reported in a number of family clusters in both a(h5n1) 20, 37, 38 and a(h7n9) infections. 34, 39, 40 nosocomial transmission of a(h7n9) infection between two patients sharing a hospital room, 41 and co-transmission of avian influenza a(h7n9) and a(h1n1)pdm09 viruses between two patients with haematological disorders sharing the same room with bed distance <1 m have been reported. 42 comparisons of the clinical features between human cases of a(h5n1) and a(h7n9) are shown in table 1 . 34, 43 closure of live poultry markets (lpm) in the mainland of china has tremendously reduced the risk of a(h7n9) infection temporarily but closure is difficult to sustain due to cultural preference for live poultry. an ecologic modelling study estimated that closure of lpm reduced the mean daily number of a(h7n9) virus infections in the four most affected cities by 97-99%. 44 a retrospective serological study of blood specimens taken during january-may and october-november in 2012 from 1544 subjects who worked in lpm, farms, slaughter houses or kept backyard poultry in eastern china before the epidemic occurred in 2013 revealed no evidence of a(h7n9) infection. 45 other novel avian influenza subtypes the first human case of avian a(h6n1) infection was reported in a 20-year-old female with pneumonia in taiwan in may 2013, with subsequent full recovery following treatment with oseltamivir. 5 the first human case of avian a(h5n6) was confirmed in a 49-year-old male in sichuan province, onset of illness to death (median, days) 21 11 43 case fatality rate in hospital 34% 53% china, in may 2014 with a fatal outcome 3 and a second case was confirmed in a 58-year-old male with history of exposure to live poultry in guangdong province in december 2014. 46 the virus was a reassortant that contained seven genes from a(h5n1) and the na gene from an a(h6n6) virus circulating in ducks. 47 (fig. 3 ). 29 the latest case was reported on 1 december 2016. 48 in addition, china has confirmed three human infections, two fatal, with avian a(h10n8) viruses that contain the internal genes from a(h9n2), as does a(h7n9). 4, 49 similar to the a(h7n9) virus, the a(h6n1) and a(h10n8) viruses have low pathogenicity in poultry and are therefore more difficult to detect in birds in contrast to the highly pathogenic a(h5n1) virus. highly pathogenic avian influenza a(h5n8) viruses belonging to clade 2.3.4.4 of the a/goose/guangdong/ 1/1996 lineage were detected in 2014 in wild birds and poultry in china, japan, south korea, netherlands, germany, italy, russia, the uk and northern ireland. the virus was detected sporadically in canada and the usa in wild birds and poultry until mid-2015. the a(h5n8) viruses were also detected in taiwan, china and in hungary and sweden in 2015. since june 2016, many more countries in both europe and asia have detected infections in wild birds and/or domestic poultry with a(h5n8). many of these recent detections were associated with mortality in wild birds. 7,50 although human infection with a(h5n8) has not been detected, the ongoing spread of this virus to different countries in recent months is worrisome and may increase the risk of human infection. visitors who have returned from areas affected by avian influenza and developed respiratory symptoms and fever within 10 days after their return should be enquired about their relevant history (travel history, occupation, contact history and clustering) to facilitate early diagnosis and treatment. it is important to obtain any history of contact with poultry (live or sick/dead poultry as a(h7n9) virus may cause no or mild symptoms in poultry in contrast to a(h5n1)), exposure to wet markets or known avian influenza cases, and laboratory exposure including staff handling with spillage. patients with influenza-like illness who present with dyspnoea, tachypnoea, evidence of hypoxaemia and pulmonary infiltrates on chest radiograph should be hospitalized. 51 early case identification and isolation precautions would facilitate clinical management which may reduce the risk of severe infection and related complications for seasonal epidemic, zoonotic and pandemic influenza, in addition to reducing the risk of influenza transmission and mitigating the impact of outbreaks on the healthcare system. common findings in severe influenza infection include low or normal white cell counts, lymphopenia and thrombocytopenia, and increased serum transaminases, lactate dehydrogenase (ldh), creatine kinase and urea/creatinine. 6, 20, 27, 51 in the influenza clinical information network (flucin) study, early elevation of c-reactive protein ≥100 mg/l was an independent predictor of severe outcome. in a study of 1770 patients hospitalized with community-acquired pneumonia (cap), procalcitonin concentration had a strong association with need for invasive mechanical ventilation (imv) or inotrope support (in 6.5% of those studied). 52 higher levels of certain biomarkers on presentation linked to inflammation, coagulation or immune function (interleukin 6 (il-6), cluster of differentiation 163 (cd163), interferon gamma-induced protein 10 (il-10), lipopolysaccharide binding protein (lbp), il-2, monocyte chemoattractant protein-1 (mcp-1) and interferon gamma-induced protein 10 (ip-10)) have been associated with disease progression in both outpatients and those hospitalized with influenza. 53 laboratory diagnosis plays an important role in the management of influenza. the narrow window of an effective antiviral intervention for both seasonal and avian influenza demands a rapid turnaround time. 6, 54 nowadays, most laboratories employ either real time reverse transcription polymerase chain reaction (rrt-pcr)-based methods or antigen detection-based assays. while the rrt-pcr approach is most sensitive and can be applied to a wide variety of specimens including the less invasive throat swab samples, it requires a certain degree of laboratory setup and technical skills, which are not readily available in many healthcare settings. the antigen-based rapid influenza diagnostic test (ridt), although less sensitive, is still the test of choice particularly as a point-of-care test. our experience on a few ridt suggested that the sensitivity could be lower for patients who presented more than 2 days after the onset of illness, for young children compared to elderly, and for cases with pneumonia compared to those without. 55 it is therefore crucial to evaluate the performance with respect to the setting that ridt will be applied. the specimen of choice is critical. while in most situations, nasopharyngeal aspirate/swab is the preferred choice of specimen for seasonal influenza, falsenegative results may occur in patients with severe pneumonia where the viral load remains high in the lower respiratory tract (lrt) but has become undetectable in the upper respiratory tract (urt). 56 similarly, lrt specimens such tracheal aspirates or sputum are the specimen of choice for diagnosis of avian influenza infection in humans as the viral load in the urt is typically low. the development and availability of more affordable and reliable molecular diagnostic tests including multiplex pcr would facilitate future clinical management of patients with severe cap due to viral or bacterial aetiology. 57 the advantages and limitations of different diagnostic tests for influenza are summarized in table 2 . 55, 57 the m2 inhibitors (amantadine and rimantadine) and the nai (oseltamivir, peramivir, zanamivir and laninamivir) are the main antiviral agents approved for the prevention of and treatment for influenza. in general, antiviral treatment should be commenced as soon as possible for any patient hospitalized with confirmed/ suspected influenza with severe, complicated or progressive illness, and also for outpatients at higher risk for influenza complications. 58 the m2 inhibitors (adamantanes) are not effective against influenza b viruses and recently circulating influenza a(h3n2) and influenza a(h1n1)pdm09 viruses, which are resistant due to an s31n mutation in the m2 ion channel. however, as some avian influenza a(h5n1) strains are still susceptible, 59 combination of an adamantane with an nai may enhance antiviral activity for susceptible isolates. 60 oseltamivir is effective in reducing mortality (or: 0.17; p = 0.04) in influenza a(h5n1) infection when given before the onset of respiratory failure, 61 and may provide some survival benefit (49% reduction in mortality) when treatment is started within 6-8 days after symptom onset. 62 several observational studies have shown that treatment with oseltamivir for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes, especially when antiviral treatment has been initiated within 2 days of illness onset but even as late as 4-5 days after symptom onset. 63, 64 a systematic review has shown that in prophylactic studies, oseltamivir could reduce the proportion of symptomatic influenza whereas in treatment studies it modestly reduced the time to first alleviation of symptoms by 16.8 h, but it caused nausea, vomiting, headaches and renal/psychiatric side effects. 65 a meta-analysis of randomized controlled trials (rct) has shown that oseltamivir in adults with influenza shortens time to clinical symptom alleviation by 21%, reduces risks of lrt complications and hospitalization, but increases the occurrence of nausea and vomiting. 66 oseltamivir resistance is infrequent in a(h5n1), and <3% in circulating a(h1n1)pdm09, a(h3n2) and b viruses but it is important to monitor for antiviral resistance when managing patients with severe influenza. 67 all h7n9 viruses are amantadine resistant due to the s31n substitution in the m2 ion channel protein. in patients hospitalized with severe a(h7n9) infection, reduction of viral load following oseltamivir treatment was associated with improved outcome, whereas the emergence of virus resistant to nai harbouring an r292k mutation was associated with poor outcomes and lack of response to oseltamivir and peramivir, and reduced susceptibility to zanamivir and laninamivir (50-and 25-fold rises in inhibitory concentration by 50% (ic 50 ), respectively). two patients with severe a(h7n9) infection and r292k mutation requiring extracorporeal membrane oxygenation had received systemic corticosteroid treatment which might have contributed to treatment failure and a fatal outcome. 68 the recommended treatment duration of oseltamivir is generally 5 days, but longer treatment (10 days, then guided by clinical and virological testing as recommended by the who) is advisable for critically ill patients with respiratory failure with persistent viral replication in the lrt. 51, 58, 69 whether higher than standard dose of nai would provide greater antiviral effects in such patients requires further investigation. one rct of hospitalized patients (76% being children) revealed no clinical or virological advantages when comparing double dose of oseltamivir against standard dose. 70 no additional benefit of higher dose oseltamivir treatment was observed in adults hospitalized with influenza a, although a faster virological response was noted in influenza b. 71 however, an rct of 18 critically ill patients with a(h1n1)pdm09 in canada found that therapy with a triple dose of oseltamivir was associated with higher proportions of viral clearance at 5 days than standard therapy (78% vs 11%; p = 0.015). 72 intravenous (i.v.) peramivir treatment was associated with viral rna decline as well as culture and rna negativity, which occurred at similar rates to those on oral oseltamivir by day 5 among adult patients hospitalized for influenza-associated lrt complications. 73 the various nai dosages for adults with adjustment for renal failure are shown in table 3 . 58 zanamivir and laninamivir have quite similar profiles of drug susceptibility. one example is that h275y mutation, which confers high level resistance to oseltamivir carboxylate and reduced susceptibility to peramivir in n1-containing viruses, does not reduce susceptibility to zanamivir and laninamivir most ridt are chromatographic immunoassays (some are fluorescence-based immunoassays); applicable to np swabs, nasal and/or throat swabs and np aspirates/washes (training and protection equipment are required). performance is best if applied within 48-72 h from onset before a significant drop in viral load (up to 4-5 days in selected populations). lower sensitivity for a(h1n1) pdm09 virus has been reported. viral cell culture detects viable viruses, including those contained in the live-attenuated influenza vaccines (laiv). isolates can be subjected to phenotypic resistance assays (e.g. neuraminidase enzyme inhibition assay). viral load, specimen quality, transport, storage and processing techniques may affect test performance. pcr assays can either provide universal detection of influenza a virus by targeting the matrix (m) gene or subtype-specific virus detection (e.g. h1n1pdm09, h3n2, h5n1 and h7n9) by targeting the haemagglutinin (ha) gene. viruses that cannot be subtyped may indicate a novel strain. newer molecular-based point-of-care tests may improve accessibility and reduce processing time and technical demands; some may allow detection of multiple viruses. cost-effectiveness of pcr is variable, depending on the circumstances. some multiplex pcr platforms may provide detection of >14 respiratory viruses (e.g. rsv, human metapneumovirus, parainfluenza virus, rhinovirus and coronavirus) and atypical pathogens (e.g. mycoplasma pneumoniae and chlamydophila pneumoniae). dfa, direct fluorescent antibody test; ifa, immunofluorescence assay; np, nasopharyngeal; ridt, rapid influenza diagnostic test; rsv, respiratory syncytial virus. significantly. 74 intravenous zanamivir was used widely on a compassionate ground for late treatment of critically ill adults with influenza a(h1n1)pdm09 and those with suspected or proven oseltamivir resistance. 75 there were no drug-related trends in safety parameters and in a subset of 93 patients with positive pcr tests at baseline for influenza, showed a median decrease in nasopharyngeal viral rna load of 1.42 log 10 copies/ml after 2 days of treatment. 76 intravenous zanamivir was used with a favourable outcome in a patient with severe a(h7n9) infection complicated by pneumonia which did not respond to oseltamivir initially. 77 favipiravir, which is an inhibitor of a viral rna-dependent rna polymerase, has strong antiviral activity against nai-resistant and sensitive influenza viruses. 67, 78 systemic corticosteroids and other immunomodulating agents for severe influenza systemic corticosteroid has been used frequently for the treatment of influenza-related ards. however, a meta-analysis of data predominantly related to treatment of severe influenza a(h1n1)pdm09 has shown that systemic corticosteroid was associated with an increase in mortality (or: 3.06, 95% ci: 1.58-5.92). pooled subgroup analysis of adjusted estimates of mortality from four studies found or of 2.82 and 95% ci of 1. 61-4.92. 79 in comparison to controls, high-dose corticosteroids (>150 mg/day methylprednisolone eqv) was associated with increased risks in 30-day mortality (38.5% vs 7.7%, p = 0.021) and 60-day mortality (50% vs 15.4%, p = 0.022) and longer viral shedding (15 days vs 13 days, p = 0.039) in patients with influenza a (h7n9) viral pneumonia while there was no difference between low dose (25-150 mg/day methylprednisolone) and controls. 80 in a study of 2649 adults hospitalized with influenza in hong kong, singapore and beijing during 2008-2011, 23.1% had received systemic corticosteroids. bacterial superinfections increased risk of death (adjusted hazard ratio (hr): 2.2, 95% ci: 1.5-3.1). systemic corticosteroids increased risks of superinfections (from 2.7% to 9.7%) and deaths when controlled for indications (adjusted hr: 1.7, 95% ci: 1.1-2.6). 81 among adults with severe sepsis not in septic shock, use of low-dose hydrocortisone versus placebo did not reduce the risk of septic shock within 14 days. the findings do not support the use of hydrocortisone in these patients without septic shock. 82 the role of low-dose systemic corticosteroid needs further investigation by a properly planned rct. 83 in a study of critically ill patients infected with influenza a (h1n1)pdm 09 requiring imv, addition of a mammalian target of rapamycin inhibitor, sirolimus 2 mg/day for 14 days to oseltamivir and prednisolone (n = 19), was associated with a higher frequency of liberation from imv (84.2% vs 47.4%, p = 0.04), a shorter duration of imv (13.8 days vs 33 days, p = 0.03) and a higher chance of achieving lrt viral rna negativity by day 7 (75% vs 33%, p < 0.05) than without addition of sirolimus (n = 19). 84 the role of sirolimus plus oseltamivir versus oseltamivir alone in a larger sample size should be examined without systemic corticosteroid. a recent study of adults hospitalized for a(h3n2) has shown that a triple combination treatment of 2 days of clarithromycin 500 mg, naproxen 200 mg and oseltamivir 75 mg twice daily (bd), followed by 3 days of oseltamivir reduced both 30-and 90-day mortalities and length of hospital stay versus oseltamivir 75 mg bd without placebos for 5 days as control. 85 confirmatory studies of the role of this triple combination would be of interest. exploratory post hoc meta-analysis of studies of severe acute respiratory syndrome (sars) and severe influenza showed a significant reduction in the pooled odds of mortality following convalescent plasma versus placebo or no treatment (or: 0.25; 95% ci: 0.14-0.45). 86 thus, convalescent plasma and hyperimmune globulin may 88 nosocomial opportunistic airborne transmission of a(h3n2) has been implicated in a medical ward with imbalanced airflow and use of non-invasive ventilation (niv) for an index patient with acute exacerbation of chronic obstructive pulmonary disease (copd). 89 standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. 90 the main infection prevention and control measures for managing influenza are droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving). droplet precautions should be added to the standard precautions when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection (table 4 ). 90 a study that measured the amount of influenza a(h1n1)pdm09 rna in aerosols in the vicinity of h1n1-positive patients undergoing aerosol-generating procedures (agp) has shown that bronchoscopy and respiratory/airway suctioning were the most definite procedures to produce aerosols above background baseline values. 92 thus, it is important for healthcare workers to take airborne precautions and perform agp preferably in an isolation room with negative pressure. in order to reduce room contamination in the hospital setting, major health organizations have recommended the application of a minimum room ventilation rate of 6 air changes per hour (ach) in existing facility whereas a higher ventilation of 12 ach is recommended for new or renovated construction, especially when caring for patients receiving mechanical ventilation and during agp. 91, 93 as single circuit niv may lead to room contamination by exhaled aerosols, 94, 95 it has been recommended to apply niv via a helmet and double circuit tubes for patients with mild to moderate respiratory failure during the influenza a(h1n1) pandemic in 2009. 96 however, it is important to ensure a good seal at the neckhelmet interface to prevent nosocomial transmission through environmental contamination by exhaled aerosols. 97 post-exposure prophylaxis with a daily dose of nai (e.g. oseltamivir 75 mg daily for adults) for 10 days is recommended for unprotected close contacts of patients with seasonal influenza who are at risk of complicated influenza. 98 who does not recommend routine post-exposure antiviral chemoprophylaxis for a(h7n9) virus. however, for some asymptomatic persons in which a substantial unprotected or prolonged exposure to an ill patient with a(h7n9) infection has occurred, initiation of empiric post-exposure antiviral treatment (e.g. oseltamivir 75 mg orally bd for 5 days), on the presumption that influenza virus infection has occurred, may be considered. this is likely to be limited to healthcare or other settings involving substantial exposure of those at higher risk for complications from table 4 infection prevention and control measures when assessing patients with complicated influenza 51,90,91 1. clinicians should pay attention to cases of community-acquired pneumonia and patients' travel history for early case detection 2. clinicians should remain vigilant against avian influenza and elicit any relevant clinical and epidemiological information from patients (fever, travel history, occupation, contact history and clustering) 3. standard, contact and droplet precautions are recommended for routine management of patients hospitalized for influenza. droplet precaution (by wearing a surgical mask within 1 m of the patient) and contact precaution (by wearing gown and gloves on entering the room and removing them on leaving) when providing care to all patients with symptoms of acute respiratory infection. contact precautions and eye protection should be added when caring for probable or confirmed cases of avian influenza infection 4. risk assessment should be conducted before performing agp. for cases with severe influenza, it is advisable to perform agp in an airborne isolation room. when an airborne isolation room is not available, the following minimum hourly averaged ventilation rates should be provided for natural ventilation: a. 160 l/s/patient (hourly average ventilation rate) for airborne precaution rooms (with a minimum of 80 l/s/patient) (note that this only applies to new healthcare facilities and major renovations); b. 60 l/s/patient for general wards and outpatient departments; and c. 2.5 l/s/m 3 for corridors and other transient spaces without a fixed number of patients 5. in view of the increasing influenza activity during winter season and the emerging threat of avian influenza, all healthcare workers and visitors are recommended to wear surgical masks when entering patient care areas and strengthen hand hygiene agp, aerosol-generating procedure. influenza virus infection, including patients with severe immunosuppression, neonates and infants, pregnant and early post-partum women, elderly adults, persons with co-morbidities and other highly vulnerable patients; or, unprotected healthcare workers, especially those involved in agp. 99 annual seasonal influenza vaccinations are recommended for the high-risk groups, including residents of institutions for elderly and disabled, any age with chronic illness, age > 65 years; 6 months to 6 years, poultry workers in countries affected by avian influenza, healthcare workers especially those frequently in contact with high-risk persons and household contacts of high-risk persons. while vaccination rates among healthcare workers are highly variable in different countries, 100 there is no conclusive evidence that seasonal influenza vaccination of healthcare workers may protect their patients in terms of reduction in risks of mortality and influenza-like illness. 101 a survey in hong kong assessing the acceptability of an additional ad hoc influenza vaccination among the healthcare professionals following seasons with significant antigenic drift has shown that the strongest factors associated with accepting an additional influenza vaccine included immunization with influenza vaccines in the past 3 years, higher perceived risk of contracting influenza and higher perceived severity of the disease impact. 102 the current influenza vaccines induce a strainspecific immunity that is not ideal for this rapidly mutating virus. a universal influenza vaccine capable of inducing a broad cross-protection across strains is needed. to this end, the m2e antigen, a linear peptide that is conserved across influenza a strains, is being evaluated a vaccine target. 103 the other approach is to prepare virus-like-particles based on ha, na and m1 proteins. 104, 105 virus-like particles with a cocktail of seasonal and potential pandemic strains have been prepared and demonstrated promising results. 106 as a(h7n9) is now endemic in over half of the provinces in mainland china and will continue to cause recurrent zoonotic disease in the winter months, public health measures to control the source such as some rest days every month for the poultry workers to thoroughly clean the lpm environment, and banning the stock of live poultry in markets overnight are important. separation of live ducks and geese from landbased (i.e. non-aquatic) poultry in lpm systems can reduce the risk of emergence of zoonotic and epizootic viruses at source. in the long run, it is advisable to adopt a complete transition from sale of live poultry in wholesale and retail lpm to centralized slaughter and sale of chilled or frozen poultry. 107 seasonal influenza epidemics and periodic pandemics are important causes of morbidity and mortality. apart from influenza a(h5n1) virus and sporadic human cases of a(h5n6), a(h10n8) and a(h6n1) infections, infection due to a(h7n9) virus, especially the recent upsurge in the number of human infections in mainland china, is of great concern. the widespread poultry outbreaks due to a(h5n8) in many countries increases the risk of human infection. early isolation and treatment with an nai for adults hospitalized with severe influenza is associated with lower mortality and better clinical outcomes. whether higher than standard doses of nai may provide greater antiviral effects in such patients would require further investigation. high-dose systemic corticosteroids were associated with worse outcomes in patients with severe influenza complicated by ards. there is an urgent need for developing more effective antiviral therapies for the treatment of influenza infections. university of hong kong. professor p.k.s.c. is a renowned clinical virologist with special interest in tumour virology and human respiratory viruses. he has published more than 330 scientific papers, and attained an h-index of 51. n.l. is professor in infectious diseases in the chinese university of hong kong. his main research interests include respiratory viral infections (influenza, rsv and coronavirus) and cap, focusing on clinical trials, patient outcomes, virokinetics and host response, and infection control in the hospital settings. he is currently working on novel antivirals and adjunctive therapies against these severe diseases. clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus human infection with a novel avian-origin influenza a (h7n9) virus who china statement on h5n6 clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study human infection 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avian influenza a(h7n9) virus: post-exposure antiviral chemoprophylaxis of close contacts of a patient with confirmed h7n9 virus infection and/or high risk poultry/environmental exposures vaccination of healthcare workers: a review influenza vaccination for healthcare workers who care for people aged 60 or older living in long-term care institutions ad hoc influenza vaccination during years of significant antigenic drift in a tropical city with 2 seasonal peaks: a cross-sectional survey among health care practitioners novel vaccines against influenza viruses formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins influenza virus-like particles comprised of the ha, na, and m1 proteins of h9n2 influenza virus induce protective immune responses in balb/c mice influenza virus-like particle can accommodate multiple subtypes of hemagglutinin and protect from multiple influenza types and subtypes interventions to reduce zoonotic and pandemic risks from avian influenza in asia additional supplementary information can be accessed via the html version of this article at the publisher's website. phylogenetic tree of haemagglutinin (ha) genes of h7n9 isolates collected from human infections. key: cord-336168-hvp13ell authors: yazdanbakhsh, maria; kremsner, peter g. title: influenza in africa date: 2009-12-15 journal: plos med doi: 10.1371/journal.pmed.1000182 sha: doc_id: 336168 cord_uid: hvp13ell maria yazdanbakhsh and peter kremsner argue that there needs to be better awareness, surveillance, and clinical management of common febrile diseases in africa, especially influenza. whereas in europe and north america most of the influenza cases are reported between december and march, in tropical and subtropical regions such as in brazil [1] or in hong kong [2] cases are seen throughout the year. epidemic peaks in the tropical areas mostly occur in between those found in the northern and southern hemispheres. a recent survey over 7 years in brazil showed that annual peaks of influenza cases occurred in association with the rainy seasons [3] . important reports on spatial and temporal data that describe the global circulation of influenza highlight the fact that there is virtually no data from africa [4, 5] . indeed, until recently, the burden of influenza in africa was believed to be negligible. however, sporadic reports from the gambia [6] , senegal [7] , congo [8] , madagascar [8] , kenya [9] , ivory coast [10] , and from gabon [11] , have indicated that influenza is circulating and may be causing epidemics regularly. the study in gabon recorded extremely high levels of antibodies to influenza a h3n2 virus in schoolchildren [11] . the haemagglutination inhibition (hi) antibodies to this influenza a virus at titers of 1,530 (ranging from 80 to 17,920) indicated that the virus had been circulating within the community in the recent past. in addition, almost all children, had anti-h1n1 hi titers above 40, while 40% showed antibodies to influenza b with hi titers of 40 or above, again highlighting the fact that multiple influenza virus strains are present in the region. the recent swine flu pandemic provides an interesting example. in the who influenza a (h1n1 swine flu) update of may 2009, many countries, but none in africa, reported virus victims [12] ; whereas two reports appeared in october 2009 that showed data on confirmed swine flu cases from south africa [13] and kenya [14] , indicating that the virus was circu-lating in africa, but because of the lack of a rigorous surveillance system, it was not reported as readily. clinically, influenza is not distinguishable from most other infectious diseases with fever in the tropics. in this context malaria is of particular interest when considering the african continent. in tropical africa, malaria is an important infectious disease and is still thought to be the main cause of febrile episodes in children. however, the threshold of clinical manifestation of malaria is strongly influenced by the endemicity of plasmodium falciparum infection in an area: in very low transmission areas, any microscopically detectable parasitemia would indicate malaria, whereas in regions of high transmission, a certain parasitemia needs to be reached to lead to malaria at least from 5 years of age on. recent, mainly unpublished observations show that there is a considerable drop in malaria incidence [15] and in p. falciparum prevalence rate [16] in some african countries. despite this reduction, the old habit of treating every child with fever with antimalarials continues. as fever due to many infectious diseases wanes after a few days without treatment, the belief that medical staff are successfully treating malaria cases lingers on. a recent study in lambarene, gabon, illustrates the extent of the problem and the unresolved conundrum. in and around lambarene all febrile children are still treated with antimalarials, mainly amodiaquine-artesunate [17] . in a study of 1,000 the essay section contains opinion pieces on topics of broad interest to a general medical audience. consecutive children presenting with fever at our research center in lambarene, the results from the thick blood smears indicated that only about 5% had malaria. recent serological tests have shown that parts of the febrile illnesses in lambarene are due not only to influenza, but also to dengue fever, chikungunya disease, and streptococcal pneumonia [18] . in addition, in tanzania where malaria is considered to be highly endemic, d'acremont and coworkers refer to recent data indicating that only 10%-40% of under-5year-old patients with fever have malarial parasites in rural areas [19] . thus, in lambarene and perhaps elsewhere in africa, the majority of febrile cases may be unnecessarily exposed to antimalarial drugs, with the well-recognized negative consequences. it is acknowledged that the problem of diagnosing influenza-like illness is already challenging in resource-rich settings apparent from data collected on the ongoing pandemic of influenza a (h1n1, swine flu). examining symptomatic individuals with recent history of travel to countries where the h1n1 virus was circulating indicated that other respiratory viruses such as rhinovirus, coronavirus, or parainfluenzavirus were responsible for influenza-like illness [20] . therefore, not surprisingly, yet often ignored, there is simultaneous transmission of different respiratory viruses and bacteria in addition to malaria that lead to febrile illnesses in africa and elsewhere in the tropics. the task of diagnosing and treating febrile illnesses properly in resource-poor tropical settings is daunting. yet, with attention to upgrading clinical research in africa focused on combatting the well known diseases such as aids, tuberculosis, and malaria, we need to start taking a few steps towards implementing programs that deal with influenza-like illness. thus in each of the northern, western, eastern, central, and southern african regions one well-established research center could be identified to act as a surveillance center. already in senegal and south africa such centers exist, but there is a need for identifying new ones in other regions and upgrading and intensifying activities in already existing ones. training should ensure that epidemiological data can be gathered on attack rates, clinical spectrum of illness, and risk factors, while molecular diagnosis of collected samples confirms influenza and identifies the strain/subtypes. in close collaboration with who centers, the behavior of the influenza virus would then be monitored properly on the african continent. contrary to common belief, excellent clinical research centers are developing in africa with good epidemiologists, information technology infrastructure, and laboratory equipment. there is no information on influenza vaccine efficacy in tropical africa. the question of whether the immune system of populations living in tropical african environments would react similarly to a vaccine developed mainly for populations restricted to certain geographical areas of the world needs to be considered. helminthic infections, malaria, and other chronic parasitic infections along with nutritional status lead to altered functioning of the immune system [11] . not only th2 responses [21] but also regulatory t cells are expanded during many parasitic infections [21, 22] and are thought to affect responses to unrelated antigens. interestingly, a study of meningococcal vaccination of infants in ghana showed lower titres of mena and menc bactericidal antibodies than in other studies in africa [23] . in a recent trial in gabon, schoolchildren from a rural and a semi-urban setting were vaccinated with influenza vaccine [11] . clear immunological differences were seen in response to the vaccine in rural versus semi-urban children. weaker th1 and pro-inflammatory cytokine responses to influenza virus antigens were seen in vaccinated rural children compared to the semi-urban vacinees. antibody levels following vaccination increased in all, but to a different extent in subjects from rural than in semi-urban areas. the antibody response following vaccination to a-h1n1 and b virus strains were significantly lower in rural compared to urban schoolchildren. the clinical relevance of such findings remains unanswered, even 30 years after studies of mcgregor in the gambia examining influenza-like illness and reporting differences in antibody reactivity in african populations and populations of european descent [24] . therefore it is important to undertake clinical trials with this vaccine in different regions of africa. the end point in these studies should be the occurrence of influenza cases to assess the efficacy of the vaccine in africa and learn about a threshold for a protective hi titer. taken together, data from sporadic studies suggest that influenza is prevalent in africa and the disease may have considerable impact on morbidity and mortality on the continent. a raised awareness of the presence of common febrile diseases such as influenza is essential for the clinical management of patients. to this end proper surveillance systems should be set up in already existing and well-established clinical research centers to understand the epidemiology of influenza in africa, which in turn may help the processes of decision making regarding influenza vaccination on the continent, which may have a high impact on health in africa. seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics influenza-associated hospitalization in a subtropical city seasonality of influenza in the tropics: a distinct pattern in northeastern brazil global patterns in seasonal activity of influenza a/h3n2, a/h1n1, and b from 1997 to 2005: viral coexistence and latitudinal gradients the global circulation of seasonal influenza a (h3n2) viruses etiology of serious infections in young gambian infants epidemiological and virological influenza survey in dakar, senegal: 1996-1998 influenza virus strains in nairobi, kenya results of two-year surveillance of flu in abidjan cellular and humoral responses to influenza in gabonese children living in rural and semi-urban areas influenza a (h1n1) -update 20 interim report on pandemic h1n1 influenza virus infections in south africa introduction and transmission of 2009 pandemic influenza a (h1n1) virus-kenya mortality rates from malaria in children under 5 fall sharply in 10 countries the limits and intensity of plasmodium falciparum transmission: implications for malaria control and elimination worldwide amodiaquine-artesunate versus amodiaquine for uncomplicated plasmodium falciparum malaria in african children: a randomised, multicentre trial community acquired pneumonia in children in lambarene, gabon time to move from presumptive malaria treatment to laboratory-confirmed diagnosis and treatment in african children with fever a variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza a(h1n1)v virus strain immune regulation by helminth parasites: cellular and molecular mechanisms upregulation of tgf-beta, foxp3, and cd4+cd25+ regulatory t cells correlates with more rapid parasite growth in human malaria infection a phase ii, randomized study on an investigational dtpw-hbv/hib-menac conjugate vaccine administered to infants in northern ghana the epidemiology of influenza in a tropical (gambian) environment key: cord-353871-mzw600ys authors: kowalczyk, d.; cieślak, k.; szymański, k.; brydak, l. b. title: the activity of influenza and influenza-like viruses in individuals aged over 14 in the 2015/2016 influenza season in poland date: 2017-02-15 journal: respiratory system diseases doi: 10.1007/5584_2016_202 sha: doc_id: 353871 cord_uid: mzw600ys infections in every epidemic season induced by respiratory viruses, especially by the influenza virus, are the cause of many illnesses and complications which often end in death. the aim of this study was to determine the activity of influenza and influenza-like viruses in individuals aged over of 14 in poland during the 2015/2016 epidemic season. a total of 5070 specimens taken from patients were analyzed. the presence of the influenza virus was confirmed in 40.2% of cases, among which the subtype a/h1n1/pdm09 (62.6% positive samples) predominated. the analysis of confirmed influenza and influenza-like viruses in individuals divided into four age-groups demonstrate that the highest morbidity was reported for the age ranges: 45–64 (13.1%) and 26–44 (12.6%) years. an increase in the number of influenza type b cases (23.7% positive samples), which was the main cause of morbidity in the age group 15–25 years, was noticeable. given the epidemiological and virological data, the 2015/2016 season in poland was characterized by increased activity of the influenza virus compared to the previous season. in the 2015/2016 season, there were more than 3.8 million cases and suspected cases of influenza and influenza-like illness, more than 15,000 hospitalizations, and up to 140 deaths. influenza is an infectious viral disease of the respiratory system, caused by influenza viruses belonging to orthomyxoviridae. there are three types of the influenza virus: a, b and c, among which infections caused by type c are asymptomatic. in every epidemic season, infections are induced by influenza virus type a and b, with usually a different course of illness for either type of virus, which also depends on the patient's age and the immune system's efficiency (brydak 2008) . influenza a virus is divided into subtypes, depending on the combination of the surface antigens hemagglutinin (ha) and neuraminidase (na). there are 18 types of ha and 11 types of na described (wu et al. 2014) . improperly treated infections caused by influenza viruses can lead to complications and consequently to death. therefore, it is important to confirm the presence of influenza virus in a short period of time, using molecular biology methods (bednarska et al. 2016b) , which enables to apply antiviral drugs, i.e., neuraminidase inhibitors, soon after onset of symptoms (fiore et al. 2011; brydak 2008) . the 2015/2016 influenza season was characterized by an increase in the number of cases and suspected cases of the influenza and influenza-like viruses, compared to the 2014/ 2015 season. there has also been a significant increase in deaths due to complications of influenza, the number of which was up to 135 in adult patients, compared with the mere 11 fatal cases in the 2014/2015 season. it is meaningful that in adult patients qualified to high-risk groups, e.g., those over 65 years of age, influenza may lead to exacerbations of chronic diseases and may be characterized by an acute fulminant course (mastalerz-migas et al. 2015; brydak et al. 2009 ). however, the percentage of vaccinated population was only 2.39% in this age range in poland in the epidemic season in question, despite the fact that the majority of local governments make the vaccination available free of charge for people over 50 years of age (brydak et al. 2012) . the aim of the present study was to analyze the activity influenza and influenza-like viruses in people over the age of 14 in the 2015/2016 influenza season in poland, according to the new reporting system (bednarska et al. 2016a the isolated material was analyzed using the rt-pcr to confirm the presence of influenza a and b viruses. a transcriptor one-step rt-pcr kit (qiagen; venlo, the netherlands) was used for the rt-pcr reaction. a 20 μl reaction mix, consisting of rnase free water, buffer, primer f (5 mm), primer r (5 mm), and the transcriptor enzyme mix, was incubated with 5 μl rna isolated from a sample. for each tested sample, an internal control amplification was performed. (h3n2), and b/phuket/3073/2013 viruses, and rnase-free water was used for negative control. before the amplification step, isolated rna were reverse transcribed into cdna at 45 c for 15 min. the cdna was analyzed as follows: initialization step of 1 cycle at 95 c for 2 min, followed by 45 cycles of amplification consisting of denaturation at 95 c for 15 s, annealing at 55 c for 30 s, and elongation at 72 c for 20 s. the presence of influenza-like viruses was confirmed in two steps by rt-pcr using rv12 ace detection kit (seegene; seul, south korea . 1) . there also were confirmed cases of influenza b in individuals over 14 years of age. this was actually the dominant type of influenza virus in persons aged 15-25. however, the highest number of cases of influenza virus type b was found in the persons aged 26-44 (n ¼ 201), far fewer in those aged 15-25 years (n ¼ 95), 45-64 years (n ¼ 85), and over 65 years (n ¼ 89) (fig. 2) . in the 2015/2016 influenza season, there were just 16 cases of influenza-like viruses reported, with rsv (n ¼ 6), piv-3 (n ¼ 4), and adv (n ¼ 3) reported most frequently and with individual cases of piv 1, hcov and rhv a/b virues. the number of 3,864,731 cases and suspected cases of influenza and influenza-like viruses in the 2015/2016 influenza epidemic season in poland was comparable to that present in the preceding season (niph-nih 2015/2016). however, the number of hospitalizations and deaths due to complications increased dramatically. the number of deaths increased from 11 in 2014/ 2015 to 140 in 2015/2016 (table 1) . despite a comparable number of cases and suspected cases of influenza and influenza-like viruses, the number of confirmed cases increased to 40.2% in 2015/2016 up from 21.2% in 2014/ 2015 (bednarska et al. 2016) . there also was a difference in the most frequent contagion between the two seasons, with subtype a/h1n1/pdm09 (62.6%) predominating in 2015/2016 as opposed to a/h3n2/ predominating the season before in persons over 14 years of age (hallmann-szelińska et al. 2016) . the present study demonstrates that subtype a/h1n1/pdm09 predominated in all age groups, except for 15-24 years old persons in whom virus type b was dominant (fig. 2) . in both compared seasons, the highest incidence of influenza was reported in the age-groups of 45-64 and 26-44 years and the lowest one in the 15-24 years old persons ( fig. 1) (bednarska et al. 2016b) . interestingly, the number of confirmed cases of influenza virus was the highest in the same age-groups of 45-64 and 26-44 years in the past 2013/2014 season (bednarska et al. 2015) . we also found that the activity of rsv and piv-3 was increased in 2015/2016 compared with that in the season before (niph-nih 2015/ 2016). the following strains were included into the influenza vaccine in the 2015/2016 season: a/california/7/2009(h1n1)pdm09, a/switzerland/9715293/2013(h3n2) and b/phuket/3073/ 2013. a particular attention should be paid to the a/california/7/2009(h1n1)pdm09 strain which was a vaccine component since the 2010/ 2011 season, and also caused most influenza cases in persons over 14 years of age in the currently evaluated season. given that the percentage of vaccinated population in this age range in poland ranks at a dismally low level and has a decreasing trend from 1.95% in 2011 to 1.37% in 2015 (epimeld 2016) , it can be judged that that the recorded number of influenza cases reflects the immunodeficiency to this viral strain (brydak 2015) . an increased number of confirmed cases of infection in the currently evaluated season was mainly caused by the influenza virus and to a lesser extent by influenza-like viruses, which may lead to a severer disease course, complications, and in consequence to death. the death as a sequelae of influenza was due usually to complications arising from the underlying chronic comorbidities, especially cardiovascular and respiratory disorders that ended up in fulminant pneumonia. the present findings of 40.2% of confirmed cases of influenza and influenza-like viruses in the 2015/2016 influenza season show a near doubling of laboratory confirmations compared with past seasons (bednarska et al. 2016b; bednarska et al. 2015) . that result points to substantial improvements in infection surveillance and imbued nuances in the virological and epidemiological procedures despite a drastically low and inexplicably decreasing percentage of adult, mostly middle-aged, population getting vaccinated against influenza each epidemic season in poland. the virological data presented in this article seek to call repeat attention to the need to vaccinate against influenza as the most effective method of preventing the infection and its severe complications, death included; a need advocated by the who, major medical societies, and 142 national influenza centers worldwide. evaluation of the activity of influenza and influenza-like viruses in the epidemic season novelties in influenza surveillence in poland antigenic drift of a/h3n2/virus and circulation of influenza-like viruses during the 2014/2015 influenza season in poland influenza, pandemic flu, myth or real threat? rythm, warsaw prophylaxis of influenza in general practice antibody response to influenza vaccine in coronary artery disease: a substudy of the flucad study implementating an influenza vaccination programme for adults aged 65 years in poland vaccinations in poland in 2015. available from antiviral agents for the treatment and chemoprophylaxis of influenza: recommendations of the advisory committee on immunization practices (acip) virological characteristic of the 2014/2015 influezna season based on molecular analysis of biological material derived from i-move study immune efficacy of first and repeat trivalent influenza vaccine in healthy subjects and hemodialysis patients bat-derived influenza-like viruses h17n10 and h18n11 acknowledgments funded by niph-nih thematic subject 5/em.1. the authors would like to acknowledge physicians and employees of vsess participating in sentinel and non-sentinel programs for their input into the influenza surveillance in poland. the authors declare no conflicts of interest in relation to this article. key: cord-292856-7hjzzxtm authors: viasus, diego; oteo revuelta, josé a.; martínez-montauti, joaquín; carratalà, jordi title: influenza a(h1n1)pdm09-related pneumonia and other complications date: 2012-10-31 journal: enfermedades infecciosas y microbiología clínica doi: 10.1016/s0213-005x(12)70104-0 sha: doc_id: 292856 cord_uid: 7hjzzxtm abstract influenza a(h1n1)pdm09 virus infection was associated with significant morbidity, mainly among children and young adults. the majority of patients had self-limited mild-to-moderate uncomplicated disease. however, some patients developed severe illness and some died. in addition to respiratory complications, several complications due to direct and indirect effects on other body systems were associated with influenza a(h1n1)pdm09 virus infection. the main complications reported in hospitalized adults with influenza a(h1n1)pdm09 were pneumonia (primary influenza pneumonia and concomitant/secondary bacterial pneumonia), exacerbations of chronic pulmonary diseases (mainly chronic obstructive pulmonary disease and asthma), the need for intensive unit care admission (including mechanical ventilation, acute respiratory distress syndrome and septic shock), nosocomial infections and acute cardiac events. in experimentally infected animals, the level of pulmonary replication of the influenza a(h1n1)pdm09 virus was higher than that of seasonal influenza viruses. pathological studies in autopsy specimens indicated that the influenza a(h1n1)pdm09 virus mainly targeted the lower respiratory tract, resulting in diffuse alveolar damage (edema, hyaline membranes, inflammation, and fibrosis), manifested clinically by severe acute respiratory distress syndrome with refractory hypoxemia. influenza a(h1n1)pdm09-related pneumonia and other complications were associated with increased morbidity and mortality among hospitalized patients. influenza a(h1n1)pdm09 virus infection was associated with significant morbidity, mainly among children and young adults. the majority of patients had self-limited mild-to-moderate uncomplicated disease. however, some patients developed severe illness and some died. in addition to respiratory complications, several complications due to direct and indirect effects on other body systems were associated with influenza a(h1n1)pdm09 virus infection. the main complications reported in hospitalized adults with influenza a(h1n1)pdm09 were pneumonia (primary influenza pneumonia and concomitant/secondary bacterial pneumonia), exacerbations of chronic pulmonary diseases (mainly chronic obstructive pulmonary disease and asthma), the need for intensive unit care admission (including mechanical ventilation, acute respiratory distress syndrome and septic shock), nosocomial infections and acute cardiac events. in experimentally infected animals, the level of pulmonary replication of the influenza a(h1n1)pdm09 virus was higher than that of seasonal influenza viruses. pathological studies in autopsy specimens indicated that the influenza a(h1n1)pdm09 virus mainly targeted the lower respiratory tract, resulting in diffuse alveolar damage (edema, hyaline membranes, inflammation, and fibrosis), manifested clinically by severe acute respiratory distress syndrome with refractory hypoxemia. influenza a(h1n1)pdm09-related pneumonia and other complications were associated with increased morbidity and mortality among hospitalized patients. © 2012 elsevier españa, s.l. all rights reserved. r e s u m e n si bien la mayoría de los pacientes infectados por el virus de la gripe a(h1n1)pdm09 tuvieron enfermedad no complicada, autolimitada, leve a moderada, la infección se caracterizó por una morbilidad significativa, especialmente entre niños y adultos jóvenes, de forma que algunos pacientes desarrollaron una enfermedad grave y algunos murieron. la infección por virus de la gripe a(h1n1)pdm09 se asoció no sólo con complicaciones respiratorias, sino también con complicaciones debidas a los efectos directos e indirectos sobre otros sistemas del organismo. en los pacientes adultos hospitalizados las complicaciones principales fueron neumonía (neumonía primaria por gripe y neumonía bacteriana concomitante/secundaria), exacerbaciones de enfermedades pulmonares crónicas (principalmente enfermedad pulmonar obstructiva crónica y asma), necesidad para la admisión en unidad de cuidados intensivos (incluso ventilación mecánica, síndrome de dolor respiratorio agudo y shock séptico), infecciones nosocomiales y acontecimientos cardíacos agudos. en los animales de experimentación infectados con virus de la gripe a(h1n1)pdm09 el nivel de replicación del virus a nivel pulmonar era más alto que el de los virus de la gripe estacional. los estudios anatomopatológicos de muestras de autopsia mostraron que el virus de la gripe a(h1n1)pdm09 actúa principalmente sobre el tracto respiratorio inferior, provocando lesión difusa del alveolo (edema, membranas hialinas, inflamación y fibrosis), lo que se traduce clínicamente en un síndrome de distrés respiratorio agudo grave con hipoxemia refractaria. la neumonía y otras complicaciones relacionadas con la gripe por virus a(h1n1)pdm09 se asociaron a una mayor morbilidad y mortalidad en los pacientes hospitalizados. © 2012 elsevier españa, s.l. todos los derechos reservados. since the 16th century, influenza pandemics have been described all over the world, at intervals ranging from 10 to 50 years and with varying degrees of severity. in april 2009, a novel influenza virus now known as influenza a(h1n1)pdm09 virus caused an outbreak of respiratory disease in mexico 1 and spread rapidly worldwide, resulting in the first influenza pandemic of this century. spain was the first country in europe to report a laboratory-confirmed case of influenza a(h1n1)pdm09 virus infection. 2 the number of hospitalizations and deaths due to influenza a(h1n1)pdm09 increased continuously until december 2009. it was recently reported that influenza a(h1n1)pdm09 virus infection during the 2010-2011 influenza season was associated with higher morbidity than that observed during the pandemic period. 3, 4 children and young adults accounted for most cases of influenza a(h1n1)pdm09 virus infection. the majority of patients had selflimited, mild-to-moderate uncomplicated disease. however, some patients developed severe illness and some died. common symptoms included cough, fever, sore throat, myalgia and headache. some cases experienced gastrointestinal symptoms (nausea, vomiting and/or diarrhea). 5 the major complications of influenza were those involving the lower respiratory tract, mainly pneumonia. 6 in addition, secondary bacterial infections, rhabdomyolysis with renal failure, seizures, and worsening of underlying conditions such as cardiovascular disease were also reported. 5, 6 influenza a(h1n1) pdm09-related pneumonia and other complications were associated with increased morbidity and mortality. the purpose of this article is to summarize the experience of the spanish network for the research in infectious diseases (reipi) with regard to influenza a(h1n1)pdm09-related pneumonia and other complications. we also performed a literature review regarding complications associated with influenza a(h1n1)pdm09. the most frequent serious complications of influenza are pulmonary, and fall into four categories: primary influenza pneumonia, secondary bacterial pneumonia, pneumonia due to unusual pathogens or in immunocompromised hosts, and exacerbations of chronic pulmonary diseases. 6 interestingly, in experimentally infected animals, the level of pulmonary replication of the influenza a(h1n1)pdm09 virus was higher than that of seasonal influenza viruses. 7 the frequency of complications in the reipi cohort of hospitalized patients with influenza a(h1n1)pdm09 virus infection is detailed in table 1 . in the reipi cohort, 585 patients (median age 40 years) required hospitalization. chest radiography was obtained on 542. a total of 234 patients (43.1%) had pneumonia, of whom 210 underwent one or more bacterial microbiologic studies. pneumonia was primary viral in 174 of these patients and concomitant/secondary bacterial in 36. 8 similarly, in a study performed in united states, 9 of 451 hospitalized patients on whom chest radiographs were performed, 195 (43%) had pneumonia (bacterial infections were reported in 13 patients with pneumonia). in other studies, the reported frequency of pneumonia in hospitalized patients ranged between 23% and 66%. [10] [11] [12] pneumonia was associated with high morbidity, as assessed by the length of hospital stay and the rates of intensive care unit (icu) admission and in-hospital complications, including mortality. 8, 9 pathological studies on autopsy samples from 100 patients with fatal influenza a(h1n1)pdm09 virus infection revealed that the virus targeted the lower respiratory tract, resulting in diffuse alveolar damage (edema, hyaline membranes, inflammation, and fibrosis), as manifested clinically by severe acute respiratory distress syndrome (ards) with refractory hypoxemia. however, a significant proportion of influenza a(h1n1)pdm09 case-patients in that report also showed viral localization along with inflammation or other histopathological changes in trachea, bronchi, or bronchioles. 13 these pathological data have been found in other studies. 14 the first conclusive evidence that the influenza virus could cause pneumonia came during the 1958 to 1959 pandemic. pathologic findings in pure influenza pneumonia include necrotizing bronchitis, hyaline membranes, intra-alveolar hemorrhage and edema, and interstitial inflammation. 6 in the reipi cohort of hospitalized patients with primary viral pneumonia, 124 (71.3%) were below the age of 50 and 98 (56.3%) were males. nearly 50% of patients had underlying medical comorbidities, mainly chronic pulmonary disease (21.8%), immunosuppression (12.1%), diabetes mellitus (12.1%) and chronic cardiac disease (8.6%). obesity (bmi > 30) was documented in 20.1% and pregnancy in seven women. the most frequent clinical features reported were fever, cough, arthromyalgia and dyspnea. pleuritic chest pain was present in 21% of patients and gastrointestinal symptoms in 15%. findings on physical examination included diffuse rales and wheezing. radiographs revealed multilobar infiltrates in 63.2%. forty-one (23.6%) required icu admission and in-hospital mortality was 4.6%. 8 it is significant that most patients requiring icu admission during the pandemic had respiratory failure due mainly to primary influenza pneumonia. [15] [16] [17] in a spanish study of patients requiring icu admission, more than half (55.1%) of subjects with primary viral pneumonia were male and the mean age was 43 years. mechanical ventilation was used in 70.2% of the patients, 60.2% with invasive modes and 23.1% with noninvasive. obesity was the most frequent comorbidity (40.1%), followed by chronic obstructive pulmonary disease (copd) (12.1%), diabetes (11%) and asthma (10.5%). overall mortality was 17.7%. 18 it has long been recognized that influenza infection is closely associated with an increased incidence of bacterial pneumonia. 19 in previous pandemics, secondary bacterial pneumonia was considered when a typical viral influenza infection was followed by near resolution, subsequently complicated 4 to 14 days later by a recurrence of fever, dyspnea, productive cough, and pulmonary consolidation. in contrast, concomitant bacterial pneumonia was considered when a bacterium was isolated during the first days of influenza virus infection onset. 20, 21 this classification had important implications for the etiologic agents identified in these patients. during pandemic (h1n1) 2009, bacterial pneumonia was infrequent in mexico and california. 11, 22 however, in studies of autopsy specimens, shieh et al 13 reported bacterial co-infection in 26 of 100 patients with fatal influenza a(h1n1)pdm09. other studies found the frequency of bacterial pneumonia in patients requiring icu admission to range between 20.3% and 32.1%. 15, 16 in the reipi cohort, the prevalence of concomitant/secondary bacterial pneumonia was 17.2%. streptococcus pneumoniae was the most frequent causative pathogen of bacterial coinfection in this cohort 8 and in other studies. 23, 24 several reports identified methicillin-resistant staphylococcus aureus as the etiologic agent for secondary/concomitant bacterial pneumonia during the pandemic. 25 other pathogens isolated were haemophilus influenzae, streptococcus spp., legionella pneumophila, pseudomonas aeruginosa, acinetobacter baumannii, and aspergillus sp. 26 studies have reported an association between bacterial coinfection and disease severity. 8, 24 one study 27 found that 70% of patients with influenza a(h1n1)pdm09 virus infection and invasive group a streptococcus died, compared with an overall mortality rate of 2-6% for hospitalized influenza a(h1n1)pdm09 patients in other studies. investigators have sought to determine the clinical features and factors associated with concomitant/secondary bacterial pneumonia. compared with patients with primary viral pneumonia, patients with bacterial pneumonia in the reipi cohort were more likely to have chronic liver disease, purulent sputum, tachycardia, pleural effusion, leukocytosis, and c-reactive protein (crp) levels above 80 mg/l at hospital admission. conversely, interstitial bilateral infiltrates in chest x-rays were more frequent in patients with primary viral pneumonia ( table 2 ). 8 moreover, dhanoa et al. 28 reported that age >50 years, presence of comorbidity, liver impairment, development of complications, supplemental oxygen requirement, leukocytosis and neutrophilia were clinical factors associated with bacterial co-infection. interestingly, studies have found that procalcitonin and crp both alone and in combination can detect pneumonia of mixed bacterial infection in this context. 29, 30 furthermore, patients with co-infection at icu admission were older and presented a higher apache (acute physiology and chronic health evaluation) ii score and sofa (sequential organ failure assessment) score compared with patients with primary viral pneumonia. 31 bacterial pneumonia presents distinctive radiographic features because it is often associated with pleural effusion, lymphadenopathy and lobar consolidations. conversely, the characteristic imaging findings in primary viral pneumonia are ground-glass opacities with areas of consolidation. 32,33 infectious agents are recognized as a major pathogenic factor in exacerbations of chronic pulmonary diseases. the relevance of viral infections has been studied in exacerbations of copd and asthma. patients were stratified into the following risk groups according to the curb-65 score: low risk (≤1 points, groups 0 and 1) and high risk (≥2 points, groups 2 and 3). rhinovirus, coronavirus, respiratory syncytial virus, and influenza are the main viral pathogens that cause exacerbations. 6, 34, 35 chronic pulmonary diseases, mainly copd and asthma, are frequent comorbidities reported in hospitalized patients with influenza a(h1n1)pdm09 virus infection. 8, 9, 11 information about clinical features and prognosis from these groups of patients during pandemic is scarce; most of the information available comes from hospitalized asthmatic children. 36 in the reipi cohort, 49 (8.4%) patients had copd and 104 (17.8%) had asthma. pneumonia was documented in 17 copd patients. of the 32 copd patients without pneumonia, 21 (65.6%) had exacerbation of pulmonary disease (evidence of wheezing at hospital admission). among patients with copd exacerbation, five required icu admission (three needed mechanical ventilation); there were no deaths. bacterial co-infection was documented in only one copd patient. moreover, pneumonia was documented in 24 asthma patients (chest x-rays were performed in 100 asthma patients). of the 76 asthma patients without pneumonia, 45 (59.2%) had exacerbations of pulmonary disease. among patients with asthma exacerbation, seven required icu admission (two needed mechanical ventilation) and none died. bacterial co-infection was not documented in these patients. characteristics of icu patients and clinical outcomes of the reipi cohort were similar to those described elsewhere. [15] [16] [17] 37 although our mortality rate was lower than that reported in the earliest studies of hospitalized patients, it was similar to that of other studies. 38, 39 severe disease occurred in 75 patients (12.8%), of whom 71 required icu admission and 13 died. among the 71 patients requiring icu admission, 53 had pneumonia, 52 underwent mechanical ventilation, and 23 developed ards. fifty-two (73.2%) of the 71 icu patients had chronic comorbid conditions, mainly chronic pulmonary disease (33 patients), chronic heart disease (10), diabetes mellitus (10) and immunosuppression (7) . only 2 of 98 pregnant women required icu admission. 40 in-hospital mortality was 2.2% (13 of 585 patients). the median time from hospital admission to death was nine days (range 1-33). among the 13 patients who died, nine were under 50 years of age, eight were women, 12 had comorbid conditions, two had morbid obesity, 11 had multilobar pneumonia, and five had bacterial coinfection. causes of death were respiratory failure/acute respiratory distress syndrome (3 out of 13 patients), shock/multiorgan failure (4 patients), decompensated comorbid conditions (4) and nosocomial infection (2) . 40 in the reipi cohort, independent factors associated with severe disease were younger age, chronic comorbid conditions, morbid obesity and bacterial co-infection. conversely, early oseltamivir therapy was a protective factor. 40 in another study, 15 investigators identified all patients with confirmed influenza a(h1n1)pdm09 virus infection who were admitted to australian or new zealand icus during winter 2009. interestingly, the number of icu admissions due to influenza a(h1n1)pdm09 was 15 times higher than that due to viral pneumonitis in previous years. infants and younger adults were found to be at particular risk of icu admission. pregnant women, obesity, and indigenous australian and new zealand populations also appeared to have an increased risk. in-hospital mortality exceeded 16%. furthermore, in icu patients in canada, 16 influenza a(h1n1)pdm09 affected primarily young, female, and aboriginal patients without major comorbidities; 28-day mortality was 14.3%. chronic lung disease, obesity, hypertension, and diabetes were the most common comorbidities. critical illness occurred rapidly after hospital admission and was associated with severe oxygenation failure, a need for prolonged mechanical ventilation, and the frequent use of rescue therapies such as extracorporeal membrane oxygenation. factors associated with icu admission or mortality during pandemic (h1n1) 2009 are detailed in table 3 . it is important to note that we documented that influenza a(h1n1) pdm09 was not associated with poorer outcomes in hospitalized pregnant women compared with non-pregnant women of reproductive age in a context of early diagnosis and antiviral therapy. 41 similarly, in the reipi cohort, well controlled on haart hiv patients had a similar clinical outcomes and prognosis to that of non-hiv patients. 42 interestingly, cap-specific scores demonstrated moderate usefulness for predicting icu admission and/or mortality in hospitalized patients with influenza a(h1n1)pdm09 complicated by pneumonia in the reipi cohort and other studies. 8, 47 consistent with these data, severity assessment tools (general severity of illness and cap-specific scores) undervalued prognosis and should not be used as instruments to guide decisions on patients requiring icu admission. 48 a limitation of these scores is that age is the variable with the most weight, and most patients affected by pneumonia during pandemic were younger adults. in addition, other risk factors for severe influenza a(h1n1)pdm09 such as obesity were not included in these scores. in addition to respiratory complications of viral influenza, several other complications due to direct and indirect effects on other body systems have been reported to be associated with influenza a(h1n1) pdm09 virus infection. influenza virus frequently exacerbates underlying heart problems and has been associated with triggering myocardial infarction. 49 in the reipi cohort, 12 patients had concurrent acute cardiac events during hospitalization (nine had acute heart failure, five had arrhythmias and one had acute coronary syndrome). among these patients, seven were over 50 years old, ten had comorbid conditions (mainly chronic heart disease and copd) and five were current smokers. regarding outcomes, two had pneumonia, ten required icu admission, and one died. interestingly, other studies documented myocarditis, pericarditis, electrocardiographic abnormalities and left ventricular systolic dysfunction concurrent with influenza a(h1n1) pdm09 virus infection. [50] [51] [52] it is well known that influenza has neurological manifestations and complications. neurological complications of influenza include encephalopathy, encephalomyelitis, transverse myelitis, aseptic meningitis, focal neurological disorders, and guillain-barre syndrome. most cases occur in children. 6 in a recent study on hospitalized pediatric patients with influenza a(h1n1)pdm09 infection, 53 the most common manifestation was seizure with underlying neurological disease followed by encephalopathy with or without neuroimaging changes. in another study, the primary influenza-associated neurologic complications were encephalopathy/ encephalitis, seizures, meningitis, and guillain-barre syndrome. 54 in table 3 factors associated with intensive care unit admission or mortality in patients with influenza a(h1n1)pdm09 virus infection 15 the reipi cohort of adult patients, two developed neurological complications: one had meningoencephalitis and the other acute visual disturbances related to oseltamivir treatment. nosocomial infections were reported in 16 patients in the reipi cohort, mainly catheter-associated bacteremia and nosocomial pneumonia. in this regard, influenza viruses have been reported to cause immune defects. 55 in addition, our data 56 and those of other studies 57 suggest that patients with influenza infection receiving corticosteroids present higher rates of nosocomial infections and sepsis. other complications reported during influenza were myositis and rhabdomyolysis, reye's syndrome, and preterm delivery. psychiatric complications after influenza infection are considered controversial. 6 acute kidney failure was also reported, with some patients requiring renal replacement therapy. risk factors for acute kidney injury included age, obesity, chronic kidney disease, and elevated creatine kinase. 58 complications of influenza a(h1n1)pdm09 virus were frequent and involved numerous organ systems. the main complications were pneumonia, icu admission (ards, septic shock, mechanical ventilation), exacerbations of chronic pulmonary diseases, nosocomial infections, and acute cardiac events. most hospitalized patients with pneumonia had primary viral pneumonia; mortality, though low, occurred mainly in patients with this complication. younger age, comorbidities, morbid obesity and bacterial co-infection were risk factors for severe disease. influenza a(h1n1)pdm09related complications were associated with increased morbidity and mortality. this study was supported by the ministerio de ciencia e innovación, instituto de salud carlos iii, (ministry of science and innovation, carlos iii health institute), programa de investigación sobre gripe a/h1n1 (influenza a/h1n1 research program) (grant: gr09/0014), and co-financed by european regional development fund "a way to achieve europe", spanish network for the research in infectious diseases (reipi rd06/0008). dr. viasus is the recipient of a research grant from the institut 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induces functional alterations in peripheral blood lymphocytes effect of immunomodulatory therapies in patients with pandemic influenza a (h1n1) 2009 complicated by pneumonia current concepts in sars treatment influenza a infection and acute kidney injury: incidence, risk factors, and complications all authors declare that they have no conflicts of interest in this article. key: cord-295531-zojb3cew authors: huggett, kathryn d.; knoop, floyd c. title: influenza a date: 2008-01-10 journal: xpharm: the comprehensive pharmacology reference doi: 10.1016/b978-008055232-3.60922-5 sha: doc_id: 295531 cord_uid: zojb3cew the influenza viruses, which contain single-stranded rna, are classified into 3 types, a, b, and c … the influenza viruses, which contain single-stranded rna, are classified into 3 types, a, b, and c dolin (2000) , murray et al (2002) , treanor (2005) . the virus has 8 segments and an enveloped nucleocapsid. type a subtypes vary in type of hemagglutinin (h1, h2, or h3 subtype), the protein that is important for host cell attachment. antibodies directed at the hemagglutinin provide human immunity to infection. in addition, the virus possesses either n1 or n2 neuraminidase (n subtype), which also assists viral entry into the host cell and elicits the production of neutralizing antibodies de jong et al (2003) . influenza is a highly communicable disease transmitted by aerosolized droplet-nuclei produced by coughing and/or sneezing, and by direct contact. all age groups are susceptible to this infection, although the very young, elderly, individuals with chronic medical complications, such as those with compromised pulmonary and cardiovascular conditions, kidney impairment, diabetes or immunosuppressive disorders, and pregnant women in the 2 nd and 3 rd trimester are at increased risk. although the reservoir is primarily human, influenza a viruses have been documented in pigs, horses, seals, ducks, chickens, and whales. certain subtypes of influenza a are species specific, although all subtypes have been documented in birds. prominent human subtypes include h3n2, h2n2, h1n1 and h1n2. the influenza a virus has 15 distinct h and 9 distinct n subtypes, although h1, h2, and h3 and n1 and n2 are prominently associated with disease outbreaks in humans. subtypes of influenza a viruses that are prominent in one species may on occasion infect and cause disease in another. in 1998, for example, the h3n2 subtype crossed from humans to pigs, causing serious disease in the swine population. in addition, influenza a viruses from birds can be transmitted to humans directly or indirectly by an intermediate host. in this way the 8 genetic elements of influenza a virus may reassort to form a new subtype (h*n*) against which humans not immune schweiger et al (2002) . in this situation a worldwide pandemic can occur. documented instances of avian influenza viruses infecting humans are numerous. influenza a is a single-stranded rna virus that causes an acute and highly contagious upper respiratory disease. the influenza a virus is a member of the family of viruses termed orthomyxoviridae. these viruses have a single negative strand of rna that consists of 8 separate segments, a nucleocapsid that is helical, and a viral specific rna polymerase. influenza viruses exit cells by budding from the host cell plasma membrane (enveloped), and possess two types of spike protein, neuraminidase [n] and hemagglutinin activity [h] , that emanate from the lipid envelope. the virus has an irregular but spherical shape and measures 80-120 nm in diameter. influenza illness is uncomplicated in an otherwise healthy and nonelderly adult. most fatalities (0.1-1.0%) occur due to secondary bacterial infection of the respiratory tree. although the most common cause of bacterial pneumonia is due to pneumococci, the most serious is due to staphylococcal infection. haemophilus influenzae, streptococcus pneumoniae, and streptococcus pyogenes are other infrequent causes of pneumonia. the mortality rate is higher among the elderly, pregnant women, and those with other medical complications. influenza a infection is often confused with the common cold (http://www.niaid.nih.gov/ factsheets/cold.htm), which may have similar signs and symptoms (http://familydoctor. org/517.xml). influenza a infection resembles numerous other mild febrile diseases but stands out due to cough, muscle aches, malaise, and ''sudden onset''. other differential diseases include infections caused by rsv (respiratory syncytial virus), adenoviruses, rhinoviruses, parainfluenza viruses, measles virus, severe acute respiratory syndrome (sars) coronavirus, mycoplasma pneumoniae, chlamydia pneumoniae (twar agent), bordetella pertussis, salmonella typhi, and streptococcus pyogenes. influenza may be difficult to diagnose in the absence of an epidemic. the etiologic agent of influenza, or the flu, is the influenza virus. the three types of virus are classified as a, b, and c. all types contain a single-stranded rna that has 8 segments packaged into an enveloped nucleocapsid. the influenza a virus is spread by droplet-nuclei from sneezing and coughing patients. the incidence parallels other upper respiratory infections that occur in northern frigid winter months (late autumn to early spring). influenza a epidemics are cyclic and occur every 2-3 years. new epidemics occur because of antigenic drift or minor changes in the h and/or n antigens, thereby allowing the virus to avoid the immune system. the cdc collects and reports relevant surveillance data http://198.246.96.2/flu/weekly/fluactivity. htm. influenza a virus causes necrosis of the respiratory epithelium in the tracheobronchial tree and nasal turbinates. desquamation of the ciliated epithelium, edema, hyperemia, increased secretions, and congestion may therefore result in a secondary bacterial infection. laboratory findings include leukopenia and often proteinuria. the influenza a virus influenza a is a highly contagious disease marked by the acute onset of fever (102-104 f), severe aches and pains, prostration, inflammation of the mucous membranes of the respiratory tract, and severe headache. in addition, the affected individual will show signs of coryza, nonproductive cough, flushed face, and sore throat. in children, nausea, vomiting, and otitis media are often reported. fever should subside in 3-4 days and recovery is usually complete in one week, although malaise and cough may continue for 2 weeks or more. the normal incubation period is 1-4 days, with an average of 2 days. adults are contagious from the day prior to symptoms for up to 5 days after the onset of illness. immunocompromised patients shed virus for weeks or months. the treatment for influenza a infections is aimed at the control of virus spread by immunization stohr (2003), weycker et al (2005) . chemoprophylaxis for pregnant women and compromised patients can be accomplished with amantadine, rimantadine, zanamivir, or oseltamivir hayden (2001), schmidt (2004) . antipyretics and analgesics are typically used for the symptomatic relief of fever and myalgia. older nursing-home residents should be administered only 100 mg/day of rimantadine. a reduction in dosage to 100 mg/day should be considered for all persons aged 65 years, if they experience possible side effects when taking 200 mg/day. *** zanarnivir is administered through inhalation by using a plastic device included in the medication package. patients will benefit from instruction and demonstration of correct use of the device. zanarnivir is not approved for prophylaxis. }}} a reduction in the dose of oseltamivir is recommended for persons with creatinine clearance <30 ml/min. the dose recommendation for children who weigh 15 kg is 30 mg twice a day. for children who weigh >15-23 kg, the dose is 45 mg twice a day. for children who weigh >23-40 kg, the dose is 60 mg twice a day. and, for children who weigh >40 kg, the dose is 75 mg twice a day. 1-40) . the terminology for influenza viruses is directly related to the type of virus, the origin or place it was first located, and the year of its discovery. the immunity invoked by influenza vaccine generally lasts 6-12 months. amantadine amantadine is active against influenza a viruses but not influenza b. it was approved in 1966 for chemoprophylaxis and in 1976 for treatment and chemoprophylaxis of influenza type a virus in both adults and children 1 year of age. amantadine may cause central nervous system side effects (13%), including anxiety, nervousness, insomnia, light headedness, and concentration difficulty, and gastrointestinal side effects (1-3%), including nausea and anorexia, at dosages of 200 mg/day. because its effects on pregnant women and their fetuses are unknown, it should be used during pregnancy only if the potential benefit justifies the risks. drug resistance develops in approximately one-third of patients, with cross resistance to rimantadine. maximum blood levels are attained within 4 hours of administration of a single 100 mg dose. the peak excretion rate of amantadine is 5 mg/hr, and the mean half-life of the excretion rate is approximately 15 hours. clearance of amantadine is reduced 2-3-fold in adults with renal insufficiency. in otherwise healthy elderly adults (65 years) the renal clearance is reduced and plasma levels therefore increased. the drug, like rimantidine, influences viral uptake by preventing membrane fusion and affects maturation of the hemagglutinin (h) to lower the infectivity of progeny virus. it is 70-90% effective in preventing illness in healthy adults and, if administered for prophylaxis within 2 days of illness, can reduce flu symptoms and shorten the duration of the illness by 1 or 2 days. rimantadine rimantadine is effective against influenza a but not influenza b viruses. it was approved in 1993 for the treatment and chemoprophylaxis of influenza a infections in adults and prophylaxis in children. some consider it appropriate for the treatment of disease in children. rimantadine may cause central nervous system side effects (6%), including anxiety, nervousness, insomnia, light headedness and concentration difficulty, and gastrointestinal side effects (1-3%) such as nausea and anorexia, at dosages of 200 mg/day. because its effects on pregnant women and their fetuses are unknown, it should be used during pregnancy only if the potential benefit justifies the risks. drug resistance develops in approximately one-third of patients, with cross resistance to amantadine. rimantadine is 70-90% effective in preventing illness in healthy adults and, if administered for prophylaxis within 2 days of illness, can reduce flu symptoms and shorten the duration of the illness by 1-2 days. zanamivir is a neuraminidase inhibitor that is active against both influenza a and b viruses. it was approved in 1999 for the treatment of uncomplicated influenza infections in patients aged 7 years. guidelines and recommendations for its use can be found at: http://www.cdc.gov/flu/professionals/treatment/ 0405antiviralguide.htm. as its effects on pregnant women and their fetuses are unknown, it should be used during pregnancy only if the potential benefit justifies the risks. resistance to zanamivir during treatment may occur, but does not happen frequently. oseltamivir oseltamivir is a neuraminidase inhibitor that is active against both influenza a and b viruses. it was approved in 1999 for the treatment of uncomplicated influenza infections in patients aged 1 year. in 2000 it was approved for the chemoprophylaxis of influenza infections in persons aged 13 years. guidelines and recommendations for its use can be found at: http://www.cdc.gov/flu/ professionals/treatment/0405antiviralguide.htm. as its effects on pregnant women and their fetuses are unknown, it should be used during pregnancy only if the potential benefit justifies the risks. resistance to oseltamivir during treatment may occur, but does not happen frequently. nasal spray. vaccine bacteria from fermented milk were incorporated into a nasal spray and used as a preventive treatment for influenza in mice hori et al (2001) . killed lactobacillus casei were used in an intranasal spray to stimulate the immune response in the respiratory tract. treated mice had one-tenth the amount of virus in their nasal cavities after virus challenge, resulting in a four times greater survival rate. the mouse model for influenza is used for the evaluation of vaccines and antiviral drugs sidwell and smee (2000) . in addition, the ferret is a natural host for this virus, with the ensuing illness closely resembling the human condition maher and destefano (2004) . basic information on the diagnosis, clinical findings, complications, prevention and treatment of influenza, including vaccine safety recommendations, can be found at: http://www3.accessmedicine.com/content.aspx?aid=17572#17572 information on the common cold and flu can be located at: http://familydoctor.org/517. xml further information on the common cold and flu can be located at: http://www.niaid.nih. gov/factsheets/cold.htm a chapter on antiviral chemotherapy can be found at: http://www.med.sc.edu:85/lecture/ chemo.htm detailed information on influenza viruses can be located at: http://www3.accessmedicine. com/content.aspx?aid=74879&searchstr=influenza+a+virus#74879 overview of the who global influenza programme haemagglutination-inhibiting antibody to influenza virus antiviral therapy for influenza: a clinical and economic comparative review population-wide benefits of routine vaccination of children against influenza strategies for use of a limited influenza vaccine supply effect of intranasal administration of lactobacillus casei shirota on influenza virus infection of upper respiratory tract in mice antigenic drift and variability of influenza viruses in vitro and in vivo assay systems for study of influenza virus inhibitors the ferret: an animal model to study influenza virus book citations harrison's principles of internal medicine medical microbiology mandell, douglas, and bennett's principles and practice of infectious diseases goodman and gilman's the pharmacological basis of therapeutics key: cord-313062-lpxmmbpy authors: amini, rachid; gilca, rodica; boucher, françois d.; charest, hugues; de serres, gaston title: respiratory syncytial virus contributes to more severe respiratory morbidity than influenza in children < 2 years during seasonal influenza peaks date: 2019-02-23 journal: infection doi: 10.1007/s15010-019-01287-5 sha: doc_id: 313062 cord_uid: lpxmmbpy purpose: to compare the frequency and the severity of influenza and respiratory syncytial viruses (rsv) infections among children < 24 months hospitalized with respiratory symptoms. methods: data from a prospective study conducted during the peak of five influenza seasons in the province of quebec, canada were used. results: we detected higher frequency of rsv compared to influenza viruses (55.3% vs. 16.3%). radiologically confirmed pneumonia was significantly more frequent in children with rsv (39%) than those with influenza (18%) and the clinical course was more severe in rsv than influenza-infected children, especially among infants < 3 months. conclusion: even during peak weeks of influenza season, we found a higher burden and severity of rsv compared with influenza virus disease in hospitalized children < 24 months. respiratory syncytial virus (rsv) and influenza virus infections are considered as leading cause of hospitalization among infants and young children [1, 2] . in a recent study, the average hospitalization rate attributable to rsv infection has been reported to be 5.2 per 1000 children under 2 years with seasonal variation from 2.5 to 9.9 per 1000 children [3] . among the same age group, a systematic review has estimated influenza-associated hospitalization rates from 1 to 5 per 1000 children [1] . for both viruses, the highest hospital admission rates were among infants < 3 months with an average rate of 17.9 and 3.3 per 1000 children, respectively, for rsv and influenza [3, 4] . most studies assessing the influenza and rsv-associated hospitalization burden focus on the entire respiratory viruses season. little information is available about the role of influenza viruses relative to rsv during periods of high influenza circulation among young children. the aim of this study is to compare the frequency and disease severity of influenza and rsv infections among children < 24 months hospitalized with respiratory symptoms during the peaks of five influenza seasons. in the province of quebec, canada, a prospective surveillance study with virologic assessment for influenza and other respiratory viruses was conducted during the peak weeks of influenza circulation among patients admitted for acute respiratory symptoms to four acute-care hospitals since 2012-2013. peak weeks are defined as consecutive weeks with ≥ 15% of weekly specimens testing positive for influenza as monitored by the provincial sentinel laboratory network. all patients hospitalized for fever/feverishness or cough or sore throat have a nasal specimen collected with a flocked swab. specimens are tested by the luminex xtag ® rvp fast multiplex, a polymerase chain reaction (pcr) assay which can detect 17 respiratory viruses (rv) [5] . patients admitted for ≥ 24 h to the hospital floor are invited 1 3 to participate in the study; those who give their informed consent are included. demographic and clinical information of patients including chronic conditions associated with increased risk of influenza complications is collected using a standardized questionnaire. in our study, patient's chronic conditions information was retrieved by nurses from medical records and we did not ascertain diagnosis criterions of the asthma condition identified through the chart review among the children < 2 years. as it is difficult to diagnose asthma in these young children and confirm it as definite bronchial asthma, we used the term "asthma-like condition". additional details are presented elsewhere [5] . the peak weeks of the 2012-2013 and 2014-2015 to 2017-2018 influenza seasons were included in the analysis. the 2013-2014 season was excluded, because only influenza viruses were tested during this season. proportions were compared with the chi-square test or the fisher's exact test where appropriate. continuous variables were compared using wilcoxon and kruskal-wallis nonparametric tests. during the peaks of five influenza seasons lasting from 8 to 10 weeks, 546 children were enrolled in the study. nearly, a third were infants < 3 months (34%, n = 188), 15% were aged 3-5 months (n = 83) and 6-11 months (n = 84), and 35% were aged 12-23 months (n = 191). except for the 2017-2018, where the epidemic peak was characterized by the concomitant circulation of influenza a and b, all the other peak weeks of the study period corresponded mainly to the influenza a peak ( at least one respiratory virus was identified in 94.1% of children. rsv was detected in more than half of children (55.3%), and the presence of both influenza and rsv was observed in 1.6% of cases. despite being during the peak weeks of the influenza epidemic, influenza viruses were detected in one of six children (16.3%). influenza a (h3n2) was the predominating subtype (60%) and influenza a (h1n1) pdm09 and influenza b were detected in 24% and 15% of specimens, respectively. other respiratory viruses (orv) were detected in 20.9% of children: entero/ rhinoviruses (7.1%), coronaviruses (5.5%), human metapneumovirus (3.3%) and parainfluenza viruses (2.7%), and other viruses (adenovirus and bocavirus) (2.2%). among the young infants < 3 months, the clinical course was less severe in human metapneumovirus (n = 3) than in rsv-infected children based on the presence of radiologically confirmed pneumonia (0% vs. 17%), length of stay (median of 3 days vs. 4 days) and intensive care unit admission (0% vs. 8.6%), although differences are not statistically significant due to the small sample size. rsv was more frequently detected among infants aged 3-5 months (73.5%) than in those < 3 months (53.7%), 6-11 months (53.6%), and 12-23 months (49.7%) (p = 0.003). conversely, influenza was more frequent among older children aged 12-23 months (22.5%) than in those < 3 months (14.4%), 3-5 months (8.4%), and 6-11 months (14.3%) (p = 0.02). there was no significant difference by age group in high-risk conditions for influenza complications, consultation delay, or influenza vaccine use between influenza and rsv-infected children ( table 1 ). the distribution of the main reasons for admission was similar between influenza and rsv infections among children aged 3-5 months and 12-23 months. among infants < 3 months, influenza-infected cases were more often admitted for fever than rsv patients (74% vs. 26%), while patients with confirmed rsv were more frequently hospitalized for dyspnea or bronchiolitis compared to influenza patients. the 2014 who severe acute respiratory illness (sari) case definition for influenza surveillance was met by more than 70% of children with influenza or rsv infections, with a higher proportion (83-89%) among children ≥ 6 months than youngest children and no significant difference between influenza and rsv-infected patients ( table 1 ). fever (reported or measured) and cough were the two symptoms included in this case definition. infants < 3 months with influenza had significantly higher rate of fever than those with rsv (100% vs. 63%, p < 0.0001) and significantly less cough (54% vs. 99%, p < 0.0001). we did not found in our analysis any specific sign or symptom discriminating between influenza and rsv infection in children < 24 months. among these children, those with rsv had a significantly higher proportion of radiologically confirmed pneumonia than influenza patients (39% vs. 18%, p < 0.01) ( table 1) . the median length of stay (los) in < 24-month-old children was similar (3 days) for both influenza and rsvinfected children. among infants < 6 months, median los was longer for rsv (4 days) than influenza (3 days, p < 0.0001). while no child with influenza was admitted to the intensive care unit (icu), 3.6% (n = 11) of rsv-infected children required it and most of them (82%, 9/11) were aged < 3 months (table 1) . no death was reported among enrolled children. during peak weeks of influenza season with mostly no overlapping rsv peak weeks, we found a significantly higher burden of rsv compared with influenza virus disease in hospitalized children < 24 months. our results underline the importance of testing young children with respiratory symptoms for a panel of respiratory viruses and the limited value of etiologic diagnoses based on clinical definition and epidemiologic data. in addition, our findings of a substantially greater rsv frequency among hospitalized young children even during influenza peak weeks underscore the need of prevention and control approaches that address rsv infections. to the best of our knowledge, this is the first study that describe the role of rsv relative to influenza viruses during the peak weeks of influenza epidemics among hospitalized young children. other prospective surveillance studies from the united states and europe documented the burden of influenza and rsv among enrolled children during whole influenza seasons or calendar years [6] [7] [8] . conducted mainly among children < 5 years in early 2000s, these studies reported similar patterns of high rsv detection (20-38%) relative to influenza (3-15%). a recent study from south africa reported that during a 5-year period (2011-2016), rsv was more often detected (24%) than influenza viruses (6%) in children < 5 years [9] . we found the highest rate of rsv among infants aged 3-5 months with rsv detection in nearly three over four patients (73.5%) followed by infants < 3 months (53.7%). similar to our results, other reports found that infants aged 3-5 months had the highest proportion of rsv detection [3, 10] , while the highest rsv frequency in the south african study was among infants < 3 months [9] . the clinical presentation of rsv infection in our study revealed the predominance of cough over the fever in particular among infants < 3 months. conversely, young infants infected with influenza had a significantly higher rate of fever than children with rsv infection. these results are consistent with previous reports on the rsv and influenza clinical features in young children [8, 9] . we also found that a radiologically confirmed pneumonia was more frequent in rsv infection than in influenza infection, which is in line with other publications [7, 8] . there was no difference in the median hospital duration (3 days) between rsv and influenza-infected children < 2 years, a finding similar to the south african study that reported a non-significantly different median los (4 days) for rsv and influenza-infected children < 5 years [9] . however, there were differences by age group in our study: infants < 6 months hospitalized with rsv infection had significantly longer hospital stay compared to those with influenza infection (median los of 4 days vs. 3 days). a longer average duration of hospitalization among rsv-infected infants has also been previously reported [7, 11] . in this study, rsv infection was associated with higher but not significantly different icu admission rates than influenza infection (3.6% vs. 0%, p = 0.07) among children < 2 years. a comparable rate of 3.1-4.3% of icu admissions associated with rsv has been reported in other prospective studies [6, 9] . there were no deaths among children included in our analysis, which is consistent with recent studies reporting the relatively low case-fatality rate (< 1%) associated with pediatric influenza or rsv infections [12, 13] . there are several limitations to this study. first, rsv seasonal epidemics overlapped differently with epidemic curves of influenza during the study period. both adults and children contribute to the quebec provincial sentinel data for respiratory viral surveillance. it is likely that children disproportionately drive rsv findings in the community and conversely that adults predominate influenza circulation data. even if rsv and influenza circulation in both adults and children may not coincide exactly with that of children, the overall rsv circulation relative to that of influenza is likely to be reflected in the sentinel surveillance curves we have presented. second, we defined influenza peak weeks as those during which the percentage of positive specimens tested was ≥ 15% as monitored by the provincial sentinel laboratory network. this definition was based on provincial data indicating that weeks with ≥ 15% of influenza-positive specimens represented more than 74% of total influenza-positive tests during entire influenza season, and this peak week definition was used in line with previously published works [14, 15] . during the peak influenza weeks of the 5 year study period, rsv and influenza had varying degree of seasonal overlap (fig. 1) . relatively complete rsv and influenza peak weeks' overlap was evident during the two last seasons (2016-2017 and 2017-2018), with more than half (56%) of the total of rsv positive tests during the peak influenza included in our study. however, after excluding these two seasons, we found similar results of higher frequency of rsv compared to influenza infections (data not presented). third, there was no information about rsv risk factors for hospitalization such as history of prematurity, specific comorbidity (cystic fibrosis, neuromuscular diseases, etc.) as our original data focused on depicting the epidemiology of influenza infections. the low frequency of influenza detection is unlikely due to vaccination given the low rate of influenza vaccination among pregnant women (~ 16%) and children < 2 years (17-19%) in quebec [16, 17] , and little evidence is available on effectiveness of current influenza vaccines for children < 2 years [18]. finally, influenza antiviral treatment data were not recorded systematically all seasons, preventing us from analyzing its impact on influenza duration of hospitalization. in conclusion, even during the peak weeks of influenza, more than half of hospitalizations for respiratory infections in children < 2 years of age was due to rsv, with a clinical course more severe than influenza notably among infants < 3 months. conflict of interest this work was a part of a study supported by the ministère de la santé et des services sociaux du québec. r. g. received research funding from ministère de la santé et des services sociaux du québec for this study and research grants from sanofi pasteur and pfizer for unrelated studies. all other authors report no potential conflicts. ethical statement institutional review board approval was obtained from all participating hospitals for the first two years; an exemption was obtained for the rest of years given the change of the design and objectives from a research study into a surveillance project. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and metaanalysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study respiratory syncytial virus-associated hospitalizations among children less than 24 months of age the burden of influenza hospitalizations in infants from other respiratory viruses are important contributors to adult respiratory hospitalizations and mortality even during peak weeks of the influenza season population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children prospective population-based study of viral lower respiratory tract infections in children under 3 years of age (the pri. de study) comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children the role of hiv in influenza-and respiratory syncytial virus-associated hospitalizations in south african children trends in chronologic age and infant respiratory syncytial virus hospitalization: an 8-year cohort study respiratory syncytial virus-and influenza virus-associated hospitalizations in infants less than 12 months of age defining the epidemiology and burden of severe respiratory syncytial virus infection among infants and children in western countries hospitalization for influenza a versus b. pediatrics epidemiology of influenza-associated hospitalization in adults when should a diagnosis of influenza be considered in adults requiring intensive care unit admission? results of populationbased active surveillance in toronto key: cord-351990-aham72b9 authors: radin, jennifer m.; hawksworth, anthony w.; kammerer, peter e.; balansay, melinda; raman, rema; lindsay, suzanne p.; brice, gary t. title: epidemiology of pathogen-specific respiratory infections among three us populations date: 2014-12-30 journal: plos one doi: 10.1371/journal.pone.0114871 sha: doc_id: 351990 cord_uid: aham72b9 background: diagnostic tests for respiratory infections can be costly and time-consuming. improved characterization of specific respiratory pathogens by identifying frequent signs, symptoms and demographic characteristics, along with improving our understanding of coinfection rates and seasonality, may improve treatment and prevention measures. methods: febrile respiratory illness (fri) and severe acute respiratory infection (sari) surveillance was conducted from october 2011 through march 2013 among three us populations: civilians near the us–mexico border, department of defense (dod) beneficiaries, and military recruits. clinical and demographic questionnaire data and respiratory swabs were collected from participants, tested by pcr for nine different respiratory pathogens and summarized. age stratified characteristics of civilians positive for influenza and recruits positive for rhinovirus were compared to other and no/unknown pathogen. seasonality and coinfection rates were also described. results: a total of 1444 patients met the fri or sari case definition and were enrolled in this study. influenza signs and symptoms varied across age groups of civilians. recruits with rhinovirus had higher percentages of pneumonia, cough, shortness of breath, congestion, cough, less fever and longer time to seeking care and were more likely to be male compared to those in the no/unknown pathogen group. coinfections were found in 6% of all fri/sari cases tested and were most frequently seen among children and with rhinovirus infections. clear seasonal trends were identified for influenza, rhinovirus, and respiratory syncytial virus. conclusions: the age-stratified clinical characteristics associated with influenza suggest that age-specific case definitions may improve influenza surveillance and identification. improving identification of rhinoviruses, the most frequent respiratory infection among recruits, may be useful for separating out contagious individuals, especially when larger outbreaks occur. overall, describing the epidemiology of pathogen specific respiratory diseases can help improve clinical diagnoses, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and treatments. acute respiratory infections make up a huge proportion of disease burden in the united states and globally, with an estimated 94 037 000 disability adjusted life years and 3.9 million deaths worldwide each year [1] . respiratory infections are often difficult to diagnose clinically due to nonspecific and overlapping symptoms. additionally, diagnostic tests can be time-consuming and costly and often require trained and well-equipped laboratories, making laboratory confirmation of each case impractical. however, laboratory results from various surveillance populations can be paired with clinical, demographic, and seasonality variables to create models that can give timely predictions of disease outcomes. preventive measures and treatments to reduce respiratory disease burden can also be improved through routine surveillance by gaining a better understanding of the percent positivity of pathogens among acute respiratory cases, seasonality, and coinfection occurrence. currently, limited respiratory disease etiology studies have been done in the united states [2, 3, 4] , despite many being done in other countries [5, 6, 7, 8, 9, 10, 11, 12, 13, 14] . additionally, few viral etiology studies have collected clinical signs and symptoms and assessed their association with a broad range of respiratory pathogens [9, 13, 14] . most descriptive studies and predictive models for respiratory diseases have focused on identifying influenza using clinical signs and symptoms [15, 16, 17, 18, 19, 20] , however few have been age-stratified and therefore may have missed some important differences in clinical presentation by age. understanding us-specific disease burden and seasonality is important, since disease incidence, distribution, and seasonality may vary between populations, regions, and climates. this study aimed to describe characteristics associated with specific respiratory pathogens, as well as the etiology, seasonality, and coinfection rates among three us populations: military recruits, department of defense (dod) beneficiaries, and civilians living near the us-mexico border. the results of this study can help improve timely and more accurate diagnosis, inform treatment plans, establish baselines of infection, identify outbreaks, and help prioritize the development of new vaccines and future treatments. participants fri and sari surveillance was conducted between october 2011 and march 2013 among three surveillance groups in the united states: civilians near the us-mexico border, dod dependents, and military recruits. separate seasonality data from the same populations was available for january 2012 through december 2013. recruits are typically young and healthy adults who enter into an 8-12 week ''boot camp'' training program, which involves strenuous, and physically demanding activities and living in high-density barracks. during the first week of training, recruits receive a series of vaccinations, including influenza (seasonally) and adenovirus. most training centers also administer at least one dose of bicillin to incoming trainees as prophylaxis against streptococcus bacteria. the dod dependent population is made up of the families of active duty and retired military personnel. this population consists of all ages and has good access to health care through the tricare health care program. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research. the research conducted in the recruit and dod beneficiary populations underwent nhrc irb approval (nhrc protocols 31230 and nhrc.1999.0002) and written consent or parental guardian consent for minors was obtained for all participants. the data collected from the border population was part of a surveillance program run by the us centers for disease control and prevention (cdc) and was considered non-research by the nhrc irb. nhrc staff members only provided diagnostic support and only received non-personally identifiable data. case definitions were slightly different for each of the three populations. in the recruit population, an fri case was defined as a person who sought medical care and had an oral temperature $38.0uc (100.5uf) and either cough or sore throat. for the beneficiary population and border populations, the same fri case definition was used; however, a fever $37.8uc (100.0uf) or subjective fever was used. additionally, inpatients who met sari case definition at select border sites were also sampled. the sari case definition included people who presented with either fever $37.8uc (100.0uf) or feeling feverish/chills, in addition to cough, and hospital admission, with onset in the last 10 days. additionally, children under age five were included if they were admitted to the hospital with clinical suspicion of pneumonia. nasal or combination nasal/pharyngeal swabs and questionnaire data were collected from a convenience sample of up to 20 patients per week per site who sought medical attention, met the fri or sari case definition, and provided written informed consent. specimens were placed in universal transport medium [21] , preserved at 280uc, and later transported on dry ice to the reference laboratory at the nhrc every one to two weeks for testing. additionally, the following demographic and clinical signs and symptoms were collected from each fri and sari case: sex, age, study population, month of illness, pneumonia, sore throat, cough, nausea, shortness of breath, congestion, pink eye, body ache, headache, temperature, number of days of symptoms before seeking care, and date of seeking care. samples were extracted using the roche magna pure lc extraction system following manufacturer's instructions. samples were then tested for a broadspectrum panel consisting of standard pcr gel tests: hmpv (single plex), covnl63 (single plex), and cov229e and covoc43 (multiplex). real-time polymerase chain reaction (pcr) assays were run on the applied biosystems 7500 fast real-time pcr system testing for influenza a, influenza b, adenovirus, rsv, rhinovirus, and a bacterial multiplex consisting of m. pneumoniae, c. pneumoniae, and b. pertussis. the rhinovirus primers used in our study have been found to correctly identify 87 of the 100 distinct rhinovirus subtypes and to accurately distinguish rhinoviruses from enteroviruses [22] . all pcr tests were done using in-house primers, except for the influenza a and influenza b which were done using cdc primers. additionally, viral culture testing for parainfluenza was done for a systematic sample of specimens not positive for influenza or adenovirus. the frequencies and means of clinical and demographic characteristics across participants positive for each of the six viruses, bacterial infections, co-infections and no/unknown pathogen were compared. (table 1) . additionally, characteristics of influenza, other pathogen, or no/unknown pathogen were compared among age stratified groups of dod beneficiaries and us-mexico border populations ( table 2) and characteristics of rhinovirus, other pathogen, and no/ (24) 14 (15) 4 (5) 6 (10) 4 (3) 13 (27) 19 (43) unknown pathogen were compared among military recruits (table 3) . chi-square tests for categorical variables and analysis of variance (anova) tests for continuous variables were used to identify signs, symptoms, associated with each pathogen group from all populations. variables that were univariately associated with the pathogens (p,0.15) were investigated further in a multinomial logistic regression model. variables with a p,0.05 were considered in the final adjusted model (tables s1-s2 in s1 file). coinfections were coded as ''other pathogen'' even if one pathogen was influenza or rhinovirus. statistical analysis was conducted using sas software (version 9.3, sas institute, inc., cary, north carolina). proc logistic with link5glogit was used for the multinomial modeling and proc anova and proc freq were used for anova and chi-squared analyses, respectively. during the 18 months of the study, october 2011 through march 2013, 1444 patients met the fri or sari case definitions and were enrolled in this study, consisting 406 (28%) from the fri/sari border, 423 (29%) from the dod beneficiary, and 615 (43%) from the fri recruit populations. the percent positivity for each pathogen out of all specimens tested was rhinoviruses (16%), influenza (14%), rsv (6%), adenovirus (2%), coronaviruses (5%), bacterial (2%), hmpv (1%), and coinfections (6%). influenza a (h3n2) was the most common influenza subtype, making up 54% of influenza specimens, followed by influenza b (31%), and ph1n1 (13%). among coronaviruses, covoc43 was the most commonly identified strain, making up 67% of coronavirus specimens, seasonal patterns were apparent for influenza, rhinovirus, and rsv, whereas more consistent low levels of infection were seen for adenovirus and bacterial infections. the recruit population had a more constant number of fri cases sampled than the other two populations throughout the study period. overall, they also had a greater percent positivity for rhinovirus compared with the other two populations, but lower percent positivity for influenza and rsv. rhinovirus appeared to have two peaks: one in spring and one in summer/fall among the border and beneficiary populations and consistently higher levels among the recruit population. in all populations influenza and rsv peaked in the winter (fig. 1) . influenza a typically peaked before influenza b. rhinovirus and bacterial infections were more frequently isolated from recruits and men who make up the majority of the recruit population; whereas influenza and rsv were less frequent among these groups compared with the dod beneficiary and border populations. fewer rhinovirus and more rsv pathogens were identified from sari cases compared to fri. pneumonia and shortness of breath were more frequent among rhinovirus cases and less common among influenza cases. however, time to seeking care was shorter for influenza and hmpv and longest for bacterial infections. influenza, rsv, adenovirus, and hmpv cases all had higher temperatures compared with no/unknown pathogens, and rhinovirus has significantly lower temperature (table 1) . descriptive statistics for influenza, other, and no/unknown pathogen among dod beneficiaries and us-mexico border populations showed different clinical presentation across three age groups. among 0-4 year olds, influenza was more frequent among us-mexico border populations compared to dod beneficiaries. in the youngest age group, sore throat and fever were also more frequent among those positive for influenza compared to other or no/unknown pathogen and among 5-24 year olds, cough, fever, and short time to seeking care were more frequent. finally, among the 25 and older age group, sore throat, nausea, congestion, pink eye, body ache, and head ache were more frequently seen in the influenza group compared to the other groups. interestingly, fever was the least frequent among influenza positive participants in the oldest age group (table 2) . clinical presentation among recruits also differed by those with laboratory confirmed rhinovirus, other pathogen, or no/unknown pathogen. a higher percentage of males were diagnosed with rhinovirus or other pathogen compared to the other and no/unknown pathogen. additionally, participants with rhinovirus had higher percentages of pneumonia, shortness of breath, congestion, and less fever and longer time to seeking care than the other groups and participants with either rhinovirus or other pathogen had higher cough than the no/unknown pathogen group (table 3) . building on the descriptive statistics, we found that fever was predicative of influenza compared to no/unknown pathogen among 0-4 year olds. among 5-24 year olds, fever, cough, and short time to seeking care were predicative of influenza compared to no/unknown pathogen. finally, among those 25 years and older, sore throat and nausea were predictive of influenza compared to no/ unknown pathogen. (table s1 in s1 file). among the recruit population, cough epidemiology of respiratory infection and less fever were predictive of rhinovirus, and cough, fever, and less shortness of breath were predictive of other pathogen compared to no/unknown pathogen. (table s2 in s1 file). at least one pathogen was identified in 51% of all fri and sari cases. coinfections were found in 81 (6%) of all fri/sari cases tested, among which were 76 double, four triple, and one quadruple coinfections. the most frequent pathogen associated with a coinfection was rhinovirus, followed by rsv and covoc43. the top three most common coinfections were rhinovirus and rsv (15% of all coinfections, 11 cases), rhinovirus and adenovirus (13% of all coinfections, 10 cases), and rhinovirus and covoc43 (12% of all coinfections, 9 cases). interestingly, our study found an overall coinfection percent positivity of 6% which declined with age: 11% of 0-4 year olds, 5% of 5-24 year olds, 3% of 25-49 year olds and 2% of 50+ year olds. (table 4 ). in order to reduce respiratory disease burden, it is necessary to gain a better understanding of the percent positivity, coinfection rates, and seasonality of specific respiratory pathogens. additionally, a predictive clinical model that uses symptoms, seasonality, and patient demographics can also help improve prevention efforts and patient treatment. timeliness is especially important for influenza antivirals, which work best if given within the first 48 hours of symptoms. however, rapid diagnostic tests have poor sensitivity [23] and multiplex pcr tests are impractical for most clinical settings. additionally, treatment with antibiotics can often be incorrectly prescribed for viral infections, leading to increased antibiotic resistance. therefore, creating models to better predict the type of pathogen using symptoms and characteristics easily collected by a clinician at the time of visit could improve treatment accuracy and help protect the effectiveness of existing antibiotics and antivirals. many respiratory etiology studies have been done outside the united states among patients with both severe, lower respiratory illness [24, 25, 26] , as well as more mild, upper respiratory disease [7, 8, 9, 10, 11, 12] . these studies are important because viral etiologies vary across populations and regions, depending on factors such as population susceptibility, age, circulating strains, climate, comorbidities, and vaccination coverage. our study adds to the very limited number of etiology studies done in the united states [2, 3, 4] by examining viral etiology among three different us populations: military recruits, dod dependents, and a us-mexico border civilians and including all ages. our study is also unique in that it collected clinical signs, symptoms, and demographics for each case tested with the broad-spectrum respiratory panel. similar studies have been limited and have used smaller sample sizes, focused on one age group, and did not test for as many respiratory pathogens. identifying population-specific baselines of infection enables us to identify elevated rates, which may indicate an outbreak or the start of a pandemic. recognizing associated symptoms can help determine the most likely pathogen, as was seen in 2009 with the pandemic influenza (h1n1) strain first identified in brawley and san diego, california, in two of the populations in this study [27] . there were several key differences of infection among the three populations. the recruit population had consistently higher levels of rhinovirus and bacterial infections than the other groups, which may be reflective of close living conditions and a younger age group, mostly 18-24 years old. this population is also highly vaccinated for influenza and showed the smallest amount of influenza infection compared with the other two populations. the higher frequency of influenza among 0-4 year olds in the us-mexico border population (61%) compared to beneficiaries (39%) may be reflective of different exposures or differences in vaccination uptake. however, the overall frequency of influenza found between the two groups was similar. adenovirus, which historically had a large impact on recruits, was also low as a result of resumption of the adenovirus vaccines in october 2011 [28] . rsv, which usually infects young children, was more commonly found in the dod dependent (48%) and border populations (48%) compared with the recruits (4%). overall, rhinoviruses were the most common respiratory pathogen identified (236 cases, 16%), followed by influenza (197 cases, 14%), rsv (85 cases, 6%), and coronaviruses (66 cases, 5%), which coincides with other etiology studies [13] . the results of the age-stratified influenza, other pathogen, and no/unknown results showed some interesting differences across age groups. most interesting was the relatively low percentage of influenza positive participants with fever (42%) in the 25 and older year age group, compared to the 0-4 (74%), and 5-25 year olds (76%). fever may sometimes be masked by the consumption of antipyretics, although this group also complained of high headache (81%) and body ache (90%). the presentation differences of influenza by age could have some important implications for influenza surveillance, as the standard influenzalike illness and sari case definitions require [29] . consequently, an age-stratified case definition may be more appropriate as the standard case definitions may be underestimating the burden of influenza in older age groups. circulating strains may also influence clinical presentation across age groups. (table 2, table s1 in s1 file). in our 5-24 year olds we found fever, cough, and short time to seeking care to be predictive of influenza infection. unfortunately, previous studies assessing the diagnostic accuracy of fever, cough, and acute onset to predict flu have found them to have low sensitivity (20%), and high specificity (96%) [18] . another study, found that sore throat and fever in participants less than 5 years old had a sensitivity of 51% and specificity of 54% and cough and fever among those greater than 5 years had a sensitivity of 80% and specificity of 42% [30] . although identifying predictive symptoms can be useful, it is important to recognize that diagnostic accuracy may still be low due to overlapping symptoms of many respiratory infections and may change due to fluctuations in circulating strains, age of the population and comorbidities. ( table 2, table s1 in s1 file). rhinovirus was the most frequently identified pathogen among recruits, although it typically does not present with severe illness or acute onset, which is reflected in the long time to seeking care among rhinovirus positive participants. interestingly, we also found the lowest percentage of fever among participants positive for rhinovirus compared to any other respiratory pathogen (table 1) . these findings are similar to other studies [13, 14, 31] and could have important implications for designing respiratory disease surveillance systems to capture outbreaks of viruses in the same family. for example, the recent outbreak of enterovirus 68 has similar symptoms of low fever [32] . (table 3, table s2 in s1 file). understanding coinfections can also be useful for preventing respiratory disease. our study found around 30% to 40% of coronavirus and adenovirus infections occurred as coinfections and they most frequently occurred with rhinovirus. similarly, other studies have also found the highest ratio of coinfections among adenovirus and coronaviruses [33, 34] , and have found rhinovirus to be part of the most frequently occurring coinfections [26, 33] . these results suggest that infection with some viruses, such as rhinoviruses, could create opportunistic environments for colonization with other viruses and bacteria. interestingly, coinfections were most common among the youngest age group, newborn to four years, which did not have the highest rhinovirus rate. targeting rhinovirus infection through creation of new vaccines or treatment could have more far-reaching benefit in protecting a person from other infections. respiratory coinfection rates have varied across studies and are likely influenced by age, type of case definition used to enroll participants, and pathogens tested. previous studies with a median age of 2-3 years old have found coinfections among sari and acute respiratory tract infections of 17% and 19%, respectively [26, 33] . a third study among childcare attendees in the us found even higher coinfection rates of 47% [4] . finally, a study from scotland that tested respiratory specimens from all age groups found a coinfection rate of 5% [34] . these studies coincide with ours and show high coinfection rates among children. declining coinfection rates with age may be a result of overall increased immunity to a broad range of respiratory pathogens as people get older. one limitation of this study is that it only captured people with fri/sari who sought medical care. military recruits may be less likely to seek care than other groups due to concern over losing training time or having to restart the program. therefore, the etiology of more mild infections may be underrepresented for these two groups. additionally, the case definitions were slightly different for the three populations, which may have influenced which pathogens were identified in each group. although this study involved three different us populations, the results of this study may be less generalizable to the general us public who are not associated with the military or living on the us-mexico border. despite this, signs and symptoms from these pathogens should be similar across other populations in similar age groups and with similar vaccination coverage. additionally, we found that seasonality of infection for recruits was similar to that of the border and beneficiary populations for several pathogens, but with different intensity. consequently, illness surveillance in recruits, which made up the largest proportion of our study population, can be beneficial in informing disease trends in the general public. although we tested for many pathogens, there are likely still circulating viruses and bacteria for which we did not test, such as bocavirus, covhku1, or potentially unrecognized viruses; therefore, these cases were likely incorrectly classified as part of the ''no pathogen'' group. additionally, the timing of sample collection in the course of illness could impact whether or not viruses were identified by pcr. symptom collection may also be biased in the youngest age group of 0-4 year olds, who may not be able to articulate how they are feeling. despite this, our study was part of a well-established existing surveillance program that consistently collected and tested a substantial number of specimens at many different sites across the united states. in the future, additional years of surveillance data will continue to improve our understanding of seasonality. although nothing will replace the accuracy of laboratory diagnostics, a broader understanding of seasonality, clinical presentation, demographics, and coinfection rates of pathogen specific respiratory diseases can improve diagnosis and treatment, by informing clinicians on appropriate antiviral and antibiotic treatment during the patient's visit. this can ultimately reduce the number of lost work days and transmission. additionally, describing baseline of disease and seasonality for specific pathogens can improve our ability to detect outbreaks. identifying the percent positivity of each pathogen, coinfection rates, and risk factors for disease will help inform vaccination programs, and possible investment in the development of future vaccines or treatments. supporting information s1 file. tables s1 and s2. of defense, or the us government. approved for public release; distribution is unlimited. u.s. government work (17 usc 105) . not copyrighted in the u.s. this research was conducted in compliance with all applicable federal and international regulations governing the protection of human subjects in research (protocols nhrc 31230 and nhrc.1999.0002). world health organization (who) the frequency and seasonality of influenza and other respiratory viruses in tennessee: two influenza seasons of surveillance data viruses associated with acute respiratory infections and influenza-like illness among outpatients from the influenza incidence surveillance project epidemiology of multiple respiratory viruses in childcare attendees viral etiology of severe pneumonia among kenyan infants and children acute respiratory infection and influenza-like illness viral etiologies in brazilian adults epidemiological analysis of respiratory viral etiology for influenzalike illness during 2010 in zuhai, china influenza-like illness sentinel surveillance in peru acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective population based study of disease burden viral etiology of influenza-like illnesses in cameroon viral etiology of influenza-like illnesses in antananarivo rapid 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detection of human rhinovirus low sensitivity of rapid diagnostic test for influenza community-acquired pneumonia distinguished from influenza infection based on clinical signs and symptoms during a novel (swine) influenza a/h1n1 pandemic multicentered study of viral acute lower respiratory infections in children from four cities of argentina, 1993-1994 respiratory viral coinfections identified by a 10-plex real-time reverse-transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness-south africa initial identification and characterization of an emerging zoonotic influenza virus prior to pandemic spread history of the restoration of adenovirus type 4 and type 7 vaccine, live oral (adenovirus vaccine) in the context of the department of defense acquisition system world health organization. who surveillance case definitions for ili performance of case definitions used for influenza surveillance among hospitalized patients in a rural area of india rhino/enteroviruses in hospitalized children: a comparison to influenza viruses severe respiratory illness associated with enterovirus d68 -missouri and illinois epidemiology of respiratory viral infection using multiplex rt-pcr in epidemiology and clinical presentations of four human coronaviruses 229e, hku1, and oc43 detected over 3 years using a novel multiplex real-time pcr method we thank maria rosario araneta, patrick blair, daisy cabrera, johnnie conolly, jennifer cortinas, larivhie delacruz, daniel edgeworth, maria fierro, james key: cord-335948-qkfxfmxb authors: ampofo, william k.; baylor, norman; cobey, sarah; cox, nancy j.; daves, sharon; edwards, steven; ferguson, neil; grohmann, gary; hay, alan; katz, jacqueline; kullabutr, kornnika; lambert, linda; levandowski, roland; mishra, a. c.; monto, arnold; siqueira, marilda; tashiro, masato; waddell, anthony l.; wairagkar, niteen; wood, john; zambon, maria; zhang, wenqing title: improving influenza vaccine virus selectionreport of a who informal consultation held at who headquarters, geneva, switzerland, 14–16 june 2010 date: 2011-08-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00277.x sha: doc_id: 335948 cord_uid: qkfxfmxb • for almost 60 years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations (2005), recognition of the continuing threat posed by avian h5n1 and the aftermath of the 2009 h1n1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy‐making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year‐round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non‐antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays – for example based upon advanced recombinant dna and protein technologies – may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large‐scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well‐characterized banks of age‐stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high‐throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x‐ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non‐mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies – such as live‐attenuated, quadrivalent or non‐ha‐based vaccines – has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high‐growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. • for almost 60 years, the who global influenza surveillance and response system (gisrs) has been the key player in monitoring the evolution and spread of influenza viruses and recommending the strains to be used in human influenza vaccines. the gisrs has also worked to continually monitor and assess the risk posed by potential pandemic viruses and to guide appropriate public health responses. • the expanded and enhanced role of the gisrs following the adoption of the international health regulations (2005) , recognition of the continuing threat posed by avian h5n1 and the aftermath of the 2009 h1n1 pandemic provide an opportune time to critically review the process by which influenza vaccine viruses are selected. in addition to identifying potential areas for improvement, such a review will also help to promote greater appreciation by the wider influenza and policy-making community of the complexity of influenza vaccine virus selection. • the selection process is highly coordinated and involves continual year-round integration of virological data and epidemiological information by national influenza centres (nics), thorough antigenic and genetic characterization of viruses by who collaborating centres (whoccs) as part of selecting suitable candidate vaccine viruses, and the preparation of suitable reassortants and corresponding reagents for vaccine standardization by who essential regulatory laboratories (erls). • ensuring the optimal effectiveness of vaccines has been assisted in recent years by advances in molecular diagnosis and the availability of more extensive genetic sequence data. however, there remain a number of challenging constraints including variations in the assays used, the possibility of complications resulting from non-antigenic changes, the limited availability of suitable vaccine viruses and the requirement for recommendations to be made up to a year in advance of the peak of influenza season because of production constraints. • effective collaboration and coordination between human and animal influenza networks is increasingly recognized as an essential requirement for the improved integration of data on animal and human viruses, the identification of unusual influenza a viruses infecting human, the evaluation of pandemic risk and the selection of candidate viruses for pandemic vaccines. • training workshops, assessments and donations have led to significant increases in trained laboratory personnel and equipment with resulting expansion in both geographical surveillance coverage and in the capacities of nics and other laboratories. this has resulted in a significant increase in the volume of information reported to who on the spread, intensity and impact of influenza. in addition, initiatives such as the who shipment fund project have facilitated the timely sharing of clinical specimens and virus isolates and contributed to a more comprehensive understanding of the global distribution and temporal circulation of different viruses. it will be important to sustain and build upon the gains made in these and other areas. • although the haemagglutination inhibition (hai) assay is likely to remain the assay of choice for the antigenic characterization of viruses in the foreseeable future, alternative assays -for example based upon advanced recombinant dna and protein technologies -may be more adaptable to automation. other technologies such as microtitre neuraminidase inhibition assays may also have significant implications for both vaccine virus selection and vaccine development. • microneutralization assays provide an important adjunct to the hai assay in virus antigenic characterization. improvements in the use and potential automation of such assays should facilitate large-scale serological studies, while other advanced techniques such as epitope mapping should allow for a more accurate assessment of the quality of a protective immune response and aid the development of additional criteria for measuring immunity. • standardized seroepidemiological surveys to assess the impact of influenza in a population could help to establish well-characterized banks of age-stratified representative sera as a national, regional and global resource, while providing direct evidence of the specific benefits of vaccination. • advances in high-throughput genetic sequencing coupled with advanced bioinformatics tools, together with more x-ray crystallographic data, should accelerate understanding of the genetic and phenotypic changes that underlie virus evolution and more specifically help to predict the influence of amino acid changes on virus antigenicity. • complex mathematical modelling techniques are increasingly being used to gain insights into the evolution and epidemiology of influenza viruses. however, their value in predicting the timing and nature of future antigenic and genetic changes is likely to be limited at present. the application of simpler non-mechanistic statistical algorithms, such as those already used as the basis of antigenic cartography, and phylogenetic modelling are more likely to be useful in facilitating vaccine virus selection and in aiding assessment of the pandemic potential of avian and other animal influenza viruses. • the adoption of alternative vaccine technologies -such as live-attenuated, quadrivalent or non-ha-based vaccines -has significant implications for vaccine virus selection, as well as for vaccine regulatory and manufacturing processes. recent collaboration between the gisrs and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants for vaccine production, the development of qualified cell cultures and the investigation of alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of the global influenza vaccine supply. • the who gisrs and its partners are continually working to identify improvements, harness new technologies and strengthen and sustain collaboration. who will continue in its central role of coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support efforts to improve the vaccine virus selection process, including through the convening of periodic international consultations. the historic initiative to establish a global network to detect and identify new and potentially dangerous influenza viruses predates the adoption of the who constitution in 1948. with memories of the 1918-1919 influenza pandemic still vivid, and the ever-evolving threat posed by influenza recognized, the who global influenza surveillance and response system (gisrs) was formally established in 1952. influenza thus became one of the first diseases to highlight the importance of international monitoring and collaboration in protecting human health. following the re-emergence of human cases of highly pathogenic avian h5n1 influenza in 2003 and the adoption of the international health regulations (2005), the gisrs was strengthened and its role in protecting public health enhanced. in addition to tracking the course and impact of annual influenza epidemics and monitoring the evolution of seasonal influenza viruses, the gisrs also acts as a global alert mechanism for the emergence of influenza viruses with the potential to cause a human pandemic. the network provides support to both seasonal and pandemic influenza preparedness and response activities in areas such as diagnostics, vaccine development, virological surveillance and risk assessment. it also acts as the focus of who efforts to former who global influenza surveillance network (gisn), which has been renamed as who global influenza surveillance and response system (gisrs) since 24 may 2011, when the world health assembly resolution wha 64.5 was adopted. assist member states in strengthening their national capacity for the surveillance, diagnosis, characterization and sharing of influenza viruses. as a key player in global influenza risk assessment and response, the gisrs continues to evolve and expand, and as of december 2010 consisted of 135 national influenza centres (nics) in 105 countries, six who collaborating centres (whoccs), 11 who h5 reference laboratories and four who essential regulatory laboratories (erls). the gisrs also works to ensure the successful coordination of who activities with those of external agencies such as the global outbreak alert and response network (goarn), national regulatory authorities, academic and veterinary institutes, and the pharmaceutical industry. the first formal who recommendations on influenza vaccine composition were issued in 1971. since 1998, separate and appropriately timed recommendations for the northern and southern hemispheres have been issued each year in february and september, respectively. these biannual recommendations are based upon the virological and epidemiological information generated by the gisrs and play a crucial role in the development, production and availability of effective influenza vaccines. the continuing threat posed by avian h5n1, the aftermath of the 2009 h1n1 pandemic, the increased knowledge of influenza, and the development and availability of new technologies provide a timely opportunity to review the complex processes and issues involved in influenza vaccine virus selection and to identify potential areas for improvement. this who informal consultation represents the latest step in an ongoing process of gisrs strengthening and was convened with the following objectives: • to review the current vaccine virus selection process, including its constraints and limitations; • to identify opportunities for improving influenza surveillance and representative virus sharing; • to assess the potential for improving the assays and technologies used for vaccine virus selection; and • to assess the potential impact of new vaccine technologies on the vaccine virus selection process. participants were drawn from a broad and highly diverse range of institutes and sectors including the following: whoccs, nics, who erls, who h5 reference laboratories, national regulatory authorities, public health agencies, academia, influenza vaccine manufacturers, and veterinary laboratories and organizations. the primary goal of the gisrs vaccine virus selection process (annex 1) is to generate and analyse the data needed to recommend the influenza vaccine viruses that will most closely match the influenza viruses likely to be circulating during forthcoming influenza seasons. current vaccine technologies and production schedules mean that decisions on vaccine composition have to be made almost a full year in advance of the peak of seasonal influenza activity. as a result, the process relies upon the earliest possible detection of emerging antigenic variants and the most up-to-date information on their potential future epidemiological significance. information must therefore be collected year round on the continuous evolution and global circulation of human influenza viruses to provide a sound basis for the biannual who recommendations on the composition of influenza vaccines for use in the northern and southern hemispheres. for countries in equatorial regions, epidemiological considerations influence which recommendation (february or september) individual national and regional authorities consider more appropriate. national influenza centres (nics) play a vital role in this complex process. their core activities include collating epidemiological information, diagnosing cases of influenza a and b infection, and identifying the subtype or lineage of the viruses responsible. the primarily molecular diagnosis of infection using rt-pcr techniques is based upon standardized primers and probes provided by the gisrs. viruses must also be isolated to allow their antigenic identification using the type-and subtype-specific reference reagents provided in annually distributed who kits. further detailed characterization may include sequence analyses to monitor genetic changes and assessment of virological traits such as resistance to antiviral drugs. sequence data are shared within the gisrs using public databases such as genbank and gi-said epiflu. nics in some settings then attempt to relate potentially important virological changes observed with clinical and epidemiological information and trends and may even conduct serological studies to evaluate the immune status of the population. weekly reports on the virological characteristics and epidemiology of circulating viruses are submitted to the who flunet -an internet-based data-query and reporting tool. information on the virus subtypes and lineages is collated, together with observations of potential clinical or epidemiological importance, and regular summaries of the geographical spread, intensity and impact of influenza are produced by who. if human infection with an avian or other animal influenza virus is suspected, a suitably equipped nic or other national influenza reference laboratory can conduct preliminary diagnostic testing using rt-pcr protocols and ⁄ or reagents for h5, h7 and h9 subtypes provided by who. such rt-pcr testing does not require high-level biocontainment facilities. however, it is expected that the detection of any unusual influenza a virus distinct from known circulating viruses, especially one suspected to be of animal origin or unsubtypable using current who reagents, will immediately be reported to who and collaboration urgently initiated with a who collaborating centre (whocc). if the required laboratory biosafety facilities and procedures are not available, then virus isolation should not be attempted in the national laboratory and the sample should be promptly sent to a whocc. the routine and timely sharing of representative circulating influenza viruses and unusual viruses with a whocc is an essential step in the vaccine virus selection process. the criteria for forwarding viruses include their temporal, geographical and age-group distribution, severity of cases and virological characteristics such as unidentified subtype and antiviral drug resistance. whoccs are then responsible for the systematic antigenic characterization of the thousands of viruses forwarded each year by nics and other laboratories, and for the detailed genetic characterization of a selected subset. such detailed antigenic and genetic characterization is a necessary step in monitoring virus evolution and detecting any distinct antigenic variants that may necessitate updating the seasonal vaccine composition. the process also allows for the identification and characterization of animal viruses causing sporadic human infections, assessment of the risk they pose and the potential development of candidate vaccine viruses as part of pandemic preparedness. of prime importance in immunity to influenza is the production of antibodies to the virus haemagglutinin (ha) protein. such antibodies can neutralize the infectivity of viruses, and their level in the blood has been shown to correlate with the level of protection against infection with a homologous virus. as a result, influenza vaccine virus selection has primarily been based upon the antigenic characterization of virus ha using the haemagglutination inhibition (hai) assay. hai tests provide a visual readout of the ability of specific antibodies to prevent the attachment of ha to red blood cells (rbcs) and thus prevent their agglutination. antigenic drift in the ha of circulating viruses in response to host immunity reduces the effectiveness of vaccines and is therefore the major consideration when recommendations are made on the composition of influenza vaccines. the hai test is likely to remain the assay of choice for the antigenic characterization of virus ha for the foreseeable future. strain-specific antisera are produced by infecting previously unexposed ('naive') ferrets with either vaccine viruses, reference viruses representative of circulating viruses or viruses that appear in hai tests to be potential antigenic variants. the resulting sets of reference viruses and antisera are then used to evaluate the antigenic characteristics of the has of recent isolates. where antigenic differences are detected, these are likely to affect human immunity against the new variants. the hai test is a surrogate for the more complicated and time-consuming virus neutralization assay used to clarify antigenic relationships when observed variations in hai titre reflect, for example, changes in receptor binding rather than differences in antigenicity. a subset of between 10% and 20% of all viruses received is selected for genetic sequencing and more detailed analysis -principally of their ha and na components. this subset is selected to include representative circulating viruses, as well as apparent antigenic variants and viruses from severe or fatal cases. phylogenetic analyses are carried out to better understand the evolution of circulating viruses, their degree of genetic heterogeneity and the emergence of new genetic clades. antigenic or other phenotypic variants may thus be defined in terms of separate genetic clades with distinct amino acid signatures. relating the locations of amino acid substitutions to antigenic, receptor-binding or glycosylation sites on the 3d structure of the ha molecule then helps to identify the individual substitutions associated with phenotypic (antigenic) changes. identifying such amino acid signatures also facilitates global monitoring of the emergence, distribution and impact of different genetic variants. this is particularly helpful when data on emergent variants are limited at the time of a who vaccine consultation. comparisons of the sequences found in clinical specimens and virus isolates are also useful in revealing amino acid substitutions which result from passage in different substrates, mainly mdck cells and eggs. up-todate sequence data are shared within gisrs and made publically available via the gisaid epiflu database. complete genome sequencing is necessary to identify animal (including avian) viruses causing human infection and is important in detecting the emergence of reassortant viruses among co-circulating human viruses or between human and animal viruses. whoccs maintain panels of reference reagents for all influenza a subtypes. these include h5 (especially h5n1), h9 and h7 avian viruses and various h1n1 and h3n2 swine viruses, as well as viruses present in other animals such as horses and dogs. whoccs also collaborate with the who erls in serological studies of representative human sera from previously vaccinated individuals. sera are provided by vaccine manufacturers and are used in hai tests to assess whether or not the antibodies induced by current vaccines are likely to be effective against currently circulating viruses. the results the principal criteria used to decide whether or not to recommend changes to influenza vaccine components include: • the emergence of an antigenically and genetically distinct variant among circulating viruses (including a novel influenza a virus with the potential to cause a pandemic); • evidence of the geographical spread of such a distinct variant and its association with outbreaks of disease, indicating its future epidemiological significance; • the reduced ability of existing vaccine-induced antibodies to neutralize the emergent variant; and • the availability of suitable candidate vaccine viruses. to facilitate collaborative studies by the whoccs and who erls and ensure that appropriate potential candidate vaccine viruses are identified in advance of the who vaccine composition consultation, the most recent virological and epidemiological data are shared and discussed via teleconferences held 6 and 2 weeks before the who consultation. a summary of each teleconference is promptly distributed to keep all nics and vaccine manufacturers informed of the developing situation. in addition, potential candidate vaccine viruses are provided to manufacturers. during the formal biannual consultations, the technical advisory group considers the cumulative antigenic and genetic data on the viruses characterized by whoccs. the data are set against the broader epidemiological context collated by who and are supported by serological data from whoccs and who erls, as well as by additional information provided by nics. hai data obtained in the different centres using a wide variety of reference viruses and ferret antisera are correlated using common reference reagents. in recent years, antigenic cartography has been used to collate and statistically visualize the degree of antigenic variation. the interpretation of hai data may, however, be complicated by the influence of changes in the receptor-binding properties of natural viruses or by the selection of variants during isolation and passaging in different cell or egg substrates. comparisons with sequence data are made to relate any differences in antigenicity with specific ha genetic clades and to more precisely define the identity of antigenic variants. the results of virus neutralization tests, which usually correspond to those of hai tests, are used to clarify the true antigenic relationships between different viruses. if the antigenic data, supported by genetic and serological data, indicate that a new antigenic variant is spreading globally, then a change in that component of the seasonal vaccine is considered to be warranted. the implementation of a rec-ommendation to update a vaccine component is, however, contingent upon the availability of suitable vaccine viruses. only after all the factors have been taken into account is a decision taken on whether or not to recommend a change in influenza vaccine virus composition. the decision is announced at an information meeting immediately following each who consultation and published on the who web site and in the who weekly epidemiological record. since the re-emergence of human cases of highly pathogenic h5n1 avian influenza in 2003, who has also regularly reviewed the available antigenic and genetic data on human and avian viruses in relation to the epidemiology of h5n1 influenza among birds. to support the development of safe and effective human h5n1 vaccines, who has coordinated the development of a number of candidate attenuated vaccine viruses (annex 1) and made them available to vaccine producers. clinical trials have been conducted to evaluate the immunogenicity of different h5n1 vaccine formulations and the breadth of antibody responses elicited. in addition, as part of pandemic preparedness, who has coordinated the ongoing development and updating of an inventory of h2, h7 and h9 candidate vaccine viruses. important constraints on the vaccine virus selection process include the tight timelines involved (annex 1), particularly in the northern hemisphere, where since recent years seasonal influenza activity tends to start increasing in middle or late january in general. as a consequence, decisions often have to be made relatively early in the influenza season. in addition, post-infection ferret antisera against potential antigenic variants are urgently required to define their antigenic relationships to previously circulating viruses. panels of recent isolates must also be prepared to assess the degree to which they are neutralized by antibodies in the sera of previously vaccinated individuals. finally, potential new candidate vaccine viruses must be prepared and evaluated for their suitability in vaccine production. ensuring the timely availability of viruses with suitable growth properties is a crucial step in ensuring that sufficient quantities of vaccine can be produced in time for administration prior to the next influenza season. although cell culture has steadily replaced the use of embryonated eggs for the primary isolation of viruses, candidate vaccine viruses must still be isolated directly in eggs according to current regulatory requirements. the limited availability of egg isolates, particularly of recent h3n2 viruses which generally grow poorly in eggs, has led to the establishment of cooperative research and development agreements (cra-das) and similar agreements between the vaccine industry and a number of whoccs to increase the availability of egg isolates for vaccine use. the gisrs vaccine virus selection process necessarily involves a series of collaborative steps, including the selection of prototype antigenic variants and suitable vaccine viruses, and the provision of standardizing reagents by the who erls. the process thus impacts directly upon the subsequent authorizing of vaccine composition by national and regional regulatory authorities and upon the large-scale production of vaccine by manufacturers. mismatches have occasionally occurred as a result of the emergence of variant strains shortly after the recommendations have been made, highlighting one of the unavoidable consequences of current vaccine development and production constraints. nevertheless, retrospective studies have shown that with very few exceptions who vaccine virus recommendations have closely matched the influenza viruses that have circulated during the following influenza season. in addition, following the out-of-season emergence of the pandemic a(h1n1) 2009 virus, this closely integrated system demonstrated its unique ability to very rapidly orchestrate the development and provision of appropriate (suitably attenuated) candidate vaccine viruses for pandemic vaccine production. global influenza surveillance has always presented a major challenge as it is a highly demanding public health need with a significantly uneven distribution of surveillance capacity worldwide. since the outbreak of severe acute respiratory syndrome (sars) in 2003, the re-emergence of h5n1 infection in humans and the 2009 h1n1 pandemic, it has become ever clearer that surveillance and the prompt sharing of viruses and information are central to the broad range of influenza preparedness and response activities. although the known impact and the awareness of seasonal influenza vary in different parts of the world, the threat posed by avian h5n1 viruses has galvanized influenza surveillance efforts in all countries. improving surveillance and acquiring the capacity to detect and report unusual cases of influenza are essential components of global pandemic planning and are enshrined in the international health regulations (2005) . successful efforts to increase the capacity of nics and other laboratories have been made, and in a number of settings the development, revision and adoption of guidelines on strengthened national, regional and global surveillance and collaboration is under way. global influenza surveillance has also been strengthened through expanded geographical coverage and the collection of more data of better quality. for example, in africa there are now 25 influenza laboratories in 21 countries, including 12 recognized nics, almost all of which have the capacity to conduct rt-pcr diagnosis of influenza infection. in less than two years, the percentage of african countries with an nic increased from 17% to 26% with the number of countries with no influenza laboratory markedly decreasing. global, regional and national training workshops, assessments and donations have all led to significant increases in trained personnel, equipment procurement and laboratory capacity, resulting in the increasingly widespread use of molecular techniques such as real-time rt-pcr and genetic sequencing. recent who capacity-building activities have included bsl-3 training courses for nics to promote safe practices when working with highly pathogenic influenza viruses, and courses on virus isolation, gene sequencing and antiviral resistance detection. increased participation in both internal and external quality assurance programmes such as the who external quality assessment project (eqap) has contributed to marked improvements in laboratory proficiency. these and other efforts enabled a more effective response to the emergence of the 2009 h1n1 pandemic in many countries. however, the pandemic also revealed significant limitations in the analysis and integration of epidemiological and virological surveillance data. in addition, few early seroprevalence surveys were conducted to allow for the timely assessment of the extent and impact of the pandemic. the pandemic also revealed significant gaps in laboratory infrastructure and personnel, equipment procurement and funding, particularly in developing countries. improvements and training in areas such as web-based integration and analyses of clinical, epidemiological and virological data are being implemented but care must be taken to ensure that such activities are not conducted at the expense of detection, characterization and virus-sharing activities in less well-resourced settings. identified research priorities in influenza surveillance and response include evaluation of the temporal and geographical circulation of influenza viruses and of the burden of influenza. in all settings, establishing a sound evidence base will support the development or updating of national, regional and global policies, plans and guidelines. this in turn could lead to greater acceptance of the use of influenza vaccines, particularly seasonal vaccines, and assist in the development of vaccination policies. the primary requirement of nics will remain the prompt diagnosis of influenza infection and the timely sharing of clinical specimens and virus isolates -especially those obtained from unusual, severe or fatal cases -backed up by appropriate epidemiological and clinical information. procedures should be in place to ensure that the increasingly predominant use of molecular diagnostic techniques, particularly real-time rt-pcr, does not adversely affect the timely isolation and for-improving influenza vaccine virus selection ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 6, 147-152 warding of viruses. improved communication between nics and whoccs on how best to facilitate prompt virus sharing, including discussion of the constraints faced, could improve coordination and avoid potential delays. a more systematic approach to engaging nic information and expertise would also lead to significant benefits. such an approach is likely to be facilitated by a number of developments in the use of who web-based tools. for example, nics with enhanced capabilities currently strengthen the collaborative characterization of viruses and aid early assessment of the significance of genetic and antigenic changes by sharing detailed virological information (especially ha sequences) on selected viruses, either directly or via public databases. as technologies advance, national patterns of seropositivity to circulating influenza viruses may also become available on a more timely basis and could thus guide vaccine use. this is particularly important given the increasing emphasis now placed on assessing vaccine effectiveness. comprehensive nic summary reports forwarded just prior to each who consultation also provide highly beneficial additional data to inform who recommendations on vaccine composition. to overcome logistical and other obstacles to the safe and efficient shipping of clinical specimens and virus isolates to whoccs, a who shipment fund project was established. the project provides support to nics and other influenza laboratories in all countries by arranging the transport of specimens and isolates along a guaranteed cold chain, especially in settings where there are severe financial and infrastructural constraints. as a direct result of the project, and associated 'infectious substances shipping' workshops conducted in all who regions, there has been a significant increase in the number of countries sharing specimens and isolates, especially following the outbreak of the 2009 h1n1 pandemic. furthermore, the expansion and harmonization of the information currently provided in the accompanying standard shipping form to include information such as clinical outcome, patient vaccination status or recent travel history would greatly enhance understanding of the epidemiological context associated with the spread of viruses. a better understanding of the diversity and evolution of animal influenza viruses is essential for evaluating the pandemic risk posed by subtypes currently causing sporadic human infections (such as h5n1 and h9n2) and informing the selection of candidate vaccine viruses. the emergence of h5n1 in particular led to the establishment in 2005 of the oie-fao network of expertise on animal influenza (of-flu) -a worldwide network of approximately 20 laboratories and institutions that coordinates the global surveillance of animal influenza. a number of joint who-offlu tech-nical initiatives on influenza at the human-animal interface have been conducted (including successful collaboration during the 2009 h1n1 pandemic) and reciprocal participation in annual meetings has taken place. there remains, however, considerable scope for improved coordination and collaboration with the animal influenza surveillance sector, especially in the collection and analysis of antigenic and genetic data, the timely exchange of representative viruses and reference reagents, and the conducting of serological studies of human exposure to zoonotic infection. influenza is an important disease of many avian and mammalian species with serious economic consequences for livestock industries and has potential adverse impacts on human food supplies. despite this, animal influenza surveillance coverage is limited with a shortage of epidemiological data on the circulation of various viruses in different countries. efforts are now under way to establish triggers for initiating enhanced surveillance that go beyond animal disease notification and sporadic human infections. although there is increasing understanding of the interrelationships between animal and human influenza and the need for 'integrated' surveillance, full collaboration at both national and global levels is currently constrained by a number of practical, funding, regulatory and policy issues. maintaining a regular dialogue based upon the mutual interests of the different networks will be an important public health activity and may also help to enhance the sustainability of animal influenza surveillance in particular settings. a more formal collaborative mechanism might allow for the improved integration of animal virus data into the who candidate vaccine virus selection process. increased awareness of the content and extent of use of animal influenza vaccines would also aid understanding of their impact on virus evolution. a range of laboratory assays and other techniques provide the complementary information on changes in the antigenic and genetic characteristics of influenza viruses needed to select the most appropriate influenza vaccine viruses. however, inherent limitations in the biological assays used and significant variations in the results obtained by different laboratories complicate the collation and definitive interpretation of data. because the hai test outlined previously is a simple, rapid and reproducible surrogate assay for virus neutralization, it is widely used to measure the antigenic relationships between different viruses as well as antibody responses to infection or vaccination. in addition, the test provides the basis of the only current quantitative correlate of protection against infection (serum hai antibody titre ‡40) used to standardize inactivated vaccines. however, variations in the physical characteristics of rbcs obtained from different species and differences in the receptor-binding properties of different viruses influence both the sensitivity and the utility of the assay. furthermore, changes in receptor-binding affinity or specificity associated with adaptation, antigenic drift or the isolation and passage of viruses in eggs and cell culture may also affect hai titres. standardization between laboratories has also proved difficult, and the assay is currently not suitable for use in a fully automated system. a range of practical refinements such as attempts to develop 'synthetic' rbcs (for example using glycan-coated beads) have been unsuccessful. given the currently limited knowledge of the principal natural receptors for influenza viruses, such approaches are unlikely to circumvent the virus-dependent shortcomings of assays based upon natural rbcs which are therefore likely to remain the primary approach to antigenic characterization for the foreseeable future. recent developments based on the use of panels of recombinant ha do offer alternative or supplementary microtitre or microarray binding-assay formats for assessing antibody specificity and antibody inhibition of the ha-glycan receptor interaction. although such approaches are relatively expensive and require a high degree of skill to implement, they are potentially highly suited to automation and in time may reduce the need for virus isolates. in addition, such formats can readily be adapted to incorporate biosensor technologies to provide more quantitative analyses of binding characteristics. a number of such assays are currently being validated using ferret and human antisera. the contribution of antibodies against virus na in conferring protection following natural infection or vaccination is still not well understood. studies of na antigenic variation have been limited, and the na content of influenza vaccines is not currently standardized. although neuraminidase inhibition (nai) assays were conducted more routinely in the past, these were cumbersome to perform and were complicated by the relatively low levels of antibodies against na in post-infection ferret sera and by interference from antibodies against ha. a number of different nai microtitre assay formats have recently been developed. these have been used to correlate antigenic changes with sequence variations in the na component, provide more precise information on the evolution of na and assess na antibody responses following vaccination. improved understanding of antigenic drift in na and of the role of anti-na antibodies in conferring immunity might have significant implications for both vaccine virus selection and vaccine development. microneutralization (mn) assays -based on measuring virus replication, cell viability or na activity -provide an important adjunct to hai tests in antigenic characterization. mn assays are generally more sensitive and measure a broader repertoire of functional antibodies that neutralize viral replication, with potential advantages in the evaluation of human serological responses. in addition, comparisons of mn and hai tests for measuring antibody responses in vaccinated individuals have shown a consistent degree of correlation and have confirmed the utility of mn assays in analyses of human antibody responses to h3 vaccine components. techniques for simplifying assay formats and making them more readily applicable to the routine testing of low-titre viruses are under investigation, and efforts are under way to use mn assays for h1 and b viruses. this should facilitate the use of mn assays to overcome the variable nature of interactions between viruses and rbcs, and hence in interpreting 'anomalous' hai results which complicate vaccine virus selection. pseudotype virus neutralization assays may also offer some advantages in scale and standardization over conventional mn assays for measuring serological responses to particular viruses, especially highly pathogenic viruses. furthermore, ongoing improvements in automation will potentially enable the more labour-intensive mn assay to be applied to large-scale serological analysis. epitope mapping using genome fragment phage display libraries provides another powerful technique for further dissecting the fine specificity of antibody responses to vaccination and infection and should allow for a better assessment of the quality of a 'protective' immune response and aid the development of additional correlates of immunity. to encourage the performance of seroepidemiological surveys to assess the impact of influenza in a population, countries should be supported in establishing well-characterized serum banks of age-stratified representative sera as a national, regional and global resource. current advantages of the gisrs serological activities undertaken in support of vaccine virus selection include the use of shared serum panels and common antigens, with frequent consensus obtained from participating whoccs and who erls. limitations include the large variability of hai data, a requirement for antibody standards and a need for mn or other assays to resolve inconsistencies. the availability of antibody standards would not only enhance the comparability of serological data generated in different laboratories and countries but also facilitate the comparison of antibody responses to different vaccines. increasing attention to influenza vaccine effectiveness studies will lead to the availability of more real-time data for comparing clinical benefit with the degree of antigenic improving influenza vaccine virus selection ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 6, 147-152 relatedness of vaccine and circulating viruses. such studies, especially those based upon laboratory-confirmed outcomes, should provide evidence of the specific benefits of vaccination. consistent studies providing estimates of vaccine efficacy over successive influenza seasons should improve understanding of the effects of small rather than major antigenic differences between vaccine and circulating viruses on clinical outcomes and should help to allay concerns arising from a perceived vaccine mismatch caused by the emergence of virus clades exhibiting little or no antigenic drift. recent advances in high-throughput genetic sequencing could potentially lead to a greatly enhanced understanding of the genetic changes occurring in influenza viruses and the evolutionary interactions that occur between co-circulating viruses. in-depth analyses of the precise mechanisms involved in the evolution and epidemiology of influenza would require advanced bioinformatics tools to comprehensively mine the data produced. such an approach should reveal, for example, the broader genetic changes that underlie antigenic variation in ha and thus allow for a better understanding of the relationship between genetic evolution and antigenic drift. increased information from x-ray crystallography on the structural features of the has of recent viruses and specific mutants, together with developments in computer modelling, should assist in attempts to predict the likely influence of amino acid substitutions on the antigenic and receptor-binding properties of new variants. further development of high-throughput laboratory systems for integrated and automated genetic and phenotypic analyses -from initial sample accession to data management -offers the intriguing prospect of a futuristic standardized virtual network for virus characterization in an epidemiological context. as such systems will have broad implications, not only for vaccine virus selection, but also for the organization and conduct of global influenza surveillance, it is extremely important that their development and deployment are integrated with the activities of the who gisrs. numerous mathematical modelling techniques have now been used to gain insights into the mechanisms that underlie both the evolution and the epidemiology of influenza viruses. for example, exploratory models have been developed to generate and test various hypotheses to explain the relatively restricted diversity of influenza viruses in terms of constrained antigenic repertoire, and to explore the underlying nature of immunity. they have also been used to improve understanding of the extent of between-subtype and between-type competition and of the potential conse-quences of such interactions for trends in the incidence of seasonal influenza viruses. phylogenetic models have also been used to identify changes in selective constraints in relation to antigenic drift and inter-species transmission. when based upon the amino acid substitutions associated with mammalian host adaptation, such models may aid assessment of the pandemic potential of avian and other animal viruses. phylodynamic modelling based upon available sequence data, supplemented with antigenic data, has already been successfully used to trace the emergence of new antigenic and genetic variants and track their geographical spread. however, in the absence of greatly improved understanding of the underlying evolutionary and biological mechanisms and other processes involved, the capacity of current mathematical modelling techniques to predict the timing and nature of future antigenic and genetic changes is limited. the intrinsically stochastic nature of influenza evolution may make such predictive modelling extremely challenging. where changes occur over short time scales, the application of simpler non-mechanistic statistical algorithms, such as those used as the basis of antigenic cartography, is likely to be more useful in facilitating vaccine virus selection than attempts to develop predictive models from the existing complex dynamical models of influenza evolution and transmission. such predictive models might presently be better suited for use in understanding the possible long-term effects of vaccination, optimizing the timing and location of focused surveillance efforts and predicting the possible consequences of the emergence of a novel virus. eventually, these models should be able to take advantage of integrated immunological and antigenic surveillance data to develop predictions of short-term dynamics in specific locations. all new influenza vaccine technologies have implications for vaccine virus selection and for regulatory and manufacturing processes. however, any potential requirement to tailor the virus selection process to specific types of vaccine is unlikely to be a crucial issue, especially if advances in vaccine technology and speed of production lead to greater flexibility in the timing of recommendations. although live-attenuated vaccines are not yet universally licensed, the current vaccine composition recommendation process is used. however, antibody response is not a good correlate of protection for such vaccines and the identification of a true correlate might affect the requirement for annual updating. several quadrivalent vaccines are also now under development that contain representative strains of the two influenza b virus lineages (b ⁄ victoria and b ⁄ yamagata) together with influenza a(h1n1) and a(h3n2) viruses. this raises a number of issues that could affect vaccine supply, including the possibility of two poorly growing vaccine viruses; the likely variable impact of a fourth component on vaccine yields and timing of manufacture; the prioritization of influenza b lineage viruses in the context of both trivalent and quadrivalent vaccine production; and the need for a fourth set of reagents. adjuvanted vaccines have been licensed with the primary aims of inducing better immune responses in certain age groups and allowing 'antigen sparing'. although there has been no specific intention to provide a broader spectrum of immunity to circumvent the need for annual vaccine updates, different products are likely to show a different breadth of response. providing recommendations in relation to product-specific cross-reactivity over successive influenza seasons is unlikely to be a feasible option for the who gisrs. in addition, various types of recombinant vaccines are now under development, including protein subunit, dna, vector and vlp vaccines -none of which are presently licensed. in the case of non-ha-based vaccines, different guidelines will apply and all such vaccines are likely to impact the current vaccine virus selection process in various ways depending upon their precise type and mechanism of protection. the level of protection afforded by immunity to na is receiving continued interest. currently, this component is included as part of the candidate vaccine virus and is selected on the basis of its sequence but not antigenicity. standardization of the na component would require antigenic characterization during the virus selection process, while antigenic changes in na in the absence of a corresponding change in ha antigenicity may on its own necessitate the updating of vaccine composition. for all such vaccines, ha variant selection may become less crucial than it is for current vaccines. although high-growth reassortants have been used to manufacture influenza a vaccine components for many years, their yields have been variable and there is continued need to identify the molecular determinants of high yield to engineer a more reliable and reproducible production process. reverse genetics, now used in the united states to produce virus reassortants for live-attenuated vaccines, has also been used to produce attenuated candidate h5n1 vaccine viruses suitable for inactivated vaccine manufacture. this approach was, however, less successful than classical reassortment in obtaining a suitable 2009 h1n1 pandemic vaccine virus, emphasizing the need for further investigation of the applicability of reverse genetics in the routine provision of suitable vaccine viruses. following the licensing of cell culture vaccines, the feasibility of isolating seasonal vaccine viruses in qualified 1 cell lines is being evaluated in a collaboration involving a number of whoccs and who erls under cradas with vaccine manufacturers. these studies should provide the basis for the introduction of a universal qualified cell culture system for providing mammalian cell-derived seasonal influenza candidate vaccine viruses. this would result in a greater choice of candidates, especially for recent h3n2 viruses, and may provide greater flexibility in responding to the 'late' emergence of a variant necessitating a vaccine composition change. such virus isolates would not be subject to undesirable egg-selected changes and would potentially provide a better match to the natural virus. however, the relative merits of egg and cell culture candidate vaccine viruses have still to be rigorously evaluated. guidance on quality assurance aspects has already been published by the european medicines agency (ema). the finalization of new ema regulatory guidelines may be accompanied by a who technical document on harmonizing regulatory approaches worldwide and the engagement of other regulatory authorities in vaccine-manufacturing nations. vaccine manufacturers and the who erls are also collaborating in an evaluation of cell culture-based reagents for use in single radial immunodiffusion (srid) potency testing, due for completion in early 2011. in addition, despite international consensus on the key quality specifications for 2009 h1n1 pandemic influenza vaccines, reagents to calibrate the majority of candidate vaccines using conventional potency tests only became available immediately prior to the initiation of clinical trials. in some cases, candidate vaccines were available ahead of the reagents. although national authorities proved flexible in accepting the use of validated alternative potency tests to allow clinical trials to proceed, newer methods such as high-performance liquid chromatography (hplc) and mass spectrometry are now being evaluated. the gisrs has a long history of success in recommending influenza vaccine compositions that have closely matched the combination of viruses circulating during subsequent influenza seasons. based upon the voluntary participation of its many constituent partners, the gisrs enjoys strong institutional and governmental support. global influenza surveillance is the foundation of the vaccine virus selection process. efforts to enhance and strengthen national, regional and global laboratory capacity for virological surveillance and representative virus sharing must continue. as part of this, improved integration of virological and disease surveillance data will be a key aim and will help to build the foundations for future studies of the impact and burden of influenza worldwide. to strengthen the pandemic influenza preparedness, collaboration between the gisrs and veterinary laboratories and organizations such as offlu in relation to zoonotic influenza infection has been greatly enhanced and has included the development of appropriate candidate human vaccine viruses from animal viruses. however, there remains considerable scope for improvement in this area, including the more timely exchange of information, viruses and reagents, and strengthened technical collaboration at all levels. although antigenic characterization of promptly forwarded virus isolates will remain the central criterion for selecting influenza vaccine viruses in the foreseeable future, technological developments (such as advanced recombinant dna and protein technologies, and highthroughput sequencing and advanced bio-informatics tools) will inevitably impact current gisrs surveillance and virus selection activities. in the interests of global public health, it will be important to integrate into the gisrs system appropriate information and data generated by various networks using emerging technologies. antigenic cartography has been adopted by the gisrs in recent years as a means of integrating hai data from different laboratories to allow for statistical comparison and visual display. the development of new statistical algorithms to complement the use of antigenic cartography may further facilitate vaccine virus selection. greater emphasis should be placed on conducting human serological studies which incorporate the use of antibody standards to improve the comparability of results. such studies would improve current understanding of the prevalence and spread of influenza, and complement the development of improved epidemiological models. greater collaborative effort is needed to generate randomly sampled, representative and integrated serological, epidemiological and evolutionary data that provide snapshots of host and viral populations suitable for modelling hypotheses on virus evolution and host immunity. the application of advanced techniques for dissecting the fine specificity of antibody responses to vaccination and infection should also lead to improvements in understanding the quality of a 'protective' immune response and aid in the development of additional correlates of immunity. recent collaboration between the gisrs and external partners including academic institutions and vaccine manufacturers has resulted in the increased availability of egg isolates and high-growth reassortants. new approaches to the generation of high-growth vaccine viruses involving the use of reverse genetics and qualified cell cultures will continue to be evaluated and developed, as will alternative methods of vaccine potency testing. who will continue to support these and other efforts to increase the reliability and timeliness of global influenza vaccine supply. new vaccine types currently under development may allow more flexibility in the timing of recommendations on vaccine virus composition. conversely, alterations to the virus selection process and additional information may be needed in relation to new-generation vaccine types with different compositions and mechanisms of protection. the who gisrs vaccine virus selection process lies at the heart of global efforts to address the constantly evolving threat posed by influenza. for decades, this highly collaborative and complex process has ensured a continued supply who writing group of effective seasonal vaccines and was able to respond very rapidly to the emergence of the 2009 h1n1 pandemic. if the current limitations and constraints inherent in the process are to be overcome, ongoing efforts by the who gisrs and its partners must continue to identify improvements, harness new technologies and strengthen collaboration. who will continue in its central role of developing and coordinating worldwide expertise to meet the increasing public health need for influenza vaccines and will support this process through the convening of periodic international consultations on improving influenza vaccine virus selection. annex 1: process of influenza vaccine virus selection and development 2 the diagram shows that the individual steps in the selection of candidate vaccine viruses and development of standardizing reagents for seasonal influenza and for a potential h5n1 influenza pandemic are essentially equivalent. for seasonal vaccines the timelines are: • steps 1-4: the collection, isolation and thorough antigenic and genetic characterization of recent virus isolates continues throughout the year; • step 4a: comparisons of the recognition of representative recent viruses by vaccine-induced antibodies in human sera are conducted 2-3 weeks before the biannual who vaccine consultation meetings; • steps 5, 6a and 7a: candidate viruses for vaccine use are reviewed and selected, and high-growth reassortants prepared and characterized following identification of (potential) antigenic variants -these steps are not solely dictated by the recommendations of the who biannual vaccine virus consultations. • step 8: evaluation of their growth properties is conducted in a timely manner around the time of the who vaccine virus consultations and prior to authorization of vaccine composition by national authorities. • step 9a: preparation of the standardizing reagents for new vaccine components is initiated once the particular vaccine virus has been selected following the who recommendation. the who informal consultation for improving influenza vaccine virus selection, 14-16 june 2010, was organized by the virus monitoring and vaccine support (vmv) unit of who, with participation from who collaborating centres on influenza, erls, national influenza centres, national control laboratories, national regulatory authorities, academic and veterinary institutions, influenza vaccine manufacturers and other collaborating organizations. in accordance with who policy, all members of the writing group that assisted who in the development of this meeting report had completed the who declaration of interests for who experts. these declarations were then evaluated by the who secretariat prior to the consultation. the members of the writing group declared the following personal current or recent (within the last 4 years) financial or other interests relevant to the subject area: group on data needs). plos curr influenza 2009 (revised 23 december 2009). rrn1135. northern hemisphere season. writing committee of the world health organization consultation on northern hemisphere influenza vaccine composition for public workshop on immune correlates of protection against influenza a viruses in support of pandemic vaccine development use of antigenic cartography in vaccine seed strain selection influenza vaccine strain selection and recent studies on the global migration of seasonal influenza viruses who. a description of the process of seasonal and h5n1 influenza vaccine virus selection and development. draft version antigenic and genetic characteristics of influenza a(h5n1) and influenza a(h9n2) viruses and candidate vaccine viruses developed for potential use in human vaccines strategies to improve global influenza surveillance: a decision tool for policymakers global influenza surveillance network: laboratory surveillance and response to pandemic h1n1 improving the process of vaccine virus selection determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the octet rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza a h1n1 virus epidemic in milwaukee, wisconsin a practical influenza neutralization assay to simultaneously quantify hemagglutinin and neuraminidase-inhibiting antibody responses development of rapid, automated diagnostics for infectious disease: advances and challenges high-throughput dna sequencingconcepts and limitations high-throughput laboratories for homeland and national security current challenges in implementing cell-derived influenza vaccines: implications for production and regulation establishment of retroviral pseudotypes with influenza hemagglutinins from h1, h3, and h5 subtypes for sensitive and specific detection of neutralizing antibodies new tests for an old foe: an update on influenza screening ecological and immunological determinants of influenza evolution population dynamics of rapid fixation in cytotoxic t lymphocyte escape mutants of influenza a epochal evolution shapes the phylodynamics of interpandemic influenza a (h3n2) in humans rambaut a et al. the genomic and epidemiological dynamics of human influenza a virus the generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types h1n1 influenza pandemic: insights from modelling. the who informal network for mathematical modelling for pandemic influenza h1n1 2009 (working ne win immunization, vaccines and biologicals (ivb) global influenza programme (gip) who regional office for south-east asia (searo) global influenza programme (gip) who regional office for europe (euro) global influenza programme (gip) global influenza programme (gip) keiji fukuda, health, safety and environment (hse) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) global alert and response (gar) global influenza programme (gip) global influenza programme (gip) who regional office for the americas (amro) laszlo palkonyay, immunization, vaccines and biologicals (ivb) global influenza programme (gip) global influenza programme (gip) global influenza programme (gip) ludy suryantoro, health, safety and environment (hse) global influenza programme (gip) immunization, vaccines and biologicals (ivb) global influenza programme (gip) acknowledgements ª 2011 blackwell publishing ltd. the world health organization retains copyright and all other rights in the manuscript of this article as submitted for publication. key: cord-312493-wbhji81g authors: tay, ee laine; grant, kristina; kirk, martyn; mounts, anthony; kelly, heath title: exploring a proposed who method to determine thresholds for seasonal influenza surveillance date: 2013-10-11 journal: plos one doi: 10.1371/journal.pone.0077244 sha: doc_id: 312493 cord_uid: wbhji81g introduction: health authorities find thresholds useful to gauge the start and severity of influenza seasons. we explored a method for deriving thresholds proposed in an influenza surveillance manual published by the world health organization (who). methods: for 2002-2011, we analysed two routine influenza-like-illness (ili) datasets, general practice sentinel surveillance and a locum medical service sentinel surveillance, plus laboratory data and hospital admissions for influenza. for each sentinel dataset, we created two composite variables from the product of weekly ili data and the relevant laboratory data, indicating the proportion of tested specimens that were positive. for all datasets, including the composite datasets, we aligned data on the median week of peak influenza or ili activity and assigned three threshold levels: seasonal threshold, determined by inspection; and two intensity thresholds termed average and alert thresholds, determined by calculations of means, medians, confidence intervals (ci) and percentiles. from the thresholds, we compared the seasonal onset, end and intensity across all datasets from 2002-2011. correlation between datasets was assessed using the mean correlation coefficient. results: the median week of peak activity was week 34 for all datasets, except hospital data (week 35). means and medians were comparable and the 90% upper cis were similar to the 95(th) percentiles. comparison of thresholds revealed variations in defining the start of a season but good agreement in describing the end and intensity of influenza seasons, except in hospital admissions data after the pandemic year of 2009. the composite variables improved the agreements between the ili and other datasets. datasets were well correlated, with mean correlation coefficients of >0.75 for a range of combinations. conclusions: thresholds for influenza surveillance are easily derived from historical surveillance and laboratory data using the approach proposed by who. use of composite variables is helpful for describing influenza season characteristics. influenza infection remains a significant public health problem, resulting in considerable global morbidity and mortality [1] [2] [3] . in temperate regions of australia, seasonal influenza outbreaks usually occur between late autumn and early spring and are associated with an increase in disease burden and utilisation of health service [3, 4] . due to differences in circulating viruses, population immunity and environmental factors, the onset, duration and severity of a season may differ from year to year [2, 5] . ongoing monitoring of influenza is therefore needed to determine the onset and severity of seasons and to monitor changes in disease trends. surveillance which involves laboratory testing can add to data on virus characteristics. influenza thresholds have been developed to indicate a level of disease activity that would signal the start or end of a season or provide an alert to an unusually severe or atypical season. the onset of a season may stimulate diagnosis, enhance case detection, promote awareness of the need for patient cohorting or isolation in hospitals, remind people about vaccination and encourage early prescription of anti-viral medication, especially in vulnerable populations [6, 7] . in the setting of a particularly severe season or pandemic, thresholds may inform the appropriate allocation of resources [8] . many influenza surveillance systems around the world have incorporated the use of thresholds. these include australia, new zealand, europe and united states (us) [9] [10] [11] [12] . methods using a variety of surveillance systems have been developed to establish thresholds for influenza activity. the methods vary in their complexity and can use either short-term or longer historical data to create time-varying or fixed thresholds. there is currently no gold standard or consensus for calculating thresholds. the simplest method uses visual inspection of historical data to create a fixed threshold used throughout the year [13] . other methods include regression models [14] [15] [16] [17] , time series methods [15] , calculation of means and medians [18] [19] [20] and adaptation of industrial control processes such as shewhart charts [21] , cumulative sum (cusum) [15, 18, 22] and the exponentially weighted moving average [23] . the us centre for disease control and prevention (cdc) calculates the baseline for their influenzalike-illness (ili) surveillance by adding two standard deviations to the mean percentage of ili visits during non-influenza weeks for the previous three seasons, with non-influenza weeks defined as periods with less than 2% of the year's total positive specimens for influenza for ≥ 2 consecutive weeks [24] . in victoria, the general practice sentinel surveillance (gpss) for ili has historically relied on thresholds determined by inspection [13] . in 2012 a novel but simple method for defining thresholds was proposed by the world health organization (who) as part of the development of global standards for influenza surveillance [7] . the proposed method aligns several years of historical data on the median week of peak activity and assigns thresholds based on means and standard deviations of aligned data. to our knowledge, this method has not yet been field tested. the aim of this study was to explore the feasibility of the who method for the calculation of influenza thresholds using a range of existing surveillance and laboratory data sources in one surveillance system. we used these data sources to compare the onset, duration and intensity of influenza seasons. victoria is a state with a population of over 5 million people located in the south-eastern part of australia. it has a temperate climate with annual seasons of influenza occurring occur between late autumn (may) and early spring (october). it has a well-established influenza surveillance system that monitors influenza activity using syndromic surveillance of ili presentations to sentinel general practitioners (gp) and a medical locum service; laboratory-confirmed influenza; hospital admissions for influenza; and more recently google flu trends and the influenza complications alert network (flucan), which monitors hospitalised patients from sentinel australian hospitals, including four victorian hospitals [9] . four independent surveillance data sources were used: (i) the victorian gpss, (ii) sentinel data from the melbourne medical deputising service (mmds), (iii) routine laboratoryconfirmed influenza (lab data) from the victorian infectious diseases reference laboratory (vidrl) and the (iv) victoria admitted episode dataset (vaed) for hospital admissions. the gpss is an annual surveillance system for ili and laboratory confirmed influenza that was established in 1993, with laboratory support added in 1998 [25] . surveillance extends from week 18 to 44 each year during the influenza season. the number of participating general practitioners (gp) has varied from 40 to 100 since the scheme's establishment. the ili definition used is based on the nationally agreed case definition of cough, fever (measured or reported) and fatigue [13] . approximately 48% of ili patients seen by sentinel gps were swabbed and of these, influenza virus was detected from an average of 34% (18%-47%) of the swabbed ili patients tested from 2003 to 2011 [9, 26] . an alternative source of community sentinel ili surveillance is the mmds, an out-of-hours medical locum service that covers an approximate 45km radius from central melbourne. gps from the deputising service consult with patients in their own home or aged care facility. the diagnosis made by the attending doctor is recorded electronically and de-identified summary data are available on a password protected website within 24 hours. ili data are extracted weekly based on a previously developed search algorithm [27] . there is no laboratory support for the mmds surveillance which has nonetheless been shown to provide equivalent information to surveillance data from sentinel general practitioners [28] . lab data from vidrl consist of laboratory detections of influenza viruses from all routine respiratory samples sent to vidrl, tested using an in-house respiratory multiplex reverse transcriptase polymerase chain reaction (rt-pcr) test that identifies influenza viruses, adenovirus, picornavirus, respiratory syncytial virus, parainfluenza virus, coronavirus and human metapneumovirus [29] . many of the samples are referred from major adult teaching hospitals in victoria [30] . the vaed is a hospitalisation dataset on all patients admitted to public and private acute care hospitals in the state of victoria. the clinical information coded for each episode of care is based on the international classification of diseases and related health problems, tenth revision, australian modification (icd-10-am). we extracted records containing influenza codes j09-11 in primary or secondary diagnostic fields. in addition, a further two composite variables were created from the product of ili and lab data. these were the gpss composite = proportion of ili cases in gpss x proportion of laboratory samples tests positive for influenza in gpss exploring a who method for influenza thresholds plos one | www.plosone.org mmds composite = proportion of ili cases in mmds x proportion of routine laboratory tests positive for influenza in lab data. while the gpss proportion and laboratory testing are part of the same system, the mmds is independent of routine clinical tests referred to vidrl. however, both mmds and routine vidrl clinical testing focus on older age groups and were matched based on age profiles [30, 31] . the metrics used for threshold calculations were the weekly gpss ili proportion per 1000; the weekly mmds ili per 1000; the proportion of laboratory test positive for influenza using the total number of tests for influenza as the denominator (lab test positive influenza); the weekly proportion of influenza admissions in the population using the mid-year estimated resident population in victoria as the denominator and expressed per 100,000 population [32] ; and the product of weekly ili and lab data per 1000. we preferred the use of test positive influenza to count data to compensate for changes in testing behaviour over time [33] . depending on availability, data were extracted from 2002 to 2011 for gpss and mmds, 2003 to 2011 for lab data and 2005 to 2011 for vaed. for mmds, lab data and vaed, data were complete for all 52 weeks but only weeks 18 to 44 were available for gpss. for vaed, only aggregated data containing admissions of more than five counts were provided to protect privacy and confidentiality of individuals. we defined the three levels of influenza threshold based on the terminology used in the who manual and the existing victorian surveillance thresholds that had been adapted from the united kingdom: seasonal, average and alert [13, 34] . seasonal threshold defines the start and end of an influenza season. the two intensity thresholds, termed average and alert thresholds, describe relative seasonal intensity. the who manual did not prescribe a specific method for determining the seasonal threshold, that is, the start of the season. we used the method of inspection of the complete data for the six datasets to determine the seasonal threshold. for each dataset this was done independently by four of the co-authors (et, kg, am, hk) and differences were resolved by discussion. for the four datasets that provided data for the whole year (mmds, lab data, vaed and mmds composite), we calculated the 95% confidence interval (ci) of the metrics used for each dataset for the period defined as out-of-season, that is, the values below the seasonal threshold, that had been determined by inspection. we also explored the seasonal threshold using the 95th percentile of out-of-season data without assuming data were normally distributed. we then compared the seasonal threshold set by inspection with the 95% ci and 95th percentiles of the average out-of-season values. average and alert thresholds were calculated for each dataset using a variation of the who protocol [7] (figure 1 ). we first determined the median week of peak occurrence using historical data, excluding the pandemic year of 2009 which was atypical from both surveillance and testing perspectives [33] . we then aligned the transmission peaks around the median week of peak occurrence ( figure 1a ) and calculated the weekly mean and standard deviations for each week centred on the median week of peak occurrence ( figure 1b and c) . the who protocol suggests the use of the normal distribution to assign thresholds based on the mean and standard deviation of the aligned data for weekly counts. however, we believed data were unlikely to be normally distributed for all years and tested this by inspection and formally using the shapiro-wilks test for normality for gpss, mmds, lab data and the vaed for each year during season [35] . in addition to the mean and standard deviations, we explored the thresholds using the median and 90th and 95th percentiles. the average threshold was determined by a comparison of the peak weekly mean and median, while the alert threshold was determined by a comparison of the peak weekly upper 90% and 95% ci upper limits with the 90th and 95th percentiles. we also performed log transformation of all datasets and calculated the corresponding geometric mean and 90/95 ci upper limit. once the seasonal thresholds were assigned, we determined the start and end of each season independently for all datasets, each defined as the two consecutive weeks where the seasonal threshold was crossed. we used the average and alert thresholds to categorise the influenza seasons, based on the threshold range of peak seasonal activity, specifically seasonal-average, average-alert or alert. comparisons were made for the onset, duration and intensity of a season across all datasets from 2005-2011 based on data availability. we also compared how all seasons compared to the average season created using aligned historical data described above. finally, to determine how correlated the six datasets were, we calculated the correlation coefficient for each year from 2005 to 2011 for a combination of datasets, from which a mean correlation coefficient and its corresponding 95% confidence limits was derived. data were analysed using microsoft® office excel 2003 and stata version 10.0 (stata corp., college station, tx, usa). the study was approved as a quality assurance project by the melbourne health office of research. gpss and mmds data in this study were collected, used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009. during the study period, the highest number of ili or influenza cases annually ranged from 56 to 208 per week for the gpss; 33 to 164 per week for the mmds; 24 to 135 per week for test positive influenza; and 28 to 204 per week for influenza admissions. testing for normality of the weekly count data for each year suggested no seasonal surveillance data had a classical normal distribution graphically (data not shown). data were consistent with a normal distribution by formal testing for 5/10 seasons in the gpss, for 5/10 seasons in the mmds, for 2/9 seasons in lab data and for 2/6 seasons in the vaed. when data were log-transformed, the number of seasons with normal distribution increased (8/10, 8/10, 5/9 and 3/6 respectively). the threshold parameters from the adapted who method are summarised in table 1 . the median week of peak occurrence for all datasets was week 34 except for the mmds composite and vaed. the values assigned for seasonal thresholds by inspection were similar to the 95% ci upper limit and 95 th percentile of the average out-of-season values for all datasets. for the average threshold, we found the peak mean values for the gpss, test positive influenza and vaed were similar to the median but the peak mean was higher for the mmds and mmds composite due to the high call out proportion in 2003. we therefore use the peak mean to define (table 1) . to set the alert thresholds, the peak 90% ci upper limit was used as we found the parameter to be similar to the peak 95 th percentile across all six datasets (table 1 and figure 2 ). the geometric means and 90% ci upper limits from log-transformed data also produced similar parameters (data not shown). using the gpss dataset as an example, figure 3 compares how an annual season compares against the average season calculated using ten years of historical data. onset, end and duration of influenza season across all datasets. for the seven years where data were available for all datasets, gpss assigned seasons tended to start much earlier for most years compared to other datasets. the use of the gpss composite suggested a later start to the season. season onset according to the vaed generally lagged behind other datasets for most years and was variable for the mmds ( table 2) . for most pre-pandemic years, there was generally good agreement for defining the end of a season across datasets. however, a divergent trend, not reflected in other datasets, was noted in the vaed from 2009 onwards ( table 2) . the vaed assigned seasons ended much later, resulting in a longer assigned seasonal duration. category of influenza season. there was agreement in describing the intensity of influenza seasons in 3/7 years prior to 2009 (table 2 and figure 4 ). during the pandemic year, the season intensity varied according to different data sources and from 2009 onwards, peak seasonal influenza activity was between the seasonal and average thresholds (or seasonalaverage) for all datasets except the vaed. datasets were found to be well correlated, with mean correlation coefficients of >0.75 for a range of combinations (table 3) . correlations between the vaed and other datasets improved once the vaed was aligned with other datasets to correspond to the one week lag in median week of peak occurrence. thresholds for influenza surveillance were easily derived using a simple method proposed by the who. the method was adapted to a non-parametric approach that produced similar findings to the suggested protocol based on the normal distribution. log transformation of the data produced comparable findings to both approaches. comparison of thresholds derived from different datasets revealed variations in defining the start of a season but relatively good agreement in describing the end and intensity of influenza seasons, except in the hospital data after the pandemic year. as the who protocol does not prescribe a method for defining the seasonal threshold, we used the simplest method of visual inspection but showed that the levels were consistent with variation in out-of-season virus circulation. numerous other approaches exist, based on more complex statistical techniques but many of these approaches usually require a pre-determined threshold to be nominated [15, 21, 23] , again often by inspection. in the setting of the average and alert thresholds for our datasets, we used the peak mean values to set the intensity thresholds as per the who protocol after observing the data agreement between the peak weekly means and medians and 90% ci upper limit and 95 percentiles. however, the median and 90 or 95 percentiles may be a more appropriate option when data are not normally distributed and no transformation has been performed. in practice, another point of consideration for the setting of the alert threshold may be a level of influenza activity that corresponds to an increased demand on the health care system [7] . this would be dependent on local health care systems. we also incorporated a two week consecutive rule into the definition of the onset and intensity of a season to reduce the number of false positive signals. based on the seasonal thresholds, we found inconsistencies in defining the start and end of a season across the datasets. given the variations in timeliness of influenza reporting [31] , we would expect the onset of ili surveillance to precede laboratory confirmed influenza and hospital admissions, and that both the ili surveillance systems might coincide with one another. by incorporating a laboratory component to the ili measure, the use of the composite variable appeared to improve the specificity and agreement between vaed and ili surveillance data. the finding is consistent with emerging literature that these composite variables may be a better proxy indicator of influenza incidence than either ili or lab data alone [36, 37] . the use of composite variables in surveillance warrants further investigations. in comparing the intensities of an influenza season, there was good agreement across all datasets, except for the vaed after 2009. the number of hospital admissions coded for influenza has increased both in and out of the influenza season, with the duration of the season prolonged due to the late end signal. these changes were not reflected in the ili or lab datasets. while there may be a number of possible explanations, such as changes in testing behaviours or disease coding, a recent study investigating the increase in out-ofseason influenza in australia suggests a genuine increase in influenza activity, combined with increased testing that occurred following the pandemic [38] . this may reflect an increased awareness of influenza in hospitalised patients among health professionals after the pandemic. additionally, surveillance data at vidrl indicate that approximately 40% of patients with an influenza-like illness were swabbed prior to 2009 [26] but this rose to 70% during the 2009 pandemic [39] and has since remained at about this proportion [9] . the measurement of the intensity a season was based on the peak of influenza activity and whether or not the thresholds were exceeded for two weeks. this reflects only a single dimension of measurement and does not take into account how long the influenza activity remained within a particular category or the rate of increase in the number of cases. for example, a short-term acute rise in influenza cases that marginally exceed the alert threshold does not correspond to a gradual or persistent elevated level of activity that may represent a higher disease burden. finally, when we compared the current parameters to the previous threshold based on seven years of historical ili data from 1994-2000 in victoria, the baseline threshold for the gpss was lower at 2 per 1000 cases compared with the revised threshold of 4 and the alert threshold was higher at 35 per 1000 cases compared with the revised threshold of 24 [13] . these differences indicate the need of regular review of surveillance-derived thresholds. in conclusion, this study has shown that the proposed who threshold protocol is simple to implement and could be easily adapted for any influenza surveillance system with adequate historical data. however, the study was based in a region with a temperate climate, and its application in the tropics would require further work. further exploration of the proposed who method in another temperate region would be of interest. while thresholds are useful as a warning system, they should always be interpreted with other available information. influenza (seasonal) factsheet epidemiology of influenza the annual impact of seasonal influenza in the us: measuring disease burden and costs global influenza seasonality: reconciling patterns across temperate and tropical regions how to deal with influenza: worthwhile surveillance system is in action world health organization (2012) who global surveillance standards for influenza. geneva: global influenza programme, surveillance and monitoring team, world health organization resource allocation during an influenza pandemic virological surveillance: influenza weekly updates centers cdcfor disease control and prevention (2013) flu activity & surveillance: weekly us influenza surveillance report weekly influenza surveillance overview in: control establishing thresholds for influenza surveillance in victoria can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data a routine tool for detection and assessment of epidemics of influenza-like syndromes in france a statistical algorithm for the early detection of outbreaks of infectious disease epidemic features affecting the performance of outbreak detection algorithms influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method modelling influenza epidemic -can we detect the beginning and predict the intensity and duration? detection of epidemics in their early stage through infectious disease surveillance do cusums have a role in routine communicable disease surveillance? detecting the start of an influenza outbreak using exponentially weighted moving average charts overview of influenza surveillance in the united states laboratory-supported influenza surveillance in victorian sentinel general practices estimation of influenza vaccine effectiveness from routine surveillance data a medical locum service as a site for sentinel influenza surveillance influenza-like illness surveillance using a deputising medical service corresponds to surveillance from sentinel general practices laboratory diagnosis and surveillance of human respiratory viruses by pcr in h1n1 swine origin influenza infection in the united states and europe in 2009 may be similar to h1n1 seasonal influenza infection in two australian states in a comparison of data sources for the surveillance of seasonal and pandemic influenza in victoria influenza surveillance in australia: we need to do more than count the use of thresholds to describe levels of influenza activity an analysis of variance test for normality (complete samples) improving the estimation of influenza-related mortality over a seasonal baseline predicting the epidemic sizes of influenza a/h1n1, a/h3n2, and b: a statistical method the significance of increased influenza notifications during spring and summer of 2010-11 in australia pandemic influenza h1n1 2009 infection in victoria, australia: no evidence for harm or benefit following receipt of seasonal influenza vaccine in 2009 we gratefully acknowledge the ongoing support of general practitioners and their practice staff participating in the general practice sentinel surveillance system and the continued involvement of the melbourne medical deputising service in influenza surveillance in victoria. we thank the victorian government department of health for provision of hospital admissions data and staff at the viral identification laboratory at vidrl for provision of laboratory data. we acknowledge the world health organization global influenza program for the threshold methodology. sentinel surveillance in victoria is supported by the victorian government department of health. ee laine tay is a post-graduate scholar in the master of philosophy in applied epidemiology program, the field epidemiology training program in australia. conceived and designed the experiments: et kg hk am. analyzed the data: et kg mk hk. contributed reagents/ materials/analysis tools: am. wrote the manuscript: et hk. provided critical revisions to the article: et kg mk am hk. key: cord-333527-66dfphxq authors: brown, lawrence h; aitken, peter; leggat, peter a; speare, richard title: self-reported anticipated compliance with physician advice to stay home during pandemic (h1n1) 2009: results from the 2009 queensland social survey date: 2010-03-16 journal: bmc public health doi: 10.1186/1471-2458-10-138 sha: doc_id: 333527 cord_uid: 66dfphxq background: one strategy available to public health officials during a pandemic is physician recommendations for isolation of infected individuals. this study was undertaken during the height of the australian pandemic (h1n1) 2009 outbreak to measure self-reported willingness to comply with physician recommendations to stay home for seven days, and to compare responses for the current strain of pandemic influenza, avian influenza, seasonal influenza, and the common cold. methods: data were collected as part of the queensland social survey (qss) 2009, which consisted of a standardized introduction, 37 demographic questions, and research questions incorporated through a cost-sharing arrangement. four questions related to respondents' anticipated compliance with a physician's advice to stay home if they had a common cold, seasonal influenza, pandemic (h1n1) 2009 influenza or avian influenza were incorporated into qss 2009, with responses recorded using a balanced likert scale ranging from "very unlikely" to "very likely." discordance between responses for different diseases was analysed using mcnemar's test. associations between demographic variables and anticipated compliance were analysed using pearson's chi-square or chi-square for linear-by-linear association, and confirmed using multivariate logistic regression; p < 0.05 was used to establish statistical significance. results: self-reported anticipated compliance increased from 59.9% for the common cold to 71.3% for seasonal influenza (p < .001), and to 95.0% for pandemic (h1n1) 2009 influenza and 94.7% for avian influenza (p < 0.001 for both versus seasonal influenza). anticipated compliance did not differ for pandemic (h1n1) 2009 and avian influenza (p = 0.815). age and sex were both associated with anticipated compliance in the setting of seasonal influenza and the common cold. notably, 27.1% of health and community service workers would not comply with physician advice to stay home for seasonal influenza. conclusions: ninety-five percent of people report they would comply with a physicians' advice to stay home for seven days if they are diagnosed with pandemic (h1n1) 2009 or avian influenza, but only 71% can be expected to comply in the setting of seasonal influenza and fewer still can be expected to comply if they are diagnosed with a common cold. sub-populations that might be worthwhile targets for public health messages aimed at increasing the rate of self-imposed isolation for seasonal influenza include males, younger people, and healthcare workers. in late march 2009 an outbreak of a new strain of influenza a (h1n1), swine-origin influenza virus (s-oiv) or "swine flu," was reported in north america [1, 2] . this disease quickly spread across the globe, and the world health organization declared a pandemic on 11 june 2009 [3] . the first cases of pandemic (h1n1) 2009 influenza in australia were reported in may 2009, coinciding with the onset of the annual influenza season. as of 01 january 2010, 37,553 cases of pandemic (h1n1) 2009 influenza had been confirmed in australia, with 191 deaths [4] . at the time the australian cases peaked, there was no approved vaccine for pandemic (h1n1) 2009 virus; traditional public health measures were critical to containing the outbreak. one strategy available to public health officials is physician recommendations for self-imposed isolation of infected individuals; specifically, to stay home for at least seven days. such public health measures, however, only work if patients are willing to comply [5] [6] [7] . this study was undertaken during the height of the australian pandemic (h1n1) 2009 outbreak to measure self-reported willingness to comply with physician recommendations to stay home for seven days, and to compare responses for the current strain of pandemic influenza, avian influenza (h5n1), seasonal influenza, and the common cold. data for this study were collected as part of the queensland social survey (qss) 2009. qss is an annual statewide survey conducted by the population research laboratory (prl) in cquniversity australia's institute for health and social science research. through a costsharing arrangement, qss enables researchers and policy-makers to incorporate questions into the survey. queensland is the second largest australian state by land area, and the third most populous state. qss uses a computer-assisted telephone interviewing (cati) system and trained interviewers to randomly sample households across queensland, including metropolitan brisbane (south east queensland) and the rest of the state (other queensland). to ensure equal representation of males and females, households are randomly pre-determined to provide a male or female respondent; if a person of that sex is not available then the household is not included in the survey. qss 2009 consisted of a standardized introduction, specific questions incorporated by researchers and the university, and 37 demographic questions. the questions were pilot tested by trained interviewers in 92 randomly-selected households, with modifications to the questions guided by both responses from the pilot study subjects and feedback from the interviewers. final interviewing was conducted between 20 july 2009 and 19 august 2009, between the hours of 10:30 am to 2:30 pm and 4:30 pm to 8:30 pm on weekdays, and between the hours of 11:00 am and 4:00 pm on weekends. four questions related to respondents' anticipated compliance with a physician's advice to stay home if they had a viral respiratory illness were incorporated into qss 2009. the four questions were: • if you had a common cold and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so? • if you had the regular flu, but not swine or bird flu, and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so? • if you had the swine flu and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so? • if you had the avian or bird flu and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so? responses were recorded using a 4-point likert scale ranging from "very unlikely" to "very likely." responses were subsequently dichotomized as "yes" (very likely or likely) and "no" (very unlikely or unlikely) and crosstabulated in a 2 × 2 table. because the data are essentially repeated measures of likelihood to comply under different circumstances, discordance between responses for the different diseases was analysed using mcnemar's test. bivariate associations between relevant demographic variables and anticipated compliance were analysed using chi-square or fisher's exact test; where demographic variables were recorded as ordinal data, analyses utilizing chi-square for linear-by-linear association were conducted to identify any significant trend effects. subsequently, multivariate logistic regression was conducted to identify covariates and interaction effects, and to adjust for confounding. each variable was entered into or removed from the logistic regression model using both forward and backward methods to identify significant covariates, the remaining variables were then individually entered into the model to identify potential confounders. the final model included significant covariates, potential confounders and significant interaction effects. for all analyses, p < 0.05 was used to establish statistical significance; for the multivariate analysis, adjusted odds ratios (aor) and their 95% confidence intervals (ci) are reported. qss 2009 had a target sample size of 1,200 subjects, with 800 subjects from south east queensland and 400 from other queensland; thus the a priori estimated sampling error at the 95% confidence level was ± 2.9% overall, ± 3.6% for the south east queensland sub-sample, and ± 5.1% for the other queensland sub-sample. panel at cquniversity (h09/06-037) and the incorporation of the influenza-related questions was approved by the human research ethics committee at james cook university (h3456). qss 2009 contacted or attempted to contact 3,112 households; 1,536 subjects declined participation, 142 households could not be contacted, and 129 were otherwise ineligible. thus, the final sample for qss 2009 included 1,292 respondents; 860 from south east queensland and 432 from other queensland for an overall response rate of 41.5%. the sample was nearly equally divided between males and females (50.2% vs. 49.8%). younger people (aged 18 -34 years) were under-represented in the sample and older people (aged > 55 years) were over-represented in the sample, otherwise the demographics of the participants reasonably approximated that of the general population [8] as shown in table 1 . responses to the four questions concerning anticipated compliance with a physician's advice to stay home are shown in table 2 bivariate associations between demographic variables and anticipated compliance with a physician's advice to stay home for the four viral diseases are shown in additional file 1: table s1 . as anticipated compliance in the setting of pandemic (h1n1) 2009 and avian influenza was near universal, there were no significant associations between demographic variables and anticipated compliance. for the common cold and seasonal influenza, however, there were a number of significant associations. respondents who were male, younger, employed (versus unemployed), and had a higher level of education were less likely to report anticipated compliance with stay home advice for both a common cold and seasonal influenza. married/partnered people and those who lived in south east queensland were also less likely to comply with advice to stay home for a common cold. people who lived in urban areas, and people employed in the health and community services sector were more likely than others to comply with advice to stay home for seasonal influenza, although 27.1% of health and community service workers would be unlikely to comply with such advice. in multivariate analysis, only sex and age remained significantly associated with anticipated compliance, and there was no interaction effect between these two variables. (additional file 2: table s2 ) females were more likely than males to report anticipated compliance for both the common cold (aor = 1.650; ci: 1.143-2.381) and seasonal influenza (aor = 1.911; ci: 1.300-2.811). nearly every respondent in this study reported they would comply with a doctor's advice to stay home for seven days if they were diagnosed with pandemic (h1n1) 2009 influenza, and the same level of compliance could be expected in the setting of avian influenza. these findings are similar to those that have been previously reported; our study adds data in the context of an actual, rather than hypothetical, pandemic. prior to the current pandemic, eastwood et al read a brief description of a pandemic influenza outbreak analogous to the 1918 spanish flu to australian telephone survey participants, and found 97.5% of respondents would stay home for seven to ten days if they were told they might have had contact with the disease [7] . similarly, barr et al [9] reported 85% of australians would be at least moderately willing to isolate themselves from others during an influenza pandemic. blendon et al [10] reported 94% of americans would comply if they contracted a pandemic influenza and public health officials recommended they stay at home for seven to ten days. in a more recent survey from june of 2009, blendon et al [11] identified 236 respondents who reported that they themselves or someone in their household had experienced flu-like symptoms, and 75% of those with symptoms had stayed home. other studies have also found support for explicit government action to contain pandemic influenza, including "encouraging" people to work from home, and quarantining infected individuals [5, 12] . interestingly, digiovanni et al [13] reported that compliance with quarantine measures during the 2003 severe acute respiratory syndrome (sars) outbreak in toronto, canada was affected more by compliance monitoring, fighting boredom and stress, and minimizing stigmatization than with any actual threat of enforcement. from a public health planning perspective, the more useful data from this study may be that regarding the level of compliance with stay at home advice that can be anticipated for seasonal influenza, and the relative lack of compliance that can be expected for the common cold. seasonal influenza is a more common disease, each year leading to approximately 18,000 hospitalizations and costing around $115 million in australia; the burden in the united states is much greater with the direct costs of influenza-related medical care exceeding $10 billion [14] . yet, these data confirm that people do not view seasonal influenza with the same level of concern as pandemic strains of influenza. while it is encouraging that respondents appear to differentiate between seasonal influenza and the common cold, the questions in this survey presumed a physician diagnosis. large numbers of people do not seek medical care for mild to moderate respiratory illness, and it is not practical to expect lay people to reliably differentiate between a common cold and influenza. public health efforts to encourage people to self-isolate for influenza-related illnesses may be more successful if they target symptoms (i.e., "cough and fever") rather than specific diagnoses. this study did find some significant associations between demographic characteristics and likelihood to comply with stay at home advice for seasonal influenza that might be useful for targeting public health efforts to increase compliance. males were less likely to report anticipated compliance with stay home advice for both a common cold and seasonal influenza, and this is consistent with other studies from australia [7, 9] . males have also previously been reported to feel less susceptible than females do to pandemic influenza, [5] although this study found no differences between males and females for anticipated compliance in the setting of pandemic (h1n1) 2009 or avian influenza. increasing age was associated with increased anticipated compliance with stay at home advice for both the common cold and seasonal influenza, while increasing education and income were associated with decreased anticipated compliance for both diseases. although the associations for education and income did not withstand multivariate analysis, the finding is consistent with previous work and both variables were retained as potential confounders in the final logistic regression model. like males, wealthier and better educated people tend to view themselves as less susceptible to influenza, while older people tend to view themselves as more susceptible [5] . many influenza-related public health campaigns target older populations; targeting stay at home messages to wealthier and better educated populations might be a novel but worthwhile effort for containing seasonal influenza. employed respondents were less likely than unemployed respondents to report anticipated compliance with stay home advice for both a common cold and seasonal influenza. this association, also, did not withstand multivariate analysis, but it is an intuitive finding. even in the setting of pandemic influenza, many people would have to forgo income in order to stay home [10] . for example, a survey of key decision makers at u.s. businesses found 74% of the businesses provided for paid employee sick leave, but 15% of businesses did not provide for any employee sick leave, whether paid or unpaid [15] . still, this study found no difference in anticipated compliance rates in the setting of pandemic (h1n1) 2009 or avian influenza. this is consistent with the findings of barr et al [9] who reported similar rates of "willingness to comply with health protective behaviours" between employed (69.5%, 95%ci: 65.5%-73.5%) and unemployed (71.8%, 95%ci: 67.7%-76.0%) survey respondents in the setting of pandemic influenza. eastwood et al, [7] however, reported the contrary, finding that employed people who were unable to work from home would be less likely to self-isolate in the setting of pandemic influenza. how closely the level of actual compliance approaches the level of self-reported anticipated compliance may well be affected by issues related to income, financial security, and employer leave policies. a particularly novel and important finding of this study was that more than one-quarter of health and community service workers reported they would not comply with a physician's advice to stay home if they had seasonal influenza. this may represent a misplaced sense of duty. previous research has demonstrated that most healthcare workers (hcws) would not abandon their responsibilities during an influenza pandemic, [16, 17] but isolating one's self when one has symptoms or a diagnosis of disease is a different proposition than simply refusing to work. despite evidence of the efficacy of vaccinating hcws, [18] [19] [20] [21] [22] [23] influenza vaccination rates among hcws are low, [24] which presents a risk of hcw-to-hcw as well as hcw-to-patient transmission if infected hcws report to work. notably, as the 2003 sars outbreak subsided and precautions were relaxed, a second wave of the disease including 90 cases of nosocomial infections emerged; 42.5% of those nosocomial infections were associated with exposure to an infected hcw. seventeen nurses contracted sars, and 12 (70.6%) had worked with a symptomatic co-worker within 10 days of developing symptoms. indeed, having worked with a symptomatic co-worker was associated with increased risk (rr = 1.88) of an hcw developing the disease [25] . we are not aware of any previous reports measuring anticipated self-isolation among hcws with influenza. public health officials and health facility supervisors must impress upon health workers the clinical and ethical importance of protecting both patients and other staff from exposure to employeeborne influenza, including seasonal influenza [26] . this study was limited in that it relied upon a telephone survey to collect data, but telephone surveys have been previously used to gather information regarding public perceptions of risk and willingness to comply with containment strategies for influenza, [5, 7, [9] [10] [11] [12] and even to assess for the prevalence of influenza [27] . the response rate for this survey was 41.5%; while this may indicate some response bias the sample was fairly representative of the general population, and the overall survey was not specific to influenza. that is, there is no reason to suspect that any potential respondent's decision about whether to participate in the survey would be related to their anticipated compliance with a physician's advice to stay home. a more important limitation of the study is that it measured self-reported anticipated behaviour in the context of a physician diagnosis of disease. actual behaviour may differ, particularly since many individuals with mild to moderate viral respiratory syndromes do not seek physician care. also, other factors including perceived severity of illness, social norms, and financial considerations could affect compliance. thus, the rates of anticipated compliance reported by respondents to this survey must be viewed as a best-case scenario, and actual compliance might be lower. still the results, both in terms of anticipated compliance and associations with demographic factors, are consistent with those of other studies [5, 7, [9] [10] [11] [12] . finally, early in the australian pandemic (h1n1) 2009 experience there was a perceived association between international travel and increased risk, [28] but qss 2009 did not inquire as to respondents' individual travel history or exposure to international travellers. ninety-five percent of people report they would comply with a physicians' advice to stay home for seven days if they are diagnosed with pandemic (h1n1) 2009 or avian influenza, but only 71% can be expected to comply with the same advice in the setting of seasonal influenza and fewer still (60%) can be expected to stay home if they are diagnosed with a common cold. sub-populations that might be worthwhile targets for public health messages aimed at increasing the rate of self-imposed isolation for seasonal influenza include males and younger people. notably, more than one-quarter of health and community service workers report that they are unlikely to comply with stay home advice for seasonal influenza; thus they too may be an appropriate (although counter-intuitive) target for influenza-related public health campaigns. additional file 1: table s1 -bivariate associations between demographic variables and anticipated compliance with physician's advice to stay home for seven days for common cold and three strains of influenza. a group on influenza: pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico severe respiratory disease concurrent with the circulation of h1n1 influenza who announces pandemic alert phase 6, of moderate severity public support for government actions during a flu pandemic: lessons learned from a statewide survey legal rights during pandemics: federalism, rights and public health laws -a view from australia knowledge about pandemic influenza and compliance with containment measures among australians pandemic influenza in australia: using telephone surveys to measure perceptions of threat and willingness to comply public response to community mitigation measures for pandemic influenza influenza a(hin1)/swine flu survey iii what australians know and believe about bird flu: results of a population telephone survey factors influencing compliance with quarantine in toronto during the 2003 sars outbreak influenza-related disease: the cost to the australian healthcare system business preparedness: novel influenza a (h1n1) will they just pack up and leave?" -attitudes and intended behaviour of hospital health care workers during an influenza pandemic how would australian hospital staff react to an avian influenza admission, or an influenza pandemic? influenza vaccination of health care workers in long-termcare hospitals reduces the mortality of elderly patients influenza vaccination for healthcare workers who work with the elderly (review) the effects of influenza vaccination of health care workers in nursing homes: insights from a mathematical model modeling the effects of influenza vaccination of health care workers in hospital departments applying network theory to epidemics: control measures for mycoplasma pneumoniae outbreaks keeping vulnerable children safe from pertusis: preventing nosocomial pertusis transmission in the neonatal intensive care unit risk of respiratory infections in health care workers: lessons on infection control emerge from the sars outbreak phase 2) of severe acute respiratory syndrome (sars) in toronto, canada what happened? semmelweis revisited: the ethics of infection prevention among health care workers telephone survey to assess influenzalike illness swine flu and travellers: an australian perspective the authors declare that they have no competing interests. key: cord-341626-04svm6le authors: assink, m.d.m.; kiewiet, j.p.; rozenbaum, m.h.; van den berg, p.b.; hak, e.; buskens, e.j.; wilschut, j.c.; kroes, a.c.m.; postma, m.j. title: excess drug prescriptions during influenza and rsv seasons in the netherlands: potential implications for extended influenza vaccination date: 2009-02-11 journal: vaccine doi: 10.1016/j.vaccine.2008.11.070 sha: doc_id: 341626 cord_uid: 04svm6le influenza and respiratory syncytial virus (rsv) infections are responsible for considerable morbidity, mortality and health-care resource use. for the netherlands, we estimated age and risk-group specific numbers of antibiotics, otologicals and cardiovascular prescriptions per 10,000 person-years during periods with elevated activity of influenza or rsv, and compared these with peri-season rates. data were taken from the university of groningen in-house prescription database (www.iadb.nl) and virological surveillance for the period 1998–2006. during influenza and rsv periods excess antibiotic prescriptions were estimated for all age groups. in the age groups 0–1 and 2–4 years, excess antibiotic prescriptions during periods with elevated rsv activity (65% and 59% of peri-seasonal rates) exceeded the surpluses estimated during the influenza-activity periods (24% and 34% of peri-seasonal rates) while for otologicals excess prescriptions were higher for influenza (22% and 27%) than for rsv (14% and 17%). among persons of 50 years and older, notably those without medical high-risk conditions, excess prescriptions for cardiovascular medications were estimated during the influenza periods at approximately 10% (this was also already seen in persons aged 45–49). our results may have implications for influenza vaccination policies. in particular, extension of influenza vaccination to groups of non-elderly adults and young children may lower excess prescriptions during these influenza periods for all three types of drug prescriptions investigated. in many countries annual influenza vaccination has been recommended for the elderly and persons with high-risk medical conditions. in the netherlands until 2007, the age-threshold for such vaccination was 65 years for non-high-risk groups. in 2008, this threshold was lowered to include all persons aged 60 years and older [1] . in addition in the netherlands, all high-risk groups are vaccinated, including individuals suffering from chronic conditions, respiratory diseases, cardiac diseases, diabetes mellitus, renal failure, those being immunocompromised and individuals aged less than 18 years of age on chronic salicylates use [1] . vaccination rates among these high-risk groups under 65 years of age ranged from 66% to 83% in the netherlands in 2005 [2] . persons of 65 years and older with a medical indication showed higher vaccination coverages compared to those of the same age without such an indication, at 90% and 76% in 2005, respectively [2] . influenza may cause acute bronchitis and pneumonia in high-risk groups and elderly and may lead to exacerbations of underlying chronic medical conditions such as cardiovascular diseases, asthma and diabetes, potentially leading to hospitalisations and death. prevention of influenza infection by vaccination is of high importance for these groups with an increased risk for complications from influenza infection [3] [4] [5] . although some countries recommend routine influenza vaccination among children aged 6 months to 2 years, clinical data about the impact of vaccination are limited [1, 6] . yet, vaccination of all healthy children in this age group could be cost-effective or even cost-saving for some societal settings [6] . in the netherlands, universal influenza vaccination of such children is in debate, however yet the vaccine efficacy and effectiveness for this specific age group is not considered to be sufficiently demonstrated [7] [8] [9] . therefore, the dutch health council concluded in 2007 , not yet to start with influenza vaccination of these groups [1] . comparable conclusions were made for healthy children aged 2 years and older. although vaccination was shown to be effective for these children, influenza was considered not to cause serious morbidity or mortality in the netherlands in those groups [1] . next to influenza virus a and b, respiratory syncytial virus (rsv) has been shown to cause similar types of complications, including complications in the respiratory tract. rsv is often recognized as a cause of morbidity and mortality among both children and adults, contributing to a major burden of illness [10] [11] [12] [13] [14] [15] [16] [17] . no preventive vaccine is yet marketed for rsv. in general, it is difficult to estimate the individual contributions of influenza and rsv to the aforementioned disease burdens accurately as influenza and rsv co-circulate during winter seasons. for burden of disease often the label influenza like illnesses (ili) is considered to primarily comprise disease due to influenza infections, however known to potentially comprise various other agents inclusive rsv, with the identification of the relative contributions of both viruses being strongly hampered [18] [19] [20] [21] . in particular, even further viral agents, such as adeno-, parainfluenza-, and corona-viruses may contribute to the burden of ili [21, 22] . for exactly analysing the burdens of disease of influenza and rsv separately, which is the goal of our current paper, ili is therefore an inappropriate concept. therefore, we chose to analyse isolates exactly related to the respective causal agents. given the potential complications of influenza and rsv, one may expect excess health-care resource utilization during epidemics [23] . studies estimating such excess resource utilization have previously been directed to hospital admissions and gp-visits, but there is as yet hardly any information on the association between the occurrence of influenza and rsv and the use of medications among the general population [24] . drug-use for specific complications of influenza and rsv could certainly temporarily be elevated. in particular, prescriptions for antibiotics, otologicals and cardiovascular medication may be elevated, dispensed for otitis media, cardiac complications, respiratory illness and other pulmonary complications. to address this topic, we investigated the association between weekly reported rsv and influenza isolates in the netherlands and the weekly number of prescription drugs dispensed by dutch pharmacies, in particular those for antibiotics, otologicals and cardiovascular drugs. from the preventive point of view, high influenza-associated drug-use may justify more extended use of preventive measures, such as influenza vaccination. in particular, it may enhance the health-economic profile of extended influenza vaccination for currently yet unvaccinated groups. as such, our research may contribute to discussions as to whether, for example, young children should be vaccinated or whether the age limit of the vaccination program should be lowered further. during 1998-2006, the dutch working group on clinical virology gathered data from 20 laboratories throughout the netherlands, testing clinical specimens for respiratory viruses, including rsv and influenza a and b. the number of patients who tested positive for rsv or influenza was reported on a weekly basis. in the absence of any drastic changes in recruiting and testing of specimens the weekly time series can validly be conceived as representing the actual time trend, without requiring any corrections to be applied on the data. to exclude weekly random fluctuations a 3-week moving average was used for presentation ( fig. 1) and for defining the specific weeks exhibiting elevated activity for influenza and rsv. two methods for defining weeks with elevated activity of influenza and rsv were applied. the first method defined a week with elevated activity as one with a moving average of more than two times the gross overall average weekly number from week 40 in 1998 onwards to week 39 of 2006 (week 1 obviously being the 1st week in january; week 40 generally being considered as the start of the influenza season). following jansen et al., the second method defined the weeks with elevated activity as those weeks from any period of at least 2 consecutive weeks, with each individual week accounting for over 5% of the season's total number of influenzaor rsv-positive specimens [18] . from both methods, it appeared that periods consisting of a number of subsequent weeks resulted, rather than individual weeks or short periods of, for example, 2 weeks only. such periods would be expected -and were indeed found -to be between week 40 and week 20 in the next year (the non-summer season) [18, 25] . the weeks that contained both influenza and rsv activity were excluded for defining the periods with elevated activity, given the difficulty to separate out the individual influences in these weeks with combined activity. as a result, only those weeks with either elevated influenza or rsv were analyzed. furthermore, a periseason was defined, containing the weeks from week 40 up to and including week 20 of the next year, which did not belong to the influenza or the rsv periods (seasons). the weeks from week 21 up to and including week 39 were labelled "summer". information on drug-use in the population was provided by a university of groningen in-house prescription database (www.iadb.nl). the database iadb.nl contains prescription, demographic and population data of 500,000 persons adherent to 50 pharmacies in the north and east of the netherlands. for analytical purposes, the prescriptions were divided over 5-year age categories. the age category 0-4 years was further divided in the ages 0-1 and 2-4, to enable analysis of infants separately. for presentation the following categorization was applied: 0-1, 2-4, 5-19, 20-49, 50-54, 55-59, 60-64 and 65+. the exact age of any person was determined every year on the 1st of october, close to the period in which in the netherlands the invitations for influenza vaccination for risk groups are sent out by the gps, supposedly just prior to the season with increased risk for influenza epidemics from october onwards to may. the annual total population sizes were based on estimates for the 1st of january by the local authorities in the places where the pharmacies are located. persons who received one of the studied drugs were divided in high-risk and low-risk groups for influenza. the respective populations belonging to both groups were estimated using prescriptions as a proxy, for those medications that are consistent with highrisk indications as specified by the dutch health council [1] and the dutch gps (http://nhg.artsennet.nl) and that are uniquely prescribed for these indications [26] . for example, dornase alfa, a drug prescribed for cystic fibrosis, was included to define persons with respiratory diseases. other medications for cystic fibrosis, such as acetylcysteine, are also prescribed for cough and were therefore not included. table 1 lists these drugs specifically. for heart medication, diuretic sulfonamides were included. other diuretics used for lowering blood pressure were excluded, as high-blood pressure is not a high-risk identified condition. furthermore, only calcium antagonists with atc-code c08d were included as only these are assumed to have cardiac effects. beta blockers and raas system agents were excluded as these are not exclusively prescribed for cardiac diseases, but also, for example, for high-blood pressure only. for renal diseases, medication for the treatment of hyperkalemia and hyperphosphatemia, and antianemic preparations and sulfonamides were included as these are often prescribed for dialysis or renal insufficiency. to identify immunocompromised patients, immunosuppressives were included, which can be prescribed, for example, for patients after organ transplantation. also hiv medications were included as also hiv-patients are listed as high-risk group. as mentioned, in the netherlands, influenza vaccination is recommended to those at the increased risk of complications based on specific medical conditions. to determine which specific patients should be labelled as belonging to the high-risk group, the recommendations of the dutch health council [1] and the guidelines of the dutch gps (http://nhg.artsennet.nl, accessed 24th september 2007) for influenza and high-risk indications were used [26] . in particular, persons were labelled belonging to the high-risk group if they had two or more prescriptions from the same group of medications included on the list, on two different dates in the year before the first of october. the latter was supposed to guarantee that the condition would be chronic in that specific year (note that a person's risk status may change from year to year). the population size of the high-risk group was subsequently determined on the 1st of october by counting the number of high-risk persons in iadb.nl; the rest of the population was assumed at low risk. we specifically directed our analysis to the drug groups of antibiotics, otologicals and cardiovascular drugs, as these drugs may be considered for those complications of both viral infections that have yet been published in the scientific literature [23, 24, [27] [28] [29] . from iadb.nl, prescriptions of antibiotics (atc-code j01), otologicals (atc-code s02) and medications for the cardiovascular system (atc-code c) were selected. antibiotics are commonly prescribed for the treatment of acute otitis media (aom), in particular for young children in which aom accounts for approximately half of all antibiotics courses delivered [30] [31] [32] . otologicals may also be prescribed for aom, despite that they are not recommended by the dutch college of general practitioners (nhg) [29] . in addition, more than average numbers of antibiotics may be prescribed for elderly persons with ili during periods with elevated activity, as particularly this group may develop acute respiratory illnesses (for example, pneumonia) as a complication of the viral infection (trimethoprim and nitrofurantoin were excluded from the analysis as they are prescribed primarily for urinary tract infections) [33] . for antibiotics and otologicals both initial and next prescriptions within the same year were considered. it is well known that influenza-related complications are more prevalent among persons with cardiovascular and other chronic diseases than in persons without such underlying conditions [4, 34] . in persons of 65 years and older with high-risk conditions an increased rate of hospitalisation for cardiac problems has been reported [17] . some further recent studies suggest that there might also be an association between cardiovascular problems and influenza epidemics among groups without any cardiovascular history yet, such as the elderly or even among those aged below 65 years of age [23, 35] . for analysing cardiovascular medications, only first prescriptions were considered as our current interest was to investigate whether the viral infections were related to new cardiovascular disease and/or exacerbations of existing -yet untreatedbackground cardiovascular conditions, rather than identify chronic medications for cardiovascular diseases (note that chronic cardiovascular medication use was used as a criterion for assigning persons to the high-risk group). a prescription was defined as a first prescription when a person had not had a prescription for the same drug or a drug from the same subgroup in the year before that specific prescription. after the various periods of elevated activity were determined, the number of prescriptions in the weeks belonging to the influenza and rsv periods/seasons were compared to the number of prescriptions in the peri-season. for comparative purposes also results for comparing with the summer season are presented. this was done in every age group for the total population, the high-risk group and the low-risk group. incidence rates were calculated and corresponding 95%-confidence intervals were estimated [36] . the incidence rate (i) was calculated as the weekly number of prescriptions per 10,000 person-years: with p i being the number of prescriptions in week i of a season (influenza, rsv, peri or summer), n the number of weeks of that specific period of interest and n the population size (per age group, for high risk or for low risk). the division by 52 is to transfer personweek estimates into person-years (for 2 years 53 instead of 52 had to be used). microsoft office excel 2003 was used for processing the data, calculations and graphics. during the study period, the average weekly number of influenza-positive and rsv-positive specimens were 11.77 and 36.90, respectively. the first method for defining the periods of elevated activity using two times the average number of isolates per week as a lower limit (23.54 for influenza and 73.80 for rsv) resulted in table 2 , showing the weeks which were labelled as belonging to the influenza, rsv, peri-and summer periods. the second method, defining the periods with elevated activity as at least two consecutive weeks in which each week accounted for over 5% of the season's total number of rsv-or influenza-positive specimens yielded comparable results (data not shown). hereafter, only for those results where both methods differed, the results of both methods are presented otherwise the results of only the first method are shown. obviously, periods with elevated activity and the peri-season changed from year to year, both in length (the summer season of course always ranged from week 21 up to and including week 39 of the next year). in some years overlap in influenza and rsv activity weeks were seen, in which case weeks were excluded. table 3 person-years during periods of elevated activity per age group for the total population, high-risk group and low-risk group. table 4 excess drug prescriptions for the periods with elevated activity compared to the peri-season, shown as numbers of prescriptions per 10,000 person-years (as % of peri-seasonal levels). the number of persons was insufficient for valid estimation. a result not statistically significant. b although borderline significant not shown here for the whole age-group as further 5-year age-group specific analyses revealed that significance was related only to a significant and clinically relevant surplus for the age category 45-49: 113.12 (12.95%) and 122.78 (17.06%) for the total population and low-risk group, respectively. table 3 presents the number of person-years for the influenza, rsv, peri-season and summer periods per age and risk group. for infants and children aged 2-4, the high-risk group was very small with 393 person-years in the influenza period and 454 personyears for the rsv period, and therefore only the figures for both low-and high-risk groups taken together were used for these ages. furthermore, in all age groups the number of person-years in the high-risk group was lower than the numbers in the low-risk group. we also note, as expected, that the older age groups contained relatively more person-years in the high-risk group than the younger age groups. fig. 1 shows the number of positive findings for rsv and influenza per week. the annual influenza and rsv epidemics are clearly seen, as is the overlap in some years. also the number of (first) prescriptions per 10,000 persons for antibiotics, otologicals and cardiovascular medication is plotted for the total population. in particular, for antibiotics a clear pattern is visible in which during influenza and rsv activity periods a peak in antibiotic use occurs. additionally, we notice that in the younger age groups amoxicillin was the mostly prescribed antibiotic, whereas from the age group 5-19 onwards other antibiotics were mostly prescribed (data not shown). the excess prescriptions per 10,000 person-years during the influenza and rsv periods are presented in table 4 , as compared to the peri-season. we noted that both methods used for estimating the activity periods showed similar results. the number of prescriptions for antibiotics was significantly elevated during the activity periods in each age group. for the age groups 0-1 and 2-4, excess prescriptions were highest in the rsv periods, in the older age groups surpluses were higher during the influenza periods. for influenza, the low-risk group showed higher excess prescriptions compared to the high-risk group. prescription of otologicals was significantly elevated during the periods of elevated activity, however only in the youngest age groups of infants and children aged 2-4 years. in contrast to antibiotics, excess prescriptions were higher during influenza periods than during rsv periods. for first cardiovascular medications, the prescription rate in the rsv periods was lower than in the peri-season, although not significant. during the influenza period, higher prescription rates were found for those aged 50 years and older. when the age group of 20-49 was analyzed in 5-year age categories separately, also a significant difference for the age category 45-49 was seen. fig. 2 shows the prescription rates per 10,000 person-years and confidence intervals for selected aggregated age groups for the different periods per year separately, as well as for the aggregated years. the incidence rates of antibiotics were increased during the influenza and the rsv periods in comparison with the peri-season (and the summer season). this increase was noticeable for every year. for otologicals, the incidence rate was increased but the confidence intervals were wide and therefore not every year rendered a statistically significant difference during the influenza and rsv periods. for first cardiovascular prescriptions also an increase was noticeable during influenza periods as compared to the peri-season, but again not for every year a significant difference was found. finally, we note from fig. 1 that elevations in the influenza and rsv periods are not necessarily followed by relatively lower levels in the peri-and/or summer seasons, suggesting that surpluses detected are actual extra prescriptions that are not "neutralized" by subsequent dips in prescriptions. statistically significant excess antibiotic prescriptions during periods with elevated activity of influenza and rsv were found for both viruses in all age groups, each year investigated and irrespective of the method used for exactly defining the influenza or rsv activity periods. for otologicals during both influenza and rsv-active periods, statistically significant surpluses were found in young children only. oppositely, excess cardiovascular drug prescriptions were identified in adults and elderly in periods with elevated influenza activity. we generally found a tendency for higher percentages of surpluses in low-risk groups than in high-risk groups. for antibiotics and cardiovascular drugs, this can probably be explained by the fact that individuals belonging to a high-risk group have a higher likelihood of being vaccinated against influenza, lowering the chance of infection and secondary bacterial or cardiovascular complications. this tendency also applied to low-risk elderly as compared to highrisk elderly, despite the fact that this whole group is recommended for vaccination. some choices in our research should be noted. specifically, the lower limit for the weekly moving-averaged number of isolates for weeks to be labelled as active was chosen by two different methods. the first used a cut-off of two times the average number of influenza or rsv isolates per week. this limit was chosen to achieve continuous periods per year of weeks subsequently labelled as active, i.e. to guarantee epidemic periods rather than fluctuations. the second method was used to be in concordance with a previously performed dutch study using the weeks which accounted for 5% or more of the season's total number of influenza or rsv isolates [18] . in general, both methods resulted in similar results. by using the influenza and rsv activity periods that excluded weeks of combined activity, possible major influence by the presence of the respective other virus was reduced. however, the exclusion of weeks in which both influenza and rs viruses were active was only necessary in 4 out of the years included in this analysis. also, in methodology excluded weeks were not included in counting person-years, so the effect of excluding those weeks maybe limited. yet, possible influence of any other respiratory virus obviously remains present. however, the impact of these viruses is probably limited, as they may have long periods of marginally increased activity rather than a clear seasonal pattern [18] . furthermore, complications are expected to be milder compared to influenza and rsv infection [18] . we compared our data for the activity periods with the periseason, which provided a more conservative estimation of the surpluses than if we would have compared with the summer period. yet, some underestimation of the surpluses may be introduced in this way. we do feel, however, that comparison with the peri-season is most appropriate, as other potential influences concerning the climate and possible other viruses that circulate in non-summer periods are probably comparable between the peri-season and the activity periods. every year, rsv-positive specimens reached a relatively small and intensive peak around week 52. earlier studies have shown similar tendencies, with rsv isolates peaking every year around the same time [14, 15, 37] . this suggests the presence of potential common strict seasonal factors which might increase both the number of isolates and prescriptions; however probably not invalidating the associations and surpluses found in our analyses, which are truly seen and are in line with other studies [14, 15, 37] . persons were labelled as belonging to the high-risk group based on specific medication profiles. in particular, persons belonging to the high-risk group were selected based on prescriptions that corresponded rather uniquely to the high-risk indications. prescriptions potentially meant for other indications, not labelled as high risk, were consistently excluded. still, it is possible that these selection criteria unjustly labelled individuals as belonging to the high-risk groups. also, some individuals may have been incorrectly excluded and labelled as non-high-risk. finally, one may hypothesize that excess prescriptions are merely shifts in time of extra prescriptions later to be outweighed by dips in prescriptions (sometimes referred to as "the harvesting effect"). visual inspection of our data however did not give any reason to support this hypothesis in our study. additionally, a formal statistical comparison of the number of prescriptions during the peri-seasons and during the first 5 weeks after the active seasons did not show any peak-dip pattern (data not shown). previously, various investigations have been performed on the association between influenza and rsv epidemics, on the one hand, and hospitalisations, mortality and outpatient visits, on the other [14, 17, 18, 24, 33, [37] [38] [39] [40] [41] [42] [43] [44] . below, we briefly compare the outcomes, knowing that the validity of making such comparisons between studies is limited due to differences in outcome measurement, statistical models, study period, and health-care system concerned. previously, only one study investigated excess antibiotic use during influenza-activity periods in the general population [24] . this study did show that otherwise healthy children get more prescriptions for antibiotics during these influenza periods. however, the surplus reported was relatively low compared to our findings [24] . two other studies estimated the excess antibiotic prescriptions during both rsv and influenza-active seasons, focussing on specific target groups [33, 43] . the first study focussed on patients suffering from chronic lung disease, showing the highest surpluses for the youngest age groups [43] . the latter study showed higher surpluses due to influenza as compared to rsv for those living in nursing homes [33] . both results are in line with our findings for antibiotics [33, 43] . several other studies indicate that, in general, both during rsv and influenza-activity periods, infants and elderly show the highest morbidity and mortality rates [14, 18, 24, 37, [39] [40] [41] [42] [43] [44] . this is certainly in line with our findings on excess antibiotic and otological prescriptions among the youngest age groups and cardiovascular medication surpluses among the older age groups. in contrast to other studies which showed higher morbidity and mortality among the oldest age groups compared with non-elderly adults, our study shows that the elevation in the prescriptions of antibiotics in the oldest age groups (65 years of age and older) is not higher than in the two younger age groups (55-59 and 60-64). yet, if compared with other adult age groups, a small increase could be seen. for influenza, this slightly deviating finding compared to non-dutch settings, might be explained by the high vaccination coverage among elderly in the netherlands. a recent dutch study showed that rsv-related excess hospitalisations were considerably higher as compared to those due to influenza [18] . comparably, a study performed for england and wales showed greater excess rates for complications during rsvactive periods among the youngest age groups as compared to influenza; similar rates were found for all other age groups [37] . five other studies focussing on excess morbidity, mortality and hospitalisation among children confirmed these results, showing that rsv was responsible for higher hospitalisation rates than influenza [14, [39] [40] [41] [42] . for the netherlands, jansen et al. recently showed excess hospitalisation for cardiovascular complications among the 50-64 years old, during influenza-active periods but not during rsv-active periods [18] . in line with their findings, we showed a significant surplus in first cardiovascular medication prescriptions during influenzaactive periods, but not during rsv-active periods for those aged 45 years and over. elevated hospitalisation and prescription rates during influenza periods in persons aged around 50 years and beyond suggests that influenza may cause cardiovascular diseases or that it may aggravate existing non-diagnosed cardiovascular diseases in older adults. another hypothesis explaining this increase might be that increased cardiovascular problems during influenza periods are related to the increased use of analgesics during those periods to alleviate influenza symptoms [45] . further research is definitely needed into this topic. thus, in general, our results seem to be comparable with most other studies relating elevated viral activity to the use of health-care resources, morbidity and mortality. all studies consistently show that the highest excess rates for the youngest age groups are mostly due to rsv, whereas those for influenza are seen in elderly. vaccination may prevent part of the excess prescriptions we have found. for example, healthy children are not recommended to be vaccinated against influenza in the netherlands, while their vaccination might prevent part of the surplus prescriptions found for this group. in particular, vaccination may prevent influenza infection and potential subsequent bacterial super infection(s) and thus avert antibiotics prescribed for the prevention and treatment of such bacterial super infections. additionally, reducing the prescription of antibiotics may also be important from the perspective of limiting the development of antibiotic resistance. such reasoning could be an additional motivation for vaccinating yet uncovered groups against influenza [23] . an effective vaccine against rsv may potentially even prevent more antibiotic prescriptions, especially in young children [37] . yet the introduction of a vaccine again rsv is not expected in the very near future [18] . during influenza-and rsv-active periods, elevations in antibiotic prescriptions were identified in all age groups. for otologicals, such an elevation was shown in the age groups of 0-1 and 2-4 years, both during influenza-and rsv-active seasons. by vaccinating young children against influenza, a part of these prescriptions for antibiotics and otologicals may be prevented. in persons of 50 years and older an elevation of prescriptions for cardiovascular medication was shown during the period of elevated influenza activity only, in particular for the low-risk population. also for antibiotic prescriptions, the excess found was higher in the low-risk population than in the high-risk population, possibly indicating the effectiveness of the vaccination program in the highrisk group, in which a relatively high coverage rate is reached in the netherlands. influenza vaccination: revision of the indication, (griepvaccinatie: herziening van de indicatiestelling the impact of influenza on the health and health care utilisation of elderly people influenza vaccination as secondary prevention for cardiovascular disease: a science advisory from the american heart association/american college of cardiology rapid reference influenza technical report of the scientific panel on vaccines and immunisation. infant and children seasonal immunisation against influenza on a routine basis during inter-pandemic period efficacy and effectiveness of influenza vaccines in elderly people: a systematic review influenza vaccines in healthy children influenza vaccines in healthy children immunopathology of rsv infection: prospects for developing vaccines without this complication the use of health economics to guide drug development decisions: determining optimal values for an rsv-vaccine in a model-based scenario-analytic approach hospitalization for respiratory syncytial virus infection in young children: development of a clinical prediction rule anticipated costs of hospitalization for respiratory syncytial virus infection in young children at risk mortality in children from influenza and respiratory syncytial virus respiratory syncytial virus or influenza? respiratory syncytial virus infection in elderly adults influenza-and rsv-associated hospitalizations among adults influenza-and respiratory syncytial virus-associated mortality and hospitalisations respiratory illness associated with influenza and respiratory syncytial virus infection the causes and diagnosis of influenza-like illness incidence of influenza and associated illness in children aged 0-19 years: a systematic review respiratory viruses and influenza-like illness: a survey in the area of rome clinical effectiveness of influenza vaccination in persons younger than 65 years with high-risk medical conditions: the prisma study the effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children the duration and magnitude of influenza epidemics: a study of surveillance data from sentinel general practices in england, wales and the netherlands effectiveness of inactivated influenza vaccine in preventing acute otitis media in young children: a randomized controlled trial influenza vaccination in the prevention of acute otitis media in children nhg-standaard otitis media acuta bij kinderen (m09) prevalence of various respiratory viruses in the middle ear during acute otitis media role of respiratory viruses in children with acute otitis media primary care management of respiratory tract infections in dutch preschool children influenza-and respiratory syncytial virus-associated morbidity and mortality in the nursing home population association of influenza vaccination and reduced risk of recurrent myocardial infarction influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly epidemiological studies: a practical guide morbidity profiles of patients consulting during influenza and respiratory syncytial virus active periods influenza and respiratory syncytial virus morbidity among 0-19 aged group in yunus emre health center mortality associated with influenza and respiratory syncytial virus in the united states contribution of respiratory syncytial virus, influenza and parainfluenza viruses to acute respiratory infections in hospitalization attributable to influenza and other viral respiratory illnesses in canadian children population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease the annual impact of seasonal influenza in the us: measuring disease burden and costs understanding the nsaid related risk of vascular events mark rozenbaum was supported by an unrestricted educational grant of sanofi pasteur msd (hoofddorp, netherlands). key: cord-324181-nyrpg3ud authors: baker, jeffrey; block, stanley l.; matharu, balpreet; burleigh macutkiewicz, laura; wildum, steffen; dimonaco, sophie; collinson, neil; clinch, barry; piedra, pedro a. title: baloxavir marboxil single-dose treatment in influenza-infected children: a randomized, double-blind, active controlled phase 3 safety and efficacy trial (ministone-2) date: 2020-05-19 journal: pediatr infect dis j doi: 10.1097/inf.0000000000002747 sha: doc_id: 324181 cord_uid: nyrpg3ud baloxavir marboxil (baloxavir) is a novel, cap-dependent endonuclease inhibitor that has previously demonstrated efficacy in the treatment of influenza in adults and adolescents. we assessed the safety and efficacy of baloxavir in otherwise healthy children with acute influenza. methods: ministone-2 (clinicaltrials.gov: nct03629184) was a double-blind, randomized, active controlled trial enrolling children 1–<12 years old with a clinical diagnosis of influenza. children were randomized 2:1 to receive either a single dose of oral baloxavir or oral oseltamivir twice daily for 5 days. the primary endpoint was incidence, severity and timing of adverse events (aes); efficacy was a secondary endpoint. results: in total, 173 children were randomized and dosed, 115 to the baloxavir group and 58 to the oseltamivir group. characteristics of participants were similar between treatment groups. overall, 122 aes were reported in 84 (48.6%) children. incidence of aes was similar between baloxavir and oseltamivir groups (46.1% vs. 53.4%, respectively). the most common aes were gastrointestinal (vomiting/diarrhea) in both groups [baloxavir: 12 children (10.4%); oseltamivir: 10 children (17.2%)]. no deaths, serious aes or hospitalizations were reported. median time (95% confidence interval) to alleviation of signs and symptoms of influenza was similar between groups: 138.1 (116.6–163.2) hours with baloxavir versus 150.0 (115.0–165.7) hours with oseltamivir. conclusions: oral baloxavir is well tolerated and effective at alleviating symptoms in otherwise healthy children with acute influenza. baloxavir provides a new therapeutic option with a simple oral dosing regimen. a nnual influenza epidemics are estimated to cause 3-5 million cases of severe illness and up to 650,000 deaths globally among all ages. 1 influenza infects all childhood age subsets and is associated with substantial morbidity, 2 with the youngest at particular risk of viral injury, secondary bacterial infections and complications. 3, 4 mortality in children varies across seasons and depends on viral subtype, preexisting immunity and presence of underlying disease. 5, 6 recent estimates for children from 92 countries, the majority of whom were <5 years old, are 9000-106,000 (median: 44,888) influenza-associated deaths annually. 7 in addition, children play a central role in influenza dissemination in the community because of their relative susceptibility to infection, high illness attack rates, prolonged viral shedding, and high contact rates between others in the household and community. 2, 8 annual vaccination is the most effective control measure for prevention of seasonal influenza and related complications. 9, 10 vaccination is recommended in the united states for individuals ≥6 months of age, who have no contraindications. 11 however, the need for vaccine reformulations each year, difficulties in producing these within short timeframes, and variable uptake and efficacy across countries mean that this strategy has limitations, 12, 13 and needs to be complemented by the availability of effective antiviral treatments. in many countries, there are currently only 2 classes of antivirals approved for the treatment of influenza in children: m2 blockers and neuraminidase inhibitors. 3 widespread, stable and transmissible resistance has rendered m2 blockers essentially ineffective. 14, 15 although neuraminidase inhibitors are effective in the treatment of influenza in children, there are restrictions for some of these based on age and mode of administration. a simplified dosing regimen (ie, single oral dosing), with better overall antiviral activity and favorable safety and tolerability, is considered desirable for the treatment of influenza in children. baloxavir marboxil (baloxavir) is a novel, first-in-class, capdependent endonuclease inhibitor. [16] [17] [18] baloxavir was first approved in japan in february 2018, followed by the united states in october 2018. in the united states, the initial indication included singledose, oral treatment of acute uncomplicated influenza in patients ≥12 years old, who have been symptomatic for ≤48 hours, which was expanded in october 2019 to include those at high risk of developing influenza-related complications. 17 these approvals were based on the clinical efficacy and safety of baloxavir versus placebo and oseltamivir in 2 pivotal phase iii trials (capstone 1 and capstone 2, respectively). 19, 20 in these studies, baloxavir showed significant improvements in time to alleviation of influenza symptoms (capstone 1) and time to improvement of influenza symptoms (capstone 2) compared with placebo, and faster reduction in infectious viral titers compared with placebo and oseltamivir in adults and adolescents. 19, 20 we report the safety and efficacy results of single oral dose baloxavir treatment in otherwise healthy children 1-<12 years old with acute influenza from ministone-2 (clinicaltrials.gov identifier: nct03629184), a phase iii, randomized, active controlled trial. this was a global, multicenter, double-blind, randomized, active controlled trial of the safety, pharmacokinetics and efficacy of a single oral dose of baloxavir versus twice-daily (for 5 days) oral oseltamivir, in otherwise healthy children with influenza. the trial enrolled children 1-<12 years of age with influenza (who were otherwise healthy) during the 2018/2019 northern hemisphere influenza season, including sites in the united states, south america and europe. enrolled children had a clinical diagnosis of influenza infection consisting of fever (tympanic temperature of ≥38°c) at screening and at least one respiratory symptom (either cough or nasal congestion). the time interval allowed between the onset of symptoms and screening was ≤48 hours. the trial excluded children with severe influenza symptoms requiring inpatient treatment and those with concurrent infections requiring systemic antiviral therapy at screening. acetaminophen was permitted for severe symptoms. children were considered "otherwise healthy" if they met none of the following exclusion criteria: any immunosuppressive disorder (including human immunodeficiency virus infection), uncontrolled renal, vascular, neurologic, or metabolic disease, hepatitis, cirrhosis, or pulmonary disease, known chronic renal failure, active cancer at any site, or a history of organ transplantation. children were enrolled in parallel to 2 cohorts: 1-<5 years old (minimum of 20 children) and 5-<12 years old (minimum of 40 children). using a permuted block randomization method, children were assigned in a 2:1 ratio to receive a single dose of oral baloxavir on day 1 (2 mg/kg for those weighing <20 kg and a single dose of 40 mg for those weighing ≥20 kg), or oral oseltamivir twice daily according to prescribing information (30 mg for patients weighing ≤15 kg, 45 mg for >15-≤23 kg, 60 mg for >23-≤40 kg, and 75 mg for >40 kg) on days 1-5. 21 doses of baloxavir in this study were chosen based on population pharmacokinetic analyses performed using data from a phase 3 study of children in japan (japiccti-173811), and evaluated with respect to their ability to match adult drug exposure. children in both groups received a 5-day regimen (baloxavir and a matching oseltamivir placebo or oseltamivir and matching baloxavir placebo). following randomization, the first dose of the trial regimen was administered under direct observation, and participants were followed until day 29. the primary endpoint was safety, defined as the incidence, severity and timing of adverse events (aes) during the 5-day treatment period and a 24-day follow-up period. vital signs (blood pressure, respiratory rate, heart rate and tympanic temperature) were measured at scheduled visits [days 1, 2, 4, 6, 10, 15 and 29 (if there were abnormal findings or aes since the previous measurement)]. blood was collected for hematology and chemistry assessment on day 1 and day 6, and nasopharyngeal swabs were performed for viral quantification (using infectious titer) on days 1, 2, 4, 6, 10, as well as days 15 and 29 if considered appropriate by the treating physician. parents completed the canadian acute respiratory illness and flu scale (carifs) 22 questionnaire at scheduled visits (day [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] , and responses were used to measure secondary efficacy endpoints including time to alleviation of signs and symptoms (ttass) of influenza [defined as when a score of 0 (no problem) or 1 (minor problem) was reported for cough and nasal symptoms on the carifs questionnaire, return to normal health and activity, and return to afebrile state (tympanic temperature ≤37.2°c), remaining for at least 21.5 hours]. subgroup analyses of ttass based on virus subtype were also performed. other secondary efficacy endpoints were duration of fever [defined as time to return to afebrile state (≤37.2°c)], duration of all symptoms, time to return to normal health and activity, frequency of influenza-related complications (all of which were predefined and required investigator confirmation: death, hospitalization, pneumonia, bronchitis, sinusitis, otitis media, encephalitis/ encephalopathy, febrile seizures or myositis) and proportion of children requiring antibiotics. secondary virologic endpoints included time to cessation of viral shedding by virus titer and change from baseline in influenza virus titer. 23 exploratory virologic endpoints included frequency of treatment-emergent amino acid substitutions (using sanger sequencing). 24 baseline samples from both treatment groups were also tested for coinfections (viral and bacterial pathogens), using the biofire filmarray respiratory panel 2 assay. 25 a sample size of 80 children in the baloxavir arm (120 in total) provided a probability of ≥90% that ≥1 child would experience an ae with a background incidence rate of 3%. a sample size of 120 children provided a probability of 80.1% that ≥1 child would experience an ae with an incidence rate of 2%. because the study was not powered for a statistical comparison between treatments, results are descriptive. the safety population comprised children who received any portion of a single dose. the intent-to-treat influenza-infected (itti) population was used for all efficacy analyses and comprised children who had a laboratory reverse transcriptase-polymerase chain reaction confirmation of influenza infection from any swab sample collected at baseline or during the study. the number of children in the itti population was continuously monitored to ensure an adequate number of influenza-infected children were recruited. data were summarized using descriptive statistics and kaplan-meier plots where applicable (sas version 9.4). this study was conducted in accordance with the declaration of helsinki and good clinical practice (ich e6) guidelines, and was approved by all relevant institutional review boards and/ or ethic committees at each center. all parents/caregivers of participants gave written informed consent and child consent was obtained where applicable. this study was performed between november 20, 2018 and august 27, 2019 at 36 sites across 6 countries (usa, poland, spain, costa rica, mexico and russia). of the 176 children enrolled, 117 (66.5%) and 59 (33.5%) patients were randomized into the baloxavir and oseltamivir groups, respectively. in total, 169 children (96%) completed the trial (fig. 1) . the predominant influenza a subtype at baseline assessed by reverse transcriptase-polymerase chain reaction was h3n2 for both groups, followed by h1n1pdm09. numbers were low for influenza b in both groups (n = 5 for baloxavir and n = 2 for oseltamivir). in the safety population, 49.1% of children were vaccinated against flu, with similar proportions vaccinated in each treatment group. demographics and baseline characteristics were similar between treatment groups (table 1 ). there were no notable differences between the treatment groups in the use of concomitant medications during the study; the most frequently reported class in both was analgesics, most commonly acetaminophen. other concomitant medications were predominantly symptomatic treatments, and were reported in very few children. a total of 122 aes was reported in 84 children (48.6%) during the study (between day 1 and 29) and most aes resolved or were resolving by study end (95.1%). the overall incidence of aes was similar between the baloxavir group (46.1%) and the oseltamivir group (53.4%; table 2 ). the incidence of aes considered related to study drug was low in both groups, 2.6% (3/115) for baloxavir compared with 8.6% (5/58) for oseltamivir. the most common aes in both groups were gastrointestinal disorders (vomiting or diarrhea), experienced by 12 children (10.4%) for baloxavir and 10 (17.2%) for oseltamivir. no deaths, serious aes or hospitalizations were reported during the study. two children experienced aes which led to withdrawal from treatment in the baloxavir group, including accidental overdose of oseltamivir placebo and grade 2 rash occurring on day 4 that resolved after 24 hours without treatment in a child who received an accidental under-dose of baloxavir (4 mg instead of 40 mg). no aes led to withdrawal in the oseltamivir group. all aes observed in the study were grade 1 or 2, except for 3 grade 3 aes. these were abdominal pain on day 8 in the baloxavir group, and severe vitamin d deficiency at baseline (undiagnosed preexisting conditions, determined by raised alkaline phosphatase levels on day 1) in 2 children in the oseltamivir group. there were no clinically meaningful changes from baseline in any laboratory parameters or vital signs. the ttass based on the carifs questionnaire was similar between treatment groups, with a median of 138.1 [95% confidence interval (ci): 116.6-163.2] hours for baloxavir and 150.0 (95% ci: 115.0-165.7) hours for oseltamivir ( fig. 2 and table 3 ). for children infected with h3n2, median ttass was similar between the baloxavir and oseltamivir groups, while for those infected with h1n1pdm09, ttass was numerically lower for baloxavir than oseltamivir; table 3 ). the number of children with influenza b was too low to allow meaningful interpretation of the data. the median duration of fever was similar between the baloxavir and oseltamivir groups: 41.2 (95% ci: 24.5-45.7) versus 46.8 (30.0-53.5) hours, respectively (table 3) the overall incidence of influenza-related complications meeting predefined diagnostic criteria was low overall: 6 (7.4%) children in the baloxavir group and 3 (7.0%) in the oseltamivir group. the most frequent complication was otitis media in both groups [3 (2.6%) for baloxavir, 4 (6.9%) for oseltamivir], with single cases of bronchitis, pneumonia and sinusitis (baloxavir) and febrile seizure (oseltamivir). the proportion of children requiring antibiotics was low in both groups: 2 children in each group received antibiotics for otitis media and 2 children in the baloxavir group received antibiotics for pneumonia and sinusitis. as previously observed in phase ii and phase iii studies, 19, 20 baloxavir was associated with a more rapid decline in infectious viral titer compared with oseltamivir. the mean reduction from baseline in influenza virus titer on day 2 (24 hours posttreatment) was considerably greater for baloxavir than oseltamivir [−3.59 (standard deviation = 1.34) vs. −1.79 (1.54) log 10 median tissue culture infectious dose/ml, respectively]. thereafter, the mean change from baseline was similar in the 2 treatment groups, plateauing on day 2 for baloxavir and from day 4 for oseltamivir. the median time to cessation of viral shedding by virus titer was shorter for baloxavir than oseltamivir by 51.6 hours [24.2 hours (95% ci: 23.5-24.6) vs. 75.8 hours (68.9-97.8); table 3 ]. in total, 32 (25.8%) children in the itti population had a coinfection with another infectious pathogen at baseline [25/81 (30.9%) in the baloxavir group and 7/43 (16.3%) in the oseltamivir group; table 1 ]. for baloxavir, most children with a coinfection had a subtype of coronavirus, followed by rhinovirus/enterovirus and respiratory syncytial virus. for oseltamivir, most children had rhinovirus/enterovirus. no results for coinfections after baseline are available. in an exploratory genotyping analysis of the polymerase acidic (pa) gene using sanger sequencing in the 81 influenza-positive children (itti) treated with baloxavir, none of the children had preexisting pa/i38x mutations in baseline samples; pa/i38x mutations have previously been shown to be the most common determinant of reduced susceptibility to baloxavir. 26 in 57 of the 81 children, sequencing was possible for both baseline and posttreatment samples, whereas 24 children had no detectable virus after treatment, or such low levels that sequencing was not possible. treatment-emergent pa/i38x substitutions were detected in 11 of 57 children (19.3%); 9 had influenza a subtype h3n2 and 2 had subtype h1n1pdm09. however, when the 24 children with no detectable virus or such low levels that sequencing was not possible (suggesting no pa/i38x substitutions) are also included in this calculation, the rate of pa/i38x substitutions is 13.5% (11/81 baloxavir has a novel mechanism of action, preventing the formation of new virions by blocking replication early in the influenza life cycle. 16, 17 two previous japanese studies evaluating the use of baloxavir in children <12 years old have been completed; however, both were small, open-label studies (japiccti-163417 24 and japiccti-173811). ministone-2 is the first global, phase iii, randomized active controlled study designed to investigate the safety and efficacy of a single dose of baloxavir in children. baloxavir safety was the primary objective of this study, and the overall findings were unremarkable, with no new safety signals identified, confirming that baloxavir is well tolerated in children. rates of aes were generally similar between groups and there were no serious aes or deaths reported. the most common aes in both groups were gastrointestinal related (diarrhea/vomiting). the safety population comprised children who received any portion of a single dose of treatment. the safety population comprised children who received any portion of a single dose of treatment. *including american indian or alaska native (n = 1 for baloxavir), asian (n = 1 for baloxavir), native hawaiian or other pacific islander (n = 1 for oseltamivir), multiple (n = 4 for baloxavir) and unknown (n = 5 for baloxavir, n = 1 for oseltamivir). †taken from the itti population. ‡one child had influenza a/h1n1pdm09 and influenza b coinfection. §in the baloxavir marboxil group, 23 children with influenza a had viral coinfections: coronavirus (11), rhinovirus/enterovirus (8) and respiratory syncytial virus (4). in the oseltamivir group, 5 children with influenza a had viral coinfections: rhinovirus/ enterovirus (3) and coronavirus (2) . in the baloxavir marboxil group, 2 children with influenza b had viral coinfections: rhino/enterovirus (1), and both adenovirus and rhinovirus/enterovirus (1). in the oseltamivir group, 1 child with influenza b had a coinfection: rhinovirus/enterovirus. no results for coinfections after baseline are available. treatment-related aes were less frequent in the baloxavir group than the oseltamivir group. most aes were considered by investigators to be related to influenza infection. in both treatment groups, most aes were either mild or moderate in severity and most resolved or were resolving by the end of the study. the data showed that ttass, a key efficacy endpoint for the study, were similar between the baloxavir and oseltamivir treatment groups. all other secondary efficacy endpoints were similar between treatment groups, and there were no differences in the incidence of influenza-related complications or the use of antibiotics. as in previous studies, 19, 20 the antiviral activity of baloxavir was shown to be favorable compared with oseltamivir. the rapid decline of infectious virus titer after 24 hours and reduced time to viral shedding are consistent with those seen previously in completed phase ii and phase iii studies, confirming the marked virologic effects achieved with a single dose of baloxavir. 19, 20 the further potential clinical value of an antiviral with such favorable virologic properties has been recently demonstrated in a postexposure prophylaxis study. 27 moreover, further studies are being conducted to determine whether transmission from infected individuals is reduced by baloxavir, 28 and whether, in combination with a neuraminidase inhibitor, baloxavir can offer benefit in patients hospitalized with severe influenza (clinicaltrial.gov identifier: nct03684044). multiple viral infections are frequent in hospitalized children with respiratory tract disease, and may be linked to greater severity in symptoms and longer lengths of hospital stays. 29, 30 in contrast, although respiratory tract infections are commonly diagnosed and treated in the community, few studies report on the frequency of coinfection in this setting. a quarter of the itti population had a coinfection with another respiratory viral pathogen at baseline. given that no data are available after baseline, there is a possibility that both baseline coinfections and later-onset coinfections contributed to a longer ttass than may have been observed in the absence of coinfections. in this study, there were no bacterial coinfections at baseline, and only 3 cases of otitis media (2 requiring antibiotics) observed in the baloxavir arm following treatment; therefore, these results may potentially indicate a reduction in acute otitis media complications following baloxavir treatment, compared with the 1 in 4 children in whom this usually develops without antiviral treatment. 31 this could contribute to a reduction of the overuse of antibiotics for prevention of this complication, 32 particularly in younger children, similar to the effect observed with oseltamivir. 33 however, these speculative results should be interpreted with caution in the absence of a specific analysis of the reduction in acute otitis media complications following baloxavir treatment. all influenza antivirals can lead to the emergence of resistant variants with reduced susceptibility; 34 however, in acute infections such as influenza, these variants are usually transient. an amino acid substitution at position 38 in the rna polymerase (pa/i38x) has been shown to be the most common determinant of reduced susceptibility to baloxavir. 35 the rate of pa/i38x viral variants in this study was consistent with that observed in 2 previous the itti population comprised children who had a laboratory reverse transcriptase-polymerase chain reaction confirmation of influenza infection from any swab sample collected at baseline or during the study. *the number of children with influenza b was too low to allow meaningful interpretation of the data. †children with a positive virus titer on day 1 were included in this analysis. ‡children whose virus titers did not reach the limit by the last observation time point were censored. completed japanese studies in children (japiccti-163417 24 and japiccti-173811) including higher prevalence rates in children <5 years old, which has also been documented for oseltamivir. 35 in this study, clinical benefit was observed regardless of i38x variants. however, the approach taken to calculate resistance rates is conservative, generally including patients in whom virus is detectable after a certain time (usually days 3-5), but excluding those who have cleared the virus before sampling. potent antivirals would be expected to clear the virus more rapidly, leaving fewer patients with samples for resistance analysis. this can potentially lead to an overestimation of resistance rates. in conclusion, this study showed that a single oral dose of baloxavir is well-tolerated, effective at alleviating influenza signs and symptoms, and results in rapid elimination of the virus in children with uncomplicated, acute influenza. importantly, it presents a new therapeutic option with a simplified and convenient singledose oral regimen, of particular value for children. influenza (seasonal) fact sheet influenza: a global perspective secondary bacterial infections associated with influenza pandemics factors affecting immune responses to the influenza vaccine the burden of seasonal and pandemic influenza in infants and children global seasonal influenzaassociated mortality collaborator network. estimates of global seasonal influenza-associated respiratory mortality: a modelling study risk factors of influenza transmission in households vaccines for seasonal and pandemic influenza strategies to achieve the healthy people 2020 annual influenza vaccine coverage goal for health-care personnel: recommendations from the national vaccine advisory committee influenza vaccines: challenges and solutions new vaccines against influenza virus the evolving history of influenza viruses and influenza vaccines incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern the origin and global emergence of adamantane resistant a/h3n2 influenza viruses baloxavir marboxil, a novel cap-dependent endonuclease inhibitor potently suppresses influenza virus replication and represents therapeutic effects in both immunocompetent and immunocompromised mouse models xofluza (baloxavir marboxil) prescribing information in vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase pa subunit baloxavir marboxil for uncomplicated influenza in adults and adolescents phase 3 trial of baloxavir marboxil in high-risk influenza patients (capstone-2 study) tamiflu (oseltamivir) prescribing information canadian acute respiratory illness and flu scale (carifs): development of a valid measure for childhood respiratory infections detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture baloxavir marboxil in japanese pediatric patients with influenza: safety and clinical and virologic outcomes multicenter evaluation of biofire filmarray respiratory panel 2 for detection of viruses and bacteria in nasopharyngeal swab samples characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil presented at options x for the control of influenza presented at options x for the control of influenza does respiratory virus coinfection increases the clinical severity of acute respiratory infection among children infected with respiratory syncytial virus? multiple simultaneous viral infections in infants with acute respiratory tract infections in spain influenza in children reducing antibiotic use in influenza: challenges and rewards impact of oseltamivir treatment on the incidence and course of acute otitis media in children with influenza emerging influenza antiviral resistance threats five years of monitoring for the emergence of oseltamivir resistance in patients with influenza a infections in the influenza resistance information study. influenza other respir viruses the authors thank all study participants and investigators who participated in the study, wenyi liu and alan j. mueller-breckenridge for outstanding support with statistical/scientific analyses, and viroclinics biosciences bv for analyses of swab samples. qualified researchers may request access to individual patient level data through the clinical study data request platform (https://vivli.org/). further details on roche's criteria for eligible studies are available here (https://vivli.org/members/ourmembers/). for further details on roche's global policy on the sharing of clinical information and how to request access to related clinical study documents, see here (https://www.roche.com/research_and_development/who_we_are_how_we_work/clinical_trials/our_commit-ment_to_data_sharing.htm). key: cord-342519-tjr6dvtt authors: souza, thiago moreno l.; salluh, jorge i. f.; bozza, fernando a.; mesquita, milene; soares, márcio; motta, fernando c.; pitrowsky, melissa tassano; de lourdes oliveira, maria; mishin, vasiliy p.; gubareva, larissa v.; whitney, anne; rocco, sandra amaral; gonçalves, vânia maria c.; marques, venceslaine prado; velasco, eduardo; siqueira, marilda m. title: h1n1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis date: 2010-11-30 journal: plos one doi: 10.1371/journal.pone.0014158 sha: doc_id: 342519 cord_uid: tjr6dvtt background: the novel influenza a pandemic virus (h1n1pdm) caused considerable morbidity and mortality worldwide in 2009. the aim of the present study was to evaluate the clinical course, duration of viral shedding, h1n1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe h1n1pdm infections during the winter of 2009 in brazil. methods: we performed a prospective single-center cohort study in a cancer center in rio de janeiro, brazil. hospitalized patients with cancer and a confirmed diagnosis of influenza a h1n1pdm were evaluated. the main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of h1n1pdm susceptibility to oseltamivir. results: a total of 44 hospitalized patients with suspected influenza-like illness were screened. a total of 24 had diagnosed h1n1pdm infections. the overall hospital mortality in our cohort was 21%. thirteen (54%) patients required intensive care. the median age of the studied cohort was 14.5 years (3–69 years). eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. a total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant h1n1pdm virus. viral evolution was observed in sequentially collected samples. conclusions: prolonged influenza a h1n1pdm shedding was observed in cancer patients. however, oseltamivir resistance was not detected. taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation. the emergence of the novel influenza a/h1n1 pandemic virus (h1n1pdm) significantly affected the utilization of healthcare resources and increased morbidity and mortality in children and young adults [1, 2] . from april through september 2009, during the fall/winter in the southern hemisphere, brazil experienced the first wave of the h1n1pdm virus, and by the end of december 2009, over 1600 h1n1pdm-related deaths had been reported in brazil [3] . emerging data on the clinical course of severe h1n1pdm infection have allowed the identification of high-risk groups, which include pregnant women and patients with morbid obesity [4, 5] . however, an analysis of the impact of this novel virus in a highly susceptible population, such as cancer patients, through clinical and virological perspectives, needs to be highlighted [6, 7, 8, 9, 10, 11] . the atypical clinical presentation of influenza infections in cancer patients, which delays clinical suspicion, antiviral treatment and adequate prevention of viral transmission, is a major challenge for clinical management in this population [12] . cancer patients are more likely to suffer from severe seasonal influenza infections [12, 13, 14] and prolonged viral shedding, as has been reported for an h3n2 seasonal virus [15] . prolonged shedding and the development of oseltamivir resistance in cancer patients infected with the h1n1pdm virus have not been thoroughly evaluated. data on these aspects could have major implications for the clinical management and infection control practices for h1n1pdm-infected cancer patients [16] . because the analysis of this novel viral infection in cancer patients is an important component of the 2009 pandemics, we conducted a prospective cohort study aimed at evaluating the clinical course of influenza infection, the duration of viral shedding, h1n1pdm evolution and the emergence of antiviral resistance in hospitalized cancer patients with a severe h1n1pdm infection in a reference cancer center during the winter of 2009 in brazil. during the study period, 44 hospitalized cancer patients with a suspected influenza infection were screened, and 24 had a confirmed influenza a diagnosis using a rapid indirect immunofluorescence (ifi) test or world health organization (who)recommended real-time rt-pcr (rrt-pcr) ( figure 1 and table s1 ). among these, 20 patients were confirmed to be positive for the h1n1pdm virus using rrt-pcr ( figure 1 and table s1 ). the remaining four patients were positive for influenza a using ifi only. considering the pandemic case definitions with reference to international guidelines [17] , these last four cases were categorized as h1n1pdm-confirmed cases. altogether, these 24 cases constituted the study population. all of the respiratory samples collected from the 20 rrt-pcr-confirmed patients were inoculated in cell cultures. we recovered the virus from 13 individuals after at least two passages in mdcks, constituting 15 isolated samples. these isolates were also analyzed for oseltamivir resistance using a functional assay. patients diagnosed with h1n1pdm were young (median age = 14.5, range 3-69 years). in total, 14 (58.3%) were under 18 years old, and 17 (70.8%) were less than 50 years old. hematologic cancer occurred in 75% (18) of the patients, whereas solid tumors occurred in 25% (6) patients (tables s2 and s3) . a total of 22 (,92%) patients had received immunosuppressive therapy in the previous 30 days. among these individuals, 18 patients (75%) were on chemotherapy, 14 (58.3%) received systemic corticosteroids and 1 (4%) received radiation therapy (table 1 and s4) . no patient received erythropoietin (epo) or immunomodulatory agents. a total of 14 patients (58.3%) presented febrile neutropenia (,500 neutrophils/mm 3 ) at the time h1n1pdm was diagnosed. the median duration of neutropenia after the onset of viral disease was two days (ranging from one to six days; table s5 ). according to the brazilian national cancer institute's protocol, all patients that presented neutropenia received g-csf until normalization of neutrophil counts. the clinical characteristics and comparisons among groups are shown in table 1 and tables s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, and s11. the overall mortality in our cohort was around 21% (n = 5), and four patients (n = 16.6%) had at least one comorbidity besides cancer. of these patients with comorbidities, one died. no pregnant or morbidly obese patients were identified. a total of 23 (95.8%) patients were treated with oseltamivir, and the median time from the initial symptoms to the initiation of therapy was three days (0-15 days; tables 1, s2 and s6). one patient that died due to severe acute respiratory failure 24 h after clinical suspicion of h1n1pdm infection never received antiviral treatment. oseltamivir was used for a median of seven days (0-19 days), and double doses (150 mg bid for adults and twice the recommended dose per kg for children) were administered for 11 (47.8%) patients (table s6) . a total of 11 (47.8%) patients received oseltamivir for more than seven days. six patients (25%) received this antiviral within 48 h of clinical suspicion. all patients that died received oseltamivir more than 48 h after the onset of the illness. however, when we compared the mortality of patients that received oseltamivir either within or after 48 h of the onset illness, no significant difference was observed (0/6 vs. 5/18, p = 0.28). no differences in prolonged viral shedding were observed between these two groups. at presentation, all patients were treated with broad-spectrum intravenous antimicrobial agents to combat community-acquired pneumonia and/or febrile neutropenia [18] , and five (20.8%) had concomitant positive cultures (tables s7 and s8) . hypoxemia was frequent, and the median pao2/fio2 on the first arterial blood gas evaluation was 192 mmhg (range: 64-367 mmhg). overall, 13 patients (five adults and eight children) were admitted to the icu. six patients were directly admitted from the emergency department, and the other seven patients were transferred from other hospital wards (table s2) . ventilatory support was given to 12 patients (table 1 and s9). invasive mechanical ventilation was performed in 10 patients (76.9%), and non invasive ventilation (niv) was performed in 3 patients (23.1%; table 1 ). among the niv patients, one required subsequent endotracheal intubation and mechanical ventilation, and all three patients were discharged from the hospital. extra-pulmonary organ failure occurred in eight patients (33.3%; table 1 and s9). of the 13 critically ill patients, 12 were treated with oseltamivir, and treatment was initiated 48 h after the first signs/symptoms of viral infection in 5 of them. adjunct or non-conventional supportive therapies for ards were performed for 12 of the 13 patients that entered the icu (92.3%). a total of 10 patients (76.9%) received systemic corticosteroids (eight due to previous use and two for shock and persistent ards); five (38.5%) were ventilated in a prone position, and four (30.8%) required recruitment maneuvers. no patient received extra-corporeal membrane oxygenation. although prolonged influenza a shedding has been observed for a cancer patient infected with the h3n2 seasonal virus [15] , more detailed data on h1n1pdm secretion in severely ill cancer patients are required. we evaluated viral shedding in 10 mechanically ventilated patients by collecting sequential respiratory samples at different time-points after the onset of illness. the duration of viral shedding was considered to be the time frame from the initial symptoms to the last h1n1pdm-confirmed sample. five (50%) patients in this group showed viral shedding for at least 11 days during oseltamivir treatment ( figure 2 and table s12 ). the median duration of h1n1pdm shedding was 23 days (ranging from 11 to 63 days; figure 2 and table s12 ). most importantly, the maximum duration of h1n1pdm shedding in our investigation was 63 days, followed by 44 days for another patient. to our knowledge, these periods constitute the longest registered cases of h1n1pdm shedding described to date. all rrt-pcr-positive samples from these patients with the longest viral shedding durations (5645 and 5899) were culturable, meaning that these were infectious viruses ( figure 2 and table s12 ). in addition, the last h1n1pdm-confirmed samples from these patients were only detected using cell culture assays, suggesting the presence of low viral loads in these specimens ( figure 2 and table s12 ). these patients still shed the virus for an additional 25 to 40 days after cessation of the antiviral treatment ( figure 2 ). to date, no significant variation has been detected at the amino acid level in the hemagglutinin (ha) of the 2009 pandemic virus [19] . therefore, we examined the genetic diversity of the h1n1pdm virus recovered from these severely ill patients by performing sequence analysis of the viral ha gene. no significant divergence was found in the h1n1pdm isolates collected during the onset of illness from the four patients with prolonged shedding ( figure s1 ). to evaluate the genetic characteristics of the isolates from patients with prolonged viral shedding, we sequenced two consecutive samples from a single individual (5645s2/09 and 5645s3/09) and compared their ha sequences to other h1n1pdm viruses from mild, severe and fatal cases from different countries. we observed that samples collected a month apart (5645s2/09 and 5645s3/09) clustered together and displayed a relatively large branch length from other h1n1pdm viruses ( figure 3 ). this result may have occurred because strain 5645s2/09 diverged from ca/ 04 in the amino acid residues l52s, l70p, p100s, c153l, t214a, q293r and i321v (h1 numbering). additionally, isolate 5645s3/ 09, which was sequenced from amino acid residues 167 to 413, also had the mutations t214a, q293r and i321v fixed in the viral population a month after the initial sampling. in the isolate 5645s3/09, another mutation was also acquired, d238p, suggesting continuous viral evolution. although we cannot determine the role of these mutations in viral pathogenesis by this result alone, the retention of the changed residues over time strongly suggests viral persistence rather than re-infection. considering that influenza resistance to antivirals is likely to emerge in immunocompromised individuals and that, with respect to the h1n1pdm virus, only a few studies have detected oseltamivir resistance [20] , we investigated the emergence of antiviral resistance in h1n1pdm samples isolated in cell culture. thus, oseltamivir carboxylate ic 50 values were measured for 15 h1n1pdm samples isolated from 13 patients. we found that 10 isolates were sensitive (0.8560.27 nm), and 5 displayed high ic 50 values for the antiviral used (165.13642.26 nm). these samples were sent to the who collaborating center at the cdc in atlanta for resistance confirmation. we found that the isolates with high ic 50 values were endowed with a neuraminidase (na) activity that was cross-resistant to oseltamivir, zanamivir and peramivir, suggesting the presence of a co-pathogen endowed with na activity within these isolates. co-infections with other respiratory viruses (coronavirus (229, 43 and 63), parainfluenza (1, 2, 3 and 4), human metapneumovirus, parechovirus, rhinovirus, rsv a/b, adenovirus and enterovirus) or atypical bacteria (mycobacterium pneumonia) were not identified in these samples, suggesting that no other viral source of na activity was responsible for these high ic 50 values. however, we co-isolated a streptococcus sp. from the samples with high ic 50 values. the na activity of this bacterial strain displayed a phenotype resistant to nais. in further testing with bacteria-free h1n1pdm isolates, the virus' ic 50 were consistent with a sensitive isolate. pyrosequencing analyses were also performed and revealed that the other high-ic 50 samples had the wt h275 residue and thus did not contain this oseltamivir resistance marker. viruses isolated from the initial onset of illness were a/california/07-like viruses ( figure s2 ). prolonged influenza shedding in cancer patients has been observed for seasonal strains [15] . regarding the h1n1pdm virus, it has been shown that prolonged virus shedding in cancer patients may occur, although such a phenomenon has only been documented through cases involving a single studied patient [21, 22, 23, 24, 25] . here, we prospectively and systematically collected information from a cohort of hospitalized cancer patients with severe h1n1pdm infections. these patients presented high mortality, prolonged viral shedding and h1n1pdm evolution without the emergence of oseltamivir resistance. this is the first study to address viral shedding and resistance in cancer patients with h1n1pdm infections; thus, it may provide insight into the role of cancer patients as potential human reservoirs for this pandemic virus. unlike previous reports, our population was composed of hospitalized, severely immunocompromised cancer patients [26] . most of them were young, had hematologic malignancies and received chemotherapy and systemic steroids in the weeks that preceded the h1n1pdm infection. the patients were treated with oseltamivir in the early course of the infection (the median time to antiviral initiation was three days). a total of 13 patients (54%) required intensive care and presented severe respiratory distress. in these patients, the mortality rates were higher (38%) than those observed for general icu patients suffering from h1n1pdm infections [2, 27] as well as for non-critically ill cancer patients [26] . however, the outcomes were not different from those reported for cancer patients requiring mechanical ventilation [28, 29] . interestingly, during the influenza season, 14 patients (58.3%) with febrile neutropenia were identified as h1n1pdm cases, a condition that is not usually investigated in this scenario. however, febrile neutropenic cancer patients have an increased risk of developing respiratory distress and multi-organ failure. therefore, screening for respiratory viruses and prompt initiation of oseltamivir treatment should be considered in these patients. febrile neutropenia indicates a poor prognostic with respect to a patient's outcome, but neutropenia duration in our cohort of patients was less than seven days. thus, prolonged viral shedding might not have a correlation with neutropenia. we observed the persistence of h1n1pdm in 5 out of 10 patients studied for this purpose. in these individuals, viral shedding continued for at least 11 days, despite the use of oseltamivir. the median duration of viral shedding in our population was 23 days, and two pediatric patients with acute lymphoblastic leukemia showed even longer virus secretions (44 and 63 days; figure 2 and table s10), although it is difficult to determine whether viral persistence was due to cancer per se or to acute lung injury and mechanical ventilation. influenza shedding is not considered to last long, and it disappears seven days after the onset (2.4 and 4.5 days for oseltamivir-and placebo-treated groups, respectively) [30] . studies aimed at monitoring 2009 h1n1pdm virus shedding using randomized trials with appropriate controls, such as outpatients and hospitalized or immunocompromised individuals, have not yet been conducted. because we were also unable to establish agematched controls with or without immunosuppression, since this study was conducted during the peak of the first wave of the 2009 pandemics in brazil, we compared our work to other studies on h1n1pdm shedding in general, hospitalized or immunocompromised populations [21, 22, 23, 24, 25, 31, 32, 33, 34, 35, 36] . in table 2 , we summarize the cohort used in each study, whether or not they were immunocompromised, their underlying diseases and the number of patients analyzed for viral shedding in each of these studies. we compared the maximum periods of shedding among these different populations and the number of patients that secreted the virus for more than seven days. these data would be more informative and relevant from a public health point of view because the duration of the quarantine for h1n1pdm was approximately that long [21] . we found ( table 2 ) that in households in hong kong [31] and canada [34] , the maximum periods of h1n1pdm shedding ranged from 8 to 11 days. these periods were not different from what was observed with military cadets [22] and during the containment phase of the pandemics in vietnam [23] (table 2) . regarding h1n1pdm shedding among infants, hien et al. showed that children five to nine years old could secrete the h1n1pdm virus for five to six days, which is markedly lower than what is observed for the seasonal influenza virus [30, 33] . compared to our results, we observed higher periods of viral shedding in two seven-year-old patients with acute lymphoblastic leukemia (table 2 and figure 2 ). however, a single report on two travelers in france showed that these apparently immunocompetent individuals secreted the h1n1pdm virus for 14 and 28 days [25] (table 2 ). although these periods of time are comparable to the time frame of virus secretion in hospitalized patients in china [23] and our work, the study from felury et al. might be as biased as ours by the small size of the cohort ( table 2) . influenza-infected immunocompromised individuals may have prolonged influenza shedding [15, 21, 32, 36] ; however, more insights are necessary to better comprehend the dynamics of the h1n1pdm virus in these individuals. mora et al. showed that in hiv-1-infected individuals, co-infection with the h1n1pdm virus might lead to an outcome not different from the one expected for immunocompetent subjects, although no systematic analysis of viral shedding was performed [36] . a similar conclusion was also drawn for transplant recipient individuals, whose longest periods of viral shedding did not exceed 11 days [32] (table 2 ). in our study, we found periods of h1n1pdm shedding similar to what the cdc observed for leukemia patients [21] (table 2) . although the small size of these cohorts of immunocompromised individuals [21, 32] , including ours, may require a more conclusive and mechanistic analysis, these observations may stimulate further systematic studies to understand or gain insight into factors associated with prolonged h1n1pdm shedding. in addition, it might give insights on basic studies on influenza pathogenesis. our results highlight the need for closer surveillance of cancer patients with h1n1pdm infections until the detection of the first negative sample. we hypothesize that follow-up protocols aimed at monitoring the persistence of viral shedding in cancer patients may be relevant if patients are submitted to immunosuppressive therapies in the days or weeks prior to or following an h1n1pdm infection. next, we sought to determine viral evolution during prolonged shedding. we found that some amino acid changes persisted from the initial symptoms until 30 days thereafter, suggesting that these patients had viral persistence rather than re-infection. in addition, an extra amino acid change (d238p) was found in the viral ha sequenced a month after the onset of illness, suggesting continuing viral evolution. although some of the mutations that we found (l52s, l70p, p100s, c153l, t214a, d238p, q293r and i321v) in strains 5645s2/09 and 5645s3/09 have not been previously described, other amino acid residue changes that were detected in our study (p100s and t214a) have been found in h1n1pdm viruses throughout the world without a significant link to viral pathogenesis or antigenic variation [37, 38, 39] . influenza viruses resistant to antiviral drugs have been reported in immunocompromised patients, [21, 40] and this resistance might be associated with prolonged viral shedding [41, 42] . notably, five isolates from two patients had high ic 50 values to neuraminidase inhibitors (nais). an na activity that was multiresistant to nais was identified and could be due to the presence of a streptococcus strain found in throat swabs and tracheal aspirates. pyrosequencing analyses of samples with high ic 50 values revealed that these specimens were h275 wild-type sensitive viruses. these results reinforce the need for additional genotyping assays to confirm the identification of putative resistant strains identified using functional assays. in addition, our findings show the need for investigating other sources of na activity in virus isolates with odd ic 50 values. the apparent paradox of prolonged viral shedding without antiviral resistance could be explained by either the inability of the immunocompromised host to effectively clear the h1n1pdm virus [12, 43] or inefficient absorption of the drug [44] . because we combined both clinical and molecular virology data, our results might contribute to the discussion on the adequate duration and type of anti-h1n1pdm treatment in immunocompromised patients with a protracted course. in these patients, the use of parenteral systemic or inhaled antivirals should also be investigated. although our work further investigates the unique dynamics of h1n1pdm virus infection in immunocompromised hosts, some caveats must be noted. because our investigation started during a new pandemic, the clinical evaluation and management protocols changed during the course of the study as new data emerged from the literature and from updated recommendations [45] . as the pandemic reached its peak in south america, the establishment of a larger and more diverse cohort with age-matched controls with or without immunosuppression became complex. thus, more indepth multivariate and mechanistic clinical analyses were limited. moreover, no recommendations for monitoring viral persistence were available; therefore, only a subset of severely ill patients admitted to the icu was evaluated. despite that, an important connection between clinical and laboratory information was studied, revealing the continuous evolution of h1n1pdm ha sequences and the stability of the na gene in severely ill patients [14] . in conclusion, this study provides evidence that severe h1n1pdm infection is associated with significant morbidity and mortality in cancer patients. in these patients, viral persistence without the emergence of antiviral resistance may occur during the clinical course of the disease. this result has major implications for the clinical management of h1n1pdm infections and infection control strategies. our study may provide insights into h1n1pdm shedding and might contribute to the development of new guidelines to manage cancer patients with h1n1pdm infection. the ethics committee (comitê de é tica em pesquisa; cep; http://www.inca.gov.br/conteudo_view.asp?id = 2380) at the instituto nacional de câncer (inca), rio de janeiro, brazil, headed by dr. adriana scheliga approved the study under protocol #18/ 2010 and waived the need for informed consent. this was a prospective cohort study conducted in the hospital do câncer-i, instituto nacional de câncer (hc-i-inca), rio de janeiro, brazil, from july 8 th to october 1 st , 2009. the hci-inca is a 160-bed comprehensive cancer center primarily for the population of rio de janeiro and neighboring states. the present study was strictly observational, and every clinical decision was at the discretion of the attending physician. all patients with a definite diagnosis of cancer requiring hospital admission for any reason and who displayed influenza-like illness were evaluated. patients in complete remission from cancer for more than five years were not considered. data were collected using a standardized case report form that included demographic data, clinical presentation, comorbidities, cancer status, use of immunosuppressive therapies, time course of acute illness, need for intensive care, use of antivirals, adjunctive therapies, advanced life support and in-hospital mortality (supporting information; si). patients were included if they had a fever (.37.8uc) and/or respiratory influenza-like illness and confirmed influenza a h1n1pdm diagnosis (by at least one of three assays, ifi, rrt-pcr or cell culture, and according to case definitions from the who [46] ). patients were treated according to the brazilian public health guidelines [47] . nasopharyngeal dacron-swab specimens were collected from all patients and placed onto transport medium (hanks solution with 100 u/ml penicillin and 100 mg/ml streptomycin) at the initial evaluation. tracheal aspirates were also obtained if the patient required tracheal intubation. patients' clinical samples were directly tested for a panel of respiratory viruses using an ifi assay for influenza a (respiratory virus panel; biotrin, mount merrion, co. dublin, ireland.). specimens were also sent to the brazilian national influenza center (ioc/fiocruz) for h1n1pdm confirmation using rrt-pcr, which was performed in accordance with the current guidelines from the who/cdc [46] . viral shedding was evaluated in the subset of patients that remained under mechanical ventilation for longer than seven days and in those patients with persistent hypoxemia and pulmonary infiltrate. these patients received a specific number to which their sample number was appended. that is, the first sample was ''s1'', and subsequent specimens were numbered consecutively (table s12 ). viral secretion was evaluated using both cell culture and rrt-pcr assays until it was negative. virus isolation was performed in madin-darby canine kidney (mdck) cells and/or embryonated eggs (see text s1 and table s12 ). the functional antiviral assay was performed using the na-star kit (applied biosystems, ca), according to the manufacturer's instructions. the rt-pcr protocol for sequencing using the sanger method and pyrosequencing [48] are presented in the si, as well as the phylogenetic analysis. blood samples were routinely sent for bacterial culturing, as were tracheal aspirates if the patient was intubated. standard descriptive statistics were used to describe the study population. continuous variables were reported as the mean 6 standard deviation or median (range) as appropriate. univariate analysis was used to identify factors associated with hospital mortality. two-sample t-tests and a chi-square or fisher's exact test were also used. two-tailed p values ,0.05 were considered statistically significant. text s1 clinical investigation and influenza virus assays. figure s1 phylogenetic tree of ha gene from the classical swine, eurasian swine, american avian and human seasonal lineages. the bootstrap probability is indicated for each interior branch, all values below 80% are hidden. the scale bar indicates the number of amino acid changes per site. colored circles indicate the samples from our study. this tree is unrooted. each influenza ha lineage is displayed beside their respective clade. found at: doi:10.1371/journal.pone.0014158.s014 (0.12 mb tif) figure s2 phylogenetic tree of na gene from the followed-up cohort. the bootstrap probability is not indicated for each interior branch since it is below 85%. the scale bar indicates the number of amino acid changes per site. the tree is rooted by california/ 07/2009 na sequence. found at: doi:10.1371/journal.pone.0014158.s015 (0.18 mb tif) critically ill patients with 2009 influenza a(h1n1) in mexico critically ill patients with 2009 influenza a(h1n1) infection in canada influenza pandêmica (h1n1) 2009 -update 10 available h1n1 novel influenza a in pregnant and immunocompromised patients influenza in immunosuppressed populations: a review of infection frequency, morbidity, mortality, and vaccine responses how i treat influenza in patients with hematologic malignancies nosocomial swine influenza (h1n1) pneumonia: lessons learned from an illustrative case severe novel influenza a (h1n1) infection in cancer patients h1n1 influenza 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households: a prospective, randomized comparison of oseltamivir treatment with or without postexposure prophylaxis comparative epidemiology of pandemic and seasonal influenza a in households h1n1 influenza in hospitalized transplant recipients a clinical virological and epidemiological analysis contagious period for pandemic (h1n1) effects of early oseltamivir therapy on viral shedding in 2009 pandemic influenza a (h1n1) virus infection influenza a pandemic (h1n1) 2009 virus and hiv pandemic potential of a strain of influenza a (h1n1): early findings pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets evolutionary trends of a(h1n1) influenza virus hemagglutinin since 1918 recovery of drug-resistant influenza virus from immunocompromised patients: a case series influenza virus reactivation after remission with oseltamivir treatment in a patient undergoing nonmyeloablative bone marrow transplantation characterization of multidrugresistant influenza a/h3n2 viruses shed during 1 year by an immunocompromised child influenza infections after hematopoietic stem cell transplantation: risk factors, mortality, and the effect of antiviral therapy h1n1 pneumonitis treated with intravenous zanamivir diretrizes para o enfrentamento à pandemia de influenza a (h1n1): ações da atenção primária à saúde cdc protocol of realtime rtpcr for influenza a(h1n1) protocolo de manejo clínico e vigilâ ncia epidemiológica da influenza detection of molecular markers of drug resistance in 2009 pandemic influenza a (h1n1) viruses by pyrosequencing we are indebted to the instituto nacional de câncer, fiocruz, faperj and cnpq for their support. the authors thank marc-alain widdowson (cdc/ccid/ncird) for critical review of the manuscript. alicia fry (cdc atlanta epidemiology intelligence officer) was also extremely helpful in discussions on virus shedding. key: cord-312461-5qzpo6l1 authors: adalja, amesh a.; watson, matthew; toner, eric s.; cicero, anita; inglesby, thomas v. title: characteristics of microbes most likely to cause pandemics and global catastrophes date: 2019-08-30 journal: global catastrophic biological risks doi: 10.1007/82_2019_176 sha: doc_id: 312461 cord_uid: 5qzpo6l1 predicting which pathogen will confer the highest global catastrophic biological risk (gcbr) of a pandemic is a difficult task. many approaches are retrospective and premised on prior pandemics; however, such an approach may fail to appreciate novel threats that do not have exact historical precedent. in this paper, based on a study and project we undertook, a new paradigm for pandemic preparedness is presented. this paradigm seeks to root pandemic risk in actual attributes possessed by specific classes of microbial organisms and leads to specific recommendations to augment preparedness activities. the recent global experience with severe infectious disease epidemics has triggered much interest in understanding the broader pandemic threat landscape. a substantial proportion of pandemic and biological threat preparedness activities have focused on list-based approaches that were in part based on pandemic influenzas of the past, historical biological weapon development programs, or recent outbreaks of emerging infectious diseases (e.g., sars, mers, ebola) (centers for disease control and prevention 2017; casadevall and relman 2010) . but such an approach inherently fails to account for agents not currently known or those without historical precedent. for that reason, preparedness activities that are limited to these approaches may hamper preparedness and lessen resilience. the purpose of this study was to analyze the characteristics of pathogens that could be capable of causing a global catastrophic biological risk (gcbr). these would be events in which biological agents-whether naturally emerging or reemerging, deliberately created and released, or laboratory engineered and escaped-could lead to sudden, extraordinary, widespread disaster beyond the collective capability of national and international governments and the private sector to control. if unchecked, gcbrs would lead to great suffering, loss of life, and sustained damage to national governments, international relationships, economies, societal stability, or global security (schoch-spana et al. 2017) . given the severe potential public health consequences of pandemic events, there needs to be a vital interest in developing and maintaining a flexible, rapid, and robust response capability. anticipating the forms of microbial threats that might cause future pandemics can help strengthen preparedness and response capacities. this paper proposes a framework for considering future pandemic threats and provides recommendations for how this framework should inform pandemic preparedness. review of the published literature and previous reports: the project team surveyed the current biomedical literature on the topic of emerging infectious disease characteristics, the pathogenic potential of microbes, and related topics. the literature review was microbe-and species-agnostic, encompassing all classes of microorganisms and host species. the literature review was accomplished with extensive pubmed searches on these subjects. relevant us government policy and strategy were reviewed. interviews: the project team interviewed more than 120 technical experts who work in and are intimately knowledgeable about this field. interviewees were drawn from academia, industry, and government. our goal was to ascertain the experts' views about the essential traits needed for a pathogen to become a gcbr, to contextualize historical outbreaks in light of these traits, and to determine which currently known infectious disease agents possess such characteristics. pandemic pathogen meeting: the project team completed a preliminary analysis that synthesized the results of our literature review and expert interviews. those findings were used to design and facilitate a meeting held on november 9, 2017, that included many of those who had been interviewed for this project. the meeting was held at the johns hopkins center for health security in baltimore, md. the purpose of the meeting was to gain additional insight and input into the project analysis, examine assumptions, and test possible recommendations. participants included representatives of us and foreign academic institutions, the federal government, and other independent subject matter experts. this paper is based on the findings of the project and is modification of the project report (johns hopkins center for health security 2018). when a pathogen has the capacity to cause a pandemic, it will possess several attributes that other microbes, capable of causing only sporadic or limited human infections, will lack. these traits can be divided into several categories: spread via respiratory transmission; capable of spread during incubation period prior to symptom onset; no preexisting host immunity; and other possible intrinsic microbial characteristics. many of these characteristics have been captured and are reflected, in equation form, by casadevall (casadevall 2017) . microbes have varied routes of transmission, ranging from blood and body fluids to vector-borne to fecal-oral to respiratory (airborne and respiratory droplet). while each mode of transmission is capable of causing large outbreaks if sustained human-to-human transmission is possible and left unchecked, certain modes of transmission are more amenable than others to intervention. for example, the transmission of an infectious disease caused by blood and body fluid transmission can be halted with infection control measures such as gloves or gowns. of the various modes of transmission, the respiratory route is the mechanism most likely to lead to pandemic spread. this is chiefly due to the fact that interventions to interrupt this method of spread are more difficult to implement when the simple and universal act of breathing can spread a pathogen. the prolific spread of influenza, pertussis, measles, and rhinoviruses is testament to this fact (herfst et al. 2017) . by contrast, although pathogens spread by the fecal-oral route, such as vibrio cholera and the hepatitis a virus, can generate explosive outbreaks, even a modicum of sanitary infrastructure can quench the outbreak. vector-borne outbreaks are a special case of a non-respiratory-spread agent. indeed, the only postulated extinction of a mammalian species by an infectious organism, the christmas island rat, was caused by a vector-borne trypanosome (wyatt et al. 2008) . for most of the agents that use this class of transmission, the spread is limited by a geographically and climatologically restricted vector habitat. humans can protect against vectors, and they can change where they live, but the christmas island rat could not. these factors have generally served to limit the pandemic potential of microbes that are spread by vectors. exceptions to this general limitation of vector-borne viruses include microbes spread by anopheles and aedes mosquitoes. pathogens spread by these mosquitoes have higher pandemic potential, given the geographic breadth of their spread. for example, most of sub-saharan africa is hospitable to the malaria-transmitting anopheles mosquitoes, while residents in 75% of us counties-as well as half the world's population-are regularly exposed to aedes mosquitoes that serve as vectors for high viremia flaviviruses and alphaviruses. such phenomena are borne out by the prolific spread of dengue, chikungunya, and zika (sinka et al. 2012 ; centers for disease control and prevention 2017). the onset and duration of the period when a person is contagious during an infection also play a major role in spread. diseases that are contagious during a late stage of infection, when infected people are very sick and therefore have more limited opportunities for spread, may be delimited in their spread. on the other hand, diseases that are contagious prior to symptom development, during the incubation period, or when only mild symptoms are present have greater opportunities for spread as infected individuals are able to conduct their activities of daily living with little or no interruption. modeling studies with simulated outbreaks have shown that the presence or absence of this timing of transmission factor can be decisive in whether an outbreak can or cannot be controlled. if a microbe is contagious before a person is seriously ill while the disease is still incubating, then there is higher potential for pandemic spread. historical examples reinforce this idea, as the only human infectious disease to be vanquished from the planet-smallpox-was one that was not contagious during the incubation period (fraser et al. 2004) . by contrast, a microbe such as the influenza virus, which is contagious prior to symptom development and has a wide range of clinical severity, is able to infect widely and is not amenable to control (brankston et al. 2007 ). microbial pathogenicity cannot, in reality, be separated from host characteristics. as elucidated by pirofsky and casadevall's host damage framework, disease is a complex interplay between a host immune system and a microbe (pirofski and casadevall 2008) . in congruity with this paradigm, host features and microbial pathogenicity are discussed together. for a microbe to cause a gcbr-level pandemic, it will be necessary for a significant proportion of the human population to be immunologically naïve to the agent so that the microbe would have a high number of susceptible humans to infect. additionally, large quantities of a sufficiently effective countermeasure (vaccine or antimicrobial agent) would not be available. immunologic naïveté would be expected with a zoonotic pathogen. the microbe, correspondingly, would have to possess the ability to evade the host immune response through virulence factors, immunological camouflage, or other features that allow a productive infection to ensue. additionally, human receptors that are utilized by a pandemic-causing microbe would likely be widespread in the population, facilitating permissive infection in the majority of humans. receptors may also provide target organ tropism for the agent, allowing severe disease to occur (e.g., lower respiratory tract and central nervous system). case fatality rates (cfrs) need not be inordinately high to cause a gcbr-level event, as evidenced by the 2.5% cfr reported for the 1918 influenza pandemic-the event closest to an actual human gcbr in the modern era (taubenberger and morens 2006) . a low but significant cfr adheres to the host density threshold theorem. according to this commonly held theorem, a microbe that kills too many of its hosts will run out of susceptible hosts and be extinguished (cressler et al. 2016) . while this may be true of pathogens that are closely linked to one host species, it is not applicable to sapronotic diseases such as amebic encephalitis and cholera (in certain contexts), which can infect and kill without jeopardizing future transmission or survival. indeed, many extinction-level amphibian infectious diseases are sapronotic in nature, such as the chytrid disease of salamanders and frogs (fisher 2017) . additionally, a gcbr-level event may not confer direct mortality. reproductive effects (i.e., in the manner of rubella or zika) or carcinogenic effects (e.g., htlv-1) could, in many ways, be highly detrimental to the future of humanity, as they could lead to significant curtailment of lifespans and diminishing birth rates, which could ultimately result in significant population collapse (rasmussen et al. 2017; tagaya and gallo 2017) . given the right context, any microbial organism could evolve or be engineered to be a gcbr. however, the most likely cause of a gcbr presently is a virus, with rna viruses being the most probable (woolhouse et al. 2013 ). historically, bacterially caused infections such as plague have had incredible impacts on the human species (raoult et al. 2013 ). however, the development of antibacterial therapies, beginning with the sulfonamides in 1935 and then penicillin in 1942, has severely limited the ability of this class of microbes to cause a gcbr-level pandemic. in addition, the relatively slower speed of replication and accumulation of mutations also disadvantages this class over viruses. for example, a human infected with the hepatitis c virus (an rna virus) produces trillions of virions per day, whereas the doubling time of yersinia pestis, the cause of plague, is 1.25 h (neumann et al. 1998; deng et al. 2002) . the public health crisis of multiple-drug-resistant bacteria, such as carbapenem-resistant enterobacteriaceae (cre) and others, is very alarming (logan and weinstein 2017) . the spread of these bacterial agents, for which few if any treatments exist, threatens the entire practice of modern medicine, from cancer chemotherapy to joint replacement therapy. however, these organisms, which have variable attributable mortality, tend to be unable to efficiently infect human hosts that are not compromised or hospitalized. as such, the risk to the general public is constrained. large outbreaks of cholera and plague have represented true public health emergencies in yemen and madagascar, but their spread reflects severe infrastructure deficiencies caused by war and supply constraints rather than true global pandemic risk (qadri et al. 2017; roberts 2017 ). fungi represent prolific pathogens outside of the mammalian species. outbreaks of chytrid fungal disease in frogs and salamanders as well as snake fungal disease represent true existential threats to affected species (fisher 2017) . however, fungi are largely thermally restricted, and only limited members of this class of microbes can infect warm-blooded organisms such as mammals (casadevall 2012) . indeed, a fungal filter is hypothesized to have existed and may be partly responsible for mammalian warm-bloodedness. the success of the mammalian-adapted fungus that causes white-nose syndrome in bats is facilitated by the lower body temperature that occurs during their hibernation (foley et al. 2011) . human infections with fungi tend to be severely damaging only in an immunocompromised host. the human innate immune system contends with countless fungal spores that are present in every breath of air. as such, many endemic fungal diseases, such as histoplasmosis or coccidioidomycosis, do not cause harm in the majority of immunocompetent humans infected. even newly emerging fungi such as candida auris and cryptococcus gattii are largely subjected to this limitation (chowdhary et al. 2017 ; centers for disease control and prevention 2010). one of the most widespread fungal outbreaks-the exserohilum fungal meningitis outbreak-was abetted by direct injection of a contaminated medical product into the spinal region of humans, which is not a usual mechanism of infection (casadevall and pirofski 2013) . without thermal adaptation (which might be feasible with deliberate manipulation), fungi, many of which are sapronotic and do not rely on or need mammalian hosts, will not constitute a pandemic threat to humans. prions-transmissible infective proteins-are one of the most fascinating and understudied of infectious agents. these agents, which are responsible for diseases such as kuru and new variant creutzfeldt-jakob disease (vcjd, the human form of "mad cow disease") in humans, cause scrapie, chronic wasting disease, and bovine spongiform encephalopathy in other mammalian species (chen and dong 2016) . though highly damaging to humans and other species they infect, prions require specific conditions for spread. new variant creutzfeldt-jakob disease was to date the most highly publicized outbreak of a human prion disease; it resulted in 229 human cases tied to the consumption of beef products primarily in england in the 1990s and the 2000s (hilton 2006) . other modes of transmission of cjd tied to iatrogenic spread via contaminated surgical instruments or cadaveric hormone products ceased once protective measures were put in place (bonda et al. 2016 ). kuru, a geographically restricted prion disease, was spread via human cannibalism in papua new guinea, and the outbreak abated once that practice was ended in the 1960s (liberski et al. 2012) . the transmission characteristics of prion diseases are such that very extraordinary circumstances, on a par with human cannibalism or massive food contamination, must be present for a gcbr-level risk to be present for humans. additionally, and almost by definition, such an event would be slow-moving (prions were once known as "slow viruses"). protozoal organisms have the distinction of being the only infectious disease to have caused the extinction of a mammalian species. the christmas island rat, unable to outrun its vector, was felled by a vector-borne trypanosome (t. lewisi) during the early twentieth century on the australian island (wyatt et al. 2008) . human forms of trypanosomiasis have not risen to such a level of concern. human protozoal infections have exerted tremendous pressure on the species, and it is hypothesized that half of all humans who have lived died of malaria, which still kills approximately half a million humans annually (world health organization 2017). however, the development of antimalarial compounds and vector avoidance strategies has proved successful when they are able to be employed appropriately, and they have relegated malaria to a pathogen whose impact is amenable to control. nonetheless, one aspect of malaria is of particular concern: the development and spread of artemisinin-resistant forms, which render treatment extremely challenging with little to no effective antimalarial agents left for use. largely confined to specific regions of asia, such as cambodia and myanmar, this organism poses severe treatment challenges and, if artemisinin-resistant forms were to spread to africa, could represent a continent-wide catastrophic biologic risk (haldar et al. 2018 ). ameba, ectoparasites, and helminths all have limited pandemic risk, as they are constrained by pathogenicity, transmissibility, or both. clonally transmissible tumors-such as the notable devil facial tumor disease in tasmanian devils-are rare occurrences in humans, with restricted modes of transmission (maternal-fetal and organ transplantation). space-adapted organisms (e.g., salmonella that originates on earth but spends time in the space station before coming back to earth) can exhibit enhanced virulence; however, they still are susceptible to antibiotic treatment and normal control measures: there is no evidence they pose greater epidemic risk than normal salmonella (wilson et al. 2007 ). an alien microbe species that is obtained on mars or meteorites and brought back to earth, one of the focuses of the planetary protection program at the national aeronautics and space administration (nasa), was not deemed by our interviewees and meeting participants to be likely to pose a threat. and if such a species were found, it would be unlikely to be adaptable to an earthlike planet environment, as adaptations to its home planet's markedly different environments would likely preclude adaptations to earth. even though the chances of serious biological risk posed by such a sample return are deemed to be low, there are many uncertainties, and the highest level biocontainment procedures are being considered for specimens that might harbor such non-earth-based organisms (national research council 2009). traditionally, viruses have been ranked at the highest level of pandemic risk, and dedicated preparedness efforts often focus solely on viruses. a disproportionate focus on viruses is justified, however, based on several aspects unique to the viral class of microbes. the high rate of replication of viruses-for instance, over 1 trillion hepatitis c virions are produced per day in a human infection-coupled with the mutability inherent in such short generation times gives viruses an unrivaled plasticity. this plasticity allows for host adaptability, zoonotic spillover, and immune system evasion. the lack of a broad-spectrum antiviral agent-like ones available for bacterial and even fungal organisms-also confers a special status on viruses. with no off-the-shelf treatment available to contain a viral outbreak, and likely no vaccine, containment efforts, at least in the early stages, will likely need to be made in the absence of a medical countermeasure (zhu et al. 2015) . there is a strong consensus that rna viruses represent a higher pandemic threat than dna viruses (kreuder johnson et al. 2015) . this assessment is derived from the fact that the stability of rna as a genomic material is less than that of dna, giving more genomic pliability to the rna viruses. dna viruses such as smallpox do challenge this assumption, and concern exists surrounding the related risks of monkeypox viruses, which are increasingly spreading in the absence of a smallpox vaccine campaign (kantele et al. 2016) . as monkeypox outbreaks continue to occur with longer chains of transmission, employing smallpox vaccines in target populations might be considered. another aspect of viral characterization is the location of replication. viruses with greater capacity for widespread have been shown in studies to be more likely to replicate in the cytoplasm of a cell (pulliam and dushoff 2009; olival et al. 2017) . this is postulated to be due to the higher affinity a virus must have for a particular type of host in order to be permitted entry into its nucleus, and this greater affinity would limit its zoonotic potential because it would be likely to be strongly tied to its usual host. in general, it is dna viruses that tend to have a nuclear replication cycle, while rna viruses have a cytoplasmic cycle. strikingly, smallpox-a dna virus with proven ability to cause pandemics-is a cytoplasmic replicator, while influenza-an rna virus with proven ability to cause pandemics -has a nuclear replication cycle. the exceptions to these rules argue against any overly strict adherence to them. other factors that may increase a virus' potential to cause a global catastrophic risk include a segmented genome (as exemplified by influenza viruses), a comparatively smaller genome size, and high host viremia (e.g., vector-borne flaviviruses). for example, the flu virus' segmented genome makes novel genetic assortment an eventuality, while a large genome may prevent nimble mutations. however, with each characteristic it is impossible to find a general rule, as exceptions abound. among currently studied viruses, the influenza a viruses are widely judged to pose the greatest pandemic risk based on historical outbreaks and viral characteristics (silva et al. 2017; imai et al. 2017 ). analysis of influenza risks is made in the centers for disease control and prevention (cdc)'s influenza rapid assessment tool (irat) which ranks h7n9 as the most concerning influenza virus strain (centers for disease control and prevention 2017). there are several viral groups other than the orthomyxoviruses (which include the h7n9 strain of influenza a) that are spread by respiratory routes, possess rna genomes, and merit enhanced attention: paramyxoviruses (especially these three genera: respirovirus, henipavirus, and rubulavirus), pneumoviruses, coronaviruses, and picornaviruses (especially these two genera: enterovirus and rhinovirus). based on our analysis and their inherent characteristics, these viral groups are the most likely source of a gcbr-level threat. there are efforts under way to construct viral catalogs of as many viruses as possible. the explicit aim of these projects is to reduce the uncertainty of outbreaks by extensively cataloging as many viral species as possible, so that a virus that causes a disease is less likely to be truly unknown. at the meeting and interviews for this project, a number of experts expressed concern that, while efforts to catalog and broadly sequence viruses in the animal world would provide new scientific discovery, we should not expect that it will identify the source of the next pandemic or that it can change the work being done for pandemic preparedness. broad viral sequencing would uncover many novel viruses. however, the vast majority of discovered viruses will not have the ability to infect humans let alone the prospect of widespread in the population. only a few viruses possess this ability. this work should be pursued with the objective of fundamental viral scientific discovery, rather than the goal of near-term improvement in pandemic preparedness. in the clinical practice of medicine, syndromic diagnosis-that is, making a nonspecific diagnosis, such as "sepsis," "pneumonia," or "viral syndrome," with little to minimal laboratory testing-is the norm. specific diagnosis (i.e., sending patient samples for definitive laboratory diagnosis) is often eschewed if it does not affect clinical management, is costly, and is not revealed with routine tests, and/or if the patient recovers. this practice has become enshrined not only in resource-poor areas in which access to diagnostic testing may be limited, but also in resource-rich areas, like north america and western europe, where specific diagnoses are viewed as superfluous. however, the yield from pursuing an etiologic diagnosis in infectious syndromes such as atypical pneumonia, sepsis, encephalitis, meningitis, and clinically significant fevers of unknown origin may be considerable, as it will provide important insight into the ongoing torrent of threats posed by the microbial world. by causing an infection with enough severity to come to medical attention, the culpable microbes have already established that they are damage-causing pathogens to humans-a feat that only a sliver of the microbial world can accomplish (woolhouse et al. 2016 ). many of these microbial diagnoses cannot be made through the routinely ordered diagnostics. therefore, a special effort would need to be made to get to a microbial diagnosis. if that were to be done more frequently and at a more strategic level around the world, it would provide an opportunity to develop new situational awareness regarding which microbes are circulating and infecting humans-information that is clinically valuable in its own right and more attuned to uncovering gcbr-level pathogens than broad viral cataloging. such efforts should not be limited to exotic "hot spots" of disease emergence but should be practiced in localities that are broadly representative of where these conditions occur. particular hot spots of emergence due to the presence of unique risk factors may be higher yield overall, but they should not be the sole sites of investigation. infectious disease emergence can occur anywhere, as evidenced by the 2009 h1n1 pandemic, which was first recognized as the etiology behind a mild pediatric upper respiratory infection in california and west nile fever emerging in cases of undifferentiated encephalitis in the new york city metropolitan area in the late 1990s (centers for disease control and prevention 2009; nash et al. 2001) . such a program would have significant cost and infrastructure implications in resource-constrained regions, so it would be most logical to set up sentinel or strategic sites for pursuing this level of microbial diagnosis in ways that are broadly representative. in developed nations such as the usa, these programs are available but underutilized because of lack of awareness or perceived lack of value by clinicians, for whom it will often not likely change therapeutic decisions. many participants in the project voiced the view that any microbe's pandemic potential could be substantially enhanced by human factors and poor preparedness, which could exacerbate a pathogen's spread or damage-causing potential. specific issues identified included gaps in hospital preparedness, medical countermeasure manufacturing capacity, medical countermeasure manufacturing locations, impacts on critical workforce members, and cascading effects on vital programs such as food production. for example, concentration of intravenous fluid manufacturing plants in puerto rico created massive shortages after a hurricane took the plants offline in 2017 (wong 2017) . the inability of hospitals to surge to meet enhanced patient needs for ventilators or icu beds is another potential constraint. human factors could also take the form of mistaken actions that are based on political considerations but are not supported by an evidence-based medical rationale, or scientific mistakes based on human error, such as misidentifying a microbe or misinterpretation of scientific or epidemiologic data. for example, early in the sars outbreak, mistakes regarding the etiology of the viral agent occurred, and the 2014 west african ebola outbreaks were initially thought to be cholera, delaying response efforts for months (world health organization 2014). some participants in this study were of the view that such factors as these could outweigh any intrinsic property possessed by a microbe or any physiologic vulnerability possessed by a human. magnification by human error could cause delays in response or awareness, allowing a pathogen to spread wider and deeper into the population and rendering containment more difficult, sowing panic, and severely stressing the healthcare infrastructure of a region. the majority view, however, was that intrinsic microbial characteristics are the main driver of a microbe's ability to cause a pandemic. pandemic preparedness should place a high priority on preparing for rna viral threats, given their frequent spread by respiratory route, cytoplasmic replication, and high mutability. surveillance, science, and countermeasure development programs and efforts should logically allocate significant resources to this class of microbes. except for influenza and certain coronaviruses, there are not major preparedness efforts being made for other viruses in this class of microbes. while rna viruses were at the top of the list of concerns, other classes of microbes, such as bacteria, fungi, and protozoa, should not be completely dismissed given characteristic that pose special concerns. cultivating and maintaining expertise in the epidemiology, surveillance, and pathogenicity of all classes of microbes, with explicit incorporation of a one health approach-which incorporates and integrates information from infectious diseases of plants, amphibians, and reptiles-will help foster the broad capacities needed for emerging pandemic and global catastrophic biological risks. pathogen-based lists, both usa and global, based on influenza precedents, historical biological weapon programs, and emerging infectious diseases were responsible for galvanizing early activities in the field of pandemic preparedness and have helped drive many important contributions. but these lists could create a sense of confidence regarding the prediction of future pandemic threats. lists can become frozen in the minds of those in the field and may be viewed as exhaustive rather than as starting points. additionally, inclusion in lists could also be sought for political (and not epidemiologic) reasons if inclusion carries with it the prospect of enhanced funding for a long-neglected endemic problem. one of the chief rationales behind this project was to attempt to move away from a strict list-based approach when considering pandemic threats and to develop a framework grounded in the facts of a microbe's biology and epidemiology. we recommend that risk assessment be rooted in the actual traits that confer pandemic or global catastrophic biological risks as opposed to a pathogen's presence on some earlier developed list. as respiratory-borne rna viruses have been identified as possessing heightened pandemic potential, it is important to strengthen surveillance activities around these viruses where they currently exist and establish them where they are not yet in place. currently, of the respiratory-borne rna viruses, only influenza and certain coronaviruses receive high priority for surveillance. while some efforts to understand coronaviruses, in the wake of sars and mers, exist, there is no systematic laboratory surveillance of coronavirus infections in humans. similarly, no such program exists for rhinoviruses, parainfluenza viruses, rsv, metapneumoviruses, and similar viruses. since this class of viruses is most likely to hold the future pandemic pathogen, constructing an influenza-like surveillance approach that better characterizes the prevalence, patterns, and geographic distribution of these viruses should be a priority. such an approach would focus on human infections, characterizing the epidemiology, virologic features, antiviral susceptibility (if applicable), and clinical manifestations in a fashion that mimics the extensive influenza surveillance conducted by the cdc and other international entities. currently, outside of anti-influenza antivirals, there is only one fda-approved antiviral for the treatment of respiratory-spread rna viruses (ribavirin). of the six fda-approved influenza antivirals-amantadine, rimantadine, baloxavir, zanamivir, oseltamivir, and peramivir-all target influenza viruses specifically and have no activity outside influenza, with two influenza a-specific agents (amantadine and rimantadine) rendered virtually obsolete because of resistance. the other antiviral agent (inhaled ribavirin) is approved for the treatment of respiratory syncytial virus (rsv) but has very limited use due to poor efficacy and major toxicity concerns for both rsv and parainfluenza viruses. there are currently no approved antivirals for any other respiratory-spread rna viruses in the world. prioritization of antiviral compounds against this group of viruses may lead to acceleration of drug development and (government and nongovernment) incentivizing programs. such antiviral compounds would have an advantage over many other emerging infectious disease countermeasures: these viruses exact a considerable toll in the form of community infections each year, providing a basis for a traditional pharmaceutical market as well as one for emerging infectious disease. pursuing not only broad-spectrum rna antivirals, but also those specifically targeted to specific viruses such as rsv, would increase the likelihood of yield. nontraditional molecules, such as monoclonal antibodies and immunomodulators, should also be investigated for a role in the treatment and prevention of rna virus respiratory infections (walker and burton 2018) . such adjunctive treatments may lead to improved clinical outcomes. to date, only one virally targeted monoclonal antibody is fda-approved: pavalizumab for prevention in high-risk infants. as with the above discussion regarding antivirals, the need for vaccines against respiratory-borne rna viruses should also be prioritized. currently, aside from influenza, for which a moderately effective but technically limited vaccine exists, there are no other vaccines for respiratory-borne rna viruses. experimental vaccines targeting rsv have made it into late clinical development only to fail. several important initiatives in this realm do exist and could be augmented to move beyond specific targets that have already been recognized. for example, the coalition for epidemic preparedness innovations (cepi) has selected a coronavirus (mers-cov) and a paramyxovirus (nipah) for vaccine development incentivizing (røttingen et al. 2017) . such a program could, in potential future initiatives, select additional vaccine targets from this group of viruses and even encourage the development of broadly protective vaccines against groups of viruses-for example, a vaccine that protects against all four strains of human parainfluenza viruses, both mers and sars covs, and both hendra and nipah viruses. additionally, the heightened interest at the national institutes of health (nih) in a universal influenza vaccine in the wake of the moderately severe 2017-18 influenza season should be channeled to provide significantly increased resources to this endeavor (paules et al. 2017) . as certain avian influenza viruses are of the highest threat tier, a universal influenza vaccine (even one that just protects against a strains) could substantially hedge against an influenza virus attaining gcbr status. as was evident during the 2009 influenza pandemic and subsequent influenza seasons, the treatment of influenza is suboptimal, despite evidence-based guidance. the status of the treatment for other respiratory viruses is even less defined. while there currently is not a robust antiviral armamentarium against these viruses, there are important clinical questions that occur with their treatment that merit further study. for example, what adjunctive therapies are useful? what coinfections may be present? at what stage of illness are rescue oxygenation devices warranted? as many of these viruses are highly prevalent in the community and are frequently encountered by clinicians in both outpatient and inpatient settings, finding answers to these questions would render clinicians more adept at dealing with pandemic versions of these viruses. with respect to influenza, there is a growing literature on the use of antiviral agents in combination with anti-inflammatory agents such as nonsteroidal anti-inflammatory agents (nsaids) and macrolide antibiotics (hung et al. 2017) . untangling the nuances of these treatment effects in order to develop robust guidance would have an impact on the ability to cope with an influenza-driven gcbr. because of the higher likelihood that a gcbr-level threat might emerge from the group of rna viruses with respiratory-spread, special attention to research on these agents is warranted if such research could increase pandemic risks. while much research on this class of viruses would be low risk and managed by appropriate approaches to biosafety, experimentally engineered antiviral resistance, vaccine resistance, or enhanced transmission, for example, would raise major biosafety and biosecurity concerns. the 1977 appearance of the h1n1 influenza a strain was thought to have resulted from laboratory escape (zimmer and burke 2009) . it is important to understand the kinds of work being performed with these agents and, in particular, to know of experiments that are being done or are being proposed that would result in increased pandemic risks. those experiments should have their own special review and approval process that is consistent with the risks and assesses the risks and benefits of this work before approval or funding of this work. as unknown infectious syndromes abound in all locations, and any given infectious syndrome may have as its etiology a potentially unknown or unappreciated microbe, specific diagnosis should be a routine endeavor. atypical pneumonias, central nervous system infections, and even upper respiratory infections often are treated without any etiologic agent being identified. as diagnostic technologies and devices improve in breadth, speed, and ease of use, the increasing uptake of these devices will provide a new opportunity to enhance situational awareness of an infectious syndrome in any location where they are deployed. such devices are currently being used in research projects in the developing world. the more routine use of devices, such as multi-analyte molecular diagnostic devices, has the capacity to provide a fuller picture of the microbiological epidemiology of any given syndrome, illuminating what has heretofore been biological dark matter (doggett et al. 2016; kozel and burnham-marusich 2017) . coupled with heightened surveillance of respiratory-borne rna viruses, the ability to capture an early signal of a potential pandemic pathogen will be greatly enhanced. to date, certain considerations have limited the uptake and use of these devices: cost, perceived lack of clinical impact, and constraints on hospital resources such as isolation beds. impacts on hospitals might be noted in laboratory testing volume as well as costs. however, when these devices are viewed in the context of pandemic preparedness, the cost-effectiveness calculation should change. these considerations could be moderated if they are considered part of a hospital's emergency preparedness activities and not exclusively as clinical (they also have benefit for antibiotic stewardship activities in both inpatient and outpatient settings). in fact, the use of these devices should be considered on a par with mechanical ventilators, vaccines, antivirals, and antibiotics in the context of pandemic preparedness. pilot projects demonstrating the feasibility of procuring such devices for infectious disease emergency preparedness could be conducted. understanding the microbial characteristics most importantly regarding the risks of pandemic or global catastrophic biological threats can help strengthen pandemic preparedness activities. while rna viruses pose the greatest risks, there are characteristics of other microbial classes that cause special concerns and are important to consider in scientific research agendas and in public health preparedness efforts. this analysis leads to a series of recommendations related to disease surveillance, antiviral and vaccine development, clinical research, and research oversight. taken together, assessment of key microbial class characteristics plus the focused actions that follow this assessment can broadly help improve preparedness for pandemic and global catastrophic risks. human prion diseases: surgical lessons learned from 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malaria vectors the exceptional oncogenicity of htlv-1 influenza: the mother of all pandemics passive immunotherapy of viral infections: 'super-antibodies' enter the fray space flight alters bacterial gene expression and virulence and reveals a role for global regulator hfq hospitals face critical shortage of iv bags due to puerto rico hurricane. the guardian rna viruses: a case study of the biology of emerging infectious diseases assessing the epidemic potential of rna and dna viruses ground zero in guinea: the ebola outbreak smouldersundetected-for more than 3 months historical mammal extinction on christmas island (indian ocean) correlates with introduced infectious disease broad-spectrum antiviral agents historical perspective-emergence of influenza a (h1n1) viruses key: cord-346906-1wmp43ti authors: lewandowski, kuiama; xu, yifei; pullan, steven t.; lumley, sheila f.; foster, dona; sanderson, nicholas; vaughan, alison; morgan, marcus; bright, nicole; kavanagh, james; vipond, richard; carroll, miles; marriott, anthony c.; gooch, karen e.; andersson, monique; jeffery, katie; peto, timothy e. a.; crook, derrick w.; walker, a. sarah; matthews, philippa c. title: metagenomic nanopore sequencing of influenza virus direct from clinical respiratory samples date: 2019-12-23 journal: j clin microbiol doi: 10.1128/jcm.00963-19 sha: doc_id: 346906 cord_uid: 1wmp43ti influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. we therefore set out to apply the oxford nanopore technologies sequencing method to metagenomic sequencing of respiratory samples. we generated influenza virus reads down to a limit of detection of 10(2) to 10(3) genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (p = 4.7 × 10(−5)). applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [c(t)] values, <30) and 6/13 samples with low viral titers (c(t) values, 30 to 40). no false-positive reads were generated from 10 influenza virus-negative samples. thus, nanopore sequencing operated with 83% sensitivity (95% confidence interval [ci], 67 to 93%) and 100% specificity (95% ci, 69 to 100%) compared to the current diagnostic standard. coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. however, at high influenza virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. we also detected a human coronavirus coinfection in one clinical sample. while further optimization is required to improve sensitivity, this approach shows promise for the nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses. adults, infants, young children, pregnant women, those with underlying lung disease, and the immunocompromised (9) . the burden of disease disproportionately affects low-/middle-income settings (10) . influenza virus diagnostics and surveillance are fundamental to identify the emergence of novel strains, to improve the prediction of potential epidemics and pandemics (4, 8) , and to inform vaccine strategy (11) . diagnostic data facilitate real-time surveillance, can underpin infection control interventions (12, 13) , and can inform the prescription of neuraminidase inhibitors (nai) (9) . currently, most clinical diagnostic tests for influenza virus depend on detecting viral antigen or on pcr amplification of viral nucleic acid derived from respiratory samples (14) . these two approaches offer trade-offs in benefits, as follows: antigen tests (including point-of-care tests [poct] ) are typically rapid but have low sensitivity (15) (16) (17) , while pcr is more time-consuming but more sensitive (9) . irrespective of the test used, most clinical diagnostic facilities report a nonquantitative (binary) diagnostic result, and the data routinely generated for influenza diagnosis have limited capacity to inform insights into epidemiological linkage, vaccine efficacy, or antiviral susceptibility. on these grounds, there is an aspiration to generate new diagnostic tests that combine speed (incorporating the potential for poct [18, 19] ), sensitivity, detection of coinfection (20, 21) , and generation of quantitative or semiquantitative data that can be used to identify drug resistance and reconstruct phylogeny to inform surveillance, public health strategy, and vaccine design. the application of oxford nanopore technologies (ont) sequencing to generate full-length influenza virus sequences from clinical respiratory samples can address these challenges. ont offers a "third-generation," portable, real-time approach to generating long-read single-molecule sequence data, with demonstrated success across a range of viruses (20, (22) (23) (24) . to date, nanopore sequencing of influenza virus has been reported using high-titer virus from an in vitro culture system, producing full-length genome sequences through direct rna sequencing (25) , or using targeted enrichment by either hybridization of cdna (26) or influenza virus-specific pcr amplification (27) . we therefore aimed to optimize a metagenomic protocol for detecting influenza viruses directly from clinical samples using nanopore sequencing. we determine its sensitivity compared to that of existing diagnostic methods and its accuracy compared to short-read (illumina) sequencing, using clinical samples from hospital patients during an influenza season and samples from a controlled laboratory infection in ferrets. further optimization is required before the nanopore method can be rolled out as a diagnostic test, but we highlight the potential impact of this technology in advancing molecular diagnostics for respiratory pathogens. study cohort and sample collection. we collected respiratory samples from the clinical microbiology laboratory at oxford university hospitals nhs foundation trust, a large tertiary referral teaching hospital in southeast england. we worked with anonymized residual material from throat and nose swabs generated as a result of routine clinical investigations between january and may 2018. samples were collected using a sterile polyester swab inoculated into 1 to 3 ml of sterile viral transport medium (vtm), using a standard approach described on the cdc website (28) . during the study, respiratory samples submitted to the clinical diagnostic laboratory were routinely tested by a pcr-based test using the genexpert assay (cepheid) to detect influenza a and b viruses and respiratory syncytial virus (rsv). the workflow is shown in fig. 1 . samples from patients in designated high-risk locations (hematology, oncology, and critical care) were tested using the biofire filmarray (biomérieux) to detect an expanded panel of bacterial and viral pathogens. quantitative data (cycle threshold [c t ]) were generated by the genexpert assay, and we used the influenza virus c t value to estimate the viral titers in clinical samples. using the genexpert assay, up to 40 pcr cycles are performed before a sample is called negative (i.e., positives have a c t value of ͻ40). quantification was not available for the biofire results. for methodological assessment, we focused on four categories of samples, as follows: positive pool, negative pools, individual positive samples, and individual negative samples. for the positive pool, we pooled 19 throat swab samples that had tested positive for influenza a virus in the clinical diagnostic laboratory to provide a large enough sample to assess reproducibility (fig. 1b) . for the negative pools, we generated three pools of throat swab samples that had tested negative for influenza virus (consisting of 24, 38, and 38 individual samples) (fig. 1b) . for the individual positive samples, we included 40 individual samples (35 throat swabs and 5 nasal swabs) that had tested positive for influenza a or b virus, selected to represent the widest range of genexpert assay c t values (13.5 to 39.3; valid test result range, 12 to 40). for the individual negative samples, we selected 10 individual throat swab samples that were influenza virus negative. quantification of viral rna in samples. we quantified viral titers in hazara virus stocks and pooled influenza a virus-positive throat swabs by quantitative reverse transcription-pcr (qrt-pcr), using previously described assays and standards (29, 30) . optimization of methods. prior to establishing the protocol detailed in full below, we assessed the impact of two possible optimization steps, centrifugation versus filtration and reduced time for cdna synthesis. for centrifugation versus filtration, we investigated two approaches to deplete human/ bacterial nucleic acid from our samples, i.e., filtration of the raw sample via a 0.4-m filter (sartorius) before further processing versus using a hard spin (16,000 ϫ g for 2 min). cdna libraries for this comparison were produced as described previously (20) . for the reduced time for cdna synthesis, to assess the possibility of time saving in the cdna synthesis steps, we compared performance of the previously described protocol (20) to that of a modified version with two alterations, first using superscript iv (thermo fisher) in place of superscript iii (thermo fisher) for reverse transcription, with the incubation time reduced from 60 min to 10 min at 42°c, and second, reducing the cdna amplification pcr extension cycling time from 5 min to 2 min. positive control. prior to nucleic acid extraction, each sample was spiked with hazara virus virions to a final concentration of 10 4 genome copies per ml as a positive internal control. this is an enveloped negative-stranded rna virus (genus orthonairovirus, order bunyavirales) with a trisegmented genome of 11,980, 4,575, and 1,677 nucleotides in length (genbank accession numbers kp406723 to kp406725). it is nonpathogenic in humans and would therefore not be anticipated to arise in any of our clinical samples. cultured virions from an sw13 cell line were provided by the national collection of pathogenic viruses (ncpv; catalog no. 0408084v). nucleic acid extraction. samples were centrifuged at 16,000 ϫ g for 2 min. the supernatant was eluted without disturbing the pelleted material and was used in nucleic acid extraction. total nucleic acid was extracted from 100 l of supernatant using the qiaamp viral rna kit (qiagen) eluting in 50 l of h 2 o, followed by a dnase treatment with turbo dnase (thermo fisher scientific) at 37°c for 30 min. rna was purified and concentrated to 6 l using the rna clean & concentrator-5 kit (zymo research), following the manufacturer's instructions. randomly amplified cdna was prepared for each sample using a sequence-independent single-primer amplification (sispa) approach, adapted from our previously described workflow (20) , based on the round a/b methodology (23) . for reverse transcription, 4 l of rna and 1 l of primer a (5=-gtttcccactggaggata-n9-3=, 40 pmol/l) (23) were mixed and incubated for 5 min at 65°c and then cooled to room temperature. first-strand synthesis was performed by the addition of 2 l superscript iv first-strand buffer, 1 l of 12.5 mm dinucleoside triphosphates (dntps), 0.5 l of 0.1 m dithiothreitol (dtt), 1 l h 2 o, and 0.5 l superscript iv (thermo fisher) before incubation for 10 min at 42°c. second-strand synthesis was performed by the addition of 1 l sequenase buffer, clinical sample collection (orange), clinical diagnostic testing (yellow), sample processing and sequencing using oxford nanopore technologies (blue), and processing of sequence data (purple). (b) outline of pooled influenza virus-positive samples into an influenza virus-negative background to generate various titers of influenza virus (from 0 to 10 6 genome copies/ml), undertaken in triplicate, and spiked with a standard titer of hazara virus control at 10 4 genome copies/ml. flua, influenza a virus. 3.85 l h 2 o, and 0.15 l sequenase (affymetrix) prior to incubation for 8 min at 37°c, followed by the addition of 0.45 l sequenase dilution buffer and 0.15 l sequenase and a further incubation at 37°c for 8 min. amplification of cdna was performed in triplicate using 5 l of the reaction mixture as input to a 50-l accutaq la (sigma) reaction mixture, according to the manufacturer's instructions, using 1 l primer b (5=-gtttcccactggaggata-3=) (23) , with pcr cycling conditions of 98°c for 30 s, 30 cycles of 94°c for 15 s, 50°c for 20 s, and 68°c for 2 min, followed by 68°c for 10 min. amplified cdna was pooled from the triplicate reaction mixtures, purified using a 1:1 ratio of ampure xp beads (beckman coulter, brea, ca), and quantified using a qubit high-sensitivity double-stranded dna (dsdna) kit (thermo fisher), both according to the manufacturers' instructions. nanopore library preparation and sequencing. multiplex sequencing libraries were prepared using 250 ng of cdna from up to 12 samples as input to the sqk-lsk108 or sqk-lsk109 kit and barcoded individually using the exp-nbd103 native barcodes (oxford nanopore technologies) and a modified one-pot protocol (https://www.protocols.io/view/one-pot-ligation-protocol-for-oxford-nanopore-libr -k9acz2e). libraries were sequenced on flo-min106 flow cells on the minion mk1b or gridion device (oxford nanopore technologies), with sequencing proceeding for 48 h. samples were batched according to the genexpert c t value (see file s1 in the supplemental material). illumina methods. nextera xt v2 kit (illumina) sequencing libraries were prepared using 1.5 ng of amplified cdna, as per the manufacturer's instructions, and sequenced on a 2 ϫ 150-bp paired-end illumina miseq run by the genomics services development unit of public health england. bioinformatic analysis. nanopore reads were base called using guppy (oxford nanopore technologies, oxford, uk). output base called fastq files were demultiplexed using porechop v0.2.3 (https:// github.com/rrwick/porechop). the reads were first taxonomically classified against the refseq database using centrifuge v1.0.3 (31) . the reads were then mapped against the reference sequence selected from the centrifuge report using minimap2 v2.9 (31, 32) . a draft consensus sequence was generated by using a majority voting approach to determine the nucleotide at each position. the resulting draft consensus sequences were subjected to a blast search against an influenza virus sequence database that included ͼ2,000 h1n1 and h3n2 seasonal influenza virus sequences between 2018 and 2019 and were downloaded from the influenza research database (33) . the reads were again mapped against the reference sequence using minimap2 v2.9, and the number of mapped reads was calculated using samtools v1.5 (34) and pysam (https://github.com/pysam-developers/pysam). the subtype of the influenza a virus derived from each clinical sample was determined by the subtypes of the ha and na reference sequences. a consensus sequence was built using nanopolish v0.11.0 (35, 36) and the margin_cons.py script (37) (https://github.com/zibraproject/zika-pipeline). for the illumina data, reads were quality trimmed to a minimum score of q30 across the read with trimmomatic (38) . bwa-mem v0.7.15 (39) was used to align the reads to reference genomes using mem defaults. samtools v1.4 was used to compute the percent reads mapped and coverage depth (34) . mapping consensuses for illumina sequencing were generated using quasibam (40) . maximum likelihood phylogeny was generated for the ha gene segment using raxml v8.2.10 (41), in which a general time-reversible model of nucleotide substitution and a gamma-distributed rate variation among sites were applied. sequence alignments were performed by using muscle v3.8 (42) . ferret study. we applied our sequencing approach to residual samples collected in a previous time course experiment undertaken in a controlled laboratory environment (43) . we tested ferret nasal saline wash samples from three independent animals over an 8-day time course, from 3 days prior to first exposure with influenza h1n1pdm09 virus and at days 1, 2, 3, and 5 postinfection. sampling and plaque assays of the viral titer were described previously (43) . ethics approval. the study of anonymized discarded clinical samples was approved by the london-queen square research ethics committee (17/lo/1420). ferret samples were residual samples from an existing study (43) for which the project license was reviewed by the local animal welfare and ethics review board of public health england (porton) and subsequently granted by the home office. data availability. following the removal of human reads, our sequence data have been uploaded to the european bioinformatics institute (https://www.ebi.ac.uk/) under bioproject number prjeb32861. method optimization to increase the proportion of viral reads derived from throat swabs. our method protocol is shown in fig. 1a . we first sequenced five influenza a virus-positive and five influenza virus-negative throat swabs, each spiked with hazara virus control at 10 4 genome copies/ml. using a sequence-independent single-primer amplification (sispa) approach (20), followed by nanopore sequencing, we produced metagenomic data dominated by reads that were bacterial in origin, with extremely few viral reads detected. passing the sample through a 0.4-m filter prior to nucleic acid extraction increased the detection of viral reads by several orders of magnitude (fig. s1 ). filtration is relatively expensive, so we also assessed the alternative approach of adding a rapid-centrifugation step to pellet bacterial and human cells, followed by nucleic acid extraction from the supernatant. we used a pooled set of influenza a virus-positive samples (concentration, 10 6 genome copies/ml) to provide a large enough sample to assess reproducibility, with the hazara virus control spiked in at 10 4 genome copies/ml. enrichment for influenza virus and hazara virus was similar for filtration versus centrifugation, based on read mapping to the viral genome (fig. s2 ). as centrifugation is simpler and less expensive, we selected this approach for all further testing. method optimization to reduce time for cdna synthesis. synthesis of tagged randomly primed cdna and its subsequent amplification via sispa (20) required lengthy reverse transcription and pcr steps (1 h and 3 h 45 min), respectively. optimizing these stages upgraded the reverse transcriptase from superscript iii to superscript iv (thermo fisher), reduced the incubation time to 10 min (processing time reduction, 50 min), and reduced the pcr extension time within each cycle from 5 min to 2 min (1 h 30 min processing time reduction). comparing this final method with our original protocol, using triplicate extractions from the pooled set of influenza a virus-positive samples demonstrated no significant loss in performance in the more rapid protocol (fig. s3) , and we adopted this approach as our routine protocol, giving a wet-lab processing time of ϳ8 h. consistent retrieval of hazara virus by nanopore sequencing. starting with an influenza a virus-positive sample pool (10 6 genome copies/ml), we made three volumetric dilution series using three independent influenza virus-negative pools (fig. 1b) . the total quantity of cdna after preparation for sequencing was consistently higher in all samples using negative pool 3 as the diluent ( fig. 2a) , indicating the presence of a higher concentration of nonviral rna within pool 3. this is likely due to host cell lysis or higher bacterial presence and demonstrates the variable nature of throat swab samples. we consistently retrieved hazara virus reads from all three dilution series by nanopore sequencing, independently of influenza virus titer in the sample (fig. 2b) . sequencing from dilution series 1 and 2 gave a consistent proportion of total reads mapping to the hazara virus genome, across dilutions and between the first two pools, with mean ϯ standard deviation values per pool of 1.4 ϫ 10 3 ϯ 660 reads per million (rpm) of total reads and 1.2 ϫ 10 3 ϯ 350 rpm, respectively. the pool 3 dilution series generated 260 ϯ 340 rpm hazara virus reads across samples and showed a decreasing trend associated with increased dilution factor as increasingly more nonviral rna was introduced from this high-background pool. limit of influenza virus detection by nanopore sequencing from pooled samples. nanopore sequencing of the triplicate sispa preparations of the influenza a virus-positive pool produced mean ϯ standard deviation of 5.3 ϫ 10 4 ϯ 3.6 ϫ 10 4 rpm mapping to the influenza a virus genome (fig. 2b) . across the dilution series, the proportion of influenza virus reads was strongly associated with influenza virus titer (p value ϭ 4.7 ϫ 10 ϫ5 ) but was also influenced by which negative pool was used for dilution, consistent with the pattern observed for the hazara virus control. sequencing the negative controls (pools with no influenza virus spike) generated no reads mapping to influenza virus. at influenza virus titers of ͻ10 3 copies/ml, influenza virus reads were inconsistently detected across the samples (fig. 2b) , suggesting that the limit of detection is between 10 2 and 10 3 influenza virus copies/ml. retrieval and reconstruction of complete influenza virus genomes from pooled/spiked samples. for the hazara virus control (10 4 genome copies/ml spike), genome coverage was 81.4 to 99.4% (at 1ϫ depth) for pools 1 and 2. coverage in the high-background pool 3 was more varied (21.5 to 96.5%; fig. 3a ). influenza a virus genome coverage at 10 6 copies/ml was ն99.3% for each segment in all samples (fig. 3a) . at 10 4 genome copies/ml of influenza virus, a mean 1ϫ coverage per segment was 90.3% for pools 1 and 2 but was substantially reduced in the high-background pool 3 to 5.7% (fig. 3a) . at influenza virus titers of ͻ10 4 copies/ml, coverage was highly varied across genome segments. however, when present at 10 3 copies/ml, 2/3 pools had sufficient data for correct subtyping as h3n2 (table 1) . having demonstrated our ability to retrieve influenza virus sequences from pooled influenza virus-positive material diluted with negative samples, we next applied our methods to individual anonymized clinical samples, with 40 samples testing influenza virus positive and 10 samples testing influenza virus negative in the clinical diagnostic laboratory. data yield varied between flow cells (range, 2.5 ϫ 10 6 to 13.2 ϫ 10 6 reads from up to 12 multiplexed samples). within flow cells, barcode performance was inconsistent when using a stringent, dual-barcode, demultiplexing method (21) . from each clinical sample, the range of total reads generated was 1.0 ϫ 10 5 to 2.4 ϫ 10 6 (median, 3.8 ϫ 10 5 reads) (table s1) . reads mapping to either the influenza a or b virus genome were present in all 27 samples with a c t of ͻ30 (range, 6 to 274,955 reads). at a c t of ͼ30, 6/13 samples generated influenza virus reads (range, 6 to 92,057 reads) (difference between sensitivity at a c t threshold of 30, p ͻ 0.0001; fig. 4 ). the highest c t value at which any influenza virus reads were detected was 36.8 (sample 37; 17 reads of influenza a virus). no reads classified as influenza virus were obtained from sequencing the 10 genexpert assay-negative samples (table s1 ). based on this small data set, sensitivity is 83% and specificity is 100% (95% ci, 67 to 93% and 69 to 100%, respectively). there was a strong correlation between c t value and both the reads per sample classified as influenza virus (r 2 ϭ 0.60) and the number of influenza virus reads per million reads (r 2 ϭ 0.62) (fig. 4) . the consensus genome sequences generated (at 10ϫ minimum depth) covered over 90% of the influenza virus genome for 17 samples, with another two generating over 80% coverage. the highest c t value of a sample from which ͼ90% of an influenza virus genome sequence was generated was 27.5 (fig. s4) . (table s1 ). four (8%) of 50 samples generated no detectable hazara virus reads, two with high numbers of influenza virus reads (for sample 1, c t of 13.5 and 1.5 ϫ 10 5 influenza b virus reads, and for sample 6, c t of 18.4 and 1.5 ϫ 10 4 influenza a virus reads) acting to dilute the control signal. the other two samples contained no detectable influenza virus reads (for sample 34, c t of 35.9, and for sample 46, influenza virus negative). the lack of control detection therefore indicates a loss of assay sensitivity due to high levels of background nucleic acid present in some samples. comparison of nanopore and illumina sequencing. we selected a subset of 15 samples from across the viral titer range and resequenced on an illumina miseq platform. the proportions of reads generated that mapped to the influenza virus genome were similar between the two sequencing technologies (fig. s5) . from 4 of the samples, nearly complete genomes were obtained. a comparison of consensus sequences derived from nanopore and illumina sequencing showed 100% concordance, except one sample that showed 7 nucleotide differences (identity, 99.94%) (table s2) . influenza virus phylogeny. we reconstructed the phylogeny using consensus sequences for the ha gene (fig. 5) . this demonstrates closely related sequences, as expected within one geographic setting in a single influenza season. detection of other rna viruses in clinical samples. within the 50 clinical samples sequenced, we found limited evidence for the presence of other rna viruses. sample 6 produced 109 reads mapping to human coronavirus in addition to ͼ1.5 ϫ 10 4 influenza a virus reads, suggesting coinfection. we also derived ͼ4.0 ϫ 10 4 reads from human metapneumovirus from an influenza virus-negative sample, providing a nearly complete genome (99.8% coverage) from one sample (fig. s1 , sample i), further detailed previously (44) . animal time course study. finally, we used samples collected from a previous animal experiment (43) to test the reproducibility of our methods across a time course model of influenza a virus infection (three ferrets swabbed preinfection [day ϫ3] and then sampled at days 1, 2, 3, and 5 following laboratory infection with influenza a virus). the proportion of viral reads present at each time point was highly congruent with viral titer (titer is shown in fig. 6a and sequencing reads in fig. 6b ). we generated consensus genome sequences from nanopore data at days 2, 3, and 5 postinfection; these were 100% concordant with illumina-derived consensus sequences from the same cdna (table s2) . to our knowledge, this is the first report of successfully applying metagenomic nanopore sequencing directly to respiratory samples to detect influenza virus and generate influenza virus sequences. the approach demonstrates excellent specificity. sensitivity varies by viral titer but is comparable to that of existing laboratory diagnostic tests for c t values of ͻ30. our optimized protocol depletes human and bacterial nucleic acids and reduces the time from sample to sequence. this method has the potential to be further optimized and validated to improve sensitivity for influenza virus, identify other rna viruses, detect drug resistance mutations, and provide insights into quasispecies diversity (45, 46) . at a population level, these sequence-based diagnostic data can, in addition, provide phyloepidemiological reconstruction, insights into transmission events, the potential to estimate vaccine efficacy (47) , and approaches for public health intervention (48) . whole-genome viral sequencing, coupled with phylogenetic analysis and appropriate clinical metadata, can contribute to the accurate tracking of outbreaks across time and space (49) . the metagenomic method employed here produced ͼ90% complete genomes for 17/27 samples with a c t value of յ30 (fig. s4) , demonstrating the ability of metagenomics to produce sufficient data for influenza virus diagnostics and genome characterization, while also detecting and sequencing other common rna viruses. despite time reductions in wet-laboratory processing, this method requires further modification to simplify and accelerate the protocol if it is to become viable as a near-to-patient test. high error rates are a recognized concern in nanopore sequence data, and cross-barcode contamination can create challenges when low-and high-titer samples are batched (21) . to avoid these problems, we batched samples according to c t value and applied stringent barcode demultiplexing criteria; however, this reduces the total data available for analysis, typically by ϳ50% but with variation between sequencing runs (21) . for future primary diagnostic use, it would be preferable to sequence samples individually using a lower-throughput flow cell, e.g., ont flongle (each paired with a negative-extraction-control sample, for which a prior spike with hazara virus, using the same methods described here, would remain appropriate). careful optimization of laboratory and bioinformatic methods is required to resolve individual sequence polymorphisms, particularly for drug resistance alleles. infectious diseases society of america (idsa) guidelines (9) recommend nasal/ nasopharyngeal specimens for influenza diagnosis, but throat swabs are easier to collect in clinical practice and therefore account for the majority of diagnostic samples processed by our clinical microbiology laboratory. further work is needed to investigate the sensitivity and specificity of our protocol for a wider array of respiratory sample types (also including bronchoalveolar lavage fluid, sputum, and saliva), which may contain different degrees of contaminating bacterial and/or human reads. loss of assay sensitivity due to the presence of high-level background dna from either the host or bacterial origin is a fundamental issue for metagenomic approaches, even in cell-free sample types such as cerebrospinal fluid (50) . this challenge is exacerbated in throat swabs, as seen in our data. our use of hazara virus as an internal positive control allows us to identify those samples in which sensitivity has dropped to ͻ10 4 viral genome copies per ml. in our test set, 8% of samples showed insufficient sensitivity for hazara virus; however, half of these contained a high titer of influenza virus, so only 4% were true sensitivity failures. this figure is in line with the reported 6% failure rate due to high background for rna virus detection from a clinically validated metagenomic sequencing assay for pathogen detection in cerebrospinal fluid (50) . at the higher c t values in our clinical samples (c t , 30 to 40), the sensitivity of nanopore sequencing was reduced compared to that of the current pcr-based test (genexpert assay; cepheid). further optimization will be required to maximize the diagnostic yield from this group of samples without sacrificing specificity. the correlation between c t value and nanopore reads confirms semiquantitative output. using samples from the ferret influenza virus model, collected under standardized laboratory conditions, we demonstrated excellent reproducibility of viral read proportions at a given viral titer across biological replicates. however, we observed heterogeneity in output between clinical samples as well as between nanopore flow cells, suggesting that the current platform is not yet sufficiently reliable for reproducibly generating quantitative data. in addition, the detection of positive controls can be impaired in high-background samples. future application of this method will involve real-time laboratory testing of respiratory samples, running the platform head to head with existing clinical diagnostics to further assess sensitivity and specificity, and using influenza virus sequence data to investigate transmission events. identifying instances of nosocomial transmission may shed light on health care-acquired infection, thus helping to improve infection control practice. assessment of diversity within deep-sequence data sets provides an opportunity to investigate the relationship between within-host polymorphisms and clinical outcomes. long-read sequences confer the potential advantage of identifying viral haplotypes and ascertaining the extent to which significant polymorphisms are transmitted together or independently (24) . we have shown that the method is robust for the identification of commonly circulating influenza virus strains in human populations, but further investigation is required to ascertain the extent to which it performs reliably in other (avian and animal) strains. comparison with existing/alternative approaches. the current standard assay for influenza diagnosis employed within the large tertiary referral teaching hospital in which this study was performed is the genexpert assay (cepheid), which detects influenza a and b viruses and respiratory syncytial virus (rsv). wider testing is performed on a subset of samples using the biofire filmarray respiratory panel (biomérieux), targeted at 20 common respiratory pathogens (17 viruses and 3 bacteria). these assays have the advantages over a metagenomic approach of higher sensitivity, shorter handling times, simpler laboratory workflow, and very rapid time to result (30 and 65 min, respectively). compared to a metagenomic approach, their limitations are that no sequence data informative for molecular epidemiology and drug resistance typing are generated for the target pathogens, and that the assay will only detect the small number of pathogens targeted. periodic refinement of such assays is required in the event of newly emergent pathogens or diverse strains of established pathogens leading to assay escape. this is not an issue affecting metagenomic sequencing, which has the ability to detect all rna viruses in a sequence-independent manner. at the time of undertaking this laboratory work, the materials costs of the metagenomic sequencing were ϳ£140 per sample when multiplexing six samples per flow cell and purchasing 48 flow cells together. the cost of the current influenza a/b and rsv genexpert test is ϳ£56, and the biofire rp panel costs ϳ£139 per sample. the existing tests have the significant advantage of short handling times and simple processing, whereas the metagenomic sequencing requires ϳ8 h and skilled laboratory staff. alternative approaches to generate sequence data include amplicon-based sequencing of the influenza virus genome (51, 52) . however, this approach detects only the target pathogen, requiring multiple assays or more complex multiplex primer schemes to add targets and capture diverse strains of the original target. the use of short-read illumina sequencing instead of nanopore sequencing for metagenomic sequencing of influenza virus (53) provides the current gold standard of sequence quality and some potential cost savings per base of sequence generated. however, our data show that at a relatively modest minimum coverage depth of 10ϫ, nanoporegenerated consensus viral genome sequences are 99.95 to 100% identical to illumina sequences. of the 13 samples compared, 12 samples were 100% concordant. the few bases that differed between the technologies in a single sample appear to be clear in each case and a genuine disagreement between the long-and short-read approaches, rather than simply being due to the higher per-base error rate of nanopore per se. larger and more diverse data sets will be required to set more rigorous thresholds for base calling. currently, it is wise to bear such potential issues in mind when comparing genome sequences generated by different technological platforms. the per-read differences in base accuracy are also compensated for by the increased read length, providing further confidence in the case of individual read taxonomic assignment. consideration of relative costs must also take into account other costsaving attributes. these include the substantially lower infrastructure and startup costs of nanopore sequencing, the unique ability to interrogate sequence data as they are generated in real time, and the potential for the portable minion device to be utilized near to patient, potentially decreasing turnaround time, particularly for high-virus-load samples which may be identified within minutes. the throughput of the nanopore flow cells allows for a small number of samples to be run immediately rather than requiring samples to be batched to reach a number sufficient to cost efficiently run a greaterthroughput short-read sequencing device. in the future, the use of an even lowerthroughput flow cell with the ont flongle adaptor may allow individual samples to be run per cell, offering quicker turnaround per sample and minimizing cross-sample contamination. a summary table comparing the different approaches is included in supplemental material (table s3) . limitations of the method. the current limitations of metagenomic methods are their sensitivity in the context of low-pathogen-titer samples. pcr-based methods measure the absolute count of viral genome copies present within a sample. metagenomic sequencing measures the proportion of total rna that is viral. metagenomic sequencing is therefore affected by the level of nontarget rna within a given sample, whereas pcr is not. as demonstrated here ( fig. 2b and table 1 ), detection of as little as 10 2 genome copies per ml is possible from throat swab samples (a level comparable with pcr-based methods), but variation in the level of background nucleic acids between individual samples makes detection at this level inconsistent. further development of methods to deplete host and bacterial rna within the samples is required to improve the performance of the assay at c t values of ͼ30. enrichment of pathogen sequences within libraries through either target capture or amplification is also an effective method to reduce the limit of target detection (54, 55) but requires the same a priori knowledge of both which pathogens are to be targeted and the full range of circulating viral diversity as other targeted methods discussed above, albeit with increased tolerance for diversity over pcr-based methods. a further limitation compared to alternative sequencing technologies is the lack of confidence in determining the presence of minority variants due to the limited per-read accuracy, although we expect this to be addressed in future iterations of the ont sequencing. in summary, while substantial further work is needed, our methods show promise for generating influenza virus sequences directly from respiratory samples. the "pathogen-agnostic" metagenomic sequencing approach offers an opportunity for simultaneous testing for a wide range of potential pathogens, providing a faster route to optimum treatment and contributing to antimicrobial stewardship. longer term, this approach has promise as a routine laboratory test, providing data to inform treatment, vaccine design and deployment, infection control policies, and surveillance. supplemental material is available online only. supplemental file 1, pdf file, 0.3 mb. the study was funded by the nihr oxford biomedical research centre. computation used the oxford biomedical research computing (bmrc) facility, a joint development between the wellcome centre for human genetics and the big data institute supported by health data research uk and the nihr oxford biomedical research centre. the views expressed in this publication are those of the authors and not necessarily those of the nhs, the national institute for health research, the department of health, or public health england. p.c.m. is funded by the wellcome trust (grant 110110). d.w.c., t.e.a.p., and a.s.w. are nihr senior investigators. the ecology and adaptive evolution of influenza a interspecies transmission the influenza of 1918: evolutionary perspectives in a historical context origins and evolutionary genomics of the 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dynamics of human influenza a virus a naturally protective epitope of limited variability as an influenza vaccine target metagenomic sequencing at the epicenter of the nigeria 2018 lassa fever outbreak the ability of single genes vs full genomes to resolve time and space in outbreak analysis laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid whole genome sequencing of influenza a and b viruses with the minion sequencer in the clinical setting: a pilot study minion nanopore sequencing of an influenza genome a metagenomic analysis of pandemic influenza a (2009 h1n1) infection in patients from north america enhanced virome sequencing using targeted sequence capture capturing sequence diversity in metagenomes with comprehensive and scalable probe design key: cord-304870-j9kadxu9 authors: chen, gongbo; zhang, wenyi; li, shanshan; zhang, yongming; williams, gail; huxley, rachel; ren, hongyan; cao, wei; guo, yuming title: the impact of ambient fine particles on influenza transmission and the modification effects of temperature in china: a multi-city study date: 2016-10-11 journal: environ int doi: 10.1016/j.envint.2016.10.004 sha: doc_id: 304870 cord_uid: j9kadxu9 background: there is good evidence that air pollution is a risk factor for adverse respiratory and vascular health outcomes. however, data are limited as to whether ambient fine particles contribute to the transmission of influenza and if so, how the association is modified by weather conditions. objectives: we examined the relationship between ambient pm(2.5) and influenza incidence at the national level in china and explored the associations at different temperatures. methods: daily data on concentrations of particulate matter with aerodynamic diameter < 2.5 μm (pm(2.5)) and influenza incidence counts were collected in 47 chinese cities. a poisson regression model was used to estimate the city-specific pm(2.5)-influenza association, after controlling for potential confounders. then, a random-effect meta-analysis was used to pool the effects at national level. in addition, stratified analyses were performed to examine modification effects of ambient temperature. results: for single lag models, the highest effect of ambient pm(2.5) on influenza incidence appeared at lag day 2, with relative risk (rr) of 1.015 (95% confidence interval (ci): 1.004, 1.025) associated with a 10 μg/m(3) increase in pm(2.5). for moving average lag models, the significant association was found at lag 2–3 days, with rr of 1.020 (95% ci: 1.006, 1.034). the rr of influenza transmission associated with pm(2.5) was higher for cold compared with hot days. overall, 10.7% of incident influenza cases may result from exposure to ambient pm(2.5). conclusions: ambient pm(2.5) may increase the risk of exposure to influenza in china especially on cooler days. control measures to reduce pm(2.5) concentrations could potentially also be of benefit in lowering the risk of exposure and subsequent transmission of influenza in china. coinciding with china's rapid economic development and urbanization, air pollution has become a severe problem, and poses a major threat to the health of the chinese population. recent estimates suggest that annually air pollution in china kills n 1.5 million people each year, about 17% of the nation's annual deaths (rohde and muller, 2015) . one component of air pollution, in particular, is of increasing concern to public health experts; compared to other components, ambient particulate matter with aerodynamic diameter b 2.5 μm (pm 2.5 ) is able to penetrate deep into the lung and into the circulatory system (s feng et al., 2016) . exposure to pm 2.5 is associated with a wide range of diseases including cardiovascular and respiratory disease (arnold, 2014) . in 2013, the global burden of disease group estimated that exposure to ambient pm 2.5 to be the 12th leading risk factor for the global burden of disease and responsible for 2.9 million deaths and 69.7 million disability adjusted life-years, worldwide (forouzanfar et al., 2015) . despite substantial reductions in the burden of communicable disease in china over the past two decades, it remains a leading cause of death in the country (wang et al., 2008) . of particular concern, both nationally and globally, is the increasing threat posed by emerging infectious diseases (e.g., severe acute respiratory syndrome [sars] and the highly pathogenic avian influenza [hpai] ) in the chinese population, both of which pose a significant public health threat (wang et al., 2008) . few studies have examined the possible effect of ambient pm 2.5 concentrations on risk of communicable disease in china, but those that have reported a positive association between ambient environment international 98 (2017) 82-88 pm 2.5 and localised transmission of influenza (c. huang et al., 2016; liang et al., 2014) . however, until now there has been no large-scale examination of the association due to past unavailability of pm 2.5 data and incident cases of influenza at the national level. neither has there been any detailed exploration of the possible effect of temperature on the transmissibility by ambient pm 2.5 of the influenza virus. in this study, we collected daily data on ambient pm 2.5 levels, weather conditions and influenza incidence from 47 cities in china in order to reliably determine the association between pm 2.5 and influenza incidence and the potential modification effects of ambient temperature on the relationship. 2.1. data collection 2.1.1. influenza data daily data of influenza cases for 47 cities in china from september 9, 2013 to december 31, 2014 were obtained from the china information system for disease control and prevention (cisdcp). china has established a web-based notifiable infectious disease management system linked with health administrative datasets, the disease control institution and the medical and health institutions at five levels: township, county (district), prefecture, province and national (wang et al., 2008) . influenza cases were defined according to technical guides for prevention and control of influenza issued by china ministry of health (http://www.moh.gov.cn/zwgkzt/s9491/200802/38820.shtml): sudden onset of fever ≥ 38°c, cough or sore throat, and absence of other diagnoses. as influenza is a notifiable disease (class c), sentinel hospitals are required to collect nasopharyngeal swabs for each identified case which are subsequently sent to designated laboratories for virus isolation and further identification and results are submitted online within 24 h (liang et al., 2014; shu et al., 2010) . ambient pm 2.5 concentrations were measured during the same time period as influenza data at 76 stations ( fig. s1 in appendix) of the china atmosphere watch network (cawnet) administered by the china meteorological administration in 47 cities. hourly pm 2.5 concentrations were measured using grimm edm 180 environmental dust monitors. further details regarding methods of measurements and the monitoring instruments used have been previously reported (wang et al., 2015) . to link pm 2.5 data with daily influenza case data, hourly pm 2.5 concentrations (c hour ) were converted to daily pm 2.5 concentrations (c daily ¼ ∑ 24 1 c hour =24). city-level average concentrations were calculated if there were two or more stations in one city. daily meteorological data were obtained from the china meteorological data sharing service system of the china meteorological administration (http://data.cma.gov.cn). daily temperature, relative humidity, atmospheric pressure, wind speed, and hours of sunshine, were collected from 70 weather stations in 47 cities during the same period as the data for pm 2.5 and influenza incidence. the average value for each meteorological variable was calculated if there were two or more weather stations within a city. the pm 2.5 -influenza association was assessed using a two-stage analytic approach which has been widely applied in previous studies (gasparrini, armstrong, and kenward, 2012; gasparrini et al., 2015; guo et al., 2014) . in the first stage, the city-specific pm 2.5 -influenza association was examined and in the second stage, a random effect metaanalysis was used to pool the associations at the country level. a time series poisson regression model was used to examine cityspecific estimates allowing for over-dispersed case counts. seasonality was controlled for using a natural cubic spline with seven degrees of freedom for time per year. a categorical variable was used to control for the confounding effect of day of the week. as weather conditions are associated with health outcomes and the impact can last for several days (guo, barnett, pan, yu, and tong, 2011; peng, dominici, and louis, 2006) , we controlled for the potential confounding effects of five meteorological variables (daily mean temperature, relative humidity, air pressure, wind speed, and hours of sunshine) with moving average of the current day and the previous seven days using a natural cubic spline with three degrees of freedom for each of parameters (guo et al., 2013) . to understand the characteristics of the lag associations between pm 2.5 and incident influenza, the associations were examined using a single lag model (from lag 0 to lag 7), and moving average lag model, separately. the city-specific effect estimates of influenza associated with a 10 μg/m 3 increase in pm 2.5 were calculated. to examine whether ambient temperature modified the associations between pm 2.5 and influenza, a stratified analysis was conducted according to daily temperature (for cold days, moderate cold days, moderate hot days, and hot days), by including an interactive term between pm 2.5 and temperature levels (as a categorical variable) in the city-specific regression model. the cold days, moderate cold days, moderate hot days, and hot days were classified by the quartile of 0-7 days' moving average of temperature in each city during the study period. to further examine statistical significance of different effects of pm 2.5 for temperature subgroups, a meta-regression was conducted with the effect estimates of stratumlevel analyses as dependent variable and categorical variable of daily temperature as dependent variable . a meta-analysis was used to pool the city-specific effect estimates obtained from the first-stage model. the meta-analysis was fitted using a random effect model by maximum likelihood, to obtain the national pooled estimates for each lag type, respectively. the pm 2.5 -influenza associations were expressed as the relative risk (rr) and 95% confidence interval (ci) of influenza associated with a 10 μg/m 3 increase in pm 2.5 . considering the modified effects of ambient temperature on pm 2.5incident influenza associations, the attributable fractions were calculated by pooled effect estimates (β) and pm 2.5 concentrations for each temperature strata according to the formulas reported by evans et al. (2013) . we used the following model to assess the daily influenza cases attributed to pm 2.5 for each city: where 'i' is the day of the incident case of influenza; 'ai' is the number of incident influenza cases attributed to pm 2.5 on day 'i'; and 'influenza i ' is the observed influenza cases on day 'i'; the rr is calculated by exp.(β) × moving average concentration of pm 2.5 on day 'i-i 1 ' and day 'i-i 2 ' accounting for the effects of pm 2.5 at lag i 1 -i 2 days; β is pooled effect estimate for different temperature stratum in corresponding to temperature on day 'i'. 95% ci of pooled β were used to calculate the 95% ci of attributable fraction with the above equation. the overall population attributable fraction was assessed by dividing the sum of all city-specific attributable cases by the total number of incident cases. in addition, the attributable fractions were also calculated for each temperature stratum. sensitivity analyses were performed on the parameters for the cityspecific model to test the robustness of our results. we varied the number of lag days to 15 days for meteorological variables, to examine whether using seven lag days was sufficient to control for their effects on influenza. we modified the degrees of freedom for meteorological variables (3-6 df) and for time per year (6-10 df). to further test the robustness of results, multi-pollutant models were conducted by controlling for the effects of ambient no 2 and so 2 . r software (version 3.2.2, r development core team 2009) was used for all data analysis. the "mvmeta" package was used to fit the meta-analyses (gasparrini et al., 2012) . the spatial distribution of ambient pm 2.5 concentrations between chinese cities was geographically heterogeneous (fig. 1) . the highest levels of pm 2.5 (n 64.90 μg/m 3 ) were generally located in cities in the western and northern regions of china and were more inland, in contrast to cities with low levels of pm 2.5 (b 33.50 μg/m 3 ) which were located in the more southern, outer regions of china. among the 47 cities, the city of shijiazhuang in hebei province had the highest pm 2.5 concentration (98.14 μg/m 3 ), while the lowest was recorded in the city of xilingele in inner mongolia (11.90 μg/m 3 ). overall, there were 76,902 incident cases of influenza cases during the time period of the study. the highest incidences of influenza occurred in cities including beijing, shanghai, guangzhou and chongqing, and the lowest incidences were observed in cities in the north eastern and central parts of china (fig. 1) . the highest daily count of new cases of influenza (n = 32) was observed in guangzhou city, guangdong province whereas the lowest was in xianning city, hubei province. a summary of daily pm 2.5 concentrations, incident influenza and the weather conditions for each city are shown in table s1 -s3 of the appendix. table 1 shows the correlations between ambient pm 2.5 and the meteorological variables under investigation. pm 2.5 concentrations were negatively correlated with temperature, relative humidity, wind speed and hours of sunshine significantly (r = − 0.31,-0.03, − 0.25 and −0.12, respectively), but were positively correlated with atmospheric pressure significantly (r = 0.06). there was a strong correlation between relative humidity and hours of sunshine and weak correlations were observed between other meteorological factors. fig. 2 shows the pooled results from the meta-analysis for different single lag models (lag 0 to lag 7). the effect of pm 2.5 on influenza was significant at lag days 2 and 3 (rr: 1.015 and 1.013; and 95% ci: 1.004, 1.025 and 1.003, 1.023, respectively). thus, a moving average lag model at lag 2-3 days was performed to examine the cumulative effect across these two days. fig. 3 shows the effect of ambient pm 2.5 on influenza at lag 2-3 days for each individual city and the overall pooled estimate at the national level. the effect estimates varied by city, with strong and significant effects observed in several cities in southwest china and central china including guilin, xianning, kunming and guangzhou. however, weak and insignificant effects were observed in cities located in northeast china and south china including shenyang, changchun and shenzhen. for the pooled estimate, the increased risk of influenza was significantly associated with an increase of 10 μg/m 3 in pm 2.5 at lag 2-3 days (rr: 1.020, 95% ci: 1.006, 1.034). fig. 4 shows the results for the stratified analyses by temperature levels per 10 μg/m 3 increase in pm 2.5 at lag day 2, lag day 3 and lag days' 2-3. significant associations at lag days' 2-3 for cold days were observed (rr: 1.027, 95% ci: 1.010, 1.044), and a weaker, non-significant association was observed for hot days (rr: 0.982, 0.943, 1.023). for different lag types, the strongest effects were observed for cold days and the weakest effects for hot days. the effects on moderate cold and moderate hot days were similar. table 2 shows the number and fraction of incident influenza that might related to ambient lag days' 2-3 pm 2.5 moving average concentrations. overall, approximately 10% of incident cases of influenza during the study period may result from exposure to ambient pm 2.5 (paf 10.72% [95% ci: 3.47%, 17.04%]). when stratified by temperature, the highest attributable fractions occurred on cold days (16.26, 95% ci: 6.67%, 24.53%). for moderate cold days and moderate hot days, the attributable fractions were similar. the effect of pm 2.5 on incident influenza was stronger on cold days than hot days at lag 2-3 days (table s4 in appendix). our findings were robust to a range of sensitivity analyses including increasing the number of lag days to 15 days for meteorological variables and when modifying the degrees of freedom for meteorological variables (3-6 df) and for time per year (6-10 df). our findings remained robust after controlling for the effects of ambient so 2 and no 2 (fig. s2 in the appendix) . in recent years, public health experts have warned about the adverse acute and chronic health effects caused by severe haze and air pollution levels in china (xu, chen, and ye, 2013) . however few studies have focused on the possible association between pollution and communicable diseases. to our knowledge, this is the first study to examine the relationship between ambient pm 2.5 and influenza incidence at the national level utilising pm 2.5 monitoring data from 76 sites and influenza data obtained from surveillance systems from 47 cities throughout china. our findings suggest that increased ambient pm 2.5 concentrations are associated with incident case of influenza at lag days 2 and 3 and that the effect is most apparent at lag day 2 when using single lag models. there was also evidence of a negative interaction with temperature such that the association between pm 2.5 and incident influenza was stronger at cooler temperatures. overall, assuming cause and effect, approximately one in every ten cases of incident influenza in china between september 2013 and december 2014 may result from exposure to ambient pm 2.5 . evidence for an association between ambient pm 2.5 and the incidence of influenza at the national level is limited but our results are consistent with previous findings based on data from individual cities in china. one study conducted in beijing reported that ambient pm 2.5 concentrations were associated with the occurrence of influenza and that there was a time-lag effect (liang et al., 2014) . we observed that the lagged effects of pm 2.5 on the incidence of influenza can be explained by the incubation period of the influenza virus consistent with previous studies (lessler et al., 2009) . a study from beijing showed that the effects are strongest with a 2-day moving average of pm 2.5 , while another study from nanjing reported that the strongest effects are for the current day and for the 2-day moving average (c feng et al., 2016; huang et al., 2016) . the difference in lag days of pm 2.5 may reflect the use of different methodologies and data sources. there are plausible mechanisms for a causal association between pm 2.5 and incident influenza although it is beyond the scope of this study to address this directly. for example, previous studies have examined the ability of fine particles to transmit viruses and have shown that fine particles which have the influenza virus attached, can accomplish long-range transportation under certain weather conditions such as dust storm days (chen et al., 2010) . fine particles with viruses attached can also be inhaled resulting in the direct delivery of the viral agents to the respiratory epithelial cells (chen et al., 2010; jaspers et al., 2005) . other studies which have examined how pm 2.5 exposure may impact on respiratory function and the inflammatory response, have reported that exposure to pm 2.5 is associated with dysfunction of the pulmonary tracheal cilia and decreased activity of alveolar macrophages (wong et al., 2009; xing, xu, shi, and lian, 2016) which in turn may enhance an individual's susceptibility to viral agents. although there are some underlying theories explaining the seasonal patterns of influenza, there has been no consideration given to the possible modifying role of air pollution on the association. air pollution exhibits a similar seasonal variation to influenza outbreaks; for example, in winter, higher concentrations of pm 2.5 have been reported possibly due to increased human activity and domestic heating leading to increased emissions (kulshrestha, satsangi, masih, and taneja, 2009) . several studies have reported that ambient temperature may modify the associations between air pollution and health (kim, lim, and kim, 2015; stafoggia et al., 2008) , but whether this is also true for influenza has not been hitherto investigated. in our study, we observed stronger associations at cold temperatures, whereas there was no effect of high temperatures on the magnitude of the association between pm 2.5 and incident influenza. these results are consistent with earlier findings including the results of an experimental study that indicated that most frequent viral transmission occurred at 5°c and was halted at 30°c. it may be speculated that this reflects the inhibitory effect that cold air has on mucociliary clearance of the respiratory tract and greater viral stability in a low-temperature environment (lowen, mubareka, steel, and palese, 2007) both of which would enhance the transmissibility of the influenza virus. however, further studies are necessary before greater inference can be made. disease surveillance systems in china have substantially improved since the sars epidemic in 2003. the cisdcp is now the world's largest internet-based disease reporting system collecting real-time information about cases and virus subtypes and it provides essential and realtime information for policy makers (wang et al., 2008) . influenza cases reported in the system were defined according to national criteria and identified by both routine clinical and laboratory examination. however, under-reporting of influenza cases may have occurred, as some individuals with influenza may not have attended hospital and therefore not been captured by the hospital reporting system (c feng et al., 2016) . another limitation of the current study is that we did not have information on some demographic and behavioural factorssuch as age, gender and cigarette smokingwhich may be associated with incident influenza . however, the inability to include these covariates in the model is unlikely to have significantly confounded the association due to the lack of an association between these variables with pm 2.5 . mortality and morbidity attributed to influenza has significant global health and economic consequences (who, 2015) . although most cases of influenza result in full recovery without serious complications, influenza contributes to the disease burden of communities and increases the risk of complications to the infection in the most vulnerable population subgroups (livingston and bernstein, 2015) . as a part of an integrated strategy for infectious disease control and prevention, the potential effect of environmental factors on disease transmission and infection should be taken into consideration. findings from this study suggest that ambient pm 2.5 , particularly on cold days, increases the transmissibility of the influenza virus. thus, following a period of heavy haze, preventive measures to prevent an increase in cases of influenza may be warranted both domestically and in neighbouring east asian countries given the effect of emissions in china on surrounding countries (kan, 2014) . future studies that focused on other components of air pollution (e.g. no) and environmental factors (e.g., meteorology, climate and geography) are necessary to more fully understand the possible impact on infectious disease transmission (ng and gordon, 2015; silva, viana, müller, livi, and dalcin, 2014) . studies are in need to establish the relationships between particle attachment and survival of influenza virus, and also methods should be developed to quantify airborne influenza virus and measure the concentration of influenza virus in ambient air especially for extremely low virus concentrations (chen et al., 2010) . we set the number of cases to zero on hot days due to there being no significant effect of pm 2.5 on influenza incidence on hot days (see fig. 4 ). pooled relative risks and 95% confidential intervals of influenza incidence associated with an increase of 10 μg/m 3 in pm 2.5 at lag 2 day, lag 3 day, and moving average of lag 2-3 days, stratified by temperatures. disease burdens associated with pm 2.5 exposure: how a new model provided global estimates ambient influenza and avian influenza virus during dust storm days and background days. environ estimates of global mortality attributable to particulate air pollution using satellite imagery impact of ambient fine particulate matter (pm 2.5 ) exposure on the risk of influenza-like-illness: a time-series analysis in beijing the health effects of ambient pm 2.5 and potential mechanisms global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990-2013: a systematic analysis for the global burden of disease study multivariate meta-analysis for nonlinear and other multi-parameter associations mortality risk attributable to high and low ambient temperature: a multicountry observational study the impact of temperature on mortality in tianjin, china: a case-crossover design with a distributed lag non-linear model the burden of air pollution on years of life lost in beijing, china, 2004-08: retrospective regression analysis of daily deaths global variation in the effects of ambient temperature on mortality: a systematic evaluation acute effects of air pollution on influenza-like illness in nanjing, china: a population-based study diesel exhaust enhances influenza virus infections in respiratory epithelial cells globalisation and environmental health in china temperature modifies the association between particulate air pollution and mortality: a multi-city study in south korea metal concentration of pm 2.5 and pm 10 particles and seasonal variations in urban and rural environment of agra incubation periods of acute respiratory viral infections: a systematic review seasonality and temperature effects on fasting plasma glucose: a population-based longitudinal study in china pm 2.5 in beijing -temporal pattern and its association with influenza prevention of influenza in children influenza virus transmission is dependent on relative humidity and temperature influenza burden and transmission in the tropics model choice in time series studies of air pollution and mortality air pollution in china: mapping of concentrations and sources dual seasonal patterns for influenza respiratory viral infections and effects of meteorological parameters and air pollution in adults with respiratory symptoms admitted to the emergency room does temperature modify the association between air pollution and mortality? a multicity case-crossover analysis in italy emergence and control of infectious diseases in china spatial and temporal variations of the concentrations of pm 10 , pm 2.5 and pm 1 in china spatiotemporal analysis for the effect of ambient particulate matter on cause-specific respiratory mortality in beijing a manual for estimating disease burden associated with seasonal influenza modification by influenza on health effects of air pollution in hong kong the impact of pm 2.5 on the human respiratory system haze, air pollution, and health in china the work of y.g. was supported by the career development fellowship of australian national health and medical research council (#app1107107). the work of g.c. was supported by china scholarship council (csc) (201506210060). this study was supported by centre for air quality and health research and evaluation. we thank china atmosphere watch network and the china information system for disease control and prevention who provided data on environmental factors and influenza incidence, respectively. the authors declare they have no actual or potential competing financial interests. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.envint.2016.10.004. key: cord-338674-tnnd1s57 authors: yin, j kevin; lahra, monica m; iskander, mary; lambert, stephen b; heron, leon; nissen, michael d; rost, laura; murphy, jennifer; sloots, theo p; booy, robert title: pilot study of influenza vaccine effectiveness in urban australian children attending childcare date: 2011-06-10 journal: j paediatr child health doi: 10.1111/j.1440-1754.2011.02098.x sha: doc_id: 338674 cord_uid: tnnd1s57 background: influenza outbreaks in the childcare setting are a significant cause of excess winter morbidity. this study explored methods of follow up and sample collection for a proposed randomised controlled trial of influenza vaccination in children attending childcare. methods: the study was conducted in four sydney childcare centres during 2007. healthy children aged 6–59 months eligible for vaccination were recruited in two centres, with another two acting as controls. data on influenza‐like illness (ili: ≥37.8°c plus at least one respiratory symptom) occurrence were collected weekly. in those children with an ili, parents were asked to collect nasal swabs and send via surface mail for viral polymerase chain reaction. vaccine efficacy (ve) for ili was estimated overall and for subgroups aged 6–23 and 24–59 months using the formula ve = 1 − relative risk (rr). results: sixty‐three per cent (151/238) of eligible children had parents give consent. sixty‐three children received influenza vaccine and 88 participated as controls. of 26 specimens returned, a virus was detected in 18 (69%); none with influenza. two symptomatic children had positive near‐patient influenza tests in general practice (one a vaccine failure). the rr with 95% confidence interval in all children and those aged 6–23 months were less than one, 0.56 (0.32–1.02) and 0.46 (0.15–1.45), respectively. conclusions: this study demonstrated the feasibility and utility of parent‐collected and mailed respiratory specimens for ve research in the childcare setting. two‐thirds of parent‐collected swabs proved positive for at least one virus. finding ways to reduce reluctance of parents to submit samples could improve the representativeness of samples collected and the power of the study. no evidence was found for influenza ve, but point estimates were in the direction of protection. influenza is a seasonal, vaccine-preventable disease which causes excess morbidity and mortality during winter in temper-ate climates. the health and economic costs associated with childhood influenza are substantial. 1 for example, in australia during 2002-2005, there were reports of 25 433 hospitalisations and four deaths for influenza and pneumonia among children aged under 5 years. 2 the annual cost due to influenza-related diseases in australia is estimated to exceed $115 million. 3 the world health organization recommends annual influenza vaccination as the cornerstone for prevention and control. efficacious influenza vaccines have been available for over 50 years, and yet, routine use in childhood remains the what is already known on this topic 1 children in childcare are more likely to contract influenza and transmit infection to their siblings, parents, extended families and child-care workers. 2 usa, canada and western australia currently have a routine influenza vaccine policy in place that includes children 6 months of age and older. 3 evidence for the effectiveness of influenza vaccine in children aged less than 24 months is limited and high quality, appropriately powered, randomised controlled trials are needed. 1 it is feasible to follow children weekly for 3 months to obtain swabs for influenza-like illness. 2 two-thirds of parent-collected swabs were positive for at least one virus demonstrating the utility of this approach for future studies. reluctance of parents to submit swabs for analysis may be a limitation of this approach. exception in most countries. the effectiveness of influenza vaccine for children in childcare has been demonstrated for children aged ն24 months. [4] [5] [6] [7] [8] although the us advisory committee on immunisation practices has recommended children aged 6-23 months to be vaccinated with influenza vaccine since 2004, 9 there is ongoing debate about vaccine effectiveness in this age group. three recent systematic reviews [10] [11] [12] concluded that either influenza vaccine was not effective in children յ24 months of age or that there were insufficient data to form a conclusion. influenza is transmitted from person to person through contact and respiratory droplets; however, the droplets do not remain suspended in the air for long nor do they travel far. 13 transmission of influenza generally requires close contact with an infected person or contact with a contaminated surface or object. 14, 15 the childcare setting provides enhanced opportunities for transmission of infections including influenza as there is prolonged close interaction between young children and the sharing of toys and other objects. further to this, young children are particularly susceptible to infection as they are immunologically naïve to many viruses. commercial childcare in australia is available in two broad categories: daycare centres (dcc) for children aged 6 weeks until 6 years and pre-school centres (psc) for children aged 3 to 6 years. commercial childcare usage in australia is increasing. the median attendance time for australian children who use childcare is 10 h per week, but 13% attend 35 h a week or more. 16 children in childcare are known to be more likely to contract respiratory illnesses, including influenza, [17] [18] [19] [20] [21] and are considered to be major transmitters of influenza to their siblings, parents, extended families and care workers. 6, 8, 22, 23 the 2007 influenza season in australia ran from late may until october and notifications peaked during august. 24 australia witnessed antigenically drifted influenza virus (a/brisbane/ 59/2007 (h1n1)-like and a/brisbane/10/2007 (h3n2)-like), and it was the most severe influenza season since a national influenza reporting system was established in 2001. 25 with this study, the primary process issues of conducting influenza vaccine research in the childcare environment were evaluated, including recruitment, retention, vaccination and specimen handling. while this pilot study was not powered to assess an efficacy end point, preliminary vaccine efficacy (ve) data were also examined. from july to august 2007, children aged 6-59 months attending four childcare centres in new south wales were recruited for this study: two dcc caring for children aged 0-59 months and two psc caring for children aged 36-59 months. the four dcc were chosen by convenience (proximity to the children's hospital at westmead) with equal number of children between dcc and psc. one dcc and one psc were allocated to influenza vaccination, and one dcc and one psc were allocated to be controls. this study was approved by the royal alexandra hospital for children ethics committee, and informed parental consent for participation was obtained prior to study procedures. the par-ticipating children were evaluated in two age groups based on age at enrolment: 6-23 months and 24-59 months. the influenza vaccine administered was a 2007 southern hemisphere preparation, purified, inactivated, split vaccine (vaxi-grip junior, provided by sanofi pasteur, lyon, france), incorporating: children were administrated the vaccine according to the standard recommended dose and schedule for age. 26 as all children at the centres randomised to receive vaccine were influenza vaccine naïve, each received two doses of vaccine 1 month apart -0.25 ml intramuscular dose for those less than 36 months of age and 0.5 ml intramuscular dose for those aged 36-59 months at the time of their first dose. vaccines were administered between 11 july 2007 and 19 september 2007. we defined influenza-like illness (ili) as an illness with fever >37.8°c and with one or more respiratory symptoms (cough, blocked nose or runny nose) to maximise sensitivity. as a protective level of antibody is usually detectable within 2 weeks of the second dose of vaccine, 13,27 ili surveillance was commenced in vaccinated children at this time point. in control children, ili surveillance was arbitrarily commenced from the week ending 26 august 2007: at this time, just over half (32/62) of the children eventually fully vaccinated had received vaccine, and from that week, the ratio of child-weeks of follow up in vaccinated and unvaccinated children was similar (fig. 2) . parent education for ili surveillance was provided at study entry. households received a weekly email or telephone call from 30 july until 21 october 2007 (12 weeks) to monitor the study children for ili symptoms. parent training for the collection of nasal swabs was conducted by study nurses after the second immunisation. nasal swabs were collected using the virocult collection system (mw950) consisting of a rayon swab on a plastic shaft, with viral transport medium-soaked foam pad in the base of the transport tube (copan italia, brescia, italy), and were returned to the queensland paediatric infectious diseases laboratory using a pre-addressed, postage-paid envelope. returned specimens were tested using previously reported, real-time polymerase chain reaction assays with reverse transcription for rna viruses. a total of 16 viruses were investigated: human rhinoviruses (hrv), 28 influenza a, influenza b, rsv, adenoviruses, hmpv, parainfluenza viruses i, ii and iii, 29 bocavirus 30 hpyv-wu, hpyv-ki, 31 and human coronaviruses: oc43, 229e, nl63 32 and hku1. 33 while it was not part of the study protocol, some children had near-patient influenza tests performed by their general practitioners and these were reported by parents to study staff. ili incidence rates were calculated using child-weeks in the denominator. rate ratios (rr) and 95% confidence intervals were calculated comparing vaccinated and unvaccinated groups. these values were used to estimate ve using the formula ve = (1 -rr) ¥ 100%. comparisons were performed in three age groups, 6-59 months (all children), 6-23 months and 24-59 months. the average ages of the children in the vaccine and control centre were 43.4 (7.7-65.4) and 44.1 (7.9-66.0) months, respectively, while the proportions who were males were 50.8 and 58.0%. data on non-enrolled children were not recorded. there were 239 children in total attending the four childcare centres, and 151 children were enrolled giving a recruitment rate of 63%. complete information was available for analysis in 150 children, with one vaccinated child lost to follow up during the study period (fig. 1 ). there were 481 (62 children) and 792 (88 children) child-weeks of follow up in vaccine and control centres, respectively. a total of 59 ilis were identified in all study children, and weekly ili incidence rates are provided (fig. 2) . of the 26 swabs received during the study period, 18 (69%) had at least one virus identified, with bocavirus being the most common virus found in six swabs (table 1) , followed by hrv in five swabs. one swab contained three viruses (hrv, adenovirus and bocavirus). thirteen of these swabs were from 12 vaccinated children, with the other half from 13 controls. two positive near-patient influenza tests (one a vaccine failure) were reported by parents to the study staff: one was from an unvaccinated child in a control centre; the other child was vaccinated with the test done 14 days post-second vaccination ( table 1) . there were a total of 59 ilis identified with efficacy point estimates in the direction of protection for all age-groups but not significant ( table 2 ). the key findings of this pilot study were that a high recruitment rate could be achieved, that recruited families were tolerant of regular weekly follow up over an extended period (3 months) and that there was no evidence of protective efficacy, but point estimates of ve for the less-specific end point of ili were in the direction of protection. it is inevitable that ili would include non-influenza infections which cause respiratory signs and symptoms, especially as we used a sensitive definition (at the expense of specificity), so it is not surprising that a range of other viral pathogens were identified in our study. a population-based surveillance study showed that less than 10% of hospitalised children aged յ59 months with ili had confirmed influenza infection. 34 our study has some limitations. the childcare centres were not randomised. the commencement midway through an influenza season limited the number of influenza cases identified. less than half the episodes in children of ili (26 out of 59, 44%) had a respiratory sample sent. this reduced sampling is probably due to the added burden on parents of sample collection (and posting) while a child is ill. in addition, as the childcare centres were in the suburbs with relatively lower socio-economic indexes for areas 35 (and also involved larger families), this, too, may have limited parental cooperation. the participants were only followed from the 2nd half of august when the 2007 season was peaking, so some may have thought that sample collection in september or october was too late. given the limited data that were collected on symptomatology and the relatively small number of specimens, it was not possible to do an extensive analysis comparing symptoms by virus type to address if there are differences in symptoms among various viruses. furthermore, the demographic data (e.g. sex, age range) of those who did not participate in the study were not collected; therefore, it was not possible to identify if there was any recruitment bias. greater efforts are required for future studies in (i) improving the proportion of swabs collected and sent by initiating ili follow up before the influenza season starts and findings better ways to overcome parents' reluctance in submitting swabs; (ii) obtaining more detailed data on symptomatology of respiratory infection; and (iii) collecting de-identified demographic data on those who are not enrolled in the study. this study showed no evidence for influenza ve. there was only a suggestion of protection in that all the point estimates were in that direction. trivalent, live, cold-adapted influenza vaccine (caiv-t) may be a better option for young children and has been demonstrated to have significantly higher efficacy than inactivated vaccine among young children during moderate 36, 37 and high attack-rate influenza seasons. 38 caiv-t was also able to provide protection even when the circulating influenza virus was an antigenically distinct strain. 37 there were two children with positive results for influenza a from the near-patient test. this type of test is known to have only moderate sensitivity but high specificity, so a positive test is unlikely to be false. 39 week ending date (2007 older children (24-59 months) are more likely to have developed natural immunity through previous infection and are more likely to have developed better personal hygiene. for these reasons, older children are less susceptible to influenza as well as other non-influenza infections that may cause ili. this pilot study has shown the feasibility and value of parentcollected (and mailed) nasal sample for influenza ve research in childcare. two-third of parent-collected swabs proved positive for at least one virus. means of lessening reluctance of parents to submit respiratory samples from their children need to be found to improve the representativeness of samples collected and the power of the study. no evidence was found for influenza ve, but point estimates were all in the direction of protection. the cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children vaccine preventable diseases and vaccination coverage in australia influenza-related disease: the cost 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chain reaction assays community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens census of population and housing: socio-economic indexes for areas (seifa), australia live attenuated versus inactivated influenza vaccine in infants and young children various factors associated with the manifestation of influenza-like illness trivalent live attenuated intranasal influenza vaccine administered during the 2003-2004 influenza type a (h3n2) outbreak provided immediate, direct, and indirect protection in children laboratory diagnosis of human seasonal and pandemic influenza virus infection key: cord-287554-2lqy2ix9 authors: amarelle, luciano; lecuona, emilia; sznajder, jacob i. title: tratamiento antigripal: fármacos actualmente utilizados y nuevos agentes en desarrollo date: 2017-01-31 journal: archivos de bronconeumología doi: 10.1016/j.arbres.2016.07.004 sha: doc_id: 287554 cord_uid: 2lqy2ix9 resumen la gripe es una enfermedad contagiosa altamente prevalente y con significativa morbimortalidad. el tratamiento disponible con fármacos antivirales, de ser administrado de forma precoz, puede reducir el riesgo de complicaciones severas; sin embargo, muchos tipos de virus desarrollan resistencia a estos fármacos, reduciendo notablemente su efectividad. ha habido un gran interés en el desarrollo de nuevas opciones terapéuticas para combatir la enfermedad. una gran variedad de fármacos han demostrado tener actividad antiinfluenza, pero aún no están disponibles para su uso en la clínica. muchos de ellos tienen como objetivo componentes del virus, mientras que otros son dirigidos a elementos de la célula huésped que participan en el ciclo viral. modular los componentes del huésped es una estrategia que minimiza el desarrollo de cepas resistentes, dado que estos no están sujetos a la variabilidad genética que tiene el virus. por otro lado, la principal desventaja es que existe un mayor riesgo de efectos secundarios asociados al tratamiento. el objetivo de la presente revisión es describir los principales agentes farmacológicos disponibles en la actualidad, así como los nuevos fármacos en estudio con potencial beneficio en el tratamiento de la gripe. abstract influenza is a very common contagious disease that carries significant morbidity and mortality. treatment with antiviral drugs is available, which if administered early, can reduce the risk of severe complications. however, many virus types develop resistance to those drugs, leading to a notable loss of efficacy. there has been great interest in the development of new drugs to combat this disease. a wide range of drugs has shown anti-influenza activity, but they are not yet available for use in the clinic. many of these target viral components, which others are aimed at elements in the host cell which participate in the viral cycle. modulating host components is a strategy which minimizes the development of resistance, since host components are not subject to the genetic variability of the virus. the main disadvantage is the risk of treatment-related side effects. the aim of this review is to describe the main pharmacological agents currently available and new drugs in the pipeline with potential benefit in the treatment of influenza. ácido acetil salicílico otros agentes con actividad antiinfluenza ouabaína pandémicos, con alta mortalidad y gran impacto sobre la salud pública 5 . la vacunación es fundamental en la prevención de la enfermedad y sus complicaciones, principalmente para grupos de riesgo como niños, ancianos, portadores de enfermedades respiratorias crónicas y embarazadas. el tratamiento disponible con fármacos antivirales, de ser administrado de forma precoz, puede reducir el riesgo de complicaciones severas; sin embargo, muchos tipos de virus desarrollan resistencia a fármacos, reduciendo su efectividad, por lo que ha habido un gran interés en los últimos años en el desarrollo de nuevas opciones terapéuticas para combatir la enfermedad. la presente revisión describe los principales agentes farmacológicos disponibles en la actualidad y analiza nuevos medicamentos en estudio con potencial beneficio en el tratamiento de la gripe 5-7 (tabla 1). los virus de la gripe pertenecen a la familia orthomyxoviridae y son clasificados como a, b o c. los virus influenza a circulan en diversas especies como humanos, equinos, porcinos y aves, mientras que los de tipo b afectan solo a humanos. el cuadro gripal causado por los tipos a y b es indistinguible; por el contrario, la c causa síntomas respiratorios leves [8] [9] [10] . la estructura del virus influenza a consta de una envoltura lipídica que proviene de la célula huésped y lleva ancladas las glucoproteínas hemaglutinina (ha) y neuraminidasa (na), antígenos de superficie usados para clasificar a los virus (por ejemplo, h1n1, h3n2, h5n1). en la membrana se encuentran también las proteínas de matriz m2 y m1, mientras que en el centro de la partícula viral se halla el complejo ribonucleoproteico (segmentos de arn viral y las proteínas polimerasa básica 1, polimerasa básica 2 [pb2] y polimerasa ácida [pa]), la nucleoproteína (np), la proteína de exportación nuclear y la proteína no estructural 2. el genoma es de arn de cadena simple y segmentado en 8 partes, que dan origen a entre 8 y 12 proteínas del virus [11] [12] [13] [14] [15] [16] . el anclaje a la célula huésped se da a través de la unión de la ha del virus con el ácido siálico de las glucoproteínas o glucolípidos de la membrana celular 17 . las especies de influenza que afectan a humanos reconocen preferentemente el ácido siálico unido a galactosa por una unión ␣2,6 (sa ␣2,6gal), que abunda en las células epiteliales del tracto respiratorio 18, 19 . una vez el virus se une al receptor de membrana, ingresa a la célula huésped por endocitosis y se libera en el citoplasma celular fusionando su membrana con la del endosoma. la ha viral es clave en este paso, ya que al ser escindida por proteasas del huésped 20,21 expone una región conocida como «péptido de fusión» que interactúa con la membrana del endosoma y determina la fusión de las membranas liberando el contenido del virión al citoplasma celular [22] [23] [24] . un paso importante para este proceso es la acidificación del endosoma que ocurre a través de un canal iónico formado por la proteína viral m2. por este canal ingresan protones y favorecen la separación de la proteína de matriz m1 y el complejo ribonucleoproteico del virus, que es así liberado al citoplasma para su posterior importación nuclear [25] [26] [27] . una vez en el núcleo, el arn viral es transcrito a arn mensajero (arnm), que es poliadenilado, hecho fundamental para la expresión de las proteínas virales 28 . la traducción de las proteínas virales es llevada a cabo por la maquinaria de la célula huésped, y una vez sintetizadas las proteínas pa, pb y np son importadas al núcleo para facilitar la transcripción y replicación del arn viral. los complejos de ribonucleoproteína son luego exportados del núcleo, para lo cual son fundamentales la proteína no estructural 2, la proteína de exportación nuclear y la m1. las proteínas virales ha, na y m2 son glucosiladas en el retículo endoplásmico y transportadas a través de la red golgi hasta la membrana celular 29 . las ribonucleoproteínas y los 8 segmentos virales una vez transportados a la membrana celular son empaquetados para generar los nuevos brotes virales. varias proteínas estructurales del virus, como ha, na y m2, contribuyen al proceso de brote por su interacción con la membrana lipídica celular 29 . el proceso finaliza cuando las membranas se fusionan en la base del brote y el virus se escinde mediante la acción de la na, que cataliza la remoción del ácido siálico de las glucoproteínas de superficie 30 . de los 5 fármacos antiinfluenza actualmente disponibles, solo 3 son recomendados por la food and drug administration de los estados unidos para esta temporada: oseltamivir (tamiflu ® ) vía oral, zanamivir (relenza ® ) vía inhalatoria y peramivir (rapivab ® ) vía intravenosa, todos inhibidores de la na 31 . los inhibidores del canal iónico m2, amantadina y rimantidina, no están recomendados dado que los virus circulantes poseen una gran tasa de resistencia a estas y que no poseen efecto en los virus influenza tipo b ni c 32 . los derivados del adamantano, como amantadina y rimantadina, son fármacos con conocido efecto antiinfluenza a y han sido la primera opción para el tratamiento de la gripe durante muchos años. su acción consiste en la unión a un bolsillo específico de la proteína viral m2, estabilizando su conformación cerrada e impidiendo que después de la fusión, el virus pueda liberar el complejo ribonucleoproteico al citoplasma para seguir adelante con el ciclo viral 33 . también se ha demostrado que la amantadina puede afectar el ph de las vesículas que transportan las glucoproteínas virales, interfiriendo así con el proceso de ensamblaje 34 . a causa de la resistencia viral a estos fármacos se han desarrollado nuevos compuestos estructuralmente relacionados, como los azoloadamantanos 35, 36 . sin embargo, no hay fármacos de este grupo con actividad probada contra todos los virus circulantes resistentes a amantadina. poseen actividad contra influenza a y b; oseltamivir y zanamivir son los más antiguos, y los de las nuevas generaciones son peramivir y laninamivir. estos fármacos impiden la escisión del ácido siálico inhibiendo la liberación del virión, evitando que las nuevas partículas virales se diseminen a otras células 37 . la resistencia global a los inhibidores de la na es menos del 2% de los virus circulantes en 2013-2014; sin embargo, se han identificado virus en comunidades localizadas (japón, china, australia) que poseen alta resistencia a estos fármacos 38 . es prometedor el desarrollo de nuevas moléculas inhibidoras de la na, con actividad frente a cepas resistentes a los fármacos actualmente disponibles [39] [40] [41] [42] . en la práctica clínica los inhibidores de la na son los únicos antigripales actualmente recomendados; entre ellos, oseltamivir es el más utilizado, dada su buena biodisponibilidad para uso oral. sin embargo, existe un intenso debate sobre la interpretación de los estudios clínicos que avalan su eficacia. un metaanálisis realizado por el grupo cochrane en 2014 evidenció un modesto beneficio del tratamiento con oseltamivir en casos de gripe leve, con una reducción de la duración de los síntomas de 7 a 6,3 días en adultos y una reducción de los síntomas en niños sanos; sin embargo, no mostro beneficio en niños asmáticos. la tasa de hospitalización de los pacientes con oseltamivir fue similar a la de los no tratados 43 . de un metaanálisis publicado en lancet en 2015 surge que para los casos leves de infección por influenza confirmada, el tratamiento con oseltamivir disminuye el tiempo de síntomas, las infecciones respiratorias bajas y las admisiones hospitalarias. en ambos estudios se reportan las náuseas y los vómitos como principales efectos secundarios 44 . en cuanto a las presentaciones severas de la gripe, si bien no existen trabajos clínicos aleatorizados, estudios observacionales indican un beneficio del uso de oseltamivir con una reducción significativa de la tasa de mortalidad para casos graves [45] [46] [47] . las guías de práctica clínica recomiendan el inicio precoz de antivirales para el tratamiento de casos graves de influenza confirmada que requieren hospitalización o en pacientes con riesgo de complicaciones severas, como menores de 2 o mayores de 65 años, portadores de enfermedad pulmonar crónica, inmunosuprimidos, obesos mórbidos, mujeres embarazadas o puérperas de menos de 2 semanas. la quimioprofilaxis posexposición se puede considerar en personas con alto riesgo de complicaciones o no inmunizados, y siempre iniciarla en las primeras 48 h posexposición. el fármaco de primera línea es oseltamivir, mientras que el uso de zanamivir inhalatorio, nebulizado o intravenoso está indicado en caso de cepas resistentes a oseltamivir o mala absorción intestinal. la dosis indicada de oseltamivir es de 75 mg 2 veces por día para tratamiento y una vez por día para quimioprofilaxis, y para zanamivir inhalatorio, de 10 mg 2 veces por día para tratamiento y una vez por día para quimioprofilaxis; este último no está recomendado en casos de asma o epoc. la duración del tratamiento debe ser de 5 días, pudiendo prolongarse en casos graves, mientras que para la profilaxis se recomienda 10 días tanto para oseltamivir como para zanamivir 32, 48, 49 . existe una considerable cantidad de fármacos que han sido ensayados por su potencial antiviral, pero aún no están disponibles para su uso en la clínica. describiremos los grupos más importantes. fármacos activos ante componentes del virus inhibidores de la unión y la fusión viral. ligandos sintéticos de la ha viral: este grupo comprende una variedad de moléculas capaces de ser reconocidas por la ha viral, interfiriendo así en la interacción virus-célula. ejemplos de ello son mbx2329 (un amino éster de fenil alquilo) y mbx2546 (una sulfonamida), que inhiben la entrada del virus influenza a mediante su unión al tallo de la ha, interfiriendo con la fusión viral 50 . también se han estudiado péptidos sintéticos análogos al ácido siálico que son reconocidas por la ha viral e inhiben su acción 51 , y antagonistas del péptido de fusión que interrumpen el cambio conformacional de ha necesario para la correcta fusión del virus a la célula 52 . arbidol: fármaco sintético actualmente aprobado para el tratamiento de la gripe en rusia y china, pero no en ee. uu. dada la insuficiente evidencia clínica 49 . se trata de una molécula hidrofóbica que penetra la membrana lipídica de virus encapsulados e interacciona con los fosfolípidos de membrana y proteínas transmembrana ricas en residuos aromáticos de la envoltura viral, interfiriendo con los procesos de entrada y fusión a la célula huésped 53, 54 . un estudio clínico multicéntrico aleatorizado (arbitr) mostró en pacientes con influenza tratados con arbidol, un acortamiento de la enfermedad con reducción de la severidad y de la emisión de virus 55 . se han identificado cepas de influenza resistentes a arbidol por mutaciones en la subunidad ha2, permitiendo que el virus prosiga con el proceso de fusión a la membrana del endosoma 56 . la síntesis de compuestos estructuralmente relacionados abre una puerta a nuevos antivirales, tal es el caso de indoles sintéticos que en estudios in vitro presentan mayor potencia antiviral que arbidol sobre ciertos subtipos de influenza a 57 . anticuerpos anti-ha: se han desarrollado anticuerpos monoclonales contra sitios altamente conservados de ha, capaces de neutralizar cepas de virus influenza. como ejemplo, el anticuerpo ch65, descrito a partir de la muestra de un paciente con vacuna antiinfluenza en 2007, ha demostrado ser eficaz in vitro contra un amplio espectro de cepas de influenza h1n1, gracias a su unión en el bolsillo de unión de la subunidad ha1 58 . el anticuerpo d1-8 ha mostrado eficacia para neutralizar diferentes cepas de virus influenza h3n2 in vitro e in vivo, logrando en ratones una mayor supervivencia comparado con el tratamiento con oseltamivir 59 . también los anticuerpos dirigidos contra el tallo de la proteína ha pueden ser eficaces contra el virus influenza. el hb36.6 ha mostrado eficacia contra diferentes cepas de h1n1 y h5n1 in vitro, mientras que en ratones demostró reducir la replicación viral y mejorar la supervivencia 60 . inhibidores de la polimerasa viral. la polimerasa viral es una proteína altamente conservada entre las distintas cepas de influenza, siendo un objetivo terapéutico de interés. favipiravir (t-705): es una molécula capaz de convertirse dentro de la célula en un análogo nucleósido, el ribonucleótido t-705-4-ribofuranosil-5'-monofosfato, que inhibe la actividad de la arn polimerasa viral sin afectar la síntesis celular de arn ni adn 61 . se ha demostrado su eficacia frente a distintas cepas de influenza [62] [63] [64] . inhibidores de pb2: para la traducción del arnm viral se requiere un iniciador 5'cap, que el virus «roba» del pre-arnm del huésped. en este proceso es esencial la unión de la subunidad pb2 a la caperuza 5' del pre-arnm del huésped y la subsecuente escisión del extremo 5' por la proteína pa con actividad endonucleasa 65, 66 . los fármacos en estudio que interfieren con este proceso tienen un gran potencial como antivirales 67 . tal es el caso de vx-787, que ha demostrado actividad in vivo contra diversas clases de influenza a, siendo eficaz tanto en profilaxis como en tratamiento 68,69 . inhibidores de la nucleoproteína. la nucleoproteína se une al arn viral y forma parte del complejo ribonucleoproteico. es fundamental para la síntesis del arn viral y también participa en la exportación nuclear de las ribonucleoproteínas virales y en el tráfico citoplasmático 70 . nucleozina: es el fármaco más estudiado de este grupo. se ha demostrado que posee actividad antiviral en diferentes tiempos del ciclo del virus: en fases tempranas inhibe la transcripción de arn viral y la síntesis proteica, y en fases tardías bloquea el tráfico citoplasmático de las nuevas ribonucleoproteínas 71 . recientemente se han desarrollado análogos a la nucleozina con potencia antiviral ante diferentes especies de influenza a 72 . naproxeno: es un inhibidor de la ciclooxigenasa 2 cuya actividad antiinfluenza ha sido identificada recientemente. se ha evidenciado que forma complejos con la np e inhibe competitivamente la unión de np al arn. tiene potencia in vitro e in vivo contra virus influenza a 73 . también se han sintetizado derivados de naproxeno con potencial efecto antiviral, que están en estudio 74 . inhibidores de la proteína ns1. ns1 es una proteína multifuncional que se une al arnm y regula pasos posteriores a la transcripción: se une al arn de cadena doble bloqueando la inhibición de la transcripción por parte de la cinasa pkr 75,76 e inhibe la exportación nuclear de arnm poliadenilado del huésped [77] [78] [79] . además, ns1 protege al virus de la defensa celular antiviral inhibiendo la respuesta mediada por interferón [80] [81] [82] . se han desarrollado distintos fármacos que inhiben la actividad de ns1 83 . por ejemplo, nsc125044 y sus derivados 84 , entre ellos el compuesto jj3297, afectan la replicación del virus in vitro restableciendo el efecto antiviral del interferón 85 . la baicalina ha demostrando actividad antiinfluenza in vitro e in vivo, ejerciendo su efecto mediante la alteración del dominio de unión de la proteína ns1 86 . el conocimiento de las funciones celulares necesarias para el ciclo viral ha permitido identificar objetivos terapéuticos para el diseño de fármacos antivirales. modular la respuesta del huésped es una estrategia que minimiza el desarrollo de cepas resistentes, dado que estos no están sujetos a la variabilidad genética que tiene el virus. por otro lado, el riesgo de efectos secundarios asociados al tratamiento es una desventaja. investigaciones sobre la interacción entre proteínas virales y del huésped describieron una extensa red con más de 130 interacciones entre 10 proteínas virales y 87 proteínas humanas 87 . análisis más recientes identificaron 91 factores del huésped cuya inhibición previene la replicación viral in vitro 88 . del análisis informático de base de datos de fármacos surgen cientos de moléculas que interactúan con factores del huésped esenciales para la replicación viral; la mayoría de estas son fármacos actualmente utilizados con otros fines terapéuticos 89 . anclaje y fusión viral. das181 (fludase ® ): es una proteína desarrollada fusionando el dominio catalítico de na con una secuencia de anclaje a la superficie de las células del epitelio respiratorio, cuya función es escindir los residuos de ácido siálico de la superficie celular, evitando así la entrada del virus a la célula. ha demostrado actividad antiinfluenza a y b en experimentos con animales; sin embargo, estudios clínicos no han mostrado eficacia en la mejoría de los síntomas de la gripe 90, 91 . aprotinina: es un polipéptido que inhibe las proteasas del huésped que escinden la ha viral. interfiere así con el proceso de unión y fusión del virus a la célula, teniendo actividad antiinfluenza in vitro e in vivo 92, 93 . últimamente se han desarrollado otros compuestos sintéticos, inhibidores de las serín proteasas del epitelio respiratorio con eficacia in vitro antiinfluenza 94 . endocitosis y fusión. glicirrizina: es un compuesto que ha mostrado actividad antiinfluenza a al alterar la estabilidad de la membrana lipídica de la célula huésped, impidiendo el proceso de endocitosis de virus encapsulados [95] [96] [97] . lj001: es una molécula de similar mecanismo de acción que glicirrizina y afecta el proceso de fusión del virus con la célula. posee efecto antiviral de amplio espectro contra virus encapsulados, incluyendo influenza a 98 . bafilomicina a1 y concanamicina a: antibióticos macrólidos que inhiben la acción de la h + atpasa vacuolar que bombea protones desde el citoplasma celular al interior del endosoma 99, 100 . más recientemente se ha estudiado la salifenilhalamida, otro inhibidor de la h + atpasa vacuolar, mostrando eficacia antiviral 101 . brote y salida del virus. la enzima farnesil difosfato sintasa, que participa en la síntesis de compuestos lipídicos de la membrana plasmática, es inhibida por la proteína viperina, cuya expresión es inducida por interferón. se ha demostrado que dicha inhibición altera la composición de la membrana plasmática celular, interfiriendo con el ensamblaje y brote de las partículas virales. la enzima farnesil difosfato sintasa ha sido propuesta como potencial objetivo para el desarrollo de fármacos con actividad antivirus influenza 102 . transcripción y transporte del arn viral. la proteína chaperona hsp90 participa en la importación nuclear del arn viral y en el ensamblaje de la polimerasa del virus, y se ha demostrado que se une a la proteína pb2, transportándose al núcleo junto con ella y modulando la interacción entre proteína polimerasa básica 1 y pa 103 . inhibidores de hsp90 como geldanamicina y su derivado sintético 17-aag han demostrado actividad contra virus influenza en cultivos celulares 104 . ribavirina y su precursor viramidina: son antivirales de amplio espectro. ribavirina (virazole ® ), usada en el tratamiento del virus de la hepatitis c, es un análogo del nucleótido guanosina y su principal mecanismo de acción es la inhibición competitiva de la enzima inosina 52 monofosfatasa deshidrogenasa de la célula huésped, afectando la biosíntesis de gtp y, por lo tanto, la síntesis del arn viral y la producción de proteínas virales 105 . a pesar de haber obtenido resultados positivos en animales 106 , los estudios clínicos no han demostrado eficacia que avale su uso 107, 108 . exportación del complejo ribonucleoproteico viral y procesos postranscripcionales. verdinexor (kpt-335): es una molécula que inhibe la proteína exportina 1 necesaria para la salida del complejo ribonucleoproteico del virus del núcleo al citoplasma. se ha demostrado que posee actividad antiviral frente a diferentes cepas de influenza 109 . nitazoxanida: es un fármaco antiparasitario con actividad ante diferentes cepas de virus influenza a, ya que inhibe la maduración de la ha viral 110 . un estudio clínico aleatorizado demostró una reducción de los síntomas en pacientes con infección por influenza no complicada 111 . vías de defensa intracelulares. la vía raf/mek/erk pertenece a la familia de las mitogen activated protein kinases -mapk-y es activada por los virus influenza. su bloqueo inhibe la exportación nuclear de ribonucleoproteínas por su interacción con la proteína viral de exportación nuclear 112, 113 . experimentos in vivo muestran que la administración del compuesto u0126, un inhibidor de mek, reduce la carga viral en tejido pulmonar y mejora la supervivencia de ratones infectados con influenza 114 . la asociación de inhibidores de mek con oseltamivir aumenta la eficacia antiviral in vitro de este último 115 . la vía del factor de transcripción nf-äb regula la expresión de citocinas antivirales cuya activación ha sido asociada con un incremento en la replicación de virus influenza 116, 117 . el ácido acetil salicílico que inhibe la ikk2, cinasa activadora de ib, tiene efecto antiviral, alterando la exportación nuclear de ribonucleoproteínas virales 118 . la modulación de esta vía es promisoria para el desarrollo de nuevas terapias. otros agentes con potencial actividad antiinfluenza. gran variedad de fármacos utilizados para otras enfermedades han sido identificados como inhibidores de la replicación del virus influenza mediante ensayos in vitro. por ejemplo, se ha demostrado que los inhibidores de la na,k-atpasa poseen actividad antiviral frente a distintos tipos de virus adn y arn. los glucósidos cardiotónicos, inhibidores específicos de la na,k-atpasa, tienen una potente actividad in vitro ante citomegalovirus y otros herpes virus; particularmente la ouabaína ha demostrado inhibir la síntesis de proteínas virales 119, 120 . el virus del ebola también puede ser inhibido in vitro por la ouabaína 121 ; en los coronavirus el principal mecanismo inhibitorio de la ouabaína está dado por la activación de la vía de señalización intracelular de la cinasa src y la inhibición del proceso de fusión 122 . los reovirus también son susceptibles a la acción de la ouabaína por alteración de la fusión viral 123 . la digoxina, otro inhibidor de la na,k-atpasa, es activo in vitro frente al vaccinia virus 124 . recientemente se ha encontrado que diferentes miembros de la familia de glucósidos cardiotónicos inhiben la replicación del virus de influenza a in vitro 125, 126 . en resumen, el tratamiento 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efficient uncoating of influenza a virus cores after penetration polyadenylation and reverse transcription of influenza viral rna assembly and budding of influenza virus a comprehensive map of the influenza a virus replication cycle food and drug administration structure and mechanism of the m2 proton channel of influenza a virus amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the ph of glycoprotein transport vesicles synthesis and anti-viral activity of azolo-adamantanes against influenza a virus flipping in the pore: discovery of dual inhibitors that bind in different orientations to the wildtype versus the amantadine-resistant s31 n mutant of the influenza a virus m2 proton channel inhibition of influenza virus infections in mice by gs4104, an orally effective influenza virus neuraminidase inhibitor global update on the susceptibility of human influenza viruses to neuraminidase inhibitors in vitro characterization of a-315675, a highly potent inhibitor of a and b strain influenza virus neuraminidases and influenza virus replication activity of the neuraminidase inhibitor a-315675 against oseltamivir-resistant influenza neuraminidases of n1 and n2 subtypes design, synthesis, and biological evaluation of crenatoside analogues as novel influenza neuraminidase inhibitors design, synthesis, and molecular docking of novel pyrrolooxazepinediol derivatives with anti-influenza neuraminidase activity oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials effectiveness of antiviral treatment in human influenza a(h5n1) infections: analysis of a global patient registry outcomes of adults hospitalised with severe influenza antiviral therapy and outcomes of influenza requiring hospitalization in ontario phe guidance on use of antiviral agents for the treatment and prophylaxis of seasonal influenza (2015-16). version 6.0 world health organization. who guidelines for pharmacological management of pandemic influenza a(h1n1) 2009 and other influenza viruses. geneva: world health organization new small molecule entry inhibitors targeting hemagglutinin-mediated influenza a virus fusion sialic acidmimic peptides as hemagglutinin inhibitors for anti-influenza therapy new influenza a virus entry inhibitors derived from the viral fusion peptides mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol synthesis of a new antiviral agent, arbidole clinical efficacy of arbidol (umifenovir) in the therapy of influenza in adults: preliminary results of the multicenter double-blind randomized placebocontrolled study arbitr] russian characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol design of inhibitors of influenza virus membrane fusion: synthesis, structure-activity relationship and in vitro antiviral activity of a novel indole series broadly neutralizing human antibody that recognizes the receptorbinding pocket of influenza virus hemagglutinin a broadly neutralizing human monoclonal antibody directed against a novel conserved epitope on the influenza virus h3 hemagglutinin globular head a computationally designed hemagglutinin stem-binding protein provides in vivo protection from influenza independent of a host immune response mechanism of action of t-705 against influenza virus t-705 (favipiravir) activity against lethal h5n1 influenza a viruses effects of the combination of favipiravir (t-705) and oseltamivir on influenza a virus infections in mice combinations of favipiravir and peramivir for the treatment of pandemic influenza a/california/04/2009 (h1n1) virus infections in mice differential activation of the influenza virus polymerase via template rna binding cap and internal nucleotides of reovirus mrna primers are incorporated into influenza viral complementary rna during transcription in vitro the cap-binding site of influenza virus protein pb2 as a drug target discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 preclinical activity of vx-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase pb2 subunit the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication nucleozin targets cytoplasmic trafficking of viral ribonucleoprotein-rab11 complexes in influenza a virus infection design, synthesis, and in vitro biological evaluation of 1h-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza a agents targeting virus nucleoprotein structure-based discovery of the novel antiviral properties of naproxen against the nucleoprotein of influenza a virus structure-based design of novel naproxen derivatives targeting monomeric nucleoprotein of influenza a virus binding of influenza a virus ns1 protein to dsrna in vitro binding of the influenza virus ns1 protein to double-stranded rna inhibits the activation of the protein kinase that phosphorylates the elf-2 translation initiation factor nucleocytoplasmic transport: the influenza virus ns1 protein regulates the transport of spliced ns2 mrna and its precursor ns1 mrna influenza virus ns1 protein inhibits pre-mrna splicing and blocks mrna nucleocytoplasmic transport the influenza virus ns1 protein is a poly(a)-binding protein that inhibits nuclear export of mrnas containing poly(a) influenza a virus lacking the ns1 gene replicates in interferon-deficient systems influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i the role of n-terminustruncated ns1 proteins of influenza a virus in inhibiting irf3 activation novel influenza virus ns1 antagonists block replication and restore innate immune function design, synthesis, and evaluation of novel small molecule inhibitors of the influenza virus protein ns1 novel inhibitor of influenza non-structural protein 1 blocks multi-cycle replication in an rnase l-dependent manner antiviral activity of baicalin against influenza virus h1n1-pdm09 is due to modulation of ns1-mediated cellular innate immune responses a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection influenza virus-host interactome screen as a platform for antiviral drug development genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery a phase ii study of das181, a novel host directed antiviral for the treatment of influenza infection sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection aprotinin, a protease inhibitor, suppresses proteolytic activation of pandemic h1n1v influenza virus aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice identification of the first synthetic inhibitors of the type ii transmembrane serine protease tmprss2 suitable for inhibition of influenza virus activation effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores the broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and hiv-1 envelope glycyrrhizin inhibits influenza a virus uptake into the cell a broad-spectrum antiviral targeting entry of enveloped viruses requirement for vacuolar proton-atpase activity during entry of influenza virus into cells inhibitory effect of bafilomycin a1, a specific inhibitor of vacuolar-type proton pump, on the growth of influenza a and b viruses in mdck cells the proton translocation domain of cellular vacuolar atpase provides a target for the treatment of influenza a virus infections the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts involvement of hsp90 in assembly and nuclear import of influenza virus rna polymerase subunits hsp90 inhibitors reduce influenza virus replication in cell culture mechanism of action of 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide (virazole), a new broad-spectrum antiviral agent aerosol therapy of influenza infections of mice and primates with rimantadine, ribavirin, and related compounds comparative clinical and laboratory evaluation of the prophylactic capacity of ribavirin, amantadine hydrochloride, and placebo in induced human influenza type a efficacy and safety of aerosolized ribavirin in young children hospitalized with influenza: a double-blind, multicenter, placebo-controlled trial verdinexor, a novel selective inhibitor of nuclear export, reduces influenza a virus replication in vitro and in vivo thiazolides, a new class of anti-influenza molecules targeting viral hemagglutinin at the posttranslational level effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b/3 trial influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade mek inhibition impairs influenza b virus propagation without emergence of resistant variants antiviral activity of the mekinhibitor u0126 against pandemic h1n1v and highly pathogenic avian influenza virus in vitro and in vivo combination of mek inhibitors and oseltamivir leads to synergistic antiviral effects after influenza a virus infection in vitro nf-kappab-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation active nf-kappab signalling is a prerequisite for influenza virus infection acetylsalicylic acid (asa) blocks influenza virus propagation via its nf-kappabinhibiting activity inhibitors of the sodium potassium atpase that impair herpes simplex virus replication identified via a chemical screening approach human cytomegalovirus inhibition by cardiac glycosides: evidence for involvement of the herg gene elucidation of the ebola virus vp24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function atp1a1-mediated src signaling inhibits coronavirus entry into host cells conformational changes required for reovirus cell entry are sensitive to ph identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity the cardioactive glycoside ouabain inhibits influenza a viral replication key: cord-325325-xw7627x9 authors: skeik, nedaa; jabr, fadi i. title: influenza viruses and the evolution of avian influenza virus h5n1 date: 2007-10-02 journal: int j infect dis doi: 10.1016/j.ijid.2007.07.002 sha: doc_id: 325325 cord_uid: xw7627x9 although small in size and simple in structure, influenza viruses are sophisticated organisms with highly mutagenic genomes and wide antigenic diversity. they are species-specific organisms. mutation and reassortment have resulted in newer viruses such as h5n1, with new resistance against anti-viral medications, and this might lead to the emergence of a fully transmissible strain, as occurred in the 1957 and 1968 pandemics. influenza viruses are no longer just a cause of self-limited upper respiratory tract infections; the h5n1 avian influenza virus can cause severe human infection with a mortality rate exceeding 50%. the case death rate of h5n1 avian influenza infection is 20 times higher than that of the 1918 infection (50% versus 2.5%), which killed 675 000 people in the usa and almost 40 million people worldwide. while the clock is still ticking towards what seems to be inevitable pandemic influenza, on april 17, 2007 the u.s. food and drug administration (fda) approved the first vaccine against the avian influenza virus h5n1 for humans at high risk. however, more research is needed to develop a more effective and affordable vaccine that can be given at lower doses. summary although small in size and simple in structure, influenza viruses are sophisticated organisms with highly mutagenic genomes and wide antigenic diversity. they are species-specific organisms. mutation and reassortment have resulted in newer viruses such as h5n1, with new resistance against anti-viral medications, and this might lead to the emergence of a fully transmissible strain, as occurred in the 1957 and 1968 pandemics. influenza viruses are no longer just a cause of self-limited upper respiratory tract infections; the h5n1 avian influenza virus can cause severe human infection with a mortality rate exceeding 50%. the case death rate of h5n1 avian influenza infection is 20 times higher than that of the 1918 infection (50% versus 2.5%), which killed 675 000 people in the usa and almost 40 million people worldwide. while the clock is still ticking towards what seems to be inevitable pandemic influenza, on april 17, 2007 the u.s. food and drug administration (fda) approved the first vaccine against the avian influenza virus h5n1 for humans at high risk. however, more research is needed to develop a more effective and affordable vaccine that can be given at lower doses. # 2007 international society for infectious diseases. published by elsevier ltd. all rights reserved. antigenic drift is a relatively minor antigenic change that occurs frequently within the ha or na of the virus, and is usually responsible for epidemic disease. 5, 6 antigenic shift brings up new viruses with different ha or na antigens. this can be achieved by mutation or genetic reassortment (two different influenza virus strains swap their genes giving rise to a hybrid strain), which usually leads to pandemic disease. 7 the 1918 influenza pandemic was caused by the h1n1 subtype that mutated from a purely avian virus. it was the worst pandemic by far. it killed at least 40 million people worldwide, including 675 000 people in the usa. the average life expectancy in the usa decreased by more than 10 years at that time. [8] [9] [10] [11] the 1957 pandemic was caused by the h2n2 subtype, a product of genetic reassortment in hosts infected with both an avian and human influenza virus. it killed about 70 000 people in the usa. [12] [13] [14] the 1968 pandemic was caused by the h3n2 subtype, a product of genetic reassortment. it killed approximately 34 000 people in the usa. 14 avian influenza is a contagious disease of animals caused by viruses that normally infect only birds, and less commonly, pigs. with the fact that both human and mammalian receptors are present in different proportions in tissues of both species, the bird influenza virus may infect humans. when mutations or reassortments occur, this could lead to the emergence of a fully transmissible strain, as in the 1957 and 1968 pandemics. finally, they can transmit not only to humans but also to other mammals. 15 in birds, the low pathogenic form causes only mild symptoms such as decreased egg production. the high pathogenic form can affect multiple organs and the mortality rate can reach 100%, often within 48 hours. the presence or absence of symptoms and their severity caused by low pathogenic viruses depend on the type of bird species affected. the same holds for the highly pathogenic ones: in wild and domestic ducks they can be asymptomatic, whereas they are lethal in terrestrial birds. 15 the first clinical respiratory illness of h5n1 avian influenza occurred in hong kong in 1997, when 18 human cases were reported during a poultry outbreak. it broke the species barrier to infect humans, cats, and tigers. [16] [17] [18] so far it has affected many countries in asia, africa, and europe. according to the cases reported to the world health organization (who) appearing on the who website on june 29, 2007, the h5n1 virus has already infected 317 humans and has killed 191 patients worldwide, with a mortality rate exceeding 50%. 19 avian influenza h5n1 also affects domesticated poultry, including chickens, ducks, and turkeys. the contribution of migratory birds like wild ducks, geese, swans, and hawks (saudi arabia) to the spread of the h5n1 virus from asia to europe and africa is still controversial, since basically all wild birds positive for h5n1 virus have been found dead or very sick, thus unlikely to have been able to fly over long distances. at the same time, more evidence supports the role of the international illegal poultry trade. it is possible that this trade contributed to the 2006 h5n1 outbreak in a large commercial farm in kaduna state in the northern part of nigeria. on the other hand, the country is known to lie along a flight route for birds migrating from central asia. the birds shed the virus in their saliva, nasal secretions, and feces. infection can spread among birds by contact with infected birds or with their excretions. people can get infected by direct or close contact with infected poultry or surfaces contaminated with secretions and excretions from the infected birds. human exposure occurs most often during slaughtering, de-feathering, butchering, or preparation for cooking. raw poultry or eggs can also transmit the disease. there are no reported cases through properly cooked poultry. 15, 20 human-to-human transmission was suggested in a family in thailand, when an 11-year-old girl who had been in contact with an infected bird, and her mother who had provided her with nursing care, both died of the avian influenza. the girl's aunt, who had also had close contact with the girl, survived the infection after treatment with oseltamivir. h5n1 was confirmed in the mother and the aunt. 21, 22 recently there have been fears of other human-to-human transmission cases after seven family members died of the avian influenza in indonesia. who confirmed that partial human-to-human transmission occurred. 20 other potential modes of transmission of h5n1 virus include contamination of hands from infected fomites and exposure to untreated poultry feces used as fertilizers. further possible but not proven modes of transmission are oral ingestion of contaminated water during swimming and direct intranasal or conjunctival inoculation during exposure to water. 23 the virulence of the avian influenza viruses in mammals is not well understood. as opposed to the earlier h5 viruses, the more recently circulating h5 viruses appear to be more pathogenic in mammals and birds. this is a feature that may precede the emergence of reassorted h5 strains with pandemic potential. 17, 24 although virulence determinants are polygenic traits, the possible major contributing factor is the hemagglutinin molecule. the acquisition of a multibasic amino acid sequence at the cleavage site of a hemagglutinin belonging to the h5 or h7 subtypes enables its widespread cleavage by ubiquitous tissue proteases, resulting in multi-organ infection and high pathogenicity. 25, 26 h5n1 viruses are relatively resistant to host antiviral cytokines, which leads to the production of high levels of cytokines and an excessive pro-inflammatory response causing tissue injury. 27, 28 this suggests that the severity of human h5n1 infection may be related to the excessive pro-inflammatory responses that exacerbate tissue injury. 23, 29 autopsies of current human cases of h5n1 influenza have revealed necrotizing hemorrhagic pneumonia, similar to that found in the 1918 influenza cases. like the 1918 virus, h5n1 is associated with unusually high death rates in humans; in fact the case death rate is 20 times higher than that of the 1918 virus (50% vs. 2.5%). 30 continuous evolution of the h5n1 virus has been suggested by changes in the internal gene constellation, expanded host range, increased pathogenicity, and greater environmental stability. 31, 32 symptoms and signs according to one study, symptoms of h5n1 infection include typical influenza-like symptoms: fever (100%), cough and sore throat (67%), myalgias (30%), pneumonia (58%), diarrhea and vomiting (50%), and congestion of the conjunctiva (0%). laboratory findings include elevated serum aminotransferases and pancytopenias. 23, 33 complications include acute respiratory distress syndrome (ards), pulmonary hemorrhage, myocarditis, pericarditis, encephalitis, multi-organ failure with renal dysfunction, and sepsis. most deaths have been related to respiratory failure. 23 the spectrum of disease is wide. two children from the same family in vietnam presented with diarrhea and encephalopathy, without any signs of respiratory compromise. 34, 35 the incubation period for h5n1 may be longer than other known human influenza viruses. 23 in 1997, most cases occurred within two to four days after exposure, while reports of cases from 2004 suggest that longer intervals of up to eight days may be possible. 16, 36 diagnosis diagnosis can be made with clinical findings, plus recent history of exposure to dead or ill poultry, and can be confirmed with serologic tests. travel history is also very important. rapid antigenic testing kits are not able to subtype influenza a, and the standard serologic test (ha inhibition test) is insensitive. [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] diagnosis can be confirmed by haspecific pcr assay or by viral culture of a nasopharyngeal aspirate obtained within three days of the onset of symptoms. 23 elisa and western blotting are useful for epidemiologic surveillance studies and retrospective diagnosis. 37 radiologic findings include diffuse, multi-focal or patchy infiltrates and segmental or lobar consolidation with air bronchograms. 36 the differential diagnoses of avian influenza include atypical pneumonia, human influenza, respiratory syncytial virus, severe acute respiratory syndrome (sars), and upper respiratory tract infections associated with conjunctivitis (e.g., adenovirus). high-risk individuals, such as patients with a history of travel within 10 days of symptom onset to a country with documented h5n1 avian influenza in poultry and/or humans, and patients who have radiographically confirmed pneumonia, ards, or any other severe respiratory illness for which an alternate etiology has not been established, should be tested. for low-risk individuals, testing should be considered in patients with a history of contact with domestic poultry or with a known or suspected human case in an h5n1-affected country within 10 days of symptom onset, documented fever of 38 8c, and one or more of the following: cough, sore throat, shortness of breath. 23, 36 treatment whenever feasible, while the number of affected persons is small, patients with suspected or proven influenza a (h5n1) infection should be hospitalized in isolation for clinical monitoring, appropriate diagnostic testing, and antiviral therapy. 38 the majority of h5n1 isolates are still sensitive to amantadine. however, more recent viruses isolated in thailand and vietnam have amino acid substitutions within the m2 protein, which confer resistance to amantadine and rimantadine. 39, 40 these viruses are susceptible to the neuraminidase inhibitors in animal models. 41 oseltamivir should be started as soon as possible. in an animal study where mice were inoculated with h5n1 virus isolated from a patient who did not survive the infection, the mice that were treated for eight days with oseltamivir had significantly better survival rates in all dose ranges when compared to animals that were treated for only five days. 39 prior animal studies demonstrated that five days of oseltamivir was effective against another h5n1 variant. 42 thus, treatment dose and duration may vary according to the pathogenicity and virulence of the virus. 39 an oseltamivir-resistant h5n1 strain (his274tyr) has been reported in two cases in vietnam. both patients died of the infection despite early initiation of treatment in one case and proper dosage (75 mg twice daily) in both cases. therefore, the use of higher doses, longer durations of treatment, or combination therapy may deserve further evaluation. 43 improper use of personal stockpiles of oseltamivir may promote resistance and should be strongly discouraged. 44 zanamivir is another neuraminidase inhibitor, and was the first neuraminidase inhibitor to be approved for influenza treatment. it is also recommended for the treatment of h5n1 infection, together with oseltamivir. corticosteroids have been used frequently in treating patients with h5n1 infection with uncertain effects. other agents like peramivir, long-acting topical neuraminidase inhibitors, ribavirin, 45, 46 and possibly interferon alpha, which has both antiviral and immunomodulatory activities, are under investigation. 47 according to a recent report, patients with spanish influenza pneumonia who received influenza-convalescent human blood products may have experienced a clinically important reduction in the risk of death. convalescent human h5n1 plasma could have similar benefits and should be studied in clinical trials. 48 although immunization with human influenza vaccine will not protect against avian influenza strains, it should be considered in poultry workers, and also be given to those traveling to affected areas, two weeks ahead of departure, to prevent co-infection and reassortment. 23 according to centers for disease control and prevention (cdc) and who recommendations, people who live in affected areas should avoid all direct contact with poultry and surfaces contaminated with poultry feces and secretions, should avoid eating undercooked eggs and poultry, should wash hands carefully and frequently, and should seek medical attention if they become ill within 10 days of suspected contact. 15, 20 post-exposure prophylaxis with oseltamivir 75 mg daily for 7 to 10 days should be recommended to household contacts of patients. 23, 49, 50 although the risk of transmission from person influenza viruses and the evolution of avian influenza virus h5n1 to person appears low, quarantining of close contacts to patients for a week after last exposure and monitoring for symptoms may help to reduce transmission rates. 23 if available, negative pressure rooms should be used for patient isolation. healthcare workers should wear n-95 masks (non-oilproof respirators with at least 95% efficiency in filtering particles more than 3 mm in diameter), gloves, long sleeved cuffed gowns, and eye protection when within 3 feet of patients. 51 quarantine and depopulation or culling of affected poultry is the preferred way of eradication. the use of inactivated h5n1 vaccines in chickens is an additional step but should be done with caution. 52 vaccine currently, there is no commercially available vaccine against h5n1 virus. due to safety and technical reasons, and since h5n1 is highly virulent and lethal to eggs, traditional methods of production are not feasible. furthermore, it is impossible to predict whether the currently circulating h5n1 strain will cause the next pandemic. if successfully produced, vaccines would likely be the most important health tools to decrease morbidity, mortality, and the economic effects of pandemic influenza. resistance to oseltamivir makes vaccines even more important. 23, 53 the h5n1 viruses can be divided into clade 1 and clade 2; the latter can be further subdivided into three subclades. these clades and subclades probably differ sufficiently in their antigenic structure to warrant the preparation of different vaccines. studies in ferrets suggest that vaccination against one clade will not protect against infection with another clade, though it will protect against influenza-associated death. 54 a new vaccine prepared from an egg-grown recombinant influenza a virus, composed of the hemagglutinin and neuraminidase genes from a human h5n1 isolate inserted into a laboratory-adapted human influenza a strain, achieved only 54% presumably protective micro-neutralization titers of 1:40 following use of the maximum tested dose (90 mg). this dose is 12 times that of the seasonal influenza vaccine, making mass production untenable with current manufacturing capacity. current seasonal vaccines contain 15 mg ha/ strain/dose. whether this vaccine could induce cross-protection against other h5n1 strains is unclear. furthermore, the death of a chinese woman infected with an h5n1 strain markedly different from those now being used to develop vaccines has recently been reported. [55] [56] [57] studies of different dose levels of vaccines administered with adjuvants like aluminum hydroxide are urgently needed to improve immunogenicity and increase the number of doses available (if lower doses are effective). [57] [58] [59] recently, promising progress has been made in industry-supported research in france and the uk, where the safety and immunogenicity of a monovalent, inactivated split-virion vaccine, derived from a highly pathogenic strain of h5n1 influenza, has been investigated; this vaccine was administered with and without a fixed dose of aluminum hydroxide as an adjuvant. on day 42, 67% of subjects who received the highest dose ha with adjuvant exhibited detectable antibody titers, and 61% had positive ha-inhibition responses. 60 other promising approaches to vaccine development involve dna, adenovirus vectors, and cell manufacturing techniques to increase the speed and capacity of vaccine production. 61, 62 on april 17, 2007, the u.s. food and drug administration (fda) approved a human vaccine against the h5n1 influenza virus, marking the first such approval in the usa. should h5n1 develop the ability to spread readily from person to person, this vaccine may provide early limited protection in the months before a vaccine tailored to the pandemic strain of the virus can be developed and produced. the vaccine will be kept in a federal stockpile and will be made available only through public health officials; it is approved for those aged 18 to 64 years who are at increased risk for h5n1 exposure. 63 for a pandemic to occur, three conditions must be met: a new influenza virus subtype must emerge, this must infect humans and cause serious disease, and finally spread easily among humans. 20 the first two conditions have already been met, and there are new suggestions of human-to-human transmission in thailand and indonesia. however, there is no evidence to-date of the easy human-to-human transmission that is key for a pandemic. [20] [21] [22] [23] with the emergence of influenza virus h5n1, the threat of an influenza pandemic seems to be real and inevitable, but no one can predict when it might happen. according to a study by the congressional budget office, the consequences of a severe pandemic could, in the usa, include 200 million people infected, 90 million clinically ill, and 2 million dead. the study estimated that 30% of all workers would become ill and 2.5% would die, resulting in a decrease in the gross domestic product of 5%. furthermore, 18 to 45 million people would require outpatient care, and the economic cost would total approximately $675 billion. 64 the world is facing the real threat of another influenza pandemic with a virus that has a great pathogenicity and a mortality rate in humans exceeding 50%. despite the high level of technology and ongoing research, at the present time there is no highly effective vaccine against avian influenza h5n1 virus that can be manufactured commercially on a large scale for use at low doses. as is always said, ''a dime of prevention is better than a pound of treatment''. responsibility should be taken at all levels and preventative steps should be implemented immediately. healthcare providers should be up-to-date with the pandemic risk and educate their patients. such information can be found at www.cdc.gov/flu. other sources of information include www.defra.gov.uk, www.fda.gov, www.who.int, and www.eurosurveillance.org. conflict of interest: no conflict of interest to declare. harrison's principles of internal medicine evolution and ecology of influenza a viruses global epidemiology of influenza: past and present influenza virus neuraminidase inhibitors influenza virus antigens and 'antigenic drift analysis of antigenic drift in recently isolated influenza a (h1n1) viruses using monoclonal antibody preparations influenza--a model of an emerging virus disease enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus the genetic archaeology of influenza characterization of the reconstructed 1918 spanish influenza pandemic virus transmissibility of 1918 pandemic influenza the threat of an avian influenza pandemic on the origin of the human influenza subtypes h2n2 and h3n2 avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics questions and answers about avian influenza (bird flu) and avian influenza a (h5n1) virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus avian h5n1 influenza in cats avian influenza h5n1 in tigers and leopards cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who avian influenza frequently asked questions probable person-to-person transmission of avian influenza a (h5n1) transmission of avian influenza viruses to and between humans avian influenza a (h5n1) infection in humans h5n1 influenza: a protean pandemic threat molecular basis for high virulence of hong kong h5n1 influenza a viruses pb2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of hong kong h5n1 influenza a viruses in mice lethal h5n1 influenza viruses escape host anti-viral cytokine responses induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? re-emergence of fatal human influenza a subtype h5n1 disease avian influenza: an emerging pandemic threat avian influenza in hong kong antigenic differences between h5n1 human influenza viruses isolated in 1997 and antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma human disease from influenza a (h5n1) detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays world health organization. who interim guidelines on clinical management of humans infected by influenza a (h5n1) virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 influenza virus in mice update: influenza activity--united states and worldwide, 2003-04 season, and composition of the 2004-05 influenza vaccine genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses oseltamivir resistance in influenza a (h5n1) infection the run on tamiflu--should physicians prescribe on demand? in vitro inhibitory effects of combinations of anti-influenza agents ribavirin aerosol treatment of influenza absence of interferon in lungs from fatal cases of influenza meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? management of influenza in households: a prospective, randomized comparison of oseltamivir treatment with or without postexposure prophylaxis effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial protecting healthcare staff from severe acute respiratory syndrome: filtration capacity of multiple surgical masks preparation of a standardized, efficacious agricultural h5n3 vaccine by reverse genetics confronting the avian influenza threat: vaccine development for a potential pandemic immunization with reverse-genetics-produced h5n1 influenza vaccine protects ferrets against homologous and heterologous challenge safety and immunogenicity of an inactivated subvirion influenza a (h5n1) vaccine lethal avian influenza a (h5n1) infection in a pregnant woman in anhui province, china vaccines against avian influenza--a race against time immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use pandemic preparedness: lessons learnt from h2n2 and h9n2 candidate vaccines safety and immunogenicity of an inactivated split-virion influenza a/vietnam/1194/2004 (h5n1) vaccine: phase i randomized trial development of adenoviral-vector-based pandemic influenza vaccine against antigenically distinct human h5n1 strains in mice from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza fda approves first u.s. vaccine for humans against the avian influenza virus h5n1 a potential influenza pandemic: an update on possible macroeconomic effects and policy issues key: cord-336465-qrok21qo authors: perez, luis e.; merrill, gerald a.; delorenzo, robert a.; schoenfeld, thomas w.; vats, abhay; moser, michael j. title: evaluation of the specificity and sensitivity of a potential rapid influenza screening system date: 2013-01-31 journal: diagnostic microbiology and infectious disease doi: 10.1016/j.diagmicrobio.2012.09.005 sha: doc_id: 336465 cord_uid: qrok21qo abstract influenza remains a serious worldwide health threat with numerous deaths attributed to influenza-related complications. it is likely that transmission of influenza and both the morbidity and mortality of influenza could be reduced if inexpensive but reliable influenza screening assays were more available to the general public or local medical treatment facilities. this report provides the initial evaluation of a pilot system designed by lucigen corp. (middleton, wi, usa) as a potential rapid near point-of-care screening system for influenza a and influenza b. the evaluation of specificity and sensitivity was conducted on stored nasal swab samples collected from emergency department patients presenting with influenza-like symptoms at a large military academic hospital and on de-identified nasal swabs and isolated rna from a local epidemiology laboratory. the gold standard for assessment of specificity and sensitivity was the luminex® respiratory viral panel. seasonal influenza epidemics result in about 3-5 million cases and around 0.25-0.5 million deaths each year worldwide (who, 2009 ). in the united states, up to 300,000 people are hospitalized annually and 10,000-40,000 die from influenza-related complications (cdc, 1999) . diagnosis of influenza a and b by clinical manifestations alone is difficult because of overlapping symptoms from a variety of pathogens including rhinovirus (arden and mackay, 2010) , coronavirus (renois et al., 2010) , parainfluenza virus (lau et al., 2005) , respiratory syncytial virus (freymuth et al., 2004) , adenovirus (lina et al., 1996) , metapneumovirus (debur et al., 2010) , enterovirus (lina et al., 1996) , and streptococcus pyogenes (yamada et al., 2010) . use of antibiotics to treat upper respiratory tract infections without proper diagnosis is a common practice in the united states (franck and smith, 2010; linder et al., 2003) . for instance, in a national ambulatory network study of 52,135 upper respiratory tract episodes identified, 65% received antibiotics (gill et al., 2006) , although respiratory viruses are responsible for approximately 80% of respiratory infection (mahony, 2008) . use of antibiotics, which are of little benefit for viral infections, can play a major role in the development and spread of antibioticresistant microorganisms and substantially increase health care costs. thus, development of a rapid, cost-effective, and accurate screening system for influenza virus available to the general population and/or local physicians and medical treatment facilities would likely reduce the misuse of antibiotic therapy for treatment of influenza infections. rapid identification of influenza infections would further reduce transmission of virus including nosocomial infections, allow for timely antiviral therapy, and likely reduce the morbidity and mortality of influenza-related complications thereby lowering health care costs. currently, there are several methods used to diagnose influenza a and b including viral culture, lateral flow immunoassays (lfias), direct fluorescent antibody tests (dfas), and nucleic acid tests (nats). diagnosis of influenza a or b through viral culture is effective but is labor intensive, time consuming, and may require highly trained personnel and several days to provide confirmed results (balada-llasat et al., 2011; ruest et al., 2003) . current lfia methods for influenza a or b have high specificity (90-100%), are cost effective, and results can be acquired within 15 to 30 min, but, unfortunately, these tests have been shown to exhibit variable sensitivity ranging from as low as 10% up to 100% (louie et al., 2009) . dfa influenza assays demonstrate sensitivities of 60% to 80%, and results can be obtained within 2 h, but require high level of technical proficiency to perform and culture to confirm the results (cram et al., 1999) . nats offer high sensitivity and specificity for the diagnosis of influenza a or b (jenny et al., 2010; louie et al., 2009; pabbaraju et al., 2008) . however, use of nats in the clinical sector may be prohibited because of high cost and delayed reporting of results as compared to lfia and dfa. the lack of an affordable, near point-of-care, sensitive, and specific screening system for influenza virus created a void that was addressed by lucigen corp. (middleton, wi, usa) by designing a first-generation system to test the feasibility of a nucleic acid lateral flow system for testing both influenza a and b. the pyroscript® influenza a and b reagents require the use of a nucleic acid lateral flow (nalf) device for the detection of influenza a or b-amplified rna molecules. nalf devices work in a manner analogous to lateral flow immunoassays. a visual result can be obtained within 2 h after sample collection allowing rapid interpretation of results without the need of complex or expensive instruments. the pyroscript® influenza screening reagents could be the alternative solution for a rapid influenza a and b test in the clinical sector. this research was conducted under an internal review boardapproved protocol which allowed for the collection of clinical samples from patients presenting with influenza-like respiratory symptoms in the emergency department (ed) of brooke army medical center after informed consent was obtained. transfer of de-identified specimens (stored virus or, in the case of h1n1 novel 2009 [swine flu] specimens, isolated viral rna) from a local epidemiology reference laboratory was also approved. twenty nasal swab samples collected from ed patients with influenza-like symptoms, 5 archived de-identified streptococcus pyogenes bacterial specimens, and 75 deidentified specimens transferred from the local epidemiology laboratory were investigated. although the viral samples obtained from the epidemiology reference laboratory were de-identified (all personal health information and personal identifiers removed), they were supplied with the etiologic agent of each sample identified. the laboratory personnel performing the extractions, luminex rvp assay, and lucigen pyroscript tests were blinded to the clinical results until the conclusion of all testing. these samples included 24 novel influenza a h1n1 rna specimens and 12 influenza a h1n1 seasonal, 12 influenza a h3n2 seasonal, 15 influenza b seasonal; 5 adenovirus, 4 parainfluenza 3 viral specimens, and 3 negative samples (no virus detected). the 5 streptococcus pyogenes culture-positive throat swab samples were confirmed via polymerase chain reaction (pcr) with specific primers for the ubiquitous pros gene as described before (livezey et al., 2011) and were included in the study as influenzanegative controls. all samples were stored at −80°c. the objective of this preliminary study was to assess the specificity and sensitivity of the influenza a and b nalf systems as a potential rapid screening assay. this system, because of its simplicity and independence from expensive or complicated instrumentation, could be used in near point-of-care settings. analysis time was considered, but did not include sample preparation time since our samples were stored specimens. for our convenience and reproducibility purposes, as well as for comparison to the food and drug administration approved xtag® (luminex molecular diagnostics, toronto, ontario, canada) respiratory viral panel (rvp) test, nucleic acid isolation was performed by an automated system (nuclisens® easymag® system, biomérieux, marcy l'etoile, france) as described in bolotin et al. (2009) . the purified nucleic acids obtained were stored at −80°c. the gold standard for the specificity and sensitivity assessment was the results of the fda approved rvp, a nucleic acid test. the rvp test had a sensitivity of 98.2% and a specificity of 96.4% in a study of 247 clinical samples when compared to dfa and culture results (mahony et al., 2007) . all viral isolates were amplified, hybridized, and detected with the luminex® is-200 instrument (luminex, austin, tx) using the xtag rvp kit following the vendor's protocol. briefly, 5 μl of purified nucleic acid from each sample was reverse transcribed in a 25-μl multiplex reverse transcription polymerase chain reaction (rt-pcr) tube. a multiplex target specific primer extension (tspe) reaction was prepared per sample using 5 μl of treated rt-pcr. after tspe, 3.5 μl of reaction product of each reaction was added directly to a micro-well containing 20 μl of rvp luminex bead mixture for hybridization. phycoerythrin reporter (100 μl) was added to each well containing hybridization product to identify the presence of a virus by assessment of the fluorescence in a luminex is-200. results generated were analyzed using the software component of the kit (tdas rvp-i). in addition, all novel h1n1 2009 rna samples were assayed by a microarray technique (electrasense® assay, combimatrix, corp., mukilteo, wa, usa) to positively identify the h1(sw)n1 variant (straight et al., 2010) . the pyroscript® influenza reagents (lucigen) were tested for all samples in this study. one reaction per test was prepared on ice in a 200-μl pcr tube as follows: 6.25 μl nuclease-free water, 12.50 μl pyroscript isothermal 2× master mix, 1.25 μl 20× primer mix, and 5 μl of nucleic acid sample. the reaction was mixed and centrifuged briefly before incubating for 40 min at 72°c in a thermal cycler (eppendorf 5417r, hamburg, germany) preheated to 72°c. the reaction was stopped at 4°c on ice until the 200-μl pcr tube containing specific amplicons was loaded in a cartridge of a type i best™ cassette (biohelix corporation, beverly, ma, usa) for detection according to the manufacturer's instructions. the influenza a or b-amplified molecules entered the dna strips via capillary action. when the cassette was closed, the pcr tube released the 6-carboxy fluorescein (fam) and biotin-labeled amplicons (flu a or flu b reagent) and detected via gold nanoparticle-labeled anti-fam antibodies. results were visually read after 10-15 min, photographed, and results recorded. each ed sample was routinely tested using a rapid antibody assay (binaxnow, alere, waltham ma, usa), dfa, and viral culture by the bamc clinical laboratory (data not shown). all seasonal influenza a samples were previously identified by the local epidemiology laboratory as influenza a h1n1 and h3n2 subtypes, respectively, as per their standard protocol (data not shown). influenza b samples were similarly identified by the epidemiology laboratory. all 12 influenza a h1n1 seasonal viral samples were confirmed by the luminex rvp assay as influenza a h1. similarly, all 12 influenza a h3n2 seasonal viral isolates were confirmed as influenza a h3 by the rvp assay. two additional samples collected at the bamc ed were identified by the rvp as influenza a (no-subtype) and were classified as novel h1n1 influenza a, although this was not confirmed by pcr or microarray. six of the 20 ed samples were identified by the luminex rvp assay as positive for virus: 2 influenza a no-subtype (as discussed above), 2 rhinovirus, 1 parainfluenza 3, and 1 metapneumovirus. in addition, all 15 influenza b, 5 adenovirus, and 4 parainfluenza 3 samples from the epidemiology laboratory were also confirmed by the luminex rvp assay. fourteen of the 20 ed samples, plus the 3 samples from the epidemiology laboratory with no virus detected, and the 5 streptococcus pyogenes bacterial samples were used as the 22 negative controls for both the influenza a and influenza b calculations (see table 1 a total of 3 false-positive (positive by the lucigen system but negative by the gold standard luminex rvp assay) influenza a and 6 false-positive influenza b results were observed with the pyroscript influenza tests. in the case of influenza a, all samples not identified by the rvp assay as influenza a are considered as true negatives for calculation of the influenza a specificity. conversely, all samples not identified by the rvp assay as influenza b are considered as true influenza b negatives. thus, the specificity of the pyroscript influenza reagents are 50/53 (94.3%) for influenza a and 84/90 (93.3%) for influenza b tests. the recent 2009 influenza pandemic caused about 55 million human cases of novel 2009 influenza h1n1 with approximately 246,000 h1n1-related hospitalizations and about 11,160 h1n1related deaths in the united states alone from april to december of 2009 according to estimates from the centers for disease control and prevention (cdc, 2010) . the development of easy-to-use, costeffective, and reliable new diagnostic methods accessible to primary health care providers for screening patients is needed for the rapid detection of influenza a and influenza b. rapid screening for influenza at primary health care facilities would aid in sequestering infected patients from the general population to limit transmission and would reduce the unnecessary use of antibiotics provided to influenza patients misdiagnosed as having bacterial respiratory illnesses. the pyroscript influenza reagents in combination with the cartridge of type i best™ cassette tested in our study demonstrated excellent outcome parameters with greater than 90% specificity and sensitivity for both influenza a and b tests. similar isothermal amplification tests in combination with type i best™ cassette have been developed and used to rapidly detect other pathogenic microorganisms including herpes simplex virus types 1 and 2 (kim et al., 2011) , mycobacterium tuberculosis (motre et al., 2011) , human immunodeficiency virus (tang et al., 2010) , and toxigenic clostridium difficile (chow et al., 2008) . the 24 novel influenza a h1n1 samples were initially identified by the rvp at the epidemiology laboratory as influenza a no-subtype. novel influenza a h1n1 is detected with the rvp assay as "influenza a no-subtype" due to recognition of the matrix gene, combined with lack of detection of either the seasonal h3 or the h1 target genes. investigators have reported that during the 2009 influenza season all "influenza a no-subtype" cases detected with the rpv test were most likely novel influenza a h1n1 (ginocchio and st george, 2009; vinikoor et al., 2009) . all 24 samples were confirmed via rt-pcr as novel influenza a h1n1 (data not shown) prior to being received at bamc. of these 24 rna samples tested at bamc by the electra-sense®, 23 were confirmed as influenza a h1(sw)n1 variants (straight et al., 2010) . the novel influenza a h1(sw)n1 rna sample not detected by this microarray technique was also reevaluated by the luminex rvp assay, and no viral etiology could be identified. therefore this sample was eliminated from this study. extraction of nucleic acids for the detection of influenza virus with automated robotic systems has been used by other investigators (belongia et al., 2010; bolotin et al., 2009; bose et al., 2009; yang et al., 2011) , as was done in this study using the nuclisens® easymag® system. the easymag® system requires at least 1 h to extract nucleic acids from 24 clinical samples including sample preparation time and 10-min viral/bacterial lysis time. automated nucleic acid extraction systems are excellent instruments for use in reference laboratories where a larger number of samples are processed but are bulky and expensive to use in small laboratories or physician's offices. alternative nucleic acid extraction methods must be incorporated into future pyroscript influenza screening systems to fulfill the time efficiency requirement of a rapid screening assay. in addition, the requirement to screen for influenza a and b separately with the tested pyroscript influenza system requires 2 reactions with different reagents and 2 detection cassettes. ideal future versions of the pyroscript influenza screening system should also allow for simultaneous screening of both viruses and might include other respiratory virus targets. the objective of this study was to determine the sensitivity and specificity of the pyroscript influenza system. loss of sensitivity occurs if the test assay does not produce a positive result when the gold standard assay is determined to be positive. the 91.8% influenza a and 93.3% influenza b sensitivity resulted from failure to identify 5 rvp-positive samples as positive by the pyroscript assay. there could be several reasons for the pyroscript not being able to identify some of the true positives. one such possibility could be due to stored rna deterioration with time. since insufficient sample remained for additional retesting by the rvp assay, the possibility cannot be excluded that the 3 h1n1 novel, the seasonal influenza a, and the seasonal influenza b samples that appear as false negatives could be from long-term storage-induced rna degradation and/or freeze-thaw cycles (botling et al., 2009; ginocchio et al., 1997) . loss of specificity occurs due to a false-positive result (positive by the pyroscript assay but not the rvp). in this study, a total of 9 false positives were observed. one possibility for these false-positive results could be that the primers for rvp and pyroscript assay are not identical. an additional possibility is that there may have been a mutation in the viral genome where the rvp primers/probes bind which did not affect the isothermal amplification employed in the pyroscript tests. another possibility could be nonspecific amplification by the pyroscript assay. in conclusion, the pyroscript influenza reagents in combination with the cartridge of type i best™ cassette are self-contained disposable detection devices and do not require special instrumentation or complicated specimen handling to obtain a visual result within 2 h after nucleic acid isolation. as such, the pyroscript influenza a and b tested in this study show the potential to fill the screening void for rapid influenza diagnosis. future development of the pyroscript influenza reagents, screening system, and the implementation of alternative nucleic acid extraction methods to provide a true point-of-care nat for human influenza diagnostics is warranted. newly identified human rhinoviruses: molecular methods heat up the cold viruses evaluation of commercial resplex ii v2.0, multicode-plx, and xtag respiratory viral panels for the diagnosis of respiratory viral infections in adults clinical characteristics and 30-day outcomes for influenza a 2009 (h1n1) validation of the taqman influenza a detection kit and a rapid automated total nucleic acid extraction method to detect influenza a virus in nasopharyngeal specimens rapid semiautomated subtyping of influenza virus species during the 2009 swine origin influenza a h1n1 virus epidemic in impact of thawing on rna integrity and gene expression analysis in fresh frozen tissue centers for disease control and prevention (cdc). cdc estimates of 2009 h1n1 influenza cases, hospitalizations and deaths in the united states application of isothermal helicase-dependent amplification with a disposable detection device in a simple sensitive stool test for toxigenic clostridium difficile diagnostic testing for influenza: review of current status and implications of newer treatment options impact of human metapneumovirus infection on in and outpatients for the years 2006-2008 in southern brazil antibiotic use for acute upper respiratory tract infections in a veteran population epidemiology and diagnosis of respiratory syncytial virus in adults use of antibiotics for adult upper respiratory 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streptococcus gene expression in humans with pharyngitis using a microarray point-of-care testing for pandemic influenza and biothreats development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbead-based assay detection of respiratory viruses by molecular methods improving isothermal dna amplification speed for the rapid detection of mycobacterium tuberculosis comparison of the luminex xtag respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection a novel electrochemical device to differentiate pandemic (h1n1) 2009 from seasonal influenza nucleic acid assay system for tier ii laboratories and moderately complex clinics to detect hiv in low-resource settings influenza a virus subtyping: paradigm shift in influenza diagnosis influenza (seasonal), fact sheet no invasive group a streptococcal infection in pregnancy comparison of commercial systems for extraction of nucleic acids from dna/rna respiratory pathogens the authors would like to acknowledge the help of dr. john a. ward with the statistical consultations he provided and suzanne mccall for her laboratory and technical support. key: cord-304569-o39kl5k4 authors: nguyen-van-tam, jonathan s title: from the editor's desk date: 2015-04-23 journal: influenza other respir viruses doi: 10.1111/irv.12311 sha: doc_id: 304569 cord_uid: o39kl5k4 nan it is now just over 1 year since i took over the editorship of influenza and other respiratory viruses (irv). in that time, there have been many changes to the journal and, of course, in the wider world of respiratory viruses. i'd like to begin by thanking my predecessor and the journal's founding editor, dr. alan hampson. alan set up, nurtured and grew irv into a vibrant and successful publication before officially handing over the reins in early 2014. he remains on the team as one of our senior editors. indeed my task as editor-in-chief would be impossible without the fantastic and dedicated team of senior editors, associate editors and professional staff at wiley, who support me, and all of whom deserve many personal thanks. the challenges posed by respiratory viruses remain as important as ever for global public health, and clinical practice. in the aftermath of the 2009 pandemic, governments and public health agencies are wrestling with 'pandemic fatigue', austerity programmes (that limit the appetite and ability to invest in pandemic preparedness), and a false impression among some politicians that since the 2009 pandemic was rather mild and 'not much to write home about', pandemic preparedness is in fact 'no big deal'. in amongst this mix are real issues pertaining to the ongoing controversy about the effectiveness of antiviral drugs, [1] [2] [3] ; and the fact that current vaccine manufacturing platforms can only offer commercial quantities of pandemic vaccine some four to 6 months after a novel virus has emerged, thus substantially reducing the overall public health benefits, even though vaccines themselves are effective. 4, 5 most public health agencies and individual experts, consider the potential pandemic threat posed by influenza a(h5n1) to be undiminished. but in addition, influenza a (h7n9) is now, if anything, considered a higher potential risk. 6 recent human cases of influenza a(h10n8), 7 and a (h5n6), 8 further remind us that the risk assessment landscape for influenza is constantly evolving and, in turn, this demands constant vigilance from the public health and scientific communities. if one shifts the focus away from influenza, the ongoing mers-cov outbreak in the middle east, is also of substantial concern, because despite its likely introduction into humans via close contact with dromedary camels, 9 nosocomial transmission appears to be a central concern, 10,11 case-fatality is high, household transmission is also described, 12 and there are currently no vaccines or specific therapies available. finally enterovirus d68 seems to be emerging as a potentially important respiratory pathogen in children. 13 seasonal influenza too, should not be overlooked as an ongoing problem. in the northern hemisphere winter of 2014-15 we have experienced substantial influenza a(h3n2) activity. unfortunately this has coincided with a poorly matched h3n2 vaccine component that has resulted in low effectiveness against the circulating h3n2 strains in the community. 14 this has recently been linked to excess winter mortality across europe in the population aged 65 years and over, 15 illustrating that we need more broadly protective vaccines, especially in the elderly. from the journal's perspective, to be in the best possible position to respond to these emerging and sometimes fastmoving threats, we have made some recent changes that enable us to publish findings with minimal delay. for papers of significant importance, we can offer a rapid peer-review scheme, which should enable us to make a decision on a manuscript within 14 days (or less). we have also improved our arrangements for all accepted articles (whether fasttracked or not). following acceptance, we can now publish all articles, as unedited manuscripts, online within 1 week of acceptance. we have also reconsidered the arrangements for review articles that we publish. we recognise that systematic reviews and meta-analyses have become a central part of evidence synthesis in modern science and medicine. with this has come the setting of standards for the conduct of such work: the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines; 16,17 and we now require any systematic reviews that we accept to conform to these principles. however, we shall continue to accept expert commentary articles alongside as these continue to be relevant and useful to our readers. hopefully these changes, along with our now wellestablished open access format will maintain the journal as a vibrant publication that is relevant, and highly accessible, to scientists and clinicians working in the field of respiratory virus infection for the foreseeable future. authors have the option to request rapid peer-review. papers considered for rapid peer-review will need to be of immediate relevance, interest, or importance to scientists, clinicians, public health practitioners or policy makers, usually in relation to a current or evolving event related to respiratory virus activity. in general, papers that report data more than 6 months old are unlikely to be considered eligible. if your paper qualifies for rapid peer-review, the journal will aim to have your paper turned round within 14 days. there is no additional charge for authors with rapid peer-review. step #1 -authors will need to contact the editorial office irv.eo@wiley.com, at least 1 week before online submission with an abstract; list of authors; please ensure a prisma checklist is completed if your paper is a systematic review article; details of research funding and disclosures of potential conflicts of interest should also be included. step #2 -the editors will confirm whether your paper will qualify for rapid peer-review. the decision is final and non-negotiable. step #3 -submit the paper via the scholarone system. accepted articles have been accepted for publication and undergone full peer review but have not been through the copyediting, typesetting, pagination and proofreading process. accepted articles are published online a few days after final acceptance, appear in pdf format only, are given a digital object identifier (doi), which allows them to be cited and tracked, and are indexed by pubmed. effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data oseltamivir for influenza in adults and children: systematic review of clinical study reports and summary of regulatory comments oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials effectiveness of h1n1 vaccine for the prevention of pandemic influenza in scotland, uk: a retrospective observational cohort study effects of vaccine program against pandemic influenza a(h1n1) virus, united states pandemic influenza viruses--hoping for the road not taken avian influenza a h10n8--a virus on the verge? evidence for camel-to-human transmission of mers coronavirus mers-cov outbreak in jeddah -a link to health care facilities hospital outbreak of middle east respiratory syndrome coronavirus transmission of merscoronavirus in household contacts high frequency of enterovirus d68 in children hospitalised with respiratory illness in norway low effectiveness of seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the united kingdom: 2014/15 mid-season results excess mortality among the elderly in european countries preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015 statement key: cord-332485-8tfgl8rp authors: marcorelles, p; freymuth, f; rambaud, c; gardach, c; legrand-quillien, m.c; lagarde, n title: décès brutal et infection à virus influenza a chez un enfant de deux ans : étude d’un cas autopsique date: 2002-02-15 journal: arch pediatr doi: 10.1016/s0929-693x(01)00693-5 sha: doc_id: 332485 cord_uid: 8tfgl8rp influenza a virus infections are common in childhood and infancy and are often underdiagnosed while serious or letal forms are rare. case-report. – we describe a case of sudden death in a two-year-old boy. pathologic findings at autopsy were consistent with myxovirus influenzae a virus infection and the virus was isolated by post mortem pcr. conclusion. – in the case of sudden death in infants, especially if pathologic findings are compatible with a viral infection, pcr may allow identification of the causative virus. loppement normal, sans maladie pulmonaire chronique. en janvier 2000 pendant l'épidémie de grippe, il a présenté durant plusieurs jours une rhinopharyngite avec fébricule. le troisième jour, il a eu un épisode de vomissements et de diarrhée. il était alors apyrétique. son comportement était habituel, il avait joué et ne s'était pas plaint. le lendemain, il apparaissait abattu et il est devenu rapidement obnubilé. les parents ont appelé le médecin traitant qui a constaté une hypothermie à 36,5°c et une tachycardie. il n'y avait pas de difficulté respiratoire ni de raideur méningée. son état était jugé suffisamment sérieux et inhabituel pour demander un transfert vers l'hôpital le plus proche. brusquement, vers 11 heures, l'enfant a présenté un arrêt cardiorespiratoire brutal durant le transport. la réanimation réalisée a été inefficace. l'autopsie a été réalisée 48 heures plus tard. les seules anomalies macroscopiques étaient des poumons congestifs très lourds (320 g) et une splénomégalie (60 g, sans débord). en microscopie il existait des lésions diffuses de tout l'arbre respiratoire. la plus remarquable, et qui expliquait le décès, était l'alvéolite hémorragique majeure diffuse aux deux champs pulmonaires réalisant une inondation alvéolaire (figure 1). il n'y avait pas de membrane hyaline ni d'inflammation alvéolaire. un infiltrat mononucléé peu abondant était présent dans le tissu interstitiel péribronchique. il existait un infiltrat inflammatoire majeur de tout le tractus laryngotrachéal [7, 8] : le virus grippal a été mis en évidence dans 20 % des cas même en période interépidémique. les lésions autopsiques ont été comparées à celles décrites depuis la pandémie de 1918 [7, 9, 10] : ces lésions morphologiques sont peu connues et essentiellement respiratoires. l'examen macroscopique trouve un tractus respiratoire rouge et des poumons lourds avec des pétéchies. les lésions microscopiques prédominent au niveau pulmonaire et comportent une alvéolite hémorragique dans 98 % des cas, très souvent associée à un infiltrat mononucléé interstitiel et à une congestion ; mais il existe rarement un infiltrat à polynucléaires en dehors des surinfections. des membranes hyalines, du fait du caractère lésionnel de l'oedème, responsable d'un syndrome de détresse respiratoire aiguë, peuvent s'y ajouter. au niveau du larynx et de la trachée, il existe une réaction inflammatoire à polynucléaires neutrophiles dans 93 % des cas, des érosions de la muqueuse et des suffusions hémorragiques de la paroi. d'autres lésions ont été signalées : des lésions discrètes de péricardite ou de myocardite, un oedème et un infiltrat mononucléé méningé. un large spectre d'atteinte du système nerveux central a été rapporté avec les virus influenza a [11, 12] . les signes principaux comprennent la somnolence, un syndrome parkinsonnien transitoire et des encéphalites de deux types : aiguës virales ou postvirales apparaissant à distance. le virus influenza a a un tropisme cérébral très sélectif pour certains groupes de neurones. les lésions cérébrales sont morphologiquement discrètes. dans l'observation de takahashi et al. [12] où les prélèvements ont été faits rapidement postmortem, le virus a été mis en évidence par pcr dans le cerveau, le poumon et le foie fournissant des arguments pour une propagation virale hématogène. il n'y avait pas de méningite à l'analyse du lcr. dans le diagnostic de la grippe, l'utilisation de la pcr a permis d'augmenter d'environ un tiers les résultats positifs par rapport aux techniques conventionnelles d'immunofluorescence et de culture [13] . ceci est d'autant plus vrai en période postmortem, car le taux de détection virale par ces techniques conventionnelles est très diminué. cet outil semble intéressant à utiliser pour rechercher les virus influenza dans le lcr lorsque surviennent des manifestations encéphalitiques, car la culture virale peut être négative [14, 15] . il apparaît donc important de réaliser des recherches virales dans toute mort subite en apparence inexpliquée et surtout des virus influenza quand il existe une alvéolite hémorragique diffuse. si cette atteinte pulmonaire explique le décès, son origine virale doit être mise en évidence car ceci permet de rassembler des observations qui restent exceptionnelles et d'insister sur les mesures de prévention chez le jeune enfant. formes respiratoires graves de la grippe chez l'enfant infection à virus influenza a chez l'enfant the sudden infant death syndrome and epidemic viral disease recent influenza virus a infections in forensic cases of sudden unexplained death pathological findings in cases of sudden death of incertain cause in infants (sids) and adults and influenza-a-infection the 1918 influenza virus : a killer comes into view pathologic findings in lungs of 5 cases from which influenza virus was isolated influenza virus and neurological diseases influenza virus-induced encephalopathy : clinicopathologic study of an autopsied case comparison of three non-nested rt-pcr for the detection of influenza a viruses probable post-influenza cerebellitis pcr on cerebrospinal fluid to show influenzaassociated acute encephalopathy or encephalitis key: cord-354151-psog34u3 authors: van asten, liselotte; bijkerk, paul; fanoy, ewout; van ginkel, annemarijn; suijkerbuijk, anita; van der hoek, wim; meijer, adam; vennema, harry title: early occurrence of influenza a epidemics coincided with changes in occurrence of other respiratory virus infections date: 2015-12-11 journal: influenza other respir viruses doi: 10.1111/irv.12348 sha: doc_id: 354151 cord_uid: psog34u3 background: viral interaction in which outbreaks of influenza and other common respiratory viruses might affect each other has been postulated by several short studies. regarding longer time periods, influenza epidemics occasionally occur very early in the season, as during the 2009 pandemic. whether early occurrence of influenza epidemics impacts outbreaks of other common seasonal viruses is not clear. objectives: we investigated whether early occurrence of influenza outbreaks coincides with shifts in the occurrence of other common viruses, including both respiratory and non‐respiratory viruses. methods: we investigated time trends of and the correlation between positive laboratory diagnoses of eight common viruses in the netherlands over a 10‐year time period (2003–2012): influenza viruses types a and b, respiratory syncytial virus (rsv), rhinovirus, coronavirus, norovirus, enterovirus, and rotavirus. we compared trends in viruses between early and late influenza seasons. results: between 2003 and 2012, influenza b, rsv, and coronavirus showed shifts in their occurrence when influenza a epidemics occurred earlier than usual (before week 1). although shifts were not always consistently of the same type, when influenza type a hit early, rsv outbreaks tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza virus type b tended not to occur at all. occurrence of rhinovirus, norovirus, rotavirus, and enterovirus did not change. conclusion: when influenza a epidemics occured early, timing of the epidemics of several respiratory winter viruses usually occurring close in time to influenza a was affected, while trends in rhinoviruses (occurring in autumn) and trends in enteral viruses were not. it has been suggested that annual epidemics of different viral infections can interfere with each other, but clear trends over long time periods and underlying mechanisms are not known. [1] [2] [3] [4] a few population-level studies in europe were based on observations in one respiratory season only (the 2009 h1n1 pandemic) in which the annually recurring influenza epidemic occurred relatively early. with the occurrence of several early influenza a seasons in recent years, an exploration of longer time trends of different viruses was considered useful to gain more insight in the suggested relationship between circulating viruses. understanding viral shifts and potential drivers thereof is relevant for further understanding whether certain viruses might promote or inhibit (pandemic) influenza spread and whether influenza vaccination could potentially affect trends in other respiratory viruses. in europe, influenza epidemics generally occur in winter, with the official start of the epidemic when influenza-like illness (ili) incidence in primary care sentinel surveillance exceeds an epidemic threshold (in combination with influenza a virus detection in clinical specimens collected from a subset of those ili patients). 5 in the netherlands, the influenza epidemic threshold has been calculated at an ili incidence of 5á1/10 000 for minimally two consecutive weeks which is usually not exceeded before the turn of the year, [5] [6] [7] [8] but the timing of the first exceedance (i.e., start of the epidemic) can vary between november and march (based on data from 1970 to 2006). 9 an extremely early influenza season was the 2009/2010 season when the influenza a (h1n1)pdm09 pandemic strain appeared and the ili epidemic threshold was reached by early october (week 41). such early occurrence may affect the circulation of other seasonal pathogens, and theories on possible interference between outbreaks of different respiratory viruses have been postulated to be a possible cause of delays in expected seasonal outbreaks of other respiratory viruses. 1-3 while those earlier population-level studies focused mainly on the possible interaction between influenza a virus and rhinovirus circulation, there may also be a relationship between influenza a and other prevalent viruses. therefore, we investigated trends in several common viruses for which laboratory data were available from national surveillance in the netherlands for a longer time period of up to 10 years including both respiratory and enteral viruses. we investigated trends in the reporting of common infectious respiratory and enteral viruses for which laboratory data were available for the 10the data were available from the "weekly virological records system" of the dutch working group on clinical virology giving the number of positive laboratory diagnoses by year and week, but not providing data on the denominator nor on age or gender of the patient. submitting laboratories are associated with either hospitals or regional laboratories to which both gps and hospitals submit samples. the estimated proportion of all positive diagnostics captured by this national surveillance varies between the monitored pathogens and was estimated between 38% (for rotavirus) and 73% (for influenza virus) in a 2002 study of five pathogens. 10 the types of tests used can differ between the submitting laboratories and over time. respiratory viruses were detected in throat swabs mainly by pcr-based methods in respiratory specimens from patients with respiratory disease symptoms. enteric viruses were detected in fecal samples by eia-based methods (rotavirus, norovirus) and by pcr-based methods (norovirus). enterovirus was detected mainly by pcr and in the early years also by culturing, in throat swabs, cnf, and in fecal specimens collected from patients suspected for an enterovirus infection. cross-reaction between rhinovirus and enterovirus pcrs for throat swab samples cannot be excluded completely. when collected from patients suspected for enterovirus infection, typing of enteroviruses indicated that this was a minority. when collected from patients with respiratory symptoms, enteroviruses might be misidentified as rhinovirus, as occurred during enterovirus d68 outbreaks. 11 to determine whether an influenza a season occurred relatively early, we used both the influenza sentinel surveillance data and the laboratory data from the weekly virological records system. as general practitioner (gp) influenza sentinel surveillance is the current gold standard for influenza surveillance in the netherlands (with an epidemic threshold), early influenza a seasons were first identified using published dates of the influenza epidemics. we combined this information with visual inspection (as there is no threshold available) of trends in influenza a laboratory diagnoses reported in the weekly virological records system for confirmation of the early ili epidemics and for identification of any additional early influenza a seasons according to those laboratory data. a pre-defined cutoff point for the identification of early influenza seasons is not available. using the published ili epidemic periods, we identified two relatively early influenza a seasons wherein influenza epidemics started before the turn of the year (i.e., before week 1): the 2003/2004 season (epidemic starting in week 49 lasting to week 4) and the 2009/2010 season (epidemic starting in week 41 lasting to week 51). 12, 13 these two early epidemics were also confirmed by visible early increases in influenza laboratory data reported in the weekly virological records system ( figure 1 ). after visual inspection of the laboratory data, the 2010/2011 season was additionally selected as an early season as influenza a diagnoses were clearly on the rise before week 1 ( figure 1 ). while the influenza epidemic in the 2008/2009 season did not start early (week 1) 8 according to the ili sentinel surveillance system, in the laboratory surveillance data this season seemed neither clearly early nor clearly late and was therefore excluded (influenza a laboratory diagnoses in this season are represented in appendix 1). as most of the viruses under study peak in winter, we defined year-long seasons running from july to june rather than using calendar years (january to december). for comparability between the seasons, we presented the number of detected viruses per week as a percentage of the total number of detections during the study period per each respective virus. due to the intensified testing that occurred during the a (h1n1)pdm09 pandemic, the total number of influenza a virus laboratory diagnoses in the pandemic season was 13á1 times higher than the average number of influenza a virus laboratory diagnoses in the 2003-2012 seasons, excluding the pandemic season. to facilitate the inclusion of such a high peak into the presented graphs, the number of influenza a virus laboratory diagnoses during the 2009/ 2010 season was scaled down (reduced by a factor 13á1, changing the height but not the shape of the epidemic in that year). as the number of influenza b virus laboratory diagnoses was very low during the pandemic, such downscaling was not performed for the influenza b virus laboratory diagnoses. smoothing of time series was performed by calculating 3-week moving averages (average of current, previous, and next week's value). correlation coefficients between different viruses were calculated using spearman's rank correlations for non-normally distributed data. we also shifted the time series forwards and backwards in time (à10 to +10 weeks) and compared whether the time shift with maximal correlation differed between early and late influenza seasons. the highest numbers of laboratory reports were available for rsv and the lowest for influenza b (20 282 and 1309 reported diagnoses during the total study period, table 1 ). time series of all considered pathogens are shown in figure 1 . almost all of the included respiratory viruses (influenza a and b virus, rsv, coronavirus) except rhinovirus showed very clear seasonality in their reporting over time. the numbers increase, peak, and are elevated during winter season (december-february) or occasionally in very early spring (march-april) ( figure 1 and appendix 2a-d). however, while influenza b virus diagnoses also displayed such winter seasonality, outbreaks did not occur each winter (five of the ten observed years showed clear influenza b epidemics, while the other 5 years showed very low levels or even near-absence). in our data, rhinovirus levels showed less clear seasonality than the other respiratory viruses. they tended to be elevated over broad periods ( figure 1 ) and seemed to be present year-round with less pronounced elevations during autumn/winter/spring. generally, rsv van asten et al. epidemics preceded influenza a virus epidemics, which in turn preceded influenza b epidemics (in the years that influenza b epidemics occurred). increases in coronavirus also usually preceded increases in influenza a (but not in the 2009/2010 season). the timing of rhinovirus levels relative to influenza a virus in laboratory diagnoses was less clear (due to seemingly year-round presence of rhinoviruses in specimens submitted for laboratory diagnoses). the two gastrointestinal viruses in the study (norovirus and rotavirus) both also presented with winter seasonality, often with norovirus preceding rotavirus epidemics ( figure 1 and appendix 2c). norovirus and influenza a virus epidemics usually overlapped, but the norovirus epidemics had broader peaks, and numbers tended to start increasing earlier than influenza a virus diagnoses levels and decreased after the disappearance of influenza a virus. also, rotavirus outbreaks usually occurred before influenza a outbreaks started. enteroviruses, of which there are many types, can cause respiratory illness, but they also cause other illness. 14 enterovirus data were sparse until 2005, but in the 2006-2012 time period, enterovirus levels generally peak in summer (june-august). visually, we assessed whether seasons with early influenza a virus epidemics (as reported in the weekly virological records system) coincided with shifts in the reporting of other common pathogens or shifts in temperature and humidity trends. occurrence of seasons with relatively early influenza a virus circulation compared to seasons with later circulation of influenza a virus is shown in figure 2 (early seasons in color, late seasons in gray). for the other viruses, the same grouping was made (based on early and late influenza seasons), with their trend during early influenza a seasons also given in color ( the correlation coefficients between influenza and other viruses were stratified by timing of the occurrence of the influenza epidemic (early versus late, table 3 ). as the different viruses might differ in the timing of their occurrence relative to influenza a occurrence, the correlation coefficients were calculated at different time lags (shifted between 10 weeks earlier up to 10 weeks later) to determine the time shift with the maximum correlation with influenza a. when comparing seasons with early versus later occurrence of influenza epidemics, the maximum correlation coefficient was observed at different lags for all viruses. this difference in time lag relative to influenza a occurrence was smallest for influenza b, indicating that influenza b circulation relative to influenza a tends to occur at a more stable delay than for the other viruses. this suggests that changes in timing of influenza a circulation will be accompanied by changes in timing of influenza b circulation. however, such an overall correlation coefficient (calculated over multiple seasons combined) obscured the visual observation that in van asten et al. two of the three early influenza seasons, influenza b virus was almost absent (figure 2 ). viruses that showed a shifted trend of reporting during years with early influenza a epidemics were of respiratory nature with clear winter seasonality and with epidemics occurring relatively close in time to influenza a virus epidemics. although per individual virus the shifts were not consistently of the same type or direction, generally said, in years when influenza a hit early, rsv tended to be delayed, coronavirus outbreaks tended to be intensified, and influenza b virus tended not to circulate at all. viruses that act on other organ systems (enteral) seemed not to be affected by the timing of influenza a outbreaks. in our data, rhinovirus showed little seasonality, but rather year-round presence, and therefore, no association with timing of influenza a could be observed. changes in seasonal patterns of respiratory pathogen laboratory reports have been observed in other, shorter, studies. in hong kong, after the early occurring a(h1n1) pdm09 pandemic, increases were observed in adenovirus and parainfluenza virus detections, while the usual rsv summer peak disappeared in data from hospitals and clinics. 15 rhinovirus and coronavirus were not included. in another study in beijing, all respiratory virus epidemics, except rhinovirus, were delayed after the pandemic influenza epidemic (data came from fever clinics which screen patients prior to being assigned to a specific hospital department). 16 however, both regions have different climates from the netherlands (and from each other) complicating comparisons. other european observations have also been reported regarding the effect of rhinovirus on influenza virus circulation. while those reports hypothesize on viral interference between influenza virus and rhinovirus circulation (with rhinovirus delaying influenza spread), 1-3,17 we (like the beijing study 16 ) did not observe clear-cut trends in rhinovirus reports in our laboratory diagnoses data. the relationship previously reported between rhinovirus and influenza virus might perhaps be age specific because in one study, the reduced likelihood of a(h1n1)pdm09 detection in rhinovirus-positive samples 3 was observed in a pediatric population, as was the reduced likelihood of detecting eight viruses in rhinovirus-positive samples in another study in children, 17 while our data were not age specific and we did not have such information on concurrent infections. besides the reported possible interaction between rhinovirus and influenza a virus, much less is known about interaction between and with other (respiratory and nonrespiratory) viruses. our data suggest that respiratory viruses may impact each other's seasonality (in this study focused on the relationship between influenza a virus and other respiratory viruses), albeit through unknown mechanisms, as also hinted at by the beijing and hong kong data. 15, 16 virtually all research suggesting interaction between respiratory viruses in human populations has focused on observational (ecological) studies such as ours, most including only one or a few seasons of data while our study included a 10-year period. only one prospective cohort study has been published (based on one season) which showed rhinovirus and coronavirus to interrupt the a(h1n1)pdm09 pandemic. 18 our results could not confirm their finding of coronavirus inhibiting influenza a virus circulation, but we observed more coronavirus laboratory reports when influenza a virus circulated early. in contrast to most of the other studies, we could investigate whether patterns recurred due to our longer study period. this revealed that coronavirus laboratory reporting was more intense in 2009/2010 when it did not overlap with influenza a virus circulation. however, it was also more intense in the early influenza a year thereafter (2010/2011) when it actually completely overlapped with the influenza a epidemic hinting that direct biological inhibition of either virus by the other might be unlikely and thus apparent interaction reported by others may have depended on indirect factors or may be spurious. these variations by year in our data illustrate how results from shorter observational studies have to be interpreted with caution, especially when they focus only on 1 year or one season. however, also in our longer study we have to remain cautious; to refute biological inhibition between coronavirus and influenza, it would be helpful to know whether the coronavirus and influenza virus specimens were from the same age group. unfortunately, as in other observational studies, surveillance artifacts could not be ruled out for any of the viruses in our study. these artifacts concern increases and decreases in laboratory testing over time that may not necessarily be related to changes in virus circulation in the population. like previous studies, we did not have information on issues potentially underlying such changes, such as shifts in laboratory testing policy, availability and use of new diagnostic tools, variations in testing intensity due to previous or new outbreaks, and possible outsourcing of certain tests. furthermore, information on age was not available in our data, possibly diluting trends that might have been visible were data investigated age specifically, or alternatively creating trends and shifts that might otherwise have been absent in certain age groups. if certain age groups are more likely to be hospitalized (such as infants with rsv infection) and/or to be sampled than other age groups and thus overrepresented in the laboratory data, our observed trends might not be representative for the total population if virus circulation trends actually differ between age groups. future ecological analyses should preferably include age-specific data as this may further clarify the true occurrence of shifted seasonality. comparison between the different studies (including ours) is also made difficult by unknowns other than age, such as illness severity and influenza vaccination status. as other ecological-type studies have suggested, also our observed shifts in the occurrence of influenza b virus, rsv, and coronavirus during early influenza a seasons suggest that viral interaction might play a role in the occurrence of virus epidemics. [1] [2] [3] 17 with our data, the precise direction of potential interactions is not clear, that is, which virus(es) impacts which. there are several types of virus-virus interactions, described in a review by da palma, 19 ranging from (i) direct virus-virus interactions (e.g., when nucleic acids or proteins of one virus physically interact with the genes or gene products of a coinfecting virus); (ii) indirect interactions resulting from alterations in the host environment; to (iii) immunological interactions. however, with the current data we cannot determine whether the viruses impacted each other biologically. further, there are other issues that can impact the timing and magnitude of seasonal epidemics such as environmental, social, and behavioral phenomena that may also be the potential drivers of observed shifts in reporting patterns instead of viral inter-action (alone). 20 to further attempt to disentangle these issues, future ecological studies should preferably include age-specific virological data over multiple seasons and better yet, cohort studies could be performed that (i) address the order of infection by different viruses (by serial sampling of subjects during the study period regardless of symptoms) as proposed by others; 17, 21 (ii) include persons from the same household as was done by pascalis et al.; 18 and (iii) include multiple years and thus multiple seasons per virus. there are several examples highlighting the importance of understanding drivers of viral shifts. as suggested by greer et al., further studies are relevant in light of the discussion whether rhinovirus promotes (pandemic) influenza spread through coughing and sneezing 22 or whether it may paradoxically provide natural protection due to inhibition of influenza infection in those already infected with rhinovirus. 17 further studies may also provide input to the question whether influenza vaccination can render persons more susceptible to other respiratory viruses due to the lack of temporary nonspecific immunity induced by actual influenza infection. 23 canadian and australian researchers hypothesize that through this same mechanism of induced temporary non-specific table 3 . correlation coefficients between number of weekly laboratory diagnoses of influenza a and weekly counts of other pathogens at different time lags (10 weeks later to 10 weeks earlier)* *bolded numbers indicate the largest correlation coefficient (with a negative correlation for enterovirus due to opposite seasonality with influenza a virus). immunity, the circulation of a seasonal influenza strain preceding a pandemic strain might decrease the susceptibility to that ensuing pandemic strain. 24 not evaluated in our study but possibly also playing a role in virus shifts or virus-virus interactions is the variation in the circulation of influenza a subtypes from year to year. evaluation of such an effect might require evaluation of longer time series as the first general impression from our data was that there was no clear consistent association with subtype as the three early influenza years were not all dominated by the same influenza a type ( studying the association of climatic factors (an example of environmental phenomena potentially affecting virus circulation) with influenza [26] [27] [28] was beyond the scope of this study, although covering the timespan of the study, a visual assessment of temperature and humidity trends showed no clear visual association between early influenza occurrence and early occurrence of low temperatures or low humidity. although two of the three early ili seasons (2009/2010, 2010/ 2011) seem to have longer and colder cold spells (average weekly temperatures below 0°c) that also occur earlier in the winter season than in the other years, the first mentioned season (the 2009/2010 pandemic year) actually showed the cold spell occurring after the influenza season instead of at the beginning or during the influenza season (appendix 2e). unlike earlier studies, we included several non-respiratory pathogens, for which the results showed no shifts in trends, and therefore, interaction with influenza a virus seems unlikely. as norovirus, rotavirus, and enterovirus affect other organ systems, direct interaction may not be expected. however, sporadic examples do not rule out such unexpected interactions as others have suggested that live polio vaccine (against an enteral virus) might prevent otitis media 29 (a respiratory infection), and decrease infantile diarrhea mortality 30 (gastro-intestinal mortality). another issue is that, like others, we investigated shifts in virus circulation within the same seasons. whether early occurrence of influenza a may affect the circulation of seasonal pathogens in seasons thereafter is not known. in conclusion, when influenza hit early we observed shifts in patterns of several respiratory pathogens that occur close in time with influenza a. further research is needed to understand whether this was caused by (in)direct biological interaction between viruses or other underlying mechanisms such as human behavior and environmental factors or whether these observed shifts were just random occurrences. understanding these phenomena is of value in understand-ing or predicting the timing and magnitude of viral epidemics, providing knowledge which can be used for early warning and the allocation of healthcare resources. does viral interference affect spread of influenza? interference between outbreaks of respiratory viruses rhinoviruses delayed the circulation of the pandemic influenza a (h1n1) 2009 virus in france impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method 08 in the netherlands: antigenic variation, oseltamivir resistance and vaccine composition for the continue morbiditeits registratie peilstations nederland de beste tijd voor griepvaccinatie multi-centre study in the netherlands examining laboratory ability to detect enterovirus 68, an emerging respiratory pathogen de mexicaanse grieppandemie van 2009: een overzicht met focus op nederland the enteroviruses: problems in need of treatments the impact of pandemic influenza a (h1n1) 2009 on the circulation of respiratory viruses 22 ª 2015 the authors. influenza and other respiratory viruses published by influenza a/h1n1 2009 pandemic and respiratory virus infections do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? intense co-circulation of noninfluenza respiratory viruses during the first wave of pandemic influenza ph1n1/2009: a cohort study in reunion island a systematic approach to virus-virus interactions seasonality of viral infections: mechanisms and unknowns respiratory viruses in hospitalized children with influenza-like illness during the h1n1 2009 pandemic in sweden coinfection can trigger multiple pandemic waves increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine seasonal influenza vaccination and the risk of infection with pandemic influenza: a possible illustration of non-specific temporary immunity following infection recent developments with regard to influenza; flu-watchers in action] actuele ontwikkelingen betreffende influenza; griepspotters in actie driving factors of influenza transmission in the netherlands environmental role in influenza virus outbreaks spatiotemporal characteristics of pandemic influenza viral interference induced by live attenuated virus vaccine (opv) can prevent otitis media effect of the administration of oral poliovirus vaccine on infantile diarrhoea mortality we would like to thank the contributing laboratories and the dutch working group for clinical virology (nwkv) for virological surveillance. key: cord-334424-z7ygy25e authors: mccaw, james m; howard, peter f; richmond, peter c; nissen, michael; sloots, theo; lambert, stephen b; lai, michael; greenberg, michael; nolan, terry; mcvernon, jodie title: household transmission of respiratory viruses – assessment of viral, individual and household characteristics in a population study of healthy australian adults date: 2012-12-11 journal: bmc infect dis doi: 10.1186/1471-2334-12-345 sha: doc_id: 334424 cord_uid: z7ygy25e background: household transmission of influenza-like illness (ili) may vary with viral and demographic characteristics. we examined the effect of these factors in a population-based sample of adults with ili. methods: we conducted a prospective cohort study in community-dwelling australian adults nested within an influenza vaccine effectiveness trial. on presentation with ili, participants were swabbed for a range of respiratory viruses and asked to return a questionnaire collecting details of household members with or without similar symptoms. we used logistic and poisson regression to assess the key characteristics of household transmission. results: 258 participants from multi-occupancy households experienced 279 ili episodes and returned a questionnaire. of these, 183 were the primary case in the household allowing assessment of factors associated with transmission. transmission was significantly associated in univariate analyses with female sex (27% vs. 13%, risk ratio (rr) = 2.13 (1.08, 4.21)) and the presence of a child in the house (33% vs. 17%, rr = 1.90 (1.11, 3.26)). the secondary household attack proportion (shap) was 0.14, higher if influenza was isolated (rr = 2.1 (1.0, 4.5)). vaccinated participants who nonetheless became infected with influenza had a higher shap (incidence rr = 5.24 (2.17, 12.6)). conclusions: the increased shap in households of vaccinated participants who nonetheless had confirmed influenza infection supports the hypothesis that in years of vaccine mismatch, not only is influenza vaccine less protective for the vaccine recipient, but that the population’s immunity is also lower. improved characterisation of the determinants of household transmission of influenza-like illness (ili) remains an important public health priority, particularly in light of the past decade's events in which we have witnessed the emergence of severe-acute-respiratory-syndrome (sars) and the 2009 h1n1 influenza pandemic. the evidence base for pandemic influenza public health interventions such as home-quarantine, provision of antiviral agents for post-exposure prophylaxis, school-closure and vaccination builds upon an appropriate understanding of the patterns and timing of infection within the household unit [1] [2] [3] [4] [5] [6] . while influenza viruses, rhinoviruses (hrvs), adenoviruses, respiratory syncytial virus (rsv) and parainfluenza viruses (pivs) are the most common aetiological agents in acute-respiratory-infection (ari) episodes [7, 8] , in 30 -40% of all ari episodes no known respiratory virus can be identified [9, 10] . this is despite discovery of a number of previously undescribed viruses since 2001 from clinical specimens from the human respiratory tract (human metapneumovirus [11] , sars coronavirus [12] , coronavirus nl63 [13] , coronavirus hku1 [14] , novel rhinoviruses [8] , human bocaviruses [15] and k1 and wu polyomaviruses [16, 17] ). reflecting the need to improve our understanding of household transmission of ari, the literature examining factors associated with household transmission of influenza [6, [18] [19] [20] [21] has expanded significantly since the 2009 h1n1 influenza pandemic [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] , including a systematic review and meta-analysis [32] . donnelly et al. estimated the serial interval for all ili (without laboratory confirmation) from case reports during the 2009 pandemic [25] . only two studies of which we are aware explicitly consider the impact of virus type on infectiousness. principi et al. found less onwards transmission to household members from influenza-negative than influenza-positive children presenting to a hospital emergency department [33] . similarly, in a cohort study of ari in young children, lambert et. al. observed significant heterogeneity in the proportion of participants' households in which one or more illness events were observed (ranging from 13% for isolation of hmpv from the child to 61% for isolation of influenza) [34] . here we report on the household transmission of a range of viruses in a cohort study of healthy, community-dwelling adults reporting symptoms of influenza-like illness (ili). the study population was sourced from a large, industry sponsored placebocontrolled phase iv efficacy trial of a licensed seasonal trivalent influenza vaccine (fluvax w , csl ltd), conducted prior to the 2009 pandemic between march and november 2008. in a previous article [35] we have described the viral aetiology of ili in the cohort, and examined the influence of virus type, host and spatiotemporal factors on disease symptomatology. full details of subject recruitment and selection for the primary phase iv vaccine efficacy trial have been described previously [35] . briefly, across 23 study sites in australia and new zealand, 7544 healthy adults aged ≥18 to <65 years were recruited for a placebocontrolled trial of a licensed trivalent influenza vaccine (fluvax w , csl ltd) in 2008 (clinicaltrials.gov #nct00562484). study participants were randomized to receive either placebo or vaccine in a 1:2 ratio prior to the southern hemisphere 2008 influenza season. from an available pool of 5624 participants from the primary study at 12 study sites, we consider the 581 persons (adults) who experienced at least one ili episodemeeting the case definition of at least one respiratory symptom (cough, sore throat, runny nose or nasal congestion) and at least one systemic symptom (fever (oral temperature ≥ 37.8°c), feverishness, chills or myalgia) [35] , and who provided written informed consent for participation in the nested cohort-study which required contribution of a valid biological sample (copan tm dry flocked swab). samples were tested for a range of respiratory viruses using a combination of multiplexed and uniplexed conventional and real-time polymerase chain reaction (pcr) assays [35] . of those, 258 were members of multi-occupancy households, allowing investigation of transmission within the household. using a non-specific and sensitive ili definition, they reported an episode of ili on 279 occasions. for each episode they returned a study questionnaire (additional file 1) detailing respiratory symptoms (see [35] for details), health seeking behaviour (health care provider consultations, hospital admission, time off work), household characteristics (number of adults (≥18 years) and children (<18 years)) and temporally associated symptoms of ili (if any) in other household members. from herein, we consider the illness episode as the primary unit of analysis. the virology results were classified into 5 virus groups [35] : influenza (influenza a, influenza b), coronaviruses (oc43, 229e, nl63, hku1), picornaviruses, other viruses (parainfluenza viruses (1, 2, 3), adenoviruses, human metapneumovirus (hmpv), bocaviruses, rsv and ki and wu polyomaviruses) or none, where none indicates that no 'tested-for' virus was detected in the participant's sample, as opposed to a missing sample or inconclusive result. study participants' vaccination status (as determined by the primary phase iv trial intervention), physical location (i.e. study site), and socio-demographic characteristics were also recorded. the relevant outcome measure for this sub-analysis was evidence of transmission within households based on experience of symptomatic illness in at least one other member of the study participant's household. study participants were asked to complete the diary on the day following cessation of their own symptoms. they recorded the date of onset of symptomatic ilis in household members from between 14 days prior to the study participant's illness through to the day of diary completion. note that the primary case in the household may or may not be the study participant. for household ili events in which the participant was the primary case (183 of 279), transmission may or may not have occurred in the household and so it is statistically valid to develop univariate and multivariate explanatory models. for participants who reported recurrent ili episodes during the study in which the same virus was isolated, we exclude all but the first episode. events with co-introduction, defined as onset of symptoms in the participant and one or more household members on the same day, were also excluded. following these exclusions, 177 episodes remained for analysis. for household ili events in which the participant was not the primary case (95 of 279), while we do know who the introducer was for these events (via the questionnaire data), other household ili events initiated by that introducer that did not involve the participant are unobserved. that is, any events in which a child (or for that matter, any other adult member of the household) introduced an infection that did not infect the study participant are not captured by the study protocol. this observation necessitates the exclusion of all household ili events in which the participant was not the primary case from the analyses. in the one remaining household ili event, the status of the participant (primary or not) was unknown, so the episode was excluded from the analysis. for the 177 episodes in which the participant was the primary case, logistic regression models were used to explore associations between host, demographic or virus variables with any observation of within-household transmission (outcome variable = transmission in household for each recorded ili episode in a study participant). the secondary household attack proportion (shap) was calculated as the proportion of potentially exposed household members (assumed susceptible) experiencing illness, averaged over all recorded episodes. we present descriptive statistics for the shap and its variation by virus, participant and demographic variables. poisson regression models were used to assess the influence of virus, participant and demographic variables on the number of secondary cases within a given household, offset against the number of potentially exposed household members (outcome variable = number of secondary cases in household for each recorded ili episode in a study participant). vaccination status of participants was not included in the primary logistic and poisson statistical analyses due to its known mitigating effect on the likelihood of influenza acquisition [35] . investigation of the influence of prior immunisation on influenza transmission in 'breakthrough cases' was explored in a secondary analysis by inclusion of a statistical interaction term between vaccination and influenza-identification status. we make an empirical calculation of the mean time between the onset of symptoms in the primary case and the onset of symptoms in the household contacts (the serial interval), for all household ili events, events in which the participant was the primary case, and events in which influenza was isolated from the participant's virological sample. all statistical analyses were conducted in stata/ic 11.1. figure 1 reports characteristics of the 258 multioccupancy households in which transmission did and did not occur. 28 study participants reported two or more ili episodes during the course of the study. for two participants, who both experienced two episodes, picornavirus was isolated on both occasions. we only retain the first episode for each participant. the distribution of household size is dramatically different based on the absence or presence of children within the household ( figure 2 ). in households without children, the distribution is left-skewed (mean household size = 3.02, standard deviation (sd) = 1.18, skewness = 1.45), while in households with children there is minimal skew (mean household size = 4.18, sd = 1.11, skewness = 0.103). table 1 summarizes the descriptive statistics (and logistic model results) associated with presence or absence of transmission in the household for the 177 household ili events in which the participant was the primary case. there is marked co-linearity between the variables 'presence of child in household' , 'age-category' and 'household size'. for example, respondents aged 35 -44 years had significantly greater odds of having a child in the household than those aged 18 -24 years (or 53.2 (13.0, 217)), while no participant aged more than 55 years lived with a child. the relationship between the household size distribution and presence or absence of children is depicted in figure 2 . we retained 'presence of child in household' in the final multivariate model for transmission due to its strong predictive role, intuitive appeal, presumed causal role in our observed (univariate) association with age-category, and previous research indicating an association between transmission and children [18, 22, 23, 29, 36] . in the multivariate model, the observed increased risk of transmission with female sex remains (or = 2.45 (1.01, 5.93), p = 0.047). presence of children in the household is both the strongest and most statistically significant factor associated with transmission (or = 2.63 (1.18, 5.88), p = 0.018). within 258 multi-occupancy households, 177 primaryparticipant introductions gave rise to 54 secondary cases among 391 potentially exposed individuals, a secondary household attack rate (shap) of 0.138. of 102 exposed children, 22 in households in which the participant was female, 41 secondary infections were reported among 238 exposed household members (shap = 0.172), compared with 13 secondary cases among 153 contacts in households in which the participant was male (shap = 0.085), a riskratio of 2.03 (1.12, 3.66), p = 0.016 (2-sided fisher's exact). in households with children, 27 secondary infections were reported among 162 exposed household members (shap = 0.167), compared with 27 secondary cases among 229 contacts in households without children (shap = 0.118), a risk-ratio of 1.41 (0.863, 2.32), p = 0.182. a multivariate poisson regression model ( table 2 ) was used to consider the influence of virus group and demographic characteristics on the number of reported secondary cases within a given household, offset against the number of potentially exposed household members. in correspondence with the logistic regression model for transmission, we include presence of children in the in a secondary analysis, we considered the influence of prior vaccination on the reported number of secondary household cases among participants testing positive for influenza compared with all other participants. in a poisson model for secondary attacks including a statistical interaction between influenza detection (true/false) and vaccination status (placebo/vaccine), the irr for influenza positive cases in those receiving placebo was 1.69 (0.421, 6.80), p = 0.459. the factor increase (interaction term) for the irr for vaccinated participants was 3.10 (0.608, 15.8), p = 0.174, yielding a net irr for vaccinated influenza-positive participants relative to vaccinated influenza-negative participants of 5.24 (2.17, 12.6), p < 0.001). under the simplifying assumption that the introducer of infection into the household is responsible for all subsequent infections, we may calculate an empiric serial interval, the time from symptom onset in one individual until symptom onset in another. we first consider infections to be related if symptoms are reported within 14 days following onset in the primary case. across all virus-type isolations, we calculate a mean serial interval of 6.0 days (sd = 3.6) for all household ili events (where the study participant was the primary case or otherwise), and 5.1 days (sd = 3.2) for the events in which the participant was the primary case. for the five events in which the primary participant had virologically confirmed influenza and transmission occurred, the mean serial interval was 4.5 days (sd = 1.6). if we limited the maximum serial interval to seven days, the mean was reduced to 4.0 days (sd = 1.7) for all household ili events and 3.9 days (sd = 1.9) for events in which the in our main analysis, we made two assumptions that we now subject to a sensitivity analysis. of the 183 events in which our participant was the primary case, 6 were classified as co-introductions as (at least) one other household member recorded symptoms beginning on the same day. as the latent period for respiratory infections may vary from individual to individual, here we exclude a further 4 episodes in which there was a 1 day interval from onset of symptoms in the study participant to onset of symptoms in another household member. the resulting multivariate models (equivalent to tables 1 and 2 ) are materially unchanged, with the expected slight reduction in statistical power (data not shown). a second assumption made was that, for participants who reported multiple ili episodes during the study period, we only excluded the latter ili episode where the same respiratory pathogen was isolated on both occasions. however, if we conservatively exclude all ili episodes except for the first (10 episodes excluded (by virus type: 6 "none", 1 "picornavirus", 2 "influenza", 1 "coronavirus")), again we find no material change in either the logistic or poisson analyses (data not shown). this study, notable in its consideration of a broad range of respiratory pathogens in addition to influenza, demonstrates that household transmission of ili is most strongly associated with host and demographic factors: female sex and the presence of children within the household (tables 1 and 2) . the observation that female sex may be associated with increased transmission in the absence of children (rr = 2.33 (0.919, 5.90), p = 0.059) is novel, perhaps suggesting that females are fundamentally more infectious, and not simply more connected to children (in terms of both their susceptibility compared with males if a child introduces infection, and their infectiousness to children if they are the primary household case). behavioural differences whilst ill may drive such an observation. alternatively, mechanisms by which influenza pathogenesis is sex dependent have been investigated [37] ; whether or not differences extend to infectiousness and susceptibility is not clear. barbara et al. have recently identified that the reporting of respiratory symptoms may be linked with risk perception [38] and hence gender [39] . clearly, we cannot exclude the possibility of gender difference in the reporting of within household transmission. the association between transmission and the presence of children within the household is consistent with many other studies [18, 22, 23, 29, 36] . the logistic and poisson model findings (tables 1 and 2 ) are consistent with an increased susceptibility for children. this is further supported by the observed increased shap in children compared to adults (0.216 compared to 0.110, a risk-ratio of 1. 95 (1.19, 3.19 ). the shap in adults did not differ by whether or not their household contained children, suggesting that other 'indirect' effects of children are less likely. as our study design limited the analysis to household events with an adult introducer, we were unable to assess the hypothesis that children may be more infectious than adults. our poisson regression analysis on the number of secondary cases given that the participant was the primary case ( table 2) indicates that isolation of influenza in the introducer of infection to the household is associated with an increase in the number of secondary cases. we explored this finding more deeply using a statistical interaction model. while somewhat limited by sample size, we found that in placebo recipients identification of influenza was not significantly associated with an increase in the number of secondary cases (irr = 1.69 (0.421, 6.80), p = 0.459), while in vaccine recipients the irr (relative to identification of any other virus, including 'none') was 5.24 (2.17, 12.6) , p < 0.001. note that our previous analysis confirms that vaccination is associated with a reduced probability of influenza virus identification [35] . additionally, 'breakthrough' influenza cases have similar symptoms compared to unvaccinated individuals [35] . we therefore suggest that our finding of increased transmission may be explained by infection with an influenza virus mismatched to the vaccine-strain (known to be in circulation during the year of study [40] ), which furthermore may be relatively antigenically novel and to which household members may be expected to have heightened susceptibility. with no virological samples available from other household members and the small number of vaccinated participants who were infected with influenza we are unable to explore this hypothesis further. across all virus types isolated and all household ili events, and assuming that all secondary cases within the household are directly infected by the introducer, we calculate a serial interval of 6.0 days. restricting to events in which the participant was the primary case and in which influenza was isolated, we calculate a serial interval of 4.5 days. this simple approach, as taken by others [22, 29] , cannot account for two important factors: community importation and infection of household members by other non-introducing members (i.e. tertiary cases). while others have partially accounted for these effects [25, 29, 41] , a mechanistically-motivated statistical model is required to fully account for such possibilities, for example as introduced by cauchemez et al. [23] who determined a serial interval for influenza of 2.6 days (sd = 1.3) compared to 2.9 days if calculated directly from empirical observations. with just 5 events, application of these more advanced model-based techniques is not justified for our data. of primary interest for this sub-analysis focussed on transmission is the complication introduced by the monitoring and assessment of ili in an individual rather than a household. ideally, a protocol such as that suggested by klick et al. would have been employed [42] . the lack of virological assessment of household secondary cases and the broad nature of the question used to establish the secondary case count in each house also contributes to uncertainty with regards to our assignment of temporally associated ili to within-household transmission. both of these limitations were an unavoidable consequence of the nesting of the data-collection protocol within a randomized placebo-controlled trial. furthermore, due to the requested timing for completion of the questionnaire, we cannot exclude the possibility that late onset of secondary (or tertiary etc.) cases may have been missed, particularly if a participant's experience of symptoms was of short duration. similarly, because the study protocol and analyses effectively assume that individuals are infectious until the end of their symptoms, any systematic differences (by virus type) in this relationship may influence the results. however the prompt to return the diary upon symptom cessation was in an effort to ensure timely reporting of questionnaire information to minimise recall bias. conversely, our poisson model implicitly assumes independence among household members, attributing all household infections to the primary case. more advanced model based methods that account for tertiary (and subsequent) cases and community introduction would be warranted with more complete data sources. as with all protocols based purely on symptomatic presentation (as opposed to active surveillance for nonclinical signs of infection such as virological or immunological measures [24, 42] ), we are unable to account for potential sub-clinical infection routes, with potential impact for our assessment of whether or not transmission did occur, the primary case status of our participants and determination of the size of the susceptible pool within a given household. conversely, taking a nonsimulation approach to analysis, we are unable to discount our estimate for the shap due to the effects of community introduction into the household, or account for community introduction and tertiary cases in our estimate for the serial interval [23] . our study sample had an over-representation of females (166 of 258 (64.3%) individuals for the 279 captured episodes; 105 of 167 (62.9%) individuals for the 177 primary-participant introductions). furthermore, it should be noted that the study population were originally volunteers in a randomized controlled trial and as such more likely to represent a group who were more concerned with their health than the general population. eligibility was restricted to healthy adults without recognized risk factors for severe influenza infection. in the context of a literature focussed on the transmission characteristics of laboratory confirmed influenza, our study is the only one that we know of to systematically explore the relationship between transmission and virus aetiology. the analyses suggest that influenza is more transmissible than other causative agents of ili, at least when introduced to the household by an adult. host and demographic factors are also of importance. further studies combining active surveillance of all household members with specimen collection and testing for a range of respiratory pathogens are warranted to elucidate these relationships. additional file 1: illness visit questionnaire. competing interests pcr has previously served on a scientific advisory board regarding influenza vaccines for csl ltd and has received a grant for an investigator initiated epidemiological study of otitis media from glaxosmithkline australia. he has also received travel support for himself and staff employed by the vaccine trial group to attend and present data at scientific meetings from baxter, glaxosmithkline, sanofi and pfizer. mdn has received travel grants from wyeth australia to present independent research at international meetings, and currently and previously has been the principal investigator for clinical trials sponsored by abbott, baxter, csl, gsk, medimmune, merck, novartis, sanofi-pasteur, wyeth, and pfizer. ml is an employee of csl limited and has an equity interest in the company. authors' contributions jmc, ph and jmv conducted the statistical analyses, provided the primary interpretation of the results and wrote the manuscript. pr was principal investigator on the vaccine efficacy trial within which the sub-study was conducted. tn, jmv, ts, mn, sl and pr conceived the sub-study and secured funding for its conduct, in partnership with csl limited represented by ml and mg. jmv coordinated conduct of the study at multiple sites and oversaw collation of the questionnaire data. ts, mn and sl oversaw conduct of and reporting of the virological testing at the queensland paediatric infectious diseases laboratory. ml was medical monitor for the main vaccine study and a partner investigator on the sub-study, as was mg. all authors contributed to critical revision of the manuscript and have seen and approved the final version of the manuscript. reducing the impact of the next influenza pandemic using household-based public health interventions the transmissibility and control of pandemic influenza a (h1n1) virus effective, robust design of community mitigation for pandemic influenza: a systematic examination of proposed us guidance estimating antiviral effectiveness against pandemic influenza using household data modeling targeted layered containment of an 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household transmission household transmission of pandemic (h1n1) assessing secondary attack rates among household contacts at the beginning of the influenza a (h1n1) pandemic in ontario, canada shedding and transmission of novel influenza virus a/h1n1 infection in households-germany transmissibility of seasonal and pandemic influenza in a cohort of households in hong kong household transmission of 2009 pandemic influenza a(h1n1): a systematic review and meta-analysis burden of influenza in healthy children and their households community epidemiology of human metapneumovirus, human coronavirus nl63, and other respiratory viruses in healthy preschool-aged children using parent-collected specimens virus detection and its association with symptoms during influenza-like illness in a sample of healthy adults enrolled in a randomised controlled vaccine trial. influenza other respi viruses the effect of age on transmission of 2009 pandemic influenza a (h1n1) in a camp and associated households mechanisms of sex disparities in influenza pathogenesis agreement between self-report and medical records on signs and symptoms of respiratory illness gender differences in risk perception: theoretical and methodological perspectives annual report of the national influenza surveillance scheme estimation of the serial interval of influenza optimal design of studies of influenza transmission in households. i: case-ascertained studies submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank study staff at the 12 sites for the recruitment of participants and collection of and processing of samples for this sub-study. we thank dale key: cord-324001-m7ys95z7 authors: kobinger, gary p.; meunier, isabelle; patel, ami; pillet, stéphane; gren, jason; stebner, shane; leung, anders; neufeld, james l.; kobasa, darwyn; von messling, veronika title: assessment of the efficacy of commercially available and candidate vaccines against a pandemic h1n1 2009 virus date: 2010-04-01 journal: j infect dis doi: 10.1086/651171 sha: doc_id: 324001 cord_uid: m7ys95z7 background. the emergence and global spread of the pandemic h1n1 2009 influenza virus have raised questions regarding the protective effect of available seasonal vaccines and the efficacy of a newly produced matched vaccine. methods. ferrets were immunized with the 2008–2009 formulations of commercially available live attenuated (flumist; medimmune) or split-inactivated (fluviral; glaxosmithkline) vaccines, a commercial swine vaccine (flusure; pfizer), or a laboratory-produced matched inactivated whole-virus vaccine (a/mexico/indre4487/2009). adaptive immune responses were monitored, and the animals were challenged with a/mexico/indre4487/2009 after 5 weeks. results. only animals that received the swine or matched vaccines developed detectable hemagglutinationinhibiting antibodies against the challenge virus, whereas a t cell response was exclusively detected in animals vaccinated with flumist. after challenge, all animals had high levels of virus replication in the upper respiratory tract. however, preexisting anti—pandemic h1n1 2009 antibodies resulted in reduced clinical signs and improved survival. surprisingly, flumist was associated with a slight increase in mortality and greater lung damage, which correlated with early up-regulation of interleukin-10. conclusions. the present study demonstrates that a single dose of matched inactivated vaccine confers partial protection against a pandemic h1n1 2009 virus, and it suggests that a higher dose or prime-boost regimen may be required. the consequences of mismatched immunity to influenza merit further investigation. 1414,000 confirmed cases and ∼5000 deaths worldwide, and the real numbers are likely to be considerably higher, because countries are now only required to confirm severe cases by laboratory diagnosis [1] . even though most patients experience a disease similar to seasonal influenza, reports of severe cases are increasing [2] [3] [4] . studies in different animal models reveal more efficient spread of the pandemic h1n1 2009 viruses to the lower respiratory tract and demonstrate increased virulence of some field isolates, suggesting that the genetic makeup of the respective strain may significantly contribute toward disease outcome [5, 6] . this observation, in combination with reports of more frequent incidents of severe disease in the southern hemisphere [7] , also increases concerns about the fall, which is typically the period of the most severe influenza activity in the northern hemisphere [8, 9] . the rapid spread of the virus in countries with high seasonal influenza vaccine coverage suggests that there is little to no cross-protection conferred by these vaccines [10] . at the same time, the presence of neutralizing antibodies and the generally milder course of dis-ease observed in individuals 160 years of age are indicative of a protective effect of prior infection with antigenetically related viruses [6] . as this pandemic unfolds, and especially in light of the emerging resistance to available antivirals [11] , assessment of the safety and efficacy of available seasonal vaccines as well as matched candidate vaccines is becoming increasingly urgent. the current study evaluates in ferrets-the preferred preclinical animal model for influenza vaccine testing-3 commercially available influenza vaccines from the 2008-2009 season and 1 fully matched laboratory-produced inactivated whole pandemic h1n1 2009 virus vaccine. immune responses were monitored, and the animals were challenged 5 weeks after vaccination with a pandemic h1n1 2009 influenza isolate that exhibits moderate to high virulence in ferrets. viral loads, morbidity, mortality, and postchallenge immune responses were documented for 2 weeks. groups of five 16-to 20-weekold ferrets without antibodies against circulating influenza strains were immunized with one of the 2008 seasonal inactivated split vaccines (fluviral; glaxosmithkline) or the cold-adapted live attenuated vaccine (flumist; medimmune), a swine influenza vaccine (flusure; pfizer), or a matched laboratory-produced inactivated vaccine (ph1n1inact). the latter vaccine consisted of a madin-darby canine kidney (mdck) cell-produced whole-virus preparation of a/mexico/ indre4487/2009 (mx10; h1n1) that was isolated during the ongoing h1n1 influenza outbreak, purified by ultracentrifugation, subsequently inactivated by addition of formalin to a final concentration of 0.1%, and incubated for 3 days at 4њc (fisher scientific). the animals received the recommended dose of the respective commercial vaccines or a dose containing 15 mg of hemagglutinin (ha) of the experimental vaccine. with the exception of flumist, which was inoculated intranasally, all vaccines were injected in the gluteal muscle at the recommended dose for humans or pigs, respectively. five weeks later, the animals were challenged intranasally with 50% tissue 5 10 culture infectious doses (tcid 50 ) of mx10. clinical signs, body temperature, and weight were assessed daily, and animals were euthanized based on clinical evaluation or at the end of the study on day 16. virus quantification and pathology. nasal washes were collected on days 1, 3, 6, 9, and 16 after challenge, and virus titers were quantified by limiting dilution. in brief, 10-fold serial dilutions were incubated on mdck cells with 6 replicates per dilution. at 72-96 h after infection, the plates were scored for cytopathic effect, and the tcid 50 virus titers were calculated using the method of reed and muench [12] . rna was isolated, and viral copy numbers were quantified using real-time reverse-tran-scription polymerase chain reaction (rt-pcr). tissues preserved in rnalater were homogenized using a bead mill homogenizer for extraction of total rna. rna was isolated from nasal washes and swabs, by use of the qiaamp viral rna mini kit (qiagen), and from tissues, by use of the rneasy mini kit (qiagen). the h1n1 virus was detected by quantitative real-time rt-pcr performed using the lightcycler 480 rna master hydrolysis probes (roche) assay targeting the ha gene (nucleotide position 714-815; genbank accession number gq160606). reaction conditions were as follows: at 63њc for 3 min; at 95њc for 30 s; and 45 cycles at 95њc for 15 s and at 60њc for 30 s with the use of a lightcycler 480 (roche). the low detection limit for this h1n1 assay is 0.1 pfu/ml. the primer sequences are as follows: haf, ggatcaagaagggagaatgaactatt; har, aatgcata-tctcggtaccactagattt; and hap, ccgggagacaaa-ataacattcgaagcaac. after euthanasia, necropsy was performed for all animals, and photographs of their lungs were taken before the lungs were harvested for histopathologic analysis. lungs were inflated by slow injection of ∼5 ml of phosphate-buffered saline (pbs; invitrogen) in the trachea, and formalin-fixed and paraffinembedded tissue sections were stained with hematoxylin-eosin. immune response assessment. serum samples were collected on days 3, 7, 10, 14, and 21 after vaccination and were analyzed for the presence of hemagglutination-inhibiting (hai) antibodies against mx10 and the seasonal h1n1 strain a/brisbane/59/2007. hai antibody titers are expressed as the reciprocal of the highest serum dilution that inhibits hemagglutination of turkey red blood cells. on day 10 after vaccination, heparinized blood was collected for proliferation assays. in brief, peripheral blood mononuclear cells were isolated by ficoll-hypaque (ge healthcare) gradient purification and cultivated in the presence of overlapping peptide pools covering the nucleocapsid (np), neuraminidase (na), and ha proteins of the related h1n1 strain a/brevig mission/1918. the proliferation response was measured by adding 5-bromo-2-deoxyuridine (brdu) to the peptide-exposed peripheral blood mononuclear cells after 72 h. the next day, cells were fixed, and brdu incorporation was quantified by immunostaining performed using a chemoluminescent substrate (roche). the proliferation index is expressed as the ratio of brdu incorporation measured for the respective influenza peptide pool and for an ebola virus peptide as negative control. messenger rna profiles of cytokines, including interferon (ifn)-a, ifn-g, interleukin (il)-6, and il-10, were generated from nasal wash rnas isolated on days 1, 3, 6, and 9 after infection and from rna isolated from the right and left lung, respectively, of animals euthanized on day 9. the assays were performed using the primers and method outlined elsewhere [13] . hai antibody titer kinetics were monitored for 21 days after immunization, because titers of 140 reciprocal dilutions remain the reference standard that is predictive of protective immunity elicited by influenza vaccine candidates [14] . hai antibody titers against the pandemic h1n1 2009 mx10 isolate were detected at day 7 in ferrets receiving the laboratory-produced matched vaccine ph1n1inact or the swine influenza vaccine flusure, reaching titers 140 on day 7 or 10, respectively. flusure or ph1n1inact did not generate detectable hai antibody titers against the seasonal h1n1 strain a/brisbane/59/2007 included in conventional seasonal influenza vaccines, such as fluviral or flumist. in contrast, between day 7 and day 10, both seasonal vaccines elicited hai antibody titers against a/brisbane/59/2007 that were 140, whereas no cross-reactive response to mx10 was detected before challenge ( figure 1b) . none of the vaccines elicited an ifn-g enzyme-linked immunospot response upon stimulation with overlapping peptides covering the np, na, and ha proteins of h1n1 strain a/brevig mission/1918. these proteins share 94%, 87%, and 86% amino acid identity with the respective mx10 proteins, which may have contributed to the weak response observed. however, increased proliferation activity in response to np peptide pools was detected in animals immunized with flumist, indicating a cross-reactive t cell response ( figure 1c) . correlation of presence of hai antibodies with milder disease and improved survival. upon intranasal challenge with mx10, all vaccinated animals displayed a 1-day delay in the onset of fever, and they then followed a course comparable to that of nonvaccinated controls (figure 2a) . clinically, animals immunized with the swine vaccine flusure demonstrated more complete protection with mild and transient signs of disease, less weight loss in the first week than in the other groups, and 100% survival. the matched ph1n1inact vaccine also resulted in reduced weight loss, clinical signs of disease, and improvement of the survival rate from 50% to 80%, compared with observations in naive controls ( figure 2b-d) . in contrast, there were no statistically significant differences in recorded clinical signs of disease, weight loss, or survival rate between the animals given fluviral and the control groups, over the course of the experiment after challenge ( ). the group of ferrets vac-p 1 .05 cinated with flumist showed a slight improvement in average body weight at days 5 and 6 after challenge. however, weight loss increased from day 6 to day 9, at which point clinical signs of disease, including nasal seromucous exudates, shallow and labored breathing, and reduced activity forced euthanasia for 4 of the 5 animals given flumist, resulting in a small increase in the mortality rate, compared with that noted for the unvaccinated control group. this increase in the mortality rate associated with mismatched flumist immunization was also observed in a second experiment with 4 animals, although the increase was not statistically significant (data not shown). at the respective times of euthanasia, gross pathological evaluation of lungs demonstrated minimal lesions in all 5 animals vaccinated with flusure, including 1 animal euthanized on day 9, without reaching experimental end points to obtain, on a timely basis, tissue samples matched to those obtained from the other groups ( figure 3 ). animals vaccinated with ph1n1inact showed a slight improvement, with smaller lesions noted in 3 of the 5 ferrets. three of 4 control animals had severe lesions with hepatization, hemorrhages, and widespread alveolitis and bronchiolitis, which were comparable to lesions observed in 4/5 or 5/5 ferrets vaccinated with fluviral or flumist, respectively. all groups reached nasal infectious titers of ∼ tcid 50 at day 1 after challenge, with the exception 6 10 of the animals immunized with the swine vaccine flusure, which had a 10-fold lower titer ( figure 4a ). the group vaccinated with flumist maintained relatively high levels of virus replication through day 3, whereas the other groups experi-enced titer decreases of у20-fold. with the exception of one animal in the control group, no infectious viral particles were detectable by titration in the nasal washes of animals on day 6 or later, although viral rna could be detected by real-time rt-pcr until the end of the experiment ( figure 4b ). animals immunized with ph1n1inact or flusure experienced the lowest ifn-a levels and up-regulation of ifn-g during the early stage of the infection. moderately elevated levels of il-6 were detected later in the course of disease-at day 9 exclusively in these 2 groups, which showed evidence of protection ( figure 5 ). in contrast, the group vaccinated with flumist exhibited the highest average of mrna transcripts for ifn-a, up to day 6, and for ifn-g, at day 3, possibly reflecting a better cellular response. of interest, at day 3, levels of il-10 transcripts were significantly higher in the upper airway of animals vaccinated with flumist, and at day 9 after infection, they were slightly increased in the lower airway, compared with observations in control animals and animals vaccinated with ph1n1inact or flusure ( figure 5 ). no differences in tumor necrosis factor-a and il-8 levels were observed between the groups (data not shown). the availability of an efficient vaccine is essential to alleviate the effect of the ongoing influenza pandemic. a possible intervention strategy to mitigate the 2009 fall influenza season in the northern hemisphere was to initially perform mass immunization with the seasonal vaccine, followed by mass immunization with the fully matched pandemic h1n1 2009 vaccine as soon as would become available. to direct a concerted public health response to control the spread of the virus, the efficacy of a newly produced matched inactivated vaccine, as well as that of already available inactivated and live attenuated vaccines, has to be assessed. toward this end, we compared the antibody and cellular responses elicited by 2 seasonal vaccines (fluviral and flumist), the commercial swine vaccine flusure, and a laboratory-produced matched inactivated whole-virus preparation. we found that only the swine and matched vaccines resulted in production of hai antibodies against the pandemic h1n1 2009 virus, whereas only flumist triggered a crossreactive cellular response. intranasal challenge with the virulent mexican isolate mx10, similar to mx/4482, which also leads to a 50% mortality rate among naive animals [6] , revealed that none of the vaccines was able to confer complete protection after only one immunization. however, flusure was associated with the best reduction in morbidity and complete protection from mortality, whereas the matched inactivated vaccine resulted in moderate clinical improvement and reduced mortality. as was expected from undetectable hai antibody titers, animals vaccinated with fluviral or flumist did not experience a beneficial effect, compared with unvaccinated control animals. the partial protection observed in animals vaccinated with one dose of the matched inactivated vaccine, despite the detection of an hai antibody response within the protective range, indicates that protection from aggressive isolates may require more than a single immunization, which would put an additional strain on vaccine availability. the use of a more virulent challenge strain enables assessment of vaccine efficacy in a worst-case scenario. however, the disease severity associated with currently circulating pandemic h1n1 2009 strains in most patients is more similar to that associated with seasonal influenza [3] . it is thus possible that a single 15-mg dose of a matched inactivated vaccine will be sufficient to confer protection against most pandemic h1n1 2009 strains, especially in individuals with some levels of cross-protection due to previous influenza infection. the efficiency of a commercially available swine vaccine indicates that this product would be adequate to protect animals, including pig herds, which could minimize interspecies transmission and maybe limit evolution of the virus. the flusure vaccine consists of an inactivated h1n1 and h3n2 type a field isolate formulated with amphigen (pfizer) as an adjuvant [15] , indicating that the use of this and other adjuvants merits a more in-depth evaluation in the context of the development of improved human influenza vaccines. a curious observation is the more severe cases of disease and the higher mortality rate noted for animals vaccinated with flumist, which correlated with slightly more infectious virus in the nasal washes of this group at day 3. this correlation figure 5 . relative quantification of cytokine messenger rna (mrna) induction. changes in cytokine mrna levels were determined by semiquantitative real-time reverse-transcription polymerase chain reaction in nasal wash rna or rna isolated from lung tissue harvested on day 9. ten nanograms of rna were used for each reaction, and the fold change was calculated using the comparative cycle threshold (ddct) method. columns denote the mean of all values obtained for the respective group, and error bars denote the standard error. ctl, nonimmunized control group; dpi, days post infection; ifn, interferon; il, interleukin. between the infectious viral load in the upper respiratory tract and the clinical outcome was in fact observed for all groups in the present study, confirming previous reports that the infectious viral load in the airway is predictive of levels of protection in vaccinated ferrets after challenge with respiratory viruses, including severe acute respiratory syndrome-associated coronavirus and influenza [16, 17] . on the other hand, data from this study also indicate that weight loss was, on average, more severe for the unvaccinated control animals than for any other vaccinated group of animals. the present study was designed to evaluate protective efficacy after vaccination and not the possible subtle negative effects caused by immunization with mismatched influenza antigens. larger study group sizes will be necessary to conclusively address this question with an appropriate degree of statistical confidence. antibody-mediated enhancement of influenza infection, including subtype-crossreactive, nonneutralizing antibodies, has been previously described in cultured cells [18] [19] [20] . this mechanism has never been directly associated with a worsened clinical condition in animal models of influenza infection, although a recent study reported that maternally derived antibodies possibly enhanced swine influenza virus-induced pneumonia in pigs [21] . these observations further support the need for a more detailed evaluation of the efficacy of influenza vaccine in controlled exper-imental conditions where various levels of preexisting immunity to mismatched influenza antigens could be studied. all animals demonstrated a strong induction of the proinflammatory cytokine il-6 at day 6; this level remained elevated at day 9 only in the 2 groups of ferrets showing noticeable protection. animals vaccinated with flumist, the only group that mounted a strong cross-reactive cellular response, had the highest ifn-g response. however, this response was not sufficient to control the disease. in fact, the strong expression of il-10, an anti-inflammatory cytokine, detected in that group on day 3 may have suppressed an appropriate inflammatory response, including il-6 expression, and temporarily favored virus replication, as previously demonstrated in pigs infected with foot-and-mouth disease [22] . there are reports showing the negative effect of il-10 on influenza virus-infected mice and pigs [23, 24] , and increased il-10 production correlated with a low antibody response in elderly individuals after influenza vaccination [25] . evaluating the response of cytokines, including il-6 and il-10, at early time points in patients may help predict unfavorable outcome and allow for better allocation of resources to individuals requiring more intensive clinical intervention. the present study reports the immune responses and protective efficacy of commercially available vaccines and one lab-oratory-produced matched vaccine with regard to prevention of pandemic h1n1 2009 infection in ferrets. the findings of this study may help to guide ongoing preparations for the influenza season in the northern hemisphere. world health organization (who) pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico swine influenza a (h1n1) strikes a potential for global disaster severe respiratory disease concurrent with the circulation of h1n1 influenza emergence and pandemic potential of swine-origin h1n1 influenza virus transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice influenza a(h1n1)v in the southern hemisphere-lessons to learn for europe? influenza activity in europe during eight seasons (1999-2007): an evaluation of the indicators used to measure activity and an assessment of the timing, length and course of peak activity (spread) across europe surveillance for influenza-united states, 1997-98, 1998-99, and 1999-00 seasons emergence of a novel swine-origin influenza a (h1n1) virus in humans oseltamivir-resistant novel influenza a (h1n1) virus infection in two immunosuppressed patients a simple method of estimating fifty percent endpoints severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction a trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets adenovirus-based vaccine prevents pneumonia in ferrets challenged with the sars coronavirus and stimulates robust immune responses in macaques investigation of the biological indicator for vaccine efficacy against highly pathogenic avian influenza (hpai) h5n1 virus challenge in mice and ferrets antibody-mediated growth of influenza a nws virus in macrophagelike cell line p388d1 infection enhancement of influenza a nws virus in primary murine macrophages by anti-hemagglutinin monoclonal antibody subtype cross-reactive, infectionenhancing antibody responses to influenza a viruses the immune response and maternal antibody interference to a heterologous h1n1 swine influenza virus infection following vaccination immunosuppression during acute infection with foot-and-mouth disease virus in swine is mediated by il-10 alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs il-10 deficiency unleashes an influenza-specific th17 response and enhances survival against high-dose challenge high interleukin-10 production is associated with low antibody response to influenza vaccination in the elderly we would like to thank eric poeschla, for providing the flumist doses, and naveed zafar janjua and nicholas svitek, for help with literature review or the cytokine real-time reverse-transcription polymerase chain reactions, respectively. key: cord-331244-zaguyxm5 authors: stephenson, iain; nicholson, karl g; wood, john m; zambon, maria c; katz, jacqueline m title: confronting the avian influenza threat: vaccine development for a potential pandemic date: 2004-07-30 journal: lancet infect dis doi: 10.1016/s1473-3099(04)01105-3 sha: doc_id: 331244 cord_uid: zaguyxm5 sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. sporadic human infection with avian influenza viruses has raised concern that reassortment between human and avian subtypes could generate viruses of pandemic potential. vaccination is the principal means to combat the impact of influenza. during an influenza pandemic the immune status of the population would differ from that which exists during interpandemic periods. an emerging pandemic virus will create a surge in worldwide vaccine demand and new approaches in immunisation strategies may be needed to ensure optimum protection of unprimed individuals when vaccine antigen may be limited. the manufacture of vaccines from pathogenic avian influenza viruses by traditional methods is not feasible for safety reasons as well as technical issues. strategies adopted to overcome these issues include the use of reverse genetic systems to generate reassortant strains, the use of baculovirusexpressed haemagglutinin or related non-pathogenic avian influenza strains, and the use of adjuvants to enhance immunogenicity. in clinical trials, conventional surfaceantigen influenza virus vaccines produced from avian viruses have proved poorly immunogenic in immunologically naive populations. adjuvanted or whole-virus preparations may improve immunogenicity and allow sparing of antigen. few infectious diseases cause such a huge annual toll of morbidity, mortality, and economic loss as influenza. in addition, influenza can unpredictably emerge to cause pandemics. the 1918 spanish pandemic spread around the world within 9 months causing up to 40 million deaths. 1 the transmission of avian influenza h5n1 to at least 32 people during the 2004 asian h5n1 epizoonotic period 2,3 prompted concerns that the next pandemic is imminent. as highlighted by the recent severe acute respiratory syndrome (sars)coronavirus outbreak, international air travel increases global vulnerability to infectious respiratory pathogens. our ability to combat influenza and its complications depends primarily on vaccination. annual influenza vaccine production is a wellplanned process that takes up to 6 months. current facilities may not be suitable for rapid bulk manufacture of avian influenza virus vaccines in response to a world threat. influenza viruses are enveloped negative-sense rna viruses with a segmented genome belonging to the orthomyxoviridae family. they are classified on the basis of their core proteins into three distinct types: a, b, and c. 4 influenza a viruses infect a range of mammalian and avian species, whereas type b and c are essentially restricted to human beings. influenza a viruses are responsible for annual epidemics and occasional pandemics, whereas influenza b viruses cause outbreaks every 2-4 years, but are not associated with pandemics. the main antigenic determinants of influenza a and b viruses are two surface glycoproteins: the neuraminidase and the haemagglutinin, both capable of eliciting immune responses in human beings. the haemagglutinin is involved with receptor binding and membrane fusion. the neuraminidase facilitates cleavage of virus progeny from infected cells, prevents viral aggregation, and aids movement through the mucosal respiratory-tract epithelium. virus strains are classified according to host species of origin, geographic site and year of isolation, serial number, and, for influenza a, by serological properties of subtypes of haemagglutinin and neuraminidase. influenza a h1 and h3 subtypes circulating in human beings evolve and undergo antigenic variability continuously. a lack of effective proofreading by the viral rna polymerase leads to a high rate of transcription errors that can result in aminoacid substitutions in surface glycoproteins. virus variants with substitutions in the antibody-binding sites can evade humoral immunity and reinfect individuals. this is termed "antigenic drift". the segmented viral genome allows for a second type of antigenic variation. if two influenza viruses simultaneously infect a host cell, genetic reassortment may generate a novel virus with new surface or internal proteins. pandemic influenza viruses arise by this process of "antigenic shift", when a virus with a new haemagglutinin subtype emerges and spreads efficiently in a naive human population. comparisons of pandemic and interpandemic influenza are shown in table 1. bird populations. 5, 6 avian influenza a viruses generally do not cause disease in these natural hosts. the principal site of influenza virus replication in aquatic birds is the gastrointestinal tract resulting in high faecal viral titres and viral transmission in migratory feeding areas. despite the range of virus subtypes, only a few haemagglutinin (h1, h2, h3) and neuraminidase subtypes (n1, n2) have established in human beings and have caused widespread respiratory disease. the haemagglutinin of human influenza viruses preferentially binds to sialic acid receptors containing ␣2,6-galactose linkages, whereas avian influenza viruses preferentially bind to those containing ␣2,3-galactose linkages. these binding preferences correlate with the predominance of sialic acid ␣2,6-galactose linkages on human epithelial cells, and ␣2,3-galactose linkages on avian intestinal epithelial cells. [7] [8] [9] although the molecular mechanisms responsible for receptor-binding specificity are poorly defined, it is believed that haemagglutinin of avian origin must acquire human receptor-binding specificity to generate influenza strains capable of sustained human-to-human transmission. site-directed mutagenesis studies have shown that only one or two aminoacid mutations are required for this change. 10 limited passage in human beings of a virus possessing an avian haemagglutinin, such as occurring in asia currently, 2,3 may be sufficient to generate such a change. during the 20th century, an h1n1 virus in 1918, an h2n2 virus in 1957, and an h3n2 virus in 1968 caused influenza pandemics. human-avian reassortant viruses seem to have caused the pandemics of 1957 and 1968. the 1957 h2n2 virus differed by three genes (haemagglutinin, neuraminidase, and the rna polymerase pb1) from the h1n1 virus that infected people between 1918 and 1957. the 1968 h3n2 virus differed by two genes (haemagglutinin and pb1) from the h2n2 virus that infected people between 1957 and 1968. in both cases, the h2 and h3 haemagglutinin genes were contributed by avian viruses. 11 sequence analyses of early h2 and h3 isolates indicate receptor-binding specificity was altered by a single aminoacid substitution soon after human introduction. 10 since pig trachea contains receptors for both avian and human influenza viruses, and can support replication of viruses of both human and avian origin, it has been suggested that genetic reassortment between avian, swine, and human influenza viruses may occur in pigs, and that they represent a "mixing bowl" for the evolution of human pandemic strains. 12 haemagglutinin and neuraminidase of h5n1 viruses isolated from human beings, poultry, and wild ducks have distinguishable properties. 13 chickens seem to support a separate natural reservoir of influenza viruses, indicating a possible role as intermediate hosts in zoonotic transmission. some avian h9 viruses established in poultry are capable of two-way transmission between domestic ducks, where they are able to generate multiple reassortants with other cocirculating viruses. 14 these reassortant viruses have haemagglutinin receptor-binding sequences potentially capable of human infection, suggesting that new viruses may emerge directly from the avian pool. the close proximity of people to high concentrations of waterfowl, poultry, and swine in southeast asia, and avian influenza activity, has identified this region as a hypothetical influenza epicentre (figure 1). 15 avian influenza viruses generally do not replicate efficiently in human beings, even after experimental infection. 16 before 1997, direct transmission of avian influenza viruses to the human respiratory system was not considered possible. however, it is now recognised that at least some subtypes of avian influenza viruses can replicate within the human respiratory tract. although it is unclear whether the recent reported increase in transmission of avian influenza to people (table 2) is the result of heightened surveillance, the geographical expansion of h5n1 poultry outbreaks across asia is an unprecedented and new event. more than 30 confirmed cases of transmission of avian h5n1 virus to human beings has increased the possibility that an avianhuman reassortant virus may emerge to effectively transmit among people. caused by immunity in population result pandemic influenza a antigenic shift: emergence of novel or little or no background immunity high attack rates, excess mortality and re-emerging subtype of influenza a (maybe partial immunity in older morbidity in all age groups people if re-emerging virus) interpandemic influenza antigenic drift: evolution of existing little immunity in infants. partial variable outbreaks or epidemics influenza (a or b) strains immunity in adults by cross-reacting with variable morbidity and antibody to previously seen mortality, usually in elderly and young and related strains (h3 greatest severity) the first association of avian influenza viruses with respiratory illness in human beings was during 1997 when six deaths from 18 human cases of highly pathogenic influenza h5n1 occurred during an outbreak among live-bird markets (table 2) . [18] [19] [20] all viral genes were of avian origin, indicating that h5n1 had crossed the species barrier without adaptation or reassortment with human viruses. despite the elimination of ducks, geese, and quail (sources of h5n1 and its donor genes) and cleaning days in the markets, h5n1 viruses have subsequently reemerged in hong kong poultry markets, 32 although hong kong remains free from infection in the 2004 h5n1 outbreak across asia. 29, 33 in late 2002, highly pathogenic h5n1 viruses were isolated from dead waterfowl in hong kong (personal communication, m peiris, university of hong kong), which was notable since h5n1 viruses do not typically cause disease in waterfowl. in february 2003, h5n1 reemerged in hong kong in two family members returning from a trip to china. 24 in 2004, pathogenic h5n1 viruses are causing extensive poultry outbreaks in asia with 23 deaths (68%) of 34 human cases reported in vietnam and thailand. 2, 3, 30 in hong kong in 1999, and again in 2003, influenza h9n2 viruses were isolated from children with mild respiratory illnesses. 22, 28 as with h5n1, no human-to-human transmission was evident. 23 additional human h9 infections in china, 34 and detection of antibody to h9 in human serum samples in china and hong kong 35 suggest that further human h9 infection has occurred. eurasian h9 viruses circulating since the late 1990s have been classified into three phylogenetic sub-lineages: g1, y280 (g9-like), and y439. 36, 37 those established in live-bird 38 and have also transmitted to swine in hong kong and china. [38] [39] [40] some avian h9 viruses have acquired receptor-binding characteristics typical of human strains, increasing the potential for reassortment within both human and pig respiratory tracts. 41 in early 2003, outbreaks of highly pathogenic avian influenza h7 among poultry occurred in the netherlands and extended to belgium and germany. 26, 27 more than 80 workers involved in control of the outbreak developed viral h7n7 conjunctivitis and a few developed respiratory illness. evidence of limited human-to-human spread, and a fatal respiratory infection, highlighted a significant threat to human health. more recently, cases of avian influenza (h7n3) infections have been reported in two cullers during control of an h7n3 poultry outbreak in canada. 30 avian influenza (h10n7) seems to have crossed the species barrier from poultry to people for the first time. in egypt in april 2004 two infants presenting with mild febrile respiratory symptoms had h10n7 influenza viruses isolated from respiratory samples. 31 as well as h5, h7, and h9 viruses, the h6 subtype has acquired the ability to infect chickens and is rapidly becoming endemic in poultry populations. phylogenetic analyses of h6n1 viruses isolated from wildfowl, showing high nucleotide homology of the internal genes to human h5n1 and h9n2 viruses, suggests these subtypes can transfer genetic material between each other and are potential sources of new strains. 42 h2n2 viruses were responsible for the 1957 influenza pandemic and circulated as the only human subtype until 1968 when it was replaced by h3n2 subtype. the h2 haemagglutinin gene, along with the neuraminidase and pb1 genes, were derived from avian sources. 11 since people born after 1968 lack immunity to the h2 subtype, h2 viruses pose a pandemic threat to this susceptible population. h2 viruses continue to circulate in wild ducks, 43 although the conditions required for the re-emergence of human h2 influenza viruses are unclear. although the virulence of avian influenza viruses has been well studied in avian species, their virulence in mammals is not well understood. infection with avian influenza a viruses in birds causes a wide spectrum of disease ranging from subclinical to overwhelming systemic illness (figure 2). both h5 and h7 subtypes have the ability to evolve into highly pathogenic forms. although virulence is a polygenic trait, a major contributing factor in birds is the haemagglutinin. cleavage of the haemagglutinin into two subunits is essential for viral infectivity. 44 haemagglutinin from strains of low pathogenicity is cleaved by proteases limited to the respiratory tract of mammalian species and the intestinal tract of avian species. by contrast, haemagglutinin from highly pathogenic viruses can be cleaved by proteases present in a range of tissues, resulting in multi-system infection. structural features at the cleavage site determine the cleavability of the haemagglutinin. the acquisition of multiple basic aminoacids in highly pathogenic h5 or h7 haemagglutinin enables cleavage of the protein by these ubiquitous proteases and confers virulence. carbohydrate side chains in the vicinity of the cleavage site may also affect the access of the proteases and hence virulence. 45 other factors in addition to the haemagglutinin cleavage site are likely to contribute to virus pathogenicity in mammals. virulence of mouse-adapted influenza a viruses have been associated with the interferon antagonist properties of the ns1 protein 46 or the ability of the neuraminidase glycoprotein to sequester circulating plasminogen and promote haemagglutinin cleavage. 47 balb/c mice [47] [48] [49] [50] and ferrets 51 are useful mammalian hosts for the evaluation of human h5n1 pathogenesis. in these mammalian hosts, the multibasic aminoacid motif in the haemagglutinin is necessary but not sufficient for virulence. 47, 52, 53 a single aminoacid substitution in pb2 is associated with high pathogenicity of human h5n1 viruses in mice 52 although substitutions in other gene products are likely to play a part. 49 in human h5n1 infection, disease progression to respiratory failure is unusually severe, with features of haemophagocytosis, leucopenia, and multiple organ failure. 3, 19, 30 in-vitro infection of human macrophages with 1997 h5n1 human viruses induces high levels of cytokines compared with some human virus strains. 54 in pig respiratory epithelial cells, the 1997 h5n1 human viruses were also shown to be relatively resistant to the inhibitory effects of host antiviral cytokines such as interferons. 55 thus, the severity of h5n1 infection in people is likely to be related to the induction of excessive proinflammatory responses that exacerbate tissue injury. it has been suggested that the nonstructural gene has a role. 54 gene sequence analyses of the 1918 pandemic virus, which displayed enhanced virulence in human beings has not yet uncovered molecular determinants previously associated with influenza virus virulence. 56, 57 the molecular determinants and gene constellations that confer virulence of avian, swine, and human viruses, and the circumstances under which virulent phenotypes emerge remains unclear. understanding the basis of virulence is important for vaccine design, particularly of live vaccines, so that viruses can be attenuated. current inactivated influenza vaccines are produced from virus grown in embryonated hens' eggs, and are of three types: whole-virus, "split-product", or subunit "surface-antigen" formulations. whole-virus vaccines are associated with increased adverse reactions, especially in children, and are little used. most influenza vaccines are split-product vaccines, produced from detergent-treated, highly purified influenza virus, or surface-antigen vaccines containing purified haemagglutinin and neuraminidase. 58 vaccines are usually trivalent, containing 15 g each of two influenza a subtypes (h1n1 and h3n2) and one influenza b strain. vaccines elicit a relatively strain-specific humoral response, have reduced efficacy against antigenically drifted viruses, and are ineffective against unrelated strains. the who reviews vaccine composition biannually and updates antigenic content depending on prevalent circulating subtypes to provide antigenically well-matched vaccines. protective efficacy of 70-95% in healthy young adults is obtained when there is a good antigenic match between the vaccine and circulating strains. 59 vaccination of the elderly is associated with 19-63% reductions in hospitalisation for pneumonia and influenza, 17-39% reductions for all respiratory conditions, and 27-75% reductions in all-cause mortality. 60 new influenza vaccines must elicit protective immunity. the haemagglutinin-inhibition test is most commonly used for the detection of antibody to influenza, although single radial haemolysis (srh) may also be used, since both are correlated with immune protection. 61, 62 in the european union, interpandemic influenza vaccines should fulfil certain criteria, prepared by the committee for proprietary medicinal products (cpmp), 63 which are usually assessed by haemagglutinin-inhibition tests in limited annual clinical studies (panel 1). in the event of pandemic influenza, vaccine demand would soar. savings made using monovalent rather than trivalent vaccine (15 g haemagglutinin per dose instead of 45 g) would possibly be offset by a two-dose schedule, increased demand, and difficulties with production of egg-grown viruses. new developments include the use of mammalian cell lines to culture influenza virus for vaccines to provide increased flexibility of production at times of heightened demand. 64 immunopotentiating effects of adjuvants and whole-virus vaccine may increase antigenicity, allowing dose content reduction enabling maximum efficient use of limited supplies. the population immune status in a pandemic situation differs from that seen during the interpandemic period. at the onset of the previous pandemics, younger adults were immunologically naive to the new strains, whereas older populations may have been primed by previous infections of related strains that circulated in earlier times. global immune susceptibility to avian influenza subtypes would be expected. the quantity of antigen required to elicit satisfactory immune responses in naive individuals is unclear since few studies have been done after the emergence of a novel virus. current events suggest the urgent need to develop a clearly defined strategy for clinical assessment of safety and immunogenicity of pandemic vaccines. in 1976 and 1977, the emergence of influenza a/new jersey/76 and a/ussr/92/77 (h1n1) triggered pandemic alerts, and afforded the opportunity for vaccine trials in immunologically naive and primed populations. a series of whole-virus vaccine studies [65] [66] [67] [68] [69] reported differences between naive populations (those aged 24 years and not exposed to previous h1n1 strains) and primed populations (older than 24). in naive patients, if one dose of vaccine was administered, large doses (in excess of 60 g haemagglutinin) were required to fulfil cpmp criteria. however, if two doses of vaccine were given, lower antigen doses (5 g) were needed. whole-virus vaccine was significantly more immunogenic than subunit or split-product vaccines. in primed patients, as is the case during interpandemic periods, no difference in immunogenicity between whole-virus vaccine and subunit or split-product vaccines was reported. however, a consistent finding was that whole-virus vaccine was associated with increased reactogenicity, particularly in children, who developed febrile complications even with low doses. 67 although licensed for use in human influenza vaccines, aluminium salts are rarely used since studies have indicated little clinical benefit. 70 however, encouraging findings were more recently reported in a study of whole-virus a/singapore/1/57 (h2n2) vaccine in immunologically naive people. 71 monovalent alumadjuvanted vaccine containing either 7·5, 3·8, or 1·9 g h2 haemagglutinin per dose was compared with unadjuvanted whole-virus 15 g vaccine. although a single dose of any vaccine was unable to elicit responses associated with protection, a second dose of vaccine boosted responses to mean geometric increase in antibody >2·5 (>2 in the у60 years group) number of seroconversions or significant rises in anti-haemagglutinin antibody (ie, four-fold increase in post-vaccination hi titre or 50% increase in srh zone) should be >40% (>30% in the у60 years group) proportion of patients achieving a seroprotective hi titre of у1/40, or srh titre of >25 mm 2 post vaccination should be >70% (>60% in the >60 years group) rates that fulfilled cpmp licensing criteria across all doses, suggesting that up to an eight-fold reduction in antigen content could be achieved with the addition of alum (figure 3). since influenza a viruses possess a segmented genome, simultaneous infection of eggs with two different viruses may result in reassortment of segments to produce a desired vaccine seed strain. the influenza a virus components of annual influenza vaccines are typically derived from egggrown reassortment viruses that have the relevant haemagglutinin and neuraminidase genes of the antigenically relevant strain, and the six remaining gene segments from a/puerto rico/8/34 (h1n1). these pr/8/34 segments confer high growth properties in eggs favoured for inactivated vaccine production. 58 this process requires large numbers of eggs, and in many companies lacks the flexibility to respond rapidly to a pandemic event. highly pathogenic h5 and h7 viruses cannot be grown in large quantities because they are lethal to chicken embryos. 72 such pathogenic strains also impose regulatory and safety issues. as the multibasic sequence cleavage site is believed to contribute to the pathogenesis of human h5n1 infection, vaccine preparation from wild-type h5n1 virus would require heightened biocontainment to protect workers and eliminate the possibility of environmental contamination and infection of susceptible animals. thus, several approaches have been attempted for avian influenza vaccine development. these include: (1) the production of inactivated vaccine from wildtype virus; (2) the selection of an antigenically related nonpathogenic vaccine strain; (3) the use of baculoviruses to express recombinant haemagglutinin; (4) dna-based vaccines; (5) the use of plasmid-based reverse genetics systems to construct vaccine seed strains possessing attenuated haemagglutinin; and (6) plasmid-based reverse genetic systems to construct attenuated donor strain recombinants. reverse genetics is likely to produce the most rapid response in an emerging pandemic. much of the preclinical and clinical development of highly pathogenic avian influenza vaccines has used h5 virus as a model. after the 1997 h5n1 outbreak, inactivated vaccines from wild-type a/hong kong/156/97 (h5n1) virus were prepared in the uk and the netherlands. 73, 74 whole-virus vaccine was effective in protecting mice against lethal challenge with h5n1 virus. 73 conventional surface-antigen vaccine was poorly immunogenic in chickens and did not protect against lethal dose challenge, although an iscom formulation (antigen as immune-complex stimulators) boosted immune responses and protected against lethal h5n1 challenge. 74 because the use of highly pathogenic strains has safety restrictions, the selection of a surrogate non-pathogenic virus capable of evoking crossreactive immunity to the 1997 hong kong h5n1 viruses was investigated. both a/duck/hokkaido/ 67/96 (h5n4) and a/duck/singapore/97 (h5n3) have haemagglutinin proteins antigenically similar to a/hong kong/ 156/97 (h5n1) and were used in experimental vaccines. 72, 73 although antibody titres to h5n1 induced by a/duck/ singapore (h5n3) vaccine were four-fold lower than the homologous strain, 73 inactivated whole-virus vaccine 72,73 and alum-adjuvanted subunit vaccine 48 were capable of protecting mice against lethal h5n1 challenge. although attempts to reassort a/duck/singapore (h5n3) with a/pr/8/34 to produce a high-growth virus suitable for vaccine production failed, 73 conventional and mf59-adjuvanted a/duck/ singapore/97 (h5n3) surface-antigen vaccines were clinically assessed in a randomised phase i trial. 75 two doses of 7·5, 15, or 30 g h5 haemagglutinin were given 3 weeks apart. antibody responses were measured by haemagglutinininhibition, virus microneutralisation, and srh. although both vaccines were well tolerated, non-adjuvanted vaccine was poorly immunogenic, with only one of 11 (9%) recipients seroconverting by haemagglutinin-inhibition and srh h5n1, and four (36%) by microneutralisation and srh h5n3 after two 30 g doses. the addition of mf59 gave significantly higher antibody responses (figure 4), and two doses achieved seroconversion rates of 13/31 (42%), 29(94%), 31(100%), and 26(84%) by haemagglutinin-inhibition, microneutralisation, srh h5n3, and srh h5n1, respectively. antibody titres by srh to h5n1 were about half those to h5n3, showing the need for close antigenic matching between vaccine and pandemic strains to ensure maximum vaccine efficacy. antibody responses after h5n3 revaccination 16 months later were boosted significantly above those achieved after two doses. 76 it is desirable for vaccines to boost responses after initial priming, since second waves occur 3-9 months after the first pandemic wave of infection. 77 one problem in assessing vaccine responses was the insensitivity of the haemagglutinininhibition test, routinely used in assessment of influenza vaccines, for the detection of antibody to h5 when compared with neutralisation tests. 78, 79 haemagglutinin-inhibition relies on the ability of antibody to disrupt the haemagglutinin-sialic-acid-receptor binding interaction between virus and erythrocytes. if the erythrocytes used in the test are not optimised for expression of ␣2,3galactose linkages that are necessary for avian influenza virus binding, the test is insensitive. 80 however, there are no recognised clinical correlates of immune protection for neutralisation antibody. it was only possible to assess vaccine responses with respect to cpmp licensing criteria after development of a specific srh assay. 81 a modified haemagglutinin-inhibition test, using enzymatically altered turkey erythrocytes or horse erythrocytes, was developed to increase the sensitivity for detecting antibody to avian influenza virus antigens. 82 an alternative to egg-derived vaccines involves the use of haemagglutinin protein expressed in insect cells by recombinant baculovirus. potential difficulties include the use of uncleaved rather than cleaved haemagglutinin and differences in glycosylation in insect cells that may effect immunogenicity. nonetheless, antibody responses to 15-45 g doses of recombinant h1 and h3 antigens are similar to those induced by licensed vaccines. 83, 84 baculovirus-derived h5 and h7 haemagglutinin vaccines protect against lethal virus challenge in chickens, even when the haemagglutinin sequence homology differs by up to 16%. 85 however, when clinically assessed in people, a recombinant baculovirus-expressed h5 vaccine was suboptimal. even after two doses of 90 g, only 52% subjects seroconverted by microneutralisation, suggesting improvements in immunogenicity are needed. 86 reverse genetics systems can be used to generate attenuated avian influenza viruses, and are likely to prove pivotal in pandemic vaccine development. the appropriate haemagglutinin and neuraminidase genes from a virus can be cloned and, if necessary, the haemagglutinin may be attenuated by removal of the multi-basic cleavage site sequence, and inserted into plasmids. the plasmids are transfected into a cell line together with plasmids encoding the internal genes from the a/pr8/34 virus to generate an appropriate non-pathogenic vaccine seed strain. vaccine candidates expressing the target haemagglutinin from highly pathogenic viruses could potentially be produced within weeks of an emerging event. recombinant h5 viruses showing desirable properties for h5 vaccine formulation-including loss of egg lethality, virulence, and infectivity in animal models-have been produced, 87, 88 although one attenuated recombinant virus showed limited neurovirulence in mice. 87 reverse genetics is thus capable of generating attenuated viruses from pathogenic strains suitable for vaccine production with only limited enhancement of biosecurity measures and using pre-existing equipment and facilities. it should also be possible to prepare panels of reassortant viruses and vaccine seed candidates containing target genes of potential pandemic viruses in advance of any specific threat. however, there are regulatory, safety, and legal problems to overcome before the technology can be used for vaccine development. mammalian cell lines (eg, vero cells) used for transfection must be of certified quality for human vaccine production. viruses generated by reverse genetics may be considered to be "genetically modified organisms", imposing local and national safety regulations regarding research and development. in addition, intellectual property rights on reverse genetics technology are held, and licences may need to be granted for commercial use of vaccines. inactivated influenza vaccines are poor inducers of cytotoxic t-cell (ctl) responses, which aid in recovery from influenza infection. it has been suggested that crossreactive immunity to several influenza virus subtypes could be induced by ctl responses to conserved epitopes in internal proteins. 89 dna vaccines integrate gene sequences for the antigenic protein of interest into bacterial plasmids that are inoculated into the host. the expression of plasmid dna produces the antigen in its natural configuration, which is more likely to stimulate neutralising antibody and undergo hla class i expression inducing ctl responses. 90 h5 and h7 haemagglutinin-expressing dna vaccine protects mice and chickens against lethal dose virus challenge. 91,92 dna vaccine expressing conserved internal proteins including nucleoprotein give partial protection to mice against h5n1 infection. 93 intradermal dna influenza vaccines are beginning clinical evaluation. iscom-formulated h1n1 vaccine can induce ctl responses and greater longer-lasting antibody responses than conventional vaccine. 94 it offers broad protection against virus challenge with h2, h3, h9, and virulent h5 viruses in mice. iscom vaccines are tolerated in human beings and induce broad ctl and rapid humoral responses. 95 mucosal delivery of inactivated influenza h3n2 vaccine adjuvanted with modified heat-labile enterotoxin from escherichia coli induces b-cell-dependent heterosubtypic immunity against lethal h5n1 virus challenge in mice. 96 in the absence of an antigenically matched vaccine, alternative vaccine strategies that induce crossreactive immunity by iscom, dna, or mucosal vaccines may provide useful firstline defence against an emerging pandemic strain. since h9 viruses do not have a multibasic haemagglutinin cleavage site, they show low pathogenicity for avian species and may be grown to high titres in eggs. both g1-like (a/hong kong/1073/99) and y280 (g9-like; a/hong kong/2018/03) h9n2 viruses are capable of human infection. 22, 28 for an effective h9 vaccine strategy, an understanding of the relative immunogenicity and crossprotection induced by these lineages is required. although infection of mice with g1 or g9 group h9 viruses did not evoke detectable crossreacting neutralising antibody, they were protected from subsequent rechallenge with the homologous or heterologous virus lineage. 97 whole-virus g1 h9 vaccine produced crossreactive antibody responses to both g1 and g9 viruses, and protected mice against rechallenge with either virus. by contrast, whole-virus g9 h9 vaccine induced homologous antibody titres only and was able to protect against g9 challenge, but showed reduced protective efficacy against the heterologous g1 lineage. here, a single dose of h9 vaccine induced adequate immune responses in mice, by contrast with findings with h5n1 vaccine that required a two-dose schedule to elicit adequate immune responses. 74, 97 since some y280 (g9-like) h9 viruses do not grow well in eggs, an a/pr/8/34 reassortant has been produced. 98 one dose of inactivated g9/pr8 vaccine protected mice against g9 challenge. two doses of vaccine increased antibody responses capable of protecting mice against both g1 and g9 h9 challenge. whole-virus and subunit a/hong kong/1073/99 (h9n2) vaccines were clinically evaluated in the uk. 99 60 adults were randomly assigned two doses, administered 3 weeks apart, of 7·5, 15, or 30 g h9 haemagglutinin content. although well tolerated, whole-virus vaccine was more reactive, in keeping with h1n1 vaccines. there was little detectable crossreactive immune response to an antigenically distinct g9 h9 virus (unpublished findings). more than 40% of the prevaccination serum samples showed reactivity to h9n2 by neutralisation and haemagglutinin-inhibition, suggesting preexisting crossreacting antibody from exposure to earlier haemagglutinins. it is unlikely that h9 influenza has circulated widely in the uk. further serological testing correlated h9 reactivity with antibody responses to h2, but not h1 or h3 haemagglutinin. people with baseline reactivity to h9 were born before 1969, and thus had been potentially exposed to h2 during its period of circulation in human beings. subjects were immunologically divided into naive and primed recipients. in truly naive subjects, one dose of either vaccine was poorly immunogenic. although whole-virus vaccine was more immunogenic than subunit vaccine, two doses still left a significant number of vaccinees with serological responses below the protective threshold (table 3) . among primed individuals, one dose of either vaccine boosted anti-h9 responses, fulfilling cpmp criteria. since the second dose was of questionable value, to preserve limited vaccine supplies during the first wave of an emerging pandemic, different schedules in different populations could be considered. a german study among 18-60 year-olds reported one dose of 15 g whole-virus vaccine a/hong kong/1073/99 (h9n2) capable of fulfilling at least one cpmp criterion and that a second dose of vaccine significantly improved responses. 71 however, age-related responses or the effect of pre-existing reactivity to h9n2 were not analysed. in keeping with the h2n2 experience, alum-adjuvant allowed a reduction in h9 content to 1·3 g per dose while maintaining immunogenicity. intranasally delivered live, attenuated cold-adapted influenza vaccines elicit systemic and local mucosal immune responses and display protective efficacy. 100 attenuated cold-adapted strains are generated by reassortment between a wild-type virus expressing target haemagglutinin and neuraminidase, and a cold-adapted donor such as influenza a/ann arbor/6/60 (h2n2). donor strains are cold adapted, temperature-sensitive, and attenuated. these properties are associated with polygenic mutations. these live attenuated viruses display high levels of phenotypic and genotypic stability and are not transmissible to close seronegative contacts. 101 both attenuated h5n1 and h9n2/ann arbor cold-adapted recombinant viruses have been generated and are seen to be non-pathogenic in mammalian and chicken models. 98, 102 concerns over the generation of a reassortant between a live virus vaccine containing an avian influenza virus and a co-infecting human strain, and the possibility of spontaneous genetic change may limit the use of such vaccines in the interpandemic period. while current intramuscular influenza vaccines are effective at inducing relatively strainspecific serum haemagglutination-inhibition igg, they are poor at stimulating secretory iga in nasal wash fluid. 103, 104 as secretory iga exhibits potential heterotypic crossreactivity to influenza virus strains at the point of entry, 104,105 live attenuated virus vaccines may offer wider protection against vaccinedrifted variants that could be advantageous once a pandemic is underway. specific influenza antiviral agents are available for early treatment and prophylaxis of influenza. 106 the adamantanes, amantadine and rimantadine, have been available for more than 30 years and inhibit strains of influenza a including nonhuman subtypes. however, rapid emergence and transmission of drug-resistant virus after treatment may render prophylaxis ineffective. the genetic basis for resistance seems to be single aminoacid substitutions in the viral m2 ion channel. the h5n1 strains isolated from poultry and human cases in 2004 had genotypic changes in the m2 gene associated with resistance, 29 suggesting these agents would be of little clinical value should these strains become capable of humanto-human transmission. neuraminidase inhibitors, such as zanamivir and oseltamivir, are effective in prevention studies 106 and are highly active against a broad range of influenza a viruses of both human and avian origin, including amantadine-resistant strains. although strains with reduced susceptibility to neuraminidase inhibitors have been isolated after sequential passage of virus in presence of drugs, clinically significant resistant strains have not, as yet, been identified. antiviral drugs may be of benefit in protecting individuals in essential services whilst waiting for an effective vaccine to be prepared; however, supply and cost issues would limit their effect on the course of a pandemic. a sufficiently large supply of antivirals to curb pandemic influenza would require international or need for robust surveillance programmes in human and animal populations and sharing of information between animal and human surveillance systems surveillance information to be open and shared in a timely fashion to assess potential threats potential pandemic strains come from animal reservoir. improved understanding of the antigenic and molecular associations between potential pandemic strains of same subtype improved understanding of immunogenicity against drifted avian influenza strains is required as the ability to generate broad crossprotective immunity is desirable in vaccine candidate. intellectual property rights of attenuated viruses produced by reverse genetics must be addressed in advance because licences for commercial use may be required vaccine virus candidate needs to be able to grow well in eggs (or approved cell culture) to improve ability to respond rapidly to emerging threat improved understanding of virulence determinants in mammalian models to be able to attenuate viruses used for vaccine manufacture safety of virus handling for workers involved in preparation of vaccine to assess and approve mammalian cell lines of human vaccine quality ensure that reagents from animal sources are transmissible spongiform encephalopathies compliant "reverse genetics" generated viruses are labelled as genetically modified organisms-implications for national and local regulatory authorities clinical assessment of vaccines derived by reverse genetics ability to organise antigenicity studies rapidly in response to emerging threat may require prepared approved protocols that can be readily adapted. improvement in assessment of antibody responses to avian influenza vaccines to establish licensing criteria standardisation of assays for detection of neutralising antibody to avian influenza establish correlates of immune protection of neutralising antibody whole-virus vaccine more immunogenic than subunit or split-product vaccine in immunological naive populations (h1n1, h9n2) two doses of vaccine required in immunologically naive populations, the first to prime and the second to boost responses (h1n1, h2n2, h5n3, h9n2) in primed populations, a single dose of vaccine can potentially induce responses associated with protection (h1n1, h5n3, h9n2) addition of adjuvants such as mf59 and aluminium salts have the potential to significantly enhance immunogenicity and spare antigen use (h5n3, h9n2 and h2n2) avian haemagglutinin (h5 and to a lesser extent h9) seems to be less immunogenic in people than h1 and h2 assessment of antibody responses to avian influenza may require additional serological methods other than the standard haemagglutinininhibition test (h5n3) potential crossreactivity with pre-existing antibodies complicates interpretation of immune responses in people (h9n2) need to develop understanding of improving vaccine candidates to enhance heterosubtypic crossreactivity and protection need to assess vaccine candidates in advance of pandemic to identify difficulties and establish dosing schedules in different populations national stockpiling before the onset of such an event; this would require considerable expense, and vaccination is likely to remain the principal means of combating pandemic influenza. although pandemic planning and understanding is greater since the first h5 outbreak in 1997, our ability to respond rapidly remains less than optimal. the 2004 asian h5n1 epizoonotic outbreak indicates the urgent need for vaccines against avian influenza viruses. however, regulatory and safety considerations confront their development (panel 2). it is necessary to improve our understanding of the virulence determinants in mammalian systems to be able to attenuate viruses to select appropriate and safe vaccine strains that can generate broad crossreactivity. national and international authorities must urgently confront regulatory issues to allow production and clinical assessment of newly generated virus vaccine candidates. there are important observations from our clinical experience with vaccines for pandemic influenza (panel 3). despite increased reactogenicity, the greater immunogenicity of whole-virus vaccines could be beneficial in a pandemic. vaccines containing avian h5 and h9 haemagglutinin seem to be less immunogenic in human beings than vaccines based on h1 and h2 haemagglutinin. whether this is a general event associated with avian subtypes is at present unclear. enhancement with mf59 or alum salts may provide best antigen use and enhance immunogenicity. overall, so far, clinical trials of avian influenza vaccine candidates have given disappointing results. it remains to be seen how plasmid-derived reverse-genetics influenza vaccines will perform once regulatory hurdles have been overcome. we have no conflicts of interest. chronicle of influenza pandemics avian influenza (h5n1) in 10 patients in vietnam the textbook of influenza evolution and ecology of influenza a viruses characterisation of a novel influenza haemagglutinin h15: criteria for determination of influenza a subtypes 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this review. search terms were "avian influenza", "influenza vaccine'', "pandemic influenza", "h5 influenza", "h9 influenza", "h7 influenza", "influenza vaccines", "pathogenesis" and "virulence". english language articles were reviewed. key: cord-339638-yrxoj1hl authors: goldman, ran d.; mcgregor, sophie; marneni, shashidhar r.; katsuta, tomohiro; griffiths, mark a.; hall, jeanine e.; seiler, michelle; klein, eileen j.; cotanda, cristina parra; gelernter, renana; hoeffe, julia; davis, adrienne l.; gualco, gianluca; mater, ahmed; manzano, sergio; thompson, graham c.; ahmed, sara; ali, samina; brown, julie c. title: willingness to vaccinate children against influenza after the covid-19 pandemic date: 2020-08-07 journal: j pediatr doi: 10.1016/j.jpeds.2020.08.005 sha: doc_id: 339638 cord_uid: yrxoj1hl objectives: to determine factors associated with parents who plan to vaccinate their children against influenza next year, especially those who did not vaccinate against influenza last year using a global survey. study design: a survey of caregivers accompanying their children 1-19 years-old in 17 pediatric emergency departments (ed) in six countries at the peak of the covid-19 pandemic. anonymous online survey included caregiver and child demographic information, vaccination history and future intentions, and concern about the child and caregiver having covid-19 at the time of ed visit. results: of 2422 surveys, 1314 (54.2%) caregivers stated they plan to vaccinate their child against influenza next year, an increase of 15.8% from the prior year. of 1459 caregivers who did not vaccinate their children last year, 418 (28.6%) plan to do so next year. factors predicting willingness to change and vaccinate included child’s up-to-date vaccination status (adjusted odds ratio (aor)=2.03, 95% confidence interval (ci) 1.29 – 3.32 p = .003); caregivers’ influenza vaccine history (aor=3.26, 95% ci 2.41 – 4.40 , p< 0.010), and level of concern their child had covid-19 (aor=1.09, 95% ci 1.01 – 1.17, p=0.022). conclusions: changes in risk perception due to covid-19, and prior vaccination, may serve to influence decision-making among caregivers regarding influenza vaccination in the coming season. in order to promote influenza vaccination among children, public health programs can leverage this information. to determine factors associated with parents who plan to vaccinate their children against influenza next year, especially those who did not vaccinate against influenza last year using a global survey. pediatric emergency departments (ed) in six countries at the peak of the covid-19 pandemic. anonymous online survey included caregiver and child demographic information, vaccination history and future intentions, and concern about the child and caregiver having covid-19 at the time of ed visit. of 2422 surveys, 1314 (54.2%) caregivers stated they plan to vaccinate their child against influenza next year, an increase of 15.8% from the prior year. of 1459 caregivers who did not vaccinate their children last year, 418 (28.6%) plan to do so next year. factors predicting willingness to change and vaccinate included child's up-to-date vaccination status (adjusted odds ratio (aor)=2.03, 95% confidence interval (ci) 1.29 -3.32 p = .003); caregivers' influenza vaccine history (aor=3.26, 95% ci 2.41 -4.40 , p< 0.010), and level of concern their child had covid-19 (aor=1.09, 95% ci 1.01 -1.17, p=0.022). changes in risk perception due to covid-19, and prior vaccination, may serve to influence decision-making among caregivers regarding influenza vaccination in the coming season. in order to promote influenza vaccination among children, public health programs can leverage this information. year, including children (1) . despite the wide availability of seasonal influenza vaccines and clear guidelines for who should be immunized, vaccine uptake remains low in most countries (2) . with the unprecedented coronavirus sars-cov-2 illness, now called the coronavirus disease 2019 (covid19) , which currently lacks a publicly available vaccine, public health authorities worldwide recommended numerous strategies to reduce spread, including reducing physical encounters and wearing masks. these measures have had variable success in curtailing the spread of the virus around the world. vaccines are likely to provide the best protection from contracting the illness and there are currently more than 100 projects centered on the development of a vaccine and many have entered clinical trials (3). there is a high likelihood that community transmission of covid-19 will continue into the next influenza epidemic (4), complicating diagnoses and further increasing the burden on health care systems (5) . to mitigate these issues, vaccinating large parts of the population against influenza in late 2020 is a key goal of public health officials. the objective of this timely survey study was to determine, during the covid-19 peak pandemic, caregiver intentions towards influenza vaccination of their children, as well as themselves. in order to better understand how covid-19 has influenced attitudes towards j o u r n a l p r e -p r o o f influenza vaccination, we specifically aimed to describe characteristics of caregivers who intend to immunize their children in 2020-2021 despite the child not receiving influenza immunization in the prior year. understanding caregivers' attitudes can help public health officials plan targeted messaging to parents in order to promote influenza vaccination in the upcoming season. this study is part of a larger covid-19 parental attitude study (covipas) study, surveying caregivers of children presenting for emergency care, in the era of covid-19. caregivers who arrived to 17 pediatric emergency departments (ed) in the usa (seattle, tacoma, los angeles, dallas, atlanta), canada (vancouver, toronto, saskatoon, edmonton, calgary), israel (shamir), japan (tokyo), spain (barcelona), and switzerland (zurich, bern, geneva, bellinzona) were asked to participate, using posters in waiting areas and patient rooms. for infectious control purposes, respondents used their own smartphones to complete the survey by logging into a secured online platform based on redcap metadata-driven software (vanderbilt university). several irbs (in switzerland and spain) provided a waiver of consent such that responding to the survey was considered consent to participate. languages available to complete the study were english, french, german, italian, spanish, hebrew, and japanese. although sites joined recruitment in a staggered fashion, surveys were obtained between march 27 and june 30, 2020. only one caregiver per family was asked to complete the survey. vaccination against influenza is available for children over 6 months in all countries that this survey took place. the study-specific questionnaire was developed to include questions regarding demographic characteristics, information about the ed visit, and attitudes around covid-19. respondents were also asked about their child's vaccination history excluding influenza. children were presented in this report, were evaluated a priori by 10 individuals representing the target group and 10 healthcare providers working in the ed environment. the final version of the survey is based on the feedback and test clarity generated from these 2 groups. basic descriptive statistics and frequencies were used to describe all variables. we compared survey data from caregivers who stated whether they plan to immunize or not immunize their children against influenza in the coming year. we then compared caregivers that did not immunize for influenza their children last year and compared those that said they do or do not plan to immunize their children for influenza next year. to determine which factors were significantly associated with caregiver decision-making to vaccinate next year despite not doing so last year, we used bivariate analyses: mann-whitney test was used for comparing nonnormal continuous variables, independent t-tests were used for comparing normallydistributed continuous variables, and chi-squared or fisher exact tests were used for categorical variables. multivariable logistic regression was used to estimate the adjusted odds ratio of agreeing to immunize against influenza in the coming year using all the variables that showed a level of p<0.100 in the bivariate analyses. all analyses were conducted with r version 3.5.1. a p-value less than 0.05 in the multivariate analysis was considered statistically significant. results a total of 2785 surveys were completed online. seven (0.3%) were excluded because the surveys were incomplete, seven (0.3%) were completed by the patient, 343 (12.3%) were for a patient less than one year of age, three (0.1%) for patients over 19 years old, and three (0.1%) with an unspecified patient age ( figure 1 ). some participants abstained from answering certain questions, accounting for a small number of unknown responses to each question. the final study sample included 2422 respondents: 2350 parents (97.0%), 67 other caregivers, and 5 respondents who did not specify their relationship to the child. median age of caregivers was 40.0 (standard deviation (sd) = 7.6) years and median age of the child was 8.3 (sd = 4.6) years. a total of 1314 (54.3%) respondents stated they intend to vaccinate their children against influenza in the coming 12 months, an increase of 15.9% compared with those that reported an influenza vaccine for their child in the past 12 months ( figure 1 ). as many as 42/2422 (1.7%) respondents did not specify their intention to vaccinate their child and 1394/2393 (58.3%) respondents stated they planned to obtain the vaccine for themselves next influenza season, compared with 974 (40.6%) in the past year (table i) . table 1 provides demographic information including a comparison between caregivers who plan to vaccinate for influenza in the coming season and those who do not. the highest likelihood of planning to vaccinate the child next year was if the caregiver planned to vaccinate themselves in the upcoming year (1213/2422; 50.1% of children). caregivers were more likely to suggest they will immunize next year if they had education beyond high school, if the child had a chronic illness or took medications regularly, if the child was up-to-date on their vaccines j o u r n a l p r e -p r o o f other than influenza according to their country-specific vaccination schedule, or if the child or caregiver received influenza vaccine the prior year. of 1459 caregivers who did not vaccinate their children against influenza in the last year, 418 (28.6%) indicated they plan to vaccinate next season, 1025 (70.3%) did not vaccinate last year and will not vaccinate this year, and 16 (1.1%) abstained from answering. only 38/2422 (1.6%) vaccinated their child last year but do not plan to do so next year. most caregivers were not concerned about their child having covid-19 when coming to the eds in the six countries where this survey took place, and we found a significant correlation between level of concern and plan to vaccinate against influenza (p-value= 0.037; figure 2 [available at www.jpeds.com]). table 2 describes characteristics of caregivers who did not vaccinate the child last year, but plan to vaccinate their child against influenza in the coming year. caregivers were more likely to change from non-vaccination last year to vaccination in the coming year when they had education more than high-school, they took the vaccine themselves or planning to get vaccinated themselves next year, had a child with an up-to-date vaccination schedule excluding influenza vaccines or were worried their child may have covid-19 or influenza during the visit in the ed. in the multivariate logistic regression analysis (table 3) behavioral changes related to covid-19, including social distancing, hand washing, and wearing a mask, will impact influenza spread in the coming year (3) and it is unclear how relaxation of social distancing measures will influence the spread of influenza. co-infection of covid-19 and other respiratory pathogens, including influenza, occurred in a significant subset of covid-19 patients (8, 9) and a case report from china describing co-infection with covid-19 and influenza a virus represent the difficulty in differentiating other causes of respiratory illness from covid-19 (10) . the director of the us center for disease control predicted that "we're going to have a flu epidemic and coronavirus epidemic at the same time" and that the combination of the two will be "more difficult and potentially complicated" (4). combined influenza and covid-19 pandemics could result in considerable morbidity and mortality, stressing the health system j o u r n a l p r e -p r o o f (3, 7) and a global interest (as seen through google trends) in pneumococcal and influenza vaccines during february-march, 2020 of the covid-19 pandemic ( has been reported (11) . in our global survey of primarily parents attending pediatric eds, a significant shift in parents' plan to vaccinate against influenza in the season following covid-19 pandemic was noted. a total of 54.3% survey respondents plan to vaccinate for influenza and 29.0% (418/1443) of caregivers that did not vaccinate last year report a change in plan to do so next year, adding to the protection of their children and reducing the chance of transmission to others. we report that caregivers who plan to vaccinate themselves are very likely to vaccinate their children and predictors for caregivers to plan to vaccinate after not doing so last year include children that have up-to-date vaccinations other than influenza, if the caregiver received influenza vaccine last year, and if they were worried their child had covid-19. seasonal influenza epidemics result in tens of millions of cases, and we have recently reported that caregivers in 14 eds are likely to provide their children with a covid-19 vaccine if it becomes available (12) . ensuring influenza vaccine acceptance and uptake in children may also serve public health in promoting covid-19 vaccines, when those become available (7). more than 70 independent barriers are associated with vaccine hesitancy and include psychological barriers (such as perceived risk, utility, and social benefit); contextual barriers (such as access to health care services); and lifestyle barriers (such as smoking, drinking, and physical activity) (13) . trust in healthcare providers' advice and mainstream medicine, influence of social network and social norms, knowledge sources about vaccines, and general views toward health are key factors in parental vaccine decision-making (14) . vaccine risk-perception j o u r n a l p r e -p r o o f of parents is often complicated by cognitive bias and personal experience (15) and in our cohort, caregivers were most likely to report plans to vaccinate their children against influenza next year if they plan to vaccinate themselves, meaning they trust the medical system and more specifically put faith in vaccines. of interest, caregivers put more value and enhance the perceived risks associated with taking action, such as a child having a severe reaction to a vaccine, than the risk of an omitted action, like the chance of contracting a disease (16) . it is important for primary care providers to highlight the safety and efficacy of the flu vaccines to parents. past behavior is a strong factor in vaccinating children and those having been vaccinated in the past against seasonal influenza being more likely to be vaccinated against pandemic influenza (17) . in our study, the influenza vaccination status of a child last year was strongly associated with a caregiver's plan to vaccinate next year (p <0.001). similarly, in a study from england, among 1001 parents with half their children vaccinated to influenza, vaccine uptake was associated with the child having previously vaccinated against influenza (18) . understanding factors that influence a change in influenza vaccine behaviour from year-to-year may guide public health efforts to increase uptake. fogel and hicks recently coined the term "flu-floppers," describing patients who received the influenza vaccine in some years but not others. they suggested that many appear to randomly alternate vaccination status from year to year (19) . in a year of covid-19 pandemic it is likely that public health worries influence parents to plan on vaccinating their children and only 4% of children vaccinated last year are likely not to be vaccinated next year (38/930 families) compared w 28.6% (417/1460) "flipping" towards vaccinating. those who stop yearly influenza vaccinations for their children most commonly cited a perceived lack of effectiveness of the vaccine (29%, n=41) (19) . public health officials will need to ensure clear messaging on the safety of influenza vaccines next year, especially in face of covid-19, to influence parental decision making to convert into vaccinating children. in a web-based survey of 500 parents from an online panel representative of the us population, the main drivers of parents' decision to vaccinate their child against influenza were prevention of influenza, reduction of influenza symptoms, and doctor recommendation; barriers to vaccination included the risk of adverse effects and the perceived low risk of influenza (20) . similarly, a review of 64 studies reported an association between vaccination uptake and perceived vaccine safety, general positive attitudes towards vaccination, positive vaccine recommendations, social influences and trust in the healthcare profession, and perceiving fewer practical difficulties with vaccination (21) . we similarly found that caregivers vaccinating their children according to the schedule, and thus likely have trust in the idea of vaccination, are likely to accept the influenza vaccination for their child next year. level of caregiver concern that the child had covid-19 was associated with the change in action towards planning to vaccinate children against influenza. in a systematic review of 37 studies involving the 2009 h1n1 pandemic influenza outbreak, the degree of threat experienced and perceptions of vaccination as an effective coping strategy was associated with stronger intentions and higher uptake of vaccination and concern about the disease as well as believing oneself to be at risk to h1n1 influenza were important factors (17) . fear of the child j o u r n a l p r e -p r o o f being sick with covid-19 is a significant predictor of caregivers changing their mind to start vaccinating next year, which is supported by the fact that correlations were found between online searches for influenza vaccines and covid-19, especially in "hotspots" of covid-19, and increased interest in influenza vaccine compared with similar time periods in non-pandemic years (11) . although caregivers with higher than high school education are more likely to vaccinate their children against influenza (79% vs 71.4%, p<0.01), including those not vaccinating last year (78.5% vs 71.4%, p=0.005), when other factors were assessed in the multivariate analysis, higher education was associated with lower likelihood of families changing their mind to start vaccinating (or 0.71, 95% ci 0.52-0.96), p=0.028). level of parental education is associated with both high and low uptake of vaccines (22) , likely due to education serving as a surrogate marker for other factors impacting adherence. our study has a number of limitations. first, the population of caregivers responding to the survey does not represent all parents in the sites where the study was conducted, as we administered the survey in a pediatric ed setting and only a subset of families (likely <5% of visitors to 17 the pediatric eds) filled out the survey. we also relied on caregiver possession of a smartphone/tablet/computer to complete the survey online, which may have limited a small percentage of caregivers from responding. secondly, we relied on the accuracy of caregiver responses to the status of both influenza and non-influenza vaccinations, because no patient records were checked for confirmation. finally, as in any survey, caregivers may share their j o u r n a l p r e -p r o o f opinions about vaccinations, but act differently once cities are back to a more normal activity, which highlights the importance of public health measures to encourage and ensure adequate vaccination uptake. in summary, we report propensity of caregivers to vaccinate their children against influenza at a higher rate in the next influenza season, with 29% of those that did not vaccinate last year reporting a plan to do so next year. caregivers who plan to vaccinate themselves, who received influenza vaccine last year, who were worried their child had covid-19, and whose children j o u r n a l p r e -p r o o f centre for disease control and prevention. estimated influenza illnesses, medical visits, hospitalizations, and deaths in the united states-2018-2019 influenza season us department of health & human services centre for disease control and prevention. flu vaccine coverage, united states 2018-19 influenza season us department of health & human services covid-19 and the next influenza season the covid-19 vaccine development landscape a looming double threat parental hesitancy about routine childhood and influenza vaccinations: a national survey the dual epidemics of covid-19 and influenza: vaccine acceptance, coverage, and mandates co-infection in sars-cov-2 infected patients: where are influenza virus and rhinovirus/ enterovirus? rates of co-infection between sars-cov-2 and other respiratory pathogens co-infection with sars-cov-2 and influenza a virus in patient with pneumonia silver lining of covid-19: heightened global interest in pneumococcal and influenza vaccines, an infodemiology study paper 2 -covid vaccine -not yet published barriers of influenza vaccination intention and behavior -a systematic review of influenza vaccine hesitancy underlying factors impacting vaccine hesitancy in high income countries: a review of qualitative studies parental decision-making on childhood vaccination refinement of outcome bias measurement in the parental decision-making context factors associated with uptake of vaccination against pandemic influenza: a systematic review: vaccine psychological factors associated with uptake of the childhood influenza vaccine and perception of postvaccination side-effects: a cross-sectional survey in england flu-floppers": factors influencing families' fickle flu vaccination patterns parents' decision-making regarding vaccinating their children against influenza: a web-based survey a systematic review of factors affecting vaccine uptake in young children understanding vaccine hesitancy around vaccines and vaccination from a global perspective: a systematic review of published literature key: cord-333722-ndth5zne authors: liu, qiang; zhou, yuan-hong; yang, zhan-qiu title: the cytokine storm of severe influenza and development of immunomodulatory therapy date: 2015-07-20 journal: cellular & molecular immunology doi: 10.1038/cmi.2015.74 sha: doc_id: 333722 cord_uid: ndth5zne severe influenza remains unusual in its virulence for humans. complications or ultimately death arising from these infections are often associated with hyperinduction of proinflammatory cytokine production, which is also known as ‘cytokine storm'. for this disease, it has been proposed that immunomodulatory therapy may improve the outcome, with or without the combination of antiviral agents. here, we review the current literature on how various effectors of the immune system initiate the cytokine storm and exacerbate pathological damage in hosts. we also review some of the current immunomodulatory strategies for the treatment of cytokine storms in severe influenza, including corticosteroids, peroxisome proliferator-activated receptor agonists, sphingosine-1-phosphate receptor 1 agonists, cyclooxygenase-2 inhibitors, antioxidants, anti-tumour-necrosis factor therapy, intravenous immunoglobulin therapy, statins, arbidol, herbs, and other potential therapeutic strategies. newly emerging and re-emerging viral threats have continued to challenge medical and public health systems and incur economic costs to both individuals and countries. the influenza virus is a main cause of those threats and is responsible for millions of severe cases and 250 000-500 000 deaths each year. 1 the scenario can be even worse during a pandemic year. the most virulent influenza, the 1918 h1n1 spanish flu, infected approximately 5% of the world's population and killed 2%. 2 the case fatality rates for the 1957 h2n2 asian influenza, the 1968 h3n2 hong kong influenza, and the 2009 h1n1 pandemic influenza were reported to be lower, with an estimated rate of 0.2% or less. 3 most alarmingly, between 1997 and 2014, several unprecedented epizootic avian influenza viruses (e.g., h5n1, h7n9, and h10n8) crossed the species barrier to cause human death. they pose an increasing threat of human-to-human transmission. 4,5 these infections in humans are accompanied by an aggressive pro-inflammatory response and insufficient control of an anti-inflammatory response, a combination of events called 'cytokine storm'. in the event of influenza infection, the severity of disease is the result of the interplay between viral virulence and host resistance. in mild infection, the host has a limited or moderate resistance, so the disrupted homeostasis is restored rapidly. however, for infections caused by the 1918 h1n1 or the h5n1 influenzas virus, the resistance became hyperactive, resulting in an excessive inflammatory reaction known as the cytokine storm phenomenon. 6 several experimental studies and clinical trials suggested that cytokine storm correlated directly with tissue injury and an unfavorable prognosis of severe influenza. 7 however, our understanding of the mechanism that promotes a cytokine storm remains limited. in this review, we focus on the potential mechanisms responsible for severe influenza-induced cytokine storm and the therapeutic strategies that might be used to improve the clinical prognosis of these infections. viruses that proliferate within these cells can infect other cells, including alveolar macrophages. 9 inflammatory responses are triggered when infected cells die by apoptosis or necrosis. 3 the initial response of the organism to harmful stimuli is acute inflammation and is characterized by increasing blood flow, which enables plasma and leukocytes to reach extra-vascular sites of injury, elevating local temperatures, and causing pain. 3 the acute inflammatory response is also marked by the activation of pro-inflammatory cytokines or chemokines. 9,10 these pro-inflammatory cytokines or chemokines can lead to the recruitment of inflammatory cells. 9, 10 then, an increasing expression of inflammatory, antiviral, and apoptotic genes occurs accompanied by abundant immune cell infiltration and tissue damage 7, 11 (figure 1 ). at the same time, regenerative processes and resolution of the damage are initiated. in most cases, function can be completely restored by this reparative process. 12 however, for severe inflammation associated with cytokine storm, more serious pathological changes are observed, such as diffuse alveolar damage, hyaline membrane formation, fibrin exudates, and fibrotic healing. 10 these are signs of severe capillary damage, immunopathologic injury, and persistent organ dysfunction. 10 moreover, the severe inflammatory cytokines/chemokines can spill over into the circulation and result in systemic cytokine storms, which are responsible for multi-organ dysfunction. 7 the inflammatory response begins when the pathogen-associated molecular pattern (pamp) from the virus is recognized by the pattern recognition receptors (prrs) of innate immune cells. 13, 14 then, specific pro-inflammatory cytokines are expressed after the downstream signaling cascades of prrs are triggered by (1) viruses infect lung epithelial cells and alveolar macrophages to produce progeny viruses and release cytokines/chemokines (mainly contains interferons). (2) cytokine/chemokine-activated macrophages and virally infected dendritic cells lead to a more extensive immune response and the initiation of cytokine storm. (3) released chemokines attract more inflammatory cells to migrate from blood vessels into the site of inflammation, and these cells release additional chemokines/cytokines to amplify cytokine storm. cytokine storm q liu et al stimuli. 14 researchers have a great interest in exploring the association between polymorphisms of prrs and host susceptibility to cytokine storm, which may help explain why some individuals, but not others, seem relatively resistant to cytokine storm. 15 severe cytokine storm, with markedly higher levels of proinflammatory cytokines including interferons (ifns), tumor necrosis factors (tnfs), interleukins (ils), and chemokines, has been detected in patients hospitalized with severe influenza infections. 16 severe cytokine storm is rarely observed in seasonal and other mild influenza, 17 indicating that high cytokine/ chemokine levels correlate strongly with disease severity. the interferon family has a critical role in the innate immune response to viruses. 18 a number of proteins with antiviral or immunomodulatory properties are produced once the ifn signaling pathway is activated. 18 although overproduction of ifn in the early stage of infection likely leads to irreversible lung damage in h5n1-infected mice, the ifn signaling pathway may also be important in restricting the dissemination of h5n1 viruses. 18, 19 tnf-a is a key cytokine in cytokine storm and is likely to account for the escalation in severity. 12 however, tnf receptor 2/2 mice, or mice treated with anti-tnf-antibodies, have no changes in survival when compared with controls following a challenge with the h5n1 virus. 12 il-1 and il-6 are the main pro-inflammatory cytokines released by hosts during viral infections. il-1 is expressed in the early stages of infection, followed by an increasing expression of il-6. 7 il-1 receptor signaling is responsible for acute the immunopathology of tissue, and il-1 receptor 2/2 mice were shown to have a worse outcome after h5n1 infection. this suggests that the pathways are protective. 7 unlike pro-inflammatory cytokines, chemokines often have specific chemotactic activities that enable monocytes and tlymphocytes to migrate from blood vessels into the site of inflammation. 9 for example, il-8 and monocyte chemoattractant protein (mcp)-1 are major chemotactic factors for neutrophils and monocytes, respectively. it should be noted that the levels of il-8, interferon-induced protein (ip)-10, mcp-1, macrophage inflammatory protein (mip)-1, and monokines induced by ifn-c (mig) were abnormally elevated in some fatal cases of h5n1 influenza infection and that il-8 had the highest level among them. 20 mip-1 2/2 mice exhibit inefficient viral clearance but reduced mortality and lung damage. 9 several studies 9,21 also investigated the roles of several chemokine receptors, such as the mip-1 receptor ccr5 and the mcp-1 primary receptor ccr2, in severe influenza infections. interestingly, ccr5 2/2 mice displayed an excessive inflammatory response and increased mortality, while ccr2 2/2 mice displayed a decreased inflammatory response and mortality but developed a significantly elevated viral load. 21 this evidence partly suggests that the pathology of severe influenza is mediated by cytokine response but not viral load. generally, the cytokine response induced directly by the influenza virus is a sprawling network, which is amplified by autocrine and paracrine mediator cascades. pathways associated with prrs, ils, ifns, tnfs, cyclooxygenase (cox)-2, and c-jun n-terminal kinase (jnk) are activated to induce the transcription of nf-kb and the formation of inflammasomes. 7 a cytokine/chemokine-driven feed-forward inflammatory circuit may be responsible for the escalation of cytokine storm. 22 however, the key factors of this network, especially those specific to the pathogenesis of severe influenza, are still unknown. by using network-based systems biology approaches, jin et al. 22 have made a successful attempt in elucidating the network properties of severe influenza. their study demonstrated that tlr2, il-1b, il-10, and nuclear factorkappa b have obvious differences between the normal and inflammatory networks. 22 complications arising from severe influenza are associated with inflammatory cells. monocytes/macrophages are the main cells recruited into the alveolar space as an initial response to viral infection. 3,9 they then increase their cytokine production and chemoattract additional immune cells into the lesion area. 3,9 nevertheless, they are also susceptible to influenza viral infection. depleting the monocytes/macrophages does not prevent immunopathology, indicating their important role in viral clearance. 3, 9 both cd4 and cd8 t cells are responsible for the immunopathology and viral clearance of infection. a lethal lung injury can be triggered by the transfer of antigen-specific cd8 t cells into transgenic mice expressing the influenza ha antigen. however, a lethal lung injury cannot be triggered in mice with defects in the epithelial early growth response-1 (egr-1), suggesting that egr-1 is a critical regulator of the immunopathology of cd8 t cells. 23 cd4 t cells (including th1, th2, th17, and treg) have been identified to contribute to both immunopathology and viral clearance of influenza infection. severe respiratory disease of influenza is often characterized by the early secretion of th1 and th17 cytokines. 24 tregs (regulatory cd4 (1) foxp3 (1) t cells) are key managers in controlling the degree of cellular immune responses to viral infections. particularly, the proliferation of memory cd8(1) cells can be effectively controlled by the memory tregs in an ag-specific manner that is mhc class ii dependent. 25 as the outcome of severe influenza is determined by both viral virulence and host resistance, the use of immunomodulatory therapy in combination with conventional antiviral therapy is highly warranted. in fact, several studies have confirmed that the mortality and organ injury of severe influenza can be reduced by immunomodulatory agents, with or without the combination of antivirals. moreover, many of them are relatively inexpensive and easily produced drugs, which could potentially be widely used in an influenza pandemic. here, we review the properties of these agents (table 1) . corticosteroids are a class of steroid hormones that exhibit anti-inflammatory activity via binding to the cytoplasmic corticosteroid receptor, which regulates transcription of antiinflammatory genes. 26 thus, corticosteroids have been widely used for anti-inflammatory treatment. during the 2009 h1n1 influenza pandemic, nearly 40% of patients in france were treated for acute respiratory distress syndrome (ards) using adjuvant systemic corticosteroids. 27 during the 2013 h7n9 avian influenza outbreak in china, 62.2% of patients received systemic corticosteroid treatment. 28 however, the evidence supporting the use of corticosteroids in severe influenza is inconclusive. an experimental study has shown that mice infected with h5n1 influenza have a similar mortality between the corticosteroid-treated group and the control group. 29 a clinical trial even identified that systemic corticosteroids were responsible for an increased long-term mortality. 27 in contrast, another independent research study reported that systemic use of corticosteroids alleviated the 2009 h1n1 pandemic influenza-associated pneumonia without adverse outcomes. 30 thus, the use of corticosteroids for severe influenza is controversial and still needs further observations. peroxisome proliferator-activated receptors (ppars), including ppar-a, ppar-b, and ppar-c, are critical regulators of inflammation. the ppar-a agonist, gemfibrozil, has been proposed to treat severe influenza due to ability to inhibit tnf, il-6, and ifn-c. 31 however, another research study 32 reported that gemfibrozil administered 48-h post-infection had no effects on the mortality of h5n1 avian influenza-infected mice. this suggests that the pharmacological mechanism of gemfibrozil to treat severe influenza still needs further characterization. there is little research on the use of ppar-b agonists to treat severe influenza, perhaps focusing on their trophic effects on oligodendrocytes in vitro. only bezafibrate was shown to have partial protection in patients with influenza-associated encephalopathy. 33 however, ppar-c agonists (e.g., rosiglitazone and pioglitazone) are considered to be the most promising candidates to improve the clinical outcome of severe influenza. 26 these thiazolidinediones can not only downregulate the inflammatory response to viral pneumonia but also increase the survival of influenza-infected mice. 34 moreover, the benefits of ppar-c agonist treatment were found to be higher than gemfibrozil. 26 in addition, a natural ppar-a and ppar-c agonist, biochanin a, which is extracted from red clover, has been confirmed to have similar immunomodulatory effects as gemfibrozil for the treatment of influenza in vivo. 35 3.3. sphingosine-1-phosphate receptor 1 agonists in recent years, there has been an increasing interest in developing novel agents with anti-immunopathologic injury activity through the sphingosine-1-phosphate (s1p) receptor pathway. 36 five specific s1p receptors have been found to regulate downstream signaling pathways. however, only s1p1, which is located mainly on pulmonary endothelial cells, exhibits cytokine-storm-blunting activity by suppressing both innate cellular and cytokine/chemokine responses. 36 for example, cym-5442 and rp-002 have been reported to protect mice from lethal infection with severe influenza by blunting cytokines and innate immune cell recruitment. 8 particularly, in murine models infected with the 2009 h1n1 pandemic influenza, s1p1 receptor agonists alone reduced over 80% of deaths from lethal infection compared to 50% protection offered by the antiviral neuraminidase inhibitor, oseltamivir. 36 furthermore, a combined therapy with the two agents can achieve an optimal protection of 96%. 36 this is by far the most promising result in improving the outcome of severe influenza using an immunomodulatory strategy. selective cox inhibitors, such as celecoxib and mesalazine, have been widely used in clinics for their antipyretic, analgesic, and anti-inflammatory properties. the monotherapy of celecoxib in a murine model of influenza does not considerably modulate disease severity. 37 however, the use of cox-2 inhibitors in combination with neuraminidase inhibitors has been shown to improve the survival of mice infected with h5n1 influenza. 32 a triple combination therapy of zalamivir, celecoxib, and mesalazine significantly reduced the mortality and levels of cytokines/chemokines of infected mice. 32 these data demonstrate that cox-2 inhibitors may provide additional benefits when combined with antivirals. as reactive oxygen species (ros) play a central role in inflammatory responses and viral replication, antioxidants that exert antiviral and anti-inflammatory effects may also be effective for the treatment of cytokine storm induced by severe influenza. 38 n-acetylcysteine (nac), a modified form of the amino acid cysteine, was shown to inhibit both h5n1 replication and h5n1-induced production of pro-inflammatory molecules (e.g., il6, ccl5, cxcl8, and cxcl10) in lung epithelial cells. 39 glycyrrhizin, an inhibitor of hydroxysteroid dehydrogenase, was shown to inhibit h5n1 replication and pro-inflammatory gene expression. 40 interestingly, an investigation showed that vitamin c was beneficial for patients suffering from severe avian influenza. 41 in fact, many plant-derived antioxidants (e.g., polyphenol, flavonoids, etc.) could also reduce the damage of epithelial cells and the mortality of mice caused by lethal influenza. [42] [43] [44] however, current evidence indicates that monotherapy using antioxidants had a limited effect on cytokine storm, and a combination with antivirals would still be needed. 45 3.6. anti-tnf therapy although we still do not fully understand the complex nature of cytokine storm, tnf is considered to be a key cytokine for acute viral diseases (e.g., influenza virus, dengue virus, and ebola virus). 7 indeed, experimental studies have shown that tnf not only affects the balance of the local microenvironment, but it also exerts broad systemic effects after entering into the circulation. 46 thus, anti-tnf strategies may be a reasonable way to treat severe influenza. studies have reported that treatments using tnf-neutralizing monoclonal antibodies or soluble tnf receptor fusion proteins can reduce the cytokine production and inflammatory cell infiltrates in influenzainfected murine lungs. 47, 48 however, no improved survival rates were observed in these studies. 47, 48 thus, the evidence for the clinical use of anti-tnf therapy in severe influenza is currently inconsistent. further clinical trials to evaluate the efficacy of anti-tnf strategies are still needed. intravenous immunoglobulin (ivig) therapy uses concentrated globulin preparations from pooled human plasma for the treatment of acute infections. the mechanism by which ivig suppresses harmful inflammation has not been definitively identified. it is believed to involve multiple immunomodulatory effects by blocking fc receptors, which are associated with tolerance to self and severity of the inflammatory state. 49 this strategy has been used in the treatment of viral-induced cytokine storm and was confirmed to have improved the outcome in infections, such as sars and the 2009 h1n1 pandemic influenza. 50,51 a meta-analysis suggested that early administration of blood products could have reduced anywhere from 26% to 50% of patients' deaths from pneumonia during the 1918 h1n1 influenza pandemic. 52 evidence of a beneficial effect of ivig therapy has been obtained in the 2009 h1n1 influenza infections. a multivariate analysis of the 22 patients who received either hyperimmune or normal ivig within five days of symptom onset found that hyperimmune ivig treatment was independently responsible for the reduced mortality of infection. 50 these results suggested that passive immunotherapy with hyperimmune globulin is a potential strategy for the treatment of severe influenza. some inexpensive generics, such as statins, angiotensin receptor blockers (arbs), and angiotensin-converting enzyme inhibitors (aceis), were also proposed as potential immunomodulatory agents to reduce inflammation caused by the influenza virus. 53 statins are competitive inhibitors of the enzyme 3hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase. they exert multiple immunomodulatory effects, such as (i) modulating the activation of immune effector cells via inhibition of ros, (ii) antagonizing high mobility group box 1 protein (hmgb1) to inhibit pro-inflammatory cytokine expression, and (iii) suppressing ccr2 gene expression. 53 in murine models infected with h1n1, h3n2, or h5n1 influenza virus, the combination of statins and caffeine alleviated the lung injury, inhibited viral replication, and appeared to have similar efficacy as oseltamivir or ribavirin. 54 however, an independent research study showed that statins cannot prevent patients infected with the 2009 h1n1 pandemic influenza from developing severe disease. 55 no direct evidence was shown that aceis or arbs might be effective in the therapy of severe influenza-induced cytokine storm. however, these agents can inhibit the inflammatory response induced by angiotensin ii and improve the survival of mice in several experimental models of acute lung injury. 56 furthermore, clinical trials also confirmed that these agents can reduce the risk of pneumonia and pneumonia-related mortality. 57 thus, both the pharmacodynamic effects and mechanisms of these generics deserve further research. 30 ineffective as monotherapy in h5n1 influenza-infected mice. 29 increased long-term mortality in influenza-infected patients with pneumonia. 27 ciglitazone and troglitazone decreased the mortality of influenza-infected mice. 34 bezafibrate partially protected patients with influenza-associated encephalopathy. 33 gemfibrozil also decreased the production of il-1, il-6, and ifn-c, but has no effects on the mortality of h5n1-infected mice when administered 48-h post-infection. 31,32 s1p1 receptor 1 agonists reduced mortality of 2009 pandemic h1n1 influenza-infected mice over 80%, compared with 50% protection of oseltamivir. 36 ineffective as monotherapy in h5n1 influenza-infected mice, while effective when in combination with neuraminidase inhibitors. 32 antioxidants n-acetylcysteine and glycyrrhizin inhibited h5n1 replication and pro-inflammatory gene expression in vitro 39,40 but ineffective as monotherapy in vivo. 45 effective in reducing the cytokine production and inflammatory cell infiltrates in influenza-infected murine lung but ineffective in improving survival of infected mice. 47 combined with caffeine or antivirals, alleviated lung injury and inhibited viral replication in h1n1, h3n2, and h5n1 influenza-infected mice. 54 ineffective in protecting 2009 pandemic h1n1-infected patients. 55 increased survival of influenza-infected mice by 75%. 58 increased survival for influenza-infected mice by 40%, while a combination with pioglitazone improved survival by 60%. 59 ox40 imparted a survival signal to the t cell via upregulating anti-apoptosis gene expression and eliminated weight loss in influenza-infected mice. 60 participated in a negative feedback loop in the jak and epidermal growth factor receptor pathway to protect against severe cytokine storm during severe influenza. 61 decreased mortality, pro-inflammation, and inflammatory cell counts of influenza-infected mice. 62 reduced the mortality, lung lesion formation, and inflammation of severe influenza-infected mice. 64 favorable in laboratorial data but limited clinical data for severe influenza. [65] [66] [67] [68] [69] [70] [71] several other immunomodulatory agents are being assessed for their efficacy, both in vitro and in vivo. lin and colleagues 58 have confirmed that ccr inhibitor-treated mice had a significant increase in survival (75% versus placebo) after being infected with influenza. adenosine 59-monophosphate-activated protein kinase (ampk) is an enzyme that exerts antiinflammatory effects after activation. the ampk activator aminoimidazole carboxamide ribonucleotide (aicar) was reported to increase survival by 40% in influenza-infected mice, while combination with pioglitazone improved survival by 60%. 59 ox40 (cd134) could impart a survival signal to t cells by upregulating anti-apoptosis gene expression, which plays a critical role in t-cell-mediated immunopathology in the lung during viral infection. 60 the ox40-immunoglobulin fusion protein (ox40-igs) treatment was shown to block the interaction of ox40 with its ligand on antigen-presenting cells and eliminate weight loss and cachexia without preventing viral clearance in influenza-infected murine models. 60 it should be noted that some inhibitors of interferon c signaling, such as suppressors of cytokine signaling (socss), were shown to have negative regulatory properties and participated in a negative feedback loop in the jak and epidermal growth factor receptor tyrosine kinase pathways. 61 these proteins are also potential agents that protect against cytokine storm during severe influenza. 61 all these findings offer a great deal of encouragement for treating influenza-induced cytokine storm with immunomodulatory agents. some antibiotics and antivirals are also known to possess anti-inflammatory effects and immunomodulatory properties in addition to their anti-pathogenic actions. both in vitro and in vivo studies have provided ample evidence for the immunomodulatory and anti-inflammatory activity of macrolides (e.g., erythromycin, clarithromycin, roxithromycin, and azithromycin). 62 macrolides can interfere with the replication cycle of influenza virus, resulting in the inhibition of viral production from infected cells. moreover, macrolide treatment of influenza virus-infected mice increased survival, suppressed inflammation, and reduced inflammatory cell counts. 62 arbidol is an antiviral that has complicated mechanisms. both membranefusion-inhibition and immunomodulatory activity may contribute to its effects. 63 our current research confirmed that post-treatment with arbidol-reduced mortality, lung lesion formation, and viral-induced inflammation by modulating the expression of pro-inflammatory cytokines in influenzainfected mice. 64 these data suggest that arbidol might also be effective in the treatment of severe influenza infections in humans. herbs may also be a potential choice for patients hospitalized with severe influenza. several chinese herbal prescriptions were recommended and authorized by the chinese government during the 2009 h1n1 and 2013 h7n9 pandemics. 65, 66 systematic reviews for clinical trials of these herbs used in influenza treatment have revealed that few herbal medicines showed a positive effect on viral shedding, but they had a positive effect on resolution or relief of symptoms. 67, 68 moreover, many herbs exhibit beneficial immunomodulatory effects for the rapid recovery of viral infections and might be effective treatments for infection with severe influenza. 69 we have reported that extracts from jiawei-yupingfeng-tang (a traditional chinese herbal formula) can alleviate influenzainduced lung lesions with both antiviral and immunomodulatory activity. 70 we also have confirmed that epigallocatechin gallate (egcg), a green tea-derived polyphenol, can inhibit the pathogenesis of influenza-infected cells due to its antioxidant activity. 71 polyphenols, triterpenoids, and flavonoids, all from herbs, may potentially be active components in protecting against cytokine storm during severe influenza (unpublished data). however, confirmation in a larger series of clinical studies is required. the persistent outbreaks of avian influenza in asia and parts of africa suggest that severe influenza, such as avian influenza, poses a major threat to public health. many severe-influenzainfected patients died from overwhelming viral pneumonia and serious complications caused by cytokine storm. in this review, we have highlighted the pathology of cytokine storm and, in particular, how an enhanced broad immune response can sometimes worsen the outcome of disease. although the precise molecular events surrounding cytokine storm have not been clarified, immunomodulatory strategies and novel approaches in targeting the host's response to severe influenza have been advocated. considering that these agents work on different intracellular pathways, they might ideally be used in combination to obtain a better outcome. based on the promising results mentioned above, combination therapies pairing s1pr and ppar agonists, cox-2 inhibitors, and antioxidants with conventional antiviral agents are promising treatments that deserve further study in randomized clinical trials. other approaches, especially those therapeutic strategies that can target signaling pathways, either to suppress redundant immune responses or reduce viral replication, will be particularly noteworthy. host response to influenza virus: protection versus immunopathology into the eye of the cytokine storm endothelial cells are central orchestrators of cytokine amplification during influenza virus infection a question of selfpreservation: immunopathology in influenza virus infection integrated clinical, pathologic, virologic, and transcriptomic analysis of h5n1 influenza virus-induced viral pneumonia in the rhesus macaque structured regulation of inflammation during respiratory viral infection innate immune responses to influenza a h5n1: friend or foe? recognition of microorganisms and activation of the immune response a calculated response: control of inflammation by the innate immune system genetic insights into sepsis: what have we learned and how will it help? avian influenza a (h5n1) infection in humans cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus-associated encephalopathy disease severity is associated with differential gene expression at the early and late phases of infection in nonhuman primates infected with different h5n1 highly pathogenic avian influenza viruses lethal dissemination of h5n1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia contrasting effects of ccr5 and ccr2 deficiency in the pulmonary inflammatory response to influenza a virus characterizing and controlling the inflammatory network during influenza a virus infection role of alveolar epithelial early growth response-1 (egr-1) in cd8 1 t cellmediated lung injury th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza antigen-specific memory regulatory cd4 1 foxp 31 t cells control memory responses to influenza virus infection immunomodulatory therapy for severe influenza early corticosteroids in severe influenza a/h1n1 pneumonia and acute respiratory distress syndrome clinical findings in 111 cases of influenza a (h7n9) virus infection effect of dexamethasone on acute respiratory distress syndrome induced by the h5n1 virus in mice systemic corticosteroids and early administration of antiviral agents for pneumonia with acute wheezing due to influenza a(h1n1)pdm09 in japan increased survival after gemfibrozil treatment of severe mouse influenza delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus bezafibrate upregulates carnitine palmitoyltransferase ii expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy ppargamma activation as an anti-inflammatory therapy for respiratory virus infections using complementary and alternative medicines to target the host response during severe influenza dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection pharmacologic inhibition of cox-1 and cox-2 in influenza a viral infection in mice drugs to cure avian influenza infection -multiple ways to prevent cell death n-acetyl-l-cysteine (nac) inhibits virus replication and expression of pro-inflammatory molecules in a549 cells infected with highly pathogenic h5n1 influenza a virus glycyrrhizin exerts antioxidative effects in h5n1 influenza a virusinfected cells and inhibits virus replication and pro-inflammatory gene expression ascorbic acid role in containment of the world avian flu pandemic inhibition of influenza a virus replication by resveratrol effects of flavonoid-induced oxidative stress on anti-h5n1 influenza a virus activity exerted by baicalein and biochanin a nrf2 expression modifies influenza a entry and replication in nasal epithelial cells n-acetylcysteine synergizes with oseltamivir in protecting mice from lethal influenza infection pathogenesis of septic shock in pseudomonas aeruginosa pneumonia inhibition of the cytokine response does not protect against lethal h5n1 influenza infection role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice mechanisms of action of intravenous immunoglobulin in autoimmune and inflammatory diseases hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza a(h1n1) infection analysis of deaths during the severe acute respiratory syndrome (sars) epidemic in singapore: challenges in determining a sars diagnosis meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? treating influenza with statins and other immunomodulatory agents evaluation of the efficacy and safety of a statin/caffeine combination against h5n1, h3n2 and h1n1 virus infection in balb/c mice effect of immunomodulatory therapies in patients with pandemic influenza a (h1n1) 2009 complicated by pneumonia potential mechanisms of at1 receptor blockers on reducing pneumonia-related mortality population-based study of statins, angiotensin ii receptor blockers, and angiotensin-converting enzyme inhibitors on pneumonia-related outcomes ccr2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection peroxisome proliferatoractivated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice a critical role for ox40 in t cell-mediated immunopathology during lung viral infection suppressor of cytokine signaling 4 (socs4) protects against severe cytokine storm and enhances viral clearance during influenza infection macrolides: from in vitro anti-inflammatory and immunomodulatory properties to clinical practice in respiratory diseases arbidol as a broad-spectrum antiviral: an update antiviral and antiinflammatory activity of arbidol hydrochloride in influenza a (h1n1) virus infection guidelines for management of avian h7n9 influenza infection guidelines for management of pandemic (h1n1) chinese medicinal herbs for influenza: a systematic review chinese herbal medicines for the treatment of type a h1n1 influenza: a systematic review of randomized controlled trials immunomodulatory effects of a traditional chinese medicine with potential antiviral activity: a self-control study jiawei-yupingfeng-tang, a chinese herbal formula, inhibits respiratory viral infections in vitro and in vivo amelioration of influenza virus-induced reactive oxygen species formation by epigallocatechin gallate derived from green tea we acknowledge research funding from the national nature science foundation of china (grant nos. 81403163 and 81402404) and yi chang scientific and technological bureau (grant nos. a14301-04 and a14301-10). key: cord-355374-e8k72955 authors: clemens, e. bridie; van de sandt, carolien; wong, sook san; wakim, linda m.; valkenburg, sophie a. title: harnessing the power of t cells: the promising hope for a universal influenza vaccine date: 2018-03-26 journal: vaccines (basel) doi: 10.3390/vaccines6020018 sha: doc_id: 355374 cord_uid: e8k72955 next-generation vaccines that utilize t cells could potentially overcome the limitations of current influenza vaccines that rely on antibodies to provide narrow subtype-specific protection and are prone to antigenic mismatch with circulating strains. evidence from animal models shows that t cells can provide heterosubtypic protection and are crucial for immune control of influenza virus infections. this has provided hope for the design of a universal vaccine able to prime against diverse influenza virus strains and subtypes. however, multiple hurdles exist for the realisation of a universal t cell vaccine. overall primary concerns are: extrapolating human clinical studies, seeding durable effective t cell resident memory (trm), population human leucocyte antigen (hla) coverage, and the potential for t cell-mediated immune escape. further comprehensive human clinical data is needed during natural infection to validate the protective role t cells play during infection in the absence of antibodies. furthermore, fundamental questions still exist regarding the site, longevity and duration, quantity, and phenotype of t cells needed for optimal protection. standardised experimental methods, and eventually simplified commercial assays, to assess peripheral influenza-specific t cell responses are needed for larger-scale clinical studies of t cells as a correlate of protection against influenza infection. the design and implementation of a t cell-inducing vaccine will require a consensus on the level of protection acceptable in the community, which may not provide sterilizing immunity but could protect the individual from severe disease, reduce the length of infection, and potentially reduce transmission in the community. therefore, increasing the standard of care potentially offered by t cell vaccines should be considered in the context of pandemic preparedness and zoonotic infections, and in combination with improved antibody vaccine targeting methods. current pandemic vaccine preparedness measures and ongoing clinical trials under-utilise t cell-inducing vaccines, reflecting the myriad questions that remain about how, when, where, and which t cells are needed to fight influenza virus infection. this review aims to bring together basic fundamentals of t cell biology with human clinical data, which need to be considered for the implementation of a universal vaccine against influenza that harnesses the power of t cells. countless examples exist for influenza a viruses causing havoc on public health, from perpetual seasonal epidemics, worldwide pandemics, and zoonotic infections from animal reservoirs, yet our current vaccine methods do not arm us against the diversity of influenza viruses. influenza vaccines are the most widely used vaccines in the world, with over 500 million doses used annually [1] , due to seasonal epidemics and the recommendation of annual vaccination. however, the efficacy of the inactivated influenza vaccine (iiv) is moderate to poor, and is impacted by antigenic drift [2] , mismatch [3, 4] , pandemic emergence due to reassortment [5] , and egg adaptations during vaccine production [6] , which can all lead to reduced protection and increased incidence of infections. the efficacy of the live attenuated influenza vaccine (laiv)-mainly recommended for use in children-has also dropped in recent years [7] , possibly due to thermal stability issues [8] or antigen competition during priming [9] . overall, these factors have culminated in reduced public confidence in influenza vaccines [10] . current vaccine stockpiles for avian influenza viruses h5n1 and h7n9 have reduced immunogenicity compared to seasonal influenza viruses [11, 12] , requiring multiple doses, the use of adjuvant, and may not match future emergent versions of these viruses [13] . the 2009 h1n1 pandemic showed that we are only able to respond after the fact, as the monovalent pandemic vaccine became available after the peak of human infections, leaving the majority of the population to "ride out the storm" and public outcry at the spectre of the pandemic severity predictions. vaccine production methods have been significantly ramped up in the wake of the 2009 pandemic, but the timing of virus isolation, distribution, and large-scale production will encounter similar issues in future pandemics. overall, a substantial revitalisation of the current vaccination program is needed to combat influenza viruses, overcome vaccine production limitations, and pre-arm ourselves against diverse and divergent influenza a viruses. vaccination educates our adaptive immune system-specifically t and b cells-for a faster, stronger, and more specific response upon re-encounter with the matching antigen. however, current iivs and laivs are not efficient in inducing t cell immunity, potentially contributing to their limited efficacy and breadth of reactivity against diverse influenza viruses. importantly, current inactivated influenza vaccines tend to prevent the induction of cross-reactive cd8 + t-cells, which would otherwise be elicited by natural influenza virus infections and are our primary protection in case of a vaccine mismatch or pandemic outbreak [14] (figures 1 and 2) . t cells express a clonal t cell receptor (tcr), which recognizes linear fragments of viral peptides that are 9-15 amino acids long, presented by the major histocompatibility complex (mhc) molecules on the surface of infected cells or antigen presenting cells. access to the mhc presentation pathway utilises endogenously (direct presentation) or exogenously (cross-presentation) derived peptides generated by the cleavage of viral proteins by the immunoproteasome. this process has important implications for vaccine approaches that need to consider the design and delivery of antigen to these pathways for the activation of t cells. viral entry and translation are required for access to mhc i processing machinery; however, peptide presentation can precede virus replication. the protein composition and immunodominance hierarchies of the subsequent t cell responses predominantly reflect abundant virion proteins expressed during infection and the timing of that expression [15] . memory t cells can provide very effective heterosubtypic immunity, whereby t cells are able to recognise different and even unrelated influenza viruses due to peptide homology or the conservation of linear peptide sequences between different strains and subtypes of influenza [16] [17] [18] [19] [20] [21] . heterosubtypic t cell immunity is especially important when there is no existing antibody response, which can occur during seasonal antigenic drift, zoonotic infection, or a pandemic situation. the protective efficacy of cross-reactive cd8 + t cells has been demonstrated in extensive animal studies of mice [22] [23] [24] and ferrets [25] . for example, mice primed with h3n2 virus, boosted with h1n1, and finally challenged with a high dose of lethal h7n7 virus have no detectable virus replication [26] . thus, unlike the majority of b cells, t cells are capable of expanding immunological protection against diverse influenza viruses. although antibody-mediated immunity elicited by natural infection or current vaccine strategies is capable of providing sterilizing protection, this protection is generated primarily against the hemagglutinin (ha) and neuraminidase (na) epitopes on the virus surface. immune escape is more common for surface antigens via modifications of glycans and amino acid substitutions at key antigenic sites than internal proteins which are constrained by functional limitations of viral fitness [27, 28] . lee et al. identified 72 t cell peptide epitopes that are cross-recognised between h5n1 and seasonal h3n2 viruses by cd4 + and cd8 + t cells, and only one was derived from the ha surface protein [17] . the majority of t cell epitopes are derived from internal proteins, which have a conservation rate of >90% between different iav strains and subtypes, whilst ha and na surface proteins are only 40-70% conserved between different iav subtypes [29] . indeed, t cells that are cross-reactive against highly pathogenic h7n9 and h5n1 avian influenza viruses can be found in the peripheral blood of unexposed healthy volunteers [17, 19, 20] . furthermore, robust and expedient recruitment of such cross-reactive t cell memory correlates with improved infection outcomes, faster recovery, and survival from h7n9 infection [30] . while influenza a and b viruses share many features, there is little sequence identity between analogous proteins (range 7-37% amino acid identity), with the exception of the polymerase basic 1 (pb1) protein (58% amino acid identity) [31] . despite this, broadly neutralizing monoclonal antibodies against highly conserved ha and na epitopes of influenza a and b viruses have been reported (reviewed in [31, 32] ). in addition, a conserved cd4 + t cell epitope in the ha fusion peptide of influenza a and b viruses can elicit cross-reactive cd4 + t cells in humans, and a number of well-characterized cd4 + and cd8 + t cell epitopes in the pb1 protein show complete amino acid conservation or only slight variation amongst influenza a and b viruses [31, 32] . this hints at the possibility of truly universal pan-influenza a and b virus t cell immunity that could be harnessed by vaccination. the area of research on t cell cross-reactivity beyond influenza a viruses currently remains underexplored. t cells are represented by a family of phenotypically diverse subtypes in terms of function, location, and magnitude. cytotoxic cd8 + t cells recognize viral peptide in the context of mhc class i (mhci), which is expressed on the surface of all nucleated cells, allowing immune surveillance. cd8 + t cells mediate the direct lysis of virus-infected cells and produce anti-viral cytokines, effectively and directly reducing viral load and the length of infection. meanwhile, helper cd4 + t cells recognize viral peptides within mhc class ii (mhcii), which is expressed by professional antigen presenting cells, b cells, macrophages, dendritic cells, and other cd4 + t cells. helper cd4 + t cells are a critical cornerstone of establishing effective immune memory against influenza virus infection, and are necessary for the establishment of cd8 + t cell memory responses [33] and high avidity class switched antibodies (reviewed in [34] ). in addition, it has been proposed that mhcii is also expressed by type ii alveolar pneumocytes during infection [35] , enabling cd4 + t cells to target a minor population of infected cells in the lung tissue by direct cytotoxic mechanisms themselves. t cells express a clonally diverse and highly specific tcr on their cell surface. the tcr consists of an α and β chain heterodimer, with the fine specificity of antigen recognition provided by somatic recombination and non-germ-line encoded additions to the complementarity determining regions (cdrs) of each chain. the estimated diversity of the human t cell repertoire is 2 × 10 7 distinct tcrs [36] , whilst the tcr diversity of an individual epitope-specific t cell response can be oligoclonal or composed of up to 40 different distinct tcr sequences [37, 38] . unlike antibody responses, tcrs do not undergo affinity maturation and the thymus involutes after puberty, greatly diminishing the output of naïve t cells as we age. the capacity for heterologous immunity by t cell responses is imperative for protection against the huge array of possible pathogens encountered in a lifetime, which out-numbers the tcr repertoire; therefore, an overlap in tcr specificities is a necessity. heterologous t cell protection to different epitopes has been reported in mice between lcmv, vaccinia virus, and pichinde virus [39] ; and in humans between influenza and hepatitis c virus [40] ; papilloma viruses and coronaviruses [41] ; influenza and epstein-barr virus (ebv) [42, 43] ; dengue subtypes [44] ; and as discussed above, between different strains of influenza and variants of immunodominant influenza-derived epitopes [45] . t cells are derived from the bone marrow progenitor cells, mature and develop in the thymus, undergo positive and negative selection processes, then emigrate as non-self-reactive immature cells in the periphery for immune surveillance for cells expressing altered or non-self-antigens. immature naïve t cells circulate between secondary lymphoid organs surveying for cognate antigen, while mature memory subsets-depending on their phenotype and priming signals-can reside in the tissue parenchyma (t cell resident memory, trm), the lymph node (t cell central memory, tcm) and periphery (t cell effector memory, tem). t cell recognition of peptide-mhc in the context of further signalling (e.g., co-stimulation and inflammatory cytokines) leads to t cell activation, proliferation, and differentiation, amplifying the effective army of t cells against infection. the number of naïve epitope-specific cd8 + t cells has been estimated in the mouse model for immunodominant and subdominant epitopes at 39-72 cells/mouse and 247-315 cells/mouse, respectively [38] . the efficiency of recruitment from the naïve pool of t cells to respond during acute infection is dependent on numerous factors, such as: the peptide context, mhc allele restriction, antigen presenting cell, the timing of antigen expression during the virus life cycle, and the avidity of the tcr and peptide-mhc interaction ( [46] , and reviewed in [47] ). after tcr engagement, recruitment, and activation, naïve t cells then amplify up to 10,000-fold during the acute stages of infection. the rapid expansion and peak magnitude of virus-specific t cells coincides with drastic reduction in viral titres from days 5-9 of primary virus infection, whereas influenza-specific antibodies peak and plateau from days 14-20 post infection. following antigen clearance at about 14 days post influenza infection, the antigen-specific t cell pool contracts, whereby the most differentiated cells undergo activation-induced cell death by apoptosis, leaving behind a stabilised memory pool [48] . the kinetics of t cell responses in human influenza infection are more variable, with some studies reporting a rapid peak in virus-specific t cells at one-week post-infection with h1n1 that subsequently contracts within 3-4 weeks after infection [22, 49] , while others suggest a more protracted response dynamic, peaking 3 weeks after infection with only modest decline in cell numbers by day 78 and returning to baseline by day 700 [50] . to what extent these variations in dynamics reflect differences in virus strain, infection severity, or individual characteristics of the host response remains to be determined. the t cell memory pool remaining after infection is estimated to have a half-life of 8-15 years in humans, and remarkably can be detected after 70 years post small-pox vaccination [51] , but the lifespan of influenza-specific t cells has not been tracked past 10 years [52] due to repeated encounters by influenza virus infection making tracking difficult with time. multiple doses of laivs-and not iivs or subunit influenza vaccines-can induce long-lived, broad, protective immune responses in mice [53] [54] [55] . therefore, laiv appears to be more immunogenic in animal studies. however, human responses against laiv are disparate depending on age, and laivs only seem more effective than iiv in children, not adults, which coincides with boosted cellular immunity [56, 57] . this is possibly a result of children's more naïve immune states resulting in a robust primary cellular immune response [58] [59] [60] [61] [62] [63] [64] [65] [66] . laiv-induced humoral and cellular immunity could be maintained for at least one year [67] , but the longevity beyond 1 year has not been studied so far. the laiv vaccine currently has limited efficacy, with a reported vaccine efficacy of only 3% for laiv vs. 65% for iiv reported in 2017 [7] . however, laiv was a sincere attempt at a t cell-inducing vaccine by nasal delivery, but appears too mild, with reduced tissue tropism and inflammation [68] and has so far failed to increase cellular immunity or improve vaccine efficacy in adults. therefore, future universal vaccines must improve upon laiv by either the use of more immunogenic vaccine vectors (such as e1 deleted adenovirus [69] or mva [70] ), adjuvants (interleukin-15 (il-15) [29] , pam2cys [71] , or mf59 [72] ), or less attenuated influenza viruses (such as the use of codon bias mutants [73] , ns1 mutants [74] , or ha-signal peptide viruses [75] ). lessons should be learned from the limited efficacy and immunogenicity of laiv in human studies in the development of next-generation universal vaccines. t cells do not operate in isolation, but in synergy with multiple immune mechanisms to provide broad heterosubtypic protection ( figure 1 ). heterologous protection mediated by cross-reactive cd8 + t cells was shown to be dependent on an interaction with macrophages and fcr-dependent non-neutralizing antibodies [76] , similar to the inexplicable protection rates beyond t cell response magnitude observed in the laiv thailand/philippines clinical trial [77] . in addition, the timing of recruitment of memory cd8 + t cells, cd4 + t cells, nk cells, or neutralizing antibodies impacted h7n9 infection outcomes [30] . further cell types also contribute to heterologous protection, such as mucosal associated invariant t (mait) cells [78] and fcr + cells to recruit t cell responses [76] . thus, the next generation of t cell vaccines should combine multiple immune responses for heterologous protection. effective heterologous immunity against zoonotic influenza (h7n9) viruses requires synergy of multiple immune arms [30, 76, 78] . without the recruitment of two or more immune arms, protective immunity is diminished, as modelled on outcomes of infection from h7n9-infected patients. although multiple arms are likely to be activated at the same time, hospitalized patients clearly demonstrate that different arms had a more prominent role if one arm fails to respond. mait: mucosal associated invariant t. cd4 + t cells are phenotypically diverse, and depending on their surface receptors and cytokine expression, can be further characterised into numerous subsets: th1, th2, treg, t follicular helper (tfh), th17, th22, and th9 (reviewed in [79] ). the cd4 + t cell field is less developed than the cd8 + t cell field (reviewed in [80] ) for influenza research, due to the availability of mhci tetramers and transgenic mouse strains. however, continued efforts using adapted methods such as peptide scanning or whole virus for identification of influenza-specific cd4 + t cells have revealed their intrinsic necessity for heterologous protection against influenza virus infection [49] . characteristically, th1 type cd4 + t cells can secrete ifn-γ and are necessary for the formation of cd8 + t cell memory capable of recall and tfh cd4 + t cells for the induction of high affinity class switched b cell memory [81, 82] . induction of ha-stem-specific antibodies requires highly developed b-cell receptor sequences that have undergone affinity maturation, likely to be coordinated by cd4 + t follicular helper (tfh) cells [83] . immunization with current iivs activates tfh cells, correlating with an increase in the number of peripheral antibody-secreting and memory b cells [84] . an emerging body of work has demonstrated the critical role tfh cd4 + cells play in shaping the antibody profile, whereby conserved nucleoprotein (np)-specific cd4 + tfh cells compete with ha-specific tfh cells, resulting in reduced h7-ha antibody recall responses after priming with the h1-ha [85] [86] [87] . therefore, the order of priming for establishing immunodominance hierarchies for cd4 + tfh responses can impact the development of ha-stem antibodies in the germinal centre. the interplay of protein-specificity and order of immune memory or "imprinting" is important in the context of human immune memory, which is heterogeneous and individualistic depending on a subject's exposure background. imprinting of ha-subtype specific cd4 + t cell and b cell memory can have an epidemiological impact. ha-imprinting was recently described in an epidemiological model explaining the age association of h7n9 and h5n1 infections, and providing >75% protection from severe infection caused by these viruses. this was attributable to an individual's first ha-group encounter resulting in an age distribution of cases born after 1968 for h5n1 and before 1968 for h7n9 corresponding to the switch from a phylogenetic group 1 (including h1, h2, and h5 ha subtypes) to group 2 (includes h3 and h7 ha subtypes) ha during the 1968 hong kong h3n2 pandemic [88] . furthermore, in addition to helper functions, influenza-specific cd4 + t cells may be imperative to protection from heterologous influenza infection. in a transgenic mouse model, cd4 + t cells have also been found to be directly cytotoxic during influenza infection [89] , and human influenza-specific cd4 + t cells can express granzymes, perforin, ifn-γ, and exhibit killing function correlating with reduced severity of infection and viral loads [49] . furthermore, a t cell-inducing vaccine mouse model identified that depletion of memory cd4 + t cells but not the cd8 + t cell compartment removed heterologous protection from lethal influenza challenges [29] . new roles continue to emerge of the key role influenza-specific cd4 + t cells play during heterologous infection. overall, influenza-specific cd4 + t cells are a cornerstone of effective influenza responses, and their role is a dynamic interplay on the recall of heterologous t and b cell responses and infection outcomes ( figure 1 ). the long-term role of imprinting cd4 + t cell memory responses by universal vaccines will have an important implication in future vaccine design, and is not yet fully explored. epidemiological studies during past influenza pandemics, where there is an absence of neutralizing antibodies, have suggested that t cell-mediated immunity provided some heterosubtypic protection [90] [91] [92] . however, the complexity involved in conducting such studies (i.e., obtaining the right cohort that does not have pre-existing antibody response and sampling prior to infection) means that direct evidence for the role of t cells during influenza virus infections in humans has been limited. as such, a major challenge in the universal vaccine field is to determine the strength of a t cell-mediated "correlate of protection". unlike animal models, influenza virus infections in humans can result in a wide spectrum of symptoms that reflect either upper or lower respiratory tract involvement. the clinical disease and epidemiology is also distinct between seasonal and avian influenza cases. thus, in practise, the definition of "protection" in human studies has varied from study to study, and has generally meant a reduction in virus shedding or manifested symptoms. these symptoms have either been self-reported [93] [94] [95] , observed [49] , or clinically determined [96] [97] [98] . in seasonal influenza, the earliest direct evidence that cd8 + t cells can mediate protection by facilitating virus clearance after infection was shown in a human challenge study [99] . thirty years later, sridhar et al. [94] took advantage of an existing community-wide cohort to show this within the context of natural infections during the h1n1 2009 pandemic. they demonstrated that the magnitude of a subset of ifn-γ + il-2 − cd8 + t cells was most strongly correlated with reduced disease severity after infection. these cells were also shown to be late-effector memory t cells (cd45ra + ccr7 − ). this was in contrast with the findings from an earlier experimental h3n2 or h1n1 human challenge study, where cd4 + t cells were correlated with reduced viral load and disease symptoms instead of cd8 + t cell memory responses [49] . this discrepancy was attributed to differences in experimental designs (i.e., virus challenge using laboratory-grown viruses and hemagglutination inhibition (hai) negative subjects versus natural pandemic infection), and it serves to further highlight the challenges associated with studying t cell responses in humans. because t cells recognize highly conserved viral proteins, there has been great interest in investigating their protective role during human cases of avian influenza virus infections. while numerous studies have demonstrated the presence of cross-reactive t cells against avian influenza viruses in vitro [17] [18] [19] [20] 86] , actual evidence of protection against infection or disease in the field is still lacking since the sporadic and virulent nature of avian influenza cases precludes any systematic cohort studies. studies to date have been observational, and were based on a relatively small number of cases [30, 100] during the h7n9 outbreak in china in 2013. these studies suggested that the early induction and recruitment of memory cd8 + t cells was associated with improved prognosis [30, 100] . in contrast to cellular immunity data (which are scarce), there have been numerous studies investigating cytokine profiles in infected humans as prognostic markers and signatures of correlates of protection. pro-inflammatory cytokines such as interleukin (il)-6 and chemotactic regulators of innate immune cells such as il-8, mcp-3, and mcp-1 are commonly induced during the acute phase of disease [93, 101, 102] . however, cytokines that are important in inducing th1 and th2 responses have also been reported to be depressed in severely ill individuals [97, 98, 100] . of particular interest is il-12, a regulator of downstream th1-responses. concentrations of this cytokine were lower in severe seasonal and avian influenza cases, which incidentally also had lower levels of peripheral cd8 + t cells [97, 100] compared to mild influenza cases. however, the role of this cytokine needs to be further validated, since data from murine models suggests that high levels of il-12 cytokine can suppress the formation of lung cd8 + memory t cells in the airways [103] . the state of current knowledge of influenza t cell responses in humans is still trying to catch up with the lessons we have learned from animal models. in vitro and murine studies have demonstrated that t cells can mediate lung pathology (reviewed in [104] ), primarily through hypersecretion of soluble factors such as ifn-γ and tnf-α, causing direct lysis or "bystander damage" to the lung milieu [105] . although immune-mediated pathology has certainly been suggested as an important component of severe influenza infections in humans [98, 106] , a definitive causal role for t cells that is uncoupled from innate immunity or viral factors is still lacking. indeed, cytokines associated with cellular innate immunity appear to be better predictors of influenza disease severity [93, 101, 102] . most studies of t cell responses in humans have been conducted within the peripheral compartment due to the ease of sample access. however, peripheral and airway mucosal immunity are not always in concordance for respiratory infections [93, 102] . furthermore, viral replication can persist in the lower respiratory tract without detection in the upper respiratory tract, where nasal swabs are often collected [107, 108] for detection of viral rna. airway samples are difficult to collect-particularly those from the lower respiratory tract. nasal swabs contain mucosal inhibitors and have low cellularity, and are inappropriate for cellular immunity studies, while nasal aspirates are rare in human influenza studies. furthermore, sampling the lower respiratory tracts through bronchoalveolar lavage is often ethically precluded in healthy adults, and therefore such studies of local respiratory tissue t cell immunity are limited to severe infection cases that compares infection outcome rather than protection from infection or disease [93, 97] . in the few rare studies where such data are available, the number of influenza-specific cd4 + and cd8 + t cells, as well as the magnitude of cytokine response, are far greater in the lungs compared to the blood [98, 109, 110] . homologous virus challenge in ferrets that received the monovalent a/viet nam/1203/2004 (h5n1) vaccine showed that the cd8 + t cells in the airways, but not the blood, correlated with early viral clearance [111] . elegant studies in murine models have further identified a t cell population in the airways (tissue resident memory t cells, see section below) that are phenotypically and functionally distinct from those in the blood [112] and that have also been identified in humans [113, 114] . however, as it is likely that the peripheral compartment will continue to be a proxy of evaluating t cell responses in humans, the extent of how accurately the peripheral t cell responses reflect the airways, and crucially, how they translate to short-and long-term protection are important considerations for effective t cell-mediated vaccine development. because most adults have some baseline level of detectable influenza-specific t cells, some efforts have been made to quantitatively identify the t cell-mediated protective threshold as the "correlate of protection". in a study involving over 2000 children in thailand and the philippines, forrest et al. reported that after receiving two-doses of laiv, hai-seronegative children that had ≥100 spot-forming unit (sfu)/10 6 pbmc in an ifn-γ-elispot assay (using inactivated vaccine components as antigens) were protected against symptomatic disease during subsequent infection [77] . this study also made two notable observations: (i) the protective thresholds for the study populations in these two countries were different, and (ii) other mechanisms of protection not attributed to the measured response were noted with increasing vaccine dosage. thus, in attempting to provide a "quantitative" correlate of protection, this study also highlighted the population heterogeneity in the t cell compartment and the importance of other immune-mechanisms (i.e., those mediated by non-ifn-γ + cells, or non-hai-reactive antibodies) that were missed by the experimental approach. in a more recent study, hayward et al. [95] chose to focus on the immunodominant response against the influenza np-, rather than the ha-specific t cell response, and reported that ≥20 sfu/10 6 pbmc was associated with reduced viral shedding in community-acquired influenza. finally, sridhar et al. [94] used statistical modelling to predict that every 10-fold increase in the ifn-γ + il-2 − cd8 + t cell frequency (enumerated by elispot) is associated with a 7-fold reduction in the risk of developing febrile influenza. because of the different assays used across these three studies, it is difficult to interpret the importance of these quantifiable findings until they are further corroborated. it is worth noting that these studies evaluated symptomatic cases, and evidence for the role of t cells in asymptomatic cases-particularly within the community setting-is still lacking. clinical evidence thus far supports the role of existing t cells-particularly the cd8 + population-in reducing symptom severity. however, the heterogeneity in experimental design (i.e., study definition, endpoints used, and the use of different or unqualified assays to measure t cell responses, and elispot methods are not directly comparable between different studies) seems to suggest the phenomenon of "the blind men and the elephant", whereby each study is measuring the t cell response as an immune correlate in different ways, sometimes providing disparate conclusions. integrating the myriad clinical findings to identify a single quantifiable trait of t cells as the "correlate of protection" remains a major challenge. while some of these issues are surmountable with sophisticated study designs and further research, others-such as airway t cell responses-may remain challenging to address in humans. thus, in addition to understanding the basic biology, the field must also work on developing a robust system that includes standardized and qualified t cell assays for use in human studies. to date, vaccines intended to elicit t cell immunity against viral infection have generated disappointing levels of protection. this has provoked the recent reassessment of variables important for the successful generation of t cell-mediated immunity with the quantity and location of the memory t cell population now considered of critical importance. t cells require contact with their target cell to mediate their cytolytic function; in other words, they act locally. therefore, any effective t cell-based vaccine targeting the earliest stages of infection would require the deposition of significant numbers of memory t cells locally within the mucosal tissue. non-lymphoid tissues house a large proportion of the memory t cell pool [115] . previously, it was thought that these were simply circulating memory t cells trafficking through the tissue as part of their immunological surveillance. however, it is now accepted that the majority of the cells present within the tissue are in fact resident and represent a distinct memory t cell population [116] . these peripherally deposited memory t cells-termed tissue resident memory (trm)-play a critical role in local immune protection by directly killing pathogen-infected cells [117, 118] , releasing cytokines that render the surrounding/local environment non-permissive for pathogen replication [119] , and promoting the recruitment of other immune cells from the circulation [120] . both cd4 + and cd8 + t cell lineages can form trm, and can be distinguished from circulating memory t cells due to the expression of key surface markers. cd4 + trm upregulate cd69 and cd11a expression [121, 122] , while cd8 + trm also express cd69 as well as cd103, the α chain of the αeβ7 integrin [116] , although cd103-negative cd8 + trm cells have also been detected [123] ; thus, alternative markers for trm are needed. not all memory t cell subsets are equally protective against influenza virus infection, with only the trm pool being shown to be absolutely indispensable for providing optimal heterosubtypic immunity ( figure 2 ). following secondary encounter with influenza virus, both cd4 + [121, 122] and cd8 + [124] lung trm rapidly acquire effector function and respond swiftly, mediating enhanced viral clearance and survival to lethal influenza infection. why do trm provide superior protection against influenza virus infection? simply, they are in the right place at the right time! influenza virus gains entry into the body by inhalation and initiates its replication cycle within the respiratory tract. the early stages of infection, when virus titres are low in the infected host, provide the ideal window of opportunity for effective immune responses to limit disease progression. trm deposited along the respiratory tract are perfectly situated to combat the earliest stages of an influenza infection. human lung harbours a large number of memory t cells [114] , a significant proportion of which express the molecular signature and phenotypic profile of trm cells. the vast majority of influenza-specific memory cd4 + and cd8 + t cells present within human lung tissue adopt a trm phenotype [114, 122, [125] [126] [127] [128] . differentiation of these influenza-specific cells into trm is important for their long-term maintenance, as we find that the size of the influenza-specific cd8 + t cell population persisting within the lung directly correlates with the efficiency with which these cells differentiate into trm [128] . influenza virus-specific trm were shown to be highly proliferative and polyfunctional [123, 128, 129] . molecular profiling revealed that human lung trm constitutively express high transcript levels of numerous cytotoxic mediators and deployment-ready mrnas encoding effector molecules, which is reflective of these cells being poised for rapid responsiveness [123, 129] . influenza virus-specific cd8 + trm in human lung tissue also maintain diverse tcr profiles-a feature important for effective t cell function and protection against the generation of viral-escape mutants [128] . influenza-specific pulmonary trm are a core component of the natural heterosubtypic immunity developed following exposure to influenza virus ( figure 2) . thus, influenza vaccines designed to impart optimal heterosubtypic immunity should evoke this memory t cell subset. defining parameters that promote trm formation along the respiratory tract is a critical step towards the development of such a vaccine. so, what do we currently know about the factors that drive trm? exposure to interleukin-15 (il-15) [130] and transforming growth factor-β (tgfβ) [131, 132] , as well as down-regulation of krüppel-like factor 2 (klf2), s1p1r [133] , t-bet and eomes expression [130] , and up-regulation of hobit [134] have been proposed as universal trm developmental requirements. in some tissues (including the lung [125, 135, 136] and brain [137] ), trm development is also dependent on local cognate antigen recognition. can we rationally design vaccines to specifically evoke trm? in 2012, iwasaki and colleagues [138] published the first vaccination regime that specifically evoked trm at a site of potential viral exposure-they coined this vaccination regime "prime-pull". this approach relied on two steps: conventional parenteral vaccination to elicit systemic t cell responses (prime), followed by the topical application of inflammatory agents to lure (pull) the cells into the tissue where they could differentiate into trm [138] . while the "prime-pull" vaccination strategy has proven effective at lodging highly-protective trm pools in the skin [118] and reproductive tract [138] , this approach is not effective at depositing trm cells within the lung [131] . this is because trm differentiation in the lung-unlike skin and reproductive tract-requires local cognate antigen recognition [125, 135, 136] . as such, an extension of the "prime-pull" vaccination regime which incorporates the local delivery of cognate antigen was developed as a pulmonary trm vaccination strategy. several groups using a variety of modified "prime-pull" vaccination approaches, including (i) intranasal immunization with influenza peptide/protein alone [135, [139] [140] [141] or loaded onto dendritic cells [142] , (ii) intranasal delivery of non-replicative influenza virus [143] , or (iii) intranasal immunization with monoclonal antibodies linked to influenza antigens that target antigen to respiratory dendritic cells [131] , were all able to successfully generate lung cd8 + trm that were highly protective against influenza virus challenge. collectively, these vaccination studies demonstrate that the key to inducing pulmonary trm is the local (intranasal) administration of the vaccine antigen ( figure 2 ). such strategies might also be beneficial for the optimization of anti-influenza antibody responses through induction of mucosal secretory iga in the respiratory tract. vaccination strategies that deposit influenza virus-specific trm cells in the lung provide exquisite protection against heterosubtypic influenza challenge [131, 143] . unfortunately, this protection is transient. unlike populations in the skin and intestinal mucosa, trm cells in the lung undergo attrition [124] as a result of increased apoptosis [144] . mouse models confirm that within 7 months of their deposition, influenza virus-specific lung trm decay below a numerical threshold, leaving the lung susceptible to reinfection [124] . this observed decay of lung trm cells in mice is no reason to completely dismiss their potential to provide long-term immunity in humans, as just because mouse lung trm decay does not necessarily mean that human lung trm also undergoes this rapid rate of attrition. recent studies by ahmed and colleagues [145] , which utilize in vivo deuterium labelling to assess human memory cd8 + t cell turnover and longevity, show that the longevity of circulating memory t cells subsets in humans does not reflect the lifespan of these cells in mice. it will be important to determine the lifespan and turnover rate of human lung trm if these cells are to be incorporated into vaccines against respiratory pathogens, provide durable vaccine memory, and determine the vaccine schedule to maintain protection. nonetheless, while lung trm erode, their counterparts in the upper respiratory tract form a stable long-lived memory t cell pool [142] . influenza-specific nasal tissue trm are effective at limiting local replication of influenza virus and can block the transmission of influenza virus from the upper respiratory tract to the lung, and in doing so, prevent the development of severe pulmonary disease [142] . these cells have the potential to provide long-term immunity against influenza virus. to protect globally against influenza virus, we need to think locally! trm located along the respiratory tract are perfectly situated to mediate rapid protection following the inhalation of influenza virus, and are capable of providing potent protection against this inhaled pathogen. influenza vaccines designed to impart heterosubtypic immunity should evoke this memory t cell subset. how best to induce influenza virus-specific trm along the respiratory tract after vaccination will be one of the challenges to address in the coming years. the major antigenic targets of influenza-specific t cells are epitopes derived from highly conserved internal virus proteins, providing the basis for heterologous or "universal" immunity across multiple unrelated strains of influenza. studies assessing the relative contributions of different influenza virus proteins to human t cell responses have identified np, matrix protein 1 (m1), and pb1 as the major targets [16, 49, 95] , suggesting that a t cell vaccine may only need to focus on a few key viral proteins to achieve similar coverage to natural infection. the focus on priming t cell memory to conserved protein targets may also have implications for reducing interference with ha-imprinting in future vaccine design [88] . t cell responses to influenza virus differ between individuals, primarily due to the expression of diverse mhc alleles (known in humans as human leucocyte antigens, hlas) that determine the array of viral peptides presented to t cells for recognition. given the polymorphic nature of hla alleles [146] and the fact that each individual expresses six hla class i (hla-i; hla-a, -b, and -c) and six hla class ii (hla-ii; hla-dr, -dq, and -dp) alleles, there is potential for vast diversity in the epitopes presented for recognition during infection. despite this, t cell responses usually focus only on a few peptide + hla epitopes, with responses to the same epitopes typically observed across individuals expressing the same hla allele. responses to different epitopes often arrange into reproducible immunodominance hierarchies, wherein they can be defined as immunodominant (large) or subdominant (small) in magnitude. the relative size of a given epitope-specific t cell response reflects a complex interplay of virus and host factors (reviewed in [47] ), including the particular combination of hla alleles expressed [147] , and does not necessarily relate to the number of naïve epitope-specific precursors available. approaches combining in vitro infection of cell lines with mass spectrometry analysis have allowed direct identification of the array of peptides presented at the cell surface by individual hla alleles following influenza infection. to date, these results show the presentation of a reasonably small selection of virus peptides (nine for hla-a*02:01 and up to six for b*07:02), mainly derived from internal virus proteins, of which only a few (one and two peptides, respectively) consistently induce immune responses [148, 149] . understanding why some presented peptides are targeted while others are seemingly ignored remains a key question that may lead to strategies for optimizing the breadth of vaccine-induced t cell responses. hla-i allele expression is an important predictor of cross-reactive influenza-specific cd8 + t cell immunity, with a recent study identifying five alleles (a*02:01, a*03:01, b*57:01, b*18:01, and b*08:01) capable of eliciting robust cd8 + t cell responses against immunogenic np and m1 peptides that are conserved across all human influenza a virus, including the novel avian-derived h7n9 virus [18] . strong representation of these "protective" hla alleles in a population is therefore predictive of universal memory t cell responses with the potential to protect against multiple circulating and novel influenza a virus strains. as hla profiles are heritable and strongly influenced by ethnicity, the extent of this universal immunity shows an expected ethnic bias, with higher coverage anticipated in caucasian populations with enrichment of these key alleles (57%, based on hla allele expression) as compared to indigenous alaskans and indigenous australians (16%) [18, 150] . whilst certain hla alleles may confer a protective advantage through universal immunity to multiple influenza strains, the expression of other alleles may increase susceptibility to severe influenza disease. the h7n9 study mentioned above also identified four hla-i alleles (a*01:01, a*24:02, a*68:01, and b*15:01) that target unique h7n9 np and m1 peptide variants that are unlikely to elicit cross-protective immunity between seasonal and h7n9 infection [18] . thus, upon infection with h7n9, individuals with these hla alleles will need time to activate and amplify new primary cd8 + t cell responses to distinct h7n9 peptide variants rather than recalling t cell responses generated against seasonal influenza viruses, potentially resulting in longer time to recovery and greater risk of severe disease compared to individuals with pre-existing cross-protective cd8 + t cell memory. hertz et al. showed that hla-a*24 alleles also have low binding preference for peptides from conserved regions of the 2009 pandemic h1n1 (ph1n1) virus, and carriage of these alleles correlated with low influenza-specific t cell responses in ph1n1-infected patients [151] . moreover, at a population level, carriage of hla-a*24 alleles or the hla-a*68:01 allele was associated with increased mortality to ph1n1. these "risk" hla alleles may serve as useful markers to identify individuals or populations that are more likely to have low magnitude and less cross-reactive memory t cell responses, placing them at greater risk of severe influenza disease. interestingly, hla-a*24 and a*68:01 alleles are highly prevalent in certain indigenous populations [152, 153] , which could explain why these groups experience a higher burden of influenza disease and mortality to seasonal and pandemic influenza [151] . however, risk hla alleles may also have indirect effects on susceptibility to influenza through associations with other autoimmune or metabolic disorders. for example, hla-a*24 is associated with diabetes [154, 155] , a disease with increased incidence in indigenous populations [156] . nevertheless, the notion of protective and/or risk hla alleles has been demonstrated previously for human immunodeficiency virus (hiv) and hepatitis c virus (hcv) [157] [158] [159] [160] , and is clearly emerging as a factor in influenza infection linked in part to the capacity of certain hla alleles to present conserved (protective) or variable (risk) influenza peptides. ethnicity is a key determinant of risk for severe influenza disease with indigenous populations worldwide experiencing a disproportionate burden of morbidity and mortality caused by infection with influenza viruses. during the 2009 h1n1 pandemic, higher hospitalization and morbidity rates were observed for indigenous people in canada, the united states, new zealand, and australia [161] [162] [163] [164] [165] . likewise, indigenous populations experienced higher mortality rates during the 1918 pandemic (8.5% compared to 2.5%, worldwide [165] ). higher influenza virus infection rates in indigenous populations may be related to crowded living conditions, increased rates of chronic disease (lung, heart, and metabolic) and co-morbidities that exacerbate the severity of infection. nevertheless, recovery from influenza depends strongly on the ability to mobilize multiple arms of the immune system-in particular, an early cd8 + t cell response [30] . the severity of influenza disease and prolonged hospitalisation periods observed for indigenous people may therefore reflect a lack of pre-existing protective cd8 + t cell immunity that promotes rapid recovery. as mentioned, hla-a*24 alleles-a known risk factor for severe ph1n1 disease [151] -are enriched within several global indigenous populations [152, 153] , indicating a possible hla-related deficiency in t cell responsiveness to influenza that may contribute to the vulnerability of these communities. notably, the hla profile of indigenous australians is relatively restricted and quite distinct from non-indigenous australians, with predominant use of hla-a*34:01, 24:02, 02:01, 11:01, and hla-b*13:01, 56:01/02, 15:21, 40:01/02 [153] . as such, the dominant cd8 + t cell responses in indigenous australians are likely to focus on different peptide+hla epitopes compared to other ethnicities. since studies have mostly focused on identifying and characterising immunogenic peptides for common and widely expressed hla alleles such as hla-a*02:01, immunogenic influenza epitopes are yet to be defined for most (71%) of the hla-i alleles found in indigenous australians. however, analysis of hla-a*02:01-restricted m1 58-66 -specific cd8 + t cells found comparable magnitude, functional quality, and clonal characteristics in indigenous australians and non-indigenous australians, suggesting that a*02:01 + indigenous australians (representing 10-15% of this population) have robust cross-protective t cell immunity to any influenza a virus [45, 153] . however, accurately determining the extent and quality of cd8 + t cell immunity in indigenous australians and other indigenous populations worldwide will require the identification of prominent t cell targets for the relevant indigenous hlas. it will be of great interest to see how effectively these hlas elicit influenza-specific cd8 + t cell immunity. based on hla profile, targeting responses to a few prominent hlas in indigenous australians could achieve high levels of population coverage [153] , but may necessitate a tailored vaccine, as many of these hla alleles occur rarely in other ethnicities. clearly, much needs to be done to improve our understanding of influenza virus infection in indigenous populations before we can design better protocols to protect these populations, which are at greater risk of severe influenza disease and death. despite the challenges of hla diversity and possible confounding associations with metabolic and autoimmune disorders, understanding t cell responsiveness to influenza across a broad range of hla profiles will be an important part of designing and testing the efficacy of future vaccines. although response magnitude-typically measured in the periphery-is undoubtedly a critical aspect of effective anti-influenza t cell immunity [49, 94] , polyfunctionality in the t cell response is also linked with improved outcome to infection [166, 167] . hla genotype may be an important intrinsic factor shaping the magnitude and functional profile of t cell responses. hla-b alleles are significantly better than hla-a alleles at generating robust polyfunctional (ifn-γ and il-2) cd8 + t cell responses to hiv, cmv, ebv, and influenza [168] . while this reflects a general enhanced effect for hla-b alleles, there also appears to be hierarchy of association with polyfunctionality amongst hla-b alleles. the combination of hla alleles expressed can also impact the number and tcr repertoire of epitope-specific precursors, while altered peptide presentation, modulation of surface expression, and competition for overlapping peptides can shape the activation and proliferation of these precursors during infection in the context of different hla profiles [147, [169] [170] [171] [172] [173] [174] [175] . it is therefore very difficult to predict t cell response hierarchies to an epitope across the hla-diverse human population. however, focusing on the largest immunodominant t cell responses may not be all-important, as smaller subdominant t cell epitopes can also contribute to influenza virus clearance, and while they may be underutilized following natural infection, they could be harnessed by vaccination to achieve their full antiviral potential and provide a broad combined response that does better than natural infection [38] . increasing the breadth of the antiviral response by targeting subdominant epitopes may also reduce the potential for mutational escape in immunodominant epitopes [148, 176] . while an effective influenza vaccine should capitalize on the ability of protective hla alleles to present universally conserved viral components by self-selecting peptides for presentation and elicit broad spectrum t cell immunity, singling out specific peptides as vaccine targets could come at the cost of population-wide coverage across diverse hla profiles. approaches that incorporate whole protein antigens are more likely to provide coverage in the human setting of diverse hla types and avoid concentrated immune pressure that may drive the emergence of escape mutations in targeted epitopes. furthermore, strategies could be used to optimize peptide presentation by risk hla alleles through modifications of extra-epitopic processing sites by vaccine design. upstream extra-epitopic sequences have already been shown to alter cd8 + t cell responses to the immunodominant a2-m1 58-66 cd8 + t cell response, possibly via changes to the cleavage pattern of the m1 protein that influence the extent of m1 58-66 peptide presentation [177] . an ideal influenza t cell-based vaccine would therefore induce an overall robust response comprised of multiple cross-reactive epitope-specific t cell populations with high functional quality and engage multiple immune mechanisms for greater synergy of protection ( figure 2 ). it would encompass the importance of hla coverage even for minor ethnicities with rare hla types, and importantly, leverage our knowledge of key epitopes and cross-reactive t cell responses to do better than nature. influenza is an rna virus, and uses its own error-prone rna-dependent rna polymerase for replicating its genome. therefore, the virus is able to adapt rapidly, and is infamous for antigenic drift and resistance to anti-virals under selection pressure. virus escape from immune-mediated control undermines effective vaccination, and will also need to be considered for a t cell-inducing vaccine. while t cell-targeted proteins and peptides are more highly conserved than the antibody targets in the surface ha, variation and adaptation in t cell epitopes derived from influenza viruses have already been identified. cd8 + t cell escape has been observed in some t cell epitopes of naturally circulating influenza viruses [45, [177] [178] [179] , which raises the concern for vaccine-mediated t cell escape. as t cells recognize viral peptides within host mhc, viruses can mutate at mhc-anchor residues to reduce presentation [180] [181] [182] [183] [184] . alternatively, exposed residues can form contacts with the tcr for recognition, and variation at tcr contact sites can reduce recognition by existing t cell responses [176, 185] . however, due to tcr diversity, establishing a new t cell response to the new peptide variant is often subsequently possible [169, 176, 182, 183, 186, 187] . mutation of viral peptides at mhc anchor residues or tcr contacts can impact viral clearance [183] and the recall of established heterosubtypic memory t cell responses [176, 180] . however, this concept remains under-appreciated, and could undermine t cell-targeted vaccine-mediated control. our previous preliminary data from the isolation of viral rna from the lungs of infected mice shows that influenza escape can occur very early after the infection of an individual mouse and is common across individual mice, and thus is not a rare event [180] . mutations at anchor sites and tcr contacts for cd8 + t cell influenza epitopes were readily identified, and reverted in the absence of epitope-specific immune pressure [180] , which may suggest hla frequencies in the population will determine the rate of t cell-mediated immune escape. population sequence studies of drift in the np protein have identified positive selection pressure [28] , and furthermore, at the epitope level, anchor mutations were identified in the hla-b*27:05-and b*08:01-restricted np 383-391/380-388 epitope [179] . other evidence of immune escape in np and m1 cd8 + t cell epitopes showed that epitopes are functionally constrained, limiting the extent of variation possible, but t cells posed an evolutionary pressure on influenza virus [28, 188] . the influenza m1 58-66 epitope reproducibly selects a dominant public trav27/trbv19 + tcrαβ in hla-a*02:01 + donors that can cross-recognize naturally occurring m1 58-66 peptide variants, seemingly limiting the establishment of mutant strains within the circulating pool of human influenza a viruses and conferring universal immunity to influenza in a*02:01 + individuals [45] . conversely, in the immunodominant hla-b*07/b*35:01 restricted np 418-426 epitope, a different pattern of sequential viral variation has generated over 20 different peptide variants at tcr contact sites [189] , with t cell evasion occurring for selected peptides [169, 186] , resulting in the need for priming multiple t cell sets for the coverage of diverse np 418 variants. in this situation, successive waves of exposure to np 418-426 variants may drive diverse and unique tcrαβ repertoire recruitment with variable patterns of cross-reactivity against individual variants that favours, rather than controls, the emergence of additional mutants [45, 189] . therefore, pre-emptive priming of t cell subsets to common escape variants could circumvent immune escape [176] , and thus could be incorporated into future vaccine strategies that will need to consider contrasting patterns of epitope variation and immune selection. a tailored mosaic peptide vaccine might be the best way to circumvent influenza virus escape, but on a population level for vaccine rollout would be impractical considering hla selection for full-length proteins and immune competition during t cell priming, this area of research needs further exploration. the h5-derived np protein is already able to be interchanged in the leningrad laiv backbone, demonstrating the possibility of a mosaic approach to incorporating t cell peptide variants with current methods [190] . furthermore, a recent study demonstrated that hiv-infected children, as opposed to hiv-infected adults, were able to generate hiv variant-specific cd8 + t cell responses limiting the selection of escape-epitope variants early in infection [191] . a superior immune response against natural influenza virus infections has also been observed in children aged 4-14, but is not well understood [192] , identifying a key window for vaccination of the t cell compartment to do better than nature. together, these studies suggest that including common tcr escape variant epitopes in a t cell-inducing influenza vaccine might be most effective in young children and could prevent vaccine-mediated t cell escape. overall, more research is needed to establish whether combining t cell epitopes, tcr escape variants, and timing of vaccination will benefit t cell-inducing influenza vaccines. in addition, little is known about cd4 + epitope variation during influenza infection due to a paucity of defined epitopes. furthermore, data is also lacking on individual human viral quasi-species generated during infection, with only one example of sequencing of t cell peptides from a longitudinal infected paediatric patient-derived viral rna [180] . this area of research will need to be expanded to enable a vaccine design that thwarts viral escape. while the recent licensure of recombinant ha protein-based vaccines can overcome manufacturing delays and problems with egg-adaptations, they represent only a marginal improvement of an old method based on inducing ha-targeted antibodies. this approach will not improve the breadth of vaccine coverage, and thus new and novel approaches towards influenza vaccination should be considered. t cell immunity has the strongest potential as the immune correlate capable for true universal pan-influenza immunity (table 1) . current efforts on the clinical development of pandemic vaccines to utilise t cells do not match this potential, and should be further prioritised. vaccines are currently being developed to utilize t cell-based immune control with the potential for "universal" protection from influenza virus infection. a number of strategies are in clinical development [193] (figure 3) , including the use of vectors such as modified vaccinia ankara (mva) and simian adenovirus encoding the internal np and m1 proteins of influenza, adenovirus 5 vectored vaccines containing the ha alone, and recombinant peptide approaches for mosaic of conserved peptides or np with m2e proteins. viral vectors have different safety profiles to recombinant proteins and inactivated vaccines, but maybe the most promising approach for robust immune responses with the potential to do "better than nature". viral vectors can be replicating (such as vaccinia, adenovirus 5, and simian adenovirus) and not safe for use in the immunocompromised and elderly. non-replicating vectors (including mva, e1-deleted adenoviruses, influenza minus ha signal peptide) can also be used, and are safe in everyone. promising results have been reported for the mva+np/m1 vaccine, boosting robust t cell responses in adults and the elderly [70] and protecting from influenza challenge [194] . furthermore, t cell-inducing vaccine strategies have also been assessed in ferret transmission models, specifically the ha minus signal peptide vaccine, which shows reduced transmission time and peak viral loads from vaccinated ferrets to naïve ferrets [75] . t cell-inducing vaccines for targeting pandemic potential viruses are a significantly under-utilised resource, with only 13% of pandemic potential vaccines in clinical development capable of inducing robust t cell responses ( figure 3a ). the majority (83%) of avian h5n1 and h7n9 pandemic potential vaccine candidates are being produced as inactivated vaccines, with the addition of adjuvant to increase immunogenicity and potentiate t cell responses. adjuvants are used in 76% of non-t cell-targeted vaccines, while only 13% of t cell-targeting vaccines use adjuvant ( figure 3a ). the methods of delivery for t cell-inducing vaccines typically do not require exogenous adjuvant because viral vectors are self-adjuvanting, but their use is constrained in some populations, such as the elderly, young children, and pregnant women. [193] , including those that use adjuvant or are reactive against avian influenza viruses (h5, h7, or h9 subtypes). vaccines missing input or not defined were excluded. (b) proportion of total vaccines from (a). * denotes vaccines which are designed to stimulate t cell response. squalene-oil-in-water-based adjuvants (i.e., mf59 and as03) have been licensed for use with some inactivated influenza vaccines in europe and the us, and have been reported to improve cd4 + t cell responses after vaccination, presumably through the robust stimulation of innate immune responses, leading to enhanced antigen presentation [195] [196] [197] . the mf59 iiv [72] can induce igg isotype switching in a cd4 + t cell-independent manner, while still inducing a robust cd8 + t cell response. this relationship may be exploited in the context of cd4 + t cell imprinting. there is also a suggestion that these adjuvants can improve cd8 + t cell responses. the vaccination of ferrets with mf59 or as03-adjuvanted, inactivated h5n1 vaccines elicited a stronger cd8 + t cell response upon virus challenge compared to unadjuvanted vaccines [111] . however, these adjuvants' effects on the cd8 + t cell populations in humans is limited [198] . care must also be taken when applying inferences from animal data to the human setting, as the majority of animal studies use naïve animals for testing vaccine responses, whereas only the very youngest in the human population are naïve to influenza virus. consensus on an acceptable level of protection for t cell vaccines -need to consider the context of pandemic preparedness and threat of zoonotic infections. -whilst immunity induced may not be sterilizing, a vaccine that reduces symptoms, the length and severity of infection, prevents deaths and hospitalizations, and potentially decreases transmission in the community may bring sufficient health and economic benefits to be worthwhile. targeting heterologous t cell responses that induce broad protection against diverse influenza virus strains and subtypes. -greater knowledge of influenza epitopes recognised by cd4 + and cd8 + t cells. -need to define universally conserved virus components that elicit broad spectrum t cell immunity for incorporation in a t cell vaccine. providing population-wide coverage across diverse hla profiles and ethnicities. -approaches that incorporate whole protein antigens or complete virus rather than selected peptides. -employ strategies to optimize peptide presentation and boost responses to subdominant epitopes (e.g., modification of extra-epitopic processing sites). -consider tailored vaccine design for ethnicities with unique or 'risk' hla profiles that place them at high risk of severe influenza disease. circumventing virus escape of t cell immunity. -spread immune pressure over multiple epitopes through use of full proteins or whole virus. -pre-emptively prime t cell subsets to common escape variants at a young age. -combine with other immune mechanisms such as induction of stem antibodies to reduce immune pressure on t cell epitopes. establish local immunity at the site of infection seeding durable, effective trm memory populations in the lung and upper respiratory tract. -requires local (i.e., intranasal) administration of antigen. -further work needed to determine the lifespan and stability of human lung and airway trm, and their potential to provide durable vaccine memory. synergize multiple immune mechanisms combining long-lasting broadly-reactive t and b cell immunity. -t cell vaccines should be used in combination with improved antibody vaccine targeting methods and induce multiple responses (e.g., peripheral cd8 + t cells, lung-resident cd8 + trm, cd4 + t fh cells, cd4 + t h1 , memory b cells and cross-reactive stem-specific antibodies) -in a combinatorial vaccine, it will be important to consider the long-term effects of vaccine imprinting on t cell and b cell responses, particularly in the face of ever-evolving influenza viruses. in addition to broadly protective t cell vaccines, various vaccination strategies to elicit ha-stem-specific antibodies are currently under development (reviewed in [199] ). however, high concentrations of ha-stem-specific antibodies are required to induce sterilizing immunity [200] . failure to induce high enough ha-stem-specific antibody titres in some individuals combined with the continuous immune pressure on this region in the rest of the population could eventually lead to unforeseen ha-stem escape mutations. a universal influenza vaccine strategy will greatly benefit from an additional layer of long-lasting broadly-reactive immunity in the form of a t cell component to dampen the severity and limit the spread of an influenza virus that managed to escape the ha stem-specific antibody response. reciprocally, the induction of additional ha-stem-specific antibodies with vaccination could help prevent vaccine-mediated t cell escape, as most natural influenza viruses will be neutralized before they establish an infection, limiting their exposure to cd8 + t cell immune pressure. a true universal influenza vaccine would combine the best of both worlds, as a "one-two" punch against influenza viruses. this review has highlighted some under-appreciated aspects of influenza-specific t cell immunity for consideration in the design of a universal vaccine of the future (table 1 ). this includes some caveats, such as the potential short half-life of t cell resident memory, t cell immune escape, and the impact of antigen presentation timing for immunodominance hierarchies in the context of hla alleles and population-wide coverage. further research is needed in the t cell field to identify their protective capacity and the optimal vaccine design for safe delivery, resulting in the longest duration effective memory population. while statistical models have predicted that a t cell-inducing vaccine is likely to be more effective in antibody naïve t cell-primed subjects for h7n9 infection than seasonal h3n2 viruses [201] , wider efficacy trials are needed in combination with clinical data. another challenge faced by t cell-inducing vaccines is that they will need to improve the standard of care provided by the current inactivated vaccines. inactivated vaccines and vaccine vectors containing influenza viral proteins elicit different immune correlates of protection, and have so far not been compared head-to-head in a large-scale efficacy trial. unfortunately, this is not in the foreseeable future when the role of t cells mediating protection from infection are still being fundamentally discussed. the use of t cell-inducing vaccines will require the standardisation of assays to assess t cell memory. the hai assay is a relatively simple rudimentary measure of virus-specific antibodies which has been in use for over 70 years, and is now well standardised [202] . in contrast, the study of influenza-specific t cell memory responses from human subjects is undoubtedly more technically-demanding and resource heavy compared to serology approaches. there is currently some concerted effort towards standardizing and streamlining experimental approaches for t cell assays in vaccine studies [203] , although it will likely take some time before a standard t cell assay is identified. while t cells have a vast potential for breadth of recognition against influenza viruses, the field is in its relative infancy compared to antibody-based vaccines. a quantum leap will be needed in adapting vaccine production, safety and efficacy trials, and standardisation of assays for their assessment on a large efficacy trial scale for their realization. the authors declare no conflict of interest. the 2003 world health assembly resolution on seasonal influenza vaccination coverage and the 2009 influenza pandemic have had very little impact on improving influenza control and pandemic preparedness influenza activity-united states, 2014-15 season and composition of the 2015-16 influenza vaccine h3n2 mismatch of 2014-15 northern hemisphere influenza vaccines and head-to-head comparison between human and ferret antisera 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trivalent influenza vaccine in a randomised trial of adults aged 65 and older cellular immune responses of older adults to four influenza vaccines: results of a randomized strategies to induce broadly protective antibody responses to viral glycoproteins heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine prior population immunity reduces the expected impact of ctl-inducing vaccines for pandemic influenza control standardization of hemagglutination inhibition assay for influenza serology allows for high reproducibility between laboratories workshop report: immunoassay standardisation for "universal" influenza vaccines. influenza other respir key: cord-335960-biwnqa3f authors: luke, anthony; d'hemecourt, pierre title: prevention of infectious diseases in athletes date: 2007-07-31 journal: clinics in sports medicine doi: 10.1016/j.csm.2007.04.006 sha: doc_id: 335960 cord_uid: biwnqa3f the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. this article reviews preventive strategies for infectious disease in athletes, including immunization recommendations and prophylaxis guidelines, improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, insect-borne disease precautions, and prevention of sexually transmitted diseases. a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. i nfections in sports can be serious medical problems. they can affect individual athletes, resulting in morbidity and decreased performance [1] . they can also be spread to other athletes, putting them at risk for similar disease and complications. the sports medicine physician may face challenging issues regarding infectious diseases when dealing with teams or highly competitive athletes who have difficulties taking time off to recover. one must treat the individual sick athlete and take the necessary precautions to contain the spread of communicable disease to the surrounding team, staff, relatives, and other contacts. the authors aim to translate recommendations in public health to practice in a sports medicine setting. sports physicians have an opportunity to see athletes during adolescence and adulthood who might not come in for routine health maintenance except when related to their sports. primary prevention of infectious disease, the ideal goal, deals with avoiding the development of the disease before infection occurs. immunizations are an example of primary prevention and have been the most successful public health programs for disease prophylaxis. secondary prevention for infection control involves prevention of spread to others. athletes are often exposed to many different people, travel to compete in various environments locally and internationally, and engage in higher-risk activities, often in close contact with others [2] . the authors discuss the preventive strategies for infectious disease in sport, including (1) a review of immunization recommendations and prophylaxis guidelines, (2) improvements in personal hygiene and prevention of spread of infectious organisms by direct contact, (3) insect-borne disease precautions, and (4) prevention of sexually transmitted diseases (stds). a special emphasis on immunizations focuses on pertussis, influenza, and meningococcal prophylaxis. athletes may share personal items (eg, towels, water bottles, and soap) and equipment (eg, weights). they may live in dormitories or in hotel rooms while traveling, which leads to close contact and high exposure to teammates. they participate in higher-risk activities [2, 11] . fewer athletes practice safe sex, which can lead to stds in homosexual and in heterosexual individuals. athletes also use more illicit drugs and alcohol, which can place them at risk of intravenous needle exposure [12, 13] . steroids, hormones, and vitamins are other substances that some athletes are injecting [14, 15] . tattoos are popular among athletes, which are another source of needle infection risk. active immunization involves the administration of all or part of a microorganism or a modified product of that microorganism, such as an antigen or protein. this administration stimulates an immune response to develop protection against future exposure to the infection [16] . although most vaccines are over 90% effective, they are not guaranteed to promote immune protection [16] . immunizations typically involve inactivated vaccines or live, attenuated viruses. inactivated vaccines include killed virus or bacterial proteins to stimulate one's immune system to develop antibodies to any similar virus or bacteria. more side effects are associated with the live, attenuated viruses-typically local pain and, rarely, hypersensitivity reactions to vaccine constituents. a mild febrile illness, a recent exposure to an infectious disease, pregnancy, breastfeeding, nonspecific allergies, and family history of an adverse event after immunization, including seizures, are not contraindications for immunization [16] . sports medicine physicians need to consider the following indications for immunizations (tables 1 and 2) : (1) routine health maintenance; (2) catch-up immunizations for failed or missed immunizations; (3) immunizations of high risk groups (ie, splenectomy, chronic disease, immunocompromised); (4) travel to an endemic area; (5) close contact with an infected individual, or (6) recent potential exposure to an infectious agent. when doing preparticipation physical examinations, it is sometimes assumed that athletes have received all their immunizations. proof of immunizations is required by schools and colleges, although exceptions can be given to individuals refusing to receive immunizations. in a study surveying 69,115 minnesota children who entered kindergarten in 1992, by 19 months of age, 73% of students had received the measles, mumps, and rubella (mmr) vaccine, and only 39% had received their fourth dose of diphtheria, tetanus, and pertussis vaccine (dtap) [17] . vaccination rates can vary substantially by age, race/ethnicity, and neighborhood [17] . white, non-hispanic students usually have higher vaccination rates than children of other racial/ethnic groups. it has been estimated that 79% of whites, 76% of hispanics, and 71% of african americans are fully immunized [18] . although mmr vaccines have been administered for many decades and incidences of disease are presently low [7] , the diseases can still occur in the adult population. between 1986 and 1989, 6% of the measles cases occurred in college students [19] . enzyme-linked immunosorbent assay tests for antibodies to mmr are available to detect for immunization status [19] . in a series of 256 students, 53 (21%) were found to be seronegative to measles alone, 13 (5%) were seronegative to rubella alone, and 5 (2%) were seronegative to measles and rubella. eighty-six percent of the individuals seronegative to measles had previously received a dose of measles vaccine. following a second injection, conversion to seropositive status rose to 97% and 100% for measles and rubella, respectively. these data support the need for a two-dose vaccine schedule [19] . pneumococcal vaccine is administered to prevent streptococcus pneumoniae infections. a conjugate heptavalent is given to during the first 2 years of life. a polysaccharide vaccine is provided to high-risk individuals older than 2 years against 23 types of streptococcus pneumonia that account for 90% of invasive disease [20] . high-risk groups include patients who have asplenia, sickle cell disease, diabetes mellitus, cirrhosis, immunocompromised states, chronic cardiac or pulmonary disease, or age 65 years or older. immunity following vaccination is successful for periods of 5 to 10 years, requiring booster injections [20] . hepatitis b is a blood-borne virus transmitted through sexual contact and parenteral exposure to blood and blood components [14] . hepatitis b has a greater risk for transmission in sports than hiv. the risk of hiv transmission is estimated to between 1 in 1 million games and 1 in 85 million games [14, 21] . the risk arises if bleeding and skin exudates from an infected individual come into contact with open wounds in another athlete, particularly during contact and collision sports. there are no confirmed cases of spread of hiv through sports [14] ; however, 5 out of 10 high school sumo wrestlers at one club developed hepatitis b [22] . another case series reported on 11 of 65 american football players who developed hepatitis b over a period of 19 months [23] . contact through open wounds, cuts, and abrasions were the suspected routes of transmission. although hepatitis a is a considered immunization in athletes who are traveling to endemic areas, routine vaccination for hepatitis b is recommended for all individuals after birth using single or combination vaccines [24] . a three-dose immunization schedule is typically used after 18 years of age, with injections at 0 months, 1 month, and 6 months, although there is an optional four-dose schedule [25] . the licensed vaccines have had 90% to 95% efficacy of preventing hepatitis b, with immunity lasting 15 years or longer [25] . immunizations for hepatitis b should be checked during the preparticipation physical examination, and catch-up immunizations recommended to the individual (see table 2 ). if individuals are uncertain about their immunization status, serologic testing for antibody to hepatitis b surface antigen can determine immunity. when athletes are known to be infected with hepatitis b, secondary prevention includes education on personal hygiene, appropriate management of open wounds, proper use of protective equipment, safe sex practices using a condom, and avoidance of intravenous blood transmission (eg, through needle sharing and illicit drug use). bordetella pertussis, which is responsible for whooping cough, is a gram-negative coccobacillus transmitted by way of airborne droplets [26] . although tetanus and polio have been controlled well with the use of vaccines [7] , the rate of pertussis cases has been increasing in adolescents and adults despite routine immunizations [27] . most cases occur in patients 10 years or older [28] . the infection is most concerning for infants because immunity is not complete until older ages. the spread to infants is typically from adults. pertussis usually presents with nonspecific upper respiratory tract infection symptoms for 1 to 2 weeks (catarrhal stage), after which the paroxysmal and sometimes uncontrollable cough develops [26] . the cough is not necessarily always followed by the classic ''whooping'' sound, and pertussis should be considered with any persistent, prolonged cough. the whole-cell pertussis vaccine is estimated to be approximately 85% effective [29] . this vaccine is still recommended for use in the routine immunization of young children; however, the immunity provided begins to decline at 4 to 12 years following vaccination, which makes adolescents and adults susceptible [27] . rare adverse reactions from the vaccine include hypotonic, hyporesponsive episodes, high fever, seizures, and anaphylaxis [26] . two acellular vaccines have been introduced that are as effective as whole-cell vaccines and have fewer adverse reactions [30] . these vaccines are combined with tetanus toxoid and reduced diphtheria toxoid (dtap). the centers for disease control and prevention (cdc) recommends use of these dtap boosters rather than the tetanus-diptheria (td) booster starting after 11 to 12 years of age [31] . for pertussis, individuals are most contagious during the first 1 to 2 weeks during the catarrhal stage but should be considered contagious until 3 weeks after the paroxysmal stage ends or after taking antibiotics for 5 days [32] . diagnosis of pertussis infection is best performed through polymerase chain reaction assay (sensitivity, 94%; specificity, 97%) or through direct fluorescent antibody testing (sensitivity, 52%; specificity, 98%). nasal swab cultures (sensitivity, 15%; specificity, 100%) are routinely performed; however, they have high false negative rates and take 7 to 12 days to yield results [33] . physicians in the united states are legally required to report cases of pertussis to state public health departments [26] . it is estimated that 80% of susceptible household contacts will be infected after close contact [26] . antibiotic prophylaxis is recommended for close contacts of persons who have pertussis to prevent outbreaks [34] . preferred drugs are azithromycin for 5 days, clarithromycin for 7 days, or trimethoprim-sulfamethoxazole or erythromycin for 14 days, which are similar for prophylaxis and treatment [34] . influenza presents with constitutional symptoms of fever, chills, malaise, fatigue, and myalgia in addition to upper respiratory tract symptoms of a sore throat, cough, and rhinitis. rarely, more serious conditions can occur, including encephalopathy, transverse myelitis, myocarditis, and pericarditis [9] . immunogenicity is determined by hemaglutinins and neuraminidases on the virus surface. antigenic drift can occur that can mutate the virus into different strains. transmission occurs by way of respiratory droplets. the virus has an incubation period of usually 2 days (range, 1-4 days), and adults are infectious from the day before symptoms begin to approximately 5 days after the illness starts [9] . symptoms usually last a week, although less likely, symptoms can last longer than 2 weeks. these symptoms can be very disruptive for treatment and challenging for the athlete to keep training and competing. a case series of 81 students, mostly healthy adolescents at a ski school in austria, reported a severe outbreak of influenza a, leading to an attack rate of 49%, with 69% becoming ill within 2 days of the outbreak. two students were hospitalized with pneumonia and 1 died [35] . influenza vaccines contain strains of antigenically equivalent strains of influenza similar to those annually recommended: influenza a (h3n2), influenza a (h1n1), and a b virus. depending on the emergence and spread of new strains, other virus strains can be added to update the vaccine [9] . the efficacy of influenza vaccine is approximately 70% to 90% for individuals under age 65 years [36] . vaccination for influenza should occur in the fall (october or november), at the beginning of the flu season (box 2) [9] . antibodies develop approximately 2 weeks after vaccination [9, 37] . inactivated influenza vaccine is generally appropriate for all populations requiring influenza vaccine. three influenza vaccines were available in the united states for the 2006 to 2007 season: fluzone (manufactured by sanofi-pasteur); fluvirin (manufactured by novartis); and fluarix (manufactured by glaxosmithkline). the typical dose is 0.5 ml administered intramuscularly, usually in the deltoid muscle. live, attenuated influenza vaccine (laiv) is approved for use in healthy, nonpregnant individuals aged 5 to 49 years. the laiv is administered by way of a nasal spray once in each nostril (flumist, manufactured by medimmune). individuals who have a hypersensitivity or anaphylactic reaction to components of the flu vaccine or to eggs should not be vaccinated [9] . adults reported having a 19% reduction in severe febrile illnesses after laiv compared with placebo [38] . side effects from laiv increased in adults within 7 days of immunization compared with placebo and consisted mainly of nasal congestion (44.5% versus 27.1%) and sore throat (27.8% versus 17.1%), which lasted, on average, 2 days. less common complaints were tiredness, cough, and chills. there was no significant difference in the number of mild febrile illnesses between immunization and placebo groups [39] . injections can be scheduled to occur at the optimum time during the athlete's competitive schedule to minimize concern about side effects. when inactivated influenza vaccine shortages occurred in previous years, the vaccine was recommended for high-risk groups as priority; however, the general recommendation now is to offer the immunization annually to anyone who wishes to reduce the likelihood of being ill with influenza or transmitting the virus if they should become infected [9] . although this policy cannot be directly translated into a benefit for the athlete, depending on the level of athlete, the use of the laiv may also be beneficial to prevent lost time from sport. influenza vaccine has been suggested for competitive athletes and essential personnel, especially before international events occurring during the influenza season [4, 40] . treatment with antiviral medications can reduce the duration of uncomplicated influenza a and b illness by approximately 1 day when administered within 2 days of illness onset [41, 42] . recommended antiviral treatment should be given for 5 days [9] . four antiviral agents are currently available: amantadine, rimantadine, zanamivir, and oseltamivir [9] . the influenza a virus, however, has become resistant to amantadine and rimantadine, which are presently not recommended to be used as first-line drugs [43] . zanamivir (ralenza, dry powder taken by orally inhaled route) and oseltamivir (tamiflu, capsule or oral suspension) are neuraminidase inhibitors and can be used to treat patients and to control influenza outbreaks in closed settings. although typically used in nursing homes, an outbreak in a dormitory may require chemoprophylaxis [9] . there are limited data to suggest that serious complications from influenza, such as lower respiratory tract infections, may be reduced [44] . the use of antiviral medications for prophylaxis of influenza is unclear and is not yet recommended for routine seasonal control [45] . the use of oseltamivir, however, has been recommended in specific cases, especially if there is high risk of spread such as household contacts and if individuals have not been immunized [46] . oseltamivir was used to treat 36 of 188 patients, including 13 athletes during the 2002 salt lake city winter olympics, with medications given to close contacts, which was believed to limit the spread of influenza [47] . clinical history and physical examination are still the mainstays for diagnosing influenza. rapid swab tests are available and take approximately 30 minutes to detect the influenza virus. the tests are less sensitive (72%-95%) and specific (76%-86%) than the traditional viral cultures [48] . they have moderate sensitivities for influenza antigens and are more likely to produce false negative rather than false positive results [48, 49] . direct and indirect fluorescent antibody staining tests are also available, but they are ordered more in hospitals because they take 2 to 4 hours to obtain results [49] . viral cultures are still the ''gold standard'' for confirming the presence of influenza and identifying the strains and subtypes [9] . neisseria meningitides is a serious concern, especially for the adolescent and college populations. an alarming trend during the 1970s demonstrated an increase in meningitis deaths in college students, with living in dormitories being a risk factor. the disease can be spread by asymptomatic carriers. students living in dormitories were 9 to 23 times more likely of getting infected than those living in other types of accommodations [50] . freshmen who lived in dormitories had an elevated risk of meningococcal disease (odds ratio, 3.6; 95% confidence interval, 1.6-8.5; p â¼ .003) compared with other college students [51] . aside from the risk of death, 11% to 19% of survivors of meningitis have serious sequelae such as neurologic disability, limb loss, and hearing loss [50] . routine vaccination with meningococcal vaccine is recommended for college freshmen living in dormitories and for other populations at increased risk. the cdc advisory committee on immunization practices recommends routine vaccination of young adolescents (11-12 years old) with meningococcal vaccine (mcv4) at the preadolescent health care visit [50] . therefore, sports medicine physicians may be faced with higher frequency of checking for meningococcal immunization status for high school and college athletes. a tetravalent conjugate vaccine (menactra, sanofi pasteur) is available against neisseria meningitidis isolates a, c, y, and w-135 in a 0.5-ml single-dose vial. over the age of 11 years, 75% of the meningococcal infections are caused by strains c, y, or w-135 (cdc, unpublished data, 2004) [50] . another vaccine, menomune (aventis pasteur limited), has been licensed since 1981 and has a similar immunogenicity profile to menactra and is delivered subcutaneously as a 0.5-ml dose. menactra and menomune have serum bactericidal protection ranging from 89.4% and 94.4% for strain w-135 and 73.5% and 79.4% for strain y, respectively [50] . revaccination may be necessary for individuals at high risk after 5 years [52, 53] . common side effects with mcv4 were local pain in just over 50% of patients, followed by swelling, induration, and redness in approximately 10.8% to 17.1%. fever was reported in 5.1% of children 18 years old or younger and in 1.5% of adults [50] . close contacts are at high risk and should be treated with chemoprophylaxis ideally within 24 hours of identifying the index patient [50] . the goal of treatment is to reduce nasopharyngeal carriage of n meningitidis. after more than 14 days after the onset of illness in the index patient, chemoprophylaxis is not necessary [50] . a single dose of ciprofloxacin (500 mg orally) or ceftriaxone (250 mg by intramuscular injection), or rifampin (600 mg twice a day for 2 days) is recommended for adults. children between 1 month and 18 years old may take rifampin (10 mg/kg every 12 hours for 2 days), or ceftriaxone (125 mg intramuscularly) if younger than 15 years [50] . one dose of azithromycin (500 mg) was also shown to eradicate n meningitidis and may represent another treatment option [54] . human papillomavirus (hpv) is associated with 99% of cervical cancers and anogenital, head and neck, and nonmelanoma skin cancers. it is an std and can be diagnosed by abnormal cervical cell changes seen on pap smear [55] . this is a common infection, especially in sexually active adolescents and university students [56] . primary prevention is now possible with two new vaccines: a bivalent vaccine against hpv types 16 and 18 and a quadravalent vaccine against types 6, 11, 16, and 18. the vaccines have a three-dose schedule: 0, 1, and 6 months (bivalent vaccine) and 0, 2, and 6 months (quadravalent vaccine). at 4.5 years, the bivalent vaccine was effective for producing a persistent antibody response against hpv 16 and 18, with more than 98% seropositivity and 96.9% effectiveness (95% confidence interval, 81.3-99.9) in reducing the number of reported abnormalities on pap smear, colposcopy, or both [57] . routine vaccination with three doses of quadrivalent hpv vaccine is recommended for girls 11 to 12 years old but can be started in girls as young as 9 years. girls and women aged 13 to 26 years who have not been vaccinated previously or who have not completed the full vaccine series are recommended to receive a catch-up series. the vaccine is intended to be administered before potential exposure to hpv through sexual contact [58] . secondary prevention involves checking the affected individual's partners for signs of genital warts and other stds. regular cervical screen is recommended. use of condoms and education on spread is important. hpv infection persists for life; however, the degree and duration of contagiousness is yet unknown [59] . athletes traveling need to consider the endemic diseases in the geographic location where they are competing. they should be aware of risks of acquiring common diseases, their accommodations (urban versus rural), local foods, and customs. immunizations should ideally be planned 4 or more months in advance to allow for adequate time to administer vaccines (table 3 ). there are many resources for information about prevention of infectious diseases for travelers (table 4) . mosquito-borne disease a number of arthropods, such as mosquitoes and ticks, can transmit diseases. mosquito-vector diseases include west nile virus, yellow fever virus, and dengue virus. west nile virus, a flavivirus, has demonstrated a seasonally endemic epidemiology with geographic variation in the united states, especially in california, arizona, and colorado [7, 61] . this disease typically presents between july and october, although cases have presented between april and december. the prevention of mosquito bites is the cornerstone of prevention. an athlete in an endemic area should wear an insect repellant such as deet (n,n-diethyl-mtoluamide), picaridin (kbr-3023), or oil of lemon eucalyptus (p-menthane-3,8 diol). deet and permethin may be applied to the clothing [62] . if a sunscreen is used concomitantly, the insect repellant should be applied on top of this and removed at the end of the day. long-sleeved shirts that are tucked into long pants are also useful. tick-borne diseases include rickettsial diseases, lyme disease, babesiosis, tickborne relapsing fever, and occasionally, tularemia and q fever (table 5) . certain athletes who participate in rural outdoor activities are more susceptible to tick bites. these sports include cross-country running, training in multiple sports in rural areas, and recreational outdoor sports such as fishing and hiking. children are more at risk to tick bites. three more common rickettsial illnesses are rocky mountain spotted fever, human monocytotropic ehrlichiosis, and human granulocytotropic anaplasmosis [60] . the infectious organisms responsible for these illnesses maintain their lifecycles in mammals and ticks. their prevalence reflects the geographic locations and the seasonality of the tick abundance. their season is usually from april to september, but they can present throughout the year. newer rickettsial diseases are emerging. these potentially lethal diseases are difficult to diagnose because they often mimic viral syndromes. as many as 60% to 75% of patients are initially misdiagnosed [63, 64] . with rocky mountain spotted fever, more than 50% of cases are reported in the five states of north carolina, south carolina, tennessee, oklahoma, and arkansas [65] . the presentation most often manifests as a sudden febrile illness with headache, myalgia, and a maculopapular rash that spreads in a centripetal pattern. rickettsia rickettsii has a predilection for endothelial cells and can cause a diffuse vasculitis and an untreated mortality of 10%. the diagnosis is based on clinical presentation, with epidemiologic, geographic, and seasonal considerations. laboratory testing may be supportive with thrombocytopenia and mild liver enzyme elevation. serologic testing is supportive only on a delayed basis with acute and convalescent titers. human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis can also present with acute headache, fever, and myalgia. laboratory evaluation often demonstrates leukopenia, thrombocytopenia, and transaminase elevation. common tick-borne illnesses that have been reported in the northeast united states are lyme disease and babesiosis, which are transmitted by the tick ixodes scapularis [66] . babesiosis can cause a febrile illness and possibly life-threatening anemia and thrombocytopenia. lyme disease is a rickettsial disease caused by borrelia burgdorferi. as such, concurrent disease may be caused by the same tick bite (see table 4 ). there are no proven vaccines for these tick-borne illnesses, but all are preventable by careful vigilance and protection. the key to prevention is to understand the regional epidemiology and seasonality of the diseases. vaccination for lyme disease (lymerix) was originally approved; however, the manufacturer took the vaccine off the market due to declining sales. there was a 49% efficacy after two doses and a 76% efficacy after three doses [67] ; however, the protection diminished after 2 years. ticks thrive in a wooded environment and at the edge of woods with surrounding high vegetation. ticks are uncommon in well-mowed lawns. relative tick-free zones can be created by placing wood chips or gravel around recreational areas to separate the woods [68] . other landscape management tips include removing clippings and leaves, keeping stone walls clean of leaves, and restricting the use of groundcover, such as pachysandra, where pets and children may play. widening woodland trails andkeeping in the center of the trail while walking may be helpful. when traveling in wooded areas, light-colored clothing is helpful to identify the tick. long pants tucked into tightly woven socks and closed shoes minimize exposure. deet at 10% to 25% should be applied to the skin. permethrin may be applied only to the clothing. clothes should be removed and cleaned and dried after exposure. the clothes dryer is effective in killing ticks. one should carefully check for ticks in the nymphal phase that may be the size of a pin head. careful inspection should be done of the hair, ears, axilla, belly button, and legs. children and pets should also be checked. it is also important to monitor pets that may travel in the woods and return indoors. the technique of tick removal is critical. tweezers with fine tips should be used close to the skin and pulled directly away. squeezing the body may allow contamination of the disease into the host [69] . lyme disease is not contracted until at least 24 hours of tick adherence [70] ; however, ehrlichiosis may transmit in less than 24 hours. preventive antibiotics are generally not indicated because less than 5% of bites are lyme infected, especially with a flat tick. after a high-risk exposure (when the tick has been engaged for more than 24 hours and is engorged), a single dose of 200 mg of doxycycline is believed to be effective [71] . most infectious diseases are spread from contact with the microorganism directly or indirectly from the infected individual. athletes frequently interact with teammates, coaches, athletic trainers, and physicians and share equipment, water bottles, towels, and supplies. this interaction is particularly a concern, with the recent outbreaks of methicillin-resistent staphylococcus aureus (mrsa) infections among sports teams [72, 73] . three categories of potential risk factors for spreading infection have been suggested: ''sharing'' (sharing soap/towels/water bottles with teammates), ''skin injury'' (cuts, abrasions), and ''close contact'' (locker adjacent to infected teammate, living on-campus) [74] . good personal hygiene can help reduce colonization of bacteria. bacterial counts can range from 5000 to 5 million colony-forming units per square centimeter on the hands [75] . universal body fluid precautions-for example, using disposable gloves when examining the oral cavity or wounds and frequent hand washing-can reduce the risk of infection. mrsa is transmitted from an infected patient to the gloves of a health care worker in approximately 17% (9%-25%). physicians, in particular, have a low compliance for using gloves and washing their hands [76] . proper surgical hand washing is recommended to be 15 to 30 seconds with soap, a 30-second rinse with water, followed by complete drying with a towel. the use of rinses and gels with concentrations of 50% to 95% alcohol take 15 seconds to use and are effective at killing organisms [75] . the use of chlorhexidine soap has been useful for reducing mrsa infections. viruses and bacteria can exist on equipment. mrsa was found in the taping gel and whirlpool in the training facilities of a professional football team [72] . using diluted bleach (1 part bleach in 9 parts water) to cleanse training areas and equipment is recommended [8] . routine cleaning schedules for shared equipment should be established and recorded. for upper respiratory tract infections, isolation of those who have had close contact with someone who has a confirmed or suspected infection, especially those who have active symptoms such as persistent fever and cough, is an effective and practical method of avoiding contact [8] . any athlete who has a scratch, abrasion, or laceration or who has potentially infectious skin lesions such as vesicular or weeping skin lesions should be removed from play until the area can be securely covered with occlusive bandages or dressings to prevent leakage of blood or serous fluid [77] . uniforms with fresh blood should be removed and replaced immediately after stopping any bleeding. bleach diluted with tap water in a 1:10 ratio can be used to wash equipment that has had contact with blood or body fluid. body substance precautions should be taken by health care professionals at all times when treating open wounds. one type of bacteria that has become more common in the hospital and a community-acquired infection is mrsa. although contact sports such as wrestling and football have been commonly associated with mrsa spread, this infection has also been discovered in minimal-contact sports such as fencing [78] . three factors are associated with mrsa spread in sports. first, even with sports that have minimal contact, there are often abrasions and chaffing from clothing and hot environments. second, equipment is often shared and there is potential for transmission of bacteria. third, many sports have sufficient skin-to-skin contact to transmit organisms. subsequently, health care providers should strongly encourage good overall and hand hygiene in addition to covering all wounds and limiting shared equipment. it is crucial to have an ample supply of soap and water and alcohol-based hand cleansers. athletes, staff, and coaches should be educated in proper first aid for wounds, in recognition of wounds that are potentially infected, and in seeking medical attention for lesions that have concerning signs, especially large pustules or boils. athlete's foot, tinea pedis, is a common ailment not only during the hot summer months but also during the winter months with indoor sports. a number of prevention items include washing feet daily; drying between the toes; wearing cotton, nonsynthetic socks; wearing bathing shoes in public showers; and wearing sandals in warmer weather. jock itch, tinea cruris, is best prevented by showering immediately after athletic endeavors and wearing cotton briefs. a good talc powder may be used for prevention of athlete's foot and jock itch. ring worm, tinea corporis, is best prevented by avoiding contact. contact athletes such as wrestlers should not participate until any lesions have cleared or can be safely and effectively covered. athletes may manifest risk-taking behavior and subsequently be at increased risk for stds [2] . the preparticipation examination affords the opportunity for the clinician to address these concerns. the cdc has emphasized the five intervention strategies [79] , which include education on sexual behavior, identification of asymptomatic individuals, diagnosis and treatment of infected individuals, counseling of sexual partners of persons who have an std, and pre-exposure vaccination when applicable. individuals at risk should be questioned about partners regarding number and same or opposite sex. information about the type of sexual activity, the use of protection, and history of previous stds should also be identified. preventive measures for an std include abstinence if an individual or partner is actively infected and undergoing treatment. pre-exposure prophylaxis is relevant in several situations. hepatitis b vaccine is recommended in all individuals potentially exposed to stds. hepatitis a vaccine is recommended for all men who have sex with men or users of illicit drugs (injectable and noninjectable). for girls and women aged 9 to 26 years, the new quadrivalent vaccine for hpv types 6, 11,16, and 18 is recommended due to the higher associated risk of cervical cancer. most condoms are made of latex and are quite effective in std prevention. in one study of partners of hiv-infected individuals, partners were 80% less likely to seroconvert than those who did not use condoms [80] . the male condom can also reduce the transmission of gonorrhea, chlamydiosis, and trichomiasis [81] . there may be some added protection against herpes simplex virus 2 and a 70% reduction of hpv transmission [82, 83] . when an individual is allergic to latex, certain polyurethane condoms are likely just as effective, although they may break more readily. conversely, natural-membrane condoms such as lambskin are too porous to be used for std prevention. only water-based lubricants should be used with latex condoms because oil bases will weaken the latex. the female condom is a double-ringed polyurethane sheath that is used vaginally and during anal receptive intercourse that is effective in limited trials in preventing hiv/stds [84, 85] . spermicides and nonbarrier contraception have no role in std prevention. finally, providers should encourage patients who have stds to notify their partners. often, this notification is pursued by the public health department. in the event of exposure to hiv by sexual exposure or needle stick, hiv prophylaxis is often undertaken and should be immediate. prophylactic treatment usually involves the hospital infectious disease division to determine the best combination therapy. education is paramount in public health and in the prevention of infectious diseases. athletes are a high-risk population often due to their increased exposure to different people and environments and, sometimes, their outgoing lifestyle behaviors. primary prevention can be promoted through accurate immunizations; appropriate, planned health maintenance; good hygiene practices; and behavior modification to minimize high-risk activities. secondary prevention can be achieved through vigilant surveillance for reportable illnesses, proper education and containment for reducing spread if an illness occurs, and timely prophylaxis with medications and immunizations in certain cases. virus infections and sports performance-a prospective study lifestyles and health risks of collegiate athletes: a multi-center study an outbreak of measles at an international sporting event with airborne transmission in a domed stadium influenza 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transmission risk behaviors and sexually transmitted diseases among women living with hiv: the willow program effect of condoms on reducing the transmission of herpes simplex virus type 2 from men to women condom use and the risk of genital human papillomavirus infection in young women use-effectiveness of the female versus male condom in preventing sexually transmitted disease in women use of reality ''female condoms'' for anal sex by us men who have sex with men. hivnet vaccine preparedness study protocol team key: cord-289285-aof7xy13 authors: michaelis, martin; geiler, janina; naczk, patrizia; sithisarn, patchima; leutz, anke; doerr, hans wilhelm; cinatl, jindrich title: glycyrrhizin exerts antioxidative effects in h5n1 influenza a virus-infected cells and inhibits virus replication and pro-inflammatory gene expression date: 2011-05-17 journal: plos one doi: 10.1371/journal.pone.0019705 sha: doc_id: 289285 cord_uid: aof7xy13 glycyrrhizin is known to exert antiviral and anti-inflammatory effects. here, the effects of an approved parenteral glycyrrhizin preparation (stronger neo-minophafen c) were investigated on highly pathogenic influenza a h5n1 virus replication, h5n1-induced apoptosis, and h5n1-induced pro-inflammatory responses in lung epithelial (a549) cells. therapeutic glycyrrhizin concentrations substantially inhibited h5n1-induced expression of the pro-inflammatory molecules cxcl10, interleukin 6, ccl2, and ccl5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with h5n1 replication and h5n1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). glycyrrhizin also diminished monocyte migration towards supernatants of h5n1-infected a549 cells. the mechanism by which glycyrrhizin interferes with h5n1 replication and h5n1-induced pro-inflammatory gene expression includes inhibition of h5n1-induced formation of reactive oxygen species and (in turn) reduced activation of nfκb, jnk, and p38, redox-sensitive signalling events known to be relevant for influenza a virus replication. therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of h5n1 disease. highly pathogenic h5n1 influenza a viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . at least for the first wave of an h5n1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . therefore, antiviral therapy for influenza a viruses including highly pathogenic h5n1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] . the neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza m2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . however, the use of both drug classes is limited by the emergence of resistant virus strains. in seasonal influenza strains, the majority of h3n2 viruses and a great proportion of h1n1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . moreover, a drastic increase in oseltamivir-resistant h1n1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . preliminary data from the united states predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating h1n1 strains to be oseltamivir resistant [14] . h5n1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza a virus strains and treatment-resistant h5n1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . more-over, parenteral agents for the treatment of seriously ill patients are missing. glycyrrhizin, a triterpene saponine, is a constituent of licorice root. it has been found to interfere with replication and/or cytopathogenic effect (cpe) induction of many viruses including respiratory viruses such as respiratory syncytial virus, sars coronavirus, hiv, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . the severity of human h5n1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . delayed antiviral plus immunomodulator treatment reduced h5n1-induced mortality in mice [31] . therefore, antiinflammatory and immunomodulatory effects exerted by glycyrrhizin may be beneficial for treatment of h5n1. also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza a virus replication and virus-induced pro-inflammatory responses [32] [33] [34] . stronger neo-minophagen c (snmc) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in japan for the treatment of chronic hepatic diseases and is marketed in japan, china, korea, taiwan, indonesia, india, and mongolia. here, we investigated the influence of snmc on h5n1 replication, on h5n1-induced cytokine expression, on h5n1-induced cellular oxidative stress, and on critical h5n1-induced cellular signalling events in human pneumocytes (a549 cell line). glycyrrhizin (stronger neo minophagen c) was obtained from minophagen pharmaceuticals co., ltd. (tokyo, japan). the influenza strain a/vietnam/1203/04 (h5n1) was received from the who influenza centre (national institute for medical research, london, uk). the h5n1 influenza strain a/thailand/ 1(kan-1)/04 was obtained from prof. pilaipan puthavathana (mahidol university, bangkok, thailand). virus stocks were prepared by infecting vero cells (african green monkey kidney; atcc, manassas, va) and aliquots were stored at 280uc. virus titres were determined as 50% tissue culture infectious dose (tcid 50 /ml) in confluent vero cells in 96-well microtiter plates. a549 cells (human lung carcinoma; atcc: ccl-185, obtained from lgc standards gmbh, wesel, germany) were grown at 37uc in minimal essential medium (mem) supplemented with 10% fbs, 100 iu/ml of penicillin and 100 mg/ml streptomycin. human monocytes were isolated from buffy coats of healthy donors, obtained from institute of transfusion medicine and immune haematology, german red cross blood donor center, johann wolfgang goethe-university, frankfurt am main. after centrifugation on ficoll (biocoll)-hypaque density gradient (biochrom ag, berlin, germany), mononuclear cells were collected from the interface and washed with pbs. then, monocytes were isolated using magnetically labeled cd14 microbeads (miltenyi biotec gmbh, bergisch gladbach, germany) following the manufacturer's instructions. monocytes were cultivated in imdm supplemented with 10% pooled human serum, 100 iu/ml of penicillin, and 100 mg/ml streptomycin. the cellular viability was assessed on confluent cell layers with celltiter-gloh luminescent cell viability assay (promega gmbh, mannheim, germany) according to the manufacturers' protocol. cell viability was expressed as percentage of non-treated control. to determine intracellular np localisation, h5n1-infected a549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, mallinckrodt baker b.v., deventer, the netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in pbs) directed against the influenza a virus nucleoprotein (np) (millipore, molsheim, france). an alexa fluor 488 goat anti-mouse igg (h&l) (invitrogen, eugene, oregon, usa) was used (1 h incubation, 1:1000 in pbs) as secondary antibody. nuclei were stained using 49,6-diamidino-2phenylindole (dapi) (sigma-aldrich chemie gmbh, munich, germany). fluorescence was visualised using olympus ix 1 fluorescence microscope (olympus, planegg, germany). for flow cytometric analysis, the same antibodies were used. the cytopathogenic effect (cpe) reduction assay was performed as described before [34] . confluent a549 cell monolayers grown in 96-well microtitre plates were infected with influenza a strains at the indicated multiplicities of infection (mois). after a one hour adsorption period, cells were washed to remove non-detached virus. the virus-induced cpe was recorded at 24 h post infection (p.i.). unless otherwise stated, a549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. for time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus. total rna was isolated from cell cultures using tri reagent (sigma-aldrich, munich, germany). real time pcr for h5 was performed using described methods [35] . the following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-fam-tca aca gtg gcg agt tcc cta gca-tamra. the fraction of cells with fractional dna content (''sub-g1'' cell subpopulation) indicates cytotoxicity. sub-g1 cells are considered to be dead (usually apoptotic) cells. cells were fixed with 70% ethanol for two hours at 220uc. the cellular dna was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (facscalibur, bd biosciences, heidelberg, germany). caspase activation was measured using the caspase-glo 8, 9, or 3/7 assays (promega, mannheim, germany) following the manufacturer's instructions. cell culture supernatants were collected and frozen at 280uc. cytokines/chemokines were quantified by specific elisa duo sets (r&d systems gmbh, wiesbaden, germany) following the manufacturer's instructions. nfkb activity was investigated in h5n1 (moi 0.01)-infected cells by quantification of the nfkb subunits rel a (p65) and nfkb1 (p50) from nuclear extracts using the transam tm transcription factor dna-binding elisas (active motif, rixensart, belgium). nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; bd biosciences, heidelberg, germany). monocytes (1610 6 in 100 ml of imdm with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. after a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with pbs containing 0.3% tritron x-100. then, nuclei were stained with 49,6-diamidino-2phenylindole (dapi). the upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with pbs. the number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an olympus ix 1 fluorescence microscope (olympus, planegg, germany). cells were lysed in triton x-sample buffer and separated by sds-page. nuclear extract were prepared using the nuclear extract kit (active motif, carlsbad, ca, usa) following the manufacturer's instruction. proteins were detected using specific antibodies against bactin (sigma-aldrich chemie gmbh, munich, germany), jnk, phosphorylated jnk, p38, or phosphorylated p38, (all purchased from new england biolabs gmbh, frankfurt am main, germany) and were visualised by enhanced chemiluminescence using a commercially available kit (amersham, freiburg, germany). reactive oxygen species (ros) were detected using the image-it live green reactive oxygen species kit (molecular probes, distributed by invitrogen, karlsruhe, germany). two groups were compared by t-test. more groups were compared by anova with subsequent student-newman-keuls test. the a549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type ii pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. if not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect a549 cell viability (data not shown) but clearly decreased cpe formation in a549 cells infected with the h5n1 influenza strain a/thailand/1(kan-1)/04 at mois of 0.01, 0.1 or 1 ( figure 1a ). similar results were obtained in a549 cells infected with strain a/vietnam/1203/04 (h5n1) (suppl. figure 1a) . staining of a549 cells for influenza a nucleoprotein 24 h after infection with strain h5n1 a/thailand/1(kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza a nucleoprotein positive cells ( figure 1b) . to examine the influence of glycyrrhizin on virus progeny, a549 cells were infected with the h5n1 influenza strain a/ thailand/1(kan-1)/04 at moi 0.01 or moi 1 and infectious virus titres were determined 24 h post infection ( figure 1c ). while glycyrrhizin in concentrations up to 50 mg/ml did not affect h5n1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (moi 0.01: 13-fold reduction, moi 1: 10-fold reduction). next, influence of glycyrrhizin on h5n1 replication was confirmed by the detection of viral (h5) rna using quantitative pcr. only glycyrrhizin concentrations $100 mg/ml significantly reduced figure 1b) or h5n1 a/vietnam/1203/04-infected (suppl. figure 1c ) a549 cells (moi 0.01) 24 h post infection. time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( figure 1d ). addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect h5n1 replication. for investigation of h5n1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: cxcl10 (also known as interferon-cinducible protein 10, ip-10), interleukin 6 (il6), interleukin 8, (il8; also known as cxcl8), ccl2 (also known as monocyte chemoattractant protein 1, mcp-1), and ccl5 (also known as rantes). a549 cells were infected with h5n1 a/thailand/ 1(kan-1)/04 or h5n1 a/vietnam/1203/04 at moi 0.01, 0.1, or 1. glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. cytokine expression was detected 24 h post infection by elisa. glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in h5n1-infected cells in a dose-dependent manner (figure 2, figure 3a ). effects were more pronounced at lower mois. notably, expression of all cytokines except il8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml figure 3a ) although these glycyrrhizin concentrations had no effect on h5n1 replication in a549 cells (figure 1, figure s1 ). cytokine expression by influenza a virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza a virus pathogenicity [5, 39] . in a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of h5n1 a/thailand/1(kan-1)/04 (moi 0.1)-infected a549 cells through 8 mm filters. monocyte migration towards supernatants of h5n1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. treatment of h5n1infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( figure 3b ). influenza viruses including h5n1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( figure 4a-c) . glycyrrhizin concentrations $100 mg/ml inhibited h5n1 a/thailand/1(kan-1)/04 (moi 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in a549 cells as determined 24 h post infection ( figure 4a-c) . lower glycyrrhizin concentrations did not affect h5n1-induced apoptosis. the detection of cells in sub-g1 phase resulted in similar findings ( figure 4d ). substances that inhibit h5n1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of rnp complexes [34, 42] . in accordance, glycyrrhizin also interfered with nuclear export rnp at moi 1 ( figure s2 ). similar results were obtained in moi 0.01 h5n1 a/thailand/1(kan-1)/04infected cells ( figure s3 ). influence of glycyrrhizin on h5n1-induced activation of nuclear factor kb (nfkb), p38, and on h5n1-induced cellular reactive oxygen species (ros) formation activation of nfkb, p38, and jnk have been associated with influenza a virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . while glycyrrhizin did not influence nfkb activity in non-infected a549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited nfkb activation in h5n1-infected cells ( figure 5a ). moreover, glycyrrhizin inhibited h5n1-induced phosphorylation of the mapks p38 and jnk ( figure 5b ). in addition to their roles during influenza a virus replication and virus-induced cytokine/chemokine expression, nfkb, p38, and jnk are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . antioxidants had been already found to interfere with influenza a virus-induced signalling through nfkb, p38, and jnk, with influenza a virus replication, and with influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] . since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with h5n1-induced ros formation. indeed glycyrrhizin exerted clear antioxidative effects in h5n1 (moi 0.01)-infected cells ( figure 5c ) causing significant reduction of ros formation already at a concentration of 25 mg/ml ( figure 5d ). here, we show that glycyrrhizin inhibits the replication of highly pathogenic h5n1 influenza a virus, h5n1-induced apoptosis, and h5n1-induced expression of pro-inflammatory cytokines in lung-derived a549 cells. after intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . therefore, the glycyrrhizin concentrations found to interfere with h5n1 replication and h5n1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. notably, although higher glycyrrhizin concentrations were needed to interfere with sars coronavirus replication [22] than with h5n1 replication, beneficial results were reported in glycyrrhizin (snmc)-treated sars patients in comparison to sars patients who did not receive glycyrrhizin [23] . notably, investigation of different glycyrrhizin derivatives against sars coronavirus led to the identification of compounds with enhanced antiviral activity [53] . therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs. experimental results suggested that glycyrrhizin might be able to affect seasonal influenza a virus disease by antiviral and immunomodulatory effects [26, 27] . mice were prevented from lethal h2n2 infection by glycyrrhizin although no influence on virus replication was detected. the mechanism was suggested to be induction of interferon-c in t-cells by glycyrrhizin [54] . moreover, glycyrrhizin was shown to influence seasonal influenza a virus replication through interaction with the cell membrane [25, 28] . however, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. since glycyrrhizin addition during the adsorption period did not influence h5n1 replication in our experiments it appears not likely that membrane effects contribute to anti-h5n1 effects detected here in lower concentrations. our results rather suggest that glycyrrhizin interferes with h5n1-induced oxidative stress. influenza a virus (including h5n1) infection induces ros formation. antioxidants were found to inhibit influenza a virus replication and influenza a virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . here, glycyrrhizin interfered with h5n1-induced activation of nfkb, p38, and jnk representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza a virus replication and influenza a virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . glycyrrhizin 50 mg/ml significantly reduced h5n1-induced activation of nfkb. in addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with h5n1-induced ros formation and with phosphorylation of the redox-sensitive mapks p38 and jnk. in our model, activation of p38 appears to be critical for h5n1-associated redox signalling since p38 inhibition had been shown before to mimick effects of the antioxidant n-acetyl-cysteine (nac) [34] . interestingly and in contrast to glycyrrhizin, nac failed to inhibit h5n1 replication or h5n1-induced cytokine/chemokine expression in therapeutically relevant concentrations. glycyrrhizin diminished h5n1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with h5n1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. therefore, h5n1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ros formation than h5n1 replication. indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . in a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with h5n1-induced pro-inflammatory gene expression but not with h5n1 replication in human monocyte-derived macrophages [57] . in addition, other immunomodulatory treatment regimens that did not influence h5n1 replication reduced mortality in h5n1-infected mice [31, 58] . therefore, glycyrrhizin represents a potential additional treatment option that interfers with both h5n1 replication and h5n1induced expression of pro-inflammatory cytokines in lung cells. interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic cd8 + t-lymphocytes. global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper nk cell cytolytic activity [60] . in this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] . in conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation snmc) interfere with highly pathogenic h5n1 influenza a virus replication and h5n1-induced proinflammatory gene expression at least in part through interference with h5n1-induced ros formation and in turn reduced activation of p38, jnk, and nfkb in lung cells. since we used the clinical formulation snmc effects of other ingredients like glycin or cystein cannot be excluded. vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza a virus pandemic [61] . anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . therefore, glycyrrhizin may complement the arsenal of potential drugs for the 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the authors thank mrs. kerstin euler, mrs. gesa meincke, and mrs. christina matreux for technical support. key: cord-345020-ai5tib7h authors: price, o. h.; sullivan, s. g.; sutterby, c.; druce, j.; carville, k. s. title: using routine testing data to understand circulation patterns of influenza a, respiratory syncytial virus and other respiratory viruses in victoria, australia date: 2019-06-17 journal: epidemiol infect doi: 10.1017/s0950268819001055 sha: doc_id: 345020 cord_uid: ai5tib7h several studies have reported evidence of interference between respiratory viruses: respiratory viruses rarely reach their epidemic peak concurrently and there appears to be a negative association between infection with one respiratory virus and co-infection with another. we used results spanning 16 years (2002–2017) of a routine diagnostic multiplex panel that tests for nine respiratory viruses to further investigate these interactions in victoria, australia. time series analyses were used to plot the proportion positive for each virus. the seasonality of all viruses included was compared with respiratory syncytial virus (rsv) and influenza a virus using cross-correlations. logistic regression was used to explore the likelihood of co-infection with one virus given infection with another. seasonal peaks were observed each year for influenza a and rsv and less frequently for influenza b, coronavirus and parainfluenza virus. rsv circulated an average of 6 weeks before influenza a. co-infection with another respiratory virus was less common with picornavirus, rsv or influenza a infection. our findings provide further evidence of a temporal relationship in the circulation of respiratory viruses. a greater understanding of the interaction between respiratory viruses may enable better prediction of the timing and magnitude of respiratory virus epidemics. influenza, respiratory syncytial virus (rsv) and other respiratory viruses are the cause of substantial morbidity and mortality, with children under 5 years of age and the elderly disproportionately burdened [1] . both influenza and rsv display distinct seasonality, however, the exact timing and magnitude of their annual epidemics remain difficult to predict [2] . a better understanding of the epidemiology of these pathogens is useful for the prevention and control of future epidemics and for optimising clinical management of patients [3] . moreover, this knowledge may inform prediction models used to estimate the timing and magnitude of influenza epidemics [2] . interference between respiratory viruses has been well documented. during peaks of influenza epidemics, the spread of other respiratory viruses, particularly rsv, appears to be limited [4] [5] [6] . delays in outbreaks of influenza during the 2009 pandemic in europe were linked to the annual rhinovirus epidemic associated with the beginning of the school year [7] [8] [9] . in turn, the influenza pandemic was observed to interfere with seasonal epidemics of rsv in france [10] and israel [11] , rsv and metapneumovirus in germany [12] , seasonal influenza in hong kong [13] and all respiratory viruses except rhinovirus in beijing [14] . studies investigating viral interference since the pandemic are sparser, though two studies reported that the timing and magnitude of respiratory virus epidemics were affected by the timing of the seasonal influenza a peak [15, 16] . collectively, these observations suggest interference may prevent respiratory viruses reaching their epidemic peaks concurrently, but also underscore the complexity of these interactions. the exact nature of interactions between different respiratory viruses remains unclear, although they are proposed to be driven by the innate immune system. once a viral infection is established, interferon production is believed to confer temporary immunity to neighbouring cells against infection by other respiratory viruses [17] . in vitro, infection with rsv is blocked by competitive infection of influenza a if the host is not infected with the two viruses simultaneously [18] . similarly, ferret models have shown that influenza a infection may prevent successive infection with rsv [19] and that coinfection with different influenza subtypes is dependent upon the order in which the viruses infect the host [20] . despite this apparent interference, viral co-infections do occur, albeit with insufficient frequency to maintain an epidemic-level spread of the co-infecting viruses. a recent study reported infrequent co-detection of rhinovirus with other viruses [21] , despite observations that rhinovirus continues to be shed for several weeks postresolution of symptoms [22] . negative associations have also been observed between the detection of influenza a, rsv, parainfluenza virus or coronavirus and co-detection of other respiratory viruses [8, 23, 24] , providing further evidence for a refractory period after initial infection during which the host is less likely to be infected by subsequent exposure to another respiratory virus. we used routine diagnostic testing data of specimens from both the community and hospitals at the victorian infectious diseases reference laboratory (vidrl) between 2002 and 2017 to describe relationships between respiratory viruses, with a focus on influenza a and rsv. from may 2002 to december 2017, 58 114 clinical specimens were collected from communities and hospitals and tested by polymerase chain reaction (pcr) for respiratory virus infection at vidrl. there were no inclusion criteria regarding symptoms, but it is assumed that testing was deemed clinically relevant. multiple specimen types were tested, but the majority were nose/throat swabs (64.2%) or nasopharyngeal aspirates (17.1%). the respiratory panel included nine viruses: adenovirus, influenza a, influenza b, parainfluenza virus, picornavirus (virus family includes rhinoviruses and enteroviruses), rsv, coronavirus (from 2010), human metapneumovirus (from 2012) and influenza c (from 2012). data were de-identified, but the date of birth, postcode of residence and sex of the patient were provided. data exclusions are shown in figure s1 . data from outbreaks, research and non-victorian residents were excluded (n = 10 325) as they followed different sampling methods. specimens collected from the same patient within 14 days were considered part of the same infection: where both specimens were positive for the same virus or both were negative, they were counted as one episode; where there were both positive and negative results, only the positive result was retained; and when two specimens were positive for different viruses, they were collapsed to represent one episode of co-detection. as a result, 8612 records were excluded. data from 2009 (n = 4232) were excluded as the influenza pandemic led to changes in referral and testing practices. data from 2016 to 2017 (n = 1293) were also excluded as the introduction of in-house testing at some referring hospitals led to a substantial decrease in samples tested by vidrl. demographic data of patients were compared using pearson's χ 2 test. weekly proportions positive for each virus were calculated to allow comparability and assess differences in virus epidemics between seasons. we compared our data to influenza notification rates in victoria obtained from the national notifiable diseases surveillance system (nndss) [25] to assess the representativeness of inter-seasonal peaks we observed. to assess timing and magnitude of epidemics, the proportion of positive specimens and the peak week of the epidemic were considered: those in the lowest quartile were considered early or small and those in the highest quartile were considered late or large. seasonality of viruses was assessed visually by time series analysis and for further investigation each virus was compared with influenza a and rsv using cross-correlations that estimated the association between peaks in epidemic curves at a lag or lead of up to 15 weeks. fisher's exact test was used to investigate any negative association between virus pairs among specimens with co-detections. multivariate logistic regression, adjusted for age category (<5, 5-19, 20-64 and ⩾65 years), sex and season, was used to produce odds ratios (or) and 95% confidence intervals for these associations and the chi-square test used to assess trend. adjustment for multiple comparisons was not performed [23, 26] . the significance level for all tests was set at p < 0.05. all data extraction, exclusion and analyses were performed in stata (version 14.2, statacorp, college station, texas). there were 33 652 pcr results from 2002 to 2015 (excluding 2009) included in this study. of these, 11 154 (33.1%) were positive for at least one of the nine viruses tested for (table 1) . picornavirus (rhinovirus) was detected most frequently (n = 5363, 33.1% of the positive specimens), followed by influenza a (n = 2259, 20.3%) and rsv (n = 1487, 13.3%). the proportion of tests positive for most viruses remained relatively stable (fig. 1) . however, there was a higher positivity rate of rsv pre-2009 (p < 0.001). the positivity rate of influenza a peaked and troughed; a year with a big epidemic was usually followed by a year with a smaller epidemic. of the influenza a-positive samples, 57.9% were a(h3n2), 23.7% were a(h1n1) and the remaining not subtyped. in most seasons, one subtype predominated, although in 2005 and 2014, the subtypes were observed to circulate with similar magnitude and timing and in 2013 they circulated as two distinct peaks of comparable magnitude. the rate of picornavirus detection increased from 2006 to 2010 and then decreased from 2011 to 2015 returning to a level similar to that observed at the beginning of the study period. more specimens tested were collected from males (53.9%) ( table 1) . as a proportion of total tests per sex, females were more likely to test positive for influenza a (p < 0.001) and metapneumovirus (p = 0.015), while males were more likely to test positive for picornavirus (p = 0.003) (table s1 ). there was no significant difference in sex distribution for the other viruses. patients residing in rural areas were significantly more likely to have a positive test than those in urban areas (p < 0.001). the same pattern was seen individually for rsv, parainfluenza virus and adenovirus. however, patients from urban areas were more likely to test positive for influenza a and metapneumovirus. associations between remaining viruses and area of residence were not significant. respiratory virus tests were most frequently requested in winter (june-august; n = 11 750, 34.9%) ( table 1 ) and were most likely to be positive in winter (p < 0.001). six of the nine viruses were most frequently detected in winter, but parainfluenza virus and metapneumovirus were most frequent in spring (september-november) and picornavirus was most frequent in autumn (march-may). tests positive for picornavirus were distributed relatively evenly across the seasons, so although the modal week was in autumn, a peak was less distinct compared to other viruses. the median age of positive tests was lower than that for all tests (36.9 (iqr: 2.4-61.5) and 45.3 (22.6-64.3) years, table 1 . demographic and temporal information for included specimens (table s1 ). notably, the median age of patients from whom specimens were collected increased fairly steadily from 27.6 years in 2002 to 61 years in 2015 (table s2) . time series analysis demonstrated annual seasonal peaks for influenza a and rsv (fig. 1) . peaks occurred most frequently in winter, with occasional peaks in late autumn (rsv) and early spring (influenza a). although influenza a virus circulation during summer in victoria is expected to be minimal, we observed increased influenza positivity rates in many summers during the study period, one of which was larger than its preceding winter peak (2013-2014). these inter-epidemic peaks were reflected in victorian notification rates (obtained from nndss) in summers from 2010 to 2011 onwards, visible as influenza activity not reaching zero as it had in previous summers (fig. 2) . seasonal peaks were also observed among the other viruses, except picornavirus, although they did not occur every year. picornavirus remained endemic throughout the year for the duration of the study period. cross-correlations were performed to ascertain whether the timing and magnitude of other viruses may differ relative to influenza a activity. results revealed a moderate to strong correlation between epidemic curves of influenza and rsv in 9 of 13 years. on average, where correlated, a seasonal epidemic of rsv occurred 6 weeks earlier than that of influenza a (table 2) , although there were 3 years where the epidemics occurred at similar times (2002, 2005 and 2006) . as a sensitivity analysis, we performed further cross-correlations to assess whether influenza subtype affected these interactions (table s3 ). the lag calculated for influenza a overall was consistently similar to that of the predominant influenza a subtype in a given season. in some years the lag calculated suggested influenza a(h1n1) circulated prior to rsv, however in these years the number of samples positive for influenza a(h1n1) was <10. no consistent pattern emerged when considering timing and magnitude of influenza a and rsv seasons ( table 2) : an early epidemic of one virus sometimes resulted in a later than usual epidemic of the other, but this was not always the case. likewise, a season with a high magnitude of infections with one virus did not necessarily result in a season with a low magnitude of the other. generally, influenza b epidemics occurred at a similar time to influenza a and in years that influenza a circulated early (2002, 2005, 2011, 2012) , influenza b activity was low (data not shown). co-detections of respiratory viruses occurred in 6.4% (n = 823) of positive samples. exploratory data analysis using univariate logistic regression suggested co-detections were more likely to occur in children under 5 years, males and during winter. odds of co-detection decreased as age increased. using the <5 year age group as the reference category, the ors (adjusted for sex and season) and corresponding 95% confidence intervals for co-infection co-detections occurred most frequently with adenovirus (40%), influenza c (39%) and coronavirus (20%), though the number of influenza c infections was small (n = 18) (fig. 3) . co-detections were rarest with picornavirus (10%) and influenza a and b (6%) infections. analysis of co-infections using fisher's exact test found a pattern of a negative association between detection of influenza a, rsv or picornavirus and co-detection of another virus (table 3) . these three viruses were involved in the highest number of significant negative associations (n = 5, table 3 ). no positive associations between viruses were considered statistically significant. multivariate logistic regression (adjusted for age group, sex and season) was used to further investigate the probability of co-detection given infection with influenza a, rsv and picornavirus (table 4 ). significant negative associations were observed for co-detection with all viruses where influenza a was detected and all but one virus for rsv and picornavirus detections (influenza b and human metapneumovirus, respectively). we used multi-year routine pcr testing data to establish patterns of respiratory virus circulation in victoria, australia. picornavirus (rhinovirus) was most frequently detected. children aged <5 years and those living in rural areas experienced a high burden of infection. time series analyses indicated the annual occurrence of epidemics for influenza a and rsv and less recurrent epidemics for influenza b, coronavirus and parainfluenza virus. picornavirus was observed to be endemic throughout the period of analysis. rsv epidemics generally began in autumn and peaked early winter, while influenza a began mid-winter and peaked late winter. the higher incidence of rsv observed pre-2009 may be a result for timing and magnitude of epidemic curves, proportion of specimens positive and the peak week of the epidemic were considered: those in the highest quartile were considered late or large; those in the lowest quartile were considered early or small. of the higher proportion of children under 5 years in our sample pre-2009, as rsv is considered the most important respiratory illness-causing pathogen in infants [27] . we observed summer peaks of influenza a in some years which was somewhat unexpected in a temperate climate but was only reflected in state-wide notification data after 2009. it is possible that inter-epidemic peaks we observed are a result of denominator data, while the increase in notifications resulted from a rise in testing after the 2009 pandemic [28] . in years that epidemics occurred, influenza b, coronavirus and parainfluenza virus peaked in winter and metapneumovirus in spring. like previous studies [15, 16] , time series analyses and crosscorrelations established distinct circulation patterns of rsv and influenza a. the two viruses rarely reached their epidemic peak concurrently, with rsv peaking an average of 6 weeks before influenza a. influenza a subtype did not affect cross-correlations: in seasons where significant correlation was observed, the lag calculated for influenza a overall was similar to that of the predominant subtype. in some seasons, influenza a(h1n1) appeared to circulate prior to rsv. however, in these seasons the number of a(h1n1) positive samples was <10, so the results should be interpreted with caution. an investigation into seasonal relationships between epidemic curves of other viruses was limited by the small proportion of positive tests. the endemic nature of picornavirus appeared to be unaffected by the circulation of other respiratory viruses, which supports previous observations of rhinovirus activity (most common species of picornavirus) [15, 16] . this may be a result of increased stability of the non-enveloped picornavirus during warmer months compared to other viruses, like influenza, which are restricted by temperature and humidity [29] . we also investigated the distribution and incidence of respiratory virus co-detection. improved availability and sensitivity of diagnostic tests has resulted in more regular detection of co-infections [30] , though the impact of viral co-infection on clinical severity remains unclear [31, 32] . a prospective household transmission study during the 2009 influenza pandemic reported that the infection wave caused by influenza a(h1n1)pdm09 was interrupted by a wave of non-influenza respiratory virus infections [33] . it found individuals infected with influenza a (h1n1)pdm09 were less likely to be infected by non-influenza respiratory viruses than non-infected individuals (rr: 0.32). further, there was a significant decrease in the duration of viral shedding in co-infections (of any respiratory viruses) compared to single infections. these observations suggest that such interactions may modulate influenza attack rate during outbreaks, thus shaping the epidemic and highlighting the importance of better understanding co-infections in the context of viral interference. we found co-detections of respiratory viruses in 6.4% of positive specimens, which falls in the 5.0-62.0% range of previous studies [31] . like other studies, we found co-infection was less likely with increasing age [34, 35] , which may be a consequence of pre-existing immunity or decreased viral shedding with increasing age [30] . we found adenovirus and coronavirus most likely to be part of a co-infection and influenza a and b least likely, corroborating results of a previous study [36] . while our data did not include patients' symptoms, immunological [37] and clinical [24, 38] data suggest that the effect of co-infection on clinical severity depends on the specific pathogens co-infecting the patient. infection by rhinovirus may result in temporary immunity of the host to infection by other respiratory viruses due to the production of cytokines [17] , thus resulting in a negative association between rhinovirus infection and co-infection with another virus [23] . moreover, it is believed to be the driver of epidemiological interaction between respiratory viruses at the population level, which is visible when two viruses may not reach their epidemic peak during the same period. while rhinovirus has been the focus of other studies investigating co-detection, we found that influenza a, rsv and picornavirus all had significant negative associations with co-detection of other viruses. results of further investigation by logistic regression adjusted for covariates that are predictors of codetection (sex, age and season) were compatible with influenza a, rsv and picornavirus conferring temporary immunity against infection by another respiratory virus. however, we cannot make causal inferences from the design used and therefore, cannot eliminate the role of other environmental factors. our study has some limitations. all inferences we made and indeed the majority of inferences made in other studies investigating respiratory virus interference are based on ecological data. with such data, we cannot determine whether events observed are the result of a biological mechanism, nor can we infer the direction of the postulated interaction, i.e. which virus impacts which. to make any form of causal inference, a prospective study that serially samples participants over multiple respiratory virus epidemics would be required. furthermore, we did not adjust for other potential drivers of viral interference, such as environmental (e.g. temperature, humidity), social and behavioural factors. also, we cannot rule out the possibility that our observations were the result of surveillance artefacts, that is, changes in testing patterns that are not a result of genuine fluctuations in viral circulation. while we excluded specimens isolated from outbreaks or for surveillance and samples from 2009 and 2016-2017 when testing patterns were obviously altered, we cannot be certain we controlled completely for this unknown. type/ subtype data for other viruses may have also improved the resolution of our findings, as other studies have noted variances in the timing of epidemics caused by different types of parainfluenza virus [4, 6] and rhinovirus [21] and there is scant information available for rsv. additionally, our study sample was drawn from patients ill enough to seek healthcare. as some viruses (such as picornavirus) are more likely to result in asymptomatic associations considered significant are bolded. the top cell represents the p-value for each measure of association and the bottom cell the or (and corresponding 95% ci) for infection. a no co-detections with these two viruses occurred infection than others, the distribution of viruses in our sample may differ from that in the population. finally, the referral base of paediatric samples for vidrl is limited as most victorian paediatric samples are forwarded to a children's hospital. given there is a high burden of respiratory virus infection in children, this may have limited our analyses. a strength of our data is that it spanned 16 years. accurately monitoring seasonal variation in respiratory virus epidemics has the potential to improve our understanding of interaction and interference between different respiratory viruses, although this remains challenging as surveillance systems for non-influenza respiratory viruses are limited in both scope and funding. our study confirms the existence of temporal relationships in the circulation of some respiratory viruses in victoria and provides further evidence to support the postulated effects of viral interference on magnitude and timing of 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non-influenza respiratory viruses during the first wave of pandemic influenza ph1n1/2009: a cohort study in reunion island community-acquired respiratory viruses and co-infection among patients of ontario sentinel practices epidemiology of viral respiratory infections in australian working-age adults (20-64 years multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferongamma response viral etiology and the impact of codetection in young children presenting with influenza-like illness acknowledgements. we thank the laboratory staff members from victorian infectious diseases reference laboratory who undertook respiratory virus testing.financial support. the who collaborating centre for reference and research on influenza is supported by the australian government department of health.conflict of interest. none. key: cord-309381-cb80ntxs authors: nogales, aitor; l. dediego, marta title: host single nucleotide polymorphisms modulating influenza a virus disease in humans date: 2019-09-30 journal: pathogens doi: 10.3390/pathogens8040168 sha: doc_id: 309381 cord_uid: cb80ntxs a large number of human genes associated with viral infections contain single nucleotide polymorphisms (snps), which represent a genetic variation caused by the change of a single nucleotide in the dna sequence. snps are located in coding or non-coding genomic regions and can affect gene expression or protein function by different mechanisms. furthermore, they have been linked to multiple human diseases, highlighting their medical relevance. therefore, the identification and analysis of this kind of polymorphisms in the human genome has gained high importance in the research community, and an increasing number of studies have been published during the last years. as a consequence of this exhaustive exploration, an association between the presence of some specific snps and the susceptibility or severity of many infectious diseases in some risk population groups has been found. in this review, we discuss the relevance of snps that are important to understand the pathology derived from influenza a virus (iav) infections in humans and the susceptibility of some individuals to suffer more severe symptoms. we also discuss the importance of snps for iav vaccine effectiveness. influenza a viruses (iav) belong to the orthomyxoviridae family, and they contain a single-stranded (ss) negative-sense viral (v)rna genome formed by eight segments that are encapsidated into particles with an envelope ( figure 1a) . each of the vrna segments contains a long central coding region flanked at 5 and 3 termini by non-coding regions (ncrs), which work as promoters to initiate viral rna synthesis (transcription and replication). moreover, the packaging signals playing a role in the efficient encapsidation of the viral segments into nascent virions, are located at the 3 and 5 end of the coding regions ( figure 1b ) [1] . structurally, vrnas form viral ribonucleoprotein complexes (vrnps), where vrnas are coated with multiple subunits of the viral nucleoprotein (np) and are associated with the heterotrimeric polymerase, which contains the polymerase basic 2 and 1 (pb2 and pb1, respectively) and acidic (pa) proteins ( figure 1a ) [2] [3] [4] . each vrnp acts as an independent transcription-replication unit using an uncommon mechanism among negative-sense rna viruses, given that viral rna synthesis occurs in the infected-cells nucleus. vrnas are used as templates by the viral polymerase to synthesize two positive-sense rna molecules, the complementary rnas (crnas), from which the same viral polymerase synthesizes more copies of genomic vrna, and the mrnas for viral protein synthesis [1] [2] [3] [4] [5] [6] . the small iav genome encodes for up to 16 viral proteins through the viral envelope is decorated with the two viral glycoproteins hemagglutinin (ha) and neuraminidase (na) at a ratio of approximately four to one, respectively [10, 11] . ha envelope protein mediates virus entry by binding to sialic acid-containing cell receptors, and then fusing endosomal and viral membranes during endocytosis [12, 13] , while na is required for viral release from infected host cells, and it acts as a receptor destroying enzyme, cleaving terminal sialic acid residues from glycoproteins present at the cell surface [14] [15] [16] . the matrix 2 (m2) protein is also found in the viral membrane, although in much lower abundance than ha or na glycoproteins. m2 is a small transmembrane protein that forms a proton-selective ion channel in the viral envelope. m2 promotes uncoating of the vrnps after membrane fusion and the protein has also an essential role in viral assembly and release [17] . under the viral envelop, there is an inner shell composed of the matrix 1 (m1) protein, which interacts in the virion with the vrnp and the ha and na proteins. m1 apart from being a membrane-associated scaffold factor of the virion, acts as a crucial factor for different viral processes during infection, including virion assembly and budding [18] [19] [20] . the nonstructural (ns) gene or segment 8 of iav encodes an mrna transcript that is alternatively spliced to express two viral proteins, the nonstructural protein 1 (ns1), produced from a continuous primary transcript, and the nuclear export protein (nep), which is produced by an alternatively processed transcript, using a weak 5 splice site. nep is also located in the virion and may interact with m1 in the viral particle [21] [22] [23] ( figure 1a) . during the infection, nep is responsible for the nuclear export of synthetized vrnp, ensuring that the vrnps are available for packaging [24] . moreover, nep has also other functions during iav infection, contributing to viral budding and to regulate viral rna synthesis. ns1 is a multifunctional protein and a key viral factor that counteracts the host antiviral responses. ns1 has been shown to inhibit the production of interferon (ifn), the activity and expression of multiple interferon-induced genes (isg) and the processing and nuclear transport of host mrnas causing cellular shut-off [25, 26] . segment 3 of iav also encodes two proteins, the polymerase component pa and pa-x. pa is translated directly from the pa mrna, whereas pa-x is translated using a +1 frameshift mechanism from the same open reading frame (orf) [9] . synergistically with ns1, pa-x is also able to block the cellular antiviral responses by inhibiting host protein expression. moreover, the pa-x protein has been shown to modulate host inflammation, immune responses, apoptosis, and virus pathogenesis [25] [26] [27] [28] [29] [30] . human iav infections cause contagious respiratory diseases associated with mild to severe respiratory illness or even death, and they are considered as an important public health threat worldwide, which also results in significant economic losses [31] [32] [33] . iav are divided into multiple subtypes, based on the ha and na glycoproteins. currently, there are 18 ha (h1 to h18) and 11 na (n1 to n11), but the growing iav surveillance programs and sequencing technologies could increase the number of subtypes in the following years. iav can infect a wide range of avian and mammalian species, although the natural reservoirs of iav are shorebirds and wild waterfowls [34] [35] [36] [37] . among all the ha and na subtypes, only h3n2 and h1n1 iav subtypes are circulating in human beings and they are responsible for annual recurrent epidemics that affect the entire world [38, 39] . seasonal influenza infections are prevented and controlled through annual vaccination campaigns to decrease iav infections and viral transmission as well as to reduce their negative impact in the global economy. however, although vaccination remains the most effective approach to protect the population from seasonal infections, the effectiveness of current vaccination approaches is suboptimal [16, [31] [32] [33] [39] [40] [41] [42] [43] [44] . thus, the production of improved prophylactic approaches, including universal vaccines, are highly desired. concerns associated with iav are further aggravated by the adaptive capacity of the viruses to infect new hosts or escape to the immune system, as well as their ability to transmit efficiently in the population and the limited therapeutic options to treat viral infections [14, 16, 25, 45] . because of the ability of iav to modify their genome using two main evolutionary mechanisms, antigenic drift and shift, viruses encoding novel antigenic proteins to which the population has limited or no preexisting immunity can be generated [10, 31, 37, 40] . for that reason, seasonal vaccines have to be reformulated yearly to guarantee that the viral glycoproteins (ha and na) in the vaccine match seasonal viruses circulating worldwide [38, 43, 46] . in addition, iav variability can lead to the generation of new virus strains with pandemic potential. for example, the first iav pandemic of this century occurred in 2009 and it is estimated that in approximately one year, the pandemic 2009 h1n1 (ph1n1) iav infected more than 600,000 human beings, causing near 16,000 deaths in over 200 countries [40, 41] . in addition, although only h1n1 and h3n2 are circulating in humans, the avian h5, h7, and h9 subtypes eventually cross the species barrier to infect humans, representing a new and serious public health problem [13, 37, [47] [48] [49] . the cellular defense mechanisms provided by the innate immune system are a formidable barrier to inhibit virus infections [50] and involve the recognition of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs). this recognition leads to the activation of signaling pathways and the production and secretion of ifns of type i (ifnα and ifnβ) and iii (ifnλ2 or il-28a, ifnλ3 or il-28b, and ifnλ1 or il-29) , and chemokines and cytokines involved in inflammatory processes [50] . iav rnas are mainly recognized by the endosomal, membrane-associated prr toll-like receptors (tlrs) 3 (double-stranded rnas, dsrnas) or 7/8 (ssrnas), respectively [50, 51] , by the cytoplasmic prr retinoic acid-inducible gene i (rig-i), which detects dsrna and 5 -triphosphates of the negative ssrna viral genome [50, 52] , generated during replication of multiple viruses, by the nod-like receptor family member nod-, lrr-and pyrin domain-containing 3 (nlrp3), which recognizes various stimuli (see below) [53] and by the absent in melanoma 2 (aim2) protein, recognizing not well-characterized influenza stimuli [54] . the result of prr detection of viral pamps is the activation of multiple transcription factors, such as the nuclear factor kappa β (nf-κb), the activator protein 1 (ap-1), and ifn regulatory factors (irf)-3 and irf-7, which are responsible for the transcription of ifns [50, 55, 56] and pro-inflammatory cytokines [57] . secreted type i and iii ifns signal through different receptors in a paracrine or autocrine way to induce the transcription of ifn-stimulated genes (isgs), several of which counteract viral replication [50, 56, 58] . just as an example mentioned below, ifitm3 is an isg playing antiviral roles against influenza virus infection and other viruses [59] . type i and iii ifns signaling pathways lead to the post-translational phosphorylation of the signal transducer and activator of transcription (stat) 1 and 2 transcription factors [60] , being the tyrosine kinase 2 (tyk2) and janus protein tyrosine kinase 1 (jak1) critical for the phosphorylation [61] . moreover, stat1 is phosphorylated by ikkε during ifn signaling and this step is important for the ifn-inducible innate immune response [62, 63] . upon phosphorylation, stat1 and stat2 associate with irf-9 forming the heterotrimeric isg factor 3 (isgf3) complex [60] . this heterotrimeric complex then translocates to the nucleus, and binds to ifn-stimulated response elements (isres) located in the promoters of isgs, up-regulating their expression [60, 64] . inflammatory cytokines, such as interleukins (il)-1a il-1b and tumor necrosis factor (tnf)-α contribute to the proliferation and migration of different immune cells, such as monocytes, macrophages, neutrophils, and natural killer (nk) cells, to the infected tissue. nk cells have the ability to kill virus-infected cells, are important for the activation of a protective cytotoxic t lymphocyte (ctl) response [65] , and nk-cell ifn-γ production is augmented by t-cell il-2 production in recall responses [66] . neutrophils and resident alveolar macrophages are also important for virus clearance, due to their ability to destroy infected cells [67] . in addition, cytokine signaling improves dendritic cells (dc) maturation, increasing the induction of adaptive immune responses by antigen presentation and co-stimulation [68, 69] . these adaptive immune responses initiated upon innate immune activation are required for protection and viral clearance [70] . nlrp3 is expressed by myeloid cells such as macrophages, monocytes, neutrophils, and dendritic cells [71] or by human bronchial epithelial cells [72] . upon stimulation, nlrp3 activates the inflammasome system, activating caspase-1 and leading to pro-inflammatory processes through the processing and activation of proil-1b, proil-18, and proil-33 [73] . nlrp3 senses iav dsrna [74] , and pb1-f2 protein [75] . furthermore, protein flux through the viral m2 ion channel activity in the trans-golgi network activates nlrp3, leading to inflammasome activation [76] . in addition to nlrp3 activation, iav activates the inflammasomes through aim2, increasing iav-induced lung injury and mortality [54] . the complement system is an important branch of innate immunity that plays an essential role in the clearance of pathogens. the complement system is triggered by three main pathways, the classical, the lectin, and the alternative pathways [77] . the first two pathways are activated with the help of pattern recognition molecules, whereas the alternative pathway is activated spontaneously. interestingly, it is known that viruses are recognized by the three pathways. in the classical pathway, the c1 complex recognizes antigen-antibody complexes, which are formed on the pathogen surface. c1qbp (complement c1q binding protein) can bind to the globular heads of c1q molecules, activating the classical pathway [78] . on the other hand, in the lectin pathway, the mannan-binding lectin (mbl)/ficolin/mannan-binding lectin serin protease (map) complex recognizes specific carbohydrates on the pathogen surface. complexes activated after the classical and lectin pathways, cleave c4 and c2, resulting in the generation of c4bc2a (c3 convertase). in the alternative pathway, spontaneous hydrolysis of native c3 results in the formation of c3b-like c3 that binds factor b and after cleavage by factor d forms the initial c3 convertase [77] . the three pathways converge at the cleavage of c3 into c3a and c3b by c3 convertases (c4b, 2a and c3b, bb). then, the c3b molecules formed bind covalently to the c3-convertases forming the c5-convertases that cleave c5 into c5a and c5b. cd55 blocks c3 and c5 activation by preventing the formation of new c3 and c5 convertases [79] . c5b starts the formation of c5b-9 or the membrane attack complex (mac). next, c8 binds to the membrane attached trimer and begins binding and polymerization of c9 that is inserted into the membrane, inducing virolysis [77] . unregulated complement activation could play a central role in the acute lung injury (ali) pathology induced by highly pathogenic viruses, including severe acute respiratory syndrome (sars) coronavirus and avian iav h5n1, and h7n9 [80] . in virus-induced acute lung diseases, high levels of chemotactic, and anaphylatoxic c5a can be generated as a result of excessive complement triggering and causing a "cytokine storm". accordingly, the blockade of c5a signaling has been involved in treating the ali induced by highly pathogenic viruses [80] . currently, particular attention is being paid to single nucleotide polymorphisms (snps) that are loci within the genome of an organism in which two or more alleles can exist. snps affect a single nucleotide or base pair and they are one of the most frequent types of genetic variations in the genome [81] [82] [83] . snps need to be presented into the population with a frequency equal to or greater than 1% to be considered as polymorphisms. there are multiple types of snps, depending on their location that can be in different regions of the genes such as promoters, exons, introns or utrs ( figure 2 ). snps in coding regions are classified as synonymous, when a nucleotide substitution does not change the amino acid sequence of the encoded protein, although other effects, such as changes in mrna structure or folding may account for variation in protein expression. on the other hand, non-synonymous snps are divided in missense or nonsense. in the first case, nucleotide substitution results in the change of one amino acid for another, affecting the protein sequence coded by a gene and therefore may lead to its dysfunction. in contrast, nonsense mutations are produced when instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. in summary, snps may influence gene regulation, the structure and stability of rna, the expression of rnas or proteins, the conformation and function of proteins, etc. thus, the identification of snps in genes and the analysis of their effects may lead us to better understand gene function or their impact on human health [84] . in fact, snps that are or could be important for multiple human pathologies, such as cancer, diabetes, heart disease, schizophrenia, blood-pressure homeostasis, and autoimmune or metabolic diseases, have been identified [85] [86] [87] [88] [89] [90] [91] . moreover, some described snps increase the human susceptibility to getting infected by viruses, bacteria or other pathogens [84, 86, [92] [93] [94] [95] [96] [97] . advanced sequencing and bioinformatics technologies have allowed the identification of a large number of human snps whose information is accessible in the databases. nevertheless, the biological significance and function for most of the snps found in the human genome remain unknown. currently, the scientific community recognizes the importance of this kind of genome variations that can act as biological markers and assist researchers in multiple aspects, such as: (1) locate genes associated with multiple diseases, (2) anticipate an individual's response to a specific infection, (3) predict population responses to several treatments such as drugs or vaccines, (4) design individualized therapies, (5) identify markers for medical testing, (6) perform pharmacogenetic studies, etc. this review focuses on the role of known snps on iav infection, as well as their impact on the effectiveness of vaccines against iav. instead of substituting one amino acid for another, the altered gene contains an early stop codon in the orf or a stop codon is abrogated, producing an elongated protein. this type of mutations results in shortened or elongated proteins leading typically to nonfunctional proteins. the functional role of snps in coding areas of the genome can be easily analyzed by studying the gene products. however, most snps fall within non-coding genome regions, therefore, predicting their effects is challenging. for example, snps in the promoter regions could affect their activity and regulation producing changes in gene expression levels. snps in utrs or intron regions have been related with an effect in protein translation or the production of splice variants of transcripts, leading to longer or shorter protein sequences, respectively. an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. nonsynonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). an snp is a variation on a single nucleotide which may occur at some specific point in the genome and that causes variations in dna sequences between members of the same species. (b) types of snps: dna variation can be located in non-coding or coding regions. snps within a coding sequence can be synonymous if they do not produce an amino acid change (silent mutation), or non-synonymous if they affect the protein sequence. non-synonymous changes can be divided into missense (producing an amino acid change in the protein) or nonsense (producing a truncated or longer protein). risk factors, including underlying co-morbidities, age, and pregnancy, affect iav susceptibility, but do not explain all the conditions under which serious iav-associated disease can occur, making likely that snps in viral and host genes affect iav susceptibility and the outcome of the disease. in fact, there are some examples of the presence of snps in host genes affecting influenza severity (table 1) , which will be discussed in this review. snps affecting iav disease have been found in genes recognizing viral components, in transcription factors important for ifn production and signaling, in isgs with antiviral activities, and in genes involved in inflammation. tlr3 recognizes dsrna, one of the iav replication intermediate products, and in turn activates ifn production, leading to an antiviral response. a missense mutation (f303s) of the tlr3 gene was found in one out of three patients developing iav-associated encephalopathy (iae), a neurological consequence of severe viral infection [98] . assays in tissue culture cells showed that a tlr3 receptor encoding the missense f303s mutation was impaired in activating the transcription factor nf-κb, and in triggering downstream signaling via the ifnβ receptor, indicating that this genetic polymorphism could lead to increased iav replication [98] . in a study of 51 italian children diagnosed with iav h1n1 infection, an additional tlr3 snp (rs5743313, genotype c/t) was identified [99] . this tlr3 snp was found in all the children developing iav-associated pneumonia (18 cases). however, the snp was found in significantly less proportion in children with milder disease, suggesting a link between tlr3 and iav pathogenicity. furthermore, in a multicenter study involving 275 adult cases of avian h7n9 and ph1n1 iav, in mainland china and hong kong, the tlr3 cc rs5743313 snp was associated with fatal cases [100] . in addition to iav, there are other examples of snps in tlr3 or tlr3 signaling genes affecting viral infections. for instance, susceptibility to chikungunya virus (chikv) infection is highly increased in human and mouse cells with defective tlr3 molecules [101] . furthermore, tlr3 snps, rs3775292, and rs6552950, leading to unknown functional consequences, were associated with an increased risk of chikv disease occurrence [101] . patients with impaired tlr3-mediated responses show an elevated susceptibility to herpes simplex-1 virus (hsv-1)-mediated encephalitis by encoding tlr3-deficient alleles [102, 103] , or by encoding deficient traf3, tbk1 and trif molecules, leading to impaired tlr-3 signaling [104] [105] [106] . in a saudi arabian population, the tlr3 rs78726532 snp was strongly associated with hepatitis b (hbv) and hepatitis c (hcv) virus infections when compared to that in healthy control subjects [107, 108] . the tlr3 rs5743314 c allele was also associated with hcv-related liver disease progression (cirrhosis and hepatocellular carcinoma) [107] . however, the functional effects of these snps seem to be unknown. rig-i detects dsrna and 5 -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation [52] . a caucasian male patient with severe iav h1n1 infection during the 2009 swine flu pandemic showed two heterozygous variants (one in each chromosome): p.r71h (snp rs72710678) and p.p885s (snp rs138425677), located, respectively, in the caspase activation and recruitment domain (card) and rna binding domains of rig-i [109] . these variants significantly decreased the recognition function of rig-i, and therefore, patient cells proved impaired antiviral responses to rig-i ligands and elevated proinflammatory responses to iav, providing evidence for dysregulation of the innate immune response and increased immunopathology [109] . these results suggest that these rig-i polymorphisms may have contributed to severe iav outcome in this patient and reinforce that rig-i variants should be evaluated in future studies of host factors affecting ssrna virus infections. irf-7 is a transcription factor that increases interferon (ifn) production in response to viruses [110] [111] [112] . a patient suffering from an unusual life-threatening disease after ph1n1 infection encodes homozygous null mutations in the irf-7 factor. both irf-7 alleles from this patient encode mutations c.1228t>g/t (f410v) and c.1261c>t/c (q421x), which are mutations decreasing the ability of irf-7 to induce the transcription of ifn genes after iav infections [113] . these findings suggest that irf-7-dependent production of type i and iii ifns is required for controlling iav infections in humans. the rare allele a of two irf-7 snps, rs12272434 and rs12290989, both located at exon/intron boundaries, were significantly associated with impaired levels of ifnα production by human plasmacytoid dendritic cells (pdcs) in response to human immunodeficiency virus 1 (hiv-1) infection [114] . therefore, these polymorphisms may affect the ability of human subjects to control hiv-1 infections, reinforcing the role of irf-7 in controlling viral infections. however, the effect of these snps should be further studied. irf-9 is a transcription factor essential for ifn signaling and the transcriptional induction of isgs [60] . stat1 and stat2, when phosphorylated, associate with irf-9 to form a heterotrimeric isg factor 3 (isgf3) complex [60] , which translocates to the nucleus, and binds isres present in the promoters of isgs, up-regulating their transcription [60, 64] . a homozygous, loss-of-function mutation in irf-9 was described in a child born to first-cousin algerian parents and living in france affected by a severe pulmonary influenza infection [115] . in particular, the homozygous mutation (c.991g>a) occurred in the final nucleotide of exon 7 and disrupted the essential splice site at the boundary of exon 7 and intron 7, leading to deleted irf-9 proteins. the consequence of this mutation was an impaired activation of irf-9, and therefore, an impaired transcription of isgs, many of which show antiviral activities [115] . similarly, a family in which several members showed a surprising susceptibility to infection by different viruses, including iav, also showed to be irf9 deficient [116] . the index patient, a boy with 10 years born at term from healthy consanguineous parents (first cousins of portuguese origin and residing in venezuela) encoded a homozygous splicing mutation in the irf9 gene. the mutation, c.577+1g>t, was located in the donor splice site of introns 5 and 6, leading to transcripts lacking exon 5. irf9 protein expression was undetectable in cells transfected with the c.577+1g>t irf9 construct, suggesting that either the protein was quickly degraded or the mrna was not translated. again, irf9-deficient cells showed a profound defect in inducing the expression of multiple isgs [116] . collectively, these findings show that human irf9-and isgf3-dependent type i and iii ifn responsive pathways are essential for controlling viral infections, including iav. the antiviral protein ifitm3 is an isg which abrogates the release of iav content from late endosomes into the cytoplasm [59] . in addition, ifitm3 promotes the survival of mouse lung-resident cd8+ t cells following iav challenge, which may help clear the infection [117] . furthermore, mice in which the expression of ifitm3 is abolished, showed severe disease after iav infection, compared to parental mice [118] . one of the clearest associations of snps in genes affecting influenza severity is located in the isg ifitm3. the human ifitm3 gene is encoded by two exons and is predicted to encode two splice variants that differ in the first amino-terminal 21 amino acids. different studies have described the effect of ifitm3 snps in influenza disease severity. northern european patients infected with iav ph1n1 2009 virus requiring hospitalization showed over-representation of the snp rs12252 in the ifitm3 gene, in which the majority t allele is replaced for a minority c allele [118] . this leads to an alteration of the first splice acceptor site, originating an ifitm3 protein lacking the first 21 amino acids (n∆21) due to the protein starting from an alternative start codon. according to these results suggesting that this snp could affect influenza disease, the minority (cc) variant rendered homozygous cells more susceptible to iav infection, and this susceptibility correlated with decreased levels of ifitm3 protein expression in comparison to the majority (tt) variant cells [118] . furthermore, cells expressing the n∆21 protein showed an impaired ability to restrict viral replication when compared to wild-type ifitm3 cells [118] . this data is consistent with previous results which show that the amino-terminal 21 amino acids of ifitm3 are relevant for attenuating vesicular stomatitis virus (vsv) replication in vitro [119] . moreover, the cc genotype was found in 25% of chinese patients showing mild disease after ph1n1 virus infection compared to 69% in patients developing a severe ph1n1 virus infection. in addition, the cc genotype was estimated to confer a six-fold increased risk for severe infection than the ct and tt genotypes [120] , reinforcing the idea that ifitm3 is a factor affecting human iav disease [121] . in another study, over-representation of the ifitm3 cc genotype was detected among fatal cases of chinese patients infected with iav ph1n1 and h7n9 viruses [100] , and in a more general study, including twelve studies published before february 2018 with more than 16,000 subjects, revealed increased risk of severe influenza in both the east asian and white populations in the subjects encoding the ifitm3 cc genotype [122] . another important snp (rs34481144) associated with risk of severe influenza in humans from the united states (us) infected with seasonal iavs is located in the 5 -utr of the ifitm3 gene [123, 124] . this snp affected ifitm3 expression being the risk allele associated with lower mrna expression. the mechanism for this lower mrna expression involves the decreased irf-3 binding and increased binding of the transcriptional repressor ccctc-binding factor (ctcf) in promoter-binding assays for the risk allele [123] . moreover, the risk allele disrupted a cpg site that becomes differentially methylated in cd8+ t cell subsets, leading to less cd8+ t cells in the airways during natural influenza infection in the carriers of the risk allele, and suggesting that a critical role for ifitm3 may be to promote immune cell persistence at mucosal sites [123] . interleukins 1a and 1b (il-1a and il-1b, respectively) are inflammatory cytokines that play critical roles in recruiting immune and inflammatory cells and developing adaptive immune responses. furthermore, accumulating evidence suggests that both cytokines play central roles in innate immunity against viral infections [125] . the frequencies of snp (allele c) located 31 base pairs upstream from the transcription start site (rs1143627), on the il-1b promoter were associated with increased risk of influenza disease in chinese subjects [126] . this nucleotide change is localized in a tata-box motif of il-1b and modulates the transcription activity of il-1b by binding to multiple transcription factors [127] . the allele t of rs1143627 enhanced il-1b protein expression, as indicated by several reports [128] . people carrying allele t showed a higher il-1b expression, which could lead to increased ifnγ production, which promotes virus clearance [129] . in contrast, expression of il-1b may be decreased in individuals who carry allele c, leading to a weaker immune response during viral infection. in addition, a t allele in il-1a gene (snp rs17561) increased the risk of iav ph1n1 susceptibility, as observed in chinese subjects [126] . the snp rs17561 introduces a nonsynonymous mutation (a114s) in il-1a protein, suggesting that this genetic variant may lead to a functional variation in host susceptibility to ph1n1. nevertheless, the molecular mechanism needs to be evaluated and the real risk of these alleles should be analyzed in larger populations. tnf-α is a pro-inflammatory cytokine which orchestrates the host´s defense. a minor allele (a) at position -238 of tnf (snp rs361525) was more frequent in greek patients infected with ph1n1 virus compared to control subjects [130] , and developing pneumonia was more uncommon in greek and mexican subjects with no copies of the minor allele compared to subjects with at least one copy of the minor allele [130, 131] , leading to the hypothesis that this snp allele could be linked with an elevated susceptibility to infection with the ph1n1 virus [124, 130] . decreased tnf-α expression was observed in subjects encoding the minor allele at position -238 [92] . this may explain how snps leading to lower production of tnf-α may predispose to more severe clinical symptoms following iav infections. however, the tnf-α rs 1800629 minor a allele, associated with higher levels of tnf-α expression, was associated with susceptibility to japanese encephalitis virus infection in an indian population [132] . the tnf-α rs 1800629 minor a allele was a risk factor to develop liver cirrhosis and hepatocellular carcinoma following hbv infection in a han chinese population [133] , suggesting that the protective or deleterious roles of tnf-α expression may vary depending on the infecting virus. chemokine receptor 5 (ccr5) is expressed mainly on macrophages, t cells, and dendritic cells. ccr5 mediates leukocyte chemotaxis in response to its ligands, including mip-1a, mip-1b, and rantes. it can help direct multiple immune cell subsets, including regulatory t cells or th17 cells to sites of infection, supporting the antiviral immune response. evidence in humans support that homozygosity for the ccr5-∆32 allele, a naturally occurring polymorphism of ccr5 encoding a 32-bp deletion, prevents its expression on the cell surface, and is linked with an elevated susceptibility to west nile virus (wnv) [134] and with increased severity of illness among patients infected with ph1n1 [135] , although this evidence is modest due to the limited number of subjects analyzed. in contrast, homozygous carriers of the ∆32 mutation are resistant to hiv-1 infection because this molecule, absent in the cell surface in subjects encoding the deletion, is a molecule normally used by hiv-1 to enter cd4+ t cells [136] . cd55 is an important complement regulatory protein which blocks c3 and c5 activation by preventing the formation of new c3 and c5 convertases, two proteases involved in inflammation and complement activation. consequently, cd55 protects cells from complement attack and decreases amplification of the complement cascade [79] . the cd55 snp (rs2564978, genotype t/t) was significantly associated with severe iav infection in chinese patients infected with ph1n1 2009 virus [137] and was associated with increased death risk in greek patients [138] . the rs2564978 snp of cd55 is located in the minimal promoter region [139] and individuals with this genotype showed significantly lower levels of cd55 expression in comparison to those with the more frequent allele [137] . therefore, patients who carry the t/t genotype may have more robust complement activation during iav infection, resulting in enhanced inflammation and disease severity [47, 79] . according to these results, the polymorphism rs2564978 in gene cd55 was linked to disease severity in adult chinese cases of avian (h7n9) and human ph1n1 iav in another study [100] . however, these findings need to be confirmed in bigger cohorts. c1qbp can bind to the globular heads of c1q molecules, activating the classical pathway of complement [78] . an increased risk of severe disease after iav infection was found in patients homozygous for the minor allele of the snp rs3786054 in european and mexican populations [138, 140] . however, the effect of this snps on gene expression and function is undescribed. soluble pattern-recognition molecules, forming part of the innate immune system, can neutralize iav infection. particularly, the serum mannose-binding lectin (mbl), several secreted human c-type lectins of the collectin family, collectin 11, and the pulmonary surfactant proteins (sp) -a1, -a2, and -d (sftpa1, sftpa2, and sftpd, respectively), may neutralize iav infectivity in vitro [141] . mice lacking sp-a or sp-d were more susceptible to iav infection, indicating that sps exert relevant roles against iav infection [142] [143] [144] . two frequent sp-a2 (sftpa2) missense alleles (rs1965708-c, leading to the mutation q223k and rs1059046-a, leading to the mutation t9n) were associated with acute respiratory failure, mechanical ventilation, and acute respiratory distress syndrome after infection with ph1n1 2009 virus in a spanish population [145] . in addition to c-type lectins, s-type lectins have been described, such as galectins, which recognize galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. importantly, intranasal treatment of galectin-1 enhanced survival of mice infected with iav by reducing viral load, apoptosis, and inflammation in the lung [146] . moreover, galectin-1 knockout mice showed increased susceptibility to influenza virus infection than wild-type mice [146] . to study human genetic susceptibility to avian iav h7n9 infection, a genome-wide association study involving 106 heavily-exposed healthy poultry chinese workers and 102 iav h7n9 patients was performed [147] . functional variants of galectin-1 gene, including rs4820294 and rs13057866, causing increased expression levels of galectin-1 expression, may confer more protection from iav h7n9 infection to the carriers of these variants [147] . the cleavage of the iav ha by host proteases is critical for viral infectivity. tmprss2 is a type ii transmembrane serine protease family member, which was shown to activate ha proteins of multiple human iavs in tissue culture cells. furthermore, deletion of tmprss2 in mice impairs the spread of h1n1 influenza viruses, including the ph1n1 2009 swine iav [148] . in addition, bodyweight loss and survival after h3n2 iav infection were less severe in tmprss2 mutant mice compared to wild type mice [148] . the genetic predisposition to severe ph1n1 2009 influenza virus was evaluated in chinese human subjects, finding that the gg genotype of rs2070788, leading to increased expression of tmprss2, was a risk variant to severe ph1n1 influenza [149] . furthermore, rs2070788 and rs383510, both of them associated with increased gene expression, were significantly associated with the susceptibility to iav h7n9 [149] . table 1 . single nucleotide polymorphisms associated with susceptibility and severity of influenza infections. recognizes dsrna, triggering ifn production. rs not annotated; f303s (nonsyn). rs5743313 (ncr). [98] [99, 100] rig-i detects dsrna and 5 -triphosphates of the negative ssrna iav genome, leading to innate immune responses activation. rs72710678; r71h (nonsyn). rs138425677; p885s (nonsyn). [109] irf-7 transcription factor that increases ifn production in response to viruses. rs786205223; f410v (nonsyn) rs375323253; q421x (nonsyn) [113] irf-9 transcription factor essential for ifn signaling and the transcriptional induction of isgs. c.991g>a occurred in the final nucleotide of exon 7 and disrupted the essential splice site at the boundary of exon 7 and intron 7 (nonsyn). c.577+1g>t, was localized in the donor splice site of introns 5 and 6 and led to transcripts lacking exon 5 (nonsyn). [115] [116] ifitm3 isg which abrogates the release of iav content from late endosomes into the cytoplasm. ifitm3 increases the survival of mouse lung-resident cd8 + t cells after iav infection, which can help clear the infection. rs12252, leading to an alteration of the first splice acceptor site, leading to an ifitm3 protein lacking the first 21 amino acids (nonsyn). rs34481144, is located in the 5 -utr and affects ifitm3 expression with the risk allele showing lower mrna expression (ncr). [ 100, 118, 120, 122] [123] il-1b inflammatory cytokine involved in the development of adaptive immune responses. furthermore, accumulating data has suggested that il-1a and il-1b have critical roles in innate immunity against viral infections. rs1143627, located 31 base pairs upstream from the transcription start site, on the il-1b promoter. this nucleotide change is located in a tata-box motif of il-1b, affecting the transcription activity of il-1b (ncr). [128, 129] galectin-1 recognizes galactose-containing oligosaccharides present in the cellular plasma membranes and in viruses, such as iav. -rs4820294 (ncr). -rs13057866 (ncr). [147] tmprss2 type ii transmembrane serine protease family member which activates ha proteins of diverse human iav in tissue culture cells. deletion of tmprss2 in mice impairs the spread of h1n1 influenza viruses, including the ph1n1. moreover, body weight loss and survival were less severe in tmprss2 mutant mice compared to wild type mice after infection with h3n2 iav. -rs2070788, localized in an intron (ncr). -rs383510, localized in an intron (ncr). [149] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). currently, iav vaccines are the main strategy to prevent iav infection, though their effectiveness is suboptimal in many cases. notably, the efficacy of vaccines against iav infections can fluctuate and there is a significant immune response variability across the population. factors such as previous exposure to iav infections or vaccines, age, and the closeness of the match between the vaccine and circulating strains are important to explain differences in vaccine effectiveness between seasons and group populations [44, 46, [150] [151] [152] . however, multiple reports have demonstrated that the host genetic background and polymorphisms on key immune response genes modulate the immune response to infection or vaccination [153] [154] [155] [156] [157] [158] [159] [160] . therefore, new insights into iav-host interaction and immune response modulating factors could allow us to design better vaccination strategies. snps may modify the humoral immune response after iav vaccination. therefore, their impact on the immune responses induced after iav vaccination are being analyzed [153] [154] [155] [156] . the major histocompatibility complex (mhc) is localized in chromosome 6 of the human genome, it includes multiple genes and exhibits considerable diversity between populations. moreover, in this genomic region, there is a higher presence of snps than in other sections of the genome. mhc class i and class ii molecules have an essential role in the adaptive immune system in response to infections. both classes of proteins bind peptide fragments derived from pathogens to be presented on the cell surface for recognition by appropriate t cells [97, 161, 162] . in those genes, the human leukocyte antigens (hla) class i and ii are important because of their role in the immune system. gelder et al. studied whether hla class ii polymorphisms modulate anti-iav antibody responses to vaccination in a united kingdom population [154] . for that, a cohort of hla-typed donors at risk was investigated, and hemagglutination-inhibition (hai) titers were evaluated before and 28 days after the administration of seasonal trivalent influenza vaccine. a correlation between hla class ii alleles and iav hai titers in the influenza risk group was found. moreover, a positive association between non-responsiveness to influenza vaccine and hla-drb1*07 and a negative association with hla-drb1*13 and hla-dqb1*0603-9/14 [154] was reported, suggesting that polymorphisms in hla class ii molecules affect antibody responses to iav vaccination. these findings are important because they could potentially identify individuals who may not be protected by current vaccination approaches. in another study, poland et al. analyzed the immunogenetic relationships between hla, cytokine and cytokine receptor gene polymorphisms in the induction of antibodies in response to inactivated seasonal vaccines [156] . authors did not find statistically significant associations between hla class ii alleles and iav hai titers. however, they established a positive association of some hla class i alleles and iav h1n1 hai titers, including hla-a*1101, a*6801, b*3503, b*1401, and c*0802. in contrast, they did not find associations between the hla-a, b or c alleles and hai antibody titers for iav h3n2. in addition, when authors evaluated a panel of 586 cytokine and cytokine receptor snps, they identified several significant associations between snps, in regulatory or coding regions of cytokine (il-6, il-12b) or cytokine receptor (il-1r, il-10rb, tnfrsf1a) genes and variations in hai antibody titers for iav h1n1 [156] (table 2) . notably, snps from three genes, il-6 (rs1800796), il-12b (rs3212227) and il-1r1 (rs3732131) revealed links with iav h1n1-induced antibody responses in an allele dose-related way. the presence of snp allele c or g in the il-12b or il-1r1genes, respectively resulted in reduced hai titers. however, high hai titers in the presence of minor snp allele g in the il-6 gene were observed [156] . snps associations between cytokine or cytokine receptor genes and iav h3n2 hai titers were also identified ( table 2) . for example, a variant ga for non-synonymous snps within the il-12 receptor gene (rs2307153; d465g) and tnf receptor 2 gene (rs5746026; k232e) displayed associations with lower hai titers, while a minor allele t variant (rs12722605) located in the 3 region of the il-2 receptor gene was related with high antibody titers ( table 2 ). these data suggest that host snps affect responses to influenza vaccine. mannose-binding lectin 2 (mbl-2) is a protein that binds n-acetylglucosamine, mannose, and fucose on different microorganisms and activates the lectin complement pathway [163, 164] . tang et al. studied the presence of snps in subjects who received an inactivated influenza vaccine. for that, authors classified the vaccine recipients in poor, normal or adverse responders. they observed that the g to a snp in the codon 54 allele (rs1800450) in mbl-2 was associated with a decreased risk for the development of adverse or poor responses (table 2 ) [165] . in addition, they did not find a significant association between responses and either tnf-α or il-10 promoter snps among the 3 response groups [165] . cytokine expressed as a response to infections or tissue injuries. it plays an important role in host defense through the stimulation of acute-phase responses. -rs1800796 (ncr). -rs2069861 (ncr). iiv [156] il-12b cytokine that serves as a crucial inducer of th1 cell development. rs3212227, located in 3´utr (ncr). iiv [156] ifn-b1 cytokine released as part of the innate immune response against infection by viruses or other pathogens. rs1364613 (ncr). iiv [156] tnfrsf1a cytokine receptor, its interaction with tnf-α control cell survival, apoptosis, and inflammation. rs4149621 (ncr). iiv [156] il-1r1 cytokine receptor involved in inflammatory and immune responses. rs3732131, located in 3´utr (ncr). iiv [156] il-10rb cytokine receptor that mediates the activation of the jak/stat signaling pathway leading to the expression of isg. rs3171425, located in 3´utr (ncr). iiv [156] il-2ra this cytokine receptor is important for the signaling pathway leading to immune cell differentiation and function. -rs2228150 (syn). -rs12722605 (ncr). iiv [156] il-10ra cytokine receptor that is involved in the inhibition of the synthesis of several proinflammatory cytokines. -rs4252249 (syn) -rs4252243 (ncr). iiv [156] il-12rb2 cytokine receptor that plays a role in th1 cell differentiation. rs2307153; d465g (nonsyn). iiv [156] il-1rn cytokine receptor which modulates a variety of immune and inflammatory responses related with il-1. -rs315952 (syn). -rs315951 located in 3´utr (ncr). iiv [156] tnfrsf1b cytokine receptor involved in the recruitment of anti-apoptotic proteins. rs5746026; k232e (nonsyn) iiv [156] mbl-2 this calcium-dependent protein that plays an important role in innate immunity, and activates the lectin complement pathway. rs1800450; g54d (nonsyn) iiv [165] il-28b (ifnl3) type iii ifn molecule, with brad functions in antiviral responses rs8099917 (ncr). iiv [153] syn-synonymous, nonsyn-nonsynonymous, ncr-non-coding region (intron, regulatory regions, promoter or utr). iiv: inactivated seasonal vaccine. il-6, interleukin 6. il-12b, interleukin 12. ifn-b1, interferon beta 1 (ifnβ). tnfrsf1a, tnf receptor superfamily member 1a. il-1r1, interleukin 1 receptor type 1. il-10rb, interleukin 10 receptor subunit beta. il-2ra, interleukin 2 receptor subunit alpha. il-10ra, interleukin 10 receptor subunit alpha. il-12rb2, interleukin 12 receptor subunit beta 2. il-1rn, interleukin 1 receptor antagonist. tnfrsf1b, tnf receptor superfamily member 1b. mbl-2, mannose binding lectin 2. il-28 b or ifnl3, interferon lambda 3. other snps that are not related with immune responses have been also linked to vaccine effectiveness. egli et al. revealed that the presence of the t/g or g/g genotype (rs8099917, minor-allele) in il-28b (ifnλ3), a type iii ifn, was linked with increased seroconversion in recipients of an inactivated influenza vaccine (table 2 ) [153] . moreover, iav-stimulated b-and t-cells from the minor-allele carriers exhibited increased hla-dr and il-4 expression, respectively. in addition, the expression of il-28b, but not il-28a or il-29, mrnas was significantly reduced in the rs8099917, minor-allele carriers. authors also reported that the il-28b rs8099917 polymorphism affected humoral responses to the iav vaccine, and had a strong outcome on cellular immune responses by modulating the th1/th2 cytokine response [153] . these findings are important because they will help to predict which individuals could not be protected by present vaccines and they can also be used to design personalized vaccine strategies to optimize the immune reaction. the sequencing of the human genome together with the development of novel bioinformatic tools have made possible the identification of multiple snps. more information is available for the scientific community in the databases. in addition, the identification and study of the human genome variability has opened the opportunity to investigate their association with the risk of developing multiple human diseases facilitating their diagnosis or the susceptibility to infections caused by viruses or other pathogens. moreover, the knowledge and analysis of genomic variability will be a valuable tool to predict the outcome of prophylactic or therapeutic interventions, including vaccines and drugs. the analysis of human snps and their association with iav infections or vaccination outcomes have just begun. however, current research and data reflect the importance to obtain a better understanding of these relations and the mechanisms underlying the effect of snps in the human immune system. in the future, this knowledge 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genetic polymorphisms on vaccine induced antibody response immunogenetics of seasonal influenza vaccine response immunological variation due to genetics of inflammatory snps and age and impact on disease manifestation genetics and vaccines in the era of personalized medicine host genetic factors can impact vaccine immunogenicity and effectiveness the genetic background influences the cellular and humoral immune responses to vaccines the major histocompatibility complex: a paradigm for studies of the human genome the nature of selection on the major histocompatibility complex a journey through the lectin pathway of complement-mbl and beyond the role of mannose-binding lectin in health and disease host single-nucleotide polymorphisms and altered responses to inactivated influenza vaccine we apologize for those publications we could not refer due to space limitations. the authors declare no conflict of interest. key: cord-353869-l53ms3q8 authors: friesen, robert h. e.; koudstaal, wouter; koldijk, martin h.; weverling, gerrit jan; brakenhoff, just p. j.; lenting, peter j.; stittelaar, koert j.; osterhaus, albert d. m. e.; kompier, ronald; goudsmit, jaap title: new class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets date: 2010-02-08 journal: plos one doi: 10.1371/journal.pone.0009106 sha: doc_id: 353869 cord_uid: l53ms3q8 background: the urgent medical need for innovative approaches to control influenza is emphasized by the widespread resistance of circulating subtype h1n1 viruses to the leading antiviral drug oseltamivir, the pandemic threat posed by the occurrences of human infections with highly pathogenic avian h5n1 viruses, and indeed the evolving swine-origin h1n1 influenza pandemic. a recently discovered class of human monoclonal antibodies with the ability to neutralize a broad spectrum of influenza viruses (including h1, h2, h5, h6 and h9 subtypes) has the potential to prevent and treat influenza in humans. here we report the latest efficacy data for a representative antibody of this novel class. methodology/principal findings: we evaluated the prophylactic and therapeutic efficacy of the human monoclonal antibody cr6261 against lethal challenge with the highly pathogenic avian h5n1 virus in ferrets, the optimal model of human influenza infection. survival rates, clinically relevant disease signs such as changes in body weight and temperature, virus replication in lungs and upper respiratory tract, as well as macroand microscopic pathology were investigated. prophylactic administration of 30 and 10 mg/kg cr6261 prior to viral challenge completely prevented mortality, weight loss and reduced the amount of infectious virus in the lungs by more than 99.9%, abolished shedding of virus in pharyngeal secretions and largely prevented h5n1-induced lung pathology. when administered therapeutically 1 day after challenge, 30 mg/kg cr6261 prevented death in all animals and blunted disease, as evidenced by decreased weight loss and temperature rise, reduced lung viral loads and shedding, and less lung damage. conclusions/significance: these data demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant animal model of influenza and justify clinical development of this approach as intervention for both seasonal and pandemic influenza. a novel class of human monoclonal antibodies against influenza has been recently discovered [1] . these antibodies bind to the membrane-proximal stem of haemagglutinin, the major viral surface protein, and neutralize the influenza virus by blocking its fusion with the host cell [2] . a panel of antibodies with a similar mode of action was reported subsequently by sui et al. [3] . due to the high conservation of their recognition site, this class of antibodies has shown the ability to neutralize a broad spectrum of influenza subtypes, including h1, h2, h5, h6 and h9 [1] , and can be expected to also neutralize viruses from subtypes h4, h8, h11-h14 and h16, as well as their future antigenic drift variants [2] . one of these antibodies, cr6261, was investigated in mice and shown to be protective when given before and after lethal challenges with h1n1 and h5n1 virus, suggesting that it has potential as the first-ever broad-spectrum monoclonal antibody for prophylaxis and treatment of influenza virus infections [1] . according to the world health organization, seasonal influenza causes up to 500,000 deaths worldwide each year [4] . immunologically naïve infants, immunocompromised individuals and the elderly are particularly susceptible to illness caused by seasonal influenza viruses, with 90% of deaths occurring in the latter group [4, 5] . in addition, subtypes of influenza a viruses that have not previously circulated among humans occasionally cross from animal reservoirs, raising the spectre of a pandemic. over the past decade, the most prominent pandemic threat appeared to be posed by highly pathogenic avian influenza viruses of subtype h5n1. however, it was a new strain of human h1n1 that emerged in mexico and the united states in march and april 2009 and rapidly spread across the globe that caused the who to declare a pandemic on june 11th [6, 7] . although the virulence of this virus is currently moderate, particularly compared to the case fatality rate of over 60% of human h5n1 infections, this may change over time. meanwhile, the threat from highly pathogenic avian h5n1 viruses persists as they continue to circulate and evolve in bird populations. preventive vaccination has historically been the primary means of influenza control, but this approach has important limitations. vaccines typically elicit a potent neutralizing antibody response only to the specific viral strains they contain, and closely related viruses [8, 9] . furthermore, influenza vaccines have suboptimal immunogenicity and efficacy in the groups at highest risk of severe disease: the very young, the elderly and immunocompromised individuals [10] . the current therapeutic regimen for influenza a is limited to two classes of drugs: the adamantanes (amantadine and rimantadine) and the neuraminidase inhibitors (oseltamivir and zanamivir). adamantanes rapidly elicit viral resistance, and resistance rates are high among h3n2 viruses and certain clades of h5n1 viruses [11] [12] [13] [14] . the use of oseltamivir, the leading antiviral influenza drug, has been limited by the sudden and widespread emergence of resistance among circulating h1n1 influenza strains [15, 16] . oseltamivir resistance has also been observed during treatment of h5n1 infection [17, 18] . zanamivir is still effective against h1n1 viruses and resistance to this drug is less likely to arise [19] . however, its use is limited to patients who can actively use an inhaled drug, which excludes young children, impaired older adults, or patients with underlying airway disease [16] -once again, the group of patients most vulnerable to serious complications from influenza infection. in the absence of reliable antiviral drugs and vaccines, development of alternative strategies for influenza prophylaxis and therapy is urgently required. in order to assess whether cr6261 may be a viable option for human treatment, we evaluated the prophylactic and therapeutic efficacy of this human monoclonal antibody in a highly stringent lethal h5n1 influenza ferret model. the ferret is the most suitable disease model for human influenza infection as it displays very human-like disease [20] [21] [22] . four groups of 6 ferrets each received 30, 10, 3 or 1 mg/kg cr6261 via an intravenous injection and were challenged the next day with the highly pathogenic avian a/indonesia/5/2005 (h5n1) virus. a control group of 6 ferrets received 30 mg/kg of the irrelevant isotype-matched antibody cr3014. all ferrets that received 30 or 10 mg/kg cr6261 survived, compared to only 33.3% of the control animals ( figure 1a) . a further reduction of the dose to 3 mg/kg was clearly correlated with a lower survival rate of 66.7%. though not statistically significant different from survival observed in control animals, there is a 50% mortality reduction. the lowest dose of 1 mg/kg cr6261 was not associated with survival benefit compared to control animals. survival times differed significantly between groups receiving 30 or 10 mg/kg cr6261 and the control group (p = 0.020). moribund animals showed general depression, anorexia and lethargy, and exhibited clinical signs of respiratory disease, including dyspnoea. animals treated at efficacious dose levels (30 and 10 mg/kg) did not loose body weight, whereas the mean weight loss in the control group was 10.5% by the time the ferrets died or were euthanized ( figure 1b ). an exception was one control ferret that succumbed to infection within 48 hours after challenge and lost hardly any weight before. ferrets that received 3 or 1 mg/ kg cr6261 showed similar declines in body weight as the control animals. one day after challenge the maximum body temperature was observed; for each ferret the maximum body temperature is depicted in figure 1c . the groups treated with 30 or 10 mg/kg had mean temperatures of 39.4uc and 40.7uc, respectively, which were significantly lower than the mean of 41.7uc observed in the control group (p,0.001 and p = 0.015, respectively). the mean temperature observed in animals treated with the lower doses of cr6261 (41.3uc and 41.5uc for 3 and 1 mg/kg, respectively) did not differ significantly from the control group. the mean temperature in animals observed 3 days before challenge was similar across the 5 groups, ranging from 37.6uc to 38.4uc. individual body temperature varied considerably within one healthy ferret over 24 hours (standard deviation 0.7uc). ferrets treated with 30 or 10 mg/kg cr6261 did not shed infectious virus in the upper respiratory tract at any time, whereas animals in the control group did ( figure 1d ). treatment with 3 and 1 mg/kg did not prevent shedding, but reduced the proportion of ferrets with infectious virus in nasal and/or throat swabs. after necropsy, all ferrets were assessed for viral load in the lungs (figure 1e), and the animals that received 30 mg/kg cr6261 or control antibody were also assessed for viral load in the brain, liver, spleen, blood and kidney. the levels of virus replication in the lungs of ferrets treated with 30 and 10 mg/kg cr6261 were 3.9 and 2.9 log 10 tcid 50 /g lower than that in the control group (both p,0.001). no infectious virus was detected in any of the other organs of the ferrets that received 30 mg/kg cr6261, whereas infectious virus was found in the brain of 2, the liver of 3, and the spleen of 5 of the 6 control animals (data not shown). there was no significant difference in lung viral load in the groups receiving 3 or 1 mg/kg cr6261 compared to the control group (p = 0.74 and p = 0.91, respectively). histopathological results were in accordance with the findings described above; animals that received 30 and 10 mg/kg cr6261 showed much less pulmonary lesions such as primary atypical pneumonia, subacute bronch(iol)itis, emphysema and congestion, or showed such lesions at a lower grade of severity, compared to animals from the other groups. bronchiolitis obliterans was not observed in any animal that received 30 mg/kg cr6261. compared to this higher-dose group, animals that received 10 mg/kg cr6261 showed more regenerative response (diffuse grey/red area and bronchioloalveolar hyperplasia) in the lungs, and more inflammatory changes in the trachea. pulmonary oedema was not observed in animals that received 30 or 10 mg/kg cr6261, but was observed frequently in all other groups. these findings were in agreement with mean lung weights, which were lowest in the animals treated with 30 or 10 mg/ kg cr6261 (6.3 g and 7.6 g, respectively) and significantly lower in this group than in control animals (15.0 g; both comparisons p,0.001). no significant difference in lung weight was found between the groups receiving 3 or 1 mg/kg cr6261 (14.1 g and 15.4 g, respectively) and the control group (15.0 g). these findings show that, in a dose dependent way, prophylactically administered cr6261 confers protection against lethal h5n1 challenge, prevents morbidity and viral dissemination and reduces pulmonary pathology. to assess the therapeutic efficacy of the monoclonal antibody cr6261, two groups of 10 ferrets were challenged as above and given 30 mg/kg of cr6261 either 4 or 24 hours later. a comparator group of 10 ferrets received 30 mg/kg of the control antibody 4 hours after challenge. survival rates in the groups receiving cr6261 at 4 and 24 hours after challenge were 100%, whereas only 20% of the animals in the control group survived (p,0.001) (figure 2a ). mean decline in body weight at the end of the experiment was 6.2% in the group of ferrets that received cr6261 4 hours after challenge ( figure 2b) , which was significantly less (p = 0.025) than the 10.1% observed in control animals. animals treated 24 hours post challenge showed a mean body weight loss of 8.4%, which was not significantly different from the control animals (p = 0.427). the group of ferrets treated with cr6261 4 hours post challenge had a mean maximum temperature of 40.0uc, compared to 41.8uc in the control group (p,0.001). in line with the rapid rise in temperature after challenge observed in the prophylaxis experiment ferrets treated with cr6261 24 hours after challenge showed a mean maximal temperature of 41.5uc before cr6261 was administered (p = 0.15 versus 41.8uc of the control group, figure 2c ). ferrets treated with cr6261 at 4 hours post challenge did not shed infectious virus in the upper respiratory tract throughout the study ( figure 2d ). in the group treated with cr6261 at 24 hours post challenge, one ferret had a low concentration of infectious virus (2.8 log 10 tcid 50 ) in the throat on day one, but no virus was detected on subsequent days. in contrast, all animals in the control group shed virus during one or more days. accordingly, the mean viral loads in the lungs of ferrets treated with cr6261 at 4 and 24 hours post challenge were considerably lower than that in the control group (differences were 3.9 and 4.5 log 10 tcid 50 /g, respectively, both comparisons p,0.001; figure 2e ). the lungs of animals that received cr6261 at 4 hours post challenge showed less pulmonary lesions (alveolar oedema, bronchiolitis obliterans, congestion, emphysema, bronchioloalveolar hyperplasia and primary atypical pneumonia), or showed such lesions at a lower grade of severity, compared to the lungs of animals from the other two groups. animals of the control group were most affected by primary atypical pneumonia. these findings were in accordance with the observation that the mean lung weights of ferrets treated with cr6261 at 4 hours post challenge were lower compared to the control group (5.7 g versus 14.9 g, p,0.001; figure 2f ). animals that received cr6261 at 24 hours post challenge showed most regenerative response (bronchioloalveolar hyperplasia) in the lungs, suggesting damage to the lung parenchyma with subsequent regenerative response. the mean lung weight in this group was significantly higher than that of the group receiving cr6261 at 4 hours post challenge (8.4 g versus 5.7 g), but lower than that of the control group (p,0.001). from the study outset, one animal had been added to each of the two treatment groups to be sacrificed for gross-pathology and histology on lungs as soon as 50% of the control animals died. the purpose was to test for possible bias due to differences in the timing of death. infectious virus titres in the lungs of the treated ferrets sacrificed at day 3 were identical to those in treated animals sacrificed at the end of the study (open circles in figure 2e) . similarly, there were no differences in lung weight and pathology between animals sacrificed at day 3 or after day 5 ( figure 2f ). this indicates that the results were not biased by differences in the timing of euthanasia or spontaneous death. cr6261 represents a new class of human monoclonal antibodies that exhibits immediate and potent efficacy for the prevention or treatment of influenza in a clinically relevant model for severe disease. the results presented here also confirm previously reported data demonstrating the prophylactic and therapeutic efficacy of cr6261 in mice challenged with h5n1 influenza viruses. together with the h1n1 challenged mice data published earlier [1] these findings indicate that cr6261 is effective across a broad spectrum of influenza viruses, including seasonal and potentially pandemic strains. passive immunotherapy would be particularly beneficial for the groups at highest risk of severe disease due to seasonal influenzathe elderly and immuno-compromised-but may also be indispensable for the general public in the event of a pandemic disease outbreak caused by high-risk pandemic candidates such as h2, h5, h6 and h9. monoclonal antibodies against influenza viruses have been studied for decades, but their potential-and thus development-as 'passive' immunotherapy for influenza has been inhibited by the lack of monoclonal antibodies with broad neutralizing activity. this lack is due to the tolerance of influenza virus for genetic changes in the most immunogenic regions on its surface. the recent discovery of broadly neutralizing human monoclonal influenza antibodies [1] and the demonstrated efficacy of a representative of this novel class of antibodies against lethal viral challenge in a clinically relevant model, as presented in this paper, create an opportunity for the prevention and treatment of influenza infections, regardless of the causal strain. the possibility of a potent and broadly neutralizing agent that would equip clinicians and public health workers to deal effectively with the influenza viruses of the future represents a paradigm shift in the approach to influenza control. influenza illness observed in the ferrets infected with h5n1 virus in this study closely resembles influenza in humans with h5n1 infection, who present with fever, cough, shortness of breath, and radiological evidence of pneumonia [23] . besides respiratory symptoms, gastrointestinal symptoms such as diarrhoea, vomiting and abdominal pain are often present. in severe cases, the pneumonia rapidly progresses to acute respiratory distress syndrome and multiorgan failure. high viral loads, extrapulmonary virus dissemination and hypercytokinaemia are associated with fatal outcome (reviewed in [24] [25] [26] ). autopsies of patients who succumbed to influenza a (h5n1) virus infection have shown diffuse alveolar damage, patchy interstitial lymphoplasmacytic infiltrates, bronchiolitis with squamous metaplasia and pulmonary congestion with various degrees of haemorrhage [27] [28] [29] . the clinical signs observed in the control animals of this ferret study correspond to the most severe influenza pathology in humans [23] . the efficacy of cr6261 in preventing these clinical signs in the prophylactic and therapeutic ferret model presented here, together with the efficacy shown in mice after lethal challenges with different viruses, strongly indicate that cr6261 can be expected to be efficacious against disease caused by the other, less virulent viruses it neutralized in vitro [1] . in a meta-analysis of experimental influenza infection of placebo-treated and untreated healthy volunteers, carrat et al. [30] studied 1280 participants who were challenged with either influenza type h1n1, h3n2 or b. interestingly, viral shedding was highly correlated with the presence of clinical symptoms such as fever, runny nose, sore throat, sneezing, cough and shortness of breath. the authors concluded from their meta-analysis that subjects with symptomatic illness shed virus in amounts 100 to 1000 fold higher than subjects who were not ill. the fact that cr6261 after intravenous administration instantaneously reduces viral shedding indicates that in man cr6261 might reduce clinical symptoms in subjects infected with influenza virus. in addition, studies in guinea pigs showed that a reduction in nasal wash titers correlate with a decreased efficiency of viral transmission by aerosol [31] . the ability of cr6261 to abolish shedding of virus in pharyngeal secretions strongly suggests that antibodies like cr6261 might prevent or reduce virus spreading at the onset of an epidemic influenza outbreak in nursing homes or in case of a pandemic. in this study, we assessed the efficacy of the human monoclonal antibody cr6261 against a highly pathogenic avian h5n1 virus, as this provides a stringent disease model for severe influenza. however, the potential use of this antibody is not limited to viruses of this subtype or to other avian strains that may pose a pandemic threat. cr6261 has been shown to recognize h1 viruses that have emerged over a time span of 90 years from the h1n1 virus which caused the 1918 'spanish flu' pandemic to the latest brisbane viruses. since the epitope is conserved cr6261 is predicted to bind to future antigenic drift variants. this means that cr6261 could be used to protect against all h1 influenza viruses, including the ones resistant to oseltamivir. use of cr6261 in combination with effective medication against h3, such as oseltamivir or zanamivir, would effectively protect against all seasonal influenza viruses, without a need for prior knowledge of the virus subtype or strain. in the present study ferrets were challenged with an inoculum which is probably much higher compared to the viral exposure in naturally infection in humans. the rapid deterioration in ferrets with death occurring within 3 days underpins this hypothesis since humans exposed to h5n1 develop the first symptoms 2-4 days after the last exposure and even periods of up to 8 days have been reported [25] . the clinical signs in this model are quite extreme, but important for establishing the efficacy of the antibody as proof of concept. the data of these two experiments demonstrate the prophylactic and therapeutic efficacy of this new class of human monoclonal antibodies in a highly stringent and clinically relevant ferret model of human influenza. the human monoclonal antibody cr6261 was isolated from the igm+, cd27+ b cell repertoire of a healthy individual who was recently vaccinated with the seasonal influenza vaccine, using phage display selection on recombinant h5 haemagglutinin [1] . cr3014, an isotype-matched antibody with the ability to neutralize sars corona virus-which has similar tissue/organ tropism as that of h5 viruses-was used as a control antibody [32] . both antibodies were produced on per.c6h cells. the study was performed with outbred ferrets (mustela putorius furo, female, age approximately 8 months, schimmel farms, uddel, the netherlands). ferrets were screened for the presence of serum antibodies against aleutian disease virus, circulating seasonal influenza virus strains (a/h1n1, a/h3n2 and b) and the challenge virus (h5n1, a/indonesia/5/2005), and only seronegative animals were used in the study. the animals were housed in study groups of 6 (prophylactic experiment) or 10 (therapeutic experiment). antibodies were administered by intravenous injection in the jugular vein. viral challenge was performed intratracheally with 10 5 tcid 50 a/indonesia/05/2005 in 3 ml of pbs [33] . clinical observations were performed twice a day on days of intervention and once daily on other days. in the prophylactic experiment, animals were weighed 2 weeks before viral challenge (day 214), immediately prior to antibody administration (day 21), and after challenge (days 2, 4, and 5 or on the day of premature death). in the therapeutic experiment, animals were weighed on day 214 and day 22, immediately prior to challenge and antibody administration, and on days 2, 4 and the last study day. body temperature was recorded every 15 minutes throughout both experiments using a device (dst micro-t, star-oddi, reykjavik, iceland) implanted in the peritoneal cavity 14 days before challenge. the animal experiments were carried out in the central animal facilities of the netherlands vaccine institute (nvi, bilthoven) under conditions that meet the dutch legal requirements for animal experimentation and are in accordance with the 'guide for the care and use of laboratory animals', the recommendations of the institute for laboratory animal research (us national institutes of health), and association for assessment and accreditation of laboratory animal care international (aaa-lac) standards. viral titrations were performed as described elsewhere [34] . briefly, pharyngeal and nasal swabs were collected from all animals at day 21, day 2, day 4, and day 5. individual swabs were homogenized and resuspended in 3 ml medium and stored at 280uc until analysis. viral titres were determined by virus titration on madine darby canine kidney (mdck) cells. viral shedding from the upper respiratory tract was analysed by calculating the proportion of ferrets with detectable levels of infectious virus in nasal and/or throat swabs relative to the number of living ferrets. after necropsy, the cranioventral, craniodorsal, caudoventral and caudodorsal sections of the right lung were collected from each animal, weighed, homogenized and resuspended in 3 ml medium, and stored at 280uc until analysis. viral titres were determined after thawing of the tissue sections followed by homogenization and resuspension by virus titration on mdck cells. in addition, viral titres in tissues of brain, spleen, liver, kidney, and plasma from animals that received either 30 mg/kg of cr6261 or cr3014 one day prior to challenge were determined using the same method. a complete macroscopic post-mortem examination was performed on all animals. this included examination of the external surfaces and all orifices; the thoracic, abdominal and pelvic cavities with their associated organs and tissues; and the neck with its associated organs and tissues. lungs were weighed and all lung lobes were inspected and lesions described. the left lung (including trachea) was collected during autopsy, inflated with 10% neutral buffered formalin for fixation/histology and microscopic examination. paraffin embedded tissue sections (left cranial lobe, left caudal lobe, right cranial-, middle-and caudal lobes and accessory lobe) were stained with haematoxylin and eosin and assessed by light microscopy for aspects like congestion, emphysema, presence of foreign body, haemorrhagy, bronchioloalveolar hyperplasia and inflammation, and oedema. prior to blood sampling, the taking of nose and throat swabs and euthanasia, the ferrets were anaesthetised with ketamin (25 mg/ kg; i.m.). for implantation of temperature sensors, antibody administration and viral challenge, the animals were anaesthetized with a mixture of ketamin (12.5 mg/kg; i.m.) and domitor (7.5 mg/ kg; i.m.), followed by antisedan (0.5 mg/kg; i.m.). survival times after viral challenge were analysed using the logrank test and survival proportions using the fisher's exact test. body weight expressed as percentage change from baseline was calculated at the end of the study period (or earlier in the event of earlier death). maximum body temperatures were observed during day one and these values were subsequently used for calculating the mean maximum body temperature for each group. variables were analysed in an one-way analysis of variance (anova) with post-hoc testing to compare to the control group using dunnett's adjustment for multiple comparisons. lung weight and lung viral titres were compared across arms using anova, with day of necropsy entered as a covariate. differences between treatment groups were estimated using marginal means, with sidak's adjustment for multiple comparisons. statistical analyses were performed using spss version 15.0 (spss inc. usa). statistical significance level was set at a = 0.05. heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells antibody recognition of a highly conserved influenza virus epitope structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses fact sheet 211: influenza. world health organization vaccines against epidemic and pandemic influenza swine influenza a (h1n1) infection in two children-southern california world now at the start of 2009 influenza pandemic the contents of the syringe interim within-season estimate of the effectiveness of trivalent inactivated influenza vaccine-marshfield, wisconsin, 2007-08 influenza season prevention and control of influenza; recommendations of the advisory commitee on immunization practices (acip) surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide amantadine-resistance among h5n1 avian influenza viruses isolated in northern china adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states high levels of adamantane resistance among influenza a (h3n2) viruses and interim guidelines for use of antiviral agents-united states, 2005-06 influenza season drug resistance. a 'wimpy' flu strain mysteriously turns scary global transmission of oseltamivir-resistant influenza crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants oseltamivir resistance during treatment of influenza a (h5n1) infection medical management of influenza infection animal models for the study of influenza pathogenesis and therapy ferrets and the challenges of h5n1 vaccine formulation animal models in influenza vaccine testing factors associated with case fatality of human h5n1 virus infections in indonesia: a case series update on avian influenza a (h5n1) virus infection in humans human infection with highly pathogenic h5n1 influenza virus human h5n1 influenza: current insight into pathogenesis h5n1 infection of the respiratory tract and beyond: a molecular pathology study the comparative pathology of severe acute respiratory syndrome and avian influenza a subtype h5n1-a review apoptosis and pathogenesis of avian influenza a (h5n1) virus in humans time lines of infection and disease in human influenza: a review of volunteer challenge studies blocking interhost transmission of influenza virus by vaccination in the guinea pig model human monoclonal antibody as prophylaxis for sars coronavirus infection in ferrets crossprotection against lethal h5n1 challenge in ferrets with an adjuvanted pandemic influenza vaccine comparison of rna hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro we thank andrea dingemans for her critical review of the manuscript, willem van aert and geert van amerongen for excellent technical assistance, james simon for supervising the viroclinics input, vera teeuwsen for preparing the protocols, and elina van herwijnen for independent data check, and jaco klap for statistical support. key: cord-327180-yw8rzrb7 authors: prateepko, tapanan; chongsuvivatwong, virasakdi title: patterns of perception toward influenza pandemic among the front-line responsible health personnel in southern thailand: a q methodology approach date: 2009-05-28 journal: bmc public health doi: 10.1186/1471-2458-9-161 sha: doc_id: 327180 cord_uid: yw8rzrb7 background: thailand has joined the world health organization effort to prepare against a threat of an influenza pandemic. regular monitoring on preparedness of health facilities and assessment on perception of the front-line responsible health personnel has never been done. this study aimed to document the patterns of perception of health personnel toward the threat of an influenza pandemic. methods: q methodology was applied to a set of 385 health personnel in charge of influenza pandemic preparedness in the three southernmost provinces of thailand. subjects were asked to rank 33 statements about various issues of influenza pandemic according to a pre-designed score sheet having a quasi-normal distribution on a continuous 9-point bipolar scale ranging from -4 for strongly disagree to +4 for strongly agree. the q factor analysis method was employed to identify patterns based on the similarity and dissimilarity among health personnel. results: there were three main patterns of perception toward influenza pandemic with moderate correlation coefficients between patterns ranging from 0.37 to 0.55. pattern i, health personnel, which we labeled pessimistic, perceived themselves as having a low self-efficacy. pattern ii, which we labeled optimistic, perceived the threat to be low severity and low vulnerability. pattern iii, which we labeled mixed, perceived low self-efficacy but low vulnerability. across the three patterns, almost all the subjects had a high expectancy that execution of recommended measures can mitigate impacts of the threat of an influenza pandemic, particularly on multi-measures with high factor scores of 4 in all patterns. the most conflicting area was vulnerability on the possible impacts of an influenza pandemic, having factor scores of high (3), low (-4), and neutral (0) for patterns i, ii, and iii, respectively. conclusion: strong consistent perceptions of response efficacy against an influenza pandemic may suggest a low priority to convince health personnel on the efficacy of the recommended measures. lack of self-efficacy in certain sub-groups indicates the need for program managers to improve self-confidence of health personnel to participate in an emergency response. an influenza pandemic is a significant natural health threat that has periodically occurred over the past 300 years [1] . its severe impacts to global human health, healthcare service, society, and economy were evidently documented during the previous pandemics [2, 3] . for a coming one, influenza experts have agreed that this threat is inevitable and possibly imminent [4] . if the next pandemic occurs, it is expected that 20% of the global population will become ill, nearly 30 million will be hospitalized and a quarter of these would die within a few months of its attack [5] . to mitigate the impacts of this threat, the world health organization (who) has recommended that all countries should consider this threat as very important and urged them to make preparations a high national priority. thailand occasionally has had serious outbreaks of avian influenza a (h5n1) since early 2004, in both poultry and humans. in response to these outbreaks and a possible future influenza pandemic, the national committee on avian influenza control and influenza pandemic preparedness has issued a national strategic plan for influenza pandemic preparedness. beyond preparedness, the perception of each individual is also a fundamental factor that contributes to the spread, prevention, and control of infectious diseases. for example, during the severe acute respiratory syndrome (sars) epidemics, the perceptions toward this disease had an effect on the preventive health behaviors (e.g., hand hygiene, mask wearing) and that consequently contributed to containing the outbreaks [6] [7] [8] . for a current threat of an influenza pandemic, sporadic perception surveys among health workers have been done in developed countries [9] [10] [11] [12] . yet this issue has not been explored in developing countries, particularly in the southeast asian region where it is more likely to be a source of the next pandemic [13] . southern thailand experienced a probable sars case in 2003, but there has been no reported case of avian influenza a (h5n1) in both poultry and human. however, the region faces a serious problem of ethnic violence. this unrest has led to the loss of over 2,600 lives and more than 7,000 injuries in the past 5 years. it is possible that the local health systems may have deteriorated due to the unrest leading to loosening of preparedness against the threat of an influenza pandemic. we have therefore conducted a study to investigate the preparedness. the current report is confined to perceptions related to the threat of an influenza pandemic with the objective to document the patterns of perception of health personnel toward this threat in southern thailand. as health personnel are key persons for influenza pandemic preparedness and con-trol, it is hoped that understanding their patterns of perception will allow control programs to properly improve the training. it may also be useful for other developing countries where an influenza pandemic is a serious threat, but the personnel are not fully prepared. q methodology, which basically originated from the theory of factor analysis [14] was applied. while conventional factor analysis is used in scale development and tries to group items or variables, q method tries to group subjects. therefore, people of the same group or having the same factor will have a similar pattern of chosen statements. the implication would be that it would be easy to put people of the same factor into the same intervention program. this method was taken into our study because this is a scientific and systematic study of human subjectivity, involving perceptions, attitudes, and opinions [15, 16] . furthermore, it is also unique since it mixes the strong points of both qualitative and quantitative research techniques, compared to traditional surveys [17, 18] . in doing q, the flow of communication surrounding the study topic (concourse) is firstly formed to get a wide range of ideas toward that topic. this is generally collected from various sources (e.g., scientific papers, books, news, interviews, focus group discussions, etc.). it is commonly presented in the form of statements. afterward, a q sample (a representative set of statements) is selected from the concourse and developed to be more meaningful, which represents various issues of the study topic and eventually is compiled into the instrument. the study subjects are then asked to rank the representative statements and place them into a score sheet, which is designed in a continuous scale ranging from strongly disagree to strongly agree, following a standardized instruction based on the judgment of each subject. this is known as the q sorting procedure. the sheets that are completely ranked by each subject (qsort) are finally correlated and analyzed by q (subjectwise) factor analysis, and the factors are then interpreted. in our study, 100 statements on various issues of an influenza pandemic were initially gathered from scientific articles, newsletters, and books to form a concourse. the protection motivation theory (pmt) was used as a basis for grouping and developing the statements into four domains: perceived severity, perceived vulnerability, perceived response efficacy, and perceived self-efficacy, by refining, clarifying, and combining the raw statements to be more meaningful and more understandable. to catch various aspects of an influenza pandemic and keeping the total number of the statements suitably manageable by our subjects, we included eight refined statements in each of such four domains with one additional item added to make the total number of the statements equal 33 (qsample). these statements were then placed into the score sheet (figure 1 ), and forced to follow a quasi-normal distribution, that is, 2-3-4-5-5-5-4-3-2. the reliability of this instrument was tested with cronbach's alpha. each statement was randomly assigned a number from 1 to 33 for the subjects to arrange and place into the score sheet. to get more understandability, the statements were pilottested with 25 health personnel and were then revised as appropriate before the study. the study was conducted in the three southernmost provinces of thailand: yala, pattani, and narathiwat, during april to october 2007. apart from the problems of ethnic violence, the area is in a remote part of the country where the logistic problems will be easily visible. the area is also close to malaysia, so cross-border diseases have a high chance of spread due to the movement of populations. the research protocol was approved by the ethics committee of the faculty of medicine, prince of songkla university, prior to conducting the study. a list of all health facilities in the study area was obtained from the local health offices. health personnel designated by each facility to be responsible for influenza pandemic preparedness were identified. these included a numbers of doctors, nurses, pharmacists, laboratory personnel, public health specialists, public health administrators, and junior health workers. all were invited to participate in the study. the selected personnel were sent a set of documents, which included a cover letter, an overview describing the study importance and objective, a set of 33 statements (q sample), a standardized step-by-step set of instructions for responding to the study, and a score sheet. following the initial mailing, two phases of follow up were performed: a sequence of telephone calls at one month, with nonresponders contacted by the first author after three months. each consenting subject was asked to rank the 33 statements about different issues concerned with an influenza pandemic into the levels of agreement and disagreement based on their own judgments. each participant was requested to place two statements in the columns of strongly disagree (-4) and strongly agree (+4), three in disagree (-3) and agree (+3), four in -2 and +2, five in -1 and +1, and five statements in the neutral response column (0). however, if they thought that our distribution did not represent their real perceptions, they were encouraged to sort such statements accordingly. each q-sort was considered as complete if all 33 statements were placed into the score sheet without repetition of the statements. the data from each completed score sheet were entered and analyzed in pqmethod 2.11 (free software). betweensubjects correlation matrix was computed and a q (subject-wise) factor analysis by principle components analysis (pca) method was performed using a varimax rotation technique. factors that could explain more than 5% of the variance were adopted and retained into the final solution. a participant who had absolute factor loadings of larger than ± 0.45, which suggests high significance (p < 0.01) with the group, was included into that particular factor. in each factor, the ascending sorted normalized scores (z-scores) of assigned number of each statement were returned into the score sheet from right to left order (figures 2, 3, and 4) . each final score sheet thus displays the pattern of the defined factor. comparisons among patterns were based on the factor scores and the mean values of the domain of the statements. for visualization of the patterns, the domains of each statement were linked to different colors or grey shadings in the final q-sort models that are shown in figures 2, 3 , and 4. since the cells in the extreme score regions reflect strong perceptions in the domains, they are the primary target for comparing similarity and dissimilarity of each group of health participant score sheet figure 1 participant score sheet. strongly agree [4] [4] [5] [5] [5] pattern i. pessimistic with perceived low self-efficacy figure 2 pattern i. pessimistic with perceived low self-efficacy. personnel's perceptions on the threat of an influenza pandemic. after consultation with an expert in instrument development, 33 statements listed in table 1 of a total 385 health personnel, 271 (70%) persons completed the score sheet. there were no statistically significant differences between responders and non-responders in terms of gender, age, religion, educational level, total period of working, job classification, experience of getting training on influenza pandemic preparedness and perceived levels of knowledge about an influenza pandemic, public health measures against an influenza pandemic and impacts of an influenza pandemic. however, the nonresponders had a lower educational level than those of the responders (35% vs. 18%, respectively). the basic characteristics of the 271 respondents are presented in table 2 . q factor analysis gave three factors that met our criteria with the percentages of explained variance being 30.1%, 8.7%, and 5.5%, respectively. after varimax rotation, 90 subjects were classified into factor i (in other words, the first pattern composites of 90 health personnel), 40 into factor ii, and 62 into factor iii. the other 79 subjects were not classified into any factor because all their loading values were less than 0.45 or had high loading on more than one factor. the composite reliability of each factor was 0.99, with the corresponding standard errors of factor scores being 0.05, 0.08, and 0.06. the correlation coefficients between the three factors were 0.37 (factor i vs. ii), 0.54 (factor i vs. iii), and 0.55 (factor ii vs. iii), indicating a moderate similarity among the patterns. the three patterns had scores for each specific statement distributed into the q-sort model or composite factor array and are displayed in figures 2, 3 , and 4. the same information is displayed in table 1 . factor scores of statement 1 were 2, -1, and -4 as shown in the first row of table 1 . in the q-sort model, statement 1 is in column +2 of figure 2 , and column -1 of figure 3 , and column -4 of figure 4 . from table 1 , statement number 9 has a common factor score of 4 for all three patterns. this indicates that all three patterns of health personnel strongly perceived that multimeasures must be performed during an influenza pandemic. statement number 12 was also in columns +4 of figures 3 and 4 , and +3 of figure 2 , which is related to response efficacy on multilevels of responsibility for preparedness against the threat. in contrast, statement 4 was the most dissenting issue with factor scores of 3, -4, and 0. health personnel classified as pattern i quite strongly perceived that thailand will have possibly high impacts from an influenza pandemic if and when one occurs, but those classified in pattern ii strongly disagreed, and those in the remaining group were neutral. the right extremes of all three q-sort models are consistently filled with three black cells (statements 2, 9, and 12) out of 5 cells of that region. this indicates that all three pattern ii. optimistic with perceived low severity and low vul-nerability figure 3 pattern ii. optimistic with perceived low severity and low vulnerability. pattern iii. mixed with perceived low self-efficacy but low vulnerability figure 4 pattern iii. mixed with perceived low self-efficacy but low vulnerability. means of factor scores for each component of the pmt are displayed in table 3 . all groups had positive perceived response efficacy of the measures. patterns i and iii, however, perceived low self-efficacy, in contrast to high perceived self-efficacy of pattern ii. optimistic personality of pattern ii was also expressed as perception of low severity and low vulnerability where the pattern i has isolated neutral perception of severity with a moderate level of perceived vulnerability. finally, more mixed appraisal is found in pattern iii, the group who perceived a low level of vulnerability but a very high level of severity. we identified three main patterns of health personnel in southern thailand based on the perception toward a threat of an influenza pandemic. pattern i was pessimistic (strongly perceived response efficacy, but perceived low self-efficacy). pattern ii was optimistic (strongly perceived response efficacy, but perceived low severity and low vulnerability). pattern iii was mixed (strongly perceived response efficacy, but perceived low vulnerability and low self-efficacy). a high perception on response efficacy was predominantly found in all health personnel groups. perceptions on vulnerability were more varied. the majority of our health personnel perceived low selfefficacy toward an influenza pandemic. self-efficacy is one important component of coping appraisal of the pmt [19] . it has powerful influence on human's feeling, thinking, motivation, and behavior [20] [21] [22] . previous metaanalyses provided evidence for self-efficacy having the largest effect size and was the strongest predictions of protection motivation [23, 24] . people with low self-efficacy usually believe that tasks are harder than they can handle. this can lead to limit task planning, increase stress, reduce the low level of attempt, and having a tendency to avoid duties and activities [20] [21] [22] . balicer et al. reported that nearly a half of local health workers may be unwilling to report to duty during a pandemic event [9] . however, that study did not identify different patterns of health workers as our study has done. another conventional survey conducted among a general population (rather than health workers) in developed countries of europe and asia on avian influenza risk perception showed a similar result. the level of self-efficacy among the respondents was also low and the authors concluded that a low level of self efficacy may obstruct any interventions [25] . the most dissenting issue among our health personnel toward this threat was on vulnerability of possible impacts in the country (statement number 4). naturally, the occurrence and severity of an influenza pandemic cannot be predicted [26] . fifteen per cent of our health pershowed that more than half did not consider that the risk of an imminent influenza pandemic was more than a possibility [10] . both perceived severity and perceived vulnerability are components of threat appraisal of the pmt [19] . perception of low level of severity and vulnerability or low levels of appraised threat of an influenza pandemic may inhibit motivation of health personnel to engage in protective behavior [27, 28] . however, the effect sizes of such two components in previous meta-analyses were small to medium and barely predicted of protection motivation and behavior compared to the components of coping appraisal (response efficacy and self-efficacy) [23, 24] . perception of response efficacy was stronger than other domains. this may be influenced by past experiences of the country, which after employing on multi-sectors and multi-measures could successfully suppress avian influenza a(h5n1) [29] . this study used a wide range of front-line health personnel responsible for influenza pandemic preparedness. thus, it may reflect the problems specific to this area with acceptable accuracy. the study was confined to the three southernmost provinces of thailand where avian influenza a (h5n1) has never occurred. our study subjects might be different from those in other regions of the country where the infected cases of that avian influenza in both humans and poultry have been reported, and intensive avian influenza controls have been fully activated. the study subjects were also predominated by personnel from health centers and community hospitals in rural areas. the threat of a pandemic may be less compared to in urban areas. the study was based on q methodology which had never been employed among local health workers; thus, the data have to be interpreted with caution. approximately 30% of the respondents were not able to be classified into any of the three groups determined by our factor analysis. the patterns are therefore far from ideal. the statements about influenza pandemic that were used in our study should be improved to be more specific for health workers in future work. despite the above limitations, this study highlights important findings. strong consistent perceptions of implementing recommended measures against an influenza pandemic can remove or mitigate impacts of this threat, and may suggest a low priority to convince health personnel on the efficacy of the measures. perception of low self-efficacy in certain subgroups who gave low scores on the statements related to self-efficacy on an influenza pandemic indicates the need to improve self-confidence of health personnel to participate in an emergency response by the control program. potter cw: a history of influenza 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subjectivity of human mind q methodology: definition and application in health care informatics cognitive and physiological processes in fear appeals and attitude change: a revised theory of protection motivation self-efficacy: toward a unifying theory of behavioral change human agency in social cognitive theory self-efficacy: the exercise of control a meta-analysis of research on protection motivation theory prediction and intervention in health-related behavior: a meta-analytic review of protection motivation theory avian influenza risk perception blaser mj: pandemics and preparations protection motivation theory effects of components of protection motivation theory on adaptive and maladaptive coping with a health threat grotto i: a systematic analytic approach to pandemic influenza preparedness planning this study was part of the first author's thesis to fulfill the requirement for phd in epidemiology at prince of songkla university (psu). we sincerely acknowledge all health personnel who participated in the study. appreciative thanks to the graduate school, psu, and the disease control department, ministry of public health, thailand for supporting the study. we also wish to thank dr. alan geater, dr. vorasit sornsrivichai, mr. edward mcneil, the epidemiology unit, faculty of medicine, psu, and mr. darrell beng, adelaide, south australia. the authors declare that they have no competing interests. tp designed this study, was the principal investigator of the project, performed data analysis, and drafted the manuscript. vc provided supervision, suggestion, and development on manuscript writing. all authors have contributed to revision of the draft version and have read and accepted the final version of this manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/9/161/pre pub key: cord-297742-0pfrk5uk authors: simusika, paul; tempia, stefano; chentulo, edward; polansky, lauren; mazaba, mazyanga lucy; ndumba, idah; mbewe, quinn k.; monze, mwaka title: an evaluation of the zambia influenza sentinel surveillance system, 2011–2017 date: 2020-01-13 journal: bmc health serv res doi: 10.1186/s12913-019-4884-5 sha: doc_id: 297742 cord_uid: 0pfrk5uk background: over the past decade, influenza surveillance has been established in several african countries including zambia. however, information on the on data quality and reliability of established influenza surveillance systems in africa are limited. such information would enable countries to assess the performance of their surveillance systems, identify shortfalls for improvement and provide evidence of data reliability for policy making and public health interventions. methods: we used the centers for disease control and prevention guidelines to evaluate the performance of the influenza surveillance system (iss) in zambia during 2011–2017 using 9 attributes: (i) data quality and completeness, (ii) timeliness, (iii) representativeness, (iv) flexibility, (v) simplicity, (vi) acceptability, (vii) stability, (viii) utility, and (ix) sustainability. each attribute was evaluated using pre-defined indicators. for each indicator we obtained the proportion (expressed as percentage) of the outcome of interest over the total. a scale from 1 to 3 was used to provide a score for each attribute as follows: < 60% (as obtained in the calculation above) scored 1 (weak performance); 60–79% scored 2 (moderate performance); ≥80% scored 3 (good performance). an overall score for each attribute and the iss was obtained by averaging the scores of all evaluated attributes. results: the overall mean score for the iss in zambia was 2.6. key strengths of the system were the quality of data generated (score: 2.9), its flexibility (score: 3.0) especially to monitor viral pathogens other than influenza viruses, its simplicity (score: 2.8), acceptability (score: 3.0) and stability (score: 2.6) over the review period and its relatively low cost ($310,000 per annum). identified weaknesses related mainly to geographic representativeness (score: 2.0), timeliness (score: 2.5), especially in shipment of samples from remote sites, and sustainability (score: 1.0) in the absence of external funds. conclusions: the system performed moderately well in our evaluation. key improvements would include improvements in the timeliness of samples shipments and geographical coverage. however, these improvements would result in increased cost and logistical complexity. the isss in zambia is largely reliant on external funds and the acceptability of maintaining the surveillance system through national funds would require evaluation. seasonal influenza virus infections are responsible for an estimated 291,243-645,832 respiratory deaths globally every year [1] . influenza infections have been described in zambia [2] since the 1990s, but because of limited laboratory diagnostic capacity prior to 2008 [3] , little was known about the contribution of influenza viruses to the respiratory disease burden, rendering difficult public health planning for the prevention and control of influenza-associated illness. at present, no influenza treatment or immunization guidelines are available in zambia, and no measures for mitigating the transmission and disease burden associated with influenza infection are in place. public health surveillance is the ongoing, systematic collection, analysis, interpretation, and dissemination of data regarding a health-related event for use in public health action to reduce morbidity and mortality and to improve health [4] . data disseminated by a public health surveillance system can be used for immediate public health action, program planning and evaluation, and formulation of research hypotheses [5] . the lack of a routine influenza surveillance program in zambia placed the country at a disadvantage as such a system would serve as an ever-ready early warning system with the potential to detect and confirm the etiology of respiratory disease outbreaks, including seasonal and pandemic influenza viruses, and trigger a response. the ministry of health (moh) recognized a need to gather influenza epidemiological and virological data from zambia that would not only help decision makers to formulate policies targeted toward influenza prevention in zambia but also to provide a more comprehensive understanding of the dynamics of influenza viruses worldwide and in african tropical zones in particular. in addition, virological data would help to identify seed viruses for the production of seasonal influenza vaccines that can be effectively used for the southern hemisphere. in recognition of these needs, in 2008 the zambia ministry of health established an influenza sentinel surveillance system (isss) targeting outpatients with influenza-like illness (ili) and in-patients with severe acute respiratory illness (sari). the world health organization (who) recommends that established influenza surveillance systems undergo a comprehensive evaluation periodically, beginning 1-2 years after implementation in order to ascertain how well the system fulfills its purposes [6, 7] . such evaluations are useful to identify shortfalls, improve performance and provide evidence of data reliability for policy making and public health interventions. however, despite recent progress in describing the epidemiology and burden of influenza in sub-saharan africa [8] [9] [10] [11] [12] [13] [14] [15] [16] , only a few countries have implemented a comprehensive evaluation of their surveillance systems [17] [18] [19] [20] [21] . to ensure that the national isss objectives are met, that they are in line with who requirements and that reliable data are generated for public health interventions, we conducted an evaluation of the performance of the zambia ili and sari isss from january 2011 to december 2017. the inception and consolidation of the surveillance system occurred during 2008-2010. the outputs of the surveillance systems during this period have been previously published [3] and they are not included in this review. overview of the influenza sentinel surveillance system during [2011] [2012] [2013] [2014] [2015] [2016] [2017] in zambia, an isss was first established in 2008 in lusaka province (the most populated province of the country where the capital city is located) [3] and subsequently expanded to the copperbelt province (the second most populated province). the objectives of the zambia-isss were to: (i) monitor the temporal trends of influenza virus circulation; (ii) monitor the circulating influenza virus types and subtypes annually, including pandemic strains; (iii) assess the proportion of patients meeting the ili and sari case definition attributable to influenza virus infection; (iv) assess risk factors for influenza-associated severe illness; (v) assess the burden of influenza-associated illness; and (vi) obtain and share clinical samples for annual selection of influenza virus strains for influenza vaccine formulation under the who-global influenza surveillance and response network. during 2011-2017, ili influenza sentinel surveillance was conducted in three urban outpatient clinics targeting outpatients with ili and three large referral hospitals targeting inpatients with sari ( fig. 1, panel a) . both pediatric and adult cases were investigated. a case of ili was defined as an outpatient of any age presenting with a recorded temperature ≥ 38°c and cough or sore throat of duration of ≤7 days [6] . a case of sari was defined as a hospitalized person who had illness onset within 7 days of admission and who met age-specific clinical inclusion criteria. a case in children aged 2 days to < 5 years included any hospitalized patient with cough or difficulty breathing and at least one of the following danger signs: unable to drink or breastfeed, lethargic, vomits everything, convulsion, chest indrawing or stridor in a calm child. a case in persons aged ≥5 years included any hospitalized patient with fever (≥38°c), cough and shortness of breath or difficulty breathing [6] . all patients with sari were eligible for enrollment; whereas 5 patients with ili (the first patient every day from monday to friday) were targeted for enrollment per week and per facility. activities at the sentinel sites situated in lusaka were coordinated by a dedicated surveillance officer from the national influenza center (nic). for sites situated in the copperbelt province, supervision was conducted by a focal point at the provincial medical office. due to high staff turnover, training was conducted once a year while supervisory visits to sites were done at least quarterly. clinical staff at sentinel sites tasked with surveillance implementation were remunerated according to time spent on surveillance activities. the zambia isss is co-funded by the zambia-moh, the u.s. centers for disease control and prevention (u.s. cdc) and who. data were collected from eligible cases using who adapted and standardized case investigation forms (cif) by trained nurses in each sentinel site. for patients meeting the sari case definition the number of patients eligible for enrollment was also collected. a combination of nasopharyngeal and oropharyngeal swabs were collected from consenting patients, placed in universal transport medium (copan, murrieta, california, usa) and transported to the university teaching hospital virology laboratory, the who-recognized nic in lusaka, via a cold chain for testing. samples with respective cif forms collected from sites situated in lusaka were sent to the laboratory within 48 h, while those from the copperbelt province were refrigerated and sent within two weeks of collection [22] . verbal informed consent was obtained from all patients prior to data and specimen collection. for children aged < 15 years, verbal consent was obtained from a parent or legal guardian. at the nic, samples and accompanying cifs were logged by recording key data in the laboratory log book. specimens were tested for the presence of influenza viruses using the u.s. cdc real-time reverse-transcription polymerase chain reaction (rt-pcr) protocol for characterization of influenza viruses [3] . during 2011-2012, all specimens from children aged < 5 years with sari were tested for other respiratory viruses including parainfluenza virus (piv) types 1, 2 and 3, respiratory syncytial virus (rsv), adenovirus, rhinovirus, human metapneumovirus (hmpv), coronavirus (oc43, nl63, 229e, hku1) and bocavirus using an ftd multiplex realtime rt-pcr assay [23] . since 2012, rt-pcr influenzapositive samples with a cycle threshold value < 28 were cultured at the nic, after which further viral identification of the samples was done using haemagglutination (ha) and haemagglutination inhibition (hai) testing. confirmed virus isolates were shared with who collaborating centers for further characterization; both viral isolates and clinical specimens were shared twice a year. data from the cif including demographics and clinical information, exposure risk and past medical history were captured in a customized influenza sentinel surveillance ms access® database with inbuilt data validation checks. data entry was done daily as forms were received. monthly reports of the total number of samples received and processed (including positive specimens by subtype) were reported to the ministry of health public health units. a separate patient line-list was also sent monthly to the sentinel sites; the results report included total samples processed by sentinel site and total confirmed influenza infections. weekly influenza surveillance data and influenza-positive specimens were reported to who afro using an excel template. virological and epidemiological data were reported to who flunet / fluid. the evaluation of the isss was based on cdc guidelines [5] and focused on the performance of the system from january 2011 to december 2017. the objective of the evaluation was to determine: (i) whether the surveillance system was designed and operated in such a way as to be capable of detecting and monitoring seasonal influenza viruses, including pandemic strains; (ii) the usefulness of the collected data to assess influenza disease burden in the general population; (iii) the impact of the system in informing public health intervention and policies; and (iv) the ability of the system to contribute to the annual selection of influenza strains for vaccine development. the performance of the system was assessed using nine surveillance attributes, namely: (i) data quality and completeness for selected key variables, (ii) timeliness, (iii) representativeness, (iv) flexibility, (v) simplicity (vi) acceptability, (vii) stability; (viii) utility; and (ix) sustainability. for consistency and comparability of findings we used the evaluation method and scoring system utilized for influenza surveillance evaluations conducted in other african countries [17] [18] [19] [20] [21] . each of the above mentioned attribute was evaluated using pre-defined quantitative or qualitative indicators. a total of 38 indicators were developed for the evaluation. the number of indicators evaluated for each attribute is provided in table 1 ; whereas the individual indicators and the calculations and data sources used to evaluate each indicator are provided in tables 2-5 . data for calculation of the indicators for data quality and completeness, timeliness, stability and utility were obtained from various sources including the main influenza sentinel surveillance database, the laboratory database, annual reports and other documents and records. in order to assess simplicity, acceptability, stability and utility, a selfadministered questionnaire as shown in additional file 1 below was designed targeting staff involved in surveillance at the sentinel sites. the questionnaire was designed to capture data based on staff perceptions of the program. data collected from the surveillance system were also compared with who minimum data collection standards for ili and sari surveillance [6] . each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total. a scale from 1 to 3 was used to provide a score for each quantitative indicator as follows: < 60% (from the above calculation) scored 1 (weak performance); 60-79% scored 2 (moderate performance); ≥80% scored 3 (good performance). for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus. the scores assigned to each indicator were averaged for all indicators evaluated for each attribute to provide a mean score for each surveillance attribute. an overall score for the surveillance system was obtained by averaging the scores of all evaluated indicators for each quantitative indicator we first obtained the proportion (expressed as percentage) of the outcome of interest over the total [18] [19] [20] [21] . for instance, for the indicator on completeness of laboratory testing (one of the indicators used to evaluate the data quality and completeness attribute) we divided the number of samples with available influenza results by the total number of samples collected and received by the laboratory. subsequently, similar to other influenza surveillance evaluations conducted in africa, we used a scale from 1 to 3 to provide a score for each quantitative indicator as follows: < 60% (as obtained in the example above) scored 1 (weak performance); 60-79% scored 2 (moderate performance); ≥80% scored 3 (good performance) [18] [19] [20] [21] . for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus. thereafter, the scores assigned to each indicator were averaged for all indicators evaluated within each attribute to provide an overall score for each surveillance attribute assessed in this study. an overall score for the surveillance system was obtained by averaging the scores of all evaluated indicators as previously described [18] [19] [20] [21] . all data generated by the surveillance system during the review period were included in the evaluation. the analysis was implemented using stata version 14.2 (sta-tacorp, college station, texas, usa). this surveillance evaluation was deemed non-research by the zambia-moh and the us cdc. consent to participate was voluntary and verbally obtained, because this was a non-research and the procedure was nonrisks to client ethical clearance was deemed unnecessary by the university of zambia biomedical research ethics committee. from january 2011 to december 2017, 10,958 patients were enrolled in the isss. of these, 5111 (46.6%) and 5847 (53.4%) had ili and sari, respectively. influenza results were available for 10,212 of 10,958 (93.2%) enrolled patients; 4776/5111 (93.5%) and 5436/5847 (93.0%) among patients with ili and sari, respectively. influenza viruses were detected in 894/10,212 (8.7%) specimens; in 601/4776 (12.6%) and 293/5436 (5.4%) specimens among patients with ili and sari, respectively. of the 894 influenza-positive specimens, 86 (9.6%) were influenza a(h1n1)pdm09, 342 (38.2%) were influenza a(h3n2), 6 (0.7%) were influenza a not-subtyped and 459 (51.3%) were influenza b viruses. the weekly number of influenza-positive specimens is provided in fig. 1, panel b . the questionnaire was completed by 43/44 (97.7%) surveillance staff at sentinel sites. of these, 35 (81.4%) were nurses, 5 (11.6%) were clinical officers, 2 (4.7%) were paramedics and 1 (2.3%) was a laboratory technician. the overall mean score for the isss in zambia was 2.6 (moderate to good performance) ( table 1) . during the evaluation period, of the seven data indicators, six had good performance and one had moderate performance ( table 2 ). approximately 70% of identified sari cases were enrolled in the surveillance program, while the enrollment target was 100%. the enrollment target for ili cases was exceeded (140% of the target of 5 cases per week and per facility). more than 90% of cif were accurately completed for key variables (including site, age/date of birth, sex, date of consultation/admission, date of symptoms onset, date of sample collection and signs and symptoms included in the case definitions) and more than 90% of samples collected had available influenza results and a positive ribonucleic protein result (an indicator of presence of human mucosal cells in the sample). the proportion of collected variables included in the who minimum data collection standard was 82.3%. information on the use of antivirals and the presence of some underlying medical conditions were not included in the patients' cifs. the mean score for data quality and completeness was 2.9 (moderate to good performance) ( table 1) . during the evaluation period, one timeliness indicator had good performance and one had moderate performance (table 2 ). approximately 25% of samples were not received or tested within the expected time period. 86.3% of the samples collected in lusaka province were received within the expected time period, whereas only 67.3% of the samples collected in the copperbelt province met the expected timeline. no significant difference in the proportion of samples positive for influenza was observed between the sites situated in lusaka (9.0% 650/ 7207) and ndola (8.1%; 244/3005) (p = 0.1429). the mean score for data timeliness was 2.5 (moderate to good performance) ( table 1) . during the evaluation period, one indicator had good performance and one had weak performance (table 2) . age representativeness was strong, with patients enrolled across all age groups. geographic representativeness was poor, with sentinel sites situated only in 2/10 (20.0%) provinces. the mean score for representativeness was 2.0 (moderate performance) ( table 1) . during the evaluation period, both indicators had good performance ( table 2 ). the isss was used as a platform to monitor the circulation of respiratory viruses other than influenza (even though only for two years). the mean score for flexibility was 3.0 (good performance) ( table 1) . during the evaluation period, of the seven indicators, six had good performance and one (time of enrollment of sari/ili cases) had moderate performance ( table 3 ). the six indicators used to assess the perception of surveillance personnel at sentinel sites to implement different surveillance activities were rated well by responding personnel. over 31% of staff, however, reported that surveillance procedures from the identification of cases to the final packaging of samples took > 30 min, showing only moderate performance. the mean score for simplicity was 2.8 (moderate to good performance) ( table 1) . during the evaluation period, all four evaluated indicators had good performance (table 3 ). more than 90% of surveillance staff was satisfied or very satisfied with the surveillance reports or the feedback of laboratory results. surveillance activities did not pose a heavy workload on surveillance staff in addition to the required clinical work. the mean score for acceptability was 3.0 (good performance) ( table 1) . during the evaluation period, of the eight indicators, five had good performance and three had moderate performance (table 4 ). all surveillance sites provided samples weekly during the evaluation period. the main aspects that affected stability were elevated frequencies of electricity cuts and generator failures. approximately 30% of surveillance staff did not receive annual refresher training during the last year and approximately 30% of expected weekly reports were not sent to the zambia moh within the expected time frame (one week from the end of the reported period). the mean score for stability was 2.6 (moderate to good performance) ( table 1) . during the evaluation period, of the four indicators, three had good performance and one had moderate performance (table 5 ). in terms of international utility the zambia isss reported regularly to the who flunet, generated samples that were shared with the who collaborating centers and contributed to regional studies. in terms of domestic utility, 4/5 (80.0%) key national objectives of the isss were met at the time of this evaluation. the mean score for utility was 2.7 (moderate to good performance) ( table 1) . during the evaluation period, the two indicators had poor performance (table 5 ). only 17% of the isss budget was provided by the zambia moh and the remaining by international agencies. the mean annual running cost of the system was approximately $310,000 ($105 per sample collected and tested in 2017). whereas, a sustainability plan was drafted in 2015, the document was not finalized and implemented at the time of this evaluation. the mean score for sustainability was 1.0 (weak performance) ( table 1 ). we evaluated the performance of the zambia isss over a 7-year period, 3 years from its inception. overall, based on the evaluated indicators, the system performed satisfactorily with a mean system score of 2.6 (moderate to good performance). the system demonstrated its utility according to its objectives by (i) monitoring the temporal trends of influenza virus circulation; (ii) monitoring the circulating influenza virus types and subtypes annually and enabling the detection of pandemic influenza a(h1n1)pdm09 in 2009 [3] and in subsequent years, (iii) assessing the contribution of seasonal influenza viruses to mild (ili) and severe (sari) respiratory illness; (iv) assessing the burden of influenza-associated illness [14] ; and (v) generating isolates to contribute to the annual influenza vaccine formulation. nonetheless, the isss was not able to identify groups at increased risk of influenza-associated severe illness (as the necessary variables were not collected in the cif), one of the objectives of the surveillance system. strengths of the system included the quality of data generated, its flexibility to monitor viral pathogens other than influenza viruses [24] , its potential expandability, its stability over the review period, and its relatively low cost. identified weaknesses were poor geographic representativeness, lack of timeliness in shipping samples from remote sites, and low sustainability in the absence of external funds. while quality and completeness of collected key epidemiological and virological data was good overall, an under-enrollment of sari cases was noted. many factors could have affected site performance including low staff numbers, high turnovers rates of staff as well as participant refusals. it should be noted that nurses employed by the moh and tasked with surveillance activities also provided direct patient care. having dedicated surveillance staff may increase enrollment rates. however, hiring new staff would decrease the sustainability of the surveillance system. each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total (indicator value). a scale from 1 to 3 was used to provide a score for each quantitative indicator as follows: < 60% (from the above calculation) scored 1 (weak performance); 60-79% scored 2 (moderate performance); ≥80% scored 3 (good performance) each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total (indicator value). a scale from 1 to 3 was used to provide a score for each quantitative indicator as follows: < 60% (from the above calculation) scored 1 (weak performance); 60-79% scored 2 (moderate performance); ≥80% scored 3 (good performance) the surveillance system performed moderately in terms of timeliness in transporting samples from the field to the laboratory. timeliness was mainly affected by the delays experienced from sites located outside lusaka. conversely, when samples were received at the central laboratory the processing time was overall satisfactory. while timeliness of shipping of samples from sites situated outside lusaka province could be improved by using couriers on a more regular basis this may increase costs. consideration should be given to timeliness of the system and its geographical representativeness especially in the african setting where more regular shipments of samples from areas distant from central locations are not only affected by cost, but also by logistical constraints. the geographic representativeness of the system was weak. establishing additional surveillance sites in other key areas of the country could provide a better representativeness of the circulating influenza viruses. however, it is unclear whether expanding the number of surveillance sites would improve the virological and epidemiological understanding of influenza in zambia. in a study that included surveillance data from 15 african countries, the detection rate of influenza virus among sari and ili cases was similar across the participating countries, suggesting that similarities of the burden of publications and reports 80.0% b 3 • circulating influenza types/ subtypes, including pandemic strains (yes) [3] • proportion of ili/sari illness attributable to influenza virus infection (yes) [3] • risk factors for influenzaassociated severe illness (no) • burden of influenza-associated illness (yes) [14] sustainability each quantitative indicator was evaluated as the proportion (expressed as percentage) of the outcome of interest over the total. a scale from 1 to 3 was used to provide a score for each quantitative indicator as follows: < 60% (from the above calculation) scored 1 (weak performance); 60-79% scored 2 (moderate performance); ≥80% scored 3 (good performance). for indicators for which a proportion over a total could not be obtained (qualitative indicators) a score was assigned based on the same scale using expert consensus b indicator value calculated by dividing the number of achieved outcome by the total number of outcome considered (i.e. 4/5 = 80.0% or 1/4 = 25.0%) influenza-associated illness across different geographical areas of the continent may be expected [15] . increasing the number of surveillance sites in zambia should be considered in light of increased cost, logistical constraints (e.g. regular supervision and transportation of specimens) and impact on laboratory testing capacity and potentially on data quality. instead of expanding the geographic coverage of the system, efforts could be targeted to improve timeliness of sample shipment and enrollment of patients meeting the sari case definition at the existing sentinel sites as well as collecting more detailed epidemiological information on enrolled cases (e.g., relevant risk factors for influenza-associated severe illness and in-hospital outcomes that are recommended by the who guidelines for global influenza surveillance, but are currently not fully captured by the system). in particular, the collection of additional information on underlying medical conditions may enable the continuous monitoring of high risk groups for influenzaassociated severe illness. this would also align the zambia isss to the minimum data collection requirements suggested by who [6] . the flexibility of the system was demonstrated by its ability to investigate the viral etiology of pathogens other than influenza [24] . while the recruitment of dedicated surveillance officers may not be justifiable for a vertical influenza surveillance program it may be considered for an integrated multi-pathogen surveillance platform. this could be potentially cost-beneficial for a sentinel surveillance system where a limited number of surveillance officers may be needed in selected sentinel sites as compared to national passive surveillance programs. integrated and well-run multi-pathogen surveillance systems, if adequately staffed may provide high quality and timely epidemiological and virological data with a "modest" financial investment. the national burden of illness attributable to specific pathogens can be estimated from sentinel surveillance data through special studies as demonstrated in zambia and other african countries for influenza [10] [11] [12] [13] [14] [15] [16] . this would be particularly relevant for policy makers if multiple pathogens are included so as to provide the relative burden associated with different etiological agents. risk factors for influenzaassociated severe illness (e.g. hospitalization or death) have also been assessed using sentinel surveillance systems [23, [25] [26] [27] and this could be implemented also in zambia for influenza and other pathogens. the stability of the system was demonstrated by its ability to enroll and process samples every week of the review period. this may be related to the simplicity and acceptability of the system and the availability and use of standard operating procedures at sites. nonetheless, tasking of surveillance staff with clinical and surveillance work may have affected the enrollment rate as previously discussed. in conclusion, the system performed satisfactorily over recent years. it contributed to the body of knowledge on the burden of influenza [3, 14] and other respiratory viruses [24] among common respiratory syndromes in zambia and globally through sharing of influenza data [8, 28] and virus isolates. nonetheless, despite its moderate cost, the isss in zambia is largely reliant on external funds and the acceptability of maintaining the surveillance system through national funds would require evaluation. improvement of the system would entail the improvement of the timeliness of sample shipments, enrollment of patients meeting the sari case definition and an increased geographical coverage; 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severity of influenza illness in malawian adults: a prospective cohort and parallel case-control study global role and burden of influenza in pediatric respiratory hospitalizations, 1982-2012: a systematic analysis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all members involved in ili and sari surveillance for data collection and their participation to the evaluation. all authors take responsibility for the integrity of the data and the accuracy of the data analysis. in addition, all authors have read and approved the manuscript in its current state. this work was supported by the zambia ministry of health and the us centers for disease control and prevention (co-operative agreement number: nu511p000828-05-02). the ministry of health, provided surveillance personnel, laboratory space, reagents and equipment to perform tests and sites for sample data collection and personnel in drafting and analyzing of the manuscript. us cdc provided funds and support for reagents and surveillance sites including technical support to analyze the data, training of key personnel on evaluation of surveillance systems and writing of the manuscript. all data generated or analyzed during this study are included in this published article. all authors declare that they have no commercial or other associations that may pose a conflict of interest. ethical approval and consent to participate this surveillance evaluation was deemed non-research by the zambia-moh and the us cdc.consent to participate was voluntary and verbally obtained, because this was a non-research and the procedure was non-risks to client ethical clearance was deemed unnecessary by the university of zambia biomedical research ethics committee. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the us centers for disease control and prevention, usa or the zambia ministry of health. not applicable. key: cord-297022-zs5m36cp authors: kwong, jeffrey c.; stukel, thérèse a.; mcgeer, allison j.; manuel, douglas g. title: appropriate measures of influenza immunization program effectiveness date: 2007-01-22 journal: vaccine doi: 10.1016/j.vaccine.2006.09.080 sha: doc_id: 297022 cord_uid: zs5m36cp groll and thomson's evaluation of the effectiveness of ontario's universal influenza immunization campaign used per capita cases of laboratory-confirmed influenza. we argue that these data are susceptible to various biases and should not be used as an outcome measure. laboratory data are traditionally used to identify the presence of influenza activity rather than to identify levels of influenza activity. a better measure of viral activity is the proportion of influenza tests positive; whereas the weekly proportion of tests positive was relatively consistent, a marked increase over time in the numbers of laboratory-confirmed cases paralleled an increase in the number of tests performed. regardless, for evaluating universal influenza immunization program effectiveness, other established and available measures employed in previous studies describing the epidemiology of influenza should be used instead of laboratory data. in their evaluation of ontario's universal influenza immunization campaign, groll and thomson state that there is a lack of high-quality influenza outcome data in ontario, so instead they examined the effectiveness of the program using per capita cases of laboratory-confirmed influenza [1] . these laboratory data are traditionally used by public health agencies to identify the presence of influenza activity -based on exceeding case or proportion positive test thresholds -and to characterize circulating strains, but there are good reasons why they are not used to identify levels of influenza activity. the most important reason that per capita cases is a suboptimal outcome measure for evaluating the effect of the immunization program is that it is susceptible to ascertainment bias [2] . heightened awareness of influenza and other respiratory infections have led to increased requests for testing. given the increased attention in recent years to both interpandemic and pandemic influenza, as well as emerging diseases such as the severe acute respiratory syndrome (sars), it is not surprising that more tests for influenza may be ordered by physicians and public health professionals in their diagnostic work-up of patients with acute respiratory illnesses and investigations of respiratory outbreaks, respectively. evidence for increased testing over time is illustrated in fig. 1 . this figure plots weekly surveillance data (obtained from the same source as groll and thomson) for influenza a and b for ontario from 1993 to 2004, and compares the number of tests performed for influenza (using viral culture or direct antigen detection) with the number of cases of lab-confirmed influenza. annual peaks corresponding to influenza season are apparent for both number of tests performed and number of cases. it is also fairly evident that number of tests performed for influenza have increased over time, with a sudden increase coinciding with and persisting since the sars outbreak that occurred in ontario in the spring of 2003 [3, 4] . the increase in numbers of laboratory-confirmed cases seen over time parallels the increase in number of tests performed. a better measure of viral activity is the proportion of influenza tests positive (the number of cases of lab-confirmed influenza divided by the number of tests performed). this is illustrated in fig. 2 , which compares the number of cases of lab-confirmed influenza with the proportion of tests for influenza that were positive. again, annual peaks corresponding to influenza season are evident for both number of cases and proportion of tests positive. however, the peaks for the proportion of tests positive are fairly consistent over time, varying between 0.15 and 0.45, whereas the peaks for the number of cases increase dramatically over time, from less than 50 to over 1000 cases per week. the relative consistency of the proportion of tests positive over time coupled with the increase in lab-confirmed cases suggests that the increase in lab-confirmed cases is attributable to more tests being performed. on an unrelated note, there was an error in fig. 1 in groll and thomson's paper: the y-axis title should be cases per 1,000,000 population and not per 100,000 population. for december 2003, there were 2394 cases of influenza reported out of a population of 12,256,645 as of 1 july 2003. this would be a monthly incidence of 195 per 1,000,000 cases and accurately reflects the data in the graph. that only 2 cases of influenza per 10,000 people are identified during months of influenza activity, when the actual rates of disease approach 5 per 100 people, further raises concern that any laboratory-derived measures of influenza activity are vulnerable to ascertainment and sampling biases. groll and thomson did examine potential ascertainment bias but -considering the level of concern and importance to the study findings -they did not go far enough. they should have disclosed more information about laboratory testing and performed more analyses to examine bias. better still, they should have used other established and available measures employed in previous studies describing the epidemiology of influenza, such as hospitalizations, mortality, emergency department use and ambulatory physician visits. incidence of influenza in ontario following the universal influenza immunization campaign a dictionary of epidemiology identification of severe acute respiratory syndrome in canada public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto mount sinai hospital, canada douglas g. manuel institute for clinical evaluative sciences, canada * corresponding author at: institute for clinical evaluative sciences, room g1 06 key: cord-322906-zef971xp authors: hochman, assaf; alpert, pinhas; negev, maya; abdeen, ziad; abdeen, abdul mohsen; pinto, joaquim g.; levine, hagai title: the relationship between cyclonic weather regimes and seasonal influenza over the eastern mediterranean date: 2020-08-12 journal: sci total environ doi: 10.1016/j.scitotenv.2020.141686 sha: doc_id: 322906 cord_uid: zef971xp abstract the prediction of the occurrence of infectious diseases is of crucial importance for public health, as clearly seen in the ongoing covid-19 pandemic. here, we analyze the relationship between the occurrence of a winter low-pressure weather regime cyprus lows and the seasonal influenza in the eastern mediterranean. we find that the weekly occurrence of cyprus lows is significantly correlated with clinical seasonal influenza in israel in recent years (r = 0.91; p < .05). this result remains robust when considering a complementary analysis based on google trends data for israel, the palestinian authority and jordan. the weekly occurrence of cyprus lows precedes the onset and maximum of influenza occurrence by about one to two weeks (r = 0.88; p < .05 for the maximum occurrence), and closely follows their timing in eight out of ten years (2008–2017). since weather regimes such as cyprus lows are more robustly predicted in weather and climate models than individual climate variables, we conclude that the weather regime approach can be used to develop tools for estimating the compatibility of the transmission environment for influenza occurrence in a warming world. furthermore, this approach may be applied to other regions and climate sensitive diseases. this study is a new cross-border inter-disciplinary regional collaboration for appropriate adaptation to climate change in the eastern mediterranean. the world health organization (who) has estimated that in 2012 approximately 12.6 million deaths (23% of all deaths worldwide) were attributed to changeable environmental factors, of which many could be potentially influenced by ongoing climate change (who 2016) . in addition, the lancet commission on health and climate change determined that "climate change could be the greatest public health threat of the 21 st century" (watts et al., 2015; . there is clear evidence that climate change in the last 50 years has affected human health, partly by altering the epidemiology of climate sensitive diseases (e.g., patz et al., 2005; mirsaeidi et al., 2016) . specifically, climate change leads to alterations in the mean, variability, seasonality and/or extremes in one or more climatic variables such as temperature, precipitation, humidity, aerosols etc. these changes influence the dispersal of pathogens, the transmission environment and the host's resilience (vittecoq et al., 2017) . health effects related to climate change tend to emerge as seasonal and geographical alterations in the spread of disease (wu et al., 2016; dennis and fischer 30% of the burden of infectious diseases in europe in the period 2009-2013 (cassini et al., 2018) . caini et al. (2018) analysed the timing of seasonal influenza maximum occurrence, showing that it was delayed by 2.8d/year in western europe from 1996 to 2016, while it was shortened by 3.5d/year in eastern europe. regarding israel, caini et al., (2018) identified a progressive delay of maximum incidence by 2.8d/year. the reasons for such changes have not yet been fully explained, but ongoing climate change is among the leading candidates. moreover, a recent study revealed intense inter-seasonal influenza activity during 2018/9 ( barr et al., 2019) , reinforcing the need for year-round surveillance of influenza, even in areas with strong seasonality patterns like the eastern mediterranean. a plethora of studies have indicated that the timing of seasonal influenza varies across latitude, thus suggesting that meteorological conditions play an important role in the transmission of the disease (soebiyanto et al., 2010; 2014; tang et al., 2010; baumgartner et al., 2012; shaman and karspeck 2012; yang et al., 2012; tamerius et al., 2013; yaari et al., 2013; chong et al., 2019) . a few studies have demonstrated that absolute humidity modulates the airborne survival and transmission of the influenza virus (shaman and kohn 2009; shaman et al., 2010; 2011) . soebiyanto et al. (2015) have investigated the association between climatic variables and seasonal influenza in temperate and sub-tropical regions, including israel. they have provided evidence that an increase in influenza activity is related to a decrease in temperature and specific humidity. recently, zhao et al. (2018) has shown that influenza specific combination of variables that leads to enhanced incidence of infectious diseases, e.g. low temperatures and rainy/moisty or dry conditions in the case of influenza (axelsen et al., 2014; guo et al., 2015; chong et al., 2020) . indeed, there is growing evidence that large scale climatic modulations such as the el-niño or la-niña may influence the onset and peak of seasonal influenza in many regions across the globe (oluwole 2015 (oluwole , 2017 chun et al., 2019) . in order to potentially predict the compatibility of the transmission environment for climate sensitive infectious diseases, it is necessary to obtain skilful predictions of several meteorological variables at the same time, as clearly shown in the current covid-19 pandemic (ma et al., 2020) . however, one caveat of using individual climatic variables is that seasonal, decadal and multi-decadal weather and climate forecast models struggle in predicting these variables individually, especially in regions distant from the onset of the el-niño (weisheimer and palmer 2014 ). however, model forecasts are generally more robust in predicting weather regimes occurrences and timing (weisheimer and palmer 2014; grams et al., 2017) . a weather regime can be considered as a capsule containing much of the information on the transmission environment, including the synergistic relations between individual climatic variables like temperature, precipitation, humidity and wind (lamb 1950; 1972; stein and alpert 1993; alpert and sholokhman, 2011; santos et al., 2016) . moreover, a weather regime approach also has the advantage of retaining the physical relationship between the individual climatic variables. south-eastern part of the eastern mediterranean region (alpert et al., 2004a) . the semi-objective synoptic classification describes well the local weather and has many implications (alpert et al., 2004a, b; saaroni et al., 2010a, b; hochman et al., 2018a, b; 2019a, b; 2020a, b) . for example, hochman et al. (2018b) have provided evidence that by the end of the 21 st century, the duration of the summer is projected to extend by 49% (+ ~ 60 days), while the winter is expected to be shortened by 56% (-~ 60 days) under the "business as usual" greenhouse gas scenario (rcp8.5). the authors (hochman et al., 2018b) concluded that these alterations may lead to substantial changes in the timing of seasonal health hazards including seasonal influenza. high-quality climatic and infectious disease information is sparse in some regions of the world, e.g., in several of the eastern mediterranean countries. in this respect, google trends is a freely accessible tool that may provide insights into population for this study, we set a cross-border inter-disciplinary regional collaboration, composed of climatologists, epidemiologists and public health professionals from the palestinian authority, israel and germany (hochman et al., 2020c) . the purpose of j o u r n a l p r e -p r o o f journal pre-proof this study is to investigate the potential link between weather regime occurrences and climate sensitive infectious diseases, and discuss in how far this relationship can help to inform decisions in the health sector. as a case study, the weekly occurrences of an eastern mediterranean weather regime -cyprus lows -together with precipitation, temperature and humidity, are related to seasonal influenza in israel, the palestinian authority and jordan. climatic data were acquired from the national centre for environmental prediction/national centre for atmospheric research (ncep/ncar) reanalysis archive (kalnay et al., 1996) . we the student t-test is used to check for statistical significance of the correlation coefficients at the 5% significance level. finally, we investigate the ability of the weather regime approach in depicting the onset and maximum occurrence of the influenza season. the onset (maximum) is defined as the first week of the season with above zero (one) normalized incidence rates or weekly occurrence of cyprus lows. it should be noted that all datasets are open access for reproducibility and transparency and the modeling framework was validated as recommended by walters et al. (2018) . data from the icdc weekly reports of positive specimens for influenza at the trends data (fig. 2 -4 ; table s1 ). the reason may be that the individual variables have a strong impact on human behavior, which the google trends tool was designed to detect (nuti et al., 2014) . in terms of seasonality, google trends estimates the onset of influenza at week 35 and the maximum occurrence at week 2 (fig. 5) . in this case, the influenza onset and maximum precede all the climatic variables, with closer relation to weekly precipitation amounts ( fig. 3 and 5) . the observed seasonal occurrence of influenza in all three regions using google trends is very similar (fig. 5 ). the large similarity between the three countries suggests that indeed the regional environmental factors may play an important role. when analyzing the individual years in terms of ili and flu+ relationship to cyprus low occurrence, some year-toyear variability is identified (fig. s1 ). for example, differences in the seasonality of influenza between years with a relatively low occurrence of cyprus lows (e.g., 2013) compared to years with a higher occurrence (e.g., 2009). still, the tight relationship between cyprus lows and influenza is retained. next, we quantify the relationship between the meteorological variables and seasonal influenza occurrence in israel. as a first choice, a stepwise multiple linear regression model is adopted for the relation between the predictors, i.e., cyprus lows j o u r n a l p r e -p r o o f and four individual meteorological variables (temperature, precipitation, specific humidity and relative humidity) and the predictand (flu+) for 2008-2017. it is found that the number of cyprus lows per week can explain 82% of the variance with a root mean square difference of 0.14. adding the other meteorological variables only marginally contributes to a higher explained variance (table 1) occurrence is identified using the student t-test at the 5% significance level (fig. 6 ). furthermore, a close inspection of figure 6 suggests that the seasonal variability of cyprus lows closely follows the seasonal variability of influenza occurrence, especially close to maximum occurrence of influenza. in addition, we tested other potential regression models for predicting flu+ from the weekly occurrence of cyprus lows. only marginal improvement is shown for increasing order of polynomial or sine non-linear models (table s2 ). in this study, we analyze the relationship between weather regimes and seasonal influenza over the eastern mediterranean for the period 2004-2017. we find that cyprus low weekly occurrence has the highest significant correlation (r = 0.91; p < 0.05) with weekly clinical influenza data over israel with respect to individual climate variables such as precipitation, temperature, specific humidity and relative humidity. influenza when performing a complementary analysis for the palestinian authority, jordan and israel using google trends data. the weekly occurrence of cyprus lows precedes the onset and peak of influenza occurrence with a lag of about 1 to 2 weeks, and a correlation for maximum occurrence of r = 0.88 (p < 0.05). the evolution of both curves matches in eight out of ten years. this is an important finding, since the influenza virus has ~3 days incubation period, and ~3 additional days until a patient visits a clinic and a few more days for positive virus identification. the role of weather in the spread of influenza is not yet fully understood. thus, it is not possible to explicitly determine the biological mechanism relating cyprus lows and influenza occurrence. however, the environmental setting given by the typical cold weather associated with the cyprus low may affect directly or indirectly influenza through the host and/or the pathogen, i.e., the epidemiological triangle (e.g., fuhrmann 2010). for example, the host susceptibility to infection during cyprus low days may increase due to seasonal hormonal changes, which may be related to a reduction in exposure to sun light. for example, some studies suggest that low levels of vitamin d may weaken the immune response (cannel et al., 2006) , while high levels may reduce the risk of both influenza and covid-19 infections and death (grant et al., 2020; merzon et al., 2020) . furthermore, rapid changes in weather, journal pre-proof which may influence both the host susceptibility and the pathogen survival, can also increase the risk of an influenza epidemic (liu et al., 2020) . indeed, the winter season in the eastern mediterranean is typically associated with rapid changes in weather governed by transitions from cyprus lows to high-pressure systems (alpert et al., 2004b) . the strongest variability in weather regime transitions is especially evident in early winter (hochman et al., 2020b) , which also corresponds to the onset of the influenza season. in addition, the ability of the influenza virus to cause infection is higher when the air is sufficiently cold (polozov et al., 2008) . finally, studies have provided evidence that fluctuations in the duration of social contacts may relate to weather conditions (e.g., smieszek 2009; willem et al., 2012) . indeed, indoor social contacts during cyprus low days may be more frequent and longer lasting. the present results suggest that the weather regime approach can be used to develop a tool for estimating the compatibility of the influenza transmission environment in a changing climate. this is particularly important, since weather and climate model forecasts are generally more robust in predicting weather regimes occurrences and timing than individual climatic variables, especially at regions distant from the el-niño (e.g., weisheimer and palmer 2014; grams et al., 2017) . this can be achieved by applying the methodology to sub-seasonal, seasonal, annual, decadal and even multi-decadal scales using climate model predictions / projections (marotzke et al., 2016; vitart and robertson, 2018) . we envisage that this novel approach could be applied to other regions (soebiyanto et al., 2015; chun et al., 2019) and infectious illnesses, such as vector-borne diseases and infectious gastroenteritis or even the sars-cov-2 and its associated disease covid-19. unfortunately, climatology tools and data are under-utilized in public health j o u r n a l p r e -p r o o f journal pre-proof (fuhrman 2010; hochman et al., 2020c) . this study exemplifies the potential for inter-disciplinary collaboration. the present results and methodology can potentially be helpful for public health in practical terms, i.e., better understanding of the correlation between weather regimes and influenza may improve vaccination policy and medical resources allocation. for example, health systems may roughly estimate the timing of seasonal influenza surge and improve the timing of seasonal vaccination campaigns for the general population, as well as for vulnerable populations including the elderly, poor, women, children, disabled, refugees and chronically ill patients. in addition, we suggest that the utilization of google trends and other social media information in real-time may be beneficial in sparsely monitored regions, such as several eastern mediterranean countries. improved understanding of the relationship between internet searches and actual illness patterns may help countries with limited monitoring of diseases to plan timely health promotion, including seasonal vaccination and campaigns for preventive measures, as well as plan allocation of medical resources. however, this type of information should not replace investments in traditional data gathering and analysis, but rather serve as a complement to it (lazer et al., 2014) . as a caveat, we note that seasonal influenza occurrence, as most other infectious diseases, may be influenced by: vaccination effectiveness, public awareness, biological and socio-economic factors etc., which are not easily quantified and measured (caini et al., 2018) . we further note that laboratory confirmed influenza cases may also suffer from inaccuracies. in fact, a recent review article that tested the quality of different laboratory tests for viral respiratory infections, including influenza found that the pooled sensitivity of the different tests was 90.9% (95% confidence interval of 88.7% -93.1%) and specificity of 96.1% (95% confidence interval of 94.2% -97.9%; vos j o u r n a l p r e -p r o o f journal pre-proof et al., 2019) . in addition, laboratory tests are indeed just the tip of the iceberg with respect to the actual incidence rates of influenza or any other infectious disease. climatic changes and associated health risks know no borders. the challenges climate change pose to society and especially to public health, can be properly met only with regional collaborations as clearly revealed in the covid-19 pandemic (otu et al., 2020) . this manuscript was prepared as part of a jordanian, palestinian, israeli and german collaboration towards the establishment of a regional climate change adaptation center (rccac). the collaborators are committed to this important regional trans-national cooperation, which will benefit the people of this vulnerable part of the world. epidemic influenza and vitamin d impact of infectious diseases on population health using incidence-based disability-adjusted life years (dalys): results from the burden of communicable diseases in europe study, european union and european economic area countries latitudes mediate the association between influenza activity and meteorological factors: a nationwide modelling analysis in 45 japanese prefectures from association between meteorological variations and activities of influenza a and b across different climate zones: a multi-region modelling analysis across the globe weather regimes and analogues downscaling of seasonal precipitation; a case study over israel the dynamics of cyclones in the twentyfirst century; the eastern mediterranean as an example dynamics and predictability of cold spells over the eastern mediterranean. climate dynamics (under review) forecasting seasonal influenza with a state-space sir model tackling covid-19: can the african continent play the long game? impact of regional climate change on human health progressive ordering with decreasing temperature of the phospholipids of influenza virus factors governing the inter-annual variation and the long-term trend of the 850-hpa temperature over israel links between the rainfall regime in israel and location and intensity of cyprus lows. international approach absolute humidity modulates influenza survival, transmission, and seasonality absolute humidity and the seasonal onset of influenza in the continental united states absolute humidity and pandemic versus epidemic influenza forecasting seasonal outbreaks of influenza a mechanistic model of infection: why duration and intensity of contacts should be included in models of disease spread modeling and predicting seasonal influenza transmission in warm regions using climatological parameters. plos (2017) modeling the spread of avian influenza viruses in aquatic reservoirs: a novel hydrodynamic approach applied to the rhône delta (southern france) rapid molecular tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review of diagnostic accuracy and clinical impact studies modelling the global spread of diseases: a review of current practice and capability health and climate change: policy responses to protect public health the lancet countdown: tracking progress on health and climate change the 2018 report of the lancet countdown on health and climate change: shaping the health of nations for centuries to come on the reliability of seasonal climate forecasts a nice day for an infection? weather conditions and social contact patterns relevant to influenza transmission impact of climate change on human infectious diseases: empirical evidence and human adaptation modelling seasonal influenza: the role of weather and punctuated antigenic drift relationship between humidity and influenza a viability in droplets and implications for influenza's seasonality developments and prospects in synoptic climatology the effects of synoptic weather on influenza infection incidences: a retrospective study utilizing digital disease surveillance this cross-border collaboration is a part of the track ⅱ initiative of the aravainstitute for environmental studies, israel. as such, we would like to thank robin twite and dr. yara dahdal for initiating cross-border collaboration. we would like to thank the team of the icdc and maccabi healthcare services, who routinely collect key: cord-314607-bcocsjij authors: memish, ziad a.; assiri, abdullah m.; alshehri, mohammed; hussain, raheela; alomar, ibrahim title: the prevalance of respiratory viruses among healthcare workers serving pilgrims in makkah during the 2009 influenza a (h1n1) pandemic date: 2011-12-23 journal: travel med infect dis doi: 10.1016/j.tmaid.2011.11.002 sha: doc_id: 314607 cord_uid: bcocsjij despite the high risk of acquiring respiratory infections, healthcare workers who treat pilgrims at hajj have not been studied in previous research on respiratory diseases during hajj. the objective of this study was to determine the prevalence of different respiratory viruses among healthcare workers who treated pilgrims during hajj 2009, the year of the influenza a h1n1 pandemic. a cross-sectional study was performed just before and after hajj (25–29 november, 2009). nasal and throat swabs were tested for 18 respiratory virus types and subtypes. a total of 184 healthcare workers were examined. most were men (85%) with an average age of 41 years. before the hajj, rates of seasonal influenza vaccination were higher (51%) than rates of pandemic influenza a h1n1 vaccination (22%). after the hajj, participants reported high rates of maintaining hand hygiene (98%), cough etiquette (89%), and wearing a face mask (90%). among all the viruses tested, only two were detected: rhinovirus was detected in 12.6% and coronavirus 229e in 0.6%. rhinovirus was detected in 21% of those who had respiratory symptoms during hajj. influenza a (including h1n1), influenza b. respiratory syncytial virus, other coronaviruses, parainfluenza viruses, human metapneumovirus, adenovirus, and human bocavirus were not detected. the finding of high rates of rhinovirus infection corresponds to their frequent occurrence in adults. none of the participants had influenza a h1n1 2009, possibly because it was also infrequent among the 2009 pilgrims. the prevalance of respiratory viruses among healthcare workers serving pilgrims in makkah during the 2009 influenza a (h1n1) pandemic keywords hajj; viral; respiratory; healthcare workers; h1n1 summary despite the high risk of acquiring respiratory infections, healthcare workers who treat pilgrims at hajj have not been studied in previous research on respiratory diseases during hajj. the objective of this study was to determine the prevalence of different respiratory viruses among healthcare workers who treated pilgrims during hajj 2009, the year of the influenza a h1n1 pandemic. a cross-sectional study was performed just before and after hajj (25e29 november, 2009). nasal and throat swabs were tested for 18 respiratory virus types and subtypes. a total of 184 healthcare workers were examined. most were men (85%) with an average age of 41 years. before the hajj, rates of seasonal influenza vaccination were higher (51%) than rates of pandemic influenza a h1n1 vaccination (22%). after the hajj, participants reported high rates of maintaining hand hygiene (98%), cough etiquette (89%), and wearing a face mask (90%). among all the viruses tested, only two were detected: rhinovirus was detected in 12.6% and coronavirus 229e in 0.6%. rhinovirus was detected in 21% of those who had respiratory symptoms during hajj. influenza a (including h1n1), influenza b. respiratory syncytial virus, other coronaviruses, parainfluenza viruses, human metapneumovirus, adenovirus, and human bocavirus were not detected. the finding of high rates of rhinovirus infection corresponds to their frequent occurrence in adults. none of the participants had influenza a h1n1 2009, possibly because it was also infrequent among the 2009 pilgrims. ª 2011 elsevier ltd. all rights reserved. healthcare workers are exposed to many respiratory infections when they see patients, and they may transmit these infections to their patients or colleagues. for example, during the outbreak of severe acute respiratory syndrome (sars) in 2003, attack rates were more than 50% in healthcare workers. 1 healthcare workers who see pilgrims during hajj (the annual muslim pilgrimage to makkah) may be at higher risk of acquiring respiratory and other infections. 2, 3 because of these risks, special immunization requirements have been proposed to protect healthcare workers, 4 although healthcare workers have not been included in previous studies of respiratory diseases during hajj. 2, 5 hajj is the largest annual mass gathering in the world; it brings more than two million people from different countries together in a small, confined area. the extreme overcrowding of pilgrims during hajj reaches about 7 persons per meter; combined with fatigue and extremely hot weather during much of the year, this crowding may increase the risk of transmitting air-and droplet-borne infectious diseases, particularly respiratory viruses. 3,5e7 an estimated one in three pilgrims experience respiratory symptoms. 6 several transmissible bacterial and viral respiratory pathogens have been reported among pilgrims, notably meningococci of all serotypes, streptococcus pneumoniae, gram-negative organisms, atypical organisms, mycobacterium tuberculosis, influenza a and b viruses, rhinoviruses, respiratory syncytial virus (rsv), parainfluenza viruses, enteroviruses, and adenoviruses. 5,6,8e10 in april of 2009, a novel influenza a strain (h1n1 2009 strain) in mexico spread globally. 11, 12 the toll was particularly heavy in saudi arabia, which ranked fourth of 22 countries in the eastern mediterranean region in deaths and probable h1n1 cases. 13 the 2009 hajj took place in november, six months later, and presented a public health challenge for infection control authorities in saudi arabia. 14 several practices to minimize disease transmission among pilgrims and healthcare workers were instituted even before the beginning of hajj season, since pre-hajj data showed low acceptance rates of h1n1 vaccine among healthcare workers. 15, 16 this study evaluated the prevalence of viral respiratory pathogens among healthcare workers during the 2009 hajj which coincided with the influenza a h1n1 2009 pandemic. healthcare workers, including physicians, nurses, health inspectors, and others, who served pilgrims during the 2009 hajj season were included. three-fourths of the healthcare workers in the study were from the saudi ministry of health (moh), and the rest were from medical missions other than moh. most of the healthcare workers had treated pilgrims previously and had been practicing medicine for more than 10 years. the main religious activities of the 2009 hajj season started on 25th november 2009 with a visit to the holy kaaba and continued for 5 or 6 days at different holy sites in mina, arafat, and muzdalifa. the current study was a cross-sectional study performed in two phases. the first phase was conducted during the week before the start of hajj on november 25th, and the second phase was conducted in the week following the end of hajj on november 30th. healthcare workers were asked to answer a questionnaire and provide nasal and throat swabs both before and after hajj. the pre-hajj questionnaire was about demographics (age, sex, occupation, and nationality), medical history (chronic disease and smoking), vaccination history (including h1n1 and seasonal influenza), and knowledge of h1n1 influenza (symptoms, transmission, and prevention). the post-hajj questionnaire included questions about exposure to infections during hajj and compliance with infection control practices (hand hygiene, cough etiquette, and wearing a mask). nasal and throat swabs were collected using the same method during both phases of the study. nose and throat swabs were collected in viral transport media using dacron swabs on stainless steel wire and plastic shafts, respectively (remel, microtest m4rt, usa). immediately after collection, samples were transported to the jeddah regional laboratory where they were stored at à80 c until tested. nucleic acid was extracted using the x-tractor gene, corbett from qiagen using 25101 vx dna/rna purification protocol. the multiplex pcr using micro fluid arrays and luminex x-map system, with xtag respiratory viral panel fast assay (manufactured by luminex molecular diagnostics, inc, toronto, on, canada, distributed by abbott molecular, wiesbaden-delkenheim, germany) was used for nucleic acid testing for 18 circulating respiratory virus types and subtypes: influenza a, influenza a h1, influenza a h3, influenza b, rsv, coronavirus 229e, coronavirus oc 43, coronavirus nl63, coronavirus hku1, parainfluenza 1, parainfluenza 2, parainfluenza 3, parainfluenza 4, human metapneumovirus, rhinovirus, adenovirus, and human bocavirus. the xtag data analysis software for rvp fast(tdas rvp fast) analyzed the data and provided a report summarizing which viruses were present. the rvp fast detects influenza b, influenza a h1 seasonal, and influenza a h3 only. if any other subtypes are present, it will indicate the presence of influenza a matrix protein only. therefore the samples in which influenza a matrix protein was detected were run separately by a singleplex pcr to detect h1 2009 pandemic strain using artus inf/h1 lc/rg rt-pcr kit (qiagen) and for avian influenza a h5n1 (subtype asia) by lightmix kit (tib, molbiol,gmbh, berlin, germany), according to the manufacturer's instructions. any strain in which rna was not detected for these four influenza a types (i.e., h1, h3 seasonal in rvp fast and independent singleplex pcrs for h1 2009 pandemic strain, and h5n1 [subtype asia]) was labeled as unsubtypeable influenza a virus. demographics, medical history, vaccination history, knowledge of h1n1 influenza, and compliance with infection control practices are presented as frequencies. the prevalence of respiratory viruses is presented as number of viruses per 1000 healthcare workers. differences in the prevalence of respiratory viruses before and after the hajj were examined using non-parametric paired statistics a total of 184 healthcare workers who treated pilgrims during the 2009 hajj season were included in the study. of these, 161 answered the (main) pre-hajj questionnaire and 104 answered the (short) post-hajj questionnaire. a total of 120 combined nasal and throat swabs were obtained during the pre-and post-hajj periods. demographic and clinical characteristics of the sample are shown in table 1 . the majority of the healthcare workers were males (85%) with an average age of 40.9 ae 9.2 years (range 23e59 years), non-saudi (71%), physicians (75%), with more than 10 years of medical experience (60%) as well as previous experience of serving in hajj medical services (83%). most of the healthcare workers (93%) described their own health as very good to excellent. chronic disease, namely hypertension, diabetes, and asthma were present in 15% and 11% were current smokers. compliance of healthcare workers with pre-hajj vaccination and infection control is shown in table 2 . eighty four percent of them got at least one vaccine before hajj. the coverage of hepatitis b, meningococcal and seasonal influenza vaccines were relatively high (73%, 67% and 51%, respectively), while the coverage of h1n1 vaccine was considerably low (22%). the main reasons described for not getting the vaccine were worries about the side effects, (42%), non-availability (34%), and fear of developing h1n1 symptoms (22%). approximately 50% of the healthcare workers did not get seasonal influenza vaccine in the past year due to the belief of being healthy (29%), lack of knowledge about the place to get the vaccine (22%), and the assumption that influenza is not a serious illness (18%). compliance with hand hygiene was noted in 98%, cough etiquette in 89% and wearing face mask in 90% of the healthcare workers. the exposure risk as defined by being within 1 m from a person with ili was reported in 61%, handling biological specimens in 34% and examining patients in 76%. about 20% of them got sick or injured during hajj. background knowledge of the healthcare workers about h1n1 2009 is shown in tables 3 and 4 . eighty five percent believed that h1n1 is a serious disease, 80% were worried about catching h1n1 influenza during hajj and 75% were aware of the main symptoms of h1n1 influenza. the main source or vehicle of h1n1 transmission as recognized by them were contact with people infected with h1n1 (86%), contaminated fomites (72%) and air (65%). appreciable level of knowledge about measures to avoid h1n1 infection were noted as described by maintenance of hand hygiene (91%), wearing a mask (76%), cough/sneeze etiquette (76%), staying away from sick people (65%), using hand sanitizer (63%), avoiding crowds/public gatherings (62%) and taking h1n1 vaccine (58%). among the 18 circulating respiratory virus types and subtypes, only two were detected in the healthcare workers in the pre-and post-hajj period: rhinovirus (n z 21, 12.6%) and coronavirus 229e (n z 1, 0.6%) . rhinovirus was detected more before the hajj (n z 14, 11.7%) than after (n z 9, 7.5%), but the difference was not statistically significant. the only isolate of coronavirus was detected in the post-hajj period. two healthcare workers had rhinovirus detected both before and after the hajj. rhinovirus was detected in 21.1% of those who had respiratory symptoms and 30.0% of those who got sick during hajj. no other respiratory viruses were detected in any of the samples. the prevalence (per 100 persons) of respiratory viruses according to age, sex, profession, smoking, vaccine, sickness, and wearing a mask is shown in fig. 1 . the prevalence was slightly higher in healthcare workers who got sick during hajj, in nurses, and in those who did not wear masks than in smokers; however, the difference was not statistically significant. hajj, the annual pilgrimage of muslims is a time of a unique mass gathering event in makkah. around two million people are confined to small area and the chances of having infections acquired by respiratory tract are increased. al-tamami et al, 10 during the 2001 hajj, found 23 cases of meningitis of all types, mainly in indians, whose ages ranged from 1 to 70 years, and in twice as many women as men. balkhy et al, 6 in 2004, studied 500 symptomatic pilgrims, 10.8% of whom had positive viral cultures. of these, influenza b accounted for 50%, followed by herpes simplex virus (21.4%), rsv (12.9%), parainfluenza (7.4%) and influenza a (5.6%). a comparative study of respiratory tract infections in symptomatic uk and saudi pilgrims by rashid et al. in 2008 8 found infections in 25% of uk pilgrims but in only 13% of saudi pilgrims. half of the infections in uk pilgrims were due to rhinoviruses, followed by influenza virus, parainfluenza, and rsv. the saudi pilgrims had higher infection rates with influenza virus (78.5%) than with rhinovirus (21.4%). in 2009, alborzi et al. 9 also reported that 32.5% of patients tested had viral pathogens: influenza in 25 (9.8%), parainfluenza in 19 (7.4%), rhinovirus in 15 (5.9%), adenovirus in 14 (5.4%), enterovirus in 5 (2%), and rsv in 4 (1.6%) and coinfection with two viruses in 1 patient (0.4%). the current study evaluated the prevalence of respiratory viruses in healthcare workers who saw pilgrims after the h1n1 2009 pandemic had been declared. we tested for 18 respiratory virus types and subtypes in the healthcare workers and found primarily rhinoviruses and a single coronavirus 229e. rhinoviruses were more prevalent after the hajj (11.7%) than before (7.5%). none of the healthcare workers tested positive for any influenza virus, including the h1n1 2009 pandemic strain. this is explained by the fact that among more than two million pilgrims in 2009, the ministry of health reported only 100 cases of h1n1 and 5 deaths. 17 the high case-fatality ratio may be because pilgrims were committed to completing hajj and delayed seeking medical care until their condition had worsened. 17, 18 since the overall number of cases among pilgrims was low, therefore, the chances of transmitting it to healthcare workers were very small. rhinoviruses are present in about two-thirds of persons with common colds and probably are responsible for more human infections than any other agents. 19, 20 they are common in all age groups, occur throughout the year, and are present worldwide. 21 louis et al. 22 found that rhinovirus was responsible for half of the respiratory infections in residents and staff in a long-term care facility for elderly persons, although in community-dwelling elderly, they cause 63% of respiratory infections. 23 renois et al 24 found rhinoviruses to be most prevalent in cases of influenza-like illness in infections with one agent (25%) as well as in coinfections with influenza a h1n1 viruses (50%). our finding that rhinoviruses are the most prevalent viruses in healthcare workers during the 2009 hajj are consistent with the other studies of rhinoviruses in the general population, in patients with influenza-like illness, and also in pilgrims. arruda et al. 25 studied the natural history of rhinovirus infections in adults during autumn and found that among 346 persons with colds, 82% (283) had rhinovirus infections and 8% had coronavirus oc43 and 229e. these findings agree with our finding of rhinovirus as the predominantly isolated virus (12.6%), followed by coronavirus 229e (0.6%), in a group of subjectively healthy healthcare workers. arruda et al. isolated a high percentage of viruses because the subjects were symptomatic, while in our study rhinovirus was detected in 21% of participants with respiratory symptoms and in 30% who got sick during hajj. rhinoviruses spread efficiently in families, in school groups, among university students, and on military bases. 26, 27 linde et al. 28 found an increase in the proportion and number of rhinovirus diagnoses that roughly parallels a decrease in influenza diagnoses, after the summer holidays and start of schools. they hypothesize that a rhinovirus epidemic could interfere with the spread of pandemic influenza in a warm and humid climate, which decreases the spread of influenza by aerosol. a similar phenomenon may be responsible for the frequent isolation of rhinoviruses in the present study. rhinoviruses may protect the host from being infected by other viruses such as influenza a virus, parainfluenza virus, adenoviruses, coronaviruses, bocavirus, metapneumovirus, and rsv. 29 rhinovirus shedding is commonly limited to 10e14 days in immunocompetent subjects. 24 however viral rna may be present from days before symptoms occur to five or more weeks after they go away. 30, 31 the influenza a h1n1 vaccination rate in healthcare workers has been reported to be lower than the seasonal influenza vaccination rate 16, 32, 33 and was 22% versus 51% in the current study. these results are also comparable to the data obtained from the united states for the same period: where vaccination coverage for h1n1 in healthcare workers was 37% and for seasonal influenza it was 62%. 32, 33 in conclusion, we found that rhinoviruses were the most frequently isolated viruses in a group of subjectively healthy middle-aged healthcare workers who treated hajj pilgrims during the 2009 influenza a h1n1 pandemic. respiratory symptoms were present in 21% of the healthcare workers in which the virus was detected. none of the participants had influenza a h1n1 2009, despite that only 22% of them were vaccinated against h1n1 vaccine, possibly because it was also infrequently found among pilgrims. usa, dr abduraman abudawod and dr. nedal almasri for their assistance in the data collection. risk of respiratory infections in healthcare workers: lessons on infection control emerge from the sars outbreak acute respiratory tract infections among hajj medical mission personnel, saudi arabia health risks at the hajj association of national health occupational physicians (anhops) respiratory tract infection during hajj influenza a common viral infection among hajj pilgrims: time for routine surveillance and vaccination mecca bound: the challenges ahead viral respiratory infections at the hajj: comparison between uk and saudi pilgrims viral etiology of acute respiratory infections among iranian hajj pilgrims risk factors of bacterial meningitis in makkah during hajj 1421 h: a pilot study outbreak of swine-origin influenza a (h1n1) virus infection e mexico, marcheapril update: swine influenza a (h1n1) infections e california and texas world health organization. the regional office of eastern mediterranean global public health implications of a mass gathering in mecca, saudi arabia during the midst of an influenza pandemic establishment of public health security in saudi arabia for the 2009 hajj in response to pandemic influenza a h1n1 acceptance and adverse effects of h1n1 vaccinations among a cohort of national guard health care workers during the 2009 hajj season prevention of pandemic influenza after mass gatherings e learning from hajj pandemic h1n1 influenza at the 2009 hajj: understanding the unexpectedly low h1n1 burden human rhinoviruses: the cold wars resume acute respiratory symptoms in adults in general practice the seasonality of rhinovirus infections and its implications for clinical recognition rhinovirus outbreak in all long term care facility for elderly persons associated with unusually high mortality acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems frequency and natural history of rhinovirus infections in adults during autumn the seattle virus watch. v. epidemiologic observations of rhinovirus infections, 1965e1969, in families with young children rhinoviruses in seattle families, 1975e1979 does viral interference affect spread of influenza? do rhinoviruses reduce the probability of viral co-detection during acute respiratory tract infections? persistence of rhinovirus and enterovirus rna after acute respiratory illness in children picornavirus infections in children diagnosed by rt-pcr during longitudinal surveillance with weekly sampling: association with symptomatic illness and effect of season influenza vaccination of health-care personnel: recommendations of the healthcare infection control practices advisory committee (hicpac) and the advisory committee on immunization practices (acip) should healthcare workers have the swine flu vaccine key: cord-329653-5nkrrqqw authors: patrick, jennifer r.; shaban, ramon z.; fitzgerald, gerry title: influenza: critique of the contemporary challenges for pandemic planning, prevention, control, and treatment in emergency health services date: 2011-04-08 journal: australas emerg nurs j doi: 10.1016/j.aenj.2011.03.001 sha: doc_id: 329653 cord_uid: 5nkrrqqw the 2009 h(1)n(1) influenza pandemic was a major challenge to health services around the world. previous experiences with severe acute respiratory syndrome (sars) and avian influenza a (h5n1) prompted initiation of formal pandemic planning. essential and desirable features of pandemic plans include preparation for surveillance, investigation of cases, treatment modalities, prevention of community spread, maintenance of essential services, research and evaluation, and implementation, testing and revision of the plan. the experience of 2009 h(1)n(1) influenza pandemic for emergency departments and their staff was problematic. the pace of the pandemic, coupled with untested pandemic plans, presented a unique range of challenges. in this paper, the contemporary challenges with respect to pandemic influenza prevention, control, and treatment are examined. the lessons learned are critical to our response to future pandemics, which are inevitable. the experience with severe acute respiratory syndrome (sars) and avian influenza a (h 5 n 1 ), as well as knowledge of influenza pandemics last century, prompted initiation of formal pandemic planning. pandemics evolve rapidly, and are complex and unpredictable. in 2004, after the sars experience, the world health organization (who) identified the essential and desirable features of pandemic plans, which included: (i) preparation for surveillance; (ii) investigation of cases; (iii) treatment modalities; (iv) prevention of community spread; (v) maintenance of essential services; (vi) research and evaluation, and implementation; and (vii) testing and revision of the plan. 1 this paper will critique the current literature with respect to contemporary challenges for pandemic influenza prevention, control and treatment. influenza viruses are myxoviruses, with three main genera, influenza a, b and c, which are capable of causing infection in humans. 2, 3 only influenza a causes epidemics or pandemics in humans. 3, 4 one antigen (haemagglutinin or h) on the outer coat of the virus anchors the virus to cells, and another (neuraminidase or n) helps it both enter and exit cells. influenza a subtypes are named according to which subtypes of h and n they possess. these antigens alter over time by a process of drift, or repeated minor mutations that occur over time, or shift, or major change in the antigens, which occurs when two different influenza viruses are simultaneously in a host and recombine. [2] [3] [4] [5] influenza may be transmitted by aerosols, large droplets, and direct and indirect contact. 6 the relative importance of these modes is considered debatable. 7 the virus can survive on non-porous surfaces for up to 48 h and on unwashed hands for 30 min. 8 both seasonal and pandemic h 1 n 1 influenza 2009 outbreaks failed to demonstrate significant airborne transmission over long distances, but aerosol transmission may occur in confined spaces, especially when a large airborne infectious burden is present. 7, 9 what is pandemic influenza, and what is the difference between it and seasonal influenza the who and the department of health and ageing (doha) state an epidemic occurs when there are more cases of a disease than is normal and a pandemic is declared when a worldwide epidemic occurs. 10, 11 the who monitors influenza globally and is the body that declares the commencement and end of pandemics. 11 influenza pandemics occur when a virus to which people have little or no immunity develops, and efficient human-to-human transmission exists. 2,3 pandemics may persist for months, years or decades, have rapid transmission, disease occurrence is outside usual seasonal patterns, and attack and mortality rates across age groups are unpredictable. 4 the declaration of a 'pandemic influenza' has major effects on resource allocation within government and non-government health agencies. it also has profound effects in the function of societies, including disruption and closure of schools and workplaces, as well as restrictions on travel and social gatherings. however, the criteria by which an outbreak of an infectious disease may be declared a pandemic are neither fixed nor well defined. 12, 13 they depend on a variety of factors including the relative incidence of an outbreak across jurisdictions, the severity of the infection, and cross-border or transnational cooperation with respect to the epidemiology of the disease. if a pandemic is defined merely by the spread of a new influenza virus strain around the world, the effects of having the formal declaration in place may disproportionately affect the function of society in the event of a mild illness. criteria also need to be developed for declaring a pandemic 'over'. in the case of pandemic h 1 n 1 2009, countries around the world ceased epidemiological tracking of the infection when it became ubiquitous. thus there is a need to establish international consensus on the formal definition and criteria for pandemic influenza to allow appropriate response to an outbreak, and to determine when declarations of pandemic may be lifted. pandemic plans require large-scale surge capacity in healthcare systems and the community. surge capacity is the ability to manage a sudden, unexpected increase in patient volume (i.e., numbers of patients) that would otherwise severely challenge or exceed the current capacity. 14 pandemics have health, economic, political, and social impacts. 15, 16 internationally, healthcare systems have few surplus resources. a 2007 us study identified that regardless of planning, few health services had staff, equipment, and facilities to implement them. 17 there is no reason to believe that australia was in any better case. in australia, doha developed the australian health management plan for pandemic influenza (ahmppi) 18 which was tested though exercise cumpston in 2006 19 and exercise sustain in 2008. 15 australian jurisdictional plans complement and augment the ahmppi. [20] [21] [22] [23] [24] [25] [26] [27] [28] exercise cumpston 06 19 gave recommendations on the ahmppi. these included calling for streamlined decision making processes, flexible response according to disease severity and local resources, improved communications, public health education, a national surveillance framework, clarification of quarantine, border control, and emergency legislation, and involvement of primary care providers in planning. 19 two years later, exercise sustain 08 15 identified the differences between pandemic and disaster responses, and stressed the impossibility to plan for all eventualities given the unpredictability of new viruses. it addressed local community empowerment, recommending planning with community leaders and groups. social distancing was identified as an important prevention strategy. development of a productive relationship between the media and public health agencies, with an emphasis on timeliness, transparency, and honesty was explored, with health professionals considered to have most credibility for message delivery. australian state and territory pandemic plans additionally recommend the establishment of flu clinics, separate influenza triage, and designated 'flu hospitals' to facilitate quarantine and allow eds and general practitioners to maintain their core business. these plans aimed for staff protection, including priority vaccination for at-risk staff, pre-and post-exposure prophylaxis, the use of personal protective equipment and access to pandemic stockpiles, with those exposed or ill expected to self-isolate. [20] [21] [22] [23] [24] [25] [26] [27] [28] other identified needs include extra funding, pre-defined triggers for plan implementation and deactivation, anticipatory training of key personnel 29 and suspension of non-essential and/or non-emergency hospital functions during the crisis. 14 ventilators, medications and personal protective equipment were to be stockpiled. increased cleaning, security and crowd management capacity was identified in the plans, along with the need to increase morgue and laboratory capacity. more non-medical staff are required for administration, communication, transportation, security, cleaning and garbage disposal, and for crowd control. surge capacity applies across the entire community, with health, government and community groups required to act cooperatively. when individual hospitals reach capacity they need to have means to move patients and/or services to other hospitals or centres. [29] [30] [31] healthcare worker illness needs to be planned for with use of agency, retired staff, and volunteers. 14 seasonal influenza seasons have highlighted a lack of surge capacity in emergency departments (eds). 32 the literature shows that pandemic planning and management are complex, expensive and difficult. in spite of exercises cumpston 06 and sustain 08, australian pandemic plans were criticised during the h 1 n 1 influenza 2009 pandemic for most of the reasons identified as problematic during those exercises. [33] [34] [35] remedying these problems goes beyond their recognition, and requires sustained and systematic investment on the part of government, the health system, health professionals, and the general public. overwhelming evidence exists that it is not possible to prevent pandemics, and when they occur they can cause substantial morbidity, mortality and social disruption. 1, 36 when the 2009 h 1 n 1 influenza isolate was identified in mexico, their borders were closed at great economic cost to the country. 37 however, the virus had already spread. air travel allows rapid and efficient spread during the incubation period when people are asymptomatic, rendering border closure and controls such as thermal scanning inefficient as a means of prevention. 38 surveillance, identifying the onset, nature and size of the outbreak and populations affected is fundamental to pandemic control, identifying spread and trends, determining at-risk populations, allowing for targeted interventions, and monitoring of their effectiveness. 38 surveillance of influenza-like illness (ili) in mexico allowed for identification and early warning of pandemic h 1 n 1 influenza 2009, 39 although there can be little doubt that the virus was circulating for some months prior to formal identification. 40 it is difficult to clinically differentiate patients with influenza from those with other viral respiratory infections. 41 rapid antigen point-of-care testing, was shown to have low accuracy for known pandemic h 1 n 1 influenza 2009, 42 and reverse transcriptase polymerase chain reaction (rt-pcr) provided the best method of testing. 43 obligatory testing of all with respiratory disease ceased during the sustain phase of the pandemic, as positive diagnosis would not change treatment or isolation, and it would have been a pointless expense to keep testing. 34 the literature shows testing was problematic and expensive. immunisation is a primary tool for pandemic influenza control, however it takes time to develop and manufacture vaccines. 16, 29 in the interim, community mitigation strategies including social distancing, cough and sneeze etiquette, and frequent thorough hand hygiene may be used. 29, 38 atrisk groups need targeted information and intervention. 38 contact tracing of healthcare worker and patients' contacts has been recommended to identify those with risk factors for severe disease who may benefit from antiviral prophylaxis. 7 a recent study in australia of the knowledge and attitudes the general public held towards pandemic h 1 n 1 influenza 2009 44 found low levels of public anxiety, a high degree of belief in government preparedness, vaccination and quarantine as effective public health measures, antiviral medication as moderately effective, and a low level of belief in hand hygiene as a preventative measure. quarantine was cited as being problematic with respect to work and food shopping. the literature shows that hand hygiene education and vaccination development are required for pandemic influenza control. social distancing is effective in infection control as a form of isolation. at the commencement of the 2009 pandemic, health department officials advised people with ili to present to their local ed 45 causing overwhelming presentations by those with ili and those fearful they may have had contact. 34, 46, 47 in a pandemic, general practitioner and hospital waiting rooms are a potential site for cross infection to already unwell people if people present with a highly infectious illness. this happened during the sars outbreak in canada in 2003. 48 the literature indicates that these patients are best treated in other sites. overcrowded living conditions contribute to rapid influenza spread. 16 this contributed to the disproportionately high impact of pandemic h 1 n 1 influenza 2009 in indigenous communities in the northern territory. 49 school closures have been used as a means of social distancing. however a western australian survey of parents whose children were in schools closed during the h 1 n 1 influenza 2009 pandemic 50 revealing that 74% of these students participated in out of home activities including sporting events, shopping, outdoor recreation and parties while their schools were closed. this paper questions the efficacy of school closure in preventing infection spread, highlighting that students were congregating elsewhere. an example of effective isolation practice was seen during the simultaneous outbreak of pandemic h 1 n 1 influenza 2009 and seasonal (h 3 n 2 ) influenza which was contained on two australian cruise ships. 51 the conditions encouraging contagion on the ships included living and socialising in partially enclosed close proximity, and people from both hemispheres (i.e., opposite flu season) coming together. prior to the outbreak, the ship procedures already included point-of-care testing, antiviral medication and isolation for ili patients. however, not all ill passengers sought medical care, presumably because they had mild illness or were unwilling to be isolated while holidaying. after disembarkation, passengers discovered their need to be quarantined via media reports, word of mouth, from ship staff or public heath staff. almost all complied with quarantine requirements, and only one case of infection passed from a passenger to another person was found, with no further community spread. thus, an epidemic on board ships was prevented from contributing to the 2009 pandemic. many people continue to work when they are ill. in the tropics influenza is under-recognised and under-diagnosed, which can cause people to inadvertently infect families, colleagues and patients. 52 studies have shown that many healthcare workers either go to work or intend to work when they have influenza. [53] [54] [55] one hospital in the usa experienced high levels of infection in their eds at the peak of h 1 n 1 influenza 2009 pandemic, with no commensurate increase in sick leave. 56 this finding was reflected in a recent australian study. 34 thus, the literature indicates that healthcare workers need further education on this matter. australian healthcare guidelines recommend contact and droplet precautions for influenza control. 57 contact precautions include surface cleaning, hand hygiene, the use of gloves and gowns, single-use equipment wherever possible, and masks and eye protection when splashes may occur. additionally, droplet precautions add patient isolation or cohorting and minimising patient transfers. particulate respirators, eye protection, and impervious gowns and gloves, are advised for all aerosol-generating procedures, which should be performed in a negative pressure room if available. 7, 41, 58, 59 australian guidelines recommend surgical masks for those entering an infectious area, coming within 1 m 57 or 2 m 7 of an infectious patient. where tolerated, masking the patient is more effective than masking the health care workers. 60 personal protective equipment is vital to healthcare workers who cannot be protected in any other way prior to effective vaccine development. in 2009, slow distribution of personal protective equipment from the national stockpile to frontline workers was a problem, with general practitoner practices running out of stock and being unable to replace it early in the pandemic. 35 while the cdc recommends the use of respiratory protection of at least the equivalent of n95 masks 61 for health care workers managing patients with ili, canadian research has found n95 masks are no more protective in influenza than normal surgical masks. 62 inconsistencies in recommendations between authorities must be resolved to improve staff compliance and confidence. 63 thus the literature calls for better distribution of personal protective equipment during an influenza pandemic and identifies research is required into appropriate mask usage. public education is vital to stop influenza spread. materials used must target the audience and be culturally appropriate. in the hunter new england area, focus groups identified challenges and potential solutions for limiting pandemic influenza in indigenous communities. 64 these included having a local resource person with an understanding of the disease, provision of clear, simple, culturally appropriate information, access to health services and knowledge of how obtain these without infecting others, sensitivity to the importance of family and cultural gatherings, and aboriginal people having a say in how support is provided. pregnant women were targeted and encouraged to take precautions against acquiring infection and accept vaccination. 65 doha developed a website 10 which gave detailed advice for individuals, households, healthcare providers, businesses and communities, and provided a telephone hotline. this allowed people to receive the best possible advice in a timely fashion. the media are vital to spreading public health messages, but can be sensationalist, ill-informed, and may desensitise the public to the issue through information overload or precipitate 'moral panic'. 34, 38 a single, knowledgeable, authoritative voice has the best chance of conveying information effectively. 34 while vaccination is vital to halt the spread of pandemics, time is needed for development and production. 38 an american study found that most of the people they surveyed would not accept a new, not fully tested vaccine approved under emergency use authorization. 66 healthcare worker immunisation rates in australia are reportedly between 20% and 50%. 67 reasons include low perception of personal risk, poor knowledge of how immunisation works, doubts about vaccine efficacy and/or safety, self-perceived contraindications, and inconvenient access. 68 in spite of the hong kong experience of sars, a study conducted prior to and repeated during the outbreak of pandemic h 1 n 1 influenza 2009 reported no significant increase in intention to receive immunisation, with participants citing fears of side effects and doubts of efficacy. 69 this was echoed in a more recent australian study 34 with widespread perception that the pandemic h 1 n 1 influenza 2009 vaccine was rushed into production and not comprehensively tested. 34, 70 low acceptance was also reported in greece. 71 in the usa there was low public willingness to accept vaccination under an emergency order. 66 when released in australian, presentation in multi-dose vials posed a problem, with insurance companies initially refusing to cover administering general practitioners. 72 the use of multi-dose vials for mass immunisation was part of the ahmppi, and known associated risks can be minimised by adherence to clear guidelines on their use. 73 alternative vaccination sites, including retail outlets and workplaces, have been proposed to increase public uptake of influenza vaccination. 74 british columbia has proposed regulatory changes to allow pharmacists to administer vaccines. 75 treatment for pandemic influenza is a matter of conjecture until the actual virus is identified and the clinical profile emerges. most pandemic h 1 n 1 influenza 2009 cases required simple supportive treatment including rest, fluids, and antipyretics, 34 however advanced oxygenation therapy including high-frequency oscillation ventilation, nitric oxide or extracorporeal membrane oxygenation was required for some with severe disease. stockpiled ventilators were too old or too simple to provide complex ventilation strategies. [76] [77] [78] [79] debate occurred about the efficacy of oseltamivir for the treatment and prevention of pandemic h 1 n 1 influenza 2009. the cochrane review concluding it merely shortened the duration of symptoms by 24 h if given within 48 h of disease onset, and thus should only be given to those sufferers with known risk factors for severe disease 80-82 especially as side effects include gastrointestinal symptoms and headaches, with rare cases of delirium and psychosis (most frequently in children and adolescents), raised liver enzymes, and allergic reactions. 83 guidelines recommend oseltamivir administration within 48 h of disease onset. 83 adherence to this would have seen many treated for a disease they did not have in the h 1 n 1 influenza 2009 pandemic, as overwhelmed pathology services saw extended time lags between specimens being sent for testing and the arrival of results. 33 generally speaking, humanity was better prepared for the h 1 n 1 influenza 2009 pandemic than for any other pandemic in history. while the disease itself was less virulent than expected, it confirmed the unpredictability of pandemic influenza, and its ability to cause significant impacts on health systems and the community. the h 1 n 1 influenza 2009 pandemic highlighted unresolved challenges identified in both exercise cumpston 06 and exercise sustain 08, which we as a profession and society must address. public health challenges include developing means of increasing acceptance of influenza vaccination by both the general public and healthcare workers, provision of targeted education for the indigenous population and other at-risk groups, improving public knowledge of social distancing and personal hygiene measures in the prevention of transmission, and improving dissemination of information during a pandemic, especially via the media. we can use the evidence to refine pandemic plans and promote community well-being during an influenza pandemic. jennifer r. patrick and gerard j. fitzgerald have no competing interests or conflict of interests to declare. ramon z. shaban is editor-in-chief of the australasian emergency nursing journal but had no role in the editorial review of this manuscript whatsoever, and has no other competing interests or conflict of interests to declare. world health organization. who checklist for influenza pandemic preparedness planning department of health and ageing. pandemic influenza -types of influenza microbiology and infection control for health professionals s principles and practice of infectious diseases a/h1n1 influenza virus the basics communicable disease control handbook asid (hic-sig) position statement: infection control guidelines for patients with influenza-like illnesses, including pandemic (h1n1) influenza 2009, in australian health care facilities. emja influenza in the acute hospital setting clinical management of human infection with pandemic (h1n1) 2009: revised guidance department of health and ageing. pandemic 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operational plan special counter disaster plan: human pandemic influenza hospital response to pandemic influenza, part 1: emergency department response nsw human influenza pandemic sub plan queensland department of the premier and cabinet tasmanian action plan for human influenza pandemic national strategic plan for emergency department management of outbreaks of novel h1n1 influenza. american college of emergency physicians surge capacity: a proposed conceptual framework pandemic influenza and critical infrastructure dependencies: possible impact on hospitals flu outbreak overwhelm eds highlight lack of surge capacity australia's influenza containment plan and the swine flu epidemic in victoria pandemic (h1n1) 2009 influenza outbreak in australia: impact on emergency departments the general practice experience of the swine flu epidemic in victoria -lessons from the front line challenges and opportunities in pandemic influenza planning: lessons learned from recent infectious disease preparedness and response efforts what mexico taught the world about pandemic influenza preparedness and community mitigation strategies communicable disease control an introdction. melbourne: ip communications new influenza a(h1n1) virus infections: global surveillance summary pandemic (h1n1) 2009 influenza community transmission was established in one australian state when the virus was first identified in north america summary of the australasian society for infectious diseases and the thoracic society of australia and new zealand guidelines: treatment and prevention of h1n1 influenza 09 (human swine influenza) with antiviral agents poor clinical sensitivity of rapid antigen test for influenza a pandemic (h1n1) 2009 virus detection of influenza a(h1n1)v virus by real-time rt-pcr the community's attitude towards swine flu and pandemic influenza h1n1 influenza 09-managing the 'moral panic hospitals inundated as swine flu panic spreads flu planners fear ers flooded with the not-so-sick sars outbreak in the greater toronto area: the emergency department experience disproportionate impact of pandemic (h1n1) 2009 influenza on indigenous people in the top end of australia's northern territory household responses to pandemic (h1n1) 2009-related school closures outbreaks of pandemic (h1n1) 2009 and seasonal influenza a (h3n2) on cruise ship influenza in the tropics self-reported anticipated compliance with physician advice to stay home during pandemic (h1n1) 2009 results from the 2009 queensland social survey incidence and recall of influenza in a cohort of glasgow healthcare workers during the 1993-4 epidemic: results of serum testing and questionnaire will they just pack up and leave?'' -attitudes and intended behaviour of hospital health care workers during an influenza pandemic which health care workers were most affected during the spring 2009 h1n1 pandemic australian guidelines for the prevention and control of infection in healthcare centers for disease control & prevention. interim guidance on infection control measures for 2009 h1n1 influenza in healthcare settings, including protection of healthcare personnel infection prevention and control in health care for confirmed or suspected cases of pandemic (h1n1) 2009 and influenza-like illnesses preventing the spread of influenza a h1n1 2009 to health-care workers outbreak of swineorigin influenza a (h1n1) virus infection -mexico surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial hospitals and the novel h1n1 outbreak: the mouse that roared reducing the risk of pandemic influenza in aboriginal communities centers for disease control & prevention. pregnant women need a flu shot public willingness to take a vaccine or drug under emergency use authorization during the 2009 h1n1 pandemic influenza vaccination among healthcare workers influenza vaccination of health care workers in hospitals--a review of studies on attitudes and predictors willingness of hong kong healthcare workers to accept pre-pandemic influenza vaccination at different who alert levels: two questionnaire surveys toll of second swine flu wave could be high low acceptance of vaccination against the 2009 pandemic influenza a(h1n1) among healthcare workers in greece insurance row threatens swine flu vaccinations h1n1 and the use of multi-dose vials in mass vaccination alternative vaccination locations: who uses them and can they increase flu vaccination rates? ministry of health services british columbia. proposed changes allow pharmacists to give injections the experiences of health care workers employed in an australian intensive care unit during the h1n1 influenza pandemic of 2009: a phenomenological study critical care doctors want escalated pandemic planning novel h1n1 influenza: the impact on respiratory disease and the larger healthcare system preparing for pandemic (h1n1) 2009 neuraminidase inhibitors-the story behind the cochrane review what can we learn from observational studies of oseltamivir to treat influenza in healthy adults? neuraminidase inhibitors for preventing and treating influenza in healthy adults: systematic review and meta-analysis module name=product%20info&searchkeyword=oseltamivir+phosphate &previouspage=â�¼/search/quicksearch. aspx&searchtype=&id=55420001 2#an-adversereactions5230 key: cord-346063-7u1a198p authors: de clercq, erik; neyts, johan title: avian influenza a (h5n1) infection: targets and strategies for chemotherapeutic intervention date: 2007-05-04 journal: trends pharmacol sci doi: 10.1016/j.tips.2007.04.005 sha: doc_id: 346063 cord_uid: 7u1a198p in an avian flu pandemic, which drugs could be used to treat or prevent infection with influenza a (h5n1) virus? foremost are the viral neuraminidase inhibitors oseltamivir and zanamivir, which have already been used to treat human influenza a (h1n1 and h3n2) and b virus infections. the use of the m2 ion channel blockers amantadine and rimantadine is compounded by the rapid development of drug resistance. although formally approved for other indications (i.e. treatment of hepatitis c), ribavirin and pegylated interferon might also be useful for controlling avian flu. combined use of the currently available drugs should be taken into account and attempts should be made to develop new strategies directed at unexplored targets such as the viral proteins hemagglutinin, the viral polymerase (and endonuclease) and the non-structural protein ns1. as has been shown for other viral infections, rna interference could be a powerful means with which to suppress the replication of avian h5n1. the outbreaks of avian influenza a (h5n1) in southeast asia, the increasing geographic distribution of this epizootic virus and its ability to transfer to humans and cause severe infection (i.e. pneumonia) have aroused serious concerns regarding the control measures that should be undertaken to curb a potential pandemic of the disease [1] [2] [3] . in the wake of such a pandemic, several preventive and therapeutic strategies have been formulated [1] , the most prominent being the stockpiling of neuraminidase inhibitors -in particular, oseltamivir (tamiflu1). the current avian h5n1 originated in 1997 in hong kong and has spread (through birds) to southeast asia and other countries, with occasional transmission to humans ($200 human cases, more than half of which were fatal). whether the current avian h5n1 will evolve further to cause a pandemic through either mutation of the current h5n1 virus ('antigenic drift') or reassortment of an avian influenza virus with a human (or other non-avian) influenza virus ('antigenic shift') [4] is unpredictable at present. we have recently reviewed the antiviral agents that are active against influenza viruses and that could be used, either therapeutically and/or prophylactically, in an influenza virus pandemic, whether it be human, avian, equine, porcine or other [1] . here, we address which strategies could be undertaken and which targets could be envisaged to combat avian h5n1 infections specifically. the replicative cycles of human (h1n1, h2n2 and h3n2) and avian (h5n1) influenza viruses [1, 5] follow a similar 'scenario' [1] (figure 1 ). after binding to sialic acid receptors, influenza virions are internalized by receptormediated endocytosis. the low ph in the endosomes triggers the fusion of the viral and endosomal membranes, and the influx of h + through the m2 channel releases the viral rna genes in the cytoplasm ('uncoating'). adamantane derivatives block this uncoating step. the rna replication and transcription steps [which require repeated cycles of (à)rna $ (+)rna polymerization reactions] occur in the nucleus and can be blocked indirectly by inosine 5 0 monophosphate (imp) dehydrogenase inhibitors (e.g. ribavirin), which suppress the biosynthesis of gtp, or directly by rna polymerase inhibitors (e.g. t705). the translation of viral mrna to proteins could be prevented by interferon and small interfering (si)rnas. packaging and budding of virions occur at the cytoplasmic membrane, and neuraminidase inhibitors block the release of newly formed virions from the infected cells [6] . the anti-influenza-virus compounds -both those that are currently available as antiviral drugs and those that are still being developed -can be divided into different classes according to their molecular target of interaction: (i) neuraminidase inhibitors (e.g. zanamivir, oseltamivir and peramivir); (ii) m2 ion channel blockers (e.g. amantadine and rimantadine); (iii) imp dehydrogenase inhibitors (e.g. ribavirin and viramidine); (iv) interferon and sirnas; (v) rna polymerase (or endonuclease) inhibitors (e.g. t705 and flutimide). representative congeners belonging to classes (i), (ii), (iii) and (v) are shown in figure 2 . whereas viral hemagglutinin (ha) is needed for the influenza virus to interact with its receptor bearing n-acetylneuraminic acid (nana, sialic acid), viral neuraminidase, which cleaves nana, enables the progeny virions to leave the infected cells and spread to other host cells. by blocking the release of virus particles, neuraminidase inhibitors prevent this spread of virus [6, 7] . of the neuraminidase inhibitors, oseltamivir has received most attention as 'the' drug that should be stockpiled for therapeutic use in humans infected with avian flu. given its ease of administration (orally, 75 mg twice daily for five days) and systemic availability, oseltamivir can certainly be recommended as the drug of choice to be used against h5n1. it should not be ignored, however, that the use of oseltamivir can lead to the development of resistance. resistance to oseltamivir in clinical isolates of human influenza a has been associated with mutations at positions 119 (e119v), 152 (r152k), 198 (d198n), 274 (h274y) and 292 (r292k) of the neuraminidase. in particular, the r292k mutation has been associated with resistance of the (human) influenza a virus to oseltamivir [8] . recently, resistance of h5n1 to oseltamivir was shown to be caused by the h274y mutation. the patient from whom the oseltamivir-resistant h5n1 strain was isolated recovered from the disease, and the virus was found to be less pathogenic (in ferrets) than the parent strain [9] . however, two other patients from whom the h274y mutant h5n1 virus was isolated during oseltamivir treatment died from the infection [10], indicating a possible association of this mutation with death. interestingly, probenecid was found to prevent the renal secretion of the parent compound of oseltamivir (an oral prodrug form), thus markedly increasing the systemic exposure of oseltamivir [11] . combining oseltamivir with probenecid might be an important therapeutic option in severely ill patients and might enable the use of the standard dose (75 mg twice daily) of oseltamivir, as currently recommended. from studies of the highly pathogenic a/vietnam/1203/04 strain of h5n1 in mice [12] , it could be inferred that a prolonged oseltamivir regimen (i.e. eight-or ten-day, rather than five-day, treatment) and a higher dose (150 mg, instead of 75 mg, twice daily) might be required for the most beneficial antiviral effect. alternatively, intravenous administration of oseltamivir might be considered a therapeutic option, particularly in severely ill patients. the other currently available neuraminidase inhibitor is zanamivir, which must be administered through an inhalation device. there is low systemic availability of zanamivir following administration, including low drug levels in the lower respiratory tract (where most of the replication of current h5n1 viruses seems to take place) [13] . however, zanamivir is active against the oseltamivir-resistant h5n1 h274y variant. although not yet demonstrated for avian influenza, neuraminidase-based resistance is more likely to develop against oseltamivir than against zanamivir [14] . combination of the two drugs might be considered if it is further corroborated that the resistance patterns of zanamivir and oseltamivir do not overlap. if needed urgently (e.g. in severely ill patients), zanamivir and oseltamivir should be administered parenterally (i.e. intramuscularly or intravenously) and, hence, the adequate formulations should be made available for this purpose. several other neuraminidase inhibitors such as peramivir (rwj270201) and a315675 have been described [15, 16] . interestingly, both peramivir and a315675 have proven to be effective against a panel of five zanamivirresistant and six oseltamivir-resistant influenza a and b viruses [17] ; again, this indicates that resistance to neuraminidase inhibitors might not overlap. also, the structure of the influenza a (h5n1) neuraminidase has recently been resolved [18] , which should help the rational design of inhibitors. in initial clinical studies, oral administration of peramivir did not offer robust protection against infection with human influenza a virus [19] ; further studies with parenteral formulations of peramivir are warranted. meanwhile, long-acting dimeric inhibitors of influenza virus neuraminidase have been developed, which offer the prospect of a new type of anti-influenza drug that could be administered as a once-weekly dose to prevent infection [20] . do any antiviral agents other than neuraminidase inhibitors have potential in the control (prevention or therapy) of influenza a (h5n1) infections? the adamantane derivatives amantadine and rimantadine are specifically active against influenza a. they interfere with the viral uncoating process through a direct interaction with the matrix (m2) protein, which functions as a channel for hydrogen ions (protons). amantadine and rimantadine, however, are notorious for rapidly leading to drug resistance, which compromises their potential usefulness, if used as single agents, in the treatment of h5n1 infections. this concern has been highlighted by recent data about adamantane resistance among influenza a (h3n2) viruses isolated in the usa [21] and worldwide [22] . of concern with regard to the potential use of the adamantane derivatives against influenza a (h5n1) is that, of the two clades of currently circulating h5n1 viruses [clade 1 (vietnam, thailand and cambodia) and clade 2 (indonesia)], the whole of clade 1 is amantadine resistant, and drug resistance has also been noted in strains from clade 2 [23, 24] . several new adamantane derivatives that are effective against influenza a (h3n2), such as 2-(1-adamantyl)-2methylpyrrolidine [25] , have been synthesized; however, these compounds have not yet been evaluated for their activity against h5n1 viruses. the broad-spectrum antiviral agent ribavirin has been used as an inhibitor of influenza a and b virus infections for >30 years [26] . ribavirin is particularly active against (à)rna viruses, including the orthomyxoviruses and paramyxoviruses. the paramyxovirus respiratory syncytial virus (rsv) is the only (à)rna virus infection for which ribavirin has been formally approved (as an aerosol). although oral ribavirin has not been successful in the treatment of influenza a (h1n1) infection [27] , it is used, in combination with (injectable) pegylated a-interferon, to treat hepatitis c virus (hcv) infection. recently, viramidine -the carboxamidine analogue of ribavirin -was shown to have similar efficacy to ribavirin against influenza virus infections and, considering its lower toxicity, viramidine might warrant further evaluation as a possible therapy for influenza a virus, including h5n1, infections [28] . ironically, interferon was discovered, almost 50 years ago, with influenza virus as the inducer [29] . in fact, baron and isaacs described the absence of interferon from the lungs in fatal cases of influenza [30] . since then, interferon and its use have come a long way, and pegylated a-interferon, in combination with ribavirin, has become the standard therapy for hcv infection. therefore, extensive experience has been accumulated with this combination that could be readily implemented in the therapy of avian flu, for which the duration of treatment would be much shorter than for hcv. when using interferon for the prophylaxis or treatment of influenza, one should, however, take into account the fact that interferon alone might cause flu-like symptoms. based on the principle of rna interference, sirnas that are specific for highly conserved regions of the viral nucleoprotein or acidic polymerase have been found to protect mice against a lethal influenza virus (i.e. h5n1) challenge in vivo [31] . delivery systems compatible with human use have demonstrated the potential use of sirnas for prophylaxis and therapy of influenza virus infections in humans [32] . similarly, sirnas have proven to be a powerful new means with which to combat other respiratory virus infections such as those involving rsv and severe acute respiratory syndrome (sars) [33, 34] . more than ten years ago, 2 0 -deoxy-2 0 -fluoroguanosine was described as an inhibitor of influenza virus transcription, although it has not been studied further in this capacity [35] . also, a substituted pyrazine (t705) was reported that has potent in vitro activity against influenza a, b and c, and is more effective than oseltamivir at protecting mice that have been experimentally infected with influenza a/ pr/8/34. t705 has not yet been evaluated against avian h5n1 but, considering its target of action, it can be assumed to be effective. this pyrazine is apparently recognized by cellular enzymes as a nucleobase and converted to the ribofuranosyl-5 0 -triphosphate metabolite, which -in turn -inhibits the viral polymerase [36] . a polymerase inhibitor can be expected to have a resistance profile that does not overlap with those of the other known influenza virus inhibitors. another possibility for selectively inhibiting influenza virus replication is to target the cap-dependent endonuclease activity of the polymerase complex. in the mid-1990s, several inhibitors of influenza endonuclease (e.g. flutimide) were reported as selective inhibitors of influenza virus a and b replication, but they have not been developed further [37] . in view of the need for additional anti-h5n1 drugs, it might seem mandatory to explore further the viral polymerase (and/or endonuclease) as an antiviral target. recently, new compounds such as thiadiazolo[2, 3-a]pyrimidine and pyrimidinyl acylthiourea were reported to inhibit influenza a (h1n1) virus replication at a low (<0.1 mm) concentration [38] . however, the mechanism and target of action of these compounds have not been elucidated. in addition, a sialidase fusion protein [das181 (fludase)] has been reported that, at subnanomolar concentrations, inhibits both human and avian influenza virus replication [39] . the potential of fludase in the (topical) treatment of influenza a virus infections remains to be established. a plausible, but not yet proven, option for the prophylaxis or treatment of influenza a (h5n1) virus infection is the combination of pegylated interferon and ribavirin, which could be complemented with amantadine (or rimantadine). this triple-drug combination has shown efficacy in the review trends in pharmacological sciences vol. 28 no.6 treatment of hcv infection [40] and might be worth exploring further for the treatment and/or prevention of influenza virus infections. even if based only on the currently available drugs, there are several double-, tripleand quadruple-drug combinations that could be envisaged for the prevention and treatment of avian h5n1 (figure 3 ) -including the combination of oseltamivir and zanamivir (because their resistance profiles overlap only partially), the combination of these neuraminidase inhibitors with m2 ion channel blockers, and further extension of these combinations to include pegylated interferon and ribavirin. the compounds shown in figure 3 are active against influenza through different mechanisms and might, when combined, synergize their antiviral action while reducing the risk of the emergence of drug-resistant virus variants. in addition to viral rna polymerase and/or endonuclease, mentioned earlier as potential targets for new anti-influenza-virus agents, there are some other clues regarding the virulence of h5n1 viruses in humans [41] that could be considered as points of attack for chemotherapeutic intervention. first, the amino acid at position 627 in the viral polymerase protein pb2 is mutated from glutamic acid to lysine in h5n1 viruses, and this might represent an adaptation of h5n1 viruses for efficient replication in mammalian cells [42] . second, the ha of h5n1 viruses contains an unusual stretch of basic amino acids (rrrkkrg) that is cleaved by ubiquitous intracellular proteases (including furin). recombinant h5n1 viruses that lack these basic amino acids are not virulent in mice [40, 43] . third, the cterminal domain of the non-structural protein ns1 of avian h5n1 viruses contains a sequence motif (esev/epev) that can be considered a virulence factor because it binds to human host proteins and disrupts their morphology and functioning [44] . in ferrets, however, the c-terminal sequence is not required for the virulence of h5n1 viruses [45] . therefore, the role of the esev/epev motif and other molecular determinants of the virulence of h5n1 viruses must be explored further [41] . several drugs are available that could be used, either alone or in combination, for the prophylaxis and treatment of an influenza a (h5n1) pandemic. this includes compounds that are already used against influenza a virus infections -such as amantadine, rimantadine, zanamivir and oseltamivir -in addition to compounds that are used for infections with other viruses, such as ribavirin and pegylated interferon, which are used to treat hcv. attempts to design and develop further new antiviral drugs should be intensified, whether based on known molecular targets such as neuraminidase and/or the viral uncoating process or on relatively unexplored targets such as the viral endonuclease or the viral rna polymerase, which, in principle, could be targeted by both nucleoside and non-nucleoside inhibitors. the latter approach can be thought of as analogous to the nucleoside and non-nucleoside inhibitors of the hiv reverse transcriptase and of the hcv rna polymerase. the who and six medical journal publishers have launched the health internetwork access to research initiative, which enables nearly 70 of the world's poorest countries to gain free access to biomedical literature through the internet. the science publishers, blackwell, elsevier, harcourt worldwide stm group, wolters kluwer international health and science, springer-verlag and john wiley, were approached by the who and the british medical journal in 2001. initially, more than 1500 journals were made available for free or at significantly reduced prices to universities, medical schools, and research and public institutions in developing countries. in 2002, 22 additional publishers joined, and more than 2000 journals are now available. currently more than 70 publishers are participating in the program. gro harlem brundtland, the former director-general of the who, said that this initiative was ''perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries''. for more information, visit www.who.int/hinari review antiviral agents active against influenza a viruses avian influenza viruses and their implication for human health avian influenza a (h5n1) infection in humans the origins of pandemic influenza -lessons from the 1918 virus orthomyxoviridae: the viruses and their replication antivirals and antiviral strategies neuraminidase inhibitors for influenza resistant influenza a viruses in children treated with oseltamivir: descriptive study avian flu: isolation of drug-resistant h5n1 virus oseltamivir resistance during treatment of influenza a (h5n1) infection the anti-influenza drug oseltamivir exhibits low potential to induce pharmacokinetic drug interactions via renal secretion -correlation of in vivo and in vitro studies virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/ 1203/04 influenza virus in mice avian flu: influenza virus receptors in the human airway drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase influence of virus strain, challenge dose, and time of therapy initiation on the in vivo influenza inhibitory effects of rwj-270201 in vitro characterization of a-315675, a highly potent inhibitor of a and b strain influenza virus neuraminidases and figure 3. potential double-, triple-and quadruple-drug combinations of currently available anti-influenza-virus compounds. drug combination regimens are aimed at synergizing antiviral action, diminishing (toxic) side effects and/or reducing the risk of developing drug resistance. influenza virus replication susceptibilities of antiviral-resistant influenza viruses to novel neuraminidase inhibitors the structure of h5n1 avian influenza neuraminidase suggests new opportunities for drug design efficacy and tolerability of the oral neuraminidase inhibitor peramivir in experimental human influenza: randomized, controlled trials for prophylaxis and treatment potent and long-acting dimeric inhibitors of influenza virus neuraminidase are effective at a onceweekly dosing regimen adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern world health organization global influenza program surveillance network (2005) evolution of h5n1 avian influenza viruses in asia evolution and adaptation of h5n1 influenza virus in avian and human hosts in indonesia and vietnam heterocyclic rimantadine analogues with antiviral activity broad-spectrum antiviral activity of virazole: 1-b-d-ribofuranosyl-1,2,4-triazole-3-carboxamide lack of effect of oral ribavirin in naturally occurring influenza a virus (h1n1) infection in vitro and in vivo influenza virus-inhibitory effects of viramidine virus interference. i absence of interferon in lungs from fatal cases of influenza protection against lethal influenza virus challenge by rna interference in vivo inhibition of influenza virus production in virusinfected mice by rna interference inhibition of respiratory viruses by nasally administered sirna using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque inhibition of influenza virus transcription by 2 0 -deoxy-2 0 -fluoroguanosine mechanism of action of t-705 against influenza virus a novel antiviral agent which inhibits the endonuclease of influenza viruses a novel class of potent influenza virus inhibitors: polysubstituted acylthiourea and its fused heterocycle derivatives sialidase fusion protein as a novel broadspectrum inhibitor of influenza virus infection triple antiviral therapy with amantadine for ifn-ribavirin nonresponders with recurrent posttransplantation hepatitis c clues to the virulence of h5n1 viruses in humans pb2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of hong kong h5n1 influenza a viruses in mice molecular basis for high virulence of hong kong h5n1 influenza a viruses large-scale sequence analysis of avian influenza isolates lethality to ferrets of h5n1 influenza viruses isolated from humans and poultry in 2004 this work is part of the activities of the virgil european network of excellence on antiviral drug resistance and the coordinated action of rivigene, which is supported by the 6th framework programme of the eu. the dedicated editorial assistance of christiane callebaut is gratefully acknowledged. key: cord-323987-gh1m05gi authors: dziąbowska, karolina; czaczyk, elżbieta; nidzworski, dawid title: detection methods of human and animal influenza virus—current trends date: 2018-10-18 journal: biosensors (basel) doi: 10.3390/bios8040094 sha: doc_id: 323987 cord_uid: gh1m05gi the basic affairs connected to the influenza virus were reviewed in the article, highlighting the newest trends in its diagnostic methods. awareness of the threat of influenza arises from its ability to spread and cause a pandemic. the undiagnosed and untreated viral infection can have a fatal effect on humans. thus, the early detection seems pivotal for an accurate treatment, when vaccines and other contemporary prevention methods are not faultless. public health is being attacked with influenza containing new genes from a genetic assortment between animals and humankind. unfortunately, the population does not have immunity for mutant genes and is attacked in every viral outbreak season. for these reasons, fast and accurate devices are in high demand. as currently used methods like rapid influenza diagnostic tests lack specificity, time and cost-savings, new methods are being developed. in the article, various novel detection methods, such as electrical and optical were compared. different viral elements used as detection targets and analysis parameters, such as sensitivity and specificity, were presented and discussed. from humanity's common illnesses, the most frequent are acute respiratory infections (aris). influenza plays a role of the most serious virus causing aris and is the most often detected in lung infections. other causatives of aris can be rhinovirus, parainfluenza, adenovirus, enterovirus, respiratory syncytial virus (rsv) and others [1, 2] . influenza wears the name of 'the mother of all pandemics'. in particular, influenza a type has the ability to cause worldwide epidemics. in the last hundred years, virus attacks were documented four times: the spanish flu (h1n1) in 1918, asian flu (h2n2) in 1957, hong kong influenza (h3n2) in 1968 and swine influenza (h1n1) in 2009. it seems obvious to expect an influenza pandemic to return [3, 4] . the illness affects groups of all ages, and this pattern is not common for most viruses. there is a high possibility for every human to suffer from influenza. groups infected are mostly schoolchildren, the elderly and patients with serious medical conditions. infections of respiratory viruses, mainly influenza, and rsv, occur seasonally in winter months (from december to march) in europe, what is less common in hot climates where rhinovirus is seen in fall and spring, while adenovirus infects all year [2] . influenza spreads very easily among the population and shows high attack rates. in the us, it is the 8th highest cause of death, infects around 0.4 to 0.6 billion of children and 0.2 to 5.0 billion adults worldwide [5] , kills 0.5 to 1 million people annually with numbers still increasing [6, 7] . the influenza virus construction is comparatively simple. it mainly contains 8-segmented rna and surface proteins with highly immunogenic properties. distinguishable are three types-influenza a, b, and c, all belonging to the orthomyxovirus family. the a-type is mostly responsible for pandemics in the 20th-21st century. two glycoproteins cover the viral surface: haemagglutinin (ha) and neuraminidase (na) in a ratio of four to one [8] . based on surface composition, distinguishable are 18 h (h1-h18) and 11 n (n1-n11) subtypes forming potentially 198 combinations [3] . influenza a infects the human population, birds, pigs, dogs, horses and more [9] . genetic recombination is possible through the segmented genome. reassortment of genes is highly important in the epidemics. human population does not have the immunity against mutants with new ha and na antigens on the virion surface. there is a possibility of interspecies transmission without genetic reassortment, like in the case of h1n1 virus between swine and humans (and conversely) or h9n2 from poultry to humans. in other cases, rna segment reassortment occurs if at least two influenza viruses infect a single hosts cell [10, 11] . animal influenza viruses which occasionally infecting humans are called zoonotic influenza viruses (in direct and indirect contact) [12] . this high possibility of genetic variation can have subsequently pandemic effects. most of the new influenza viruses are mutants forming from antigenic drift [11] . the b-type influenza virus has similar biological properties to the a-type one. however, through electron microscopy, they are indistinguishable in size and shape. influenza b infects mainly humans and rarely other species. the antigenic drift occurs less often than in the a-type virus [8, 9] . the c-type influenza virus naturally infects humans but is less frequently detected, causes mild pediatric infections and sometimes affects adults [13] . it differs from a and b types through a shorter genome (1 segment less), and its major surface glycoprotein is hemagglutinin-esterase-fusion (hef), functioning as h and n together [14] . additional minor protein m2 is categorized as a single-pass integral membrane protein. it plays the role of a proton-selective ion channel, ph sensitive [15] . recently, new influenza virus genus was isolated from pigs and cattle and specified as d virus. it shows many similarities to c type virus. however, its structural differences make it a danger to public health due to the ability of binding human tracheal epithelia [9] . some studies have shown that 94-97% of workers exposed to cattle breeding have specific antibodies against influenza d, what means a risk of zoonotic infections. real-time polymerase chain reaction (rt-pcr) assay is believed to be adequate for influenza d virus infection diagnosis [16] . the influenza virus has a diameter of around 100 nm [17] . influenza a virus proteins (ha, na, and m2) are localized externally on the surface, more specifically they protrude above the lipid membrane. the infection starts with virus linking to the host respiratory epithelial cells. it recognizes and binds to sialic acid receptors via h proteins. sialic acids are nine-carbon acidic monosaccharides mainly found at the end of many glycoconjugates. the terminal carbon-2 can bind to carbon-3 or carbon-6 of galactose, forming different α-linkages and steric configurations. in human population dominate α-2,6 bonds, while α-2,3 are also common; however, the latter are more common in other species (ducks, birds). so there is a possibility of human infection by avian influenza, but less efficiently [18, 19] . the next step is neuraminidase activity. sialic acid is rifted from the cell's surface, what enables the influenza virus release and distribution in the respiratory tract. the na protein plays a role in replication of a and b influenza types. the m2 protein is essential after cell entry through uncoating of influenza a virus [13] . influenza incubation lasts 1 to 4 days, then the virus sheds and symptoms appear. viruses circulate in a host for 5 to 10 days but decrease 3-5 days after the first symptoms [20] . viral rna genome is segmented thus recombination between different strains is possible. this process is called antigenic shift; however, it is sporadic and occurs less than once per decade [3] . as a result, surface glycoproteins undergo some variations (minor changes in amino acid sequence like point mutations in genes). the ability of influenza virus to progressive antigenic change forces updates of vaccines composition [21] . influenza glycoproteins are an excellent target for virus detection due to many copies of ha (around 300) and na (around 50) on one viral particle [21, 22] . the 'gold star' are commercial ridts showing results in around 15 min. ridts are immunoassays that can identify influenza viral nucleoprotein antigens in respiratory specimens. monoclonal antibodies target viral nucleoprotein using immunochromatographic or immunoassay techniques. observed results are color changes or some other optical signals [29] . for example 'alere and influenza a&b' test uses an isothermal nucleic acid amplification, and 'bd veritor' uses chromatographic techniques as influenza detection methods. the mode of action of ridt is presented in figure 1 . the 'gold star' are commercial ridts showing results in around 15 min. ridts are immunoassays that can identify influenza viral nucleoprotein antigens in respiratory specimens. monoclonal antibodies target viral nucleoprotein using immunochromatographic or immunoassay techniques. observed results are color changes or some other optical signals [29] . for example 'alere and influenza a&b' test uses an isothermal nucleic acid amplification, and 'bd veritor' uses chromatographic techniques as influenza detection methods. the mode of action of ridt is presented in figure 1 . the main advantage is the possibility of providing the test in a physician's office in an effortless way with approximately moderate sensitivity (50-80%) [30] and high specificity (90%), but only in a qualitative way (positive/negative). thus, false positive and false negative results should be considered, especially during high influenza activity season [29] . false-negatives occur more often than false-positives. negative results cannot exclude virus infection [31] . undeniably, this kind of tests contributes to shortening the time of infection diagnosis, especially when the algorithm of influenza detection is long and laboratory procedures and requirements are high [32, 33] . ridts performance is better in children than adults (around 13% greater) due to higher viral loads and longer viral shedding [34] . tests differ in the virus type detected and with distinguishing between types, it means detection of only a type, a and b types with or without distinguishing between them [35] . sensitivity for b-type virus detection is lower than a-type. none of ridts can distinguish influenza a subtypes. in some tests, the results standardization is possible due to analyzing devices. they are available in cassette, card and dipstick formats, with a visual inspection or automated readers [36] . ridts belong to ii class of devices which need general and special controls. they were the main advantage is the possibility of providing the test in a physician's office in an effortless way with approximately moderate sensitivity (50-80%) [30] and high specificity (90%), but only in a qualitative way (positive/negative). thus, false positive and false negative results should be considered, especially during high influenza activity season [29] . false-negatives occur more often than false-positives. negative results cannot exclude virus infection [31] . undeniably, this kind of tests contributes to shortening the time of infection diagnosis, especially when the algorithm of influenza detection is long and laboratory procedures and requirements are high [32, 33] . ridts performance is better in children than adults (around 13% greater) due to higher viral loads and longer viral shedding [34] . tests differ in the virus type detected and with distinguishing between types, it means detection of only a type, a and b types with or without distinguishing between them [35] . sensitivity for b-type virus detection is lower than a-type. none of ridts can distinguish influenza a subtypes. in some tests, the results standardization is possible due to analyzing devices. they are available in cassette, card and dipstick formats, with a visual inspection or automated readers [36] . ridts belong to ii class of devices which need general and special controls. they were reclassified by food and drug administration (fda) from i class with low risk due to many failures in the h1n1 pandemic in 2009. also, fda evaluated some criteria for ridts that must be adhered [31] . as the market is enriched in commercial ridts, many authors have compared their sensitivities and specificities in influenza a/b detection. ryu et al. [34] have compared three digital ridts 'sofia influenza a + b fluorescence immunoassay', 'bd veritor system flu a + b assay' and 'buddi influenza a and b test' with conventional 'sd bioline'. results have shown for influenza a: buddi, sofia, veritor, and bioline sensitivity on level 87.7%, 94.5%, 87.7%, and 72.6%, respectively and specificity: 100%, 97.7%, 96.5%, and 100%, respectively, which are only partially accurate with producers assurance. for influenza b, sensitivities and specificities were on similar levels. none of the tests showed cross-reactivity with other respiratory viruses. the sofia test with the fluorescence reader had the best sensitivities. ridts with digital readout systems showed many similarities to conventional assays like small sample volume (less than 150 µl) and short analysis time (around 15 min) but exhibited much better sensitivities, even one order of magnitude lower limits of detection (lods). 'gold sign flu' and 'quick navi-flu' two ridts basing on immunochromatography were compared by akaishi et al. [37] they were applied for nasopharyngeal swabs as appropriate for the influenza a and b antigens detection. quick navi-flu demonstrated better sensitivities and specificities. advanced were short detection time (less than 10 min for most probes) compared to fluorescence-based tests. another type of point-of-care (poc) test 'the cobas ® liat ® influenza a/b' was evaluated by melchers et al. [38] the molecular assay based on rt-pcr showed results in 20 min. detection time of immunofluorescent pads was shorter, but sensitivities varied from 50% to 70% and often had to be confirmed by pcr methods. the cobas ® liat ® longer analysis time was balanced with an automated system, higher sensitivities (96%) and specificities (100%) compared to another test (diagenode). this assay was faster than others fda-approved rt-pcr-based tests with analysis time ≥1 h. two authors evaluated and compared different ridts on a wide range of patients for influenza and rsv detection and distinction. gómez et al. [39] tested 209 breath samples (nasopharyngeal swabs or aspirates) from both adults and children and moesker et al. [40] used >500 swabs only from pediatric patients (aged 0-5 years). fluorescence immunoassays (fias) with europium dye 'sofia ® influenza a + b' and 'sofia ® rsv' were compared to reference methods, rt-pcr, and cell culture. automatic analyzers showed higher sensitivities and specificities than other market ridts [39] . comparison of 'influenza ab ® ' and 'binaxnow rsv ® ' tests with a traditional rt-pcr confirmed the lower accuracy of ridts than in virus isolation and rt-pcr methods [40] . pediatric specimens were showing higher sensitivities as children have higher viral titers thus better ridts responses. in the literature, ridts evaluation or comparison to each other or the reference points are trending in the last few years. besides the examples described above, available are also 'alere i influenza a&b' nucleic acid amplification versus 'xpert flu/rsv' [41] , 'the bd veritor™ system flu a + b' versus the 'sd bioline assay' [34] , maripoc ® test [42] , becton dickinson test [43] , 'flu a + b' vs real-time-pcr [44] , and many more [45] [46] [47] [48] [49] [50] [51] , focusing on saliva specimens or nasopharyngeal swabs [52] . immunofluorescence method is an antigen detection with fluorescent microscope usage. the results are received in 2-4 h with high specificity and moderate sensitivity. distinguishable are direct (dfa) and indirect (ifa) fluorescent antibody staining assays for influenza a and b detection. subtyping of influenza a virus is not possible [53] . dfa assays are believed to have an easy procedure and short response time, so they are popularly used for influenza diagnosis. in the test, respiratory epithelial cells from nasopharyngeal swabs are directly stained with fluorescent-labeled antibodies and examined under fluorescent microscope. sensitivities are around 60-80% levels. on the usa market, only two approved tests by fda can be found, which are the d3 fastpoint l-dfa and bartels viral respiratory screening and identification kit [54] . dfa tests do not win in the influenza diagnosis with molecular assays which are giving higher sensitivities. from serological tests, the most commonly used are hemagglutination inhibition assay (hai), microneutralization/virus neutralization assay (vn), single radial hemolysis (srh), complement fixation assay, enzyme-linked immunosorbent assay (elisa) and western blotting [55] . these kinds of tests are generally not recommended because of paired serum samples necessity. the first swab must be collected as soon as possible at the beginning of an infection and the second about 2-4 weeks after. there is also a problem with the test availability. the results from a single serum specimen are not interpretable. although the assay is cheap and simple, the sensitivity is unsatisfactory [28, 53] . hai is generally used for influenza antibodies detection, which inhibits the interaction between h glycoprotein and red blood cells receptors. the test can be performed on inactivated viruses and is positive when four-fold or more rise of specific antibody titer is observed. the rise is between acute and convalescent serum samples and is measured by hemagglutination inhibition [55] . in the srh method, the formed complex of antigen-antibody induces the measurement of hemolysis areas which are proportional to antibodies quantity. there is no pretreatment of the serum needed. the technique is commonly used in natural infections and vaccinations [56] . the srh gives higher sensitivities than the hai assay. the vn measures virus-specific antibodies induction and their ability for virus neutralization. as a result, viral cells infection is prevented. this method is also routinely used in natural infections and vaccinations in influenza seasons. the vn assay gives higher sensitivities than the hai assay, but there are some restrictions in the diagnostic application (certified laboratories necessity). this assay requires infectious, active viruses [55] . elisa tests [57] are performed since the 1990s with high sensitivity and specificity. they are available in two forms: paper strips and microtiter plates. despite the big popularity of the test, the major disadvantage is still the lower sensitivity compared to tests based on nucleic acid-techniques. in a conventional test, the influenza virus is detected through specific antigen-antibody interaction and immunocomplex-enzyme linkage, resulting in color change [58] . some research groups are working on enhancing the sensitivity of these tests by using gold and europium nanoparticles with positive results. the europium nanoparticle-based immunoassay (enia) detects 29 strains of influenza a virus and some b virus subtypes with 16-times higher sensitivity than commercial elisa assay [59] . the viral culture method was introduced in the 1940s and is believed to be the most traditional and the gold standard for influenza diagnosis [31] . influenza viruses are recovered in clinical samples through propagation in mammalian cells or embryonated eggs. the principle is inoculation of permissive cell lines or embryonated eggs with infectious samples, propagation for a week (up to 10 days), observation of the cytopathic effect, and checking virus infection by various methods: immunofluorescence microscopy, antibody staining or erythrocytes hemadsorption [54] . these methods base on pcr and detection of specific dna/rna sequences of the virus. nats offer higher sensitivity than antigen-based tests and in much shorter time. currently available are reverse transcriptase-pcr (rt-pcr), sequencing-based tests like next-generation sequencing (ngs), ligase chain reaction, dna microarray tests, simple amplification-based assay (samba), nucleic acid sequencing-based amplification (nasba), loop-mediated isothermal amplification-based assay (lamp) and more. general majority of nats is performed within 2-4 h with influenza a subtypes information. commercially available are 26 fda licensed nats for influenza virus detection [60] . rt-pcr allows identification of influenza viral rna in respiratory specimens and is believed to be the most powerful influenza identification assay all over the world. it uses nested primers to detect and subtype influenza viruses [13] . results of the analysis offer very high specificity and the sensitivity is believed to be the highest of all conventional detecting methods [31] . the test procedure contains viral rna extraction from a specimen, rna reverse transcription to single-stranded complementary dna (sscdna) by reverse transcriptase enzyme and product amplification with fluorescent detection. some molecular assays using rt-pcr technique cannot only distinguish a and b types of influenza viruses but even identify specific seasonal influenza a subtypes, like h1n1 or h3n2 [61] . rt-pcr method compared to cell culture and elisa shows 103-and 106-times higher sensitivity, respectively. additionally, this sensitivity is not dependent on the patients' age. the procedure of combining multiple primers sets in 'multiplex rt-pcr' method enables detection of several respiratory viruses in one reaction. the main disadvantage is the 1-8 h long reaction time and diagnosis costs, as rt-pcr is the most expensive test kind [13, 60] . except conventional rt-quantitative pcr (rt-qpcr), specialists are working on speeding up the analysis, like one-step high-speed droplet-rt-pcr, getting results within 14 min [62] . samba method involves isothermal nucleic acid amplification with the three-step procedure: viral rna extraction, dna amplification by isothermal dna polymerase and dipstick system detection. results are obtained in around 2 h. this test is appropriate for avian and human seasonal influenza and gives high sensitivity (100% and 97.9% for influenza a and b) [63] . nasba is an isothermal amplification assay, pcr-independent. in one reaction it uses three enzymes: rnase h, t7 rna polymerase, avian myeloblastosis virus reverse transcriptase (amv-rt). single-stranded dna probes are used to capture viral rna sequences, then separated on a microfluidic chip. the test is suitable for seasonal influenza, in outbreaks of aris. moore et al. [64] showed 100% sensitivity on 19 clinical samples. this method is applicable for detecting other viruses, like hiv, rsv, sars [30] . lamp approach is used for the detection of many viruses like sars, adenovirus, rhinovirus, influenza virus and others. it uses dna polymerase or rna reverse transcriptase and two sets of primers which can recognize six distinct regions in viral complementary dna (cdna) [30] . the target gene is amplificated and determined by photometrical methods (color change when sybr green added). the sensitivity is similar to rt-pcr assay [65] . rt-lamp variety proposed by parida et al. [66] showed a ten times higher sensitivity compared to an rt-pcr method, what is 0.1 tcid 50 /ml (tissue culture infection dose at 50% end point). ngs is one of the most influencing techniques in genetic and medicine fields. ngs compared to the original sanger sequencing extremely speeds up the analysis due to automation possibilities. the next advantage is decreasing the costs from $100 million for human genome sequencing in 2001, by sanger method, to $2400 nowadays. some other technique, illumina platform, offers one million bases sequencing for the cost from $0.05 to $0.15 [67, 68] . in general, illumina platform is basing on the amplification of nucleic acid fragments on solid substrate or bridge amplification. whitehead et al. [69] applied illumina ngs platform to create sequence-function map to optimize influenza-binding proteins. this technique has big chances to develop new effective influenza inhibitors. another platform, roche 454 life sciences, developed ngs platforms based on pyrosequencing. it has found application in influenza a case for molecular markers identification, gene coding m2 protein mutation identification or single nucleotide polymorphism detection in the gene coding hemagglutinin [54] . except listed above, there are more ngs platforms, like pacific bioscience, ion proton, complete genomics and more, which use different sequencing techniques and have both advantages and limitations, vary on costs, read length, analysis time and error rate. for example, the chembio portable lateral flow platform can be performed outside the laboratory giving results in 25 min. the luminex analysis requires fewer reagents, offers fewer errors and a wide linear range [55] . among methods mentioned, general diagnostic tests for influenza base on virus culture (conventional and shellvial), detection of viral nucleic acid (pcr) or antigens (by neuraminidase enzymatic activity, fluorescent antibody or enzyme/optical immunoassay) and serologic tests. from them, culture methods can be excluded due to specialized laboratory requirement. also, serologic tests are impractical, need two adequate specimens and are time-consuming. rapid results can only give detection of nucleic acids or specific viral antigens and promote practical and useful diagnosis [21] . however, rt-pcr is still considered as time-consuming and expensive, and the elisa test does not offer high sensitivity [70, 71] . due to various limitations in the conventional detection methods, new diagnostic approaches are being developed. main trends for influenza virus detection are: (i) modifications of traditional 'gold star' methods like pcr, ridts, elisa what results in analysis time shortening, costs lowering, lod and limit of quantification (loq) improvement, (ii) conjugating of traditional methods and creating new platforms, micro-biochips and others, (iii) introducing known solutions to new ones, like smartphone-based analysis control with results data insertion into google maps, (iv) reuse of the functions of known devices, like glucometer, smartphone cameras, (v) the most common used detection methods: spectral/optical, electrical, (vi) and entirely new approaches. some of the approaches for influenza virus are presented in figure 2 . detection limits were shown in plaque forming units (pfu), g/l, viral copies, m, hemagglutinating units (hau), and tcid 50 units. they were difficult to compare as relied on different quantification methods. moreover, influenza have many components acting as target analytes, which in general are in different quantities/ratios in one viral particle or between influenza types/subtypes. exemplary are dna/rna (quantified in copies/ml or m), ha (quantified in hau) or whole viral particles (quantified in pfu). comparison of most lods between newly developed biosensors and different conventional methods were given. give detection of nucleic acids or specific viral antigens and promote practical and useful diagnosis [21] . however, rt-pcr is still considered as time-consuming and expensive, and the elisa test does not offer high sensitivity [70, 71] . due small, micro-size devices are trending in point of care (poc) tests. electromechanical systems on micro-or nano-scale have chemical, biological and medical applications. this kind of assays gives high efficiencies, small amounts of used materials and low waste production. they also speed up the analysis time and makes influenza diagnosis laboratory-independent [72] . another critical feature is the devices' portability. for example, using microfluidic rt-pcr with a continuous-flow and disposable electrical printed (dep) chips an influenza virus of swine-origin can be detected in a 15 min analysis [73] . combimatrix corporation developed influenza a microarray detecting all known so-far virus subtypes in less than 5 h [74] . biochips are common. not only for influenza disease but, i.e., for tobacco mosaic virus (tmv), human rhinovirus serotype 2 (hrv2) and others [75] . small, micro-size devices are trending in point of care (poc) tests. electromechanical systems on micro-or nano-scale have chemical, biological and medical applications. this kind of assays gives high efficiencies, small amounts of used materials and low waste production. they also speed up the analysis time and makes influenza diagnosis laboratory-independent [72] . another critical feature is the devices' portability. for example, using microfluidic rt-pcr with a continuous-flow and disposable electrical printed (dep) chips an influenza virus of swine-origin can be detected in a 15 min analysis [73] . combimatrix corporation developed influenza a microarray detecting all known so-far virus subtypes in less than 5 h [74] . biochips are common. not only for influenza disease but, i.e., for tobacco mosaic virus (tmv), human rhinovirus serotype 2 (hrv2) and others [75] . the invention of user-friendly techniques brought measurements to smartphone systems. the study of yeo et al. [76] showed the performance of a smartphone-based rapid fluorescent diagnostic system (srfds) created for the h9n2 virus diagnosis in chickens. the authors used oropharyngeal (op) and cloacal (cl) samples and compared their method with real-time rt-pcr. the limit of detection of srfds was 7.5 pfu/ml, what is 138-fold higher than in conventional colloidal-gold-based avian influenza rapid diagnostic test. the specificity was 100% and the sensitivity 99.44% for op and 95.23% for cl specimens, making this test comparable to rrt-pcr. pretreatment swab was transferred to the sample pad, after 15 min reaction time the smartphone camera was used as a detector, by using filtration of the excitation light by the emission filter in the light-emitting diode (led) module. the results were displayed on the smartphone screen, as a ratio of a control and test lines on a strip and coordinated with the location on google maps, to check for non-/infected areas. another group, wu et al. [77] , also have used smartphone assistance for influenza a detection. an automated and portable paper-based microfluidic system was developed. the chip consisted of two modules: the storage module with reagent chambers and the reaction module with the absorbent pad and nitrocellulose membrane functionalized with specific monoclonal antibodies. the smartphone was used for image capturing from the membrane by camera and for processing the image with an algorithm to the application developed with java. the smartphone was used as a guide to the microcontroller, connected via bluetooth. it enhanced multiple reaction steps performance, collected the results and sent it to medical agencies if necessary. another idea was to use already existing techniques and standardized analyzers. zhang et al. [78] designed an electrochemical assay which used a glucometer. glucose-containing substrate (sg1) exposed to influenza virus (or neuraminidase) released glucose which is determined amperometrically. samples were analyzed directly, without further preparation. the result was the detection of 19 strains of influenza viruses (h1n1 and h3n2) in 1 h. this method offers user-friendly, fast and inexpensive detection. when classifying influenza diagnosis methods by used measurement techniques, dominating are optical [79] and electrical [80] . they are believed to be fast, easy, adequate (providing qualitative and quantitative analysis) and relatively inexpensive. the influenza virus gold electrode electrochemical sensor was proposed by horiguchi et al. [81] immobilization of ha specific receptor (6 -sialyllactose) onto gold allowed label-free h1n1 detection. quartz crystal microbalance (qcm) technique and electrical detection were developed for biosensor measurements. qcm gave 2 −4 hau sensitivity with 10 min detection time and electrical detection gave sensitivity of 2 −6 hau and 30 min analysis time. the sensitivity was higher than conventional immunochromatographic technique (ict). different type of bioreceptor, anti-m1 antibodies was developed for influenza a detection. nidzworski et al. [82] modified boron-doped diamond (bdd) electrodes with 4-aminobenzoic acid and anti-m1 antibodies in self-assembled monolayer (sam) approach. by electrochemical impedance spectroscopy (eis) measurements authors achieved the lowest limit of detection (1 fg/ml) compared to previously reported methods. the assay offered easy sample pretreatment, short incubation time (<5 min) and non-interference of bacteria and yeast which might be present in patients swab. another antibodies immobilization method was presented by cheng et al. [83] simple technique of ac electric field application on electrodes induced positive dielectrophoresis. subsequently, viral particles were attracted to the immunosensor. commercially available surface acoustic wave (saw) electrode chip of 50 mm length size was used. the detection limit was 0.25 pg/ml with very fast response time 30 s. the authors have achieved the sensitivity of 90% and specificity of 70%. apart from electrodes mentioned above, there is a wide range of electroactive materials for biomolecule recognition. available are carbon-based materials like carbon paste, glassy carbon [84, 85] , graphene-oxide [86] , reduced graphene oxide [87] , boron-doped diamond (presented in figure 3 ) [88] . there are also noble metal-based materials like silver, gold [89, 90] , platinum, zinc, cadmium, and others [91] . moreover, every listed material can undergo multiple methods of surface modification, not only with nanoparticles [91] [92] [93] [94] [95] [96] [97] , but hybrids and biological materials, like aptamers [98] [99] [100] , sialic acid [88] or its derivatives [101] , composites [84, 85, 102] , nanohybrids [103] and dyes [86] . for influenza biosensing the most often used measurement techniques are differential pulse voltammetry [85, 91, 103] , cyclic voltammetry [102] , electrochemical impedance spectroscopy [84, 104] , and amperometry [86, 105] . interesting research of mubarok et al. [106] showed whole blood analysis for neuraminidase activity label-free detection. the cleavage of glycosidic linkage of self-synthetized n-acetyl-2-o-(4aminophenyl)-α-neuraminic acid (ap-neu5ac) in the presence of na released p-aminophenol molecules. as p-aminophenol showed electroactivity, the electrical signal onto a bare glassy carbon (gc) electrode was recorded. lod of na activity was 5.6 ng/ml. the sensor was applicable also for urine, saliva and nasal swabs. currently, many authors construct glycan-based biosensors [5, 19, 107] as they are natural viral receptors with selectivity for pathogenic subtypes. they form a compact layer on the measurement surface, so-called glycocalyx, reaching even 100 mm concentration. simple electrode modification might exclude time-consuming and expensive antibodies assay. for example, lod for h3n2 was achieved on 13 viral particles per 1 µ l level [107] . for glycan-derivatives sams the most often used electrochemical techniques are impedance spectroscopy and amperometry. except influenza proteins mentioned in '2. influenza pathogenesis' section, some non-structural proteins pa-x, pb1-f2, ns1, ns2 and others were recently found [108] they were also identified as pathogenic for the host organism [109] [110] [111] . electrochemical eis biosensor for pb1-f2 was developed by miodek et al. [112] the authors used antibody-antigen approach and modified gold electrode with a specific anti-pb1-f2 antibody with three steps. firstly, pyrrole and ferrocene derivatives were electrochemically polymerized onto the au surface. the next step was biotin/streptavidin linkage and the closing step was biotinylated antibodies immobilization. for confirmation of proper sensor modification the surface plasmon resonance (spr), atomic force microscopy (afm) and cyclic voltammetry (cv) techniques were used. by differential pulse voltammetry (dpv) two linear ranges were observed, 50-300 nm and 0.5-1.5 mm of pb1-f2 what was probably caused by two specific antibody sites. lod was on 0.42 nm level. from optical/spectral methods the most common are fluorescence, ultraviolet/visible (uv/vis) spectroscopy, surface enhanced raman spectroscopy (sers) [113, 114] and others [79] . recently developed influenza sensor by liu et al. [115] used fluorescence for neuraminidase detection. the macrocyclic dye substrate (squaraine-derived core blocked on ends with sialic acid) reacted with viral na releasing the blockages. subsequently, the free core was encapsulated with there are also noble metal-based materials like silver, gold [89, 90] , platinum, zinc, cadmium, and others [91] . moreover, every listed material can undergo multiple methods of surface modification, not only with nanoparticles [91] [92] [93] [94] [95] [96] [97] , but hybrids and biological materials, like aptamers [98] [99] [100] , sialic acid [88] or its derivatives [101] , composites [84, 85, 102] , nanohybrids [103] and dyes [86] . for influenza biosensing the most often used measurement techniques are differential pulse voltammetry [85, 91, 103] , cyclic voltammetry [102] , electrochemical impedance spectroscopy [84, 104] , and amperometry [86, 105] . interesting research of mubarok et al. [106] showed whole blood analysis for neuraminidase activity label-free detection. the cleavage of glycosidic linkage of self-synthetized n-acetyl-2-o-(4aminophenyl)-α-neuraminic acid (ap-neu5ac) in the presence of na released p-aminophenol molecules. as p-aminophenol showed electroactivity, the electrical signal onto a bare glassy carbon (gc) electrode was recorded. lod of na activity was 5.6 ng/ml. the sensor was applicable also for urine, saliva and nasal swabs. currently, many authors construct glycan-based biosensors [5, 19, 107] as they are natural viral receptors with selectivity for pathogenic subtypes. they form a compact layer on the measurement surface, so-called glycocalyx, reaching even 100 mm concentration. simple electrode modification might exclude time-consuming and expensive antibodies assay. for example, lod for h3n2 was achieved on 13 viral particles per 1 µl level [107] . for glycan-derivatives sams the most often used electrochemical techniques are impedance spectroscopy and amperometry. except influenza proteins mentioned in '2. influenza pathogenesis' section, some non-structural proteins pa-x, pb1-f2, ns1, ns2 and others were recently found [108] they were also identified as pathogenic for the host organism [109] [110] [111] . electrochemical eis biosensor for pb1-f2 was developed by miodek et al. [112] the authors used antibody-antigen approach and modified gold electrode with a specific anti-pb1-f2 antibody with three steps. firstly, pyrrole and ferrocene derivatives were electrochemically polymerized onto the au surface. the next step was biotin/streptavidin linkage and the closing step was biotinylated antibodies immobilization. for confirmation of proper sensor modification the surface plasmon resonance (spr), atomic force microscopy (afm) and cyclic voltammetry (cv) techniques were used. by differential pulse voltammetry (dpv) two linear ranges were observed, 50-300 nm and 0.5-1.5 mm of pb1-f2 what was probably caused by two specific antibody sites. lod was on 0.42 nm level. from optical/spectral methods the most common are fluorescence, ultraviolet/visible (uv/vis) spectroscopy, surface enhanced raman spectroscopy (sers) [113, 114] and others [79] . recently developed influenza sensor by liu et al. [115] used fluorescence for neuraminidase detection. the macrocyclic dye substrate (squaraine-derived core blocked on ends with sialic acid) reacted with viral na releasing the blockages. subsequently, the free core was encapsulated with macrocyclic tetralactam what caused spectral changes. red-shift in absorbance and fluorescence emission of squaraine were observed in the na presence. results were visible with naked eye. with some optimization, this portable sensor might be used in influenza diagnosis in poc or for effective determination of antiviral inhibitor drugs. the authors claimed possibilities of differentiation for classes of mammalian, bacterial and viral neuraminidase and suitability for quantitative analysis. another technique, sers, was used in magnetic immunosensor for avian influenza detection [116] . sam approach of 4-mercaptobenzoic acid (4-mba) molecules chemisorption on the gold nanoparticles (aunps) was used for further influenza a igg antibodies immobilization. the sandwich-type biosensor structure enabled qualitative and quantitative analysis. h3n2 was detected at lod 10 2 tcid 50 /ml with linearity range 10 2 -5 × 10 3 tcid 50 /ml. the assay had a potential for poc use due to time efficiency, sensitivity, and portability. a similar study by park et al. [117] has shown sers-based assay in a lateral flow strip. the principle was exchanging aunps from the commercial kit with sers-active nanotags. this innovation enhanced precision and sensitivity of the raman signal. the au-nanotags-influenza-antibodies complex was captured onto an antibodies-modified strip with positive probes giving two red strains (control line and sers-test tine). negative probes gave only one response on the control line. lod was calculated as 1.9 × 10 4 pfu/ml and this value was one order of magnitude higher than standard colorimetric kits. a different detection-type, immunochromatographic test (fict), for avian influenza a was proposed by yeo et al. [118] the authors have chosen fluorescent red dye 53 which intensity increased intensity by additional fluorescent phosphor groups linkage. the assay was to europium-based fict and standard ridt. influenza a virus from nasopharyngeal swabs was detected by a portable fluorescent strip reader. analysis time was only 15 min, the lod was estimated at 20 hau/ml, and the linearity range was 20-640 hau/ml. fict was 4-times more sensitive than europium-based fict and 16-times more sensitive than the rapid diagnostic test. another use of dye, 3,3 ,5,5 -tetramethylbenzidine (tmb), was in colorimetric immunosensor assays. lin et al. [58] similarly used antibodies in complex with streptavidin/biotin linker, liposome and antigen. innovative was horseradish peroxidase (hrp) encapsulation in liposome. after the addition of hydrogen peroxide (h 2 o 2 ) and tmb to the complex, lysis of liposomes occurred. hrp catalyzed the decomposition of h 2 o 2 generating · oh radicals which oxidized tmb and gave a color change to the solution. the detection was possible by the naked eye and spectrophotometric technique. lod of h5n1 was 0.04 ng/ml with linearity from 0.1 to 4.0 ng/ml. compared to standard elisa where very small absorbance and visual no color change was observed with the concentration below 4.0 ng/ml, the authors approach showed much more sensitivity. the modification of tmb-based assay was proposed by ahmed et al. [119] for spectrophotometric h3n2 virus detection authors have used gold nanoparticles-carbon nanotubes (aunps-cnts) hybrids, which showed the high catalytic activity of tmb oxidation. the complex of aunps-cnts-tmb-h 2 o 2 in the influenza presence showed a change in color. blue tone intensity varied depending on the virus concentration. the complex absorbed the light in λ max = 450 nm which was measured using a microplate reader. this method had a limit of detection 385 times lower (3.4 pfu/ml) than conventional elisa. ahmed et al. [120] similarly have used (+)aunps in tmb-based method achieving h1n1 virus detection at even lower levels of 10.79 pg/ml and for h3n2 of 11.62 pfu/ml improving sensitivity to 500-times higher than elisa. next optical detection technique upconversion luminescence resonance energy transfer (lret) was used by ye et al. [121] . the authors detected avian influenza h7 virus subtypes. the biosensor contained donor fluorophores (bagdf 5 :yb/er upconversion nanoparticles, ucnps) and acceptor fluorophores (aunps). lret measurement was activated by hybridization between complimentary oligonucleotides which were linked to nps. the target h7 gene was conjugated to acceptor nps and complimentary genes to donor nps. hybridization process decreased the fluorescence of ucnps and enhanced light absorption of aunps. upconversion spectra were registered after 2 h probes incubation. lod was 7 pm of the hemagglutinin gene and linear response from 10 pm to 10 nm. glycan-based methods mentioned in the electrical assays also have found application in optical biosensors. zheng et al. [122] used glycan-functionalized gold nanoparticles (ggnps) that bounded and aggregated on the viral surface. the authors have differentiated fourteen influenza strains and distinguished them from a human respiratory syncytial virus. the principle was different ha-binding preferences depending on the configuration of c-c bond in sialic acid receptors (described in section 2. 'influenza pathogenesis'). the one-step procedure, mixing virus with ggnps and 90 min incubation resulted in a color change from red to purple and was measured spectrophotometrically. except for natural glycans, he et al. [123] have synthesized influenza virus na resistant sialosides, with c-, s-and triazole linkage ends and printed onto a glass surface. these molecules could capture eight virus strains at very low concentration. caught viruses gave fluorescence intensity rise. lod was on 35 ceid 50 (chicken embryo infectious dose). adegoke et al. [124] as first evaluated the synthesis of cdznsetes quantum dots (qd) with the one-pot hot-injection method. they were used for near-infrared-emitting viral rna detection. the fluorescence signal was measured after 3 min incubation of qd and h1n1 probe. low lod level (1 copy/ml) confirmed much better sensitivity than ridts. the standard influenza virus detection method (rt-pcr) was modified by hmila et al. group [125] . the novel assay development was motivated by new mutations in ha and na of tunisian poultry. the risk of economic losses and human infections significantly increased. the authors have used an aptamer-real time-pcr. advanced was using one step selex procedure for h9n2 specific ssdna aptamers selection and later use as ligands for virus capture. the assay was adequate for direct swabs with no need for sample pretreatment and showed rapid, label-free results and high sensitivity. conventional elisa showed lod on 1.00 × 10 5 tcid 50 /ml and this assay 1.00 × 10 2 tcid 50 /ml. another group [126] has performed a new platform in china where standard pcr was conjugated with mass spectrometry-electrospray ionization (esi-ms) for identification of respiratory viruses. the authors have used nasopharyngeal aspirates for the analysis and have compared their method with dfa and later confirmed by rt-pcr plus sequencing. pcr-esi-ms showed higher sensitivities because detected more viruses in patients' specimens and was adequate for co-infections determination. the method could detect many virus families like coronaviruses, adenoviruses, alphaviruses and others. pcr-esi-ms was believed as faster and more automatic than conventional fda but required expensive equipment and had no practical usage in poc devices. for rapid and accurate influenza detection with high sensitivity and short time, eboigbodin et al. [127] have proposed the assay combining two techniques. reverse transcription (rt) of cdna to rna in single-step reaction and strand invasion-based amplification (siba ® ) in isothermal conditions allowed to detect both influenza a and b in only 15 min where traditional rt-pcr required more than 50 min. beneficial was rna usage for detection instead of dna as it is major influenza genetic material. 100 copies of h1n1 rna were detected with 100-times higher sensitivity than rt-pcr. modification of lamp method was proposed by ge et al. [128] lamp is believed to be the most willingly used nucleic acid-based isothermal amplification assay. however, differentiation of products containing multiple targets is still challenging as currently existing methods (e.g., electrophoresis) require specialized equipment. simple colorimetric method with invasive gold nanoparticles reaction was used. nps aggregation and dispersion resulted in solution color change. the sensor could subtype h1 an h3 with lod of 10 rna copies and influenza b with lod 100 rna copies. coordination of two trending viral detection methods, electrical and optical was proposed by sepunaru et al. [129] virus tracking was achieved by uv-vis spectroscopy. influenza particles were able to absorb silver nps and showed maximum absorbance at 401 nm. moreover, the tagged virus could be absorbed on the gc electrode and gave an electrical response. chronoamperometric signal of silver nps oxidation process was proportional to virus quantity. this assay enabled viral and bacterial infection distinction. sakurai et al. [130] have improved the common antigen-detection rapid influenza test known as immunochromatography (ic). ic could be accomplished in less than 20 min, however, had low sensitivity (around 60%) and lod on 10 3 pfu which is lower than pcr-based methods. the improvement was based on antibodies conjugation with fluorescent beads what enhanced the sensitivity 100-times. moreover, the assay was more accurate for early infections detection than ic. gouma et al. [131] have recently invented the novel isoprene sensor for an influenza virus ( figure 4 ). the authors have claimed that infected patients generate more volatile products compared to healthy ones. volatile products come from the alveolar and airway epithelium as well as leukocytes infiltrating the lungs, like volatile organic compounds (vocs) and nitric oxide (no). they were used as biomarkers to detect the disease. the constructed device was a portable 3-sensor array microsystem offering rapid non-invasive screening. the measurement needed to be conducted as fast as the disease was (potentially) present to observe biomarkers changes in time. the sensor was believed to give satisfying specificity and sensitivity. more precisely, the sensor could detect three gases: isoprene, ammonia, no in the temperature control conditions. it measured resistance changes of h-wo 3 material with exposure to no, no 2 , methanol, and isoprene at 350 • c. improvement was based on antibodies conjugation with fluorescent beads what enhanced the sensitivity 100-times. moreover, the assay was more accurate for early infections detection than ic. gouma et al. [131] have recently invented the novel isoprene sensor for an influenza virus ( figure 4 ). the authors have claimed that infected patients generate more volatile products compared to healthy ones. volatile products come from the alveolar and airway epithelium as well as leukocytes infiltrating the lungs, like volatile organic compounds (vocs) and nitric oxide (no). they were used as biomarkers to detect the disease. the constructed device was a portable 3-sensor array microsystem offering rapid non-invasive screening. the measurement needed to be conducted as fast as the disease was (potentially) present to observe biomarkers changes in time. the sensor was believed to give satisfying specificity and sensitivity. more precisely, the sensor could detect three gases: isoprene, ammonia, no in the temperature control conditions. it measured resistance changes of h-wo3 material with exposure to no, no2, methanol, and isoprene at 350 °c. jiang et al. [132] have invented an influenza sensor using a saw platform. the piezoelectric linbo3 wafers were coated with sio2. in this technique, the propagation of acoustic waves was changing at the measured material, depending on analytes located on the surface. love waves detected antigens through specific antigen-antibody interaction. the authors have used two reagents that were effective for immobilizing ha antibodies on the measurement surface. they were triethoxysilybutyladehyde (altes) and triethoxysilylundecanal ethylene glycol acetal (actes), which were end-functionalized with carboxylic acid and aldehyde. lod for h1n1 ha antigen was 1 ng/ml. oyama et al. [133] have developed their point-of-care testing chip. it was based on antigenantibody interactions with fluorescence detection. the authors applied absorbing polymer providing continuous sample flow and separation of bounded and free antibody residues. a glass fiber sheet was chosen as a flow medium. antibodies were fluorescently labeled by dylight488 and dylight650. the achieved sensitivity was more than thousand times better compared to immunochromatographic jiang et al. [132] have invented an influenza sensor using a saw platform. the piezoelectric linbo 3 wafers were coated with sio 2 . in this technique, the propagation of acoustic waves was changing at the measured material, depending on analytes located on the surface. love waves detected antigens through specific antigen-antibody interaction. the authors have used two reagents that were effective for immobilizing ha antibodies on the measurement surface. they were triethoxysilybutyladehyde (altes) and triethoxysilylundecanal ethylene glycol acetal (actes), which were end-functionalized with carboxylic acid and aldehyde. lod for h1n1 ha antigen was 1 ng/ml. oyama et al. [133] have developed their point-of-care testing chip. it was based on antigen-antibody interactions with fluorescence detection. the authors applied absorbing polymer providing continuous sample flow and separation of bounded and free antibody residues. a glass fiber sheet was chosen as a flow medium. antibodies were fluorescently labeled by dylight488 and dylight650. the achieved sensitivity was more than thousand times better compared to immunochromatographic commercial assays (lod above 104 ng/ml). krishna et al. [134] have developed giant magnetoresistance (gmr) sensor, which adapted monoclonal antibodies to influenza h3h2v nucleoprotein with magnetic nanoparticles (mnps). in the virus presence, mnps were bonded to the sensor, and the resistance change was measured. lod was 1.5 × 10 2 tcid 50 /ml. this assay was applicable for nasal swabs. except for the methods mentioned above, many authors have proposed novel ideas. for example, shelby et al. [18] invented the magnetic relaxation nanosensor (mrns) which was selectively binding to hemagglutinins. the influenza virus variants detection was at 1.0 nm concentration level. kirkegaard et al. [135] invented screen-printed aptasensor (pedot) for impedance-based influenza a detection. ozcelik et al. [136] evaluated the optofluidic wavelength division multiplexing method for single-virus detection. the authors could distinguish three subtypes of influenza a using two approaches. first was virus labeling with different color labels, and second was the combination of two different colors for every viral strain. lee et al. [137] presented quantitative h7n9 virus screening without dna amplification, basing on single-particle dual-mode total internal reflection scattering (sd-tirs) with transmission grating (tg). chan et al. [138] claimed that the flow graphene transistor-based dna sensors have not been explored yet, so they proposed microfluidic integrated reduced graphene oxide (rgo) transistor for the h5n1 influenza virus gene detection. zhang et al. [139] elaborated a label-free optical sensor for influenza serotyping. the authors have used the pattern recognition method based on arrayed imaging reflectometry (air) platform. when antireflective chip condition was perturbed due to binding to, e.g., an antibody, the reflected light changes were quantitatively detected. katayama et al. [140] created an influenza virus biosensor based on two methods: electrochemiluminescence (ecl) of modified au electrode combined with immunoliposome encapsulating ru (ii) complex. the authors have achieved sensitivity higher than elisa; the detection range was from 2.7 × 10 2 to 2.7 × 10 3 pfu/ml. li et al. [141] combined complement fixation and luminol chemiluminescence for rh7n9 detection, which is a recombinant avian influenza virus protein. this assay could be completed in 2.5 h with a linear detection from 0.25 fg/ml to 25 ng/ml. tran et al. [142] detected influenza a viruses using carbon nanotubes field effect transistor (cntfet) dna sensor. the response time was less than 1 min, and the detection range was linear from 1 pm (lod) to 10 nm. the sensor signal recovery was 97% after 7-month storage in ph-controlled conditions. the most interesting biosensors were compared in table 2 . rt-siba rna 100 copies -<30 min [127] mrt-lamp-cirn rna 10 1 /10 2 copies 10 4 -10 0 rna copies/µl~3 0 min [128] other novel ideas vp-viral particles, nir-near-infrared fluorescence, ha-p-hemagglutinin peptide, rp-recombinant protein, lsv linear sweep voltammetry, sers + lfa-surface-enhanced raman scattering-based lateral flow assay, rt-siba-reverse transcription strand invasion-based amplification, mrt-lamp-cirn-reverse transcription loop-mediated isothermal amplification-based assay coupled with cascade invasive reaction, cft + cl-complement fixation test+ luminol chemiluminescence. the scope of this article was presenting progress in the influenza sensing area, discussing the chosen materials and recognition elements, measurement techniques, sensitivities, specificities and further applications in poc devices. from all the presented influenza virus diagnosis methods, the most used in laboratories are 'gold standard' conventional methods. however, the majority of these require long time analysis (6-24 h for rt-pcr) [40] but compensating it with high sensitivities and the most reliable results. that is why the need for improvement for 'gold standards' increase significantly. despite the fact of low and variable sensitivity, ridts are the main improvement in influenza diagnostics, especially in pandemic seasons. they are pivotal in the emergency rooms by decreasing numbers of unnecessary antibiotics dosages. performing ridts at physicians' offices relieves laboratories work and enables the focus on specimens designed for culture or other time-consuming methods. moreover, the increased interest in specific laboratory diagnosis is due to minimizing the antibiotics resistance of patients and improves the influenza recognition system. as influenza is a public-health threat and influenza a is pandemic, portable, fast and accurate tools are in high demand in the medical industry to control virus outbreaks and spreads. also, this kind of devices needs constant updates due to genetic assortment of human, swine and avian influenza. new h/n genes are produced, against which human population lacks immunity. generally understood the 'biosensors' field has attracted many scientists. from a wide range of proposed assays, it is possible to choose these with reasonable cost, good selectivity and sensitivity, and practical application in poc. for authors of this article, the electrochemical sensors win the competition. it seems that they meet the need for rapid and accurate influenza diagnosis. they offer a vast number of electrode materials and target detection molecules with practically endless modification methods. furthermore, these sensors successfully can be expanded for other pathogen detection by changing the kind of probe immobilized on the electrode surface. funding: this research was funded by the ncbr, techmatstrateg1/347324/12/ncbr/2017; ventures program of the foundation for polish science, co-financed by the european union, regional development fund; the project "research and development work on an innovative, ultra-sensitive, fast and cheap micro-test for detecting the influenza virus-flusensor", rpma.01.02.00-14-6231/16 as part of action 1.2 "enterprise r&d activity" of the regional operational programme of mazowieckie voivodeship for 2014-2020, co-financed by the european regional development fund, priority axis i "use of r&d activity in economy"; the project "research and development works to develop a multisensor prototype-an innovative micro-sensor for identifying bacterial or viral causes of upper respiratory tract infections" as part of action 1.1 "enterprise r&d projects" sub-action 1.1.1 "industrial research and development activities carried out by enterprises" of the smart growth operational programme 2014-2020, co-financed by the funds from the european regional development fund. the authors declare no conflict of interest. 13-viral diseases chapter 18-viral upper respiratory infection past pandemics | pandemic influenza (flu) | cdc. available online highly specific and rapid glycan based amperometric detection of influenza viruses rapid diagnostic assay for intact influenza virus using a high affinity hemagglutinin binding protein influenza (seasonal) the biology of influenza viruses an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza d virus: basis for its broad cell tropism structure and function of the influenza virus genome rates of spontaneous mutation among rna viruses review of rapid diagnostic tests for influenza sense rna viruses-negative sense rna viruses influenza a 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detection of single influenza viruses tagged with silver nanoparticles fluorescent immunochromatography for rapid and sensitive typing of seasonal influenza viruses novel isoprene sensor for a flu virus breath monitor saw sensor for influenza a virus detection enabled with efficient surface functionalization a sensitive point-of-care testing chip utilizing superabsorbent polymer for the early diagnosis of infectious disease giant magnetoresistance-based biosensor for detection of influenza a virus screen-printed all-polymer aptasensor for impedance based detection of influenza a virus optofluidic wavelength division multiplexing for single-virus detection direct quantitative screening of influenza a virus without dna amplification by single-particle dual-mode total internal reflection scattering a microfluidic flow-through chip integrated with reduced graphene oxide transistor for influenza virus gene detection a label-free optical biosensor for serotyping "unknown" influenza viruses detection of influenza virus by a biosensor based on the method combining electrochemiluminescence on binary sams modified au electrode with an immunoliposome encapsulating ru (ii) complex combining complement fixation and luminol chemiluminescence for ultrasensitive detection of avian influenza a rh7n9 detection of influenza a virus using carbon nanotubes field effect transistor based dna sensor key: cord-310182-muybvyqa authors: fan, victoria y; jamison, dean t; summers, lawrence h title: pandemic risk: how large are the expected losses? date: 2018-02-01 journal: bull world health organ doi: 10.2471/blt.17.199588 sha: doc_id: 310182 cord_uid: muybvyqa there is an unmet need for greater investment in preparedness against major epidemics and pandemics. the arguments in favour of such investment have been largely based on estimates of the losses in national incomes that might occur as the result of a major epidemic or pandemic. recently, we extended the estimate to include the valuation of the lives lost as a result of pandemic-related increases in mortality. this produced markedly higher estimates of the full value of loss that might occur as the result of a future pandemic. we parametrized an exceedance probability function for a global influenza pandemic and estimated that the expected number of influenza-pandemic-related deaths is about 720 000 per year. we calculated that the expected annual losses from pandemic risk to be about 500 billion united states dollars – or 0.6% of global income – per year. this estimate falls within – but towards the lower end of – the intergovernmental panel on climate change’s estimates of the value of the losses from global warming, which range from 0.2% to 2% of global income. the estimated percentage of annual national income represented by the expected value of losses varied by country income grouping: from a little over 0.3% in high-income countries to 1.6% in lower-middle-income countries. most of the losses from influenza pandemics come from rare, severe events. few doubt that major epidemics and pandemics will strike again and few would argue that the world is adequately prepared. since the 2013-2016 ebola virus disease outbreak in western africa, the united states national academy of medicine 1 and several other groups [2] [3] [4] have pointed to gaps, and the need for greater investment, in preparation against epidemics and pandemics, of ebola virus disease and other infectious diseases. attempts to justify greater investment have mostly been based on estimates of the industrial and macroeconomic losses attributable to influenza pandemics. [5] [6] [7] [8] [9] [10] [11] we have recently extended the loss assessment to include a valuation of the lives lost as a result of the increases in mortality resulting from influenza-pandemic risk. 12 the inclusion of such a valuation increased the estimated loss attributable to modelled pandemic risk several fold. below, we discuss our method and summarize our findings. box 1 presents the definition of several of the terms we are using in this paper. most previous economic studies on global influenza pandemics have focused on income losses, through reductions in the size of the labour force and productivity, increases in absenteeism and, importantly, as the result of individual and social measures that interrupt transmission, but disrupt economic activity. while measures such as the per-capita gross national income include the effect of pandemics on income, they also exclude the value of changes in mortality risk to individuals. if, in assessments of investments in pandemic preparedness and mitigation, we neglect this dimension of loss, we will underestimate the value of such investments, relative to alternative uses of public finances. the broader approach that we recently applied, to the assessment of economic losses attributable to pandemic influenza, factors in the intrinsic loss associated with increases in mortality. 12 in effect, this approach assigns a dollar value to small changes in mortality probabilities, using values derived from empirical studies of how individuals and societies actually value changes in mortality risk. [13] [14] [15] [16] this approach has already been employed extensively in environmental economics 13, 14 and has also been used in global health, by the lancet commission on investing in health. 15, 16 past literature we searched google scholar and pubmed® for studies on the economic losses from influenza. almost all of the previous studies examined economic losses in terms of income and ignored the value of, and the loss associated with, mortality risk. the world bank, for example, generated estimates of global income losses under different influenza pandemic scenarios. 10, 11 it found that a pandemic of the same severity as the 1918 influenza pandemic might reduce global gross domestic product by about 5% and that the disruptive effects of avoiding infection would account for about 60% of that reduction. another study of the consequences of a range of pandemic severities included an extremely severe scenario that would lead to income losses of over 12% of gross national income worldwide, including losses of over 50% of the gross national incomes of lower-income countries. 5 we found other integrative estimates of the magnitude of pandemic risk in two partially proprietary sources. 17, 18 several studies have examined specific dimensions of the economic impacts of annual influenza, such as direct costs, e.g. medical and hospitalizations costs, and indirect costs, e.g. lost earnings due to illness and productivity costs. there are examples of such studies based in the americas, 6, 7, [19] [20] [21] [22] asia 8, 23 and europe. 24, 25 other models have added an estimated value of the intrinsic undesirability of nonfatal illness or of pandemic fear, as seen in the population response to severe acute respiratory syndrome in asia. 8 media coverage may also lead populations to overreact to mild pandemics. 9 abstract there is an unmet need for greater investment in preparedness against major epidemics and pandemics. the arguments in favour of such investment have been largely based on estimates of the losses in national incomes that might occur as the result of a major epidemic or pandemic. recently, we extended the estimate to include the valuation of the lives lost as a result of pandemic-related increases in mortality. this produced markedly higher estimates of the full value of loss that might occur as the result of a future pandemic. we parametrized an exceedance probability function for a global influenza pandemic and estimated that the expected number of influenza-pandemic-related deaths is about 720 000 per year. we calculated that the expected annual losses from pandemic risk to be about 500 billion united states dollars -or 0.6% of global income -per year. this estimate falls within -but towards the lower end of -the intergovernmental panel on climate change's estimates of the value of the losses from global warming, which range from 0.2% to 2% of global income. the estimated percentage of annual national income represented by the expected value of losses varied by country income grouping: from a little over 0.3% in high-income countries to 1.6% in lower-middle-income countries. most of the losses from influenza pandemics come from rare, severe events. economic losses of pandemic risk victoria y fan et al. we found only two articles that included estimates of the loss from the elevated mortality associated with influenza pandemics. 8, 19 of the 10 studies included in a recent systematic literature review on the costs of influenza, 26 only one 19 took account of the value of mortality risks. one strand of economic research has examined the intrinsic value of mortality risks, which is commonly expressed as the so-called value of a statistical life. this value is derived either from questionnaires that canvass how much compensation an individual would demand, to accept a small increase in the probability of their death, 1 or from quantitative studies of the labour market that investigate the trade-offs between small fatality risks and income. 2, 27 beyond influenza, the value of mortality risks has been included in estimating the costs of vaccine-preventable diseases 28 and in evaluating the economic burdens posed by rheumatic heart disease. 29 far more studies have assessed the burden of specific environmental risk factors. 13, 14 the value of a statistical life, which is sometimes expressed as the value of a standardized mortality unit (smu), i.e. an increase in the annual risk of death of 1 in 10 000, varies by both the age and income of the individual involved. 15, 16, 27 in general, the value of mortality is elastic to age and to income, i.e. younger individuals place a higher value on mortality than older individuals, and higher-income individuals generally value mortality more than lower-income individuals. the main findings of our recent study appeared consistent when, in robustness and sensitivity checks, we used estimates that were unconditional on age and estimates with varying income elasticity with respect to the value of mortality. 12 given the uncertain nature of an influenza pandemic, in terms of both when it may occur and how large the mortality risks will be, we applied an expected-loss framework that accounts for the uncertainty over a long period of time. 7 an expected-loss framework incorporates information on the risk of an uncertain event, e.g. a pandemic, with information on the severity or value of that event, e.g. the increase in mortality. although it has been estimated that the 2013-2016 ebola virus disease outbreak led to about 11 300 deaths, 30 the death toll from a severe influenza pandemic might be 2500 times higher than this. 12 in any given year, however, the risk of a severe influenza pandemic is much smaller than that of an ebola epidemic. the use of an expected-loss framework allows policy-makers to compare the expected losses associated with events with relatively high annual probability, but low mortality, e.g. an ebola outbreak, with those of events with relatively low probability but high mortality, e.g. the 1918 influenza pandemic. expected-loss frameworks are commonly used, by actuaries in the insurance industry, to calculate the size of premiums, e.g. for flood or health insurance. to value the consequences of uncertain events appropriately, the insurance industry estimates so-called exceedance probability functions. these functions generate estimates of the probability that, over a specified time frame, losses from an uncertain event, e.g. an influenza pandemic, would exceed any specified level. for our analysis, we developed an exceedance probability function for a global influenza pandemic. to parameterize the function, we turned to historical data on global influenza pandemics since the 1700s. [31] [32] [33] [34] six pandemics in this period led to excess mortality rates ranging between 0.03% and 0.08% of world population. in 2017, this range would be the equivalent of between 2 million and 6 million excess deaths globally. a modelling exercise for the insurance industry concluded that the annual risk of an influenza outbreak on the scale of the 1918 pandemic lies between 0.5% and 1.0%. 18 for more severe pandemics, we fitted a parametrized exceedance probability function to modelled data that had been previously reported. 18, 35 following common practice in the insurance industry, we defined risk, r(s), in terms of the annual probability of a pandemic having a severity exceeding s smus and the return time for s as the expected number of years before a pandemic of at least severity s will occur. if t(s) is the return time, then t(s) = r(s) −1 . for example, if the annual probability of a pandemic of severity at least s is 1%, then its return time will be 100 years. if we had access to a function r(s) showing exceedance probability as a function of severity, our analysis could proceed using the expected value of severity of all pandemics. because r(s) is the complementary cumulative of the density for s, we would have expected value of: we calibrated this model using historical estimates of the frequency and severity of influenza pandemics, which we obtained from our literature search on pubmed® and google scholar. for mortality data relating to the 1918 influenza pandemic, we also searched the libraries at harvard university and the university of hawai'i for historical documents and life tables. studies were restricted to those with abstracts in english. the consequences of a pandemic, in terms of lost income or lost lives. the expenditures made to prepare for -or recover from -a pandemic. excess death attributable to a given influenza pandemic (expressed in this paper in standardized mortality units or smus -a unit of 1 per 10 000 per year). the estimated probabilities that, in any given year, pandemics of varying degrees of severity will occur. defined in the probabilistic sense as the sum, across severities, of the losses associated with a pandemic of any given severity multiplied by the probability that a pandemic of that severity will occur in the coming year. note: much of this nomenclature accords with that of the insurance industry. policy & practice economic losses of pandemic risk victoria y fan et al. like other economic studies of pandemic influenza, we identified two main influenza pandemic scenarios in terms of aggregate mortality: moderate and severe. our review classified the 1918 pandemic as severe. as the world population in 1918 was about 1830 million and historical data indicate that there were at least 20 million pandemicrelated deaths in that year, the excess death rate associated with the pandemic was at least 1.1%. a closer examination of the data from india indicate that the true global rate was probably far higher than 1.1%, the pandemic led to 14 million deaths in india [36] [37] [38] and it seems implausible that india accounted for 73% of all of the pandemic-related deaths at a time when it had 18% of the world population. however, to be conservative, we estimated an expected annual excess mortality rate of 0.93 smus. in the corresponding model for moderate pandemics, we used a global expected excess mortality rate of 0.05 smus, as seen in historical moderate pandemics. 39 our calibration pointed to a very fat-tailed distribution. 12 thus, compared with an exponential function, the hyperbolic family of complementary cumulative distributions provided more natural candidates for r(s). we parameterized the hyperbolic function in terms of its expectation and the fatness of its tail. 40 thus: 12 the estimated proportion of annual national income represented by the losses varied according to country income grouping, from a little over 0.3% in high-income countries to 1.6% in lower-middle-income countries ( table 1) . the expected-loss framework distinguishes between the loss associated with a certain event that occurred, e.g. the mortality that occurred as a result of the 1918 influenza pandemic, and the expected loss associated with an uncertain event over a period of risk exposure. the expected loss combines both the risk of a moderate or severe pandemic and the losses from that event should the event occur. the expectedloss framework thus produces estimates of expected losses of an uncertain event, rather than actual losses of a certainly occurring event. we estimated the expected number of pandemic-related deaths to be about 720 000 per year. this level of mortality is on a similar scale to that attributable to other, more certain, causes of death, including other major infectious causes of death. 12 importantly, we concluded that most of the expected loss from influenza pandemics results from extreme events. another effort to estimate exceedance probability functions indicated that, among all pathogens that can cause a pandemic, influenza virus was likely to be the predominant cause of pandemic-related mortality. 18 the implication is clear: any efforts at pandemic preparedness need to be most strongly focused on influenza and on preparation for a severe scenario. our results present losses much higher than those found in studies limited to income losses. income losses have been estimated to represent around 15% and 50% of the total economic losses associated with a severe pandemic and a mild pandemic, respectively. 5, 11 in previous studies, across modelled pandemics of all severities, mean income losses were estimated to be us$ 80 billion per year 5,11 , i.e. about 16% of our estimate of total pandemicrelated costs. in terms of the percentage of global income, our estimate of total pandemicrelated losses (0.6%) falls within the corresponding intergovernmental panel on climate change's estimates of the costs of global warming (0.2-2.0%). 43 however, the magnitude of future global warming and the associated economic losses are still uncertain. 44, 45 the same is true for future pandemics. many of the hundreds of studies on the potential costs of climate change 46 have been hampered by the wide variation in estimates of the so-called social cost of carbon. 47 if this cost is set at about us$ 120 per tonne, the cost of the carbon dioxide emissions in 2013 would have been about 1% of global income. 46, 48 as in many previous attempts to estimate the economic losses associated with a pandemic, many previous attempts to estimate the social costs of carbon have focused on national income accounts, without any explicit valuation of the increases in mortality resulting from climate change. the mortality-associated costs of climate change may be relatively small, however, since the slowness of climate change should allow for compensatory human adaptation. our study had several limitations. first, we ignored the intrinsic undesirability of nonfatal illness and/ or pandemic fear. intense media coverage may lead populations to overreact to mild pandemics. second, our estimates of future pandemic risk and severity, and the economic estimates based on these epidemiological estimates, are relatively crude partly because pandemics remain rare and uncertain events. future modelling should lead to improved estimates over time. third, the assignment of monetary value to small changes in mortality risk and, particularly the relationship between valuation of such risk and both individual income and age at death, remains controversial. however, the results of sensitivity analyses, in which we applied a range of assumptions on these parameters, indicated that our main findings were reasonably robust. in addition to pathogens of pandemic potential, an expected-loss framework may also be applied usefully to malaria and other diseases that have fluctuating incidence. as cases of the disease become rarer as the result of effective interventions, malaria becomes less visible politically and financially, and policy-makers in some countries may have responded by reducing control efforts prematurely. policy-makers, and the societies they serve, could benefit by using an expected-loss framework to estimate the losses associated with uncertain and rare events across the full range of potential outcome severities. this could lead to appropriate and beneficial adjustments to each policy-maker's sense of risk and sense of value and to improved national policies on epidemic and pandemic preparedness. a recent united states national academy of medicine report argued that, given the risks we estimated, policy attention has fallen short. 49 il est nécessaire d'investir davantage dans la préparation contre les grandes épidémies et les pandémies. les arguments en faveur de cet investissement s'appuient en grande partie sur les estimations des pertes au niveau du revenu national que pourrait entraîner une grande épidémie ou une pandémie. récemment, nous avons élargi ces estimations pour y inclure la valeur des pertes faisant suite à des hausses de mortalité dues à des pandémies. cela a donné des estimations nettement plus élevées de la valeur totale de la perte que pourrait occasionner une future pandémie. nous avons paramétré une fonction de probabilité de dépassement pour une pandémie mondiale de grippe et avons estimé que le nombre escompté de décès dus à cette pandémie de grippe était d' environ 720 000 par an. nous avons calculé que les pertes annuelles découlant du risque de pandémie représentaient environ 500 milliards de dollars des états-unis, soit 0,6% du revenu mondial par an. cette estimation rejoint (dans la fourchette inférieure) celles du groupe d' experts intergouvernemental sur l' évolution du climat quant à la valeur des pertes dues au réchauffement de la planète, qui vont de 0,2% à 2% du revenu mondial. le pourcentage estimé du revenu national annuel représenté par la valeur escomptée des pertes variait selon la catégorie de revenu des pays: d'un peu plus de 0,3% dans les pays à revenu élevé à 1,6% dans les pays à revenu intermédiaire-tranche inférieure. la plupart des pertes découlant de pandémies de grippe sont dues à des événements rares et graves. требуется увеличение инвестиций в подготовку к борьбе с крупными эпидемиями и пандемиями. аргументы в пользу таких инвестиций в значительной степени основаны на оценках потерь в национальном доходе, которые могут возникнуть в результате крупной эпидемии или пандемии. недавно авторы расширили эту оценку, включив в нее количество людей, погибших в результате увеличения смертности, связанной с пандемией. это привело к более высоким оценкам полного ущерба, который может возникнуть в результате будущей пандемии. авторы параметризовали функцию вероятности превышения смертности для глобальной пандемии гриппа и подсчитали, что прогнозируемое число смертей от гриппа и пандемии составляет около 720 000 в год. мы подсчитали, что ожидаемые ежегодные потери из-за риска пандемии составляют около 500 млрд долларов сша (или 0,6% мирового дохода) в год. эта цифра находится в пределах оценки (ближе к нижней границе), полученной межправительственной группой экспертов по изменению климата для оценки риска глобального изменения климата, которая составляет от 0,2 до 2% от глобального дохода. предполагаемый процент годового национального дохода, представленный ожидаемой величиной потерь, варьировался по группам стран в зависимости от уровня дохода: от немногим более 0,3% в странах с высоким уровнем доходов до 1,6% в странах с низкими и средним доходом. большинство потерь от пандемии гриппа происходят по причине редких тяжелых явлений. hay una necesidad no satisfecha de invertir más en la preparación para grandes epidemias y pandemias. los argumentos a favor de dicha inversión se basan, en gran parte, en las estimaciones de las pérdidas en los ingresos nacionales que podrían darse como resultado de una gran epidemia o pandemia. recientemente, ampliamos el cálculo para incluir la valoración de las vidas perdidas como resultado del aumento de la mortalidad relacionado con la pandemia. esto dio como resultado unas estimaciones notablemente más altas del valor de la pérdida que podría resultar de una futura pandemia. hemos parametrizado una función de probabilidad de excedencia para una pandemia de gripe mundial y estimado que el número esperado de muertes causadas por una pandemia de gripe es de aproximadamente 720 000 por año. calculamos que las pérdidas anuales esperadas del riesgo de pandemia son de unos 500 000 millones de dólares estadounidenses, o el 0,6 % de los ingresos mundiales, por año. esta estimación se encuentra dentro, pero cerca del mínimo, de las estimaciones del panel intergubernamental del cambio climático sobre el valor de las pérdidas por el calentamiento global, que oscilan entre el 0,2 % y el 2 % de los ingresos globales. el porcentaje estimado de los ingresos nacionales anuales representado por el valor esperado de las pérdidas varió según la agrupación de ingresos del país: de poco más del 0,3 % en los países con ingresos altos al 1,6 % en los países con ingresos medios o bajos. la mayoría de las pérdidas por pandemias de gripe provienen de casos raros y severos. assessment of economic vulnerability to infectious disease crises from panic and neglect to investing in health security: financing pandemic preparedness at a national level financing of international collective action for epidemic and pandemic preparedness in search of global governance for research in epidemics global macroeconomic consequences of pandemic influenza. sydney: lowy institute for international policy the economic impact of pandemic influenza in the united states: priorities for intervention. emerg infect dis economic losses of pandemic risk victoria y fan et al total economic consequences of an influenza outbreak in the united states. risk anal valuation of the risk of sars in taiwan. health econ on sars type economic effects during infectious disease outbreaks. washington: world bank world bank evaluating the economic consequences of avian influenza. washington: world bank the loss from pandemic influenza risk the cost of air pollution. health impacts of road transport valuing mortality risk reductions from environmental, transport, and health policies: a global metaanalysis of stated preference studies global health 2035: a world converging within a generation the income elasticity of the value per statistical life: transferring estimates between high and low income populations boston: air worldwide pandemics: risks, impacts, and mitigation the annual impact of seasonal influenza in the us: measuring disease burden and costs. vaccine direct medical cost of influenza-related hospitalizations in children economic impact of influenza. the individual's perspective economic costs of influenza-related work absenteeism. value health the cost of influenza in thailand the macroeconomic impact of pandemic influenza: estimates from models of the united kingdom, france, belgium and the netherlands the possible macroeconomic impact on the uk of an influenza pandemic influenza cost and cost-effectiveness studies globally-a review the value of individual and societal risks to life and health during the ' decade of vaccines, ' the lives of 6.4 million children valued at $231 billion could be saved. health aff (millwood) the economic impact of rheumatic heart disease in developing countries geneva: world health organization the next influenza pandemic: can it be predicted? jama a history of influenza the chronicle of influenza epidemics introduction to pandemic influenza through history modelling a modern-day spanish flu pandemic the population of india and pakistan influenza in india 1918: excess mortality reassessed estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis observations on mortality during the 1918 influenza pandemic characterizing the amount and speed of discounting procedures expert judgments of pandemic influenza risks preparing for the next pandemic climate change temperature impacts on economic growth warrant stringent mitigation policy. nat clim chang updating estimation of the social cost of carbon dioxide global problems, smart solutions: costs and benefits using and improving the social cost of carbon economic aspects of global warming in a post-copenhagen environment the neglected dimension of global security: a framework to counter infectious disease crises the neglected dimension of global security -a framework for countering infectious-disease crises we thank peter sands and bradley chen. vyf has a secondary appointment with harvard t h chan school of public health, boston, united states of america. key: cord-324007-hapzf0fl authors: mcgeer, allison j. title: diagnostic testing or empirical therapy for patients hospitalized with suspected influenza: what to do? date: 2009-01-01 journal: clin infect dis doi: 10.1086/591852 sha: doc_id: 324007 cord_uid: hapzf0fl accumulating evidence supports the use of specific diagnostic tests and antiviral therapies for seriously ill patients with influenza. among available diagnostic tests, reverse-transcriptase polymerase chain reaction is faster than culture and more sensitive than commercial antigen assays. current neuraminidase inhibitors were approved on the basis of their efficacy in ambulatory patients, but seriously ill patients who receive these agents are less likely to die, even when treatment is initiated >48 h after symptom onset. for patients hospitalized with suspected influenza, it is unclear which circumstances warrant diagnostic testing and which warrant the use of empirical therapy. rapid antigen assays may reduce the unnecessary use of other tests and medications but are relatively insensitive, thus eliminating many patients with influenza as candidates for treatment. empirical antiviral therapy ensures that all patients receive treatment promptly, at a cost equivalent to that of diagnostic tests alone, but results in the receipt of treatment by many patients without influenza. for patients hospitalized with suspected influenza, clinicians need to combine these approaches in order to optimize patient care. trials of early therapy (!48 h after symptom onset) with neuraminidase inhibitors (nais) in healthy adults and children. these trials demonstrated that therapy was associated with a significant reduction in the duration and severity of illness and a 40%-60% reduction in the percentage of patients who developed complications or required hospitalization [2] [3] [4] [5] [6] [7] . similar reductions in complications and hospitalization associated with early therapy with oseltamivir have been identified in subsequent observational cohort studies of nursing home residents during influenza outbreaks, in 2 studies in which administrative databases were examined, and in cohort studies of immunocompromised patients [5, [8] [9] [10] . however, there is a difference between the early treatment of otherwisehealthy outpatients and the treatment of patients who require hospital admission. the question becomes, what data do we have on the impact of antiviral therapy for the treatment of influenza in patients requiring hospitalization? there currently are 3 cohort studies that have examined the impact of treatment on patients with community-acquired illness severe enough to warrant hospitalization. in a cohort study by falsey et al. [11] [12] [13] , viral testing was done systematically for patients admitted to rochester general hospital with underlying cardiopulmonary disease and respiratory tract infection, congestive heart failure, exacerbation of chronic lung disease, or acute respiratory viral illness during 4 winter seasons, from 1999 to 2003. of the 193 patients with influenza, 53 received treatment with amantadine or rimantadine, and 15 received treatment with nais. in-hospital mortality among these patients was 6%. the investigators were unable to find an effect of treatment with antiviral drugs. however, it is important to recognize that the study was not powered to detect a clinically significant difference: an analysis based on a cohort of 200 patients has !30% power to detect a 50% reduction in mortality. furthermore, the authors indicated that it appeared that more severely ill patients were more likely to be treated with an antiviral drug, a bias that would decrease the probability of finding a treatment effect. in a retrospective study of adult patients with influenza who were admitted to the prince of wales hospital in hong kong during the 2004-2005 influenza season with fever and respiratory and systemic symptoms, a clinically and statistically significant reduction in length of hospital stay was associated with treatment with oseltamivir [14] . in north america, less severely ill patients would have been screened from this cohort and sent home, but, in hong kong, most patients with fever and respiratory and systemic symptoms were admitted to a hospital regardless of disease severity and were screened for severe acute respiratory syndrome coronavirus and other respiratory viruses. patients who had been symptomatic for р2 days received empirical therapy with oseltamivir; patients who had been symptomatic for у2 days received treatment at the discretion of their physician. in this study, a total of 356 patients were admitted to the hospital, and 257 received treatment with oseltamivir. of those receiving treatment, 161 received treatment within 2 days of symptom onset. patients who received treatment experienced a median reduction in their length of hospital stay of 2 days-an ∼30% reduction-relative to that of patients who did not receive treatment or who received treatment 12 days after symptom onset ( ) [14] . p ! .0001 further support for the use of specific antiviral therapy for hospitalized patients comes from data collected prospectively in a cohort study of patients in toronto, which showed a surprisingly large reduction in mortality even when therapy was started 148 h after symptom onset [15] . this study correlated mortality with specific antiviral therapy over 2 influenza seasons (2004-2005 and 2005-2006) in toronto. of 327 adult patients with laboratory-confirmed influenza who were admitted to a hospital, 106 patients (32%) received treatment with oseltamivir. overall in-hospital mortality in this cohort was 10.7%. although the observation of a treatment effect was not anticipated, owing to the small sample size, patients who received treatment with oseltamivir had a risk of death (or) of 0.21 ( ), corresponding to a point estimate of a 79% reduc-p p .03 tion in mortality, compared with patients who did not receive treatment. given the limitations of this study's methodology, it is not possible to state unequivocally that oseltamivir treatment reduces mortality among patients admitted to a hospital with influenza. on the other hand, because of the established treatment effect in healthy outpatients, the apparent magnitude of the treatment effect in compromised and seriously ill patients, and the established safety of oseltamivir, a placebo-controlled trial to determine the efficacy of antiviral therapy for the treatment of severe influenza may no longer be ethically justifiable. of note, over the 3 seasons of surveillance in the toronto invasive bacterial diseases network (tibdn) study, about onethird of patients were hospitalized within 48 h of symptom onset. in addition, the treatment effect seen in this cohort was not different for patients treated р48 h or 148 h after symptom onset. these data confirm the findings of ison et al. [16] , who demonstrated that hospitalized patients receiving treatment with rimantadine and zanamivir shed influenza virus for several days after hospital admission. both the fact that one-third of patients present early and the fact that treatment may be effective when initiated 148 h after symptom onset in hospitalized patients emphasize how important it is to improve our understanding of severe influenza illness. another interesting finding from the tibdn study is that, during the 2006-2007 influenza season, 2 of 21 patients admitted to an intensive care unit after out-of-hospital cardiac arrest tested positive for influenza [15] . this raises a concern that influenza may trigger ventricular arrhythmias and sudden death and supports the results of a number of cohort studies suggesting that influenza vaccination is protective against sudden death. this accumulating evidence for a treatment effect suggests that it is prudent to establish a policy of antiviral treatment for patients who are seriously ill with influenza. such a policy will have a low risk of adverse events and a low risk of increasing selective pressure for the development of resistant strains of influenza virus. oseltamivir has no proven serious adverse effects [6, 7, 17] . the neurobehavioral adverse events reported primarily in japanese adolescents may have been an effect of either influenza or treatment; only further study will resolve this question. reassuringly, the rate of neurobehavioral adverse events reported to the us food and drug administration by japan is !1 case per 100,000 prescriptions [18] . for patients within the age range typically admitted to an intensive care unit, that rate is probably low enough to be of very limited clinical relevance. with regard to increasing selective pressure for the development of resistant virus strains, it is important to remember that nais are active against only influenza virus. if a patient treated with nais does not have influenza, no selective pressure is being applied, and antiviral resistance will not be increased in the patient population. this differs from the effect of empirical antibiotic therapy: every time an antibiotic is used, selective pressure is applied to the host's normal flora, whether or not the antibacterial is active against the pathogen being targeted. past influenza-management guidelines have not offered much guidance regarding antiviral treatment for patients admitted to a hospital. nonetheless, recent guidelines suggest that antiviral therapy should be offered to patients with severe illness and to those who are most likely to develop complications and/ or to die. for example, the association of medical microbiology and infectious diseases canada/canadian pediatric society recommends antiviral treatment for individuals with severe illness and for those most likely to develop complications from influenza or to die prematurely as a result [19] . the american academy of pediatrics recommends antiviral treatment for high-risk children and for other children with moderate to severe disease [20] . italian guidelines recommend prioritizing therapy for patients at risk of complications, ensuring that patients take the drug as early as possible, and using antiviral therapy only at the seasonal peak of influenza prevalence [21] . a decision to treat with antiviral therapy requires that physicians either rapidly make a reasonably definitive diagnosis of influenza or choose to treat empirically when the probability of influenza is above a certain threshold. the former is obviously preferable, when possible. among adults, acute respiratory illness with fever and early cough has a positive predictive value for influenza of 170% during influenza season [22] . however, many adults, particularly those who are elderly or those who have a significant underlying illness, do not mount an adequate febrile response and may not present with early cough. thus, clinical features alone cannot be used to diagnose influenza. commonly available diagnostic tests for influenza are shown in table 1 [23] [24] [25] [26] . of the available laboratory tests, rt-pcr is preferred for its speed, sensitivity, and specificity but is not currently available to a majority of clinicians. in toronto, most hospital laboratories provide eia testing. on average, eia sensitivity is 50%-70%, compared with that for viral culture, and specificity is ∼95%, depending on the laboratory and the test. however, the use of viral culture as the gold standard for sensitivity may be outdated. table 2 compares the sensitivity of viral culture to that of rt-pcr [26] [27] [28] [29] . the proportion of virus detected by culture in the different studies ranges from 50% to 90%, or ∼70% for purposes of estimation. thus, actual rapid eia sensitivity is not 60% but is closer to 60% of 70%, which is ∼42%. other rapid tests with sensitivities ranging from 24% to 90%, relative to that for viral culture, share the same limitation [24] . the lack of a rapid and sensitive clinical diagnostic test for influenza is problematic. although a number of new and sensitive molecular techniques are being investigated, such tests will not become available for several years [30] . given the limitations of currently available diagnostic tests, empirical therapy is an option worth considering. empirical therapy has the advantage of offering earlier treatment, which is likely to be more effective. it also may be the most costeffective option, because laboratory testing actually may be more expensive than therapy (which costs ∼$60 for a 5-day course). however, empirical therapy has the disadvantage of resulting in many more patients receiving treatment than actually have influenza. the prevalence of influenza in some recent patient cohorts is shown in table 3 [11, 15, 28, 29] . these studies looked at data from different groups of patients, in different years and at different times of year. seasonal and yearto-year variations in the underlying incidence of influenza explain much of the difference observed. overall, these studies suggest that, during most influenza seasons, 10%-15% of adult patients with pneumonia and/or febrile respiratory illness are likely to have influenza virus infection [29] . thus, empirical therapy will result in 5-15 patients without influenza receiving treatment for every patient with influenza. this ratio is similar to that reported in recommendations for the use of empirical therapy for atypical bacterial infection in pneumonia and, thus, is worthy of consideration [31, 32] . on the other hand, the testing of patients provides infor-mation to clinicians that enables more-directed therapy. evidence from both pediatric and adult studies indicates that testing for influenza results in reduced use of antibiotics and possibly reduced use of some other diagnostic tests [12, 33, 34] . such observations suggest that the information is immediately useful to clinicians. however, false-positive test results occur and may mislead clinicians. the benefits and limitations of laboratory diagnostic testing versus empirical antiviral therapy for influenza are summarized in table 4. as a practical matter, each infectious disease specialist must weigh the uncertainties of diagnosis and the effects of treatment to determine the best option for each patient under his or her care. the accumulating evidence suggests that, for patients with acute cardiorespiratory illness requiring hospital admission during influenza season, consideration should be given to either prompt laboratory diagnostic testing and treatment for influenza virus-infected patients or empirical antiviral therapy for influenza. the best choice is made on a case-by-case basis and depends on the severity of illness in the patient being admitted (since earlier therapy for pneumonia is more effective), the probability of influenza virus infection in the individual patient, and the sensitivity of the rapid diagnostic tests available. it is hoped that the introduction of rt-pcr testing into hospital laboratories and the accumulating information from cohort studies and trials of antiviral therapy among severely ill patients with influenza will soon result in a better understanding of effective diagnosis and therapy and in improved outcomes for severely ill patients. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) use of oseltamivir during influenza outbreaks in ontario nursing homes risk of pneumonia and other complications of influenza-like illness in patients treated with oseltamivir effect of oseltamivir on the risk of pneumonia and use of health care services in children with clinically diagnosed influenza impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations symmonds-abrahams m. neuraminidase inhibitors for preventing and treating influenza in children neuraminidase inhibitors for preventing and treating influenza in healthy adults prevention and treatment of influenza in highrisk groups: children, pregnant women, immunocompromised hosts, and nursing home residents safety and tolerability of oseltamivir prophylaxis in hematopoietic stem cell transplant recipients: a retrospective case-control study use of oseltamivir to control influenza complications after bone marrow transplantation respiratory syncytial virus infection in elderly and high-risk adults impact of rapid diagnosis on management of adults hospitalized with influenza pulmonary complications of interpandemic influenza a in hospitalized adults factors associated with early hospital discharge of adult influenza patients antiviral therapy and outcomes of influenza requiring hospitalization in ontario safety and efficacy of nebulized zanamivir in hospitalized patients with serious influenza the safety of oseltamivir in patients with influenza: analysis of healthcare claims data from six influenza seasons post-marketing adverse event reports review of central nervous system/psychiatric disorders associated with the use of tamiflu the use of antiviral drugs for influenza: recommended guidelines for practitioners antiviral therapy and prophylaxis for influenza in children italian guidance on antiviral use for management and prevention of seasonal influenza [abstract p1318 does this patient have influenza? influenza (flu): influenza symptoms and laboratory diagnostic procedures role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics laboratory diagnosis of human seasonal and pandemic influenza virus infection testing of diagnostic methods for detection of influenza virus for optimal performance in the context of an influenza surveillance network diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza incidence and characteristics of viral community-acquired pneumonia in adults surveillance for laboratory confirmed influenza requiring intensive care unit admission in toronto, canada expert consultation on diagnosis of h5n1 avian influenza infections in humans a worldwide perspective of atypical pathogens in community-acquired pneumonia treatment of community-acquired pneumonia-idsa guidelines impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial the impact of rapid antigen testing for influenza on paediatric hospital admissions in a large uk emergency department: a retrospective observation financial support. biocryst pharmaceuticals, inc., provided educational grant support to develop this article and the symposium on which it is based, "antiviral therapy for influenza: challenging the status quo" (san diego), 4 october 2007.supplement sponsorship. this article was published as part of a supplement entitled "antiviral therapy for influenza: challenging the status quo," jointly sponsored by the institute for medical and nursing education and international medical press and supported by an educational grant from biocryst pharmaceuticals, inc.manuscript preparation. margery tamas of international medical press (atlanta) provided assistance in preparing and editing the manuscript.potential conflicts of interest. a.j.m. has served as a consultant to biocryst pharmaceuticals, inc., has received funding for investigator-initiated research under contract with hoffman-laroche and biocryst pharmaceuticals, inc., and is a member of the speakers' bureau for gilead pharmaceuticals. key: cord-327516-i25whxt2 authors: horby, peter title: improving preparedness for the next flu pandemic date: 2018-07-25 journal: nat microbiol doi: 10.1038/s41564-018-0206-7 sha: doc_id: 327516 cord_uid: i25whxt2 pandemic influenza remains the single greatest threat to global heath security. efforts to increase our preparedness, by improving predictions of viral emergence, spread and disease severity, by targeting reduced transmission and improved vaccination and by mitigating health impacts in lowand middle-income countries, should receive renewed urgency. g iven its potential to cause an acute global health crisis with many millions of deaths, pandemic influenza can rightly be considered the greatest single threat to global health security. yet despite this threat, influenza has recently been eclipsed in the popular and scientific consciousness by less common infections, such as those caused by ebola and zika viruses. the centenary of the 1918 influenza pandemic, which is estimated to have killed 50 million people, is an opportune time to remind ourselves that the greatest risks often lay in the mundane. much like we need reminding that driving is far more dangerous than flying, familiarity can breed contempt. the appearance of a human influenza pandemic depends on the emergence of a novel virus that can readily infect and transmit between people. the mostly likely source of a pandemic virus is the pool of influenza viruses that infect animals such as wild birds (the natural reservoir of influenza a viruses), domestic poultry and pigs. the scale of pig and poultry farming has increased massively over the past 50 years, with the estimated global number of pigs and chickens having increased roughly twofold and fivefold, respectively, from the early 1960s until now (from 400 million to around 1 billion pigs; and 4 billion to 20 billion chickens) 1 . swine influenza is endemic in pig populations, with co-circulation of multiple subtypes and the intermittent introduction of new strains from avian or human sources, whilst poultry populations are affected by an increasing variety of influenza viruses. human infections with a diverse range of zoonotic influenza viruses are now being detected ( fig. 1) , some of which are associated with a high case fatality rate, and some of which have mutations that confer resistance to the major classes of influenza antiviral drugs. although it is hard to be sure as surveillance and genetic sequencing capabilities have been increasing, we seem to be experiencing a notable increase in the genetic exchange and diversification of animal influenza viruses 2 . determining which of these viruses will cause the next pandemic, when it will happen and how bad it will be remains incredibly challenging. however, there are warning signs to be heeded and areas in which our preparedness could be strengthened to make sure that we are best placed to identify and swiftly confront the next influenza pandemic. influenza is probably one of the most studied viruses, yet fundamental gaps exist in our ability to predict the transmissibility and virulence of novel influenza viruses. the recent lifting by the us national institutes of health of their moratorium 3 on funding of so-called 'gain-of-function' experiments (laboratory experiments where viruses are genetically altered to assess the effect on properties such as transmissibility and virulence) is good news, as it allows us to explore the limits of evolution and the genotypic predictors of phenotype 4 . however, there are limits to the predictive value of laboratory experiments and animal models, and when a new pandemic virus does emerge, which inevitably it will, the most important measure will be the disease severity per infected person. this information is crucial for understanding the potential impact of the pandemic and appropriately calibrating the political and public health response. estimating the severity per infected person requires robust and real-time data on the number of people infected and the proportion within this group that develop severe disease 5 . such data are surprisingly hard to gather and interpret because care-seeking and care-giving behaviour can change as awareness of a pandemic and pressures on healthcare change. this is an area that requires investment in methodologies and tools for gathering the necessary input data, including potential modifying factors such as care-seeking behaviour, and feeding that data into analytic frameworks for estimating the severity per infected person. mathematical models of disease transmission have become a routine tool for evaluating and predicting the behaviour of epidemics. these models are attractive to public health officials as they provide a quantitative answer to many questions, but most importantly to: "how bad is this and how bad might it get?" a promising approach to even faster and more reliable characterization of outbreaks is 'modeldriven data collection' , which tells us "how much data to collect and when to collect it" in order to improve the predictive power of the models and to maximize the efficiency of data collection 6 . this concept should be tested by developing and piloting modeldriven data collection systems for seasonal or zoonotic influenza, to see what and how much data are needed to provide estimates of the severity per infected person that are sufficiently reliable to be actionable. the world population is around four times the size it was in 1918, and the mobility of this population is massively increased 4 . if a potential pandemic influenza virus acquires the ability to readily transmit between humans, it will spread with alarming speed and essentially become unstoppable. aeroplane transportation is the greatest facilitator of rapid global spread 7 , yet airport screening has limited ability to prevent importation and at best can lead to a short delay (less than two weeks) in the onset of local transmission 8, 9 . at a national level, measures to limit local transmission, such as school closure, can have some effect in reducing community transmission and can mitigate pressures on the healthcare system by reducing peak incidence. the effect is, however, dependent on timing and coordination and therefore requires access to real-time surveillance data, such as absenteeism 10, 11 . the simultaneous use of face comment masks and hand hygiene together, targeting both aerosol and contact transmission, can reduce spread under laboratory conditions, although the effectiveness of these measures as a community-wide intervention is unproven 12 . whilst neuraminidase inhibitors (nais) have been demonstrated to be effective in reducing symptomatic influenza and intra-household transmission when used prophylactically, there are no data on the effectiveness or cost effectiveness of nai use in reducing community-wide transmission 13 . therefore, the stockpiling of nais by individual countries could permit prophylactic use that may reduce the local impact of a pandemic, but at the global level nais are not likely to have a significant role in reducing pandemic influenza transmission. around half of the world's population lacks full access to essential health services 14 . it is therefore not surprising that the burden of influenza is greater in low-and middleincome countries (lmics) than in highincome countries. in the 2009 pandemic, the estimated death rate in africa was 2-4 times that of other regions 15 , whilst mortality rates for influenza-associated acute lower respiratory tract infection in children younger than 5 years are three times higher in low-income countries than in high-income countries 16 . the influenzaassociated death rate in the elderly is also likely to be substantially increased in lmics compared to higher-income countries 17 . this burden of influenza in lmics is often unrecognized, particularly in rural areas 18, 19 . any assessment of our ability to mitigate the health impact of the next influenza pandemic must consider what will be available for the large and vulnerable populations living in lmics. the therapeutic efficacy of nais is a matter of some debate, but can probably be summarized as a proven but modest effect on the duration of symptoms in patients with mild influenza, and a probable but unproven small reduction in severe outcomes such as pneumonia and death. given the weak evidence of an impact on severe outcomes, the nai oseltamivir has recently been 'downgraded' to the complementary part of the who essential medicines list, only to be used for critically ill patients hospitalized with influenza 20 . it is, therefore, hard to see nais having a significant global impact on the rate of hospitalization or death in any future pandemic. in addition to the weak evidence of an impact on severe outcomes, the existence of viral genetic variants that confer considerable resistance to nais make it clear that new antivirals for influenza are needed. there are candidates in clinical development but progress is slow, and companies have favoured evaluating new candidate drugs in patients with uncomplicated influenza, since this is the bigger market. what is needed is a large influenza clinical trials network that can evaluate multiple candidates and combination therapies (including antiviral combinations and host-directed therapies) in an adaptive platform trial, as has been achieved for cancer, for example with the i-spy2 platform trial 21 . however, at this time, interventions other than antivirals can probably offer more health benefit globally. the two simple interventions that can save lives are antibiotics and oxygen. secondary bacterial infections, largely due to streptococcus pneumoniae, are thought to have made a substantial contribution to influenzaassociated pneumonia and death in 1918 (ref. 22 ). despite the widespread availability of antibiotics, only around half of children with pneumonia in lmics receive care from a healthcare provider who can give appropriate antibiotics 23 . although oxygen therapy is considered an essential medicine, reliable access to oxygen is absent in much of the world due to technical and cost challenges 24 . the other intervention likely to have a major impact is pneumococcal conjugate vaccines (pcvs). these vaccines have been shown to reduce the risk of influenza-associated pneumococcal disease 25 , and an analysis in china suggests that routine pcv immunization prior to a pandemic would have a major impact on mortality and would be cost saving 26 . globally, only 37% of children received three doses of pcv in 2015, although coverage is actually higher in low-income than highincome countries due to support from agencies such as gavi, the vaccine alliance 27 . effective, safe and affordable interventions, such as antibiotics, oxygen and pcvs, will save lives during an influenza pandemic and are available now. such interventions should be made available to all. whilst current influenza vaccines meet an important need and their wider use should be promoted, traditional influenza immunization approaches and the predominant egg-based production methods seriously limit their value as a tool in pandemic preparedness. there has certainly been progress in the speed of development and breadth of candidate vaccine viruses, and in the diversification and expansion of production capacity -global pandemic influenza vaccine production capacity is at its highest level ever, and may be sufficient to immunize 43% of the current global population with two doses, or 86% with one dose 28 . nevertheless, the time frame to produce and evaluate new vaccines against novel antigenic variants and to switch vaccine production is still too slow to have a substantial impact on the first wave of a pandemic. only time will tell if the notional pandemic vaccine production capacity will ever be realized, and a pandemic vaccine will find its way into people's arms in time. substantial efforts are ongoing to optimize the use of existing vaccine technologies and to develop novel approaches, including, most importantly, work to realize the aspiration of a broadly protective, universal influenza vaccine. these efforts are of major global importance and must continue to be supported. if we do not keep our eye on the ball and continue to aggressively pursue improvements in pandemic influenza comment preparedness across the whole range of disciplines, including basic science, vaccinology, clinical evaluation and therapeutics, public health and health service delivery, we will be culpable of neglecting the obvious risks in favour of the more exotic and dramatic. whilst the recently updated world health organization public health research agenda for influenza has some value, it does not constitute a strategic plan of action. as we have seen being recently developed for other high-threat pathogens, such as the viral haemorrhagic fevers and middle east respiratory syndrome coronavirus, what is needed is a blueprint for action against pandemic influenza that is comprehensive, detailed, endorsed, funded and monitored. ❐ peter horby 1,2 notice announcing the removal of the funding pause for gain-offunction research projects vaccine trialist group the author declares no competing interests. key: cord-328290-kbysppgb authors: beckmann, christiane; hirsch, hans h. title: diagnostic performance of near-patient testing for influenza date: 2015-03-31 journal: j clin virol doi: 10.1016/j.jcv.2015.03.024 sha: doc_id: 328290 cord_uid: kbysppgb background: rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. direct antigen detection (dad) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (nat) is more sensitive, but also more time-consuming. objectives: to evaluate the performance of a rapid isothermal nat and two dads. study design: during february–may 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal nat (alere™ influenza a&b) as well as the dad sofia(®) influenza a + b and binaxnow(®) influenza a&b for detection of influenza-a and -b virus. respifinder-22(®) a commercial multiplex nat served as reference test. serial 10-fold dilutions of influenza-a and -b cell culture supernatants were examined. another 225 patient samples were tested during december 2014–february 2015. results: compared to respifinder-22(®), the isothermal nat alere™ influenza a&b, and the dad sofia(®) influenza a + b and binaxnow(®) influenza a&b had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. alere™ influenza a&b showed 85.7% sensitivity and 100% specificity in the second cohort. isothermal nat was 10-100-fold more sensitive compared to dad for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/ml. the average turn-around time (tat) of isothermal nat and dads was 15 min, but increased to 110 min for alere™ influenza a&b, 30 min for binaxnow(®) influenza a&b, and 45 min for sofia(®) influenza a + b, when analyzing batches of 6 samples. conclusion: simple sample processing and a tat of 15 min render isothermal nat alere™ influenza a&b suitable for sequential near-patient testing, but the tat advantage is lost when testing of larger series. community-acquired respiratory virus isothermal pcr turn-around time (tat) antigen detection nucleic acid amplification testing (nat) a b s t r a c t background: rapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. direct antigen detection (dad) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (nat) is more sensitive, but also more time-consuming. objectives: to evaluate the performance of a rapid isothermal nat and two dads. study design: during february-may 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal nat (alere tm influenza a&b) as well as the dad sofia ® influenza a + b and binaxnow ® influenza a&b for detection of influenza-a and -b virus. respifinder-22 ® a commercial multiplex nat served as reference test. serial 10-fold dilutions of influenza-a and -b cell culture supernatants were examined. another 225 patient samples were tested during december 2014-february 2015. results: compared to respifinder-22 ® , the isothermal nat alere tm influenza a&b, and the dad sofia ® influenza a + b and binaxnow ® influenza a&b had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. alere tm influenza a&b showed 85.7% sensitivity and 100% specificity in the second cohort. isothermal nat was 10-100-fold more sensitive compared to dad for influenza virus culture supernatants with a lower limit of detection of 5000-50,000 copies/ml. the average turn-around time (tat) of isothermal nat and dads was 15 min, but increased to 110 min for alere tm influenza a&b, 30 min for binaxnow ® influenza a&b, and 45 min for sofia ® influenza a + b, when analyzing batches of 6 samples. conclusion: simple sample processing and a tat of 15 min render isothermal nat alere tm influenza a&b suitable for sequential near-patient testing, but the tat advantage is lost when testing of larger series. © 2015 elsevier b.v. all rights reserved. rapid and sensitive diagnostic tests for influenza virus permit timely treatment decisions and infection control measures in emergency rooms, hospital wards, and nursing homes [1] [2] [3] . the current methods used for the laboratory diagnosis of influenza include virus isolation by cell culture, direct antigen detection (dad), and nucleic acid amplification testing (nat). virus isolation by cell culture permits identification of infectious virus with fairly high sensitivity, but is time-and resource-consuming and largely limited to specialized laboratories. dad has been widely used because of its short turn-around time (tat), but is limited by its lower sensitivity, especially in adult patients and those presenting several days after symptom onset [4, 5] . nat is characterized by high sensitivity and specificity, semi-quantification in real-time formats, and its amenability to automation and multiplexing [6] . however, nat requires a molecular diagnostic laboratory with skilled personnel, appropriate instrumentation, but rarely delivers results with tat of <6 h. to evaluate the performance of the isothermal pcr for influenza (alere tm influenza a&b) and two dads (sofia ® influenza a + b and binaxnow ® influenza a&b) versus a commercial multiplex nat (respifinder-22 ® ). between february 2014 and may 2014, we tested a first cohort of 211 consecutive nasopharyngeal swabs from patients with influenza-like illness by the isothermal nat alere tm [6, 8] . discordant samples were resolved by inhouse qnat for influenza [7] with a tat of 6 h. total nucleic acids were extracted from 200 ul of the respiratory sample using the corbett cas-1200 system (qiagen hilden, germany). in total, 57/436 samples (13.1%) were positive for influenza-a the performance of all three rapid tests compared to the reference method for influenza-a and -b combined is shown in table 1 . using multiplex nat as reference, alere tm influenza a&b, sofia ® influenza a + b, and binaxnow ® influenzaa&b had sensitivities of 82.3%, 59.3% and 29.6%, and specificities of 99.7%, 98.9% and 100%, respectively. specificity of the test could be confirmed with influenzanegative samples that had been tested with respifinder-22 ® (n = 373). multiplex nat detected 14 other pathogens in these samples including rhinovirus/enterovirus, coronavirus oc43, nl63, 229e and hku1, human bocavirus, rsv a and rsv b, adenovirus, parainfluenza-3, parainfluenza-4, human metapneumovirus and the bacteria bordetella pertussis and mycoplasma pneumoniae. none of the 11 false-negative samples were positive for another pathogen. overall, the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and ä values (interobserver agreement) for influenza-a and -b were slightly higher for specimens from pediatric patients (89.5%). the limit of detection for the tests was evaluated using serial dilutions of fresh cell culture supernatant of influenza-a h3n2 and h1n1 and influenza-b grown on llc-mk2 cells (fig. 1) . for influenza-a, the detection limit of the alere tm influenza a&b according to qnat was approximately 5,000 geq/ml (h3n2), 50,000 geq/ml (h1n1), and 10,000 geq/ml for influenza-b. overall, the sensitivity of both dads (sofia ® influenza a + b and table 1 performance the dilutions were directly tested using the alere tm influenza a&b (alere, triangles), sofia ® influenza a + b (sofia, circles), and binaxnow ® influenza a&b (binax, squares). in parallel, the dilutions were extracted and quantified by qnat as described in study design [7] . solid symbols, positive testing; open symbols, negative testing; lines, lower limit of detection of indicated rapid test; hatched area, limit of detection of the influenza qnat. binaxnow ® influenza a&b) were about 1-2 orders of magnitude lower than for alere tm influenza a&b. in our prospective study, the overall sensitivity and specificity of the alere tm influenza a&b assay was 82.3% and 99.7% for influenza. several factors may have played a role: 1. a large number of the samples (n = 211) from the first cohort were obtained towards the end of the influenza season between february and may 2014, which decreases the positivity rate and pretest probability. 2. 13/436 samples were analyzed retrospectively from frozen samples, but the manufacturer recommends using fresh samples. by excluding these 13 samples from analysis, the sensitivity increased to 86.5% and the specificity to 100%. possibly, tests using high-grade extraction-amplification procedures are less affected by testing frozen samples, even with lower amounts of influenza virus, than alere tm influenza a&b. of those 13 samples 5 resulted in discordant results with the alere tm influenza a&b assay, with viral qnat loads between 30 and 18,000 geq/ml. 3. influenza-a and -b from cell culture estimated the limit of detection of around 5,000 to 50,000 geq/ml for alere tm influenza a&b. therefore, patients with lower viral loads might not be identified. an independent conclusion about the assay performance. other studies report a sensitivity of alere tm influenza a&b of 73%-99% for influenza-a, 97%-100% for influenza-b; and specificity of 95%-100% for influenza-a, and 100% for influenza-b [9] [10] [11] . in cohort-1, alere tm influenza a&b yielded 1 false-positive result among 211 samples, and no false-positives among 225 samples of cohort-2. the rate corresponds to 0.23% or 1 among 436 symptomatic patients, which would be unjustifiably treated or isolated. conversely, antiviral treatment would be withheld in 373 patients tested negative. while the data suggest that testing of patients improves timely delivery of antivirals, 15 per 100 infected would not receive this treatment. the clinical impact may differ depending on the clinical scenario: hospitalized patients are likely to be confirmed during the course of their disease, whereas outpatients are less likely to be re-tested unless symptoms persist. the current study indicates that isothermal nat as provided by the alere tm influenza a&b is significantly faster than conventional qnat (15 min versus 6-8 h) or multiplex nat (16 h) and comparable to dad. as the sample preparation is equally simple, the alere tm influenza a&b could potentially be performed outside of the molecular diagnostic laboratory setting e.g., in a physician's office or in the emergency room. this could save transport time to the laboratory. however, care needs to be taken that the individuals operating the system are properly trained and certified. in the single sample testing, however, running larger numbers of more than 6 patient samples becomes challenging, and the tat advantage of isothermal pcr is lost. taken together, the alere tm influenza a&b can reduce the tat compared to multiplex nat or qnat. the sensitivities and specificities were comparable to conventional nat reference tests and superior to the two dad assays. the alere tm influenza a&b may be attractive for first-line rapid testing of individual patients presenting to health care, followed by qnat or multiplex nat, when information on relative amounts or the presence of other viruses is needed. none. none declared. not required. rapid diagnosis of influenza infection in older adults: influence on clinical care in a routine clinical setting impact of rapid diagnosis on management of adults hospitalized with influenza ecil-4): guidelines for diagnosis and treatment of human respiratory syncytial virus, parainfluenza virus, metapneumo virus, rhinovirus, and coronavirus rapid influenza diagnostic test use and antiviral prescriptions in outpatient settings pre-and post-2009h1n1 pandemic influenza viruses: update on epidemiology, clinical features, treatment and vaccination comprehensive diagnostics for respiratory virus infections after transplantation or after potential exposure to swine flu a/h1n1: what else is out there? outcome of influenza infections in outpatients after allogeneic hematopoietic stem cell transplantation a retrospective analysis of nosocomial viral gastrointestinal and respiratory tract infections multicenter clinical evaluation of the novel alere i influenza a&b isothermal nucleic acid amplification test evaluation of the alere i influenza a&b nucleic acid amplification test by use of respiratory specimens collected in viral transport medium evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b we thank our colleagues of the division infection diagnostics and the technicians of the molecular diagnostics and virus isolation laboratories for support and assistance. key: cord-290100-wnjjqqn5 authors: wong, samuel y.s.; kung, kenny; wong, martin c.s.; wong, carmen; tsui, wendy; chan, king; liang, jun; lee, nelson l.s.; cheung, annie w.l.; wong, eliza l.y. title: primary care physicians’ response to pandemic influenza in hong kong: a mixed quantitative and qualitative study date: 2012-07-11 journal: int j infect dis doi: 10.1016/j.ijid.2012.03.015 sha: doc_id: 290100 cord_uid: wnjjqqn5 objectives: the current study was conducted to use a developed framework to appraise the public primary care response to pandemic 2009 influenza a h1n1 virus in hong kong in 2009. methods: a cross-sectional survey was conducted of 300 doctors working in public primary care clinics. in addition, a qualitative study was conducted in two selected general outpatient clinics (gopcs) with 10 doctors between september and december 2009. results: we found that there was an increase in clinical service demand for public primary care doctors and that there was lower compliance with hand washing as compared to the wearing of masks among gopc doctors during the study period. conclusions: since hand hygiene and influenza vaccination are effective methods to prevent the spread of influenza infection, future studies should explore the reasons for non-compliance with these preventive behaviors among doctors. more education and training in dealing with influenza a h1n1 infection may be needed. in april 2009, the influenza a h1n1 virus surfaced and spread rapidly across the globe. on june 11, 2009 , the world health organization (who) declared a pandemic caused by the influenza a h1n1 virus. in any healthcare system, primary care is at the forefront of the response to any emerging epidemic. since the outbreak of severe acute respiratory syndrome (sars), 1 there has been a growing recognition of the need for an integrated preparedness approach to deal with public health threats, to include acute clinical care, public health, and emergency management systems. 2 preparing for health threats is particularly important in primary care -the first point of contact for patients entering the healthcare system. in 2008, patel et al. 3 developed a framework that can be used to facilitate the systematic planning of the primary care response to pandemic influenza and to appraise the coverage of key elements in the preparedness to deal with a pandemic. this framework consists of four functional domains that include clinical care, the internal and macro-environment of the primary care/general practice, and the public health responsibilities of doctors. these functional domains were first identified by the authors through a review of the peer reviewed and gray literature that included strategies relevant to general practice at the time of an influenza pandemic, and the framework was later validated through interviews with general practitioners and practice nurses and senior decision-makers. it was subsequently used to evaluate 89 publicly available jurisdictional plans in five countries. 3 since the provision of primary care is organized differently in different countries, studying how each primary care system responds to a pandemic may generate transferable learning for other primary care systems. recent research has studied the knowledge, attitudes, and practices, or vaccination acceptability in dealing with an influenza a h1n1 pandemic among primary care practitioners in singapore, 4 australia, 5 france, 6 and the netherlands. 7 however, few studies have been conducted using a previously developed framework to evaluate the response of primary care to an influenza a h1n1 pandemic before the escalation of the pandemic alert and during the influenza a h1n1 pandemic. the current study was conducted to use a developed framework to appraise the public primary care response to pandemic 2009 influenza a h1n1 virus in hong kong in 2009. to better understand the responses of primary care doctors to the influenza a h1n1 pandemic, both a quantitative survey and a qualitative study were conducted. the peak activity of the pandemic h1n1 virus in hong kong occurred during july through september 2009. this was also the time when our survey study was conducted. from july 20, 2009 to august 18, 2009 , 300 questionnaires were mailed to doctors who worked in 54 general outpatient clinics (gopcs) distributed across five major geographical clusters in hong kong: 11 in the new territories east cluster, 8 in the new territories west cluster, 6 in the kowloon central cluster, 23 in the kowloon west cluster, and 6 in the hong kong west cluster. in hong kong, the hospital authority manages all government run gopcs in primary care in seven geographic clusters. the role of the gopcs is to provide access to quality clinical care in the form of primary care services to the financially vulnerable, the elderly, and patients with chronic diseases. most gopcs are located in the community and are often the first point of contact with public clinics among the elderly and the financially vulnerable. ethics approval for this study was granted by the survey and behavioural research ethics committee of the chinese university of hong kong. using an anonymous questionnaire, primary care doctors who worked in these clinics were asked to report four domains of primary care practice with respect to a published framework for planning to cope with pandemic influenza in primary care. the questionnaire was developed to include five major domains of primary care practice that have been found to be relevant in the planning to manage pandemic influenza. these include: (1) changes in clinical services and clinical care for influenza; (2) changes in the internal environment of primary care practice such as preventive behaviors of doctors, including hand washing and wearing a mask; (3) changes in the macro-environment of primary care practice such as the use of guidelines, training, or measures; (4) public health responsibilities such as in primary care; and (5) the impact of influenza a on quality of life, assessed using three questions: ''has influenza affected the quality of your life?'', ''did you feel depressed in the past 2 weeks?'', and ''did you feel emotionally stressed in the past 2 weeks?''. the questionnaire had a multiple choice design and the respondents could tick one or more of the alternatives. moreover, the primary care practitioners were asked about whether they would be willing to be vaccinated when a vaccine was available. the survey also included questions on demographics such as age and sex, as well as educational background, postgraduate qualifications, and the type of clinic the doctor worked in. a sample of the questionnaire is provided as supplementary material. the questionnaire was piloted on 30 primary care practitioners before the actual distribution, and modifications were made as a result of the comments and suggestions received from the primary doctors. to further explore the responses of primary care physicians to pandemic influenza in hong kong, individual interviews were conducted among physicians at two randomly selected gopcs. the interviews were conducted using a discussion guide covering two areas: workload during pandemic influenza and the acceptance of influenza vaccination. to allow a greater expression of views on these sensitive issues, individual interviews were used instead of focus groups. ten individual interviews were conducted in the selected gopcs until data saturation was reached. descriptive statistics are presented. chi-square tests were used for the analyses of categorical variables, and analysis of variance was used for continuous variables. an analysis was performed to explore the relationships between having encountered patients with suspected influenza a h1n1 and the responses to the five domains in the clinical services questionnaire using chi-square statistics. all statistical analyses were performed using spss for windows v. 16 .0 (spss, chicago, il, usa), and the level of significance was set at 5%. for the qualitative study, the content of individual interviews was transcribed and coded using nvivo 7.0. data were analyzed by two independent researchers (elyw and awlc) based on the discussion guide. during the analysis, data within themes were scrutinized for agreement in views across the range of participants. of the 300 questionnaires sent to the primary care doctors of gopcs, 126 were completed and returned, giving a response rate of 42%. demographic information for the doctor respondents is shown in table 1 . since there is no primary care registry in hong kong, we were unable to compare the demographic information of the respondents to those of the rest of the primary care doctors in hong kong. when compared to the findings of the 2009 health manpower survey on all registered doctors, we found a higher proportion of female doctors in our survey than in the health manpower survey (57% male and 43% female as compared to 72% male and 28% female). the mean age of doctors in our survey was 36 years as compared to a median age of 45 years in the health manpower survey. in our survey, 96% of doctors worked full time and 77% had a postgraduate qualification, with 55% having a diploma in family medicine, 10% a masters in family medicine, and 44% a fellowship in family medicine. with respect to changes in clinical services, 59% of participating gps noticed a higher demand for services. moreover, the majority (86%) stated that influenza a h1n1 had affected their clinical practice; 88% of these doctors reported testing patient temperature as a routine procedure and 76% insisted every patient wear a mask during consultations. among all respondents, 63% stated that they had encountered a patient suspected of influenza a h1n1. among the suspected cases, around 20% were laboratory confirmed. with respect to changes in the internal environment of practice, 94% of participants stated that they always wore a mask during consultations before the influenza a h1n1 epidemic and this percentage increased to 99% during the epidemic. similarly, 45% of participants stated that they washed their hands between or before patient encounters before the epidemic, while at the time of the survey (during the pandemic), 54% of them stated that they washed their hands between or before patient encounters (p < 0.001). other precautions, in addition to hand washing and the wearing of a mask, included asking all cleaning staff to wear masks (76%), cleaning the work surfaces with antiseptics (70%), and asking staff to check their temperature before going to work (60%). moreover, 73% of the clinics required nursing/reception staff to wear masks before the epidemic, and during the pandemic, 97% of clinics required reception staff to wear masks. among the respondents, 42% stated that they would have the influenza a h1n1 vaccination when it was available. the majority stated that they would not have the influenza vaccination and the reasons given for this were: (1) they did not trust the effectiveness of the new vaccine (57%), and (2) they did not think it is necessary (30%). the majority of participating doctors stated that the intranet and other communication channels at the clinics they worked for (56%) were their most likely sources of updated information on influenza a h1n1, followed by correspondence from the government or semi-government organizations (38%). with respect to the macro-environment of primary care practice, 71% of doctors who participated in this survey used guidelines to assist them in making clinical decisions and 56% of these doctors had received training on the use of guidelines. however, 62% continued to want more professional education regarding how to deal with h1n1 influenza. the majority of doctor participants were satisfied with the measures the government had implemented to prevent influenza a h1n1 from spreading in the community and more than half would have liked to have had more involvement in the management of influenza a h1n1 in the community (57%). with respect to public health responsibilities, more than half (59%) of the doctors had not participated in surveillance activities associated with acute respiratory infections. among those who had, 58% reported suspected cases of influenza a h1n1 to the government. on a scale of 0-100 for 'not affected at all' to 'extremely affected', around 29% of doctor respondents scored 50 or above on the scale with the question, ''has influenza a h1n1 affected the quality of your life?''. on a scale of 0-100 for 'not depressed at all' to 'extremely depressed', around 14% of doctor respondents scored 50 or above on the scale with the question, ''did you feel depressed in the past 2 week?''. on a scale of 0-100 for 'not stressed at all' to 'extremely stressed', 27% of doctor respondents scored 50 or above on the scale with the question ''did you feel emotionally stressed in the past 2 weeks?'' 3.1.6. relationships between having encountered patients with suspected influenza a h1n1 and the five domains of practice in our analyses on the relationships between having encountered patients with suspected influenza a h1n1 and clinical service (table 2) , a significantly higher proportion of doctors who had encountered patients with suspected influenza a reported seeing more patients than those who had not encountered such patients (71% vs. 38%; p < 0.001). they were also more likely to have used guidelines when making clinical decisions (77% vs. 60%; p = 0.055) and were more likely to have participated in surveillance activities (54% vs. 16%; p < 0.001). in addition, they were also more likely to have felt emotionally stressed in the past 2 weeks when compared to those who had not encountered a suspected influenza a infection (mean score: 38.3 vs. 22.4; p = 0.001). in terms of training, they were more likely to have a postgraduate qualification (83% vs. 67%; p = 0.052) and were more likely to be a family medicine specialist (47% vs. 26%; p = 0.055). twelve individual interviews were conducted among physicians in two selected gopcs between september and december 2009. two themes were explored: increased workload and attitudes towards influenza vaccination. the majority commented on the increased workload and the long working hours during the influenza pandemic, because they had to work on current pre-booked chronic cases and also new fever cases. in addition, there was no quota limit for 'walk in fever case'. a typical comment was as follows: ''though the office hour is until 5 p.m. but there are still a lot of patients lining up at 5 p.m., we must accept them. . .unlimited quota but limited manpower. . .'' (c1) also, some physicians revealed that they were too busy to wash their hands between patient consultations: ''there are many patients walking in for fever consultation, the clinic is open during lunch time as well. . .and we are even too busy to wash hand. . .'' (c3) the majority of the physicians agreed to having an influenza vaccination, which was regarded as a healthcare professional's responsibility. however some participants hesitated to receive the swine flu vaccination because of queries regarding efficacy and side effects. some expressed the view that the swine flu vaccination was not necessary because they had already contracted swine flu: ''i will receive the vaccination because it is healthcare professional's responsibility. . .to protect ourselves. . .to protect public. . .'' (c7) ''vaccination for swine flu is still new. . .i will wait to see the efficacy and side effect from more clinical trial'' (c11) ''vaccination may not be necessary. . .because some of us have been contracted from swine flu already. . .even my family members have contracted already'' (c33) in this survey, we found that a significant proportion of public doctor respondents reported an increase in workload as a result of the influenza a h1n1 pandemic. indeed, one fifth of them stated that they had increased their office hours to cope with the higher demand for services. with regard to changes in the internal environment of practice, the majority of doctors (99%) who responded to the survey reported that they had always worn a mask during consultations in the past 3 days, although the proportion who had always washed their hands between/before consultations in the past 3 days was much lower (54%). the lower proportion of doctors who reported always having washed their hands between/before consultations in the past 3 days could be a concern, as previous studies have demonstrated that hand hygiene is an effective method to prevent the transmission of influenza and that hand washing is one of the effective methods to reduce an influenza pandemic. [8] [9] [10] the reasons for the lower self-reporting of hand washing between patients when compared to wearing masks are unknown, but we can speculate that this could be due to the fact that more time and effort is required for washing hands between patients as compared to wearing a mask. this is further supported by findings from our qualitative study, which showed that doctors might have been too busy to wash their hands between patients. almost half of the respondents (42%) reported that they were willing to have the influenza a vaccination, and for the other respondents the two most common reasons for refusal to have the vaccine were not trusting the effectiveness of the new vaccine and not thinking it necessary. these findings were further confirmed by the data from our qualitative study. moreover, the additional reason of thinking that they had already been infected with influenza a h1n1 might have further reduced the willingness to accept the influenza a h1n1 vaccination. these findings among doctors in our study are similar to those recently reported in hong kong among hospital healthcare workers, which showed community nurses having the lowest willingness to accept vaccination. 11 starting in 2010, all seasonal influenza vaccines have included the swine flu component. whether this will help to ease some fear is unknown. of note, current available surveillance data suggest no increase in adverse events with swine flu vaccines compared to seasonal flu vaccines. 12 acceptability of pandemic influenza vaccination was found to be higher in studies conducted in general practice in france and the netherlands, at 60% and 80%, respectively. 6,7 similar to findings from previous studies on the same issue, the major concerns for both nurses and doctors with respect to influenza a h1n1 vaccination were fear of side effects and concerns regarding the efficacy of the new vaccine. 6, 7, [11] [12] [13] [14] annual vaccination against influenza has been considered to be the best way to reduce sickness and death from influenza in high-risk populations, to reduce absenteeism from work in healthy adults, and to minimize healthcare-associated transmission of influenza. [15] [16] [17] with respect to the use of guidelines in dealing with pandemic influenza and the need for further training and education for dealing with pandemic influenza, we found that more than half of the doctors who worked in public clinics were likely to use guidelines (71%), but at the same time wanted to have more training on how to deal with pandemic influenza (62%). the high proportion of public doctor respondents who reported using guidelines could likely be due to the use of an intranet within large organizations, or more standardized behavior among employees in the same organization, although this is not supported by findings from our community nurses. despite the reported use of guidelines by healthcare workers, a significant proportion continued to state the need for more training in dealing with influenza a h1n1. with regard to assuming public health responsibilities, the rate among public doctors was 41%. we could speculate that this is due to the more standardized instructions or supervision among doctors who work in the same organization as compared to doctors who work in private settings who may work solo or work in small groups. not surprisingly, among doctors who had encountered a suspected influenza a h1n1 infection, a higher demand for services and higher emotional distress in the past 2 weeks were reported when compared to those who had not encountered a suspected influenza a h1n1 infection. increased stress related to dealing with novel infections has been reported previously and our findings are consistent with previous findings. 4, 18 moreover, we found that doctors who reported having more training in family medicine were more likely to have reported suspected influenza a h1n1 infections. whether this is due to the greater training received, which increased their awareness and alert in detecting or suspecting influenza a h1n1 infections in patients, is unknown, but as a group we failed to find any significant differences in terms of protective behaviors such as washing hands or wearing masks in the past 3 days, or having an influenza vaccination, between those with and without postgraduate qualifications. a major limitation of the survey was the low response rate. thus, we are unsure about the representativeness of the results from these studies. an additional limitation is that we have only analyzed self-reported behaviors and attitudes. we did not directly observe their behavior and there could have been a social desirability bias for respondents to have a 'better' response in order to look good, especially among those in public clinics. public community doctors responded that they would like to have more education and training for dealing with pandemic influenza. future policies from the government should look into the educational needs of healthcare workers to increase their confidence in dealing with influenza pandemics. another finding was the lack of willingness of healthcare workers to have the pandemic influenza vaccination. future work to determine effective interventions to increase uptake, which may include designing interventions based on the ecological model, is called for; the need to address low influenza vaccination rates in this high-risk group is urgent in the context of a pandemic response. another key finding was the low level of compliance of the doctor respondents with hand washing between patients. due to the possibility of social desirability bias, we believe that the rate of hand washing may have been even lower than that reported in this study. as shown in the results, this low rate of hand washing between patients could have been a bigger problem. more education may be needed or more research is needed to explore the reasons for the lack of hand washing among frontline doctors, which may put both doctors and patients at risk of cross-infection. monitoring community responses to the sars epidemic in hong kong: from day 10 to day 62 public health preparedness: a system level approach general practice and pandemic influenza: a framework for planning and comparison of plans in five countries a crosssectional study of primary care physicians in singapore on their concerns and preparedness for an avian influenza outbreak attitudes amongst australian hospital healthcare workers towards seasonal influenza and vaccination positive attitudes of french general practitioners towards a/h1n1 influenza pandemic vaccination: a missed opportunity to increase vaccination uptakes in the general public? high vaccination rates for seasonal and pandemic (h1n1) influenza among healthcare workers in dutch general practice facemasks and hand hygiene to prevent influenza transmission in households physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base willingness of hong kong healthcare workers to accept pre-pandemic influenza vaccination at different who alert levels: two questionnaire surveys press release: review of pandemic vaccines underway influenza vaccination among primary healthcare workers which determinants should be targeted to increase influenza vaccination uptake among healthcare workers in nursing homes? prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the effectiveness of vaccination against influenza in healthy, working adults influenza vaccination of healthcare workers and vaccine allocation for healthcare workers during vaccine shortages longitudinal assessment of community psychobehavioral responses during and after 2003 outbreak of severe acute respiratory syndrome in hong kong this study was supported by the research fund for the control of infectious diseases (rfcid), food and health bureau, hong kong sar government.conflict of interest: the authors declare that they have no competing interests. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijid.2012.03.015. key: cord-341923-jwckbdnb authors: to, kelvin kai-wang; li, iris wai-sum; hung, ivan fan-ngai; cheng, vincent chi-chung; yuen, kwok-yung title: pathogenesis of pandemic h1n1 2009 influenza virus infection and the implication on management date: 2010-04-28 journal: front med china doi: 10.1007/s11684-010-0030-9 sha: doc_id: 341923 cord_uid: jwckbdnb the pandemic h1n1 2009 influenza virus has caused the first influenza pandemic of the 21st century, leading to disproportionate fatalities in the low-risk population despite the generally mild nature of the illness. advances in science and technology have allowed very detailed study on the pathogenesis of this novel virus, and many have already been published in less than a year after the start of the pandemic. information generated from cell lines, animal models, and clinical data analysis has provided us with greater understanding of the behavior of this virus and the associated host response. the new knowledge will allow us to formulate scientifically sound and evidence-based management plans. the emergence of human cases of h5n1 influenza virus infection in 1997 [1] and severe acute respiratory syndrome (sars) coronavirus infection in 2003 [2] has alerted the world of the devastating consequences of novel human respiratory viruses. in anticipation of a pandemic influenza, which may cause even greater morbidity and mortality worldwide, antiviral stockpiling and clinical trials for h5n1 vaccines were started in many developed countries [3] . as expected, the current pandemic h1n1 2009 influenza virus, which was first detected in march 2009, has spread worldwide [4, 5] . although most patients infected with this novel strain present with mild upper respiratory tract symptoms [6] , the higher fatality rate in children and young adults clearly differentiate this virus from seasonal influenza virus [7, 8] . moreover, obese patients [9] and pregnant or postpartum women [10] [11] [12] [13] are at increased risk of complications requiring intensive care or even death. these distinct clinical observations pose important scientific questions on our understanding of the differences in pathogenesis between this pandemic virus and the seasonal influenza virus. the marked advances in science and technology since our last pandemic in 1968 allow an unprecedented opportunity to do very detailed study on the pathogenesis of this infection. results from these studies, which have important potential implications on the treatment of 2009 h1n1 virus, are reviewed and discussed in this paper. in contrast with the seasonal influenza virus, the 2009 h1n1 virus had greater predilection for the lower respiratory tract, as demonstrated by studies in ferrets and mice [14] [15] [16] . in fact, some patients may have the virus detected exclusively from the lower respiratory tract specimens [17] . this may be partially explained by the broader receptor binding properties. binding of influenza virus to host cell surface glycoproteins or glycolipids is mediated by viral hemagglutinin (ha), and amino acid substitution in ha could affect the binding. in the 1918 h1n1 virus, both asp190glu and asp225gly (h3 numbering) of the ha altered the binding specificity to avian type receptor [18] [19] [20] . for h2 and h3 subtypes, avian to human adaptation can be achieved by substitutions at positions 226 and 228 [21] . by constructing theoretical ha-glycan structural complexes, it was found that lys145, which is unique in the 2009 h1n1 virus, together with lys133 and lys222, may form a positively charged lysine fence at the base of the binding site and favor the binding to both α2-3 and α2-6 glycans [22] . in addition, asp190, a residue critical in the binding to α2-6 glycans in the 1918 h1n1 virus, was postulated to be stabilized by a unique set of residues ser186, thr187, and ala189. in human seasonal influenza virus, asp190 substitution reduced the binding to α2-3 glycans, but in the 2009 h1n1 virus, this interaction was rescued by the lysine fence. this prediction was confirmed by carbohydrate microarray analysis, which showed that the 2009 h1n1 virus could also bind to the α2-3 sialic acid residue [23] , similar to h5n1 virus [24] . however, direct glycan binding assay from another research group showed that the ha of 2009 h1n1 virus could bind to α2-6 sialic acid residue but minimally to α2-3 sialic acid residue [15] . this apparent contradiction may be related to the selection of different sialyl probes and the differences in binding assays. the ability to bind α2-3 glycan is important since this may be associated with the binding to lower respiratory tract in human [25] . in vitro studies showed that the 2009 h1n1 virus could replicate in human cell lines originating from extrapulmonary tissues, including gastrointestinal, genitourinary, neuromuscular, immune cells, and conjunctiva [26, 27] . viral load was greatest in colorectal adenocarcinoma (caco-2) and rhabdomyosarcoma (rd) cell lines, consistent with the clinical observations of gastrointestinal symptoms and rhabdomyolysis [28] [29] [30] [31] . however, the level of viral replication in cell lines was similar to seasonal h1n1 virus except in the human conjunctiva, where 2009 h1n1 virus had better replication [26] . the broad viral tropism in cell lines is in accordance with the detectable virus in extrapulmonary specimens, including blood, urine, and stool from patients infected with 2009 h1n1 virus [32, 33] . since receptor binding is critical in the pathogenesis of influenza virus infection, mutations at the binding site of the ha may alter the virulence. an asp222gly (h1 numbering) substitution in ha of 2009 h1n1 virus was found to be more common among fatal human cases [34] . strains carrying asp274glu substitution in ha have also been found in the united states, spain, and italy, but the clinical data from these cases were not available [35] . analysis using informational spectrum method bioinformatics platform has found that position 274 may be associated with change in binding specificity. however, the binding property of this mutant virus has not been determined, and further studies are required to verify the clinical significance of this substitution. severe disease can be attributed to specific virulence factors of the virus that may lead to excessive inflammation, as in the case of h5n1 and 1918 h1n1 viruses. known virulence factors in h5n1 virus include lys627 of pb2, which increases the viral replication; an asn66ser substitution in pb1-f2, a protein that induces apoptosis, enhances inflammation in mice, and predisposes to secondary bacterial infection; ns1, which antagonizes the antiviral effect of interferon; and a multibasic ha cleavage sequence, which allows cleavage by ubiquitous proteases [36] . for the 1918 h1n1 virus, a full-length pb1-f2 has been postulated to be an important virulence factor [37] . however, these virulence factors are absent from the 2009 h1n1 virus: a glutamic acid is found at residue 627 of pb2; pb1-f2 is truncated due to the presence of stop codon at position 12; a deletion of a pdz ligand domain is found in ns1; and a single basic amino acid is found at the ha cleavage site [36, 38] . even the introduction of known virulence-determining mutations into pb2 of the 2009 h1n1 virus did not enhance virulence [39] . however, the sequence analysis of the 2009 h1n1 virus pb2 genes has identified ser590 and arg591, which is rare in seasonal influenza [40] . in that study, it was demonstrated that the paired substitution was associated with an increase in polymerase activity and enhanced viral replication. the level of viral load at the pathophysiological focus of infection represents the dynamic interaction between the virus and host immunity. this level is the result of viral replication, and its destruction by the host immune response may be an important determinant of disease severity. a higher viral load in respiratory tract samples was associated with fatal disease from h5n1 [41] and with disease severity in infection due to respiratory syncytial virus and human metapneumovirus [42] . however, when compared with seasonal influenza virus infection, the nasopharyngeal viral load was slightly lower in patients with 2009 h1n1 virus infection [32] . for severe cases of 2009 h1n1 virus infection, the initial viral load was found to be similar to that of patients with mild disease, but there was a slower decline in viral load [33] . therefore, the temporal rate of clearance of virus, rather than the initial viral load, is an important determinant of severity. this is in accordance with a study conducted in mice, which showed that effective host innate immune response only occurred after 48 h of infection, which is the time when viral load started to decrease [43] . the reassortment between the triple-reassortant swine influenza virus (which contains genes originated from h1n1 avian virus, h3n2 seasonal human virus, and h1n1 classical swine virus) and the eurasian "avian-like" swine influenza virus (originated from h1n1 avian virus) resulted in the 2009 h1n1 virus that is antigenically distinct from recent seasonal influenza h1n1 viruses [36, 38, 44] . serological surveys have been conducted worldwide to determine the level of pre-existing immunity in the population. in the united states, 34% of patients born before 1950, but only 4% born before 1980, had a microneutralization titer≥160 [45] . in that study, it was determined that a microneutralization titer of 80-160 corresponded to a hemagglutination inhibition titer (hai) of 40, which was associated with > 50% reduction in the risk of infection [46] . in england, the level of crossreactive antibody increased significantly with age, with 31.3% in those over 80 years old having a hai of ≥32 [47] . similar findings were reported from finland, where 55.6% of people born before 1919 had hai ≥40, but only present in 21.2% born between 1920 and 1930 and < 1% in those born after 1940 [48] . the level of pre-existing cross-reactive antibody is much lower in studies conducted in asia. in a serological survey performed in guangxi, china, only 1.7% of serum samples had a hai ≥40, and none were over 60 years old [49] . vaccine studies showed that only 4% of participants aged 61 years or above had a hai of ≥40 before vaccination [50, 51] . in japan, preexisting neutralizing antibody was only present in those born before 1920 [14] . most of these prevalence studies showed that the level of cross-reactive antibody tends to be higher in the older population. the higher rate of crossreactive antibody in the older population was proposed to be due to cross reactivity generated from exposure to the 1918 h1n1 virus or the immediate descendants. monoclonal antibodies generated against the ha sites sa of the 1918 h1n1 virus had neutralizing activity against the 2009 h1n1 virus and reduced the replication of 2009 h1n1 virus in the lungs of mice [52] . furthermore, antibodies generated by the 1976 swine origin h1n1 virus vaccine also protected mice infected with the 2009 h1n1 virus [53] . the lack of exposure to these viruses may partially explain why younger adults have higher clinical attack rate and more serious disease for the 2009 h1n1 influenza than seasonal influenza [54] [55] [56] . despite the fact that severe disease disproportionately affected young adults, still, most infected patients in this age group had mild disease. antibody levels cannot fully explain the situation, because protective immunity is dependent not only on the level of antibody but also on the t cell immunity. it has been found that 69% of cd8 + t cell epitopes and 41% of cd4 + t cell epitopes are conserved in the 2009 h1n1 virus when compared with seasonal influenza virus [57, 58] . furthermore, memory t cell response, as measured by interferon-g secretion after stimulation by t cell epitopes, was present for both conserved and nonconserved epitopes. similarly, a study using blood samples from donors with no history of 2009 h1n1 virus infection showed that the 2009 h1n1 virus peptides could induce specific t cell response [59] . these results are consistent with a previous study in human volunteers that demonstrated the importance of t cell immunity in the absence of cross reactive antibodies [60] . these studies have highlighted the role of t cell immunity, especially in patients without protective antibody. if the cell-mediated immune response, such as cytotoxic t lymphocyte, can be recruited early enough, the viral load can be controlled at a lower level at the early stage of the disease, and the viral load may drop faster, which would result in less cell death and cytokine activation. in addition to t cells, natural killer (nk) cells are also involved in the defense against influenza [61] . the major nk activating receptors involved in nk cell cytotoxicity, nkp46, was able to recognize the ha of 2009 h1n1 virus with subsequent direct killing [62] . this is in contrast with h5n1 virus, in which nkp46 could not elicit direct killing of the virus, and this may be one of the reasons why h5n1 virus is the most pathogenic influenza virus for human. hypercytokinemia has been associated with the disease severity of several respiratory viruses, including sars coronavirus [63] and h5n1 virus [41] . in vitro studies in human macrophages infected with 2009 h1n1 virus showed that the induced proinflammatory cytokine levels were similar to cells infected with seasonal influenza virus but much lower than those of h5n1 virus [64] . weaker induction of interferons, cxcl10, and tnf-α was found in dendritic cells infected with 2009 h1n1 virus or seasonal influenza virus than those infected with mouse adapted h1n1 and h3n2 viruses [65] . another study showed comparable cytokine response in bronchial epithelial cells and alveolar type i-like pneumocytes infected by 2009 h1n1 virus or seasonal h1n1 virus [26] . in a macaque model of 2009 h1n1 virus infection, there were persistently elevated levels of monocyte chemoattractant protein-1 (mcp-1), macrophage inflammatory protein 1 alpha (mip-1α), il-6, and il-18 in the inflammatory lungs [14] . ifn-g was overexpressed in the lung of mouse model coinfected with influenza virus and staphylococcus aureus and in the lung tissue of fatal 2009 h1n1 human cases [66, 67] . for patients with 2009 h1n1 virus infection, levels of proinflammatory cytokines or chemokines were higher in patients with most severe disease who died or developed acute respiratory distress syndrome than in those with disease of moderate severity or mild symptoms. granulocyte colony-stimulating factor (g-csf), mcp-1, tnf-α, il-1α, il-6, il-10, and il-15 levels were higher in patients with most severe disease than those with moderate or mild severity during the initial three days after symptom onset, with il-6, il-10, and il-15 levels persistently elevated through the course of illness [33] . on the other hand, il-17 levels were found to be lower in the more severe groups for samples collected within three days of symptom onset but did not differ significantly if samples were collected later than four days after symptom onset. in another study comparing single serum samples collected at a median of five days after symptom onset, highest levels of il-6, il-15, and il-12p70 were found in the most severe cases, and il-17 level was noted to be similar between the critical cases and noncritical hospitalized cases but higher than in outpatients or controls [68] . high levels of proinflammatory cytokines may be mediated by a sustained elevation of toll-like receptor 3 (tlr-3), as seen in human postmortem lung tissues [67] . taken together, immunodysregulation is a key factor in patients with severe disease, but the degree of cytokine activation may be much less than the more pathogenic avian h5n1 virus. the severity of 2009 h1n1 virus infection can range from asymptomatic infection to fatal disease. host genetic makeup has been proposed to be one of the determinants. since different strains of inbred mice exhibited variable susceptibility to infection by seasonal influenza virus [69] , understanding the host determinants of influenza viral replication can facilitate the development of anti-influenza treatment directed at host factors instead of viral targets. using yeast two-hybrid analysis and genome wide expression profiling, 616 human factors have been proposed to be involved in viral-host interaction for seasonal influenza virus, which can be broadly divided into those that are involved in viral replication and those involved in the regulation of ifnβ [70] . another study has identified interferon-inducible transmembrane proteins to be critical for the antiviral action of interferon type i and ii in seasonal influenza viruses [71] . using genome-wide rna interference screen, many host factors were shown to be associated with the replication of 2009 h1n1 virus [72, 73] . the significance of certain host factors has been confirmed by either knockdown mutants that could not produce the specific host factor or by the inhibition of the factor using small molecule inhibitors. these proteins included son dna binding protein (trafficking of influenza virons during early infection), cdc-like kinase 1 (splicing of viral m2 messenger rna), and p27 (required during late stage of viral replication) [72] . in another study, the inhibition of gene expression using sirna confirmed the importance of 12 genes in viral replication of 2009 h1n1 virus, which included those involved in viral entry (cd81, mid1ip1, arcn1, atp6v0c, map2k3, fgfr4, gsk3b, and cse1l), postviral entry steps including transcriptional regulation (camk2b) and ha cleavage (prss35), and nuclear trafficking (cse1l, prss35, and camk2b). the mechanism of inhibition on viral cycle is unknown for sumo2 and gabbr1 [73] . an australian study demonstrated the association between immunoglobulin g2 (igg 2 ) deficiency and severe disease [74] . in that study, patients with severe h1n1 infection, as defined by admission to intensive care unit (icu) for respiratory and/or vasopressor support, had significantly lower levels of igg 2 than patients with disease of moderate severity. most of these patients also had low levels of igg 2 in their convalescent sera. since the half-life of igg 2 is three weeks [75] , the authors suggested that these patients were likely to have underlying igg 2 deficiency rather than a low igg 2 level that was precipitated by the 2009 h1n1 virus infection, and patients with underlying igg 2 deficiency may not be able to mount early innate immune response to influenza, thus being predisposed to severe disease. most fatal cases of 2009 h1n1 virus infection suffered from respiratory failure [76] , and lung pathologies represented the most prominent findings in autopsy studies. the main pathological features in the lung included diffuse alveolar damage (dad), necrotizing bronchiolitis, and dad with intense alveolar hemorrhage [33, 67, 77] , which were similar to those reported for fatal h5n1 cases and previous pandemics [1, 78] . pulmonary artery microthrombi were present in 15/55 (27.2%) of cases from the two series. this phenomenon has been described in patients with seasonal influenza, and one postulation was that the thrombosis may have been triggered by the transient appearance of anticardiolipin antibody [79] . in addition to the lower respiratory tract, tracheitis was also found. evidence of secondary bacterial infection was seen in up to 55% of postmortem cases [80] . bacterial pathogens included streptococcus pneumoniae, staphylococcus aureus, streptococcus pyogenes, haemophilus influenzae, and streptococcus mitis [81] . the coinfection by communityacquired methicillin-resistant staphylococcus aureus (ca-mrsa) has led to a fatal disease in a 42 year-old immunocompetent man [82] . in this patient, there were areas with typical changes of bacterial pneumonia, namely, patchy consolidation with polymorphs inside alveolar cells. in other areas, there were features typical of viral pneumonitis, which were alveolar spaces with necrotic debris and detached pneumocytes or atypical pneumocytes with mildly enlarged nuclei and granular to vacuolated cytoplasm. influenza virus infection may predispose to bacterial infection by destruction of the physical barrier that exposes potential binding sites for the bacteria, impaired mucociliary clearance, immunodysregulation, upregulation of receptors, and expression of bacterial genes that encode toxin [83] . conversely, s. aureus specific protease, which cleaves influenza ha, may increase viral replication [84] . extrapulmonary pathology was prominent in some patients [33] . lymphoid aggregate and myofibril necrosis of the myocardium, typical features of viral myocarditis, were found. in one case series, reversible cardiac dysfunction occurred in 4.9% of 2009 h1n1 virus infection [85] , and these cases may have myocarditis. hemophagocytosis, a frequent histopathological finding in patients with severe infection by intracellular pathogens, was evident in lymph nodes of some cases [86] . splenic vein thrombosis was found in a patient who has suffered from multiorgan failure. hepatic necrosis was found in the autopsy of a pregnant woman [67] . viral load studies informed us that the speed of viral clearance, but not the initial viral load, was important in predicting severity. antiviral drugs have been considered to be a cornerstone in the treatment of influenza [87] . the current strain of pandemic influenza is universally resistant to amantadine or rimantadine, due to ser31asn mutation in the m2 gene [88] . most remain sensitive to neuraminidase inhibitors, including oseltamivir, zanamivir, and peramivir. in retrospective studies, delayed treatment with oseltamivir was associated with icu admission or death [12, 89] . this may be explained by a faster viral load reduction in patients who have received oseltamivir within two days of symptom onset [90] . however, the reduction in viral load was slow, with more than 50% of patients still having detectable virus in nasopharyngeal aspirate after five days of treatment regardless of the timing of starting oseltamivir. furthermore, oseltamivir-resistant strains have emerged worldwide [91] [92] [93] [94] [95] [96] [97] . as of 3 february 2010, there have been 225 oseltamivir-resistant strains reported to the world health organization [97] . all of these strains contained the his275tyr mutation. most of these were unrelated, but clusters have been found in the community and in the hospital [97, 98] . oseltamivir-resistant strains may exist as quasi-species, which may hinder the detection of the resistant strains [93] . oseltamivir resistance may also emerge in patients taking oseltamivir as postexposure prophylaxis [99] . treatment with intravenous zanamivir has been successful in a 10-year old girl with acute lymphoblastic leukemia and another 22 year-old woman with hodgkin's disease and infected with an oseltamivirresistant strain [100, 101] , but the virus was still detectable 5 and 13 days after intravenous zanamivir, respectively. furthermore, one of the two cases was started on high doses of steroid concomitantly. thus, the cause of the improvement is still uncertain. although zanamivir resistance has not been detected in the current pandemic strain, it has been reported in seasonal influenza a virus [102] . intravenous peramivir is also available, but the ic 50 for oseltamivir-resistant strains is high and unlikely to be useful in oseltamivir-resistant cases [103] . in addition to neuraminidase inhibitors, other classes of antiviral agents against influenza are developing, including adamantanamine derivatives, inosine monophosphate (imp) dehydrogenase inhibitors, rna polymerase inhibitors, and sirnas [104] . in addition to drugs that target the virus directly, drugs that alter host factors are also developed. in vitro studies have shown that das181, a sialidase fusion protein which removes cell-surface sialic acid residues from respiratory epithelia, has potent antiviral effect on 2009 h1n1 virus, including oseltamivir-resistant strains [105] . it has been shown that standardized extracts from echinacea purpurea has activity against the 2009 h1n1 virus, including oseltamivir-resistant strains, but the mechanism of action is unclear [106] . more effective antiviral drugs will be urgently needed to decrease the viral burden and hence may improve the prognosis in patients with severe disease. as the virus can be found in extrapulmonary tissues, optimal antiviral agents should be able to achieve high concentrations in these tissues. oral oseltamivir has good absorption, but it cannot be used in patients who cannot tolerate oral feeding or in those who have poor gastrointestinal absorption. although zanamivir has good antiviral activity, even against oseltamivir-resistant strains, nebulized, or inhaled route would achieve very low systemic concentration. therefore, the only currently available options are the parenteral forms of zanamivir or peramivir. unfortunately, resistance in seasonal h1n1 virus was reported even before these parenteral agents were marketed [101] . it is important to remember that early treatment of mild cases by antiviral drugs would decrease viral load more rapidly with earlier resolution of symptoms by one more day, but whether early treatment of those with risk factors for severe disease will prevent progression and death is uncertain. since the viral load has a plateau by the time of presentation for most severe cases, the efficacy of these parenteral antiviral drugs remains to be determined in formal clinical trials of patients with severe disease. apart from antiviral treatment, antibacterial coverage is also necessary because of the high prevalence of bacterial coinfection. empirical antibiotics should be able to cover potential bacterial pathogens, especially s. aureus, including ca-mrsa, s. pneumoniae, and s. pyogenes. diagnostic tests for bacterial copathogens should be employed to guide antibiotics therapy [87] . as persistent cytokine activation is evident in patients with severe disease, strategies to damn down the deleterious inflammatory response are required. pooled immunoglobulin in the form of convalescent plasma or hyperimmune globulin may serve both antiviral and immunomodulatory function. in a meta-analysis of the use of convalescent plasma in the 1918 influenza pandemics, there was lower mortality rate in the convalescent plasma group [107] . convalescent plasma was also shown to be effective in a patient with severe h5n1 infection [108] . however, the effectiveness in 2009 h1n1 virus infection has not been reported. pooled immunoglobulin was also successful in two patients with severe disease and igg 2 deficiency [74] . a study with a mouse model showed that monoclonal antibody was more effective than oseltamivir in the treatment of h5n1 and seasonal h1n1 virus infection [109] . hyperimmune globulin may be a useful strategy. in a mathematical model, it has been shown that passive immunotherapy is a feasible option even if there is a low percentage of donors [110] . corticosteroid has been a matter of debate. a study involving patients in the icu has reported success using corticosteroid [111] . it has been suggested that a low dose of steroid is helpful in severe cases to compensate for relative adrenal insufficiency [112] . the addition of celecoxib and mesalazine to antiviral drug has improved survival in mice [113] . other potential immunomodulators include fibrates and statins [114] . however, these agents have not been tested in the setting of pandemic influenza and await further studies to establish the efficacy. with the advance in medical science and technology, knowledge regarding the 2009 h1n1 virus has expanded at an unprecedented pace. further characterization of viral and host determinants of severe disease would be important. the understanding of the pathogenesis will form the basis for formulating scientifically sound strategies in clinical management. clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study pandemic influenza preparedness in the asia-pacific region emergence of a novel swine-origin influenza a (h1n1) virus in humans world health organization. transmission dynamics and impact of pandemic influenza a (h1n1) 2009 virus clinical investigation group of china. clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china critically ill patients with 2009 influenza a(h1n1) in mexico pediatric hospitalizations associated with 2009 pandemic influenza a (h1n1) in argentina intensive-care patients with severe novel influenza a (h1n1) virus infection -michigan h1n1 influenza a and pregnancy outcomes in novel influenza a (h1n1) pregnancy working group. h1n1 2009 influenza virus infection during pregnancy in the usa h1n1) working group. severe 2009 h1n1 influenza in pregnant and postpartum women in california barboza p; epidemic intelligence team at invs. epidemiology of fatal cases associated with pandemic h1n1 influenza vitro and in vivo characterization of new swine-origin h1n1 influenza viruses transmission and pathogenesis of swine-origin 2009 a(h1n1) influenza viruses in ferrets and mice pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets preferential lower respiratory tract infection in swine-origin 2009 a(h1n1) influenza a single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission human and avian influenza viruses target different cell types in cultures of human airway epithelium extrapolating from sequencethe 2009 h1n1 'swine' influenza virus receptor-binding specificity of pandemic influenza a (h1n1) 2009 virus determined by carbohydrate microarray haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to human-type receptors avian flu: influenza virus receptors in the human airway tropism and innate host responses of the 2009 pandemic h1n1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract differential susceptibility of different cell lines to swine-origin influenza a h1n1, seasonal human influenza a h1n1, and avian influenza a h5n1 viruses rhabdomyolysis associated with 2009 influenza a(h1n1) melting muscles: novel h1n1 influenza a associated rhabdomyolysis novel h1n1-associated rhabdomyolysis leading to acute renal failure gastrointestinal manifestations among chilean patients infected with novel influenza a (h1n1) 2009 virus viral load in patients infected with pandemic h1n1 2009 influenza a virus delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection world health organization. preliminary review of d222g amino acid substitution in the haemagglutinin of pandemic influenza a (h1n1) 2009 viruses identification of hemagglutinin structural domain and polymorphisms which may modulate swine h1n1 interactions with human receptor emergence and pandemic potential of swine-origin h1n1 influenza virus resurrected pandemic influenza viruses antigenic and genetic characteristics of swine introduction of virulence markers in pb2 of pandemic swine-origin influenza virus does not result in enhanced virulence or transmission adaptive strategies of the influenza virus polymerase for replication in humans fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus cutting edge: stealth influenza virus replication precedes the initiation of adaptive immunity origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus determinants of immunity to influenza infection in man incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study high frequency of cross-reacting antibodies against 2009 pandemic influenza a (h1n1) virus among the elderly in finland serologic survey of pandemic (h1n1) 2009 virus a novel influenza a (h1n1) vaccine in various age groups safety and immunogenicity of 2009 pandemic influenza a h1n1 vaccines in china: a multicentre, double-blind, randomised, placebo-controlled trial naturally occurring human monoclonal antibodies neutralize both 1918 and 2009 pandemic influenza a (h1n1) viruses protection of mice against lethal challenge with 2009 h1n1 influenza a virus by 1918-like and classical swine h1n1 based vaccines australia's winter with the 2009 pandemic influenza a (h1n1) virus comparison of adult patients hospitalised with pandemic (h1n1) 2009 influenza and seasonal influenza during the "protect" phase of the pandemic response complications of seasonal and pandemic influenza pre-existing immunity against swine-origin h1n1 influenza viruses in the general human population preexisting immunity to pandemic (h1n1) assessment of seasonal influenza a specific cd4 t cell responses to 2009 pandemic h1n1 swine-origin influenza a virus cytotoxic tcell immunity to influenza influenza and the challenge for immunology killing of avian and swine influenza by natural killer cells characterization of cytokine/chemokine profiles of severe acute respiratory syndrome cytokine profiles induced by the novel swine-origin influenza a/ h1n1 virus: implications for treatment strategies pandemic h1n1 2009 influenza a virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons a postinfluenza model of staphylococcus aureus pneumonia lung pathology in fatal novel human influenza a (h1n1) infection th1 and th17 hypercytokinemia as early host response signature in severe pandemic influenza host genetic background strongly influences the response to influenza a virus infections a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication association between severe pandemic 2009 influenza a (h1n1) virus infection and immunoglobulin g(2) subclass deficiency catabolism of gamma g-globulin and myeloma proteins of the subclasses gamma g1 and gamma g2 in a healthy volunteer critical care services and 2009 h1n1 influenza in australia and new zealand iner working group on influenza. pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico the pathology of influenza virus infections anti-cardiolipin autoantibodies and pulmonary embolism. a case for a common cause pulmonary pathologic findings of fatal 2009 pandemic influenza a/h1n1 viral infections bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states fatal co-infection with swine origin influenza virus a/h1n1 and community-acquired methicillin-resistant staphylococcus aureus interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness synergistic role of staphylococcal proteases in the induction of influenza virus pathogenicity reversible cardiac dysfunction associated with pandemic-2009-h1n1 influenza a. chest intracellular microbes and haemophagocytosis clinical management of human infection with pandemic (h1n1) 2009: revised guidance update: drug susceptibility of swine-origin influenza a (h1n1) viruses pandemic influenza a (h1n1) virus hospitalizations investigation team. hospitalized patients with 2009 h1n1 influenza in the united states the natural viral load profile of patients with pandemic swine-origin influenza a h1n1 2009 (ph1n1) and the effect of oseltamivir treatment oseltamivirresistant novel influenza a (h1n1) virus infection in two immunosuppressed patients oseltamivirresistant 2009 pandemic influenza a (h1n1) virus infection in two summer campers receiving prophylaxis-north carolina oseltamivir-resistant influenza a pandemic (h1n1) 2009 virus first cases of spread of oseltamivir resistant swine flu between patients are reported in wales detection of an oseltamivir-resistant pandemic influenza a/h1n1 virus in hong kong oseltamivir-resistant pandemic (h1n1) 2009 influenza in a severely ill patient: the first australian case update on oseltamivir-resistant pandemic a (h1n1) 2009 influenza virus vietnam h1n1 investigation team. a community cluster of oseltamivir-resistant cases of 2009 h1n1 influenza emergence of oseltamivir-resistant pandemic h1n1 virus during prophylaxis intravenous zanamivir for oseltamivir-resistant 2009 h1n1 influenza h1n1 pneumonitis treated with intravenous zanamivir zanamivirresistant influenza viruses with a novel neuraminidase mutation emergence and spread of oseltamivir-resistant a(h1n1) influenza viruses in oceania developing new antiviral agents for influenza treatment: what does the future hold? novel pandemic influenza a(h1n1) viruses are potently inhibited by das181, a sialidase fusion protein anti-viral properties and mode of action of standardized echinacea purpurea extract against highly pathogenic avian influenza virus (h5n1, h7n7) and swine-origin h1n1 (s-oiv) meta-analysis: convalescent blood products for spanish influenza pneumonia: a future h5n1 treatment? treatment with convalescent plasma for influenza a (h5n1) infection pre-and postexposure use of human monoclonal antibody against h5n1 and h1n1 influenza virus in mice: viable alternative to oseltamivir logistical feasibility and potential benefits of a population-wide passive-immunotherapy program during an influenza pandemic h1n1 influenza a virusassociated acute lung injury: response to combination oseltamivir and prolonged corticosteroid treatment a rationale for using steroids in the treatment of severe cases of h5n1 avian influenza delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus confronting an influenza pandemic with inexpensive generic agents: can it be done? key: cord-290031-vffa1bu0 authors: richmond, heather; rees, natasha; mchale, sharon; rak, aaron; anderson, jonathan title: seasonal influenza vaccination during a pandemic date: 2020-07-31 journal: human vaccines & immunotherapeutics doi: 10.1080/21645515.2020.1793713 sha: doc_id: 290031 cord_uid: vffa1bu0 in the northern hemisphere, the persistence or reemergence of coronavirus circulation into the 2020–2021 influenza season threatens to overwhelm health-care resources and systems and increase mortality and morbidity. data from australia show that stay-at-home policies have reduced both influenza and coronavirus cases early in the season, thus “flattening the curve.” however, influenza vaccination is critical to ensure the reduction in co-infection. several policies, such as vaccination strategies to accommodate physical distancing measures, change population recommendations, and timing and location of vaccination have been implemented to increase influenza vaccine uptake during the pandemic. this commentary explores those policies. the persistence or reemergence of coronavirus circulation into the 2020-2021 influenza season threatens to overwhelm health-care resources and systems and increase mortality and morbidity. learnings from australia, where concomitant responses to both influenza and coronavirus are ongoing, offer insights that may assist influenza vaccination planning and implementation in the upcoming northern hemisphere influenza season and beyond. these include changes to the timing and location of vaccine administration to accommodate social distancing, policies to ensure optimal management of public demand, access and uptake of available vaccines across the season, and the need for communications to be clear, frequent, and aligned among all stakeholder groups. data from the southern hemisphere show that stay-at-home policies have reduced both influenza and coronavirus cases early in the season, thus "flattening the curve." however, as physical distancing measures such as shelter in place and lockdown restrictions are eased, influenza infections are likely to emerge and endure over several months. in recognition of this, the australian government introduced several policy changes to increase influenza vaccine uptake and implemented new vaccination strategies to accommodate physical distancing measures, changing not only who gets a vaccine but also when, where, and from whom. australia provides free influenza vaccine each season for atrisk populations, with strong uptake in these groups. 1 at the beginning of the pandemic, the australian government recognized the importance of maintaining health-care visits and preventive care. additionally, the government issued a requirement that by may 1, 2020, all individuals visiting, working, or living in an aged care/nursing/long-term care or assisted living facility must receive an influenza vaccine. the policy was also extended on april 1, 2020, to apply to childcare centers. these policy changes were designed to further protect vulnerable populations and recognized health-care workers and caregivers as critical target populations for influenza vaccination. influenza vaccines are manufactured and delivered in a timeframe that allows providers to start vaccination prior to peak circulation of the influenza virus. policy changes in australia, together with strong communication from public health agencies and media reporting about the risk of coinfection and importance of influenza vaccination, generated significant demand for influenza vaccines early in the season. as a result, the australian government asked seqirus to go back into production requiring education to the public about the need to extend the duration of the immunization program and urging the need to continue vaccination late into the season. 2 social and physical distancing requirements throughout the vaccination period in australia have yielded novel approaches to vaccine administration within the traditional clinic settings and through alternative models for administration supporting safe, effective vaccination. these locations have included pharmacies, carparks/parking lots, offices, church or community halls, or parks and outdoor areas that would allow people to maintain a safe distance while being vaccinated. planning for these activities occurred expeditiously, as local solutions, rather than being organized by public health authorities. immunization advisory groups, providers, and influenza vaccine manufacturers have worked together to share learnings and optimize vaccination during this pandemic. to achieve high immunization rates, the australian government worked with industry to align delivery dates of vaccine to immunization sites upon availability, although demand was so great at times that short-term shortages occurred. the centers for disease control and prevention (cdc), the white house coronavirus taskforce, and the who's european office have stated the need to use the influenza vaccine as a tool in the fight against co-infection. 3 receiving an influenza vaccine reduces the number of infected patients, thereby helping to "relieve pressures" in hospitals treating patients with coronavirus. in the european union, seasonal influenza causes 4−50 million symptomatic cases each year. 4 in the us, the cdc estimates that in any given influenza season, 140,000-810,000 hospitalizations occur due to influenza. 5 the opportunity to increase influenza vaccination rates in the 2020-2021 season, to reduce the burden on health-care systems if a second coronavirus surge occurs, may lead to permanent, improved policy changes for influenza vaccination campaigns for years to come. as the influenza season approaches, public health stakeholders have already begun to consider the potential impact of concurrent influenza with coronavirus and potential changes to target populations. for example, the united kingdom and certain regions of italy are considering expanding influenza recommendations for people under 65 y of age, while the german government is considering a recommendation for voluntary lifespan immunization. other countries are looking to increase the health and social care worker vaccination. with changes to vaccine recommendations or mandates, it is critical that recommending bodies (national immunization technical advisory groups -nitags) coordinate with medical and health professional societies to manage and adequately communicate these changes to all stakeholders involved in influenza vaccination. manufacturers are currently experiencing significant demand for influenza vaccines from many northern hemisphere countries. 6, 7 meeting sudden, increased demand can be challenging as manufacturing preparations begin at least 12 months before each season. nonetheless, manufacturers are responding by increasing and extending current production; however, the ability to dramatically increase supply in a short period of time is limited, and additional doses that are produced will require acceptance of later delivery compared to "normal" seasons. policy changes and strong public education can potentially optimize influenza vaccine coverage as doses become available throughout the season but need to be sustained into future seasons to maintain vaccine uptake and achieve the required on-time supply of doses, including influenza vaccines that are specifically designed for certain populations. clear and simple messaging about the influenza vaccine helps to play a role in spreading the word about the importance of influenza vaccination, particularly with the prospect of changes in demand, population recommendations, timing, and administration locations. in some countries, the coronavirus pandemic may result in many people seeking influenza vaccination who have not been vaccinated previously. these people may have new or additional questions that need to be considered. the opportunity for providers to communicate the right vaccine for the right individual within an opportune time frame may be advantageous for those at higher risk for co-infection. further, as we have seen in australia, the timing of vaccination will need to be extended well into the influenza season to align with vaccine availability, reaching as many as possible prior to the peak of influenza circulation. therefore, the message to vaccinate early -without creating perceived shortagesand throughout the season, is vital to the effectiveness of the 2020-2021 campaign. by working together, health officials, medical authorities, and medical journalists can help deliver effective messages about influenza vaccination during the pandemic, while being open to new and evolving questions based on the situation. clear, consistent, frequent, and honest communication from public health officials to all influenza vaccine providers, and to the public, is the only way to manage expectations and ensure that continued attempts to "flatten the curve" and protect patients from influenza and coronavirus are achieved. the case for seasonal influenza vaccination is very strong, yet vaccination rates remain suboptimal in most countries. 8 influenza vaccine policy changes during the coronavirus pandemic have the potential to improve coverage but are limited by the ability of manufacturers to significantly increase supply doses in a short period of time. sustaining these policy changes will not only help to reduce seasonal influenzarelated deaths and hospitalizations but assist government preparedness for the next pandemic, particularly an influenza pandemic, by balancing vaccine supply/demand at higher levels. influenza disease is an annual public health threat with or without the concurrent pandemic. influenza vaccination is an ongoing public health tool to reduce disease now and in the future. the potential for coronavirus to persist or resurge during the upcoming influenza season is generating significant demand for influenza vaccines, which may outstrip available supply, particularly early in the season, with limited time and ability to produce additional vaccine. there is a current need to reconsider the timing of vaccination and differentiation among influenza vaccines to ensure that the best vaccine is available for each member of the population. increasing influenza vaccine uptake within the constraints of social distancing requirements may also require modification of timing, location, and provider -as the australian example illustrates. clear and succinct communications designed by multiple stakeholders (including industry), which are then delivered cohesively and with repetition, should be an annual component to influenza preparation. policy changes helping to drive increased demand are welcomed and should be sustained in the interests of public health and as an additional pandemic preparedness measure. planning for the 2020-2022 season must begin now given the lead times required for the manufacturing of influenza vaccine. no potential conflicts of interest were disclosed. stepping up flu vaccine supply -seqirus to supply additional two million flu vaccines to australia what if flu and covid-19 overlap? australia is trying to avoid that scenario. healthline. 11 researchers report 21% covid-19 co-infection rate. center for infectious disease and policy (cidrap) european centre for disease prevention and control (ecdc): factsheet about seasonal influenza disease burden of influenza. estimated range of annual burden of flu in the u.s. since higher flu vaccination rates could help expose new viruses like covid-19 earlier, expert says. the guardian flu shot makers gear up-and get creative-for a critical vaccination season world health organization. regional office for europe. influenza vaccination coverage and effectiveness key: cord-307607-8xn9jtmh authors: sargin, seyid ahmet title: potential anti-influenza effective plants used in turkish folk medicine: a review date: 2020-08-31 journal: j ethnopharmacol doi: 10.1016/j.jep.2020.113319 sha: doc_id: 307607 cord_uid: 8xn9jtmh ethnopharmacological relevance: due to the outbreaks such as sars, bird flu and swine flu, which we frequently encounter in our century, we need fast solutions with no side effects today more than ever. due to having vast ethnomedical experience and the richest flora (34% endemic) of europe and the middle east, turkey has a high potential for research on this topic. plants that locals have been using for centuries for the prevention and treatment of influenza can offer effective alternatives to combat this problem. in this context, 224 herbal taxa belonging to 45 families were identified among the selected 81 studies conducted in the seven regions of turkey. however, only 35 (15.6%) of them were found to be subjected to worldwide in vitro and in vivo research conducted on anti-influenza activity. quercetin and chlorogenic acid, the effectiveness of which has been proven many times in this context, have been recorded as the most common (7.1%) active ingredients among the other 56 active substances identified. aim of the study: this study has been carried out to reveal the inventory of plant species that have been used in flu treatment for centuries in turkish folk medicine, which could be used in the treatment of flu or flu-like pandemics, such as covid 19, that humanity has been suffering with, and also compare them with experimental studies in the literature. materials and methods: the investigation was conducted in two stages on the subject above by using electronic databases, such as web of science, scopus, sciencedirect, proquest, medline, cochrane library, ebsco, highwire press, pubmed and google scholar. the results of both scans are presented in separate tables, together with their regional comparative analysis. results: data obtained on taxa are presented in a table, including anti-influenza mechanism of actions and the active substances. rosa canina (58.7%) and mentha x piperita (22.2%) were identified as the most common plants used in turkey. also, sambucus nigra (11.6%), olea europaea (9.3%), eucalyptus spp., melissa officinalis, and origanum vulgare (7.0%) emerged as the most investigated taxa. conclusion: this is the first nationwide ethnomedical screening work conducted on flu treatment with plants in turkey. thirty-nine plants have been confirmed in the recent experimental anti-influenza research, which strongly shows that these plants are a rich pharmacological source. also, with 189 (84.4%) taxa, detections that have not been investigated yet, they are an essential resource for both national and international pharmacological researchers in terms of new natural medicine searches. considering that the production of antimalarial drugs and their successful use against covid-19 has begun, this correlation was actually a positive and remarkable piece of data, since there are 15 plants, including centaurea drabifolia subsp. phlocosa (an endemic taxon), that were found to be used in the treatment of both flu and malaria. plants have always been the primary choice for preventing and treating various diseases faced by human beings, and contain specific or broad-spectrum active compounds for almost any type of disease (alaoui-jamali, 2010) . people living in turkey have also benefited from plants in the prevention and treatment of various diseases for centuries. people living in rural areas still have an especially rich medicinal plant repertoire (ertuğ, 2004) . although herbal cures such as rosehip tea, peppermint-lemon tea and garlic-lemon tea, which are used to prevent and treat flu outbreaks, are well known by the local people, the vast majority of them and their antiinfluenza effects have not yet been adequately investigated in vitro by the related industries (bekut et al., 2018) . in virus classification, influenza viruses are rna viruses that comprise 4 of the 7 genera of the family orthomyxoviridae (kawaoka, 2006) , while human rhinoviruses (hrvs) are within the genus enterovirus and only english and turkish words were used in the search engines. if they exist, their english translations were reviewed for the studies conducted in different languages, such as chinese, korean and french. in this context, approximately 700 articles conducted between january 1977 and february 2020 throughout turkey were excluded since they did not meet the inclusion criteria and a consensus has been provided among the 81 works on the determination of medicinal plants used by local people for centuries. the list of selected plants from these studies is presented in table 1 . the studies determined to be within the scope of plant screening were reviewed, compared and carefully selected according to the following criteria. accordingly, a study should: • be carried out in an area within the borders of turkey. • performed on ethnobotanical or ethnopharmacological concept layout. • include scientific names and local names of the plants used. in addition, the criterion for choosing the book sources was either the writer having an academic title or the work having been cited. if neither of these were in case, the work was not taken into consideration. the screening of the resulting plants in the world literature was carried out considering the following criteria. accordingly, a study should be: • an experimental (in vitro or in vivo) study, not a review. • included the scientific name of the plant in its title. in case of writing only the english name of the plant, it is obligatory to include the scientific name in the text. • carried out under the headings of "anti-flu, anti-influenza or antiviral activities against influenza". if it contains the active compound(s), it becomes preferable and the mechanism of action is recorded. table 1 contains the scientific names of plants, their families, local names, english common names, parts used, forms used, and references. the validation of the scientific names of the specified plant taxa was provided by the book turkey plant list (vascular plants) (güner et al., 2012) , the international plant names index (ipni: http://www.ipni.org) and the plant list (http://www.theplantlist.org). english common names of the taxa are placed in the table using the following databases or search engines: eppo global database j o u r n a l p r e -p r o o f , lebanon flora (http://www.lebanon-flora.org), springer link (https://link.springer.com/article), flora of israel online (http://flora.org.il), altervista flora italiana (http://luirig.altervista.org/flora), and plants of the world online (http://www.plantsoftheworldonline.org). taxa for which common english names could not be found have been noted as endemic to turkey, or containing irano-turanian elements. finally, the plants were arranged in alphabetical order according to family names. in order to prove the scientific validity of the ethnobotanical data obtained, the research data of the experimental studies regarding the taxa in the list, as found in the world literature, are shown in a separate table (table 4 ). in this table, the mechanism of action, active compounds and used parts are also included, in addition to the researched taxa and their references. great care has been taken to ensure that the findings obtained in these screening studies belong to experimental studies (in vitro or in vivo), not a review. after obtaining the total list of plants with anti-influenza potential in turkish folk medicine, a comparison was made to determine the similarity percentages in similar studies conducted in neighboring and nearby countries (table 2) . to avoid distraction from the subject integrity, not all studies in those countries were included in our comparison. therefore, only the study with the richest content and the highest percentage of similarity from each country was included in the comparison list. studies with a similarity percentage >10% were eliminated in the primary elections. ozturk et al. (2017a) 13 5.8 southeastern anatolia sargin and büyükcengiz (2019) 13 5.8 mediterranean tuzlacı and doğan (2010) 13 5.8 eastern anatolia tuzlacı and erol (1999) 13 5.8 mediterranean ertuğ (2004) 11 4.9 aegean güneş and özhatay (2011) 11 4.9 eastern anatolia kılıç (2016) 11 4.9 eastern anatolia kilic and bagci (2013) 11 4.9 eastern anatolia guzel and guzelsemme (2018) 10 4.5 mediterranean ozturk et al. (2017b) 10 4.5 mediterranean saraç (2005) 10 4.5 all regions tetik et al. (2013) 10 4.5 eastern anatolia yeşilyurt et al. (2017b) 10 4.5 marmara akgül et al. (2016) nacakcı and dutkuner (2015) 9 4.0 mediterranean özçelik (2016) 9 4.0 mediterranean akan and bakır sade (2015) 8 3.6 southeastern anatolia akbulut et al. (2019) 8 3.6 aegean kurt and karaoğul (2018) 8 3.6 black sea paksoy et al. (2016) 8 3.6 central anatolia sargin et al. (2013a) 8 3.6 aegean yılmaz (2019) 8 3.6 aegean demirci and özhatay (2012) 7 3.1 southeastern anatolia kaval et al. (2014) 7 3.1 eastern anatolia kocabaş and gedik (2016) 7 3.1 mediterranean maranki and maranki (2016) 7 3.1 all regions tuzlacı and eryaşar-aymaz (2001) 7 3.1 marmara ugulu et al. (2009) 7 3.1 aegean tanker et al. (1998) the demand for new antimicrobial agents, especially antivirals, is constantly increasing. this demand arises from the lack of antiviral agents in the market and the emergence of resistant mutants to existing drugs (vijanyan et al., 2004) . throughout our existence, human beings have always been in search of healing from plants in the fight against winter diseases, but clinical studies have to this point been limited. although the following work is relatively new in turkey, they are promising for future study: duman et al. (2018) elicited in vitro antiviral activity of ribes uva-crispa l and ribes multiflorum kit ex schult, which are naturally grown in turkey, use the methanol and aqueous extracts of the leaves and fruits; dogan et al. (2020) revealed anti-rsv effects of ribes uva-crispa juicy fruit and leaf methanol extracts against the respiratory syncytial virus (rsv) (the cause of a worldwide viral infection), and emphasized their advantages to synthetic drugs; finally, adem et al. (2020) found that natural polyphenols, such as hesperidin, routine, diosmin and apiin were more effective than nelfinavir in treating covid-19. the plants (table 3) , which have been used by locals in turkey for centuries for the prevention and treatment of influenza and its adverse effects -from colds to sudden deaths from respiratory failure -need to be investigated in this way. today, much more research is needed, as outbreaks such as sars, avian influenza, swine influenza and covid-19 threaten the existence of human beings every year. distribution of 81 studies by region was performed as follows: 13 in the mediterranean (16.0%), 11 in eastern anatolia (13.6%), 10 in the marmara and aegean region (12.3%), 8 in the black sea (12%), 7 in central and southeastern anatolia (11.1%), and 15 general studies across all regions (18.5%). the regional distribution of 921 total citations received was as follows: mediterranean: 150 (16.3%), eastern anatolia: 141 (15.3%), aegean: 109 (11.8%), marmara: 98 (10.6%), central and southeastern anatolia: 82 (8.9%), black sea: 75 (8.1%), and general studies covering all regions: 184 (20.0%). the reason why the studies conducted in the mediterranean and eastern anatolia regions were highly cited may be due to the fact that there are more plant options, which is the result of having a higher rate of biodiversity and endemism in these regions (güner et al., 2012) compared to others, that the locals can use in the treatment of influenza. in addition, the topographic structure of the region, and the fact that the region is isolated from city centers in winter conditions (doğanay and orhan, 2016) may have been a factor for the people living in these rural areas to choose mostly natural treatment methods. it has been determined that 224 plants, selected from 81 studies composing of 57 articles, 13 books, seven theses, three chapters and one congress report in total, belonging to 43 families. these plant taxa most commonly belong to the lamiaceae (88 taxa, 39.3%), compositae (32 taxa, 14.3%), rosaceae (21 taxa, 9.4%), malvaceae (13 taxa, 5.8%), and other families (70 taxa, 31.3%). the most preferred outcome of the lamiaceae family may be due to the turkish people's preference for flu treatment, as it is the family that contains the highest dosage of essential oils (askun et al., 2012) . the second family, compositae, is known as turkey's most common family (guner et al., 2012) . infusions prepared from taxa with capitula flower structures such as its representative chamomile are widely used by local people. therefore, this was an expected result. according to studies conducted in different regions of turkey ( fig. 1) , the most common genera are sideritis (16 taxa, 7.1%), salvia (12 taxa, 5.4%), thymus (12 taxa, 5.4%), and origanum (10 taxa, 4.5%). this finding may indicate that these genus members are more effective in anti-influenza treatment than other genera. in addition, they are the most favored medicinal tea for the locals of turkey, and even without natural nationwide distribution, it is possible to find these products in almost every public market, herbal and spice shop (ertug 2004; dogan 2012) . some species, such as thyme (thymus spp.), melissa (melissa officinalis), lavender (lavandula angustifolia), cassidony (lavandula stoechas) and sage (salvia officinalis), are today being grown in home gardens, balconies or on small farms by rural people for folk medicine use, or for trade and household income (güneş, 2017; ekşi et al., 2020) . like thyme, melissa, lavender, and sage, among the identified plants, 145 were wild (64.7%), 49 were wild and cultivated (21.9%), 27 were endemic (12.1%) and 3 (allium cepa, allium sativa and malus domestica) were cultivated (1.3%). these parameters are shown in a column in table 3 ; wild taxa as "w", cultivated "c", cultivated & wild "cw" and endemic "e". most of the plant pieces used are aerial parts (41.1%), flowers/flowering branches/petals (30.8%), leaves (25.0%), fruits (17.4%), seeds/cones (8.5%), roots/bulbs/tubers (6.7%), and other parts (stems, buds, barks, whole parts, resins, tars, cupula, bracts, fruit stalks, essential oils and fixed oils) (14.3%). those parts were mostly used as infusions (78.6%), decoction/boiling (19.2%), raw eating/swallowing/salad (12.9%), molasses/jam/syrup/juice (7.6%), lotion/drop/cataplasm/vapor compression (6.3%) and other consumption types (roasting, mouthwash, tincture, mixture and pastes) (5.4%) and powdered for spice use (3.1%). the taxa having with the most usage types are citrus spp (7 types, 3.1%), rosa canina and rubus sanctus (5 types, 2.2%) and vitis vinifera (4 types, 1.8%), while the taxa with the maximum number of consumption parts belong to rosa canina and tilia tomentosa (6 parts, 2.7%), and juniperus oxycedrus (5 parts, 2.2%). additionally, rosa canina (with 5 different types of use and 6 different parts) have appeared as the most efficient plants in terms of the total of both part and usage type (table 3) . 16 taxa, such as rosa canina (with 46 references and 56.8%) and mentha x piperita (with 17 references and 21.0%) (fig. 2 , red color), have been identified as the most frequently cited plants. the reason why these herbs are highly cited may be a reflection of their stronger protective and therapeutic effects against flu; this may be the result of the experience gained in turkish folk medicine for centuries. we would obviously see this when comparing similar studies between 17 geographically close countries (fig. 2 , blue color). the emergence of the data presented in table 2 in a similar manner as in figure 2 confirms the superior efficacy of these plants, with 76.7% similarity. as a matter of fact, similar results were obtained from studies conducted in 17 neighboring countries, comparing with the taxon list presented in the study, including especially rosa canina (11 countries with 64.7%), sambucus nigra (8 countires with 47.1%) and mentha x piperita (6 countries with 35.3%). while the similarity was seen mostly in iraq (75.0%), bosnia and herzegovina (73.3%), and cyprus (73.1%), the least similarity was seen in montenegro (31.8%) and israel (38.1%). this may due to the fact that, besides the resemblance of landforms, climate and vegetation, we lived together with the cultures of those countries during the ottoman period for about 500 years. the reason for the low similarity in israel and montenegro may be due to the geographical distance as well as the difference of social-cultural habits, religious rituals, topography and flora (table 2) . it was not very surprising that matricaria chamomilla emerged as the plant used most in influenza treatment in 12 countries (70.6%) since the spreading area of this plant is very wide and it is very easy for the public to access and use (fig. 2) . (2020) lamiaceae boiss. (2006) tuzlacı (2006) experimental research studies carried out in the world in terms of anti-influenza activities have been determined only for 35 out of 224 taxa (15.6%). still, among these studies, the active substances were detected for only 18 taxa (8.0%); for the remaining 17 taxa (7.6%), it was observed that they had not been specified (table 4 ). in table 4 , only "the parts used in research" were given as an idea for these taxa for which active gradients had been "not specified". it is noteworthy that no investigation has been conducted for 189 (84.4%) taxa yet (they are highlighted in bold in table 3 ). among these 35 taxa, the most common active chemicals are quercetin and chlorogenic acid (7.1%), mentofin (5.4%) and 1,8-cineole (3.6%). the most preferred mechanisms in research are inhibition of viral replication by inhibiting viral nucleoprotein synthesis or polymerase and neuraminidase activity (40.4% out of the 47 mechanisms in total), blocking the receptor site of the viruses by inhibition of neuraminidase, reducing the hemagglutination, or blocking hemadsorption (31.9%), inhibition of the virus-induced cytopathic effect by blocking hemadsorption (21.3%), and stimulating and boosting of the immunity (6.4%). the reason that the six taxa at the end of the list are shown as a line separated from the alphabetical sequence is that there was no significant result for virus inactivation in the experimental studies conducted for them (table 4) . according to screening results found in the global literature, the most preferred plants in experimental antiinfluenza studies are sambucus nigra (14.3%, out of 35 taxa), olea europaea (11.4%), followed by eucalyptus camaldulensis, e. globules, melissa officinalis and origanum vulgare (8.6%). the reason for this may be that these plants are easily accessible in nature or from the virtual market environment, and can be obtained for less money. additionally, eucalyptus trees in turkey are also known as "malaria trees", as the infusion prepared from its leaves is used against malaria in traditional medicine (baytop, 1999; ertuğ, 2004) . although its effectiveness against covid-19 has not been fully proven by clinical trials, the widespread use and mass production of chloroquine and similar malaria drugs are permitted in many countries, and positive results continue to be achieved (millán-oñate et al., 2020; touret and de lamballerie, 2020) . this correlation of data has been positive and unexpected because there are fourteen more plants, including centaurea drabifolia subsp floccosa (an endemic taxon), which have been detected in this study to be used in the treatment of malaria. these fifteen plants are presented in table 3 by adding the "*" sign to the end of their scientific names. the percentage of compatibility of the plant parts belonging to these 35 (15.6%) taxa found between the investigation results in the world literature and ethnobotanical results of the study was found to be 92.9%. this result may prove the fact that for centuries, the locals have been equally justified in their preferences of plant usage. taxa containing quercetin, which has a typical polyphenol structure with anti-influenza activity, are hypericum perforatum, morus alba and papaver rhoeas (kim et al., 2010; liu et al., 2016; kim and chung, 2018) (table 4) . it is not accidental that we detected quercetin and chlorogenic acid as the most common active gradients in our screening records, because these compounds are found to be the most effective compounds used in the treatment of influenza. supporting these findings, kumar et al. (2003) stated in a study of mice that quercetin ( fig. 2a) may be useful as a drug to reduce oxidative stress caused by influenza virus infection in the lungs, and to protect them from the toxic effects of free radicals. in another study, wu et al. (2016) stated that quercetin, which shows inhibitory activity in the early stage of influenza infection, offers a future therapeutic option for developing effective, safe and affordable natural products for the treatment and prophylaxis of influenza virus infections. moreover, nile et al. (2020) , in an investigation of the antiviral and cytotoxic effects of quercetin 3-glucoside (q3g) from dianthus superbus, q3g (fig. 2b) found that this substance showed strong antiviral activity against influenza a and b viruses. therefore, they emphasized that it could be developed and used as a natural anti-influenza drug. on the other hand, chlorogenic acid (cha) is a caffeoylquinic acid constituent (fig. 2c) found in many vegetables and fruits traditionally used in turkish folk medicine, such as cydonia oblonga, crataegus monogyna, morus alba, hypericum perforatum, eucalyptus globules (baytop, 1999; ding et al., 2017; kim and chung, 2018) . indeed, many researchers including ding et al. (2017) and ren et al. (2019) have pointed out that cha acts as a neuraminidase blocker to inhibit influenza a virus at both in vitro and in vivo levels, thus they stated that cha is potentially beneficial in the treatment of influenza. among the researches, the taxa containing the most active compounds in terms of anti-influenza activity were glycyrrhiza glabra (11 chemicals with 31.4% out of the 35), papaver rhoeas (7; 20.0%), morus alba (5; 14.3%) and punica granatum (4; 11.4%) ( table 4 ). glycyrrhiza glabra (licorice) is among the oldest and most popular traditional herbal medicines worldwide (grienke et al., 2014) . also, its roots are one of the most frequently used parts for treating respiratory tract infections in turkish folk medicine (baytop, 1999; ertuğ, 2004) . hence, the roots may have appeared to have the greatest number of active ingredients in the screening. this result overlaps with the findings of grienke et al. (2014) because they had emphasized that the accumulation of the plant components exhibits 3d similarities to known flu neuraminidase inhibitors (which are key enzymes in viral replication and the first-line drug target to fight influenza) according to their basis of a shape-focused virtual screening. therefore, this finding may be pointing out that this plant is more effective and specific than other taxa in terms of anti-influenza activity. active compounds identified (and used parts) alcea olea europaea l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson (2012) j o u r n a l p r e -p r o o f 24 olea europaea l. not specified (fruits-essential oil) both in influenza a/h1n1 and hrv14, replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants (thymbra capitata, origanum dictamnus and salvia fruticosa in extra-virgin olive oil) origanum vulgare l β-carotene and linoleic acid (aerial parts) decrease influenza virus activation by inhibiting the hemagglutination mancini et al. (2009) origanum vulgare l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson (2012) origanum vulgare l. not specified (essential oil) linalool (essential oil) linalool (essential oil) reduce visible cytopathic effects of influenza a/ws/33 virus activity by > 52.8%. choi (2018) papaver rhoeas l. kaempferol-3-sophoroside, kaempferol-3neohesperidoside, kaempferol-3-sambubioside, kaempferol-3-glucoside, quercetin-3-sophoroside, luteolin, chelianthifoline sambucus nigra l. not specified (fruits) exhibit a specific neuraminidase-inhibiting effect krawitz et al. (2011) silybum marianum (l.) gaertn. silymarin (seeds) reduces cytopathic effect (cpe) and inhibits viral mrna synthesis with no cytotoxicity song and choi (2011) thymbra capitata (l.) cav. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. thymbra capitata (l.) cav. apigenin, thymol (aerial parts-essential oil) both in influenza a/h1n1 and hrv14, replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants ( the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. (2015) olea europaea l. * not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. (2015) origanum acutidens (hand.-mazz.) ietsw. * none of the extracts inhibited the reproduction of influenza a/aichi virus in mdck cells sökmen et al. (2004) rosmarinus officinalis l. * carnosic acid (aerial parts) inhibit both a-and b-type hrsv, while it does not affect the replication of influenza a virus shin et al. (2013) teucrium polium l.* not specified (aerial parts) no significant effects on influenza virus infectivity derakhshan (2015) * the taxa that have no significant result for virus inactivation. j o u r n a l p r e -p r o o f in addition, 9 medicinal exotic herbs were detected to have been traditionally used in the treatment of influenza and sold in herbal and public markets. zingiber officinale (ginger), curcuma longa (turmeric), syzygium aromaticum (cloves), piper nigrum (black pepper) and cinnamomum verum (cinnamon) are examples of these plants. information on which parts, methods, and how often these plants are used in flu treatment is given in table 5 . the citrus species presented in table 3 are actually exotic species. for several centuries, they have mainly exhibited a distribution in the aegean and mediterranean coasts in turkey's flora. citrus limon (lemon), c. sinensis (orange), c. reticulata (tangerine), c. paradisi (grapefruit) and c. x aurantium (citrus) are among these types. eucalyptus camaldulensis and e. globulus (eucalyptus trees), another plant that has settled in the flora, are of australian origin and have been used in forestry, roadside landscaping, drying of the marshes and folk medicine practices, such as combating malaria, since the ottoman era (özgün, 2013) . the point we should especially emphasize here is that, while herbal products to be released for the treatment of influenza are determined by world health organisation (who) and the european phytotherapy scientific cooperative (escop), and controlled by the turkish government, these standard practices are not yet available for fresh or dried plant taxa that are traditionally consumed and sold in public markets and herbalist shops in turkey. besides, it can never be ignored that medicinal plants are very successful in preventing and treating influenza if used according to the prescriptions specified in their pharmacopoeia. thus, it is necessary to record traditional-empirical practices with proven trial-and-error methods urgently, to demonstrate their activities and active ingredients in vitro or in vivo studies, and to enlighten the public by adding optimal tariffs to their pharmacopoeia by the relevant official standard institutions. in our study, it was also determined that 27 endemic plants were used effectively in influenza treatment and collected from nature. the unconscious collection of endemic and endangered species in the red list of the international association for nature conservation (iucn) should be more carefully monitored using laws, media and educational tools and methods, and the necessary precautions should be urgently taken. j o u r n a l p r e -p r o o f although the first choice for influenza control and reducing the effects of epidemics is a vaccine, it is also known that it is not the fastest and most effective option since modifications in viral proteins require annual adaptation of the influenza vaccine formulation, as noted by nachbagauer and palese (2020) . considering the side effects and complications of antiviral medicines, the search for more effective remedies for fast-spreading pandemic influenza strains continues intensively all over the world today. due to their easy production, low cost, water-solubility, low toxicity and selective effects, medicinal plants, especially herbal essential oils and antiviral compounds found in their aqueous extracts are the most studied natural ingredients in recent times (grienke et al., 2009) . therefore, natural products such as traditional herbs show great promise in the development of potentially effective new antiviral drugs. particularly, recent studies on phytochemicals, such as quercetin, chlorogenic acid, mentofin, and linalool abundantly found in many plants and vegetables, eliminate the efforts and huge costs of finding lots of antiviral vaccines that need to be renewed every year and allow us to be more optimistic about the successful management of the next influenza outbreaks. turkey has remarkable potential for serious research on this topic due to having vast ethnomedicinal experience and the richest flora of europe and the middle east. this study, conducted in this regard, is the first nationwide ethnomedical screening study conducted on flu treatment with plants in turkey. in particular, we would like to emphasize that the most common detected genus members, such as sideritis (16 taxa; 7.1%), salvia (12; 5.4%), thymus (12; 5.4%), and origanum (10; 4.5%) may be more efficient in terms of the anti-influenza targeting than other genera for the interest of the sectors that are researching new natural drug sources. through this study, we strongly recommend these 35 (15.6%) plants, which have proved their high antiinfluenza activities and inhibition potentials in the experimental studies, to be subject to clinical research and for widespread use in the near future. also, with 189 (84.4%) taxa detections that have not been investigated yet, it is an important resource for both national and international pharmacological researchers. clinical research and evaluation studies required for standard compliance for human use, starting especially with the fifteen plant taxa whose use records against both malaria and influenza were presented in this study, can be begun. with a possible mass production of one or more malaria-like drugs, a significant contribution can be provided to the indigenous people living in that region and to the national economy. therefore, more experimental studies are urgently needed to understand the true value of these plants. based on the data to be obtained, we believe that the future extension of anti-influenza studies, including plant taxa that are frequently used in turkish folk medicine, would be a more effective option. adem, s., eyupoglu, v., sarfraz, i., rasul, a., ali, m., 2020. j o u r n a l p r e -p r o o f (2010) (2010) potential of selected lamiaceae plants in anti (retro) viral therapy 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and food security centre for science technology of the non-aligned and other developing countries determination of antiviral activity and cytotoxicity of selected sage (salvia l.) species burdur ili bitki envanteri (ekonomik, nadir ve endemik bitkileri). sistem ofset sağlıklı bir yaşamdır yabancı otlar: türk mutfak kültürü üzerine araştırmalar osmanlı ağaç kültüründe yeni ve egzotik bir tür: okaliptüs. çağdaş türkiye tarihi araştırmaları dergisi ethnomedicinal uses of the wild vascular plants from european turkey (turkish thrace) ethnopharmacological survey of medicinal plants in ulukışla (niğde-turkey) local knowledge of medicinal plants and wild food plants among tatars and romanians in dobruja traditional uses of medicinal plants in solhan (bingöl-turkey) ethnobotanical study on medicinal plants in bingöl (city center) (turkey) antiviral activity of the oseltamivir and melissa officinalis l. essential oil against avian influenza a virus (h9n2 anti-influenza a virus effect of 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as a basis for antiviral drug development quercetin as an antiviral agent inhibits influenza a virus (iav) entry i̇yileştiren bitkiler an ethnobotanical survey in selected districts of the black sea region (turkey) plants used as folk medicine in some settlements of the marmara region ethnobotanical features of datça peninsula (muğla) inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (sambucus nigra l.) during an outbreak of influenza b panama eaten raw, infusion, decoction, jam, marmalate tuzlacı and erol (1999) , saraç (2005) , koçyiğit and özhatay (2006) , özhatay et al. (2009 özhatay et al. ( ), ugulu et al. (2009 j o u r n a l p r e -p r o o f (2017) 13 5.8 black sea ozturk et al. (2017a) 13 5.8 southeastern anatolia sargin and büyükcengiz (2019) 13 5.8 mediterranean tuzlacı and doğan (2010) 13 5.8 eastern anatolia tuzlacı and erol (1999) 13 5.8 mediterranean ertuğ (2004) 11 4.9 aegean güneş and özhatay (2011) 11 4.9 eastern anatolia kılıç (2016) 11 4.9 eastern anatolia kilic and bagci (2013) 11 4.9 eastern anatolia guzel and guzelsemme (2018) 10 4.5 mediterranean ozturk et al. (2017b) 10 4.5 mediterranean saraç (2005) 10 4.5 all regions tetik et al. (2013) 10 4.5 eastern anatolia yeşilyurt et al. (2017b) 10 4.5 marmara akgül et al. (2016) 9 4.0 central anatolia bulut et al. (2017a) 9 4.0 aegean cansaran and kaya (2010) 9 4.0 black sea güner and selvi (2016) 9 4.0 marmara nacakcı and dutkuner (2015) 9 4.0 mediterranean özçelik ( (2010), güneş and özhatay (2011) , i̇şler (2017) lamiaceae (2010) alcea rosea l. not specified ( kim and chung (2018) nigella sativa l. not specified (seeds) enhance immune responsiveness and suppress pathogenicity of influenza viruses in turkeys umar et al. (2016) olea europaea l. not specified (leaves) blokes the receptor site of the viruses mehmood et al. (2018) olea europaea l. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson (2012) j o u r n a l p r e -p r o o f sambucus nigra l. not specified (fruits) exhibit a specific neuraminidase-inhibiting effect krawitz et al. (2011) silybum marianum (l.) gaertn.silymarin (seeds) reduces cytopathic effect (cpe) and inhibits viral mrna synthesis with no cytotoxicity song and choi (2011) thymbra capitata (l.) cav. carvacrol (essential oil) shows significant antiviral activity. olive oil was included in formulations to ameliorate its potential cytotoxic effects. vimalanathan and hudson (2012) thymbra capitata (l.) cav. apigenin, thymol (aerial parts-essential oil)both in influenza a/h1n1 and hrv14, replication cycle and progeny virus production were significantly decreased after the treatment with capeo (an essential oil combination based on three aromatic plants ( cota tinctoria (l.) j.gay* not specified (aerial parts) no correlation was found between antiviral activity and fatty acid contents of the extracts. orhan et al. (2009) ficus carica l.* not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. (2015) olea europaea l. * not specified (fruits) the results indicated that the prepared emulsions could elicit a little degree of immunity, but they could not inhibit the anamnestic response and infection. najjari et al. (2015) origanum acutidens (hand.-mazz.) ietsw. * none of the extracts inhibited the reproduction of influenza a/aichi virus in mdck cells sökmen et al. (2004) rosmarinus officinalis l. * carnosic acid (aerial parts) inhibit both a-and b-type hrsv, while it does not affect the replication of influenza a virus shin et al. (2013) teucrium polium l.* not specified (aerial parts) no significant effects on influenza virus infectivity derakhshan (2015) * the taxa that have no significant result for virus inactivation.j o u r n a l p r e -p r o o f key: cord-289439-jrvl0ykn authors: nelson, martha i.; lloyd-smith, james o.; simonsen, lone; rambaut, andrew; holmes, edward c.; chowell, gerardo; miller, mark a.; spiro, david j.; grenfell, bryan; viboud, cécile title: fogarty international center collaborative networks in infectious disease modeling: lessons learnt in research and capacity building date: 2018-10-23 journal: epidemics doi: 10.1016/j.epidem.2018.10.004 sha: doc_id: 289439 cord_uid: jrvl0ykn due to a combination of ecological, political, and demographic factors, the emergence of novel pathogens has been increasingly observed in animals and humans in recent decades. enhancing global capacity to study and interpret infectious disease surveillance data, and to develop data-driven computational models to guide policy, represents one of the most cost-effective, and yet overlooked, ways to prepare for the next pandemic. epidemiological and behavioral data from recent pandemics and historic scourges have provided rich opportunities for validation of computational models, while new sequencing technologies and the ‘big data’ revolution present new tools for studying the epidemiology of outbreaks in real time. for the past two decades, the division of international epidemiology and population studies (dieps) of the nih fogarty international center has spearheaded two synergistic programs to better understand and devise control strategies for global infectious disease threats. the multinational influenza seasonal mortality study (misms) has strengthened global capacity to study the epidemiology and evolutionary dynamics of influenza viruses in 80 countries by organizing international research activities and training workshops. the research and policy in infectious disease dynamics (rapidd) program and its precursor activities has established a network of global experts in infectious disease modeling operating at the research-policy interface, with collaborators in 78 countries. these activities have provided evidence-based recommendations for disease control, including during large-scale outbreaks of pandemic influenza, ebola and zika virus. together, these programs have coordinated international collaborative networks to advance the study of emerging disease threats and the field of computational epidemic modeling. a global community of researchers and policy-makers have used the tools and trainings developed by these programs to interpret infectious disease patterns in their countries, understand modeling concepts, and inform control policies. here we reflect on the scientific achievements and lessons learnt from these programs (h-index = 106 for rapidd and 79 for misms), including the identification of outstanding researchers and fellows; funding flexibility for timely research workshops and working groups (particularly relative to more traditional investigator-based grant programs); emphasis on group activities such as large-scale modeling reviews, model comparisons, forecasting challenges and special journal issues; strong quality control with a light touch on outputs; and prominence of training, data-sharing, and joint publications. in 2018 the fogarty international center celebrates its 50th year as the only part of the us national institutes of health focused singularly on global health. the fogarty international center (fic) uses high return-on-investment approaches to maximally impact global health, including (a) establishing strong international collaborative research networks, (b) building capacity for research in lowand middle-income countries (lmics) through training, and (c) liaising with a wide range of other us government agencies and private partners with a shared interest in advancing global health. here, we describe two flagship fic-led programs that identified critical gaps in infectious disease research and formed global networks of researchers to address the scientific questions most needed to inform policy and outbreak response. the 2001 foot-and-mouth disease outbreak in the uk, the 2009 influenza a/h1n1 pandemic, and the 2013-2016 epidemic of ebola in west africa underscore the continual threat of emerging and re-emerging acute infections. knowledge of the mode of spread of a pathogen and the subpopulations at highest risk of transmitting the pathogen and experiencing severe disease can inform key decisions about social distancing and prioritization of therapeutics and vaccines. designing disease models rooted in empirical data, enhancing existing tools for data analysis, and teaching collaborators around the world how to apply these tools represent highly cost-effective ways to prepare for the next infectious disease outbreak. past disease events have highlighted the capabilities of infectious disease modeling and molecular epidemiology to effectively guide outbreak responses, but also revealed persistent challenges in making predictions in data-scarce and rapidly changing field settings (woolhouse, chase-topping et al. 2001; keeling, woolhouse et al. 2003; heesterbeek, anderson et al. 2015; woolhouse, rambaut et al. 2015; dudas, carvalho et al. 2017) . the increasing availability of large, electronic datasets, or "big data," presents new opportunities for scientists to understand drivers of disease. electronic health records, social media, satellite imagery, and cell phone records provide highly granular information on human behavior, the environment, transmission patterns, and disease burden (lazer, kennedy et al. 2014; bansal, chowell et al. 2016; simonsen et al., 2016) . recent advances in next-generation sequencing techniques have markedly increased the amount of pathogen genetic data available for study, providing almost real-time insights into how pathogens from different locations are genetically related and infer transmission patterns (dudas, carvalho et al. 2017) . increasingly, researchers are devising methods to collect data even in resource-limited settings. the highly portable minion sequencer demonstrated how real-time wholegenome sequence data could be generated in remote locales during the zika epidemic (quick, loman et al. 2016; faria, quick et al. 2017) . mobile phone records and remotely-sensed nightlights present additional strategies to track human movements and contacts in resourcelimited settings wesolowski, eagle et al. 2012; wesolowski et al., 2016b) . since its inception in 2001, the division of international epidemiology and population studies (dieps) has played a central role in promoting the growth of a vibrant and multi-disciplinary infectious disease modeling community through its misms and rapidd programs. together, these programs have strengthened infectious disease modeling on a global scale by developing research and training networks, facilitating collaboration and data sharing, developing and disseminating new methodologies, and building a stronger collective voice for evidence-based policymaking. most critically, these programs have been catalysts for other larger us agencies and governments worldwide to expand support for infectious disease modeling research and to incorporate models into policy decision-making. the misms project was initiated in 2001 to build global research capacity for the study of influenza viruses using computational methods (http://misms.net, box 1). the acronym originally referred to the multinational influenza seasonal mortality study, although the program quickly expanded beyond mortality studies. misms has a mission to promote research, training and inform policy, more specifically (i) to characterize the global epidemiological and evolutionary dynamics of influenza in different host species, and (ii) to empower influenza researchers globally to study influenza and inform policymaking in their own countries. towards this end, 18 technical training workshops were conducted on six continents during 2007-2018. these workshops focus on reviewing the state of influenza research and epidemiology in different global regions, and hands-on teaching of time series analysis of contemporary and historical outbreaks, control measures, mathematical transmission models, and phylogenetic approaches (fig. 1) . misms supports interdisciplinary approaches by fostering collaborations between historians, demographers, ecologists, evolutionary biologists, vaccine manufacturers, microbiologists, epidemiologists, and policymakers, as evidenced by the rich diversity of publications (fig. 2 ). none of the last three pandemics -influenza a/h2n2 in 1957, a/ h3n2 in 1968, and a/h1n1 in 2009 -originated in a high-income country. building capacity for influenza research in low-and middleincome countries is an essential but underfunded part of global pandemic preparedness and a key goal of the misms program. a central feature of misms workshops has been partnership with local organizations to help identify talented scientists in under-resourced settings. workshops are held either in low-or middle-income countries, or the program provides funding for researchers from these countries to attend workshops in high-income countries (box 1). the 2009 misms workshop in dakar, senegal, was co-organized with institut pasteur and was the first influenza workshop ever held in africa. in addition to periodic workshops that provide intense short-term training to 30-100 participants, misms staff also host junior and mid-career researchers from central america, europe, africa, and asia for further training at the nih for periods ranging from a few months to several years. these scientists gain deep expertise in epidemiological and phylogenetic approaches and establish research programs to inform policy in their home countries. organizing workshops in under-studied tropical settings brought critical attention to the burden of influenza and the variability of seasonal patterns in less connected locales and warm, humid climates bloom-feshbach, alonso et al. 2012) . increased recognition of the global burden of influenza in tropical and semi-tropical regions also has encouraged uptake of routine annual vaccination in many middle-income countries. however, several misms studies rooted in epidemiological and virological data have pointed out that the semi-annual schedule is designed for wealthier countries in the temperate regions in the northern and southern hemispheres, and are suboptimal for many tropical countries that experience influenza at different times of the year, or have multiple peaks (alonso et al., 2007; de mello, de paiva et al. 2009; tamerius et al., 2011; bloom-feshbach, alonso et al. 2012; green, andrews et al. 2013; alonso et al., 2015a,b; ayora-talavera, flores et al. 2017) . misms research has informed whether a tropical country should opt for the northern or southern hemisphere vaccine formulation -or in some cases both. large countries with heterogenous influenza patterns and climate, such as china, mexico and brazil, may require different vaccine recommendations for their northern and southern regions (alonso et al., 2007; de mello, de paiva et al. 2009; ayora-talavera, flores et al. 2017) . additionally, misms research demonstrating the low impact of senior vaccination programs in the us and italy paved the way for expanded vaccination why have rapidd and misms workshops been so successful? since 2007, rapidd has sponsored 118 workshops on topics ranging from gain-of-function experiments to the latest developments in particle filtering methods. these workshops have had a remarkable track record of enabling constructive syntheses of current frontiers in the science of disease modeling, and in spurring innovation and analysis of case studies to advance those frontiers. they also have earned a strong reputation in the field as important venues for scientific discourse. here we summarize distinctive elements of those workshops that we believe have contributed to their success. • lightweight and nimble proposal process, steered by two generous and visionary scientists that had deep expertise in the field. the program 'let a thousand flowers bloom' by accepting workshop proposals on a rolling basis with a two-page initial proposal. for promising proposals, program leadership then iterates with proposers to refine the focus on rapidd goals, ensure solid deliverables, and optimize the participant list. • small size and balanced composition. research workshops are generally kept to 10-20 participants, to ensure free-flowing and natural discussion. age structure is key, with emphasis on including junior scientists (especially postdocs) so there are people with the time and incentive to do follow-up work. • flexible structure. adapt meeting structure to meet scientific needs. some workshops focus on conceptual or methodological issues, others on particular disease systems. some of the best workshops had a central theme combined with data in hand and time to analyze it. • keep workshops short (typically 1.5-3 days) and maximize the value of in-person meetings. pre-workshop teleconferences (among leaders or the whole group) help to reach consensus on goals and scope, so talks are on target and preliminary work can get done. avoid overloading the schedule with formal talks, to ensure ample time for discussion and breakout groups; some participants can act as synthesizers or reactors rather than giving talks. relaxed interactions on a hike or at a pub usually pay off. • designed for follow-through. before the meeting, specify deliverable and timelines, and identify who will lead these efforts (often rapidd postdocs or workshop organizers). get key pieces such as data sets and foundational analyses in place beforehand. require workshop reports within a few months, summarizing the scientific content of the meeting, progress toward deliverables, and new opportunities that have emerged. support follow-up when warranted, including 'working meetings' of project leaders, but also be prepared to stop threads that are not productive or not aligned with program priorities. since 2005, misms has convened 18 training workshops on all 5 continents and trained several hundreds of scientists on computational methods for infectious diseases. the unique characteristics of these workshops include: • the primary output of these workshops is technology transfer; i.e. capacity building in infectious disease analytics. participants are not expected to become modeling experts after a short workshop, but instead they should understand principles of data analysis and modeling (to the point that they are able to understand a disease modeling article). • misms workshops are typically regionally-focused and organized in tandem with a local university, research institute, or ministry of health. regional participants are identified based on an exhaustive pubmed search (especially in early years of the misms program), word-ofmouth, and existing misms contacts (particularly as the program matured). public health experts from who, cdc, and local institutions, are always invited. • workshops are sometimes organized in conjunction with a larger influenza scientific meeting to decrease travel costs and optimize participation. • weeklong workshops include 2 days of general scientific session and 3 days of hands-on training. the general session is meant to expose the state of the art of influenza epidemiology in the region and highlight success stories in influenza modeling and misms collaborations. the hands-on training session demonstrate methods in time series analysis, transmission models, and evolutionary analyses. • workshops are open to scientists and public health experts who do not have prior training in infectious disease modeling, e.g. veterinarians, clinicians, virologists, lab technicians, policy makers, etc. • flexible travel support for participants based on abstract selection process; focus on participants bringing data that may be amenable to modeling, those who have a clear training plan, and those from underserved areas. • training portion of the workshop is limited to 30 participants, for 5-8 faculty, to facilitate one-on-one interactions. • participants are encouraged to bring their own data; at least a day is devoted to small-group analyses of these data. sample datasets are always available for demonstration purposes and for those unable to bring data (or with sparser datasets). • while the workshops are focused on influenza, participation is open to scientists working on related infectious diseases, recognizing that relevant (or specific) influenza data may be scarce in some countries, and that computational skills are cross-cutting. • workshop deliverables include joint publications and supplementary issues that spur collaborative work (for instance, supplementary issues on big data for infectious disease surveillance or historical pandemics bansal, chowell et al. 2016; simonsen et al., 2016)) • similarly to rapidd, there is opportunity for follow-up beyond the workshop, primarily via longer visits to nih for more extensive training with misms staff, and occasionally, through remote collaborations. workshops can also be organized in the same region a few years later to gauge progress. misms staff provides support for completion of analytical work and manuscripts, where workshop participants are recognized as first authors. programs targeted at children who are important foci of transmission (simonsen et al., 2003; simonsen, reichert et al. 2005; rizzo, viboud et al. 2006; simonsen et al., 2009) . global vaccine strategies also require an understanding of how viruses migrate long distances between countries, and how viruses evolve antigenically to require vaccine strain updates (ferro, budke et al. 2010; adler, eames et al. 2014) . active areas of misms research include a deeper understanding of influenza migration pathways at different spatial scales, climatic and demographic drivers of influenza seasonality, and the mapping between vaccine match and population-level protection, all of which can affect vaccine policy (box 2). the global network of collaborators that grew out of the misms program is a unique resource to leverage in the event of the next pandemic influenza or another infectious disease threat. as a case in point, in 2009, the presence of misms-trained researchers in mexico facilitated early reporting of severity and striking age-shifts in mortality patterns that are signatures of new pandemic viruses (chowell et al., 2009a , 2009c ). during the critical early stages of the pandemic, rapid reporting of these epidemiological patterns informed use of limited resources, including vaccine and m.i. nelson et al. epidemics 26 (2019) 116-127 therapeutics, as well as interventions such as school closures (chowell et al., 2009a , 2009c , chowell, echevarria-zuno et al. 2011 . misms collaborations also focus on "archeo-epidemiology" studies, or studies of past influenza pandemics and other historic scourges . such studies have provided estimates of influenza transmission intensity, age-structured mortality patterns, spatial dynamics, and the distribution of mortality across multiple pandemic waves as far back as the 1880s (andreasen, viboud et al. 2008; bloom-feshbach et al., 2011; simonsen et al., 2011) . quantitative studies of past patterns can help predict the course of future pandemics and inform the range of plausible scenarios for preparedness, including elevated mortality occurring several years after the initial policy impact of rapidd and misms: select success stories. • vaccination: misms studies in large and climatologically-diverse countries have identified geographic differences that affect annual influenza vaccination programs. because of the timing of influenza epidemics and composition of circulating strains in southern china and southern brazil, southern provinces should use the southern hemisphere influenza vaccine, while the northern part of these countries should use the northern hemisphere vaccine (miller, viboud et al. 2008; chowell, viboud et al. 2009; yu, alonso et al. 2013; alonso et al., 2015a,b ). • surveillance: misms work on the phylodynamics of swine influenza viruses highlight the importance of undersampled "source" locations with large pig populations and intense swine flows to other regions, such as russia. surveillance efforts should target these regions that are expected to generate important global diversity of swine viruses ). • pandemic control measures: mexico implemented nationwide social distancing measures to control the 2009 influenza pandemic (combination of mandatory school closure, restaurant closure, and cancellation of large gatherings) during an 18-day period in late april and early may 2009. misms researchers were among the first to work with the ministry of health to assess the dynamics of the outbreak and the impact of interventions. by fitting transmission models to surveillance data, they found that social distancing reduced influenza transmission by one-third, lending support to these costly measures. beyond the 2009 pandemic, this works suggests that social distancing interventions could be implemented to mitigate severe outbreaks and alleviate the pressure on healthcare, for limited periods of time and until other interventions (such as vaccination) can kick in (chowell, bertozzi et al. 2009; chowell, viboud et al. 2009; chowell, echevarria-zuno et al. 2011 ). • addressing data gaps: on the rapidd front, modelers thought creatively to find ways to fill data gaps that were essential obstacles to resolving policy-relevant health problems. for example, a common problem in livestock diseases is the lack of information on farm locations and animal movements, especially in the us. these are key ingredients to design transmission models, evaluate interventions, and help prepare for potential outbreaks such as foot-and-mouth disease. the rapidd network designed a method to leverage veterinary records and mandatory licenses filed by farmers. further, much simulation work was done based on the richer uk dataset informing farm locations, animal movements, and observed foot-and-mouth disease outbreaks, to strengthen us models and remedy data gaps. • gain-of-function studies and pandemic risk prediction: as the debate on gain-of-function studies intensified, rapidd organized a twoworkshop series on "assessing the outbreak potential of nonhuman influenza viruses using sequence-based risk approaches'" and "modeling and predicting influenza phenotypes" to explore modeling of pandemic potential. gain-of-function studies indicated that an avian influenza virus undergies a series of genetic changes for human adaptation, which are required for efficient transmission among mammals. the workshops assembled global experts to synthesize current knowledge about the three key steps of the adaptation process: switch of the hemagglutinin surface protein to mammalian alpha-2,6 sialic acid binding, enhancement of ph-and temperature-dependent stability of the hemagglutinin, and adaptation of the viral polymerase to function in mammalian cells. these workshops spurred modeling work to infer pandemic potential based on ferret transmission experiments, and further work is underway to explore whether mapping of genotype-tophenotype traits is possible, despite rampant epistatic interactions across the influenza genome (buhnerkempe et al., 2015a) . other avenues for policy-oriented research include the optimization of global influenza surveillance strategies to identifies early precursors of pandemic strains. • vaccination decline in the aftermath of ebola: another example of the link between rapidd research and policy is a study on the precipitous decline in childhood immunization during the 2014-2015 ebola outbreak (takahashi et al., 2015) . rapidd modelers estimated that around one million children in liberia, sierra leone and guinea were vulnerable to measles following the suspension of vaccination campaigns during the ebola outbreak. the study concluded on the urgent need to mount an aggressive vaccination campaign as soon as the ebola outbreak subsided. this advice was heeded by public health authorities, as measles vaccination campaigns targeting several million children were launched in sierra leone and guinea in october 2015 and in liberia a few months earlier. emergence of a pandemic virus (box 2). misms also has worked actively to bridge human and veterinary research communities and understand the emergence of pandemic viruses at the animal-human interface. two workshops held in ames, iowa, usa, in collaboration with us department of agriculture, focused on swine influenza, while another held in padua, italy, in collaboration with istituto zooprofilattico sperimentale delle venezie, focused on avian influenza, providing training opportunities in an active area of influenza research. improvements to genetic sequencing technologies have reduced costs worldwide and advanced the field of 'phylodynamics', in which epidemiological dynamics are inferred from pathogen sequence data. in the early 2000s, new computational packages, including bayesian evolutionary analysis sampling trees (beast), provided the capacity to infer detailed evolutionary, demographic and spatial patterns from sequence data (lemey et al., 2009a,b; lemey, rambaut et al. 2010) . however, in-depth studies of influenza virus evolution require large numbers of viral sequences carefully sampled over time and space, which are unavailable in many low-and middle-income countries. to address this gap, fogarty's misms staff partnered with the national institute of allergy and infectious diseases (niaid) and national library of medicine (nlm), to provide whole-genome sequencing of influenza virus collections free of cost via the influenza genome sequencing program (box 3). when this sequencing project began in 2005, there were fewer than 100 complete influenza virus genomes available on genbank. as of 2017, almost 20,000 influenza virus genomes have been sequenced through this project and made publicly available. a strong linkage between the influenza genome sequencing program and fogarty's misms network of global researchers, and their viral collections, has helped address important gaps in influenza sequence availability and in turn transmission dynamics. the program targeted understudied aspects of influenza virus evolution, including large regions of the viral genome not typically sequenced, under-sampled populations in tropical settings, influenza b virus, and non-human mammalian hosts (box 3). the program fostered a global culture of data-sharing that facilitates large-scale comparative studies across subtypes, regions, and species, advancing our fundamental understanding of influenza virus evolution and ecology. despite increased availability of influenza genetic and epidemiological data, there are still important gaps, particularly regarding surveillance in africa and south america and at the animal-human interface (viboud, nelson et al. 2013) , and poor integration of different data streams (e.g., epidemiological, antigenic and genetic information), representing target areas of future misms efforts. because influenza is unique in combining long-term pandemic risk with a continued need for annual vaccine updates to reduce the burden of seasonal influenza, the field has traditionally enjoyed far better attention than other respiratory infections. as genomic and epidemiological data for other respiratory pathogens increases, a natural extension for misms would be to broaden its scope beyond that of influenza. recognizing this potential, misms collaborators have embarked on modeling studies of pneumococcus and respiratory syncytial virus, and their interactions with influenza (weinberger, simonsen et al. 2012; shrestha, foxman et al. 2013; weinberger, harboe et al. 2014; shrestha, foxman et al. 2015; weinberger, klugman et al. 2015) . with additional resources, a more systematic broadening of the scope of the misms study to other important human respiratory pathogens would provide fresh opportunities to enhance understanding of multiple disease systems while drawing on an established global research network and a wealth of computational tools. development of infectious disease models deeply rooted in empirical data can improve planning for, and response to, infectious disease threats; however, the link between modeling groups and policy makers remains tenuous in many countries (heesterbeek, anderson the influenza genome sequencing project, a large-scale collaboration between nih institutes and j. craig venter institute, transforms our understanding of influenza virus evolution. et al. 2015 ; metcalf, edmunds et al. 2015) . fogarty played an important role in promoting the field of modeling population-based impact of biological threats within the u.s. government. immediately following the events of september 11 th , 2001 and before the anthrax-laced letters that began just one week later, dieps helped establish intergovernmental networks to model and address biological threats. these efforts contributed to launch an extramural modeling funding program on models of infectious disease agents (midas) managed by the nih national institute of general medical studies and nascent funding to nih fogarty to study bioterror threats. in 2007, the rapidd program was established with funds from the department of homeland security to build a hub-and-spokes network of infectious disease modelers working at the interface of policy and academic research, in close collaboration with government. the program's mandate was to advance us capacity in infectious disease modeling by pioneering new methodologies, studying key emerging disease threats, and training the next generation of scientists (box 4). rapidd departed from traditional grant-based programs in its flexibility and concentration on working group and workshop activities, and on developing research capacity. a key success of rapidd since 2007 has been to promote collaborative research and synergies between modeling groups beyond core participants of the program (box 1). rapidd research has been at the forefront of responding to the greatest infectious disease crises of recent years, particularly the 2013-16 ebola epidemic in west africa and the zika epidemic in the americas. modeling work helped to identify key routes of ebola transmission and characterize effectiveness of intervention policies (team, aylward et al. 2014 , camacho, kucharski et al., 2015a , 2015b , kucharski, camacho et al., 2015 team, agua-agum et al. 2015) . further work, in collaboration with who's consortium for ebola modeling, identified risk factors for transmission including severe symptoms, death, non-hospitalization, older age, and travel history (international ebola response team et al., 2016) . modeling work further demonstrated the unintended consequences of the ebola-associated healthcare disruption on childhood immunization, and stressed the need to promote supplemental vaccination campaigns, particularly for measles (takahashi et al., 2015) . further, rapidd collaborators have developed predictive maps of the spread of the zika virus in the americas, driven by environmental conditions and population mobility (bogoch, brady et al. 2016) . another rapidd modeling study analyzed factors shaping the efficacy of screening air travelers for emerging pathogens, including ebola and mers-coronavirus . taken together, careful literature surveys of prior modeling work, in addition to primary research done by rapidd, form a substantial body of "case law" for infectious disease modeling, which can be used as reference to understand and model outbreaks caused by new pathogens with similar properties. as an important demonstration of this principle, prior modeling work on dengue provided the groundwork for rapid development of regional and global models for the spread of zika virus (bogoch, brady et al. 2016 ). during 2007-2015, rapidd functioned primarily via parallel working groups focused on four areas identified as having important research gaps: (i) methodological and data issues, (ii) zoonoses and pathogen emergence, (iii) vector-borne infections, and (iv) disease dynamics in small mammals. the working groups conducted primary research and led multi-disciplinary workshops on emerging research frontiers (box 1). since 2007, rapidd organized 118 workshops across a wide range of topics, with participation from thousands of scientists and government employees globally. workshops produced large-scale reviews of the state-of-the-art of disease modeling in particular areas, and opinion pieces discussing new frontiers of model-data synthesis (katz, plowden et al. 2004; buhnerkempe et al., 2015b; frost, pybus et al. 2015; gog, pellis et al. 2015; heesterbeek, anderson et al. 2015; lloyd-smith et al., 2015a,b; metcalf et al., 2015a, b) . working group members then pursued follow-up research to address the identified gaps by developing new modeling approaches or new applications of models to understudied but important diseases. the direct link between ra-pidd research and policy was ensured by periodic presentations to the us white house scientific offices. overall, since 2007, rapidd has made substantive contributions across a remarkable range of public health challenges (box 2), from strategies to deploy current and future vaccines to the unique role of bats as zoonotic reservoirs. while a comprehensive review of the more than 1000 rapidd publications is beyond the scope of this review (fig. 3, table 1 ), below we single out a few achievements of the program. many of the most important emerging disease threats involve zoonotic pathogens originating from animal reservoirs. by conducting a seminal review of 442 published modeling studies of 85 zoonotic pathogens, rapidd researchers identified critical areas in need of further attention ). the review highlighted the marked predominance of studies of directly transmitted infections with simple life cycle (e.g., influenza, sars), over infections involving multiple hosts with complex life cycles (vector-borne diseases, protozoans, food-borne infections). it identified a dearth of modeling research on looming threats including ebola, chikungunya and yellow fever viruses, which was regrettably prescient in light of subsequent epidemics around the world (who, 2017). crucially, the review noted that very few studies linked dynamics across the animal-human interface, where the defining process of zoonotic spillover occurs, and few box 4 : training the next generation of scientists: rapidd post-doctoral fellows. • outstanding fellows handpicked and hired for 2 years minimum, and up to 4. • comfortable prestigious fellowship, with independent and flexible funding for travel. • strong impetus to work with the larger rapidd network of outstanding scientists, particularly by organizing workshops and working groups, monthly webex presentations, periodic policy-oriented seminars at the white house, and annual rapidd network meeting. • post-doctoral fellows helped drive major collaborative reviews of modeling gaps (eg, zoonoses and emerging infections, vector-borne diseases) -a superb bonding exercise leading to high-profile publications in their chosen fields. • community of rapidd fellows learned from each other on topics ranging from communication skills to methodological issues. • of the 13 rapidd fellows, 11 are in tenured or tenure-track academic positions, 1 works for the us government and 1 for a private company (as of july 2018). m.i. nelson et al. epidemics 26 (2019) 116-127 models incorporated evolutionary processes. to address these gaps, the zoonosis working group launched a series of methodological developments to link models across the animal-human interface, analyze the stuttering chains of transmission that foreshadow emergence events, and describe the evolutionary processes linked to pathogen emergence, with key applications in monkeypox, mers-coronavirus and influenza h5n1 and h7n9 viruses rimoin, mulembakani et al. 2010; blumberg and lloyd-smith, 2013a, b; park, loverdo et al. 2013; blumberg, enanoria et al. 2014; chowell, blumberg et al., 2014; kucharski and althaus, 2015; kucharski, mills et al. 2015; lloyd-smith et al., 2015a,b; plowright, parrish et al. 2017) . similarly, the rapidd vector-borne disease working group's review of 388 studies of mosquito-borne pathogen transmission determined that most models were still primarily derived from mid-20th century methods pioneered by ross and macdonald smith et al., 2014) . in response, the group developed more refined models to capture important features of mosquito-borne infections, including mosquito life history, heterogeneous biting and fine-scale spatio-temporal variation in transmission. applying these methods to malaria and dengue highlighted the impact of incorporating realistic mosquito biology and biting heterogeneities on control efforts perkins, garcia et al. 2014; reiner, le menach et al. 2015) . in recent years, a major focus of rapidd research has been infectious disease forecasting, responding to a rising need in the public health community, particularly for influenza and emerging infections yang, cowling et al. 2015; yang et al., 2015a; yang, olson et al. 2016 ). in addition to rapidd's work on evaluating the impact of interventions in the midst of the west african ebola epidemic, rapidd launched an after-the-fact forecasting challenge using synthetic epidemiological data sets to assess model performances in a controlled environment (merler, ajelli et al. 2017; viboud et al., 2017a, b) . this unprecedented group effort revealed that the most accurate disease forecasts stemmed from ensemble predictions combining outputs from different models, since even the best models have weaknesses, and that prediction accuracy does not scale with model complexity. the forecasting challenge also strengthened communication and collaboration between modeling groups, laying the foundation for more effective response for the next public health crisis (viboud et al., 2017a (viboud et al., , 2017b . influenza forecasting represents an area of rich synergy between the rapidd and misms programs. a major aim of ongoing influenza research is to predict the emergence and trajectory of new seasonal strains, and hence improve the imperfect semi-annual vaccine strain selection process. forecasting work in this area was greatly enhanced by a 2016 workshop, organized in collaboration with who, that highlighted the need for better flow of genetic and antigenic information from public health agencies to the institutions and mathematical modelers to improve prediction accuracy and horizon (morris, gostic et al. 2017) . further, rapidd and misms collaborators were the first to analyze the potential benefits of broadly cross-protective influenza vaccination programs (arinaminpathy, ratmann et al. 2012; subramanian, graham et al. 2016 ). this is a particularly fruitful area of overlap between rapidd and misms, as universal influenza vaccine candidates enter pre-clinical trials, and an ideal opportunity to integrate network members' expertise on relevant evolutionary and immunological concepts with the global source-sink dynamics so unique to influenza russell, jones et al. 2008 ). although the four rapidd working groups had separate areas of focus, several synergistic themes rapidly emerged (box 5). one dominant theme was the spatiotemporal dynamics of pathogens, encompassing major strides in spatial dynamics and mapping of vector-borne infections, a broad effort to advance data-driven models for foot-andmouth disease in the us and abroad, and cutting-edge syntheses of novel and traditional data streams informing human demographics and mobility to produce a new generation of spatio-temporal epidemic models. another major rapidd theme on pathogen evolution -entailing phylodynamics, virulence evolution, host jumps and drug resistance -has generated methodological breakthroughs and important applications to influenza, dengue, malaria, and more. focused workshops re-examined current thinking on optimal drug treatment to minimize resistance evolution (read, day et al. 2011; kouyos, metcalf et al. 2014) , and the potential to predict influenza pandemic risk from viral sequence data lipsitch, barclay et al. 2016) . bibliometric analysis of the rapidd and misms programs. rapidd publications are based on any mention of the rapidd program in acknowledgments; this includes fellows and faculty funded by rapidd and/or participation to rapidd workshops that led to publications. misms publications are based on influenza research articles published by staff of the fogarty international center, as listed in affiliations. data collection performed on july 1, 2018. since 2007 rapidd has advanced the methods and applications of data-driven modeling of infectious diseases, with a clear focus on policy-relevant research, and has fostered a new generation of talented researchers in this field (box 4). the program's remarkable success arises from strong collaborations developed among the many participants over the years, its flexibility and agility in convening workshops on emerging research frontiers, and from providing freedom and unparalleled networking opportunities for talented post-doctoral scientists. looking forward, pending availability of funds, rapidd will continue its research emphases on emerging infections and vectorborne diseases, while growing new areas of focus on topical challenges such as on modeling the dynamics of anti-microbial resistance, and predicting the impact of next-generation vaccines such as for respiratory syncytial virus, malaria, typhoid, dengue, and the universal influenza vaccine. the fogarty-led misms and rapidd programs have been catalysts in advancing state-of-the-art global infectious disease modeling and training with tangible impacts in building capacity globally and guiding policies, such as defining age priority groups for epidemic and pandemic influenza vaccines, addressing optimal delivery of influenza vaccines in tropical and low-income countries, and highlighting gaps in measles vaccine coverage following the ebola outbreak in west africa. in addition to influencing policy, a major success of the misms and rapidd programs is reflected by the outstanding professional trajectories of their alumni, domestically and internationally, and the excellence and international scope of their publications (table 1 , box 4). of the more than 1400 publications of the misms and rapidd programs, which have been cited over 50,000 times, 28% include authors from lmics. many of the rapidd and misms trainees and collaborators are now established as senior researchers. both programs rely on global multi-disciplinary networks to identify and address research questions at the forefront of infectious disease transmission and evolution. both programs build off the "big data" revolution that provides increasingly detailed and abundant information on disease patterns and host behavior, leveraging a surge of pathogen genetic sequence data, exquisitely detailed epidemiological information derived from digital and social media, and human mobility and demographic proxies derived from mobile phones or remote sensing. new methodological approaches are needed to handle such a vast and diverse amount of information. further, both programs have long histories of promoting data sharing and development of publicly available methodological tools, with key contributions to the influenza genome sequencing program and database, the beast package for phylogenetic and associated analyses, and development of new transmission modeling packages (pomp, tsir) and spatial analysis software (epipoi) (alonso and mccormick, 2012; becker and morris, 2016; king, 2018) the misms and rapidd programs are also well connected with parallel efforts at nih and cdc and in the broader scientific community. for instance, within nih, there are strong synergies between rapidd and midas, a funding program in infectious disease modeling managed by the national institute for general medical sciences, and between misms and the centers of excellence in influenza research and surveillance (ceirs), funded by the national institute of allergy and infectious diseases. in parallel, there are strong ties between rapidd and misms and the cdc's influenza division, involving joint workshops, forecasting challenges (e.g., (merler, ajelli et al. 2017; viboud et al., 2017a viboud et al., , 2017b ) and post-doctoral exchanges. further, there is a tight link between misms and rapidd and national and global public health agencies, with regular participation of collaborators in the white house pandemic preparedness working group, us government modeling coordination groups, and who modeling groups. several of the misms workshops have been co-organized with, and in support of, foreign government agencies (fig. 1) , while many international government representatives have taken part in misms and ra-pidd workshops. despite these successes, some challenges are worth noting. the most key aspects of diseases dynamics investigated by rapidd. problematic is the lack of sustainable long-term funding for these programs more than 10 years after they were created, even though their combined annual costs are under 2m$, which is negligible relative to the costs of public health crises (see (office of science, 2016; world bank and ecohealth alliance, 2018) for a discussion of the value of infectious disease preparedness and modeling). it is worth nothing however that the low cost of computational modeling does not include funding for generation of biomedical, epidemiological and surveillance data, which are essential to support modeling work. and with their extended duration and distributed network structure, the rapidd and misms programs fall outside standard funding streams, despite their extraordinary efficiency in propelling leading-edge science and addressing problems of national interest (box 2). as the ebola and zika outbreaks clearly demonstrate, infectious disease threats are not in retreat advances in multi-pathogen diagnostics and sequencing will facilitate real-time molecular epidemiology during future outbreaks, but will require people on the ground and international partners who can rapidly interpret large amounts of data. development of new vaccines (e.g., respiratory syncytial virus, universal influenza) and the growing challenge of anti-microbial resistance will require improved models to optimize public health policies and understand pathogen evolutionary responses. in conclusion, the misms and rapidd programs illustrate the power of scientific diplomacy and collaborative research networks, with demonstrable successes in improving research and control of infectious diseases. we see no shortage of policy-relevant research themes for these networks to explore, including the emergence of new threats at the animal-human interface, microbial interactions within and between hosts, integration of traditional and novel data streams into ever more sophisticated models, short-and long-term disease forecasts, projections of the impact of novel vaccines, and anti-microbial resistance. fogarty-led disease modeling programs will continue to strengthen capacity in lmics for outbreak analysis, and in turn help control emerging infectious disease threats domestically and internationally. this article is dedicated to our fogarty colleague, dr. ellis mckenzie, who was instrumental in launching, growing and shepherding the rapidd program from 2007 until his death in 2016. ellis played a key role in promoting use of infectious disease models in government and mentored countless infectious disease modelers. the establishment and success of the program would have been impossible without ellis's gentle and inspired guidance. the rapidd program is indebted to the leadership, vision, and guidance of two outstanding and incredibly generous scientists who shepherded this program since 2007, bryan grenfell and ellis mckenzie. we acknowledge support from the department of homeland security and fic for the rapidd program, and the hhs pandemic threat unit, office of global affairs and fic for the misms program. we are grateful to felix wu, amherst college, for performing a thorough bibliometric analysis of the rapidd and misms publications and generating maps of co-authors. we are thankful to kate skoczdopole for continued 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viruses to fight other viruses: the influenza vaccine case date: 2020-04-22 journal: embo mol med doi: 10.15252/emmm.202012059 sha: doc_id: 304485 cord_uid: vouu56rr undoubtedly, vaccination is one of the health interventions showing major impact on humankind. vaccines remain one of the most effective and safest ways to tackle infections. the current coronavirus pandemic is not an exception, and we all hope that ongoing international efforts will succeed in developing a vaccine soon. in this scenario, the present work published in this edition of embo molecular medicine by demminger and colleagues (demminger et al, 2020) is timeliness to exemplify the steps needed to develop effective vaccines. t his study focuses on influenza, a seasonal pandemic with major annual impact on the health systems worldwide. this infection is associated with high mortality and morbidity in very young and old individuals. stories in the recent news have highlighted the unusual deadly influenza pandemic lasting from 1918 to 1920, the so-called spanish flu, highlighting the devastating impact of these viral infections. the current influenza prophylaxis is based on immunization with trivalent or quadrivalent influenza vaccines containing antigens from influenza a and b viruses. however, this vaccine is mostly strain specific and the process to produce it is timeconsuming. in addition, evidence indicates it does not provide protection against emerging zoonotic influenza a virus strains. overall, the need to develop a novel vaccine conferring protection as broad as possible is widely acknowledged. one observation key for this work is the discovery of protective antibodies against the surface glycoprotein hemagglutinin (ha) acting via interference with viral replication or via fc-receptor-mediated cellular cytoxicity (dilillo et al, 2016; wu & wilson, 2017) . demminger and colleagues decided to exploit an adeno-associated virus (aav) vector to express influenza antigens to elicit protective antibody responses. the selection of aav as vector offers several advantages. aav cannot replicate in humans, and data indicates they do not induce any pathophysiology. they are easy to produce under good manufacturing practice standards and can be re-administered if needed. of note, aav has been already tested to express influenza antigens and showed to confer protection (lin et al, 2009; sipo et al, 2011; xin et al, 2001) . demminger and colleagues developed aav vectors expressing wild-type ha, the surface exposed neuraminidase (na), or chimeric ha (cha) containing head regions from influenza a virus subtypes, or ha headless constructs. constructs were based on proteins encoded by the pandemic influenza virus a/california/7/2009 (h1n1) pdm (cal/7/9). control assays confirmed the robust expression of all antigens. next, they assessed the immunogenicity in mice challenged intranasally testing side-by-side empty aav vector (expressing gfp) and whole-inactivated virus (wiv) as controls. as expected, all aav constructs expressing influenza antigens did elicit reactive antibodies that correlated with the total serum igg titers. interestingly, aav vectors induced antibodies against a panel of different influenza viruses. somewhat unexpectedly, wiv vaccination also elicited broadly reactive antibodies. interestingly, neutralizing antibodies were found only in the sera of mice challenged with aav containing ha, or cha, and these antibodies were specific for the virus used for prime immunization. of significant therapeutic relevance, authors demonstrated that aav vector vaccination induced strong fc-activating antibody response against cal/7/9 but also against the mouse-adapted heterologous influenza virus pr8. these positive results encouraged authors to test whether aav vectors do protect mice from homologous (cal/7/9) and heterologous (pr8) challenges. although the mouse model has its limitations in terms of translational potential, it offers the opportunity to obtain first proof-of-concept information. as read-outs of infection, demminger and coworkers used weight loss and survival of mice over a 14-day period. mice vaccinated with the aav construct were protected, and preliminary data suggest that three doses of the vaccine were better than two doses. although these findings are sound, it is important to note that determination of viral titers in the tissues over time should be determined to solidify the read-outs based on animal behavior, clinical parameters, and/or survival. some control experiments revealed a reduction in viral titers in vaccinated animals. in a remarkable tour de force, authors went on to assess the protective efficacy of the aav vectors in ferrets. this animal model reproduces very closely the course of human influenza. in these experiments, authors also introduce a group immunized with a commercial quadrivalent influenza vaccine of season 2017/18. animals were vaccinated intranasally three times in 4week interval whereas the control group immunized with the commercial vaccine were given two doses by the intramuscular route. it can be argued that the different experimental regime of the groups is not suitable for a comparison between the different vaccine formulations. however, the aim of this work was to demonstrate whether the aav vectors do confer protection and not whether they are better than the commercial vaccine. in a 3-day infection protocol, ferrets vaccinated with the aav vectors showed clear signs of recovery assessing serous nasal exudate, congestion, frequent sneezing, wheezing, and depression. notably, only in those ferrets vaccinated with the aav-ha construct and the commercial vaccine was there a reduction in the load of virus although no sterile immunity was induced. virus clearance was associated with a significant reduction in pathophysiology and induction of neutralizing antibodies. this elegant study follows a comprehensive design from in vitro characterization of the aav vectors to animal pre-clinical studies probing two different research models, one of them of significant translational potential (fig 1) . the use of research models approximating human disease, ferrets in this case, should be a golden standard approach in any work focusing on vaccine development. the value of the mouse model cannot be disregarded but, certainly, the time is ripe to probe models with increasing translational value. this work also reinforces the utility of aav vectors as platform to develop vaccines. aav vectors are already approved by the ema and fda for its use in humans and have been tested in pre-clinical immunization studies against infectious diseases. demminger and colleagues demonstrate that they should be considered as a valid platform for a broadly protective influenza vaccine. i anticipate that this comprehensive work will lead to significant advances in our quest to develop effective influenza vaccines and may result in similar research to tackle other infections. i will encourage the vaccinology field to consider and set clear parameters that should be determined in any research like the one presented in this issue of embo molecular medicine (demminger et al, 2020) . from clear guidelines on power calculation for the in vivo work to the biological read-outs that should be analyzed. among others, i will urge colleagues to consider essential a comprehensive immune profiling at the relevant mucosae coupled with the quantification of cytokines and chemokines. technology such as mass cytometry could improve significantly the breath of data obtained while, perhaps, uncovering new biology in terms of immune populations relevant for vaccine development. after developing aav vectors expressing influenza surface exposed antigens, authors followed an elegant pipeline probing their vectors first in mice and subsequently in ferrets, research model that accurately recapitulates human influenza infections. vaccines were administered via the intranasal route, key to develop protective antibodies. adeno-associated virus-vectored influenza vaccine elicits neutralizing and fcgamma receptor-activating antibodies broadly neutralizing anti-influenza antibodies require fc receptor engagement for in vivo protection a new genetic vaccine platform based on an adeno-associated virus isolated from a rhesus macaque vaccine protection against lethal homologous and heterologous challenge using recombinant aav vectors expressing codon-optimized genes from pandemic swine origin influenza virus (soiv) a perspective on the structural and functional constraints for immune evasion: insights from influenza virus a novel recombinant adeno-associated virus vaccine induces a longterm humoral immune response to human immunodeficiency virus aav vector expressing influenza surface exposed antigens testing in ferrets (model for human influenza infections) 2. key: cord-330512-nu8q72l9 authors: iskander, john; strikas, raymond a.; gensheimer, kathleen f.; cox, nancy j.; redd, stephen c. title: pandemic influenza planning, united states, 1978–2008 date: 2013-06-17 journal: emerg infect dis doi: 10.3201/eid1906.121478 sha: doc_id: 330512 cord_uid: nu8q72l9 during the past century, 4 influenza pandemics occurred. after the emergence of a novel influenza virus of swine origin in 1976, national, state, and local us public health authorities began planning efforts to respond to future pandemics. several events have since stimulated progress in public health emergency planning: the 1997 avian influenza a(h5n1) outbreak in hong kong, china; the 2001 anthrax attacks in the united states; the 2003 outbreak of severe acute respiratory syndrome; and the 2003 reemergence of influenza a(h5n1) virus infection in humans. we outline the evolution of us pandemic planning since the late 1970s, summarize planning accomplishments, and explain their ongoing importance. the public health community’s response to the 2009 influenza a(h1n1)pdm09 pandemic demonstrated the value of planning and provided insights into improving future plans and response efforts. preparedness planning will enhance the collective, multilevel response to future public health crises. influenza pandemics occur when an animal influenza virus to which humans have no or limited immunity acquires the ability, through genetic reassortment or mutation, to cause sustained human-to-human transmission leading to community-wide outbreaks (1) . the existence of a pandemic is currently determined by the extent of disease spread, not by the lethality of the disease caused by the novel virus (2) . during the twentieth century, influenza pandemics occurred in 1918, 1957, and 1968 . the 1918 pandemic, known as the "spanish flu" pandemic, was unique in that the highest number of deaths was among young, healthy persons. excess mortality in the united states during the 1918 pandemic was estimated at 546,000 deaths (3) . the pandemics in 1957 and 1968, although associated with death rates greater than those for seasonal influenza epidemics (3), were far less devastating than the 1918 pandemic. before 1976, public health planning for pandemics primarily occurred in response to detection of a novel influenza virus. this reactive mode continued despite the framework outlined in 1960 by us surgeon general l.e. burney for responding to the next pandemic. that framework involved recognition of the pandemic (i.e., surveillance), manufacture and distribution of vaccine, and identification of research needs (4) . large-scale infectious disease response planning may have been hampered by the tacit assumption that the government's public health resources were better directed to other priorities. in january 1976, a novel swine-origin influenza virus emerged among soldiers at fort dix, new jersey (5); 1 soldier died, and an estimated 230 were infected. the emergence of influenza virus of swine origin at fort dix led to the decision to mount a national immunization program (6) . the following events occurred subsequent to this decision: congress funded vaccine production and liability indemnification of manufacturers, vaccine was produced, a mass immunization campaign commenced, and 45.65 million persons were vaccinated in the united states (7) . initial fears that the virus would cause a pandemic did not materialize: sustained transmission did not occur outside of fort dix. the vaccination campaign began in october 1976 and was halted in december because of initial reports of a rare association between the so-called "swine flu" vaccine and guillain-barré syndrome; the association was later confirmed (7) . an influential policy review of the "swine flu affair" (i.e., the campaign to immunize the us population against a possible epidemic) identified several critical needs for future planning: 1) a more cautious approach to interpreting limited data and communicating risk to the public, 2) greater investment in research and preparedness, 3) clearer operational responsibilities within the federal government, 4) clear communication between planners at all levels of government, 5) strengthened local capacity for plan implementation, and 6) improved mechanisms for program evaluation (8) . in november 1977, separate from the fort dix outbreak, a strain of human influenza a(h1n1) virus reemerged in the former soviet union, northeastern china, and hong kong, china, even though the virus had not circulated since 1957. this strain primarily affected young persons, and caused mild illness (9) . the virus was found to be closely related to a 1950 a(h1n1) strain but dissimilar to the 1957 strain, suggesting that this 1977 outbreak strain had been preserved since 1950 (9) . the confluence of fears of a possible pandemic in 1976 followed by the reemergence of a new strain of circulating seasonal influenza virus in 1977 led to focused pandemic planning efforts in the united states. the primary purpose of this article is to describe us pandemic planning during 1978-2008, just before the onset of the influenza a(h1n1) pdm09 pandemic in april 2009. we believe that understanding the historical and policy context within which the a(h1n1)pdm09 pandemic occurred is helpful in assessing the implications of pandemic planning for responses to future pandemics and for ongoing infectious disease preparedness efforts. we conducted searches of the medical literature and key websites (e.g., www.pandemicflu.gov) for peer-reviewed manuscripts and published governmental plans relevant to pandemic planning during 1978-2008. we also consulted authors' personal files and the internet for records of speeches, national and international conference proceedings, and other unpublished original source documents. in addition to published survey data concerning local and state response planning (10,11), we sought unpublished data from the association of state and territorial health officials, the national association of county and city health officials (naccho), and the council of state and territorial epidemiologists (cste). a historical overview of key milestones in us pandemic planning is provided in the table. in 1977, a federal interagency working group on influenza was formed at the request of the deputy assistant secretary for health in the department of health, education, and welfare, partly in recognition of the need for greater cooperation across government "silos." the interagency group included representatives from the center for disease control (cdc; renamed centers for disease control and prevention in 1992), the national institutes of health, the food and drug administration (fda), and the department of defense. under cdc leadership, the work group drafted the first us pandemic plan, which was released in 1978 and included recommendations for annual influenza immunization of persons at high risk, strengthening of surveillance, expanding research, and establishing a planning and policy mechanism (12) . the plan was revised in 1983 to include a new recommendation to develop means to distribute and use influenza antiviral drugs (r.a. strikas, pers. comm.). even before completion of the pandemic plan, participants of a 1977 conference on influenza, held by the secretary of the department of health, education, and welfare, recommended continued federal support for influenza vaccination, particularly to increase vaccination levels of persons at high risk, to improve pandemic preparedness. in addition, cdc implemented a federally funded seasonal influenza immunization program, which purchased 3.4 and 2.4 million vaccine doses for the 1978-79 and 1979-80 influenza seasons, respectively, of which ≈1 million and >1.4 million doses, respectively, were administered. initial plans were to purchase 8-9 million doses of vaccine. however, budget constraints limited vaccine purchases and ended the program after 1980 (13, 14) . the next major event leading to further us pandemic planning was 1986 legislation creating the national vaccine program office (nvpo), which was given a mandate to coordinate federal vaccine-related activities. at the options for the control of influenza ii meeting held in 1992, a consensus report identified the core components of pandemic preparedness: surveillance, vaccines, antiviral drugs, nonmedical/personal hygiene measures, communications, and enhanced annual seasonal influenza vaccination programs (15) . in 1993, nvpo formed the federal interagency group on influenza pandemic preparedness and emergency response (grippe). the group, which included nonfederal consultants and representatives from cdc, fda, the national institutes of health, and the department of defense, drafted a pandemic planning framework that was published in 1997 (16) and updated by federal staff in 2002 (17) . the grippe-initiated planning documents emphasized the need for enhancements to influenza surveillance, vaccine production and distribution, antiviral drugs, influenza research, and emergency preparedness. perhaps the most consequential outcome of grippe was the creation of a core group of public health experts dedicated to pandemic planning. global events helped accelerate interest in pandemic planning. in 1997, hong kong recorded the first outbreak of avian influenza a(h5n1) virus infections in humans. virus was transmitted from infected chickens directly to humans, and 6 of 18 persons with confirmed infection died. in late 1997, >1.5 million chickens were culled throughout hong kong as part of successful efforts to stem the outbreak (18) . this event, combined with the 2003 reemergence of a(h5n1) virus, led to concerns that the next pandemic would be caused by spread of a(h5n1) virus through asia into africa and europe. in the united states, despite the crucial role of state and local authorities in implementing pandemic plans, a 1995 cste survey indicated that <60% of state health departments perceived the need for a state-specific plan (10) . through a cooperative agreement between cdc and cste, a state and local planning effort was begun in the fall of 1995. the state project steering committee included the grippe co-chairs and representatives from cdc, nvpo, cste, and the association of public health laboratories. a meeting of >40 state and local health officials convened in september 1996 in atlanta and identified 4 "pillars" deemed most critical for state and local pandemic preparedness efforts: 1) surveillance, 2) vaccine delivery, 3) communication and coordination, and 4) emergency response. from this meeting and subsequent subgroup meetings dedicated to the 4 pillar areas, critical elements of draft state and local guidelines were developed by january 1997. four states (connecticut, missouri, new mexico, and new york) and 1 local area (east windsor township, new jersey) were selected by the state project steering committee-primarily on the basis of the identification of a key project leader within each jurisdiction-and funded to pilot test the draft guidelines; 1 additional state, maine, volunteered to test the draft guidelines without cste support. these 5 states conducted pilot tests during february and march 1998 and submitted results to cste. findings were discussed on april 7-8, 1998 , at a meeting in atlanta. the major outcomes from pilot testing were the following recommendations: 1) a fifth pillar area, guidance for use of antiviral drugs, should be added to the guide; 2) the format of the guidelines should be more in concert with the national plan (18) ; and 3) all states should receive the revised guidelines to enable development of state-specific plans (r.a. strikas, pers. comm.). these 3 issues were discussed at the association of state and territorial health officials/nac-cho annual meeting in september 1998 and incorporated into the state and local pandemic influenza planning guidelines (r.a. strikas, pers. comm.), which were then further revised. california, maryland, minnesota, and south carolina were funded through cste to develop state plans and submitted their model plans in april 2000. a national pandemic influenza steering committee was subsequently formed; it was comprised of immunization (11) . throughout this process, all states received the same nominal level of funding support, which was typically used to convene a statewide stakeholders meeting. elements critical to the planning process included technical support provided by the national steering committee and the identification of a key public health professional within each state who assumed responsibility for leading and coordinating planning efforts. arkansas, arizona, and oregon concurrently developed plans of their own accord; west virginia, tennessee (1999), and pennsylvania (1999) had already developed plans. ultimately, funds were sought for every state to develop a plan. at this early stage in the planning process, the importance of disseminating information to the broader public health community was recognized. on february 25, 1999, and july 13, 2000, cdc presented satellite videoconferences on influenza pandemic preparedness for states and local areas, which were viewed by >7,000 and ≈6,000 participants, respectively. state and local public health staff engaged in development of pandemic plans participated in the broadcasts. at a meeting of state and local planners sponsored by cste and cdc in atlanta on september 12-13, 2000, detailed discussions were held regarding 1) a scenario of how an influenza pandemic might affect states in 2001; 2) how states should enhance surveillance; 3) how vaccination priorities should be determined, and 4) other national and federal pandemic planning issues, such as infection control, patient triage, and antiviral drug usage (r.a. strikas, pers. comm.). after the september 11, 2001, terrorist attacks on the united states, public health preparedness emerged as a priority of the federal government. in 2001, bioterrorism emergency funding support was provided to all states to assist in the nation's response to the anthrax attacks. the 2003 reemergence of avian influenza a(h5n1) infections in humans fundamentally altered the scale of pandemic preparedness. as the a(h5n1) virus spread to more countries in east and southeast asia during 2004-2005, concern grew among senior policymakers and public health experts that the world was on the verge of an influenza pandemic. a(h5n1) infection in humans primarily resulted from exposure to ill poultry and had a case-fatality rate of ≈60%. substantial federal funding was provided for federal-level planning, procurement of countermeasures (e.g., vaccines and antiviral drugs), development of countermeasures, and state and local pandemic preparedness efforts (20) . state health departments eventually received $550 million to prepare for an influenza pandemic. additional high-level policy engagement by the us federal government included the national strategy for pandemic influenza, which was announced in november 2005 (21) , and the white house's national implementation plan, which was published in may 2006 and addressed federal planning and response strategies: international transport and border control; protection of human and animal health; and security and continuity of operations issues (22) . in 2006, the biomedical advanced research and development authority (barda) was established within the department of health and human services in response to the growing need for a centralized effort to coordinate research, development, and procurement of countermeasures against potential natural or intentional public health emergencies (23). barda preparations for a possible a(h5n1) pandemic included development of a stockpile of influenza vaccines produced by using strains circulating in poultry and wild birds in asia (24). in addition, the us government began to purchase influenza antiviral medications for the strategic national stockpile sufficient to treat 25% of the us population. additional investments were initiated to procure ventilators and personal protective equipment, such as respirators. the us government also initiated an advanced development agenda for vaccines, therapeutics, and diagnostics. barda co-invested with industry to modernize vaccine production methods, with the 5-year aim of creating the capacity to produce sufficient vaccine to protect the entire us population within 6 months of the onset of an influenza pandemic (22) . the us government invested in modernizing diagnostic technologies for public health laboratories. in september 2008, fda approved specific pcr tests for a panel of influenza diagnostics to be used in cdc reference laboratories in the united states and department of defense laboratories around the world. this diagnostic test panel will detect and identify a(h5n1) infections and distinguish novel influenza virus infection from infection with seasonal a, b, and a(h1) and a(h3) influenza viruses. barda and cdc awarded contracts in november 2006 for development and evaluation of clinical point-of-care rapid diagnostics to identify seasonal influenza viruses and a(h5n1) viruses (25) . beginning with its first published pandemic plan in 1999 (26) , the world health organization globally promoted pandemic planning among member states, with continued planning efforts thereafter (27) . the international partnership on avian and pandemic influenza was formed to coordinate support for developing countries' efforts to control the spread of a(h5n1) virus and to prepare for an influenza pandemic. this international body convened a series of meetings beginning in january 2006; these efforts generated hundreds of millions of dollars in pledges to support global pandemic preparedness and promoted a level of visibility and readiness that would not otherwise have been possible. in addition to direct financial assistance, the us government provided technical assistance to help countries develop capacities for rapid response, laboratory diagnosis, and surveillance. the federal government recognized that the foundation for domestic pandemic response rests with state and local governments; thus, the 2005 department of health and human services strategy and the white house strategy and implementation plan called for major efforts in planning, exercising, and refining state and local preparedness. the 2006 pandemic and all-hazards preparedness act called for a review of comprehensive state pandemic preparedness plans. the federal government reviewed and scored the plans and released the results to the public in january 2009 (28); preparedness levels varied across states and across the domains that were scored. in 2008, as part of its local health profile survey, naccho queried local health departments about emergency preparedness and planning activities they had undertaken during the past year (29): 89% of 2,332 responding health departments said they had developed or updated pandemic influenza preparedness plans, and 86% said they had participated in tabletop drills or exercises. in addition, 76% had updated their written response plan on the basis of a postexercise after-action report, 72% had participated in a functional drill, and 49% had participated in a full-scale drill or exercise. a total of 68% of local health departments had reviewed existing state legal authorities for isolation and quarantine, and 46% had assessed the emergency preparedness competencies of staff. only 1% of local health departments did none of the above. the 4 pandemic planning pillars-surveillance, vaccine and antiviral drug delivery, emergency response, and communication-are a solid foundation for pandemic preparation. although state pandemic plans may have different structures, reliance on these pillars has remained more or less constant across jurisdictions and over time. the major contemporary developments in these core areas are summarized below. surveillance, including rapid detection of human infection with novel influenza viruses, remains a cornerstone of pandemic response. this need has been recognized since the early stage of state-and local-based planning (10) . improvements in diagnostic technology have enabled confirmation of infection with novel influenza viruses within hours rather than weeks. human infection with a novel influenza virus became a nationally notifiable disease in 2007, and since then, an increased number of infections have been detected (30). virologic surveillance is also used to determine which seasonal viruses are circulating and thus provides information for seasonal vaccine strain selection. systems to measure the effect of seasonal influenza (i.e., pediatric deaths, hospitalizations, and syndromic surveillance) have also been enhanced. these systems have been further adapted to measure the effect of pandemic influenza (31) . the need to maintain ongoing surveillance for novel influenza viruses (e.g., viruses of swine or avian origin) in humans and animals exemplifies the one health concept (32) . in recognition that vaccine might be in short supply during the early phase of a pandemic, federal vaccine allocation guidelines were published in 2008 (33) . these guidelines laid the groundwork for the pandemic vaccine priority-group recommendations put forth during the 2009 a(h1n1)pdm09 pandemic (34) . antiviral medications are critical to a pandemic response, particularly in the interval between recognition of the pandemic and the availability of vaccine. plans for using these countermeasures have stressed the need for early treatment of affected persons and assumed that the drugs would be scarce. it was recognized at the 1996 cste meeting that close coordination between emergency response staff and public health authorities is needed to develop and implement effective state and local influenza response plans. this recognition has strengthened over time. although, states were initially not allowed to use bioterrorism funds awarded in 2001 to support pandemic planning, key emergency management concepts, including the all-hazards approach and unified incident command, were eventually integrated into planning efforts (35) . communication, more than ever, is a fundamental component of any response effort. timely, transparent, and proactive communication is critical, particularly in the early stages of a confirmed or suspected outbreak, when factual information is limited and the public demand for information and guidance is high. continuous media coverage and the evolving role of social media (36) must be used to enhance communication to and from the public, particularly concerning new or evolving recommendations for disease control. pandemic planning since 2005 had a direct and obvious effect on the response to the 2009 influenza a(h1n1) pdm09 pandemic; however, pandemic preparedness has been a feature of public health since the late 1970s. coordinated state and federal planning processes have been a consistent feature of that planning. the pillars of pandemic planning response have remained conceptually constant: surveillance; vaccination and delivery of other medical countermeasures; emergency response coordination; and communications. although the 2009 a(h1n1)pdm09 pandemic spread globally within a matter of weeks, a 1918-like pandemic did not materialize. nonetheless, this most recent pandemic resulted in ≈12,500 deaths in the united states, ≈90% of which occurred in persons <65 years of age (37) . in the wake of this pandemic, the challenge in preparedness is to sustain the interest of private and public sectors in planning for a large-scale outbreak that may have a much more severe effect at a time that cannot be predicted. recent assessments of state level epidemiology capacity revealed potentially critical gaps in personnel and training needed for a rapid response to an epidemic (38) . there will be a need for continued commitments to support state, local, and national planning for the next infectious disease emergency. a comprehensive, coordinated, and effective response cannot be built at the time of a crisis. for future planning and response efforts, sufficient resources are required to sustain the public health response infrastructure developed during the past decade. an effective response to a pandemic requires at least 4 distinct elements. first, material resources, such as vaccines, antiviral drugs, and personal protective equipment are essential. second, a commitment to planning, exercising, and refining plans is necessary. third, a sufficiently large and robustly trained workforce is the basis of any response. fourth, a commitment to improvement is crucial. this concept extends from continuously improving plans and training to ensuring that scientific advances are incorporated into procurement and planning. one of the main lessons from the history of influenza is to expect the unexpected. plans and training should be flexible and designed to respond to various levels of disease severity or newly identified pathogens. benefits from pandemic preparedness will enhance our collective public health response to the next infectious disease crisis. dr iskander is a senior medical consultant in the office of the associate director for science, cdc. world health organization. pandemic influenza preparedness and response: a who guidance document us department of health and human services. about pandemics pandemic versus epidemic influenza mortality: a pattern of changing age distribution influenza pandemic: preparedness plans of the public health service swine influenza a outbreak from the national institute of allergy and infectious diseases of the national institutes of health, the center for disease control, and the bureau of biologics of the food and drug administration: a status report on national immunization against influenza guillain-barré syndrome following vaccination in the national influenza immunization program preparing for avian influenza: lessons from the "swine flu affair historical perspective-emergence of influenza a (h1n1) viruses preparing for pandemic influenza: the need for enhanced surveillance influenza pandemic preparedness interagency work group on pandemic influenza. a plan for pandemic influenza. department of health, education, and welfare influenza immunization program review and update of influenza grant programs pandemic planning: conclusions and recommendations influenza pandemic preparedness plan for the united states influenza pandemic preparedness action plan for the united states: 2002 update outbreak of avian influenza a(h5n1) virus infection in hong kong in 1997 influenza pandemic planning: review of a collaborative state and national process continuation guidance for cooperative agreement on public health preparedness and response for bioterrorism-budget year five, program announcement 99051 national strategy for pandemic influenza national strategy for pandemic influenza: implementation plan ) of the vaccines and related biological products advisory committee report to congress: pandemic influenza preparedness spending planning for the next pandemic of influenza world health organization. who strategic action plan for pandemic influenza us government departments, agencies, and offices. assessment of states' operating plans to combat pandemic influenza national association of county and city health officials update: influenza a (h3n2)v transmission and guidelines-five states surveillance for influenza during the 2009 influenza a (h1n1) pandemic-united states confronting zoonoses through closer collaboration between medicine and veterinary medicine (as 'one medicine') us department of health and human services and us department of homeland security. guidance on allocating and targeting pandemic influenza vaccine use of influenza a (h1n1) 2009 monovalent vaccine: recommendations of the advisory committee on immunization practices (acip) influenza pandemic preparedness pandemics in the age of twitter: content analysis of tweets during the 2009 h1n1 outbreak estimating the burden of 2009 pandemic influenza a (h1n1) in the united states assessment of epidemiology capacity in state health departments-united states key: cord-299207-lw0cv74b authors: upadhyay, ranjit kumar; kumari, nitu; rao, v. sree hari title: modeling the spread of bird flu and predicting outbreak diversity date: 2007-05-08 journal: nonlinear anal real world appl doi: 10.1016/j.nonrwa.2007.04.009 sha: doc_id: 299207 cord_uid: lw0cv74b avian influenza, commonly known as bird flu, is an epidemic caused by h5n1 virus that primarily affects birds like chickens, wild water birds, etc. on rare occasions, these can infect other species including pigs and humans. in the span of less than a year, the lethal strain of bird flu is spreading very fast across the globe mainly in south east asia, parts of central asia, africa and europe. in order to study the patterns of spread of epidemic, we made an investigation of outbreaks of the epidemic in one week, that is from february 13–18, 2006, when the deadly virus surfaced in india. we have designed a statistical transmission model of bird flu taking into account the factors that affect the epidemic transmission such as source of infection, social and natural factors and various control measures are suggested. for modeling the general intensity coefficient [formula: see text] , we have implemented the recent ideas given in the article fitting the bill, nature [r. howlett, fitting the bill, nature 439 (2006) 402], which describes the geographical spread of epidemics due to transportation of poultry products. our aim is to study the spread of avian influenza, both in time and space, to gain a better understanding of transmission mechanism. our model yields satisfactory results as evidenced by the simulations and may be used for the prediction of future situations of epidemic for longer periods. we utilize real data at these various scales and our model allows one to generalize our predictions and make better suggestions for the control of this epidemic. bird flu also known as avian influenza is an infection caused by a virus known as orthomyxoviridae in virus classification [55] . influenza virus has only one species in it, which is called influenza a virus. these influenza viruses occur naturally among birds. wild birds worldwide carry these viruses in their intestine but usually do not get sick from them. however, avian influenza is very contagious among birds and can make some domesticated birds including chickens, ducks and turkeys very sick and kill them [20, 24, 30, 43] . infected birds shed influenza viruses from their saliva, nasal secretions, etc. susceptible birds become infected when they come in contact with the contaminated surfaces. domesticated birds may become infected with avian influenza viruses through direct contact with infected waterfowl or other infected poultry [24, 28, 38] or through contact with surfaces (such as dirt or cages) or materials (such as water or food) that have been contaminated with the virus [1, 15, 39, 45, 49] . infection with bird flu viruses in domestic poultry causes two main forms of disease that are distinguished by low and high extremes of virulence. the "low pathogenic" form may go undetected and usually causes only mild symptoms (such as ruffled feathers). however, the highly pathogenic form spreads more rapidly through flocks of poultry. this form may cause diseases that affect multiple internal organs and has a mortality rate that reaches 90-100% often within 48 hours [8] . in 2006, the high security animal disease laboratory in bhopal confirmed genetic signatures of the virus in eight samples of dead poultry tissues that it had received from navapur in nandurbar district near the maharashtra-gujarat border. for more than two years, the virus has ravaged poultry and caused human illness and death in many southeast asian countries and china. between april [12, 14, 15, 23, 26, 40, 42] . avian influenza or bird flu can result in immediate and severe disaster, for example, the outbreak in usa [48] in 1983-1984 led to destruction of more than 17 million birds at a cost of nearly us $56 million [21] . similar case again happened in hong kong in 1997-1998 [12, 15, 38, 39, 42] . therefore rapid and effective measures must be taken to stop the spread of epidemics. the most effective measures to prevent the transmission of bird flu are rapid destruction of all infected or exposed birds, proper disposal of carcasses and excrement, the quarantining and rigorous disinfectioning of farms and timely use of vaccine [4, 25, 35, 36, 50] . the information relating to the spread of this epidemic in canada may be obtained from the website [3] . in general, the influenza virus or flu virus can be classified into three categories: types a, b and c, which are distinguished by differences in two major internal proteins. influenza virus type a is the most significant epidemiologically and the most interesting from an ecological and evolutionary stand point, because it is found in a wide variety of bird and mammal species [9, 41] and can undergo major shifts in immunological properties. type b is largely confined to humans and very little is known about type c. type a virus is responsible for causing bird flu, which was first found in italy in 1878. type a virus is further divided into subtypes based on differences in membrane proteins ha and na, which are the most important targets for the immune system. the notation hhnn is used to refer to the subtype comprising the hth discovered ha proteins and the nth discovered na protein. the subtype h5n1 virus of type a virus is the main cause of the bird flu [12, 15, 23, 31, 42] . subtype is further divided into strains; each genetically distinct virus isolated is usually considered to be a separate strain [11, 33] . according to the research of world health organization (who), the transmission mode of bird flu can be divided into following two types [21] : • spread from one farm to another within a country and • spread from country to country. generally, the virus resides in bird droppings, contaminated soil and airborne virus. contaminated equipments, vehicles, food, cages and clothing like shoes can carry the viruses from farm to farm. some evidence suggests that flies can also act as mechanical vectors [18] . wet markets where live birds are sold under crowded and sometimes unsanitary conditions can be another source of spread. these constitute the main cause of the former transmission. export and import of poultry products are the main cause of the latter transmission, since they can carry the viruses for long distances freely when artificial factors are prevented. migratory birds can also be a cause of transmission between the countries [5, 27] . efforts have been made on the study of avian influenza and most of the recent papers focus on topics such as the route of transmission, physiological and biological properties, etc. the bird flu virus of low pathogenicity can mutate into highly pathogenic one after a short time; the virus is sensitive to temperature change (it was found that the virus survives for shorter time at a higher temperature). this kind of influenza is able to transmit to humans under some circumstances (http://www.who.int/mediacentre/factsheets/fs277/en/) [6, 7, 21, 44, 47, 52] ; however, no sufficient and clear evidences of human-to-human transmission have been found up to now [21] . among these researches, an important approach to study bird flu is to establish a statistical transmission model, from which the general trends of epidemics can be predicted, and the effect of various control measures can be assessed [13] . to cite successful examples, model for foot-and-mouth disease has successfully controlled the epidemic in 2001 in uk [46] . in 2003, model for severe acute respiratory syndrome (sars) has reached the similar objectives [16, 34] . likewise, we expect our statistical transmission model of bird flu will also reach its objective. in order to study the patterns of the spread of epidemic, we have made an investigation of outbreaks of the epidemic in one week during february 13-18, 2006 till it reached india. the actual data for outbreaks of bird flu in a week have been taken from an article of the indian express published on february 19, 2006. on february 18, 2006 the lethal strain of h5n1 virus surfaced in india in the trivial pocket of navapur in nandurbar district of northern maharashtra. it was the sixth day of our study. on monday, the first day, outbreaks occurred in five countries of africa and eastern europe namely nigeria, greece, slovenia, romania and bulgaria. on the second day bird flu hit four nations of southeast asia, central asia and europe namely indonesia, iran, austria and germany. on the third day, outbreaks took place in two european countries, hungary and italy. on thursday, new cases of bird flu were found in three european and african countries, switzerland, denmark and egypt. reaching france on friday, bird flu, at last, on saturday surfaced in india ( fig. 1) . we investigated many related reports on bird flu and collected some important information that became a base for our model, which is described below. in general, the outbreak of an infectious disease is dependent upon three necessary conditions as the source of an infection, the route of transmission and the herd susceptibility [21] . other social and natural factors also play an important role in the transmission of infection, for example, the control measures and the change in temperature. the source of infection that led to the outbreak is not clear. in some researcher's view migratory birds are thought to be carrying the virus [5, 27] . if migratory birds had brought the virus one would have expected outbreaks well before february as bird's migrations were over by around november. so we consider in our problem the source of outbreaks of bird flu to be the transportation of infected poultry as globalization has turned the chicken into the world's number one migratory bird species. we have assumed that it is mainly the human activities of commerce and trade that this epidemic has spread. for implementing this we have considered the concerns expressed by scientists from max plank institute for dynamics and self organization, germany [19] . they have recognized the statistical characteristics of human travel independent of the means of transportation people use for the modeling of the spread of epidemics as published in the article [19] . also, we acquired some of the important information about bird flu such as, the virus h5n1 is sensitive to temperature changes, and the virus survives for shorter time at a high temperature. also there are many effective control measures to block the virus transmission such as compulsory vaccination, culling of all infected or exposed birds [2] . indeed, in the absence of such control mechanisms this avian influenza may pose a big threat to global health care [10, 17, 29, 32, 37, 51, 53, 54] . we know the major factors that play an important role in the transmission of bird flu are the way the infected poultry products are transported, air temperature, the control measures (for example, culling the poultry in the infected form, introducing compulsory vaccination to enhance the resistibility of poultry in the non-infected farms forbidding live birds being sold under crowded and unsanitary conditions, etc.), migratory birds and other infected transportation vehicles (which means the vehicles carry infected poultry or bird dropping or contaminated soil, etc.). also there are some other factors not considered in our model, viz. bird flu transmitting to human beings, viruses of low pathogenicity mutating into high pathogenicity after some time, etc. since these elements will not contribute much to the usual transmission of bird flu [22] . how do these factors affect the transmission? the infected animals are the source of the infection, higher air temperature can drastically cut down the lifetime of the virus, and transportation of infected poultry is the route of transmission. control measures such as active and effective actions play an important role in preventing and destroying the epidemics, which can effectively block the route of transmission of the infection, diminish the source of infection and promote the resistibility of susceptible poultry [21] . so we must take all the major factors into account in the formulation of our transmission model. these factors may be reflected in the following parameters: n(n) is the total number of regions of outbreak on the nth day. d(n) is the lifetime of the virus regarding the nth day since the beginning of the epidemic which implicitly corresponds to air temperature. i (n) is the resistibility of the poultry on the nth day since from the beginning of the epidemic many of the above control measures objectively promote the resistibility of poultry and even human beings. f (r) is the distribution of the probability that infected poultry products are transported a distance r. the following are necessary assumptions for our model: let p (n, r) represent the probability for a new outbreak to take place, then p (n, r) ∝ n (n), p (n, r) ∝ d(n), p (n, r) ∝ 1/i (n), considering the above assumptions, we obtain our proposed model for transmission of bird flu as under: where is taken close to 0.6 [22] and r ∈ [0, 1] is a random float number. in this section, we have discussed the methodology that we have used for simulation experiments. we first discuss, one by one the various parameters taken in our model. also we explain, how we have computed these parameters and finally how we have given the methodology used to predict a new outbreak with the help of a flowchart suggested by li et al. [21] . • in our model, we have used the random float number r which we have generated in our program by using random number generator. why do we need r in our model? as we know the outbreak of bird flu is a probabilistic instance and not a deterministic one, even though f (r), d(n), n (n) contribute much to p (n, r), we cannot definitely assure that there will be a certain outbreak of bird flu in a region but can only say that there is enormous possibility or danger for an outbreak to take place. so an additional random parameter r is introduced to reflect the uncertainty. . by means of fitting the above data by curve fitting method, we obtained an approximate formula as follows: where t represents the air temperature. in our simulation, we studied the epidemic for one week till it reached india, that is for a short duration. hence the temperature change may be taken as a linear approximation regarding the epidemic duration, that is, in which t 1 and t 2 are two constants that can be determined by fitting the average temperature of the various countries through which the virus reached india. in this model t 1 = 0.2 and t 2 = 2.2286 (started on february 13, 2006) . so the relation between the lifetime of the virus and the epidemic duration shall be a compound form of (2) • i (n) stands for the resistibility of poultry on the nth day. obviously the resistibility will increase with the artificial interventions and the control measures. we assume the increase abides by law similar to sigmoid function 1/(1+e −x ). thus i (n) assumes the following form: apparently, this is a modified sigmoid form. when n = 0, i (0) = 1, and when n = ∞, i (n) = b; which indicates the resistibility is impossible to approach a very big number [21] . • f (r) is the distribution of the probability that infected poultry products are transported a distance r. the basic idea for taking the above mentioned form of f (r) stems from howlett [19] . the researchers at max planck institute for dynamics and self organization have used the dispersal of dollar bills within the united states as a proxy measurement of human movement (since people cannot be tracked while banknotes travel with the people). they analyzed the data on the peregrinations of more than half-a-million us dollar bills recorded over a five-year period on an online bill-backing system (http://www.wheresgeorge.com) and give a simple model that only depends upon two parameters. since the poultry products are also imported and exported very frequently, to various countries, we can assume its transport to be very similar to the human movement. so the probability that initialize s, n 0 , n, n(n), count=0 determine k(n) count=0 calculate probability p(n, r) infected poultry product is transported from the farm may assume a distance r, for r larger than 10 km with close to 0.6. this distribution behaves like a power law. this function decreases as r grows larger, meaning that transportation of poultry over a long distance is less common than short ones. however, it does not decrease as fast as other common probability distributions, which means that transportation over long distances are still common enough to have a significant effect. poultry products make many short journeys, but the occasional long haul ensures that they disperse widely. • a threshold value s is necessary which acts as a criterion: when p (n, r) is greater than s there will be an outbreak; otherwise not. however, how to determine the possible number of outbreaks per day denoted by k(n)? intuitively, k(n) shall be in direct proportion to n (n); however, since only the nearest several outbreaks have notable contribution to the probability of a new outbreak a number of distant outbreaks contribute little, so dependent relation of k(n) upon n (n) is of the form [21] where a 1 and 0 < b>1, that is, k(n) increases slowly with the augment of n (n). thus the method for predicting an outbreak is explained with the help of an algorithm given in fig. 2 . we provide a short description of the algorithm. suppose the epidemic has begun, we compute the number of actual outbreaks on the nth day. first we generate k(n) according to (6) . then we calculate each p (n, r) according to (1); when p s, a new outbreak will take place, otherwise not. so, the total number of new outbreaks is always less than k(n). in our model, there are six parameters , a, b, b, c and s which are all adjustable. so one may argue that too many adjustable parameters may not be an advantage for a "good" model. however, we must analyze independence of these parameters. here s is not an independent parameter, rather dependent on f (r), hence on . parameter a controls the initial possible number of outbreaks the epidemic may abort if a is too small and overflow if a is large. so there shall be a proper intermediate value for a. b denotes the general trend of the outbreaks, the total number of outbreaks will grow too rapidly to be practical if b is a big number and may be too flat if b is too small. b determines the ultimate resistibility, which reflects the final degree of stringency of artificial interventions; the greater b is the more stringent the interventions are. as of c, it reflects the average degree of stringency throughout the epidemics; the smaller c is the more stringent the control measures are. in other words, b determines the final height of the curve i (n) and c controls the shape or the process of i (n). therefore each parameter has a definite meaning and a specific role and has little overlap regarding the role, so the model is reasonable. for conducting simulations we have assumed distances: < 500 miles as 10 units, 500-1000 miles as 11 units, 1000-1500 miles as 12 units and 1500-2000 miles as 13 units and so on. the parameters are initialized as the beginning: n 0 = 6 refers to the total number of days (for which study is conducted) the epidemic lasts. n(1) = 5 means there are in all five cases of new outbreaks on the first day. the other parameters a, b, b, c and s will be determined in the simulation by comparing with the actual epidemic data. results of simulation are shown in figs it is easy to see that the simulation is roughly in accordance with the actual situation. the parameters related to the artificial interventions include b and c. if b is large, the epidemic can take place few times in most simulations; if b is very small, the epidemic can always happen and most times the overflow can happen. as for c, if it is small enough, the epidemic hardly takes place, otherwise it can almost happen. therefore the effective, powerful and stringent control measures are the key to stop the epidemics. it should be pointed out that the parameters in the model are independent of each other, since each of them plays an independent role. these parameters have definite meaning, so the result of simulation can hardly coincide with the actual situation of the epidemic if the parameters cannot be adjusted to proper values. simulation results are presented in table 1 . it is clear from the table that the probability of new outbreak to occur decreases with the increase of distance from the source countries. the probability of occurrence of outbreak in india is very low because the flu virus reaches on the sixth day and also the country is far from the source country. the probability of occurrence of outbreak in indonesia is also very low even on the second day. this is due to very large distance from the source country and also due to some other factors (like temperature). figs. 3 and 4 show the pattern, that the probability of outbreak to take place follows, depending upon the distance and number of days. these graphs show that the probability of outbreak is inversely proportional to the distance from the source country as well as the day on which the flu virus reaches the country. bird flu is a highly pathogenic epidemic that can result in serious disaster in many areas. immediate and effective control measures are of great importance in preventing the transmission of avian influenza. so it is challenging to study it from various angles, and develop statistical-mathematical transmission models [21] . in this research, we put forward a statistical transmission model, which exhibits satisfactory results verified through simulations. these results may be used for the prediction of the future situation of the epidemics. meanwhile (as many control measures have been taken into account in the formulation of the model) if the actual effect of these measures are assessed, then useful and effective control measures can be proposed for the prevention of the epidemic. from figs. 3 and 4 , it is found that the probability of outbreak is inversely proportional to both the distance from the source country and the number of days at a fixed distance. our ongoing investigations relate to the question of how to assess the actual control measures and assign the 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characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness isolation and characterization of prevalent strains of avian influenza viruses in china probable person-to-person transmission of avian influenza a (h5n1) evolution and ecology of influenza a viruses microbial adaptation and change: avian influenza h5n1 outbreaks and enzootic influenza world health organization, cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who world health organization, evolution of h5n1 avian influenza viruses in asia world health organization, influenza pandemic preparedness and response, geneva: the organization fields virology the third author's research has been supported by a grant from the foundation for scientific research and technological innovation (fsrti)-a constituent division of sri vadrevu seshagiri rao memorial charitable trust, hyderabad 500 035, india. key: cord-299359-s8j78naz authors: sundaram, maria e.; mcclure, david l.; vanwormer, jeffrey j.; friedrich, thomas c.; meece, jennifer k.; belongia, edward a. title: influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness date: 2013-09-15 journal: clin infect dis doi: 10.1093/cid/cit379 sha: doc_id: 299359 cord_uid: s8j78naz background. the test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve). recent studies have suggested that influenza vaccination increases the risk of noninfluenza respiratory virus infection. such an effect could create bias in ve studies using influenza-negative controls. we investigated the association between influenza infection, vaccination, and detection of other respiratory viruses among children <5 years old and adults ≥50 years old with acute respiratory illness who participated in seasonal studies of influenza vaccine effectiveness. methods. nasal/nasopharyngeal samples collected from 2004–2005 through 2009–2010 were tested for 19 respiratory virus targets using a multiplex reverse-transcription polymerase chain reaction (rt-pcr) platform. vaccination status was determined using a validated registry. adjusted odds ratios for influenza and vaccination status were calculated using three different control groups: influenza-negative, other respiratory virus positive, and pan-negative. results. influenza was detected in 12% of 2010 children and 20% of 1738 adults. noninfluenza respiratory viruses were detected in 70% of children and 38% of adults without influenza. the proportion vaccinated did not vary between virus-positive controls and pan-negative controls in children (p = .62) or adults (p = .33). influenza infection was associated with reduced odds of vaccination, but adjusted odds ratios differed by no more than 0.02 when the analysis used influenza-negative or virus-positive controls. conclusions. influenza vaccination was not associated with detection of noninfluenza respiratory viruses. use of influenza-negative controls did not generate a biased estimate of vaccine effectiveness due to an effect of vaccination on other respiratory virus infections. the case vs test-negative control study design is the basis for observational studies of influenza vaccine effectiveness (ve) [1] [2] [3] [4] [5] [6] . cases and controls are recruited at the time of presentation of acute respiratory illness (ari) in clinic and hospital settings. individuals presenting with ari who test positive for influenza are considered cases, whereas those who test negative for influenza are considered controls. this study design is convenient to implement and inherently accounts for potential confounding due to differences in healthcareseeking behavior between vaccinated and unvaccinated individuals [1] [2] [3] . it has recently been suggested that influenza vaccination may increase the risk of non-influenza respiratory virus infection by decreasing temporary nonspecific immunity [7, 8] . one proposed mechanism involves activation of the innate immune response following influenza infection, leading to a temporary reduction in the risk of infection with a different respiratory virus. by reducing the risk of influenza infection, the influenza vaccine could paradoxically create an increased risk of infection with other noninfluenza respiratory viruses. if this phenomenon occurs, it could lead to biased estimates of influenza vaccine effectiveness in studies using laboratory-confirmed influenza cases and influenza-negative controls. in this scenario, the risk of noninfluenza viral illness would be higher in vaccinated than unvaccinated individuals, and an 'influenza-negative' control group would therefore have a higher proportion of vaccinated individuals compared to the source population. this could theoretically contribute to overestimation of true ve (ie, bias away from the null); therefore, a key assumption of the test-negative control design of influenza vaccine effectiveness studies is that the proportion of noninfluenza viral illness does not differ by influenza vaccination status [9] . the goals of this study were to determine if influenza vaccination is associated with detection of noninfluenza respiratory viruses and to determine if vaccine effectiveness estimates differ when different control groups are used in the analysis. to achieve these goals, we analyzed available data from members of a community cohort who saw a physician for acute respiratory illness and consented to participate in a study of influenza vaccine effectiveness over 6 influenza seasons. the vaccine effectiveness study enrolled individuals of all ages (with some variation by season), but this analysis was restricted to children <5 years old and adults ≥50 years old. for participants in these age groups, multiplex reverse transcription polymerase chain reaction (rt-pcr) testing was subsequently performed to detect other respiratory viruses, providing an opportunity to investigate the relationship between influenza vaccination and infection with other viral pathogens. young children and older adults are among the most vulnerable individuals to influenza infection and complications [10, 11] and calculating influenza vaccine effectiveness in these groups is therefore of high importance [12] . the marshfield clinic research foundation has conducted seasonal studies of influenza vaccine effectiveness in a wisconsin population cohort since the 2004-2005 season. the details of the seasonal studies have been reported elsewhere [2, 3] . briefly, patients with ari were recruited during each influenza season in primary care clinics, urgent care, emergency department, and an acute care hospital. symptom eligibility criteria varied by season but included fever/feverishness or cough during most seasons. individuals with illness duration ≥10 days (2004-2005 through 2006-2007) or >7 days (2007-2008 through 2009-2010) were excluded to minimize false negative rt-pcr results. after obtaining informed consent, a nasal swab (children <12 years old) or a nasopharyngeal swab (adolescents and adults) was obtained and placed in viral transport media for influenza testing. symptoms and onset date were assessed during the enrollment interview. real-time rt-pcr was performed each season to identify influenza cases. after testing was complete, aliquots of samples in viral transport media were frozen at −80°c. this study was reviewed and approved by the marshfield clinic institutional review board. during each season, all study participants (or parents) provided informed consent for influenza testing. multiplex rt-pcr testing to detect additional viruses was subsequently approved by the irb with a waiver of informed consent. archived samples were tested for the presence of respiratory virus nucleic acid using a multiplex respiratory virus panel (genmark dx esensor respiratory viral panel). this multiplex panel tested for respiratory syncytial virus (rsv) a and b, human rhinovirus, human metapneumovirus, parainfluenza viruses 1-4, influenza a and b (including subtypes of influenza a), coronaviruses oc43, nl63, hku1, and 229e, and adenoviruses b and e. nucleic acid was extracted from the swabs using the roche magnapure 2.0 system and was then amplified using rt-pcr with target-specific primers. target-specific signals were determined by voltammetry, a process that generates electrical signals from ferrocene-labeled signal probes. the multiplex assay has been validated for influenza and rsv a and b against singleplex assays approved by the centers for disease control and prevention (cdc) and found to have 97%, 93%, and 98% sensitivity and 97%, 99%, and 99% specificity for influenza a, influenza b, and rsv, respectively (unpublished data). cases were defined as laboratory-confirmed influenza based on a positive real-time rt-pcr test for influenza a (h3n2), a (h1n1), a (h1n1)pdm09, or type b. three different control groups were constructed for analytical purposes. control group 1 included all individuals testing negative for influenza (influenza-negative controls); group 2 included individuals negative for influenza but positive for at least one other respiratory virus in the multiplex panel (virus-positive controls); and group 3 included individuals negative for all respiratory viruses in the multiplex panel ( pan-negative controls). the first control group is the standard definition used in test-negative case-control designs, whereas the second and third control groups represent subsets of the first group. vaccination status and dates were determined by a real-time, internet-based registry used by all immunization providers serving the local population (www.recin.org). cases and controls were considered vaccinated if seasonal trivalent inactivated influenza vaccine (tiv) or live attenuated influenza vaccine (laiv) was received at least 14 days before ari symptom onset. children who were partially vaccinated according to the advisory committee on immunization practices (acip) definition (ie, received 1 of 2 recommended doses) were excluded [13] . separate analyses were conducted for children <5 years old and adults ≥50 years old, and only the first enrollment was included for each individual in a given season. vaccination status was compared for virus-positive controls and pan-negative controls using logistic regression. odds ratios describing the association between vaccination and influenza infection were determined separately for each of the 3 case-control groups by logistic regression. logistic regression models included influenza status as the outcome and vaccination status as the primary exposure variable with adjustment for potential confounders, including gender, influenza season, age (continuous), presence of a chronic disease conferring increased risk for influenza complications [14] , and interval in days from symptom onset to swab collection. sas 9.2 (sas institute, cary, nc) was used for all statistical analyses. over 6 influenza seasons, 1616 children and 1568 adults contributed 2010 and 1738 unique observations, respectively. influenza was detected in 251 (12.5%) children and 343 (19.7%) adults. among those without influenza, a respiratory virus was detected in 1411 (70.2%) of children and 659 (37.9%) of adults. in children without influenza, the most commonly detected single viruses were rsv (n = 435), human rhinovirus (n = 298), human metapneumovirus (n = 150), and parainfluenza (n = 113); 271 (13.5%) of swabs from children were positive for ≥2 noninfluenza respiratory viruses. in children, the most common coinfection was rsv with rhinovirus (n = 73). in adults without influenza, the most commonly detected single viruses were rsv (n = 170), human rhinovirus (n = 126), human metapneumovirus (n = 125), and coronavirus (n = 122); 47 (2.7%) of swabs from adults were positive for ≥2 noninfluenza respiratory viruses. in adults, the most common coinfection was rsv with coronavirus (n = 8). the virus-positive controlgroup and pan-negativecontrolgroup did not differ with regard to age, gender, vaccination status, or interval from symptom onset to swab in either children or adults ( table 1 ). the proportion of children with a high-risk health condition did not differ between control groups; there were more adults with a high-risk health condition in the pan-negative control group than in the virus-positive control group (table 1) . in univariate analyses, no association was found between influenza vaccination and single virus detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. single infection with parainfluenza virus was less common in vaccinated children (4.6%) than vaccinated children (6.7%; p = .03). among adults, there was a significant association in the opposite direction: parainfluenza was detected in 4.6% of vaccinated and 2.6% of unvaccinated adults (p = .04). for both children and adults, the adjusted odds of influenza infection were significantly lower in the vaccinated group relative to any of the 3 control groups, indicating significant vaccine effectiveness ( table 2 ). among adults, the adjusted odds ratio (aor) ranged from 0.53 to 0.58; among children the aor ranged from 0.50 to 0.56 depending on which control group was used. in both children and adults, the aor using 'influenza-negative controls' (ie, the current standard for ve studies) varied by no more than 0.02 compared to the aor using other virus-positive controls, and all aor confidence intervals overlapped. the adjusted odds of being vaccinated did not differ significantly in the pan-negative control group vs the other virus-positive control group, either in children (p = .96) or in adults (p = .89). results were similar when the analysis was repeated after excluding 14 children who received laiv. there were no adults in this analysis who received laiv. detection of a noninfluenza respiratory virus by multiplex rt-pcr was not associated with influenza vaccination status over a period of six influenza seasons. in both children and adults, the virus-positive control group and the pan-negative control group were similar in terms of age distribution, gender, and influenza vaccination status. there was no association between influenza vaccination and detection of rsv, adenovirus, human metapneumovirus, human rhinovirus, or coronavirus. only parainfluenza infection was significantly associated with influenza vaccination, but the association was in opposite directions for children and adults. the p values were not adjusted for multiple comparisons, and the inconsistency between children and adults suggest that this association is the result of a type 1 error. to assess the potential impact of noninfluenza respiratory viruses in vaccine effectiveness studies, we calculated the aor for influenza vaccination in cases and controls using 3 different control groups. among children, the aors were 0.52 and 0.50, respectively, using influenza-negative controls and virus-positive controls. this corresponds to an absolute difference of 2% in the vaccine effectiveness estimate, with broadly overlapping confidence intervals. nearly all vaccinated children in this analysis received tiv, and the results were similar when laiv recipients were excluded. among adults ≥50 years old, the adjusted odds ratios were nearly identical (0.56 and 0.57, respectively) when using influenza-negative controls and viruspositive controls, with overlapping confidence intervals. other studies have found evidence of an association between influenza vaccination and infection with a non-influenza respiratory virus. in 2008, a study of influenza vaccine effectiveness in australian children 6 to 59 months old found that influenza vaccination was associated with detection of other respiratory viruses [15] . nasal swabs from 289 children were tested by rt-pcr for influenza, rhinoviruses, rsv, parainfluenza virus 1-3, human metapneumovirus, and enteroviruses. adjusted vaccine effectiveness was 58% (95% ci, 9-81) using influenza-negative controls and 68% (95% ci, 26-86) using other virus-positive controls. the authors noted the higher point estimate of vaccine effectiveness using virus-positive controls, although there was substantial overlap in the confidence intervals for these 2 point estimates. the authors speculated that the difference between point estimates could be due to false negative rt-pcr tests in some children (ie, pan-negative controls) due to inadequate sample collection. they also mentioned the possibility that influenza vaccination could increase the risk of infection with other viruses, but they viewed this as biologically implausible. the relationship between influenza vaccination and infection with noninfluenza viruses was also investigated in a clinical trial of tiv in children [7] . in that study, tiv recipients (n = 69) had a 4-fold increase in risk of infection with noninfluenza respiratory viruses, compared to placebo recipients (n = 46); the incidence of seasonal influenza did not differ significantly between the tiv and placebo groups. the authors acknowledged that the association with noninfluenza viruses could be spurious, but they suggested that 'receipt of tiv could increase influenza immunity at the expense of reduced immunity to noninfluenza respiratory viruses, by some unknown biological mechanism' [7] . they identified temporary nonspecific immunity as a possible explanation for this observation. according to this proposed mechanism, unvaccinated children were more likely to acquire influenza infection, and the abbreviations: aor, adjusted odds ratio; ci, confidence interval; uor, unadjusted odds ratio. a all models adjusted for gender, influenza season, age (in years), presence of high-risk health condition, and interval (days) from symptom onset to swab collection. resulting innate immune response provided temporary, nonspecific protection against infection with other respiratory viruses. this was described further in a letter that raised concerns about bias in studies of vaccine effectiveness that use rt-pcr confirmed cases and influenza-negative controls [8] . these 2 studies had relatively small sample sizes and were limited to a single influenza season. however, one of the studies was a randomized, placebo-controlled trial, a study design that is less susceptible to bias and confounding compared to observational studies. we were unable to replicate the previously reported association in children or adults with a larger sample size over multiple influenza seasons. however, we cannot rule out the possibility that vaccination may alter susceptibility to non-influenza viruses in some circumstances. there is limited biological evidence to support an effect of nonspecific immunity across virus families or species and limited evidence to support the role of vaccination in such immunity. cross-reactivity of the t-cell response has been described before for different subtypes of influenza a [16] [17] [18] , although cross-reactivity generated by the innate immune system likely lasts only 10-14 days [19] and may not apply to interactions between viruses of different families or species. other researchers have also suggested that outbreaks of different respiratory viruses may interact or interfere with each other on a population level [20] [21] [22] . this mechanism may help to explain effects not seen in our analysis but which have been reported elsewhere [7, 8] . in conclusion, our findings do not support the hypothesis that influenza vaccination is associated with an increased risk of infection with noninfluenza respiratory virus. further research may aid in determining the biological plausibility of temporary nonspecific immunity generated by infection with specific respiratory viruses. finally, we found no difference in vaccine effectiveness estimates using test-negative vs other virus-positive control groups. the results of this analysis strongly support the validity of case vs test-negative control study designs that are currently used in multiple countries to estimate influenza vaccine effectiveness in the outpatient setting. estimating vaccine effectiveness against laboratory-confirmed influenza using a sentinel physician network: results from the 2005-2006 season of dual a and b vaccine mismatch in canada effectiveness of inactivated influenza vaccines varied substantially with antigenic match from the 2004-2005 season to the 2006-2007 season influenza vaccine effectiveness in wisconsin during the 2007-08 season: comparison of interim and final results moderate influenza vaccine effectiveness in i-move: a european network to measure the effectiveness of influenza vaccines early estimates of seasonal influenza vaccine effectiveness in europe among target groups for vaccination: results from the i-move multicentre case-control study increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine virus interference and estimates of influenza vaccine effectiveness from test-negative studies the test-negative design for estimating influenza vaccine effectiveness mortality due to influenza in the united states-an annualized regression approach using multiple-cause mortality data the burden of influenza in children efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) centers for disease control and prevention (cdc). prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) vaccine effectiveness against laboratory-confirmed influenza in healthy young children: a case-control study virus specificity of human influenza virus-immune cytotoxic t cells seasonal influenza vaccination and the risk of infection with pandemic influenza: a possible illustration of non-specific temporary immunity following infection cytotoxic t-cell immunity to influenza immunity to respiratory viruses interference between outbreaks of respiratory syncytial virus and influenza infection interference between outbreaks of epidemic viruses does viral interference affect spread of influenza? acknowledgments. we gratefully acknowledge the staff of data managers, research coordinators, interviewers, laboratory technicians, and others who contributed to the vaccine effectiveness study and this analysis. we also thank alicia fry, mark thompson, and frank sifakis for manuscript review and feedback.financial support. medimmune, llc, provided funding for multiplex reverse-transcription polymerase chain reaction testing.potential conflicts of interest. m. e. s., j. k. m., and e. a. b. receive research funding from medimmune, llc. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-304023-s22wi0t0 authors: basile, l.; torner, n.; martínez, a.; mosquera, m.m.; marcos, m.a.; jane, m. title: seasonal influenza surveillance: observational study on the 2017–2018 season with predominant b influenza virus circulation date: 2019-10-30 journal: vacunas doi: 10.1016/j.vacun.2019.09.003 sha: doc_id: 304023 cord_uid: s22wi0t0 introduction: influenza is a common respiratory infectious disease affecting population worldwide yearly. the aim of this work is to describe the 2017–2018 influenza season and how it affected elderly population in catalonia despite moderate vaccine coverage among this age group. methods: influenza surveillance based on a primary care sentinel surveillance, virological indicators systematic sampling of ili attended and severe influenza confirmed cases (shlci) admitted to hospital. analysis of data by chi-squared, anova, multiple regression and negative control test or case to case for vaccine effectiveness assessment in primary care and shlci respectively. results: moderate-high intensity and early onset season with predominance of influenza b virus (ivb) (63%) followed by an increase of circulation of influenza a virus (iva). a total of 419 iv from primary care samples. vaccine effectiveness (ve) in primary care setting was 14% (95%ci: 0–47%). 1306 severe cases (adjusted cumulative incidence 18.54/100,000 inhabitants (95%ci: 17.54–19.55)). the highest proportion of severe cases were in the >64 (65.1%) (aor 15.70; 95%ci: 12.06–20.46; p < 0.001) followed by 45–64 yo (25.4%) (aor 6.03; 95%ci: 4.57–7.97). ve in preventing intensive care unit (icu) admission was 35% (95%ci: 10–54%). final outcome death while hospitalized occurred in 175 shlci cases with a case fatality rate of 13.4%. conclusions: 2017–2018 influenza season was an unusual epidemic season with an early onset, great predominance of influenza b (yamagata strain) virus with a high hospitalization rate of severe cases among elderly stressing the need to upgrade vaccine uptake in this age group. vigilancia de la gripe estacional: estudio observacional sobre la temporada 2017-2018 con circulación predominante del virus de la gripe b r e s u m e n introducción: la gripe es una enfermedad infecciosa respiratoria común que afecta cada año a una proporción importante de la población mundial. el objetivo de este trabajo es describir la temporada de influenza 2017-2018 y cómo afectó a la población anciana en cataluña a pesar de la cobertura moderada de vacunas en este grupo de edad. métodos: vigilancia de la gripe basada en la vigilancia centinela de atención primaria (ap), indicadores virológicos por muestreo sistemático semanal de pacientes con síndrome gripal (sg) atendidos en ap y casos graves de gripe confirmada grave ingresados ??en el hospital. las estadísticas utilizadas para el análisis fueron el test anova, la prueba de chi-cuadrado, el análisis de regresión múltiple y la prueba de control negativo y caso a caso para la evaluación de la efectividad de la vacuna (ev, por sus siglas en inglés) en ap y casos graves hospitalizados, respectivamente. resultados: la temporada 2017-2018 se caracterizó por presentar una intensidad moderadamente alta, con inicio temprano y de larga duración. el predominio del virus de la gripe b (vgb) (63%) seguido por un aumento de la circulación del virus de la gripe a (vga). un total de 419 iv de muestras de ap. la ve para prevención de la infección en casos con sg en ap fue del 14% (ic 95%: 0-47%). se registraron 1.306 casos graves (incidencia acumulada ajustada 18,54/100.000 habitantes (ic 95%: 17,54-19,55)). la proporción más alta de casos graves fue en > 64 años (65,1%) (or: 15,70: ic 95%: 12,06-20,46; p < 0,001) seguido del grupo de 45-64 años (25,4%) (or: 6,03; ic 95%: 4,57-7,97). la ve en la prevención de ingreso en la uci fue del 35% (ic 95%: 10-54%). se registraron 175 defunciones con una tasa de letalidad del 13,4%. influenza is a common respiratory infectious disease that affects an important proportion of population worldwide every year. clinical manifestations can vary from a relatively mild disease characterized by sudden onset of fever, headache, myalgia and cough to a severe disease due to complications such as pneumonia and death especially in people at high risk, mostly aged 65 or older. 1, 2 of the four types of seasonal influenza viruses (iv) (a, b, c and d), a and b viruses cause seasonal epidemics of disease. most cases of human influenza are clinically diagnosed as influenza-like illness (ili) making differentiation from other winter circulating respiratory viruses such as rhinovirus, respiratory syncytial virus, parainfluenza and adenovirus difficult. 3 yet those cases requiring hospitalizations because of a more severe outcome are laboratory tested and confirmed as severe cases of influenza infection that need to be hospital admitted. surveillance of influenza is essential for the early detection of epidemics, annual updates of vaccine components and evaluation of new variants or subtypes of iv. although seasonal influenza vaccine does not display high effectiveness, especially when there is a high rate of mismatching between circulating and vaccine strain composition, it is the best preventive measure for preventing deaths and hospitalizations in at risk population. 4 the 2017-2018 influenza season presented a predominant circulation of influenza virus type b during the first epidemic weeks with a high rate of severe influenza hospitalizations, especially among the elderly. the aim of this work is to describe the 2017-2018 influenza season according to the pidirac sentinel influenza surveillance system and how it affected elderly population in catalonia despite moderate vaccine coverage among this age group. the surveillance of influenza in catalonia started during the season 1988-1989 and was modified from season 1999-2000, in order to achieve major geographical and major representativeness coverage, in the obtaining of samples for the viral study. primary care surveillance: the number of sentinel doctors participating in the season was of 60 physicians (35 general practitioners and 25 pediatricians), located in 43 primary care centers, distributed across several regions and covering 1.03% of the population of catalonia. to achieve morbidity and virological indicators, these practitioners collect pharyngeal and nasal samples during the entire epidemic season, and forward data on attended morbidity. systematic sampling of the first 2 weekly ili attended by each sentinel physician was carried out. a panel of respiratory viruses is investigated: influenza virus a (iva), influenza b (ivb) and c virus (ivc), and other respiratory viruses: respiratory syncytial (rsv), parainfluenza 1, 2, 3 and 4 (piv) viruses, adenovirus (adv), coronavirus (cov), enterovirus (ev), human rhinovirus (hrv), human metapneumovirus (hmpv) and human bocavirus (hbov). morbidity indicators were collected automatically on a daily basis from sentinel practitioners: home calls performed, number of ili cases diagnosed including data on gender, age and vaccination status. the thresholds for weekly incidence rates of ili to assess intensity of epidemic activity (baseline, low, moderate, high and very high) were obtained by the mobile epidemics method (mem) 5 since 2010, severe hospitalized laboratory confirmed influenza cases (shlci) were included in sentinel seasonal influenza surveillance to assess severity of influenza epidemics. shlci case definition is as follows: • anyone presenting with ili that requires hospital admission because of pneumonia, acute respiratory distress, multiorganic impairment, septic shock, icu admission or • anyone who develops the latter symptoms during their hospital stay having being admitted to the hospital for any reason other than influenza or the influenza epidemic activity in catalonia during the 2017-2018 season was moderately high, with an early onset by the end of 2017 and a long duration. the ili weekly incidence curve shows that the duration of the epidemic wave was longer that the former season, lasting 13 weeks, with a maximum incidence of 413.3/100,000 inhabitants (fig. 1 younger than 5 years old the age group with the highest disease burden (6921.96/100,000 h.). comparing with the previous season, the cumulative incidence rates were higher in all age groups as shown in table 1 . the predominant virus was ivb virus followed by an increase of circulation influenza ah3n2 and a(h1n1)pdm09, after peak of epidemic activity was attained on week 3 (fig. 1) . a total of 419 iv were isolated from the 977 samples collected from the primary care sentinel network, 50.2% of them were pediatric samples, with a positivity rate of 67.5% (332/492) to respiratory viruses included in the panel for the virological assessment and of these 57.8% (192/332) were positive to iv. the positivity rate for all respiratory viruses in adult samples (>14 yo) was 64.3% (313/485); of these 72.5% (227/313) corresponded to influenza iva. statistically significant difference was observed in the iv positivity in the >14 yo (or 0.52: 95%ci: 0.37-0.72; p < 0.001). the distribution of iv according to type showed predominance of the ivb with 63% vs. iva (37%). the distribution of the type/subtype of iv is shown in fig. 2 . according to the phylogenetic analysis of the iv circulating during the season, 80% of the ah3n2 virus characterized belonged to the genetic group of the a/hongkong/4801/2014like (also designated like a/bolzano/7/2016) which was included in the seasonal anti-influenza vaccine. the remaining 20% belonged to the a/singapore/infimh-16-0019/2016 strain not included in the seasonal influenza vaccine. all a(h1n1)pdm09 strains belonged to a/michigan/45/2015 (h1n1)pdm09-like, included in the seasonal anti-influenza vaccine. an influenza a(h1n1)pdm09 (a/michigan/45/2015) strain presented the h275y resistance mutation to oseltamivir. the ivb strains identified correspond to the b/phuket/3073/2013 (lineage yamagata), strain not included in the trivalent anti-influenza vaccine. thirty four (8%) of the laboratory confirmed iv had been vaccinated. vaccine effectiveness (ve) to prevent infection (6) cases may present more than one risk factor. before 48 h of onset of symptoms vs. before 48 h of admission. statistical difference was observed in icu admission for those patients who received antiviral treatment up to 48 h after hospital admission (32.4%) in contrast to those who did so more than 48 h after admission (67.6%) (or: 0.64; 95%ci: 0.47-0.88; p < 0.01) as to risk factors the most frequent was cardiovascular disease (38%) followed by chronic obstructive pulmonary disease (26.1%) and diabetes (25.3%). seven shilc cases were pregnant women, 5 (72%) of whom were not vaccinated (table 3) . a clear predominance of ivb was observed (60.3%: 788 cases) during the season, followed by a late increase in circulation of iva virus which accounted for 39.7% (518 cases) of shlci. by subtypes, shlci iva viruses were mainly a h3n2 (63.5%) (fig. 3) . all ivb strains studied were identified as b/phuket/3073/2013 -yamagata lineage. of the influenza a h3n2 isolates subtyped, 76.6% were identified as a/singapore/16-0019/2016(h3n2) strain not included in the seasonal vaccine. all a (h1n1)pdm09 strain analyzed belonged to the a/michigan/45/2015(h1n1) strain included in the seasonal vaccine. ve in preventing icu admission rendered 35% (95%ci: 10, 54) ( table 4) . pneumonia was the main complication occurring in 63.9% (829) shlci cases, 48.1% (382) of which presented bacterial coinfection. acute respiratory distress syndrome accounted for 46% (596) and multiorganic failure for 7.3% (95) cases. five influenza associated encephalitis occurred unvaccinated shlci patients aged 42-to 67 years old. three were caused by ivb and 2 by iva (1 a(h1n1)pdm09 and 1 not subtyped). final outcome death while hospitalized occurred in 175 shlci cases with a case fatality rate of 13.4%. the average age of the deceased was 78.76 years (sd 12.9) and the median age was 82 years (rank inter-quartile 71-88 years), 93.7% had at least one risk factor for developing severe complications and death from their influenza infection (not taking into account old age as a risk factor), 57.3% of them were not vaccinated. ivb was identified in 70% (122) influenza virus infection is still a topic of great relevance to global health. 1 the high percentage of hospitalizations (65.1%) and mortality in the age group of over 64 (17.5%), especially those over the age of 80, where mortality is higher (33.3%), reflects the increase in the life expectancy of the population. this fact makes it necessary to deepen in the knowledge of how they affect aging, their interaction with the most prevalent chronic diseases in the elderly and their immune response in order to apply preventive measures that offer a better protection to this population group. 10 the 2017-2018 influenza season showed a moderate, early-onset activity (approximately 2 weeks) with longer epidemic duration than usual (13 weeks). the pattern of atypical circulation with circulation of initial influenza b viruses and a rebound of influenza a virus could be responsible for this longer duration of the activity. the prevalence of virus b/phuket/3073/2013 -yamagata lineage not included in the trivalent vaccine of the 2017-2018 season and the contribution of virus circulation a/singapore/16-0019/2016 (h3n2), also not included in the vaccine as well as the high proportion of elderly cases (65% >64 years old) may have been the cause of the great number of shlci cases. this fact has also been reported in the spanish influenza surveillance network 2017-2018 report. 11 at the primary care level, the most affected were those under 15 years of age with a cumulative incidence of 6921.96 in the 0-4 age group or 4065.04 in the 5-14 age group. the ve has shown low values in >64 age group. however, it should be taken into account that vaccination has an impact on the reduction of hospitalization and admission to icu. in severe case hospitalizations due to severe influenza, >64 age group presented the highest hospitalization rates (60.95/100,000) compared to other age groups, which highlights the need to increase vaccine coverage in this age group to prevent mortality. 12 the delay in the administration of antiviral drugs, from the onset of symptoms in those patients with a risk condition of complication identified such as elderly people or people with underlying chronic illnesses, is likely to be a bad prognosis of the flu vaccine. this fact evidences the need to administer early treatment (within 48 h of admission) to be effective. 13 most frequent influenza infection complication is pneumonia, yet in this season five influenza encephalitis were registered among adult shlci patients. acute encephalopathy syndromes as a complication of influenza infection has been previously described by other authors in children and adults, but our observation in unvaccinated adult population with risk factors to develop influenza complications underscores the need for higher vaccination coverage in at risk population despite age. [14] [15] [16] there are some limitations to be taken into account, such as the fact that the total hospitalization burden including non-severe hospital admitted cases with laboratory confirmed influenza is not included. this data would be desirable to be included in influenza surveillance in order to have a broader assessment of influenza burden of disease in the population and in turn have a more exhaustive treatment and vaccine effectiveness estimation. in conclusion the 2017-2018 influenza season in catalonia was, as in the rest of spain, 10 an unusual epidemic season with an early onset, great predominance of influenza b (yamagata strain) virus circulation followed by a second wave of influenza a (subtypes h3n2 and (h1n1)pdm09) that resulted in a high hospitalization rate of severe cases especially among the elderly. the low vaccine coverage among >64 age group stresses the need to upgrade vaccine uptake in this age group as well as in those of any age with a risk factor for presenting influenza related complications. analysis of data and samples are performed under the epidemiologic surveillance of influenza and acute respiratory infections established for public health surveillance purposes, there is no need for individual signed consent. nevertheless verbal consent is requested by each physician upon sampling. no funding received. the authors declare there is no conflict of interest. vaccine effectiveness in older individuals: what has been learned from the influenza-vaccine experience estimates of us influenza-associated deaths made using four different methods. influenza other respir viruses influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method. influenza other respir viruses pla d' informació de les infeccions respiratòries agudes a catalunya (pidirac) european centre for disease prevention and control. severe influenza surveillance in europe estratègia de vigilància dels casos greus de grip hospitalitzats estimating influenza vaccine effectiveness with the test-negative design using alternative control groups: a systematic review and meta-analysis severe influenza in 33 us hospitals, 2013-2014: complications and risk factors for death in 507 patients informe de la vigilancia d ela gripe en españa. temporada 2017-18 factors associated with 30-day mortality in elderly inpatients with community acquired pneumonia during 2 influenza seasons. hum vaccines immunother effectiveness of antiviral treatment in preventing death in hospitalized cases of severe influenza over six influenza seasons the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia case report acute influenza virus-associated encephalitis and encephalopathy in adults: a challenging diagnosis neurologic complications of influenza b virus infection in adults key: cord-309860-otx45b8x authors: conway, nicholas t.; wake, zoe v.; richmond, peter c.; smith, david w.; keil, anthony d.; williams, simon; kelly, heath; carcione, dale; effler, paul v.; blyth, christopher c. title: clinical predictors of influenza in young children: the limitations of “influenza-like illness” date: 2012-09-03 journal: j pediatric infect dis soc doi: 10.1093/jpids/pis081 sha: doc_id: 309860 cord_uid: otx45b8x background: influenza-like illness (ili) definitions have been infrequently studied in young children. despite this, clinical definitions of ili play an important role in influenza surveillance. this study aims to identify clinical predictors of influenza infection in children ≤5 years old from which age-specific ili definitions are then constructed. methods: children aged 6–59 months with a history of fever and acute respiratory symptoms were recruited in the western australia influenza vaccine effectiveness (waive) study. clinical data and per-nasal specimens were obtained from all children. logistic regression identified significant predictors of influenza infection. different ili definitions were compared for diagnostic accuracy. results: children were recruited from 2 winter influenza seasons (2008–2009; n = 944). of 919 eligible children, 179 (19.5%) had laboratory-confirmed influenza infection. predictors of infection included increasing age, lack of influenza vaccination, lower birth weight, fever, cough, and absence of wheeze. an ili definition comprising fever ≥38°c, cough, and no wheeze had 58% sensitivity (95% confidence interval [ci], 50–66), 60% specificity (95% ci, 56–64), 26% positive predictive value (95% ci, 21–31), and 86% negative predictive value (95% ci, 82–89). the addition of other symptoms or higher fever thresholds to ili definition had little impact. the centers for disease control and prevention definition of ili (presence of fever [≥37.8°c] and cough and/or sore throat) was sensitive (92%; 95% ci, 86–95), yet lacked specificity (10%; 95% ci, 8–13) in this population. conclusions: influenza-like illness is a poor predictor of laboratory-confirmed influenza infection in young children but can be improved using age-specific data. incorporating age-specific ili definitions and/or diagnostic testing into influenza surveillance systems will improve the accuracy of epidemiological data. population surveillance is used to guide preventative strategies for influenza such as choosing strains for seasonal influenza vaccine constitution and early identification of pandemic or epidemic antigenic drift variants [1] . early diagnosis of influenza disease also influences clinical decision making, especially when managing those at higher risk of severe disease [2] . influenza disease surveillance usually includes a combination of community-and hospital-based syndromic surveillance and routinely collected data concerning morbidity and mortality, with only some including laboratory confirmation of influenza infection. consistent with other countries, australian children <5 years old experience the highest laboratoryconfirmed influenza notification rate (3.4 times the rest of the population in 2008), the highest rate of general practice consultations with influenza-like illness (ili) ( 50 000/100 000 population in 2008), and the greatest morbidity [3] . in the united states, children <5 years comprised 28% of ili presentations in the centers for disease control and prevention (cdc) national network during the 2007-2008 influenza season [4] . the value of these data using ili surveillance depends on a reliable and robust definition of ili as well as a clear understanding of how ili activity relates to influenza. the definition of ili varies between countries and surveillance systems, but it usually includes the presence of fever and symptoms of acute respiratory tract infection [5] [6] [7] . influenza-like illness is a poor predictor of actual influenza infection in adults, despite attempts to improve the accuracy of the definition [8] , but there is limited data on the reliability of ili in predicting influenza infection in children [9] [10] [11] [12] . in this study, we have used data collected from young children recruited as part of a community and hospital influenza surveillance program to assess the clinical predictors of influenza infection in children 5 years old. we developed age-specific ili definitions and tested their diagnostic accuracy against existing definitions and parental opinion. the western australia influenza vaccine effectiveness (waive) study is an observational study designed to measure influenza vaccine effectiveness in children. the study design has been described elsewhere [13, 14] . in brief, children 6-59 months of age were eligible for participation if they presented with symptoms suggestive of an acute respiratory infection to selected general practices (2008 only), emergency departments (eds; 2008 and 2009), and pediatric inpatient facilities (2008 and 2009 ). the recruitment period was for the duration of the winter influenza season as determined by local population surveillance. children were eligible for enrollment in 2008 whether they had a history of fever (by parental report) or a measured temperature >37.8°c on presentation in addition to any acute respiratory symptoms within 72 hours before recruitment. to enhance recruitment in 2009, children were enrolled whether they had a history of fever or a measured temperature of >37.5°c on presentation plus the presence of any acute respiratory symptoms within 96 hours of recruitment. the study received approval from the relevant local human research ethics committees. on enrollment by trained research staff, parents were asked to complete a questionnaire detailing demographics, medical history, and presenting symptoms. temperature measured at enrollment was recorded by research staff. per-nasal swabs (copan diagnostics inc., murrieta, ca) placed in viral transport medium or per-nasal aspirates were collected. influenza testing on nasal swabs was performed by polymerase chain reaction (pcr) directed at hemagglutinin and matrix gene targets in multiplex real-time assay [14, 15] , and by conventional cell cultures [14] . in addition, samples underwent pcr directed at other common respiratory viruses including respiratory syncytial virus (rsv), parainfluenza viruses 1-3, human metapneumovirus, rhinoviruses, adenoviruses, coronaviruses (other than severe acute respiratory syndrome coronavirus), and bocavirus [14] . presence of influenza a or b detected by pcr or culture was collapsed into 1 dichotomous dependent outcome: influenza present or absent. the predictor variables of interest fell into 2 groups: (1) demographic factors (age, sex, race [indigenous or other], deprivation quintile, influenza vaccination status, prematurity [<37 completed weeks gestation], birth weight, past medical history, child care usage, household composition, and household smokers); and (2) symptomatology (recorded temperature and presence or absence of parentally reported: cough, coryza, wheeze, breathing difficulties, earache, sinusitis, sore throat, irritability, rash, diarrhoea, vomiting, lethargy, poor feeding, sleep disturbance, fever, and pallor). vaccination status was verified via the australian childhood immunisation register (acir) [16] . where acir conflicted with parental report, the family doctor was contacted to establish the number of trivalent influenza vaccine (tiv) doses given previously. because they are not normally distributed, categorical variables were created for age, household composition, and duration of fever and respiratory symptoms. postal addresses were geo-coded before conversion to deprivation quintiles using data produced by the australian bureau of statistics [17] . statistical analysis was performed using spss 16.0.0 (spss inc., chicago, il). after initial univariate analysis, variables were analyzed simultaneously within both groups (demographic factors and symptoms) by forced entry into a multivariable logistical regression model. factors found to be significant (p < .05) at the group stage were then entered into a model encompassing both groups. various definitions of ili were then constructed based on the significant predictors of influenza infection. sensitivity, specificity, and positive predictive value (ppv) and negative predictive value (npv) and their respective binomial 95% confidence intervals (cis) were then calculated. in addition, positive and negative likelihood ratios (lr+ and lr-) were also calculated. ninety-five percent cis for likelihood ratios were calculated in the manner described by simel et al [18] . new ili definitions were compared with definitions used by the cdc ( presence of fever (100 o f [37.8°c ]) and a cough and/or sore throat in the absence of a known cause other than influenza [5] ) and parents' response to the questionnaire question: "do you think your child has influenza ('flu')?" nine hundred and forty-four subjects were recruited (315 subjects in 2008 and 629 subjects in 2009). the majority were recruited within the hospital setting (general practice, 153 subjects; eds, 619 subjects; inpatient wards, 172 subjects). twenty-five subjects were withdrawn (specimens not processed, 8; invalid consent, 8; incorrect age, 3; other, 6), resulting in a total sample population of 919. altering the eligibility criteria in 2009 did not result in any additional recruits that were ineligible by 2008 criteria: 17 subjects had a temperature recorded >37.5°c yet <37.8°c; however, all of these subjects also had a history of fever within the previous 72 hours. respiratory viruses were identified in 711 subjects (77.4%). rhinovirus (n = 239), rsv (n = 210), and influenza virus (n = 179) were most frequently detected. of those in whom influenza was detected, 131 had influenza a (including 97 subtyped as influenza a/h1n1 2009) and 48 had influenza b. cultures proved positive for 59% (77 of 131) of those with influenza a and 58% (28 of 48) of those with influenza b. influenza was detected in 23.5% (74 of 315) of recruits in 2008, compared with 17.4% (105 of 604) of eligible recruits in 2009 (p = .03). the median age was 22 months and 526 of 919 (57%) were male. chronic comorbidities were uncommon: 140 children (15%) had a comorbid condition, of which asthma was the most common (89 of 919, 10%). premature births (<37 weeks gestation) accounted for 122 of 919 (13%) subjects. nearly one-half of all children enrolled (430 of 919, 47%) had received the recommended schedule for seasonal influenza vaccination (ie, 2 doses of tiv in the first year of vaccination followed by 1 dose in subsequent years [19] ) with a further 126 of 919 (14%) having received 1 dose in total ( table 1) . the most common symptoms reported by parents were cough (794 of 919, 86%), coryza (801 of 919, 87%), poor feeding (686 of 919, 75%), sleep disturbance (657 of 919, 71%), and irritability (607 of 919, 66%), although wheeze and respiratory distress were less prevalent (410 of 919, 45% and 413 of 919, 45%, respectively) ( table 2 ). for those that tested positive for influenza, the average temperature at enrollment was 39.5°c (range, 37-43.0; standard deviation . five hundred and ninety-eight parents recorded a response to the question, "do you think your child has influenza ('flu')?" seventy-eight of the 333 (23%) answering "yes" proved positive for influenza, whereas 24 of the 265 (9%) answering "no" were positive for influenza (p < .001). age >2 years, lack of tiv, lower birth weight, sharing a home with 2 or more other children, and being cared for by a single adult were significant demographic predictors of influenza infection on univariate analysis (table 3) . with the exception of the number of adult household members, all of these variables were significant when entered simultaneously within this group, and so they were entered into the final model. the symptoms that were significant univariate predictors of influenza infection were as follows: raised temperature, fever for >3 days, presence of cough, absence of wheeze, respiratory distress, and rash. fever, presence of cough, and absence of wheeze remained significant when entered simultaneously within the symptom group, and so they were retained for the final model. with the exception of number of children in the house, all variables entered into the final model remained significant (see table 3 ). based on the results of the regression equation, ili was defined in this population using various combinations of the following criteria: presence of cough, absence of wheeze, and incremental thresholds of fever (see figure 1 and table 4 ). a complete data set for each of these variables was available for 798 cases, which were used for subsequent calculations of the performance of ili definition. the presence of cough alone was highly sensitive (93%; 95% ci, 84-96) yet lacked specificity in diagnosing influenza infection (14%; 95% ci, [12] [13] [14] [15] [16] [17] . if the definition of ili comprised cough and the absence of wheeze, sensitivity was reduced (60%; 95% ci, 52-68) but specificity improved (59%; 95% ci, 55-63). the addition of fever 38°c to this definition resulted in a small the above analyses were repeated with data stratified according to vaccination status (no tiv vs any tiv) and age group (2 years vs >2 years) (see supplementary digital content). the observed ppvs in the unvaccinated group were significantly greater than the vaccinated group. subgroup analysis of each vaccination group stratum by year of illness was undertaken with no appreciable difference noted (data not shown). for those 2 years, an ili definition that included fever 38°c, cough, and the absence of wheeze had a significantly lower sensitivity and ppv and a higher specificity compared with those >2 years. the reliability of parental opinion was unaffected by vaccination status or age group of child. young children and infants with seasonal influenza can present with a wide variety of symptoms and may not yet be developmentally capable of verbalizing symptoms to their caregivers [20] . current definitions of ili are largely derived from adult studies and lack validation within the pediatric setting [5] [6] [7] . fever is a common presenting symptom for young children with influenza. when all children with acute respiratory illness (irrespective of presence or absence of fever) are tested, 95% of those who test positive for influenza had a history of fever [21, 22] . systematic review of the literature has failed to find any particular combination of additional symptoms that can reliably predict influenza infection [8] . only 2 studies in this systematic review enrolled preschool children: in 1 study, 18 of 610 cases with fever and respiratory illness were <5 years old [9] , whereas the other did not stratify by age [10] . later, ohmit and monto [11] reported a ppv of 64% in a subset of 221 children <5 years old presenting with cough and fever (>38.2°c). the generalizability of these figures is limited, however, because the study design excluded children with rsv infection, which would have falsely elevated the incidence of influenza infection in the study population [11] . a further prospective pediatric study assessing the predictive nature of an ili diagnosis in children with fever and symptoms suggestive of respiratory infection (n = 128; age <17 years) did not stratify results by age, making it less generalizable to younger children [12] . this study recruited young children presenting with acute respiratory symptoms during 2 successive winter influenza seasons, which included the first wave of pandemic influenza a/h1n1 2009. in this population, we have found that an existing ili definition in common usage (cdc definition) proved highly sensitive yet lacked specificity in identifying those with influenza infection. a surveillance system that uses this ili definition in isolation would therefore grossly overestimate influenza prevalence by virtue of the large number of false positives generated. in contrast, we found that an ili definition comprising fever 38°c, cough, and absence of wheeze achieved a greater balance between sensitivity and specificity in the study population. however, ili was a poor predictor of influenza infection regardless of which definition was tested. children with ili had a 20%-30% probability of actually having influenza infection, whereas those without ili (regardless of definition tested) had a 10%-15% probability of testing positive for influenza. each definition tested had positive and negative likelihood ratios approaching 1, indicative of little appreciable change in the odds of an individual having disease if they met ili definition criteria or not. furthermore, parental prediction of influenza infection in their children compared favorably with use of an ili definition, underlining the poor diagnostic accuracy of ili overall. decisions regarding the investigation and management of children suspected of having influenza take into account a number of factors, including the known incidence of influenza at that time, the likelihood of severe disease, and the availability of antiviral medication [2] . however our findings would suggest that, due to the inaccuracy of syndromic definitions, clinicians should maintain a low threshold for influenza testing in children with possible influenza. this is especially important for children with moderate to severe illness and/or those requiring hospitalization. similarly, diagnostic testing is required to obtain accurate influenza surveillance in young children. ideally, this should include testing by highly sensitive and specific methods such as pcr, although immunofluorescent antigen detection tests are sufficiently sensitive for use on nasopharyngeal aspirates. the rapid antigen tests have been used in some studies [23, 24] , but they are less sensitive than laboratorybased tests (especially for influenza a/h1n1 2009); their performance is influenced by specimen type, test brand used, and the virus type and subtype; and they do not identify influenza a subtypes. these tests have been successfully incorporated into public health influenza surveillance systems in the past [25] ; however, they need to be reassessed now that influenza a/h1n1 2009 is circulating. the eligibility criteria for our study included a history of fever. this methodology is similar to a number of previous studies [9] [10] [11] [12] and highlights the need for external validation of any clinical predictor tool that is intended to be used in unselected populations. however, our findings remain relevant to the general pediatric population given that the vast majority of children with influenza present with fever [21, 22] . the diagnostic accuracy of ili differed between those vaccinated and those unvaccinated (reflecting the lower prevalence of influenza in those who were vaccinated). the pediatric population studied had high vaccination rates with tiv as a result of a state-wide campaign introduced in 2008 that provides free seasonal influenza vaccination to this age group. the high prevalence of influenza vaccination in our study population contributes to the generalizability of our findings to other countries where influenza vaccination is readily available to children <5 years of age. the reliability of ili as defined by fever (38°c), cough, and absence of wheeze was age dependent, with less sensitivity and greater specificity in those 2 years of age or younger. this is a reflection of proportionally higher numbers of children 2 years old with wheeze irrespective of influenza status (data not shown), presumably as a result of a bronchiolitic-type illness. this age-dependent manifestation of influenza infection further highlights the problems inherent in applying adult-derived ili definitions to the pediatric population. because the presence of wheeze was determined by the parents, the calculated prevalence is likely to be different from one based on a clinical definition [26] . however, because this bias is constant between those who tested positive for influenza and those who were negative, it is not expected to have affected the results. also, in practice, assessing children with an ili usually relies on parental history as well as clinical findings, making our findings relevant to real-life circumstances. to our knowledge, this is the first study attempting to construct a definition of ili for children aged 5 years and under using prospectively gathered data from a general pediatric population presenting with symptoms suggestive of acute respiratory tract infection. we have demonstrated that when predicting influenza infection in younger children, an ili definition constructed using age-specific data and comprising presence of fever (38°c), cough, and absence of wheeze results in a greater balance between sensitivity and specificity compared with a definition of ili used by current surveillance systems. the diagnostic accuracy of influenza virus surveillance systems would be enhanced by developing age-specific ili definitions aimed at the pediatric age group and/or by incorporating diagnostic testing into the system. author contributions. n. t. c. and z. v. w. analyzed the data and revised the manuscript. p. c. r., d. w. s., a. d. k., s. w., h. k., d. c., and p. v. e. devised the waive study and revised the manuscript. c. c. b. supervised the present study, analyzed the data, and revised the manuscript. financial support. the waive study is funded by the western australia department of health. potential conflicts of interest. n. t. c., p. c. r., and c. c. b. are members of the vaccine trials group, telethon institute for child health research. the vaccine trials group has received funding from vaccine manufacturers for conducting clinical trials, although not in relation to this study. p. c. r. has served on a scientific advisory board regarding influenza vaccines for csl ltd., has received travel support from baxter and glaxosmithkline to present at scientific meetings, and received institutional funding for investigator-led who global influenza surveillance network 2010-11 influenza antiviral medications: summary for clinicians annual report of the national influenza surveillance scheme -8 influenza season overview of influenza surveillance in the united states heterogeneous case definitions used for the surveillance of influenza in europe working towards a simple case definition for influenza surveillance does this patient have influenza? evaluation of clinical case definitions of influenza: detailed investigation of patients during the 1995-1996 epidemic in france diagnosing influenza: the value of clinical clues and laboratory tests symptomatic predictors of influenza virus positivity in children during the influenza season clinical predictors of influenza in children lessons from the first year of the waive study investigating the protective effect of influenza vaccine against laboratoryconfirmed influenza in hospitalised children aged 6-59 months vaccine effectiveness against laboratory-confirmed influenza in healthy young children. a case-control study duplex real-time reverse transcriptase pcr assays for rapid detection identification of pandemic (h1n1) 2009 and seasonal influenza a/h1, a/h3, and b viruses australian childhood immunisation register seifa: socio-economic indexes for areas likelihood ratios with confidence: sample size estimation for diagnostic test studies the australian immunisation handbook 9th edition clinical presentation of influenza in unselected children treated as outpatients the underrecognized burden of influenza in young children performance of six influenza rapid tests in detecting human influenza in clinical specimens accuracy and interpretation of rapid influenza tests in children enhancing public health surveillance for influenza virus by incorporating newly available rapid diagnostic tests what do parents of wheezy children understand by "wheeze"? [see comment epidemiological research from glaxosmithkline and csl ltd. d. w. s. is a director of 2 not-for-profit organizations (the influenza specialist group and the asia-pacific alliance for the control of influenza) that receive funding from vaccine manufacturers. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-260191-0u0pu0br authors: haas, w.; krause, g.; marcus, u.; stark, k.; ammon, a.; burger, r. title: „emerging infectious diseases“: dengue-fieber, west-nil-fieber, sars, vogelgrippe, hiv date: 2004-05-29 journal: internist (berl) doi: 10.1007/s00108-004-1199-2 sha: doc_id: 260191 cord_uid: 0u0pu0br some emerging infectious diseases have recently become endemic in germany. others remain confined to specific regions in the world. physicians notice them only when travelers after infection in endemic areas present themselves with symptoms. several of these emerging infections will be explained. hiv is an example for an imported pathogen which has become endemic in germany. sars and avian influenza are zoonoses with the potential to spread from person to person. avian influenza in humans provides a possibility for the reassortment of a potential new pandemic strain. outbreaks of dengue fever in endemic areas are reflected in increased infections in travelers returning from these areas. currently, west-nile-virus infections are only imported into germany. the timely implementation of diagnostic, therapeutic and infection control measures requires physicians to include these diseases in their differential diagnosis. to achieve this goal, good cooperation between physicians, laboratories and the public health service is essential. ▃ die erfahrungen mit sars zeigen, wie rasch sich ein neuer erreger buchstäblich in wochen weltweit ausbreiten kann. epidemiologie aids-erkrankungen wurden 1981 erstmals in den usa beschrieben.retrospektive untersuchungen belegen jedoch,dass die der aids-erkrankung zugrunde liegende hiv-infektion sich bereits seit anfang der 1960er jahre zunächst unbemerkt weltweit ausgebreitet hat,wobei der internationale reiseverkehr wohl eine wesentliche rolle gespielt hat.⊡ abbildung 1 zeigt die aktuelle weltweite verteilung von hiv-infektionen (prävalenz),⊡ abbildung 2 die verteilung der neuinfektionen mit hiv im jahre 2003 (inzidenz). bei den von deutschen im ausland erworbenen hiv-infektionen rangiert als infektionsregion südostasien vor subsahara-afrika,gefolgt von der karibik/lateinamerika, anderen ländern in westeuropa und nordamerika sowie osteuropa. im vergleich zu deutschland, wo ca. 80% der hiv-infizierten männer sind und der altersgipfel in den altersgruppen zwischen 25 und 40 jahren erreicht wird, sind in entwicklungsländern mit vorwiegend heterosexueller übertragung von hiv mehr als die hälfte der infizierten frauen, der altersdurchschnitt liegt unter 25 jahren. der bedeutsamste übertragungsweg für hiv sind ungeschützte sexualkontakte, je nach region kann intravenöser drogenkonsum mit spritzentausch eine erhebliche rolle spielen. in vielen ärmeren ländern muss auch mit der möglichkeit von hiv-übertragungen durch verwendung unzureichend sterilisierter instrumente im medizinischen und paramedizinischen bereich (z.b.beim tätowieren) oder durch nicht auf hiv untersuchte blutspenden gerechnet werden.übertragungen von hiv durch kontaminiertes wasser, lebensmittel oder vektoren wie stechmücken sind nicht beschrieben. insbesondere im bereich der prostitution müssen in den meisten ländern über dem durchschnitt liegende hiv-infektionsraten erwartet werden,zumal andere, zusätzlich vorliegende sexuell übertragbare infektionen das übertragungsrisiko für hiv weiter steigern. von besonderer bedeutung als hiv-infektionsrisiko sind "urlaubspartnerschaften", da in diesen eher auf den schutz durch kondome verzichtet wird. tage bis wochen nach einem infektionsereignis treten bei ca. 50-70% der infizierten symptome einer akuten virusinfektion auf.am häufigsten werden fieber,muskel-und gliederschmerzen, lymphknotenschwellungen, ein flüchtiges, meist stammbetontes virusexanthem, kopfschmerzen und durchfälle berichtet.auf grund der unspezifischen symptomatik ist bei entsprechenden beschwerden eine expositionsanamnese zu empfehlen, bei der vor allem nach sexuellen risiken (ungeschützter vaginal-oder analverkehr) gefragt werden sollte. tions in travelers returning from these areas. currently,west-nile-virus infections are only imported into germany.the timely implementation of diagnostic,therapeutic and infection control measures requires physicians to include these diseases in their differential diagnosis.to achieve this goal,good cooperation between physicians, laboratories and the public health service is essential. abstract some emerging infectious diseases have recently become endemic in germany.others remain confined to specific regions in the world.physicians notice them only when travelers after infection in endemic areas present themselves with symptoms.several of these emerging infections will be explained.hiv is an example for an imported pathogen which has become endemic in germany.sars and avian influenza are zoonoses with the potential to spread from person to person.avian influenza in humans provides a possibility for the reassortment of a potential new pandemic strain.outbreaks of dengue fever in endemic areas are reflected in increased infec-gehäuften erkrankungen bei rückkehrern wieder.west-nil-virus-erkrankungen kommen derzeit nur als importierte erkrankungen in deutschland vor.wichtig ist,diese erkrankungen frühzeitig in die differenzialdiagnostischen überlegungen des klinikers einzubeziehen,um die erforderlichen maßnahmen zur diagnostik,therapie und zum infektionsschutz rechtzeitig einleiten zu können.dies erfordert ein gutes zusammenspiel mit dem labor und dem öffentlichen gesundheitsdienst. [23] . in deutschland wurden 9 wahrscheinliche fälle bekannt, die sich alle in betroffenen ländern infiziert hatten. sars wird im wesentlichen durch tröpfcheninfektion und/oder durch schmierinfektion übertragen.die inkubationszeit beträgt 2-10 tage.das erregerreservoir ist noch nicht eindeutig geklärt,ein tierisches reservoir wird jedoch angenommen [5] . der zentrale ausgangspunkt für die weltweite verbreitung von sars war das hotel metropol in hongkong. dort hatte ein arzt übernachtet,der sich zuvor in sei ein antikörpernachweis gelingt frühestens ca.3 wochen nach dem infektionsereignis. fällt bei vorliegen der geschilderten klinischen symptomatik und klarem anamnestischen infektionsrisiko der antikörpernachweis negativ aus, ist entweder eine wiederholung der untersuchung angezeigt oder es kann ein hiv-nukleinsäurenachweis (pcr-untersuchung) versucht werden, der im rahmen einer frischen infektion ca. 1-2 wochen vor dem antikörpernachweis positiv wird. bei einem negativen antikörpernachweis 3 monate nach dem letzten möglichen expositionsereignis kann eine hiv-infektion in der regel ausgeschlossen werden. die wichtigste präventionsmaßnahme für reisende ist der verzicht auf ungeschützte sexuelle kontakte bzw. die konsequente verwendung von kondomen beim geschlechtsverkehr. aus medizinischer sicht stellt die hiv-infektion keinen hinderungsgrund für reisen dar. je nach immunstatus, reiseland und reisemodalitäten kann jedoch ein erhöhtes infektions-bzw.erkrankungsrisiko für andere erreger bestehen.während impfungen mit totimpfstoffen unbedenklich sind (der impferfolg kann bei bereits bestehendem immundefekt beeinträch[18] . insbesondere innerhalb von krankenhäusern betroffener regionen hat sich sars sehr rasch sowohl auf patienten als auch auf personal übertragen.in deutschland ist es dagegen weder innerhalb noch außerhalb der krankenhäuser zu übertragungen von sars gekommen [20] . nach gegenwärtiger erkenntnis scheint dagegen das risiko einer übertragung von sars zwischen flugpassagieren während des fluges gering zu sein [13, 21] .seit juli 2003 wurden 6 weitere sars-fälle bekannt: 2 infektionen bei laborwissenschaftlern (singapur, taiwan) und 4 infektionen in südchina, bei denen die infektionsquelle unklar ist. bei sars handelt es sich um eine schwere, infektiöse atemwegserkrankung, die sich klinisch und radiologisch entweder als atypische pneumonie oder als akutes atemnotsyndrom (engl. adult respiratory distress syndrome,ards) manifestiert. die klinische symptomatik beginnt mit influenzaähnlichen symptomen mit meist hohem fieber, später kommen husten und rasch zunehmende atemnot sowie durchfälle hinzu [15] . als erreger wurde im märz 2003 das sars-coronavirus (sars-cov) identifiziert [2] . für die frühe diagnostik steht die polymerasenkettenreaktion (pcr) zum nachweis von sars-cov in respiratorischen sekreten,im stuhl und anderen sekreten zur verfügung. dabei muss allerdings beachtet werden, dass ein negativer befund auch in der akuten erkrankungsphase eine infektion mit sars-cov nicht ausschließt. ein indirekter immunfluoreszenztest zum serologischen nachweis von igm-und igg-antikörpern gegen sars-cov und ein elisa bieten sich insbesondere für epidemiologische fragestellungen an, da entsprechende nachweise erst mehrere wochen nach infektion zu erwarten sind. die bekämpfung und eindämmung einer derartigen epidemie ist aufgabe des öffentlichen gesundheitsdienstes. hier ist zum einen eine lückenlose surveillance und eine frühzeitige verfügbarkeit umfassender informationen und empfehlungen erforderlich [10] .für die gesundheitsämter stellt eine derartige epidemie eine herausforderung dar,da sie sehr umfangreiche schutzmaßnahmen umsetzen müssen [19] .diese bestehen zum einen in der frühzeitigen identifizierung von kontaktpersonen und gegebenenfalls in der absonderung von personen nach einem festgelegten schema.dies erfordert auch eine enge zusammenarbeit mit krankenhäusern,niedergelassenen ärzten und krankentransportdiensten [11] . eine impfung wird mittelfristig nicht zur verfügung stehen. die aviäre influenza wurde zum ersten mal im jahr 1878 in italien beschrieben [16] . aviäre influenza wird durch die subtypen h5 und h7 des influenza-a-virus verursacht.das reservoir dieser erreger stellen wildvögel (insbesondere wasservögel) dar, deren infektion in der regel asymptomatisch verläuft. mutationen im hämagglutinin sind jedoch die ursache dafür, dass immer wieder h5-bzw. h7-viren auftreten, die für vögel hochpathogen sind. zwischen 1959 und 2003 wurden insgesamt 21 ausbrüche bei geflügel registriert [24] . schwere respiratorische erkrankungen durch ein rein aviäres virus nach übertragung direkt von erkranktem geflügel auf den menschen wurden erstmals bei dem ausbruch 1997 in hongkong beobachtet.bis zu diesem zeitpunkt war eine direkte übertragung von aviären influenza-a-viren auf den menschen nicht belegt worden. seit 1997 wurde in 6 situationen eine übertragung von hochpathogenen aviären influenzaviren (hpai) auf den menschen nachgewiesen (⊡ tabelle 1). davor war man davon ausgegangen, dass für eine übertragung auf den menschen eine genetische neukombination ("genetic shift") von aviären und humanen influenza-a-viren erforderlich ist. als hauptvehikel für die genetische neukombination, das sog. reassortment, des segmentierten genoms der influenza-a-viren gilt das hausschwein, da es auf seinen zellen sowohl rezeptoren für aviäre als auch humane influenza-a-viren besitzt.ein reassortment konnte als entstehungsweg der pandemischen influenzaviren der "asiatischen grippe" 1957/58 (h2) und der "hongkong-grippe" 1968-70 (h3) nachgewiesen werden. im frühjahr 2003 trat ein großer ausbruch mit h7-viren in den niederlanden auf und breitete sich nach belgien und auf einen betrieb in deutschland aus. mehr als 1000 betriebe waren betroffen, mehr als 30 mio.vögel wurden daraufhin getötet [9] . bei diesem ausbruch wurden bis ende april 266 fälle von konjunktivitis diagnostiziert. fast alle erkrankte hatten engen kontakt mit erkranktem geflügel gehabt. bei 40 (12%) der erkrankten traten zusätzlich grippeähnliche symptome ("influenza-like illness",ili) auf,bei weiteren 23 personen wurde nur eine ili beschrieben. ein tierarzt, der einen betrieb mit infizierten tieren besucht hatte, verstarb kurz darauf an der erkrankung.der nachweis von h7n7 in der bronchoalveolären insgesamt wurden bei diesem ausbruch 453 menschen mit gesundheitsproblemen identifiziert [9] . bei 89 dieser personen war der influenza-a/h7-nachweis positiv. drei kontaktpersonen von an konjunktivitis erkrankten,die an der tötung von influenza-a (h7n7) infiziertem geflügel beteiligt waren, die selbst aber nicht mit infiziertem geflügel in berührung gekommen waren,wiesen ebenfalls symptome auf.in ihren bindehautabstrichen wurden influenza-a/h7n7-viren nachgewiesen. dies wurde als ein starker hinweis für eine mensch-zu-mensch-übertragung gewertet [3, 9] . [7, 25] .in keinem der fälle kam es bisher durch die h5n1-erkrankungen zu einem ausbruch oder infektionsketten mit einer effizienten mensch-zu-mensch-übertragung. nur ein teil der infizierten reisenden entwickelt symptome, nur ein teil der erkrankten sucht einen arzt auf und auch in diesen fällen wird nicht immer eine untersuchung auf dengue-fieber durchgeführt. serologische untersuchungen haben gezeigt,dass bei etwa 7% der tropenreisenden mit fieber eine dengue-infektion vorlag [12] . wie die ersten erfahrungen zeigen,ist die surveillance auf der basis des ifsg jedoch im sinne eines frühwarnsystems gut geeignet,risikogebiete für reisende identifizieren und entsprechende reisemedizinische informationen veröffentlichen zu können. dengue-viren werden durch aedes-moskitos übertragen und sind in über 100 tropischen und subtropischen ländern verbreitet. für die großen epidemien beim menschen ist ausschließlich die virusübertragung zwischen vektor (vor allem aedes aegypti,in südostasien auch aedes albopictus) und mensch von bedeutung. die aedes-mücken sind zeitlebens, d.h. 2 bis 4 monate lang infektiös. das spektrum der klinischen manifestation reicht von inapparenten oder milden formen bis zu schweren, unter umständen letalen verläufen.die inkubationszeit beträgt im durchschnitt 4-7 tage ein wirksamer dengue-impfstoff ist bisher nicht verfügbar. deshalb kommt der prävention die entscheidende bedeutung zu. reisenden sollten gebiete mit massiven dengue-ausbrüchen meiden. eine expositionsprophylaxe gegenüber moskitostichen ist wichtig und muss aufgrund der überwiegend tagaktiven dengue-vektoren konsequent durchgeführt werden. [1] . in deutschland werden derzeit blutund plasmaspender von der spende zurückgestellt, wenn sie sich zwischen dem 1. juni und dem 30. november eines jahres auf dem nordamerikanischen kontinent aufgehalten haben und der tag der rückkehr weniger als 4 wochen zurückliegt. beim menschen nimmt die überwiegende zahl von wnv-infektionen einen inapparenten oder milden verlauf.nach einer inkubationszeit von 3-14 tagen entwickeln etwa 20% der infizierten eine fieberhafte erkrankung, die einige tage andauert. die symptome umfassen kopfund muskelschmerzen, übelkeit, exanthem und generalisierte lymphknotenschwellungen. eher selten kommt es zu einem komplizierten verlauf mit zeichen der meningoenzephalitis. dann ist ein breites spektrum an neurologischen symptomen zu beobachten.über die hälfte der patienten mit enzephalitis leidet an spätfolgen. bei den ausbrüchen in den letzten jahren in usa, rumänien, israel und russland lag die letalität bei den hospitalisierten patienten zwischen 4% und 14%. risikofaktoren für einen tödlichen verlauf sind höheres alter,diabetes mellitus und immunsuppression. bei neurologischen erkrankungen von personen, die sich in den letzten 14 tagen in endemiegebieten aufhielten, sollte unter berücksichtigung der jahreszeit und der aktuellen epidemiologischen situation differenzialdiagnostisch auch an eine wnv-infektion gedacht werden. die diagnose einer wnv-infektion kann durch den direkten virusnachweis (z. b. pcr) oder auch serologisch erfolgen (nachweis wnv-spezifischer igm-antikörper, mindestens 4facher titeranstieg zwischen akutphase-serum und rekonvaleszenten-serum; [14] ). mögliche kreuzreaktionen mit anderen flaviviren müssen berücksichtigt werden (dengue, gelbfieber, fsme, japanische enzephalitis, st.-louis-enzephalitis). dabei spielt auch die gezielte reiseanamnese eine wichtige rolle. zurzeit werden in deutschland epidemiologische untersuchungen (surveys bei vögeln, pferden, ausgewählten humanen populationen) zum vorkommen von wnv bzw.zur prävalenz von wnv-antikörpern durchgeführt, um das gefährdungspotenzial für die bevölkerung besser einschätzen zu können. estimated risk of west nile transmission through blood transfusion during an epidemic in queens identification of a novel coronavirus in patients with severe acute respiratory syndrome ausbruch von aviäre influenza durch influenzavirus a/h7n7 in den niederlanden increase in imported dengue isolation and characterization of viruses related to the sars coronavirus from animals in southern china dengue and dengue hemorrhagic fever avian influenza a (h5n1) in 10 patients in vietnam west nile fever -a reemerging mosquito-borne viral disease in transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands sars-surveillance -wurde sie den anforderungen an die surveillance neu auftretender infektionskrankheiten gerecht? prevalence of infection with dengue virus among international travellers transmission of the severe acute respiratory syndrome on aircraft west-nil-virus: prävalenz und bedeutung als zoonose-erreger the severe acute respiratory syndrome e (1878) epizoozia tifoide nei gallinacei west nile virus sars: retrospective cohort study among german guests of the hotel ‚m' , hongkong arbeitsaufwand des ögd im zusammenhang mit sars -ergebnisse einer befragung zur seroprävalenz von krankenhaus und praxispersonal nach der betreuung von sars-patienten zur übertragung von sars in verkehrsflugzeugen -erfahrungen aus deutschland are we ready for pandemic influenza consensus document on the epidemiology of severe acute respiratory syndrome (sars) avian influenza a (h5n1) da die übertragung überwiegend durch nachtaktive mückenarten erfolgt, ist bei aufenthalten in gebieten mit bekannter wnv-aktivität eine expositionsprophylaxe erforderlich. key: cord-356188-rwf78stz authors: oshansky, christine m.; thomas, paul g. title: the human side of influenza date: 2012-07-01 journal: journal of leukocyte biology doi: 10.1189/jlb.1011506 sha: doc_id: 356188 cord_uid: rwf78stz a clear understanding of immunity in individuals infected with influenza virus is critical for the design of effective vaccination and treatment strategies. whereas myriad studies have teased apart innate and adaptive immune responses to influenza infection in murine models, much less is known about human immunity as a result of the ethical and technical constraints of human research. still, these murine studies have provided important insights into the critical correlates of protection and pathogenicity in human infection and helped direct the human studies that have been conducted. here, we examine and review the current literature on immunity in humans infected with influenza virus, noting evidence offered by select murine studies and suggesting directions in which future research is most warranted. influenza infection in humans can cause a wide range of disease symptoms, from asymptomatic to serious illness, characterized by sudden onset of fever, myalgia, and respiratory indications, including nonproductive cough, sore throat, and rhinitis. in addition, children infected with influenza commonly present with otitis media, nausea, and vomiting and more frequently, complain of rhinorrhea [1, 2] . historically, the pediatric and elderly populations are considered vulnerable during influenza epidemics [3] [4] [5] [6] , and although still debated, studies suggest that children have a unique role in the spread of influenza during epidemics [7] [8] [9] . children under 2 years old are more susceptible to secondary complications [10] , and infants with severe respiratory tract viral infection are often at greater danger for pulmonary dysfunction, including wheezing, allergy, and asthma later in life [11] [12] [13] . in contrast, elderly individuals are at higher risk for developing hemorrhagic bronchitis, pneumonia, and death when infected with influenza [14] , and this population generally has a less-robust immune response following influenza immunization. although rsv infections are the leading cause of hospitalizations of children in the united states, influenza accounts for the highest incidence of infection in children under 2 years of age [15] . influenza epidemics occur annually and can cause significant morbidity and mortality worldwide. during 1976 -2007, the cdc estimated that in the united states alone, annually, 8.5% (6309) of all "deaths with underlying pneumonia and influenza causes" (74, 363) were influenza-associated, and 2.1% (23, 607) of "deaths with underlying respiratory and circulatory causes" (1,132,319) were influenza-associated [16] . furthermore, individuals with underlying respiratory and cardiac disease or diabetes mellitis, infected with influenza, are often at higher risk for developing hemorrhagic bronchitis or pneumonia and associated comorbidities. in 2008, it was estimated that there were 28,000 -111,500 deaths attributable to influenza in children ͻ5 years of age globally, with developing countries carrying the largest burden [17] . in the united states, annual influenza epidemics account for an estimated $10 billion (12% of the total economic burden) in direct medical costs [18] and an estimated $44 -$163 million in hospitalization costs for children [19] . introduction of a swine-origin influenza into the human population in 2009 quickly disseminated globally, causing the first pandemic of the 21st century. this novel h1n1 virus, hereafter referred to as a(h1n1)pdm09 [20] , disproportionately affected children and young adults [21, 22] . observed mortality increased between four and 10 times in the pediatric population during the initial wave of a(h1n1)pdm09 infection in 2009 compared with previous influenza seasons [23] , whereas those individuals born prior to 1950 had pre-existing, cross-reactive antibodies against a(h1n1)pdm09 [24] . as a result, within the population of those with laboratory-confirmed a(h1n1)pdm09, 2% were older individuals (ͼ60 years), whereas 79% were ͻ30 years old. risk factors for severe a(h1n1)pdm09 infection are similar to those for seasonal influenza but also include obesity. the majority of the severe or fatal cases have accompanying chronic illnesses, such as cardiac disease, chronic respiratory diseases, and diabetes [25] . several studies suggest that pregnant and postpartum women are at increased risk for complications from infections with a(h1n1)pdm09 [26 -28] . furthermore, severity of a(h1n1)pdm09 infection was related to the length of time between symptom onset and the initiation of antiviral treatment with na inhibitors [29] . influenza infections are associated with secondary bacterial infections, or superinfections, most notably, streptococcus pneumoniae and staphylococcus aureus (reviewed in ref. [30] ). in children, the most common secondary bacterial infections lead to acute otitis media, which is found in 50% of patients with symptomatic influenza infection, with five to six cases observed annually for every 100 children ͻ2 years of age [10] . since the emergence of a(h1n1)pdm09, approximately one-third of fatal infections are associated with bacterial coinfection [31, 32] , but this number includes cases where respiratory support is needed, adding to the risk of secondary infections. in fatal cases of those infected by a(h1n1)pdm09, diffuse alveolar damage alone, diffuse alveolar damage associated with necrotizing bronchiolitis, and diffuse alveolar damage with hemorrhage were found, and in most of the cases where necrotizing bronchiolitis was found, the individuals had bacterial coinfections [33] . vaccination remains an effective and primary preventive strategy to avert influenza infection. the world health organization and cdc recommend that children and adults over the age of 6 months receive an influenza vaccination annually [34] . currently, there are three licensed seasonal vaccines administered in the united states: 1) a tiv, administered by i.m. injection; 2) a laiv, delivered intranasally; and 3) an i.d.-administered tiv preparation [35] . each includes three circulating strains of influenza grown in eggs, reflecting annual surveillance data that predict which strains (a/h1, a/h3, and b) are most likely to be circulating the following season in northern and southern hemispheres. the i.m. tiv is approved for use in children and adults over 6 months of age, whereas the newer i.d. tiv is approved for adults aged 18 -64 years. laiv is approved for use in healthy children and adults between 2 and 49 years of age. the current influenza vaccines work to induce memory recall responses, primarily via humoral immune responses against the ha and na surface glycoproteins, and ha inhibition antibody responses following immunization correlates with protection against infection [36, 37] . the main component standardized in influenza vaccines is the ha protein, and it is well-characterized in safety trials and challenge studies that the amount of ha within the vaccine correlates with level of protection elicited by immunization [38, 39] . beginning during the northern hemisphere's 2010 -2011 influenza season, a vaccine containing 60 g ha/vaccine strain, as opposed to 15 g in other preparations, was approved as an alternative tiv for elderly individuals (over age 65 years) [35] . following vaccination, ascs rapidly proliferate upon antigen exposure, and cell numbers peak 1 week postimmunization in healthy adults and children [40, 41] . igg and iga ascs decline to low levels by 4 -6 weeks postimmunization [42, 43] . this increase in the total numbers of ascs corre-sponds to nab levels postvaccination, peaking at 4 weeks in adults and children [43] . laiv elicits a strong serum and mucosal influenza-specific antibody response [44] , and studies in young children (6 -59 months of age) showed that those receiving laiv had significantly reduced incidence of influenza infection [45] . although limited data exist, influenza-specific ascs and nab titers are increased to a greater degree following tiv immunization compared with laiv vaccination [43, 46, 47] , but the role of neutralization of laiv occurring in antigen-experienced individuals is not well understood. influenza immunization is effective, and studies in healthy children ͻ15 years of age have shown tiv efficacies ranging from 31% to 90% (reviewed in ref. [48] ). since 2010, universal influenza vaccination is recommended, i.e., for all individuals at least 6 months of age, but traditional efforts remain focused on those individuals at greatest risk of serious disease: young children, elderly, individuals with pulmonary or cardiovascular disorders, and those who are immunocompromised or pregnant [35] . vaccination programs also elicit indirect benefits, including herd immunity, by which immunized individuals protect those who are immunocompromised or other nonvaccinated individuals [49, 50] . this effect was observed in a retrospective epidemiological study in japan and the united states examining mortality and influenza vaccination rates between 1949 and 1998 [51] . as children have long been considered to have an important role in the spread of influenza during epidemics [7] [8] [9] , japanese officials legislated compulsory vaccination of school-aged children in 1977, and as the policy changed to optional immunization in 1987, it became clear that mortality rates were inversely correlated with overall vaccination coverage. however, recent reviews of vaccine studies in various populations have concluded that overestimation of the benefits of vaccination is common, particularly within the elderly population [52, 53] , but studies that take into account selection biases do suggest that vaccination is associated with lower risk of hospitalization for pneumonia or influenza, as well as all-cause mortality [52, 54, 55] . furthermore, in october 2009, the united states commenced a national influenza h1n1/2009 vaccination campaign, and it is estimated that ϳ41.2% and 43% of the u.s. population received the 2009 -2010 and 2010 -2011 influenza vaccines, respectively [56] . following implementation of this campaign, the frequency of positive influenza cultures reported to the cdc declined quickly. immunization and herd immunity resulting from prior exposure of a(h1n1)pdm09 likely resulted in decreased cases of influenza during the 2010 -2011 influenza season. undoubtedly, influenza vaccines can be improved, particularly for the most vulnerable populations. continuous epidemiological surveillance of circulating influenza strains is required for keeping pace with antigenic drift and shift (reviewed in ref. [57] ). antigenic drift is an endless battle, whereby viral variants emerge as a result of point mutations within the viral genome, escaping antibody-mediated viral neutralization. whereas many mutations occur throughout the viral genome, the point mutations within the ha or na proteins can change antibody-binding domains, such that individuals no longer have adequate, protective b cell memory responses. in contrast, major qualitative changes in antigenicity as a result of antigenic shift occur as a result of genetic reassortment and replacement of one or more influenza gene segments. this, in turn, creates a new influenza virus subtype, to which little or no immunity may exist within the population. antigenic shift remains a chief concern, in that highly pathogenic influenza viruses could potentially gain efficient human-to-human transmission. as influenza viruses frequently undergo antigenic drift and less frequently, shift, a desirable vaccine candidate would be able to induce highly specific, cross-reactive antibodies and t cell responses. one strategy used in vaccine improvement includes the use of adjuvant to increase antigen uptake by apcs and enhance cytokine production. neither of the u.s.-licensed tivs nor laiv include adjuvant, but there are several licensed vaccines available outside of the united states that use oil and water emulsions for seasonal influenza as well as for a/h5n1 (reviewed in ref. [58] ). for instance, novartis vaccines and diagnostics (switzerland) manufactures fluad using mf59 adjuvant, and glaxosmithkline biologicals (belgium) produces pandemrix, which includes as03. fluad has been used in europe since 1997 and was recently approved for use in canada in adults 65 years and older. mf59 and as03 are both squalene-based with surfactants included to stabilize the emulsions. clinical trials of mf59-adjuvanted vaccines are well-tolerated and elicit higher antibody responses in adults [59 -61] and in children [62] [63] [64] compared with unadjuvanted vaccine preparations. the concept of a universal influenza vaccine, where one vaccine elicits protection against multiple influenza subtypes, is becoming a reality. the influenza m2 ion channel is expressed at high levels on the surface of influenza-infected cells and has a conserved ectodomain (m2e) that is 24 aa in length [65] . animal studies have demonstrated improved viral clearance and cross-protection upon lethal challenge in mice following passive m2 mab transfer or m2-immunized mice (reviewed in ref. [66] ). phase i clinical safety trials evaluating vax102, manufactured by vaxinnate, have reported increased m2e antibody responses [67] . creation of a universal influenza vaccine that targets conserved regions of the ha stem region is particularly appealing, and several groups have demonstrated encouraging results using broadly neutralizing mab to regions of ha that are cross-reactive between group 1 and group 2 influenza strains [68 -70] . structural analyses indicate that these antibodies bind to conserved pockets of the a helix of the ha stalk, located below the trimeric head region [71] [72] [73] . it is therefore within the realm of possibility to focus immunization strategies to these conserved regions in the ha and m2 proteins to elicit effective antibody, and presumably t cell, responses in the case of emergent influenza viruses. still, it remains a mystery as to why these conserved epitopes have not generated a high level of cross-protection in populations repeatedly immunized or infected with diverse viral strains. factors that limit influenza vaccine efficacy in humans include age, immunocompetence, and pre-existing antibody levels from previous infections or immunizations. whereas key lessons have been learned using animal models of influenza infection, studies done using human participants are invaluable for describing the unique and often complex characteristics of the human immune response to infection and immunization. here, we describe central findings obtained from human influenza challenge studies, natural infections, and ex vivo characterization of human immune cells ( table 1 ). figure 1 indicates where questions remain for the human immune response to infection. effective control and clearance of influenza infection can involve most components of the innate and adaptive immune responses. in mammals, the cellular innate immune system acts as a first-line defense consisting of multipotent hematopoietic stem cells that differentiate into nk cells, mast cells, eosinophils, and basophils, as well as macrophages, neutrophils, and dcs, collectively known as phagocytic cells. phagocytes function as apcs, engulfing and digesting opsonized pathogens and apoptotic cells. these cells essentially act as scavengers to uptake toxic metabolic byproducts and produce myriad inflammatory mediators, which ideally, result in the killing of viruses, bacteria, and parasites. however, abundant inflammation often has a role in creating immunopathology and can contribute to diverse inflammatory diseases, including asthma [123] [124] [125] . in the absence of protective immune memory, the innate response limits initial viral replication, while the adaptive immune response develops. influenza primarily infects epithelial cells lining the respiratory tract but can infect directly or be phagoctyosed by "professional" innate immune cells, dcs, and macrophages, which mature in response to viral stimuli or signals from other infected cells. after activation, mature dcs migrate to draining lns, where they have a major role in antigen presentation to naïve t cells. influenza infection of human monocyte-derived dcs can result in decreased dc maturation from ns1 suppression of type i ifn production [126] . seasonal h1n1 viruses, including a(h1n1)pdm09, elicit weak, proinflammatory cytokine gene expression in human dcs [127] . we know from murine models that influenza contains ligands that activate several prr signaling pathways, including tlr3 and retinoic acid-inducible gene i (rig-i), which recognize dsrna, and tlr7, which recognizes ssrna [128 -130] . type i (ifn-␣/␤) and type iii (ifn-1-3) ifns mediate expression of more than 300 isgs via the jak/stat pathway and are important in the induction of antiviral responses against influenza viruses [131] . stimulation of these pathways in dcs and macrophages leads to the induction of a potent antiviral response, including production of proinflammatory cytokines il-6 and ifn-␣ within the respiratory tract by day 2 following infection, paralleling the peak of viral replication, mucus production, and disease symptoms. tnf-␣ and il-8 levels peak between day 3 and 6 after infection as symptoms begin to subside [95] . antimicrobial peptides, such as collectins, defensins, and cathelicidins, are important innate effectors in the human defense against influenza infection. collectins, or collagenous lectins, are found primarily in mucosal secretions and include sp-a and sp-d; sp-d has been shown to inhibit ha activity by binding to n-linked, high-mannose oligosaccharides in a calcium-dependent manner, while enhancing activation of neutrophils in in vitro assays [132, 133] . in murine models, sp-a and sp-d have roles in promoting viral clearance from lungs and reducing inflammatory cytokines [134, 135] , but their role during human influenza infection is less well-defined. influenza virus impairs calcium metabolism and subsequent calcium deactivation in neutrophils [136] , and interestingly, sp-d can protect neutrophils from calcium deactivation if preincubated with virus, presumably by inhibiting ha activity [132] . in humans, the only cathelicidin, human cathelicidin 18, the active form of which is ll-37, is found in diverse cell types, including neutrophils, nk cells, monocytes, b cells, ␥␦ t cells, mast cells, as well as epithelial cells lining the lungs [137, 138] . like surfactants, ll-37 shows antiviral activity against influenza and can reduce viral titers in in vitro studies [139] . defensins have a key role in host defense and act primarily to disrupt lipid membranes leading to lysis and subsequent death of bacteria, fungi, and enveloped viruses [140] . there are two classes of defensins, ␣-defensin and ␤-defensin, which were first described in models of host defense against bacterial pathogens. both types are found in the respiratory and gastrointestinal tracts. the ␣-defensins in humans include hnp-1, -2, -3, and -4, all found in high concentrations within neutrophil granules and released within the lungs in response to proinflammatory signals during infection and inflammation [141] . in vitro, hnp-1 and hnp-2 act as chemokines for human monocytes [142] , and hnp-1, -2, and -3 can induce neutrophil internalization of influenza in vitro [143] . moreover, the ␣-defensins may have a role in directly inactivating influenza virus as a/wsn preincubated with hnp-1 showed decreased infection in vitro in a dosedependent manner [144] . hnp-1 can also inhibit influenza virus replication via modulation of cellular pathways, including the pkc pathway [145] . heterogeneous populations of monocytes comprise ϳ10% of blood leukocytes in humans (4% in the mouse), and their roles in controlling pathogens and inflammation can be characterized by functional cellular subsets. human blood monocytes can be divided into three populations based primarily on cell surface expression of cd14 (lps receptor) and cd16 (fc␥riii). the first subgroup consists of "classical monocytes", characterized by cd14 expression and lack of cd16 expression (cd14 ϩ cd16 ϫ ). the cd14 ϩ cd16 ϫ monocytes comprise 80 -90% of blood monocytes and resemble murine inflammatory ly6c ϩ (gr1 ϩ ) monocytes [146] . the second subgroup is defined by high expression of cd14 and cd16 (cd14 ϩ cd16 ϩ ) and release tnf following lps stimulation [147] . the third monocytic subgroup is characterized by low expression of cd14 and high expression of cd16 (cd14 dim cd16 ϩ ), and at least one study has shown increased numbers in the blood of sepsis patients [148] . cd14 dim blood monocytes function as efficient producers of proinflammatory cytokines in response to viruses and nucleic acids [149, 150] . few studies have examined the role of monocytes during influenza infection in humans, particularly regarding the specific subsets mentioned above, but comparison of ifn-␥ production from t cells cocultured with cd64 ϩ cd16 ϫ and cd64 ϫ cd16 ϩ monocytes [119, 120] cellular immunity class i hla presents peptides from internal and external viral proteins. [121, 122] showed that cd64 ϫ cd16 ϩ monocytes (equivalent to the cd14 ϩ cd16 ϩ monocytes) induced a greater response to influenza a antigen [151] . in vitro, human dcs and macrophages treated with type i or type iii ifn prior to influenza infection result in potent antiviral activity and decreased viral replication [127] . among dc populations, cd11c ϫ cd14 ϫ hla-dr ϩ cd123 ϩ pdcs were found to produce more ifn-␣ in response to a high dose of a/pr8/8/34 (h1n1) [152] . whereas increased numbers of total cd14 ϩ cells are observed in the blood of influenza-infected individuals, decreased blood myeloid dcs and pdcs were observed (defined in this study as hla-dr ϩ cd14 ϫ cd16 ϫ cd11c ϩ and hla-dr ϩ cd14 ϫ cd16 ϫ cd123 ϩ , respectively) [153] . however, increased numbers of pdcs were observed in nasal secretions compared with healthy controls within this cohort. the ns1 protein is a well-characterized antagonist of the host's antiviral response, acting primarily to circumvent ifn-␣/␤ responses (reviewed in ref. [154] ), and in vivo models show that its deletion results in virus attenuation and higher levels of ifn [155] . type i ifn antagonism can result in inhibition of adaptive immunity by interfering with dc maturation and thus, the ability of dcs to activate t cells [126] . the ns1 protein from influenza b virus inhibits isgylation, an important host antiviral effector mechanism, by interacting with the n-terminal domain of isg15 [156] . murine models have provided several clues regarding the actual role of monocytes during the innate immune response to influenza infection. in vivo, ccr2-dependent recruitment of inflammatory monocytes is critical for host defense and contributes to the innate immune response, as well as downstream adaptive responses [157] . during influenza infection, ccr2deficient mice have reduced monocyte and increased neutrophil recruitment, but, despite higher viral lung titers, in one model of infection these mice were protected from severe pneumonitis [158, 159] . in the lungs, ccr2-mediated recruitment of tnf and inos-producing dcs augments influenzaspecific t cell responses [160] . at least one murine study suggests that pregnant mice have increased infiltration of neutrophils (cd11b high ly6g high mhcii ϫ ) and macrophages (cd11b high ly6g ϫ mhcii high ) and elevated, proinflammatory cytokine levels, despite no change in viral titers in the lungs compared with nonpregnant animals [161] . in vitro, influenza infection of murine alveolar epithelial cells resulted in monocyte production of mcp-1 (ccl2, the ligand for ccr2) and rantes, and monocyte migration was dependent on ccr2 expression [162] . although some murine models suggest that neutrophils may have little role in mediating viral clearance [163] , type i interferon (ifn-␣/␤)-dependent generation of ly6c hi monocytes following influenza infection may be necessary to limit excess neutrophil infiltration, preventing tissue damage caused by uncontrolled inflammation [164] . in addition, the newly described innate lymphoid cells have been implicated in having a role in airway integrity following influenza infection [165] . thus, innate immune cells, particularly monocytes, have at least two roles during influenza infection: to boost the innate immune response by producing proinflammatory cytokines and to enhance influenza-specific t cell responses. however, the lack of clear correspondence between murine and human monocytic subsets has made the application of these data to the human situation difficult and highlights the need for specific studies focused on human innate immune responses. during rsv and influenza infection of infants, neutrophils are recruited to the upper and lower airways in high numbers [103, 104] , and fatal cases during the a(h1n1)pdm09 pandemic with necrotizing bronchiolitis had more neutrophils within the lung infiltrate [33] . the neutrophil chemoattractant il-8, produced by bronchial epithelial cells, is elevated early during experimental human influenza infection [95] and is increased in the serum of a/h5n1-infected individuals [96] . increased il-8 levels in bals of humans are associated with the infiltration of neutrophils and may have a role in neutrophil-mediated lung injury and development of acute respiratory distress syndrome [97] . ex vivo influenza infection of pb-mcs has been shown to induce apoptosis in neutrophils [166] and peripheral blood monocytes [167] . influenza infection of neutrophils resulted in increased hydrogen peroxide production, enhanced surface expression of fas antigen and fasl, and secretion of fasl into the supernatant [166] . interestingly, this study showed that influenza infection of neutrophils resulted in increased internalization of escherichia coli, suggesting a role in controlling bacterial secondary infections in humans. moreover, standard chemotaxis assays during experimental influenza infection [168 -170] or chemokine receptor expression during ex vivo influenza infection [105] demonstrated impaired chemotaxis of monocytes and granulocytes. still, although all of these studies suggest that neutrophils can be regulated and recruited during human influenza infection, the specific protective and pathological roles that they play in vivo remain unclear. nasopharygeal aspirates of infants infected with influenza have increased levels of proinflammatory cytokines such as il-6, tnf-␣, il-10, or ifn-␥ compared with rsv-or human metapneumovirus-infected infants [98 -100] , and mip-1␣ levels are higher in infants with more severe influenza infections characterized by hypoxic bronchiolitis [101] . variability between these studies is likely a result of seasonal differences observed year-to-year. individuals infected with a/h5n1 have high viral loads detected within respiratory secretions and peripheral hypercytokinemia [96, 102] . severe disease is associated with macrophage infiltration into the lungs, and chemoattractants of monocytes and macrophages, such as ifn-inducible protein 10 (ip-10), monokine induced by ifn-␥ (mig), and mcp-1, were found to be elevated in the serum of influenza-infected individuals and were highest in those infected with a/h5n1 [96] . therefore, peripheral (or local) hypercytokinemia likely contributes to enhanced immunopathogenesis during influenza disease. although there is no difference in the duration of viral shedding in children hospitalized for a/h1n1 or a/h3n2 infections compared with those infected with influenza b virus, children with influenza a exhibited different serum cytokine profiles. serum ifn-␥ levels were similar during acute-phase infection [171] , but levels were increased 3 weeks following influenza b infection compared with influenza a infection. furthermore, serum il-4 levels were significantly higher during the acute and recovery phases of influenza a infection. influenza strain differences were also observed in umbilical cord blood lymphocytes treated with uv-inactivated viruses. uv-inactivated influenza b virus induced less ifn-␥, il-4, or il-10 relative to uv-inactivated a/h1n1, a/h3n2, and a/h2n2 [172] . together, these data strongly suggest that innate cells and their associated responses have an important role in influenza disease manifestation, by providing protection and by contributing to pathological outcomes. the murine studies cited here clearly demonstrate that the local inflammatory environment can be dramatically different from the blood profile. the vast majority of experimental data in humans is restricted to blood leukocytes, with minimal emphasis placed on sites of infection. further, we lack good, functional analysis of these cells in humans, and most studies provide correlational, cross-sectional observations. as a result, there is a significant gap in our knowledge of innate immune cells in affected tissues. the history of respiratory virus challenge studies dates back to the 1918 influenza pandemic [173] , and early studies in humans infected with influenza identified the principal correlate of protection against influenza infection as strain-specific, virus-neutralizing antibodies directed against the ha. these studies (reviewed in ref. [80] ) suggested that the presence of serum antibody is associated with protection, as illness is less frequently observed in those people with pre-existing nab [74] . subsequent studies showed that protection is also correlated with the development of serum and mucosal anti-na or anti-ha igg and iga [37, 47, [75] [76] [77] [78] , but this protection can be incomplete, as individuals with detectable nab can still be infected [79, 174] . antibodies specific for the na have been shown to reduce disease severity by restricting virus release from infected cells and enhancing viral clearance (reviewed in ref. [175] ). in addition to ha and na, antibodies are produced to np, matrix m2 protein, and pb1-f2 [176] , although the significance of these responses remains unclear. of course, antigenic shift and antigenic drift can both thwart the efficacy of serological memory. the recent circulation of a(h1n1)pdm09 has provided a modern "experiment of nature", demonstrating the importance of pre-existing antibody from prior infection or vaccination [81] [82] [83] . even prior to 2009, it was well-recognized that heterosubtypic immunity has a large role in protection against antigenic variants of influenza [84, 85] , and vaccine studies support the idea that cross-protective, adaptive immune responses occur [86] . likewise, neutralizing polyclonal antibodies cross-reactive against h5 can also be produced in individuals vaccinated against seasonal influenza, as well as those naturally infected with a(h1n1)pdm09 [87, 88] . compared with seasonal influenza infections prior to 2009, ϳ31% of b cell epitopes are conserved in a(h1n1)pdm09, with 17% of those conserved within the surface viral proteins [177] . the ha of a(h1n1)pdm09 contains sequences homologous with that of a/h1n1 (1918) demonstrated by the a/h1n1 (1918) mab 2d1, which binds to the sa antigenic site of the ha [89] . furthermore, this particular antibody can neutralize ha activity in vitro, and mab-treated mice show reduced viral titers when challenged with a(h1n1)pdm09 [90] . most neutralizing antibodies bind to the exposed regions of the ha that surround the receptor-binding site and interfere with attachment to sialic acids on the cell surface. therefore, generation of crossprotective neutralizing antibodies is elusive, as the external viral proteins are most exposed to immune pressure, but universal immunization against conserved ha stalk and m2e domains is promising [71] [72] [73] . human pbmcs include at least three major subsets of dcs: cd141 ϩ mdcs, cd1c ϩ mdcs, and pdcs (reviewed in ref. [178] ). dcs process and present antigen to t cells and therefore have an important role in regulating the adaptive immune response. depending on the local cytokine milieu, mdcs can differentiate into macrophages or tissue-specific dcs (i.e., epithelial langerhans cells or interstitial dcs). as influenza primarily infects epithelial cells lining the respiratory tract, lung-resident dcs and macrophages are particularly important for efficient development of an adaptive immune response. once activated, mdcs function as apcs to activate t cells; on the other hand, pdcs are major producers of type i ifn upon tlr activation. in response to influenza infection in vitro, human dcs and macrophages undergo maturation, secreting type i ifn and inducing t cell proliferation and ifn-␥ production [179 -181] . cellular immune responses (cd8 ϩ ctls and cd4 ϩ th cells) are important for virus clearance in murine models of influenza infection, with cd4 ϩ and cd8 ϩ t cell-depleted mice displaying delayed viral clearance. the relevance of these observations remains somewhat controversial in humans. regard-less, as in mice, the human lung contains resident respiratory virus-specific cd8 ϩ memory t cells [182] , which can effectively kill virus-infected cells. human cd8 ϩ t cells recognize antigen in the context of surface hla molecules and generally recognize a broad range of influenza epitopes, including those from structural and nonstructural influenza proteins [121] . approximately 41% of cd4 ϩ and 69% of cd8 ϩ t cell epitopes are conserved in a(h1n1)pdm09 from the prior circulating seasonal h1n1 [177] . furthermore, cd8 ϩ intraepithelial t cells can mount fast and efficient recall responses upon viral infection [183] , and immunization studies in humans have shown that t cell responses peak within 1 week after symptom onset [174] . in terms of pandemic viruses, individuals infected with a(h1n1)pdm09 were shown to have rapid, virus-specific cd8 ϩ t cell recall responses early during infection, but this ctl response wanes over time [184] . likewise, vaccinated individuals, who were then naturally infected with a(h1n1)pdm09, had t cell responses increase by twofold early after infection [174] . memory cd4 ϩ or cd8 ϩ t cells isolated from pbmcs of healthy individuals are also cross-reactive against proteins of heterologous viruses, including those from a(h1n1)pdm09 and a/h5n1 [91] [92] [93] [94] , but immunocompromised patients do not have lasting antibody or cd4 ϩ /cd8 ϩ memory responses following natural a(h1n1)pdm09 infection [185] . furthermore, ctls, isolated from a(h1n1)pdm09-or a/h5n1-inexperienced individuals with a memory cd45ra med cd62 ligand lo ccr7 ϫ phenotype, will respond robustly to peptide pools and influenza-infected cells [34, 177, 186, 187] , suggesting that t cells may provide some level of protection against conserved epitopes [110] . although nonconserved regions of the virus may effectively escape recognition by t cells [188] , the majority of t cell responses is elicited against conserved, internal viral proteins, generating much interest in vaccine or therapeutic strategies that involve t cell-mediated immunity. a frequently neglected population of lymphocytes, ␥␦ t cells comprise 1-5% of t cells in the blood of humans, the majority of which are v␥9v␦2 t cells. these ␥␦ t cells are considered to be a major innate-like t cell subset (reviewed in ref. [189] ), and in vitro studies suggest that activated human v␥9v␦2 t cells may have a role in the antiviral response by killing influenza-infected, monocyte-derived macrophages and producing high levels of ifn-␥ [190, 191] . in particular, neonates are known to have compromised cellular immune responses and represent an important target population for influenza immunization. despite being such a critical target population, there are relatively few neonatal models of respiratory infections, and for ethical reasons, it is appropriately difficult to acquire data from human neonates. in the case of rsv infection, murine neonate models suggest that the age of primary viral infection can determine the immunopathology of a secondary viral infection [192] [193] [194] . during rsv infection, younger murine neonates (յ7 days) will develop an immune response characterized by increased eosinophil and t cell recruitment to the lungs, enhanced airway hyper-responsiveness, and higher il-13 levels upon reinfection 12 weeks later, whereas older mice (ն4 weeks) develop an immune response characterized by decreased t cell and eosinophil recruitment to the lungs upon reinfection [192, 193] . likewise, a neonatal mouse model of influenza a virus showed that early infection was associated with decreased cd8 ϩ t cell recruitment and function and enhanced airway inflammation [195] . these studies highlight an important concept, whereby infection too early results not in protective immunity but immunopathogenesis. consistent with an age-dependent defect in adaptive immunity, during rsv and influenza infection of infants, few cd4 ϩ or cd8 ϩ t cells or cd56 ϩ nk cells are found in the upper and lower airways [100, 103, 104] , in contrast to the observed infiltration of lymphocytes in adult a/h5n1 influenza [106] and sars cov infections [196, 197] . moreover, fatal cases of a(h1n1)pdm09 had increased numbers of cd8 ϩ t cells and granzyme b ϩ cells in their lungs [33] . this observation may represent a correlation with higher levels of viral antigen or may indicate a pathogenic role of exuberant cd8 ϩ t cell responses. on the other hand, the elderly have been shown to have reduced ctl activity compared with younger individuals, and this can have a negative impact on immunization efficacy [111] [112] [113] . however, prevaccination t cell responses do not correlate with postvaccination antibody titers [114] , and as the elderly also have decreased antibody responses following vaccination, t cell responses may provide a better correlate of vaccine efficacy in the elderly population [115] . interestingly, unstimulated memory t cells from older individuals are in a higher state of differentiation than those of younger individuals, and it was shown that fewer senescent, influenza-specific t cells, characterized as killer cell lectin-like receptor subfamily g member 1 (klrg1) hi cd57 hi , are associated with a more robust antibody response following tiv delivery [110] . the immunodominance hierarchy in hla-a*02 individuals is often reported as directed primarily against the conserved influenza a m1 58 -66 (gilgfvftl) [198, 199] . however, studies suggest that although this cd8 ϩ t cell response to m1 58 -66 is conserved in hla-a*02 individuals, it may not be strictly immunodominant, as greater responses were observed against internal np and polymerase pb1 subunit proteins in some individuals [122, 200] . in terms of hla presentation of influenza peptides, the hla-a*02:01-restricted m1 58 -66 peptide-specific ctl response is characterized by expression of v ␤ 17 tcr use [201] , and in hla-a*02:01 individuals, v ␤ 17 ϩ ctls were the dominant cells responding to in vitro influenza exposure. in contrast, cord blood from hla-a*02:01 infants, thus having no prior influenza exposure, was characterized by a m1 58 -66 -specific cd8 ϩ t cell response that was less dependent on the presence of the cd8 ϩ v ␤ 17 ϩ t cell population [109] . therefore, maturation of the ctl response upon subsequent influenza infections is likely related to effective clearance of viral infections and has important implications for therapeutic interventions. although children beyond infancy who are considered high-risk for influenza infection, i.e., children with underlying diseases, such as asthma, sickle cell disease, and those receiving solid organ transplantation, have sig-nificant increases in serum nab and t cell responses, children with systemic lupus erythematosus are unable to mount significant t cell responses following tiv immunization [108] . these findings suggest that the pathogenesis of respiratory viral infections in infants may be associated with the absence of immunological memory or a failure to develop an appropriate, well-regulated t cell response. undoubtedly, host genetics have a role in the outcome of many infectious diseases, including influenza. mouse models have provided some clues regarding the role of the host genetic component on the inflammatory response in the respiratory tract. briefly, these studies found that influenza-induced pathology is altered depending on the inbred mouse strain used for infection. the genetic background of the host also controls cumulative and maximal viral titer, underscoring the importance of limiting viral replication early during influenza infection [111, 202] . in humans, certain populations worldwide have been found to be more prone to severe disease during influenza infection. for instance, aboriginal peoples have more severe illness and increased mortality during the a/h1n1 pandemics of 1918 and 2009 compared with those of non-aboriginal descent [29, 116 -118] . it is important to note, however, that poor socioeconomic conditions and metabolic diseases, such as diabetes, are more common in indigenous populations [203] , and this may contribute to influenza-related disease. moreover, hla alleles are proposed to have some role during influenza infection, as differential ctl responses exist in individuals with varying hla alleles [204] . gene polymorphisms located in human chromosomes 1 and 17 were also determined to be associated with severe pneumonia during a(h1n1)pdm09 infection [120] . interestingly, two of these snps are mapped to genes fcgr2a and c1qbp. the protein encoded by fcgr2a is involved in phagocytosis and clearance of immune complexes and expressed on the surface of phagocytic cells. the protein encoded by c1qbp is a critical factor involved in complement activation. notably, presence of low-avidity antibodies and subsequent formation of immune complex-mediated complement activation in the lungs of patients with severe influenza infection are associated with severe disease following influenza infection [107] . recently, "systems biology" strategies have emerged to improve vaccine efficacy, measuring expression patterns to expose protective molecular signatures following immunization or natural infection [46, 205] . confirming in vitro mouse studies, which suggest that mbl can neutralize influenza [206, 207] , human polymorphisms identified within mbl2 are associated with poor antibody responses to influenza vaccination [119] . although mbl deficiency has been observed to influence the outcome in several respiratory diseases, including pneumococcus infection, tuberculosis, and sars cov (reviewed in ref. [208] ), it did not have an obvious role in a(h1n1)pdm09 infection of naïve individuals [209] . these studies highlight the involvement of host ge-netics in development of enhanced disease, although they likely represent a small fraction of the potential regulatory host genetic elements. studies of innate and adaptive immune responses to influenza virus infection have been restricted primarily to animal models of disease and inflammation. as our immunological tools become more sophisticated, more emphasis should be placed on human immunology as opposed to relying solely on animal infection models. moreover, the vast majority of experimental data in humans is restricted to blood leukocytes, with minimal emphasis placed on the innate immune response, particularly at sites of infection. as a result, there is limited scientific knowledge regarding the specific roles of immune cells in affected tissues. understanding the innate response in particular is critical to appreciate early 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diabetes prevalence among american indians and alaska natives and the overall population-united states preferential hla usage in the influenza virus-specific ctl response systems vaccinology: learning to compute the behavior of vaccine induced immunity complement-dependent neutralization of influenza virus by a serum mannose-binding lectin human mannan-binding lectin inhibits the infection of influenza a virus without complement mannose-binding lectin deficiency and respiratory tract infection no association between mannose-binding lectin deficiency and h1n1 2009 infection observed during the first season of this novel pandemic influenza virus the authors are funded in part by u.s. national institutes of health/national institute of allergy and infectious diseases contracts hhsn266200700005c (st. jude center of excellence for influenza research and surveillance) and hhsn272200800058c (systems influenza) and the american lebanese syrian associated charities (alsac). key: cord-324301-bzrh2fni authors: zambon, maria title: influenza, respiratory syncytial virus and sars date: 2005-05-01 journal: medicine doi: 10.1383/medc.33.5.130.64960 sha: doc_id: 324301 cord_uid: bzrh2fni abstract acute lower respiratory tract infections (lrtis) are a major worldwide health problem, particularly in childhood, and are ranked first among the conditions contributing to the global burden of disease. about 30–50% of acute lrtis are viral in origin; of these, influenza and respiratory syncytial virus (rsv) are associated with the greatest disease burden in humans. vaccination against circulating human influenza strains and the use of neuraminidase inhibitor drugs have improved the options for control of influenza, but as yet there are no successful vaccines or antiviral drugs for use against rsv infection. the recent emergence of the sars coronavirus in the human population in 2003, with an ensuing global epidemic affecting more than 8000 individuals with a case fatality of about 10%, underlines the fact that respiratory viral infections of humans may originate in animals, and that many different influenza a viruses also occur naturally in animal reservoirs, representing a constant threat of zoonotic infections of humans and ensuing global pandemics. avian influenza viruses have transmitted directly to humans from domestic poultry on several occasions in the last decade, and the current extensive burden of disease from avian influenza in south east asia provides a real possibility for the emergence of a novel influenza virus pathogenic in humans. acute lower respiratory tract infections (lrtis) are a major worldwide health problem, particularly in childhood. about 30-50% of acute lrtis are viral in origin; of these, influenza and respiratory syncytial virus (rsv) are associated with the greatest disease burden in humans. the emergence of the severe acute respiratory syndrome (sars) coronavirus in 2003, and the ensuing worldwide epidemic, highlights the fact that respiratory viral infections in humans may originate in animals. many different influenza a viruses occur naturally in animal reservoirs, and present a constant threat of zoonotic infections and global pandemics. influenza viruses are small (80-120 nm diameter), contain rna and are enveloped. there are three types -a, b, and c. type a is further classified according to the properties of the surface proteins haemagglutinin and neuraminidase. all a subtypes are found in aquatic birds, which are the natural reservoir ( figure 1) ; only a few subtypes circulate in humans and other mammals. type b and c influenza have only one subtype and are restricted to humans. seasonal illness, epidemics and pandemics -influenza viruses circulating in humans (a h1n1, h3n2, b and c) cause respiratory tract disease. influenza a is generally considered to be clinically more severe than influenza b; influenza c causes only a mild illness confined to the upper respiratory tract. circulation of influenza a and b viruses in humans ( figure 2 ) causes unpredictable seasonal epidemics of disease in temperate climates, with excess population morbidity and mortality, usually occurring between october and march in the northern hemisphere and lasting about 6-8 weeks. 1 in the uk, 5000-10,000 deaths are associated with influenza a and b epidemics every year, and more than 20,000 in severe years. widespread pandemics of severe disease occur less frequently, occurring on at least three occasions in the last century (1918, 1957, 1965) , and have been associated with high mortality. clinical features -influenza a and b illness in humans ranges from subclinical or mild upper respiratory tract symptoms to more severe illness including laryngotracheitis and pneumonia or, less commonly, death from respiratory system failure. the most common presenting symptoms are cough, high temperature, joint pain and general malaise ( figure 3 ). the rapid onset and short incubation period (about 48 hours) are characteristic, though incubation can last up to 4 days. individuals at greatest risk of complications are those with pre-existing cardiac and respiratory disease, the elderly, and those with impaired immune systems ( figure 4 ). the severity of the illness reflects pre-existing host immunity and the prevailing virus strain. virus variability -protective immunity to influenza is conferred by antibodies. the ability of influenza to cause re-infections is related to the genetic mutability of the virus. in every replication round, mutant viruses are generated, some of which have a growth advantage because they can partially evade host immune responses. variants capable of causing epidemics in susceptible populations emerge by a process termed 'antigenic drift'. new influenza a drift variants arise every 2-3 years; influenza b drift variants arise every 4-5 years. influenza as a zoonosis -the segmented nature of the influenza genome allows reassortment of segments when a single host is infected with more than one virus. this occurs regularly in aquatic birds, in which almost all combinations of influenza a virus segments can be detected, but is less common in mammals. it was previously thought that, for a novel subtype of influenza to arise in humans, reassortment of two virus subtypes had to occur in a mammal that could then transmit to humans. the pig was considered a suitable 'intermediate host', because both avian and human viruses, which differ slightly in their surface receptor requirements, can replicate in pigs. however, following transmission of h5n1 directly from birds to humans on 18 occasions figure 5 ), it is evident that cross-species barriers to transmission may be less stringent than was thought. nevertheless, the requirements for adaptation of avian viruses to mammals are poorly understood. cross-species transmission of novel subtypes into susceptible human populations (antigenic shift) are thought to be the source of pandemics of influenza. zoonotic infections involving several different subtypes of influenza have occurred in the last few years, indicating the pandemic potential of influenza viruses circulating in domestic poultry. control -the presence of a large, mobile animal reservoir of influenza a virus suggests that eradication of this agent will be impossible. control strategies focus on limiting the opportunities for cross-species transmission of novel subtypes; for example: • housing domestic poultry in shelters to avoid contact with overflying migratory birds • eliminating/reducing live bird markets • housing aquatic birds and domestic poultry separately • slaughtering domestic flocks infected with highly pathogenic influenza a viruses. these measures may achieve some success in preventing zoonotic transmission of influenza a to humans, but have little impact on its annual cycle. unprecedented levels of h5 circulating in domestic poultry in south east asia present a particularly high risk for emergence of a novel pandemic influenza a strain. immunization -antibodies against haemagglutinin (and, to a limited extent, neuraminidase) can prevent disease caused by the same strain of virus. this is the basis of vaccination for influenza. currently, most vaccines used worldwide are subunit vaccines. however, the high variability of influenza virus means that antibodies to one strain confer only limited protection against drift variants. thus, influenza vaccines are given annually before the influenza season to boost pre-existing immunity, and the composition of the vaccine is updated regularly. in developed countries, the benefit of immunization has led to expansion of age-related vaccination policies. in the uk in 2000, vaccination was introduced for all individuals over 65 years of age, irrespective of pre-existing illness. vaccination rates vary considerably between countries. there is increasing interest in vaccination of children. 2 they induce broader, long-lasting immunity. child vaccination may also help to prevent transmission in the community in general. antiviral drugs -despite the preventive efficacy of vaccination, the need for treatment of severe influenza remains. amantadine (or the related compound rimantadine) was, until recently, the only anti-influenza drug available. it selectively targets a viral protein (m2) and inhibits viral replication, but its use has been limited in the last 30 years, partly because of side-effects (dizziness, confusion) that particularly affect the elderly, and also because drug-resistant mutants arise frequently and can be readily transmitted. neuraminidase inhibitors are a more recently developed, novel class of anti-influenza compounds ( figure 6 ). 3 they act on viral neuraminidase, prevent release of virus particles from infected cells, and are likely to be most efficacious when given early in illness. since 2000, the uk national institute for clinical excellence has recommended that neuraminidase inhibitors may be used for treatment and prophylaxis, with certain restrictions. mutations conferring resistance to neuraminidase inhibitors do not arise very frequently in vivo, and viruses containing them generally lose reproductive fitness and are at a disadvantage in transmission. this suggests that widespread resistance to antiviral drugs is unlikely to emerge and is most likely in treated children, probably because the viral load is highest and therefore the capacity for viral replication in the presence of drugs is greatest. rsv (figure 7) is a negative-sense, non-segmented, enveloped rna virus of 100-300 nm diameter. it is best known as a cause of bronchiolitis in infants, but can cause respiratory tract infection in all age groups. upper respiratory tract infections (urtis) and lrtis range in severity from subclinical infection to pneumonia and death. more than 60% of children have been infected with rsv by their first birthday and more than 80% by 2 years of age; thereafter, individuals are infected approximately every 3 years. rsv infections in adults are probably under-recognized, and the severity of rsv infection in the elderly may be much underestimated as a cause of pneumonia. about 5% of elderly individuals are thought to become infected with rsv every year. transmission of rsv is primarily through large aerosol droplet or secretions, causing widespread nosocomial infection. outbreaks in adult and paediatric facilities are difficult to control. rsv infection in immunocompromised individuals is severe and lifethreatening; mortality may be 50-70% in adult bone marrow transplant recipients, in whom viral shedding may be prolonged. pathophysiology -rsv infects the respiratory epithelium, leading to increased goblet cell production of mucus. dying infected ciliated epithelial cells combine with mucus to form plugs that block the airways; the consequences are most severe in very small babies with narrow airways. this leads to the characteristic signs and symptoms of atelectasis and the clinical syndrome of bronchiolitis. in rsv bronchitis and pneumonia, the peribronchiolar and interstitial infiltrate is characteristically lymphocytic. clinical features -the severity of rsv infection is related to age. in young infants, the illness is seldom asymptomatic and lasts for 1-3 weeks. early signs of infection may include difficulty in feeding, nasal congestion, cough and otitis media compatible with urti. fever is often but not invariably present in rsv infection. abnormal breath sounds, tachypnoea and hypoxaemia suggest lower respiratory tract involvement. bronchiolitis and pneumonia are the two primary manifestations of progression to lrti; they may be difficult to distinguish and can occur simultaneously. the clinical features of bronchiolitis are wheezing and hyperaeration, and these are characteristic of infants with rsv infection. in the usa, rsv is estimated to cause 4500 deaths per year in children under 2 years of age. the risk of hospitalization in otherwise healthy under-2s is 0.5-2%, and 10-20% of children admitted to hospital require mechanical ventilation. these rates are higher in the first 6 months, and may be higher still in children with underlying acquired or congenital cardiopulmonary disease. older children and adults with rsv infection or re-infection usually have a milder or asymptomatic respiratory infection with a lower likelihood of lrti. adults with rsv-associated respiratory tract infections may experience prolonged symptoms. disease in the elderly may be particularly severe; up to 50% develop pneumonia. investigations -rsv infection is often diagnosed on the basis of the clinical features. the certainty of the diagnosis is increased when rsv is known to be circulating in a seasonal epidemic. chest radiography findings are nonspecific and commonly include hyperaeration and peribronchial thickening, with areas of consolidation and interstitial infiltrates in patients with rsv pneumonia. there is a range of respiratory findings in immunocompromised adults, including pleural effusions. laboratory diagnosis depends on detection of viral antigen in respiratory secretions by immunofluorescence, rapid antigen tests or culture of the virus. serological tests are of little help in diagnosis of rsv infection, because they rely on the use of paired acute and convalescent sera. complications -premature and very young infants are more likely to suffer acute apnoeic episodes and require assisted ventilation. bronchiolitis and pneumonia are the major complications of rsv disease in young children. children with congenital heart disease or chronic lung disease, and immunocompromised children and adults, are also at risk of severe disease. pneumonia is the major complication in adults and the elderly. estimates of the incidence range from 10% in nursing homes to 55% in a hospital in-patient population; estimated mortalities in the elderly are 3-5% in the former and 10-20% in the latter. rsv infection has very high mortality (50-70%) in severely immunocompromised individuals. management -supportive care is the mainstay of management of rsv disease in infancy. maintenance of oxygenation, hydration and nutrition is essential in hospitalized patients, and ventilatory support may be necessary in severe cases. a trial of bronchodilators may be beneficial. controlled trials of corticosteroids and vitamin a supplementation have not proved efficacy in infant rsv disease. immunization -there are two subtypes of rsv -a and b. the surface glycoproteins f and g are the major antigens of the virus to which neutralizing antibodies are directed. protective immunity to rsv is complex. antibodies generated during natural infection are not necessarily protective. a high proportion of primary rsv infections occur before 6 months of age, when maternal antibody levels are highest. overall, current (controversial) data suggest that neutralizing antibody to rsv is beneficial. neutralizing antibody titres in human sera correlate inversely with the likelihood of hospitalization as a result of rsv infection, and neutralizing antibody titre correlates with a reduced risk of re-infection. immunoglobulin infusions with high neutralizing titres of antibody to rsv (rsvig, figure 8 ) have been used to treat rsv illness in normal-risk and high-risk infants. in normal infants, there is little evidence to justify rsvig for treatment of rsv infection. however, prophylaxis may be useful in high-risk infants (e.g. those with bronchopulmonary dysplasia). assessment of risk is important, because intravenous rsvig is contraindicated in congenital cyanotic heart disease. recombinant humanized rsv monoclonal antibodies are now available for intramuscular treatment and prophylaxis of rsv. early clinical data suggest that this preparation of passive antibodies may have wider application, with fewer limitations than rsvig; however, it is extremely expensive. few antiviral drugs are available for rsv. ribavirin is a guanosine analogue that has been used widely, though its precise mode of action is uncertain and many clinical studies show conflicting results. several studies have raised further doubts about the clinical effectiveness of ribavirin in infants and children at risk of severe rsv disease, and in ventilated children. in most centres, its use is now restricted to the immunocompromised or severely ill. several rsv vaccines (live attenuated, subunit and recombinant) are undergoing clinical trials. development of safe vaccines has been impeded by poor understanding of the factors governing immune protection in different age groups, 4 and early vaccination attempts in which more severe disease was seen in vaccinees. it is likely that there will be significant progress in future and that the types of vaccines suitable for different age groups may differ. transmission -sars cov spread worldwide within weeks in early 2003. the major route of transmission was respiratory, primarily through droplet, secretions and aerosol formation. about 60% of cases resulted from infection in health-care settings, emphasizing the importance of close contact and exposure to bodily secretions. (the syndrome was noticed through unusual clusters of severe respiratory illness in hospital settings.) peak virus shedding is 7-12 days after the onset of illness (figure 9 ). the virus is found in various body fluids. infectious virus has not been recovered later than 21 days after illness onset. neutralizing antibody is detected from about 10 days post-onset. risk factors for sars cov infection are: • close contact with civet cats/racoon dogs • eating/preparing civet meat • laboratory work with sars cov • contact with a known case of sars. clinical features -presentation is with fever and respiratory illness with cough and shortness of breath, progressing to acute respiratory distress syndrome and death in 10% of cases. fatalities increase significantly over the age of 40 years; mortality is up to 40% in the over-50s. onset of illness is 2-10 days post-infection, with a mean of about 5-6 days. about 60% of patients suffer later gastrointestinal symptoms of diarrhoea and vomiting. management and control -specific control measures have not yet been developed, though work is in progress on antiviral drugs and suitable vaccines. during the 2003 epidemic, various nonspecific therapeutic measures were used with variable success: • corticosteroids • antimicrobials to prevent secondary bacterial infection • positive-pressure ventilatory support • anti-inflammatory drugs • infusion of antisera from convalescent patients. the epidemic was controlled mainly through public health measures such as contact-tracing and quarantining. this policy was effective because there is little evidence of transmission before symptom onset, so infected individuals are easily identified.  • consider for prophylaxis in infants < 2 years receiving oxygen therapy for bronchopulmonary dysplasia • infants born < 32 weeks with bronchopulmonary dysplasia are likely to benefit from 6-12 months' prophylaxis • infants born > 32 weeks with bronchopulmonary dysplasia may not benefit • should not be used in cyanotic congenital heart disease • not evaluated in paediatric or adult immunocompromised patients • main emphasis in nosocomial outbreaks should be infection control; efficacy is improved in such settings • initiate treatment before onset of respiratory syncytial virus season • defer live virus vaccines (e.g. mmr) until last dose the contribution of influenza to acute respiratory infections, hospital admissions and deaths in winter the japanese experience with vaccinating school children against influenza neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir age related differences in humoral immune response to respiratory syncytial virus infection in adults confirmation of a novel corona virus as the primary cause of severe acute respiratory syndrome key: cord-332516-eaqpiq1o authors: joseph, carol; togawa, yu; shindo, nahoko title: bacterial and viral infections associated with influenza date: 2013-08-27 journal: influenza and other respiratory viruses doi: 10.1111/irv.12089 sha: doc_id: 332516 cord_uid: eaqpiq1o influenza‐associated bacterial and viral infections are responsible for high levels of morbidity and death during pandemic and seasonal influenza episodes. a review was undertaken to assess and evaluate the incidence, epidemiology, aetiology, clinical importance and impact of bacterial and viral co‐infection and secondary infection associated with influenza. a review was carried out of published articles covering bacterial and viral infections associated with pandemic and seasonal influenza between 1918 and 2009 (and published through december 2011) to include both pulmonary and extra‐pulmonary infections. while pneumococcal infection remains the predominant cause of bacterial pneumonia, the review highlights the importance of other co‐ and secondary bacterial and viral infections associated with influenza, and the emergence of newly identified dual infections associated with the 2009 h1n1 pandemic strain. severe influenza‐associated pneumonia is often bacterial and will necessitate antibiotic treatment. in addition to the well‐known bacterial causes, less common bacteria such as legionella pneumophila may also be associated with influenza when new influenza strains emerge. this review should provide clinicians with an overview of the range of bacterial and viral co‐ or secondary infections that could present with influenza illness. bacterial secondary infections or co-infections associated with cases of influenza are a leading cause of severe morbidity and mortality, especially among high-risk groups such as the elderly and young children. vaccines and antiviral and antibiotic therapies are now readily available to clinicians for control and prevention of primary and secondary bacterial infections. thus, information on the overall range, incidence and severity of influenza co-infections and secondary infections associated with different influenza strains, aetiological agents, different age groups and their underlying risk conditions is very important contextually for clinicians and public health specialists involved in implementing policy and treatment regimes for this disease spectrum. bacterial infection may be concurrent with influenza viral infection, and the resulting co-infection can lead to an enhanced pneumonic illness or may occur shortly after influenza virus has been largely cleared from the lungs, when the host appears to be more susceptible to bacterial infection. 1, 2 morbidity and mortality are recognised to be greater in cases of influenza-associated bacterial infection compared with bacterial pneumonia without influenza infection 3 with all age groups affected by this synergistic process. the annual increase in influenza activity during winter months is usually accompanied by an increase in cases of community-acquired pneumonia (cap). the most com-mon causes of cap are streptococcus pneumoniae (s pneumoniae), staphylococcus aureus (s aureus) and haemophilus influenzae (h influenzae). s pneumoniae is the most frequently isolated pathogen associated with influenza 4 , although deaths, especially in children are also associated with s aureus infection, as highlighted by the recent emergence of community-acquired methicillin-resistant staphylococcus aureus (mrsa). 5 the objective of this review is to assess and evaluate the incidence, epidemiology, aetiology, clinical importance and impact of co-infection and secondary infection associated with influenza. as there are few review articles on this topic, it also aims to integrate some newly published reports. the main focus of this review is pulmonary infection while some less common extra-pulmonary complications as shown by case reports are also included. information in this review should supplement the information available to clinicians on antimicrobial treatment therapies, including antibiotic sensitivity information, local guidelines and local antimicrobial susceptibility data as well as local availability of medicines. bacterial outcomes have been extensively studied in both influenza pandemic and epidemic periods and latterly within the context of available viral and bacterial vaccines that protect against primary and secondary infection. in addition to prevention strategies, treatment of influenza complications has also become available through antiviral and antibiotic therapies. the goal of combined therapies for prevention and treatment must surely be a reduction in the proportion of bacterial infections associated with influenza. however, the wider use of antibiotics for treatment of bacterial influenza needs to be considered alongside the corresponding requirement for appropriate use of antibiotics, in order to reduce the increasing burden of antibiotic resistance of bacterial strains implicated in co-infection with influenza. the mechanisms by which co-infection and secondary infection take place are complex. reports from past influenza pandemics show an extremely high frequency of lung colonisation by bacterial species that are commonly found in the nasopharynx. most evidence suggests that virusinduced changes in the respiratory tract prime the upper airway and lung for subsequent bacterial infection. secondary bacterial infections are facilitated by virus-induced cytopathology and resulting immunological impairment, which may be caused in part by the overproduction of inflammatory cytokines. 6 modification of the immune response either by diminishing the ability of the host to clear bacteria or by amplification of the inflammatory cascade is likely to contribute to the severity of the resulting infection. 7 animal studies using murine models have shown that influenza predisposes to bacterial pneumonia. 6, [8] [9] [10] lag times of 7-21 days have been calculated in studies for onset of bacterial infection following seasonal influenza 11 although much shorter times from onset to death have been recorded in pandemic periods. [12] [13] [14] influenza a is the dominant strain associated with co/ secondary bacterial infection with evidence that specific n2 seasonal subtypes cause more severe infection than other subtypes. 15 influenza b, although generally regarded as having less impact on morbidity and mortality in healthy persons can also cause severe secondary bacterial infection during seasonal influenza episodes, especially within younger age groups. 5, 16, 17 methods we searched the national library of medicine through pubmed using the search terms: ('influenza' and 'secondary infection'), ('influenza' and 'pneumonia') and ('influenza' and 'co-infection'). we also searched the references of the identified articles for additional articles. we included studies on both influenza type a and b, and also seasonal and pandemic influenza. the search was limited to studies of disease in humans that were published in english from 1918 to the end of 2011. we only selected studies in which pathogens were identified. we then reviewed abstracts and titles and selected studies that were relevant to the topic of interest. bacterial co-infectiona bacterial pneumonia occurring simultaneously with onset of influenza virus illness secondary bacterial infectiona bacterial pneumonia occurring after influenza illness onset or clearance of influenza virus viral co-infection -influenza virus and one or more other respiratory viruses detected simultaneously by microbiological examination of respiratory samples. the three influenza pandemics of the 20th century (1918 h1n1, 1957 h2n2 and 1968 h3n2) are all associated with secondary bacterial pneumonia. 18 this pandemic has been extensively researched, mainly due to its global impact and estimated 40-50 million deaths in an era of unknown virological cause and absence of antibiotic therapy, in order to understand its aetiological and epidemiological features. british and french army camps in 1916/ 17 were the initial setting for major outbreaks of purulent bronchitis associated with haemophilus influenzae, pneumococcus, streptococcus and staphylococcus. over a period of 2 months in 1918 at 37 large american army camps, secondary bacterial infection occurred in approximately 17% of those diagnosed with influenza of which approximately 35% were fatal. 4 oxford describes these army camp outbreaks as progenitors of the ensuing h1n1 pandemic of 1918. 19 overall, it has been estimated that in the us military, the mean influenza attack rate during the course of the 1918/ 1919 pandemic was 23%, the mean percentage of influenza cases that developed pneumonias was 16% and the mean percentage of pneumonia cases that were fatal was 34%. 4 recent re-analyses of post-mortem lung cultures from 1918 showed evidence of bacterial infection in >90% of the specimens. 18, 20 experts now support the sequential infection hypothesis and believe that bacteria were secondary invaders to pulmonary tissues weakened by the influenza virus. they suggest that the scale and range of bacterial invaders was random, and in the case of large group outbreaks, depended on the occurrence of particular bacteria in the respiratory tract of persons at the time of infection and on their occurrence in contacts. the fatal outcome of influenza pneumonia was therefore determined partly by virally depressed local and general pulmonary resistance and partly by the virulence and nature of the invading bacteria. 12 brundage explains the high transmission rates in military camps and other crowded settings as due to 'cloud adults'affected persons who increased the aerosolisation of colonising strains of bacteria to other susceptible persons. military personnel were deemed to be highly susceptible because of their closed community style living and their physically weakened state. in american civilian populations, attack rates and deaths were similar among younger adults and overall were approximately 28% for influenza with 30% of associated pneumonias being fatal. 21 studies also show that, in all age groups, deaths were strongly correlated with pneumonia cases than with influenza clinical cases alone. children had the highest rates of clinical influenza infection, whereas young adults had the highest influenza pneumonia rates and associated fatality rates. 20, 22, 23 1957 h2n2 pandemic the asian influenza pandemic was similarly characterised by waves of influenza followed by an increase in hospitalisations and deaths from pneumonia. one us study showed these were associated with s pneumoniae, h influenzae and s aureus 24 , while a dutch study of 158 asian influenza deaths documented that s aureus and pneumococci were recovered from 59% and 15% of lung cultures, respectively. 25 a british study of 140 hospitalised cases of pneumonia over a two-month period in 1957, a high proportion of whom also had evidence of confirmed influenza a infection, showed that s aureus was isolated from 27% of the cases and pneumococci and h influenzae from 15% and 4%, respectively. 26 mortality was 47% in the staphylococcal group compared with 16% in the nonstaphylococcal group: eight of the 18 staphylococcal deaths were in persons with no previous disease while seven were in cases with chronic chest disease. in the 1968 h3n2 hong kong pandemic, a three-fold increase in the incidence of staphylococcal pneumonia was found in one hospital study compared with the number of pneumonic cases in the previous year. of 128 patients with pneumonia during the pandemic influenza period, 26% were proven staphylococcal pneumonia cases and a high correlation between pneumonia and influenza infection was documented. 27 in england and wales, the national experience of the hong kong influenza in 1968/69 reported that mortality was substantially lower than in previous influenza winters. 28 however, excess respiratory deaths were recorded in the second wave of the pandemic in 1969/70 and increased by approximately 55% and circulatory system deaths by 4%. deaths in the elderly increased by 10%, in those aged 40-60 years by 8% and in younger adults by 4%. 29 in the united states, significant excess pneumonia-influenza mortality occurred in all nine geographical areas of the country in the first wave in 1968/69 and followed influenza activity by several weeks. 30 the first pandemic of the 21st century in 2009 is still being researched but so far has shown a similar pattern to previous pandemics with a high proportion of cases and deaths occurring in younger age groups compared with nonpandemic influenza seasons. a study of the first 47 deaths in new york city showed 13 (28%) had evidence of invasive bacterial co-infection. s pneumoniae was most commonly identified [8 patients (17%)], followed by s pyogenes [3 patients (6%)]. one paediatric case had post-mortem evidence of both bacteria. 13 a multi-centre review of 77 deaths in another us study between may and august 2009 found evidence of concurrent bacterial infection in specimens from 22 (29%) of the 77 patients, including 10 caused by s pneumoniae. 31 an analysis of 631 patients admitted to hospital over the five-month pandemic period in 2009 in the uk with confirmed pandemic influenza infection reported that 102 cases had radiological evidence of pneumonia and that mortality in cases with radiographic pneumonia was significantly higher than in cases without (p = 0á0008). four cases of pneumonia (4%) had positive bacteriological findings, three of whom diedtwo children with methicillin-resistant staphylococcus aureus (mrsa) and one adult with s pneumoniae in sputum. one adult had s aureus bacteraemia and survived. 32 in a study of 68 autopsy reports from a total of 457 pandemic influenza deaths in the uk, 28 (41%) reported that bacterial secondary infection was the significant complication; pneumococcus was the most common agent identified (25%). 33 in argentina, nasopharyngeal swab samples from 199 cases of confirmed h1n1 pandemic infection were tested for 33 additional microbial agents using masstag pcr methods. at least one additional agent of potential pathogenic importance was detected in 152 samples, including s pneumoniae (41%); h influenzae (68á4%); s aureus (23%); and methicillinresistant s aureus (mrsa, 4%). other viruses such as rsv, and influenza b were found in 20 samples and other bacterial pathogens in five. the presence of s pneumoniae was strongly correlated with severe disease and was present in 56á4% of severe cases versus 25% of mild cases (p = 0á0004). in subjects 6-55 years of age, the adjusted odds ratio (or) of severe disease in the presence of s pneumoniae was highly significant (p = 0á0001). this study demonstrated that the presence of s pneumoniae in nasopharyngeal swab samples could predict severe disease outcome, the risk being more acute in persons aged between 6 and 55 years. in this lowrisk age group, severity of disease could be predicted with 90á97% accuracy via a multivariable logistic regression model. 34 in the united states, those aged 5-19 years influenza and co-secondary infections ª 2013 blackwell publishing ltd experienced overall the largest relative increase in pneumococcal hospitalisations during the 2009 pandemic influenza period compared with seasonal baseline estimates for this age group and mirrored both temporal and geographical influenza activity across the country. 35 no national relative increase occurred in persons aged <5 years or aged 65 years or more, suggesting that the pandemic influenza virus was the likely cause of the increase in the younger age group. a prospective, observational, multicenter study conducted in 148 spanish intensive care units (icu) and with 645 patients, all of whom had confirmed h1n1 pandemic influenza infection showed that co-infection occurred in 113 (17á5%) of patients. s pneumoniae was identified as the most prevalent bacteria (54á8%). co-infection was associated with increased icu mortality (26á2% versus 15á5%), but cox regression analysis adjusted by potential confounders did not confirm a significant association between co-infection and icu mortality. 36 a study of 100 fatal cases of h1n1 influenza in the united states showed 26% overall were due to bacterial co-infection, mainly caused by s. pneumoniae. 37 similarly, a study of paediatric h1n1-associated mortality in the united states demonstrated that 28% of fatal cases had evidence of co-infection 38 while a study in england of 70 h1n1 paediatric deaths confirmed bacterial co-infection in 20% of the cases. 14 a french study measured levels of procalcitonin (pct)a recognised marker of bacterial infection, in patients with h1n1 influenza pneumonia admitted to hospital and was able to conclude that levels of 0á8 lg/l or more discriminated well between isolated viral and mixed bacterial and viral pneumonia. 39 this information together with clinical judgement may help to identify patients for whom antibiotic therapy may be inappropriate. the overall conclusion to date from epidemiological and clinical studies of the 2009 h1n1 pandemic is that worldwide incidence was low and infection was mostly mild. the fact that much of the 2009 pandemic occurred outside the regular season for pneumococcal disease in temperate regions may help to explain the lack of a marked increase in risk of pneumococcal infection at this time. 40 death rates although low, were more prevalent in younger age groups than the elderly and excess deaths were recorded in children by one international european study 41 and in england where the childhood mortality rate was six per million population compared with an estimate of two per million population for seasonal influenza among children aged <14 years. 14 as in the pandemics of the 20th century, s pneumoniae, h influenzae and s aureus were the main bacterial infections associated with severe infection or death in this pandemic. measures to prevent and treat their adverse impact on pandemic influenza cases in the future should now be incorporated into pandemic plans. 42 the literature on co-infections with other viruses during pandemic periods is sparse in comparison with that available for influenza-associated bacterial infections. there are no reports documenting solely viral co-infections during the 1957 and 1968 pandemics, possibly because these infections were not sufficiently severe to merit hospitalisation and/or enhanced microbiological investigation. the 2009 pandemic first appeared in some countries during their normal seasonal activity. as a result, national virological surveillance schemes were able to demonstrate the emergence of the new h1n1 strain against a background and subsequent decline of circulating seasonal h1n1 and h3n2 strains. in new zealand, 13 cases of pandemic h1n1 cases co-infected with seasonal h1n1 were detected, all with mild disease. 43 in argentina, 20 of 199 persons investigated with pandemic disease were co-infected with another respiratory virus including rsv (a or b), rhinovirus and coronavirus. seven of these cases were classed as having severe disease (hospitalisation or death with no other risk factors related to underlying disease) and 13, mild disease (ambulatory cases). 34 a us study of 173 cases of pandemic h1n1 found co-infection with other viruses in 20 cases (11á6%), rhinovirus being the most common agent. 44 in england and wales, the health protection agency's national virological surveillance scheme for community cases of influenza 45 detected 14 (3%) specimens where cases had evidence of pandemic h1n1 infection together with rsv, human metapneumovirus (hmpv), rhinovirus or parainfluenza virus (personal communication j field). seasonal influenza activity provides more opportunities for monitoring the changing epidemiology and microbiological features of influenza-related co/secondary infection but essentially mirrors that of findings in recent pandemics. maxwell first noted that bacterial pneumonia could occur during interepidemic periods when sporadic cases of influenza were investigated. 46 mccullers shows that from 1968 to 1999 excess deaths directly attributed to pneumonia and influenza (p&i deaths) from selected us cities data were more commonly associated with influenza a(h3n2) rather than h1n1 or influenza b infections. 7 meningococcal infections were observed to increase in the presence of both influenza a 47,48 and influenza b 49 , but no causal relationship was identified in a later study. 50 in sweden, pneumococcal infections were calculated to increase by 12-20% per influenza season over a ten-year study period with a lag time of 1-3 weeks for pneumococcal disease following peaks in influenza incidence. 11 in canada, a recent observational study showed that the seasonality and time lag of pneumococcal disease was only partially related to influenza seasonality. other factors such as reduced temperatures and daylight hours were important for the regular appearance of pneumococcal disease each winter. however, the study did find that influenza increased the risk of pneumococcal disease through enhancing pneumococcal invasion in colonised individuals, but had minimal impact on the transmission dynamics of pneumococcal infection. 40 transmission studies using animal models show increased incidence and severity of bacterial pneumonia after influenza infection is pneumococcal strain dependent. different strains may increase the duration of pneumococcal carriage and enhance the bacterial pneumonia. 10 in another study using infant mice, influenza virus was shown to be essential for pneumococcal transmission in a co-housed group although other indirect effects by which the virus altered the immune response of the mice were also considered to be important reasons in the dynamics and synergism between influenza and pneumococcal infection. 9 the introduction of a sevenvalent pneumococcal conjugate vaccine (pcv7) for infants has been shown in the united states to lead to a reduction in pneumococcal infections in vaccinated children and in adults through herd immunity effects. 51 a significant fall in influenza-associated pneumonia hospitalisations was observed among vaccinated children and unvaccinated adults; the vaccine acted to prevent the secondary pneumococcal pneumonia that followed influenza infection. 51 a nine-valent pneumococcal conjugate vaccine (pnccv) in south africa was shown to prevent 31% of pneumonias associated with any of seven respiratory viruses in hospitalised children. the study concluded that a significant proportion of viral pneumonia is due to bacterial co-infection and is preventable by a bacterial vaccine. 52 a study of influenza-related paediatric deaths in the united states over the 2003/04 influenza season found 24 of 102 deaths to have a bacterial cause, mainly s aureus. 53 other us studies of that season also noted a rise in reports of community-acquired s aureus infections in children and young adults, a significant proportion being mrsa infections. 54 similar findings were obtained for the 2006/07 influenza season in the united states 55 and by kallen who reported 51 cases of influenza-related s aureus, 37 of which were mrsa infections in young adults. where outcome of illness was known, deaths occurred in 24 of 47 cases. 56 finelli notes that the proportion of influenza-related s aureus paediatric deaths increased fivefold between 2004 and 2007. 5 although less common than influenza a-associated mortality, deaths do result from influenza b infection, and awareness is growing of the role influenza b co-infection may play in severity of influenza-related illness. a review of influenza surveillance data in the united states from 2004 to 2007 revealed that anywhere from 23 to 38% of the annually reported paediatric deaths attributable to influenza were from influenza b, and many of the fatal cases had evidence of bacterial co-infection. 5 england during the seasonal outbreak of influenza in 2010/ 2011 included four cases of influenza b infection, of which three were fatal. 17 case reports of three healthy women with no known risk factors and severe co-infections of s. pyogenes in two and s. pneumonia in the third that required intensive resuscitation measures recently in switzerland 16 , further underscore the potential impact bacterial co-infection with influenza b can have on morbidity and mortality. multi-viral co-infections have mainly been identified in paediatric studies. between two and six viral co-infections were reported per child in china, 57 a mix of influenza a h1 and h3 with influenza b in japanese children 58 and coinfection with influenza and human metapneumovirus (hmpv) in two successive winters 2002-2004, also in japan. 59 a one-year study in peru found 5á5% of virological surveillance samples to be positive for influenza plus additional respiratory viruses 60 while a similar finding of 3% was obtained from the community-based virological surveillance data in england and wales for the 2010/11 winter season (personal communication j field). none of these reports suggested that the cases had severe disease. the 2009 pandemic is the first pandemic where virological and microbiological tests for the disease have been conducted intensively at a global level. as a consequence, a wide range of less common pathogen pairings were encountered. in south africa, co-infection with hiv or active tuberculosis was a common finding among the investigated early fatal cases, signifying that these conditions could be associated with increased mortality risk. 61 from a tb endemic area, it has been reported that an immunocompromised cancer patient was co-infected with both tb and pandemic h1n1a rare finding. 62 in mexico, a study of 126 hiv patients with respiratory symptoms found that in the 30 patients co-infected with pandemic h1n1 virus, illness opportunistic infections were more severe and involved longer hospital stays (p = 0á0013), higher hospitalisation rates (p < 0á0001) and increased deaths (p = 0á026). deaths were also associated with delayed administration of oseltamivir (p = 0á0022). 63 in the united states, hopkins et al. reported six cases of bacterial tracheitis (bt) that were isolated in conjunction with influenza a (h1n1). no previous h1n1 cases have presented as bt in the literature to date. 64 six cases of bacterial co-infection due to legionella pneumophila were reported from italy; the authors commenting on the fact that bacterial co-infections associated with the influenza influenza and co-secondary infections ª 2013 blackwell publishing ltd a h1n1 pandemic have not been well described yet because of lack of data. 65 the first case of panton-valentine leukocidin (pvl) necrotising pneumonia due to influenza a (h1n1) and community-acquired methicillin-resistant staphylococcus aureus was reported from spain, 66 the authors recommend that other clinicians become aware of this coinfection. similarly, co-infection with dengue virus and pandemic influenza h1n1 is likely to be a feature in tropical countries where seasonal patterns of these two viruses were contemporaneous, as documented in a case report from puerto rico. 67 during seasonal influenza activity in 2006, influenza a h3n2 infection was associated with a fatal paediatric case of campylobacter jejuni infection in malaysia. 68 co-infection with campylobacter spp. has not been previously described together with influenza virus. this review has documented the adverse impact of co/ secondary bacterial infection with influenza infection, and the higher rates of severe morbidity or mortality that subsequently occur in all age groups. streptococcus pneumoniae continues to be the dominant pathogen involved in this synergistic process 7 followed mainly by staphylococcus aureus 5 and haemophilus influenzae. 12, 18 legionella pneumophila a less common bacterium was also found to be associated with influenza infection in the 2009 h1n1 pandemic. 65 pneumonia remains the single commonest cause of death in children <5 years. 69 clinicians and public health officials now have several means by which influenza-associated pneumonias can be prevented or ameliorated. control and prevention, through viral and bacterial vaccines, and prophylaxis and treatment, through the application of antiviral and antibiotic therapies all contribute to reducing the global burden of these infections. all of these measures however have limitations which impede their effectiveness. antibiotic resistance has been recognised as a growing concern for many years and during the 2009 pandemic, an increasing number of oseltamivir-resistant influenza strains were detected, particularly among immunocompromised patients with influenza infection. 70, 71 other threats to combating influenza epidemics or pandemics in the future might include timely production and administration of effective vaccines and logistical issues associated with stockpiling and distribution of antiviral and antibiotic drugs. 42, 72 most vaccine effectiveness studies select high-risk groups within which to show enhanced protective effects and typically present data on levels of protection against hospital admissions or mortality associated with influenza rather than prevention of secondary infections as a clinical end point. 73 pneumococcal vaccine effectiveness studies also focus on prevention of invasive pneumococcal disease and may or may not include information on influenza vaccine status as a confounding variable. 74 some studies have focused on the protective effect of dual influenza and pneumococcal vaccinations in the elderly. in sweden, influenza and pneumococcal polysaccharide vaccines together reduced hospital admissions for influenza, pneumonia and invasive pneumococcal disease by 32, 22 and 54%, respectively. overall mortality was also reduced by 27%. 75 a study of prior influenza vaccination in relation to its effect on severity and mortality in patients with cap during seasonal influenza periods showed that prevention of the predisposing viral illness reduced the risk for more severe secondary pneumonia. however, outside the influenza season, no significant influence of influenza vaccination status on cap severity was found. 76 most of the subjects in this study were elderly and therefore in the risk group for influenza vaccination. increasing the uptake of influenza vaccination in younger age groups could contribute to the overall prevention of influenza-attributable pneumococcal disease either directly or through protection from influenza via herd effects on other individuals. 40 the 2009 pandemic provided the first opportunity for significant international use of antivirals to prevent the spread of influenza and to treat infected individuals. given prophylactically, antivirals aid prevention of infection either in the absence of vaccine protection or within high-risk groups and may also prevent transmission within closed communities or households. when used for treatment, observational studies have confirmed that oseltamivir given within 48 hours of symptom onset (the recommended time for maximum effectiveness) improves survival in patients with severe influenza and may reduce secondary bacterial infection. 77 of 70 paediatric deaths related to the 2009 pandemic and investigated in one study, 45 (64%) had received antiviral therapy but only seven (10%) within 48 hours of onset. 14 21% of the deaths in this cohort occurred in healthy children. the authors recommend that vaccination of children should be extended to include nonrisk groups and that antiviral treatment should be given as early as possible after symptom onset. 14 antibiotic treatment should be guided by information on the likely bacterial pathogens associated with the influenza virus circulating in the community. recommendations should also defer to local cap management practice because inappropriate use of antibiotics increases antibiotic resistance. if invasive bacterial infection is suspected, early antiviral treatment and appropriate use of antibiotics should be administered. aggressive use of antimicrobial therapy early in the course of infection may reduce severe morbidity and mortality of influenza-associated bacterial infection. 6 the articles in this review suggest that influenza-related bacterial infections overall may account for up to 30% of cap cases, mainly in the elderly. 42 in the developing world, this percentage is much higher, but children are the main sufferers, and pneumonic infections are the leading cause of death in children aged <5 years. many of these deaths are preventable through immunisation, treatment and access to health care. the recent initiative of the global alliance on vaccines and immunisation, which supports global coverage of conjugate hib and pneumococcal vaccines in childhood programmes, estimates that the incidence of severe pneumonia and associated mortality in children in developing countries may be reduced by 50% through this programme. 69 however, other inequities are likely to persist between developed and developing countries with reference to access to adult respiratory vaccines, the ability to stockpile antivirals and antibiotics as part of pandemic planning and remain a major obstacle to global-improved public health goals. antibiotic usage to combat bacterial infection is recognised as adversely contributing to changes in sensitivity and resistance of these drugs with evidence that communityacquired mrsa infections are leading to high levels of morbidity and mortality in individuals with influenza, especially children. 5 if influenza vaccine coverage was extended to all children and low-risk adults aged <65 years, protection against influenza infection would be enjoyed by a larger proportion of the population, overall attack rates should fall, there should be an associated reduction in the incidence of secondary bacterial infections, a subsequent fall in the number of antibiotic prescriptions for these infections and therefore a slowing down in the rise of antibiotic resistance. obviously, this simplistic approach overlooks the vast organisation of economic and human resources needed to achieve these outcomes, but nevertheless, they remain a public health goal. interventions through vaccination have been shown to be cost saving and of cost benefit in influenza epidemic and pandemic settings, 78 but again, depend on staying one step ahead of the viruses and bacteria they seek to eradicate. the co-infections with more than one strain or subtype of influenza virus reported in this review have not yet provided any evidence of re-assortment threats or the emergence of new influenza strains. in 2001/02, a re-assortment between h1n1 and h3n2 produced a small number of cases of h1n2 infection in some countries, but circulation of the new strain was not sustained the following winter and did not confer more severe levels of illness compared with other subtypes. however, drifted strains occur on a regular basis and give rise to high levels of primary and secondary infection, particularly when there is a mismatch to strains contained within the seasonal vaccine. in conclusion, ample evidence exists to show that severe bacterial infection can be a consequence of influenza infection, both in pandemic and seasonal episodes. planning for future pandemics must therefore give equal emphasis to prevention and treatment of both these conditions, partic-ularly in high-risk groups such as the very young and the elderly. influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung respiratory viral infection predisposing for bacterial disease: a concise review disease severity in patients with simultaneous influenza and bacterial pneumonia interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness influenza-associated 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epidemic of influenza a bacterial pneumonia during the hong kong influenza epidemic of 1968-1969 national experience with hong kong influenza in the united kingdom excess deaths attributable to influenza in england and wales: age at death and certified cause national influenza experience in the usa bacterial coinfections in lung tissue specimens from fatal cases of 2009 pandemic influenza a (h1n1) -united states risk factors for hospitalisation and poor outcome with pandemic a/h1n1 influenza: united kingdom first wave predictive clinicopathological features derived from systematic autopsy examination of patients who died with a/h1n1 influenza infection in the uk 2009-10 pandemic streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic influenza impact of the 2009 influenza pandemic on pneumococcal pneumonia hospitalizations in the united states h1n1 semicyuc working group. community-acquired respiratory coinfection in critically ill patients with pandemic 2009 influenza a(h1n1) virus pandemic influenza a (h1h1) pathology and pathogenesis of 100 fatal cases in the united states deaths among children-united states can procalcitonin help identify associated bacterial infection in patients with severe influenza pneumonia? a multicentre study invasive bacterial diseases network, fisman d. evaluation of coseasonality of influenza and invasive pneumococcal disease: results from prospective surveillance higher all-cause mortality in children during autumn 2009 compared with the three previous years: pooled results from eight european countries bacterial pneumonia and pandemic influenza planning pandemic (h1n1) 2009 and seasonal influenza a (h1n1) co-infection rate and influence of respiratory virus coinfection on pandemic (h1n1) influenza disease. infectious diseases society of america 48th annual meeting, vancouver. oral abstract session hpa -health protection agency homepage -protecting people, preventing harm, preparing for threats the relation of influenza virus and bacteria in the etiology of pneumonia influenza a and meningococcal disease influenza a and meningococcal disease bacterial meningitis after influenza invasive pneumococcal and meningococcal disease: association with influenza virus and respiratory syncytial virus activity? impact of pneumococcal conjugate vaccination of infants on pneumonia and influenza hospitalization and mortality in all age groups in the united states a role for streptococcus pneumoniae in virus-associated pneumonia influenza special investigations team influenza-associated deaths among children in the united states severe community-acquired pneumonia due to staphylococcus aureus, 2003-04 influenza season severe methicillinresistant staphylococcus aureus community-acquired pneumonia associated with influenza-louisiana and georgia staphylococcus aureus community-acquired pneumonia during the 2006 to 2007 influenza season multipathogen infections in hospitalized children with acute 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community-acquired methicillin-resistant staphylococcus aureus clone usa 300: successful management of the first documented paediatric case co-infection with dengue virus and pandemic (h1n1) fatal influenza a (h3n2) and campylobacter jejuni coinfection what are the implications for childhood pneumonia of successfully introducing hib and pneumococcal vaccines in developing countries oseltamivir resistance in adult oncology and hematology patients infected with pandemic (h1n1) 2009 virus detection of oseltamivir sensitive/resistant strains of pandemic influenza a virus (h1n1) from patients admitted to hospitals in thailand bacterial pneumonias during an influenza pandemic: how will we allocate antibiotics? contribution of vaccine-induced immunity toward either the ha or the na component of influenza viruses limits secondary bacterial complications epivac study group. effectiveness of the 23-valent polysaccharide pneumococcal vaccine against invasive pneumococcal disease in people 60 years or older effects of a largescale intervention with influenza and 23-valent pneumococcal vaccines in elderly people: a 1-year follow-up influenza vaccination is associated with reduced severity of community-acquired pneumonia early versus late oseltamivir treatment in severely ill patients with 2009 pandemic influenza a (h1n1): speed is life public health and economic impact of vaccination with 7-valent pneumococcal vaccine (pcv7) in the context of the annual influenza epidemic and a severe influenza pandemic the authors have no competing interests. key: cord-318696-jheb2fnn authors: kesic, matthew j.; meyer, megan; bauer, rebecca; jaspers, ilona title: exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza a infection date: 2012-04-09 journal: plos one doi: 10.1371/journal.pone.0035108 sha: doc_id: 318696 cord_uid: jheb2fnn exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. the greater mexico city area was the primary site for the spring 2009 influenza a h1n1 pandemic, which also coincided with high levels of environmental ozone. proteolytic cleavage of the viral membrane protein hemagglutinin (ha) is essential for influenza virus infectivity. recent studies suggest that ha cleavage might be cell-associated and facilitated by the type ii transmembrane serine proteases (ttsps) human airway trypsin-like protease (hat) and transmembrane protease, serine 2 (tmprss2), whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (slpi). based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. we utilized our in vitro model of differentiated human nasal epithelial cells (necs) to determine the effects of ozone on influenza cleavage, entry, and replication. we show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. we also determined that functional forms of hat, tmprss2, and slpi are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. we also show that addition of antioxidants significantly reduces virus replication through the induction of slpi. in addition, we determined that ozone-induced cleavage of the viral ha protein is not cell-associated and that secreted endogenous proteases are sufficient to activate ha leading to a significant increase in viral replication. our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility. influenza a virus is responsible for the seasonal epidemics and reoccurring pandemics, which represents a worldwide threat to global public health [1] . it is a major cause of morbidity and mortality worldwide, as during the recent h1n1 pandemic. in the u.s. alone, over 100,000 individuals are hospitalized and over 20,000 people die every year due to influenza virus infection and related diseases [2, 3] . despite large-scale efforts in vaccination and antiviral therapies, the morbidity and mortality rates associated with influenza infections have not significantly changed in recent years [4, 5] . in the context of potentially pandemic respiratory viral infections, it is important to identify molecular targets/pathways for therapeutic intervention to protect susceptible sub-populations. epidemiologic studies show that exposure to inhaled oxidants such as cigarette smoke, diesel exhaust, and ozone can modulate immune function and increase susceptibility to respiratory viral infections [6, 7, 8, 9, 10, 11] . despite the extensive study into different factors influencing susceptibility to infection, the mechanism(s) by which inhaled oxidants modify viral pathogenesis are very complex and have yet to be fully elucidated. the ability of oxidants to cause lung injury/dysfunction is dependent, in part, on the delicate equilibrium that exists between oxidants and antioxidants. oxidative stress is caused by an imbalance between the production of reactive oxygen species (ros) and the body's ability to readily detoxify reactive intermediates. ozone is one of the most abundant components of air pollution in urban areas, and has been shown to be a potent inducer of oxidative stress causing airway inflammation and increased respiratory morbidities [8, 9, 12] . the mexico city metropolitan area (mcma), one of the most densely populated areas in the world, experiences high levels of air pollutants such as environmental ozone and particulate matter (pm) [13, 14, 15] . the mcma is located 2240 m above sea level and is surrounded by mountains. due to this geographical location, there is less available oxygen, making combustion less efficient, which produces more polycyclic aromatic hydrocarbon (pah) pollutants. for these reasons, mexico city receives higher levels of environmental ozone and various other types of photochemical smog [13] . it was here that the first influenza pandemic of the 21 st century emerged in march 2009. it was responsible for an estimated ,258,698 laboratory confirmed cases and roughly ,1,370 deaths by december 2009 [1, 16, 17] . the normal ''flu season'' occurs during the colder half of the year in each hemisphere [18] . interestingly, this outbreak emerged during the dry and warmer months when ozone levels were significantly higher [19, 20] . possible mechanisms by which oxidative stress alters the airway environment leading to broadened cellular tropism and/or susceptibility to viral infections have been proposed. [21, 22, 23] the relationship between oxidative stress and the cellular protease/antiprotease balance has been considered to be a major contributor in the development of numerous airway pathologies [24] . early studies demonstrated that the cleavage of influenza ha is essential for viral penetration into host cells and that this mechanism was mediated by cellular trypsin-like serine proteases. these proteases, in turn, are regulated by mucus antiproteases, such as slpi and a 1 -antitrypsin (a1at) [25, 26, 27, 28, 29] . recent studies have identified specific cellular proteases and antiproteases that may be involved in influenza infection, which include transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), and secretory leukocyte proteinase inhibitor (slpi) [30, 31, 32, 33, 34] . the expression of these proteases in the lung are necessary for cleavage of the viral ha surface protein, thus allowing viral fusion and entry into the host cell. studies have shown a correlation between inflammation and oxidative stress which alters expression of these proteases and antiproteases [29, 35] . specifically, hat has been shown to be released into the airway fluids under inflammatory conditions, particularly in asthmatics [31, 36] . in addition, a murine study showed slpi expression was suppressed and protease expression was increased in nrf2-deficient mice which led to increased inflammation, further demonstrating a relationship between oxidative stress and protease expression [37] . oxidative stress derived from cigarette smoke or addition of reactive oxygen intermediates has been shown to decrease antiprotease activity [35, 38] and a murine study demonstrated that decreased antiprotease expression increased influenza infectivity [39] . the imbalance of protease/antiprotease expression caused by oxidative stress has been attributed to both an increase in inflammatory cells, which release proteolytic enzymes capable of destroying lung tissue, and a functional deficit of antiproteases due to oxidation of their active site [35, 40] . in addition to influenza, studies have shown that regulated proteolysis is required for the spread/ propagation of many human viruses, including human immunodeficiency virus (hiv), nipah, ebola, severe acute respiratory syndrome coronavirus (sars-cov), and metapneumoviruses [41, 42, 43, 44, 45] . although it has been shown that oxidative stress increases severity of viral infections, the exact mechanism as to how and why this happens and the role of ozone exposure in these responses are not fully understood. while the deleterious health effects of both ozone and influenza have been well documented, few studies have looked at the effects of ozone in the context of an influenza infection. herein, we used our established cell culture model of differentiated human nasal epithelial cells (necs) [46] , exposed them to 0.4ppm of ozone, and determined the effects on susceptibility to an influenza a infection. we show that there is a delicate balance of proteases and antiproteases present in the human airway surface liquid. additionally, we found exposure to ozone disrupts this equilibrium by enhancing protease secretion while inhibiting antiprotease expression leading to increased susceptibility to viral infection. we directly show that ozone increases soluble protease expression and that they are functional for cleaving the viral ha protein and activation of the intact influenza virion. primary human nasal epithelial cells (necs) were obtained from healthy adult volunteers. this protocol was approved by the university of north carolina school of medicine institutional review board for biomedical research. in addition, written informed consent was provided by each study participants and/or their legal guardians. primary human nasal epithelial cells (necs) were obtained as previously described [6] . briefly, necs were obtained from healthy adult volunteers by gently stroking the inferior surface of the turbinate several times with a rhino-probe curette (arlington scientific, arlington, tx), which was inserted through a nasoscope. this protocol was approved by the university of north carolina school of medicine institutional review board for biomedical research. nec were expanded to passage 2 in bronchial epithelial growth medium (begm, cambrex bioscience walkersville, inc., walkersville, md) and then plated on collagencoated filter supports with a 0.4 mm pore size (trans-clr; costar, cambridge, ma) and cultured in a 1:1 mixture of bronchial epithelial cell basic medium (bebm) and dmem-h with singlequot supplements (cambrex), bovine pituitary extracts (13 mg/ml), bovine serum albumin (bsa, 1.5 mg/ml), and nystatin (20 units) . upon confluency, all-trans retinoic acid was added to the basolateral medium to establish air liquid interface (ali) culture conditions (removal of the apical medium) to promote differentiation. mucociliary differentiation was achieved after 18-21 days post-ali. we obtained the madin darby canine kidney (mdck) cell line from the american type culture collection (atcc, manassas, va). the beas-2b cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct [47] . we obtained our beas-2b cells from the american type culture collection (atcc, manassas, va). beas-2b cells were grown in keratinocyte basal medium (kbm) supplemented with 30 mg/ml bovine pituitary extract, 5 ng/ml human epidermal growth factor, 500 ng/ml hydrocortisone, 0.1 mm ethanolamine, 0.1 mm phosphoethanolamine, and 5 ng/ml insulin. the primary human necs were exposed under ali conditions to filtered air or 0.4 ppm ozone for 4 h in the exposure chambers operated by the u.s. environmental protection agency, environmental public health division. in noted experiments, 10 mm of a cell-permeable form of reduced glutathione, glutathione-ethylester (gsh-et) (sigma-aldrich st. louis. mo) or 1 mm of egcg (sigma-aldrich st. louis. mo) was added to the basolateral medium 30 min prior to ozone exposure, similar to our previous studies [6, 46] . construction of vlp expression plasmids. to generate the b-lactamase-m1 fusion expression plasmid (pcaggs-b-lacm1 pr8) the influenza a/puerto rico/8/34/mount sinai (h1n1) m1 sequence was pcr amplified from the m1 expression vector pdz-m (which was kindly provided by dr. adolfo garcia-sastre, mount sinai school of medicine) and inserted into the pcaggs vector [48, 49, 50] . the b-lactamase gene was pcr amplified from pcdna3.1 and fused n-terminally to m1 within pcaggs to create a modified b-lactamase-m1 fusion. in the modified b-lactamase, the first 24 amino acids were excluded to remove a secretion signal and his 24 was substituted with asp to create an optimal kozak consensus sequence. the pcdna3.1-blactamase construct has been described previously [51] . the influenza a/puerto rico/8/34/mount sinai (h1n1) ha (pcaggs-ha) and na (pdz-na) over-expression vectors were generously provided by dr. aldolfo garcia-sastre and have been described previously [48, 49, 50, 52] . production of vlps. to generate influenza a/pr/8/h1n1 b-lactamasem1vlps (pr8 b-lacm1 vlps), 293t cells were cotransfected with 3 mg each of pcaggs-ha, pdz-na, and pcaggs-b-lacm1 pr8 using fugeneh hd (roche applied science, indianapolis, in) according to manufacturer's instructions. the supernatant containing the vlps were collect 48 h post-transfection and clarified of floating cell debris by centrifugation at 3,000 rpm for 10 min. the vlps were concentrated once by low-speed centrifugation through an amicon ultra 100 kd centrifuge filter unit (millipore; billerica, ma), and the retentates were aliquoted and stored at 280uc. vlp entry assay. the vlp entry assay was performed as previously described [6] with modifications. briefly, target cells were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h postexposure, vlps were added to the apical surface in a total volume of 100 ml and incubated at 37uc for 3 h. the cells were washed twice with hbss to remove unbound virus and infected cells were detected by using geneblazer tm fret in vivo cell-based assay system substrate ccf2-am according to manufacturer's recommendations (invitrogen). samples were lysed in hbss by freeze-thaw method repeatedly treated for 3 cycles (frozen in liquid nitrogen for 3 min and thawed in a 65uc water bath for 3 min). viral entry was quantified by using the polarstar optima plate reader (bmg labtech, inc.). all experiments were performed in triplicate in three independent experiments. we used influenza a/bangkok/1/79 h3n2 serotype (which was kindly provided by dr. melinda beck, university of north carolina). we also obtained influenza a/malaya/302/1954 h1n1 serotype from the american type culture collection (atcc, manassas, va) for western blot analysis of the cleavage products of the viral ha protein. virus was propagated in 10-dayold embryonated hen's eggs. the virus was collected in the allantoic fluid and titered by 50% tissue culture infectious dose in madin-darby canine kidney cells and hemagglutination as described before [53] . stock virus was aliquoted and stored at 280uc until use. unless otherwise indicated, for infection about 5610 5 cells were infected with approximately 128 hemagglutination units (hau) of influenza a, which resulted in approximately 10% of the cells being infected with influenza 24 h post-infection. total rna, total protein, basolateral supernatants, and apical washes were collected 24 h post-infection. apical surface liquid was collected at 24 h post-exposure by washing the apical surface of the cells with hbss and total protein concentrations were determined by bradford protein assay (bio-rad). cell lysates were prepared at 24 h post-infection in passive lysis buffer (promega, madison, wi) with a protease inhibitor mixture (cocktail set iii; calbiochem, san diego, ca) as well as phosphatase inhibitors (0.5 mm navo4, 1 mm b-glyceropho-phate) on ice for 30 min. after centrifugation, total protein concentrations were determined by bradford protein assay (bio-rad). both the apical supernatants and cellular lysates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to nitrocellulose (schleicher & schuell biosciences, keene, nh). proteins were detected using specific antibodies to hat, tmprss2, and spli (1:1,000; santa cruz biotechnology, santa cruz, ca) or influenza a virus hemagglutinin h1 antibody (1:1,000; abcam, cambridge, ma). b-actin (1:2,000; us biological, swampscott, ma), which was used as a loading control. antigen-antibody complexes were stained with anti-rabbit or anti-mouse, horseradish peroxidase (hrp)-conjugated antibody (1:2000, santa cruz biotechnology) and detected with supersignal west pico chemiluminescent substrate (pierce, rockford, il). densitometry was performed using a fujifilm las-3000 imager (fuji film global tokyo, japan). apical surface liquid was collected by washing the apical surface of the cells with hbss and total protein concentrations were determined by bradford protein assay (bio-rad). 50 mg of total protein were immunoprecipated overnight at 4uc with 200 ng of anti-tmprss2, anti-hat, or igg isotype as a control, followed by 1 h incubation with 50 ml of protein g-agrose beads (pierce, rockford, il). cleared supernatants were incubated with 50 ul of influenza a/malaya/302/1954 h1n1 for 30 min at 32uc. virus was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred to nitrocellulose (schleicher & schuell biosciences, keene, nh). proteins were detected using specific antibodies to hat, tmprss2, and spli (1:1,000; santa cruz biotechnology, santa cruz, ca) or influenza a virus hemagglutinin h1 antibody (1:1,000; abcam, cambridge, ma). influenza virus titers in apical washes were determined by 50% tissue-culture infectious dose (tcid50) in madin darby canine kidney (mdck) cells and evaluated using agglutination of red blood cells as an indicator according to a modified protocol described before [54] . briefly, mdck cells grown in roundbottom 96-well plates were inoculated with virus-containing samples diluted in serum-free dmem containing 20 mg/ml trypsin using log10 dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . a modification to the above protocol was performed to test secreted protease activity. in these experiments, necs were exposed to either 0.4ppm of ozone or air for 4 h. 24 h postexposure, apical supernatants were collected and protein concentrations were determined by bradford protein assay. 100 mg of protein within the apical supernatant, which contained the endogenous secreted proteases, was incubated with 80 ml of virus for 30 min at room temperature. mdck cells grown in roundbottom 96-well plates were inoculated with the above samples diluted in serum-free dmem or serum-free dmem containing 20 mg/ml trypsin, as a positive control, using 10-fold dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . 10 mm phenylmethyl sulfonyl floride (pmsf) (sigma-aldrich st. louis mo) was used to inhibit trypsin protease activity in control samples. recombinant human slpi (rhslpi) (r&d systems minneapolis mn) was added to serum-free dmem containing 20 mg/ml trypsin and incubated for 60 min at room temperature prior to addition of 80 ml of virus. mdck cells grown in round-bottom 96well plates were inoculated with the above samples diluted in serum-free dmem using 10-fold dilutions. after 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. wells exhibiting hemagglutination were considered positive and virus titers were expressed as log tcid 50 . all experiments were performed in triplicate in three independent experiments. total rna was extracted using trizol (invitrogen) according to the supplier's instruction. first-strand cdna synthesis and realtime rt-pcr was performed as described previously [46, 53] using commercially available primers and probes for ha (inventoried taqman gene expression assays) purchased from applied biosystems (foster city, ca). interleukin-6 (il-6) levels necs were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h post exposure, basolateral supernatants were collected for the measurement of ldh concentration according to the manufacturer's recommendations (takara bio inc. madison, wi). released ldh concentration was expressed as optical density. measurements of il-6 concentration were determined according to the manufacturer's recommendations (bd biosciences san diego, ca). released il-6 concentration was expressed as pg/ml. to measure trans-activation of the slpi promoter, 1.2610 5 beas-2b were cotransfected with either 0.1 mg of slpi-luciferase reporter plasmid (which was kindly provided by dr. rosalia simmen, arkansas children's hospital research institute) along with 0.02 mg of thymidine kinase-renilla luciferase by using fugene 6 (roche) according to the manufacturer's recommendations. after 24 h, cells were treated with 1 mm egcg or dmso. 8 h post-treatment cells were pelleted and lysed in passive lysis buffer (promega, madison, wi). the trans-activation activity was measured as luciferase light units as described previously [55] . all transfection experiments were performed in triplicate and normalized for transfection efficiency by using renilla luciferase. unpaired student's t-test and one-way anova were used for determination of statistically significant differences. the use of the term significant in text refers to a comparison of values for which p,0.05. our group has recently demonstrated that oxidative stress increases susceptibility to influenza virus and that these responses may be mediated via increased viral entry [6, 46] . therefore, we wanted to evaluate how exposure to ozone affects viral entry and replication. we chose to examine influenza infection in human necs exposed to 0.4ppm ozone for 4 h, based on our group's recent in vivo [12, 56] and in vitro [9] studies. to assess the effects of ozone on necs, we determined ldh and il-6 levels in basolateral supernatants as markers of cytotoxicity and inflammation, respectively ( figure 1a and 1b) . as expected, exposure to 0.4ppm of ozone for 4 h causes a significant increase in both inflammation and cytotoxicity [57] . subsequently, 24 h postexposure, cells were infected with influenza a/bangkok/1/79 or a mock control. 24 h post-infection total rna was subjected to real time rt-pcr to quantitate the influenza viral hemagglutinin transcript (ha) ( figure 1c ). as depicted, we saw a significant increase in the amount of viral ha mrna produced in the cells previously exposed to ozone as compared to the filtered air control. similarly, by analyzing the apical washes for influenza viral titers 24 h post-infection, we saw a significant increase in viral titers in ozone exposed cells as compared to the control ( figure 1d ). these results demonstrate that pre-exposure to ozone significantly increased viral replication in necs. to determine the potential mechanism(s) mediating the enhancement of viral replication following acute ozone exposure, we assessed whether innate antiviral immune response mediators were modulated. specifically, we analyzed the expression of interferon-b (ifn-b), interferon-a (ifn-a), retinoic acid inducible gene i (rig-i), and toll-like recptor-3 (tlr-3). human necs were exposed to ozone and 24 h post-exposure were infected with influenza a. 24 h post-infection total rna was isolated and rt-pcr was performed to quantitate the amount of each cellular transcript. ozone alone had no significant effect on mrna expression in any of the four antiviral genes as compared to the air control (figure 2a-d) . as expected, we found substantial inductions of these four antiviral mediators 24 h post-infection with influenza a, but without significant differences in ozone exposed compared to control cells. these data demonstrate that exposure to ozone does not alter baseline expression of host antiviral immune response genes, and does not interfere with their induction in response to influenza infection. since we have shown that ozone exposure did not abrogate antiviral responses, we wanted to determine if the increase in influenza virus infection is due to the oxidative stress pathway/ mechanism induced by ozone exposure. to test this hypothesis, we treated our necs with either a potent phase-ii antioxidant inducer or a direct antioxidant. we have previously shown that oxidative stress increases susceptibility to influenza infection [6] . previous reports have shown that nrf2 gene expression and protein expression can be induced via antioxidant supplementation [58, 59, 60] , more specifically by the addition of the polyphenolic catechin, epigallocatechin-3-gallate (egcg). this compound has the ability to induce nrf2 activation which in turn up-regulates the expression of multiple phase-ii antioxidants [6, 61, 62] . based on our previous published data [6, 46] , we determined that 1 mm of egcg or 10 mm of gsh-et is sufficient to detect an increase in nrf2 expression or counteract oxidative stress induced by diesel exhaust exposure in necs. these concentrations also correlate with levels of egcg that are achievable in vivo [63, 64, 65] . necs were treated with either 1 mm egcg, 10 mm gsh-et, or dmso as a vehicle control for 30 min prior to a 4 h 0.4ppm ozone exposure. 24 h post-ozone exposure cells were infected with influenza a virus and total rna was isolated 24 h post-infection to determine the level of influenza ha transcripts by rt-pcr. as shown in figure 3a , exposure to ozone significantly increases viral ha expression. interestingly, the addition of either egcg or gsh-et significantly inhibited viral ha transcription as compared to the vehicle control. we again collected apical washes to perform viral titer assays. our results, shown on a log scale, indicate that both egcg and gsh-et significantly reduced viral replication ( figure 3b ). taken together, these results demonstrate that antioxidants, either through nrf2 activation (egcg) or by direct addition of an antioxidant (gsh-et), counteract the oxidative stress induced by ozone exposure and can suppress influenza virus replication in necs. ozone not only triggers intracellular oxidative stress, but has also been shown to directly affect and modify the cellular lipid membrane [66, 67] and transmembrane molecules [68] . since the influenza virion must first bind and enter the target cell prior to replication, we hypothesize that the increased susceptibility to influenza after ozone exposure most likely happens early in the virus lifecycle, upstream of viral replication. to determine which step(s) in the virus life cycle are affected by ozone exposure, we employed an enzymatic virus-like particle (vlp) assay. this assay quantitatively measures the amount of virus that enters the cells. similar to our previous work [6] , cells were infected with a vlp that only express the hemagglutinin (ha), neuraminidase (na), and matrix (m) proteins along with a functional b-lactamase reporter fusion (pr8 b-lacm1 vlp) prior to loading with the fluorogenic substrate ccf2-am . in cells in which the pr8 b-lacm1 vlp has entered, the ccf2-am substrate will be cleaved, disrupting fret of the substrate, and resulting in increased ccf2 emission at 447 nm. to determine the effects of ozone exposure on virus entry, necs and the cell line mdck as a control were exposed to air or ozone and 24 h post-exposure, cells were infected with pr8 b-lacm1 vlp. in this experiment, mdck cells are used as a control cell line which does not produce proteases required to activate the viral ha protein. figure 4 shows that exposure to ozone results in a significant increase in viral entry in the necs alone. in summary, these data demonstrate that acute exposure to ozone enhances viral entry, consistent with the increased viral replication shown in figure 1c . ozone exposure disrupts host protease/antiproteases balance as stated earlier, recent reports have identified two endogenous human trypsin-like serine proteases (tmprss2 and hat) that possess the ability to cleave influenza virus in vitro [30, 32, 33, 69] . viral ha proteolytic cleavage is required for viral fusion and entry into the host cell. proteases are regulated by antiproteases, such as slpi and to a lesser extent a1at. studies have shown that a disruption of the protease/antiprotease balance is a hallmark of numerous lung diseases and pathologies including copd, emphysema, asthma, and cancer [40, 70, 71, 72] . hennet et al, demonstrated a link between oxidative stress and protease expression which led to increased influenza infection in mice [39] . linking this work with our current hypothesis, we investigated the protease/antiprotease balance on the epithelial surface in the context of an acute ozone exposure. for these experiments we exposed our necs to either air or ozone for 4 h. 24 h post-exposure, both apical surface liquid and cell lysates were collected to characterize secreted/soluble and membrane-bound intracellular levels of hat, tmprss2, and slpi. western blots revealed that intracellular levels of slpi decreased slightly with ozone exposure, while hat and tmprss2 expression remained relatively unchanged ( figure 5a ). although it is well known that slpi is secreted and is present in normal human airway surface liquid, only recently was the transmembrane protease, hat, found to be secreted in an in vitro overexpression system [33] . we found the levels of the secreted forms of hat, tmprss2, and slpi to be significantly changed by ozone exposure ( figure 5b ). under normal conditions, slpi is in greater abundance than the proteases, yet 24 h post ozone exposure, we found a decrease in slpi but a significant increase in both hat and tmprss2. it is worth noting that the time at which these samples were taken corresponds to the exact time post ozone exposure when the cells were infected with influenza in the data shown in figures 1, 2 and 3, thus representing the levels of protease and antiprotease present at the time of infection. to determine if disruption in the protease/ antiprotease equilibrium is due to oxidative stress imposed by the ozone exposure, we treated our necs with the potent nrf2 inducer egcg prior to ozone challenge. figure 5c shows that egcg did increase levels of slpi which corresponds with a decrease in protease expression. finally, figure 5d demonstrate that the addition of 1 mm egcg significantly increases the transactivation of the slpi promoter which correlates with the increase in slpi protein expression displayed in figure 5c . taken together, these results indicate that ozone-induced oxidative stress disrupts the protease/antiprotease balance, in favor of protease activity. furthermore, we demonstrate that the equilibrium can be restored with the induction of nrf2 via antioxidant supplementation resulting in an increase in the antiprotease slpi. we characterized the cleavage products of the viral ha protein to determine if the proteases produced by acute ozone exposure can cleave an intact influenza virion. we first investigated where ha cleavage takes place. necs were exposed to air or ozone and 24 h post-exposure, cells were infected with influenza a/malaya/ 302/1954 h1n1. the cell lysates were analyzed by western blotting for detection of the ha cleavage products. figure 6a shows that the majority of the viral ha protein within the cell is cleaved and there is not a significant change with ozone exposure. in addition, western blot analysis of apical washings incubated with virus showed that ha0 was cleaved and that there was an increase in the cleavage of ha from the ozone exposed cells as measured by densitometry. quantification of the h2 cleavage products demonstrated that ozone exposure leads to enhanced proteolytic activation of the virion. this data demonstrate that ha cleavage can take place in both the intra-and extracellular environment and that ozone exposure increases the proteolytic activation of the virus in the airway ( figure 6b ). to characterize the function of tmprss2 and hat in ha cleavage, we depleted the proteases via immunoprecipitation prior to addition of the virus. figure 6c shows the apical washes from necs that were immunoprecitipated with anti-hat, anti-tmprss2, and igg as a control. the cleared supernatants were incubated with influenza a/malaya/302/1954 h1n1. as shown in figure 6d , there is little difference in the cleavage of the ha protein when the protease are depleted separately, but we demonstrate a significant decrease in the cleavage of ha when both proteases were removed. this data show that the protease(s) found in the apical surface liquid are able to cleave an intact influenza a virion. in addition we show that there are additional proteases secreted into the airway liquid space that are able to activate the virus. although we show that slpi, hat, and tmprss2 are secreted by necs, the question remains as to whether they are functional. to test functionality and the role they play in a viral infection, we employed a modified infectivity viral titer assay. these experiments are very similar to the viral titer assays mentioned earlier, but with a few modifications. similar to the cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, samples were collected and subjected to western blot analysis to detect slpi, hat, tmprss2, and b-actin. a) total cellular lysates were analyzed for intracellular slpi, tmprss2, and hat protein expression by western blot. membrane was stripped and analyzed for b-actin as a loading control. b) 24 h post-exposure, apical supernatants were analyzed for secreted slpi, tmprss2, and hat protein levels. c) cultures of differentiated human epithelial cells were treated with egcg or dmso as a vehicle control for 30 min before exposure to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were analyzed for secreted slpi, tmprss2, and hat protein levels. d) function activity of the slpi promoter was determined using a dual-luciferase reporter assay. beas-2b cells were co-transfected with slpi-luc and tk-renilla. cells were treated with egcg or dmso as a vehicle control. cells were harvested 8 h post-treatment and analyzed for luciferase expression. the values represent luciferase production from a representative experiment performed in triplicate. necs were obtained from four healthy volunteers (n = 4). densitometry was used to quantitate the amounts of protein, and the numbers below the gel indicate the ozone/air or egcg/dmso sample ratio. doi:10.1371/journal.pone.0035108.g005 figure 6 . secreted proteases are functional for hemagglutinin cleavage. cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. a) 24 h post-exposure, cells were infected with influenza a/malaya/302/1954. 24 h post-infection, total cellular lysates were analyzed for intracellular cleavage of the viral ha protein by western blot. b) 24 h post-exposure, apical supernatants were collected and incubated with influenza a/malaya/302/1954. cleavage of the viral ha protein was analyzed by western blot. c) cultures of differentiated human epithelial cells were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post-exposure, apical supernatants were immunoprecipitated (ip) with anti-hat, anti-tmprss2, or an isotype control (igg). protein levels were analyzed by western blot. d) apical supernatants of differentiated human epithelial cells, were immunoprecipitated (ip) with anti-hat, anti-tmprss2, or an isotype control (igg) followed by incubation with influenza a/ malaya/302/1954. cleavage of the viral ha protein was analyzed by western blot. necs were obtained from four healthy volunteers (n = 4). densitometry was used to quantitate the amounts of cleaved h2 protein, and the numbers below the gel indicate the ozone/air ratio. doi:10.1371/journal.pone.0035108.g006 assays before, we utilized the well-characterized mdck cell line which does not produce the proper proteases to activate the influenza virion, which can be overcome by the addition of exogenous trypsin. in contrast to the previous titer experiments above, we did not add exogenous trypsin to enable multicycle replication of influenza virus in these cells. these experiments allowed us to test if secreted proteases present in the apical surface liquid from necs exposed to air or ozone were able to facilitate multiple rounds of viral replication in mdcks. we show that the secreted proteases present in the apical supernatants from necs are functional and can propagate viral entry and replication. we also demonstrate that the ozone-exposed nec supernatants displayed significantly greater infection kinetics than the air control ( figure 7a ). this correlates with the increase in protease expression post ozone exposure ( figure 5b ), viral entry (figure 4) , and viral replication ( figure 1c and 1d ). in addition we show that this is a protease-mediated event, since the addition of a known serine protease inhibitor, pmsf, significantly inhibits viral replication. to demonstrate that slpi has the ability to protect airway epithelium from influenza infection by inhibiting proteolytic cleavage, we incubated various amounts of recombinant human slpi (rhslpi) to our traditional viral titer assay. briefly, we incubated our viral titer media, serum-free dmem containing 20 mg/ml of exogenous trypsin, with varying amounts of rhslpi prior to addition of influenza. as shown in figure 7b , addition of rhslpi significantly inhibits multicycle viral replication. we conclude that the addition of rhslpi directly inhibits trypsinmediated cleavage of viral ha leading to decreased infection and replication. taken together, these data demonstrate that exposure to ozone disrupts the protease/antiprotease balance resulting in greater secretion of endogenous proteases leading to increased viral cleavage/activation culminating in enhanced viral entry and replication. numerous epidemiological studies link increased ambient ozone levels with respiratory viral infections [1, 73, 74] . however, the role of ozone exposure in viral replication and pathogenesis remains to be fully defined. because ambient ozone is a major source of oxidative stress in the airway epithelium and this is the primary site for influenza infections, it is important to determine the role ozone exposure plays in viral infections. although it has been shown that ozone exposure can modulate many aspects of the immune response [8] , how these alterations influence a viral infection have yet to be elucidated. recent studies have demonstrated the importance of the protease/antiprotease balance in the context of normal lung homeostasis, and have shown that oxidative stress can disrupt this delicate equilibrium [40, 75] . it has been well documented that many lung pathologies are dependent on the regulatory interplay between oxidative stress and protease expression [40, 70, 71] . although there is speculation that mexico city's increased influenza-related morbidity and mortality rates were due to exposures to higher levels of ozone, little research has been done looking at the effects of pre-exposure to ozone in the context of an influenza infection in humans. herein, we demonstrate that acute ozone exposure disrupts the protease/ antiprotease balance resulting in increased secreted protease expression in primary human epithelial cells, resulting in increased ha cleavage and activation of the influenza virion, which correlated with enhanced viral entry and replication. numerous host cell-dependent factors can affect and control influenza virus attachment and uptake by: (i) proteolytic cleavage of viral ha by host cell-derived serine proteases [30, 33] , (ii) host cell derived innate immune defense molecules aimed at inhibiting the infectious virions [25, 76] , and (iii) antiviral mediators limiting viral replication and shedding of virus particles [6, 52] . we first focused on potential effects of ozone on host antiviral defense responses. our data showed that exposure to ozone elicited the typical pro-inflammatory and cellular damage responses as seen with increased release of il-6 and ldh. we found that the same exposure regimen also resulted in significant increase in viral replication. we initially hypothesized that this could be due to a disruption in one of the cellular antiviral response mechanism/ pathways, and assessed the transcription levels of 4 classical antiviral mediators; rig-i, ifn-a, ifn-b, and tlr-3. similar to our previous studies using exposure to diesel exhaust [46] , we found that ozone did not decrease expression of these mediators or their response when challenged with influenza. in our previous study [46] , we found that exposure to an oxidant pollutant increased influenza virus attachment and/or entry, but did not distinguish between these two steps in the viral infection cycle. therefore, we focused our attention on steps upstream of viral replication to identify mechanisms by which oxidants, such as ozone, affect influenza infectivity. to dissect specific points in the virus life cycle upstream of viral replication that could determine the role ozone exposure plays in viral susceptibility, which ultimately dictates viral pathogenesis and outcome, we utilized our previously described enzymatic virus-like particle (vlp) assay [6] . our data demonstrated that ozone exposure significantly increased influenza virus entry. this is consistent with our previous work showing that exposure to other oxidant pollutants and suppression of nrf2 increases influenza virus entry [6, 46] . similar to these previous studies, data shown here demonstrated that the mechanism(s) through which ozone exposure alters influenza virus entry is mediated by oxidative stress. since ozone directly interacts with the apical surface of the respiratory epithelium we hypothesize that ozone either directly, or via the induction of biologically active ozone reaction products, modifies either the expression or function of surface proteins, as previously been demonstrated for human surfactant protein a (sp-a) [77] . among the most prominent proteins on the apical surface potentially regulating infection are the proteases. to date, at least five different proteases have been identified in the airways of animals and humans [78] . despite many years of effort, the exact protease(s) that activates influenza has yet to be identified. numerous groups have reported that the family of type ii transmembrane serine proteases and trypsin-like proteases are responsible for viral cleavage [26, 30, 33, 79] . previous studies have examined these proteases in the context of influenza infections, specifically tmprss2 and hat. although these studies demonstrated that these proteases are capable of cleavage, the experiments were performed in cell lines such as mdcks [33] or the colorectal adenocarcinoma cell line caco-2 [32] , neither of which are natural target cells for influenza. since the cellular protease determines the tropism as well as efficiency of viral replication, we utilized fully differentiated human primary nasal epithelial cells (necs) in this study, a natural host cell for influenza virus. we show that hat and tmprss2 are not only present, but secreted from necs ( figure 5b ), and that ozone exposure enhances the release of the proteases into the apical compartment. this is consistent with reports showing that oxidative stress increases cellular protease activity [38] . since proteases in the upper airway are inhibited by slpi [78] , we focused on this antiprotease and its expression in relation to hat and tmprss2. similar to previous reports, we report that slpi is detected in the secreted airway and oxidative stress reduces the expression and function of the protein ( figure 5b ) [35, 80] . these data are consistent with our hypothesis that influenza virions can become activated in the airway surface liquid prior to binding to the epithelial surface. virion activation in the airway prior to binding would eliminate the need for a membrane-bound protease thus broadening viral tropism from airway epithelium, that contain the necessary proteases, to any mammalian cell, since 2,6-linked sialic acid is expressed by most mammalian cells ranging from lymphoid to glial cells [81, 82] .this could explain the increased morbidity and mortality associated with the mexico city 2009 h1n1 pandemic which resulted in increased infection of multiple cell types including distal airway epithelium and immune cells including alveolar macrophages [83, 84] as compared to previous seasonal outbreaks. egcg has been shown to have potent antioxidant capabilities; in part by inducing the expression of a number of antioxidant enzymes [85] . in vitro and in vivo studies have shown that this supplement induces the expression of phase ii antioxidant genes such as gstm1, which was associated with nrf2-are signaling [58, 86] . whether and how activation of antioxidants expression is involved in the potential antiviral effects of egcg is not known. previous studies have indicated that it directly binds influenza virus and therefore prevents attachment and entry into host cells [63, 87] . however, these studies were conducted in mdck cells, which are not a natural host cell for influenza and require addition of exogenous proteases to achieve viral entry [63] . iizuka et al, demonstrated that nrf2-knockout mice display protease/antiprotease imbalance leading to increased susceptibility to cigarette smoke-induced emphysema [37] . in addition, the same group showed that nrf2 expression exerts a protective effect through the transcriptional activation of antiproteases [88] . our data demonstrate that supplementation of differentiated nasal epithelial cells with egcg from the basolateral side (to eliminate direct interaction with the virus during infection) significantly increases slpi production ( figure 5c ). it is worthy to note that we show a direct inverse relationship between the levels of secreted slpi and secreted protease expression, suggesting a potential novel antiviral mechanism by which egcg could be protective against influenza infections. we next determined that the secreted proteases are functional. we demonstrate that proteases present in the apical surface liquid were able to activate the virus ( figure 6b ) and produce multiple rounds of viral replication ( figure 7a ). in addition, apical supernatants from ozone-exposed epithelial cells had significantly higher viral titers, which correlate with the increased protease expression displayed in figure 5b . in the final experiment we utilized rhslpi and show a dose dependent protective response ( figure 7b ). this demonstrates that that the antiprotease slpi has a protective affect against viral activation and replication. in conclusion, this is the first study to demonstrate that secreted proteases from primary human respiratory nasal epithelium proteolytically activate influenza virions and that exposure to a common ambient air oxidant pollutant increases these effects. we speculate that individuals with increased airway oxidative stress, whether due to an underlying medical condition or exposure to high levels of air pollutants, would develop 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oxidative injury oral absorption and bioavailability of tea catechins inhibition of the infectivity of influenza virus by tea polyphenols transcription factor nrf2 plays a pivotal role in protection against elastaseinduced pulmonary inflammation and emphysema protease inhibitors in respiratory disease: focus on asthma and chronic obstructive pulmonary disease we thank ms. luisa e. brighton for her expert technical assistance and dr. philip bromberg, m.d., for his critical review of the manuscript. key: cord-290352-0pc5eji4 authors: de jong, menno d.; hien, tran tinh title: avian influenza a (h5n1) date: 2005-10-06 journal: j clin virol doi: 10.1016/j.jcv.2005.09.002 sha: doc_id: 290352 cord_uid: 0pc5eji4 since their reemergence in 2003, highly pathogenic avian influenza a (h5n1) viruses have reached endemic levels among poultry in several southeast asian countries and have caused a still increasing number of more than 100 reported human infections with high mortality. these developments have ignited global fears of an imminent influenza pandemic. the current knowledge of the virology, clinical spectrum, diagnosis and treatment of human influenza h5n1 virus infections is reviewed herein. periodically, completely novel antigenic subtypes of influenza viruses have been introduced in the human pop* corresponding author. tel.: +84 8 9237 954; fax: +84 8 9238 904. ulation, causing large-scale global outbreaks with high death tolls. the most devastating influenza pandemic in modern recorded history, known as the "spanish flu", occurred in 1918-1919, killing up to 100 million people worldwide. other less destructive pandemics during the previous century occurred in 1957 and 1968 . avian influenza a viruses are key to the emergence of human influenza pandemics. the virus strains implicated in the 20th century's influenza pandemics originated directly from avian influenza viruses, either through genetic reassortment between human and avian influenza strains (1957 and 1968) or possibly through adaptation of purely avian strains to humans (1918) . it was long thought that the restricted host range of avian influenza viruses precluded direct transmission to humans, and that the emergence of pandemic strains required genetic reassortment between avian and human strains. however, occurrences of direct bird-to-human transmission of avian influenza viruses have increasingly been reported in recent years, culminating in the ongoing outbreak of influenza a (h5n1) among poultry in several asian countries with associated human infections. these unprecedented developments have resulted in increasing global concerns about the pandemic potential of these viruses. influenza viruses are pleomorphic, enveloped rna viruses belonging to the family of orthomyxoviridae. protruding from the lipid envelope are two distinct glycoproteins, the hemagglutinin (ha) and neuraminidase (na) . ha attaches to cell surface sialic acid receptors, thereby facilitating entry of the virus into host cells. since it is the most important antigenic determinant to which neutralizing antibodies are directed, ha represents a crucial component of current vaccines. na is the second major antigenic determinant for neutralizing antibodies. by catalyzing the cleavage of glycosidic linkages to sialic acid on host cell and virion surfaces, this glycoprotein prevents aggregation of virions thus facilitating the release of progeny virus from infected cells. inhibition of this important function represents the most effective antiviral treatment strategy to date. a third membrane protein, the m2 protein, is present in small quantities in influenza a viruses. by functioning as an ion channel, this protein regulates the internal ph of the virus, which is essential for uncoating of the virus during the early stages of viral replication. this function is blocked by the antiviral drugs amantadine and rimantadine. the genome of influenza viruses is segmented, consisting of 8 single-stranded, negative sense rna molecules, which encode 10 proteins. the rna segments are contained within the viral envelope in association with the nucleoprotein (np) and three subunits of viral polymerase (pa, pb1, and pb2), which together form the ribonucleoprotein (rnp) complex responsible for rna replication and transcription. additional proteins contained within the virion include m2 and the viral nuclear export protein (nep), which function in assembly and budding, and export of rnp from the nucleus, respectively. based on antigenic differences in np and m proteins, influenza viruses are classified as types a, b, and c. influenza b and c viruses are not divided into subtypes. all avian influenza viruses are classified as type a. further subtyping of influenza a viruses is based on antigenic differences between the two surface glycoproteins ha and na. to date, 16 ha subtypes (h1-h16) and 9 na subtypes (n1-n9) of influenza a viruses have been identified (fouchier et al., 2005) . the standard nomenclature for influenza viruses includes the influenza type, the host of origin (excluding humans), the place of isolation, the strain number, the year of isolation, and finally the influenza a subtype in parentheses (e.g. a/duck/vietnam/11/04 (h5n1)). the natural reservoir of influenza a viruses are aquatic birds, in which the viruses appear to have achieved an optimal level of host adaptation (webster et al., 1992) . transmission between birds occurs directly or indirectly through fecally contaminated aerosols, water, feed, and other materials. the spectrum of disease in birds ranges from asymptomatic infection, to mild respiratory illness, to severe and rapidly fatal systemic disease. most avian influenza viruses isolated from birds are avirulent, i.e. result in asymptomatic infection or only mild disease. avian influenza viruses capable of causing outbreaks of severe disease (fowl plague) in chickens or turkeys are classified as highly pathogenic, and are currently restricted to h5 and h7 subtypes. infection of poultry by highly pathogenic avian influenza viruses is characterized by disseminated infection, and clinically manifested by decreased egg production, respiratory signs, excessive lacrimation, edema of the head, diarrhea, neurological symptoms, and death. from the principal reservoir of aquatic birds, viruses are occasionally transmitted to other animals, including mammals and domestic poultry, causing transitory infections and outbreaks. through adaptation by mutation or genetic reassortment, some of these viruses may establish speciesspecific permanent lineages of influenza a viruses, and cause epidemics or epizootics in the new host. in the human population, the establishment of these lineages in the 20th century was preceded by influenza pandemics. transmission of viruses and transitory infections may also occur among the new hosts, e.g. between humans and pigs or chickens and humans. although all ha and na subtypes are found in aquatic birds, the number of subtypes that have crossed the species barrier and established stable lineages in mammals is limited. only three ha and two na subtypes (i.e. h1-3 and n1-2)) have circulated in humans since 1918. in horses, only two influenza a subtypes (h7n7 and h3n8) are found, while, despite susceptibility to all avian subtypes in experimental settings, the only subtypes recovered from pigs in nature are h1, h3, n1, and n2. the molecular, biological or ecological factors determining the apparent subtype-specific ability of viruses to cross species barriers and spread among a range of hosts remain largely unresolved. while interspecies transmission does occur at times, there certainly are host range restrictions. human influenza strains preferentially bind to sialic acid residues linked to galactose by the ␣2,6 linkage, while avian and equine influenza strains recognize sialic acid linked to galactose by ␣2,3 linkage (connor et al., 1994; gambaryan et al., 1997; matrosovich et al., 1997 matrosovich et al., , 2004 rogers and d'souza, 1989; . correspondingly, human respiratory epithelial cells predominantly contain ␣2,6 sialic acid-galactose linkages, while the host cells in birds and horses mainly contain ␣2,3 linkages (couceiro et al., 1993; ito et al., 1998; matrosovich et al., 2004) . respiratory epithelial cells in the pig contain both ␣2,3and ␣2,6 linkages, which explains why this animal is susceptible to both human and avian influenza viruses (ito et al., 1998) . because of this trait, the pig is widely regarded as a potential source of new pandemic strains, since it could serve as a non-selective host in which mixed infection of avian and human strains efficiently occurs, potentially resulting in new reassortant viruses, or in which purely avian strains can adapt to human receptor recognition. introduction of an influenza a virus with a novel ha gene in a population which lacks immunity to this ha has the potential to cause a pandemic when the virus posesses the ability to spread efficiently among humans. during the 20th century, this has happened three times, in 1918, 1957, and 1968 , killing millions of people worldwide. in all three pandemics, the viruses originated from avian influenza viruses. the virus strains responsible for the influenza pandemics of 1957 and 1968 both first emerged in southeastern asia, and both arose through reassortment of genes between avian viruses and the prevailing human influenza strain (scholtissek et al., 1978) . the "asian influenza" pandemic of 1957 was caused by an h2n2 virus that had acquired three genes (h2, n2, and pb1) from avian viruses infecting wild ducks, in a backbone of the circulating h1n1 human influenza strain. as the asian flu strain emerged and established a permanent lineage, the h1n1 strains soon disappeared from the human population for unclear reasons. similarly, the h3n2 virus causing the "hong kong influenza" pandemic of 1968 consisted of two genes from a duck virus (h3 and pb1) in a background of the human h2n2 strain circulating at that time. the latter virus disappeared with the emergence of the h3n2 virus and since then has not been detected in humans. sequence analysis of the hypothetical precursor strain, which immediately preceded the pandemic h3n2 virus suggested that fewer than six amino acids in ha had changed during the avian-to-human transition . interestingly, a number of these changes may reflect adaptation to the new host since they modified the area surrounding the receptor-binding pocket of ha, including a glu to leu change at position 226 which is particluarly implicated in determining specificity for human receptors. the fact that, beside one or two novel surface glycoproteins, both pandemic strains also posessed a pb1 gene of avian origin is intriguing and may suggest a role of this gene in interspecies transmission (kawaoka et al., 1989) . although millions of people died during the 1957 and 1968 pandemics, the viruses involved did not appear particularly virulent, suggesting that lack of immunity was the main reason for the excess mortality. this was different during the "spanish flu" pandemic of 1918, in which lack of immunity in the human population was combined with an apparent extremely high virulence of the virus, resulting in the demise of up to 100 million people worldwide. because the 1918 pandemic occurred before viruses were identified as the causative agents, no intact virus has been available for analysis. this and the similar lack of available human and animal influenza strains circulating before 1918 has made it difficult to determine the exact origin of the pandemic h1n1 virus and the reason for its extreme virulence. however, valuable insight has been provided by the recovery of fragments of viral rna isolated from archived autopsy specimens and tissue from alaskan flu victims buried in the permafrost (taubenberger et al., 1997) . this enabled sequence analysis of five of the eight genes (ha, na, np, m, and ns) (reid et al., 2004) . phylogenetic analyses of these genes suggest that the 1918 h1n1 virus may not have arisen by the same mechanism as the 1957 and 1968 pandemic viruses, i.e. by reassortment of avian and human influenza viruses, but perhaps by direct transmission from an avian source after adaptation in humans or another permissive mammalian host, such as the pig (reid et al., 2004) . this is supported by the observation that the 1918 pandemic strain retained the amino acid residues at positions 226 and 228 of ha predictive for binding to avian receptors (taubenberger et al., 1997) . recent chrystallographic studies showed that structural changes in the h1 ha allowed the virus to recognize human receptors despite the presence of these avian-like residues, which may explain why the virus could nevertheless efficiently infect and spread among humans (gamblin et al., 2004; stevens et al., 2004) . the possibility that the 1918 strain had retained the structure and biological properties of its avian ancestors while acquiring the ability to recognize and efficiently infect human cells may explain the high virulence of this virus. mathematical modelling studies have suggested that the transmissability of the 1918 virus was not remarkably different than regular human influenza strains, suggesting that extremely efficient spread did not account for the high morbidity and mortality (mills et al., 2004) . while part of the high mortality of the 1918 pandemic could be explained by the lack of antibiotics to treat secondary bacterial pneumonia and poor living conditions, the extremely rapid and severe clinical course implies high pathogenicity of the virus as the major cause. the molecular basis for this high virulence remains unclear. the 1918 h1 ha lacks the multibasic cleavage site characteristic of highly pathogenic avian influenza viruses (reid et al., 1999; taubenberger et al., 1997) . there are conflicting observations concerning the role of the ns gene in the 1918 pandemic strain. in mice, the presence of the complete ns or only the ns1 segment seemed to confer decreased, rather than enhanced pathogenicity of reassortant h1n1 viruses (basler et al., 2001) . in contrast, in vitro experiments in human lung cells suggested more efficient inhibition of interferon-regulated genes by h1n1 virus in the presence of the 1918 ns gene (geiss et al., 2002) . the most convincing evidence implicates ha as an important determinant of the high virulence. the presence of ha of the 1918 virus conferred high pathogenicity in mice to human strains that were otherwise non-pathogenic in this host (kobasa et al., 2004) . furthermore, these infections were associated with severe hemorrhagic pneumonia and the induction of high levels of macrophage-derived cytokines and chemokines, strikingly reminiscent of clinical observations in humans during the spanish flu pandemic, as well as of recent in vitro and in vivo observations of infections with highly pathogenic avian influenza h5n1 viruses (cheung et al., 2002; oxford, 2000; peiris et al., 2004; to et al., 2001) . in recent years, it has become clear that human infections with highly pathogenic influenza h5n1 viruses are associated with severe, often fatal disease. in may 1997, following outbreaks of influenza h5n1 among poultry on three farms in the new territories of hong kong, an influenza h5n1 virus was isolated from a 3-year-old boy in hong kong, who died of severe pneumonia complicated by acute respiratory distress syndrome and reye's syndrome (subbarao et al., 1998) . in november and december of the same year, concomittant with outbreaks of influenza h5n1 among chickens in poultry markets and on farms in hong kong, 17 additional cases of human h5n1 infections were identified, 5 of which were fatal (chan, 2002; yuen et al., 1998) . the outbreak was contained after the slaughtering of all 1.5 million chickens in hong kong. in response to the outbreak, influenza surveillance in poultry was intensified permitting early recognition of other outbreaks of avian influenza in 2001 and 2002. no further human h5n1 infections were reported until february 2003, when two laboratory-confirmed cases and one probable case were identified in one family from hong kong (peiris et al., 2004) . the daughter died of an undiagnosed respiratory infection while visiting fujian province in mainland china. upon their return to hong kong, the father and son developed severe respiratory illnesses of which the father died. h5n1 virus was isolated from both patients. in december 2003, an outbreak of highly pathogenic h5n1 virus was identified among poultry in the republic of korea (lee et al., 2005) . subsequently, outbreaks by antigenically related viruses were reported among poultry in thailand, viet nam, japan, china, cambodia, laos, malaysia, and indonesia. the reason for this apparent simultaneous occurrence of h5n1 outbreaks in many asian countries remains unclear. however, h5n1 viruses have also been found in dead migratory birds, which may suggest a role of wild birds in the maintenance and spread of h5n1 viruses in the region (chen et al., 2005; li et al., 2004) . human infections during the southeast asian outbreaks were first reported in early 2004 from viet nam and thailand, followed by still ongoing resurgences of human cases in viet nam, cambodia, and indonesia from then onwards (chotpitayasunondh et al., 2005; tran et al., 2004) . at the time of this writing (august 2005), the total number of confirmed human cases of influenza h5n1 in the 4 countries amounts to 112 (thailand: 17; cambodia: 4; indonesia: 1; viet nam: 90), of which 57 were fatal (who, 2005) . it cannot be excluded and may even be likely that additional cases have gone unnoticed in these and other affected countries due to a lack of clinical awareness, active surveillance, or diagnostic facilities (hien et al., 2004) . while many countries initially affected by poultry outbreaks in 2004 have been declared free of the virus, h5n1 virus seems to have reached endemic levels in poultry and aquatic birds in several asian countries, despite attempts to contain the outbreak by extensive culling of poultry. in these countries, continuing occurrences of bird-to-human transmissions increase the opportunity of the virus to adapt to humans and acquire the ability to spread between humans. in addition, continuing cocirculation of avian and human viruses in these countries, where humans live in close proximity with poultry and pigs, increases the risk of reassortment between both in co-infected humans or other mammalian hosts, such as the pig. the recent isolation of h5n1 viruses from pigs in china (chen et al., 2004) , and, albeit at low prevelance, the detection of h5n1 antibodies in vietnamese pigs (choi et al., 2005) , are concerning in this respect. for all these reasons, the current developments in southeast asia seem to justify the global concern that, similar to 1957 and 1968, a new pandemic influenza strain may emerge from this region in the near future. at presentation, most cases of human h5n1 infections were characterized by a severe influenza syndrome, clinically indistinguishable from severe human influenza, with symptoms of fever, cough and shortness of breath, and radiological evidence of pneumonia (chotpitayasunondh et al., 2005; tran et al., 2004; yuen et al., 1998) . abnormalities on chest radiographs at presentation included extensive, usually bilateral infiltration, lobar collapse, focal consolidation, and air bronchograms. radiological evidence of pulmonary damage could still be observed in surviving patients several months after the illness. beside respiratory symptoms, a large proportion of patients also complained of gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain, which are common in children with human influenza, but not in adults. in some cases, diarrhea was the only presenting symptom, preceding other clinical manifestations (apisarnthanarak et al., 2004; de jong et al., 2005) . unlike human infections with h7 or h9 viruses, conjunctivitis was not prominent in h5n1-infected patients. the clinical course of the illness in severe cases was characterized by rapid development of severe bilateral pneumonia necessitating ventilatory support within days after onset. complications included acute respiratory distress syndrome, renal failure, and multi-organ failure. evidence that the clinical spectrum of human h5n1 infections is not restricted to pulmonary symptoms was provided by a reported case of possible central nervous system involvement in a vietnamese boy who presented with diarrhea, followed by coma and death. influenza h5n1 virus was isolated from throat, rectal, blood, and cerebrospinal fluid specimens, suggesting widely disseminated viral replication (de jong et al., 2005) . his sister had died of a similar illness 2 weeks earlier, but no diagnostic specimens were obtained. although highly virulent h5n1 viruses have shown neurotropism in mammals such as mice and cats (keawcharoen et al., 2004; lipatov et al., 2003; tanaka et al., 2003) , these cases may be similarly rare as central nervous system manifestations associated with human influenza (morishima et al., 2002; sugaya, 2002) . genetic predisposition of the host to such manifestations may play a role. striking routine laboratory results in h5n1-infected patients, especially in severe cases, were an early onset of lymphopenia, with a pronounced inversion of the cd4+/cd8+ ratio, thrombocytopenia and increased levels of serum transaminases (chotpitayasunondh et al., 2005; tran et al., 2004; yuen et al., 1998) . high levels of cytokines and chemokines have been observed in several h5n1-infected patients, suggesting a role of immune-mediated pathology in the pathogenesis of h5n1 infections (peiris et al., 2004; to et al., 2001) . this was supported by pathological examination in two patients who died during the outbreak in hong kong, which showed reactive hemophagocytosis as the most prominent feature . other findings included diffuse alveolar damage with interstitial fibrosis, hepatic central lobular necrosis, acute renal tubular necrosis, and lymphoid depletion. although the gastrointestinal, hepatic, renal, and hematologic manifestations could suggest wider tissue tropism, there was no evidence of viral replication in organs outside the respiratory tract . however, viral replication in the gastrointestinal is strongly suggested by reported virus isolation and detection of positive strand viral rna from fecal specimens (de jong et al., 2005; uiprasertkul et al., 2005) . while many laboratory-confirmed h5n1 infections were associated with severe, often fatal disease, milder cases have also been reported, especially during the outbreak in hong kong (chan, 2002; yuen et al., 1998 ). an increasing number of milder cases also seemed to occur in viet nam, as the outbreak progressed in 2005 (who, 2005) . while increased clinical awareness and surveillance may account for such observations, progressive adaptation of the virus to humans is the dreaded alternative explanation. the occurrence of mildly symptomatic and asymptomatic infections have also been suggested during the outbreak in hong kong by seroepidemiological studies in household members of h5n1-infected patients and health care workers. in these studies, 8 of 217 exposed and 2 of 309 non-exposed healthcare workers were seropositive for h5n1-specific antibodies (bridges et al., 2002) . seroconversion was documented in two exposed nurses, one of whom reported a respiratory illness 2 days after exposure to an h5n1-infected patient. more importantly than showing the occurrence of asymptomatic infections, these data indicated that nosocomial person-to-person transmission had occurred, albeit limited to a few cases. an additional case of possible human-to-human transmission during the hong kong outbreak was suggested by h5n1-seropositivity in a household contact of a patient, who had no history of poultry exposure (katz et al., 1999) . seroepidemiological studies in health care workers involved in the care of h5n1infected patients in thailand and viet nam in 2004 have not shown evidence of person-to-person transmission, despite the absence of adequate infection control measures in the vietnamese cohort at the time of study (apisarnthanarak et al., 2005; liem and lim, 2005; schultsz et al., 2005) . during the outbreak in thailand in 2004, extensive epidemiological investigations have suggested person-to-person transmission from a child, who died of presumed h5n1 infection, to her mother who had no history of exposure to poultry and had provided prolonged unprotected nursing care to her daughter . an aunt of the child may have been infected by the same route since her last exposure to poultry before infection had been 17 days, considerably longer than the estimated incubation period of 2-10 days. there have been several similar family clusters of h5n1 cases in viet nam, which have all ignited concerns about the possibility of human-to-human transmission, but most of which could be explained by common exposure to poultry. while there has been no evidence of efficient transmission of influenza h5n1 virus between humans to date, caution and detailed investigations remain warranted in case of any cluster of infections, especially in view of the relatively rapid evolution h5n1 viruses have exhibited in recent years. in 1996, an h5n1 virus was isolated from geese during an outbreak in guangdong province in china (influenza a/goose/guangdong/1/96 (a/g/gd/96)) (xu et al., 1999) . this virus proved to be the donor of the ha gene of the reassortant h5n1 viruses causing the outbreak among poultry and humans in hong kong in 1997. the internal genes of the hong kong h5n1 viruses were closely related to those of an h9n2 virus isolated from quail (guan et al., 1999) . the origin of the na gene remains unclear, but was notable for a 19-amino acid deletion in the stalk region (subbarao et al., 1998) . such deletions may be associated with adaptation of influenza viruses to land-based poultry (matrosovich et al., 1999) . the ha gene contained multibasic sequences at the cleavage site, in accordance with its classification as a highly pathogenic strain (claas et al., 1998; matrosovich et al., 1999) . after the eradication of the 1997 hong kong strain, the goose precursor viruses continued to circulate in geese in southeastern china (cauthen et al., 2000; webster et al., 2002) . through reassortment between this virus and other avian viruses, multiple antigenically similar genotypes, that were highly pathogenic in chickens but not in ducks, emerged and again were eradicated in hong kong in 2001 and 2002 . then, in late 2002, h5n1 strains isolated from wild migratory birds and resident waterfowl in two hong kong parks showed marked antigenic drift and exhibited high pathogenicity in ducks sturm-ramirez et al., 2004) . the latter property is rarely found in nature, and had not been observed in strains isolated during previous years. an antigenically and molecularly similar virus caused the two confirmed human infections in early 2003 in a family from hong kong peiris et al., 2004) . h5n1 influenza viruses isolated from healthy ducks in southern china between 1999 and 2002 were all antigenically similar to the precursor influenza a/g/gd/96 virus (chen et al., 2004) . it is thought that these ducks played a central role in the generation of the virus responsible for the outbreaks in southeast asia since 2003. detailed genetic analyses of h5n1 strains isolated during the period 2000-2004 from poultry and humans in china, hong kong, indonesia, thailand, and viet nam, demonstrated that a series of genetic reassortment events, all traceable to the a/g/gd/96-precursor virus, ultimately gave rise to a dominant h5n1 genotype (genotype z) in chickens and ducks . this genotype is implicated in the human cases in hong kong in 2003 and the outbreaks among poultry and humans since 2004. the evolution of h5n1 viruses in recent years has been associated with increasing virulence and an expanding host range, which beside terrestrial poultry and wild birds, also includes mammals. while all h5n1 viruses isolated from ducks in china between 1999 and 2002 were highly pathogenic in chickens, an increasing level of pathogenicity was observed in mice with the progression of time: virus isolated in 1999 and 2000 were less pathogenic than those isolated in 2001 and 2002 (chen et al., 2004) . it has been suggested that the increasing ability to replicate in mammals has resulted from transmission between ducks and pigs. the expanding host range is also illustrated by successful experimental infection of domestic cats, and natural infections of tigers and leopards with recent h5n1 strains (keawcharoen et al., 2004; kuiken et al., 2004) . in summary, continued evolution of h5n1 viruses since 1997, involving multiple genetic reassortment events between a/g/gd/96-like viruses and other avian viruses and perhaps transmission between birds and pigs or other mammalian hosts, have resulted in a highly virulent genotype with an expanded host range which is now causing widespread outbreaks among poultry and humans in southeast asia. while transmission between birds and humans at present still seems inefficient, as does transmission between humans, this may change when the virus is allowed to continue its evolution through adaptation and reassortment. although virus isolation remains the gold standard of diagnosis and indispensable for virus characterization, rapid laboratory confirmation of suspected human influenza in routine diagnostic laboratories is usually performed by immunochromatographic or immunofluorescent detection of influenza virus antigens, or reverse transcriptase (rt) pcr detection of viral nucleic acids in respiratory specimens. in addition, serological evidence of human influenza a virus infection can be obtained by commercially available elisa kits which detect antibodies to conserved viral antigens, such as the nucleoprotein. in the absence of cocirculating avian influenza strains in the human population, further subtyping of influenza viruses or detection of subtype-specific antibodies are usually not done by routine diagnostic laboratories, but are restricted to reference laboratories involved in epidemiological analyses and planning of vaccine strains. however, in case of an outbreak of avian influenza, efforts to further subtype the virus, e.g. by subtype-specific rt pcr methods, should be made by routine laboratories since immediate knowledge about the infecting influenza subtype is essential for infection control and timely epidemiological investigations. dependence on reference laboratories, which in the case of many southeast asian countries affected by avian influenza outbreaks, are situated abroad, potentially results in unacceptable delays and hampers timely recognition of outbreaks and institution of adequate control measures (hien et al., 2004) . however, the reality is that diagnostic facilities in many affected countries are scarce and often not sufficiently equipped for virological diagnostics, let alone subtyping of influenza viruses. global efforts to improve diagnostic capacity in resource-poor countries may prove an important step towards the prevention and control of pandemic influenza (hien et al., 2004) . similar to human influenza viruses, avian viruses can be isolated in embryonated eggs or in cell culture, using permissive cells such as madin darby canine kidney (mdck) cells or rhesus monkey kidney (llc-mk2) cells. unlike human strains and avirulent avian strains and in accordance with their promiscuity for cellular proteases, highly pathogenic avian viruses do not require the addition of exogenous trypsine for efficient replication in cell culture. for safety purposes, the isolation of highly pathogenic avian influenza virus requires biosafety level 3 laboratory facilities or higher. cytopathic effects in cell culture are non-specific. initial identification of influenza a virus can be performed by immunofluorescent staining with monoclonal antibodies against the nucleoprotein. further ha and na subtyping is performed by subtype-specific rt pcrs of culture supernatant or hemag-glutination inhibition and neuraminidase inhibition assays using a panel of reference antisera against various subtypes. in human infections, avian influenza viruses have mostly been isolated from conjunctival swabs and respiratory specimens such as throat or nasal secretions or washings tran et al., 2004; yuen et al., 1998) . in one reported case of h5n1 infection, virus was also isolated from serum, cerebrospinal fluid, and a rectal swab (de jong et al., 2005) . detection of influenza a viral antigens in clinical specimens by direct immunofluorescence or by rapid immunochromatographic assays are widely used for diagnosis of human influenza because of their ability for rapid diagnosis. however, in patients with avian influenza, the usefulness of these assays seems limited due to low sensitivity (peiris et al., 2004; yuen et al., 1998) . in addition, some rapid antigen detection kits do not distinguish between influenza types a and b, and none of the currently available immunofluoresent and immunochromatographic assays distinguish between influenza a subtypes. however, developments of h5n1-specific rapid antigen detection tests are ongoing . rt pcr methods allow for sensitive and specific detection of viral nucleic acids and have shown to increase the diagnostic sensitivity for many viral pathogens when compared to culture or antigen detection methods. during the h5n1 outbreaks in hong kong and southeast asia, rt pcr methods for specific detection of h5n1 viral nucleic acids have proven valuable and seem the diagnostic methods of choice in case of an outbreak of avian influenza (chotpitayasunondh et al., 2005; tran et al., 2004; yuen et al., 1998) . especially when using real-time pcr technology, a reliable subtype-specific diagnostic result can be generated within a few hours after specimen collection. a disadvantage of rt pcr methods is its proneness for contamination and the consequent risk of false-positive results, which should be be minimized by proper precautions, including physical separation of laboratories for pcr preparation and amplification, and the use of the uracil-n-glycosylase system to prevent contamination by carryover of amplimers. in addition, the inclusion of an internal control in rt pcr assays is highly desirable to monitor for false-negative results due to inefficient nucleic acid extraction, cdna synthesis, or amplification. during outbreaks of avian influenza, the detection of subtype-specific antibodies is particularly important for epidemiological investigations. hemagglutination inhibition (hi) assays are the gold standard for detection of antibodies against human influenza viruses. however, their usefulness for detection of antibodies against avian viruses in mammalian species, including humans, seems limited (beare and webster, 1991; hinshaw et al., 1981; kida et al., 1994) . several studies have shown a failure to detect hi antibodies against avian viruses in mammals, even in cases where infection was confirmed by virus isolation. possible reasons for this failure include poor immunogenicity of some avian viruses and lack of sensitivity to detect low titered or less avid antibodies induced by avian viruses (hinshaw et al., 1981; kida et al., 1994; lu et al., 1982; rowe et al., 1999) . it has been demonstrated that hi testing with subunit ha, but not with intact virus, could detect antibodies against an avian h2n2 virus (lu et al., 1982) . however, neutralizing antibodies against this virus could readily be detected with intact virus. a direct comparison of hi testing with a microneutralization assay in h5n1-infected persons from the 1997 hong kong outbreak indeed showed the latter to be more sensitive . while an indirect elisa assay using recombinant ha from h5n1/97 showed at least equal sensitivity as the microneutralization assay, the specificity in adult sera was inferior, possibly due to the presence of crossreactive epitopes common to all has . based on these observations, neutralization assays are the methods of choice for detection of antibodies against avian viruses in humans. using these assays, it has been shown that the kinetics of the antibody response against h5n1 virus in patients infected during the hong kong outbreak are similar to the primary response to human influenza viruses (katz et al., 1999) . neutralizing antibodies were generally detected 14 or more days after the onset of symptoms and titers equal to or higher that 1:640 were observed 20 or more days after onset. currently, two classes of drugs are available with antiviral activity against influenza viruses: inhibitors of the ion channel activity of the m2 membrane protein, amantadine and rimantadine, and inhibitors of the neuraminidase, oseltamivir, and zanamivir. the therapeutic efficacy of amantadine in human influenza is unclear due to a paucity of reliable clinical studies, but reductions of fever or illness by 1 day have been observed in adults and children (nicholson et al., 2003) . major disadvantages of amantadine include neurotoxicity and a rapid development of drug resistance during treatment. resistance is conferred by single nucleotide changes resulting in amino acid substitutions at positions 26, 27, 30, 31, or 34 of the m2 protein. rimantadine causes less neurological side effects but is not available in most parts of the world. although several h5n1-infected patients have been treated with amantadine during the 1997 h5n1 outbreak in hong kong, the numbers were too small to draw any meaningful conclusions concerning its activity against this virus (yuen et al., 1998) . in vitro sensitivity testing of virus isolated from the first patient during this outbreak showed normal susceptibility to amantadine (subbarao et al., 1998) . strikingly, genotype z h5n1 viruses isolated from poultry and humans in thailand and viet nam in 2004 invariably showed an amantadine-resistance conferring amino acid substitution at position 31 of the m2 protein, indicating that amantadine treatment is not an option during the ongoing outbtreak in southeast asia puthavathana et al., 2005) . both oseltamivir and zanamivir have proven efficacy in the treatment of human influenza when started early during the course of illness, and are particularly effective as seasonal or postexposure prohylaxis (nicholson et al., 2003) . zanamivir has poor oral availability and is therefore administered by inhalation, which has limited its use in the elderly and may induce bronchospasm. oseltamivir can be given orally. the development of drug resistance during treatment has been reported for both drugs and is associated with mutations in the active site of neuraminidase or in the hemaglutinin. the latter mutations decrease the affinity of ha for the cellular receptor, thereby obviating the need for neuraminidase to escape the cells. data on the efficacy of neuraminidase inhibitors in avian influenza virus are scarce. the h5n1 strains implicated in the 1997 hong kong outbreak were susceptible in vitro to oseltamivir and zanamivir (govorkova et al., 2001; leneva et al., 2000) . oral oseltamivir and topical zanamivir also showed therapeutic and protective activities against hong kong h5n1 isolates in murine animal models (gubareva et al., 1998; leneva et al., 2001) . recent murine studies suggest that, perhaps due to higher virulence, higher doses of oseltamivir and longer durations of treatment are necessary to achieve antiviral effects in mice against h5n1 strains causing the southeast asian outbreak since 2004, when compared to the 1997 hong kong h5n1 strain (yen et al., 2005) . oseltamivir treatment has been given to several h5n1infected patients, but no conclusions can be made concerning its efficacy. however, the timing of antiviral treatment may not have been optimal in many cases of avian influenza so far. beneficial effects of antiviral treatment in human influenza are optimal when started within 48 h after onset of the illness. during the h5n1 outbreak in viet nam in 2004, h5n1infected patients were admitted 5 days or later after onset of symptoms (tran et al., 2004) . earlier recognition of avian influenza in humans may improve the efficacy of antiviral treatment. while several h5n1-infected patients have received steroids in addition to oseltamivir, the potential benefits of this need formal evaluation in clinical studies (tran et al., 2004) . considering the observed cytokine dysregulation in h5n1-infected animals and humans, a beneficial effect of immunomodulating agents could be hypothesized and perhaps requires further study. finally, neutralizing monoclonal antibodies have been shown effective in treating established influenza a virus infection in mice with severe combined immunodeficiency (palladino et al., 1995) . although mice are not men, this strategy deserves attention in the treatment of a severe illness such as influenza h5n1. birds infected with avian influenza excrete large amounts of virus in feces and other secretions, which contaminate the direct environment, such as dust, soil, water, cages, tools, and other fomites. avian influenza virus may remain infectious in soil, water, or contaminated equipment for weeks to months, depending on the temperature and humidity (i.e. longer in colder climates). illness in birds caused by highly pathogenic avian influenza viruses results in systemic replication and the presence of infectious virus in their eggs and many tissues and organs. transmission of avian influenza viruses between birds occurs directly or indirectly through contact with fecally contaminated aerosols, water, feed, and other materials. birdto-human transmission likely occurs via the same route, i.e. direct contact with birds or contaminated fomites. most, but not all human infections with avian influenza viruses involved handling of affected poultry or direct exposure to live poultry in the week before onset of the illness (koopmans et al., 2004; mounts et al., 1999; tran et al., 2004) . case-control studies during the 1997 h5n1 outbreak in hong kong identified visiting a stall or market selling live poultry during the week before the illness as a risk factor, whereas eating or preparing poultry products were not (mounts et al., 1999) . in cases in which no apparent direct exposure to poultry could be identified, contact with contaminated environment, such as water, has been suggested (de jong et al., 2005) . of note, it has been shown that ducks infected by the currently circulating h5n1 strain in southeast asia remain healthy but excrete large amounts of virus for prolonged periods of time (hulse-post et al., 2005) . since water in ponds and canals in which large flocks of ducks reside, is widely used for bathing and drinking in rural areas of many southeast asian countries, it may not be unlikely that such water represents a source of transmission when contaminated by infected ducks. in fact, contact with contaminated water is regarded as the most important mode of transmission between aquatic birds. a limited number of possible human-to-human transmissions of influenza h5n1 have been reported, which involved prolonged, close and unprotected contact with infected patients (katz et al., 1999; koopmans et al., 2004; ungchusak et al., 2005) . similar to human influenza, droplet and contact transmission are probably the most effective means of transmission of avian influenza virus between humans, should the virus acquire the ability for efficient spread, but airborne transmission remains a possibility. the occurrence of diarrhea in h5n1-infected patients, which may contain infectious virus, represents a potential non-respiratory route of transmission, which needs to be considered in infection control practices (apisarnthanarak et al., 2004; de jong et al., 2005; tran et al., 2004) . data concerning excretion patterns and periods of potential infectivity are lacking for human infections with avian influenza viruses. based on exposure histories, the incubation time for human h5n1-infections has been estimated 2-10 days, but it is not known whether excretion of virus occurs during this time (tran et al., 2004; yuen et al., 1998) . based on the current (lack of) knowledge, infection control measures during contact with potentially infected birds or environment, or with patients with suspected or confirmed infection should prevent contact, droplet, and airborne transmission. these measures include mask (preferably high efficiency masks, with surgical masks as a second alternative), gown, face shield, or goggles and gloves. the efficacy of neuraminidase inhibitors as seasonal or postexposure prohylaxis against human influenza is high (nicholson et al., 2003) . offering prophylactic treatment to potentially exposed people in the setting of a poultry outbreak of avian influenza, as has been done during h7-outbreaks in the netherlands and canada (koopmans et al., 2004; tweed et al., 2004) , is rational but hardly feasible during the ongoing outbreak in southeast asia for logistical and financial reasons. postexposure prophylaxis to unprotected healthcare workers and close contacts of infected patients is advisable. the potential use of specific monoclonal antibodies for prophylaxis warrants further investigation. eliminating the source of infection, i.e. infected birds, remains the most effective infection control measure. culling of all infected poultry have proven succesful during avian influenza outbreaks in hong kong, the netherlands and canada (chan, 2002; koopmans et al., 2004; tweed et al., 2004) . however, considering the geographic extensiveness of the outbreak in southeast asia, the different farming practices, and the reported h5n1 infections in migratory birds (chen et al., 2005) , it seems very unlikely that culling of poultry alone will contain the outbreak in that region. the bulk of human influenza vaccines are produced from inactivated viruses grown in embryonated eggs. vaccine production against highly pathogenic avian influenza viruses is complicated because of the requirement for high biosafety containment facilities, and the difficulty, in some cases, to obtain high virus yields in embryonated eggs because of the virus' pathogenicity (stephenson et al., 2004; wood and robertson, 2004) . several other approaches have been used in an attempt to overcome these obstacles, including the use of reverse genetics techniques, generation of recombinant hemagglutinin, dna vaccination and the use of related apathogenic h5 viruses with and without different adjuvants (nicholson et al., 2003; stephenson et al., 2004; webby et al., 2004; wood and robertson, 2004) . experimental h5n1 vaccines in which important virulence determinants were altered using plasmid-based reverse genetics, have shown protective efficacy to homologous and heterologous h5 strains in animal models and may prove an attractive approach (li et al., 1999; lipatov et al., 2005; takada et al., 1999) . studies in humans using an h5n3 vaccine developed from a 1997 apathogenic avian virus showed high rates of seroconversions to the vaccine strain and heterologous h5n1 strains after three doses, but only when the vaccine was given with the adjuvans mf59 . in animal models, baculovirusderived recombinant h5 vaccines were immunogenic and protective, but results in humans were disappointing even when using high doses (crawford et al., 1999; treanor et al., 2001) . h5 dna vaccines protected mice from infection by homologous, but not by heterologous h5n1 viruses (epstein et al., 2002; kodihalli et al., 1999) . the increasing frequency of outbreaks with highly pathogenic avian influenza viruses among poultry and direct transmission of these viruses to humans, culminating in the ongoing extensive h5n1 outbreak in southeast asia, has ignited grave concerns about an imminent influenza pandemic. indeed, two of three prerequisites for a human pandemic have been met in the southeast asian h5n1 outbreak: the emergence of an antigenically novel strain to which the population has no immunity, and the transmission of this strain to humans in whom it can cause severe disease. to date, there is fortunately no evidence of efficient spread of h5n1 virus between humans, but continued circulation of this strain, which now has reached levels of endemicity among poultry in several southeast asian countries, increases the opportunity to adapt to humans through mutation or genetic reassortment in humans or intermediate mammalian hosts. as suggested by the "spanish flu" pandemic of 1918, extremely high transmissability is no prerequisite for a severe pandemic killing tens of millions of people, and as shown by the severe acute respiratory syndrome (sars) virus epidemic in 2003, viruses can rapidly spread across the globe in the current age of intense global travel. as a consequence of all this, pandemic preparedness has become an important issue worldwide. pandemic plans, which include stock-piling of antivirals and candidate vaccines, are being developed by an increasing number of countries worldwide, and alternative methods for rapid vaccine production and potential methods enabling dose reduction of vaccines are increasingly propagated (schwartz and gellin, 2005; stephenson et al., 2004; webby et al., 2004; webby and webster, 2003; wood and robertson, 2004) . notwithstanding the importance of these efforts to prepare for a possible h5n1 pandemic, more structural and longer term global efforts are needed to allow for early recognition of novel influenza viruses or other emerging pathogens infecting humans in the future. in 2002, a who global agenda for influenza surveillance and control has been adopted, of which the main objectives are to strengthen surveillance, improve knowledge of the disease burden, increase vaccine use, and accelerate pandemic preparedness (stohr, 2003) . it is essential that these objectives are increasingly focused on the southeast asian region, which has been the source of previous pandemics and is the epicentre of the current pandemic threat. many southeast asian countries currently lack the expertise, financial means, and infrastructure for human and animal diagnostics and surveillance. global investments to improve public health care infrastructures and laboratory facilities, and to transfer clinical, epidemiological, and technical knowledge to these countries are much needed (hien et al., 2004) . the window of opportunity in the era of global travel is narrow. local capacity, and less dependence on foreign laboratories and expertise, will allow for earlier recognition and quicker responses to epidemics. in addition, local availability of clinical, scientific, and laboratory capacity facilitates and expedites clinical, virological, and epidemiological analyses needed to optimize outbreak control, infection control, and clinical managment. it also guarantees the timely availability of virus strains for monitoring virus evolution and planning of vaccines by reference laboratories. such global investments to enhance local infrastructure and expertise will increase the chances of success of containing an influenza pandemic at the source by antiviral prophylaxis and other preventive measures suggested by recent mathematical modelling studies (ferguson et al., 2005; longini et al., 2005) . seroprevalence of anti-h5 antibody among thai health care workers after exposure to avian influenza (h5n1) in a tertiary care center 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human influenza a h5n1 virus related to a highly pathogenic avian influenza virus receptor specificity in human, avian, and equine h2 and h3 influenza virus isolates influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity baculovirus-derived hemagglutinin vaccines protect against lethal influenza infections by avian h5 and h7 subtypes fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma dna vaccine expressing conserved influenza virus proteins protective against h5n1 challenge infection in mice strategies for containing an emerging influenza pandemic in southeast asia characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human h1 and h3 influenza a and influenza b viruses share a common high binding affinity for 6 -sialyl(n-acetyllactosamine) the structure and receptor binding properties of the 1918 influenza hemagglutinin cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses emergence of multiple genotypes of h5n1 avian influenza viruses in hong kong sar h5n1 influenza: a protean pandemic threat molecular characterization of h9n2 influenza viruses: were they the donors of the "internal" genes of h5n1 viruses in hong kong? characterization of influenza a/hongkong/156/97 (h5n1) virus in a mouse model and protective effect of zanamivir on h5n1 infection in mice avian influenza-a challenge to global health care structures replication of avian influenza a viruses in mammals role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia molecular basis for the generation in pigs of influenza a viruses with pandemic potential antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics avian influenza h5n1 in tigers and leopards potential for transmission of avian influenza viruses to pigs enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus dna vaccine encoding hemagglutinin provides protective immunity against h5n1 influenza virus infection in mice transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands avian h5n1 influenza in cats characterization of highly pathogenic h5n1 avian influenza a viruses isolated from south korea efficacy of zanamivir against avian influenza a viruses that possess genes encoding h5n1 internal proteins and are pathogenic in mammals the neuraminidase inhibitor gs4104 (oseltamivir phosphate) is efficacious against a/hong kong/156/97 (h5n1) and a/hong kong/1074/99 (h9n2) influenza viruses genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia recombinant influenza a virus vaccines for the pathogenic human a/hong kong/97 (h5n1) viruses lack of h5n1 avian influenza transmission to hospital employees neurovirulence in mice of h5n1 influenza virus genotypes isolated from hong kong poultry in 2001 efficacy of h5 influenza vaccines produced by reverse genetics in a lethal mouse model containing pandemic influenza at the source failure to detect hemagglutinationinhibiting antibodies with intact avian influenza virions the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties avian influenza a viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the ha receptor-binding site human and avian influenza viruses target different cell types in cultures of human airway epithelium transmissibility of 1918 pandemic influenza encephalitis and encephalopathy associated with an influenza epidemic in japan case-control study of risk factors for avian influenza a (h5n1) disease influenza a pandemics of the 20th century with special reference to 1918: virology, pathology and epidemiology virus-neutralizing antibodies of immunoglobulin g (igg) but not of igm or iga isotypes can cure influenza virus pneumonia in scid mice re-emergence of fatal human influenza a subtype h5n1 disease molecular characterization of the complete genome of human influenza h5n1 virus isolates from thailand origin and evolution of the 1918 "spanish" influenza virus hemagglutinin gene evidence of an absence: the genetic origins of the 1918 pandemic influenza virus receptor binding properties of human and animal h1 influenza virus isolates receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin single amino acid substitutions in influenza haemagglutinin change receptor binding specificity detection of antibody to avian influenza a (h5n1) virus in human serum by using a combination of serologic assays on the origin of the human influenza virus subtypes h2n2 and h3n2 avian influenza h5n1 and healthcare workers vaccination strategies for an influenza pandemic cross-reactivity to highly pathogenic avian influenza h5n1 viruses after vaccination with nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a potential priming strategy confronting the avian influenza threat: vaccine development for a potential pandemic structure of the uncleaved human h1 hemagglutinin from the extinct 1918 influenza virus the global agenda on influenza surveillance and control reemerging h5n1 influenza viruses in hong kong in 2002 are highly pathogenic to ducks characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness influenza-associated encephalopathy in japan avirulent avian influenza virus as a vaccine strain against a potential human pandemic neurotropism of the 1997 hong kong h5n1 influenza virus in mice initial genetic characterization of the 1918 "spanish" influenza virus pathology of fatal human infection associated with avian influenza a h5n1 virus avian influenza a (h5n1) in 10 patients in vietnam safety and immunogenicity of a recombinant hemagglutinin vaccine for h5 influenza in humans human illness from avian influenza h7n3, british columbia influenza h5n1 replication sites in humans probable person-to-person transmission of avian influenza a (h5n1) responsiveness to a pandemic alert: use of reverse genetics for rapid development of influenza vaccines are we ready for pandemic influenza? evolution and ecology of influenza a viruses characterization of h5n1 influenza viruses that continue to circulate in geese in southeastern china cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza latex agglutination test for monitoring antibodies to avian influenza virus subtype h5n1 genetic characterization of the pathogenic influenza a/goose/guangdong/1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 (h5n1) influenza virus in mice clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus we thank the staff of the hospital for tropical diseases ho chi minh city, the health service of ho chi minh city and the ministry of health, viet nam. menno de jong is funded by the wellcome trust, uk. key: cord-335647-dhcxj7cj authors: vanderlinden, evelien; naesens, lieve title: emerging antiviral strategies to interfere with influenza virus entry date: 2013-06-25 journal: med res rev doi: 10.1002/med.21289 sha: doc_id: 335647 cord_uid: dhcxj7cj influenza a and b viruses are highly contagious respiratory pathogens with a considerable medical and socioeconomical burden and known pandemic potential. current influenza vaccines require annual updating and provide only partial protection in some risk groups. due to the global spread of viruses with resistance to the m2 proton channel inhibitor amantadine or the neuraminidase inhibitor oseltamivir, novel antiviral agents with an original mode of action are urgently needed. we here focus on emerging options to interfere with the influenza virus entry process, which consists of the following steps: attachment of the viral hemagglutinin to the sialylated host cell receptors, endocytosis, m2‐mediated uncoating, low ph‐induced membrane fusion, and, finally, import of the viral ribonucleoprotein into the nucleus. we review the current functional and structural insights in the viral and cellular components of this entry process, and the diverse antiviral strategies that are being explored. this encompasses small molecule inhibitors as well as macromolecules such as therapeutic antibodies. there is optimism that at least some of these innovative concepts to block influenza virus entry will proceed from the proof of concept to a more advanced stage. special attention is therefore given to the challenging issues of influenza virus (sub)type‐dependent activity or potential drug resistance. human influenza a and b viruses cause significant morbidity and mortality, particularly in infants and elderly people, or those suffering from preexisting pathology or immunodeficiency. 1, 2 the united states centers for disease control and prevention estimated that, from 1976 to 2000, seasonal influenza epidemics were responsible for >200,000 annual hospitalizations and an annual average of >30,000 influenza-associated deaths in the usa. 3 approximately 90% of the influenza-associated deaths occur among adults aged ≥65 years. 4 to evade the immune response, the circulating influenza h3n2, h1n1, and b viruses continuously change their antigens, and this explains why current influenza vaccines require figure 1 . overview of the influenza virus entry and replication process. in the inset on the right, the different virion components are specified. (a) after binding of the viral ha to sialylated glycans on the host cell surface, the virus is internalized by endocytosis. (b) acidification of the endosome leads to activation of the m2 proton channel and virion acidification, resulting in virus uncoating (i.e., dissociation of the vrnps from the m1 capsid protein). the low ph inside the endosome also triggers a conformational change in the ha, leading to fusion of the viral and endosomal membranes. after vrnp release in the cytoplasm and dissociation of residual m1, nuclear localization signals in np direct the transport of the vrnps into the nucleus. (c) in the nucleus, the viral polymerase starts mrna synthesis by cleaving off 5 -capped rna fragments from host cell pre-mrnas. then, viral mrna transcription is initiated from the 3 end of the cleaved rna cap. (d) viral mrnas are transported to the cytoplasm for translation into viral proteins. ha, m2, and na are processed in the endoplasmic reticulum and the golgi apparatus, and subsequently transported to the cell membrane. (e) besides viral mrna synthesis, the viral polymerase performs the unprimed replication of vrnas. the vrnas are first transcribed into positivestranded crnas, which then function as the template for the synthesis of new vrnas. during their synthesis, vrnas and crnas are encapsidated by nps. export of the newly formed vrnps into the cytoplasm is mediated by an m1-ns2 complex that is bound to the vrnps. (f) as they reach the cell membrane, the vrnps associate with viral glycoproteins at the plasma membrane from which new virions bud off. finally, the na cleaves the sialic acid termini on viral and cell membrane glycoproteins, thereby releasing the progeny virions from the host cell. annual updating. these vaccines provide inadequate protection in some target populations (particularly the elderly). 5 besides, there is concern that the widely spread and highly pathogenic avian h5n1 influenza virus may acquire human transmissibility and become a potentially disastrous pandemic virus. the human case-fatality rate of this avian h5n1 is reported to be 59%, 6 although some investigators have raised the possibility that subclinical cases of h5n1 infections in humans may remain unnoticed. 7 for comparison, the case-fatality rate of the 1918 influenza virus was estimated >2.5%. 8 as shown in figure 1 , the influenza virus replication cycle contains several steps amenable to antiviral intervention. this review focuses on the viral entry pathway, which, given the acute onset of influenza virus infection and the inflammation associated with it, is a particularly attractive process to interfere with. we describe the current insights into the structure and functions of the viral and cellular components involved in this entry process, and the antiviral strategies that are being explored (an overview of the described compounds is given in table i ). effective antiviral drugs to prevent or treat influenza infections should at all times be available. today, two classes of anti-influenza virus drugs exist: the m2 proton channel blockers (i.e., the adamantane compounds, amantadine and rimantadine), and the neuraminidase inhibitors (nais) (oseltamivir and zanamivir). 13 the first two compounds have limited utility, since they are associated with neurological side effects, have no activity against influenza b virus, and the vast majority of circulating strains are adamantane-resistant. 13 a detailed description of their mode of action and resistance mechanisms will be given below. the obviously superior class of anti-influenza virus drugs are the nais oseltamivir and zanamivir that are active against all influenza a and b viruses. these structural analogues of sialic acid bind to the catalytic pocket of the viral na and inhibit its function in releasing the newly produced virus from the host cells. 14, 15 there is a critical difference in the na binding mode of oseltamivir compared to that of zanamivir, which explains their significantly different resistance profile. due to its larger hydrophobic side chain, oseltamivir requires rotation of the noncatalytic glu276 residue within na to create a binding space for oseltamivir. 16 by contrast, the smaller size of zanamivir enables direct binding of this compound to na. in a mutant n1 na containing a his to tyr substitution at position 274, this rotation can no longer occur, rendering the na resistant to oseltamivir binding. during the 2008-2009 season, oseltamivir-resistant h1n1 viruses were isolated all over the globe, even from untreated patients. 17, 18 in a japanese study in 2004, nine out of 50 children treated with oseltamivir carried oseltamivir-resistant h3n2 viruses. 19 fortunately, oseltamivir-resistant viruses are still sensitive to zanamivir, for which resistance has only scarcely been reported. 20, 21 on the other hand, the patient-unfriendly administration route for zanamivir (i.e., by powder inhalation device) explains why oseltamivir (which is given by oral capsules) is generally preferred in the clinical setting. inhalation of zanamivir is a priori excluded in patients suffering from severe influenza symptoms with acute respiratory distress, such as patients infected with the highly pathogenic avian h5n1 virus, or severe cases of the 2009 pandemic h1n1 virus. to address this issue, an intravenous formulation of zanamivir is under consideration. 22, 23 besides, new nais are being developed. peramivir, which has to be administered intravenously, has been licensed in japan and south korea, while, in the united states, its use was temporarily allowed during the 2009 h1n1 pandemic. 24 unfortunately, the widespread oseltamivir-resistant h1n1 his274tyr mutants show intermediate cross-resistance to peramivir. 25 another nai, laninamivir (cs-8958), was approved in japan in 2010 and is currently in phase iii trials in the united states. 26, 27 this promising compound requires only one single intranasal administration (based on its long half-life), and has a similar na binding mode and favorable resistance profile as zanamivir. 28 finally, novel nais with a sialic acidrelated or unrelated structure have been developed by rational design, but are still in the early experimental stage. [29] [30] [31] to face the emerging resistance to nais (in particular, oseltamivir), entirely novel antiinfluenza virus drugs are urgently needed. the two products that are most advanced in clinical development are the nucleobase analogue t-705 (favipiravir) and the receptor destroying protein das181. for t-705, phase iii trials in the united states are pending. its active ribosetriphosphate metabolite is recognized by the influenza virus polymerase, causing competitive inhibition of viral rna synthesis and/or lethal viral mutagenesis. 32 t-705 has broad anti-rna virus activity beyond influenza virus and is presumed (based on cell culture data) to have a high barrier for viral resistance. 33 the second agent, das181, is currently in phase ii trials. this recombinant protein is a sialidase that cleaves the influenza virus receptors in the airway epithelia. more details on das181 are provided in section 3. within the influenza virus particle, the single-stranded, negative-oriented rna genome is divided over eight viral ribonucleoprotein (vrnp) segments, which are protected by the capsid shell formed by the m1 protein, further surrounded by the viral envelope. two viral spike proteins protrude from the virion: the hemagglutinin (ha) and na, which have a leading role in viral entry and release, respectively. the ha and na glycoproteins are the main antigens against which the host immune response is raised. in the case of influenza a virus, 17 ha and 10 na subtypes are known, which are all present in aquatic birds, the natural reservoir for influenza a viruses. 34 the only exception is h17, which was isolated only recently from bats. 35, 36 the emergence of a new pandemic virus is explained by the reassortment of genome segments, which occasionally occurs upon dual infection of an animal species (such as a pig) that carries the avian-as well as the human-type influenza virus receptors. 37 the influenza virus ha ( fig. 2a) is a homotrimeric type 1 membrane glycoprotein. its membrane-distal globular head domain contains the receptor binding site (rbs), whereas the ha stem structure (which contains the fusion peptide) is responsible for intraendosomal membrane fusion. 34 in influenza virus-infected cells, ha is first synthesized as its precursor protein ha0, which assembles into a noncovalently linked homotrimer 38 and is cleaved into two polypeptides (ha1 and ha2 containing, in the case of h3, 328 and 221 amino acids, respectively), which remain covalently attached by a disulfide bond. 39 for most has, ha0 cleavage occurs at a single arginine residue and is performed by a membrane-bound or secreted serine protease that is restricted to bronchiolar epithelium, such as tryptase clara, the human airway trypsin-like protease or tmprss2. 40, 41 the has from highly pathogenic avian viruses contain a series of basic residues at their cleavage site, 42 allowing recognition by furin-like intracellular proteases that are widely distributed in avian tissues, thus explaining their systemic spread and high virulence. 43 inhibition of the cellular proteases performing ha0 cleavage is an original antiviral strategy, and peptidomimetic furin inhibitors have proven to inhibit the replication of an avian influenza virus in cell culture. 44 after ha0 cleavage, minor rearrangements lead to insertion of the fusion peptide (located at the n-terminus of ha2) into a negatively charged cavity, thus priming the ha for ph-dependent fusion. 40 posttranslational modifications of ha comprise the addition of acyl chains to the short cytoplasmic tail, 45 and n-glycosylation at several asparagine residues in the ectodomain. 39 besides masking the antigenic epitopes by sterically hindering antibody recognition, 46, 47 the n-linked glycans also function in the correct folding of ha in the endoplasmic reticulum, 48, 49 modulation of receptor binding, 50 controlling ha0 cleavage, 51 and maintaining the ha in its metastable conformation required for fusion activity. 52 the n-glycans that are most conserved among various influenza has are located at the n-terminus of ha0 (or after cleavage, ha1) 48 and in the ha stem region. 53 the 17 influenza ha subtypes are classified into two phylogenetic groups (fig. 2b) . the h1 and h5 has belong to the same clade within group 1, whereas h3 ha belongs to group 2. 35, 54, 55 although this phylogenetic classification was primarily based on ha protein sequence, comparison of available ha crystal structures indicates that the regions involved in membrane fusion show striking similarities on a group-specific basis. 54 in the first step of the infection cycle, the ha attaches, via the rbs in its globular head, to sialylated glycoproteins or glycolipids on the host epithelial cells. 56 this ha-receptor interaction is highly specific for sialylglycoconjugates and plays an essential part in the species recognition of avian versus human influenza viruses. 57 the has from human-adapted viruses, including the pandemic viruses of the h1n1, h2n2, or h3n2 subtype, preferentially bind to cell-surface glycans terminating in α2-6-linked sialyl-galactosyl residues [neu5ac(α2-6)gal], whereas avian influenza a viruses have a preference for α2-3-linked sialyl-galactosyl termini. [58] [59] [60] [61] [62] the has of influenza b viruses which, in nature, are only detected in humans and seals, show a binding preference for α2-6-linked glycans. [63] [64] [65] thus, it is important to underline that the species specificity of the ha-glycan interaction is not based on recognition of the terminal sialic acid itself, but, rather, its linkage to the vicinal galactose and the sugars beyond galactose. 66, 67 a correlation between glycan topology and species specificity was established from ha-glycan cocrystal structures as well as glycan array data. 58 with regard to the ha residues that are directly involved in sialic acid binding, these are highly conserved across different ha subtypes. these amino acids (tyr98 1 , ser136 1 , trp153 1 , his183 1 , leu194 1 ) [amino acid numbering based on the h3 ha sequence; the suffixes 1 and 2 denote location in the ha1 and ha2 subunit, respectively] lead to a fixed orientation of the sialic acid relative to the ha rbs. 68 although sialic acid is generally considered to be the primary attachment receptor, influenza virus is able to bind and enter (though considerably less efficiently) into cells of which all surface sialic acids, whether attached to glycolipids or glycoproteins, were removed by treatment with exogenous micromonospora viridifaciens sialidase. 69 hence, it has been proposed that, besides sialic acid, other receptors may be involved in influenza virus entry, which can work either independently or via a multistep process. 69, 70 which specific amino acid residues in ha govern its avian versus human receptor preference, varies among the different has, and is still incompletely understood, although α2-6 tropism is generally linked to residues asp190 1 and asp225 1 in h1 and leu226 1 in h2 and h3 has. 71 to cross the avian-human species barrier, acquisition of the human receptor binding preference is not sufficient, since additional amino acid changes are required, particularly in the influenza virus polymerase complex. 71 in a recent study in which the avian h5n1 virus was passed in ferrets, four mutations in the head domain of h5 ha, combined with the glu627lys hallmark mutation in the pb2 subunit of the polymerase complex, were able to lead to airborne transmission of this virus in ferrets. 72 a similar study with a reassortant virus carrying the ha of avian h5n1 also concluded that its avian-to-mammalian adaptation requires a combination of ha mutations to not only switch its receptor preference from α2-3 to α2-6, but also increase the stability of the ha protein. 73 when considering the ha-receptor binding as an antiviral target, the multivalent nature of this interaction may present as a challenge. this binding is highly dynamic and involves an ensemble of sialylated glycans making contact with multiple ha trimers. 74 in this manner, the avidity effects of the multivalent interaction compensate for the intrinsically low glycan binding affinity for a single binding site on ha [with a dissociation constant (kd) in the millimolar range]. 75 thus, to develop inhibitors that block the receptor binding of ha, at least three factors need to be taken into account: large sequence variation among ha subtypes and antigenic drift of ha; avian versus human-specific receptor use; and multivalent nature of the ha-receptor interaction. an ideal inhibitor would be species-and ha subtype-independent. there are three conceivable strategies for inhibiting attachment of influenza virus to its target cell: (i) an antiviral compound binding to the ha rbs; (ii) an inhibitor blocking the sialic acid-containing receptors on the epithelial cell membrane; or (iii) a receptor-destroying agent. virus-neutralizing antibodies the first and natural types of binding inhibitors are the virus-neutralizing antibodies raised during the course of an influenza virus infection. these neutralizing antibodies are predominantly directed toward the surface of the membrane-distal globular head domain of ha. 76 during the 1918 pandemic, some patients were treated with human blood products from recovering influenza patients. 77 eight controlled studies reported between 1918 and 1925 were recently reviewed, and it was concluded that the overall case-fatality rate was reduced from 37% among control patients to 16% among treated patients. treatment was most effective when initiated early (i.e., less than 4 days after pneumonia became apparent). 77 these historical data demonstrate that passive immunization with anti-ha antibodies can be considered in case a pandemic occurs. obviously, safety considerations about the use of patient-derived materials need to be addressed. an elegant method for the isolation of human antibodies was reported by simmons et al., 78 who prepared h5n1 neutralizing monoclonal antibodies from the memory b-cells of patients recovered from an h5n1 infection. two monoclonal antibodies were effective in a mouse influenza model when administered no later than 72 hr after infection. 78 an attractive new concept is the development of monoclonal antibodies that bind to the conserved rbs of ha and, hence, are endowed with heterosubtypic ha neutralizing activity. a first human monoclonal antibody directed against h1 ha, encoded ch65, was derived from plasma cells of a person immunized with the 2007 trivalent influenza vaccine. cocrystallization of its fab fragment with h1 ha revealed that this antibody acts as a sialic acid mimic since the tip of its heavy-chain complementarity determining region 3 (hcdr3) inserts in the rbs of h1 ha (fig. 2c ). 79 since ch65 was shown to neutralize 31 out of 36 h1n1 isolates covering a period of more than 30 years, and to interact with the conserved rbs itself, resistance selection by ch65 may be expected to be rare, unless associated with reduced viral fitness. 79, 80 it should however be noted that the rbs of ha is smaller than the interaction site of an antibody 81 and, therefore, ch65 forms additional interactions with rbs surrounding residues that are less conserved among the different has. the more broadly acting monoclonal antibody c05 binds to h1, h2, h3, h9, and h12 has and was isolated from a phage-display library constructed from bone marrow donated after seasonal influenza infection. cocrystallization studies demonstrated that the hcdr3 part of c05 forms a loop that inserts into the conserved rbs of ha, while its hcdr1 region makes only minimal contact with rbs surrounding and more variable residues. 82 a third cross-reactive monoclonal antibody, s139/1, neutralizes h1, h2, h3, h13, and h16 virus strains. 83 the hcdr2 region of s139/1 was shown to form multiple hydrophobic interactions within the rbs of h3 ha. the rather low affinity of this binding interaction is compensated in the bivalent igg molecule, and this avidity effect is required to broaden the neutralizing activity of s139/1 to strains of the h1, h2, h13, and h16 subtypes. 84 regarding influenza b viruses, the human monoclonal antibodies 3a2 and 10c4, reactive against b viruses of the yamagata lineage, recognize the 190-helix (residues [190] [191] [192] [193] [194] [195] [196] [197] [198] in ha1) near the rbs. 85 the human monoclonal antibodies cr8033 and cr8071 were shown to neutralize both yamagata and victoria lineage b viruses and protect mice after challenge with a lethal dose of influenza virus. 86 although the therapeutic use of an anti-influenza antibody may appear complicated, some parallel can be seen with the palivizumab antibody that is already in use for the prophylaxis of another respiratory virus, that is, respiratory syncytial virus (rsv). 87 an innovative strategy to improve the pharmacokinetics and reduce the production cost of therapeutic antibodies consists of single-domain antibody fragments (also referred to as nanobodies) derived from camelid immunoglobulins. 88 a nanobody directed to the globular head of h5 ha was shown to be effective in h5n1-infected mice. the activity of the monovalent nanobody was increased by a factor 60 when using a bivalent format, consisting of two paratope containing domains connected by a flexible linker. 89 lectins another type of immune proteins capable of catching viruses is the collagenous c-type lectins (referred to as collectins) such as the lung surfactant proteins. the role of surfactant protein d (sp-d) in the innate immune response to influenza virus is explained by its capacity to cause virus particle aggregation, thereby preventing virus attachment to the host cells. 90 besides, sp-d has various immunological effects that account for its ability to limit lung inflammation by respiratory pathogens. 91 regarding potential antiviral use, design of modified forms of the porcine sp-d lectin (which has higher anti-influenza virus activity than its human counterpart) is aided by the growing insight into how its carbohydrate recognition domain (crd) precisely interacts with the high-mannose glycans attached near the rbs of ha. 92, 93 in addition, n-linked sialoglycans attached to the crd of sp-d are considered important, since they may cause additional interactions between the sp-d and the ha rbs and enhance the antiviral effect. 90 a similar action principle, that is, binding to high-mannose carbohydrates on the viral ha, accounts for the anti-influenza virus activity of the bovine serum lectin cl-43. 94 likewise, cyanovirin-n, a lectin isolated from escherichia coli, recognizes high-mannose oligosaccharide structures on diverse viral glycoproteins, explaining its broad activity against unrelated viruses such as influenza virus and hiv. 95 cyanovirin-n was shown to inhibit influenza virus replication in cell culture as well as mouse and ferret infection models. 96, 97 although sp-d and cyanovirin-n manifest broad anti-influenza a and b virus activity, some virus strains (such as the a/pr/8/34 h1n1 strain) are known to be insensitive, due to the lack of particular asn-linked oligosaccharides on the head of their ha. 98 the location and number of glycans attached to the head of ha is quite variable, since acquisition of epitope shielding oligosaccharides is part of the viral immune escape. 46 in contrast, the glycans attached to the ha stem have a structural function in protein refolding, and the corresponding glycosylation sites are therefore more conserved. 52, 53 this implies that antiviral use of lectin compounds directed toward ha head glycans might lead to escape mutants devoid of specific glycans, although the newly exposed antigenic sites might also render the mutated virus susceptible to immunological control. 99 sialyl-containing macromolecules and sialomimetics an alternative approach to block the ha rbs makes use of receptor mimics, such as sialyl-containing macromolecules. the gangliosides sialylparagloboside (spg) and gm3 (neu5acα2-3galβ1-4glcβ1-1 ceramide) were proven to bind to ha and inhibit the virusinduced cytopathic effect, [100] [101] [102] and their antiviral activity correlated with their ha binding affinities. 101 the hydrophobic ceramide moiety of spg and gm3 was found essential, since the uncoupled trisaccharides 3 -sialyllactosamine and 3 -sialyllactose (which constitute the termini of spg and gm3, respectively) produced no effect. micelle formation of these gangliosides in aqueous solution likely causes protrusion of their sialic acid parts toward the outside of the micelles, resulting in high sialic acid density and, hence, a multivalent binding interaction with ha. 101 in a recent report, hendricks et al. described that liposomes bearing sialylneolacto-ntetraose c (lstc) can form multivalent interactions with influenza virus. 103 in contrast to monovalent lstc, these decoy liposomes are able to competitively bind influenza virus in a hemagglutination inhibition assay, and suppress influenza virus replication in cell culture and mouse models. pentadecapeptides binding to h1 and h3 has were obtained from phage-displayed random peptide libraries by serially repeated affinity selection. a docking simulation indicated that these peptides act as sialomimetics. some showed inhibitory activity against h1 and h3 influenza viruses in cell culture. 104 jeon et al. used a peptide with a sequence derived from the globular head region of ha to screen a dna library for ha-binding aptamers. the selected aptamer, a22, was proven to block the rbs of ha and inhibit influenza a viruses in vitro (i.e., cell culture) and in vivo (i.e., animal studies). 105 another macromolecule, the sulfated sialyl lipid nmso3 (fig. 3) showed antiviral activity against influenza h3n2, but not against b viruses. 106 we recently found that nmso3 inhibits influenza virus binding to cells at 4 • c. 107 although nmso3 has a strong negative charge and, hence, a direct interaction of nmso3 with the sialic acid binding residues of the ha rbs can be anticipated, the precise mode of action of this antiviral compound remains to be determined. nmso3 has broad activity against diverse viruses (in cell culture as well as animal models) 106, 108 and presents as a relevant antiviral lead compound. the opposite strategy to block binding of influenza virus to its cell receptor, is the development of sialic acid binding compounds. the ha-binding and neu5acα2-3gal-containing ganglioside gm3 was used to select potential inhibitors from a phage-displayed random peptide library. 109 two pentadecapeptides, c01 (gwwykgrarpvsava) and c03 (ravwrhs-vatpshsv), were picked out and acylated to a c18 group, in order to form a molecular assembly and promote multivalent binding. both c18-peptides provided inhibition of influenza virus infection in cell culture. their anti-influenza virus activity was comparable to that of the wheat-germ agglutinin lectin, 109 which is known to interact with sialoglycoconjugates. 110 finally, a third strategy for inhibition of virus binding is destruction of the sialylated glycan receptors. years ago, it was observed that cells are less susceptible to influenza virus infection after enzymatic removal of sialic acid from the cell surface. 111 the new anti-influenza virus agent das181 is a recombinant fusion protein, consisting of a sialidase catalytic domain derived from actinomyces viscosus and an epithelium-anchoring domain. das181 efficiently removes α2-3and α2-6-linked sialic acids and displays broad activity against influenza a and b viruses as well as parainfluenza viruses. 112, 113 since das181 acts on the host cell rather than the virus, it is assumed to have a reduced potential for generating drug resistance. after more than 30 passages in the presence of das181, influenza virus mutants were selected with low to moderate resistance to the compound (i.e., three-to 18-fold increase in antiviral ec 50 value). the resistant viruses showed an attenuated phenotype compared to the wild-type virus, yet unchanged virulence in mice. when further passaged in the absence of compound, the viruses quickly regained the wild-type sensitivity. sequencing revealed that the resistant mutants contained substitutions in the ha near its rbs, as well as in the na, leading to altered ha and na functionality. 114 the concern that desialylation of the airway epithelium might unmask certain cryptic receptors and increase the susceptibility to streptococcus pneumonia, was contradicted by mouse experiments showing that das181 treatment does not lead to an increased incidence of secondary pneumonia. 115 das181 requires topical delivery as an inhalant. it is currently in phase ii clinical trials (at once daily dosing of 10 mg during 3 days) for the treatment and prophylaxis of influenza-like illness. 116 after binding to the sialylated glycans on the cell surface, influenza virions are internalized by endocytosis. in general, viruses can be internalized by clathrin-mediated endocytosis (cme); caveolin-mediated endocytosis; macropinocytosis; or other less characterized mechanisms. 117 early electron microscopic analysis of influenza virus-infected cells showed the presence of virions in clathrin-coated pits and vesicles, providing direct evidence that influenza virus can enter the cell by cme. however, since virions were also found in smooth pits, the virus is able to follow an alternative clathrin-independent pathway. 118 further support came from investigations in which dominant negative forms of cellular endocytic regulators were expressed, or by using pharmacological inhibitors, that is, the cme inhibitor chlorpromazine, the cholesterol-depleting agents nystatin or methyl-β-cyclodextrin; or genistein, an inhibitor of caveola formation. 119 additional evidence that influenza virus exploits cme and a clathrin-and caveolinindependent route in parallel, was provided by real-time imaging. both routes appear to be equally efficient in supporting the infection once the virus is internalized. 120 only recently, the clathrin-and caveolae-independent influenza virus uptake was shown to have the characteristics of macropinocytosis. 121 influenza virus entry was completely inhibited when cells were simultaneously treated with dynasore and the amiloride derivative eipa, which inhibit dynamin-dependent cme and macropinocytosis, respectively. the sialic acid attachment sites for influenza virus possess no host cell signaling capacity and, hence, additional postattachment factors may be required for efficient viral entry. 122 since the virus fails to enter lec1 cells, a mutant cho cell line that is totally deficient in n-terminal glycosylation, it was suggested that n-linked glycoproteins may be required for efficient endocytosis of the virus. 122 besides, binding of influenza a virus to cells was found to induce lipid raft rearrangement and activation of signaling molecules, such as the epidermal growth factor receptor (egfr) or other receptor tyrosine kinases. also, it was observed that the activated egfr kinase is involved in promoting the initial virus uptake, and that virus internalization was considerably reduced in the presence of genistein, a broad inhibitor of receptor tyrosine kinases. 123 thus, the precise mechanisms for endocytic uptake of influenza virus are still not fully understood. whether any of these insights may be translated into a relevant antiviral concept is unsure. influenza virus appears to exploit endocytic routes and signaling platforms that are intimately linked to normal cell functioning and thus not readily amenable to selective antiviral intervention. for instance, the above-mentioned pharmacological agents, which were very useful to demonstrate the role of cme or macropinocytosis, only affect the viral uptake at subtoxic concentrations. one potential approach is the use of membrane fluidity modulators, which restrict the movement of membrane molecules. the neutral glycolipid fattiviracin (fv-8; isolated from streptomycetes) interferes with cell-cell fusion in hiv-infected cells and was also reported to have anti-influenza virus activity. 124 interference with cell membrane fluidity may also be the principal mode of action of glycyrrhizin (fig. 3) , the main active constituent of licorice root. glycyrrhizin has broad antiviral activity against diverse enveloped viruses, including influenza virus, herpes simplex virus (hsv), varicella-zoster virus (vzv), vaccinia virus, vesicular stomatitis virus, measles virus, hiv-1, and the sars coronavirus. 125, 126 its anti-influenza virus activity was already demonstrated in 1983. 127 more recently, a flow cytometric internalization assay was used to show that glycyrrhizin inhibits the endocytic uptake of influenza virus. 126 glycyrrhizin was further proven to decrease the fluidity of the cell membrane, an effect that was attributed to its cholesterol-related chemical structure 125 (fig. 3) . besides its antiviral effect, glycyrrhizin displays anti-inflammatory and immunomodulatory effects. 128 these combined pharmacological effects may be advantageous in the treatment of virus infections with a strong inflammatory component, such as the severe airway inflammation (cytokine storm) caused by the avian h5n1 virus. 128 in japan, glycyrrhizin is already in clinical use since many years, and based on this the compound is considered to have favorable safety with no serious side effects. 125, 126 another broad-spectrum antiviral agent interfering with membrane fusion is the aryl methyldiene rhodamine derivative lj001. this compound displays activity against a wide range of unrelated enveloped viruses, including influenza a virus, hiv-1, yellow fever virus, hepatitis c virus (hcv), vesicular stomatitis virus, and vaccinia virus. 129 time-of-addition experiments demonstrated that lj001 acts upon virus entry, since inhibition was only achieved when the compound was added before or during infection. lj001 was shown to intercalate into viral as well as cellular membranes. its potent antiviral activity and low cytotoxicity was explained by the capacity of the host cell for active and rapid biogenic repair, while disruption of the virion envelope is irreversible. lipoglycopeptides are lipophilic derivatives of glycopeptide antibiotics such as the widely used antibiotic vancomycin. some lipoglycopeptides are not only endowed with increased antibacterial activity, but also display activity against diverse viruses such as hiv, herpes viruses, or flaviviruses. 130 regarding influenza virus, we recently described the structureactivity relationship of a series of aglycoristocetin derivatives containing an aryl-substituted cyclobutenedione. 131 the lead compound sa-19, which carries a phenylbenzyl substituent, displayed strong and consistent activity against all influenza a and b viruses tested. 107 no resistance to sa-19 was observed after 15 virus passages in cell culture. this compound was shown to cause intracytoplasmic trapping of influenza virus prior to its nuclear entry, presumably by disturbing the endocytic uptake of the virus at the site of the plasma membrane. it would be relevant to see whether kistamicin a and b, two ristocetin-related glycopeptides that were reported to have anti-influenza virus activity several years ago, 132 display a similar mode of action as sa-19. this is somewhat suggested by the fact that the antiviral activity is higher for kistamicin b, which contains a lipophilic substituent analogous to that of sa-19. an alternative strategy would be to interfere with endosome acidification. upon internalization and entry into early endosomes, influenza viruses undergo an initial acidification step to ph ∼ 6. they then traffic to late endosomes, where further acidification to ph ∼ 5 provides the trigger for fusion of the endosomal and viral membranes. 133 acidification of the endosomes is accomplished by the cellular vacuolar proton atpase (v-atpase), which is potently and selectively inhibited by the macrolide antibiotics bafilomycin a1 and concanamycin a. both compounds block influenza virus entry when added within the first 10 min after infection. 134 a different type of v-atpase inhibitor, the natural compound diphyllin, produced surprisingly potent and selective inhibition of influenza virus replication in cell culture. 135 likewise, lysosomotropic weak bases such as ammonium chloride and chloroquine inhibit influenza virus entry by elevating the endosomal ph. 118, 136 chloroquine shows in vitro anti-influenza virus activity at concentrations that can, based on data from its use for malaria prophylaxis, be reached in humans. 137 however, a double-blind, placebo-controlled efficacy trial concluded that chloroquine is unable to prevent influenza virus infection, 138 and this agrees with its failure to prevent influenza virus infection in mouse and ferret models. 139 possibly, the chloroquine dose used in the clinical study may have been too low. this dose was estimated to produce blood concentrations in the range of the 50% antiviral concentrations in cell culture, and was selected so as to avoid any serious side effects. 138 thus, although bafilomycin a1 and chloroquine represent excellent tools to examine the precise mechanism of influenza virus entry, their relevance for influenza therapy is limited. as explained in the next part, the adamantane compounds amantadine and rimantadine block influenza a virus entry mainly by inhibiting the m2 proton channel. at higher (∼100 μm) concentrations, they raise the endosomal ph due to their basic character, thereby affecting ha-mediated fusion at low ph. 140 hence, amantadine-resistant viruses selected in vitro can contain mutations in either the m2 or ha protein. most of these ha substitutions render the ha less stable, and thus increase the ph at which fusion occurs. we recently observed that some h1n1 viruses such as the a/pr/8/34 strain are particularly sensitive to a subtle increase in the endosomal ph, as caused by newly synthesized amantadine analogues bearing different scaffold structures (torres, unpublished data). the low ph inside the endosomes activates the viral m2 proton channel that is embedded in the viral membrane, leading to transport of proton ions into the interior of the endosomally entrapped virus. as a result, the vrnps become dissociated from the m1 matrix protein (the so-called "uncoating" event), and the viral genome is released. 141 the m2 of influenza a virus (a/m2) is a short polypeptide of only 97 residues, assembled into a homotetrameric, integral membrane channel protein consisting of (i) a short unstructured n-terminal ectodomain (residues 1-24); (ii) a pore-forming transmembrane helix (residues 25-46) responsible for tetramerization and proton translocation; (iii) a cytoplasmic amphipathic helix (residues 47-61), involved in virus assembly and budding; and (iv) a disordered tail (residues 61-97) that interacts with the m1 matrix protein 141 (fig. 4) . activation of the m2 ion channel below ph 6 is caused by protonation of the third his37 residue in the m2 tetramer. 142, 143 the protonated imidazole ring of his37 is involved in a cation-π interaction with the indole ring of trp41. 142 these two residues, his37 and trp41, functioning as a ph sensor and gate, respectively, are critical for m2 proton channel function, and hence invariable among influenza a and b viruses. 144, 145 besides, val27 forms a valve that controls the entrance of protons, while asp44 is indirectly hydrogen bonded to the indole nitrogen of trp41 via a water cluster at the exit of the channel. thus, asp44 and val27 act as gatekeepers at opposite ends of the channel. 146, 147 comparison of the nmr and crystal structures of the a/m2 transmembrane domain obtained at neutral (ph 7.5-8), intermediate (ph 6.5), or acidic (ph 5) ph, provided a detailed insight into the low ph-induced changes in a/m2 protein conformation. [146] [147] [148] at neutral ph, the val27 valve is open, whereas the trp basket, formed by the trp41 residues at the opposing end, has a small hydrophobic opening. when the ph is reduced, the val27 valve constricts, while the trp basket opens. 147 two mechanisms for proton transport through the aqueous pore of a/m2 have been proposed. in the wire model, protons are conducted via a continuous column of water molecules. opening of the pore is achieved by electrostatic repulsion of the protonated his37 residues, which, according to this model, play only a passive role. 142, 149 in contrast, in the shuttle model, his37 plays an active role in proton transport by protonation and deprotonation, which is facilitated by imidazole ring reorientations and small-amplitude backbone fluctuations. 150, 151 in analogy to the a/m2 protein, bm2 (the m2 protein from influenza b virus) forms a homotetrameric integral membrane protein, with characteristic proton channel activity and a ph profile similar to that of its functional homolog a/m2. due to its coiled-coil structure, the transmembrane region of bm2 is able to form a stable tetramer by itself, without the c-terminal amphipathic helix that is necessary for tetramerization of a/m2. 152 except for the hxxxw motif in the transmembrane domain, with the his and trp acting as key residues for proton channel activation and gating, a/m2 and bm2 share little sequence homology. furthermore, the bm2 proton channel activity is higher than that of a/m2. 144 this higher conductance may in part be explained by two extra serine residues in the channel pore of bm2, which can facilitate proton relay. 152 the discovery that the adamantane derivatives amantadine and rimantadine (fig. 5 ) inhibit influenza a virus replication was made decades ago 153 and, in fact, was instrumental in elucidating the function of m2. 154 both amantadine and rimantadine are inactive against influenza b viruses. cocrystallization of amantadine with the transmembrane domain of a/m2 identified a drug binding site in the n-terminal channel lumen, that is surrounded by residues that are mutated in amantadine-resistant viruses (in particular, val27, ala30, ser31, and gly34). binding of amantadine apparently leads to occlusion of the channel pore, but may also affect protonation of the critical his37 residue. 146 on the other hand, a solution nmr of the a/m2 channel in complex with rimantadine revealed four equivalent binding sites, located on the lipid-facing side of the channel, between adjacent helices near the trp41 gate. in this way, binding of rimantadine could stabilize the closed state of the a/m2 tetramer. 148 finally, solid-state nmr spectroscopy of a/m2 in phospholipid bilayers showed the existence of two amantadine binding sites: a high-affinity site in the n-terminal lumen, which is occupied by a single amantadine molecule, and a low-affinity site at the c-terminal protein surface, which only becomes occupied at higher amantadine concentrations. 155 the presence of both binding sites was confirmed by molecular dynamics simulations, which further indicated that amantadine can bind inside the n-terminal lumen under low-and high-ph conditions. 156 importantly, the identification of the a/m2 binding sites for amantadine and rimantadine provided an explanation why both compounds lack activity against influenza b viruses. compared to a/m2, the bm2 pore has two more serine residues, which probably disable binding of the hydrophobic adamantane moiety within the bm2 channel. 149, 152 also, the residues that make up the low-affinity binding site for amantadine in a/m2 have uncorrelated counterparts in the bm2 protein. 152 during many years, amantadine and rimantadine have been successfully used for both prophylaxis and therapy of influenza a virus infections, though amantadine is associated with neurological side effects. 13 nowadays, their clinical utility is limited since most circulating strains are adamantane-resistant. [157] [158] [159] thirty percent of treated patients shed adamantane-resistant mutants, which replicate equally well as wild-type virus, are cross-resistant to amantadine and rimantadine, and are readily transmitted to contact persons. 13, 159 during the 2009-2010 season, 99.9% of h1n1 virus isolates were adamantane-resistant. 157 the resistance mutations are mostly located in the transmembrane region of the a/m2 protein, the most common changes being leu26phe, val27ala, ala30thr, ser31asn, gly34glu, and leu38phe. 160 attempts were made to develop new adamantane derivatives, which are able to interfere with the a/m2 ion channel activity of amantadine-resistant viruses. guided by the novel structural insights into the a/m2 binding interaction of amantadine, wang et al. recently developed spiro-adamantane inhibitors with potent activity against val27ala and leu26phe mutant a/m2 proteins. 161 these molecules have a larger size than amantadine and are therefore able to fill the upper pore of a/m2, even when its volume is increased by the val27ala or leu26phe substitution. one compound (9 161 ; fig. 5 ) showed antiviral activity against the wild type as well as the a/m2-val27ala and a/m2-leu26phe mutant viruses, and its ec 50 values were similar to that of amantadine against the wild-type virus. 161 an imidazole derivative of pinanamine, synthesized by zhao et al., showed moderate inhibition of an a/m2-ser31asn mutant virus. 162 several research groups have developed polycyclic amine compounds to achieve more potent inhibitors of a/m2. [163] [164] [165] two fine examples are the imine compound 8e 166 (fig. 5 ) and the spiro compound 4b 167 (fig. 5) , which were both reported to be ∼200-fold more potent than amantadine. although compound 8e 166 was found to be cross-resistant to amantadine when evaluated against an a/m2 mutant virus, it can serve as a novel scaffold for the design of superior m2 blockers. another study explored the size limits of polycyclic amine derivatives as potential a/m2 inhibitors. 168 surprisingly, both ring-contracted (8 168 in fig. 5 ) and ring-expanded (24 168 in fig. 5 ) polycyclic compounds were able to bind to wild-type a/m2, and some analogues showed increased binding affinity compared to amantadine itself. biochemical studies with mutant a/m2 proteins and molecular docking indicated that compared to amantadine, one of the ring-expanded derivatives showed a different binding mode to the high-affinity a/m2 binding site (i.e., the inner channel pore region delineated by val27, ala30, and ser31). amantadine not only targets the a/m2 channel, but, as a weak base, also indirectly inhibits ha-mediated fusion at concentrations at least 100-fold higher. thus, an alternative approach is to develop a compound reacting with both targets at similar concentrations. in this case, viral resistance would require the appearance of amino acid changes in two separate viral proteins, which may be expected to be a rare event. replacement of the primary amino group of amantadine by a more basic secondary or tertiary amino group, and addition of side groups on the adamantane ring system, resulted in compounds interfering with ha at lower concentrations, while the concentration affecting m2 proton channel activity was increased. however, during passage of the virus in the presence of these compounds, the escape rate was still high, yielding drug-resistant mutants with amino acid substitutions in both the ha and a/m2 proteins. 169 a reason for this high escape rate may be that the resistance mutations selected by these compounds can be located at different sites in ha or m2, without any reduction in viral fitness. the low ph inside the late endosome leads to an extensive and irreversible conformational change of the viral ha, resulting in fusion of the viral and endosomal membranes (fig. 2d) . a key role is played by the fusion peptide (defined as the 23 n-terminal residues of ha2), which is the most conserved region of ha and contains a series of hydrophobic residues. 170, 171 at neutral ph, the fusion peptide is sequestered in a pocket of ionizable residues, but upon acidification, to ph 5-6 for most influenza viruses, the fusion peptide is extruded toward the target membrane. by comparing the x-ray crystallographic structures of the ectodomain portion of ha, obtained at either neutral or acidic ph, the following rearrangements were noted to occur at low ph: (i) the globular head domain containing the rbs detrimerizes; (ii) the n-terminus of the central triple-stranded coiled coil is extended by the interhelical chain and the short α-helix, hereby releasing the fusion peptide from its buried position; and (iii) in the middle of the long αhelix two turns undergo a helix-to-loop transition to form a 180 • reverse turn, positioning the fusion peptide and viral membrane anchor at the same end. 55, 172 the actual membrane fusion proceeds through a hemifusion intermediate 173 (fig. 2d) . according to the stalk-pore model, the extruded fusion peptide inserts into the endosomal membrane. at the same time, the c-terminus of ha2, which is anchored in the viral membrane, is reoriented thereby drawing the endosomal and viral membranes together. after mixing of the outer membrane leaflets (prefusion stalk intermediate), a hemifusion diaphragm is formed. mixing of the inner and outer membrane leaflets results in the formation of a fusion pore, allowing release of the vrnps into the cytoplasm. 174 a first approach to interfere with the ha-mediated fusion process is to inhibit the acidinduced conformational change of ha, using small molecules that bind to and stabilize the neutral ph conformation. one of the first influenza virus fusion inhibitors to be reported was tert-butyl hydroquinone (tbhq; fig. 6 ), which specifically inhibits h3 viruses. 175 several years later, the binding site of tbhq within the h3 ha stem structure was identified by crystallization of the tbhq-ha complex, and was shown to lie within a hydrophobic pocket, formed at an interface between ha subunits. 55 besides several hydrophobic interactions, tbhq is hydrogen bonded with the side chain carbonyl of glu57 2 and the main chain carbonyl of arg54 2 of one monomer, and the main chain carbonyl of leu98 2 of another monomer, hereby stabilizing the nonfusogenic ha conformation. 55 during the conformational change of ha, a critical role is played by the adjacent lys58 2 , located at the c-terminus of the short αhelix and involved in the loop-to-helix transition. 55 the relevance of the hydrophobic pocket around glu57 2 for the development of fusion inhibitors active against group 2 has was further confirmed by our studies with the novel anti-influenza virus agent 4c (fig. 6 ). 176 although 4c and tbhq have very different chemical structures, we noticed a clear similarity between the ha binding mode of tbhq and that predicted for the n-(1-thia-4-azaspiro [4.5] decan-4yl)carboxamide part of 4c. however, the aromatic imidazo[2,1-b]thiazole ring of 4c allows the formation of several additional hydrophobic interactions within this cavity. the inactivity of the two compounds against group 1 viruses can be explained by analysis of ha crystal structures, which revealed that residues 56 2 -58 2 in group 1 has form an extra turn, resulting in blockage of the tbhq/4c binding site. 55 unfortunately, the antiviral activity of 4c is restricted to h3n2 viruses, since an h7n2 virus was shown to be insensitive, despite the fact that the h3 and h7 has belong to the same phylogenetic group 2. also, resistance to 4c emerged within only three passages in cell culture. 176 conversely, several fusion inhibitors targeting group 1 has have been reported in the literature, that is, bmy-27709, cl-385319, ro5464466, and stachyflin (see fig. 6 for chemical structures), which inhibit the conformational change of h1 (and, when tested, h2) ha but, unfortunately, have no activity against h3 viruses. [177] [178] [179] [180] attempts to override this subtype dependency by synthesizing novel derivatives proved unsuccessful. 181 also, initial predictions of their ha binding pocket using in silico docking did not fully correlate with subsequent data obtained after cocrystallization of the compound with ha or photoaffinity labeling. 55, 175, 177, 182 whatever their virus specificity, these small molecule fusion inhibitors were all found to readily select for resistance, at least in cell culture. two types of resistance mutations have been identified. the first are amino acid substitutions within the binding pocket, which affect the inhibitor binding to ha. alternatively, the ha stabilizing effect of the inhibitors can be counteracted by ha mutations that elevate the fusion ph, meaning that the mutant ha acquires its fusogenic conformation at less acidic ph. 55, 176 thus, further development of this type of small molecule fusion inhibitors has been hindered by their subtype-dependent anti-influenza virus activities and low barrier for resistance selection. there may, however, be other ways to inhibit the ha-mediated membrane fusion. instead of preventing ha refolding, diiodofluorescein induces the irreversible conformational change of ha. these premature rearrangements, resulting in virions with fusion-inactive has, also lead to inhibition of the fusion process. 183 furthermore, it may be possible to interfere with membrane fusion following the ha refolding event. this mode of action has been proposed for dextran sulfate, a sulfated polysaccharide ( fig. 3) with broad-spectrum antiviral activity. this agent has been reported to inhibit not only influenza a virus, but also hiv, rsv, hsv, and cytomegalovirus. [184] [185] [186] the anti-influenza virus activity of dextran sulfate, which appears to be restricted to influenza a viruses, correlates with its molecular weight, and levels off when the molecular weight increases above 10,000. 184 the anionic dextran sulfate can be assumed to form electrostatic interactions with the viral ha, which has a net positive charge at ph 7 or less. 187 this is consistent with fluorescence microscopy studies, showing the binding of fluorescein-labeled dextran sulfate to ha-expressing cells. 188 while dextran sulfate had no effect on virus binding at 4 • c, 184 it was found to inhibit the low ph-induced fusion process using a fusion assay based on octadecyl-rhodamine fluorescence dequenching. 187 no direct inhibition of the acid-induced refolding of ha was noticed. 188 however, in order to be active, the compound needed to be present during the fusion process at low ph. 187, 188 these combined data suggest that the dextran sulfate binding site might be inaccessible in the low-ph ha-membrane complex and that dextran sulfate may interfere with a step subsequent to the conformational rearrangement of ha, for instance by causing steric hindrance of the membrane mixing event. 188, 189 it remains to be investigated whether other polysulfated polysaccharides with anti-influenza virus activity (such as compound pkg-03 that was isolated from a microalga 190 ) have a similar mode of action as dextran sulfate. another high molecular weight molecule, retrocyclin 2, also acts against a wide range of viruses, including influenza virus, hiv, and hsv. [191] [192] [193] [194] retrocyclin 2 is a circular octadecapeptide belonging to the θ -defensins, which are antimicrobial peptides of the innate immune system. 195 a detailed mechanistic study showed that its inhibitory effect on influenza virus replication was based on prevention of the ha-mediated membrane fusion at low ph. 194 however, retrocyclin 2 remained effective when added after the conversion of ha to its fusogenic conformation or after hemifusion, an intermediate state in which the outer membrane leaflets have merged while the inner leaflets are still separated. thus, retrocyclin 2 was proposed to prevent the subsequent membrane rearrangements by causing cross-linking and immobilization of surface glycoproteins. 194 a similar interference with the membrane fusion process probably accounts for the broad anti-influenza virus activity of arbidol. this small molecule (fig. 3) has been licensed in russia and china for influenza virus prophylaxis and therapy. besides influenza a and b viruses, its antiviral spectrum encompasses rsv, parainfluenza virus, rhinovirus, hepatitis b virus, and hcv. 196 it is well tolerated as a drug and arbidol-resistant influenza viruses have not (yet) been isolated in the clinic. 197 however, arbidol-resistant viruses, obtained after 14 virus passages in cell culture, were shown to carry mutations in the ha2 subunit associated with an increased fusion ph. 197 arbidol may thus act by stabilizing the prefusogenic ha protein in a similar manner as tbhq and the other small molecule fusion inhibitors described above, but, unlike the latter compounds, arbidol is less subtype-specific. 197 an alternative mode of action was proposed from biochemical studies with various model membranes, showing that arbidol has membranotropic properties, particularly due to its interaction with negatively charged membrane phospholipids. 198, 199 since this membrane interaction is most pronounced at acidic (fusion) ph, arbidol could alter the membrane fluidity during the fusion process and make the bilayer less fusogenic. 196 likewise, the inhibitory effect of arbidol toward hcv entry was explained by its capacity to dually interact with cell membrane phospholipids and aromatic residues (such as tryptophan) that are present in fusion-mediating glycoproteins of hcv. this complexation would prevent the conformational changes in the viral glycoprotein required for membrane fusion. 196, 198 at this time, a dual interaction of arbidol with membrane phospholipids and the influenza virus ha is merely speculative, but this mode of action would reconcile the biochemical and virological in vitro data outlined above. in the context of in vivo studies, arbidol may also have immunostimulatory properties by inducing interferon-α, activating phagocytic macrophages, or stimulating the humoral and cell-mediated immune response. 200 as already explained, several reported fusion inhibitors suffer from subtype-dependent antiinfluenza virus activity and rapid emergence of resistance. these drawbacks could be avoided by targeting the fusion peptide, which is highly conserved among all has and contains the 23 n-terminal residues of ha2. a monoclonal antibody directed against the first 15 residues of ha2 was selected from mice immunized with an h5n1 virus. 201 in vitro, this mab 1c9 antibody inhibits syncytium formation in ha-expressing cells, indicating inhibition of the fusion process. when administered to mice, mab 1c9 provided protection against h5n1, both prophylactically and therapeutically. 201 though highly relevant, cross-reactivity with other has was not yet investigated. a recent strategy with high clinical relevance comes from the discovery of broad neutralizing antibodies directed against relatively conserved pockets in the ha stem structure. 202 already in 1993, the first antibody reacting with different ha subtypes was selected. 203 this mouse monoclonal antibody, designated c179, was shown to neutralize the h1, h2, and h5 has, all belonging to group 1. 203, 204 identification of the resistance mutations in c179-resistant viruses, obtained by virus passaging in the presence of this antibody, allowed to locate its binding site in the middle of the ha stem. c179 was proven to inhibit ha-mediated fusion in a polykaryon assay in influenza virus-infected cells, 203 and was shown to be effective in h1n1or h2n2-infected mice. 205 the first human antibodies to be identified were specific for either group 1 or group 2 has, and were obtained by systematic screening of a wide array of b-cells from influenza-vaccinated or influenza-infected individuals, or by constructing combinatorial libraries. the antibodies f10 and cr6261 show broad neutralizing activity against group 1 has, and a partially overlapping binding pocket within the ha stem. [206] [207] [208] crystallization of f10 and cr6261 in complex with h1 or h5 ha revealed that a conserved hydrophobic tip on their hcdr2 region inserts into a hydrophobic pocket adjacent to the short α-helix in the ha stem, thereby allowing interactions of the antibody with the fusion peptide. 206, 207 another monoclonal antibody, encoded cr8020, interacts with h3 and h7 has, which belong to group 2. cocrystallization of cr8020 with h3 ha identified its binding pocket lower down the ha stalk, thus in closer proximity to the viral membrane compared to cr6261. 209 stabilization of the ha prefusogenic conformation by cr6261 and cr8020 was corroborated by the finding that both antibodies prevented exposure of protease-susceptible sites in ha when the virus was incubated at low ph. 206, 209 broad coverage of all influenza a viruses could be achieved by combining a group 1 and group 2 specific antibody. further significant progress was made by corti et al., who successfully isolated a pan-influenza a neutralizing antibody that recognizes all group 1 and group 2 has, by interrogating a large number (about 100,000) of donor plasma cells. 210 cocrystallization of this fi6 antibody with an h1 or h3 ha protein revealed its interaction with a conserved epitope in the f (fusion) subdomain. 210 hence, binding of fi6 or the optimized fi6v3 antibody is assumed to increase the stability of the f subdomain, thus preventing the conformational change of ha that is required for membrane fusion. this mode of action accords with the inhibitory effect of fi6 on syncytium formation in ha-positive cells. alternatively, prevention of ha0 cleavage (at least for viruses requiring extracellular cleavage) or cross-linking of ha subunits, have been implicated in the virus-neutralizing activity of fi6. passive immunization with fi6 was shown to confer prophylactic and therapeutic protection to influenza virus-infected mice and ferrets. 210 recently, dreyfus et al. isolated the human monoclonal antibody cr9114, which neutralizes influenza a and b viruses. 86 cr9114 recognizes an epitope in the ha stem that is nearly identical to that of the group 1-specific antibody cr6261. however, subtle conformational differences explain why the cr9114 antibody has a much broader anti-ha reactivity. another ha stem-binding antibody, pn-sia28, showed antiviral activity against all group 1 viruses tested (i.e., h1n1, h2n2, h5n1, and h9n2 viruses), as well as some isolates of the h3n2 virus, which belongs to group 2. 211 however, h3n2 strains isolated after 1982 and h7n2 viruses were not inhibited. in order to localize the binding epitope for pn-sia28 in the ha stem, the authors selected escape mutants by repeated passaging of the virus in the presence of the antibody. similar attempts to generate escape mutants with some of the other broad neutralizing antibodies suggest that viruses with mutations in the corresponding ha stem regions do not readily emerge in cell culture. for instance, with the cr6261 antibody, ten virus passages were required, 208 while in other studies, no escape mutants were detected. 212 these observations seem to indicate that the conserved ha stem epitopes targeted by these broad acting antibodies are less prone to mutations due to fitness constraints. this hypothesis, however, still remains to be verified by mutational analysis. it is clear that the discovery of these broad neutralizing anti-ha stem antibodies offers entirely new perspectives for passive or active immunization against influenza a viruses. also, peptides directed against the conserved epitopes in the ha stem region have been developed, such as the hb36 peptide that interacts with the cr6261-binding epitope and recognizes several group 1 has (i.e., h1, h2, h5, and h6). 213 the concept of a therapeutic peptide used to inhibit virus fusion is validated in the hiv field by the clinical use of enfuvirtide, a 36 amino acid peptide that binds to the hiv gp41 protein. 214 after disruption of the vrnps from the matrix m1 protein and fusion pore formation, the vrnps are released in the cytoplasm and transported into the nucleus. 215, 216 how the vrnps are released from m1 is only partially understood. in the intact virion, the m1 protein forms the capsid shell located underneath the envelope, and is tightly associated with the vrnps. 217, 218 the current insights into the protein structure of m1 and its crucial role in organizing virion structure were recently reviewed. 219 once inside the endosome, the m2-mediated acidification of the virion interior leads to vrnp uncoating, possibly by inducing a conformational change in m1. 220, 221 recent studies indicate that the oligomerization state of m1 is ph-dependent and that oligomers of intact m1 dissociate into stable dimers at acidic ph. 222 the disappearance of a visible m1 layer in virions exposed to ph 5 was imaged by cryoelectron tomography. 223 after their transport through the fusion pore, the vrnps appear to be associated with some residual m1 protein, which, inside the cytoplasm, dissociates from the vrnps to finally allow their nuclear entry. 215, 224 this second dissociation process may depend on cytosolic m1 modifications, such as phosphorylation or zinc binding. 220, 225 a peptide derived from the zinc finger domain of m1 was reported to display broad and potent anti-influenza virus activity in cell culture when added within 1 hr after infection, classifying this "peptide 6" as an entry inhibitor. 226 as far as we know, no other attempts have been reported in which m1 was explored as an antiviral target. development of a potent m1 inhibitor might be challenging, due to the abundant presence of this protein in the virion. at last, the free vrnps are imported in the nucleus via the nuclear pores. each vrnp contains one of the eight vrna genome segments, which is associated with a single copy of the viral polymerase (the heterotrimer of pb1, pb2, and pa), and multiple copies of the nucleoprotein (np). 227 although these four viral proteins all contain at least one nuclear localization signal (nls), the vrnp nuclear import appears to be primarily dependent on the nls in the n-terminus of np. [228] [229] [230] due to this nls, the vrnp is recognized as a cargo by the cellular importin-α protein, and after formation of a ternary complex with importin-β, is transported into the nucleus. 231 the specificity of np (and pb2) for the different isoforms of importin-α differs for avian and human viruses, implicating a role in influenza virus adaptation. 231, 232 the viral np has both structural and regulatory functions in influenza virus replication. besides being the main structural component of the vrnps, np has a crucial role in the consecutive replicative stages, by regulating vrnp nuclear import; viral rna transcription; and nuclear export (via interaction of np with m1). 233 the conserved protein sequence of np further adds to its attractiveness as an antiviral target, since this implicates that np inhibitors could be broadly active across the different virus subtypes. 233 this is illustrated by the small molecule ingavirin, which inhibits influenza a and b viruses in vitro and in vivo. 234 ingavirin was reported to inhibit np oligomerization and subsequent nuclear import of newly synthesized np. 235 this mechanism of action is distinct from that of the np aggregating agents nucleozin and its structural analogues 3061 and "compound 3", which were independently identified by several groups [236] [237] [238] [239] (fig. 7a) . these np binding agents display anti-influenza virus activity against all influenza a viruses tested, including h1n1, h3n2, and h5n1 viruses. 238, 239 in the first report, nucleozin was proven, by fluorescence microscopy, to cause np aggregation and trap the vrnps in a perinuclear halo. 236 nucleozin was also active in the cellular vrnp reconstitution assay, which directly measures the transcriptional activity of vrnp. 236 this indicates that nucleozin not only interferes with nuclear entry, but also with other replicative processes in which np is involved. the np aggregating activity of nucleozin was confirmed by experiments in which np was cocrystallized with the related "compound 3". 239 formation of higher order np oligomers was observed, in which two np trimers are linked to each other through six molecules of "compound 3", each interacting with two antiparallel binding pockets. 239 the nitrophenyl moiety of "compound 3" interacts with one binding pocket (close to the np residues tyr289 and asn309) in an np from one trimer, while the isoxazole heterocycle binds to the other binding pocket (around the tyr52 residue) of an np in the other trimer, and vice versa 239 (fig. 7b) . these data nicely agree with the resistance mutations identified in np (i.e., tyr52cys/his, tyr289his, and asn309lys) after virus passaging in the presence of these np binding agents. 236, 238, 239 nucleozin showed a rather modest in vivo activity, protecting 50% of h5n1-infected mice. 236 however, full protection of h5n1-infected mice was obtained with "compound 5", a derivative of "compound 3" with improved solubility and metabolic stability. 239 although most available antiviral strategies are directed toward a viral protein, the possibility to block a cellular component with a critical role in virus replication receives increasing attention. 240 an antiviral targeting a host cell factor can be assumed to have reduced selectivity (i.e., window between cytotoxicity and antiviral efficacy). on the other hand, its resistance barrier could (in theory) be higher when compared to a direct antiviral compound. 241 for a virus with a high mutation rate such as influenza virus, this appears a considerable advantage. 242 two studies using genome-wide rna interference screening identified several host cell factors necessary for influenza virus replication. 135, 243 further analyses, based on a pseudotyped particle entry assay, allowed the selection of cellular factors that regulate the low ph-dependent and ha-mediated entry. among them are proteins involved in the ip3-protein kinase c (pkc) or phosphatidylinositol-3-kinase (pi3k)-akt signaling pathways; copi components (involved in endosomal trafficking); vacuolar atpases; fibroblast growth factor receptor 135 ; and son dna binding protein (important for influenza virus trafficking to late endosomes). 243 these intriguing data create new opportunities for designing antiviral concepts toward host cell factors. 242 in a proof-of-concept study, compounds such as sirolimus, podophyllotoxin, or other inhibitors of any of these host cell factors, were found to inhibit virus replication with quite remarkable selectivity. 135 the bisindolylmaleimide compounds specifically inhibit all pkc isoenzymes with a similar potency, by blocking the atp-binding site on the catalytic domain of pkc. 244 bisindolylmaleimide i has been shown to interfere with influenza a and b virus replication early in infection, probably by affecting endocytosis or vesicle transport. 245 the pkcβii isoform was proven to be critical for influenza virus entry, since accumulation of the virus in late endosomes was observed in cells expressing a phosphorylation-deficient form of pkcβii. 246 the pi3k and its downstream effector akt/protein kinase b are signaling mediators induced by influenza a virus, and their role in virus replication seems to be multifaceted. 247 upon virus attachment to the cell, pi3k is activated in a short and transient manner, promoting a step during virus entry that precedes early and late endosomal transport. 123, 247 at later stages of infection, the influenza a virus nonstructural ns1 protein induces a second phase of sustained pi3k activation to prevent premature apoptosis during viral propagation. the influenza b virus ns1 protein apparently lacks this function. 248 in contrast, accumulation of vrna leads to pi3k activation in cells infected with influenza a or b viruses. finally, pi3k signaling is also essential for efficient irf-3 activation during type i interferon (ifn) induction. 249 the antiviral effect of ifn is mediated by the interferon inducible transmembrane (ifitm) protein 3 and results in inhibition of viral genome release into the cytoplasm. 250 notwithstanding this complex function of pi3k, it was shown that inhibition of pi3k results in decreased influenza virus replication, 247 which makes this pathway a potential antiviral target. during the past years, significant advances have been made in unraveling the structure of the influenza virus proteins involved in virus entry, from its initial attachment to the sialylated receptors until its nuclear entry. the available crystal structures of ha, m2, and np enable a rational and computer-aided design of directly acting antivirals. a few m2 inhibitors with activity against amantadine-resistant viruses have already been reported. the old paradigm that the ha appears too variable to be a valid antiviral target is challenged by broad-acting macromolecule inhibitors, such as lectins or antibodies interacting with a conserved site in the ha rbs or stem region. whereas small molecules inhibiting the conformational change of ha have a restricting subtype dependency, this is not the case for compounds (such as arbidol), which interfere with the membrane fusion event itself. the recently discovered np aggregating agent nucleozin represents an entirely novel class of anti-influenza agents with clinical relevance. complementary to these structural studies, much attention is currently given to the complex cell biology of the influenza virus entry pathway, with a particular interest in the host cell factors involved. diverse compounds interfering with any of these cellular factors have been reported, setting the stage for a new type of indirectly acting antivirals with a higher barrier for resistance selection. in times of increasing resistance to oseltamivir, agents with a more favorable resistance profile are indeed urgently required. 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pro-inflammatory cytokine and chemokine expression in human macrophages a broad-spectrum antiviral targeting entry of enveloped viruses polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of hiv entry anti-influenza virus activity and structure-activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains new antiviral antibiotics, kistamicins a and b. i. taxonomy, production, isolation, physico-chemical properties and biological activities endocytosis of influenza viruses requirement for vacuolar proton-atpase activity during entry of influenza virus into cells human host factors required for influenza virus replication infectious cell entry mechanism of influenza virus in vitro inhibition of human influenza a virus replication by chloroquine chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine is effective against influenza a virus in vitro but not in vivo fusion mutants of the influenza virus hemagglutinin glycoprotein structural and dynamic mechanisms for the function and inhibition of the m2 proton channel from influenza a virus protonation of histidine and histidine-tryptophan interaction in the activation of the m2 ion channel from influenza a virus histidines, heart of the hydrogen ion channel from influenza a virus: toward an understanding of conductance and proton selectivity influenza b virus bm2 protein has ion channel activity that conducts protons across membranes functional studies and modeling of pore-lining residue mutants of the influenza a virus m2 ion channel structural basis for the function and inhibition of an influenza virus proton channel structure and mechanism of proton transport through the transmembrane tetrameric m2 protein bundle of the influenza a virus structure and mechanism of the m2 proton channel of influenza a virus the m2 proton channels of influenza a and b viruses nmr detection of ph-dependent histidine-water proton exchange reveals the conduction mechanism of a transmembrane proton channel mechanisms of proton conduction and gating in influenza m2 proton channels from solid-state nmr solution structure and functional analysis of the influenza b proton channel antiviral activity of 1-adamantanamine (amantadine) influenza virus m2 protein has ion channel activity structure of the amantadine binding site of influenza m2 proton channels in lipid bilayers computational study of drug binding to the membrane-bound tetrameric m2 peptide bundle from influenza a virus comprehensive assessment of 2009 pandemic influenza a (h1n1) virus drug susceptibility in vitro resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance surveillance of resistance to adamantanes among influenza a(h3n2) and a(h1n1) viruses isolated worldwide flu channel drug resistance: a tale of two sites molecular dynamics simulation directed rational design of inhibitors targeting drug-resistant mutants of influenza a virus m2 design and synthesis of pinanamine derivatives as anti-influenza a m2 ion channel inhibitors identification of hits as matrix-2 protein inhibitors through the focused screening of a small primary amine library synthesis of benzopolycyclic cage amines: nmda receptor antagonist, trypanocidal and antiviral activities design and synthesis of bioactive adamantanaminoalcohols and adamantanamines discovery of highly potent agents against influenza a virus synthesis and antiviral activity evaluation of some new aminoadamantane derivatives. 2 exploring the size limit of templates for inhibitors of the m2 ion channel of influenza a virus how to overcome resistance of influenza a viruses against adamantane derivatives changes in the conformation of influenza virus hemagglutinin at the ph optimum of virus-mediated membrane fusion a spring-loaded mechanism for the conformational change of influenza hemagglutinin structure of influenza haemagglutinin at the ph of membrane fusion lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion composition and functions of the influenza fusion peptide inhibition of the fusioninducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin molecular mechanism underlying the action of a novel fusion inhibitor of influenza a virus inhibition of influenza a virus (h1n1) fusion by benzenesulfonamide derivatives targeting viral hemagglutinin inhibition of influenza a virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin identification of a novel ha conformational change inhibitor of human influenza virus an approach to the identification of potent inhibitors of influenza virus fusion using parallel synthesis methodology ph-dependent changes in photoaffinity labeling patterns of the h1 influenza virus hemagglutinin by using an inhibitor of viral fusion structure-based identification of an inducer of the low-ph conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity differential inhibitory effects of sulfated polysaccharides and polymers on the replication of various myxoviruses and retroviruses, depending on the composition of the target amino acid sequences of the viral envelope glycoproteins sulfated polysaccharides are potent and selective inhibitors of various enveloped viruses, including herpes simplex virus, cytomegalovirus, vesicular stomatitis virus, and human immunodeficiency virus inhibitory effect of dextran sulfate and heparin on the replication of human immunodeficiency virus (hiv) in vitro dextran sulfate inhibits the fusion of influenza virus with model membranes, and suppresses influenza virus replication in vivo dextran sulfate inhibits fusion of influenza virus and cells expressing influenza hemagglutinin with red blood cells fusion and infection of influenza and sendai viruses as modulated by dextran sulfate: a comparative study in vitro inhibition of influenza a virus infection by marine microalga-derived sulfated polysaccharide p-kg03 theta defensins protect cells from infection by herpes simplex virus by inhibiting viral adhesion and entry retrocyclin 2: a new therapy against avian influenza h5n1 virus in vivo and vitro retrocyclin: a primate peptide that protects cells from infection by t-and m-tropic strains of hiv-1 carbohydrate-binding molecules inhibit viral fusion and entry by crosslinking membrane glycoproteins mammalian defensins in the antimicrobial immune response mechanism of inhibition of enveloped virus membrane fusion by the antiviral drug arbidol characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol biochemical mechanism of hepatitis c virus inhibition by the broad-spectrum antiviral arbidol membranotropic effects of arbidol, a broad anti-viral molecule, on phospholipid model membranes pharmacokinetic properties and bioequivalence of two formulations of arbidol: an open-label, single-dose, randomized-sequence, two-period crossover study in healthy chinese male volunteers monoclonal antibodies against the fusion peptide of hemagglutinin protect mice from lethal influenza a virus h5n1 infection a potential role for monoclonal antibodies in prophylactic and therapeutic treatment of influenza a common neutralizing epitope conserved between the hemagglutinins of influenza a virus h1 and h2 strains an epitope shared by the hemagglutinins of h1, h2, h5, and h6 subtypes of influenza a virus protection against the mouse-adapted a/fm/1/47 strain of influenza a virus in mice by a monoclonal antibody with cross-neutralizing activity among h1 and h2 strains antibody recognition of a highly conserved influenza virus epitope structural and functional bases for broad-spectrum neutralization of avian and human influenza a viruses heterosubtypic neutralizing monoclonal antibodies cross-protective against h5n1 and h1n1 recovered from human igm+ memory b cells a highly conserved neutralizing epitope on group 2 influenza a viruses a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins a human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes structural basis of influenza virus neutralization computational design of proteins targeting the conserved stem region of influenza hemagglutinin approaches for identification of hiv-1 entry inhibitors targeting gp41 pocket transport of incoming influenza virus nucleocapsids into the nucleus nuclear import of the influenza a virus transcriptional machinery identification of the domains of the influenza a virus m1 matrix protein required for np binding, oligomerization and incorporation into virions the role of nuclear import and export in influenza virus infection influenza virus assembly and budding effect of m1 protein and low ph on nuclear transport of influenza virus ribonucleoproteins unpacking the incoming influenza virus dissection of influenza a virus m1 protein: ph-dependent oligomerization of n-terminal domain and dimerization of c-terminal domain structural changes in influenza virus at low ph characterized by cryo-electron tomography influenza virus uncoating in infected cells and effect of rimantadine a small percentage of influenza virus m1 protein contains zinc but zinc does not influence in vitro m1-rna interaction antiviral activity of influenza virus m1 zinc finger peptides architecture of ribonucleoprotein complexes in influenza a virus particles the mechanism by which influenza a virus nucleoprotein forms oligomers and binds rna ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein nuclear import of influenza virus rna can be mediated by viral nucleoprotein and transport factors required for protein import differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus the influenza virus nucleoprotein: a multifunctional rna-binding protein pivotal to virus replication activity of ingavirin (6-[2-(1h-imidazol-4-yl)ethylamino]-5-oxohexanoic acid) against human respiratory viruses in in vivo experiments nebol'sin ve. effect of the antiviral drug ingavirin on intracellular transformations and import into the nucleus of influenza a virus nucleocapsid protein identification of influenza a nucleoprotein as an antiviral target design, synthesis, and in vitro biological evaluation of 1h-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza a agents targeting virus nucleoprotein high-throughput identification of compounds targeting influenza rna-dependent rna polymerase activity inhibition of influenza virus replication via small molecules that induce the formation of higher-order nucleoprotein oligomers cellular networks involved in the influenza virus life cycle targeting cell signalling pathways to fight the flu: towards a paradigm change in antiinfluenza therapy the host interactome of influenza virus presents new potential targets for antiviral drugs genome-wide rnai screen identifies human host factors crucial for influenza virus replication the bisindolylmaleimide gf 109203x is a potent and selective inhibitor of protein kinase c entry of influenza viruses into cells is inhibited by a highly specific protein kinase c inhibitor role of protein kinase c betaii in influenza virus entry via late endosomes bivalent role of the phosphatidylinositol-3-kinase (pi3k) during influenza virus infection and host cell defence influenza a virus ns1 protein activates the pi3k/akt pathway to mediate antiapoptotic signaling responses phosphatidylinositol-3-kinase (pi3k) is activated by influenza virus vrna via the pathogen pattern receptor rig-i to promote efficient type i interferon production ifitm3 inhibits influenza a virus infection by preventing cytosolic entry influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion at the ku leuven, belgium and her ph.d. as doctor in pharmaceutical sciences (2012) at the rega institute for medical research the authors acknowledge a grant from the geconcerteerde onderzoeksacties (goa/10/014). they wish to thank w. van dam, s. stevens, l. persoons, and f. de meyer for their dedicated contribution to their research work. key: cord-264335-c2hfh3dq authors: gunson, rory; maclean, alasdair; davies, eleri; bennett, susan; miller, rhona; carman, w.f. title: development of a multiplex real-time rt-pcr that allows universal detection of influenza a viruses and simultaneous typing of influenza a/h1n1/2009 virus date: 2009-10-23 journal: j virol methods doi: 10.1016/j.jviromet.2009.10.006 sha: doc_id: 264335 cord_uid: c2hfh3dq on june 11, 2009, the world health organization declared that the influenza a/h1n1/2009 virus had become the first influenza pandemic of the 21st century. rapid detection and differentiation from seasonal and avian influenza would be beneficial for patient management and infection control. it was the aim of this study to develop a real-time rt-pcr that can detect all influenza a viruses and offer simultaneous typing for influenza a/h1n1/2009. this would be a useful addition to existing diagnostic protocols for influenza a. its routine use would allow laboratories to screen out influenza a/h1n1/2009 positive samples rapidly and would reduce overall testing costs. on april 15 and 17, 2009, a novel swine-lineage influenza a (influenza a/h1n1/2009) infection was reported to the world health organisation (who) by the centers for disease control and prevention (cdc) in atlanta in two children presenting with febrile respiratory illness from adjacent counties in southern california (cdc, 2009 ; novel swine-origin influenza a (h1n1) virus investigation team, 2009). these cases were not epidemiologically linked and neither child had exposure to swine. since the original identification of influenza a/h1n1/2009 in the united states and mexico, sustained human-to-human transmission has been seen in other countries and on june 11, 2009, the world health organization declared that the virus had become the first influenza pandemic of the 21st century. although the influenza a/h1n1/2009 virus is likely to become the predominant influenza a type encountered in most countries, seasonal influenza a types may also co-circulate (kelly et al., 2009) and in some countries sporadic h5n1 infections may still occur (who report of avian influenza, 2009). determining the subtype of influenza virus is important as it has implications for patient management and infection control (meijer et al., 2009; beigel and bray, 2008; hall et al., 2009) doses of oseltamivir are necessary for successful treatment of infection (lackenby et al., 2009; white et al., 2009) . in order to detect and then type influenza a viruses most laboratories use a two tier testing system comprising of a universal influenza a screening assay complemented with a suite of subtyping assays that determine whether the sample is seasonal influenza a (human h1n1 and h3n2), avian h5n1 or the influenza a/h1n1/2009 virus. although useful for the reasons outlined above, the testing structure prolongs the time it takes to complete testing and is costly as most influenza a positive samples will have to be tested using the individual typing assays. this article describes the development of a multiplex real-time reverse transcription polymerase chain reaction (rtpcr) that allows universal detection of all influenza a viruses and simultaneously subtypes all that are influenza a/h1n1/2009. an internal control rtpcr assay was also incorporated in order to detect pcr inhibition, failed extraction/pcr and technical error. use of this assay will allow laboratories to screen respiratory samples for influenza a/h1n1/2009 virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. the influenza a/h1n1/2009 rtpcr assay was designed to target segment 6 of the na using published sequence data table 1 ) amplify a 73-bp amplicon and bioinformatics analysis using blast and clustal alignments showed no significant homology to human or other known influenza a gene sequences (fig. 1) . the highest homology is 91% to classical swine flu, e.g. a/swine/england/wvl15/1997(h1n1). with this strain there were 4 mismatches in the probe sequence, which would be expected to prevent hybridisation although this was not experimentally tested. in addition we would not expect to be screening humans infected with classical swine flu strains. the chosen primers and probe do not span any of the variable antigenic sites minimising the possibility of point mutations occurring (abed et al., 2002) . a widely used universal influenza a rtpcr was used as part of the multiplex rtpcr. the assay targets the matrix region of the virus. participation in various eqa schemes has shown this assay to detect influenza a viruses from humans and animals with high sensitivity (carr et al., 2009 ). the rtpcr assay used for the internal control targets equine arteritis virus (eav) and was taken from an existing publication (scheltinga et al., 2005) . the primer and probe concentrations for each assay were individually optimised using in house protocols. all assays, singleton or multiplex, used the primers at an optimised concentration of 1 m and the probe at 0.2 m in a 15 l reaction volume (all primers and probes are shown in table 1) . for singleton assays, one-step rtpcr was performed on 6 l of rna extract with the platinum one-step qrt-pcr kit (invitrogen) on an abi prism 7500 sds real-time platform (applied biosystems). the following thermal profile was used: a single cycle of reverse transcription for 15 min at 50 • c, 2 min at 95 • c for reverse transcriptase inactivation and dna polymerase activation followed by 40 amplification cycles of 15 s at 95 • c and 34 s at 60 • c each (annealing-extension step). for the multiplex rtpcr, one-step rtpcr was performed on 6 l of rna extract with the qiagen quantifast mulitiplex rt-pcr kit (qiagen, crawley, united kingdom) on an abi prism 7500 sds real-time platform. the following thermal conditions were used: a single cycle of reverse transcription for 20 min at 50 • c, 5 min at 95 • c for reverse transcriptase inactivation and dna polymerase activation followed by 40 amplification cycles of 15 s at 95 • c and 30 s at 60 • c each (annealing-extension step). data acquisition occurred at the annealing step of each cycle and the threshold cycle (ct) for each sample was calculated by detersequences had 100% homology with the primers and probe, whereas there were a significant number of differences between both seasonal hin1 and swine and avian n1 sequences. all reference sequences were obtained from the influenza sequence database (isd) at los alamos. sequences were aligned using bioedit. mining the point at which the fluorescence exceeded the threshold limit. please note that the qiagen quantifast multiplex rt-pcr kit was used for the multiplex rtpcr only as, unlike the invitrogen rt-pcr kit, it is specially designed to prevent competition from occurring between the pcr tests within a multiplex. such competition often results in false negative reactions or strange traces which can impede result interpretation. previous comparisons between this kit and the invitrogen kit have shown it to be superior for multiplex rtpcr (gunson et al., 2008) . the specificity of the multiplex assay was assessed using a recent world health organisation (who) panel. this panel contained examples of seasonal influenza a, h1n1 and h3n2, the influenza a/h1n1/2009 and numerous avian a/h5n1 viruses. a pool containing the following commonly encountered respiratory pathogens was also tested: influenza b, influenza c, parainfluenza 1-4, human metapneumovirus, respiratory syncytial virus, mycoplasma pneumoniae, rhinovirus (untyped) and coronaviruses 229e, oc43 and nl63. the end point detection limit of the new multiplex rtpcr was directly compared to each rtpcr in singleton format using a dilution series of an influenza a/h1n1/2009 clinical sample. this was carried out to ensure that multiplexing did not result in a reduction in end point detection limit. the final assessment comprised of assessing the multiplex rtpcr on 92 clinical samples. of the 92 samples, 40 were seasonal influenza a viruses (20 subtyped as h1n1 and 20 as h3n2) that had been submitted to the wossvc during previous respiratory seasons. the remaining 52 were samples that had been sent to the laboratory in 2009 and found to be influenza a/h1n1/2009 positive using an alternative rtpcr (carr et al., 2009) . all samples contained h1n1 at varying concentrations (ct values range from 17 to 39). total nucleic acid was extracted from the aforementioned samples using qiaamp viral rna kit (qiagen, crawley, united kingdom) on the qiagen mdx according to the manufacturer's instructions. please note that eav was added to the lysis buffer prior to extraction in order to detect pcr inhibition, extraction failure or technical error (300 l × 10 −6 (ct 23) of bhk tissue culture grown eav was added to the lysis buffer to extract 96 samples). internal control was also added to ensure that its presence did not compete with the diagnostic components of the assay and inhibit their performance. the multiplex rtpcr was evaluated using a recent panel distributed by the who comprising seasonal influenza a (h1n1 and h3n2), avian (h5n1) and influenza a/h1n1/2009 influenza a viruses ( table 2 ). the universal influenza a component detected all samples as positive. the na assay was positive in only two cases (samples 2 and 6) and did not detect the seasonal influenza a (h1n1 and h3n2) viruses or avian influenza viruses. the multiplex assay also did not detect any of the commonly encountered respiratory pathogens as positive. the end point detection limit of both singleton rtpcr assays were compared using a dilution series of a influenza a/h1n1/2009 clinical sample. the universal influenza a test detected the 10-3 dilution in one out of two occasions (table 3) whereas the na assay was slightly less sensitive detecting the 10-2 dilution. comparing the end point detection limit of each of the test components in singleton and multiplex form showed that multiplexing had no effect on test performance. the final comparison assessed the multiplex assay on 92 clinical samples. the multiplex rtpcr detected all samples as influenza a positive. the na assay detected all but 1 of the influenza a/h1n1/2009 samples as positive. the false negative sample had a ct value >38 in the universal influenza and was confirmed as positive using an alternative influenza a/h1n1/2009 assay (carr et al., 2009) . none of the 40 samples containing seasonal influenza a were positive by na assay. this article describes the development of a rapid, specific and sensitive multiplex rtpcr assay that detects all influenza a types and simultaneously identifies samples that contain the pandemic influenza a/h1n1/2009 virus. the assay also incorporates an internal control and thus is useful for detecting pcr inhibition or technical error. multiplexing was shown to have no effect on the performance of the individual test components and, because the assay utilised the qiagen quantifast multiplex rt-pcr kit, the presence of internal control within the samples did not compete with the other diagnostic components. the universal influenza a assay has been described elsewhere and the results shown here confirm it to be sensitive and able to detect a wide range of influenza a types. the na assay was specific as it did not cross-react with seasonal influenza a strains currently circulating (subtypes h1n1 and h3n2) or other commonly encountered respiratory pathogens. in addition, the n1 assay did not detect as positive any of the influenza a, subtype h5n1 viruses. close homology was observed between amplicon and classical swine influenza a (h1n1). the assay was not assessed against this virus and therefore we cannot be sure as to whether the test will detect this virus. however, four mismatches in the probe were observed which may reduce the likelihood of detecting this virus. the sensitivity of the na assay was slightly less than the frontline universal influenza a test. this was shown using the dilution series and was confirmed when it was assessed using the 52 influenza a/h1n1/2009 positive clinical samples where the na assay failed to detect one sample which was positive using the universal influenza a test and an alternative influenza a/h1n1/2009 assay. the ct value of the sample was >38 in the universal influenza a assay which, based on the results from the dilution series, was likely to be beyond the detection limit of the h1n1 assay. based on the results shown here, the multiplex rtpcr is a useful addition to existing diagnostic protocols for influenza a. its routine use would allow laboratories to screen out pandemic influenza a/h1n1/2009 positive samples rapidly. such samples are likely to be in the majority and therefore only a small number of samples would require to be tested using seasonal or h5n1 specific assays. use of this test would provide clinicians with complete results in rapid fashion and would reduce costs as far fewer samples will be sent for seasonal influenza or h5n1 typing. divergent evolution of hemagglutinin and neuraminidase genes in recent influenza a:h3n2 viruses isolated in canada current and future antiviral therapy of severe seasonal and avian influenza development of a real-time rt-pcr for the detection of swine-lineage influenza a (h1n1) virus infections update: infections with a swine-origin influenza a (h1n1) virus-united states and other countries using multiplex real time pcr in order to streamline a routine diagnostic service pandemic influenza a (h1n1)v viruses currently circulating in new zealand are sensitive to oseltamivir epidemiological characteristics of pandemic influenza h1n1 2009 and seasonal influenza infection european influenza surveillance scheme. oseltamivir-resistant influenza virus a (h1n1) emergence of a novel swine-origin influenza a (h1n1) virus in humans diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time rna pcr what is the optimal therapy for patients with h5n1 influenza key: cord-299613-5ju5fcf4 authors: arthi, vellore; parman, john title: disease, downturns, and wellbeing: economic history and the long-run impacts of covid-19 date: 2020-11-03 journal: explor econ hist doi: 10.1016/j.eeh.2020.101381 sha: doc_id: 299613 cord_uid: 5ju5fcf4 how might covid-19 affect human capital and wellbeing in the long run? the covid-19 pandemic has already imposed a heavy human cost—taken together, this public health crisis and its attendant economic downturn appear poised to dwarf the scope, scale, and disruptiveness of most modern pandemics. what evidence we do have about other modern pandemics is largely limited to short-run impacts. consequently, recent experience can do little to help us anticipate and respond to covid-19’s potential long-run impact on individuals over decades and even generations. history, however, offers a solution. historical crises offer closer analogues to covid-19 in each of its key dimensions—as a global pandemic, as a global recession—and offer the runway necessary to study the life-course and intergenerational outcomes. in this paper, we review the evidence on the long-run effects on health, labor, and human capital of both historical pandemics (with a focus on the 1918 influenza pandemic) and historical recessions (with a focus on the great depression). we conclude by discussing how past crises can inform our approach to covid-19—helping tell us what to look for, what to prepare for, and what data we ought to collect now. the health and economic toll of the covid-19 pandemic continues to expand throughout the globe, impacting countries both rich and poor. as it does so, the virus exposes the strengths and weaknesses of our healthcare systems, political institutions, media, and our economies themselves. much of the discussion to date has understandably focused on stemming the immediate costs of the covid-19 crisis: among them, mortality, business failures, job losses, and foreclosures. this pain is salient, and as such, very obviously demands an urgent response. however, there are potential outcomes of the current pandemic which, while perhaps less salient, also merit urgent attention: namely, long-run damage to human capital and wellbeing. it is to these particular long-run effects that we turn our attention in this paper. 1 the potential for long-run harm to human capital arises from two main facts about the current pandemic. first, key features of covid-19-among them its geographic reach, its relatively high ease of transmission, its comparatively low lethality, and its many emerging sequelae-have given rise to widespread and potentially lasting morbidity among its many survivors. second, the pandemic has sparked an unprecedentedly large downturn, which in its own right has the capacity to permanently scar trajectories of health and income, even for those who do not fall ill themselves. while the costs of these long-run effects may seem far away, they are latent today and could become massive down the line: burdening healthcare systems and government assistance programs, suppressing work capacity and human capital investment, and reducing economic prosperity more generally. luckily, the returns to avoiding these harms, or to acting swiftly to compensate for them before they have a chance to compound, tend to be much higher the sooner interventions can be made (see, e.g., heckman, 2007; almond & currie, 2011) . together, the potential for diffuse and latent adverse effects, and the cost-effectiveness of early remediation, suggest that in addition to any efforts to address the immediate pain of the pandemic, our eyes should also be on the future-and on actions we can take now to mitigate the long-run pain for affected cohorts, and therefore, the wider economy. but what, exactly, is the long-run human fallout of covid-19 likely to be-and who will bear the brunt of this crisis? to answer these questions, we need the sort of long-run view that only history can provide. an obvious starting point is to look to evidence from historical pandemics. despite potential differences in empirical settings and epidemiological characteristics, the sheer number and diversity of past pandemics means that covid-19 has many close historical analogues as a health crisis. for instance, while the current pandemic is frequently described as unprecedented, in many ways, its immediate effects on health are not altogether anomalous. with cases first appearing in december 2019, sars-cov-2, the pathogen behind covid-19, spread throughout the world in a matter of weeks, with deadly consequences. by the end of april 2020, worldwide cases had topped 3 million, and fatalities exceeded 200,000. as of this writing in late october 2020, and with the pandemic still spreading, cases exceed 43.8 million, and fatalities have surpassed 1.1 million. 2 for context, deaths from h1n1 (swine flu) in 2009-10 were smaller in magnitude, with estimates of over 200,000 deaths attributable to the virus (dawood et al., 2012) . while at 1 million deaths, the hong kong flu of 1968 is comparable to covid-19 in its death toll to date, the asian flu of 1957 was substantially deadlier, killing 2 million people. likewise, cholera, typhus, smallpox, measles, and tuberculosis all have had high death tolls, including during the 20th century. reaching even further back, the black death left a devasting imprint on the world, killing a third of europe's population. 3 clearly, historical pandemics offer a rich evidence base that can help shed light on the range of possible long-run effects of covid-19 through morbidity and mortality. however, there is one crucial aspect of the current pandemic that sets it apart from all but the most catastrophic historical disease outbreaks 4 : the presence of an acute public health crisis alongside massive and widespread economic disruptions. not just that-it is the fact that this health crisis has precipitated an economic one. to wit, efforts to stop the spread of the virus, alongside failures to contain it, have contributed to a dramatic slowdown of the global economy. consider, for instance, the economic dislocation experienced in the u.s., a country which quickly came to lead the world in both confirmed covid-19 cases and deaths. in march and april of 2020, roughly 20 percent of the united states' labor force filed unemployment claims. double-digit unemployment would continue through the summer. the dow jones fell by over 35 percent. for contrast, during 1957 influenza pandemic, responsible for roughly 100,000 deaths in the u.s. (glezen, 1996; simonsen et al., 1997) , unemployment peaked at 7.4 percent, and the dow fell 15 percent-certainly a recession, but nothing on the order of what we have already experienced during the covid-19 outbreak, just a few months in. the unprecedented scale of the covid-19 economic downturn relative to past pandemics is apparent in figure 1 , which shows the evolution of u.s. gdp over time, with major epidemics highlighted. notes: annual gdp per capita data for 1790 through 2019 are taken from https://www.measuringworth.com. quarterly real gdp per capita data for 2019 and 2020 are taken from https://fred.stlouisfed.org/series/a939rx0q048sbea and deflated to 2012 3 plague would continue to impact economies into the 20 th century, with an outbreak in san francisco infecting 121 individuals and killing 119 (echenberg, 2010) . the san francisco outbreak presents interesting parallels to . despite health officials identifying the plague and urging action, california's governor, henry gage, denied there was an outbreak, partly out of a desire to prevent business losses from a quarantine. it took the intervention of federal authorities and a new governor to implement proper measures to stop the spread of the plague. california officials reacted quite differently to covid-19, at least initially, swiftly imposing stay-at-home orders. a century later, the calculus of weighing an economic slowdown against the spread of disease has changed. despite a much larger population, and a much more widespread pandemic, covid-19 had claimed under 150 lives in san francisco county as of late october 2020, thanks in part to these relatively early and stringent interventions. 4 for instance, the aids crisis and the black death may be some of the only other major pandemics where mass morbidity and mortality were accompanied by dramatic and widespread economic dislocation. dollars to match the historical data. code to generate the figure and the underlying data for it can be found at open-icpsr (https://doi.org/10.3886/e125441v1). clearly, this feature of the current pandemic calls for complementary evidence if we are to understand its potential for long-run harm: there is no suitable all-in-one historical analogue for covid-19, and evidence from past pandemics alone is likely to understate the potential for damage to (or intervention in) health and welfare through income and labor-market channels. indeed, to continue with our u.s. example, the two-trillion-dollar coronavirus aid, recovery, and economic security act's closest comparison, is not to be found in past responses to health crises, but rather in the response to past macroeconomic crises-e.g., the american recovery and reinvestment act in the case of the great recession, and the new deal in the case of the great depression. federal outlays as a percentage of gdp rose from 3.3 percent at the start of the great depression to 9.8 percent by 1934. the cares act alone is equal to roughly 10 percent of 2019 gdp, and this does not account for additional relief that may be approved in the coming months. 5 asset purchases in response to the great recession increased the federal reserve's balance sheet from $0.9 billion in 2007 to $4.5 trillion in 2015. that balance sheet has gone from $4.2 trillion in february of 2020 to $7.2 trillion just four months later. 6 by nearly any metric, covid-19 has generated both an economic crisis and a government response of historic scale. studying how individuals emerged from these primarily economic disasters, and what role government fiscal interventions played in their recovery, may therefore help us flesh out the incomplete perspective we would gain from studying past health shocks alone. turning to a combination of historical crises, then-past pandemics and recessions, both-allows us to consider events that in many ways more closely mirror current circumstances, and whose contextual differences can themselves be informative of our current situation first, and most crucially, these events have had time to fully unfold: the short-, medium-, and long-run consequences of these events can be directly observed. second, the diverse array of historical events, settings, and mechanisms provides a set of reasonable analogues for covid-19, even as our understanding of covid-19 evolves. third, the economic history literature shows how much can be learned with clever analysis of even incomplete or imperfect data. missing and inaccurate health data is unfortunately directly relevant to assessing the spread of covid-19, given, for instance, current issues with testing and coordination. thus, a historical perspective allows us to use rich data to look at not only the short-term effects of crises like covid-19 on health, labor, and human capital, but also the long-term and intergenerational impacts along these dimensions for both individuals and the wider economy. in so doing, it can offer us insight on the current crisis-telling us what to look for, what to prepare for, and what data we ought to collect now. put another way, understanding the lingering health and economic impacts of these past crises offers valuable insight for anticipating and responding to the potential long-term impacts of covid-19. to examine how history can inform our view of the coronavirus pandemic and associated policy responses as they relate to long-run wellbeing, we begin in section ii by reviewing the features of covid-19 that will determine its potential health and economic impacts, and placing these features in historical context. then, in sections iii and iv, respectively, we narrow our focus to two of the closest analogues to the current pandemic-one, the 1918 influenza pandemic, which speaks to -direct‖ health-channel effects; and another, the great depression, which speaks to -indirect‖ effects through the labor market and wider economy. there, we review the economic literature on the short-and long-term effects on cohorts exposed to these massive shocks, and discuss how these short-run experiences can give rise to lasting, and sometimes hidden, damage. we conclude by discussing what economic historians and researchers of covid-19 can offer each other. before we can look to historical evidence on how covid-19's effects may unfold in the long run, it is useful to fix ideas about key features of the current crisis-its epidemiology, its demographics, and the policy responses to date. comparing these features to those seen in past pandemics offers a sense of which historical pandemics might serve as the most useful points of reference going forward. we draw here on the principles outlined by morens et al. (2009) to categorize pandemics. they point to eight characteristics common to most accepted definitions of a pandemic: 1) wide geographic extension and 2) disease movement, which speak to the disease's spatial reach; 3) high attack rates and explosiveness, 4) infectiousness, and 5) contagiousness, which speak to how it spreads; 6) severity, which speaks to its potential for population scarring and culling; and 7) minimal population immunity and 8) novelty, which speak to the scope for harm and the speed with which preventive and therapeutic responses can be marshalled. by all measures, covid-19 presents these hallmark features of a pandemic. understanding exactly how covid-19 reflects each dimension is essential for understanding the likely short-and long-run consequences of the pandemic. the widespread nature of pandemics makes their health and economic impacts particularly devastating: with effects felt everywhere, it becomes increasingly difficult to shift economic activity to, or medical resources from, unaffected areas. while the true extent and timing of covid-19 cases is yet to be determined, the evidence to date indicates that the global spread of the virus has been incredibly rapid. the earliest reported cases appeared in december 2019 in wuhan, china. that same month, the virus made it to france. by january 2020, there were confirmed cases throughout asia, europe, north america, and africa, and by the end of the month, the number of cases worldwide reached 10,000. in the months that followed, that number rose sharply-first to 85,000 in february, 750,000 in march, and over 3,000,000 by the end of april. by may 2020, only 12 sovereign states had no confirmed cases, 10 of which are island nations in oceania. covid-19 had become a truly global pandemic by the end of spring 2020, and both cases and fatalities have continued to rise across the globe in the months that followed. 7 this feature of covid-19 surely has much to do with the highly globalized nature of our modern economy. indeed, we see similar patterns in historical pandemics, reaching as far back as we have had extensive trade routes. 8 nearly every country with reliable mortality statistics displayed excess deaths from the 1918 influenza pandemic . similarly, the plague pandemic originating in canton and hong kong in 1894 spread to 77 ports across five continents (who, 2000) , and even the justinian plague of 542 reached asia, africa, and europe. even in a historical era where countries were less tightly integrated than they are today, 7 as of late october 2020, there have been nearly 44 million cases of covid-19 worldwide, and over 1 million deaths-and, as winter dawns on the northern hemisphere, and as we enter a new and possibly more lethal wave of the pandemic in many parts of the world, these numbers seem poised to rise further. 8 even though world economies are substantially more tightly integrated than in the past, even in the preindustrial era, alfani & murphy (2017) document that it was common for disease to be transmitted along trade routes or through inter-regional commercial contact. big trading centers in particular, such as amsterdam, london, and venice, frequently faced outbreaks of plague (see alfani (2013) , biraben (1975) , and curtis (2016) for the underlying studies). the only thing that truly spared an area from pandemics was isolation. 9 with the increase over the last few centuries in both global connectedness and population density, the implications for our current crisis are clear. the speed with which a disease spreads directly impacts the difficulty of containing it. indeed, it is these transmissibility-related features that account for many of the public health measures seen in response to the current crisis-some, such as early international travel restrictions, which tried to contain a disease that in many countries was already being spread locally via community transmission; and some, such as stay-athome orders and mask-wearing, which have been more effective in slowing transmission once it was too late for a containment strategy to be tenable. high attack rates and explosiveness (multiple cases appearing in a short time span) make it hard to stay ahead of a disease. these characteristics are functions of a disease's infectiousness and contagiousness: its ability to spread from person to person. covid-19 is transmitted by respiratory droplets and aerosols produced when an infected person coughs or sneezes. this has led to covid-19 having a daunting rate of transmission, with early estimates of a basic reproductive rate of between 4 and 9 (sanche et al., 2020) . 10 as a point of reference, these transmission numbers are akin to those seen for past sars, polio, mumps, yellow fever, and 1918 influenza outbreaks (see figure 2 ). the economic history of these pandemics thus provides a guide for what we might expect from the covid health crisis. for contrast, the economic history of measles-which presents far higher transmission rates, with estimates of basic reproductive rates greater than 10 (guerra et al., 2017)-offers a sense of how much worse things could be. notes: case mortality rates are for untreated patients. for covid-19, basic reproduction rates are taken from https://wwwnc.cdc.gov/eid/article/26/7/20-0282_article. all other reproduction and fatality rates are taken from https://docs.google.com/spreadsheets/d/1khcewy-d9hxlwrft9jjrq2xf6whqlmwyrxel6wjxkw8/edit#gid=0 (the data 9 the opening up of regions to trade, or conquest, has generated a large literature on the role of disease in shaping economies. see in particular the literature on the columbian exchange (nunn & qian, 2010) . 10 the basic reproductive rate is the number of expected cases directly generated by one case when all individuals in the population are susceptible to infection. like the disease's ease of transmission, the severity with which it manifests symptoms will also be a crucial determinant of both its consequences for individuals and the wider economy, and the nature and magnitude of the government response. for instance, a highly lethal pandemic may generate extensive and indiscriminate mortality; a less lethal pandemic may generate culling (selective mortality related to a specific health threshold); and an even less lethal pandemic may generate very little mortality, but substantial health scarring among survivors. if a disease is so mild that many of those who are infected remain asymptomatic, this can, in the absence of widespread testing, undermine efforts to slow transmission. likewise, rates of infection, in combination with severity considerations, will help determine whether governments intervene, or merely wait for the disease to -take its course‖ on the way to achieving herd immunity. in its april 14, 2020 covid-19 strategy update, the world health organization note that 40 percent of those infected experience moderate disease, including pneumonia, and 15 percent experience severe disease. they cite a crude clinical case fatality rate of over three percent that rises to 15 percent or higher in individuals over the age of 80. 11 as shown in figure 2 , the crude mortality rate for covid-19 in its first months is similar to that of the 1918 influenza pandemic and of measles, but far lower than the deadlier recent outbreaks of mers, ebola, sars, and lower still than the truly devastating historical toll of smallpox, which had an average case fatality rate of 30 percent (ellner, 1998) . this rich historical spectrum of pandemic severity, in turn, demonstrates that both mild and severe diseases impact the economy, albeit in very different ways. for instance, the eradication of hookworm in the u.s. south-a disease which is not fatal, but which primarily causes lethargy and anemia-improved returns to schooling, educational attainment, and incomes in areas with high prior infection rates, but did little to change to overall demographic, economic, or institutional structure (bleakley, 2007) . for contrast, the black death and other pre-modern outbreaks of plague, which had extraordinarily high death tolls, fundamentally reshaped the global economy through their effects on population size and demographic structure. 12 in this context, covid-19's wide scale and relatively low lethality will surely have a bearing on the scope, magnitude, and timescale of damages. as we will see in later sections, it suggests that we might ultimately expect to see the greatest harm only in the long run, with widespread generational scarring arising from short-run morbidity and economic disruptions. the novelty of a pandemic virus contributes to its potential for destruction: it takes time to identify a new disease, understand key features of its epidemiology, develop treatments and vaccines, and achieve a degree of population immunity. in the meantime, everyone represents a potential victim. as a novel coronavirus, 11 it is worth emphasizing that even if a patient survives covid-19, they may still face substantial harm. for instance, emerging research suggests that some covid patients may experience persistent symptoms well beyond the normal recovery period, and that others-even some with relatively mild cases-may nevertheless suffer permanent respiratory, cardiovascular, and neurological damage. 12 voigtländer & voth (2013a , 2013b argue that the increased wages due to labor shortages from plague mortality increased demand for urban products, driving a cycle of urbanization and additional disease that moved europe out of a malthusian trap into a modern world of permanently higher per capita incomes. dittmar & meisenzahl (2017) demonstrate that outbreaks of plague aided the spread of reformation laws and the expansion of public goods. for more on the economic history of plagues, see alfani & murphy (2017) . covid-19 struck a population with neither natural nor acquired immunity: wherever the virus spread, it had the potential to be devastating. with little immediate means of preventing, testing for, or treating it, some of the only short-run mitigation strategies available have been relatively brute-force ones such as lockdowns and border closures. consequently, economic shutdowns-resulting both from official government actions and from individuals taking actions to avoid exposure-have been widespread, leaving no major economies or populations spared. interestingly, because medical technology was limited for much of the past, and societies could only count on some degree of population immunity, even endemic (i.e., non-novel) diseases could have the sort of destructive potential we only typically see today in new disease variants such as the novel coronavirus. 13 for instance, in a variety of past pandemics studied by economic historians, cases of an endemic disease would sporadically rise sharply, with substantial consequences for living standards and economic organization. indeed, a large literature considers the impact of such diseases on the growth trajectories of countries over the long run, often focusing on tropical diseases like malaria, yellow fever, dengue, and others. one strand of studies considers the direct impact of disease on human capital formation (see, for example, bleakley (2003 bleakley ( , 2010 on malaria). another strand focuses on the impact of these endemic diseases on institutional development, finding that disease environments inhospitable to colonial settlers drove them to rely on extractive institutions that were ultimately harmful to economic growth (acemoglu et al., 2001) . finally, scholars have considered the way that one society's acquired immunity to an endemic disease can devastate the economy of another society lacking that immunity (diamond, 1999; mcguire & coelho, 2011; tang, 2017) . together, this historical evidence gives us a picture of what our circumstances might look like today if we are unable to adequately ramp up our capacity for disease prevention and treatment, and are instead forced to rely on acquired immunity, the nature of which for covid-19 is still poorly understood. age has been at the forefront of discussions about the disparate impact of the current crisis, and shutdown efforts have been framed in part around protecting older individuals and other vulnerable populations, such as the immunocompromised, while societies work to expand medical capacity and develop a vaccine. in this respect, covid-19 is much like many infectious disease outbreaks in the past-though young people can both transmit the disease and become ill, it is the elderly and those with poor baseline health are at greatest risk. cdc estimates put the risk of hospitalization five times higher, and the risk of death from covid-19 90 times higher, for 65 to 74 year-olds compared to individuals in their twenties. 14 likewise, despite claims in some quarters that covid-19 is -the great equalizer,‖ it is already becoming clear that socioeconomic status will be central to understanding the demographics of this crisis. one of the ways low-income populations will be affected is through differential exposure to pandemic risks. 15 individuals who continue to do their jobs in person during the pandemic-including service-industry workers with extensive contact with customers, healthcare professionals, and other frontline workers-will 13 that is, it is improved medical knowledge that has allowed us to escape from the sort of flare-ups of endemic disease so frequently observed in the past, and it is relative our lack of knowledge about covid-19, as a new virus, that makes our current situation in some ways comparable to even past endemic disease outbreaks. economic historian joel mokyr expands on this idea regarding the evolution of knowledge regarding infectious disease outbreaks in a recent op-ed: https://www.cnn.com/2020/04/23/opinions/struggle-between-people-and-microscopic-pathogens-mokyr/index.html. 14 retrieved from https://www.cdc.gov/coronavirus/2019-ncov/covid-data/investigations-discovery/hospitalizationdeath-by-age.html, august 25, 2020. 15 the mirror image of differential exposure is differential transmission risk. this is one reason why some have called for prioritizing the strategic testing of workers at highest risk of spreading the novel coronavirus to others, particularly asymptomatic ones. bear a disproportionate burden of the pandemic's health impacts. these workers are more likely to be in low-paying jobs, and are more likely to be women and minorities, than their counterparts with jobs allowing them to work from home. consider, for instance, meat and poultry workers in the u.s. the mean annual wage in the industry is only $28,000 (bls, 2018) . 16 among laborers in the food manufacturing industry, 27 percent are black and 40 percent are hispanic (eeoc, 2018). 17 three-quarters of full-time, year-round healthcare workers are female, with that share even higher among the lower paid nursing and health aide occupations, critical occupations with severe risk of exposure to this disproportionate exposure to virus for lower income groups, women, and minorities is exacerbated by differences in these groups' access to healthcare and the quality of that health care-factors that affect both vulnerability and resilience to pandemic disease. membership in more than one of these groups will tend to compound disadvantage even further. preliminary research suggests that black patients exhibiting covid-19 symptoms were six times less likely to get treatment or testing than white patients. 19 this is not unique to covid-19: similar patterns have been observed for other modern pandemics including the h1n1 influenza outbreaks (quinn et al., 2011) . the outsized impact of pandemics on minority populations and people of lower socioeconomic status has historical precedent. 20 the 1918 influenza pandemic hit the poor first and hardest (sydenstricker, 1931; mamelund, 2018) , a point we will return to in section iii. explanations for this relationship mirror modern ones: poorer populations lived in denser housing units under worse conditions, and had occupations that increased exposure to the virus. 21 moreover, low incomes constrained their ability to avoid exposure and seek treatment. 22 historical evidence shows that to escape a 19 th century outbreak of yellow fever, wealthier residents often left the city-an option unavailable to low-income workers with tenuous job security. this is a pattern that we see as well during outbreaks of plague in earlier centuries, and is part of dittmar & meisenzahl's (2017) explanation for why the black death paved the way for institutional reform: the old elites simply left town (dinges, 1995; isenmann, 2014) . this sort of regional flight is unlikely to be a central dimension along which covid-19 has differential impacts across income levels-but it does raise important issues that set the current crisis somewhat apart 16 https://www.bls.gov/oes/2018/may/oes513022.htm 17 https://www.eeoc.gov/statistics/employment/jobpatterns/eeo1/2018/national-naics3/table?naics=311&state=&cbsa= 18 figures are based on the u.s. census bureau's calculations using american community survey data (https://www.census.gov/library/stories/2019/08/your-health-care-in-womens-hands.html). 19 https://www.nytimes.com/2020/05/10/us/coronavirus-african-americans-bias.html 20 the relationship between pandemic exposure and socioeconomic status has not always been constant. as alfani & murphy (2017) note, studies of the plague in europe find the black death to have been universally deadly. however, plagues of the fifteenth and sixteenth centuries exhibited the negative relationship between socioeconomic status and mortality found in more recent pandemics (see, for example, slack (1985) , alfani (2013) , and carmichael (2014)). however, the final major plagues of the seventeenth century once again tended to have severe consequences across all classes. 21 mamelund (2018) note that in norway, the impacts of the pandemic were most severe for transport, hotel and industry workers, paralleling the observations above about covid-19. 22 these factors also translated into worse outcomes for minorities. as the exception that proves the rule, black americans fared better than white americans during the 1918 pandemic (crosby, 2003; økland & mamelund, 2019) . crosby (2003) argues that the black population had disproportionately high exposure to the early, less virulent summer wave of the pandemic due to their worse occupations and living conditions, conferring some degree of immunity to the more deadly later wave. a similar mechanism operated during historical yellow fever outbreaks, albeit resulting in advantages for native-born individuals over immigrants. individuals born in an area with endemic yellow fever and exposed at a young age often contracted a mild form of the disease, and then developed immunity. as a consequence, adult immigrants were far more likely to die in yellow fever outbreaks in the united states than either native-born whites or blacks (patterson, 1992) . saavedra (2017) exploits this pattern to demonstrate that earlylife yellow fever exposure negatively impacted adult occupational outcomes, with white males born to immigrant mothers during yellow fever pandemics less likely to become professionals than the sons of native-born mothers. from other historical pandemics: the spatial distribution of population within and across cities, the degree of interconnection between rural and urban areas, and the extent of urban health penalties. while cities are much healthier today than in the past, 23 societies today are also much denser, more urbanized, and better connected-all factors that would tend to make modern pandemics both faster to spread and harder to control than in centuries prior. and indeed, while covid-19 has hit dense metropolitan areas particularly hard, as in the past, under the current crisis, rural communities have not been spared. this is in part because of the relative ease with which people circulate between communities with our modern transportation networks, but also because of the way that the nature of modern work tends to place individuals in close contact with each other, even in less densely populated areas. to wit, major rural clusters of covid-19 in the united states have been tied to large meat and poultry processing facilities, with workers at these facilities experiencing case rates an order of magnitude higher than the general u.s. population (dyal et al., 2020) . moreover, rural areas' demographic composition (often older and less affluent) and healthcare infrastructure (often sparser) can also contribute to their difficulties with pandemic disease. to respond effectively to pandemics in the moment, and to deal with their long-run fallout, will require an understanding of its distributional effects over time and space. we explore these central consideration in depth in section iii. evaluating the policy response to covid-19 and how it compares to historical pandemics requires recognizing that information on the disease and how to stop its spread has been limited to date, and is still evolving. 24 this issue stems in part from covid-19 being a novel disease-developing treatments and vaccines takes time, and public health recommendations can change as knowledge advances. it also stems from incomplete and inaccurate data: limitations on covid-19 testing has often meant relying on mortality rates rather than case rates. incidentally, this is the same approach economic historians are often required to take. morbidity data are rare historically and, when available, may be unrepresentative and inaccurate. mortality data are both far more prevalent and reliable, even if it is morbidity that is typically more relevant to the economic impacts of a pandemic, particularly less lethal ones. for health officials today, the need to assess the spread of covid-19 through mortality data leads to the frustration of identifying the arrival of cases with a substantial lag. for the economic historian, this lag is irrelevant, but the issue remains that only those places experiencing excess mortality can be identified; diseases leading to widespread morbidity but little mortality may be equally important for the evolution of economies, but far more difficult to identify prior to modern medical records. again, this suggests that evidence from crises that have run their course can be informative of what to expect going forward. while some of the challenges in developing effective covid-control responses have stemmed from incomplete and rapidly evolving knowledge of the disease, they have also stemmed from issues of state capacity, political will, and ideology. for instance, policymakers, firms, and individuals have been hamstrung by not only limited testing and contact tracing capacity, but also by a failure at times to deploy these tools efficiently. constraints such as these are a product of both the limitations of medical technology, and broader issues of political leadership and coordination. the inability to identify and isolate individuals at risk of spreading the disease, in turn, has necessitated rather blunt policy tools, such as business closures and stay-at-home orders. in the u.s. in particular, these covid-control efforts have been aggressively decentralized, and have tended to prioritize both commerce and individual liberty-even where these might be at odds with each other, or lead to ineffective disease control. 25 to wit, business owners and public officials have struggled to gain widespread compliance with (and have often declined to enforce) precisely the sorts of behaviors-e.g., mask-wearing, social distancing-that would allow for the safe reopening of businesses. indeed, it appears that the fear and uncertainty created by the failure to control the spread of disease, in turn, has contributed to prolonging economic pain (goolsbee & syverson, 2020) . challenges such as these, related to culture and institutions, are nothing new-in fact, they characterize the u.s.'s historical experience of managing epidemic disease. in his excellent the pox of liberty, troesken (2015) lays out how the very institutional features-among them a decentralized federal system, a focus on property rights and commerce, and protection of individual liberties-that led to the u.s.'s rapid economic development also often undermined its attempts to control past outbreaks of smallpox, typhoid, and yellow fever. the examples he provides have uncanny parallels to the u.s.'s approach so far to managing covid-19. strategies to manage the spread of covid-19 have been varied, with many jurisdictions pursuing multiple complementary approaches, often including coordinated sourcing and distribution of protective equipment, reallocation of medical capacity, virus and antibody testing, contact tracing, frequent sanitizing of public facilities, social distancing, mask-wearing, managing congestion in public places by staggering timings and moving activities outdoors, limiting large gatherings, quarantining infected individuals, and minimizing the risk of disease exposure via closures of businesses and schools and broader stay-at-home orders. of these strategies, shutdowns and quarantines have been some of the most accessible, widely used, and hotly debated under covid-19. a shutdown-centered approach such as this also has strong historical precedent. in fact, closures and quarantines were some of the only tools available to societies prior to the virology advances of the 19th and 20th centuries. though the shutdown of firms has been more comprehensive under covid-19 than in many past pandemics, 26 the primary measures being taken now, such as quarantining sick individuals, restricting public gatherings, and closing schools, were all implemented during the 1918 pandemic (markel et al., 2007; hatchett et al., 2007) , albeit with a smaller scope and shorter duration. 27 likewise, when england was combatting the 25 other countries offer a contrast on one or more of these dimensions. for example, though some of these early gains have since dissipated, taking a more authoritarian and centralized approach, china and india had some initial success in containing the virus following swift and complete lockdowns, underscoring the potential importance of state capacity and centralization in pandemic control. likewise, south korea's response demonstrated the importance of relative novelty: due in part to experience with past respiratory pandemics, pre-existing public health infrastructure and greater public buy-in with mask-wearing allowed them to respond more quickly and effectively to the greater use of firm closures as a disease-control strategy today could be due, for instance, to improvements over time both in the safety net and in remote-work capabilities. this more widespread pause on non-essential activities may in turn lead to fairly different effects of covid-19 relative to past pandemics, e.g., in terms of patterns of disease transmission or total economic impact. 27 while cities employed a diverse set of non-pharmaceutical interventions (npis) during the 1918 pandemic, only a small fraction of these interventions (namely, closures of public facilities, isolation policies, bans on public gatherings, and making influenza a notifiable disease) were widely practiced across localities (hatchett et al., 2007) . moreover, markel et al. (2007) document that npis were in place for only 2 months or less in 19 out of the 43 cities in their sample; even the maximum duration in their sample (170 days, in kansas city, missouri) still falls far short of the u.s.'s experience of covid-19 npis to date. indeed, barro (2020) suggests that the short average duration of npis during the 1918 pandemic led them to be relatively ineffective in curtailing mortality. the efficacy of npis depends on factors including the type of intervention, when it is first implemented, how long it is in place, and the strictness with which it is implemented. because of this, and although npis under covid-19 have been in place longer than during the 1918 pandemic, it is difficult to say at this stage whether this necessarily means that they will have been more effective in reducing morbidity and mortality-particularly because covid-19 npis in the u.s. have tended to be implemented somewhat late in the pandemic's course, have tended to be intermittent and noncomprehensive, and have tended to be leniently enforced, all factors which undermine efficacy and which may themselves be contributing to the need for longer npi duration. plague in the 1600s, they quarantined ships from other countries, closed ale houses, and limited the number of lodgers allowed in a house, actions that would sound familiar to cruise ship passengers and restaurant owners during the covid-19 pandemic (bell, 1924) . indeed, it is striking-maybe even alarming-how little has changed about our best options for fighting pandemics, despite centuries of advances in medicine, public health, and living standards. this policy response, necessitated by factors including inadequate testing and broader uncertainty about key epidemiological parameters-even those as basic as precisely how and through whom the disease can be transmitted, and whether it is possible to become re-infected-makes the economic history of policy responses to pandemics particularly relevant for studying the current crisis. 28 even when we contemplate a world where successful covid-19 vaccines are available, history sounds a note of caution: the same underlying issues that have made mask-wearing both incomplete and fraught in settings, like the u.s., with a strong institutional commitment to liberty and rugged individualism, could also be expected in the context of covid-19 vaccination. as troesken (2015) notes, anti-vaccinationism has a long history in the u.s., bolstered by the common failure to appreciate the extent of infectiousdisease externalities (the choice not to vaccinate can be individually rational, even if people understood externalities, which they largely do not), as well as by a belief in both minority rights (individuals cannot be forced to vaccinate) and federalism (individuals preferring not to vaccinate can sort into amenable jurisdictions). 29 while troesken documents that mandatory vaccination was frequently enforced in the past via fines, 30 or by denying access to schools or other public services, it is difficult based on the nature of the u.s. covid-19 response to date to imagine such enforcement mechanisms being implemented. instead, in heterogeneous, strongly pro-individual, pro-freedom societies, we may need to rely on a stylized fact that troesken demonstrates using data from 19 th century smallpox epidemics in germany: vaccinations rates rise in pandemic years, because during pandemics, the risk of infection rises sharply, and the private costs of non-vaccination are clearly outweighed by the private benefits. for thinking about the direct effects of pandemics on the health and wellbeing of individuals in the shortand long-run, the 1918 influenza pandemic, or the -spanish flu,‖ provides a useful point of reference. covid-19 to date parallels the 1918 pandemic in several key ways, including its rate of transmission, global spread, and crude mortality rates. 31 the spanish flu was one of the most acute and widespread natural disasters in modern history. taubenberger & morens (2006) estimate that during the pandemic, roughly 500 million individuals, equivalent to roughly a third of the world's population at the time, were infected and symptomatic. case fatality rates, at over 2.5 percent, were at least 25 times as high as in other influenza pandemics, making the 1918 virus especially lethal. all told, somewhere between 50 and 100 million individuals perished globally. the death toll in the u.s. alone exceeded that from all american combat deaths over the twentieth century (almond, 2006) . 28 see for example markel et al. (2007) on the effectiveness of school closures in combatting the spread of the 1918 influenza pandemic, meyers and thomasson (2020) on the effects of polio-related school closures on educational attainment, and alfani & murphy (2017) on city-level quarantines during pre-modern plagues. 29 cultural features like distrust of scientific expertise, and of institutions more generally, may also undermine vaccine compliance. 30 troesken (2015) notes that the underlying legal theory behind vaccination non-compliance fines gained further credence after the supreme court upheld the affordable care act's insurance mandate in national federation of independent business v. sebelius, 567 u.s. 519 (2012). 31 there are challenges to drawing lessons from the 1918 pandemic. much remains unknown about the origins and epidemiology of the virus and its economic impacts are confounded by the effect of world war i. the pandemic itself was sharp, sudden, and concentrated over the span of little more than a 12-month period. the virus, an h1n1 strain similar to that which caused the 2009 swine flu outbreak, spread roughly simultaneously across europe, asia, and north america, in three distinct waves over the year beginning in spring 1918. the first of these waves, appearing in march 1918, was relatively mild. it was followed by a substantially more catastrophic one from september to november 1918, and another in the early months of 1919 (taubenberger & morens, 2006) . in some parts of the world, particularly in east asia, a further major wave of pandemic influenza hit as late as 1920 (lin & liu, 2014; ogasawara, 2018) . this sort of timing and spacing was unprecedented among influenza pandemics, as was its distinctive mortality profile. where influenza death rates by age typically follow a u-shape, with high mortality rates among the very young and the very old (as is also the case with the sars-cov-2, the virus behind covid-19), the 1918 strain followed a w-shape, with a sharp peak in mortality risk among young adults as well. 32 indeed, almost half of all influenza-related deaths during the pandemic period accrued to those aged 20-40 (taubenberger & morens, 2006) . the age pattern associated with this strain of influenza was in fact so unusual that it has been exploited as a diagnostic tool in recent studies. for instance, while the 1918 influenza pandemic is typically thought to have emerged in full force in europe around the summer of 1918, and in a milder form somewhere in the central u.s. in spring 1918, detailed age-by-month mortality statistics allow olson et al. (2005) to uncover evidence that an early -herald‖ wave of pandemic influenza was actually present in new york city well beforehand, from february to april of 1918. during this period, the age profile of excess influenza mortality had started to shift from the older ages typical of interpandemic seasons to the younger ages that characterize pandemic seasons. this underscores the value of accurate and disaggregated data in tracing the origins and spatiotemporal spread of pandemics, and the need to strengthen not only rapid-response public health infrastructure, but also that to support ongoing disease surveillance. turning to morbidity, those under the age of 35, and particularly, those aged 5-14, had disproportionately high incidence of influenza-however, the latter group had a much lower death rate from influenza and pneumonia than other ages, further sharpening the middle peak in the morbidity-adjusted pandemic mortality curve (taubenberger & morens, 2006) . age, however, was not the only major factor that contributed to pandemic mortality risk, and a range of recent studies have emerged cataloging the often interrelated features of countries, cities, and individuals that led to disparities in the immediate mortality burden of the 1918 flu. on these mechanisms, the evidence is mixed-surely in part because of diverse empirical settings and disciplinary approaches-but certain patterns do emerge. first, baseline health status mattered: both pre-pandemic pneumonia, a bacterial condition with a strong biological interaction with the influenza virus, and infant mortality rates, a proxy for population health, contributed to higher pandemic flu mortality (acuna-soto et al., 2011; clay et al., 2019) . likewise, high levels of air pollution, an environmental factor that aggravates respiratory conditions and depresses baseline health, also raised pandemic mortality. for instance, clay et al. (2019) examine evidence from a panel of 438 u.s. cities, and find that the air pollution generated by coal-fired electricity plants was a significant contributor to pandemic mortality, with effect sizes roughly half those associated with measures of population health and poverty. 33 together, they estimate that these factors accounted for approximately half of all cross-city variation in pandemic mortality. in another study, they find that both infant and all-age mortality were impacted adversely by the presence of coal-burning plants, with poor air quality responsible for 19-26 percent of total pandemic mortality in high-and medium-pollution cities, a figure equivalent to some 30,000-42,000 excess deaths beyond those attributable to the pandemic alone (clay et al., 2018) . 34, 35 second, population density and related concerns, such as housing quality and the number and composition of social interactions, were also important factors in pandemic mortality. in europe as in the u.s., the pandemic came to cities earlier, and was more devastating there, a phenomenon linked to urbanization and residential crowding (chowell et al., 2008; mamelund, 2006; murray et al., 2006) . transmission was localized, and influenza and pneumonia mortality exhibited significant and rather tight (e.g., 200-1,500 m) spatiotemporal clustering (grantz et al., 2016a,b; tuckel et al. 2006) , though proximity to high-risk population centers like wwi military bases appears to have had little effect (clay et al., 2019) . although urban centers were associated with higher pandemic mortality, the opposite population gradient prevailed when comparing among cities, or among rural areas: in both cases, smaller, less dense localities fared worse (acuna-soto et al., 2011; chowell et al., 2008) , suggestive perhaps of capacity constraints in the healthcare workforce and medical infrastructure. third, factors-such as illiteracy and foreign-born status-that might have prevented individuals from adopting public health recommendations were strong predictors of elevated mortality, often above and beyond their association with poverty. 36 higher rates of illiteracy were linked to higher rates of influenza mortality during the pandemic, across both cities and neighborhoods (clay et al., 2019; grantz et al., 2016a, b) . likewise, foreign-born status not only predicted higher pandemic mortality in hartford, connecticut, but the relationship between nativity and mortality persisted even after controlling for socioeconomic status, population density, and neighborhood ethnic composition, indicating perhaps a role for social factors, or language or cultural barriers to the adoption of relevant public health measures (tuckel et al., 2006) . crucially, the consequences of these barriers were not limited to the foreign-born: holding all else equal, native-born individuals living in areas with a higher share of foreign-born had higher mortality rates than their counterparts living alongside a lower share of foreign-born neighbors. this emphasizes the importance of neighborhood spillovers in infectious disease transmission-and, of course, demonstrates the interrelated nature of individual-and neighborhood-level mechanisms. 33 it is possible that indoor pollution and seasonality also played a role in air quality-influenza interactions, both during and outside pandemic times. for instance, influenza is generally prevalent in the winter, a time when coal smoke from home heating also tended to peak in this era (barreca et al., 2014) . 34 clay et al.'s (2018) observation that modern levels of pollution in parts of the developing world, including india and china, are on par with those in the early 20 th century u.s., sounds an ominous note in light of the current crisis-though the circumstances today (e.g., improved medical technology, the higher baseline share of trafficrelated emissions, a fall in pollution due to widespread economic shutdowns) may be just different enough to ameliorate concerns over the lethal interaction between pollution and pandemic influenza. 35 while pollution can lower baseline health by undermining the respiratory system, it is worth noting that pollution may also be associated with higher baseline health, insofar as it proxies economic activity. for instance, clay et al. (2016) find evidence of crucial tradeoffs between the income generated through industrial activity on the one hand, and the pollution generated on the other in the u.s. from the 1930s to the 1960s. in less developed localities, infant mortality followed a u-shaped pattern with respect to the expansion of coal capacity: first falling as rising incomes and cleaner residential energy sources buoyed infant health, and then rising as subsistence health needs were met and the concentration of pollution grew. the net health effects of a pandemic that dampens economic activity (and so reduces pollution), then, is therefore likely to be context-specific, depending on factors such as the level of baseline health and income, the extent of medical infrastructure, and the strength of social safety nets. 36 troesken (2015) also points to individualism and liberty as cultural/institutional values that tend to lower individual-level compliance with public health recommendations. when considering these biological, demographic, and socioeconomic factors in quick succession, it is difficult not to see the overarching hand of income in all of these mechanisms-though, to be clear, several of these studies are careful to disentangle these factors from their association with income. in theory, income gradients in pandemic mortality could arise through a number of channels, including many of those hinted at above: e.g., the tendency of those with higher incomes to have better baseline health status, rendering them biologically less vulnerable and more resilient to infection; higher-quality and lower-density housing, reducing the chances of viral transmission; better public health knowledge, the human capital necessary for individuals to effectively assimilate this knowledge and to adopt life-saving recommendations, and timelier and more robust public health interventions, all slowing the spread of illness; better access to healthcare and medical infrastructure, improving the probability of survival conditional on infection; and a greater capacity for individuals to undertake avoidant, adaptive, and compensatory behaviors, both throughout and following the pandemic. crucially, these channels can operate at both individual and institutional (e.g., city or country) levels, with both richer people and localities-and certainly, the interaction of these-theoretically better equipped to weather the crisis. the fact that some of these channels are highly correlated, of course, can make it difficult to pinpoint the underlying mechanisms: higher-socioeconomic status (ses) individuals are likelier to be both healthier, protecting them from infection, and more educated, rendering them better able to adopt public health measures; cities tend to be richer in both income and infrastructure, but they are also more heterogeneous and densely populated than rural areas. nevertheless, the literature can still shed light on the role of income on net. while some studies explicitly looking at its role in pandemic severity have shown little relationship between pre-1918 economic development and pandemic mortality (brainerd & siegler, 2003) , a great many indicate that poverty exacerbated mortality risk. for instance, murray et al. (2006) document tremendous (i.e., over thirty-fold) within-and cross-country variation in excess mortality due to the 1918 pandemic, with nearly half of this variation explained by baseline per capita income. taking a finer-grained look at these issues, grantz et al. (2016a,b) explore the socioeconomic determinants of pandemic mortality and transmissibility using detailed data from chicago. among the associations they find between health and various poverty proxies are large, statistically significant, and negative associations between census tract-level homeownership rates and mortality. these findings are consistent with the lower baseline health of lower-ses neighborhoods, their poorer access to medical care, and their lower awareness and adoption of public health recommendations. shanks & brundage (2017) add that these factors may be proxying other features of low-ses populations, such as a higher risk of sequential infections (e.g., pandemic influenza followed by a secondary bacterial infection such as pneumonia), or the larger number and lower-ses composition of their social interactions. all of these could have contributed to higher cumulative pandemic mortality through faster and more widespread disease transmission, higher incidence of infection, or higher case fatality rates. these results suggest that rather than acting as a democratizing force, the pandemic further entrenched preexisting socioeconomic disparities. the clear implication of studies documenting the immediate health effects of the 1918 outbreak is that the damage from pandemics has, and remains likely to, fall disproportionately on disadvantaged communities. apart from its effects on health, however, the pandemic also had important consequences for population dynamics. one such effect pertains to temporal and cross-disease mortality spillovers resulting from pandemic-era mortality patterns. noymer (2011) shows that the 1918 influenza pandemic hastened the decline of tuberculosis in the u.s. through a harvesting mechanism. specifically, he suggests that independent competing risks may be responsible for this phenomenon, driven by substantial age overlap in the profile of prospective tuberculosis and (pandemic-type) influenza victims. this -passive selection‖ contrasts with -active selection‖ based on biological interactions between influenza and tuberculosis. this harvesting, in turn, had long-lived implications for sex differences in post-pandemic mortality rates: because tuberculosis morbidity disproportionately affects men, and because the influenza pandemic reduced the pool of those who might die of tuberculosis in the years following, the pandemic had the effect of eroding women's longevity advantage over men. we might expect similar outcomes in the context of covid-19 given that a large share of those dying have one or more co-morbidities, though the distinct age profile of 1918 pandemic deaths versus covid-19 deaths may complicate these dynamics. studying brazil, guimbeau et al. (2020) likewise find rather larger reductions in sex ratios at birth following the 1918 influenza pandemic, consistent with the greater vulnerability of male fetuses to adverse in utero shocks-a phenomenon often seen in the literature on famines and environmental disasters. such changes in the sex ratio, or in sex-specific survival, may well have had long-run implications for marriage and labor markets. another major area in which the pandemic affected demographic behavior relates to marriage and fertility. in some cases, this was largely a function of pandemic psychology. mamelund (2004) shows that a climate of fear and uncertainty in 1918 norway, alongside social distancing efforts and peculiarities of norwegian marriage laws (which imposed a one-year waiting period before widows could remarry), led to a drop in births in 1919, as families deferred childbearing. higher rates of maternal mortality and miscarriage during the pandemic likely also contributed to a drop in birth rates. this pent-up demand for children (alongside -replacement‖ demand for children lost to the pandemic) was released after the crisis passed, resulting in a baby boom in 1920. elsewhere, as was the case in nearby sweden, changes in fertility arose from the way that pandemic mortality affected markets for marriage and labor: boberg-fazlić et al. (2017) find evidence of a drop in fertility during the pandemic, followed by a short-lived rebound in post-pandemic fertility. the net effect in the long term, however, was to reduce fertility-due in part to persistent disruptions to marriage markets (particularly in rural areas and poorer cities); the adverse effects on income; as well as to behavioral changes induced by the pandemic, including a rise in female labor supply (and so, an increase in the opportunity cost of childrearing) in regions with high male pandemic mortality rates. perhaps most noteworthy, the short-run post-pandemic fertility increase was selective in nature: a child born during this boom was more likely born to mothers who were married or who were high-ses city-dwellers. this was largely driven by postponement fertility, and particularly, selective postponement. 37 finally, pandemic-related mortality affected childbearing through its effect on survivors' incomes. donaldson and keniston (2014) show that the high pandemic death toll in some regions of india implied a substantial increase in per capita incomes, as survivors assumed the agricultural land of pandemic victims. in light of this rise in incomes, they find an increase in both the quantity and quality (given by literacy and height) of children born following the pandemic in india. phenomena such as these, which change the sex-and age-composition of the population-not to mention the average health status of successive cohorts-are likely to have long-lived effects on economic development, population health, and individual wellbeing. the lethality and peculiar age profile of the 1918 pandemic also give rise to long-run considerations. these may be especially relevant in light of covid-19, where the vast majority of people who become sick ultimately survive. 38 during the 1918 pandemic, young adults-including prime childbearing-age womenwere some of the likeliest to fall ill: in some parts of the u.s., roughly a third of all mothers (relative the about 28 percent of the general population) became infected during the crisis (almond, 2006) . moreover, 37 across settings, evidence of replacement fertility is rather more limited. 38 note however that covid-19 appears to be less prevalent, and possibly less severe, among prime-aged people. consequently, it is possible that scarring through the health channel under covid-19 may end up being less severe, and/or less widespread, than that following the 1918 influenza pandemic. scarring through the income/labor-market channel, however (explored in more detail in section iv), could well be substantially worse following covid-19 than the 1918 pandemic, given the latter's relatively mild and short-lived effects on the economy. despite the very high mortality rates from this pandemic, most of those infected ultimately survived. this left considerable scope for maternal morbidity-and, through the impact of maternal stress and illness on intrauterine hormones, nutritional resources, and other factors-for insults to fetal health. 39 in what is perhaps the seminal study in economics of the 1918 influenza pandemic's long-run effects on wellbeing, almond (2006) finds wide-ranging adverse effects on later-life human capital and labor market outcomes among u.s. cohorts exposed to the pandemic in utero. these include substantial reductions in high school completion rates, wages, and socioeconomic status, alongside large increases in the probability of living in poverty, the receipt of welfare payments, the likelihood of incarceration, andparticularly among men-the probability of physical disability. 40 that these adverse outcomes exist in spite of a pandemic-induced increase in miscarriages, stillbirths, and infant mortality rates (see, e.g., guimbeau et al., 2020; mamelund, 2004 )-all culling forces which likely resulted in a pool of survivors if anything positively selected on health-is a testament to the catastrophic extent of post-1918 scarring. almond's initial study has also since spawned a large and varied literature interrogating the long-run effects of the 1918 pandemic across a range of global settings. a first set of studies dig deeper into the u.s. case. one such study shows that 1919 birth cohorts (and in particular, those born in quarter 2 of 1919, who were in utero at the height of the pandemic), are 4 percentage points (or 10 percent) more likely to report fair or poor health than their counterparts born in surrounding years; see a statistically significant 17-35 percent increase in a range of functional limitations, including trouble hearing, speaking, lifting, and walking; and are also likelier to experience diabetes and stroke (almond & mazumdar; . others debate the possibility of pandemic-induced selection into fertility, which could confound estimates of the long-run health effects of early-life pandemic exposure. these studies ultimately conclude that the positive selection of wwi recruits, and the corresponding negative selection of pandemic-era fathers, does not substantially alter the conclusion that fetal exposure to the 1918 pandemic was a major and direct cause of these cohorts' later-life disadvantage (brown & thomas, 2018; beach et al., 2018) . a newer set of papers, focusing on non-western, and particularly, lower-income, settings, shows that the evidence on the pandemic's long-run penalties is robust across a range of empirical contexts, each with different levels of baseline income and health status, different institutional responses to the pandemic, and different degrees of involvement in wwi. for instance, as in the west, in taiwan there is evidence of permanent scarring: cohorts exposed to the pandemic in utero faced penalties with respect to educational attainment, heights, kidney disease, circulatory and respiratory issues, and diabetes (lin & liu, 2014) . in low-income settings with minimal public health intervention, even higher incomes only did so much to buffer these shocks: in a sample of high-ses children in japan, ogasawara (2018) finds that in utero exposure to the 1918 influenza pandemic reduced boys' and girls' heights by 0.28 cm and 0.14 cm, respectively-magnitudes which in other studies have been associated with substantial increases in the probability of type ii diabetes, osteoarthritis, and heart disease. the long-run results seen in japan, as in guimbeau et al. (2020) in brazil, are consistent with sex differences in resilience to adverse health shocks. 39 now quite common and influential in economic research, the conceptual framework linking early-life conditions to later-life health and wellbeing is termed the -barker‖ or -fetal origins‖ hypothesis. this hypothesis holds that certain chronic conditions stem from deficits in the fetal environment (barker, 1992) . based on this initial literature in epidemiology and medicine, which focused on evidence from historical famines, a growing literature in economics has used these ideas to model the technology of human capital formation, and to identify sensitive and critical periods for the development of a range of outcomes contributing to labor market success and general wellbeing, including cognitive and non-cognitive skills, metabolism, and longevity (heckman, 2007; almond & currie, 2011) . 40 meanwhile, swedish 1918 pandemic survivors saw reductions in life expectancy (helgertz & bengtsson, 2019) . the reduction in the health, human capital, and labor market prospects of cohorts exposed in utero also appears to have dampened their marriage market prospects in ways that continue to carry intergenerational consequences. while both men's and women's own educational attainment was lower among exposed cohorts, only exposed women appear to suffer a marriage market penalty: they marry earlier, to spouses with lower levels of education (fletcher, 2018) . these are factors generally understood to reduce household incomes, female control of household resources, and the budget share allocated to child-centric expenditure. as such, these effects could represent a mechanism-alongside, e.g., epigenetics, or the more direct role of parental education in facilitating children's access to quality healthcare and schooling-by which we see intergenerational persistence in the consequences of early-life exposure to the influenza pandemic of 1918. indeed, moderate adverse effects on educational attainment, occupational prestige, and family socioeconomic status have been documented up to the third generation, i.e., the grandchildren of those exposed in utero (cook et al. 2019) . what action, if any, did households take to shield their children from these effects, or to help them recover? while surprisingly little has been written in the context of the 1918 pandemic on questions of individual-and household-level avoidance, adaptation, and remediation, parman (2015) is a noteworthy exception. 41 drawing on linked microdata from the u.s., he finds evidence of reinforcing investments in response to the 1918 influenza pandemic: that is, families with a child in utero during the crisis shifted resources to the child's older siblings, leading the latter children to higher educational attainment. parman explicitly rules out changes in family size, birth spacing, or selectivity in any such changes, underscoring that the effects observed here are directly a function of parents reallocating limited resources away from affected children, and toward the child with a higher human capital endowment at birth. thus, household responses may have if anything compounded any early-life disadvantage associated with the 1918 shock. historical pandemics can help us think about potential long-run effects on wellbeing arising directly through the current pandemic's patterns of morbidity and mortality. but what about the impacts resulting from its disruption of daily economic life? one of the central features of the current coronavirus pandemic is the sudden, extreme, and widespread economic disruption it has caused. on this count, it has perhaps less in common with other recent pandemics. indeed, the immediate economic disruption caused by the 1918 pandemic pales in comparison to that caused by so, while this historical pandemic can 41 some work has, however, addressed the broader policy responses (and lack thereof) to the 1918 pandemic in the u.s. for instance, hatchett et al. (2007) find that cities that simultaneously implemented multiple non-pharmaceutical interventions (consisting of, e.g., isolation of sick individuals, bans on public gatherings, mandatory notification of disease, and closure of public gathering places, staggered business hours, and no-crowding rules) early in the pandemic had peak mortality rates roughly half that of cities that did not implement such interventions, and substantially lesssteep epidemic curves. no single intervention was responsible for these gains; rather, it was the combination of multiple mutually reinforcing interventions that were effective. these findings are in line with markel et al. (2007) , who emphasize the importance of early and sustained non-pharmaceutical interventions during the pandemic. while many cities were successful in taking such a multi-pronged approach to pandemic management, on the whole the u.s. policy response to the 1918 pandemic was rather weak, undermined by a preoccupation with world war i-related efforts. 42 until the covid-19 crisis, there had been relatively little work on the effects of the 1918 pandemic on economic activity, largely for lack of high-frequency, spatially disaggregated data on local economic conditions (see beach et al. (forthcoming) for an excellent overview of both the state of this literature and related empirical challenges). indeed, the precise magnitude and temporal reach of these economic effects are still being debated (see, e.g., basco et al. (2020) , barro et al. (2020) , correia et al. (2020) , lilley et al. (2020) , and velde (2020)), and a challenge for many of these studies in identifying pandemic effects on the economy remains the confounding effect of world war give us insight into long-run effects on wellbeing through the health channel (-direct‖ effects), we must look elsewhere to think about the long-run consequences of pandemics through corresponding economic downturns (-indirect‖ effects). but where to look for a suitable comparison? in some ways, episodes such as the black death or the aids crisis in sub-saharan africa would seem to present closer analogues than the 1918 influenza pandemic, as health events with massive and lasting economic ramifications. 43 the catastrophic loss of life under these pandemics fundamentally reshaped entire societies and economies, with, for instance, the resulting labor scarcity driving up the real wages of survivors, and, in some cases, precipitating other major demographic, economic, social, cultural, and institutional changes (young, 2005; alfani & murphy, 2017) . indeed, some point to the former plague as a major contributor to sustained rises in western european living standards even under a malthusian regime (voigtländer & voth, 2009 , 2013a , and to the region's rapid economic development and eventual divergence from the rest of the world over the early modern period (clark, 2007) . notes: the insured unemployment rate is based on employees covered under unemployment insurance as reported to states by employers. covid-19 cases are relative to the entire state population. unemployment data were retrieved from https://oui.doleta.gov/unemploy/claims.asp. covid-19 data were retrieved from https://github.com/nytimes/covid-19-data. the code and data needed to generate the figure are available at open-icpsr (https://doi.org/10.3886/e125441v1). in each of these pandemics, mass mortality led to rapid and dramatic changes in population density and age i. moreover, it is worth noting that the 1918-focused studies that have emerged in the wake of covid-19 tend to conflate the economic effects of the pandemic that arise from within and outside the -direct health-shock‖ channel. to disentangle these channels and use a shock of comparable magnitude, we focus primarily on the great depression when examining the long-run human effects of economic dislocation. 43 see alfani & murphy (2017) for an excellent and in-depth review of the literature on pre-industrial plagues, their long-run socioeconomic consequences, and parallels to modern pandemic control efforts. structure, which in turn affected factor prices and labor markets. thankfully, mortality rates under covid-19 are not on such a scale as to produce the sort of fallout seen with these events. 44 instead, it appears it may be a combination of factors other than the virus's actual toll on morbidity and mortality that is the source of economic dislocation in this instance. indeed, as figure 3 shows, the severity of the immediate health effects has not been a clear predictor of a locality's economic downturn. likewise, emerging evidence complicates the popular conception that pandemic-control measures themselves, such as stay-at-home orders, are primarily responsible for the downturn associated with covid-19. for instance, while gupta et al. (2020) suggest that 60% of the decline in employment in the early months of the pandemic was driven by state and local social distancing policies, kahn et al. (2020) show that the labor market effects of covid-19 to date have been broader-based than is typically thought. all u.s. states exhibited a collapse in job vacancies in march 2020, and a corresponding rise in unemployment insurance (ui) claims, irrespective of either the intensity of the virus's initial spread or the timing of stay-at-home orders. these phenomena were seen for the most part across both essential and non-essential sectors, directly-and indirectly-affected sectors, and across occupations with and without work-from-home capabilities. they conclude that -the current damage done to the economy is not solely caused by the stay-at-home orders; it is too large and pervasive.‖ exploring the drivers of the collapse in economic activity, goolsbee & syverson (2020) suggest that -individual choices were far more important [than government restrictions,] and seem tied to fears of infection.‖ these voluntary disease-avoidance strategies by individuals are likely connected to the lack of decisive and coordinated policy responses, and to broader uncertainty about this novel disease. it remains to be seen whether other plausible mechanisms may also have a role-e.g., global supply chains that allow covid-related firm slowdowns in one country or sector to propagate to others, or changes in firm production decisions under covid uncertainty. clearly, both the current crisis and our understanding of it are still rapidly evolving. what we do know, however, is that the downturn this pandemic has precipitated is substantially larger than in other modern pandemics, and unlike in some pre-modern plagues, is likely unrelated to either mortality-related changes in demography or to immediate reductions in labor supply or work capacity due to contemporaneous morbidity. as such, crises of primarily economic origin, such as historical recessions-and in particular, the great depression-may make the best analogues: while the coronavirus pandemic is a public health crisis, to be sure, it has manifested above all as a massive economic disruption, both in terms of magnitude and reach. 45 accordingly, we might want to think about its health and human capital consequences through this -livelihoods‖ channel as well. indeed, it is these effects that are likely to be most relevant to our current situation. beginning with short-term effects, we can look to a large literature on business cycles and health. these studies indicate that the net effects of downturns on morbidity and mortality will likely be highly contextdependent. this is because health is multidimensional, there are many countervailing channels through which local economic conditions can affect wellbeing, and because the particulars of the empirical 44 even while modern globalization has made disease transmission faster and harder to control, and even while increased efficiency in healthcare systems and global supply chains have complicated efforts to quickly ramp up treatment and control responses, other modern factors have made the current pandemic less dangerous to health than those that came before it-among them, improved medical technology, which has made it easier to manage secondary infections, and higher incomes, which have made human populations both less vulnerable and more resilient to infectious disease. 45 this is certainly true at least in a distributional sense. while adverse effects will certainly be severe through direct morbidity/mortality channels, these will nevertheless be relatively concentrated. for contrast, adverse spillovers from these direct health effects, and from broader disease-control efforts, will be much more diffuse, even if less acute. consider, for instance, that unlike the health-channel scarring effects of pandemics discussed in section iii, the economy-channel shocks apply to everyone to one extent or another, not just those who survive infection. setting-e.g., the size, nature, and origin of the shock; the baseline level of population health; and the strength of social safety nets-will ultimately govern which of these effects dominate (arthi et al., 2017; cutler et al., 2016) . recessions have been shown to improve health, for instance, by freeing up time for health-promoting activities such as exercise, childcare, and breastfeeding (dehejia & lleras-muney, 2004; miller & urdinola, 2010; ruhm, 2000) ; by reducing the income available to sustain unhealthy behaviors such as alcohol, tobacco, and drug abuse (ruhm & black, 2002; ruhm, 2005) ; by reallocating high-skilled but displaced healthcare workers toward higher-risk populations (stevens et al., 2015) ; and by limiting individuals' exposure to environmental and work-related hazards, including traffic accidents, on-the-job injuries, and pollution (muller, 1989; chay & greenstone, 2003; miller et al., 2009). 46 meanwhile, adverse income shocks can compromise access to basic needs such as nutrition, medical care, and housing (griffith et al., 2013; painter, 2010) ; and can cause psychological stress that in turn raises rates of self-harm and risky behaviors (eliason & storrie, 2009; sullivan & von wachter, 2009) . 47 while in theory, the net effect of local economic shocks on health is ambiguous, in practice, the bulk of the evidence drawn from modern and rich-country settings suggests that on net, total mortality rates fall during recessions (arthi et al., 2017) . in addition to setting-specific features like higher baseline health and stronger safety nets, the fact that beneficial channels tend to dominate in these settings may be in part because this evidence comes principally from small fluctuations in local economic conditions: using cross-country evidence over two centuries, cutler et al. (2016) show that mild downturns lower mortality, while large ones raise it. 48 the downturn caused by covid-19 would surely qualify as the latter. the evidence is much more mixed in developing-country and historical settings, where levels of baseline income and health are low, where safety nets are weak, and where cutting-edge medical technology is less accessible (see, e.g., baird et al. (2011) and ferreira & schady (2009) ). in such settings, even small losses in income can be devastating to health (costa, 2015; heckman, 2007) , and there is less scope for the sort of offsetting positive spillovers and behavioral changes seen in more modern and affluent settings. 49 consequently, this evidence seems to more often indicate countercyclical mortality. for instance, arthi et al. (2020a) show that even in the presence of adaptive migratory responses, the cotton famine, a major 1860s downturn in britain's cotton textile-producing regions, substantially raised mortality in cotton regions, particularly amongst the elderly (who were more sensitive to income shocks), amongst cotton households (who faced unemployment and reduced hours), and amongst those working in non-tradeables 46 the case of pollution in particular underscores how complex the interactions between health and the economy can be-all the more so during a respiratory pandemic that has precipitated an economic crisis. tied as pollution is to economic activity, a downturn that reduces pollution (and so reduces direct health hazards) also reduces income (and so raises indirect health hazards). moreover, it does so unevenly across space and demographic groups. add to this long-standing (i.e., baseline) distributional considerations around who is most exposed to environmental and pandemic hazards (see, e.g., chay & greenstone, 2005; currie et al., 2015) ; and who, conditional on exposure, is most sensitive to income shocks, environmental shocks, infectious disease shocks, or even all three simultaneously (see, e.g., hsiang et al. 2019; almond & currie, 2011) ; and a key question for assessing covid-19's effects through economy-environment interactions then becomes, from both an aggregate and distributional standpoint, whether and for whom the losses in health and human capital through the income channel are offset by the gains in health through actions taken to reduce the spread of influenza, the reduction of pollution, and the interaction of these factors. 47 see arthi et al. (2017) for a much more detailed review. note as well that under covid-19 stay-at-home orders and supply-chain disruptions, the effects through many of these mechanisms are likely to be much more extreme, since the reduction in economic activity has been much more acute (in some cases, nearly absolute). 48 this is the case even in rich countries, but especially in poor ones. in the latter, as discussed above, even smaller economic fluctuations can raise net mortality. 49 consequently, we might expect developing countries to face the greatest tension between the desire to limit the direct health costs of covid-19 on the one hand, and the desire to limit those health costs arising from the corresponding economic contraction on the other. this is especially the case if pandemic-control measures are seen as helping the former objective while harming the latter, though it is worth noting that it is still unclear the extent to which pandemic-control measures are responsible for the contraction caused by (whose livelihoods depended on the success of the local cotton industry). diverse historical evidence such as this can help us think about how the effects of the covid-19 crisis might out play out differently in other economies, particularly in the long run-something we cannot get from modern data, and especially, from modern u.s. data, alone. likewise, turning to the great depression, a more recent and thus perhaps more comparable setting to today's, stuckler et al. (2012) find at best mixed evidence of a beneficial health effect of the downturn: while there was a small reduction in all-cause mortality during this crisis, only those reductions in heart disease (small) and traffic fatalities (rather larger) could plausibly be linked to contemporaneous local economic shocks; other recession-related causes of death identified in the literature, such as suicide, rose substantially. fishback et al. (2007) similarly find that had new deal relief spending not intervened, the great depression would have created a -demographic disaster,‖ depressing birth rates and elevating death rates relative to prior trends (particularly among infants, those perhaps most vulnerable to short-run income fluctuations). their results emphasize the importance of government responses to economic crises that in turn become health crises (and vice-versa): for instance, they note that while all-cause non-infant mortality rates were largely unaffected by relief spending, such income support nevertheless did help reduce rates of certain salient causes of death such as suicide, one of the few causes of adult mortality identified in stuckler et al. (2012) as seeing a marked increase during the great depression. while current debates around covid-19 are understandably focused on the immediate impact of pandemic-induced recession conditions, the economic history literature teaches us that we should be equally-perhaps even more-concerned about the long-run scarring effects arising from this economic dislocation. indeed, this channel may be especially relevant in more modern, high-income, and robustsafety net settings where most people survive an adverse shock, only to contend with the long-term and sometimes latent fallout. some of these scarring effects stem from the immediate impact on household incomes. depression-era resource deficits have been shown to affect cohorts that were in utero at the time well into adulthood, lowering their college completion rates and later-life incomes, and raising their rates of later-life poverty and disability-adverse effects that were only more pronounced in poorer areas, and areas that received less relief spending (arthi, 2018; fishback & thomasson, 2014) . 50 meanwhile, other long-run penalties arise from disruptions to labor markets and human capital acquisition. a large contemporary literature studies the phenomenon of labor market scarring, or the idea that economic conditions at the time of labor market entry may have lasting effects on training decisions, occupational choice, career trajectories, and lifetime income. 51 this evidence, much of it taken from college graduates around the 2008 recession, is mixed: some studies suggest that the impact of initial labor market conditions diminishes over the course of an individual's career-often within the first decade-while others find that some penalties associated with early-career shocks can be cumulative and permanent (see rothstein (2019) for an in-depth review; see also, kahn (2010) ). these effects are often heterogeneous by skill level, and may be driven by mismatch in initial job placement (faberman & mazumder 2012 , liu et al. 2016 , oyer 2006 , şahin et al. 2014 , van den berge 2018), lower initial wages (which may be partially related to job mismatch; 50 the stress of adverse shocks may also be transmitted intergenerationally through epigenetic channels. see, e.g., costa et al. (2018) . likewise, there is evidence that both pandemics and recessions-as traumatic and stressful events-can shape the attitudes and preferences of those exposed during formative years in ways that can have lasting political and economic consequences (see, e.g., campante et al. (2020) ; giuliano & spilimbergo (2015) ; malmendier & nagel (2011); and schoar & zuo (2017) ). 51 while this literature focuses on adverse shocks at the time of labor market entry, note that compared to other recessions, long-run labor market scarring could even extend to a different and younger range of cohorts in the covid-19 case, because of widespread school closures. other covid-19-related mechanisms, such as the loss of parental income, would tend to compound these effects further. oreopoulos et al. (2012) ), reduced working time (cockx & ghirelli 2016) , and delays in finding employment (genda et al. 2010) , among other factors. moreover, strategic responses to these shocks, such as migration (feigenbaum 2015) , temporary exit from the labor force (hershbein 2012) , and human capital acquisition (charles et al. 2018 , barr & turner 2015 , may themselves have implications for short-and long-run labor market prospects, as separate from those arising directly from the initial shock. 52 these studies thus strongly suggest that downturns may have important -overhang‖ that may potentially -reduce prosperity for decades to come,‖ both for directly-affected cohorts and the wider economy (rothstein 2019, p. 4) . accordingly-and bearing in mind that under covid-19, peak unemployment rates for younger workers have been nearly three times the national average 53 -very-long-run and even intergenerational evidence on these issues can be especially valuable. recent work in economic history has looked to the great depression in order to offer precisely this sort of perspective. these studies show substantial and persistent penalties for all workers in severely-hit areas, but especially for new labor market entrants, who faced very different constraints and scope for adaptation than did incumbent workers. moulton (2017) , for instance, finds a substantial earnings penalty amongst less-educated american men just entering the labor market in 1930. while there are large adverse effects for those born in severely-affected states, this age-at-downturn penalty disappears in lessaffected states. likewise, examining evidence on labor force transitions using large-scale linked microdata from the u.s., arthi et al. (2020b) show that many younger workers during the depression accepted work that they otherwise might not have considered in better economic times-whether because of their now-dire need, the additional competition from older workers, or some combination of these factors. moreover, many young people seeking work were locked out of the labor market completely by their older counterparts, who now remained in the labor force (or even re-entered it) at higher rates. evidence on occupational transitions and socioeconomic mobility also suggest important career-stage gradients in scarring: younger workers were crowded out of the best local job opportunities by their older counterparts, with young workers in more rural areas pushed out of farming by older workers who retained these jobs at higher rates, and into general laborer and non-occupational positions; and those in more industrial areas being pushed into farming, the less desirable class of occupations in these areas. importantly, while both of these outcomes represent a short-run penalty for newer labor market entrants, the long-run implications for wellbeing may be very different, given the rapid urbanization and the incipient decline of the agricultural sector that was to come. indeed, by providing the impetus to leave agriculture (or by prompting higher rates of out-migration-younger labor market cohorts irrespective of sector were also likelier to have moved across state lines or into urban areas during the depression, perhaps in response to the dearth of local opportunities for inexperienced workers), the great depression may have had a small silver lining for young rural workers. however, at least in the short run, it served to hamper upward mobility-or even, to induce downward mobility. for instance, liu & fishback (2019) show that though concerns over skill depreciation and mismatch during spells of un-or underemployment animated depression-era policymakers, new deal programs largely failed to match workers to jobs that used their skills, often resulting in lower incomes and transitions into lower-skilled employment or unemployment-though at least some general human capital was maintained. meanwhile, feigenbaum (2015) finds that by 1940, intergenerational mobility had fallen for men growing up in cities severely hit by the depression. migration-in particular, the superior destination choices of the sons of richer fathers-was an important mechanism behind these results, again emphasizing the capacity of large adverse shocks to exacerbate rather than level preexisting inequalities. the history of past pandemics and economic downturns provides sobering guidance for what we might expect from the current covid-19 crisis. there is a complicated relationship between health and economic productivity that will shape the immediate and latent effects of covid-19 in both obvious and subtle ways. given that these latent effects unfold over decades and even generations, economic history is uniquely capable of providing evidence on the potential long-term costs of the pandemic. experience from both historical pandemics and historical recessions can inform our view of the possible long-run effects of covid-19, and how we might mitigate these costs. the experience of the 1918 influenza pandemic suggests that disease exposure can impact individuals throughout their lifetimes, both directly through poorer ongoing health, and indirectly through reduced investment in human capital. the costs were not limited to those individuals directly exposed; instead, they spilled over within households and across space, sectors and generations. moreover, while mortality is salient, and the saving of lives remains perhaps the primary objective during a pandemic, avoiding and compensating for morbidity is arguably as important a matter of policy concern, especially in the context of possible long-run effects. particularly in a pandemic where large shares of prime-aged people fall ill (as in the 1918 pandemic), or in pandemics where many are infected but ultimately survive (as in both the 1918 pandemic and covid-19), experiences of pandemic illness may have lasting effects over the life-course, either through the initial illness (which may, for instance, compromise fetal nutrition, reduce work capacity, or permanently damage health), or through its sequelae later in life. the great depression points to other long-term effects that are likely to emerge from the pandemic-related slowdown in economic activity: both being born or entering the labor market during the great depression led to economic penalties well into adulthood, and constraints on migration had adverse effects on individuals and firms. importantly, history shows us that these two types of harms are mutually reinforcing: damage to health tends to undermine labor market prospects in the long run, while damage to labor market prospects tends to undermine health in the long run. researchers and policymakers should therefore consider the potential for these long-run costs when weighing the short-term costs and benefits of pandemic control and fiscal intervention. history suggests potentially massive future costs for both the economy and the safety net arising from the dampened economic fortunes, chronic health issues, and foregone fertility of cohorts impacted by covid-19. given that human capital investments are generally more productive the earlier they are implemented, this suggests that policy interventions undertaken now, such as cash relief, could be especially cost-effective, and their net long-run benefits tremendous. economic history also reveals that we cannot think of the health and economic impacts of covid-19 independently of one another. past pandemics indicate that regardless of the pathology of a disease, its impacts are often a function of economic conditions. while some pandemics spared no class, many disproportionately impacted individuals of lower socioeconomic status due to a variety of factors including their occupations, living conditions, and access to healthcare. these individuals are at greater risk of exposure, face greater harms conditional on exposure, and are less able to remediate these harms. we have already seen this taking place with covid-19, and need to remain aware that the spread of the disease and the severity of its effects will be in part a function of the spatial distribution of residence, economic activity, and environmental harms. these disparate impacts of the virus itself will be compounded by the associated economic downturn. to the extent that the covid-19 economic downturn limits exposure to environmental and work-related hazards, or reduces spending on unhealthy behaviors, non-coronavirus related dimensions of health may actually improve. however, both the modern literature on developing countries and the u.s.'s experience during the great depression suggest that the severe economic downturn may compound health problems in areas with lower baseline incomes and weaker safety nets. identifying the channels through which income loss and general recession conditions impact health is necessary for properly interpreting any observed changes in population health levels during covid-19, and for designing effective policies to safeguard health. successful implementation of these policies also requires a firm understanding of history-roadblocks to public health initiatives during past pandemics associated with institutional structures and individual attitudes offer cautionary tales for our current crisis. while economic history provides useful insights for the current pandemic, the way in which the pandemic is unfolding also provides a fresh perspective with which to revisit the past. we are witnessing the actions that individuals and families, workers and firms, citizens and public officials alike, take to guard against the pandemic, and the damage it has done to the economy. we are witnessing how these responses change as new information on covid-19 emerges. the current pandemic affords us unprecedently rich and disaggregated data that, even while still evolving, can give new insights into which groups might warrant additional study in past pandemics. all of these dimensions of covid-19 can help us reshape the roadmap for studying the economic history of pandemics. one of the most important ways the covid-19 experience can shape the direction of economic history may not be in seeking out the similarities but rather focusing on differences. while the rate of transmission and severity of the effects of covid-19 have historical analogues, many relevant features of the world are meaningfully different-among them, the global nature of production; flows of people, goods, and information; urbanization; baseline living standards; medical technology; public health infrastructure; and the role of government. these differences can help us understand both past and present pandemics better; moreover, they help us understand how and why things have changed. for example, the covid-19 shutdowns have been more far-reaching, and the corresponding economic downturn more damaging, than we might have predicted from previous pandemics. can these differences explain the far greater economic costs of covid-19 relative to similarly lethal pandemics of the 20th century? this suggests an important direction for future economic history work: identifying why the nature of the response to public health crises differed, and why the resulting economic consequences were often smaller historically. engaging in this work also allows us to grapple with challenging questions about tradeoffs between population health and economic activity. these tradeoffs are incredibly difficult to tackle head on in the face of an unfolding crisis; they force unfathomable but unavoidable choices on policymakers often working with limited information. by offering insight into not just the actions but also the short-and long-run 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system: a commercial manufacturing platform for viral vaccines and gene therapy vectors date: 2015-03-20 journal: biotechnol j doi: 10.1002/biot.201400438 sha: doc_id: 326960 cord_uid: 9phlylce the baculovirus expression vector system (bevs) platform has become an established manufacturing platform for the production of viral vaccines and gene therapy vectors. nine bevs‐derived products have been approved – four for human use (cervarix®, provenge®, glybera® and flublok®) and five for veterinary use (porcilis® pesti, bayovac csf e2®, circumvent® pcv, ingelvac circoflex® and porcilis® pcv). the bevs platform offers many advantages, including manufacturing speed, flexible product design, inherent safety and scalability. this combination of features and product approvals has previously attracted interest from academic researchers, and more recently from industry leaders, to utilize bevs to develop next generation vaccines, vectors for gene therapy, and other biopharmaceutical complex proteins. in this review, we explore the bevs platform, detailing how it works, platform features and limitations and important considerations for manufacturing and regulatory approval. to underscore the growth in opportunities for bevs‐derived products, we discuss the latest product developments in the gene therapy and influenza vaccine fields that follow in the wake of the recent product approvals of glybera® and flublok®, respectively. we anticipate that the utility of the platform will expand even further as new bevs‐derived products attain licensure. finally, we touch on some of the areas where new bevs‐derived products are likely to emerge. the baculovirus expression vector system (bevs) is far from new. for thirty years researchers have been using this platform to express recombinant proteins, and thousands of proteins have been successfully expressed and purified. however, for much of this time, bevs was rele-gated to the ranks of research tool. what we have seen in the last decade is the elevation of bevs from research tool to an established manufacturing platform for production of novel biologic products. ten years ago there were only two commercial products manufactured using the bevs manufacturing platform and both of these were veterinary vaccines to prevent classical swine fever in pigs. since then, seven new products have been licensed, four of which are for humans, including vaccines and therapeutics, and many more products are in development (table 1) [1, 2] . we have passed a tipping point where bevs-derived products are becoming mainstream, and the bevs platform is being actively utilized by major players in the biotechnology industry to develop new products. although the bevs platform has distinct features that make it an attractive platform for the production of many biologics, it is not ideally suited for all products. factors such as protein complexity, post-translational modification, scale and cost must be considered collectively when selecting a manufacturing platform; these have been extensively reviewed elsewhere [2] [3] [4] . in this review, we explore the bevs platform by looking at how the system works and the advantages and limitations of the platform from a manufacturing and regulatory perspective. the opportunities to develop new products using bevs are abundant, and we review the latest developments in the gene therapy and influenza vaccine fields as examples. finally, we consider some newer areas where bevs-derived products show promise for the future. the bevs platform has been previously described [1, 2, [5] [6] [7] [8] . the platform takes advantage of baculoviruses' natural propensity to infect insect cells. in nature, there are more than 500 different types of baculoviruses, all of which have a host range restricted to invertebrates [9] . in the laboratory and for manufacturing purposes, the most commonly used baculovirus is autographa californica multiple-capsid nuclear polyhedrosis virus (acmnpv), a virus with a double-stranded dna genome of approximately 134 kb [10] . the large size of its genome gives the baculovirus ample capacity to accommodate large amounts of foreign dna, including multiple genes, an advantage over other expression vectors such as vaccinia and adenovirus [2] . to begin the bevs process, a recombinant baculovirus is constructed comprising the desired gene(s) of interest (goi) (fig. 1) . first, the goi is cloned into a transfer plasmid, typically behind the strong polyhedrin or p10 promoter that can drive protein expression to high levels in insect cells [11, 12] ; notably, these promoters are not very active in e. coli and, therefore, can be stable expression cassettes. the goi is flanked by acmnpv dna, e.g. the polyhedrin promoter on one side and a portion of the essential gene orf1629 on the other. insect cells are then co-transfected with a mixture of the transfer plasmid and parental acmnpv dna that has been linearized such that the parental polyhedrin gene and portion of orf1629 are missing, rendering it non-infectious [13] . the plasmid and parental dna undergo homologous recombination to generate de novo recombinant baculoviruses. these baculoviruses are plated and individual plaques purified to isolate a single, pure plaque of recombinant baculovirus. this plaque is subsequently passaged through multiple rounds of insect cell infection to generate a high-titer stock and establish a working virus bank (wvb) that can be utilized for protein production. for manufacturing purposes, it is important that wvbs be stable and retain integrity as virus passages are scaled up. laboratory kits such as bac-to-bac ® (life technologies) that employ bacmid technology have been developed that allow researchers to quickly and easily construct recombinant baculoviruses in e. coli rather than [14] . long-term stability of the baculovirus is an important consideration if large-scale protein production is envisioned. once a high-titer wvb has been established it is used to infect insect cells and stimulate protein production. cells are seeded in culture flasks (for small-scale production) or bioreactors (for large-scale production) and the wvb added to infect the insect cells when they are in their logarithmic growth phase. the baculoviruses reprogram the cellular machinery to produce the recombinant protein(s). following protein expression (typically 48-96 hours post-infection), the cells and/or supernatant are harvested, depending on whether the product is intracellular or secreted, respectively, and the proteins are purified according to standard techniques such as ultracentrifugation or column chromatography. many different insect cell lines have been used for bevs but the most common are derived from ovarian cells of the fall army worm, spodoptera frugiperda (e.g., sf-21, sf-9 and expressf+ ® [protein sciences corporation]), and the cabbage looper, trichoplusia ni (high five tm cells, life technologies) [5, 15, 16] . high five cells are used to manufacture the licensed human papillomavirus vaccine, cervarix ® (glaxosmithkline), and sf-21 cells are used to produce the antigen used in the prostate cancer immunotherapy, provenge ® (dendreon). expressf+ cells are used to manufacture three licensed products: flublok ® influenza vaccine (protein sciences corporation), glybera ® gene therapy for the treatment of familial lipoprotein lipase deficiency (uniqure), and ingelvac circoflex ® veterinary vaccine to protect against porcine circovirus type 2 (boehringer ingelheim vetmedica). large scale manufacturing and commercial production require specific cell line characteristics such as scalability, high yields, the ability to grow in low-cost, serum-free media, and qualification to meet regulatory agency (e.g. fda, ema, etc.) requirements for purity and safety. characteristics of the bevs platform are summarized in fig. 2 . these features as well as other important considerations about the technology are discussed below. the choice of an expression system for manufacturing is a complex decision that must balance many facets, including attainment of specific product features, the demands of the therapeutic indication, and the needs of the manufacturer. comparisons of the bevs platform to other expression systems such as bacteria, yeast, mammalian cells and plants have been made and are useful to consider when selecting a platform [2] [3] [4] . bevs employs recombinant technology, giving researchers and product developers a level of control over the production process that is not possible with other techniques. a good example is found with vaccine manufacturing. traditionally, vaccines are made by cultivating large volumes of the pathogen against which protection is desired to generate the "raw materials" required for the product. this can be accomplished by infecting substrates such as embryonated chicken eggs or mammalian cells, both of which are used to manufacture the majority of the vaccines recommended for routine immunization [1, 17] . the pathogen is either weakened and administered live as a live attenuated vaccine, or is killed or inactivated with reagents such as formalin or heat prior to being formulated into vaccine. these methods yield safe and effective vaccines; however, important shortcomings have been observed. the first concerns specificity. for example, traditional influenza vaccine production involves virus propagation in eggs. as influenza viruses are rna viruses, they have a tendency to mutate to optimize their figure 1 . the bevs platform. the bevs platform is an efficient process for producing a wide variety of proteins in a streamlined manner. a gene of interest (goi) is cloned into a transfer plasmid behind a strong promoter (green arrow) and surrounded by dna homologous to the parent baculovirus (yellow and green boxes). a library of recombinant baculoviruses (rbv) can be made using standard cloning techniques and varying the goi. construction of an rbv takes eight days. to generate protein, the appropriate rbv is scaled up (taking on average two to five weeks) and used to infect insect cells, which programs the cells to generate large quantities of recombinant protein that can subsequently be purified to high levels using standard techniques. a single insect cell line can be used to produce all proteins. protein production averages three to five weeks and yields highly pure, biologically active products. growth [18] . generally, the changes introduced into the virus sequence have little impact on vaccine efficacy. however, since the virus receptors for birds and mammals differ, these changes can be meaningful, and it has been documented that in some cases the changes have rendered egg-based influenza vaccines ineffective [19] . the bevs process does not involve pathogen growth and, therefore, avoids this complication. rather than cultivate a pathogen to collect "raw materials", a recombinant baculovirus is constructed that codes for the antigen required for protection. since accommodations for growth do not need to be made, the antigen can be an exact sequence match to the human pathogen. this has the potential to solve the ineffectiveness observed for some egg-adapted vaccines as was described by skowronski et al. (2014) [19] . moreover, specific point mutations can be introduced to enhance features such as stability. for example, it has recently been reported that purified hemagglutinin protein, the protective antigen in influenza vaccines, appears unstable in the srid potency assay due to crosslinking of specific cysteine residues in the protein [20] . by mutating these cysteine residues to non-thiol residues, it was possible to prevent cross-linking and enhance stability [21] . a second shortcoming with traditional vaccine manufacturing is process length. again, influenza serves as a good example. influenza epidemics occur every year and annual influenza vaccination is recommended by the centers for disease control and prevention [22] . in addition, pandemic outbreaks occur occasionally, the most recent of which was the 2009 h1n1 swine flu (a/california/07/2009) [23, 24] . timeliness of vaccine manufacture is critical to ensure that adequate vaccine supply is available. egg-based influenza vaccine manufacturing takes on average about six months, as the process of creating a high-producing, egg-adapted seed virus is slow [17] . this means that manufacturing of seasonal influenza vaccines must begin the winter prior to an epidemic, before that season's strain prevalence is definitively known, to guarantee adequate supply, and this has resulted in seasons where the available influenza vaccines have not matched the circulating influenza strains [25, 26] . moreover, the president's council of advisors on science and technology (pcast) reported that pandemic influenza vaccine in 2009 was not readily available until after the pandemic peaked, a major concern for public health and safety (www.whitehouse.gov/sites/default/files/microsites/ostp/ pcast-influenza-vaccinology-report.pdf). pcast identified recombinant technology and freedom from virus growth and egg adaptation as the solution to this problem. bevs-derived influenza vaccines do not require seed viruses and can be produced in as little as 45 days [5] . in addition to specificity and speed, recombinant technology offers advantages with respect to purity and product design. with respect to purity, recombinant products are free of pathogens, eggs and many chemicals (such as formalin and antibiotics) that can be undesirable or allergenic [27] . for product design, recombinant techniques make it possible to engineer proteins with desired features, such as fusion proteins that increase immunogenicity or include multiple antigens and truncated proteins with deleted domains to improve yields and ease purification [28] [29] [30] [31] [32] . one example of this application is the antigen used in provenge immunotherapy. the antigen is a fusion glycoprotein consisting of prostatic acid phosphatase (pap) linked to granulocyte-macrophage colonystimulating factor (gm-csf) and is used to stimulate autologous antigen presenting cells [33] . the gm-csf activates the antigen presenting cells and enhances cell viability while the pap serves as the target antigen. another example is the cervarix ® vaccine manufactured by glaxosmithkline. the vaccine is a bivalent virus-like particle (vlp) comprised of the human papillomavirus l1 proteins (strains 16 and 18) . by expressing c-terminally truncated l1 proteins, the manufacturers are able to prevent intracellular vlp self-assembly, which would complicate purification. instead, the l1 subunit proteins are purified to a high degree and vlp assembly is achieved in vitro [28] . the bevs platform has inherent safety measures built in that are attractive from a regulatory perspective. baculoviruses have a narrow host range restricted to specific insects and are considered safe to use as biological pesticides with no negative impact on plants, mammals, birds, fish or non-target insects [9] . people are exposed to baculoviruses daily by consuming fresh vegetables. for instance, a serving of coleslaw may contain hundreds of millions of baculoviruses [34] . baculovirus vectors have also been explored as gene therapy vectors. these studies have demonstrated that baculoviruses cannot repli-cate in mammalian cells and cannot express a gene cassette unless it is driven by a mammalian promoter [9, [35] [36] [37] [38] . a major consideration for regulatory agencies when evaluating a novel cell substrate is the potential to harbor adventitious agents that could threaten patient safety. there are very few adventitious agents that can replicate in both insect and mammalian cells. a notable exception is arboviruses that can be transmitted to humans via insect bites and can cause complications such as encephalitis and hemorrhagic fever [39] . to mitigate this risk, cells from non-biting insects such as the fall armyworm and cabbage looper have been used with bevs as noted above. nonetheless, adventitious agents have been detected in some insect cell lines. for instance, the trichoplusia ni high five cell line, bti-tn-5b1-4, used to make cervarix ® , was found to be latently infected with an alphanodavirus that was induced by recombinant baculovirus infection [40] . in addition, other insect cell lines, such as those generated from drosophila melanogaster, have been shown to harbor innate retroelements derived from retroviruses that could potentially be infectious [41] . more recently, a possible insect-specific virus sf-rhabdovirus was identified in spodoptera frugiperda cells [42] . although studies showed that this virus could not enter or replicate in human cell lines and, therefore, is unlikely to be a risk, novel cell lines will most likely need to be characterized and monitored for the presence of this virus as they are for nodaviruses, retroviruses and others. in general, because adventitious agents are a potential threat, cell substrates of all origins (including insect and others) must be thoroughly tested for the presence and infectivity of such agents before they are allowed by regulatory agencies for manufacturing use. in addition to the safety considerations just discussed, there are two important regulatory features associated with the bevs platform that should be considered. the first is that bevs is a transient protein expression system; the recombinant baculoviruses used vary based on their foreign gene cassettes but a single cell line can be used for the expression of all proteins (fig. 1) . qualifying a cell line is no small feat. it can take years of work to adequately ensure purity and safety in addition to high productivity. stable cell lines have to be independently qualified each time their genetic composition changes [43] . in contrast, cell lines used with bevs and other transient expression systems remain constant and, consequently, need to be qualified just once. a second feature is the growing number of bevsderived products that have been approved by regulatory agencies worldwide ( table 1) . as is the case for all technologies, prior regulatory approvals remove barriers for future product approvals. the technology becomes more mainstream and less novel with each approval, and the safety database of patients that are administered products without complication continues to grow. nine bevsderived products have been licensed, providing regulators confidence with the platform. the following characteristics make the bevs platform appealing for commercial manufacturing: scalability, biosafety, flexibility and existing manufacturing capacity. insect cells are grown in suspension, so if the cells and baculoviruses have been optimized for large scale and multiple passages, the culture size is only limited by the size of the bioreactor. for example, expressf+ cells have been used to produce recombinant proteins at scales ranging from two to 21 000 l [44] . the cost of goods for bevs production is largely dependent on capital costs and yields. as discussed by cox (2012), vast global bioreactor capacity (~500 000 l) already exists and presents the opportunity to minimize the investment needed to establish bevs manufacturing facilities [1] . moreover, opportunities for yield improvements are abundant and include genetic and fermentation-based approaches; these are described elsewhere [1] . unlike some other production facilities, bevs facilities can be multi-purpose and used to produce a variety of bevs-derived products, especially when disposable or single-use technology is employed [45] . this is because a single cell line can be used for production of different products. this feature is especially meaningful for regions of the world where limited manufacturing capacity exists. a single bevs facility could, for example, be used to produce vaccines for diseases endemic to a region and quickly be converted to produce pandemic influenza vaccine in an emergency. finally, because the bevs platform does not require the handling of live, potentially dangerous pathogens, requirements for biocontainment that can be very costly are reduced. the bevs platform is a versatile technology useful for the manufacture of many products; however, other platforms may be better suited for the production of certain proteins. for instance, small proteins that do not require post-translational modifications are best made in e. coli that can quickly generate high yields at low cost [4] . yeasts such as s. cerevisiae can also produce high yields of protein at low cost and are capable of some post-translational modifications [4] . insect cells are capable of many post-translational modifications but proteins that require complex post-translational modifications and folding may best be made in mammalian expression systems. for www.biotecvisions.com www.biotechnology-journal.com example, the glycosylation patterns produced by insect and mammalian cells are related, and glycoproteins produced in insect cells are often correctly folded, biologically active and immunogenic [46] . however, insect cells generate less complex n-glycans than mammalian cells and this can negatively impact biological function [46, 47] . some developments have been made to address this limitation, including engineering transgenic insect cell lines that stably express mammalian glycosylation enzymes or co-expressing such enzymes with the gene of interest in a single baculovirus [48] [49] [50] [51] . whether this is required must be assessed on a protein by protein basis. licensure of the first bevs-derived products has paved a regulatory pathway, reducing regulatory uncertainty. in this section we discuss opportunities in the areas of gene therapy and influenza vaccines spawned by the two most recent bevs-derived product approvals, glybera ® (licensed by the ema in 2012) and flublok ® (licensed by the fda in 2013), respectively. the bevs approach to gene therapy has largely involved the production of recombinant adeno-associated viruses (raavs) that house therapeutic dna. the use of raavs as a gene delivery vector has gained popularity for several reasons, including long-term gene expression, lack of pathogenicity and ability to transduce a wide variety of cells, both dividing and non-dividing [52, 53] . nine different raav serotypes (1-9) are most commonly used for raav-based gene therapy, each serotype with a different propensity for tissue-specific infection and infection kinetics [54] . recombinant aav-based gene therapies have been in development and shown promise for some time; however, a major limitation to their implementation had been the inability to scale up the manufacturing process to produce sufficient quantities of raavs. the original raav vectors were produced in mammalian tissue culture using adherent cells such as hek293 cells, which required about 5000 175-cm 2 flasks to produce enough material for a large animal study or human clinical trial (~10 15 raav particles) [55] . to overcome this limitation, scientists adopted and optimized the bevs platform for production of large scale, high titer raavs [55] [56] [57] [58] [59] [60] [61] [62] [63] . by adjusting parameters such as multiplicity of infection, cell density and fermentation mode, raav yields on the order of 10 14 vector genomes per liter have been reported [57] . the traditional bevs production strategy for raavs requires the co-infection of insect cells with three differ-ent recombinant baculoviruses: bac-rep that expresses the major aav replication enzymes (rep78 and rep52); bac-cap that expresses the aav virion coat proteins; and bac-goi that expresses the gene of interest flanked by the aav inverted terminal repeat elements that are required for the rescue, replication and packaging of the gene [55, 56] . no adenovirus helper is needed as is required for mammalian cell raav production [64, 65] . due to some genetic instability of the bac-rep construct, a streamlined two-baculovirus system has further been developed where rep and cap proteins are expressed from a single baculovirus (bac-repcap) and rep78 and rep52 are transcribed from a single mrna species that enhances stability [56] . alternatively, genetic modifications have been made to the original bac-rep and bac-cap constructs to enhance stability and improve expression [58] . an important regulatory hurdle was overcome in 2012 when glybera received marketing authorization in europe, making it the first gene therapy product approved in the western world and launching the bevs platform into the spotlight as a preferred platform for raav manufacture. glybera (alipogene tiparvovec) is comprised of the human gene lpl s447x in a bevs-derived raav serotype 1 vector and is used for the treatment of patients with lipoprotein lipase (lpl) deficiency [66] . clinical studies have shown glybera to be safe and effective [66, 67] . glybera will likely be the beginning when it comes to raav-based gene therapy. the approach is relevant to the estimated thousands of monogenic diseases [68] . treatments are actively being investigated in a diverse array of therapeutic areas and dozens of product candidates are in clinical development (summarized in table 2 ). hemophilia is one area where progress has been made (reviewed in [69] ). there are four ongoing human clinical trials involving raav serotypes 8 or 2, all designed to express factor ix for the treatment of hemophilia b (table 2 ). factor viii raav-based therapy is a target for the treatment of hemophilia a, the most common severe inherited bleeding disorder, and only a modest increase in plasma factor viii levels is expected to be required to be clinically relevant [69] . raav-based treatments for retinal degeneration, including macular degeneration and leber's congenital amaurosis type 2, are another area of intense investigation [70, 71] . retinal treatments are ideal because of cell accessibility through intravitreal and subretinal injections and the ability to assess structure and function noninvasively. serotype 2 is most commonly used for these therapies but types 2, 5, and 7-9 are all capable of infecting photoreceptors, the most prominent cell type for retinal degenerations [70] . diseases of the central nervous system (cns), such as parkinson's and alzheimer's diseases, are also actively being tested with raav-based therapies that are promising. aavs exhibit a strong preference for neuronal transduction, making them a popular gene delivery vehicle for cns therapies but vector improvements are still needed to optimize treatments (discussed in [72] ). final-ly, duchenne muscular dystrophy (dmd) is a therapeutic target where progress is being made. although the single gene affected in dmd (dystrophin) has long been known, its large size has made gene therapy a challenge. recent treatment approaches to overcome this include exonskipping, trans-splicing and micro-and mini-dystrophin delivery strategies [53, 73] . for fifty years influenza vaccine manufacturing technology remained largely stagnant. that changed with the licensure of flublok ® in 2013. flublok ® is a trivalent bevs-derived vaccine for seasonal influenza composed of 135 µg of recombinant hemagglutinin (ha) derived from the two a and one b influenza viruses selected for inclusion in the annual influenza vaccine by the world health organization; the vaccine is licensed by the fda for adults 18 and older [74] . influenza vaccines are standardized to contain a specific amount of ha, the major surface glycoprotein on the influenza virus [17] . bevs-derived recombinant ha forms trimers that in turn oligomerize into immunogenic rosettes [75] . these proteins can be purified to high levels resulting in a vaccine that has been shown to be safe and effective in clinical studies [5, [76] [77] [78] [79] . the advantages of recombinant bevs vaccines for pandemic influenza are especially important. vaccines for avian influenza viruses such as the h5, h7 and h2 subtypes are urgently needed because of these viruses' high pathogenicity and mortality rates in humans and the fact that h2 has previously demonstrated pandemic potential and human-to-human transmissibility [80, 81] . a monovalent variation of the flublok ® vaccine called panblok ® has been developed. in a phase ii study of h5 panblok ® (a/indonesia/5/05), a two-dose schedule of vaccine at doses of 3.8-45 µg ha formulated with the adjuvant glucopyranosyl lipid a/stable emulsion (gla/se) had an acceptable safety and reactogenicity profile and elicited serologic responses meeting seroconversion criteria in adults 18-49 years old [82] . moreover, an earlier study showed that people administered h5 panblok ® (a/hong kong/156/1997) in 1998 were primed for an enhanced immune response following administration of an antigenically variant vaccine strain in 2006 [83] . evaluated a different bevs-derived h5 subunit vaccine candidate (a/goose/guangdong/1/96) and showed that it protected against a lethal challenge in balb/c mice and in specific pathogen-free and commercial chickens, suggesting it could be useful as both a human and animal vaccine [84] . multi-component vlp vaccines for pandemic influenza are under development that are composed of recombinant ha, neuraminidase (na) and matrix 1 (m1) proteins produced in sf-9 cells [85] [86] [87] . evaluation of an h5n1 (a/indonesia/5/05) vlp vaccine in a phase i/ii study of adults 18-40 years old showed that two doses of unadjuvanted vaccine at 15, 45 or 90 µg ha/dose were generally well-tolerated and resulted in seroconversion [85] . similarly, a phase ii study of an h1n1 (a/california/04/2009) vlp vaccine in adults 18-64 years old showed it was safe at doses of 5, 15 or 45 µg ha/dose and elicited high rates of seroprotection (82-92%) [86] . more recently, an h7n9 vlp vaccine was developed that was comprised of ha and na proteins matched to a/anhui/1/2013 (h7n9) and m1 protein matched to a/indonesia/05/2005 (h5n1) [87] . this vaccine candidate was tested with or without saponin-based iscomatrix adjuvant in balb/c mice and was shown to protect against a lethal challenge. antibodies against both ha and na were elicited, with 3-to 4-fold higher responses in the iscomatrix groups. although the data are promising, a challenge for the development of multi-component vlp vaccines will be standardizing the vaccines for each of their components (e.g., quantity of ha vs. na vs. m1). an advantage of using recombinant technology for vaccine design is the opportunity to modularly add or subtract antigens to a formulation. inclusion of recombinant na in vlp vaccines has been shown to induce formation of anti-neuraminidase antibodies [87] . it has been noted that different vaccine compositions (e.g., vlp vs. subunit vs. whole virion) induce different immune profiles in balb/c mice [88] . while the advantages of these various profiles are not yet clear, the flexibility of the bevs platform enables catering towards different outputs. besides inclusion in vlps, recombinant na can be individually produced via bevs and may serve as a potentially efficacy-enhancing additive to influenza vaccines [89] . na immunity is infection-permissive but can reduce infection severity and duration [90] . the potential benefits of including recombinant na in influenza vaccines has recently been reviewed [90] . other opportunities have emerged with bevs as researchers begin pursuit of a so-called universal influenza vaccine. licensed influenza vaccines offer limited cross protection to heterologous influenza viruses and, thus, there is the need for annual update of seasonal influenza vaccines and concern over pandemic preparedness. a successful universal influenza vaccine would offer long-lasting and broad protection against a range of different influenza virus strains. approaches to universal influenza vaccine design include ha stalk-based constructs and chimeric ha-based vaccines that are composed of conserved stalk domains fused to "exotic" heads, usually of avian origin (these approaches are reviewed elsewhere [91] ). the bevs platform, being based on recombinant technology, offers the flexibility and genetic control required for the design and manufacture of these universal vaccine candidates. in this review we have taken a close look at the bevs platform, describing how the platform works, outlining the features and limitations of the technology and highlighting the growth opportunities that emerged from the two most recent bevs-derived product approvals. we expect this growth to continue and expand as future bevsderived products attain regulatory approval. the approvals of bevs-derived cervarix ® and flublok ® vaccines have broadened the acceptance of the platform beyond its initial veterinary borders to use in healthy adolescents and adults. human therapeutics is another area of use and gene therapy in particular is a growing area of interest; the approval of glybera drew major attention to bevs-derived raavs. baculoviruses themselves can also be used as gene delivery vectors, and other recombinant protein complexes produced using bevs are being explored for the delivery of various peptides and antigens, such as in the form of the newly characterized vault particles [92] . the speed at which recombinant proteins can be produced using bevs makes it a particularly attractive platform to design safe and effective vaccines to be available to timely combat new infectious pathogens as they arise. success with this approach has been demonstrated for influenza and can be applied to broader areas. for example, coronaviruses have been plaguing both people and animals especially in the last decade with lethal outbreaks of severe acute respiratory syndrome (sars) in 2003, middle east respiratory syndrome (mers) in late 2012, and porcine epidemic diarrhea virus (pedv) in 2013 [93] [94] [95] . all coronaviruses share a similar structure that includes the presence of the spike glycoprotein on the viral envelope that is the dominant immunogen [96] [97] [98] . multiple coronavirus bevs-derived vaccine candidates have been developed that include recombinant spike protein either alone as a subunit vaccine or together with recombinant envelope and membrane proteins as vlps and have demonstrated efficacy in animal models [29, [99] [100] [101] [102] [103] [104] [105] [106] [107] . the spike protein has been shown to be immunogenic both as full length protein and as a truncated protein containing only the extracellular domain [29, 100] , and multiple routes of vaccine administration have been examined [104] . two recently emerged threats are chikungunya virus and ebola virus, both of which can be addressed with bevs-derived vaccines. chikungunya virus causes a serious disease that involves severe joint pain and can be fatal; a recent outbreak appeared in saint martin in december 2013 and has since made its way to more than 20 countries or jurisdictions in the americas, including the continental united states [108] . chikungunya is an arbovirus, for which there are many opportunities to develop bevs-derived vaccines (reviewed in [39] ). both subunit and vlp vaccine candidates expressing the chikungunya viral envelope glycoproteins have been developed using bevs, with vlps demonstrating higher immunogenicity in mice [109] [110] [111] . ebola virus is a filovirus that causes lethal hemorrhagic fever in humans and is devastating africa in an ongoing outbreak [112] . the ebola virus glycoprotein has been shown to be the protective antigen and could be produced similar to a chikungunya virus vaccine [113] . in addition to glycoprotein-based vaccines, the bevs platform has shown early promise for the production of biotechnology journal toxin-based vaccines. for example, the c-terminal heavy chain domain of clostridial botulinum neurotoxin, a highly toxic protein that causes botulism, has been produced with bevs and has demonstrated immunogenicity and challenge protection in mice [114, 115] . recombinant toxin vaccines for other diseases such as clostridium difficile could also be possible [116, 117] . in conclusion, bevs is a versatile platform whose potential is just beginning to be realized. the technology offers speed, flexibility, specificity and safety, and the use of a single cell line to manufacture multiple products makes bevs an attractive platform to adopt. bevs can be used to develop a wide variety of products and is especially well suited for combating rapidly emerging and dangerous pathogens. with new threats continually on the rise, tools such as bevs offer an important defense. the author works for protein sciences corporation, which has a financial interest in bevs and manufactures flublok ® influenza vaccine. recombinant protein vaccines produced in insect cells vaccines for viral and parasitic diseases produced with baculovirus vectors commercial production in insect cells. one company's perspective production of recombinant proteins by microbes and higher organisms a fast track influenza virus vaccine produced in insect cells insect 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baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy adults 50-64 years of age safety and immunogenicity of a baculovirus-expressed hemagglutinin influenza vaccine: a randomized controlled trial comparative immunogenicity of recombinant influenza hemagglutinin (rha) and trivalent inactivated vaccine (tiv) among persons > or = 65 years old global alert to avian influenza virus infection: from h5n1 to h7n9 receptor specificity and transmission of h2n2 subtype viruses isolated from the pandemic of 1957 evaluation of safety and immunogenicity of recombinant influenza hemagglutinin (h5/indonesia/05/2005) formulated with and without a stable oil-inwater emulsion containing glucopyranosyl-lipid a (se+gla) adjuvant immune responses of healthy subjects to a single dose of intramuscular inactivated influenza a/vietnam/1203/2004 (h5n1) vaccine after priming with an antigenic variant a subunit vaccine candidate derived from a classic h5n1 avian influenza virus in china protects fowls and balb/c mice from lethal challenge h5n1 virus-like particle vaccine elicits cross-reactive neutralizing antibodies that preferentially bind to the oligomeric form of influenza virus hemagglutinin in humans safety and immunogenicity of a virus-like particle pandemic influenza a (h1n1) 2009 vaccine in a blinded, randomized, placebo-controlled trial of adults in mexico development of influenza h7n9 virus like particle (vlp) vaccine: homologous a/anhui/1/2013 (h7n9) protection and heterologous a/chicken/jalisco/cpa1/2012 (h7n3) cross-protection in vaccinated mice challenged with h7n9 virus influenza virus-like particles elicit broader immune responses than whole virion inactivated influenza virus or recombinant hemagglutinin baculovirus-expressed influenza vaccine. a novel technology for safe and expeditious vaccine production for human use influenza neuraminidase as a vaccine antigen advances in universal influenza virus vaccine design and antibody mediated therapies based on conserved regions of the hemagglutinin development of the vault particle as a platform technology middle east respiratory syndrome coronavirus: transmission and phylogenetic evolution severe acute respiratory syndrome vs. the middle east respiratory syndrome porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines the sars-cov s glycoprotein current advancements and potential strategies in the development of mers-cov vaccines drug targets for rational design against emerging coronaviruses baculovirus surface display of sars coronavirus (sars-cov) spike protein and immunogenicity of the displayed protein in mice models antigenic and immunogenic characterization of recombinant baculovirusexpressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design immune responses against severe acute respiratory syndrome coronavirus induced by viruslike particles in mice virus-like particles of sarslike coronavirus formed by membrane proteins from different origins demonstrate stimulating activity in human dendritic cells vaccination of mice with recombinant baculovirus expressing spike or nucleocapsid protein of sars-like coronavirus generates humoral and cellular immune responses effect of mucosal and systemic immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice chimeric severe acute respiratory syndrome coronavirus (sars-cov) s glycoprotein and influenza matrix 1 efficiently form virus-like particles (vlps) that protect mice against challenge with sars-cov assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus m and s proteins purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice chikungunya at the door -deja vu all over again? effective chikungunya virus-like particle vaccine produced in insect cells chikungunya virus-like particles are more immunogenic in a lethal ag129 mouse model compared to glycoprotein e1 or e2 subunits enhanced production of chikungunya virus-like particles using a high-ph adapted spodoptera frugiperda insect cell line estimating the ebola epidemic antibodies are necessary for rvsv/zebov-gp-mediated protection against lethal ebola virus challenge in nonhuman primates easy expression of the c-terminal heavy chain domain of botulinum neurotoxin serotype a as a vaccine candidate using a bi-cistronic baculovirus system rapid immune responses to a botulinum neurotoxin hc subunit vaccine through in vivo targeting to antigen-presenting cells development of a recombinant toxin fragment vaccine for clostridium difficile infection recombinant antigens based on toxins a and b of clostridium difficile that evoke a potent toxin-neutralising immune response reingard grabherr key: cord-298216-iq7fenxm authors: jiang, chao; yao, xingang; zhao, yulin; wu, jianmin; huang, pan; pan, chunhua; liu, shuwen; pan, chungen title: comparative review of respiratory diseases caused by coronaviruses and influenza a viruses during epidemic season date: 2020-05-13 journal: microbes infect doi: 10.1016/j.micinf.2020.05.005 sha: doc_id: 298216 cord_uid: iq7fenxm severe acute respiratory syndrome coronavirus 2 (sars-cov-2) continues to sweep the world, causing infection of millions and death of hundreds of thousands. the respiratory disease that it caused, covid-19 (stands for coronavirus disease in 2019), has similar clinical symptoms with other two cov diseases, severe acute respiratory syndrome and middle east respiratory syndrome (sars and mers), of which causative viruses are sars-cov and mers-cov, respectively. these three covs resulting diseases also share many clinical symptoms with other respiratory diseases caused by influenza a viruses (iavs). since both covs and iavs are general pathogens responsible for seasonal cold, in the next few months, during the changing of seasons, clinicians and public heath may have to distinguish covid-19 pneumonia from other kinds of viral pneumonia. this is a discussion and comparison of the virus structures, transmission characteristics, clinical symptoms, diagnosis, pathological changes, treatment and prevention of the two kinds of viruses, covs and iavs. it hopes to provide information for practitioners in the medical field during the epidemic season. in december 2019, a novel coronavirus, sars-cov-2, caused a pneumonia epidemic 44 in wuhan, hubei province of china. it erupted in many other countries in the following 45 months and eventually became a worldwide pandemic. the pneumonia was officially named 46 covid-19 by world health organization (who) [1] . so far, the pandemic is still accelerating. more than 3.7 million people were confirmed infected, 260,000 more people however, the fact is that the 1918 h1n1 virus also caused more deaths in younger people 139 [38]. h7n9 and h5n1 viruses were limited in transmission due to the similar reason 140 mentioned above with mers-cov and sars-cov. however, the mortality of h7n9 is as 141 high as 37%, and h5n1 is 53% [39, 40] . nevertheless, the high pathogenicity and high 142 mortality make them easier to be identified and controlled at the beginning of the break. 143 although h7n9 and h5n1 viruses transmitted in humans ineffectively, their geographic 144 expansion and genetic recombination still suggested the potential of forming the next 145 pandemic [41, 42] . 146 as listed in table 1 , people are easily infected with covs or iavs through direct 147 contact and airborne droplets [43] . since most of these viruses are of animal origin or use 148 animals as intermediate hosts [29, 31] , it is necessary to keep a safe distance between humans 149 and wild animals, as well as maintaining ecological balance in the world to prevent the 150 breaking of epidemics in future. 152 the major clinical symptoms are summarized in table 2 both covs and iavs can cause ards and lead to multiple organ failure and death. the published pathological data that were collected from autopsy, lung biopsies and chest 177 computed tomography (ct) scan are summarized in table 3 . for patients with hphts 344 the authors declare that they have no conflicts of interest. severe acute 457 respiratory syndrome (sars): a year in review infection of human airway epithelium by 459 human and avian strains of influenza a virus genetic recombination, and 468 pathogenesis of coronaviruses hosts and sources of endemic human clinical features of patients infected with 474 2019 novel coronavirus in wuhan, china clinical spectrum of the middle east respiratory syndrome 485 coronavirus (mers-cov) middle east respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory 490 syndrome coronavirus disease from saudi arabia: a descriptive study severe acute respiratory syndrome: historical, epidemiologic, and clinical 493 the epidemiology of severe 495 acute respiratory syndrome in the 2003 hong kong epidemic: an analysis of all 1755 patients clinical 498 features and short-term outcomes of 144 patients with sars in the greater toronto area clinical progression and viral 501 load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study a comparison of the clinical and 538 epidemiological characteristics of adult patients with laboratory-confirmed influenza a or b 539 during the 2011-2012 influenza season in korea: a multi-center study comparison of 541 clinical features and outcomes of medically attended influenza a and influenza b in a defined 542 population over four seasons seasonal 545 influenza a/h3n2 virus infection and il-1beta, il-10, il-17, and il-28 polymorphisms in 546 iranian population differences in clinical features between influenza a h1n1, a 548 h3n2, and b in adult patients prediction of h7n9 epidemic in china proinflammatory effects of the hemagglutinin protein of the 552 avian influenza a (h7n9) virus and micrornamediated homeostasis response in thp1 cells lung pathology of fatal severe 565 acute respiratory syndrome lung pathology of severe 567 acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore multiple organ infection and the 570 pathogenesis of sars molecular pathology analyses of two fatal 572 human infections of avian influenza a(h7n9) virus emerging h7n9 influenza a (novel reassortant avian-origin) 574 pneumonia: radiologic findings pathology and virology findings in 576 cases of fatal influenza a h1n1 virus infection in 2009-2010 lung pathology in 578 fatal novel human influenza a (h1n1) infection clinical features of pneumonia caused by 2009 580 influenza a(h1n1) virus in beijing pathological study of 582 archival lung tissues from five fatal cases of avian h5n1 influenza in vietnam molecular biology, and pathogenesis of avian influenza a (h5n1) 585 infection in humans apoptosis and pathogenesis of avian influenza a (h5n1) virus in humans re-emergence of fatal epidemiological features of coronavirus hku1-associated community-acquired pneumonia an outbreak of coronavirus oc43 595 respiratory infection in normandy, france coronavirus 229e-related 597 pneumonia in immunocompromised patients detection of human coronavirus nl63 in 599 young children with bronchiolitis comparison of the pathology caused by h1n1, h5n1, and 601 h3n2 influenza viruses respiratory syndrome and severe acute respiratory syndrome: current therapeutic options 605 and potential targets for novel therapies hopes rise for coronavirus drug remdesivir remdesivir for severe acute respiratory syndrome coronavirus 2 608 causing covid-19: an evaluation of the evidence /remdesivir-clinical-trials hwgcpa-g-hc hydroxychloroquine and covid-19 challenges and cares to promote rational use of chloroquine and 612 hydroxychloroquine in the management of coronavirus disease 2019 (covid-19) pandemic: a 613 timely review a rapid systematic review of clinical trials for the management of 2019 novel coronavirus disease drug repurposing approaches for the 621 treatment of influenza viral infection: reviving old drugs to fight against a long-lived simultaneous determination of amantadine and 624 rimantadine in feed by liquid chromatography-qtrap mass spectrometry with 625 information-dependent acquisition simultaneous 627 determination of amantadine, rimantadine, and memantine in processed products, chicken 628 tissues, and eggs by liquid chromatography with tandem mass spectrometry alternative regimens of neuraminidase 631 inhibitors for therapy of hospitalized adults with influenza: a systematic review of randomized controlled trials safety and effectiveness 634 of neuraminidase inhibitors for influenza treatment, prophylaxis, and outbreak control: a 635 systematic review of systematic reviews and/or meta-analyses impact of potential permissive 638 neuraminidase mutations on viral fitness of the h275y oseltamivir-resistant influenza a(h1n1)pdm09 virus in vitro, in mice and in ferrets oseltamivir (tamiflu((r))) in the environment, resistance development in influenza effectiveness of baloxavir marboxil 647 against influenza in children baloxavir marboxil: a novel cap-dependent endonuclease (cen) inhibitor for the treatment of acute uncomplicated influenza baloxavir marboxil susceptibility of 652 influenza viruses from the asia-pacific baloxavir marboxil) for the treatment of acute uncomplicated influenza favipiravir (t-705), a broad spectrum inhibitor of viral rna 656 polymerase viral polymerase inhibitors t-705 and t-1105 are potential inhibitors of zika virus replication effectiveness of favipiravir (t-705) 660 against wild-type and oseltamivir-resistant influenza b virus in mice exposure-response analyses of baloxavir marboxil in adults and adolescents including 663 novel hemagglutinin-based influenza virus inhibitors influenza a virus 667 hemagglutinin mutations associated with use of neuraminidase inhibitors correlate with 668 decreased inhibition by anti-influenza antibodies inhibitors targeting the influenza virus hemagglutinin novel hemagglutinin-based influenza virus inhibitors das181 for 676 treatment of parainfluenza virus infections in hematopoietic stem cell transplant recipients 677 at a single center severe parainfluenza virus 3 pneumonia in allogeneic hematopoietic stem cell transplant 680 recipients requiring mechanical ventilation double-blind, placebo-controlled, crossover trial of das181 (fludase(r)) in adult 683 subjects with well-controlled asthma evolution of pandemic influenza virus a(h1n1)pdm09 in 2009-2016: dynamics of receptor 686 specificity of the first hemagglutinin subunit (ha1) homology modeling study toward identifying 688 structural properties in the ha2 b-loop that would influence the ha1 receptor-binding site subunit vaccines against emerging pathogenic human the sars-cov-2 vaccine pipeline: an overview progress and prospects on vaccine development 705 against sars-cov-2. vaccines (basel) 2020 an evidence based perspective on mrna-sars-cov-2 can an effective sars-cov-2 vaccine be developed for the older 709 population? development of 711 epitope-based peptide vaccine against novel coronavirus immunoinformatics approach preliminary identification of potential vaccine targets for 714 the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies flumist vaccine: questions and answers -summary. paediatr child 728 prevention and 730 control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices -united states, 2017-18 influenza season prepandemic influenza vaccine h5n1 (split virion, inactivated, 734 adjuvanted) [prepandrix]: a review of its use as an active immunization against influenza a 735 subtype h5n1 virus acute respiratory distress syndrome. b ldh, lactate dehydrogenase table 3. the pathological changes of the patients after infection sore throat the pulmonary interstitial edema and lymphocytic infiltration key: cord-322082-80ym2rsq authors: monto, arnold s; fukuda, keiji title: lessons from influenza pandemics of the last 100 years date: 2020-03-01 journal: clin infect dis doi: 10.1093/cid/ciz803 sha: doc_id: 322082 cord_uid: 80ym2rsq seasonal influenza is an annual occurrence, but it is the threat of pandemics that produces universal concern. recurring reports of avian influenza viruses severely affecting humans have served as constant reminders of the potential for another pandemic. review of features of the 1918 influenza pandemic and subsequent ones helps in identifying areas where attention in planning is critical. key among such issues are likely risk groups and which interventions to employ. past pandemics have repeatedly underscored, for example, the vulnerability of groups such as pregnant women and taught other lessons valuable for future preparedness. while a fundamental difficulty in planning for the next pandemic remains their unpredictability and infrequency, this uncertainty can be mitigated, in part, by optimizing the handling of the much more predictable occurrence of seasonal influenza. improvements in antivirals and novel vaccine formulations are critical in lessening the impact of both pandemic and seasonal influenza. outbreaks of seasonal influenza are perennial occurrences in the temperate zones. their impact on morbidity and mortality is highly variable but in some years can occur at levels that nearly disrupt the functioning of healthcare systems [1, 2] . during such seasonal outbreaks, questions usually center around the severity and how well the vaccine is protecting. but, regardless of disruptions, their impact is quickly forgotten. by contrast, pandemics of influenza occur much less often but are viewed as more threatening because of their relative unfamiliarity and potential for catastrophic impact. even a century later, much of the concern stems from recognition of the sheer number of deaths attributable to the 1918 influenza pandemic. while estimates of death have varied greatly, recent scholarship, largely based of previously omitted data from lower-income countries, such as india, has revised global estimates upwards [3] now, the estimate of 50 million deaths is generally used as an overall global estimate, constituting nearly 3% of the world's population at the time [4] . pandemics are caused only by type a viruses. the current classification of a subtypes was developed in 1980 based on molecular evidence indicating that the previous nomenclature needed revision with, in addition, the inclusion of neuraminidase (na) [5] . table 1 shows the terminology used pre-1980 and the current terminology. years listed are either the start of virologically confirmed pandemics or consensus dates reflecting when it was thought that a new subtype had emerged based on serology [6] [7] [8] [9] . influenza viruses were first isolated in the 1930s, and the etiology and timing of previous activity were based on testing of sera from individuals who had lived through the period in question. this approach, termed "seroarcheology, " resulted in occasional controversy. most identified the 1889 influenza as caused by a2 viruses and postulated that a3 viruses had started to circulate in 1902, with no recognized pandemic occurrence. persons who lived through the 1918 pandemic were found to have antibodies against "swine" influenza viruses, now designated as a(h1n1). the more recent reconstruction of that virus confirms the overall validity of the seroarcheologic technique [10, 11] . the most remarkable epidemiological feature of the 1918 pandemic was the unexpectedly high mortality among those aged 20-39 years [3] . theories to explain this pattern abound but most involve an aberrant immune response [12] . one recent hypothesis postulates that prior infection of children in the 1889 pandemic rendered them particularly susceptible by immunologic imprinting to reinfection in 1918 when they were in their late 20s [13] . current evidence suggests that older individuals may have actually been protected in 1918. this is in contrast to the traditional belief in the w-shaped epidemic curve, in which the high mortality in the elderly was a result of the erroneous inclusion of seasonal disease from the early months of 1918 [14] . figure 1 shows the age-specific mortality in philadelphia where the pandemic shut down the city and peaked at a weekly annualized rate of 140 deaths per 1000 population [3, 15, 16] . another often overlooked but constant feature of all pandemics is the high mortality in the very young experiencing their first influenza infection [14] . these observations indicate that understanding the positive and negative effect of prior influenza exposure is critical [17] . other observations of relevance to planning efforts are indications of the usefulness of nonpharmaceutical interventions in mitigating community impact [18] . the susceptibility of pregnant women was well documented; it should not have been such a surprise during the 2009 influenza pandemic when it was rediscovered [3] . sudden death has often been emphasized as a feature of 1918, but it took, on average, 9 or more days for death to occur [19] (figure 2 ). this stresses the need for health systems to have the surge capacity necessary to handle patients with the more typical prolonged illness regardless of the severity of a pandemic. a proportion of the deaths were associated with bacterial complications. the global increase in antibioticresistant organisms is another major vulnerability [20] . most pre-1980 lists of influenza pandemics included one in 1947 despite lack of documentation of global outbreaks ( table 1 ). in that year, seasonal vaccine became ineffective and it was thought that a new subtype named "a prime" had emerged [21, 22] . this understanding was important in developing the doctrine of original antigenic sin. it is now understood that an intrasubtypic reassortment occurred in 1947, which resulted in a major antigenic change of the a(h1n1) viruses [23] . a new subtype, a(h2n2), actually emerged in 1957 when 3 gene segments coding for b these strains were identified by serology, but the specific identification is in dispute. some have the 1889 virus as h3n8 but without a different subtype identified starting in 1902, a year when there was not a clear pandemic [6] [7] [8] [9] . hemagglutinin (ha), na, and an internal component moved from an avian virus into the circulating a(h1n1) virus through genetic reassortment [24, 25] . the resulting a(h2n2) virus, which was called "asian influenza" since it emerged from china, totally replaced the a(h1n1) viruses. since this was the first true pandemic since 1918, there was immediate concern about its potential impact and great relief when it was found to resemble seasonal influenza with morbidity highest in children and mortality at the extremes of age [26, 27] (figure 3 ). in the united states, the virus emerged in the spring of 1957, but outbreaks intensified only after schools in the southern united states opened in august, underscoring the importance of children in dissemination [28] . although vaccine was available in the united states late in the first wave, it had to be reformulated because of subpotency and standardization issues, concerns still being addressed [29] ( figure 4 ). with little vaccine available, attention was paid to other ways to reduce transmission. a controlled experiment conducted at the veterans administration hospital in livermore, california, demonstrated reduced transmission from the use of ultraviolet lights [30] . that tantalizing observation has been used recently to strengthen the suggestion that small-particle aerosol transmission of influenza viruses is of importance. the a(h2n2) period lasted only 11 years until mid-1968. in july of that year, a major outbreak in hong kong signaled that another reassortment event had occurred [31] . avian influenza genes, one coding for ha and the other an internal component, replaced the existing counterparts in the circulating a(h2n2) virus; the na gene was not replaced [24, 25] . emergence of a(h2n2) and a(h3n2) viruses and later events led to the concept that "novel" influenza viruses are most likely to come from east asia. at the time, it was conjectured that reassortment (or "shift") of avian and human influenza viruses occurred in a nonhuman "mixing vessel" because humans were believed not to have the right cellular entry receptors for avian influenza viruses. pigs have receptors for both human and avian influenza viruses, and since influenza viruses replicate in these animals they were considered to be the mixing vessel [32] . this was further supported by the observation that humans, poultry, pigs, and wild birds live in close proximity in east asia, providing ample opportunity for reassortment to occur there. the a(h3n2) pandemic exhibited the same patterns of morbidity and mortality as the earlier a(h2n2) pandemic. in terms of reasons for emergence of a pandemic variant after only 11 years, it is of interest that the last outbreak of a(h2n2) in 1967-1968 was extensive, as measured by pneumonia and influenza (p and i) mortality. this indicates that a considerable percentage of the population still remained susceptible to a(h2n2) [33, 34] . however, the new a(h3n2) virus completely replaced the previous subtype, and its variants, more than 50 years later, have been responsible for the greatest proportion of mortality from influenza viruses. the first a(h3n2) pandemic wave occurred in the united states in midwinter 1968-1969 at a time typical of seasonal influenza but which in some parts of the world was delayed. there has been speculation that the delay was a result of protection from the unchanged na. even in the united states, contemporaneous studies showed reduction in infection in those with higher anti-na titers, indicating an independent protective effect beyond anti-ha [35] . the role of anti-na remains an issue in present-day efforts to improve vaccine [36] . in january 1976, an outbreak of severe influenza occurred at the us military's fort dix, new jersey. the causative virus was surprisingly found to be a variant of swine influenza, now recognized to be an a(h1n1) virus [37] . since previous serologic studies had shown that the 1918 pandemic was probably caused by swine influenza (table 1) , there was strong concern that the fort dix outbreak could herald another severe pandemic [38] . in the united states, vaccine production was begun after liability concerns of the manufacturers had been addressed. even though no further human outbreaks were detected, mass vaccinations were begun and stopped only when a relationship between the vaccine and guillain-barré was identified. this "affair" has been studied extensively in terms of potential pitfalls in pandemic response and decision making [39] . in the following year, a different a(h1n1) virus, one that had been circulating before 1957, was identified [40] . transmission of this virus, termed "russian influenza" since the reports first came from the far east of the soviet union, was unexpected because the virus had not been detected for 20 years. infections were widespread, generally mild, and limited to younger individuals; residual protection was nearly complete in older individuals [41] . this event has never been considered a true pandemic because so much of the world's population was not susceptible and because of the uncertain origin of the virus. the re-emerged a(h1n1) virus remained in persistent circulation worldwide along with a(h3n2) viruses and continued to evolve until it disappeared in 2009. before this, when a new a subtype began circulating, it completely replaced the previous one. the concept that avian influenza viruses could not directly infect humans ended in 1997 when avian a(h5n1) viruses spread directly from poultry to humans, causing a small but highly important outbreak in hong kong. this event, which raised global concern, resulted in the deaths of 6 of 18 patients with documented infection [42, 43] . once control measures, especially culling of poultry, were put into place, new cases abruptly stopped. no further human cases were detected until, in 2003, when, in conjunction with die-offs of poultry, the spread of a(h5n1) to humans occurred, mainly in southeast asia [44] [45] [46] . most human infections were the result of contact with poultry, but examples of limited human-to-human transmission were documented [47] . because human cases were often severe and resulted in respiratory failure and death, there was high global concern that a pandemic of this virus would be severe if sustained human-to-human transmission occurred [48] . the nature of the threat, arising in animals but directly of concern to humans, highlighted the generally poor coordination and often rigid separation between animal and human health authorities at national and international levels, as well as a general lack of national planning. the adoption of new international health regulations in 2005, which was strongly influenced by the emergence of sars (severe acute respiratory syndrome) and the re-emergence of a(h5n1) in 2003, constituted a major step forward [49] . in the united states, there was particular attention directed to nonpharmaceutical interventions, a result of the recognition that pandemic-specific vaccines would be available relatively late and that influenza-specific antiviral drugs, while important, would be limited in quantity. there were discussions as to whether the use of antivirals might be able to contain human transmission of an emerging virus at the source; these plans were mainly predicated on the emergence occurring in asia [50, 51] . continuing concern about outbreaks of avian influenza was interrupted when the first pandemic of the 21st century unexpectedly started in mexico in 2009 [52] . as a result of intrasubtypic reassortment, the a(h1n1) variant involved was antigenically highly distinct from previously circulating influenza a(h1n1) viruses [53] . the previous prevailing dogma was that pandemic influenza was the result of the emergence of a new virus subtype. however, the subsequent global spread indicates that a pandemic is better defined by the global population's immunological susceptibility and antigenic distance of the new virus from other influenza viruses, rather than rigid applications of virologic rules involving antigenic shift [54] . the 2009 a(h1n1) virus was associated with lower attack rates in older individuals, presumably because of prior exposure to older a(h1n1) viruses. its spread in north america in the spring extended quickly to other parts of the world, highlighting the importance of air travel in accelerating dissemination. in the united states, the spring wave slowed with the beginning of school summer vacations only to pick up again as schools opened in the autumn, reconfirming the importance of children in transmission. this most recent pandemic has been extensively documented. severe disease developed in a small proportion of healthy adults, many of whom had no underlying conditions, which was reminiscent of 1918 but at a much smaller scale [55] . particularly vulnerable groups included indigenous populations, well-documented in canada in the first spring wave [56] . this was not observed in the second wave, most likely related to modifications in response, including careful employment of antivirals. the association of severity with pregnancy was another clear reminder of the 1918 pandemic. a newly observed risk was morbid obesity [57] . the new pandemic virus completely replaced the prior circulating seasonal a(h1n1) but cocirculation of the influenza a(h3n2) virus continued. a societal issue of considerable importance, which is essential to address for future pandemics as well as for seasonal influenza planning efforts, was the perception promoted by some that the 2009 pandemic was a "fake" pandemic [58, 59] . the claim, amplified by social media, was that the public health response was a conspiracy by governments and the world health organization (who) to benefit the sale of influenza vaccines. the overall pattern of mortality, which was less extensive than in 20th-century pandemics, was an important component [55] . but perhaps the more fundamental observation is that the accusations were consistent with a broader erosion of trust within society. the need to focus on communications and trust building in all phases of a pandemic is an essential lesson for improving planning for pandemics and responding to seasonal influenza. during the past 100 years since 1918, each of the 4 influenza pandemics has presented both common and unique challenges. none has been predictable in terms of timing, location of onset ,or the causative influenza virus. those that started in 1957 and 1968 had the most similar morbidity and mortality patterns, with severe complications and deaths that were highest at the extremes of age. the practical consequences for planning are the need to direct interventions to cover such groups while recognizing that other groups may also be at a higher than usual risk [60] . the age groups most at risk may be the same as in seasonal influenza but may not. questions about "severity" are to be expected early, but determining such levels is particularly challenging. the impact on morbidity and mortality may differ, and perception of severity may also differ widely depending on place and time. during the start of events, the information available to health authorities is often limited and highly uncertain. nonetheless, severity assessments are likely to be important for justifying the use of nonpharmaceutical interventions, such as closing schools and restricting population movement. such actions, which apparently had an effect in 1918, are socially disruptive and likely to be divisive. reducing impact may benefit from using more resources early while communicating the uncertainties involved and the consequences of inaction. since 1957, vaccine has always been available late, often after the first wave. in 2009, the current system of virus sharing through frameworks already established at who worked well but vaccine was still not available widely nor equitably. new technologies, such as a universal vaccine, may eventually change this situation but not in the near term. the prepandemic use of vaccines containing known potential pandemic viruses, often with adjuvants, has been proposed, but there are significant uncertainties in choosing what viruses might go into such a vaccine or for taking the inherent risks [61, 62] . preparing for, and responding to, a pandemic is a complex phenomenon, combining science, societal beliefs, practical operational considerations, and political will. some countries and regions have continued to update plans, but others have not. this is a reflection, in part, of uncertainties following the 2009 pandemic but also what has been termed "pandemic fatigue. " the latter issue has been made worse by the repeated recognition of the pandemic potential of different avian influenza virus variants that have infected humans [63] [64] [65] . given this context, it is important to recognize that seasonal influenza occurs every year and many of the essential control measures for pandemics are based on those used for seasonal influenza. it is critical to avoid viewing pandemic and seasonal influenza as unrelated. seasonal influenza is a cause of significant morbidity and mortality, and the vaccine supply used for seasonal influenza sets, in a real-world sense, the production capacity for a pandemic. some countries that will want access to pandemic vaccine do not consider seasonal influenza as a priority. this will limit their capacities to vaccinate their most vulnerable subpopulations in a pandemic, even if vaccine is available. this situation is especially true of lower-resource countries, and continued efforts to document the impact of seasonal influenza and, concomitantly, to develop the health system capabilities needed to support a pandemic response remain high priorities. determining the possible reduction in seasonal severe disease from the use of vaccine can be evaluated in a vaccine probe study in which the vaccine is given under controlled conditions to young children in underresourced areas, similar to studies that documented the need for pneumococcal vaccine [66] . the need for all countries to have and use vaccine in a pandemic is an issue of the equitable distribution of resources on both a national and global scale. scientific advances have positioned the world to respond better to both seasonal and pandemic threats of influenza. however, to make the most of such advances before the next pandemic will still require consistent attention and both scientific and political leadership. potential conflicts of interest. a. s. m. reports consulting fees from sanofi, seqirus, and roche, outside the submitted work. k. f. has no potential conflicts to disclose. both authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. chasing seasonal influenza-the need for a universal influenza vaccine update: influenza activity in the united states during the 2017-18 season and composition of the 2018-19 influenza vaccine epidemic influenza: a survey updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic a revision of the system of nomenclature for influenza viruses: a who memorandum viral infections of humans: epidemiology and control pre-epidemic antibody against 1957 strain of asiatic influenza in serum of older people living in the netherlands immunological interrelationships of hong kong, asian and equi-2 influenza viruses in man recycling of asian and hong kong influenza a virus hemagglutinins in man initial genetic characterization of the 1918 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can neuraminidase-based immunity contribute to better influenza virus vaccines? swine influenza a at fort dix influenza a virus recycling revisited the swine flu affair: decision-making on a slippery disease. washington: us government printing office the russian flu: its history and implications for this year's influenza season tecumseh study of illness. xiii. influenza infection and disease, 1976-1981 characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 re-emergence of fatal human influenza a subtype h5n1 disease genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia world health organization international avian influenza investigative team. avian influenza a (h5n1) in 10 patients in vietnam probable limited person-to-person transmission of highly pathogenic avian influenza a (h5n1) virus in china the threat of an avian influenza pandemic world health organization. international health regulations strategies for containing an emerging influenza pandemic in southeast asia containing pandemic influenza at the source critically ill patients with 2009 influenza a(h1n1) in mexico antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans what is a pandemic? preliminary estimates of mortality and years of life lost associated with the 2009 a/h1n1 pandemic in the us and comparison with past influenza seasons canadian critical care trials group h1n1 collaborative. critically ill patients with 2009 influenza a(h1n1) infection in canada who working group for risk factors for severe h1n1pdm infection. risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis report of the review committee on the functioning of the international health regulations (2005) in relation to pandemic (h1n1) 2009. 2011 (fineberg report). available at response to the 2009 pandemic: effect on influenza control in wealthy and poor countries novel framework for assessing epidemiologic effects of influenza epidemics and pandemics investing in immunity: prepandemic immunization to combat future influenza pandemics a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases human infection with influenza h9n2 human infection with a novel avian-origin influenza a (h7n9) virus h6 influenza viruses pose a potential threat to human health vaccines as a tool to estimate the burden of severe influenza in children of low-resourced areas key: cord-282085-r3w90vg8 authors: epperly, d. e.; caney, d. n. title: covid-19 viral loads, environment, ventilation, masks, exposure time, and severity : a pragmatic guide of estimates date: 2020-10-05 journal: nan doi: 10.1101/2020.10.03.20206110 sha: doc_id: 282085 cord_uid: r3w90vg8 it is desirable to better characterize and understand how ventilation improvements in office spaces could offer significant protection against transmission of covid-19. it is also desirable to understand how ventilation in office spaces compares to outdoor settings. an attempt to find this information from online searches that included medical journals, private industry, and us government provided materials failed to find specific quantitative estimates and recommendations, which motivated this study. this study uses measured amounts of sars-cov-2 in the air of a hospital room with covid-19 patients from a published and peer-reviewed study and known influenza a challenge doses from a published and peer-reviewed study and known ashrae office ventilation standards and an outdoor air exchange model to estimate the time necessary to cause various exposure levels and resulting infection potential in various indoor and outdoor settings of both influenza a and covid-19. while these estimations have unknown error margins and cannot be considered authoritative, they may have utility in comparing various environments and relative risk factors. the estimates in this study also present an initial framework and specific quantitative examples for better understanding of the effects of ventilation on aerosolized transmission, and the immunology related to challenge doses, and the potential for low-level viral load exposure to result in some level of immunity without symptoms of illness (asymptomatic infection). specific quantitative examples of exposure viral load versus symptoms and immune response may increase public understanding and consciousness of concepts such as viral load, exposure time, challenge dose levels, shedding quantities, immune seroconversion, and re-challenge and could achieve new levels of personal hygiene that complement centuries-old adages such as wash your hands. a study entitled validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1) pdm09 dose-finding investigational new drug study 10 demonstrated that low viral load exposures caused people to develop increased immunity levels without becoming significantly sick -they "seroconverted while having minimal clinical illness and no shedding". abbreviations: mmid, mild to moderate influenza disease; tcid50, 50% tissue culture infectious dose. it can be seen from the study that symptoms increased significantly at 10^5 tcid50 exposure in an influenza a (h1n1 challenge study. shedding significantly increased at 10^6. another human challenge study of h3n2 influenza a found similar results in "a dose-finding study of a wild-type influenza a(h3n2) virus in a healthy volunteer human challenge model" 11 throughout this study, the "dose" listed above will be associated with symptom categorizations as follows: • this study considers that the common use of the phrase "covid-19 mild case" best matches the symptoms of "mild to moderate influenza disease" (mmid) in terms of the illness experience. anything without mmid would not be "flu like", but might be cold-like or sore-throat-like and this study considers that "not ill" given all the severity concerns surrounding covid-19 cases. since 20% of those in the study challenge group experienced mmid at the 10^5 exposure level, this was probably the best exposure level to use to describe "mild illness" / "covid-19 mild case". since the majority the 10^6 and above exposure levels experienced mmid, this is considered the level at which "possible severe illness" might develop -conversely, below this level, development of severe illness necessitating medical attention would be expected to be extremely rare. since covid19 and influenza a severity has many determinants, these categorizations assume good general health and no significant co-morbidities. while these "power of 10" boundaries are only approximately reflective of reality, they are helpful for estimation. per "viable sars-cov-2 in the air of a hospital room with covid-19 patients" 12 , it was found that "viable virus was isolated from air samples collected 2 to 4.8m away from the patients" in a 3.5 x 7.0 meter hospital room with "six air changes per hour" (0.1 air exchanges per minute). "estimates of viable viral concentrations ranged from 6 to 74 tcid50 units/l of air.". regardless of the number of infected patients, this study uses the mean viral concentration of 40 tcid50 units/l of air as the reference environment that contains one or more sick persons. since the ceiling height was not specified, it can be estimated from "ceilings in patient bed areas including bed rooms, bed bays and recovery areas should be a minimum of 2700mm." 13 therefore, the entire room can be estimated to be 66 cubic meters (2331 cubic feet). normalized to the outdoor air exchange model, this would be a room of 3375 cubic feet at 337.5 cfm producing 6 air exchanges per hour (0.1 air exchanges per minute). no specific mention of patient face masks was noted in the reference hospital room, so this study assumes none were in place on these patients. it appears that at least 1 of the patients was strongly positive with a 32 ct pcr test, but these 2 hospital patients may not be among the strongest level of shedding patients. they may or may not represent a "typical" hospital patient shedding scenario. this study uses an aerosol diffusion and dilution model that scales linearly with air exchange. given a reference scenario of a specific stable aerosol concentration at 6 air exchanges per hour, that concentration would be considered to diminish to half of the reference at 12 acph. additionally, this study's model of air exchanges does not consider area population densities such as "cubic feet of available air per person". perhaps obviously, a large warehouse with 2 people at 6 air exchanges per hour would likely be safer than a small room with the same 6 acph. this study uses a hospital room with specified dimensions as the reference case, so population density adjustments would need to be made for crowded clubs or sparsely populated warehouses. diffusion and dilution realities are more complex than the model used in this study and better estimates could be achieved with better models. it would be preferred to have more data regarding indoor aerosolized viral concentrations at distances of approximately 2 to 5 meters from an infected individual. having only 1 reference environment is a limitation of this study. perhaps a later work can improve upon this limitation. adult respiration rate "while resting is about 5-8 litres per minute", and "light activities minute volume may be around 12 litres" 14 and "moderate exercise may be between 40 and 60 litres per minute". in this study, the respiration rates of 12 l/m and 60l/m will be considered for the person being exposed. "according to ashrae standard 62.1, an office will require 4-10 air changes per hour depending on the occupancy and size of the office." in an example ventilation "rule of thumb" example, a room of 6912 cubic feet having 6 air exchanges per hour would require 691 cfm. 15 normalized to the outdoor air exchange model, this would be a room of 3375 cubic feet at 337.5 cfm producing 6 air exchanges per hour (0.1 air exchanges per minute). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . since no viral load challenge data for covid-19 could be found at time of writing, this study must adjust estimates from challenge data for influenza a to estimate sars-cov-2 infectivity. sars-cov-2 has been claimed to have r0 values ranging from 1.5 to 3.5 to 5.7 depending upon estimation sources. the mean of these 3 numbers is about 3.6. h1n1 influenza r0 is widely estimated to be near 1.46 to 1.48 with a mean of 1.47. the r0 means of influenza a vs covid-19 r0 calculate to a ratio of about 1 to 2.5. using infection fatality rates (ifr) of 0.1% for influenza a and 0.4% for covid-19 shows about a 1 to 4 ratio. total fatalities for a typical influenza a year vs covid-19 appear to be about a 1 to 3 ratio as of september 2020. the mean of these 3 ratios calculates to 3.2. this study chose to round up to 4 with a bias toward representing covid-19 as much more aggressive than influenza a, which creates more "conservative" estimates that result in additional "safety margin". the reader / observer is free to re-calculate the spreadsheet times based on a different "influenza a to covid-19 infectivity multiplier" as this adjustment multiplier value accuracy is a limitation of this study and is admittedly weakly supported. future studies could improve this adjustment value. based on the aerosolization model defined above, this study will consider that viral shedding levels of both influenza a and covid-19 are similar for patients of similar infection severity. thus, from the shedding perspective, influenza a and covid-19 shedding data are considered equivalent in this study. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.03.20206110 doi: medrxiv preprint it has been shown in various challenge studies as noted earlier that case severity tends to increase with increasing initial exposure viral load. patient viral loads also vary dramatically depending upon stage of illness. 16 figure 1 , it can be seen that symptoms typically became observable within 3 days of challenge. from data in that study relating to the pre-symptomatic phase, the viral load contributing to shedding at day 1.5 was about 10^3 and was 10^4 or higher during the peak symptom period. this represents a 10 fold increase between the median pre-symptomatic time period and the peak symptom period, keeping in mind that patients in this study did not experience severe symptoms that would require hospitalization, though they were studied in a clinical environment. per "viable sars-cov-2 in the air of a hospital room with covid-19 patients" 17 it is known that both patients were hospitalized which would suggest that their cases were more severe than a typical mild covid-19 case and that maximum shedding would be greater than mid-phase pre-symptomatic shedding for these same patients. patient 1 had a pcr cq [similar to ct] of 32 which is 2^4 (16x) less than that of a typical cq positive threshold of 36 or below (some use cq < 40) 18 . patient 2 had an unspecified cq. the hospitalized covid-19 patients experiencing a more severe case covid-19 than that of the influenza a study suggests that the patients tend to have longer periods of symptoms and shedding than that of the influenza a study. taking the average of the 10 fold increase from the influenza study and a 16x cq from positive threshold in the covid-19 hospital air study, this study uses a roughly 13 to 1 ratio of symptomatic to pre-symptomatic shedding multiplier. that is to say that during mid-point of pre-symptomatic period (median of time to symptoms), the shedding is about 1/13 th that of a hospitalized covid-19 patient during the mid point of their hospitalization. so the air viral load was adjusted from 40 tcid50 units / liter as measured in the hospitalized covid-19 patients to an estimate of 3 tcid50 units / liter for the mid-phase pre-symptomatic individual modeled in this study. due to rapidly rising viral loads during the pre-symptomatic phase of infection, any estimate is widely dependent upon the viral load replicating within the infected individual. when considering exposure period estimates in table 2 , the likelihood of encountering someone who is already experiencing minor symptoms, or nearly so on the illness timeline, should be considered. a study on mask efficacy suggests that the exposure period estimate in the table could be adjusted to accommodate mask usage. "surgical masks nearly eliminated viral rna detection in the coarse aerosol fraction with a 25 fold reduction in the number of viral copies, a statistically significant 2.8 fold reduction in copies detected in the fine aerosol fraction, and an overall statistically significant 3.4 fold reduction of viral copy number in the exhaled aerosols." 19 comparing a 3375 cubic foot office with 6 air exchanges per hour (per ashrae standard 62.1) with an comparable sized open space outdoor setting with a 3 mph breeze (1056 air exchanges per hour) shows that a similarly sized outdoor space has 176 times more air exchanges over any given time period than a small office or a hospital room similar to the reference cases used in this study. an open outdoor space is further superior due to its virtual "infinite ceiling". additionally, indoor spaces tend to have areas of stagnant air flow around the cubicles, furniture, and other semi-enclosed spaces within the room. table 2 considers the cases of a non-infected person in an indoor environment within 2 to 4.8 meters of 2 infected persons and estimates the exposure period related to likely outcomes. the entire table uses shedding equivalency for influenza a and covid-19 as stated previously. rows 1 through 10 consider the case of a non-infected person in the environment performing light activity functions and respirating accordingly, while rows 11 and 12 consider a person engaging in heavy exercise or otherwise respirating at 60 liters per minute. table 2 provide documentation and analysis of the "reference" environment which has 2 hospitalized influenza a patients at 4 challenge influenza a doses from the respective studies. it uses an influenza a infectivity model. from those 4 rows, it can be seen that the non-infected visitor not wearing any ppe / masks would likely remain not ill when present for less than 2 minutes, and perhaps develop a minor illness after 20 minutes, and likely having mild illness after 3.5 hours, and almost assuredly becoming ill after 34 hours. immune response and some level of immunity could begin in as short as 2 minutes, but more likely would occur after more than 3.5 hours. the visitor would be unlikely to shed any virus until around or after a 3.5 hour visit. again, this considers patients ill with influenza a using direct influenza a challenge study data. rows 5 and 6 use estimations to model the 2 hospitalized patients with visitor result as if the patients were ill with covid-19. from those 2 rows, it can be seen that the non-infected visitor not wearing any ppe / masks would perhaps develop a minor illness after 5 minutes, and likely experiencing mild illness after 52 minutes. rows 7 through 12 use estimations to model the result as if a pre-symptomatic person was infected with covid-19 but not yet far enough into the infection as to have symptoms and large amounts of aerosolized shedding that would likely be present in a hospitalized patient with symptoms requiring hospital admission. in rows 7 through 9, it can be seen that the non-infected visitor would perhaps develop a minor illness after 1 hour, and likely experiencing mild illness after 11 hours. in row 10, the estimation is adjusted to simulate an outdoor environment with a 3 mph wind as noted above. it can be seen that the non-infected visitor not wearing any ppe / masks within 2 to 4.8 meters of 2 infected persons would likely remain not ill when present for less than 20 hours. this 20 hours estimate clearly shows the contrast between indoor environments that have no special ventilation measures and outdoor settings. indoor settings with strong ventilation improvements could begin to approach those of an outdoor setting. in rows 11 and 12, the estimation is adjusted to simulate the same outdoor environment, but with the non-infected visitor exercising around 2 resting pre-symptomatics. it can be seen that a non-infected person would likely remain not ill when present for less than 4 hours and perhaps develop a minor illness after 40 hours. this implies that outdoors is low risk even without masks. while it was considered too complex to estimate the case of 2 pre-symptomatics exercising heavily with an uninfected person outdoors, it may be possible extrapolate this scenario from the table. in rows 13 through 18, the estimation is adjusted to simulate an indoor environment with 24 air exchanges per hour, which is 4 time more ventilation than the reference office. similar to the previous rows, individuals ill with covid-19 and pre-symptomatic individuals are considered. in row 19, an outdoor environment is simulated with a person exercising near individuals ill with covid-19. rows 20 through 27 add surgical mask considerations. an n-95 mask would have even better performance. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . table 2 shows the multiple potential scenarios based on a spreadsheet formula 20 . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.03.20206110 doi: medrxiv preprint from results, it can be seen that likely outcomes vary widely based on ventilation conditions and the level of shedding of the infected individual. while the estimates predict that most outdoor settings with as low as a mild 3 mph breeze are likely to result in no or minor illness, there are many determinants associated with severe cases including comorbidities such as skilled-nursing-facility resident status, cardiac health issues, obesity, cancer, immune-compromised, etc. results estimates assume good general health and no significant co-morbidities as stated earlier. in table 2 , the "likely outcome" column study calculates for the scenario where there are known to be a few infected within any given social setting. in a real population that has, for example only a 100 per 100,000 population infectionrate, that "likely outcome" would have a 1 in 1000 chance of occurring. in the other 999 of 1000 cases, since no-one is infected, no-one can become infected, so the result is not ill. this study does not consider that 1 in 1000 probability that lowers the probability of becoming ill in a "real life scenario". the estimates in this study consider the case of any number of healthy individuals being within 2 to 4.8 meters of 2 definitely infected individuals (the hospital reference case used as the baseline for all estimates) so that a "worst case probability" scenario can be estimated. the probability of infection could be computed separately based on "local infection rates" and then further adjusted based on the illness "likely outcome" for any given exposure period. all estimates are subject to prior mentioned limitations. all times are specified as days:hours:minutes. the term "office" implies a room with 6 air exchanges per hour (acph) as table 2 defines unless otherwise noted. all other aspects can be also observed in table 2 and prior commentary. as mentioned in the "mask usage" section, the term "mask" refers to a surgical mask. from table 2 , many personal and business scenarios can be considered. the border between "not ill" and "minor illness" can help determine risk profile for virtually any scenario. the term "mild illness" is a clinical definition, and for covid-19 can mean quite unpleasant flu-like symptoms including fatigue, fever, chills, muscle aches, headache, sorethroat, etc. that would not require hospitalization. the definitions of moderate and severe mean they most often require clinical attention and/or hospitalization. "minor illness" can mean "common-cold" like symptoms or slightly worse but not full "flu like" symptoms which is defined by "mild illness". the covid-19 moderate and severe cases are referred to in table 2 as "possible severe illness". see the "known challenge dose" section for more detail. to help clarify table 2 , here are a few observations that might be made: a person wearing a mask doing light work (light active) near a covid-19 hospital patient (sickhosppt, covid) wearing a surgical mask in a 6 acph patient room may encounter enough exposure to develop a minor illness in a 49 minute period (0:00:49); but would likely not develop mild illness "flu like" symptoms unless in that environment for over 8 hours (0:08:14). using n-95 and other improved ppe would have the effect of increasing those estimates. it is possible that immunity may begin developing at even shorter intervals. it is possible that by rotating medical staff work through covid-19 patient areas at exposure intervals below the low end of this range several days apart and monitoring antibody test levels could help provide a path to immunity and increased ability to work with increasing exposure level environments without illness. encountering short to increasing length exposures and avoiding lengthy exposures until antibody test titers become strongly positive may be a path to avoiding healthcare staff illness. it is likely several shortterm exposures separated by several days will increase antibody test titers, and as they reach protective thresholds, longer exposures would likely increase those antibody test titers to a level of sterilizing immunity. repeat exposure would likely maintain that sterilizing immunity indefinitely. having non-immune medical staff avoid lengthy exposure to patients that have high shedding potential based on ct counts could also help avoid medical staff illness. both wearing masks, would likely result in those within a few meters becoming infected. with better ventilation at 24 acph, this would likely extend to more than 32 hours (1:08:59). people not wearing masks doing light work in a 3 mph open area outdoor breeze with a pre-symptomatic individual nearby would likely not encounter enough exposure to become ill until after over 20 straight hours (0:20:22). people not wearing masks doing heavy exercise in a 3 mph open area outdoor wind with a sick individual nearby may encounter enough exposure to develop a minor illness "cold like" in around 3 hours (0:03:03). people not wearing masks in a typical 6 acph office with a pre-symptomatic individual nearby would remain not ill in an encounter that is less than 6 minutes. in the scenario of a hair-styling appointment, if done within a common 6 acph indoor environment with no-one wearing masks and a pre-symptomatic person, minor illness might occur in around 1 hour (0:01:09), whereas that would extend to (0:04:37) in a 24 acph environment, which would significantly reduce the chance of illness in a 1 hour appointment. wearing masks would further improve the likelihood of being not ill. in the 24 acph environment with masks even with a sick person, the other person would likely remain not ill after 19 minutes (0:0:19). in the scenario of a 2 hour movie at a theatre in a 24 acph environment without masks and with pre-symptomatic persons, minor illness might occur in around 4 hours (0:04:37). if masks were used, even if a sick person were present, minor illness might occur in 0:03:17. not wearing a mask and being in proximity to a sick person indoors for 5 minutes could result in minor illness. extending that to 52 minutes could result in mild illness. passing them briefly for less than a minute would likely leave one not ill. this scales to hours and days when outdoors -certainly of no concern for brief encounters -even without a mask. regarding brief encounters, keeping calm and moving along works even when around people who are known to be sick. for pre-symptomatics and when wearing masks, these numbers scale accordingly to even longer periods of safety -see table 2 . again, in a "real life" scenario, the probability of infection described above is on the order of 1 in 1000 when there is a 100 per 100,000 population infection-rate. it is noteworthy that once an individual has reached a target exposure level, the individual should attempt to eliminate further exposure for several days to a week so that the adaptive immune system can respond to the exposure before encountering additional exposure. general immunology and the influenza a challenge study referenced earlier has demonstrated that a person becoming sick begins to lightly shed virus about 2 days prior to symptoms and rapidly ramps infectivity to a peak that occurs about 1 day after symptoms begin; whereupon a normal immune system responds to reduce infectivity within a few days to a week or so of symptom onset. this may depend upon general individual health and care in using optimal balances of rest and light exercise during recovery. some youth and extreme athletes are able to knock down illness within hours or a couple of days. by knowing the type of individual that is most likely to be encountered (pre-symptomatic or sick) and the likelihood of encountering an ill person (e.g. 1 in 1000 or confirmed sick), one may discover a time limit estimate that meets one's personal risk tolerance. it is important to keep in mind that these are estimates and the actual times could vary widely based on a large number of factors that even include amount of sleep, eating habits, exercise, and other factors too numerous to express. personal, business, and public health guidelines must consider many factors including number of active infections per capita, general area population density, specific environment occupancy, temperature, respiration rates, loudness of vocal activation, general population health, etc. table 2 , the studies that feed it, and other related studies, may help determine reasonable health guidelines and be useful to the general public in setting individual risk tolerances. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . businesses may benefit from having this study's actionable quantitative estimates inform specific business decisions including ventilation enhancement opportunities. given diverse business environmental characteristics and the limitations of this study, it is recommended that this study help inform local entity specific and personal / individual decisions. it is not intended for use in specifying government policy. for example, a person in excellent health who rapidly and fully recovered from covid-19 would likely select different conditions than those in other categories. every person's risk tolerance and optimal exposure level is different. those already recovered may benefit from light rechallenges that preserve antibody titer levels and related sterilizing immunity 21 . every business building has attributes that may favor applying different estimates. importantly, those with co-morbidities that place them at high-risk for a severe case should target zero exposure. people who attend to these high-risk individuals should use similar precaution. those who are at lower risk for a severe case and are not in contact with high-risk individuals could opt for a more "normal" lifestyle and use table 2 to guide them. one intent of this study is to identify the estimated conceptual boundaries for "not ill" and "minor illness" (table 2) so that the "generally healthy" population that is at low-risk for a severe case could generally gravitate toward those goals such that they avoid illness and perhaps gain some level of progressive immunity during an outbreak. those who have already recovered from an outbreak could select boundaries that are more-likely to help them maintain immunity without being exposed to an excessively strong (or weak) re-challenge. in healthy individuals, properly timed and dosed re-challenges can be helpful to maintain sterilizing immunity that protects those not yet infected during months-long outbreaks 22 . estimates such as these, especially when the accuracy is improved through similar future studies, provide the structure for a powerful 21 st century data-science-based methodology for avoiding illness and maximizing immune response. raising public understanding and consciousness of concepts such as "viral load", "exposure time", "challenge dose levels", "shedding quantities", "immune seroconversion", and "re-challenge" can achieve new levels of personal hygiene that exceed centuries-old adages such as "wash your hands". it is possible future generations would have basic knowledge most of the world never imagined in the 20 th century and prior. if every high-school student read and was tested on content similar to this study as part of a health class assignment, a tremendous step forward would accompany the next generation. the authors performed all work on this study without compensation as an act of good will and statesmanship and have no beneficial affiliations with any institutions related to the work. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted october 5, 2020. . https://doi.org/10.1101/2020.10.03.20206110 doi: medrxiv preprint while the estimates in this study cannot be considered authoritative due to estimation limitations, they offer considerably greater resolution and accuracy than who and cdc recommendations such as "6 feet of distance" and "use facial coverings" which do not differentiate between exposure periods and environments. the estimates in this study could be a significant improvement over current who and cdc guidelines in that they provide specific quantitative number estimates extrapolated from peer-reviewed studies for various environments. the various environments include typical asheae office ventilation standards, one example of dramatically improved ventilation (4x), and outdoor 3 mph conditions. the estimates also consider the infected person as either very sick or pre-symptomatic, the level of exertion of those breathing in vicinity of the infected, and various likely outcomes from not ill to possible severe illness. this study considers aerosolized transmission at a distance of > 2 meters. the estimations in the table focus on aerosolized transmission and do not consider the case of droplet or fomite (surface contact) based transmission which can be averted through some distance, barriers, socially hygienic behavior (not sneezing at your neighbor), handwashing, and other non-ppe / mask interventions. while the reader may personally choose to consider the estimates for personal, business, and other guidelines, they have no merit of accuracy, assurance, guarantee, medical efficacy, or legal standing. in the absence of better information, these estimates should be considered only with additional good judgement and after further verification. human response to pathogens is widely variant and these estimations assume the normal healthy population with better than average immune response and no co-morbidities. any application of these estimates is at the risk of those applying them. there may be unforeseen errors in the estimation, extrapolation, and underlying study interpretation. there may be errors in the referenced studies. no liability may be assumed or implied. the primary purpose of this study is academic, theoretically didactive, and abstractly educational. it should be considered a simple example of estimation methods but not an authoritative reference. choice to observe any of its specific results or commentary belong solely to the reader. as additional data becomes available, this study could be revised to reference and include that additional data. the estimation methods used in this study could also be improved. together, additional data and improved estimation methods could be used to revise the spreadsheet that makes the objective computations found in table 2 . future studies could improve models and estimation accuracy. while the values in table 2 are specific, they cannot be relied upon as accurate. this study is intended to be an early prototype of what could be accomplished going forward. it is believed similar but unreleased studies with these improvements already exist but have not yet been published and widely distributed to the public. it is hoped that publication of this study accelerates publication and widedistribution of similar, yet superior, studies. aa, president, inflection technologies corporation computer science, magna cum laude, application developer at tahoe blue ltd validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1) pdm09 dose-finding investigational new drug study viable sars-cov-2 in the air of a hospital room with covid-19 patients airborne transmission of sars-cov-2: theoretical considerations and available evidence probable evidence of fecal aerosol transmission of sars-cov-2 in a high-rise building validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1)pdm09 dose-finding investigational new drug study a dose-finding study of a wild-type influenza a(h3n2) virus in a healthy volunteer human challenge model validation of the wild-type influenza a human challenge model h1n1pdmist: an a(h1n1)pdm09 dose-finding investigational new drug study viable sars-cov-2 in the air of a hospital room with covid-19 patients influenza virus aerosols in human exhaled breath: particle size, cultur-ability, and effect of surgical masks key: cord-275355-4izc5jxs authors: hayden, frederick; croisier, alice title: transmission of avian influenza viruses to and between humans date: 2005-10-15 journal: j infect dis doi: 10.1086/444399 sha: doc_id: 275355 cord_uid: 4izc5jxs nan reports of seropositivity for different avian influenza a viruses in exposed poultry workers, including the new findings reported by puzelli et al. in this issue of the journal of infectious diseases [1] , and the recent instances of cross-species transmission that caused human disease [2] raise fundamental questions regarding the routes of transmission of avian viruses to and between humans, possible differences in transmission patterns between human and avian influenza viruses, and implications for prevention in those occupationally exposed to infected animals and also in health care, household, and community settings. documentation of seropositivity for avian influenza viruses in farm workers is not a new finding [3] , and previous studies have assessed human susceptibility by intranasal inoculation of selected avian influenza viruses [4] . however, the outbreak in europe of h7n7 virus infec-tion that led to many cases of conjunctivitis and 1 death resulting from viral pneumonia [5, 6] , as well as the unprecedented epizootic caused by highly pathogenic avian influenza h5n1 virus in southeast asia, emphasize the importance of these issues. one strength of the study by puzelli et al. is the multiplicity of the serological tests used, which included a microneutralization assay with infectious virus, a hemagglutination inhibition (hi) assay, and a confirmatory western blot analysis with purified h7 hemagglutinin to exclude the possibility of nonspecific cross-reactions with antibodies to human influenza viruses. differential absorption with human influenza virus has also been utilized to confirm the presence of avian influenza virus-specific antibodies [7] . such methods are essential to document seropositivity for an avian influenza virus, particularly when concerns about extensive transmission to humans are raised, as was reported elsewhere for h7n7 virus infection in the netherlands [8] . the possibility that the latter virus caused widespread subclinical infections in poultry workers and household contacts-and, hence, manifested efficient human-tohuman transmission-was raised by 1 study that used a modified hi assay to measure antibody [8] . similarly, the recent report of asymptomatic infection by h5n1 virus in northern vietnam, as de-termined by the detection of h5n1 rna in household contacts, requires substantiation by confirmatory serological testing [9] , although culture-confirmed h7n3 illnesses have occurred without an apparent detectable serologic response [10] . even in those individuals with proven seropositivity for an avian influenza virus, it is uncertain whether they have been only exposed to antigen or are productively infected. the findings that seropositivity occurs in small numbers of poultry workers exposed during outbreaks of illness in poultry caused by some avian strains (h7n7, h7n3, and h5n1) but not others (h7n1 and h5n2) argue for actual infection and support the notion that some avian influenza viruses are more likely than others to infect humans [1] . definitive evidence for active infection would include detection of virus or viral rna at the time of exposure or illness. in any case, the recent reports of the apparently greater adaptation of h7n7 [7] and h5n1 [9] avian influenza viruses to humans than was previously recognized and the potential for reassortment during dual infection with a low or highly pathogenic avian influenza virus and a conventional human influenza virus mandate careful laboratory documentation involving multiple assays. transmission of human influenza virus occurs by inhalation of infectious droplets or airborne droplet nuclei and, perhaps, by indirect (fomite) contact followed by self-inoculation of the upper respiratory tract or conjunctival mucosa. the relative importance of these routes is debated, and there is evidence to support each of them, including transmission within health care facilities [11, 12] , in human influenza. it is likely that each route contributes to transmission under appropriate circumstances and that the manifestations of illness, respiratory tract viral loads, and, perhaps, the type of infecting influenza virus influence the likelihood of transmission by a particular route. of course, the use of measures to prevent infection, such as personal protective equipment (e.g., masks and eye protection), hand hygiene, and specific chemoprophylaxis or immunization modalities, by potentially exposed persons will alter the observed risks. transmission of avian influenza virus likely encompasses these routes, as well as others. human conjunctiva [13] and ciliated nasal epithelial cells [14] contain cellular receptors that are recognized preferentially by the hemagglutinin of avian (a2,3 linkages between the terminal sialic acid residues and galactose), rather than human (a2,6 linkages), influenza viruses. the distribution of avian-type receptors in the lower airways and other tissues of humans requires study. however, it is particularly concerning that perhaps only 2 amino acid changes in the viral receptor binding site may be required to change the tropism of the h5 hemagglutinin from avian-to human-type receptors [15] . clinically apparent infections due to avian influenza viruses of the h7 subtype typically cause conjunctivitis and demonstrate higher viral loads in the eye than in the pharynx [5, 6, 10] . the importance of the eye or nose as a site of initial infection and the importance of subsequent replication with non-h7 avian influenza viruses are unknown. in h5n1infected patients, conjunctivitis has not been a feature, and rhinorrhea has been inconsistently reported. in contrast, the frequent occurrence of diarrhea and the detection of viral rna in most fecal samples tested suggest that h5n1 virus may replicate in the human gastrointestinal tract and raise the question of whether human feces could be a source of transmission [16] . most cases of human infection due to avian influenza viruses have involved close contact with infected poultry, particularly ill or dying chickens. during the outbreak in hong kong in 1997, 1 case-control study [17] found that exposure to live poultry within a week before the onset of illness was associated with human disease, but no significant risk was related to traveling, eating or preparing poultry products, or being exposed to persons with disease caused by h5n1 virus. another study in hong kong [18] found that exposure to ill poultry and butchering of birds were associated with seropositivity for h5 avian influenza viruses. four workers who culled infected birds in japan [19] and 2 animal attendants who cared for infected tigers in thailand [20] were found to have antibodies to h5n1 virus during the outbreaks in 2004; seroconversion indicating recent infection was found in only 1 of the japanese workers. during the first wave of human infections in 2003-2004, a history of direct contact with poultry was found in 8 of 10 h5n1-infected patients in vietnam [21] and with dead chickens in 8 of 12 h5n1-infected patients in thailand [22] , whereas no clinical cases of illness were noted in those involved in mass culling of poultry. it has been estimated that 12%-61% of rural thai residents have regular contact with birds [22] . however, ∼30% of h5n1-infected patients in vietnam have not reported exposure to sick poultry [16] , which leaves the issue open to speculations about more frequent human-tohuman transmission than has been found. however, this finding might be biased, because retrospective notification of animal disease has important consequences in some countries. infection after consumption of fresh duck blood and undercooked poultry products has been suspected in some cases of illness. indeed, transmission to felids was observed after experimental feeding of infected chickens to domestic cats [23] , and feeding tigers raw infected chicken led to outbreaks of illness in thai zoos, in which felid-to-felid transmissions were also implicated [20, 24] . infected birds shed high concentrations of virus in feces [25] . direct intranasal or conjunctival inoculation while swimming in contaminated water or, perhaps, inhalation or ingestion of water could have been potential modes of transmission to some h5n1-infected patients. as for human influenza, hand contamination from fomites and self-inoculation into the eye or upper respiratory tract remain possible modes. greater adaptation of avian influenza viruses to human hosts could alter the routes of transmission and increase the likelihood of human-to-human spread. in addition to sporadic bird-to-human and suspected environment-to-human transmission, human-to-human transmission of h5n1 avian virus has been implicated by epidemiological findings in several household clusters in which similar illnesses were reported in relatives [21] and in 1 well-documented situation in which there was child-to-mother and likely childto-aunt transmission in thailand [26] . these probable human-to-human transmissions involved close contact during the critical phase of illness and were inefficient without additional chains of transmission. several household contacts also developed symptomatic h7n7 avian virus infections after exposure to ill family members in the netherlands in 2003 [6] . however, in contrast to the studies of human influenza viruses [27] , molecular epidemiological studies to rigorously establish human-to-human transmission of h5 avian viruses have not been completed. cohort studies in 1997 found that human-to-human transmission might have occurred through close physical contact but not through social contact [28] . intimate, face-to-face contact without the use of measures to prevent infection was implicated in these circumstances, and no evidence to date indicates that there has been human-to-human transmission of h5n1 avian virus by small-particle aerosol exposure. recent serosurveys in southern vietnam and thailand have not found evidence for inapparent infections in family contacts [16] . although viral rna was detected by polymerase chain reaction in swab samples from asymptomatic family contacts of ill patients in vietnam in 2005, these infections remain to be confirmed by serological testing. nosocomial transmission of h5n1 virus to health care workers (hcws) was found by serological assessment in hong kong in 1997 [29] and is suspected in a nurse exposed to an infected patient in vietnam in 2005 [16] . to date, the risk of infection in health care settings appears low, even when appropriate isolation measures have not been used [7, 30, 31] . in 2004, no illness occurred in exposed hcws or laboratory workers in vietnam [21] or in 35 exposed and unprotected hcws in thailand [30] . no serological evidence of infection was present in 83 exposed and masked hcws in hanoi [7] , and another study of 64 unprotected hcws in ho chi minh city found no illness or seroconversion [31] . however, given the potential threat and changing transmissibility of avian influenza viruses, isolation precautions within health care facilities should encompass the measures used for potentially pneumoenteric pathogens, such as severe acute respiratory syndrome-associated coronavirus [32] . in summary, observations made to date suggest that differences in the routes of transmission between human and avian influenza viruses exist. the multiple potential routes for the spread of avian influenza viruses, particularly h5n1, indicate that, in addition to protection for the respiratory tract and eyes, proper hand hygiene may be especially important in preventing infection. this applies also in emergency departments and clinics where patients with febrile illnesses who are from areas with documented h5n1 virus infections in poultry or people may be evaluated. in households in which illness has occurred, additional specific protective measures-that is, postexposure chemoprophylaxis with oseltamivir-would be advisable for known household contacts. in affected countries, public education regarding simple precautionary measures for food preparation, poultry handling, and avoidance of contaminated water are essential until effective human vaccines for h5n1 viruses become available. serological analysis of serum samples from humans exposed to avian h7 influenza viruses in italy between 1999 and avian influenza. geneva: world health organization pandemic influenza: a zoonosis? replication of avian influenza viruses in humans avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands world health organization international avian influenza investigation team, vietnam. lack of h5n1 avian influenza transmission to hospital employees final analysis of netherlands avian influenza outbreaks reveals much higher levels of transmission to humans than previously thought world health organization. who intercountry-consultation: influenza a/h5n1 in humans in asia human illness from avian influenza h7n3, british columbia influenza in the acute hospital setting transmission of influenza: implications for control in health care settings avian influenza and sialic acid receptors: more than meets the eye? human and avian influenza viruses target different cell types in cultures of human airway epithelium restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host world health organization. who consultation on case management and research on human influenza a/h5 case-control study of risk factors for avian influenza a (h5n1) disease, hong kong risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 japan: serological investigation among humans involved in the mass culling operation probable tiger-to-tiger transmission of avian influenza h5n1 avian influenza a (h5n1) in 10 patients in vietnam human disease from influenza a (h5n1) avian h5n1 influenza in cats avian influenza h5n1 in tigers and leopards laboratory study of h5n1 viruses in domestic ducks: main findings probable person-to-person transmission of avian influenza a (h5n1) assessment of hemagglutinin sequence heterogeneity during influenza virus transmission in families antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts risk of influenza a (h5n1) infection among health care workers exposed to patients with influenza a (h5n1), hong kong atypical avian influenza (h5n1) avian influenza h5n1 and healthcare workers world health organization. who interim guidelines on clinical management of humans infected by influenza a (h5n1) key: cord-345836-74d2mb70 authors: hogg, william; gray, david; huston, patricia; zhang, wei title: the costs of preventing the spread of respiratory infection in family physician offices: a threshold analysis date: 2007-11-13 journal: bmc health serv res doi: 10.1186/1472-6963-7-181 sha: doc_id: 345836 cord_uid: 74d2mb70 background: influenza poses concerns about epidemic respiratory infection. interventions designed to prevent the spread of respiratory infection within family physician (fp) offices could potentially have a significant positive influence on the health of canadians. the main purpose of this paper is to estimate the explicit costs of such an intervention. methods: a cost analysis of a respiratory infection control was conducted. the costs were estimated from the perspective of provincial government. in addition, a threshold analysis was conducted to estimate a threshold value of the intervention's effectiveness that could generate potential savings in terms of averted health-care costs by the intervention that exceed the explicit costs. the informational requirements for these implicit costs savings are high, however. some of these elements, such as the cost of hospitalization in the event of contacting influenza, and the number of patients passing through the physicians' office, were readily available. other pertinent points of information, such as the proportion of infected people who require hospitalization, could be imported from the existing literature. we take an indirect approach to calculate a threshold value for the most uncertain piece of information, namely the reduction in the probability of the infection spreading as a direct result of the intervention, at which the intervention becomes worthwhile. results: the 5-week intervention costs amounted to a total of $52,810.71, or $131,094.73 prorated according to the length of the flu season, or $512,729.30 prorated for the entire calendar year. the variable costs that were incurred for this 5-week project amounted to approximately $923.16 per participating medical practice. the (fixed) training costs per practice were equivalent to $73.27 for the 5-week intervention, or $28.14 for 13-week flu season, or $7.05 for an entire one-year period. conclusion: based on our conservative estimates for the direct cost savings, there are indications that the outreach facilitation intervention program would be cost effective if it can achieve a reduction in the probability of infection on the order of 0.83 (0.77, 1.05) percentage points. a facilitation intervention initiative tailored to the environment and needs of the family medical practice and walk-in clinics is of promise for improving respiratory infection control in the physicians' offices. there is a paucity of empirical evidence in the literature about actual intervention strategies to improve respiratory infection control practices and analyze the efficiency implications for health policy. prevention, especially within health care settings, has assumed paramount importance in the fight against respiratory infection. since influenza is typically transmitted by droplets and contact routes, there are precautions that can be taken to reduce its transmission [1] [2] [3] . interventions designed to prevent the spread of respiratory infection within family physician (fp) offices could potentially have a significant positive influence on the health of canadians. while there are costs associated with the implementation of any intervention, the benefits stemming from the outcomes of such interventions have the potential to outweigh them. however, there are few evaluations of outreach facilitation that have studied the net costs of delivering interventions of this nature that exist in the literature. an exception is a study authored by hogg, baskerville, and lemelin [4] , which consisted of a randomized, controlled, field trial of an intervention aimed at improving preventative care tailored to the needs of participating family practices. it demonstrated the effectiveness of a multi-faceted outreach facilitation in improving overall preventative care performance. it is the first analysis of cost consequences of an outreach facilitation intervention of which we are aware, and it indicated that the cost savings attributable to the reduction in inappropriate testing on the one hand, and increases in appropriate testing on the other hand, may outweigh all of the intervention costs. those authors argued that a costly intervention that achieves success may be preferred on a cost-benefit basis to a cheaper one that demonstrates very little or has no lasting effect. while based on an original and a very different application, this current study employs a similar approach to investigating the resource allocation implications of another type of outreach facilitation intervention that was designed to prevent the spread of respiratory infection within fps' offices. evidence from a systematic review has shown that influenza transmission occurs primarily by the droplet and short-distance contact routes [1] . the best practices promoted by the intervention are the droplet and contact precautions, which are described presently. from a clinical perspective, improvement in adoptions of best practices prevents the respiratory virus transmission and therefore, is likely to reduce transmission rates. our particular case consists of an outreach facilitation intervention designed to improve respiratory infection control practices in community-based fp offices. it was conducted in the city of ottawa and delivered by five public health nurses. to our knowledge, it was the first facilitator-based intervention to promote respiratory infection control guidelines. although the intervention has been documented in detail elsewhere [2, 5] , we provide a summary of the intervention and its outcomes in this paper. a total of 53 family medicine practices participated in this pre-post intervention observational study, and all 53 completed the study intervention. of the 143 participating physicians, 110, or 77 % of them, completed all or part of the pre-intervention questionnaire. the objective was to determine the effectiveness (in terms of compliance) of a short-term intervention to facilitate the incorporation of best practices in respiratory infection control in primary care offices. a mnemonic was developed for both the nurses and physicians to summarize the best practices by the acronym "masks" (mask for the patient with cough and a fever, alcohol gel hand sanitization, seating of potentially infectious patient apart from others, "kleen"-disinfection of hard surfaces and signage). the intervention commenced with the public health nurse facilitators providing the baseline audit feedback on the respiratory infection control practices in the participating family physicians' practice to physicians and to other practice staff. physicians were presented directly (and other staff either directly or indirectly through the physicians) with evidence-based best practices and a facilitative "tool kit". this tool kit contained colourful signage outlining best practices for respiratory infection control, signage demonstrating proper hand-washing techniques and use of alcohol-based gel, a reference list of major guidelines sources and web sites, four infection control articles, a box of procedural masks, wall-mounted alcohol gel dispensers with refills, alcohol gel pumps, and hospital-grade disinfectant wipes. during the five-week intervention with their assigned recruited practices, the facilitators worked independently. throughout the intervention the facilitators corresponded with the project team daily and attended scheduled weekly meetings to share information and strategies. in order to measure outcomes, four respiratory control activities for an ambulatory office were viewed as the primary indicators of effective respiratory infection control: 1) signage posted in or about the waiting room; 2) the receptionist giving masks to patients having a cough and/ or fever; 3) instructing patients having a cough and/or fever to use alcohol gel to clean their hands; and 4) requesting patients having a cough and/or fever to sit at least one meter away from others. professional nurse auditors were deployed once to obtain data before the intervention and once six weeks after the intervention. the auditor sat for an hour in the waiting room of the physicians' offices and noted the presence or absence of the four respiratory control activities listed just above. they also inquired as to how often potentially contaminated areas were cleaned with disinfectants, and if alcohol-based hand gels were used in examining rooms. the auditors were blinded to the outcome measures and aware only of data gathering requirements. in order to separate the intervention from the data collection, the physicians, office staff and facilitators were blinded from the outcomes and were not informed of the presence of the auditors. statistically significant differences between before and after the intervention were observed for all four of the primary outcome measures: 67.3% (95% ci: 54.1%-80.5%), 48.1% (34.0%-62.1%), 54.7% (38.9%-70.5%) and 34.6% (20.1%-49.0%), respectively. overall, the number of practices that applied all of the four audited primary prevention measures was 3.8% (0%-9.1%) prior to the intervention and 52.8% (38.9%-66.7%) following the intervention (p < .001), demonstrating a 49 (35.1-63.0) percentage point increase in the adoption rate of best practices. this study demonstrated that facilitation of a multi-faceted intervention by public health nurses successfully promoted best practices in respiratory infection control in primary care practices. however, it did not consider health-related outcomes before or after the intervention. we conducted a cost analysis of the respiratory infection control intervention. a standard cost-benefit analysis or cost-effectiveness analysis could not be conducted in this case due to the absence of information on health-related outcomes. as supportive information, we also attempted to evaluate a threshold value for the intervention's effectiveness that could justify the costs incurred by the intervention in terms of the potential cost savings. standard methodological approaches can be found in drummond et al. [6] and muennig [7] . we determined the explicit costs of the intervention from the perspective of the provincial government, which is responsible for financing health care in ontario. the potential cost savings for this intervention referred to the costs of medical care averted due to the improved respiratory infection control practices that reduce the probability of infection in the physicians' offices. these implicit cost savings can include the cost of health care provider visits by patients experiencing illness symptoms, the cost of medical tests and procedures, and the cost of hospitalizations that were avoided. the actual explicit costs of the intervention over 5 weeks were gathered from the public health budget rationale (2004) for the inputs of labour, auditing services, supplies, facilitator travel, and honoraria that compensated the practices for the time diverted from normal activities. labour costs referred to the salaries and benefits of the five nurse facilitators and of the 0.5 full-time-equivalent project manager. the audit costs included the costs of the audit itself, involving feedback both before and after the intervention provided to the practices, as well as the traveling costs of the auditor. supply costs referred to the costs of the tool kits provided by the facilitators for each practice. an honorarium was paid to each fp practice site for the time it spent participating in the project. in addition to those variable costs, which vary directly with the number of practices that participate, it is important to include the fixed costs of the intervention, which consisted primarily of training the nurse facilitators. the investment in training generated returns extending well beyond the 5-week period of execution of the intervention. the amortization period for recovering the cost of training is much longer than this time frame for the initial intervention, as the skills obtained from training can be utilized again in subsequent years. the initial training cost should therefore be distributed across the estimated useful life of the investment item, taking into account the discount factor. we selected a discount factor of 5 percent as recommended by other papers containing cost analyses [4, 6, 7] . in addition to the discount rate, we have to select the length of the time period over which to amortize the training cost, which should be related to the life span of the training. as desai [8] pointed out in his application to obstetrics, often the analyst must assume a length for the useful life span, but this is often initially unclear. existing research from the field of organizational behaviour indicates that the payoffs stemming from a one-time, up-front investment in employer-paid training for human resources intervention tend to decline after four years [9, 10] . due to the fact that the facilitators received their training over a two-week period, we adopted a somewhat shorter amortization period for the cost of their training by assuming that it is valid for 3 years. the cost of the training of the five nurse facilitators was amortized over a 3-year life span at a discount rate of 5% based on the training expenses that were initially incurred at the beginning of the intervention. in another scenario, we included the entire training cost into cost analysis instead of amortizing it, which would imply that the training has no value after the current season. results were also presented at discount rates of 0% and 8% in the mathematical summary which details the discounting process (see additional file 1). the other costs listed in the public health budget rationale, such as recruiting participating practices, office assistance, and projection management were not included because these were costs incurred for this particular research pilot project rather than those of the intervention. those costs would not arise in the facilitation intervention implementation if it were to be adopted on a widespread basis. all of the direct costs were presented in micro detail for the 5-week period over which the intervention was executed, both in terms of total levels and on the basis of costs per practice. as such, the cost estimates that we generated should generalize to similar projects in other geographic areas that are on either larger or smaller scales. we have made some assumptions regarding how a facilitation program might be organized in order to deal with evaluating the costs of training the facilitators. our outreach facilitation program is most likely to be effective if delivered during or just before the peak season for respiratory infections (i.e., september, october, and november). hence our training activities would ideally be applied for 3 months per year over 3 years, generating a cumulative total of 9 months of utilization. while the program would aim to introduce proper respiratory infection control practices to be followed all year round, the medical practitioners might be more interested just prior to the influenza season. therefore, although the training remains valid for years into the future, we envisaged that the program would be delivered during that 3-month period every year. we nevertheless also produced estimates based on the scenario for which the intervention is executed yearround. while the explicit costs of implementing this intervention can be assessed with accuracy, it is much more difficult to estimate the implicit cost savings because of the lack of information regarding a key event, namely the reduction in the probability of spread as a direct result of the intervention. we assume without solid evidence that improved infection control reduces the respiratory infection rate at physicians' offices, but we certainly do not know how by much the probability of infection changed after the intervention. in order to generate an accurate estimate of the total health-care costs averted by this intervention, one would require the following pieces of information: i) the incidence or frequencies of transmission at physicians' office, ii) the effect of the intervention in reducing those rates, iii) the probabilities of the various potential health outcomes that could arise given infection, and iv) the cost of the treatments associated with those outcomes. with the exception of item iv), these pieces of information were not available. drawing from several data sources in the literature, we therefore adopted an indirect approach to esti-mate the potential health-care costs that might be averted as a result of the intervention, and we attempted to make a case that the potential benefits were large relative to the explicit intervention costs. there are a range of treatments for different influenza patients according to the seriousness of the infections. the patient who is infected with influenza may rest at home, visit an emergency room, or be hospitalized. if the patient only needs care at home, he or she may request sick leave from his or her job. in such a case, cost arises from the patient's perspective or the societal perspective (from the lost output) but not from the ministry of health's perspective. another possibility is that a few patients die from influenza, but it is impossible to attach a precise value for the cost of death. therefore, we only took the intermediate events of outpatient visits and hospitalizations into account in estimating the avoided costs. the costs denominated in us dollars (as they were presented in some studies that we cited) were converted into canadian dollars by the current exchange rate [11] , and costs from data in prior years were adjusted for inflation and denominated in 2004 constant dollars using appropriate component of the consumer price index [12] where necessary. the underlying approach for the cost analysis of the intervention involves an efficacy rate, which is defined as the decrease in the probability of transmission that is attributable to the intervention. we could not evaluate this quantity, but we could evaluate the threshold value that would render the intervention beneficial, which was judged to be worthwhile if: cost savings -intervention cost > = 0. the cost savings attributable to the intervention were expressed as follows: (cost of hospitalization for a flu patient*number of flu cases avoided due to the intervention in the physician's office*proportion of the infected people who were hospitalized) + (cost of outpatient visit for a flu patient* number of flu cases avoided due to the intervention in the physician's office*proportion of the infected people who had an outpatient visit). note that infected individuals who were hospitalized or who had an out patient visit may or may not have passed through the fps' practices; there are other modes of infection besides transmission in these clinics. the second element in each of the terms in parentheses, which is a counterfactual, can be expressed as: the number of flu cases avoided in the physician's office due to the intervention = number of patients visiting the physician's office* (probability of contracting influenza in that office without the intervention -probability of contracting influenza in that office with the intervention). substituting that expression into the primary equation yields the following expression after a slight algebraic manipulation: (probability of contracting influenza in the office without the intervention -probability of contracting influenza in the office with the intervention) = intervention cost/ [number of patients visiting the physician's office*(cost of hospitalization*proportion of infected people who were hospitalized + cost of outpatient visit*proportion of infected people with an outpatient visit)]. a critical element for this calculation is transmission rates for influenza in settings such as physicians' offices. while there are articles in the literature dealing with the incidence of transmission of certain viruses within the general population, we were unable to find research pertaining to the incidence of transmission within physician offices or similar locations involving close contact with the public, such as waiting rooms, emergency rooms, and school busses. we searched for papers on medline, cinahl and embase by the key words "influenza or flu, and transmission or infection, and bus or waiting room or emergency room or emergency department or physician office", and we also asked for help from several experts in this area to search for the requisite information. we did locate some information regarding the incidence of transmission of influenza during airline flights. in our judgment, however, these figures are not reliable estimates of the rate of infection with and without the intervention that would occur in a fp's office. in light of that source of uncertainty, our approach was to calculate an estimated value for the left side of the above expression (i.e the reduction in the likelihood of infection) that represents a threshold value for the minimum efficacy of the intervention such that the potential cost savings of the intervention outweigh its costs. we solve that expression for the lowest possible value at which the net costs of the intervention would be negative. if the efficacy of the intervention is any lower than that value, its net costs would be greater than 0. table 1 presents the number of hours of intervention work activity and the percentage of total hours spent at the 53 medical practices by the facilitators. the total number of hours worked was 875 (25 days ã� 5 facilitators ã� 7 hours/day). in table 1 , it should be noted that the time spent on "other" needs to be removed from the analysis, as that labour time was not allocated to the project. therefore, the total hours for the five public health nurse facilitators spent on the intervention should be the figures listed under the "total" label minus those listed under the "other" label, which worked out to a total of 617 hours. on average, they spent approximately 11 1/2 hours at each practice for which they were responsible. given a yearly salary of one nurse facilitator of $47,876 (in the ottawa area) and an annual total of 1,950 hours worked in one year, the hourly wage rate of one nurse facilitator was $24.55. this generated a labour cost per practice of $285.80. in the 5-week intervention period, the labour costs (for time actually worked) for all five nurse facilitators combined amounted to $15,147.35 ($24.55 ã� 617 hrs). the total costs for the intervention are presented in table 2 . the third column provides the data on the costs of the outreach facilitator intervention denominated in 2004 dollars on the basis of the 5-week period during which they actually worked. the fourth column contains similar data, except that all of the costs were estimated on the table 2 were based on the assumption that the 5 facilitators would work at the same pace for an entire year, and would thus visit approximately 551 practices. the difference between these three scenarios consists of a pro-rating of all of the variable costs while holding the training costs fixed. the figures in the last column were exactly same as those in the third column except for the training cost. the training cost presented in the last column was not amortized over 3 years, which accounts for the approximately threefold increase in the training costs coupled with no change in the other costs. the intervention costs amounted to a total of $52,810.71 that was actually incurred over the 5-week intervention, $131,094.73 per flu season, or $512,729.30 per calendar year. in order to extrapolate these cost figures to other geographical areas, the distinction between the variable costs and the fixed costs plays an important role. the variable costs that were incurred for this 5-week project amount to $48,927.51, which is the sum of all of the costs listed in the third column of table 2 the (fixed) training costs must be calculated in a different fashion, however. as explained in the mathematical summary (see additional file 1), we calculated an annual value of $3,883.20 for the training costs. this figure is equivalent to $73.27 for each participating practice. had these five facilitators worked for the entire 13-week flu season, the total training costs would still be $3,883.20, but many more practices could have been involved, thus lowering the per-practice training cost to $28.14 ($3,883.20/138 practices). if these same facilitators were to be assigned to this project on a year-round basis, the per-practice training costs would become one quarter of the prior figure, or $7.05, because the nurses work 4 times longer during the year. the first element that we obtained for the expression for averted costs was the number of patients that passed through the offices of the participating physicians, and were therefore at risk of becoming infected. in pre-intervention questionnaire, physicians were asked how many patients they typically see per half-day, from which we may estimate the number of patients visiting the physician offices during a 5-week period. 103 physicians responded to the question, and the mean value was 14.55. imputing this value to all of the physicians that were covered in our intervention, approximately 104,033 patients visited the 143 participating physicians over 5 weeks. *the training cost was amortized over 3 years using 5% discount rate, and therefore the training cost for each calendar year was $3,883.20, as shown in the additional file 1. this training cost for five facilitators would be totally fixed for a 3-year period. even if these 5 facilitators were to conduct this activity for the entire flu season, and thus serve more than 53 practices, this cost would not change. 1 costs for the 5-week intervention based on the actual 5-week length of the project. 2 assume that intervention lasts 3 months (13 weeks) during flu season. all figures except the training cost were obtained by converting the 5-week totals (that apply to our particular intervention) listed in third column to weekly rates and then multiplying by 13. 3 assume that intervention lasts one year (52 weeks). all figures except the training cost were obtained by converting the 5-week totals (that apply to our particular intervention) listed in third column to weekly rates and then multiplying by 52. 4 costs for the 5-week intervention based on the actual 5-week length of the project and the training cost was not amortized. the figures for the costs of treating influenza patients were drawn from several sources. hogg, baskerville and lemelin [4] performed a cost savings analysis associated with administering influenza vaccine in the elderly. they obtained the estimated cost of an emergency room visit due to influenza from jacobs and hall, which was approximately cn $76.00 in 1999 or cn $84.00 in 2004 [4, 13] . this cost was virtually identical to the costs for an outpatient visit reported in other studies [14, 15] . we turn next to the proportion of people infected with influenza that ended up being hospitalized. an estimate of this proportion can be obtained by dividing the hospitalization rate among all subjects with influenza (regardless of where it was contracted) by the proportion of all subjects who become infected (regardless of where it was contracted). the latter quantity can be thought of as the illness or transmission rate of the influenza. in an analogous fashion, an estimate of this proportion of infected people who had an out-patient visit can be obtained by dividing the out-patient rate among all subjects with influenza by the proportion of all subjects who become infected. unfortunately, we could find no paper in the literature that provided values for the hospitalization rate or the outpatient visit rate given that a patient has influenza. in order to obtain rough estimates of these quantities, we borrowed heavily from the paper by nichol [16] that dealt with vaccination against influenza. by a systematic literature review, the author obtained estimates of 'the hospitalization rate due to influenza and its complications', 'outpatient visit rate due to influenza and its complications', and 'the influenza (and its complications) illness rate' among healthy working adults aged between 18 and 64 years. nichol also derived from the monte carlo simulation the difference of the hospitalization rate (as well as the outpatient visit rate and the illness rate) for influenza and its complications between unvaccinated and vaccinated subjects. however, the influenza's complications were widely defined in nichol's paper. in our analyses, we focused on only influenza and pneumonia associated with influenza. therefore, by assuming that vaccination is 100% effective in preventing episodes of influenza (and pneumonia associated with influenza), we used the number of vaccinated individuals as a proxy for the number of non-infected subjects. in this respect, the three difference rates reported by nichol can be interpreted as each of these three incidence rates due to influenza only (and pneumonia associated only with influenza). therefore, we used these differences, 0.026% (95% probability interval (pi): 0.011%, 0.043%), 2.5% (1.2%, 4.5%), and 5.5% (3.2%, 9.0%), as our estimates for 'the hospitalization rate due to influenza only', 'outpatient visit rate due to influenza only', and 'the influenza illness rate', respectively. when we inserted these values into the expression for the proportion of infected patients who ended up hospitalized, we obtained a value of 0.00472 (pi: 0.00344, 0.00478), and the value for the proportion of infected patients who had an outpatient visit was 0.4545 (pi: 0.375, 0.5). inserting all of the figures that we obtained above back to the primary expression for the cost savings, and combining that information with the value for the explicit costs of intervention, the efficacy of the intervention (probability of contracting influenza in the office without the intervention -probability of contracting influenza in the office with the intervention) was equal to 0.83% (pi: 0.77%, 1.05%). the implication is that the threshold value for the efficacy at which the cost savings of the intervention barely outweigh the costs was 0.83%. the goal would thus be to reduce the probability of infection occurring in fps' offices by at least 0.83%. in addition, if we included the non-amortized training cost into analysis, the threshold value rose slightly to 0.93%. the figures that entered into the calculations are presented in table 3 . this paper has provided detailed information on the costs of an outreach facilitation initiative designed to prevent the spread of infectious diseases by promoting best practices in respiratory infection control in primary care practices. we have generated accurate estimates of the explicit costs of implementing such a program on a per-practice basis, which permits the extrapolation of these unit costs to other geographical domains. we have also provided some preliminary estimates of the potential cost savings to the health-care system. due to the lack of knowledge about the frequency of respiratory infection occurring at physicians' offices, particularly an estimate of the reduction in the probability of infection attributable to the intervention, we did not have enough evidence to evaluate precisely the benefits of the intervention. as an alternative approach, we undertook a threshold analysis to estimate a threshold value of the efficacy that could render the intervention cost saving. based on our conservative estimates referring to direct savings in the form of healthcare costs averted, there are indications that the outreach facilitation intervention program would result in cost savings if it could achieve a reduction in the probability of infection at the physician offices on the order of 0.83 percentage points. this implies that if we assume that there was a 1.00% chance of contracting influenza in fp offices without intervention, to achieve the efficacy rate of 0.83%, the probability of contracting influenza in fp offices with intervention would be 0.17%, representing a large relative risk reduction in influenza transmission in fp offices. on the other hand, if we assume a higher probability of contracting influenza in fp offices without intervention, such as 5%, to achieve the targeted efficacy rate of 0.83%, the probability of contracting influenza would be approximately 4.2%, representing a smaller relative risk reduction in influenza transmission in fp offices. moreover, in addition to the direct cost savings to the health care system that may be realized, there are potential indirect cost savings associated with our intervention as well, such as the potential to avoid disastrous human loss and suffering caused by viruses such as the severe acute respiratory syndrome (sars). the scope of the influences of the infectious diseases such as sars and influenza extend far beyond the costs that were mentioned above, especially in the health-care and tourism sectors. the total costs in terms of lost production of the sars epidemic to toronto's economy had been estimated to be $1 billion, and the estimate for the economic cost for all of canada was around $1.5 billion [17] . within the health care sector, the indirect costs borne by non-sars patients were enormous. sars affected all health-care workers -especially those on the front line -and delayed "non-emergency surgeries" such as organ transplants and cancer radiation [18] . according to ontario health minister tony clement, as of june 27, 2003, sars had cost ontario's health-care system $945 million, which was spent mostly on special supplies and added health-care workers needed to protect health-care workers, as well as on constructing specialized sars clinics and isolation rooms [17] . this in turn had a huge impact on non-sars related health care system utilization, both due to diversion of resources as well as severe stress amongst the health care providers. for instance, a study comparing the periods before and during the sars outbreak in the gta and non-gta areas by woodward et al. found the greatest impact of sars on reduction in the utilization of inpatient and outpatient hospitalization, diagnostic testing, physician and emergency department visits, use of prescription drugs, intensive care bed availability, and cardiac care during april 2003 to may 2003 [19] . avoiding such negative consequences implies that our intervention may also generate implicit or indirect cost savings. the 5-week intervention costs amounted to a total of $52,810.71. the results of the cost analysis suggest that the intervention can be cost saving because the 0.83% point reduction of the probability of influenza at the physicians' offices appears to be a feasible target for the effectiveness of the studied intervention. a facilitation intervention tailored to the environment and needs of the family practice and walk-in clinics is of great promise for improving respiratory infection control in the physicians' offices. future research to conduct further economic evaluations of such an intervention based on adequate dataparticularly in relation to infection incidence rates and the ability to lower them -would aid in important public health policies and administrative decision-making on implementing preventive care guidelines. the author(s) declare that they have no competing interests. william hogg and patricia huston conceived the intervention, provided the data, participated in the study design and in critical revisions of the manuscript, and contributed to all other aspects of the study. david gray contributed to the cost evaluation study design, assisted with the calculations, and thoroughly revised the manuscript. wei zhang acquired the economic data, performed the cost evaluation analysis and interpretation, and drafted the first pass of the manuscript. all authors read and approved the final manuscript. transmission of influenza a in human beings promoting respiratory infection control practices in primary care: primary care/public health collaboration. accepted for publication in canadian family physician the inexact science of influenza prediction cost savings associated with improving appropriate and reducing inappropriate preventive care: cost-consequences analysis harnessing primary care capacity to enhance the public health response to respiratory epidemics. accepted for publication in canadian family physician methods for the economic evaluation of health care programmes oxford designing and conducting cost-effectiveness analyses in medicine and health care the cost of emergency obstetric care: concepts and issues assessing the economic impact of personnel programs on work-force productivity recouping training and development costs using preemployment agreements. employee responsibilities and rights journal daily foreign exchange rates look-up statistics canada: the consumer price index and major components for health care estimating the cost of outpatient hospital care cost benefit of influenza vaccination in healthy, working adults: an economic analysis based on the results of a clinical trial of trivalent live attenuated influenza virus vaccine the economic impact of pandemic influenza in the united states: priorities for intervention cost-benefit analysis of a strategy to vaccinate healthy working adults against influenza the economic impact of sars. cbc news online updated the toronto sars experience. division of infectious diseases utilization of ontario's health system during the 2003 sars outbreaks: an ices investigative report. institute for clinical evaluative sciences we are very thankful to dr. carmel martin, who contributed in the design and implementation of the facilitation study. we also thank three referees for their efforts and their constructive comments. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/7/181/pre pub mathematical summary -amortization of training costs. click here for file [http://www.biomedcentral.com/content/supplementary/1472-6963-7-181-s1.doc] key: cord-265138-i5m3ax7g authors: wang, xi-ling; yang, lin; chan, king-pan; chiu, susan s.; chan, kwok-hung; peiris, j. s. malik; wong, chit-ming title: model selection in time series studies of influenza-associated mortality date: 2012-06-20 journal: plos one doi: 10.1371/journal.pone.0039423 sha: doc_id: 265138 cord_uid: i5m3ax7g background: poisson regression modeling has been widely used to estimate influenza-associated disease burden, as it has the advantage of adjusting for multiple seasonal confounders. however, few studies have discussed how to judge the adequacy of confounding adjustment. this study aims to compare the performance of commonly adopted model selection criteria in terms of providing a reliable and valid estimate for the health impact of influenza. methods: we assessed four model selection criteria: quasi akaike information criterion (qaic), quasi bayesian information criterion (qbic), partial autocorrelation functions of residuals (pacf), and generalized cross-validation (gcv), by separately applying them to select the poisson model best fitted to the mortality datasets that were simulated under the different assumptions of seasonal confounding. the performance of these criteria was evaluated by the bias and root-mean-square error (rmse) of estimates from the pre-determined coefficients of influenza proxy variable. these four criteria were subsequently applied to an empirical hospitalization dataset to confirm the findings of simulation study. results: gcv consistently provided smaller biases and rmses for the influenza coefficient estimates than qaic, qbic and pacf, under the different simulation scenarios. sensitivity analysis of different pre-determined influenza coefficients, study periods and lag weeks showed that gcv consistently outperformed the other criteria. similar results were found in applying these selection criteria to estimate influenza-associated hospitalization. conclusions: gcv criterion is recommended for selection of poisson models to estimate influenza-associated mortality and morbidity burden with proper adjustment for confounding. these findings shall help standardize the poisson modeling approach for influenza disease burden studies. numerous studies have demonstrated that influenza causes substantial burden on mortality and morbidity [1] [2] [3] . reliable estimates for disease burden associated with influenza in the community are essential for public health policy-making. however the case numbers of influenza infections derived from medical records grossly underestimated the true burden [4] . during 2001 to 2009 there were only 138 deaths registered in hong kong with underlying cause of influenza infection [5] . underreporting of influenza cases was due to the fact that influenza infections usually caused relatively mild symptoms and many infected people did not seek medical care from hospital or clinic. among outpatients and inpatients with influenza-like illness, few were tested for influenza to get confirmed diagnoses. even for those with laboratory confirmed infections, influenza was rarely recorded as underlying cause of death on their death certificates. several statistical models have been used to quantify the disease burden attributable to influenza [6] . among these models, poisson regression models have become increasingly popular in recent years [7] [8] [9] . unlike most of the other models, the poisson model does not require clear seasonality of influenza to define influenza epidemic and nonepidemic periods. therefore, it is particularly suitable for tropical and subtropical regions where influenza seasonality is less clear and influenza viruses could be circulating throughout the whole year. another advantage of the poisson model lies in its ability to adjust for multiple seasonal confounders simultaneously. there are two types of confounders that are often considered in poisson models: measured confounders, such as meteorological factors, circulation of other respiratory pathogens and air pollution [10] ; and unmeasured confounders, such as seasonal change in host susceptibility and health seeking behavior [11] . however, overadjustment of confounders may result in underestimation of true effects, as some variations caused by influenza were allocated to confounders. likewise, inadequate adjustment could lead to residual confounding that causes spurious association between influenza proxy variable (such as proportions of specimens testing positive for influenza) and health outcome of mortality or hospitalization. therefore, proper adjustment of confounders is critical for obtaining reliable estimates of influenza-associated disease burden. previous studies using poisson models adjusted for unmeasured confounders by incorporating sinusoidal pairs [12] [13] [14] [15] or a smoothing function of time trend into the poisson model [8, 16, 17] . however, few studies has properly discussed on how to determine the adequacy of adjustment for seasonal confounders in the model. here we conducted a simulation study with the aim to compare the performance of several commonly adopted model selection criteria, in terms of selecting the best-fit poisson model with adequate adjustment for confounders. four model selection criteria were considered in this study: quasi akaike information criterion (qaic), quasi bayesian information criterion (qbic), partial autocorrelation functions of residuals (pacf), and generalized cross-validation (gcv). we obtained weekly all-cause mortality data from 1998 to 2008 from the census & statistics department, and daily meteorological data of temperature and relative humidity from the hong kong observatory. daily concentrations of air pollutants nitrogen dioxide (no 2 ), sulfur dioxide (so 2 ), ozone (o 3 ) and particulate matters with diameter less than 10 mm (pm 10 ) were obtained from the environmental protection department. weekly numbers of specimens positive for influenza and respiratory syncytial virus (rsv) as well as total numbers of tested specimens were obtained from the microbiology laboratory of queen mary hospital. influenza virology data of this single laboratory have been demonstrated representative of the virus activity in the entirety of hong kong [16] . we performed a simulation study by generating mortality data from a poisson model with adjustment for over-dispersion [18] . this model is similar to the models used in our previous studies on influenza-associated mortality and morbidity [17] , in which an influenza proxy variable is added to assess influenza effects. to derive a proper estimate for influenza-associated mortality or morbidity, it is important to adjust for confounding to separate the effect of influenza from those of other seasonal factors. cocirculation of rsv, together with two meteorological factors of temperature and humidity, are adjusted for as confounders in this study given their association with both health outcomes and influenza [19, 20] . weekly concentrations of four major ambient air pollutants are also included as confounders based on recent findings on the association between influenza virus and ambient air pollutants [21] . unmeasured confounding is adjusted for by including the long-term and seasonal trends of outcome variables. a typical model was as follows: y t *quasipoisson(m t ,wm t ), log (m t )~b 0 zbflu t zb 1 rsv t zs(t, df~11|k) zs(temp t , df~3) zs(hum t , df~3)zb 2 no 2t zb 3 so 2t zb 4 o 3t zb 5 pm 10t zb 6 sars t : y t denotes the weekly number of all-cause deaths which was assumed to follow a poisson distribution with an over-dispersion parameter w [22] . flu t and rsv t denote the proxy variables for influenza and rsv, which are weekly proportions of specimens testing positive for influenza or rsv. three natural cubic spline smoothing functions of s(t, df = 116k), s(temp t ) and s(hum t ) are added to adjust for time (t = 1,2,…,574), weekly mean temperature (temp t ) and relative humidity (hum t ), where the degrees of freedom (df) of time k ranges from 1 to 10 per year. we used a natural cubic spline with fixed degrees of freedom so that the locations of knots were evenly distributed [23] . no 2t , so 2t , o 3t and pm 10t denote the weekly mean concentrations of four air pollutants, respectively. to adjust for the increased mortality during the outbreak of severe acute respiratory syndrome (sars) in 2003, we added into the model a dummy variable sars for the sars period of week 1-30 of year 2003. before simulation, we first estimated the coefficients of model (1) by fitting it to the all-cause mortality data during 1998 to 2008 in hong kong. the degrees of freedom were fixed to three for weekly temperature and humidity based on our previous experience [17] and to one per year for long term and seasonal trends. mean mortality for weekm m t was then predicted from this fitted model with the b coefficient for influenza variable flu t fixed to 0.33 (i.e. mortality increasing 3.3% when the influenza positive proportion increases 10%). the over-dispersion parameter wwas also derived from this model. because there was no statistical package available for data simulation based on the over-dispersed poisson distribution, we simulated 500 mortality datasets by assuming that mortality followed a negative binomial distribution, i.e. y t *negbin(m t ,h) when h~m t w{1 [24] . given the uncertainty in degrees of freedom for unmeasured seasonal confounders, we repeated the above simulation process with the degrees of freedom of s(t, df) changing from 1 to 2,3,…, 10 per year. hence, we got a total of 5000 weekly mortality datasets and 500 for each fixed degrees of freedom for t. we then applied model (1) with degrees of freedom varying from 1-10 per year for s(t,df) to each set of 500 simulated data), and selected the best-fit model with the minimal value for each of the following model selection criteria: 1) quasi-akaike information criterion (qaic) [25] : 2) quasi-bayesian information criterion (qbic) [26] : where n is the number of observations. 3) residual autocorrelation: the sum of the absolute value of the partial autocorrelation function (pacf) of the residuals up to 5 lag weeks. 4) generalized cross validation (gcv) [26] : where tr(r) is the trace of weighted additive-fit operator corresponding to the last iteration of the local-scoring procedure; y t is the observed number of death at week t;m m t is the predicted number of death at week t; d(y t ;m m t ) is the deviance of y t fromm m t ; n is the number of observations. we calculated the bias as the average difference between the estimated coefficients of the influenza variable from the best-fit model and the true coefficient of 0.33. standard error and rootmean-square error (rmse) were defined as the standard deviation and square root of the mean square error of estimated coefficients, respectively. in this study we took rsme as the primary measure to compare the performance of different model selection criteria, as it could evaluate both accuracy and variation of the estimates [25] . the criterion that obtained the minimal rmse under the different assumptions of confounding was considered as the best criterion in selecting the model with adequate adjustment for confounders. to investigate the robustness of our results, we conducted a sensitivity analysis by changing the pre-determined coefficient of influenza variable from 0.33 to 0.1 and 0.5, which were the lower and upper boundaries of influenza effects based on our previous experience. because a short study period might offer less reliable estimates with large standard error, we did another sensitivity analysis with the data of 2003 to 2008 or those of 2006 to 2008. given that influenza effects on mortality might lag several weeks behind the increase of influenza activity [15] , we separately added the influenza proxy variables up to 3 weeks before (lag 1-3 weeks) into the models to assess any lag effects. all the analyses were conducted using r software (version 2.13.0) [27] . we applied the four model selection criteria to an empirical dataset of weekly hospitalization numbers of pediatric patients younger than 18 years. these patients were admitted into two major public hospitals on hong kong island from october 2003 through september 2008, with acute respiratory disease (ard) listed in the first five discharge diagnoses. these data were retrieved from the computerized database of the hong kong hospital authority, according to the international classification of disease 9 th revision (icd9) codes of 4602466 or 4802487. five age groups were considered: 021, 122, 225, 5210, 10218 years. this poisson model was similar to model (1), except that two proxies for adenovirus (adeno t ) three types of parainfluenza viruses (p1 t , p2 t , p3 t ) were added as confounders, because these data were only available after 2003. influenza-associated hospitalization rates were defined as the difference between the observed and expected hospitalization under the assumption of no circulating influenza viruses. these rates were separately estimated from the best-fit models chosen by each criterion, and the bias and rmse were calculated by comparing with the observed admission rates of a pediatric cohort of influenza hospitalization cases. as previously described [17] , this cohort was composed of all the pediatric patients who were recruited from the same two hospitals and diagnosed with influenza infection by immunofluorescence tests and viral culture. ethics approval for collecting specimens from pediatric patients was obtained from the ethics committee of li ka shing faculty of medicine, the university of hong kong (ec1880-02). figure 1 shows the weekly number of deaths simulated under the scenario of low and high seasonal confounding (df = 1 and df = 10 per year for the seasonal trend smoothing functions). the simulated data fluctuated within the range of 400 to 1100 with a steadily increasing annual trend. as expected, the data simulated under df = 10 was rougher and closer to the true mortality than those simulated under df = 1 (figure 1) . overall, the simulated allcause mortality data were generally comparable with the true mortality data. most models overestimated influenza effects with a few exceptions observed for the models selected by the minimal gcv ( figure 2) . estimates tended to have larger biases as the seasonal confounding of the simulated data increased. overall the models selected by pacf, qaic and qbic had the larger biases (ranging from 0.0022 to 0.2909) than did those selected by gcv (ranging from 0.0008 to 0.007) (figure 2 ). standard error of influenza coefficients was comparable between these four criteria, ranging from 0.0012 to 0.0045. rmse was similar between the models selected by pacf, qaic or qbic when the df of smoothing functions for time were less than 5 per year, but dramatically increased when the df increased to 5 or more per year ( figure 2 ). the rmse of gcv criterion remained lower than those of the other three criteria (figure 2 ). biases and rmse did not markedly change when the predetermined coefficient for influenza proxy variable of weekly positive proportions changed from 0.33 to 0.1 and 0.5 ( figure s1 ). among the four criteria, gcv still provided the smallest bias and rmse under the different simulation scenario. sensitivity analysis of a shorter study period of 2003-2008 or 2006-2008 showed slightly higher rmse than those from the whole study period, but gcv provided smaller biases and rmse compared to the other three criteria ( figure s2 ). in the models with the lag effects of up to 3 weeks, gcv still provided the smallest biases and rmse for influenza coefficients ( figure s3 ). figure 3 shows the percentage difference between the estimated excess ard hospitalization rates and directly observed admission rates of influenza cases in the pediatric cohort from 2003 to 2008. for the age groups of 021and 10218 years, the best-fit models selected by gcv provided the estimates closer to the observed rates than did those selected by qaic, qbic and pacf ( figure s4 ). estimates from the four criteria were comparable for the 122 and 225 age groups. all the poisson models respectively selected by the four criteria slightly overestimated the true rates for all the age groups, except that the pacf and gcv criteria provided the estimates smaller than the observed rates in the 5210 age group. among the four criteria, gcv had the smallest biases and rmse, whereas qaic and qbic had the largest (table 1 ). as underreporting of influenza cases is common in clinical practice, the poisson modeling approach has been widely accepted in estimating disease burden of influenza [28] . two recent studies in canada and hong kong have demonstrated the estimates of influenza-associated hospitalization derived from poisson regression models reasonably matched the numbers of patients with laboratory confirmed influenza infections [17, 29] . however, it is extremely difficult to obtain the gold standard data on influenza associated deaths to assess the validity of the statistical models, because patients with influenza infection could have died from secondary bacterial infections and exacerbation of their preexisting conditions [30, 31] . therefore, their presenting problems may not be directly linked to influenza. moreover, given the potential lag time between severe complications and primary influenza infection, influenza virus might have become undetectable in these patients in the time of admission. hence recorded influenza deaths could still seriously underestimate the true numbers of deaths due to influenza even if laboratory tests for influenza are intensively conducted. in this study we performed a simulation study to assess the performance of poisson regression models. the small biases and rmse of most estimates may give further evidence to support the validity and reliability of poisson models. in this study, we adopted a semi-parametric model with smoothing functions to adjust for potential confounders, whereas most of the other studies just used linear terms of confounding variables in their poisson models [13, 15] . this semi-parametric model is preferred over the traditional parametric model in that it does not require any pre-determined relationships between the independent and dependent variables and thereby allows us to assess both linear and nonlinear relationships. although we chose natural spline smoothing functions in this study, there are also other smoothing functions available. however, previous studies have found that having a sufficient number of degrees of freedom is more important than the type of smoothing functions for adequate adjustment of confounding in the semi-parametric model [32] . therefore, in this simulation study, we mainly focused on the determination of degrees of freedom by an appropriate model selection criterion. among the four criteria under study, gcv consistently provided the smallest biases and rmse under the different assumptions of seasonal confounders, particularly when this confounder was assumed to have a high seasonal variation. our findings were robust to the various assumptions of influenza coefficients in simulation and also to the length of study period. increase of rmse was observed when the study period was shorten, which is not surprising as using less data points would increase the variation of estimates. all the four model selection criteria were developed under the different schemes. pacf measures the autocorrelation of the residuals, whereas both qaic and qbic evaluate the relative goodness of fit of a statistical model by quantifying the relative lost of information when a given model is used to describe the reality. therefore the latter two reflect the tradeoff between accuracy and simplicity, but qaic penalizes the number of model parameters to a lesser extent than qbic does. unlike other criteria, gcv assesses the model validity by cross-validation, i.e. randomly sampling data as training and test datasets to compare the accuracy and variation of prediction. our findings that gcv outperforms the other criteria in poisson models are also in line with previous studies on air pollution [25] . we chose the best-fit model based on the adequacy of confounding adjustment in terms of providing reliable estimates for influenza effect. although many seasonal factors could confound influenza effects on mortality, we only focused on the confounding of long term and seasonal trends of mortality in the present study, as this factor affected our estimates to a greater extent than any other confounders according to our previous experience. in this study the best-fit model was selected by minimizing each of the selection criteria, but the magnitude of their difference was not assessed. some studies suggested that the models selected under the different criteria might perform equally well if the difference between these criteria were small [33, 34] . however, it is not easy and somewhat arbitrary to define the cutoff points for small difference. burnham and anderson (2002) developed a set of cutoff points for aic to select the models with meaningfully different estimates [33] . similar thresholds for the bic value were also introduced by kass and raftery [34] . however, so far there are no commonly accepted cutoff points for those selection criteria used in our study. therefore, we did not take into consideration of the difference magnitude between these values, in order to achieve the simplicity and efficiency in the model selection procedure. there are several limitations in our study. first we assessed the parameter uncertainty in poisson regression models based on the data of subtropical city hong kong, where influenza seasonality is less clear than that in the temperate regions [16] . given the well defined winter peaks of influenza in temperate regions, it can be expected that the data from these regions probably required less complex adjustment for seasonal confounders. nevertheless, the framework developed in our study can still be applied to a wide range of data. second, we did not separately estimate the effects of different influenza virus subtypes, although previous studies have demonstrated their difference in excess mortality and mutation frequency [35, 36] . unfortunately, the virus subtype data during the study period are not available to us. future studies are needed to assess the performance of these model selection criteria in assessing the disease burden associated with each subtype. by applying poisson regression models to an empirical dataset of influenza hospitalization, we demonstrate that our findings can be generalized to other health outcomes. the best-fit models were validated by comparing the estimates of age-specific excess hospitalization rates with the observed rates in a pediatric cohort undergoing intensive laboratory tests for influenza infections. consistent with the findings of our mortality simulated study, gcv criterion outperformed qaic, qbic and pacf with smaller biases and rmse. given the enormous cost in money and manpower by such a prospective cohort study, statistical modeling is relatively easier to conduct and able to provide reliable estimates for influenza associated disease burden. in conclusion, our results suggested that the gcv criteria should be recommended for selection of the best-fit model in the future disease burden studies using poisson models. standardization of this modeling procedure shall increase the reliability of estimates and facilitate the comparison across countries or regions. abbreviations: qaic, quasi-akaike information criterion; qbic, quasi-bayesian information criterion; pacf, partial autocorrelation function; gcv, generalized cross validation; rmse, rootmean-square error; (tif) figure s4 weekly numbers of observed and fitted hospitalization by age group. the fitted hospitalization data were derived from the best-fit models selected by the generalized cross validation (gcv) criterion. (tif) the burden of influenza in east and south-east asia: a review of the english language literature the underrecognized burden of influenza in young children the annual impact of seasonal influenza in the us: measuring disease burden and costs estimating influenza-associated deaths in the united states department of health, the government of the hong kong special administrative region (2012) death statistics -by sex and age, cause of death in tabulation list of the international classification of diseases a practical guide for designing and conducting influenza disease burden studies influenza-attributable mortality in australians aged more than 50 years: a comparison of different modelling approaches circulating influenza virus, climatic factors, and acute myocardial infarction: a time series study in england and wales and hong kong excess mortality monitoring in england and wales during the influenza a(h1n1) 2009 pandemic air pollutants and health outcomes: assessment of confounding by influenza confounding by season in ecologic studies of seasonal exposures and outcomes: examples from estimates of mortality due to influenza estimates of us influenza-associated deaths made using four different methods influenza-associated hospitalization in a subtropical city mortality associated with influenza and respiratory syncytial virus in the united states influenza-associated mortality in hong kong seasonal effects of influenza on mortality in a subtropical city validation of statistical models for estimating hospitalization associated with influenza and other respiratory viruses regression models for count data in r winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease influenza and the rates of hospitalization for respiratory disease among infants and young children part 4. interaction between air pollution and respiratory viruses: time-series study of daily mortality and hospital admissions in hong kong generalized linear models generalized additive models overdispersed poisson regression models for studies of air pollution and human health model choice in time series studies of air pollution and mortality generalized additive models r: a language and environment for statistical computing. r foundation for statistical computing time series methods for obtaining excess mortality attributable to influenza epidemics the need for validation of statistical methods for estimating respiratory virus-attributable hospitalization influenza virus induces bacterial and nonbacterial otitis media invasive bacterial infections in relation to influenza outbreaks advances in statistical methods for the health sciences model selection and multimodel inference: a practical information-theoretic approach bayes factors the genomic and epidemiological dynamics of human influenza a virus influenza associated mortality in the subtropics and tropics: results from three asian cities we thank the census and statistics department of hong kong for providing mortality data. the hong kong hospital authority for providing hospitalization data. the hong kong observatory for providing meteorological data, the environmental protection department for providing air pollution concentration data. we also thank ms yk chau, dr tq thach and dr hk lai for their comments. key: cord-293472-d3iwlpsr authors: afilalo, marc; stern, errol; oughton, matthew title: evaluation and management of seasonal influenza in the emergency department date: 2012-04-06 journal: emerg med clin north am doi: 10.1016/j.emc.2011.10.011 sha: doc_id: 293472 cord_uid: d3iwlpsr seasonal influenza causes significant morbidity and mortality, primarily due to increased complication rates among the elderly population and patients with chronic diseases. timely diagnosis of influenza and early recognition of an influenza outbreak or epidemic are key components in preventing influenza-related complications, hospitalizations, and deaths. emergency departments are the most frequent points of entry for most influenza cases and are well positioned to identify and manage influenza community outbreaks and epidemics. emergency departments need specific infection control measures to curb the spread of influenza in the emergency department and hospital during the influenza season. 226,000 hospitalizations, an estimated 3.1 million hospitalization days, with costs of more than $5 billion annually. 3, 8 costs related to influenza epidemics surpass $12 billion and cause millions of lost work hours each year. 9 timely diagnosis of influenza and early recognition of an influenza outbreak or epidemic are key components in preventing influenza-related complications, hospitalizations, and deaths. as the primary gateway to the health care system, emergency departments (eds) are the most frequent points of entry for patients with influenza who seek medical attention. as a result, emergency physicians are well positioned to play a pivotal role in promptly identifying and adequately managing influenza community outbreaks and epidemics. this article provides an updated overview of influenza to enhance the clinical judgment of emergency physicians and facilitate accurate decision making and diagnosis of seasonal influenza, thereby minimizing influenza's potential morbidity and mortality. the epidemiology of influenza differs globally. influenza outbreaks can occur during a specific season, referred to as seasonal influenza, or influenza activity can be present throughout the year. in northern and southern hemisphere temperate zones, influenza is highly seasonal and attacks predominantly occur during the winter months. for northern hemisphere countries like the united states and canada, seasonal influenza usually starts in november, peaks from december to march, and abates in may, 10 whereas for southern hemisphere countries like australia, the flu starts in may, peaks in june to september, and ends in november. substantial fluctuations in influenza viral transmission patterns may occur with peaks occurring much earlier or later than anticipated. 11 in contrast, tropical regions lacking a distinct winter season exhibit different patterns of activity, in which influenza viruses may be isolated year round 12 with biannual influenza outbreaks. 13 typical outbreaks usually begin suddenly, spread in the community peaking during a period of 2 to 3 weeks, and continue for an average duration of 3 months. 14 in terms of clinical signs important for the emergency physician, the first indication of onset of a flu outbreak in a community is a surge in pediatric febrile respiratory illnesses, followed by increases in adult influenza-like illnesses (ili). 15 as the predominant front line of health care systems, eds including emergency physicians are well positioned to detect local outbreaks of the flu in their early stages and notify appropriate public health authorities to take proper measures to contain the outbreak. similarly, emergency physicians can play a pivotal role in containing emerging flu pandemics by keeping abreast of global influenza epidemics. influenza viruses belong to the orthomyxoviridae class of viruses and structurally consist of an inner core and outer membrane. the core contains a nucleoprotein antigen that determines the classification of the influenza virus into its 3 basic types: a, b, or c. 16 the outer membrane contains a coat of proteins including glycoproteins. influenza a viruses are categorized based on 2 immunologically important glycoproteins: hemagglutinin (h) with 16 different subtypes (h1-h16) and neuraminidase (n) with 9 different subtypes (n1-n9). 17 for instance, the influenza a (h1n1) virus responsible for the 2009 flu pandemic expresses hemagglutinin 1 (h1) and neuraminidase 1 (n1) subtypes, whereas influenza a (h2n2) virus, which caused the influenza pandemic of 1957 to 1958, expresses hemagglutinin 2 (h2) and neuraminidase 2 (n2) subtypes. influenza b and c viruses are not subcategorized. influenza a, b, and c viruses have similar structural and biologic characteristics but differ antigenically with varying prevalence and virulence. influenza a is the most prevalent of the 3, frequently causes seasonal outbreaks and epidemics in humans, and infection with this subtype leads to more severe morbidity than influenza b and c. in addition, influenza a is the only subtype that causes pandemics. influenza a h1n1 and h3n2 are currently the predominant virus subtypes causing influenza infection in humans. since 1977, these 2 flu viruses have been circulating, causing seasonal influenza worldwide, whereas influenza a h2n2 subtype has not circulated in humans since 1968. 10 influenza b viruses circulate less widely than influenza a, causing fewer seasonal outbreaks and epidemics, whereas influenza c viruses cause only sporadic cases or minor outbreaks but not epidemics. in both cases, humans develop antibodies to these influenza viruses during childhood that provide some protection later against severe disease. 18 however, in children less than 6 years of age who have not yet acquired antibodies to influenza c, this virus can cause serious respiratory infections. 19 antigenic drift and antigenic shift influenza a viruses, more than influenza b and c viruses, have a natural tendency to periodically undergo hemagglutinin and neuraminidase antigenic changes. small point mutations in the rna gene segments that code for these 2 glycoproteins lead to minor hemagglutinin and neuraminidase antigenic changes called antigenic drifts that result in localized outbreaks. large mutations with viral gene reassortment that result in major hemagglutinin and neuraminidase antigenic changes referred to as antigenic shifts are associated with more widespread epidemics and pandemics. influenza a, because of their greater propensity for antigenic variation, is the only influenza virus type able to undergo antigenic shifts, whereas all 3 virus types (influenza a, b, and c) have the ability to undergo antigenic drifts. influenza viral infection starts with transfer of virus-laden respiratory secretions from an infected person to an immunologically susceptible host. the virus initially attaches to the epithelial cells of the upper respiratory tract and, if not neutralized by the host's immune system, the virus can continue to invade more and more cells as the virus descends the respiratory tract. after adsorption and binding of viral hemagglutinin to host cell sialic acid-conjugated glycoproteins, the virus enters the host cell. this adsorption and binding is deemed necessary for virus cell entry, 20 and is epidemiologically significant because the configuration of sialic acid-conjugated glycoproteins differs from one species to another, which may exert a crucial role in limiting transfer of influenza viruses across species. 21 once the virus has entered the host cell, it immediately disrupts normal cell function and starts replicating and releasing its viral progeny. neuraminidase is essential for viral release and propagation. 22 viral replication leads to host cell degradation and death via several mechanisms that shut off protein synthesis and release potent cytokines. 23, 24 cytokines, such as type i interferons, interleukins, tumor necrosis factor, as well as other inflammatory mediators, are thought to cause coughing and other systemic symptoms of flu. virus replication starts within 4 to 6 hours of host cell infection, and continues until about 24 hours before symptom onset. 25 the duration between incubation period, symptom onset, and virus shedding can range from 18 to 72 hours depending, in part, on inoculum dose. 26 the quantities of shed virus measured in specimens exhibit a distinct pattern and temporally correlate with symptom onset and severity of illness. virus shedding is observed starting within 24 hours before the onset of symptoms, peaks in 1 to 2 days after the onset of symptoms develop, remains high for another 1 to 2 days correlating with when the illness is most severe, and then rapidly declines, coming to an end approximately 7 to 10 days after infection. however, in certain circumstances, virus shedding can continue for weeks. two key factors that influence the duration of viral shedding are age and severity of illness. [27] [28] [29] young children, because of their relative lack of immunity, can shed virus for 10 days or more. 29, 30 patients with chronic diseases and more severe, complicated influenza shed the virus for an average of 2 days longer than uncomplicated influenza. 27, 31, 32 in elderly 31 and immunocompromised patients, 33-37 viral shedding and potential infectivity can persist for weeks, even months. multiple pathologic changes and pulmonary function abnormalities are observed during active uncomplicated acute influenza infection. bronchoscopy often shows inflammation and edema of the bronchial mucosa, most notably in the lower respiratory tract, that lead to decreased forced flow rates and increased pulmonary resistance, which may persist for weeks after clinical recovery. in patients with asthma and chronic obstructive pulmonary disease, influenza can cause acute decreases in forced expiratory vital capacity (fvc) and forced expiratory volume in 1 second (fev1). 38, 39 virus infection can advance into the lung parenchyma either via inhalation or contiguous spread from the upper respiratory tract causing primary viral pneumonia. tracheitis, bronchitis, and bronchiolitis are seen, characterized by submucosal hyperemia, edema, focal hemorrhage with bloody fluid, and loss of normal ciliated epithelium. 40 disruption of the normal epithelial barrier to infection, and abnormalities in ciliary clearance mechanisms, along with increased adherence of bacteria to virusinfected epithelial cells, predispose to bacterial superinfection. the most common pathogens responsible for bacterial infection are staphylococcus aureus, streptococcus pneumoniae, and haemophilus influenzae. the signs and symptoms of seasonal influenza are variable in severity, and dependent on the age of the patients. in adults, influenza is usually characterized by respiratory symptoms with other constitutional symptoms such as fever, myalgia, malaise, and headache. an abrupt onset is common, such that patients are often able to report the time of onset. respiratory symptoms and cough may initially be mild, but can progress causing dyspnea and pleuritic chest pain. degree of fever is variable; fever in the elderly is usually not as severe as in young patients. during the flu season, patients with influenza-like symptoms and proven influenza infection were more likely to have cough (93% vs 80%), fever (68% vs 40%), cough and fever together (64% vs 33%), and/or nasal congestion (91% vs 81%) compared to those without influenza. 41 for decreasing the likelihood of influenza, the absence of fever (likelihood ratio [lr], 0.40; 95% confidence interval [ci], 0.25-0.66), cough (lr, 0.42; 95% ci, 0.31-0.57), or nasal congestion (lr, 0.49; 95% ci, 0.42-0.59) were the only findings that had summary lrs less than 0.5. 42 patients may also present with isolated gastrointestinal or central nervous system (cns) involvement. children with influenza often do not present with the classic symptoms. they often cannot describe their symptoms, and tend to have more gastrointestinal symptoms. symptoms can mimic bacterial sepsis with high fevers, and children with influenza may present with febrile seizures. 43 in uncomplicated influenza, there are a few physical findings. there may be evidence of hyperemia of the pharynx, even with severe sore throat complaints. mild cervical lymphadenopathy and otitis media may be present, especially in younger patients. a dry cough is usually noted on chest examination with clear lungs or rhonchi, unless complicated by pneumonia. if there are no complications, fever and body aches can last 3 to 5 days, and the cough and lack of energy may last for 2 or more weeks. 44 pneumonia is the major complication of influenza and occurs especially in high-risk patients: children aged less than 5 years (especially those aged <2 years) adults aged 65 years or more persons with chronic diseases persons with immunosuppression women who are pregnant or postpartum (within 2 weeks after delivery) persons aged 18 years or younger who are receiving long-term aspirin therapy first nations/alaska natives persons who are morbidly obese (ie, body mass index [bmi] greater than or equal to 40) residents of nursing homes and other chronic-care facilities. 45 pneumonia can either be of viral or secondary bacterial cause. primary viral pneumonias are uncommon but tend to have increased symptom severity. however, during influenza outbreaks, influenza virus types a and b are responsible for more than half of all community-acquired viral pneumonia cases. secondary bacterial pneumonia is a significant complication of influenza, accounting for 25% of all influenza deaths. 46 children hospitalized with influenza-associated pneumonia have a higher risk for intensive care admission, respiratory failure, and death compared to those hospitalized with influenza without pneumonia. classically, influenza patients complicated with pneumonia have an exacerbation of fever and respiratory symptoms after an initial improvement. the most common bacterium is s pneumonia, accounting for approximately 50% of cases. staphylococcus aureus and haemophilus influenza are also important common organisms. during the 2006 to 2007 influenza season, 51 cases of community-acquired s aureus pneumonia were reported to the cdc. almost 50% of these cases had antecedent or concomitant viral illness, and just under 80% of the s aureus cultures were mrsa. the median age was 16 years, 44% had no known pertinent medical history, and approximately half of patients, for whom final disposition was known, died a median of 4 days after symptom onset. despite the selection bias in the cases reported, community-associated s aureus (ca-mrsa) influenza in the emergency department pneumonia accounts for severe pneumonia with high mortality in young otherwise healthy patients with influenza. therefore, empiric therapy for severe communityacquired pneumonia should include treatment against s aureus, including mrsa. 47 neurologic complications include encephalitis, transverse myelitis, and guillain-barré syndrome. reye syndrome has been reported in patients using aspirin after influenza infections. myositis is rare, but has been reported more commonly in children than adults. it presents in early convalescence with acute onset of pain and tenderness in the lower leg muscles severe enough to limit walking. serum creatine kinase (ck) levels transiently increase, with complete recovery generally occurring in 3 to 4 days; renal failure is rare. 48 cardiovascular involvement occurs by directly affecting the myocardium or exacerbating existing cardiovascular conditions. the frequency of myocardial involvement in influenza infection is variable, with rates of up to 10% having been reported in the literature, although this depends on the methods used to detect myocardial involvement. although many patients are asymptomatic, a significant proportion of these have electrocardiogram changes. fulminant myocarditis resulting in cardiogenic shock and death may occur. when a patient's condition deteriorates with hemodynamic compromise, cardiac involvement should be considered. the mainstay of treatment of influenza myocarditis is supportive. cardiovascular deaths also increase during influenza epidemics by increased deaths from coronary artery disease. these deaths have been shown to be reduced by influenza vaccination, which should be offered to all patients with cardiovascular disease. 49 other rare complications encountered include toxic shock syndrome in conjunction with secondary s aureus infection and parotitis. 50 influenza can be difficult to diagnose based on clinical symptoms alone because the initial symptoms of influenza can be similar to those caused by other infectious agents including mycoplasma pneumoniae, adenovirus, respiratory syncytial virus, rhinovirus, parainfluenza viruses, and legionella. it is important for the ed to develop clinical pathways to identify ili so that contagious patients can be segregated and treated effectively. the cdc defines ili as patients with temperature greater than 37.8 c (100 f) plus either cough or sore throat in the absence of a known cause other than influenza. as described, patients with influenza may have atypical presentations. fever is not always present, especially in premature infants, young infants, elderly patients, or immunosuppressed patients, and patients may present with only myalgias, headache, fatigue, or other complications. 51 the ed needs to consider the variability of clinical presentations and the prevalence of influenza in the community regarding investigation and treatment, and infection control isolation protocol. patients with suspected influenza should have standard laboratory investigations such as a complete blood count and electrolytes; the results are usually nonspecific, but leukopenia is typical and thrombocytopenia may be present. patients with physical signs that suggest meningitis should undergo a lumbar puncture. in patients with hypoxemia, the elderly, or high-risk patients with pulmonary symptoms, a chest radiograph should be performed to exclude pneumonia. dyspnea and chest pain are typically used as indicators for obtaining a chest radiograph. shortness of breath may be a useful indicator of pneumonia-complicating influenza. 52 radiological findings include bilateral interstitial infiltrates, and focal infiltrates may indicate superimposed bacterial pneumonia. diagnostic testing does not have to be performed on every patient who presents to the ed with ili, especially when there is a circulating influenza outbreak or epidemic. confirmation of influenza virus infection is not required for clinical decisions to prescribe antiviral medication. the decision to administer influenza treatment or chemoprophylaxis should be based on clinical illness and epidemiologic factors, and the start of therapy should not be delayed pending results, especially during an influenza outbreak. 51 influenza diagnostic testing is not clinically indicated when test results will not alter a patient's clinical care or influence clinical practice for other patients (fig. 1) . a positive influenza test may be used to confirm influenza virus in the community, which may affect clinical practice related to home care guidance, hospital infection control practices, future testing practices, and so forth. neither the rapid influenza test nor clinical prediction rules were superior to clinical judgment alone in the diagnosis of influenza. in one study of 258 patients with 21% confirmed influenza, the overall clinical judgment had a sensitivity of 29% (95% ci, 18%-43%) and specificity 92% (95% ci, 87%-95%), which improved to a sensitivity of 67% (95% ci, 39%-86%) and specificity of 96% (95% ci, 81%-99%) when patients presented within 48 hours. rapid influenza tests only had a sensitivity of 33% (95% ci, 22%-47%) and specificity of 98% (95% ci, 96%-99%), and a clinical prediction rule showed a sensitivity of 40% (95% ci, 27%-54%) and specificity of 92% (95% ci, 87%-95%). 53 thus, in times of high disease prevalence such as during influenza outbreaks or epidemics, most patients exhibiting ili symptoms can be diagnosed clinically as having influenza, without performing any diagnostic tests. clinicians should consult the cdc's global flu activity update (http://www.cdc.gov/flu/ international/activity.htm) for the latest updates on the international flu situation, and fluview (http://www.cdc.gov/flu/weekly/) for a summary of flu activity in the united states. diagnostic testing is ideally indicated in 2 circumstances: (1) for sporadic cases of ili, during periods of low disease prevalence, and (2) for severely ill patients. influenza should be confirmed in sporadic cases of ili to rule out another viral diagnosis, for example severe acute respiratory syndrome (sars) or coronavirus. diagnostic testing is recommended in severely ill patients because there is a greater urgency to make the correct diagnosis to provide appropriate medical management. in these 2 cases, rapid influenza diagnostic tests (ridts) and reverse transcriptase polymerase chain reaction (rt-pcr) are appropriate. the 2009 evidence-based clinical practice guidelines for the diagnosis, management, and chemoprophylaxis of seasonal influenza developed by the infectious diseases society of america recommend that the following patient populations undergo diagnostic testing for influenza if testing results will influence medical management (box 1). 54 influenza virus can be isolated from different types of specimens, including nasal, throat, or nasopharyngeal swabs, aspirates or washes, and sputum samples. nasopharyngeal specimens (swabs and aspirates) are more sensitive for detecting the virus than throat swabs or sputum specimens 54, 55 ; in one comparison of 3 rapid assays and immunofluorescence for influenza detection, sensitivity for all of these methods increased by approximately 40% when nasopharyngeal swabs instead of throat swabs were used. 56 acceptable specimens also vary depending on the specific diagnostic test ( table 1 ). the optimal time frame for collecting diagnostic specimens influenza in the emergency department confirmation of influenza virus infection by diagnostic testing is not required for clinical decisions to prescribe antiviral medications. decisions to administer antiviral medications for influenza treatment or chemoprophylaxis, if indicated, should be based upon clinical illness and epidemiologic factors, and start of therapy should not be delayed pending testing results (http://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm). respiratory specimens should be collected from an ill patient as early as possible after onset of symptoms (ideally <48-72 hours after onset) to help maximize influenza testing sensitivity. 2 influenza like-illness (history of feverishness or documented fever with either cough or sore throat), fever with other respiratory symptoms, etc. note that some persons may have atypical presentations (eg, elderly, very young infants, immunosuppressed, and patients with certain chronic medical conditions). fever is not always present (eg, premature infants, young infants, elderly, immunosuppressed). other symptoms associated with influenza include myalgias, headache, and fatigue. complications include exacerbation of underlying chronic disease, (eg, congestive cardiac failure, asthma), pneumonia, bacterial co-infection, bronchiolitis, croup, encephalopathy, seizures, myositis, and others. 3 eg, decisions on use of antibiotics or antiviral medications, on conducting further diagnostic tests, on recommendations for home care, or on recommendations for ill persons living with persons with high-risk conditions. consult infectious disease society of america, american thoracic society, association of american physicians, and advisory committee on immunization practices for antibiotic guidance. 4 persons ‡65 years or <2 years; pregnant women; persons with chronic lung disease (including asthma), heart disease, renal, metabolic, hematologic and neurologic disease; immunosuppression; and morbid obesity. 5 largely depends on the amount of viral shedding at the time of testing. in immunocompetent children and adults, in whom viral shedding is brief, specimen samples yield the best results during the first 1 to 5 days of illness. 54, 57 low viral titers in the first 12 to 24 hours following onset of clinical illness have been suggested as a cause of falsenegative results in patients tested early 58 ; similarly, specimens obtained after 5 days of illness have an increased likelihood of false-negative results because of decreased virus shedding. immunocompetent infants and young children spread virus for longer (1 week), which ideally permits the collection of specimens after 5 days of illness. 54 irrespective of age, specimen collection in immunocompromised persons can also exceed 5 days of illness because virus shedding in this patient population can last for weeks, even months. 54 in the ed, collection of serum specimens is not recommended for diagnostic purposes because results are not readily available and therefore cannot guide clinical decision making and management. 54 during influenza season (testing should be done in the following persons if the result will influence clinical management) outpatient immunocompetent persons of any age at high risk of developing influenza complications (eg, hospitalization or death) presenting with acute febrile respiratory symptoms 5 days or less after illness onset (when virus is usually being shed) outpatient immunocompromised persons of any age presenting with febrile respiratory symptoms, irrespective of time since illness onset (because immunocompromised persons can shed influenza viruses for weeks to months) hospitalized persons of any age (immunocompetent or immunocompromised) with fever and respiratory symptoms, including those with a diagnosis of community-acquired pneumonia, irrespective of time since illness onset elderly persons and infants presenting with suspected sepsis or fever of unknown origin, irrespective of time since illness onset children with fever and respiratory symptoms presenting for medical evaluation, irrespective of time since illness onset persons of any age who develop fever and respiratory symptoms after hospital admission, irrespective of time since illness onset immunocompetent persons with acute febrile respiratory symptoms who are not at high risk of developing complications secondary to influenza infection may be tested for purposes of obtaining local surveillance data throughout the year (testing should be done for the following persons) health care personnel, residents, or visitors in an institution experiencing an influenza outbreak who present with febrile respiratory symptoms within 5 days after illness onset persons who are epidemiologically linked to an influenza outbreak (eg, household and close contacts of persons with suspected influenza, returned travelers from countries where influenza viruses may be circulating, participants in international mass gatherings, and cruise ship passengers) who present within 5 days after illness onset table 3 ). b serologic testing is not recommended for routine patient diagnosis. c a fourfold or greater increase in antibody titer from the acute (collected within the first week of illness) to the convalescent phase (collected 2-4 weeks after the acute sample) indicates recent infection. data from refs. 55, 59, 64 several different laboratory testing methods for detecting influenza virus are available in the united states ( table 1) and canada ( table 2) . these methods include immunofluorescence microscopy (direct or indirect antibody staining), viral culture (conventional and rapid), rt-pcr, ridts, and serologic testing. among these testing methods, ridts with rapid processing yielding timely results that can influence clinical decision making and patient management are most pertinent for the needs of the ed. based on which type(s) of influenza virus (a and/or b) can be detected, diagnostic tests can be categorized into 3 types: (1) those that detect only influenza a; (2) those that detect either influenza a or b, but cannot discriminate between the two; and (3) tests that both detect and distinguish between influenza a or b viruses. only rt-pcr and viral culture can identify influenza strains. ridts are rapid antigen point-of-care tests capable of identifying influenza a and b viral types in respiratory specimens in approximately 10 to 15 minutes. 59 ridts are immunoassays that come in user-friendly, diagnostic kits with varying complexity that either: (1) only detect influenza a virus, (2) detect but cannot distinguish between influenza a and b viruses, or (3) both detect and distinguish between influenza a and b viruses. commercial ridts currently available in the united states and canada are listed in table 3 , and general ridt characteristics, including advantages and disadvantages, are described in box 2. ridts are valuable in the ed because they produce results in a timely and clinically relevant manner that facilitate on-site point-of-care diagnosis of influenza that, according to limited research, has led to a decrease in demand for further diagnostic tests (eg, chest radiography, blood cultures) and the use of antibiotics, thus resulting in decreased patient costs. 61 recommendations for the use of ridts were developed and promulgated by the world health organization (who) (box 3). the cdc has recently issued guidelines for clinicians on the use of ridts for the 2010 to 2011 influenza season. 59 a major drawback of ridts is their limited reliability in accurately detecting influenza virus, which depends on a wide variety of factors, including ridt sensitivity, specificity, positive and negative predictive values, type of specimen collected, and time of collection with respect to onset of symptoms. although ridts exhibit high specificities ranging between 90% and 95%, ridts have substantially lower sensitivities, ranging from about 70% to 90% in children, decreasing even further to approximately 40% to 60% or lower in adults, compared with viral culture and rt-pcr. 54, 62, 63 thus, if ridts are the only diagnostic assay used in a center, positive results can be trusted but a negative result cannot reliably exclude disease. table 4 displays specifically selected commercially available ridts and corresponding test sensitivities, specificities, and positive and negative predictive values, which also vary according to the study. time of specimen collection also influences the accuracy of ridt results: the closer within the period of viral shedding and illness the specimen sample is obtained, the more accurate the result. 60, 64 in an effort to minimize false interpretation of ridts, the cdc has published the following guidance statements that emergency physicians and other health care professionals must keep in mind when performing and interpreting ridts 59,65 : 1. the reliability of a positive ridt result increases in patients with clinical signs and symptoms consistent with influenza. 2. collection of specimens within 48 to 72 hours of illness onset increases the likelihood of producing a positive ridt result. 4. use ridts with high sensitivity and specificity (see table 4 ): a. ridt sensitivities are generally low to moderate, ranging between 10% and 70% (most are approximately 50%-70%) compared with the gold standard viral culture or rt-pcr. an ridt with low sensitivity yields false-negative results. b. ridt specificities are generally high, approximately 90% to 95%, compared with the gold standard viral culture or rt-pcr. an ridt with high specificity yields few false-positives. rt-pcr is replacing viral culture as a reference standard because it is currently the most sensitive, specific, and versatile diagnostic test available for diagnosing influenza. 66 based on nucleic acid amplification, rt-pcr can detect the influenza virus but also differentiate between virus types, subtypes, and even determine viral strain, all in approximately 4 to 6 hours. as a result, rt-pcr has become the recommended test of choice for accurately diagnosing influenza in a timely fashion. 54 during the recent pandemic, the cdc released a method through the who for rt-pcr detection of influenza a that allowed clinical and reference laboratories to standardize methodology and thus produce data that could be compared between laboratories. 67 however, the major advantage of rt-pcr lies in its ability to more readily detect influenza viruses in people with chronic lung diseases and immunosuppressed persons, who may exhibit lower levels of the virus. 68 in these susceptible patients, rt-pcr can efficiently and accurately confirm the diagnosis of influenza to support therapeutic and infection control decisions. immunofluorescence yields timely results within 2 to 4 hours, and this can be used as a screening test. however, immunofluorescence has several disadvantages, including lower sensitivity (47%-93%) 69, 70 and specificity compared with viral culture, and is labor intensive, requiring specially trained laboratory personnel who may not be available 24/7, even in large hospitals. in addition, test performance depends on an adequate specimen sample that must include respiratory epithelium cells. 54 influenza virus can be cultured either by isolation of virus in cell culture (conventional tube culture), which provides results in 3 to 10 days, or by shell vial culture, which offers the advantage of a faster turnaround time of 48 to 72 hours. 54 because of the lengthy turnaround times of either method, viral culture is not a useful diagnostic test in the ed for aiding initial clinical decision making and management. however, viral culture allows for subsequent analyses, including sensitivity testing and subtyping performed by reference laboratories. during the influenza season, viral culture is indicated primarily for confirming negative ridt and immunofluorescence results, as well as for influenza virus surveillance because it provides key information regarding influenza virus strains and subtypes. 54 during the off-season, viral culture is indicated in patients who present to the ed within 5 days of symptom onset with suspected ili, especially if the person is epidemiologically linked to an influenza outbreak. 54 serologic testing is not useful or recommended in the ed because results are not readily available and therefore cannot facilitate clinical judgment, diagnosis, or management of influenza. 57 serologic tests that include hemagglutinin inhibition, neutralization, complement-fixation, and enzyme-linked immunosorbent assay (elisa) are mainly used to establish a diagnosis retrospectively and for research purposes. 57 because most individuals have previously been infected with influenza viruses, to reliably determine antibody titers, a single serum sample collected in the ed is inadequate, but paired specimen samples (acute and convalescent sera) are needed. whenever interpreting any influenza diagnostic test, the emergency physician or other health care professional must keep in mind the limitations of these tests, especially for ridts. in addition, the clinician should be aware of the disease prevalence in the community at any given time, because the level of influenza activity is known to affect the accuracy and reliability of test results. with respect to patient management, a positive influenza test result does not necessarily rule out any overlying coinfection by additional pathogens, and in the case of initial negative influenza test results from less sensitive diagnostic methods like ridts, the clinician should contemplate additional diagnostic testing (such as rt-pcr or culture) and decide whether antiviral treatment should be initiated empirically. currently, 4 antiviral medications from 2 drug classes have been approved and are available for the treatment and prevention of influenza in the united states, canada, and most other countries. these medications include amantadine and rimantadine, which belong to the drug class adamantanes, and oseltamivir (tamiflu; roche) and zanamivir (relenza; glaxosmithkline), which belong to the class neuraminidase (na) inhibitors. adamantanes are active only against influenza a virus, whereas na inhibitors are active against both influenza a and b viruses. other antiviral pharmacologic properties are compared in table 5 . in the last several years, adamantines have become less clinically useful because of their widespread resistance to influenza a (h3n2) and 2009 (h1n1) virus strains. 71 as a result, amantadine and rimantadine are currently not recommended for the treatment or chemoprophylaxis of influenza a virus. 45 the goals of influenza pharmacotherapy are to decrease symptoms, prevent associated complications, and reduce functional disability, hospitalizations, and mortality. treatment decisions on administering antiviral therapy should take into account factors such as time since symptom onset, underlying conditions, and severity of disease. abbreviations: crcl, creatinine clearance; fda, us food and drug administration; gi, gastrointestinal. a fda has authorized treatment of s-oiv with oseltamivir in children greater than or equal to 3 months of age. b fda has authorized prophylaxis for s-oiv with oseltamivir in children greater than or equal to 1 year of age. according to the cdc 45 and other published guidelines, 54 antiviral treatment is recommended for patients infected by the influenza virus who meet the following criteria: 1. patients with laboratory-confirmed or highly suspected influenza virus infection considered high risk for developing influenza complications (box 4). treatment is recommended irrespective of illness severity or vaccination status; 2. patients with laboratory-confirmed or highly suspected influenza virus infection requiring hospitalization, irrespective of underlying illness or vaccination status; 3. patients with laboratory-confirmed or highly suspected influenza virus infection who have severe, complicated, or progressive illness; antiviral treatment should be considered for adults and children with influenza virus infection who meet the following criteria: 1. outpatients at high risk of complications (see box 4) with illness that is not improving and who have a positive influenza test result from a specimen obtained more than 48 hours after onset of symptoms; 2. outpatients with laboratory-confirmed or highly suspected influenza virus infection who are not at increased risk of complications, whose onset of symptoms is less than 48 hours before ed presentation, and who would like to shorten the duration of illness and further reduce their risk of complications; 3. outpatients with laboratory-confirmed or highly suspected influenza virus infection who are in close contact with persons at high risk of complications secondary to influenza infection; 4. patients whose onset of symptoms occurred more than 48 hours before ed presentation with persisting moderate to severe illness may also benefit from treatment. because viral titers rapidly decrease by day 3 to 4 of illness in untreated, previously healthy persons, efficacy of antiviral therapy is directly related to time of treatment initiation. 72 studies have found that early treatment, especially initiated within the first 6 hours of symptom onset, provides the greatest benefit in reducing symptoms. 72 antiviral treatment initiated within 48 hours of onset of influenza illness can lead to shorter duration of symptoms and decreased illness severity. studies administering na inhibitor antiviral medications in previously healthy patients with uncomplicated influenza resulted in a shorter duration of illness by 1 to 2 days. 28, 29, [73] [74] [75] [76] [77] [78] [79] in addition, research has shown that early initiation of treatment with antivirals can also decrease the rate of serious influenza-related complications (eg, pneumonia, respiratory failure, and death) in high risk patients. 80 in contrast, slight or no benefit has been observed in healthy people when antiviral treatment is started more than 48 hours after the onset of uncomplicated influenza. 45 as a result, influenza antiviral treatment, when clinically indicated, should be initiated in a timely fashion, preferably within 48 hours of symptom onset, and not after laboratory confirmation of influenza. in certain patient populations, antiviral treatment may still be beneficial even if given more than 48 hours after symptom onset. these patients include pregnant women, patients with severe or progressive illness requiring hospitalization, and patients at high risk for suffering influenza complications. a study by siston and colleagues 81 found that, in pregnant women, treatment with antiviral medications decreased respiratory complications and death even when initiated 3 to 4 days after symptom onset compared with 5 days or more. based on observational studies, oseltamivir decreases severe clinical outcomes in hospitalized patients with influenza. in a multivariate influenza in the emergency department analysis, treatment with oseltamivir led to a significantly decreased risk of death within 15 days of hospitalization (odds ratio [or], 0.2; 95% ci, 0.1-0.8). 82 benefits were detected even in patients whose treatment was initiated more than 48 hours after the onset of symptoms. a study by lee and colleagues 83 found that among 99 hospitalized patients (median age, 70 years) with laboratory-confirmed influenza who received oseltamivir, benefits were observed even when oseltamivir was started up to 96 hours after illness onset. influenza virus vulnerability to antiviral drugs is continuously evolving. as a result, emergency physicians need to be familiar with the most recently updated information available on antiviral resistance and recommendations on antiviral use. as of january 2011, the cdc recommends the following antiviral drugs for treatment and chemoprophylaxis of seasonal influenza ( table 6 ). 45 the most effective way to prevent and control seasonal influenza is through immunization of both health care workers and patients. 84 procedures should be institutionalized, which ensures that patients and visitors with respiratory infection symptoms follow triage procedures in the ed that effectively isolate them as rapidly as possible. in hospital entrances and the ed triage, there should be clear signage with instructions regarding respiratory hygiene and cough etiquette. face masks should be available to cover the nose and mouth when coughing or sneezing, and waste receptacles are needed to dispose of contaminated tissues. there should also be instructions on how and when to perform hand hygiene. passive signage asks patients to self-identify; the triage health care team should actively ask patients about possible symptoms while maintaining a distance of at least 1 m from them. waiting times should be minimized and closely monitored, with staffing adjustments made accordingly. during periods of increased influenza activity, facilities should consider setting up pretriage stations that facilitate rapid screening of patients for symptoms of influenza to separate those patients from others. registration can identify the charts of patients with potential influenza to expedite care. waiting rooms should be segregated into 2 areas; patients with and without respiratory symptoms. when possible, physical barriers should separate the patients. in the ed, patients should be evaluated in single treatment areas. 85 the health care worker should use personal protection equipment (ppe), including a surgical mask, and a face-shield or mask with visor attachment, if there is a high chance of splash or spray of respiratory secretions. gloves and a long-sleeved gown should be worn when entering the room of a patient with suspected or confirmed influenza. 85 the health care worker should remove all ppe just before leaving the patient's room and discard it in the hands-free waste and linen receptacle within the room. hand hygiene should be performed after removing gloves and gown, before removing mask and protection, and again after leaving the room. 86 if a patient with droplet precautions in the ed needs to be moved for investigation, the patient should wear a face mask and continue to follow cough etiquette and hand hygiene. there should be appropriate communication to other personnel about patients with suspected or confirmed influenza before transferring them to other departments (eg, radiology) and admitting units in the facility. some procedures performed on patients with suspected or confirmed influenza infection may be more likely to generate higher concentrations of infectious respiratory aerosols. these procedures include intubation and related procedures (eg, manual ventilation, open endotracheal suctioning, cardiopulmonary resuscitation, sputum induction, nebulized therapy, and noninvasive positive pressure ventilation such as continuous positive airway pressure [cpap] or biphasic positive airway pressure [bipap]). although there are limited data available on influenza transmission related to such aerosols, many authorities recommend the additional precautions to be used when such procedures are performed. 87 the number of health care workers present should be limited to only those essential for patient care and support. those present should have received influenza vaccine. there should be a low threshold for intubation rather than using prolonged aerosol-generating procedures such as bipap and cpap. the health care worker should wear respiratory protection including a fitted n95 respirator during aerosol-generating procedures. n95 respirators should be used in the context of a comprehensive respiratory protection program that includes fit testing and training as required under the respiratory protection standard (29 cfr 1910 .134) of the occupational safety and health administration (ohsa). 88 the procedures should be conducted in an airborne infection isolation room (aiir), when feasible. aiirs reduce the concentration of infectious aerosols and prevent spread into adjacent areas using controlled air exchanges and directional airflow. aiir are negative-pressure rooms relative to the surrounding areas, with a minimum of 6 air exchanges per hour. the air should be exhausted directly to the outside or filtered through a high-efficiency particulate air (hepa) filter before recirculation. there should be environmental surface cleaning following the procedure. visitors should not be present during aerosol-generating procedures. visits to patients with suspected or confirmed influenza should be controlled such that visitors should be instructed to limit their movement within the facility. facilities should provide instruction before visitors enter a patient's room on hand hygiene, limiting surfaces touch, and use of ppe. 89 visitors should be advised to contact their health care provider for information on influenza vaccination, if this has not been received; if they are high-risk patients (as described earlier), 45 chemoprophylaxis may be offered if they are in close contact with the patient. health care workers in the ed presumably receive education and training programs on preventing transmission of all infectious agents, including influenza. these programs should be updated periodically and competency should be documented. health care workers who develop fever and respiratory symptoms should be instructed not to report to work, or, if working, they should put on a face mask and promptly notify their supervisor and/or infection control personnel. health care workers should be excluded from work for at least 24 hours after they no longer have fever. those with ongoing respiratory symptoms should be evaluated to determine appropriateness of contact with patients. health care workers caring for immunocompromised patients should be considered for temporary assignment or exclusion from work for 7 days from symptom onset or until the resolution of symptoms, whichever is longer. 85 administration of antiviral treatment and chemoprophylaxis of health care workers should be considered when appropriate. early treatment with antiviral agents and vaccination are especially important for health care workers at higher risk for influenza complications, including pregnant women and women up to 2 weeks after giving birth; persons 65 years and older; and persons with chronic diseases such as asthma, heart disease, diabetes, diseases that suppress the immune system, and morbid obesity. 45 work reassignment should be considered for those at higher risk to avoid potentially high-risk exposure such as performing or assisting aerosol-generating procedures in patients with suspected or confirmed influenza. the ed should have adequate isolation facilities and clear protocols of rapid admission to the wards to prevent boarding. lastly, discharge instructions should be developed and given to every patient with influenza discharged home from the ed. the emergency department team feels that you have the seasonal flu or influenza and your symptoms are mild enough to send you home for observation and recovery of your illness. influenza is contagious, and you should use proper precautions so that you do not pass your infection on to others. when you leave the emergency department, please wear a mask and keep it on until you arrive home if you cannot keep a distance of 2 m from others. you may also wear it at home, as necessary. do not use public transportation (bus, subway) to go home. go straight home; do not make any stops on the way (eg, drug store, grocery store). if you were given a prescription, make arrangements for a family member or friend to pick it up. you should isolate yourself in your home until 7 days after the onset of illness or at least 24 hours after symptoms have resolved, whichever is longer. do not go to work, school, or public places. do not share personal items, such as towels, drinking cups, cutlery, thermometers, and toothbrushes. always use hygiene and prevention measures to avoid contamination: wash your hands frequently. cough or sneeze into the crook of your elbow rather than into your hands. use tissues and dispose in waste basket. keep your surroundings clean. while at home, it is important that you monitor your own health to be sure that your illness does not worsen. you should consult your doctor or return to the emergency department if you develop one of these symptoms: shortness of breath, difficulty breathing, chest pain, recurrent vomiting, or high fever 38.5 c (101.3 f). pay attention to the onset of any illness stay home if mild flulike symptoms occur go to a doctor with a fever more than 38 c (100.4 f) and belong to a group at risk of developing influenza complications (children less than 2 years of age, pregnant women, person 65 years old and older, and persons with chronic diseases such as asthma, heart disease, diabetes, and diseases that suppress the immune system) go to a doctor with a fever more than 38 c (100.4 f) and one of these symptoms: chest pain child who is too quiet and less active than normal, or refuses to play, or is agitated the symptoms of an influenza pandemic can be similar to those of seasonal flu (ie, fever, headache, myalgia, coryza, gastrointestinal symptoms, sore throat, or cough). in the last century, 4 influenza pandemics were caused by novel influenza viruses. the most significant was in 1918, when the so-called spanish flu killed 40 to 50 million people worldwide. 90 in 2009, there was the emergence of a novel h1n1 virus, a genetic combination of human and swine influenza viruses. because many persons have little or no immunity to a new pandemic virus, the disease can spread quickly. with the h1n1 pandemic, the rate of infection was highest among young individuals; infections were less common in persons older than 65 years, perhaps secondary to preexisting immunity against antigenically similar viruses. [91] [92] [93] there are several differences between seasonal and pandemic influenza 94 : the who is responsible for monitoring the spread of influenza worldwide, declaring a pandemic, and coordinating the global response. however, the local health care systems need to develop surveillance to detect and monitor for a pandemic strain. it is important for the emergency physician to be cognizant that patients presenting with severe ili, with epidemiologic links to southeast asia, in particular china, with no diagnosis within the first 72 hours of hospitalization may represent the patient with an emerging respiratory infection. 95 patients with severe respiratory infections are those with fever and new onset of cough or shortness of breath with radiographic evidence of acute respiratory distress syndrome or other life-threatening complications such as encephalitis. the emergency physician should enquire about the patient's travel history or any close contact with persons who have traveled 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health and long-term care novel swine-origin influenza a (h1n1) virus investigation team. emergence of a novel swine-origin influenza a (h1n1) virus in humans implications of the emergence of a novel h1 influenza virus older age and a reduced likelihood of 2009 h1n1 virus infection what is the difference between the ordinary/seasonal influenza -or "flu" -and an influenza pandemic early detection of severe emerging or re-emerging respiratory infections through severe respiratory illness (sri) surveillance. public health agency of canada appendix 1: references for table 4 manufacturer's product information or product insert comparison of binax now and directigen for rapid detection of influenza a and b evaluation of three immunoassay kits for rapid detection of influenza a and b comparison of rapid diagnostic techniques for respiratory syncytial virus and influenza a virus respiratory infections in young children comparison of a new neuraminidase detection assay with an enzyme immunoassay, immunofluorescence, and culture for rapid detection of influenza a and b viruses in nasal wash specimens application of directigen flu-a for the detection of influenza a virus in human and nonhuman specimens comparison of directigen flu-a with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza a virus comparison of rapid detection methods for influenza a and their value in health care management of institutionalized geriatric patients evaluation of a rapid enzyme immunoassay for detection of influenza a virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus evaluation of the directigen flua1b test for rapid diagnosis of influenza virus type a and b infections clinical evaluation of the zstatflu-ii test: a chemiluminescence rapid diagnostic test for influenza virus comparison of the directigen flu a1b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection comparison of lateral-flow immunoassay and enzyme immunoassay with viral culture for rapid detection of influenza virus in nasal wash specimens from children comparison of the denka seiken influ a-b quick and bd directigen flu a1b kits with fluorescent-antibody staining and shell vial culture methods for rapid detection of influenza viruses suboptimal detection of influenza virus in adults by the directigen flu a1b enzyme immunoassay and correlation of results with the number of antigen-positive cells detected by cytospin immunofluorescence comparison of four clinical specimen types for detection of influenza a and b viruses by optical immunoassay (flu oia test) and cell culture methods evaluation of a rapid optical immunoassay for influenza viruses (flu oia test) in comparison with cell culture and reverse transcription-pcr evaluation of biostar flu oia assay for rapid detection of influenza a and b viruses in respiratory specimens evaluation of an optical immunoassay for the rapid detection of influenza a and b viral antigens a flu optical immunoassay (thermobiostar's flu oia): a diagnostic tool for improved influenza management simultaneous detection and typing of influenza viruses a and b by a nested reverse transcription-pcr: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (flu oia) evaluation of diagnostic tests for influenza in a pediatric practice evaluation of a rapid test (quickvue) compared with the shell vial assay for detection of influenza virus clearance after antiviral treatment influenza virological surveillance in children: the use of the quickvue rapid diagnostic test quickvue influenza test for rapid detection of influenza a and b viruses in a pediatric population clinical evaluation of an immunochromatography test for rapid diagnosis of influenza evaluation of immunochromatography method for rapid detection of influenza a and b viruses evaluation of new rapid influenza virus detection kit-quick s-influ a/b "seiken comparison of a new lateral-flow chromatographic membrane immunoassay to viral culture for rapid detection and differentiation of influenza a and b viruses in respiratory specimens evaluation of a neuraminidase detection assay for the rapid detection of influenza a and b virus in children new point of care test is highly specific but less sensitive for influenza a and b in children and adults evaluation of an immunochromatography test using enzyme immunoassay for rapid detection influenza a and b viruses comparison of three rapid diagnostic kits using immunochromatography for detection of influenza a viruses evaluation of an immunochromatography test kit for rapid diagnosis of influenza evaluation of flow-through immunoassay for rapid detection of influenza a and b viruses key: cord-262201-4pab383g authors: wang, lei; zhang, rui-ming; liu, gui-ying; wei, bao-lin; wang, yang; cai, hong-yan; li, feng-sen; xu, yan-ling; zheng, si-ping; wang, gang title: chinese herbs in treatment of influenza: a randomized, double-blind, placebo-controlled trial date: 2010-06-22 journal: respir med doi: 10.1016/j.rmed.2010.05.015 sha: doc_id: 262201 cord_uid: 4pab383g objective: to investigate the efficacy and safety of antiwei, a traditional chinese prescription, in the treatment of influenza. methods: in a multi-center, randomized, double-blind, placebo-controlled trial, we recruited 480 adults aged 18 to 65 years within 36 h of onset of influenza-like symptoms. there were 225 patients with confirmed influenza. eligible patients were randomly assigned 6 g of antiwei (n = 360) or placebo (n = 120) twice daily for three days. all patients recorded their temperature and symptoms on diary cards during treatment. analyses were performed in both the influenza-like population and the influenza-confirmed population. results: antiwei increased patients’ recovery by 17% (p < 0.001), and reduced the severity of illness measured by the median symptom score by 50% (p < 0.001) in both the influenza-like and the influenza-confirmed populations, compared to placebo. the influenza-confirmed patients reported reductions in the severity of fever (p = 0.002), cough (p = 0.023) and expectoration (p = 0.004) after one-day of treatment with antiwei, compared to placebo. the adverse event profiles were similar for antiwei and placebo. conclusion: antiwei was effective and well tolerated in treatment of natural influenza infection in adults. antiwei represents a clinically valuable intervention in the management of influenza. traditional chinese herb; influenza; randomized controlled trial summary objective: to investigate the efficacy and safety of antiwei, a traditional chinese prescription, in the treatment of influenza. methods: in a multi-center, randomized, double-blind, placebo-controlled trial, we recruited 480 adults aged 18 to 65 years within 36 h of onset of influenza-like symptoms. there were 225 patients with confirmed influenza. eligible patients were randomly assigned 6 g of antiwei (n z 360) or placebo (n z 120) twice daily for three days. all patients recorded their temperature and symptoms on diary cards during treatment. analyses were performed in both the influenza-like population and the influenza-confirmed population. influenza caused by infection with either influenza a or b viruses, is one of the most common and important respiratory illnesses affecting all ages. uncomplicated influenza is generally resolved over a 2-to 5-day period, although cough and malaise can persist for weeks. influenza epidemics such as the influenza a (h1n1) pandemic in 2009, which is the first pandemic since 1968, are associated with increased morbidity and hospitalization rates, and excess mortality 1,2 within the most vulnerable populations, including the elderly, infants and those with underlying medical conditions. 3 although drugs such as the adamantanes (i.e., amantadine and rimantadine) and the neuraminidase inhibitors (i.e., oseltamivir and zanamivir) are available to treat or prevent influenza, recent emergence of adamantane resistant strains has rendered these agents less effective. 4, 5 in addition, sporadic cases of oseltamivir-resistant influenza a (h1n1) virus infection have been reported worldwide following the h1n1 pandemic of 2009. 6 even though vaccination strategies against influenza a (h1n1) virus are now present in china, some patients may look beyond conventional treatments and consider complementary and alternative medicines, including traditional chinese medicine (tcm), in this situation. tcm represents one aspect of chinese medical philosophy that is characterized by an emphasis on maintaining and restoring balance. chinese medicinal herbs, the most important component of tcm, are derived from plants and usually incorporate one or more herbs as the basic drugs to treat the disease. 7 supplemental use of chinese medicinal herbs were found to be beneficial in the management of severe acute respiratory syndrome (sars) in 2003 8e10 and, based on that supplementary treatment, chinese medicinal herbs might also show promise for treating or preventing influenza and influenza-like illness. some randomized controlled trials (rcts) have been published, but the evidence for treating or preventing influenza by tcm has not yet been convincingly established, due to poor reporting quality and small sample size. 11, 12 antiwei granule is a traditional chinese prescription, consisting of mahuang (herba ephedra), baimaogeng (rhizoma imperatae), gegen(radix puerariae), guizhi (ramulus cinnamoumum), kuxingren (semen armeniacae amarum.), ganjiang (rhizoma zingiberis) and gancao (radix glycyrrhizae). one gram of antiwei granule equals 3.83 g of crude drug. the resource, pharmacological actions and components in this formula are listed in table 1 . 13 according to tcm, mahuang and baimaogen in this formula, which are regarded as "the principal" herbs, could relieve exterior disorder and defervesce. gegen and guizhi, which serve as "the minister", cooperate with "the principal" to relieve exterior syndrome and defervesce. kuxingren and ganjiang are "the adjuvant drug", which can open the inhibited lung-energy, regulate the flow of qi, eliminate phlegm and relieve cough. gancao serves as "the guiding drug", which can moisten lung to arrest cough and coordinate all of the drug actions of this formula. antiwei has been used widely based on clinical experience rather than the evidence of rcts in the treatment of influenza-like illness (such as fever, headache, pain and cough) in china. 14 therefore, a prospective, multi-center, randomized, double-blind, placebo-controlled clinical trial was undertaken to investigate the efficacy and safety of antiwei granule for the treatment of naturally acquired influenza in humans. patients and ethics eligible patients, who presented with fever over 37.4 c and at least one respiratory symptom (cough, sore throat, or nasal symptom) and at least one constitutional symptom (headache, fatigue, myalgia, thirst or chills), were recruited from emergency departments from january to june, 2007. the inclusion criteria were as follows: (1) clinical diagnosis of influenza; (2) age 18 to 65 years; (3) within 36 h of onset of influenza-like illness; (4) written informed consent. the exclusion criteria were as follows: (1) any treatments after onset of influenza-like illness; (2) other confirmed upper respiratory viral infections; (3) suspected bacterial infection (based on symptoms, leukocyte count > 10.0 â 10 9 /l or neutrophil granulocyte ! 80%); (4) pregnancy or breast-feeding; (5) allergies to food additives, drugs, and any components in antiwei granule; (6) current alcoholism or drug abuse; (7) severe diseases of cardiac, respiratory, hepatic, renal, central nervous system, haematopoietic system cancer; (8) the inability to understand and complete this study; (9) current psychiatric illness or dementia; (10) clinically important chronic illnesses or known hiv infection, receiving steroids or other immunosuppressants, or having been vaccinated against influenza in the previous 12 months. we also required that patients of childbearing age use contraception. to reduce heat in cases of exterior syndrome with fever and painful stiffness of the back and nape; to relieve thirst in febrile diseases and diabetes mellitus; to arrest diarrhea in spleen insufficiency; to promote eruption for measles 16 .48 guizhi the died young stem of cinnamomum cassia presl. (family lauraceae) to induce sweating for releasing the muscles in cases of wind-cold affliction; to warm and unblock the meridians to relieve various pains due to cold and congealing blood; to stimulate menstrual discharge for treating amenorrhea to replenish qi and tonify the heat for treating arrhythmia in cases of heart qi deficiency; to tonify the spleen for treating lassitude, anorexia and loose bowels in cases of spleen insufficiency; to relieve epigastric colic and spastic pain of the limbs; to dispel phlegm and arrest cough; to clear heat and counteract toxin for treating sore throat, boils, sores and drug overdose; most frequently for modulating the ingredients in a prescription data from classified dictionary of traditional chinese medicine by zhu-fan xie. 13 the medical ethics committee of west china hospital at sichuan university approved this study (no. irb-2007-13) , and it is in accordance with the recent principles of the declaration of helsinki. 15 all patients gave informed consent and were free to withdraw from the study at any time. this was a prospective, multiple-center, randomized, double-blind, placebo-controlled trial, which was undertaken at eight chinese centers: three in north china, three in northeast china, one in northwest china, and one in west china. patients were randomized in a double-blind fashion to receive either antiwei granule or placebo. the randomization code was generated by the prco plan of the analysis system of sas (version 6.12 for windows). allocation details were sealed in an envelope, and were unknown both to investigators and participants in this study. antiwei and placebo were supplied by tasly group (tianjin, china), the herbal composition of antiwei is presented in table 1 . the placebo granule was composed of starch and bitter agents, but was visually indistinguishable from the antiwei in appearance, color, size and packaging. the subjects in the antiwei group received 6 g of antiwei granule twice a day for three days, whereas the patients in the placebo group received 6 g of placebo twice a day for three days. all patients were treated in an equivalent fashion. we assessed compliance by daily diary cards and review of the returned medication at the end of treatment. we took present, past and personal medical histories, measured vital signs such as temperature, respiration rate, heart rate and blood pressure, did physical examinations of pharynx, larynx and lung in respiratory system, and collected baseline virological samples before treatment. after accepting treatment, patients recorded their axillary's temperature every 2 h for up to 12 h on day one. on days 2 and 3, they recorded their axillary's temperature at 8am, 10am, 12am, 4pm and 8pm in a diary card. to exclude other respiratory illnesses, subjects received chest x-ray screening before beginning treatment. the presence and severity of influenza symptoms, including cough, sore throat, nasal obstruction and rhinorrhoea, headache, fatigue, myalgia, thirst and chills, were recorded once daily using a four-point scale (0 absent, 1 mild, 2 moderate, and 3 severe). the first record was performed when the patient was admitted, and the next two records (on days 1 and 3 after treatment) were done in the morning. influenza was confirmed by a direct immunofluorescent antibody assay (dfa) of nasopharyngeal swabs, which showed sensitivities of 89.7% for influenza a virus and 87.9% for influenza b virus and specificities of 99.3% for influenza a virus and 100% for influenza b virus. 16 treatment assignment was blinded during virological testing, which was done at west china hospital, sichuan university, chengdu. the primary endpoints were severity of illness (measured by the mean symptom scores for the whole treatment period) and the number of recovered patients in the intention-to treat and influenza-confirmed populations. recovery was defined as absence of fever (temperature < 37.4 c) and all nine symptoms (chills, headache and myalgia, nasal obstruction, sore throat, fatigue, cough, rhinorrhoea, expectoration, and thirst). secondary outcomes included the length of time to alleviate fever within the first 24 h after treatment, the severity of each symptom, and the rate of influenza-viruspositive conversion to negative for the influenza-confirmed population at entry. participants were required to record any unexpected signs, symptoms, or feelings during the treatment period, and routine tests of blood, urine, stool, as well as hepatic and renal functions and electrocardiogram (ecg) were performed at admission and again after treatment to assess safety in both groups. a sample size of 135 for the antiwei group and 45 for the placebo group, at a ratio of 3:1, were calculated according to published data 17 to have a power of 80% or greater to detect a difference of 10 percent in recovery rate, assuming a significance level of 0.05. sample size calculations were performed using a normal approximation to the wilcoxon rank-sum test. influenza-like and influenza-confirmed populations were both analyzed, with the influenza-like population consisting of randomized patients who received at least one dose of medication in this study. safety analyses were performed on those populations, which included all patients who received at least one dose of medication and who had at least one follow-up for safety, whether or not they were withdrawn prematurely. all analyses were performed using stata 11.0. measurement data showing a normalized distribution were described as mean ae standard deviation and analyzed with analysis of variance to determine the difference between the antiwei and placebo group. measurement data showing a non-normalized distribution were described as median and analyzed by the wilcoxon rank-sum test. frequency data were analyzed using the chi-squared (c 2 ) or fisher's tests. the primary endpoints were analyzed using the survival method. kaplanemeier curves were constructed and significance-tested using the log-rank method. for the mean symptom scores we used analysis of variance. the rate of influenza-virus-positive conversion to negative was compared between different groups with c 2 test. p 0.05 for two-tailed tests was considered to be significant. a total of 480 eligible patients were enrolled and randomly assigned to receive antiwei granule (n z 360) or placebo (n z 120). of these, 34 patients withdrew from the study early for several reasons including lost to follow-up (antiwei: n z 6; placebo: n z 7), protocol violation (antiwei: n z 15; placebo: n z 3), and refusal to continue (antiwei: n z 1; placebo: n z 2). overall, 225 (48.2%) patients had laboratory-confirmed influenza (fig. 1 ). of these, 125 (55.6%) had influenza a, 74 (32.9%) had influenza b and 26 (11.5%) had both influenza a and b ( table 2 ). all available data from these patients were included in the efficacy and safety analyses. no differences were observed between two groups in the demographics and clinical characteristics. safety of the study drug was assessed for all 480 patients. for influenza-confirmed patients, antiwei resulted in significantly more patients recovering or showing a reduction in the severity of illness. the number of patients recovering after three days of treatment was 42 of 177 (23.2%) in the antiwei group (p z 0.009) (fig. 2) compared to 3 of 48 (6.25%) in the placebo group, and this benefit was also seen in the patients with influenza-like disease (86/353 vs. 10/114, p < 0.001). similarly, antiwei resulted in significant reductions in the total symptom score after three days of treatment in the influenza-confirmed population (à15.20 ae 6.25 vs. à10.26 ae 7.72, p < 0.001) and in the influenza-like population (à15.07 ae 6.32 vs. à8.81 ae 6.99, p < 0.001) compared to placebo. after one-day treatment, the improved percentage of symptom score (difference between the symptom scores at baseline and after one-day treatment is divided by the symptom score at baseline) in fever (57.9% vs. 34.4%, p z 0.002), cough (32.9% vs. 16.3%, p z 0.023) and expectoration (32.9% vs. 16.3%, p z 0.004) was significantly reduced in the antiwei group compared to placebo in influenza-confirmed patients. there was no statistically significant reduction seen in the severity of chills (34.9% vs. 28 (fig. 3) . at the same time, the median temperature fell by 0.8 c from baseline in the antiwei group but by only 0.4 c in the placebo group (p z 0.0001). treatment benefit for fever was apparent as early as 10 h after the start of treatment (fig. 4) . antiwei treatment resulted in 20% more patients becoming afebrile within 24 h, as compared to placebo (p z 0.011). in the influenza-confirmed population, influenza virus decreased to 27.2% in the antiwei group and 58.7% in the placebo group after three days of treatment (p z 0.001) (fig. 5 ). compliance with medication was high in both treatment groups. the majority patients (n z 446) recorded taking more than 90% of medication. antiwei was generally well tolerated compared with placebo, with no increase in withdrawal rates because of adverse events. one patient given antiwei reported mild paroxysmal palpitation but this resolved within two days and did not result in withdrawal from the study. laboratory results, ecg and vital signs did not differ significantly from baseline between the two groups. most of the patients included in this study came from an epidemic region during the influenza season. 18e23 after dfa screening with nasopharyngeal swabs, the positive rate in our study was 48.2%, which is slightly lower than 61.0% in zambon's study. 24 the oral administration of 6 g of antiwei twice daily was associated with significant clinical and antiviral effects in healthy adults with naturally occurring influenza when given within 36 h of onset and was generally well tolerated. for patients with influenza-like illness, the number of recovered patients after three days of treatment in the antiwei group increased 17% and the severity of symptoms was significantly reduced 50% compared to placebo. after one-day treatment, antiwei's benefit for reducing fever was apparent and the other two key symptoms (cough and expectoration) were significantly improved by the end of treatment. at the same time, antiwei was well tolerated, with safety similar to placebo. since the introduction of tcm to the world, there have been debates regarding a role for herbal medicines in the therapy of illness such as influenza infections. according to treatise on cold-induced diseases (shang han lun), a variety of herbal formulas have been used to treat patients with infectious diseases for over 1800 years. chinese medicine is widely accepted amongst chinese populations. the use of chinese medicine in treating pandemic and endemic diseases has long been described in traditional medical books such as shang han lun, and huangdi's canon of medicine (huangdi nei jing). the prescriptions for infectious diseases were made based on the clinical presentations, rather than laboratoryconfirmed pathogens, which were beyond their knowledge at that time. only a small fraction of the thousands of medicinal plants used worldwide has been tested rigorously in randomized, placebo-controlled trials. yet randomized, controlled trials are the best way to demonstrate the efficacy of any medical, herbal or conventional therapy. 25 although herbs or herbal products, as part of complementary and alternative therapies, have been proposed, their effectiveness in treating or preventing influenza has not been established beyond reasonable doubt. 11 the extract of elderberry was used to treat the influenza in the past, 26 and two studies have demonstrated a significant benefit of herbal products in prophylaxis against viral infection. 27, 28 herbal supplement users have also reported significant improvement in influenza-like symptoms such as chills, cough, fatigue, and headache and quality-of-life measures such as mental health score, 27 as they have in our study. figure 3 improved percentage in symptom scores after oneday treatment in influenza-comfirmed patients. *p < 0.05, placebo vs. antiwei. improved percentage in symptom scores defines that difference between the symptom score at baseline and after one-day treatment is divided by the symptom score at baseline. a variety of chinese herbs have been widely used as antipyretic drugs for clearing accumulated heat in the lungs, according to the theory of tcm. one recent study by kubo t. et al. showed that there is a more antipyretic effect of mao-to, a japanese herbal medicine, for treatment of type a influenza infection in children, in comparison to oseltamivir. 29 the japanese traditional herbal medicine includes mahuang, guizhi, kuxingren and gancao, which as components were included in antiwei. in our study, the antipyretic effect was apparent within 24 h of antiwei administration, when influenza symptoms are generally most troublesome. although we did not assess economic effects, the indirect costs of influenza such as workplace absenteeism and performance at work could be improved according to the clinical benefits observed in the influenza-confirmed population and in the population with influenza-like illness. 30 studies in animal experiments and in vitro, have confirmed that antiwei can inhibit growth of a variety of virus, including influenza a and b, respiratory syncytial virus (rsv), adenovirus etc (data not shown). we now show that antiwei can significantly reduced the positive rate of influenza virus after treatment. these findings are consistent with results from studies of other antiviral drugs for influenza and other self-limiting viral diseases, for which increased therapeutic benefit is obtained when treatment is started as early as possible after symptom onset. 31 the duration of cough and expectoration was shorter and the severity of the two symptoms was generally lower in the antiwei groups (data not shown). influenza is transmitted by virus-laden secretions. cough and expectoration could be the most distressing symptoms of influenza and also contributes to the spread of infection. patients who received antiwei had significantly lower positive rate of virus on laryngopharynx swabs than those in the placebo group. such differences, together with improvement of symptoms, including cough and expectoration, may help to reduce transmission of the virus. antiwei was generally well tolerated, with almost no adverse events reported. only one patient given antiwei reported mild paroxysmal palpitation, which was resolved within two days without additional treatment. this symptom was not associated with discontinuation rates in the antiwei group compared to placebo. no other adverse effects or abnormalities in laboratory test were associated with antiwei treatment. it must be noted that the antiwei contains mahuang, which has been reported to have some side effects (such as chest pain, hypertension, arrhythmia, myocardial infarction, stroke, or death 32 ) when served as a dietary supplement for weight reduction and performance enhancement, but the use of mahuang in this study was not associated with any significant adverse events. in our prescription, 6 g of antiwei equals to 23 g of crude herbs, in which there is 2.5 g of mahuang, so the daily dose of mahuang was only 5.0 g, which is a small dosage compared with the conventional dose in other tcm treatments. we recognize that this study does have some potential limitations. we did not include follow-up after treatment, and the symptoms of influenza were observed for only 4 days, thus by the end of study many patients had not fully recovered. therefore, the duration of illness and the time of alleviation could not be exactly evaluated. in addition, the protocol specifically excluded individuals with medical conditions which would place them in high-risk populations that are often associated with more severe influenza. in conclusion, our study clearly indicated that antiwei was an effective treatment of influenza-confirmed and influenza-like illness in otherwise healthy adults, and antiwei can provide an alternative to conventional treatment for influenza or influenza-like symptoms. antiwei might also be a potent medication for use in influenza pandemics and in situations where a shortage of antiviral agents and vaccines exists. our findings provide a rationale for continuing studies of this agent in the treatment of influenza, and these should now include studies in children and in high-risk populations. further large-scale studies to investigate the other antiviral effect of antiwei, and studies of its mechanism(s) are also now required. intensive care adult patients with severe respiratory failure caused by influenza a (h1n1)v in spain the transmissibility and control of pandemic influenza a (h1n1) virus epidemiology of influenza high levels of adamantane 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medicine traditional chinese medicine in cold-pestilential pathogen and severe acute respiratory syndrome the declaration of helsinki direct detection of influenza virus antigen in nasopharyngeal specimens by direct enzyme immunoassay in comparison with quantitating virus shedding a multiple center, randomized, controlled, double-blinded and double-dummy trial of yiqing shuangjie capsule and tablet in treating acute upper respiratory tract infection with the syndrome of heat attacking the lung and weifen (chin) analysis of the antigenic and genetic characteristics of influenza virus subtype a3 circulated in sichuan province during analysis of surveillance of influenza in xinjiang from analysis of influenza surveillance in sichuan province analysis of influenza surveillance in changchun during analysis of surveillance of influenza in tianjing from analysis of influenza surveillance in changchun during diagnosis of influenza in the community: relationship of clinical diagnosis to comfirmed virological, serologic, or molecular detection of influenza herbal remedies inhibition of several strains of influenza virus in vitro and reduction of symptoms by an elderberry extract (sambucus nigra l.) during an outbreak of influenza b pannama the use of an herbal formula by hospital care workers during the severe acute respiratory syndrome epidemic in hong kong to prevent severe acute respiratory syndrome transmission, relieve influenza-related symptoms, and improve quality of life: a prospective cohort study efficacy and safety of the standardised ginseng extract g115 for potentiating vaccination against the influenza syndrome and protection against the common cold antipyretic effect of mao-to, a japanese herbal medicine, for treatment of type a influenza infection in children the impact of influenza and influenza-like illness on productivity and healthcare resource utilization in a working population efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza virus infections ephedra-containing dietary supplements in the us versus ephedra as a chinese medicine the authors would like to thank ms. feng m. and mr. she b. for their work in documenting the data, he clara, phd. and none declared. key: cord-276577-06boh550 authors: schanzer, dena l.; garner, michael j.; hatchette, todd f.; langley, joanne m.; aziz, samina; tam, theresa w. s. title: estimating sensitivity of laboratory testing for influenza in canada through modelling date: 2009-08-18 journal: plos one doi: 10.1371/journal.pone.0006681 sha: doc_id: 276577 cord_uid: 06boh550 background: the weekly proportion of laboratory tests that are positive for influenza is used in public health surveillance systems to identify periods of influenza activity. we aimed to estimate the sensitivity of influenza testing in canada based on results of a national respiratory virus surveillance system. methods and findings: the weekly number of influenza-negative tests from 1999 to 2006 was modelled as a function of laboratory-confirmed positive tests for influenza, respiratory syncytial virus (rsv), adenovirus and parainfluenza viruses, seasonality, and trend using poisson regression. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests. the sensitivity of influenza testing was estimated to be 33% (95%ci 32–34%), varying from 30–40% depending on the season and region. conclusions: the estimated sensitivity of influenza tests reported to this national laboratory surveillance system is considerably less than reported test characteristics for most laboratory tests. a number of factors may explain this difference, including sample quality and specimen procurement issues as well as test characteristics. improved diagnosis would permit better estimation of the burden of influenza. although influenza virus infection is associated with considerable morbidity and mortality [1] [2] [3] , laboratory confirmation of clinical illness is the exception rather than the rule. clinicians do not routinely seek laboratory confirmation for several reasons: diagnosis will often not alter patient management, a paucity of real-time, accurate, inexpensive testing methods [4] and because influenza is not recognized as the etiology of the clinical presentation [5] . accurate diagnosis of influenza-like illness, however, could improve clinical care through reduced use of antibiotics and ancillary testing, and more appropriate use of antiviral therapy [6] . although rapid influenza tests such as pointof-care tests are purported to generate results in a timely fashion to influence clinical care, the performance characteristics of the currently available tests are sub-optimal [7] . new technologies with improved sensitivity such as reverse-transcriptase polymerase chain reaction (rt-pcr) [8] as well as the use of more effective collection systems such as the flocked nasopharyngeal swab compared to traditional rayon wound swabs, and the recommendation to collect more ideal specimens, such as nasopharyngeal swabs rather than throat swabs are likely to improve diagnostic sensitivity [9] [10] [11] [12] . the performance characteristics of currently available tests for influenza vary considerably and the overall sensitivities of these tests when used in routine practice are also dependent on the type of specimen collected, the age of the patient and point in their illness in which they are sampled [4, 9, [13] [14] [15] . we sought to estimate the sensitivity of influenza testing based on results of a national respiratory virus surveillance system using a model-based method [1, 2, [16] [17] [18] . weekly respiratory virus identifications from september 1999 to august 2006 were obtained from the respiratory virus detection surveillance system (rvdss), public health agency of canada [19, 20] . the rvdss collects, collates, and reports weekly data from participating laboratories on the number of tests performed and the number of specimens confirmed positive for influenza, respiratory syncytial virus (rsv), para-influenza virus (piv), and adenovirus. specimens are generally submitted to laboratories by clinicians in the course of clinical care, and by clinicians participating in one of our national influenza surveillance programs, (fluwatch [20] ). indicators of influenza activity are reported year round on a weekly basis to the fluwatch program. the rvdss is supplemented by case reports of influenza positive cases [19, 21] . from the case reports, influenza a was confirmed in all age groups and sporadic cases were confirmed in the off-season months of june through september. infants and children under the age of 5 years accounted for 25% of the influenza a positive tests, and persons over the age 65 years another 35%. unfortunately, fluwatch surveillance data does not provide the total number of tests by age. testing practices are known to be varied [22, 23] . the predominant testing methods used for influenza detection varied considerably by province or laboratory and over time. for the 2005/06 season a survey of laboratory techniques in current use indicated that culture accounted for 44% of the diagnostic tests with rt-pcr, rapid antigen tests and direct fluorescent-antibody assay (dfa) accounting for 21%, 19%, and 16% respectively [23] . the weekly number of tests negative for influenza was modelled, using poisson regression, as a function of viral identifications for influenza, rsv, adenovirus and piv as well as a baseline consisting of seasonality, trend and holiday variables. the estimated baseline implicitly accounts for influenza tests on specimens taken from patients with respiratory infections due to respiratory pathogens other than the four viruses captured in the rvdss, as long as both the testing behaviour of clinicians and respiratory illnesses caused by other respiratory pathogens follow a consistent seasonal pattern as prescribed by the model (see below, the poisson regression model with a linear link function was estimated using sas [24] proc genmod: coefficients b 5 to b 9 are multipliers. the weekly number of influenza negative tests estimated to be falsely negative is given by b 5 infla w +b 6 inflb w . the weekly number of influenza negative tests attributed to rsv is given by b 7 rsvp w. , and similarly for adenovirus and piv. for each positive influenza a test, an additional b 5 tests above baseline were performed and found to be negative. by specifying a linear link, a value of 0.33, say, for coefficient b 5 , means that for every test for which influenza a was confirmed, 0.33 additional tests, on average, were performed on truly influenza a positive specimens and found to be negativewhich corresponds to a sensitivity of 75%. sensitivity was calculated as the number of influenza positive tests divided by the number of influenza positive tests plus the model-estimated number of false negative tests, or equivalently, the estimates of sensitivity for influenza a and b are given by 1/ (1+b 5 ) and 1/(1+b 6 ) respectively. the false negative rate is 1 minus sensitivity. while the null value for b 5 is zero, which indicates no statistical association between the number of influenza positive tests and the number of influenza negative tests, the corresponding null value for sensitivity is 1. for each test confirmed positive for rsv, on average b 7 tests were performed for influenza and found to be negative for influenza. these b 7 tests are attributed to an rsv infection, however the number of influenza-negative tests that actually tested positive for rsv is unknown. if all specimens had been tested for the same viruses (panel tests), 1/b 7 would correspond to the sensitivity for rsv testing, and the sensitivity for adenovirus and piv given by 1/b 8 and 1/b 9 respectively. some laboratories are known to test for viruses sequentially [22] , and so 1/b 7 -1/b 9 were not interpreted as estimates of the sensitivity for other viruses. sequential testing may occur if a rapid test for influenza is negative and the laboratory then performs pcr or culture testing. similarly in young children with a respiratory illness in the winter, rapid tests for rsv infection may be performed first, and only specimens with negative results submitted for subsequent testing for influenza or other respiratory viruses [25] . by contrast, many laboratories conduct panel tests for multiple viruses for ease of handling, decreased patient sampling, and recognition that coinfection can occur. either form of sequential testing would not bias the estimate of sensitivity applicable to test results reported to rvdss, though significant use of rapid antigen tests in the laboratories reporting to rvdss would reduce the overall sensitivity. as a single specimen may undergo multiple tests, the false-negative rate applicable to a specimen that has undergone multiple tests would be expected to be much lower than the system average for individual tests. parameters b 1. to b 4 account for trends and the seasonality of truly negative specimens (patients presenting with other acute respiratory infections). over 50,000 tests for influenza were reported to the rvdss each year, peaking in 2004/05 at 101,000. overall 10% of the influenza tests were positive for influenza, ranging from 4% to 13% depending on the season. the proportion positive for rsv, parainfluenza and adenovirus averaged 9%, 3% and 2% respectively. as seen in figure 1 , no virus was identified in 75% of specimens submitted for testing (white area under the curve). even for the winter months of december through april, one of these 4 viruses was identified on average in no more than 30% of the specimens. the strong and consistent synchronization of negative tests with influenza positive tests, as seen in figure 1 , is suggestive that false negative results contributed to the large number of negative tests during periods of influenza activity. the sensitivity for influenza a testing averaged 33.7% (with model-estimated 95% confidence intervals of 33.3-34.1) for the 1999/2000-2005/06 period. influenza b testing had a similar estimated sensitivity at 34.7 (95% ci 33.4-36.1). estimated sensitivities varied somewhat from season to season, generally ranging from 30%-40% (table 1) , and provincial level estimates, as well, were within a similar range. stratifying by province or season produced similar estimates for the sensitivity of influenza a testing: 32% (95% ci 30-34) and 36% (95% ci 33-41) respectively. estimates of sensitivity based on test results reported to the rvdss for individual laboratories with sufficient data to fit the model showed significant variation, with estimates of sensitivity ranging from 25-65%. as expected, laboratories using primarily rapid antigen tests had lower estimated sensitivities, and laboratories that used pcr methods had higher sensitivity estimates. however, information on testing procedures is limited primarily to the 2005/06 survey. as well, additional irregularities were noticed in the laboratory data and not all laboratories provided sufficient data to fit the model. figure 2 illustrates a good model fit where the weekly number of influenza negative tests is well explained by the model covariates, with a few exceptions. firstly, it is evident that additional specimens were tested during the sars period, as indicated by the period where the number of weekly influenza negative tests exceeded the expected number, or equivalently, a period of successive positive residuals. residuals typically capture random variation; hence represent tests that can not be allocated based on the specified model. in addition to the sars period, testing appears to have been elevated for a number of weeks in january 2000 during the peak of the 1999/2000 a/ sydney/05/97 (h3n2) season in which respiratory admissions were unusually elevated [26, 27] , and in december 2003, when an elevated risk of paediatric deaths associated with the a/fujian/411/02 (h3n2) strain [28] was identified in the us. as these periods corresponded to a period of heightened public awareness due to severe influenza outbreaks, parameter estimation was repeated without these data points. exclusion of these data points did not alter the sensitivity estimate for influenza. the attribution of influenza negative test results to influenza and other viruses is illustrated in figure 3 . the baseline curve is the model estimate of the number of tests that were likely truly negative for all four viruses tested. a reduction in specimen collection and testing, primarily for viruses other than influenza, is also evident over the christmas period ( figure 3) . the weekly proportion of tests confirmed positive for influenza peaked each season at 15 to 30%. accounting for the model estimated false negative rate suggests that during periods of peak influenza activity, 40-90% of tests were performed on specimens taken from persons recently infected with influenza. influenza was confirmed in only 14% of specimens sent for testing over the winter period, whereas the sensitivity estimate would imply that up to 40% of influenza tests could be attributed to an influenza infection. the corresponding figures for the whole year indicate that 10% of specimens were confirmed positive for influenza and 30% of influenza tests could be model-attributed to an influenza infection annually. despite a relatively large number of tests in the off-season, the number of influenza positive tests was almost negligible; suggesting that the false positive rate applicable to rvdss influenza testing is minimal. the model estimated sensitivity based on influenza test results reported to the rvdss of 30-40% is much lower than the standard assay sensitivities documented in the literature. standard sensitivities for diagnostic procedures used by participating laboratories ranged from 64% for rapid antigen tests to 95% for rt-pcr tests, averaging 75% for the study period [23] . as performance characteristics of specific tests are generally based on high quality specimens, the difference of approximately 40% is likely linked to any one of many operational procedures that affects the quality of the specimen and its procurement. unlike validation studies, our samples are taken from a variety of clinical settings and processed with a variety of procedures across the country. as well, variation in the indications for diagnostic testing may vary across the country. as there are many other respiratory pathogens that are not routinely tested for, or reported to the rvdss, including human metapneumovirus (hmpv), coronaviruses, and rhinoviruses for which patients may seek medical care and present with influenza like illness [29] [30] [31] [32] , a large proportion of negative test results was expected. the overall model fit, and the general consistency of the sensitivity estimates, suggests that these many respiratory viruses were reasonably accounted for by the seasonal baseline and that the strong association between the number of influenza positive and influenza negative tests on a weekly basis is indicative of a significant number of false negative results, rather than the activity of another virus or viruses exactly synchronous with influenza. the latter would bias the estimated sensitivity of the system downwards. however, to significantly and consistently bias the estimate, the degree of synchronization would have to be fairly strong, persist over the whole study period, and occur in all provinces. synchronization was not observed among the rvdss viruses (influenza a, influenza b, rsv, adenovirus and piv), and elsewhere other viruses such as rhinovirus, coronavirus and hmpv accounted for only a small proportion of the viral identifications and were not found to be synchronized with influenza [33] . as well, patients may present for care due to a secondary bacterial infection. while any specimen would likely test negative as the virus, at this point, is likely not detectable, the model would statistically attribute a negative test in this case to the primary infection; one of the four rvdss viruses or to the seasonal baseline that represents other respiratory infections, depending on the level of viral activity at the time of the test. this is not considered a source of bias. the large variation in false negative rates estimated for individual laboratories reporting to the rvdss suggests that standardization of sample procurement, testing and reporting procedures would likely reduce the overall false negative rate. the accuracy of diagnostic tests is known to be affected by the quality of the specimen [10, 11] , its handling, the timing of collection after symptom onset, and the age of the patient [14, 15] . even with the most sensitive molecular methodologies, yield was shown to be strongly related to the time since onset of symptoms [9, 14] , with a 3-fold decline in proportion positive within 3 to 5 days after onset of symptoms for both rt-pcr and culture procedures. for most laboratory tests, specimen procurement within 72 hours of from the onset of symptoms is recommended [6] , yet patients often present much later in the course of illness. estimates of the median time since onset of symptoms suggest a delay of 3 and 5 days for outpatient and inpatients respectively [15] , however these estimates are limited to patients with laboratory confirmed influenza. in addition, there are inherent differences in the performance characteristics of the currently used diagnostic tests [4, 6, 8, [34] [35] [36] [37] [38] . lack of standardization between diagnostic tests and algorithms used in different laboratories reporting to the rvdss adds to this complexity. the routine use of rt-pcr testing has only recently become available in canada (only 20% of tests used rt-pcr methods as of 2005/06 [23] ), but increased use of this modality is expected to improve accuracy. population or system level sensitivity estimates that include the effects of sample quality are limited. grijalva and colleagues [39] estimated the diagnostic sensitivity in a capture recapture study of children hospitalized for respiratory complications at 69% for a rt-pcr based system and 39% for a clinical-laboratory based system (passive surveillance of tests performed during clinical practice, and using a variety of commercially available tests). though the expected proportion of influenza tests that were due to influenza infections is unknown and variable, our model estimate of 30% appears plausible. cooper and colleagues [33] attributed 22% of telephone health calls for cold/flu to influenza over two relatively mild years, and elsewhere 20% of admissions for acute respiratory infections (including influenza) in adults aged 20-64 years were attributed to influenza, and 42% for seniors [1] . while there are limitations with this approach, there are no other simple alternatives to assist in the interpretation of the rvdss data. it would have been helpful to analyze data based on each specimen sent for testing. with only the number of weekly tests and number of positive results, we were unable to calculate the number of specimens that were actually found to be negative for all four viruses, or to estimate the extent of co-infection. coinfection, which was not accounted for in our model, could result in an under-estimation of the number of falsely negative tests, as the attribution of an influenza negative test that was actually coinfected with influenza and another respiratory virus would have to be split between the viruses. with auxiliary information associated with each specimen, model estimates of false negative rates based on, for example, test type, time since onset of symptoms, age of the patient, or clinical presentation would have allowed us to explore the reasons for the high false negative rates. as the false negative rate appears to be laboratory dependant (data not shown), this estimated range is applicable only to the rvdss for the study period. a significant reduction in the false negative rate is anticipated as methods become standardized and with the uptake of the new rt-pcr methods. as positive results, particularly for culture, are often obtained a week or more after the specimen was received, some positive results may have been reported in a different week than the test. multiple test results for a single specimen may have also contributed to reporting irregularities. these irregularities would tend to bias the estimated parameter towards zero, and hence the estimated sensitivity towards 1. considering the overall model fit and the relative severity of influenza [1] , we conclude that our estimate of sensitivity may be slightly over-estimated (number of false negatives under-estimated). poor test sensitivity contributes to the chronic underestimation of the burden of influenza in the general population. since estimates of the burden of illness drive planning for preventive and therapeutic interventions, it is important to improve all aspects leading to improved diagnostic accuracy. we have illustrated a simple method that uses the surveillance data itself to estimate the system wide sensitivity associated with the weekly proportion of tests confirmed positive. although our estimate of sensitivity is only applicable to the interpretation of the rvdss data over the study period, similar estimates for specific cohorts or laboratory procedures may help guide further investigation into the reasons for the large number of false negative test results. the capacity for improved diagnostic accuracy will ultimately improve our understanding of the epidemiology of influenza. role of influenza and other respiratory viruses in admissions of adults to canadian hospitals co-morbidities associated with influenza-attributed mortality influenzaattributable deaths: canada 1990-1999 sensitivity of diagnostic tests for influenza varies with the circulating strains accuracy and interpretation of rapid influenza tests in children role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics the limitations of point of care testing for pandemic influenza: what clinicians and public health professionals need to know genescan reverse transcription-pcr assay for detection of six common respiratory viruses in young children hospitalized with acute respiratory illness enhancing the predictive value of throat swabs in virological influenza surveillance comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients use of throat swab or saliva specimens for detection of respiratory viruses in children nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms virological surveillance of influenza-like illness in the community using pcr and serology effectiveness of reverse transcription-pcr, virus isolation, and enzyme-linked immunosorbent assay for diagnosis of influenza a virus infection in different age groups hospitalization attributable to influenza and other viral respiratory illnesses in canadian children influenza-attributed hospitalization rates among pregnant women the modelled attribution of the weekly number of specimens tested for influenza to influenza (a and b), and adenovirus, parainfluenza virus, and rsv combined is shown along with the numbers confirmed positive. the total is the number of weekly tests for influenza (most were likely panel tests). the baseline accounts for routine tests in the hypothetical absence of influenza, rvs, adenovirus and parainfluenza activity, and corresponds to the model estimate of the number of tests that were truly negative for all tested viruses. the blue area (light plus dark) corresponds to tests attributed to influenza, with the light blue area corresponding to tests confirmed positive for influenza. the purple area (light plus dark) corresponds to tests attributed to rsv, adenovirus or parainfluenza influenza-associated hospitalizations in the united states influenza in canada: 2005-2006 season influenza in canada: 2003-2004 season antiviral therapy and outcomes of influenza requiring hospitalization in ontario impact of changing laboratory diagnostics on influenza surveillance sas/stath 9 user's guide strategy for efficient detection of respiratory viruses in pediatric clinical specimens prescription for excellence: how innovation is saving canada's health care system emergency department overcrowding: ambulance diversion and the legal duty to care influenza-associated deaths among children in the united states characterization of viral agents causing acute respiratory infection in a san francisco university medical center clinic during the influenza season human metapneumovirus infections in adults: another piece of the puzzle human metapneumovirus infection in adults human metapneumovirus infection in the canadian population the contribution of respiratory pathogens to the seasonality of nhs direct calls superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children real-time pcr in clinical microbiology: applications for routine laboratory testing evaluation of three immunoassay kits for rapid detection of influenza virus a and b performance of six influenza rapid tests in detecting human influenza in clinical specimens comparison of the directigen flu a+b test, the quickvue influenza test, and clinical case definition to viral culture and reverse transcription-pcr for rapid diagnosis of influenza virus infection estimating the undetected burden of influenza hospitalizations in children the authors acknowledge the support of the national fluwatch network and all those involved in the collection and compilation of this data. special thanks to the anonymous reviewers for valuable comments. key: cord-297829-aynigoud authors: zhang, li; seale, holly; wu, shuangsheng; yang, peng; zheng, yang; ma, chunna; macintyre, raina; wang, quanyi title: post-pandemic assessment of public knowledge, behavior, and skill on influenza prevention among the general population of beijing, china date: 2014-04-13 journal: int j infect dis doi: 10.1016/j.ijid.2014.01.003 sha: doc_id: 297829 cord_uid: aynigoud background: the aim of this study was to assess the knowledge, behavioral, and skill responses toward influenza in the general population of beijing after pandemic influenza a (h1n1) 2009. methods: a cross-sectional study was conducted in beijing, china, in january 2011. a survey was conducted in which information was collected using a standardized questionnaire. a comprehensive evaluation index system of health literacy related to influenza was built to evaluate the level of health literacy regarding influenza prevention and control among residents in beijing. results: thirteen thousand and fifty-three valid questionnaires were received. the average score for the sum of knowledge, behavior, and skill was 14.12 ± 3.22, and the mean scores for knowledge, behavior, and skill were 4.65 ± 1.20, 7.25 ± 1.94, and 2.21 ± 1.31, respectively. the qualified proportions of these three sections were 23.7%, 11.9%, and 43.4%, respectively, and the total proportion with a qualified level was 6.7%. there were significant differences in health literacy level related to influenza among the different gender, age, educational level, occupational status, and location groups (p < 0.05). there was a significant association between knowledge and behavior (r = 0.084, p < 0.001), and knowledge and skill (r = 0.102, p < 0.001). conclusions: the health literacy level remains low among the general population in beijing and the extent of relativities in knowledge, behavior, and skill about influenza was found to be weak. therefore, improvements are needed in terms of certain aspects, particularly for the elderly and the population of rural districts. educational level, as a significant factor in reducing the spread of influenza, should be considered seriously when intervention strategies are implemented. pandemic influenza a (h1n1) 2009, a new strain of triplereassortant influenza a virus composed of a combination of human, swine, and eurasian avian strains, spread rapidly through more than 200 countries 1 and was the first global pandemic of the 21st century. 2,3 on august 10, 2010 the world health organization (who) declared that we had entered the post-pandemic period and the h1n1 virus had taken on the behavior of a seasonal influenza virus. 4 there was an effective control and prevention campaign during and after the 2009 influenza pandemic in beijing, which included identifying, treating, and isolating people who had the disease and educating the public about the steps that individuals could take to reduce the risk of transmission. meanwhile, health education campaigns touching on good hygiene practices and social distancing were implemented in hospitals, schools, local communities, and through mass media. 5 since the severe acute respiratory syndrome (sars) outbreak in 2003, the government of china has strengthened its surveillance and established the prevention and control system for infectious disease. 6 the level of science and technology in this field in china has since improved significantly. compared with the abundant research on how the government and institutions could improve the surveillance management and prevention system, there have been few public reports assessing the effect of these policies and the level of health literacy associated with influenza prevention in the general population. background: the aim of this study was to assess the knowledge, behavioral, and skill responses toward influenza in the general population of beijing after pandemic influenza a (h1n1) 2009. methods: a cross-sectional study was conducted in beijing, china, in january 2011. a survey was conducted in which information was collected using a standardized questionnaire. a comprehensive evaluation index system of health literacy related to influenza was built to evaluate the level of health literacy regarding influenza prevention and control among residents in beijing. results: thirteen thousand and fifty-three valid questionnaires were received. the average score for the sum of knowledge, behavior, and skill was 14.12 ae 3.22, and the mean scores for knowledge, behavior, and skill were 4.65 ae 1.20, 7.25 ae 1.94, and 2.21 ae 1.31, respectively. the qualified proportions of these three sections were 23.7%, 11.9%, and 43.4%, respectively, and the total proportion with a qualified level was 6.7%. there were significant differences in health literacy level related to influenza among the different gender, age, educational level, occupational status, and location groups (p < 0.05). there was a significant association between knowledge and behavior (r = 0.084, p < 0.001), and knowledge and skill (r = 0.102, p < 0.001). conclusions: the health literacy level remains low among the general population in beijing and the extent of relativities in knowledge, behavior, and skill about influenza was found to be weak. therefore, improvements are needed in terms of certain aspects, particularly for the elderly and the population of rural districts. educational level, as a significant factor in reducing the spread of influenza, should be considered seriously when intervention strategies are implemented. ß 2014 the authors. published by elsevier ltd on behalf of international society for infectious diseases. it has been shown that health education is directed towards improving health literacy 7 and it is expected that this would have a positive effect on influenza prevention and control in the future. there has been no investigation regarding health literacy of influenza prevention in the general population of beijing. after the 2009 pandemic, it was necessary to collect some baseline data to understand and monitor public perceptions and behaviors. we conducted a survey in six districts of beijing, china, in early 2011 to assess the influenza-related health literacy level in the general population of beijing after the 2009 influenza pandemic, and to explore the behavior and skill factors affecting the incidence level of influenza. a cross-sectional study was conducted in beijing, china, in january 2011. subjects were recruited via a multi-stage stratified cluster sampling technique. first, three urban districts and three rural districts were selected randomly from a total of 18 districts in beijing. five sub-districts/towns were then selected randomly in each of the six districts, from which five communities were selected randomly. lastly, 18 subjects for each age group (18-29, 30-39, 40-49, 50-59, and !60 years) were recruited from each community, with equal weighting of the sexes. the standardized interview questionnaire was designed to collect the following data: (1) socio-demographic characteristics (gender, age, education, occupation, and general health status); (2) knowledge about the disease and its symptoms; (3) practices towards influenza and people with influenza-like-illness (i.e., avoidance practices, cough etiquette, use of masks, hand washing, being vaccinated, health-seeking behaviors); (4) perceived ability to avoid illness; (5) attitudes towards the vaccine, and (6) comprehension of health materials related to influenza (i.e., medication instructions, educational information about influenza and the vaccine). lastly, participants were asked to gauge their ability to use a thermometer. questions were divided into three sections under the headings of knowledge, behaviors, and skills. after obtaining informed consent from the subject, the survey was administered by face-to-face interview. for the purpose of analysis, each question that was answered positively was given a score of 1 and each question that was answered negatively or was answered as 'don't know' was given a score of 0. the total score for the three sections was 24 points: the total score for 'knowledge' was 7 points, and a qualified level was considered to be 6-7 points; the total score for 'behavior' was 13 points, and a qualified level was considered to be 10-13 points; the total score for 'skill' was 4 points, and a qualified level was considered to be 3-4 points. questionnaire data were entered in duplicate using epidata software, and data were analyzed using spss 18 statistical software (spss inc., chicago, il, usa). descriptive statistics, such as percentages, means, and standard deviations, were calculated. to analyze the significance of the continuous data, an analysis of variance (anova) was applied. chi-square tests of significance were used for analyses of categorical variables regarding the qualified proportion of the three sections. the relationships among knowledge, behavior, and skill were analyzed by correlation analysis. statistical significance was accepted at p < 0.05 for all analyses. this study was approved by the institutional review board and human research ethics committee of the beijing center for disease prevention and control (cdc). a total of 13 286 adults were approached; 13 053 valid questionnaires were received, giving an effective response rate was 98.1%. the demographic characteristics of participants are reported in table 1 . the average score for the sum of knowledge, behavior, and skill was 14.12 ae 3.22, and the mean scores for knowledge, behavior, and skill were 4.65 ae 1.20, 7.25 ae 1.94, and 2.21 ae 1.31, respectively. the statistic of the total score of these three sections was found to follow an approximately normal distribution. the overall mean score for knowledge was 4.65 ae 1.20, and 23.7% of participants met the qualified standard of knowledge. both the overall knowledge score and the qualified proportion for knowledge were significantly higher in urban areas compared to rural areas (f = 63.968, p < 0.001; chi-square = 12.701, p < 0.001). the mean knowledge score fell significantly with increasing age (f = 31.064, p < 0.001) and increased significantly with higher educational levels (f = 158.175, p < 0.001) ( table 2 ). the qualified proportion in the different age groups fell significantly with increasing age (chi-square = 20.991, p < 0.001) and increased significantly with higher educational levels (chi-square = 92.145, p < 0.001) ( table 3) . males had a significantly higher mean score for behavior than females (f = 92.904, p < 0.001). the mean score of urban residents was significantly higher than that of rural residents (f = 54.786, p < 0.001). the mean score in the different age groups fell significantly with increasing age (f = 5.965, p < 0.001) and there was a significant rise with the increase in educational level (f = 99.843, p < 0.001) ( table 2) . of the participants, 11.9% met the qualified standard of behavior. males had a significantly higher qualified proportion table 3 the qualified proportions for knowledge, behavior, and skill about influenza among respondents group of behavior than females (chi-square = 13.066, p < 0.001) and there was a significant rise with increasing educational levels (chisquare = 61.584, p < 0.001) ( table 3 ). urban residents had a significantly higher mean score for skill than rural residents (f = 428.286, p < 0.001). the mean score for the different age groups fell significantly with increasing age (f = 217.352, p < 0.001) and increased significantly with higher educational levels (f = 770.232, p < 0.001). there was a significant difference among the three occupational status levels (f = 21.669, p < 0.001), with the group of students having the highest mean score (2.57 ae 1.27) and the non-working group having the lowest mean score (2.14 ae 1.30) ( table 2) . of the participants, 43.4% met the qualified standard of skill. the qualified proportion of skill in urban residents was significantly higher than in rural residents (chi-square = 125.588, p < 0.001). the qualified proportion in the different age groups fell significantly with increasing age (chi-square = 477.533, p < 0.001) and increased significantly with higher educational levels (chi-square = 1322.136, p < 0.001). there was a significant difference among the three occupational status levels (chisquare = 31.165, p < 0.001), with the group of students having the highest qualified proportion (53.9%) and the non-working group having the lowest qualified proportion (40.6%) ( table 3) . there were positive correlations between knowledge and behavior, and knowledge and skill, which were statistically significant (p < 0.001). however, the extent was weak, as the correlation coefficients were r = 0.084 and r = 0.102, respectively (table 4 ). in recent years, pandemic influenza, as a global public health problem, has caused worldwide concerns. 8, 9 many previous studies have shown that the risk of seasonal or pandemic influenza infection depends on biological characteristics, individual or collective behaviors, and the environmental context. 10 research has been done on the knowledge, attitudes, and practices (kap) related to pandemic influenza a (h1n1) 2009 among the chinese general population, 11 but a related study has not been reported from beijing. this study could provide some important information to fill the gaps in this field. it was necessary and valuable for us to conduct the study to determine the overall level of influenzarelated health literacy in the general population of beijing after the 2009 pandemic, data that provide a baseline for influenza prevention and control strategies in the future. furthermore, our assessment may help shape policy and provide information to the international community. in this study we found that the qualified proportion of urban residents was significantly higher than that of rural residents; this was considered to be associated with socio-economic factors, such as income and medical resources allocation, 12,13 as well as the ability to access health information. the socio-economic status in rural areas is significantly lower than in urban areas; rural residents are always less likely to obtain the recommended preventive healthcare services, 14 and their limited ability to acquire information via modern media systems 15 impedes the dissemination of health information among rural dwellers. in addition, most public education activities are currently carried out in the communities of urban districts, hence the public awareness of influenza-related knowledge, behavior, and skill of rural residents is lower than that of urban residents. in this study, education was found to be the most important factor influencing levels of infectious disease health literacy, and past research on the relationship between education and health has drawn similar conclusions. howard et al 16 found that if health literacy levels were similar, differences in self-reported health status by education would be about 20% lower. there is also some indirect evidence. goldman and smith 17 found that well-educated patients are better able to manage complicated self-care regimens in hiv/aids and diabetes. other studies have found that education is linked to faster adoption of new medical technologies 18 and that consumer knowledge is linked to the increased use of preventive care. 19 in this study, the qualified levels of all three sections (knowledge, behavior, and skill) in the general population were significantly higher (p < 0.001) with a higher level of education, which is similar to the nationwide health literacy level of china. 20, 21 at present, different intervention strategies aimed at populations with different levels of education should be implemented. compared with younger people, the older age groups had worse health literacy related to influenza. the qualified proportion in the older age group was significantly lower than that in the younger group. older adults have lower immunity and ability to fight off disease and are at higher risk of becoming infected with influenza viruses. the risk of influenza-related complications and deaths among the elderly are significantly higher than in younger people. 22 the results of beijing's sixth population census showed the proportion of elderly (!60 years) to be 12.5%, 23 demonstrating that beijing has already become an aging society. the statistics in this study indicated that the influenza-related health literacy level among elderly residents in beijing was low. as a high-risk and susceptible population, the elderly should be paid more attention with regard to influenza prevention and control. there were positive correlations among knowledge, behavior, and skill about influenza, but the extent was weak. this indicates that there is still a gap in knowledge, behavior, and skill. full knowledge about the prevention of influenza does not mean reasonable behavior or skill. it is necessary to carry out various types of health education program aimed at behavior and skill. the study has a few limitations. first, some data were selfreported, which could have led to problems of recall bias. second, this study was a sampling survey, which will inevitably have had a sampling bias. however, because the participants were selected from communities by strict random sampling, it is believed that they do represent the general population of beijing. thirdly, there were no baseline data for influenza-related health literacy in the general population of beijing for the pre-pandemic period, so we cannot know whether the health literacy level of people was improved after in recent years, pandemic influenza, as a global public health problem, has caused worldwide concerns. 8, 9 many previous studies have shown that the risk of seasonal or pandemic influenza infection depends on biological characteristics, individual or collective behaviors and the environmental context. 10 as there has been no related study reported in beijing, this study could provide some important information to fill gaps in this field. it was necessary and valuable for us to conduct the study to determine the overall level of influenza-related health literacy in the general population of beijing after the 2009 pandemic, data that provide a baseline for influenza prevention and control strategies in the future. furthermore, our assessment may help shape policy and provide information to the international community. in conclusion, following the h1n1 2009 pandemic, the general population of beijing has some correct knowledge, practices, and skills related to influenza, however this health literacy level is low and the extent of relativities in knowledge, behavior, and skill about influenza was found to be weak. improvements are needed in terms of certain aspects, particularly for the elderly and the population of rural districts. educational level, as a significant factor in reducing the spread of influenza, should be considered seriously when intervention strategies are implemented, and we should provide more individual health counseling and education services for residents. world health organization. global alert and response (gar): pandemic (h1n1) 2009-update 112. geneva: who pandemic potential of a strain of influenza a (h1n1): early findings the first pandemic of the 21st century: a review of the 2009 pandemic variant influenza a (h1n1) virus world health organization. h1n1 in post-pandemic period. geneva: who response to the first wave of pandemic (h1n1) 2009: experiences and lessons learnt from china china's engagement with global health diplomacy: was sars a watershed health literacy as a public health goal: a challenge for contemporary health education and communication strategies into the 21st century pandemic influenza preparedness in the asia-pacific region wide spread public misconception in the early phase of the h1n1 influenza epidemic integrative study of pandemic a/h1n1 influenza infections: design and methods of the copanflu-france cohort knowledge, attitudes and practices (kap) related to the pandemic (h1n1) 2009 among chinese general population: a telephone survey an exploratory spatial analysis of pneumonia and influenza hospitalizations in ontario by age and gender equity and health sector reforms: can low-income countries escape the medical poverty trap? are rural residents less likely to obtain recommended preventive healthcare services? the construction and development status analysis on rural informationization in beijing impact of health literacy on socioeconomic and racial differences in health in an elderly population can patient self-management help explain the ses health gradient? the effect of education on medical technology adoption: are the more educated more likely to use new drugs? the role of consumer knowledge of insurance benefits in the demand for preventive health care among the elderly ministry of health of the people's republic of china. 2009, the first time chinese residents health literacy survey. beijing: ministry of health china association for science and technology. a survey of public scientific literacy in china. beijing: china association for science and technology geneva: who the sixth national population census this study was supported by grants from the beijing nova program of beijing science and technology commission conflict of interest: the authors declare that no conflict of interest exist. key: cord-273907-58jufmx7 authors: shen, kun-ling; namazova-baranova, leyla; yang, yong-hong; wong, gary wing kin; rosenwasser, lanny j.; rodewald, lance e.; goh, anne eng neo; kerem, eitan; o’callaghan, chris; kinane, t. bernard; elnazir, basil; triasih, rina; horne, rosemary; chang, anne b.; buttery, jim; etzel, ruth a.; ouchi, kazunobu; hoey, hilary; singh, varinder; rivera, genesis c.; li, spencer s.; guan, yu; cao, ling; zheng, yue-jie; feng, lu-zhao; zhong, wu; xie, zheng-de; xu, bao-ping; lin, rong-jun; lu, gen; qin, qiang; zhu, chun-mei; qian, su-yun; liu, gang; zhao, cheng-song; wei, zhuang; zhao, yu-hong title: global pediatric pulmonology alliance recommendation to strengthen prevention of pediatric seasonal influenza under covid-19 pandemic date: 2020-09-13 journal: world j pediatr doi: 10.1007/s12519-020-00389-7 sha: doc_id: 273907 cord_uid: 58jufmx7 nan the world health organization (who) announced covid-19 as a global pandemic in march of 2020 [1] . the covid-19 pandemic may persist for a long period of time. global prevention and control becomes a complex and challenging task, and such efforts should be sustained. although general measures, such as social distancing, face masks, respiratory hygiene and hand sanitization, will bear fruits for decreasing spread of other respiratory illnesses including influenza, the specific prevention through vaccination is a key focus especially in the upcoming winter and spring seasons [2] . winter and spring are the peak seasons for influenza and other common respiratory infectious diseases. children are a high-risk group for influenza. the prevalence of influenza in children is 20-30% every year [3] , and it often enhances outbreaks in the community. the pediatric population in child care centers, kindergartens and primary schools is closely confined and is vulnerable to mass outbreaks [4] . the high infection rate of influenza in children is also one of the important reasons for the spread of influenza virus in the community [5] . if an outbreak of influenza coincides with that of covid-19, the impact on the pediatric population will be even more severe [6] . according to who, the covid-19 outbreak in early 2020 occurred at the same time as the influenza outbreak [7] , and the primary clinical manifestations of influenza and covid-19 overlap considerably. an epidemic of influenza would challenge the diagnosis and treatment in fever clinics and the supplies of personal protective equipment (ppe). therefore, the upcoming winter and spring seasons are of high alert [8] , as immense pressure will be placed on hospitals' overall diagnosis and treatment capacity and on their critical care capacity. co-infection of influenza and covid-19 can occur in patients and present with severe symptoms. this will further increase the complexity of clinical diagnosis and treatment [6] . hospitals usually have concentrated patient flows, and the risk of cross infection is increased, which threatens the health of all patients, their caregivers, and medical staff members. the who, the united nations international children's emergency fund (unicef), and various nations have called for strengthening of prevention and control of seasonal influenza while making every effort to cope with the covid-19 pandemic. these organizations vigorously advocate influenza vaccination as the primary measure for pandemic prevention and control, so as to minimize the interference of an influenza epidemic during the prevention and control of the covid-19 pandemic situation. according to the who recommendation, influenza vaccination is an effective supplementary measure against the covid-19 pandemic for countries that are about to enter the winter. all eligible individuals should receive the influenza vaccine [2] . unicef calls for influenza vaccination for children during the covid-19 pandemic to protect them from other diseases [9] . the us centers for disease control (cdc) stated that in the coming winter influenza and covid-19 may spread simultaneously and recommended vaccination for all children older than 6 months [10] . the australian government strongly recommends that people, especially children and other high-risk groups, should be vaccinated with influenza vaccine when actively coping with the possible overlap of covid-19 and influenza [11] . the british public health agency also stated that owing to the covid-19 outbreak, influenza vaccination is more important than ever before [12] . new zealand called for vaccination for high-risk target populations [13] (such as children, seniors and pregnant women) to protect communities and to utilize resources most effectively. brazil initiated early vaccination to reduce influenza cases in the midst of covid-19, with children from 6 months to 6 years of age being the priority target population [14] . thus, preschool-age and younger children are the key vaccination population, and influenza vaccination is the best preventive measure. to control the pandemic situation more effectively, the global pediatric pulmonology alliance (gppa), based on best-practices and evidence from various countries, recommends that all global and regional organizations, medical societies, and health agencies join hands in the prevention of seasonal influenza in children. measures to improve influenza vaccination include promoting public awareness about infection control measures and thorough education of the benefit of influenza vaccination. the ultimate goal is to achieve early notification, early appointment and early vaccination, which will reduce the likelihood of coincident epidemics of influenza and covid-19 this winter. it is recommended that vaccination policy, vaccine safety, and medical advice be promoted and reiterated in child care centers, kindergartens, and schools [15] . countries and regions with ample resources should implement free influenza vaccination in child care centers, kindergartens and schools, so as to increase the coverage rate of influenza vaccination among the pediatric population [16] . it is also advised that in countries or regions, where influenza vaccination may not be part of national immunization program (nip), pediatricians should check with respective health authorities or local guidelines to reach the suggested beneficiary population. (1) infants under 6 months influenza vaccine cannot be given to infants under 6 months. we recommend parents, caregivers, and family members of the infants to be vaccinated [4] , and if included in national recommendations, expectant mothers should receive influenza vaccination during pregnancy to protect themselves and protect newborns from influenza infection during their first six month of life. (2) children over 6 months and school-age children this is the priority population for influenza vaccination. all children over 6 months of age are eligible, except for those who are allergic to vaccine substances/components or who are experiencing fever or an acute infection. children with a history of egg allergy of any severity can receive any licensed, recommended, and age-appropriate influenza vaccine. children with stable chronic pulmonary, cardiovascular, renal or neurological diseases should get the influenza vaccine as early as possible [4] . children with primary immunodeficiency, hiv infection and secondary immunodeficiency (leukemia, carcinoma, receiving immunosuppressive therapy, etc.) should consult with medical professionals before using live attenuated influenza vaccines. staff should be vaccinated every year [17] . schools and kindergartens should carry out awareness and education campaigns on prevention and control of infectious diseases and on promotion of vaccination to students and parents, so as to improve vaccination rates and to prevent the outbreak of clustering epidemic [17, 18] . family members of children and frequent visitors (such as caregivers, housekeeping workers, etc.) also should be vaccinated annually [17] . pediatric medical staff and workers are in frequent and close contact with children. they should be vaccinated every year. this should also be considered for other staff groups, such as hospital cleaners and porters. this protects the pediatric health care workers, maintains health care capacity by reducing sick leave, and also helps to reduce transmission from members of staff to children and others, thereby reducing the risk of hospital-acquired infections. it is a concern that shedding of influenza virus has been shown shortly before clinical illness develops and that health care workers frequently continue to work while unwell. influenza vaccine needs to be given every year. countries and regions around the world should prepare ample vaccine supplies and should complete vaccination of the pediatric population about 2-3 months before the arrival of the influenza season [4] . although the gppa is advocating vaccinations (as described above), public health priorities may differ across nations; therefore, pediatricians should check their national guidelines for the eligible population, available vaccine products and the number of doses to administer based on age, vaccination history, and vaccine product to make a final call. the inactivated vaccine is to be injected intramuscularly (except intradermal injection preparation). children over 1 year and adults should be vaccinated in the deltoid muscle of the upper arm as the first choice. the anterolateral thigh is the best vaccination site for infants between 6 months and 1 year. patients with thrombocytopenia or other hemorrhagic diseases may have the risk of bleeding during intramuscular injection; so subcutaneous injection is recommended. injection dosage should be referenced to the product specification. (1) parents should make an appointment before vaccination and arrive at the vaccination site on time, so as to avoid crowding at the site. in conclusion, under the covid-19 pandemic, getting the influenza vaccine in time offers children a safer winter and a healthier future. world health organization. who director-general's opening remarks at the media briefing on covid-19 world health organization. who director-general's opening remarks at the media briefing on covid-19 vaccines against influenza who position paper influenza vaccination twg. technical guidelines for seasonal influenza vaccination in china feasibility of social distancing practices in us schools to reduce influenza transmission during a pandemic co-infection with sars-cov-2 and influenza a virus in patient with pneumonia influenza laboratory surveillance information by the global influenza surveillance and response system (gisrs) recommended composition of influenza virus vaccines for use in the 2020-2021 northern hemisphere influenza season united nations international children's emergency fund. vaccinations and covid-19: what parents need to know frequently asked influenza (flu) questions: 2020-2021 season influenza vaccination the national flu immunisation programme 2020/21 the official website of the new zealand government. govt begins largest ever flu vaccine campaign early. 2020 comeca-segunda-feiravacinacao-contra-gripe cdna national guidelines for public health units:seasonal influenza infection nontraditional uses of live attenuated influenza vaccine: school-located influenza vaccination guidelines for the prevention, control and public health management of influenza outbreaks in residential care facilities in australia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations author contributions feng lz, zhong w and xie zd contributed to data curation; all the other authors wrote a part of original draft. key: cord-305936-tdswzj7r authors: freitas, andré ricardo ribas; donalisio, maria rita title: excess of mortality in adults and elderly and circulation of subtypes of influenza virus in southern brazil date: 2018-01-08 journal: front immunol doi: 10.3389/fimmu.2017.01903 sha: doc_id: 305936 cord_uid: tdswzj7r purpose: in the elderly population, the influenza infection and its clinical complications are important causes of hospitalization and death, particularly, in longer-lived age. the objective of this study is to analyze the impact of influenza virus circulation on mortality in the elderly and adults, in years with different predominant virus strains. methods: we performed a time trend study to evaluated excess of mortality for pneumonia and influenza, respiratory disease, and all-causes in southern region of brazil, from 2002 to 2015. after considering other models, we opted for serfling regression. excess of death rates per 100,000 inhabitants were analyzed in specific age groups (24–59, 60–69, 70–79, ≥80 years) and by year of occurrence. mortality information were taken from brazilian mortality information system and etiological data were accessed in sentinel virological surveillance database, getting the weekly positivity of the immunofluorescence tests for influenza a (h1n1, h3n2), and b. results: in southern brazil, there is an evident seasonal pattern of all death outcomes among different age groups in the dry and cold season (april–september). the highest excess mortality rates occurs among older, particularly in years of circulation of influenza ah3n2, especially among people ≥80 years, in 2003 and 2007—years of great severity of influenza activity. after 2009, with the introduction of the pandemic influenza ah1n1, we observed a lower impact on the mortality of the elderly compared to <60 years. discussion: a cross reactivity antibody response from past exposure probably provided protection against disease in the elderly. despite not controlling for comorbidities, climate, and vaccination, for the >70 years, ratio of respiratory diseases excess mortality rates between ah1n1 (2009) and severe year of h3n2 (2007) shows protection in the pandemic year and great vulnerability during ah3n2 virus predominance. conclusion: the reduced immune response to infection, and to vaccination, and presence of comorbidities recommend a special attention to this age group in brazil. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases are some of the preventive and public health measures. introduction human influenza viruses can cause diseases through many direct and indirect pathological effects. consequences are destruction of infected cells, release of cytokines leading to fever, malaise, damage to respiratory epithelium and pulmonary parenchyma, and pneumonia. it includes secondary bacterial infections because of tissue damage and exacerbation of preexisting comorbidities such as cardiovascular and renal diseases, diabetes, or chronic lung disease (1) (2) (3) . the rates of hospitalization and mortality associated with influenza are higher among patients with chronic diseases, children under 1 year and after 65 years of age (4, 5) . with the aging population in recent decades, the raw number of hospitalizations and deaths related to pneumonia and influenza tends to increase (4) , this phenomenon has been observed also in brazil (6, 7) . however, the impact and severity of influenza virus circulation depend in part, on the strain that predominates in the season each year. due to the lack of laboratory confirmation, influenza-associated morbidity and mortality are often classified as pneumonia, other respiratory diseases, or other causes. given the difficulty of directly measuring influenza morbidity and mortality, time series models are used to elucidate disease patterns in various age groups. trends are usually determined by means of statistical inference, based on seasonal coincidence of the occurrence of certain diseases or death and laboratory confirmation of the viral circulation (4, 8) . different approaches, with and without the quantification of the proportion of viral isolates, can produce average estimates of excess deaths associated with the circulation of certain viral variants (9) (10) (11) . viral surveillance data, hospitalization, or death indicators are particularly useful for the study of influenza in the tropics, as seasonality may be less evident (11) (12) (13) . serfling regression has been used to analyze excess of mortality related with respiratory virus circulation (7, (14) (15) (16) . despite some limitations (17) , the inclusion of sinusoidal terms in weekly regression may reduce spurious correlation between influenza occurrence and death (18, 19) . it is particularly useful when no other covariables are available, and with small samples of viral sentinel surveillance data (18) . poisson regression and the generalized linear model (glm) can produce more specific estimates and support adjustments for variables (temperature, humidity, comorbidities, other circulations of viruses), although they require a more robust and consistent virological surveillance and cannot be used for pandemics (4) . in brazil, surveillance for influenza syndromes was implemented in 2000, monitoring the occurrence of respiratory viruses (influenza a and b, parainfluenza 1, 2, and 3, respiratory syncytial virus, adenovirus). the brazilian ministry of health provides vaccination coverage annually since 1999 for seniors and some risk groups, with vaccine coverage of the elderly population at around 80% in southern brazil, the region with the highest coverage of the country. despite the adequate coverage, protective titers after vaccination (hi ≥ 0) are consistently lower with poorer cell mediate and antibody responses in the elderly comparing to adults (20) . considering the vulnerability of the elderly to influenza virus infection, and the lack of studies on its repercussion in brazil, the objective of this study was to analyze the impact of different strains of influenza a virus circulation. we analyzed particularly the most predominant variants (ah1n1 and ah3n2) on excess of mortality in the adults and elderly of different age groups in a region with marked seasonality of respiratory diseases in brazil. this is a time trend study to evaluated excess of mortality from 2002 to 2015 in southern region of brazil (states of paraná, santa catarina e rio grande do sul), total area is 576,774,31 km 2 , population is 27,386,891 inhabitants with subtropical climate (köppen-geiger classification cfa). we choose these states for analysis because of the consistent seasonal pattern of influenza, as well as the availability and quality of etiological data from the virological surveillance system in that region. for the mortality rates of specific age groups (24-59, 60-69, 70-79, and ≥80 years) and death causes, we took data from brazilian mortality information system. causes are classified according to international causes of death icd-10 revision, pneumonia, and influenza (icd j 10 to j18.9), respiratory diseases (icd j00 to j99), and all-cause (excluding external causes of mortality). we obtained population of each year and age group from instituto brasileiro de geografia e estatística-ibge from the census-2010, and population estimates for the following years. etiologic information of flu-like syndrome was accessed in database of the national sentinel virological surveillance system. it has data from 128 sentinel units distributed in all regions of the country-north (21 units), northeast (26 units), southeast (34 units), south (38 units), and central west (9 units). surveillance is performed through the systematic collection of weekly samples of nasopharyngeal secretions from patients who present flu-like syndrome. reference laboratories process samples by using indirect immunofluorescence (iif), with tests for influenza a and b, parainfluenza 1, 2, and 3, respiratory syncytial virus, and adenovirus. a portion of the samples is submitted to polymerase chain reaction tests to identify the virus genotype. we calculated the laboratory positivity indicator using weekly positive results of iif divided by the total of weekly valid tests, i.e., excluding the results within inadequate samples (not enough biological material, improper storage, incorrect material in the sample) or inconclusive results (no valid results). influenza vaccination coverage (%) of southern region from 2002 to 2015 was obtained from brazilian national program of immunization data base (datasus). the criteria used to define the period of increase of influenza activity was when the positivity of the samples tested exceeded twice the annual mean of the weekly positivity of samples processed by surveillance, during two consecutive weeks. in the year 2009, we consider the period officially recognized by the brazilian ministry of health as epidemic by the influenza ah1n1pmd2009 strain, due to irregularity of the sample collection by the sentinel surveillance system at the end of epidemic. we calculated the weekly mortality rates by age group using the number of deaths per group of causes divided by the estimated population in the middle of the year multiplied by 100,000. we constructed a serfling cyclical regression model (14) for weekly data applied to each age group and causes of death (pneumonia and influenza, respiratory diseases, and all causes), as seen in others studies (7, 15) , to estimate baseline of predicted deaths in the absence of influenza epidemics. to fit regression, we used period of 13 years (from 2002 to 2015), excluding the weeks of epidemics periods. a cyclical linear regression was adjusted with the equation: where y is the mortality rate, β is the coefficients of regression, t is time in weeks, and t 2 and t 3 are variables for adjusting the secular trend of the disease. we used of sine and cosine for adjust of annual and semiannual periodic components. after adjusting a linear regression and define the expected mortality rate, we delimited 95% upper confidence limit of the baseline as the reference threshold in the absence of influenza epidemics. we calculated the excess of deaths as the observed mortality minus the expected mortality in the periods when mortality was above 95% of the confidence interval during epidemics periods. we also present ratios of excess mortality rates among years of predominant circulation of influenza strains ah3n2 (mean and years of severity), ah1n1 pre-pandemic, and ah1n1 postpandemic for each age group. for data compilation, we used microsoft office excel 2007, and for statistical analysis, spss for windows, version 24.0. results table 1 shows the proportion of positivity of the iif nasopharyngeal samples and the annual prevalence of strains of influenza in the period. before 2009, the year of entry of the pandemic strain ah1n1pmd 2009, there was a predominance of influenza ah3n2 in the years 2003 to 2007. after 2009, there is alternation of strains in the southern brazil. annual elderly vaccination coverage in southern region is high and homogeneous, around 80%, and even higher in the recent years. there is an evident seasonal pattern of deaths from pneumonia and influenza, respiratory diseases, and all-causes among the elderly in different age groups in the dry, cold months (april-september) in southern region (figure 1) . we note a progressive increase in the rates of excess deaths (of all outcomes) with increasing age, especially among those older than 70 years. in the pre-pandemic years with dominance of the ah1n1 strain, the excess of mortality rates associated with influenza were relatively low, compared to years of prevalence of ah3n2 strain ( table 2) . among those over 80 years, the ratio of excess mortality rates between 2009 and the years with dominium of h3 strains was less than one. this ratio suggests that this age group was spared in the 2009 pandemic. however, in years of predominance of strain h3, excess of mortality rate of all causes in this group were 449.6 per 100,000 (corresponding to 1,598 obits), 5, and 8.2 times greater than the same rate in years of circulation of h1n1 in pre-and post-pandemic period, respectively. among adults (24-59 years), we observe a large excess of deaths rates during the 2009 pandemic (953 obits), which correspond to 7.1 excess deaths from all causes, and 99 excess mortality from respiratory diseases associated with viral infection in every 100,000 individuals of the age group. the ratio between excess mortality rates due to pneumonia/influenza in the pandemic year (2009) and the mean rate of the period was 12 times higher among the youngest ( table 2) . rates of excess mortality by pneumonia and influenza and respiratory diseases are lower than all causes in all age groups, but particularly high in older than 80 years ( table 2) . the results highlight the great vulnerability of elderly to influenza ah3n2, especially among older than 70 years in severe years of influenza activity, like 2003 and 2007. the study also shows the lower impact of influenza ah1n1pdm 2009 in this age group compared to younger. risk of dying among the elderly in years of circulating ah3n2 influenza has been reported in several parts of the world (9, 10, 21, 22) ; however, in brazil, there are no recent estimates available. few studies analyze the circulation and impact of influenza in tropical and subtropical regions (6, 7, (9) (10) (11) . influenza b virus is also associated with severe disease (23); however, this variant did not circulate with intensity during the study years in brazil. although the elderly are the most vulnerable group to viral respiratory infections, we found relative small excess of deaths in years of circulating ah1n1 pre pandemic (2002 and 2008) . study comparing excess deaths from respiratory diseases in the elderly in latin america shows stable rates (mean of 89.4 per 100,000 inhabitants) in southern brazil between 1998 and 2008 (prepandemic flu a-h1n1), although higher in brazil than in other countries (24) . in the usa and in european countries, influenza seasons dominated by subtype ah3n2 are typically associated with mortality two to three times higher than in seasons with predominance of ah1n1 (prior to pandemic strain 2009) and of influenza b viruses (9, 10, 19, 25) . when all causes of death are studied, the overall mortality associated with influenza among elderly exceeds that observed in younger age group. it should be considered that all causes mortality is a non-specific measure and a distant outcome of influenza infection. however, it is difficult to determine which group of causes of death could better characterize the influenza burden in mortality. by choosing only the respiratory causes, we may underestimate clinical complications of pulmonary viral infection (e.g., cardiovascular). therefore, in this study, we analyzed all causes, respiratory, and pneumonia and influenza deaths. the unfavorable evolution of infection in the elderly is possibly due to the prevalence of comorbidities, deficiencies in defense mechanisms, and poor antibody response to vaccination, as cellmediated and humoral responses limit severity of disease (26) . patients with chronic diseases are more susceptible to infection due to decline of the immune function through inflammatory mechanisms, hindering the mucosal barrier, and the adaptive and innate immunological defense mechanisms (27) . the immune response to infection in the elderly tend to be delayed and weak, with prolonged inflammatory responses, which involves different types of host reaction, mainly to clearance virus. the exacerbations of these mechanisms may induce immunemediated pathology causing tissue damage (28) . cytokine high serum levels of il-6, tnf-a, ifn-g and sil-2r, chemokines ip-10, mcp-1, and monokine induced by ifn-g (mig), are associated with severe clinical cases and lung damage (29) . immunological abnormalities in people with diabetes, chronic respiratory diseases, cardiopathy, or other chronic diseases have increased risk of severe infection and bad prognosis (19) . for example, there is the consistent association of influenza infection with cardiovascular mortality, particularly acute myocardial infarction (30) . in part, it is attributed to altering endothelial function due to an acute inflammatory and procoagulant stimulus during viral infection (31, 32) . clinical complications of diabetes triggered by influenza infection cause impairment of leukocyte function and increase post-infection colonization rates resulting in poor prognosis in the elderly (33, 34) . in young people and adults, in 2009, the emerging influenza ah1n1 strain had a notable impact on the mortality of people up to 59 years in various parts of the world, including brazil (7, 25, 35, 36) . excess mortality of individuals aged 24-59 years in the state of são paulo, brazil was identified during the pandemic ah1n1 virus (7) . pregnant women adults with metabolic conditions, including obesity, chronic respiratory disease, and other chronic diseases were significantly associated with severe acute respiratory syndrome and the lethality in brazil (37) . our study showed a 41.5-fold higher rate of mortality from pneumonia and influenza in adults (24-59 years) in the pandemic year ah1n1 than the average of years with predominance of ah3n2 circulation in southern region. in addition to the clinical severity and the large portion of the affected population, pandemics affect age groups in different ways (38) . while only 10% of deaths from seasonal influenza occur among those under 65 years of age, in the pandemics of 1918, 1957-1958, and 1968 , this proportion was 95, 40, and 50%, respectively (39) . therefore, pandemics tend to affect a larger proportion of young people than seasonal influenza. in this study, higher rates of death due to pneumonia, influenza, respiratory, and all causes were observed among those aged 24-59 years in 2009. one explanation for the higher mortality observed among the youngest is that they would be more prone to the situation known as "cytokine storm, " i.e., a dysfunctional overproduction of cytokines that would lead to diffuse damage to the respiratory tract with severe and potentially lethal systemic repercussions (40) . viral replication and production of inflammatory mediators seem to be involved in the pathogenesis of infection with influenza a h1n1pmd2009, hindering the clearance of virus in lung tissue and leading to pathologic lesions (41) . another explanation for the lower mortality in the elderly is that they were exposed previously to antigens of the pandemic virus. hancock et al. (42) suggested a cross-reactive antibody response to 2009 pandemic ah1n1. similarities between ah1n1 antigen from 2009 and 1918 were detected. this last virus strain has not circulated since 1958 (39) , when the ah1n1 strain was displaced by ah2n2 (asian flu). at that time, ah1n1viral circulation occurred mainly in children, the current elderly of 2009. the emergence of the ah3n2 strain in the pandemic year 1968 (hong kong flu) affected several age groups. this new strain resulted from a large genetic mutation (shift) recombining virus material of the circulating ah2n2 with the avian h3, of asian origin, resulting in the new variant ah3n2 (38) . in 2002-2003, under selective pressure an antigenic small mutation (drift), resulted in a/fujian/411/02(h3n2) a strains emerged after a "jump" in genes evolution of hemagglutinin and neuraminidase proteins of virus surface (43, 44) . the circulation of the fujian strain had a great impact on the mortality from pneumonia in several parts of the world in 2003-2004 and 2004-2005 (22) and in brazil (45) . in 2007, a new drift resulted in influenza ah3n2 detected in south brazil (46) also affecting hospitalizations and deaths in various parts of the world (47) . we observed high rates of excess mortality in the elderly, in the years of 2003 and 2007. limitations of this study refer mainly to the ecological analysis of pooled data. we did not analyze individual information regarding comorbidities and history of vaccination that could be important confounders influencing mortality (17) . we just had the overall annual vaccination coverage which were in general, around 80% in the period. estimates of the number of deaths (all causes, respiratory, and pneumonia-influenza) supposedly related to influenza may be inaccurate in inferring the impact of respiratory viruses. correlations in time series studies may produce spurious associations, especially between all causes of death and influenza infection, due to the distance between cause and outcome, and to multiple components of the obits. serfling addresses part of this limitation by introducing sinusoidal terms in equation, since non-influenza mortality is not expected to coincide exactly with sinusoidal pattern (14, 19) . moreover, excess mortality of pneumonia, respiratory diseases, and all causes can be considered as an alert to surveillance of viral respiratory diseases, such as a sentinel indicator to be investigated (4, 48) . although all causes mortality is a non-specific indicator, it does not underestimate the complications of chronic diseases associated with influenza (4). despite the influenza component in all causes mortality is small, the indicator can be considered an indirect measure, a warning, useful in epidemiological monitoring. another limitation is the lack of robust etiologic data from virological surveillance in the years 2002-2012, which could lead to imprecision in the analyses; however, the data on the predominance strains in the southern region are reliable, and influenced the composition of the vaccine of each season. considering the option for the analysis model, serfling linear regression may produce different estimates when compared with other models (poisson, arima, and glm) (9, 10); poisson and arima models produce higher mortality estimates than serfling, and serfling higher than glm, especially among the elderly (16, 17, 21) . we chose serfling model because we do not have robust virological surveillance data, before 2013, and the study period includes a pandemic year (4) . besides, in this study, we did not analyze climatic variables (minimum temperatures and relative air humidity) that could also interfere with viral transmission and increase the impact of the disease, particularly in the elderly. in conclusion, probably previous exposures to influenza ah1n1 in the past influenced the mortality of brazilian elderly in 2009, despite the vulnerability of this age group to clinical complications. for the >70 years, we observe higher excess mortality rates (of all outcomes) in severe year of ah3n2 circulation (2003, 2007) . it is also worth noting that vaccination has been associated with the prevention of death particularly at age 65 (49) . therefore, the high elderly vaccination cover in southern brazil may have attenuated excess of mortality estimated, although the immune response is limited among those. more attention should be given to the circulation of influenza ah3n2 in subtropical regions in brazil. the reduced immune response to infection and to vaccination, and associated comorbidities recommend a special attention to this age group. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases in the region are some of the preventive and public health measures. both authors made contributions to the conception of the work, acquisition, analysis, interpretation of data, and writing the manuscript. center for 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for cardiovascular disease prevention multiple immunological abnormalities in patients with type 1 (insulin dependent) diabetes mellitus use of influenza and pneumococcal vaccines in people with diabetes mortality attributable to influenza in england and wales prior to, during and after the 2009 pandemic risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis pandemic h1n1 influenza in brazil: analysis of the first 34,506 notified cases of influenza-like illness with severe acute respiratory infection (sari) pandemic versus epidemic influenza mortality: a pattern of changing age distribution epidemiology of influenza and its control aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza a (h1n1): role of the host immune response in pathogenesis crossreactive antibody responses to the 2009 pandemic h1n1 influenza virus the evolution of human influenza a viruses from 1999 to 2006: a complete genome study molecular evolution of human influenza a/h3n2 virus in asia and europe from 2001 to molecular characterization of influenza viruses collected from young children in uberlandia, brazil-from virus influenza detectados no estado do rio grande do sul durante center for disease control and prevention. influenza activity -united states and worldwide, 2007-08 season the impact of influenza epidemics on mortality: introducing a severity index deaths averted by influenza vaccination in the us during the seasons 2005/06 through 2013/14 authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-300311-eah49b3g authors: bueving, herman j.; van der wouden, johannes c. title: what is the role of virus vaccination in patients with asthma? date: 2007-05-30 journal: curr allergy asthma rep doi: 10.1007/s11882-007-0033-z sha: doc_id: 300311 cord_uid: eah49b3g it is estimated that viruses play a role in 30% to 80% of asthma exacerbations. thus, virus vaccination in patients with asthma could play an important role in preventing asthma exacerbations and other complications. influenza is the only agent for which a routine vaccine is currently available. this article discusses whether influenza vaccination in patients with asthma, based on the available evidence, is justified. cost-effectiveness of (influenza) vaccination for patients with asthma is questionable. for the other major viruses involved, the present state of affairs is described. although progress is being made, a vaccine may be available in the near future only for respiratory syncytial virus (rsv). meanwhile, clinicians and patients should aim for an optimal treatment with the currently available asthma medication. worldwide, asthma is one of the major chronic diseases and there is evidence its incidence and prevalence is still growing [1] . today asthma is regarded as a chronic inflammatory disease of the airways instead of solely a reversible airway obstruction. the use of rescue and anti-inflammatory medication has altered the prospects of asthma patients and has improved their quality of life. thus, most asthma patients can lead a normal life without restrictions. the degree of asthma control achieved by patients is an important predictor of the likelihood of complications of the disease [2•] . however, upper and lower respiratory infections can trigger exacerbations of acute asthma. most infections have a viral origin. they play an important role in clinical complications of asthma such as dyspnea, pneumonia, and respiratory insufficiency, which result in loss of quality of life, absenteeism, use of rescue medication, use of antibiotics, physician consultation, emergency department visits, hospitalization, and sometimes death. the best way to prevent exacerbations caused by infection is vaccination. this review summarizes the current evidence on the role of viral vaccination in the protection of asthma patients. we searched medline and google scholar for relevant articles and reports published between 1999 and july 2006 on the current status of vaccination in patients with asthma. viral infections have long been associated with asthma exacerbations. studies in children report varying incidences, suggesting that between 30% and 80% of exacerbations are due to a virus [3] [4] [5] [6] . for adults data on this issue are scarcer, but adults are believed to have as many asthma exacerbations linked with viral infections as children [7, 8] . the spectrum of viruses found consists of rhinovirus, coronavirus, respiratory syncytial virus (rsv), influenza virus, and an assortment of other viruses. of these, rsv and influenza affect primarily the lower respiratory tract, whereas rhinovirus and coronavirus replicate in the upper respiratory tract. the mechanism by which these viruses cause asthma exacerbations is not yet known precisely. postulations vary from direct infection to indirect induction of inflammatory responses [9] . in case of complications in patients with asthma, laboratory proof for the presence of virus should preferably coincide with symptomatic disease. however, evidence for the rate of complications mostly comes from large observational studies. in these reports on the impact of viruses, laboratory proof is often lacking. instead, extrapolated, partly population-based, incidence rates of influenza or influenza-like illness and the occurrence of complications are used as a proxy. in these studies, hard confirmation of the responsible agent on the individual level is not possible [10] [11] [12] [13] . other studies are often based on selected populations with existing symptoms or complications, reflecting only the worst of the spectrum of disease caused by acute respiratory infections [9, [14] [15] [16] [17] ] and thus disregarding their often self-limiting nature. although asthma control achieved by asthmatics is an important predictor of the likelihood of complications of the disease [2•], asthma exacerbations do not respond to inhaled steroids nor can they substantially be prevented in this way [18, 19] . only the use of oral corticosteroids seems to be unmistakably effective [20, 21] . the availability of effective vaccines against the key viruses involved in asthma exacerbations thus could play an important role in its prevention. below, we will discuss the vaccines that are available or may become available in the near future [22] . this species consists of about 100 serotypes and is responsible for the majority of "common colds." in asthmatics human rhinoviruses are responsible for the majority of asthma exacerbations and complications of asthma [23] . prevalent serotypes change from year to year. when prevalent, the relative predominance from any serotype is not higher than 40%. due to these circumstances and the large number of serotypes, an effective vaccine is not expected in the near future [22,24•]. coronaviruses have recently been highlighted because of a disease outbreak caused by the so-called severe acute respiratory syndrome (sars) coronavirus. in adults with asthma, coronaviruses are the second most important cause of virally induced exacerbations [7, 8] . animal vaccines are being tested. despite this a vaccine for human use will probably not be available in the near future [22]. rsv is especially predominant in young childhood and causes wheezing in children. in elderly people with asthma, rsv also may play an important role [25] . a connection has been suggested between bronchiolitis caused by rsv infection in childhood and the development of asthma [26] . against this background, a vaccine for rsv may not only prevent exacerbations but may also prevent the development of asthma later on. however, in the 1960s an experimental vaccine unfortunately had serious adverse effects by increasing the clinical symptoms of naturally acquired rsv infections. several other vaccines are being tested [27] , and despite the initial problems it is believed that a vaccine for routine immunization may be available within the next decade [22] . the influenza viruses are classified in three genera, labeled a, b, and c. only types a and b cause considerable epidemics. every year influenza viruses change their genome partially, which is called an antigenic drift. because of different subtypes and antigenic drift, formerly built-up natural immunity or vaccine-initiated immunity will not provide protection throughout successive seasons. there are three types of influenza a virus known to have infected humans and to have transmitted from human to human: h1n1, h2n2, and h3n2. however, water birds carry and are infected with about 15 different h-types. infections can spread from these birds to poultry, swine, and other animals living more closely to humans. by mixing infections, new subtypes can develop (antigenic shift). if a new subtype has the ability to infect humans and disseminate, a pandemic could occur. this is an often expressed fear in the case of the h5n1 influenza virus. influenza is the only lower respiratory tract infection in humans for which a vaccine has existed for decades. inactivated vaccines, delivered by subcutaneous or intramuscular injection, are widely used for the prevention of influenza. the guidelines of most western countries advise to vaccinate patients with asthma [28] . influenza is one of the common respiratory tract infections in humans [29] and according to the world health organization a yearly public health problem [30] . every year, influenza centers worldwide report influenza activity and the occurrence of influenza-like illnesses (ili) as well as data on subsequent health care use. but, many viruses can cause ili and thus a direct relation between influenza virus and symptomatic disease is difficult to determine. in published articles isolated serologic incidence rates, rates of ili, and the occurrence of complications are often used as proxy measures for influenza-related clinical illness. confirmation of influenza at an individual level is not provided in these large observational studies [11, 12, 31, 32] . however, when influenza infection is confirmed by laboratory tests as in trials or smaller observational studies, the clinical impact of influenza seems to be limited [4,6,33,34•,35] . incidence of influenza varies from place to place and from season to season. national and international influenza centers and organizations worldwide report incidence figures. usually, these figures are measured in an indirect way and relate to excess hospitalizations or mortality due to pneumonia and influenza or excess health care use. seasonal incidence figures for ili of 20% to 30% are not uncommon. however, symptomatic disease should preferably be corroborated by a positive laboratory test [36] . only in children do such figures seem to be available for a considerable number of years [37] . based on these data, the average incidence, depending on age category, is between 5% and 9.5%. the clinical picture is usually described as mild [37] . regarding the fluctuating incidence of ili reported, it is likely that in adults, the average incidence does not exceed these figures. comparable figures on the incidence in asthmatics are even scarcer. the proportion of asthma exacerbations due to influenza reported in asthmatic children were 18% and 5% [4,34•] . no severe complications (eg, hospitalizations) were found in these two studies. the effect of influenza vaccination in preventing clinical symptoms is a much-debated item. a number of reviews indicate that (inactivated) vaccines can have an efficacy of 70% in reducing serologically confirmed cases of influenza. however, when using symptom-based outcomes the vaccines showed an efficacy of only about 25% [38] [39] [40] . moreover, in case of a mismatch between the vaccine composition and the natural virus, efficacy will be much lower or absent. over the past years, live attenuated vaccines have been developed, tested, and used for intranasal administration. the less invasive route, of course, is a benefit in administering the vaccine. besides, the extra local immune response that is activated is a potential benefit. yet, even then there still remains a striking difference between efficacy and clinical effectiveness [39] . currently, animal research is being done into the development of vaccines that induce broad-spectrum and long-lasting immune response, which, if successful, would alter the prospects of the current influenza vaccination programs. in asthmatics, observational studies report varying and sometimes even contradictory outcomes [41] [42] [43] . but clinical efficacy has not been established yet at the highest level of evidence. uncertainty still remains about the degree of protection vaccination affords against influenzarelated symptoms such as asthma exacerbations [44••] . in a recent study in children with asthma, no positive effect of vaccination was found [34• ]. however, a distinct effect on quality of life in influenza-related episodes in asthmatic children was reported [6] . as for side effects and complications, the evidence that influenza vaccination is safe and well tolerated is extensive [44••] . several studies have addressed the cost-effectiveness of influenza vaccination. we also identified one costeffectiveness study that addressed a hypothetical rsv vaccine in elderly people [45] . all cost-effectiveness studies are based on a population-wide use of the vaccines, for example, in preschool children or elderly persons. in all of these studies, the cost-effectiveness of influenza vaccination is based on several premises. often the incidence of influenza is approximated by that of ili in epidemic seasons. these figures certainly exceed the average incidence of influenza when taking into account the seasons with low or no influenza activity. other biases may be the target populations and the definition used for influenza [46] . in the elderly cost-effectiveness is globally accepted [47] . in either case, population-wide cost-effectiveness preventive options against influenza for healthy adults 14 to 60 years old do not seem to be easily achieved, and therefore some authors have suggested that the most cost-effective option is not to take any action [48] . for high-risk patients with chronic respiratory conditions, again, in the elderly cost-effectiveness was achieved; however, this was not the case for the 18 to 65 years age category [49, 50] . in children with high-risk conditions the use of influenza vaccination was calculated to be cost-effective. however, this study assumed a much higher incidence and a far more favorable effectiveness of influenza vaccination than described above [50] . the role of virus vaccination in patients with asthma at this moment and in the near future seems to be limited (table 1) . currently, vaccines are available for routine use only for influenza. however, because of a lack of clinical effectiveness, the natural antigenic variations of the influenza virus, and the low average incidence of influenza, cost-effectiveness in children and adults with asthma will not be easily achieved if vaccination has to be delivered annually. as for the other viruses that play an important role in the complications of patients with asthma, many hurdles remain to be overcome. for rsv, the first experiences with vaccines were a major disappointment. in the case of coronaviruses, attention is understandably focused on a vaccine for the prevention of sars. as for rhinoviruses, the major cause of asthma exacerbations, because of the amount of subspecies a vaccine will not be available in the near future. of course other therapies, such as antiviral medication or immunoglobulin, are used and are sometimes effective in reducing complications. but, the therapeutic use of these treatments is limited because they are often expensive and should be used only when there is proof of the agent that is causing the patient's symptoms. for prophylactic use their cost is a major problem. yet, the role of the clinician in patients with asthma experiencing the threat of an exacerbation with its complications is clear. an oral corticosteroid can be provided and kept on hand by the patient. this permits initiation of more prompt and effective treatment than is likely to occur when a patient must first go to a physician's office or emergency department, because it can be taken as soon as the response to bronchodilator therapy is incomplete [21] . moreover, patients with asthma should be treated according to national and international standards. access to and compliance with inhaled corticosteroid treatment is an important predictor of the likelihood of asthma exacerbations occurring, including those that occur during respiratory viral infections [2•]. papers of particular interest, published recently, have been highlighted as: • of importance •• of major importance incidence and prevalence of asthma and allergic rhinitis: a cohort study of finnish adolescents this review describes the epidemiology of asthma exacerbations based on multiple sources viruses as precipitants of asthmatic attacks in children community study of role of viral infections in exacerbations of asthma in 9-11 year old children asthma exacerbations in children associated with rhinovirus but not human metapneumovirus infection influenza vaccination in asthmatic children: effects on quality of life and symptoms respiratory viruses and exacerbations of asthma in adults respiratory tract viral infections in inner-city asthmatic adults viruses in asthma exacerbations the relationship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis influenza and the rates of hospitalization for respiratory disease among infants and young children the burden of influenza illness in children with asthma and other chronic medical conditions respiratory infections and the autumn increase in asthma morbidity the incidence of respiratory tract infection in adults requiring hospitalization for asthma spectrum of clinical illness in hospitalized patients with "common cold" virus infections epidemiology of respiratory viruses in patients hospitalized with near-fatal asthma, acute exacerbations of asthma, or chronic obstructive pulmonary disease population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children inhaled steroids for episodic viral wheeze of childhood early use of inhaled corticosteroids in the emergency department treatment of acute asthma early emergency department treatment of acute asthma with systemic corticosteroids treatment strategies for viral respiratory infection-induced asthma epidemiology of human rhinoviruses this interesting thesis describes the difficulties met and the progress made in rhinovirus research respiratory syncytial virus infection in elderly and high-risk adults clinical patterns and natural history of asthma progress in the development of respiratory syncytial virus and parainfluenza virus vaccines influenza vaccination in 2000: recommendations and vaccine use in 50 developed and rapidly developing countries respiratory viral infections in adults who 2003: influenza factsheet populationbased study on incidence, risk factors, clinical complications and drug utilisation associated with influenza in the united kingdom the underrecognized burden of influenza in young children the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial influenza vaccination in children with asthma: randomized double-blind placebo-controlled trial until now, the only rct investigating the clinical effectiveness of influenza vaccination in children burden of influenza in children in the community invited commentary: use of selective viral cultures to adjust nonvirologic endpoints in studies of influenza vaccine efficacy incidence of influenza and associated illness in children aged 0-19 years: a systematic review vaccines for preventing influenza in healthy adults assessment of the efficacy and effectiveness of influenza vaccines in healthy children: systematic review methodological quality of studies and patient age as major sources of variation in efficacy estimates of influenza vaccination in healthy adults: a meta-analysis clinical effectiveness of conventional influenza vaccination in asthmatic children effectiveness of influenza vaccine for the prevention of asthma exacerbations influenza vaccination in patients with asthma: effect on the frequency of upper respiratory tract infections and exacerbations vaccines for preventing influenza in people with asthma. cochrane database syst rev 2004:cd000364. systematic review gathering all available evidence from randomized controlled trials into the effects and adverse events of influenza vaccination the cost-effectiveness of a hypothetical respiratory syncytial virus vaccine in the elderly systematic review and economic decision modelling for the prevention and treatment of influenza a and b influence of clinical outcome and outcome period definitions on estimates of absolute clinical and economic benefits of influenza vaccination in community dwelling elderly persons prevention and early treatment of influenza in healthy adults is immunizing all patients with chronic lung disease in the community against influenza cost effective? evidence from a general practice based clinical prospective cohort study in utrecht, the netherlands cost-effectiveness of influenza vaccination in high-risk children in argentina key: cord-258781-peppszqx authors: ishola, david a.; phin, nick title: could influenza transmission be reduced by restricting mass gatherings? towards an evidence-based policy framework date: 2011-08-18 journal: j epidemiol glob health doi: 10.1016/j.jegh.2011.06.004 sha: doc_id: 258781 cord_uid: peppszqx introduction: mass gatherings (mg) may provide ideal conditions for influenza transmission. the evidence for an association between mg and influenza transmission is reviewed to assess whether restricting mg may reduce transmission. methods: major databases were searched (pubmed, embase, scopus, cinahl), producing 1706 articles that were sifted by title, abstract, and full-text. a narrative approach was adopted for data synthesis. results: twenty-four papers met the inclusion criteria, covering mg of varying sizes and settings, and including 9 observational studies, 10 outbreak reports, 4 event reports, and a quasi-experimental study. there is some evidence that certain types of mg may be associated with increased risk of influenza transmission. mg may also “seed” new strains into an area, and may instigate community transmission in a pandemic. restricting mgs, in combination with other social distancing interventions, may help reduce transmission, but it was not possible to identify conclusive evidence on the individual effect of mg restriction alone. evidence suggests that event duration and crowdedness may be the key factors that determine the risk of influenza transmission, and possibly the type of venue (indoor/outdoor). conclusion: these factors potentially represent a basis for a policy-making framework for mg restrictions in the event of a severe pandemic. it is well established that influenza is transmitted from person to person through close contact with an infected, symptomatic individual. the exact mechanisms by which transmission occurs are still unclear, but is believed to involve multiple routes, including respiratory droplets and direct/indirect contact [1] with secretions or fomites. aerosol transmission may also occur, but controversy surrounds this [2] and some further evidence of aerosol transmission has recently been put forward [3] . this is an important issue as the mechanism of transmission is always a key factor in infection control planning. while uncertainty persists regarding aerosols, droplet and contact transmission remain largely regarded as the most important and likely routes. transmission through these routes clearly requires physical nearness to infected persons, via either direct touch or the propulsion of large droplets across a relatively short distance. this requirement makes the consideration of mass gatherings a crucial issue. whilst there is not a ''standard'' definition of what constitutes a mass gathering, there is an understanding that these are events that involve large numbers of people (suggested minimum of 1000 people) [4] [5] [6] congregating in finite geographical areas to share an event or experience. individuals can be in very close proximity for variable periods of time and, if the event is over a number of days, may even share over-crowded and/or temporary accommodation. therefore for those infectious diseases such as influenza, where close contact is the main determinant of transmission, it appears self-evident that mass gathering events could lead to the rapid transmission of a new causative agent. in the event of an influenza pandemic, minimizing transmission of influenza has been a priority for public health action. a variety of non-pharmaceutical public health interventions to reduce close contact between infected and susceptible individuals, or the opportunities for the virus to be picked up by susceptible people such as self-isolation of patients, respiratory etiquette and hand washing have been advocated. banning or restricting mass gatherings has been seen as a logical extension of this policy, however, it is a particular concern of policy makers that the scientific evidence upon which to base guidance for mass gatherings is lacking. this is particularly important given the need to weigh any potential benefits against the economic and social disruption that banning or restricting mass gatherings could have on society. there have been other systematic reviews undertaken in recent years on the broader subject of non-pharmaceutical interventions aimed at reducing transmission of respiratory viral infections [7, 8] . these reviews considered a range of such interventions, and they explicitly recognized that there is a paucity of primary evidence regarding restriction of mass gatherings (and other ''social distancing'' measures); thus they did not primarily focus on this specific area. the aim of this review, therefore, was to attempt to reduce this evidence gap by assessing the available evidence base relating to the utility of restrictions on mass gatherings during an influenza pandemic. this is an important policy area not only because of the impact such restrictions could have on public confidence and morale, but also because of the economic and liability issues that such action might generate. the findings of the review may be able to help inform policy statements on the effectiveness of mass gathering restriction interventions that may be deployed to help reduce influenza virus spread during a pandemic. the study protocol was largely based on the university of yorkõs centre for reviews and dissemination guidance for undertaking reviews in healthcare [9] . work commenced in mid-july 2010 and the main part of it was completed in mid-october 2010. further work was carried out in june 2011 to bring the literature coverage up to date. the following specific questions were developed to capture the objectives of the review: • is there an association between mass gatherings and influenza outbreaks or spread? • are there any particular characteristics (such as size or duration) of mass gatherings that influence transmission of influenza? • does the restriction of mass gatherings reduce the spread of influenza within the community (compared with no restriction or with other interventions)? the following types of studies were included in the review: • randomized controlled trial • quasi-experimental study -non-randomized controlled study -before-and-after study • observational study -cohort study -case-control study • outbreak reports -outbreak/cluster reports -historical archival outbreak analyses • surveillance reports -major event infection surveillance reports the list above is largely in hierarchical order of study design quality [9] , but with the addition of outbreak and surveillance reports, not traditionally included in such lists, important to the topic of this review. to cover the other ''non-standard'' study design encountered, the term ''quasi-experimental'' is employed to describe controlled but nonrandomized studies [10, 11] . only studies published in english were included. relevant systematic and narrative reviews and operational description papers were utilized for useful background information. the reference lists of the systematic reviews were scanned to identify potentially relevant primary studies that could be considered for inclusion. case reports, mathematical modelling and human/non-human experimental laboratory studies were excluded from the review. outcome measures included laboratory-confirmed influenza infection (i.e., documented by virus isolation, molecular testing such as polymerase chain reaction, and serological studies) and clinical influenza-like illness as defined by the investigators, whilst recognizing that this is less specific. the search strategy focused on primary studies taking into account the issues detailed above. the term ôinfluenzaõ rather than ôpandemic influenzaõ was used to reflect the entire spectrum of influenza for which guidance would be relevant. the transmission of other respiratory viruses was also included because of the similarity of transmission and therefore the potential applicability of any results to influenza. an initial ''scoping'' search was followed by further discussions and consultation on the work plan and search strategy. the term mass gathering on its own proved inadequate as a search term as there is no clearly accepted definition of what constitutes a mass gathering. therefore, a range of additional terms were used, such as public gatherings, social gatherings, large crowds, mass events, festivals, olympics, hajj, championship, and others listed in table 1 . transit settings such as long-distance flights and cruise ships were not included, as it was decided that these settings would fit better in a separate review of evidence for influenza transmission through transport. the final search with revised terms and refined using the ''advanced search'' tool on pubmed was carried out as described in table 1 , with no time period restrictions. the initial searches included the literature up till july 2010. a further search was conducted on 31 may 2011 to bring the literature coverage up to date as part of an editorial process. in addition, secondary searches were also initially carried out in the following databases: scopus, excerpta medica database (embase), and the cumulative index to nursing and allied health literature (cinahl). these are described in table 2 . in addition to the search results, the reference lists of papers identified from the search were scanned for other potentially relevant studies. efforts were also made to identify studies other than those published in the peer-reviewed literature; the authorsõ study selection was conducted in stages. at all stages, scanning and reviews were done independently by the authors, who then harmonized their selections. papers identified by the searches were scanned and some excluded on the basis of the ôti-tleõ for relevance to the review. the abstracts of the remaining papers were then reviewed to identify studies that appeared to meet the inclusion criteria. the full text versions of all these articles were then sought and read in full. for papers that had relevant titles but no abstracts, the full text articles were also obtained, scanned for relevance, and if relevant they were read in full. in addition, some papers that did not meet the inclusion criteria (e.g. operational descriptions of interventions, commentaries, or editorial reviews of influenza transmission or pandemic influenza) were selected to provide relevant background or supplemental information. data from the selected full text papers were extracted using a pre-designed form. data elements included publication information, study characteristics, participant characteristics, the intervention (if any) and setting, outcome and results. the data extracted were used to determine the eligibility of each paper for inclusion in the review. this process resulted in a final selection of eligible articles that were then included in the review. the main body of this work was undertaken in 2010 by the uk health protection agency as part of a comprehensive influenza scientific evidence review informing the uk national pandemic influenza strategy. within this context, the work was carried out within a very tight three-month timeframe and with significantly limited resources. as a result, some of the most comprehensive steps required of a full systematic review could not be realistically accommodated. therefore, a modified process was adopted in conducting this ''rapid evidence assessment'' rather than a full, formal systematic review. rapid evidence assessments are well recognized as a pragmatic approach to include all the major elements of a full systematic review, ensuring an outcome that is as comprehensive as possible while accepting limited compromises in order to meet stipulated deadlines for feeding into the policy process [9] . due to the time constraints, it was not practical to pursue time-intensive contacts with authors or to seek translations for non-english articles, which were therefore excluded. articles lacking abstracts were largely excluded, and there was a limited search for unpublished studies. however, to try to mitigate these limitations, full draft versions of the review were submitted to national experts at the uk health protection agency and the scientific pandemic influenza committee of the uk department of health, seeking their comments and advice on any papers or documents that may have been omitted. some further work was carried out in may to june 2011 to update the literature. at the outset, a strategy was developed to assess the quality of eligible studies using the critical appraisal skills programme (casp) tools as appropriate for each type of study [12] . however, after completion of the literature search, it was clear that the types of studies and the study design types identified were not suitable for assessment by the casp tools. similarly for the outbreak reports included in the review, no suitable existing framework could be readily identified for quality assessment. the orion tool was not strictly applicable as it is specific to outbreaks of nosocomial infection [13, 14] . therefore, a modified approach was adopted, whereby each paper was categorized as having low risk, some risk, or high risk for bias with respect to the relevant review question addressed by that study. the grading was assessed based on the presence of significant methodological limitations. a high risk of bias was attributed to papers with at least three significant methodological issues as identified by the review authors, while papers with at least two issues were classified as having some risk. this approach was adapted from a system previously used by jefferson et al. [7] . the quality of the review itself was tested against checklists based on the moose [15] and prisma [16, 17] standards. within the restrictions of the time constraints already described, key gaps and issues identified by the checklists were addressed as appropriate and/or acknowledged in sections 2.5 (explaining the review context including time constraints) and 4.4 (recognizing the study limitations). the data synthesis was restricted to a narrative approach that included an analysis of the relationships within and between studies and an overall assessment of the robustness of the evidence and limitations of both the studies and the evidence review [9] . in addition, the synthesis considered the implications for policy and guidance development as well as future research. the search identified a total of 1706 papers after accounting for overlaps between the different databases. these were reviewed on the basis of ôtitleõ for relevance to the review (fig. 1) , and on this basis 1593 papers were excluded. abstracts for the remaining 113 papers were reviewed and a further 54 were eliminated. full copies of the remaining 59 papers plus 6 papers identified from scanning the reference lists of review papers, and 3 papers retrieved from the authorsõ hardcopy files, were reviewed and summarized. table 3 provides a classification of the 68 papers that were read in full text. after full text review, 24 papers were classified as meeting the inclusion criteria. these included a quasi-experimental study (a non-randomized trial) [18] , nine observational studies [19] [20] [21] [22] [23] [24] [25] [26] [27] , seven outbreak reports [28] [29] [30] [31] [32] [33] [34] , three historical outbreak archive analyses [35] [36] [37] , and four event surveillance reports [38] [39] [40] [41] . details for each of these studies are summarized in table 4 . the 44 papers that were read but not included in the review are listed in table 5 . one quasi-experimental study by qureshi et al. [18] attempted to investigate the incidence of vaccine preventable influenza-like illness among pakistani pilgrims to the hajj religious gathering in 1999. the hajj is an annual religious event that takes place over a number of days in a very small geographic area of saudi arabia usually involving 2 to 3 million pilgrims from all over the world. accommodation is at a premium during this event and many pilgrims stay in tents specifically erected for the event and that are often over-crowded. although primarily a vaccine efficacy study, the rates of influenza-like illness reported in vaccinated pilgrims were 36% compared with 62% in non-vaccinated pilgrims. however, these results were based on clinical endpoints without microbiologic confirmation; a non-randomized design was used, and the study was not designed to address the primary question of this review. nine observational studies estimated the risk of acute respiratory illness and/or influenza-like illness associated with the hajj pilgrimage by attempting to measure its occurrence in pilgrims. four [23] [24] [25] 27] of these studies confirmed the cause of illness by laboratory testing, while the other five relied on specified symptom complexes as surrogate indicators. among the nine observational studies were four cross-sectional studies. balkhy and colleagues [24] tested 500 hajj pilgrims in 2003 who presented with symptoms of upper respiratory tract infection in the second week of the event; 30 (6%) of the 500 pilgrims tested positive for influenza. rashid et al. [23] assessed the burden of laboratory-confirmed influenza and respiratory syncytial virus (rsv) infections in symptomatic british hajj pilgrims in the 2005 event. of 202 symptomatic pilgrims who underwent nasal swab testing, 28 (about 14%) had confirmed influenza (mostly a type), while only 9 (4%) had rsv infection. in the 2006 hajj, the same investigators [27] found comparable levels of laboratory confirmed influenza (10-11%) in both uk and saudi pilgrims. these relatively low levels of infection contrasted with the findings in the cross-sectional study by deris et al. [20] who based their assessment on syndromic influenza-like illness rather than laboratory-confirmed infection. they found an influenza-like illness prevalence of 40% in malaysian pilgrims who had just completed the hajj. the other five observational studies were similarly designed, involving groups of intending hajj pilgrims who were recruited in their home regions or countries prior to the event, and then re-assessed this was a well-organized systematic prospective influenza surveillance program, described by the authors as the first of its type at a large games event limitations include: -no indication of total numbers of people at the event or in the city -no indication of the background ili activity in the city or country; or whether this was during the local winter influenza season after the pilgrimage. even though three reports were described by their respective authors as ''prospective cohort'' studies [19, 21, 22] , none of these five studies included an ''unexposed'' (non-hajj attending) control group for comparison, indicating that they should be more accurately regarded as ''before and after'' studies. choudhry et al. [19] assessed saudi residents (attending the hajj from a different part of the country) and found an incidence of influenza-like illness of about 40%. three studies of french pilgrims by gautret et al. [21, 22, 26] found rates of cough of between 48% and 61%; while in el-bashir and colleaguesõ serological study of uk pilgrims [25] , respiratory symptoms occurred in more than 80%, but the seroconversion rate was 38% of 115 participants. these variable data underline the difficulties of comparing different studies that employ contrasting measures of influenza. interpretation needs to take account of the fact that studies using clinical outcomes suggest much higher levels of influenza-like conditions than the ones that involve laboratory confirmation. even among the laboratory-based studies, differences in laboratory techniques may also be significant. there were four types of outbreaks in the reports that were reviewed. the first of those was a paper by pang et al. [32] that described the experience of dealing with the sars (severe acute respiratory the next group consists of four reports of influenza outbreaks occurring at a religious event in australia and at three large, open-air music festivals in europe. all of the events lasted several days and involved crowds ranging from 100,000 to 400,000 people. it is particularly relevant that the three music festivals occurred during the 2009 influenza pandemic-two at the beginning and one later during the pandemic. the report by blyth et al. [28] described an outbreak of influenza at a large, 5-day religious event in australia during july 2008 attended by over 400,000 participants from 170 countries. over 100,000 of the pilgrims were accommodated in a variety of make-shift, overcrowded venues such as sports halls, community centers and schools. one hundred laboratory-confirmed cases of influenza were identified among attendees. seven different strains of influenza were identified (four influenza a and three influenza b), highlighting the potential for the introduction of novel influenza strains. loncarevic et al. [31] described an outbreak of influenza at a 4-day music festival in serbia during july 2009 involving over 190,000 participants, with a number of them coming from other european countries. many of the participants stayed at a large campsite where overcrowding was an issue. sixtytwo laboratory-confirmed cases of h1n1 (2009) were identified; some of which were secondary cases. although the virus was already present in serbia at the time of the festival, the sudden increase in cases, in particular the secondary cases associated with the festival, suggests possible local spreading. the authors also reported on a small outbreak of influenza at an international sporting event held in serbia in july over a period of 12 days and attended by over 500,000 spectators. seven confirmed cases of h1n1 (2009) were identified in six athletes and a volunteer helping at the games. although a much larger event, the numbers affected are considerably smaller and seem to be restricted to participants rather than those attending. gutierrez et al. [30] described a 4-day music festival in belgium during early july 2009 attended by an estimated 120,000 people from all over europe. twelve laboratory-confirmed cases of h1n1 (2009) were identified. although sporadic cases of h1n1 (2009) had been detected in belgium prior to the festival, an increase in cases was observed after the event and the decision to shift to mitigation was taken almost a week after the first festivalassociated cases were identified. the authors suggest that this festival highlights the potential seeding role for these events in the early stages of a pandemic. presentation and discussion paper on data from the 1918-1919 influenza pandemic. the substantial research report arising from the study was included in the study [37] memish botelho-nevers et al. [29] depict an outbreak of influenza at a 7-day music festival in hungary during august 2009 attended by 390,000 people from all over europe. many of the participants were located on a campsite set up for the festival. eight laboratory-confirmed cases of h1n1 (2009) were identified. cases of pandemic influenza had already been identified in hungary and at the time the community influenza-like illness rate was 7.8 per 100,000 against an estimated 3.6 per 100,000 at the music festival. the third type of outbreak report described by saenz et al. [33] involved a large, international medical conference held in iran during september 1968 at the early stages of the 1968/1969 pandemic. the 7-day conference was attended by over 1000 participants from all over the world. it was estimated that about a third of the participants developed an influenza-like illness with an overall attack rate of 36%. the virus was isolated from throat and nasal washings; this was found to be the pandemic a/hong kong/68 virus. there was evidence that close contacts of returning attendees were also affected, but none of the episodes led to a rapidly expanding focus of infection. the high attack rate raises the question of whether the indoor setting may represent a particularly high risk. lastly, there was an innovative study examining the 2009 a(h1n1) outbreak in mexico [34] . the authors described the details of 202 cases and then used a series of epidemiological tools to map the spread of the virus throughout the country. they identified two major contributory factors to the rapid spread of influenza: one was the very high population density in parts of mexico city; the other was the massive religious festival involving more than 2 million people, which took place in the cityõs iztapalapa neighbourhood at the very early stage of the outbreak, shortly before the new virus was identified. it is suggested by the authors that this temporal association between [39] low schenkel [40] low the mass gathering event and the subsequent increase in numbers of cases may reflect participants returning to their own neighbourhoods across the city, and from there to other parts of the country. inevitably for a subject of research such as mass gathering restrictions, where prospective studies present serious practical challenges, researchers have sought to utilize historical data to try to draw out major lessons for current impact. this review included three historical analyses of the non-pharmaceutical responses to the 1918-1919 influenza pandemic. markel et al. [37] examined the variety of nonpharmaceutical interventions that were deployed in 43 american cities during the 1918-1919 influenza pandemic. the combination of school closures and concurrent public gathering bans was implemented in 34 (79%) of the 43 cities and was the commonest combination of measures deployed. applied early in the pandemic, this combination was significantly associated with reductions in the weekly excess death rates. hatchett et al. [36] undertook a similar analysis on a smaller number of american cities where the timing of 19 different types of non-pharmaceutical interventions was available. they found that the early application of multiple interventions showed a trend towards lower cumulative excess mortality, but that no single intervention showed an association with improved aggregate outcomes for the pandemic. both studies suggested that for non-pharmaceutical interventions to be beneficial, they should be applied early and in a sustained manner. a further review of the u.s. public health response to the 1918 pandemic by aimone [35] gave conflicting results. this review examined the public health response in new york city. in new york during the 1918 pandemic, mass gatherings were not prohibited nor were schools closed; instead, the city opted for a policy of staggered business hours to avoid rush-hour crowding, enhanced surveillance so that cases were quickly identified and isolated, and an intensive program of health education. the reported outcome measures for new york city were comparable with those seen in other american cities, and new york city experienced one of the lowest excess death rates on the eastern seaboard of the united states. surveillance reports from four major sporting events within the last decade were considered suit-able for review [38] [39] [40] [41] . the study by gundlapalli et al. [38] reported the experience of influenza surveillance during the winter olympics at salt lake city in 2002. no indication of the numbers attending was given, but it is assumed that the numbers were large. twenty-eight cases of confirmed influenza from three clusters of influenza-like illness (ili) were identified and these were restricted to either participants in the games or support staff for the games. the clusters consisted of 12 members of a national team who trained and lived together, 8 participants of a sport and 13 law enforcement officers who worked and lived in close proximity. lim et al. [39] reported on the experience of managing the asian youth games at singapore in june 2009. these games involved over 2000 athletes and officials from 43 countries. although numbers of spectators are not given, it is assumed that the crowds were large. at the start of the games, singapore had already reported 600 confirmed cases of h1n1 (2009). six laboratory-confirmed cases of h1n1 (2009) were identified during the 8 days of the event-four on one football team. no information on the numbers of confirmed h1n1 (2009) in the population after the games was available. there was no increase in ili activity during the winter olympics in torino in 2006 [41] . the final study by schenkel et al. [40] reported on the experience of syndromic surveillance during the fifa world cup in germany during june/july 2006. at the time, germany was experiencing a very large outbreak of measles. measles is essentially spread by the respiratory route and is highly infectious. however, despite enhanced daily surveillance, no outbreaks of respiratory disease or measles associated with the world cup were detected. as stated in section 2.6, the approach to quality assessment of the reviewed papers was based on a pragmatic framework for categorizing studies into low-or high-risk for bias, depending on the presence or absence of significant methodological limitations. a high risk of bias was attributed to papers with at least two significant methodological problems as identified by the review authors (table 6 ). the evidence to help address important public health questions around mass gatherings and influenza transmission is sparse, especially in the context of an influenza pandemic. in addition, the topic does not lend itself to ease of scientific investigation and there are probably many who may feel that it is self-evident that mass gatherings facilitate the transmission of infectious diseases. this systematic search of the literature identified a limited number of studies that addressed the review questions regarding whether mass gatherings are associated with influenza transmission and whether restricting mass gatherings reduces the spread of influenza within the community. in attempting to understand and describe a situation or intervention, a common understanding or definition is essential. there is currently no generally accepted definition of what constitutes a mass gathering. however, the literature on mass gathering medical care highlights an emerging consensus amongst those providing emergency medical care at organized events. in this setting, mass gatherings are considered to be organized events with more than 1000 people in attendance [4] [5] [6] . a recently published guidance document from the world health organisation (who) expanded the term to cover any organized or unplanned event involving enough people to ''strain the planning and response resources of the (host) community, state or nation'' [42] . the major limitation in trying to define mass gatherings is that any single definition would inevitably be too simplistic as it would need to incorporate events as diverse as the hajj (lasting about 1 month and involving between 2 and 3 million people), and a football match (involving several thousand spectators over a period of about 2 h). a system for classifying mass gatherings on the basis of size and duration is lacking and may be required. in recognition of the difficulties of conducting hypothesis-based studies that directly implement and assess the effects of restrictions of mass gatherings in real life, an indirect approach was taken to address the review questions as follows: to address whether mass gatherings are associated with influenza transmission, evidence was derived from the following: -a quasi-experimental study that was primarily designed to quantify vaccine efficacy in the form of a non-randomized trial. -observational studies that assessed participants before and after exposure to mass gathering events. -reports of influenza outbreaks and other respiratory illnesses at mass gathering events. -communicable disease surveillance reports from some major events. a number of studies [18] [19] [20] [21] [22] have consistently demonstrated, over a number of years, that respiratory virus transmission occurs amongst pilgrims attending the annual hajj in saudi arabia, and it is recognized as an issue of international public health significance [43] [44] [45] [46] that could be particularly important in a pandemic situation. a significant proportion of pilgrims are affected by symptoms of either an influenza-like illness or an acute respiratory illness with the proportion affected reaching about 40% in some studies [19, 20] . the hajj is, however, a unique event with almost 3 million people converging on a relatively small geographic area for a period of at least 5 days, extending up to 4 weeks. crowd density is very high and overcrowding in the living accommodation is common. given the unusual nature of this event, the applicability of these findings to other mass gatherings is therefore limited. there is also a question regarding the fact that several of these studies are based on clinical criteria that could be due to other viral respiratory illnesses besides influenza. in a small number of outbreak studies involving influenza-like illness and confirmed influenza at large music festivals, there is varying evidence about the extent to which influenza transmission occurs. outbreaks were based on laboratory diagnosis, and transmission was confirmed in all, though they had varying infection rates ranging from roughly 3 to 25 per 100,000. two recent studies undertaken during the 2009 pandemic suggest that at the beginning of a pandemic, these gatherings may act as seeding events [30] ; indeed it has been suggested at the early stages of the 1999 pandemic a(h1n1), a huge easter season mass gathering involving 2 million people may have helped in propelling the first wave of the outbreak throughout mexico [34] . there is no compelling evidence from event surveillance reports, such as international athletic events, the world cup and the winter olympics, to indicate that these major events significantly increase community transmission of influenza. outbreaks did occur in some cases, but these always seemed restricted to the actual competitors and staff rather than the crowds attending or the wider community. together, all of these reports point to the potential for influenza outbreak and transmission in connection with large, multiple-day, open-air events. thus there is some evidence to indicate that mass gatherings may be associated with an increased risk of influenza transmission, but it seems to be very variable. the type of mass gathering event seems to be of considerable importance in terms of the risk of influenza transmission ( table 7) . most of the evidence supporting the role of mass gatherings in the transmission of influenza comes from events where there are crowds with high crowd densities (which may be theoretically estimated at >5 people per square metre), and where the participants are likely to live close together for prolonged periods, e.g. the hajj pilgrimage [18] [19] [20] [21] [22] and large musical festivals [28] [29] [30] . in these events, accommodations which are already likely crowded are also likely to be relatively basic, such as communal camp-style living, with the probable risk of suboptimal hygiene facilities. it seems apparent that events where close contact among participants extends beyond event venues and into accommodation areas are most associated with influenza. event size, per se, does not seem to be a critical factor. in contrast, there is no convincing evidence that major organized sporting events are associated with significantly increased influenza transmission in those attending the event [38] [39] [40] . an important example of this contrast comes from serbia, where two major events of different type and scale happened to coincide in 2009, providing a ''natural experimental'' opportunity for comparison [31] . in the larger event, only four event-linked confirmed influenza cases occurred at the world university games held over a 12-day period and involving almost 25,000 athletes and staff with about 500,000 spectators. however, in a relatively smaller event held in the same month and within the same country, as many as 47 event-linked confirmed cases occurred at a 4-day music festival with around 190,000 participants. furthermore, in surveillance reports from recent major international sports competitions, cases or small clusters of influenza were reported, but these were mainly among the event participants rather than among the overall population of people exposed to the events [38, 39] . this was true even for one major event that took place within a pandemic context [39] . these events showed no clear evidence of influenza transmission, indicating that influenza may not be a significant cause for concern at modern world sports events. this view is supported by the surveillance report from the 2006 world cup in germany [40] , where instances of transmission were not reported, meaning that they were either not detected or very low. this situation may in part be explained by the brief transitory nature of contact in the crowds in highly organized international sports festivals such as the olympics and the world cup, which are usually seated events with good spacing in-between seats and mostly in open-air settings with dilution of any infectious droplets that may be generated. the apparently low or absent influenza transmission at such events may also reflect the contemporary fact that many people who attend major sports championships tend to have planned their visit a long time ahead, as ticket sales usually start months or even years in advance. spectators as well as participants tend to stay in more conventional accommodations such as hotels rather than tents or other forms of portable or camp-style quarters with highly crowded conditions. it is also important to note that contemporary major events are now deploying increasingly developed systems for infectious disease surveillance and control [47] , which are crucial for early detection and containment where possible. the other situation of note relates to indoor events such as large conferences, typified by the international medical conference held over 7 days in iran during the early stages of the 1968/1969 pandemic [33] . during this meeting it was estimated that about a third of the participants developed an influenza-like illness with an overall attack rate of 36%; the pandemic virus was isolated from those cases where testing had been undertaken. this potentially highlights the role that ventilation may play in the reduction of influenza transmission and is another factor worthy of consideration. in summary, the type of mass gathering event seems to influence the risk of influenza transmission, key factors being the degree of crowdedness, the event duration and, possibly, whether the event is held indoors or outdoors. multiple-day events with crowded communal accommodations may be the links to mass gatherings most associated with influenza. to address whether mass gathering restrictions can reduce influenza transmission, the mainly relevant papers found were archival studies of the 1918-1919 influenza pandemic [35] [36] [37] and an analysis of the 2003 sars outbreak [32] . no relevant randomized controlled trials were found, reflecting the practical difficulties that such studies would involve. mass gatherings of varying dimensions were restricted at a large number of american cities during the 1918-1919 period. the reports again highlighted the difficulty of interpreting what was meant by a mass gathering, e.g. including schools, cinemas, theatres and other public places. in general, evidence suggests that these measures had a beneficial effect, especially where implemented early in the course of the outbreak [36, 37] . however, these benefits were not universal across all the cities [35] . restrictions were typically implemented as part of a set of interventions, e.g. combining quarantine and isolation policies with banning mass gatherings. as a result, it is extremely difficult to tease out the individual effects of mass gathering restrictions alone. using multivariate techniques, investigators attempted to isolate the differential effects of individual restriction measures and found indications that certain interventions (such as closures of entertainment venues) had measurable specific impact [36, 37] , but this evidence is limited. an analysis of the 2003 sars outbreak in china attempted to probe the impact of mass gathering restrictions that were applied in a contemporary setting [32] . however, as with the historical studies, it was not possible to distinguish the specific effects of mass gathering restrictions from amongst the broad range of other public health interventions that were applied. in summary, there is some evidence that when applied early and in tandem with other public health measures, such as isolation and quarantine and closures of educational institutions, mass gathering restrictions may help in reducing transmission. in a pandemic like that experienced during 2009, it is unlikely that the measures described above could be justified; however, in a much more severe pandemic, the cost-benefit equation could easily shift the other way. the application of bans on mass gatherings and other related public health measures are therefore highly dependent on an early indication of the severity as measured by its impact on individuals and society. there are two further, critical domains of uncertainty that need to be considered in the development of evidence-based guidance and policies regarding mass gatherings. the first domain relates to issues around the current understanding about how influenza is spread, and factors that can affect transmissibility (e.g. host factors, pathogen factors, environmental factors and particle size) [9] . key questions remain in these areas, which may be important in making specific recommendations regarding particular types and scope of mass gathering restrictions. the second domain impinging on the potential effectiveness of any public policy on mass gatherings includes the whole range of factors affecting adherence and compliance. for instance, the experience of the 2009 pandemic has raised significant questions around how willing people might be to comply with bans imposed on mass gathering restrictions [48] [49] [50] [51] . other challenging issues include the problematic ethical and legal frameworks for implementing restrictions for public health purposes [52, 53] , as well as considerable logistical and economic implications. if longplanned events were to be cancelled, who would be liable for the huge personal, corporate and national costs that such cancellations might incur? in considering policy recommendations within a pandemic context, the most practical approach for all but the more severe pandemics may be a strategy of encouraging voluntary restrictions. this would involve giving the public the best available information and advising rather than legislating that organizations and individuals avoid non-essential events where there is at least some evidence of transmission risk. for such a strategy to have a chance of succeeding, it would be most important to have in place a carefully and sensitively prepared communication strategy, since recommendations to avoid public gatherings are likely to run against powerful social pressures to do otherwise [54] . the success of any public health strategy-and this must be particularly true in relation to a potential or imminent pandemic situation-requires the building of trust [55] and an intelligent and purposeful engagement of the public even prior to the event [56] . there are other important issues to note. although this review has focused on mass gatherings, limiting transmission of influenza clearly requires a multifaceted approach. some studies in this review reflected such an approach; for example, in the historical outbreak investigations where restrictions on mass gatherings were combined with other non-pharmaceutical measures [32, 36, 37] . it would be prudent to apply the best evidence relating to other social distancing interventions in conjunction with any specific policies on mass gatherings. an additional question that should be considered is whether non-pharmaceutical health interven-tions such as mass gathering restrictions, actually reduce the health burden of influenza, or only modify the epidemiology by temporarily delaying the eventual impact of an event such as a pandemic. it is tempting to speculate on the possibility of a reduction in disease burden; historically, some of the american cities that implemented mass gathering restrictions during the 1918-1919 pandemic seemed to achieve significant reductions in peak morbidity and mortality that may well have reduced the overall burden. however, pragmatically, countries like the united kingdom aim to achieve a slower spread, to prolong the troughs and to flatten the peaks of the epidemic curves to buy time for countermeasures to take effect and to enable services to cope with a lower volume of activity. in this context, the most important point to emphasize is that mass gathering restrictions must only be a part of a range of interventions. the synergistic effects of multiple interventions is what would allow the best chance to achieve significant reductions in overall burden. more evidence is needed on this subject. there is a need for well-designed studies to more accurately quantify the nature of the infections causing flu-like symptoms at mass gatherings, confirm the key parameters that influence the transmission of influenza in these settings, and to directly assess the impact of mass gathering restrictions. but as ''gold standard'' randomized comparison designs are extremely difficult to apply to this kind of intervention, attention needs to focus on optimal observational study options. from a uk perspective, british people travelling abroad for mass gatherings such as the hajj or other large events could be approached for inclusion in prospective studies for comparison with appropriately matched, nontravelling controls, with care being taken to avoid drawbacks observed in existing studies. within the uk, mass gatherings ranging from indoor events in theatres and cinemas, to outdoor events such as football matches and major musical events like the glastonbury festival or travellersõ horse fairs represent potential opportunities for carefully designed prospective cohort studies to be undertaken. the biggest issue is persuading funders that given the current paucity of good studies, such further research is still needed. as previously noted, this review has examined an intervention area in which there are a limited num-ber of relevant studies and there is limited common understanding of what constitutes a mass gathering. despite these challenges, it has been possible to extract limited strands of evidence that may be useful towards policy development. also, within the boundaries of the inclusion criteria, and despite the limited work timescale, this review was able to capture all the relevant studies identified by recent systematic reviews investigating non-pharmaceutical interventions designed to limit transmission of influenza and/or other respiratory viral infections [7, 8, 57] . however, due to time constraints in carrying out this work, as described in section 2.5, while every effort has been made to be as comprehensive as possible, it is acknowledged that the rapid evidence assessment process necessarily involved some limitations. the literature search is not necessarily exhaustive and there is therefore a possibility of incomplete retrieval of all potentially relevant studies. while a range of study designs were reviewed, there was not a single randomized controlled trial that was suitable for inclusion. this is of course not surprising, given the formidable logistical, cost and ethical hurdles that make large-scale experimental epidemiological studies of the restriction of mass gatherings impractical and probably impossible. some of the included studies had significant design and quality issues as duly reflected in the individual paper summaries and the discussion, and highlighted in tables 4 and 6. a common issue with a number of the studies included in the review is that they depended on clinical symptoms like cough or syndromes such as influenza-like illness, rather than laboratory diagnosis of influenza. in such studies it is not possible to isolate the impact of influenza as opposed to other respiratory viruses, and the risk of influenza could potentially be over-interpreted. in the studies where laboratory testing was undertaken, influenza was only confirmed in minority proportions of people with respiratory symptoms, ranging between 6% and 14% [23, 24, 27] , but possibly up to a roughly estimated 40% in one study [25] . this review did not include specific ''specialized'' settings that may arguably be regarded as mass gatherings, such as groups of people travelling for periods of time on cruise ships. the authors took the view that any transit-related settings would be best considered under a separate evidence review of influenza transmission through transportation. modelling studies were also excluded, and it is acknowledged that their inclusion may offer an additional dimension in order to build a fuller picture. the application of quantitative techniques could have potentially enhanced the simple narrative approach that was adopted for the analysis. however, there are insufficient studies presenting quantitative data on this subject, and moreover, there is a high level of heterogeneity amongst the identified studies. in conclusion, there are limited data indicating that mass gatherings are associated with influenza transmission. some evidence suggests that restricting mass gatherings together with other social distancing measures may help to reduce transmission. however, the evidence is not strong enough to advocate legislation or proscription. therefore, in a pandemic situation a cautious policy of voluntary avoidance of mass gatherings would be prudent. none declared. transmission of influenza a in human beings questioning aerosol transmission of influenza aerosol transmission of influenza a virus: a review of new studies mass 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measures in a pandemic responses to pandemic (h1n1) sip 5: social distancing during a pandemic. not sexy, but sometimes effective: social distancing and non-pharmaceutical interventions influenza a(h1n1) and pandemic preparedness under the rule of international law ethical and legal challenges posed by severe acute respiratory syndrome: implications for the control of severe infectious disease threats easier said than done: behavioral conflicts in following social-distancing recommendations for influenza prevention situational awareness, health protective responses to pandemic influenza a (h1n1) in hong kong: a cross-sectional study including the public in pandemic planning: a deliberative approach physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review key: cord-273147-24fkaqlz authors: brownstein, john s; wolfe, cecily j; mandl, kenneth d title: empirical evidence for the effect of airline travel on inter-regional influenza spread in the united states date: 2006-09-12 journal: plos med doi: 10.1371/journal.pmed.0030401 sha: doc_id: 273147 cord_uid: 24fkaqlz background: the influence of air travel on influenza spread has been the subject of numerous investigations using simulation, but very little empirical evidence has been provided. understanding the role of airline travel in large-scale influenza spread is especially important given the mounting threat of an influenza pandemic. several recent simulation studies have concluded that air travel restrictions may not have a significant impact on the course of a pandemic. here, we assess, with empirical data, the role of airline volume on the yearly inter-regional spread of influenza in the united states. methods and findings: we measured rate of inter-regional spread and timing of influenza in the united states for nine seasons, from 1996 to 2005 using weekly influenza and pneumonia mortality from the centers for disease control and prevention. seasonality was characterized by band-pass filtering. we found that domestic airline travel volume in november (mostly surrounding the thanksgiving holiday) predicts the rate of influenza spread (r (2) = 0.60; p = 0.014). we also found that international airline travel influences the timing of influenza mortality (r (2) = 0.59; p = 0.016). the flight ban in the us after the terrorist attack on september 11, 2001, and the subsequent depression of the air travel market, provided a natural experiment for the evaluation of flight restrictions; the decrease in air travel was associated with a delayed and prolonged influenza season. conclusions: we provide the first empirical evidence for the role of airline travel in long-range dissemination of influenza. our results suggest an important influence of international air travel on the timing of influenza introduction, as well as an influence of domestic air travel on the rate of inter-regional influenza spread in the us. pandemic preparedness strategies should account for a possible benefit of airline travel restrictions on influenza spread. the influence of air travel on influenza spread has been the subject of numerous investigations using simulation, but very little empirical evidence has been provided. understanding the role of airline travel in large-scale influenza spread is especially important given the mounting threat of an influenza pandemic. several recent simulation studies have concluded that air travel restrictions may not have a significant impact on the course of a pandemic. here, we assess, with empirical data, the role of airline volume on the yearly interregional spread of influenza in the united states. we measured rate of inter-regional spread and timing of influenza in the united states for nine seasons, from 1996 to 2005 using weekly influenza and pneumonia mortality from the centers for disease control and prevention. seasonality was characterized by band-pass filtering. we found that domestic airline travel volume in november (mostly surrounding the thanksgiving holiday) predicts the rate of influenza spread (r 2 ¼ 0.60; p ¼ 0.014). we also found that international airline travel influences the timing of influenza mortality (r 2 ¼ 0.59; p ¼ 0.016). the flight ban in the us after the terrorist attack on september 11, 2001 , and the subsequent depression of the air travel market, provided a natural experiment for the evaluation of flight restrictions; the decrease in air travel was associated with a delayed and prolonged influenza season. the influence of air travel on the geographic spread of influenza has been the subject of a number of simulation studies [1] [2] [3] [4] . discrete time seir (susceptible-exposedinfectious-recovered) models coupled with air transportation data have been used to explain the global path of influenza epidemics [3] and pandemics [5] . however, there is surprisingly little empirical information on how airline travel influences the spread of influenza through regions, nations, and across the globe. although recent work suggests high geographical coincidence of time series of influenza mortality at the hemispheric [6] and national scale [7] [8] [9] [10] , little is known about how epidemics may be connected across large areas. analyzing spatial-temporal patterns of influenza epidemics represents a critical step toward understanding how population movement contributes to epidemic fluctuations, and will help inform the evaluation of targeted control strategies. a recent study examined the between-state progression of inter-pandemic influenza in the united states and found a strong relationship with movement of individuals to and from their workplace [10] . although this local travel may be largely responsible for spread within a region (for example, a state, where travel is dominated by personal vehicular movement), inter-regional influenza spread may be more significantly influenced by long-range airline travel, which comprises almost half of all movement at distances greater than 1,000 miles and the majority of travel at over 2,000 miles [11] . understanding the role of airline travel in large-scale influenza spread is especially important given the mounting threat of an influenza pandemic [12] [13] [14] . the decision of whether travel restrictions should be put into place when a pandemic strain emerges beyond the source is currently under consideration by the world health organization [15] . in this study, we characterize the spatial variability in the inter-regional timing of the seasonal component of influenza mortality across the united states and assess its relationship to airline volume. influenza epidemics peak each year during the winter in the northern and southern hemisphere; thus, epidemics at a particular geographic location typically display strong seasonal cycles ( figure 1 ). here, we apply signal processing methods to disease surveillance data to resolve spatial-temporal patterns in the seasonal cycle of inter-regional influenza spread across the us. based on these patterns, we examine how international and domestic airline travel may influence both the introduction of new viral strains and their spread. the black line represents the aggregated national data of p&i weekly mortality. the blue line represents the seasonal influenza curve, derived by band-pass filtering the demeaned data (two-pole, two-pass butterworth, 1/64-1/40 frequency range). for comparison with the raw data, the mean is added after filtering. the filtered time series plus mean accounts for 99.8% of the mortality, indicating that most deaths are from the mean and seasonal variation and not the high-frequency cycles. (b) lines represent the raw time series data for each of the nine geographic regions of the us. (c) lines represent the seasonal influenza curves for each of the nine geographic regions of the us, derived by band-pass filtering. doi: 10.1371/journal.pmed.0030401.g001 data on weekly mortality from pneumonia and influenza (p&i) were obtained from the centers for disease control and prevention 121 cities mortality reporting system (http:// www.cdc.gov/epo/dphsi/121hist.htm) for nine influenza seasons, from 1996-1997 to 2004-2005, representing 396,506 deaths [16] . because the strength of the seasonal cycle is weak for cities with small case counts and because some city data contain missing data points, we stacked the raw city-level data to obtain composite waveforms for each of the nine major geographic regions of the united states, as defined by the centers for disease control and prevention ( figure 2 ). if the noise in the city data is random, such stacking should improve the observation of any coherent regional signal. for each region, we characterized the seasonality of p&i mortality by filtering. we use band-pass filtering to focus on the seasonality of influenza mortality (box 1). specifically, to isolate the seasonal (annual) cycles of influenza mortality we band-pass filtered each of the regional time series using a two-pole, two-pass (zero phase) butterworth filter with low and high cutoff periods of 40 and 62 wk. prior to filtering, time series were demeaned, and tapered at the ends to zero to reduce edge effects. for each influenza year, coincidence in the timing of seasonal influenza mortality across geographic regions was estimated from the phase shift with a national seasonal curve, derived by summing of all city data and filtering. we used spline resampling to achieve daily resolution. we divided the filtered data into subsets by influenza year (week 40 of one year to week 39 of the following year). we then performed cross-correlation with the national time series for each possible comparison (nine regions times 9 y) to estimate phase shifts (lag or lead times), considering a shift range of 620 wk. the phase shift with the maximum cross-correlation served as an estimate of the relative timing of the seasonal influenza curve in a given region and a given year. we also estimated the peak date of the seasonal national curve for each year. for each year, the time required for an influenza wave to spread across the us was estimated by the variability in the seasonal phase shifts for the nine regions. we used the variation in the phase shifts from the national curve for each year as estimated by the 99% confidence interval to approximate the time to transnational spread. we modeled changes in the rate of inter-regional spread of seasonal influenza mortality as a response to yearly fluctuations in domestic airline volume. monthly estimates of the sentinel cities that report mortality due to p&i used in the centers for disease control and prevention 122 cities mortality reporting system are displayed (black dots). because the strength of the seasonal influenza cycle is weak for cities with small case counts and because some city data contain missing data points, we aggregated the raw city-level data to obtain composite waveforms by major geographic region, the aerial unit of analysis for this study. doi: 10.1371/journal.pmed.0030401.g002 box 1. time series analysis is a well known method for revealing time-dependent phenomena that are not necessarily apparent in raw data. because of the strong seasonality of influenza mortality, we used band-pass filtering to isolate the patterns around the yearly (seasonal) signal of influenza. such filtering essentially smoothes over and removes variations at short time scales (such as daily changes) and long time scales (such as biennial) to isolate the coherent seasonal patterns ( figure 1 ). essentially, we extract a range of seasonal frequencies of interest from the time series while rejecting (attenuating) frequencies outside that range. our filtering approach reflects the fact that, from a time series perspective, the seasonality of influenza mortality is nearly stationary for these data in that the peaking always occurs at similar times in the winter months. this is in contrast to other infectious diseases such as measles and dengue, where strong non-stationary signals are observed and more sophisticated analysis methods, such as wavelets and empirical mode decomposition, are appropriate. passengers on domestic flights were obtained for november to january of each influenza season [17] . this range was selected because influenza activity begins to increase in november [18] , and viral isolate collections by the world health organization and national respiratory and enteric virus surveillance system (who/nrevss) collaborating laboratories show that all regions have influenza activity as of january each year. we also investigated the effect of international airline travel on the absolute timing of the seasonal peak of national influenza mortality. monthly estimates of passengers on international flights were obtained for september to november of each influenza season [17] . we selected this range as the most likely time window in which new viral strains would be introduced each influenza season. we used the peak date from the filtered national curve as the indicator of the absolute timing of influenza mortality for a given year. we fit stepwise regression models to both time to transnational spread and peak timing using domestic and international airline travel volume, respectively. a normal response distribution was used in both cases after analysis of the residuals and statistical tests of normality, including the kolmogorov-smirnov and shapiro-wilk tests. for each model, we evaluated covariates in a stepwise fashion. our model for inter-regional influenza spread included overall domestic airline volume for october, november, and december as separate covariates. our model for influenza peak included overall international airline volume for september, october, and november as separate covariates. in each case, we included a linear trend term to account for the potential effect of improved city reporting over time. we also assessed significance after applying a bonferroni correction to adjust for the effect of testing across multiple months. in order to investigate other possible contributing factors, we also included the effect of winter severity and dominant strain in our stepwise regression model [19] . first, we collected climate data to account for the effect of winter temperature. past studies have shown that colder conditions promote human indoor crowding and thus increased virus transmission and possibly a faster course of virus spread [20, 21] . we obtained data from the national climatic data center on national average winter temperature (december-february) and included this as a term in our model [22] . in addition, we examined the effects of the temperature of individual winter months as covariates. we also calculated the minimum mean temperature for a winter period and examined its effect on inter-regional influenza spread and peak. in any given season, a number of strains of varying virulence and spatial distribution can be circulating. previous research has shown that the dominant circulating subtype is associated with the impact and rate of spread of influenza epidemics [7, 10, 19] . thus, strain variation could have an effect on our measures. in order to account for this factor, we included the dominant subtype (a/h3n2 or a/h1n1) as a categorical variable in our model. finally, previous work has shown that, at the state level, time to transnational spread is influenced by the first state to be affected [10] . therefore, to account for this potential confounding, we also included the first region with activity identified with the phase shift analysis as a covariate in the model. model fitting was performed in sas version 9.0 for windows (sas institute, cary, north carolina, united states). the p&i mortality data have limitations, including spatial and temporal variation in voluntary reporting and uncertainty about the proportion of deaths attributable to epidemic influenza. therefore, we validated mortality patterns with viral surveillance data from the who/nrevss collaborating laboratories from 1997-2005. these viral data provide time series of the percentage of positive influenza specimens for an influenza season (from week 40 of one year to week 20 of the following year). high-quality data were available at the national scale for the eight influenza seasons from 1997-1998 to [2004] [2005] , and at the regional scale for the six influenza seasons from 1999-2000 to [2004] [2005] . for each season, we calculated the national peak dates of viral activity. additionally, we calculated the yearly time to transnational spread based on peak week of regional viral activity available from 1999-2005. in order to establish the causal link between flight reductions in the us after the terrorist attack on september 11, 2001, and a delayed epidemic peak, we examined whether a similar delay occurred in europe, where flight restrictions were not imposed. we obtained weekly influenza-like illness data for france from 1996-2005 from the french sentinel network. this voluntary surveillance system, active since 1984, collects reports from general practitioners across france [23] . we estimated the peak week of yearly influenza epidemics by two methods. first, we estimate peak week by the raw time series and taking the week of highest incidence. second, we applied our filtering approach described above to estimate peak influenza activity from the smoothed time series. this smoothing could potentially provide a more robust estimate of peak date. our filtering approach reflects the fact that the seasonality is nearly stationary. spectral analyses of national influenza mortality data confirm that the yearly (;52 wk) fourier component is the dominant period and that a seasonal time series plus mean can explain 99.8% of the national mortality. our analyses do not examine the high-frequency epidemic peaks, which were found to be extremely noisy and poorly defined for many influenza seasons (for example, the 2000-2001 and 2002-2003 seasons) and may be more influenced by imperfect reporting (figure 1b) . in contrast, the peaks in the seasonal curves are coherent and well-behaved across the nine regions, and therefore should be reliable proxies of peak influenza mortality ( figure 1c) . although the sequence of infection varied among regions from year to year, certain spatial-temporal patterns emerged in the seasonal component of p&i mortality (figure 3 effect of airline volume on influenza inter-regional spread and peak we found that changes in the rate of spread and timing of seasonal influenza mortality were correlated with yearly fluctuations in monthly airline volume ( figure 4a and 4b) . an inverse correlation was found between time to transnational spread of influenza and the number of traveling domestic passengers during the november to january period (pearson correlation, r ¼ à0.69; p ¼ 0.021). though each of the three months reveal an inverse relationship, we found that domestic airline volume in november was the single significant predictor of influenza spread (r 2 ¼ 0.60; p ¼ 0.014) (figure 4 ). this relationship was especially evident for the 1997-1998 season, in which there was both the lowest airline volume (39 million passengers) and the slowest spread (26 d). a strong inverse correlation was found between the timing of an influenza season and the numbers of traveling international passengers between september and november (pearson correlation, r ¼ à0.66; p ¼ 0.027) (figure 4 ). in this case, although all three months showed an inverse relationship, international travel in september was the single month to significantly predict the seasonal national peak (r 2 ¼ 0.59; p ¼ 0.016). this is especially evident during 2001-2002, when international flight volume decreased by 27%, from 4.9 million international passengers to 3.5 million, and peak influenza mortality was delayed by 2 wk. furthermore, we also found a delayed peak during the 2002-2003 season, when international airline travel was down by 10% because of the residual effect of the events of september 11, 2001, on travel behavior. a continued trend back to baseline peak was found during the 2003-2004 and 2004-2005 seasons as international airline activity resumed its levels of before september 11, 2001 . relationships for both influenza spread and peak were still significant after application of the bonferroni correction to account for multiple testing of individual months (alpha ¼ 0.017). we did not find a significant relationship between climate and inter-regional influenza spread. although we did find a 2001-2002 warm temperature spike and a positive relationship between hot temperatures and late peaking, this relationship was not significant and dropped out of the however, the 1999-2000 season was the warmest november-february period since 1895 and yet had an earlier than average national influenza peak. we also considered winter months separately, as well as overall minimum mean winter temperature, but none were significant predictors. in addition, strain type did not account for any significant amount of the variability in transnational spread or peak time of influenza mortality. finally, first region affected was also not a significant explanatory variable (see protocol s1). viral data from the who/nrevss collaborating laboratories were used to validate seasonal patterns obtained from the filtered mortality data. we found that peaks in the seasonal mortality data occurred about a month after those in the viral data (mean delay ¼ 30.8 d; 95% confidence interval: 9.1-52.4 d). the estimated spread and peak of the filtered mortality and viral data were well correlated, with spearman rank correlations of 0.928 (p ¼ 0.004) and 0.695 (p ¼ 0.028), respectively. our validation with the viral data indicates that although the absolute scaling between influenza activity and seasonal mortality differs, the relative ordering of peak dates and time to transnational spread between these two datasets is consistent. furthermore, analysis of the viral surveillance data confirms the effect of september 11, 2001 . we found a significantly longer time to transnational spread and a delayed peak date for the 2001-2002 season. the time to transnational spread was 53 d, 60% longer than the average of 33 d, which is a statistically significant difference (p , 0.001). the national viral peak date for the 2001-2002 season was calculated at february 23, significantly later than the average of january 20 across the other seasons (p ¼ 0.012). unlike in the united states, we did not see a similarly delayed peak of influenza activity during the 2001-2002 season in france, where flight restrictions were not imposed. for estimation based on both the raw and filtered time series, the defined peak during this season was estimated at the fourth week in january, 2002. this peak week was not significantly different than that of the eight other influenza seasons (for the raw time series, mean peak was the fourth week; 95% confidence interval: 0-9 wk; for the filtered time series, mean peak was the third week; 95% confidence interval: 1-5 wk). this result provides further evidence that the delayed 2001-2002 us influenza mortality peak was linked to the flight restrictions following the events of september 11, and the subsequent depressed air travel market. this study is an empirical analysis of the spatial-temporal pattern of inter-regional influenza spread across the united states and provides evidence for factors that influence it. whereas previous simulation models have suggested that air travel may play an important role in the spread of annual influenza [1, 3] , we provide what is to our knowledge the first empirical evidence to confirm the effect airline volume on long-range spread. our findings suggest that once intro-duced, new viral strains are likely to spread rapidly across geographic regions. furthermore, though between-state movement may be driven primarily by workflows [10] , our results suggest that inter-regional spread occurs by a different mechanism, where air travel may be an important mode of long-range dissemination of influenza. we find that the effect of airline volume on regional influenza spread is largely based on travel in november. though influenza activity is highest between january and march, initial regional seeding of infection may occur earlier. our results suggest that for a non-pandemic year, travel during the thanksgiving holiday may be central to the yearly national spread of influenza in the us. similarly, we found that international airline travel influences the absolute timing of seasonal influenza mortality. the flight ban in the us after the terrorist attack of september 11, 2001 , and the subsequent depression of the air travel market provided a natural experiment for the evaluation of the effect of flight restrictions on disease spread. the importance of airline activity was highlighted by the delayed peak of influenza in 2001-2002 following the period of reduced flying activity. this finding is further validated by the absence of a similar delay in influenza activity in france, where flight restrictions were not imposed. our model suggests that september may be the critical month for entry of new influenza strains into the us from foreign countries, earlier than the established start of the us influenza season in october/november. although seasonal influenza activity usually begins to increase as early as october or november, current laboratory surveillance by the who/nrevss collaborating laboratories consistently collects viral isolates in its first week of testing (week 40; first week of october). over the last eight influenza years (1997-1998 to 2004-2005) , 0.62% (standard deviation ¼ 0.59%) of specimens on average test positive for influenza in the first week of october, indicating that the introduction of new viral strains has already occurred in september. indeed, new antigenically distinct strains result from a continuous evolutionary process of small changes in influenza surface antigens and are not limited to a given location or time period [24] , and therefore international travel in september can surely not be the only mechanism of strain introduction. while our study suggests that airline passenger volume explains about 60% of the inter-annual variation in interregional influenza spread and peak, there is still an unexplained component. the timing of seasonal influenza mortality could reflect the additional influences of climatic conditions [19, 25] rather than solely the introduction of new strains into a susceptible population by airline travel. however, we find that monthly national temperatures were not a significant predictor in our models. another issue is that strain variation could have an effect on our measures. our models included a term for dominant subtype, which was not found to be significant. recent studies have shown that influenza spreads more efficiently during seasons dominated by subtype a/h3n2 than when a/h1n1 or b dominate [7, 9, 19, 26] . for instance, the 1981-1982 and 1990-1991 seasons, which were dominated by influenza b, were substantially less synchronous than other seasons of the 1980s and 1990s [10] . interestingly, the 2001-2002 season (after september 11, 2001) , where we found delayed spread, was dominated by a/h3n2 circulation. in this case, lack of synchrony cannot be explained by dominant subtype, which suggests that other factors (including reduced airline travel) may have been responsible. in fact, our study period from 1996 to 2005 represents the longest stretch of a/h3n2 season in over 30 y (seven out of nine seasons were dominated by a/ h3n2), essentially controlling for the effect of subtype. yearly changes in public health intervention strategies, such as vaccination campaigns, and vaccine efficacy may also affect patterns of spread. improved vaccination coverage or strain match in a given season would decrease the rate of disease transmission and thus also slow the rate of spatial spread [27, 28] ; such a scenario could have potentially caused the delay that occurred in 2001-2002. studies of influenza mortality based on us multiple cause-of-death files [29] provide longer and more comprehensive time series, and enable a more detailed analysis of these multiple effects. however, yearly data become available only after 3 y, and therefore cannot be used for the current analysis. in contrast, data from the 121 cities mortality reporting system provide a more current time series of influenza mortality that is available for examining the recent fluctuations in human travel, including evaluating the effect of the 2001-2002 flight reduction on influenza spread. our study does, however, have certain limitations that are inherent in the use of mortality data from the 121 cities mortality reporting system. one limitation is associated with the voluntary design of the system. there is variability in time of filing of death reports from week to week because of changes in volunteer staff and insufficient staff to keep up with reporting during the peak of the influenza season. we observed that reporting quality varies with both time and city, as evidenced by the presence of gaps (weeks with no data) and anomalous behavior in some of the city time series. we therefore stacked the raw city data according to major geographic regions. this stacking enabled us to extract coherent regional seasonal signal from the p&i data. these results lead us to believe that the noise in the city data was random and that there were no systematic biases that would account for our findings. furthermore, p&i mortality has been validated as a good relative proxy for the severity of influenza epidemics [30] . thus, the use of these p&i data to estimate relative seasonal curves of influenza mortality should be appropriate as well. we used influenza mortality time series data, which may not correspond precisely to influenza activity. p&i mortality reflects a somewhat uncertain mixture of deaths from influenza and other respiratory diseases, and the proportion of influenza deaths may vary with time. furthermore, although influenza strikes all age groups, non-pandemic influenza mortality predominantly affects the elderly, and older age groups typically peak later, while young children peak earlier [31] . there may also be other factors related to the biology of disease progression and associated complications that cause timing differences between influenza morbidity and mortality. however, our analyses show that influenza mortality patterns correspond with trends in virological data from laboratory surveillance, which suggests that we have captured a true pattern of influenza timing and spread. although a more detailed national dataset of confirmed influenza infections and matched strain information would be ideal, our study demonstrates how analyses of imperfect influenza surveillance data can reveal important spatial-temporal trends, providing potentially vital information for disease prevention and control. the alarming spread of the highly pathogenic avian influenza a (subtype h5n1) in both wild and domestic poultry in southeast asia and europe [32, 33] , with probable human-to-human transmission [34, 35] , has intensified the debate over whether border control and travel restrictions could substantially impede the spatial spread of an emerging pandemic strain. our results suggest that limiting domestic airline volume would have a measurable impact on the rate of spread of an influenza pandemic, and particularly on spread across regions. because influenza pandemics have shown unusual spatial and demographic patterns as well as higher basic reproductive number due to lack of immunity, the relationship between air travel volume and domestic influenza spread may nonetheless be different in a pandemic scenario [36, 37] . however, our finding that international travel influences the timing of epidemic influenza should apply directly to a pandemic scenario, where the objective will be to reduce the probability of strain introduction. recent individual-based simulation models of pandemic influenza transmission have attempted to model the effectiveness of social distance measures, including travel restrictions [38, 39] . while eliminating travel in and out of affected areas along with imposing border restrictions may provide some relief by delaying spread by up to 8 wk [38] , drastic reductions in domestic travel are required to have much impact internally [38, 40] . although these simulation studies have found that these strategies may not have a significant impact on the course of a pandemic, the models lack parameterization of the underlying relationship between air travel and influenza spread based on experimental data. future work using simulation will benefit greatly from parameter estimates based on empirical findings such as those presented here. although the mechanisms driving the seasonality of influenza epidemics are still not well understood, our findings do suggest that fluctuations in airline travel have an impact on large-scale spread of influenza. at the regional level, our results suggest an important influence of international air travel on influenza timing as well as an influence of domestic air travel on influenza spread in the us. however, for the global influenza pandemic widely believed to be inevitable [41] , the efficacy of travel advisories, flight restrictions, or even complete flight bans as a control measure is still uncertain. though our results suggest a possible benefit 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logistic and legal limits, and possible consequences we gratefully acknowledge the thoughtful input of cé cile viboud, ben reis, isaac kohane, donald goldmann, gary fleisher, and jonathan abbett.author contributions. jsb, cjw, and kdm designed the study, analyzed the data, and contributed to writing the paper. background. in both the northern and southern hemispheres, influenza epidemics occur annually during the winter ''flu season.'' although the disease maps out a remarkably similar pattern in most years, little is known about the specific mechanisms by which geographic spread occurs. given the perennial possibility of influenza global epidemics (pandemics) such as occurred in 1918, 1957, and 1969 , as well as the more recent, localized outbreaks of avian influenza (''bird flu'') in which a high proportion of affected people have died, we need to understand how influenza spreads in order to limit the destructive impact of future pandemics.why was this study done? in theory, airline travel might be expected to play a role in the spread of influenza across large distances. if so, reducing or restricting air travel might be an appropriate public health intervention in the early stages of an influenza pandemic. this study was performed to identify specific effects of air travel on the annual spread of influenza in the united states.what did the researchers do and find? the researchers analyzed weekly government records on deaths from influenza and pneumonia in cities from nine regions of the us during the nine influenza seasons between 1996 and 2005. for each year, they determined the time it took for the epidemic to spread across the us and the date of the national peak in influenza deaths. they then used government estimates of passenger air travel to explore any connection with the timing of the annual flu epidemics.the analysis found that the usual time for an influenza epidemic to reach peak levels across the us was approximately two weeks, and that the national peak date fell within two days of the average date, february 17, in five of the nine seasons. in general, influenza was found to spread more slowly during years when the number of domestic air travelers, particularly during november, was lower. also, the peak of the influenza season was found to come later during years when the number of international air travelers, particularly in september, was lower. these results, based on reported deaths from pneumonia or influenza, were corroborated using data from an influenza virus surveillance program, and could not be explained by variations in winter temperatures or by different types of influenza virus circulating in different years.of note, the peak date of the us influenza season following september 11, 2001 , was delayed by 13 days to march 2, consistent with marked reductions in airline travel following the terrorist attack, and then returned to february 17 over the subsequent two influenza seasons as international airline travel returned to its previous levels. in contrast, the investigators found no delay in the 2001-2002 influenza season in france, where flight restrictions were not imposed.what do these findings mean? while this study does not demonstrate that travel restriction would be effective in altering the course of a flu pandemic, it does provides evidence that air travel plays a significant role in the annual spread of influenza in the united states. although other factors, related or unrelated to the decrease in air travel after september 11, may have affected the course of the 2001-2002 influenza season, the general findings across several years suggest that air travel affects both the peak date and the rate of spread of influenza. these findings merit consideration in the process of preparing for the next influenza pandemic.additional information. please access these web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed. 0030401.world health organization: influenza pandemic preparedness page us department of health and human services: avian and pandemic flu information site wikipedia page on influenza pandemic (note: wikipedia is a free internet encyclopedia that anyone can edit) key: cord-296998-ep46lzeo authors: pawelec, graham; mcelhaney, janet title: recent advances in influenza vaccines date: 2020-04-28 journal: f1000res doi: 10.12688/f1000research.22611.1 sha: doc_id: 296998 cord_uid: ep46lzeo seasonal influenza remains a major public health problem, responsible for hundreds of thousands of deaths every year, mostly of elderly people. despite the wide availability of vaccines, there are multiple problems decreasing the effectiveness of vaccination programs. these include viral variability and hence the requirement to match strains by estimating which will become prevalent each season, problems associated with vaccine and adjuvant production, and the route of administration as well as the perceived lower vaccine efficiency in older adults. clinical protection is still suboptimal for all of these reasons, and vaccine uptake remains too low in most countries. efforts to improve the effectiveness of influenza vaccines include developing universal vaccines independent of the circulating strains in any particular season and stimulating cellular as well as humoral responses, especially in the elderly. this commentary assesses progress over the last 3 years towards achieving these aims. since the beginning of 2020, an unprecedented international academic and industrial effort to develop effective vaccines against the new coronavirus sars-cov-2 has diverted attention away from influenza, but many of the lessons learned for the one will synergize with the other to mutual advantage. and, unlike the sars-1 epidemic and, we hope, the sars-cov-2 pandemic, influenza will not be eliminated and thus efforts to improve influenza vaccines will remain of crucial importance. despite the common perception that it is "only a flu", seasonal influenza is a powerful pathogen responsible for many hundreds of thousands of deaths every year, especially of elderly people. it is somewhat puzzling that a highly contagious pathogen responsible for an estimated average of half a million fatalities every year is faced with such insouciance by most people. indeed, globally, 300,000 to 700,000 people die from the consequences of respiratory complications of influenza each year, with a huge difference in mortality rates according to age: possibly a vanishingly small number of younger adults (0.001%) compared with 0.03% of people aged 65-74 but rising to 0.1% of people over 75 1 . although this may seem to be a low chance of death caused directly by influenza, indirect sequelae of influenza infection contribute to a deteriorated health status and frailty in the elderly. these long-term sequelae are not limited to the respiratory tract but are increasingly associated with systemic, especially cardiovascular 2 , symptoms. vaccination is a highly effective, minimally invasive, and cheap protective measure. however, there are many unsolved problems associated with the current seasonal influenza vaccines. thus, advances in effective influenza vaccination must include increasing the protective efficiency of the vaccine, especially in the elderly. this review will focus on recent advances in the science and the r&d, but one should not forget that the sociology of enhancing acceptance and uptake is of paramount importance too. predicting the next season's predominant influenza strains is a major undertaking that is always fraught with difficulty and often incorrect. recent advances in surveillance, data exchange, and bioinformatics may help to mitigate this problem. unexpectedly, help may be at hand from the surveillance of social networking sites, which may yield more topical data than public health services 3 . however, instead of chasing seasonal variations, it would be much more advantageous to develop vaccines that were effective against all influenza strains, hence the intensive efforts to develop "universal vaccines" that will protect regardless of the seasonal strain. many such efforts focus on directing antibody production away from targeting parts of the virus that are different from strain to strain (i.e. the highly variable hemagglutinin [ha] head structures) and towards generating antibodies against conserved antigens. these may be from the stem part of the molecule, which is not normally immunodominant. these antibodies should provide heterosubtypic protection, i.e. against multiple different strains 4 . however, until recently, this approach was limited to one viral group, but modifying the glycosylation state of stem regions may increase antibody accessibility and broaden the range of strains targeted 5 . it is not known whether these experiments in mice or even in ferrets reflect what would be effective in humans. in naturally acquired h1n1 infection, at least, it seems that anti-head and not anti-stalk antibodies play a predominant role, as expected 6 . nonetheless, in passive immunisation studies, monoclonal antibodies against stem antigens can be protective in human influenza challenge 7 . other approaches include active immunisation with multi-epitope protein vaccines containing several from influenza a and b, common to multiple strains of influenza virus. there is some evidence in humans that vaccination in one season may confer protection in the next season against strains that were not circulating at the time of the earlier vaccination. an analogous approach employing mixtures of synthetic peptides is also being pursued by other companies, for example 8 . an advantage of this approach, analogous to that in cancer vaccines, is to select epitopes stimulating both humoral and cellular immunity; indeed, an interesting aspect of the action of the m-001 vaccine 9 is that it does not contain any ha head epitopes and stimulates predominantly cellular responses. another approach attempts to exploit an elegant idea to focus antibody responses on the stalk by vaccinating with stalk domains engineered onto different head domains, which were shown to be protective in mice 10 . a very recent publication now reports the outcome of a phase i clinical trial concluding that high anti-stalk titres were induced and paves the way for further development of universal influenza virus vaccines 11 . a major bottleneck in influenza vaccine production is the inability to generate large amounts of vaccine quickly. one problem here resides in methodology for producing the vaccine, which requires improvement. the technique still employed by most vaccine producers is to grow the virus in hens' eggs. alternatives are being energetically sought after, including cell culture approaches and genetic engineering of viral components. there are several reasons for this, not only to speed up the cumbersome process of growing the virus in eggs (not to mention allergy problems) but also because in some cases egg-adapted viruses used to make the vaccine are not identical to the wild-type pathogen in circulation that season and do not protect 12 . thus, it was concluded that the quadrivalent vaccine "flucelvax" (grown in cultured canine kidney cells) was potentially superior to egg-based vaccines in real-life practise as well as in clinical trials 13 , and importantly a similar trivalent vaccine was reported to be effective in individuals over 60 years of age 14 . an alternative approach dispensing with the need for using live viruses has now reached fruition in the "flublok" vaccine using cultured insect cells to produce the vaccine after their infection with genetically engineered baculovirus vectors. employing a direct comparison with egg-grown virus vaccines, researchers reported that the recombinant vaccine was more efficacious, also in older adults 15 . the use of recombinant technology obviates the need for using live viruses, and once the sequence is known, production can be much more rapid, which is important for combating new and emerging strains. it may also be possible to simplify production even more by producing recombinant vaccines in plants, which can result in very rapid synthesis of viral proteins. plant-based vaccines have not yet entered clinical trials, but experiments in mice have suggested that such virus-like particles are protective, even in very old animals 16 . some investigators go even further and propose that mrna itself can be used as a vaccine without the need to produce viral proteins outside of the host at all. this results in probably the most rapid pipeline for developing prophylactic vaccines. to stabilise the rna, it can be enclosed in liposomes, as in the mrna-1851 phase i trials in miami and berlin, showing immunogenicity and good tolerance 17 . adjuvants are non-immunogenic vaccine components that enhance immunity in different ways: by a depot effect or by stimulating antigen-presenting cells, for example 18 . optimal vaccine formulations will of course depend on the route of administration. all of these considerations have been receiving a great deal of attention over the last 3 years. new data on the use of well-established adjuvants such as m59 or even alum reflect the rather surprising relative paucity of information on their action 19,20 . work on developing new adjuvants, such as "self-adjuvanting" lipid nanoparticles including toll-like receptor (tlr) ligands, is beginning to deliver encouraging results 21 . mouse models are revealing that responses to intranasal vaccines may also be enhanced by the inclusion of adjuvants such as the tlr ligand cpg 22 . increasing attention is being paid to testing new adjuvants not only in young animals but also in those of advanced age, the most susceptible group 23 . mouse models are suitable for testing the use of live attenuated viral vaccines, rather than the non-viable immunogens usually employed. thus, the effects of different interferons on outcomes can be assessed using genetically deficient mice, for example 24 . however, mice are not people, and proof of the pudding must be rcts in humans, but there remain relatively few adjuvants in common use so far and this differs in different countries [25] [26] [27] . developing better adjuvants is a high priority, especially for vaccination of the elderly 28 . current discussions centre around the practicalities and ethics of human challenge models for assessing vaccine efficacy in the most relevant possible "model" 29 . flu vaccines are mostly injected intramuscularly (i.m.), which may be suboptimal. alternative routes include intradermal (i.d.), oral, or inhaled. regarding the route of administration, oral vaccines using live attenuated viruses have not yet found widespread application since pilot studies in humans in 2016 30 , but in the meantime different approaches are being tested to protect various forms of immunogen from degradation in the stomach 31,32 . the expectation that i.d. administration might be more effective than the usual i.m. injection seems not to have been fulfilled, judging from the data thus far accumulated. for example, a comparison of recombinant vaccine followed by trivalent inactivated vaccine given i.d. or i.m. revealed no differences 33 . there were some earlier data suggesting that for the elderly the i.d. route might be more efficacious, but differences were not large 34 . other variant application routes are being examined in mice, for example, the so-called "primepull" strategy whereby i.m. priming is boosted by inhalation 35 . a very recent study employed intranasal administration of a novel adjuvanted vaccine to mimic natural influenza infection and to activate cd8 + t cells in situ in the lungs. this sophisticated approach employed 2',3'-cyclic guanosine monophosphateadenosine monophosphate (cgamp) as an "adjuvant" to activate the innate immune sensor stimulator of interferon genes (sting) targeted to lung-resident alveolar macrophages and alveolar epithelial cells (aecs). moreover, encapsulation of cgamp with pulmonary surfactants enabled stimulation of sting in aecs while sustaining the barrier of the pulmonary surfactant appropriately. in this way, intranasally administered vaccine resulted in protection against multiple strains of influenza in mice and ferrets, associated with both humoral and cellular responses 36 . current vaccines are licensed based on world health organization-approved centres that test efficacy solely in terms of standard measures of humoral immunity. subjects do not necessarily include elderly people, who may not respond as well as younger adults, and differences in responses between older men and women 37 are not taken into account. measured parameters defining responses on the basis of which vaccines are licensed may not always be appropriate: for example, the requirement for an increase in antibody titre to two of three strains in the vaccine may miss the third one that could be critical that season. also, non-response could erroneously be attributed to subjects who already have a high (protective) titre of antibody pre-vaccination and cannot increase it more. these measures of vaccine responsiveness thus cannot predict clinical protection. in this context, advances in molecular analysis of the immune response may lead to insights on individual variability and guide vaccine design and application 38, 39 . hence, better predictive biomarkers are required, particularly considering the essential component of t cell immunity, which is not usually measured in the assessment of vaccine efficacy prior to licensing. over the last 3 years, the realisation that vaccines need to be formulated to take the importance of the t cell response into account has come to the fore. this is a crucial issue not only because t cells are required to eliminate infected host cells prior to viral release but also because the epitopes recognised by t cells tend to be conserved across viral strains 40 . thus, efforts to develop new and improved vaccines increasingly aim to generate cellular as well as humoral responses 41 . moreover, the type of cellular response achieved is likely to be of major importance 42 , and this can be markedly influenced by the nature of the adjuvant 43 as well as by multiple host factors including frailty 28 and medication 44, 45 . the impact of frailty is currently being intensively investigated, with some studies clearly documenting an important influence on effectiveness 46 while others do not 47 . the reasons for such discrepancies are likely to be multifactorial and are not yet clarified but may at least partly reside in definitions of frailty and pre-frailty 48 as well as the population studied and subject selection 49 . in particular, there has been some controversy regarding whether repeated annual vaccinations with the same antigens might result in decreased responsiveness 50 , although recent studies suggest that this is not likely to be the case 51 . confounding factors could also include exposures over the life-course that may have thus far under-investigated effects, such as exposure to ionising radiation 52 . it has also been noted that obesity can dampen influenza vaccine efficiency (although, importantly, it did not decrease efficacy, i.e. the serological response) 53 . other important host factors probably include the influence of infection with hiv, even when controlled 54 , or with other persistent latent viruses. one of the latter is most notably cytomegalovirus (cmv), which is also likely to play a role, although its impact on humoral responses remains controversial 55 . this may at least partly be due to the fact that cmv infection mostly affects t cell responses, with only knock-on effects on antibody levels, especially in the elderly 56 . the main mechanism responsible may be cmv-driven impaired granzyme b responses in influenza-specific cytotoxic cd8 + t cells and higher levels of il10 57 , either human or cmv decoy derived 58 . finally, an impact of the microbiota, usually taken to refer to the gut microbiota, is emerging as an important confounding factor in responsiveness to influenza vaccination 59 . attempts to manipulate the microbiota to enhance responsiveness are being tested in clinical trials using probiotics 60 or synbiotics 61 , so far without resounding success but with some recent evidence of efficacy in the elderly 62 . an extensive "super-meta-analysis" of 28 studies published at the end of 2019 investigated the state of knowledge on the impact of "intravenous drug use, psychological stress, acute and chronic physical exercise, genetic polymorphisms, use of pre-/pro-/symbiotics, previous bacillus calmette-guã©rin vaccination, diabetes mellitus, vitamin d supplementation/deficiency, latent cmv infection and various forms of immunosuppression" on responses to influenza vaccination 63 . this study concluded that "while the inhibiting effect of several immunosuppressive host factors was evident, the enhancing effect of pro/pre/symbiotics and chronic physical exercise was doubtful and virus type-specific (a but not b)" and that "studying the host-related correlates of the influenza vaccine-induced immune response could contribute to the production of new personalized vaccines and to the development of new patient-oriented vaccination strategies in a value-based public health perspective" 63 . nonetheless, there are also detailed studies documenting improved cellular and humoral responses in elite athletes, so the degree of exercise and overall fitness may be crucial to seeing increased responsiveness 64 . thus, these issues all remain a focus of intensive research which undoubtedly carry a great deal of relevance not only for influenza vaccination but for responses to other pathogens as well. moreover, benefits of effective vaccination to prevent influenza may be felt in unrelated areas of healthcare owing to their indirect impact on other diseases as diverse as lung cancer 65 and in particular cardiovascular disease 66 . while academic research teams are working on the development of "universal influenza vaccines" and other improvements and advances, one should not forget that the regulatory pathway to the approval of these new vaccines is often referred to as the "valley of death": many new vaccines never make it through the many steps in the approval process. the cost of making a new influenza vaccine is estimated at $1 billion and takes 10-15 years. even the cost of annually refreshing the influenza strains contained in the current seasonal vaccines is estimated to be $5-18 million per year. these are significant non-scientific hurdles that need to be overcome when considering recent advances in influenza vaccines (https://www. wired.com/story/flu-vaccine-big-pharma/). nonetheless, a recent analysis suggests that despite the drawbacks of current seasonal influenza vaccines, there is a huge public health and public financial benefit to the use of influenza vaccines so that further improvements would make a big impact 67 . as vaccines produced in cells rather than eggs become more generally available, some analyses are concluding that in addition to other potential advantages (see above), they may also be more cost-effective 68 . as with other areas of medicine, a "one-size-fits-all" approach to influenza vaccination will never be optimal for every individual. in particular, the state of health and pre-exposures of the vaccinee will be highly influential in determining the success of the vaccine. in an ideal situation, prior to vaccination, the immunological history of the person would be assessed from a small blood sample. this would determine the state of humoral and cellular immunity as it pertained to influenza reactivity and the composition and nature of the vaccine modified accordingly. if the individual manifested problems regarding deficits in the presence and functions of antigenpresenting cells, steps would need to be taken to adjust adjuvants or vaccine antigens, or even replace defective antigen-presenting cells with artificial engineered constructs 69 , which might one day be possible in vivo. similarly, if the t cell or b cell repertoire lacked cells with the appropriate antigen receptors, these could be engineered in. this scenario is admittedly highly unlikely, even in the not-so-near future, but technological progress in these areas, partly driven by efforts of cancer therapy researchers 70 , has been so rapid that such an individualised approach may become feasible at some point. in the past 3 years, there has seen steady progress in the science behind the development of improved influenza vaccines, but the main hurdles have not yet been overcome. these remain the continued necessity for producing seasonal vaccines rather than a universal vaccine, the mode of production (egg, cultured cells, recombinant products), the development of better adjuvants, the focus on humoral and under-appreciation of cellular immunity, and perceived problems of immunosenescence 28,71,72 , as well as poor vaccine uptake in most countries. although practical progress has been slow, results from the last 3 years encourage the belief that significant inroads will be made over the next 3 years. estimates of global seasonal influenza-associated respiratory mortality: a modelling study impact of early life exposure to ionizing radiation on influenza vaccine response in an elderly japanese cohort. vaccine obesity impairs the adaptive immune response to influenza virus impact of aging and hiv infection on serologic response to seasonal influenza vaccination effect of latent cytomegalovirus infection on the antibody response to influenza vaccination: a systematic review and meta-analysis pubmed abstract | publisher full text | free full text cytomegalovirus seropositivity predicts a decline in the t cell but not the antibody response to influenza in vaccinated older adults independent of type 2 diabetes status pubmed abstract | publisher full text | free full text influenza vaccine-mediated protection in older adults: impact of influenza infection, cytomegalovirus serostatus and vaccine dosage pubmed abstract | publisher full text | free full text human cytomegalovirus ul111a and us27 gene products enhance the cxcl12/cxcr4 signaling axis via distinct mechanisms pubmed abstract | publisher full text | free full text antibiotics-driven gut microbiome perturbation alters immunity to vaccines in humans pubmed abstract | publisher full text | free full text | f1000 recommendation the influence of probiotics on vaccine responses -a systematic review impact of ageing and a synbiotic on the immune response to seasonal influenza vaccination; a randomised controlled trial pubmed abstract | publisher full text | free full text effects of lactobacillus coryniformis k8 cect5711 on the immune response to influenza vaccination and the assessment of common respiratory symptoms in elderly subjects: a randomized controlled tria mapping host-related correlates of influenza vaccine-induced immune response: an umbrella review of the available systematic reviews and meta-analyses. vaccines (basel) elite athletes on regular training show more pronounced induction of vaccine-specific t-cells and antibodies after tetravalent influenza vaccination than controls effect of annual influenza vaccination on reducing lung cancer in patients with chronic obstructive pulmonary disease from a population-based cohort study pubmed abstract | publisher full text | free full text | f1000 recommendation beneficial effects of vaccination on cardiovascular events: myocardial infarction a review of the cost-effectiveness of adult influenza vaccination and other preventive services editorial note on the review process are written by members of the prestigious . they are commissioned and f1000 faculty reviews f1000 faculty are peer reviewed before publication to ensure that the final, published version is comprehensive and accessible. the reviewers who approved the final version are listed with their names and affiliations.the reviewers who approved this article are: the benefits of publishing with f1000research:your article is published within days, with no editorial bias you can publish traditional articles, null/negative results, case reports, data notes and morethe peer review process is transparent and collaborative key: cord-315339-dcui85lw authors: broadbent, andrew j.; boonnak, kobporn; subbarao, kanta title: respiratory virus vaccines date: 2015-03-13 journal: mucosal immunology doi: 10.1016/b978-0-12-415847-4.00059-8 sha: doc_id: 315339 cord_uid: dcui85lw this chapter reviews the main viral pathogens of the respiratory tract, the immune responses they induce, currently available vaccines, and vaccines that are in development to control them. the main viruses responsible for acute respiratory infection in people include respiratory syncytial, influenza, human parainfluenza, human metapneumo-, human rhino-, corona-, and adenoviruses. licensed vaccines are available only for influenza virus, with vaccines against the other pathogens either in clinical trials or in preclinical stages of development. the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies are known to protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. as we breathe, we sample an estimated 10 l of air per minute (kohlmeier and woodland, 2009 ). the mucosa of our respiratory system is, therefore, in direct and continual contact with the environment and, as such, is highly exposed to microorganisms, some of which may be pathogenic. respiratory infections are among the leading causes of acute illness and mortality worldwide, being responsible for nearly 4 million deaths annually, the majority of which occur in infants and children in developing countries (girard et al., 2005) . the main viruses responsible for acute respiratory infection include respiratory syncytial virus (rsv), influenza virus, human parainfluenza virus (hpiv), human metapneumovirus (hmpv), human rhinovirus (hrv), coronaviruses, and adenoviruses. however, despite the public health importance of these infections, licensed vaccines are currently available only for influenza viruses. protective immunity against respiratory virus infection is a complex interplay between systemic and mucosal responses. however, immune responses generated during a natural infection may not provide complete protection from reinfection and may actually contribute to the pathogenesis of disease (reviewed in sections pathogenesis and immune responses to respiratory virus infection). vaccine-induced immune responses must, therefore, aim to be more protective and less pathogenic than those induced naturally. in addition, our understanding of the relative contribution of mucosal and systemic immunity to protection remains incomplete. for example, it is well known that inactivated vaccines against influenza given intramuscularly (i.m.) are protective owing to the induction of systemic humoral immunity in the absence of a robust mucosal immune response, and current guidelines for vaccine licensure require influenza vaccines to induce systemic immune responses. however, it is also evident that some intranasal vaccines are protective owing to the induction of mucosal immunity, despite less impressive systemic immune responses (reviewed in section respiratory virus vaccines). unfortunately, standardized methods of measuring mucosal immune responses are lacking, and reliable correlates of protection for vaccines that protect through mucosal immunity have not been identified. in this chapter, we review the main viral pathogens of the respiratory tract, the immune responses they induce, current vaccines, and vaccines that are in development to control them. the viruses that infect the respiratory tract belong to various families and vary in their genome composition, the presence or absence of an envelope, and their replicative cycles. the majority of respiratory viruses that are responsible for acute respiratory infection belong to the paramyxoviridae family and include rsv, hpiv, and hmpv. these viruses infect cells lining the respiratory tract by first attaching to the cell through the interaction of viral envelope glycoproteins, with one or more cellular receptors in the host cell plasma membrane. for example, the hpiv envelope protein hn binds sialic acid residues extending from host cells (schomacker et al., 2012) , and the g protein of rsv and hmpv binds to glycosaminoglycans (gags) that comprise long chains of disaccharides that form part of the cellular glycocalyx (feldman et al., 1999) . the rsv and hmpv f protein are also known to bind gags, and findings indicate that the f protein of these viruses is involved in attachment by interacting with the cellular proteins nucleolin and integrin αvβ1, respectively (tayyari et al., 2011; cseke et al., 2009) . upon binding to the host cell, the f protein undergoes a conformational change that exposes a hydrophobic fusion peptide, which is responsible for the fusion of the paramyxovirus envelope and the host cell plasma membrane. after viral fusion, the genome is released into the cytoplasm, viral genes are transcribed, and viral genomes are replicated (collins and crowe, 2007; collins and melero, 2011) . the paramyxovirus genome comprises single-stranded, negative-sense, nonsegmented rna. the viral rna (vrna) must first be transcribed into positive-sense messenger rna (mrna) before viral proteins can be translated by the host cell machinery. this is achieved by a viral rna-dependent rna polymerase (the large, l, protein) that is packaged into the virion and enters the host cell upon infection. the l protein is also responsible for genome replication, in which positive-sense complementary rna (crna) serves as an intermediate template for the production of vrna. an essential cofactor for the l protein is the phospho (p) protein, which tethers the polymerase so it can reach the bases in the vrna and also binds the n protein, which encapsidates the vrna and crna. there is also evidence that transcription is enhanced by the m2-1 protein and that the switch from transcription to replication is mediated by the m2-2 protein (collins and crowe, 2007; collins and melero, 2011) . once transcribed, viral structural proteins assemble and newly synthesized viral genomes are packaged into virions that bud from the plasma membrane. the matrix, or m protein, lines the inner surface of the viral envelope and may play a role in budding (henderson et al., 2002; teng and collins, 1998) . in addition, the hn protein of hpiv is also involved in budding and in clearing sialic acid residues from the plasma membrane (karron and collins, 2007) . to complete the replication cycle, paramyxoviruses have evolved multiple mechanisms to prevent the activation of cellular defenses in response to infection, such as the nonstructural (ns) proteins 1 and 2 of rsv (collins and crowe, 2007) and the c or v proteins of hpiv (karron and collins, 2007) . one additional protein found in paramyxoviruses is the short transmembrane glycoprotein (sh) that is anchored to the envelope and shares structural features with viroporins, a group of hydrophobic molecules that insert into the membrane of infected cells and increase their permeability to small molecules and ions (gonzalez and carrasco, 2003) . the orthomyxoviridae family includes influenza viruses, which bind to terminal sialic acid-galactose linkages by the hemagglutinin (ha) envelope glycoprotein. orthomyxovirus attachment to the host cell initiates receptor-mediated endocytosis and endosome acidification. protons are permitted to enter the influenza virion via the m2 ion channel, and acidification results in a conformational change in the ha protein, revealing the fusion peptide that initiates membrane fusion between the viral envelope and the endosome membrane (reviewed by palese and shaw (2007) ). the ha is synthesized as a precursor (ha0) that is cleaved into its active form (ha1 and ha2) by cellular proteases, and the amino acid sequence at the cleavage site determines the type of protease that is able to activate the ha. if trypsin-like proteases are required for cleavage, the virus is limited in its tissue tropism to the respiratory tract of humans and the gastrointestinal tract of birds; however, the presence of multiple basic residues at the cleavage site extends the range of proteases that can cleave the ha, resulting in a disseminated, often lethal, infection in poultry (wright et al., 2007b) . once the virus envelope has fused with the endosome, the influenza genome enters the cytoplasm. the orthomyxovirus genome comprises seven or eight segments of singlestranded, negative-sense rna, and each segment encodes one or more proteins. each segment is encapsidated in nucleoprotein (np) and forms a panhandle comprising the 5′ and 3′ ends, to which a polymerase complex is attached. together, these are known as the viral ribonucleoprotein complex. in the nucleus, orthomyxovirus vrna is either transcribed into mrna or replicated by means of a positive-sense crna intermediate. viral mrna molecules exit the nucleus and are translated in the cytoplasm by the host cell machinery. structural proteins assemble at the plasma membrane, where newly synthesized viral genomes are packaged and virions bud (palese and shaw, 2007) . how the individual segments traffic to the plasma membrane and are packaged remain active areas of research. the matrix (m1) protein lines the virion beneath the envelope and may be important for morphology and viral assembly at the plasma membrane. in addition, the neuraminidase (na) protein permits budding by cleaving sialic acid residues from the host cell plasma membrane (palese and shaw, 2007) . to complete the replication cycle, influenza viruses inhibit interferon (ifn) production and signaling. this is achieved by the ns protein 1 (palese and shaw, 2007) (section adenoviruses). the genomes of coronaviruses and rhinoviruses comprise positive-sense, single-stranded rna that can be translated by the host cell machinery in the cytoplasm (kennedy et al., 2012) . coronaviruses, which belong to the coronaviridae family, are enveloped and attach to host epithelial cells by the spike (s) envelope proteins (blau and holmes, 2001) . fusion occurs at the plasma membrane, or after endocytosis, and the genome is translated into a polyprotein, which is then posttranslationally processed into structural proteins that form viral particles and nonstructural proteins that aid in viral genome replication (lai et al., 2007) . rhinoviruses, which belong to the picornaviridae family, are not enveloped and instead have a capsid of icosahedral symmetry comprising four proteins, vp1-4 (reviewed by greenberg (2011) , kennedy et al. (2012) ). the majority of rhinoviruses bind to intercellular adhesion molecule-1 (icam-1) (greve et al., 1989) , and binding leads to a conformational change in the capsid that creates a pore, through which the genome enters the cytoplasm to be translated and replicated (bella and rossmann, 2000) . adenoviruses are nonenveloped and possess a capsid of icosahedral symmetry. at each of the 12 corners, a fiber protrudes from the capsid that makes contact with the host cell receptor to initiate receptor-mediated endocytosis. acidification of the endosome results in conformational changes in the capsid that lead to viral uncoating and the release of the double-stranded dna genome into the cell. the genome is transported into the nucleus, where it is transcribed into rna, which is alternatively spliced into monocistronic mrnas that are translated by the host cell machinery into early gene products. early gene products remodel the intracellular environment to favor viral replication and are responsible for viral replication. the late phase of the viral life cycle is concerned with the production of structural proteins in sufficient quantities to ensure adequate packaging of the newly synthesized genomes and maximizing the production of progeny virions, which are released by cell lysis (berk, 2007) . respiratory viruses can infect various parts of the respiratory tract and cause a range of illness. mild upper respiratory tract (urt) infection (urti) can be complicated by sinusitis or otitis media, and a lower respiratory tract (lrt) infection (lrti) can lead to bronchiolitis or pneumonia and possible postinfectious respiratory complications such as sensitization to asthma. the major public health impact is from lrtis, and rsv is responsible for the majority of cases in infants. hmpv, hpiv, and influenza can also lead to lrti, with hpiv3 and hmpv affecting infants almost as young as those afflicted with rsv, whereas hpiv1, hpiv2, and influenza are often diagnosed in children 6 months of age or older. rsv and influenza are also recognized as an important cause of lrtis in the elderly and in those with cardiopulmonary disease or immunosuppression (schmidt, 2011) . moreover, influenza pandemics occur at irregular and unpredictable intervals with widespread morbidity and mortality and economic consequences. in addition, although there have been no cases of severe acute respiratory syndrome (sars) since 2004, several novel coronaviruses have been identified, including the virus responsible for middle east respiratory syndrome (mers) (zaki et al., 2012) . given the clinical significance of these infections, and the fact that licensed vaccines are available only for influenza viruses, there is an unmet need for vaccines. humans are the only natural host for rsv, with infections occurring in annual epidemics during winter and spring months in temperate climates and the rainy season in the tropics (girard et al., 2005) . the virus is highly contagious, with most children being infected in the first year of life. the peak of severe disease usually occurs before 6 months of age, with the peak incidence of hospitalization in 2-to 3-month-old infants (collins and melero, 2011) . reinfection is also common. in one study, among children who had been infected in their first year of life, 47% were reinfected in their second year, and 45% in their third year of life (glezen et al., 1986) . moreover, reinfection is independent of antigenic changes in the virus, implying that the protective immunity mounted during an infection does not protect against subsequent reinfection (collins and melero, 2011) . this is of note when attempting to induce protective immune responses by vaccination. globally, there were an estimated 34 million cases of lrti caused by rsv in children under 5 years of age in 2005, with 10% requiring hospitalization (martinez et al., 1997) . in the united states, one study estimated that 2.1 million children under 5 years of age require medical attention each year owing to rsv (botosso et al., 2009) , and another study estimated that rsv was responsible for 18,000-75,000 hospitalizations per year (girard et al., 2005) . in the united kingdom, the total annual incidence of hospitalization attributed to rsv was 28.3 per 1000 children under 1 year of age and 1.3 per 1000 children between 1 and 4 years of age (waris, 1991) . more than half of the hospitalizations for rsv occur in previously healthy, fullterm infants, and children who experienced severe lrti caused by rsv were at increased risk of wheezing and asthma later in life (girard et al., 2005) . pediatric mortality from rsv in the united states was estimated to be between 5.3 per 100,000 per year in infants under 1 year of age and 0.9 per 100,000 per year in children 1 to 4 years of age in one study (cooney et al., 1975) , and in another u.s. study, rsv was estimated to be responsible for 90-1900 deaths per year (girard et al., 2005) . however, an estimated 99% of rsv-associated deaths occur in developing countries, possibly because of limited health-care resources (martinez et al., 1997) . in healthy adults, reinfection rates are approximately 5-10% per year, though hospitalizations are rare. morbidity and mortality are more pronounced in the elderly and it has been estimated that rsv causes an average of 17,358 deaths annually in the united states, with 78% occurring in individuals over 65 years of age (cooney et al., 1975) . in addition, the cost of caring for patients with severe lrti from rsv and its sequelae are substantial (girard et al., 2005) . rsv infection induces antibodies against the two main antigens, the f and g envelope glycoproteins. the g protein is the most variable protein in rsv and is the basis for the separation of strains into two antigenic groups (a and b). moreover, sites of positive selection that partially coincide with epitopes recognized by anti-gprotein monoclonal antibodies (mabs) suggest immunedriven rsv evolution (botosso et al., 2009) . however, most anti-g mabs do not neutralize infectivity (martinez et al., 1997) and the selection pressure is, therefore, weak. this favors a slow coevolution of several rsv lineages, and multiple genotypes within each group can cocirculate within the same season, with shifts in the predominance of groups a and b occurring in 1-to 2-year cycles (waris, 1991) . in contrast, the sequence of the f gene is highly conserved among rsv isolates, despite the identification of a number of neutralizing mabs against the protein that should impart a selection pressure for mutation (collins and melero, 2011) . this implies that the function of the f protein confers structural restrictions that may limit antigenic diversity. human parainfluenza viruses are also a common cause of acute respiratory infection, with 80% of children seropositive by 5 years of age (cooney et al., 1975) . as with rsv, reinfection is common (schomacker et al., 2012) . there are four serotypes of hpiv (hpiv 1-4), with each serotype associated with a broad spectrum of upper and lower respiratory symptoms. hpiv1 and 2 are, however, more frequently associated with croup (laryngotracheobronchitis), and hpiv3 is more likely to cause bronchiolitis, pneumonia, and lrti resembling disease caused by rsv (schomacker et al., 2012) . hpiv4 is a less frequent cause of clinically significant disease, though a study found hpiv4 in 10% of hpiv-positive samples in a day-care setting (fairchok et al., 2011) . hpiv lrti is a major cause of hospitalization in children under 5 years of age, second only to rsv, though infection is usually self-limiting and rarely fatal, unless an individual is immunosuppressed. severe infection may have long-term effects on lung function, but this remains unclear (schomacker et al., 2012) . hmpv belongs to the same subfamily as rsv, and two major groups (a and b) and four minor subgroups (a1, a2, b1, and b2) have been identified, based on sequence variability in the g and f glycoproteins (kroll and weinberg, 2011) . as with rsv, and hpiv, by 5 years of age, most children will have been infected with hmpv, and reinfections are common (kroll and weinberg, 2011) . the virus also has a seasonal distribution, with the main occurrence in winter and spring (kahn, 2006) . hmpv typically leads to flu-like symptoms in otherwise healthy adults, but is responsible for 5-15% of hospitalizations for lrti in children and can lead to severe disease in the elderly or immunocompromised hosts (papenburg and boivin, 2010; boivin et al., 2007) . influenza viruses are divided into types a, b, and c based on antigenic differences in the np and m genes. influenza a viruses are the most clinically significant and are divided into subtypes based on antigenic differences in the ha and na genes. to date, 16 ha and 9 na subtypes have been identified from waterfowl (palese and shaw, 2007) and 17th and 18th subtypes of ha have been identified from bats in guatemala and peru (tong et al., 2012 (tong et al., , 2013 . influenza viruses cause a spectrum of clinical illness associated with infection of the upper and lower respiratory tract, with more severe disease associated with lrti. the viruses are spread by respiratory droplets or direct contact. annual influenza epidemics have a seasonal distribution, with the main occurrence in winter months (seasonal influenza) in temperate climates (girard et al., 2005) . however, unlike rsv, hpiv, and hmpv, influenza a viruses have a broad host range that includes birds, pigs, dogs, horses, marine mammals, and humans, with the main reservoir for infection being aquatic birds (palese and shaw, 2007; wright et al., 2007b) . this broad host range, together with the segmented nature of the influenza virus genome, makes the epidemiology of influenza complex and gives rise to zoonotic infections and pandemics. pandemic influenza can arise if a novel virus emerges that readily transmits from person to person and if the majority of the population is susceptible to infection. if an avian or animal virus crosses the species barrier to circulate in humans, the population will probably be immunologically naïve and, therefore, susceptible to infection. however, the virus must be able to transmit efficiently from person to person for a pandemic to occur. as the influenza virus genome is segmented, if a host is infected with two or more influenza viruses, the potential exists for the gene segments to reassort, such that a progeny virus containing genes from each parent virus can be produced (wright et al., 2007b) . if a virus that circulates within the human population reassorts with one that is novel for humans, the resultant virus may possess genes that allow it to replicate efficiently in humans, but with glycoproteins to which the population is immunologically naïve, and a pandemic could occur. the introduction of a virus with a novel ha subtype into the human population is known as "antigenic shift" (wright et al., 2007b) . three global influenza pandemics were recorded in the twentieth century from viruses of the subtypes h1n1, h2n2, and h3n2, respectively. in 2009, another pandemic h1n1 (ph1n1) influenza virus emerged in mexico (girard et al., 2010) . this h1n1 virus was antigenically unrelated to previously circulating seasonal h1n1 viruses, and molecular studies revealed that it was a reassortant with genes derived from viruses that had been circulating in pigs: the north american h3n2 triple-reassortant, the classical swine h1n1, and the eurasian "avian-like" swine h1n1 viruses. in most countries, the median age of infection during the 2009 pandemic was estimated to be 12-17 years, and in most individuals, infection led to a mild, self-limiting urti. however, 2-5% of confirmed cases in the united states and canada required hospitalization, and the case-fatality rate was 0.15-0.25%. moreover, nearly one-third of the fatalities among hospitalized patients occurred in previously healthy individuals (girard et al., 2010) . after each pandemic, the newly emerged subtype became established and caused annual seasonal influenza epidemics. in the united states, it has been estimated that 25-50 million cases of influenza occur annually, with approximately 225,000 requiring hospitalization (lambert and fauci, 2010) . current vaccines are aimed at the circulating h1n1 and h3n2 subtypes of influenza a and the predominant circulating strain of influenza b and are therefore trivalent vaccines. however, two antigenically distinct lineages of influenza b viruses (victoria and yamagata) cocirculate, and the world health organization recommended that influenza vaccines should contain both of these lineages. clinical trials of quadrivalent vaccines containing the h1n1 and h3n2 influenza a viruses and the victoria and yamagata influenza b viruses have been conducted, and their use in the united states received an interim recommendation of the advisory committee on immunization practices for the 2013-2014 influenza season (dolin, 2013) . owing to the low fidelity of the rna-dependent rna polymerase of influenza, and immune selection pressure on the ha protein, viral replication can yield a quasi-species that may differ antigenically from the parent virus. therefore, each season, the predominant circulating strain may be antigenically distinct from the previous year. this phenomenon is known as "antigenic drift" and leads to a need to update the influenza vaccine annually (wright et al., 2007b) . sporadic infections by h5n1, h9n2, h7n7, and h7n9 subtypes of influenza have been reported in humans who were in close direct contact with infected animals. additionally, h3n2 variant viruses have infected humans, the majority of whom were in close contact with pigs (epperson et al., 2013) . so far, there has been limited transmission of these viruses between people, though there is concern that they may acquire mutations or gene segments that allow efficient spread from person to person. coronaviruses are frequent causes of the common cold, causing urtis throughout the world, in all age groups, leading to millions of days of work and school absence, physician visits, and frequent inappropriate antibiotic use (greenberg, 2011) . coronaviruses are transmitted by respiratory droplets and are reported to cause 7-30% of common colds, with a peak prevalence in late fall, winter, and early spring. the first human coronaviruses (hcov) to be recognized as significant respiratory pathogens, hcov-229e and oc43, were identified in the 1960s (greenberg, 2011) . whereas infection with the majority of coronaviruses is associated with self-limiting urt symptoms in otherwise healthy individuals, a coronavirus was identified as the agent responsible for sars in 2003 (drosten et al., 2003; ksiazek et al., 2003) . the sars coronavirus (sars-cov) emerged in the guangdong province of china in november 2002 and spread to 32 countries, leading to 8096 cases and 774 deaths worldwide by the time the outbreak was brought under control in june 2003 (who, 2004) . subsequently, heightened international surveillance for coronaviruses led to the identification of the strains hcov-nl63, nh, and hku1 in 2004 (greenberg, 2011 previously, hrvs were classified into two species, hrv-a (containing 75 serotypes) and hrv-b (containing 25 serotypes). in 2009, a novel species, hrv-c, was identified, which contains at least 50 serotypes (jacobs et al., 2013) . hrvs are spread by direct contact, hand-to-hand contact or aerosols. traditionally, they have been associated with a urti, causing between 25 and 50% of common colds (makela et al., 1998) . however, they are increasingly recognized as a cause of lrti, particularly in patients with asthma and in infants, the elderly, and immunocompromised individuals (jacobs et al., 2013) . bronchiolitis is a common clinical manifestation in hospitalized children infected with hrv, and hrv is also a common pathogen in viral community-acquired pneumonia. hrv has also been associated with exacerbations of asthma and chronic obstructive pulmonary disease (jacobs et al., 2013) . as of this writing, 52 serotypes and seven species of adenovirus have been identified. tissue tropism and clinical manifestations vary between the serotypes, and the viruses are responsible for both febrile respiratory disease and gastrointestinal illness (reviewed by lynch et al. (2011) ). adenoviruses are estimated to be responsible for 5-10% of pediatric and 1-7% of adult respiratory tract infections (ison, 2006; lee et al., 2010) . they are spread primarily via respiratory droplets, direct contact, or fomites, and more than 80% of cases are in children under 4 years of age (lynch et al., 2011) . however, epidemics have also been described in children and adults, especially in military recruits in closed or crowded settings. most individuals develop a self-limiting urt infection that may be asymptomatic, but conjunctivitis, tonsillitis, otitis media, or croup can occur. infection can also spread to cause bronchiolitis or pneumonia or disseminate to cause viral meningitis or encephalitis that can be fatal (lynch et al., 2011) . there are many factors that determine the pathogenesis of disease and clinical outcome, including factors pertaining to the virus, host genetics, host immune responses, and the environment. the site of viral replication may influence the pathogenesis of disease and outcome of infection. for example, seasonal influenza viruses usually infect the epithelium of the urt, which is consistent with the most common clinical manifestations of seasonal influenza, whereas highly pathogenic avian influenza viruses of the h5n1 subtype show a stronger tropism for the lrt than for the urt (kuiken et al., 2012) . h5n1 viruses attach abundantly to "clara" or club cells lining the bronchioles, type ii pneumocytes lining the alveoli, and alveolar macrophages in the alveoli, consistent with the clinical manifestation of diffuse alveolar damage (kuiken et al., 2012) . rsv also targets both type i alveolar and nonbasilar epithelial cells and possibly alveolar macrophages, which may contribute to lrti (van drunen littelvan den hurk and watkiss, 2012). tissue tropism is determined, in part, by the receptor preference of the virus. for example, cells lining the urt of humans predominantly possess sialic acid residues with a terminal α2, 6 linkage to galactose, whereas cells lining the human lrt have both α2, 3 and α2, 6 linkages (shinya et al., 2006) . it is believed that the ability of an influenza virus ha to preferentially bind α2, 6-or α2, 3-linked sialosaccharides therefore partly determines the tissue tropism and hence the clinical outcome. when host cells are infected, type i ifn and proinflammatory cytokines are expressed, cellular translation is suppressed, and an antiviral state is induced (discussed in section immune responses to respiratory virus infection). however, most viruses that infect the respiratory tract modulate the host response to infection by blocking ifn activation and/or signaling and inhibiting apoptosis. this prevents the host from effectively clearing virally infected cells and inducing an antiviral state in neighboring cells, thereby promoting viral replication in infected tissues that may contribute to the observed pathology. rsv is the most effective paramyxovirus at subverting host cell responses, inhibiting apoptosis and type i ifn production and signaling by means of the ns1 and 2 proteins, inhibiting nuclear factor-κb (nf-κb) activation through the binding and sequestration of cellular protein kinase r (pkr) by the viral n protein, and inhibiting the production of stress granules that can restrict viral replication (collins and melero, 2011) . influenza a viruses encode the ns1 protein that downregulates ifn production (palese and shaw, 2007) , and hpiv encodes either a c protein or a v protein that suppresses ifn induction and signaling (karron and collins, 2007) , whereas sars-cov encodes eight proteins that antagonize the ifn response by a variety of mechanisms (totura and baric, 2012) . in addition to blocking ifn activation and signaling, viruses employ other means of ensuring their replication in the face of host immunity. the rsv g protein is highly glycosylated, a feature that may inhibit the binding of antibodies, and the protein is highly variable, enabling a substantial population of immune-escape mutants to be produced during infection. additionally, a truncated, soluble form of the g protein is produced during infection, which acts as a decoy antigen that can bind rsv-specific antibodies, thus reducing their availability to neutralize virus. rsv also infects antigen-presenting cells (apcs), such as dendritic cells (dcs), and can affect their maturation and antigen-presentation functions and can lead to dysregulation of adaptive immune responses (van drunen littel-van den hurk and watkiss, 2012). a number of virulence determinants have also been identified in influenza viruses, including the ha and the polymerase complex. for example, the presence of a multibasic site in the ha gene renders viruses highly pathogenic in poultry, as they are able to replicate systemically (bosch et al., 1981; kawaoka and webster, 1988) . in addition, substitution of a glutamic acid (e) residue for a lysine (k) residue at amino acid position 627 in the pb2 protein (e627k) of the polymerase is associated with altered host range (subbarao et al., 1993) and virulence in humans and in mice that are experimentally infected with avian h5n1 and h7n7 viruses (hatta et al., 2001; munster et al., 2007; subbarao et al., 1993) . in the absence of the e627k mutation, a pb2 d701n mutation is also correlated with increased virulence (li et al., 2005) , and in the absence of both the pb2 e627k and the d701n mutations, pb2 590s and 590r were found to contribute to 2009 ph1n1 influenza virus replication and virulence (mehle and doudna, 2009 ). in addition, an n66s mutation in the pb1 f2 protein has been shown to contribute to the virulence of h5n1 and 1918 pandemic h1n1 viruses (conenello et al., 2007) , and several virulence determinants have been identified in the ns1 protein, including d92e, p42s, l103f, and i106m, reviewed by kuiken et al. (2012) . there are several host factors that are known to contribute to the severity of respiratory virus disease and the outcome of infection. for example, it is known that the more severe rsv lrti in infants is associated with prematurity, chronic lung disease, congenital heart disease, and t cell immunodeficiency. other risk factors include low birth weight, multiple births, male gender, and low titers of maternally derived anti-rsv antibodies (groothuis et al., 2011; van drunen littel-van den hurk and watkiss, 2012) . additionally, low levels of vitamin d in cord-blood of healthy neonates is correlated with an increased risk of rsv lrti in the first year of life (belderbos et al., 2011) . infants are also at a greater risk of lrt disease from hpiv infection than older children; this has been attributed to smaller airways that are more susceptible to obstruction, immature immune responses, and the presence of anti-hpiv maternal antibodies that can suppress primary antibody responses (crowe and williams, 2003; karron and collins, 2007) . in adults, immunodeficiency, immunosuppression, or old age may lead to more severe illness (collins and melero, 2011) . in addition, a number of genetic polymorphisms have also been described in host genes that may affect the outcome of respiratory virus infection and disease severity. single-nucleotide polymorphisms have been identified in genes that encode surfactant proteins, such as surfactant proteins a, b, c, and d; pattern recognition receptors (prrs), such as toll-like receptors (tlrs); chemokines and cytokines, such as rantes, il-4, -5, -6, -8, -9, -10, -13, and -18, tnf-α, tgf-β, and ifn-γ; chemokine and cytokine receptors, such as ccr5, il-4ra, and il-8ra; adhesion molecules, such as icam-1, vcam-1, and e-selectin; and hla molecules such as hla-a and -b, among others (miyairi and devincenzo, 2008; poland et al., 2008) . however, few consistent results have been obtained between studies, probably a result of differences in study design, sample size, etc., in addition to true variability. clearly the contribution of genetic polymorphisms to disease outcome is complex and remains an active area of research. as well as being responsible for viral clearance and protection against reinfection, the host innate and adaptive immune responses to respiratory viruses can lead to pathology and enhanced disease. this is especially important to bear in mind in vaccine development, as vaccine-induced immune responses must protect against infection without leading to immunopathology. excessive inflammation is an important component in the pathogenesis of respiratory virus infections. upregulation of il-8 leads to the recruitment of neutrophils to the site of infection, and although these cells may participate in virus elimination, in high numbers they can also cause pathology. upregulation of il-8 is known to correlate with rsv disease severity (goetghebuer et al., 2004) and, during an influenza infection, overproduction of cytokines such as tnf-α, il-6, il-8, and type i and ii ifns and chemokines can also result in the recruitment of immune cells to the site of infection and result in damage to lung tissue (cheung et al., 2002; de jong et al., 2006) . elevated il-8/cxcl8, mip1α and β/ccl3 and 4, rantes/ccl5, and cxcl9 have also been described in children with hpiv disease, with an association of more severe hpiv disease with high concentrations of il-8 and ip-10 (reviewed by schomacker et al. (2012) ). additionally, the rsv soluble g protein can lead to leukocyte recruitment by mimicking the chemokine fractalkine (tripp et al., 2001) and this can further exacerbate inflammation. pathogenesis can also be enhanced by an insufficiency of anti-inflammatory immune responses in the lung, such as the cytokines il-10 and tgf-β (carlson et al., 2010; lebouder et al., 2009; sun et al., 2009) , or insufficient numbers of immunosuppressive resident alveolar macrophages (rygiel et al., 2009; snelgrove et al., 2008) . dysregulation of adaptive immune responses can also lead to increased pathology, and a th2-biased cellular immune response has been implicated in the immunopathogenesis of rsv disease (van drunen littel-van den hurk et al., 2007) . various defense mechanisms have evolved in the respiratory tract to prevent and control infection. currently, there is considerable effort to develop or improve vaccines against respiratory viruses. however, achieving this goal has been complicated by an incomplete knowledge of how the immune system recognizes, contains, and eliminates respiratory viruses. this section discusses the immune responses against respiratory virus infections, from the initiation of innate and adaptive responses following primary virus infection to the recall of immune responses during a secondary infection. in addition, advances in our understanding of respiratory mucosal immunity are discussed. a common feature of respiratory virus infections is that the initial infection is established in epithelial cells lining the respiratory tract. epithelial cells, as well as alveolar macrophages and dcs, are exposed to the contents of the airway lumen and detect the presence of an invading virus through prrs (holt et al., 2008) . the recognition of pathogen-associated molecular patterns (pamps) by these receptors initiates a cascade of signals that results in the production of cytokines and chemokines. the release of these inflammatory mediators into the surrounding environment establishes a local antiviral state. in addition, chemokines provide the necessary signals for the recruitment of leukocytes to the site of infection. finally, the combination of inflammatory cytokines and prrs initiates the process of dc maturation and trafficking that is required for the induction of adaptive immune responses (holt et al., 2008) . the best described of the prrs are those of the tlr family. with respect to respiratory viruses, tlr3, 7, and 9 recognize various products of viral replication (doublestranded rna, single-stranded rna, and unmethylated cpg dna, respectively) (alexopoulou et al., 2001; diebold et al., 2004; hagglund et al., 2004; lund et al., 2003) , and tlr4 recognizes the f protein of rsv (kurtjones et al., 2000) . tlrs that recognize nucleic acids are located in late endosomes. this location optimizes the ability of tlrs to interact with viral nucleic acids while limiting their access to host-derived nucleic acids (heil et al., 2003; matsumoto et al., 2003) . although tlrs expressed on the cell surface or within the cell utilize different signaling pathways, each of these receptors can activate the transcription of ifninducing genes . viral rna is also recognized by cytoplasmic sensors such as rna helicases. the retinoic acid-inducible gene i protein interacts with 5′-triphosphate rna and is important for early cytokine production in response to numerous rna viruses (hornung et al., 2006; pichlmair et al., 2006; yoneyama et al., 2004; kato et al., 2005; pothlichet et al., 2013; graham et al., 2013) . the melanoma differentiationassociated gene 5 protein is a related helicase that recognizes polyinosinic polycytidylic acid and is crucial for innate recognition for picornaviruses and human metapneumovirus infection (banos-lara mdel et al., 2013) . similar to signaling through tlrs, the pathways utilized by rna helicases ultimately trigger ifn-regulatory factor and nf-κb activation (le goffic et al., 2007) . the key difference between these molecules and tlrs is that the rna helicases are localized throughout the cytosol, rather than being restricted to intracellular compartments. thus, pathogens such as paramyxoviruses that do not enter endosomes can trigger innate immune responses via rna helicases. the innate recognition of viral components through prrs described above leads to a program of gene expression that promotes a localized antiviral state and elicits the recruitment of inflammatory cells to the site of infection. type i interferons, including ifn-α and ifn-β, are most commonly associated with early antiviral responses in the lung, and numerous studies in mice and humans have shown that plasmacytoid dcs (pdc's) are the primary source of these cytokines after infection with viruses (asselin-paturel et al., 2001; mcgill et al., 2009) . however, there is a level of redundancy with respect to ifn production, with alveolar macrophages or pdcs predominating depending on the type of viral infection (pribul et al., 2008) . the precise contribution of pdcs to lung antiviral immunity is also controversial; several in vitro studies show that respiratory viruses, including influenza viruses, can infect pdcs and pdcs can activate virus-specific cd4 + t cells (wikstrom and stumbles, 2007; geurtsvankessel and lambrecht, 2008) , but evidence for a major role of pdcs in controlling influenza virus in vivo is absent. type i ifns produced after respiratory virus infection act in concert with prr signaling to form a feedback loop, by signaling through the ifn-α/β receptor to promote sustained production of proinflammatory cytokines such as tnf-α, il-1, and il-6 by lung-resident innate immune cells (chan et al., 2005; nakajima et al., 2013; almansa et al., 2012) . these proinflammatory cytokines and prrmediated signals also prompt alveolar macrophages, dcs, and epithelial cells to initiate a coordinated program of chemokine production after viral infection. for example, dcs secrete successive waves of chemokines after influenza virus infection, beginning with those capable of recruiting inflammatory cells such as neutrophils and natural killer (nk) cells to the lung, followed by chemokines associated with the recruitment of monocytes and t cells (piqueras et al., 2006; marois et al., 2012; teijaro et al., 2010) . the cytokines il-1β, il-18, and il-33 activate monocytes, macrophages, and neutrophils and drive the development of cd4 + t cell adaptive responses in both mice and humans. cd4 + t cell differentiation in the presence of il-1β, il-18, or il-33 results in th17, th1, or th2 effector cells, respectively (chung et al., 2009; dinarello, 1999; lasiglie et al., 2011; ohno et al., 2009 ). these cytokines are processed as a result of caspase-1 activation, and activation of caspase-1 is regulated by the inflammasome, a large mutimeric structure (martinon et al., 2002) . a subgroup of the nucleotide-binding domain, leucine-rich repeat-containing proteins (nlrps) are key mediators of the inflammasome. activation of the inflammasome can be divided into two categories: activation driven by host-and environmentderived molecules and activation driven by pathogen-associated activators, including pamps derived from bacteria, virus, fungus, and protozoa. paramyxoviruses such as rsv and orthomyxoviruses such as influenza viruses can activate the nlrp3 inflammasome (kanneganti et al., 2006; segovia et al., 2012; komune et al., 2011) , which has been shown to play a critical antiviral role in influenza virus-infected mice (thomas et al., 2009; ichinohe et al., 2009; allen et al., 2009 ). after infection of the lrt, antigen-bearing mature dcs enter the lymph nodes draining the lung, where they form stable interactions with naïve t cells specific for that antigen through t cell receptors (tcrs). signals delivered by antigen recognition, in addition to accessory signals delivered through costimulatory molecules, result in t cell priming and the clonal expansion of antigen-specific effector t cells (moon et al., 2007; obar et al., 2008) . the instructions delivered by dcs during the initial expansion phase can have a dramatic impact on the survival and function of the responding t cells. for instance, expression of fasl on dcs after influenza infection has been shown to regulate the magnitude of the cd8 + t cell response (legge and braciale, 2005) . resident cd8α + conventional dcs in the mediastinal lymph node also mediate the induction of protective immunity to influenza virus, and these cells have been found to cross-present viral antigens to cd8 + t cells without being directly infected by the virus (belz et al., 2004) . after activation and clonal expansion of antigen-specific effector t cells in the draining lymph nodes, the cells lose their preference for the lymphoid tissue and migrate via the bloodstream to the site of infection, where the antiviral mechanisms described below are exerted. analysis of chemokine expression in the lung during the adaptive phase of the immune response has shown elevated expression of numerous molecules associated with effector t cell trafficking (monick et al., 2007) . for instance, the trafficking of effector t cells during rsv infection is partially dependent on cxcr1, and this chemokine receptor may play a role in other paramyxovirus infections (harcourt et al., 2006) . the appearance of antigen-specific effector t cells at the site of virus infection is first observed around 6-7 days after infection with influenza and parainfluenza viruses in mice (pommerenke et al., 2012; kohlmeier and woodland, 2009; lawrence and braciale, 2004; roman et al., 2002) . the continual migration of effector t cells from lymphoid tissues during acute infection results in a massive increase in the number of antigen-specific cells in the lung airways and parenchyma from 7 to 10 days after influenza virus infection (flynn et al., 1998) , and the arrival of effector t cells has an immediate and dramatic impact on the viral load (kohlmeier et al., 2010) . the adaptive immune responses induced after a respiratory virus infection are shown in figures 1-3 . upon arrival at the effector site (figure 1 ), antigen-specific t cells first interact with apcs such as dcs (shen et al., 2010) . moreover, a subset of dcs, classed as cd11c hi , present a crucial t cell survival factor (il-15) to antiviral cd8 + t cells in trans (mcgill et al., 2010) . immune responses are determined by the cytokine milieu in the respiratory tract, as well as the type and level of costimulatory molecules expressed by apcs. for instance, lung dcs are biased to promote th2 responses, most likely via the production of il-6 in the absence of the th1-prone il12p70 cytokine or through the production of leukotriene ltc4 (dodge et al., 2003; barrett et al., 2011) . effector t cells employ one of the following three antiviral mechanisms ( figure 2 ). first, t cells can promote the lysis of infected cells by exocytosis of granules containing perforin and granzyme (trapani and smyth, 2002; hou and doherty, 1995) . second, t cells can induce apoptosis of infected cells by expressing cd95 (fas) ligand (fasl) hou and doherty, 1995) or tnf-related apoptosis-inducing ligand (trail) (brincks et al., 2011) . third, t cells can produce proinflammatory and regulatory mediators, such as ifn-γ, after an encounter with virally infected cells (hamada et al., 2013) . several studies suggest a crucial role for the cytolytic functions of cd8 effector t cells in influenza virus infection. the direct lysis of infected cells requires tcr-mediated recognition of processed viral antigens on the infected target cell (brincks et al., 2008; topham and doherty, 1997) . in contrast, the release of proinflammatory mediators such as ifn-γ by cd8 + t cells has only a modest impact on virus clearance and recovery. there is also evidence from models of influenza infection that infected alveolar epithelial cells may be eliminated by the host response through the action of macrophages capable of triggering apoptosis through a trail-dependent mechanism (herold et al., 2008) . effector cd4 + t cells have also been found to exhibit cytotoxic activity in vitro, but the contribution of this mechanism to virus clearance in vivo is modest (agrewala et al., 2007; graham et al., 1994) and is restricted to the cytolysis of cells that bear viral antigens presented by major histocompatibility complex (mhc) class ii molecules. such cells include cd45 + mononuclear phagocytic cells, and a few cd45 − lung parenchymal cells, such as type ii alveolar epithelial cells, that express mhc class ii molecules in either a constitutive or an inducible manner (debbabi et al., 2005) . the primary role of antiviral cd4 + t cells is to support the activation and differentiation of b cells, which leads to antibody production (topham et al., 1996; topham and doherty, 1998) as discussed below. another aspect of effector t cells is their potential to exert cytotoxic activity and simultaneously produce cytokines such as ifn-γ. for example, during an influenza infection, the interaction of primed cd8 + t cells with lung dcs elicits both cytokine production and cytotoxic phenotypes (hufford et al., 2011) . to summarize the cellular immune responses during a primary influenza infection, specific cd4 + and cd8 + effector t cells in the lung predominantly produce ifn-γ and figure 1 upon virus infection, viral antigen from dying respiratory epithelial cells is transported from the lung to the lymph node by migratory antigen-presenting cells such as mouse cd103 + dendritic cells (dcs) or human monocyte-derived dcs that can stimulate both cd4 + and cd8 + t cell responses. viral antigen can also be transferred to lymph node-resident cd8α + dcs and presented to naïve cd8 + t cells. in addition, plasmacytoid dendritic cells (pdcs) are an early source of antiviral type i interferon via recognition of viral rna. tnf-α, and cd4 + effector t cells also produce il-2 and il-10 (carding et al., 1993; pipeling et al., 2008; mayer et al., 2005) . cd8 + effector t cells localize to the respiratory epithelium and induce apoptosis of infected epithelial cells through fas-fasl interactions or the exocytosis of cytolytic granules containing perforin and granzyme tripp et al., 1995) . b cells are found interspersed in the lung interstitium and in the cervical and mediastinal/bronchial lymph nodes that drain the upper and lower respiratory tract, respectively. during a respiratory virus infection, a tertiary lymphoid structure also forms along the branching point of the bronchial tree, called the bronchus-associated lymphoid tissue (balt) (brandtzaeg, 2010) . the balt contains organized b cell areas, germinal centers, and antibody-forming cells (randall, 2010) . b cell responses can be classified into three categories: innate-like b cell responses, t-dependent b cell responses, and t-independent b cell responses. innate-like b cell responses consist of antibodies produced almost exclusively from b-1 cells, a small subset of b cells characterized by a unique developmental origin, phenotype, tissue distribution, and regulation, compared with conventional b cells (baumgarth et al., 1999; baumgarth, 2011) . t-dependent b cell responses are b cell responses that are facilitated by cd4 t cells, whereas t-independent b cell responses are not. it has been shown that cd4 t cell deficiency results in a drastically reduced humoral response to influenza virus infection in mice (mozdzanowska et al., 2005) . however, mice lacking cd4 and cd8 t cells are still protected from lethal infection mozdzanowska et al., 2005) , highlighting the importance of t-independent b cell responses. b cell responses are critical for viral clearance in primary respiratory virus infections, such as influenza (gerhard, 2001) . although control of early infection (3-6 days postinfection) is not impaired in b-cell-deficient mice, the mice fail to clear the virus and ultimately succumb to infection (graham and braciale, 1997; lee et al., 2005) . studies in influenza virusinfected mice also show that serum antibody titers are first detected around 6-7 days postinfection, at least 3 days later than responses are detected in the respiratory tract. they steadily increase for about a month, after which relatively high figure 2 during the effector phase of the response, influenza-specific cd8 + t cells elicit a cytotoxic response. tnf-α and nitric oxide (no) produced from a subset of dcs and neutrophils contribute to both viral clearance and immunopathology. lung cd11c hi dcs present the crucial t cell survival factor il-15 to antiviral cd8 + t cells in trans and promote the production of chemokines. in draining lymph nodes, cd11b + dcs contribute to the expansion of specific cd8 + t cells, and conventional dcs (cdcs) induce the th2 response. plasmablasts initially produce igm, and effector cd4 + t cells provide help for virus-specific b cell/plasmablast differentiation and proliferation. pdcs also play a detrimental role by eliminating virus-specific cd8 t cells in a process involving fasl. antibody titers are maintained for life. virus-specific antibodyforming cells reside transiently in the spleen, from around 6 or 7 days postinfection, and persist for the long term in the bone marrow (jones and ada, 1987; hyland et al., 1994) . after recovery from an infection (figure 3 ), a state of immunological "memory" ensues, in which the individual is better able to control a subsequent infection with the same pathogen (ahmed and gray, 1996) . immunological memory is maintained by both t and b cell subsets, and there are profound differences in the generation, trafficking, and maintenance of t and b cell memory. antigen-specific memory t cells persist at increased frequencies, have a reduced requirement for costimulatory signals in comparison to naïve t cells, and respond quickly to antigenic restimulation (woodland et al., 2002) . in the case of influenza and parainfluenza (piv) virus infections, it has been clearly established that both cd4 + and cd8 + memory t cell subsets respond to and control secondary infection (woodland and dutton, 2003) . b cell memory is characterized by two distinct populations: long-lived plasma cells that continually secrete antibody and memory b cells that persist in a quiescent state (bachmann et al., 1994; slifka and ahmed, 1998) . the generation of long-lived plasma cells is dependent on cognate t-b cell interaction and cd40 signaling that occurs in the germinal center (noelle et al., 1992; lee et al., 2003) . antigen-specific igg and iga antibodies are maintained long after infection and may be protective against heterologous strains of virus. for example, up to 96% of people born between 1909 and 1919 in finland had preexisting antibodies to the 2009 ph1n1 influenza virus, probably because of its relationship to the 1918 h1n1 pandemic influenza virus that circulated in the first part of the twentieth century (ikonen et al., 2010; yu et al., 2008) . the presence of crossreactive antibodies contributed to the unexpectedly low numbers of the elderly with severe illness during the 2009 pandemic, compared with seasonal influenza virus strains (monsalvo et al., 2011; o'donnell et al., 2012) . although neutralizing antibodies directed against the ha globular head are highly efficient at preventing and clearing influenza virus infection, they can also figure 3 in the memory phase, migratory lung dcs capture viral antigen retained on follicular dcs (fdcs) in tertiary lymphoid organs and present it to specific t cells in the respiratory draining lymph nodes. these stimulated t cells upregulate the expression of cd69, causing them to be retained in the lymph nodes that drain the site of primary infection. resident dcs are able to reactivate memory responses in the lymph nodes. lung dcs can promote the production of iga in a process that depends on tgf-β. the generation of b cell memory, particularly the generation of long-lived plasma cells, is dependent on cognate t-b cell interaction and cd40 signaling that occurs in the germinal center. provide a selective pressure for viral immune evasion. crossprotection against various influenza a subtypes, termed heterosubtypic immunity, requires the immune system to recognize epitopes that are conserved between subtypes. such epitopes can be found in the membrane-proximal stalk region of ha (han and marasco, 2011) or in internal proteins such as nucleoprotein or the m protein. anti-stalk antibodies do not inhibit virion binding to mammalian host cells, but inhibit fusion between the viral envelope and the endosomal membrane. they have broadly neutralizing activity and passively protect mice from lethal challenge in vivo (okuno et al., 1994; throsby et al., 2008; sui et al., 2009 ). interestingly, cross-reactive anti-ha stalk monoclonal antibodies have been generated from the acute response to 2009 h1n1 pandemic virus and also from healthy subjects vaccinated with inactivated virus (corti et al., 2011; sui et al., 2009; wrammert et al., 2008) . how to induce high-titers of anti-ha stalk antibodies in humans remains an active area of universal influenza vaccine research. cellular immune responses to cross-reactive epitopes (often expressed on internal viral proteins) also provide a substantial degree of protection against serologically distinct viruses (yewdell et al., 1985; rimmelzwaan and osterhaus, 1995) , and although these heterosubtypic cellular responses are not able to prevent reinfection, they can ameliorate disease by reducing the maximal viral load, mediating faster viral clearance, and providing a substantial degree of protection against challenge with a lethal dose of virus in animal models (hillaire et al., 2011) . there is also some epidemiological evidence that heterosubtypic cellular immunity plays a role in the response to infection with novel influenza viruses in humans; however, the protective effect appears to be weak and may wane over time (epstein, 2006; epstein and price, 2010) . it has, therefore, been suggested that protective cellular immunity could be sustained by reinfection or annual immunization. the effector mechanisms of heterosubtypic immunity remain ambiguous . in murine models of influenza a virus infection, heterosubtypic immunity is observed in the absence of antibodies that recognize influenza envelope glycoproteins and is thought to be mediated primarily by cd8 + t cells, with a relatively small contribution by cd4 + t cells mckinstry et al., 2012) . heterotypic influenza-specific cd8 + t cells have also been shown to lyse influenza virus-infected cells (nguyen et al., 1998) . however, heterosubtypic immunity has been observed in cd8 + t-cell-deficient mice, but not in mice lacking b cells , indicating that there is redundancy in the system. the same investigators found that the heterosubtypic immunity does not require ifn-γ (nguyen et al., 2000) , but does require a properly diversified antibody repertoire (nguyen et al., 2007) . the mucosal immune system has been described in detail elsewhere in this book; however, there are some features unique to the respiratory tract that are worth noting. like the immune system in general, the mucosal immune system of the respiratory tract uses innate and specific mechanisms to prevent and limit infection. innate defenses include physical and chemical factors such as the secretion of mucus, which traps microorganisms and antigens and facilitates their transport out of the body by mucociliary motion. mucosal secretions also contain chemically active substances, including acids, lactoferrin, and lysozyme, which inhibit the growth of microbes. in addition, the luminal side of the respiratory tract is physically protected by layers of epithelial cells that adhere to each other at tight junctions, using occludin and various members of the claudin family, and at adherent junctions using e-cadherin (tsukita et al., 2008) . in humans, the respiratory tract can be divided anatomically into the urt, which comprises the nose, mouth, and pharynx, and the lrt, which includes the trachea, bronchi, and lungs, with the lymphoid tissue of waldeyer's ring representing the line of demarcation. the unpaired nasopharyngeal tonsils (also called the adenoids) and the palatine and lingual tonsils constitute most of waldeyer's ring, with the tubal tonsils and lateral pharyngeal bands as less prominent components (dolen et al., 1990) . this lymphoid tissue is functionally comparable to the nasal or nose-associated lymphoid tissue (nalt) in rodents, which is composed of two paired lymphoepithelial structures beside the nasopharyngeal duct, dorsal to the cartilaginous soft palate (kuper et al., 1992; fukuyama et al., 2002) . however, rodents do not have tonsils. waldeyer's ring is more strategically situated than the nalt to generate mucosal immunity, because its elements are exposed to both airborne and alimentary antigens. in addition, human tonsils have deep antigen-retaining crypts, and tonsils express germinal centers shortly after birth, whereas the rodent nalt has a plain surface and requires an external stimulus to induce the expression of germinal centers (brandtzaeg, 2010) . tissue equivalent to waldeyer's ring has also been found in nonhuman primates (loo and chin, 1974; harkema et al., 1987) and horses (mair et al., 1987 (mair et al., , 1988 , but functional studies have not been performed in these species. the mucosa-associated lymphoid tissues (malt), including the nalt and balt, consist of follicle-associated epithelium and t-cell-and b-cell-enriched areas. the initiation of antigen-specific immune responses occurs at special gateways, which comprise microfold (m) cells located in the epithelium overlying the malt follicles. the cilia of the apical side of the m cells are shorter than those of conventional epithelial cells, and on the basal side, there is a large pocket-like structure that can hold immunocompetent cells required for the generation of immune responses such as t cells, b cells, and apcs. as lysosome development in m cells is poor, in most cases antigens pass through the cells unmodified and are taken up by dcs in the pocket (sato and kiyono, 2012) . upon encountering antigen, dcs migrate to the t cell region of the malt and present peptide antigen via mhc molecules to naïve t cells. antigen-specific t cells become primed, clonally expand, and leave the follicle to enter the circulation. they then home to effector sites to elicit mechanisms involved in viral clearance as described above. in the b cell region, a germinal center forms and antibody class switching occurs (cerutti, 2008) . a class switch to iga predominantly occurs in the malt owing to the action of the iga-associated cytokine family of tgf-β, il-2, il-4, il-5, il-6, and il-10 (mcghee et al., 1989; mestecky and mcghee, 1987; cerutti, 2008) . postswitched iga + b cells leave the malt through efferent lymph vessels under the control of the lipid mediator, sphingosine-1-phosphate, and the cells then enter the circulatory system (gohda et al., 2008; lazarus et al., 2003) and home to effector sites found in unorganized lymphatic tissue spread over the lamina propria that underlies the mucosal epithelium. in the lamina propria, b cells differentiate into plasma cells and secrete iga, igd, igm, and igg antibodies, although iga is the major mucosal antibody isotype (shikina et al., 2004; shimoda et al., 2001) . surgical removal of the murine nalt or cervical lymph nodes does not, however, abrogate cellular or antibody immune responses to experimental influenza infection, suggesting that there may be additional inductive sites other than the nalt and demonstrating that dissecting the relative contributions of anti-influenza immunity is difficult. in the urt, iga is the major mediator of immunity to influenza. in mice that had recovered from an influenza infection, immunity to reinfection was abrogated by the intranasal instillation of anti-iga antibodies, but not anti-igg or igm (renegar and small, 1991a) , and intravenous passive transfer of iga resulted in iga in nasal secretions that protected mice from intranasal challenge with influenza (renegar and small, 1991b) . after passive transfer, nasal iga titers that conferred protection were at a concentration equivalent to that seen in convalescent mice, whereas igg transudation into the urt could be detected only after 2.5 times the normal convalescent serum titer had been passively transferred (renegar et al., 2004) . moreover, recombinant iga is sufficient to prevent influenza transmission in a guinea pig model (seibert et al., 2013) . in humans, iga is present in monomeric and dimeric forms. during transcytosis through mucosal epithelial cells, an extra polypeptide secretory component is added to dimeric iga and the resulting molecule is known as secretory iga (s-iga). dimeric and s-iga are 7-10 times more efficient than monomeric iga at neutralizing influenza viruses (renegar et al., 1998) . dimeric and s-iga are represented by two subclasses, iga1 and iga2, with covalently or noncovalently joined dimers, respectively. both subclasses are detected in nasal secretions after an influenza infection; however, ha preferentially stimulates an iga1 response (brown et al., 1985) . s-iga is not considered to be inflammatory because the fc region is not available to activate immune cells or bind complement. s-iga is also resistant to proteolysis and can neutralize viruses inside epithelial cells and transport viruses that have passed the epithelial barrier to the lamina propria back to the lumen (sato and kiyono, 2012) . s-iga may therefore be useful in preventing viruses from breaching the mucosal barrier, while avoiding immunopathology by not activating inflammatory responses directly and by limiting the number of antigen-antibody complexes in the lamina propria that can trigger inflammation. it is currently thought that plasma igg serves as a backup for s-iga in the urt, whereas in the lrt, igg is the dominant antibody involved in protection (renegar et al., 2004) . the fc receptor for igg mediates transport of igg across epithelial barriers by transcytosis, permitting the transudation of igg from the serum into the lung where it is able to neutralize viruses (spiekermann et al., 2002) . this explains why passively transferred igg is effective at preventing severe disease from respiratory infections in experimental animals and why serum igg antibodies are the main correlate of protection for parentally administered inactivated influenza vaccines in humans (section respiratory virus vaccines). viruses that can cause repeated infection are typically characterized either by a failure to induce robust immunity or by significant antigenic diversity in the face of protective immune responses. influenza viruses can reinfect hosts because the antigenic sites evolve and drift to avoid neutralization by prior immunity. infection induces a strong homosubtypic neutralizing antibody response in healthy individuals that contributes to recovery and protection from repeat influenza virus infection with homologous virus or an antigenically similar virus (wrammert et al., 2008) . moreover, natural infection can lead to long-lasting immunity to the infecting virus. for example, when the influenza a h1n1 subtype reemerged in 1977, the most susceptible members of the population were those born after the time when similar h1n1 viruses had previously circulated, the 1950s (shortridge et al., 1979) . however, because influenza viruses undergo antigenic drift and shift, the effective period of protection may last only until an antigenic variant emerges. in contrast to the robust strain-specific protection after influenza virus infection, primary infection with rsv, hpiv, and hmpv provides only partial protection from reinfection. rsv commonly reinfects the host even though genetic diversity in the virus is not extreme. in healthy adults challenged every few months with the same strain of rsv, about 25% were infected each time and about half of those became symptomatic (hall et al., 1991) . these studies are now being reproduced with experimental human challenge infection (devincenzo et al., 2010) , and access to modern immunological techniques may provide some insight into the mechanism of immune evasion. most reinfections are limited to the upper respiratory tract, unless subjects are immunocompromised. reinfections may be the consequence of a highly prevalent and contagious virus, effective evasion of local and innate immunity, or a steep gradient for transudation of antibody from the serum to the nasal epithelium (graham, 2011) . alternatively, rsv infection may alter the characteristics of the adaptive immune effectors and memory. although rsv infection provides a sufficient antigenic stimulus to induce both antibody (shinoff et al., 2008) and t cell responses (heidema et al., 2008) , the durability of the antibody is poor (handforth et al., 2000; dakhama et al., 1997; collarini et al., 2009 ). the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. it seems logical to consider live attenuated vaccines delivered intranasally for protection against respiratory viruses, as they would induce a mucosal immune response. however, a systemic immune response can be protective if it is sufficiently robust, such as that induced by inactivated influenza vaccines administered by the i.m. route. moreover, achieving an appropriate balance between sufficient attenuation and immunogenicity, especially in young infants who must be vaccinated in the face of maternal antibody, is a challenge. this section describes licensed vaccines as well as vaccines that are currently in development. vaccination remains the primary strategy for the prevention and control of influenza (lambert and fauci, 2010) . as described in section immune responses to respiratory virus infection, after an influenza infection, both cellmediated immunity and systemic and mucosal neutralizing antibodies are produced. whereas cell-mediated immunity contributes significantly to the clearance of a primary influenza virus infection, and can ameliorate disease caused by reinfection, neutralizing antibodies play an important role in preventing reinfection. the goal of vaccination is to prime the immune response to limit viral replication upon subsequent infection, and inactivated, recombinant hemagglutinin and live attenuated influenza vaccines (laivs) are licensed for use, with novel vaccines in varying stages of development. this section focuses on licensed vaccines and the immune responses they elicit, as determined from clinical trial data. owing to antigenic drift in circulating viruses (discussed in section virology), an influenza vaccine from one season may not be effective in subsequent seasons. each year, the strains that are to be included in the vaccine for the next influenza season are chosen and vaccine seed viruses are generated (lambert and fauci, 2010) . for inactivated vaccines, the influenza a seed viruses are reassortant viruses, with the ha and na gene segments derived from the circulating virus and the internal protein genes derived from a vaccine donor strain that is adapted for high yield in eggs (a/puerto rico/8/34; pr8) (kilbourne, 1969) . licensed laivs also contain the ha and na from the circulating virus, combined with the internal protein genes from temperature-sensitive, cold-adapted, attenuated master donor viruses that limit replication of the vaccine viruses to the cooler upper respiratory tract (maassab, 1967) . if the yield of the vaccine virus in eggs is poor, they may be "egg-adapted" through serial passage. vaccine viruses are then amplified in hundreds of millions of eggs and purified. the inactivated vaccine viruses are treated with formalin or β-propriolactone and "split" with detergents before being formulated for clinical use, with or without thimerosal as a preservative (fiore et al., 2013) . until 2013, seasonal influenza vaccines were trivalent, containing two subtypes of influenza a viruses (h1n1 and h3n2) and one influenza b virus. however, from the 2013 winter season in the northern hemisphere, quadrivalent vaccines, containing two subtypes of influenza a viruses and two strains of influenza b viruses, have become available. it takes several months from the generation of a seed virus to the manufacture and distribution of a vaccine. typically, for seasonal influenza in the northern hemisphere, manufacturers amplify vaccine viruses and inactivate or purify them between february and late summer and formulate and distribute them for administration in the fall before the anticipated peak of the influenza season (lambert and fauci, 2010) . in 2009, the h1n1 pandemic virus emerged in april, when the manufacture of seasonal trivalent vaccines was already under way. a monovalent h1n1 vaccine was produced as quickly as possible in addition to the seasonal vaccines, but the monovalent vaccine was not available for widespread use until after the pandemic had peaked in the northern hemisphere and was not available at all during the 2009 winter season in the southern hemisphere (broadbent and subbarao, 2011; skowronski et al., 2011) . in addition, current global vaccine production capacity does not cover the high-risk population around the world (girard et al., 2005) . one means of increasing capacity is to move toward using cell culture instead of eggs for vaccine production, and several companies are investigating this. additionally, the amount of ha in each vaccine dose could be reduced and used with an adjuvant such as mf59 or as03. however, adjuvanted seasonal influenza vaccines are not yet licensed in the united states. trivalent inactivated vaccines (tivs) against seasonal influenza are administered i.m. and have an efficacy ranging from 60 to 100% in preventing influenza morbidity and mortality in healthy adolescents and adults (osterholm et al., 2012) . a trivalent laiv consisting of an a or b ann arbor cold-adapted backbone together with the ha and na of the target viruses (flumist ® , medimmune), administered by a nasal spray, is licensed in several countries, including north america and europe. in the united states, the seasonal laiv is licensed for healthy individuals between 2 and 49 years of age, but it is currently not approved for use in children under 2 years of age, and it is not approved for use in the elderly or in immunocompromised individuals (ambrose et al., 2011) . there is a substantial body of evidence from largescale randomized clinical trials demonstrating the effectiveness of laivs, and a number of clinical studies have also shown that the efficacy of laivs is equivalent or superior to that induced by i.m. vaccination with tivs in children (reviewed by luke et al. (2013) ). in adults, however, the majority of double-blind, randomized, placebo-controlled trials have shown tivs to have a greater efficacy than laivs monto et al., 2009; ohmit et al., 2006 ohmit et al., , 2008 . in the military, vaccine effectiveness was also found to be higher for tivs than for laivs, except in new recruits (eick-cost et al., 2012; wang et al., 2009 ). tivs primarily induce serum antibodies against the influenza ha glycoprotein, which are typically measured by hemagglutinin inhibition (hi) assays, which serve as a surrogate for virus-neutralization assays. in healthy individuals who are immunologically primed by previous infection or vaccination, influenza-specific antibodysecreting cells in the peripheral blood peak 1 week after vaccination and serum antibody levels peak 2 to 4 weeks postvaccination. however, in unprimed individuals, for example, children, it may take 4 weeks or longer for serum antibody levels to peak after vaccination (brokstad et al., 1995; cox et al., 1994; el-madhun et al., 1998) . the rise in serum antibody titer after tiv administration has been documented for multiple isotypes, including igm, iga, and igg, with a more pronounced rise in igg titers (moldoveanu et al., 1995) . in contrast, vaccination with an laiv leads to seroconversion more frequently in immunologically naïve individuals than in those who are immunologically primed. for example, in children, after a single dose of trivalent laiv, seroconversion rates of 16-58%, 92-100%, and 88-100% have been reported against influenza a h1n1, h3n2, and b, respectively, which increased to 77% and 61% for h1n1 after a second dose at day 28 or 60, respectively (belshe et al., 1998; lee et al., 2004) . however, in healthy adults, serum antibody titers are lower after laiv than tiv vaccination (moldoveanu et al., 1995) , and in one study only 59% of laiv recipients had an increase in serum igg titer compared to 94% of tiv recipients . protection mediated by inactivated vaccines therefore correlates with serum neutralizing antibody titers, whereas other immune mechanisms contribute to protection mediated by laiv. a higher percentage of laiv recipients have mucosal antibodies and antibody-secreting cells than those receiving tiv. one study recorded that 83% of laiv recipients had increased influenza-specific iga in the mucosa, compared to only 38% of tiv recipients . moreover, levels of iga in nasal wash specimens correlated with protection against challenge with wild-type influenza viruses . mucosal iga in adults vaccinated with laiv declined 6 months after vaccination . in addition to iga, igg has also been found in nasal secretions after laiv (moldoveanu et al., 1995) . as described (section immune responses to respiratory virus infection), iga is the major mediator of immunity to influenza infection in the urt, with igg serving as a backup. this also appears to be the case after vaccination with laiv. the majority of antibodies induced by influenza vaccines that are associated with protection are directed against the globular head of the ha. recently, neutralizing antibodies have also been identified that bind to a conserved epitope in the ha stem. the ha stem antibodies have been found to be broadly neutralizing, and there is much effort to generate vaccines that elicit these antibodies to produce a more broadly cross-protective vaccine (corti et al., 2010; ekiert et al., 2009; kashyap et al., 2008; okuno et al., 1993; sui et al., 2009 ). in addition, antibodies directed against the na protein are also generated after vaccination with both tiv and laiv (murphy et al., 1972) . anti-na antibodies restrict virus release from infected cells and reduce the severity of disease by limiting spread (murphy et al., 1972) . however, currently licensed inactivated vaccines are standardized to ha, but not na protein content. the amount of na protein varies from vaccine to vaccine, and the contribution of vaccine-induced anti-na antibodies to protection against influenza is not well understood (hassantoufighi et al., 2010) . in addition to humoral immunity, influenza-specific cd8 + cytotoxic t lymphocytes (ctls) are associated with accelerated clearance of virus and recovery from infection. however, the extent to which the cellular immune response is protective against infection is unknown because the recall response is likely to occur after the peak of viral replication (subbarao et al., 2006) , and cell-mediated immunity induced after vaccination has been less well studied than the humoral response, and the results are variable. studies have shown that immunization of healthy adults with whole-virus inactivated vaccine resulted in enhanced ctl responses in peripheral blood, whereas immunization with a subunit vaccine resulted in poor ctl responses, the duration of which varied from several months to years (ennis et al., 1977; mcmichael et al., 1981; powers and belshe, 1993) . in addition, an increase in ifn-γ-producing t cells was seen in children ages 6 months to 9 years of age who were vaccinated with inactivated vaccine; however, similar responses were not induced in adults (he et al., 2008) . h5n1-specific cd4 + t cells were also detected after a single dose of as03-adjuvanted inactivated vaccine and was amplified by a second dose of vaccine (moris et al., 2011) . in addition, numerous studies have found a significant increase in ifn-γ-producing cd4 + and cd8 + t cells after vaccination with laiv in both adults and children (basha et al., 2011; forrest et al., 2008; he et al., 2006a; hoft et al., 2011; lanthier et al., 2011) ; however, the role of cellular immune responses in laiv-mediated protection needs further investigation. more than 90% of annual influenza-related deaths in the united states occur in individuals 65 years of age or older (thompson et al., 2003) . vaccinating elderly individuals is therefore a public health priority. however, a randomized placebo-controlled trial estimated the efficacy of the tiv to be 50% for the prevention of influenza in older adults (govaert et al., 1994) . elderly individuals often have a significantly reduced antibody response to influenza vaccination (goodwin et al., 2006) . in a quantitative review of 4492 elderly subjects, 42%, 51%, and 35% seroconverted to h1n1, h3n2, and influenza b vaccination, respectively, compared to 60%, 62%, and 58% in younger subjects (goodwin et al., 2006) . the impaired ability of the elderly to mount an adequate immune response to influenza vaccines has been attributed to immunosenescence. immunosenescence is the decline in the body's ability to fight infection, mount novel immune responses, and recall responses (targonski et al., 2007) , and both innate and adaptive responses are implicated. impaired function of costimulatory molecules, altered secretion of inflammatory cytokines, and diminished function of natural killer cells, macrophages, and neutrophils have been observed in the elderly, as well as a decreased proliferative capacity of b cells and impaired t cell memory recall (sullivan et al., 2010) . in addition, thymic involution and a decline in t cell output are features of advancing age. this, together with a lifetime of exposure to a variety of pathogens, leads to a reduction in the naïve t cell pool and a relative increase in the proportion of memory t cells in the elderly compared with young adults. the most pronounced functional changes are in the cd8 + t cell subset, in which progressive exhaustion occurs (pawelec et al., 2005) , whereas the cd4 + t cell subset is less affected by replicative senescence (czesnikiewicz-guzik et al., 2008) . the tiv is standardized on the basis of the amount of ha protein, with one dose of 15 μg per strain being recommended in healthy, previously primed individuals. increasing the dose increases serum antibody response to the vaccine, and in an attempt to enhance immune responses to influenza vaccines in the elderly, a high-dose tiv (60 μg ha protein per strain) was licensed in 2009 for use in persons ages 65 years and older. postlicensure studies have shown enhanced immune responses in this age group, compared to the standard dose (sullivan et al., 2010) , and vaccine effectiveness studies are under way. in addition, an as03-adjuvanted tiv (discussed below) containing 15 μg ha protein per strain showed a 12% higher efficacy than a nonadjuvanted tiv in a phase 3 randomized clinical trial in the elderly, but the difference was not statistically significant (mcelhaney et al., 2013) . laivs are not licensed for use in the elderly at present; however, they have been evaluated in clinical studies in persons 50 years of age and older and are safe and well tolerated. in clinical trials, laivs were administered in addition to tivs, and coadministration was reported to enhance local ha-specific iga antibody responses. however, the efficacy of the combination was not greater than that of tiv alone (gorse et al., 2004; powers et al., 1989 powers et al., , 1991 treanor et al., 1996) . in individuals with chronic or immunocompromising conditions, serological responses to tiv vaccination are typically lower than in healthy adults. antibody responses against influenza were adequate in hiv-seropositive individuals who had no or minimal immunodeficiency or had responded well to antiretroviral therapy (chadwick et al., 1994; huang et al., 1987; madhi et al., 2011; staprans et al., 1995) . however, in individuals with advanced hiv disease and low cd4 t cell counts, tivs may not induce protective titers even after two doses (kroon et al., 2000; miotti et al., 1989) . laivs are not licensed for use in immunocompromised individuals. although there is a large amount of data available on the immune responses to seasonal influenza vaccines in humans, improved seasonal and pandemic influenza vaccines are evaluated first in animal models. the most commonly used models are mice and ferrets. mice immunized with inactivated influenza vaccines develop serum hi and neutralizing antibodies, the titers of which correlate with protection from subsequent challenge (luke and subbarao, 2011) . although cellular immune responses are mounted during a secondary influenza infection (woodland et al., 2002) , passively transferred antibodies protected immunosuppressed mice, suggesting that cell-mediated immunity is not essential for protection if sufficient antibody is present (virelizier, 1975) . the goal of parenteral immunization with inactivated influenza vaccines is to induce sufficient serum antibody titers to limit influenza disease. this protection is mediated by serum igg that transudes into the lower respiratory tract, neutralizing virus. passive transfer of immune serum to naïve mice reduced the replication of influenza virus in the lungs and protected recipient mice from lethal influenza pneumonitis, but did not prevent tracheitis or replication of the influenza virus in the urt (ramphal et al., 1979) . during a natural influenza infection of the urt, mucosal immune responses, including secretory iga antibodies, play an important role in controlling disease (section immune responses to respiratory virus infection). several studies have documented that higher levels of serum antibodies are required to provide protection against respiratory viruses in the urt compared to the lrt (prince et al., 1985; ramphal et al., 1979; takiguchi et al., 1992) . additionally, influenza in ferrets is primarily a urt infection and vaccination with killed or inactivated influenza viruses does not protect against influenza infection unless administered with an adjuvant (potter et al., 1972a,b) . adjuvants are probably required for parenterally administered inactivated vaccines to elicit the higher levels of serum igg antibody that are needed to restrict viral replication in the urt, in the absence of robust mucosal immune responses. in mice, laivs induce a range of systemic and pulmonary immune effectors and protect animals against challenge virus replication (chen et al., 2011; lau et al., 2011) . the magnitude of the pulmonary iga and memory cd8 + t lymphocyte responses depends on the replication efficiency of the vaccine virus in the respiratory tract, but systemic immunity, such as serum antibody titers and memory cd8 + t lymphocytes in the spleen, does not . after one dose of an h5n1 laiv that replicated in the lungs of mice and induced local immunity, influenza-specific lymphocytes in the lung contributed to the clearance of challenge virus from the lungs, whereas the contribution of serum antibody and splenic cd8 + t cells was negligible. after two doses, complete protection was achieved and was associated with maturation of the antibody response (lau et al., 2012) . taken together, the data suggest that laiv protects animals by inducing multiple arms of the immune response, including mucosal immunity and pulmonary and systemic antibody and cellular immune responses, in a manner similar to natural infection. however, inactivated vaccines aim to induce serum antibody alone. although this does not mimic a natural infection, if antibody titers are sufficiently high, the inactivated vaccine will protect against disease caused by influenza viruses. for inactivated influenza vaccines, serum anti-ha antibody titers correlate well with resistance to influenza infection in people as well as in animal models. lower antibody titers are associated with an increased risk of illness, though a specific antibody titer that can guarantee protection from infection has not been identified. an hi titer of 1:40 represents the level at which it is anticipated that approximately 50% of persons will be protected (hobson et al., 1972) , and this benchmark forms the basis of the licensing criteria for inactivated vaccines (cber, 2009) . although several papers refer to "seroprotection," there is insufficient evidence to support the use of this term for vaccines. the benchmark of an hi titer of 1:40 was defined in healthy adults who were experimentally challenged with an influenza virus (hobson et al., 1972) . however, antibody titers that correlate with protection in healthy adults may not translate to clinical improvements in influenza outcomes in the elderly (gorse et al., 2004) . in addition, laivs are effective despite inducing variable serum hi antibody titers. therefore, alternative correlates of protection are needed. as iga and cellular immune responses are generated in the lungs of mice vaccinated with laiv, in addition to systemic antibody and cellular responses, the extent to which the different arms of the immune response contribute to laiv-induced protection are beginning to be evaluated (chen et al., 2011; lau et al., 2011 lau et al., , 2012 . laiv-induced nasal wash igg and iga correlated with protection from virus replication, and in human challenge studies, either serum antibody or nasal wash iga was a predictor of protection (belshe et al., 2000b) . cellular immune responses have also been investigated as a correlate of laiv-induced protection, and one study found a correlation between ifnγ-producing t cells (measured by elispot) and protection from culture-confirmed influenza illness in young children (forrest et al., 2008) . in addition, it has been shown that laivs alter the expression of ifn-related genes, whereas tivs do not, indicating that the innate immune response plays an important role in protection mediated by laivs (nakaya et al., 2011) . adjuvants are added to vaccine formulations to enhance immune responses to the antigen in the vaccine. aluminum salts (alum) are the most commonly and historically used adjuvants worldwide. they act by capturing antigens at the injection site, so the antigen is slowly processed and presented by the immune system (the so-called depot effect), and they cause mild cell damage and inflammation that promotes a th2 immune enhancement (tetsutani and ishii, 2012) . moreover, alum particles enter host cells and bind dna, introducing it into the cytoplasm of antigen-bearing dcs, where it engages receptors that promote both mhc class ii presentation and dc-t cell interactions (mckee et al., 2013) . oil-in-water adjuvants, such as mf59 (novartis) and as03 (glaxosmithkline), are more effective at inducing high-titer antigen-specific serum antibody responses than alum and have been used with inactivated split-virion influenza vaccines in europe. these adjuvants induce broad, cross-clade humoral responses and permit dose sparing, in which comparable immune responses are induced with a reduced amount of ha in the vaccine (galli et al., 2009; leroux-roels et al., 2007) . our understanding of the mechanism of action of mf59 and as03 remains incomplete. neither appears to act via a depot effect; instead they induce a local and transient proinflammatory cytokine and chemokine response at the injection site and draining lymph nodes that recruit immune cells from the circulation. in mice, as03 induced the cytokine il-6 and chemokine cxcl1, which peaked locally by 6 h postvaccination. the neutrophil-mobilizing cytokine csf3 and lymphocyte-mobilizing cytokines ccl2, 3, and 5 were induced by 24 h postvaccination. in addition, the eosinophil-recruiting chemokine ccl11 and the cytokine il-1β were induced at low levels, and ifn-γ, csf2 (gm-csf), and tnf-α were induced at levels that were only marginally above background. ifn-α and -β were not induced. the local cytokine response was paralleled by an enhanced recruitment of monocytes and granulocytes in the draining lymph node (morel et al., 2011) . mf59 also induces local upregulation of cytokines, chemokines, and other innate immunity genes, promoting the recruitment of immune cells such as monocytes, dendritic cells, and granulocytes. however, the mechanism of action of mf59 was found to be independent of the nlrp3 inflammasome, but required myd88 for a tlr-independent signaling pathway (seubert et al., 2011) . whereas the inactivated vaccine viruses are typically disrupted with detergents to make split-virion (subunit) vaccines, inactivated whole influenza virion (wiv) vaccines have also been developed, in which the virions are left intact. these are less widely used because of increased reactogenicity and adverse events (fiore et al., 2013) . however, mice immunized with wiv vaccines consistently showed higher hi titers and virus-neutralizing antibody titers than subunit vaccines, as well as an increased production of proinflammatory cytokines by dendritic cells and ifn-α by plasmacytoid cells, resulting in a desired th1 response (geeraedts et al., 2008; koyama et al., 2010) . the approach of using inactivated whole influenza vaccines is being revisited with pandemic influenza vaccines. whole-virion inactivated h1n1 and h5n1 vaccines administered with alum are immunogenic in humans (kulkarni et al., 2012; lagler et al., 2012; lin et al., 2006; nakayama et al., 2012) . dna vaccines encoding one or several proteins of influenza viruses induce an immune response targeting the encoded proteins (fynan et al., 1993; ulmer et al., 1993; wolff et al., 1990) . dna vaccines can be produced rapidly and at low cost; however, designing dna vaccines is complex. over the years, it has been shown that numerous factors play roles in the efficiency of expression, such as the promoter, the g/c content, supercoiling, polyadenylation, and codon optimization (laddy and weiner, 2006) . in addition, safety remains a concern, as there might be a risk of integration into the host genome (klinman et al., 1997) . numerous studies have evaluated dna vaccines expressing np, m1, or ha proteins in animal models (fu et al., 1999; saha et al., 2006; tao et al., 2009; ulmer et al., 1998 ulmer et al., , 1996a . in mice, the administration of dna vaccines encoding the np protein of influenza induces a strong ctl response, which correlates with protection against challenge with homologous or heterologous viruses (ulmer et al., 1993) . in addition, one study showed that delivering the vaccine by in vivo electroporation instead of the classical epidermal route also induces protective humoral and cellular immune responses in mice, ferrets, and nonhuman primates (laddy et al., 2008) . recently, a phase 1 clinical trial with an adjuvanted plasmid dna vaccine encoding influenza h5, ha, np, and m2 elicited t cell responses against ha in the majority of the subjects and against np and m2 in some (smith et al., 2010) . licensed influenza vaccines primarily elicit an immune response to the globular "head" region of the ha glycoprotein. however, immune selection pressure leads to antigenic drift (discussed in section clinical features and epidermology), so new influenza vaccines need to be selected for each season as well as when a pandemic emerges. it remains difficult to predict which strains will circulate in the upcoming influenza season, and rates of morbidity and mortality are greater in influenza epidemics when the virus and vaccine are "mismatched" (pica and palese, 2013) . the expectation is that vaccines capable of protecting against a broad(er) spectrum of influenza viruses would result in less frequent updating of seasonal influenza vaccines and would provide a degree of preexisting immunity if a novel strain emerges. vaccines that aim to provide broad cross-protection against multiple subtypes of influenza are known as universal vaccines, and several platforms are in development, including those that target the ha, m2, np, m1, and na proteins (subbarao and matsuoka, 2013) . unlike the head region of ha, the "stalk" domain has a high degree of sequence conservation between influenza viruses, and broadly neutralizing stalk-reactive antibodies have been identified. these antibodies may inhibit phinduced conformational changes in the ha required for cellular entry, prevent the cleavage of ha0 into ha1 and ha2 (discussed in section virology), or act via antibodydependent cell-mediated cytotoxicity (adcc) or through the activation of complement . however, the ha stalk is less immunogenic than the head, and several techniques have been employed to elicit stalk-reactive antibodies, including removing the head, novel prime-boost strategies, and sequential vaccination with chimeric ha molecules that contain the same stalk region but "exotic" head domains that aim to boost antibodies to the conserved epitopes within the stalk . these strategies have been tested in mice with varying degrees of success in generating protective immune responses against heterologous influenza viruses (subbarao and matsuoka, 2013) . taken together, ha stalkbased strategies show promise as universal influenza vaccine candidates. the influenza m2 protein extends beyond the viral envelope, and antibodies against the m2 extracellular domain (m2e) may act via adcc, nk cell activity, or complementmediated lysis. in addition, the sequence of the m2e is conserved among human influenza a viruses, making it an attractive target for universal vaccines. however, the peptide is poorly immunogenic, and techniques to improve immunogenicity are under investigation in mice, including fusing the peptide to an immunogenic carrier protein or delivering the antigen in a virus-like particle (vlp) (reviewed by subbarao and matsuoka (2013) ). the influenza np and m1 proteins are also conserved among influenza a viruses and have potential for use in a universal vaccine, but as they are not exposed on the surface of virions or infected cells, they mainly induce cellular immune responses. phase 1 and 2 clinical trials with modified vaccinia virus ankara (mva) expressing np and m1 proteins report a 60% reduction in laboratory-confirmed influenza infection after experimental challenge (lillie et al., 2012) ; however, larger studies are required to confirm this. immune responses directed against the influenza na protein do not protect against infection, but rather limit the spread of infection and reduce the severity of disease. recently, baculovirus-expressed vlps containing n1 na proteins were found to induce heterosubtypic na antibodies in mice that protected them against homologous and heterologous challenge (quan et al., 2012) . a universal vaccine may therefore benefit from anti-na immune responses. it may be useful to combine several components into a universal vaccine, for example, np and m1 to induce cellular responses and ha and m2e to induce humoral responses. however, there are several challenges to the development of a universal vaccine. as outlined above, most of the viral proteins that potentially induce broad heterosubtypic immunity are poorly immunogenic and require a large dose of antigen, multiple doses, addition of adjuvants, fusion to immunogenic carriers, or the use of vectors or vlps. additionally, regulatory challenges include how to determine and define the potency of the vaccine and the need to identify immune correlates of protection and develop validated assays to measure them. in addition, clinical trials will be challenging because it is likely that efficacy will be measured in terms of amelioration of disease rather than preventing infection (subbarao and matsuoka, 2013) . given these hurdles, achieving a truly universal vaccine that protects against all types or subtypes of influenza will probably proceed in a stepwise manner, with the first step being a vaccine that is more broadly cross-protective than currently licensed vaccines. the primary target populations for rsv vaccination are young infants and the elderly, because hospitalization rates are the highest in these age groups and 78% of rsv-related deaths occur in individuals over 65 years of age (thompson et al., 2003) . there is a large body of evidence that protection against infection is conferred mainly by neutralizing antibodies (collins and melero, 2011) ; however, multiple doses of vaccine might be necessary in young infants, because of the immature immune system and the presence of maternal antibodies. in addition, protective immunity mounted during infection does not protect against subsequent reinfection. reinfections are common and are independent of antigenic changes in the virus (collins and melero, 2011) . these factors present challenges to rsv vaccination and, as yet, vaccines have not been licensed. this section discusses clinical trial data from various approaches to rsv vaccination. the first approach to vaccination was the development of a formalin-inactivated rsv vaccine (f1-rsv) that was evaluated in the 1960s in infants and young children. a concentrated f1-rsv vaccine, given i.m. with alum, was well tolerated and moderately immunogenic, but was poorly protective against infection. in fact, vaccinees who were exposed naturally to infection during the subsequent rsv season had immune-mediated enhancement of disease, with 80% of individuals requiring hospitalization and two fatalities (fulginiti et al., 1969; kapikian et al., 1969; kim et al., 1969) . subsequent studies revealed that vaccine-induced immune responses were different from those elicited after natural infection, with poor induction of neutralizing antibodies (murphy and walsh, 1988) and an exaggerated cd4 + t-cell response (kim et al., 1976) . vaccine-mediated enhancement of disease also occurred in murine models, with poor induction of neutralizing antibodies probably due to denaturation of antigen in the vaccine or a lack of antibody affinity maturation after poor tlr stimulation (delgado et al., 2009 ). in addition, an exaggerated th2 response and a loss of regulatory t cells contributed to disease (connors et al., 1992 (connors et al., , 1994 de swart et al., 2007; loebbermann et al., 2013; waris et al., 1996) . therefore, it is clear that in addition to a neutralizing antibody response, a balanced cd4 and cd8 t cell response is also desirable. after this experience, inactivated rsv vaccines were considered unsuitable for pediatric use, and alternative approaches were sought. in the 1960s, a series of live attenuated vaccines (lavs) was developed by serial passage of rsv at suboptimal temperatures (cold passage, cp) or by growth in the presence of mutagens to produce temperature-sensitive (ts) mutants. the lav did not cause disease enhancement in animal models or in clinical trials (wright et al., 2007a) . the most promising vaccine generated by this method, cpts248/404, was well tolerated and immunogenic in seronegative infants and children greater than 6 months of age, but caused mild congestion in younger infants and was deemed to be insufficiently attenuated (wright et al., 2000) . several lavs have also been developed using reverse genetics techniques and were evaluated in clinical trials. the most promising, ra2cp248/404/1030δsh (medi-559), was strongly ts and was well tolerated in infants . the first dose provided substantial reduction in replication of a second dose of vaccine given 2 months later. however, the majority of individuals did not have increases in serum antibody titers, indicating that other immune mechanisms might play a role in the protection from replication of the second dose of vaccine. as with live attenuated influenza vaccines, establishing correlates of protection for live rsv vaccines is an active area of research, made more challenging by the weak immune responses in infants and limitations on sampling. as of this writing, clinical trials are under way to further monitor tolerability and immunogenicity and the ability of candidate vaccines to induce protection against natural rsv exposure in the community (collins and melero, 2011) . genetic changes have been employed to generate candidate lavs, including deletion of the m2-2 coding sequence, which increases transcription and antigen synthesis at the expense of viral replication (bermingham and collins, 1999) , and deletion of the ns1 and ns2 genes, which increase ifn production and signaling and might limit viral replication and pathology, but increase immunogenicity (teng et al., 2000) . the use of attenuated parainfluenza virus as a vector to express rsv f and/or g protein has also been considered as a pediatric vaccine against both hpiv and rsv, as piv has the advantage of improved in vitro growth and stability compared with rsv. bovine piv3 is attenuated in humans and has been used as a vector into which the f and hn genes from human piv3 and the f and/or g protein of rsv are incorporated to make a bivalent vaccine against both hpiv3 and rsv (schmidt et al., 2002; tang et al., 2008) . one example of this approach is in clinical trials in children older than 6 months of age and who are seronegative to rsv and hpiv3 (collins and melero, 2011) . the vaccine in this study comprises a bovine piv3 vector into which the human piv3 f and hn genes and the rsv f gene have been added (tang et al., 2004) . as an alternative to bovine piv3, murine piv1 (sendai virus) is also being evaluated as a vaccine backbone into which rsv antigens are inserted (jones et al., 2009) . there is substantial antigenic crossreactivity between sendai virus and hpiv1, and the virus may be attenuated in humans. other approaches to live vaccines for rsv include the use of alphaviruses (elliott et al., 2007) or replicationdefective adenoviruses (shao et al., 2009) as vectors for rsv antigens. in addition to an rsv vaccine in infants, there is also a need for rsv vaccines in older children, adults, and the elderly. however, these individuals are likely to have been previously infected with rsv, and preexisting immunity may restrict the replication of lavs. in addition, vectored vaccine approaches should not be based on common human pathogens, as preexisting immunity to the vector may limit vaccine virus replication and interfere with the development of immunity. the use of protein-based vaccines in these populations is currently being evaluated. disease enhancement has not been documented in adults who were previously exposed to rsv, and candidate rsv f protein-based vaccines are well tolerated in healthy adults, children over 12 months of age, pregnant women, and the elderly (girard et al., 2005) . in a phase 3 clinical trial involving 289 children 1-12 years of age with cystic fibrosis, a purified f protein-3 (pfp-3) vaccine induced a fourfold rise in serum antibody titer, but was not associated with significant protection against lrti episodes compared to placebo . in another approach, a subunit vaccine of copurified f, g, and m proteins from rsv-a was tested in adults and found to induce neutralizing antibodies in 76-93% of vaccinees, but titers waned after 1 year, suggesting that annual immunization might be necessary with this vaccine (girard et al., 2005) . interestingly, in a phase 1 clinical study of 35 pregnant women vaccinated with pfp-2, anti-f antibodies were persistently elevated in newborns of the vaccinated mothers, without enhancement of disease. maternal immunization could, therefore, be a strategy to protect infants under 6 months of age, for whom the development of an rsv vaccine has been such a challenge (durbin and karron, 2003) . a recombinant postfusion form of the f protein with a deletion of the major hydrophobic regions was also produced. this subunit vaccine forms stable trimers that are recognized by neutralizing mabs. high levels of neutralizing antibodies were induced in the sera of vaccinated rodents, and the animals were protected from challenge. this may represent an improved subunit vaccine (mclellan et al., 2011; swanson et al., 2011) . another protein-based vaccine, bbg2na, has been evaluated in clinical trials. bbg2na consists of a fragment of the rsv g protein that contains a central conserved domain fused to the albumin-binding domain of streptococcal g protein, expressed in bacteria. however, this vaccine was not very immunogenic in clinical trials and was associated with hypersensitivity reactions (purpura) in some individuals (power et al., 2001) . vlps have also been evaluated for use as an rsv vaccine, and one such formulation (rsv-f particle vaccine) is currently in clinical trials (collins and melero, 2011) . it is the hope that improved methods of producing rsv antigens or using vlps will result in an effective vaccine that could be given to adults periodically, possibly with the annual influenza vaccine. however, in children, it remains to be seen whether a vaccine can be developed that is sufficiently attenuated, yet immunogenic enough to provide protection. however, even if complete protection is unrealistic, immunity that results in substantial reduction of virus replication may be sufficient to prevent severe disease. as 80% of children are seropositive for hpiv by 5 years of age, and hpiv lrti is a major cause of hospitalization in this age group (schomacker et al., 2012) , young infants and children are the target population for hpiv vaccination. protective immunity against infection is mediated primarily by mucosal and serum neutralizing antibodies; however, reinfection is common owing to a difficulty in maintaining protective titers of s-iga and igg in the respiratory lumen, thus representing a challenge to vaccine development (glezen et al., 1984) . this section reviews data from clinical trials of inactivated, live, and vectored hpiv vaccines. the first approach taken to hpiv vaccination was the use of formalin-inactivated piv3 (fi-piv3) in an intramuscular vaccine. although vaccine-induced disease enhancement seen with formalin-inactivated rsv was not observed with fi-piv3, there was no evidence of protection either (kim et al., 1969) . subsequent vaccine efforts, therefore, focused on the generation of live attenuated vaccines, and two approaches were investigated: the use of a ts hpiv3 strain and the use of a bovine piv strain. successive passage at lower temperatures has been shown to attenuate hpiv. the most promising vaccine candidate using this approach, cp45, is designated by the number of times the virus was passaged at low temperature in african green monkey kidney cells (belshe and hissom, 1982; ray et al., 1995) . the vaccine was safe and immunogenic in adults, seropositive and seronegative children, and infants 1 month of age or older (belshe et al., 2004b) . in a phase 2 clinical trial in 6-to 18-month-old children, the vaccine was well tolerated and 84% of previously seronegative vaccinees had a fourfold or greater rise in serum geometric mean antibody titer (belshe et al., 2004b) . moreover, coadministration of an rsv and cp45 piv vaccine was successful, with little evidence of interference (belshe et al., 2004a) . however, the cp45 piv vaccine was overattenuated in seropositive children and adults. bovine piv3 makes an attractive lav as it is known to be antigenically related to human piv, is attenuated in humans, and protects monkeys against challenge with hpiv3. clinical trials were conducted in adults, seropositive children, seronegative infants, and children 2-6 months of age with residual maternal antibodies. in seropositive individuals, the vaccine was overattenuated, but in seronegative children and infants, the vaccine virus was highly infectious. despite replication of the vaccine virus, serum antibody responses to hpiv3 were low, consistent with an incomplete antigenic relatedness between the human and the bovine piv hn genes (greenberg et al., 2005; karron et al., 1995) . a phase 2 clinical trial of 192 children showed that seroconversion to bovine piv3 occurred in 57-67% of vaccinated children after three doses, but that seroconversion to hpiv3 occurred in only 21-25% of vaccinees (greenberg et al., 2005) . additionally, during the study, 47% of the placebo group experienced piv3 infection, highlighting the ubiquitous nature of the pathogen. after these studies, it was concluded that bovine piv3 might better serve as a backbone for the insertion of hpiv3 genes. as described above, at this writing, one such vaccine, with hpiv3 hn and f genes and the rsv f gene inserted into a bovine piv3 backbone, is being evaluated in clinical trials in seronegative children. this vaccine candidate was highly attenuated in seropositive children and adults. additional vaccine candidates include those generated by reverse genetics. one such vaccine contains mutations in the p/c gene, which reduces the virus's ability to inhibit type i ifn induction and signaling, and another contains ts and attenuating mutations from other paramyxoviruses (sato and wright, 2008) . by the age of 5 years, most children will have been infected with hmpv. reinfections are common, and hmpv is responsible for 5-15% of childhood hospitalizations for lrti. in addition, severe disease can also be seen in the elderly or immunocompromised individuals (feuillet et al., 2012) . despite this clinical need, hmpv vaccines have not yet entered clinical trials, and the data reviewed in this section are from studies in animal models. as with rsv, vaccination of macaques and mice with inactivated hmpv led to disease enhancement after challenge and a strong th2-type immune response associated with a lack of neutralizing antibodies (herfst et al., 2008a; hamelin et al., 2007) . therefore, alternative approaches have been evaluated, including soluble protein-based subunit vaccines, live attenuated vaccines, or dna vaccines encoding viral proteins. soluble f-protein subunit vaccines have been shown to generate high titers of neutralizing antibody in serum in golden syrian hamsters and cotton rats, associated with protection against subsequent infection (cseke et al., 2007; herfst et al., 2007) . however, stable and long-term immunity was not induced in monkeys (herfst et al., 2008b) . live attenuated vaccines have been generated either by cold passage or by reverse genetics. temperature-sensitive viruses conferred complete protection against heterologous hmpv challenge in golden syrian hamsters (herfst et al., 2008a) , and a recombinant hmpv lacking a region of the sh (δ 101−103 sh), the g, or the m2-2 protein induced high titers of neutralizing antibody and protected hamsters and african green monkeys against piv and hmpv challenge (biacchesi et al., 2004; buchholz et al., 2005 buchholz et al., , 2006 . a chimeric vaccine has also been developed in which the f2 subunit of the hmpv fusion protein is inserted into a backbone of bovine piv that also contains the hpiv3 f and hn genes (tang et al., 2003) . this b/hpiv3/hmpv f2 vaccine induced neutralizing antibodies and protected hamsters and african green monkeys against piv and hmpv challenge (tang et al., 2003 . coronaviruses such as hcov-229e and oc43 are frequent causes of respiratory illness throughout the world, and sars-cov and mers-cov represent significant public health threats. however, at present there are no licensed vaccines for human coronavirus infections, though a number of vaccines are licensed against animal coronaviruses, and several vaccine platforms have been developed for sars-cov that show great promise in preclinical studies. whole sars-cov particles inactivated with β-propiolactone, formalin, uv light, or a combination of two techniques have been evaluated in mice, ferrets, rabbits, and nonhuman primates. such vaccines have been administered by a variety of routes and tested in the presence or absence of various adjuvants. in general, vaccination elicited a serum igg response in animal models, with higher doses of vaccine eliciting higher igg antibody titers ( subbarao, 2010) . moreover, some studies demonstrated that inactivated sars-cov vaccines were efficacious in protecting mice (see et al., 2006; spruth et al., 2006; stadler et al., 2005) and nonhuman primates (qin et al., 2006; zhou et al., 2005) , although many studies did not investigate protective efficacy. in addition, an inactivated sars vaccine that was evaluated in clinical trials was safe and well tolerated and induced neutralizing antibodies (lin et al., 2007) . many subunit vaccines comprise purified, recombinant sars-cov s protein, as this protein is the target for protective neutralizing antibodies elicited by sars-cov infection. a truncated soluble form of the s protein that lacks the transmembrane domain also neutralized infectivity of sars-cov (bisht et al., 2005; he et al., 2006b; zhou et al., 2006) and substantially reduced the titer of challenge virus replication in the respiratory tract of mice (bisht et al., 2005) . a trimeric spike protein vaccine (trispike) was also immunogenic in mice and hamsters and provided protection against challenge in hamsters, with reduced occurrence and severity of pneumonitis and no evidence of pulmonary consolidation or sars-cov-associated hepatic cellular necrosis (kam et al., 2007) . however, sera from animals immunized with trispike were associated with a 100-to 1000-fold increase in virus entry into fcγrii-positive (ace2-negative) human b cells, which has led to concerns about antibody-dependent enhancement of disease (kam et al., 2007) . vectored vaccines have been developed against sars-cov. in this approach, sars-cov proteins are expressed by venezuelan equine encephalitis virus replicon particles (vrps), chimeric bovine/human piv3 vectors, recombinant replication-defective human adenovirus-5 (ad-5), poxvirus mva, attenuated rabies viruses, or attenuated vesicular stomatitis virus (subbarao, 2010) . when animals were immunized with vectored vaccines expressing the s protein of sars-cov, neutralizing antibodies were induced, and vaccinated animals were protected from challenge. however, if other sars-cov proteins were expressed, animals were not protected from challenge. for example vrps expressing the sars-cov s protein provided protection from challenge in mice; however, vrp expressing the sars-cov n protein (vrp-n) did not (deming et al., 2006) . moreover, vrp-n vaccination resulted in a marked bronchiolitis, alveolitis, and interstitial accumulation of eosinophils and mononuclear leukocytes (deming et al., 2006) . in addition, chimeric bovine/ human (b/h) piv3 vectors expressing the sars-cov s protein or the s, m, and e proteins together also generated neutralizing antibodies in hamsters and protected animals from challenge, whereas b/h parainfluenza viruses expressing m, n, or e proteins were not protective . the route of administration is also important in determining protective efficacy of a vaccine. an ad-5 vector expressing the sars-cov s and n genes given i.m. had a limited effect in reducing pulmonary replication of challenge virus, despite eliciting higher serum igg titers and a greater cellular immunity than when the vaccine was given intranasally (i.n.). however, the vaccine administered i.n. induced higher mucosal iga responses than when given i.m. and provided protection from pulmonary replication, suggesting that mucosal immunity is important in mediating protection (see et al., 2006) . mice, rabbits, and monkeys immunized with a modified vaccinia virus mva-s vaccine were protected from replication of challenge virus (bisht et al., 2004; chen et al., 2005) . however, in ferrets, mva-s antibodies declined rapidly after vaccination, and the vaccine did not protect the animals from replication or spread of the challenge virus (weingartl et al., 2004) . a recombinant live attenuated sars-cov vaccine virus in which expression of the e protein was abrogated by point mutations and a deletion in the nucleotide sequence was restricted in vitro and attenuated in hamsters (dediego et al., 2007) . in addition, inactivation of the 3′-5′ exonuclease attenuated sars-cov, and the resulting virus was protective in an aged immunocompromised mouse model of sars infection (graham et al., 2012) . dna vaccines expressing sars-cov s protein (either without the cytoplasmic domain or without the cytoplasmic and transmembrane domains) also induced humoral and cellular immunity in mice and protected animals from replication of challenge virus . in addition, priming with a dna vaccine and boosting with an inactivated vaccine resulted in an increase in cd4 + t cells and a stronger antibody response (zakhartchouk et al., 2005) . the dna vaccine was well tolerated and produced cellular immune responses and neutralizing antibody in a phase 1 clinical trial (martin et al., 2008) . despite these successes in preclinical vaccine development, as the sars outbreak was declared over in 2003, the impetus was not sustained long enough for many of these products to be evaluated in clinical trials. a cautionary note about sars-cov vaccines is the association of a variety of vaccine platforms with pulmonary immunopathology in mice after challenge with the virus, despite immunogenicity and protective efficacy (tseng et al., 2012) . the relevance of the rodent model to vaccines in humans remains uncertain. adenovirus epidemics have been described in adults, especially in military recruits in closed or crowded settings (lynch et al., 2011) . live attenuated, orally administered adenovirus vaccines against serotypes 4 and 7, which cause respiratory disease, were used by the u.s. military starting in 1971 (top, 1975) . during this time the incidence of disease fell substantially. however, when the manufacturer ceased vaccine production in 1995, epidemics reemerged in military facilities (lynch et al., 2011) . in 2011, a new live attenuated adenovirus vaccine was issued to military recruits during basic training, and there has since been a reduction in the rate of febrile respiratory illness (armed forces health surveillance, 2013). the vaccine is not available to children or the civilian adult population at present, though adenoviruses are estimated to account for 5-10% of pediatric and 1-7% of adult respiratory tract infections (lynch et al., 2011) . in summary, the majority of studies evaluating respiratory virus vaccines measure serum antibody responses, because a large body of evidence indicates that, although both cellular and humoral responses contribute to the clearance of a primary infection, neutralizing antibodies protect against secondary infection. humoral responses can be readily detected after vaccination with inactivated or subunit vaccines; however, fewer individuals seroconvert after vaccination with live vaccines. alternative immune mechanisms such as mucosal antibody responses are probably responsible for protection by live attenuated vaccines, and immune correlates of protection are under investigation. because respiratory pathogens infect the host at mucosal surfaces, the induction of mucosal immunity by vaccination is desirable. vaccines that are administered by intramuscular or subcutaneous injection induce protective immunity in the systemic immune compartments, but are generally poor at inducing mucosal immune responses. mucosally delivered vaccines, in contrast, induce both mucosal and systemic immunity and are also more readily accepted because they do not require needles or syringes (levine and dougan, 1998; yuki and kiyono, 2009) . ongoing research into the molecular and cellular mechanisms of surface immunological barrier systems has provided practical strategies for the development of a new mucosal vaccine for the control of respiratory viruses. m cells, located in the follicle-associated epithelium of the malt, play a pivotal role in the uptake of luminal antigens in the respiratory tract and the induction of antigen-specific immune responses in both the systemic and the mucosal compartments . it is, therefore, logical and attractive to develop m-cell-targeted mucosal vaccines. several molecules have been found to bind preferentially to m cells, such as ulex europaeus agglutinin-1 (uea-1), which has specificity for α(1,2)-fructose (sharma et al., 1998) , and rho-protein 1 derived from reovirus, which binds to a carbohydrate structure containing α2, 3-linked sialic acids on the plasma membranes of m cells (helander et al., 2003) . moreover, intranasal vaccines conjugated to either uea-1 (manocha et al., 2005) or rho-protein induced not only strong antigen-specific plasma igg and mucosal iga responses, but also ctl immunity (wu et al., 2001) . in addition, a novel m-cell-specific monoclonal antibody has recently been identified that selectively recognizes m cells, but not goblet cells or epithelial cells. this has been used as a carrier for an m-cell-targeted mucosal vaccine (wu et al., 2001; nochi et al., 2007) . concerns have been raised about the potential induction of unwanted mucosal inflammation associated with m cell targeting (kuolee and chen, 2008) , and additional research is needed to better understand how m cells sample antigens and transcytose them to the basolateral membrane. however, most microparticles administered orally become trapped in the mucus, and only a small fraction of them enter mucosal inductive sites. new approaches that are being developed to transitionally or conditionally enhance the number and function of m cells (neutra and kozlowski, 2006) are likely to reduce these concerns. there is substantial interest in the exploitation of nanoparticle technology for drug and vaccine delivery. nanoparticles are solid particles ranging in size from 10 to 1000 nm in diameter that are often made from biodegradable materials. an antigen payload can be dissolved, entrapped, adsorbed, attached, or encapsulated into the matrix of the particle and released as the particle degrades over a period of time, which may vary from days to weeks, depending on the formulation (adair, 2009) . several types of nanoparticles have been investigated for vaccine delivery and have proven to be safe, nontoxic, and suitable for loading with antigens. these include the polyesters (poly(lactic acid), poly(glycolic acid), and their copolymers), polyorthoesters, polyanhydrides, and polycarbonates (reddy et al., 2006) . these materials can protect antigens from degradation, and the particles can be prepared in a chemically reproducible manner within a narrow size limit. in addition, some biopolymers exhibit a natural adjuvant behavior, for example, poly(lactide co-glycolide) (plga) appears to activate the maturation of dcs, possibly by providing a necessary stimulatory signal, though the exact mechanism is not fully elucidated (bennewitz and babensee, 2005; yoshida and babensee, 2004) . surface modifications of nanoparticles that change their overall charge and hydrophobicity are aimed at improving mucoadherence properties (des rieux et al., 2006) . this can be achieved by coating with stabilizers, other polymers, or surfactants. polyethylene glycol has been used for its stabilizing properties and because it can enhance the affinity of the nanoparticles for mucosal surfaces. molecules such as lectins and chitosan can also increase interaction with and transport across the mucosal surface. interestingly, chitosan is reported to be able to open tight junctions between epithelial cells, facilitating the transport of encapsulated macromolecules across the epithelial layer . nanoparticles coated with mannose (mannan) have also been produced with the aim of targeting mannose receptors on apcs, thus improving cell adhesion and uptake (cui and mumper, 2003) . a plga-coated nanoparticle vaccine encapsulating influenza virus proteins has been developed. the influenza ha protein retained its antigenicity after encapsulation, and mice vaccinated with plga particles of 2.2-10.8 μm in diameter mounted both systemic and mucosal responses, which were protective against intranasal challenge with an h3n2 influenza virus (amidi et al., 2007; moldoveanu et al., 1993) . chitosan-coated nanoparticles containing purified influenza virus have also been tested in human volunteers, in which a fourfold or greater increase in anti-ha antibodies was observed in >40% of the volunteers . moreover, a single dose of the intranasal vaccine resulted in high titers of nasal iga antibody and strong systemic antigen-specific responses, which were greater than those induced after intramuscular inoculation with soluble influenza antigen (amidi et al., 2007) . nanoparticleconjugated rsv proteins have also been shown to generate strong systemic th1 immunity in mice, associated with protection against wild-type rsv challenge (kalkanidis et al., 2006; xiang et al., 2006) . in addition, plgacoated nanoparticles incorporating hpiv hn and f glycoproteins have been shown to induce virus-neutralizing antibodies, which were protective against challenge infection in hamsters (ray et al., 1993) . studies in animal models and humans have shown that the choice of adjuvant can dramatically affect the immediate immune response and long-term protective effect of a vaccine (ogra et al., 2001; galli et al., 2009) . in addition, the quality of the immune response, especially the development of high-affinity b cells, long-lived memory b cells, and plasma cells can be influenced by the choice of adjuvant (galli et al., 2009) . although the mechanisms have not yet been fully elucidated, mucosal adjuvants can be broadly classified into two categories: those that act as immunostimulatory molecules and those that facilitate vaccine delivery (o'hagan, 2001) . the former include adjuvants that are toxin-based or cytokine-based and molecules associated with innate immunity, for example, pamps. the latter contain immune-stimulating complexes, liposomes, live attenuated vectors, and chitosan (discussed above). from a mechanistic point of view, mucosal adjuvants modulate innate immune responses in the same way as parenterally administered vaccines (lambrecht et al., 2009) , and tlr agonists constitute a major category of mucosal adjuvants. these adjuvants are based on pamps and are often formulated as oil-inwater emulsions. virosomes are virus-like particles that have been investigated for their potential as vaccines bungener et al., 2005) . they closely resemble native virus; however, they are nonreplicating and consist of reconstituted viral envelopes, generated by treatment with a detergent. they have a diameter of 100-200 nm and, as such, fall into the size range of small particles. virosomal influenza vaccines are available commercially and have been shown to induce ha-specific antibodies after i.m. administration. phase 1 clinical trials revealed the tolerability and immunogenicity of a matrix tm -adjuvanted virosomal h5n1 dna vaccine (cox et al., 2011) . influenza virosomes that incorporate the rsv-fusion protein have also been constructed and have been shown to generate virus-specific iga in the urt and lrt after i.n. administration with adjuvant in mice (cusi et al., 2002) . immune stimulating complexes (iscoms) are nonreplicating particles of ∼40 nm diameter, comprising viral glycoproteins complexed with saponin derived from the bark of the tree quillaia saponaria (gregory et al., 2013) . iscom antigens from a number of microorganisms, including viruses, bacteria, and parasites, have been shown to induce humoral and cell-mediated immunity; th1 immune responses appear to predominate . extensive studies have been carried out with influenza virus iscoms in several species, including mice and monkeys, and an equine influenza iscom vaccine is available commercially (mumford et al., 1994 ). an influenza iscom vaccine (sundquist et al., 1988) stimulated high levels of virus-specific igm and igg serum antibodies and proliferative t cell responses in macaques, and the animals were completely protected from intratracheal challenge with the virus (rimmelzwaan et al., 1997) . intranasal immunization of mice with rsv iscoms induced very high levels of long-lasting rsv-specific iga in both the urt and the lrt (hu et al., 1998) . bovine rsv iscoms inoculated subcutaneously into calves were completely protective against challenge with virulent bovine rsv, whereas calves immunized with a conventional bovine rsv vaccine developed moderate to severe respiratory disease after challenge (hagglund et al., 2004) . the iscom-vaccinated animals developed high titers of nasal and serum virus-specific igg as well as serum iga, which correlated with protection. detoxified derivatives of cholera toxin (ct) and heat-labile enterotoxin (lt) produced by enterotoxigenic escherichia coli are effective mucosal adjuvants that promote mucosal and systemic immunity to coadministered protein antigens via oral or nasal routes (freytag and clements, 1999; mestecky et al., 1999) . the results of murine studies demonstrated that both ct and lt induced the expression of b7-1 and/or b7-2 on apcs that deliver costimulatory signals to cd4 + t cells (freytag and clements, 1999; mestecky et al., 1999) , but ct acts in the presence of il-4 to induce predominantly th2 responses, whereas lt supports th1 responses with ifn-γ production. recent vaccine development efforts have focused on the nasal administration of antigen with ct and lt derivatives for the induction of mucosal iga (byun et al., 2001; fujihashi et al., 2002) . both ct and lt are ab5-type molecules, consisting of one a subunit and five b subunits. chimeric molecules, made by the spontaneous association of the a subunit of ct with the b subunit of the lt, or vice versa, are effective mucosal adjuvants for protein vaccines. the type of t helper responses induced is dictated by the origin of the b subunit (takahashi et al., 2009) . one such example is the nasal administration of an influenza ha subunit vaccine together with a chimeric mutant (m) ct-a/lt-b adjuvant. in murine studies, this adjuvanted vaccine protected animals from influenza virus challenge (kweon et al., 2002) . however, there are some concerns about the safety profile of toxin-derived adjuvants. nasal administration of mct-a/lt-b targeted neuronal tissues, though it did not affect trafficking of coadministered vaccine antigens into the neuronal tissues (kweon et al., 2002) . taken together, these findings suggest that although recombinant chimeric toxin-based molecules show promise as a new generation of mucosal adjuvants, the safety of the ct adjuvant may need to be improved. the inactivated lt-adjuvanted intranasal influenza vaccine (nasalflu) was withdrawn because of concomitant facial nerve paralysis (bell's palsy) that was noted as a potential adverse event caused by the ct adjuvant kunzi et al., 2009; garner-spitzer et al., 2009 ). several cytokines associated with innate immunity and inflammation support the generation of antigen-specific s-iga and serum igg/iga and may be of use as mucosal adjuvants. mice nasally immunized with soluble antigens, including ovalbumin or tetanus toxoid (tt) plus il-1α and il-1β developed antigen-specific antibody responses that were similar to those induced by coadministered ct adjuvant. the cytokines il-1α and il-1β promoted antigen-specific igg1 antibody responses initially, followed by igg2b, with minimal igg2a antibody responses, a pattern associated with a predominantly th2-type response (staats and ennis, 1999; thompson and staats, 2011) . furthermore, levels of antigen-specific s-iga antibody similar to those induced by ct were found in mucosal secretions of mice that received nasal il-1. these results indicate that il-1 could be a useful mucosal adjuvant. apcs are known to contribute to the creation of an appropriate cytokine environment for the growth and development of th1 or th2 responses. two well-known cytokines secreted by apcs, il-6 and il-12, can influence the outcome of th1 and th2 cell subset-mediated immune responses (vajdy et al., 1995; rincon et al., 1997) . when tt was used as an antigen, nasal coadministration of il-6 or il-12 induced antigen-specific serum igg antibody responses and promoted protective immunity against lethal challenge with tetanus toxin. interestingly, whereas nasal treatment with il-12 promoted mixed th1/th2-type responses, il-6 supported predominantly th2-type responses. in addition, il-12 but not il-6 can induce antigen-specific mucosal iga antibody (boyaka et al., 1999) . these findings suggest that il-12 could be a potent mucosal cytokine for the upregulation of antigen-specific mucosal immune responses. type i ifn affects the differentiation and function of immune cells, including t cells and dcs, and efficiently enhances a primary antibody response (marrack et al., 1999; luft et al., 1998) . it was reported that type i ifn was effective as both a systemic and a mucosal adjuvant, promoting th1-type immune responses (proietti et al., 2002) . nasal administration of influenza vaccine with type i ifn was effective at inducing serum antigen-specific igg2a and mucosal iga antibody responses and at providing full protection against influenza virus challenge (proietti et al., 2002) . it is well known that i.n. vaccination stimulates immune responses in the nalt and is effective at inducing mucosal immunity in the respiratory tract (brandtzaeg, 2011; jabbal-gill, 2010) . intranasal administration of the live attenuated influenza vaccine flumist (medimmune) has proven effective in protection against seasonal influenza virus infection and protects children against drifted influenza virus strains (belshe et al., 2000a; mendelman et al., 2004) . furthermore, owing to the development of novel technologies, both aerosol spray and droplet delivery of vaccines are attractive possibilities (jabbal-gill, 2010) . in addition to the i.n. route of vaccine inoculation, the sublingual route has been explored (shim et al., 2011; czerkinsky et al., 2011) . the sublingual epithelium harbors a dense lattice of dcs, and vaccine delivered via this route stimulates broad and disseminated mucosal and systemic immune responses, similar to intranasal inoculation . sublingual vaccination with soluble or particulate antigens promotes strong mucosal iga and systemic igg antibody responses as well as cd8 + t cell responses. overall, sublingual immunization was comparable to nasal immunization in the magnitude, breadth, and anatomic dissemination of the induced immune responses. importantly, sublingual administration did not redirect antigens and/or adjuvants to the brain . sublingual vaccination against influenza has been shown to protect mice against lung infection (song et al., 2008) . in addition, sublingual administration of inactivated influenza a/pr8 (h1n1) vaccine virus together with a mucosal adjuvant such as ct (cuburu et al., 2007) or a nontoxic mct-a/lt-b adjuvant induced both systemic and mucosal virus-specific antibody responses as well as ctl responses with protective immunity after respiratory challenge with the a/pr8 virus (kweon et al., 2002; song et al., 2008) . these studies demonstrated that sublingual administration of an inactivated influenza virus with a toxin adjuvant such as ct did not migrate to or replicate in the central nervous system. moreover, using mucosal adjuvant such as ct mobilizes dcs within the sublingual epithelium. these observations suggest that sublingual immunization may be another attractive and safe mucosal route for the administration of respiratory virus vaccines. given the global burden of respiratory tract infection, there remains an unmet need for effective methods of intervention. the most efficient means of preventing respiratory virus infections is vaccination. however, among respiratory viruses, licensed vaccines are available only for influenza. systemic immune responses can be protective in the absence of mucosal immunity if they are sufficiently robust, such as that induced by i.m. inactivated influenza vaccines; however, many vaccines in development for respiratory viruses are live attenuated viruses that are mucosally administered. although live vaccines show promise, our understanding of the mechanism of protection at mucosal surfaces is incomplete, and there is currently a lack 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sars-associated coronavirus (sars-cov) neutralizing antibodies in mice key: cord-270703-c8mv2eve authors: christensen, paul a; olsen, randall j; perez, katherine k; cernoch, patricia l; long, s wesley title: real-time communication with health care providers through an online respiratory pathogen laboratory report date: 2018-11-30 journal: open forum infect dis doi: 10.1093/ofid/ofy322 sha: doc_id: 270703 cord_uid: c8mv2eve we implemented a real-time report to distribute respiratory pathogen data for our 8-hospital system to anyone with an internet connection and a web browser. real-time access to accurate regional laboratory observation data during an epidemic influenza season can guide diagnostic and therapeutic strategies. we implemented a real-time report to distribute respiratory pathogen data for our 8-hospital system to anyone with an internet connection and a web browser. real-time access to accurate regional laboratory observation data during an epidemic influenza season can guide diagnostic and therapeutic strategies. keywords. analytics; epidemic; epidemiology; influenza; laboratory. the us centers for disease control and prevention (cdc) provides data regarding influenza activity, aggregated from state data sources that generally lag 1 or more weeks behind the date of release [1] . however, real-time data summarizing regional hospital system observations are more relevant for local clinical decision-making. clinicians frequently request updates from the microbiology laboratory on influenza test positivity, in addition to other common respiratory pathogens, during the respiratory virus season to help inform their daily practice. in addition, clinical laboratories should routinely monitor local influenza data to determine if epidemics are occurring, if continued testing is necessary, or if patients can be treated based on positive symptoms alone [2, 3] . to address these local needs in a major us metropolitan area, our clinical microbiology laboratory implemented an online dashboard to distribute respiratory pathogen data for our 8-hospital system to clinicians, epidemiologists, infection control practitioners, system leadership, and the public. the report provides easy access from any workstation or mobile device with an internet connection. development of this report began in the fall 2017, before the respiratory virus season, during which influenza reached an epidemic status across the united states that resulted in supply shortages, testing difficulties, and a widespread public health crisis [4, 5] . respiratory pathogen panel test result data were extracted from our laboratory information system (scc soft computer). the extracts included de-identified laboratory result data, including specimen collection date, facility, and result for all influenza and respiratory pathogen tests. the data were further analyzed and aggregated to produce 4 interactive charts published to a public-facing web server. the accuracy of the report was validated by comparison with data generated natively by our laboratory information system, as well as a manual review of all test results from 1 day. we gathered visitor statistics from the server log files. internet protocol addresses were mapped to internet service provider, country, city, and organization using ipinfo.io [6] . four distinct data summary analyses were performed. first, for our most commonly detected pathogens (influenza a, influenza b, respiratory syncytial virus, and rhinovirus/enterovirus), we calculated the number of positive tests for each day and week. second, we calculated the number of positive tests at each facility. third, we calculated the daily and weekly positivity rates of our respiratory pathogen molecular test. fourth, we calculated the frequency with which each pathogen was identified by our molecular test. these counts reflected anonymized and aggregated data devoid of protected health information. to present users with interactive and dynamic data, we elected to use hypertext markup language (html) [7] and javascript [8] as our visualization modality. we used chart.js [9] as the framework for producing our interactive charts. the data analyses generated javascript arrays that were stored in chart.js data structures to produce 4 charts ( figure 1 for both chart 1 and chart 3, we created radio buttons that allowed the user to toggle between a weekly or daily summary. for chart 1, we built a radio button to switch between basic view and detailed view. in basic view, the influenza a molecular and antigen results are grouped together; in detailed view, the subtypes of influenza a detected by our molecular platform are graphed separately. three distinct time intervals were supported, including the most recent 8 weeks, the past year, and 2015-present. the 4 charts were packaged into an html report and uploaded to a public-facing web server. we unveiled the report and requested informal feedback at the system infection control meeting, the system antimicrobial stewardship meeting, and the hospital infection prevention and control committee meeting. we included a link to the report in an e-mail distributed to all employees from the executive vice president of the hospital system. we updated the graphs daily. over the subsequent 9 weeks, the report was accessed 1594 times, and over the next 27 weeks, the report was accessed 839 times. approximately 66% of the originating ip addresses were from within our hospital system, and 6% were from locations outside the united states. views on mobile devices accounted for 16% of the traffic, and 20% of views were referred from the department of pathology and genomic medicine website. views peaked at 241 per hour right after the link was distributed by our executive vice president. during the first week, 64% of all hour intervals saw at least 1 page view, with an average of 8 views per hour. daily view counts decreased as the influenza season ended and stabilized at 32 views per week on average. at the height of the influenza epidemic at our hospital system, 32% (88/273 on december 22, 2017) of all influenza antigen tests and 24% (47/197 on december 27, 2017) of respiratory pathogen molecular tests were positive for influenza a or influenza b. forty-six percent (55/120 on december 25, 2017) of these molecular tests were positive for any respiratory pathogen. vendor supply stocks were limited nationwide [5] , and in january 2018, our supply of universal transport media diminished, requiring the creation of 1:3 aliquots to preserve material for sample collection. based on these data and following cdc/world health organization (who) guidelines for epidemics [2, 3] , our primary care group stopped testing for influenza and treated symptomatic patients as if they were influenza positive. our interactive website provided near real-time data, which allowed this decision to be made a week earlier than otherwise would have been possible using federal and state data. furthermore, our inpatient pharmacy was able to anticipate oseltamivir utilization and stock accordingly while remaining prepared for potential drug shortages. brief report • ofid • 3 we developed a near real-time report that presents statistics regarding respiratory pathogen testing from our microbiology laboratory. the population tested includes all inpatient and outpatient individuals across our 8-hospital system (2264 operating beds) and all patients in the associated free-standing emergency and primary care clinics. the report is available to any device with an internet connection and is updated daily to provide critical data to clinicians, epidemiologists, infection prevention and control committees, hospital leadership, and the public. we developed the site with mobile devices in mind, which allows the graphs and fonts to be readable on any platform. the user can switch between daily and weekly data aggregations using radio buttons. the time interval of interest can be modified using preconfigured buttons. data can be filtered by clicking the data labels in the chart legends. these features are possible because of our decision to develop a web-based report, as opposed to a pdf, spreadsheet, or word processing document. anecdotal feedback collected at the time of rollout was universally positive. interest in the report quickly peaked after the initial announcement but continued to be viewed daily. as the influenza season ended, infectious diseases clinicians asked that we add a rhinovirus/enterovirus trend to the website so they could track the summer respiratory virus season as well. at least 1 clinician changed ordering practice in early september 2018 after identifying a spike in rhinovirus/enterovirus positivity frequency. in november 2018, the chief physician executive and specialty physician group ceo sent an e-mail to physician leaders across the system regarding a significant uptick in respiratory syncytial virus isolates and rising influenza a pathogens detected by laboratory testing, which was identified through our website report. based on the data provided by our laboratory in this report and cdc/who guidelines for epidemics, our primary care group stopped laboratory testing for influenza and treated symptomatic patients as if they were influenza positive. access to accurate real-time data during an epidemic influenza season can guide diagnostic and therapeutic strategies. during the epidemic, there was a nationwide shortage of testing reagents [5] . earlier identification of high positivity rates by monitoring real-time data allows for an institution to implement "treat don't test" guidelines earlier, preserving test reagents for critical situations where they are most needed. in summary, our microbiology laboratory implemented a near real-time internet report to distribute respiratory pathogen data for our 8-hospital system to clinicians, hospital epidemiologists, infection control committees, system leadership, and the public. facile access to accurate real-time data during an epidemic influenza season can guide diagnostic and therapeutic strategies. the report is available at https://flu.houstonmethodist.org/. fluview: a weekly influenza surveillance report prepared by the influenza division guide for considering influenza testing when influenza viruses are circulating in the community world health organization. who recommendations on the use of rapid testing for influenza diagnosis centers for disease control and prevention. summary of the 2017-2018 influenza season labs take stock of surprising flu season ip address api and data solutions -geolocation, company, carrier info, type and more world wide web consortium. w3c html ecmascript language specification financial support. this work was supported by the department of pathology and genomic medicine at houston methodist hospital. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-277970-sb1wjd3b authors: kang, qianli; wang, yanyan; cui, qinghua; gong, lili; yang, yong; jiang, haiqiang; rong, lijun; rong, rong; du, ruikun title: screening for anti-influenza actives of prefractionated traditional chinese medicines date: 2020-10-14 journal: evid based complement alternat med doi: 10.1155/2020/4979850 sha: doc_id: 277970 cord_uid: sb1wjd3b traditional chinese medicines (tcms) have proven to possess advantages in counteracting virus infections according to clinical practices. it's therefore of great value to discover novel antivirals from tcms. in this paper, one hundred medicinal plants which have been included in tcm prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each by sequentially using cyclohexane, dichloromethane, ethyl acetate, n-butanol, and water. 500 tcm-simplified extracts were then subjected to a phenotypic screening using a recombinant iav expressing gaussia luciferase. ten tcm fractions were identified to possess antiviral activities against influenza virus. the ic50's of the hit fractions range from 1.08 to 6.45 μg/ml, while the sis, from 7.52 to 98.40. furthermore, all the ten hit fractions inhibited the propagation of progeny influenza virus significantly at 20 μg/ml. the hit tcm fractions deserve further isolation for responsible constituents leading towards anti-influenza drugs. moreover, a library consisting of 500 simplified tcm extracts was established, facilitating antiviral screening in quick response to emerging and re-emerging viruses such as ebola virus and current sars-cov-2 pandemic. influenza a virus (iav) infections usually cause acute respiratory disease with substantial morbidity and mortality [1] . according to the world health organization (who), seasonal influenza causes up to 650,000 deaths annually [2] . vaccines and antivirals are effective countermeasures in combating iav infections. however, current influenza vaccines require reformulation annually and do not always match circulating strains, while resistance to marketed anti-influenza drugs are increasingly emerging [3] [4] [5] . erefore, novel antivirals with different mechanism of action are urgently needed. historically, natural products were the source of virtually all medicinal preparations, such as tcms. more recently, natural products or their derivatives have continued to enter clinical trials, accounting for 32% of new medicines approved by the us food and drug administration (fda) between 1981 and 2014 [6] . however, traditionally naturalproduct research usually subject concentrated extract samples to screening. such extracts are complicated mixtures, and all the components may reach the biological target in the assay, bringing several technical problems, including (i) the concentrations of some components are too low to have measurable effects; (ii) interference or nuisance compounds may confound the signal from the assay; and (iii) the possibility of additive or synergistic effects of several compounds [7] . it is unlikely to isolate each compound from a crude extract in advance, since it is too onerous and uneconomical. alternatively, prefractionation strategies have been reported to solve, at least partially, these problems [8, 9] . for example, in a high-throughput extract prefractionation screening, 80% of the primary-screen hits from prefractionated samples were not observed in assays of the associated crude extract [10] . in the present study, 100 medicinal plants which have been included in tcm prescriptions for antiviral treatment were selected and prefractionated into 5 fractions each. e library consisting of 500 prefractionated tcm extracts was subsequently subjected to a phenotypic screening for antiinfluenza actives [11, 12] . as a result, ten tcm fractions were identified to have antiviral potency against iav, deserving further analysis for novel anti-influenza lead drugs. one hundred medicinal plants that have been previously used for antiviral treatment were purchased from affiliated hospital of shandong university of traditional chinese medicine (jinan, china). e botanical authentication was performed by pro. lingchuan xu (college of pharmacy, shandong university of traditional chinese medicine, jinan, china). voucher specimens of these materials were deposited for references in our lab. e samples were stored at −20°c and pulverized before use. more information of the 100 plants are provided in table s1 2.2. standard extraction preparation. each dry powder (200 g) of tcms was extracted with 1000 ml cyclohexane (cyh) firstly, and then the leftover solution was sequentially extracted using dichloromethane (dcm), ethyl acetate (eac), n-butanol (nba), and distilled water (w), according to the method described by liu et al [13, 14] . e five extracted solutions of 100 tcms were harvested separately by ultrasonic extraction at 40°c for 30 min and repeated twice followed by filtration and evaporation under vacuum. e organic fractions were dried in 45°c vacuum, and the water fractions were freeze-dried. all 500 samples were sealed in vessels and refrigerated separately. a portion of each extracts was removed, dissolved in dimethyl sulfoxide (dmso) at 50 mg/ml, and arrayed in 96well plates as a prefractionated tcms library. to illustrate the stability of extraction method, several active prefractionations were selected for analysis by hplc using a sunfire-c18 tm column (4.6 × 150 mm, 3.5 µm, waters corporation, milford, ma) by a waters 2695 system coupled with a waters 2998 pda detector. e mobile phase consisted of 0.5% formic acid water solution and acetonitrile. e flow rate was maintained at 0.8 ml·min −1 , and the injection volume was 20 µl. supplemented with 10% fetal bovine serum (fbs; gibco, carlsbad, ca, usa), 1,000 units/ml penicillin and 100 μg/ml of streptomycin (invitrogen, carlsbad, ca, usa). e replication-competent reporter influenza a virus carrying the gaussia luciferase gene (pr8-pb2-gluc) and wildtype influenza a/puerto rico/8/34 (h1n1, pr8) were propagated as previously described [11, 12, 15] . infections were performed in opti-mem containing 1.5 µg/ml n-tosyl-l-phenylalanine chloromethyl ketone (tpck)-trypsin (sigma-aldrich, st. louis, mo, usa). a phenotypic screening for antiinfluenza actives were carried out as previously described [11, 16] . in brief, mdck cells growing in white, flat-bottom, 96-well culture plates (perkinelmer, waltham, ma) were infected with pr8-pb2-gluc virus at 0.01 multiplicities of infection (moi) in the presence of test samples of 20 μg/ml. after 36-hr incubation, gluc assay was performed using pierce gaussia luciferase glow assay kit ( ermo scientific, rockford, il, usa) according to the manufacturer's instructions. mock infected cells were used as blank control. dmso and baloxavir acid (bxa) were set as negative and positive control, respectively [17] . for dose-response analysis, pr8-pb2-gluc-infected cells were treated with serially diluted samples, with final concentrations ranging from 40 μg/ml to 0.055 μg/ml. after 36-hrs incubation, the gaussia luciferase activities were determined. e cytotoxicity assay was performed as described previously [18] . mdck cells in 96-well assay plates were treated with serial diluted extracts (twofold diluted from 200 μg/ml to 1.56 μg/ml for 8 dilution series) and incubated at 37°c for 36 hrs. cell viability was assessed by cck-8 (medchemexpress, monmouth junction, nj, usa) according to the manufacturer's instructions. titer reduction assay was performed as previously described [11] . in brief, mdck cells grown in 24-well plates were inoculated with the influenza pr8 virus at an moi of 0.01. after 2-hour incubation at 37°c, inoculations were replaced by fresh opti-mem (1.5 µg/ml of tpck-trypsin) containing test extracts at 20 µg/ml concentration. at 36-hour after infection (p.i.), the supernatants were removed for titration. in order to quantify the robustness of the screen, z′ factor was calculated from the normalized signals from positive and negative control wells on each plate with the following equation: z′ � 1-3 × (sd of positive control + sd of negative control)/(mean of negative controlmean of positive control). sd represents the standard deviation. z′ value between 0.5 and 1.0 is considered robust enough for an hts assay [19] . e percent inhibition of the tested samples was calculated with the following equation: percent inhibition � (signal of negative control-signal of tested compound)/(signal of negative control-signal of positive control) × 100%. tcms have proven to possess valuable advantages in clinical practices, including the treatment of influenza virus which caused respiratory disease [20, 21] . novel anti-influenza actives from tcm samples are therefore anticipated. to this end, 100 medicinal plants which have been recorded as antiviral formula compositions were fractionated with cyclohexane, dichloromethane, ethyl acetate, n-butanol, and water sequentially, generating a library consisting of 500 prefractionated tcm extracts (figure 1, table s1 ). as a quality control, three fractions were randomly chosen for reextraction and hplc analysis. as figure s1 shows, the chromatogram of the tcm fraction replicates displayed identical compositions, suggesting that our library is of high quality and reliable for bioactive screen. previously, we generated a recombinant iav expressing gaussia luciferase, based on which a phenotypic highthroughput screening approach was subsequently established, providing a powerful tool for antiviral discovery [11, 12, 15] . a phenotypic screening was therefore carried out against the prefractionated tcm library for anti-influenza actives. e practical screening procedure is shown in figure 2 . primarily, the inhibitory potency of each tcm fraction at 20 μg/ml against iav was determined. e z′ value of each screening plate (ranges from 0.55 to 0.89) was evaluated as quality control (figure 3(a) ). as a result, 47 samples showing >80% inhibition were cherry-picked as primary hits and subjected to a second round of antiviral determination as well as cytotoxicity assay (figures 2 and 3(a) ). among the 47 primary hits, 13 were excluded due to cytotoxicity, 24 were confirmed as inactives, and the 10 leftover hit fractions were shortlisted for dose-response analysis (figure 3(b) ). since all ten hits showed dose-dependent inhibition to iav replication ( figure s2 ), the ic 50 of each hit fraction was therefore calculated, as well as the cc 50 's and selectivity index (si) ( table 1) . all ic 50 's of the hits ranges from 1.08 to 6.45 μg/ml. moreover, except ethyl acetate fraction of glycyrrhizic, of which the si is lower (7.52), all sis of the other nine hits exceed 10, suggesting high potency as antiviral actives. considering exogenous luciferase expression was used to indicate iav replication when using reporter influenza pr8-pb2-gluc virus, a gluc inhibitor should probably be identified as false positive. to better address the antiviral activities of hit fractions against iav, a conventional titer reduction assay were further performed using influenza a/puerto rico/8/34 (pr8) virus. as shown in figure 4 , all 10 hit fractions suppressed the viral replication significantly at 20 μg/ml, suggesting that all hit fractions comprise anti-influenza components by targeting influenza viral replication. notably, nba fraction of areca catechu l. and eac fraction of magnilia officinalis almost completely inhibited iav yielding. it has been well recognized that, for drug discovery using hts, diversity within biologically relevant 'chemical space' is more important than library size, while natural products provide a different, wider, and more drug-like chemical space than synthetic compounds [22, 23] . moreover, the clinical use of tcms have proven therapeutic efficacy for various diseases [24] [25] [26] , coupling the components and specific targets tightly. in this study, 100 tcm plants were selected as compositions of antiviral prescriptions by literature review [27] , and each plant was prepared into 5 simplified extracts, generating a library consisting of 500 tcm fractions ( figure 1, table s1 ). prefractionation can partially remove compounds that are likely to cause artefacts, greatly reducing the complexity of each extract, and as a consequence, increase the hit rate when subjected to an antiviral screening. in addition, since the fractions were prepared by a chromatographic method, subsequent chromatography on a target fraction can be achieved easily, in contrast to the risk that one may fail to find a responsible constituent in active crude extracts [7] . by using a hts approach based on recombinant reporter influenza pr8-pb2-gluc virus, the antiviral activity of each fraction against iav was evaluated, and 10 simplified extracts were identified as anti-influenza actives ( figure 3 ). all sis of the hits exceed 10, except with ethyl acetate fraction of glycyrrhizic, of which the si is 7.52 (table 1) . it could be proposed that these tcm extracts might contain bioactive components responsible for anti-influenza virus activity at nontoxic concentration, providing a promising source of natural influenza inhibitors. virus titer (tcid 50 /ml) results are the means ± standard deviation. all data have three multiple duplications. * * * , p < 0.0001; ns, no significant difference. e dotted line represents the detection limit. interestingly, it was elucidated previously that the antiviral effect of polygonum cuspidatum is associated with active compounds such as resveratrol and emodin, which inhibit the replication of influenza h1n1 virus directly by inducing ifn-β [28] . however, magnolia officinalis contains polyphenolic compounds that play a protective role in influenza virus-infected mice [29] . in addition, glycyrrhiza, cinnamon, areca catechu l, and spatholobus suberectus have a wide range of pharmacological activities in clinic, including influenza virus infections [30] [31] [32] [33] . ese data on the other hand suggested the accuracy and robustness of our antiviral screening results, and the hit fractions deserve further analysis for antiviral discovery. in summary, we prepared a library containing 500 prefractionated tcm extracts from 100 herbs with potent antiviral activity. as a pilot, a phenotypic screening was carried out against the library using a recombinant influenza a virus expressing gaussia luciferase, and 10 fractions possessing anti-influenza potencies were identified, including fractions of areca catechu l., glycyrrhizin, cinnamon, tripterygium wilfordii, spatholobus suberectus, polygonum cuspidatum, and magnolia officinalis. it is of great interest to investigate the bioactive components of these extracts and the mechanism of action in future. e data used to support the findings of this study are available from the corresponding author upon reasonable request. e authors declare that they have no conflicts of interest. qianli kang and yanyan wang contributed equally to this work. rr and lr conceived and designed the experiments. qk, lg, and hj collected the herbs and prepared the herbal extracts. yw and qc carried out the antiviral screening. rd and yy analyzed the data. qk, yw, and rd wrote the paper. rr and lr edited the paper. all authors contributed to the final version. mortality burden of the 1918-1920 influenza pandemic in hong kong world health organization influenza virus resistance to neuraminidase inhibitors incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern emergence of 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drugs, natural products, and molecules from combinatorial chemistry evaluation of the anti-neuraminidase activity of the traditional chinese medicines and determination of the anti-influenza a virus effects of the neuraminidase inhibitory tcms in vitro and in vivo experimental study on antivirus activity of traditional chinese medicine traditional chinese medicine and related active compounds: a review of their role on hepatitis b virus infection research and application of antiviral chinese herbal medicine, shandong science and technology press polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression protective effect of a polyphenolic rich extract from magnolia officinalis bark on influenza virus-induced pneumonia in mice uralsaponins antiviral triterpenoid saponins from the roots of glycyrrhiza uralensis in vitro antiviral activity of cinnamomum cassia and its nanoparticles against h7n3 influenza a virus immunomodulatory and antioxidant effects of total flavonoids of spatholobus suberectus dunn on pcv2 infected mice study on herbal textual evolution and flavonoids and their pharmacological of spatholobi caulis table s1 : the information of the selected 100 plants. figure s1 : comparison of hplc chromatogram of two n-butanol fractions extracted by the same method. figure s2 : the dose-response curves of ten active hits. (supplementary materials) key: cord-278807-p1crrb8n authors: antón, a.; marcos, m.a.; torner, n.; isanta, r.; camps, m.; martínez, a.; domínguez, a.; jané, m.; jiménez de anta, m.t.; pumarola, t. title: virological surveillance of influenza and other respiratory viruses during six consecutive seasons from 2006 to 2012 in catalonia, spain date: 2016-03-02 journal: clin microbiol infect doi: 10.1016/j.cmi.2016.02.007 sha: doc_id: 278807 cord_uid: p1crrb8n most attention is given to seasonal influenza and respiratory syncytial virus outbreaks, but the cumulative burden caused by other respiratory viruses (rv) is not widely considered. the aim of the present study is to describe the circulation of rv in the general population during six consecutive seasons from 2006 to 2012 in catalonia, spain. cell culture, immunofluorescence and pcr-based assays were used for the rv laboratory-confirmation and influenza subtyping. phylogenetic and molecular characterizations of viral haemagglutinin, partial neuraminidase and matrix 2 proteins were performed from a representative sampling of influenza viruses. a total of 6315 nasopharyngeal samples were collected, of which 64% were laboratory-confirmed, mainly as influenza a viruses and rhinoviruses. results show the significant burden of viral aetiological agents in acute respiratory infection, particularly in the youngest cases. the study of influenza strains reveals their continuous evolution through either progressive mutations or by segment reassortments. moreover, the predominant influenza b lineage was different from that included in the recommended vaccine in half of the studied seasons, supporting the formulation and use of a quadrivalent influenza vaccine. regarding neuraminidase inhibitors resistance, with the exception of the 2007/08 h275y seasonal a(h1n1) strains, no other circulating influenza strains carrying known resistance genetic markers were found. moreover, all circulating a(h1n1)pdm09 and a(h3n2) strains finally became genetically resistant to adamantanes. a wide knowledge of the seasonality patterns of the rv in the general population is well-appreciated, but it is a challenge due to the unpredictable circulation of rv, highlighting the value of local and global rv surveillance. respiratory viruses (rv) cause significant morbidity and mortality in the human population. most attention is given to the impact of seasonal outbreaks by human respiratory syncytial (hrsv) and influenza viruses, but the cumulative burden caused by more than 200 other known rv (picornaviruses, paramyxoviruses, coronaviruses and adenoviruses, among others) is not widely appreciated [1] . in the present study the circulation and seasonality of rv from 2006 to 2012 in catalonia (spain) are described. periods, demographic characteristics (gender and age) and nasopharyngeal samples were systematically collected for virological diagnosis from outpatients with influenza-like illness (ili) (two first ili consultations per week per physician), through the pidirac (daily information on acute respiratory illness plan of catalonia) sentinel surveillance network. ili is defined as acute respiratory tract infection presenting with sudden onset of symptoms; and at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath, according to the european centre for disease prevention and control's clinical criteria of ili [2] . the pidirac sentinel surveillance network is based on a medical sentinel network at primarycare centres coordinated by the public health agency of catalonia, that covers all seven health regions into which the catalan territory is divided. primary-care centres involved in the sampling varied from the 2006/07 season to the 2011/12, ranging from 27 in the former to 38 in the latter, and covered approximately 1% of the total population in catalonia. two independent nested multiplex rt-pcr were used to detect human influenza a (fluav), b (flubv) and c (flucv) viruses, hrsv, human adenoviruses (hadv), human parainfluenza viruses (hpiv) 1-4, human coronaviruses (hcov) 229e and oc43, human enteroviruses (hev) and human rhinoviruses (hrv) a, b and c [3, 4] . subtyping (seasonal h1, h1pdm09 and h3) of influenza a viruses isolated on mdck or mdck-siat1 (vircell, granada, spain) cell culture was performed by using the annual who influenza immunofluorescence assay, or directly from laboratory-confirmed clinical samples using a one-step multiplex real-time rt-pcr assay [5] . influenza laboratory-confirmed samples collected from patients belonging to different age groups (0-4, 5-14, 15-65, > 65 years old), from different geographical sites and in different weeks were selected for a good representativeness of the phylogenetic and molecular characterizations of circulating influenza viruses in catalonia throughout the period of study. the coding sequences of complete domain ha1 of viral haemagglutinin (ha) protein, and the partial neuraminidase (na) and matrix 2 (m2) proteins from fluav and flubv laboratory-confirmed specimens were sequenced, as well as, the coding region of the haemagglutinin-esterase (he) protein from flucv laboratory-confirmed specimens, as previously described [6] . updated amplification and sequencing protocols are available on request. phylogenetic analyses of sequences from the present study together with sequences from clade reference strains downloaded from the gisaid database (global initiative on sharing avian influenza data, available at: www.platform.gisaid.org) were carried out with mega v5.2 [7] . sequences were aligned using the muscle program, and the molecular evolutionary models of nucleotide substitutions were fitted to the multiple sequence alignments using evolutionary analyses conducted in mega v5.2 [7] . the phylogenetic trees were reconstructed using the neighbour-joining (nj) distance method as implemented in mega v5.2 [7] with the evolutionary model with the lowest bayesian information criterion score. reliability for the internal branch was assessed using the non-parametric bootstrap analysis with 1000 replicates. the amino acid substitutions of predicted influenza protein sequences were studied using mega v5.2 [7] relative to the homologous sequences of the corresponding recommended vaccine strains [8] . the potential n-linked glycosylation sites in ha1 amino acid sequences were tracked using the n-glycosite tool [9] . statistical analyses were performed using spss v17 (spss inc., chicago, il, usa). numeric variables were compared using the non-parametric mann-whitney u-test for comparisons between more than two groups. chi-squared test, fisher test, and the or and their 95% ci were calculated to assess associations between categorical variables. values of p <0.05 were considered to be statistically significant. the nucleotide sequences from the present study were submitted to the gisaid database. no ethical approval was required for this study. the seasonal hrsv outbreaks usually started early every season, before the seasonal influenza circulation. when fluav and flubv were co-detected during a season, fluav was first, with the predominance of a particular fluav subtype, and was followed by flubv later (fig. 1 ). the only exception was the 2009 influenza pandemic. a(h1n1)pdm09, which was first noted in june 2009 and which circulated showing a biphasic pattern. a first peak was detected during weeks 24-35 (summer months), and a second peak during weeks 41-49 (autumn months), before hrsv circulation and outside the usual months of influenza outbreaks (from december to march). during the first two 2009 pandemic peaks, other fluav subtypes (seasonal h1 and h3) and flubv remained almost undetected despite the large sampling done to strengthen the a(h1n1) pdm09 surveillance. during these six consecutive seasons other rv than influenza viruses were mainly detected during the cold months, often just before and after the seasonal influenza epidemics ( fig. 2) , with scarce circulation during the inter-seasonal periods. differences between the rv detection rates (see supplementary material, table s1 ) were observed just before and after the 2009 pandemic (p <0.05). the detection rates of hcov, hrv, hpiv 1-4 and hev increased after the pandemic table s2 ) [10] . phylogenetic analyses (ha1 and na) of 111/117 seasonal a(h3n2) strains (table 2; phylogenetic analyses (ha1 and na) of 121/123 a(h1n1) pdm09 strains (table 2; and supplementary material, fig. s3 ), showed that 116 strains were carrying the genetic features (s203t in ha1, and v106i and n248d in na) of strains belonging to the clade 7 described by nelson et al. [11] . strains collected during the 2009/10 season remained genetically close to those first described at the beginning of the pandemic. most of 2010/11 strains genetically evolved and fell within four different genetic subgroups based on ha sequences. in addition, at least ten strains without key genetic features of nelson's clade 7 were detected during the first two pandemic seasons, of which the latest strains (2010/11 season) showed genetic drift from the early 2009 isolates. phylogenetic analysis of ha1 sequences of 126 flubv strains revealed the co-circulation of b/victoria (54) and b/ yamagata-lineage (72) strains. an alternance in the predominant lineage, which was different from what was included in the recommended vaccine composition, was shown in three out of the six studied seasons ( table 2) . but this alternance did not seem to affect the flubv detection rates (see supplementary material, table s1 ). in fact, the highest flubv detection rate (19%) was reported during the 2010/11 season, when the predominant circulating lineage was well-matched with the lineage included in the vaccine ( table 2) . phylogenetic analyses (ha1 and na) of 51/54 b/victoria strains ( table 2; (2) in case of different phylogenetic variants, the most frequent is marked in bold letters. 1 the partial na sequences for phylogenetic and molecular characterization could not be obtained from some of these strains. 2 one intra-clade reassortant strain (iowa/19 ha; england/259 na). 3 one intra-clade reassortant strain (england/259 ha; stockholm/18 na). 4 intra-clade reassortant (brisbane/60 ha; malaysia/2506 na). for a(h3n2) subtype during the 2011/12 season (see supplementary material, fig. s2 ). phylogenetic analysis of 19 flucv strains (see supplementary material, fig. s6 ) detected during the 2009/10 and 2011/12 seasons, revealed that strains belonged to the c/ kanagawa/1/76-related and to c/sao paulo/378/82-related lineages [5] , remaining genetically similar. regarding na mutations related to neuraminidase inhibitors (nais) resistance, known genetic markers were not found in the influenza strains studied, with the only exception being the h275y mutation [10] in some 2007/08 seasonal a(h1n1) strains, as described above. some mutations within the enzyme active site or its surroundings were found in the characterized strains (see supplementary material, table s2 ), which might be associated with decreased or reduced susceptibility to nais [10, 12] , but are not yet characterized. in m2 sequences, the predominance of the genetic adamantanes-resistant a(h3n2) strains during the 2006/07 season (13/15, 87%) and later (100%) by acquiring the s31n mutation [12] were observed. all characterized a(h1n1) pdm09 strains were also carrying s31n mutation as described at the beginning of the pandemic. double mutations, s31n/ v27a and s31n/v27f, in one 2011/12 a(h3n2) strain and in one 2009/10 a(h1n1)pdm09 strain were also found, respectively. no mutations in m2 protein sequences related to antiviral resistance were found in seasonal a(h1n1) strains. our results show the significant burden of viral aetiological agents in acute respiratory infections, particularly in the youngest patient group, as well as the decline in rv detection rates as the age increases. in the adult population, viral respiratory infection might be underestimated because it is usually mild and self-limiting. gender did not seem to be related to an increased infection susceptibility, except in hrsv or hev. overall, the most frequently detected rv were fluav, hrv, hadv, flubv and hrsv, although hrsv and influenza viruses mostly circulated as seasonal outbreaks, and not continously throughout the year, such as hadv and hrv. differences in age distribution among rv were found. statistical differences between the age of patients infected by the several fluav subtypes were not found, although the means and iqr suggest that patients infected by seasonal a(h1n1) or a(h1n1)pdm09 cases were younger than those infected by a(h3n2). variations in the pattern of age-specific positive proportions between different subtypes were previously described [13, 14] . more a(h1n1)pdm09 susceptibility in younger patients was attributed to the little or no pre-existing immunity to the virus among children and young adults [15] . hrv or hcov were commonly detected in all age groups, which might be explained by their high genetic diversity or the incomplete cross-reactive immune response, leading to continuous re-infections throughout life. differences in the rv detection rates and in the ages of infected patients after the 2009 pandemic were observed as noted by other authors [16, 17] , although these have not been reported in other studies [18] . this might be a result of specific and non-specific cross-reactive immunity against other rv following the a(h1n1)pdm09 infection [17] . but a consequence of the larger sampling cannot be discarded. continuous evolution through either progressive amino acid substitutions (with changes on potential n-glycosylation sites) or by segment reassortments [19, 20] can affect (a) the antigenicity and tropism features by changes in the protective antigenic epitopes or in the receptor binding site of ha protein [21] [22] [23] [24] [25] , or (b) the susceptibility to the available antivirals through changes in the na and m2 proteins [10, 12] . driftedstrains with substantial antigenic changes, driven by the host immune response acting as an evolutionary selective pressure, lead to the annual vaccine composition update [8] . a(h3n2) strains, which circulated at varying levels throughout the study period, despite the wide community protection acquired by the natural infection and the seasonal vaccination since its appearance in 1968, belonged to several genetic subgroups, showing a great genetic heterogenity. a(h1n1)pdm09 has been genetically evolving into several phylogenetic groups since 2009, but remains antigenically similar [8] . however, close attention should be paid to future antigenic drift events in response to an increased natural or vaccine-induced immunity. regarding flubv and vaccine composition, the predominant lineage did not match the recommended vaccine lineage in half of the studied seasons. the inaccurate prediction of the predominant flubv lineage in trivalent influenza vaccines supported the formulation of a quadrivalent influenza vaccine [26] [27] [28] to enhance protection. however, flubv lineage alternance has not been reported so far, with a high predominance of b/yamagata lineage since the 2011/12 season [29] . some intra-clade reassortant strains were found. it is wellknown that viral segment reassortment is a powerful genetic mechanism for influenza evolution. as these genetic events are uncommon, these findings highlight the importance of studying at least the envelope ha and na sequences to monitor their emergence and spread, as well as, the value of local surveillance to detect these minor viral populations. throughout these six consecutive seasons the picture of the available antiviral drugs to fight against influenza infection changed considerably, and our results also showed the trends clinical microbiology and infection, volume 22 number 6, june 2016 reported worldwide [8, 30] . during the 2006/07 season both adamantane and nais were the two antiviral family drugs available. in catalonia, the first genetic oseltamivir-resistant seasonal a(h1n1) variants, which carried h275y mutation in na associated with antiviral resistance [10] , were detected in a percentage of 7% during the 2007/08 season. these strains also carried the compensatory mutations, that favoured the global spread of h275y variants despite the absence of drug selective pressure [10, 30] . according to who data, an average of approximately 24% of characterized strains in europe were shown to possess high-level oseltamivir-resistance, ranging from no detection in some countries to 68% in norway [30] . in addition, circulating a(h1n1)pdm09 and a(h3n2) strains are also resistant to adamantanes, since they carry the s31n mutation in m2 protein [12, 30] , remaining susceptible to nais. indeed, adamantanes cannot now be considered suitable for seasonal influenza treatment. in the present study, with the exception of h275y seasonal a(h1n1) strains that did not circulate since the 2009 pandemics, no other circulating influenza strains carrying genetic markers related to nais resistance were found [10] . changes within the enzyme active site or its surroundings were found in na sequences, but further phenotyping studies should be performed. there is public health concern that the antiviral resistance genetic markers could become fixed in the viral genome, as detected in a low percentage (<1%) among circulating viruses [31] . the rapid global spread of oseltamivir-resistant seasonal a(h1n1) influenza viruses without drug pressure should serve as a reminder for close local and global surveillance. a wide knowledge of the seasonal patterns of rv in the general population contributes to a better diagnosis and management of respiratory infections, but it is considered a challenge because of the unpredictable nature of rv circulation. indeed, continuous local and global surveillance of influenza and other rv must be carried out to monitor their prevalence, their genetic diversity and the emergence of antiviral resistance. the authors have no conflicts to declare. viral pneumonia influenza surveillance: influenza case definitions. european centre for disease prevention and control simultaneous detection of influenza a, b, and c viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-pcr assay simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays selection and viral load kinetics of an oseltamivirresistant pandemic influenza a (h1n1) virus in an immunocompromised patient during treatment with neuraminidase inhibitors influenza c virus surveillance during the first influenza a (h1n1) 2009 pandemic wave in catalonia mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods world health organization (who) tracking global patterns of n-linked glycosylation site variation in highly variable viral glycoproteins: hiv, siv, and hcv envelopes and influenza hemagglutinin neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods the early diversification of influenza a/h1n1pdm guidance for clinical and public health laboratories testing for influenza virus antiviral drug susceptibility in europe differences in patient age distribution between influenza a subtypes agespecific epidemic waves of influenza and respiratory syncytial virus in a subtropical city incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study impact of the 2009 influenza a(h1n1) pandemic wave on the pattern of hibernal respiratory virus epidemics relationships between a(h1n1)pdm09 influenza infection and infections with other respiratory viruses circulation of other respiratory viruses and viral co-infection during the 2009 pandemic influenza the evolution of human influenza viruses orthomyxoviridae: the viruses and their replication crystal structure of unliganded influenza b virus hemagglutinin cross-protective potential of a novel monoclonal antibody directed against antigenic site b of the hemagglutinin of influenza a viruses networks link antigenic and receptor-binding sites of influenza hemagglutinin: mechanistic insight into fitter strain propagation structural basis of preexisting immunity to the 2009 h1n1 pandemic influenza virus glycans on influenza hemagglutinin affect receptor binding and immune response the rationale for quadrivalent influenza vaccines the need for quadrivalent vaccine against seasonal influenza public health impact of including two lineages of influenza b in a quadrivalent seasonal influenza vaccine medical research council's national institute for medical research (mimr) oseltamivir-resistant influenza a(h1n1)pdm09 viruses the authors would like to thank the working group of influenza surveillance network in catalonia. this work was partially supported by fondo key: cord-275814-seirbkiq authors: tuncer, necibe; le, trang title: effect of air travel on the spread of an avian influenza pandemic to the united states date: 2014-03-31 journal: international journal of critical infrastructure protection doi: 10.1016/j.ijcip.2014.02.001 sha: doc_id: 275814 cord_uid: seirbkiq abstract the highly pathogenic avian influenza (hpai) strain h5n1, which first appeared in hong kong in 1997, achieved bird-to-human transmission, causing a severe disease with high mortality to humans [18]. according to the world health organization (who), a total of 637 cases were reported in fifteen countries, including 378 deaths, corresponding to a case fatality rate of nearly 60% [19]. avian influenza continues to be one of the deadliest diseases that jumps from animals to humans. epidemiologists believe that it is likely to cause the next major global pandemic that could kill millions of people. the 2002 outbreak of severe acute respiratory syndrome (sars) demonstrated that international air travel can significantly influence the global spread of an infectious disease. this paper studies the effects of air travel on the spread of avian influenza from asian and australian cities to the united states. a two-city mathematical model involving a pandemic strain is used to derive the basic reproduction number ( r 0 ), which determines if the disease will spread and persist ( r 0 > 1 ) or go extinct ( r 0 < 1 ). real air travel data is used to model the disease spread by individuals who are susceptible to or are infected with pandemic avian influenza. analysis of the two-city model helps understand the dynamics of the spread of pandemic influenza when the cities are connected by air travel. understanding these effects can help public health officials and policy-makers select the appropriate disease control measures. also, it can provide guidance to decision-makers on where to implement control measures while conserving precious resources. the avian influenza virus belongs to a group of viruses known as influenza a that circulate in many avian and mammalian species, including humans [2] . the highly pathogenic avian influenza (hpai) strain h5n1 achieved bird-tohuman transmission and caused a severe disease to humans with high mortality [18] . hpai first appeared in 1997 in hong kong, where it infected eighteen humans and caused six deaths. to date, a total of 637 cases in fifteen countries have been reported to the world health organization (who), including 378 deaths with a case fatality rate approaching 60% [19] . interestingly, there has been no decline in the mortality rate since the first appearance of hpai. in march 2013, china reported several confirmed cases involving the h7n9 strain that had not been seen previously in humans [20] . most patients showed flu-like symptoms, and a throat swab taken from a patient in jiangsu province revealed the presence of the h3n2 human influenza virus and the h7n9 avian influenza virus [19] . avian influenza continues to be one of the deadliest diseases that links humans and animals. indeed, it is expected to cause the next major influenza pandemic. an influenza pandemic occurs when a new strain of virus circulates among humans, potentially killing millions of people. aside from its high mortality rate, avian influenza is monitored closely for a pandemic because influenza a type viruses can evolve rapidly and jump between species. the viruses evolve through evolutionary mechanisms such as drift and shift. drift involves small, gradual changes to surface proteins while shift involves abrupt major changes to the influenza virus. shift evolution occurs through reassortment, the mixing of two influenza viruses (e.g., avian influenza and human influenza) into a new strain that carries properties from both strains. shift typically occurs when a patient gets infected with both strains of the influenza virus. recent outbreaks in china show that shift evolution can occur at any time with potentially deadly effects. vaccination is one of the best ways to prevent the spread of pandemic influenza and reduce its severity. however, producing live attenuated and inactive vaccine seed viruses against a potential pandemic-causing avian influenza strain, and subsequently performing pre-clinical tests and clinical trials can take several months, if not longer. unfortunately, it is not possible to predict which avian virus strains will cause the next influenza pandemic or when the pandemic will occur. of great concern is the fact that once pandemic avian influenza surfaces, it will spread globally in no time at all because the world is connected via air travel. the 2002 outbreak of severe acute respiratory syndrome (sars) demonstrated that international air travel accelerated the global spread of an infectious disease [14] . this paper attempts to study the effects of air travel on the spread of avian influenza. understanding the effects can help public health officials and policy-makers to select the appropriate disease control measures. also, it can provide guidance to decision-makers about where to implement control measures while conserving precious resources. this paper presents a mathematical model with spatial heterogeneity that is designed to better understand the emergence and the spread of pandemic avian influenza. spatial effects regarding disease spread can be modeled by diffusion or dispersal models. in a diffusion model, the movements of infected individuals are instantaneous and the disease spreads immediately to neighboring areas. on the other hand, in a dispersal model, the movements of individuals are discrete and usually occur between so-called patches connected by dispersion. dispersal models are more appropriate when considering travel over long distances. thus, a two-city dispersal model is developed to predict the spread of avian influenza from asia and australia to major u. s. cities via air travel. real air travel data is used to model the disease spread by individuals who are susceptible to or are infected with pandemic avian influenza. the two-city model helps identify the most effective intervention strategies at the origin and destination cities as well as help quantify the effectiveness of control measures that should applied to reduce the morbidity and mortality of a pandemic. this can provide valuable guidance to public health officials and policy-makers as they decide how, where and when to target pandemic influenza intervention strategies while efficiently using the available resources. this section describes the mathematical model, and the data and model parameters. a two-city model is used to express the spread of avian influenza from its origin in an asian or australian city to major cities in the united states. a single-city model is first specified, which is subsequently generalized to a two-city model by considering the travel of susceptible and infected individuals with the new strain of avian influenza. avian influenza, one of the most dangerous diseases that links humans and animals, is currently the foremost pandemic threat. early mathematical models that investigate the impact of a hypothetical pandemic have focused only on humans [1, 4, 5, 10, 13] . at this time, the avian influenza virus can only be transmitted from birds to humans; it is not yet capable of sustained human-to-human transmission. most of the human cases reported to the who were due to close contact between humans and domestic birds. recent mathematical models capture the bird-to-human transmission pathway of avian influenza [6] [7] [8] [9] . the simplest bird-tohuman model was introduced by iwami et al. [7] ; this model is the starting point for the single-city model presented in this paper. the notation used in our model is different, but the two models are equivalent. in our single-city model, the domestic bird population is divided into two non-intersecting classes: susceptible domestic birds denoted by s d (t) and infected domestic birds denoted by i d (t). the natural birth rate of the bird population is expressed by λ d . susceptible birds have a natural death rate μ d while infected birds die at the rate μ d þ ν d , where the additional death rate ν d is induced by hpai. susceptible humans are denoted by s(t) and infected humans by i(t). the natural birth rate of the human population is denoted by λ. susceptible humans die at the rate μ while infected humans die at the rate μ þ ν, where ν is the additional death rate due to avian influenza. the single city model, which comprises the bird and human populations, is given by the force of the infection in the domestic bird population is denoted by β d i d s d , where β d is the transmission rate at which a susceptible bird contracts avian influenza from an i n t e r n a t i o n a l j o u r n a l o f c r i t i c a l i n f r a s t r u c t u r e p r o t e c t i o n 7 ( 2 0 1 4 ) 2 7 -4 7 infected bird. the incidence rate in the human population is denoted by βi d s, where β is the rate at which a susceptible human contracts avian influenza upon contact with an infected bird. to investigate the impact of a hypothetical pandemic, we assume that avian influenza evolves to become a human-tohuman transmissible pathogen. this evolution likely occurs as a result of one of two evolutionary mechanisms: drift and shift. we assume that the mutation of avian influenza is caused by reassortment (shift mechanism) and denote the mutation rate of avian influenza into a pandemic influenza by m. the new avian influenza strain that causes a pandemic is called "pandemic avian influenza." the human population that is infected with the new strain is denoted by p(t). infection from one strain of influenza a virus provides cross-immunity to infections from other strains. this is why we assume that humans infected with pandemic avian influenza can infect susceptible humans, but not humans who are infected with avian influenza. the new incidence of pandemic avian influenza is denoted by β p ps, where β p is the transmission rate. the additional death rate due to pandemic avian influenza is denoted by ν p . table 1 lists the state variables in the model while table 2 lists the model parameters. the two-city model assumes that the pandemic originates in a city in asia or australia and spreads to a city in the united states by air travel. we only consider the air travel of susceptible and infected humans with pandemic avian influenza because the infected individuals can only contract the disease from domestic birds (that do not travel). if the travel of humans is not considered, then the pandemic is modeled within each city by a single-city model. in the following, we use subscripts to distinguish cities. the travel rate of individuals from city 1 to city 2 is denoted as k 1 and the corresponding travel rate from city 2 to city 1 is denoted by k 2 . the two-city model is given by we assume that individuals infected with pandemic avian influenza have minor flu-like symptoms and, therefore, are "well enough" to continue with their travel plans. thus, the travel rate for infected individuals is the same as that for susceptible individuals. fig. 1 shows the movements of individuals between city 1 and city 2 through the various stages of the disease. in the model, the transmission of pandemic avian influenza occurs only between individuals in a city. specifically, infected individuals in one city do not directly infect individuals in another city. this assumption is motivated by the fact that individuals in a city are much more likely to be in contact with city residents than touriststhe number of contacts between city residents is extremely large compared with the number of contacts between residents and tourists on a given day. we also assume that the transmission rates of avian influenza within the bird and human populations, and the transmission rates of pandemic avian influenza are the same for all cities. international airline flight data provided by the office of the secretary for aviation and international affairs was obtained from the united states department of transportation website [16] . tables 3 and 4 present flight data obtained from the report entitled "u.s. international air passenger and freight statistics" for the third quarter of 2011. table 3 lists the numbers of passengers who took direct flights from asian or australian cities to u.s. cities. table 4 shows the numbers of passengers who traveled from major airports in asia and australia and the corresponding u.s. shares expressed as percentages. the travel rates between city 1 (k 1 ) and city 2 (k 2 ) are estimated by dividing the total number of passengers traveling from city 1 to city 2 by the population of city 1. since time is measured in days, the result is further divided by 365 4 to obtain the travel rate of individuals of a city per day. the populations of cities considered in this work are presented in table 5 . for example, the travel rate from seoul to los angeles is estimated as similarly, the travel rate from los angeles to seoul is estimated as the lifespan of humans varies from country to country, and even from city to city. table 5 lists the life expectancies of the countries considered in this research. the natural death rate of humans is estimated as according to the who, the duration of the infectious period for hpai-infected human cases is six to seven days. therefore, the disease-induced death rate of hpai is taken to be ν ¼ 1 6:5 ¼ 0:15 days à 1 . we adjust the disease-induced death rate as a result of pandemic avian influenza using ν p ¼ 0:06, which is taken from [7] . thus, we assume that the death rate caused by the new strain of avian influenza is smaller than the death rate of hpai. the who gives the infectious period of domestic birds to be ten days, so ν d ¼ 0:1 days à 1 . the natural death rate and the disease-induced death rate are assumed to be the same for all cities. the total human population nðtþ ¼ sðtþ þ iðtþ þ pðtþ satisfies the differential equation: upon solving eq. (3), we see that the total human population is a bounded function of time: where n 0 is the initial human population. the limit of the human population is applying similar analysis to the total bird population n d ðtþ ¼ s d ðtþ þ i d ðtþ, we obtain in all the simulation results presented in this paper, the bird population is given in units of 10 7 domestic birds and the human population is given in units of 10 5 individuals. eqs. (4) and (5) are used to estimate the recruitment rates for the bird and human populations, respectively. the food and agriculture organization [3] gives the total poultry population as 20.4 billion, which corresponds to 2040 in units of 10 7 . commercial poultry is usually kept for two years, so the natural death rate of birds is μ d ¼ 1 2â365 days à 1 . therefore, we estimate the recruitment rate for the bird population as the recruitment rate of humans in each city is computed similarly using the population and life expectancy data given in table 5 . the cumulative number of hpai-infected human cases reported by the who exhibits seasonality [15] . this is likely due to factors such as temperature, rainfall, migration patterns of wild birds, survival of the hpai virus in the environment and seasonality in bird-to-bird transmission. several such mechanisms are considered in [15] to understand the reasons for the seasonality in avian influenza data; the authors report that the main culprit is bird-to-bird transmission. we use their fitted parameter values for the bird-to-bird and bird-to-human transmission rates, which are β d ¼ 5:29 â 10 à 5 and β ¼ 1:9 â 10 à 11 , respectively [15] . in the case of pandemic avian influenza, we set the humanto-human transmission rate to β p ¼ 3 â 10 à 7 . this is a purely hypothetical value because there is no good way to estimate it. the parameter is fixed by assuming that the transmission of the new pathogen is higher than the transmission of the current pathogen. currently, avian influenza is in the zoonotic stage, meaning that the only transmission pathway is from birds to humans. the new pathogen evolves into a strain that is capable of human-to-human transmission. we use the mutation rate m¼0.01 as given in [7] . all the simulations reported in section 3 use the parameter values listed in this section. we only change β d or β p to vary the simulations when necessary and the changes are mentioned whenever they are applied. epidemiologists are interested in the long-term dynamics of the spread of an infectious disease. by studying the solutions of the mathematical model, we hope to answer the question of whether the disease will die out or persist in the bird and human populations. in particular, we want to measure the severity of the hypothetical pandemic caused by avian influenza. to predict the severity of an outbreak or pandemic, a summary parameter called the reproduction number is calculated from the population model. the reproduction number i n t e r n a t i o n a l j o u r n a l o f c r i t i c a l i n f r a s t r u c t u r e p r o t e c t i o n 7 ( 2 0 1 4 ) 2 7 -4 7 r 0 is an epidemiologically important threshold value that determines whether a disease will persist or die out. the basic reproduction number gives the secondary number of infections produced by one infected individual in a totally susceptible domain during her/his infectious period. if r 0 o1, then the disease-free equilibrium is locally asymptotically stable, meaning that the disease will eventually become extinct. if r 0 41, then the disease-free equilibrium is unstable and the disease will persist in the population. determining an expression for the basic reproduction number and estimating its value are important when selecting the most effective control measures for eliminating a disease. reproduction numbers are derived by performing a qualitative analysis of a mathematical model. the procedure is described in detail in the following sections. the first step in the qualitative analysis of the model is to find constant solutions that do not change in timeequilibrium points. equilibrium solutions for the single-city model have been studied in [7] and will not be repeated here. however, we will summarize the model dynamics. the disease free equilibrium (dfe) for the single-city model is ðs n d ; 0; s n ; 0; 0þ, where s n d ¼ λ d =μ d and s n ¼ λ=μ. the basic reproduction numbers for the bird population r 0 b and the human population r 0 h , respectively, are that is, if r 0 o1 and r h o o1, then the dfe vanishes in both populations; otherwise, the disease becomes endemic. this paper focuses on the dfe of the two-city model. the equilibrium points for the two-city model satisfy the following steady-state equations, which are derived by setting the time derivatives equal to zero: we denote the dfe equilibrium by ε n ¼ ðs n d1 ; 0; s n 1 ; 0; 0; s n d2 ; 0; s n 2 ; 0; 0þ, where this is the state in which hpai and pandemic avian influenza are both wiped out in the bird and human populations. this infection-free state is stable if the reproduction numbers for the bird and human populations in both cities are less than one. the basic reproduction numbers for the bird populations in city 1 and city 2 are given by for a system with multiple infected compartments, as in the two-city model defined by eq. (2), the reproduction number is defined as the number of infections produced by one infected individual at the dfe [17] . following the next generation approach described in [17] , we classify the compartments as infected and uninfected. in our case, the infected compartments consist of birds infected with hpai, humans infected with hpai and humans infected with pandemic avian influenza. we define two matrices: (i) f, which expresses the rate of new incidences; and (ii) v, which represents the transfer of individuals between compartments: the principal eigenvalue of the next generation matrix fv à 1 is the basic reproduction number. the next generation matrix for the two-city model in eq. (2) is in which the reproduction numbers for the human populations in city 1 and city 2 are given bŷ we take the basic reproduction number for the human population to be the maximum of the reproduction numbers for the two cities: fig. 8 san francisco 3:61 â 10 à 5 tokyo 6:55 â 10 à 4 fig. 9 united states 0.0155 tokyo 6:15 â 10 à 4 fig. 10 united states 0.0156 seoul 5:18 â 10 à 4 fig. 11 united states 0.0153 hong kong 5:18 â 10 à 4 fig. 12 united states 0.0156 sydney 2:21 â 10 à 4 fig. 13 united states 0.0156 beijing 9:99 â 10 à 4 similarly, the reproduction number for the bird population is r 0 ¼ maxfr 1 0 ; r 2 0 g: hpai and pandemic avian influenza both become extinct in the bird and human populations when all the reproduction numbers are less than one. the preceding mathematical discussion leads to the following important result. theorem 1. if r 0 o1; r 0 o1, then the dfe ε n is locally asymptotically stable. if r 0 41 or r 0 41, then the dfe ε n is unstable. sensitivity analysis of the model predictions to small changes in parameter values can help determine which parameters are the best targets for controlling the disease and slowing an epidemic. let λ be a quantity that depends on two parameters of different scales, i.e., λ ¼ λðp; qþ. then, the sensitivity of λ with respect to the parameters p and q is given by the sensitivity s λ p gives the amount of change that occurs in λ in response to small changes in p. however, because p and q are parameters with different scales, the effect of changes in p and q on λ cannot be compared easily. therefore, we use an alternative measure that allows for the comparison of different scales. the normalized sensitivity, which is also called elasticity, of the quantity λ with respect to parameter p is given by the elasticity e λ p describes how the quantity λ is affected by small changes in p. a negative elasticity means that a onepercent increase in the parameter p results in an e λ p % decrease in λ. similarly, a positive elasticity means that a one-percent increase in the parameter p results in an e λ p % increase in λ. in the following section, we derive analytic expressions for the elasticities of the basic reproduction number and the prevalence of pandemic avian influenza. the elasticity values are analyzed in the context of the simulation results. this section presents the numerical simulation results for the two-city model. the two-city model was solved numerically using the ode15s built-in function provided in matlab 2011b. the parameters were set to the values described in section 2. in the simulations, a small initial infection was introduced in the origin city of the pandemic, and it was assumed that no infected cases exist initially in any of the u.s. cities. a total of fourteen simulations were conducted for the spread of the pandemic spanning a period of ten years. the reproduction number for the bird population was computed to be r 0 ¼ 1:06. the computed reproduction numbers for humans in the various cities are shown in table 6 . the fact that the bird population reproduction number is greater than one (r 0 ¼ 1:06) suggests that the dfe is unstable, and this is in agreement with the corresponding simulation results (figs. 2-15 listed in the last column of table 6 ). in each of these figures, subfigure(a) shows the number of infected individuals with pandemic avian influenza in the origin city while subfigure(b) shows the number of infected individuals with pandemic avian influenza in the destination u.s. city. in the simulations, we see that for the air travel data taken from table 3 , the numbers of infected people in u.s. cities are less than those in the origin cities. on the other hand, when we consider the data from table 4 , it is interesting to see that the situation reverses. this is not surprising because the travel rates differ considerably in the two types of data. tokyo has the busiest air traffic to the united states -5.2 million passengers in just one quarter (table 4 ). this contributes to a high travel rate because the population of tokyo is 13.2 million (k 1 ¼ 0:0043 and k 2 ¼ 0:00018). the next section investigates the effects of air travel on pandemic prevalence in more detail. 3.2. several researchers have studied the state of h5n1 infections in bird populations [11, 12, 15] . these studies suggest that h5n1 infections are at the endemic state in bird populations. as such, the reproduction numbers for the bird populations should be reduced to control the disease. if the reproduction number r 0 is kept below 1, then an h5n1 infection in a bird population is eliminated. as a consequence, "spill over" infections to human populations are also eliminated [12] . martcheva [12] conducted a sensitivity analysis of the reproduction number r 0 and reports the following results: these results suggest that the highest impact on r 0 is obtained by increasing the natural death rate for the bird population μ d . thus, one of the most effective control measures that can be applied to poultry is culling. culling increases the parameter μ d and produces the largest change to r 0 . in the pandemic scenario, the reproduction number r 0 is also an important parameter whose value should be kept below one. however, control measures applied to a bird population may be insufficient to reduce the prevalence of a pandemic. we now develop an analytic expression for the number of infected people with pandemic influenza at the equilibrium state. we call a state in which hpai dies out in the population but pandemic avian influenza persists as the "pandemic-only equilibrium" and denote it by ðŝ d1 ; 0;ŝ 1 ; 0;p 1 ;ŝ d2 ; 0;ŝ 2 ; 0;p 2 þ. the pandemic-only equilibrium satisfies the steady-state equations (eq. (6)). thus, the parametersp 1 andp 2 satisfy the following equations: heren i ¼ŝ i þp i , i¼1, 2 denotes the total population of city i at the pandemic-only equilibrium. to study the effect of the travel parameters k 1 and k 2 on the prevalence of the pandemic avian influenza, we derive the elasticities of pandemic prevalence with respect to the parameter set p ¼ ðβ p ; μ 1 ; ν p 1 ; μ 2 ; ν p 2 þ. upon implicitly differentiating the functions f ðp 1 ;p 2 þ and gðp 1 ;p 2 þ, we obtain the following equations: where the preceding discussion leads to the formulation of the following proposition. proof. at the pandemic-only equilibrium, f ðp 1 ;p 2 þ ¼ a 1p1 þ β pp 2 1 þ k 2p2 ¼ 0,p 1 is strictly positive and β pp 2 1 þ k 2p2 40, so a 1 o0. similar analysis yields a 2 o0. from eqs. (9) and (10), we obtain the following expression: which states that both ða 1 a 2 àk 1 k 2 þ and ∂p 1 =∂k 1 are non-zero as long as a 2 b 1 ðp i þàk 2 b 2 ðp i þ is non-zero for each parameter p i . from eqs. (12) through (15) , it follows that a 2 b 1 ðp i þà k 2 b 2 ðp i þ a 0. moreover, since a 1 a 2 à k 1 k 2 is continuous in all its parameters, it must have a definite signeither all positive or all negative. setting k 1 ¼ k 2 ¼ 0, we see that a 1 a 2 40 and, thus, upon solving eqs. (9) and (10), we obtain the following analytic expressions for the elasticities of pandemic prevalence: from these analytic expressions, it is clear that the only positive elasticity is for the transmission rate of pandemic avian influenza. the elasticities of pandemic prevalence with respect to parameters μ 1 , μ 2 , ν p 1 , ν p 2 are all negative. these results suggest that control measures that decrease the transmission rate and infectious period of pandemic avian influenza should be applied. examples of control measures that might be applied to stop or limit the spread of pandemic influenza are isolating infected individuals, quarantining susceptible individuals who are exposed to the virus and vaccinating individuals against the pandemic strain. these measures impact different parameters in eq. (2) . for example, isolation and quarantine affect the transmission rate β p while vaccination reduces the susceptibility of healthy individuals (thus reducing β p ) and might also decrease the infectious period (thus increasing ν p ). evaluating the efficacy of control measures is a non-trivial task for an actual infection, let alone for a hypothetical pandemic avian influenza outbreak. nevertheless, we compute the elasticities of the pandemic prevalence numerically in order to elucidate the efficacy of control measures. first, we compute the number of infected individuals with pandemic avian influenza at the pandemic-only equilibrium ðp 1 ;p 2 þ with k 1 and k 2 varying from 0 to 1. in the simulations, city 1 is the origin of pandemic influenza and city 2 is the destination city. then, the elasticities of pandemic prevalence are plotted as functions of k 1 and k 2 . to further visualize the effects of the air travel, for a fixed k 1 value, the elasticities are plotted for varying k 2 values, and vice versa. the simulation results show that, although the analytic expressions for ep 1 β p and ep 2 β p are symmetric, the effects of air travel on the elasticities differ for the origin city and destination city (see figs. 16(a) and 17(a) ). the simulation results in fig. 16 demonstrate that isolating and quarantining individuals from the origin city is appropriate when the travel rate out of the city is very small (i.e., when k 1 is small). when k 1 is large (e.g., k 1 40:2 in fig. 16(a) ), applying control measures aimed at decreasing the transmission rate of the pandemic influenza will not decrease the prevalence of infected individuals, potentially leading to wasted resources. on the other hand, in the destination city, much is gained from isolation and quarantine measures when k 2 is very small. during a pandemic, government agencies in the destination city should consider implementing disease control measures along with restricting travel (see fig. 17 ). next, we consider the elasticities of pandemic prevalence with respect to the disease-induced death rates for each city. since the elasticities are symmetric, we only show the results for origin cities. the simulation results in fig. 18 suggest that vaccination of individuals in the origin city should be considered when the outbound travel rate is very small and the inbound travel rate is very high. vaccinations in the destination city decrease the pandemic prevalence in the origin city only when the travel rate into the origin city is very high (see fig. 19 ). effects of air travel on r 0 we computed the basic reproduction numbers for each origin city and destination city,r 1 0 andr 2 0 , using the parameters described in section 2.2. in the following, we take city 1 to be tokyo and city 2 to be a city in the united states, where the transmission rate of pandemic avian influenza in both cities is β p ¼ 3 â 10 à 5 . when there is no travel between the two cities, we obtain the same reproduction numbers for each city as when the reproduction numbers are computed for each city in isolation: when k 2 is fixed and k 1 increases,r 1 0 decreases. the same is true for the reproduction number for city 2r 2 0 (see fig. 21 ). according to theorem 1, if the reproduction numbers for the cities are greater than one, then the disease will always persist. for example, the reproduction numbers for two cities arer 1 0 ¼ 1:40 andr 2 0 ¼ 8:87 when hpai is in the endemic state in the bird population (current state). this scenario is illustrated in fig. 22(a) , where the higher reproduction number leads to higher prevalence of the disease. in a situation where the reproduction number is greater than one in one city and less than one in another city, then the disease still persists in both cities (see fig. 22(b) ). next, we investigate the effects of air travel on the elasticities of the reproduction numbers for various cities. if the cities are isolated and there is no travel that links them, then the elasticities of the reproduction numbers r 1 0 ; r 2 0 with respect to the parameters β p , λ 1 , λ 2 , μ 1 , μ 2 , ν p 1 and ν p 2 are obtained from the analytic expressions given in eq. (16) : when the two cities are connected via air travel, then the elasticities of the reproduction numbers r 1 0 ; r 2 0 with respect to the parameters β p , λ 1 , λ 2 , μ 1 , μ 2 , ν p 1 and ν p 2 are obtained from their analytic expressions. the elasticities of the reproduction number for city 1 are the elasticities of the reproduction number for city 2 are as can be seen in the above expressions, the elasticities of the reproduction numbers with respect to the parameter β p are not affected by the travel rates. however, the elasticities with respect to parameters λ 1 and λ 2 are affected drastically. consider, for example, fig. 23(a) and (b) , which illustrate the effects of air travel on er is no air travel, the reproduction number is very sensitive to changes in λ 1 (er 1 0 λ1 ¼ 1). when the two cities are connected via air travel, then the elasticity er 1 0 λ1 drops below 0.04. on the other hand, when the two cities are isolated, the sensitivity of the city 1 reproduction number to λ 2 is nonexistent, but when the cities are connected, the sensitivity increases above 0.9. the same reasoning holds for city 2. the elasticity of er 1 0 νp 1 is à0:99 when there is no air travel. this is a very high sensitivity in the opposite direction, meaning that control measures that decrease the infectious period (e.g., vaccination) result in a large reduction of the basic reproduction number for city 1. when the two cities are connected, but the travel rate from city 1 is assumed to be zero (i.e., k 1 ¼ 0 and k 2 40), then the elasticity er increases as k 2 increases and decreases as k 1 increases. as fig. 24(a) suggests, control measures focused on reducing the infectious period are a waste of time and money if the travel rate out of city 1 is high and the travel rate into city 1 is low. in such a case, the best strategy is to vaccinate individuals in city 2 (see fig. 24(b) ). the two-city model presented in this paper was developed to express and reason about the spread of pandemic influenza from cities in asia and australia to the u.s. via air travel. data obtained from the u.s. department of transportation and who were used to estimate fixed model parameters such as the travel rate, birth rate and natural death rate. model parameters such as transmission rates and hypothetical pandemic death rates were obtained or estimated from fitted values in the literature. the reproduction numbers computed for the bird and human populations (r 0 and r 0 ) are important threshold values. if either r 0 41 or r 0 41, then the disease-free equilibrium is unstable, meaning the disease will continue to persist within the populations. in all the cases considered, although the computed r 0 values are small (10 à 2 or less), the r 0 value is 1.06, which suggests that the dfe is unstable; this agrees with the numerical simulation results. the sensitivity analysis involving elasticities helps understand the effects of various parameters on r 0 and pandemic prevalence. the results show that culling, which increases the natural death rate of birds and significantly affects the r 0 value, is the most effective control measure that can be applied to poultry. additionally, control measures such as isolation, quarantine and vaccination should be applied to the human population to reduce pandemic prevalence. isolation and quarantine decrease the transmission rate, while vaccination affects the transmission rate as well as the infectious period. the effectiveness of the control measures, however, depends strongly on the air travel rate. for instance, if the travel rate out of a city is large (i.e., greater than 0.2), then isolating and quarantining infected and susceptible individuals are not very effective at reducing pandemic prevalence. an expected result is that air travel has a large impact on r 0 . when cities are not connected, the elasticities of the reproduction numbers with respect to recruitment rates are constant (i.e., equal to one). however, when the cities are connected, the elasticities are strongly impacted by air travel. specifically, when the travel rates are positive, the reproduction number for the origin city is very sensitive to changes in destination city recruitment rate, and vice versa. generally, the effects of air travel on the elasticities are different for the origin city and destination city. however, upon examining the effect of the induced pandemic death rate, we discovered that when the travel rate out of an origin city is small, vaccination and other similar control measures in the origin city are ineffective, but vaccination in the destination city is much more effective. thus, combining disease control measures and restricting travel to the appropriate cities is a promising strategy for eradicating pandemic avian influenza. we hope that this paper will stimulate renewed research on understanding the dynamics of the spread of pandemic disease. such research can help public health officials and policy-makers select the appropriate disease control measures, and also provide valuable guidance on where to implement control measures while conserving precious resources. this research was supported by nsf grant dms-1220342. strategies for containing an emerging influenza pandemic in southeast asia fields virology, lippincott-raven food and agriculture organization mitigation strategies for pandemic influenza in the united states assessing the impact of airline travel on the geographic spread of pandemic influenza prevention of avian influenza epidemic: what policy should we choose avian-human influenza epidemic model avian flu pandemic: can we prevent it avian-human influenza epidemic model with diffusion containing pandemic influenza at the source an avian influenza model and its fit to human avian influenza cases avian flu: modeling and implications for control pandemic influenza: risk of multiple introductions and the need to prepare for them the effect of global travel on the spread of sars modeling seasonality in avian influenza h5n1 reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission world health organization, influenza at the human-animal interface world health organization, influenza at the human-animal interface, summary and assessment as of world health organization, overview of the emergence of and characteristics of the avian influenza a (h7n9) virus key: cord-270910-xb746mv5 authors: lebrun-harris, lydie a.; mendel van alstyne, judith a.; sripipatana, alek title: influenza vaccination among u.s. pediatric patients receiving care from federally funded health centers date: 2020-07-24 journal: vaccine doi: 10.1016/j.vaccine.2020.07.021 sha: doc_id: 270910 cord_uid: xb746mv5 introduction: during the 2018–2019 influenza season, vaccination coverage among u.s. children was 62.6%. the purpose of this study was to estimate the prevalence of influenza vaccinations among pediatric patients seen in u.s. health centers, and to explore potential disparities in vaccination coverage among subpopulations. funded by the health resources and services administration (hrsa) within the u.s. department of health and human services, these health centers provide primary and preventive care to underserved and vulnerable individuals and families in order to reduce health disparities based on economic, geographic, or cultural barriers. methods: cross-sectional data, analyzed in 2019, came from the most recent waves of the health center patient survey (2009, 2014). the sample consisted of children ages 2–17 years receiving care from hrsa-funded health centers. the outcome of interest was selfor parent-reported receipt of influenza vaccine in the past year. multivariable logistic regression was used to estimate the adjusted prevalence rate ratios for the association between demographic characteristics (age, sex, race/ethnicity, poverty level, urban/rural residence, geographic region), health-related variables (receipt of well-child check-up, asthma diagnosis), and influenza vaccination. results: influenza vaccination coverage among pediatric health center patients increased from 46.6% in 2009 to 67.8% in 2014. in the adjusted model for 2014, there were few statistically significant differences in vaccination coverage among subpopulation groups, however american indian/alaska native children had 31% increased vaccination coverage compared with non-hispanic white children (aprr: 1.31, 95% ci: 1.02–1.60) and children living in the south had 26% decreased vaccination coverage compared with those living in the northeast (aprr: 0.74, 95% ci: 0.54–0.93). conclusions: influenza vaccination coverage among pediatric health center patients in 2014 exceeded the national average (as of 2018–2019), and few differences were found among at-risk subpopulations. hrsa-funded health centers are well-positioned to further increase the vaccination rate among children living in underserved communities. vaccinations, extolled as one of the greatest public health achievements of the 20th century, continue to be a critical tool for mitigating death and disease in the united states (u.s.) and globally. for a single-year birth cohort, it is estimated that routine childhood vaccination prevents nearly 20 million cases of disease in the u.s., including over 40,000 deaths [1] . moreover, avoided morbidity and mortality due to vaccination is estimated to save $13.5 billion in direct costs and $68.8 billion in total societal costs over the cohort's lifetime [2] . despite the remarkable outcomes provided by recommended vaccines they remain underutilized among children, due to barriers to accessing vaccination services, vaccination exemptions, and parental vaccine hesitancy [3] [4] [5] . in the u.s. each fall, the centers for disease control and prevention and the american academy of pediatrics recommend https://doi.org/10.1016/j.vaccine.2020.07.021 0264-410x/published by elsevier ltd. seasonal influenza vaccination for everyone six months of age and older, excluding those with medical contraindications [6, 7] . and each year, public health departments and primary care providers work to implement this recommendation. historically, underserved populations, such as those living below the federal poverty line, and those who are uninsured or underinsured, have lower seasonal influenza vaccination rates [8] [9] [10] , leaving those individuals and their communities more susceptible to influenza and related complications. young children are at particularly high risk for severe influenza complications [11, 12] . estimates from the 2018-2019 influenza season based on the national immunization survey-flu indicate that the childhood influenza vaccination rate was nearly 63%-an increase of almost 5 percentage points from the 2017-2018 influenza season [13] . while this marks a positive shift, the coverage rate remains below the national benchmark of 70% [14] . health care providers caring for the underserved are uniquely positioned to help prevent seasonal influenza infections through vaccination and to increase community immunity in the areas they serve. health centers funded by the health resources and services administration (hrsa) within the u.s. department of health and human services, under section 330 of the u.s. public health service act [15] provide high quality, accessible, and affordable primary and preventative health care to underserved and vulnerable populations across the u.s. in order to reduce health disparities based on economic, geographic, or cultural barriers. in 2018, hrsa-funded health centers served over 28 million patients, including over 8.7 million children ages 0 through 17 years, which represents one in nine u.s. children [16, 17] . health centers operate with a sliding scale fee structure for patients, based on their income level. with over 91% of patients living at or below 200% of the federal poverty guideline [16] , the health center program provides a model of low cost, high quality health care that can contribute to improving utilization of preventative health services including seasonal influenza vaccination [18] . given the program's reach among children within underserved communities, health centers are cornerstones in promoting public health in general and influenza community immunity in particular [19] . to date, there have been no studies examining influenza vaccine uptake among patients seen in hrsa-funded health centers. only one previous study, published in 2003, has examined pediatric vaccinations in the context of health centers, however it focused on the childhood immunization schedule rather than seasonal influenza vaccinations. that study found few disparities in childhood immunizations, and the authors hypothesized that enabling services provided by health centers facilitated access to timely vaccinations [20] . in 2010, the advisory committee for immunization practices (acip) recommended universal seasonal influenza vaccinations among those six months of age and older [21, 22] . thus, this study sought to examine the rates of pediatric influenza vaccinations among health center patients before and after the acip recommendation. the current study contributes to the literature by examining, for the first time, the prevalence of influenza vaccine administration among children served by hrsa-funded health centers. the strengths of the study include the use of hrsa datasets which are nationally representative of u.s. individuals who receive primary and preventive care from health centers, as well as the analysis of several sociodemographic characteristics to explore potential disparities in influenza vaccine uptake across pediatric subpopulations in underserved communities. based on previous studies which have documented minimal disparities in health care access and utilization among health centers [15, [23] [24] [25] , we hypothesized that we would find few disparities in pediatric influenza vaccinations in these settings. we conducted secondary analysis of data from the health center patient survey, a nationally representative survey of people who receive care from u.s. health centers funded by hrsa. the survey is conducted periodically (every 4-6 years) by hrsa's bureau of primary health care and is designed to capture information on patient sociodemographic characteristics, health conditions, health behaviors, access to and utilization of health care services, and satisfaction with care. survey questions are based on other established national health surveys, including the national health interview survey, national ambulatory medical care survey, medical expenditure panel survey, and national health and nutrition examination survey. sample selection is based on a stratified three-stage random sampling design. first, health center organizations are sampled, stratified by funding stream, size, u.s. census region, urban/rural location, and number of care delivery sites per health center organization. then, up to three care delivery sites per health center organization are sampled. finally, individual patients from each site are sampled. patients are eligible for the survey if they had at least one medical visit to the health center site in the past year. surveys are conducted through computer-assisted personal interviews by trained field interviewers. for children ages 12 and under, parents or other knowledgeable caregivers respond to the survey; children ages 13 and over respond to the survey themselves, after parental/caregiver assent is obtained. the 2014 health center patient survey was most recently fielded survey, when a total of 7,002 patients were surveyed between october 2014 and april 2015. among patients who were screened and determined to be eligible, 91% completed an interview. the previous fielding was the 2009 health center patient survey, which included a total of 4,562 patients surveyed between september and december 2009; in this fielding, 98% of patients who were screened and deemed eligible completed an interview. institutional review board approval for the original data collections in both 2009 and 2014 was obtained from research triangle international, the organization that administered the survey on behalf of hrsa. for the current study, conducted in 2019, the analytic sample of pediatric health center patients included 1,093 children between the ages of 2 and 17 years from the 2014 survey, and 464 children from the 2009 survey. we examined receipt of annual influenza vaccine, as assessed by parent-or self-reported responses to two questions. the first question asked about influenza vaccination injection: ''during the past 12 months, {have you/has name} had a flu shot? a flu shot is usually given in the fall and protects against influenza for the flu season. the flu shot is injected in the arm. do not include an influenza vaccine sprayed in the nose." the second question asked about influenza vaccination nasal spray: ''during the past 12 months, {have you/has name} had a flu vaccine sprayed in {your/his/her} [26] nose by a doctor or other health professional? this vaccine is usually given in the fall and protects against influenza for the flu season." for this study, we combined responses to both questions to create a dichotomous outcome measure to capture the receipt of an annual influenza vaccine (yes vs. no). sociodemographic covariates of interest included patient age, sex, race/ethnicity, family poverty level, urban/rural residence, and u.s. census region. we also included two health-related variables, specifically, receipt of a well-child check-up in the past 12 months and lifetime diagnosis of asthma (an indicator of a highrisk population). we examined the rates of influenza vaccination among pediatric health center patients in 2009 versus 2014. we also stratified vaccination rates by age group (2-5 years, 6-11 years, 12-17 years) to determine whether rates declined with age, as is found in national trends [27] . we limited subsequent analyses to the 2014 sample of pediatric patients, the most recent year of health center data available, to assess potential disparities in receipt of influenza vaccines among this population after the acip issued its 2010 recommendation for universal vaccination. we first examined the distribution of sociodemographic and health characteristics for patients, and conducted bivariate analyses and chi-square tests of independence with design-based f statistics to assess the associations between receipt of influenza vaccine and each of the characteristics of interest. we set statistical significance at p < 0.05, and calculated 95% confidence intervals for each estimate. finally, we conducted multiple logistic regression to examine the independent associations between each covariate of interest and receipt of an annual influenza vaccine. results are presented as adjusted prevalence rate ratios (prrs), which represent the likelihood that children with specific characteristics of interest received an annual influenza vaccine, relative to other children in meaningful reference groups while holding all other correlates constant. about 28% of the sample had missing income data, therefore we created a separate category for ''missing income" in order to retain these observations. we conducted all analyses using stata/se, version 15.1, and employed weights based on the survey's sampling design to produce estimates that adjusted for the complex sampling design and were representative of the underlying population. the overall rate of influenza vaccination among pediatric patients seen in hrsa-funded health center increased from 47% in 2009 to 68% in 2014, a 45% increase over 5 years (fig. 1) . increases in vaccination coverage were seen across all three age groups. the largest relative increase occurred among youth 12 to 17 years, from 41% in 2009 to 64% in 2014, a 54% increase (23 percentage points). in both 2009 and 2014, there was an inverse doseresponse relationship between vaccination and patient age, with the youngest age group (2-5 years) having the highest rates of vaccination and the oldest age group (12-17 years) having the lowest rates. in 2014, 29% of hrsa-funded health center pediatric patients in the analytic sample were between the ages of 2 and 5 years, and another 33% were between 12 and 17 years, with the remainder between 6 and 11 years (table 1 ). there were slightly more male patients (55%) than female patients. the most common racial/ethnic group among pediatric patients was hispanic (53%), followed by non-hispanic white (22%) and non-hispanic black (18%). about 60% of the sample lived below the federal poverty level. over 40% of pediatric patients lived in rural areas, and slightly more lived in the west (36%) and south (29%) compared with the northeast (20%) and the midwest (15%). almost 80% of children had received a well-child check-up in the past year, and about 29% had a lifetime diagnosis of asthma. in unadjusted analyses, only two characteristics were associated with receipt of annual influenza vaccination among health center pediatric patients in 2014 (table 2) . specifically, a larger proportion of children living in urban settings received a influenza vaccine than those living in rural settings (74% vs. 59%, p = 0.0137). in addition, children living in the northeast and the west had higher rates of influenza vaccination than those living in the south and the midwest (range: 53%-81%, p = 0.0066). there were no statistically significant associations between influenza vaccination and child age, sex, race/ethnicity, federal poverty level, well-child check-up, and asthma diagnosis. after adjusting for all covariates simultaneously in the multiple logistic regression, the association between urban/rural residence and influenza vaccines was no longer statistically significant (table 3) . however, children living in the south had a 26% decreased prevalence of influenza vaccine receipt, compared with those living in the northeast (aprr = 0.74, 95% ci: 0.54-0.93). in addition, american indian/alaska native children had a 31% increased prevalence of influenza vaccine receipt relative to non-hispanic white children (aprr = 1.31, 95% ci: 1.02-1.60). among u.s. children ages 2-17 years receiving care from hrsafunded health centers, there was a 21 percentage point increase in annual influenza vaccinations between 2009 (prior to the 2009 acip recommendation) and 2014, from 47% to 68% of patients. the 2014 influenza vaccination rate among this traditionally underserved population exceeded the 2013-2014 rate seen among u.s. children nationally (59%) by almost 10 percentage points. the age-related pattern seen in health center pediatric patients, showing lower vaccination rates with increasing age, was similar to the pattern in the general u.s. population [28] . several subpopulations of health center patients exceeded the national benchmark of 70% uptake, including young children (ages 2-5 years), american indian/alaska native and hispanic children, and those living under the federal poverty level, in urban locations, and in the northeast and west. one possible explanation for the increased influenza vaccinations among these subgroups is that health centers may deliver childhood vaccines through the federal vaccines for children program, which provides free or low-cost vaccines for uninsured, underinsured, and medicaid-insured children, as well as american indian/alaska native children [29] . another potential explanation is that hrsa implemented new activities during this timeframe to promote continuous quality improvement among its grantees, including publicly disseminating clinical performance data and implementing a pay-for-performance program [30] . additional studies are needed to assess potential differential impacts of these quality improvement efforts on specific subpopulations. although these results demonstrate a vast improvement in influenza vaccinations among this population within a 5-year span, there is a continuing need to further increase vaccination rates. plans-rubió's (2012) study of community immunity against influenza viruses suggests that 80% vaccination coverage is required in healthy persons (and 90% in high-risk persons) to establish a sufficient network of protection in the u.s [31] . results of this study indicate that hrsa-funded health centers are effectively providing equitable access to seasonal influenza vaccination to a wide spectrum of medically underserved pediatric patients. health centers have a long history of reducing health care disparities by providing access to primary and preventive care to medically underserved and vulnerable populations [15, [23] [24] [25] . the 2014 health center data on influenza vaccinations corroborated this pattern, revealing few disparities with the exception of statistically significant lower rates among pediatric patients in the south (53%, compared with 81% in the northeast) and significantly higher rates among american indian/alaska native patients (83%, compared with 59% among non-hispanic white patients). previous studies have identified several institutional factors which may contribute to health centers' ability to provide equitable access to services across a broad range of clinical domains, such as cancer screening, chronic condition management, prenatal care, and well-visit check-ups. these institutional factors include the provision of supportive services (e.g., care coordination, health education, translation, transportation), early and widespread implementation of electronic health records, and recognition as patient-centered medical homes [26, 32, 33] . strengthening access to and quality of primary care broadly through these efforts may translate to increased use of specific services including influenza vaccinations, and fewer missed opportunities for influenza vaccinations. indeed, yue and colleagues (2019) found that adult health center patients who used support services had a 16 percentage point higher likelihood of receiving an influenza vaccine compared with those who did not use support services [32] . given the demonstrated and potential efficacy of these factors in improving health care provided by health centers, hrsa has invested hundreds of millions of dollars to accelerate and optimize the adoption of health information technology, as well as incentivize the adoption of the patient-centered medical home model of care through its quality improvement awards [30] . while previous studies have found positive effects of these innovations on access to care, quality of care, and disparities [26, 32, 33] , additional analyses may examine their potential impact specifically on pediatric influenza vaccine uptake. our finding of lower rates of pediatric influenza vaccination among health center patients in the south was consistent with patterns identified using the national immunization survey and the national health interview survey [28, 34, 35] , suggesting opportunities to geographically target interventions and resources to improve the uptake of influenza vaccines in this region. results from this study can be leveraged to inform efforts by hrsafunded primary care associations and health center controlled networks that work with health centers in the south [36, 37] . additional research is needed to better understand the underlying factors that lead to lower influenza vaccinations among children seen in health centers in the south. racial/ethnic disparities in childhood influenza vaccination are well established in the literature, although the findings are mixed. studies of young children (under age 3) have found that white children have higher vaccination coverage than black or hispanic children [38, 39] , while national estimates of all children up to age 17 show that white children have similar vaccination rates to black children and lower rates than hispanic children [27, 40] . in contrast, our study found no statistically significant differences between these three racial/ethnic groups among the population of pediatric patients served by hrsa-funded health centers. the lack of disparities found within these settings may be a reflection of the health center program's mission to provide culturally competent, patient-centered, comprehensive primary care regardless of individuals' ability to pay, as well as supportive services that promote access to health care [41] . in particular, hrsa requires that health centers incorporate cultural competency activities to address the unique needs of the populations they serve. this includes arranging interpretation and translation services for patients with limited english-speaking ability, providing resources and training to staff on delivering culturally sensitive services and bridging cultural differences, and regularly conducting needs assessments to improve service delivery with a particular focus on patient population characteristics that impact health status or health care access/utilization (e.g., social factors, physical environment, cultural/ethnic factors, language needs, housing status). in addition, health centers are directed by governing boards that must be composed of a patient majority; at least 51% of board members must be patients at the health center and must reflect the population served in terms of demographic factors such as race/ethnicity and gender. we also found that the 2014 influenza vaccination rate among american indian/alaska native pediatric health center patients was notably higher than the national average for american indian/alaska native children in 2013-2014 (83% vs. 66%) [27, 40] . researchers using the national health interview survey have also reported higher influenza vaccination coverage among american indian/alaska native children compared with other racial/ethnic groups [35] , which may reflect clinical recommendations to target vaccination efforts for this subpopulation [42] . our results showed that about 43% of pediatric health center patients lived in rural areas, twice the proportion of children living in rural settings in the u.s. in general [43, 44] . although rural pediatric patients were less likely to receive influenza vaccinations than urban patients in the unadjusted analysis, there were no differences based on urban/rural status in the adjusted analysis, indicating that health centers are successfully serving areas with lower access to care overall. there are study limitations worth mentioning. first, while the results are generalizable to the population of underserved pediatric patients seen in hrsa-funded health centers, they are not representative of the general u.s. child population. although the findings apply only to a specific subgroup, they represent 8.7 million u.s. children ages 17 and under, a not insignificant number. second, because the health center patient survey includes patients of all ages, the sample of pediatric patients is relatively limited, and the small sample sizes for certain subgroups may have limited our ability to detect statistically significant differences. third, although influenza vaccines are recommended for infants starting at 6 months, it was not possible to examine vaccination among health center patients younger than 2 years because of how age was coded in the dataset; therefore we were unable to obtain estimates of influenza vaccination for this age group. in addition, two influenza vaccine doses are recommended for children 6 months through 8 years in their first vaccination season but the health center patient survey does not ask whether children received 1 or 2 doses or whether it was their first time receiving a vaccine, therefore it was not possible to distinguish between the proportion of children with full versus partial influenza vaccination coverage. another limitation is potential recall bias. specifically, parents may not accurately remember details of their child's influenza vaccinations, and children 13 years and over who respond for themselves on the survey may be less reliable in reporting their health care utilization in general and in particular may be less aware that they received an influenza vaccine. under these circumstances, our study may underestimate the prevalence of annual influenza vaccination. additionally, the survey includes limited parental or household information, such as parents' education level or health literacy, so it was not possible to assess the association between several potentially relevant family characteristics and pediatric patients' influenza vaccine uptake. in particular, children ages 13 and over were not asked about their health insurance coverage, so we were unable to examine influenza vaccination rates based on insurance status. however, previous research has cited a positive correlation between state-level medicaid reimbursement and influenza vaccination rates among poor children across three separate influenza seasons [45] , which may be particularly relevant for improving vaccination rates among health center patients, given that nearly half are medicaid or chip beneficiaries [16] . similarly, the study did not examine the role of knowledge, attitudes, and practices toward vaccines among parents of pediatric health center patients, which might influence the decision to obtain influenza vaccinations. recent literature illustrates that parental concerns about seasonal influenza vaccine exist, including beliefs that the vaccine causes influenza illness, the vaccine is unnecessary because influenza illness is mild, and the vaccine is not effective [46] [47] [48] [49] . beyond concerns specific to influenza vaccine, more generalized concerns may influence decisions to vaccinate. concerns over safety and vaccine ingredients, number of vaccines administered, mistrust of the mainstream medical system, and the belief that vaccination should be a personal choice persist, and are often shaped by a parent's social sphere of influence [50] [51] [52] [53] . finally, the health center patient survey was last conducted in 2014-2015, so the data are now several years old. however, examining the 2014 data is still informative in terms of comparing to national averages and providing a baseline for comparing pediatric influenza vaccine rates in the future. the next wave of the survey is planned to be conducted in summer/fall of 2020, which will provide valuable information to ascertain whether hrsa-funded health centers have continued to increase influenza vaccine uptake among their pediatric patients, and to examine any current disparities impacting subpopulations of interest. furthermore, future research should use these anticipated data to examine how the covid-19 pandemic may impact patients' perceptions of vaccines and influence vaccine uptake. additional research is needed to further explore patient, family, provider, and organizational factors that may influence influenza vaccination among children receiving care at hrsa-funded health centers. notwithstanding the limitations mentioned above, this study provides the first nationally representative estimates of influenza vaccination rates among pediatric patients receiving care from hrsa-funded health centers, both overall and for subpopulations based on demographic and health-related factors. results reveal opportunities to increase pediatric influenza vaccination in health centers so that all subpopulations can attain and surpass the national benchmark of 70% coverage. possible strategies to improve influenza vaccine coverage include partnering with state and regional primary care associations, which are tasked with providing programmatic, clinical, and financial training and technical assistance to safety-net providers, to support health centers in developing immunization strategies tailored to their localized communities [36] . additionally, health information technology can be used to better facilitate important vaccine conversations, including patient and provider reminders and prompts in electronic health record systems and patient portals [54] [55] [56] . other potential vehicles for improvement include parent education, proactive appointment scheduling, and strong provider recommendations [48, 57] . findings suggest that hrsa-funded health centers can serve as critical providers in engaging and serving diverse constituencies, reducing disparities in influenza vaccination, and increasing immunity among the nation's most underserved communities. the authors have no financial disclosures. the views expressed in this article are those of the authors and do not necessarily reflect the official policies of the us department of health and human services or the health resources and services administration, nor does mention of the department or agency names imply endorsement by the us government. simply put: vaccination saves lives economic evaluation of the routine childhood immunization program in the united states vaccination coverage among children aged 19-35 months -united states vaccination coverage for selected vaccines and exemption rates among children in kindergarten -united states, 2017-18 school year department of health and human services office of disease prevention and health promotion. healthy people 2020 midcourse review prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices-united states surveillance of vaccination coverage among adult populations -united states health insurance is associated with preventive care but not personal health behaviors influenza vaccination among us children with asthma influenza vaccination coverage of vaccine for children (vfc)-entitled versus privately insured children centers for disease control and prevention national center for immunization 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health center patients organizational factors associated with disparities in cervical and colorectal cancer screening rates in community health centers centers for disease control and prevention national center for immunization and respiratory diseases. early-season flu vaccination coverage-united states influenza vaccination coverage among us children from health resources and services administration bureau of primary health care. primary care associations health resources and services administration bureau of primary health care racial disparities in vaccination for seasonal influenza in early childhood complete influenza vaccination trends for children six to twenty-three months health resources and services administration bureau of primary health care. what is a health center new census data show differences between urban and rural populations number of children key facts about the number of children how to improve influenza vaccination rates in the u.s. j prevent med public health = yebang uihakhoe chi flu-floppers": factors influencing families' fickle flu vaccination patterns vaccine hesitancy and influenza beliefs among parents of children requiring a second dose of influenza vaccine in a season: an american academy of pediatrics (aap) pediatric research in office settings (pros) study partnering with parents to remove barriers and improve influenza immunization rates for young children an assessment of parental knowledge, attitudes, and beliefs regarding influenza vaccination what is 'confidence' and what could affect it?: a qualitative study of mothers who are hesitant about vaccines mott children's hospital. national poll on children's health. mott poll report: do parents have selective hearing about flu vaccine for children vaccine safety: myths and misinformation factors influencing african-american mothers' concerns about immunization safety: a summary of focus group findings association between patient reminders and influenza vaccination status among children using reminder/recall systems to improve influenza immunization rates in children with asthma the impacts of email reminder/recall on adolescent influenza vaccination association between provider recommendation and influenza vaccination status among children the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2020.07.021. key: cord-299364-t549rf3o authors: noh, ji yun; choi, won suk; lee, jacob; kim, hye lim; song, joon young; cheong, hee jin; kim, woo joo title: clinical performance of the sofia™ influenza a+b fia in adult patients with influenza-like illness date: 2015-10-31 journal: diagnostic microbiology and infectious disease doi: 10.1016/j.diagmicrobio.2015.05.016 sha: doc_id: 299364 cord_uid: t549rf3o abstract the sofia™ influenza a+b fia demonstrated 74.0% sensitivity and 95.4% specificity for influenza a in patients with influenza-like illness in 2012–2013 season. it yielded higher sensitivity than sd bioline influenza ag a/b/a(h1n1/2009) (54.1%) for influenza a (p <0.01). the sofia™ influenza a+b fia might be useful for rapid diagnosis of influenza. a rapid diagnosis of influenza allows for early decision making and timely intervention in patients with influenza-like illness (ili). it is also advantageous in the selection of an appropriate antiviral treatment, the reduction of antibiotics use, and the avoidance of unnecessary diagnostic examination, which, in turn, reduces medical costs (bonner et al., 2003; nitsch-osuch et al., 2013) . rapid influenza diagnostic tests (ridts) have been widely used in clinical practice to diagnose influenza because they are easy to use and provide results within 10-15 minutes (cdc, 2013; cho et al., 2013) . however, the results of conventional ridts are limited in reliability due to various and unsatisfactory performances (cdc, 2013; cho et al., 2013) . the sensitivity of an ridt varies depending on the type of test and has been shown to be affected by multiple factors: study population, elapsed time from symptom onset, viral titer, sample type and status, circulating influenza virus, and epidemic size. the clinical performances of ridts have either been evaluated in a clinical field or by using frozen samples, with reported sensitivities ranging from 40.3% to 73.3% compared to pcr-based detection (choi et al., 2011; leonardi et al., 2013; stripeli et al., 2010; sutter et al., 2012) . the sofia™ influenza a + b fia (quidel corporation, ca, usa) is a novel fluorescent immunoassay used to detect influenza a and b within 15 minutes using the sofia analyzer (lewandrowski et al., 2013) . in this study, the clinical performance of the sofia™ influenza a + b fia was prospectively evaluated and compared with the performance of the sd bioline influenza ag a/b/a(h1n1/2009) (standard diagnostics, yongin, south korea) in cases of ili in adult patients. from december 23, 2012, to april 11, 2013, a prospective study was conducted in 3 teaching hospitals in south korea. two nasopharyngeal swabs were obtained from adult patients (≥18 years) with ili who visited an emergency department or outpatient clinic. ili was defined as an acute respiratory infection with measured fever of ≥38°c and a cough that occurred within 7 days. among 2 nasopharyngeal swabs, 1 flocked swab was randomly placed in 900 μl of viral transport medium (vtm) (bd, nj, usa) and was used to perform promptly ridts at patients' bedsides. after agitating the flocked swab thoroughly in a vial, samples were tested using both sofia™ influenza a + b fia and sd bioline influenza ag a/b/a(h1n1/2009) simultaneously. all procedures were conducted according to the manufacturers' protocols (quidel, n.d.; standard diagnostics, n.d.) . the other flocked swab was immediately placed in 3 ml of vtm (bd), and samples were kept at −70°c until use. total rna was extracted automatically by nimbus (hamilton robotics, reno, nv, usa) as per the manufacturer's protocol. real-time reverse transcription polymerase chain reaction (rt-pcr) was performed using anyplex™ ii rv16 detection (seegene, seoul, south korea) to detect influenza a and b viruses. among the samples tested, those positive for influenza a virus were selected and tested to differentiate subtype using seeplex® influenza a/b onestep typing (seegene) and the powerchek™ influenza sih1/h3/h5 real-time rt pcr kit (kogenebiotech, seoul, south korea). sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of each ridt were calculated using the results of rt-pcr as a gold standard. a mcnemar test was performed to compare (fig. 1) . the sensitivities of both ridts for influenza a virus were higher for patients who had visited the hospital within 2 days of symptom onset than for patients who visited hospital more than 2 days after symptom onset ( in a previous study, the sensitivity of the sofia™ influenza a + b fia was reported to be 78.1% during the 2011-2012 influenza season, when implemented at the bedsides of infants and children (rath et al., 2012) . using stored samples, it displayed a sensitivity of 82.2% for influenza a, compared to real-time rt-pcr; however, the mean age of the study population was younger (21.3 years old) than ours (lee et al., 2012) . in another prospective study, the sensitivity of sofia™ influenza a + b fia for the influenza a virus, compared to real-time rt-pcr, was 85% and 69% using nasal or nasopharyngeal swabs, respectively (lewandrowski et al., 2013) . although more than 90% of those patients were younger than 22 years old, this study showed a lower sensitivity for detection of influenza a using nasopharyngeal swab than our results (lewandrowski et al., 2013) . however, direct comparison of the performance of ridts between these studies should be limited in interpretation because the clinical performance of ridts can be affected by multiple factors, some of which are uncontrollable. in our study, the sensitivities of both ridts for influenza a virus were higher in patients who visited the hospital within 2 days of symptom onset, compared to those who visited hospital 2 days after the symptom onset. this might be due to high viral titer during the early infection phase, and this finding is consistent with a previous study (choi et al., 2011) . however, in a previous report, the sensitivity of ridt was low during the very early period (within 3 hours of symptom onset) and the time interval from onset to consultation was shorter in ridt false-negative group (5.5 hours) than in true-positive group (11.5 hours) (harada et al., 2012) . time from symptom onset to hospital visit was recorded in the unit of day in our study; thus, analysis based on hours was not available. this study has some limitations. first, we used limited commercial rt-pcr kits as a gold standard. the sensitivities to detect influenza viruses vary with each commercial kit. second, samples were used promptly for sofia™ influenza a + b fia after specimen collection. however, samples for pcr were eluted in 3 ml of vtm and were frozen at −70°c and used after thawing. there is a chance that influenza virus rna was diluted in vtm. freezing and thawing can reduce viral titer in clinical samples. these limitations can produce false negatives in pcr, causing false positives of the sofia™ influenza a + b fia in this study. also, there is a chance that uneven distribution of influenza virus between 2 nasopharyngeal swabs could affect the results. only adult patients were included in this study and relatively lower viral titer in adult than children could affect low sensitivity of ridts. however, viral titer was not determined. during the study period, influenza b virus rarely circulated; thus, we did not evaluate the performance of ridts for influenza b. in addition, direct comparison of sensitivity against influenza a(h1n1)pdm09 between sofia™ influenza a + b fia and sd bioline influenza ag a/b/a(h1n1/2009) is limited in interpretation. because the sofia™ influenza a + b fia distinguishes influenza a and b, whereas the sd bioline influenza ag a/b/a(h1n1/2009) has 3 lines to differentiate influenza a, b, and influenza a(h1n1)pdm09. despite limitations, this study is valuable as a prospective study on the performance of the sofia™ influenza a + b fia in the clinical field during the 2012-2013 influenza season in northern hemisphere. due to its ability to provide relatively high sensitivity in the detection of the influenza a virus, it will be one of viable tools for the rapid diagnosis of influenza in clinical practice. impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial evaluation of 11 commercially available rapid influenza diagnostic tests-united states evaluation of five rapid diagnostic kits for influenza a/b virus the clinical usefulness of the sd bioline influenza antigen test(r) for detecting the reliability of a rapid test for the clinical diagnosis of influenza a/h1n1 2009 evaluation of sofia fluorescent immunoassay analyzer for influenza a/b virus comparison of conventional lateral-flow assays and a new fluorescent immunoassay to detect influenza viruses detection of influenza a and b viruses with the sofia analyzer: a novel, rapid immunofluorescence-based in vitro diagnostic device influence of rapid influenza test on clinical management of children younger than five with febrile respiratory tract infections early detection of influenza a and b infection in infants and children using conventional and fluorescencebased rapid testing standard diagnostics. sd bioline influenza ag a/b/a(h1n1) pandemic. available at performance of rapid influenza testing in hospitalized children performance of five fda-approved rapid antigen tests in the detection of 2009 h1n1 influenza a virus test sensitivities were compared between the sofia™ influenza a + b fia and the sd bioline influenza ag a/b/a(h1n1/2009) (a) influenza a virus (b) a/h3n2 influenza virus this work was supported by a grant of the tepik (transgovernmental enterprise for pandemic influenza in korea), which is part of the korea healthcare technology r&d project by ministry of health & welfare, republic of korea (grant number a103001) and dow biomedica (seoul, south korea). the funders had no role in study design, data collection or analysis, decision to publish, or preparation of the manuscript. this study was supported in part by dow biomedica (seoul, korea). key: cord-258496-h264umt1 authors: jaakkola, kari; saukkoriipi, annika; jokelainen, jari; juvonen, raija; kauppila, jaana; vainio, olli; ziegler, thedi; rönkkö, esa; jaakkola, jouni jk; ikäheimo, tiina m title: decline in temperature and humidity increases the occurrence of influenza in cold climate date: 2014-03-28 journal: environ health doi: 10.1186/1476-069x-13-22 sha: doc_id: 258496 cord_uid: h264umt1 background: both temperature and humidity may independently or jointly contribute to the risk of influenza infections. we examined the relations between the level and decrease of temperature, humidity and the risk of influenza a and b virus infections in a subarctic climate. methods: we conducted a case-crossover study among military conscripts (n = 892) seeking medical attention due to respiratory symptoms during their military training period and identified 66 influenza a and b cases by pcr or serology. meteorological data such as measures of average and decline in ambient temperature and absolute humidity (ah) during the three preceding days of the onset (hazard period) and two reference periods, prior and after the onset were obtained. results: the average temperature preceding the influenza onset was −6.8 ± 5.6°c and ah 3.1 ± 1.3 g/m(3). a decrease in both temperature and ah during the hazard period increased the occurrence of influenza so that a 1°c decrease in temperature and 0.5 g decrease per m(3) in ah increased the estimated risk by 11% [or 1.11 (1.03 to 1.20)] and 58% [or 1.58 (1.28 to 1.96)], respectively. the occurrence of influenza infections was positively associated with both the average temperature [or 1.10 per 1°c (95% confidence interval 1.02 to 1.19)] and ah [or 1.25 per g/m(3) (1.05 to 1.49)] during the hazard period prior to onset. conclusion: our results demonstrate that a decrease rather than low temperature and humidity per se during the preceding three days increase the risk of influenza episodes in a cold climate. respiratory tract infections (rti) are the most common infections worldwide, and hence pose a considerable economic burden to healthcare services. there is substantial evidence on the seasonal variation of respiratory morbidity and mortality, which results in increased use of health services and hospital admissions during the winter months [1] . influenza epidemics constitute a serious public health problem associated with increased morbidity and mortality, especially in high risk populations. worldwide, these annual epidemics result in about three to five million cases of severe illness, and about 250 000 to 500 000 deaths [2] . seasonal variation of influenza a and b outbreaks is a well-known phenomenon and it explains a substantial proportion of excess winter morbidity and mortality [3] . meteorological parameters, in particular temperature and humidity, may contribute to the observed seasonal variation in the occurrence of influenza episodes. studies from temperate and tropical climates have demonstrated that low temperature [4] and humidity increase the risk of seasonal influenza onset in the winter [5] [6] [7] [8] . low temperature and dry air was recently reported to increase influenza and pneumonia mortality [9] . furthermore, experimental studies have shown that both low temperature and humidity favor the spread of influenza viruses [10] [11] [12] [13] . although surrounded by controversy, available scientific evidence suggests that either inhalation of cold air or cold stress cause pathophysiological responses that may contribute to an increased susceptibility to viral infections [14] . low humidity and influenza, on the other hand, may be connected through changes in the virus stability and transmission [13] . the association between temperature, humidity and the occurrence of influenza has remained unclear. our previous study conducted in a northern climate demonstrated that the occurrence of respiratory tract infection is associated with both low temperature and humidity [15] . however, to our knowledge; no corresponding studies examining the association between influenza virus infections, temperature and humidity have been conducted in cold climates with subfreezing temperatures. the objective of the present study was to examine the relations between temperature, humidity and the risk of influenza virus infections in a subarctic climatic zone in northern finland. for this purpose, we conducted a case-crossover study among young army conscripts during their military training period. our hypothesis was that decreased daily temperature and humidity during outdoor training and associated with physical exercise would increase the risk of influenza a and b virus infection. the study population included 892 military recruits (mean age 19.6 ± 0.8 years) from the two intake groups enrolling in military service in july 2004 and in january 2005 in the kajaani garrison in northern finland (64°n, 27°e). the follow-up period was from july 2004 to the end of december in 2005. the total number of men in the intake group was 1,836 in july 2004 (participation 82%) and 1,861 in january (75%). both intake groups served part of their military training during the winter season. the study protocol was approved by the medical ethics committee of the kainuu central hospital. the conscripts were described of the study, the performed assessments, possible inconvenience, the confidentiality and storing of data both in written and oral form. in addition, they were informed that participation is voluntary and that they can discontinue their participation at any time. in addition, it was clearly indicated that all potential participants who declined to take part of the study were eligible for treatment and were not disadvantaged in any other way by not participating. the conscripts provided their written consent following this information. this study focussed on influenza a and b cases, because they can be identified on the basis of severe symptoms which lead to a contact with medical service. influenza a and b also have similar seasonal variation indicating identical environmental determinants of the onset. influenza c was excluded because of milder and more variable symptoms compared with influenza a and b as well as different type of seasonal pattern which indicates different environmental causes. conscripts who sought medical attention for acute respiratory infections at the military primary health care clinic of the kainuu brigade were assessed initially by a nurse and then examined by a physician for diagnosis and treatment, if considered necessary. they were also asked to fill in a questionnaire assessing outdoor training and symptoms during the three preceding days. at that time sputum (n = 379) samples were collected. in addition, acute and convalescent serum samples were obtained (n = 520 pairs). the diagnosis of influenza was based on a positive pcr test or a significant increase in influenza a or b antibodies in paired serum samples. nucleic acids were extracted from 100 μl of sputum sample. viral rna was reverse transcribed into cdna and influenza a and b viruses were detected by amplifying cdna in real-time multiplex pcrs [16] . serum samples were tested for igg antibodies to influenza a, and b viruses by enzyme immunoassay using extracts of madin darby canine kidney cells (mdck) infected with either influenza a/beijing/353/89, or influenza b/panama/45/ 90 viruses, respectively. uninfected mdck cells were served as control antigen. a fourfold or greater increase in antibody titers between acute-and convalescent-phase sera was considered significant. exposure assessment was based on meteorological data obtained from the kajaani airport meteorological station; the nearest station of the finnish meteorological institute located approximately 15 km from the garrison. average daily temperature and absolute humidity (ah) were calculated based on eight separate measurements conducted at three-hour intervals per day. in addition, an average of the three preceding days (e.g. a 72 h period) from the visit to the clinic (day 0) were calculated and used in the regression models (see below). we also separately examined the maximal changes, as well as the starting level from where temperature and ah changed. a maximum decline in temperature and ah was calculated as the largest change (maximum versus minimum) occurring in these parameters within the three day period (e.g. day −3 versus day −2, day −3 versus day −1, day −2 versus day −1 etc.). the meteorological conditions of the day the conscripts visited the clinic for influenza were not included to the analyses. the present study utilized a case-crossover design which is most suitable for studying relations with the following characteristics: 1) the individual exposure varies within short time intervals; 2) the disease has abrupt onset and short latency for detection; and 3) the induction period is short [17] . for ambient temperature and ah the hazard period was defined as three days preceding the visit to the clinic for a respiratory infection (and when virological samples were collected) on the basis of the estimated incubation period of 1-2 days for influenza [18] . a symmetric bidirectional selection of two reference periods shortly before and after the hazard period was utilized [19] . this means that for temperature and humidity, threeday periods seven days before and after the visit to the clinic for influenza were used in the models. this type of symmetrical selection of reference periods controls for temporal and seasonal confounding [19] . means in temperature and ah between the hazard and reference periods were compared with one-way anova. conditional logistic regression models were used to calculate the exposure odds ratios (or) for the hazard period compared with the reference periods. the maximal change in temperature and ah was adjusted for its initial level in the analyses. the phreg procedure was applied using the discrete logistic model and forming a stratum for each matched set. thus the or represented the risk of the onset of influenza in relation to the level or change of temperature and ah. statistical analyses were performed by sas version 9.2. for windows (sas institute, inc.; cary, nc). the total number of cases was 66; 57 influenza a episodes and 9 influenza b episodes. the incidence of influenza a was 105/1000 person years and influenza b 16.5/1000 person years. the symptoms preceding the identification of influenza started on average three days before seeking medical attention. 69% (45/65) reported outdoor training, 76% (33/43) physical exercise and 72% (31/43) feeling cold during the previous three days. the daily average temperature, absolute humidity and the number of influenza episodes during the study period are presented in figure 1 . as the total number of days with episodes was 28, we utilized meteorological information from a total of 84 days. during the study period the daily average temperatures ranged from −22.8 to +22.0°c and ah from 0.8 to 13.8 g/m 3 . most influenza cases occurred between january and march 2005 with only a few episodes occurring during the autumn months. figure 2 demonstrates mean daily temperature and ah during the hazard and reference periods for the entire study population (n = 66). the figure demonstrates that the occurrence of influenza is preceded by decreases in both temperature and humidity. table 1 shows the means, their average before the decline, as well maximal declines in temperature and ah separately for the hazard and reference periods. the mean temperature was slightly higher during the hazard compared to the reference periods (p < 0.05). also, the maximal decline during hazard period was greater for both temperature and humidity compared with the reference periods (p < 0.001). of note, the temperature but not ah within the hazard period before they declined was also higher (p < 0.05) compared with the reference periods. figure 1 incidence of influenza episodes, mean daily temperature (°c) and mean daily absolute humidity (ah) (g/m 3 ) during the study period. table 2 shows the odds ratios and their 95% confidence intervals (ci) for the relations between onset of influenza and the average temperature, ah and their maximal changes. the risk of contracting influenza was positively associated with mean temperature and ah. a decrease in both temperature and ah (maximal change) during the three days prior to seeking medical consultation increased the risk of influenza. according to these results, a 1°c decrease in temperature and 0.5 g decrease per m 3 in ah increased the estimated risk by 11% (or 1.11; 95% ci 1.03 to 1.20) and 58% [or 1.58; 95% ci 1.28 to 1.96). there was a cluster of 13 cases during the same day and the impact of the cluster was assessed in the sensitivity analysis. the exclusion of the cluster did not substantially change the associations between temperature and humidity. however, due to the small sample size the confidence intervals are wider and associations nonsignificant. there is consistent evidence that wintertime cold temperatures increase respiratory morbidity and mortality [1, 15, 20, 21] . we conducted a case-crossover study to assess the relations between daily temperature and humidity igure 2 mean daily temperature (°c) and absolute humidity (g/m 3 ) during the hazard and reference periods starting 7 days before and after the beginning of the hazard period. values represent means of the observed cases of influenza (n = 66). for the logistic regression analyses the mean temperature and ah was calculated from the three preceding days (day-3 to 0) of the onset of an influenza infection and similarly for the reference periods 7 days before and after the infection. a maximum decline in temperature and ah was calculated as the largest change (maximum versus minimum) occurring in these parameters within the three day period (e.g. day −3 versus day −2, day −3 versus day −1, day −2 versus day −1 etc.) and similarly for the reference periods. values represent mean ± sd. one-way anova, a p < 0.05, b < 0.001. and the risk of influenza infections in subarctic climatic zone among military conscripts. our results indicate that the risk of influenza is related to a decrease in both temperature and humidity, but the influenza risk in general may be reduced at very low temperatures. this could be explained by the effects of temperature and humidity on both the micro-organisms and humans. on one hand the virulence of influenza is expected to be stronger near zero than at subfreezing temperatures, but on the other hand a decrease in temperature makes airways more susceptible to the onset of respiratory infections. our novel finding was that influenza risk is reduced at very low temperatures. this observation is supported by the fact that 74% of the influenza infections occurred at a temperature range of +5 to −10°c (and 38% between +5°c to −5°c) which is not especially cold for the climate. also our previous study from the same population showed that the occurrence of respiratory tract infections was the highest when temperature was at or slightly below 0°c [15] . similarly very low ah reduced the risk of influenza, which was demonstrated by a slightly higher ah during the hazard (3.09 g/m 3 ) compared with the reference periods (2.82 and 2.57 g/m 3 ). our results from subfreezing environmental conditions differ from previous experimental studies which have shown that low temperature (incubation at +21 − +24°c) increased the survival time and accelerated the transmission of the influenza virus [10, 12] . lowen [13] showed that influenza transmission increases when guinea pigs are housed at low temperature (+5°c) conditions and relates this to the effect of temperature on the physical barriers of the host. also low humidity may improve influenza transmission due to altered function of the respiratory tract of the host [22, 23] or enhanced stability of the virus particle [10] [11] [12] [13] . especially for conditions of low temperature and dry air the viability of influenza a is the highest when rh is below 50% [24] . furthermore, in conditions of low absolute humidity (such as in a cold climate) droplet nuclei are small sized and remain airborne for extended periods of times increasing the opportunity for transmission of pathogens they carry and favouring the spread of influenza [25] [26] [27] [28] . the reason why we observed a reduced risk of influenza at low temperature and ah could indicate that the optimal temperature and humidity for the viability, transmission and replication of influenza occurs at higher environmental temperatures and humidity levels. this assumption is supported by a recent epidemiologic study from us which showed that influenza mortality increased when ah decreased below 6 g/kg and peaked when temperature was −1.1°c [29] . though, this study used monthly means in their data analyses instead of daily averages used in the present study. we observed that a decrease in temperature and ah increased the risk of influenza. for temperature the risk was associated with a higher initial levels before the decline. this seemingly contradictory finding could be explained by a novel idea: we should consider the effects of temperature on the viral agent separately from that of the host. higher temperatures approaching zero degrees may favour transmission and survival of the virus itself, but a decline in temperature and humidity may make the host more susceptible through body cooling and/or drying of the respiratory tract. a sudden decrease in temperature and humidity could be related to an increased experience of cold stress and altered airway function increasing the susceptibility to viral agents. military wintertime training is associated with significant cold exposure and heavy physical exercise which could result in aggravated airway cooling and functional changes of the airway epithelia favouring influenza virulence and other respiratory tract infections [15] . it has been suggested that even cooling of the body surface could elicit a reflex vasoconstriction in the nose and upper airways and inhibit the respiratory defence and convert an asymptomatic subclinical viral infection into a symptomatic clinical infection [14] . alternatively, breathing cold air causes cooling of the upper respiratory tract and results in vasoconstriction [30] . also inhaling larger volumes of air with low ah causes drying of the mucosal membrane [22] , which can even lead to epithelial damage [23] . these effects could depress the ciliary movement in the respiratory tract and increase the susceptibility to infections. although no conclusive evidence exists, the seasonal variation in immune responses could further contribute to host susceptibility to infections [31] . our novel finding suggests that a combination of relatively warmer temperature and higher humidity followed by a sudden decline in these meteorological parameters have the strongest impact on the risk of influenza. it should be remembered that both temperature and humidity are the odds ratios (95% confidence interval) were calculated per 1°c temperature and per 0.5 g /m 3 absolute humidity decreases. *adjusted for the initial level before the decline. interconnected for which it is not meaningful to evaluate their independent roles in the present study. to our knowledge none of the previous research assessing influenza morbidity originates from very cold climates, such as this study. however, the association between temperature and humidity and the occurrence of influenza has been demonstrated in epidemiological studies examining onset [4] [5] [6] [7] [8] and outcomes, such as mortality [3, 7, 9, 29 ]. an increased influenza risk with low air temperatures has been observed in temperate [4, 7, 9] or tropical countries [5, 32] . furthermore, low humidity [7, 9] , or its decrease [8] increases the occurrence of influenza. a recent study demonstrated that low wintertime outdoor ah is also reflected as reduced indoor humidity and could predict increased influenza virus survival [33] . an inverse correlation between ah and temperature was shown at the onset of infection in a study modeling influenza-like illness and weather factors from three european countries [34] . mortality studies from moderate climates, such as the us have demonstrated that low temperature [9] and humidity [7, 9] during the prior few weeks were associated with increased wintertime influenzarelated mortality. especially low ah is suggested to explain a larger portion of the observed mortality [6, 29] . these studies support our finding that both temperature and ah are associated with the risk of influenza. however, a direct comparison of the results between these studies is not possible because of the different modes of influenza transmission between tropical, moderate or cold climates [35] . the significance of meteorological parameters for explaining the seasonality of influenza is under debate [36] [37] [38] and it has been suggested that temperature and humidity could be accessory or modulating factors for example in the timing of the onset of an influenza epidemic [3] . in addition, the importance of either temperature or humidity on the occurrence of influenza is being discussed [6, 29, 35] . due to the close dependence of these physical quantities it may be difficult, or not even meaningful, to examine their independent roles. potential other explanatory factors for the seasonality of influenza include alterations in host susceptibility (e.g. immunity), viral mutations, as well as the effects of weather on social contact patterns [39] during the wintertime which enhances influenza spread. we used a case-crossover design which eliminates confounding by stable individual characteristics and by determinants of influenza risk including crowding and annually occurring respiratory infection epidemics. the fact that, compared to the general population, military conscripts are more frequently exposed to low temperatures for prolonged periods, gives additional strength to this study. in addition, physical exercise may aggravate respiratory cooling and the onset of influenza. military service in finland is mandatory for young men, and conscripts thus represent the ordinary population of this age group. as study limitations, we were not able to control for person-to-person transmission, which is likely to occur in a military setting and would facilitate the spread of the virus. in principle, time progression could also be analysed, by using calendar time as an effect modifier, and could provide additional information. however, unfortunately the amount of data was not sufficient for such analyses. furthermore, a non-zero reinfection probability would affect the transmission dynamics, but its detailed analysis was not possible with the current data. clustering of influenza cases to a relatively narrow temperature and ah range could reduce the width of the confidence intervals, but would not likely affect the point estimates of the meteorological variables on influenza. however, we conducted a sensitivity analysis which showed a similar association (not significant) after excluding the possible effect of an influenza epidemic. yet, larger samples from equal climatic conditions and follow-up of multiple influenza seasons would be useful to strengthen our findings. one methodological limitation was due to difficulties in the collection of sputum samples which may lead to underdiagnoses of cases. given the gap in knowledge, our results provided a good starting point and a priori hypothesis for further studies. wintertime influenza epidemics can cause serious public health and economic problems including worker absenteeism and productivity losses, burdening of health care services due to hospitalisation, and in the worst case, deaths. an improved understanding of influenza virus transmission is of importance in order to enhance the accuracy of surveillance systems, to have more precise predictions on influenza epidemics and pandemics in the future, and eventually to develop better disease-control intervention strategies. in conclusion, our study suggests that a decline in temperature and humidity is associated with the occurrence of influenza infection episodes in young healthy men during wintertime in a subarctic climate. however, very low temperatures and absolute humidity may even reduce the occurrence of influenza infections. further studies are needed to confirm causality and establish threshold values between temperature, humidity and influenza infections. our findings are important from a public health perspective and should be considered when planning appropriate cold risk management strategies. exposure to cold and respiratory tract infections influenza (seasonal) influenza and the winter increase in mortality in the united states, 1959-1999 environmental factors and seasonal influenza onset in okayama city, japan: case-crossover study seasonal oscillation of human infection with influenza a/h5n1 in egypt and indonesia absolute humidity modulates influenza survival, transmission, and seasonality absolute humidity and the seasonal onset of influenza in the continental united states absolute humidity as a deterministic factor affecting seasonal influenza epidemics in japan a retrospective study airborne micro-organisms: survival tests with four viruses virus survival as a seasonal factor in influenza and poliomyelitis survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids influenza virus transmission is dependent on relative humidity and temperature eccles r: an explanation for the seasonality of acute upper respiratory tract viral infections cold temperature and low humidity are associated with increased occurrence of respiratory tract infections rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 case-crossover design in air pollution epidemiology incubation periods of acute respiratory viral infections: a systematic review reference selection strategies in casecrossover analyses of air pollution exposure data: implications for bias effects of cold weather on mortality: results from 15 european cities within the phewe project preventing cold-related morbidity and mortality in a changing climate nasal mucosal temperature after exposure to cold, dry air and hot, humid air epithelial shedding is associated with reactions to cold, dry air relationship between humidity and influenza a viability in droplets and implications for influenza's seasonality review of aerosol transmission of influenza a virus transmission of influenza a in human beings inactivation of influenza a viruses in the environment and modes of transmission: a critical review aerosol transmission of influenza a virus: a review of new studies absolute humidity, temperature, and influenza mortality: 30 years of county-level evidence from the united states cold air-provoked respiratory symptoms: the mechanisms and management cold exposure: human immune responses and intracellular cytokine expression seasonal influenza activity in hong kong and its association with meteorological variations predictors of indoor absolute humidity and estimated effects on influenza virus survival in grade schools the role of weather on the relation between influenza and influenza-like illness global influenza seasonality: reconciling patterns across temperate and tropical regions seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us seasonal variation in host susceptibility and cycles of certain infectious diseases influenza seasonality: underlying causes and modeling theories a nice day for an infection? weather conditions and social contact patterns relevant to influenza transmission decline in temperature and humidity increases the occurrence of influenza in cold climate the finnish defense forces are acknowledged for their contribution to the study. the scientific advisory board of defense provided partial funding to the study. the kainuu central hospital is acknowledged for enabling the implementation of this study. furthermore, the authors express their gratitude to the finnish meteorological institute for providing the temperature data. jj was supported by the research council for health, the abbreviations ah: absolute humidity; or: odds ratio; ci: confidence interval. the author(s) declare that they have no competing interests.authors' contributions kj participated in the analyzing and interpretation of the study, drafted the manuscript, and is the principal author of the manuscript. as participated in the analyzing and interpretation of the study and participated in writing the manuscript (special expertise: microbiology). jj participated in the analysis and interpretation of data (special expertise: statistical analyses) and writing of the manuscript. rj participated in the conception and design of the study, implemented the experimental protocol and collection of data, participated in the analysis and interpretation of data and in writing the manuscript (special expertise: otorhinolaryngology). jk participated in the analysis and interpretation of data (special expertise: virological analyses) and writing of the manuscript. ov participated in the analysis and interpretation of data (special expertise: microbiology) and writing of the manuscript. tz conducted the virological analyses, participated in the analysis and interpretation of data (special expertise: virology) and writing of the manuscript. es conducted the virological analyses, participated in the analysis and interpretation of data (special expertise: virology) and writing of the manuscript. jjk participated in the analysis and interpretation of data (special expertise: environmental health, case-crossover analysis) and writing of the manuscript. tmi participated in the analysis and interpretation of data (special expertise: environmental effects on human health and performance) and writing of the manuscript. all authors have approved the final version of the manuscript. key: cord-310956-qwe4ndvb authors: qian, yan‐hua; su, jing; shi, ping; he, en‐qi; shao, jie; sun, na; zu, rong‐qiang; yu, rong‐bin title: attempted early detection of influenza a (h1n1) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia date: 2011-04-18 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00248.x sha: doc_id: 310956 cord_uid: qwe4ndvb please cite this paper as: qian et al. (2011) attempted early detection of influenza a (h1n1) pandemic with surveillance data of influenza‐like illness and unexplained pneumonia. influenza and other respiratory viruses 5(6), e479–e486. background to collect disease information and provide data for early detection of epidemics, two surveillance systems were established for influenza‐like illness (ili) and unexplained pneumonia (up) in wuxi, people’s republic of china. objectives the current study aims to describe the performance of these surveillance systems during 2004–2009 and to evaluate the value of surveillance data in detection of influenza epidemics. methods two national ili sentinel hospitals and three up sentinel hospitals provided data to the surveillance systems. the surveillance data from hospital‐based outpatient clinics and emergency rooms were compared by year. the ili data of 2009 were further modeled based on previous data using both a control chart method and a moving average regression method. alarms of potential epidemics would be raised when the input surveillance data surpassed a threshold. results in 2009, the proportions of ili and respiratory illness with fever (one surveillance syndrome of the up system) to total patient visits (3·40% and 11·76%, respectively) were higher than the previous years. the surveillance data of both systems also showed developing trends similar to the influenza a (h1n1) pandemic in 2009. when the surveillance data of 2009 were fitted in the two detection models, alarms were produced on the occurrence of the first local case of influenza a (h1n1), outbreaks in schools and in general populations. conclusions the results indicated the potential for using ili and up surveillance data as syndromic indicators to detect and provide an early warning for influenza epidemics. influenza, commonly known as the flu, is a severe infectious disease accounting for between 250 000 and 500 000 deaths worldwide every year. millions of people died in the influenza pandemic of 1918, one of the worst epidemic disasters in recorded human history. 1 the life style of modern society, including overcrowded cities and rapid global transportation, has resulted in dissemination of influenza and generation of novel virus strains. in the past year, the world health organization (who) announced a level-6 alert for the influenza a (h1n1) pandemic, the highest level of disease outbreak, because of the sustained spread of this new virus on a global scale. 2 the world was fortunate not to have experienced another devastating influenza epidemic, because the fatality rate of the new virus strain was similar to seasonal influenza virus. 3 however, there remains an inevitability of a new influenza pandemic, which could be highly infectious and as deadly as the 1918 influenza pandemic. thus, it is necessary, although challenging to detect the onset of a new influenza epidemic, to allow the government to make timely and proper interventions to reduce the social and economic impact from influenza epidemics. the influenza surveillance system long has been playing an essential role in collecting disease information and providing evidence for prevention and control strategies and measures. two surveillance systems were established in wuxi for influenza-like illness (ili) and unexplained pneumonia (up) after the severe acute respiratory syndrome (sars) outbreak. the ili system was implemented for influenza surveillance, and the up system was designed to track lower respiratory illness with pneumonia symptoms. although the disease surveillance systems appeared decades later than in developed countries, their performance is by no means inferior to define the distribution of circulating strains in the community and detect disease aberration. here we described and compared the accumulated ili data from 2004 to 2009 and up data from 2007 to 2009. to further evaluate the effectiveness of these surveillance systems in early warning of influenza epidemics, we monitored ili data between 2004 and 2008 by both a control chart method and the serfling method and tested goodness of fit using influenza a (h1n1) data of 2009. wuxi is a prefecture-level city of jiangsu province, located in eastern china, with a population of 6ae2 million. wuxi has seven districts and two county-level cities (jiangyin and yixing). wuxi has a typical subtropical monsoon climate and seasonal influenza usually peaks once a year. 4 there are two surveillance systems in wuxi. one is ili surveillance system and the other is up surveillance systems. both systems collected data from sentinel hospital-based outpatient clinics and emergency rooms. all the selected sentinel hospitals are general hospitals and available to every resident in wuxi city including jiangyin and yixing county. the participating doctors in referral sentinel hospitals were provided with detailed diagnosis instructions for sample selection. as required by the chinese center for disease control and prevention (cdc), two hospitals (children's hospital and the no. 3 people's hospital of wuxi) were selected as the national sentinel hospitals for ili surveillance. the number of patient visits ranged from 8000 to 12 000 ⁄ week. ili was defined as follows: fever ‡38°c, either cough or sore throat, and lack of evidence of other laboratory-confirmed diagnosis. pharyngeal swab specimens collected from patients with ili were sent to cdc laboratories for subsequent isolation and identification. the proportion of patient visits for ili was calculated as the number of patients with ili divided by the total number of patients and was reported through the influenza surveillance system of china. the up surveillance was performed in three hospitals (the no. 2 people's hospital of wuxi, the people's hospital of jiangyin county, and the people's hospital of yixing county). this system was built with special attention to lower respiratory illness, especially pneumonia caused by unknown reagent like sars. routinely collected data included respiratory illness with fever (riwf, also known as acute respiratory illness) and classified pneumonia. emergency measures would be taken in case of any up. pneumonia was diagnosed according to international classification of diseases (10th version, j00-j99). unexplained pneumonia was defined as follows: fever ‡38°c, pneumonia-like characteristics in diagnostic imaging, decreased white blood cell count or decreased lymphocyte differential count in early clinical stage, and no improvement in or even worsening of patient's condition after regular antibiotic treatment for 3-5 days. the total number of patient visits ranged from 11 000 to 14 000 ⁄ week, among whom an average of 3ae5-3ae7% were diagnosed as classified pneumonia. weekly numbers of riwf and classified pneumonia were reported to wuxi center for disease prevention and control. we retrospectively studied ili data from 2004 to 2009 and up data from 2007 to 2009, respectively. all the data were organized by microsoft office excel 2007. statistical analysis was performed by using spss software (version 17.0, spss inc., chicago, il, usa). the kolmogorov-smirnov test was used to check the normality of the data. the chisquare test was used for the comparison of different ratios and proportions. the ili data during 2004-2008 were used in statistical modeling for influenza detection. both a control chart method 5-7 and a moving average regression method 8, 9 were applied to produce the threshold value. alarms of potential epidemics were raised when the input surveillance data of 2009 surpassed the threshold. the control chart method was originally developed to determine and control the situation for manufacturing processes and was later implicated in surveillance analysis. 10 in this study, a long-term average weekly proportion of ili (p) and its standard deviation (sp) were calibrated using all the surveillance data. the data higher than p + 2sp were referred to as being out of statistical control and were excluded. such data were usually observed in epidemics of seasonal flu and could not generally represent a stable and predictable situation over time. adjusted average weekly proportion of ili (p') and standard deviation (sp') were calculated with those data in the state of statistical control. the threshold was defined as p' + 2sp'. the moving average regression method was originally proposed by serfling and was used in our study to generate a time-varying threshold for influenza. 8, [11] [12] [13] [14] a linear secular trend was estimated from the surveillance data through 2004-2008 by using the regression analysis tool of excel. the secular trend was then removed from the original surveillance data, so the adjusted data represented only seasonal change and could be compared by year. the time-varying threshold was calibrated as the 5-year moving average plus 1ae6 sd (standard deviation) (p = 0ae05) or 2ae0 sd (p = 0ae01) from the mean. the first imported case of the influenza a (h1n1) was reported on june 25, 2009 (the 26th week of the year). the first local case was reported on august 16 (week 33). the first school case was reported on september 3 (week 36), and transmission spread quickly when students came back to school in september. the influenza epidemic spread widely during september to december in the general population, with 132 confirmed cases in september, 89 cases in october, 146 cases in november, and 113 cases in december. the last identified patient was reported on february 12, 2010. the epidemic timeline is shown in figure 1 . the surveillance data collected from the two sentinel hospitals showed an earlier peak of the proportion of ili in 2009 when compared with the same period in 2007 and 2008 ( figure 2 ). the ili proportion aberrantly increased to 3ae72% in the 22nd week of 2009, higher than 3ae61% in 2007 and 2ae74% in 2008. this increase in ili proportion appeared 4 weeks earlier before the first imported case of influenza a (h1n1) and started to decline after week 28. simultaneously with the appearance of the first local case, the proportion of ili started to increase rapidly beginning in week 34, indicating the early stage of the influenza epi-demic. the proportion of ili peaked in the general population at week 39 (4ae74%), when the schools also experienced a peak of illness. figure 3 ). the proportion of riwf to total patient visits (11ae76%) was also significantly higher than that in the previous 2 years (9ae91% and 10ae14%, respectively), while the proportion of pneumonia to riwf was similar in all 3 years (3ae53-3ae69%) ( table 1 and figure 3 ). the proportion of riwf began to increase in week 32, around the time of the first imported influenza a (h1n1) case. when we examined the 2009 epidemic season, the trend of up surveillance data was similar to the trend of ili data (figures 2 and 3) . therefore, the data observed from up surveillance system may also reflect the epidemic situation of influenza. the basic design of outbreak detection is that an alert is generated when the current data surpass a threshold. therefore, determining a threshold is essential and crucial for the performance of an early warning system. to obtain the optimal sensitivity and specificity, both control chart method and moving average regression method were used to calibrate the threshold. the control chart method was used to determine an influenza threshold by week. a centerline threshold value was drawn based on the weekly proportion of ili from 2004 to 2008. when the 2009 data were fitted into the chart, alarms of aberrant higher proportions of illness appeared in week 18, week 20 to week 29, week 31 to week 41, week 45 to week 47, and week 49 (figure 4) . these results were consistent with the true observations, including the first imported case in week 26, the first local case in week 33, the outbreak in school beginning in week 36, and the outbreak peak in general population in week 45. in the moving average method, a time-varying threshold for influenza was generated. the ili counts for 2009 were higher than the moving average baseline in week 18, week 20 to week 21, week 24, and week 32 to week 50 ( figure 5 ). more precisely, the ili counts were higher than mean + 1ae6 sd in week 34 to week 37, week 39, and week 45 to week 47, and the periods from week 34 to week 37 and week 46 to week 47 showed the highest ili counts (above mean + 2ae0 sd). the aberration points were consistent with the reported date of the first local case, the outbreak in schools, and the peak in the general population. the early detection of disease outbreaks has long been important to public health. the importance of a surveillance network at the global level has been unprecedentedly emphasized because of the emergence of newly infectious diseases, pandemics, and the threats of bioterrorism. 15 the monitoring of surveillance data plays an essential role in detecting signals for a potential outbreak. for diseases or illnesses with enormous social impact, timeliness of detection is extremely valuable. with these types of diseases or illnesses, indicators like hospital consultations rather than mortality or morbidity are used in modeling, because they tend to provide earlier outbreak indication. 16, 17 for influenza, ili is commonly used as an indicator to predict the occurrence of epidemics. 13, 18, 19 unlike other diseases, the value of routinely collected ili data for influenza detection has been carefully studied and confirmed. 17, 18, 20 in this current study, weekly counts of consultations for ili and up were monitored for influenza detection. the distribution of sentinel ili data in 2009 ( figure 2) were consistent with the real spread of the pandemic and showed aberrant increase close to or earlier than the reference data. these results confirmed the capability of ili surveillance system to detect influenza. although the up surveillance system was designed to detect sars instead of influenza, the data are useful because ili and pneumonia usually have similar symptoms such as fever, cough, and breathing difficulty. compared with the reference data (figure 1 ), the sentinel up data (table 1 and figure 3 ) also showed similar epidemic trend. however, the efficiency of early detection was not high using the up surveillance system as significantly increased proportion of riwf was observed in sentinel hospitals only after the first local case was reported. a reasonable explanation might be because of the diagnostic criteria. ili was used specifically to indicate influenza, while riwf includes pneumonia and many other kinds of respiratory illnesses besides ili. the nature of the up system could not distinguish between the respiratory complaints related to influenza and those caused by other pathogens. the population at risk could be another reason for not being able to detect outbreaks because the ili sentinel sites and up sentinel sites cover different areas. nevertheless, the sentinel up data provide additional information to detect influenza epidemic and can add confidence to declare an epidemic alarm. at the beginning of the 2009 pandemic flu period, asia was in especially high alert of epidemic influenza and china implemented aggressive policies trying to delay illness spread by early identification and isolation of every confirmed h1n1 patient. the increased proportion of ili and riwf before the first imported h1n1 case could be explained as a surveillance artifact because of the heightened concern of individuals. with the development of the epidemic, control strategies were changed to intensive treatment of the seriously ill and self-isolation of mild cases. people who had mild flu symptoms were encouraged to take anti-viral medicines and stay at home instead of seeking medical care in hospitals. all the sentinel hospitals collected data as usual without any strengthened effort. the higher proportion of ili and riwf observed after week 29 was more likely due to the high incidence of h1n1 influenza in general population rather than surveillance artifact. a variety of statistical methods have been developed to monitor surveillance data, including time series, regression, cumulative sum (cusum), and hidden markov models. 9, 11, 14, [21] [22] [23] [24] the performance of these models is usually evaluated from the following characteristics: sensitivity, specificity, and timeliness. 21, 25 sensitivity is the ability to find a real outbreak alert; specificity is the ability to exclude false alarm; timeliness is the ability to raise an alarm within the shortest amount of time from the onset of the peak of the season. most approaches of early detection produce alerts based on the theory of signal detection. 25 the optimal sensitivity and specificity depend on a proper level of threshold, but there is always a trade-off between a high threshold for good specificity and a low threshold for good sensitivity. the calibration of a threshold is a complex determination for each system. both the control chart method and the serfling method are time series approaches and have their own advantages in reducing the variance caused by epidemic data or secular trend. 14, 21 the control chart method optimizes the threshold calibration by excluding the unexpected data that are beyond the controlled scope. 6 the serfling method provides reasonably accurate estimates by adjusting the threshold line with secular regression trend. 8 the us centers for disease control and prevention (atlanta, ga, usa) developed an early aberration reporting system (ears) based on cusum method, which can use only recent data and is supposed to incorporate the threshold calculation by regression method. 21, 22 however, a study in hong kong showed that the regression method and the time series method are superior to the cusum method in a small city with fewer sentinel sites, more variable data, and a more abrupt peak in activity. 14 as a result, the cusum method was not used in our study. the control chart method detected all four remarkable signals, namely the occurrence of the first imported case, the first local case, outbreaks in schools, and the peak in the general population ( figure 4) . it also produced two false-positive alarms, one in the beginning and the other at the end of the epidemic period. when using mean + 1-ae6 sd as the threshold, the serfling method detected the outbreaks in schools and the peak in the general population, with a false-positive signal in the interval; when raising the threshold to mean + 2ae0 sd, only the outbreaks in schools could be detected in a timely manner, and the second alarm was 1 week after the peak in the general population ( figure 5 ). the comparison of the two methods indicated that the control chart method had better sensitivity and timeliness and the serfling method had better specificity. nevertheless, considering that the false-positive alerts of the control method appeared outside the epidemic period, this fact might be taken in favor of a more sensitive detection. the same opinion was seen in gault's paper. 18 a final decision probably should be made combining the results of both methods. the control chart method can generate sensitive and timely alerts, and serfling method can offer secular trend to increase specificity and avoid wasting medical resources. in developing countries like china, surveillance systems like the ili and up systems have only recently been implemented. we performed this study after the 2009 pandemic flu in attempt to make full use of these routinely collected background data. the results are theoretically helpful for healthcare workers to take prevention and control measures when influenza epidemic occur, including preparing antiviral drugs and other medical supply needs, and sending health education messages. so far no such actions, which we already took during the epidemic period, have been applied based on our study. future studies will be carried out to improve the performance of these surveillance systems, such as adding more sentinel sites, covering larger population, improving the quality of indicators, and trying different detection algorithms. studies of decision making are also needed before these approaches could be used in practice. the decision theory provides mathematical methods to estimate the benefits of true alarms and the costs of false alarms for getting an optimal threshold. 25 this analysis was beyond the scope of our current study, and we will investigate such methods in our future work. in conclusion, this study described the surveillance data of ili and up during 2007-2009 and the epidemic progress of influenza a (h1n1) pandemic in wuxi, china. the surveillance data of both systems were correlated with the influenza epidemic and therefore valuable in monitoring influenza activity and generating early warning methods. the control chart method and serfling method had their respective merits and faults in data analysis. further studies could be considered to optimize the threshold of warning. the principle is to improve performance of surveillance systems and allow timely precautionary measures to be implemented in vulnerable populations. emerging infections: pandemic influenza world now at the start of 2009 influenza pandemic mortality from pandemic a ⁄ h1n1 2009 influenza in england: public health surveillance study guideline for prevention and control of influenza statistical quality control methods in infection control and hospital epidemiology, part ii: chart use, statistical properties, and research issues statistical quality control methods in infection control and hospital epidemiology, part i: introduction and basic theory study on early warning based on influenza surveillance data in guangdong province methods for current statistical analysis of excess pneumonia-influenza deaths time-series forecasting use and interpretation of statistical quality control charts influenza-associated hospitalizations in the united states impact of influenza vaccination on seasonal mortality in the us elderly population can syndromic thresholds provide early warning of national influenza outbreaks? methods for monitoring influenza surveillance data trends and directions of global public health surveillance the impact of influenza epidemics on hospitalizations value of icd-9-coded chief complaints for detection of epidemics performance of a syndromic system for influenza based on the activity of general practitioners, france situational uses of syndromic surveillance sensitivity, specificity and predictive values of health service based indicators for the surveillance of influenza a epidemics comparing aberration detection methods with simulated data a simulation model for assessing aberration detection methods used in public health surveillance for systems with limited baselines review of an influenza surveillance system, beijing, people's republic of china disease surveillance using a hidden markov model the emerging science of very early detection of disease outbreaks key: cord-263464-fdosch11 authors: nuvey, francis sena; edu-quansah, elijah paa; kuma, george khumalo; eleeza, john; kenu, ernest; sackey, samuel; ameme, donne; abakar, mahamat fayiz; kreppel, katharina; ngandolo, richard bongo; afari, edwin; bonfoh, bassirou title: evaluation of the sentinel surveillance system for influenza-like illnesses in the greater accra region, ghana, 2018 date: 2019-03-14 journal: plos one doi: 10.1371/journal.pone.0213627 sha: doc_id: 263464 cord_uid: fdosch11 background: influenza-like illness (ili) is a medical diagnosis of possible influenza or another respiratory illness with a common set of symptoms. the deaths of four schoolchildren, during a pandemic influenza outbreak in december 2017 in ghana, raised doubts about the ili surveillance system’s performance. we evaluated the ili surveillance system in the greater accra region, ghana, to assess the system’s attributes and its performance on set objectives. methods: cdc guidelines were used to evaluate the data of the ili surveillance system between 2013 and 2017. we interviewed the surveillance personnel on the system’s description and operation. additionally, routinely entered ili data from the national influenza center provided by the six sentinel sites in accra was extracted. we sampled and reviewed 120 ili case-investigation forms from these sites. surveillance activities were examined on system’s performance indicators, each being scored on a scale of 1 to 3 (poorest to best performance). results: all population and age groups were under ili surveillance over the period evaluated. overall, 2948 suspected case-patients, including 392 (13.3%) children under-five were reported, with 219 being positive for influenza virus (predictive value positive = 7.4%). the predominant influenza subtype was h3n2, recorded in 90 (41.1%) of positive case-patients. the system only met two out of its four objectives. none of the six sentinel sites consistently met their annual 260 suspected case-detection quota. samples reached the laboratory on average 48 hours after collection and results were disseminated within 7 days. of 120 case-investigation forms sampled, 91 (76.3%) were completely filled in. conclusions: the ili surveillance system in the greater accra region is only partially meeting its objectives. while it is found to be sensitive, representative and timely, the data quality was sub-optimal. we recommend the determination of thresholds for alert and outbreak detection and ensuring that sentinel sites meet their weekly case-detection targets. introduction influenza-like illnesses (ili), often also called acute respiratory infection or flu-like syndrome, are acute viral infections of the respiratory tract with similar signs and symptoms to influenza. ili is a syndrome and affected persons may become infectious before, during or after the onset of symptoms. the pathogen can be transmitted both directly (by droplets) and indirectly through contact with contaminated fomites. children, the elderly and pregnant women, as well as persons with chronic illnesses or immunosuppression are at the highest risk for morbidity and mortality from ilis [1, 2] . according to the world health organization (who), most influenzas in the global circulation are of zoonotic origin. sub-types implicated in epidemics include h1n1, h5n1, h7n9, h7n7 and h3n2 [3] . influenza has a global annual attack rate of 5-10% in adults and 20-30% in children, causing between 3-5 million cases of severe illness and about 500,000 deaths yearly [4] . pandemics of influenza have had high fatality rates in the past and robust surveillance systems are key to global efforts to prevent similar outbreaks [5] . due to the epidemic-prone nature of influenza pathogens and their high propensity for mutations, the world health organization (who) recommends strict adherence to infection control and prevention measures including increased handwashing during peak flu seasons [2] . in ghana, laboratory-based surveillance for respiratory tract infections (rtis) only tests for influenza in suspected cases. rtis remain a major cause of morbidity and mortality in ghana ranking second among the top 10 diseases seen at outpatient departments (opds) in healthcare facilities across the country, in 2016 [6] . respiratory diseases may occur because of an invasion of a susceptible host by microbes including bacteria and viruses. three main types of influenza viruses exist, namely; influenza a, b, and c but epidemics are often linked to the influenza a strain [7] . influenza surveillance data between 2012 and 2014 in ghana indicated 1041 positive influenza cases out of a total 8601 respiratory samples tested, with 6 different subtypes [influenza a (h3, h1n1, h1, h5) and influenza b (victoria, yamagata)] identified [8] . ghana began influenza surveillance in 2007 to obtain data on strains in circulation. the national influenza center (nic) was formally recognized by the who in 2010 after the 2009 pandemic influenza [9] . the nic is a member of the who global influenza surveillance and response system (gisrs) and is located in the department of virology of the noguchi memorial institute of medical research (nmimr). in collaboration with the ghana health service (ghs) and the ministry of defense (mod), it currently operates sentinel surveillance for influenza in 27 sites across all regions in ghana with support from the u.s. naval medical research unit no. 3 (namru-3), centers for disease control and prevention (cdc) and who [8] . ili surveillance is conducted all year round across the sentinel sites. the ili surveillance system aims to detect early unusual events indicating a change in circulating influenza sub-types, identify and monitor vulnerable groups for influenza, determine influenza thresholds and detect antiviral resistance. in december 2017, an outbreak of influenza in a school in the ashanti region of ghana was followed by the death of four children (case fatality rate = 5.2%) [10] . this raised concerns about the effectiveness of the influenza surveillance system, particularly that in the greater accra region of ghana, which recorded no alerts over the past five years. we evaluated the effectiveness of the ili sentinel surveillance system to determine if its objectives are being met and to assess its attributes and usefulness. the findings of this study are important in order to give useful recommendations to improve the current system. we described the attributes and effectiveness of the ili sentinel surveillance system in the greater accra region (gar) of ghana using a descriptive cross-sectional survey. we extracted and evaluated routinely recorded ili data between january 2013 and december 2017. the gar is one of ten regional demarcations in ghana with a population of about 5 million people [11] . as the most densely populated region in ghana, it is mainly an urban settlement and lies in the southeastern part of the country along the coast of the gulf of guinea. it is the administrative capital of ghana with over 500 public and private health care facilities. six of these facilities in the region conduct ili surveillance including four ghs facilities, one military and one quasi-government facility (see fig 1) . they fall under the three main levels of healthcare delivery in ghana; primary, secondary and tertiary. the manhean health center provides primary health services, while secondary healthcare providers involved in ili surveillance are tema polyclinic, achimota hospital and university of ghana hospital, legon. lastly, the greater accra regional hospital (garh) and 37 military hospital provide tertiary care. data on ili from all six sentinel facilities in gar was extracted and abstracted from the nic database into microsoft office excel format. additionally, we selected three sentinel facilities, based on the different levels of care they provided, and obtained permission for site visits. these sentinel sites; garh, achimota hospital and manhean health center, were visited for at least one week each and their records were collected and reviewed. the researchers interviewed all personnel directly involved in ili surveillance and also partook in surveillance activities while observing practices. the staff members were interviewed using a structured questionnaire and guided interviews on their knowledge on ili surveillance activities. forty case investigation forms were randomly sampled from each of the three facilities, using microsoft excel. the unique serial numbers on case investigation forms for each sentinel site were used to retrieve the randomized forms for examination. in addition, we collected and reviewed ili registers at the sites. the evaluation was conducted using the cdcs guidelines for evaluating public health surveillance systems [12] . nine attributes: simplicity, flexibility, data quality, acceptability, sensitivity, predictive value positive, representativeness, timeliness and stability, usefulness and the utility of the system to achieve its objectives were evaluated. the indicators for each attribute or characteristic measured were scored one point each. based on the evaluator's assessment, an indicator is scored zero (0) if the key finding from evaluation do not support the indicator assessed in relation to the attribute. a score of one (1) is given to each indicator that supports the attribute assessed. the assessment scores were then summed and divided by the total number of indicators used in evaluating each attribute. attributes with relative scores of more than twothirds of the total score, were considered as major strengths of the system and scored 3 overall. those with less than one-third were major weaknesses (overall score = 1). scores between one-third and two-thirds were relative strengths (overall score = 2). scores were on a scale of 1 to 3: poorest to best performance, respectively. the evaluation was done within the framework of integrated disease surveillance and response matrix implemented by the ghana health service and therefore did not have to receive formal review by ethical review committees. the field epidemiology and laboratory training programme and national influenza center in ghana approved the study. permission was sought and obtained from the public health directorate of ghana health service and authorities in the sentinel facilities before commencement of the evaluation. all respondents provided informed, written consent and were assured of confidentiality. the surveillance system was found to be utilizing the syndromic approach by screening suspected cases and thereafter conducting further laboratory confirmatory tests on collected nasopharyngeal or oropharyngeal specimen. a reverse transcriptase-polymerase chain reaction (rrt-pcr) is used to confirm real influenza cases from among those suspected using the influenza case definition and determine the influenza virus sub-type. we found that data on patients meeting the ili case definition (s2 table) from the sentinel sites are collected together with nasopharyngeal or oropharyngeal specimen. specimen are stored in sample bottles with virus transport media (vtm) in a cold chain system and submitted to nmimr for laboratory confirmation. each site is required to suspect five cases weekly using the case definition. thus, the expected annual total for each site is 260 suspected cases. firstly, clinicians at the opds suspect cases and trained personnel (i.e. nurses, laboratory scientists and surveillance officers) collect the specimen for storage in designated refrigerators at the disease control units. case detection is, in most cases, directly done by ili surveillance team members at the opds. socio-demographic, epidemiologic and clinical data are collected on each case using case investigation forms. the nic supplies the sentinel sites with the tools including case investigation forms, vtm, and specimen bottles, for the system's operation. they contact the sentinel sites via phone calls and visit the sentinel sites in the gar at least twice every week to pick up specimen. the nic reports results from the polymerase chain reaction laboratory test to each sentinel site, the national surveillance department (nsd), and who via e-mail. data is entered into the flu-net system, a who (gisrs) global database specifically designed for influenza surveillance, weekly. sentinel sites receive printed laboratory results from the nic averagely every 7 days. at the facility level, health information units (hiu) enter the data into the district health information management system 2 (dhims 2). ili data entry into dhims 2 started in 2017. the ghs, nic and other stakeholders periodically hold review meetings to discuss influenza activities and publish the data generated by the system in reports and journals. the information and data flow in the ili surveillance system is further illustrated in fig 2. of the 2948 suspected cases tested, 219 (7.4%) cases were tested positive for influenza viruses. s1 table) . utility of the ili sentinel surveillance system in attaining set objectives. the ili surveillance system has four objectives: early detect events that show change in severity or patterns of influenza infection or emergence of new strains, determine influenza thresholds, identify and monitor at risk groups and detect antiviral resistance in circulating strains. the system met two out of its four objectives over the period evaluated. it was able to detect and characterize influenza viruses in circulation, as from influenza a and b lineages, and established the groups of persons most at risk of influenza infection. it however did not set thresholds, which is the minimum number of suspected cases above which the system is alerted, and was not performing antiviral resistance testing. surveillance system attributes. usefulness: data generated by the system informed the choice of vaccines used in controlling influenza outbreaks in ghana. in addition, who and cdc use the system's information to monitor influenza activity globally, through sharing of confirmed influenza samples. tables 1 and 2 show the key findings on the indicators and scores for all the qualitative and quantitative attributes evaluated respectively. sensitivity: the ili system in gar has been able to confirm influenza cases in each of the years evaluated (s1 table) . evidence of aggregate data from ghana can also be found in the who flunet system, available free at http://apps.who.int/flumart/default?reportno=1. predictive value positive (pvp): the predictive power of the ili case definition is tested using rrt-pcr. overall pvp for the period was 7.4% (range 4.7%-14.8%). see s3 table for yearly pvps over the evaluated period. simplicity: surveillance personnel interviewed identified case definition to be simple. only opds partake in ili surveillance and follow-up of confirmed cases done within 5 days. however, specialized training is required for specimen collection and laboratory confirmation, data collected on each suspected case is comprehensive and laboratory testing is not done at sentinel sites. flexibility: modification of the ili system in 2016, enabled detection of severe acute respiratory infections (sari) with no difficulty. furthermore, addition of other common respiratory symptoms to case definition at the sites did not disrupt the system. data quality: key surveillance information was completely provided in 76% (91/120) of case investigation forms sampled and data extracts were comparable at sentinel facilities and the nic. in spite of this, only two sites: garh and achimota hospital, had 2017 influenza data table 1 entered in the dhims-2 platform and laboratory results were not entered in ili registers at the three sites. acceptability: on average, five out of the six sentinel sites detect influenza cases each year (83%) and about 80% of case investigation forms sampled were completely filled out by ili personnel. all sentinel staff interviewed reported satisfaction with feedback from nic and test results from collected specimen are on average tested within 2 days. most of the sites (5/6) however, do not meet the case detection quota for each year with some recording as low as three suspected cases per year only (see fig 3) . representativeness: age and sex distribution of total cases detected reflect the general distribution of ghana's population. the median age of cases was 30 years (range: 3 weeks to 90 years). females constituted 60% (1775/2948) of cases. timeliness: it takes on average 10 days between symptom onset and detection at facilities. the majority of specimens are tested within 48 hours after collection and results are disseminated within 7 days. this conforms with the who set standard timelines for influenza surveillance. stability: data flow in the system conforms to set standards; testing is routinely done within 48 hours after case detection and results are released within 7 days to the sites on average. the system is sustainable with donors mainly funding its operation. there was not a time influenza surveillance in the greater accra region, ghana during the period evaluated when no site recorded cases, but all sites detected cases only for 60% of the period. influenza causes considerable morbidity and about 500,000 deaths per year globally. the disease is highly infectious with high pandemic potential. therefore, worldwide surveillance systems to record influenza-like illnesses (ili) in real time and detect possible influenza outbreaks are essential to prevent and control epidemics. ghana's ili surveillance system since its inception in 2007, aims to early detect changes in circulating influenza, identify vulnerable groups to influenza infection, determine influenza thresholds and detect antiviral resistance to influenza viruses. the late detection of a pandemic influenza outbreak in ghana raised doubts about the ili surveillance system's performance. our study provides evidence, that the ili sentinel surveillance system in the greater accra region (gar), ghana, is only partially meeting its objectives because it did not have thresholds for alerting the health system and does not perform antiviral resistance testing. it is sensitive and timely in detecting influenza cases but of low predictive value positive (pvp). it is representative of the population under surveillance and flexible to modifications. the system is fairly stable and acceptable to key stakeholders; the quality of data is relatively high. predominant influenza subtype in circulation is influenza a (h3n2) virus. the sentinel sites consistently failed to meet their case detection quotas annually over the period evaluated. despite these apparent weaknesses in the ili system, the good performance of the laboratory component is commendable and key to detection of novel influenza viruses for prompt response. even though the who's standards for influenza surveillance alluded to the possibility of resource limitation hindering achievement of all influenza surveillance system objectives, it advocates for influenza surveillance systems capable of collecting the minimum amount of data needed for decision making [5] . for the past two decades of influenza surveillance in ghana, the system proved its utility by its ability to detect and classify circulating strains as well as providing key information for public health action. similar findings were made in the south african [13] and madagascar [14] influenza surveillance systems. even though the pvp is low, it generally conforms to other syndromic surveillance systems with broad case definitions but specific for respiratory diseases in this case, aimed at maximizing influenza case detection [15] . nevertheless, the unmet objectives, lack of thresholds and antiviral resistance testing, of the system require attention. the lack of antiviral resistance testing to detect the emergence of treatment resistant strains and the absence of thresholds preventing the issuing of alerts, are a major drawback. this situation is not specific to the gar alone but is found in all sites in ghana. in addition, the mainly conservative management of clinical signs and symptoms, of confirmed influenza cases in the health system, without the use of recommended antiviral agents, may partly explain the absence of antiviral susceptibility testing. these shortfalls may, however, be caused by a lack of capacity or resources (antiviral drugs and laboratory reagents) at the sentinel facilities to determine thresholds for influenza alerts and test for antiviral resistance, as was observed in evaluations done in other settings [16, 17] . threshold establishment for disease surveillance is paramount to be able to alert the health system early when outbreaks occur for prompt public health action [1] . a study to test the moving epidemic method in determining thresholds for ili and sari surveillance systems in europe, was able to detect epidemic periods with few or no false alarms in different countries [18] . other methods employed in cambodia [19] and australia [20] had similar findings. the nic must take the lead using the who manual [21] , to choose an epidemic threshold determination method and train focal persons in the facilities to use it, to ensure this key information is available. even though implementing an integrated (human and animal) surveillance could be costeffective and improve case detection and response [22] , discussions with ili surveillance personnel at sentinel sites revealed the absence of a link between influenza surveillance in humans and animals. human infection with zoonotic influenza strains is possible and may cause mild to severe form of the disease. there have been recorded episodes of pandemics in humans over a century due to cross-species transmission of zoonotic respiratory viruses including influenza viruses, notably spanish flu (1918), severe acute respiratory syndrome (2003) and swine flu (2009), resulting in high morbidity and mortality globally [23, 24] . thus, the importance of a one-health approach in the surveillance and response with control of pandemics in an increasingly globalized world is evident. research findings in africa, europe and the americas, have shown incremental gains derived when interventions are integrated between human and animal health systems [25] . the nic and veterinary service department in ghana should collaborate to formalize protocols for engaging each other to integrate influenza surveillance systems in humans and animals, using a one health approach, as this can provide additional key information on possible cross-species transmission in the country and enhance savings. the usage of one laboratory to test for both human and animal pathogens in canada was shown to cost about 30% less than the combined original operational costs of testing individually in both laboratories [26] . in ghana, the timeliness of the system in case detection, laboratory confirmation and result dissemination is commendable. this strength may be as result of the weekly visits nic makes to sentinel sites to collect specimen, supply virus transport media in specimen bottles and transfer results. the nic should further take advantage of these visits and collaborative meetings to ensure each site meets weekly case detection quotas. focal persons at the various sites must take the lead. in spite of the usefulness and fair performance of ili surveillance in gar on indicators evaluated, it is only partially meeting its set objectives. it is sensitive in detecting circulating influenza types, representative of the population under surveillance and timely. however, sentinel sites do not consistently meet annual case detection quotas. there is the need to address shortfalls in the system's objectives as well as improving case detection at sentinel facilities. this would ensure that the successes chocked are not undermined, thereby preventing increasing morbidity and mortality related to influenza infections. failure of the system to address these shortfalls would also affect ghana's contribution to the who global influenza surveillance and response system. considering the zoonotic character of most influenza viruses, it is important that a one health approach is adopted with influenza surveillance in ghana. we propose strict adherence to case detection targets by individual sentinel sites and determination of alert thresholds for the system to allow for a more effective monitoring of influenza activity in the region for prompt public health actions. supporting information s1 table. ili case definitions used for screening and enrolment by the ili sentinel surveillance technical guidelines for integrated disease surveillance and response in the african region world health organization. community case management during an influenza outbreak: participant's handbook influenza at the human-animal interface; sumary and assessment influenza (seasonal) fact sheet [internet]. who. world health organization world health organization. global epidemiological surveillance standards for influenza virological surveillance of influenza-like illness among children in ghana ghana health service/ministry of health. interim report: surveillance response to influenza type a (h1n1) outbreak in kumasi academy, asokore -mampong municipality population projections by districts updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group evaluation of two influenza surveillance systems in south africa evaluation of the influenza sentinel surveillance system in madagascar overview of syndromic surveillance: what is syndromic surveillance? morb mortal wkly rep strategy to enhance influenza surveillance worldwide a summary of influenza surveillance systems in australia influenza surveillance in europe: establishing epidemic thresholds by the moving epidemic method establishing seasonal and alert influenza thresholds in cambodia using the who method: implications for effective utilization of influenza surveillance in the tropics and subtropics exploring a proposed who method to determine thresholds for seasonal influenza surveillance world health organization. who interim global epidemiological surveillance standards for influenza creating a framework towards integrated health syndromic surveillance and response in africa global burden of influenza: contributions from resource limited and low-income settings sars, the first pandemic of the 21st century one health: the theory and practice of integrated health approaches. croydon: cab international the world bank. people, pathogens and our planet: towards a one health approach for controlling zoonotic diseases we acknowledge support received from the national influenza center and noguchi memorial institute of medical research, and afrique one alliance. in addition, we would like to thank the physicians, nurses, surveillance officers, laboratory scientists and health information officers who contribute to the influenza-like illnesses sentinel surveillance system in the greater accra region of ghana, as well as the staff and cohort 11 residents of the ghana field epidemiology and laboratory program. key: cord-306983-6w2fvtfy authors: wang, siye; le, trong quang; kurihara, naoki; chida, junji; cisse, youssouf; yano, mihiro; kido, hiroshi title: influenza virus—cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza date: 2010-10-01 journal: j infect dis doi: 10.1086/656044 sha: doc_id: 306983 cord_uid: 6w2fvtfy background. severe influenza is characterized by cytokine storm and multiorgan failure with edema. the aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. methods. weanling mice were infected with influenza a wsn/33(h1n1) virus. the levels of proinflammatory cytokines, tumor necrosis factor (tnf) α, interleukin (il) 6, il-1β, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. the effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. results. influenza a virus infection resulted in significant increases in tnf-α, il-6, il-1β, viral hemagglutininprocessing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokineand trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. conclusions. the influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza. cular hyperpermeability and multiorgan failure in severe influenza remains unclear. significant increases in levels of proinflammatory cytokines such as tumor necrosis factor (tnf) a, interleukin (il) 6, and il-1b (ie, cytokine storm) affect host survival both positively and negatively [5] [6] [7] . the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and multiorgan failure [8] . in addition, influenza a virus infection upregulates several cellular proteases, including ectopic trypsin [9] and matrix metalloprotease (mmp) 9 [10] . ectopic trypsin, like tryptase clara [11] , mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin [12] , which is crucial for viral entry and replication [13] [14] [15] [16] [17] and subsequent tissue damage in various organs [9, 16, 17] . influenza a virus infection significantly upregulates trypsin in endothelial cells and in hippocampal neurons [9] . because trypsin efficiently converts pro-mmp-9 to active mmp-9 [18] , induction of both proteases synergistically degrades basement membrane proteins, potentially destroying tight junctions and the blood-brain barrier, followed by multiorgan failure [19, 20] . the aim of the present study was to define the pathogenic impact of cytokine storm in influenza a virus infection and the molecular mechanisms by which proinflammatory cytokines cause vascular dysfunction in animal models and in human vein endothelial cells. the results pointed to the role of the influenza virus-cytokine-protease cycle as one of the main mechanisms of vascular dysfunction in severe influenza. specified pathogen-free 3-week-old weanling c57bl/6crslc female mice were obtained from japan slc. under ketamine anesthesia, 250 or 500 plaque-forming units (pfu) of influenza a/wsn/33(h1n1) [21, 22] in 15 ml of saline or saline alone as the vehicle was instilled intranasally in mice. mice ( ) also received inhibitors against nuclear n p 10 factor-kappa b (nf-kb), such as pyrrolidine dithiocarbamate (10 mg/kg) and n-acetyl-l-cysteine (10 mg/kg) [23, 24] , and inhibitor against activator protein 1, nordihydroguaiaretic acid (2.5 mg/kg) [25] , intraperitoneally. these inhibitors were administrated once daily for 4 days immediately after viral infection (day 0). virus titers were determined in madin-darby canine kidney cells [11] . all animals were treated in accordance with the guidelines of the animal care committee of the university of tokushima. cell culture. human umbilical vein endothelial cells (lonza) were grown using the protocol supplied by the manufacturer. the cells were infected by influenza a virus wsn at a multiplicity of infection of 0.5 or treated with recombinant human il-6, tnf-a, and il-1b (10 ng/ml of each) (peprotec) in the presence or absence of antibodies against these cytokines (abcam). evaluation of vascular permeability. vascular permeability was analyzed by the evan's blue extravasation method [26] . one hour after intraperitoneal injection of 400 ml of 2% (w/ v) evan's blue dye in saline, the whole body was perfused with saline through the cardiac ventricle. the leakage of dye was detected macroscopically and by fluorescence microscope. enzyme-linked immunosorbent assay (elisa). the levels of il-6, tnf-a, and il-1b in tissue homogenates and plasma were measured using cytokine elisa kits (bd biosciences). western blotting and gelatin zymography. tissues were homogenized with 3 volumes of tris-hcl, ph 6.8, containing 2% sodium dodecyl sulphate and 0.5 m nacl, and centrifuged at 12,000 g for 30 min. human endothelial cells were lysed in radioimmune precipitation buffer (nacalai tesque) at 4њc. these extracts (30 mg protein) were electrophoresed and transferred to polyvinylidene difluoride membranes. rabbit antizonula occludens-1, anti-occludin antibodies (zymed), and anti-actin antibody (chemicon) were used. immunoreactive bands were detected using chemiluminescence (amersham bio-sciences). for gelatin zymography, the extracts (50 mg protein) were subjected to electrophoresis on 10% gelatin zymogram gels (invitrogen) as reported previously [9] . immunohistochemical staining. immunohistochemical staining was conducted as described elsewhere [9] . lung and brain sections were reacted overnight with polyclonal antibodies against human influenza a, b virus (takara) at 4њc, washed, and then reacted for 1 h at room temperature with a biotinylated second antibody. the sections were counterstained with mayer's hematoxylin. permeability assay. human endothelial cells grown to confluence on 12-well tissue culture plates with falcon cell culture inserts (1.0 mm), were exposed to the cytokines for 12 h in the presence or absence of 50 mm of aprotinin (nacalai tesque). changes in the monolayer permeability were analyzed and quantified as clearance of fluorescein isothiocyanate-dextran from the upper chamber to lower chamber as reported previously [27] . total rna was isolated from human endothelial cells using an rneasy mini kit (qiagen) and reverse transcribed using oligo primers and superscript iii rt (gibco brl) for complementary dna synthesis. the following primer pairs were used to amplify human trypsin (hprss): hprss (forward primer [f], 5'-atccaggtgagactgggagagc-aca-3', nucleotide (nt) 222-246, and reverse primer [r], 5'-gtagaccttggtgtagactccaggc-3', nt 692-716) and those of viral ns1 as reported elsewhere [28] . rt-pcr and quantification of gene expression by real time-pcr were performed using fast start sybr green master (roche diagnostics) on an abi prism 7300 system [28] . human endothelial cells were cultured on glass chamber slides until confluence. after washing twice with calcium-and magnesiumfree phosphate-buffered saline (pbsϫ), the cells were incubated with cytokines (10 ng/ml for each cytokine) for 10 h with or without pretreatment for 30 min with 20 mm protease-activated receptor (par) 2 antagonist peptide, fsy-nh 2 [29] , or 50 mm aprotinin at 37њc. the cells were also activated with 1 mg/ml trypsin or 10 mm par-2 agonist peptide [30] for 30 min. the cells were also treated for 5 h with 10 nm calcium ionophore a23187 (calbiochem) or 2 mm cacl 2 . for imaging, 10 mm fluo-3/am (invitrogen) was introduced into the cells by incubation for 30 min. the cells were then washed twice with pbsϫ and incubated with 5 mm glucose in pbsϫ at 37њc. intracellular calcium ([ca 2+ ] i ) levels were analyzed using a confocal laser scanning microscope (model cm1900; leica). statistical analysis. results are presented as mean value ‫ע‬ standard error of the mean (from 3-5 independent experiments). differences between groups were examined for statistical significance by the paired t test or 1-way analysis of variance. the wilcoxon test for comparisons of kaplan-meier survival curves was used. a p value !.05 was considered to be statistically significant. a virus wsn to study the pathogenic effects of cytokine storm on vascular dysfunction. the levels of tnf-a and il-6 in the lungs, the site of initial virus infection, were increased persistently for 6 days, and levels of il-1b peaked at days 4-6 after infection ( figure 1a ). because these cytokine responses are associated with activation of the transcription factors nf-kb and activator protein 1 [7, [31] [32] [33] , we treated mice once daily for 4 days with anti-oxidant inhibitors: pyrrolidine dithiocarbamate and n-acetyl-l-cysteine against nf-kb activation, and nordihydroguaiaretic acid against activator protein 1 activation. pyrrolidine dithiocarbamate and nordihydroguaiaretic acid significantly suppressed the upregulation of tnf-a and il-1b ( ), and n-acetyl-l-cysteine suppressed tnf-a ( p ! .001 p ! ) and il-6 ( ) at day 4 after infection ( figure 1a ). .001 p ! .01 gelatin zymography showed upregulation of ectopic trypsin in mice lung, brain, and heart during infection for 6 days ( figure 1b ). trypsin induction was inhibited by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid, probably via blockade of nf-kb and activator protein 1 binding in the promoter region of the gene (s. r. talukder, unpublished data). viral rna replication in various organs at day 4 after infection was suppressed by 11 order of magnitude by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid ( figure 1c ). suppression of viral multiplication and induction of cytokines and trypsin by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid significantly improved the survival of mice at day 14 after infection (ie, the late stage of infection) ( figure 1d ). the kinetics of viral replication monitored by viral ns1 gene showed that the level of viral rna was the highest at day 4 after infection and decreased at day 6 in these organs (figure 2a ). to determine the pathogenesis of tissue injury, the viral protein accumulation in the lung and brain at day 4 after infection was analyzed by immunohistochemical staining ( figure 2b ). viral protein was detected in alveoli and terminal bronchioles in the lung and was also detected in the brain, particularly in the hippocampus, neocortex, brainstem, and brain capillaries. vascular hyperpermeability is one of the main complications of organ injury in severe influenza. vascular permeability was analyzed by infiltration of evans blue dye in the lung and brain after infection ( figure 2c and 2d) . in contrast to no dye infiltration in uninfected animals, infected mice showed a progressive increase in vascular permeability in the lung and brain at day 4 after infection. fluorescence microscopy showed leakage of dye from the blood vessels in these organs ( figure 2d ). to elucidate the mechanisms underlying vascular dysfunction in the brain, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 and transmembrane occludin, and the matrix protein laminin were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 after infection, which were partly rescued by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, or nordihydroguaiaretic acid ( figure 2e ). no other tight-junction protein, claudin-5, or matrix fibronectin and type iv collagen was affected (data not shown). to clarify the linkage between upregulated cytokines and trypsin and vascular hyperpermeability after viral infection, the relationships among these findings were examined in human endothelial cells. viral infection significantly increased tnf-a and il-6 levels (2.7fold and 7.1-fold, respectively) but not il-1b levels in the culture media in a time-dependent manner over a 24-h period ( table 1) . influenza a virus infection upregulated human trypsin/ hprss gene by approximately 2-fold in the cells after infection for 6-12 h ( figure 3a ). to analyze the linkage between cytokines and trypsin in the cells, changes in the expression of hprss gene were analyzed after exposure to 10 ng/ml tnfa, il-6, and il-1b instead of viral infection for 6 h ( figure 3b ). all tested cytokines tended to upregulate hprss expression levels, especially tnf-a ( ) and il-1b ( ), although p ! .01 p ! .05 less effectively than did viral infection, and the upregulation was inhibited by simultaneous treatment of the respective neutralizing antibodies (100 ng/ml) with these cytokines (p ! for tnf-a; for il-1b). .05 p ! .01 treatment of the cells for 12 h with tnf-a, il-6, and il-1b markedly suppressed tight-junction protein zonula occludens-1 levels and occluding levels slightly, and loss of these proteins were abrogated by simultaneous treatment of the cells with 50 mm of the nonpermeable trypsin inhibitor aprotinin ( figure 4a ). furthermore, cytokine treatment disrupted the continuous and linear arrangement of zonula occludens-1 among the cells, and aprotinin inhibited the disruption ( figure 4b ). accordingly, treatment with cytokines, especially il-1b and tnf-a, tended to increase endothelial cell monolayer permeability and this effect was blocked by 50 mm of aprotinin ( ) ( figure p ! .05 4c) . these findings suggest that cytokines upregulate trypsin in vascular endothelial cells and that secreted trypsin plays an (original magnification, ϫ200) . b, immunoreactive deposits in the lung (original magnification, ϫ200). c, viral antigen (arrowheads) in epithelial cells of respiratory bronchioles and infiltrated leukocytes in alveoli (original magnification, ϫ400). d, no immunoreactive deposits in the brain before infection (original magnification, ϫ200). e, virus antigen in the cornu ammonis (ca) 1 and ca-2 and in the stratum granulosum of the dentate gyrus (dg) of the hippocampus (original magnification, ϫ200). f, virus antigen (arrowheads) in the enlarged image of ca-1 (original magnification, ϫ400). scale bars are 100 mm.c, vascular permeability in the lung and brain analyzed by evan's blue dye extravasation before (wsn-d0) and after infection at day 4 (wsn-d4). d, fluorescent micrographs of evan's blue leakage from capillaries in the brain and lung before and after infection at day 4. e, loss of tight-junction proteins, zonula occludens (zo) 1 and occludin, and laminin in the brain analyzed by western immunoblotting at day 4 after infection and its restoration by pyrrolidine dithiocarbamate (pdtc), n-acetyl-l-cysteine (nac), and nordihydroguaiaretic acid (ndga) treatments. the levels before infection are shown as control (ctr). important mechanistic role in the loss of zonula occludens-1 and increased permeability. 2+ ] i through g protein-coupled receptors, leading to cytoskeletal reorganization in the microvascular endothelium and consequent increase in permeability and tissue edema [34] . trypsin receptor par-2 is a g protein-coupled receptor activated by trypsin and tryptase and plays an important role in increasing [ca 2+ ] i [35] . to investigate the mechanisms underlying vascular hyperpermeability in severe influenza, we treated human endothelial cells with cytokines, trypsin, and par-2 agonist peptide in pbsϫ and then measured [ca 2+ ] i ( figure 5 ). marked [ca 2+ ] i mobilization was found after treatment of the cells with trypsin, par-2 agonist peptide, and cacl 2 for 10 h, whereas calcium ionophore a23187 decreased [ca 2+ ] i . treatment with tnf-a, il-1b, and il-6 also increased [ca 2+ ] i , and the mobilization was suppressed by pretreatment of the cells with par-2 antagonist, fsy-nh 2 , or aprotinin for 30 min. these results suggest that [ca 2+ ] i mobilization by proinflammatory cytokines through activation of trypsin and its receptor par-2 is one of the main mechanisms underlying increased endothelial cell permeability. the present study reports several new observations: (1) proinflammatory cytokines, tnf-a, il-1b, and il-6, when upregulated by influenza a virus infection, induce trypsin expression in various organs and human endothelial cells; (2) the upregulated trypsin induces [ca 2+ ] i mobilization via activation of the par-2, followed by loss of zonula occludens-1 and vascular hyperpermeability; (3) inhibitors of nf-kb and activator protein 1 effectively suppress the upregulation of proinflammatory cytokines and trypsin and improve the survival rates of infected mice. based on these results, we propose the influenza viruscytokine-protease cycle hypothesis as one of the mechanisms of vascular dysfunction in multiorgan failure with cytokine storm in severe influenza and influenza-associated encephalopathy ( figure 6 ). the significance of proinflammatory hypercytokinemia, or cytokine storm, in the pathogenesis of influenza a virus infigure 6 . the hypothesis of influenza virus-cytokine-protease cycle, which may affect the pathogenesis of vascular hyperpermeability and tissue destruction in severe influenza. ap-1, activator protein 1; bbb, blood-brain barrier; par-2, protease-activated receptor 2; zo-1, zonula occludens-1. fection remains unclear. the positive effects include that cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, the negative effects of excess cytokines include the fact that the hyperinflammatory process evoked by viral infection [8, 36] may become harmful through intracellular activation of nf-kb, activator protein 1, and the janus kinase-signal transducers and activators of transcription signaling pathways [31-33, 37, 38] . the in vivo experiments presented here showed that nf-kb and activator protein 1 inhibitors markedly suppress the expression of cytokines and trypsin, viral replication, and endothelial dysfunction and result in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production and activation of vasodilatory mediators, such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs [8] (figure 6 ). the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyperpermeability fol-lowing the cytokine storm remain unclear. tnf-a upregulation alters the cellular redox state, reduces the expression of 4 complex i subunits by increasing mitochondrial o 2 ϫ production and depleting adenosine triphosphate (atp) synthesis, decreases oxygen consumption (resulting in mitochondrial damage) [8, 39] , and increases [ca 2+ ] i [40] . atp depletion dissociates zonula occludens-1 from the actin cytoskeleton and thereby increases junctional permeability [41] . the present results allow us to propose a new mechanism of junctional permeability regulation: upregulated trypsin by influenza a virus and/or proinflammatory cytokines induces increase in [ca 2+ ] i and loss of zonula occludens-1 in endothelial cells via par-2 signaling. in contrast to the marked upregulation of cytokines in the lungs ( figure 1a) , upregulation of cytokines in the brain was mild (data not shown), which suggests that the bloodbrain barrier destruction is the result of systemic effects of cytokines produced in the lung in severe influenza. anti-cytokine antibodies and trypsin inhibitors may effectively suppress junctional permeability. endothelial dysfunction induced by the influenza virus-cy-tokine-protease cycle in the early stage of severe influenza may also affect various circulating factors, coagulation factors, and complement systems, as well as vascular interacting cells, such as neutrophils, macrophages, and lymphocytes. multiorgan failure is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1 and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and the formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway [42] . in addition to these data, upregulated trypsin and proinflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of influenza a virus infection. further studies are required on the role of the influenza virus-cytokine-protease cycle in the pathogenesis of multiorgan failure, particularly in the late stage of viral infection. influenza: emergence and control influenza a and b virus infection in infants and young children during the years 1957-1976 influenza a virus associated with acute encephalopathy pcr on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis systemic cytokine responses in patients with influenza-associated encephalopathy induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression inflammatory cytokines in vascular dysfunction and vascular disease identification of trypsin i as a candidate for influenza a virus and sendai virus envelope glycoprotein processing protease in rat brain matrix metalloprotease-9 and tissue inhibitors of metalloproteinases i in influenza-associated encephalopathy isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelia clara cells. a possible activator of the viral fusion glycoprotein 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nf-kb and ap-1: involvement of the ras dependent pathway increased ap-1 and nf-kb activation and recruitment with the combination of the proinflammatory cytokines il-1b, tumor necrosis factor alpha and il-17 in rheumatoid synoviocytes tnf-induced mitochondrial damage: a link between mitochondrial complex i activity and left ventricular dysfunction acute encephalopathy associated with influenza and other viral infections molecular structure and assembly of the tight junction identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury we are grateful to mayumi shiota for expert assistance. key: cord-292794-okh6i4l1 authors: wang, bin; yu, hai; yang, fu-ru; huang, meng; ma, ji-hong; tong, guang-zhi title: protective efficacy of a broadly cross-reactive swine influenza dna vaccine encoding m2e, cytotoxic t lymphocyte epitope and consensus h3 hemagglutinin date: 2012-06-27 journal: virol j doi: 10.1186/1743-422x-9-127 sha: doc_id: 292794 cord_uid: okh6i4l1 background: pigs have been implicated as mixing reservoir for the generation of new pandemic influenza strains, control of swine influenza has both veterinary and public health significance. unlike human influenza vaccines, strains used for commercially available swine influenza vaccines are not regularly replaced, making the vaccines provide limited protection against antigenically diverse viruses. it is therefore necessary to develop broadly protective swine influenza vaccines that are efficacious to both homologous and heterologous virus infections. in this study, two forms of dna vaccines were constructed, one was made by fusing m2e to consensus h3ha (mha), which represents the majority of the ha sequences of h3n2 swine influenza viruses. another was made by fusing m2e and a conserved ctl epitope (np147-155) to consensus h3ha (mnha). their protective efficacies against homologous and heterologous challenges were tested. results: balb/c mice were immunized twice by particle-mediated epidermal delivery (gene gun) with the two dna vaccines. it was shown that the two vaccines elicited substantial antibody responses, and mnha induced more significant t cell-mediated immune response than mha did. then two h3n2 strains representative of different evolutional and antigenic clusters were used to challenge the vaccine-immunized mice (homosubtypic challenge). results indicated that both of the dna vaccines prevented homosubtypic virus infections completely. the vaccines’ heterologous protective efficacies were further tested by challenging with a h1n1 swine influenza virus and a reassortant 2009 pandemic strain. it was found that mnha reduced the lung viral titers significantly in both challenge groups, histopathological observation showed obvious reduction of lung pathogenesis as compared to mha and control groups. conclusions: the combined utility of the consensus ha and the conserved m2e and ctl epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse model. this may provide a basis for the development of universal swine influenza vaccines. swine influenza virus (siv), member of genus influenza a virus of the family orthomyxoviridae, is a common and important causative pathogen involved in the porcine respiratory disease. mortality of siv-infected pigs is low, but morbidity may approach 100%. clinical signs of swine influenza include high fever, lethargy, anorexia, coughing, labored breathing and nasal discharge. synergistic or secondary infections with opportunistic organisms may increase the severity of clinical disease [1] . beyond the veterinary implications, influenza virus infections in pigs also present an important public health risk. since pigs express sialic acid receptors for both mammalian and avian strains of influenza viruses on their tracheal epithelial cells [2] , they could potentially serve as "mixing vessels" for the generation of new reassortant strains of influenza viruses that have pandemic capacity. the recently emerged 2009 pandemic h1n1 which resulted in over 18,449 deaths is an example (http://www.who.int/csr/don/2010_08_06/en/ index.html). genetic analyses revealed that this pandemic h1n1 influenza virus is a triple reassortment of multiple strains of viruses circulating in the north america and eurasia swine population [3, 4] . therefore, siv-infection control would be of benefit to both reduce the economic losses of swine industry and human health. vaccination is considered to be the most effective method to control sivs. currently, commercially available swine influenza vaccines are adjuvanted, wholevirus killed vaccines. although the vaccines reduce the severity of disease and the extent of virus shedding in pigs after challenge, they do not provide consistent protection from infection [5] . the vaccines function by targeting the surface glycoprotein hemagglutinin (ha), the most variable influenza virus antigen. protective efficacy depends on the antigenic match degree between vaccine and circulating strains. however, influenza viruses continuously evolve by increasing the mutations in epitopes (antigenic drift) or by reconstituting the genome with other strains (antigenic shift). the increased incidence of avian-like or human-like siv reassortants, which exhibit great genetic diversity and thus antigenic diversity with classical sivs, has been documented [6] [7] [8] , resulting in the "antigenic mismatch" between vaccine and the circulating strains. in addition, each of the dominant subtypes circulating in swine population worldwide, i.e. h1n1, h3n2 and h1n2, has developed multiple genetic clusters based on the phylogenetic analysis of ha genes [9] [10] [11] [12] . previous studies have demonstrated that some viruses in different clusters showed only limited cross-reactivity [10, 12] , suggesting that the genetic and antigenic heterogeneities within each subtype may reduce the vaccines' effectiveness [13] [14] [15] [16] . vaccines that induce broad protective immunity against diverse siv clusters and even subtypes, or limit the spread of the viruses between pigs and humans, especially for pandemic strains, are therefore needed. consensus has of different strains within one subtype were previously studied for eliciting cross-cluster protection [17] , but ha induces predominately a subtype-specific humoral immune response. in contrast, the conserved viral antigens, such as m2e and internal np, can generate heterosubtypic immunity protective against diverse virus strains and subtypes [18] [19] [20] [21] . unlike ha, the np-induced cell-mediated immune responses do not prevent infection, but reduce the severity of illness and accelerate the virus clearance. it is reasonable to propose that the combined utility of the consensus ha and conserved viral proteins will confer complete protection against homosubtypic and, at least partial, heterosubtypic challenge. in the present study, two types of swine influenza dna vaccines were constructed: (1) fusing matrix 2 ectodomain (m2e) to consensus ha of h3 subtype siv (mha, means m2e + ha) and, (2) fusing m2e and a conserved ctl epitope, np147-155, to consensus h3 ha (mnha, means m2e + np147-155 + ha). then their protective efficacy and broadness against divergent h3n2 and h1n1 siv challenges were tested in mice. the dna vaccines elicited m2e-and ha-specific antibodies to increase the expression level, the m2e, ctl epitope and consensus h3 ha gene was optimized for codon usage, rna structure and gc content. then the codonoptimized consensus ha, linked with m2e, was cloned into eukaryotic expression vector with or without np147-155 to generated two dna vaccines ( figure 1 ). after nucleotides sequencing, the dna vaccine plasmids were transfected into human embryonic kidney (hek) 293 t cells,the expression of each chimeric protein was confirmed by indirect immunofluorescence assay (ifa). ifa showed that both m2e and ha genes expressed in vitro (data not shown). then the two dna vaccines, as well as empty vector, were coated with gold particles and delivered into the skin with a gene gun. humoral immunity was analyzed by detecting the presence of antigen-specific antibodies. as can be seen in figure 2 , each of the constructs evoked a substantial ha-specific igg response after the booster injection, suggesting that the two vaccines were adequately delivered and expressed in mice. no significant difference in serum igg antibody levels were observed between mha and mnha group (p > 0.05), indicating that the addition of a 9-mer length ctl epitope did not influence the vaccine's antibody-inducing ability significantly. but the antibody titer of m2e was lower than that of ha (p < 0.001). this is not surprising, because m2e contains only 24 amino acids and it was reported that m2e is less immunogenic than ha [22] . hi assays were then performed on both homosubtypic and heterosubtypic virus to assess the ability of inducing relevant and cross-reactive functional antibodies. as detailed in table 1 , both of the vaccine immunization groups developed hi antibodies against homologous viruses, but induced very low levels of hi antibodies against heterologous viruses. the ability of the clt epitope-containing vaccine, mnha, to induce cellular immune response was determined by ifn-γ elispot assay. a synthesized peptide np147-155 was used for restimulating splenocytes isolated from 3 mice of each group. results showed that, after restimulation, the number of activated ifn-γ secreting cells from mice immunized with mnha was significantly higher than that from mice immunized with mha and empty vector (p < 0.001, figure 3 ). both mha and mnha vaccination conferred complete protection against homologous challenge, but mnha showed more significant cross-protection against heterologous challenge than mha did our previous study indicated that the wild type of swgd164 strain (h1n1) replicates poorly in mice (unpublished data). to make the virus be suitable for vaccine efficacy assessment in vivo, we constructed a recombinant virus (r164) by using reverse genetics. r164 contains ha and na from swgd164, and the internal proteins from pr8. balb/c mice were infected intranasally with two homologous strains belonging to different clusters within h3 subtype 21 days after the last immunization [9] . mice lungs were taken 3 days post infection for virus titration and histopathologic changes observation. results demonstrated that there was no detectable virus load in all the vaccine-immunized mice, while empty vector control group showed high lung viral titers ( figure 4 ). in addition, histopathologic observation showed no obvious histopathologic changes in vaccinated mice. in contrast, the empty vector control group exhibited histopathological damages including the dropout of mucous epithelium cells, interstitial edema, hyperemia, hemorrhage, and inflammatory cell infiltration ( figure 5 ). since m2e and np147-155 are conserved in different subtypes of influenza viruses, we hypothesized that the dna vaccine can confer partial protection against heterosubtypic challenge. to test this, the vaccines' protective ability was studied in mice challenged with h1n1 sivs, which are also circulating in swine population worldwide. the immunized mice were challenged with heterologous g11 and rpan09, respectively. lung viral titers determination and pathological analysis were performed as described above. results indicated that all the mice that had been vaccinated with mha had a detectable virus level, although showed a reduction in mean viral titers in both challenge groups compared with vector control, the reduction did not reach significance (p = 0.06 for rpan09 group and p = 0.67 for g11 group, figure 4 ). histopathological analysis exhibited corresponding results, i.e., mice of mha group had lung pathology as almost severe as vector inoculated group. in mnha group, although the mice also exhibited detectable gly-gly-gly-gly-ser-gly-gly-gly-gly-ser-gly-gly-gly-gly-ser linker. virus shedding, the mean viral titers had been significantly reduced in both rpan09-and g11-challenged groups compared to vector-immunized control (p < 0.001), showing average viral titer reduction of 3.4 and 2.1 logs, respectively ( figure 4 ). as expected, pathology damage of mice lung of mnha group was less pronounced than that of mha and vector control group, moderate histopathological lesions, mainly vascular congestion and various degrees of hemorrhage were observed ( figure 5 ). collectively, these findings indicate consensus ha in combination with m2e elicits cross-cluster immunity, and ctl response contributes to the control and clearance of infection and reduces pathogenesis. although the viruses are highly and continuously variable because of antigenic drift and shift, influenza is a vaccinepreventable disease. the conventional inactivated human influenza vaccines are updated annually with the world health organization-recommended h1n1, h3n2, and influenza b strains in order to antigenically match the viruses predicted to be the most likely to cause the next epidemic. unlike human influenza vaccines, strains used for swine influenza vaccines preparation are not regularly replaced, making the vaccines provide limited protection against antigenically diverse sivs. a universal vaccine designed based on the conserved viral proteins that confers broad protection is an attractive solution to counter the features of highly variable of not only human but also swine influenza viruses. many studies developed universal vaccines against human influenza, whereas broadly responsive swine influenza vaccines, to our knowledge, have not been reported. the virus envelope glycoprotein ha is the most abundant surface protein, antibodies against ha can provide protection by blocking virus attachment and entry. studies from several groups have demonstrated the antiviral efficacy of consensus ha as an "intra-subtype" universal vaccine. the study of chen et al. [17] showed that the consensus h5 ha-based dna vaccine elicited only moderate neutralization activities toward the h5n1 clade 2.1 and clade 2.3 viruses, and provided not complete, albeit significant, protection against clade 2.1 virus, suggesting that consensus ha alone may be not enough to induce complete protection against all strains within one subtype, much less heterosubtypic strains. however, ideally, universal vaccines should be capable of inducing protection against both homosubtypic and heterosubtypic challenges. to this end, we linked m2e and ctl-inducing epitope np147-155 to consensus h3 ha to construct a universal swine influenza vaccine. the reasons we chose m2e and np147-155 are: (1) both of them are highly conserved among different siv strains, np147-155 are even conserved among human, avian and swine figure 4 protection of mice from homologous and heterologous challenges. mice lung viral titers at day 3 after challenges were determined in eggs from an initial dilution of 1:10 in phosphate-buffered saline and expressed as eid 50 /ml. the limit of virus detection was 1.5log. influenza strains (table 2) , they were added in view of their ability to broaden the protective scope of the vaccine. (2) np147-155 induces a vigorous ctl response [23] . studies indicated that specific ctls provide some level of crossprotection against antigenically distinct viruses of not only the same subtype but also different subtypes [24, 25] . previously we have shown that swhlj1 and swgd164 had evolved into 2 independent lineages based on the phylogenetic analysis of the ha gene. to see whether the two strains were antigenically distinct, cross-hi assay was performed by using r164-specific anti-serum. it was found that the titer of cross-reacting hi antibodies (swhlj1-reacting) was significantly lower than that of r164-reacting hi antibodies (p < 0.001, data not shown), thus suggesting that they belong to different antigenic clusters. results of present study demontrated that both mha and mnha vaccination provided complete protection against these two viruses challenges, cross-cluster protections were therefore suggested. while mnha, but not mha, induces immune responses partially protect against heterologous influenza infection, suggesting the echanced t-cell mediated response is not required for homologous protection. results also further confirm that ctl responses play important roles for heterologous protection, meaning that strengthening ctl responses are promising ways for universal influenza vaccines development. it was established that the 2009 pandemic h1n1virus is pathogenic and is readily transmitted in pigs [26] [27] [28] [29] . although the new h1n1 virus is now considered to be post-pandemic, the possibility that it recombines with other influenza viruses in pigs then yielding a novel potential epidemic or pandemic strain still exists [30] . here in this study, the mice were partially protected from the reassortant 2009 pandemic virus challenge after the immunization of mnha. the vaccine immunization inhibited the pulmonary viral replication significantly in mice, thus could accelerate the virus clearance, and reduce the potential of transmission and the risk of recombination. although the heterologous protection elicited by the dna vaccine was not complete, this methodology has a large potential for improvements. the present study lays the foundation for universal swine influenza vaccine development, and call for further investigations in which the heterologous immune response should be further enhanced, such as the addition of molecular adjuvants [31, 32] and/or more copies of conserved viral protein encoding genes [33] , and the usage of dna-prime protein/virus-boost immunization schedule [34, 35] . a previous study conducted by heinen et al. constructed a dna vaccine containing m2e and full length of np genes, but ha encoding gene was not included. their data showed that the m2e-np dna vaccination produced enhancement of disease after challenge [36] . although the mechanism underlying which is unclear, we think the ha may play roles in this discrepancy between the results of heinen et al.'s and ours. another possible reason should be taken into consideration is that gene gun immunization induces more strong immune response that the conventional needle injection method, which was used in heinen et al.'s study [37] . it is necessary to study whether the dna vaccines we developed here will confer protection in pig models. also, it would be interesting to study how, if does, the ha protein influence the vaccines' efficacy. the combined utility of the consensus ha and the conserved m2e and ctl epitope can confer complete and partial protection against homologous and heterologous challenges, respectively, in mouse models. this may provide a promising strategy for universal swine influenza vaccine development. viruses, cell and mice medicine, south china agricultural university. r164, containing swgd164 surface glycoproteins and pr8 internal proteins, was prepared by using reverse genetics as described [38] . these viruses were propagated in allantoic cavities of 9 to 10-day old spf embryonated chicken eggs and stored at −80°c until use. fifty-percent embryo infectious dose (eid 50 ) titers were determined by serial titration in embryos and calculated by the method of reed and muench [39] . hek 293 t cells were maintained in dulbecco's modified eagle's medium (dmem, sigma) supplemented with 10% fetal bovine serum in humidified 5% co 2 atmosphere at 37°c. spf female balb/c mice (4-6 weeks of age) were purchased from shanghai slac laboratory animal co., ltd., and maintained with free access to sterile food and water. all animal studies were conducted in accordance with the ethical guidelines and were approved by the ethical committee of shanghai veterinary research institute, chinese academy of agricultural sciences. a total of 162 ha and 105 m2 sequences with full-length of h3n2 subtype siv were, respectively, downloaded from genbank database and aligned by megalign program supplemented in dnastar package (dnastar madison, wi). the consensus sequences were created based on the most common amino acid in each position of the alignment. after the consensus m2 sequence was generated, the first 24 amino acids were selected as m2e motif [40] . then codons of the consensus ha, as well as m2e and an immunodominant cytotoxic t lymphocyte (ctl) epitope of the np protein, np147-155 (tyqrtralv) [41] , were optimized for mammalian expression and synthesized by genscript corporation (nanjing, china). the (g4s)3linked chimeric orf encoding m2e, ctl epitope and consensus ha was pcr-amplified by introducing 5' sma i and 3' nhe i restriction sites for ligation into the pcaggs vector, under the control of the cytomegalovirus (cmv) enhancer and chicken β-actin promoter (designated as mnha). similarly, mha was designed and constructed, except that the ctl epitope was omitted from the nterminal end of the consensus ha ( figure 1 ). after the recombinant plasmids being identified by nucleotide sequencing, they were propagated in e. coli bacteria and purified using mega purification kit (qiagen, valencia, ca) for in vitro transfection as well as in vivo animal immunization. the final dna preparations were resuspended in nuclease-free water and stored at −20°c until further use. hek 293 t cells were seeded in 6-well plates and transfected at 80-90% confluence with 4 μg of mha, mnha or empty vector using lipofectamine 2000 transfection reagent (invitrogen), as recommended by the manufacturer. after 48 h, the transfected cells were scraped from the culturing plate, washed with pbs, then spotted onto a glass slide, air dried, and fixed with pre-chilled acetone. upon removal of the residual solvents from the slides, the cells were incubated with anti-m2e and -ha polyclonal antibodies for 1 h at 37°c. a secondary alexa fluor 568conjugated goat anti-mouse igg antibody was then used to detect the primary antibody. fluorescence images were scanned using an inverted microscope after the samples were mounted by glycerol. gene gun immunization was performed as previously described [42, 43] . mice received two nonoverlapping abdominal deliveries of antigen encoding plasmid-or empty vector-coated gold beads (1 μm) at the shaved skin with a 3-wk interval. with each shot, 2 μg of dna immobilized onto 0.5 mg gold particles was delivered at a helium discharge pressure of 450-500 psi with a helios gene gun (bio-rad). each test group contained 43 mice with experiments organized as follows: (1) 3 mice of each group were used to complete ifn-γ elispot assays on day 21 post the 2nd immunization; (2) the left 40 mice of each group were divided as 4 subgroups, each comprised of 10 mice, then intranasally challenged with 10 6 eid 50 of two h3n2 (swhlj1 and r164) and two h1n1 (g11 and rpan09) strains, respectively, at 21 days after the final immunization. serum samples were collected from orbital bleeds on day 14 post last immunization. m2e and ha antibody titers were measured using indirect elisa. elisa plates were coated overnight with synthetic m2e peptides or inactivated swhlj1 in 0.1 m carbonate buffer (ph 9.6) at 4°c and blocked with 5% non-fat milk for 2 h at 37°c. a series of two-fold dilution of sera (starting dilution 1:500 for ha and 1:100 for m2e, 100 μl/well) were incubated at 37°c for 1 h, followed by three washes with pbst. then hrp-conjugated goat anti-mouse igg (zhongshan biotechnology co., ltd beijing, china) and 3,3'-5,5'-tetramethyl benzidine (tmb) was used for m2-and ha-specific antibody detection and color development, respectively. the resulting optical density (od) at 450 nm was determined with a plate reader after the reaction was stopped with 2 m h 2 so 4 . hi assays were performed using 0.5% chicken red blood cells (rbcs) with 4 ha units of homologous (swhlj1 and r164) and heterologous (g11 and rpan09) virus and receptor-destroying enzyme (rde, sigma) treated serum, as previously described [44] . the reciprocal of highest dilution of serum that gave complete inhibition of hemagglutination was considered the hi titer. each assay was performed in triplicate. a titer of less than 10 (starting serum dilution) was assigned for serum samples that did not inhibit hemagglutination. the elispot assays were performed using mouse ifnγ elispot kits following methods recommended by the manufacturer (dakewe biotech, pr china). briefly, singlecell suspensions of freshly isolated spleen lymphocytes were seeded into the plates (10 6 /well) pre-coated with anti-ifn-γ monoclonal antibody. cells were stimulated with synthesized np147-155 peptide at a final concentration of 5 μg/ml in a 37°c humidified incubator with 5% co 2 . phytohemagglutinin (pha, 5 μg/ml) and medium alone were used as positive and negative controls, respectively. after a 36 h culture, plates were washed and incubated for 1 h with biotinylated anti-mouse ifn-γ antibody. streptavidin-horseradish peroxidase was then added, ifn-γ spots were developed with 3-amino-9 -ethylcarbazole (aec) and counted using an automated elispot reader. results were expressed as spot-forming cells (sfc) per million cells. on day 3 post-infection, mice from each group were sacrificed to collect lungs for virus titration and pathologic examination to determine the protective ability of dna vaccines. for virus titration, the whole lungs were homogenized in 1 ml of sterile phosphate-buffered saline (pbs) containing 0.1 mg/ml of streptomycin and 100 iu/ml of penicillin. the homogenates were diluted 10-fold serially after being centrifuged, then inoculated into embryonated chicken eggs. infection within individual eggs was confirmed using a standard hemagglutination assay, the viral titers were determined by the method of reed and muench. lung tissue histopathologic sections were made as described elsewhere [45] . briefly, the removed lungs were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. five micrometer sections were cut and stained with hematoxylin and eosin (h&e), and reviewed for histopathologic changes. 2006bad06a04/03), and the 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1918 pandemic influenza virus experimental inoculation of pigs with pandemic h1n1 2009 virus and hi cross-reactivity with contemporary swine influenza virus antisera novel reassortment of eurasian avian-like and pandemic/2009 influenza viruses in swine: infectious potential to humans coadministration of dna encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice coexpressed rig-i agonist enhances humoral immune response to influenza virus dna vaccine complete protection against a h5n2 avian influenza virus by a dna vaccine expressing a fusion protein of h1n1 ha and m2e immunization of pigs against influenza virus infection by dna vaccine priming followed by killed-virus vaccine boosting importance of a prime-boost dna/protein vaccination to protect chickens against low-pathogenic h7 avian influenza infection vaccination of pigs with a dna construct expressing an influenza virus m2-nucleoprotein fusion protein exacerbates disease after challenge with influenza a virus the relative immunogenicity of dna vaccines delivered by the intramuscular needle injection, electroporation and gene gun methods a dna transfection system for generation of influenza a virus from eight plasmids a simple method of estimating fifty per cent endpoints sequence comparison between the extracellular domain of m2 protein human and avian influenza a virus provides new information for bivalent influenza vaccine design enhanced recognition of a modified peptide antigen by cytotoxic t cells specific for influenza nucleoprotein distinct contributions of vaccine-induced immunoglobulin g1 (igg1) and igg2a antibodies to protective immunity against influenza selected strategies to augment polynucleotide immunization efficacy of nucleoprotein and haemagglutinin antigens expressed in fowlpox virus as vaccine for influenza in chickens pathogenesis of pandemic influenza a (h1n1) and triple-reassortant swine influenza a (h1) viruses in mice submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the authors would like to acknowledge drs. wen-bao qi and ming liao (college of veterinary medicine, south china agricultural university) for providing rpan09 virus. we also gratefully acknowledge dr. yan-jun zhou (shanghai veterinary research institute) for providing laboratory reagents. this study was supported by grants from china international sci & tech cooperation program (2010dfb33920), shanghai basic research program (10jc1417300), national scientific supporting program of china data of experimental and control groups were presented as the averages ± standard error (se) and evaluated by anova method, where statistically significant results were defined as having a p value of less than 0.05. the authors declare that they have no competing interests. key: cord-292709-4hn55wui authors: nor, mohd basri mat; richards, guy a.; mcgloughlin, steve; amin, pravin r. title: pneumonia in the tropics: report from the task force on tropical diseases by the world federation of societies of intensive and critical care medicine date: 2017-12-31 journal: journal of critical care doi: 10.1016/j.jcrc.2017.11.004 sha: doc_id: 292709 cord_uid: 4hn55wui abstract the aetiology of community acquired pneumonia varies according to the region in which it is acquired. this review discusses those causes of cap that occur in the tropics and might not be readily recognizable when transplanted to other sites. various forms of pneumonia including the viral causes such as influenza (seasonal and avian varieties), the coronaviruses and the hantavirus as well as bacterial causes, specifically the pneumonic form of yersinia pestis and melioidosis are discussed. in general, community acquired pneumonia (cap) is caused by pathogens that are common to all geographical areas; s. pneumoniae, viruses, chlamydia, mycoplasma, legionella and less commonly s. aureus and k. pneumoniae. however some organisms are endemic to specific regions and early recognition and awareness of these is critical to the diagnosis and to a favourable outcome. this is no less the case in residents of or travellers to tropical regions. this review discusses those that are most prevalent and which can cause potentially lethal infections. in adults, respiratory viruses account for 10% to 40% of cap and the most common of these are influenza, parainfluenza, adenovirus and respiratory syncytial viruses (rsv) [1, 2] . influenza viruses which are classified by their core proteins (i.e. a, b or c) and belong to the family orthomyxoviridae, cause predominantly respiratory disease in humans. influenza type a and b account for n 50% of viral pneumonia in adults whereas influenza c infections generally cause mild respiratory disease and are not thought to cause epidemics [3] . the close contact between humans and animals in tropical areas may enhance the genetic reassortment of influenza viruses which when disseminated into the human population may result in pandemics [4] . other important respiratory viruses in the tropics that can cause severe pneumonia are influenza a h1n1, avian influenza viruses (h5n1, h7n9), severe acute respiratory syndrome associated coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) [5, 6] . standard, droplet and contact precautions are recommended for these selected acute respiratory infections and whenever possible patients should be placed in airborne infection isolation rooms. influenza viruses have been associated with annual epidemics and intermittent pandemics throughout the world. despite the absence of a winter season in the tropics, consistent seasons of infection have nevertheless been observed. the composition of the antigenic surface glycoproteins of the influenza virus, hemagglutinin (h) and neuraminidase (n) are used for subtyping, resulting in names like h3n2 and h1n1. antigenic drift represents the minor changes of h and n side chains and is responsible for seasonal epidemics. influenza pandemics occur less frequently and these result from major changes in antigenic structure in the envelope glycoproteins (antigenic shift) resulting from reassortment of various viruses such as swine, equine and human varieties. contemporary geographic distributions show that east, south and southeast asia influence unduly the evolution of seasonal influenza a (h3n2), exporting most of the evolutionarily strains that ultimately spread globally. the obvious role of asia in h3n2's evolution has been ascribed to the seasonal nature of influenza in temperate climates [7] . seasonal influenza is an acute respiratory illness caused by influenza a or b viruses. given that these infections including the pandemic variety h1n1 now occur in all parts of the world they will not be discussed in any detail in this treatise. avian influenza viruses (e.g. h5n1 and h7n9) have emerged relatively recently and cause disease in humans and currently remain a potential threat, particularly in the southeast asia [5, 8] . the first association of avian influenza h5n1 with clinical respiratory disease was in 1997 in hong kong, as a human infection transmitted from birds. later h5n1 re-emerged in humans in 2003 as a highly pathogenic virus resulting from antigenic drift to which a larger number of species were vulnerable and conferring resistance to adamantine antivirals [9] . as yet, human to human transmission is rare and the vast majority of cases are related to contact with birds. travellers who have a history of recent exposure to birds in affected areas and who present with otherwise unexplained ards should be screened. h5n1 has been reported from 16 countries and is currently most prevalent in egypt [9] . as of august 2017, from 859 laboratory confirmed cases of influenza a h5n1, 453 (53%) patients have died [10] . 3.1.1.1. clinical. following exposure, the incubation period is seven days or less. clinical characteristics include fever, respiratory illness, pneumonia, diarrhoea and encephalopathy. laboratory abnormalities may include leukopenia, lymphopenia, thrombocytopenia and elevated serum aminotransferases. complications include multi-organ failure, pulmonary haemorrhage, pneumothorax and pancytopenia. radiographic findings include diffuse or patchy infiltrates and segmental or lobar consolidation. progression to respiratory failure is associated with diffuse bilateral ground-glass infiltrates. 3.1.1.2. diagnosis. a comprehensive travel and epidemiological history is critical in suspected cases. patients who meet clinical and epidemiological criteria should be tested for h5n1 avian influenza infection. diagnosis can be established by rrt-pcr or viral culture of respiratory specimens. serological testing is not helpful in the acute setting but useful for retrospective diagnosis [11] . 3.1.2.1. introduction. another avian influenza virus, h7n9, derived from reassortment of at least four avian influenza viruses, has caused severe pneumonia in some patients. it emerged in 2013 and originated from eastern china [12] . additional cases have been detected in mainland china, hong kong, macao, taiwan and malaysia. similar to h5n1 this virus occurs primarily in bird handlers or following recent exposure to live poultry or potentially contaminated environments. to date, there is no evidence of sustained human-to-human transmission. the incubation period has been estimated to be from 3 to 7 days, but can be as long as 10 days. presenting signs and symptoms may include fever, cough, dyspnoea, headache, myalgia and malaise. patients present with lrti which may progress rapidly to pneumonia and potentially acute respiratory failure, ards, septic shock, multi-organ failure, rhabdomyolysis and encephalopathy. severe illness and fatal outcome have been frequently observed in pregnant women, older persons and those with chronic illnesses [13, 14] . as of mid-august 2017, a total of 1557 laboratory-confirmed cases have been reported to who including 605 (39%) deaths [10] . real-time reverse-transcriptase polymerase reaction (rrt-pcr) for avian influenza a h7n9 is the preferred diagnostic test, since rapid antigen tests may be insensitive for novel or avian influenza strains. nasopharyngeal swabs or aspirates should be obtained for testing [11] . vaccination is the best method of protection during a pandemic however if not available or if a patient contracts influenza then antiviral therapy is indicated in certain circumstances. currently two classes of antiviral drugs are available for the treatment and prevention of influenza: the neuraminidase inhibitors (oseltamivir, zanamivir and peramivir) and the adamantines (amantadine and rimantadine) [15] . the adamantines block m2 protein channels and inhibit the uncoating of the influenza virus after it enters the host cells. they are only active against influenza a not b viruses and due to a marked increase in resistant isolates, many countries do not recommend that they be used except in combination with neuraminidase inhibitors in selected case. therefore, adamantines are not recommended for treatment of novel influenza a virus infections [16] . neuraminidase inhibitors are recommended to treat patients hospitalised with seasonal influenza, inclusive of h1n1, and avian influenza (h5n1 and h7n9) infections. in adults, the dose of oseltamivir is 75 mg orally twice daily; inhalational zanamivir is 10 mg (two inhalations) twice daily; and peramivir is 600 mg iv once daily. in patients with severe influenza, there are insufficient data to recommend the use of inhaled zanamivir or iv peramivir as empirical therapy. as such empirical treatment with oseltamivir should be started as soon as possible for all hospitalised cases associated with severe disease, and for confirmed and probable outpatient cases. early therapy (especially within 48 h of illness onset) can shorten the duration of the symptoms in high-risk patients and reduce secondary complications associated with influenza. the recommended duration of antiviral therapy is five days for uncomplicated disease. in severely ill hospitalised or immunocompromised patients, longer courses of treatment (e.g. 10 days) should be considered. in these circumstances, a higher dose of oseltamivir has been recommended by some experts (150 mg twice daily) in adults with normal renal function. however, oral oseltamivir has been reported to be adequately absorbed in critically ill patients and higher dosing may not provide additional clinical benefit. in obese patients, studies have demonstrated adequate exposure to oseltamivir carboxylate (active metabolite of oseltamivir) with the 75 mg twice daily dosing regimen. limited data suggest that oseltamivir administered by oro/naso-gastric tube is well absorbed in critically ill patients, including in those on continuous renal replacement therapy and ecmo [16] . for patients who cannot absorb or tolerate oral oseltamivir, the use of iv peramivir or investigational iv zanamivir should be considered. it is possible that novel influenza a viruses may become resistant to oseltamivir and peramivir during treatment but remain susceptible to zanamivir. in resistant infection, zanamivir is the preferred agent and can be delivered by inhalation or by the intravenous route. corticosteroid treatment is controversial with some reports showing benefit and others harm. in all cases of severe influenza whatever the type bacterial coinfection is possible and as such, appropriate antimicrobial treatment directed toward those organisms causing bacterial acute community acquired pneumonia, and mechanical ventilation as required can reduce the mortality rate. extracorporeal membrane oxygenation (ecmo) has been utilised in some patients with ards and one study showed a possible mortality benefit in patients who were referred to a specialised ecmo centre [17] . ongoing monitoring for antiviral resistance among avian influenza a viruses is crucial. some evidence of antiviral resistance has been reported in highly pathogenic avian influenza (hpai) "asian h5n1 viruses" and "asian h7n9 viruses". future treatment strategies may make use of new generation broadly reactive monoclonal antibodies. in addition, a viral rna polymerase inhibitor, favipiravir is registered for the treatment of pandemic influenza in japan [3] . potentially life-threatening coronavirus infections are caused by the severe acute respiratory syndrome associated coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov) [6] . cases of sars were first noted in guangdong province, china in november 2002. the illness spread to 30 countries in north america, south america, europe and asia before the outbreak was contained. sars-cov is a novel coronavirus that jumped the species barrier from civet to man. based upon clusters of cases in hong kong and canada, sars-cov is transmitted by means of close person-to-person contact via droplets or fomites. it has been suggested that airborne and faecal-oral transmissions is also possible [18] . the incubation period is two to seven days and approximately 95% will develop symptoms by 10 days. this is followed by an influenza like (ili) prodrome (stage 1) of high fever (n 38°c), malaise, headache, myalgia and chills without upper respiratory tract symptoms. about 10% to 20% of patients have diarrhoea. the prodrome may last for three to seven days after which involvement of the lrt (stage 2) occurs and this begins with a non-productive cough and dyspnoea. this may progress to acute hypoxaemia with radiologic progression to pneumonia. about 20% of patients progress to stage 3 and develop features of ards that may require mechanical ventilation. the chest radiograph shows diffuse interstitial infiltrates characteristics of ards. risk factors for poor outcomes include older age, underlying comorbidities such as diabetes, atypical symptoms and elevated lactate dehydrogenase on admission [18, 19] . the diagnosis is based on clinical, epidemiological and laboratory criteria and case definitions have been developed by the centers for disease control and prevention. the reverse transcriptase polymerase chain reaction (rt-pcr) and serum antibodies as measured by enzyme-linked immunosorbent assay (elisa) testing, are two reliable tests for diagnosis. however serologic testing during the acute illness has limited value since antibodies typically develop several weeks into the illness. there is no specific treatment recommended except for supportive care. the benefit or otherwise of antiviral drugs or glucocorticoids has not been established for treatment of sars [20] . mers is a newly recognized highly lethal respiratory illness caused by mers-cov. mers-cov is a zoonotic virus and studies have shown that humans are infected through contact with infected dromedary camels or infected people. the disease was first reported in saudi arabia in june 2012 [21] . so far, all cases have been linked through travel to, or residence in, countries in and near the arabian peninsula. outside the arabian peninsula, the largest known outbreak of mers occurred in the republic of korea in 2015 which was also linked to a traveller returning from the arabian peninsula. a total of 186 confirmed patients with mers-cov infection across 16 hospitals were identified. eightytwo (44.1%) of the cases were patients exposed in hospitals, 61 (32.8%) were caregivers, and 25 (13.4%) were hospital staff [22] . a total of 83.2% of the cases were linked to five super-spreaders, all of whom presented with pneumonia and were in contact with hundreds of people. since 2012, although 27 countries have reported cases of mers-cov, 80% have been reported by saudi arabia. mers-cov can infect anyone and patients have ranged in age from b1 to 99 years old. the incubation period for mers is usually about 5 to 6 days, but can range from 2 to 14 days. most patients present with a severe acute respiratory illness manifested by shortness of breath but preceded by fever, cough, myalgia and arthralgia. rapid progression to pneumonia may occur within the first week, often requiring mechanical ventilation and other organ support such as renal dialysis [23] . patients in the korean outbreak had fever and chills as the most common symptoms but gastrointestinal symptoms including diarrhoea and nausea/vomiting have also been reported. as of august 2017, 35% of laboratory-confirmed mers-cov cases reported to who have died. studies have shown that older age and pre-existing comorbidities are important risk factors for death [23, 24] . those with mild symptoms (such as cold-like symptoms) or no symptoms at all usually recover. case definitions have been developed by who, the centers for disease control and prevention (usa) and the ministry of health of saudi arabia. confirmation of the diagnosis is by rrt-pcr performed on respiratory secretions. currently, there is no vaccine to prevent mers-cov infection and no proven antiviral therapy. management includes preventive daily hygiene measures and travel precautions. thereafter symptomatic and supportive therapy is required [25] . the hantavirus is a member of the bunyaviridae family and the genus includes n 50 different viruses that manifest with a wide spectrum of clinical illnesses [26] . the pathogenesis of hantavirus is related to the targeting of vascular endothelial cells, alveolar macrophages and follicular dendritic cells, as well as the renal tubular epithelium [27] . the induction of vascular or capillary leak is a key component of the pathogenicity [28] . transmission to humans is via contact with, or inhalation of aerosolised urine, saliva or faeces of the rodent host (family muridae). as it is predominately a rural disease, risk factors include farming, land development and camping, however due to the method of inoculation, it is usually acquired indoors [27] . person to person transmission has been documented previously in some subtypes so respiratory precautions are recommended for health care workers. it is estimated that over 30,000 cases of hantavirus occur globally each year, with the majority in asia [29] . it causes two major clinical syndromes namely hantavirus pulmonary syndrome (hps) and haemorrhagic fever with renal syndrome (hfrs) with the former occurring predominately in the americas [26] . reports have indicated that there is an increasing incidence of hps in south america [30] . the case definition of the hps as per the centers for disease control and prevention (cdc) is: 'a febrile illness with bilateral diffuse interstitial edema that may resemble the acute respiratory distress syndrome, with respiratory compromise requiring supplemental oxygen developing within 72 hours of hospitalisation, in a previously healthy person' [31] . the incubation period is two to three weeks followed by a number of phases that include [29] ; a prodrome with nonspecific viral symptoms however also with thrombocytopenia, lasting for 1 to 5 days; and the pulmonary phase in which symptoms of respiratory failure predominate progressing extremely rapidly (over 8 to 24 h), to shock, coagulopathy, pulmonary oedema, bronchorrhea, arrhythmia and death. lastly in the recovery phase patients often have a period of significant diuresis as endothelial function recovers and fluid redistribution occurs, with urine outputs of 300 to 500 ml per hour for 24 h [27] ; thereafter there is a convalescent phase which can be prolonged over weeks to months [28] . hantavirus is diagnosed by serology, with hantavirus igm positive by the time symptoms are present. if an appropriate history of exposure is obtained, the presence of thrombocytopenia, hemoconcentration, n10% immunoblasts and a lack of toxic granulation has a high sensitivity and specificity for hantavirus [28] . chest radiograph abnormalities consistent with interstitial oedema are usually present on admission which worsen as respiratory function declines [27] . advanced respiratory and haemodynamic monitoring and support, may be necessary, inclusive of mechanical ventilation and extracorporeal membrane oxygenation (ecmo) which is often a challenge in lics [28] . it is recommended that intravenous fluids are minimised with the early institution of vasopressor and inotropic support as aggressive fluid resuscitation has been shown to increase the risk of respiratory failure [28] . unfortunately, no specific antiviral therapy has been demonstrated to be effective. burkholderia pseudomallei is a gram negative pathogen endemic to south east asia, northern australia, india, south china and taiwan and cases have been documented in brazil and elsewhere in south america, papua new guinea, fiji, and new caledonia [32] . despite being the most common cause of fatal community acquired bacteremic pneumonia in north east thailand and darwin, it is seldom recognized when imported to areas where it is not endemic [33] . the prevalence varies in affected regions and may be related to climactic conditions, being particularly influenced by rainfall, having reached record rates (50.2 cases per 100,000 people) after heavier than usual precipitation in 2009-2010 in northern australia [33] . global mortality estimates are extremely high, similar to deaths from measles and much higher than leptospirosis and dengue, infections that receive considerably more attention. in fact the mortality may be even higher, as diagnosis is low in endemic areas primarily due to the low resource settings in which it occurs, as laboratory facilities are generally poor to non-existent [34] . infection occurs from inoculation from contaminated soil or water, through abrasions, ingestion or inhalation with subsequent haematogenous spread. those with chronic diseases such as diabetes, renal failure and cystic fibrosis or those on immunosuppressive agents appear to be more susceptible [35, 36] . most of the infections in endemic areas are asymptomatic with a significant number having antibodies but no history of disease [37] . however at least 50% of cases present with pneumonia of varying severity ranging from a nodular infiltrate with septic shock which has a mortality of up to 90%, to a lobar consolidation presenting with less florid features. the former often has little in the way of respiratory symptoms, presenting with fever, hypotension and organ dysfunction [35] . the more indolent form may mimic tuberculosis radiologically, most commonly involving the upper lobes, however it may also occur in the lower lobes in which case pleural effusions and empyema may occur [32, 37] . pulmonary and diffuse metastatic abscesses (involving spleen, kidney, prostate and liver), osteomyelitis, and arthritis may also occur [35] . the diagnosis is dependent on obtaining a positive culture. melioidosis must be considered in the differential of all febrile patients that have visited endemic regions, as antibiotics used routinely for community acquired pneumonia are not effective, and inappropriate therapy in severe disease increases mortality. although b. pseudomallei grows readily, laboratories may misidentify the organism as a pseudomonas which may lead to confusion and potentially incorrect therapy prior to the availability of the antibiogram. certain specific culture media may enhance growth however these are not always available outside of endemic areas [38] . serological tests are available and a positive indirect hemagglutination test (iha) or enzyme-linked immunosorbent assay (elisa) in a traveller may raise the suspicion of melioidosis but definitive diagnosis is based on culture positivity [33, 39] . treatment for severe disease is administered as an initial intensive intravenous phase with combinations of ceftazidime or meropenem/ imipenem with cotrimoxazole for at least 10 days as recommended in thailand or 14 days as recommended in australia. thereafter an oral eradication phase consisting of cotrimoxazole or amoxicillin/ clavulanate is administered for 12-20 weeks (thailand) or 3 months (australia) [40] . yersinia pestis is a gram negative coccobacillus and is notorious historically as the cause of 'the black plague'. y. pestis is likely to have caused three major pandemics which led to significant loss of life. in the first in the 6th century ad it is thought that in some regions, between 50 and 60% of the population may have perished [41] . according to the who, currently there are high rates of plague in the democratic republic of congo, madagascar and peru. the disease has three clinical syndromes: bubonic, pneumonic or septicaemic [41] . pneumonic plague leads to purulent, exudative bronchopneumonia that is usually fatal if untreated [42] . the yersinioses are zoonotic infections in which humans are accidental hosts via contact with an infected animal, usually with the flea as the vector. the infection can be acquired via a bite from a flea, scratches or bites from infected animals or rodents or inhalation of infected respiratory secretions. the infecting organisms are carried via lymphatics to the local lymph node which and this is followed by a severe inflammatory reaction [43] . there were 3248 cases of plague reported to the who between 2010 and 2015 with 584 deaths with 95% of cases from africa. areas affected by plague include north america, the former soviet union, africa, asia and south america [44] . the majority of cases are of the bubonic form (80 to 95%) which manifests with fever, chills, malaise, dizziness and associated lymphadenitis causing intense pain and swelling in a lymph node region. if untreated, dissemination occurs to the lungs or meninges in 50% of cases causing pneumonia or meningitis. septicaemic plague can occur without the bubonic illness in 10 to 20% of cases and is a severe illness that can lead to profound multi-organ failure [43] . pneumonic plague can either be primary or secondary. the former develops from aerosol exposure to y. pestis, with symptoms developing 1 to 6 days after inoculation [45] . this can be caused by person to person spread via exposure to respiratory droplets and is the cause of outbreaks in extended families. secondary pneumonic plague is due to dissemination of y. pestis into the lungs during either the bubonic or septicaemic phases and occurs in approximately 10% of patients [41] . recent data suggested that pneumonic forms are increasing in incidence accounting for 23% of global plague cases [44] . a high index of suspicion is important in order to make the diagnosis, which is then confirmed by serology or culture or by more recently developed rapid diagnostic tests [46, 47] . a high percentage of blood cultures are positive in septicaemic plague and gram stain and culture of sputum or lymph node aspirate is the usual means of diagnosis demonstrating small gram negative coccobacilli [48] . the mortality rate of plague if untreated is between 50 and 90% with pneumonic plague having a mortality of 100% if untreated and 50% if treated. previously streptomycin was the preferred antibiotic however in most cases gentamicin is now preferred. alternative agents include doxycycline and tetracycline. fluoroquinolones are the preferred agents for post-exposure prophylaxis [45] . cap is usually managed according to national guidelines that are directed toward those organisms seen most frequently in each region. the conditions described above may not be seen frequently if at all in other parts of the world and as such may not be treated appropriately and may also result in considerable spread. all intensivists should be aware of these conditions and make sure that a good travel history is obtained in each and every case. management of infections in critically ill returning travellers in the intensive care unit-i: considerations on infection control and transmission of resistance viral infection in community-acquired pneumonia: a systematic review and meta-analysis influenza-a model of an emerging virus disease community acquired pneumonia in the tropics community-acquired pneumonia explaining the geographical origins of seasonal influenza a (h3n2) community-acquired pneumonia cumulative number of confirmed human cases of avian influenza a(h5n1) reported to who monthly risk assessment summary. influenza at the human animal interface interim guidance for specimen collection, processing, and testing for patients with suspected infection with novel influenza a viruses associated with severe disease in humans human infection with a novel avianorigin influenza a (h7n9) virus human infection with avian influenza a h7n9 virus: an assessment of clinical severity clinical findings in 111 cases of influenza a (h7n9) virus infection optimizing antiviral therapy for influenza: understanding the evidence interim guidance on the use of antiviral medications for treatment of human infections with novel influenza a viruses associated with severe human disease extracorporeal membrane oxygenation for ards in adults severe acute respiratory syndrome severe acute respiratory syndrome sars: systematic review of treatment effects centres for disease control and prevention. centres for disease control and prevention. middle east respiratory syndrome (mers) middle east respiratory syndrome coronavirus outbreak in the republic of korea epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study mortality risk factors for middle east respiratory syndrome outbreak, south korea middle east respiratory syndrome hantavirus infection: a global zoonotic challenge hantavirus pulmonary syndrome hantavirus cardiopulmonary syndrome hantavirus infections for the clinician: from case presentation to diagnosis and treatment hantaviruses and cardiopulmonary syndrome in south america case definitions for infectious conditions under public health surveillance centers for disease control and prevention burkholderia pseudomallei and burkholderia mallei: melioidosis and glanders melioidosis: a review predicted global distribution of burkholderia pseudomallei and burden of melioidosis the epidemiology and clinical spectrum of melioidosis: 540 cases from the 20 year darwin prospective study management of infections in critically ill returning travellers in the intensive care unit-ii: clinical syndromes and special considerations in immunocompromised patients development of antibodies to burkholderia pseudomallei during childhood in melioidosis-endemic northeast thailand comparison of ashdown's medium, burkholderia cepacia medium, and burkholderia pseudomallei selective agar for clinical isolation of burkholderia pseudomallei workshop on treatment of and postexposure prophylaxis for burkholderia pseudomallei and b. mallei infection pneumonic plague: the darker side of yersinia pestis early emergence of yersinia pestis as a severe respiratory pathogen clinical problem-solving. sick as a dog development and testing of a rapid diagnostic test for bubonic and pneumonic plague fast and simple detection of yersinia pestis applicable to field investigation of plague foci practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of america none. none declared. key: cord-288487-hs3wfffs authors: lambert, stephen b; allen, kelly m; carter, robert c; nolan, terence m title: the cost of community-managed viral respiratory illnesses in a cohort of healthy preschool-aged children date: 2008-01-24 journal: respir res doi: 10.1186/1465-9921-9-11 sha: doc_id: 288487 cord_uid: hs3wfffs background: acute respiratory illnesses (aris) during childhood are often caused by respiratory viruses, result in significant morbidity, and have associated costs for families and society. despite their ubiquity, there is a lack of interdisciplinary epidemiologic and economic research that has collected primary impact data, particularly associated with indirect costs, from families during aris in children. methods: we conducted a 12-month cohort study in 234 preschool children with impact diary recording and pcr testing of nose-throat swabs for viruses during an ari. we used applied values to estimate a virus-specific mean cost of aris. results: impact diaries were available for 72% (523/725) of community-managed illnesses between january 2003 and january 2004. the mean cost of aris was au$309 (95% confidence interval $263 to $354). influenza illnesses had a mean cost of $904, compared with rsv, $304, the next most expensive single-virus illness, although confidence intervals overlapped. mean carer time away from usual activity per day was two hours for influenza aris and between 30 and 45 minutes for all other ari categories. conclusion: from a societal perspective, community-managed aris are a significant cost burden on families and society. the point estimate of the mean cost of community-managed influenza illnesses in healthy preschool aged children is three times greater than those illnesses caused by rsv and other respiratory viruses. indirect costs, particularly carer time away from usual activity, are the key cost drivers for aris in children. the use of parent-collected specimens may enhance ari surveillance and reduce any potential hawthorne effect caused by compliance with study procedures. these findings reinforce the need for further integrated epidemiologic and economic research of aris in children to allow for comprehensive cost-effectiveness assessments of preventive and therapeutic options. respiratory virus infections are a major cause of morbidity and healthcare usage in children, resulting in substantial costs for families and society [1] [2] [3] [4] [5] . given their ubiquity, there has been surprisingly little research examining the costs associated with childhood respiratory infections that has involved collecting primary data from families. even for influenza, the most studied of all respiratory viruses, cost-of-illness and vaccine cost-effectiveness evaluations in children have tended to rely on assumptions or use retrospectively collected estimates, often from surveys, for resource utilisation, such as carer time away from work in seeking healthcare or caring for an ill child [6] [7] [8] [9] . there are three pieces of evidence required by those developing health policy in assessing whether to recommend or implement a publicly-funded vaccination program, or any intervention, against respiratory viruses: epidemiology of the targeted illness, the efficacy of the intervention, and the cost-effectiveness of the intervention [10] . all interventions to prevent or treat infections will be associated with a cost of implementation, but cost-effectiveness is determined not only by the cost of the intervention, but also by costs arising from the illness. getting these data for respiratory viruses, particularly information on indirect costs incurred by families, requires a conjunction of epidemiologic and economic research [11] . the prospect of new and improved influenza vaccines [12] , the hope of new vaccines against other respiratory viruses [13] , development of novel therapeutic possibilities [14] , and the possible use of nonpharmaceutical interventions to contain virus transmission [15] [16] [17] [18] all underline the need to more critically weigh the costs and benefits of prevention and treatment for common respiratory tract viruses. we present here findings from a community-based cohort study using parent-collected specimens for etiologic assignment and diary recording of impact data. these data have been used to calculate virus-specific costs of illness from a societal perspective, including often neglected indirect costs. the study cohort and acute respiratory illness surveillance details of recruitment, composition, and maintenance of the dynamic study cohort have been published elsewhere [19] . ethics approval for the study was given by the royal children's hospital ethics in human research committee, melbourne, and written informed consent was obtained from parents before participation. this dynamic cohort consisted of one healthy child less than five years of age at time of recruitment from each study family. children involved in this study were recruited from a number of sources. in victoria, australia, maternal and child health nurses (mchns) provide support to families during the early childhood years, particularly on issues to do with general health and vaccination. based on a model used by our group for community vaccine studies [20] , mchns from 26 local councils assisted with recruitment by providing study information to parents of eligible children. advertising material for the study was placed in child care and playgroup centers and, because of proximity, we also used bulletin boards and staff e-mail lists at the royal children's and the royal women's hospitals in melbourne. details about the study child and household demographics were collected at an enrolment home visit, including annual gross household income collected in 2003/2004 australian dollar values (aud$). income was separated into four bands, roughly dividing the study households into quartiles: band 1, less than $52,000 (24% of study households); band 2, $52,000 to $77,999 (28%); band 3, $78,000 to $103,999 (23%); and band 4, $104,000 or greater (25%). the approximate proportions for australian households during the same period were: band 1, 54%, band 2, 20%, band 3, 13%, and band 4, 13% [21] . parents undertook daily respiratory symptom surveillance of the study child using a diary card and collected a combined nose-throat swab (nts) and completed a summary impact diary when the child had an acute respiratory illness (ari). for this study we used a sensitive ari definition that had previously been used in an influenza vaccine efficacy study [22] and our pilot study [23, 24] . symptoms were classified as category a (fever, wheezing, shortness of breath, pulmonary congestion or moist cough, pneumonia, or ear infection) and category b (runny nose or nasal congestion, sore throat, cough, muscle aches, chills, headache, irritability, decreased activity or lethargy or weakness, or vomiting). an ari of interest required one category a or two category b symptoms on a single day [22] . other than pneumonia, which we asked parents to record only if supported by a health care professional's diagnosis, no illness or symptom details, including a report of otitis media, were validated by study staff or health care professionals. a new ari could not commence unless there were three symptom free days since the end of the previous ari. this meant an ari could contain no more than two consecutive symptom-free days. study families were asked to continue normal healthcare seeking behaviour and treatments, and were not alerted about the start of the influenza season or asked to alter surveillance during the winter season. pre-stamped envelopes were provided and families were asked to return all completed study documents (daily symptom diary, impact diaries) at the end of each month. ari duration was calculated using symptom diary data and aris were classified by study staff as being simple (no fever or otitis media recorded), or occurring with fever, otitis media, or with both fever and otitis media [22] [23] [24] . the nts was couriered to the victorian infectious diseases reference laboratory (vidrl) where it was tested for a number of common respiratory viruses using a polymerase chain reaction (pcr) method for adenoviruses and reverse transcription (rt) pcr for rna viruses: influenza a virus, influenza b virus, respiratory syncytial virus (rsv), parainfluenza viruses i, ii, and iii (pivs), and picornaviruses [19] . a letter outlining these test results was sent to parents when these details became available. at completion of the study all specimens were transported to the queensland paediatric infectious diseases (qpid) laboratory where they were tested for human metapneumovirus (hmpv) and human coronavirus nl63 (hcov-nl63) using rt-pcr [19] . a summary impact diary was used to collect details of resources used during the study child's ari and was based on an impact diary used in a pilot study [23, 24] , with some simplification. the units of resource use requested were: ▪ health care visits: number and timing of primary care (general practice) visits, hospital presentations and admissions, and visits to other providers (such as naturopaths, homeopaths); ▪ use of prescribed antibiotics; ▪ laboratory tests performed to investigate the illness; ▪ carer time consumed during the illness seeking health care; and ▪ excess carer time during the illness spent caring for the ill child. we did not collect information about some items that were shown not to be major cost drivers in the pilot study: non-antibiotic prescription medication, over-the-counter and other medication, paid childcare for other children whilst normal carers were spending time caring for the ill study subject, and travel costs seeking health care. the average total cost for these items in the pilot study [23] was aud$16 per ari. time values were captured in hours and minutes. parents were not given instructions about when or how frequently they should capture time data during an ari. for both carer time spent seeking healthcare and excess time spent caring for an ill child, time was recorded as a total value for the ari in two categories: time away from work and time away from usual, non-work activities. all costs were incurred over a 380 day period between 17 january 2003 and 31 january 2004. costs are reported in this manuscript using australian dollar values, with 2003 taken as the reference year for reporting unit prices. the mean exchange rates for major currencies during the study were: united kingdom (uk) pound £1 = aud$2.49, euro €1 = aud$1.73, and united states (us) $1 = aud$1.50 [25] . discounting costs for time preference is not routinely considered for periods of time less than 12 months, and as this study period barely exceeds this time frame, no costs have been discounted. details of the source and value for all costs are provided ( table 1) . applied costs were retrieved, where possible, from published sources, and where no standard published cost was available we used costs derived from the pilot study. resource costs were allocated as being borne by either the 'patient and family' sector, the 'healthcare' sector, or the 'employer' of absent staff. the proportions of time away from work seeking healthcare or time away from work caring for an ill child that were incurred by either the patient and family sector or met by an employer were not collected, and these values have been derived from the same proportions in the pilot study, based on 202 illnesses (table 1 ) [23] . we applied a sex-weighted hourly wage rate derived from the australian bureau of statistics average weekly full-time adult total earnings for all reported times [26] . we calculated mean costs (total and by categories) with 95% confidence intervals (95% ci) and median costs with interquartile ranges for aris in study children. data were analysed using stata 9.2 for windows (statacorp, texas, usa). there were 234 children, one from each study family, progressively enrolled in the study and we identified 730 aris in 56,397 child-days of follow-up [19] . of these, 487 aris (67%) had at least one specimen and an impact diary available, 41 (6%) had an impact diary returned but no specimen, 56 (8%) had at least one specimen returned but no impact diary available, and 146 (20%) had neither a specimen nor impact diary returned. children aged between one and two-years of age contributed the most person-time to the study (28% of all child-days) and had the highest acute respiratory illness (ari) incidence rate (0.56 aris per child-month). contribution by males and females was equivalent, and children who attended some form of out-of-home care were responsible for 67% of all person-time [19] . the 523 illnesses with a diary returned that did not involve a hospital admission had a total cost of $161,454 (table 2) , and mean cost of $309 (95% ci $263 to $354). as our particular interest is in the cost of communitymanaged aris, that is, those illnesses that do not require the mean and median costs by virus identification, including co-identification and specimen availability, are provided ( table 2 ). the differences between the mean values and the median values demonstrate the right-skewed nature of these data, similar to other health-related costs [27] . whilst confidence intervals overlap, the point estimate of the mean cost of an influenza a ari, $904, is three times higher than the next most expensive single virus ari: rsv $304. of the 51 aris where more than one virus was identified, influenza a virus was present in four: two illnesses with co-identification with a picornavirus alone, one illness with hcov-nl63 alone, and one illness with a picornavirus and piv. these four illnesses had a mean cost of $499. there were no illnesses where influenza b virus was identified. three children had received influenza vaccine in the year prior to the study and none had an influenza-positive ari. as the difference in mean cost between the most expensive (rsv: $304) and least expensive (hmpv: $219) noninfluenza single virus ari falls within a comparatively narrow band ($85) we collapsed these data into a single category for further comparisons ( there was little difference in the mean duration of influenza a illnesses and other single virus illnesses, but coidentifications were 2.2 and 3.6 days longer than each of these respectively ( table 4 ). the mean delay between illness onset and a result letter being sent was shortest in influenza illnesses at 6.3 days (table 4 ). in this study we present the costs associated with community-managed respiratory viral infections in healthy preschool aged children. these costs are based on the direct recording of impact information captured by parents when the study child was unwell. the study has a unique combination of features including a sensitive definition for ari, parent-collected specimens, laboratory testing for respiratory viruses using sensitive molecular methods, and, based on findings from our pilot study, comprehensive collection of costs, including the previously neglected indirect cost, time away from a usual, non-work activity. we found, from a societal perspective, the point estimate for the mean cost of all aris ($309; 95% ci $263 to $354) was not dissimilar to the mean value we calculated from the pilot study ($241; 95% ci $191 to $291) [23] using the same ari definition and a slightly modified impact diary. the use of pcr testing for diagnosis on collected specimens allowed us to assign impact and costs to specific viral agents. for all but influenza a illnesses, the cost of community-managed aris in healthy preschool-aged children fell within a relatively narrow $85 range. despite overlapping confidence intervals, the finding of most note in this study was the dramatically higher point estimate of the mean cost of influenza a aris, being three times higher than illnesses caused by rsv and the other common respiratory viral infections of childhood. the presence of fever and/or otitis media generally increased the mean cost of illness; but despite having a high prevalence of fever, a longer mean duration, and higher primary care usage [19] , adenoviral infections, for example, did not have the cost burden of influenza infections. this highlights the pivotal contribution of excess carer time away from usual non-work activity to total costs, making it the key cost driver for all aris in children and differentially amplifying the total costs of influenza illnesses. whilst the confidence intervals for mean cost of influenza a aris and other single virus aris overlap, due to the relatively small number of influenza illnesses available for costing, we believe it is unlikely that chance could account for such an extreme difference. the availability of preventive vaccines and specific therapeutic options makes influenza the most studied of respiratory viruses in all age groups; no other virus is more predictably disruptive year-on-year than annual interpandemic influenza [2] [3] [4] [5] . studies conducted in the second half of last century [28] [29] [30] [31] and recent observation [32, 33] and intervention [34] [35] [36] ideally further studies in other countries should be conducted to allow for an examination of how impact and cost data vary with the nature of the healthcare system, local virus epidemiology, and other societal factors, including household structure. despite lower mean costs than influenza illnesses and the lack of population-based prevention options, the importance of working towards the prevention of other respira-tory viral infections is obvious. picornavirus aris, though typically milder and more difficult to be certain of a causal association with illness [37, 38] , were associated with the highest overall costs of any viral group totalling over $50,000 or one-third of all costs, for the 12-month study period. in the absence of specific vaccines and therapies for other viruses, the application of nonpharmaceutical interventions at a population level, such as improved hand and respiratory hygiene, may have an important place in reducing illness due to respiratory viruses [16] . our findings reinforce the importance of virus testing in such studies to accurately estimate epidemiology and costs [11] . these data add to accumulating evidence that laboratory confirmation of influenza, in particular, is required, rather than less specific influenza-like illness (ili) definitions or hospital coding. other recent studies have found laboratory-confirmed influenza hospitalizations were two to four times more costly [39] [40] [41] than shown in previous studies using coding-based estimates [6, [42] [43] [44] . when ili definitions or coding are used, rather than laboratory confirmation, a lack of specificity results in influenza illnesses being mixed with other agents, thereby considerably diluting cost differences [45, 46] . a direct comparison of parent-collected nts specimens with collection of a more invasive specimen, such as a nasopharyngeal aspirate, by a healthcare worker at the time of an ari was beyond the scope of this study. any reduction in sensitivity caused by the type of specimen used is likely to minor: our finding that 74% of all specimens collected from children in this study were positive for at least one virus is within the range of values from recent home visit studies which also used pcr for diagnosis and nasopharyngeal aspirates (69%) [47] or nasal lavages (83%) [48] . there are clearly some issues about the cost of illnesses caused by respiratory viruses in children unresolved by our study, and some issues that need to be considered before interpretation. despite being a relatively large cohort the number of illnesses on which to make costing estimates for some virus types is quite small. further community-based estimates are required to not only confirm our findings but to improve precision around point estimates. compared with the australian population, households with lower incomes were under-represented in our study sample, and, despite overlapping confidence intervals around income band point estimates of mean costs, this may have lead to an overestimation of total costs. however, this may be balanced somewhat by the over-representation of households from the top income band which had a relatively lower mean ari cost ($272). this pattern of household income distribution was similar to that found in the pilot study [23] . for this study we sought to make our study sample more representative of the general community by focusing our recruitment efforts in local council areas with a higher proportion of lower income households. we have no empiric data available that would allow us to quantify the effect of any potential bias resulting from this skewed sample. other recent burden studies do not report similar household level income data to allow for comparison [49] [50] [51] . it may be the case that lower income households are under-represented as they do not have the spare capacity required, in time or other resources, to allow for study involvement. we received impact diaries for just over 70% of all aris identified by daily symptom surveillance. aris without a diary were more likely to be shorter and without fever or otitis media; any information bias resulting from this would likely be in the direction of inflating mean illness costs. our study only captured information from a single season with higher than normal influenza activity with h3n2 influenza a (drifted strain subtype a/fujian/411/ 2002-like) being the predominant circulating type [52] . variations in incidence and severity year-by-year for all respiratory viruses make it difficult to directly translate our findings to other years. we believe documenting all time spent on caring for an ill child is important, even when taken away from a usual activity. we appreciate that applying standard wage rates to leisure time is not a straightforward issue in economics. this approach values carer leisure time and non-paid working time in a similar way to a worker's time consistent with neoclassical theories of labour economics [53] . in attaching value to leisure time and using sex-weighted wage rates, we have made our assumptions explicit, and provided sufficient detail (table 3 ) so that others can adjust unit prices using different approaches. previous burden data [49] have been used to assess the cost-effectiveness (c/e) of using influenza vaccine in children [54] . if our cost values, incorporating these indirect costs, were used in the numerator of c/e calculations, there is a distinct possibility of double counting [55] . double counting is likely where the denominator is a utility measure that incorporates a quality assessment (such as the quality adjusted life year or qaly), and most economists would see leisure time as a logical component of the qaly. there is also debate [53] about the inclusion, measurement, and valuation of lost working time in economic evaluations, with the debate centring on whether in practice qaly instruments capture income effects related to absenteeism. for all illnesses where a specimen was tested, parents received a result letter by mail. the delay between illness onset and posting the letter was shortest for influenza illnesses, but for most illnesses parents would have been aware of the result before illness end. pandemic influenza was not being widely discussed in australia during 2003, but interpandemic influenza does receive media coverage annually encouraging vaccine uptake, and this may have caused parents to overestimate key parameters associated with their child's influenza-positive illness. however, if such a bias was in operation it might also be expected that time values for illnesses where no virus was identified may be relatively understated when compared to aris with one or more viruses present. we did not find such a phenomenon; aris with no virus identified had a higher mean cost than those with a single virus present, and for the key cost driver of excess carer time away from a usual activity, no cause illnesses had higher values than both single and multiple virus aris. despite the impact of respiratory viral infections in children there are relatively few burden comparisons available that collect primary data from ill children. an italian study examining the impact of hmpv, rsv, and influenza in children less than 15-years of age presenting to an emergency department found hmpv illnesses to be significantly more burdensome than rsv, having a similar impact to influenza [50] . in our study hmpv was the least expensive single-virus illness. this finding may be due to the different nature of illnesses that result in hospital presentation or hospital admission, compared with community managed illness. of the 730 aris in this study only 4.0% (n = 29) prompted hospital presentation, with less than 1% (n = 5) requiring admission. an excellent community-based finnish study describing the burden of influenza in children 13-years of age or younger over two seasons, with 2231 child-seasons of data, also contrasts this imbalance between community-managed and hospitalized cases of influenza, with only three emergency department referrals and one hospital admission in 131 children less than three years of age with influenza [49] . this study differed from ours in that whilst it used laboratory confirmation, it did not employ more sensitive molecular diagnostics [56] , families were required to visit the study clinic when the study child had fever or signs of respiratory infection, indirect costs did not include nonwork time away from a usual activity, and the study did not provide a comparison with other viral acute respiratory illnesses [49] . the findings from the finnish study reinforce the need to follow children for aris over more than one season, with different rates of influenza infection from year-to-year in each age group. these differences extended to changes in likelihood of infection between age groups: for example, the rate of laboratory-confirmed influenza increased by one-third from season one (2000) (2001) to season two (2001) (2002) for children less than three years of age, but the rates for three to six year olds and seven to 13 year olds fell 47% and 86%, respectively. a german study, recruiting children less than three years of age with lower respiratory tract infection (lrti) through office and hospital-based paediatricians, collected cost of illness from a societal perspective, including loss of work days by caregivers [51] . this study showed that non-hospitalized cases of influenza lrti had twice the cost of piv lrti and were one-third more costly than rsv lrti, with this difference made up entirely by indirect costs [51] . whilst methods vary, previous cost effectiveness studies of influenza vaccine in children are characterised by two findings: first, that cost-effectiveness is unsurprisingly enhanced by taking a societal perspective through the inclusion of indirect costs [5, 6, 8, 43, 54, 57] . our findings reinforce the importance of indirect costs [51] , and highlight a previously inadequately measured layer of burden -carer time away from a usual, non-work activity. second, the potential cost-effectiveness of implementing a vaccination program is improved by flexible or non-individual based delivery programs [6, 43] . vaccine delivered through pharmacies for a small service fee -improving access and negating the time and costs associated with a primary care visit -or large school-based programs, are likely to be acceptable to parents and providers. it is likely that the cost benefits of preventing influenza in children would extend beyond the targeted age-group [58] , similar to the indirect effects in older age groups seen following the introduction of childhood conjugate pneumococcal vaccination in the us [59] . our study reinforces the costly impact of all respiratory viruses, but particularly interpandemic influenza, on children, their families, and society. efforts to further explore the costs associated with community-managed illness over a number of seasons for all respiratory infections are needed. similar to recent hospital-based findings, using laboratory-confirmation to specifically identify influenza appears to increase the cost of illness many fold; a finding that may make population-based vaccination programs a more cost-effective proposition. we believe the use of parent-collected specimens may have important effects in reducing bias in both the epidemiologic and impact data collected. not requiring parents to either present with their ill child to a health clinic or host a home visit by study staff may result in enhanced ari surveillance, but more importantly, allows for the reporting of impact data uncontaminated by compliance with study procedures, thereby reducing any impact a hawthorne effect may have. further studies that collect primary, integrated epidemiologic and economic data, particularly indirect costs, directly from families about community-managed aris in children, are required. such data would allow for a 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impact of community-acquired and nosocomial lower respiratory tract infections in young children in germany higher than normal seasonal influenza activity in victoria the role and estimation of productivity costs in economic evaluation costeffectiveness of influenza vaccination of healthy children avoiding double-counting in pharmacoeconomic studies diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza the efficacy, effectiveness and cost-effectiveness of inactivated influenza virus vaccines herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children control and prevention: direct and indirect effects of routine vaccination of children with 7-valent pneumococcal conjugate vaccine on incidence of invasive pneumococcal disease -united states medicare statistics: average patient contribution per service medicare benefits schedule book operating from 1 november 2002 medicare benefits schedule book operating from 1 november 2003 content/health-pbsgeneral-pubs-manual-appendix3.htm] 65. national hospital cost data collection national hospital cost data collection the work was supported by project grants from the victorian department of human services, the murdoch children's research institute, and the university of melbourne. stephen lambert was the recipient of a national health and medical research council public health postgraduate scholarship. we thank all children and families who volunteered to participate in the study. this study would not have been possible without the generous terence nolan and stephen lambert have, in the past five years, received research grants for epidemiological and vaccine related research from csl limited, medimmune, gsk biologicals, wyeth, and merck. kelly allen and robert carter have no competing interests to declare. all authors were involved in the study design and approach and sbl and tmn developed the original protocol. kma and sbl were responsible for the day-to-day conduct of the study. sbl performed the analysis and drafted the article. all authors contributed to and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge http://respiratory-research.com/content/9/1/11 key: cord-260525-bohv78hi authors: mei, yang; weinberg, samuel e.; zhao, lihui; frink, adam; qi, chao; behdad, amir; ji, peng title: risk stratification of hospitalized covid-19 patients through comparative studies of laboratory results with influenza date: 2020-07-31 journal: eclinicalmedicine doi: 10.1016/j.eclinm.2020.100475 sha: doc_id: 260525 cord_uid: bohv78hi background: the outbreak of coronavirus disease 2019 (covid-19) in december 2019 overlaps with the flu season. methods: we compared clinical and laboratory results from 719 influenza and 973 covid-19 patients from january to april 2020. we compiled laboratory results from the first 14 days of the hospitalized patients using parameters that are most significantly different between covid-19 and influenza and hierarchically clustered covid-19 patients. findings: compared to influenza, patients with covid-19 exhibited a continued increase in white blood cell count, rapid decline of hemoglobin, more rapid increase in blood urea nitrogen (bun) and d-dimer, and higher level of alanine transaminase, c-reactive protein, ferritin, and fibrinogen. covid-19 patients were sub-classified into 5 clusters through a hierarchical clustering analysis. medical records were reviewed and patients were risk stratified based on the clinical outcomes. the cluster with the highest risk showed 27·8% fatality, 94% icu admission, 94% intubation, and 28% discharge rates compared to 0%, 38%, 22%, and 88% in the lowest risk cluster, respectively. patients in the highest risk cluster had leukocytosis including neutrophilia and monocytosis, severe anemia, increased red blood cell distribution width, higher bun, creatinine, d-dimer, alkaline phosphatase, bilirubin, and troponin. interpretation: there are significant differences in the clinical and laboratory courses between covid-19 and influenza. risk stratification in hospitalized covid-19 patients using laboratory data could be useful to predict clinical outcomes and pathophysiology of these patients. funding: national institute of diabetes and digestive and kidney disease, department of defense, and national heart, lung, and blood institute. we searched pubmed and google scholar on may 15, 2020 using keywords "covid-19, influenza" without restrictions of time or language. there were no studies directly comparing the clinical and laboratory courses between western patients with covid-19 and influenza. one study compared and influenza patients at presentation in china. we next searched "risk stratifications, covid-19, laboratory results". many studies reported risk factors associated with covid-19 using clinical outcomes as end points. there were no studies using hierarchical clustering approach to stratify laboratory data to predict risks in correlation with clinical outcomes. we report one of the first comparative studies between covid-19 and influenza patients in the united states on the temporal changes of laboratory results. we also used a unique hierarchical clustering analysis through laboratory data that are distinctive between covid-19 and influenza and revealed 5 different covid-19 risk clusters. this approach could be useful to prospectively stratify risk groups and predict pathophysiology in hospitalized covid-19 patients. there were significant clinical and laboratory differences between covid-19 and influenza over the course of hospitalization. these differences, combined with risk stratification following temporal changes of laboratory results, are important for differential diagnosis and prognosis of patients with severe viral respiratory infections. future research on the studies of pathophysiology of different covid-19 risk clusters would be helpful to understand the disease. coronavirus disease 2019 (covid-19) rapidly disseminated in most countries. the causative agent, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is a novel beta-coronavirus with phylogenetic similarity to sars-cov [1] . to this date, in mid may 2020, the virus has infected over four million individuals worldwide and has claimed close to 30 0,0 0 0 lives. the mortality has been predominantly attributed to progressive pneumonia, leading to acute respiratory distress syndrome. other organ failures and coagulopathy associated with covid-19 has been observed in many studies, particularly in the hospitalized patients [ 2 -4 ] . the pandemic of covid-19 overlaps with 2019-2020 flu season, which makes it difficult to differentially diagnose and manage patients with acute respiratory symptoms. currently published clinical and laboratory data on covid-19 are largely limited to studies with small sample size and mostly originated from china. studies on temporal tracking of laboratory results in correlation with the severity of the disease are scarce. the most commonly reported laboratory abnormalities in covid-19 include lymphopenia, prolonged prothrombin time (pt), and elevated lactate dehydrogenase (ldh) [3] . a study showed that a predictive model using laboratory values had a stronger discriminatory power predicting mortality in hospitalized patients compared to the clinical models [5] . laboratory manifestations in the western population and their correlation with the disease course of the hospitalized covid-19 patients, especially their differences from those in influenza patients, are unclear. in this study, we compared the laboratory manifestations of covid-19 patients admitted to northwestern medicine health system and compared these findings to a cohort of influenza patients. we explored laboratory parameters that could predict the mortality in the hospitalized patients. we retrospectively collected demographic and clinical data from 973 patients with covid-19 and 719 patients with influenza (including influenza a and b) from northwestern medicine health system during the flu season of 2020 from january to april 2020. these represent all the positive cases during this period. most of the patients were adult over 20 years of age (968 patients with covid-19 and 702 patients with influenza) since the pediatric patients were managed at other institutions. 5 covid-19 and 17 influenza patients less than 20 years of age were still included in the analysis but were not representative of the wider population under 20 years of age. data collected include laboratory results from the day of presentation to day 14 of admission and clinical information until early may 2020. many patients, especially those with influenza, did not have a full range of data sets since they were not hospitalized. we compared these laboratory results between patients with covid-19 and influenza and identified the most significantly different parameters between these two diseases. we then set the criteria to select patients in that at least 7 days of data points in each of these parameters have to be met for further analysis. 154 and 23 hospitalized covid-19 and influenza patients, respectively, fit the criteria and their data in these parameters were applied for a hierarchical clustering analysis. five patients in the covid-19 cohort were not grouped in any clusters, therefore excluded from further analyses. patients in different clusters from the analysis were compared for their demographic information, including age and sex, underlying medical conditions, and clinical outcomes including fatality rate and length of hospital stay. the study protocol was approved by the institutional review board at northwestern university. the requirement for informed consent was waived approved by the review board. samples were collected using a nasopharyngeal swab or by bronchoalveolar lavage (bal). the swabs were then placed into a collection tube with 3 ml of virus preservation solution. for realtime reverse transcriptase polymerase chain reaction (rt-pcr) assay, total rna was isolated using the qiaamp minelute virus spin kit (qiagen, valencia, ca, usa). the rt-pcr assay was developed in the molecular diagnostic laboratory at northwestern memorial hospital, using primers, probes, reagents and procedure designated by the center for disease control and prevention (cdc). the viral rna was amplified using a one-step procedure using taqpath 1-step rt qpcr master mix and cdc designed primers and probes (2019-ncov kit, idt coralville, ia). the products were amplified on the quant studio 6 flex system (thermo scientific, waltham, ma, usa) or xpert xpress sars-cov-2 system (cepheid, sunnyvale, ca, usa). nasopharyngeal swabs were tested for influenza viruses with two assays: xpert xpress flu/rsv from cepheid incorporation (sunnyvale, ca, usa) for testing patients in the emergency room and filarray respiratory panel 2 from biomerieux (marcy-l'étoile, france) for inpatient testing. the xpert xpress flu/rsv assay is a real-time rt-pcr-based assay for the detection and differentiation of influenza a and b viral rna. the test was performed with the cepheid genexpert system. the assay targets unique gene sequences that encode influenza a matrix (m), influenza a basic polymerase (pb2), and influenza a acidic (pa) proteins for influenza a virus and influenza b matrix (m) and influenza b nonstructural (ns) proteins for influenza b virus. the filmarray respiratory panel 2 (rp2) test consists of automated nucleic acid extraction, reverse transcription, nucleic acid amplification, and result analysis in approximately 45 min per specimen. the clinical and laboratory information were extracted from electronic medical records of northwestern medicine health system. routine complete blood counts (cbc), including counts on red blood cells (million per microliter, m/ μl), hemoglobin (gram per deciliter, g/dl), white blood cells (thousand per microliter, k/ μl), neutrophils (thousand per microliter, k/ μl), monocytes (thousand per microliter, k/ μl), lymphocytes (thousand per microliter, k/ μl), and platelets (thousand per microliter, k/ μl), were performed on peripheral blood samples by sysmex sn-10 automated hematology analyzer. routine blood biochemical parameters were performed on peripheral blood samples using instruments and assays listed in table s1 . there were 154 covid-19 patients with at least 7 days of data points in parameters presented in fig. 3 for clustering analysis. there were only 23 influenza patients with complete 7 days of data points, which prevented meaningful clustering assay. besides these patients, there were random missing data in other patients in figs. 1 , 2 , and s2 due to the fact that many patients were not hospitalized or had limited number of laboratory tests performed. a two-tailed, unpaired t test was used to compare laboratory parameters between patients with covid-19 and influenza. the heat map and clustering of 154 covid-19 patients were analyzed by online morpheus (broad institute, boston, ma). hierarchical clustering was performed with one minus spearman rank correlation with average linkage within patients. the clinical outcomes were compared among the identified clusters using logistic regression for binary outcomes (icu use, intubation, discharged alive, and other respiratory infections) and cox proportional hazards model for time-to-event outcomes (in-hospital death and death censored hospitalization stay). because some of the events were rare, firth's bias reduction method based on penalized likelihood was used [6] . the survival curve and death-censored hospital stay curve were estimated using kaplan-meier method, and the significance was assessed using the log-rank test. p < 0 ·05 was considered statistically significant. statistical analyses were performed using the prism software (version 8 ·0) and r (version 3 ·5 ·1). the corresponding author has funding support from the national institute of diabetes and digestive and kidney diseases, national heart, lung, and blood institute, and the leukemia and lymphoma society. these sponsors had no roles in study design, data collection and analyses, interpretation of the data, or writing of the manuscript. we collected clinical and laboratory data from 973 patients with covid-19 and 719 patients with influenza (including influenza a and b) from january to april 2020. most of the patients were over 20 years of age. the influenza cohort included 274 males and 445 females with the diagnostic samples collected between january 1, 2020 and april 3, 2020. the covid-19 cohort includes 507 males and 465 females with the samples collected between march 14, 2020 and april 21, 2020. we first compared the differences in the prevalence of the diseases in different age groups. consistent with prior reports, influenza was more prevalent in younger patients [7] whereas covid-19 was more common in older patients [ 4 , 8 ] ( figure s1 ). the fatality rate was higher in covid-19 patients (35/973, 3 ·5%) than influenza patients (8/719, 1 ·1%, p = 0 ·0015). we evaluated clinical and laboratory results at presentation. compared to influenza patients, patients with covid-19 presented with significantly higher levels of red blood cell (rbc) count, hemoglobin, and platelet count. the level of lactate dehydrogenase (ldh) was significantly higher in patients with influenza. troponin was higher in covid-19 patients. the other laboratory parameters remained no statistically significant differences between influenza and covid-19 (table s2) . we compiled and temporally tracked all available laboratory results in these patients from the day of presentation to day 14 of the disease process. overall, there were multiple parameters that showed significant differences between covid-19 and influenza ( fig. 1 ) . in hematologic parameters, the white blood cell (wbc) count gradually increased in both diseases until day 8-9 when influenza patients started to decrease. however, wbc counts continued to increase in patients with covid-19, which was mainly due to the increased absolute neutrophil count (anc). the absolute lymphocyte count (alc) in covid-19 patients was stable at the borderline reference range during this period. in comparison, influenza patients showed more fluctuating lymphocyte counts. hemoglobin and rbc in both groups gradually reduced over time. however, covid-19 patients dropped more rapidly and plummeted to the same levels as influenza patients in the later stages of disease course. the platelet counts were significantly higher in covid-19 patients, although thrombocytopenia was rare during this clinical course in both diseases ( fig. 1 a) . we separated the patients in each group based on sex and age ( < 60 or ≥60) ( fig. 1 b) . wbc increased in all subgroups of covid-19 patients with the increase most significant in older ( ≥60 years old) male patients. the same was observed for anc. interestingly, wbc in younger ( < 60 years old) female influenza patients increased significantly in the early stages of disease course followed by a gradual decrease, which could be seen in most of the leukocyte lineages. more important, the differences in the level of anemia were primarily contributed by male covid-19 patients. in addition, older male patients were more anemic at presentation and during the course. serum biochemical tests also revealed statistically significant differences between covid-19 and influenza ( fig. 2 ) . alanine aminotransferase (alt) and blood urea nitrogen (bun) were started at around the higher reference range in patients with both diseases. while their levels remained relatively constant in patients with influenza, alt and bun gradually increased and became significantly higher in the late stages of disease course in covid-19 patients. this was most prominent in male patients whereas the differences were not significant in female patients ( fig. 2 a) . the level of d-dimer started at increased levels in both diseases. the level continued to climb in patients with covid-19 in both males and females, although there was no statistical significance due to inadequate data points in influenza patients ( fig. 2 b and s2a ). the levels of c-reactive protein (crp), ferritin, and fibrinogen in covid-19 patients were significantly increased and reached the highest level at 6-8 days, which were followed by gradual decrease ( fig. 2 b) . notably, clinical data of these parameters in influenza patients were not as comprehensive as those in the covid-19 patients, which compromised statistical analyses when divided based on age and sex. ldh was significantly higher in influenza patients ( fig. 2 c) . the levels of alkaline phosphatase and bilirubin were slightly higher in influenza patients than covid-19 patients over the 14-day course, although there was no statistical significance. the other parameters tested, including aspartate transaminase (ast), creatinine, prothrombin time (pt), partial thromboplastin time (ptt), and troponin, did not show differences between influenza and covid-19 groups ( figure s2b ). these analyses reveal that overall, covid-19 patients tend to have higher hemoglobin and red blood cell counts but more rapid decline, higher white blood cell and neutrophil counts especially in the late stages of disease course, and higher alt, bun, and d-dimer. however, it is unclear whether there are any correlations among these parameters in covid-19 patients, and whether there are specific groups in these patients that may show distinct clinical outcomes. to answer these questions, we performed a hierarchical clustering analysis to compare various laboratory parameters in individual patient over the 14-day course. for the laboratory parameters, we focused on complete blood count, d-dimer, bun, and alt since they were significantly higher in patients with covid-19 compared to those with influenza. we chose 154 covid-19 and 23 influenza inpatients with at least 7 days of data points in each of these parameters to analyze. five clusters (1 to 5 from far left to far right, respectively) were revealed in covid-19 patients whereas no distinct clusters were identified in influenza patients due to lack of sufficient patients ( fig. 3 ) . cluster 1 showed severe worsening anemia, increased red blood cell distribution width (rdw), leukocytosis (including neutrophilia and monocytosis), higher bun, creatinine, d-dimer, alkaline phosphatase, bilirubin, and troponin ( fig. 4 a) . these parameters were much milder in other clusters, especially in clusters 3 and 4. the level of crp was high in cluster 2 ( fig. 4 b) . other parameters, including lymphocyte and platelet counts ( fig. 4 a) , alt, fibrinogen, ferritin, pt, ptt, ldh, oxygen saturation, and maximum temperature remained no differences among these clusters (fig. s3) . we further investigated the clinical manifestation of patients in these different covid-19 clusters. cluster 1 showed the highest mortality rate (27 ·8%), which was followed by group 2 and 5 (12 ·5%). group 3 and 4 had the lowest mortality rate (9% and 0%, respectively) ( table s3a and fig. 5 a) . there were also significantly increased rates of icu admission (odds ratio 18 ·85, 95% ci 4 ·20-180 ·23), intubation (odds ratio 40 ·07, 95% ci 8 ·62-391 ·26), and other respiratory infections (odds ratio 26 ·10, 95% ci 6 ·79-127 ·49) in cluster 1 compared to cluster 4 (table s3b) . notably, there was no significant differences in age distributions in these clusters although males were predominant in most of the clusters, including clusters 3 and 4. the majority of the patients in all clusters had pre-existing conditions, mostly hypertension and type 2 diabetes (table s3c ). compared to cluster 4, the other groups showed significantly increased length of hospital stay, especially for cluster 1 ( fig. 5 b) . overall, our study reveals significant differences in the laboratory parameters between the hospitalized covid-19 and influenza patients. importantly, we found that the hospitalized covid-19 patients with the worst prognosis often show worsening anemia, increasing rdw, worsening neutrophilia and monocytosis, and significantly higher levels of bun, creatinine, d-dimer, alkaline phosphatase, bilirubin, and troponin. these laboratory parameters could be useful clinically to predict the outcomes of the patients. the overlap of the flu season with the pandemic of covid-19 complicates the clinical management of patients with respiratory symptoms. studies of direct comparison of clinical and laboratory results between these two infectious diseases are rare. a recent report compared hospitalized patients with acute respiratory distress syndrome (ards) caused by covid-19 and influenza h1n1. the authors concluded that ards patients with covid-19 had lower severity of illness at presentation and lower mortality adjusted by sequential organ failure assessment scores [9] . however, the study used data from two independent institutes, which could lead to certain bias. it is also unclear the differences on the clinical and laboratory results during the hospitalization of these patients. to address this question, our study showed that there were significant differences between hospitalized patients with covid-19 and influenza when we followed their temporal changes of laboratory results. compared to influenza patients, the most significant differences over the course of 14 days of hospitalization in covid-19 patients were worsening anemia, worsening leukocytosis, and an increase in d-dimer, bun, and alt. instead of comparing clinical endpoints to evaluate risks as performed in most of the published studies, we stratified the hospitalized covid-19 patients through clustering of their laboratory results that were most significantly different from influenza patients (i.e. complete blood count, d-dimer, bun, and alt) during the first 14 days of hospitalization. the major differences in these clusters were red cell indices, neutrophil count, monocyte count, and bun. surprisingly, lymphocyte count did not show differences as prominent as the other parameters among these patients. lymphopenia was shown to be common and correlated with a worse prognosis in covid-19 patients [10] . a recent meta-analysis showed a 3fold higher risk of developing severe covid-19 with lymphopenia [11] . however, the studies involved in this meta-analysis were all data from patients in china. the findings in our cohort may represent distinctive features in the western population. in fact, the majority of the covid-19 patients in our study showed lymphocyte counts within the reference range over the 14-day hospitalization (low level reference 1 ·0 × 10 9 /l), even in patients in cluster 1 with the worst prognosis. reviewing of the clinical record, including the record after the initial 14 days, revealed that cluster 1 had the worst outcomes including high fatality, high icu admission and intubation rate, and prolonged hospital stay. compared to cluster 4 with the best outcome, patients in cluster 1 showed features consistent with previously published reports, including high wbc, bun, creatinine, alkaline phosphatase, bilirubin, and troponin [ 4 , 5 , 8 , 12 ] . however, severe anemia was not reported as a prognostic feature. a recent report showed no differences in hemoglobin between survival and non-survival covid-19 patients in china [13] . therefore, worsening anemia represents another distinctive feature of worse prognosis in our cohort, especially for male patients. notably, anemia was associated with neutrophilia and monocytosis in cluster 1, which could also be related to additional respiratory infections that were more frequent in these patients. these laboratory features associated with a worse prognosis were more prominent in males. indeed, of the 13 deceased patient in all clusters, 10 were males. the majority of the patients in all 5 clusters had more than 1 comor-bidity, which is not a confounding factor in risk stratification in this study. it would be interesting to further investigate whether different risk clusters of covid-19 correlate with the pathophysiology in these patients. for example, cluster 1 may represent patients who were showing a clotting/vascular system pathology with multiorgan dysfunction (elevated d-dimer, and abnormal cardiac, renal, and liver function tests). cluster 2 may represent cytokine storm or secondary bacterial pneumonia (elevated crp) without involvement of other organs. future studies on specific organ systems in correlation with other clinical manifestations, including the level of inflammatory cytokines, may be useful to confirm the underlying pathology in patients in these clusters. some other clinical and laboratory results that were reported to be distinctive between mild and severe covid-19 patients were not informative when we evaluate the risks in our cohort. there were no differences in daily minimum oxygen saturation or maximum temperature among the clusters. in the laboratory data, there were no difference in alt, fibrinogen, pt, ptt, ferritin and ldh over the 14-day course. these laboratory parameters could be important cohort. * p < 0 ·05, * * p < 0 ·01, * * * p < 0 ·001, * * * * p < 0 ·0 0 01 in differentiating hospitalized and non-hospitalized patients, which is not the focus of this study. our study has its limitations. first, the patients were limited to a single healthcare system in chicago metropolitan area. second, due to the lack of sufficient number of influenza patients with over 7 days of laboratory data sets in our hierarchical analysis, we could not perform the same risk stratification in these patients. it remains to be determined whether similar laboratory patterns as in covid-19 cluster 1 are also present in severe hospitalized influenza patients. third, because of the same reason, several laboratory parameters did not show statistical significance between covid-19 and influenza, although they appeared to be apparently different. overall, our findings provide values to predict risk groups for further management in hospitalized covid-19 patients in the western population. further prospective studies using independent groups will be informative to confirm these findings. this work was supported by national institute of diabetes and digestive and kidney disease (niddk) grant r01-dk124220 (p.j.), department of defense grant ca140119 (p.j.), and national heart, lung, and blood institute (nhlbi) grant r01-hl148012 (p.j.). p.j. is a scholar of the leukemia and lymphoma society. deidentified participant data will be available upon request to the corresponding author with publication. dr. ji reports grants from national institute of diabetes and digestive and kidney diseases, grants from national heart, lung, and blood institute, grants from leukemia and lymphoma society, during the conduct of the study. all other authors declare no relevant conflicts of interest. supplementary material associated with this article can be found in the online version at doi: 10.1016/j.eclinm.2020.100475 . a novel coronavirus from patients with pneumonia in china abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical and laboratory predictors of in-hospital mortality in patients with covid-19: a cohort study in wuhan, china bias reduction of maximum likelihood estimates seasonal incidence of symptomatic influenza in the united states covid-19 in critically ill patients in the seattle region -case series comparison of hospitalized patients with ards caused by covid-19 and h1n1 lymphopenia predicts disease severity of covid-19: a descriptive and predictive study lymphopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a systemic review and meta-analysis clinical, laboratory and imaging features of covid-19: a systematic review and metaanalysis clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china key: cord-293299-gdew0ueo authors: jordan, william s.; dowdle, walter r.; easterday, bernard c.; ennis, francis a.; gregg, michael b.; kilbourne, edwin d.; seal, john a.; sloan, frank a. title: influenza research in the soviet union—1974 date: 1974-12-17 journal: j infect dis doi: 10.1093/infdis/130.6.686 sha: doc_id: 293299 cord_uid: gdew0ueo nan tamination procedures must be responsible. a means for interruption of such transmission must be sought. (g) risk figures for patients of hb agpositive physicians, surgeons, and dentists should be developed. if carriage of hg ag is hazardous to patients, the risk should be defined, and appropriate measures developed for reduction of the risk. (h) the effect of the season on dissemination of herpesviruses should be studied. in addition, the cytomegalovirus group should be evaluated for antigenic variants, since there may be considerable variation in potential for disease among different members of this group. (i) measures for the increase of specific resistance to representatives of the herpesvirus group should be evaluated. while the effectiveness of specific attenuated or inactivated vaccines may appear to be unlikely on theoretical grounds, transfer factor and other cellular immunological approaches should be studied. (3) personnel, physicians, and nurses must be trained in hospital epidemiology, surveillance, and control procedures, to implement and to increase the available knowledge. in addition, innovative approaches to the dissemination of knowledge concerning the usage of antimicrobial agents, hazards of various invasive procedures, and hospital control practices must be encouraged. william l. hewitt selected fields of health and medical science. the initial problems selected were malignant neoplasms, cardiovascular diseases, and environmental health. at its second session, the u.s.-u.s.s.r. joint committee for health cooperation agreed to further exploration in the field of influenza and other viral diseases; recommendations regarding areas of collaboration would be considered at the third session of the joint committee in moscow in june 1974. on behalf of the development of these recommendations, a delegation of scientists from the united states visited the soviet union during february 17-27, 1974, for discussions with soviet scientists in leningrad and moscow. the delegation was larger than the small group that had visited several institutes in these two cities in january 1973 [1] , and was able to collect additional information on soviet research. this summary presents a synthesis of information collected by members of the visiting delegation regarding soviet research in the five principal areas considered for possible joint collaboration. a long historical tradition for the use of livevirus vaccines in the soviet union dates from smorodintsev's experimental infection of volunteers with influenza virus in 1937 [2] . that the success of immunization with live virus has been limited is evidenced by continued research on new methods for attenuation and administration of virus and by the present emphasis on research in chemoprophylaxis. furthermore, mass immunization is not yet undertaken on more than a city or regional basis, and no more than 50 million doses of vaccine are used each year. the eventual goal is the achievement of mass immunization of about 70% of the population. such immunization would be nonselective, except that the vaccination of children would be emphasized. there is little interest in the use of inactivated vaccine by parenteral administration, although such vaccine is now being produced experimentally and is to be utilized, if approved, in hyperimmunization of volunteers for the production of immunoglobulin against influenza virus. administration and control. the development of new or modified vaccines is chiefly under the aegis of the all-union research institute of influenza in leningrad, although the institute for viral preparations in moscow also undertakes limited research and development in addition to its production activities. the initial testing of a new vaccine in volunteers is under the control of the institute involved in its development. protocols are first reviewed by an institute expert committee, then by a review council before testing of vaccines in volunteers. experimental vaccines are preliminarily tested under standard protocols for mycobacteria, mycoplasmas, and hemadsorbing viruses. initial testing in volunteers is principally for reactogenicity. testing is stepwise in relation to volunteer susceptibility and is restricted to young adults. thus, the live vaccine is successively tested in volunteers (five per group) with high, low, and undetectable levels of serum antibody, and then finally in groups of 20 antibody-negative subjects. application of the vaccine to nonvolunteer populations in field trials requires the permission of the ministry of health. selection of strains for the production of vaccines is made in the november through january period by a committee under the ministry of health; results of testing of the first three production batches in a total of 60 volunteers are submitted to the laboratory of biologic control, which is part of the tarasevich control institute for biological preparations of the ministry of health. allantoic fluid vaccine is distributed to sanitary-epidemiologic stations that arrange for and direct its use in organized population groups, such as industrial workers. neither allantoic nor tissue culture vaccines are available directly to polyclinics or other medical facilities. production. experimental vaccines are produced by the institute involved in their development. thus, smorodintsev's allantoic fluid vaccines are produced at the all-union research institute of influenza, while tissue culture vaccines originated by the institute for viral preparations are prepared there. on the other hand, the smorodintsev vaccine approved for general use is produced by the institute for viral preparations. about 12 million of the 45-50 million doses of influenza vaccine produced each year are produced in the latter institute. production of the remainder appears to be distributed among institutes in other cities. chick embryos used at both the all-union research institute and the institute 687 for viral preparations are furnished from flocks under supervision of the veterinary department. however, the all-union research institute minimizes the problem of avian leukosis, citing the negative evidence of 10,000 children who received mumps vaccine and were followed for 12 years, plus the impracticality of testing when 15 million eggs are used annually. the institute for viral preparations reported negative rip (resistance-inducing factor) tests on periodic testing of eggs. research. research in influenza vaccines appears to be entirely committed to live virus immunization. the primary site of this research is the all-union research institute of influenza, although some vaccine-related research is undertaken elsewhere, primarily at the institute for viral preparations. a significant amount of the genetic and molecular virologic research at the all-union research institute is oriented to problems of viral attenuation and virulence markers. until recently, the attenuation of viruses for use in the production of vaccine has been empirically achieved by serial passage in chick embryos and periodic testing in volunteers for reactogenicity. attention is now being directed to other means of attenuation and to the effects of employing a changed route of administration of low-passage, virulent virus for the reduction of toxic effects. preliminary experiments have attempted the recombination of freshly isolated virulent strains with attenuated "laboratory" viruses for the production of so-called "biological recombinants" that possess the antigens of the wild-type parent but are changed in other properties, such as virulence for mice. cold adaptation of viruses by passage at 25 c is also under study. a rapid-passage method in which allantoic fluids are harvested and passaged every 24 hr is also under investigation as a means of shortening the time required for attenuation. the greatest emphasis at present is on the use of the peroral (po) route of administration of live vaccine to children as a method of reducing symptoms associated with intranasal administration. comparative studies of the two routes have been done principally at the all-union research institute with allantoic fluid vaccines. these studies indicate that multiplication of virus is reduced after po adminstration, as evidenced by recovery of virus in low concentration only on the fifth day and a delay in antibody response when compared with that to intranasal vaccine. immunogenicity is also reduced by po administration, so that three separate applications of the virus at 14-day intervals are required to achieve a reasonable rate of seroconversion. at the institute for viral preparations, dr. a. k. alexeeva is investigating po live-virus vaccine prepared in tissue culture. the commitment to live influenza vaccines extends also to the immunoprophylaxis of other diseases, including infections with parainfluenza and respiratory syncytial viruses, adenoviruses, and mycoplasma. the ultimate research objective is the suppression of respiratory diseases, particularly in children, through the use of "combined immunization. " as previously reported [1] , passive immunotherapy is carried out through administration of y-globulin derived after specific immunization of man. earlier interest in the use of high-titered y-globulin intranasally as a prophylactic agent seems to have been abandoned. rather, y-globulin given parenterally was reported to be efficacious in alleviating high fever and other evidences of "toxicity," particularly in children. no new data on efficacy were provided. the y-globulin is prepared at the pasteur institute in leningrad from serum drawn from volunteers hyperimmunized with virus vaccines. the y-globulin is approved for use and is available at pharmacies without prescription at a cost of 3.75 rubles/2 ml. surveillance. most epidemiologic data presented covered the period from 1957 to the present, with by far the greatest emphasis on influenza a. the soviet union experienced major epidemics of influenza in 1957, 1959, 1962 (mixed a and b), 1965, 1967, 1968, and 1970 , reflecting roughly a two-to three-year cycle. the epidemics of 1957, 1962, and 1968 appear to have caused the greatest morbidity. sudden onset of countrywide outbreaks during the late fall or early winter months have characterized most epidemics, and the northern areas and urban populations have been most frequently and most severely affected. apparently most large cities (population of more than 200,000) experience relatively uniform increased morbidity during epidemics, with little asynchronous occurrence countrywide. influenza is a disease primarily of children most often affecting those between the ages of nine and 15 years. surveillance of influenza and other acute respiratory diseases (ard) is based on morbidity reports originating at the polyclinic or hospital level. large factories, mills, etc., also have clinics and provide data for the system. physicians are required to report cases seen at clinics or on house calls. report cards are filled out daily, collected by trained health personnel, tabulated, and sent to district sanitary-epidemiologic stations; from here reports are sent to successively higher administrative units: the rayon, the oblast, then to the republics, and finally to the ministry of health. although this system (which also applies to other reportable communicable diseases) is the formal, regular, national method of morbidity reporting, two other systems of influenza and ard reporting exist that serve somewhat different functions but utilize the same basic polyclinic source of data. these systems are operated by the all-union research institute, leningrad, and the ivanovsky institute, moscow. for the last two or three years the former institute has operated a surveillance system for influenza-ard consisting of regular daily morbidity reporting (by cable or telephone) from the capitals of the 15 republics. thirty-seven other cities submit regular reports weekly. another 100 cities are supposed to report weekly, but perhaps only 30-40 of these do so regularly. the morbidity data received by .the all-union research institute are identical to data reported in the national system. however, this institute receives these data either one day or one week before the regular system; it uses them primarily to determine the probability of influenza morbidity in the 52 cities under close surveillance. in 1957 the ivanovsky institute began receiving morbidity data every 10 days from 30 towns throughout the soviet union. the number of towns reporting grew to 55 but is now only 20. these 20 cities report the same data to the all-union research institute; the cities are distributed widely throughout the soviet union and have populations of 200,000 or more. fourteen of these cities report each week; six cities report by cable daily. all report the total number of patients seen at polyclinics or at home who are diagnosed as having influenza or ard plus the same morbidity data for children up to seven years of age. during influenza epidemics the ivanovsky institute also receives daily by cable the total number of hospitalized cases of influenza and the total number of deaths attributable to influenza. the daily and weekly morbidity data are considered provisional, and each month the 20 cities send an adjusted tabulation of cases which is final. a monthly summary of these data, along with laboratory results, is mailed to each city. a quarterly report is compiled and sent to the world health organization for publication. physicians are required to report all cases of influenza and ard they diagnose, and most do so; many ill persons are never seen by physicians. as a result of several apartment house surveys after two major epidemics, a rough estimate of overall morbidity of 35 % was obtained. approximately 50%-60% of these cases were seen by a physician. laboratory surveillance. the all-union and ivanovsky institutes receive regular laboratory reports from the 53 and 20 reporting cities, respectively. although it was not clearly confirmed, it appears that the gamaleya institute presently serves as a resource and administrative headquarters for most if not all of the laboratories that provide input to all three institutes. serologic tests are performed at the gamaleya institute on sera received weekly from 20 sanitary-epidemiologic stations. sera are obtained from patients of all ages presenting at clinics with noninfectious diseases, from blood donors, from military populations, and from children attending day-care centers and schools. 2, and 3) , and mycoplasma infections also are diagnosed by direct fluorescent antibody staining of nasal smears. the reagents are thought to be prepared at the all-union research institute and distributed to its stations. some of the stations also perform serodiagnostic tests, but many forward paired sera to the gamaleya institute for examination. paired sera are said to be difficult to obtain. some stations may attempt virus isolation in tissue culture; many apparently employ embryonated eggs for isolation of influenza viruses. computer modeling. soviet epidemiologists have devoted considerable effort to the 'development of a model for the prediction of registered numbers of cases of influenza or ard for 128 geographic units within the soviet union. since registered morbidity rather than a more direct measure of real influenza-ard morbidity is used, the model probably has more applicability for health planners, who need to anticipate increased requirements for delivery of health care associated with epidemics, and for industrial organizations that face future production losses, than for those concerned with anticipating changes in the health status of the population per se. critical to the quality of the data input is the proportion of the cases registered. many behavioral factors affect the proportion of cases registered. among these is probably the cost of iiiness. although both inpatient and outpatient services are provided at no direct cost to the patient, sick leave benefits do not always cover absences from work because of illness. for the first five years of employment, benefits cover 50% of the employee's salary; for the next three, the percentage increases to 80%; only after eight years on the job does sick leave cover the employee's entire salary during illness. thus, influenza imposes an indirect cost on many in terms of lost income. some may be reluctant to declare themselves ill because they do not want to incur this loss. once an influenza-ard epidemic has been identified in one location, the model enables one to predict future registrations for 128 observational units, encompassing 99 % of the soviet population. the model serves two roles: (1 ) prediction of daily registrations in a large number of locations; (2) evaluation of the effects of specific public programs (e.g., vaccination). the former application is particularly important in the soviet union, since physicians from all types of specialties and even medical students in advanced stages of training are mobilized to treat influenza during epidemics. although data from the morbidity registration system in the soviet union provide the basic information for this model, data from the 52 all-union reporting centers appear to be used as well, particularly for obtaining early warning of the location of the initial epidemic outbreak in the country. the model's theoretical structure was developed at the gamaleya institute by l. v. rvachev. rvachev's academic training is in mathematics and physics; the model is based on principles from physics. the basic equations have been presented by baroyan et al, [3] ; a much more rigorous theoretical development of the model has been published by rvachev [4] . the formal structure of the model will not be presented here, but brief mention will be made of the independent variables and parameters that serve as the basis for predictions. movement of persons among geographic areas provides the mechanism for the spread of influenza-ard. the model contains a large number of transportation coefficients (based on sales of airline, bus, and railroad tickets) relating interlocational flows of people. these data have been assembled through rather painstaking efforts on the part of staff. one of the soviet scientists estimated that it took three man-years to secure this information alone. two epidemic-specific parameters reflect the initial (i.e., at the time of epidemic outbreak) density of influenza-susceptible individuals and the speed of transmission within a location. these are estimated from data on registered morbidity in the location in which the initial outbreak occurs. it appears that data must be obtained for five to 14 days. rvachev maintains that the estimates are maximal likelihood estimates. once the epidemic-specific parameters have been estimated, predictions can be made for all locations in the soviet union. the interval between the date predictions are made and the peak of the influenza-ard epidemic in the median location is about 1.5 months. these two parameters are said to embody virological and immunological factors associated with the epidemic. the model, however, does not consider such factors explicitly. other variables and parameters in the model are the area's population and frequency distribution of length of influenza illness, obtained by averaging of data from a monograph by zhdanov et al. [5] . the predictive ability of the model has been evaluated with use of both historical data (for outbreaks in 1957, 1959, 1962, 1965, 1967, 1969, and 1970 ) and data generated for two years since the model was developed. the soviets show extensive plots of actual vs. predicted values of influenza-ard morbidity; these plots indicate that the model predicts quite well. timing of the epidemic and its intensity (measured by the number of registered cases in the location) have been used to assess predictive accuracy. a prediction is considered "accurate" if two conditions are satisfied. first, the day on which influenza-ard reaches its highest level must be within five days of the day on which the peak is predicted to occur. second, the following inequality must be satisfied: 0.7 < (highest predicted registered morbidity in location) / (highest actual registered morbidity in location) < 1.5. using this definition of accuracy, recent tests have shown the model to be "accurate" 80% of the time. compared with similar models in other fields, the model for prediction of influenza-ard epidemiology provides reasonably good predictions of the dependent variable. however, at present it provides very little time between the date on which predictions are made and the date on which epidemics occur. this interval might, of course, be lengthened if the epidemic-specific parameters were estimates based on influenza data from another country, at a time before the epidemic reached the soviet union. for this reason, there is some interest among the soviets in having the united states and other countries implement the model. discussions along this line are now underway with the german democratic republic and bulgaria. research on the ecology of influenza viruses is done at the ivanovsky institute under the direc-tion of d. k. lvov in collaboration with l. y. zakste1skaya. dr. lvov indicated that most of the work had been done since 1970. while there were earlier reports on animal influenza viruses, there appears to have been no organized program until 1970. dr. lvov has been investigating the ecology of arthropod-borne viruses for some time; he now is using some of the same procedures and materials in studying the ecology of influenza. most of his efforts have been directed toward avian species. thus far, most of these studies have been conducted in. the far east of the soviet union (specifically, the kamchatka region and the komandorskiye islands). other areas have included the eastern arctic, west central arctic, murmansk regions, byelorussia, and the caspian sea. all of the areas studied so far have been on the perimeter of the country. while the field aspects have been directed by drs. lvov and a. a. sazanov, the antigenic and serologic analyses and virus characterizations have been done under the direction of dr. zakstelskaya. viruses characterized as h3n2 have been isolated from the chicken, pig, dog, calf, and wild birds (terns and herons) in various parts of the soviet union. in the far east, antibody to the h3 antigen has been found in wild birds, fur seals, cattle, and mink. while there is serological evidence for the h3n2 virus in the far east, the only isolations of that virus from wild birds (terns and herons) have been in the caspian sea area. other information includes: (1) the demonstration of a/equine/miamij63 (heq2neq2) antibody in bird sera, (2) the demonstration of a/swine/ iowa/30 (hsw1nl) antibody in seal and some bird sera, (3) the presence of fowl plague virus (hav1) in chickens in 1970 and the presence of that antibody in wild birds in 1972, and (4) the isolation of swine influenza virus (a/swine/ tartu/1/70) from pigs in 1970. a virus (hav7n2) was isolated from terns in the caspian sea area in june 1973. it is also interesting to note that newcastle disease virus was isolated from wild birds (species not indicated) on kamchatka in 1972, the same year it was isolated from wild ducks in california by workers in the united states. several soviet scientists are interested in antiviral chemoprophylaxis and therapy, including use of 691 interferon and interferon inducers against influenza, and there is some overlap of interests by scientists at various institutions. however, there was little evidence of formal collaboration in this area of influenza research in the soviet union. laboratory research with anti-influenza drugs. dr. g. a. galegov of the ivanovsky institute, a biochemist, leads a group of scientists interested in testing antiviral agents. he hopes to achieve additive therapeutic effects without additive toxicity by using combinations of drugs or interferon plus a drug. dr. galegov has been a coauthor of several publications in this area [6] and is currently seeking an agent that will interfere with the rna polymerase of influenza virus. he is studying a disulfide agent that decreases the rna polymerase activity by about 40%. the name of the agent and the concentration required to produce this effect were not stated; no studies have been performed with it in animals. his laboratory apparently does not perform animal experiments with anti-influenza drugs but does measure their antiviral effects in tissue culture while attempting to delineate mechanisms of action. development of new anti-influenza drugs. very little information was acquired in this area. the two new chemical agents used in the soviet union to prevent influenza, oxolin and bonaphton, were supplied to clinicians for testing in volunteers by dr. pershin of the institute for chemo-therapeutic preparations in moscow; unfortunately, he was not a participant in the discussions. dr. d. m. zlydnikov of the all-union research institute had used these drugs to prevent or treat influenza but did not know what effect they had on influenza virus in vitro or on influenza infection in animal models. dr. zlydnikov suggested that dr. pershin might have done some studies in cell cultures or animals. the scientists who are concerned with influenza chemoprophylaxis and chemotherapy obtain the agents used from manufacturing facilities, such as dr. pershin's institute in moscow, or from outside the soviet union. the clinical investigators who used these agents in volunteers apparently had not done independent in vitro or animal studies. clinical studies. the research performed in the large and active volunteer unit at the all-union research institute of influenza can be briefly summarized. (l) amantadine. the observation made by scientists in the united states that amantadine was partially effective for prophylaxis against influenza type a2 infection in human volunteers was confirmed. no information was obtained about its mechanism of action in tissue culture or in animals. this drug has been used widely in epidemiologic field trials and is now approved for general use. the package insert states: "for the purpose of individual prophylaxis of influenza, the 0.25 % ointment is used in the form of daily two fold application to the nasal mucosa during the period of elevation and maximum development of the epidemic outbreak of influenza (for 25 days) or upon contact with an influenza patient." (3) bonaphton (called 6 bromonaphthoquinone-l, 2 ). dr. zlydnikov obtained this agent from dr. pershin and studied its effect in human volunteers challenged with partially attenuated influenza virus [a 2/hong kong/68 (h3n2)]. the drug was given the day before challenge and daily for six days after challenge. controls received a placebo. safety was studied in a total of 363 volunteers in many experiments. efficacy studies were limited to evaluation of clinical morbidity; no virus isolation or antibody studies were performed. a regimen of 50 mg given po two times daily, from the day before to six days after challenge, was said to decrease morbidity (e.g., 38.4% of 13 volunteers given bonaphton vs. 78.6% of 14 volunteers given placebo). large epidemiologic field trials were done, and approval for general use was obtained. as was the case with oxolin, no data were presented about the mechanism of bonaphton's effect, and it was not learned whether animal studies had been performed with this drug. (4) interferon and interferon inducers. soviet scientists are interested in the use of interferon, either by aerosolization of exogenous human interferon (widely used to treat influenza) or by induction with viral vaccines or chemical inducers. these studies were reviewed in detail in 1973 [1] ; no new information has been acquired. some interesting research is being done by two laboratories in the general area of nonspecific resistance to influenza. drs. t. g. orlova and l. m. mentkevich at the ivanovsky institute have studied the effects of interferon induction by viruses (including influenza) and nuc1eotides in irradiated cba mice, which received transplants of either syngeneic bone marrow or rat bone marrow [7] . induction of interferon by all inducers except influenza virus appeared to be related to production of interferon in bone marrow cells as measured by species specificity. influenza virus induced mouse interferon in irradiated mice that had received rat bone marrow transplants, a fact suggesting the possibility of extramedullary interferon induction by influenza. few laboratories in the soviet union appear to be concerned primarily with basic genetic studies of influenza viruses. recently, as noted, laboratories in the all-union research institute have begun to apply techniques of cold adaptation and recombination to attenuation of strains used for production of live-virus vaccine. in the laboratory of dr. d. b. golubev, antigenic and other variation of the neuraminidase antigens of h3n2 influenza viruses have been under study; his demonstration of differing temperature inactivation and temperature optima of these neuraminidases provides interesting genetic markers. this laboratory has continued studies on the possible relation between temperature optima of neuraminidase and virulence. a lower temperature optimum was described as characteristic of attenuated strains. dr. golubev also reported that, by use of the cf test and purified neuraminidase (n2) proteins, three major antigenic subgroups could be distinguished among influenza strains isolated since 1957. he described these groups as repre-senting "semi-shifts"; the three groups include strains isolated from 1957 to 1965, from 1967 to 1971, and from 1972 to the present, respectively. each semi-shift was accompanied by a change in neuraminidase inactivation temperature. each semi-shift also occurred at the time of a major epidemic in the soviet union. at the ivanovsky institute of virology, studies of influenza virus rna polymerase have been undertaken by dr. galegov, but these studies apparently are in their early stages. dr. u. z. ghendon's group at the institute for viral preparations has worked extensively with temperature-sensitive mutants of fowl plague virus since shifting attention from polioviruses to influenza. results of the assortment of the fowl plague ts mutants into five complementation groups have been published. more recently, ghendon has found evidence of variation in rna polymerase activity among wildtype strains of virus associated with epidemics in december 1971, january 1973, and early 1968. in contrast to observations of workers in the united states, ghendon has evidence against the association of rna virion polymerase activity with the ribonuclear protein. dr. f. i. yershov of the ivanovsky institute is attempting to establish persistent in vitro infections with influenza viruses. such studies may relate to possible mechanisms for survival of the virus during epidemic periods and for antigenic variation. work on such persistent infections with influenza viruses has just begun; major effort in the past involved tick-borne encephalitis virus and parainfluenza viruses. this report, without critique, has attempted to summarize objectively information helpfully provided by soviet colleagues. because of the press of time, important contributions may well have been missed by such a brief survey, and the possibility is acknowledged that some of the presented facts may have been misunderstood and are, therefore, misrepresented. apologies are offered for all such errors, with the expectation that they will be corrected as the collaboration recommended by the delegations from the united states and the soviet union becomes a reality. to these observers, it appears that the recognition given by the ministry of health of the soviet union to influenza as an important national 693 health problem exceeds that given by health authorities in the united states. the soviet investment in research on influenza probably exceeds investment in the united states. the development of a national system of morbidity surveillance and of computer forecasting as a means of allowing local health authorities to prepare for an increased demand for medical services recognizes the impact of the disease in the community. such morbidity reporting is superior to that in the united states and provides a hard-data base for the concern evidenced by the soviet ministry. clearly, neither country has solved the complex problem of influenza prevention; there is much to be gained through mutual cooperation. influenza and interferon research in the soviet union investigation of volunteers infected with the influenza virus possibilities for computer modeling of influenza epidemics in the u report on a visit to bulgaria, december 1-29, 1972. world health organization uchenie 0 grippe review of problems in viral chemotherapy interferon production and infection caused by influenza virus in radiation chimeras key: cord-268593-rvxxv1dn authors: wang, mingyang; veit, michael title: hemagglutinin-esterase-fusion (hef) protein of influenza c virus date: 2015-07-28 journal: protein cell doi: 10.1007/s13238-015-0193-x sha: doc_id: 268593 cord_uid: rvxxv1dn influenza c virus, a member of the orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. humans are the main reservoir of the virus, but it also infects pigs and dogs. very recently, influenza c-like viruses were isolated from pigs and cattle that differ from classical influenza c virus and might constitute a new influenza virus genus. influenza c virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein hef that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of hemagglutinin (ha) and neuraminidase (na) of influenza a and b viruses. here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. the main focus is on the structure of the hef protein as well as on its coand post-translational modification, such as n-glycosylation, disulfide bond formation, s-acylation and proteolytic cleavage into hef1 and hef2 subunits. finally, we describe the functions of hef: receptor binding, esterase activity and membrane fusion. electronic supplementary material: the online version of this article (doi:10.1007/s13238-015-0193-x) contains supplementary material, which is available to authorized users. influenza c virus was first isolated during an epidemic of respiratory illness in 1947. since the virus showed no cross reactivity with antisera against influenza a and b viruses it was classified as a new genus of the orthomyxoviridae, named influenza c virus (francis et al., 1950; taylor, 1949 taylor, , 1951 . influenza c virus usually causes inflammation of the upper respiratory tract, especially in children from two to six years of age. clinical symptoms, such as cough, fever, malaise are typically mild. only occasionally the virus spreads to the lower respiratory tract and causes bronchitis, bronchiectasie and broncho-pneumonia (gouarin et al., 2008; kauppila et al., 2014; matsuzaki et al., 2007; matsuzaki et al., 2006; muraki and hongo, 2010) . although influenza c virus infections occur primarily in a pattern of sporadic cases or in limited outbreaks (joosting et al., 1968; minuse et al., 1954) , serological studies indicated that this virus is widely distributed around the world and that the majority of humans develope antibodies against the virus early in life (matsuzaki et al., 2006; salez et al., 2014) . in a serological study carried out in france, 60%-70% of the population was found to be previously exposed to the virus, the highest rates of positive samples was found in the 16-30 years age group . in a 6-year tracking study in hospitalized children in spain, influenza c infections accounted for 13% of influenza-positive cases (calvo et al., 2013) . the results indicate intense circulation of influenza c virus in the human population. the primary host and reservoir of influenza c virus are humans, but there is evidence that this virus possesses the ability to also infect animals (muraki and hongo, 2010) . serological studies showed that antibodies against influenza c virus are widely present in dogs and especially in pigs (brown et al., 1995; horimoto et al., 2014; manuguerra et al., 1993; ohwada et al., 1987; yamaoka et al., 1991; youzbashi et al., 1996) . in 1981, a number of influenza c virus strains were isolated from pigs in beijing and these strains could be transmitted from pig to pig under experimental conditions (guo et al., 1983) . in 2011, an influenza c-like virus was isolated from clinically ill pigs exhibiting influenza-like symptoms (c/oklahoma/ 1334/2011) and also from cattle (d/bovine/oklahoma/660/ 2013) which subsequently turned out to be the main reservoir of this newly discovered virus (collin et al., 2014 (collin et al., , 2015 hause et al., 2013) . phylogenetic analysis showed that these strains have only 50% overall amino acid homology to human influenza c viruses, a divergence similar to that described between influenza a and b viruses (hause et al., 2013) . accordingly, no cross reactivity was observed between these strains and human influenza c virus antisera. this new strain has a broader cell tropism than human influenza c virus and is capable of infecting and transmitting by direct contact in both pigs and ferrets. it also encodes a novel mechanism for generating the m1 protein and, importantly, is unable to reassort with human influenza c virus and generate viable progeny. based on these differences to influenza c virus it was suggested that this virus warrants classification as a new genus of influenza virus, named influenza d virus (collin et al., 2014; hause et al., 2013) . influenza c virus particles exhibit two morphologies, either spherical with a diameter of 80-120 nm or filamentous with the same diameter but with lengths in µm range (waterson et al., 1963) . already during the budding process at the plasma membrane, filamentous particles may aggregate via their long axes into 500 μm long cord-like structures, which are all covered by a layer of surface projections (nishimura et al., 1990) . studies using reverse genetics showed that an amino acid exchange at residue 24 of the m1 protein (ala to thr) that reduces membrane association of this intrinsically hydrophobic protein eliminates cord formation and also affects virus morphology (muraki et al., 2004; nishimura et al., 1994) . another unique characteristic of influenza c virus particles observed by electron microscopy is a reticular hexagonal structure, which is formed by the hef protein and discussed in more detail below (apostolov and flewett, 1969; flewett and apostolov, 1967; herrler et al., 1981) . the influenza c virus genome consists of negative-sense, single-stranded rna (desselberger et al., 1980) , but in contrast to influenza a and b virus only seven (not eight) gene segments are present in virus particles (see fig. 1 for the structure of a virus particle and fig. 2 for the structure of viral genome segments). the longest three segments encode the proteins pb2, pb1 and p3 that form the heterotrimeric polymerase complex (yamashita et al., 1989) . the protein encoded by the third segment is named p3 (instead of pa as in the case of influenza a virus) since it does not contain negative charges at neutral ph. the fourth segment encodes the glycoprotein hef, the only spike of the viral membrane (herrler et al., 1988a) . the fifth segment encodes the nucleoprotein np that associates with the viral genome segments along its whole length and builds, together with the polymerases the viral ribonucleoprotein complex (vrnps) (nakada et al., 1984b) . the sixth segment encodes two proteins, the matrix protein m1, a peripheral membrane protein that covers the viral envelope on its inside, and cm2, a short transmembrane protein supposed to exhibit proton-channel activity required for dissociation of ribonucleoprotein (rnp) complexes from m1 and thus release of uncoated rnps into the cytoplasm where they are imported into the nucleus to start viral replication. m1 and cm2 are generated by alternative splicing, but in a different manner as described for influenza a virus. whereas in influenza a virus m1 is translated from a unspliced mrna, m1 of influenza c virus is generated from a spliced mrna. removal of an intron generates the stop codon uga such that a protein containing 242 residues is translated (yamashita et al., 1988) . the unspliced mrna encoding cm2 translates into a long precursor protein (374 residues), named p42. p42 contains an internal signal peptide (residues 239-259) which co-translationally targets the protein from the cytosol to the er and presents it to the translocon. here residues c-terminal to the signal peptide are translocated into the lumen of the er until translocation is stopped by a second hydrophobic region (residues 285-308) that functions as the transmembrane region (tmr) of cm2. the signal peptide is then cleaved by signal peptidase yielding the cm2 protein (115 residues) and the p31 protein (259 residues). p31, which is identical in sequence to m1 (except the 18 c-terminal amino acids), is rapidly degraded after cleavage from p42 suggesting that it does not plays any functional role for the viral life cycle (hongo et al., 1999; pekosz and lamb, 1998) . whether cm2 is a proton channel has not been directly demonstrated by biophysical assays, but it alters the intracellular ph in transfected cells and its transmembrane domain can substitute for that of the influenza a virus m2 protein (stewart and pekosz, 2012) . the seventh vrna encodes the two non-structural proteins ns1 and ns2 that are also generated via mrna splicing (nakada et al., 1985; nakada et al., 1986) . the unspliced mrna is translated into the ns1 protein (246 residues) and the spliced mrna translates the shorter ns2 protein (182 residues). the n-terminal 62 residues of ns1 and ns2 are identical in sequence, splicing then generates a shift in the orf such that the remaining residues are translated from a different reading frame (alamgir et al., 2000) . all orfs are flanked by non-coding (nc) sequences, which are more variable in length than those of influenza a and b virus (crescenzo-chaigne et al., 2008) . non-coding sequences are divided into conserved and non-conserved sequences. the first twelve nucleotides at each 3′ end (3′-ucguu/cuucgucc-5′) as well as the last eleven nucleotides at each 5′ end (5′-agcaguagcaa-3′) are conserved between genome segments (desselberger et al., 1980; robertson, 1979) and are partially complementary to each other, which enables the single stranded rna to form a "panhandle" structure (cheong et al., 1999; desselberger et al., 1980) . this peculiar structure serves as the promotor for transcription of crnas and vrnas, and is required for the endonuclease activity of the viral polymerase complex figure 1 . scheme of influenza c virus and influenza a/b virus particles. proteins having the same function are depicted with the same symbol. note that influenza c virus has only one spike protein, the hemagglutinin-esterase-fusion glycoprotein hef that combines the functions of both hemagglutinin (ha) and neuraminidase (na) from influenza a and b virus. pb1, pb2, p3 and pb1, pb2, pa are the polymerase proteins of influenza c virus and influenza a/b virus, respectively, that build together with the nucleoprotein np and the viral rna-segments the ribonucleoprotein complexes (vrnp). m1 is the matrix protein and m2 and cm2 the proton-channel. segment six and seven encode two proteins which are generated by splicing. influenza c virus possesses 7 minus-senses, singlestrand and segmented rna. each segment possesses 12 conserved nucleotides at 3′ terminal and 11 conserved nucleotides at 5′ terminal. a poly u motif is close to 5′ terminal and it transcripts into mrna poly a tail. each of the longest 5 segments possesses only 1 open-reading-frame (orf) and encodes pb2, pb1, p3, hef and np, respectively. (crescenzo-chaigne et al., 2008; fodor et al., 1994; hsu et al., 1987) . a uridine-rich region located at position 17 to 22 at the 5′ end of each segment is the template for the poly a tail present at the 3′ end of each mrna (desselberger et al., 1980) . while influenza a and b virus contain the two glycoproteins hemagglutinin (ha) and neuraminidase (na) inserted into the viral membrane, influenza c virus possesses only one spike designated hemagglutinin-esterase-fusion (hef) protein which combines the functions of both ha and na (herrler et al., 1988a; herrler and klenk, 1991) . like ha, it recognizes and binds to a receptor on the cell surface to initiate virus entry. however, the receptor determinant is not n-acetyl-neuraminic acid, but another derivative of neuraminic acid, namely n-acetyl-9-o-acetylneuraminic acid (rogers et al., 1986) . hef also catalyzes fusion of the viral envelope with endocytic vesicles by a mechanism that is believed to be similar to the well characterized fusion activity of ha. finally, hef is the receptor-destroying enzyme, which is the function of the neuraminidase (na) in influenza a and b virus. hef does not cleave the terminal sialic acid residue from carbohydrates, but has an esterase activity that removes the acetyl group from position c-9 of n-acetyl-9-oacetylneuraminic acid (herrler et al., 1985) . this function is probably required to release freshly budded virus particles from infected cells, which would otherwise be trapped at their plasma membrane if the receptor would still be present. interestingly, hef can substitute for both ha and na to support influenza a virus replication if its gene is equipped with the packaging signals from influenza a virus (gao et al., 2008) . after a description of the structure and the modifications of hef its three functional activities will be discussed in more detail below. all full-length hef protein sequences present in the influenza virus database (http://www.ncbi.nlm.nih.gov/genomes/ flu/database/nph-select.cgi) contain 641 amino acids (aa, excluding signal peptide), except hef from one strain which is one amino acid shorter. hef (like ha) is a typical type 1 transmembrane protein with a short n-terminal, cleavable signal peptide (14 amino acids), a long ectodomain (612 aa), a transmembrane region (26 aa) and a very short cytoplasmic tail (three aa). hef present in infectious virus particles is composed of two subunits, the n-terminal 432 amino acids are the hef1 polypeptide, the remaining sequence including the hydrophobic fusion peptide, the transmembrane domain (tmd) and the cytoplasmic tail is called hef2 (nakada et al., 1984a; pfeifer and compans, 1984) . hef proteins of the novel influenza c-like viruses (influenza d virus) contain a very similar number of amino acids (664 including signal peptide) as hef from influenza c virus, are predicted to also adopt a type i membrane topology, but the amino acid identity with hef is only ∼53% (hause et al., 2013) . according to a phylogenetic analysis of their hef genes the existing strains are divided into six genetic and antigenic lineages, taylor/1233/47, aichi/1/81, sao paulo/378/82, kanagawa/1/76, yamagata/26/81 and mississippi/80 (matsuzaki et al., 2003; muraki and hongo, 2010; muraki et al., 1996; speranskaia et al., 2012) . however, there is very little sequence variation (table s1 ) and thus influenza c virus was considered to be monosubtypic and stable in evolution, but reassortment between strains within the influenza c genus occur frequently which leads to the appearance of new strains better adapted to their host (matsuzaki et al., 2003; peng et al., 1994) . we aligned the hef sequences from the six influenza c virus lineages to reveal amino acid identity and other common characteristics (table s1 ). all residues important for the structure of hef, such as glycosylation sites, cysteine residues (with one exception, cys 332 in the taylor lineage, numbering of hef excluding the signal peptide), the n-terminal region of hef2 containing the hydrophobic fusion peptide and the amino acids of the receptor-binding and receptor destroying domain of hef are invariant. in general (and in contrast to the highly variable ha proteins of influenza a and b virus), only a few amino acid residues are not conserved through all lineages of hef. 35 of them are located in hef1 and seven in the smaller subunit hef2. there are three small regions in hef1 where many of the variable amino acids are clustered; residues 61-65 contain four amino acid substitutions, residue 165-172 six exchanges and residues 190-195 five substitutions (table s1 ). in the crystal structure of hef the variable regions are located in loops at the surface of the trimer; residue 165-172 and 190-195 near the receptor binding site at the top of the molecule and residues 61-65 near the esterase domain (fig. 4) . these amino acids have been shown to be antibody epitopes that gradually change due to antigenic drift (matsuzaki et al., 1992) . initial studies using electron microscopy showed that the hef spike forms a mushroom-shaped trimer consisting of a membrane-near stalk and a globular head (herrler et al., 1981; hewat et al., 1984) . x-ray crystallography of the bromelain-cleaved ectodomain of hef then revealed the high resolution structure (4.5 å) of the hef trimer. although there is only 12% amino acid identity between ha and hef, the overall structure of both molecules as well as folds of individual segments are quite similar, except an additional bulge, which is located at the lower part of the globular domain and contains the esterase region that is not present in ha (fig. 3) . similar to ha, the receptor-binding region is located at the top of the head domain, which consists only of hef1 residues. the stalk is formed by three 60 å long αhelices that contain the whole hef2 sequence and n-terminal residues 1-40 and c-terminal residues 367-432 of hef1. the fusion peptide at the n-terminus of hef2 is located around 35 å above the membrane, but in contrast to ha, the first four residues are exposed at its surface and not buried within the trimer (rosenthal et al., 1998; zhang et al., 1999) . the detailed structure of the receptor binding site and the catalytic center of the esterase activity will be discussed in more detail in the last paragraph. hef is synthesized on membrane-bound ribosomes and the primary translation product is subjected to a series of co-and post-translational modifications, most of them are required for proper folding and/or functioning. already during translocation of hef into the lumen of the er the n-terminal signal peptide is cleaved, carbohydrates are attached and intramolecular disulfide linkages are formed and probably remodeled. these co-translational modifications affect folding of the molecule and its trimerization, processes which are (at least in ha and other viral glycoproteins) a prerequisite for exit of cargo from the er (doms et al., 1993) . later on a long chain fatty acid is attached to a cysteine located at the end of the transmembrane region and hef is proteolytically cleaved into the subunits hef1 and hef2, a process that is essential for virus replication. timing of both modifications has not been analyzed for hef, but in ha palmitoylation occurs prior to proteolytic cleavage (veit and schmidt, 1993) . hef, like ha, contains only asparagine-linked carbohydrates; o-glycosylation does not occur (herrler and klenk, 1991; hongo et al., 1986a) . the composition of the carbohydrate chains has not been precisely determined, but apparently, some of them are not terminally glycosylated since they are not processed to an endo-h resistant form (pekosz and lamb, 1999) . the location of the individual glycosylation sites in the crystal structure of hef is depicted in fig. 4 . seven of the eight highly conserved n-glycosylation sequons (asn-x-ser/thr) are used. one is located in hef2 and six in hef1, three in the globular head and two in the hinge region that connects the stalk with the head. the site at position 589 is not glycosylated, probably because it is located too close to the membrane-spanning region and cannot be accessed by the oligosaccharide transferase (herrler and klenk, 1991; nakada et al., 1984a; pfeifer and compans, 1984) (fig. 4 and (h1n1)) and 1flc (hef from influenza c virus strain c/jhb/1/66 strain). hef1 and hef2 subunits of one monomer are drawn in red and green, respectively. the other two monomers are labelled in yellow and blue, respectively. evolution (skehel and wiley, 2000) , their distribution is quite similar to that of hef, i.e. the majority is located in the larger subunit. glycosylation of hef is crucial for proper folding of the glycoprotein by protecting it from proteolytic degradation and hence important for the presentation of antigenic epitopes (hongo et al., 1986b) . there are 15 cysteine residues in hef1, twelve of them form six intrachain disulfide linkages that stabilize the globular head domain. their location is depicted in the crystal structure of hef in fig. 5 . cysteine 332 is not required for proper folding and functioning of hef since it is exchanged by a tyrosine in the taylor lineage of influenza c virus. two cysteines, cys373 and cys399 that do not form a disulfide linkage in the mature protein are located at the hinge that connects the globular head with the stalk region. the remaining cysteine in hef1 forms an interchain disulfide bond with the only cysteine residue in the ectodomain of hef2, which is located at the bottom of the trimer. a similar distribution of disulfide bonds is present in ha of influenza a virus, i.e. one disulfide bond connects ha1 with ha2; the majority are intrachain bonds, three or four in ha1 and just one in ha2 (segal et al., 1992; skehel and wiley, 2000) . the rare occurrence of disulfide-bonds in hef2 and ha2 allows this subunit to perform the large conformational changes that catalyze membrane fusion. hef (at least from the strain c/jhb/1/66) is subject of a complicated folding procedure involving the formation and remodeling of intramolecular disulfide bond (szepanski et al., 1994) . in virus-infected cells freshly synthesized hef has an apparent molecular weight (mw) of 80 kda as demonstrated by both reducing and non-reducing sds-page. this corresponds to the predicted molecular weight of the glycosylated form of the protein (sugawara et al., 1981) . subsequently hef is converted to a form with a mw of 100 kda, which appears after non-reducing, but not after reducing sds-page, indicating that intramolecular disulfidebond formation causes the decrease in electrophoretic mobility (herrler et al., 1981) . besides reduction of disulfide bonds, proteolytic cleavage also converts the 100 kda into the 80 kda form suggesting that the 100 kda form possesses a strained conformation (herrler et al., 1979; szepanski et al., 1994) . when hef from c/jhb/1/66 was expressed from cdna in the absence of the other viral proteins, conversion into the 100 kda form was either very inefficient or not observed at all suggesting that the interaction of hef with other viral proteins is required for folding (szepanski et al., 1994) . expressed hef is not transported to the cell surface, which is in line with the established paradigm that proper folding is a prerequisite for exit of proteins from the er and hence transport to the plasma membrane (doms et al., 1993) . the defect in disulfide bond formation and surface transport was partially overcome by either deleting its short cytoplasmic tail (arg-thr-lys), replacing it by the longer cytoplasmic tail of influenza a virus ha or exchanging the two basic amino acids to acidic or hydrophobic residues (oeffner et al., 1999; szepanski et al., 1994) . in contrast, hef proteins from the strains c/california/78, c/ann arbor/1/50 and c/taylor/1233/47 were efficiently transported to the plasma membrane in the absence of other viral proteins (pekosz and lamb, 1999; vlasak et al., 1987) . in addition, conversion of an 80 kda to a 100 kda band was not obvious by sds-page, although heterogeneity of bands after non-reducing sds-page suggests that remodeling of (table s1 ). threonine residue 270, which is exchanged by isoleucine in a virus variant that has acquired the ability to grow in mdckii cells, is also marked as sticks. (this residues is termed thr284 in the publication (szepanski et al., 1992) since the 14 amino acid long signal peptide was included in the numbering). figure was created with pymol from pdb file 1flc. hef1 and hef2 subunits are drawn in red and green, respectively. disulfide bonds also occurs (pekosz and lamb, 1999) . the reason for this strikingly different behavior of hef proteins is unknown, but either subtle amino acid differences between hef proteins of different virus strains or between the cloned hef-gene and the gene present in virus particles of c/jhb/ 1/66 have been discussed (pekosz and lamb, 1999) . there are also other indications that folding of hef is more complicated than folding of ha. whereas has from several influenza a virus strains have passed the medial-golgi (determined as acquisition of endo-h resistant carbohydrates), around 15 min after synthesis and are rapidly (t 1/2 : 30 min) and completely transported to the cell surface (engel et al., 2012) , intracellular transport of hef is slow and incomplete. half times of more than 60 min for acquisition of endo-h resistant carbohydrates and for transport to the plasma membrane have been reported and only a fraction (70%) of all synthesized molecules appear at the cell surface (pekosz and lamb, 1999) . in addition, hef exhibits intrinsic temperature sensitivity. expression levels of hef at the plasma membrane are two times higher at 33°c compared to 37°c and, probably as a consequence, membrane fusion is more efficient at 33°c than at 37°c. since trimerization of hef is also reduced at 37°c the underlying cause of reduced cell surface exposure is slower and less efficient folding of hef at higher temperatures (takashita et al., 2012) which is reminiscent of temperature sensitive mutants of ha of influenza a virus . the temperature sensitivity of hef is probably an adaption of the virus to replicate only in the upper respiratory tract that has, due to contact with inhaled air before it is warmed up, a lower temperature than the lower respiratory tract. in the lab (cell culture and chicken embryos) influenza c virus is also amplified at 33°c where it grows to higher titers than at 37°c (crescenzo-chaigne and van der werf, 2007; o'callaghan et al., 1977; pachler et al., 2010; wagaman et al., 1989) . however, other proteins also influence the temperature preference for virus replication since the polymerase also exhibits a higher activity at 33°c than at 37°c (nagele and meier-ewert, 1984 ). another common modification of viral glycoproteins is the covalent attachment of fatty acids, usually palmitate (c 16:0) in a thioester-type linkage to cysteine residues located either at the cytosol-facing end of the transmembrane region or in the cytoplasmic tail (veit, 2012; veit et al., 2013) . hef of influenza c virus is unique in this aspect, since it contains mainly stearic acid linked to cysteine 652 (veit et al., 1990; veit et al., 1996) (fig. 6) . this longer chain fatty acid (c 18:0) was initially identified by chromatographic determination of hef-bound, [ 3 h]-labelled fatty acids, but results were recently confirmed by mass-spectrometry with c-terminal anchoring fragments of hef purified from virus particles (kordyukova et al., 2008) . these studies revealed also that influenza b virus ha possessing two cytoplasmic cysteines contains only palmitate, whereas has of influenza a virus having one transmembrane and two cytoplasmic cysteines contain both palmitate and stearate, but the latter is exclusively attached to the cysteine positioned in the transmembrane region (kordyukova et al., 2008; naeve and williams, 1990; naim et al., 1992; steinhauer et al., 1991; veit et al., 1990; veit et al., 1991) (fig. 6) . it was originally proposed that the different length of the cytoplasmic tails of ha (11 aa) and hef (3 aa) could be the reason for different fatty acid selection (veit et al., 1996) , but a recent comprehensive mutagenesis study with ha revealed that the location of a cysteine relative to the transmembrane region is the decisive factor for selective attachment of stearate (brett et al., 2014) . enzymes that attach palmitate and stearate to ha or hef (or to other viral glycoproteins) have not been identified so far, but likely candidates are members of the family of dhhcproteins, polytopic membrane proteins with the glu-his-his-cys motif in one of their cytoplasmic loops, that are known to acylate cellular proteins (greaves and chamberlain, 2011) . acylation of ha of influenza a virus is essential for virus replication, since (depending on the virus strain) either virus mutants with more than one acylation site deleted show drastically impaired growth or could not be created at all by reverse genetics (chen et al., 2005; wagner et al., 2005; zurcher et al., 1994) . recombinant virus lacking the acylation site of hef could be rescued, but viral titers were reduced by one log relative to wild type flu c (our unpublished results). the resulting virus particles have a regular protein composition and no changes in their morphology were obvious by electron microscopy, but their hemolytic activity is reduced indicating a defect in membrane fusion. this is in accordance with results on several ha subtypes showing that (i) the stearoylated cysteine at the end of the transmembrane span is less important for virus replication compared to the two cytoplasmic palmitoylated cysteines and that (ii) acylation affects opening of a fusion pore (sakai et al., 2002; ujike et al., 2004; wagner et al., 2005) . however, for h2 subtype ha it was reported that acylation does not influence ha's membrane fusion activity, but plays an essential role for virus particle assembly (chen et al., 2005; zurcher et al., 1994) . several studies illuminated the essential role of palmitoylation for association of ha with rafts, cholesterol and sphingolipid-enriched nanodomains on the cellular plasma membrane that serves as the viral assembly and budding site (engel et al., 2010; levental et al., 2010; melkonian et al., 1999; veit and thaa, 2011) (fig. 6) . interestingly, hef is apparently not a component of rafts, at least it does not associate with detergent-resistant membranes, their controversial biochemical correlate indicating that virus particles buds from the bulk phase of the plasma membrane (zhang et al., 2000) . after synthesis of the precursor hef0 a yet unknown protease hydrolyses the peptide bond between arg432 and ile433, which is located in the stem region of the trimeric spike and thus in a similar position as the cleavage site in ha (klenk et al., 1975; lazarowitz and choppin, 1975; rosenthal et al., 1998; sugawara et al., 1981) linkage, which is also located in the stalk region of the protein. proteolytic cleavage is an essential prerequisite for the membrane fusion activity of hef (and also of ha) since it enables the protein to get activated by low ph (herrler et al., 1981; kitame et al., 1982; ohuchi et al., 1982) . hef proteins from all influenza c virus strains contain a monobasic cleavage site and are in this respect similar to has from human, porcine, equine and low pathogenic avian influenza a viruses (herrler and klenk, 1991; neumann and kawaoka, 2006) . polybasic cleavage sites that are present in ha of highly pathogenic avian influenza a viruses and processed by the ubiquitous protease furin are not found in any hef protein. consequently, replication of influenza c virus is limited to the site of virus infection, the respiratory tract. spread to other tissues or even systemic infection, as observed for highly pathogenic avian influenza virus having a multibasic cleavage site between ha1 and ha2, does not occur with influenza c virus (horimoto and kawaoka, 1994; stieneke-grober et al., 1992) . multiple replication cycles of influenza c virus in tissue culture are enabled by addition of trypsin, whereas embryonated eggs produce infectious virus with cleaved hef (herrler et al., 1979; sugawara et al., 1981) . the enzyme catalyzing proteolytic cleavage of hef has not been identified so far, but since both ha and hef can be cleaved by trypsin at similar concentrations in vitro (5∼20 µg/ml) it seems likely that they are also activated by the same (or very similar) enzymes inside cells (herrler et al., 1979; nerome et al., 1979) . likely candidates are members of the family of type ii transmembrane serine proteases, such as hat (human airway trypsin-like protease), tmprss2 and tmprss4 (transmembrane protease serine s1, members 2 and 4). these enzymes are expressed at the plasma membrane of human bronchial/ tracheal epithelial cells where they cleave ha from various, but not all subtypes (bottcher-friebertshauser et al., 2013) . electron microscopy revealed another unique feature of influenza c virus particles not observed for influenza a and b virions. hef trimers on the surfaces of both spherical and filamentous particles are arranged in a reticular structure that has been described to consist mainly of hexagons (compans et al., 1977; flewett and apostolov, 1967; waterson et al., 1963) . the regular polymeric reticular structure can be observed not only on the surface of intact viral particles, but also when hef is removed from the membrane, either by limited proteolytic digestion or by spontaneous release (herrler et al., 1981) . these results indicate (i) that the hexagonal arrangement is an intrinsic feature of hef and does not require other viral proteins such as m1 and (ii) its formation likely involves lateral interaction between the ectodomains of hef; the tmr and cytoplasmic tail are not required to maintain the structures. which amino acids form lateral interactions between hef trimers and which function it serves for virus replication has not been investigated. one might speculate that the formation of a regular arrangement of hef trimers on the plasma membrane might induce membrane curvature, i.e. it acts like an extrinsic coat that might help to sculpt a virus particle out of the membrane. however, the lateral arrangement of exclusively hexagons would result in the formation of a flat structure without any curvature. thus, in order to create and cover a spherical particle, hef must form a precisely defined arrangement of pentagons and hexagons. virus budding might be reinforced by the matrix protein m1 that has been shown to form virus-like particles when expressed in the absence of other viral proteins (muraki et al., 2004) . m1 might execute a pushing force by oligomerization at the inner site of the plasma membrane. how these two assumed activities of hef and m1 are coupled is not obvious since the cytoplasmic tail of hef is very short, only three amino acids, and might thus not be able to bind to m1 with high affinity. ha of influenza a and b virus and hef of influenza c virus use different neuraminic acid derivatives as receptor (fig. 7) . hef binds to n-acetyl-9-o-acetylneuraminic acid (9-o-ac-neu5ac), which can be present on both glycolipids and glycoproteins to function as viral receptor (herrler et al., 1988a; rogers et al., 1986) . likewise, hef binds to its receptor regardless of whether 9-o-ac-neu5ac is attached via an α-2,3 or α-2,6 linkage to the following galactosyl residue (rogers et al., 1986) . in contrast, ha uses terminal n-acetylneuraminic acid (neu5ac) and the glycosidic bond of neu5ac influence the host specificity. avian influenza viruses usually bind to neu5ac-α2,3-gal while mammalian influenza viruses usually bind to neu5ac-α2,6-gal (thomas and noppenberger, 2007; trebbien et al., 2011) . the unique receptor specificity of influenza c virus has been used as an efficient tool to detect 9-o-ac-neu5ac on the surface of various cells (martin et al., 2003; muchmore and varki, 1987; zimmer et al., 1994) . there is some evidence that the abundance of 9-o-ac-neu5ac in cultured cells influences the tropism of influenza c virus. influenza c virus is usually grown in mdck i cells, whereas another subline of madin-darby canine kidney cell, mdck ii cells are (due to insufficient number of receptors) resistant against virus infection. a mutant of influenza c virus with the ability to replicate in mdck ii cells has an amino acid exchange from threonine to isoleucine at position 270 (see fig. 4 for the location of thr270 in the crystal structure of hef) that apparently increases the affinity of hef for its receptor (szepanski et al., 1992) . using reverse genetics it was recently confirmed that the exchange from threonine to isoleucine is necessary and sufficient to enable influenza c virus to grow in mdck ii cells (crescenzo-chaigne and van der werf, 2007). the crystal structure shows that hef binds to 9-o-ac-neu5ac in a similar pattern as ha binds to neu5ac. the binding elements consist of an α-helix, a loop and an extended strand (fig. 8a) . the key residues for binding hef to 9-o-ac-neu5ac are shown in fig. 8b . tyr127, thr170, gly172, tyr227 and arg292 form hydrogen bonds with hydroxyl-groups of the ligand, and some other residues form the structural support of the receptor binding site. the hef binding site also contains a unique hydrophobic pocket that accommodates the acetyl methyl group (rosenthal et al., 1998) . some coronaviruses, such as the prototype member mouse hepatitis virus (mhv) and human and bovine coronavirus, contain a hemagglutinin esterase (he) protein that also uses 9-o-ac-neu5ac as receptor (mayr et al., 2008; schwegmann-wessels and herrler, 2006; vlasak et al., 1988) . the crystal structure of he from bovine coronavirus revealed that the ligand is bound in an opposite orientation compared to hef and ha (zeng et al., 2008) (fig. 8c ). in accordance with its receptor binding specificity, hef is an esterase that cleaves acetyl from the c9 position of terminal 9-o-ac-neu5ac residues to release virus particles from infected cells (herrler et al., 1988a; herrler et al., 1985; mayr et al., 2008) (fig. 7) . the esterase activity of hef belongs to the class of serine hydrolase, where the −oh group of a serine residue performs a nucleophilic attack on the carbonyl-group of the substrate. since the −oh group is not sufficiently nucleophilic it is activated by two other amino acids that together build the typical catalytic triad of serine hydrolases, the amino acids serine, histidine and aspartic acid. the base histidine polarizes and deprotonates the −oh-group of serine to increase its reactivity whereas aspartic acid aligns and polarizes the histidine (charge relay system) (herrler et al., 1988b; kraut, 1977; pleschka et al., 1995) . crystallography in the presence of two non-hydrolysable receptor analogues of hef revealed that serine 57, aspartic acid 352 and histidine 355 are the key residues for the acetylesterase activity of hef (rosenthal et al., 1998) (fig. 8d ). prior to that it has already been shown that mutation of ser57 and his355 completely abolished the enzymatic activity of hef, essentially confirming the data from crystallography, but mutation of other residues in the vicinity, i.e. asp247, asn266 and his354 also affected the hydrolytic activity of hef (pleschka et al., 1995) . ser57 is positioned for nucleophilic attack on the carbonyl carbon of the 9-o-ac-neu5ac group. the carbonyl oxygen of the substrate points into an 'oxyanion hole' formed by the . cellular receptors and receptor-destroying activity of influenza c virus and influenza a and b virus. the structure of cellular receptors for hef from influenza c virus (n-acetyl-9-o-acetylneuraminic acid) and ha from influenza a and b virus (nacetylneuraminic acid) are shown. both neuraminic acid derivatives are the terminal sugars in carbohydrate chains attached to glycolipids or glycoproteins located at the cellular surface. subtypes of influenza a virus ha discriminate between an α2-6 and α2-3 linkage to the second galactosyl residue, a property that (partially) explains species specificity. hef of influenza c virus apparently recognizes n-acetyl-9-o-acetylneuraminic acid independent of its linkage to the next sugar. hef has also esterase activity that cleaves acetyl from the c9 position. in influenza a and b virus the receptor-destroying activity is performed by the na protein, which hydrolyzes the glycosidic bond between sialic acid and galactosyl residues. the cleaved bonds are indicated by a red line. side chain of asn117 and the nh -groups of gly85 and ser57 (rosenthal et al., 1998) . arg 322 of hef forms two hydrogen bonds with the sialoside carboxylate group (fig. 8d) . the structure of the esterase site is quite similar between hef and coronavirus he. the catalytic triad of he consists of the same amino acids, i.e. ser40, his329 and asp326; ser40 also forms an oxyanion hole with the side chains of gly75 and the nh group of asn104 (zeng et al., 2008) . membrane fusion between the viral envelope and endocytic vesicles is the crucial step to release the viral genome into the cytoplasm of the cell (hamilton et al., 2012; skehel and wiley, 2000) . there are two essential requirements for both hef and ha to catalyze membrane fusion: (i) the precursor proteins hef0 and ha0, must be cleaved into the subunits hef1 (ha1) and hef2 (ha2). (ii) the proteins must then be exposed to acidic ph to become fusogenic. this was initially demonstrated for influenza viruses by a simple membrane fusion assay, hemolysis of erythrocytes that occurs only if virus particles containing cleaved ha or hef are exposed to acidic ph (formanowski et al., 1990; huang et al., 1981; kitame et al., 1982; lenard and miller, 1981; maeda and ohnishi, 1980; ohuchi et al., 1982) . biochemical assays subsequently revealed that low ph initiates a conformational change since molecules become susceptible to proteolytic (rosenthal et al., 1998) and (c) from reference (zeng et al., 2008) with permission. digestion (formanowski et al., 1990) . the low ph is thought to cause protonation of specific amino acids that triggers the following large scale rearrangement of the proteins. histidines might play this role since their pkas match the ph of endosomes (5.5-6). for ha of influenza a virus specific histidine residues have been identified (mair et al., 2014) , but similar studies have not been performed with hef. in both influenza a and c virus threshold ph values that initiate membrane fusion differ from strain to strain by about 0.7 ph units. this does not necessarily mean that different histidines are the relevant target of protonation, but that (between strains) variable amino acids in the vicinity of a specific histidine affect its pka. for influenza c virus ph values required to cause hemifusion (measured as lipid mixing, range of 5.6-6.1) are 0.3-0.6 ph units higher than ph values for full fusion (measured by hemolysis, range of 5.1-5.7) (formanowski et al., 1990) . interestingly, kinetic studies with influenza c virus revealed a lag phase before onset of fusion that is not observed with influenza a and b virus (formanowski et al., 1990) . it is likely that the lag phase reflects dispersion of the lateral arrangement of hef spikes on the viral membrane that might hinder hef's conformational change. accordingly, when virus particles are treated with low ph before electron microscopy, hef spikes are less well ordered and the typical hexagonal structure disappeared (hewat et al., 1984) . the molecular details of the subsequent refolding of hef have not been revealed, but it is believed that they are similar to the well characterized conformational changes of ha that were elucidated by a comparison of the crystal structure of ha at neutral ph with the structure of a ha fragment after low ph treatment (bullough et al., 1994) . the first conformational change removes the fusion peptide from its buried location at the bottom of the stalk and exposes it at the surface of the molecule such that it can insert into the endosomal membrane ("jackknife mechanism"). a second conformational change then bends the ectodomain thereby drawing the fusion peptide towards the transmembrane region. this leads to a close apposition of viral and endosomal membranes, hemifusion with exchange of lipids, opening of a fusion pore and eventually complete merger of both lipid bilayers. the second conformational change requires so-called "heptad repeats", amphipathic helices which interact to form a stable 6-helix coiled coil domain (cross et al., 2009; harrison, 2008; kemble et al., 1994; skehel and wiley, 2000) (fig. 9a and 9b ). with the software "multicoil scoring form" (http://groups.csail.mit.edu/cb/ multicoil/cgi-bin/multicoil.cgi), a highly probable heptad repeats domain was found between amino acids 500 and 540, residues that encompass the long α-helix of hef2. they are thus in a similar position as the heptad repeats that form the six-helix bundle coiled-coil in the low ph structure of ha (fig. 9c ). this region of hef thus might convert from a long, uninterrupted helix into two smaller, antiparallel helices, which are connected by a loop and form the coiled-coil domain that stabilizes the fusion conformation (bullough et al., 1994) (fig. 9a and 9b) . the fusion peptides of ha and hef have similar, but also different features. the first 23 amino acids of ha2 (glfgaiagfieggwtgmidgwyg, sequence of h1 subtype) is highly conserved between subtypes and contains hydrophobic, aromatic, but also some negatively charged residues. it is also characterized by gxxg and gxxxg motifs that are known to mediate interactions between transmembrane segments. in a lipid environment the fusion peptide of ha forms a boomerang-like structure (aa 1-20) or a (tighter) helical hairpin (aa 1-23) (han et al., 2001; lorieau et al., 2012) . the sequence at the n-terminus of hef2 (ifgid-dliigllfvaiveagigg) is not conserved to that of ha2. however, if the first six residues, which are not buried within the trimeric stalk, are not taken into account some sequence homology between ha and hef is apparent. the fusion peptide of hef has a similar amino acid composition as that of ha, but glycine residues do not form gxxg or gxxxg motifs. the structure of hef's fusion peptide in lipid micelles is not known, but in the hef trimer it already adopts a looplike structure. although influenza c virus is currently not a serious threat to humans, it might be nevertheless fruitful and revealing to study its biology. whereas the receptor-binding and receptor-destroying activities of hef are now well characterized and its fusion activity is likely to be similar to that of ha, the mechanism of virus assembly and budding is largely unexplored and might be different for influenza a and c virus. if it is confirmed by more sophisticated methods that hef does not associate with membrane rafts (zhang et al., 2000) , it is likely that influenza a and c virus bud at different sites of the apical plasma membrane, membrane rafts in the case of influenza a virus (gerl et al., 2012; rossman and lamb, 2011) and the bulk phase or other domains in the case of influenza c virus. since rafts are believed to enrich viral proteins and deplete many cellular proteins they represent the first concentration step in the assembly of a virus particle that contains very little cellular proteins (veit and thaa, 2011) . one might speculate that the regular arrangement of hef trimers might substitute for the concentration of ha in rafts, i.e. its formation might displace cellular proteins from the viral assembly site. a regular arrangement of hexagons and pentagons might then help to shape a virus particle out of the plasma membrane. for influenza a virus it has been demonstrated that virus scission is achieved by the m2 protein that is targeted to the edge of the assembly site and inserts an amphiphilic helix into the inner leaflet to induce membrane curvature (rossman et al., 2010) . whether cm2 plays a similar role for release of influenza c virus has not been investigated, but the bioinformatic tool heliquest (http://heliquest.ipmc.cnrs.fr/) predicts the presence of an amphiphilic helix at the beginning of the cytoplasmic tail of cm2. the recent developments of reverse genetics systems for influenza c virus make some of the mentioned questions amenable to experimental verification (crescenzo-chaigne and van der werf, 2007; muraki et al., 2007; pachler et al., 2010) . thus, further studies might reveal common and different principles of influenza virus budding that might be helpful to combat the disease. . probable mechanism for hef-mediated membrane fusion. (a) probable conformational change of hef2 during membrane fusion. left part: structure of the hef2 subunit at neutral ph. the hr is located in the long α-helix of hef2 and thus in a similar position as the hr in ha. right part: hypothetical structure of the hef2 subunit at acidic ph. (b) hypothetical scheme for the hef-catalyzed membrane fusion mechanism. upper left part: hef binds to its receptor via its hef1 subunit (brown) and is endocytosed. upper right part: acidification of the endosome causes a conformational shift in hef2. the fusion peptide, which was (partially) buried in the stalk is exposed and inserts into the endosomal membrane. lower, left part: the middle part of the hr domain (green) changes its conformation from a helix to a loop, which causes bending of the molecule and close apposition of viral and cellular membrane allowing the exchange of lipids (hemifusion). lower, right part: interactions between the fusion peptide and the tmd of hef might cause opening of a fusion pore. (c) prediction of a heptad repeat (hr) in hef by online software "multicoils scoring form". phylogenetic analysis of influenza c virus nonstructural (ns) protein genes and identification of the ns2 protein further observations on the structure of influenza viruses a and c activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium site-specific s-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate serological studies of influenza viruses in pigs in great britain 1991-2 structure of influenza haemagglutinin at the ph of membrane fusion prospective study of influenza c in hospitalized children influenza virus hemagglutinin (h3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: m1 proteins of two subtypes differ in their ability to support assembly structure of influenza virus panhandle rna studied by nmr spectroscopy 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in france natural infection of dogs by influenza c virus: a serological survey in spain recombinant influenza c hemagglutinin-esterase as a probe for sialic acid 9-o-acetylation location of neutralizing epitopes on the hemagglutinin-esterase protein of influenza c virus frequent reassortment among influenza c viruses clinical features of influenza c virus infection in children a nationwide epidemic of influenza c virus infection in japan in 2004 influenza c virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. many raft proteins are acylated, while few are prenylated type c influenza virus. i. studies of the virus and its distribution selective inactivation of influenza c esterase: a probe for detecting 9-o-acetylated sialic acids the molecular virology and reverse genetics of influenza c virus evolution of the haemagglutinin-esterase gene of influenza c virus identification of an amino acid residue on influenza c virus m1 protein responsible for formation of the cordlike structures of the virus a mutation on influenza c virus m1 protein affects virion morphology by altering the membrane affinity of the protein fatty acids on the a/japan/305/57 influenza virus hemagglutinin have a role in membrane fusion influenza-c-virion-associated rna-dependent rna-polymerase activity effects of altering palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins complete nucleotide sequence of the influenza c/california/78 virus nucleoprotein gene influenza c virus rna 7 codes for a nonstructural protein the influenza c virus ns gene: evidence for a spliced mrna and a second ns gene product (ns2 protein) established cell line sensitive to influenza c virus host range restriction and pathogenicity in the context of influenza 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influenza c virus deacylation of the hemagglutinin of influenza a/aichi/2/68 has no effect on membrane fusion properties the influenza c virus cm2 protein can alter intracellular ph, and its transmembrane domain can substitute for that of the influenza a virus m2 protein and support infectious virus production influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease effects of various proteases on the glycoprotein composition and the infectivity of influenza c virus a single point mutation of the influenza c virus glycoprotein (hef) changes the viral receptor-binding activity post-translational folding of the influenza c virus glycoprotein hef: defective processing in cells expressing the cloned gene intrinsic temperature sensitivity of influenza c virus hemagglutininesterase-fusion protein studies on survival of influenza virus between epidemics and antigenic variants of the virus a further note on 1233 influenza c virus avian influenza: a review distribution of sialic acid receptors and influenza a virus of avian and swine origin in experimentally infected pigs influence of acylation sites of influenza b virus hemagglutinin on fusion pore formation and dilation palmitoylation of virus proteins timing of palmitoylation of influenza virus hemagglutinin association of influenza virus proteins with membrane rafts the hemagglutinating glycoproteins of influenza b and c viruses are acylated with different fatty acids site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins palmitoylation of influenza virus proteins the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza c viruses detection of influenza c virus by using an in situ esterase assay acylationmediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity the fine structure of influenza a, b and c viruses prevalence of antibody to influenza c virus among pigs in hyogo prefecture evidence that the matrix protein of influenza c virus is coded for by a spliced mrna comparison of the three large polymerase proteins of influenza a, b, and c viruses distribution of influenza c virus infection in dogs and pigs in bavaria structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution x-ray crystallographic determination of the structure of the influenza c virus haemagglutinin-esterase-fusion glycoprotein influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins modification of sialic acids by 9-o-acetylation is detected in human leucocytes using the lectin property of influenza c virus mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation the work in the authors' laboratory on influenza virus is supported by the german research foundation (sfb 740, tp c3). mingyang wang is a recipient of a phd fellowship from the china scholarship council.abbreviations 9-o-ac-neu5ac, n-acetyl-9-o-acetylneuraminic acid; c, cys, cysteine; cm2, influenza c ion-channel matrix protein 2; ct, cytoplasmic tail; er, endoplasmic reticulum; ha 0 , hemagglutinin precursor; ha 1 , ha 2 , hemagglutinin subunits; hef, hemagglutininesterase-fusion protein; hef 1 , hef 2 , hemagglutinin-esterase-fusion protein subunits; kda, kilodalton; mdck i, madin-darby canine kidney cell type i; mdck ii, madin-darby canine kidney cell type ii; m1, matrix protein 1; na, neuraminidase; neu5ac, n-acetylneuraminic acid; orf, open reading frame; rnp, ribonucleoprotein complex; s, ser, serine; sds-page, sodium dodecyl sulfate polyacrylamide gel electrophoresis; tmd, transmembrane domain; tpck-trypsin, tosylphenylalanine chloromethyl-ketone treated trypsin; wt, wild type. mingyang wang and michael veit declare that they have no conflict of interest. this article does not contain any studies with human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution 4.0 international license (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. key: cord-265751-q1ecpfyg authors: shahani, lokesh; ariza-heredia, ella j.; chemaly, roy f. title: antiviral therapy for respiratory viral infections in immunocompromised patients date: 2017-01-16 journal: expert rev anti infect ther doi: 10.1080/14787210.2017.1279970 sha: doc_id: 265751 cord_uid: q1ecpfyg introduction: respiratory viruses (influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus) represent the most common causes of respiratory viral infections in immunocompromised patients. also, these infections may be more severe in immunocompromised patients than in the general population. early diagnosis and treatment of viral infections continue to be of paramount importance in immunocompromised patients; because once viral replication and invasive infections are evident, prognosis can be grave. areas covered: the purpose of this review is to provide an overview of the main antiviral agents used for the treatment of respiratory viral infections in immunocompromised patients and review of the new agents in the pipeline. expert commentary: over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. advancements in novel antiviral therapeutics with high resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. the spectrum of immunocompromised hosts has expanded over the past decade owing to prolonged survival of patients with various malignancies and advances in both solid-organ and hematopoietic stem cell transplantation. novel immunosuppressive therapies create diverse immune deficits that generate a substrate for opportunistic infections [1] . these patients are defined by higher susceptibility to infections by organisms with lower native virulence than in immunologically normal hosts. influenza, parainfluenza, respiratory syncytial virus, coronavirus, human metapneumovirus, and rhinovirus represent the most common cause of respiratory viral infections in immunocompromised patients [1] . most of these infections are seasonal, and the viruses cause a wide range of upper respiratory tract infections (urtis) and lower respiratory tract infections (lrtis). however, adverse outcomes are far more likely in immunocompromised patients than in nonimmunocompromised individuals and include progression to pneumonia, respiratory failure, and increased mortality rates (1) (2) (3) (4) . in fact, the lrti rates and mortality rates for hematopoietic stem cell transplant (hsct) recipients and patients with hematological malignancies reportedly range from 10% to 50% [2] [3] [4] [5] . longterm complications associated with respiratory viral infections, such as airflow obstruction and bronchiolitis obliterans, have developed in hsct and lung transplant recipients [6, 7] . figure 1 highlights the high rates of progression to lrti and death among immunocompromised patients with common respiratory viral infections. the management of viral infections is challenging because viruses are intracellular parasites that use many of the host's own pathways to replicate and propagate. therefore, antiviral agents need to target specific viral components to avoid potential damage to host cells. figure 2 highlights the life cycle of viral replication and site of action of various antiviral agents. advances in the treatment of respiratory infections have been made over the past decades. table 1 highlights the current available agents for treatment of respiratory viral infections and table 2 lists the agents currently in the pipeline for these different viruses. the purpose of this review is to provide an overview of the main antiviral agents that are used in the management of respiratory infections in immunocompromised patients focusing on its clinical relevance and our experience, as well as to provide an update on the current investigational agents in the pipeline. the influenza virus is among the most common human respiratory viruses and belongs to the orthomyxoviridae family. four types of influenza viruses are a, b, c, and d. human influenza a and b viruses cause seasonal epidemics of disease almost every winter in the united states. influenza type c infections cause a mild respiratory illness and are not thought to cause epidemics. influenza d viruses primarily affect cattle and are not known to infect humans [17] . influenza a viruses are grouped into subtypes based on antigenic characteristics of 2 proteins on their surfaces, hemagglutinin (ha) and neuraminidase (na) with 18 different ha subtypes and 11 na subtypes. influenza a viruses can be further broken down into different subtypes. the most common subtypes of influenza a virus affecting humans are h1n1 and h3n2. influenza b viruses are not grouped into subtypes but can be further broken down into lineages. currently, circulating influenza b viruses belong to 1 of 2 lineages: b/yamagata and b/victoria [18] . the seasonal prevalence of influenza infections in immunocompromised patients, including solid-organ transplant and hsct recipients, closely parallels the community-wide prevalence, with peaks from december to february, with influenza b activity sometimes seen in april and may [19] . however, the illness has the potential to be more severe in this population than in healthy host [20] . without treatment reported, mortality rate range from 25% to 40% in immunocompromised patients, and is related to complications including pneumonia, and bacterial and fungal superinfections [21] . in a retrospective study, we identified profound lymphocytopenia (absolute lymphocyte count <200 cells/ ml), age greater than 65 years, and neutropenia (absolute neutrophil count <500 cells/ml) as potential risk factors associated with progression from urti to lrti [22] . early antiviral therapy within the first 48 h after presentation has been associated with improved prognosis in several studies [23] [24] [25] . the two main groups of antivirals used to treat influenza are m2 inhibitors (amantadine and rimantadine), which only act against influenza a, and na inhibitors active against influenza a and b: oseltamivir, zanamivir, and peramivir. m2 inhibitors inhibit the ion channel of the m2 protein in the influenza a virus, leading to defects in uncoating and assembly of the virus ( figure 2 ). the influenza virus enters its host cell via receptor-mediated endocytosis; thereafter, it is localized on endocytotic vacuoles. the m2 proton channel transports the ions needed for acidification of the influenza virus inside the vacuoles. this acidification is required for dissociation of the m1 protein from the ribonucleoprotein complexes and the onset of viral replication [26, 27] . the recommended dose of amantadine is 200 mg given once daily or 100 mg given twice daily (duration of therapy is generally 5 days) [8] . the most common side effects of these agents are gastrointestinal (nausea and vomiting) and effects on the central nervous system, including anxiety, insomnia, impaired thinking, confusion, lightheadedness, and hallucinations [8] . resistance of influenza a infection to m2 inhibitors results from mutations of the pore-lining residues in the ion channel, keeping adamantine and rimantadine from entering the channel [28] . according to data from the world health organization collaborating center for surveillance, epidemiology, and control of influenza at the us center for disease control and prevention (cdc), the rates of m2 inhibitors resistance have increased from 0.4% in 1994-1995 season to 12.3% in 2003-2004 [29] . however, during the 2005-2006 season, rates as high as 92% were reported for the influenza a (h3n2) virus [30] . recent cdc data demonstrate high prevalence of m2 inhibitors resistance in all influenza a (h3n2) and influenza a (h1n1) pdm09 virus isolates tested [31] . currently, the current advisory committee on immunization practices (acip) guidelines for treatment of influenza infections, do not recommend the routine use of amantadine and rimantadine in the usa for therapy or chemoprophylaxis for currently circulating influenza a virus strains [8] . nais block the active site of neuraminidase, resulting in uncleaved sialic acid residues on the host cell surface and viral envelopes ( figure 2 ). uncleaved sialic acid bound to viral ha causes viral aggregation on the host cell surface, which reduces the amount of virus released [8] . nais are virustatic, not virucidal, and early administration of them is a key factor in the development of resistance to the virus and their effectiveness. treatment with these inhibitors should neither be delayed while awaiting the results of diagnostic testing nor withheld from infected patients with indications for therapy who present more than 48 h after the onset of symptoms, particularly patients needing hospitalization [8] . in particular, the nais zanamivir and oseltamivir are first-line agents for treatment of and prophylaxis for influenza. oseltamivir is an oral nai usually prescribed as 75 mg orally twice daily (renally adjusted). the recommended duration of antiviral therapy is 5 days [8] . however, a longer duration (10 days) may be considered for severely ill patients or influenza a(h1n1) virus strains h275y substitution leads to resistance [10, 11] zanamavir nai intravenous zanamivir available through compassionate use program single dose of 600 mg administered intravenously [8] influenza a (h1n1) with both an h275y and an e119d or e119g na substitution lead to resistance to zanamivir [12, 13] . peramivir nai longer duration of 5 days in high-risk patients [9] . [8] . this antiviral therapy is most likely to provide the most benefits when initiated within the first 48 h after an infection occurs, so treatment should be initiated as soon as possible [8, 32] . some experts recommend higher dose of orally administered oseltamivir (e.g. 150 mg twice daily in adults with normal renal function) for the treatment of influenza infection in immunocompromised patients and those who are hospitalized and severely ill [8] . however, no clear evidence indicates that doubling the dose of oseltamivir is a more effective treatment than administering the normally prescribed dose in hospitalized patient, with or without severe illness [33, 34] . in a randomized trial of hospitalized patients with severe influenza, mortality rates were similar for patients who received oseltamivir at the double and standard doses [33] . however, 4 patients on the standard dose arm who were infected with influenza a (h1n1) virus without the h275y substitution at baseline acquired this substitution while on treatment. although no inferences can be made so far due to the small number of patients, using higher dose to prevent resistance and clinical failure in severely ill patients or immunocompromised patients still need to be determined in future studies [33] . in addition, a prospective study of adults hospitalized with influenza a and b infections treated with a single or double dose of oseltamivir twice daily demonstrated no differences between the groups in viral clearance, fever duration, oxygen supplementation, or hospitalization length [34] . patients receiving antiviral medications whose infections do not respond to treatment may have infections with antiviral-resistant influenza viruses. authors reported oseltamivir resistance, sometimes occurring within 1 week after treatment initiation, in immunocompromised patients with influenza a (h1n1) viral infections in the 2009 pandemic (pdm09) [35] . genotypic and phenotypic antiviral susceptibility testing are currently available to check the presence of mutations conferring resistance [36] . the more common emergence of resistance to oseltamivir in immunocompromised patients probably partly owes to prolonged viral shedding despite the use of antiviral therapy [37] . use of infection control measures is vital to reduce the risk of oseltamivir-resistant virus transmission in immunocompromised patients [32] . the pooled incidence rate of oseltamivir resistance for seasonal influenza a(h1n1) infections was estimated to be about 2.6% by a systematic review in 2009 [38] . however, in europe during the 2007-2008-winter season, rates of influenza a(h1n1) resistance were higher (up to 68%) [39] . authors reported that a specific substitution of the seasonal influenza a (h1n1) virus strains h275y (histidine-to-tyrosine substitution in neuraminidase), caused resistance in most of these cases [10, 11] . most circulating influenza a (h3n2) and influenza a (h1n1) pdm09 are still susceptible to oseltamivir and zanamivir. of the influenza a (h1n1) pdm09 infections tested during the 2013-2014 influenza season, 98.2% were susceptible to oseltamivir, and 100% were susceptible to zanamivir [40] . usually mild and limited to the first 2 days of treatment, nausea and vomiting are the most common reported toxic effects of oseltamivir, occurring in about 15% of recipients [41, 42] . zanamivir is administered via an inhaler in a dose of 2 inhalations (each inhalation of 5-mg) twice a day [27] . the chemoprophylaxis dosage of zanamivir is 10 mg (2 inhalations) administered once a day [8] . in randomized trials, this treatment shortened the duration of influenza symptoms by 1-3 days [43] [44] [45] [46] . use of intravenous (iv) zanamivir was evaluated in a recent phase iii clinical trial where the efficacy and safety of 300 mg or 600 mg of intravenous zanamivir twice daily were compared to 75 mg of oral oseltamivir twice daily for the treatment of hospitalized patients with influenza infections. the preliminary analysis showed no statistical difference in the time to clinical response (primary outcome variable) between iv zanamivir at 600 mg and oseltamivir, or between iv zanamivir at 600 mg or 300 mg [47] . it is currently available for compassionate use from its manufacturer via a us fda emergent investigational new drug application, and in a compassionate-use program in europe [48, 49] . zanamivir is currently the therapy of choice for oseltamivir-resistant influenza infections. however, the literature contains few cases of influenza virus with zanamivir resistance. infections with the h1n1 influenza strain possessing both an h275y na substitution (oseltamivir resistance) and an e119d (with aspartic acid replacing glutamic acid at position 119) or e119g (with glycine replacing glutamic acid at position 119) na substitution are resistant to zanamivir [12, 13] . the main adverse reactions to zanamivir are related to bronchospasm. use of inhalation powder to treat influenza infection is not recommended for patients with underlying airway issues (i.e. chronic obstructive pulmonary disease, asthma [8] . also, as it contains a lactose carrier, it can clog ventilator tubing nebulizers and mechanical ventilators [50] . peramivir is active against influenza a and b and was approved by the fda in 2014 for treating uncomplicated influenza infections in adults [51] . it is the first nai approved for iv use and is administered as a single iv dose of 600 mg because of its strong and prolonged affinity for the na in influenza virus. peramivir use should be considered for patients who are unable to tolerate oral or enteric drugs [51, 52] . use of a single dose of 600 mg of peramivir administered intravenously, alleviated influenza symptoms an average of 21 h sooner and fever approximately 12 h sooner than in patients given a placebo in a published report [52] . a study of patients at high risk for complications (including patients with diabetes, with chronic respiratory disease, or receiving immunosuppressive therapy), given peramivir for up to 5 days demonstrated shorter durations of illness than in patients given a single dose, and hence a longer duration of treatment for immunocompromised patients is suggested [9] . also, an open-label, randomized study of high-risk patients during the 2009 influenza a (h1n1) pdm09 demonstrated that use of peramivir (300 mg twice daily or 600 mg once daily) for 5-10 days reduced viral shedding and produced clinical improvement [53] . authors have reported cross-resistance of oseltamivir and peramivir in immunocompromised patients infected with influenza a (h1n1) virus containing the h275y variant [54] [55] [56] . therefore, patients infected with influenza a virus with a suspected or documented h275y substitution should not receive peramivir [14] . diarrhea is the most common reported adverse effect of peramivir [52] . more serious reactions associated with the central nervous system have included delirium and abnormal behavior leading to injury in patients with influenza who received oseltamivir or peramivir. primarily reported among children, these neurological events often began abruptly and resolved rapidly [57, 58] . the contribution of treatment with neuraminidase inhibitors to these events has yet to be established [59] , as some of these adverse events may have been related to the influenza infection rather than its treatment. authors have frequently reported neuropsychiatric symptoms in children with influenza infections; these symptoms were not always associated with the treatment with neuraminidase inhibitors [60-64]. das181 (ansun biopharma, san diego, ca, usa) is a recombinant fusion protein with a sialidase derived from actinomycoses viscosus that cleaves sialic acid receptors in host cells ( figure 2 ) [65] . this protein binds to cells and efficiently removes cell-surface sialic acid residues from respiratory epithelium, inhibiting viral infection. considering that das181 targets the host cells rather than the virus, it is less likely than virus-targeted drugs to induce treatment resistance. das 181 is administered via inhalation and has exhibited preclinical activity against numerous strains of influenza (a and b) and parainfluenza viruses (pivs) [65, 66] . in a phase ii double-blind, placebo-controlled clinical trial assessing influenza viral load and patient safety in otherwise healthy influenza-infected participants, an inhaled das181 dosage of 20 mg per day reduced viral loads and viral shedding in the multiple-dose group more than in patients taking a placebo as measured using quantitative polymerase chain reaction (p < 0.05); however, there was no difference in alleviation of flu-like symptoms between the placebo and the treatment arms. overall, das181 was well tolerated for up to 7 days when administered via daily inhalation for 5-7 days except for thrombocytopenia and liver test abnormalities in some instances. favipiravir (t705; toyama chemical, tokyo, japan) is an investigational antiviral drug that functions as a nucleotide analog and inhibitor of the viral rna polymerase of influenza. favipiravir is active against a broad range of influenza a, b, and c viruses, including highly pathogenic avian a (h5n1) and novel avian a (h7n9) viruses [67] , as well as influenza viruses resistant to treatment with nais or m2 inhibitors [68] . studies of preclinical cellular and mice models have demonstrated synergy of favipiravir with oseltamivir [69, 70] . this drug is currently being tested in phase iii clinical trials in the usa, europe, and latin america [69, 70] . laninamivir (cs-8958; biota pharmaceuticals, alpharetta, ga, usa) is a long-acting nai administered via a dry-powder inhaler. a phase iii randomized controlled trial demonstrated the superiority of a single inhalation dose of laninamivir octanoate to a 5-day course of oral oseltamivir in adults with seasonal influenza [71] . the drug is potentially effective against oseltamivir-resistant viruses and is currently available in japan. laninamivir has been demonstrated to be effective in reducing transmission of influenza infection from patients to household contacts. in a randomized trial, household contact of patient with influenza infection were randomly assigned to receive a single dose of laninamivir, 2 doses of laninamivir given daily for 2 days, or a placebo. family members in the laninamivir groups were less likely to develop clinical influenza as compared to the placebo group [72] . daiichi sankyo company, ltd. in japan plans to study the drug for the prevention of influenza, in single inhalation dose, in both adult and children [73] . jnj-63623872 (vx-787; janssen pharma, titusville, usa) is a nonnucleoside inhibitor targeting pb2, an influenza rna polymerase protein, inhibiting production of viral mrna, and preventing cell death [74] . it demonstrated activity against all influenza a strains tested in vitro. human studies have demonstrated significant decrease in virus shedding, when administered at a loading dose of 900 or 1200 mg on the first day followed by 600 mg once daily for 4 days [75] . a phase iib trial evaluating the dosing and frequency of the drug in healthy patients with uncomplicated influenza infection is currently under way [76] . nitazoxanide (nt-300; romark laboratories, florida, usa), an antiparasitic agent, appears to inhibit the maturation of influenza virus ha [77] . in a phase iib/iii trial, the treatment with nitazoxanide 600 mg twice daily for 5 days was associated with reduction in symptoms duration and viral titers among patients with acute uncomplicated influenza infection [15] . nitazoxanide has also shown synergistic effects in vitro with nais [78] and a current phase iii trial to investigate the efficacy of this synergism has been completed and results are awaited [79] . medi8852 (astrazeneca, gaithersburg, maryland, usa) is a monoclonal antibody targeting the highly conserved epitope in the ha stalk of influenza a virus [80] . it is currently being evaluated in a phase ib/iia clinical trial for safety and efficacy of a single intravenous dose in combination with oseltamivir, and as a monotherapy in adult patients with confirmed acute, uncomplicated influenza a infections [81] . vis410 (visterra, inc., cambridge, ma, usa) is a neutralizing human igg1 anti-ha antibody, which binds to a conserved region of the ha stalk of the influenza virus [82] . in mice, it resulted in 100% protection from influenza infection when administered prophylactically [83] . coinfections with bacterial pathogens and influenza infection may lead to significant morbidity and mortality. bacterial coinfection is associated with an increase in disease severity, hospital admission and even mortality, with streptococcus pneumoniae and staphylococcus aureus been the most common pathogens in such setting followed by, haemophilus influenzae, and group a streptococci [84] . a recent meta-analysis by klein et al. (2016) noted that older age, a higher apache ii (acute physiology and chronic health evaluation ii) score, diabetes mellitus, and sepsis were risk factors predisposing to coinfections [84] . the american college on immunization practices (acip) recommends simultaneous antiviral and antibiotic treatment for severely ill patients with influenza infections [8] . consistent with the acip guidelines, the infectious disease society of america (idsa) guidelines recommend appropriate use of diagnostic tests as guidance for targeted antibacterial therapy for hospitalized patients. recommended antibacterial therapy includes cefotaxime, ceftriaxone, and respiratory fluoroquinolones. treatment with vancomycin, linezolid, or other agents directed against methicillin-resistant staphylococcus aureus (mrsa) is recommended for patients with confirmed or a compatible clinical presentation of mrsa infection (i.e. shock and necrotizing pneumonia) [85] . respiratory syncytial virus (rsv), an enveloped, singlestranded rna virus of the family paramyxoviridae, frequently causes seasonal upper respiratory viral infections in infants and young children. symptomatic rsv reinfections in immunocompetent adults often consist of urtis lasting 2-5 days. in immunocompromised patients such as hsct and solid-organ transplant recipients, rsv infections may progress to severe and life-threatening lrtis [86] . investigators at the university of texas md anderson cancer center developed an immunodeficiency scoring index for rsv that accounts for major risk factors that identify hsct recipients who are at high risk for progression of rsv infection to an lrti and rsv-associated mortality [87] . age, neutropenia, lymphocytopenia, graft-versus-host disease, use of myeloablative conditioning regimens, use of corticosteroids, a recent hsct, and pre-engraftment are the main risk factors that are weighed in this index to categorize patients into prognostic risk groups [67] : low (0-2), moderate (3) (4) (5) (6) , and high (7) (8) (9) (10) (11) (12) risk. the authors reported a statistically significant trend of higher incidence of lrti-and rsv infection-associated mortality as the risk increased from low to moderate to high (p < 0.001). patients in the high-risk group demonstrated greatest benefit of ribavirin-based therapy at the urti stage and were at the highest risk for progression to lrti and death in the absence of antiviral therapy. we suggest using the immunodeficiency scoring index for rsv to identify high-risk patients who would benefit from treatment with aerosolized ribavirin. as seen in hsct recipients, researchers noted an association between a low lymphocyte count (mean, 580 cells/mm 3 ) and rsv infection progression to an lrti in solid-organ transplant recipients with lung transplant recipients having the highest risk of adverse outcomes [88] . ribavirin is a nucleoside analog that resembles guanosine. as a monophosphate, ribavirin inhibits the dehydrogenase enzyme, which is essential for the synthesis of guanosine triphosphate, and reduces the cellular deposits of guanidine necessary for viral growth. it inhibits the initiation and elongation of rna fragments resulting in inhibition of viral protein synthesis (figure 2 ) [89] . aerosolized ribavirin is the only fda-approved treatment of severe rsv-lrtis in hospitalized infants and young children with underlying compromising conditions (prematurity, cardiopulmonary disease, or immunosuppression) [90] . rsv infections markedly increase morbidity and mortality rates in hsct recipients. ribavirin-based antiviral therapy is recommended by european guidelines for leukemia patients and hsct recipients at high risk of complications [16, 91] . in a systematic review of the literature by shah et al. [92] and based mainly on retrospective studies, any form of ribavirinbased therapy (alone or in combination with immunomodulators) prevented urtis from progressing to lrtis (from 45% to 16%) and improved mortality rates (from 70% to 35%) when compared to no therapy in adult hsct recipients [92] . whether the benefits of aerosolized ribavirin versus the oral form justify its use in immunocompromised patients remain subject of controversy, especially given the recent drastic increase in the cost of the aerosolized form [93] . researchers have systematically reviewed the use of oral ribavirin to treat various respiratory viral infections, including rsv infections [94] . the authors concluded that mortality rates were highly variable and often dependent on the underlying severity of illness rather than the effects of oral ribavirin; however, there were not randomized or control studies available for evaluation [94] . in 2004, khanna et al. [95] reported that oral ribavirin had a good safety profile in 34 rsv-infected patients with upper or lower respiratory tract infection but could not draw a strong conclusion regarding its efficacy. the doses recommended in the european conference on infections in leukaemia (ecil-4) guidelines included a loading dose of 600 mg followed by 200 mg every 8 h the first day, 400 mg every 8 h the second day, and then escalation daily to a maximum of 30 mg/kg/day [16] . the iv formulation of ribavirin has been beneficial in some cases of rsv infection, but further trials are needed [96, 97] . various other therapies such as, intravenous immunoglobulin (ivig), rsv hyperimmunoglobulin, and palivizumab (a monoclonal rsv igg), have been used for treatment and prevention of rsv infections in immunocompromised patients with mixed results. early studies demonstrated that ribavirin in combination with rsv ivig (respigam; medimmune, gaithersburg, md, usa), an hyperimmune globulin preparation with high concentrations of rsv-neutralizing antibodies, offered a mortality advantage over ribavirin alone in rsv-infected pediatric hsct recipients with lrtis [98] . however, production of rsv ivig has since then been discontinued because of the introduction of alternatives such as palivizumab, an engineered anti-rsv monoclonal antibody. palivizumab is currently approved for prophylaxis for rsv infection in a select group of high-risk infants with bronchopulmonary dysplasia, infants with a history of premature birth (â�¤35-week gestational age), and children younger than 24 months with hemodynamically significant congenital heart disease during the rsv infection season [99] . the american academy of pediatrics recommends a palivizumab dose of 15-mg/kg body weight administered monthly throughout the rsv infection season (first dose administered prior to commencement of the season and a maximum of 5 doses per season) [99] . kassis et al. demonstrated the utility of palivizumab for prophylaxis in a hsct unit following an rsv infection outbreak. palivizumab was useful in preventing rsv infection in 16 rsv-negative patients considered to be at high risk for complications from rsv infection when combined with strict infection-control measures [100] . in contrast, palivizumab failed to demonstrate any impact on progression to lrti or mortality in a case series of 40 allogeneic hsct recipients infected with rsv [101] . given the questionable efficacy and high cost of palivizumab, mainly for adult patients, routine use of it is not encouraged in the adult immunocompromised population [102] . in adult hsct recipients with rsv pneumonia, uncontrolled studies suggested that use of combination therapy with ribavirin and ivig improved survival [103, 104] . additional studies of rsv-infected lung transplant recipients demonstrated that combined treatment with ribavirin (nebulized or iv) with ivig and/or corticosteroids reduced mortality rates, length of mechanical ventilation, and incidence of bronchiolitis obliterans [105] . although combined use of ribavirin and ivig has not been supported by a randomized trial, this expensive treatment is reserved for select patients with rsv-related lrtis and severe immune deficiency [103, 104] . aln-rsv01 (alnylam pharmaceuticals, cambridge, ma, usa) is small-interfering rna (sirna) that inhibits rsv replication by interrupting synthesis of the viral nucleocapsid protein, and treatment with this compound has demonstrated promising results in phase ii clinical trials [106] . rna interference is a natural process and sirnas induce sequence-specific degradation of mrna and thus reduce expression of the corresponding protein [106] . in a randomized, double-blind, placebocontrolled trial, researchers administered prophylactic aln-rsv01 as a nasal spray before experimental inoculation in healthy adults wild-type for rsv and observed a 38% reduction in the number of infections [106] . in a phase iia randomized, double-blind, placebo-controlled trial of adult lung transplant recipients with confirmed rsv urtis, use of aerosolized aln-rsv01 (0.6 mg/kg) daily for 3 days significantly reduced mean cumulative daily symptom scores (p = 0.035) and the incidence of progressive bronchiolitis obliterans syndrome by day 90 more so than in patients given a placebo (6% vs. 50 %; p = 0.027) [107] . also, a recent phase iib trial with lung transplant recipients demonstrated a trend of decreasing new or progressive bronchiolitis obliterans (bos) incidence (14% vs. 30%; p = 0.058) at 180 days. the treatment effect was enhanced with initiation of aln-rsv01 use fewer than 5 days after symptom onset [108] . whether further development of this compound would be pursued remains unknown at the present time. mdt-637 (microdose therapeutx, monmouth junction, nj, usa and gilead sciences, foster city, ca, usa) and the gs-5806 (gilead sciences, foster city, ca, usa) are both antiviral fusion inhibitors. oral gs-5806 has shown safety and tolerability in healthy adults [109] . currently, two phase iib trials are underway to evaluate the antiviral effects, pharmacokinetics, safety, and tolerability of gs-5806 in hsct recipients with either rsv uri or lrti [110, 111] . mdt-637 is delivered as a dry-inhalation powder and has been evaluated in a phase ii trial to assess safety and tolerability in healthy adults [112] . al-8176 (alios, south san francisco, ca, usa) is a nucleoside inhibitor of the l-protein [113] and has demonstrated efficacy in human challenge studies [114, 115] . l-protein is an rna-dependent rna polymerase of rsv, and its inhibition impact future viral replication [113] . in a randomized, double-blind, placebo-controlled phase ii challenge study conducted in healthy adult volunteers who were infected intranasally with rsv, al-8176 was well tolerated and demonstrated significant reduction in rsv viral loads (p < 0.0002) and improvement in symptom scores (p < 0.02) when compared to placebo [114, 115] . polyclonal high-titers rsv immunoglobulin (ri-001; adma biologics, inc., hackensack, nj, usa) is being tested in patients who are immunocompromised to prevent progression of urtis to lrtis. preliminary results are pending [116] . motavizumab is a newly developed monoclonal antibody targeting a highly conserved antigenic site on the fusion glycoprotein of rsv. it had antiviral effects in hospitalized children but was not superior to palivizumab in seasonal rsv prophylaxis in preterm infants with chronic lung disease of prematurity at-risk for rsv related lrti, hospitalization or death [117] . in 2010, fda antiviral drugs advisory committee declined the request for licensure of motavizumab. the concerns raised included the lack of additional benefits of motavizumab over palivizumab and the additional risk of cutaneous hypersensitivity reactions [118] . parainfluenza virus (piv) is a single-stranded, enveloped rna paramyxovirus comprising 4 antigens that share serotypes, although most clinical piv infections are caused by types 1, 2, and 3. in the general population, most clinical piv infections are caused by piv-3 followed by piv-1 and piv-2 [119] . although piv infections often occur year round, peak seasonal activity reportedly occurs from late september to december for piv-1 and during the spring and summer months for piv-3 [119] . piv most commonly affects the upper respiratory tract after an incubation period of 1-4 days and is commonly associated with urtis in children. in immunocompromised patients, authors described progression to lrti in about 37% of hsct recipients and piv-infected patients with hematological malignancies [120] . the risk factors for progression from piv-urti to piv-lrti include lymphocytopenia, neutropenia at the onset of infection, use of corticosteroids during piv-urti, and respiratory coinfections [120] . risk factors for piv-related mortality include lymphocytopenia, young age (<2 years), refractory or relapsed underlying hematological malignancies, an acute physiology and chronic health evaluation ii score greater than 15, respiratory coinfections, and steroid use at infection onset [120] . no antiviral agents are licensed to treat piv, so its management is limited to supportive care. in some instances, physicians have used oral or aerosolized ribavirin with or without ivigs for the treatment of piv lrti in immunocompromised patients with various outcomes [121] . new antiviral agents and vaccines in the pipeline may change the paradigm of piv infection management, particularly in immunocompromised patients. although, as described above, clinical providers have used oral and aerosolized ribavirin to treat piv [122] , the available data on their use for this infection remain controversial. two recent systematic reviews on hsct recipients and hematological malignancy patients demonstrated that ribavirin was not significantly more effective at preventing the progression of urti to lrti or piv-associated mortality than was supportive care alone [120, 123] . also, in lung transplant recipients with piv infections, use of oral ribavirin for 14 days at 15-20 mg/kg/day in 2 divided doses (dose length) was associated with some benefits, including a lower rate of bronchiolitis obliterans syndrome within 6 months after development of the infection than that in a non-ribavirin group (5% vs. 24%; p = 0.02) [124] . given, the lack of clear evidence of a positive outcome in piv-infected patients as well as the absence of control studies, justified recommendation for the use of ribavirin for the treatment of piv in immunocompromised patients cannot be made. as described above, das181 is a novel sialidase fusion protein with activity against piv in vivo and in vitro because it effectively cleaves sialic acid from respiratory epithelial cells, preventing piv entry into the cells (figure 2 ) [125] . das 181 have been administered on a compassionate-use basis for severe piv infections in immunocompromised patients, with apparent clinical benefits and antiviral effects [126] . in a case series, 4 pediatric hsct recipients with piv detected in respiratory specimens (2 from the upper respiratory tract and 2 from the lower respiratory tract) received inhaled das181 for 5-10 days. oxygen requirements and respiratory rates improved in all 4 patients, and their viral loads decreased within 1 week after therapy initiation [127] . in a similar case series, 16 hsct recipients received das181 daily to treat piv infections (14 lrtis and 2 urtis). of the 16 patient, 9 had complete clinical response, and 4 patients had a partial response to das181 therapy. of 7 patients with virological and spirometric data, 5 had reduction in piv viral load in nasopharyngeal secretions and 4 had improved forced expiratory volumes by the end of treatment [128] . in an ongoing phase ii double-blind, placebocontrolled trial, investigators are examining the effects of das181 in immunocompromised patients with piv-related pneumonia [129] . a recent report described the use of das181 in 13 hsct recipients: 56% of them had responses to therapy, and 24% had partial responses. they also had greater than a 1-log reduction in piv viral load [130] . bcx2798 and bcx2855 (biocryst pharmaceuticals, inc., birmingham, al, usa) are new antiviral hemagglutinin neuraminidase inhibitors and have been evaluated in mouse models of infection with a virus similar to piv, recombinant sendai virus [131] . bcx2798 and bcx2855 have demonstrated antiviral activity against piv-3 by markedly reducing pulmonary viral titers and mortality rates in rats when given intranasal within 24 h after development of infection [132] . human studies of these two inhibitors have yet to be undertaken. human rhinoviruses (hrvs) are positive-sense, single-stranded rna viruses with icosahedral symmetry. they are characterized into three genetically distinct groups designated a, b, and c within the genus enterovirus and family picornaviridae. the viral capsid that encases the rna genome is made up of four proteins: vp1, vp2, vp3, and vp4. the remaining nonstructural proteins are involved in viral genome replication and assembly [133] . hrv infections are responsible for more than one half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work in the usa [134] . peak incidence occurs in the early fall, with a smaller peak in the spring [135] . both peak incidences are associated with urti, otitis media, and sinusitis [133] . a recent study of patients going to the emergency room with influenza-like illnesses who also had hematological malignancies demonstrated that 40% of the patients (110/272) presented with hrv infections. researchers found that the severity of hrv infection in these patients was similar to that of h1n1 influenza in the 2009 pandemic. nearly 40% of patients with hrv-associated respiratory symptoms were admitted to the hospital, 29% had lrtis, and 11% needed intensive care unit admission [136] . other studies, including those with hsct recipients, have replicated these results [137, 138] . markers for increased immunosuppression and illness severity in patients with hrv infections, including neutropenia (absolute neutrophil count â�¤500 cells/âµl), hypoalbuminemia (serum albumin level â�¤3.2 mg/dl), and infections with a respiratory co-pathogen(s) were associated with progression to hrv-related pneumonia [138] . in contrast, parody et al. [139] described a much lower rate of progression to lrti (13%) in a similar patient population. use of a different case definition for hrv infection may explain the disparity in the prevalence of lrtis in these two reports. chronic hrv infection has occurred in lung transplant patients [140] . furthermore, in a study of 36 adult lung transplant recipients, 13% of all bronchoalveolar fluid specimens obtained from 15 (42%) symptomatic patients over a 2-year period were positive for hrv [141] . currently, treatment of hrv infection consists of supportive care. antiviral medications for hrv are under investigation. the viral capsid was the initial viral protein targeted in the development of drugs to inhibit viral replication. these drugs work by binding to the hydrophobic pocket of the viral capsid, resulting in a conformational change, increasing the stability of the virion and interfering with its ability to interact with the cellular receptor [142] . vapendavir (aviragen therapeutics, alpharetta, ga, usa) is an oral agent that binds to the hrv vp1 capsid protein and prevents the release of viral rna into the target cells. vapendavir exhibits antiviral activity against hrv-a and hrv-b serotypes; however, activity against hrv-c is not yet known. a phase ii randomized, double-blind, placebo-controlled study of asthmatic adults with hrv urtis showed lower severity scores for cold symptoms, greater mean reductions in asthma scores, and higher evening peak expiratory flow in those given vapendavir than placebo [143] . pleconaril (viropharma, exton, pa, usa) was the first developed capsid-binding anti-hrv agent. two phase iii multicenter studies in the usa and canada randomized 2,096 healthy subjects with self-diagnosed colds into groups receiving pleconaril at 400 mg orally twice daily or placebo for 5 days. in the primary-efficacy population, which consisted of 1,363 subjects with hrv rna detected in nasal secretions, pleconariltreated subjects experienced a 1-day reduction in the mean duration of illness compared to the placebo group (7.3 days versus 6.3 days; p = 0.001) [144] . in another study, researchers found an association between hrv susceptibility to pleconaril and clinical outcomes [145] . the fda declined licensing of pleconaril owing to concerns of development of resistant virus strains. additionally, interactions among cytochrome p-450 3a, hormonal contraception, and antiretroviral therapy for human immunodeficiency viral infection may reduce the effectiveness of pleconaril [146] . rupintrivir (agouron pharmaceuticals, inc., san diego, ca, usa) is an in vitro 3c protease inhibitor that acts against many hrvs and enteroviruses. rupintrivir reduced viral loads and respiratory symptoms in healthy volunteers with experimentally induced rhinovirus colds and was well tolerated by the participants [147] . however, in trials of patients with natural infections, rupintrivir failed to reduce viral loads or symptom severity [148] . inhaled interferon-beta (sng001, synairgen plc, southampton, england) was tested in a phase ii, placebo controlled randomized trial of adult asthmatics receiving inhaled corticosteroids and with a history of deterioration with colds, and was associated with significant improvement in asthma symptoms, 65% fewer moderate exacerbations, improved morning peak expiratory flow rates, and reduced use of relief bronchodilators [149] . human metapneumovirus (hmpv) is an enveloped, negativesense, single-stranded rna virus. it is the first human member of the metapneumovirus genus in the pneumovirinae subfamily within the paramyxoviridae family. first identified in the netherlands in 2001, serological studies of antibodies against hmpv indicated that the virus has circulated in humans for at least 50 years [150] . phylogenetic analysis has identified two genotypes of hmpv: hmpv a and hmpv b [151] . hmpv uses a fusion mechanism to penetrate target cells. the fusion process consists of insertion of the hydrophobic fusion peptide into the target cell membrane and refolding of the f protein. this step requires the interaction of two specific domains: heptad repeats a and b [152] . investigators have studied this process for development of it as a potential antiviral treatment. hmpv causes respiratory infections and has a seasonal distribution comparable with those of influenza and rsv infections [153] . although immunocompromised patients acquire hmpv infections at the same frequency as immunocompetent individuals, they are at higher risk for severe infections. this higher risk likely can be attributed to poor viral clearance [153, 154] . a recent systematic review in hsct recipients and hematologic malignancy patients estimated the incidence of progression of hmpv-urti to lrti at 34% and an associated mortality rate of 6% [154] . factors associated with this progression in hsct recipients include early onset of infection after transplantation, steroid use, and a low lymphocyte count [155] . to date, treatment of hmpv infections has been mainly supportive. researchers have investigated several treatment regimens. standard immunoglobulin preparations have inhibited replication of hmpv in vitro [156] , and approaches such as use of selective immunoglobulins and fusion inhibitors have demonstrated antiviral activity in vitro and in animal studies. administration of oral or aerosolized ribavirin with or without polyclonal ivigs has been advocated for the treatment of severe hmpv infections and is currently used in some centers for highrisk patients [156] [157] [158] [159] [160] , although most data are still anecdotal. fusion inhibitors target the initial steps of viral fusion and penetration into the human cell. fusion inhibitors with sequence similarity with the hra and hrb domains of the viral fusion protein have demonstrated important role in viral inhibition. balb/c mice inoculated with lethal intranasal hmpv challenge were completely protected from clinical symptoms and mortality if they simultaneously received the hra2 peptide [152] . hr-1 peptides also have demonstrated effectiveness as viral inhibitors [161] . researchers developed mab 338 (medimmune, gaithersburg, md, usa) to target hmpv fusion proteins. it appeared to effectively neutralize hmpv in golden syrian hamster models and reduce the pulmonary viral titers, thereby limiting severe acute manifestations and bronchial hyper-reactivity [162] . a human monoclonal antibody fragment (human fab ds7) with biological activity against the fusion protein of hmpv demonstrated prophylactic and therapeutic potential against severe hmpv infections when tested in cotton rats [163] . human coronavirus (hcov) is a single-stranded, enveloped rna virus belonging to the family coronaviridae. in temperate climates, hcov infection is transmitted primarily during the winter and is a well-recognized cause of urtis during the respiratory viral season [164] . usually mild in immunocompetent hosts, hcov infection in immunocompromised populations may progress to lrti [16] . emerging hcovs, such as severe acute respiratory syndrome-associated hcov in 2002-2003 and the more recently identified middle east respiratory syndrome in 2012-2013, have prompted a further impetus to develop therapeutics against this infection because current antiviral agents are lacking and treatment of it remains palliative. discovery and in vitro evaluation of hcov therapy is ongoing, including investigation of entry inhibitors, human monoclonal antibodies, and proteosome inhibitors [165] [166] [167] [168] . respiratory viral infections continue to be major clinical problems in immunocompromised patients. high clinical suspicion and the use of rapid diagnostic tests remain crucial, as early treatment is associated with improved outcomes and reduced transmission. several advances in the prevention and treatments of influenza infection have occurred in recent decades. inadequate efficacy of the influenza vaccine as well as the emergence of antiviral resistance, which appears to occur more commonly in immunocompromised patients than in healthy host, underline the difficulties in management of respiratory infections in immunocompromised individuals. rsv and piv infections continue to be associated with high morbidity and mortality, and further advancements in prevention of and therapy for respiratory viral infections are needed. the impact of rhinovirus, coronavirus, and metapneumovirus infection in patients with compromised immune systems is becoming evident as new, widely available molecular testing improves the recognition of these viral infections. over the past decade, important diagnostic advances, specifically, the use of rapid molecular testing has helped close the gap between clinical scenarios and pathogen identification and enhanced early diagnosis of viral infections and understanding of the role of prolonged shedding and viral loads. respiratory viral infections can be complicated for both clinicians and immunocompromised patients. future studies that identify and validate scoring systems to ascertaining patients at highest risk for complications of respiratory viral infections including lrti, are of utmost importance. also, identification of long-term complications after respiratory viral infections in immunocompromised patients and devising interventions for prevention will be of the utmost value. last, advancement in novel antiviral therapeutics with high-resistance thresholds and effective immunization for preventable infections in immunocompromised patients are needed. to curtail the impact of respiratory viruses on our immunocompromised patients, we should focus on prevention of exposure and progression to worse outcomes. multiple interventional modalities should be studied from stimulation of the innate immune system, response to immunizations, to new antiviral therapies, to avert infection and progression to lower tract respiratory infections. one of the main challenges for immunocompromised patients is the ability to clear infections with subsequent complications associated with worsening infections, prolonged shedding, risk of resistance and coinfections. treatment targeting not only viral replication, but also the immune response to these infections may offer better outcomes. last, understanding the role of the microbiome and virome, and its implications on transmission as well as development of infection will be key for development of new strategies. â�¢ respiratory viruses are the most frequent cause of respiratory infections in immunocompromised patients, and are associated with higher rate of progression to pneumonia, respiratory failure and death. â�¢ high prevalence of m2 inhibitors resistance detected in influenza a (h3n2) and 2009 h1n1 virus strains preclude their use for prophylaxis or empiric treatment of seasonal influenza â�¢ neuraminidase inhibitors are the first line agents for treatment of influenza and treatment is most likely to provide the most benefit when initiated within the first 48 h of illness â�¢ zanamivir is currently the therapy of choice for the treatment of oseltamivir-resistant influenza infection â�¢ an immunodeficiency scoring index for rsv, that accounts for the number of risk factors, can be used to identify hsct recipients who are at high risk for progression to rsv lrti and rsv associated mortality â�¢ ribavirin-based therapy (alone or in combination with immunomodulators) can be effective in preventing progression from urti to lrti and may improve mortality in highly immunosuppressed adult hsct recipients â�¢ the safety and efficacy of das181 in immunocompromised patients with piv pneumonia, is currently being studied in an ongoing phase 2 double-blind, placebo-controlled trial. â�¢ vapendavir binds to the hrv capsid protein, preventing the release of viral rna into the target cells and has demonstrated favorable results in asthmatic adults with hrv urtis. â�¢ antiviral agents for hmpv and hcov are still under study in vitro or in animal models this paper was not funded. ej ariza-heredia has received research grants from oxford immunotec. rf chemaly has received research grants from gilead, gsk, and ansun pharmaceuticals and honoraria from adma biologics and gilead. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. papers of special note have been highlighted as either of interest (â�¢) or of considerable interest (â�¢â�¢) to readers respiratory infections in immunocompromised patients â�¢ the paper describes the immune defect in immunocompromised patients and their increased susceptibility to respiratory infections viral infections in immunocompromised patients the clinical features and outcome of 2009 h1n1 influenza infection in allo-sct patients: a 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coverage in thai patients with type 2 diabetes mellitus 2010-2018: experience from a tertiary diabetes center in bangkok date: 2020-05-11 journal: j clin transl endocrinol doi: 10.1016/j.jcte.2020.100227 sha: doc_id: 300900 cord_uid: 0wfsr4iw trends in influenza and pneumococcal vaccine coverage in thai patients with type 2 diabetes mellitus 2010-2018: experience from a tertiary diabetes center in bangkok background: routine vaccination is an important part of preventive services in treating patients with type 2 diabetes (t2dm). there are no available data in temporal trends of vaccination coverage rates in both influenza and pneumococcal vaccines among thai patients with t2dm. aim: this study aimed to elucidate influenza and pneumococcal vaccination trends and to identify factors that affect vaccination rates in those patients. method: a retrospective study of randomly medical records stratified by 13 diabetologists was conducted in patients with t2dm from 2010-2018 at theptarin hospital, a private multi-disciplinary diabetes center in bangkok. adherence to influenza and pneumococcal vaccinations according to current guidance on adult immunization in thailand had been studied. the rate of both vaccinations from each diabetologist had also been recorded. results: a total of 2,114 medical records (female 51.7%, mean age 65.2±12.8 years, bmi 26.5±4.6 kg/m(2), a1c 7.1±1.3%, median duration of diabetes 13 years) were retrospectively reviewed covering a 9-year period. we audited 3,554 selected outpatient visits for influenza and pneumococcal vaccinations rates as key performance index in each year. the overall vaccination rate was 39.6% for influenza, 17.4% for the pneumococcal vaccine, and only 13.7%, for both vaccines. the trends of influenza vaccination rates increased from 32.9% in 2010 to 52.2% in 2018 but the trends of pneumococcal vaccination rates were relatively stable at less than 20%. the rate of both vaccinations varied considerably from 0-44% among our diabetologists. age ≥ 65 years, duration of dm ≥ 15 years, the presence of chronic respiratory disease, and moderate to severe charlson comorbidity index (cci) score were positively associated with both received vaccinations. conclusions: the completeness and timeliness of influenza and pneumococcal vaccinations were unsatisfactory in thai patients with t2dm. more efforts are needed to increase both influenza and pneumococcal vaccination rates. a retrospective study of randomly medical records stratified by 13 diabetologists was conducted in patients with t2dm from 2010-2018 at theptarin hospital, a private multi-disciplinary diabetes center in bangkok. adherence to influenza and pneumococcal vaccinations according to current guidance on adult immunization in thailand had been studied. the rate of both vaccinations from each diabetologist had also been recorded. results: a total of 2,114 medical records (female 51.7%, mean age 65.2±12.8 years, bmi 26.5±4.6 kg/m 2 , a1c 7.1±1.3%, median duration of diabetes 13 years) were retrospectively reviewed covering a 9-year period. we audited 3,554 selected outpatient visits for influenza and pneumococcal vaccinations rates as key performance index in each year. the overall vaccination rate was 39.6% for influenza, 17.4% for the pneumococcal vaccine, and only 13.7%, for both vaccines. the trends of influenza vaccination rates increased from 32.9% in 2010 to 52.2% in 2018 but the trends of pneumococcal vaccination rates were relatively stable at less than 20%. the rate of both vaccinations varied considerably from 0-44% among our diabetologists. age ≥ 65 years, duration of dm ≥ 15 years, the presence of chronic respiratory disease, and moderate to severe charlson comorbidity index (cci) score were positively associated with both received vaccinations. vaccination is one of the most effective interventions to control transmission, decrease morbidity and mortality of seasonal influenza [1] . patients with type 2 diabetes mellitus (t2dm) are a key target of routine annual influenza vaccination and periodically pneumococcal vaccination as epidemiologic studies suggested that these patients are at high risk for complications, hospitalization, and death from influenza and pneumococcal disease [2] . an increased incidence of pneumococcal infection followed by influenza infection had been observed [3] . as a result, both annual influenza vaccination and adherence to pneumococcal vaccination are an important part of preventive services in treating patients with t2dm from various organizations [4] [5] [6] . although it has been recommended that persons with chronic diseases and those at increased risk of influenza complications should be vaccinated yearly, only a small proportion of high-risk individuals all over the world actually receive influenza vaccination especially in asian countries [7] . the world health organization (who) proposed in 2005 that the influenza vaccination rate should attain coverage rate of 75% among the high-risk groups [8] . however, the current median influenza coverage rate is merely 50.3% among european countries [9] . when compared with western countries, influenza vaccination coverage rate remains considerably low in most asian countries [10, 11] . while influenza vaccination has been focused as key to prevent influenza pandemic in the future, the importance of pneumococcal vaccination should also receive attention in the immunocompromized patients including people with diabetes. invasive pneumococcal diseases (such as meningitis and bacteremia) and pneumonia from streptococcus pneumoniae are associated with increased mortality among individuals with diabetes [12] . therefore, the advisory committee on immunization practices (acip) recommends pneumococcal vaccination in individuals with diabetes [13] . there are currently two types of pneumococcal vaccines -pneumococcal conjugate vaccine (pcv13) and pneumococcal polysaccharide vaccine (ppv23). guidelines for pneumococcal immunization across the world are complex and vary greatly between countries in terms of age groups and risk groups recommended for vaccination, as well as which vaccine should be administered. in thailand, our current national guidance on adult immunization still reflects the previous version of acip recommendation which suggested routine pcv13 for adults ages 65 years and older and followed with ppv23 one year later [14] . for people with diabetes younger than 65 years, only ppv23 is suggested to be administered and repeated every 5 years. despite various campaigns and free influenza vaccination for high-risk groups from the thai government, annual influenza vaccination uptake in the general thai population remained suboptimal at the rate of lower than 30% [15] . there are no available data in temporal trends of vaccination coverage rates in both influenza and pneumococcal vaccines among thai patients with t2dm. based on the available data among patients with t2dm in east asian countries [16] [17] [18] , the annual influenza vaccination rate was only 30-60% and pneumococcal vaccination rate was less than 20%. in an effort to further improve both vaccines coverage rates, it is necessary to understand the current trends and associated factors of vaccination coverage rates. the primary objective of this study is to determine the trends of influenza and pneumococcal vaccine uptakes in individuals with t2dm who attended a routine outpatient diabetes clinic at our hospital. the secondary objective is to identify factors that affect vaccination rates in those patients. the current study is a retrospective analysis of randomly sampled paper-based medical records, stratified by 13 diabetologists, each with annual medical records of 350-500 samples. it was conducted in patients with t2dm from 2010-2018 at theptarin hospital, a private multi-disciplinary diabetes center in bangkok, thailand. this study was a part of our annual quality improvement program which has been carried out since 2010 in order to improve various aspects of diabetes care and benchmark results in each diabetologists. over 2,000 registered t2dm patients follow-up regularly at our diabetes center. patients with age < 15 years, patients with type 1 diabetes mellitus (t1dm) and patients with other types of diabetes were excluded. the data on patient characteristics, smoking status, glycemic control, pattern of diabetes treatments, adherence to influenza and pneumococcal vaccinations according to current guidance on adult immunization in thailand were collected [14] . we used medical records as a source to assess the receipt of influenza and pneumococcal vaccinations. the vaccination venue could be either inside our hospital or other healthcare services. because influenza vaccination is provided for free in people with diabetes in the government healthcare programs since 2008, some patients might receive free influenza vaccination elsewhere. the presence of comorbidities was determined by the charlson comorbidity index (cci) [19] which composed of 19 medical conditions. each comorbidity category has an associated weight (from 1 to 6) and the sum of all the weights results in a single comorbidity score for each patient. a score of zero indicates that no comorbidities was found. the higher the score, the more likely the predicted outcome will result in higher mortality. patients were divided into three groups: mild, with cci scores of 1-2; moderate, with cci scores of 3-4; and severe, with cci scores ≥5 [20] . the rate of both influenza and pneumococcal vaccinations from each diabetologist over the study period was also collected. this study was approved by the institutional review board (irb) committee of theptarin hospital (ec no.6continuous variables were presented as mean ± standard deviation (sd) a total of 2,114 medical records (female 51.7%, mean age 65.2±12.8 years, bmi 26.5±4.6 kg/m 2 , a1c 7.1±1.3%, median duration of diabetes 13 years) with 3,554 selected outpatient visits were selected based on results from random sampling method as shown in figure 1 . this number of medical records represented 20% of all visits during the study period. in this retrospective study, 40% of all selected medical records had been audited for more than once (ranging from 2-7 times) during the 9-year period. insulin usage rate was 23.9% and moderate to severe cci (cci≥3) were presented in 78.8% of all patients. the details of audited patients were presented in table 1 . the overall vaccination rate was 39.6% for influenza, 17.4% for the pneumococcal vaccine, and only 13.7% for both vaccines. when stratified by each diabetologist, the rate of both influenza and pneumococcal vaccinations varied considerably from 0-44% during the study period. as shown in figure 2 , influenza vaccination rates show an increasing trend going from 32.9% in 2010 to 52.2% in 2018 but pneumococcal vaccination rates remain relatively stable at less than 20%. when the vaccination coverage rates were stratified by age groups, younger patients consistently received influenza and pneumococcal vaccinations less than elderly patients as revealed in figure 3 . however, the rate of both influenza and pneumococcal vaccinations remained suboptimal in t2dm patients aged ≥85 years with only one-third of these patients having received both vaccines. in t2dm patients with severe comorbidities (cci≥5), only 47.0% of these patients received influenza vaccination and only 24.3% of these patients received pneumococcal vaccination as shown in figure 4 . when stratifying the rate of vaccinations by bmi category, the influenza vaccine coverage rate was lowest in patients with bmi < 18.5 kg/m 2 when compared with other bmi categories but the pneumococcal vaccine coverage rate was lowest in patients with bmi ≥ 30 kg/m 2 as shown in figure 5 . in the univariate analysis, factors associated with influenza vaccination were older age, female, duration of dm ≥ 15 years, current smoking, the presence of chronic pulmonary disease, insulin usage, and moderate to severe cci. however, only older age, long-duration of dm ≥ 15 years, the presence of chronic pulmonary disease, and moderate to severe cci remained significant in the multivariate analysis as shown in table 2 . these four factors also remained significant in the multivariate analysis of associated factors in pneumococcal vaccinations as shown in table 3 . based on multivariate analysis, t2dm patients with cci≥3 were more likely to receive influenza vaccine (or = 1.86; ci 95% 1.49-2.33) and pneumococcal vaccine (or = 4.97; ci 95% 3.06-8.09). our main findings from this study were that the trends of influenza vaccination rates increased over the 9-year study period but remained suboptimal as half of t2dm patients did not receive influenza vaccination. for pneumococcal vaccination, the rate of vaccination was relatively stable at less than 20% througout the study period. the rate of both influenza and pneumococcal vaccinations varied considerably from 0-44% among our diabetologists. the strongest predictive factors to receive both influenza and pneumococcal vaccinations were the presence of moderate to severe comorbiditis. our results implied that t2dm had largely been ignored as an indicator for influenza and pneumococcal vaccines unless the patients have complex comorbidities. these results highlight the need for pro-active vaccination service to younger patients with t2dm. in thailand, the ministry of public health has been providing seasonal influenza vaccine to people with chronic illness free of charge since 2008. however, eligible patients must receive influenza vaccine from the public sector or private providers who participate in the national health insurance plan [21] . despite having this program in place for over a decade, the uptake of influenza vaccination among thai people remained suboptimal. for pneumococcal vaccine, the patients must cover the cost of this vaccine themselves. therefore, the pneumococcal vaccine coverage rate is much lower than in other countries [22, 23] . beside socio-economic status, attitudes and beliefs of eligible patients toward vaccination play a major role in the decisionmaking process [24, 25] . adult vaccination is often undervalued and there is a lack of assertiveness from healthcare professionals when compared with childhood vaccination. despite more opportunities to get vaccinated in patients with t2dm when visiting routine outpatient clinics, vaccination rates for these populations have been suboptimal all over the world. vaccination coverage has been set as one of the key measures for successful diabetes program and also a key public health action to prevent the spread of infectious diseases [4] . physicians and related healthcare professionals should take a role to review immunization histories and to provide vaccinations for their patients. our present results suggest that active hospital-based vaccination in adult patients with t2dm needs improvement and more effective reminder systems for attending diabetologists should be implemented. based on our study, the different vaccine coverage rates by different age groups imply that different targeted interventions should be employed. younger t2dm patients should receive more information and be more aware of the importance of vaccines as one of the core components in diabetes care. however, healthcare professionals rarely offer information or discuss routine vaccinations with people with diabetes in busy clinic settings. insufficient information to patients from healthcare professionals, and/or lack of assertiveness by treating physicians had been cited as one of the main barriers to increase vaccination rates [25] . attitudes towards vaccination have changed over time especially in vaccine-hesitant individuals [26] . some patients who refused vaccines in the past might change their minds to actively receive vaccines during pandemic period or if their physician insists on the importance of vaccine as a part of their diabetes care. vaccine hesitancy exists across all socioeconomic strata of the population. patient-oriented and trust-worthy information should be offered to these people. our findings are also in keeping with a recent study from france that falling vaccine coverage rate could be observed after a flu pandemic [27] . as the trend of influenza vaccination rate in our cohort continuously increased until the peak rate at almost 60% in 2015 and then the trend dropped the year after. there was a peak period of seasonal flu activity in thailand during 2015-2016. this phenomenon is explained by the possibility of vaccine hesitancy effect regarding its effectiveness against the pandemic [28] . another possible explanation is our hospital policy to start collecting influenza vaccination as a key quality improvement program since 2015. therefore, it could affect the surge of vaccination rate in the first year after implementation. however, these observations underscore the need for sustainable interventions to increase vaccination uptake. even among european union countries, none could achieve the target for influenza vaccination of 75% in people living with chronic disease (the median uptake rate was only 50%) [9] . global efforts to increase seasonal influenza vaccine coverage rate must involve more proactive strategies from healthcare providers around the world to prevent the future influenza pandemic. during influenza outbreaks, pneumococcal vaccines may help prevent secondary pneumococcal infections [29] . the rate of pneumococcal vaccination uptakes in patients with t2dm are variable and difficult to benchmark when compared with influenza vaccine coverage rate. in contrast to some higher income countries such as south korea which the coverage rate of ppv dramatically increased after the national policy to give free vaccine in all elderly koreans aged ≥65 years [17] , pneumococcal vaccine has to be paid out-of-pocket in thailand. although pediatricians generally recommend pcv in infants, it has not been included in the national immunization program and the vaccine coverage is still low in children [30] . pcv13 vaccine has been approved for adults in thailand in immunocompromized conditions and people with diabetes age ≥ 65 years old since 2011 but lack data to establish costeffectiveness in general people. as a result, less than 5-10% of thai people with t2dm received pneumococcal vaccination from a previous study in thailand [31] . as shown in this study, the coverage rate of our patients with severe comorbidities was almost 10 times greater than patients with mild comorbidities. this data implies that the importance of pneumococcal it is important to note that our study has several limitations. first, this study was conducted only in a private hospital in bangkok. the findings could not be generalized to other patients with t2dm in thailand. the socioeconomic factor influences the decision to receive both vaccines in many patients. in private settings, the patients have to pay the cost of influenza and pneumococcal vaccine for themselves. therefore, this study may have underestimated the rate of influenza vaccination if patients did not inform the treating physicians that they were already received influenza vaccination from other places. for the pneumococcal vaccination, our vaccine coverage rate may be overestimated from the patients with higher socio-economic status in private settings. similarly, the health beliefs and knowledge of influenza among various diabetologists in our hospital could affect the overall rate of vaccination in our study. but our study could serve as a reference for future studies on vaccinations among thai people with diabetes. second, data on vaccination in some patients were documented by treating physicians as self-report data from their patients. the recall bias from these patients could affect the results. however, an earlier study reported that self-reporting may be the only effective and feasible way to gather data from diverse population [33] . additionally, this study was conducted based on medical audits over the 9-year period with a uniform format. it would not affect greatly the trend of vaccination coverage rates. third, some factors such as education level, economic status, previous history of vaccine-preventable hospitalizations could not be retrieved from our medical records which might affect the associated factors in receiving both vaccines [34] . finally, the sample size of audited medical records was not planed as this study was a part of quality improvement program since 2010. the stratification by characteristics that may impact the results improves the sample quality but a convenience sample may not reliably infer to a population. in conclusion, the completeness and timeliness of influenza and pneumococcal vaccinations were unsatisfactory in thai patients with t2dm as reported from previous studies around the world. to the best of our knowledge, this study provides an insight into preventive care services for thai patients with t2dm. even though the positive trend had been observed for influenza vaccination, the pneumococcal vaccination rates especially in younger patients remained severely suboptimal. our findings suggest that younger t2dm patients are overlooked when compared with elderly patients. an increase in vaccination coverage rates should be encouraged as a key quality improvement initiative [35] . use of health information technology for identification of unvaccinated patients, promoting a positive attitude toward vaccination to patients, and periodic assessment with giving feedback to treating physicians should be employed to help achieve higher vaccination rates. the data used to support the findings of this study are available from the corresponding author upon request. the authors declare that there is no conflict of interests 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evaluation of their impact on patient's influenza vaccine uptake characteristics associated with the uptake of influenza vaccination among adults in the united states vaccine hestiancy around the globe: analysis of three years of who/unicef joint reporting form data trends in seasonal influenza vaccine coverage of target groups in france trajectories of seasonal influenza vaccine uptake among french people with diabetes: a nationwide retrospective cohort study comparison of dual influenza and pneumococcal polysaccharide vaccination with influenza vaccination alone for preventing pneumonia and reducing mortality amongthe elderly: a meta-analysis cost-utility analysis of 10-and 13-valent pneumococcal conjugate vaccines: protection at what price in the thai context? the effectiveness of holistic diabetic management between siriraj continuity of care clinic and medical outpatient department practical recommendations for the management of diabetes in patients with covid-19 validation of self-report of influenza and pneumococcal vaccination status in elderly outpatients influenza vaccination rate and its association with chronic diseases in china: results of a national cross-sectional study a review of the key factors to improve adult immunization coverage rates: what can the clinician do? the authors wish to thank dr.tinapa himathongkam for excellent language editing and dr. krittadhee karndumri for assisting in statistical analysis. parts of this manuscript had previously been presented as a poster in international diabetes federation (idf) meeting 2019, busan, south korea. no source of funding was applied in this retrospective study. key: cord-257491-tsdwsyjs authors: cieślak, k.; szymański, k.; kowalczyk, d.; brydak, l. b. title: influenza and influenza-like viruses in children in the epidemic season 2015/2016 in poland date: 2016-12-31 journal: influenza and respiratory care doi: 10.1007/5584_2016_178 sha: doc_id: 257491 cord_uid: tsdwsyjs influenza is an infectious disease caused by influenza a and b viruses. children are the group which is the most exposed to influenza and influenza-like infections. they are considered as carriers of influenza infections in the population. in the epidemic season 2015/2016 more than 8000 samples were tested, of which over 30 % specimens were collected from patients aged 0–14 years. in 42.3 % cases the influenza or influenza-like viruses were confirmed. the most common subtype was a/h1n1/pdm09. analysis of positive specimens was categorized into three smaller groups 0–4, 5–9, 10–14. differences in the frequency of virus detections in younger age groups appeared. this study has shown that children are a very important group in the spread of the influenza virus in the population. a higher percentage of vaccinated children would decrease the number of infected patients in the whole population. influenza is a disease caused by viruses a, b, and c belonging to the family orthomyxoviridae. cases of infection with influenza type c are much rarer. therefore, laboratory diagnostics of influenza viruses are based on the confirmation of the presence of a and b influenza viruses (brydak 2012) . the main symptoms of influenza infection are sudden onset of disease, fever, chills, cough, sore throat, muscle pain, headache, and general malaise. the course of influenza infection in children is further characterized by symptoms from the gastrointestinal tract. it is worth noting that the symptoms above mentioned are not specific to influenza infection, but also for infections caused by other respiratory viruses (influenza-like viruses), which makes the diagnosis difficult (who 2014) . children are a group particularly affected by influenza virus infection. their immune system is not yet fully formed. for this reason, children are considered a higher risk group and vaccination from the 6th months of life up to 18 years of age is recommended (hallmann-szelińska et al. 2015) . despite the recommendation presented at the beginning of 2015, vaccination coverage rate in polish children amounts to merely 0.73 % (epimeld 2016) . this is outrageously low compared to the percentage of vaccinated children in other european countries. the basic methods of detection of influenza viruses from material taken from a patient belong to the molecular biology. a quick diagnostic confirmation of the virus, especially in children, enables a rapid implementation of antiviral treatment, and thus reduces the chance of serious complications from influenza, including fatal course of disease. in the influenza season 2015/2016, five deaths of children under the age of 14 years caused by influenza were reported (niph-nih 2016). children are exposed not only to influenza infection, but also to influenza-like viruses, which circulate at the same time, and resemble the influenza infection. the most frequently diagnosed influenza-like viruses include parainfluenza 1, 2, 3, human respiratory syncytial viruses a and b (rsv a, rsv b), human corona viruses 229e/ nl63 and oc42 (hcov), human rhinovirus (rv), human adenovirus (adv), and human metapneumovirus (hmpv) (brydak 2008) . in the first 3 months of the epidemic season 2015/2016 (october-december, 2015) there were 406 more cases of confirmed and suspected cases of influenza in children up to 14 years of age compared with the preceding epidemic season in poland (niph-nih 2016). the increased number of influenza infection could augur a higher activity of the virus in the months ahead as well. in the present study, therefore, we seek to determine the overall activity of influenza and influenza-like viruses in children under 14 years of age in the epidemic season 2015/2016 in poland. the study protocol was approved by an institutional ethics committee and the study was conducted in accordance with the principles for biomedical human research as set by the declaration of helsinki. the material for testing were swabs taken from the nose and throat, collected within the framework the non-sentinel and sentinel influenza surveillance programs (who 2009). there were 3376 specimens taken from children up to 14 years of age tested in the epidemic season 2015/2016 in poland. the specimens were analyzed at the department of influenza research, national influenza centre at national institute of public health -national institute of hygiene in warsaw, poland, as well as in 16 voivodeship sanitary epidemiological stations throughout the country. the national influenza centre served as the reference laboratory. the patients were divided into three age groups: 0-4, 5-9, 10-14 years, according to the division introduced in the reporting system updated in 2014 . for rna isolation, maxwell 16 total viral nucleic acid purification kit (promega corporation; madison, wi) was used. the isolation was performed according to the instructions of the manufacturer from 200 μl of sample suspended in saline and rna was eluted using 50 μl of rnase-free water. real-time rt-pcr (h1n1) pdm09, a/switzerland/9715293/2013, and b/phuket/ 3073/2013. the negative control was the rnasefree water provided in the kit. before the amplification process, cdna was subjected to a reverse transcription (30 min 50 c). the dna was subjected to a process of initiation (1 cycle 95 c for 2 min), followed by 45 cycles of amplification: denaturation at 95 c for 15 s, annealing at 55 c for 30 s, and elongation at 72 c for 20 s. to confirm the genetic material of influenza-like viruses, rt-pcr reaction using rv12 ace detection kit (seegene; seul, south korea) was performed. influenza a virus, influenza b virus, adv, rsv a, rsv b, hmpv, hcov, piv-1, piv-2, piv-3, and rv could be detected. random hexamer-primed cdna synthesis products were generated using the revertaid first strand cdna synthesis kit (thermo fisher scientific; carlsband, ca), according to the of manufacturer's instructions. amplicons were detected by gel electrophoresis. there were 8542 specimens tested during the epidemic season 2015/2016, in poland, of which 3376 were collected from children 0-14 years of age. in 1428 (42.3 %) out of the 3376 specimens the genetic material of influenza and influenza-like virus was detected. influenza a virus was confirmed in 46.0 % of cases, influenza b in 41.8 % of cases, and influenza-like viruses in 12.2 % of cases (fig. 1) . the dominant subtype of influenza a virus was a/h1n1/pdm09 (57 %), while 40 % of cases were represented by unsubtyped influenza a virus. the smallest group consisted of the a/h1n1/ and a/h3n2/ subtypes, which accounted for 2 % and 1 % respectively (fig. 2) . confirmations of the influenza virus in the three junior age groups: 0-4, 5-9, 10-14 years of age demonstrate that in children of 0-4 years of age infections caused by both influenza b and a were common. the youngest patient in this age group was a 14-day old baby. the age group of 5-9 years of age was dominated by influenza b infections. the number of confirmations of infections with all subtypes of influenza a virus was distinctly lower in this group. in the age group of 10-14 years of age, the rate of influenza virus infections (158 cases) was markedly lower than the 601 cases in children aged 0-4 and the 494 cases in children aged 5-9). in this oldest age group infections with influenza b virus also dominated (fig. 3) . out of the genetically confirmed influenzalike viruses, rsv virus was detected exclusively in the group of 0-4 years of age. other viruses were only sporadically present (fig. 4) . the results also confirm the frequent presence of influenza and influenza-like viruses in children aged 0-14 years. in addition, in the youngest age-group of 0-4 years, the presence of also virus type b was confirmed, which shows that young children have a poorly developed immune system, making them more vulnerable to disease. large gatherings of people contribute to influenza infections. children due to daily stays in kindergartens and schools are particularly susceptible to infection and also are carriers of infection (hallmann-szelińska et al. 2015 . the rsv virus is a leading cause of respiratory tract infections in infants. this infection usually runs a mild course in older children and adults, but is severe in infants who often require hospitalization (brydak 2008) . due to an extremely low percentage of vaccinated children, the number of cases of influenza steadily increases from season to season. this widely spreading tendency could be overcome through the implementation of vaccination against influenza from the sixth month of age, which is recommended by the advisory committee on immunization practices (acip) (cdc 2016) and by other associations, including the american academy of pediatrics (aap 2016). unfortunately, few parents undertake a decision to vaccinate their children, which in case of infection may end up with lifethreatening complications. the same holds true for vaccination of pregnant women, which actually provides immunization for both mother and child. a confirmed case of influenza in a 14-day old child we observed in the present study pointedly shows that the mother was not vaccinated during pregnancy, although such vaccinations are recommended in poland in the preventive vaccination plan. a study on the association between the number of vaccinated school children and influenza morbidity in the elderly, conducted in japan in 2001, demonstrates that influenza vaccination of children has resulted in a reduction of morbidity and mortality in the elderly (reichert et al. 2001 ). children are a special segment of population that has a substantial impact on the spread of influenza virus. therefore, a need for influenza vaccination in children should not be underestimated. aap backs new acip recommendation on influenza vaccine novelties in influenza surveillance in poland influenza, pandemic flu, myth or real threat? rythm warsaw influenza-an age old problem centers for disease control and prevention. vaccine recommendations of the acip weekly influenza surveillance preventive vaccinations in poland influenza in children aged under 14 years in poland viral infections in children in the 2014-2015 epidemic season in poland influenza and influenza-like illness in poland the japanese experience with vaccinating schoolchildren against influenza who regional office for europe guidance for sentinel influenza surveillance in humans ho regional office for europe guidance for sentinel influenza surveillance in humans information about influenza (seasonal) acknowledgments this work was funded by niph-nih thematic subject 5/em.1. the authors' thanks go to physicians and employees of sanitary epidemiological stations participating in sentinel and non-sentinel programs for their input into the influenza surveillance in poland. the authors declare no conflicts of interest in relation to this article. key: cord-296277-paqu1t1e authors: fragaszy, ellen b; warren-gash, charlotte; wang, lili; copas, andrew; dukes, oliver; edmunds, w john; goonetilleke, nilu; harvey, gabrielle; johnson, anne m; kovar, jana; lim, megan sc; mcmichael, andrew; millett, elizabeth rc; nazareth, irwin; nguyen-van-tam, jonathan s; tabassum, faiza; watson, john m; wurie, fatima; zambon, maria; hayward, andrew c title: cohort profile: the flu watch study date: 2016-03-03 journal: int j epidemiol doi: 10.1093/ije/dyv370 sha: doc_id: 296277 cord_uid: paqu1t1e nan influenza is a common, highly contagious respiratory virus which infects all age groups, causing a range of outcomes from asymptomatic infection and mild respiratory disease to severe respiratory disease and death. 1 if infected, the adaptive immune system produces a humoral (antibody) and cell-mediated (t cell) immune response to fight the infection. 2 influenza viruses continually evolve through antigenic drift, resulting in slightly different 'seasonal' influenza strains circulating each year. population-level antibody immunity to these seasonal viruses builds up over time, so in any given season only a proportion of the population is susceptible to the circulating strains. occasionally, influenza a viruses evolve rapidly through antigenic shift by swapping genes with influenza viruses usually circulating in animals. this process creates an immunologically distinct virus to which the population may have little to no antibody immunity. the virus can result in a pandemic if a large portion of the population is susceptible and the virus is easily spread. 1 international influenza surveillance is typically based upon cases seeking medical care. [3] [4] [5] however, this focus greatly underestimates the true community burden of seasonal influenza: the majority of cases are mild and self-limiting, with asymptomatic infections accounting for 25% to 75% of all infections. 6, 7 effective influenza control requires knowledge of disease burden and factors affecting influenza transmission. existing parameters for mathematical models of influenza interventions are largely derived from household cohort studies conducted in the usa between 1948 and 1981. [8] [9] [10] since then there have been profound social changes affecting population contact and mixing patterns that are likely to impact on influenza transmission. these changes include more women working, more children attending day care, more commuting and international travel and increased vaccine coverage. evolutionary changes to circulating viruses may affect transmission dynamics, patterns of clinical illness and the adaptive immune responses elicited. 1, 11 rapid advances in laboratory methods have also occurred, providing unique opportunities to investigate immune correlates, both humoral and t cell based, with influenza infection rates and disease severity. 11, 12 the initial flu watch cohort, funded by the uk medical research council (mrc), began in 2006 as a collaboration between epidemiologists at the centre for infectious disease epidemiology at university college london (ucl), virologists and mathematical modellers from the health protection agency (hpa, now public health england), immunologists at the mrc human immunology unit at oxford university and the mrc general practice research framework (gprf). it aimed to estimate community burden of influenza and influenza-like illness, generate up-to-date knowledge of demographic, social and behavioural factors affecting influenza transmission, measure antibody and t cell immune responses to influenza and to use knowledge generated to inform modelling parameters. in addition, a pandemic preparedness cohort was envisioned, in which participants already familiar with the study consented to be re-contacted in the event of a pandemic, to allow rapid redeployment of the study. when the 2009 influenza ah1n1 pandemic arose, further funding was secured jointly from the mrc and wellcome trust, allowing continued follow-up and an expansion in cohort size. new collaborators for this phase included the mrc centre for outbreak analysis and modelling, the wellcome sanger institute, the primary care research network and additional epidemiology and public health experts from the hpa. additional study aims were to inform the national and international response to the current and future pandemics. specific objectives were to examine clinical profiles of illness, estimate population infection denominators and case fatality risk, describe epidemiological characteristics of the infection in real-time, monitor changes in population behaviour, and investigate access to services, attitudes to and uptake of antivirals and vaccine, and immunity to infection in order to inform vaccination policy and development. during the pandemic, flu watch also provided control data and samples for studies of severe influenza (mosaic) and studies of influenza infection risk in people working with pigs (cosi). 13, 14 who is in the cohort? households were recruited from registers of 146 volunteer general practices (gp) across england, who formed part of the mrc gprf or (from the 2009 pandemic onwards) the primary care research network. participants were selected from gp lists by computer-based random number generation. gps sent invitation letters inviting the randomly selected person and their household to participate. although it was recognized that this would bias invitations towards larger households, such as those with children, this was accepted as the role of children in influenza transmission was an important research question. weighting by the inverse of household size in analyses was planned to account for this sampling design. to be eligible to participate, the whole household had to agree to take part in follow-up over the coming winter, with adults aged 16 years agreeing to have blood samples taken. exclusion criteria included household size > 6 people, individuals with terminal illness, severe mental illness or incapacity and heavy involvement in other ongoing research. gps reviewed invitation lists and removed anyone meeting these criteria, before sending letters. cohorts were recruited to allow follow-up of participants over six influenza seasons-the 2006/07, 2007/08 and 2008/09 periods of seasonal influenza circulation, the summer and winter waves of the 2009 pandemic and the first post-pandemic season 2010/11. from season 3 (2008/09) onwards, previous participants were invited to take part again. in season 1, invitation letters were sent to 2300 households from 42 practices, and 602 individuals from 243 households agreed to participate. in subsequent seasons the response rate was not monitored as practices (rather than the university study team) sent the invitation letters and not all returned data on numbers sent. compared with the english population, young adults, non-white ethnic groups, people living in socially deprived areas and those living in the north of england, west midlands and london were under-represented in the flu watch cohort (table 1) . the basic cohort design baseline/pre-season phase a baseline visit was made to the household at enrolment, during which a research nurse collected blood samples for serological and t cell analysis from all adults aged 16 years or older. blood sampling was optional for those aged 5-15 years and not done in those under 5 years of age. visits occurred in the evenings, as bloods had to be couriered overnight to oxford for early morning analysis of t cells. the serum samples collected we recentrifuged, frozen and later batch-tested for influenza antibodies by the hpa. nurses assisted families with a series of laptop-based surveys collecting information on basic demographics, health and chronic illness, respiratory hygiene, household structure and relationships, accommodation, contacts and activities. households received participant packs containing paper illness diaries, thermometers and nasal swab kits including instructions on their use and the viral transport medium to be stored in the refrigerator. in order to obtain reliable measures of the number of illnesses, we actively contacted participants every week with automated telephone calls to assess the presence or absence of respiratory illness in each household member. for each respiratory illness, participants were reminded to fill in a prospective paper illness diary. these collected information on illness onset date, temperature and presence and severity of symptoms such as feeling feverish, headache, muscle aches, cough and sore throat. diaries also collected data on contact patterns and activities before and during illness. participants took a nasal swab on day 2 of any respiratory illness for polymerase chain reaction (pcr) analysis of influenza, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), rhinovirus, coronavirus, adenovirus and parainfluenzavirus. during the first season, swabbing was limited to periods of influenza circulation. the sanger institute genetically sequenced some of the viral isolates from the summer and winter waves of the pandemic (seasons [4] [5] . in addition, all participants completed one-off activity and contact paper diaries on at least 1 pre-determined weekday and 1 weekend day during the active follow-up period. these diaries collected information on where participants were (i.e. at home, at work etc.), whether they had contact with crowds and the number, duration and age groups of personal contacts throughout the day. at the end of follow-up, nurses made a final household visit to take a follow-up blood sample (for paired serology) and assist participants with an exit survey. nurses also checked participants' medical records for information on chronic illnesses, influenza and pneumococcal vaccinations, prescriptions, gp consultations, hospitalizations and deaths. the cohort evolved over time to maximize system reliability, minimize the number of data sources and allow increased recruitment during the pandemic. in season 3 we offered participants the option of moving from paper illness diaries with weekly automated phone calls to weekly emailed surveys with or without optional sms reminders. for the pandemic and post-pandemic cohort, most surveys moved to a custom-built website for self-completion. in order to achieve real-time monitoring of illnesses during the pandemic, participants were emailed a link to a retrospective online weekly survey and provided with laminated wipe-clean charts at home to record daily symptoms as a memory aid. in season 3 there were additional one-off surveys collecting data on indoor and outdoor temperature and humidity, travel patterns and non-response to weekly surveys. during seasons 5 and 6 we added questions to existing surveys on attitudes towards influenza vaccination and antivirals. in season 6 we included quality of life questions. 15 the cohort design evolved with the emergence of the novel h1n1 pandemic strain during season 3.we continued active follow-up through the uk summer wave of the pandemic (season 4). for the uk winter wave of the pandemic (season 5), the study split into three separate cohorts: t cell (comprising both previous and newly recruited participants), serology and virology (both comprising new participants). for the t cell cohort, continuing participants used the spring blood sample from season 3 as a baseline sample. they also gave a pre-vaccination blood sample to allow distinction of antibody rises caused by infection rather than vaccination. this was particularly important for the winter wave of the pandemic, as we anticipated widespread vaccination. the serology cohort was identical but lacked t cell samples. for the virology cohort, no blood samples were taken. this allowed for rapid recruitment of a large number of participants (n ¼ 1778) to increase the accuracy of weekly estimates of illness rates during the pandemic, with minimal nurse time required. all nasal swabs were tested for influenza a and b, rsv and hmpv but, due to the large number of samples generated during the pandemic, only a selection in seasons 5 and 6 were tested for other viruses. retention of enrolled participants throughout the cohorts was good. figure 1 displays the number of enrolled participants each week, with arrows pointing out the staggered starts and exits of the cohorts along with other important dates. loss to follow-up came in two main varieties: nonresponse to weekly contact and loss to follow-up for paired blood samples. we obtained weekly responses from 87.3% of followup weeks overall, which increased to 88.4% if we exclude periods when there were technical difficulties with our automated phone calls (1 week in season 1 and 4 weeks in season 2). response completeness generally increased after the introduction of email and online surveys in season 3 ( table 2 ). only 12.4% of households were classified as poor responders (responding to < 70% of follow-up weeks). poor response appeared to be more common as deprivation increased. we obtained paired blood samples from 80% of participants required to provide them and from 27% of participants aged 15 and under, for whom blood samples were optional (table 3) . the three main clinical outcomes were: (i) influenza-likeillness (ili), defined as a respiratory illness with cough and/or sore throat and fever > 37.8 c;(ii) pcr-confirmed influenza illness; and (iii) influenza seroconversion, defined as a 4-fold titre rise in strain-specific antibody titres in unvaccinated individuals. table 4 summarizes the data and biological samples collected during baseline, active follow-up and post-season phases. we additionally linked participants' data to small area statistics such as the index of multiple deprivation and rural/urban indicators. 16, 17 details of the t cell methodology have been described previously. [18] [19] [20] what has been found? key findings and publications our first publication provided comprehensive national estimates of clinical and sub-clinical disease burden in the community regardless of consultations, and allowed comparison between seasonal and pandemic influenza. 2 we found that on average, influenza infected 18% of unvaccinated people each winter and up to 75% of these infections were asymptomatic. approximately 25% of infections were pcr confirmed and only 17% of people with pcr-confirmed disease sought medical attention; figure 2 indicates how the primary care-based surveillance underestimated the burden of infection in the community. results were similar between pandemic and seasonal influenza, although people infected with the 2009 pandemic strain had less severe symptoms than those infected with seasonal h3n2 strains. our second publication provided strong evidence that naturally occurring, cross-protective t cell immunity protects those infected with influenza against developing disease in seasonal and pandemic periods. 16 this protection was independent of baseline antibodies and protective levels of influenza-specific t cells were found in 43% of the population. these findings help explain why such a large proportion of infections remain asymptomatic and have implications for the development of cross-protective 'universal' vaccines based on this response. in order to evaluate different methods of collecting data during a pandemic, we compared prospectively collected flu watch data on illnesses and vaccine uptake with retrospectively collected data from the health survey for england. 21 we found that retrospectively collected data underestimated disease burden but accurately estimated vaccine uptake when compared with prospectively collected data. current work includes an analysis of occupational exposure to pigs as a risk factor for human infection with swine and human influenza viruses; age as a predictor of t cell responses; and a comparison of serological pandemic infection rates from flu watch and the health survey for england. we believe the poor response in this season may be due to summer holidays. what are the main strengths and weaknesses? flu watch is a large community cohort study broadly representative of the population of england. it is the first modernday household study of influenza transmission in a temperate climate, comparable to the landmark tecumseh studies of the 1960s and 70s. 22 a major strength is the inclusion of different household types (rather than just households with children, as in earlier studies) which allows influenza infections to be explored across the whole of society. we used highly active methods of surveillance for influenza and other respiratory 23 or from populations with high vaccination rates which greatly limits interpretation (1968 h3n2 pandemic). 22 historical data on laboratory-confirmed rates of seasonal influenza mainly come from historical community studies of families in the usa between 1948 and 1981. 10, 22, 24, 25 flu watch is a good example of collaboration between disciplines (epidemiology, immunology, virology and primary care) and partners. the study provides a rich source of data on social, behavioural and biological factors affecting influenza transmission, enabling exploration of many research questions. limitations include delays in obtaining funding, ethics and r&d approval across multiple sites, resulting in delayed recruitment during the pandemic and fewer participants overall. although the initial response to invitation letters was low, it is unclear if this would bias results. ideally, cohorts would have had pre-and post-influenza season bleeds, but recruitment periods were not perfectly streamlined with influenza seasons so adjustments for bleed timings were made during analysis. the study design and data collection methods evolved in response to experience and changing questions. whereas this optimized and streamlined methods, it also increased complexity of data management. can i get hold of the data? where can i find out more generate novel data on antibody and t cell immunity, to inform influenza control initiatives. • a total of 5484 participants were recruited from 2205 households randomly selected from registers of participating general practices. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study sources of uk flu data, influenza surveillance in the united kingdom. page last reviewed 09 overview of influenza surveillance in the united states, page last reviewed 13 world health organization. who interim global epidemiological surveillance standards for influenza time lines of infection and disease in human influenza: a review of volunteer challenge studies estimating pathogenspecific asymptomatic ratios seattle virus watch 4. comparative epidemiologic observations of infections with influenza-a and influenza-b viruses, 1965-1969, in families with young children tecumseh study of illness .13. influenza infection and disease, 1976-1981 a study of illness in a group of cleveland families .1. plan of study and certain general observations preexisting influenzaspecific cd4þ t cells correlate with disease protection against influenza challenge in humans diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza mechanisms of severe acute influenza consortium (mosaic) from pandemic to policy: combating swine flu euroqol: the current state of play natural t cell mediated protection against seasonal and pandemic influenza: results of the flu watch cohort study rural and urban area classification for output areas induction of multifunctional human immunodeficiency virus type 1 (hiv-1)-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dna-and modified vaccinia virus ankara-vectored vaccines expressing hiv-1 gag coupled to cd8þ t-cell epitopes rapid effector function in cd8þ memory t cells response definition criteria for elispot assays revisited population-level susceptibility, severity and spread of pandemic influenza: design of, and initial results from, pre-pandemic and hibernating pandemic phase study using cross-sectional data from the health survey for england (hse) the tecumseh study of respiratory illness. ix. occurrence of influenza in the community a study of illness in a group of cleveland families. xvi. the epidemiology of influenza, 1948-1953 the virus watch program: a continuing surveillance of viral infections in metropolitan new york families. 3. preliminary report on association of infections with disease studies of the community and family: acute respiratory illness and infection we acknowledge the contribution of alison bermingham, now sadly deceased, of the respiratory virus unit, centre for infections, public health england. we thank all flu watch participants and nurses for their support of the study. peripheral blood mononuclear cells (pbmc) separated, part of the sample was immediately tested against pools of peptides representing each of the virus proteins in an ex vivo ifn-celispot assay. 18, 19 the rest of the sample was frozen down for more detailed peptide mapping studies using ifn-celispots and/or in vitro culture and testing by intracellular cytokine staining to determine cd8/4 restriction. post-season t cell analysis was only conducted in seasons 1 and 3. key: cord-302529-43pd2qsp authors: el moussi, awatef; pozo, francisco; ben hadj kacem, mohamed ali; ledesma, juan; cuevas, maria teresa; casas, inmaculada; slim, amine title: virological surveillance of influenza viruses during the 2008–09, 2009–10 and 2010–11 seasons in tunisia date: 2013-09-19 journal: plos one doi: 10.1371/journal.pone.0074064 sha: doc_id: 302529 cord_uid: 43pd2qsp background: the data contribute to a better understanding of the circulation of influenza viruses especially in north-africa. objective: the objective of this surveillance was to detect severe influenza cases, identify their epidemiological and virological characteristics and assess their impact on the healthcare system. method: we describe in this report the findings of laboratory-based surveillance of human cases of influenza virus and other respiratory viruses' infection during three seasons in tunisia. results: the 2008–09 winter influenza season is underway in tunisia, with co-circulation of influenza a/h3n2 (56.25%), influenza a(h1n1) (32.5%), and a few sporadic influenza b viruses (11.25%). in 2010–11 season the circulating strains are predominantly the 2009 pandemic influenza a(h1n1)pdm09 (70%) and influenza b viruses (22%). and sporadic viruses were sub-typed as a/h3n2 and unsubtyped influenza a, 5% and 3%, respectively. unlike other countries, highest prevalence of influenza b virus yamagata-like lineage has been reported in tunisia (76%) localised into the clade b/bangladesh/3333/2007. in the pandemic year, influenza a(h1n1)pdm09 predominated over other influenza viruses (95%). amino acid changes d222g and d222e were detected in the ha gene of a(h1n1)pdm09 virus in two severe cases, one fatal case and one mild case out of 50 influenza a(h1n1)pdm09 viruses studied. the most frequently reported respiratory virus other than influenza in three seasons was rsv (45.29%). conclusion: this article summarises the surveillance and epidemiology of influenza viruses and other respiratory viruses, showing how rapid improvements in influenza surveillance were feasible by connecting the existing structure in the health care system for patient records to electronic surveillance system for reporting ili cases. identification and characterization of circulating influenza viruses is essential to detect the emergence of antigenic drift variants causing influenza epidemics and novel a subtypes with the potential to cause an influenza pandemic. thus, virological surveillance of influenza provides a basis for selection of the virus strains to be included in the annual formulation of influenza vaccines [1] . surveillance data from the african continent has increased substantially in the past five years [2] [3] [4] [5] , but they are still insufficient to allow for a thorough understanding of influenza virus circulation patterns on the continent and their associated morbidity and mortality, or to inform influenza control strategies [6, 7] . the primary objective of this study was to develop or strengthen influenza sentinel surveillance systems in line with who standards in selected north african countries. in the past decade, information of epidemic strains from tunisia was largely unknown due to lack of any systemic study. in this present study we are reporting the activity and circulation of influenza viruses during three seasons (2008-2009, 2009-2010 and 2010-2011 ) as a part of global influenza surveillance network, which was expanded to tunisia since 1980. a subset of sentinel primary care physicians participating in virological surveillance schemes in the community submits respiratory samples for virological testing from patients presenting in primary health care with an ili, as well as all regional emergency centres and hospitals that take on surveillance of influenza from community, hospitalized and fatal cases. the surveillance of influenza and other respiratory viruses is undertaken by 268 primary care centres for adult and pediatric patients ( fig. 1 ) distributed in 24 governorates covering 2.7% of general tunisian population (table 1) . sentinel physicians report weekly the total number of patient visits to their facilities for influenza-like illness (ili) and acute respiratory infection (ari) within four age categories (0-4 years, 5-14 years, 15-64 years, and 65+ years). sentinel physicians are asked to collect respiratory specimens from patients with symptoms of ili or ari. ili was defined as an outpatient with fever (38uc) and cough or sore throat with onset less than five days prior to presentation in the absence of a specific diagnosis. ari was defined as an outpatient with sudden onset of respiratory signs including cough, difficulty breathing, rhinitis and general symptoms such as fever, headache, fatigue, and myalgia less than five days prior to presentation. the physicians sent specimens for influenza testing and basic demographic data from a subset of patients with ili each year during october through may to the national influenza centre (nic) situated at the charles nicolle's hospital tunis, provided that the number of consultants is 10% above the total number of consultants in the sentinel centre. in addition, according to world health organization (who) recommendations, severe acute respiratory infection (sari) related to influenza viruses has been added to existing outpatient surveillance systems to fully describe the spectrum of disease related to influenza and to identify individuals at highest risk for severe disease. as a result, various existing routine influenza surveillance systems in tunisia were enhanced or supplemented to gain a rapid understanding of this novel virus, to monitor its spread and impact, and to evaluate the uptake, impact and effectiveness of the various countermeasures that were implemented. during each season, oro-pharyngeal and naso-pharyngeal swabs were collected from ili and sari patients enrolled under the virological surveillance system and placed in viral transport medium (vircell, spainh). oro-pharyngeal and nasopharyngeal swabs collected from the same patient were placed in one cryovial, stored at 4uc at the sentinel site, and transported daily to the nic. the samples taken from severe cases were from children and/or adult hospitalised in the care unit or patients hospitalized with severe acute respiratory infection were additionally tested for other respiratory viruses. for all participants, respiratory specimens collection were performed after informed consent, under the supervision of local sanitary authorities. it is just an informal statement of the study and members of the ethic committee have previously always approved the work of the nic. in fact, patients included in this study were of all ages and consulted the sentinel clinics or were hospitalized in state or private hospitals for influenza like symptoms infection. the consent was verbal because the patients, or parents in the case of minors, accept the test for influenza viruses since it is free and safe. until now, written consent is judged not necessary by the ethics committee. samples were analysed for diagnostic purposes using the real time pcr cdc protocol [8] for detection of influenza viruses and using xtagh respiratory viral panel fast (abbott molecular, germany) and the luminexh technology for other respiratory viruses. all influenza positive samples were sub-typed using specific real-time pcrs for influenza a(h1n1)pdm09, a/h3n2 and for influenza b viruses using ''influenza virus b real time rt-pcr kit'', ''subtype h1 of influenza virus a real time rt-pcr kit'' and ''subtype h3 of influenza virus a real time rt-pcr kit''(shanghai zj bio-tech co., ltd). every year, we realized the exchange of influenza strains with who collaborating centre for influenza in london. in the pandemic year, we succeeded to cultivate some tunisian strains of influenza a(h1n1)pdm09 and influenza b in national influenza centre madrid spain. in fact, cell culture virus isolation will be implemented in the national influenza centre in tunisia in the next few years. a representative number of influenza viruses were genotypically characterized by analysis of the nucleotide sequence of partial haemagglutinin ha1 chain (931 nucleotide residues) and partial neuraminidase (836 nucleotide residues) genes in order to know if circulating viruses were well-matched with vaccine viruses and check for the most frequent amino acid key changes related to neuraminidase inhibitors resistance respectively. all viruses analysed were amplified and sequenced according to the protocol of national influenza centre madrid [9] . tunisian sequences were aligned with other sequences from reference influenza a viruses available at the ncbi influenza virus resource (http://www.ncbi.nih.gov/genomes/flu/ swineflu.html) and global initiative on sharing avian influenza data database (http://www.platform.gisiad.org) using clustal w program implemented in mega version 4 under default conditions [10] . the nucleotide sequence data reported in this work were deposited in the genbank nucleotide sequence database with accession numbers jn037697 to jn037779. were associated with influenza a/h3n2 infection. despite the importance of these preliminary results, our surveillance system had limitations in season 2008-2009, and the rate of influenza virus detection remained low (,10%). in fact, the specimen collection and storage techniques may not always have been optimal. in addition, the identification of influenza viruses was performed primarily using immunofluorescence assays which are less sensitive for the detection of influenza viruses than viral culture the curve of cases of influenza viruses spread out over 11 weeks (from wk 1 to wk 11). there is a decrease of this curve during two weeks (wk 5 and wk 6) (in january 24-30 th and january 31 st -february 6 th ). this descent of curve is probably due to the disturbances in the schooling, the transport and the work in this period. two peaks have been reported in the wk 4 and wk 9 (58.3% and 69.2%).we observe the decrease of influenza a (h1n1)pdm09 in the sentinel samples since week 11. nine influenza-associated deaths were confirmed (ranging 19 to 57 years old) in an ante mortem or post mortem specimen. five of these fatal cases were pregnant women with an underlying clinical risk factor (mean 32 year old). it is important to note, that all deaths were associated with influenza a(h1n1)pdm09 infection. in fact, pregnancy was identified as a particular risk group for adverse influenza outcome during previous pandemics, and also during seasonal influenza [11, 12] . two respiratory disease outbreaks in closed settings were reported during the 2010-2011 season; one in the intensive care unit of charles nicolle's hospital concerning eight members of the medical staff and the second in the intensive care unit of rabta's hospital of tunis when a patient 67 year old diabetic died after infection. ten deaths were virologically confirmed in two separate outbreaks with influenza a(h1n1)pdm09 detected. it was expected that the virus would behave as a seasonal virus and continue to circulate in the population. its behaviour, however, could not be reliably predicted [13] , although it was considered likely that the virus would continue to cause serious disease in a minority of those infected in younger age groups and people in high-risk groups [14] . severe cases were defined as any condition or clinical presentation requiring hospital admission for clinical management according to who guidance criteria [15] . overall, influenza activity in tunisia in 2010-2011 reached a level higher than that seen in the winter of the 2008-2009 season, but lower than during the first wave of the pandemic in the summer of 2009. in fact, in season 2010-2011 over 50% of sentinel specimens were tested positive for influenza. this intensity of influenza activity was similar to that observed during the peak of the 2009-2010 'pandemic' season. this may reflect a greater intensity of influenza circulation resulting from the introduction of a novel virus into naïve human populations, and/or improvements in the sensitivity of laboratory diagnostic methods to detect influenza in use in tunisia. however the percentage of consultants for ili or ari in the sentinel centres in tunisia in 2010-2011 season was lower (8.3%) than the percentage of consultants in the pandemic year (30.3%) (fig. 3) . clinical consultation rates for ili or/and ari were declining overall, whereas the percentage of consultants in the next year of the pandemic was not correlated to virological analysis of influenza viruses in tunisia. despite the increased number of samples obtained in season 2010-2011 under the enhanced surveillance system, there were limitations to our ili surveillance system. this is probably due to the participating clinicians may have not correctly identified all ili or sari cases. also it is possible due to a sub-declaration of the clinical services in the sentinel centres in comparison with the last year in which the medical crow was more motivated by the ministry of health because of the pandemic. the same situation has been reported in england by who european influenza network [16] . in fact, both the burden of severe respiratory infection and the proportion due to viral etiologies including influenza are largely undocumented in africa highlighting the need for continued development of respiratory illness surveillance in this continent [17] . during the 2008-2009 season, 420 specimens were collected from patients at sentinel sites. of those, 80 (19%) were positive for influenza viruses. 45 influenza a/h3n2 (perth lineage) (56.25%), rsvs (26.8%) and 32 hmpvs (21%). in season 2010-2011, a total of 160 specimens were tested positive for respiratory virus and the most frequent respiratory viruses were: rsvs (97/160; 60.6%) and rhinovirus/enterovirus (37/60; 61.6%). the most common nonflu pathogen circulating in three seasons causing the lower respiratory tract infections leading to hospitalisation especially in children was rsv (207/457; 45.29%). in 2010 in chile there have been more cases of acute ari in children but this is attributable to epidemics of respiratory syncytial virus infections (rsv) rather than influenza [18] . this emphasises the importance of countries being able to test for a suite of respiratory pathogens, not just influenza. this data contribute to a better understanding of the circulation of influenza viruses and other respiratory viruses especially in north-africa. phylogenetic analysis of the ha1 nucleotid sequence of 23 influenza a(h1n1)pdm09 viruses from mild, severe (patients hospitalized with severe pneumonia and severe acute respiratory syndrome) and fatal cases, shows that all viruses characterised in tunisia during season 2009-2010 were outside the seven genetic groups described in the european centre for disease prevention and control (ecdc) report [19] . a total of 27 ha genes of influenza a(h1n1)pdm09 viruses regardless of whether they were from 2011 from mild, severe cases and from influenza cases admitted to intensive care units were also sequenced and analysed [20] . this genetic diversity of tunisian strains compared to a/california/7/2009 was consistent with expected patterns of virus evolution. additional substitutions in the position 222 of ha gene were found in 4 tunisian sequences out of 50 viruses (8%). d222g substitution observed in a total of three viruses analysed (6%). most of viruses with this mutation were found in severe cases (2/3), one of them from a fatal case [21] . although most of studies have demonstrated the presence of d222g substitution in severe cases, it was also reported in mild cases [22] [23] [24] [25] . d222e substitution was found in one out of 50 viruses studied (2%). this sample was taken from a patient with severe clinical syndrome. in fact, d222g mutation has been considered relevant for the acquisition of a hypervirulent phenotype during the 1918 influenza pandemic [26] , while the role of d222e in virulence has been ruled out [27] . the clinical significance of d222e mutation is still unclear. analysis of sequences of neuraminidase gene of influenza a(h1n1)pdm09 from 18 severe cases, not received any antiviral treatment, did not show presence of any of the known mutations associated to neuraminidase inhibitors resistance. influenza b viruses were grouped as victoria lineage or yamagata lineage on the basis of the ha gene sequence (fig. 6 [28] . most of yamagata strains were detected in severe cases (9/13; 69%). inspite of lower mutation rate and lower pathogenicity than influenza a, influenza b infection contributes to significant proportion of acute respiratory infection among tunisian people. notably, in season 2008-2009 influenza a/h3n2 viruses, followed by influenza b, have been the predominant influenza viruses circulating in tunisia. in season 2009-2010, pandemic influenza a(h1n1)pdm09 viruses were the predominant circulating viruses but, in contrast to the 2010-2011 season, there is a higher rate of co-circulation with influenza b viruses. the circulation of other winter viruses such as human respiratory syncytial virus and the particularly cold weather were also identified during three seasons in tunisia. unlike the vast majority of influenza b viruses circulating in the world during season 2010-2011, which were from the b/victoria lineage, most of influenza b tunisian strains belong to the b-yamagata lineage, not included in the 2010-2011 vaccine. therefore a virus strain belonging to the b-yamagata lineage was indeed recommended for the vaccine composition of the northern hemisphere for the season 2012-2013. appearance of d222g substitution in ha of a(h1n1)pdm09 viruses circulating in tunisia might be related with severe respiratory disease. mutations associated with resistance to neuraminidase inhibitors oseltamivir and zanamivir were not detected in the neuraminidase gene. to this end proper surveillance systems should be set up in already existing and wellestablished national influenza centres to understand the epidemiology of influenza and other respiratory viruses in north africa, which in turn may help the processes of decision making regarding influenza vaccination on the continent, which may have a high impact on health in africa. pandemic h1n1 and seasonal h3n2 influenza infection in the human population show different distributions of viral loads, with substantially effect the performance of rapid influenza tests influenza surveillance among outpatients and inpatients in morocco one health concept for strengthening public health surveillance and response through field epidemiology and laboratory training in ghana afriflu-international conference on influenza disease burden in africa surveillance and management of influenza on the african continent improving influenza surveillance in sub-saharan africa world health organization. cdc protocol of realtime rtpcr for swine influenza a(h1n1) substitutions in position 222 of haemagglutinin of pandemic influenza a (h1n1)2009 viruses in spain mega 4: molecular evolutionary genetics analysis (mega) software version 4.0 severe forms of influenza a (h1n1) 2009 in pregnant women: experience of the university hospital of fez, morocco and literature review pandemic influenza a(h1n1) 2009 virus in pregnancy world health organization. weekly epidemiological record 18 world health organization. who recommendations for the post-pandemic period clinical management of human infection with pandemic (h1n1)2009: revised guidance world health organization. epidemiological and virological situation update of the 2010/2011 influenza season in the who european region seasonal influenza epidemiology in sub-saharan africa: a systematic review global influenza epidemiology overview for europe -week 30 &id = 919&rootfolder = %2fen%2factivities%2fsciadvice%2flists%2fecd c%20reviews. accessed summary for europe. community network of reference laboratories (cnrl) for human influenza in europe genetic diversity of influenza a(h1n1)2009 virus circulating during the season 2010-2011 in spain haemagglutinin d222g mutation found in a fatal case of pandemic (h1n1) flu in tunisia virulence associated substitution d222g in the hemagglutinin of 2009 pandemic influenza a(h1n1) virus affects receptor binding altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a(h1n1) 2009 influenza virus observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome evolutionary trends of a(h1n1) influenza virus hemagglutinin since 1918 molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza h1n1 2009 viruses associated with severe and fatal infections transmission of hemagglutinin d222g mutant strain of pandemic (h1n1) 2009 virus european surveillance centre for disease prevention and control. surveillance report. weekly influenza surveillance overview. main surveillance developments in week the authors gratefully acknowledge who centre for influenza in london and centers for disease control and prevention (cdc) for the collaboration. we also thank dorra arab ennigrou and ines laaribi in the national influenza centre-tunis; mónica gónzalez-esguevillas, nieves cruz, ana calderón, noelia reyes, manuela lopez-valero, mar molinero and silvia moreno in the national influenza centre-madrid for excellence technical assistance. key: cord-272655-qeojdpez authors: remolina, yuly andrea; ulloa, maría mercedes; vargas, hernán; díaz, liliana; gómez, sandra liliana; saavedra, alfredo; sánchez, edgar; cortés, jorge alberto title: viral infection in adults with severe acute respiratory infection in colombia date: 2015-11-17 journal: plos one doi: 10.1371/journal.pone.0143152 sha: doc_id: 272655 cord_uid: qeojdpez objectives: to identify the viral aetiology in adult patients with severe acute respiratory infection (sari) admitted to sentinel surveillance institutions in bogotá in 2012. design: a cross-sectional study was conducted in which microarray molecular techniques for viral identification were used on nasopharyngeal samples of adult patients submitted to the surveillance system, and further descriptions of clinical features and relevant clinical outcomes, such as mortality, need for critical care, use of mechanical ventilation and hospital stay, were obtained. setting: respiratory infections requiring hospital admission in surveillance centres in bogotá, colombia. participants: ninety-one adult patients with acute respiratory infection (55% were female). measurements: viral identification, intensive care unit admission, hospital stay, and mortality. results: viral identification was achieved for 63 patients (69.2%). comorbidity was frequently identified and mainly involved chronic pulmonary disease or pregnancy. influenza, bocavirus and adenovirus were identified in 30.8%, 28.6% and 18.7% of the cases, respectively. admission to the intensive care unit occurred in 42.9% of the cases, while mechanical ventilation was required for 36.3%. the average hospital stay was 9.9 days, and mortality was 15.4%. antibiotics were empirically used in 90.1% of patients. conclusions: the prevalence of viral aetiology of sari in this study was high, with adverse clinical outcomes, intensive care requirements and high mortality. acute respiratory infection is one of the main causes of hospitalization and death worldwide, although identification of the aetiological agent is not achieved in a majority of cases. instead, the infections are treated empirically and often successfully with antimicrobial therapy. nonetheless, the roles of viruses in the aetiology of these infections are becoming clear, especially after the 2009 pandemic of the new influenza a subtype h1n1 [1] . the presence of a virus does not imply either a more benign clinical course or that systemic inflammatory responses or complications will be absent [2] . due to its implications for public health, the efforts in reinforcing and improving the epidemiological surveillance of respiratory infections have increased. under this initiative, countries have developed surveillance systems by following cases of influenza-like illness and severe acute respiratory infections (saris), which are clinically diagnosed among patients with fever, coughing or sore throat, difficulty breathing and the need for hospitalization [3] . the main aims of surveillance have been to provide information on circulating viruses and the susceptibility of influenza to available antivirals and also to promote and define vaccination needs in different populations. the true impact of viral infections in the aetiology of acute respiratory disease requiring hospitalization is unknown [4] . the aims of this study were to identify viral aetiologies in hospitalized adult patients with sari in bogotá in 2012 and to describe the characteristics and clinical outcomes among these patients. this study was performed in colombia's capital city of bogotá, which is located in the andes in south america, near the equator and 2,600 metres above sea level. a total of 7 tertiary care hospitals performing sentinel surveillance of sari during 2012 participated in the study. such hospitals forwarded all respiratory samples from patients with sari to the district health department. sari was defined as any respiratory infection with a possible viral and/or bacterial origin requiring inpatient management and a clinical presentation of fever of less than 14 days after onset and higher than 38°c, shortness of breath, cough, hypoxia and systemic compromise (systemic inflammatory response syndrome or organ failure), depending on symptom severity [5] . the samples were taken via nasopharyngeal aspiration or swab. the samples were sent, together with the required basic epidemiological data collection form, through the epidemiological surveillance system (sistema de vigilancia epidemiológica nacional-sivigila). samples were sent in a viral transport medium to the public health laboratory of the district health department, where they were stored between 4 and 8°c in refrigerators intended for this purpose. need for intensive care, the use of mechanical ventilation and the length of hospital stay. inclusion criteria consisted of patients over 18 years of age who provided a respiratory sample in 2012 at one of the hospitals performing sentinel surveillance. patients with incomplete medical records from the sampling institution were excluded. the following research and ethics committees of each participating institution approved the study: , with the exception of one institution that requested written informed consent to access medical records (fundación cardioinfantil, instituto de cardiología). written consent was obtained from 25 participants or their proxies (in case of death) from that institution (39 patients were not found, did not respond or refused to provide written consent). written informed consent, in the cases in which was requested and obtained, was kept at universidad nacional de colombia. a total of 288 nasopharyngeal aspiration or swab samples were taken during 2012 from adult patients of the 7 hospitals. the sample was randomly selected, with the only inclusion criteria being patients older than 18 years of age with sari reported to the surveillance system during that year. a sample size calculation found that 117 respiratory samples were needed for a prevalence of 25%, with the poorest accepted result of 20%, 90% power and a 95% confidence level. however, because 150 molecular tests could be processed, the sample was increased to 150 patients. subsequently, the medical records were reviewed, and after the approval and authorisation of the research and ethics committee of each institution, the information was gathered from each hospital. a microarray diagnostic assay using clart 1 pneumovir equipment by genomica (madrid, spain) was used to identify the viruses involved [6] . this assay detects and characterises viruses that most frequently cause respiratory symptoms in humans. the following viruses were analysed: respiratory syncytial virus (rsv) a and b, influenza a (h1n1, h3n2, 2009 a/h1n1 pdm), b and c virus, parainfluenza virus subtypes 1, 2, 3, 4a and 4b, metapneumovirus a and b, adenovirus, enterovirus, rhinovirus, coronavirus subtype 229e and bocavirus. this kit amplifies specific fragments of the viral genome via a reverse transcription polymerase chain reaction (pcr) or via a pcr with hybridisation detection using specific capture probes [7] . the following variables were collected from the patients' medical histories according to international definitions: age, gender and comorbidities such as chronic obstructive pulmonary disease (copd), diabetes mellitus and heart failure. the inclusion criteria took into account the systemic inflammatory response syndrome (sirs), which was defined as a heart rate over 90 beats per minute, a breathing rate of over 20 breaths per minute, leukocytosis of over 12,000 cells per millilitre or leukopenia of fewer than 4,000 cells per millilitre and fever; pneumonia among sari patients was diagnosed from radiological findings of consolidation or alveolar infiltrates. the confusion, urea, respiratory rate and blood pressure (curb)-65 scores for pneumonia were applied using the criteria of age greater than or equal to 65 years, impaired consciousness, blood urea nitrogen (bun) over 20 mg/dl, breathing rate above 30 breaths per minute and a systolic arterial pressure under 90 mmhg or a diastolic pressure under 60 mmhg [8] . additionally, severe pneumonia was considered among patients complying with the following major and minor criteria of the american thoracic society/infectious disease society of america (ats/idsa): shock, need for mechanical ventilation, thrombocytopenia (platelets under 100,000), an arterial oxygen partial pressure to fractional inspired oxygen ratio (pao 2 /fio 2 ) ratio, multilobar compromise, impaired consciousness, leukopenia (under 4,000) and bun over 20 mg/dl [9] . shock was also considered when the systolic arterial pressure was below 90 mmhg or when the diastolic arterial pressure was under 60 mmhg. moderate oxygenation impairment was defined by a pao 2 /fio 2 below 220 mmhg and over 160 mmhg, and severe oxygenation impairment was defined by an index less than or equal to 160 mmhg. institutions that identified viral antigens following institutional protocols performed and interpreted these tests in their hospitals. colonisation of the airway was defined as the presence of microorganisms in a gram test or in a culture of the airway. gram tests and sputum cultures or other respiratory samples were performed based on clinical decisions made by the doctor from the institution. a viral co-infection diagnosis was made when 2 or more different viruses were identified in the same sample. the chi-square test and fisher's exact test were used to compare categorical variables, and the comparison of continuous variables was performed with either student's t test or the mann-whitney u test on a case-by-case basis. the variable analysis was performed using stata (ver. 11.0), and p values less than 0.05 were considered significant. odds ratios (or) with corresponding 95% confidence intervals (ci) were used to analyse outcomes. in 2012 in bogotá, 288 samples from respiratory secretions were collected in the sentinel surveillance system from patients over 18 years of age. through randomised sampling, 150 cases of sari were selected from those respiratory samples (fig 1) . the medical histories of 99 patients with sari reported through the surveillance system were examined. these histories were obtained from 7 bogota hospitals in 2012 according to the following distribution: 45 (45.5%) were from the san rafael university hospital clinic, 23 (23%) were from fundación cardioinfantil, 10 (10%) were from suba hospital, 9 (9.1%) were from el tunal hospital, 6 (6%) were from santa clara hospital, 3 (3%) were from san ignacio university hospital, and 3 (3%) were from the occidente de kennedy hospital. of the 99 patients, 8 showed disease progression beyond 14 days and were dismissed from the final analysis (fig 1) . of the 91 patients included in the analysis, 50 (55%) were female; the patients' average age was 50.6 years (range, 18 to 95 years). table 1 shows the most frequent comorbidities identified. the average length of disease progression was 5.1 days, with a minimum duration of hours and a maximum duration of 14 days (table 2) . radiographical abnormalities were observed in 65 patients. the following radiological findings of chest images were described in the medical histories, in order of frequency: 32 patients had interstitial infiltrates (35.1%), 27 had alveolar infiltrates (29.6%), 26 had consolidation (28.6%), 21 had multilobar compromise (23.1%), and 19 had pleural effusion at admission (20.9%). pleural effusion was found in 15.3% of the patients with alveolar infiltrates and 13.2% of the patients with interstitial infiltrates. we among the sari patients, 82 (90.1%) were treated with antibiotics; patients received between 1 and 7 antibiotics, with an average of 2.3 antibiotics per patient. antibiotics were started a median of 1 day after admission (range, 0 to 21 days, 95% ci: 0-7 days). the beta-lactam group was the most frequently used. oseltamivir was used in 72.5% of cases (66 patients), with a minimum duration of 1 day, a maximum duration of 10 days and an average duration of 4.2 days. oseltamivir was started a median of 1 day after admission (range, 0 to 21 days, 95% ci: 0-9 days). at least one virus was identified in 63 patients (table 3) . influenza virus was the most common and was isolated in 28 patients (30.8%), of whom 21 (75%) had influenza a and 7 (25%) had influenza b (table 3) . of the parainfluenza virus subtypes identified, five cases were piv 3, and one case was piv 1. in the group with more than one viral infection ( influenza and bocavirus were identified throughout the year, though cases of influenza were commonly found in may, august and september, and cases of bocavirus were commonly found in june and november. influenza b was identified only in the second semester, while the two cases of influenza a/h2n3 were seen in may. adenovirus was seen only during the rainy season (april to may and august to november); most cases of metapneumovirus, parainfluenza and rsv were seen only in the first semester, specifically in april and may; the two cases of coronavirus were identified in may (fig 2) . patients with and without viral detection did not differ in terms of comorbidity, with the exception of the frequency of diabetes mellitus. diabetes was detected in 4.8% of patients with viral detection and 20.8% of patients without viral detection (fisher's exact test, p = 0.034). of the patients with pneumonia, a virus was identified in 28 patients (73.7%). severe pneumonia was diagnosed in 62.5% of patients without viral detection and 60.1% of patients with viral detection. no significant differences were observed between the groups with and without viral detection with regards to sirs, neutropenia during hospitalization or shock. of the 22 patients with sari and a history of copd, viruses were identified in 16 (72.7%), 5 of whom had viral co-infections. the viruses were distributed as follows: 6 cases of bocavirus, 4 of adenovirus, 3 of influenza a, 3 of parainfluenza, 3 of rsv, 2 of metapneumovirus, 1 of coronavirus and 1 of rhinovirus. of the 91 patients, 13 were active smokers and 9 (69.2%) had a viral infection, with bocavirus being the most frequently isolated (33.3%) virus. with regards to complications, pleural effusion developed in 12.5% of patients without viral identification and 19% of patients with viral identification; closed thoracotomy was required in 4.2% of patients without viral identification and 3.2% of patients with viral identification; inpatient infection was present in 8.3% and 3.2% of patients without and with viral identification, respectively; and underlying disease complications were found in 25% of patients without viral identification and 38.1% of patients with viral identification. these differences were not statistically significant. patients with chronic pneumopathy showed more complications in their underlying pathologies (77.3% vs. 20.3%, or: 13.5; ci 95%: 3.5-52.7). a total of 14 (15.4%) patients died. however, two cases included either an inadequate sample for pcr or no pcr amplification; thus, the outcome analysis was performed for 12 patients. mortality was reported for 4 cases (16.7%) in the group without viral identification and 8 cases (12.7%) in the group with viral identification (p = 0.23, or: 0.72; 95% ci: 0.17-3.68) ( table 4) . mechanical ventilation was required for 33 patients (36.3%); this intervention was invasive in 24 (26.4%) patients, non-invasive in 13 (14.3%) patients and invasive and non-invasive in 4 (3.6%) patients. this outcome occurred among 7 patients (29.2%) without viral identification and 23 patients (36.5%) with viral identification (p = 0.41, or: 1.4; 95% ci: 0.46-4.59). the average length of mechanical ventilation was 7 days, with a minimum of 1 day and a maximum of 20 days. thirty-nine (42.9%) patients were admitted to the intensive care unit (icu); 3 had unsuitable samples or non-amplified pcr results, 9 (37.5%) were from the group without an isolated virus, and 27 (42.9%) were from the group with an isolated virus (p = 0.21, or 1.25; 95% ci: 0.43-3.75). one case with viral identification was readmitted to the unit with an identification of influenza subtype ah1n1. the average length of icu stay was 8.4 days, with a range between 1 and 21 days. the group with an isolated virus had an average icu stay of 3.8 days, with a range between 1 and 21 days, and the group without an isolated virus had an average stay of 2.8 days, with a range between 1 and 13 days (p > 0.05). the average hospital stay in the sari group was 9.9 days, with a range between 1 and 45 days, with average stays of 10.6 and 9.8 days for patients with and without a virus, respectively (p = 0.6). among patients without a virus, the hospital stay varied between 1 and 45 days, while patients with a virus had hospital stays that varied between 1 and 28 days. of the 8 patients from the viral infection group who died, bocavirus was isolated in 5, influenza was isolated in 4 (all cases of influenza a), metapneumovirus was isolated in 3, and rsv was isolated in 1. of the patients with bocavirus isolation, 19.2% died. death occurred in 14.3% of those in whom influenza was identified, 50% of those in whom metapneumovirus was identified and 20% of those in whom rsv was identified. among patients with viral identification who died, 5 (62.5%) had viral infections of 2 or more viruses (p = 1.7, or: 2.4; 95% ci: 0.46-18.89), and fewer had only one virus detected. no statistically significant differences were observed in the outcomes of the remaining patients with mixed viral infection, which does not confirm higher morbidity in patients with more viruses isolated. the diagnosis of acute respiratory infections is common and apparently easy to perform; however, the determination of the infection's causal agent is more complex, as current diagnostic tools are limited and rarely available in primary health care centres or even in hospitals in much of the world [10] . the role of viruses and their prevalence is a matter of debate and discussion, and findings vary worldwide. the results from studies in new zealand, spain and more recently in the united kingdom report prevalence rates of 28% [11] , 18% [12] ,and 44% [13] , respectively. in our study, viruses were identified as the most frequent causal agents of sari requiring hospitalization in 2012, with most cases showing a high rate of viral co-infection, a high degree of morbidity, prolonged hospital stays and frequent needs for icu management and mechanical ventilation. the definition of sari used in this study is of great utility from an epidemiological surveillance perspective, which is known as syndrome surveillance or surveillance of syndromes [14] . nonetheless, the results reported here demonstrate its clinical relevance and potential utility in this field, as it appears to identify potentially severe patients and those with high complication rates; thus, the definition's routine use should be considered during the performance of clinical duties. furthermore, current challenges in the epidemiological surveillance of viral respiratory tract infections include the early and fast identification of aetiological agents, especially at the beginnings of outbreaks, and the optimal and timely management of a large number of samples [14] . our study suggests that molecular technology makes it possible to closely follow circulating viruses in these groups of patients. in contrast to the epidemiological definition used, the curb-65 index of pneumonia was not useful, as only 10 (26%) scored for general hospitalization and none scored for icus using these criteria. this finding contrasts with the high number of our patients who required icu care, which agrees with findings in the literature that applied this scale in cases of viral pneumonia during the influenza a subtype h1n1 pandemic of 2009; these results demonstrate its low value for detecting either severity or the need for icu admission in patients with viral pneumonia [15] . these findings represent a marked difference in severity stratification between influenza-related pneumonia and pneumonia caused by other aetiological agents, indicating the importance of clinical judgement in this scenario [16] . the application of these scales in non-influenza viral pneumonia has yet to be assessed. moreover, a recent multicentre study of adults with radiographically confirmed pneumonia has shown that viruses are now the most commonly identified pathogens. human rhinovirus and influenza virus are more frequently found than streptococcus pneumoniae [17] . together, viruses represent a quarter of patients and more than half of the pathogens identified. the mortality rate was relatively high for patients both with and without communityacquired pneumonia. a study performed in the united states reported a low mortality rate in patients hospitalized for viral infection; nonetheless, bacterial co-infection increased both the morbidity and the mortality [18] . this variable should be considered for our patients. countries with marked seasons report a correlation of influenza with high mortality due to respiratory infections, which is usually related to community-acquired pneumonia [19] . this seasonal pattern of acute respiratory infections, especially viral infections due to influenza, is dependent on temperature, humidity and host factors, such as serum vitamin d levels [20] . colombia is located in the tropics and thus lacks seasons; however, this study confirms that these infections, especially those caused by influenza and rsv infections, increase during the rainy season in countries at these latitudes [21] . this pattern shows the importance of epidemiological surveillance, especially during the seasons when viruses circulate, because it may increase control and prevention strategies such as timely vaccinations. techniques based on identifying nucleic acids, such as those used in this study, can obtain more rapid and precise results for diagnosing viral infections in order to provide appropriate and managed medical care [22] . there is limited information on the diagnostic utility of these new tests. a study by sultankulova et al. compared viral isolation of influenza a with dna [23] . microarray technology showed a higher sensitivity (99.5%) and similar specificity (98.5%) to real-time pcr. however, another study with the same microarray kit used in this study showed a high specificity (100%) and low sensitivity (52%) in the clinical scenario of atypical pneumonia [24] . a study in japan using near patient automated microarray technology showed not only a higher sensitivity and specificity compared to immunochromatographic antigen detection (the gold standard used was virus isolation) but also quicker results for children infected with influenza and rsv [25] . a recent multicentre study in the united states using real-time pcr technology was able to precisely and reproducibly detect all adenovirus infections in a group of children and adults with respiratory infections, which increased the possibility of establishing a clear diagnosis [26] . taken together, use of molecular technology for the diagnosis of viral infections can improve detection and identify the cases in which antibiotic use might be inadequate. the use of antibiotics in acute respiratory infections is indiscriminate and excessive, and according to worldwide literature, is employed in more than half of all respiratory infections [27] . this study determined that 90% of the cases are treated with antibiotics, although most of the infections were viral in origin. additional strategies, such as the measurement of procalcitonin, can identify patients who would not benefit from antibiotic therapy [28] . this study has several limitations. although it was multicentre in design, the information was gathered from only one city in colombia. one important limitation is that it is a retrospective study; thus, it was not possible to control viral and bacterial sampling, and there was limited access to relevant data, such as previous influenza or pneumococcal vaccination history or co-morbidity measurement. the sample size was smaller than expected, although the prevalence of viral infection was higher than expected. together, all of these limitations lead to difficulties in establishing significant comparisons among the groups and in appropriately assessing the impact of co-morbidity and bacterial co-infection in the outcomes, especially in relation to the role of pneumococcus [29] . other limitations include the difficulty of defining the actual roles of certain viruses, such as bocavirus and rhinovirus, in respiratory infection, which are also detected in asymptomatic patients according to descriptions in the literature [4] . the empirical use of antibiotics in most cases, without using additional tools to confirm bacterial infection, is also a limitation. viral pneumonia epidemic viral pneumonia and other emerging pathogens guia opertativa para la vigilancia centinela de eti e irag what is the role of respiratory viruses in community-acquired pneumonia?: what is the best therapy for influenza and other viral causes of community-acquired pneumonia? protocolo de vigilancia en salud publica-infección respiratoria aguda (ira) caracterización de virus causantes de infecciones respiratorioas humanas mediante identificación genomica para diagnostico in vitro respiratory viruses in children admitted to hospital intensive care units: evaluating the clart(r) pneumovir dna array defining community acquired pneumonia severity on presentation to hospital: an international derivation and validation study infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the current epidemiology and clinical decisions surrounding acute respiratory infections incidence and characteristics of viral community-acquired pneumonia in adults viral community-acquired pneumonia in nonimmunocompromised adults adults hospitalised with acute respiratory illness rarely have detectable bacteria in the absence of copd or pneumonia; viral infection predominates in a large prospective uk sample surveillance for emerging respiratory viruses pandemic influenza (h1n1) 2009 pneumonia: curb-65 score for predicting severity and nasopharyngeal sampling for diagnosis are unreliable severity of influenza a 2009 (h1n1) pneumonia is underestimated by routine prediction rules. results from a prospective, population-based study community-acquired pneumonia requiring hospitalization among u.s. adults bacterial complications of respiratory tract viral illness: a comprehensive evaluation estimates of mortality attributable to influenza and rsv in the united states during 1997-2009 by influenza type or subtype, age, cause of death, and risk status roles of humidity and temperature in shaping influenza seasonality epidemiology and seasonality of respiratory tract virus infections in the tropics utilization of nucleic acid amplification assays for the detection of respiratory viruses comparative evaluation of effectiveness of iavchip dna microarray in influenza a diagnosis microorganisms in respiratory tract of patients diagnosed with atypical pneumonia: results of a research based on the use of reverse transcription polymerase chain reaction (rt-pcr) dna microarray method and enzyme-linked immunosorbent assay the clinical utility of a near patient care rapid microarray-based diagnostic test for influenza and respiratory syncytial virus infections in the pediatric setting multi-center evaluation of the adenovirus r-gene us assay for the detection of adenovirus in respiratory samples antibiotic prescribing in ambulatory care settings for adults with colds, upper respiratory tract infections, and bronchitis procalcitonin to initiate or discontinue antibiotics in acute respiratory tract infections a role for streptococcus pneumoniae in virus-associated pneumonia we acknowledge the following institutions for their participation in this study: suba hospital, hospital de occidente kennedy, santa clara hospital (gerson arias), el tunal hospital (diana marcela bejarano), san ignacio university hospital (sandra valderrama), fundación cardioinfantil (alvaro arango), and hospital san rafael (carlos restrepo). key: cord-276037-0bxwv6b7 authors: bias, harald; quarcoo, david; meier-wronski, claus; wicker, sabine; seybold, joachim; nienhaus, albert; groneberg, david a; roux, andres de title: self-reported adverse reactions in 4337 healthcare workers immunizations against novel h1n1 influenza date: 2011-08-17 journal: bmc res notes doi: 10.1186/1756-0500-4-297 sha: doc_id: 276037 cord_uid: 0bxwv6b7 purpose: the use of the 2009 h1n1 vaccine has generated much debate concerning safety issues among the general population and physicians. it was questioned if this is a safe vaccine. therefore, we investigated the safety of an inactivated monovalent h1n1 pandemic influenza vaccine methods: we focused on the h1n1 pandemic influenza vaccine pandemrix(® )and applied a self reporting questionnaire in a population of healthcare workers (hcws) and medical students at a major university hospital. results: in total, 4337 individuals were vaccinated, consisting of 3808 hcws and 529 medical students. the vaccination rate of the employees was higher than 40%. the majority of individuals were vaccinated in november 2009. in total, 291 of the 4337 vaccinations were reported to lead to one or more adverse reactions (6.7%). local reactions were reported in 3.8%, myalgia and arthralgia in 3.7%, fatigue in 3.7%, headache in 3.1%. conclusions: our data together with available data from several national and international institutions points to a safe pandemic influenza vaccine. various infectious diseases play a major role in occupational health. next to classical diseases such as tuberculosis [1] , new diseases including sars [2] [3] [4] or the novel influenza a h1n1/2009 virus [5, 6] are endangering occupational health in the last years. the novel influenza a h1n1/2009 virus was first identified in mexico and led to pandemic warning of the who in june 2009 [7] . novel influenza a h1n1/2009 virus is often called "swine flu" and represents a result of the reassortment of different influenza viruses [8, 9] . it was reported that the hemagglutinin (ha) gene of a h1n1/2009 was similar to that of swine flu viruses which are present in united states pigs since the year 1999. by contrast, the matrix protein (m) and neuraminidase (na) genes are found in european swine flu isolates. phylogenetic analysis of the pandemic h1n1/2009 virus shows that six genome segments stem from a triple-reassortant virus circulating in north american swine, seeded from human, avian and classical swine lineages. concerning the pandemic, a first outbreak of influenza-like illness occurred in mexico and the usa in april 2009 and the us centers for disease control and prevention reported seven cases of novel a/h1n1 influenza by this time. by april 24 the who issued a health advisory on the outbreak of "influenza like illness in the united states and mexico". despite measures by the mexican government against the spread of the virus, the number of confirmed cases raised to 2,099 by may 7 2009 [10] . one month later, on june 11, 2009, the who officially declared a h1n1 pandemic [11] . this was the first pandemic since the 1968 hong kong flu pandemic. the who alert level was lifted to phase 6. the safety of influenza vaccine is in the focus of research since many years [13] [14] [15] [16] [17] . due to a special debate on the safety of the pandemic influenza vaccines [18] , it was the objective of the present study to analyse the safety using a self reporting questionnaire approach in the acute event of a pandemic and a novel vaccine which was debated for its safety by the general population and healthcare worders (hcws). we chose a population of healthcare workers (hcws) and medical students after vaccination with pandemrix ® since according to german federal recommendations hcws had a top priority for vaccination [19] . after the declaration of the pandemic, the german federal commission for vaccination (stiko) recommended the vaccination for hcws. the local occupational health service of the university hospital berlin charité was supplied with 6000 pandemrix ® doses on october 26, 2009 . the active antigen of pandemrix ® derived from the a/ california/7/2009 (h1n1). pandemrix ® also contains an immunologic adjuvant as03 which consists of dl-αtocopherol (vitamin e), squalene and polysorbate 80. thiomersal (thimerosal) is added as a preservative. other components are formaldehyde, sodium deoxycholate, and sucrose [20] . the vaccine is generated in hen's eggs and therefore also contains traces of egg proteins. a single dose of the vaccine was injected by occupational physicians with the recommended dosage and vaccination procedure. in the situation of a pandemic and the acute supply of a novel vaccine which included the immunologic adjuvant as03 and thiomersal (thimerosal), and a public debate about the safety of this vaccine, the sample selection method based on the assessment of all individuals that were vaccinated after informed consent at the occupational medicine centre of germany largest university hospital. no specific further sample selection methods were applied. in total, 3808 hcws and 529 medical students were vaccinated with pandemrix ® after informed consent. the observed vaccination period began on october 26, 2009 and ended at december 30, 2009. all individuals that were vaccinated after informed consent. applying a cross-sectional study design, a self reporting questionnaire (tab. 1) was used that consisted of questions relating the time of the vaccination, the start of symptoms, the duration of symptoms, the consultation of a physician and the incapacity for work with duration. the symptoms included in the questionnaire are presented in appendix 1. the self reporting questionnaires were received until 2010-01-31. in total, 3808 hcws and 529 medical students were vaccinated. the peak of vaccinations was november 13 2009 with 459 vaccinations at one day ( figure 1 ). overall, 291 of the 4337 vaccinated individuals returned the questionnaire and reported adverse reactions. this is a rate of 6.7%. the majority of reported adverse reactions was found in the age between 30 and 39 years. (figure 2 ) the most frequently reported local site reactions were: local pain/pruritus or the sensacion of heat at the injection site in 3.8% out of the 4337 vaccinations, myalgia or arthralgia in 3.7%, induration or erythema at the injection site in 2.6%, lymph node swelling in 0.9%, skin rash in 0.3% and ecchymosis at the injection site in 0.1% ( figure 3) . the presence of systemic adverse reactions were reported as follows: fatigue in 3.7%, headache in 3.1%, flu-like symptoms in 2.3%, shivering in 1.9%, temperature > 38°c in 1.3%, dizziness in 1.1%, increased perspiration in 1.1%, gastrointestinal symptoms in 1.0%, drowsiness in 0.9%, , insomnia in 0.7%, formication in 0.3%, further some severe reportable adverse reactions were observed (0.5%, figure 3 ) as one case of facial nerve paralysis, one case of rheumatoid arthritic symptoms and one case of skin alteration which was reported to the local health authorities and the paul-ehrlich-institute. the mean duration of symptoms lasted 3.5 days, the maximal duration of symptoms was reported with 40 days. overall, 42 hcws (0.97%) were not able to work due to the adverse reactions ( figure 4) . the resulting leave of absence was 2.7 days in mean with a maximum absence of 14 days in one case. in total, there were 115 days of absence recorded. personal protective equipment is important to prevent transmission of novel a/h1n1 as stated earlier by shine et al [21] . however, vaccination is the most effective means of preventing influenza transmission and associated morbidity and mortality. it is most important to realize that an effective measure against a pandemic is to have vaccinated and well-informed health care workers. unfortunately, the a/h1n1 vaccination coverage was extremely affected by an ongoing public discussion about potential side effects. therefore, we analysed self reporting questionnaires concerning adverse reactions in 4337 hcws and medical students. of course, this study -that was initiated in the acute event of a pandemic and a safety discussion -has a variety of limitations. apart from the paucity of demographic data, self reporting questionnaires are largely limited since there may be a number of individuals who do not return the questionnaire despite adverse reaction manifestation. however, there was a need to assess potential adverse reactions since the general population and the hcws asked for data about the new vaccine. therefore, we decided to undertake a self reporting study despite that fact that the extent of underreporting of side effects can not be examined precisely in the chosen design. it is noteworthy that with our self reporting system we found a rate of 6.7% (291 of 4337 vaccinations). this frequency differs slightly from data from other studies but points to a safe vaccine in terms of acute adverse reactions. a prospective, randomised study with 178 participants by vajo et al. concluded that all adverse events were rare, mild, and transient. using the vaccine fluval p, the most frequent reactions in this study were pain at injection site (eight cases) and fatigue for 1-2 days after vaccination (three cases) [22] . concerning the vaccination rate we can report a rate of 4337 of about 10 000 employees of the hospital. this is a vaccination rate of over 40%. in a parallel study in frankfurt/main, the influenza vaccination rates of the hcws of the university hospital frankfurt were measured. in this study, we were also able to show that the 2009 vaccination rate (seasonal influenza [40.5%], swine flu [36.3%]) was better than the average annual uptake of influenza vaccine in the german health care system (approximately 22% for seasonal and 15% for swine flu) [23] . in meantime, a number of studies were published that also addressed safety issues of the h1n1 vaccination in healthcare workers [24] [25] [26] . i.e. an inactivated, splitvirus, unadjuvanted ah1pdm vaccine, manufactured in japan, was given to hcws from october 19, 2009 . a retrospective cohort study was conducted and and severe adverse events were rare [24] . a recent study using monovalent vaccination (panenza; sanofi pasteur, val de reuil cedex, france) among hcws in a university hospital setting in thailand also reported a low rate of side effects. the most common adverse reaction was fatigue/uncomfortable feeling (24%) [25] . it can be summarized that our data points to a safe pandemic influenza vaccine in our 4337 vaccinations. it needs to be taken into account that the use of self reporting questionnaires leads to differing results concerning the frequency of adverse reactions, of course. therefore, this mode of reporting should be interpreted cautiously and only applied in the acute event of pandemics/novel vaccines that are administered without the usual safety testings. prevalence of latent tuberculosis infection among health care workers in a hospital for pulmonary diseases treatment and vaccines for severe acute respiratory syndrome geographic dependence, surveillance, and origins of the 2009 influenza a (h1n1) virus reassortment patterns in swine influenza viruses historical perspective-emergence of influenza a (h1n1) viruses world health organization declares a (h1n1) influenza pandemic trivalent inactivated influenza vaccine safety in children: assessing the contribution of telephone encounters misperceptions regarding influenza vaccine safety for individuals with chronic medical illness safety of trivalent inactivated influenza vaccines in adults: background for pandemic influenza vaccine safety monitoring active influenza vaccine safety surveillance: potential within a healthcare claims environment feasibility of telemonitoring for active surveillance of influenza vaccine safety in the primary care setting in the netherlands swine flu agitates the adjuvant debate healthcare workers should get top priority for vaccination against a/h1n1 flu, who says novel h1n1 influenza and respiratory protection for health care workers safety and immunogenicity of a 2009 pandemic influenza a h1n1 vaccine when administered alone or simultaneously with the seasonal influenza vaccine for the 2009-10 influenza season: a multicentre, randomised controlled trial influenza a (h1n1) 2009: impact on frankfurt in due consideration of health care and public health effectiveness and safety of pandemic influenza a (h1n1) 2009 vaccine in healthcare workers at a university hospital in japan high coverage and safety of influenza a (h1n1) 2009 monovalent vaccination among health care personnel in thailand immunogenicity and safety of a china-made monovalent pandemic (h1n1) 2009 influenza a vaccine in healthcare workers in guangzhou, china self-reported adverse reactions in 4337 healthcare workers immunizations against novel h1n1 influenza the authors would like to thank the medical director of the charité, prof. dr. u. frei for his support during the h1n1 vaccination campaign and all involved hcws. authors' contributions hb carried out the study, participated in the analysis and drafted the manuscript. dq participated in the analyzing and drafting process. cm-w and sw participated in the pilot study design. js and an participated in the design of the study and the statistical analysis. dag and adr conceived of the study, and participated in its design and coordination and helped to draft the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. submit your manuscript at www.biomedcentral.com/submit key: cord-269324-zh1a3gwh authors: mubareka, samira; palese, peter title: human genes and influenza date: 2008-01-01 journal: j infect dis doi: 10.1086/524067 sha: doc_id: 269324 cord_uid: zh1a3gwh nan why some individuals resist infection or recover quickly, whereas others experience severe disease associated with infection, is a fundamental question that medicine has struggled to answer. pathogens and host immune factors have been extensively investigated for many infectious diseases, to address these questions. however, limited information is available concerning the influence of host genetics on the response to viral infections. genetic determinants have the potential to play a role at numerous points during the course of viral infection, including viral attachment and entry, replication, disease progression and development of severity, and, finally, transmission. in this issue of the journal, albright et al. [1] propose that the severity of influenza illness may have a heritable component. to investigate this hypothesis, the authors used as a resource the utah population data base, which contains data from founding families and their descendants, comprised primarily of members of the church of jesus christ of latter-day saints (i.e., mormons), thus representing a relatively ethnically homogeneous population. albright et al. [1] estimated the relative risk (rr) of death for relatives of 4855 individuals (spanning 3 generations) who died of influenza. a substantial proportion of deaths occurred during the 1918 influenza pandemic, when a total of 1937 deaths were documented between 1918 and 1921, and 1293 additional deaths occurred between 1922-1932, followed by a dramatic decrease in the number of deaths occurring annually. the rr of death for first-degree relatives was 1.54 (p ͻ .0001). the rr was higher for spouses (1.98) and for secondand third-degree relatives (1.22 and 1.16, respectively). the timing of the deaths of third-degree relatives suggests that the deaths were not the result of a common exposure. to control for shared environment, the rr of death for spouses' relatives was compared and was found to be lower for first-, second-, and third-degree relatives. excess relatedness among individuals dying of influenza was estimated using the geneological index of familiality, which demonstrated that relatedness among these individuals, including individuals who died during the 1918 pandemic, was greater than expected (p ͻ .001). the analysis was repeated with close relatives excluded (to control for shared environment), and the results were consistent with previous findings. consistent results were not observed when the same analysis was repeated for individuals with diphtheria-associated deaths. for such individuals, excess relatedness was demonstrated; however, when close relationships were excluded, no excess relatedness was detected. specific genes responsible for the host immune response have been invoked as major determinants of the clinical course of hiv-associated disease and hepatitis b and c virus infections [2, 3] . however, there is very little information with respect to genetic determinants as they relate to severe influenza. over the past decade, a greater understanding of the immune evasion strategies of influenza virus has developed. this knowledge can be used to propose several candidate genes that may be responsible for severe illness. clinical and animal studies indicate that cytokine dysregulation is associated with acute respiratory distress syndrome and death among hosts infected with avian influenza virus (h5n1) [4 -6] . toll-like receptors (tlrs), particularly tlr3 (which recognizes double-stranded rna) and tlr7 and tlr8 (which recognize single-stranded rna), are central to antiviral innate immunity [7] . singlenucleotide polymorphisms in tlr genes are not uncommon and vary among populations [8] . tlr4 has been implicated in the innate immune response to respiratory syncytial virus (rsv) infection, and polymorphisms in the tlr4 gene have been associated with severe bronchiolitis in rsv-infected infants, although the significance of the asp299gly polymorphism appears to be a matter of ongoing debate [9 -12] . a missense mutation in the tlr3 gene (f303s) conferring loss of function in in vitro assays has been identified in a japanese child with influenzaassociated encephalopathy [13] . interestingly, better survival rates have been demonstrated for tlr3 knockout mice than for wild-type mice, despite the tlr3 knockout mice having higher lung virus titers after influenza virus infection [14] . tlr genes (in particular, tlr3) would therefore be worthy candidate genes to investigate. in addition, rna helicases, such as retinoic acid inducible gene i (rig-i), recognize double-stranded rna and, thus, contribute to the antiviral state of an infected cell [7] . inhibition of rig-1 by nonstructural protein 1 (ns1) of influenza virus antagonizes interferon (ifn) production and is a means by which the virus evades the host immune system [15] . therefore, the rig-i gene should also be considered to be a candidate for attempts to elucidate aberrations in the immune system that may lead to severe influenza illness. other host gene products involved in ifn production and induction antagonized by the influenza virus ns1 are worthy of consideration [16] . changes in genes involved in other aspects of immunity, such as the complement system, have also been associated with recurrent viral upper respiratory tract infections, and they should not be overlooked [17] . mannose-binding lectins are part of the innate immune system, binding pathogenassociated carbohydrates and activating the complement system through the classical pathway. mutations within the mbl gene have been associated with severe acute respiratory syndrome (sars) [18] . susceptibility to the sars coronavirus has been associated with changes in the 2'5'oligoadenylate synthetase 1 (oas1) and myxovirus resistance 1 (mxa) genes as well [19] . all of these genes would be excellent candidates for an analysis to iden-tify determinants of severity of disease after influenza virus infection. the findings of the study by albright et al. [1] raise many complex questions. the outcome of influenza illness is likely to be dependent on environmental, nutritional, demographic, immunological, and (especially) virologic factors. undoubtedly, host factors, including comorbidities, will also have a bearing on outcome, and the relative contribution of different genetic determinants is likely to vary depending on environmental and virologic contexts. a significant proportion of the data collected in this study emanates from the influenza pandemic of 1918, which occurred in a setting very different from that of interpandemic cases. in addition, the use of death certificates imposes significant limitations. during the 1918 pandemic, anyone dying with respiratory symptoms was likely to have been given a code denoting "pneumonia and influenza," which introduced a potential bias. one could argue that errors related to certification of death would be random rather than systematic and should have minimal bearing on the results. nonetheless, death certificates are notoriously inaccurate, and the absence of microbiological and/or serologic data is a significant limitation of this study. there are also a number of potential confounding factors in this investigation. other heritable factors, such as cardiopulmonary conditions or cystic fibrosis (the most common autosomal recessive lethal disorder in whites), are possible examples. other examples would include common conditions that have a genetic component, such as asthma and coronary artery disease. there is reasonable evidence that the influenza-associated mortality rate is higher for individuals with established coronary artery disease (than for those with other chronic conditions) [20] . coinfections may also confound the data, and the contribution of coinfecting pathogens to mortality cannot be excluded, bearing in mind that genetic susceptibility to such organisms as the pneumococcus has been proposed [21] . environmental and geographic factors, such as socioeconomic variables and environmental and occupational (e.g., mining) exposures, may also cluster in families, and such factors as poverty, malnutrition, and overcrowding may span generations. in addition, access to health care may have been limited for certain families in remote settings, thus contributing to the lethality of influenza virus infection, particularly in individuals at the extremes of age. the implications of this study by albright et al. [1] are that heritable factors for severe influenza illness may well exist and that elucidation of these factors remains a challenge. identification and characterization of genetic determinants of the outcome of infection with influenza virus would lead to further insight into the pathophysiology of the virus and its immune evasion strategies. one would hope that this insight would translate into identification of prognostic factors and improved management of individuals at risk for adverse outcomes after influenza virus infection. identification of genetic determinants of outcome after influenza virus infection would require a substantial number of severe cases of influenza (in which severity of illness is not the result of other factors, such as underlying cardiovascular disease) to gain sufficient power and produce interpretable data. resources stemming from the sequencing of the human genome are now available and render this challenging task more feasible. such approaches as the genomewide and candidate gene study designs are reviewed by burgner et al. elsewhere [22] . the latter approach may be more feasible, given the growing knowledge regarding ifn antagonism and immune evasion by influenza viruses. one must bear in mind that findings may not apply across all host or virus populations. in the event of a pandemic, however, early identification of and intervention for individuals at higher risk for severe illness and death resulting from influenza due to a predisposing genetic determinant would be invaluable. evidence for heritable predisposition to death due to influenza human genes that limit aids a comparative review of hla associations with hepatitis b and c viral infections across global populations acute respiratory distress syndrome induced by avian influenza a (h5n1) virus in mice role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia toll-like receptors and rna helicases: two parallel ways to trigger antiviral responses genetic polymorphisms of viral infection-associated toll-like receptors in chinese population involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus tlr-4 and cd14 polymorphisms in respiratory syncytial virus associated disease association between common toll-like receptor 4 mutations and severe respiratory syncytial virus disease common human tolllike receptor 4 polymorphisms-role in susceptibility to respiratory syncytial virus infection and functional immunological relevance a missense mutation of the toll-like receptor 3 gene in a patient with influenza-associated encephalopathy detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia inhibition of retinoic acid-inducible gene i-mediated induction of beta interferon by the ns1 protein of influenza a virus type 1 interferons and the virus-host relationship: a lesson in détente homozygous deletion of the cyp21a-tnxa-rp2-c4b gene region conferring c4b deficiency associated with recurrent respiratory infections association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphisms of oas1 and mxa genes: a case-control study influenza vaccination as secondary prevention for cardiovascular disease: a science advisory from the american heart association/american college of cardiology mbl genotype and risk of invasive pneumococcal disease: a casecontrol study genetic susceptibility to infectious diseases: big is beautiful, but will bigger be even better? key: cord-307813-elom30nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: 2018-07-10 journal: front immunol doi: 10.3389/fimmu.2018.01547 sha: doc_id: 307813 cord_uid: elom30nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu 1918 h1n1, asian flu h2n2 1957, hong kong h3n2 flu 1968, and more recently the pandemic of h1n1 2009 (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the 2015 h1n1 outbreak in india (1, 2) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m2 ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups (3) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported (4, 5) . high level of resistance (up to 91%) to m2 blockers has been reported in h3n2 virus strain in american isolates (6) . resistance has also been reported in h5n1 virus (7) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h1n1)pdm09 in india 2015 associated with oseltamivir drug resistance (8, 9) . in addition, a large cluster of influenza a(h1n1)pdm09 viruses in japan was found to have increased oseltamivir and peramivir drug resistance (5) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h5n1 and h7n9 virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death (10) (11) (12) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated (13) . in this review, we will discuss the latest studies (in the past 5 years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: (1) entry, (2) genome nuclear import, (3) replication and protein synthesis, and (4) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed (14, 15) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps (16, 17) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis (18) . macropinocytosis was revealed as an alternative entry pathway for iav (19) , which subsequently enters the canonical endocytic pathway (20, 21) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph 6.5 to 5.0, mediated by v-atpase proton pump (22) , takes place as the endosome matures (23, 24) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha1 and ha2 from precursor molecule ha0 (25, 26) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m2 proton channel (27) , which in turn promotes the dissociation of m1 layer from both the viral envelope (24) and the viral ribonucleoprotein (vrnp) complex (28) . interestingly, a sharp decrease in ph from neutral to an acidic ph of 5.0 as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m1 from the np in the vrnp complex, destabilization of m1 layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome (24) . proteolytic cleavage of ha0 to ha1/ha2 is an important step in iav replication. this cleavage relocates ha2, converting previously uncleaved ha0 to a metastable conformation that induces membrane fusion at acidic ph (29) . inefficient cleavage and activation of ha leads to low infectivity (30) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position 329. human airway epithelium serine proteases hat and tmprss2 were identified as the host factors for cleavage at this residue (31) . aprotinin, purified from bovine lung (32) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery (33) . its potential as an anti-iav drug has been recognized for over a decade (34) and has been shown to reduce the infectivity of a broad spectrum of iav strains (34, 35) both in vitro (26) and in vivo (36) . once withdrawn from the western drug market due to its association with mortality (33), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia (36) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to 1996 (37) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities (35) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer (38, 39) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia (38) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h5 and h7 subtypes, are reported to have ha cleavage sites rich in basic residues (30) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin (40) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h7 virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h1n1 virus bearing single cleavage site (41) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm (24) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro (42) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a1, when used at high concentrations (10-100 nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase (43, 44) . however, prominent cytotoxicity to host cells was also observed at such concentrations (44) . interestingly, lower concentrations (0.1 nm) of bafilomycin a1 lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a1 is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect (45) . in contrast to bafilomycin a1, diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects (46) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir (46) . since drug resistance to these widely administered antivirals is of major public health concern (13), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes (47, 48) . while humans express ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10, only ifitm 1, 2, and 3 are both immune-related as well as interferon (ifn)-inducible (48) , and have been observed to restrict the replication of different viruses, including iav (49) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes (50) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place (51) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase 2 (acp2)-mediated niemann-pick c2 activity and impaired the membrane fusion of iav and influenza b virus (ibv) (52) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm3 exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion (53) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm (54, 55) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek 293t cells and (less permissive) hela cells, mirna-33a has been identified as a negative regulator for iav infection via the inhibition of archain 1 (arcn1, also known as δ-copi) (56). arcn1 is a subunit of the copi complex that is required for intracellular trafficking and endosome function (57) , depletion of which has been reported to inhibit iav infection (14) . despite impaired iav internalization caused by arcn1 depletion via sirna (56, 58), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means (58) . it is hypothesized that the long-term (lasting days) perturbation on arcn1 by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection (58) . the potential of targeting arcn1 for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place (59) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus (60), while recent studies report that vrnp complexes utilize the importin-α-importin-β1 (impα-impβ1) system for nuclear import (59, 61) and lacking of importin-α7, in an importin-α7 knockout mouse model were found to be resistant to iav infection (62) . ivermectin has long been clinically administered for the treatment of parasitosis (63) , but has recently come to attention as a potential inhibitor of impα/β (64) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv-1 (64) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited (65) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex (59) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment (66, 67) . cdc2-like kinase 1 (clk1) is a kinase which regulates alternative splicing of pre-mrna (68) . inhibition of clk1 by the chemical tg003 or knockdown of clk1 is shown to cause a decrease in m2 mrna generation and disrupt downstream m2 protein expression, prominently reduced iav propagation (15) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg003 in vitro (69) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m1 mrna (70) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions (71) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners (72) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals (73) . nuclear rna export factor 1 (nxf1) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf1 in hek 293t cells revealed prominent viral mrna nuclear retention in host cell nucleus (74) . protectin d1 (pd1), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects (75) . pd1 production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd1 was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf1 was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd1 in regulating nxf1 in nuclear export of viral rna. natural pd1 expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor-4a (eif4a) family plays an important role in protein translation (76, 77) . eif4a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro (78) , with inhibition of iav mrna translation (79) . the eif4a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro (80) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development (81) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding (82) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others (83) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir (84) . nitazoxanide has a high barrier of resistance to iav (83) and other viral strains resistance to neuraminidase inhibitors (85), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials (83). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus (59), assembled (86) , and transported to the plasma membrane [apical in polarized cells (87) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1547 exportin 1 (xpo1, also known as crm1) is well known for its function in the nuclear export of protein (88) and rna, including viral rna (89) . similar to hiv (89, 90) , iav viral rna does not directly bind to xpo1 but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns2) and the vrnp complex have been proposed as the nuclear export complex (91) . cellular xpo1 has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo1 inhibitor, revealing that in vitro inhibition of xpo1 led to nuclear retention of vrnp complex (92, 93) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects (94) . verdinexor (also known as kpt-355) is a new bioavailable selective inhibitor of xpo1. it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments (95, 96) . it is worth mentioning that delayed administration of verdinexor at day 4 post-infection was still deemed beneficial, with reduced viral load in vivo (96) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within 48 h of symptom onset) (97) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp2392-e10, which binds and inhibits the function of xpo1, can suppress iav replication in vitro (98) further strengthens the concept of iav intervention by targeting xpo1. viral m1 protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m1 protein links vrnp complex to viral nuclear export protein nep which interacts with xpo1 for nuclear export (59) . thus, viral m1 protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge (99) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m1 protein (72) , increases the self-association of m1, and hinders m1 nuclear import (100) . csa also promotes the cypa-dependent degradation of viral m1 protein (72, 101) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m1 protein stability and function. recently, cd151, a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo (102) . knockdown of cd151 in primary human nasal epithelial cells resulted in the nuclear retention of host xpo1, viral np, nep, and m1 proteins, with an increased survival rate observed in iavinfected cd151 knockout mice. co-immunoprecipitation assays suggest that cd151 interacts with viral np, m1, and nep proteins (102) ; however, the exact domains involved in interaction and the mechanism of cd151 function in nuclear export remain unclear. given that a small molecule inhibitor for cd151 is now under development (103) , more data revealing the role of cd151 in iav infection and subsequent use in targeting cd151 as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u0126, probably mediated via myosin (light chain) (104), a known motor protein, impairs the nuclear export of vrnp complexes (105) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo (106) and in vitro (105) . the replication of ibv (107) as well as borna disease virus (108) was shown to be inhibited by u0126, suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u0126 has little effect when administered orally (106) . another mek inhibitor, ci-1040 (also known as pd184352) was shown to have high potency against iav in vitro (106). ci-1040 has completed phase ii clinical trials as an anti-tumor drug, with the application of ci-1040 as a potential anti-iav drug candidate recently revisited. unlike u0126, ci-1040 is orally bioavailable and oral administration of ci-1040 at 48 h post-infection protected 60% of the iav-infected mice, while the oseltamivir-treated group experienced a 100% death rate (109) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci-1040 as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor 2 (fpr2) located at the host cell surface was identified as an erk stimulator (110) . antagonizing fpr2 promoted the survival of iav-infected mice (110) . furthermore, fpr2 antagonists have been described to possess antiviral activity against not only iav but also ibv infection (111) , promoting the idea that antagonizing fpr2 to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab11a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis (112) , a property utilized by many rna viruses, including iav (87, (113) (114) (115) . iav progeny virus production was found to be significantly reduced in rab11a + knockdown human cell lines (116) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab11a knockdown cells (114, 115) ; in cells expressing deletion mutant of rab11 family interacting proteins (87) ; as well as cells treated with chemicals to interfere microtubule (114) . direct interaction of vrnp complex with rab11a has also been verified (114, 115) , demonstrating the dependence of vrna complex transport on rab11a + vesicles and the microtubule network during viral replication. since rab11a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif13a, a kinesin-3 family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab11a + vesicles (117) . kif13a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif13a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab11a or kif13a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability (118) . histone deacetylase 6 (hdac6) was found to deacetylate α-tubulin, one of the subunits of microtubule (119) . a study has demonstrated that hdac6 is involved in iav replication (120) . inhibition of hdac6 by tubacin or knockdown of hdac6 gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac6. this data suggests that activation of hdac6 by its stimulant could be a potential approach to anti-iav therapy, despite hdac6 stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes (121) and intercellular connections (122) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m1 as well as m2, are also suggested to play an important role in the initiation of the budding process (123, 124) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a6 (anxa6), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane (55), ultimately causing a reduction in egression of progeny virion from infected cells (54) . this reduction could be reversed by the addition of exogenous cholesterol (55) . similar to anxa6 overexpression, addition of a hydrophobic polyamine, u18666a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication (55) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) (123) , it was demonstrated that anxa6 overexpression or u18666a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m2 protein, suggesting that iav m2 clustering (which provides membrane curvature for scission) is mediated by cholesterol (125) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells (126) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α2-adrenergic receptors (α2-ars) have been recently identified as a key host factor involved in iav replication (127) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α2-ar-mediated signaling. in vitro stimulation of α2-ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α2-ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise (61) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin (46) , ha maturation inhibitor nitazoxanide (85) , fpr2 antagonists (111) , and xpo1 inhibitor verdinexor (96) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table 1 summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail (128) (129) (130) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il-6, il-8, tumor necrosis factor (tnf)-α, and ccl2 as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns (128, 129) . type i ifns are known inducer for the upregulation of death receptor 5, which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes (128) . il-8 and ccl2 produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection (131) with transmigration of neutrophils carry out by adhesion molecules, such as cd11a, cd11b, and cd18 (132) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin (132) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav (133) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd8 + t-cells in the infection site by secreting and leaving a trail of cxcl12 (134) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production (135) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd11c low b220 + plasmacytoid dc (pdc) and two types of conventional dcs (cd103 + cd11b low and cd103 − cd11b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells (129) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen (129) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively (130) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr7 and ccl19/ccl21 (130, 136) for antigen presentation via mhc class i and ii to naïve cd8 + and cd4 + t-cells, respectively (137) (138) (139) (140) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd8 + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd8α + dcs which are the most efficient antigen-presenting cells to cd8 + t-cells (137) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation (141) . within the lymph node, naïve cd8 + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il-12, type i ifns, and il2 (142, 143) , and the help from activated cd4 + t helper cells (144). differentiated ctls downregulate their lymph node homing receptor ccr7 and upregulate ccr4 and cxcr3 for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd4 + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd28 expressed on the t-cells and the ligand for cd28 (cd80 and cd86) expressed on dcs playing an important role (144). activation of cd4 + t-cells lead to differentiation into different effector cells subsets, including the classical th1 and th2, and the more recently identified regulatory t cells, follicular t helper cells, th9, and th17 subsets (144). th1 cells regulate to the differentiation of ctls as mentioned whereas th2 cells contributes to the activation of b-cells through cd40l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center (145) . detailed functions of regulatory t cells, follicular t cells, th9, and th17 cells are discussed elsewhere (144, 145). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere (146) (147) (148) (149) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure 1 . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development (150, 151) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients (152) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h5n1 and h7n9) (153, 154) or suppressing (h1n1, h3n2) (155) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of 1918, which claimed the lives of 20-50 million of the 500 million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted 1918 iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present (156) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection (131) . neutrophils characteristically control microbial infections by generating bactericidal (157) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin (131) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h3n2 strain hkx31 displayed neutrophil-mediated viral clearance via phagocytosis (132, 158) . depletion of neutrophils has found to enhance viral load in the iav-infected animals (158) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr8, an iav strain highly pathogenic to mice (159) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium (157) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro (160) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones (161) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir (161) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h7n9 and severe h1n1 infection increased alveolar epithelial cell permeability (162) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome (162) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net (159) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines (163) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine (164) have been suggested to inhibit netosis. despite it has been reported that during iav h1n1 infection, pad4 knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice (165) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors (166) . similar to t-helper cells, they are classified into subsets by their ability to produce type 1 (th1), type 2 (th2), and type 3 (th17 and th22) cytokines. previous studies confirmed the involvement of ilcs of group 2 linage (ilc2) in iav infection and airway inflammation (166, 167) . on the positive side, during the recovery phase of iav infection, ilc2 expresses amphiregulin which promote airway epithelium repair (166, 168) , thus facilitating the recovery of the infected lung. on the other hand, in response to il-33 produced by macrophages, dcs, and nkt cells, ilc2 secretes il-5 and il-13 and induce airway hyper-responsiveness. recruitment of eosinophils by il-5 to the lung also mediates airway inflammation (166) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients (166) , it will be of particular interest to investigate the role of ilc2 in iav infection, particularly in asthma patients. ilc1s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells (169) . it was recently appreciated that tissue-resident ilc1s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav (170) . interestingly, ifn-γ was found to suppress ilc2 activity and reduce il5 production which exacerbates disease severity during influenza a(h1n1) pdm09 infection (171) . this data may highlight a link between ilc1 and ilc2 and suggesting ilc1 can suppress ilc2 activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i2, corticosteroids, and testosterone have been reported to suppress ilc2 activity (172, 173) . in addition to il-33, the epithelial cytokines il-25, thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d2 were found to activate ilc2 (173) . the therapeutic potential of these ilc2 activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection (174) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox1-5 and dual oxidase (duox) 1 and 2. ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated (174) . dual oxidase1 and 2 are found to be host-protective (174, 175) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex (175) . in addition to altering viral mrna splicing, ros generated by doux2 has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox2 was silenced in vitro (176) . increased viral replication was also observed in mice with doux silenced (175) , further depicting the protective role of doux in iav infection. unlike doux, nox2 activation could be harmful to host. iav infection was reported to induce nox2-dependent endosomal ros production (177) . ros could target the conserved cys98 on toll-like receptor (tlr) 7, and inhibit tlr7-mediated type i ifn expression during a mild iav h3n2 infection in vivo (177) . iav-infected mice treated with specific nox2 inhibitor, cholestanol-conjugated gp91ds-tat, were found to have reduction in endosomal ros production, restored tlr7 activity, and displayed a decreased viral load (177) . in addition to nox2, nox4dependent ros production has also been reported to activate mapk/erk signaling (178) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox4 knockdown resulted in a reduction of viral replication in vitro (178) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial (175, 176) , while nox2 and nox4 are harmful during iav infections (177, 178) . finally, ns1 (not to be confused with iav ns1 protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox1, nox2, and nox4. a study demonstrated that ns1 suppresses iav-induced nox2 and significantly inhibits iav virus replication (179) . besides cholestanolconjugated gp91ds-tat and ns1 aforementioned, apocynin, a phagocytic nox2 inhibitor as well as ros scavenger (180) (181) (182) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs1 and socs3 in vitro (154) and reduce peri-bronchial inflammation and viral titer in vivo (183) . interestingly, ebselen, another nox2 inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav (184) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections (185, 186) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase 3, 7, 8, and 10 (187) (188) (189) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav (153, 190) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd8 + t-cell response (191) . in addition to recruiting monocytic cells to the infection site, cd8 + t-cells response was observed to deteriorate lung pathology (192) and damage healthy, non-infected lung epithelial cells (193) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il-15 and il-6 in balf (192) , which promote the survival of and proliferation of cd8 + t-cells (194, 195) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein-1 was observed in tnf −/− mice infected with sub-lethal dose of iav (196) . in addition, decreased cd8 + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl-2 was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice (192) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented (197) . tnf has been observed to stimulate the expression of cxcl2 in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage (198) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy (199, 200) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare (201) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) (202, 203) , contributing to neural damage (204) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain (205) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il-6, lung injury, and edema (205) . the protective role of il-6 was demonstrated in mice challenged with sub-lethal iav infection. il-6-deficient mice displayed exacerbated pulmonary damage (206, 207) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells (207) . iav suppresses the anti-apoptotic mcl-1 and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance (206) . addition of il-6 restored the expression of mcl-1 and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il-6 knockout mice during mild iav infection. il-6 has also been shown to induce the proliferation of lung il-10 + regulatory t cells and il-27, which act to limit excessive proliferation of cd8 + t-cells and subsequent cd8 + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology (208) . despite the apparent protective role of il-6, high levels of il-6 in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il-6 used as a marker for prognosis (199-201, 209, 210) . the role of il-6 in regulation of bbb permeability was reported (211) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il-6 as a form of therapy in severe iav infection should be considered. one such option is the anti-il6 antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il-6 due to severe iav infection has yet to be conducted. on the other hand, in a case of h1n1 virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il-6 from the bloodstream (212) has showed beneficial to the patient (213) . more research is required to confirm whether the removal or neutralization of il-6 could be a potential therapy for severe iav infections. the activation of cd8 + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd8 + t-cell inflicted host cell damage. il-6 mediates il-27 induction (208) . il-27 acts to suppress cd8 + t-cells and reduce morbidity through il-10 and regulatory t-cells (208) . much like other immunomodulatory approaches, the timing for applying il-27 should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il-27 to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il-27 administration during the recovery phase (5-10 days post-infection) accelerated recovery and improve lung immunopathology (214) . notably, il-27 could also suppress th17 responses and increases susceptibility to secondary s. aureus infection (215) . therefore, co-administration of antibiotics should be considered when utilizing il-27 as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns1 inhibits the production of ifns by antagonizing irf-3, a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection (216, 217) . in addition to inhibiting ifn expression, the induction of socs3 inhibits ifns signaling by suppressing cytokine signaling has been documented (155) . the recognition of 5′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs3, which in turn represses type i ifns expression (155) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one (218) . while type i ifn has been demonstrated to inhibit iav replication in vitro (219) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged (219) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts (10-100 units per mice daily); with higher dosages (1,000-10,000 units per mice daily) shown to increase morbidity (220) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged 50 or above (221) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice (222) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr5-mediated epithelial cell death by type i ifn (223) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn (224) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection (225) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication (176, 222, 224) without the risk of previously reported type i ifns-mediated immunopathologic side-effects (222, 224, 226) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo (227) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns (222) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox-2 by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav (228) (229) (230) . in addition, the use of zanamivir in tandem with a specific cox-2 inhibitor was shown to increase the survival rate of mice lethally infected with avian h7n9 iav, when compared to mice treated solely with zanamivir (229) . activated cox-2 regulates downstream prostaglandin production. one such example is pge2, a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge2 was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication (231) . the use of chemicals ah6809 and gw627368x to antagonize pge2 downstream signaling molecules ep2 and ep4 respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep2 and ep4 antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg2 production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed (232) . the long history of clinical tcm use supports the clinical feasibility of peg2 inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into 3 groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) (233) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection (234) (235) (236) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed (237) (238) (239) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr1-10, while mice have been identified to express functional tlr1-9 as well as tlr11-13 (240) . most tlrs-with the exception of tlr3utilize myd88 as an adaptor protein during signal transduction. tlr3 utilizes trif as an adaptor. tlr4 is known for its ability to utilize either myd88 or trif, with the choice of adaptor dependent on its sub-cellular location (241) . different tlrs, such as tlr3, 7, and 8 (240) as well as tlr2, tlr4, and most recently tlr10 (235) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr10 being an exception (242) (243) (244) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress (245) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr4, activating a tlr4-trif-traf6-nf-κb signaling cascade to eventually trigger the release of il-6, ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s100a9 upon intracellular prr ddx21 recognition of iav subsequently induces the activation of tlr4, further contributing to iav-induced mortality (246) . since tlr4 has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr4 could potentially reduce the severity of iav infections. eritoran (e5564) is a specific tlr4 antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment (247) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day 6 after infection beyond the recommended therapeutic time window (within 48 h after the first display of clinical symptom) for use of oseltamivir (248), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr4 antagonist, fp7 (249), alongside a newly developed decoy peptide 2r9 that has been shown to disrupt tlr2, 4, 7, and 9 signaling via tirap, has been shown to protect mice from lethal iav infection (250) . these results support the potential use of tlr4 antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr2-mediated signaling through the use of an anti-tlr2 antibody, significantly protected against lethality when administered on day 2 and 4 post-iav infection (251) . a study also demonstrated that h5n1-infected tlr3 knockout mice had better survival than h5n1-infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung (252) . an increasing number of tlr antagonists are now under development (253, 254) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il-6 and ccl-5 in vitro by down-regulating upstream tlr3 expression (255) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav (256) . this was determined to be caused by the downregulation of tlr7. these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table 2 summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients (257) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly (258) and immunosuppression (257) . in addition, viruses rely on host metabolism to perform essential functions during replication (259) (260) (261) (262) . these processes exert a large energy demand on the host within a very short period of time (263) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells (264) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez235, which inhibited the iav-mediated c-myc induction. administration of bez235 2 days prior to infection and up to 4 days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro (264) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive (265) , were found to induce drastic alterations in metabolic levels and programs (263) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases (263, 265, 266) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor 1α and the induction of cytokines such as il-1β (267) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection (268) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains (252) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance (73, 83, 107) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m2 blockers, are recommended to be administered within 48 h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe (96, 109, 214, 251) , even up to 6 days post-infection (248) , providing a clear clinical advantage over na inhibitors and m2 blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the 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cord-270772-zshjrc87 authors: to, kelvin kai-wang; zhou, jie; chan, jasper fuk-woo; yuen, kwok-yung title: host genes and influenza pathogenesis in humans: an emerging paradigm date: 2015-06-14 journal: curr opin virol doi: 10.1016/j.coviro.2015.04.010 sha: doc_id: 270772 cord_uid: zshjrc87 the emergence of the pandemic influenza virus a(h1n1)pdm09 in 2009 and avian influenza virus a(h7n9) in 2013 provided unique opportunities for assessing genetic predispositions to severe disease because many patients did not have any underlying risk factor or neutralizing antibody against these agents, in contrast to seasonal influenza viruses. high-throughput screening platforms and large human or animal databases from international collaborations allow rapid selection of potential candidate genes for confirmatory functional studies. in the last 2 years, at least seven new human susceptibility genes have been identified in genetic association studies. integration of knowledge from genetic and phenotypic studies is essential to identify important gene targets for treatment and prevention of influenza virus infection. influenza virus is one of the most common seasonal respiratory viruses affecting humans, leading to 250 000-500 000 deaths every year [1] . the 2009 pandemic a(h1n1) virus (a[h1n1]pdm09 virus) was estimated to cause 201 000 respiratory deaths in the first 12 months, with most deaths occurring in patients aged <65 years [2, 3 ] . the avian influenza viruses a(h5n1), a(h7n9), a(h10n8), a(h5n6), together with the severe acute respiratory syndrome and middle east respiratory syndrome coronaviruses, are the most virulent respiratory viruses affecting humans [4 ,5-7] . most patients with influenza virus infection develop mild upper respiratory tract infection. life-threatening complications include severe viral pneumonia or secondary bacterial pneumonia, acute respiratory distress syndrome, pulmonary embolism, myocarditis, encephalopathy, reye's syndrome, hemophagocytic syndrome, multiorgan dysfunction and exacerbation of underlying chronic cardiovascular and respiratory diseases [3 ,4 ] . currently available antivirals and vaccines have limited efficacy in the treatment and prevention of influenza virus infection [8] . understanding the pathogenesis of influenza virus infection in human is important in designing novel strategies to improve the management of influenza. to cause infection, influenza virus needs to evade the host immunity, enter and replicate inside host cells, and disseminate to other cells or organs. host damage can be a result of direct virus-induced damage, immune-mediated damage and/or secondary bacterial infection [9] [10] [11] . virologists have extensively studied the role of viral components in the viral life cycle and in the pathogenesis of influenza virus infection in humans [12] . specific amino acid changes in the viral proteins have been associated with increased disease severity in humans or adaptation of avian influenza viruses in humans [13] . clinical risk factors for severe influenza have been well described ( figure 1 ) [3 ] . however, specific human genes regulating the influenza virus life cycle or virus-induced inflammatory responses are less well described. traditionally, the study of host genes depends on prior knowledge of a candidate gene. the importance of the candidate gene is verified using single gene knockout/ knockdown or gain-of-function studies with reverse genetics in vitro or in vivo. for example, chinese and japanese patients with influenza-associated encephalopathy (iae) were found to have elevated (c16:0 + c18:1)/ c2 acylcarnitines ratios. carnitine palmitoyltransferase 2 (cpt2) variants f352c and v368i were found to be overrepresented in these iae patients when compared with the general population. these mutations render this enzyme susceptible to inactivation at high temperature occurring in febrile patients [14, 15] . high-throughput screening platforms have allowed researchers to systematically screen a large number of genes associated with influenza virus infection in vitro, in animals or in humans. the availability of deep sequencing data of the human genome allows researchers to compare genetic variations between influenza patients and the general population [16 ] . these technological advances, combined with in vitro or bioinformatics analysis, have revealed several genes associated with severe influenza ( figure 2 and table 1 ). in this review, we focus on specific host genes that have been shown to be directly related to the pathogenesis of human influenza identified or with updated knowledge in the past 2 years. four types of surfactant proteins are found in human pulmonary surfactant. both surfactant protein a2 (sftpa2) and surfactant protein d (sftpd) are collectins and exhibit antiviral activity against influenza virus [17] . sftpa2 variants (rs1965708-c, rs1059046-a, and haplotype 1a[0]) have been associated with more severe respiratory deterioration [18] . surfactant protein b (sftpb) is a hydrophobic protein responsible for the structural integrity of the pulmonary alveoli [19] . in a cohort of 84 chinese patients with severe and mild a(h1n1)pdm09 infection who were matched for age, sex and underlying conditions, the snp rs1130866-c was significantly associated with severe disease. the association between rs1130866-c and severe disease was verified in a second cohort patients with a(h1n1)pdm09 infection, in which multivariate analysis was performed to adjust for potential confounding factors [20 ] . the snp rs1130866-c has been associated with glycosylation at the amino acid residue 129 of sftpb, which reduces secretion of sftpb [21] . it remains to be determined whether sftpb has direct antiviral activity against influenza virus. surfactant protein c (sftpc) is a lipoprotein with antiviral activity against respiratory syncytial virus [22] , but its antiviral activity against influenza virus or its genetic variants associated with severe influenza has not yet been reported. lectin, galactoside-binding, soluble, 1 (lgals1), also known as galectin 1, can bind to influenza viruses and inhibit viral replication [23] . carriage of lgals1 rs4820294/rs2899292 haplotype gg was associated with protection from a(h7n9) virus infection in humans. furthermore, rs4820294/rs2899292 haplotype gg was correlated with higher levels of lgals1 mrna and protein expression in lymphoblast cell lines [24 ] . therefore, the differential lgals1 expression may contribute to the distinct susceptibility of some individuals to human a(h7n9) influenza. upon influenza virus infection, type i interferons are produced by host cells to limit viral replication. the host factors -age -pregnancy, obesity, underlying conditions -pre-existing level of immunity -genetic susceptibility function of type i interferons is mediated via the expression of many interferon stimulated genes (isgs). several isgs are especially important in the pathogenesis of influenza virus infection. interferon-induced transmembrane proteins (ifitm), including ifitm1, ifitm2 and ifitm3, were identified to have direct antiviral activity against influenza virus in a genomewide sirna screen [25] . a human long noncoding rna, which reduces ifitm3 gene expression, enhanced influenza virus replication in a transgenic mouse model [26 ] . ifitm3, which is located in endosome, inhibits viral replication by blocking the fusion of the viral and the host membrane [27, 28] . interestingly, amphotericin b, an antifungal, was found to increase viral replication by interfering with the blockage of membrane fusion by ifitm3 [29] . the c/c genotype of rs12252 is associated with a 21-amino-acid truncation at the n-terminal of the ifitm3 protein, which alters the localization of the ifitm3 from endosomal compartment to the cell periphery [30] . several studies have looked into ifitm3 snp rs12252 in the susceptibility to severe influenza in humans. the genotype rs12252-c was over-represented in caucasian and chinese patients with severe a(h1n1)pdm09 virus infection [31 ,32] . in patients with a(h7n9) infection, those carrying rs12252-c/c genotype had higher mortality [33] . however, the importance of rs12252-c allele in severe influenza has been challenged. firstly, in a study using pseudotyped influenza a viruses, transfection of a549 cells with plasmids expressing the truncated form of ifitm3 could reduce viral replication similarly to those with plasmids carrying the full length ifitm3 [34] . secondly, in a study comparing 34 patients with severe a(h1n1)pdm09 virus infection and >5000 controls, no significant difference in the rs12252 genotype frequency was found [35] . interferon regulatory factor 7 (irf7) is a transcription factor regulating the expression of type i interferons [36] . influenza a virus replicates to high titers in madin-darby canine kidney cells with knockdown of the irf7 gene [37] . murine tracheal epithelial cells deficient in irf7 have impaired expression of interferons after influenza virus infection [38] . in a study comparing the genetic variants in 534 healthy individuals, irf7 snp rs12805435 host genes and influenza pathogenesis to et al. 9 host genetic determinants of influenza virus disease severity identified in humans. host genes that have been associated with severe influenza are highlighted in red. was found to be associated with the induction of antiviral genes in dendritic cells in response to influenza virus infection [39] . in a 7-year-old girl without known immunodeficiency who suffered from severe influenza virus infection, irf7 mutation was identified using whole exome sequencing [40 ] . the induction of type i and type iii interferon genes in dendritic cells and pulmonary epithelial cells were found to be impaired in this girl. post-translational cleavage of influenza virus hemagglutinin by host protease is a prerequisite for the virus-host membrane fusion and thereby, for virus infectivity, tissue tropism and virus pathogenicity [41] . transmembrane protease, serine 2 (tmprss2), a type ii transmembrane serine protease, cleaves and activates the viral hemagglutinin during influenza virus infection [42] . three independent studies assessed the role of tmprss2 in influenza-infected mice. tmprss2 knockout mice were resistant to influenza a(h7n9) and a(h1n1) virus infection, but there were discrepant results regarding susceptibility to a(h3n2) virus infection [43] [44] [45] . by integration of a pilot genomewide association study and the lung expression quantitative trait loci (eqtl) dataset, a tmprss2 intronic snp rs2070788 was prioritized for further studies [46] . the genetic predisposition of rs2070788 to severe a(h1n1)pdm09 was validated in 409 a(h1n1)pdm09 patients including 162 severe cases and 247 mild controls. in the functional study, a regulatory snp rs383510 in high linkage disequilibrium with rs2070788 was uncovered as the causal variant underlying the genetic association. genetic predispositions of rs2070788 and rs383510 to severe influenza were also validated in an a(h7n9) patient cohort. serpine1, an isg, encodes plasminogen activator inhibitor 1 (pai-1). a549 cells over-expressing serpine1 inhibited the spread of influenza a virus when compared to cells without serpine1 overexpression [47 ] . pai-1 inhibits the extracellular cleavage of hemagglutinin from ha0 into ha1 and ha2. serpine1 knockout mice had higher viral titers and more severe disease than wild type mice. in human fibroblast cell lines derived from patients carrying rs6092-a allele which results in intracellular retention of pai-1, influenza virus replication was enhanced when compared with infection in cell lines deriving from patients carrying rs6092-t/t genotype. cytokines are important in the defense against influenza virus infection. however, excessive cytokine response is associated with severe influenza [9, 32] . tumor necrosis factor-a (tnf-a) has been considered a proinflammatory cytokine, and treatment with anti-tnf-a improved the survival of a(h1n1)-infected mice [48] . however, tnfknockout mice had more severe disease [49] . furthermore, the soluble form of tnf-a has been shown to be required for the control of cd8 + t cell response [50] , which suggests that tnf-a is required for the control of infection. a network based approach combining mouse, human and in vitro data showed that the tnf pathway is important in influenza virus infection [51 ] . tnf-238a and tnf-308g alleles have been associated with severe influenza in studies comparing a(h1n1)pdm09 patients and healthy controls [52, 53] . however, these associations were not found in another study comparing fatal cases and the general population [54] . interleukin-10 (il-10) is persistently elevated in patients with severe influenza virus infection [9] . il-10 knockout mice were shown to have more rapid viral clearance and improved survival [55] . blockage of il-1b could ameliorate inflammation of influenza-virus infected human pulmonary endothelial cells and lung fibroblasts [56] . il-10-592c, il-10-1082a allele and il-10-1082 a/a genotype have been associated with severe disease [53] . in another study involving 167 patients with a(h1n1)pdm09 virus infection and 192 healthy controls, rs17561 of il1a and rs1143627 of il1b gene were associated with susceptibility to infection [57] . however, the associations of snps in il-10, il-1a and il-1b genes with influenza virus disease severity were based on single cohorts, and further studies are necessary to confirm the significance of these polymorphisms in influenza. the interaction between the host antibody fc region and fc receptors is important in the immune defense against influenza virus infection. fc fragment of igg, low affinity iia, receptor (fcgr2a) gene encodes fcg receptor iia (fcgriia), which binds immune complexes. the association between fcgr2a polymorphism and severe a(h1n1)pdm09 virus infection was first identified in a european cohort [58] , although such association was not found in a chinese cohort [59] . fcgriia signaling was found to be important in immune complex-mediated platelet activation during influenza virus infection [60] . interestingly, in recent studies of broadly neutralizing antibodies against influenza viruses, the ability to form immune complex by the interaction between fc region of the antibody and fcg receptors may mediate antibodydependent cellular cytotoxicity, which is important for in vivo efficacy of the antibody [61, 62] . tlr3 is a major sensor of viral double stranded rna. f303s mutation of tlr3 was found to be associated with influenza-associated encephalopathy [63] , and snp rs5743313-c/t genotype was associated with a(h1n1)pdm09 patients with pneumonia [64] . the importance of tlr3 has been subsequently confirmed in a knockout mice study [65] . the future on the study of host genes in humans this review has summarized host genes that have been shown to be important for the pathogenesis of influenza in humans. with high-throughput assays, a large number of genes or genetic variants were found to be associated with viral replication or disease severity in animals or in humans. the current challenge is how we can select specific host genes that are important for the treatment or prevention of influenza virus infection. discrepancies in the results from similar studies also raise the suspicion whether certain genes are indeed important. for example, out of 925 host factors which affect influenza virus replication that were identified during in vitro genomewide sirna knockdown screening, only 69 genes were present in at least two of these screens [66] . in human genetic association studies, many genetic variants identified to be associated with disease severity in one study could not be reproduced in other studies (table 1) . several strategies can be used to improve our ability to find genes that are relevant to influenza pathogenesis in humans. in human genetic association studies, it is important to limit confounding factors as much as possible. for example, the cases and controls should be matched for age, sex, ethnic group and comorbidities, and multivariate analysis can be utilized to assess whether the genetic variant is an independent risk factor. the identified genetic variants should be validated in other independent cohorts. the functional significance of these genetic variants can be refined by eqtl analysis [67] . pathway analysis helps to identify pathways that may not be apparent when analyzing individual genes. interactome screens, which identify host genes that interact with viral proteins, may increase the likelihood of identifying genes that are potential host targets for treatment [68] . the establishment of genetically diverse mice population has allowed the identification of susceptibility genes by correlating genetic variations to disease phenotypes [69 ] . in addition to genetic data, host response to influenza has also been assessed using transcriptomic, proteomic, metabolomic and lipidomic analysis of cell cultures, animals or humans [70 ,71] . these data reflect biological regulation of host factors at different levels. an integration of these 'omics' data will improve the accuracy of identifying the susceptibility genes [72 ] . although studies have identified several genetic polymorphic genes that predispose to severe influenza, it is possible that these susceptible individuals may have very different susceptibility gene combinations. in the future, it is possible that whole genome sequencing will allow more accurate identification of susceptibility gene combinations in any particular individual (figure 3 ). in addition to comparing severe and mild influenza cases, another approach to identify host susceptibility genes is to examine patients who are resistant to influenza virus infection. in a study of a patient with a congenital disorder of glycosylation but without serological evidence of influenza virus infection, virus production was only found in 1 of 3 macrophage cultures derived from this patient [73] . the authors of this study postulated that the lack of glycosylation of viral surface proteins may affect virus production. understanding the role of host factors in virus infections have led to host-targeted antivirals. das181 removes sialic-acid containing receptors from respiratory epithelial cells and prevents virus attachment to host cells. in a phase ii clinical trial, das181 has been shown to reduce nasal or pharyngeal viral load in influenza patients [74] . antivirals and anti-inflammatory agents targeting host factors and vaccine strategies improving host response have shown promise in animal models. for example, sphingosine-1-phosphate agonist, protectin d1 and anti-leptin antibody have been shown to protect mice against lethal influenza virus infection [70 , 75, 76] . imiquimod, a tlr7 agonist, has been shown to expedite and augment antibody response after influenza virus vaccine in both animal and human studies [77, 78] . an integration of in vitro, animal and clinical data will allow researchers 12 engineering for viral resistance papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest world health organization: influenza (seasonal) estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study two years after pandemic influenza a/2009/h1n1: what have we learned? viral lung infections: epidemiology, virology, clinical features, and management of avian influenza a(h7n9) emergence in china of human disease due to avian influenza a(h10n8) -cause for concern? middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome clinical, virological, and histopathological manifestations of fatal human infections by avian influenza a(h7n9) virus influenza a viruses: new research developments cross-species transmission and emergence of novel viruses from birds thermolabile phenotype of carnitine palmitoyltransferase ii variations as a predisposing factor for influenza-associated encephalopathy fatal viral infectionassociated encephalopathy in two chinese boys: a genetically determined risk factor of thermolabile carnitine palmitoyltransferase ii variants nucleic acids research database issue and molecular biology database collection the role of surfactant in lung disease and host defense against pulmonary infections surfactant protein a genetic variants associate with severe respiratory insufficiency in pandemic influenza a virus infection diseases of pulmonary surfactant homeostasis ifitm proteins restrict viral membrane hemifusion amphotericin b increases influenza a virus infection by preventing ifitm3-mediated restriction the n-terminal region of ifitm3 modulates its antiviral activity by regulating ifitm3 cellular localization ifitm3 restricts the morbidity and mortality associated with influenza this study has demonstrated the importance of ifitm3 using in vitro and animal experiments, and identified a ifitm3 genetic variant to be overrepresented in hospitalized influenza patients when compared to the general population interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with severe influenza in chinese individuals early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal h7n9 infection ifitm3 polymorphism rs12252-c restricts influenza a viruses ifitm3 and susceptibility to respiratory viral infections in the community the irf family transcription factors at the interface of innate and adaptive immune responses high yield production of influenza virus in madin darby canine kidney (mdck) cells with stable knockdown of irf7 type i and type iii interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia common genetic variants modulate pathogen-sensing responses in human dendritic cells this is the first report of the use of whole exome sequencing in identifying genes associated with susceptibility to severe influenza the hemagglutinin: a determinant of pathogenicity influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts the host protease tmprss2 plays a major role in in vivo replication of emerging h7n9 and seasonal influenza viruses tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice tmprss2 is essential for influenza h1n1 virus pathogenesis in mice the identification of tmprss2 as the susceptible gene for severe illness of 2009 pandemic a(h1n1) influenza and infection of a(h7n9) influenza a serpin shapes the extracellular environment to prevent influenza a virus maturation this study demonstrated the importance of serpine1 gene in influenza virus pathogenesis by showing the differences in the phenotype of cells isolated from patients with different genetic variant in serpine1 gene inhibition of the inflammatory cytokine tumor necrosis factor-alpha with etanercept provides protection against lethal h1n1 influenza infection in mice negative regulation of lung inflammation and immunopathology by tnf-alpha during acute influenza infection soluble, but not transmembrane, tnf-alpha is required during influenza infection to limit the magnitude of immune responses and the extent of immunopathology prioritizing genes responsible for host resistance to influenza using network approaches this study incorporated in vitro, animal and human data to prioritize genes for future research on genetic susceptibility to severe influenza role of tumor necrosis factor gene single nucleotide polymorphisms in the natural course of 2009 influenza a h1n1 virus infection plasma cytokine levels and cytokine gene polymorphisms in mexican patients during the influenza pandemic a(h1n1)pdm09 a pilot study of host genetic variants associated with influenza-associated deaths among children and young adults a detrimental effect of interleukin-10 on protective pulmonary humoral immunity during primary influenza a virus infection induction of interleukin-1 beta (il-1beta) is a critical component of lung inflammation during influenza a (h1n1) virus infection genetic variants in il1a and il1b contribute to the susceptibility to 2009 pandemic h1n1 influenza a virus genetic variants associated with severe pneumonia in a/h1n1 influenza infection the lower serum immunoglobulin g2 level in severe cases than in mild cases of pandemic h1n1 2009 influenza is associated with cytokine dysregulation influenza virus h1n1 activates platelets through fcgammariia signaling and thrombin generation broadly neutralizing influenza hemagglutinin stem-specific antibody cr8020 targets residues that are prone to escape due to host selection pressure broadly neutralizing hemagglutinin stalk-specific antibodies require fcgammar interactions for protection against influenza virus in vivo a missense mutation of the toll-like receptor 3 gene in a patient with influenza-associated encephalopathy toll-like receptor 3 gene polymorphisms and severity of pandemic a/h1n1/2009 influenza in otherwise healthy children highly pathogenic avian influenza a h5n1 and pandemic h1n1 virus infections have different phenotypes in toll-like receptor 3 knockout mice genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery the role of regulatory variation in complex traits and disease influenza virus-host interactome screen as a platform for antiviral drug development modeling host genetic regulation of influenza pathogenesis in the collaborative cross the lipid mediator protectin d1 inhibits influenza virus replication and improves severe influenza this study showed that treatment targeting lipid mediators can improve the survival of mice with severe influenza patientbased transcriptome-wide analysis identify interferon and ubiquination pathways as potential predictors of influenza a disease severity methods of integrating data to uncover genotype-phenotype interactions glycosylation, hypogammaglobulinemia, and resistance to viral infections a phase ii study of das181, a novel host directed antiviral for the treatment of influenza infection endothelial cells are central orchestrators of cytokine amplification during influenza virus infection leptin mediates the pathogenesis of severe 2009 pandemic influenza a(h1n1) infection associated with cytokine dysregulation in mice with dietinduced obesity recombinant influenza a virus hemagglutinin ha2 subunit protects mice against influenza a(h7n9) virus infection immunogenicity of intradermal trivalent influenza vaccine with topical imiquimod: a double blind randomized controlled trial ifitm3 limits the severity of acute influenza in mice proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium human h-ficolin inhibits replication of seasonal and pandemic influenza a viruses a functional variation in cd55 increases the severity of 2009 pandemic h1n1 influenza a virus infection thermal instability of compound variants of carnitine palmitoyltransferase ii and impaired mitochondrial fuel utilization in influenza-associated encephalopathy tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells enrichment of variations in kir3dl1/s1 and kir2dl2/l3 among h1n1/09 icu patients: an exploratory study killer-cell immunoglobulin-like receptors (kir) in severe a (h1n1) 2009 influenza infections chemokine receptor 5 big up tri, open32 allele in patients with severe pandemic (h1n1) the ccr5delta32 allele is not a major predisposing factor for severe h1n1pdm09 infection characterization in vitro and in vivo of a pandemic h1n1 influenza virus from a fatal case key: cord-294323-mryiqmsw authors: kumar, binod; asha, kumari; khanna, madhu; ronsard, larance; meseko, clement adebajo; sanicas, melvin title: the emerging influenza virus threat: status and new prospects for its therapy and control date: 2018-01-10 journal: arch virol doi: 10.1007/s00705-018-3708-y sha: doc_id: 294323 cord_uid: mryiqmsw influenza a viruses (iavs) are zoonotic pathogens that cause yearly outbreaks with high rates of morbidity and fatality. the virus continuously acquires point mutations while circulating in several hosts, ranging from aquatic birds to mammals, including humans. the wide range of hosts provides influenza a viruses greater chances of genetic re-assortment, leading to the emergence of zoonotic strains and occasional pandemics that have a severe impact on human life. four major influenza pandemics have been reported to date, and health authorities worldwide have shown tremendous progress in efforts to control epidemics and pandemics. here, we primarily discuss the pathogenesis of influenza virus type a, its epidemiology, pandemic potential, current status of antiviral drugs and vaccines, and ways to effectively manage the disease during a crisis. influenza viruses belong to the family orthomyxoviridae and are the leading cause of severe respiratory illness across the world. they are enveloped viruses containing a single-stranded, negative-sense rna genome, and they account for a large number of deaths each year. in an electron microscope, influenza a and b viruses look similar and are virtually indistinguishable. they are either spherical (100 nm in diameter) or filamentous (often in excess of 300 nm in length) in form [1] . of the four influenza virus types (a, b, c and d), influenza a virus (iav) causes the most severe disease and infects a variety of animals, including humans, pigs, horses, sea mammals, and various bird species (reviewed in reference [2] ). type a mutates more rapidly and exhibits a higher degree of variability in its antigenicity and virulence than the other influenza types [3, 4] . it can cause zoonotic infections and adapts easily to humans, leading to a sustained human-to-human transmission, which favors the emergence of novel strains. in this review, we have focused primarily on the contemporary aspects of influenza a virology and new prospects for its treatment and prevention. the genome of influenza a and b viruses consists of eight single-stranded viral rna (vrna) segments, while influenza c virus has a seven-segment genome. each segment codes for one of the viral proteins, which include the major surface glycoproteins hemagglutinin (ha) and neuraminidase (na), the nucleocapsid protein (np), three subunits of the viral rna-dependent rna polymerase (rdrp) (pa, pa-x, pb1, pb2, pb1-f2), the matrix proteins (m1, m2) and the nonstructural proteins ns1 and ns2 [5] . all influenza a viruses are classified based on their surface glycoproteins, ha and na. ha is responsible for binding to sialic acid (sa) (n-acetyl neuraminic acid) at the termini of glycans, which act as receptors on the host cell plasma membrane, while the na, a type ii integral membrane glycoprotein with sialidase enzymatic activity, is involved in the final step of the replication cycle and helps in release of mature virions. the two surface glycoproteins, ha and na, are present in a ratio of 4:1 [3] . co-evolutionary adaptation between ha and na allows them to perform the complimentary functions of sa binding (by ha) and sa removal (by na). the segmented nature of the genome and the high frequency of mutations during replication in multiple hosts is responsible for regular epidemics and occasional pandemics. the two major factors in influenza epidemics and pandemics are genetic drift and genetic shift [6] . genetic drift occurs due to point mutations in the influenza virus genome, as the viral rna polymerase, unlike dna polymerase, lacks a proofreading function making coding errors and multiple mutations more likely. a genetic shift occurs when two or more different influenza virus strains infect the same cell in a host, leading to recombination of genetic materials, an event that occasionally generates a new strain with a novel combination of hemagglutinin and neuraminidase. these genetic shifts lead to pandemics when the novel strain acquires the capacity for sustained efficient human-to-human transmission. to date, 18 novel hemagglutinins (h1-h18) and 11 neuraminidases (n1-n11) have been identified [7] . most of the combinations of h and n types (144) are found in wild birds, which serve as reservoirs for influenza viruses and pose a severe risk, because they can be infected with multiple strains and serve as potential mixing vessels. h17-18 and n10-11 have not been detected in birds but have been found in bats [7, 8] . iavs that infects birds have an ha receptor-binding specificity for α2-3 sa, while has from iavs that infect humans have a higher specificity for α2-6 sa, with the major exception of the highly pathogenic avian influenza (hpai) strain h5n1, which has a preference for α2-3 sa. the differences in preferred cellular binding sites allow different strains of influenza virus to infect either birds or humans, thereby creating lineages that are host specific, and so far, only h1n1, h2n2, h3n2, h5n1, h7n7 and h9n2 viruses are known to infect humans. however, the respiratory epithelial cells of pigs (swine) express both α2-3and α2-6-linked sialic acids and can therefore support infections with both avian and human influenza virus strains. this makes pigs a mixing vessel for producing novel strains with the ability to infect humans, and some of these strains can cause fatal infections ( fig. 1) [9] . these novel reassortant viruses, due to a lack of existing immunity in human population, can lead to pandemic situations, as witnessed in the year 2009. the human population remains at risk of an influenza pandemic each year due to the high mutation rate of the virus. influenza pandemics have occurred several times, with inter-pandemic intervals averaging approximately 40 years [10] . type a has been responsible for several widespread fig. 1 mechanisms for the emergence of pandemic influenza virus strains. the virus keeps circulating among own species and sometimes jump the species barrier to generate a novel strain of pandemic potential pandemics since the 16 th century. the three major pandemics were the spanish flu , the asian flu (1957), and the hong kong flu , which resulted in a large number of deaths [11] (fig. 2) . the 1918 (h1n1) pandemic has been recorded as the worst pandemic in history. it infected 500 million people globally, caused approximately 675,000 deaths in the united states [12] , and killed up to 50-100 million people worldwide [13] . the viral genome reconstructed from the lung tissues of several victims demonstrated that it was an avian-descended h1n1 virus [14] . waterfowl, of the order anseriformes, such as ducks, swans and geese, serve as reservoirs of all iavs. charadriiformes, including shore birds, gulls, and terns, also harbor influenza virus, but of a different gene pool from those of the anseriformes, and the two remain the most important orders for the transmission and spread of hpai [15] . influenza viruses from these birds are able to infect other bird species, such as chickens, as well as mammals, and they adapt to a new host by accumulating mutations through genetic drift or genetic shifts [12] . due to the unavailability of any iav sequences from prior to 1918, the possibility of involvement of an intermediate host in the emergence of the virus in humans during the 1918 pandemic remains an unresolved mystery [16] . however, the virus was readily transmitted to pigs, as was also observed during the 2009 pandemic of h1n1 [17] . most of the deaths resulted from respiratory complications, such as bronchopneumonia with bacterial invasion and progressive cyanosis and collapse. scientists believe that the pathogenicity of the 1918 h1n1 virus was amplified by concomitant infection of influenza virus with bacteria such as s. pneumoniae and s. pyogenes [18] . the 1918 pandemic spread in three rapid waves within an approximately 9-month period. the large number of deaths could also be attributed to several other factors, such as unpreparedness for an influenza virus strain of pandemic potential and the lack of effective vaccines to prevent influenza and antibiotics to treat secondary bacterial pneumonia. after the pandemic period, the virus kept accumulating mutations for several years and disappeared in 1957, only to reappear in circulation in 1977 [2] . following the spanish flu in 1918, another influenza pandemic occurred in 1957 and was called the asian flu. the 1957 pandemic was caused by the h2n2 strain of iav and resulted in ~115,700 excess deaths. the overall impact on mortality was one-tenth of that observed during the 1918 spanish flu (h1n1) pandemic [19] . this new influenza strain was detected in february 1957 in yunnan province of china, and by april, the virus had spread to hong kong, followed by singapore, taiwan, japan and the rest of the world by the summer of 1957 [20] . in the usa alone, this strain caused [21, 22] . "original antigenic sin" is a phenomenon where a prior exposure to an antigen leads to an optimal immune response to the related antigen. thus, during the asian flu pandemic, individuals, except those who were 60 years and older, had no prior exposure to the h2n2 strain and therefore had no previous immunity, leading to a large susceptible population in the united states becoming infected [21] . the effectiveness of an influenza vaccine may decrease if the antigenic distance between the vaccine and circulating strains increases. also there is a possibility that the original antigenic sin could make people who are vaccinated, more susceptible to the virus than those who are not vaccinated [23] . in the year 1968, a new influenza virus strain (h3n2) that differed from the asian pandemic strain (h2n2) by its ha glycoprotein but had the same na glycoprotein, replaced the h2n2 strain that had been circulating in all countries since 1957, and this led to the third pandemic causing a large number of deaths [24] . the h3n2 strain, which was first isolated in hong kong in july 1968 [25] , was highly transmissible but caused disease milder than the asian flu. the virus mainly spread due to international air travel and resulted in an increase in the mortality rate in united states during the pandemic season (1968/1969), especially in persons <65 years old [25] . the h3n2 strain caused an estimated 98,100 excess deaths over the 4-year period 1968-1971 [19] . the 1957 (h2n2) and 1968 (h3n2) influenza pandemic viruses were avian-human reassortants in which avian gene segments were introduced into a human-adapted virus that was already in circulation [26] . the spring of 2009 again marked the emergence of a novel subtype of influenza a virus (pandemic h1n1-2009), which caused the first pandemic of the 21 st century. the newly emerged virus subtype spread worldwide with unprecedented speed and proved its ability to be transmitted from human to human [27] . the health authorities gained momentum, and strict surveillance programs started globally to combat the threat by this novel virus [28] , which was a fourth-generation descendant of the 1918 h1n1 virus [29] . the world health organization (who) declared a pandemic in june 2009, and the phase ended by august 2010. according to who, 50.7% of subtyped influenza a viruses collected globally from july 11-17, 2010 and reported on july 28 were the pandemic h1n1-2009 strain [30] . by october 2009, around 191 countries had reported more than 375,000 laboratory-confirmed cases of pandemic (h1n1) 2009 and more than 4500 deaths [31] . the rates of hospitalization and death varied among countries. according to a study done from 15 april 2009 through 23 january 2010 in the usa, 272 pediatric deaths were found to be associated with laboratory-confirmed pandemic h1n1-2009 [32] . while the rate of hospitalization was higher in children, the adult population aged 65 years of age or older showed the lowest rate [33] . according to one study, the death toll due to this novel pandemic h1n1-2009 strain was 18,631, as declared in the who reports. however, they reported that the actual mortality burden due to the pandemic was substantially higher and that the number of cases was underreported in africa and asia [34] . the 2009 influenza pandemic came to an end, and just like any other pandemic strain, the pandemic h1n1-2009 strain has been considered a seasonal strain since july 2010. iavs are also widely distributed in avian species (ducks, geese, swans, gulls, terns, etc.) around the world and are predominantly maintained in asymptomatic infections termed "low-pathogenic avian influenza" (lpai). around 105 different species of birds have been documented to harbor iavs [35] . the virus predominantly infects the epithelial cells of the intestinal tract [36] and is subsequently excreted in the faeces. iavs are known to cross species barriers and be transmitted to other species. a recent example could be seen in the harbour seals (phoca vitulina) of the north-european coastal waters, where h10n7 (lpai) infection caused high mortality [37] . there are also instances where lpai has jumped from birds to long-finned pilot whales (globicephala melas) and balaenopterid whales [38] . influenza viruses circulating in mammalian species including dogs (canis lupus familiaris) and horses (equus ferus caballus) are also thought to be derived from avian influenza viruses [39] . strains of subtypes h3n8 and h3n2 are currently circulating amongst dogs [39] while h3n8 virus has long been circulating amongst horses [39] , and bactrian camels (camelus bactrianus) [40] . the lpai subtypes h5 and h7 can subsequently evolve into highly pathogenic avian influenza (hpai) viruses by insertion of a multi-basic cleavage site in the viral ha [41] . recent years have seen the occurrence of two such hpai strains of subtypes h5n1 and h7n9 in asian countries that resulted in a high fatality rate and hospitalization [42] . an h7n9 strain is currently in circulation and is the cause of significant public health concern in china [42, 43] . another hpai strain of subtype h5n6 was first reported to infect humans in april 2014 in sichuan province and again in december 2014 in guangdong province, followed by four more cases in december 2015 in china [44] . the recent presence of h5n8 and h5n5 infections in various duck species also poses a threat of evolution of this lineage of hpai h5 viruses in the future [45] . hpai viruses have also been documented in some non-primate mammals living in captivity, such as tigers (panthera tigris), cats (felis catus), leopards (panthera pardus), and owston's palm civets (chrotogale owstoni) [46] . influenza virus primarily spreads from one person to another through respiratory droplets when the infected person comes in close contact with a healthy person (generally within a distance of a meter). the virus can survive for 24 to 48 hours on hard, non-porous surfaces and thus may also spread when a person comes in contact with any such surface or item contaminated with the respiratory droplets from an infected person [47] . a typical influenza infection is often characterized by sudden onset of fever, chills, headache, malaise and myalgia, followed by prominent upper respiratory tract symptoms, such as rhinorrhea, cough, sore throat and inflammation of the upper respiratory tract. apart from these, gastrointestinal symptoms such as nausea, vomiting and diarrhea are very common [48] . however, the duration of illness in cases of pandemic h1n1-2009 infections were found to be slightly longer than that of seasonal influenza infections [49] , and gastrointestinal symptoms, especially diarrhea, appeared to be more prominent than in seasonal influenza [50] [51] [52] . the incubation period of influenza virus from the time of infection to appearance of symptoms typically varies from 1 to 4 days [53] , but it may extend up to 7 days in some cases [54, 55] , and weakness and fatigue can sometimes last for weeks. an infected person typically sheds virus one day prior to the appearance of symptoms, which spreads infection before the sick can be isolated, and the virus continues to be shed until the symptoms resolve. the peak viral load is generally observed on the day of the onset of symptoms and gradually decreases with time. children and younger adults often shed the virus for 10 days or more [56] , while an immunocompromised person may shed the virus for weeks [57] . the virus can be detected in easily clinical specimens such as nasal/throat swabs and nasopharyngeal aspirates. there are also reports of viral load detection in urine and stool of infected patients [58, 59] . accurate diagnosis and prompt treatment with antiviral drugs can have positive effects on human health and reduce the economic burden of influenza illness each year. however, because several other respiratory viruses, including adenoviruses, rhinoviruses, respiratory syncytial virus (rsv), coronaviruses, metapneumoviruses and parainfluenza viruses, can cause common symptoms of influenza-like-illness (ili), many cases are misdiagnosed as influenza [60] . proper specimen collection is of paramount importance, regardless of the diagnostic method used. nasopharyngeal specimens are always preferred over throat swabs or other specimens [61] . the best time to collect the clinical specimen is on the second or third day of symptoms (when viral shedding is at its peak), as the results obtained will be more reliable than when samples are obtained earlier or later in the course of disease [28, 51, 55, 61, 62] . there are a number of methods available for influenza diagnosis including rapid antigen tests, viral culture, serology, conventional reverse transcription polymerase chain reaction (pcr), reverse transcription loop-mediated isothermal amplification (rt-lamp), real-time reverse transcription polymerase chain reaction (rt-pcr) and immunofluorescence assay. for rapid antigen (influenza) tests, the preferred specimens are nasopharyngeal or nasal swabs or throat swabs collected within 3-4 days of infection for more accurate testing. these tests provide results in less than 15 minutes with 40-70% sensitivity [63] when compared with viral culture (3-10 days) or rt-pcr, which has greater than 90% specificity and is moderately fast. therefore, false negative results are more common than false positive results during influenza seasons when bedside rapid antigen tests are used [63, 64] . these rapid antigen tests can differentiate between seasonal influenza a and b types, but they are unable to detect pandemic h1n1-2009 viruses exclusively [30] . health care professionals, during the time of year when outbreaks of ili are common, can perform tentative diagnosis of influenza using various commercially available rapid immunoassay kits, but due to the limitations of rapid viral tests, confirmatory laboratory testing should be done to determine the treatment of choice [64] . while virus culture is believed to be one of the most accurate methods for identifying viral strains and subtypes, it can sometimes be an impractical choice for physicians who usually need to initiate antiviral drug therapy within 48 hours of the onset of symptoms [59] . the virus culture method also becomes a secondary choice during pandemic situations when a large number of infected people rush to hospitals for diagnosis and treatment [51, 62] . the most sensitive diagnostic tool available to date is the real-time reverse transcription polymerase chain reaction (rrt-pcr) test [62, 65] . rrt-pcr detects the viral rna with high sensitivity in a few hours and requires relatively little effort. it targets the matrix gene to detect influenza viruses and the ha gene, not only to broadly distinguish between influenza a from b types but also to detect different strains of influenza a viruses (h3n2, h1n1, h1n1pdm09, etc. with high sensitivity and specificity [62] . the taqman chemistry is the most commonly used, as it gives high accuracy and specificity; however, it also comes with the burden of slightly higher costs when compared to the sybr green chemistry. the sybr green chemistry is cost effective, as it does not require dual-labelled probes like the taqman chemistry and is highly sensitive. the sybr green chemistry however, has not been widely used for clinical diagnostics, as it uses an intercalating dye that can produce fluorescence with any mis-amplified dna, thus compromising the specificity of the test [66] . a new diagnostic method named rt-smartamp assay was developed in japan during the 2009 h1n1 pandemic to reduce the time required for detection. the rt-smartamp assay includes reverse transcription and isothermal dna amplification in one step, and rna extraction and pcr are not required. an exciton-controlled hybridizationsensitive fluorescent primer specifically detected the ha segment of the pandemic h1n1-2009 influenza a virus within 40 minutes without cross-reacting with seasonal a (h1n1), a (h3n2), or b-type virus. it was found to be an efficient method for detection of iav in patient's swab samples in early stages of infection [67] . a recent study demonstrated the diagnostic potential of recombinant scfv antibodies generated against the hemagglutinin protein of influenza a virus for diagnosis and treatment of human influenza a virus infections. in that study, an elisa was developed that demonstrated 83.9% sensitivity and 100% specificity for h1n1 influenza a viruses and promised to be a cheaper alternative to the costly rrt-pcr test [68] . at research institutes and in reference or hospital laboratories, where sophisticated equipment is available, electron microscopy, cytology and histology may also be used to diagnose influenza virus infections. effective management of influenza lies in following good health practices and preventive measures laid down by health authorities. appropriate treatment of the patients can be done after accurate and timely diagnosis, and this can further reduce the inappropriate use of antibiotics and antiviral therapy. usually, ??antiviral?? therapy is preferred, as bacterial co-infection usually occurs only after viral infection. the first line of antiviral therapeutics that are chosen are inhibitors of viral proteins. the antiviral drugs currently available against influenza viruses are adamantane derivatives (amantadine and rimantadine) and neuraminidase (na) inhibitors (zanamivir, oseltamivir and peramivir). a viral infection can be inhibited at several crucial steps, such as entry, signaling, assembly, and egress (fig. 3) . adamantane derivatives inhibit virus multiplication by interfering with the transmembrane domain of the matrix protein (m2) of influenza type a viruses and also interferes in viral assembly during viral replication [69, 70] (fig. 3) . amantadine was approved for clinical use in 1966, and rimantadine was approved in 1993 [71, 72] . in the united states, three fda-approved neuraminidase inhibitor antiviral drugs are currently recommended by the us centers for disease control and prevention (cdc): oseltamivir (available under the trade name tamiflu), zanamivir (trade name, relenza), and peramivir (trade name, rapivab). the recently circulating influenza a and b viruses in the usa are susceptible to neuraminidase inhibitors, but amantadine and rimantadine are not recommended because of resistance to these drugs and also because they are not effective against influenza b viruses. all of these drugs are partially licensed against influenza in various countries. controlled clinical trials have shown sufficient effectiveness of these classes of drugs, which also prevent influenza-related illness. oseltamivir and zanamivir are recommended for all individuals with suspected or confirmed influenza requiring hospitalization and patients in high-risk groups, such as children under the age of two years, adults 65 years or older, pregnant women, immunosuppressed individuals, and ??women who have given birth within the previous two weeks??. in addition to the antiviral drugs that are available for treating influenza infections, there are new alternatives with better therapeutic potential, which studies suggest may prove to be beneficial in the near future. the longacting inhaled neuraminidase inhibitor (nai) cs-8958 (also known as r-118958) has shown promising results in murine models of influenza treatment [73] . a polymerase inhibitor, t-705 (toyama chemical), whose mechanism of action is the inhibition of the viral rna polymerase, has not only been found effective against all three influenza virus types (a, b and c) but is also effective to some extent against other rna viruses, including hemorrhagic fever viruses [74] . another drug, das181, which is a fusion construct that includes the sialidase from actinomyces viscosus, targets the viral attachment process during the early stages of replication of influenza virus [75] . another recent study showed that chlorogenic acid (cha) has antiviral properties and shows an inhibitory effect on a/puertorico/8/1934(h1n1) (ec 50 = 44.87 μm), a/ beijing/32/92(h3n2) (ec 50 = 62.33 μm), and oseltamivirresistant strains in the late stage of the infectious cycle by blocking neuraminidase activity [76] . cha (100 mg/kg/d) administered as an intravenous injection, showed 60% and 50% protection from death against the h1n1 and h3n2 strains, respectively, by reducing the viral titers and alleviating virus-associated inflammation in lungs of infected mice [76] . there are several other novel influenza antiviral drugs under clinical development in the united states, such as avi-7100, which is a 20-mer phosphorodiamidate morpholino oligomer (pmo) iv formulation that hampers the translation and splicing of mrna derived from the matrix gene [77] . cr6261 and cr8020 are monoclonal antibodies that bind to the conserved stalk region of ha and inhibit the entry and fusion stages [78] . ev-077 is a dual thromboxane receptor antagonist and thromboxane synthase inhibitor that inhibits virus replication by inhibiting the increase of prostanoids that is associated with influenza virus infections. the drug prevents inhibition of the host immune response by the virus, thus increasing virus replication [79] . a recent study also showed the anti-influenza activity of a natural product, aureonitol, a compound obtained from fungi that has shown inhibitory effects against both influenza a and b virus replication. aureonitol inhibits influenza virus hemagglutination and thus impairs virus adsorption [80] . the use of antiviral drugs is preferred during pandemic situations until an effective and sufficient vaccine is available. however, these drugs have a number of side effects, and viruses tend to develop resistance against these drugs over the course of time. the emergence of antiviral-drug-resistant seasonal influenza a viruses is a major concern. initially both amantadine and rimantadine were successful in inhibiting iav infection, and the efficacy was around 90% [81] . the first adamantine-resistant viruses were reported during the 1980 epidemic, and since then, the number has continued to increase [82] . surveillance for adamantane resistance among a (h3n2) viruses from 1991 to 1995 revealed that the global frequency of resistance was as low as 0.8% [83] . another study conducted in 2004 showed that this global resistance frequency increased to 12.3%, and a year later it reached 96%, 72%, and 14.5% in china, south korea, and the united states, respectively [84] . the alarming increase in drug-resistant h3n2 strains in the usa in 2005 led the us-cdc to issue a public health warning recommending clinicians not to prescribe adamantanes for the remainder of the 2005 and 2006 season [85] . most of the adamantineresistant h3n2 isolates (98.2%) were found to contain an s31n mutation in the m2 transmembrane domain, while l26f, v27a, and a30t mutations accounted for the rest (1.8%) [86] . starting in 2005, the number of cases of resistance increased exponentially, and from 2005 to 2006, almost 90.6% of h3n2 strains and 15.6% of h1n1 were adamantane resistant [87] . in the usa alone, 96.4% of h3n2 isolates and 25% of h1n1 isolates were adamantane resistant [84, 87] , and the resistance-conferring mutation was s31n in the m2 gene in both h1n1 and h3n2 strains. the pandemic h1n1-2009 strain [88] as well as the h5n1 and h7n9 strains that caused fatal zoonotic infections in humans in 2003 and 2013, respectively, were also observed to have the same s31n mutation in the m2 gene [89, 90] . by 2013, almost 45% of all the iav isolates were resistant to adamantanes [91] . the neuraminidase inhibitors (nais) are the second class of anti-influenza drugs, and the only one currently being used worldwide. these drugs target the surface protein na to produce an antiviral effect [92] . the nais oseltamivir (tamiflu) and zanamivir (relenza) are most effective if administered within 36-48 hours of the onset of symptoms [93] . zanamivir was approved for prophylaxis and treatment of iav infection in humans in july 1999, followed by oseltamivir in october 1999 [94] . the global neuraminidase inhibitor susceptibility network (nisn), established in 1999, reported that during the period 1996-1999, all human influenza isolates were found to be susceptible to nais; however, in the later years 2005 and 2007, the frequency of oseltamivir resistance in global h1n1 isolates increased slightly by 0.4% and 0.6%, respectively [95] . in 2007-2008, there was a significant 7% global rise in oseltamivir-resistant h1n1, but not h3n2 strains, and all oseltamivir-resistant h1n1 isolates from that season were sensitive to zanamivir [96] . in the 2008-2009 season, more than 90% of the globally circulating h1n1 subtypes were found to be oseltamivir resistant [97] . in the year 2009, the circulating nai-resistant h1n1 strains were replaced by the novel pandemic h1n1-2009 strain, which, fortunately, was sensitive to nais [98] . these influenza a (h1n1 and h5n1) viruses have shown resistance due to mutation of a histidine to a tyrosine at residue 274 of the na (h274y), which confers a high level of resistance to oseltamivir but has no effect on susceptibility to zanamivir or to the adamantanes [99] . although antiviral drugs against influenza are readily available worldwide, the administration of vaccines remains at the forefront for managing influenza virus infections because prevention is still better and more cost-effective than cure. the administration of influenza and pneumonia vaccines is one of the highest priorities in primary-care medicine [100] . since their first introduction in the 1940s, influenza vaccines have come a long way [101] . these early vaccines were inactivated whole-virus vaccines that were generated in embryonated chicken eggs and inactivated by treatment with formalin. the genetic drift in viral genome has made it necessary to formulate new vaccines each year. although separate vaccines are now available for individual influenza viruses, a universal influenza vaccine has not yet been developed due to the highly variable nature of the surface glycoproteins ha and na. who maintains surveillance of circulating strains of influenza viruses in both the northern and southern hemisphere, and influenza vaccines are formulated annually to be administered to healthy individuals and those at higher risk of complications prior to the start of the flu season. there are currently several types of influenza vaccines available, of which the major types are conventional inactivated influenza vaccines and live attenuated influenza virus (laiv) vaccines. the conventional inactivated influenza vaccines consist of purified virus particles that have been inactivated by treatment with formalin or β-propiolactone. live attenuated influenza vaccines are made using virus strains that are cold adapted, temperature sensitive, and attenuated to prevent them from causing illness. laiv vaccines have been successfully made that can be administered via nasal spray (flumist). they have shown high efficacy in children when compared to inactivated vaccines [102] , as the laiv activates mucosal, systemic humoral, and cellular immunity, just like natural influenza viruses. in the usa and canada, an laiv vaccine is licensed under the trade name flumist, while in europe it is licensed under the trade name fluenz. since laiv vaccines have been observed to be less effective in adults, inactivated split vaccines are recommended for adults. traditional trivalent vaccines containing two influenza a strains (h1n1 and h3n2) and one influenza b strain sometimes show limited protection due to a lineage mismatch between the vaccine b strain and the circulating b strain. to minimize the limitation in protection by trivalent vaccines, the fda, for the first time in 2009, considered the inclusion of an additional influenza b strain, thus making a quadrivalent vaccine. both live-attenuated quadrivalent influenza vaccines and inactivated quadrivalent influenza vaccines are known to confer significant protection against the drifted circulating influenza b viruses [103] . apart from the traditional vaccine approaches, other approaches includes the development of dna vaccines against different influenza virus antigens [104] , the development of a possible universal influenza vaccine targeting the ha stalk domain [105, 106] , and the use of influenza-virus-like particles as vaccines [107] . the long delivery time frame for egg-based vaccines can be a critical factor during a pandemic, and therefore, cell-culture-based vaccines (e.g., optaflu, flucelvax, preflucel, and celvapan) are also being used to overcome this issue [108] . due to the increasing burden of vaccine formulations and cases of antiviral-drug-resistant influenza virus isolates turning up every year, it has become necessary to search for alternatives to the current treatment and prevention strategies. the last few decades have seen a tremendous effort being made to develop inhibitors and blockers of vital genes of influenza viruses. novel drugs have been formulated against the viral nucleoprotein [109] and non-structural proteins [110] . several studies have also been performed using sirna and antisense oligonucleotides as gene silencing tools to inhibit influenza virus replication in cell lines, chicken embryonated eggs, and mice [111] [112] [113] . the potential of sirnas as antivirals was first recognized in 2011, when this approach was used against the viral transcription factor, p (phosphoprotein), and viral f (fusion) protein of rsv [114] . in 2003, chen's laboratory published the first report of the use of sirnas against np, pa, pb1, pb2, m, and ns genes that showed various degrees of inhibition of multiple subtypes of influenza viruses [113] . a study conducted using antisense oligonucleotides against the 3' ncr of vital segments of the iav genome showed significant inhibition of viral replication. the designed antisense molecules were tested against the a/pr/8/34 (h1n1), a/ udorn/307/72 (h3n2), and a/new caledonia/20/99 (h1n1) strains of iav and were found to reduce the cytopathic effect caused by these viruses for almost 48 hours postinfection in cell lines and to increase the survival of experimental mice [112] . ribozymes (rz) and dnazymes (dz) are yet another class of gene-silencing tools that have been demonstrated to control iav replication [115, 116] . a study conducted on the a/pr/8/34 (h1n1) strain showed that rz and dz along with antisense molecules accomplish a synergistic cleavage of the matrix (m1) gene of influenza virus, thus inhibiting virus replication in host cells [117] . another recent study conducted on influenza b virus also confirmed the role of dz in inhibiting iav replication [70] . the designed dz showed a significant 52% inhibition of the b/yamagata/1/73 strain of influenza b virus [70] . in another recent study, the authors revealed that self-assembling influenza nanoparticle vaccines could elicit broadly neutralizing h1n1 antibodies. they genetically fused the viral hemagglutinin to ferritin, a protein that naturally forms nanoparticles, and showed that this influenza nanoparticle vaccine generated more than tenfold higher ha inhibition antibody titres than those induced by the licensed inactivated vaccine [118] . another prospective approach to achieving high virus-neutralizing activity is the use of monoclonal antibodies and recombinant antibody fragments [119, 120] . several host-cell molecules have been known to play a crucial role during influenza virus infection, thereby representing targets for designing inhibitors of virus-cell interactions. one such target is the vacuolar proton-atpase, which when inhibited, renders viral m2 ion channels inactive [121] . a few other studies have also focused on inhibitors of cellular proteases [122] that block the proteolytic activation of ha and blockers of the cellular ubiquitin-proteasome system [123] . in the past few years, there has been a remarkable increase in the number of studies describing the use of a new class of influenza-virus-neutralizing antibodies targeting conserved sites in the ha stem. these molecules have shown varying levels of cross-reactivity toward group 1 [124, 125] , group 2 [126, 127] and group 1 & 2 viruses [128, 129] . despite these efforts, antibodies that can react with the stem region of both group 1 and 2 subtypes are rare and do not cover all subtypes. in view of this, in a recent study, a broad spectrum human monoclonal antibody (mab-medi8852) was developed, which unlike other stem-reactive antibodies, used a rare heavy chain vh (vh6-1) gene and carried a low level of somatic mutations [130] . medii8852 was effective in mice and ferrets and was better than oseltamivir. its broad neutralizing capability makes this molecule a good candidate for development as an immunotherapy for influenza-virusinfected humans [130] . these alternative approaches, which, when backed up with clinical trials, will provide promising tools for managing influenza virus infections effectively. influenza viruses have successfully evolved with striking survival strategies. they circulate worldwide with established lineages in avian and mammalian species. of the four influenza virus types (a, b, c and d), influenza a viruses are the most virulent and have the potential to cause both epidemics and pandemics due to genetic drift and genetic shift, respectively. types b, c and d are not known to cause pandemics. influenza a virus has a wide range of hosts, which provides opportunities to cross species barriers, thus increasing the chances of an influenza pandemic. the virus circulates around the world and causes annual outbreaks resulting in about 3-5 million cases of illness and up to 500,000 deaths [131] . two classes of antiinfluenza drugs (adamantanes and nais) are available, of which only the nais are currently effective against circulating strains of iav. vaccination is one of the best approaches to prevent influenza infections annually. however, due to frequent mutations in the surface glycoprotein ha, iav acquire enough mutations each year to escape the protectivity of the annually formulated vaccines, and some of them show a high level of resistance against antiviral drugs [132] . alternative approaches such as the use of sirna, antisense nucleotides, dz and rz have gained importance in the past few decades and have shown promising results in cell lines and mouse models [69, 70, 112, 113, 115, 117] . several other studies are still being performed to develop a universal influenza vaccine that can neutralize all types of iav. influenza viruses have evolved in parallel to humans to establish successful infections and continue to pose a significant threat to both life and economy. the health authorities invest large amounts of money into annual vaccine formulations, and the virus acquires several mutations to render those vaccines ineffective within a year. thus, new alternative approaches to combating antiviral resistance and the development of universal vaccine formulations are currently needed in order to manage future influenza threats. influenza viruses have been the cause of annual epidemics throughout the world. the a subtypes occasionally cause pandemics that lead to the death of millions of people. the history of influenza suggests that the virus is highly unpredictable in its ability to jump species barriers and cause threatening situations for mankind. health authorities across the world have influenza preparedness plans that are based on combined surveillance data received from both the southern and northern hemisphere. advancements in science have brought together several antiviral therapeutic strategies combined with novel drugs that can be used to 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vesicular stomatitis virus propagation by establishing an antiviral state heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine broadly cross-reactive antibodies dominate the human b cell response against 2009 pandemic h1n1 influenza virus infection preexisting human antibodies neutralize recently emerged h7n9 influenza strains a highly conserved neutralizing epitope on group 2 influenza a viruses a potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza a virus a neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza a hemagglutinins structure and function analysis of an antibody recognizing all influenza a subtypes epidemiology and pathogenesis of influenza influenza virus resistance to neuraminidase inhibitors key: cord-268296-w0i7rhru authors: barros, eliana nogueira castro de; cintra, otavio; rossetto, erika; freitas, laís; colindres, romulo title: patterns of influenza b circulation in brazil and its relevance to seasonal vaccine composition() date: 2015-11-25 journal: braz j infect dis doi: 10.1016/j.bjid.2015.09.009 sha: doc_id: 268296 cord_uid: w0i7rhru data on the burden of disease and circulation patterns of influenza b lineages for brazil are limited. this review aims to describe the pattern of influenza b occurrence in brazil to have a better understanding of its epidemiology and its relevance when considering seasonal influenza vaccine composition. a review of the data including analysis of international and local surveillance data as well as information from online search of databases using medical subject headings terms in conjunction with screening of abstracts from scientific events was performed. based on international epidemiologic surveillance data, moderate levels of influenza b disease (19%; 2006–2014) were observed. of these nine years, it was possible to compare data from three years (2007, 2008 and 2013) which have information on the circulating influenza b lineage. co-circulation of influenza b lineages was observed in all these three influenza seasons, of which, during one season, a high degree of mismatch between the vaccine lineage and the predominant circulating lineage (91.4% [2013]) was observed. local surveillance data reveal a distinct and dynamic distribution of respiratory viruses over the years. data from published literature and abstracts show that influenza b is a significant cause of disease with an unpredictable circulation pattern and showing trends indicating reemergence of the b/victoria lineage. the abstracts report notable levels of co-circulation of both influenza b lineages (2000–2013). mismatch between the southern hemisphere vaccine and the most prevalent circulating viruses in brazil were observed in five influenza seasons. the evidence on co-circulation of two influenza b lineages and mismatched seasons in brazil indicates the benefit of quadrivalent influenza vaccines in conferring broader seasonal influenza protection. additionally, improving influenza surveillance platforms in brazil is important for monitoring disease trends and the impact of introducing seasonal influenza vaccination. influenza is a highly infectious acute viral illness resulting in significant morbidity as well as healthcare resource utilization. in healthy individuals influenza is generally self-limiting, but can often cause complications. 1, 2 there are 3 types of seasonal influenza viruses -a, b, and c. influenza a causes moderate to severe illness and affects individuals of all age groups. influenza b can cause disease of similar severity as influenza a, and even though the morbidity is higher in children, all age groups can be affected. 3, 4 the influenza b virus is more stable than influenza a, with less antigenic drift and consequent immunologic stability, and does not undergo the process of antigenic shift. influenza c is rarely reported as a cause of human illness, probably because most cases are subclinical. 5 both influenza a and b cause annual epidemics worldwide and are estimated to result in 3-5 million cases of severe illness, and 250,000-500,000 deaths. 6 influenza vaccination is the most important prophylactic intervention against infection. until the 2012-2013 influenza season, use of trivalent inactivated influenza vaccines, containing two influenza a strains (a[h1n1] and a[h3n2]) and one influenza b lineage (b/yamagata or b/victoria) was recommended for use in immunization programs by the world health organization (who). 6 as influenza viruses undergo frequent changes in their surface antigens, new influenza vaccines are designed annually to match the circulating virus subtype expected for the next influenza season. 7 the selection of the influenza b lineage is considered critical in determining the effectiveness of vaccination programs. 7 unfortunately, the correct prediction of the predominating circulating b lineage is quite difficult, often leading to inaccuracies in prediction, causing a mismatch between the recommended vaccine lineage and the circulating influenza b lineage. prior studies have raised concerns that mismatches can result in lower vaccine effectiveness, due to the absence of cross-protection between antigenically distinct influenza b lineages, leading to more influenza cases, 8, 9 and an increase in influenzarelated medical resource utilization and costs. 2, 10 in addition to this, matias et al. 11 have found that influenza b-associated mortality could serve as a surrogate marker of disease severity. in the latin america and caribbean region, seasonal influenza causes high morbidity placing a substantial economic burden on healthcare systems and society. 12 data on the burden of influenza disease for brazil are limited, most likely due to underreporting. 12 between 2000 and 2008, data from the influenza surveillance system in brazil revealed that influenza-like illness (ili) led to a total of 4.39-16.92% of hospital consultations, and in 2008, of all positive reported influenza cases, 43.29% (95% ci: 37.59-49.13) were influenza b. 12 the ministry of health (moh, "ministério da saúde") of brazil promotes annual national influenza vaccination campaigns. over the years, there has been a gradual expansion of the recommended groups for annual influenza immunization in brazil. 13 since 1999, influenza vaccination was introduced for elderly people aged above 65 years and other groups vulnerable to complications (patients with co-morbidities). in the year 2000, individuals 60 years or older were included for vaccination. 14, 15 during 2011-2012, in addition to elderly people, vaccination was extended to children aged six months to those aged below two years, pregnant women, healthcare professionals, and indigenous people. in 2013, women after child birth, individuals with chronic disease and transplant, and individuals in detention facilities were included for annual influenza vaccination. in 2014, children aged 2-4 years were also included in the recommended target at-risk groups for vaccination. the information system of national immunization program for brazil ("programa nacional de imunizaç ão") 13 reported that across all target vaccination groups, overall mean vaccination coverage of 86.8% was reached in 2014. 13 during all influenza vaccination campaigns in brazil, trivalent vaccines were used according to who recommended vaccine composition for south hemisphere. 16 although high vaccination coverage levels have been reached in these target vaccination groups, little is known about the effectiveness of vaccination programs in brazil. 14, 17 a number of factors, in particular vaccine coverage, is known to influence the effectiveness of influenza vaccination programs. however, the brazilian moh data shows vaccine coverage to be high in almost all years since the introduction of vaccination. importantly, the extent to which the vaccine recommended influenza b virus lineage matches the influenza virus lineage circulating in the population during an influenza season is known to impact the effectiveness of seasonal influenza vaccination programs. 14 data on laboratory surveillance of the influenza b virus in brazil are limited, specifically data on the burden of disease and circulation patterns of influenza b lineages. the present integrative review of publicly available data aims to consolidate findings on the pattern of influenza b occurrence in brazil to have a better understanding of influenza b epidemiology and its relevance to seasonal vaccine composition. different sources were used to retrieve information on epidemiological surveillance. we referred to international data sources to check who recommendations on the vaccine composition in the southern hemisphere, 18 and information on circulating influenza lineages for brazil, the south america region and globally from the who/flunet database which provides data through its network -global influenza surveillance and response system (gisrs) laboratories. 19 consolidation of available national epidemiological information on influenza in the epidemiological bulletins of the brazilian moh was also performed. bulletins available from 2009 to epidemiological week 35 of 2014 were considered. 15 in brazil, two types of surveillance exist: sentinel surveillance of ili and universal severe acute respiratory syndrome (sars) surveillance. ili is defined as fever followed by cough or sore throat and symptoms onset within the last seven days. sars is defined as fever followed by cough or sore throat and experiencing dyspnea, requiring hospitalization. oxygen saturation levels lower than 95%, respiratory distress, or respiratory rate increase, are also considered in sars. the sentinel ili surveillance, which has a network of units distributed throughout the country's geographic regions, is used in the identification and characterization of circulating respiratory viruses in viral isolations. the universal sars surveillance, which was implemented post-identification of the influenza a(h1n1) 2009 pandemic strain (pdm09), monitors hospitalized cases and deaths. in both systems, data are collected by means of standardized forms and entered into the online information systems: influenza epidemiological surveillance system called sivep-gripe for ili cases, and the national information system for notifiable diseases known as sinan influenza web for sars cases. results from tests performed at the 27 public health central laboratories ("laboratórios centrais de saúde pública, lacen") are routinely included in these systems. the diagnostic kits currently available identify the influenza a(h1n1)pdm09 viral strain (determined by who), influenza a(h3n2), influenza a not subtyped, and influenza b virus. the antigenic characterization of the circulating influenza virus lineage is performed by three laboratories in brazil ("instituto evandro chagas" -pará, "instituto adolfo lutz", são paulo and "instituto oswaldo cruz", rio de janeiro). these laboratories are part of a network of 140 national influenza centers acknowledged by who as members of the gisrs. an integrative literature review was performed using online database tools such as pubmed/medline, lilacs and scielo. searches on online databases were conducted using the following search strategy built for the pubmed database (via medical subject headings [mesh] controlled vocabulary) and adjusted for other databases according to their specificities: "(((((influenza [title/abstract]) or influenzae)) or ((orthomyxoviridae [mesh major topic]) or influenza, human [mesh major topic]))) and brazil". studies on influenza b, with brazil as the place of study and those with virological influenza b information were considered. articles published between 2007 and 2014 were considered and a language constraint was not applied. articles and abstracts with general influenza reports or without subtyping (a or b) were not included. one reviewer screened titles and abstracts for relevance, as the defined inclusion criteria were restrictive. an additional research was conducted in abstracts of the annals of scientific events related to the area of research. a total of 10 events were considered. 20-29 all authors agreed with the selected publications. data on viral lineage isolation and characterization, vaccination campaign, viral lineage surveillance and strain match-mismatch from different sources were collected and consolidated. statistical analysis was not performed, and thus all data are descriptive. the pattern of hospitalized sars cases from the universal sars surveillance showed that in 2013, although influenza a(h1n1)pdm09 was predominant, 22.5% of sars cases positive for influenza (1337/5935, with 85 deaths) were caused by the influenza b virus. regional variation was observed between the regions -south region (29%) and north region (12%). in 2014 (up to epidemiological week 35) 6% of sars cases positive for influenza (75/1307, with 9 deaths) were caused by the influenza b virus (table 1) . a total of 722 articles were identified, of which 602 articles were excluded after the review of titles as they were related to influenza other than influenza b. subsequent to the screening of titles, abstracts of the 120 potentially relevant articles were reviewed and, as a result, 108 articles were excluded when the eligibility criteria were not met (fig. 2) . in all, 12 articles presenting evidence on influenza b virus circulation in brazil for the years 1996-2013 were included. [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] details for each of the studies are shown in supplementary file 1. two studies detailed characteristics of an influenza outbreak in february 2012 on cruise ships. 32, 35 fernandes et al. 35 reported findings from the first influenza outbreak detected by brazilian public health authorities in a vessel cruising in south america. of 11 hospitalized cases of acute respiratory illness, there were six cases with influenza b virus detected in the nasopharyngeal isolates. 35 in another study by borborema et al. 32 , it was shown that the influenza b virus was the cause of the outbreak (detected in seven individuals with respiratory illness). other reports from the online literature review were primarily from retrospective descriptive studies. freitas 36 showed that influenza b was attributable to moderate levels of disease during 2000 and 2010 (3-17%), and its occurrence was notable in the south and central-west regions. paiva et al. 37 confirmed the reemergence of the b/victoria viruses in são paulo, brazil, during the years 1996-2012. in table 2 , based on who/flunet reports, consolidated annual data on the lineages that were part of the vaccine, the proportion of predominant circulating lineages and the proportion of lineage mismatch is shown for south america and brazil. data for brazil was available for nine years; of these, it was only possible to compare data from three years (2007, 2008 and 2013) which have information available on the circulating influenza b virus lineage. in brazil, co-circulation of both influenza b lineages was detected in all these three influenza seasons, and a high level of mismatch (91.4%) was observed in one year (2013) ( table 2 ). in the same year for south america 52.0% (2013) of mismatch was observed. that in 2013, a high degree of mismatch between the vaccine and the predominating circulating lineage (91.4%) occurred, and during the other two influenza seasons, a partial mismatch was reported. the three reviewed abstracts, which specifically report findings on influenza b mismatch, corroborate this unpredictable behavior of influenza b disease in brazil for many other seasons for which data were not available in the international epidemiological surveillance data. significant levels of co-circulation of both influenza b lineages (b/victoria and b/yamagata) and lineage mismatch between the vaccine and circulating lineage was reported for brazil in five influenza seasons (2002, 2005, 2008, 2010, and 2013) . [42] [43] [44] previous studies suggest that even with a partial mismatch over the years, due to the unpredictability of influenza b lineage circulation, the disease burden (in terms of clinical cases, hospitalizations and health care resource utilization) and societal burden can be considerable. 2, 9 for example, a mismatch in the 2007-2008 influenza season in the united states was estimated to have costed health care providers and society well over $100 million and $1 billion, respectively. 45 in another study conducted in taiwan, it was reported that an epidemic predominated by influenza b/yamagata lineage occurred during the 2011-2012 season during which the trivalent influenza vaccine contained influenza b/victoria lineage. expectedly, the morbidity and mortality of this vaccine mismatched epidemic was substantial (influenza b: 60.7% and 66.9% of confirmed influenza cases and deaths, respectively; 87.2% of samples showed the presence of vaccine opposite lineage). 46 surveillance data on the age distribution of influenza cases is available only for 2013. this data showed that influenza b virus was predominantly observed in younger individuals aged 5-29 years, with the highest proportions in children aged 5-9 years and 10-19 years. even though this observation was restricted to only one year with 90% mismatch it is in line with previous reports wherein influenza b has been reported to affect more schoolchildren and the appearance of the new b lineage reinforced this pattern. 4 an implication of this finding is that children and adolescents might benefit the most from the addition of a second influenza b lineage to the seasonal influenza vaccine. moreover, it could be useful for the entire population as it is widely considered that vaccinating children can reduce influenza illness in family members and other susceptible populations in the community by reducing the risk of exposure and subsequent influenza infection and related complications. 47 11 that influenza b-associated mortality could potentially be a valid indication of disease severity. thus the evidence presented here for one year underscores the importance of a universal influenza vaccination strategy compared to targeted immunization as universal immunization at high vaccine coverage levels could potentially interrupt transmission reducing the occurrence of outbreaks and severe disease. our literature review confirms the re-emergence of b/victoria lineage in brazil during the years 2000-2002. these data are in line with that of motta et al. 48 which shows the re-emergence of the victoria-lineage viruses in the northern hemisphere and in the south and south east regions of brazil where previously the b/yamagata lineage was the predominating circulating lineage and the vaccine recommended lineage. other studies confirm that the b/yamagata was the major circulating lineage until the 1980s, when b/victoria lineage viruses appeared; since then, drift variants of both influenza b lineages have been co-circulating worldwide. 49, 50 as influenza b viruses can co-circulate during an epidemic allowing the re-emergence of old lineages due to re-assortment between the different strains, 4,48 there is a need to improve influenza laboratory-based surveillance in brazil. importantly these data also highlight the unpredictability of influenza b circulation, making it difficult to consistently predict which b lineage will predominate during a given influenza season. due to the little cross-protection conferred between the two antigenically distinct influenza b lineages, a vaccine lineage mismatch with the circulating lineage could result in additional burden in terms of health care resource utilization and can adversely impact the society. this burden could, in particular, be substantial during an outbreak. to avoid this additional burden, a plausible option would be to switch from the use of trivalent influenza vaccines to quadrivalent vaccines in national campaigns. 51 quadrivalent influenza vaccines are expected to offer broader protection against influenza b disease. the who recently updated recommendations beginning with the 2013-2014 influenza, now recommending the use of a quadrivalent vaccine composition with the addition of a second influenza b lineage as well as the two influenza a strains and one influenza b lineage contained in the trivalent vaccines. 6, 52 some limitations of this review require consideration. besides the inherent limitations of each of the studies included in the literature search and abstracts, there are other important limitations. statistical and quality analysis of the articles was not performed. also, the interpretation of data is constrained by the gaps in laboratory surveillance in which there were no influenza b data available for many years with respect to age, severity, and seasonality. as we present data over many years through which the surveillance system has gradually improved, the number of positive samples is still small, and the more recent years are likely to be overrepresented. additionally, sentinel surveillance data cannot be truly representative for the whole population as the system of sentinel units involves different levels of care. moreover, there is no standard protocol for patient selection for sample collection which may have led to a selection bias of certain age groups. despite these limitations, the surveillance system for influenza and other respiratory viruses which has improved over the years has proven useful to describe influenza b circulation patterns for brazil and demonstrate that influenza b epidemiology is changing in line with observations reported from other countries. 4 influenza surveillance systems, and laboratory-based and epidemiological studies of influenza b in brazil, while limited, have improved over the years. there is a need to strengthen and extend influenza surveillance for influenza b sample subtyping to determine the behavior pattern of influenza b lineages. the findings from this integrative data review provide evidence of the unpredictable nature of influenza b circulation in brazil, the increasing frequency of co-circulation of both influenza b lineages, and mismatch between the circulating influenza b lineage and the composition of the seasonal influenza vaccine for the region. this data is suggestive of the additional benefit that quadrivalent influenza vaccines, containing both influenza b lineages (b/yamagata and b/victoria), could offer over the use of the currently available trivalent vaccines for the prevention of seasonal influenza in brazil. all authors participated to the conception/design of the review, performed or supervised the analysis, and interpreted the data. otavio cintra, eliana nogueira castro de barros, laís freitas and erika rossetto collected or assembled the data. erika rossetto wrote the preliminary report of the review findings. all authors read and approved the final manuscript. eliana nogueira castro de barros, otavio cintra and romulo colindres are employees of the gsk group of companies. romulo colindres and otavio cintra report ownership of stock options and/or restricted shares. laís freitas reports that she is working for gsk vaccines, but is employed by shift de gestão em serviç os. erika rossetto has no conflict of interest. population-based study on incidence, risk factors, clinical complications and drug utilisation associated with influenza in the united kingdom influenza-related health care utilization and productivity losses during seasons with and without a match between the seasonal and vaccine virus b lineage comparison of influenza a and influenza b virus infection in hospitalized children editorial commentary: changing epidemiology of influenza b virus public health foundation influenza (seasonal) world health organization.who position paper: influenza vaccines estimating vaccine effectiveness against laboratory-confirmed influenza using a sentinel physician network: results from the 2005-2006 season of dual a and b vaccine mismatch in canada influenza vaccine: the challenge of antigenic drift impact of influenza b lineage-level mismatch between trivalent seasonal influenza vaccines and circulating viruses estimates of mortality attributable to influenza and rsv in the united states during 1997-2009 by influenza type or subtype, age, cause of death, and risk status. influenza other respir viruses burden of influenza in latin america and the caribbean: a systematic review and meta-analysis. influenza other respir viruses ministério da saúde departamento de informática do sus (datasus) effectiveness of the brazilian influenza vaccination policy, a systematic review ecological study on mortality from influenza and pneumonia before and after influenza vaccination in the northeast and south of brazil. cadernos de saúde pública world health organization. who recommendations on the composition of influenza virus vaccines. influenza ministério da saúde secretaria de vigilância em saúde. oordenação geral de doenças transmissíveis world health organization. influenza vaccine viruses and reagents european society for paediatric infectious diseases 24. international congress on infectious diseases options for the control of influenza world society for pediatric infectious diseases patterns of influenza infections among different risk groups in brazil acute respiratory infection and influenza-like illness viral etiologies in brazilian adults molecular characterization of influenza b virus outbreak on a cruise ship in brazil 2012 applications of a duplex reverse transcription polymerase chain reaction and direct immunofluorescence assay in comparison with virus isolation for detection of influenza a and b impact of respiratory infections by influenza viruses a and b in pediatrics patients from federal university of parana, brazil influenza b outbreak on a cruise ship off the sao paulo coast, brazil sentinel surveillance of influenza and other respiratory viruses, brazil evolutionary pattern of reemerging influenza b/victoria lineage viruses in sao paulo, brazil, 1996-2012: implications for vaccine composition strategy etiologia viral das infecç ões respiratórias agudas em população pediátrica no instituto fernandes figueira/fiocruz/rj análise filogenética e padronização da técnica de eletroforese em gel com gradientes desnaturantes (dgge) para caracterização das linhagens do vírus influenza b identificadas durante as epidemias de vigilância de oito vírus respiratórios em amostras clínicas de pacientes pediátricos no sudeste do brasil the epidemiology and antigenic characterization of influenza viruses isolated in curitiba, south brazil mismatch between vaccine strains and circulating influenza b viruses in different regions of brazil: 2001-2013. options for the control of influenza. cape town influenza virus surveillance conducted by the institute adolfo lutz influenza b circulation during twelve influenza seasons (2001-2013) in a tertiary hospital in sao paulo, brazil. options for the control of influenza. cape town the economic value of a quadrivalent versus trivalent influenza vaccine surveillance and vaccine effectiveness of an influenza epidemic predominated by vaccine-mismatched influenza b/yamagata-lineage viruses in taiwan, 2011-12 season assessing optimal target populations for influenza vaccination programmes: an evidence synthesis and modelling study the reappearance of victoria lineage influenza b virus in brazil, antigenic and molecular analysis genetic analysis and evaluation of the reassortment of influenza b viruses isolated in taiwan during the reappearance and global spread of variants of influenza b/victoria/2/87 lineage viruses in the world health organization. influenza recommended composition of influenza virus vaccines for use in the 2015 southern hemisphere influenza season the authors would like to thank andreza madeira macario (epidemiologist to inovatec) for intellectual contribution to the study, amrita ostawal for medical writing services (consultant publications writer to gsk vaccines) and bruno dumont (business & decision life sciences on behalf of gsk vaccines) for editorial assistance and publication coordination. supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.bjid.2015.09.009. r e f e r e n c e s key: cord-266204-ipa017wz authors: poland, g. a.; ovsyannikova, i. g.; kennedy, r. b. title: personalized vaccinology: a review date: 2018-08-28 journal: vaccine doi: 10.1016/j.vaccine.2017.07.062 sha: doc_id: 266204 cord_uid: ipa017wz abstract at the current time, the field of vaccinology remains empirical in many respects. vaccine development, vaccine immunogenicity, and vaccine efficacy have, for the most part, historically been driven by an empiric “isolate-inactivate-inject” paradigm. in turn, a population-level public health paradigm of “the same dose for everyone for every disease” model has been the normative thinking in regard to prevention of vaccine-preventable infectious diseases. in addition, up until recently, no vaccines had been designed specifically to overcome the immunosenescence of aging, consistent with a post-wwii mentality of developing vaccines and vaccine programs for children. it is now recognized that the current lack of knowledge concerning how immune responses to vaccines are generated is a critical barrier to understanding poor vaccine responses in the elderly and in immunoimmaturity, discovery of new correlates of vaccine immunogenicity (vaccine response biomarkers), and a directed approach to new vaccine development. the new fields of vaccinomics and adversomics provide models that permit global profiling of the innate, humoral, and cellular immune responses integrated at a systems biology level. this has advanced the science beyond that of reductionist scientific approaches by revealing novel interactions between and within the immune system and other biological systems (beyond transcriptional level), which are critical to developing “downstream” adaptive humoral and cellular responses to infectious pathogens and vaccines. others have applied systems level approaches to the study of antibody responses (a.k.a. “systems serology”), [1] high-dimensional cell subset immunophenotyping through cytof, [2,3] and vaccine induced metabolic changes [4]. in turn, this knowledge is being utilized to better understand the following: identifying who is at risk for which infections; the level of risk that exists regarding poor immunogenicity and/or serious adverse events; and the type or dose of vaccine needed to fully protect an individual. in toto, such approaches allow for a personalized approach to the practice of vaccinology, analogous to the substantial inroads that individualized medicine is playing in other fields of human health and medicine. herein we briefly review the field of vaccinomics, adversomics, and personalized vaccinology. vaccines have been one of the most effective public health strategies in preventing infectious diseases. a decade ago, we described the idea of vaccinomics and adversomics, based on the immune response network theory [5, 6] , which utilizes immunogenetics/imunogenomics and systems biology approaches to understand the basis for inter-individual variations in vaccineinduced immune responses in humans, as well as the basis for adverse side effects from vaccines [7] . vaccinomics and adversomics explore the influence of genetic and non-genetic regulation on the heterogeneity of vaccine-induced immune responses at both the personal and population levels [5] . in particular, vaccinomics and adversomics utilize high-throughput, high-dimensional systems biology approaches, which aim to predict variations in protective and maladaptive innate and adaptive immune responses to vaccines [1] [2] [3] [4] 6, 8] . in this regard, the basis of personalized (and predictive) vaccinology is the assessment of an individual's genetic background, sex, as well as other factors that may impact vaccine immunogenicity, efficacy, and safety [8] [9] [10] [11] . we and others have widely published on the applicability of the tools and concepts of vaccinomics, including immunogenetics and immunogenomics, to the knowledge-based directed development of new and improved vaccine candidates [12] [13] [14] [15] . the application of these concepts is likely to allow for explanation, quantification, and prediction of vaccine-induced protective immune responses-including the http://dx.doi.org/10.1016/j.vaccine.2017.07.062 0264-410x/ó 2017 elsevier ltd. all rights reserved. development of predictive immune signatures in response to vaccines. indeed, we have previously published what we believe is the first draft of a mathematical model and predictive equation describing the non-random events that lead to a pre-determined immune response [6] : b i x iþe y = measure of immune response b o = intercept b i = coefficient for the ith variable x i and indicates the amount of change in y for a 1 unit change in x i ε = random deviations from the model we recognize that such an equation, given the current state of the science, is incomplete and cannot yet predict immune responses. but we present it as an early directional attempt to quantify such an equation. such an approach begins to move us into a 21st-century model of directed vaccine development and an advanced understanding of how, and by what mechanisms, vaccines and vaccine adjuvants trigger both useful and maladaptive innate and adaptive immune responses. we believe that vaccinomics and adversomics represent approaches counter to the standard methods of vaccine development until recently. historically, vaccine development has been empirical, despite many emerging and re-emerging complex, hyper-variable pathogens-many with elaborate immune escape mechanisms. in addition, vaccine coverage rates continue to suffer as society is risk-averse toward vaccines and demands levels of safety that may not be achievable. finally, the ''one-size-fits-all" approach to the practice of vaccinology ignores the complexity and diversity of the human immune system and host genome. thus, the promise of vaccinomics and related paradigms is to identify specific immune response profiles, immunosignatures, and biomarkers that predict vaccine safety and/or efficacy, and which may lead to new vaccine candidates. vaccinomics provides the opportunity to examine not only immune response genes likely to be involved in vaccine response, but also the possibility of identifying the influence of new (uncharacterized) genes on vaccine-induced immunity. in turn, the identification and directed study of such genetic variants allows recognition, often at the molecular level, of the effects of differential binding, processing, and expression/presentation of antigenic viral peptides used in vaccine development, identification of the differential range of presented peptides (genetic restriction), altered secretion patterns (cytokines) in response to vaccines or vaccine adjuvants, altered transcription of important genes (signaling molecules) and gene products, altered binding of virus/antigens by membrane-based receptors (tlrs, other), differential receptor function, expression, and affinities, and the impact of epigenetics on vaccine-induced immune responses. we have utilized this knowledge in our own laboratory to create a research-oriented paradigm of ''discover-validate-characterize-apply," which may be used in new candidate vaccine development ( fig. 1 ) [6] . in this paradigm, we have been able to utilize vaccinomics approaches to discover genetic variants that are significantly associated with subsequent downstream immune responses, validate that such variants are indeed associated, then seek to characterize the mechanism whereby such effects occur and, finally, apply this knowledge-often in functional studies that confirm the effect on immunity. such knowledge can be exploited in developing immune strategies to enhance or circumvent genetic restrictions, for example, in triggering vaccine-associated immune responses, by ''reverse engineering" around a given genetic or other obstacle to generating protective immune responses. there are a growing number of studies reporting unbiased genome-wide assessments of genetic variation and its influence on adaptive (humoral and cellular) vaccine-induced immune responses across multiple viral and bacterial vaccines. for example, candidate and gwas immunogenetic and phamacogenetic studies have identified polymorphisms in hla, kir, mica, and btn genes associated with immune responses to pathogens causing disease in humans, such as hepatitis c [16] , mycobacterium leprae [17, 18] , human immunodeficiency virus [19] , and measles [20] [21] [22] . similar studies have identified novel genes impacting immune responses to vaccines, including hepatitis b, rubella, influenza a, smallpox, anthrax, and mumps [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . our gene association studies of measles-mumps-rubella (mmr) vaccines have demonstrated that inter-individual variations in measles vaccine virus-induced humoral and cellular responses are significantly associated with polymorphisms in immune response genes and, together with hla alleles, explain 30% of the inter-individual variability in humoral response [5, [34] [35] [36] . these findings, which illustrated the importance of key hla alleles in the adaptive humoral immune response to measles vaccine, led to the identification of naturally processed and presented measles-derived peptides isolated from specific hla polymorphisms associated with vaccine non-and hyper-response [37, 38] . these peptides containing specific components (adjuvants and biodegradable nanoparticles) are now being utilized in a reverse-engineering strategy to develop peptide-based candidate measles vaccines. likewise, homan et al. have attributed diminished protection to differential hla presentation of t and b cell epitopes between vaccine and wild type strains of mumps virus [39] . this diminished efficacy could theoretically be overcome by incorporating defined critical immunogenic peptides into an improved vaccine. tlr genes represent an important link between the innate and the adaptive immune system [40, 41] . as an example, we have demonstrated that measles vaccine-induced humoral responses are significantly associated with coding polymorphisms in the tlr2 (rs3804100) and tlr4 (rs5030710) genes [42] . for the rubella vaccine and tlr3 gene, a tlr3 gene snp rs5743305 was associated with rubella-specific gm-csf production [43] . our recent mumps vaccine study has identified and replicated tlr4 snps associated with a 45% decrease in antibody titer, and a tlr5 snp associated with a 64% increase in t cell response (unpublished data). these data strongly suggest that robust tlr activation by measles, mumps, and rubella viruses is crucial for optimal vaccine response. supporting these findings is a study demonstrating that an inactivated mumps vaccine containing a protollin-based tlr2/4 adjuvant is highly immunogenic in a mouse model; it led to superior total igg levels, higher neutralizing antibody titers, greater mucosal iga production, and enhanced th1/th2 cytokine secretion [44] . one potential application of this finding is to identify the specific and critical interactions between tlrs (and other genes) and virus, leading to advances in our knowledge of the precise mechanisms driving immunity to mmr vaccine. significant sex differences in humoral and cellular immune responses to vaccines are apparent [45, 46] . additionally, local and systemic adverse rates are generally higher in females versus males. protective antibody responses are significantly higher in females than males after vaccination against influenza, yellow fever, measles, mumps, rubella, hepatitis a and b, herpes simplex (hsv) 2, rabies, smallpox, and dengue viruses [47] [48] [49] [50] [51] [52] [53] [54] [55] . sex-based differences in humoral immune responses are observed through various age groups [47] [48] [49] [50] [52] [53] [54] [55] [56] [57] , suggesting that sex steroid hormones are not the singular mediators of sex differences in humoral immune responses to vaccines [45, 58] . this suggests that genetic, or other, factors may be an important driver of sex-related differences in humoral immune response [59] . despite significant evidence of immune response differences between the sexes, for the most part, vaccine studies have not examined and analyzed immune response outcomes by sex [60, 61] . in fact, little information is known about potential mechanisms for sex-based effects, which should be a priority for vaccine research studies. discovery of specific factors involved in sex-based differences in immune response may allow the identification of new correlates of vaccine immunogenicity. in a cohort of 556 older (ages 50-64) and 558 younger (ages 18-49) previously vaccinated individuals, the seasonal trivalent influenza vaccine induced >1.5-fold higher a/h3n2-specific hai antibody titers in women than men across both age groups [47] . similarly, a study of standard seasonal influenza vaccine and high-dose influenza vaccine responses in a sex-balanced cohort of 414 elderly subjects (ages 65-95) demonstrated significantly higher rates of seroconversion in females than in males [48] ; however, no significant differences in antibody measures were found between males and females after seasonal influenza vaccination in another cohort of 158 older adults (ages 50-74) [62] . a study by furman et al. examining gene expression, serum cytokines/ chemokines, cell subsets, and phosphorylation events found several serum markers (lept, il-1ra, crp, gm-csf, and il-5) to be more highly expressed in females than males after influenza vaccine [51] . this same report used a systems biology approach to identify a gene cluster involved in lipid biosynthesis that is regulated by testosterone and significantly correlated with poor humoral responses following influenza vaccination in men [51] . these data suggest that this gene cluster (e.g., genes involved in lipid metabolism) could be an important driver of sex-related differences in humoral immune response. this collective knowledge could substantially assist future personalized vaccine development efforts through the generation of new knowledge and the identification of targets and biomarkers that predict vaccine responses in specific populations (e.g., females vs. males; young vs. old; obese vs. lean). further research is needed to clarify the effects of sex on immune response. identification of molecular immune signatures of sex differences in innate and adaptive immune responses to vaccines may provide evidence necessary for additional efforts in designing personalized vaccination and vaccinomics approaches (i.e., in which males and females might be vaccinated differently using different doses or different vaccines) to provide equal protection while reducing side effects [46, 63, 64] . a significant global public health issue is the aging of the population. as individuals age, immunosenescence develops, leading to poorer immune responses to vaccines. immunosenescence is an age-related dysregulation of the immune system due to ageassociated changes in innate and adaptive immune system components, which leads to impaired immunity and protection following immunization or infection [65] [66] [67] . published data reveal that innate and adaptive immunity is decreased with age, but the systems-level mechanisms for these findings are unclear [66, 68] , particularly in regard to influenza and other viral vaccine responses where the morbidity, mortality, and associated healthcare costs are greater in older individuals [11] . major signs of innate immune dysfunction commonly observed in the elderly include, but are not limited to, altered cytokine secretion; decreased nk cell activity; reduced tlr expression; and a chronic inflammatory state (elevated levels of il-1b, mcp-1, tnf-a, and serum il-6) known as ''inflamm-aging" [8, [69] [70] [71] . age-related humoral immune dysfunction, for example, might be overcome through optimal stimulation of innate and/or th cell-specific genes, which may be different in males and females. for example, adjuvanted zoster subunit vaccine (hz/su) reduced the risks of herpes zoster, and postherpetic neuralgia in immunocompetent persons 70 years of age and older [72] . this hz/su vaccine contains varicella zoster virus glycoprotein e and a novel as01 b adjuvant system aimed to improve and preserve with age zoster-specific cd4+ t cell responses [73] . a tlr4 agonist gla-se (glucopyranosyl lipid adjuvant formulated in a stable emulsion) has been shown to enhance th1 responses to influenza vaccine in older adults [74] , suggesting a potential mechanism for targeting innate receptor agonists (e.g., tlrs) that enhance innate immune responses against influenza. given the substantially diminished efficacy of influenza and other vaccines with age and the importance of developing improved vaccines [75] , data from vaccinomics studies could be used to inform directed and rational development of next-generation influenza vaccines-potentially circumventing immunosenescence-related factors. systems biology approaches provide a unique opportunity to identify biomarkers likely to be involved in immune responses to vaccination [1] [2] [3] [4] 8, 76, 77] . fourati et al. applied a systems vaccinology approach to examine gene signatures and molecular pathways of age-related hyporesponse to hepatitis b vaccine (hbv) in naïve older adults [78] . they observed the b cell signaling pathway (and higher memory b cell frequencies) and inflammatory pathway (and increased frequencies of activated pro-inflammatory innate cells) were strongly correlated with higher and low antibody responses to hbv, respectively. this signature, including serum cytokine profiling and flow cytometric correlates of response, predicted the antibody response to hbv with up to 65% accuracy [78] . this study demonstrates that a systems biology approach can be used to predict age-related immune response to vaccination. obesity is another major public global health concern. in the us, 68% of adults and nearly 32% of children and adolescents are now overweight or obese [79] . weight gains across all countries have been demonstrated to be associated with increasing socioeconomic status. obesity has been shown to be a predictor of impaired immunogenicity (e.g., decreased antibody response) to hepatitis b, tetanus toxoid, rabies, and influenza vaccines [80] [81] [82] [83] , and as such can be considered a marker, or state, of immunosuppression at its extremes. these data suggest that obesity is correlated with poorer vaccine-induced immune responses in humans, and further research is required to understand the immune mechanisms that are altered in obesity. as individuals age, their circulating leptin levels rise with a concomitant reduction in leptin signaling; this results in leptin resistance, which is a finding associated with obesity [84] . leptin resistance has been shown to adversely affect the immune response in obese subjects, including responses to influenza virus [85, 86] . for example, obese individuals demonstrate decreased activation of influenza-specific cd8+ t cells compared to healthyweight persons, including decreased production of ifn-c and granzyme b, suggesting that influenza vaccination may not be as effective in the obese population as in healthy-weight individuals [87] . given only moderate seroprotection of influenza and other vaccines in obese older adults [83] , and the importance of developing improved influenza vaccines [75] , systems biology studies designed to identify the mechanisms for improved immune response are needed. in fact, data from vaccine studies could be used to inform directed and rational development of personalized vaccines that optimally stimulate innate and adaptive immune responses in males and females and overcome immune deficiencies induced by obesity [88] . careful vaccine studies comparing lean and obese persons could provide foundational data used to improve vaccine-induced protection in the obese, a subpopulation with an elevated risk for serious vaccine-preventable illnesses and suboptimal vaccine-induced protective responses [10] . adversomics utilizes tools-much like those used in vaccinomics-to identify, characterize, and predict adverse, or maladaptive, immune responses to vaccines [6, 89, 90] . the promise of adversomics would be to develop or identify either predictors or immune signatures of maladaptive immune responses that lead to harm rather than benefit, and to better understand the generation and mechanisms of such maladaptive immune responses. we have asked the question, as have other scientists, ''does it make sense in the 21st century to give the same vaccine, dose, and at the same frequency to everyone, regardless of age, weight, gender, race, genotype, and medical condition?" for example, we give adult males and females the same dose, and the same number of doses of vaccines, ignoring the findings that females nearly always have superior humoral immune responses to males for all vaccines studied, and yet experience significantly more side effects-more adverse events, of greater duration, and of higher intensity [47, 55, 60] . while the field is young in implementation, research has already revealed associations between specific genes or snps and adverse immune outcomes. for example, associations between cytokine gene expression and fever after smallpox vaccine have been identified [91] . other studies have demonstrated correlations between smallpox vaccine-induced fevers and il1a and il18 snps [92] . other smallpox vaccine-induced adverse events such as fever, rash, and enlarged lymph nodes have been significantly associated with mthfr, irf1, and il4 snps haplotypes [93] . while smallpox vaccine is not used in the general population, such studies stand as examples of the usefulness of vaccinomic approaches. finally, other recent studies have identified generic fever gene networks (tnfa) after vaccine administration [94] , and relationships between mmr vaccine administration and snps in ifi44l, cd46, scn1a, 2a, and tmem16 (ano3) genes [95] . despite the tremendous success of vaccines, vaccinologists face several current challenges, including difficulty in developing vaccines for hypervariable viruses (hiv, rhinovirus, hepatitis c virus, coronavirus) and complex pathogens (malaria, mycobacterium tuberculosis); newly emerging pathogens, such as zika virus (zikv); complications imposed by aging and immunosenescent populations; inadequate understanding of the neonatal and newborn immune systems; increasingly immune deficient or immunocompromised populations due to hiv, cancer, or medications; sexbased differences in vaccine response and adverse-event rates; enhanced scrutiny of vaccine safety; and as noted global increases in age and weight. in addition, vocal and active anti-vaccine groups whose messages are not easily countered by facts or scientific studies have materially and detrimentally affected vaccine coverage rates [96] [97] [98] . vaccinomic approaches can be utilized to better understand these issues; this information can then be used to inform new approaches, new understandings, and new vaccine candidates. just as new technologies have created exciting new opportunities in personalized medicine, they have brought with them novel challenges in addition to those mentioned above. in order for the full potential of personalized vaccines to be achieved, we must overcome additional challenges, such as the need for the following: larger genotype:phenotype datasets (often in the many thousands to ten thousands) integrating increasingly diverse high-throughput, highdimensional data types biomarkers that can reliably distinguish which product patients receive based on the likelihood of their response or an adverse side effect vaccines with different mechanisms of action may require a move away from humoral correlates of protection for licensure; in this regard, correlates of protection based on cellular immune outcomes are likely to play an important role in future vaccines more sophisticated biostatistical and bioinformatics approaches that can identify patterns and causative networks within terabyte levels of extremely high dimensional data types from the economic side: methods of technology transfer and funding mechanisms to move novel vaccines developed through vaccinomic approaches into low and middle-income countries who often most need specific vaccines (malaria, others) we have seen the shift from ''vaccinology 1.0," which is the empirical ''isolate-inactivate-inject" paradigm, to ''vaccinology 2.0"-the use of recombinant technology and novel adjuvants. however, even this paradigm is limited by our incomplete mechanistic understanding of adjuvants and innate immunity. as we adopt approaches such as those listed above, we envision a movement of the field into an era of ''vaccinology 3.0," during which we expect to see the use of vaccinomics and systems-level approaches to develop new vaccines; innovative vaccine-antigen packaging methods; and adjuvant development targeted at the innate response pathways best suited for a given pathogen. a common reaction to this paradigm of personalized vaccinology is questioning cost and economics. at one level, such considerations are simply ''too soon" in the development of the science to effectively answer. however, like progress being made in individualized medicine, it is likely that being able to provide the right vaccine to the right patient-for the right reasons and at the right dose-will lead to improved medical outcomes and reduced costs at the population level. personalized vaccinology is the goal of applying the concept of personalized medicine to vaccines. rapid strides in omics technologies and foundational work applying systems biology, computational immunology and reverse vaccinology have facilitated modern approaches to vaccine design and development enabling us to create vaccine formulations for new and re-emerging pathogens. egg-based influenza vaccines take >6 months to create. the recent licensure of cell culture-based influenza vaccines demonstrate that rapid, scalable processes can now be implemented in order to create vaccine against emerging influenza strains (e.g., h1n1, h5n1, h7n9, h9n2, h7n8) within weeks [99] and can be safely administered to individuals with egg allergies [100] . the ebola outbreak in liberia, sierra leone, and guinea in 2015 provides an example of the need to rapidly develop vaccine candidates [101] . dna vaccines, virus-like particle vaccines, and replicating/nonreplicating viral vector vaccines have all been created and tested. among the most promising are a replication-competent, recombinant vesicular stomatitis virus vector expressing the glycoprotein of ebola zaire (rvsv-zebov), [102] a variety of adenovirusvectored vaccines expressing ebola glycoprotein, [103, 104] a modified vaccinia virus ankara-based vaccine encoding the ebola zaire glycoprotein (mva-bn-filo), [105, 106] and dna-based vaccinesone expressing glycoproteins from both zaire and sudan, and the other expressing the marburg glycoprotein [107] . although the rvsv-based vaccine elicits high titers of neutralizing ab, it is contraindicated in children and those with compromised immune systems. viral vector vaccines present the problem of developing robust immunity to the vector as well as the target immunogen, limiting their usefulness to a single vaccination. the availability of vaccines in multiple vector backbones opens up the possibilities for prime-boost vaccination strategies for ebola, similar to those that have been applied to hiv, malaria, and tuberculosis [108] [109] [110] [111] . in this regard, a prime-boost regimen using the mva-based vaccine as the booster vaccination has shown considerable promise [101] . another example of modern vaccine development being applied to a new pathogen can be seen with the response to zika virus. a purified, formalin-inactivated vaccine (zikv piv) has been developed by the walter reed army institute of research (wrair) [112] and is being evaluated in several clinical trials (nct02963909, nct02952833, nct02937233), while other inactivated vaccines are in preclinical development [113] . two variants of a plasmid dna vaccine containing the prm-env proteins have been developed by niaid and one of the formulations is currently in a phase i clinical trial (nct02840487) [114] . inovio pharmaceuticals developed their own plasmid dna vaccine (also expressing prm-env), which is currently in two clinical trials (nct02809443, nct02887482). rna-based vaccines [115] and a variety of subunit and viral vector-based vaccines are also in development [113, 116, 117] . dna and rna-based vaccines can be rapidly made at minimal costs compared to other formulations and are fairly stable, without the cold-chain requirements of live virus-based vaccines. subunit vaccines are typically safer than whole virus-based products, which represents an active area of investigation not only for pathogens with no existing vaccines, but also for improving on established vaccines. our group and others have identified pathogen-derived epitopes as preliminary steps in the development of safe, stable, and effective peptide-and protein-based vaccines for smallpox, influenza, measles, tuberculosis, staphylococcus, and myriad other viral and bacterial pathogens [38, [118] [119] [120] [121] [122] . parallel efforts by different groups to create new vaccines result in a spectrum of potential products that can be uniquely tailored to specific population groups. live viral vaccines rapidly inducing robust immunity can be used in healthy individuals where time is of the essence (e.g., in outbreak scenarios), while inactivated or subunit vaccines can be used in vulnerable populations such as pregnant women or those with immunocompromising conditions, or in young children where the presence of maternal antibody interferes with whole virus vaccines. vaccines based on different viral vector backbones can be combined into effective primeboost regimens. vaccines with specific adjuvants may be most appropriate for the elderly in order to overcome immunosenescence, or in the very young in order to compensate for immune system immaturity. we, along with increasing numbers of other scientists, believe that personalized vaccinology will revolutionize the practice of vaccinology to the benefit of human health. as part of the development of this field of science, vaccinomics and adversomics will allow us to develop molecular immune signatures of adaptive and maladaptive immune responses to vaccines, develop early biomarkers of vaccine response in vaccine trials, identify who should get what vaccine and at what dose, and increase safety and public confidence in vaccines by reducing the likelihood of serious adverse events related to vaccines. in many ways, however, personalized vaccinology is most challenged by the difficulty in moving the field away from the post-wwii population-level paradigm of ''one dose of every vaccine for everyone," toward an individualized or personalized approach based on the unique factors relevant to a given individual. in his book, the structure of scientific revolutions [123] , thomas kuhn recognized that ''we wrongly believe scientific progress is a process of linear accretion of knowledge, that science is predicated on the belief that the scientific community understands what the world is like, and that we suppress or resist 'fundamental novelties' because they are seen as subversive to our firmly held beliefs of what the world is like." later in his book, he suggests that ''new advances always have and always will reveal that science and medicine includes bodies of belief incompatible with beliefs we hold today, and that advancements come when we reject a time-honored scientific theory in favor of another incompatible with it." these cognitive biases have, in our opinion, been manifest in our discussions with scientific colleagues as we developed this field of science. schopenhaur, the german philosopher, suggested that new discoveries are at first ridiculed, then opposed, and finally accepted as self-evident. vaccinomics and adversomics appear to be moving from the ridiculed and opposed steps, and into the not-yet quite self-evident phase of the continuum. part of the challenge is that often the concept of personalized vaccinology suggests to the reader that a unique vaccine will be developed for each individual. while that is one tactic being used in the cancer-vaccine field, it is neither necessary nor practical for the prevention of infectious diseases. rather, the personalized vaccinology approach would suggest the development of specific vaccines based on factors that relate to overcoming the potential for poor immunogenicity and the potential for adverse events. an excellent example is influenza vaccines. a mere decade or so ago, only a trivalent injectable influenza vaccine was available. quadrivalent vaccines were unavailable. for with one exception, everyone received the same vaccine and dose, regardless of age, weight, immunosuppression state, etc. at the current time in the us, multiple influenza vaccines are available so that the right vaccine, for the right patient, can be given at the right time. for example, laiv (live attenuated influenza vaccine) can be used in younger subjects or the needle-phobic. high-dose or mf59-adjuvanted vaccines can be chosen for the elderly. recombinant vaccines can be chosen for those with egg allergy, and so on. this is the approach that should be taken with all vaccines. in some cases it may mean merely adjusting the dose based on weight, gender, or age. in other cases it may mean utilizing an adjuvanted or non-adjuvanted vaccine based on immune status. other examples include the recently licensed mf59 adjuvanted influenza vaccine (fluad ò ), which has demonstrably higher immunogenicity and efficacy than its nonadjuvanted counterparts, [124] [125] [126] or the highly effective as01adjuvanted zoster glycoprotein e vaccine, which does not contain live virus and may be more broadly suitable for administration to older individuals [72, 73] . thus, the movement toward a new paradigm of vaccine practice, based on a personalized approach, is occurring in the 21st century based on new scientific knowledge, market demand, safety considerations, immunogenicity concerns, public health trends (age, obesity, other), and the simultaneous pull of individualized medicine in other medical arenas. the net result is likely to be higher vaccine coverage rates, increased public confidence in vaccines, improved immunogenicity and adverse event rates, and a reduction or elimination in the morbidity and mortality related to vaccine-preventable diseases. as a result, we anticipate a new era of personalized ''predictive vaccinology," whereby we abandon a ''one size and dose fits all vaccine approach" in order to design and develop new vaccines, and acquire the ability to make the following predictions for each individual: whether to give a vaccine based on likelihood of response (and perhaps need); the likelihood of a significant adverse event to a vaccine; and the number of doses likely to be needed to induce a protective response to a vaccine [63] . current vaccine development is largely empirical. vaccines are tested by trial and error, are mass produced, and given to the entire population using the same antigen dose, route of administration, number of vaccinations, and at the same age. in contrast, the new vaccine-development paradigm begins with the ''discovery" of new knowledge by integrating unbiased, comprehensive analysis of the genome, transcriptome, proteome, metabolome, microbiome, and immunome-along with the assessment of multiple measures of immune function-in order to under-stand and evaluate perturbations of the immune system. findings are then ''validated" in replication cohorts or additional model systems. the new knowledge is then ''applied" to the creation of new vaccine formulations that can undergo additional testing to start a new round of ''discovery," or can move into clinical trials in order to develop vaccine products engineered to elicit (or avoid) specific effects on the immune system. each product is tailored to specific subgroups such that robust, protective immunity can be elicited in the old and young, lean and obese, or male and female, while avoiding inappropriate immune responses due to genetics, metabolism, race, gender, malnutrition, immunosuppression, and other host factors or underlying conditions. r01ai033144, and contract no. hhsn266200400025c (n01ai40065). the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. the authors would like to acknowledge the contributions of the centers for disease control and prevention (cdc), which provides financial support to the world health organization initiative for vaccine research (u50 ck000431). dr. poland is the chair of a safety evaluation committee for novel investigational vaccine trials being conducted by merck research laboratories. dr. poland offers consultative advice on vaccine development to merck & co. inc., avianax, dynavax, novartis vaccines and therapeutics, emergent biosolutions, adjuvance, seqirus, and protein sciences. drs. poland and ovsyannikova hold three patents related to vaccinia and measles peptide research. dr. kennedy has received funding from merck research laboratories to study waning immunity to mumps vaccine. these activities have been reviewed by the mayo clinic conflict of interest review board and are conducted in compliance with mayo clinic conflict of interest policies. dissecting polyclonal vaccine-induced humoral immunity against hiv using systems serology cytometry by time-offlight shows combinatorial cytokine expression and virus-specific cell niches within a continuum of cd8 + t cell phenotypes highresolution myogenic lineage mapping by single-cell mass cytometry metabolic phenotypes of response to vaccination in humans heterogeneity in vaccine immune response: the role of immunogenetics and the emerging field of vaccinomics vaccinomics, adversomics, and the immune response network theory: individualized vaccinology in the 21st century pharmacology, vaccinomics, and 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vaccine (tiv) in the elderly research reported in this publication was supported by the national institute of allergy and infectious diseases of the national institutes of health under award number u01ai089859, r37ai048793, key: cord-007876-s5y6gyut authors: nan title: selected epidemics & emerging pathogens – respiratory illnesses – an overview date: 2017-09-12 journal: dis mon doi: 10.1016/j.disamonth.2017.03.016 sha: doc_id: 7876 cord_uid: s5y6gyut nan as discussed earlier, infections remain a leading cause of death worldwide, as well as in the united states [1] . of concern, some of these infections are associated with vaccine preventable pathogens, such as s. pneumoniae, and influenza [1,2], the morbidity and mortality of which could thus be significantly reduced or prevented were immunization rates higher, in both the developed and developing countries. in addition to well studied pathogens, novel and more virulent ones continue to be identified, that are capable of causing human and animal illness. these include various avian influenza strains [2] [3] [4] [5] [6] , metapneumovirus [7] , multidrug resistant tuberculosis [8] , more aggressive coronaviruses [9] [10] [11] [12] , and others. respiratory syncytial virus (rsv) is an underappreciated respiratory pathogen. while it is well known as the most common etiology of lower respiratory tract infections in children, resulting in nearly 2.1 million outpatient visits among those less than 5 years of age, and over 57,000 hospitalizations in the same cohort, it is estimated that rsv causes 177,000 hospitalizations, and 14,000 deaths among adults over 65 years of age [13] [14] [15] . rsv clinically is variable in presentation, referable to age, patient health and comorbidities. young healthy individuals typically experience mild, cold-like symptoms, with recovery expected in one to two weeks. infants present with bronchiolitis, adults mild upper respiratory infections (uri). severe pneumonia may occur, especially the elderly who have comorbidities, and/or impaired cellular immunity. aerosolized ribavirin can be used for rsv in infants. risk benefit must be balanced when considering the use of ribavirin in adults. new pathogens are being discoveredsome through unknown means, and others through natural adaptation. globalization, population shifts and the changing ecology, including encroachment of previously unexplored regions has altered the longstanding epidemiology of infectious diseasescausing spread where once continents and oceans contained the pathogen. influenza viruses are ubiquitous in the animal population, with a wide array of natural hosts, and possess the capacity through the phenomenon of reassortment to infect an expanded range of hosts, including humans, as well as acquire greater pathogenicity [16] [17] [18] [19] [20] [21] [22] . pandemic influenza viruses can thus emerge [16, [18] [19] [20] [21] [22] . it has long been recognized that influenza viruses exchange genetic material, (reassortment) either emerging as a new strain, as we continue to see with h5n1 [16, [23] [24] [25] , h1n1 [26] [27] [28] , and now the latest h7n9 [3, 4, 29, 30] . but this likely holds true for other viruses, as recently demonstrated with a novel coronavirus, most recently referred to as middle east respiratory syndrome (mers cov) [11, 12] . given the proximity of people to animals, through occupation and avocation, the human-animal interface becomes a significant risk for human illness from influenza viruses, as has been seen in several outbreaks, including h5n1 since 1997 [16] . there are a multitude of respiratory pathogens worth describing. however, with the recent emergence of yet another highly pathogenic avian influenza -h7n9 [29, 30] and novel coronavirus (mers cov) which appears more deadly than sars cov [11, 12] , it seems worthwhile to discuss these infectious agents. it is hoped that lessons learned from these latest outbreaks can be applied towards preparedness against a wide range of pulmonary threats, and enhance our infection control capacity. regardless which emerging pathogen we discuss -avian influenza h5n1, h7n9, mers cov, swine flu or other viruses, it is important to recognize that preparedness efforts as a response to a potential pandemic caused by swine or avian influenza, sars or mers, can also enhance awareness, promote vaccine use, advances in diagnostic and treatment capabilities toward other significant infectious disease worldwide. controlling infectious diseases can be challenging given the large number of disease causing pathogens, their capacity for adaptation to environmental changes and antimicrobial therapies, and opportunities for spread. respiratory contagions remain an issue of enormous concern in the containment of infections, especially with overcrowding and other population, social, and travel determinants. the spread of pulmonary infections occurs readily from both the upper and lower respiratory tracts (figure 1 ) how respiratory illnesses spread [6, [26] [27] [28] droplets proximity (less than 6 feet social distancing especially) environment (overcrowding for example) fomites mucosa prior immunity (or lack thereof) poor hygiene inherent transmissibility of pathogen (ro) in the next section we will discuss influenza viruses, including the newest pathogenic one h7n9, and coronaviruses. although traditionally they caused mild respiratory illness, from 2003 there are more highly pathogenic cov illness causing ones, as seen with sars coronavirus, and mers coronavirus. the term "influenza" describes an acute viral disease of the respiratory tract often referred to colloquially as "the flu." it is caused by viruses belonging to the orthomyxovirus family, which includes the genera of influenza virus a, b, and c. these are defined by the antigenicity of their nucleocapsid and matrix proteins ( figure 2 ) [1-8]. influenza a viruses are negative sense, single-stranded rna viruses, with an 8 segment genome that encodes for 10 proteins [3, 5, 9] , and are usually associated with more severe human illness, epidemics, and pandemics. these viruses are further classified or subtype based upon two surface proteinshaemagglutinin (h), which attaches the viral particle to the host cell allowing entry, and neuraminidase (n) which facilitates the spread of progeny virus. the neuraminidase protein is the target for the class of antiviral therapy referred to as neuraminidase inhibitors [1, 2, 4, 6, 7, [9] [10] [11] [12] [13] [14] . there are 16 h proteins, and 9 n subtype proteins the combination of which make up all the known influenza a subtypes [11, 12] . important to recognize from the perspective of pathogenicity, and potential contagion, even pandemic threats are the concepts of "antigenic drift" and "antigenic shift." "antigenic drift" refers to the various mutations and changes in surface antigenicity of surface proteins as a response to host immunity. h9n2 viruses that were isolated in chickens from china were noted to undergo antigenic drift, in order to evolve into distinct antigenic groups [15] [16] [17] [18] [19] [20] [21] this may have resulting in some of the immunization failure noted during china's long term vaccination program for chicken farms [15, 20] . "antigenic shift" is an event that can lead to the creation of a novel virus against which humans have little or no immunity. recognizing influenza has a segmented genome, shuffling of gene segments can occur if two different subtypes of influenza a virus coinfect the same cell. conditions that favor the emergence of such "shifts" have been surmised to involve humans living in proximity to farm animals (humananimal interface), especially poultry and pigs [15, 19] . of note, swine are susceptible to infection from both avian and mammalian host preference influenza viruses [15, 19] . in southern china dogs were noted to have serology positive avian origin h9n2 influenza. ferrets and other mammals have been noted to have avian infection as well. such an expanded host range reveals a potential threat whereby a pathogenic influenza virus with low human to human transmission can intermingle and share genetic material with an influenza virus possessing high capability for human spread. to date, h9n2 is endemic in eurasia, and has caused significant morbidity and mortality in poultry, as well as lost egg production [15] . of note, co-infection with other pathogens is possible in the animal, as well as human host range. for example, h9n2 has been shown to make poultry more susceptible to secondary infection, especially e coli, with a resulting fatality rate estimated at 10% [15] . the expansion of host range is not uncommon in influenza viruses. for example, if an h3n2 and an h5n1 virus co-infect a human or pig, it is possible a new virus such as h5n2 could emerge; a hybrid could combine the high virulence of h5n1 with the efficiency of human to human transmission found in the "parent" human virus. [13, 21] . according to the world health organization (who) influenza at the human-animal interface report for 20 december 2016 -16 january 2017 [22] there are new human influenza infections reported. these include influenza a (h7n2), a (h7n9), and a (h9n2). h9n2 is a low pathogenicity virus, with wide distribution, and seropositivity is not uncommon in occupationally exposed poultry workers [15] [16] [17] [18] [19] [20] [21] 23] . because it circulates at the human avian interface, the potential for new reassortants, not the least of which would be a new epidemic or pandemic subtype underscores the importance of better infection control, and human protective measures, including occupational hygiene. in rural areas, domiciles shared with flocks, undercooking of birds, eating raw eggs and poultry blood are also risk factors. during this period, china also reported to who 100 laboratory confirmed human cases of h7n9, the majority from mainland china [22] . to date 918 laboratory-confirmed human cases of h7n9 were reported, with at least 359 deaths. antigenic and genetic characterization of h9n2 swine influenza viruses in china world health organization influenza at the human animal interface summary 16 the avian influenza h9n2 at avian human interface: a possible risk for future pandemics lyme disease -time for a new approach avian influenza a (h5n1) infection in humans radiological findings of chest in patients with h7n9 avian influenza from a hospital human infection with a novel avian-origin influenza a (h7n9) virus avian influenza a (h7n9) virus serosurvey of human metapneumoviruses in croatia respiratory syncytial virus infection in elderly and high risk adults influenza: lessons from past pandemics, warnings from current incidents avian influenza: critical considerations for primary care physician cdc statistics on leading causes of death malaria in the post genome era the changing epidemiology of malaria elimination: new strategies for new challenges replication cycle of chikungunya: a re-emerging arbovirus cdc yellow book travel health information 〈https knowledge and use of preventive measures for chikungunya virus among visitors virgin island national park pre-travel health advice-seeking behavior among us international travelers departing from boston logan international airport travel health knowledge, attitudes, and practices among united states travelers risk factors for infection in international travelers: an analysis of travel-related notifiable communicable diseases leading causes of death -number of deaths for leading causes of death avian influenza: critical considerations for primary care physician influenza h7n9 and h9n2 viruses: coexistence in poultry linked to human h7n9 infection and genome characteristics pandemic and avian influenza a viruses in humans: epidemiology, virology, clinical characteristics, and treatment strategy h9n2 influenza virus in china: a cause of concern avian influenza a (h7n9) virus serosurvey of human metapneumovirus infection in croatia multidrug-resistant tuberculosis: a menace that threatens to destabilize tuberculosis control the biology of coronaviruses coronaviruses post sars: update on replication and pathogenesis hadi mohamed rae. outbreak of middle east respiratory syndrome coronavirus (mers cov) in saudi arabia: a retrospective study severe respiratory illness caused by a novel coronavirus in a patient transferred to the united kingdom from the middle east respiratory syncytial virus infection in elderly and high risk adults national foundation for infectious diseases respiratory syncytial virus (rsv) infection rsv pneumonia, a community-acquired infection in adults avian influenza: the next pandemic influenza: lessons from past pandemics, warnings from current incidents characterization of the reconstructed 1918 spanish influenza pandemic virus changing concepts of airborne infection of acute contagion diseases; a reconsideration of classic epidemiologic theories probable person to person transmission of avian influenza a h5n1 avian influenza information for physicians proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells characterization of the reconstructed 1918 spanish influenza pandemic virus avian influenza virus h5n1: a review of its history and information regarding its potential to cause the next pandemic differential use of antivirals for treatment of patients with influenza a (h1n1) pdm09 in germany. influenza other respir viruses asthma and severity of 21009 novel h1n1 influenza: a population -based case-control study pediatric emergency research networks (pern) h1n1 working group. predictors of severe h1n1 infection in children presenting within pediatric emergency research networks (pern): retrospective case-control study the genesis and source for the h7n9 influenza viruses causing human infections in china human infection with a novel avian origin influenza a (hh7n9) virus key: cord-277217-jh4qmoso authors: ortiz, justin r.; jacob, shevin t.; eoin west, t. title: clinical care for severe influenza and other severe illness in resource‐limited settings: the need for evidence and guidelines date: 2013-08-27 journal: influenza and other respiratory viruses doi: 10.1111/irv.12086 sha: doc_id: 277217 cord_uid: jh4qmoso the 2009 influenza a (h1n1) pandemic highlighted the importance of quality hospital care of the severely ill, yet there is evidence that the impact of the 2009 pandemic was highest in low‐ and middle‐income countries with fewer resources. recent data indicate that death and suffering from seasonal influenza and severe illness in general are increased in resource‐limited settings. however, there are limited clinical data and guidelines for the management of influenza and other severe illness in these settings. life‐saving supportive care through syndromic case management is used successfully in high‐resource intensive care units and in global programs such as the integrated management of childhood illness (imci). while there are a variety of challenges to the management of the severely ill in resource‐limited settings, several new international initiatives have begun to develop syndromic management strategies for these environments, including the world health organization's integrated management of adult and adolescent illness program. these standardized clinical guidelines emphasize syndromic case management and do not require high‐resource intensive care units. these efforts must be enhanced by quality clinical research to provide missing evidence and to refine recommendations, which must be carefully integrated into existing healthcare systems. realizing a sustainable, global impact on death and suffering due to severe influenza and other severe illness necessitates an ongoing and concerted international effort to iteratively generate, implement, and evaluate best‐practice management guidelines for use in resource‐limited settings. the 2009 influenza a (h1n1) pandemic highlighted the essential role of hospital care of the severely ill in the response to public health emergencies. early reports from mexico and canada of intensive care units (icus) filled to capacity with patients with severe respiratory infections helped to calibrate the early, aggressive global public health response. [1] [2] [3] the critical care medical community and public health reacted with unprecedented coordination to describe severe disease, to disseminate data on the epidemiology and care of pandemic patients, and to convey the impact of the outbreak on health systems. [4] [5] [6] [7] [8] while the worst-case scenario of a 1918-scale pandemic was avoided, there were reports of patients with severe disease stressing critical care services in communities throughout the world. 2, [9] [10] [11] these reports show that severely ill patients can divert resources and impact the balance of care delivery even in hospitals where the overall capacity is not exceeded. this problem is particularly acute in resource-limited settings where there is decreased capacity to manage severe illness. while building resource-intensive and highly technological icus is not feasible for many parts of the world, much can be done to improve care for severely ill patients in more austere environments. in this review, we discuss the inter-related issues of pandemic influenza, severe seasonal influenza, and severe illness more generally. given the burden of severe illness in resource-limited settings, it is vital to improve capacity to care for severely ill individuals in these environments. estimates of 2009 h1n1 pandemic influenza morbidity and mortality differ considerably among countries. 1, [12] [13] [14] in highresource countries, large epidemiologic studies have shown that the overall incidence of pandemic influenza requiring hospitalization was comparable to interpandemic seasons, [15] [16] [17] [18] but there were important demographic groups that experienced substantial increases in severe influenza disease. these groups included young adults, pregnant women, obese persons, and indigenous persons. 13 in low-and middleincome countries, where laboratory testing for clinical care or for public health surveillance was limited, the full impact of the 2009 pandemic has been harder to measure. [19] [20] [21] however, modeling studies estimate that there were disproportionately increased pandemic deaths in resource-limited regions. 22 compared with the americas, the risk of pandemic respiratory and circulatory mortality was 1á4 times higher in southeast asia and 2á4 times higher in africa. 22 these data underscore the importance of improving clinical management of influenza disease in resource-limited settings. a 2010 who consultation on the clinical care of pandemic influenza noted that there were limited clinical data and guidelines for the management of severe manifestations of viral infection in resource-limited settings. 10 in light of the apparent paucity of data about supportive management of severe influenza-specific illness, the who public health research agenda for influenza commissioned the international respiratory and severe illness center (intersect) at the university of washington to perform a systematic review on non-antiviral, supportive management of persons with severe 2009 pandemic influenza a (h1n1). a 2012 update of this review, limited to randomized controlled trials, controlled prospective cohort studies, and systematic reviews/meta-analyses found only seven pertinent studies, mostly of adults. 23 one study found benefit of convalescent plasma infusion for severe illness, 24 three studies found no benefit of corticosteroids for severe respiratory disease, [25] [26] [27] and three studies had mixed results on the benefit of extracorporeal lung support for severe respiratory disease. [28] [29] [30] no study identifying a therapeutic benefit from an intervention was applicable to healthcare delivery in resourcelimited settings. while 2009 pandemic influenza gained attention worldwide, the burden of seasonal influenza is perhaps less widely recognized. yet, the contribution of seasonal influenza to severe illness is substantial, especially in resource-limited settings. a variety of data support this assertion. among children younger than 5 years of age in sub-saharan africa and south asia, influenzainfection and co-infections are commonly identified during hospitalizations for acute lower respiratory infections (alri). [31] [32] [33] [34] the only systematic review and metaanalysis that estimates the global burden of severe influenza disease was recently published. 35 the study, which analyzes only early childhood disease, reports that 99% of influenza-related cases of severe alri among children younger than 5 years occur in low-and middle-income countries, and that 13% of all alri in this age group are associated with influenza virus infection. the study dispels the myth that influenza is only a problem in temperate regions. the authors calculated that the incidence rate of severe influenza-associated alri in early childhood is similar in developing countries as compared with industrialized, temperate countries (both are around 1-2 episodes per 1 000 child-years). 35 this pediatric study underestimates the overall burden of severe influenza, as research has demonstrated that influenza mortality among adults is considerably higher than in children. 36 in the united states, approximately 100 children die annually from influenza virus infection, as compared with greater than 32 000 influenza-attributable deaths among persons older than 65 years. 36 the burden of influenza among adults may be even higher in developing settings. recent data from south africa suggest that the risk of influenza mortality among the elderly in that country is greater than in the united states. 37 further, countries with a high prevalence of hiv/aids and minimal availability of highly active antiretroviral therapy may also experience substantial risk of influenza mortality among non-elderly adults. 38 thus, beyond pandemic planning and response, it is critical to optimize care of severe seasonal influenza in resource-limited settings. more generally, the global burden of severe illness is poorly understood. in the united states, acute respiratory distress syndrome (ards) and sepsis are two of the most common severe illnesses requiring critical care. 39, 40 among adults worldwide, extrapolated data suggest that 15-19 million cases of sepsis and 1á15-5á5 million cases of ards occur annually. 2 however, considering that about 60% of the global burden of respiratory mortality is in children <5 years of age, 41 these estimates are a substantial underestimate of the total global burden of severe illness. the vast majority of severe illness occurs in low-and middle-income countries, 42 yet there are currently little clinical data or evidence-based management guidelines to improve hospital care for patients in these settings. 43 in intensive care medicine, severe illness treatment is syndromic in approach. clinical management of two common syndromes encountered in intensive care units -ards and sepsisfollow standardized guidelines. 44, 45 these syndrome-focused guidelines facilitate the rapid recognition and treatment for life-threatening conditions, even before specific etiologies are identified. this approach also promotes the widespread adoption of research-proven interventions such as low tidal volume lung protective ventilation for ards. 40, 46 similarly, several studies suggest that sepsis outcomes may be improved by the use of protocolized sepsis care pathways. 47, 48 syndromic management is therefore an important tool in improving care of severely ill patients. in resource-limited settings, where advanced diagnostic equipment may not be available, syndromic management of severely ill patients using readily available tools offers a practical and feasible strategy for care. evidence of the benefit of syndromic management of hospitalized patients in resource-limited settings comes from the who integrated management for childhood illness (imci) program. imci guidelines were developed using existing clinical evidence and expert opinion to standardize healthcare provider training and care delivery in resourcelimited settings. 49 the imci guidelines are designed to identify children in need of care by presenting signs and symptoms, and they do not require diagnostic tests that are likely unavailable in most austere settings. studies evaluating training and implementation of imci guidelines have shown a substantial impact on improved management and survival related to childhood pneumonia and other common illnesses. [50] [51] [52] [53] [54] for example, a cluster randomized trial in kenya evaluating the efficacy of a multi-faceted quality improvement intervention for the management of severely ill children resulted in improved quality of clinical care when compared with a less comprehensive approach. 50 similarly, in a pediatric outpatient and emergency unit in malawi, directed trainings based on imci to improve triage and emergency care resulted in streamlined healthcare delivery and a 10% decrease in pediatric in-hospital mortality. 51 modifications of imci have also been shown to improve pneumonia outcomes when implemented in the community setting. [55] [56] [57] moreover, economic analyses have found imci protocolized care to be cost effective and comparable to preventive interventions such as routine childhood pneumococcal conjugate immunization. 58 it is important to highlight that clinical management guidelines may have only modest effects on important outcomes in resource-limited settings for several reasons, including an incomplete evidence base and numerous challenges to implementation. 59 further, some studies have shown limited or no effects of knowledge translation activities, such as clinical guideline implementation. 60, 61 nevertheless, even modest treatment effects on a high burden disease can have a massive impact when widely implemented. the imci experience demonstrates the potential benefits of syndromic management for persons with severe respiratory infections in resource-limited settings. global disparities in access to care for severe illness are substantial. 43, 62, 63 who reported in 2009 that poor clinical outcomes of pandemic influenza were associated with delays in seeking health care, limited access to supportive care, and "rapidly progressive overwhelming lung disease which is very difficult to treat." 9 disparities in access to critical care may partly explain some of the pandemic influenza mortality differences reported among countries. 64, 65 for example, during the early phase of the h1n1 pandemic, reported icu mortality in middleincome mexico was twice that of high-income canada. 2, 3, 14 access to typical health technologies used to manage critically ill patients such as pulse oximetry, invasive hemodynamic monitoring, blood gas analyzers, and mechanical ventilation may be limited or absent in resource-limited settings. 66, 67 hospital care is often delivered by nurses and non-specialist doctors who may have limited time, resources, training, and access to information to manage severely ill patients, 67,68 particularly during a public health emergency like the 2009 influenza pandemic. from the few surveys of icu resources in resource-limited settings, data suggest that many hospitals are ill equipped to dedicate sufficient personnel and supplies required by patients with severe illness. [69] [70] [71] [72] alarming shortages of reliable electricity, clean water, and supplemental oxygen have been reported from hospitals in sub-saharan africa and southeast asia. 73, 74 developing country health systems may be weak and hospital support from government ministries is often lacking. 67 thus, strategies to improve care delivery in low-and middle-income countries must address challenges of improving access to care, training and retention of healthcare providers, supply chain management, and strengthening healthcare systems. 42 any new healthcare intervention designed to improve management of severe influenza disease must be integrated into the current health system structure and strengthen healthcare delivery overall if it is to be successfully adopted and remain sustainable. despite the absence of sophisticated equipment and abundant resources, including icus, it is likely that many lives in resource-limited settings can be saved by promoting the basic tenets of severe illness management. 42, 67, 75 examples include simple triage systems to rapidly identify ill patients, protocolized supplemental oxygen therapy managed by nonphysician staff, infection source control, and early antimicrobial therapy for sepsis based on local antimicrobial clinical care for severe influenza ª 2013 blackwell publishing ltd susceptibility testing, prompt fluid resuscitation for septic shock, and standardized infection control measures such as hand cleaning. several recent global initiatives have developed guidelines for the syndromic management of severe influenza and other severe illness in resource-limited settings. during the h1n1 influenza pandemic, the who assembled a group of experts to generate a document addressing management of severe respiratory distress and shock in resource-limited settings. 76 more recently, similar advice was produced by the who for the clinical management of novel coronavirus from outbreaks in 2012 in the middle east (publication pending). the who integrated management of adult and adolescent illness (imai) program, a sister initiative to imci, created a comprehensive manual for the care of hospitalized patients by clinicians in district hospitals that includes sections on severe illness management. 77 the european society of intensive care medicine also recently produced sepsis management guidelines targeted to resource-limited settings. 78 these largely expert opinion-based documents fill major gaps in management guidelines but require quality clinical research to provide missing evidence and refine best-practice recommendations. a reminder of the importance of evidence generated by studies in at-risk populations was provided by the unexpectedly harmful effect of fluid boluses for kenyan children with severe infection in the feast (fluid expansion as supportive therapy) trial. 59 severe illness, influenza-related and otherwise, causes a profound burden of disease in resource-limited settings. quality clinical management is an essential element in mitigating this burden. in support of this, a recent call to action by unicef, and several governmental and nongovernmental organizations, focuses global attention on treatment of preventable childhood deaths, including respiratory infections. 79 syndromic management approaches to the care of severe illness seem practical and feasible. however, there are few data to guide the optimal management of severely ill patients in these resource-limited settings. while several recent initiatives now provide long awaited guidance for clinicians in resource-limited settings, a sustainable, global impact on outcomes due to severe influenza and other severe illness will require an ongoing and concerted international effort to implement, evaluate, and refine these guidelines. epidemiological characteristics and underlying risk factors for mortality during the autumn 2009 pandemic wave in mexico critically ill patients with 2009 influenza a(h1n1) in mexico 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settings: lessons learned and future directions clinical management of adult patients with complications of pandemic influenza a (h1n1) 2009: emergency guidelines for the management of patients with severe respiratory distress and shock in district hospitals in limited-resource settings world health organization. imai district clinician manual: hospital care for adolescents and adults. guidelines for the management of illnesses with limited resources. geneva: world health organization recommendations for sepsis management in resource-limited settings we would like to thank danielle clark, sherry dodson, terri hough, tim nguyen, kristina rudd, joanne rich, and nahoko shindo for their assistance with this article. the authors report no financial competing interests. key: cord-339230-cc7gcy5b authors: smith, amber m.; mccullers, jonathan a. title: secondary bacterial infections in influenza virus infection pathogenesis date: 2014-07-16 journal: influenza pathogenesis and control volume i doi: 10.1007/82_2014_394 sha: doc_id: 339230 cord_uid: cc7gcy5b influenza is often complicated by bacterial pathogens that colonize the nasopharynx and invade the middle ear and/or lung epithelium. incidence and pathogenicity of influenza-bacterial coinfections are multifactorial processes that involve various pathogenic virulence factors and host responses with distinct siteand strain-specific differences. animal models and kinetic models have improved our understanding of how influenza viruses interact with their bacterial co-pathogens and the accompanying immune responses. data from these models indicate that considerable alterations in epithelial surfaces and aberrant immune responses lead to severe inflammation, a key driver of bacterial acquisition and infection severity following influenza. however, further experimental and analytical studies are essential to determining the full mechanistic spectrum of different viral and bacterial strains and species and to finding new ways to prevent and treat influenza-associated bacterial coinfections. here, we review recent advances regarding transmission and disease potential of influenza-associated bacterial infections and discuss the current gaps in knowledge. pneumonia is a leading cause of death in the united states and worldwide [(centers for disease control deaths and mortality); world health organization]. multiple respiratory viruses and bacteria can cause an infection that leads to severe pneumonia, and it is now recognized that a high proportion of community-acquired pneumonia is caused by coinfections. pathogens including influenza viruses, parainfluenza viruses, respiratory syncytial virus (rsv), human metapneumovirus (hmpv), streptococcus pneumoniae (pneumococcus), staphylococcus aureus, and group a streptococcus (s. pyogenes, or gas), and others, alone or in various combinations, cause millions of ambulatory care visits for pneumonia and thousands of deaths each year in the united states. the resulting economic burden is greater than 17 billion dollars (file and marrie 2010) . in addition, otitis media is the leading reason for visits to a pediatrician (2.4 million visits per year) (centers for disease control deaths and mortality, centers for disease control estimated burden of acute otits externa)], further increasing the health care cost of these pathogens. although otitis media has classically been considered a bacterial disease, an increasing amount of evidence suggests that viral infections are a common cause and a great deal of acute otitis media (aom) results from coinfections with two or more pathogens (heikkinen 2000) . of the multiple viruses and bacteria that participate in coinfections of the lung and middle ear, one of the most important is the influenza virus. although influenza is a major public health threat on its own, bacterial coinfections complicating influenza contribute greatly by exacerbating disease severity. detailed descriptions of fatal cases date as far back as the eighteenth century (laennec 1923) indicating that viral-bacterial coinfections have been recognized as being prevalent for hundreds of years. since then, further study has taken place. the most infamous event was during the ''spanish flu '' pandemic in 1918-1919 where more than 95 % of the 50+ million deaths were complicated by a bacterial coinfection (morens et al. 2008) . although significant improvements regarding health care have been made, new pathogenic strains emerge and complications from bacterial coinfections continue. approximately 50-70 % of severe or fatal cases in the 1957 h2n2 and 1968 h3n2 pandemics and nearly one-third of those in the 2009 h1n1 pandemic had bacterial complications (louria et al. 1959; weinberger et al. 2011) . furthermore, when a bacterial coinfection was identified, mortality was high despite appropriate antibiotic use in the majority of cases (domínguez-cherit 2009; kumar 2009; jain et al. 2009; palacios et al. 2009 ). today, it is well recognized that bacterial pneumonia complicates disease initiated by respiratory pathogens like influenza viruses. pneumococcus remains the most frequently identified bacterial pathogen associated with influenza infections and the most common cause of communityacquired pneumonia (cap) despite use of the pneumococcal conjugate vaccine (pcv) in children and adults (nelson et al. 2008) . however, over the last decade, s. aureus dominated influenza-associated childhood fatalities in the us and accounted for *75 % of deaths from bacterial coinfections. s. aureus has likely become a more common cause of fulminant coinfections due to the emergence in some countries of the methicillin-resistant clonotypes usa300 and usa400 (mrsa). it is unclear why these strains are more likely lead to secondary pneumonia with influenza than other circulating strains. there is currently no vaccine for s. aureus. group a streptococcus only occasionally complicates viral infections (chaussee et al. 2011 ) and, when present, falls behind pneumococcus and s. aureus in prevalence. there is little systematic surveillance of bacterial coinfections during seasonal influenza, but this continued threat to public health has led to increased research on the co-pathogenesis of pneumonia due to influenza viruses and bacterial pathogens [reviewed in (short et al. 2012a; bosch et al. 2013; metzger and sun 2013; mccullers 2014) ]. this research has significantly improved the current state of knowledge of influenza coinfections through the use of animal models and, more recently, through the use of theoretical models. key questions regarding transmission, invasion, and pathogenicity remain unanswered. identifying how a bacterial coinfection renders mild influenza infections fatal is key to effectively combating pneumonia and preparing for future influenza pandemics. during the 1918 influenza pandemic, the armed forces of several countries made detailed accounts of infectious disease-related illnesses since their efforts during world war i were severely impacted (brundage and shanks 2007; shanks et al. 2010) . this led to the first animal studies confirming that bacteria contribute to disease during influenza virus infections by using filtered and unfiltered human sputum (wherry and butterfield 1921) . these experiments were followed in 1931 by shope, who conducted controlled experiments in pigs with a swine influenza virus and haemophilus influenzae suis (shope 1931) , and in the 1940s by francis and torregrosa, who used a mouse model with the mouse-adapted influenza a/puerto rico/8/1934 virus together with pneumococcus, s. aureus, or h. influenzae (francis and de torregrosa 1945) . since then, a variety of animal models have been used to study coinfections (fig. 1) . the sequential viral-bacterial mouse model of pneumonia, which we characterized in detail in 2002, remains the most useful and well-defined system for investigating coinfections, particularly given the lack of comprehensive data from natural infections in humans. in the initial model, sublethal doses of pr8 and of a type 2 laboratory strain of pneumococcus (d39) reproducibly caused severe secondary bacterial pneumonia when given intranasally in balb/c mice (mccullers and rehg 2002) . the influenza virus infection had to precede the bacterial challenge to observe synergistic disease. an interval of 3-14 days between inoculations fig. 1 animal models of influenza-associated bacterial coinfections. several different animal models have been used to study the effect that influenza viruses have on bacterial transmission and colonization and on invasive diseases, such as acute otitis media and pneumonia (wherry and butterfield 1921; shope 1931; francis and de torregrosa 1945; berendt et al. 1975; rarey et al. 1987; hajek et al. 1999; hirano et al. 1999; mccullers and rehg 2002; okamoto et al. 2003; seki et al. 2004; peltola et al. 2006; montgomery et al. 2008; small et al. 2010; diavatopoulos et al. 2010b; lee et al. 2010; jamieson et al. 2010; mccullers et al. 2010; loving et al. 2010; iverson et al. 2011; kudva et al. 2011; ayala et al. 2011; chaussee et al. 2011; short et al. 2011; mina et al. 2013; mchugh et al. 2013; redford et al. 2014) with the organisms resulted in the most severe disease, and peak severity occurred when pneumococcus was given 7 days postviral infection. simultaneous administration of the two pathogens had only additive effects on morbidity, rather than the synergistic effects observed during the sequential infection. this model was later improved by engineering pneumococcal strains to express luciferase, which allows for quantitative bioluminescent imaging to track progression of the infection in live mice (mccullers and bartmess 2003) . multiple strains of influenza, including the 2009 h1n1 pandemic virus, can prime mice for secondary pneumonia (wanzeck et al. 2011 ), but the doses necessary to have comparable results differ in a strain-dependent fashion. in addition, other viruses (e.g., rhinovirus, adenovirus, coronavirus, parainfluenza virus, hmpv, and rsv) have been used within the same model framework (reviewed in (bosch et al. 2013) . a variety of clinical outcomes can be modeled with different pneumococcal strains, including pneumonia with and without bacteremia, sepsis with secondary seeding of organs leading to pneumonia, otitis media, and sinusitis (peltola et al. 2005; mccullers et al. 2007; smith et al. 2007) . furthermore, multiple bacterial species can synergize with influenza viruses to cause disease (fig. 1 ). the mouse model for influenza-bacterial coinfections has several limitations. for example, viruses that replicate well in mice are required to produce robust and reproducible effects, a limitation that affects the certainty with which conclusions can be extrapolated to humans. this is mitigated somewhat by using different species of mice, including the c57bl/6 strain, which behaves similarly to the balb/c strain (karlstrom et al. 2011) , and the dba/2 strain, which is highly permissive to a variety of human influenza strains (alymova et al. 2011 ). in addition, a ferret model can be used to confirm results found using the mouse model or to answer questions about strain-related differences in pathogenesis since ferrets are susceptible to most human viruses and exhibit a disease course similar to humans (peltola et al. 2006; mccullers et al. 2010) . chinchillas and weanling ferrets can also be infected with a variety of pneumococcal strains (hajek et al. 1999; mccullers et al. 2010) , although the disease manifestations do not map precisely to the mouse model. there are limited data with other viruses and bacteria in the ferret model, but unpublished experience from our laboratory has shown that s. aureus will not cause respiratory infections in ferrets even when the animals are preinfected with influenza. another limitation of the mouse model is the poor transmission potential for respiratory viruses or bacteria between mice, thus requiring the use of alternate models such as neonatal mice (diavatopoulos et al. 2010a) early animal models of aom utilized the chinchilla due to their large and accessible middle ear spaces (hajek et al. 1999) . these studies demonstrated that the greatest incidence of aom occurred in animals receiving bacteria 4-8 days following influenza (hajek et al. 1999) , similar to the data concerning timing of pneumonia. more recently, juvenile and infant mouse models have been developed so that diseases of young adults and children, respectively, can be mimicked (mccullers et al. 2007; diavatopoulos et al. 2010a) . similar models are used to investigate the effects that influenza viruses have on bacterial colonization (tong et al. 2001; nakamura et al. 2011) . studying viral-bacterial interactions in animal models has significantly increased our knowledge about the transmission and pathogenicity of coinfections. however, age, gender, weight, and exposure to anesthesia all contribute to susceptibility to infection in animals in these models, so extreme care must be taken in pathogenesis studies to control all these variables. in addition, studies must carefully select pathogen strains, inoculum sizes, and the sequence and timing of infections since all influence the progression of bacterial pneumonia following influenza. influenza viruses readily transmit from person to person via small or large respiratory droplets from a sneeze or cough. successful transmission and infection typically begins 1 day prior to developing symptoms, which can last up to 7 days in adults and 21 days or more in children (world health organization writing group et al. 2006) . while influenza viruses can spread by large droplets up to six feet away, pneumococcal transmission is thought to require close contact of individuals. recent evidence, however, suggests that this distance can be lengthened if the individual is virus infected. in fact, epidemiological studies found connections between upper respiratory tract (urt) infections, likely of viral origin, and an increase in bacterial transmission and carriage prevalence (gwaltney et al. 1975; garcía-rodríguez and fresnadillo martínez 2002; pettigrew et al. 2008; murphy et al. 2009; ansaldi et al. 2012) . influenza virus' impact on pneumococcal transmission was recently illustrated in the ferret model where transmission events and recipient acquisition increased while the distance necessary for successful bacterial acquisition decreased ). both bacterial titers and disease severity intensified in the contact ferrets. this relationship was further examined in the infant mouse model, where influenza virus replication and nasopharyngeal bacterial growth were deemed essential for pneumococcal transmission between littermates (diavatopoulos et al. 2010b; short et al. 2012b ). these outcomes may not be seen with all influenza-pneumococcal pairings since all observed effects were both viral and bacterial strain dependent. for instance, h3n2 influenza viruses enhance pneumococcal sinusitis and aom and induce bacterial colonization and disease more frequently than h1n1 or influenza b viruses (peltola et al. 2006; short et al. 2013b) . similarly, colonization and aom development were greater with pneumococcal serotype 19f compared to serotype 7f (mccullers et al. 2010) . although the precise mechanisms responsible for enhancing the transmission profile that influenza viruses provide pneumococci are currently unknown, it is likely due to an increase in pathogen density and frequency of secretion events (e.g., sneezing and coughing) in the infected individual combined with a decrease in immunity and resistance from natural barriers breaking down in the person who is newly exposed. during an influenza virus infection, the respiratory tract environment is primed for efficient bacterial invasion. natural physiological barriers are compromised and a heightened state of inflammation is reached. numerous factors dictate whether an individual develops a mild or serious infection. the time between exposure to the virus and the bacteria and the pathogen strain and inoculum size all influence influenza coinfection pathogenesis. in addition, many of the virulence factors expressed by each viral and bacterial pathogen act in strain-specific and sitespecific manners and can favor different outcomes. the extensive, and growing, list of possible mechanisms ( fig. 2 ) emphasizes the need to understand how each interacts and how to effectively combat the disease. as an influenza virus infection progresses, respiratory tract damage accumulates and primes the damaged and undamaged areas for bacterial colonization due to disrupted mechanical clearance mechanisms and exposed receptors. airway damage from overexuberant inflammatory responses and disruption of specific immune responses to viral pathogens leave the airways suitable for invasion by bacterial pathogens (reviewed in short et al. 2012a; bosch et al. 2013; metzger and sun 2013; mccullers 2014) . the host depends on the mucociliary apparatus in the lung and nasal passages to clear invading pathogens, but viral insults can damage the respiratory epithelium and inhibit this mode of removal (pittet et al. 2010) . receptors [e.g., plateactivating factor receptor (pafr) (cundell et al. 1995; miller et al. 2007 )] permissive to attachment of bacterial invaders become exposed in these inflamed areas, as shown by autopsy studies in humans and in vivo infections in mice (giles and shuttleworth 1957; oseasohn et al. 1959; herzog et al. 1959; plotkowski et al. 1986 plotkowski et al. , 1993 louie et al. 2009 ). additional adhesion sites in the lung appear as the viral lesions begin to heal. pneumococcus, h. influenzae, and s. aureus, in particular, all use bacterial adhesions to bind exposed laminin, type i and iv collagen, and fibrin/fibrinogen deposition in areas of incomplete healing (fainstein et al. 1980 ). injured or differentiating cells also provide new sites on apical receptors [e.g., asialylated glycans or integrins) for both s. aureus and pseudomonas aeruginosa (reviewed in puchelle et al. 2006) . this increased attachment within the lung, trachea, and nasopharyngeal surfaces may be mediated, at least in part, by viral neuraminidase (na) activity (hirano et al. 1999; peltola et al. 2005) , which facilitates bacterial adherence by exposing host cell receptors and providing decoy receptors when sialylated mucins fig. 2 influenza-bacterial interaction during coinfections. numerous alterations of the respiratory epithelium and host immune responses occur during influenza virus infection that predisposes a host to coinfection with bacterial pathogens. as influenza virus infects and kills host cells, epithelial surfaces become exposed and permissive to bacterial attachment. physical barriers (e.g., mucociliary transport) are damaged, pathogen detection is decreased, antimicrobial peptides (amps) are downregulated, receptors are upregulated, virus production is enhanced, bacterial transepithelial migration is permitted, and repair mechanisms are lost. several host responses are also dampened, altered, or removed. alveolar macrophages, neutrophils, dendritic cells, and nk cells have altered cytokine profiles and become impaired and/or depleted. these changes result in a heightened inflammatory environment with decreased bacterial surveillance and eradication are disrupted. some bacteria, like pneumococcus, use their own nas to access receptors and cleave sialic acids to avoid host defenses and prevent mucociliary clearance, replacing, or complementing antecedent viral infections (camara et al. 1991) . although decreased mucociliary transport impacts bacterial access to the middle ear, receptor-mediated mechanisms may not be as relevant in aom. in neonatal mice infected with influenza virus, bacteria can be seen localizing to inflammatory infiltrates, rather than to the epithelium (short et al. 2011 ), suggesting that different mechanisms are driving the enhanced bacterial replication. it has been hypothesized that viral-mediated destruction still has a role, but in the context of nutrient availability rather than receptor upregulation (short et al. 2011) . the influenza glycoprotein hemagglutinin (ha), which binds sialic acid residues on host cell surfaces and aids viral internalization, has an indirect effect on influenza-pneumococcal synergy within the middle ear (short et al. 2013b ). the ha specificity is sufficient to produce differential viral replication and bacterial localization. here, h3 viruses have higher replicative ability than h1 viruses, even across various nas, but the effect is site specific and does not depend on cell tropisms (short et al. 2013a ). it is likely that similar mechanisms dictate each type of infection with both the ha and na having specific roles, but the interactions remain complex. it does, however, help to explain the differential outcomes that different influenza strains have on pneumococcal coinfection. various bacterial species frequently colonize the nasopharynx and reach a balance with the mucosal immune response such that they are not harmful to the host. in this state, bacteria exist in either biofilms or move between intracellular and extracellular states. most often, colonizing strains remain in the upper airways since movement to the lower respiratory tract is inhibited by physical barriers (e.g., mucociliary mechanisms) and by immune responses (e.g., resident immune cells, complement, and mucosal antibodies). in this manner, the breakdown of physical barriers and disruption of host responses can result in bacteria emerging from biofilms (marks et al. 2013) . most invasive infections and pneumonia occur within a short period of time after a new strain is acquired from the environment rather than from a long-term carriage isolate disseminating to other sites in the body. this is likely due to colonizing strains being limited by systemic immunity, such as pathogen-specific serum igg, which prevents successful invasion of the lower airways but may tolerate carriage [reviewed in (mccullers et al. 2010) ]. in the mouse model, bacteria are delivered directly to the lung, which mimics a direct-inhalation scenario. it is unclear whether this scenario is physiologically relevant in humans, or whether some period of colonization of the nasopharynx must occur prior to invasion and dissemination [reviewed in (mccullers 2014) ]. the inoculum size and volume, and the length of anesthesia all influence how much bacteria reach the lower airways and thus the disease model being studied. both the ferret and mouse aom models, on the other hand, use colonization as a prerequisite for pneumonia or aom, so a more natural infection for this site can be instigated. influenza viruses and live-attenuated influenza vaccines (laiv) can result in prolonged bacterial colonization and enhanced bacterial replication within the nasopharynx in both mice and ferrets (peltola et al. 2006; nakamura et al. 2011; short et al. 2012b; mina et al. 2013 ). this may be mediated by type i interferon responses and bacterial toxins, such as pneumococcal pneumolysin (nakamura et al. 2011) . nasopharyngeal colonization can result in bacterial migration to the middle ear via the eustachian tube. in mice aom models, animals become colonized with pneumococcus within 72 h after inoculation and can experience recurrent episodes when virus infected (mccullers et al. 2007 ). of those that developed aom (*70 %), resolution occurred within 48 h but colonization persisted for nearly 30 days (mccullers et al. 2007) . in the chinchilla model, animals infected with influenza virus experience negative middle ear pressure and eardrum inflammation associated with epithelial damage and cellular and mucosal debris accumulation in the eustachian tube (giebink et al. 1987) . similarly, both virus-mediated inflammation and hearing loss are observed in neonatal mice and ferrets (rarey et al. 1987; short et al. 2011) . however, in contrast to the findings in chinchillas, minimal eustachian tube damage is observed and bacterial localization specificity suggests that invasion techniques observed in other models are unlikely to be relevant to bacterial aom (short et al. 2011 ). nevertheless, the increased pathology may support bacterial replication in the middle ear. if bacteria are successful in migrating to the lungs, a combination of increasing bacterial burden and an accompanying, intense inflammatory response may result in the host developing pneumonia. the early stages of pneumonia are marked by capillary congestion and fluid in the alveolar regions, which provides a medium where pneumococci can readily grow [reviewed in (mccullers 2001) ]. as blood vessels become permeable, inflammatory cells are allowed to enter the lung, receptors become upregulated and bacteria easily adhere to, invade, and kill epithelial cells. the combined effects result in significant inflammation, a hallmark of pneumonia. during coinfections, the host is in a relative state of immune dysregulation with heightened inflammatory and anti-inflammatory responses [reviewed in (short et al. 2012a; bosch et al. 2013; metzger and sun 2013; mccullers 2014) ] likely due to expression of various pathogenic factors. bacterial cytotoxins, like the pneumococcal pneumolysin (tuomanen et al. 1995; kadioglu et al. 2008) , s. aureus panton-valentine leukocidin (pvl) (niemann et al. 2012 ) and b. pertussis toxin (pt) (ayala et al. 2011) , are known to influence host inflammation and may work in concert with viral cytotoxins. these bacterial factors may intensify the cell death and inflammatory signaling resulting from pores formed by the influenza cytotoxic protein, pb1-f2 (chen et al. 2001) . the pb1-f2 protein of some influenza viruses increases pathologic effects by causing cell death, increasing viral replication, and altering inflammatory responses to primary viral infections and to bacterial coinfections (conenello et al. 2007; mcauley et al. 2007 mcauley et al. , 2010a smith et al. 2011a . pb1-f2 can act in a proapoptotic fashion due to its mitochondrial targeting sequence and ability to form pores when interacting with membrane-based proteins (chen et al. 2001; gibbs et al. 2003; chanturiya et al. 2004; zamarin et al. 2005; danishuddin et al. 2010; mcauley et al. 2010a ). this likely results in the death of epithelial cells and immune cells, which may balance the high replicative ability and support rapid spread through cell monolayers thereby contributing to virulence in vivo (zamarin et al. 2006; mcauley et al. 2010a, b; smith et al. 2011a; varga et al. 2011 varga et al. , 2012 . kinetic analyses suggest that this mechanism impacts viral loads during the later stages of the influenza infection, but is overshadowed by more prominent mechanisms during secondary bacterial infections (smith et al. 2011a . these cellular effects have been mapped to a specific set of amino acids in the c-terminal end of the protein, which are found in most of the early twentieth century h1n1 strains (mcauley et al. 2010a) . although rare (hai et al. 2010) , a serine at position 66 (i.e., '66s polymorphism') impacts virulence with highly pathogenic strains with full-length pb1-f2s (e.g., 1918 h1n1, h5n1) but not less pathogenic strains with truncated pb1-f2s (e.g., 2009 h1n1) (conenello et al. 2007 (conenello et al. , 2011 hai et al. 2010; varga et al. 2011 varga et al. , 2012 . the 66s polymorphism facilitates binding of pb1-f2 to the mitochondrial antiviral-signaling (mavs) protein adaptor protein and subsequent inhibition of interferon production (varga et al. 2011 (varga et al. , 2012 . as a result, viral virulence in primary infection and secondary bacterial infection models is severely exacerbated. the most relevant pb1-f2 mechanism may be its ability to modulate the immune response during influenza infections and coinfections. the high proinflammatory activity of pb1-f2 intensifies disease in animal models, particularly with respect to induction and severity of bacterial coinfections (mcauley et al. 2007 (mcauley et al. , 2010a alymova et al. 2011; weeks-gorospe et al. 2012) , and is marked by a large influx of immune cells and cytokine storm (conenello et al. 2007; mcauley et al. 2007 mcauley et al. , 2010a . pathogenic pb1-f2s, such as that from the 1918 pandemic strain, elevate neutrophils and macrophages and contribute to the pathologic tissue destruction observed during bacterial coinfections (mcauley et al. 2007 ). this is likely due to regulation of the type i interferon response (le goffic et al. 2010; conenello et al. 2011; varga et al. 2011 varga et al. , 2012 and apoptotic monocytes infected with influenza (chen et al. 2001; gibbs et al. 2003; zamarin et al. 2005) . specific molecular signatures that facilitate this inflammatory environment have been identified (mcauley et al. 2007 (mcauley et al. , 2010b . amino acids 62l, 75r, 79r, and 82l in the c-terminal portion of pb1-f2 of select strains are positively associated with inflammation and hypercytokinemia in infected animals and negatively correlated with survival. the precise mechanism and contribution of these signatures singly or in combination is unclear. a key prerequisite for bacterial invasion into respiratory epithelium is the induction of inflammation. several immune responses are activated and act to control bacterial pathogens that invade the lung (joyce et al. 2009; koppe et al. 2012) . a robust initial response is sufficient to immobilize bacterial invaders before full establishment and uncontrolled growth puts the host in a harmful inflammatory state. the degree of attack and the initial replicative ability within mice are dose-dependent and occur in the lung only when alveolar macrophages become overwhelmed with bacteria (smith et al. 2011b ). for small inocula of bacteria, resident macrophages provide the first line of defense and result in rapid elimination of bacterial pathogens while maintaining homeostasis, which is represented by a low inflammatory state (knapp et al. 2003; dockrell et al. 2003 smith et al. 2003 , 2011b . however, given a large invasion or a compromised host state (e.g., influenza virus infected), bacterial outgrowth occurs and an inflammatory response is launched. neutrophils appear first and are followed shortly by inflammatory macrophages (jonsson et al. 1985; fillion et al. 2001; knapp et al. 2003) . the inflammatory influx in the lungs results from bacterial recognition by antigen presenting cells (apcs), and subsequent cytokine and chemokine production. bacterial phagocytosis by these cells is only efficient if ample complement proteins are available to opsonize the pathogen or if typespecific antibody is made available by b-cells. respiratory viruses compromise many aspects of the early detection and response to bacterial pathogens like pneumococcus or s. aureus [reviewed in (short et al. 2012a; bosch et al. 2013; metzger and sun 2013; mccullers 2014) ]. viruses and bacteria also activate many of the same cytokines, inflammatory cells, and pattern recognition receptors (e.g., tlr4) that can synergize during coinfections and generate inflammation (navarini et al. 2006; joyce et al. 2009; karlstrom et al. 2011; kuri et al. 2013 ). interference of immune responses occurs through various manners, such as by viral expression of multifunctional proteins like the influenza virus ns-1 (hale et al. 2008 ) and pb1-f2 (mcauley et al. 2007 . depending on the stage of influenza, the innate, cellular, and anergic responses may differentially synergize. since phagocytic cells are critical in creating a bactericidal environment, it is not surprising that these cells are impacted by viral and bacterial mechanisms when secondary infections occur. the activity of neutrophils and macrophages is dampened along with their cytokine production as natural killer (nk) cells become impaired during influenza virus infection and undergo additional suppression during coinfections as a result of heightened tnf-a expression (small et al. 2010) . further functional suppression of these cells occurs when type i interferons (ifna,b) place epithelial cells in antiviral states and alter their chemotactic functions (joyce et al. 2009; shahangian et al. 2009; le goffic et al. 2010; conenello et al. 2011; nakamura et al. 2011; tian et al. 2012; li et al. 2012) . for instance, neutrophil chemoattractants kc and mip-2 (shahangian et al. 2009 ) and macrophage chemoattractant ccl2 (nakamura et al. 2011 ) all become downregulated, which inhibits recruitment of immune cells leading to inefficient bacterial clearance. ifn-a,b may also decrease th-17 cytokines il-17, il-22, and il-23 during s. aureus coinfection, which increases inflammation and decreases viral and bacterial clearance (kudva et al. 2011) . production of interferon-c increases during influenza resolution and can downregulate bacterial scavenger receptors (e.g., marco) on macrophages leaving phagocytic cells suppressed and cytokine profiles altered sun and metzger 2008) . additional proinflammatory [e.g., il-1b, tnfa, il-6, and il-12 (seki et al. 2004; smith et al. 2007; shahangian et al. 2009; nakamura et al. 2011; mchugh et al. 2013) ] and anti-inflammatory cytokines [e.g., il-10 (van der sluijs et al. 2004) ] become inflated and further compound downstream events like macrophage and neutrophil recruitment and dendritic cell function during influenza-pneumococcal coinfection (sun and metzger 2008; shahangian et al. 2009; wu et al. 2011; nakamura et al. 2011; kuri et al. 2013) . even viral and bacterial pathogens themselves can induce apoptosis of phagocytic cells (colamussi et al. 1999; engelich et al. 2001; kobayashi et al. 2003 kobayashi et al. , 2010 mcnamee and harmsen 2006) , thus leaving the infected areas severely damaged. many of these responses require time to activate, thus the susceptibility of a host to bacterial pathogens following influenza virus infection indicates an effect that may be fully realized during viral preinfection. indeed, novel analyses of coinfection kinetics identified and detailed the dominant mechanism driving influenza-pneumococcal synergy as a direct viral-dependent reduction in bacterial phagocytosis by alveolar macrophages . we predicted that this phagocytosis is reduced by 85-90 % at day 7 of the influenza virus infection. we later confirmed that this was a major driver of influenza-pneumococcal synergy with a mouse model by labeling and tracking these cells before and during influenza infections . our experiments showed that the resident macrophage population declines as influenza progresses, suggesting that influenza virus directly depletes these cells rather than simply reducing their function. furthermore, bacterial outgrowth correlated to the level of depletion, which offers new insight into why the timing of bacterial infection has a profound impact on disease outcome . as the vigorous antiviral inflammatory response begins to subside, a new state of innate immune activation that may alter responsiveness to new pathogenic insults is reached. the lung becomes repopulated with resident alveolar macrophages as recruited macrophages proliferate and differentiate. in an attempt to return the lung to homeostasis, wound-healing processes coordinate an antiinflammatory response characterized by il-10 (van der sluijs et al. 2004; hussell and cavanagh 2009) and suppress pathogen recognition systems [reviewed in (metzger and sun 2013) ]. during the recovery phase, the host becomes immunologically desensitized both locally and systemically (van der sluijs et al. 2004; didierlaurent et al. 2008) , which can last for several weeks and prolongs the opportunity for bacterial invasion. the degree and length of this suppression is viral strain-dependent (ludewick et al. 2011) , and occurs through diverse mechanisms. for instance, alveolar macrophages with high expression of homeostatic moieties such as cd200r, a regulatory antiinflammatory ligand (barclay et al. 2002; minas and liversidge 2006; snelgrove et al. 2008; jiang-shieh et al. 2010) , become desensitized when expression of cd200 on apoptotic immune cells increases and open the airways to bacterial invasion (goulding et al. 2011) . in conjunction, absence of cd200r in mice inhibits bacterial outgrowth and prevents migration of bacteria to exogenous sites, such as the blood, in influenza-infected mice (goulding et al. 2011) . elevated glucocorticoid levels also cause sustained immunosuppression, as was demonstrated in a model of listeria monocytogenes coinfection (jamieson et al. 2010) . the inflammatory nature of influenza-pneumococcal coinfections extends to middle ear invasions. middle ear inflammation, regardless of pathogenic origin, is sufficient to induce aom. influenza viruses can initiate this inflammation (abramson et al. 1981 (abramson et al. , 1982 short et al. 2013b) , cause hearing loss and instigate bacterial growth within the ear cavity in a viral strain-dependent, but cell tropism independent, manner (short et al. 2013a) . inflammation in aom is characterized by an influx of neutrophils and expression of key proinflammatory genes (i.e., pro-il-1b, il-1a, and cxcl2) (abramson et al. 1981 (abramson et al. , 1982 short et al. 2011 short et al. , 2013b . chinchilla studies indicate that immunosuppression, rather than inflammation, is the key mechanism contributing to enhanced pneumococcal replication since influenza viruses can inhibit neutrophils and render clearance ineffective (abramson et al. 1981 (abramson et al. , 1982 . however, it has also been hypothesized that the enhanced bacterial growth results from nutrients becoming available in areas damaged by this response (short et al. 2013b) . more experiments are clearly necessary to elucidate the underlying relationship between viral replication and inflammation in aom. the mechanisms discussed thus far have detailed how the virus affects host responses to invading bacteria. the relationship is somewhat complementary, however, since rebounds in viral load and reduced viral clearance are consistently observed in animal models (mccullers and rehg 2002; iverson et al. 2011; weeks-gorospe et al. 2012; . the rapidity of the viral reply suggests a fast-acting mechanism, which may occur through direct interactions of the two pathogens, bacterial interference with antiviral immunity, or virulence factors synergizing . kinetic studies suggest that pneumococci directly interact with influenza-infected epithelial cells to cause a sudden release of virus , but experimental studies have not been crafted to confirm this prediction. the precise effects of bacterial virulence factors are likely type specific as s. aureus may be capable of cleaving influenza hemaggluttinin to enhance invasion of host cells and thus impact viral load (tashiro et al. 1987) . it is feasible that additional, although unknown, bacterial virulence factors have immune-modulatory effects and could interfere with viral clearance, such as t-cell-mediated infected cell clearance or other innate immune components. determining if bacterial gene products from various species have similar effects during infection and, if so, how they complement viral factors is an important, but largely unexplored, area. another interesting area for investigation in animal models is the idea that the respiratory and gastrointestinal microbiome affects the development of antiviral responses to pathogens (ichinohe et al. 2011; licciardi et al. 2012; abt et al. 2012; wang et al. 2013) . understanding how commensal species influence host immune status may help explain the heterogeneity in responses to pathogenic invasions. the outcome of influenza virus coinfection is often severe despite appropriate vaccination and treatment. in addition, antiviral and antimicrobial resistance is increasing (musher et al. 2002; levy and marshall 2004; hayden 2006 ) and many treatment options have the potential to cause adverse effects on the host (mccullers and english 2008; karlstrom et al. 2011) . with the high prevalence of viral-bacterial coinfections in some situations, discovering treatment options that can prevent or treat both the influenza virus infection and the secondary bacterial infection are of utmost importance. several antiviral drugs targeted against various influenza virus components have been or are currently being developed [reviewed in (hayden 2013) ]. neuraminidase inhibitors (nais) are one class of drugs that have become the pillar of influenza treatment in recent years. nais act to block virus from budding out of infected cells, thereby preventing the spread of virus to neighboring cells (moscona 2005) . nai therapy can prevent secondary bacterial pneumonia in animals infected with influenza. mice given nai treatment within 72 h postinfluenza infection or prophylactically experienced improved survival due to delayed development of and progression of pneumonia with nai treatment, decreased viral loads, and reduced secondary bacterial infections. treatment in the later stages of influenza virus infection (i.e., 5 days) reduces bacterial invasion without any impact on viral loads, signifying that mechanisms independent of replication inhibition are in play (mccullers 2005) . their action may directly, or indirectly, lessen the effects of viral virulence factors, prevent receptor exposure, reduce use of sialic acids as catabolic substrates, and/or activate immunological components in a viral straindependent manner [reviewed in (mccullers 2011)] . although nai resistance remains problematic and may be reduced with combination therapy, treatment with multiple nais can inhibit antiviral efficacy (duval et al. 2010) . nais in combination with antibiotics, on the other hand, can facilitate recovery from influenza and alter the coinfection pathogenesis in infected animals (mccullers 2004) . other antivirals [e.g., peramivir and laninamivir (nais), favipiravir (rna polymerase inhibitor)] that are not yet licensed may provide benefit to coinfected hosts, but these have yet to be tested in animal coinfection models. it is important to note that nais specifically target influenza virus na and do not inhibit bacterial nas with clinically relevant doses (nishikawa et al. 2012 ). while nais may reduce incidence of bacterial pneumonia, and thus antibiotic requirements, nas derived from invasive or commensal bacteria may antagonize their effectiveness (nishikawa et al. 2012) . thus, nai therapy could have differential bacterial-dependent effects as well. unlike antivirals, which interrupt disease progression by preventing viral spread, antibiotics work to eliminate pathogens directly. some antibiotics, however, kill bacteria through mechanisms that can have harmful repercussions. for instance, therapy with cell wall active agents (e.g., ampicillin), the mainstay of treatment of community-acquired pneumonia in children (bradley et al. 2011) , causes significant inflammation and lung injury in animal models (karlström et al. 2009 ). the characteristic inflammation in secondary bacterial infections is due to immune cells responding to the release of bacterial components, such as cell wall components, during lysis (karlstrom et al. 2011) . thus, alternative treatments that eliminate pathogens while preserving host integrity are desirable. antibiotics that reduce neutrophil influx or cytokines and thus circumvent the inflammatory tissue damage are beneficial in coinfected animals (karlstrom et al. 2011; liu et al. 2013) . in particular, protein synthesis inhibitors (e.g., clindamycin) and macrolides (e.g., azithromycin) have anti-inflammatory properties in addition to bactericidal activity, thereby clinically curing mice with influenza-associated pneumococcal pneumonia (karlström et al. 2009 ). nevertheless, antibiotic treatment alone is suboptimal. anti-inflammatory agents, such as corticosteroids (e.g., dexamethasone), in conjunction with antibiotic therapy improve beta-lactam-induced immunopathology and mortality in animals with severe pneumonia. however, giving dexamethasone prophylactically during influenza infections negatively impacts adaptive immunity and results in reduced viral clearance . thus, use of immune-modulatory approaches may be best reserved for severe infections where inflammation is driving poor outcomes but avoided in primary viral infections where detrimental effects on the host response may result. given the benefit of anti-inflammatory treatment and the importance of inflammation in viralbacterial coinfections, better success may occur if specific inflammatory pathways or pathogenic factors are targeted [reviewed in (mccullers 2011) ]. vaccination remains fundamental to prevention of influenza and bacterial infections. data from animal models indicate that vaccinating against influenza viruses effectively circumvents bacterial associated pneumonia (huber et al. 2010; chaussee et al. 2011; mina et al. 2013 ) but may support colonization and replication in the urt (mina et al. 2013 ). an important caveat of current vaccines against influenza viruses is that partial protection of related strains may not be sufficient to alleviate bacterial complications. on the other hand, antibacterial vaccines are important to block the specific bacteria being targeted and to prevent clinically severe influenza infections by reducing the coinfection component. vaccinating animals against some bacteria (e.g., pneumococcus, h. influenzae) prevents invasive diseases caused by these pathogens, but protection is limited to vaccine-specific types and efficacy may be lost in influenza virus infected animals (mina et al. 2013 ). in addition, vaccines against other coinfecting bacteria, such as s. aureus, are not currently available. interestingly, vaccination with a liveattenuated b. pertussis vaccine can protect against lethal challenges with influenza virus by controlling cytokine responses that lead to virus-mediated inflammation (li et al. 2010) . although the vaccine has not yet been approved for use in humans, it is promising and may benefit as a prophylactic agent against infection with influenza viruses. unraveling the relationships between pathogen replication and interactions and the resulting airway alterations and inflammation that are driving coinfection host pathology and disease is complicated. kinetic models are a robust means of analyzing experimental results and explaining biological phenomena without testing every scenario experimentally. they have proven valuable in the identification and characterization of mechanisms driving influenza virus infections, pneumococcal infections, and bacterial coinfection establishment and severity. a growing body of work modeling in vivo influenza virus infections has improved our knowledge about the viral life cycle, viral control by the host, pathogenic differences in strains, and efficacy of antiviral treatment [reviewed in (smith and ribeiro 2010; beauchemin and handel 2011; smith and perelson 2011) ]. these models have characterized the spread of virus during early infection and yielded estimates of strain-specific viral infection and production rates, infected cell life spans, and infectious virus half-life, all of which are not amenable to experimental investigation. most of these studies model data from humans or large animals where only nasal wash titers are available and, thus, are restricted to studying nasopharyngeal infections (baccam et al. 2006; saenz et al. 2010; canini and carrat 2010; pawelek et al. 2012) . a few models, however, take advantage of data collected in the mouse model, including pathogen and immunological measurements, to study invasive lung infections (handel et al. 2010; miao et al. 2010; smith et al. 2011a ). viral load dynamics can be accurately modeled using target-cell limitation (fig. 3) , through undefined mechanisms, as the primary means of viral control while excluding specific immune responses (baccam et al. 2006; smith et al. 2011a) . it is important to note that these models do not discount the fact that fig. 3 kinetics of influenza-pneumococcal coinfection. model schematic and equations that result in the observed kinetics of influenza virus infection followed by pneumococcus given 7 days postinfluenza infection . during primary influenza, susceptible epithelial (target) cells (t) become infected at a rate bv per cell. infected cells (i 1 ) first undergo an eclipse phase at rate k per cell prior to entering a state (i 2 ) in which virus is produced. productively infected cells are lost, through apoptosis, viral cytopathic effects, or removal by immune cells, at a rate d per cell. virus (v) is produced at rate p per cell, which is significantly increased by bacterial presence (ap z ) (boxed), and cleared at rate c. invading pneumococci (p) proliferate at maximum rate r with a tissue capacity k p cfu/ml. bacteria are cleared via phagocytosis by alveolar macrophages (m a ) at rate cf per cell, which is significantly reduced by virus presence /v=ðk pv þ vþ ð þ(boxed). with this kinetic description, viral titers increase exponentially, peak and begin to decline prior to bacterial invasion. once bacteria are present, a viral rebound occurs and bacteria grow exponentially before reaching a maximum capacity. the potential increase in bacterial adherence to virus-infected cells and any accompanying cell death has little effect are excluded here immunological factors may drive influenza virus pathogenesis, but that this information can simply not be extracted from viral loads miao et al. 2011) . quantifying the effect that host factors have on viral replication has been restricted by the limited amount of data detailing the innate immune responses [reviewed in (smith and perelson 2011)] . as more data arise, new quantitative descriptions of influenza virus kinetics will be developed and will undoubtedly aid experimental interpretation. kinetic models depicting bacterial infections are an exciting new tool being used to study pathogenesis (smith et al. 2011b) . capitalizing on data obtainable in the mouse model system, we characterized the dose-dependent innate immune control of a pneumococcal invasion and quantified the contributions of alveolar macrophages, neutrophils, inflammatory macrophages, cytokines, and damage to bacterial pathogenesis (smith et al. 2011b) . model analysis revealed the exact thresholds for bacterial establishment, growth, and eradication with alveolar macrophages playing a central role. the dose-dependent invasive ability of pneumococci was solely dependent on the number and phagocytic ability of resident macrophages initially present. thus, any alterations to resident cells, such as death from an antecedent viral infection, would result in immediate pathogenic invasion. while the rapid neutrophil influx could facilitate bacterial removal, pneumococcal-induced neutrophil apoptosis hindered complete eradication. this process was also dependent on alveolar macrophages and whether they were engaged damage control rather than bacterial clearance. inflammatory macrophages had little effect on clearance but still contributed to respiratory tract damage. through this model, we successfully captured the biochemical, cellular, immunological interactions of pneumococci with the host and identify the critical processes driving pathogenesis. modeling the interactions of two pathogens requires the combination of previously developed single infection models. thus far, the only model depicting a coinfection is one that we formulated for influenza-pneumococcal coinfection using the models discussed above (fig. 3) . we quantitated the enhanced bacterial growth and viral rebound and evaluated prior hypotheses about the interaction between influenza, pneumococcus, and the host. careful model development and analyses showed that any enhanced bacterial adherence to epithelial cells, with respect to both invasion and cell death, was negligible compared to the viral-induced impairment of alveolar macrophages. while the mechanism for this prediction is not available through initial modeling efforts, it did pinpoint the process driving influenza-pneumococcal synergy that should be subject to further examination in the laboratory . in fact, the decrease in phagocytosis by alveolar macrophage was later determined to be a result of influenza virus directly killing these cells . remarkably, both our models ) and experiments agreed that these cells diminished to 85-90 % of their baseline level within 7 days. receptor-mediated mechanisms may still drive the synergy, although not in the context of enhanced invasion. our model predicts that bacterial interaction with virus-infected epithelial cells releases virus and thus increases viral loads postbacterial invasion. bacterial proteases or nas may liberate virus in the same manner as viral na. s. aureus proteases can activate influenza virus ha cleavage and enhance viral invasion into host cells (tashiro et al. 1987) . furthermore, some commensal bacteria [e.g., s. mitis (nonaka et al. 1983; beighton and whiley 1990) , certain s. pneumoniae (scanlon et al. 1989) , actinomyces naeslundii and a. viscosus (moncla and braham 1989) , porphyromonas gingivalis (moncla et al. 1990) , and s. oralis (homer et al. 1996) ] that secrete nas or exogenous na can rescue influenza virus replication if viral na is missing or inhibited (liu and air 1993; hughes et al. 2000; nishikawa et al. 2012) . thus, it is feasible that other na possessing bacterial species can act in the manner predicted by our model. to uncover the underlying mechanism, a combination of in vitro and in vivo experiments with viruses and bacteria that exhibit differential expression of na is necessary . it is becoming better appreciated that pneumonia is frequently caused by coinfecting pathogens. viral-mediated mechanisms are also important in other invasive infections, such as otitis media. the underlying relationship between viral and bacterial density, inflammation, and the host microbiome during influenza coinfections is exceptionally complex. even with the growing body of work detailing various aspects of viral-bacterial coinfections, determining the precise contributions of each interrelated factor is challenging. furthermore, studying coinfections has become problematic due to the numerous site-, pathogen-, time-, and hostspecific variations to consider. thus, it is necessary to employ the next generation of analyses using a mixture of animal models and kinetic models with the goal of obtaining results translatable to infections in humans. some important areas for consideration 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world health organization global burden of disease world health organization writing group, bell d, nicoll a et al (2006) non-pharmaceutical interventions for pandemic influenza, international measures successive influenza virus infection and streptococcus pneumoniae stimulation alter human dendritic cell function influenza virus pb1-f2 protein induces cell death through mitochondrial ant3 and vdac1 influenza a virus pb1-f2 protein contributes to viral pathogenesis in mice prevalence of pb1-f2 of influenza a viruses acknowledgments this work was supported by nih grant ai100946 (ams) and alsac (jam). we thank betsy williford and klo spelshouse (sjcrh biomedical communications) for assistance with figure illustration. key: cord-302141-gd663uag authors: vousden, nicola; knight, marian title: lessons learned from the a (h1n1) influenza pandemic date: 2020-10-12 journal: best pract res clin obstet gynaecol doi: 10.1016/j.bpobgyn.2020.08.006 sha: doc_id: 302141 cord_uid: gd663uag influenza in pregnancy is a common condition that is associated with increased risk of hospital admission. women with comorbidities are at greater risk of severe outcomes. there are substantial gaps in our knowledge of the impact of severe influenza on perinatal outcomes, especially in low and middle-income countries, but preterm birth, fetal death, infant respiratory infection and hospital admission may be increased. thus, influenza is major burden on health services. immunisation is cost-effective, safe and effective at preventing influenza in pregnant women and their infants but policies and uptake vary worldwide. operational challenges and concern over the safety, efficacy and necessity of the immunisation are common and there is a lack of evidence on how to overcome these barriers. this review identifies learning points relevant to the current covid-19 pandemic through describing the epidemiology and impact of seasonal and a(h1n1)pdm09 influenza in pregnancy, alongside the effectiveness and use of immunisation. influenza is a highly infectious acute viral infection which affects the respiratory tract. in the majority of the population influenza causes mild symptoms which are self-limiting. however, influenza can cause severe illness and even death, with 290,000 to 650,000 respiratory deaths being attributed to influenza annually (1) . it is mainly caused by influenza types a and b which occur in seasonal patterns with epidemics in winter in the northern hemisphere (1) . novel strain of the influenza a virus, termed a(h1n1)pdm09 (h1n1 for brevity in this review), led to a global pandemic estimated to cause between 100 000 to 400 000 deaths globally in the first year. influenza pandemics are impossible to predict and can cause a spectrum of mild to severe disease. whilst the majority of severe cases are in vulnerable groups such as those with chronic medical conditions, healthy persons are more likely to experience serious disease from pandemic than seasonal influenza. this places significant sustained strain on health services and may result in significant economic loss (2) . the aim of this review is to describe the current evidence around the epidemiology and impact of seasonal and h1n1 influenza in pregnancy, the effectiveness and use of influenza immunisation with the aim of identifying learning points from past pandemic relevant to the current covid-19 pandemic. seasonal influenza has been reported to affect between 483 to 1097 pregnant women per 10,000 (3) . these estimates are based on serological testing in women with and without symptoms of infection and it is estimated that approximately 25% develop symptoms (4, 5) . studies reporting the j o u r n a l p r e -p r o o f in nearly every instance (94%) there were delays in appropriate referral, testing and treatment as influenza was not considered a possible diagnosis when pregnant women presented with respiratory illness. this report therefore recommended the promotion of influenza vaccination to pregnant women at any stage of pregnancy and that influenza should be considered early on presentation to health care facilities in order to test and initiate treatment (10) . there are two antivirals used to treat influenza in pregnancy, oseltamivir (tamiflu) and zanamivir; both are neuraminidase inhibitors. a 2014 cochrane systematic review reported that, aside from reduced time to alleviate symptoms, there was little evidence of benefit of treatment in the general adult population (11) . however, pregnant women were specifically excluded from most trials included in the review. evidence from observational studies suggests that compared to those that did not receive treatment, treatment with neuraminidase inhibitors in pregnancy reduces length of hospital stay (12) and the risk of icu admission (6, 13) and early treatment reduced the risk of death compared to those who received late treatment (6) . therefore, uk guidelines recommend that antiviral treatment should be commenced as early as possible in pregnant women with signs of influenza (14). more recently, the 2017 confidential enquiry into maternal deaths and morbidity in the uk described a further death of a pregnant woman from influenza following delay of diagnosis and initiation of treatment. this highlights the necessity for these recommendations to remain on the international agenda even outside of pandemic periods (15) . "a previously healthy woman in the early third trimester of pregnancy was admitted with a respiratory illness during the peak of the influenza season. neither her gp nor the hospital team considered the diagnosis of influenza. it was not considered until a week into her admission by which time she was receiving intensive respiratory support on the critical care unit. antiviral medication was not commenced empirically but only following a positive tracheal aspirate which confirmed h1n1, almost two weeks after the start of her illness. she continued to deteriorate despite ecmo and died. she had not been immunised. it was unclear whether immunisation had been offered". reproduced with permission from 'saving lives, improving mothers' care: lessons learned to inform future maternity care from the uk and ireland confidential enquiries into maternal deaths and morbidity 2009-2012'. in 2015, the who taskforce for influenza in pregnancy undertook a systematic review of the impact of influenza on adverse birth outcomes (16) . this review identified 21 low and very low-quality comparative studies, 20 of which were undertaken in high-income countries, again showing the limited evidence about the impact of influenza other contexts such as in the presence of maternal malnutrition (17) . the two highest quality studies identified that severe h1n1 influenza increased the risk of preterm birth (or 2.39, 95% ci 1.64 -3.49 (18) and or 4.0 , 95% ci 2.71 -5.90 (19) ), whereas, a further study reported no association when women with mild to moderate h1n1 were included (16) . more recently, two studies reported no association between predominantly mild h1n1 influenza and risk of preterm birth (or 0.77, 95% ci 0.32 -1.88 (20) , ahr 1.0, 95% ci 0.98 -1.1(21)), although the risk was increased among women with pre-existing medical conditions (ahr 1.5, 95% ci 1.1 -2.2) including for spontaneous preterm birth (ahr 1.7, 95% ci 1.1 -2.6) (21). furthermore, a national study of women hospitalised with seasonal influenza found that whilst a higher proportion gave birth at <37 weeks, the risk was not significantly increased compared to women without influenza (22) . the who systematic review reported, from five studies, that maternal influenza did not significantly affect the risk of the infant being small for gestational age (or 1.24, 95% ci 0.96 -1.59) (16) . however, the two of the studies based on seasonal influenza reported an increased risk, including the only study of women with severe influenza (aor 1.66, 95% ci 1.11 -2.49) (23), whereas the three studies reporting pandemic influenza found no association (16) . similarly, a recent prospective cohort study found no association between predominantly mild h1n1 influenza and risk of the infant being small for gestational age (or 1.30, 95% ci 0.74 -2.81) (20) . a number of studies have reported the impact of maternal influenza on fetal death, but these are generally very low quality with variable definitions and low case numbers (16) . the two highest quality studies identified by the 2015 systematic review were undertaken during the 2009 pandemic. both reported an increased risk of fetal death following severe (aor 4.2, 95% ci 1.4 -12.4) (19) and mild to moderate maternal influenza (ahr 1.91, 95% ci 1.07 -3.41) (24) . however, a recent national study of women hospitalised with seasonal influenza in the uk found no increased risk of fetal death but an increased risk of admission to neonatal icu (aor 1.86, 95% ci 1.1 -3.42) (22) . similarly, a recent norwegian registry cohort reported that seasonal influenza was not associated with increased risk of fetal death, whereas pandemic influenza significantly increased the risk (ahr 0.90, 95% ci 0.64 -1.27 and 1.75, 1.21-2.54 respectively). this study also explored the impact of gestation at time of influenza and found that the risk of fetal death was highest following influenza-like illness in the first trimester (ahr 2.28, 95% ci 1.45 -3.59) (25) . the risk of congenital anomalies has also been reported to be increased (aor 2.00, 95% ci 1.62 -2.48), including neural tube defects, hydrocephaly, congenital heart defects (26) . as infants have no prior exposure or immunity to influenza they are highly susceptible to influenza illness which results in significant utilisation of health services (27) . a 2017 systematic review undertaken by the who influenza working group reported that there was limited evidence reporting influenza morbidity and mortality outcomes in infants, especially in lmic (28) . the evidence that is available suggests that infant influenza under 6 months of age is associated with increased rates of hospitalisation, severe acute lower respiratory infection (28) and higher death rates (28, 29) . several large prospective cohort studies, predominantly undertaken in the pandemic period, have reported that within the pregnant population there are factors which increase the risk of hospitalization or severe outcomes from influenza (13, 22, 30, 31) . pregnant women with a high bmi j o u r n a l p r e -p r o o f are more likely to be admitted to hospital with influenza (aor 1.9 95% ci 1.15-1.93) and more likely to need intensive care (aor 2.93, 95% ci 1.99 -4.31) (30), however this association was not seen in a recent population cohort with seasonal influenza (22) . whilst not conclusive across all studies, women from black or other minority ethnic groups have also been reported to be at increased risk of hospital admission with h1n1 influenza (aor 1.6, 95% ci 1.1 -2.3) (30). this is in keeping with other studies reporting that indigenous women were at greater risk of icu admission from h1n1 in australia and new zealand (aor 2.3, 95% ci 1.4 to 3.7)(31) and pregnant women admitted to hospital with h1n1 in california were more likely to be hispanic than non-pregnant women (13) . a high proportion of pregnant women with severe outcomes from influenza are reported to have underlying medical conditions (range 33.8% to 55.3%) (6, 13) . the risk of hospital admission with pandemic influenza in pregnancy has also been reported to be increased in multiparous women, those with multiple pregnancies and in women under 25 years old that smoked. (30) there is some evidence that the risk of complications from influenza may be greater in the second and third trimester than the first trimester of pregnancy. women with a gestation of less than 20 weeks have been reported to have double the risk of icu admission with h1n1 compared to non-pregnant women (rr 2.4, 95% ci 1.3 -4.6), whereas those 20 weeks or more had a 13-fold greater risk of admission (rr 13.2, 95% co 9.6 -18.3) (31). similarly in the uk, 86% of pregnant women admitted to icu admission with h1n1 were in their third trimester as shown in figure 1 (30), and in the us 49% of icu admissions and 64% of deaths from h1n1 were in their third trimester (6). <<figure 1 here>> in 2012 the world health organisation released a position statement recommending that pregnant women should be prioritised in vaccination programmes for seasonal influenza and that they should j o u r n a l p r e -p r o o f be vaccinated at any stage of pregnancy. this was made on the basis of the risk of severe disease, safety and efficacy of the vaccine in preventing influenza in women and their infants and operational feasibility (32). prior to this, there has been international effort to increase the availability and capacity to deliver influenza vaccines especially in low-and middle-income countries in the event of a pandemic (33). global production capacity increased substantially from 2006 to 2016 and the who continues to work to increase access in low-resource countries through the pandemic influenzas preparedness framework (34) . influenza vaccines have been used in pregnant women in the us since the 1950s but uptake increased dramatically following the 2009 pandemic. it is widely accepted as safe throughout pregnancy (35) and effective at preventing seasonal influenza in pregnant women and their infants (36) (37) (38) (39) (40) . a pooled analysis of three randomised controlled trials (rct) undertaken in nepal, mali and south africa demonstrated that maternal immunisation prevents 50% (95% ci 32 -63%) of confirmed influenza in pregnancy and up to six months postpartum. the benefit was greater when immunisation was given at or after 29 weeks of gestation (71%, 95% ci 50 -83% compared to 30%, 95% ci 2 -52% before 29 weeks') (41) . however, the mechanism behind this response was unclear as previous studies have shown that antibody response to influenza immunization might decline as pregnancy progresses, therefore the authors reported the study may be underpowered for this outcome. some studies have reported potential benefit in reduced risk of adverse pregnancy outcomes following maternal immunisation, including in preterm birth, (42, 43) low birth weight (43, 44) and stillbirth (45) . however, the findings differ between countries, for example, an rct undertaken in nepal showed a 15% reduction in low birth weight infants in immunised mothers (95% ci 3 -25%) (38) , whereas two studies undertaken in africa showed no difference (37, 40) . overall, a pooled j o u r n a l p r e -p r o o f analysis found that maternal immunisation did not significantly affect the risk of preterm birth, low birthweight or still birth (41) . this apparent lack of effect may be expected given the heterogenous nature of studies on birth outcomes. currently influenza vaccines are not licensed for infants under 6 months of age (46) . therefore, maternal immunisation offers the sole route of protection for infants through transplacental transfer of antibodies from mother to fetus (28) . recent systematic reviews have found that maternal immunisation reduces the risk of laboratory confirmed influenza in infants by 34% (95% ci 15-50%) (47) to 48% (95% ci 33 -59%) and associated hospital admissions by 72% (95% ci 39 -87%) (46) , with the greatest benefit in infants in the first two months of age (41, 46) . a pooled analysis of three rcts undertaken in lmic reported that maternal influenza immunization is also 20% effective at protecting young infants against severe pneumonia (48) and reduces the risk of hospital admission with all cause acute lower respiratory illness by 44% (95% ci 1 -68%) (46) . influenza immunization in pregnancy has been demonstrated to be cost-effective for both seasonal and pandemic influenza (49) (50) (51) . a study in the uk found that immunising pregnant women against seasonal influenza was associated with an incremental cost-effectiveness ratio of around £23,000. this assumed that the vaccine is well-matched to the circulating strain and is delivered largely by nurses in primary care settings during routine antenatal care. delivery by midwives or in separate appointments would be associated with additional cost (52) . further studies have suggested that immunisation targeted to women with co-morbidities would be cost-saving (53). one study in mali estimated that maternal influenza immunisation could be highly cost-effective in low-income countries if programmatic costs are kept low, for example through delivery in combination with existing vaccine programmes (54) . however, overall there is little evidence on the j o u r n a l p r e -p r o o f economic burden of influenza and thus the value of seasonal influenza immunisation in pregnancy in lmic (55) . the limited availability of incidence and impact data in this setting, with differing access to care and underlying comorbidities such as hiv and malnutrition, undoubtedly underlies this problem. indeed in 2013, the global alliance for vaccines and immunisation (gavi) considered and rejected an investment in maternal influenza immunisation programmes in lmic due to limited data about the local burden of disease and anticipated impact (17) , which was also reflected in a report by bill and melinda gates in 2015 (56) . more recent, studies demonstrating the impact on infants in lmic may go some way towards meeting this challenge in the future (46, 48) . a 2014 report identified that worldwide, 42% of who member states had influenza immunization policies that targeted pregnant women. this varied widely by region from 6% in africa to 64% in europe (57) . countries with national policies were more likely to be high or upper-middle income classification (57) . since the 2009 pandemic, immunisation is increasingly recommended for all pregnant women regardless of trimester or underlying comorbidities as shown in figure 2 a 2013 systematic review of the uptake of influenza vaccination in pregnant women identified a number of characteristics that increased the likelihood of immunisation. these included women that were of white ethnicity and non-smokers (65) , which have been confirmed in more recent studies (22, 66) . several studies have reported an association with higher socioeconomic status such as professional employment or higher education (65) but not all studies are conclusive (22) . the characteristics of pregnant women associated with influenza vaccination uptake during seasonal campaigns and the h1n1 pandemic have been reported to be similar (65) . the reasons for differing coverage are complex, interrelated and specific to different contexts. in lower-resourced countries, limited vaccine procurement impedes coverage (58) . where there is j o u r n a l p r e -p r o o f adequate policy and procurement in place, a number of other barriers have been cited. the circulating influenza strain is constantly changing; therefore, the strain of vaccine is changed annually in both the northern and southern hemisphere to match the circulating strain. this, along with diminishing immunity over time, means that vaccination in every pregnancy is recommended. in temperate climates there are clear seasonal patterns of influenza transmission, so the vaccine is targeted in annual campaigns at the start of these periods. this has considerable operational implications for example, in procurement, cold chain storage and health system capacity (56) . it is also recommended that vaccine delivery occurs through pre-existing healthcare contacts, for example through routine antenatal care (67) as easy access to vaccination services improve the uptake (65) . in many lmic, coverage of antenatal care means the opportunities for vaccine provision may be fewer. argentina's success has been attributed to joint effort between operational and central health teams with the support of scientific societies and opinion leaders and a committed campaign for community awareness including media communications (63) . in the uk, vaccines can be provided by primary care, hospital based maternity care or community pharmacies. whilst this provides multiple opportunity for provision, it means accurate data capture is challenging and local policies differ, with immunisations rarely being provided in the same contact as antenatal care. in light of the sars-cov-2 pandemic, in the 2020/21 influenza season all nhs hospitals will be asked to offer vaccinations to pregnant women attending maternity appointments with the aim of improving coverage (68). in the uk there is variation in the proportion of women immunized against influenza based on their estimated due date. women in their first or third trimester during the main influenza immunization programme in november are less likely to be immunized (22) . whilst in the first trimester this may represent the delay in recognising or booking pregnancy, women in their third trimester have substantial contact with hcp and opportunity for immunisation. the most commonly cited reasons j o u r n a l p r e -p r o o f for not being immunised include: the perception that influenza is a mild disease and that the risk of infection was low and lack of accurate knowledge that the vaccine is effective and safe (65, 66, 69) . data from public health england show that only a small proportion of pregnant women in the uk refuse or decline immunisation (5·7%) (64) . similarly, a systematic review identified that often women were not aware they should receive the vaccine (65) . provision of information on immunisation by hcp has consistently been shown to increase immunisation uptake by as much as 100-fold (70) (71) (72) . pregnant women who were recommended immunisation by hcp were also more likely to believe it to be safe and effective (65) . however, evidence from surveys of hcp found that only 62% of midwives had received training on immunisation in pregnancy and 60% were confident in giving advice. only 9% gave immunisations despite the majority being happy to do so (73) . whilst there is considerable evidence about the barriers to immunisation in pregnancy, there is little evidence to support interventions to overcome them. the national institute for health and care excellence (nice) undertook a systematic review in 2018 which identified few studies in pregnancy. a number of interventions including education resources /information by healthcare professionals, message reminder services and multi-component interventions including vaccine champions and interactive educational materials have been studied, with predominantly negative findings (74). we have described a number of characteristics that increase the risk of severe outcomes with influenza in pregnancy. it is important to note the similarities with those hospitalised with sars-cov-2, where over half of pregnant women admitted to hospital with sars-cov-2 infection in pregnancy were from black or other ethnic minority groups (56%), two thirds were overweight or obese (69%) and a third had pre-existing co-morbidities (34%) (75) . this suggests that these groups are at greater j o u r n a l p r e -p r o o f risk of future viral pandemic illness, and pandemic preparedness plans should take this into account. there may also be a role in optimising health pre-pregnancy to reduce the risks associated with seasonal influenza, alongside promotion of immunisation and a lower threshold for seeking health care and admitting women from these risk groups, as has been recommended for women of bame groups by the royal college of obstetricians and gynaecologists (rcog) in the sas-cov-2 pandemic (76) . despite proven safety and efficacy, influenza immunisation policy and uptake vary globally. for policymakers to make informed decisions about immunisation programmes, reliable data are essential. it is therefore vital that pregnant women are included in immunisation trials, yet they are often systematically excluded (77) . a recent media report suggested that the first two covid-19 vaccines to enter large-scale testing in the us will not include pregnant women (78), similarly pregnant women or those planning to become pregnant are currently excluded from both national vaccine trials in the uk (79). maternal immunisation has the potential to protect not just the mother but also the baby prior to delivery, and in the first few months of life, as shown with the influenza vaccine. research should therefore also aim to assess the impact of immunisation throughout this vulnerable period in order to fully inform investment decisions. should a covid-19 vaccine become available for use in pregnancy, implementation programmes will need to take into account the challenges faced in delivery of other immunisations. however, there is a lack of evidence identifying ways to overcome barriers to vaccination of pregnant women. strategies could consider ease of access, ideally within routine antenatal care with training of maternity care providers, and dissemination of accessible information on the risks and benefits to the mother, fetus and infant. communication strategies would need to remain flexible as j o u r n a l p r e -p r o o f information arises and may differ as risk groups are identified for example targeting language barriers (80) . influenza in pregnancy is a relatively common condition that can cause increased risk of hospital admission, especially in the later stages of pregnancy. whilst the risk of severe morbidity and mortality from influenza in pregnancy is not increased compared to the general population, it still causes excess hospital admissions and preventable deaths. some studies suggest that women with a raised bmi, of black or other minority ethnic group, or with underlying medical conditions are at greater risk of severe outcomes. data on the perinatal impact are very heterogenous but there is evidence that severe influenza may increase the risk of preterm birth and fetal death or intensive care admission. in infants under 6 months, limited evidence suggests that influenza increases risk of hospitalisation and severe lower respiratory infections. there are substantial gaps in our knowledge of the impact of influenza, especially in lmic. future pandemic strategies in pregnancy should consider mechanisms for collecting and communicating data. influenza vaccines are cost-effective, safe and effective at preventing influenza in pregnant women and their infants. however, the majority of evidence relates to high-income settings. pregnant women should be included in vaccine trials, which should also examine the impact on infants. policies on immunisation in pregnancy vary worldwide and many countries have poor uptake. there are many interrelated reasons for this, but operational challenges and concern over the safety, efficacy and necessity of the immunisation are common. there is insufficient evidence on how to overcome these barriers. j o u r n a l p r e -p r o o f • influenza immunisation should be promoted to all pregnant women at any stage of pregnancy. • antiviral treatment should be commenced as early as possible in pregnant women with signs of influenza. • pandemic preparedness plans should consider mechanisms to collect and communicate data and how to deliver essential maternity care safely. • there is a lack of epidemiological, and therefore cost-effectiveness, studies of influenza in pregnancy undertaken in low and middle-income countries. current surrogates of hospital and icu admission may inadequately describe morbidity in this setting. • vaccine trials should include pregnant women and should explore the longer-term impact of maternal immunisation on infant outcomes. • future research should explore how to overcome barriers to immunisation in pregnancy. influenza (seasonal) factsheet incidence of influenza virus infection among pregnant women: a systematic review community studies of influenza: new knowledge, new questions. the lancet respiratory medicine comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study. the lancet respiratory medicine pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states severity of 2009 pandemic influenza a (h1n1) virus infection in pregnant women pregnancy as a risk factor for severe outcomes from influenza virus infection: a systematic review and meta-analysis of observational studies pregnancy as a risk factor for severe influenza infection: an individual participant data meta-analysis saving lives, improving mothers' care: lessons learned to inform future maternity care from the uk and ireland confidential enquiries into maternal deaths and morbidity neuraminidase inhibitors for preventing and treating influenza in healthy adults and children. the cochrane database of systematic reviews benefit of early initiation of influenza antiviral treatment to pregnant women hospitalized with laboratory-confirmed influenza department of health and royal college of obstetricians and gynaecologists improving mother's care-lessons learned to inform maternity care from the uk and ireland confidential enquiries into maternal deaths and morbidity maternal influenza and birth outcomes: systematic review of comparative studies influenza epidemiology and immunization during pregnancy: final report of a world health organization working group maternal and neonatal outcomes among pregnant women with 2009 pandemic influenza a(h1n1) illness in florida, 2009-2010: a population-based cohort study /h1n1 infection: national cohort study risk of pregnancy complications and adverse birth outcomes after maternal a(h1n1)pdm09 influenza: a norwegian population-based cohort study the relationship between 2009 pandemic h1n1 influenza during pregnancy and preterm birth: a population-based cohort study incidence, risk factors and impact of seasonal influenza in pregnancy: a population-basede case control study effect of respiratory hospitalization during pregnancy on infant outcomes risk of fetal death after pandemic influenza virus infection or vaccination seasonal and pandemic influenza during pregnancy and risk of fetal death: a norwegian registry-based cohort study influenza and congenital anomalies: a systematic review and meta-analysis global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis. the lancet incidence of laboratoryconfirmed influenza disease among infants under 6 months of age: a systematic review influenzaassociated deaths among children in the united states the 2015 global production capacity of seasonal and pandemic influenza vaccine world health organisation. pandemic influenza preparedness framework safety of influenza vaccines in pregnant women effectiveness of maternal influenza immunization in mothers and infants maternal immunisation with trivalent inactivated influenza vaccine for prevention of influenza in infants in mali: a prospective, active-controlled, observer-blind, randomised phase 4 trial. the lancet infectious diseases year-round influenza immunisation during pregnancy in nepal: a phase 4, randomised, placebo-controlled trial. the lancet infectious diseases clinical infectious diseases : an official publication of the infectious diseases society of america influenza vaccination of pregnant women and protection of their infants efficacy, duration of protection, birth outcomes, and infant growth associated with influenza vaccination in pregnancy: a pooled analysis of three randomised controlled trials. the lancet respiratory medicine fetal death and preterm birth associated with maternal influenza vaccination: systematic review the effects of influenza vaccination during pregnancy on birth outcomes: a systematic review and meta-analysis the safety of inactivated influenza vaccines in pregnancy for birth outcomes: a systematic review maternal influenza immunization and birth outcomes of stillbirth and spontaneous abortion: a systematic review and meta-analysis. clinical infectious diseases : an official publication of the infectious diseases society of america influenza vaccination during pregnancy for prevention of influenza confirmed illness in the infants: a systematic review and meta-analysis the effectiveness of influenza vaccination in pregnancy in relation to child health outcomes: systematic review and meta-analysis maternal influenza immunization and prevention of severe clinical pneumonia in young infants: analysis of randomized controlled trials conducted in nepal, mali and south africa. the pediatric infectious disease journal clinical infectious diseases : an official publication of the infectious diseases society of america vaccination against pandemic influenza a/h1n1v in england: a real-time economic evaluation cost-effectiveness of seasonal inactivated influenza vaccination among pregnant women the cost-effectiveness of vaccinating pregnant women against seasonal influenza in england and wales an incremental economic evaluation of targeted and universal influenza vaccination in pregnant women costeffectiveness of maternal influenza immunization in bamako, mali: a decision analysis a systematic review of the social and economic burden of influenza in low-and middle-income countries path to impact: a report from the bill and melinda gates foundation convening on maternal immunization in resource a global review of national influenza immunization policies: analysis of the 2014 who/unicef joint reporting form on immunization how close are countries of the who european region to achieving the goal of vaccinating 75% of key risk groups against influenza? results from national surveys on seasonal influenza vaccination programmes an analysis of national target groups for monovalent 2009 pandemic influenza vaccine and trivalent seasonal influenza vaccines in 2009-10 and 2010-11 pandemic h1n1 (swine) influenza vaccine uptake amongst patient groups in primary care in england. department of health, health protection authority pandemic a(h1n1) 2009 influenza vaccination survey, influenza season pandemic influenza vaccination during pregnancy: an investigation of vaccine uptake during the 2009/10 pandemic vaccination campaign in great britain maternal immunization in argentina: a storyline from the prospective of a middle income country seasonal influenza vaccine uptake in gp patients: winter season determinants of uptake of influenza vaccination among pregnant women -a systematic review determinants of influenza and pertussis vaccination uptake in pregnancy: a multicenter questionnaire study of pregnant women and healthcare professionals. the pediatric infectious disease journal department of immunization, vaccines and biologicals, world health organisation, geneva; 2017. 68. department of health & social care and public health england cross-sectional study on factors associated with influenza vaccine uptake and pertussis vaccination status among pregnant women in germany knowledge and attitiudes of pregnant women and their providers towards recommendations for immunization during pregnancy midwives' influenza vaccine uptake and their views on vaccination of pregnant women increased awareness and health care provider endorsement is required to encourage pregnant women to be vaccinated vaccination in pregnancy: attitudes of nurses, midwives and health visitors in england flu vaccination: increasing uptake, evidence reviews for increasing uptake in clinical risk groups. national institute for health and care excellence characteristics and outcomes of pregnant women admitted to hospital with confirmed sars-cov-2 infection in uk: national population based cohort study royal college of obstetricians and gynaecologists and the royal college of midwives covid-19) infection in pregnancy: information for healthcare professionals consider pregnancy in covid-19 therapeutic drug and vaccine trials. the lancet covid-19 vaccine trials will exclude pregnant women for now university of oxford. covid-19 vaccine study (cov002) oxford, united kingdom2020 pandemic influenza and pregnant women: summary of a meeting of experts the authors have no conflict of interests to declare. key: cord-283537-49ic7p3u authors: chong, ka chun; goggins, william; zee, benny chung ying; wang, maggie haitian title: identifying meteorological drivers for the seasonal variations of influenza infections in a subtropical city — hong kong date: 2015-01-28 journal: int j environ res public health doi: 10.3390/ijerph120201560 sha: doc_id: 283537 cord_uid: 49ic7p3u compared with temperate areas, the understanding of seasonal variations of influenza infections is lacking in subtropical and tropical regions. insufficient information about viral activity increases the difficulty of forecasting the disease burden and thus hampers official preparation efforts. here we identified potential meteorological factors that drove the seasonal variations in influenza infections in a subtropical city, hong kong. we fitted the meteorological data and influenza mortality data from 2002 to 2009 in a susceptible-infected-recovered model. from the results, air temperature was a common significant driver of seasonal patterns and cold temperature was associated with an increase in transmission intensity for most of the influenza epidemics. except 2004, the fitted models with significant meteorological factors could account for more than 10% of the variance in additional to the null model. rainfall was also found to be a significant driver of seasonal influenza, although results were less robust. the identified meteorological indicators could alert officials to take appropriate control measures for influenza epidemics, such as enhancing vaccination activities before cold seasons. further studies are required to fully justify the associations. hong kong, a city located in the south china sea, has a humid subtropical climate with winter (december-february) temperatures that usually range from 10 to 20 °c, warm springs and autumns, and hot summers (june-september) with daytime temperatures in the low to mid 30 s and nighttime temperatures in the high 20 s. in temperate regions, influenza has a clear seasonal pattern with an exponential increase in infections in the winter, which is followed by a fade-out period of a few months. in subtropical regions, there is no sufficient understanding of the seasonal pattern of influenza and its relationship with meteorological factors. the number of epidemic peaks can differ across various subtropical regions, with the peaks usually occurring at different periods within a year [1] [2] [3] . according to the world health organization (who), influenza epidemics result in 250 to 500 thousand deaths worldwide annually [4] . in hong kong, the influenza hospitalization rate and the pneumonia and influenza (p&i) associated mortality were estimated to be 29 and 4.1 per 100,000 person-years respectively [5, 6] . hong kong was also regarded as an epicenter of pandemic influenza in southeast asia. insufficient information about viral activity creates difficulties in forecasting the disease burden and thus hampers official preparation efforts. despite numerous researches that have discovered meteorological factors associated with various activities of influenza, little is known about the drivers of transmission or its seasonal variations for different climates. a small variation in influenza transmission could result in amplification and damping of infection oscillations over time and thus sustain a seasonal pattern [7] . possible drivers of influenza transmission include meteorological variations [8] , susceptible numbers [9, 10] , and social mixing [3] . recently, shaman et al. employed a mathematical model to demonstrate that the seasonal pattern of influenza in the united states could be drawn based on the process of simulations driven by the absolute humidity [8] . this finding motivated a further investigation of potential meteorological drivers for subtropical climates. in this study, meteorological determinants that could drive the seasonal variations of influenza in hong kong were investigated by a mathematical model. we hypothesized that the transmission rates in a population-level model, as well as the infection oscillations of seasonal influenzas, are affected by meteorological factors. identification of the drivers will help to improve the understanding of influenza transmission and to alert officials to implement preemptive control measures for seasonal influenza. data on deaths from p&i from 2002 to 2009 in hong kong were obtained from the hong kong census and statistics department (figure 1 ). the mid-year population (nyear) from 2002 to 2008 was collected from the hong kong census and statistics department [11] . we separated each wave of p&i deaths by year from week 35 to week 34 of the following year. as the wave of 2007 was stopped earlier, it would start from week 35 until week 23 of 2008. the 2008 wave is from week 24 to week 16 of 2009, in order to prevent the overlap of cases after the outbreak of the 2009 h1n1 pandemic. the weekly average of meteorological parameters: air temperature, relative humidity, total rainfall, total solar radiation, wind direction, and wind speed from 2002 to 2009 were collected from the hong kong observatory. the time series of the data is shown in figure 2 . actual vapor pressure (e) was calculated as a metric for absolute humidity by the teten's formula [12, 13] : where es(ta) is the saturation vapor pressure (hpa), rh is the relative humidity (%), and ta is the air temperature (°c). the es was calculated as follows: ta ta ta e s 7 . 237 27 . 17 exp 105 . 6 ) ( the saturation vapor pressure in the teten's formula can also be obtained by the integration of clausius-clapeyron equation and is acceptable for most meteorological purposes [12, 14] . as the wind data was in the polar coordinate scale, we develop wind velocity variables in the cartesian scale that encompasses wind direction and wind speed, thus preventing the problem of northerly bearings being split at true north. two parameters of wind velocity (east-to-west and north-to-south) were used as metrics for wind data in the analysis. we extended the susceptible-infected-recovered (sir) model from chowell et al. [15] to describe the dynamic system of seasonal influenza. in this model, a population is comprised of four compartments: susceptible (s(t)); infectious (i(t)); recovered (r(t)); and dead (d(t)), at each time point t. the sir model consists of four differential equations that describes the rates of subject movements for each of the time steps. we assumed homogeneous mixing, meaning that each individual has the same chance of contacting another individual within the population. in the compartmental model, once susceptible individuals in compartment s(t) get infected, they will move to compartment i(t) and stay there for the infectious period. when the infectious period is over, the individuals in compartment i(t) will recover and move to compartment r(t) or will die and move to compartment d(t). in this model, the time-varying transmission rate per individual is βt and the force of infection for time t is βti. we denote s(t), i(t), r(t), and d(t) as s, i, r, and d as the subpopulations in each compartment for time t. the deterministic system of equations are as follows: we assumed that the length of the generation interval (gi) follows an exponential distribution with mean = 1/(γ + δ). suppose cfp is the average case fatality proportion, the mortality rate is δ = (cfp/(1 − cfp)) and δi is the influenza deaths generated by the differential equations. to make the model coefficients more comparable to each other, meteorological variables are transformed by subtracting the mean and divided by the standard deviation (sd) over their sampling period. let xt i be a particular i-th independent variable (e.g., air temperature at time t), the transformed form would be: where i x is the sample mean and σ i x is the sd for the sampling period. the meteorological effects are related to βt using the following linear component: where n is the number of independent variables. the model will determine the significant drivers to the influenza transmission rate. in the differential equations, we assumed a 5-day length of gi [16] and a 0.2% cfp [17] . to account for the variation of partial immunity to the seasonal influenza, we followed previously published procedures [15, 18] . the initial number of susceptibles in equation (3) was calculated by where d(0) was set to be the number of p&i deaths in the first epidemic week. thus, the initial number of recovered individuals can be calculated as rather than fixing a value, mid-year population (nyear) was used for each wave, so as to reduce the impact from natural mortality and birth. the weekly p&i death data was fitted to model generated deaths (i.e., δi) and the meteorological time series data consisted of the variables (xt i ) for each epidemic wave. parameters i(0), b0, b1,…,bn could be estimated by least-squares fitting to the data. as weekly data was used, t was measured in weeks. statistically significant meteorological parameters (p-value of bi < 0.05) were declared as potential drivers to seasonal variations of influenza. a stepwise variable selection approach was adopted and the best fitted model was chosen as the one with all statistically significant variables and the lowest akaike information criterion (aic) [19] : where m is the number of data points, p is the total number of parameters, and sse is the sum of square errors. instead of sampling, all possible parameter combinations were assessed by a grid search. as the absolute humidity was derived from the temperature and relative humidity, they could not be included in the same variable pool during the stepwise variable selection, due to the co-linearity problem. the variable set with temperature, relative humidity, plus other variables and the set with absolute humidity plus other variables were separately adopted in the variable selection in order to draw two final models. the best fitted model was then chosen based on aic value (lower being better). adjusted r-square (adj-r 2 ) is the measure of proportion of variance explained by the model after the parsimony adjustment. the difference of adj-r 2 between the null model and fitted model (δadj-r 2 ) was interpreted as the proportion of variance explained by the meteorological factors. we conducted a sensitivity analysis addressing two aspects: (1) model parameters: sensitivity analysis was performed by varying the length of the gi for 3 days and 7 days [16] and the cfp for 0.1% and 0.4% [17] . (2) model structure: in addition to the linear form of equation (5), a multiplicative exponential form was also adopted in model fitting to test whether this would produce different results: table 1 summarizes the results of the best fitted models with lowest aic and all statistical significant meteorological parameters. compared with the null models (βt = constant), models with meteorological parameters had better goodness of fit in terms of their aic (figure 3 ). no null model was found to be the best fitting model after the stepwise variable selection. with the exceptions of the 2003 and 2005 p&i waves, adj-r 2 was always greater than 40%, indicating that models with selected meteorological parameters explained more than 40% of the variance in p&i mortality after the adjustment of number of parameters. except for 2004, the models with significant meteorological factors could account for more than 10% of variance in addition to the null model (i.e., δadj-r 2 > 10%). for the 2008 epidemic, the meteorological parameters accounted for more than 50% of the variability in the p&i data. nevertheless, the p&i data of the 2004 epidemic could not be well explained by the best fitted models. as shown in table 1 , air temperature and rainfall were the most common significant variables driving the seasonal variations of the p&i waves from 2002 to 2008. the air temperature was negatively associated with the time-varying transmission rate βt in six of the seven epidemics; for one sd decrease in temperature, the transmission rate would increases by 4.1, 9.8, 2.6, 2.9, 5.2, 3.7 (×10 −9 ) for the years 2002, 2003, 2004, 2006, 2007 and 2008 respectively. moreover, rainfall was positively associated with the transmission intensity in five of the seven epidemic waves. when there was a sd increase in rainfall, the transmission rate would increases by 10.7, 4.5, 2.3, 9.8, 5.5 (×10 −9 ) for the years 2003-2006, and 2008 respectively. a negative association was found for 2007. surprisingly, relative humidity and absolute humidity did not show much contribution to the variance of βt among all the p&i epidemics. a sensitivity analysis was conducted to test the impact of our results from different parameter settings. in brief, varying the cfp (0.1% and 0.4%) and gi (3 and 7 days) only produced a slight effect on the goodness of fits. as shown in figures 4 and 5 , the best fitting curves were highly similar. in terms of aic and adj-r 2 , no significant differences were produced as a result of using different cfp and gi settings (tables 2 and 3 ). the fitness of the models with either cfp = 0.1% or gi = 3 days were worse than the other models in several epidemic waves. the effect of temperature was only slightly sensitive to the variation in cfp and gi. in most situations, air temperature continued to be identified as a common driver of seasonal variations. when gi = 3 days, air temperature significantly drove the variations in all epidemics. whereas the effect of rainfall was moderately sensitive to variations in cfp and gi. rainfall was identified as the significant driver of four of the studied influenza epidemics. the decrease of rainfall's significance may be due to the model variance shared with relative humidity. the meteorological variable selection was not sufficiently sensitive, even if the model structure was changed to the exponential form (table s1 ). recent studies have demonstrated that environment factors account for a proportion of the seasonality, as well as infection oscillations, of influenzas in temperate regions [8, 9] . here we used a mathematical model to explore the potential meteorological drivers for seasonal oscillations of influenza in a subtropical city, hong kong. through modulating the transmission rates by the meteorological factors in an infectious disease model, the seasonal variations of influenza infections could be well-depicted. according to our results, although no meteorological parameters dominated the seasonal variations for all epidemics, air temperature significantly modulated the fluctuations of transmission rates for most of the epidemics between 2002 and 2009. rainfall was also found to be a significant driver for most of the epidemics, although its direction of association was not unidirectional and it was moderately sensitive to changes in the model parameters. in many laboratory and epidemiological studies, air temperature is often found to be associated with influenza transmissions [1, [20] [21] [22] [23] [24] . an epidemiological study from chan et al. [1] found that temperature and relatively humidity were associated with the activity of seasonal influenza in hong kong; a cold and humid climate was related to higher activities of both influenza a and b. lowen et al. [21] conducted an experimental study using a guinea pig model to demonstrate that cold temperature favored to the spread of the influenza virus. our study extended these findings by showing that cold temperature was associated with the mechanism driving seasonal oscillations at a population level. this is perhaps due to prolonged survival of viral particles under colder conditions. nevertheless, the effect of temperature could be confounded by other factors [25] . for example, a decrease in temperature could enhance crowding at indoor activities, and would thus increase the contact, aerosol and droplet transmission intensity. in our study, we could not identify any strong evidences that absolute humidity drove the seasonal variability in hong kong, even though experimental and modeling studies have shown that absolute humidity was related to viral survivorship and was capable of driving the seasonality of influenza in temperate regions [9, 10, 25] . one possible explanation for this is that the absolute humidity in hong kong was high all year around, compared to temperate areas. like other tropical and subtropical regions, use of air conditioning is common in hong kong when the temperature is high. one could argue that, using air conditioning would lower the indoor absolute humidity and thus modulate the survivorship of the influenza virus. the effect from air exchange would indeed offset the impact of disease transmission. in addition to cold temperature, experimental studies have indicated that a low relative humidity could enhance the influenza transmission [21] . according to our results, relative humidity was not identified as a significant driver for the seasonal variation of influenza infection. this might be accounted for by the relative unpopularity of indoor heating in hong kong compared to temperate regions. moreover, the predominant mode of influenza virus spread was proposed to be different between temperate and tropical regions [26] . relative humidity would be more insensitive for transmissions by the contact route than by the aerosol route. previous studies shown that rainfall could be used as a predictor to forecast influenza infection rates for sub-tropical regions, but not in all temperate regions [27] . these authors also indicated that rainfall was correlated with seasonal influenza transmission in hong kong [20] , and this finding was in line with other tropical areas [28] . nevertheless, there remains no clear and definitive explanation for the mechanism of rainfall driving the influenza seasonality. although low temperature and dry air have been proven to be favorable for survival of viral particles [22] , no study has investigated the relationships between rainy conditions and bulk aerosol transport. one plausible mechanism is that rainfall could affect human social behaviors, such as indoor activities, and therefore influence the number of contacts and the risk of exposure to contaminated environments or infected individuals. in our study, we found that rainfall significantly drove some epidemics but that its direction of association was not unidirectional, likely due to the problem of multicollinearity, which has been investigated in our association analysis (table s2) . solar radiation could cause seasonal variations in vitamin d photosynthesis that may affect immune responses as well as playing a role in the influenza seasonality [29, 30] . the preventive efficacy of vitamin d supplementation against influenza infections has also been demonstrated in trial studies [31] . our study did not identify solar radiation as a driver for the seasonality of influenza infection. this result was not surprising because the effect of solar radiation on the population of the subtropics is not as well documented as in temperate regions. in addition, influenza a would be more likely affected by vitamin d status than influenza b [31] . this factor might confound our findings when p&i data was adopted in the study. nevertheless, the role of solar radiation in seasonality remains controversial because it has been difficult to explain the influenza dynamic in outdoor environments; most transmissions occur in indoor environments through airborne transmission or contact [32] . rather than pooling all of our data, the purpose of conducting the analysis by seasons was to investigate the meteorological effects independent of the between-season variations. the between-season effect was made up by "nuisance variables", which could result from variations of reporting rate and other potential factors that affected the susceptibility numbers [10] , such as the vaccination effectiveness. although the partial immunity of the seasonal influenza was adjusted in our analysis, some factors are difficult to measure and interpret. analysis by seasons could confound the relationship between meteorological factors and transmission rate, and thus generate inconsistency for the estimated coefficients (e.g., a negative association of rainfall in 2007). an additional analysis was conducted using the pooled data and the results were summarized in supplementary table s3 . by this approach the main finding was unchanged (i.e., a low temperature drove the influenza transmission). it should be noted that between-season effect accounted for 20% of the total variance in addition to the meteorological determinants. in our study, there is undoubtedly some degree of correlation between meteorological variables. hence, we additionally conducted a correlation analysis in which the pearson correlation coefficients and variance inflation factors (vif) were drawn from the pooled data, with the results summarized in supplementary table s2 . although positive correlations were found between temperature and solar radiation, and between rainfall and humidity, no serious effect of multicollinearity was found for any of the predictors based on a simple rule-of-thumb (i.e., all vifs were less than 3). one limitation of our study is that we only investigated the environmental drivers for disease transmission and could therefore not completely rule out confounding factors. according to some studies [8] [9] [10] 22] , some seasonal changes of host behavior (e.g., international travel [33, 34] and school holidays [35] ) might also affect the transmission dynamics. it has been shown that the closure of kindergartens and primary schools was able to reduce the disease transmission rate by around 25% for the 2009 influenza a/h1n1 pandemic. nevertheless, its effect upon the seasonal variation of influenza is controversial. some studies [10, 36] pointed out that no substantial effect on the transmission reduction could be detected when schools were closed. in addition, our results were undoubtedly affected by the demographics of the population (e.g., age and gender). subjects with different clinical status, such as chronic obstructive pulmonary disease, may also have confounded the likelihood of p&i deaths. the sufficiency of details to address these issues requires a huge effort in data collection, which remains difficult to achieve at this stage. further research is warranted to investigate the effects of seasonal social/behavior patterns. a limitation to our study is that the use of p&i mortality to represent the influenza activity may not be completely adequate and could potentially bias the findings. although some studies have preferred using p&i mortality [15] , we also analyzed the p&i excess mortality to test the robustness of the study finding. we adopted the traditional serfling approach to estimate the excess mortality [37, 38] . the serfling method is a linear regression model using harmonic terms to calculate the expected mortality in the absence of influenza virus activity. the number of excess deaths attributable to influenza was estimated as the difference between the observed and the upper 95% limit of the prediction interval of baseline deaths. the details were noted in supplementary table s4 . from the results, the principal finding was unchanged (i.e., air temperature remained a significant driver of the seasonal patterns). it should be noted that no climatic variables can be fitted into the year 2003 due to few p&i excess mortality. this might be due to the mitigation measures for the severe acute respiratory syndrome (sars) epidemic that potentially also reduced the number of influenza cases [39] . undoubtedly, it has been widely recognized that the disease severity of influenza, in terms of excess p&i deaths and hospitalization, tended to be higher in the influenza a dominant seasons than in those with influenza b as the dominant virus strains [5] . moreover, some influenza b epidemics resulted in increased hospitalizations but not increased mortality [40] . as a result, our findings might be less precise for mild influenza seasons and may not reflect the general influenza experience in hong kong. this is a common limitation in studies that employed mortality surveillance. in addition to death data, influenza-like illness (ili) surveillance has been commonly adopted as a proxy for influenza activity. nevertheless, the definition of ili failed to document significant influenza-associated morbidity and mortality [5] . as such, ili is a poor indicator of influenza activity when adopted in areas with a less defined pattern of seasonality [41] . laboratory surveillance data would be a better indicator for influenza activity but large efforts would be required to gather and collate such data. the data used in this study was only applied the retrospective fitting. further studies would have to be conducted to validate these results. for example, more data is required to extend the model application to projecting the sir curve for model validations [8] . plausible causality and potential interactions should also be justified, such as direct and indirect effects of air temperatures [22] . nevertheless, our study represents an initial step towards identifying potential meteorological determinants for driving the seasonal variations of influenza in a subtropical region. this study identified the potential meteorological drivers for the seasonal variations of influenza in a subtropical city, hong kong. results show that the cold air temperature was a significant driver for increasing the transmission intensity of seasonal influenza from 2002 to 2009. rainfall was also found to be a significant driver for some seasons, although this result was less robust. an accurate would enable officials to take appropriate control measures for influenza epidemics, such as maintaining sufficient indoor temperature and enhancing vaccination activities prior to the cold seasons. further laboratory and epidemiological studies are required to validate and justify the associations proposed here. seasonal influenza activity in hong kong and its association with meteorological variations influenza in tropical regions 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humidity on seasonal influenza in tropical areas the seasonality of pandemic and non-pandemic influenzas: the roles of solar radiation and vitamin d influenza, solar radiation and vitamin d. dermatoendocrinology randomized trial of vitamin d supplementation to prevent seasonal influenza a in schoolchildren a note on the inactivation of influenza a viruses by solar radiation, relative humidity and temperature modeling the impact of air, sea, and land travel restrictions supplemented by other interventions on the emergence of a new influenza pandemic virus assessing the impact of airline travel on the geographic spread of pandemic influenza school closure and mitigation of pandemic (h1n1) effects of school closures, 2008 winter influenza season methods for current statistical-analysis of excess pneumonia-influenza deaths the impact of influenza epidemics on mortality: introducing a severity index respiratory infections during sars outbreak the impact of influenza epidemics on hospitalizations clinical signs and symptoms predicting influenza infection this study was supported by the project (2012zx09303012-002) from the shenzhen research institute, the chinese university of hong kong, shenzhen, china. the authors thank the hong kong observatory, the hospital authority and the environmental protection department for providing the datasets for this study. the authors also thank external reviewers for their expertise advice on constructing the manuscript. william goggins and maggie haitian wang collected the data and conducted the data analysis. ka chun chong conducted the data analysis, summarized the results, and wrote the manuscript. benny chung ying zee initiated the research and revised the manuscript. all the authors read and approved the final version of the manuscript. the authors declare no conflict of interest. key: cord-266100-1rktb6yq authors: darwish, ilyse; miller, chris; kain, kevin c.; liles, w. conrad title: inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza date: 2012-01-13 journal: int j med sci doi: 10.7150/ijms.3880 sha: doc_id: 266100 cord_uid: 1rktb6yq in vitro, nitric oxide (no) has been shown to have antimicrobial activity against a wide range of viruses, including influenza a virus. therefore, we hypothesized that inhaled nitric oxide (ino) would increase survival in vivo by reducing the viral load in c57bl/6 mice infected with a lethal dose of influenza a/wsn/33 (h1n1; wsn/33) virus. no was delivered to influenza-infected mice either continuously or intermittently at 80 or 160 ppm, respectively, using both prophylactic and post-infection treatment strategies. murine survival and weight loss were assessed, and lung viral load was quantified via plaque assay. here, we report that ino administered prophylactically or post-influenza infection failed to improve survival of infected mice. no difference in lung viral load was observed between experimental groups. although no has antiviral activity against influenza a virus in vitro, ino therapy provided no apparent benefit when used for treatment of influenza a virus infection in vivo. influenza a viruses infect approximately 5-15% of the population, resulting in 250-500 thousands deaths each year (1) . the most widely used class of drugs for treatment of clinical influenza is the neuraminidase inhibitors, including oseltamivir and zanamivir. the clinical impact of these drugs is limited by the development of antiviral drug resistance. specifically, decreased efficacy of neuraminidase inhibitors has been reported against seasonal h1n1 influenza and 2009 novel swine-origin h1n1 influenza, as well as avian influenza h5n1 virus (2) (3) (4) (5) (6) (7) (8) (9) . in addition, initiation of antiviral therapy in influenza a virus-infected individuals beyond the first 48-72 hours after the onset of influenza symptoms is asso-ciated with greater mortality and decreased antiviral efficacy compared with treatment initiated within 48-72 hours of symptom onset (10) (11) (12) (13) (14) (15) . these caveats underscore the need to develop novel and effective influenza therapeutic strategies. further investigation of other intervention strategies which have shown promising results against influenza a viruses in vitro but have not been investigated in vivo are warranted. nitric oxide (no) is an important cellular signalling molecule synthesized from l-arginine by no synthase (nos). there are three types of nos: constituent and calcium-dependant isoforms that are principally present in endothelial and neuronal cells (enos and nnos, respectively), and the inducible or ivyspring international publisher calcium-independent isoform, inos (16) . in the airways, nos is present in a variety of cells, including macrophages, vascular endothelial cells, airway epithelial cells and neurons where nos activity is known to mediate neurotransmission, smooth muscle contraction and mucin secretions. no is also a well known biological mediator in the host response to infection (16, 17) . various inflammatory stimuli such as lps and cytokines including ifng and tnf can cause high and sustained no production by inos; depending on the species, strain, infection dose and pathogen entry route, inos activity can result in proor anti-inflammatory responses, cytotoxicity, or cytoprotection [reviewed in (16) ]. in vitro, no antimicrobial activity has been demonstrated against a variety of viruses including ectromilia virus, vaccinia virus, herpes simplex type 1 viruses, coronavirus, and influenza a and b viruses (18) (19) (20) (21) (22) . in these studies, administration of the no donor s-nitroso-n-acetylpenicillamine (snap) to virus-infected cells significantly reduced viral burden. a human trial for treatment of severe acute respiratory syndrome (sars) found inhaled no (ino), at 30 ppm or less, decreased the spread and intensity of lung infiltrates and improved arterial oxygen saturation (23) . severe cases of influenza infection are often associated with multisystem organ failure and hypoxemic respiratory failure, including acute lung injury/acute respiratory distress syndrome (ali/ards) requiring advanced mechanical ventilatory support (24, 25) . affected individuals may receive 'rescue' therapies, including ino, in an attempt to improve outcome (25) . however, ino administration for ards secondary to viral pneumonia has not been specifically reported to improve clinical outcome (24, 25) . the objective of this study was to determine whether ino administration could reduce viral load and improve survival in a murine model of severe influenza. inhaled no delivery would provide a safer and easier delivery method rather than administration of no donors, as ino is approved for treating term and near-term neonates with hypoxemic respiratory failure up to a dose of 80 parts per million (ppm) (26, 27) . it has been reported that exogenous gaseous no (gno) at a high dose of no less than 160 ppm and with five hours of continuous exposure, can elicit a non-specific antimicrobial response against a broad range of microorganisms in vitro (28) . in vivo, 160 ppm ino treatment would result in no binding to hemoglobin to form methemoglobin, resulting in reduced oxygen transport and hypoxemia, as well as the potential for elevated levels of the harmful no metabo-lite no2. however, miller et al. (29) has shown that gno in an intermittent delivery regimen of 160 ppm for 30 min every 3.5 hours can prevent methemoglobinemia and reduce the potential of host cell toxicity in vitro and in vivo (miller c, personal communication), while retaining antimicrobial properties in vitro. we evaluated the ability of ino to improve survival of influenza a/wsn/33 (mouse-adapted h1n1 strain; wsn/33) infected mice. experimental c57bl/6 mice were inoculated intranasally with an 80-100% lethal dose of wsn/33 (1000 pfu). at 5 days post-infection, the majority of mice in all experimental groups experienced weight loss ( fig. 1a and 2a ). at 7 days post-infection, mice began to reach euthanasia criteria (≤80% of day 0 weight), and by day 10 post-infection, most mice were euthanized ( fig. 1b and 2b ). if 20% weight loss was not met by day 10 post-infection, the infection typically resolved, and surviving mice gained weight. weight loss over the course of infection was accelerated in mice administered continuous ino at 80 ppm starting 1 hour prior to inoculation compared to infected control mice receiving compressed room air (p < 0.001) (fig. 1a) . continuous ino administration at 80 ppm starting 1 hour prior to inoculation significantly decreased survival of wsn/33-infected mice compared to infected control mice administered compressed room air (p < 0.01). during the course of infection, 100% of continuous ino treated mice were euthanized compared to 80% of infected control mice (fig. 1b) . intermittent ino administration at 160 ppm for 30 min intervals every 3.5 hours starting either 1 hour prior to or 4 hours post-infection resulted in similar weight loss kinetics (fig. 2a) and consequent survival kinetics (fig. 2b ) of infected mice compared to infected control mice administered compressed room air. as gaseous no (gno) at high concentrations has been shown to decrease the viral load of infected cells in vitro (miller c, personal communication), we examined whether ino could reduce the viral load of influenza virus-infected mice. ino was administered starting 1 hour prior to influenza wsn/33 infection and continued either continuously at 80 ppm or in-termittently at 160 ppm for 30 min every 3.5 hours until mouse lungs were harvested at peak influenza viral load in the lungs (determined to be day 5 post-infection based on preliminary studies, data not shown). since ino was administered both prior to and for 5 days post-infection, we were able to test whether ino at intermediate (80 ppm) or high concentration (160 ppm) could prevent either viral entry or viral replication in vivo, and thereby reduce viral load. continuous ino at 80 ppm, intermittent ino at 160 ppm, and compressed room air administration yielded similar lung viral loads of infected mice on day 5 post-infection ( fig. 3a and b, respectively) . therefore, both continuous and intermittent ino administration failed to reduce lung viral load of infected mice, compared to infected control mice administered compressed room air. ino therapy is currently fda approved for the treatment of term and near-term neonates with hypoxemic respiratory failure associated with clinical or echocardiographic evidence of pulmonary arterial hypertension (26, 27) . variable findings have been reported for ino efficacy when administered at 1 ppm and up to 80 ppm. for its indicated use, ino has been found to increase vasodilation, improve oxygenation, reduce length of mechanical ventilation, reduce oxygen requirement, and decrease length of stay in the intensive care unit (27, 30, 31) . however, systematic reviews have failed to demonstrate that ino therapy reduces overall mortality (32, 33) . systematic reviews and meta-analysis of randomized controlled trials have shown that ino, when used therapeutically in the management of ards, results in a transient improvement in arterial oxygenation but does not reduce mortality (34) (35) (36) . moreover, ino therapy for ards may increase the risk of ino treated patients developing renal dysfunction (35, 36) . despite this, 39% of critical care specialists surveyed reported using ino for the management of patients with ards in ontario, canada (37) . typically, ino is administered at initial doses of 5-20 ppm in randomized controlled trials and observational studies for neonatal hypoxic respiratory failure (27) . although fda-approved at concentra-tions up to 80 ppm, no specific dose of ino has been proven more advantageous than another (27, 34) . rather, methemoglobinemia, defined as 7% methemoglobin in davidson et al. (38) , was more likely to occur. methemoglobinemia may account for the decrease in survival observed in our study with continuous ino administration at 80 ppm. no2 concentrations were measured daily over the course of infection and kept below 2 ppm as is acceptable in humans, however, lung toxicity may still explain these results as the toxic threshold in mice may be lower. on the other hand, given previous in vitro findings by mcmullen et al. (28) , 80 ppm may also have been too low of a concentration to provide an antiviral effect. a high dose of no at 160 ppm was administered intermittently, not to target the airway vessels specifically, but rather to induce an antimicrobial effect while avoiding the harmful effects of high dose continuous ino delivery. ino administered to influenza infected mice in this manner, either prophylactically or therapeutically, failed to improve survival of infected mice, change the course of weight loss, or decrease the lung viral load, compared to control mice receiving compressed air. therefore, although administration of high dose intermittent ino may have reduced the harmful side-effects of no, antimicrobial activity was not observed in vivo. in conclusion, despite the demonstrated antimicrobial activity of no against influenza a virus in vitro, the results of this study do not support the use of ino as a prophylactic or treatment strategy to reduce viral burden or improve clinical outcome in severe influenza in vivo. furthermore, it may be difficult to achieve viricidal concentrations of no in the airways using ino at concentrations that are safe in the living host. animal use protocols were reviewed and approved by the university health network ontario cancer institute animal care committee, and all experiments were conducted in accordance with institutional guidelines in an animal biosafety level 2 facility. female c57bl/6 mice, 9-11 weeks old, were obtained from jackson laboratories (bar harbor, me, usa) and maintained under pathogen-free conditions with a 12-hour light cycle. on day 0, while under light isofluorane anesthesia, experimental mice were infected via nasal instillation with 10 3 plaque forming units (pfu) of influenza a/wsn/33 (h1n1; wsn/33,) (stock kindly provided by dr. eleanor fish, university health network/university of toronto) in 50 μl pbs. weight was recorded daily for a maximum of twelve days post-infection, and mice were sacrificed when euthanasia criteria was met (greater than 20% weight loss). lung tissue was harvested for analysis on day 5 post-infection. prophylactic or post-infection ino therapy was initiated either 1 hour prior to or 4 hours post-infection, respectively. mice were placed in flow-through chambers with free access to food and water and received either compressed room air, continuous no at 80ppm +/-5ppm mixed with compressed room air, or intermittent no for 30 min every 3.5 hours at 160ppm+/-5ppm mixed with compressed room air. soda lime (200 g) was supplied to each chamber, and gas flow was maintained at 10-12 l/min to scavenge and minimize no2 levels, respectively. no2 levels were limited to <2 ppm for continuous ino therapy and <8 ppm for intermittent ino therapy. no and no2 levels were measured using an aeronox machine (pulmonox medical, ab, ca). lungs were harvested and frozen at -80°c. lungs were thawed, weighed, and homogenized in 1 ml pbs for 30 sec using a tissue miser homogenizer (fisher scientific, on, ca). lung homogenates were spun at 10,000xg for 10 min, aliquoted, and stored at -80°c for viral yield titration. influenza wsn/33 viral yield in lung homogenates was quantified by plaque assay in mdck canine kidney epithelial cells (atcc, va, usa). cells were maintained in eagle's mem (atcc, va, usa) supplemented with 10% fetal bovine serum and antibiotics. mdck cells were cultured at 37°c with 5% co2. cells were plated at a concentration of 8x10 6 cells/plate in 6 well culture plates. 12-24 hours later, medium was removed and mdck cells were washed twice with pbs. 10-fold dilutions of lung homogenates were added to mdck cells in 500 μl eagle's mem, in duplicate, and incubated at 37°c with 5% co2 for 1 hour with plates rocked every 15 min. after incubation, 1 ml of serum-free 2x eagle's mem supplemented with 8 μl/ml trypsin, 60 μl/ml of 7.5% sodium bicarbonate and 20 μl/ml antibiotics, combined with 1 ml of 1.2% agarose, was added to each well. once the agarose set, plates were incubated at 37°c for 42-72 hours until syncitia were observed. plates were fixed with carnoy's fixative (3:1, methanol:glacial acetic acid) for 30 min then stained with 0.1% crystal violet in 20% ethanol to visualize plaques. viral load is expressed as plaque forming units per gram of lung tissue (pfu/g). log-rank tests were performed on kaplan-meier survival curves. significant differences in weight loss between groups were assessed by two-way analysis of variance (anova) and bonferroni post-tests were performed. a student's t-test was carried out on viral yield data to assess significant differences (p ≤ 0.05) between experimental groups. surveillance for neuraminidase inhibitor resistance among human influenza a and b viruses circulating worldwide from influenza season week 39 ending influenza a (h1n1) virus resistance to oseltamivir weekly update on oseltamivir resistance to influenza h1n1 avian flu: isolation of drug-resistant h5n1 virus oseltamivir resistance during treatment of influenza a (h5n1) infection dual resistance to adamantanes and oseltamivir among seasonal influenza a (h1n1) viruses: 2008-2010 temperature-sensitive mutants of influenza virus. iv. induction of interferon in the nasopharynx by wild-type and a temperature-sensitive recombinant virus early administration of oral oseltamivir increases the benefits of influenza treatment factors associated with case fatality of human h5n1 virus infections in indonesia: a case series hospitalized patients with 2009 h1n1 influenza in the united states delayed clearance of viral load and marked cytokine activation in severe cases of pandemic h1n1 2009 influenza virus infection early versus late oseltamivir treatment in severly ill patients with 2009 pandemic influenza a (h1n1): speed is life nitric oxide and the immune response antimicrobial reactive oxygen and nitrogen species: concepts and controversies (review) no inhibitions: antimicrobial properties of nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus inhibition of influenza virus replication by nitric oxide inhibition of viral replication by ifng induced no synthase evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type 1 replication inhalation of nitric oxide in the treatment of severe acute respiratory syndrome: a rescue trial in beijing nonventilatory strategies for patients with life-threatening 2009 h1n1 influenza and severe respiratory failure critically ill patients with 2009 influenza a (h1n1) infection in canada approval of nda 20-846 inomax nitric oxide gas. usa: fda evidence-based clinical practice guideline: inhaled nitric oxide for neonates with acute hypoxic respiratory failure the antimicrobial effect of nitric oxide on the bacteria that cause nosocomial pneumonia in mechanically ventilated patients in the icu gaseous nitric oxide bactericidal activity retained during intermittent high-dose short duration exposure low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. clinical inhaled nitric oxide research group early compared with delayed inhaled nitric oxide in moderately hypoxaemic neonates with respiratory failure: a randomised controlled trial inhaled nitric oxide for respiratory failure in preterm infants inhaled nitric oxide in preterm infants: a systematic review inhaled nitric oxide for acute hypoxemic respiratory failure in children and adults: a meta-analysis effect of nitric oxide on oxygenation and mortality in acute lung injury: systematic review and meta-analysis inhaled nitric oxide for acute respiratory distress syndrome and acute lung injury in adults and children: a systematic review with meta-analysis and trial sequential analysis survey of interventions for the prevention and treatment of acute respiratory distress syndrome inhaled nitric oxide for the early treatment of persistent pulmonary hypertension of the term newborn: a randomized, double-masked, placebo-controlled, dose-response, multi-center study. the i-no/pphn study group cm consults and has minority shares in a variety of companies developing unrelated nitric oxide products. id, kck, and wcl have declared that no conflict of interest exists. key: cord-288938-4bheqtk5 authors: hönemann, m.; martin, d.; pietsch, c.; maier, m.; bergs, s.; bieck, e.; liebert, u.g. title: influenza b virus infections in western saxony, germany in three consecutive seasons between 2015 and 2018: analysis of molecular and clinical features date: 2019-10-08 journal: vaccine doi: 10.1016/j.vaccine.2019.08.027 sha: doc_id: 288938 cord_uid: 4bheqtk5 background: the impact of annual influenza epidemics and prevailing strains varies worldwide and regional. the majority of vaccines used contained two influenza a strains and only one influenza b strain (trivalent vaccine). aim: the aim of the study was to compare laboratory confirmed influenza b cases during three consecutive years with respect to vaccination history, clinical symptoms and molecular virology. methods: partial ha gene sequences were analyzed for lineage determination and complete ha sequence in cases with reported vaccination and in fatal cases. clinical data were retrieved from patient charts. findings: during the 2015/16 season, 75 influenza b cases were retrieved; 11 in 2016/17, and 274 in 2017/18. the frequency of yamagata-lineage strains increased from 7.6% to 100%. no difference was detected in the relative frequency of co-morbidities in season 2017/18. 37.7% of the adult patients and 4.5% of pediatric patients were vaccinated against influenza. interpretation: phylogenetically, yamagata strains clustered similarly in 2017/2018 when compared to the previous two influenza seasons. while the relative frequency of influenza b cases differed, the clinical symptoms remained similar. conclusion: world health organization recommendations for the use of tetravalent vaccines that contain two influenza b strains (yamagata and victoria) in addition to the two influenza a strains (h1n1 and h3n2) should be implemented in national vaccination guidelines. funding: this research was partially supported by the association of sponsors and friends of leipzig university. influenza b virus of the orthomyxoviridae family [1] has a segmented single stranded, negative sense rna genome. first isolated in 1940, influenza b virus diverged into two lineages, victoria and yamagata, in the late 1970s with similar clinical [2] but different phylodynamic properties [3] . although infections in pigs and seals were observed, there is no known animal reservoir for influenza b [4] . thus, while influenza a infections can be zoonotic, influenza b virus only circulates in the human population. the narrow host range and slower evolution [5] are thought to be the main contributing factors to the less frequent occurrence of influenza b epidemics [6] . in 2017/18 influenza b predominance was reported in countries throughout the northern hemisphere. it was the leading influenza type in europe and canada [7] [8] [9] and the second most common type after influenza a/h3n2 in the usa [10] . in germany, after the seasons of 2001/2, 2005/6 and 2015/16, it was only the fourth season of the 21st century with a dominance of influenza b [11] . as the prevailing lineage of influenza b changes frequently, the world health organization (who) recommended to include both lineages in a tetravalent vaccine in 2012 [12] . the key component of the vaccine is the viral hemagglutinin (ha) that covers the viral surface in a trimeric form. the major antigenic regions are the 120-loop, 150-loop, 160-loop, and 190helix [13] and are located on the ha1 subunit which forms the globular head domain. it is responsible for recognition and binding of sialic acids on the surface of the target cells. the ha2 subunit is the main component of the ha stalk. although some antibody vaccine j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / v a c c i n e cross reactivity is observed [14, 15] , vaccine efficiency between the two lineages might be reduced in seasons in which the formulation of the trivalent influenza vaccine does not match the circulating strain due to antigenic differences [16, 17] . additionally, there is growing evidence that vaccine effectiveness is already waning within a season thus reducing immunological protection even if the vaccine contained the matching strain in the previous season [18] . the aim of this study was to analyze clinical and molecular features of laboratory-confirmed influenza b cases during three consecutive seasons between 2015 and 2018. a comparison between patient characteristics was done in relation to the combined seasons of 2015/16 and 2016/17 when appropriate. additionally, molecular epidemiology of the viral hemagglutinin (ha) gene was performed on selected isolates and amino acid changes were mapped to the major antigenic domains of the ha protein. specimens and clinical data. 11,956 respiratory samples, including nasal aspirates, supernatants from nasal and pharyngeal swabs, throat rinsing fluid, tracheal secretions, and bronchoalveolar lavage fluids, of 7681 patients were tested for respiratory virus infections. testing was initiated at the discretion of the lineage determination. na sequences of a 123 bp-long amplicon of the ha gene [19] were assessed (bigdye terminator sequencing kit v1.1 and abi 3500 genetic analyzer, applied biosystems, foster city, usa). the sequenced region corresponds to nucleotide positions 966-1088 and 963-1085 of the complete ha gene of the victoria lineage (b/brisbane/60/2008) and the yamagata lineage (b/phuket/3073/2013), respectively. sequences with nucleotides g16, g17, g28, g55, c64, g73, and a112 belonged to the victoria-lineage. viruses with nucleotides a16, a17, a28, a55, t64, t73, and g112 belonged to the yamagata-lineage. phylogenetic analysis. na sequencing of the complete ha was performed according to who guidance [20] . in cases of low influenza b concentration an alternative protocol generating three instead of two overlapping fragments was used [21] . phylogenetic trees of the 1710 nucleotides large ha coding region were constructed at nucleotide level with the mega software version 6 using the maximum-likelihood method. bootstrap analysis was performed with 1000 replicates [22] . complete ha sequences were submitted to genbank (accession numbers mk459565 -mk459656). statistical analysis. statistical analysis was performed using ibm spss statistics for windows, version 24.0 (armonk, ny: ibm corp.). continuous values were expressed as mean or median (range) and categorical data as frequencies (percentages). student's t-test or mann-whitney u test was performed to compare means. chi-square or fisher's exact test were performed for categorical variables. all tests were two-tailed. a p-level of < 0.05 was considered significant. relative and absolute numbers of influenza types detected between 2015 and 2018 are shown in table 1 . for season 2015/16 the maximum number of cases occurred between weeks 10 and 14 of 2016. the peak occurrence in patients below 18 years was in week 12 while it was week 14 for the adult patients (fig. 1a) . the eleven cases of the season of 2016/17 occurred table 2 study population and clinical features of influenza b infected cases. (table 2) . for the pediatric patients there were no significant differences between season 2017/18 and 2015-2017 except for the combined rate of comorbidities and risk factors for a severe course of the influenza. however, this could not be attributed to a single analyzed factor. when looking at the adult population, there were no significant differences between season 2017/18 and the previous seasons except for a higher proportion of female patients and a lower proportion of viral coinfections in 2017/18. most differences were noted between the age groups. 32.3% of the adult patients were vaccinated, mostly with the trivalent vaccine, whereas 95.5% of the pediatric patients were unvaccinated. the rate of comorbidities was significantly higher in the adult population. furthermore, the clinical course of the disease was more severe in the adult population as higher rates of icu admissions, invasive ventilation, and fatal outcomes were observed. ha phylogeny. sequence analysis of the whole hemagglutinin gene was performed for 16 victoria and 76 yamagata strains and consistently confirmed the results of the initial lineage determination. included were all strains of patients with a known trivalent (n = 33) or tetravalent (n = 7) vaccination, randomly selected patients without known vaccination (n = 51), and of all patients that had a fatal outcome within 30 days of the influenza b detection (n = 27). except for one strain of 2017, all victoria strains originated from the 2015/2016 season and belonged to the 1a clade. all of the strains were phylogenetically closely related and did not cluster based on vaccination history or fatal outcome (fig. 3a) . all analyzed yamagata strains belonged to clade 3. all of the strains were phylogenetically closely related, but strain phylogeny indicates a discrete drift from season 2015/16 to season 2017/18. again, no clustering with regard to vaccination history or fatal outcome was observed (fig. 3b) . amino acid changes. the detected amino acid changes (aachanges) within the ha protein are depicted in table 3 the influenza season of 2017/18 was dominated by the yamagata-lineage of influenza b in europe. in germany, it was the largest season in terms of case numbers since the establishment of the nationwide surveillance system in 2001 [11] . large scale epidemics or pandemics are usually caused by influenza a. this is attributed mostly to the broader host range and faster rate of evolution of influenza a [6] . influenza b infections usually occur with a temporal delay towards the end of an influenza adominated season [23] . the exceptional impact of influenza b infections in 2017/18 with regard to public health was therefore surprising. however, seasons with a high proportion of influenza b infections occur infrequently. specifically, in germany, the mild seasons of 2001/2 and 2005/6 and the severe season of 2015/16 have been dominated by influenza b with a share of up to 78% of all influenza cases. the high severity of the season 2017/18 with an overall estimate of 9 million excess consultations in germany [11] may be attributed to several factors. according to the ecdc, the influenza season of 2017/18 started earlier than most of previous ones [9] . while this is true for the epidemic phase of the two influenza b heavy seasons in this study, the time frame for isolated cases was comparable. in the season of 2015/16 the epidemic phase started in week 5 of 2016 and lasted until week 15. in 2017/18 the epidemic threshold was crossed in week 52 of 2017 until week 14 of 2018 [11] . another factor contributing to the severity of the season might be the patient population itself. both the attack rate and the relative illness ratio of influenza b are highest for children and decrease with age [24, 25] . however, the major difference that was seen for the season 2017/18 was the higher proportion of adult patients. the age-subgroups themselves, being pediatric patients and adult patients, were not different in their clinical characteristics between 2017/18 and the two previous seasons. however, the adult population that was seen had a high proportion of comorbidities and risk factors that might have contributed to a fig. 3 (continued) severe outcome of the influenza disease, as it is known for example for cardiac insufficiency [26] , additionally to age itself [2, 27, 28] . a shift towards the adult population thus increases the impact on the public health system and may explain the severity of this season with a high number of excess consultations. an interpretation of the presented clinical data, however, needs to be done with caution as there may be a bias towards severe disease courses in a hospital setting. a fatality rate of 11.2% of the adult population of this study can thus not be transferred to the general population. a third potential factor may be the circulating influenza type. the yamagata-lineage of influenza b was predominantly found in the season of 2017/18. in contrast to the victoria lineage, which has a faster evolution with nearly each season representing a phylogenetic bottleneck, the biology of the yamagata lineage is different. in long term studies of the ha of the yamagata lineage a long co-circulation of yamagata variants was observed [3, 13, 29] . in accordance with these observations the analyzed strains of this study were phylogenetically closely related. furthermore, the detected amino acid changes were scattered throughout the ha and to the best of our knowledge no substitution is known for increased virus pathogenicity. since 2000, changes in the antigenic sites are dominantly located in the 120-loop and 190-helix regions, indicating main targets for immune pressure. with regard to the vaccine strain b/phuket/3073/2013 the main antigenic regions were only affected in 9 strains and occurred mainly in the 120-loop region, followed by the 160-loop region for the analyzed yamagata strains. in contrast, all analyzed victoria strains had at least one amino acid change in the 120-loop region with regard to the vaccine strain b/brisbane/60/2008. it is therefore surprising that the yamagatalineage that circulated in the population for three consecutive seasons was able to cause such a major epidemic. table 3 amino acid changes of the victoria-and yamagata-strains with regard to the respective vaccine strain. amino acids are numbered with regard to the ha0 protein, and ha1 as well as ha2 subunits are indicated. the vaccine strains to which the isolates were compared are given in red for each lineage. isolates from this study are labeled as vaccinated with trivalent vaccine (triangle), vaccinated with tetravalent vaccine (square), randomly selected case without vaccination history (circle), fatal cases (black filled symbols). symbols represent the main antigenic sites, 120-loop (a; amino acids 116-137, 38, 56, 75, 177, 179-181), 150-loop (b; amino acids 141-150), 160-loop (c; amino acids 162-167) and 190helix (d; amino acids 194-202). *for the yamagata-lineage, 48 strains, that were identical at amino acid level, are depicted as a pool and contain patients with the following characteristics: 15xs; 15xd; 3xh, 1xj, 14x4. a reason for this might be the interplay between the circulating influenza strains causing infections and the choice of the vaccine components or type of vaccine. in the season of 2015/16 influenza a/h1n1 pdm09 and the victoria-lineage of influenza b caused the majority of influenza infections. in 2016/17 influenza a/h3n2 was the main cause of flu disease. when looking at the influenza b component of the broadly used trivalent influenza vaccine there was a switch from b/phuket/3073/2013-like (yamagata-lineage) to b/ brisbane/60/2008-like (victoria-lineage) in the season 2016/17, effectively causing a mismatch in each of the three studied seasons. in 2017/18 the only strain that was not immunologically covered by recent epidemics or the current vaccine composition was the influenza b yamagata-lineage. an increased proportion of non-immune individuals in the adult population thus may have contributed to the observed shift in the age distribution of the infected patients. cross-reactive antibodies can be isolated from vaccinated individuals [30, 31] but the contribution to the immunity against influenza b is not well defined. some degree of lineage crossprotection has been reported upon vaccination with trivalent influenza virus vaccines. however, it seems to be limited to certain age groups and the vaccine efficacy is consistently superior for influenza b strains belonging to the vaccine lineage [32, 33, 16] . the capability of an individual to generate a broad antibody response against epitopes that include regions conserved between the two lineages, e.g. in the ha stalk, seems to be of major importance. thus, the factors influencing lineage cross-protection are complex and not easy to predict as also the immune memory may have a substantial impact on the targeted epitopes [34, 35] . in 76% of the patients with a vaccination history in this study the trivalent vaccine, with an expected moderate level of vaccine efficiency [7] , was used. the majority of the vaccinated patients were adults. however, a major improvement to influenza disease burden may only be accomplished by extending vaccine recommendations to younger age groups that are immunologically more naïve and thus more susceptible to infections [36, 25] . who recommendations for the use of tetravalent vaccines that contain two influenza b strains (yamagata and victoria) in addition to the two influenza a strains (h1n1 and h3n2) should be implemented in national vaccination guidelines as it was done in germany as a result of the severity of the 2017/18 influenza season. the biology of influenza viruses clinical characteristics and severity of influenza infections by virus type, subtype and lineage: a systematic literature review the contrasting phylodynamics of human 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protective b cell responses to influenza age, influenza pandemics and disease dynamics this research was partially supported by the association of sponsors and friends of leipzig university (vereinigung von förderern und freunden der universität leipzig). the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.08.027. key: cord-263277-m4too6ob authors: guzmán, carlos alberto title: next generation influenza vaccines: looking into the crystal ball date: 2020-08-21 journal: vaccines (basel) doi: 10.3390/vaccines8030464 sha: doc_id: 263277 cord_uid: m4too6ob influenza infections are responsible for significant number of deaths and overwhelming costs worldwide every year. vaccination represents the only cost-efficient alternative to address this major problem in human health. however, current vaccines are fraught by many limitations, being far from optimal. among them, the need to upgrade vaccines every year through a time-consuming process open to different caveats, and the critical fact that they exhibit poorer efficacy in individuals who are at high risk for severe infections. where are we? how can knowledge and technologies contribute towards removing current roadblocks? what does the future offer in terms of next generation vaccines? seasons. on the other hand, they presented the pros and cons of protein-based vaccines produced using a baculovirus-based approach, which eliminates the constraints resulting from relying on influenza virus replication. nevertheless, practical considerations, like manufacturing capacity to serve the huge demand for influenza vaccines and considerably higher production costs still represent a bottleneck. in their review article, the authors also presented a comprehensive summary of the current efforts to improve the influenza vaccines produced using standard existing technologies. in this regard, the use of adjuvants to improve the overall efficacy of vaccines for specific subpopulation groups is discussed, as well as the exploitation of nanotechnologies to increase the immunogenicity of protein-based vaccines. an elegant approach enables the engineering of recombinant viruses for vaccine production, which co-expresses the vaccine relevant hemagglutinin together with a helper hemagglutinin [10] . this strategy prevents the emergence of egg-adaptive mutations and reduces the overall production times. they also address the use of peptide-based vaccines as a potential alternative. although promising, the presented drawbacks suggest that considerable time and effort will be required to make it a viable approach for implementation in the influenza vaccine field. the review by clemens et al. further elaborates on the potential and limitations of harnessing t cells for the development of influenza vaccines [11] . in this particular context, animal studies support the potential of exploiting this strategy to generate universal or at least broader next generation vaccines providing heterosubtypic protection by stimulating long-lasting t cell resident memory cells. nevertheless, this approach faces several major constraints. as described in the review, a universal vaccine based on peptides should mimic more closely the immune responses observed after natural infections by inducing both cross-reactive peripheral and tissue resident cd8 t cells in addition to cross-reactive antibodies and cd4 t follicular and helper cells. however, as described in the review, special vaccination strategies will be needed to promote the seeding of tissue resident memory t cells in the respiratory tract and prevent their attrition. furthermore, universal protection will require addressing human leucocyte antigen (hla)-restriction by the inclusion of appropriate peptides. such vaccines should not only stimulate multiple cross-reactive epitope-specific t cells, but also encompass epitopes recognized by rare hla types from minor ethnicities. finally, current influenza vaccines are approved dependent on the accepted correlates for protection based on the stimulation of functional antibodies recognizing the hemagglutinin. large-scale clinical studies will be needed to define similar robust correlates for a conceptually novel class of t cell influenza vaccines. new disruptive technologies, such as the use of nucleic acid vaccines (e.g., dna or rna), which render unnecessary the production of proteins, can be also exploited to develop next generation influenza vaccines. in this regard, rna vaccines represent a cutting-edge approach, which dramatically reduces production times, ensure the fidelity of the encoded sequences, and are insulated from shortages of eggs or the constraints attached to cell factories. this renders this technology particularly appealing for potential emerging influenza pandemics. preclinical studies in different animal species and first-in-man studies also demonstrated the intrinsic value of this approach [12, 13] . however, these studies, as well as some of the trials carried out in the context of the covid-19 pandemic [14, 15] showed that the higher reactogenicity of rna vaccines respect to what observed for influenza vaccines developed using conventional standard technologies might be an issue for the global acceptance of an rna-based prophylactic vaccine. in the review by scorza and pardi, the authors summarize the current state of the art in terms of preclinical and clinical studies, as well as the major roadblocks that need to be overcome for implementation [16] . in this context, they describe the strategies exploited to increase the half-life, stability, cellular uptake and translatability of both self-amplifying and non-replicating mrna. a distinctive strength of this class of vaccines is their rapid scalable egg-independent production. of particular interest is the analysis presented in this review benchmarking the strengths and limitations of this class of vaccines in terms of the world health organization guidelines for the preferred product characteristics of next generation influenza vaccines [17] . the availability of innovative technologies will certainly positively affect the development of next generation influenza vaccines. however, influenza vaccines based on both traditional and emerging technologies face the major constraint represented by the immune history of the individual vaccinees. the review article from lewnard and cobey analyze the specific constraints and provide examples on how the dynamics of immune memory can affect vaccine effectiveness [18] . it is critical to understand the underlying mechanisms driving these processes, which go far beyond the potential impact of the original antigenic sin during immune imprinting. this will allow dissecting differential patterns of vaccine effectiveness in vaccinees depending on immune imprinting and memory. immediately after birth, human beings lose their naivety, and previous infection and vaccination events condition responsiveness, for good or bad, from season to season, according to age and the antigen contact history. the rational design of next generation influenza vaccines able to confer protection in all age and vulnerable groups is an ambitious and challenging venture. different approaches can be exploited to stimulate the production of broadly protective vaccine responses [19] . among them, the more conserved structures of the virus are used as an antigenic target, such as the stalk region of the hemagglutinin, or the matrix protein 2, which is highly conserved between influenza subtypes. an alternative approach relies on the exploitation of in silico tools to develop consensus sequences for computationally optimized broadly reactive hemagglutinin antigen variants (cobra), which are able to stimulate broadly reactive antibodies [20] . the use of some of these approaches might require the development of alternative tools to assess vaccine efficacy. current surrogated correlates of protection, which are accepted for the standard influenza vaccines, may not be relevant for next generation vaccines. in fact, the stimulated clearance mechanism might go far beyond traditional neutralization by blocking the virus binding to the receptor (e.g., the inhibition of viral membrane fusion and maturation, antibody-dependent and -independent cytotoxicity, the inhibition of viral replication or spreading). interestingly, the adjuvantation of conventional influenza vaccines has also proven effective, not only for improving responsiveness in certain subpopulation groups [21] , but also at broadening responsiveness to other influenza subtypes [22, 23] . a critical aspect for the development of a vaccine conferring broad cross-protective immunity against influenza is the determination of vaccine efficacy. the clinical trials required to achieve this goal may prove a roadblock. however, the availability of controlled human infection models for the influenza virus is an effective alternative to address this issue [24] . whether these approaches can lead to a true universal influenza vaccine or to next generation vaccines with a broader efficacy profile, rendering possible to boost against seasonal influenza every 5-10 years instead of every year, or to be prepared for a broad range of strains with a potential to cause pandemics, remains to be elucidate. however, the crystallization of any of these two scenarios will represent a significant improvement with respect to the current situation. it is important to highlight that regardless of the fact that upcoming next generation vaccines are formulated with well known standard antigens or innovative components promoting cross-protective immunity, or that they will be based on well established or emerging technology platforms for vaccine formulation or delivery, the key problem in vaccine-mediated immunity against influenza will remain. namely, key influenza-specific issues of interference due to pre-existent immunity and overall poor responsiveness to vaccines in vulnerable individuals (e.g., the elderly, the very young, those affected by co-morbidities) often result in unpredictable poor responsiveness to vaccination. therefore, the problem of poor protection against influenza infection post vaccination is, by and large, defined by a knowledge gap in key aspects of human immunology, such as the underlying mechanisms involved in poor individual responsiveness, and how to tailor vaccines according to the needs of subpopulation groups to generate broadly protective and long-lasting immunity. this current knowledge gap can be addressed by performing comprehensive and standardized profiling at baseline and post immunization of responders and non-responders to influenza vaccination in different age groups, both in healthy individuals as well as in those affected by co-morbidities known to affect responses to vaccination. the in-depth analysis and comparison of the obtained immunological and molecular signatures using artificial intelligence tools will enable the identification of the putative underlying mechanisms. these mechanisms will provide intervention targets to develop more efficient tailored vaccination strategies, as well as potential predictive biomarkers of responsiveness to develop innovative diagnostics for the stratification and follow-up of vaccinees. funding: this work was supported in part by grants from the eu (transvac2-730964; incentive-874866). the author declares no conflict of interest. global seasonal influenza-associated mortality collaborator network. estimates of global seasonal influenza-associated respiratory mortality: a modelling study drugs for influenza treatment: is there significant news? front factors that influence the immune response to vaccination immunogenicity 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vaccines 2020, 8, 464 key: cord-293234-ouykx6g5 authors: puig-barberà, j.; díez-domingo, j.; arnedo-pena, a.; ruiz-garcía, m.; pérez-vilar, s.; micó-esparza, j.l.; belenguer-varea, a.; carratalá-munuera, c.; gil-guillén, v.; schwarz-chavarri, h. title: effectiveness of the 2010–2011 seasonal influenza vaccine in preventing confirmed influenza hospitalizations in adults: a case–case comparison, case-control study date: 2012-08-24 journal: vaccine doi: 10.1016/j.vaccine.2012.07.006 sha: doc_id: 293234 cord_uid: ouykx6g5 introduction: we estimated influenza vaccine effectiveness (ive) to prevent laboratory-confirmed influenza-related hospitalizations in patients 18 years old or older during the 2010–2011 influenza season. methods: we conducted a prospective case-control study in five hospitals, in valencia, spain. study subjects were consecutive emergency hospitalizations for predefined conditions associated with an influenza-like illness episode <8 days before admission. patients were considered immunized if vaccinated ≥14 days before influenza-like illness onset. cases were those with a real time reverse transcriptase polymerase chain reaction (rt-pcr) positive for influenza and controls were rt-pcr positive for other respiratory viruses. adjusted ive was estimated as 100 × (1 − adjusted odds ratio). to account for indication bias we computed adjusted ive for respiratory syncytial virus related hospitalizations. results: of 826 eligible hospitalized patients, 102 (12%) were influenza positive and considered cases, and 116 (14%) were positive for other respiratory viruses and considered controls. adjusted ive was 54% (95% confidence interval, 11–76%). by subgroup, adjusted ive was 53% (4–77%) for those with high-risk conditions, 59% (16–79%) for those ≥60 years of age, and, 54% (4–79%) for those ≥60 years of age with high-risk conditions. no influenza vaccine effect was observed against respiratory syncytial virus related hospitalization. conclusion: influenza vaccination was associated with a significant reduction on the risk of confirmed influenza hospitalization, irrespective of age and high-risk conditions. yearly seasonal influenza epidemics are associated with excess morbidity and mortality [1] . vaccination against influenza is considered the most effective strategy for preventing influenza [2] . as a consequence of antigenic drift, influenza vaccines are to be a produced every year [3] . despite this achievement, vaccine effectiveness varies from season to season and can be very low one in four influenza seasons [4] . this is due to the unpredictable antigenic distance between vaccine's and the circulating strains [5] . as a consequence, evidence on influenza vaccine effectiveness has been difficult to obtain and is disputed [4, [6] [7] [8] . the reappraisal of the evidence on influenza vaccine effectiveness is possible by the availability of reverse transcriptase polymerase chain reaction (rt-pcr) to diagnose influenza infection [9] . rt-pcr has allowed the development of the test-negative approach for measuring influenza vaccine effectiveness. in test-negative case-control studies, cases are rt-pcr positive for influenza, and controls those negative for influenza. this approach has been advocated for its practicability, comparability between cases and controls, and the use of laboratory confirmed outcomes [8, 10] . various authors have used the test-negative casecontrol study [11] [12] [13] [14] [15] [16] [17] [18] . under conditions of concurrent circulation an appropriate test-negative control group are patients testing positive for other respiratory viruses, ensuring similarity on quality of sample collection and specificity of outcomes [10, 15, 19] . using a prospective case-case comparison approach, we have estimated seasonal influenza vaccine effectiveness (ive) to prevent laboratory confirmed influenza-related hospitalizations in adults. during the 2010-2011 influenza season, we performed a prospective case-control study in five hospitals in valencia, spain. the five hospitals provided care to 975,174 inhabitants 18 years of age or older. the influenza season was defined by the weeks with positive specimens for influenza on enrolled patients. it began on 12 december 2010 (week 50) and ended on 19 march 2011 (week 11) . patients with confirmed influenza by a rt-pcr test were considered cases and patients with an rt-pcr confirmed infection for other respiratory viruses were considered controls. influenza vaccines were offered free of charge to health district inhabitants older than 6 months of age with high-risk conditions and to 60 years old or older. three vaccine formulations were used. subunit trivalent non-adjuvanted vaccine (influvac ® , abbot-solvay, illinois, usa; batch numbers v4, v20, v23) offered to subjects less that 60 years of age, virosomal trivalent subunit vaccine (inflexal ® -v, crucell, leiden, the netherlands; batch numbers 300187601, 300189301, 300194401) offered to subjects 60 years old or older, and an mf59 tm -adjuvanted trivalent subunit vaccine (chiromas ® , novartis vaccines and diagnostics, massachusetts, usa; batch numbers 104603, 104702, 104802, 105001) offered by licensure requirements to those 65 years old or older. subunit trivalent non-adjuvanted was offered in the five health districts included in the study; virosomal vaccine was used in two, and the mf59 tm -adjuvanted vaccine was used in three. the strains included in the influenza vaccine for the 2010-2011 season were a/california/7/2009 (h1n1)-like, a/perth/16/2009 (h3n2)like, and b/brisbane/60/2008-like [20] . we established an active surveillance system. full-time field researchers identified, monday to saturday, patients who were hospitalized, coming from the emergency department, in the previous 24-48 h. patients whose indications for admission were any of a predefined set of conditions, described as possibly associated with a recent influenza infection [14] , were invited to participate. patients were excluded if institutionalized, not permanent residents, with reported egg allergy, had been hospitalized in the previous 30 days, or if they had had a previous laboratory confirmed influenza infection. patients were included if they reported an ili episode, defined as at least one of these four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of these three respiratory symptoms (cough, sore throat, shortness of breath), sudden onset was not a requisite for inclusion [21] , less than 8 days preceding their arrival at the emergency department. the ethics research committee of the centro superior de investigación en salud pública (csisp) approved the study. all study subjects gave written informed consent before enrollment. a nasopharyngeal and a pharyngeal swab were obtained from each included patient. samples were introduced into vials with viral transport medium and kept at −20 • c until sent to the reference laboratory. four multiplex real-time rt-pcr/pcr qualitative amplifications were performed: multiplex # 1 for influenza virus type a [22] and influenza virus type b [23] ); multiplex # 2 for coronavirus, metapneumovirus, and bocavirus [24] [25] [26] ; multiplex # 3 for respiratory syncytial virus (rsv), adenovirus and parainfluenza virus [27] ; and multiplex # 4 for rhinovirus [28] . negative results for viruses were only considered if human ribonucleoprotein gene amplification was positive. laboratory procedures to prevent pcr contamination were followed and a series of multiplex assays #1 to #4 negative controls without sample nucleic acid were included in all runs. information was obtained on age, sex, indications for inclusion, hospitalization date, time elapsed from symptoms onset to swabbing, presence of major underlying medical conditions, long-term treatments, contact with children, smoking habits, occupation, number of physician encounters in the last three months, number of hospitalizations in the last year, prescription of antivirals, intensive care unit admission, death in hospital and length of stay. functional status, measured by barthel index [29] , was obtained in study subjects 65 years old or older. social class was assigned according to occupation [30] . influenza vaccination status was obtained by asking the patient if he or she had received the current season's influenza vaccine, on which month, and if the vaccine had been administered at least two weeks before the onset of symptoms. in addition, vaccination status was independently ascertained by a researcher blinded to patient characteristics, who consulted valencia's population-based vaccine information system. a patient was considered immunized with the 2010-2011 influenza vaccine if the vaccine was registered as administered 14 or more days before the date of ili onset or if the patient recalled the month when the vaccine was administered and if it had been administered more than two weeks previous to current ili episode onset. information related to the administration of the 2009-2010 seasonal influenza vaccine, the a(h1n1) pandemic vaccine and previous 23-valent polysaccharide plain pneumococcal vaccinations was obtained from the vaccine information system. ive was defined as 100 × (1 − adjusted odds ratio [or]) [31, 32] . the adjusted or was obtained using a logistic regression model using stepwise background selection of the variables, with a criterion of p < 0.1 to remain in the model, starting with a fully saturated model, including being immunized with current season's vaccine, sex, age (in 10 years of age intervals), socioeconomic class, number of high-risk conditions, obesity (imc ≥ 40), smoking antecedents, number of physician encounters in the last three months, hospitalizations in the last year, pneumococcal vaccination, antiviral prescription, epidemiological week and time from symptoms onset to swabbing. we defined four groups for ive estimation: (a) all cases and controls enrolled (overall group); (b) all cases and controls with high-risk conditions regardless of age; (c) cases and controls 60 years old or older and (d) cases and controls 60 years old or older with high-risk conditions. to validate our estimates, we computed ive against rsv-related hospitalization following the same design and analysis strategy followed for influenza-related hospitalization, but in this instance cases were those positive for rsv and controls those positive for the other respiratory viruses, including influenza. the significance in differences in the distribution of covariates, between cases and controls, was estimated using the chi-squared, or fisher's test, for categorical variables; and the t-test, or kruskal-wallis test, for continuous variables; p < 0.05 was considered significant. all probabilities were 2-tailed. all analyses were performed with stata 12.1 (statacorp, college station, tx). we identified 2286 eligible patients, 826 complied with all inclusion criteria, 102 (12%) were positive for influenza and considered cases; 116 (14%) were positive for other respiratory viruses and considered controls (fig. 1) . swabs were performed 7 days or less after onset of symptoms in 93% of study subjects. time from onset to swabbing was similar between cases and controls, median was in both instances four days, p = 0.28. hospitalization rate associated with any of the respiratory viruses assessed was 22 per 100,000 18 years old or older. by age group, hospitalization rate associated to respiratory viruses was 5, 25, 44, and 90 per 100,000 18-49, 50-64, 65-74, and 75 years old or older, respectively. influenza-related hospitalization rate was 4, 17, 14, and 28 per 100,000 18-49, 50-64, 65-74 and 75 years old and older. type and number of virus identified were: h1n1pdm09, 74 (34%); rsv, 59 (27%); rhinovirus, 22 (10%); coronavirus, 18 (8%); influenza b, 16 (7%); parainfluenza virus, 6 (3%); mixed infections, 10 (5%); h3n2, 7 (3%); and human metaneumovirus, 6 (3%). including mixed infections, influenza accounted for 47% (n = 102) of all respiratory viruses identified. influenza subtypes identified were h1n1pdm09 (n = 78; 76%), influenza b (n = 17; 17%), and h3n2 (n = 7; 7%). influenza viruses circulated concurrently with rsv, rhinovirus and coronavirus (fig. 2 ). there were no differences regarding emergency admission diagnoses between cases and controls; with the exception of heart failure, that was 3% in cases compared to 10% in controls (p = 0.057) ( table 1 ). in 17% of influenza patients the presenting complain was not for a respiratory condition (table 1) . the duration of symptoms previous to admission was evenly distributed between cases and controls, p = 0.65 (table 1 ). there were no differences in the percentage of sudden onset of symptoms, malaise, myalgia, headache, sore throat or shortness of breath; the only significant difference was the frequency of fever in influenza cases, p = 0.003 (table 1) . when compared to controls, cases were younger, had fewer high-risk conditions, were of higher social class, more frequently smokers, and consulted their general practitioners or had been hospitalized in fewer occasions (table 2 ). there were no differences between cases and controls aged 65 years or more in their barthel index scores (table 2) . when restricting the comparison, between cases and controls, by the presence of high-risk conditions, the differences that remained significant were age, 23-valent pneumococcal vaccination, and having been vaccinated with the previous or current season influenza vaccines (table 2) . when restricted to those 60 years old or older, age and influenza vaccination with the previous or current seasonal influenza vaccine remained as significant differences between cases and controls ( table 2 ). compared to 36.3% cases, 66.4% controls were immunized with the 2010 influenza seasonal vaccine; p < 0.0001 (table 2) . controls also had more often been vaccinated with the 23-valent pneumococcal polysaccharide vaccine (p = 0.015), 2009 seasonal influenza (p < 0.0001), and 2009 pandemic (p = 0.0162) vaccines (table 2) . when high-risk conditions or age were taken into account, no differences were observed in the percentage of cases and controls vaccinated with the pandemic vaccine, and only in those with table 2 cases and controls a characteristics and vaccination history by group: overall, highrisk conditions, and 60 years old or older. high-risk conditions, irrespective of age, there was a difference (p = 0.05) in pneumococcal vaccination (17% cases vs. 29% controls) ( table 2) . this difference was not observed between cases and controls 60 years old or older ( table 2 ). according to the presence of high-risk conditions or age, the percentage of controls who had been vaccinated with the 2010 vaccine compared to cases remained significantly higher (table 2) . current influenza season vaccination was highly associated with previous influenza-seasonal vaccination (p < 0.0001) and age 60 or older (p < 0.0001). adjusted vaccine effectiveness to prevent confirmed influenzaassociated hospitalization was 54% (95%ci, 11-76%) ( table 3) . for the subgroup analysis, in those with high-risk conditions influenza vaccine effectiveness estimate was 53% (95%ci, 4-77%); for those 60 years old or older, it was 59% (95%ci, 16-79%); and for those 60 years old or older with high-risk conditions it was 54% (95%ci, 4-79%) ( table 3 ). the overall adjusted or of rsv-associated hospitalization of 2010 seasonal influenza vaccination was 1.2 (95%ci, 0.6-2.4); for those with high-risk conditions it was 1.4 (95%ci, 0.7-3.0); for those 60 years old or older, 1.5 (95%ci, 0.5-3.1); and for those 60 years old or older with high-risk conditions, 1.7 (95%ci, 0.8-3.7). subjects vaccinated experienced a risk of influenza-related hospitalization two times lower compared to the unvaccinated. the vaccine preventive effect was specific for influenza. three recent systematic reviews of studies reporting influenza vaccine efficacy or effectiveness [4, 7, 33] reach the conclusion that evidence on ive to prevent influenza in older adults is scarce, elusive or non-existent. osterholm et al. [4] looked for studies published between january 1967 and february 2011 with outcomes confirmed by rt-pcr or viral culture, as estimates based on serologic outcomes [34] overestimate inactivated vaccine efficacy [9] . they identified only two observational studies in older adults [16, 35] . talbot et al. [16] studied three consecutive seasons (2006-2009), using a test-negative case-control design, and reported a pooled adjusted ive of 61% for influenza-related hospitalizations; however, these estimates were only significant when pooled over three seasons. more recently, castilla et al. [18] conclude that ive for preventing laboratory-confirmed influenzarelated hospitalization in adults 60 years of age or older, during the 2010-2011 influenza season, is 58% to 59%. influenza vaccine effectiveness depends closely on the match of the vaccine strain to the circulating strain [4, 5] . during the 2010-2011 influenza season, 36% of hospitalized subjects with confirmed influenza had been immunized with the seasonal vaccine. this was in clear contrast to what was observed during the 2009-2010 autumn pandemic wave, when, in presence of a good match between the circulating and the vaccine strain, vaccine failures were rare [14] . the percentage of vaccine failures observed during the 2010-2011 influenza season can be interpreted considering that 20% of specimens collected in europe showed a reduced activity against the a/california/7/2009 vaccine virus strain [36] . we tried to minimize selection bias by an enrollment strategy based on an active surveillance system, the use of broad eligibility requirements for inclusion, completeness of inclusion, and enrolling subjects without previous knowledge of their vaccination or case-control status. we reduced classification bias by the use of two independent sources to ascertain vaccination, performing rt-pcr for influenza diagnosis, and by the case-case comparison. patients' recall is considered a valid source of influenza vaccination status [37] , but is limited by recall bias and uncertainty on date of vaccine administration, or type of vaccine administered. record of vaccination reliably indicates immunization, but absence of record is not informative. we estimate electronic vaccine information system sensitivity as 90%, and specificity as 99%, during the 2009-2010 autumn pandemic wave [38] . with the data collected in the present study, and using a capture recapture method [39], completeness of ascertainment was 93% for electronic vaccine information system, 98% for patient recall, and 99% for both sources. we aimed to reduce classification bias considering a study subject as vaccinated or non-vaccinated adding the information provided by both sources. rt-pcr is the preferred diagnostic test for influenza [9] ; but, case status misclassification may contribute to underestimation of ive because of false-negative rt-pcr results [10, 15, 40] . to maximize rt-pcr sensitivity, we included patients with onset of symptoms seven or less days before hospitalization. pcr positivity is, with a similar swabbing strategy, 88% and 70%, at 4 and 6 days after symptoms onset [40] . a non-differential misclassification of true positives as negatives cannot be ruled out and underestimation of vaccine effectiveness is to be expected if a test-negative design is used. this was minimized by case-case comparison [10, 15] . although nasopharyngeal aspirate is considered the best specimen for detection of influenza viruses [41] , we opted for pharyngeal (throat) and nasopharyngeal swabbing to reduce patients discomfort and performance easiness. in children nasal swabs are comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses, except rsv [42] . in adults, swabbing has been used to study respiratory virus disease [43, 44] , and ive [14, 18, 40, 45, 46] . we obtained a yield of positives similar to other studies on hospitalized adults [16, 43] , and the timing of the epidemic wave and types and subtypes we identified were consistent with those reported by spain's surveillance system [47] . swabbing is a reliable and convenient alternative to obtain specimens for rt-pcr testing [42] , and accounting for days elapsed from symptoms onset to swabbing, should limit the effect of misclassification of true positives as negatives [10, 15] . the case-case analysis approach design assures comparability of controls to cases [10, 15] . in a case-case comparison approach, cases and controls should mainly differ in the exposure (and its correlates) associated to the outcome of interest [19] . all this is even more plausible if influenza and other respiratory viruses cocirculate concurrently (fig. 2) . 5.4. impact of age as a confounder and age-related protection due to previous exposure age effect was taken into account by adjustment, and by performing an analysis restricted to those 60 years old or older. vaccine effectiveness could be explained in the elderly by acquired protection due to distant exposure to similar h1n1 strains. we consider this pre-existing protection bias in our results as debatable. first, we observed the third h1n1pdm09 wave, this repeated circulation levels exposure to h1n1pdm09 over the age range [48] . second, age-specific h1n1pdm09 influenza-related hospitalization rates were in our population two to seven times higher in the 75 years or more age group. third, seroepidemiology studies [48] [49] [50] have described the persistence of protective antibody titers against h1n1pmd09 only in a small fraction of subjects 80 years old or older [48] [49] [50] . fourth, when t cell epitopes are compared between h1n1pdm09 and seasonal h1n1, 41% and 69% for cd4+ and cd8+, respectively, are conserved [51] , hence a less dependent on age protection for severe episodes should be expected in those 18 years old or older [14] . fifth, vaccine effectiveness did not differ when age was considered. the main weakness of our study was the number of influenzarelated hospitalization. although we were able to assess adjusted ive on large groups, this was done with broad confidence intervals, and we did not attain a sufficient number of cases to provide robust ive estimates by virus strain or vaccine type. we report ive estimates with a low probability of bias and the current vaccines provided a significant health benefit. any single ive study results are difficult to generalize. variability of the factors involved, such as circulating strains, vaccine types and composition, match between vaccine's and circulating strains, population characteristics, and outcomes measured are limitations to generalizability. future studies should be planned, after taking into consideration the strengths and limitations exposed, to attain the necessary statistical power to obtain robust ive estimates by virus antigenic subtypes, comparing the different vaccines available, and for relevant high-risk groups. the impact of influenza epidemics on hospitalizations influenza vaccines. who position paper the annual production cycle for influenza vaccine efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis influenza vaccine: the challenge of antigenic drift influenza vaccination: policy versus evidence a systematic review of the evidence on the effectiveness and risks of inactivated influenza vaccines in different target groups estimating the effect of influenza vaccines efficacy studies of influenza vaccines: effect of end points used and characteristics of vaccine failures influenza seasonal vaccine, preliminary mid-season effectiveness estimates: reason for concern, confounding or are we following the right track? effectiveness of inactivated influenza vaccines varied substantially with antigenic match from the 2004-2005 season to the 2006-2007 season association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada cyceva study team. estimating the influenza vaccine effectiveness in elderly on a yearly basis using the spanish influenza surveillance network -pilot case-control studies using different control groups and pandemic vaccines, to prevent influenza hospitalizations during the autumn 2009 influenza pandemic wave in castellón, spain. a testnegative, hospital-based, case-control study vaccine effectiveness against laboratory-confirmed influenza in healthy young children: a case-control study effectiveness of seasonal vaccine in preventing confirmed influenza-associated 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infections in adults: another piece of the puzzle the diagnosis of viral respiratory disease in older adults estimates of pandemic influenza vaccine effectiveness in europe, 2009-2010: results of influenza monitoring vaccine effectiveness in europe (i-move) multicentre case-control study vigilancia de la gripe en españa, temporada 2010-11. (desde la semana 40/2010 hasta la semana 20/2011) incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study serologic survey of pandemic (h1n1) 2009 virus, guangxi province high frequency of cross-reacting antibodies against 2009 pandemic influenza a (h1n1) virus among the elderly in finland preexisting immunity against swine-origin h1n1 influenza viruses in the general human population the authors thank the staff of the hospital la plana, in vila-real; arnau de vilanova, in valencia; la ribera, in alzira; san juan, in alicante; and hospital de elda, in elda. as well, we thank all the study participants and their families.we wish to express our recognition to prof juan garcía-de-lomas for his support and contribution on the laboratory methods section. we also acknowledge the dedication and commitment of researchers in the field begoña escribano-lópez, verónica alcarria-garcía, ester huet-trujillo, ángela lópez-doménech, montserrat cano-armenteros m and consuelo calvo-mas.funding: the study was funded in part by contract code grt109 between sanofi-pasteur and centro superior de investigación en salud pública (csisp). sanofi-pasteur did not participate in the design or conduct of the study, collection, management, analysis, or interpretation of the data, writing the report, and the decision to submit the report for publication. key: cord-326177-zzsaf3bl authors: khatri, mahesh; richardson, levi arthur; meulia, tea title: mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model date: 2018-01-29 journal: stem cell res ther doi: 10.1186/s13287-018-0774-8 sha: doc_id: 326177 cord_uid: zzsaf3bl background: mesenchymal stem (stromal) cells (mscs) mediate their immunoregulatory and tissue repair functions by secreting paracrine factors, including extracellular vesicles (evs). in several animal models of human diseases, msc-evs mimic the beneficial effects of mscs. influenza viruses cause annual outbreaks of acute respiratory illness resulting in significant mortality and morbidity. influenza viruses constantly evolve, thus generating drug-resistant strains and rendering current vaccines less effective against the newly generated strains. therefore, new therapies that can control virus replication and the inflammatory response of the host are needed. the objective of this study was to examine if msc-ev treatment can attenuate influenza virus-induced acute lung injury in a preclinical model. methods: we isolated evs from swine bone marrow-derived mscs. morphology of msc-evs was determined by electron microscopy and expression of mesenchymal markers was examined by flow cytometry. next, we examined the anti-influenza activity of msc-evs in vitro in lung epithelial cells and anti-viral and immunomodulatory properties in vivo in a pig model of influenza virus. results: msc-evs were isolated from msc-conditioned medium by ultracentrifugation. msc-evs were round-shaped and, similarly to mscs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens i and ii. incubation of pkh-26-labeled evs with lung epithelial cells revealed that msc-evs incorporated into the epithelial cells. next, we examined the anti-influenza and anti-inflammatory properties of msc-evs. msc-evs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25–5 μg/ml. msc-evs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. the anti-influenza activity of msc-evs was due to transfer of rnas from evs to epithelial cells since pre-incubation of msc-evs with rnase enzyme abrogated the anti-influenza activity of msc-evs. in a pig model of influenza virus, intratracheal administration of msc-evs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. the histopathological findings revealed that msc-evs alleviated influenza virus-induced lung lesions in pigs. conclusions: our data demonstrated in a relevant preclinical large animal model of influenza virus that msc-evs possessed anti-influenza and anti-inflammatory properties and that evs may be used as cell-free therapy for influenza in humans. results: msc-evs were isolated from msc-conditioned medium by ultracentrifugation. msc-evs were round-shaped and, similarly to mscs, expressed mesenchymal markers and lacked the expression of swine leukocyte antigens i and ii. incubation of pkh-26-labeled evs with lung epithelial cells revealed that msc-evs incorporated into the epithelial cells. next, we examined the anti-influenza and anti-inflammatory properties of msc-evs. msc-evs inhibited the hemagglutination activity of avian, swine, and human influenza viruses at concentrations of 1.25-5 μg/ml. msc-evs inhibited influenza virus replication and virus-induced apoptosis in lung epithelial cells. the antiinfluenza activity of msc-evs was due to transfer of rnas from evs to epithelial cells since pre-incubation of msc-evs with rnase enzyme abrogated the anti-influenza activity of msc-evs. in a pig model of influenza virus, intratracheal administration of msc-evs 12 h after influenza virus infection significantly reduced virus shedding in the nasal swabs, influenza virus replication in the lungs, and virus-induced production of proinflammatory cytokines in the lungs of influenza-infected pigs. the histopathological findings revealed that msc-evs alleviated influenza virus-induced lung lesions in pigs. (continued on next page) conclusions: our data demonstrated in a relevant preclinical large animal model of influenza virus that msc-evs possessed anti-influenza and anti-inflammatory properties and that evs may be used as cell-free therapy for influenza in humans. keywords: mesenchymal stem cells, extracellular vesicles, influenza, acute lung injury, stem cell therapy, large animal model background influenza a viruses (iav) cause an acute respiratory disease in humans and animals. annual outbreaks and occasional pandemics of influenza result in millions of deaths, suffering, and economic losses. in the us alone, since 2010 influenza viruses have caused 140,000-710,000 hospitalizations resulting in 12,000-56,000 deaths annually (https://www.cdc.gov/flu/about/disease/burden.htm). the elderly, infants, and people with underlying conditions are at high risk of influenza-associated mortality. in addition to seasonal and pandemic viruses, highly pathogenic avian influenza (hpai) h5n1 virus has been repeatedly transmitted directly from avian species to humans. in humans, h5n1 virus is associated with severe disease resulting in multi-organ failure and high mortality rates [1, 2] . as of 30 october 2017, hpai h5n1 viruses have caused 860 human infections resulting in 454 deaths since 2003 (http://www.who.int/influenza/human_animal_interface/2017_10_30_tableh5n1.pdf?ua = 1) severe cases of influenza cause significant mortality due to their ability to induce cytokine-mediated immune lung pathology with features of moderate to severe acute respiratory distress syndrome (ards) [3] . influenza virus infections are generally controlled by annual vaccination. however, these vaccines provide limited protection against new reassortants which are genetically different from the vaccine virus. in the event of a pandemic, generation of a new vaccine containing circulating viruses takes approximately 6 months. moreover, influenza viruses continually undergo mutations resulting in the generation of new viral strains that can become resistant to currently available antiviral drugs. thus, alternative therapies capable of inhibiting influenza virus replication and attenuating the inflammatory response of the host are needed. mesenchymal stem (stromal) cells (mscs) are multipotent cells that were first identified in bone marrow (bm) as plastic adherent fibroblast-like cells. mscs possess multilineage differentiation, and immunomodulatory and tissue repair properties [4] . due to these properties mscs are attractive as cellular therapy for inflammatory and autoimmune diseases, and regenerative medicine. several studies of ards in animal models have shown beneficial effects of msc administration, and clinical trials have shown the feasibility of msc administration in patients with ards [5] [6] [7] . therapeutic studies are underway. similarly to ards, severe influenza virus infections in humans and animal models show acute inflammatory response and lung damage [8] . as mscs suppress inflammation and have tissue repair and regenerative ability, ards and influenza are appropriate targets for msc therapy. however, msc therapy in mice models of influenza show inconsistent results [9] [10] [11] [12] . also, we and others have shown that influenza virus infects mscs and that infection may alter the immunoregulatory and differentiation properties of mscs [13] [14] [15] . several studies indicated that the beneficial actions of mscs are due to release of paracrine factors since only a few transplanted mscs engraft at the site of injury [16] . recently, extracellular vesicles (evs) that include exosomes (exo) which are released from multivesicular bodies and microvesicles (mvs) that are shed from the cell surface, and apoptotic bodies were identified in msc secretions [17] . in this paper evs, exo, and mvs will be collectively referred to as evs. msc-derived evs have similar expression of surface molecules and contain msc-specific proteins, mrnas, micrornas (mirnas), organelles, and lipids [18] [19] [20] . in diseased tissue, msc-evs interact with injured cells and transfer proteins, mrna, and bioactive lipids from mscs to injured cells resulting in tissue repair [21, 22] . in rodent models, these evs were as therapeutically efficacious as mscs in e. coli endotoxin-induced acute lung injury (ali) and e. coli-induced severe pneumonia in mice [20, 23] . due to their close similarity in anatomy, physiology, and immunology to humans, pigs are used as a large animal preclinical model for several human diseases, including respiratory diseases and regenerative medicine [24, 25] . in addition, pigs are naturally infected with influenza virus as the respiratory epithelium of pigs expresses receptors utilized by avian and mammalian influenza viruses [26] . influenza virus pathogenesis and clinical signs in pigs are also similar to those observed in humans. thus, pigs are a suitable large animal model to study human influenza virus pathogenesis and to test the efficacy of therapeutics including mscs or their derivatives for influenza. mscs from femur bones of 2-to 6-week-old commercial pigs were isolated as described previously [27, 28] . briefly, the tip of each bone was removed and the marrow was harvested by inserting a syringe needle into one end of the bone and flushing with dulbecco's modified eagle's medium (dmem; gibco). the bm cells were filtered through a 70-μm nylon mesh filter (bd, falcon, usa) and mononuclear cells were obtained by density gradient centrifugation over ficoll-hypaque. cells (1-5 × 10 5 /cm 2 ) were plated in 75-cm 2 cell culture flasks in dmem containing 10% fetal bovine serum (fbs; gibco) and 1% antibiotic solution (gibco) (c-dmem). cultures were incubated at 37°c in a humidified atmosphere containing 95% air and 5% co 2 . the nonadherent cells were removed after 72 h of culture and cells were passaged when they were 90% confluent by treating them with 0.25% trypsin containing 0.02% edta. mscs passaged between three and five times were used for the generation of msc-evs for in vitro and in vivo experiments. for the isolation of msc-evs, mscs were cultured in c-dmem in t225 flasks and msc-evs were isolated as previously described [23, 29, 30] . briefly, cells when 80% confluent were washed with serum-free dmem and cultured in dmem containing 0.5% bovine serum albumin. after 48 h, conditioned medium (cm) from msc cultures was collected and centrifuged at 3000 rpm for 20 min to remove the cellular debris. next, cm was ultracentrifuged at 25,000 rpm for 70 min at 4°c. the ev pellet was washed with dmem by ultracentrifugation at 25,000 rpm for 70 min at 4°c. the ev pellet was then suspended in phosphatebuffered saline (pbs; 10 μl pbs/million mscs) and further dilutions were made in serum-free dmem. the protein content of evs was determined by microbicinchoninic acid protein assay kit (thermofisher scientific). msc-ev rna was isolated using the rnaeasy kit (qiagen) and rna concentration was determined by nanodrop (thermofisher scientific). evs (10 μl) were applied to a formvar/carbon-coated grid for 5 min; after blotting, the grid was stained with 2% aqueous uranyl acetate for 1 min. after blotting, the grids were air-dried and examined under tem (hita-chi, h-7500, japan). mscs and evs were examined for surface expression of mesenchymal markers and swine leukocyte antigen (sla)-i and sla-ii, and evs were also examined for ev markers (cd9, cd63, and cd81) by flow cytometry as described previously [31] . evs (30 μg in 50 μl pbs) were incubated with 10 μl of 4-μm diameter aldehyde/sulfate latex beads for 15 min at room temperature followed by the addition of 1 ml pbs and incubation was further continued for 2 h with gentle shaking. the reaction was stopped by incubation for 30 min in 100 mm glycine. mscs were detached by treatment with 0.25% trypsin-edta and single cell suspensions of msc and evcoated beads were stained with the following primary antibodies: mouse anti-pig cd29, mouse anti-human cd90 (bd biosciences), mouse anti-pig cd44 and sla-i and sla-ii (vmrd). for the detection of ev markers, ev-coated beads were stained with the following primary antibodies cross-reactive with pig: mouse antihuman cd9 (genetex), mouse anti-human cd63 (bd biosciences), and mouse anti-human cd81 (bd biosciences) for 20 min at 4°c in dark. cells and beads were washed three times and incubated with secondary antibodies conjugated with allophycocyanin or phycoerythrin for 30 min. appropriate isotype and secondary antibodies were used as controls for nonspecific binding. cells and beads were acquired by c6 flow cytometer (bd accuri cytometers) and analyzed using cflow® plus software (accuri) as described previously [32] . we examined whether msc-evs had the ability to enter cells using pig lung epithelial cells (mk1-osu; lecs). mk1-osu is a spontaneously immortalized cell line established in our laboratory. this cell line was derived from the distal trachea and proximal lung tissue of a 5to 6-week-old pig. these cells express α2-3and α2-6linked sialic acids (receptors for avian and mammalian influenza viruses, respectively), and support the replication of swine, avian, and human-origin influenza viruses (unpublished results). msc-evs were labeled using pkh-26 red fluorescent cell linker kit (sigma-aldrich) as per the manufacturer's instructions. lecs cultured overnight in a 24-well plate were incubated with labeled msc-evs for 24 h at 37°c. to confirm the internalization of evs inside the cells, the cell cytoplasm was stained using polyclonal rabbit anti-human β-tubulin as the primary antibody (thermofisher scientific) and alexa 488-conjugated goat anti-rabbit secondary antibody (thermofisher scientific). cell nuclei were stained with 4′,6-diamidino-2-phenylindole (dapi; thermofisher scientific life sciences). the cells were examined under a fluorescence microscope (olympus, japan). for flow cytometry, a single cell suspension was obtained by treating the cells with 0.25% trypsin-edta and washing with pbs. cells were acquired by c6 flow cytometer (bd accuri cytometers) and incorporation of pkh-26 labeled evs in cells was analyzed by cflow® plus software (accuri). the effect of msc-evs on the ability of influenza virus to hemagglutinate red blood cells was examined by hemagglutination inhibition assay. two-fold dilutions of msc-evs (starting from 10 μg/ml) in 25 μl volume were mixed with 8 hemagglutination (ha) units of swine (swine/tx/98; h3n2 and swine/mn/08; h1n1), avian (gull/md/1995; h9n5 and chicken/ny/h7n2), and human (human/ca/09; h1n1) influenza viruses and incubated for 20 min at 37°c followed by the addition of 50 μl 1% turkey red blood cell suspension. plates were incubated for 30 min at room temperature and the concentration of msc-evs capable of inhibiting ha activity of different influenza viruses was determined. to examine the effect of msc-evs on influenza virus replication, two sets of experiments were conducted. in the first set, we incubated swine/mn/08; h1n1 (swiv) equivalent to moi 1 with 10 μg/ml msc-evs for 20 min at room temperature. lecs cultured overnight were then infected with swiv alone or virus-msc-ev mixture for 1 h at 37°c. cells were washed with pbs and were incubated with or without msc-evs. after 8 h, influenza virus nucleoprotein (np) was detected using anti-np monoclonal antibody in an immunofluorescence assay (ifa). cells expressing np protein were counted in at least five microscopic fields and virus titers in culture supernatants were determined by titration in mdck cells [8] . at 24 h after infection, apoptotic cells were detected by tunel assay using apoptag fluorescein apoptosis detection kit (emd millipore). apoptotic cells were counted in five microscopic fields and data are expressed as mean ± sd number of apoptotic cells/ microscopic field. in the second set of experiment, lecs were infected with swiv at an moi 1 for 1 h at 37°c, followed by washing with pbs, and lecs were incubated with or without msc-evs for 8 h. influenza-infected cells were detected by the expression of np by ifa. in some experiments, msc-evs were treated with 1 u/ ml rnase for 1 h at 37°c, 1 u/ml rnase was then added to stop the reaction and msc-evs were washed by ultracentrifugation [29] . lecs were washed with pbs and fixed with 80% acetone for 10 min at -20°c. the expression of influenza virus np in infected cells was detected using mouse anti-np antibody as a primary antibody and alexa 488-conjugated goat anti-mouse igg as a secondary antibody. nuclei were stained with dapi (thermofisher scientific life sciences). five-week-old conventional large white-duroc crossbred pigs were obtained from the osu herd. maintenance of pigs and all experimental procedures were conducted in accordance with the guidelines of the institutional laboratory animal care and use committee, the ohio state university (protocol #2014a00000040). at 8 weeks of age, nine pigs (mean weight 12.1 ± 1.3 kg) were divided into three groups of three pigs each. pigs in group 1 were inoculated intranasally with dmem and these pigs served as controls. pigs in groups 2 and 3 were similarly inoculated with swiv (5 × 10 6 tcid 50 per pig). pigs had a hemagglutination inhibition (hi) antibody titer of 1:12 ± 4 against swiv before infection. in our previous study, infection of 8-week-old commercial pigs with swiv (5 × 10 6 tcid 50 per pig) induced extensive lung lesions [33] . twelve hours after swiv infection, pigs in groups 2 and 3 were administered intratracheally with dmem and msc-evs (80 μg/kg body weight (bw)), respectively. in human clinical trials for ards [6] , mscs between 1 and 10 × 10 6 cells/kg bw were used. in our study, ev dose per kg bw was calculated by the protein content of evs produced by 10 × 10 6 mscs cultured for 48 h. in a previous study [23] intratracheally administered evs were effective in treating e. coli-induced severe pneumonia. based on this observation, in this study evs were administered by the intratracheal route. pigs were monitored daily for clinical signs and nasal swabs were collected at 1 and 3 days postinfection (dpi) for virus titration in mdck cells. at dpi 3, pigs were euthanized and lungs were harvested. lung lysate was prepared from the left lung for virus quantification and cytokine analysis, and the entire right lung was fixed in 10% buffered formalin for microscopic examination. the time-point dpi 3 was selected based on our earlier publication showing higher virus replication and proinflammatory cytokine production at dpi 3 compared with dpi 6 [8] . paraffin-embedded sections of lung tissues were stained with hematoxylin and eosin and examined with the help of a trained pathologist who was not aware of the experimental design. the slides were examined for bronchiolar epithelial changes, peribronchiolar inflammation, and interstitial pneumonia, and lesions were scored from 0-3 as described previously [34, 35] . a 10% (w/v) lung homogenate was prepared from left apical lobe lung tissue and virus titer was determined by titration in mdck cells as described [33] . virus titers were calculated by the reed and muench method. lung lysates from pigs were prepared and levels of tumor necrosis factor (tnf)α, cxcl10, and interleukin (il)-10 in lung lysates were determined by enzyme-linked immunosorbent assay (elisa) as described previously [8] . in vitro data on msc-ev-mediated inhibition of swiv replication were analyzed by student's t test. microscopic lung lesions, virus titers, and cytokine concentrations between groups of pigs were compared using kruskal-wallis test. p values < 0.05 were considered statistically significant. we isolated mscs from the bm of 2-to 6-week-old pigs that showed characteristic features of mscs, such as adherence to a plastic surface, fibroblast-like morphology (fig. 1) , self-renewal potential, and high in vitro proliferation capacity and differentiation potential (data not shown). colony-expanded mscs showed the expression of the mesenchymal markers cd29, cd44, cd90, and sla-i, but sla-ii was not detected on these cells. msc-evs were round-shaped and, similarly to mscs, evs isolated from bm-mscs also expressed mesenchymal markers; however, unlike mscs, they lacked the expression of both sla-i and ii (fig. 1) . msc-evs also expressed ev-specific markers such as cd9, cd63, and cd81 (fig. 2) . we also determined the rna and protein concentration in msc-evs. msc-evs contained 113 ± 37 ng/100 μl evs (n = 5) total rna and 79 ± 1 μg/100 μl evs (n = 4) total protein. additionally, msc-evs showed the ability to incorporate into lecs. evs stained red with pkh-26 dye were found inside the cytoplasm of cells when examined under a fluorescent microscope. incorporation of msc-evs in lecs was also confirmed by flow cytometry (fig. 3) . to determine if msc-evs possess anti-influenza activity, we first examined if evs inhibited the ha activity of influenza viruses. eight ha units of swine/tx/98; h3n2, swine/mn/08; h1n1, gull/md/1995; h9n5, chicken/ ny/h7n2, and human/ca/09; h1n1 influenza viruses were incubated with different concentrations of msc-evs and the ha activity of these viruses was examined. msc-ev concentrations between 1.25 and 5 μg/ml completely inhibited the ha activity of these viruses (table 1) . we examined msc-evs derived from bm-mscs for anti-influenza activity. swiv (sw/mn/08; moi of 1) was incubated with msc-evs (10 μg/ml) for 20 min at room temperature. lecs were infected with ev-swiv mixture or virus only. at 8 h after infection, iav-np was detected by ifa ( fig. 4a and c) . significantly reduced (p < 0.05) influenza replication was detected when cells were infected with swiv pretreated with msc-evs. consistent with reduced influenza virus replication in lecs treated with msc-evs, msc-evs also significantly (p < 0.05) inhibited the apoptosis of influenza-infected lec ( fig. 5a and b) . in another set of experiments, porcine lung epithelial lecs were infected with swiv for 1 h, washed, and incubated with media without or with msc-evs (10 μg/ml). at 8 h after infection, iav-np was detected by ifa. the addition of msc-evs after virus entry significantly (p < 0.05) reduced influenza replication in lecs (fig. 6) . we examined the anti-influenza and immunomodulatory effect of msc-evs in influenza virus-induced ali in pigs. eight-week-old pigs were infected intranasally with 5 × 10 6 tcid 50 of swiv. twelve hours after swiv inoculation, dmem or msc-evs (80 μg/kg) were administered intratracheally in influenza-infected pigs. at days 1 and 3 after ev administration, we collected the nasal swabs, and at day 3 after ev administration the pigs were euthanized and bronchoalveolar lavage (bal) and lungs were collected. lungs were homogenized to make 10% lung lysate for determining virus titers, and cytokines were examined in the lung lysate. swiv induced extensive lung lesions in infected pigs as determined by infiltration of inflammatory cells, thickened alveolar walls, and collapsed alveoli, whereas lungs of pigs administered with msc-evs showed minor infiltration of inflammatory cells. the mean microscopic lung lesion score in swiv + dmem inoculated pigs was 7.3 ± 1.5 compared with 2.6 ± 1.5 in the swiv + ev administered group (fig. 7a-d) . consistent with the inflammatory lesions in lungs, the levels of total protein in the bal of swiv + ev (174 ± 20 μg/ml) administered pigs were also lower than the levels in swiv + dmem (270 ± 34 μg/ml) inoculated pigs (fig. 7e) . we detected the influenza virus shedding in nasal swabs at 1 and 3 days after ev administration. virus shedding was 100-fold lower in the ev-administered group at 3 days post-ev administration as compared with swiv + dmem inoculated pigs (fig. 8) . similarly, virus titers were also 100-fold lower in the lung lysate of ev-administered pigs. these data suggest that msc-evs inhibit influenza virus replication and shedding in pigs. next, we examined if msc-evs also modulated the inflammatory cytokine production in lungs. in the msc-ev administered group, the levels of tnfα were 251 ± 46 pg/g lung lysate compared with 386 ± 40 pg/g in pigs inoculated with swiv + dmem. similarly, levels of cxcl10 in swiv + ev administered pigs were 3259 ± 469 pg/g compared with 4456 ± 495 pg/g in lung lysates of swiv + dmem inoculated pigs. the levels of the anti-inflammatory cytokine il-10 were slightly higher in the swiv + ev administered group compared with the swiv + dmem inoculated pigs (fig. 9) . these findings suggest that msc-evs possess anti-influenza and anti-inflammatory properties and attenuated influenza virus-induced ali in a pig model. we isolated and characterized evs from porcine bm-mscs using protocols as described for human mscderived evs [23, 29, 30] . as for human msc-evs, we also used tem to determine the size and morphology of msc-evs. size distribution of human msc-evs was also determined by nanotracking but this technique was not used in this study. human msc-derived evs expressed mesenchymal markers and lacked the expression of mhc molecules [36] . in our study, pig msc-derived evs also expressed mesenchymal markers and the expression of both sla-i and ii was not detected in evs, suggesting that pig msc-evs are similar to human msc-evs and will be useful in understanding the mechanisms of msc-ev-mediated therapeutic effects in a pig model of human diseases. a number of studies have demonstrated the beneficial effects of msc-evs in vitro and in animal models of lung, kidney, and liver diseases [37, 38] . furthermore, several studies have shown that evs exert their therapeutic effects by transferring their mrna to diseased cells [20, 29, 39, 40] . in this study, the anti-influenza virus activity of msc-evs in lung epithelial cells was mediated by their mrna content as pretreatment of evs with rnase enzyme abrogated the anti-influenza virus activity of evs. in a recent study, qian and colleagues [41] demonstrated that exosomes (exo) isolated from umbilical cord-derived mscs inhibited replication of hepatitis c virus (hcv) in human dermal fibroblast 7 cells. these authors further identified four msc-exo-specific mirnas with anti-hcv activity. msc-ev-specific mirnas have also been implicated in the regulation of cell survival, differentiation, and immunomodulation [42, 43] . future studies will focus on identifying msc-ev-specific mrna and mirnas that may play a role in regulating the anti-influenza activities of msc-evs. one of the mechanisms by which influenza viruses cause extensive lung damage and mortality is due to the induction of unregulated inflammatory response [44, 45] . similarly to mscs, msc-derived evs also possess immunomodulatory activity [46] [47] [48] [49] [50] [51] [52] . in this study, msc-ev inoculated pigs showed significantly less lung inflammation and decreased levels of proinflammatory cytokine and chemokine production. additionally, increased levels of anti-inflammatory cytokine il-10 were observed in msc-ev administered pigs. these data suggest that, in addition to anti-influenza activity, msc-evs also exert their immunomodulatory activities and suppress the inflammatory response in influenza. similar to our findings, msc-evs caused decreased production of inflammatory cytokines and increased production of il-10 in a mouse model of ards [20] . monsel and colleagues [23] demonstrated that msc-evs express cyclooxygenase (cox)-2 mrna, the enzyme that induces prostaglandin e2 (pge2) synthesis. pge2 secreted by mscs has been shown to reprogram proinflammatory monocyte-macrophages (m1) to the anti-inflammatory (m2) type that produces high levels of il-10 [53] . additionally, msc-evs interact with immune cells and cause the production of transforming growth factor (tgf)β and t-regulatory cells (tregs) [48] . tregs promote virus clearance and recovery in influenza virus-infected mice [54, 55] . in future studies, we will examine whether msc-ev treatment induces the generation of m2-type macrophages and tregs in a pig model of influenza virus. mscs mediate their immunomodulatory and tissue repair functions by the release of numerous therapeutic soluble factors [43, 56] . indoleamine 2,3-dioxygenase (ido) is produced by mscs and modulates the functions of immune cells and inhibits influenza virus replication [57] . similarly, ll37, an antimicrobial peptide, is produced by mscs and has been shown to inhibit the growth of several species of bacteria and also inhibits replication of influenza virus [58, 59] . additionally, ll37 was also shown to regulate the immunomodulatory activities of mscs [60] . currently, data on whether ll37 and ido are transferred from mscs to their secreted evs are not available and were not examined in this study. it may be possible that these factors may be involved in mediating the antiinfluenza and anti-inflammatory activities of msc-evs in our influenza model. recently, a few studies examined the therapeutic potential of mscs in influenza in mice with conflicting results [9] [10] [11] [12] . the liles and matthay groups reported that mouse and human mscs failed to prevent influenza virus-induced mortality and ali in mice [10, 11] . more recently, two publications have shown that mscs reduced influenza virus-induced ali in mice infected with hpai h5n1 and h9n2 without affecting the virus replication [9, 12] . we and others have shown previously that influenza virus infected mscs and that infected mscs produced inflammatory cytokines [13] [14] [15] . of the in vivo studies that examined the therapeutic efficacy of mscs in influenza virus-induced ali, none examined the infection of administered mscs with influenza virus. replicating virus in influenza virus-infected mice may infect the injected mscs and virus-infected mscs may not be functionally effective in inhibiting virus replication and inflammation. in in-vitro studies, influenza virus infection caused the apoptosis and lysis of infected mscs [13] . inactivated mscs have been shown to retain some of their immunomodulatory functions. heat-inactivated mscs modulated the functions of monocytes/macrophages but had no effect on t or b cells [61] . in studies which demonstrated the protective effects of transplanted mscs in influenza virus-infected mice, anti-inflammatory fig. 8 effect of msc-ev administration on virus titers in nasal swabs and lungs of pigs infected with swiv. eight-week-old pigs were mock infected or infected with swine/mn/08; h1n1 (swiv). after 12 h, pigs were administered intratracheally with dulbecco's modified eagle's medium (dmem) or msc extracellular vesicles (evs). nasal swabs were collected from infected pigs at 1 and 3 days after ev administration (1dpev and 3dpev). at 3 days after ev administration, pigs were euthanized and lungs were harvested. lung tissues were homogenized to prepare 10% lung lysate. influenza virus shedding in nasal swabs and virus titers in lungs were determined by titration in mdck cells. values in each bar indicate mean virus titers of three pigs ± sd fig. 9 effect of msc-ev administration on cytokine production in lungs of swiv-infected pigs. eight-week-old pigs were mock infected or infected with swine/mn/08; h1n1 (swiv). after 12 h, pigs were administered intratracheally with dulbecco's modified eagle's medium (dmem) or msc extracellular vesicles (evs). three days after ev administration, pigs were euthanized and cytokine production in lung lysate was analyzed by elisa. each bar represents mean concentrations of cytokines ± sd from three pigs. il interleukin, tnf tumor necrosis factor effects observed in these studies may likely be mediated by influenza-infected dead or apoptotic mscs. future experiments will be required to confirm the infection of transplanted msc by influenza virus and to examine their interaction with immune cells in animal models of influenza virus. msc-evs were recently identified and were as equally effective as mscs in treating endotoxin and e coli-induced ali in rodent models [20, 23] . here, we demonstrated that msc-evs attenuated influenza virus-induced ali in a pig model. importantly, administration of msc-evs was safe since no gross pathological lesions were observed in any of the internal organs of the pigs. additionally, msc-ev treatment was also found to be safe in human patients treated for graft-versus-host disease (gvhd) and chronic kidney disease [50, 62] . thus, the findings of this study may help in the planning of future clinical trials in humans using msc-evs as a cell-free therapy for ards. msc-evs offer several advantages over mscs as a therapy for human diseases. although mscs have been proven to be effective and safe in treating several human conditions in animal models and clinical trials, safety concerns such as pulmonary embolism, uncontrolled differentiation, and tumor formation are still associated with the use of mscs as a therapy in humans [63, 64] . on the other hand, use of evs as a therapy was found to be safer than mscs [20, 50] . evs are stable in circulation due to their lipid membrane, have no risk of aneuploidy, and are well tolerated in recipients due to their small size and lack of expression of mhc molecules. moreover, evs can be stored at -80°c without losing their biochemical activity [65, 66] . thus, evs have the potential to become a safe and effective cell-free therapy for human diseases. however, a number of challenges, such as standard protocols for the production of clinical grade evs, their dosing, quality control, and storage conditions, still need to be addressed before msc-evs can be advanced to the clinic. this study has certain limitations: 1) an additional control group consisting of evs derived from fibroblasts is needed to confirm that the beneficial effects in influenza are specifically mediated by msc-ev; and 2) administration of evs by the intravenous route would 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stem cells maintain immunomodulatory capacity umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases caveats of mesenchymal stem cell therapy in solid organ transplantation concise review: workshop review: understanding and assessing the risks of stem cell-based therapies in vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model recent developments in cellular immunotherapy for hsct-associated complications we thank dr. juliette hanson, megan strother, ronna woods, and sara tallmadge for veterinary and animal care. we also thank dr. chun-ming lin for his help with the histopathological examination of lung sections, dr. kuldeep chattha for his assistance with flow cytometry, and santosh dhakal and sydney bartolet for their help with animal inoculations and necropsy. this research was partly supported by the national institute of health (grant #r21hl125191 to mk). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-304089-u2abo951 authors: trombetta, claudia maria; perini, daniele; mather, stuart; temperton, nigel; montomoli, emanuele title: overview of serological techniques for influenza vaccine evaluation: past, present and future date: 2014-10-13 journal: vaccines (basel) doi: 10.3390/vaccines2040707 sha: doc_id: 304089 cord_uid: u2abo951 serological techniques commonly used to quantify influenza-specific antibodies include the haemagglutination inhibition (hi), single radial haemolysis (srh) and virus neutralization (vn) assays. hi and srh are established and reproducible techniques, whereas vn is more demanding. every new influenza vaccine needs to fulfil the strict criteria issued by the european medicines agency (ema) in order to be licensed. these criteria currently apply exclusively to srh and hi assays and refer to two different target groups—healthy adults and the elderly, but other vaccine recipient age groups have not been considered (i.e., children). the purpose of this timely review is to highlight the current scenario on correlates of protection concerning influenza vaccines and underline the need to revise the criteria and assays currently in use. in addition to srh and hi assays, the technical advantages provided by other techniques such as the vn assay, pseudotype-based neutralization assay, neuraminidase and cell-mediated immunity assays need to be considered and regulated via ema criteria, considering the many significant advantages that they could offer for the development of effective vaccines. the human influenza virus is one of the most important infectious diseases in the world [1] . it is rooted in the past and has periodically decimated the world's population since ancient times via pandemics. influenza virus infects all age groups but children and adults over the age of 65 are most at risk. vaccination is recommended for these age groups, and also for anyone with high-risk conditions due to complications of influenza and those with chronic medical conditions (metabolic, cardiac, pulmonary or kidney diseases, as well as immunocompromised patients) [2, 3] . generally the same recommendation is extended for nursing home and health workers. vaccines and antiviral drugs are the two primary methods of implementing influenza prophylaxis. they are the only effective ways to prevent infection and treat illness [4] , and their availability plays a key role in the event of a pandemic. evidence has been obtained from the relatively mild 2009 h1n1 influenza pandemic which demonstrated that vaccines and antiviral drugs were not readily available in time for more than 90% of the world's population [5] . antiviral drugs can give responsive protection against influenza virus and are useful for containment at the beginning of a pandemic, but in the long term, infection control depends on vaccination [6] . vaccination remains the most efficacious method to control seasonal infections and the most important strategy to prepare for a possible pandemic [7] . the degree of protection elicited by vaccination depends on interplay between vaccine composition and circulating influenza viruses, the age of the vaccine recipient and their previous exposure to influenza. currently, inactivated vaccines are the most effective means to counteract influenza infection [8] . they show a 60%-100% ability to prevent morbidity and mortality in low-risk target populations, such as healthy adolescents or adults, but may have little effect in younger (naï ve) or older (decreased immune function) populations [9] , as well as over time, due to low antigenic match [10] . in order for a vaccine to be marketed, it is necessary to evaluate its immunogenicity -the capacity of the vaccine to induce an immune response. studies have shown that antibodies directed against viral haemagglutinin (ha) are an important correlate of protection [11, 12] . serological techniques commonly used to quantify influenza-specific antibodies include the haemagglutination inhibition (hi), single radial haemolysis (srh) and virus neutralization (vn) assays. hi and srh are common, easy to perform and reproducible techniques, whereas vn is more laborious, less immediate, does not allow for simultaneous analysis of a large number of samples but is advantageous due to the possibility of detecting all functional antibodies that interfere with infection [13] and are of low antibody titres [14, 15] . recent studies have also been focused on the activity of neuraminidase (na) [16, 17] and on cellular immunity induced by vaccines [18, 19] . each vaccine needs to fulfil the criteria which include exclusively srh and hi assays, issued by the committee for proprietary medicinal products (cpmp) in order to be licensed [20] (table 1) . table 1 . committee for proprietary medicinal products (cpmp) criteria. seroconversion (hi): if pre-vaccination serum is negative, then post-vaccination serum must have a titre ≥40; if pre-vaccination serum is positive, then at least a fourfold titre increase is required. seroconversion (srh): if pre-vaccination serum is negative, then post-vaccination serum haemolysis area must be ≥25 mm 2 ; if pre-vaccination serum is positive, then there must be at least a 50% increase in haemolysis area. seroprotection (hi): a serum sample is considered seroprotected when it shows an hi titre ≥40 or an srh titre >25mm 2 . seroconversion rate: proportion of subjects showing seroconversion. seroprotection rate: proportion of subjects showing seroprotection. seroconversion rate > 30% mean geometric increase > 2. 5 mean geometric increase > 2.0 seroprotection rate > 70% seroprotection rate > 60% the cpmp criteria refer to two different target groups-healthy adults between 18-60 and those over 60-but other age groups that may receive such vaccines have not been included. in 2006, the committee on immunization practices (acip) updated their influenza vaccine recommendations and introduced categories such as children between 6-9 and 24-59 months, pregnant women, healthcare providers, patients with chronic disease and immunosuppressed patients [21] . despite extensive research having been conducted in recent years to identify correlates of protection for these two age groups, little is currently known about protection in the event of hospitalization, secondary bacterial infections, chronic illness and even less about protective correlates in specific risk groups, even if they may be important for society [22, 23] . a correlate of protection is an important milestone in the development of a new vaccine but despite this, it still remains a very confused concept. some definitions have been given, such as the one offered by plotkin and gilbert [24] , which states that "a correlate reflects a statistical relation between an immune marker and protection but does not necessarily imply causal agency of the marker" and that proposed by qin et al. [25] , suggesting correlates predict "protection for new settings and describe the data requirements for rigorous validation of an immunological measurement at each level". the concept is based on the immunogenic capacity of the vaccine to produce an antibody and/or cell-mediated immune response in its recipients [26] . studies conducted by hobson [11] and others [12, 27, 28] have established that ha antibody titre is correlated with protection against influenza infection. generally an hi antibody titre of 40 is defined as 50% protective against influenza infection compared to ha titre < 10. correlates of protection can be absolute or relative. even if the ideal scenario is for a correlate to be absolute, meaning that protection is almost guaranteed by a definitive threshold of response (such is the case for diphtheria, tetanus and rubella), many correlates of protection are relative. in these instances, protection is usually conferred by a certain response level, but this can vary between vaccinated individuals and disease can occur in some vaccinees regardless of a theoretically protective correlate response [29] . for instance, with inactivated vaccines, the consensus derived from several studies is that an hi antibody level of 1:40 against ha should be considered protective [30] . however, this level is only 50%-70% protective so this correlate of protection should be regarded as relative rather than absolute [30] . coudeville et al. [12] developed a model, using a meta-analytical approach, in order to estimate the level of protection against influenza associated with any hi titre level. the results suggest that the relationship between titre and protection is identified by a curve rather than a threshold. the clinical protection shows a progressive and significant increase at titres up to 100 (which included the common threshold of 1:40) but advantages become marginal beyond 150. pertaining to correlates of protection for influenza vaccines, it is also important to distinguish between young and elderly vaccinees, due to the fact that igg serum antibodies only correlate well with protection for adults under the age of 50 [30] . humoral immune responses raised against influenza viruses or related vaccines are mediated by several factors, such as age, the simultaneous presence of other diseases and the contemporaneous use of medicines that may affect immune function. several studies have been conducted on the immune response to influenza vaccines and conflicting results were obtained. some show that vaccination induces a lower hi antibody response in elderly compared to young recipients, while others report no discrepancy between age brackets or indeed report a contrary result. protective immune indicators against influenza in at-risk groups must yet be defined since, even in this case, some studies have reported a reduced humoral response in risk groups, while other studies have shown that the humoral response is comparable to healthy control subjects [26] . it is also necessary to distinguish between vaccine efficacy and vaccine effectiveness. often the distinction between these terms is ignored and they are used interchangeably, which can result in widespread confusion and misconception of "vaccine efficacy" [31] . in fact, "vaccine efficacy" is measured precisely as the ability of a vaccine to prevent disease in vaccinated individuals, with emphasis on the exact levels of vaccine-induced disease reduction [32] . "vaccine effectiveness" refers to how well a vaccine protects against influenza when routinely used in the community, as opposed to in a randomized control trial. this is evaluated by observational studies and represents the reduction of infection frequency in vaccinated individuals compared to those who have not been vaccinated, assuming that the vaccine has induced said reduction [33] . confusion also surrounds the topic of "surrogates of protection". consistent definitions have been published by both plotkin and quin, detailing a surrogate of protection: "as an immune marker that can substitute for the clinical end point and thus, can be used to reliably predict vaccine efficacy". however, according to quin, a surrogate may or may not be considered as a causal agent of protection, whereas plotkin considers a surrogate of protection to be an immunological measurement performed when unable to ascertain a true correlate but stresses that there is no direct causality assumed with a surrogate [29] . another relevant concept defines surrogates of protection as correlates able to predict the level of protective efficacy of a vaccine by comparing immunological measurements of vaccinated and unvaccinated individuals [34] . a general surrogate of protection needs to be adequately specific in several circumstances in order to be generalized to untested groups [35] . the hi assay is based on the ability of antibodies, if present in the serum, to prevent agglutination between erythrocytes and viral haemmaglutinin [36] . the antibody titre is expressed as the reciprocal of the highest serum dilution showing complete inhibition using 4 hau units/25 µl or 8 hau units/50 µl [8, 37] . the starting dilution is generally 1:10 and the lower limit of a detectable antibody titre is 10. when the titre of antisera is under a detectable threshold, due to a low or non-existent amount of antibodies, this is conventionally expressed as 5, half the lowest detection threshold [38] . as previously mentioned, an antibody titre of 40 is generally considered as a protective threshold level, beyond which there is a 50% or greater reduction in the possibility of contracting influenza infection [11, 39] . an hi titre equal to or greater than 40 is used as an immunological correlate of protection and is regarded as the best currently available parameter for predicting protection from natural infection, according to fda guidelines for pandemic influenza vaccines [40] . the problem raised by black et al. [41] , however, is that this correlate is well defined in adults but not in children although it also applies to serology of samples from this age group. children show a reduced capacity for cellular immunity and have little to no previous exposure to either influenza or vaccination. this situation necessitates the definition of a protective correlate specific to this category. more importantly, previous exposure to the influenza virus or vaccination can play a crucial role in reaching the protective threshold level in adults. in fact, even if all those who receive the vaccine are seroprotected (a subject is defined as seroprotected if the antibody level is above a certain cut-off [38] ), this result may not correspond to a 50% vaccine-induced reduction of risk of influenza infection, due to the presence of protective antibodies in some subjects prior to vaccine administration. therefore, an hi titre of 40 is not a threshold of protection induced by the vaccine but protection induced by antibodies in general [42] . this is the reason why, in vaccine assessment, seroprotection is complemented by the two further criteria, seroconversion and mean geometric increase which help discriminate high antibody titres prior to vaccination. black et al. [41] conducted a study in children aged between 6 and 72 months who had not previously been vaccinated in order to evaluate the relationship between hi antibody titre and clinical protection from influenza illness. the subjects were divided into 3 groups and each received two doses, one month apart, of either an mf59 adjuvanted trivalent inactivated influenza vaccine (ativ, fluad®, novartis vaccines, siena, italy), a subunit trivalent inactivated influenza vaccine (tiv, glaxosmithkline, rixensart, belgium) or a saline placebo. clinical cases for influenza like illness were confirmed by reverse transcription polymerase chain reaction testing for influenza. for the recipients of the adjuvanted vaccine, results confirmed that a cut-off of 1:110 measured 21 days after the second vaccine dose could be used as a marker of 50% protection in the juvenile sample population. titres of 1:215, 1:330 and 1:629 correspond to protection levels of 70%, 80% and 90% respectively (table 2) . importantly, similar results could not be achieved in the tiv vaccine group. therefore, it is evident that a higher hi titre is required in naï ve subjects compared to those with a previous history of influenza infection in which other protective factors may come into play. a further consideration could be to assess whether the hi assay is indeed the best option to evaluate influenza vaccines for children [43] . vaccination in children must also overcome other issues. in addition to defining protective correlates specific to the immune responses elicited by this age cohort, it is necessary to define the method and quality of vaccine administration, in addition to the most suitable vaccine evaluation techniques. the virus neutralization (vn) is a helpful assay for diagnostics and basic research that enables the observation of the humoral immune response against a virus [44] . in the vn assay, the highest serum dilution that induces a 50% inhibition of virus growth is identified as a neutralizing titre, based on the amount of virus in negative control wells [45] . the sample titre is defined as the maximum dilution factor at which antibodies can be identified. for instance, if there are detectable antibodies in a sample at a dilution of 1:40, the sample will have a titre ≥40 [46] . vn is a particularly useful technique for serology of avian strains of influenza a, and also influenza b viruses, as several studies have detailed the unsuitability of hi to detect antibody responses against these viruses [15, [47] [48] [49] [50] . because of the insensitivity of detecting h5-induced antibody responses by hi, the quantification of functional neutralizing antibody responses is the analytical aim of vn immunogenicity studies [51] . in comparison to hi, the vn assay identifies a wide range of neutralizing antibodies because it detects antibodies that neutralize the virus via entry/replication inhibition in mammalian cells whereas hi only measures antibodies directed against viral haemagglutinin that act by preventing erythrocyte agglutination. as has been suggested in animal model studies, the prevention of infection is predicted by antibody-mediated neutralisation while disease prevention is correlated to hi [52] [53] [54] [55] [56] . conventional neutralization tests are based on the inhibition of cytopathic effect in madin-darby canine kidney (mdck) cell cultures, resulting in laborious and slow tests. a vn assay with microtitre plates, in combination with a downstream elisa to detect virus-infected cells, is faster, producing results in just two days [37] . vn assays are also able to detect antibodies at low titres [14, 52] and can distinguish between pre-and post-vaccination titres, especially in the instance of small (less than two-fold) differences between titres, when compared to hi [57] . one of the major drawbacks of the vn assay platform is the necessity to handle wild-type virus and the associated costs of high-level biocontainment facilities (i.e., biosafety level 3 laboratory) when studying the serology of highly pathogenic strains, such as h5 and h7. in this instance, the use of influenza ha pseudotypes as surrogates for wild-type virus is a safer alternative, that may also have increased throughput capability and ease of standardization benefits (see section 5) . other limitations of the vn assay include extensive training requirements for laboratory personnel, issues with throughput/simultaneous screening of large panels of sera and importantly, problems with standardising cell preparations, virus inoculations and incubation times [15, 49] . incidentally, inter-laboratory variation is significant with vn assays due to the lack of common reference protocols, discrepancies with assay endpoint determination and limited knowledge of correlates of protection. currently, no protective correlates have been defined for vn in animal or human models, and due to the assay's variability, a vn titre equivalent to an hi titre of 40 is highly specific for each antigen-laboratory combination and therefore cannot be generalized [57] . according to the world health organization (who), h5n1 infection can be confirmed by vn when one of the following criteria is met: a "fourfold or greater rise in antibody titre against a (h5n1) in paired sera (acute and convalescent) with the convalescent serum having a titre of 1:80 or higher or antibody titre of 1:80 or more in a single serum collected at day 14 or later after onset of symptoms and a positive result using a different serological assay" [58] . scientific studies have uncovered a more complicated situation concerning vn, both in the case of h5n1 infection and in the case of other influenza infections, such as h1n1. some studies of h5n1 employ a titre ≥80 as an efficacy endpoint for avian influenza vaccines [59, 60] , whereas others consider a seroprotection cut-off of 1:20 to be suitable, on the basis of correlation with an srh area of 25 mm 2 [13, 45, 51] . based on previous research regarding h1n1, allwin et al. [61] suggest a seropositive threshold of a 1:64 virus-neutralising serum dilution. other studies agree in considering a titre ≥10 as the minimum detection limit and a titre ≥40 as a significant response [52, 54, 55] . the titre ≥10 could be interpreted as an effective indicator of population exposure to the virus by either natural infection or vaccination [46] . regardless of the lack of clearly defined criteria for vn, many studies have shown that the assay correlates well with hi ( figure 1 ) [62] [63] [64] with the exception of h7 virus subtypes [64] . okuno et al. [65] have demonstrated a good correspondence between hi and vn titres in some circumstances and also report a heightened reliability of vn when compared to hi (figure 2 ). they recorded both sets of titres raised against the seasonal influenza strains a/yamagata/12/86 (h1n1), a/fukuoka/c29/85 (h3n2) and a/shisen/2/87 (h3n2). the results showed a good correlation between titres calculated for a/yamagata/12/86 (h1n1) and a/fukuoka/c29/85 (h3n2), but the vn titres were lower than those for hi against a/shisen/2/87 (h3n2). an explanation of this is that, over recent years, most schoolchildren have received a multivalent vaccine including a/yamagata/12/86 (h1n1) and a/fukuoka/c29/85 (h3n2) strains, but not a/shisen/2/87 (h3n2), since it was a candidate vaccine strain from 1988. the vn assay can be developed for any influenza a subtype [15] and has also been demonstrated for influenza b viruses. it is able to distinguish between viruses that belong to the same lineage, as well as to different sublineages [49] . although vn assay results are not strictly necessary in obtaining committee for medical products for human use (chmp) approval, the ema recommends the quantification of neutralizing antibodies able to inhibit viral attachment, entry and release of progeny virions [66] . this recommendation further reinforces the necessity to revise existing guidelines. a pseudotype virus has the "core" of one virus (e.g., a retrovirus) and the outer "envelope" protein(s) of another (e.g., the ha/na of influenza virus). the core virus has deletions in its genome making it replication-deficient (allowing it to be used under bsl1/2 containment), and harbours a reporter transgene (e.g., luciferase or green fluorescent protein (gfp)). the envelope glycoprotein permits entry into susceptible target assay cells (e.g., human embryonic kidney 293 t cells (hek293t)/mdck) via interaction with sialic acid. during target cell transduction, the pseudotype virus genome becomes integrated into the cell genome, resulting in reporter gene expression. thus, the number of transduced cells can be accurately quantified (via a luminometer, fluorescent microscope or fluorescence-activated cell sorting (facs)) and the subsequent inhibitory effects of functional antibodies (directed against the ha1 head and the ha2 stalk) in serum determined [67] [68] [69] . figure 3 below shows a schematic representation of the production and assay of influenza ha pseudotyped retroviruses. pseudotype production of highly pathogenic avian influenza (hpai) strains (which have a polybasic cleavage site in the ha) routinely involves the transfection of three plasmids into 293t producer cells: retroviral gagpol plasmid, ha-expressing plasmid, and retroviral vector plasmid incorporating the reporter gene. additionally, for the production of low pathogenic avian influenza (lpai) strains (single arginine at cleavage site), an additional plasmid expressing a protease (either transmembrane protease serine 2 ((tmprss2) or human airway trypsin-like protease (hat)) is necessary [70, 71] . the pseudotype neutralization assay is also amenable to multiplexing provided that the two desired subtypes fall into different ha groups (ha group 1 comprises h1, h2, h5, h6, h8, h9, h11, h12, h13, h16 and h17; ha group 2 comprises h3, h4, h7, h10, h14 and h15). this has recently been successfully achieved with avian influenza strains h5 and h7 to study the immunogenicity of a bivalent h5/h7 chicken vaccine [73] . when evaluating sera for the presence and magnitude of neutralizing antibodies directed against two viruses (h1 and h3 for seasonal, h5 and h7 for pandemic, for example), there are distinct advantages to the use of a multiplex assay rather than individual subtype assays, which are in routine usage. in a multiplex format, inter-assay variability is likely to be reduced, since only a single serum dilution series needs to be performed and the same preparation of target cells (e.g. mdck) is used for the two viruses. also, the antibody response to one virus may serve as an internal "serostandard" for the antibody response to the second virus and vice versa, as two separate luciferase reporters (firefly and renilla) are employed for this assay [73] . a pseudotype-based assay is more readily amenable to high-throughput processing of large serum panels, which will enable effective and informative larger-scale studies to be carried out. there are also beneficial safety and financial implications to the use of this assay, since antibody responses against two viruses are performed on the same serum samples with the entire process performed outside of high bsl facilities, which by their nature are expensive to maintain and require a high level of user training. the pseudotype-based neutralization assay is both "serum-sparing" and "antigen-sparing" as only 2-5 µl of serum is required per assay compared with the significantly larger volume required for traditionally employed influenza serology assays, and very small volumes of pseudotype virus (<1 µl virus/well) are required. it is therefore possible to measure antibody responses against large panels of sera more rapidly and more accurately than using traditional influenza serology assays (hi, vn, srh). pseudotype-based antibody assays have been shown to have broad utility for the detection of neutralizing antibody responses in avian and human sera, from natural infection and pre/post-vaccination against both avian and human influenza viruses [67, [74] [75] [76] [77] [78] [79] [80] . alberini et al. [74] undertook a comparative serological study using pseudotype neutralization, vn, hi and srh. from this study, a pseudotype titre corresponding to a vn titre of 80 was extrapolated to give 1:357. further large-scale comparative studies with both seasonal and pandemic vaccines are required to complement this. particular interest should be paid also to the potential of pseudotype neutralization assays to study antigenic evolution of influenza viruses. the continuous rapid evolution of influenza viruses, driven by error-prone replication and increasingly by immune pressure, significantly influences the sensitivity of available serological assays for this virus. it can also limit the efficacy of human and avian influenza vaccines and the susceptibility of these viruses to anti-viral drugs, via the emergence of drug resistance. as substitution rates are significantly higher in influenza ha and na genes compared with internal genes, retroviral and lentiviral pseudotypes bearing ha and na envelope glycoproteins devolved from the rest of the virus are ideal tools to monitor the effects of antigenic drift on serological outcomes, and can be used for accurate sequence-directed, highly sensitive, low-containment assays for measuring antibody responses against influenza ha. it is relatively straightforward to update the pseudotype-based ha neutralization assay to measure responses against newly emerging influenza viruses [69, 71, 74, 78, 81] . upon availability of the viral rna/cdna, ha/na genes can be sequenced, readily pcr-amplified and cloned, or custom synthesized, and retroviral pseudotypes prepared for use in neutralization assays. site-directed mutagenesis of the ha can subsequently be used to "fine-tune" the neutralization assay in order to track the evolutionary progression of the circulating virus at the genetic, and more importantly for serology, at the antigenic level. therefore, these assays can be continually and easily updated to measure the immunogenicity of current and new vaccines and therapeutics, and for sero-surveillance studies in new outbreak locations. another considerable advantage of pseudotype-based assays is the capacity for adequately evaluating the immune response induced by currently licensed influenza vaccines. in fact, antibodies elicited by such vaccines are predominantly haemagglutination-inhibition (hai)-competent antibodies that target the globular head of ha, thus inhibiting pseudotype entry into target 293t cells [67, 82] . these antibodies predominantly confer homosubtypic/strain-specific protection and only rarely confer heterosubtypic protection. however, recent research by many groups is centered on the elicitation of antibodies directed against the stalk of the influenza ha that have been shown to confer broad protection across a range of subtypes. more specifically, pseudotype-based assays have been shown to be highly efficient for the measurement of broadly neutralizing antibodies directed against the ha2 stalk of influenza, making them ideal serological tools for the study of cross-reactive responses against multiple influenza subtypes with pandemic potential [81, 83, 84] . the microneutralization assay can also measure ha2 stalk responses, albeit with lower sensitivity. this is likely due to the fact that accessibility to the target epitope is limited by the tight packing of ha molecules in wild-type viruses as compared with pseudotype viruses [75] . the hi assay does not measure any ha2 antibodies. this underscores the importance of conducting comparative serological investigations to more accurately dissect the antibody response against novel vaccines and for sero-epidemiological studies, as each assay measures different (but occasionally overlapping) antibody responses [69, 74, 85] . single radial haemolysis (srh) is a serological technique developed in 1975 [86] that combines the advantages of the single radial diffusion (srd) and hi assays [87] . this technique takes advantage of antibody diffusion within a gel for the determination of antibodies that might be present in analysed sera. the haemolysis, mediated by complement and induced by the antibody-antigen complex, produces easily identifiable "zones of haemolysis", whose size is proportional to the concentration of antiinfluenza antibodies present in the sera [88] . srh has been used to detect antibodies not only against the influenza viral hemagglutinin but also against numerous other viruses such as coronaviruses, parainfluenza virus, dengue virus and japanese encephalitis virus [89] [90] [91] [92] [93] [94] [95] [96] . the greatest advantage of srh is its safety as, unlike vn assays which require wild-type influenza virus, srh is performed with inactivated virus. this aspect is particularly advantageous in the case of h5n1 because the serological tests can be safely carried out under bsl-2 containment [97] . srh is inexpensive, rapid, reliable, reproducible and the quantitative, unbiased results are available after an overnight incubation [89, 91, [98] [99] [100] [101] . other significant advantages to the srh assay include the ability to simultaneously and rapidly test a large number of samples without pre-treatment (excluding complement inactivation), and the requirement for only a small volume of sera [93] . for influenza a viruses, there is a good correlation between the results obtained by srh and hi-an hi titre of 40 with human sera corresponds to an srh titre of 19-33 mm 2 , while for influenza b viruses, srh is consistently more sensitive than hi [102] . a study conducted by morley et al. [88] shows that the results obtained with srh and hi correlate strongly with those obtained with the vn, suggesting that the antibodies detected with these techniques may have the ability to neutralize the virus. in fact, given all these features, srh seems to be a technique particularly suitable for detecting rises in influenza antibody titres [103] . following the emergence of avian influenza viruses capable of causing infection in humans, and limited use of hi assays for these viruses due to an underestimation of the human immune response raised against these pathogens [104] [105] [106] (figure 4 ), srh has been widely used as a sensitive and specific technique for the detection of human antibodies directed against avian influenza viruses in clinical trials [107] [108] [109] . this technique is officially recognized by the ema and, in order for a vaccine to be licensed, it must meet two specific parameters for srh in two age groups: adults from 18 to 60, and seniors over the age of 60. for the first age group, a vaccine can be licensed when showing a number of seroconversions or significant increase in ha antibody titre > 40%, or when the proportion of subjects with an area ≥25 mm 2 is higher than 70%, or when the mean geometric increase > 2.5 [20] (table 1) . as for the elderly, the frequency of seroconversions or titre increase must be > 30%, or the proportion of subjects with a titre ≥25 mm 2 higher than 60%, or a mean geometric increase of >2.0. clinical studies conducted in children and/or adolescents show that, for these age groups, the parameters used to evaluate the immunogenicity of the vaccine are the same as for adults [110, 111] . this situation again raises the need to revise the criteria for licensing vaccines for juvenile and adolescent age groups. . antibody titres measured by hi, vn and srh assays after a vaccination with a non-adjuvanted vaccine or an mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine. at each visit, the hi assay was shown to underestimate antibody responses when compared to vn and srh assays [106] . neuraminidase, in addition to ha, is the other major surface glycoprotein of the influenza viruses. the primary role of na is apparent in the later stages of the virus life cycle and involves viral release and subsequent spread from infected cells [112] . na is immunogenic but not all antibodies directed against na are able to block its enzymatic function [113] . na-targeted antibodies do not have neutralizing capacity defined as an ability to prevent infection, but are instead able to significantly reduce viral replication within, and egress from, host cells. these characteristics imply that na affects both disease occurrence and severity, as well as the spread of infection [114, 115] . unfortunately, the role of antibodies against na in immunity has not been studied sufficiently and conflicting results have been reported by various authors [116] . the factors that have limited the study of na are increased surface expression of viral ha compared to na (1:4/1:2) [117] , antigenic competition leading to a greater ha response upon simultaneous antigen presentation [118] and the lack of a reliable detection technique [119] . the studies conducted on na utilise a wide variety of techniques. some studies have measured post-vaccination neuraminidase inhibition (ni) activity using a fluorescent substrate (mun), and results reveal that data obtained using this type of test are rapid, sensitive, reproducible and quantitative, and allow for the distinction between infected and vaccinated animals [120] . others have developed a high-throughput screening assay (accelerated viral inhibition with na avina) that is able to quantify viral replication through the evaluation of na activity in cell culture wells. this technique has proven to be advantageous because it is not necessary to use specialized reagents and is relatively simple to perform. the required amount of virus for this assay is low and therefore it should be possible to carry out the assay with virus prepared in eggs, without necessitating a virus concentration step. its characteristics make it particularly appropriate for predicting vaccine efficacy and may be extremely useful in establishing the immunogenicity of seasonal and pandemic influenza vaccines [121] . currently, the most used technique for the quantification of antibodies against neuraminidase is the enzyme-linked lectin assay (ella). originally developed by lambré [122] , the assay is based on the ability of neuraminidase to cleave sialic acid residues from a substrate (usually fetuin). the desialylation uncovers sugar residues present of the surface of fetuin, which are otherwise masked and unreachablesuch residues are then recognized by peanut lectin labeled with hrp. however, the potential presence of antibodies against na in experimental samples would inhibit this process. possible sources of na for ella assay are: purified na, mismatched viruses (containing ha not circulating in humans) and pseudotyped viruses. the ella test is performed in microtitre plates, is able to evaluate specific antibody to na and thus could be useful to investigate na antigenic drift, as well as to measure the na antibody response to vaccines. safety, better sensitivity than the traditional thiobarbituric acid (tba) assays and no hazardous reagents make it a promising assay for the study of na antibody response [123, 124] . however, the research conducted on na is complicated by its instability and the lack of commercial vaccines with controlled na activity [125] . moreover, the instability of na activity at certain concentrations, even a few months after production, and a variation of about 40-fold in the na activity of different virus preparations made with similar strains have been observed, making the production of vaccines with a uniform content of na difficult [126] . additionally, the demand for a validated assay that can be used to measure antibody responses that can "neutralize" na after vaccination is increasing [125] . a study conducted by monto and kendal [127] describes the effects linked to pre-existing antibodies directed against the na during the onset of an influenza outbreak with a new ha subtype. in 1968, a hong kong type a influenza a (h3n2) virus arose, with a discrepant haemagglutinin but an unchanged neuraminidase compared to previously circulating asian strains (h2n2). this study showed that individuals who had pre-epidemic antibodies against na had a lower rate of influenza than those who lacked existing anti-na antibodies. the relationship between the decrease of infection-rate and the increase of pre-existing anti-na antibody levels provides evidence of how antibodies directed to na could be protective during an influenza epidemic. the immunological response to na could be of particular importance during an influenza pandemic where the majority of the population would be ha-naï ve but could have previous immunity to na [124] . in fact, the results of some studies have shown that prior infection with influenza h1n1 2009 (h1n1pdm09) could provide immune protection against h5n1 virus, as their na protein head region sequences are more conserved [119, 128] . in particular, antibodies directed against these conserved regions of na could help to provide a significant level of protection against the disease [129] . a further study in naï ve ferrets showed that cross-protection against h5n1 observed in adjuvanted seasonal trivalent or pandemic (h1n1) vaccines can be attributed to both ha and na but the production of antibodies directed against viral na provides a strong correlate of protection in this model [130] . the development of sufficiently powerful vaccines to maintain high levels of anti-na antibodies in the population, to improve adaptive immune recognition during an antigenically similar influenza epidemic, is increasingly promising [127] . in 1974, couch et al. [131] demonstrated that an inactivated, recombinant influenza virus vaccine was able to induce a monospecific antibody response to na. this vaccine revealed itself to be antigenic, essentially non-reactogenic, and able to induce significant protection against disease. this protection was provided by a previous infection with an influenza virus that posessed an antigenically identical na. the currently administered whole-virus and subvirion vaccines are standardized purely on their ha content [132] , despite containing both ha and na glycoproteins, meaning that na content variation is not controlled between vaccine batches [133] . it has been estimated that for the currently available vaccines, all tivs contain na antigen capable of inducing an antibody response directed against na (antibodies that inhibit na) while the live attenuated influenza vaccines (laiv) induce a lower response against n1 and n2, compared to that obtained with the tivs [16] . although there are no analyses or content standards regarding na antigen in approved influenza vaccines, studies show the need to include an immunogenic amount of na in vaccines [134] . adequate incorporation of na in vaccines provides the potential to improve homologous immunization against influenza and to elicit expanded heterovariant immunity in the case of the emergence of an epidemic virus with unexpected antigenic changes [135] , in other words the presence of na could provide a stronger and broader immune response to influenza viruses. in addition, the incorporation of na in vaccines could lead to a reduction of the ha dose necessary to induce a protective immune response [136] . another advantage of na is its slower antigenic evolution, which is evidenced by a greater stability in nature [137] . this characteristic implies that na would be able to induce longer-lasting immunity than that provided by ha or conventional vaccines [138] . whereas it is necessary to almost annually refresh the virus strains included in vaccines (since the antibody response is predominantly strain specific) [139] , the advantage that the presence of an adequate amount of na could offer should not be underestimated. in addition, it has also been shown that when ha and na are provided in equal amounts, and as purified proteins separated from other viral proteins, they are equivalent from an immunogenic viewpoint. one approach in order to elude "the antigenic competition" of the two antigens might be to even out the mixture, that is, to modify the amounts of both antigens in such a way that there is no competition. these changes may be of particular interest in the case of vaccines administered to humans, because antigen balancing may result in a more holistic immune response against influenza. the use of purified, viral na protein, would result in a lower toxicity than vaccines with whole, live or inactivated viruses and would not need adjuvants [138] . kilbourne et al. [114] demonstrated the immunogenicity of a purified, non-adjuvanted influenza virus (n2) neuraminidase vaccine. a single dose of this preparation was non-reactogenic and immunogenic in primed human subjects. in the light of recent studies and experience of the h1n1 pandemic in 2009, which epitomised the unpredictability of human influenza [139] , a sustained research focus is required to investigate the protective effects and duration of antibodies elicited against na [140] . the immunity induced by current vaccines is predominantly based on antibodies capable of neutralizing pathogens [141] . the dominant role of antibodies and antibody data requests from the regulatory agencies in releasing an influenza vaccine have focused mainly on the antibody response, often overlooking cell mediated immunity [142] which is also necessary for defence against many pathogens [143] . even if cell-mediated immunity does not appear to contribute significantly to the prevention phase of the infection, it plays an important role in viral clearance after influenza infection and may also prevent complications associated with influenza [10] . in particular, t lymphocytes play a crucial role in mediating the cellular immune response, by providing a helper antibody response and intervening directly in reducing viral replication [144] . in a mouse model, it has been shown that cytotoxic t lymphocytes (ctl) are protective against influenza viral infection and that cross-reactive ctl responses could be potentially protective against influenza viruses that present antigenic drift and that are not neutralized by antibodies [145] . in the context of t lymphocytes, cd8 + t cells play an important role in virus clearance [146] and are the principal mediators of what is termed "heterosubtypic immunity" understood as a type of protection against viruses that differ serologically, provided by the cellular response to cross-reactive epitopes [147] . whilst knowledge of the role of cd4 + t cells in heterosubtypic immunity is still lacking [148] , it has been shown that cd4 + t cells play a crucial role not only in clearance but also in the recall of cd8 + t cell responses, as well as in the maintenance of cd8 + memory cells [146] . the advantage of cell-mediated immunity (t cells) is the ability of this kind of immunity to target internal proteins common to heterologous viral strains thus providing the vaccine with the capacity to induce a protective immune response against a wider range of viral strains [149] . an integration of current vaccination strategies with a "t-cell based vaccine" strategy may turn out to be highly effective [150] . in particular, the np protein represents the major target structure for cross-reactive ctl [151] . in addition to the features listed above, the results of a study conducted by murasko et al. [152] also consider a further important aspect of cell-mediated response and the important role it could play in the control of influenza in an elderly population group. assuming that neutralizing antibodies are the most significant defence prior to infection, cytokine t lymphocytes eradicate the virus-infected cells. in young people, both cell-mediated and immune responses provide a defence against influenza infection, while in the elderly, the antibody response is insufficient and the role of cell-mediated response becomes fundamental in the eradication of the virus. beyond neutralizing antibodies, non-neutralizing antibodies have been the object of recent studies, due to their ability to provide some protection against influenza infection [153, 154] . jegaskenda et al. [155] developed a novel assay able to assess the specificity and function of antibody dependent cellular cytotoxicity (adcc) specific to influenza. the adcc antibodies against influenza virus have been identified also in absence of detectable neutralizing antibodies. therefore, the vaccines that promote cellular immunity may be an important alternative in combating potentially lethal and highly pathogenic influenza viruses, as well as providing help in preventing an influenza pandemic by promoting strong memory ctl activity, in conjunction with the antibody-based approach already underway [149] . the influenza virus has decimated populations throughout the world since ancient times [156] and still remains one of the most serious and persistent health problems for humanity, due to high rates of morbidity and significant economic cost [157] . the characteristic that makes influenza viruses exceptional is their ability to evade host immunity and cause recurrent annual outbreaks, and at irregular intervals major global pandemics, due to the introduction of antigenically new viruses in a immunologically naï ve human population [158] . vaccination has historically been a pillar for infection control [159] and provides both direct and indirect effects [160] . the direct effects are exhibited in a decreased susceptibility towards the disease by recipients, which implies a reduction in the probability of disease contraction. indirect effects include a reduced risk of disease in both vaccinated and unvaccinated persons. therefore, when an uninfected person comes into contact with someone infected, the likelihood of disease transmission is lower, compared to in the absence of vaccination. in order for a vaccine to be licenced, it must meet at least one of three criteria required by the ema, depending on the age group [20] . children and adolescents represent a crucial point because the vaccination is also recommended for these cohorts [21] , but they are not yet included as age groups in the official criteria used to authorize a vaccine. the question raised by black et al. [41] is an essential starting point in analysing the current situation regarding juvenile vaccination, as data for this age cohort showed that the conventional hi titre of 40 provided only 22% protection from clinical infection. it is increasingly necessary to have adequate criteria specific for children, in order to licence a vaccine. also, given that children and adolescents are widely vaccinated in the event of a pandemic, it would be optimal to administer a vaccine type and dosage specific to this age group, so as to curtail outbreak severity. the criteria currently in use require revision in light of the aforementioned situation, and because criteria validity may be discrepant between seasonal and pandemic influenza vaccines [161] . furthermore, it has yet to be confirmed whether correlates of protection provided by egg-derived vaccines can also be applied to cell-culture derived vaccines [162] . another aspect that should not be underestimated concerns vaccine formulations and in turn, the related serological correlates. at present, correlates of protection for influenza vaccines lack adequacy with regards to laiv vaccines, and thus guidelines cannot be applied for these formulations [123, 163] . given the importance of vaccination in order to protect humanity from disease, a critique of the current situation is necessary and criteria required for official vaccine dissemination need to be updated. malleability of criteria between vaccine types and target populations would also be desirable, given the complicated state of play within vaccine administration. each individual target population has different immunologic conditions and varying levels of prior exposure to the virus, making it inevitably necessary to expand upon the two age groups currently considered in official ema criteria. besides extending vaccination to additional age groups, the technical support provided by the vn, na activity and cell-mediated immunity (cmi) could prove useful. vn assays using 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prototype pandemic influenza (h5n1) vaccine: a phase ii, double-blind, randomized trial efficacy, safety, and immunogenicity of a vero-cell-culture-derived trivalent influenza vaccine: a multicentre, double-blind, randomised, placebo-controlled trial guideline on influenza vaccines prepared from viruses with the potential to cause a pandemic and intended for use outside of the core dossier context; committee for human medical products all authors contributed to the writing and revisions of the manuscript. the authors declare no conflict of interest. key: cord-276015-id15u3br authors: beran, jiří; šalapová, eva; špajdel, marian title: inosine pranobex is safe and effective for the treatment of subjects with confirmed acute respiratory viral infections: analysis and subgroup analysis from a phase 4, randomised, placebo-controlled, double-blind study date: 2016-11-07 journal: bmc infect dis doi: 10.1186/s12879-016-1965-5 sha: doc_id: 276015 cord_uid: id15u3br background: inosine pranobex (isoprinosine®) is an immunomodulatory drug approved in several countries for the treatment of viral infections. this study compared the efficacy and safety of inosine pranobex versus placebo in subjects with clinically diagnosed influenza-like illness, including subjects with laboratory-confirmed acute respiratory viral infections. subgroup analyses evaluated the efficacy of inosine pranobex compared to placebo in otherwise healthy (without related ongoing disease) subjects that were less than 50 years of age and healthy subjects that were at least 50 years of age. the effect of body mass index (bmi) was evaluated in subjects less than 50 years of age. methods: a total of 463 subjects were randomly assigned to receive inosine pranobex (n = 231) or placebo (n = 232) in this phase 4, randomised, double-blind, multicentre study. the primary efficacy endpoint was time to resolution of all influenza-like symptoms present at baseline to none. safety was evaluated through analysis of adverse events, vital signs, and physical examinations. results: the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant but showed a faster improvement in subjects in the inosine pranobex group versus those in the placebo group hazard ratio = 1.175; (95 % ci: 0.806–1.714). p-value = 0.324. in the subgroup analysis for subjects less than 50 years of age, statistically significant differences in time to resolution of influenza-like symptoms that favoured the inosine pranobex group over the placebo group were observed in those without related ongoing disease and those who were non-obese (bmi <30 kg/m(2)). the differences between the inosine pranobex and placebo groups in subjects at least 50 years of age without related ongoing disease and in subjects less than 50 years of age who were obese (bmi ≥30 kg/m(2)) were not statistically significant. inosine pranobex was generally well tolerated, and no deaths were reported. conclusions: the study results indicate the safety of inosine pranobex for the treatment of subjects with confirmed acute respiratory viral infections and confirm the efficacy of inosine pranobex versus placebo in healthy non-obese subjects less than 50 years of age with clinically diagnosed influenza-like illnesses. trial registration: ewo-iso-2014/1, eudract 2014-001863-11; date of registration: 29 apr 2014; detail information web link: https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-001863-11/results electronic supplementary material: the online version of this article (doi:10.1186/s12879-016-1965-5) contains supplementary material, which is available to authorized users. acute respiratory infection is a serious infection that is responsible for approximately 3.9 million deaths per year and is one of the leading causes of morbidity and mortality worldwide [1] . acute respiratory infections are categorized as either upper or lower respiratory infections and are caused by well-recognised viral pathogens, including but not limited to influenza virus (types a and b), parainfluenza virus, respiratory syncytial virus (rsv), metapneumovirus (types a and b), coronavirus, rhinovirus, enterovirus, reovirus, bocavirus, and adenovirus, and bacterial pathogens, primarily streptococcus pneumoniae and haemophilus influenza [2, 3] . influenza is an acute viral respiratory infection that affects 5 % to 15 % of the global adult population per year and results in approximately 0.25 to 0.5 million deaths and 3 to 5 million cases of severe illness worldwide annually [4, 5] . influenza-like illnesses (ili) are considered a subset of acute respiratory infections and result in the sudden onset of symptoms such as fever (body temperature greater than 38°c), cough, and sore throat in patients [5] . physicians have difficulty with the treatment of ili because determining the aetiology is generally not possible solely on a clinical basis. current pharmacological interventions for the prevention and treatment of respiratory viral infections are primarily limited to vaccines and antivirals for influenza. available antiviral treatments for influenza infection are m2 ion channel inhibitors (eg, amantadine and rimantadine) and neuraminidase inhibitors (eg, oseltamivir and zanamivir) [6] . if the infection is caused by bacterial pathogens, treatment can involve antibiotics and also medications that provide symptomatic relief. however, in case of viral aetiology, only medication for symptomatic treatment can be provided. [7] . vaccination for seasonal influenza remains one of the standard approaches for prevention of the disease. however, immunisation rates for seasonal influenza remain low in many european countries even though the influenza vaccine is part of the national immunisation schedule in these countries [8] . moreover, the vaccination is effective only when most of the circulating influenza viruses in a given season are similar to the viruses that were included in the influenza vaccine. the vaccine does not offer any clinical benefit against other pathogens that circulate during a season [9] . inappropriate use of antibiotics for the treatment of acute respiratory infections has been observed during winter months, even though the majority of infections are caused by viral pathogens and are self-limiting. this practice may result in side effects and the development of antibiotic resistance in pathogens as well as an increased cost burden for the healthcare system [3, 10, 11] . inosine pranobex (isoprinosine®), a combination of the p-acetamidobenzoate salt of n,n-dimethylamino-2-propanol and inosine in a 3:1 molar ratio, is an immunomodulatory antiviral drug that has been licensed since 1971 in several countries worldwide for the treatment of viral infections [2, 12] . inosine pranobex stimulates a nonspecific immune response that is independent of the specific viral antigen responsible for the ili. in clinical studies, inosine pranobex has been shown to induce a type 1 t helper cell-type response in mitogen-or antigenactivated cells, and this response initiates t-lymphocyte maturation and differentiation and potentiates induced lymphoproliferative responses (13) (14) (15) . similarly, the drug modulates t-lymphocyte and natural killer cell cytotoxicity and cd8+ suppressor and cd4 + -helper cell functions and increases the number of immunoglobulin g and complement surface markers (14, 15) . inosine pranobex also increases cytokine interleukin (il)-1 production and il-2 production and upregulates the expression of the il-2 receptor in vitro [13, 14] . the safety profile of inosine pranobex has been established through clinical trials for several indications and populations [2, [14] [15] [16] . a rapid increase in the number of mononuclear cells after the first dose of inosine pranobex was observed in 75 % of the subjects, and this increase was consistent with clinical observations of rapid resolution of common cold symptoms [15, 17] . this phase 4 study aimed to compare the efficacy and safety of inosine pranobex with placebo in subjects with laboratory-confirmed acute respiratory viral infections in order to evaluate the clinical use of inosine pranobex for the treatment of acute respiratory viral infections. the primary efficacy endpoint was comparison between inosine pranobex and placebo groups in terms of the time to resolution of all influenza-like symptoms present at baseline to none. in a subgroup analysis of subjects with clinically diagnosed ili, the study further evaluated the efficacy of inosine pranobex compared to placebo in healthy (without related ongoing disease) subjects less than 50 years of age and in those at least 50 years of age. the study also evaluated the effect of body mass index (bmi) in subjects who were less than 50 years of age and were non-obese (bmi <30 kg/m 2 ) or obese (bmi ≥30 kg/m 2 ). in addition, the study evaluated the efficacy of inosine pranobex in subjects less than 50 years of age for the time to resolution of all influenza-like symptoms present at baseline to mild or none (i.e. score of 1 or 0 on the influenza-like symptoms assessment scale). this was a phase 4, randomised, placebo-controlled, double-blind, multicentre study in subjects with clinically diagnosed ili, including subjects with laboratoryconfirmed acute respiratory viral infections due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus 1 or 3. the study was conducted at 25 study sites in the czech republic (14 study sites) and slovakia (11 study sites), with enrolment occurring between december 2014 and april 2015, and the last subject visit was on 03 june 2015. detailed primary efficacy endpoints are provided in the additional file 1. male and nonpregnant female subjects aged 18 to 75 years with a clinical diagnosis of ili were included in this study. influenza-like illnesses were defined as an oral temperature of at least 38°c observed at the study site with at least 1 respiratory symptom of cough, sore throat, or nasal obstruction and at least 1 constitutional symptom of fatigue, headache, myalgia, or feverishness. the respiratory and constitutional symptoms were required to be considered by the subject as moderate or severe in intensity (a score of more than 1 on the 4point influenza-like symptoms assessment scale). the subjects were required to have experienced the onset of ili no more than 36 h prior to screening, where onset is defined as the time when the subject experienced fever and at least 1 respiratory symptom and at least 1 constitutional symptom. the full inclusion criteria and influenza-like symptoms assessment scale are detailed in the additional file 1. subjects were excluded from participation in this study if they met any of the following criteria: had an immunosuppressive disorder or were receiving immunosuppressive therapy; were undergoing treatment with xanthine oxidase inhibitors or uricosuric agents or treatment with thiazide or loop diuretics; had chronic renal dysfunction or severe liver function impairment; were lactose intolerant; had cancer in a nonremission stage; were undergoing treatment with zidovudine; were pregnant or lactating/ breastfeeding; had received a dose of inosine pranobex, oseltamivir, zanamivir, amantadine, or rimantadine during this occurrence of ili; or had been administered an investigational drug or investigational vaccine within 30 days prior to screening. detailed exclusion criteria are provided in the additional file 1. a sample size of 258 subjects (129 subjects in each treatment group) in the modified intent-to-treat (mitt) analysis set with a total of 430 randomly assigned subjects was calculated using the log-rank test (inputting the median survival times). sample size calculations were performed using pass software version 12 (ncss, llc, kaysville, utah, usa) and considered a statistical power of 80 % to detect a clinically relevant difference between 3.5 days in the treatment group and 5 days in the placebo group. a 20 % dropout rate was also considered, which meant that a final sample size of 206 subjects (103 subjects in each treatment group) would be required for the study. however, because of the challenges faced during enrolment, which included a late influenza alert and an unexpectedly mild influenza season, the decision was made to continue enrolment until 30 april 2015. a total of 463 subjects were randomly assigned, and of these, only 137 subjects met the criteria for inclusion in the mitt analysis set. all analyses were conducted using sas® software version 9.2 (sas institute inc, cary, north carolina, usa). all statistical tests were 2-sided hypothesis tests performed with a 5 % level of significance, which resulted in 95 % (2-sided) confidence intervals (cis). no adjustments for multiplicity were made. the hazard ratios (hrs) and 95 % cis were estimated using a proportional hazards model (hr >1 indicated a benefit to inosine pranobex compared with placebo). the safety analysis set consisted of all subjects who received at least 1 dose of any study drug. all analyses using the safety analysis set grouped subjects according to the treatment that the subjects had actually received. the mitt analysis set consisted of all randomly assigned subjects who had a positive laboratory confirmation of acute respiratory viral infection due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus 1 or 3. this set was used for the primary and secondary efficacy analyses. detailed primary efficacy endpoints are provided in the additional file 1. all analyses using the mitt analysis set grouped subjects according to the randomised treatment. the intent-to-treat (itt) analysis set included all subjects who were randomly assigned to receive double-blinded study drug and was used for the subgroup analyses. on day 1, eligible subjects were randomly assigned to receive either inosine pranobex or placebo in a 1:1 allocation ratio with no stratification. the active drug and matching placebo tablets were provided in identical cartons that were identified with a kit number, such that all study site staff and subjects remained blinded throughout the study. only personnel with access to the interactive web response system and clinical supplies were unblinded and had access to the treatment assignments; all other parties involved in the study were fully blinded. inosine pranobex or placebo 500-mg tablets were selfadministered by the subjects for 7 days (2 tablets orally 3 times daily). the first dose was taken immediately after randomisation at the study site, and the remaining doses were to be self-administered at home. doses were taken approximately 8 h apart but consistent with the subject's lifestyle, ie, scheduling of dosing did not disturb the subject's usual sleep patterns. the subjects were provided with kits containing (randomised) medication sufficient for 1 subject for 7 days of treatment. subjects were instructed to consume no more than 42 tablets for the specified duration and were required to return the excess study drug tablets at the end-of-treatment (eot) visit. adherence to study drug administration was good and was monitored as part of the study. the total study duration was 21 days (±3 days) for each subject and consisted of a 7-day dosing period (day 1 to day 7), 1 day for the eot visit (day 8 + 1 day), and a 13-day follow-up period (day 21 ± 3 days) after the eot visit. prior to randomisation on day 1, a nasopharyngeal swab sample was collected to test for the presence of influenza a or b virus, rsv, adenovirus, and parainfluenza virus 1 or 3 using the appropriate polymerase chain reaction analyses. the results were used to identify the subjects to be included in the mitt set and consequentially identify the subjects to be included in the primary endpoint analysis. a detailed procedure for the study visits is included in the additional file 1. the primary efficacy endpoint was the time to resolution of all influenza-like symptoms present at baseline to none (ie, a score of 0, defined as the complete absence of symptoms, on the influenza-like symptoms assessment scale [details provided in additional file 1]). the secondary endpoints included time to resolution of respiratory symptoms (cough, sore throat, and nasal obstruction); time to absence of fever (oral temperature of ≤37.5°c for at least 2 consecutive readings that were at least 12 h apart); time to resumption of normal activity (ie, score of 0 on daily activities assessment scale); and frequency of viral respiratory infection complications, defined as hospitalisation, death due to ili or complications of ili, or requirement of antibiotic treatment for secondary bacterial infection. a subgroup analysis was conducted for time to resolution of all influenza-like symptoms present at baseline to none in subjects with clinically diagnosed ili. this was conducted in subjects less than 50 years of age and subjects at least 50 years of age without related ongoing disease (related ongoing disease was any medical condition with the preferred terms of asthma, bronchitis, chronic bronchitis, or chemical bronchitis that was ongoing at the start of the study). in addition, an analysis was conducted in subjects less than 50 years of age who were non-obese (bmi <30 kg/m 2 ) or obese (bmi ≥30 kg/m 2 ). an additional analysis was conducted for time to resolution of all influenza-like symptoms to mild or none for subjects less than 50 years of age. safety was evaluated during the study through analysis of adverse events (aes), vital signs, and physical examinations. the study was performed in accordance with ethical principles that have their origin in the declaration of helsinki, international council for harmonization e6 (r1), and all applicable regulations. study was approved before study start by two multicentre ethics committees (mec). one mec in the university hospital brno approved study for all study centres in the czech republic and the second one mec of košice regional office approved study for all study centres in slovakia. all potential subjects signed an informed consent form prior to randomisation on day 1 before any study-related procedures were performed. the study included a total of 463 subjects who were randomly assigned to receive either inosine pranobex (n = 231) or placebo (n = 232), and 98.5 % of subjects completed the study (fig. 1 -flow chart) . there were 7 subjects who discontinued the study; the reasons included protocol noncompliance (n = 2), aes (n = 2; rhinopharyngitis and pleuropneumonia), and withdrawal of consent (n = 2). overall, 137 subjects (29.6 %) had positive nasopharyngeal swab test results and were included in the mitt analysis set (inosine pranobex, n = 71; placebo, n = 66). the details of the demographic and baseline characteristics are presented in the additional file 1. the demographic characteristics were similar between the 2 treatment groups. the overall mean age was 41.9 years, and the overall mean bmi was 26.450 kg/m 2 (range: 12.07 to 45.11 kg/m 2 ). the majority of subjects in this study were less than 65 years of age. at baseline, most of the subjects presented with at least 1 influenza-like symptom (cough, sore throat, nasal obstruction, fatigue, headache, myalgia, or feverishness) with a score of 1, 2, or 3 in severity. medical histories were reported for 172 subjects (74.5 %) in the inosine pranobex treatment group and 178 subjects (76.7 %) in the placebo group, and the most commonly reported medical histories were vascular disorders and surgical and medical procedures. medical histories of gastrointestinal disorders (10.8 % in the inosine pranobex group and 11.2 % in the placebo fig. 1 flow-chart of enrolment, placement in treatment and placebo arms, division into the different subgroups and mitt and itt analysis sets group) and hepatobiliary disorders (10.4 % in the inosine pranobex group and 11.6 % in placebo group) were also reported. overall, the mean reported dose compliance was high, and it was similar between treatment groups (97 % in each treatment group). in the majority of subjects in both treatment groups, dose compliance ranged from 80 to 120 % (inosine pranobex, n = 217 [94.8 %]; placebo, n = 220 [93 %]). the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant but showed a trend towards improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: 1.175; 95 % ci: 0.806, 1.714; p = 0.324) (fig. 2) . a substantial decrease in the proportion of subjects with all influenzalike symptoms was observed in the inosine pranobex group after 9 days while a decrease to similar proportions occurred only after 11 days for subjects in the placebo group. the differences in time to resolution for the secondary endpoints also showed a similar trend towards improvement for subjects in the inosine pranobex group compared with subjects in the placebo group, but these secondary endpoint differences were not significant. the detailed results are provided in the additional file 1. in the subgroup analysis, for subjects less than 50 years of age without related ongoing disease, the difference in time to resolution of all influenza-like symptoms between treatment groups was statistically significant (p = 0.050) and showed faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: 1.234; 95 % ci: 0.969, 1.571) (fig. 3 and table 1 ). however, for subjects at least 50 years of age without related ongoing disease, the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant (hr:0.887; 95 % ci: 0.0.604, 1.303; p = 0.488). for non-obese (bmi <30 kg/m 2 ) subjects less than 50 years of age, the difference in time to resolution of all influenza-like symptoms between treatment groups was statistically significant (p = 0.018) and showed a faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: 1.307; 95 % ci: 1.010, 1.691). however, for obese (bmi ≥30 kg/ m 2 ) subjects less than 50 years of age, the difference in time to resolution of all influenza-like symptoms in subjects less than 50 years of age, the difference in time to resolution of all influenza-like symptoms to mild or none between treatment groups was statistically significant (p = 0.009) and showed a faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: 1.298; 95 % ci: 1.035, 1.627). the proportion of subjects experiencing treatmentemergent aes (teaes) and the number of the majority of teaes were mild or moderate in severity and were unrelated to study drug, in the opinion of the investigator. overall, 4 severe teaes were reported in 3 subjects (0.6 %). severe teaes of rhinopharyngitis, maxillary sinusitis, and vertebrogenic pain syndrome were reported in 1 subject (0.4 %) each in the inosine pranobex group, and the severe teae of pleuropneumonia was reported in 1 subject (0.4 %) in the placebo group. no deaths were reported during the study. overall, 6 subjects (2.6 %) in the inosine pranobex group and 7 subjects (3.0 %) in the placebo group experienced teaes that led to study drug discontinuation. three treatment-emergent serious aes (saes) were reported in 2 subjects (0.4 %). severe rhinopharyngitis and severe vertebrogenic syndrome were reported in 1 subject in the inosine pranobex group, and 1 subject in the placebo group reported severe pleuropneumonia. these saes resulted in permanent discontinuation of the study drug and discontinuation of both subjects from the study. all saes resolved, and none of the saes in the opinion of the investigator were related to the study drug. the mean changes in vital sign and physical examination values from baseline were small, and no clinically the "bold data" are statisticly significant significant trends were observed between treatment groups. no pregnancies were reported during the study. this was a phase 4, randomised, placebo-controlled, double-blind, multicentre study that evaluated the efficacy of inosine pranobex in subjects with clinically diagnosed ili, including subjects with laboratory-confirmed acute respiratory viral infections due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus 1 or 3. the study also evaluated the efficacy of inosine pranobex in subgroups of subjects less than 50 years of age who were without related diseases (such as asthma, bronchitis, chronic bronchitis, and chemical bronchitis) and who were non-obese (bmi <30 kg/m 2 ) or obese (bmi ≥30 kg/m 2 ) as well as a subgroup of subjects at least 50 years of age without related ongoing disease. in addition, a subgroup analysis was conducted in subjects less than 50 years of age for time to resolution of all influenza-like symptoms to mild or none. in the current study, the analysis of the primary endpoint of time to resolution of all influenza-like symptoms showed a faster improvement in subjects treated with inosine pranobex compared with subjects administered placebo, although the difference between treatment groups did not reach the threshold of statistical significance. the results were similar for the secondary efficacy endpoints of time to resolution of respiratory symptoms (cough, sore throat, and nasal obstruction), time to absence of fever, and time to resumption of normal activity. the difference in the occurrence of viral respiratory infection complications between treatment groups was not statistically significant. immunosenescence, ie, the age-related decline of the immune system, and obesity play an important role in the efficacy of the immune response to pathogens [18, 19] . older subjects show a diminished immune response to pathogens, which increases their risk for severe infection and compromises their ability to adequately combat viral infections. this phenomenon was observed with split-virus influenza vaccines; a low response to the vaccine was observed in older adults, whereas the vaccine was effective in younger subjects. this low response resulted in increased susceptibility to influenza and associated complications in older adults compared to younger adults who typically benefit from a higher response [20, 21] . obesity has also been identified as an independent risk factor for increased susceptibility to influenza virus infection; this susceptibility results from diminished cd4+ and cd8+ t-cell responses and lower influenza vaccine antibody levels [19, 22, 23] . obesity may also increase the risk of pneumonia or other infections by restricting lung volume [24] . immunosenescence and obesity can bias efficacy studies because of the impaired response of the immune system to pathogens, as the risk of complications is increased in such individuals. in the subgroup analysis of the current study, in subjects less than 50 years of age who were without related ongoing disease and in those less than 50 years of age who were non-obese (bmi <30 kg/m 2 ), statistically significant differences in time to resolution of influenzalike symptoms favoured the inosine pranobex group over the placebo group. statistically significant differences were not observed between the inosine pranobex and placebo groups in subjects at least 50 years of age without related ongoing disease or in subjects less than 50 years of age who were obese (bmi ≥30 kg/m 2 ). thus, the efficacy of inosine pranobex was improved in nonobese subjects compared with obese subjects, probably because the immune system in the former is more capable of defending against pathogens and is not negatively affected by obesity-related complications. older patients have a decreased immune response to pathogens as a result of immunosenescence; therefore, they may take longer to recover from illnesses such as influenza and antiinfluenza drugs may not be as effective. in an additional analysis, in subjects less than 50 years of age, statistically significant differences in time to resolution of influenzalike symptoms to mild or none favoured the inosine pranobex group over the placebo group, thus indicating that the improvement of symptoms was better with inosine pranobex than placebo in this subset of subjects (hr: 1.298; 95 % ci: 1.035, 1.627). a substantial decrease in the proportion of subjects with all influenza-like symptoms was observed in the inosine pranobex group after 9 days while a decrease to similar proportions occurred only after 11 days for subjects in the placebo group. this difference could be a result of the time necessary for activation of the immune system, as inosine pranobex acts indirectly by stimulating the immune system and does not directly attenuate the symptoms. this result is also consistent with the results observed in a study in healthy volunteers in which inosine pranobex showed immunomodulating properties through an increase in serum levels of interferon-γ, il-2, il-10, and tumour necrosis factor-α from 7 to 10 days [13] . the approved dose and treatment duration of inosine pranobex (two 500-mg tablets orally 3 times daily) were used in this study, and the administered treatment did not vary according to weight or symptom duration. from the subgroup analysis, the posology of inosine pranobex (3 g/day orally) in this study is most suitable for subjects less than 50 years of age without related ongoing disease and subjects less than 50 years of age who were non-obese (bmi <30 kg/m 2 ). for certain subjects, such as those at least 50 years of age and those who are obese, different dosing strategies could be more appropriate; varying the dosing regimen requires further evaluation. the use of a placebo-only group in this study was justified because ili is generally mild and self-limiting and no other treatments are approved for acute respiratory viral infections other than influenza. in addition, the use of influenza-specific antivirals (neuraminidase inhibitors or amantadine) is not a component of routine medical management of ili in many countries, including the countries in which the study was conducted. performing a high-quality efficacy trial for ili is challenging because of epidemiologic considerations from the influenza outbreak period; the influenza season cannot be predicted in advance and can vary from year to year [25] . furthermore, the enrolment of subjects with symptoms that have been present for less than 36 h is necessary, as the first 36 h is the period of maximal viral replication and antiviral medication is expected to have the most benefit during this time. the current study was anticipated to be performed in 1 influenza season in the northern hemisphere, between 01 october 2014 and 30 april 2015, and enrol the required number of subjects in each group during this timeframe. however, enrolment was challenging, as it could not be commenced because of a low attack rate and was only started after a late alert was issued by national public health authorities in the first week of december 2014 regarding the statistically higher incidence of acute respiratory viral infections. a low density of circulating viruses was present until the end of january 2015, which resulted in the enrolment of subjects until 30 april 2015 in order to maximize the number of completed subjects. approximately 60 % of the randomised subjects were expected to have a positive laboratory confirmation of acute respiratory tract infection. however, only 137 subjects met the criteria for inclusion in the mitt analysis set, which was 121 subjects fewer than the 258 subjects expected. the effects of the absence of a significant influenza outbreak adversely affected the statistical power, thus reducing the power of the study, which could potentially explain the lack of statistical significance for the primary and secondary efficacy endpoints. a slightly longer duration of treatment or a different dosing strategy may have influenced the observed efficacy of inosine pranobex, as proof of therapeutic effect requires a high attack rate, ie, a higher number of sick patients during one influenza season. the attack rate is difficult to predict, and studies, which will adjust the sample size calculations to account for the possibility of a below-average flu season or studies with longer duration, e.g., those that include 2 or more influenza seasons to account for lower than predicted attack rates, are necessary to achieve the desired results [25] . in addition, 10.8 % of enrolled subjects in the inosine pranobex group reported a medical history of gastrointestinal disorders and 10.4 % reported hepatobiliary disorders. pharmacokinetic parameters were not measured in this study, but it is possible that the presence of these disorders at baseline may have affected the absorption, distribution, and metabolism of the study drug and may have influenced the study results, including the efficacy results. furthermore, age, comorbidities, and the obesity of enrolled subjects, particularly in subjects more than 50 years of age, may also have affected the outcome of the study. the safety analysis demonstrated that inosine pranobex treatment was well tolerated, and no major differences in safety profiles were observed between treatment groups. treatment-emergent saes were reported in 2 subjects, and none were considered to be related to study drug by the investigators. no subjects died during the study. no significant changes were observed in vital signs and physical examinations in either study group. the study results indicate the safety of inosine pranobex for the treatment of subjects with confirmed acute respiratory viral infections and confirms the efficacy of inosine pranobex versus placebo in healthy non-obese subjects less than 50 years of age with clinically diagnosed influenza-like illnesses. the results of this study were affected by epidemiologic considerations, which included a late influenza alert and a low density of circulating viruses. further studies may be important to define predictors of treatment success, including the potential of different dosing strategies in certain patient populations, such as those with underlying conditions that may impact drug plasma levels and related drug effects. additional file 1: the primary efficacy assessment; secondary efficacy endpoints; detailed primary endpoint; inclusion and exclusion criteria; procedures and table s1 . influenza-like symptoms assessment scale; table s2 . demographic and other baseline characteristics (itt analysis set); secondary endpoints; table s3 . time to resolution of respiratory symptoms and resumption of normal activity (mitt analysis set); table s4 . treatment-emergent adverse events (safety analysis set); table s5 . treatment-emergent ae occurring in at least 3 % of subjects overall by system organ class per treatment (safety analysis set); figure s1 . flow-chart of enrolment, placement in treatment and placebo arms, and division into the different subgroups for analysis; table s6 . time to resolution of all influenza-like symptoms between treatment groups; influenza: brave call to action the results of studying of inclusion of inosine pranobex into the therapy of acute respiratory viral infections in children surveillance of acute respiratory infections in mumbai during 2011-12 influenza (seasonal) influenza and other respiratory viruses detected by influenza-like illness surveillance in leyte island, the philippines current and future antiviral therapy of severe seasonal and avian influenza symptomatic treatment of the common cold with a fixed-dose combination of paracetamol, chlorphenamine and phenylephrine: a randomized, placebo-controlled trial seasonal influenza vaccine provision in 157 countries (2004-2009) and the potential influence of national public health policies efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis esac project group. european surveillance of antimicrobial consumption (esac): outpatient antibiotic use in europe antibiotics in the treatment of upper respiratory tract infections in poland. is there any improvement multicenter randomized study of inosine pranobex versus acyclovir in the treatment of recurrent herpes labialis and recurrent herpes genitalis in chinese patients isoprinosine affects serum cytokine levels in healthy adults immunomodulatory effects of inosine pranobex on cytokine production by human lymphocytes efficacy of immunomodulators in children with respiratory diseases in environmentally poor areas inosine pranobex -cytotoxic activities and effect of on replication of human parainfluenza viruses (hpiv-2, hpiv-4), entroviruses (ca16, ev71) and adenoviruses (hadv-2, hadv-5) in vitro isoprinosine induces a rapid lympho-mononuclear response in adult participants effects of aging on human leukocytes (part ii): immunophenotyping of adaptive immune b and t cell subsets obesity is associated with impaired immune response to influenza vaccination in humans immunosenescence: implications for response to infection and vaccination in older people effects of aging on human leukocytes (part i): immunophenotyping of innate immune cells overweight and obese adult humans have a defective cellular immune response to pandemic h1n1 influenza a virus obesity and influenza infection severity the impact of obesity on the immune response to infection challenge of conducting a placebo-controlled randomized efficacy study for influenza vaccine in a season with low attack rate and a mismatched vaccine b strain: a concrete example all authors acknowledge support from dr richard south, dr sandra palleja, and priyanka tiwari of ppd for their involvement in the main stages of the trial and preparation of the clinical study report and manuscript. all authors also thank saurabh aggarwal, phd, senior medical writer at ppd, for his significant contributions to this manuscript. the design of the study, collection, analysis, and interpretation of data and writing the manuscript was funded by ewopharma international. the datasets generated during and/or analysed during the current study are available on eudract website: https://www.clinicaltrialsregister.eu/ctr-search/ trial/2014-001863-11/results. patient data are provided as summary data in anonymized form. authors' contributions jb participated in the entire study as an investigator at one of the centres. he evaluated the data and clinical study report and contributed to the manuscript. es participated as the medical officer of the study and contributed to the manuscript. she is a medical director in ewopharma international. ms participated as a biostatistics specialist. he assessed the data and clinical study report, suggested additional analyses, and contributed to the manuscript. all authors read and approved the final version of manuscript. es is an employee of ewopharma international. jb received grants for performing the study at his centre. ms has no competing interests. not applicable. the study was performed in accordance with ethical principles that have their origin in the declaration of helsinki, international council for harmonization e6 (r1), and all applicable regulations. study was approved before study start by two multicentre ethics committees (mec). one mec in the university hospital brno approved study for all study centres in the czech republic and the second one mec of košice regional office approved study for all study centres in slovakia. all potential subjects signed an informed consent form prior to randomisation on day 1 before any study related procedures were performed. key: cord-284087-g2jfnxja authors: falcone, valeria; bierbaum, sibylle; kern, winfried; kontny, udo; bertz, hartmut; huzly, daniela; panning, marcus title: influenza virus a(h1n1)pdm09 hemagglutinin polymorphism and associated disease in southern germany during the 2010/11 influenza season date: 2013-02-09 journal: arch virol doi: 10.1007/s00705-013-1610-1 sha: doc_id: 284087 cord_uid: g2jfnxja a novel influenza a virus emerged in early 2009 to cause the first influenza pandemic of the 21(st) century. understanding the evolution of influenza virus is crucial to determine pathogenesis, vaccine efficacy, and resistance to antiviral drugs. in this study, we investigated the molecular evolution of influenza virus a(h1n1)pdm09 in the 2010/11 influenza season in southern germany by sequence analysis of the influenza virus hemagglutinin gene from 25 patients with mild, moderate, and severe disease. phylogenetic analysis revealed co-circulation of different genetic groups. the d222g mutation, which had previously been observed in severe cases, was not detected. immunocompromised patients were not affected more severely than non-immunocompromised patients (p>0.05), although longer shedding was observed in some of them. interestingly, additional mutations and potential glycosylation sites were detected in samples from the lower respiratory tract in two patients, but not in the corresponding upper respiratory tract specimens. the h275y mutation in the influenza virus neuraminidase gene, known to confer resistance to the neuraminidase inhibitor oseltamivir, was detected in one patient. the first influenza pandemic of the 21 st century emerged in mexico in march 2009 and was caused by a novel influenza a(h1n1)09 virus (a(h1n1)pdm09). in general, the course of the pandemic was moderate from a public-health perspective. however, a particular proportion of the human population including immunocompromised patients, children and pregnant women was at risk to develop severe disease. hemagglutinin (ha) and neuraminidase (na) represent two major surface glycoproteins of influenza virus. the ha determines host-receptor tropism and constitutes the key immunogenic site for the human immune response. since the beginning of the 2009 pandemic, a(h1n1)pdm09 rapidly evolved, and seven different clades/groups characterized by distinct molecular markers in the ha gene have been reported [23] . the d222g mutation in the receptor-binding domain of the ha gene has been linked to severe cases [10] . of note, another mutation involving the same residue (d222e) was also observed but could not be associated with more severe cases [1] . understanding the molecular evolution of influenza virus is therefore crucial to identify mutations that might be associated with a more virulent phenotype. the second surface glycoprotein, na, facilitates the release of newly synthesized virions from infected cells [5] . neuraminidase can be blocked by antiviral drugs such as oseltamivir and zanamivir, which act by interfering with the release of progeny virus, thereby preventing new rounds of infection. a single point mutation at position 275 in the neuraminidase glycoprotein, resulting in a histidine-to-tyrosine shift, can confer resistance to oseltamivir [13] . aim of this study was to investigate the molecular evolution of a(h1n1)pdm09 in the 2010/11 influenza season in southern germany by sequence analysis of the ha gene of mild, moderate, and severe influenza cases. particular attention was paid to immunocompromised patients. moreover, the occurrence of resistance to oseltamivir, the only drug currently recommended for prophylaxis in high-risk groups, was determined. respiratory specimens were obtained from paediatric and adult patients hospitalized at the freiburg university medical centre as well as from outpatients. all had influenza-like illness, including fever, cough, and/or sore throat, as judged by the treating physician. respiratory specimens comprising nasopharyngeal aspirates (npa), tonsillo-pharyngeal flocked swabs collected in 0.5 ml viral transport medium (copan, brescia, italy), or bronchoalveolar lavage fluids (bal) were analysed at the department of virology. testing of patient samples was approved by the institutional review board of freiburg university. in brief, nucleic acids were extracted using a qiaamp minelute virus spin kit (qiagen, hilden, germany) on a qiacube robot (qiagen) according to the manufacturer's instructions. in 2010/2011, a broadly reactive multiplex pcr approach was chosen to detect influenza viruses a and b as well as other relevant respiratory viruses. samples were analysed using ftd respiratory pathogens version 08/2010 (fast-track diagnostics, junglinster, luxemburg) as recommended. the assay utilizes 5'nuclease technology (taqman) and employs a 5-tube multiplex one-step realtime rt-pcr approach. in tube 1, real-time rt-pcr for influenza virus a, a(h1n1)pdm09, influenza virus b, and rhinovirus are combined. tube 2 contains reagents for parainfluenza viruses 2, 3, and 4, and brome mosaic virus (bmv) as a pcr inhibition control; tube 3, for coronavirus 229e, coronavirus nl63, coronavirus oc43, and coronavirus hku1; tube 4, for respiratory syncytial virus a/b, adenovirus, parechovirus, and enterovirus; and tube 5, for parainfluenza virus 1, human metapneumovirus a/b, human bocavirus, and mycoplasma pneumoniae. the bmv inhibition control was added to each patient sample before nucleic acid extraction. an agpath-id one-step rt-pcr-kit (invitrogen, karlsruhe, germany) was used for rt-pcr on an abi 7500 real-time machine (applied biosystems, wiesbaden, germany). cycling conditions were as follows: 50°c for 15 min, 95°c for 10 min followed by 40 amplification cycles of denaturation at 95°c for 8 s and combined annealing/extension at 60°c for 34 s. for the detection of the oseltamivir-resistance-associated mutation h275y, a commercially available assay (tib-molbiol, berlin, germany) was used according to the manufacturer's instructions. the ha gene was amplified directly from clinical samples using the superscript tm iii one-step rt-pcr system (invitrogen, karlsruhe, germany). in brief, a reaction volume of 50 ll contained 1x reaction buffer, 2 mm mgso 4 , 0.5 lm each primer, 2 ll enzyme mix, and 5 ll of purified nucleic acids. the primer sequences were as follows: h1n1_ha_f1, ccg caa atg cag aca cat ta; h1n1_ha_r1, ccc att aga gca cat cca gaa [12] . cycling conditions in a veriti 96-well thermal cycler (applied biosystems, weiterstadt, germany) were 50°c for 30 min and 94°c for 2 min, followed by 33 amplification cycles at 95°c for 15 s, 51°c for 30 s, and 68°c for 30 s. pcr products were purified using a qiaquick pcr purification kit (qiagen) as recommended. purified pcr products were sequenced directly using primers h1n1_ ha_f1 (ccgcaaatgcagacacatta) and h1n1_ha_ fseq_902 (cagacacccaagggtgctat). sequences were aligned using bioedit (mega 4.1). nucleotide sequence alignments were done using a clu-stalw method with mega 4.1. phylogenetic trees were constructed using the maximum-parsimony method. ncbi genbank accession numbers for the sequences determined here are jx413801 to jx413833. amino acids were numbered starting after the dtlc signal peptide. potential n-linked glycosylation sites were predicted using the free software netnglyc 1.0. data were analyzed using spss software version 19 (spss, chicago, usa). data were compared by fisher's exact test, and p-values were deemed significant at the 0.05 level. in order to detect molecular changes in the ha gene of influenza virus, 33 a(h1n1)pdm09 ha sequences representing 25 individual patients were analysed. as shown in table 1, 14 of the 25 patients (56 %) were immunocompromised or immunosuppressed, suffering from different underlying diseases. virus isolates on mdck siat-1 cells were obtained from 28 of the 33 respiratory samples (data not shown) [16] . the median age of the patients was 40 years (95 % confidence interval [ci], 22.9-44.3 years]. based on clinical criteria, 11/25 (44 %) were regarded as mild (median age, 6 years; range, 1-55 years), 10 (40 %) as moderate (median age, 53 years; range 0.1-76 years) and 4 (16 %) as severe cases (median age, 47 years; range, 0.2-55 years) according to zarychanski et al. [27] . all severe cases were hospitalized patients known to be at risk for severe disease (3 of these 4 patients were immunocompromised, and one of them was obese). only 1 of the 11 immunocompetent patients and 3 of the 14 immunocompromised patients developed severe disease (fisher's exact test, p=0.60). in 7 (21 %) of the 33 samples (corresponding to 5 [20 %] of the 25 patients), co-infection with another respiratory virus was detected. coronavirus oc43 and/or nl63 were detected in 3 of these 5 patients, rsv and human bocavirus were co-detected in the npa of one, and rsv alone was detected in a pharyngeal swab from another patient. clinically, the patient with rsv and bocavirus coinfection presented with mild disease; the remaining patients displayed moderate (n=3) to severe (n=1) symptoms. none of the patients showed co-infection with mycoplasma pneumoniae as assessed by multiplex pcr. prolonged shedding (i. e., c 2 weeks) of influenza virus in the respiratory tract was observed in 6 of the 25 patients (24 %; 3 with severe, 2 with moderate, and 1 with mild disease). a total of 5 out of 6 patients were immunocompromised, and 5 of the 25 patients (4 of them immunocompromised) received oseltamivir therapy. the ha sequences were closely related to each other and to the reference strain a/california/07/2009 (a/cal/h1n1/ 09). the differences between german sequences and a/cal/h1n1/09 ranged from 5 to 10 amino acids ( table 2 ). all of the viruses analysed displayed the amino acid changes p83s and s203t in the ha1 region as well as i321v and e 374 k in the ha2 region. phylogenetic analysis showed simultaneous co-circulation of influenza virus of groups 4, 5, 6, and 7 in southern germany ( figure 1 ). genetic groups were named according to the ecdc technical document of august/september 2011. in detail, 16 of the 33 sequences (48 %) belonged to genetic group 6, characterized by the double mutation d97n and s185t. moreover, 3 of these 6 showed an additional s84i mutation (as influenza a/delaware/ af21764/2010), and one of them had a p159s mutation. of note, this particular strain was isolated from an influenzavaccinated individual. ten of the 33 sequences (30 %) belonged to genetic group 5, characterized by the mutations d97n, r205k, i216v and v249l. in this group, three also had the h138q mutation. as observed by piralla et al. [18] , two sub-clusters were seen within this group. group 4, characterized by amino acid mutations n125d included four sequences, all obtained from the same patient. finally, 3 of the 33 sequences (9 %) clustered within group 7 and were characterized by the mutations s185t, s143g, and a197t. double mutations characteristic of group 3 (a134t, s183p), and group 2 (n31d, s162n) were not observed. severe, moderate, and mild cases were scattered throughout the phylogenetic tree. some of the observed amino acid substitutions involved the major antigenic sites of the ha molecule (table 2) . five potential n-glycosylation sites, typical of a/cal/ h1n1/09, corresponding to residues 23, 87, 276, 287, and 481, were also detected in the german sequences. however, in two patients, two additional glycosylation sites were detected at residue 119 (lysine to asparagine) in one case and at residue162 (serine to asparagine) in the other case. as shown in table 2 , jx413805 and jx413803 correspond to sequences obtained from the upper and lower respiratory tract of the same patient. interestingly, the k119n mutation, representing an additional potential n-glycosylation site, was present only in the bal sample. moreover, the cycle threshold (ct) value of the h1n1 similarly, in another severe case, the ct value for the bal sample obtained 20 days after onset of disease was lower (ct 21) than that for the nasopharyngeal swab (ct 29). also in this case, two additional amino acid changes (k130r and m344l, genbank accession number jx483122) were detected in the bal sample. treatment with oseltamivir was reported in 5 of the 25 patients (4 immunocompromised patients and 1 obese patient). the oseltamivir-resistance-associated mutation h275y was detected in 1 (20 %) of the 5 treated patients, a one-year-old child with a genetic disorder, who was treated for 21 days with oseltamivir. he recovered fully from ca1 s t t t t t t t t t t t t t t t t t t t t t t 205 ca2 s t t t t t t t 186 sb a t 190 sb s g 197 a 203 ca1 s t t t t t t t t t t t 205 ca2 influenza despite development of oseltamivir resistance and prolonged shedding of resistant virus for [ 2 weeks. in this molecular study, we were able to show that influenza viruses circulating in southern germany in the first post-pandemic season differed relatively little from each other and from the vaccine strain a/cal/h1n1/09. some of the amino acid substitutions that were observed involved the major antigenic sites of the ha molecule. five classical antigenic sites (sa, sb, ca1, ca2, and cb) have been described in the ha of seasonal influenza h1n1 virus [3, 24] , all located in the globular head of ha. recent studies have also described some important antigenic sites in the stem region of ha [25] . all viruses analysed here had the amino acid changes p83s and s203t in the ha1 region and i321v and e374k in the ha2 region. as reported by others [8, 18, 19] , and as published in the ecdc report released in august/september 2011 [23] , co-circulation of different genetic groups was observed. group 6, characterized by mutations d97n and s185t, was the dominant h1n1 lineage (48 %), followed by group 5 (33 %; d97n, r205k, i216v), group 4 (12 %; n125d) and group 7 (9 %; s185t, s143g, a197t). although a number of mutations have been reported in circulating a(h1n1)pdm09, they have not significantly affected virus antigenicity and pathogenicity as demonstrated by in vitro studies [26] . clinically, the mutations d222g and n have been associated with a more virulent phenotype [10] . however, recent studies show that within the current a(h1n1)pdm09 ha framework, the effect of the 222 mutation on receptor binding appears to be less dramatic when compared to the 1918 influenza a(h1n1) virus ha framework, since the binding preference for a2-6 sialylglycans is still maintained [26] . also, other mutations, such as the double mutation n125d and e374k, have been associated with a more virulent phenotype. this double mutation has been associated with several breakthrough infections despite influenza vaccinations and was identified in some fatal cases [2] . moreover, it was associated with decreased antibody recognition in vaccinated individuals [21] . the german ha sequences carrying this double mutation originated from an unvaccinated immunocompromised individual with severe illness. due to the lack of serum specimens, we could not analyze the ability of antibodies to recognize the hemagglutinin of a(h1n1) pdm09 in our study. however, immune escape from the vaccine strains might be an issue of concern. no d222g change was observed here. in our study, no association of a specific amino acid change with severe illness could be observed. however, in two patients, differences between the ha sequence could be detected in viruses isolated either from the upper respiratory tract (urt) or the lower respiratory tract (lrt). in particular, two additional mutations (k130r-m344l in one case and k119n-i216k in the other case) were identified only in the lrt. moreover, analysis of potential glycosylation sites revealed that the k119n mutation provides an additional potential glycosylation site. human influenza viruses carrying the k119n mutation show improved growth in eggs and appear to exhibit enhanced virulence in the mouse model [9, 14] . glycosylation at position 119 is essential for improved virus protein yield in eggs [14] . egg adaptation of human influenza viruses is known to increase their affinity for the 2,3-sialic acid (sa) receptor and concomitantly impairs their ability to bind to 2,6-sa [11] . although it is clear that influenza virus tropism depends on several viral and host factors and not only on ha specificity, the presence of the k119n mutation exclusively in the virus isolated from the lrt might indicate a more efficient binding/replication of viruses carrying this mutation to/in cells expressing 2,3-sa. importantly, 2,3-sa is known to be found in abundance in the lower respiratory tract [20] . of note, the ct value of the a(h1n1)pdm09 real-time rt-pcr from the nasopharyngeal swab was higher than that of the bal sample carrying the mutation (ct 26 versus ct 19) . since bal samples are usually significantly more diluted than nasopharyngeal swabs, the observed difference suggests a real replication advantage of the strain harboured in the lrt. virus histochemistry studies could be used to analyze the pattern of binding of mutants to human respiratory tissue, as already suggested [6, 22] . moreover, analysis of growth curves of the two isolates in different cell lines may reveal a possible replication advantage, at least in cell culture. many patients with influenza have more than one viral agent, with reported co-infection frequencies as high as 20 % [4] . we detected co-infection with another respiratory virus in 7 out of 33 samples (21 %), corresponding to 5 of the 25 patients (20 %). the most frequent co-infecting agents were coronaviruses (5 of 7 samples), followed by rsv (2 of 7 samples) and human bocavirus (1/7). disease resulting from co-infection with influenza virus and coronaviruses has been reported to be more severe [4] , although the number of influenza virus and coronavirus co-infections is low [4, 15, 17] . here, patients co-infected with coronaviruses showed moderate to severe disease, whereas co-infection with rsv and human bocavirus in one child resulted in mild disease. finally, the oseltamivir-resistance-associated mutation h275y was detected only once in our study population. although oseltamivir has been widely used in the 2009 pandemic and thereafter, cases of resistance have remained scarce to date. however, prolonged shedding of high levels of resistant influenza virus in individual cases poses the threat of spread into populations that are at risk and finally into the general population as resistant viruses retain fitness [7] . in concordance with other studies, our results underline the importance of monitoring influenza virus evolution and development of resistant viruses. of note, the lrt might harbour more virulent variants than the urt. immune escape of influenza virus in subsequent influenza seasons is an issue of concern, making continuous surveillance essential. severe outcome of influenza a/h1n1/09v infection associated with 222g/n polymorphisms in the haemagglutinin: a multicentre study a new pandemic influenza a(h1n1) genetic variant predominated in the winter influenza season in australia the antigenic structure of the influenza virus a/pr/8/34 hemagglutinin (h1 subtype) rate and influence of respiratory virus co-infection on pandemic (h1n1) influenza disease influenza hemagglutinin and neuraminidase membrane glycoproteins influenza virus receptor specificity: disease and transmission characteristics of a widespread community cluster of h275y oseltamivir-resistant a(h1n1) pdm09 influenza in australia genetic diversity of the 2009 pandemic influenza a(h1n1) viruses in finland adaptation of pandemic h1n1 influenza viruses in mice observed association between the ha1 mutation d222g in the 2009 pandemic influenza a(h1n1) virus and severe clinical outcome early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals first sequence-confirmed case of infection with the new influenza a(h1n1) strain in germany neuraminidase inhibitors for influenza an additional oligosaccharide moiety in the ha of a pandemic influenza h1n1 candidate vaccine virus confers increased antigen yield in eggs frequency of detection of upper respiratory tract viruses in patients tested for pandemic h1n1/09 viral infection mdck-siat1 cells show improved isolation rates for recent human influenza viruses compared to conventional mdck cells streptococcus pneumoniae coinfection is correlated with the severity of h1n1 pandemic influenza segregation of virulent influenza a(h1n1) variants in the lower respiratory tract of critically ill patients during the 2010-2011 seasonal epidemic genetic characterization of the influenza a pandemic (h1n1) 2009 virus isolates from india avian flu: influenza virus receptors in the human airway minor changes in the hemagglutinin of influenza a(h1n1)2009 virus alter its antigenic properties seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses nucleotide sequence of the haemagglutinin gene of a human influenza virus h1 subtype screening of random peptide library of hemagglutinin from pandemic 2009 a(h1n1) influenza virus reveals unexpected antigenically important regions structure and receptor binding properties of a pandemic h1n1 virus hemagglutinin correlates of severe disease in patients with 2009 pandemic influenza (h1n1) virus infection acknowledgments we are grateful to nadja besazza for excellent technical assistance. we are grateful to dieter neumann-haefelin and markus hufnagel for critical reading of the manuscript. this work was partially supported by the german bundesministerium für bildung und forschung (bmbf, contract #01es0830). key: cord-292963-8wzyfb2j authors: zeng, zheng; huang, xiang-rong; lu, pu-xuan; le, xiao-hua; li, jing-jing; chen, de-ming; yuan, jing; li, guo-bao; liu, ying-xia; zhou, bo-ping title: imaging manifestations and pathological analysis of severe pneumonia caused by human infected avian influenza (h7n9)() date: 2015-03-02 journal: radiol infect dis doi: 10.1016/j.jrid.2015.02.003 sha: doc_id: 292963 cord_uid: 8wzyfb2j objective: to investigate the imaging and pathological findings of severe pneumonia caused by human infected avian influenza (h7n9), and therefore to further understand and improve diagnostic accuracy of severe pneumonia caused by human infected avian influenza (h7n9). methods: the relevant clinical and imaging data of 19 cases, including 10 males and 9 females, with pneumonia caused by human infected avian influenza (h7n9) was retrospectively analyzed. one of the cases had received percutaneous lung biopsy, with the clinical, imaging and pathological changes possible to be analyzed. results: the lesions were mainly located at lower lobes and dorsal of lungs, involving multiple lobes and segments. ground-glass opacities and/or pulmonary opacities were the more often imaging manifestations of severe pneumonia caused by human infected avian influenza (h7n9) in early and evolving phases (19/19,100%). by biopsy following percutaneous lung puncture, exudation of slurry, cellulose, rbc and neutrophils, formation of hyaline membrane, squamous metaplasia and organizing exudates were observable at the alveolar space. some of alveoli collapsed, and some responded to show compensatory emphysema. conclusion: the imaging features of severe pneumonia caused by human infected avian influenza (h7n9) include obvious ground-glass opacity and pulmonary consolidation, mainly at lower lobes and dorsal of lungs, with rapid changes. the cross-analysis of imaging and pathology preliminary can elucidate the pathological mechanisms of ground-glass opacities and pulmonary consolidation of severe pneumonia. such an intensive study is beneficial to prompt clinicians to observe and evaluate the progress of the disease. in addition, it is also in favor of managing the symptoms and reducing the mortality rate. human infected avian influenza (h7n9) is an acute respiratory infection caused by h7n9 subtype of avian influenza a virus [1] . the disease was firstly reported in the spring of 2013 in yangtze river delta, china [2] . a total of 23 cases of human infected avian influenza (h7n9) was admitted to our hospital from december, 2013 to april, 2014, with 19 cases suffering from severe pneumonia. in this study, we retrospectively analyzed imaging and pathological manifestations of the disease to shed light on its clinical diagnosis, therapeutic efficacy evaluation and prognosis. the clinical, radiological and pathological data of 19 cases with severe pneumonia caused by human infected avian influenza (h7n9) from december 18, 2013 to april 18, 2014 in shenzhen third people's hospital, china, were collected. all the patients were tested positive to nucleic acid of h7n9 subtype of avian influenza virus by cdc of guangdong province and shenzhen city, china, in line with the diagnostic criteria of severe human infected avian influenza. the 19 cases included 10 males and 9 females, aged 31e82 years with a median of 55 years. two patients had preexisting hypertension; 3 had hypertension and diabetes; 1 had tuberculosis; and 1 had right pulmonary embolism. fever was the most common symptom, found in all 19 cases (100%), and ardent fever (39 c or above) showed up in 15 patients (78.9%). cough was also the most common symptom, occurring in all 19 cases (100%), with expectoration in13 cases (68.4%) and 1 case coughing up dark red bloody sputum. anhelation occurred in 11 cases (57.9%). all patients were admitted to our hospital at d 4e14 after onset, averagely 8.4 days, and received antiretroviral and respiratory supporting therapies. six patients assured of a history of contact to live poultry, by another 5 patients denied a history of contact to live poultry. the epidemiologic data of the other 8 patients was not available. wbc count was detected to have a decrease in 7 cases, being from 2.20 â 10^9/l to 7.11 â 10^9/l, with a mean of 4.69 â 10^9/l. neutrophil percentage increased in 13 cases, but remained normal level in 6 cases, being from 63.0% to 92.9%, with a mean of 76.8%. according to the diagnostic and treatment protocol for human infected avian influenza a (h7n9) established by national council on health and family planning commission, p. r. china (edition, 2014), the cases with any one of the following criteria can be diagnosed as severe. (1) chest x-ray demonstrates lesions with multiple lobes involved or lesions progress more than 50% within 48 h; (2) difficulty breathing, with more than 24 breaths per minute; (3) severe hypoxemia, with oxygen flow at 3e5 l per minute and spo2 92%; (4) shock, ards or mods (multiple organ dysfunction syndrome). biopsy following percutaneous lung tissue puncture was performed in one case to observe the pathological changes. the pathogenic bacteria was observed after pas and masson staining. acid-fast bacteria staining was performed to detect possible infection of mycobacterium tuberculosis. conventional chest x-ray was performed using philips didi th/vr, with the tube voltage 102 kv and automatic tube current. bedside chest x-ray was performed using hitachi sirisu130hp mobile dr, with a tube voltage 100 kv and automatic tube current. ct scanning was performed using toshiba tsx-101a 64-slice spiral ct, with a tube voltage 135 kv, automatic tube current, a pitch of 0.9, a matrix of 512 â 512, an fov of 320 mm â 320 mm, a thickness of 5.0e6.0 mm, and an interval of 1 mm. ct scanning was from the apex to the bottom of lungs continuously. lung window was reconstructed using conventional 1 mm and high resolution of 5 mm after the scanning. all the images were independent analyzed by two radiologists with a title above associate chief-physician, and consensus was reached after consultation and discussion. the images were analyzed in terms of distribution and range of lesions, morphology of the lesions as well as changes of mediastinum and pleura. (1) ct manifestations at the early phase (d 1e4 after onset) the lesions more often onset from a lower lung lobe (17/ 19), only 2 cases had their lesions onset from an upper lung lobe. poorly-defined patches of shadows and fragmental ground-glass opacities were demonstrated by ct scanning. changes of pulmonary interstitium were observed, including interlobular septal thickening, acinar nodules, and other changes. chest ct scanning demonstrated rapid progress of the lesions within a short period of 1e2 days, with rapid expansion, fusion, formation of large patchy opacities, and the lesions were demonstrated to involve multiple lobes (fig. 1 ). (2) ct manifestations at the evolving phase (d 5e10 after onset) the lesions of 18 cases involved both lungs (18/19, 94.7%), and only 1 case showed lesions with unilateral lung involved (1/19, 5.3%). the lesions of 19 cases involved median lobe (lingual lobe) or lower lobe (19/19, 100%). the lesions of 18 cases involved 4 to 6 lung segments (18/19, 94.7%). ground-glass opacity was demonstrated by ct scanning in all 19 cases (100%), which were poorly defined in fragments and large patches. pulmonary consolidation was also demonstrated in all 19 cases (19/19, 100%), which was more commonly found at the lower lung lobes to cross segments or lobes with higher density. air bronchogram was observed in the area of consolidation. air sacs in lungs were demonstrated in 3 cases (3/19, 15.8%), which were round with smooth lining, different sizes, various shapes and could be absorbed. lymphadenectasis was demonstrated in 1 case (1/19, 5.3%), with several enlarged lymph nodes in the mediastinum, the larger one in front of the tracheal eminence, and the smaller one having a diameter of about 15 mm.pleural effusion was demonstrated in 13 cases (13/19, 68.4%), mostly in a small quantity, 2 cases of unilateral and 11 cases of bilateral ( fig. 2) . (3) ct manifestations at the absorbing phase (d 11-after onset) all the patients were admitted to our hospital at d 4e14 after onset (mean, 8.4 days), and by chest ct scanning, the lesions began to be absorbed at d 7e19 after onset, averagely at d 12.0 after onset. interval between onset and beginning of absorption ranged from 1 day to 12 days, with an average of 3.7 days. the group of 19 patients received antiretroviral and symptomatic therapies. in 1 patient, the lesions began to be absorbed at d 12 after admission to hospital, and in the remaining 18 patients, the lung lesions began to be absorbed at d 3 after hospitalization. during absorption, the range of fig. 1 . a female patient aged 39 years was definitively diagnosed with severe pneumonia caused by human infected avian influenza a (h7n9). 1a) chest ct scanning at d 4 after onset demonstrates large patchy consolidation at the right lower lung lobe, with observable air bronchogram; 1b) chest ct scanning at d 6 after onset demonstrates substantial progress of the lesions, with large patchy consolidation and poorly defined patchy ground-glass opacities at lower lobes of both lungs and the right middle lung lobe. consolidation gradually reduced with decreased density, consolidation and recruitment of lung. the lesions at the upper and median lung lobes were absorbed earlier than those at the dorsal part of lower lung lobe and the subpleural lesions. the earlier emerging lesions were absorbed later, and vise versa. finally, the residual lesions were more often located at dorsal part of the lower lung lobe and under the pleura. in this group of patients, 18 were cured after hospitalization for 9e50 days (averagely 20.5 days). by ct scanning, consolidation disappeared in 14 cases (14/19, 73.7%); multiple patches of shadows were shown in 16 cases (16/19, 84.2%); lamellar ground-glass opacities were shown in 9 cases (10/19, 52.6%); linear shadows in 17 cases (17/19, 89.5%); latticed shadows and subpleural line in 7 cases (7/19, 36.8%); air sacs or pseudocavity, paraseptal emphysema, scar emphysema, subpleural bulla in 5 cases (5/19, 26.3%) (fig. 2) . death occurred in 1 case due to respiratory and renal failure (fig. 3 ). one case of this group received biopsy following percutaneous lung tissue puncture at the advanced period of the condition. histopathology mainly showed fibrotic changes. necrosis and abscission of some alveolar epithelia as well as reactive hyperplasia of alveolar epithelium were observed (fig. 3ced) , but with no viral inclusions within epithelial cells. slurry, cellulose, rbc, effused neutrophils, formation of hyaline membranes, squamous metaplasia and organizing exudates were observed in alveolar space (fig. 3e ). some pulmonary alveoli were shown with atrophy or compensating emphysema. pulmonary interstitial fibrosis, sometimes leukomonocyte, phlogocyte and reactive hematophagocyte were observable. formation of hyaline thrombus and dic were shown in capillary of pulmonary interstitium, sometimes with vascular occlusion (fig. 3f) . empsyxis and secondary infection was sometimes found (fig. 3 ). human infected avian influenza a (h7n9) is a newly emerging infectious disease caused by h7n9 subtype of avian influenza a virus. the disease was firstly reported in china in 2013, with main manifestations of flu-like symptoms such as fever, cough, and so on [3] . severe pneumonia prompts critical condition, often in combination with ards, infectious shock, and so on. chest ct scanning and diagnostic imaging are the important means for its clinical diagnosis and therapeutic evaluation [4] . this group of 19 cases with severe pneumonia caused by human infected avian influenza a (h7n9) had received more than three times of chest ct scanning. after comprehensive analysis, the ct manifestations of these patients include: (1) the lesions are more often found at the lower lung lobes, with 17 cases of this group showing lesions at 1 or 2 lower lung lobes. with progress of the disease, the lesions rapidly progress to involve multiple segments and lobes of both lungs, often 3 or more lung lobes up to all segments and lobes of both lungs. pulmonary opacities are mainly located at the dorsal part of lungs. (2) ground-glass opacities and pulmonary opacities are the cardinal imaging infestations to severe pneumonia caused by human infected avian influenza a (h7n9). when the condition is mild or at its early stage, the lesions are mainly ground-glass opacities. at the evolving phase, the percentage of pulmonary opacities increases, with air bronchogram in the pulmonary opacities. ground-glass opacities mainly distribute at the anterior border of pulmonary opacities, showing as sporadic patchy opacities, even as "white lung" when in severe condition. the patient may develop significant hypoxemia, respiratory distress syndrome and respiratory failure. (3) pleural effusion is also one of the imaging features of human infected avian influenza a (h7n9), with 13 cases of this group showing pleural effusion, and its incidence rate is higher than the previous report [5] . it may be related to the fact that all patients of this group suffered from severe pneumonia. the pleural effusion may be caused by systemic inflammatory response triggered by direct involvement of pleura by virus and/or virus evoked cytokine storm. (4) pulmonary interstitial fibrosis is the main findings at the recovery phase, with all cases of this group showing as small patchy shadow under the pleura and/or at dorsal part of lower lobes. fibriform cords, latticed shadows, fragmented ground-glass opacities and other pulmonary fibrosis are also ct changes. at the same time, in this group, subpleural paraseptal emphysema and ulotic emphysema, subpleural bullas and localized bronchiectasis are observable. pathological findings in the cases of severe pneumonia caused by human infected avian influenza a (h7n9). influenza virus is categorized into orthomyxovirus, and it is a single minus strand, segmental rna virus that is enveloped [4] . the hemagglutinin in the tunica external of influenza virus a plays a key role in the pathomechanism of human infected avian influenza a (h7n9). inflammatory factors may be mediate systemic inflammatory response syndrome and ards. therefore, severe pneumonia is demonstrated as extensive ground-glass opacities and obvious consolidation by ct scan whose underlying mechanism is damaged pulmonary alveoli and pulmonary capillaries caused by virus, extensive effusion of pulmonary interstitium and pulmonary parenchyma [6] . diffuse alveolar damage, interstitial fibrosis and air cavity extension, infiltration of leukomonocyte and plasma cells were demonstrated by histopathology. therefore, the ct manifestations of severe pneumonia caused by human infected avian influenza a (h7n9) can be explained by the histopathologic findings as its pathomechanism. and, the main pathological changes of body organs are the result of phagocytosis of red blood cells, white blood cells and platelets by macrophages, known as reactive hemophagocytic syndrome. it can also explain different degrees of reduction of peripheral blood counts by clinical laboratory tests at the early and evolving phases of severe pneumonia cause by human infected avian influenza a (h7n9). and the varying degrees damage of cd4t lymphocytes indicates immune responses. 3. the differential diagnosis of severe pneumonia caused by human infected avian influenza a (h7n9) severe pneumonia caused by human infected avian influenza a (h7n9) should be distinguished from influenza a (h1n1 or h5n1) and severe acute respiratory syndromes, and other conditions. pneumonia caused by human infected avian influenza a (h1n1, h5n1 and h7n9) is all caused by influenza a virus, all with flu-like symptoms. the conditions are demonstrated as multiple ground-glass opacities in different sizes and consolidation by chest ct scan [7e10]. compared to pneumonia caused by influenza a (h1n1), the lesions caused by h5n1 and h7n7 occupy a larger range and develop more rapidly, and air bronchogram is more common. the disease progressions of influenza h5n1 is rapid, following by h7n9 and h1n1. by ct scans, pneumonia caused by influenza a (h5n1) is mainly displayed as large patchy ground-glass opacities and consolidation at both lungs. the lesions distribute widely and progress rapidly [7, 8] . in some cases, the lesions are erratic and absorbed slowly, with obvious pulmonary interstitial fibrosis and a mortality rate of about 59%. onset from middle lobe and lower lobe of lungs, the lesions in the cases of human infected avian influenza a (h7n9) are mainly displayed as ground-glass opacities and lung consolidation, which change rapidly and are absorbed slowly, too [2, 6, 11] , with a mortality rate of about 36%. the lesions are mainly displayed as multiple patchy ground-glass opacities fig. 3 . a male patient aged 34 years was definitively diagnosed with severe pneumonia caused by human infected avian influenza a (h7n9), and this is the case that received biopsy following percutaneous lung tissue puncture. 3aeb) chest ct scanning at d 12 after onset shows large patchy consolidation and ground-glass opacities in all lobes of both lungs, with poorly defined boundary. air bronchogram is observable in the lesions, and mediastinal emphysema is demonstrated; 3c) the lung tissue is demonstrated to be degenerated and necrotic, with a few lymphocytes infiltrated; 3d)reactive hyperplasia of the alveolar epithelium and pulmonary interstitial fibrosis are demonstrated; 3e)the alveolar epithelium is demonstrated to be dropped, with light red liquid, cellulose and a few lymphocytes in alveolar space; 3f) alveolar epithelial cellsare demonstrated with hyperplasia, with some alveolar epithelium dropped and vascular occlusion observable. and patchy or large patchy high-density consolidation in the cases with influenza a (h1n1), with segmental atelectasis and pleural effusion [10, 12] as well as a mortality rate of about 6%. only based on radiological findings, the identification of infections by these different influenza viruses is challenging. and the differential diagnosis should be made in combination to epidemiological and etiological findings. chest ct scans also display sars as ground-glass opacities and lung consolidation, which progress rapidly with involvement of both lungs in about 50% of the patients, more often at middle and lower lung lobes. the lesions mainly distribute in the peripheral pulmonary tissue, with detectable interlobular septal thickening by high-resolution ct as broken paving-stones. accompanying bronchiolectasis and a small quantity pleural effusion are also detectable [13] . these findings resemble to those of human infected avian influenza a (h7n9), but the interstitial changes in the cases of human infected avian influenza a (h7n9) are not obvious. the lesions of human infected avian influenza a (h7n9) show up in a more extensive range, with no obvious coverage at the peripheral pulmonary tissue. epidemiological history and laboratory tests for its pathogen are the key to identify the two conditions. generally, severe pneumonia caused by human infected avian influenza a (h7n9) is demonstrated with ground-glass opacities and lung consolidation by chest ct scan. these lesions onset from middle and lower lobes of both lungs, and the condition may develop into pulmonary cavity or aerothorax, hydropneumothorax. the differential diagnosis for identification of influenza a virus (including h7n9, h5n1 and h1n1) is challenging based only on radiological findings. and their identification should be made in combination to epidemiological and etiological findings. cross analysis of radiology and pathology is a way to further understand severe pneumonia caused by human infected avian influenza a (h7n9). and such a study is beneficial to clinical observation and evaluation of the condition, and is of great significance for disease control and mortality reduction. national health and family planning commission. diagnostic and treatment protocol for human infections with avian influenza a (h7n9). edition characteristics of the imaging manifestations and dynamic changes in patients with severe pneumonia caused by h7n9 avian influenza virus epidemiology and imaging manifestations of avian influenza clinical and imaging diagnosis of emerging infectious disease. beijing: people's medical publishing house preliminary imaging findings of novel reassortant avian-origin influenza a (h7n9) pneumonia the correlation of ct manifestation, viral load and cd4þt lymphocyts in patients with h7n9 avian influenza pneumonia ct manifestation and dynamic changes of grave pneumonia in adults caused by h5n1 subtype of human avian influenza virus imaging features of pneumonia caused by highly pathogenic h5n1 subtype human avian influenza virus human avian influenza types and manifestations in ct images of pneumonia caused by influenza a (h1n1) imaging manifestations of the severe cases of pneumonia caused by h7n9 subtype human avian influenza virus correlative study of semi-quantitative score of chest ct findings and viral load in novel influenza a (h1n1) virus infection imaging diagnosis of sars key: cord-275150-d63noia4 authors: ye, chuchu; zhu, weiping; yu, jianxing; li, zhongjie; fu, yifei; lan, yajia; lai, shengjie; wang, yuanping; pan, lifeng; sun, qiao; zhao, genming title: viral pathogens among elderly people with acute respiratory infections in shanghai, china: preliminary results from a laboratory‐based surveillance, 2012‐2015 date: 2017-07-06 journal: j med virol doi: 10.1002/jmv.24751 sha: doc_id: 275150 cord_uid: d63noia4 acute respiratory infections (aris), with viral pathogens as the major contributors, are the most common illnesses worldwide, and increase the morbidity and mortality among the elderly population. the clinical and pathological features of elderly people with aris need to be identified for disease intervention. from january 1, 2012 through december 31, 2015, respiratory specimens from patients above 60 years old with aris were collected from the outpatient and inpatient settings of six sentinel hospitals in pudong new area. each specimen was tested via multiplex polymerase chain reaction (pcr) for eight target viral etiologies including influenza, human rhinovirus (hrv), human para‐influenza virus (piv), adenovirus (adv), respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human coronavirus (hcovs), and human bocavirus (hbov). a total of 967 elderly patients with aris were enrolled, including 589 (60.91%) males, and the median age was 73 years old. 306 (31.64%) patients were tested positive for any one of the eight viruses, including 276 single infections and 30 co‐infections. influenza was the predominant virus (14.17%, 137/967), detected from 21.35% (76/356) of the outpatients and 9.98% (61/611) of the inpatients. influenza infections presented two annual seasonal peaks during winter and summer. compared with non‐influenza patients, those with influenza were more likely to have fever, cough, sore throat, and fatigue. this study identified influenza as the leading viral pathogen among elderly with aris, and two seasonal epidemic peaks were observed in shanghai. an influenza vaccination strategy needs to be advocated for the elderly population. those with influenza were more likely to have fever, cough, sore throat, and fatigue. this study identified influenza as the leading viral pathogen among elderly with aris, and two seasonal epidemic peaks were observed in shanghai. an influenza vaccination strategy needs to be advocated for the elderly population. influenza virus, public policy, respiratory track acute respiratory infections (aris) are the most common illnesses worldwide. they cause nearly four million deaths per year, at a rate of more than 60 deaths/100 000 people. 1,2 among all the individuals with aris, the elderly suffer disproportional morbidity and mortality rates, mostly due to viral respiratory infections. [3] [4] [5] the rates of aris increase with age in adults and are highest among individuals aged >65 years. 6 during the past decades, some nations, including china, have faced a rapid increase in the proportion chuchu ye, weiping zhu, and jianxing yu contributed equally to this work. each specimen was tested for the following eight viral to have fever (307, 86.24%), sore throat (132, 37.8%), headache (97, 27.25%), fatigue (110, 30.90%), and runny nose (89, 25.00%) than inpatients (p = 0.000, table 1 ). of the 967 enrolled patients, 306 (31.64%) tested positive for at least one of the eight target viruses. of these patients, 276 presented with a single infection, whereas 30 (9.80%) were infected with more than one virus. the predominant etiologies of the respiratory viruses in singleinfection cases were influenza (137, 44.77%), hrv (44, 14.38%) and piv (23, 7.52%). no hbov positive samples were detected. influenza was also the most frequently detected pathogen among multiinfection cases; influenza plus hrv and influenza plus hmpv were the most frequent pathogen combinations among co-infections, and these combinations were detected in five specimens (16.67%), followed by influenza plus rsv (4, 13.33%). two patients were infected with three pathogens (fig. 1 ). of the 356 outpatients, 41.85% tested positive for at least one virus. this rate was much higher than that among inpatients (25.70% [157/611], p = 0.000). the most common viruses were influenza and hrv; 21.35% (76/356) and 6.18% (22/356) of outpatients tested positive for these viruses, respectively, whereas 9.98% (61/611) and 3.60% (22/611) of inpatients did so, respectively. outpatients were more likely to test positive for influenza than inpatients (21.35 vs. 9.98%, p = 0.000). the proportion of patients with co-infection was similar among outpatients (11, 3.09%) and inpatients (19, 3.11%, table 2 ). the positive percentage of influenza virus presented two seasonal peaks, with a primary peak in the winter (january-february) and a secondary peak in the summer (july-august), with the positive percentage of 36.09% and 20.13% respectively. the winter and summer peaks among outpatients were much higher than those among inpatients (46.75 vs. 27.17%, p = 0.008; 32.14 vs. 13.09%, p = 0.000, fig. 2 were infected with a(h1n1)pdm09, and one was infected with similar studies have demonstrated that influenza causes the great majority of aris, and the impact of influenza on the elderly is substantial. more than 2/3 of influenza-related hospitalizations and more than 80% of the influenza-related deaths in the united states are estimated to involve the elderly. 13, 14 in addition to their increased morbidity and mortality rates, the clinical features of influenza among showed that the vaccine effectiveness (ve) against a(h3n2) was lower than that against the a(h1n1)pdm09 strain over those years. 19 vaccination is the most effective strategy to prevent influenza. although elderly might experience a declining immunogenicity after vaccination due to the immunosenescence, 20 they are still an important target population for influenza vaccination because of its highest influenza-related death burden. seasonal influenza vaccination is recommended for this population worldwide. 21, 22 a quantitative review of 31 vaccine antibody response studies showed that the protection rates of influenza vaccine against a(h1n1), a(h3n2), and b-type influenza in the elderly were 69%, 74%, and 67% respectively. 23 according to a study conducted in brazil, no influenza virus was identified during the influenza season among a group of elderly patients with aris, with an influenza immunization coverage of 62.30%. 24 influenza vaccines specifically targeting this population have been developed and might facilitate additional efforts to prevent influenza among the elderly. 25, 26 the elderly of several chinese cities (ie, beijing and ningbo) are offered free annual influenza vaccinations. 19, 27, 28 however, similar public health policies have not been offered in shanghai yet. previous researches had showed that most of the h7n9 cases were older people with the median age over 60 years. 29, 30 one case infected with a(h7n9) was identified by our surveillance system, which was helpful to increase the timeliness of both clinical treatment and public health disposal. several limitations exist in this study. first, because the surveillance system has been operating for only 4 years, and the quantity of the specimens was inadequate at the beginning period, seasonal trends cannot be well described yet. however, the predominant viral pathogen in elderly with aris and the characteristics of these individuals was clearly revealed by our preliminary results, which provide primary background information. second, sampling bias might be caused due on convenience sampling method. beside, although some inpatients were excluded because they previously visited outpatient clinics, the bias caused by repeated visits remains in our study, especially among inpatients. to improve future surveillance, more information (eg, detailed clinical symptoms, complications, vaccines, and the treatment histories of the patients) should be collected to evaluate the disease burden associated with target respiratory viruses and determine the risk factors. the findings of the current study showed that influenza is the leading viral pathogen among both elderly inpatients and outpatients with aris. furthermore, two seasonal epidemic peaks were identified in shanghai, china. an influenza vaccination strategy should be advocated for the elderly population to reduce the disease burden of influenza among this population at high risk for complicated diseases. acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden who (world health organization). world health report estimates of world-wide distribution of child deaths from acute respiratory infections infectious aetiologies in elderly patients hospitalised with non-pneumonic lower respiratory tract infection mortality associated with influenza and respiratory syncytial virus in the united states randomised controlled trial and health economic evaluation of the impact of diagnostic testing for influenza, respiratory syncytial virus and streptococcus pneumoniae infection on the management of acute admissions in the elderly and high-risk 18-to 64-year-olds the new aging society: demographic transition and its effects on old-age insurance and care of the elderly in china who (world health organization). world health statistics (whosis) pneumonia burden in elderly patients: a classification algorithm using administrative data clinical analysis of community-acquired pneumonia in the elderly medical and economic burden of influenza in the elderly population in central and eastern european countries area disease estimation based on sentinel hospital records community-acquired pneumonia influenza-associated hospitalizations in the united states clinical features of influenza a virus infection in older hospitalized persons surveillance of 16 respiratory viruses in patients with influenza-like illness in nanjing the clinical and etiological characteristics of influenza-like illness (ili) in outpatients in global epidemiological surveillance standards for influenza influenza vaccine effectiveness against medically-attended influenza illness during the 2012-2013 season in beijing prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices, united states, 2015-16 influenza season who (world health organization). influenza-vaccine use technical guidelines for the application of seasonal influenza vaccine in china antibody response to influenza vaccination in the elderly: a quantitative review viral etiology among the elderly presenting acute respiratory infection during the influenza season immunosenescence: influenza vaccination and the elderly correlates of protection against influenza in the elderly: results from an influenza vaccine efficacy trial review of 10 years of clinical experience with chinese domestic trivalent influenza vaccine anflu ® influenza vaccine effectiveness in preventing hospitalization among beijing residents in china comparative epidemiology of human infections with avian influenza a h7n9 and h5n1 viruses in china: a populationbased study of laboratory-confirmed cases epidemiology of human infections with avian influenza a(h7n9) virus in china viral pathogens among elderly people with acute respiratory infections in shanghai, china: preliminary results from a laboratory-based surveillance the authors would like to thank the staff of the six sentinel hospitals in pudong new area for their assistance with throat swab acquisition and data collection. we also thank dr. luzhao feng and dr. zhibin peng from chinese center for disease control and prevention for their assistance. this study was supported by the fund of key discipline construction of health system in pudong new area (pwzx2014-14) . key: cord-261282-r1nprlne authors: chughtai, a. a.; wang, q.; dung, t. c.; macintyre, c. r. title: the presence of fever in adults with influenza and other viral respiratory infections date: 2016-10-03 journal: epidemiol infect doi: 10.1017/s0950268816002181 sha: doc_id: 261282 cord_uid: r1nprlne we compared the rates of fever in adult subjects with laboratory-confirmed influenza and other respiratory viruses and examined the factors that predict fever in adults. symptom data on 158 healthcare workers (hcws) with a laboratory-confirmed respiratory virus infection were collected using standardized data collection forms from three separate studies. overall, the rate of fever in confirmed viral respiratory infections in adult hcws was 23·4% (37/158). rates varied by virus: human rhinovirus (25·3%, 19/75), influenza a virus (30%, 3/10), coronavirus (28·6%, 2/7), human metapneumovirus (28·6%, 2/7), respiratory syncytial virus (14·3%, 4/28) and parainfluenza virus (8·3%, 1/12). smoking [relative risk (rr) 4·65, 95% confidence interval (ci) 1·33–16·25] and co-infection with two or more viruses (rr 4·19, 95% ci 1·21–14·52) were significant predictors of fever. fever is less common in adults with confirmed viral respiratory infections, including influenza, than described in children. more than 75% of adults with a viral respiratory infection do not have fever, which is an important finding for clinical triage of adult patients with respiratory infections. the accepted definition of ‘influenza-like illness’ includes fever and may be insensitive for surveillance when high case-finding is required. a more sensitive case definition could be used to identify adult cases, particularly in event of an emerging viral infection. respiratory infections are common and one of the leading causes of morbidity and mortality, particularly in the extremes of age [1] [2] [3] . influenza a and b, human rhinoviruses (hrv), respiratory syncytial virus (rsv), adenoviruses (adv) and parainfluenza virus (piv) are common respiratory viruses in adults and children [1] [2] [3] [4] [5] . of respiratory infections, influenza is the most well studied viral infection, and is commonly reported (around 50%) as the cause of epidemics of respiratory infection, including nosocomial outbreaks [6] . influenza virus is commonly isolated from febrile paediatric and elderly patients presenting with influenza-like illness (ili) and acute respiratory illness (ari) symptoms [1] . the accepted clinical case definition of ili includes fever, which may be suitable for identifying paediatric cases, but less so for adults. fever is thought of as the most common presenting symptom of influenza in hospital emergency departments; however, the presence of fever depends on the age of person and the type of virus [7] [8] [9] [10] . it is known that fever is less common in adults than children with influenza, and that adults may have atypical presentations [5, 6, 11] . in a matched case-control study in finland, 317 laboratoryconfirmed influenza cases and 353 controls with respiratory symptoms were recruited in children aged 413 years. fever was present in 89·8% (317/353) and 35·7% (126/353) of cases and controls, respectively [12] . in contrast to this, fever is not a common presentation in adults with laboratory-confirmed influenza. monto et al. [13] examined clinical trial data of 3744 adult ili cases (defined as body temperature 537·8°c or patients subjective feeling of feverishness) and of those 2470 (66%) had laboratory-confirmed influenza. fever (537·8°c) was reported in 68% of laboratory-confirmed influenza cases, compared to 40% other ili cases [13] . during a randomized clinical trial (rct) around the efficacy of facemask and hand hygiene in the household setting, 44% (15/34) of secondary cases with influenza a and 32% (8/ 25) of cases with influenza b had fever history [14] . the rate of fever was 66% (137/207) in hospitalized influenza cases in a us study [15] . another us study showed that less than half (42·4%) of healthcare workers (hcws) with laboratory-confirmed influenza presented with fever [5] . fever is a less common presenting symptom in elderly people which may lead to diagnostic and treatment delays [16] . in patients admitted with myocardial infarction, 9% had unrecognized and undiagnosed influenza on testing at admission, highlighting the low level of clinical suspicion of influenza [17] . the rate of fever also varies between influenza strains, being more common in influenza a strains than b, and higher in h3n2 [7] [8] [9] [10] 18] . fever is a commonly reported symptom during influenza outbreaks and pandemics due to novel and more virulent nature of strains. in china 67·4% of the patients infected by influenza a(h1n1)pdm09 had fever [19] . in another study in beijing 465 suspected ili cases were tested and of those 318 (68%) were positive for influenza virus (pandemic h1n1-165 and seasonal influenza h3n2-153) and all had history of fever [20] . the aim of this study was to compare the rates of fever in adult subjects with confirmed influenza and other respiratory virus infections and examine predictors of fever. we analysed a dataset of laboratory-confirmed viral respiratory infections collected from three clinical trials of hcws where active surveillance for respiratory viral illness was conducted in prospective follow up [21] [22] [23] . the same methods, data collection forms and outcome measures, were used across the three studies, allowing the data to be pooled [21] [22] [23] . two studies were conducted in beijing china: trial 1 (2008/2009) and trial 2 (2009/2010) and another study (trial 3) was conducted in hanoi, vietnam in 2010/2011 [21] [22] [23] . in all clinical trials, participants were asked to complete diary cards on a daily basis to collect information on number of working hours, patients seen, mask use hours, high-risk procedures performed and appearance of respiratory symptoms. thermometers were given the participants to measure their temperature daily and at symptom onset. symptomatic cases were asked to complete sick patient follow-up forms and detailed information was collected on the following symptoms: chill or fever, cough, congestion, runny nose, sore throat, sneezes, lethargy, loss of appetite, abdominal pain, muscle or joint aches. swabs of both tonsils and the posterior pharyngeal wall were collected on the day of reporting. in all rcts, fever was defined as having body temperature 538°c. clinical respiratory illness (cri) and ili were in the primary outcomes in three clinical trials. cri was defined as two or more respiratory symptoms or one respiratory symptom and a systemic symptom and ili was defined as fever 538°c plus one respiratory symptom [21] [22] [23] . we analysed data from all subjects with a positive isolation of a respiratory virus by multiplex polymerase chain reaction (pcr). descriptive analysis was conducted for rates of fever by virus type. a logistic regression analysis was used to determine the predictors of fever. a multivariable log binomial model was fitted, using a generalized linear model to estimate relative risk (rr). all variables were included in initial model. in the final model, we included only those variables that were significant (p < 0·25) in initial analysis. a backward elimination method was used to remove the variables that did not have any confounding effect, i.e. could not make meaningful change (±10%) in the rr of the comparison arm. finally we estimated the rates of cri and ili in the laboratory-confirmed viral respiratory infections and laboratory-confirmed influenza infections. the data was analysed using sas v. 9.4 (sas institute inc., usa). the demographic characteristics of 158 cases with laboratory-confirmed viral infections are presented in table 1 . ninety (57%) cases were from china and 68 (43%) were from vietnam. the mean age of hcws was 32·8 years and most participants were nurses (65%) and female (87%). most cases were non-smokers (92%) and had not received influenza vaccine (86%). viruses isolated included rhinovirus (n = 75, 47%), rsv (n = 28, 18%), influenza (n = 13, 8%), piv (n = 12, 8%), human metapneumovirus (hmpv; n = 7, 4%), coronavirus (n = 7, 4%) and adv (n = 1, 1%). more than one virus was isolated in 15 cases (9·5%), including nine cases with influenza co-infection. fever was documented in 23·4% cases (37/158) with a positive laboratory viral diagnosis. table 2 details rates of fever (538°c) associated with individual viral respiratory infections. hrv was the most common infection and 25·3% (19/75) of these had a fever. in 28 cases of rsv, four (14·3%) had fever; 8·3% (1/12) of piv and 30% (3/10) of influenza a cases had fever. seven cases of coronavirus and hmpv each were confirmed and of those two (28·6%) had fever. when cases with influenza and a co-infection were included, 36·4% (8/22) had fever. in univariate analysis, country, gender and smoking were significant predictors of fever. country and smoking remained significant predictors in multivariate analysis while gender became non-significant. fever rate was significantly higher in hcws in vietnam compared to hcws in china [rr 2·99, 95% confidence interval (ci) 1·24-7·20]. smokers were around five times more likely to have fever compared to non-smokers (rr 4·65, 95% ci 1·33-16·25). virus type was not associated with fever in univariate analysis; however, after adjusting for other variables, rates of fever were significantly higher in hcws co-infected with more than one virus compared to all other viruses excluding influenza (rr 4·19, 95% ci 1·21-14·52) ( table 3) . cri symptoms were present in 84·8% (137/158) of hcws with laboratory-confirmed viral infections and 90·9% (20/22) laboratory-confirmed influenza infections. the corresponding rates of ili in the two groups were 9·5% (15/158) and 13·6% (3/22), respectively. we have shown, using prospectively collected data, that the rate of fever in adults with confirmed viral respiratory infections is much lower than described in children [1, 9] . the standard clinical case definition of ili requires fever to be presentthe majority of influenza cases in this series would have been missed using the ili definition. this has implications for effective triage, early antiviral treatment and preventive measures for adults with influenza, particularly during outbreaks and pandemic situations. for other respiratory infections, clinical case definitions need to be more sensitive, or >75% of cases will be missed. the main implication for future surveillance, measurements and research studies is that the ili case definition in adults may be highly insensitive. for some types of surveillance systems, this may not be an issue, but for diagnostic screening in event of an emerging viral infection (such as for triage and implementation of infection control protocols) [24, 25] , a more sensitive case definition is needed. rates of fever in influenza and other viral respiratory infections in this study were lower compared to other studies which report fever in around 50-70% adult cases [1, 5, 13, 15] . however, this variation may be due to different study base, case definition and viral strains, as well as the prospective measurement of incident infections. many research studies use fever as an inclusion criterion for laboratory testing [13, 26] . while this may be suitable for studies in children, it is not adequately sensitive for studies of adults, as we have shown the majority of confirmed cases will be missed. the cut-off point for fever could be another factor in sensitivity. some studies have set lower cut-off points for fever, and report higher rates of fever in laboratory-confirmed influenza cases [13, 27] . carrat et al. collected data of cases presented in 35 general practices in france and collected nasal swabs from suspected influenza cases and defined fever as 537·8°c. they found fever in influenza a(h3n2), influenza a(h1n1) and influenza negative cases in 95·2%, 77·5% and 72·7%, respectively. applying a cut-off of 538·2°c, the corresponding rates are 82·2%, 59·3% and 43·9% [27] . symptoms of feverishness (subjective feeling of fever) are included in ili definitions in some cases [13] . previous studies report high rates of fever in children compared to the adults [11] . low rates of fever in adults may also be due to protection via cross-reactive antibodies due to age-dependent differences in the immunity [7, 28] . continued exposure to influenza throughout life may result in a broader protection with age. infection may provoke a stronger immune response in children with minimal to no exposure history compared to adults. therefore influenza infection history might help explain potential differences in clinical symptom severity (and presence of fever) between children and adults. a recent study reported high rates of influenza and other respiratory virus in afebrile hcws with only respiratory symptoms [5] . of 22 laboratory-confirmed influenza cases in this study, only three (13·6%) had ili symptoms, which is very low compared to other studies. in a prospective influenza surveillance study, ili symptoms were present in 48% of adults and 61% of children with laboratory-confirmed influenza virus [1] . cri symptoms were present in 90·9% (20/22) of laboratory-confirmed influenza cases in this study. a highly sensitive definition of influenza may be required to diagnose most of adult influenza cases in the clinical setting to ensure rapid treatment and isolation, and prevention of nosocomial transmission. inclusion of ili cases may overestimate the proportion of febrile cases in influenza surveillance given fever is included in the definition. pre-symptomatic and asymptomatic influenza cases will also be missed, although infectivity and transmissibility of these cases is yet to be proven [29] . longitudinal studies, where all participants are tested, provide similar estimates around rates of fever as in our study [14] . we propose a more sensitive clinical case definition without fever as a requisite criterion. clinical signs and symptoms are less studied for other viral respiratory infections, but available evidence suggests that other respiratory viruses are associated with a lower rate of fever compared to influenza [5, [30] [31] [32] [33] . putto and colleagues [30] examined the clinical records of 258 children (>3 months) in a large hospital in finland, including adv (25 cases), influenza a and b (74 cases), piv (99 cases) and rsv (60 cases). fever (539·0°c) was recorded in 68% cases with adv, 84% influenza a virus, 65% influenza b, 41% piv-1, 50% piv-2, 47% piv-3, and 52% rsv. van den hoogen and colleagues estimated the prevalence and clinical symptoms of hmpv infection, in the netherlands and fever was reported in 61% of the hmpv-positive cases [31] . in hong kong, hmpv was found in 5·5% (32/587) of children admitted in hospitals and all had fever [32] . manoha et al. examined nasal wash specimens from 931 hospitalized children and found hmpv (6%), rsv (28·5%), rhinoviruses (18·3%), influenza a (6%), piv-1 (0·2%) and piv-3 (0·3%). fever was reported in 39·2% cases with hmpv, 37·8% cases with rsv and 30·2% with rhinovirus [33] . of the 210 elderly patients with influenza and 145 with rsv, fever was reported in 65% and 50%, respectively [3] . a us study also reported low rates of fever in hcws infected with coronavirus 229e (13·5%), coronavirus hku (11·4%), coronavirus nl63 (31·3%) and rsv (12·9%) and all cases of hmpv were without fever [5] . rate of fever for all other viruses (excluding influenza) was 21·5% (28/130) in this study. co-infection with more than one virus was the strongest predictor of fever for adults with confirmed viral respiratory infections in the present study. previous studies also show high rates of fever in cases with dual respiratory viral infections compared to single viral infection [34, 35] . rates of hospitalization and icu admission are also reported to be higher in cases with dual respiratory viral infections [36] [37] [38] . increased severity of symptoms in co-infection cases might be due to an altered immune response [34] . around 10% (15/158) of cases in our dataset were infected with more than one virus. drews et al. reviewed the data of eight prospective epidemiological studies and reported the rate of co-infection was 5% [37] . studies in children generally report higher rates of co-infection cases (17-20%) [34, 35, 38, 39] . clinicians should consider the possibly of co-infection if a patient presents with fever; however, further epidemiological and clinical studies are required. smoking was also a significant predictor of fever in this study. smoking increases the risk of viral and bacterial infections through changes in respiratory epithelial and altered immune response [40] [41] [42] [43] .the risk of influenza also increases several times in smokers, compared to non-smokers [40] . atypical clinical presentation of influenza and other respiratory infections in adults could be due to altered structural and immune response associated with active/passive smoking and other environmental hazards. the mechanism by which smoking increases the risk of fever is not clear. high rates of fever in smokers may also be due changes in immunoglobulin levels which could increase viral load. the severity of symptoms generally increases when high viral load is detected in the blood [44] . the difference in fever rates between china and vietnam may be due to prevalence of viruses and co-infection. rsv was the most commonly isolated pathogen from china (31%), followed by rhinovirus (20%) and influenza virus (13%). in contrast to this hrv was the most commonly isolated pathogen from vietnam (85·3%). the number of cases with co-infection were also different in the two countries -13 (14%) in china and two (3%) in vietnam. in multivariate analysis, we adjusted for country and type of virus. limited data are available regarding the prevalent viruses circulating in china during the study period. for the trial 1 period, all influenza was influenza a(h1n1)pdm. for the trial 2 period, 21·3% were h1n1pdm, 2·9% were h3n2, 3·0% were influenza b victoria, 2·6% were influenza b yamagata, 71·2% were influenza a unsubtyped (y. zhang, beijing centre for disease prevention and control, personal communication). we could not obtain data on the viruses circulating in vietnam during the study period. there are some limitations to this study. we did not subtype the influenza strains, and studies show that the rate of fever also varies between influenza strains [7] [8] [9] [10] 18] . fever data was self-reported but self-measured in three trials using a traditional glass and mercury thermometer. lower fever rates in chinese hcws in this study might be due to due to differences in circulating viruses (and their pyrogenicity) between the two countries when the studies were conducted. a japanese study of children with influenza reported a tendency towards shorter duration of fever with increasing age in children [18] ; however, age and other demographic characteristics were not significant in that study. compared to children, this study shows that adults are less likely to have fever with a respiratory viral infection, even influenza. the implication of this finding is that for rapid treatment and reducing the risk of transmission of infection, clinicians should be aware that a diagnosis of viral respiratory infection, even influenza, is possible in the absence of fever. many of these infections are transmissible even when infected persons are asymptomatic or presymptomatic, and greater vigilance for respiratory symptoms in hcws could reduce nosocomial transmission of respiratory viral infections. the absence of fever should not preclude a differential diagnosis of influenza or other respiratory viruses in adults. influenza surveillance in communitydwelling elderly compared with children viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings respiratory syncytial virus and influenza a infections in the hospitalized elderly respiratory viruses transmission from children to adults within a household influenza among afebrile and vaccinated healthcare workers clinical manifestations and consequences of influenza tecumseh study of illness. xiii. influenza infection and disease differing virulence of h1n1 and h3n2 influenza strains influenza a and b virus infections in children differences in clinical features between influenza a h1n1, a h3n2, and b in adult patients seasonal influenza in adults and children -diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america signs and symptoms predicting influenza in children: a matched case-control analysis of prospectively collected clinical data clinical signs and symptoms predicting influenza infection viral shedding and clinical illness in naturally acquired influenza virus infections is influenza an influenza-like illness? clinical presentation of influenza in hospitalized patients fever in the elderly ischaemic heart disease, influenza and influenza vaccination: a prospective case control study natural course of fever during influenza virus infection in children clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china clinical predictors for diagnosing pandemic (h1n1) 2009 and seasonal influenza (h3n2) in fever clinics in beijing a cluster randomised trial of cloth masks compared to medical masks in healthcare workers a cluster randomized clinical trial comparing fit-tested and non-fit-tested n95 respirators to medical masks to prevent respiratory virus infection in health care workers a randomized clinical trial of three options for n95 respirators and medical masks in health workers hospital triage system for adult patients using an influenza-like illness scoring system during the 2009 pandemic -mexico pandemic influenza triage tools: user guide predicting influenza infections during epidemics with use of a clinical case definition evaluation of clinical case definitions of influenza: detailed investigation of patients during the 1995-1996 epidemic in france influenzavirus infections in seattle families, 1975-1979. i. study design, methods and the occurrence of infections by time and age does influenza transmission occur from asymptomatic infection or prior to symptom onset? fever in respiratory virus infections prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients children with respiratory disease associated with metapneumovirus in hong kong epidemiological and clinical features of hmpv, rsv and rvs infections in young children single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection single, dual and multiple respiratory virus infections and risk of hospitalization and mortality dual respiratory virus infections multiple simultaneous viral infections in infants with acute respiratory tract infections in spain multiple versus single virus respiratory infections: viral load and clinical disease severity in hospitalized children cigarette smoking and infection smoking and the outcome of infection cigarette smoke extract suppresses human dendritic cell function leading to preferential induction of th-2 priming the associations of race, cigarette smoking, and smoking cessation to measures of the immune system in middle-aged men correlation of rhinovirus load in the respiratory tract and clinical symptoms in hospitalized immunocompetent and immunocompromised patients the authors thank the staff at the beijing centre for disease control and national institute of hygiene and epidemiology. thanks are also due to the staff from the hospitals in china and vietnam which participated. 3m helped in fit testing during the first rct in china, no financial support was provided. the second rct in china was funded through the australian national health & medical research council of australia (grant no. 630787). funding to conduct the vietnam trial study was received from the australian research council (arc) (grant no. lp0990749). dr abrar chughtai had testing of filtration of masks by 3m for his phd. professor c. raina macintyre has held an australian research council linkage grant with 3m as the industry partner, for investigator-driven research. 3m have also contributed supplies of masks and respirators for investigator-driven clinical trials. she has received research grants and laboratory testing as in-kind support from pfizer, gsk and bio-csl for investigator-driven research. the remaining authors have no competing interests to declare. key: cord-263353-4mnsjbib authors: maman, issaka; badziklou, kossi; landoh, essoya d; halatoko, afiwa w; nzussouo, talla n; defang, gabriel n; tamekloe, tsidi a; kennedy, pamela j; thelma, williams; kossi, komlan; issa, zoulkarneiri; kere, abiba b title: implementation of influenza-like illness sentinel surveillance in togo date: 2014-09-20 journal: bmc public health doi: 10.1186/1471-2458-14-981 sha: doc_id: 263353 cord_uid: 4mnsjbib background: the emergence of avian influenza a/h5n1 in 2003 as well as the pandemic influenza a (h1n1) pdm09 highlighted the need to establish influenza sentinel surveillance in togo. the ministry of health decided to introduce influenza to the list of diseases with epidemic potential. by april 2010, togo was actively involved in influenza surveillance. this study aims to describe the implementation of ili surveillance and results obtained from april 2010 to december 2012. methods: two sites were selected based on their accessibility and affordability to patients, their adequate specimen storage capacity and transportation system. patients with ili presenting at sentinel sites were enrolled by trained medical staff based on the world health organization (who) case definitions. oropharyngeal and nasopharyngeal samples were collected and they were tested at the national influenza reference laboratory using a u.s. centers for disease control and prevention (cdc) validated real time rt-pcr protocol. laboratory results and epidemiological data were reported weekly and shared with all sentinel sites, ministry of health, division of epidemiology, who and cdc/namru-3. results: from april 2010 to december 2012, a total of 955 samples were collected with 52% of the study population aged between 0 and 4 years. of the 955 samples, 236 (24.7%) tested positive for influenza viruses; with 136 (14.2%) positive for influenza a and 100 (10.5%) positive for influenza b. the highest influenza positive percentage (30%) was observed in 5–14 years old and patients aged 0–4 and >60 years had the lowest percentage (20%). clinical symptoms such as cough and rhinorrhea were associated more with ili patients who were positive for influenza type a than influenza type b. influenza viruses circulated throughout the year with the positivity rate peaking around the months of january, may and again in october; corresponding respectively to the dry-dusty harmattan season and the long and then the short raining season. the pandemic a (h1n1) pdm09 was the predominantly circulating strain in 2010 while influenza b was the predominantly circulating strain in 2011. the seasonal a/h3n2 was observed throughout 2012 year. conclusions: this study provides information on influenza epidemiology in the capital city of togo. influenza-like-illnesses (ili) is a significant source of morbidity and mortality worldwide [1] . the world health organization (who) estimates that globally influenza accounts for between 3 and 5 million severe cases and 250.000 to 500.000 deaths annually, with most deaths occurring among elderly populations [2] . in temperate regions, ili is reported throughout the year with a marked increase in cases recorded during winter periods [3] . however, in tropical and subtropical regions where viral transmission occurs throughout the year, the data on the burden of influenza-like-illness are limited. nevertheless there is some evidence of a slight increase in cases during the rainy season [4, 5] . the emergence of new highly pathogenic influenza a/ h5n1 viruses in 2003 [6] , their wide circulation in wild and domestic birds and its association with human infections which involves high mortality, has raised global concern about the risk of another influenza pandemic. the emergence of novel human pandemic influenza a (h1n1) in april 2009 [7, 8] and its rapid worldwide spread has motivated the monitoring of influenza and has enhanced preparedness to counter a possible emerging pandemic. in the african region, countries in collaboration with international partners (e.g. who, cdc, namru-3, etc.) put efforts together to establish influenza surveillance capacities as part of the broader strategy for integrated disease surveillance and response (idsr) [9, 10] . while most countries in asia, north america and europe have wellestablished influenza surveillance, few such systems have been established in sub-saharan africa [5, 11] . influenza surveillance helps in understanding the epidemiology and impact of the disease; therefore providing information about seasonality and the groups at high risk of influenza infection. furthermore, the identification and characterization of circulating viruses will help to provide influenza isolates for monitoring changes in viral antigens and the development of vaccines. thus influenza surveillance provides data for pandemic influenza monitoring and planning as well as for decision-making [12] [13] [14] . in togo, the first suspected cases of human avian influenza a/h5n1 were reported between 2007 and 2008 in the maritime region, a few kilometers from the capital city lomé, which has a population of more than 2 millions. between april and december 2009, cases of ili were observed and were suspected to be pandemic influenza given the emergence of the novel human pandemic influenza a (h1n1) pdm09. with no ongoing influenza surveillance, our country was not yet ready to confirm and effectively monitor the severity of the disease. the lack of molecular laboratory technology to detect influenza viruses significantly reduced our ability to manage and control the pandemic. therefore, the ministry of health (moh) in collaboration with the institut national d'hygiène (inh) decided to add influenza to the list of diseases with epidemic potential to be monitored and reported through the idsr program. by april 2010, togo was actively involved in ili surveillance with the support of united state government through the centers for disease control (cdc) and the naval medical research unit-3 (namru-3). this study aims to describe the implementation of ili surveillance and results obtained from april 2010 to december 2012. the ili surveillance system constitutes a collaborative partnership between several togolese institutions within the ministry of health (moh). the departments involved in this surveillance are the division of epidemiology, the national influenza reference laboratory (nil) hosted by the institut national d'hygiène (inh), and the sentinel sites located at the hôpital de bè and military health services in the capital city lomé (figure 1) . a protocol for influenza surveillance was written with the technical support of cdc and namru-3 experts. the ili sentinel surveillance sites were selected based on their accessibility and affordability to patients with low socioeconomic status, the qualifications of medical staff, adequate specimen storage capacity, and an established transportation system to the national influenza reference laboratory (nil). the first site was hôpital de bè, established in april 2010 and located in district n°3. this site was chosen for its geographical location in an area of high population density and high consultation rate. this hospital hosts a pediatric unit and a general medicine ward. the second site established in december 2011, is under the management of the military health services and located in district n°5; its selection was based on the essential role of the armed forces in case of a pandemic and their ability to serve both military and civilian populations. this military site is composed of three units and is attended by military personnel, their families as well as civilians. the two sentinel sites are located in the capital city of togo where approximately 20% of the country's population lives. lomé has two rainy seasons and two dry seasons: the long rainy season (april to june) and the short rainy season (mid-september to october). the long dry season extends from december through march, while the short dry season lasts for two months (july to august). lomé is a coastal city that borders the atlantic ocean to the south, ghana to the west, benin to the east ( figure 1) and is at the crossroad with considerable commercial exchange of goods and movements of population. the who case definition [15] that was used, defined ili as "any person with a sudden onset of fever (≥38°c) and cough or sore throat accompanied or not by general symptoms such as myalgia, prostration, headache or malaise". this definition was used during 2010-2011 period. in 2012, the definition was changed to "any person with a sudden onset of fever (≥38°c) or history of fever and cough or sore throat accompanied or not by general symptoms such as myalgia, prostration, headache or malaise". at both sentinel sites, from monday to friday physicians enrolled the first two outpatients who met the case definition and samples were collected during consultation. the study population included every outpatient, between april 2010 to december 2012, presenting at any of the sentinel sites and meeting the ili case definitions regardless of age or sex and who consented to participate in the surveillance. this population represents a wide cross-section of ethnic and socioeconomic groups. samples collected were nasopharyngeal and oropharyngeal swabs and were placed in the same tube containing a viral transport medium (vtm). they were stored between 2 to 8°c at the bacteriology laboratory of the sentinel site prior to delivery to the nil within 48 hours. before samples were transported, laboratory personnel at the sentinel site conducted quality control checks of information on patients' case report forms. the nil provides the sentinel sites with logistical and material support such as swabs, viral transportation media, cryovials, cool boxes, and ice packs. a quota of 20 samples was targeted from each sites and transported twice a week (tuesday and thursday) to the nil. review meetings with all stakeholders were organized two or three times per year as part of a strategy to in 2007 at sigbéhoué (district des lacs), adétikope (district du golfe) and agodekê (district de zio). in 2008 at agbata (district des lacs). all theses foci were located at few kilometers from the capital city, lomé. improve the surveillance system by identifying strengths and areas of concern during these meetings. socio-demographic (age, sex, date of birth, residential area, travel history) and clinical (date of onset, date of consultation, previous treatment, vaccination status, co-morbidities) data were collected from all patients using a case report form (crf) during consultation. epidemiological data were stored in a single database with laboratory data using a single identification number for each patient. each week, nil provided reports on the distribution of total samples collected, as well as on the number of confirmed influenza cases to the moh, to the sentinel sites, who flunet, cdc, and namru-3. samples collected were analyzed at the national reference influenza laboratory at inh. from every sample, three aliquots were made, two of which were stored at −80°c for external quality control and further analysis (if not subtyped) at namru-3 in cairo, egypt. the other one was kept between 2 to 4°c for rna extraction followed by influenza virus detection by real time rt-pcr within 72 hours after sample reception. for the testing of influenza viruses, rna extraction was performed from 140 μl of naso and/or oro-pharyngeal cells contained in the vtm by using a qiamp viral rna mini kit (qiagen) following the manufacturer's protocol. for detection and typing, it was run on an abi 7300 machine, the real time rt-pcr using the ambion enzyme agpath one-step (ambion, applied biosystems) that amplifies influenza a and b. the u.s. cdc provided the protocol used to detect influenza viruses [16] . in order to determine the quality of the sample, the presence of human ribo-nucleoprotein (rnp) was assessed for each specimen tested. socio-demographic and clinical epidemiological data were entered into a database created using epi-info software version 3.5. data analysis was conducted using spss software version 16.0 (spss inc., chicago, il). student t-test was used for comparison of mean age and the pearson chi-square or fisher exact test to compare laboratory results by age groups and clinical symptoms. the protocol was approved by the moh as part of the monitoring of diseases with epidemic potential and therefore did not require ethical review. verbal consent was obtained from all patients. a total of 955 patients were enrolled in this study. seven hundred and twenty seven (76%) patients were enrolled from the hôpital de bè and 228 (24%) from the military health service site (table 1 ). there was no significant difference in the proportion of females compared to males enrolled in this study (49.9% vs. 50.1%; p = 0.37), and the gender distribution at the two sites was similar. most of patients (65%) were under 15 years of age, while less than 6% were 45 years or older. patients who presented at the hôpital de bè were significantly older (mean age = 17.1 years) compared to those who were seen at the military health service (mean age = 10.6 years with 71% of patients aged less than 5 years) (p = 0.0001). approximately 2% of the patients reported having received influenza vaccination within the last year. of the samples collected for ili surveillance, 236 (24.7%) tested positive for influenza viruses. of these, 136 (14.2%) tested positive for influenza a virus and 100 (10.5%) for influenza b virus ( table 2 table 2 ). the proportion of influenza positive cases varied between different age groups with a higher proportion of influenza a detected in the 15-29 year-old group (20%) than other age groups (p = 0.01; table 3 ). significantly, the pandemic influenza a (h1n1) pdm09 was more often detected in patients aged 5-14 (p = 0.003) and 15-29 (p = 0.03) years than in other age groups. seasonal a/h3n2 was predominant in patients aged 30-44 years (15%; p = 0.0003) and was the only influenza a subtype detected among patients who were 60 years or older. ili was observed throughout every year with irregular peak activity occurring twice annually during the months of; may and november in 2010; may and october in 2011; april/august, october in 2012 ( figure 2) . however, the number of patients/samples enrolled was not consistent. influenza a virus was detected predominantly in 2010 and correlated with the ili peak. the first cases of pandemic influenza a (h1n1) pdm09 were only confirmed in may. during the ili peak, the influenza positivity rate was 26% in may and 30% in november with the pandemic influenza strain, the most subtype detected. the pandemic virus remained predominant between october 2010 and april 2011 (figure 2 ). during the ili peaks in 2011, the influenza positive rate ranged from 20% to 60% with the predominance of influenza b virus activity in may representing 93% of all virus detected (28/30). the second peak was correlated to the seasonal influenza a/h3n2 activity in october with 80% of viruses detected (8/10). from october 2011, there was a co-circulation of influenza type a and type b with low activity of pandemic strain until september 2012 while the seasonal influenza a/h3n2 was detected throughout the year 2012. fever (85%), cough (87%), and rhinorrhea (76%) were the major symptoms for all age groups although sore throat (38%) and headaches (14%) were also recorded ( table 4 ). ili patients who tested positive for influenza were more likely to present with cough (p = 0.004) and headaches (p = 0.03) but were less likely to present with difficulty breathing (p = 0.02) compared to those who tested negative for the influenza virus. among symptoms, only cough was more common in patients testing positive for influenza a than those who tested positive for influenza b (p = 0.003). rhinorrhea was more common in patients with seasonal a/h3n2 than in those with pandemic influenza a (h1n1) pdm09 (p = 0.0004). due to the lack of ili surveillance in togo, there was no information about the epidemiology of ili or influenza viruses until 2010. the first samples collected were processed in may 2010 and the presence of pandemic influenza a(h1n1) pdm09 virus was confirmed in togo one year after the novel pandemic influenza occurred in mexico (april 2009). this is the first report that describes the epidemiology of influenza in togo using data from the ili sentinel surveillance system. during the two and half year period of ili sentinel surveillance, influenza viruses were detected in 236 (25%) of 955 samples. the average percentage positive in this study was higher than the positivity rate observed in 15 other african countries between 2006 and 2010 [17] . however, during the same timeframe, other countries in the temperate climate region: madagascar (40%), morocco [18] . there are several reasons to explain the difference in the percentage positive observed between countries including the temporal distribution of these viruses, the sample collection method, the number of samples collected and the geographical distribution of sentinel sites. most of our data includes post pandemic influenza a (h1n1) pdm09; this is a different picture compared to other african countries (2006) (2007) (2008) (2009) (2010) and to the south american region. the sample collection method was different from one country to another. in our study, we used two swabs (one oro-pharyngeal and one nasopharyngeal) for each enrolled patient and put both swabs in the same cryotube, thereby increasing the viral load and enhancing rt-pcr detection while in some other countries samples were collected either with nasopharyngeal [19] or oro-pharyngeal swab only [18, 20] . in addition, the number of samples tested in most of the other countries was quite high compared to our sample numbers and they were collected from many sites ranging from only 3 to as many as 22. we only used two sentinel sites as our catchment area. the influenza positivity rate varied by year with the highest rate obtained in 2010. since the number of samples collected during this year was very low (87 samples) than the two subsequent years, the rate could be influenced. nevertheless, our percentage positive was similar to that of ghana and rwanda in africa [17] and that of taiwan [21] , but at different periods of time. table 3 distribution of influenza viruses confirmed and ili patients influenza a was predominant in 2010 with pandemic influenza a (h1n1) pdm 09 in our study; this observation was similar to that of other countries in west africa [17] . however, in 2010, the situation was different in other subregions with predominance of influenza b in central/south and north africa [17, 22] and seasonal influenza a/h3n2 in east africa [17] . this difference could be explained by the fact that circulation of pandemic influenza a (h1n1) pdm09 was delayed in west africa and occurred one year after it was predominantly circulating in other african subregions [17, 23] . while two years is not sufficient time for an adequate description of the seasonality of influenza virus transmission, we did observe trends in the lomé commune region. the influenza b virus showed a peak activity during the rainy seasons (may and october) and the pandemic influenza a (h1n1) pdm09 was more frequent during the long dry season while the seasonal a/h3n2 was detected across both seasons. although the seasonality of influenza viruses in african countries is not yet clear, we observed that our trends were similar with the influenza peaks, which have often been associated with the rainy season activity in other tropical countries [24] [25] [26] . in our study, influenza cases were highest (30%) in the 5-14 year age group but also high among other age groups, with lowest percent positive (20.3%) among 0-4 and > 60 years (20.0%). this distribution is consistent with the observation in the study conducted in 15 countries of africa during 2006 to 2010 and in peru [17, 18, 20] in which young children and adults were shown to have the highest influenza viral disease. contrary to our study, a study from venezuela [27] showed higher detection rates in 0-4 year olds. the percentage positive of influenza a was significantly higher in ili patients in the 15-29 age groups. therefore, we found that pandemic influenza a (h1n1) pdm09 was detected significantly among 5-29 years old. this finding is consistent with other studies in the african region [17, 22, 28] that have found that pandemic influenza a (h1n1) pdm09 is most commonly identified in school-age children and young adults. while pandemic influenza a (h1n1) pdm09 appeared more often in older children, seasonal influenza a/h3n2 appeared more likely to infect adults in the 30-44 year-age category. our finding was similar to the observation from a study conducted in peru [18] , where the author found that the seasonal influenza a/h3n2 virus was detected with adults of 45 to 59 years. in conclusion, our results are consistent with studies from africa and south american regions which observed that seasonal influenza a/h3n2 affected a wide range of age groups with predominantly 30 to 60 years old while the pandemic influenza a (h1n1) pdm09 and influenza b virus infections occurred more frequently among older children and young adults. we observed that clinical symptoms were associated with influenza viruses. the influenza type a was more frequently detected than type b in patients presenting with cough and rhinorrhea. this result is consistent with the observation of a study from venezuela [27] . in contrast with this study were pandemic influenza a (h1n1) pdm09 was associated with ili patients with cough, our study showed that ili patients with rhinorrhea were associated with seasonal a/h3n2. our study had some limitations. our data were collected only from 2 sites in an urban area in the capital city of togo and could not be generalized to the population. the percent influenza positivity and age distribution of positive cases were influenced by the low number of samples collected which may be attributable to the non availability of a physician to collect nasal and orpharyngeal swabs. the low number of samples may have also contributed to the high positivity rate. in addition, this low proportion may not be representative to better describe the distribution of influenza cases in the age groups. physician time limitations were due to the time consumption and their workload (number of patients viewed in consultation at the outpatients' department). children were over-represented in this study thus introducing a bias, as the number of adults was not comparable to children under 5 years old. some possible reasons to explain this bias include the fact that the military health service has three units but only the family health care center was functional when added as a site in december 2011. because this unit is a pediatric health center, the high number of children enrolled from this site can account for the observed figures. at the hôpital de bè site, we observed that many patients, mostly adults were not enrolled as ili patients due to the lack of recorded fever (≥38°c), suggesting that we should be considering history of fever as one of the enrollment criteria for ili. this study focused exclusively on outpatients thus limiting our ability to examine the severity of the influenza viruses in hospitalized cases. since our influenza surveillance system had challenges in collecting samples of severe acute respiratory infection (sari) and the lack of data on hospitalizations with patient follow-up we excluded discussions on sari from this study. to improve our influenza surveillance system, it will be necessary to expand the system in other regions by including sari surveillance for severe disease to give a complete picture of influenza burden and epidemiology in our country. these data provided information on the epidemiology of influenza in the lomé commune region in the capital city of togo. some efforts are needed to allow better understanding of influenza burden and epidemiology by expanding sentinel sites in other regions and including sari surveillance. future studies will also be focused on identifying the etiologic agents for the 75% of ili cases that were negative for influenza viruses. retrospective analyses of these stored samples will be necessary to identify other respiratory viruses circulating, including respiratory syncytial virus (rsv), coronaviruses, human metapneumovirus (hmpv) and rhinoviruses. who: the global burden of disease: 2004 update who: influenza fact sheet available the global impact of influenza on morbidity and mortality seasonality of influenza in the tropics: a distinct pattern in northeastern brazil epidemiology and seasonality of respiratory tract virus infections in the tropics who: affected areas with confirmed cases of h5n1 avian influenza who: pandemic (h1n1) epidemiology and factors associated with fatal cases preparedness for highly pathogenic avian influenza pandemic in africa who regional office for africa: integrated disease surveillance in the african region: a regional strategy for communicable diseases influenza in africa: uncovering the epidemiology realities and enigmas of human viral influenza: pathogenesis, epidemiology and control who: recommended composition of influenza virus vaccines for use in the 2013 southern hemisphere influenza season manual for the laboratory diagnosis and virological surveillance of influenza who: cdc protocol of real time rt-pcr for detection and characterization influenza 2009 a (h1n1) pdm virus influenza surveillance in 15 countries in africa influenza-like illness sentinel surveillance in peru influenza surveillance among children with pneumonia admitted to a district hospital in coastal kenya challenges of establishing routine influenza sentinel surveillance in ethiopia nationwide surveillance of influenza during the pandemic (2009-10) and post-pandemic (2010-11) periods in taiwan sentinel surveillance for influenza-like illness, severe acute respiratory illness and laboratory-confirmed influenza in kinshasa, democratic republic of congo pandemic influenza a virus subtype h1n1 circulation in west africa epidemiological and virological influenza survey in dakar, senegal: 1996-1998 influenza in outpatient ili case-patients in national hospital-based surveillance seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics sentinel surveillance of influenza-like illness in two hospitals in influenza viruses in nigeria, results from the first 17 months of a national influenza sentinel surveillance system implementation of influenza-like illness sentinel surveillance in togo we would like to thank all sentinel staff at the hôpital de bè and the military health services for their essential role in the ili sentinel surveillance. we are grateful to the ministry of health and the division of epidemiology for their support and coordination. we would like also express our sincere thanks to the team of the national influenza reference laboratory for their efforts in collecting samples, clinical data and detection by rt-pcr of influenza viruses. we wish to thank mr koffi akolly, field epidemiologist for designing the map of the figure 1 . the influenza sentinel surveillance was successful established with the technical and financial support of cdc and namru-3. we are grateful also to cdc reviewers for their precious analyses and revision of this paper prior it's submission for publication. the authors have declared that no competing interests exist.authors' contributions im contributed to the study design, statistical analyses of data and wrote the paper. kb and edl contributed to the study design, interpreted analysis and review the manuscript. awh and tat contributed in the study design and review the manuscript. tnn, gnd, wt and pjk provide technical advice for study protocol, methodology, revised critically the manuscript for important scientific content and have given final approval for the version to be published. zi and kk were involved in literature review and revising the manuscript. abk contributed to the facilitation of the project, participated in its design, coordination and review the paper. all authors read and approved the final manuscript. key: cord-260690-h5pjv2dw authors: druce, julian; tran, thomas; kelly, heath; kaye, matthew; chibo, doris; kostecki, renata; amiri, abdul; catton, mike; birch, chris title: laboratory diagnosis and surveillance of human respiratory viruses by pcr in victoria, australia, 2002–2003 date: 2004-11-12 journal: j med virol doi: 10.1002/jmv.20246 sha: doc_id: 260690 cord_uid: h5pjv2dw respiratory viruses were identified by the polymerase chain reaction (pcr) in more than 4,200 specimens collected during 2002 and 2003 in victoria, australia from patients admitted to hospitals or participating in an influenza surveillance program. influenza viruses and picornaviruses were important causes of morbidity in both years. additional testing of picornavirus‐positive samples suggested that rhinoviruses but not enteroviruses were more likely to be associated with respiratory symptoms, irrespective of the season in which they circulated. detection of influenza viruses was strongly associated with the clinical symptoms of cough, fever, and fatigue; but each of the other respiratory viruses occasionally caused these symptoms or was responsible for symptoms severe enough to require hospitalization. human coronaviruses hcov‐oc43 and hcov‐229e circulated at low levels throughout the study period with peak activity in winter, but overall did not circulate as widely as has often been reported for these agents. evidence for the human metapneumovirus (hmpv) was only sought in the second year of the study and revealed low‐level circulation of this virus, mainly in the cooler months among the very young and adult populations. the detection rate of all viruses declined with increasing age of the patient, particularly in hospital patients. infection with more than one respiratory virus occurred in a small number of patients; picornaviruses were most commonly implicated in these dual infections. j. med. virol. 75:122–129, 2005. © 2005 wiley‐liss, inc. viruses are known to be common causes of respiratory illness throughout the world, with seasonal variation that is more marked in temperate rather than tropical climates. these viruses cause illnesses that can range from a brief upper respiratory tract infection to severe systemic illness resulting in death. the risk of infection is often related to age. influenza, for example, is known to have its highest attack rates amongst the very young and the very old [mcintyre et al., 2002] . respiratory syncytial virus (rsv) affects mainly the young, although less typical infections amongst adults are recognized [hall, 2001] . the risk of infection also depends on pre-existing medical conditions and immunocompetence. historically, laboratory diagnosis of respiratory infection has been slow because of its reliance on virus isolation or serological techniques. the use of immunofluorescence-based assays has improved turnaround times, but a nasopharyngeal aspirate is required for optimal results and the method suffers from a limited number of viruses being detectable with the reagents available. the availability of new classes of drugs active against both influenza a and b viruses , preclinical and clinical trials of drugs targeted at respiratory viruses other than influenza [hayden et al., 2003; cianci et al., 2004; uckun et al., 2004] , and the potential for new vaccines [power et al., 2001] has encouraged further a shorter diagnostic turnaround time for the detection of respiratory viruses. an increasing number of diagnostic laboratories use polymerase chain reaction (pcr)-based assays to rapidly identify a wide range of viruses in specimens collected from a variety of respiratory sites [billaud et al., 2003; echavarria et al., 2003; vuorinen et al., 2003] . the results of a 2-year retrospective study of respiratory viruses in more than 4,200 specimens obtained from individuals living in victoria, australia are presented. the seasonality of the viruses detected, their occurrence by age group, their presentation in hospitalized patients compared to those participating in an influenza surveillance program, and the occurrence of dual infections were investigated. specimens were received from individuals included in a victoria-wide influenza surveillance program operating between may and september each year [watts et al., 2003] or from patients who had been admitted to victorian hospitals with a respiratory illness (or acquired their infection nosocomially during their admission) ( table i) . clinical material sent for laboratory testing from hospitalized patients included nose swabs, throat swabs, nasopharyngeal aspirates (npas), endotracheal aspirates, bronchoalveolar lavages (bals), bronchial washings, lung biopsies, sputum samples, and pleural fluids. nose and throat swabs (nts) pooled into viral transport medium were received from surveillance patients. they were only taken from individuals with symptoms of 3 days or less of fever, cough, and fatigue. when multiple samples from a single patient were sent for testing, only one result was included in the analysis. a total of 333 additional respiratory specimens, including 20 from asymptomatic laboratory staff was used to validate the pcr assays for influenza a virus (h1 and h3 subtypes), influenza b virus, rsv, parainfluenza viruses (at least one of types 1-3), picornaviruses (a mixture of enteroviruses and rhinoviruses), and adenoviruses (each of different serotype) (table iii) . pcr assays were developed and validated on clinical material (nts, npas, and bals) in parallel with the existing techniques of virus isolation [lewis and kennett, 1976] and/or immunofluorescence (performed using a bartels viral respiratory screening and identification kit (trinity biotech, county wicklow, ireland) ). the process included the design and evaluation of primers; optimization of nucleic acid extraction conditions; establishment of optimum pcr amplification conditions; evaluation of applicable specimen types; determination of assay sensitivity compared to conventional assays; specificity testing using clinical material likely to be negative (including asymptomatic staff volunteers); or material previously shown to be positive for respiratory viruses by conventional assays or by sequencing of an amplified product where no other confirmatory method was available. because of the lack of immunofluorescence and routine isolation methods to confirm pcr positivity for human metapneumovirus (hmpv), human coronavirus type oc43 (hcov-oc43), and human coronavirus type 229e (hcov-229e), sequencing of the pcr-amplified products of these viruses was carried out with the same second round primers used to derive the product. sequences obtained were confirmed by comparison with those in the national center for biotechnology information (genebank) database. prior to testing, each specimen was spiked with a low copy number of bovine diarrheal disease virus (bvdv) to act as an internal control for the nucleic acid extraction, reverse transcription, and pcr amplification steps. the approach of using a non-human virus as an internal inhibition control has been reported previously [druce et al., 2002] . viral nucleic acid was extracted using one of two commercially available methods. the preferred method used a magnapure lc automated extraction robot with a total nucleic acid isolation kit (roche diagnostics, mannheim, germany). this involved the extraction of viral nucleic acid from 200 ml of the clinical material in viral transport medium, with elution into a final volume of 50 ml of the supplied elution buffer. specimens containing inhibitors of the pcr as shown by failure of the bvdv internal control to amplify were reextracted through a column (highpure viral nucleic acid extraction column; roche diagnostics) and retested. ten microliters of rna was linearized for 10 min at 658c, added to 12 ml of reverse transcription master mix consisting of random hexamers (roche diagnostics), dntps (amersham biosciences, buckinhamshire, uk), and amv-rt enzyme and buffers (promega, madison, wi) and incubated for 30 min at 428c followed by 10 min at 1008c. the cdna product was stored at 48c until tested. adenovirus dna extracted by either of the above methods survived this process (results not shown). a panel of nested pcr assays capable of detecting 11 respiratory viruses was developed using primers reported by others or designed in-house. tube 1 included influenza a virus (h1 and h3 subtypes) [zhang and evans, 1991] , influenza b virus [zhang and evans, 1991] , adenoviruses [allard et al., 1992] , and the bvdv internal control [cleland et al., 1999] . tube 2 included rsv [osiowy, 1998 ] and picornaviruses (with primers specific for all enteroviruses and rhinoviruses) [ireland et al., 1993] . tube 3 included parainfluenza virus types 1, 2, and 3. tube 4 included hcov-229e and hcov-oc43, and tube 5 included hmpv only. when required, a separate pcr assay was used to distinguish enteroviruses from rhinoviruses when the picornavirus component of tube 2 was positive [zoll et al., 1992; steininger et al., 2001] . a negative control of nuclease-free water was included in every assay and a virus positive control consisting of low copy number cdna (or dna for adenovirus) included in the relevant pcr targeting that virus. the primers used for first and second round amplifica-tions, and their gene/product targets, are listed in table ii . for first round amplification, 3 ml of cdna template was added to 40 ml of a mastermix containing 500 nm first round primers (100 nm for the picornavirus primers), 1.8 mm mgcl 2 , 2 mm dntps and 0.3 u of taq polymerase (qiagen, hilden, germany). the first round pcr conditions consisted of one cycle of 948c for 5 min followed by 35 cycles of 948c (30 sec), 558c (30 sec), 728c (60 sec), then 728c for 5 min. the conditions for tube 2 were reduced to 20, 20, and 30 sec, respectively, for cycles 2-36. for second round amplification, 2 ml of the first round product was transferred to fresh mastermix containing second round primers. the second round pcr amplification conditions were the same as for round 1 except the 35 cycle component for tube 2 was reduced to 25 cycles and the annealing temperature reduced to 508c. separation of amplified material and molecular weight markers (roche diagnostics) was performed by electrophoresis for 30 min at 80 ma on a 2% agarose gel prestained with ethidium bromide. gels were photographed using a gel doc 2000 (biorad, hercules, ca). a total of 4,254 patient specimens were examined in 2002 and 2003 (table i) . in both years, more specimens were received from hospitalized patients than from those participating in the influenza surveillance program conducted during may-september. there were more specimens received from females (53.6%) than males (46.4%) participating in the surveillance program. conversely, 59.7% of specimens from hospitals were from male patients. the overall respiratory virus detection rate was higher in specimens received from surveillance patients than hospital patients in both years (54% and 49% in 2002 and 2003, respectively, for surveillance patients compared to 30% and 33%, respectively, for hospital patients). experiments carried out during the development and validation process showed that it was not possible to incorporate all the required primers into a single tube assay without significant loss of sensitivity for some viruses (results not shown). overall, the pcr assays developed were able to detect at least 0.1 tissue culture infectious dose (50%) using the methods described (results not shown). the sensitivity of the influenza a virus and influenza b virus pcr components was 86% and 87%, respectively, compared to the combined conventional assays of immunofluorescence and virus isolation (table iii) . however, additional testing using matrix specific primers (not shown) confirmed that the influenza virus pcr positive/conventional test negative samples were true positives. the true sensitivity for influenza a was therefore 89.5% (95% ci, 66.9-98.7) and specificity 100% (95% ci, 98.9-100). for influenza b the sensitivity was 90.0% (95% ci, 67.1-98.8) and specificity 100% (95% ci, 98.9-100). a total of 44 specimens were positive for picornaviruses by pcr but negative by virus isolation, the only conventional test available. positive results for picornaviruses were confirmed using enterovirus-and rhinovirus-specific primers, giving an assay sensitivity for picornaviruses of 100% (95% ci, 92.5-100) and specificity 100% (95% ci, 98.7-100). although based on small numbers in some instances, the sensitivity of the pcr assays for each of rsv, parainfluenza viruses, and adenoviruses was 100% (table iii) . the 20 specimens collected from asymptomatic volunteer staff were negative in each of the pcr assays. figure 1 . all viruses for which a pcr was available were detected during this time. overall, respiratory viruses were detected in 1,583 (37%) of 4,254 specimens received for testing. the virus detection rates for 2002 and 2003 during the surveillance period (may-september) and the official winter months of june-august are shown in table iv . a greater proportion of viruses were detected in surveillance patients in both years during these overlapping times. the average winter detection rate of 40% in hospitalized patients decreased to 22% in the summer of 2002/3. influenza a virus was the most common virus detected (fig. 1) . discrete winter peaks occurred in both years, with activity for this virus being greater in 2003 than 2002. identification of influenza a virus was rare outside the peak incidence times of may-september. picornaviruses were the second most common virus detected after influenza a virus (fig. 1) and were detected in every month of the year, although they were most common in the cooler months. although the screening pcr did not discriminate between rhinoviruses and enteroviruses, a subgroup of 36 picornavirus-positive specimens, 10 collected during august 2003 (winter) and 26 collected during december 2002 and january-february 2003 (summer), were rhinovirus positive when retested using a genus-specific pcr. rsv circulated in discrete winter peaks in both years, with little activity outside these times (fig. 1) . the circulation of coronaviruses was similar to rsv, although the peak incidence in both years was approximately one month later for the coronaviruses (compare fig. 1b and c) . the most commonly detected coronavirus was hcov-oc43, with sporadic appearance of hcov-229e mainly in autumn and spring. parainfluenza viruses were detected in low numbers throughout both years, with a tendency for increased detection validation of the hcov-oc43, hcov-229e, and hmpv assays was by sequencing of the amplified product as described in the ''patients and methods.'' b when these are included as true positives (see ''results''), the sensitivity for influenza a was 89.5% (95% ci, 66.9-98.7) and specificity 100% (95% ci, 98.9-100); sensitivity for influenza b was 90.0% (95% ci, 67.1-98.8) and specificity 100% (95% ci, 98.9-100); and sensitivity for picornaviruses was 100% (95% ci, 92.5-100) and specificity 100% (95% ci, 98.7-100). in october and november. adenoviruses were detected in low numbers throughout the year. in 2003, when testing was available, hmpv detection was highest in the cooler months of june-september. the proportions of each of the respiratory viruses detected according to age-groups and whether the spe-cimens were tested as part of the influenza surveillance program or for hospital patients is shown in table v . irrespective of the source of the specimens, individuals aged 10 years and under were more likely to have a respiratory virus detected. in adults, the rate of detection decreased with increasing age and this was more pronounced in the hospitalized patients. the most common viruses detected in hospital patients, irrespective of age, were influenza a and picornaviruses. in hospitalized children under 6 years of age, and hospitalized adults, the picornavirus detection rate was higher than that for influenza. in surveillance patients, influenza a and b viruses together accounted for at least 60% of the viruses detected in all age groups. sixty three patients (4%) were infected with 2 viruses. the most common viruses implicated in these dual infections were picornaviruses and adenoviruses (table vi) . picornaviruses were present with another virus in 37 patients (representing 59% of the cases of dual infection) while adenoviruses were present in 21. these two viruses were detected together in 12 specimens. there was insufficient clinical information supplied to assess the influence of dual infections on the degree of morbidity compared to patients infected with a single virus. this study reports virological testing of specimens from hospitalized patients and community-based patients who were part of victoria's annual winter influenza surveillance program. the overall detection rate was influenced by the source of specimen referral, with a higher proportion of specimens from surveillance than hospitalized patients yielding a recognized respiratory virus. the introduction of nucleic acid detection techniques into the diagnostic laboratory has decreased the time to diagnosis of many viral infections and also enabled the detection of one or more pathogens in a single specimen using individually directed pcrs or multiplexed assays [billaud et al., 2003; echavarria et al., 2003; vuorinen et al., 2003] . these assays have also increased the proportion of specimens in which an aetiologic agent can be detected, as evidenced by the recent discoveries of hmpv [van den hoogen et al., 2001] and coronavirus hcov-nl63 [van der hoek et al., 2004] , agents which do not replicate reliably in cell lines used in many diagnostic laboratories. as expected in our study, influenza viruses were the most common agents identified, although viruses other than influenza sometimes caused an influenza-like illness. picornaviruses were an important cause of morbidity in both hospital and surveillance patients. although the picornavirus pcr detected both enteroviruses and rhinoviruses, all the picornavirus-positive specimens that were analysed further, including those detected during summer, were identified as rhinoviruses. although some pcr-based assays have (22) n/a n/a n/a n/a n/a n/a year 1,414 427 (30) 1,681 557 (33) the combined detection rate for each of the viruses from both hospital and influenza surveillance patients is shown in the last row. indicated a low asymptomatic carriage rate for picornaviruses [johnston et al., 1993] , it is likely that the rhinoviruses detected by the current assays are the causative agents of the illness. further studies are needed to analyze the severity of clinical illness associated with dual infections involving rhinoviruses. however, the ability of rhinoviruses to produce symptoms sufficiently severe to be mistaken for influenza infection or sometimes requiring hospitalization is noteworthy. of the viruses associated regularly with lower respiratory tract infection, rsv was the most common. although rsv was detected most commonly in hospitalized children as expected, it was also detected regularly in adults. rsv has been shown previously to contribute to community and hospital cases of influenza-like illnesses [zambon et al., 2001] . the role of the newly identified hmpv was investigated only in 2003. this virus was associated mainly with hospitalized cases, suggesting that it may be clinically distinguishable from influenza infection in the community setting. it was less common in older children and teenagers than in infants, young children and adults. a similar epidemiological pattern has been reported in other parts of the world [falsey et al., 2003] , but more detailed studies on the epidemiology of this virus are warranted. coronaviruses, which have been reported to cause up to 35% of all cases of the common cold [mcintosh, 1997] , and are associated with hospitalizations among older adults [falsey et al., 2002] , accounted for a few cases of influenza-like illness, particularly in younger children, but otherwise were present in only 6% of surveillance patients and 12% of hospitalized patients. the role of hcov-oc43 in causing discrete outbreaks in elderly residents of aged-care facilities has been described [birch et al., in press] , and further studies on the impact of the newly recognized hcov-nl63 are needed. because the diagnostic testing algorithm involved multiple targets, it was anticipated that some samples would contain more than one virus. most of the viruses were identified on a prolonged background of adenovirus and picornavirus activity throughout the 2 year period. it is not surprising therefore that these viruses were most commonly detected as part of dual infections. rhinoviruses have been previously shown to occur commonly with rsv in cases of acute bronchiolitis in infancy and to increase the risk for severe disease [papadopoulos et al., 2002] . the availability of pcr technology now provides the opportunity for prospective studies on the epidemiology and clinical impact of dual infection with respiratory viruses. because of the lack of clinical information supplied at the time of specimen receipt, we were unable to link many of the virological findings with clinical symptoms or outcome for hospitalized patients. however, the influenza surveillance program shows that other viruses, especially rhinoviruses, are often associated with symptoms of cough, fever, and fatigue. we found relatively low levels of coronaviruses in both years, in contrast to previous reports. this study contributes to the demonstration of the importance of pcr in the investigation of the epidemiology and laboratory diagnosis of respiratory viral infections. pcr assays provide an opportunity to identify new viruses and detect those which do not replicate reliably in cultured cells. they also provide a rapid and sensitive means of diagnosis which may impact on the treatment and duration of hospitalization in many cases. hcov-oc43 hmpv adenovirus ---12 1 3 4 1 influenza a ---7 6 1 4 -influenza b ---4 ----picornavirus ----4 7 3 4 rsv ----1 ---parainfluenza -----1 -1 hcov-oc43 --------hmpv ------- detection of adenoviruses in stools from healthy persons and patients with diarrhoea by two-step polymerase chain reaction detection of rhinovirus and enterovirus in upper respiratory tract samples using a multiplex nested pcr human coronavirus oc43 causes influenza-like illness in residents and staff of aged care facilities in melbourne orally active fusion inhibitor of respiratory syncytial virus use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis c virus effectiveness of neuraminidase inhibitors in treatment and prevention of influenza a and b: systematic review and meta-analysis of randomized controlled trials utility of a multiplex pcr assay for detecting herpesvirus dna in clinical samples rapid detection of adenovirus in throat swab specimens by pcr during respiratory disease outbreaks among military recruits rhinovirus and coronavirus infection-associated hospitalizations among older adults human metapneumovirus infections in young and elderly adults respiratory syncytial virus and parainfluenza virus efficacy and safety of oral pleconaril for treatment of colds due to picornaviruses in adults: results of 2 double-blind, randomized, placebocontrolled trials improved detection of rhinoviruses in nasal throat swabs by seminested rt-pcr use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms comparison of rhinovirus-sensitive hela cells and human embryo fibroblasts for isolation of rhinoviruses from patients with respiratory disease clinical virology vaccine preventable diseases and vaccination coverage in australia direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical specimens by multiplex reverse transcription-pcr assay association of rhinovirus infection with increased disease severity in acute bronchitis safety and immunogenicity of a novel respiratory virus vaccine (bbg2na) in healthy young adults early detection of acute rhinovirus infections by a rapid reverse transcription-pcr assay phenyl phosphoramidated derivatives of stavudine as anti-hiv agents with potent and selective in vitro antiviral activity against adenovirus a newly discovered human pneumovirus isolated from young children with respiratory tract disease identification of a new coronavirus five years' experience of reverse transcriptase polymerase chain reaction in daily diagnosis of enterovirus and rhinovirus infections establishing thresholds for influenza surveillance in victoria contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study pcr detection and differentiation of influenza virus a, b, and c strains general primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections key: cord-279615-yne753y6 authors: jelley, lauren; levy, avram; deng, yi‐mo; spirason, natalie; lang, jurissa; buettner, iwona; druce, julian; blyth, chris; effler, paul; smith, david; barr, ian g. title: influenza c infections in western australia and victoria from 2008 to 2014 date: 2016-07-23 journal: influenza other respir viruses doi: 10.1111/irv.12402 sha: doc_id: 279615 cord_uid: yne753y6 background: influenza c is usually considered a minor cause of respiratory illness in humans with many infections being asymptomatic or clinically mild. large outbreaks can occur periodically resulting in significant morbidity. objectives: this study aimed at analyzing the available influenza c clinical samples from two widely separated states of australia, collected over a 7‐year period and to compare them with influenza c viruses detected in other parts of the world in recent years. patients/methods: between 2008 and 2014, 86 respiratory samples that were influenza c positive were collected from subjects with influenza‐like illness living in the states of victoria and western australia. a battery of other respiratory viruses were also tested for in these influenza c‐positive samples. virus isolation was attempted on all of these clinical samples, and gene sequencing was performed on all influenza c‐positive cultures. results and conclusions: detections of influenza c in respiratory samples were sporadic in most years studied, but higher rates of infection occurred in 2012 and 2014. many of the patients with influenza c had coinfections with other respiratory pathogens. phylogenetic analysis of the full‐length hemagglutinin–esterase–fusion (he) gene found that most of the viruses grouped in the c/sao paulo/378/82 clade with the remainder grouping in the c/kanagawa/1/76 clade. influenza c is often ignored compared with the other human pathogens in the orthomyxoviridae family (influenza a and influenza b). with respect to other influenza types, it causes a milder disease with fewer complications and, as it has not been included in routine testing, there is little new information being generated about its impact. in etiologic studies of respiratory illnesses that have included influenza c testing, it usually accounts for a low proportion of acute respiratory pathogens identified. for example, a canadian study 1 found 2.32% of respiratory samples tested from children identified influenza c, while a study in japan spanning 18 years 2 (1996-2013) found influenza c in 0.43%-1.73% of samples from children; this result was similar to a spanish study that reported influenza c in 0.7% of children's samples. 3 low rates were reported in a retrospective study scottish study that screened 3300 respiratory samples collected from children and adults during the period from august 2006 to june 2008 with only six positive influenza c-positive samples (0.2%) identified (with 4/6 from children ≤2 year) compared to 3.2% influenza a detections and 0.9% influenza b detections. 4 higher rates of influenza c infection have been reported in a nigerian study, where 4.8% of the respiratory samples from children were positive for influenza c 5 and finnish study among young adult male military recruits, 6 in which influenza c was identified in 4.2% of all samples. influenza c can also be identified in a significant proportion of children hospitalized with lower respiratory tract infections, as reported by shimizu et al. 7 2015, who found approximately 10% of children had influenza c present in samples collected from four japanese hospitals during 2009-2010. often these cases of influenza c also have other viruses or bacteria detected in the specimen making it difficult to attribute the contribution of influenza c infections to the clinical illness manifestations. 5, 8 in contrast to most viral etiology studies, serological studies often find high seroprevalence rates in the community which rise rapidly up to 10 years of age to around 50%-60% of the population, indicating that widespread transmission has occurred among children in many different countries. 9 unlike influenza a and b infections where early treatment with neuraminidase inhibitor drugs can be effective, there are no effective antiviral treatments for influenza c. this study analyzed influenza c viruses detected in respiratory samples collected from two influenza illness surveillance programs operating in the state of western australia (wa) from 2001 to 2014: one covering patients of all ages presenting to general practitioners with an influenza-like illness (ili), and the other covering young children presenting with a respiratory illness to a metropolitan pediatric hospital emergency department or a general practitioner. these were compared with influenza c viruses detected from routine screening of respiratory samples from melbourne, victoria from 2011 to 2014. this report is the first to isolate and characterize (including sequencing) influenza c viruses collected in australia. isolation of influenza c viruses was performed using madin-darby canine kidney (mdck) cells (atcc ccl-34) using maintenance media (dmem coon's basal media containing sodium bicarbonate (3%) with the addition of 2 mmol/l glutamine, 1% non-essential amino acids, 0.05% nahco3, 0.02 mol/l hepes, 4% penicillin and streptomycin, 2 μg/ml amphotericin b and 4 μg/ml trypsin). samples were incubated for up to 5 days at 33°c without co 2 , and virus growth was quantified by determining the hemagglutination titer (ha) with 0.5% fowl and turkey rbc. 8,12 prior to sequencing or culturing, due to the age of some of the clinical samples, a real-time pcr assay was designed and run so that samples could be retested at the who centre to confirm that viral rna was still detectable. primers and a minor groove binding probe were designed using an alignment of influenza c hemagglutinin-esterase-fusion (he) gene sequences from genbank. sequences were downloaded and aligned using the megalign program of dnastar (madison, wi, australia), and he primers and probes were obtained (geneworks, adelaide, sa, australia) and used for the real-time pcr of clinical samples and also for the confirmation of influenza c virus isolates (table 1) jelley et al. purified rna extracted from influenza c-positive samples was amplified using the bioline mytaq one-step rt-pcr kit (bioline, australia) using in-house-designed he gene-specific primers supplied by bioneer (melbourne, australia; table 1 ). rt-pcr was per for the 16 influenza c-positive samples from the spn(wa) gp surveillance, the age range was 1-40 years with the median age of 22 years (25% male, 75% female), while for the 63 influenza c-positive waive samples the age range was 0.6-5.1 years with a median age of 1.7 year (46% male, 54% female). note that the waive program only recruits children aged between 6 months and 59 months who attended the emergency department or were admitted to the princess margaret hospital, perth (wa) with an ili (defined by the presence or history of fever and a respiratory symptom). this study group was therefore made up exclusively of young children and they had higher rates of influenza c than the spn(wa) group (table 1) . during the waive study, the overall prevalence of influenza c cases was 1.6% and varied from 0% (2009) table 3 ). the most common multiple infections along with influenza c were with human enteroviruses/ rhinoviruses (13 samples), influenza a (seven samples; four with influenza a h1n1pdm09 and three with influenza a(h3n2)) followed by rsv (six samples) and adenovirus (six samples) ( hemagglutinin-esterase-fusion gene sequence was obtained for all samples that yielded influenza c virus isolates. thirty-five influenza c full he sequences (28 from perth/wa and seven from victoria) and one partial he sequence from perth were obtained from the virus isolates. these sequences were compared with publically available he sequences from reference viruses including those that represent the six antigenically distinct influenza c groups from recently circulating influenza c viruses (fig. 1) victorian samples were evenly distributed in both of these clades (fig. 1) . the bootstrap values for all of these clade assignments were high. the this is the first report characterizing influenza c viruses obtained from australia. influenza c viruses were detected sporadically in two widely in the waive study, 11.1% vs 16.8%, respectively. the frequency of large influenza c epidemics in australia is unknown, but looking at the waive data (table 2 ) there is some indication that they may occur approximately every 2 years, as the influenza c cases detected were the highest in 2010, 2012, and 2014. in japan, it also appears that influenza c epidemics could be detected every couple of years. 14 overall, we were moderately successful in isolating influenza c, with 51.5% of original clinical samples yielding isolates. these isolates and 2012 from children of less than 10 years of age. in a paediatric clinic of the university of milan, a study over 4 years (2008-9 to 2011-2) in children under 15 years of age with influenza c and radiographically confirmed community-acquired pneumonia, five cases were identified with two viruses of the c/kanagawa/1/76-lineage and three from the c/sao paulo/378/82-lineage. 18 clearly, viruses from these two influenza c lineages have been the dominant clades seen in many countries in recent years even though they appear to have changed very little based on their he phylogeny, since they were first reported. unfortunately, our sample size was not large enough to be able to gain an in depth understanding of influenza c within australia. nor can it determine the level of reassortment that has occurred as the six internal genes have yet to be sequenced. however, odagiri et al. detection of influenza c virus by real time rt-pcr assay. influenza other respir viruses epidemiological information regarding the periodic epidemics of influenza c virus in japan (1996-2013) and the seroprevalence of antibodies to different antigenic groups prospective study of influenza c in hospitalized children detection of influenza c virus but not influenza d virus in scottish respiratory samples specific viruses detected in nigerian children in association with acute respiratory disease influenza c virus infection in military recruits-symptoms and clinical manifestation influenza c virus and human metapneumovirus infections in hospitalized children with lower respiratory tract illness isolation and characterization of influenza c viruses in the philippines and japan influenza c virus high seroprevalence rates observed in 3 different population groups effectiveness of trivalent flu vaccine in healthy young children the detection and multiplication of influenza c virus in tissue culture new algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of phyml 3.0 the dominant antigenic group of influenza c infections changed from c/sao paulo/378/82-lineage to c/kanagawa/1/76-lineage in yamagata isolation of influenza c virus recombinants hemagglutinin-esterase-fusion (he) protein of influenza c virus phylogenetic analysis and seroprevalence of influenza c virus in mie prefecture, japan in 2012 influenza c virus-associated community-acquired pneumonia in children. influenza other respir viruses the melbourne who collaborating centre for reference and research on influenza is supported by the australian government department of health. the authors thank all staff from pathwest additional supporting information may be found online in the supporting information tab for this article. key: cord-260728-4w23kwzu authors: timmermans, ans; melendrez, melanie c.; se, youry; chuang, ilin; samon, nou; uthaimongkol, nichapat; klungthong, chonticha; manasatienkij, wudtichai; thaisomboonsuk, butsaya; tyner, stuart d.; rith, sareth; horm, viseth srey; jarman, richard g.; bethell, delia; chanarat, nitima; pavlin, julie; wongstitwilairoong, tippa; saingam, piyaporn; el, but sam; fukuda, mark m.; touch, sok; sovann, ly; fernandez, stefan; buchy, philippe; chanthap, lon; saunders, david title: human sentinel surveillance of influenza and other respiratory viral pathogens in border areas of western cambodia date: 2016-03-30 journal: plos one doi: 10.1371/journal.pone.0152529 sha: doc_id: 260728 cord_uid: 4w23kwzu little is known about circulation of influenza and other respiratory viruses in remote populations along the thai-cambodia border in western cambodia. we screened 586 outpatients (median age 5, range 1–77) presenting with influenza-like-illness (ili) at 4 sentinel sites in western cambodia between may 2010 and december 2012. real-time reverse transcriptase (rrt) pcr for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ili-specimens negative for influenza were cultured, followed by rrt-pcr for enterovirus and rhinovirus (ev/rv) and ev71. influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from june to november. isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in cambodia. influenza vaccination coverage was low (<20%). western cambodian h1n1(2009) isolate genomes were more closely related to 10 earlier cambodia isolates (94.4% genome conservation) than to 13 thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the thai references. most genes showed signatures of purifying selection. viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus a4, a6, a8, a9, a12, b3, b4 and echovirus e6 and e9 using nested rt-pcr methods. a single specimen of ev71 was found. despite proximity to thailand, influenza epidemiology of these western cambodian isolates followed patterns observed elsewhere in cambodia, continuing to support current vaccine and treatment recommendations from the cambodian national influenza center. amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is also needed. acute respiratory infection (ari) is the leading cause of morbidity and mortality in cambodia [1] . previous studies have attributed the etiology of acute viral respiratory infections in cambodia to rhinovirus, respiratory syncytial virus (rsv), parainfluenza virus (piv), influenza virus a and b, human metapneumovirus (hmpv), bocavirus, adenovirus, enterovirus, and coronavirus [2] . influenza has been described to be one of the most important causes for hospitalization of children with an ari [3] . it was suggested that some southeast asian countries, where influenza a infection is present year round, play an important role in the global spread of influenza that could trigger annual epidemics in temperate regions [4, 5] . until the establishment of the national influenza center (nic) at institut pasteur du cambodge (ipc) and a national influenza-like-illness (ili)-sentinel surveillance system in 2006, little was known about influenza circulation in cambodia. since then, the sentinel surveillance system has, together with event-based surveillance, demonstrated evidence of seasonal, pandemic and avian influenza. previously published data revealed that thailand and cambodia, which are positioned geographically in the northern hemisphere, demonstrate differing influenza epidemiology with regard to seasonality, with thailand exhibiting a northern hemisphere transmission pattern while transmission in cambodia typically occurs from june to december, similar to the southern hemisphere [6] . the rainy season in western cambodia is june to november and the dry season is december to may. the more recent data suggests that influenza seasonality is more similar in thailand and cambodia than previously thought. [5] . depending on the type of genes and the specific residues, phenotypic effects of random or neutral mutation of the virus can be positive, purifying or neutral [7] . influenza virus efficiently escapes from host antibodies through an accumulation of mutations/single amino acid changes (antigenic drift) at the antigenic sites (epitopes) in surface glycoproteins of the hemagglutinin (ha) gene, and to a lesser extent, neuraminidase (na) genes [8] . the antigenic sites are five somewhat overlapping regions (designated a to e for the h3 strains [9] [10] [11] and ca1, ca2, cb, sa, and sb for the h1 strains [12] ). antigenic mapping by smith et al. [13] showed that 11 antigenic clusters of viruses emerged during the 35-year period following the introduction of the a/h3n2 virus in humans in 1968 and that major jumps (or "cluster transitions") occur between antigenically distinct clusters of viral sequences roughly every 3 years (antigenic shift) [13] . until 2009, the cambodian national sentinel surveillance system only covered 8 out of 24 provinces based on a network of urban sentinel-sites at referral hospitals. no data existed about circulating influenza strains and other respiratory pathogens in western-cambodia along the thai-cambodia border, which is a poor and rural area with large volume of crossborder traffic. between 2010 and 2012, the armed forces research institute of medical sciences (afrims) established four additional influenza sentinel surveillance sites in four border provinces in western cambodia, in collaboration with the cambodian communicable disease control (ccdc) department and the ipc. this study describes the circulation of influenza and non-influenza respiratory viruses and the genetic diversity of influenza viruses in western cambodia along the thai-cambodia border which has provided information for treatment, prevention, and control strategies for these populations. the ili and event-based surveillance systems are public health activities organized by the ministry of health in cambodia and as such have a standing authorization from the national ethics committee. samples were all anonymized for the purpose of this study. the study was approved by scientific review committee at afrims in thailand, the institutional review boards at the walter reed army institute of research (wrair) in the united states and the national ethics committee for health research (nehcr) in cambodia. written informed consent was obtained from volunteers or parents or legal guardians of children enrolled in the study. between may 2010 and december 2012, we collected specimens and surveillance data for influenza and other viral respiratory pathogens from a subset of outpatients presenting with influenza-like-illness (ili) at four sentinel sites-located in five health centers and hospitals in battambang, oddar meanchey, pailin and banteay meanchey provinces in cambodia (fig 1) . fig 1 was created using arcview gis version 3.1 (http://arcview.software.informer.com [14, 15] ) sentinel health centers and hospitals were selected based on sufficient overall patient volume and patients with upper respiratory infections (uri), geographical representativeness and ease of specimen transport to the study laboratory. sites were established sequentially: battambang in 2010 (2 sites), oddar meanchey and pailin in 2011, and banteay meanchey in 2012. due to the cross-sectional design of our study in a region for which no previous baseline respiratory surveillance existed, no minimal sample size was calculated. nevertheless, and in accordance with national and world health organization influenza surveillance guidelines [16], we aimed to collect a cross-section of data distributed over time and space. for every site -and pending on local epidemiology and health facility workload, 5 to 10 ili specimens were collected each week (monday to friday) for 52 weeks per year and largely from the first two subjects meeting the predefined inclusion criteria. demographic, clinical data and respiratory specimens were collected from patients who met the case definition for ili defined as persons of 1 year of age or older, arriving at the health center or hospital within 5 days of fever onset with a fever (axillary 38.1°c or oral 38.6°c) and cough or sore throat in the absence of other diagnoses. patients younger than 1 year, who could not provide consent or those with nasal lesions or epistaxis were excluded. demographic data included gender, occupation, presenting signs and symptoms, self-reported influenza vaccination history, medication use and travel within the last 7-days. parents reported for children, which in our study are defined as all study volunteers younger than 18 years old. descriptive statistics of demographic and clinical data including frequencies and cross-tabulations were entered in a study database by double data-entry using microsoft excel 2013 and analyzed using ibm spss statistics 21.0 (spss, ibm inc.) up to three respiratory swabs per volunteer were collected by trained staff with standard procedures. one nasal swab was tested on-site with quickvue influenza a+b (quidel, san diego, calif.) using monoclonal antibodies specific for influenza a and b virus. a combined nasal and throat specimen was placed in a universal transport medium (utm, copan diagnostics, corona, ca, usa), stored between 2-8°c or in liquid nitrogen and shipped to the afrims-cnm (cambodia national malaria center) laboratory at the battambang referral hospital for testing for influenza virus. all ili-specimens were typed and subtyped for influenza a and b at the afrims-cnm laboratory in battambang. influenza-positive specimens were forwarded to the virology unit at institut pasteur du cambodge (ipc) in phnom penh and the who collaborating center (whocc) for reference and research on influenza in melbourne, australia. nucleotide sequence and phylogenetic analysis of influenza viruses was conducted at the virology branch at the walter reed army institute of research (wrair), silver spring, maryland, u.s. influenza-negative specimens were sent to the department of virology at afrims in bangkok, thailand for viral culture and further characterization of other respiratory viruses. rna was extracted from an aliquot of 140 μl of each combined nasal and throat specimen using qiagen viral rna mini kits (qiagen, hilden, germany). the influenza genome was amplified and detected using a standardized the real-time reverse transcription (rrt)-pcr assay on a smartcycler1 ii platform (cepheid, sunnyvale, ca), to test for influenza a and/or b types. samples positive for influenza a and/or b were further tested to confirm influenza a and/or b positivity and to determine influenza a subtypes. the hemagglutinin-gene (ha) specific primers and probes were used for subtyping of human influenza a subtypes h1, h3, h5 and pandemic h1 (ph1, previously known as swh1). nucleoprotein-gene (np) specific primers and probe for universal swine influenza was also used to confirm influenza a subtype ph1 detection in some samples. the 25 μl reaction mixture consisted of 5 μl of rna eluate and 20 μl of the master mixture which included 0.5 μl enzyme mixture (superscript iii rt/platinum1 taq dna polymerase-life technologies), 0.5 μl rnaseout™ recombinant ribonuclease inhibitor (invitrogen™, carlsbad, ca), 12.5μl 2x reaction mix, 0.5μl of both primers and 0.5μl of probe, and 5μl rnase-free water (see s1 table for primer sequences). thermocycling reaction conditions for influenza typing were as follows: initial reverse transcription at 50°c for 30 min, followed by denaturation and pcr activation by taq inhibitor activator at 95°c for 2 min and 45 cycles of pcr amplification (<95°c for 15 sec then 64°c for 30 sec). for influenza a subtyping: reverse transcription at 50°c for 30 min, pcr activation by taq inhibitor activator at 95°c for 2 min and 45 cycles of pcr amplification (<95°c for 15 sec then 62°c for 30 sec). after influenza typing by rrt-pcr, two 500 μl aliquots of every influenza-positive specimen was transferred into cryogenic vials for transport in liquid nitrogen to ipc in phnom penh. specimens testing positive for influenza by rrt-pcr were inoculated onto madin-darby canine kidney (mdck) cells for isolation of influenza strains at the virology unit of ipc. the influenza strains were analyzed by a hemagglutination inhibition test using reference antigens and anti-sera provided by the whocc for reference and research on influenza in melbourne, australia. the na-star1 kit (life technologies, carlsbad, ca, usa), a chemiluminescent neuraminidase (na) inhibition assay which utilizes a 1,2-dioxetane derivative of sialic acid as substrates, was used for na inhibitor susceptibility testing on a subset of isolates, randomly selected across specimen collection sites and dates. the concentration of drug required to inhibit 50% of the na activity (ic 50 ) was calculated using the non-linear curve-fitting function in the graphpad prism 4 package (graphpad software, inc., la jolla, ca, usa). the average ic 50 (nm) (± standard deviation) of two independent determinations was calculated for each virus. outliers of more than 2 standard deviations from the overall mean were retested twice. a subset of isolates were forwarded to whocc in melbourne for confirmation of strain analysis by ha inhibition test and na inhibitor susceptibility using an na enzyme inhibition assay with a fluorescent substrate munana [2'-(4-methylumbelliferyl)-α-d-n-acetylneuraminic acid sodium salt hydrate]. the typical range of ic 50 was calculated as the mean ic 50 ± 3 standard deviations using a panel of well-characterized reference strains kindly provided by the who collaborative center for reference and research on influenza, melbourne, australia. isolates with ic 50 values within or close to the typical ic 50 range were considered to be sensitive isolates. ic 50 values outside of the typical range and between 50 and 200 nm were isolates with mildly reduced sensitivity and ic 50 values well outside the typical range and greater than 200 nm were considered as isolates with highly reduced sensitivity. viral rna segments, extracted from mdck supernatant were sequenced at ipc in phnom penh from 15 samples collected in 2011 and from 10 samples collected in 2012 for both pandemic influenza (ph1n1) and influenza h3n2 (h3n2), representing our four study sites with varying success (s2 table) . cleaned nucleotide sequence data was sent in fasta-format to the bioinformatics section at the virology branch of the wrair in silver spring, maryland, usa. sequences for ph1n1 and h3n2 sequence datasets respectively were combined with references available from the influenza viral resource at genbank [17] and the gisaid epiflu database [18] representing global diversity of ph1n1 from 2009-2013. sequences were aligned using muscle version 3.8.31 [19] implemented in seaview version 4.4.2 [20] and manually inspected for accuracy. best models were determined from alignments using jmodeltest2 version 2.1.4 [21] . maximum likelihood (ml) phylogenetic trees were generated using phyml version 3.0 as implemented in seaview version 4.4.2 [22] . see table 1 for a detailed description of datasets and models used in analysis. ml phylogenies were annotated using mega version 5 and figtree version 1.4.0 [23] . all influenza sequences used in analysis have been deposited into genbank under accessions ku299790-ku299957. alignments were analyzed for diversity, percent nucleotide similarity, antigenic variation and selection using mega version 5 [24] and the hyphy package [25] implemented through the datamonkey webserver [26] [27] [28] . annotation was conducted using genbank's annotation tool in the influenza viral resource [17] and a literature review. all specimens tested negative by influenza pcr were cultured for isolation of respiratory virus and detection by cytopathic effect (cpe) and/or with either immunofluorescence assay (ifa), or direct fluorescence assay (dfa). a subset of 164 culture-negative specimens (collected between may 2010 and april 2012), where we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old as compared with previous studies (1%) in cambodia [2] , were tested for enterovirus and rhinovirus by two separate nested rt-pcr methods adapted from coiras et al., 2004 and singh et al., 2002 [29,30] , one for simultaneous detection of pan-enteroviruses and rhinoviruses, and the other specific for enterovirus 71 (ev71). pcrproducts positive for enterovirus or rhinovirus were sequenced for nucleotide analysis (fig 2; s3 table) . cell culture of influenza-negative specimens enterovirus ev71 detection included single-step first-round rt-pcr and semi-nested pcr (primers are in s3 table) . the ev71 type specific primers were modified from the previous study by singh et al. 2002 [30] . detection of pan-enteroviruses and rhinoviruses (pan-ev/rv nested rt-pcr) is similar to the procedure for ev71 including 2 rounds of pcr as reported before [29] . the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between 5 0 non-coding region (5 0 ncr) and vp4/vp2 regions that was previously described by coiras et al. 2004 [29] . ev/rv-2n was modified from primer 2-ev/rv [29] for using in nested pcr reaction. single-step first-round rt-pcr reaction was the same as described above except that 0.25 pmol of each forward and reverse primer (1-ev/rv and 2-ev/rv) was used (s3 table) . the rt step was performed at 42°c for 60 min, followed by 35 cycles of thermocycling 94°c for 30 seconds, 50°c for 1 min, and 72°c for 1 min. the reaction was further incubated at 72°c for 10 min. five micro-liter of 1:20 diluted first round pcr products were added to the nested pcr reaction with the same reagents as in the first-round but without dtt and amv-rt and the primers were 0.25 pmol of each forward and reverse primer, 3-ev/rv and ev/rv-2n. the reaction was incubated at 95°c for 5 min followed by 35 cycles of thermocycling 94°c for 30 seconds, 52°c for 1 min, and 72°c for 1 min. the pcr products (9 μl each) were subjected to electrophoresis in agarose gels, with 100-bp dna ladder serving as a molecular marker. a negative control of rna-free water and a positive control of cdna template from the ev71 reference strain (atcc 1 no. vr-1432 tm ) were included in each experiment. the pcr products from the positive samples were sequenced on both strands with the pcr primers (s3 table) . the specific bands from the first or second rounds pcr were purified using qiaquick gel extraction kit (qiagen, germany) before sending for direct sequencing. sequencing service was performed by ait biotech (singapore). the sequences from both strands were combined for analysis and edited with sequencher (gene code corp., usa). homology searches were through nucleotide blast program [31] along with the percentage of sequence identity of the two given sequences. 586 ili-patients (median age 5 year, range 1-77) were enrolled from five sites from may 2010 to december 2012; among these, 168 (29%) tested positive for influenza by rrt-pcr (table 2) . most frequent symptoms reported were fever (100%, inclusion criterion), cough (100%,), runny nose (90%), congestion (77%), sore throat (75%,) and headache (76%). although gender distribution was similar in ili-patients, more females were positive for enterovirus (71%) or rhinovirus (64%). patients with influenza were slightly older (median age 7 years old) and presented more often with a sore throat (78%). compared with other ili-cases, patients testing positive for enterovirus reported body pain (20% vs. 44%) and headache (50% vs. 77%) less frequently, although chills (50% vs. 32%), vomiting (35% vs. 16%) and abdominal pain (29% vs. 19%) were more common. dyspnea was uncommon, yet most often seen in patients positive for adenovirus (14%). diarrhea was observed in ili-patients (6%-12%) except those infected with enterovirus (0/17). influenza vaccination coverage in 2010 and 2011 approximated 20% in response to the influenza a/ph1n1(2009) pandemic yet decreased to zero in 2012 by subjects' reports. five (table 3 and figs 2 and 3a). vaccination coverage during the entire study period was 11% (63/583). the probability of developing influenza among ili-patients who had been vaccinated (20/63) and who had not (148/520) was similar and not significantly different (0.32 vs. 0.28, p = 0.28). all ic 50 -values of influenza a and b isolates tested from 2011 were within the susceptible range for both oseltamivir and zanamivir (table 3) . overall, at least 1 respiratory virus was detected in 258 out of 586 (44%) ili-specimens collected between may 2010 and december 2012 (fig 2) ; of which most were influenza (168, 29% of ili-cases), followed by rhinovirus (33, 6%), adenovirus (24, 4%), non-polio enterovirus (17, 3%) and parainfluenza virus (piv) (16, 3%). all of the 418 ili specimens that tested negative for influenza a or b by rrt-pcr were sent for viral culture. forty (10%) were culture positive; 24 adenovirus, 11 piv1, 2 piv2, 3 piv3 were isolated (figs 2 and 3b ). no rsv, hmpv or coronaviruses were identified. pan-ev/rv and ev71 nested rt-pcr (s4 table) detected 17 samples positive for enterovirus, which included coxsackievirus a (n = 10, 1 a4, 3 a6, 1 a8, 1 a9, 4 a12), echovirus (n = 5, 2 e6, 3 e9) and coxsackievirus b (n = 2, b3, b4). a single specimen was found to be negative during the first round of rt-pcr but positive by second round pan-ev/rv and ev71 nested rt-pcr. the 662 base pairs pcr product fragment obtained from pan-ev/rv nested rt-pcr was sequenced using the rv/ev inner primers. the 528 bases obtained from sequencing were found to be 99% identical to the enterovirus a71/homo sapiens/vnm/208/2011 strain (accession: kj686294). when the 227 bp pcr product obtained from ev71 nested rt-pcr was sequenced using the ev71 inner primers, 131 bases was obtained. this sequence was found to be 99% identical to enterovirus a71/cambodia: banteay meanchey 2012 strains (accessions: kp308459, kp308453, kp308450, kp308448, kp308430, kp308427, kp308410, kp308406). out of the 33 specimens positive with rhinoviruses, only 18 could be serotyped. of these 18, most were rhinovirus a (n = 11), followed by rhinovirus b (n = 4) and rhinovirus c (n = 3) (fig 4b) . amplicons were sequenced and sequences were analyzed using genbank's blast tool to identify the virus species. pandemic influenza a (ph1n1). all sequences were highly related with no more than 1.67% divergence between the strains detected in the study and respective seed viruses included in the vaccine composition (a/california/7/2009, a/brisbane/10/2010, and a/christchurch/ 16/2010; s5 table, fig 5) for any of the segments analyzed. when compared to the a/california/7/2009 vaccine strain, all cambodian viruses on average had 11 amino acid changes within the ha segment. amino acid changes found in the ha gene can be found in table 4 . no evidence for amino acid substitutions leading to changes in glycosylation was seen in the ha dataset; however, several changes among sequenced samples occurred in antigenic sites, polymorphic sites or had a recorded effect in in vitro analyses per the literature (s6 table) . for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or table) . for the mp gene, all samples contained the s31n mutation that may confer amantadine resistance as determined by the genbank influenza viral resource annotation tool [32] . no features or changes of interest were seen within the np or ns segment. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/california/7/2009) as well as to the yearly defined groups (2011 and 2012). the mp and np segments showed the fewest substitutions (table 4 ). there were no samples within the ha amino acid analysis that had unique substitutions relative to the global sequences. however, the na gene had unique amino acid substitutions not found with the global references; the function of these substitutions is unknown (s7 table) . selection pressure was determined using the dn/ds statistic. values of dn/ds > 1 are indicative of positive or adaptive selection, values <1 are indicative of purifying or negative selection and a value = 1 indicates neutral or no selection ongoing in the dataset. all samples were under purifying selection for all segments with exception to the ns segment of the 2011 cambodian samples, which had a dn/ds value of 1.0 suggesting neutral selection (s8 table) . for ph1n1 there was no evidence of geographic clustering of sequences within sampling site (colored branches in fig 5) ; nor was there evidence of reassortment among the sequences sampled. sequences (references and samples) from cambodia 2011 distributed throughout the trees; however, the majority of 2011 sequences, including those corresponding to strains detected in this study appeared within a 2011 specific cambodian clade. however, this clade was only evident in ha and na phylogenies and did not appear in np, mp or ns phylogenies (s1-s5 figs). influenza a h3n2 (h3n2). all sequences were highly related with no more than 1.77% divergence between the samples and the furthest related respective vaccine (s9 table, fig 6) for any of the segment. the majority of segments were more closely related to the a/victoria/361/ (1) table) . there were several amino acid changes among the sequenced samples occurring in antigenic sites, polymorphic sites or which resulted in a change of glycosylation (table 5 ). for the na gene, several amino acid substitutions resulted in addition/loss of glycosylation sites, or were found in polymorphic or antigenic/catalytic sites (epitopes) ( table 5 ). for the mp gene, it was noted that all samples contained the s31n mutation that may confer adamantane resistance as determined by the genbank influenza viral resource annotation tool [32] . no features or changes of interest were seen within the ns segment for any sample. the ha and na segments contained the most amino acid substitutions when compared to the reference (a/victoria/361/2011) as well as to the yearly defined groups (2011 and 2012). the mp and ns segments showed the fewest substitutions (s10 table) . there were several amino acid substitution within the ha amino acid analysis that were unique relative to the global sequences (and vaccine references); two of which occurred in antigenic regions (table 5 ) and five additional changes of unknown effect or location significance (s11 table) . one amino acid substitution in the na gene was found to be unique in antigenic site a (table 5 ) and 7 unique substitutions of unknown effect or location significance were also found (s12 table) . all 'unique' aa substitutions were not found with the global sequences or vaccine references. all samples were under purifying selection for all segments with the exception to the ns segment. when samples were separated based on the respective years of collection, the ns gene showed evidence of neutral selection (dn/ds~1). however, when all samples were combined to increase sample size, it showed overall evidence of purifying selection (dn/ds < 1; s10 table) . overall, for h3n2, there was no evidence of geographic clustering of sequences within the same sampling site except samples from oddar meanchey in which all but one sample clustered into the ohio 2012 vaccine cluster (green branches in fig 6) . this clustering was visual only, not statistically significant due to small sample size, though the cluster was well supported (cluster node value 81, value obtained using associated likelihood ratio test (alrt); analogous to bootstrap analysis). there was no evidence of reassortment among the samples analyzed and all samples were consistently grouping with either the a/ohio/02/2012 or a/victoria/361/2011 vaccines. there was one exception; sample a/cambodia/v1221301/2011 consistently fell outside of both the ohio 2012 and victoria 2011 vaccine clusters (fig 7) for all segments analyzed. this is to the best of our knowledge the first influenza surveillance study in remote border areas in western cambodia. over the time course of 31 months, spanning 3 influenza seasons including the last few months of pandemic a/ph1n1(2009) influenza circulation, seasonal influenza virus was the most commonly detected respiratory virus in this predominantly pediatric population of subjects who presented with ili (fig 3) . despite detection of human highly pathogenic avian influenza (hpai) h5n1 virus in western cambodia since early 2011 [36] [37] [38] , h5n1, typically detected in south-central cambodia, was not found at our sentinel sites. influenza subtypes varied by year with a/ph1n1(2009) being predominate in 2010, influenza b in 2011, and influenza a/h3n2 in 2012. we did not expect to detect any h5n1 in our laboratory surveillance system as most suspected h5n1 cases would have been screened out by the health centers based on reported exposure to dead or diseased poultry. the specimen of one patient with reported h5n1 exposure in 2011 was investigated in our laboratory and tested negative for h5n1. table 5 . changes in glycosylation, antigenic and polymorphic sites linked to amino acid (aa) substitutions and sub-antigenic/catalytic sites (epitopes) where aa substitutions were found for hemagglutinin (ha) and neuraminidase (na) segments of h3n2. [39] . our data revealed that the influenza b did not match well with strains included in vaccine composition of trivalent vaccines; therefore, if vaccination is to be implemented, the quadrivalent influenza vaccine that contains the two influenza b lineages, rather than the trivalent vaccine, may be more suitable. aside from a limited quantity of oseltamivir, stockpiled by the government for management of severe disease by pandemic or avian influenza [40] , use of antivirals for influenza is very low in cambodia [40] . as a result, all strains we detected were susceptible to neuraminidase inhibitors (nis) such as oseltamivir and zanamivir, which can be partially explained by the absence of these medications in cambodia. due to widespread adamantane resistance, we did not conduct susceptibility testing on this class of antiviral drugs; however, we did find the s31n mutation conferring adamantane resistance in all of our influenza isolates tested. phylogenetically, all our samples clustered together within each of the two subtypes (a/ ph1n1 or a/h3n2), which is not unusual given the location and specimen collection dates compared with the reference sequences used. our western cambodian ph1n1(2009) isolates were more closely related, based on full genome analysis, to 10 earlier isolates from cambodia (94.4% genome conservation) than to the 13 thai isolates (75.9% genome conservation) or the california 2009-vaccine reference. however, it was also noted that amino acid changes were shared with the thai references suggesting a possibility of mixing between thai and cambodian influenza as expected. isolates from battambang, being western cambodia's major transportation hub, showed the highest diversity of amino acid changes. analysis of sequence data from our influenza patients revealed a random or neutral mutation of the influenza genomes as expected. koel et al. [41] discovered that periodic major antigenic change in influenza a/h3n2 virus was caused mainly by single amino acid substitutions, which occurred at only seven out of 131 possible amino acid positions in ha at antigenic sites immediately adjacent to the receptor binding site (rbs). these substitutions were located in antigenic sites a (position 145) and b (positions 155, 156, 158, 159, 189, and 193) , with none in sites c, d, or e. we did not see these changes within antigenic sites a or b in our dataset. the ha and na genes in our h1 and h3 isolates showed signatures of purifying selection meaning that the virus keeps these genes conserved by removing random mutations as they occur. these single amino acid mutations were found at non-epitope sites of ha [41] that have not been associated with major antigenic changes on their own. however, it is possible that some of these mutations may constitute permissive or compensatory mutations that would be important in enabling co-mutations that could affect viral fitness, and as such, have an incentive to remain fixed under purifying selection. permissive mutations have been described in the spread of oseltamivir resistant influenza a/h1n1 virus that carry the h274y mutation and are increasingly being recognized as a major force in evolution [42, 43] . glycosylation (addition of oligosaccharide chain to the surface protein) is another common form of protein modification. alteration of glycosylation sites can affect folding and conformation changes in the surface glycoprotein, hereby impacting virus survival and transmissibility. in addition, glycosylation can affect interaction with receptors and cause a virus to be more [or less] recognizable by the innate host immune system and antibodies [44] . none of the changes in glycosylation in our isolates have been reported in the literature as having an effect on viral structure or function; but more data may be needed. mapping sites of mutation and glycosylation on epitopes provides a better understanding of antigenic drift and is important for improving vaccine strategies [45] . overall, and despite adequate storage and transportation of our specimens, culture yield was low in our dataset (~10%) with isolation of only adenovirus and parainfluenza. this may be due to an exclusion of children younger than 1 year, the outpatient setting with less severe disease and possibly a later presentation to the health facility (which was possible up to 5 days after fever onset) resulting in a lower viremia. previous work among hospitalized patients has shown presence of human coronavirus, human bocavirus, hmpv and rsv in cambodia [2] but these were not detected by our culture techniques. therefore, the number of non-influenza viruses in our population is possibly underestimated. additional testing is ongoing at afrims in order to elucidate true disease burden of non-influenza viruses on the population. in comparison with previous work in cambodia [46] , we found a relatively higher proportion (9.3% versus 1.3%) of ili-patients testing positive for adenovirus over 2 dry seasons in 2011 and 2012 which may be explained by our younger study population who may be disproportionately affected by this virus. piv-3 (0.9%), which in previous dry seasons was described as the most common parainfluenza-virus in cambodia [2] , was the least common type during our surveillance period, whereas piv-1 (5.6%) was most common, particularly during november 2011 to march 2012. this is not consistent with previous reports for cambodia [46] . the lack of presence of piv-3 in our study population may be due to an outbreak of piv-1 during the surveillance time frame and the fact that we did not include children under 1 year old, an age when 40% of piv-3 is usually detected [47] . by employing a highly sensitive and specific nested rt-pcr assay for enteroviruses and rhinoviruses, we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old compared with previous studies (1%) in cambodia [2] which could be partially explained by the predominantly outpatient population in our study sample. this is likely an underestimation of the true prevalence in this population since we excluded children less than one year of age, an age group more likely infected by enteroviruses at rates exceeding those of older children and adults by several fold [48] [49] [50] . the most common enteroviruses found among children under 5 years old in our population were coxsackieviruses (7.3%) and echoviruses (3%). although we did not find coxsackievirus a16, the most common etiologic agent for hand-foot-mouth-disease (hfmd), we found other coxsackieviruses that can cause hfmd such as coxsackievirus a6 (n = 3) and a12 (n = 4). coxsackievirus a6 is known to cause either an atypical rash resembling eczema herpeticum or chickenpox (united states 2011-2012 and in europe since 2009) or nail loss one to two months after onset of symptoms (finland 2009, taiwan 2010, japan 2011). coxsackievirus a12 was one of the enteroviruses implicated in hfmd outbreaks in china in 2008 and 2009 [51] . while these patients reported symptoms of fever (38.1-39°c), runny nose, chills, cough, congestion and abdominal pain, they did not report symptoms specific to hfmd such as herpangina and skin rash. we did not detect in our study ev71 virus genotype c4, which was the principal etiologic agent causing the 2012 hfmd outbreak across southeast-asia that manifested as a life-threatening neuro-respiratory syndrome in 78 children under 3 years old from 14 provinces in cambodia [52] . although we did not have ev71 test results from specimens collected during the peak of the outbreak, our data suggests that ev71 virus was not present yet in the border areas of western cambodia before april 2012, the time that the first cases were found elsewhere in cambodia. regarding testing for non-influenza viruses, the use of the less sensitive viral culture as the first-line test supplemented by ifa on only a subset of influenza-negative ili-specimens underestimates overall extent of transmission as well as co-infections. exclusion of children under 1 year of age may also contribute to underestimation. our data was derived from a passive surveillance system with convenience sampling. while we attempted to select surveillance sites representative of the nearby geographic locations, we had no accurate figures of the catchment populations or denominators at our respective sentinel sites. our staggered method of adding sentinel sites suggests that not all sentinel sites enrolled patients evenly over 3 influenza seasons. similarly, with a small sample size and limited number of sampling locations, no inferences could be made with regard to temporal or spatial flow of influenza strains in our phylogenetic analysis. now with a more established system in place, our data may provide a useful baseline for future molecular evolution studies of influenza in cambodia and in the region. despite proximity to thailand, influenza activity, seasonality, antigenicity and anti-viral susceptibility in western cambodian isolates followed patterns observed elsewhere in cambodia rather than thailand. this supports earlier recommendations from the cambodian nic to use the northern hemisphere influenza vaccine on a southern hemisphere vaccination schedule. additionally, use of the quadrivalent versus the trivalent vaccine should improve coverage of influenza b strains circulating in cambodia. neuraminidase inhibitors may still be used for treatment and chemoprophylaxis for seasonal influenza given little evidence for resistance. the amino acid mutations at non-epitope sites, in particular of ha, require further investigation in light of the increasingly important role of permissive mutations in the evolution of influenza virus. further research to clarify the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in cambodia is needed. table. samples collected for genetic analysis in this study including virus subtype, sample origin, specimen type, sampling date and segments successfully sequenced. (docx) s3 table. primers for rt-pcr and nucleotide sequencing for ev/rv and ev71: the enterovirus 71 type specific primers were modified from the previous study by [53] . the modifications were made by following the alignments of vp1 sequences of different ev71 strains collected during 2002-2011 from thailand, taiwan, philippines, vietnam, and china available in genbank including the sequences with the accession no. jn191177-9, fj969151, fj969163, jq 621835, jq621841, am490141-63, jq315092, and jx203305. the primers for simultaneous detection of enteroviruses and rhinoviruses were designed in the polyprotein gene, between 5 0 non-coding region (5 0 ncr) and vp4/vp2 regions that was previously described by coiras et al. 2004 [29] . ev/rv-2n was modified from primer 2-ev/rv [29] for using in nested pcr reaction. (docx) s4 table. total number of viruses identified by pan-enteroviruses and rhinoviruses (ev/ rv) pcr and nucleotide sequencing † (docx) s5 table. ph1n1 average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s6 table. changes in glycosylation, antigenic and polymorphic sites linked to aa substitutions and sub-antigenic/catalytic sites (epitopes) where amino acid (aa) substitutions were found for for the ha and na segments of ph1n1. aa substitution nomenclature is as follows; reference amino acid (a/california/7/2009), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s7 table. unique ph1n1 amino acid (aa) changes of unknown function in specific samples for the na gene as compared to a/california/7/2009. aa substitution nomenclature is as follows; reference amino acid (a/california/7/2009), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s8 table. ph1n1 sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s9 table. h3n2 average percent nucleotide sequence identity between vaccine strains and cambodia isolates gene segments. (docx) s10 table. h3n2 sample amino acid substitution summary and selection analysis by group per segment analyzed. (docx) s11 table. unique h3n2 amino acid changes of unknown function to specific to samples for the ha gene as compared to a/victoria/361/2011. aa substitution nomenclature is as follows; reference amino acid (a/victoria/361/2011), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s12 table. unique h3n2 amino acid changes of unknown function to specific to samples for the na gene as compared to a/victoria/361/2011. aa substitution nomenclature is as follows; reference amino acid (a/victoria/361/2011), amino acid site, sample amino acid. amino acids are numbered from the start codon of the segment (atg:methionine). (docx) s13 world health organization (2012) cambodia health service delivery profile acute viral lower respiratory tract infections in cambodian children: clinical and epidemiologic characteristics burden of interpandemic influenza in children younger than 5 years: a 25-year prospective study the genomic and epidemiological dynamics of human influenza a virus influenza seasonality and vaccination timing in tropical and subtropical areas of southern and south-eastern asia influenza activity in cambodia during codon bias and frequency-dependent selection on the hemagglutinin epitopes of influenza a virus electron microscopy of influenza virus structural identification of the antibody-binding sites of hong kong influenza haemagglutinin and their involvement in antigenic variation the hemagglutinin membrane glycoprotein of influenza virus the structure and function of the hemagglutinin membrane glycoprotein of influenza virus antigenic variation in influenza virus mapping the antigenic and genetic evolution of influenza virus why arcview 3.x is till in use world health organization (2008) a practical guide to harmonizing virological and epidemiological infuenza surveillance the influenza virus resource at the national center for biotechnology information a global initiative on sharing avian flu data muscle: a multiple sequence alignment method with reduced time and space complexity seaview version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building more models, new heuristics and parallel computing jmodeltest 2: more models, new heuristicssea-view version 4: a multiplatform graphical user interface for sequence alignment and parallel computingphylogenetic tree building figtree v1. 3.1: tree figure drawing tool mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods hyphy: hypothesis testing using phylogenies datamonkey 2010: a suite of phylogenetic 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isolated during 1960 and 1961 coxsackievirus b5 infection and aseptic meningitis in neonates and children an epidemic of echovirus 18 meningitis aedes hensilli as a potential vector of chikungunya and zika viruses complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in we gratefully thank all the volunteers for their study participation and study site and provincial health department staff in battambang, oddar meanchey, banteay meanchey and pailin for collecting the data and specimens. we would like to acknowledge the thai-cambodian afrims field team for their dedication and commitment: mr key: cord-298551-ua90xoak authors: bennet, rutger; hamrin, johan; wirgart, benita zweygberg; östlund, maria rotzén; örtqvist, åke; eriksson, margareta title: influenza epidemiology among hospitalized children in stockholm, sweden 1998–2014 date: 2016-06-14 journal: vaccine doi: 10.1016/j.vaccine.2016.04.082 sha: doc_id: 298551 cord_uid: ua90xoak background: influenza remains a common reason for the hospitalization of children. there is a need for long term studies that are also population based. we describe the epidemiology of severe influenza in a defined population 1998–2014. method: retrospective study of annually collected data of virologically confirmed influenza in hospitalized children 0–17 years living in the catchment area (230,000 children). we gathered information about comorbidity and complications from case records, and compared influenza a, b and a(h1n1)pdm09 with respect to these factors. results: a total of 922 children with influenza were hospitalized. the mean rate remained unchanged at 22.5–24.2 per 100,000 children per year. there were two major outbreaks: influenza a(h3n2) in 2003–2004 and the a(h1n1) pandemic in 2009–2010. the proportion of children with influenza b increased from 8% during the first half of the study period to 28% during the second half. the highest admission rate was found in children <3 months of age, 169 per 100,000. children with influenza b were older than those with influenza a. comorbidity was found in 34%, complications in 41%, and 11% needed intensive care management. the mortality rate was 0.17/100,000 children. conclusion: influenza remains an important reason for the hospitalization of children, especially during the first years of life. the increasing proportion of influenza b may have to be considered when recommending influenza vaccines. influenza is common among children, especially before 5 years of age [1] . we here describe the influenza a, b and a(h1n1)pdm09 epidemiology in the 0-17-year-old population of northern stockholm during 17 seasons, 1998/99-2013/14, and its implications for the immunization of children. in stockholm, children with certain chronic diseases are eligible for free seasonal influenza immunization. this was introduced in 2004 and originally included children with chronic pulmonary or heart disease, but after the pandemic 2009 was expanded to include also chronic liver or renal diseases, immunosuppression, diabetes mellitus, extreme obesity (body mass index > 40), neuromuscular disease affecting breathing capacity, and children with multiple dysfunctions/handicaps. during the pandemic, adjuvanted monovalent h1n1 vaccine (pandemrix ® , glaxosmithkline biologicals, rixensart, belgium) was offered to all children from 6 months of age. however, except during the pandemic season, when 60% of all children accepted the offer, the vaccine uptake has been low (<5%), perhaps due to fear of side effects or unawareness of the severity of influenza [2] [3] [4] . in order to increase the knowledge base for vaccination recommendations, in addition to data that can be obtained from administrative databases, we here provide population-level influenza burden data based on observed, virologically confirmed hospitalizations. this is a retrospective study of annually gathered information about influenza in children (<18 years hospitalized at astrid lindgren children's hospital, stockholm, sweden during 1998-2014. the hospital is a tertiary referral center with surgery and a pediatric intensive care unit (picu) with resources for extracorporeal membrane oxygenation (ecmo), but only children resident in the catchment area were included in the study. we obtained population data from the stockholm area database at statistics sweden (www.scb.se). during the study period, the number of children in the primary catchment area increased by 17%, from 197,945 to 230,877. the number of person years for a specific age group during a certain year was considered equal to the number of children on december 31. we extracted information about risk factors, complications and intensive care management from the hospital charts. from 2009 we have also been able to extract information from the vaccine register in stockholm county [3] . risk factors were neuromuscular disease, chronic lung disease, immunosuppression, and chronic conditions such as kidney or liver disease, or inborn errors of metabolism. if a child had both neuromuscular and chronic lung disease, the factor judged most important for the clinical course was counted. recurrent wheezing/"asthma" in children younger than two years and uncomplicated preterm birth (>30 gestational weeks) were noted but not considered risk factors. we counted sinusitis, tracheitis and presumed bacterial pneumonia (with or without empyema), but not otitis media as focal complications. chest x-ray was always performed when pneumonia was suspected. if perihilar infiltrates and/or hyperinflation were present, a diagnosis of bronchitis or bronchiolitis was made. bacterial and viral pneumonia were differentiated mainly by c-reactive protein (crp) levels, with a cut-off value of 40 mg/l. neurologic complications were seizures; either primary or secondary (in children with underlying neurologic disease), and encephalitis confirmed by electroencephalography. other complications included a few rare conditions such as myocarditis. dehydration alone was not considered a complication. as a routine, children admitted from our pediatric emergency ward with respiratory symptoms or fever without localizing signs, including febrile convulsions, are examined for viral etiology. during the winter season samples are primarily investigated for respiratory syncytial virus (rsv) and influenza viruses. if they are found negative, viral investigation is extended to other respiratory viruses (adenovirus, bocavirus, coronavirus, enterovirus, human metapneumovirus, parainfluenza virus 1-3, and rhinovirus) [5] . all cases in this report were virologically confirmed by the laboratory. influenza virus was detected with immunofluorescence and viral isolation prior to october 2007, when these methods were replaced by real-time polymerase chain reaction (rt-pcr) [5, 6] . before the switch to rt-pcr the diagnostic sensitivity of the three methods was evaluated in 585 samples at our laboratory [5] . the rt-pcr analysis has since then been improved (in 2009 and 2011 by the inclusion of new probes for influenza a(h1n1)pdm09 and in 2014 by the redesign of the influenza b probe). rsv has since 2009 been diagnosed with a rapid point-of-care test, with negative specimens further investigated for both rsv and influenza with rt-pcr. the chi-square test, the mann-whitney u-test, and exact clopper-pearson binomial confidence intervals were employed as appropriate. for multivariate analysis, we used a generalized linear model with the logit function in statistica ® v. 10, with influenza subtype (non-pandemic a or b) as the dependent variable, and age and presence or absence of risk factors and complications as independent variables. there were 922 children (56% male) with confirmed influenza during the study period. 557 had non-pandemic influenza a, 179 had influenza b, and 186 had influenza a(h1n1)pdm09, out of whom 93 belonged to the 2009-2010 pandemic. during all winter seasons there were influenza epidemics with varying severity. all but two influenza epidemics occurred during rsv epidemics after january 1 and there were then always more children with rsv during a given week (fig. 1 ). the two largest outbreaks 2003-2004 and 2009-2010 peaked before january 1 and preceded the rsv epidemics. influenza b was present in all but two seasons and dominated during three seasons, all of which occurred during the second half of the study period. co-infections (with any simultaneously circulating respiratory virus) were identified in 11% of the patients. this is a minimum figure, since some children, for instance those with a positive point-of-care test for rsv, were not examined for additional viral etiology such as influenza. when the period comprising the first eight seasons was compared with the second period, excluding the pandemic and thus also comprising eight seasons, there were significantly more cases of influenza b during the latter period (p < 0.01). however, the overall rate of influenza cases in children <17 years remained unchanged at 22.5 (95% c.i. 20.1-24.9) and 24.2 (21.9-26.5) per 10 5 person years, respectively. the cumulative age distribution is shown in fig. 2 . children with influenza b were older (as also shown in table 1 ) than those with influenza a. influenza a(h1n1)pdm09 is presented separately for the pandemic year 2009-2010 and the post pandemic years, since the pandemic patients were significantly older (p < 0.001). the yearly incidence rates in different age groups varied considerably, with median (range) for children <5 years 59 (19previously known risk factors were found in 312/922 (34% , table 1 ), the most important being neuromuscular disease (131 cases) and chronic lung disease (40 cases). the frequencies of recurrent wheezing (not included among risk factors) and preterm birth were comparable to their occurrence in the general child population at 6% and 2.6% (24/922), respectively. complications were seen in 380/922 (41%). most were related to the respiratory tract where pneumonia dominated (117) out of which half (58) were considered bacterial in origin. other bacterial superinfections, such as sinusitis (including periorbital cellulitis) were diagnosed in 22 children. approximately 50% of the children had a blood culture taken, which was positive in 12 (6 streptococcus pneumoniae, 3 staphylococcus aureus, 2 streptococcus pyogenes and 1 neisseria meningitidis). neurological complication was the second most common with encephalitis (33) and primary or secondary seizures (104). risk factors and complications were more common among children with influenza b than in those with influenza a in the univariate analysis (p < 0.05), but when included with age in the multivariate model, only age remained significant (p < 0.001, table 2 ). children with recurrent wheezing had a low rate (1/59) of picu admission. their median duration of hospital stay was 2 days, which is equal to that of other children without risk factors. intensive care admission was required for 105/922 children (11%); 41/610 (6.7%) without risk factors and 57/171 (33%) with neuromuscular or chronic lung disease (p < 0.001). two children were treated with ecmo. six children (0.17/10 5 person years) died, 1 <1 year, 2 1-9 years, and 3 >10 years old. three of these were previously healthy, whereof two had an influenza b infection. the causes of death were encephalitis, myocarditis and bacterial pneumonia in the previously healthy children, and non-specific complications in the three children with co-morbidity. this is a report of children hospitalized for influenza a or b in a defined population in the northern stockholm area 1998-2014, covering the pre-pandemic period, including the 2003-2004 outbreak, the 2009 pandemic, and four post-pandemic seasons. there was a strong seasonal variation and the epidemiologic pattern was often in accordance with what has been seen in other countries, such as the large outbreak of a(h3n2) in 2003-2004 [7] . with the exception of the influenza a(h1n1)pdm09 pandemic with a peak in late autumn 2009, the epidemic peaks occurred during winter and coincided with rsv, with co-infections detected in 11%. as we have reported earlier, rsv and influenza co-infections do not have a more severe course or more late wheezing, but tend to follow the expected clinical course of the rsv infection [8] . most population based long-term studies are from the north american continent, but one study from finland covers a 16 year period up to 2004 [9] . there have been both single center and multicenter studies [10, 11] . some were population based whereas others represented tertiary centers without a defined population. many of these studies have covered the periods before the 2009 pandemic and have later been extended to also include the pandemic year [12, 13] . several studies described the pandemic and a few have included the first post pandemic season [14, 15] . no study has like ours covered both pre-and post-pandemic seasons, when the novel influenza a(h1n1)pdm09 continued to circulate. our findings are in accordance with these studies, all pointing to the fact that influenza in children is still a problem. neuromuscular disease and chronic lung disease were the dominating risk factors. in our setting recurrent wheezing/"asthma" and uncomplicated preterm birth were not important risk factors. bacterial superinfection, mostly pneumonia, was the most common complication. in most studies the distinction between bacterial and viral pneumonia has not been discussed. in our study 40% were considered to be viral [16] . neurologic complications, such as seizures and encephalitis, were the second most common category. intensive care was needed for 11% of all children, which is in agreement with most other studies [12, 13, 17] . the need for intensive care was highest (18%) in children with risk factors. the case fatality rate noted by us was similar to that reported from other western countries [18] . our age distribution and incidence rate calculations are similar to, although in the lower range, of what has previously been reported [19] . 30% of the children were below one year and 72% below five years of age, resulting in an incidence of 99/10 5 and 59/10 5 , respectively. the highest rate, 169/10 5 , was observed among the youngest (<3 months) infants, as in a recent study from the usa [17] . an important part of our study is the comparison of different influenza types. in several studies comparisons have been done between seasonal influenza and (h1n1)pdm09 [14, 15] . we studied influenza a(h1n1)pdm09 and the pandemic year separately from the following years, because of the marked difference of age distribution. during the pandemic, children were older in accordance with what has been presented from several other centers [12, 15, 20] . during the following seasons, however, the average age was actually somewhat lower than in children with other influenza a types. one contributing factor could be the high vaccine coverage (60%) with the adjuvanted vaccine (pandemrix) during the pandemic in sweden, which has been shown to give protection for at least two subsequent seasons [3] . in addition to the emergence of pandemic influenza, also the burden of influenza b has come into focus in recent years [21] . we found influenza b especially during the second half of the study period. previous epidemiologic studies used different methods for estimating the number of influenza cases, such as excess of cases with influenza-like illness during influenza seasons, virus isolation, and rapid methods like immunofluorescence and pcr [22] . at our laboratory immunofluorescence and viral isolation were replaced by the more sensitive pcr in october 2007. to what extent this has contributed to our observed increase of influenza b can be discussed. the initial pcr system had a higher sensitivity for influenza b compared to immunofluorescence and virus isolation, while the sensitivity for influenza a was lower. the influenza a pcr has since then been continually improved, and in a review by mahoney et al. it is stipulated that well optimized rt-pcr assays have 5-10% higher sensitivity than virus isolation [23] . one limitation of our study is its retrospective nature. however, the same pediatricians have been responsible for the care of the infected children during the entire period. the same level of care has been provided from the picu with availability of ecmo treatment during the entire period. furthermore, in our study "retrospective" means yearly reviews and documentation of the previous season as a preparation for planning of the following season. our study is a summary of these reviews. another important factor, as mentioned, is the introduction of new tests that could be more sensitive. on the other hand, the use of rapid tests precluded the detection of coinfections -found in more than 20% in an earlier study from our hospital [8] . the effect of influenza vaccine in children has been the subject of several reviews. in contrast to the known effect of trivalent influenza vaccine (the only one used during the studied period except for the pandemic year) in healthy children >18 months, less is known about its effect in younger children and in those with risk factors. during the 2009-2010 pandemic when 69% of the swedish childhood population was vaccinated with a monovalent adjuvanted vaccine an effect of 91% was demonstrated [2] . as also found in an english study, the protective effect was still present in children with comorbidity, albeit smaller than in children without risk factors [24] . even though the immunization of children with risk factors is free, few parents (<5%) take the opportunity to vaccinate their children. the reason for the low uptake of the vaccine is probably multifactorial. in a recent questionnaire study in the usa two factors were highlighted: fear of side effects and the belief that influenza is not a serious threat to children [4] . in conclusion, our population based study demonstrates that influenza in young individuals is still a major problem, especially but not exclusively in those with risk factors. we hope to be able to use this information to encourage more parents to immunize their children. there was a significant increase of influenza b during later years, primarily affecting older children with risk factors. the reason for this increase is probably multifactorial, but it points to the need for increased protection against influenza b, e.g. through the development of the quadrivalent vaccine. it may be important to note that in our study half of the children who died had no risk factors and that influenza b was the cause in 2/6 cases. none. global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis 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influenza hospitalization in children comparison of children hospitalized with seasonal versus pandemic influenza a complications and associated bacterial coinfections among children hospitalized with seasonal or pandemic influenza the burden of seasonal and pandemic influenza in infants and children risk factors for mechanical ventilation in u.s. children hospitalized with seasonal influenza and 2009 pandemic influenza a influenza-associated pneumonia in children hospitalized with laboratoryconfirmed influenza the burden of influenza hospitalizations in infants from influenzaassociated pediatric deaths in the united states vaccination of healthy children against seasonal influenza: a european perspective children with asthma hospitalized with seasonal or pandemic influenza the burden of influenza b: a structured literature review influenza and the rates of hospitalization for respiratory disease among infants and young children molecular diagnosis of respiratory virus infections age-specific effectiveness of an oil-inwater adjuvanted pandemic (h1n1) 2009 vaccine against confirmed infection in high risk groups in england key: cord-010786-w3kjc6so authors: ghaderi, sara; berg-hansen, pål; bakken, inger johanne; magnus, per; trogstad, lill; håberg, siri eldevik title: hospitalization following influenza infection and pandemic vaccination in multiple sclerosis patients: a nationwide population-based registry study from norway date: 2019-12-23 journal: eur j epidemiol doi: 10.1007/s10654-019-00595-2 sha: doc_id: 10786 cord_uid: w3kjc6so patients with multiple sclerosis (ms) are at increased risk of infections and related worsening of neurological function. influenza infection has been associated with increased risk of various neurological complications. we conducted a population-based registry study to investigate the risk of acute hospitalization of ms patients in relation to influenza infection or pandemic vaccination in norway. the entire norwegian population in the years 2008–2014 was defined as our study population (n = 5,219,296). information on ms diagnosis, influenza infection and vaccination were provided by norwegian national registries. the self-controlled case series method was used to estimate incidence rate ratios (irrs) with 95% confidence intervals (95% ci) in defined risk periods. 6755 ms patients were identified during the study period. average age at first registration of an ms diagnosis was 51.8 years among men and 49.9 years among females (66.9%). the irr for emergency hospitalization among ms patients the first week after an influenza diagnosis was 3.4 (95% ci 2.4–4.8). the irr was 5.6 (95% ci 2.7–11.3) after pandemic influenza, and 4.8 (95% ci 3.1–7.4) after seasonal influenza. pandemic vaccination did not influence risk of hospitalization [irr within the first week: 0.7 (95% ci 0.5–1.0)]. among ms patients, influenza infection was associated with increased risk for acute hospitalization while no increased risk was observed after pandemic vaccination. influenza vaccination could prevent worsening of ms-related symptoms as well as risk of hospitalization. multiple sclerosis (ms) is a chronic neurological disease with unknown aetiology and unpredictable progress. immunological mechanisms are thought to play an important role in the development of ms due to interaction between environmental and genetic risk factors [1] . ms patients are at increased risk of infections, as disease-modifying therapies may alter the normal function of the immune system, or due to underlying changes in immune response [2] . although hospitalization rates among ms patients have declined over the past few decades, they remain generally higher than in the general population [3] [4] [5] . ms-related factors and infections are the main causes of hospitalizations among ms patients. increased exacerbation rates in ms patients have been reported after influenza illness [6] similar to what has been observed after systemic or general viral infections [7] [8] [9] . viral infections have been shown to be associated with clinical activity of ms [2, [8] [9] [10] , hence infectious diseases such as influenza may induce relapses and cause acute data from the norwegian patient registry has been used in this publication. the interpretation and reporting of these data are the sole responsibility of the authors, and no endorsement by the norwegian patient registry is intended nor should be inferred. worsening of neurologic function, while vaccination against influenza may prevent such adverse events [11, 12] . previous studies have mainly focused on influenza vaccination and risk of ms relapse [12, 13] . in this large register-based study we provide a comprehensive assessment of hospitalization risk after specifically influenza infection. we aim to examine risk of acute hospitalization among ms patients up to 3 months following influenza infection and pandemic vaccination in a large population-based cohort consisting of 6755 ms patients using nationwide populationbased norwegian registries. the study population consisted of all norwegian residents during the period 2008-2014 (n = 5,219,296) as registered in the norwegian national registry. this registry provided information on date of birth, date of emigration, and death. we linked information from several national health registries and databases. all registries contain the personal identification number (pin) which is unique for all citizens, enabling linkage of information at the individual level. information from hospitals and specialist care were obtained from the norwegian patient registry (npr). the npr is an administrative database linked to the reimbursement system with mandatory reporting [14] . diagnoses are reported according to the international classification of disease, version 10 (icd-10). the npr provided information on hospitalizations with icd-10 code g35 ("multiple sclerosis"). information on influenza diagnoses in primary care was retrieved from the norwegian directorate of health which reimburses consultations in emergency outpatient clinics and general practice. influenza diagnoses in primary care were based on the international classification of primary care, second edition (icpc-2) code r80 ("influenza"). the criteria for using the r80 code include specific symptoms (e.g. rapid onset, chills/fever and fatigue) and ongoing influenza epidemic or influenza in the community. these criteria have been described in detail previously [15, 16] . in addition to the clinical diagnoses of influenza, we obtained information on pandemic influenza infections from the norwegian surveillance system for communicable diseases to which laboratory-confirmed pandemic influenza (but not seasonal influenza) was reported [17] . dates of pandemic vaccinations were obtained from the norwegian immunisation registry [18] . the pandemrix ® vaccine was offered to the whole population. notification of pandemic influenza vaccinations to the norwegian immunisation registry was mandatory. information on medical prescriptions with ms specific medications dispensed from norwegian pharmacies was provided by the norwegian prescription database (norpd) from 2004 onwards [19] . the anatomical therapeutic chemical classification system (atc) codes for ms related medications were l03ax13 copaxone ® (glatiramer acetate), l04aa27 gilenya ® (fingolimod), l04aa31 aubagio ® (teriflunomide), n07xx09 tecfidera ® (dimethyl fumarate), l03ab07 avonex ® , rebif ® (interferon beta 1a), l03ab08 betaferon ® , extavia ® (interferon beta 1b), l03ab13 plegridy ® (peginterferon beta 1 a). medication with atc codes l04aa23 tysabri ® (natalizumab) and l04aa34 lemtrada ® (alemtuzumab) are administered in hospitals only, and were not included. the norpd does not hold individual-level information on medication administered during hospitalizations. an ms patient was defined by having at least one ms diagnosis registered in specialist care, combined with at least one dispensed ms medication (54% of patients). if lacking information on medication use, it was required to have at least two registrations of an ms diagnosis (46% of patients). our outcome was defined as acute hospitalizations with an ms diagnosis registered as the cause of hospitalization. timing of influenza was examined in relation to hospitalizations with ms. individuals were considered as having an influenza infection if they were diagnosed with influenza in primary care, or had a laboratory-confirmed diagnosis. we included information on influenza between 1st of january, 2008 (start of follow-up) to 15th of may, 2014 (end of last influenza season in the study). pandemic influenza was defined as a diagnosis of influenza within the main wave of the h1n1 influenza pandemic, which in norway lasted from 1st of october, 2009, to 31st of december, 2009 [16, 20] . seasonal influenza was defined as a diagnosis of influenza during the influenza surveillance periods in norway (september to may) for the period 2008-2014, excluding the pandemic season (from september 2009-may 2010). the season starting september 2014 was not included due to short follow-up time. the pandemic vaccination campaign began on 19th of october, 2009 and vaccine was offered to the whole population free of charge. about 40% of the population was vaccinated and more than 97% of pandemic vaccinations were administered before 31st of december, 2009 [21] . we applied the self-controlled case series (sccs) method to estimate incidence rate ratios (irrs) of acute hospitalization with ms diagnosis in various risk periods following a diagnosis of influenza infection or vaccination, compared with a background period [22] . this method uses information from cases only. the strength of this method is that patients serve as their own controls, thus confounding from all unmeasured factors that do not vary in the study period is eliminated. to avoid surveillance bias, only acute hospitalizations were included. we analysed persontime and timing of hospitalization according to defined time periods (0-1, 2-3, 4-6, 7-9, and 10-12 weeks after influenza infection and vaccination). this study included the influenza seasons from 1st january, 2008 until 15th of may, 2014. individuals were followed until outcome of interest, death, emigration, or the end of study on 31st of december, 2014, whichever occurred first. the sccs approach was applied in four separate models. in model i, we estimated the irr of acute hospitalization among ms patients following a diagnosis of influenza, including any type of influenza (any influenza seasons including the pandemic) in the period from 1st of january, 2008 through 15th of may, 2014. in model ii, we estimated irr of acute hospitalization among ms patients only during the main pandemic period (1st of october, 2009 through 31st of december, 2009). in model iii, the irr of acute hospitalization among patients was estimated following a diagnosis of seasonal influenza infection in the period from 1st of january, 2008 through 15st may, 2014, excluding the pandemic season (from september 2009-may 2010). in model iv, we estimated irr of acute hospitalization following pandemic vaccination (1st of october, 2009 through 30th of april, 2014). irr was estimated using conditional poisson regression and adjusted for age at hospitalization. in an effort to evaluate vaccine safety in patients using different types of ms medications, as suggested in previous studies [23, 24] , we studied the risk of acute hospitalization among ms patients using interferon beta versus non-interferon beta medications. only ms patients with available data on medication were included in this sub-analysis. all analyses were performed by using the stata 13 software (statacorp. 2013. stata statistical software: release 13. college station, tx: statacorp lp.). during 2008-2014, there were 6755 ms patients fulfilling the study criteria. details on ms patients' characteristics is summarized in table 1 . more women (67.1%) than men were registered with ms. average age at first registration with an ms diagnosis in specialist care was 51.8 years among men and 49.9 years among women ( fig. 1 ). during the whole study period, an influenza diagnosis (seasonal and pandemic combined) was registered for 12.7% of ms patients, compared with 12.9% in the general population (table 2) . overall, 60.7% of ms patients received the pandemic vaccine, 57.4% among men and 62.3% among women. in the general population (individuals without an ms diagnosis), 37.3% were vaccinated with the pandemic vaccine, 34.2% among men and 40.4% among women. hospitalizations for ms registered at the same day as the diagnosis of influenza infection was high compared with the number of hospitalizations on subsequent days. there were no differences in the patterns of hospitalizations before and following pandemic vaccinations (fig. 2) . the irr of acute hospitalization within 1 week after an influenza infection (model i) was elevated when compared with the other time periods (irr 3.4, 95% ci 2.4-4.8) ( table 3) . similar findings were observed following pandemic influenza (irr 5.6, 95% ci 2.7-11.3) (model ii) and following seasonal in a sub-analysis on risk of acute hospitalization, we compared ms patients using interferon beta (2592 patients) with those using non-interferon beta medications (2743 patients). the sub-analyses yielded similar results as the main analyses. in this nationwide registry-based study from norway with individual level data. we observed an increased risk of acute hospitalizations with ms within the first week after a diagnosis of seasonal or pandemic influenza infection. pandemic vaccination was not associated with risk of acute hospitalization among ms patients. a major strength of the current study was the availability of information from several independent health registries for the complete norwegian population of more than 5.2 million individuals. further, data on hospitalization, influenza infection and vaccination were prospectively collected and recorded independently in separate databases, by different procedures and systems, which minimizes the risk of bias due to differential reporting and selection. in norway, the public health care system is financed through governmental funding and all hospitalizations are free of charge. ms is a serious condition which is most likely treated in hospitals, meaning that information on hospitalization is recorded in the national registries. norwegian health registries and databases have high quality with mandatory reporting and minimal loss to follow-up. another strength of the study was the sccs method accounting for any factor or characteristic that is not measured but remains constant over the observation period, such as genetics, gender, socio-economic status, and lifestyle related factors [25] . a limitation of this registry-based study is that ms diagnoses were not validated. diagnosis of ms is a prolonged and challenging process and some patients could be misdiagnosed [26] . however, a recent validation study on individuals residing in nordland county in norway and registered with ms in npr examined the medical record of these individuals and reported that 91.5% of patients had a confirmed ms diagnosis [27] . we believe that our inclusion criteria for ms patients (patients with at least two registered diagnoses in specialist care or combination of information on diagnoses in specialist care with information on use of ms medications) provided a highly reliable identification of ms patients. it is likely that we mostly included patients with the relapsing-remitting form with or without secondary progression. primary progressive ms cases accounts for 10-15% of the overall population with ms, and these patients may be missed or under-represented in our study [6, 28] . another weakness of the study is under-reporting of influenza infections in primary care, as only those seeking medical attention for influenza are registered. it has been estimated that around 5-10% of the population is infected with influenza during a regular season [29] . during the 2009 pandemic outbreak less than 4% of the general norwegian population were diagnosed with influenza by a primary care physician [15, 30] , however the clinical attack rate of influenza was estimated to be approximately 25-30.0% [21] . in our study sample, the overall proportion in the general population with an influenza diagnosis during the 2009 pandemic outbreak was 2.5%. among ms patients, 4% were diagnosed with pandemic influenza. this supports that ms patients were more likely to seek medical help when having influenza, possibly due to the seriousness of ms disease and the potential risks associated with having an influenza infection. it could also reflect that ms patients are more susceptible to influenza than healthy persons [2] . however, as those with very mild influenza or milder symptoms of ms may have been less likely to seek health care, our results may reflect associations with more severe influenza symptoms, or associations in patients with more severe ms. another possible weakness is the potential misclassification when using influenza diagnoses from primary care. however, our results remained similar to the main analysis when including only cases with laboratory confirmed pandemic influenza. in accordance with previous studies, there was an increased risk of acute hospitalization among ms patients after an influenza diagnosis [6, 7, 9, 10] , and no association between pandemic vaccination and risk of acute hospitalization was found [11] [12] [13] . more than six thousand ms patients were included in our study, which to our knowledge, is the largest study of seasonal and pandemic influenza infection among ms patients. although we could not identify true ms relapses in our data, we believe studying acute hospitalizations may provide an indication for worsening msrelated symptoms and relapses. previous studies did not focus on influenza infection and mostly combined several types of infections [7] [8] [9] , and these were based on substantially smaller number of patients, and more selected study populations. in previous studies, worsening ms-related symptoms among patients has been reported following infection [2, 7, 8] . disease-modifying therapies alter the immune function and may potentially increase the risk of infections [2] . a population-based swedish study reported higher hospital admission due to infections, including influenza. however, type of influenza and time from infection to hospitalization was not reported [4] . a questionnaire study among 233 dutch ms patients, focused on effect of influenza illness and exacerbation of ms symptoms and reported a significantly higher rate of exacerbations among ms patients with influenza compared with those without influenza [6] . our results support that within the first week following influenza infection, the risk of acute hospitalization was high among ms patients. a longitudinal study in 73 patients with relapsing-remitting multiple sclerosis assessed the contribution of upper airway infections to the risk of exacerbations [7] . they reported a significantly increased risk of exacerbation 2 weeks before until 5 weeks after the onset of a clinical infection (rate ratio 2.1), which also is in accordance with our study. risk of relapse after influenza vaccination (seasonal and pandemic) has been addressed in previous studies which are comparable to ours and no change in risk has been reported [11] [12] [13] . we observed that a higher proportion of ms patients were vaccinated against pandemic influenza compared with the general population. ms patients were defined as a risk group and were especially recommended pandemic vaccination. in our study, we did not observe any change in risk of acute hospitalization following pandemic vaccination which is in concordance with previous findings on safety of vaccination. vaccination is considered as a possible trigger for autoimmune disease activity such as ms, however, previous studies as well as the current indicate that immunization is safe and may prevent influenza infection and potential complications. for ms patients, type and timing of vaccine (with inactivated influenza virus or live attenuated) combined with ms specific medications (interferon-beta versus non-interferon-beta medications) should be considered carefully [9] . in our sub-analyses, stratified by interferon-beta and non-interferon-beta medications, we found no differences in risk estimates related to vaccination. studies of influenza vaccination and risk of relapse have mainly been based on case reports and questionnaires. such study designs are prone to selection/recall bias, and small study samples which may undermine the internal and external validity of a study. we acknowledge that underreporting of influenza infection is highly likely in our study. however, we utilized timing of events in the sccs model in which patients serve as their own control, which enable us to address effects of both seasonal and pandemic influenza. sccs models, to our knowledge, have not previously been applied in this context. careful consideration of vaccination, taking the patient's personal history into account, could help preventing infections and worsening of ms-related symptoms. in this nationwide registry-based study we found an increased risk for acute hospitalization among ms patients within the first week after influenza diagnosis. influenza infections may cause worsening of ms-related symptoms and trigger new relapses and should be prevented when possible. we observed no excess risk of acute hospitalization following pandemic vaccination. our study indicates that ms patients could benefit from influenza vaccination, as reducing the risk of influenza infections could prevent worsening of ms-related symptoms and possibly new relapses. funding the work was supported by the norwegian institute of public health and by the research council of norway through its centres of excellence funding scheme [grant number 262700]. the sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation, review, or approval of this manuscript. multiple sclerosis infection risk in patients on multiple sclerosis therapeutics dramatically changing rates and reasons for hospitalization in multiple sclerosis hospital admission due to infections in multiple sclerosis patients hospital admissions and ms: temporal trends and patient characteristics effects of influenza vaccination and influenza illness on exacerbations in multiple sclerosis prospective study on the relationship between infections and multiple sclerosis exacerbations the risk of relapses in multiple sclerosis during systemic infections immunization panel of the multiple sclerosis council for clinical practice guidelines temporal relationship between environmental influenza a and epstein-barr viral infections and high multiple sclerosis relapse occurrence seasonal and h1n1v influenza vaccines in ms: safety and compliance vaccines and multiple sclerosis: a systematic review immunizations and risk of multiple sclerosis: systematic review and meta-analysis comparison of data from the norwegian patient register and the cancer registry of norway encephalitis after influenza and vaccination: a nationwide population-based registry study from norway risk of fetal death after pandemic influenza virus infection or vaccination the norwegian instiute of public health the norwegian immunisation register-sysvak the nordic countries as a cohort for pharmacoepidemiological research narcolepsy and hypersomnia in norwegian children and young adults following the influenza a(h1n1) 2009 pandemic effect of vaccines and antivirals during the major 2009 a(h1n1) pandemic wave in norway-and the influence of vaccination timing tutorial in biostatistics: the self-controlled case series method immunogenicity and predictors of response to a single dose trivalent seasonal influenza vaccine in multiple sclerosis patients receiving disease-modifying therapies influenza vaccination in patients with multiple sclerosis is possible with some considerations self controlled case series methods: an alternative to standard epidemiological study designs undiagnosing" multiple sclerosis: the challenge of misdiagnosis in ms validation of the multiple sclerosis diagnosis in the norwegian patient registry ocrelizumab versus placebo in primary progressive multiple sclerosis influenza-fact sheet about seasonal influenza burden of medically attended influenza in norway ethical approval the study was approved by the regional committee for medical and health research ethics, south-east region, norway, and the norwegian data protection authority. key: cord-271172-y48dovux authors: potter, christopher william title: chapter 25 respiratory tract viruses date: 1998-12-31 journal: principles of medical biology doi: 10.1016/s1569-2582(97)80009-8 sha: doc_id: 271172 cord_uid: y48dovux summary respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the epidemiology, pathogenesis, clinical disease, and treatment. virus infections of the respiratory tract affect all people in all places at all times throughout life. over 140 viruses are involved: the average child under the age of five years or adult in the developed countries will experience four to five and two to four infections each year, respectively; and although many infections are relatively mild, some are life-threatening. douglas and edelstein (1988) have said that, "about three hundred million cases of acute respiratory disease occur in the united states each year, accounting for about one hundred and fifty million visits to physicians. the cost of these illnesses exceeds one billion dollars exclusive of time lost for work": this statistic, adjusted for population, can be directed at any country. the size of the problem demands much of a general physician's time, while the importance has focused the attention of epidemiologists, microbiologists, pharmaceutical companies and many others. the viruses which cause respiratory tract infections in man include both rna and dna viruses in six families: this does not include many viruses which can cause respiratory symptoms, but where the principal tissue or organ of infection is other than the respiratory tract. these families are listed in table 1 , together with data on the finer classification into sub-families, genera, or sub-genera, the number of distinct types of virus in each group and the number of these that cause human respiratory infections. thus, there are six families encapsulating 21 sub-divisions and over 240 viruses, of which over 140 cause respiratory tract infections in man, while the remainder cause other types of infection or infections in other species. the exact numbers are complicated by the rhinoviruses where more than 100 serotypes are known, but many more probably exist, and by influenza a and b virus which exhibit continuing genetic change producing new antigenic variants in most years. the respiratory tract can be considered to be stratified horizontally into levels, beginning with the nasal passages and descending sequentially to the throat, trachea, bronchi, bronchioles, and the alveoli: it is convenient to divide the respiratory tract in this manner, since many viruses have a predilection for infection of a particular level, and these associations are shown in table 2 . thus, the common cold, limited to the nasal passages, is caused commonly by rhinoviruses and virology. rhinoviruses belong to the family picornaviridae and are distinguished from other members of this family by a higher particle density, acid lability which limits intestinal infection, relative heat stability, resistance to a variety of detergents and survival for several hours or even days outside the body. virus particles contain an internal rna which is single-stranded with positive polarity surrounded by a capsid of 60 capsomeres arranged in icosahedral symmetry: particles are approximately 30 nm in diameter. rhinoviruses replicate in human cells and less well in the cells of other primates: the conditions for growth are a relatively low ph of 7.0 to 7.2, a low temperature of 33 °c and aeration of cell cultures; and under these conditions cytopathic effects can be observed. the growth cycle takes some 11 to 17 hours; and each infected cell yields 10-200 virus particles. cross-neutralization tests indicate over 100 distinct serological types, but some sharing of epitopes does exist (hamparian et al., 1987) . epidemiology. the frequency of respiratory infections is 2 to 4 each year for adults, with a higher incidence in children: most are conmion colds of which some 30% are due to the rhinoviruses, and a number of serotypes can circulate concurrently with no evidence of cross-immunity. infections tend to be most common in spring and autumn, and are spread by large and small droplets of nasal secretions from infected persons, by direct spread from nasal secretions via hand to hand contact and auto-inoculation of nasal or conjunctival mucosa or via inanimate objects: infection can be initiated by very little virus, and the viruses can survive for long periods outside the body. peak titers appear in nasal secretions 1 to 3 days after the onset of symptoms, and virus persists for 5 to 7 days; however, virus secretion can continue for 2 to 3 weeks. a serum antibody response is seen in 30 to 90% of affected patients and a local iga response in 70 to 100%: immunity lasts 2 to 4 years, but the multiplicity of circulating serotypes gives litde or no protection against clinical disease (gwaltney, 1982) . pathogenesis. the pathogenesis of rhinovirus infection is not understood: viruses grow to high titer in the nasal epithelium, but biopsies of antigen-positive nasal mucosa cells show litde pathological change. infection causes progressive loss of ciliated epithelial cells, an inflammatory cell infiltrate, edema, hyperemia and a seromucinous exudation (hendley, 1983) . virus replication is predominantly in the nasal mucosa: other cell types such as in the nares and throat are relatively resistant. clinical features. after an incubation period of 1 to 4 days during which virus titers increase, symptoms of nasal obstruction and discharge, sneezing and coughing occur; a sore throat with erythema and headache may be experienced, but fever and systemic symptoms are usually absent. the symptoms usually improve after 2 to 3 days; but symptoms may last for 7 days, and in 25% of cases for 14 days. complications include sinusitis and otitis media; rhinoviruses have been isolated from the middle ear, but more often a bacterium has been found in conjunction with virus. small particle infection can cause tracheobronchitis and bronchiolitis, and exacerbations of asthma and chronic bronchitis are commonly precipitated by rhinovirus infection. diagnosis. rhinoviruses can be recovered from infected persons by inoculating nasal secretions onto monkey or human cells and incubating these in a roller drum at 33 °c to maximize the cytopathic effects; this appears 2 to 6 days after infection (al-nakib and tyrrell, 1988) . isolates are serotyped using a battery of neutralizing antisera in various combinations, but this is seldom carried out except for research purposes; and the lack of any specific treatment means that laboratory diagnosis is seldom attempted. treatment. there is no specific treatment for rhinovirus infections, and the treatments recommended are to relieve symptoms: nasal decongestants can be used to relieve obstruction; gargles will relieve the sore throat; and analgesics can be used if necessary. if the cough is severe, suppressants such as codeine or dextromethorphan can be given. the multiplicity of antigenic types makes vaccine development impractical, and volunteer studies have shown that inactivated vaccines have litde protective effect. locally applied interferon a suppresses virus replication, but long-term use is cytotoxic causing irritation, bleeding, and ulceration in some patients. virology. coronaviruses, within the family coronaviridae are divided into four antigenic groups containing a total of 13 serotypes of which two are infectious agents of man. the singular structure and method of replication distinguish these viruses. virus particles are 80 to 160 nm in diameter, and contain a large singlestranded rna with positive polarity and a nuclear protein coiled internally to form the nucleocapsid. this core is surrounded by a lipid bilayer into which are inserted three distinct glycoproteins which radiate from the surface: these are a receptor binding glycoprotein which initiates cell fusion and induces neutrahzing antibody production; a glycoprotein with combined neuraminidase and hemagglutinating activity; and a glycoprotein which binds to the internal nucleocapsid and effects virus budding. coronaviruses can be cultivated in organ cultures of human embryonic trachea or less successfully in tissue cultures of human cells. the serotypes are distinguished by neutralization tests, or more commonly by immunofluorescence or elisa tests. epidemiology. coronavirus infections usually occur in the winter and early spring months, with epidemics every 2 to 3 years: the two serotypes causing human infection tend to be exclusive, and 5 to 30% of common colds are due to these viruses. infection is spread by small particle aerosols; many infections are asymptomatic, and may not induce an immune response, but clinical infections invariably induce detectable antibody; however, antibody titers may not be sustained, and reinfection with the same serotype can occur within months (isaacs et al., 1983) . almost 100% of adults have detectable antibody to both serotypes. pathogenesis and clinical disease. these features are similar to those described for rhinoviruses; however, the incubation period is usually longer at 2 to 5 days, the illness persists for 2 to 18 days, some 25% of patients exhibit a mild fever but throat infection and cough is less frequent. complications of infection are unusual, but isolated cases of pneumonia are recorded; and these viruses cause exacerbations of asthma and chronic bronchitis. the pathogenesis of infection is little understood, but the evidence suggests a slow and progressive destruction to the ciliated epithelium. diagnosis and treatment. diagnosis of coronavirus infection is both difficult and unsatisfactory: organ cultures are the most sensitive for virus isolation but are rarely available, and tissue culture systems are relatively insensitive. direct demonstration of virus in cells in respiratory secretions by immunofluorescence or elisa are used in some laboratories, but most diagnoses are made retrospectively by demonstrating a significant rise of antibody following infection (schieble and kapikian, 1979) . as for rhinovirus infection, treatment is symptomatic: no antiviral drugs have been developed and no vaccine is available for coronavirus infections. experimentally, a-interferon has been shown to ameliorate symptoms and reduce virus replication. some 50% of common colds, characterized as an afebrile illness localized to the nasal epithelium and presenting with nasal obstruction and discharge, sneezing and coughing are caused by rhinoviruses and coronaviruses; the remainder are caused by a number of other viruses including echoviruses, coxsackie a and b viruses. respiratory syncytial virus, influenza c and parainfluenza viruses ( table 2) . as these infections are relatively mild and a large number of agents are involved, exact laboratory diagnosis is seldom undertaken except for research purposes, and there are no specific treatments. virology. there are 47 serotypes of human adenoviruses in six subgenera in the genus mastadenovirus in the family adenoviridae: eight serotypes in three subgenera cause respiratory infection in man (table 2 ). virus particles contain single-stranded dna surrounded by at least 11 polypeptides. the outer capsid is made up of 252 capsomeres arranged in an isocosahedral of 20 triangles with 12 vortices giving a particle diameter of 60 to 80 nm: 240 capsomeres are hexons with six neighbors and are composed of a single protein with common epitopes for all serotypes; and the remaining 12 capsomeres are pentons with five neighbors, each composed of a base with a fiber-like projection radiating outwards and are serotype specific. human adenoviruses only replicate sequentially in human cells. virus particles attach to cell receptors via the penton fiber and then to other cell receptors by the hexons: following entry, cellular dna and protein synthesis are inhibited; replication takes place in the cell nucleus; newly-formed pentons are toxic to cells; and virus is released after cell death. the division of human adenoviruses into six subgenera is based on the sequence homology of the dna genome, dna fragment analysis following restriction enzyme treatment, oncogenicity for newborn hamsters, the molecular mass of the internal proteins, length of fibers and precent g + c in the viral dna. the division into serotypes is based on neutralization tests which show no cross-reactivity. this classification is important, since it is consistent with the association of the viruses with various human infections (wadell, 1984) . epidemiology. adenoviruses cause both sporadic and epidemic infections: five per cent of acute respiratory infections and ten per cent of febrile infections of children, together with 3 and 7% of these infections in adults, are caused by these viruses; and some 10% of all pneumonias in children are due to adenovirus infection. transmission is by respiratory droplets, whilst in children fecal/oral transmission is important: fecal excretion can continue for weeks and months after acute infection. by the age of 10 years, 50% of children have antibody to serotypes 1,2, and 5; antibody to other serotypes is less common, but in total the results reflect the importance of these viruses in human infection. pathogenesis. adenoviruses, spread by droplet infection, impinge on epithelial cells in the pharynx or in the lower respiratory tract to enter and kill cells by a combination of inhibition of cellular metabolism, virus replication and the toxic effects of the penton: the results are extensive desquamation of affected areas, causing sore throat, necrotizing bronchitis, bronchiolitis and interstitial pneumonia. in lymphoid cells, infection causes hypertrophy: affected cells can harbor latent virus for months, years or throughout life; and although 50% of excised tonsils and adenoids contain latent virus, there is no evidence that this can exacerbate, except possibly in pertussis syndrome. clinical disease. adenoviruses cause some 5% of all acute respiratory disease (ard) seen in children aged five years or less (brandt et al., 1969) . these are predominantly due to serotypes 1,2 and 5 (subgenera b); and the frequency of these infections in children means adult infections are unusual. spread is by droplet infection: after an incubation period of 2 to 4 days, symptoms begin with pharyngitis, cough, nasal congestion and coryza, whilst fever, exudative tonsillitis, malaise, headache and myalgia are often seen. infection tends to progress over a 2 to 3 day period, and resolves in 5 to 8 days. infection may extend to cause laryngotracheobronchitis, bronchitis and pneumonia, the last being particularly important in young children and the cause of some deaths. sporadic infections of both children and adults are also caused by adenovirus types 3 and 7 (subgenera c): these present as above, but laryngotracheobronchitis and pneumonia are more commonly seen, with 40 to 50% showing radiological evidence of pneumonia; and with conjunctivitis, when the infection is known as pharyngo-conjunctival fever, and that occasionally is seen as epidemics among children and in families. the respiratory tract complications are particularly severe in immunocompromised patients. probably due to overcrowding and fatigue, adenovirus types 3,4 and 7 cause epidemic ard in military recruits: some 80% of recruits can become infected, 20 to 40% require hospitalization for lower respiratory tract infection and pneumonia, and deaths are recorded. adenoviruses have been frequendy isolated from patients with whooping cough syndrome in conjunction with bordetella pertussis', this is usually reported as adenovirus type 5. however, there is little evidence that adenoviruses can produce this syndrome alone: the presence of adenovirus may be due to reactivation from tonsillar tissue by bordetella pertussis to complicate the infective process. diagnosis. adenoviruses are present in throat swabs, throat or nasal washings or feces of infected persons. virus growth is best in tissue culture of human embryonic kidney, but hela and kb cells are a more available alternative. growth is recognized by a characteristic cytopathic effect; however, this may take days or weeks to develop, but virus can be demonstrated early in infected cells by immunofluorescence with specific antibody. isolates can be identified as adenoviruses with antisera to the common epitopes of the hexon using complement fixation, immunofluorescence or elisa tests; and specific serotypes identified by neutralization tests. there are no effective antiviral agents for the treatment of adenovirus infection, and treatment is limited to the relief of symptoms. in addition, there is no adenovirus vaccine; however, the problem of ard in military recruits is of such importance that a vaccine has been developed: live adenovirus types 4 and 7, the main viruses responsible for ard, are given orally as a coated enteric preparation; this establishes an intestinal infection, induces an immune reaction and, in bypassing the respiratory tract, does not cause symptoms (tarafuji etal, 1979) . virology. epstein-barr virus (eb v) is a member of the family herpetoviridae, and the only member of the sub-family gammaherpesvirinae to cause infection in humans: the virus is distinguished from other herpetoviridae in replicating or establishing a latent infection in p-lymphocytes, and the potential for promoting tumorgenicity of these cells. the virus is relatively large at 150 to 200 nm diameter: particles are composed sequentially of linear double-stranded dna and an internal core of proteins; a capsid of 162 capsomeres arranged in an icosahedral form; a protein tegument; and a trilaminar envelope derived from the cellular membrane into which are inserted spikes of several virus-specific glycoproteins. infection is primarily of the epithelial cells of the oropharynx, and via the complement receptor cd21: virus infection from these cells spreads to p-lymphocytes where a few cells undergo lytic infection while the majority support a latent infection that leads to cell proliferation. the mechanism for this latency is not fully understood, but is believed to be due to the absence or low-level of host cell transcriptional factors that are essential for virus replication: the result is a persistent, life-long p-lymphocyte infection. epidemiology serological studies from most developed countries have indicated that eb v infection is common, and 80 to 90% of adults have been infected: in developing countries, infection occurs earlier and is more common. once infected, a subject will remain a virus excretor for months, years and probably life, with continuous virus production from a variable number of p-lymphocytes in the oral cavity. infection is accompanied by an intense immune response; and it is the balance between virus production and immune status which determines the extent of virus secretion at any one time. the immune response is not sufficient to resolve the illness, as infected lymphocytes appear to be resistant to cytotoxic t cells and there is a down-regulation of hla antigen expression. the immortalization of p-lymphocytes is polyclonal and leads to cell proliferation; this can be further exaggerated in immunosuppressed patients, such as hiv or malaria infected persons, which in turn can lead to a chromosome translocation where the c-myc oncogene comes adjacent to a strong promoter: the outcome is a malignant cell transformation giving rise to burkitt's lymphoma (lenair and bornkamm, 1987) . pathogenesis. infection is from saliva of previously infected subjects: since virus production is low, transmission requires close contact, and peaks of infection are seen at ages 1-6 and 14-20 years corresponding to the ages of early and adolescent intimacy. the exact site of infection remains unknown, but waldeyer's ring, rich in lymphocytes, epithelial cells or the salivary glands are suggested by various authors. virus-infected (3-lymphocytes are disseminated throughout the body via the blood stream, and antigen-positive cells can be detected in most organs and tissues. infection is accompanied by an intense immune reaction to virus and to the proliferating p cells which includes virus-specific antibody, heterophile antibody, autoantibodies and rheumatoid factor (robinson and stevens, 1984) ; indeed, the clinical disease is due to the nature and the intensity of the immune responses. the changing pattern of the immune state regulates virus secretion, and the infectivity of the patient for others. clinical features. following contact with infected saliva, there is an incubation period of 30-45 days followed by a short prodromal illness of headache, malaise and fatigue: after this, the definitive symptoms of glandular fever occur. most patients complain of a sore throat with hyperemia and hyperplasia of the lymphoid tissue; exhibit an exudate over the pharynx; have a fever which lasts for some 10 days; and have cervical or general lymphadenopathy: fever and malaise can persist for weeks or months; secondary infections are common; blood dyscrasias occur; splenic enlargement is common, but rarely leads to rupture; and a mild hepatomegaly is seen in 5 to 10% of patients associated with a transient jaundice. mild rubilliform skin rashes can occur; however, ampicillin causes a maculopapular rash and is not used in patients with sore throats. oral cavity obstruction due to massive enlargement of tonsils, adenoids and epiglottis may require emergency treatment. fatalities are recorded, but are usually associated with immunocompromised patients. diagnosis. the symptoms of glandular fever usually alert the physician who can confirm his suspicions by a number of laboratory tests. the virus is difficult to cultivate: in vitro growth is only seen in lymphocytes, but only a few cells support virus replication; however, virus protein or dna can be demonstrated in infected cells by western blotdng or dna hybridizafion tests, respectively. serological tests include the demonstration of serum igm antibody to virus capsid proteins using an elisa test, or the detecuon of the heterophile antibody response using the paul bunnell test. atypical monocytes can form up to 20% of the peripheral blood leucocytes. treatment and control. both interferon and acyclovir have been shown to diminish virus secretion during treatment, but relapses occur when treatment is stopped: more importantly, neither treatment significantly ameliorates symptoms, and are therefore not recommended. in the absence of specific therapy, treatment is supportive: the sore throat can be treated with analgesics; and some suggest corticosteroids limit the duration of illness, possibly by the effect on the immune response. the importance of ebv in initiating burkitt's lymphoma in african children where the incidence is 1:5000, has focused attention on developing an ebv vaccine; such vaccines are undergoing clinical trials at the present time. although febrile sore throats are caused by adenovirus and ebv infection, a large number of other viruses can produce the same clinical symptoms; these include influenza a, b, and c, numerous serotypes of coxsackie and echoviruses and parainfluenza viruses. collectively, these latter viruses are responsible for more than half the febrile sore throats which occur; and collectively, viruses are responsible for over 90% of the febrile sore throats caused by infectious agents. virology. influenza viruses are the only viruses of the genus orthomyxovirus in the family orthomyxoviridae, affecting man, birds, horses, pigs, and other species. the viruses are approximately spherical with a diameter of 80 to 120 nm. each virus particle consists of single-stranded rna of negative polarity segmented into eight fragments of varying size. the rna is closely associated with a nuclear protein (np) and the polymerase enzyme complex to form a helical structure: the np takes one of three antigenic forms which allows influenza viruses to be classified into types a, b, and c. surrounding a nuclear protein is the matrix or membrane protein, and this in turn is surrounded by a lipid bilayer. inserted into the bilayer, and radiating from the surface, are two virus glycoproteins. the most numerous glycoprotein is the hemagglutinin (ha) which is the receptor binding component; the classification of influenza types into subtypes is based principally on the different antigenic forms of the ha (hi, h2, h3) molecule. the second glycoprotein is the neuraminidase (na) which is important in both cell infection and in facilitating the release of newly formed virus from the surface of infected cells; the na is antigenically variable (n,,n2) and this variation is used in the subtype classification of the viruses. influenza viruses grow in human and monkey cells and in the amniotic and allantoic cavity of embryonated hens' eggs. absorbed virus is uncoated and the virus rna together with the polymerase enzyme complex pass to the cell nucleus. replication, which is dependent on cell rna synthesis, produces virus components which pass to the cell membrane for assembly, and are then budded from the cell surface: the complete cycle takes 8 to 10 hours. epidemiology. influenza holds a unique position among the viruses causing respiratory tract infection, since it commonly and dramatically causes local outbreaks or widespread epidemics, and these occur in most parts of the world and in some countries in most years. epidemics occur suddenly and without warning, and the number of people infected range from few hundreds to hundreds of thousands: although short-lived, epidemics can infect up to 70% of a population, with clinical disease occurring in 50% of infected subjects, and deaths due direcdy or indirectly to influenza number from 1000 to 25,000 per 50 million persons per year in developed countries. the importance of this infection in causing morbidity and mortality is reflected in the enormous scientific effort made to understand the virus, the nature of the disease and to devise methods for control. when influenza virus isolates are cross-referenced to patients, time and place, several patterns are seen. firstly, most pandemics and widespread epidemics are caused by influenza a viruses; influenza b viruses are associated with self-limiting epidemics which occur in families or small communities; and influenza c virus is associated with sporadic infections, mainly among young children. influenza a exhibits the greatest antigenic diversity; influenza b exhibits some variation; and influenza c is relatively stable. secondly, the recorded patterns of influenza a epidemics during the past century exhibit two phenomena. every 10 to 15 years since records began in 1890, influenza virus has undergone major antigenic changes in the ha molecule, termed antigenic shift: the emergence of these new subtypes has resulted in the pandemics seen in the years 1933 (hi), 1947(h1), 1957(h2) and 1968(h3); and caused the epidemics which followed until the next new subtype emerged. these subtypes are distinct, and immunity to one provides no protection against infection by others. the origin of new subtypes cannot be by simple mutation from previously existing strains, since many genetic changes occur and intermediary strains are not found. two theories for the origin of new subtypes are advanced. firstly, two influenza viruses, one of human origin and the other probably of avian origin, infect the same cell: due to the segmental nature of the virus genomes, reassortant virus is easily produced combining the properties to infect man and the ha glycoprotein of the non-human strain. the new subtypes can now cause pandemic infection in populations with no previous immunity. an alternative theory is that the various subtypes circulate sequentially over a period of 70 to 80 years, since on two occasions antibody to new serotypes has been detected in sera from elderly people years prior to the emergence of that new subtype to cause pandemic infection: where the viruses survive between times is unknown. between the times of antigenic shift, epidemics occur in most years and the strains which cause them exhibit antigenic drift; these strains belong to the same subtype, but do not cross-react completely, and infection by one strain does not induce solid immunity to later emerging strains. this sequential accumulation of mutations arises naturally, and is selected by antigenic pressure in the immune and partially-immune population. in addition to antigenic drift and shift, viruses isolated from different places at the same time, and even viruses from different individuals in the same epidemic, can exhibit antigenic differences; this is known as inter-and intra-epidemic variation, and both underline the difficulty in matching vaccine virus to epidemic strains, and contributes to the disappointing low levels of immunity induced by inactivated influenza vaccines. the degree of crossprotection is directly related to the degree of cross-reaction of the virus ha, and this is shown in table 3 . influenza b viruses exhibit antigenic drift but not antigenic shift. pathogenesis. the pathogenesis of influenza has not been agreed among researchers, and many features are not understood. virological investigations indicate that infection is from virus inhaled as droplets on to the epithelial cells of both the upper and lower respiratory airway. histological studies of nasal exudate and tracheal biopsies indicate that the major site of infection is the ciliated columnar epithelial cells which become progressively rounded and swollen, and exhibit vacuolation with loss of ciliation; the progression usually begins in the tracheal bronchial epithelium and then ascends. the result is the widespread destruction of the ciliated epithelium down to the basement membrane which itself is not affected; the lesions become increasingly permeable with polymorphonuclear infiltration and edema. because of the generalized symptoms of uncomplicated influenza, viremic spread has been suspected; however, there is no conclusive evidence that viremia occurs. in contrast, virus infections have been associated with ecg and eeg changes; some unconfirmed observations of virus antigen in brain and heart tissue have been published; and infection can be associated with viral encephalitis, particularly among children. these findings suggest dissemination of either virus or virus products from the respiratory tract; virus is known to grow in leucocytes and the release of pyrogens or cytokines offers an explanation for some systemic symptoms. clinical disease. the symptoms of influenza tend to be constant regardless of the subtype or strain of virus; however, the clinical features of influenza in young children may vary from that of adults, with croup a more common symptom in children and sore throats more common in adults. droplet infection is followed by an incubation period of approximately 24 to 96 hours: the onset of illness is usually abrupt. symptoms include fever, headache, photophobia, shivering, dry cough, malaise, aching of muscles and a dry, ticking throat which can lead to the voice becoming husky or lost. the eyes are often watery, burning and painful on movement. fever is usually continuous, and typically lasts some 3 days: in a percentage of patients, a second rise in temperature may occur, usually smaller than the first, which gives the infection a biphasic fever curve. the cough may persist for several days; the nose can be blocked or show a purulent discharge; and myalgia is most severe in leg muscles, but also may involve the other extremities. acute illness usually resolves within 7 days, but patients frequently complain of feeling listless for weeks, and depression is a common residual complaint. infections caused by influenza b resemble closely those caused by influenza a; in contrast, influenza c is usually a mild upper respiratory tract infection. the complications of infection include tracheobronchitis and bronchiolitis: these patients exhibit a productive cough and chest tightness, and crepitations are commonly heard but the lungs are usually radiologically clear. these complications are most conmionly seen in patients with obstructive bronchitis and in older people, and death from influenza can result in such patients. pneumonia in patients with influenza virus infection can be primary or secondary. in viral pneumonia, patients developed a persistent fever and leucocytosis, dyspnea, hypoxia, and cyanosis; this follows the acute symptoms described above. sputum specimens show no bacterial cause, and a proportion of these patients die of diffuse hemorrhagic pneumonia. more commonly, pneumonia following influenza is due to secondary bacterial infection, principally with staphylococcus aureus, but also with streptococcus pneumoniae, hemophilus influenzae and other bacterial species: this complication is a major cause of death among elderly people and those with underlying disease such as congestive heart failure and chronic bronchitis. in addition, patients with diabetes, renal disease, alcoholism and those who are pregnant also have an increased susceptibility to secondary bronchopneumonia. influenza is also associated with myalgia, a common feature of acute disease, but clinical myositis and myoglobinuria can occur: the symptoms develop after the onset of respiratory infection, when muscles become painful and tender, but without neurological symptoms. an important complication of influenza infection is the syndrome known as reye's syndrome characterized by encephalopathy and fatty liver degeneration; this is chiefly seen at age 8 to 15 years, and among those hospitalized the mortality can be as high as 50%. the association of reye's syndrome following infection by influenza a or b or other viruses has been fully demonstrated, but the pathogenesis remains obscure (carey et al., 1976) : researchers have highlighted the association of virus infection with treatment of fever with high concentrations of aspirin, and for this reason aspirin should not be given to patients in this age group. more conjectural is the association of influenza infection in pregnancy with congenital abnormality; this is not justified with our current knowledge. further complications reported are ketoacidosis in diabetic patients, acute viral encephalitis in children, guillain-barre syndrome, sudden infant death syndrome and toxic shock syndrome resulting from the dual association of staphylococcus aureus and influenza infection. diagnosis. influenza viruses can be recovered from throat washings or swabs by inoculating tissue cultures of kidney tissue from rhesus monkeys, chicks, and a variety of other species. after incubation, newly produced virus can be detected in supernatant fluids by the ability to agglutinate erythrocytes (hemagglutination), or the adherence of erythrocytes to virus particles assembled on the cell surface (hemadsorption). influenza virus can also be cultured in the amniotic cavity of embyronated eggs: after incubation, high titers of virus are found in the amniotic fluid, and are detected by hemagglutination. the viruses are recognized as influenza a, b, or c by complement fixation tests using extracts of infected cells containing high concentrations of np antigen, and type-specific antisera. further identification of influenza isolates into sub-types and strains is dependent upon antigenic differences in the ha. this is determined by hemagglutination inhibition (hi) tests against antisera raised in experimental animals against a range of virus subtypes and strains: the titer of each antisera against homologous virus is known prior to testing, and the pattern of hi titers found against an unknown influenza virus determines the strain and type. however, this is a highly specialized typing system which is the responsibility of who reference laboratories who constantly type new isolated viruses in a worldwide endeavor to detect new virus variants as they arise. proof of influenza infection can also be obtained by demonstrating a rise in specific complement fixating or hi antibodies in sera collected early after the onset of symptoms and 14 to 21 days later. control and treatment. the constant, almost annual antigenic changes seen in influenza virus a and to a lesser extent influenza b means that vaccines need to be developed for each new epidemic strain. at present, vaccines are produced by inoculating virus into embryonated eggs, purifying and inactivating the resultant virus growth to give a whole virus, disrupted virus or virus subunit (ha and na) vaccines (potter, 1982) . to date, no live attenuated virus vaccines are available. inactivated vaccines produce few reactions, but most are mild and ephemeral; induce serum antibody in the majority of subjects, but immunity in only 60 to 90% of vaccinees. due to the severity and fatalities from influenza, vaccine is offered annually to at-risk patients: these include persons aged 65 yrs of age, patients with a history of chronic chest or heart disease, and patients with asthma, renal dysfunction and metabolic disorders. in some years, such as when a new subtype is recognized, key personnel in industry and social services should be offered vaccine. at present, the treatment of influenza is symptomatic: patients are advised to remain in bed for 2 to 3 days until the acute symptoms subside; symptoms of headache and fever are treated with paracetamol; codeine linctus can relieve the cough; insomnia may be treated by barbiturates or promethazine; and antibiotics are indicated when chest complications are present or suspected. the use of prophylactic antibiotics in patients with chronic chest disease is common, but not recommended. the compound amantadine, and the analog rimantidine, are active against influenza: an oral dose of 100 mg per day given to people in contact with influenza decreases the chance of infection by some 70%, and given to patients with clinical disease can reduce both the length and severity of disease (dolin et al., 1976) . virology. parainfluenza viruses of the genus paramyxovirus in the family paramyxoviridae are distinguished by the size and shape of the nucleocapsid, biochemical similarity, antigenic cross-reactivity and the presence of a surface glycoprotein with combined hemagglutination and neuraminidase activity. virus particles consist of single, non-segmented, negative-strand rna, and three internal proteins surrounded by a lipid bilayer with a fourth protein, into which are inserted the hemagglutinin/neuraminidase molecule and a fusion protein which both radiate from the surface of the virion particle. the complete virion has a diameter of 150-200 nm. the viruses replicate in primary human and monkey cells with assembly of new virus particles taking place in the cytoplasm and release by budding: the effect on the cells is lytic with cytopathic effect and occasionally syncytia formation. viruses can be detected by hemadsorption of guinea-pig erythrocytes to infected cells through virion particles budding through the cell membrane. four serotypes cause respiratory infection in man, and are individually recognized by various tests including hemagglutination inhibition, hemadsorption inhibition and neutralization tests. epidemiology. parainfluenza viruses types 1, 2, and 3 are the major cause of tracheobronchitis and croup in young children; type 3 is frequently associated with pneumonia; and type 4 causes mild upper respiratory tract infections. infection is by droplets and requires only a small dose of virus; virus from infected persons is shed for 3 to 10 days, but in some cases can continue for 3 to 4 weeks. infection by all types is worldwide with peak numbers occurring in the winter months: epidemics are frequently recorded, and reinfection common (chapman et al., 1981) . over 50% have antibodies to one or more of these viruses by age 2 years, and over 75% by age 4 years. pathogenesis. mild infections are mostly of the nose and throat with minimal involvement of the lower respiratory tract; more extensive infection by types 1 and 2 involves the larynx, trachea, and bronchi with pneumonia occurring in some 15 to 20% of patients; and type 3 infection causing a higher incidence of bronchiolitis and pneumonia. virus replication has a lytic effect on the epithelial cells, whilst in the trachea and bronchi infection causes excess mucus production leading to atelectasis and pneumonia. infection induces an ige antibody response in serious cases which in turn initiates histamine release: it is thought that these responses are important in the pathogenesis of infection. clinical features. following an incubation period of 2 to 4 days, primary infection in children is seen as a rhinitis and pharyngitis with erythema: some evidence of bronchitis is commonly seen with hoarseness, cough with croup and bronchitis with rhonchi. fever is recorded and lasts 2-3 days. in more severe cases, infection extends to produce a heightened fever, a laryngotracheobronchitis with a barking cough and croup which lasts for 48 to 72 hours; symptoms may worsen to cause air hunger and cyanosis, sternal and intercostal retractions, airway obstruction and glottic and subglottic narrowing (parrott et al., 1962) : if pneumonia develops, the cough is productive, and radiological examination may show interstitial and perihylar infiltration. diagnosis. viruses present in throat washings can be cultivated on monkey kidney cells: after replication, virus particles can be demonstrated on the surface of infected cells by hemadsorption. alternatively, virus can be detected directly by immunofluorescence tests on respiratory secretions (ray and minnich, 1987) . diagnosis based on serological tests is less satisfactory: primary infection induces a type-specific antibody response detectable by hemagglutination inhibition, complement fixation, or neutralization tests, but subsequent infections induce a heterotypic response. control and treatment. much research has been carried out on the development of a parainfluenza virus vaccine but none is available at the present time: inactivated vaccines induce serum antibodies, but only partial immunity. treatment is symptomatic: children may be nursed in plastic tents supplied with cool moistened oxygen for 2 or 3 days to relieve respiratory symptoms; severe obstruction may require endotracheal intubation or tracheostomy; and accumulative and excessive tracheobronchial secretion may require bronchoscopy aspiration. antibiotics are used where investigations indicate secondary bacterial infection. the use of corticosteroids is contentious, but aerosolized preparations of the antiviral compound ribavirin may be valuable. some 50% of cases of laryngotracheitis and croup in children under age three years are due to parainfluenza virus infection; the remaining cases are due to influenza a and b, respiratory syncytial virus (rsv) and various serotypes of coxsackie and echoviruses. the severity of infection indicates laboratory investigation; thus, the contribution of these latter agents to the syndrome is well-documented. acute bronchitis, or more commonly tracheo-bronchitis since contiguous respiratory compartments are usually involved, has been associated with adenovirus type 3, 4 and 7 infection in both children and adults, but other serotypes have been identified. among the other infections causing bronchitis are rhinoviruses and rsv virus in children, and measles and influenza a virus infections in children and adults: these infections may precede secondary bacterial infection. exacerbation of chronic bronchitis is frequently associated with virus infections; these include a wide range of viruses, but are most commonly caused by rsv, rhinoviruses, and parainfluenza viruses. virology. respiratory syncytial virus (rsv) belongs to the genus pneumovirus within the family paramyxoviridae; and is distinguished by the form of a nucleocapsid, replication entirely in the cell cytoplasm and the absence of hemagglutinin and neuraminidase glycoproteins. the virus particle is structurally similar to that of other members of the family consisting of a single, non-segmented, negative strand of rna and three internal proteins surrounded by a lipid bilayer with two associated proteins: inserted into the outer aspect of the lipid bilayer are spikes of an attachment protein and a fusion protein. the diameter of the virion is 120 to 300 nm. rsv replicates in the cytoplasm of a range of human and animal cells: cell death is principally the result of cell fusion, and the formation of multinucleate syncytia indicates the presence of virus and gives the virus its name. antigenic variants are known, and this has resulted in recognition of two subgroups. epidemiology. rsv causes annual epidemics in the winter months in most countries which are indicated by an abrupt rise in the number of pediatric admissions to hospital (glezen and denny, 1973) . infection is spread by large droplets and therefore require close contact; by hand from nasal and conjunctival secretions; or via inanimate objects and self-inoculation: the virus is highly infectious, and 50% of children are infected by 1 year of life, and all children by age 2 years, with recurrent infection common. the virus causes 75% of all bronchiolitis and 25% of all pneumonia cases seen in children under 1 year of age: the mortality rate is 0.5 to 2.5% with most in children with underlying heart or respiratory disease. infection is essentially an upper respiratory tract infection in children aged less than 6 weeks or over 6 months; however, between these age limits 30% of infections involve the lower respiratory tract. recovery is accompanied by serum antibody and a cell-mediated immune response which protects against subsequent lower respiratory tract infection; the local iga antibody response is ephemeral, allowing further upper respiratory tract infections in later life. pathogenesis. after an incubation period of 3 to 6 days, infection begins in the nasopharynx with virus titers reaching a maximum at 2 to 3 days, declining between 3 to 6 days but can be detected in some patients for 3 weeks. spread to the lower respiratory tract is by cell-to-cell interaction in the respiratory epithelium and via aspirates. cell infection is cytopathic following cell fusion, causing inflammation and necrosis with associated plugging of the airways; but other factors are involved in the disease which are not fully defined: these include immunopathology due to antibody production, the formation of antigen-antibody complexes, delayed hypersensitivity reactions, an exaggerated cytotoxic t-cell response and an ige response as described for parainfluenza virus infection (welliver et al., 1984) . clinical disease. in children aged less than 6 weeks or greater than 6 months infection is usually seen as an upper respiratory tract (urt) infection with rhinitis, a mild fever, sneezing, and wheezing; some 40% of children exhibit a lower respiratory tract involvement with tachypnea, rales, and rhonchi. more severe lower respiratory tract (lrt) infection may occur, but this is more common at age 6 to 24 weeks: following the mainly urt infection, patients develop a bronchiolitis with dyspnea, severe tachypnea, and intercostal and substernal retraction; and in most severe cases an added pneumonia occurs with hypoxia and cyanosis. radiological appearances vary from normal to that of a bacterial pneumonia, but clinical severity is not mirrored by the radiological changes. the infection lasts 6 to 12 days with patients showing improvement after 3 to 4 days, but in severe cases symptoms may persist for several weeks. diagnosis. aspirates or nasal secretions contain virus that can be detected by inoculating tissue cultures which show syncytia formation following virus replication, or by direct tests for virus antigen using immunofluorescence tests. infection induces a rise in serum antibody detected by complement fixation or neutralization tests. treatment. patients with lrt infection commonly require hospitalization for supportive therapy: reduction of fever and hydration is commonly adequate, but in more severe cases oxygen may be required to assist breathing; mechanical removal of respiratory secretions may be necessary, and blood gases should be monitored. the infection responds to treatment with the antiviral compound ribovirin, and administration of this compound as a small particle aerosol has proven effective (hall et al., 1983) . no effective vaccine has been developed despite 20 years of dedicated research. studies have shown that 50 to 90% of bronchiolitis cases are caused by rsv; and characterized by necrosis and sloughing of the bronchiolar epithelial leading to the plugging of small airways, obstruction and atelectasis (hall et al., 1986) . however, other viruses more appropriately associated with other compartments of the respiratory tract, can produce the same pathological changes and clinical symptoms. thus, bronchiolitis has been associated with infections by influenza viruses a and b, and adenoviruses; and in young children with parainfluenza virus infection: these virus infections are described under separate headings. pneumonia, characterized by radiological changes, physical signs and pathology, is uncommonly related to infection by any virus; however, three reservations should be admitted. firstly, cases of acute pneumonia due to adenovirus and influenza viruses, although unusual, are well-documented, and fatalities have been recorded following these infections. secondly, severe infection by viruses in higher compartments of the respiratory tract, can extend to cause pneumonia: these include adenovirus and influenza viruses again, and rsv and parainfluenza viruses in young children. thirdly, primary pneumonia is a rare presentation by measles, chicken pox (varicella/zoster) and cytomegalovirus (cmv) infection: although unusual in normal subjects these are more commonly seen in immunocompromised persons, where the infection can be devastating. pneumonia in the immunocompromised by measles, chicken pox or cmv is usually an extension of typical infection to involve the lungs, but can present without a rash or with an atypical rash in patients with no history of infection. patients develop cough and chest pains, and more seriously dyspnea and cyanosis; x-rays may show evidence of viral pneumonia with atypical, patchy consolidation; and deaths are recorded in 40% or more of immunocompromised patients. pathologically, the alveoli contain edema fluid with macrophages, but few polymorphonuclear cells; and typically and diagnostically, giant multinuclear cells. the viruses can be grown from the bronchial secretions; however, since these are rapidly progressing infections and suggested treatments are available for two of these viruses, quicker methods such as the direct demonstration of virus by immunofluorescence or elis a tests on cells in secretions are needed. there is no treatment for measles virus pneumonia; acyclovir or ganciclovir are reported to be of value in the treatment of cmv; and acyclovir will prevent pneumonia in chicken pox patients, but has no proven value in treating established pneumonia. respiratory tract infections are among the commonest of illnesses, and most individuals will experience two to five infections during each year of their lives. the illnesses vary from relatively mild common colds caused by rhinoviruses and coronaviruses, to severe bronchiolitis and pneumonia caused by adenoviruses and influenza viruses and respiratory syncytial virus (rsv) in infants: the former is associated with little morbidity and no mortality, while influenza is responsible annually for between 1 and 25 thousand deaths per 50 million population. over 140 viruses cause respiratory tract infections, with the added complications of influenza viruses where new antigenic variants are recognized almost annually; and immunity to infection by one virus strain offers little or no protection to infection by others. knowledge of the mechanisms of spread of respiratory viruses is largely understood and has helped in infection control; however, the clinical signs and symptoms of infection tend not to be diagnostic of the causative agent; and although vaccines have been developed for the more serious infections such as influenza and some adenovirus infection, none are available for other important infections. treatment is largely symptomatic, but the compounds ribovirin for rsv infection and amantadine for influenza virus infection have been shown to be effective. much remains to be discovered before more effective measures can be implemented to limit the enormous costs incurred by these infections. the number of viruses involved is large, and the spectrum of illness complex: in the present chapter, the viruses are described, together with the features of the 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study of the multistep development of cancer: proposal for a new scenario inactivated influenza virus vaccine efficiency of immunofluorescence for rapid diagnosis of common respiratory viruses production of autoantibodies to cellular antigens by human b cells transformed by epstein-barr virus coronaviruses. in: diagnostic procedures for viral, rickettsial and chlamydial infections simultaneous administration of live, enteric-coated adenovirus 4, 7 and 21 vaccines: safety and immunogenicity molecular epidemiology of human adenoviruses defective regulation of immune responses in respiratory syncytial virus infection rhinoviruses in seattle families 1975-1979 the tecumseh study of respiratory disease vi. frequency of the relationship between outbreaks of coronavirus infection world-wide epidemiology of human adenovirus infections influenza seminars in respiratory infections parainfluenza virus bronchiolitis: epidemiology and pathology respiratory syncytial virus epidemiology of respiratory syncytial virus infection in washington d.c. ill composite analyses of eleven consecutive yearly epidemics respiratory syncytial virus: brief review key: cord-287824-zg5akivn authors: chan, yinghan; ng, sin wi; mehta, meenu; anand, krishnan; kumar singh, sachin; gupta, gaurav; chellappan, dinesh kumar; dua, kamal title: advanced drug delivery systems can assist in managing influenza virus infection: a hypothesis date: 2020-09-24 journal: med hypotheses doi: 10.1016/j.mehy.2020.110298 sha: doc_id: 287824 cord_uid: zg5akivn outbreaks of influenza infections in the past have severely impacted global health and socioeconomic growth. antivirals and vaccines are remarkable medical innovations that have been successful in reducing the rates of morbidity and mortality from this disease. however, the relentless emergence of drug resistance has led to a worrisome increase in the trend of influenza outbreaks, characterized by worsened clinical outcomes as well as increased economic burden. this has prompted the need for breakthrough innovations that can effectively manage influenza outbreaks. this article provides an insight into a novel hypothesis that describes how the integration of nanomedicine, with the development of drugs and vaccines can potentially enhance body immune response and the efficacies of anti-viral therapeutics to combat influenza infections. apart from covid-19, influenza is another infectious disease that ranks high as one of the 62 deadliest, characterized by a remarkably high rate of transmission that could cause a rapid 63 spread. it is estimated that influenza kills approximately 500 thousand people yearly [4, 8] . 64 killed virus vaccine as an intramuscular injection and attenuated live vaccine as a nasal spray, 65 are the two most widely known vaccines for this deadly virus [9] . in the recent years, an 66 increasing trend of influenza outbreaks have been observed, prompting medical researchers to 67 design and develop suitable vaccines and novel therapeutic modalities [10] . despite the 3 68 availability of vaccines that may protect individuals from well-matched strains, it is well-69 known that the influenza virus has high mutation rates, resulting in frequent mismatches due 70 to antigenic drift and shift, thereby, necessitating the development of a new vaccine every few 71 years [11] . however, the development of a new vaccine is time-consuming. in addition, 72 vaccine-development remains mostly applicable to developed countries, attributing to the cost 73 factor involved. moreover, long term use of standalone anti-influenza drugs and vaccines are 74 often associated with adverse reactions and other shortcomings, that limit their effective 75 clinical applications [12] . for instance, although the neuraminidase inhibitor oseltamivir has 76 been widely employed as an anti-influenza drug, it was found that the drug does not offer 77 benefits in patients with pre-existing medical conditions [12, 13] predict possible chronic and other unforeseen in-vivo effects [61, 62] . hence, it is hoped that 327 this hypothesis will trigger further exploration into nanomedicine-based approach to elucidate 328 the in-depth mechanisms involved, along with their safety, to pave way for a paradigm shift in 329 influenza management approaches. the authors declare that they have no known competing financial interests or personal 338 relationships that could have appeared to influence the work reported in this paper. single-371 dose mucosal immunization with a candidate universal influenza vaccine provides 372 rapid protection from virulent h5n1, h3n2 and h1n1 viruses exploiting nanotechnology to target viruses universal influenza vaccines: from viruses to 378 nanoparticles nanotherapeutic anti-influenza solutions: current knowledge and future challenges influenza antivirals and resistance: the next 10 384 years? influenza viruses -antiviral therapy and resistance influenza vaccines: evaluation of the 389 safety profile immunogenicity and 392 protection of oral influenza vaccines formulated into microparticles celastrol-loaded liquid crystalline nanoparticles as an anti-inflammatory intervention 396 for the treatment of asthma nanomaterials designed for antiviral drug delivery transport across biological barriers dramatically increase zanamivir absolute bioavailability in rats: implications for an 403 nanotechnologies for boswellic acids the potential 408 of sirna based drug delivery in respiratory disorders: recent advances and progress targeting 411 neutrophils using novel drug delivery systems in chronic respiratory diseases emerging 414 complexity and the need for advanced drug delivery in targeting candida species albumin nano-encapsulation of piceatannol enhances its anticancer potential in 418 colon cancer via downregulation of nuclear p65 and hif-1α. cancers (basel) nanomedicine for infectious disease applications: 421 innovation towards broad-spectrum treatment of viral infections nanoparticles in influenza 424 subunit vaccine development: immunogenicity enhancement. influenza other respi 425 nanoparticle-based 427 vaccines against respiratory viruses organic and inorganic nanoparticle vaccines for prevention of 430 infectious diseases formulation and characterization of glibenclamide and quercetin-434 loaded chitosan nanogels targeting skin permeation patented therapeutic 437 drug delivery strategies for targeting pulmonary diseases increasing 440 complexity and interactions of oxidative stress in chronic respiratory diseases: an 441 emerging need for novel drug delivery systems nanotechnology-based antiviral therapeutics rutin loaded 446 liquid crystalline nanoparticles inhibit lipopolysaccharide induced oxidative stress and 447 apoptosis in bronchial epithelial cells in vitro biopolymer 450 encapsulated live influenza virus as a universal cd8+ t cell vaccine against influenza 451 virus. vaccine anti-455 bacterial activity of inorganic nanomaterials and their antimicrobial peptide conjugates 456 against resistant and non-resistant pathogens perspectives and 459 advancements in the design of nanomaterials for targeted cancer theranostics microparticles as vaccine adjuvants and delivery systems investigation of 465 tunable acetalated dextran microparticle platform to optimize m2e-based influenza 466 vaccine efficacy molecular assembly and application of biomimetic microcapsules hybrid inorganic-organic capsules for eficient intracellular delivery of novel sirnas 472 against influenza a (h1n1) virus infection biodegradable polyelectrolyte/silica composite microcapsules as carriers for small 476 application of dendrimers for the treatment of 479 infectious diseases nanoparticle vaccines against infectious diseases delivery vehicles for active phytoconstituents emerging trends 492 in nanomedicine for topical delivery in skin disorders: current and translational 493 oligonucleotide 495 therapy: an emerging focus area for drug delivery in chronic inflammatory respiratory 496 diseases emerging trends in 498 the novel drug delivery approaches for the treatment of lung cancer interactions 501 with the macrophages: an emerging targeted approach using novel drug delivery 502 systems in respiratory diseases gene delivery by pamam dendrimer 505 conjugated with the nuclear localization signal peptide derived from influenza b 506 identification of biomarkers and genetic approaches toward chronic obstructive 510 pulmonary disease inhibition of influenza a virus infection in vitro by saliphenylhalamide-loaded 513 porous silicon nanoparticles enhanced inhibition of influenza virus infection 516 by peptide-noble-metal nanoparticle conjugates development of an adjuvanted nanoparticle vaccine against influenza virus, an 520 in vitro study pulmonary surfactant-biomimetic 523 nanoparticles potentiate heterosubtypic influenza immunity protein nanoparticle immunization induces broad cross-protection against 527 different influenza viruses in mice virus-mimetic polymer 530 nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine porous gold nanoparticles for 533 attenuating infectivity of influenza a virus inhibition of h1n1 influenza virus infection by zinc oxide nanoparticles: 537 another emerging application of nanomedicine phase 3 pivotal trial of nanoflu tm in older adults editorial: advances and challenges in nanomedicine nanoethics: from utopian dreams and apocalyptic nightmares towards a 543 more balanced view the authors declare no conflict of interest, financial or otherwise. key: cord-268693-td6kvmlq authors: martins, leila droprinchinski; da silva, iara; batista, wellington vinicius; de fátima andrade, maria; dias de freitas, edmilson; martins, jorge a. title: how socio-economic and atmospheric variables impact covid-19 and influenza outbreaks in tropical and subtropical regions of brazil date: 2020-09-16 journal: environ res doi: 10.1016/j.envres.2020.110184 sha: doc_id: 268693 cord_uid: td6kvmlq covid-19 has been disturbing human society with an intensity never seen since the influenza epidemic (spanish flu). covid-19 and influenza are both respiratory viruses and, in this study, we explore the relations of covid-19 and influenza with atmospheric variables and socio-economic conditions for tropical and subtropical climates in brazil. atmospheric variables, mobility, socio-economic conditions and population information were analyzed using a generalized additive model for daily covid-19 cases from march 1(st) to may 15(th), 2020, and for daily influenza hospitalizations (2017-2019) in brazilian states representing tropical and subtropical climates. our results indicate that temperature combined with humidity are risk factors for covid-19 and influenza in both climate regimes, and the minimum temperature was also a risk factor for subtropical climate. social distancing is a risk factor for covid-19 in all regions. for influenza and covid-19, the highest relative risks (rr) generally occurred in 3 days (lag=3). altogether among the studied regions, the most important risk factor is the human developed index (hdi), with a mean rr of 1.2492 (95% ci: 1.0926-1.6706) for covid-19, followed by the elderly fraction for both diseases. the risk factor associated with socio-economic inequalities for influenza is probably smoothed by influenza vaccination, which is offered free of charge to the entire brazilian population. finally, the findings of this study call attention to the influence of socio-economic inequalities on human health. the covid-19 pandemic has been destructive to human lives, economy and social relations around the world. in just a few months, it quickly spread to all countries and killed more than 800,000 people, consuming tens of millions of jobs (worldometer website) . covid-19 is caused by sars-cov-2, which can be transmitted by two main ways: contact (surface contact, i.e. fomites and contact with a contaminated person) and by air, via droplets and aerosol (kutter et al., 2018; patients et al., 2020; setti et al., 2020; stadnytskyi et al., 2020; tellier et al., 2019; world health organization, 2020) . influenza, another respiratory virus, has also caused tens of thousands of deaths worldwide each year. the influenza virus, which can also cause severe acute respiratory syndrome (sars), has forms of transmission similar to although there is no consensus on the relevance of each mode (brankston et al., 2007; killingley and nguyenvan-tam, 2013; krammer et al., 2018; kutter et al., 2018; lee, 2007; tellier, 2006) . therefore, the transmission of both is susceptible to climate conditions and, in the case of influenza, seasonality is already described in the literature (alonso et al., 2007; carleton and meng, 2020; li et al., 2015; moriyama et al., 2020; petrova and russell, 2018; russell et al., 2008; shaman and kohn, 2009; shimmei et al., 2020; sobral et al., 2020) . the world pandemic caused by covid-19 has demanded a huge effort of the scientific community to identify the relevant factors and their associations with virus transmission. as the virus spreads to new areas, more studies are required to understand the disease under the socioeconomic and climate conditions of different countries, especially those in africa and south america (belser, 2020; marson and ortega, 2020; wilder-smith et al., 2020) . besides social distancing, the influence of climate variables on sars-cov-2 spread capacity has already been j o u r n a l p r e -p r o o f explored by some studies (ma et al., 2020; qi et al., 2020; tobías and molina, 2020; xie and zhu, 2020) , as well as other factors (domingo and rovira, 2020; sarmadi et al., 2020; urrutia-pereira et al., 2020) . however, the relations of climate variables were based only on subtropical regions, which makes the pandemic view somewhat early and narrow. even for influenza, only a few studies were conducted in tropical regions and a deep investigation of its relation with environmental variables is still needed (alonso et al., 2007; krammer et al., 2018) . however, such previous studies can provide important insights on , some studies analyzed the influence of temperature, humidity and rainfall (auler et al., 2020; prata et al., 2020; rodrigues et al., 2020; tenório and lansac-tôha, 2020) for some brazilian cities. some factors cannot be ignored to address the spread of respiratory viruses as sars-cov-2 and influenza. for instance, the effects of population age, hygiene measures, social distancing, socioeconomic conditions and climate. therefore, in this study, we investigated the associations of covid-19 cases and the number of hospital admissions by influenza with atmospheric variables, social distancing and socio-economic conditions for different climate regions in brazil. besides, we compare the results of both diseases, aiming to establish a robust relation between influenza and covid-19. brazil is the unique huge country that extends from the equator to the subtropics (5 o 16`19`` n and 3 o 45`07`` s, with an area of 8 516 000 km 2 ) and thus has different climate zones (fig. 1) . the socio-economic conditions also show wide variations inside the country; thus, it is a perfect j o u r n a l p r e -p r o o f area to study the influence of these factors on the dissemination of covid-19 and influenza, two respiratory viruses with similarities in the modes of transmission. in addition, the inherent bias associated with the data record can be minimized using the information of the same country, mainly for covid-19, which is a new disease. another important feature used was to choose regions that do not represent the main gates for people and goods entering the country. the only exception was the state of amazonas, the home of the manaus free trade zone. however, we kept the region in the study, since it is a very particular tropical area, where the main city of manaus is surrounded by the largest tropical forest in the world. the amazonas state has a low population density (2.23 inhabitants km -2 ), but 51% of the population is living in the capital manaus. j o u r n a l p r e -p r o o f five brazilian states representing different climate regimes were chosen (fig. 1) . the state of amazonas (am) has a tropical rainforest climate, with annual average temperature and rainfall of 27.4 °c and 2145 mm, respectively. this type of climate is classified as af (tropical without a dry season), following the köppen climate classification (alvares et al., 2013) . maranhão (ma) and ceará (ce) have a tropical savanna climate, predominantly aw (tropical with a dry winter) and as (tropical with a dry summer), respectively. they have a similar annual pattern of rainfall and temperature, with low-temperature variations during the year, but with well-defined seasonality for the monthly rainfalls. for both states, the rainy season occurs between february and may, while the driest period occurs between august and november. espírito santo (es), located in southeast brazil, has also a tropical savanna climate (aw) in most of its area, but with monthly rainfall not changing significantly over the year, as in ma and ce. there is no welldefined dry season and only a slightly rainy period from november to january. the state of paraná (pr) has well-defined seasons (summer, fall, winter and spring), with humid subtropical (cfa and cfb) oceanic climates, without a dry season. curitiba is the main city of the state, and the average temperature and monthly rainfall are 17.1 °c and 130 mm (climate-data website). the meteorological data were compiled from the national institute of meteorology stations (instituto nacional de meteorologia -inmet, see supplementary material), which consisted of the average daily maximum and minimum temperature (°c), rainfall (mm), relative humidity (%), wind speed (m s -1 ) and insolation (hours), from january 1 st , 2017 to may 15 th , 2020 (inmet website). as the wind speed is not available for all stations, it was not included in the final regression analysis. by the way, no statistical significance was found for this variable in our analysis. should be shared between the three levels of government: federal, state and municipal administrations. however, only the last two moved to do something in their own way, without any coordination or effort from the ministry of health. as a measure of the recognized effect of social distancing on covid-19 dissemination, we used the community mobility reports trends, an indicator provided by google (google covid19 website). this mobility reports show the relative percentual changes in the averaged categorized places concerning baseline days (jan 3 rd -j o u r n a l p r e -p r o o f feb 6 th , 2020), named as mobility. these data present a good linear correlation with a similar product provided by apple. as socio-economic indicators, we used: access to clean water supply, the elderly fraction (≥ 60 years old), and the human development index (hdi) which, in summary, is a measure of three key dimensions related to development: income, education and health. the number of tests performed was also considered in the regression analysis. the indicators were obtained from the brazilian institute of geography and statistics and the ministry of regional development (ibge and snis websites). table 1 shows the values of these variables for each state, besides information concerning the population, case fatality rate (cfr), and the number of intensive care units (icu) available for the studied states. a descriptive analysis of atmospheric variables and health data was performed. a generalized additive model for location, scale and shape (gamlss), combined with the negative binomial distribution, a distribution belonging to the exponential family (hastie and robert, 1990; wedderburn, 1974; stasinopoulos et al., 2018) , were used to investigate the potential influence of the atmospheric and socio-economic variables on covid-19 cases and influenza hospitalizations, as well to determine the most important variables among those analyzed. the model systematic part is given by: where, x ji are the explanatory variables from j=1, 2,…,6; and from time (day) from i=1,…,n: maximum and minimum temperatures, relative humidity, rainfall frequency, insolation and social distancing measured by mobility (only for covid-19). rainfall was added to the model as a binary variable (0 without rain, 1 with rain), since the interest was to observe the effect of its occurrence and not of intensity. hdi is the human development index, water corresponds to the percentage of homes with access to clean water supply in the state, elderly is the fraction of the population aged ≥ 60 years, and test is the number of tests done per 100,000 inhabitants. these variables allowed to observe the scenarios between each studied brazilian state since the socioeconomic and population characteristics are quite variable. after contamination, there is a latency period of the virus in the organism. the incubation period for viruses is quite variable; in general, the interval between exposure and the onset of symptoms varies from 2 to 7 days (lauer et al., 2020; lessler et al., 2009 . therefore, for this study, lags from 0 to 7 days were considered. exposure-response curves were also plotted at lag 3 (3 days of interval between exposure and the onset of symptoms and their notification in this study), using the distributed lag non-linear model (dlnm), with reference values centered on the median of the variable (gasparrini, 2011) . finally, the values of relative risks (rr), at 95% confidence (ci), were calculated for each variable in the states. statistical analyses were performed using the software r 3.6.1 (r core team, 2019). were also those with the highest percentage of relative humidity (mean above 75%). the state of pr, with the lowest temperatures and humidity, presented the most dispersed values for maximum and minimum temperatures. in terms of relative humidity, the greatest dispersion was observed for ma. insolation was significantly higher for pr, around 8 hours, which is almost the double of the average for the other states. the social distancing proposed by the local authorities reduced urban mobility by up to 50%, comparing with the baseline (jan 3 rd -feb 6 th , 2020). the reduction in mobility was not significant, but much worse for am, where the adhesion to social distancing was lower and mobility was reduced by only 20% in the studied period. j o u r n a l p r e -p r o o f probably due to . the cfr values presented in table 1 are higher when compared to statistics reported to other countries (banerjee et al., 2020; verity et al., 2020) . the rr calculated for covid-19 cases is shown in social distancing, measured by mobility changes in this study, also presented a well-marked rr for all states, which corroborates the influence of social distancing measures as recommended by the who and already observed for brazil (aquino et al., 2020; tenório and lansac-tôha, 2020; valenti et al., 2020) . the comparison of the exposure-response curves of mobility and rr at lag 3 is presented in figure 4 . for ce, es, ma and pr, exposure-response curves of mobility and rr present a similar behavior. without any reduction in mobility (100% on the mobility axis of in all states analyzed in this study, a one-week interval was observed between exposure and the onset of covid-19 notifications, but shorter intervals, usually 3 days, were also observed. this time lag estimated here is in the set of variables and it is the interval between the exposure and the notification. the first symptoms after exposure to sars-cov-2 normally occur after 2 to 7 days, but it can extend up to 14 days, which is considered the quarantine time for covid-19 (jiang et al., 2020; lauer et al., 2020) . socio-economic conditions (hdi) proved to be the most important risk factor among the states. this suggests a difficulty for people to understand the importance of hygiene measures and social distancing on the spread of covid-19. in addition, the index indirectly measures the difficulty j o u r n a l p r e -p r o o f that people have to keep social distancing due to the daily need to search for resources for their livelihood. therefore, income and education levels are the main relevant factors. the access to the health care system is another important factor related to socio-economic conditions that can also help explain the high number of deaths in am, ce, and ma, the states with less icu available by inhabitant (see table 1 ). the fraction of elderly, one additional information captured by the hdi, is also an important risk factor, as already noticed and documented by health systems around the world (banerjee et al., 2020; yi-ru wang, 2020; verity et al., 2020) . the elderly are the most vulnerable group to covid-19. the statistical description of atmospheric variables from 2017 to 2019 is presented in the supplementary material (fig. s.3) . the prevalence of influenza hospitalizations (icd j09-j11) is presented in figure 5 . the state of ma presented the highest prevalence of influenza. for ma and ce, a peak of prevalence is observed in march/april, which differs from pr and es, with a peak in may/june, and am, with a peak not clear. the prevalence of influenza in brazil is quite variable, and the fatality rate is 0.4/100,000 inhabitants (for 2019), according to the brazilian ministry of health. this variation is dependent on several factors, including climate conditions, which show wide variations across brazil. influenza presents a well-defined seasonality (fig. 5 and fig. s.4) , as observed for other parts of the world. however, it is not possible to attribute the same association to climate, as observed in other parts of the world. j o u r n a l p r e -p r o o f table 3 presents the influenza rr and ci for the analyzed variables. as mentioned before, the wind speed was previously analyzed, and the results indicate that this variable is not significant. maximum temperature and humidity are the main atmospheric risk factors for hospital admissions by influenza in all analyzed climate regimes. besides, ce and es also presented significant rr for minimum temperature and insolation (protective effect), while pr for rainfall frequency. the results do not indicate hdi as an important factor when comparing the states, although clean water supply is a risk factor, indicating that other socio-economic inequalities should be explored, besides those measured by hdi. an important aspect for all brazilian states is the annual influenza vaccination program of the brazilian unified health system (sus, http://www.saude.gov.br/sistema-unico-de-saude), that provides free influenza vaccine for vulnerable groups (elderly > 60 years old, pregnant women, children under 5 years old, chronic patients, health workers and indigenous population). therefore, the vaccine is a protective factor j o u r n a l p r e -p r o o f for the population, independently of income, which can explain this result for hdi. on the other hand, age is the most important risk factor for influenza, as demonstrated by an rr of 1.7151 (95% ci: 1.6742-1.7574). the set of variables presented a pronounced rr at lag 3 for hospitalization due to influenza, which is in agreement with the latency time for the virus (lessler et al., 2009; o'shea et al., 2019) . the regression analysis performed from march 1 st to may 15 th of years 2017-2019 for influenza provide similar results of those obtained using the total daily data from 2017-2019 (table 3) , with the elderly as the most important factor, with significant rr for temperature and humidity, and no significant rr for hdi (see table s .2). the rainfall and minimum temperature were risk factors in am, which is consistent with the year station and the annual prevalence of influenza. comparing socio-economic characteristics, hdi showed statistical significance for covid-19 and influenza, and amazonas was the state with the highest significance for this variable (see the p-value in table s .3). the percentage of elderly people was statistically more significant in ma for covid-19 while, for influenza, it was pr. in the case of access to clean water supply, the highest statistical significance was observed in the state of ce for both influenza and covid-19. the two states with the lowest number of tests per 100,000 inhabitants, ma and pr, showed higher significance (α = 0.001). hdi and the elderly are important factors for covid-19 cases, while the elderly fraction is remarkably the main factor for influenza in the set of variables and states. as previously argued, this difference may be due to the influenza vaccination provided by sus every year for vulnerable groups. as the vaccine is free and is systematically provided by sus, the existing inequalities measured by hdi are not highlighted in influenza, in an opposite way of what j o u r n a l p r e -p r o o f happens in the case of covid-19, for which no vaccine is available yet. however, other inequalities such as access to clean water supply is a risk factor. temperature and humidity are factors significantly associated with both diseases, which suggests that seasonality may be a factor attributable to both viruses. this explains the high number of cases in the tropical climate. the exposure-response curves for covid-19 and influenza at lag 3 for maximum temperature and relative humidity for am (tropical climate) and pr (subtropical climate) are presented in temperature presents similarities in the risks for covid-19 (fig 6 a and b) and influenza (fig 6 c and d) , with a risk factor above percentile 75 th for both diseases. humidity is also observed in having the same way, as can be seen on exposure-response curves for am (fig. 6) . the same result is observed in the case of pr for both diseases. however, for humidity, these similarities in the curves are not so clear, but the risk is around the mean (tables 2 and 3 ) and a protective factor is observed between 25 th and 75 th percentiles for influenza and 75 th and 99 th percentiles for covid-19. anyway, it is important to note that the covid-19 period in pr presented atypical meteorological conditions (drier) concerning the climatology of the corresponding period. seasonality; temperature and humidity are the main related atmospheric variables. therefore, we believe that covid-19 also has a similar annual cycle like influenza, with tropical regions presenting a different cycle than subtropical regions, since the variability of temperature and humidity is significantly different between these regions. in this study, we evaluated the influence of atmospheric and socio-economic conditions on the spread of covid-19 and influenza in tropical and subtropical states in brazil. covid-19 presented a different epidemiological profile in tropical and subtropical brazilian climates. seasonality showed similarities with the behavior of influenza in previous years. despite the relevance of social distancing, indicated in this study by mobility, the climate had an important influence on both covid-19 and influenza suggesting that, for the tropical region (am, ma, and ce), covid-19 arrived in a favorable time. this favorable climate condition, identified by an abnormal rainy season that influenced humidity, was observed mainly in am and ma and may have contributed to a large number of cases. on the other hand, covid-19 arrived in unfavorable periods and atypical meteorological conditions for pr state, i.e. under much drier and sunnier conditions than normal, with several hours of solar radiation that contributed to the control of covid-19 spread in the state. age is the most important risk factor for influenza, while for covid-19 the hdi is the most important factor to explain the differences among regions in the number of covid-19 cases followed by age and social distancing. j o u r n a l p r e -p r o o f finally, the dataset has limitations associated with sub notifications of cases and deaths by covid-19. similar limitations can be extended to the social distancing measure, analyzed through the mobility changes since it cannot represent with accuracy how far people are staying away from each other. besides, hospital admission for influenza obtained from sus cannot represent the total population, as well as the data available for covid-19 that is yet limited to a few months, which can influence the results. however, we believe that these results can be extended to other similar regions and call attention to the influence of socioeconomic inequalities on the spread of the pandemic. seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics köppen's climate classification map for brazil evidence that high temperatures and intermediate relative humidity might favor the spread of covid-19 in tropical 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cities of brazil covid-19 transmission in mainland china is associated with temperature and humidity: a time-series analysis regional determinants of the expansion of covid-19 in brazil temperature and latitude analysis to predict potential spread and seasonality for covid-19. ssrn electron association of covid-19 global distribution and environmental and demographic factors: an updated three-month study sars-cov-2rna found on particulate matter of bergamo in northern italy: first evidence absolute humidity modulates influenza survival, transmission, and seasonality association between seasonal influenza and absolute humidity: time-series analysis with daily surveillance data in nacional de informações sobre saneamento, ministério do desenvolvimento regional association between climate variables and global transmission of sars-cov-2 the airborne lifetime of small speech droplets and their potential importance in sars-cov-2 transmission gamlss : a distributional regression review of aerosol transmission of influenza a virus recognition of aerosol transmission of infectious agents: a commentary social distancing and movement constraint as the most likely factors for covid-19 outbreak control in brazil social distancing and movement constraint as the most likely factors for covid-19 outbreak control in brazil covid-19 in brazil: "so what is temperature reducing the transmission of covid-19 ? covid-19 and air pollution: a dangerous association? social distancing measures could have reduced estimated deaths related to covid-19 in brazil estimates of the severity of coronavirus disease 2019: a model-based analysis high temperature and high humidity reduce the transmission of covid-19 updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan quasi-likelihood functions , generalized linear models , and the gauss -newton method can we contain the covid-19 outbreak with the same measures as for sars? modes of transmission of virus causing covid-19 : implications for ipc precaution recommendations association between ambient temperature and covid-19 infection in 122 cities from china the authors would like to acknowledge the organizations and institutes for providing the dataset.we would also like to thank the national council for scientific and technological development (conselho nacional de desenvolvimento científico e tecnológico -cnpq), process grant no. 306862/2018-2. key: cord-011757-11r3dnse authors: van wijhe, maarten; ingholt, mathias mølbak; andreasen, viggo; simonsen, lone title: loose ends in the epidemiology of the 1918 pandemic: explaining the extreme mortality risk in young adults date: 2018-09-06 journal: am j epidemiol doi: 10.1093/aje/kwy148 sha: doc_id: 11757 cord_uid: 11r3dnse in the century since the 1918 influenza pandemic, insights have been sought to explain the pandemic’s signature pattern of high death rates in young adults and low death rates in the elderly and infants. our understanding of the origin and evolution of the pandemic has shifted considerably. we review evidence of the characteristic age-related pattern of death during the 1918 pandemic relative to the “original antigenic sin” hypothesis. we analyze age-stratified mortality data from copenhagen around 1918 to identify break points associated with unusual death risk. whereas infants had no meaningful risk elevation, death risk gradually increased, peaking for young adults 20–34 years of age before dropping sharply for adults ages 35–44 years, suggesting break points for birth cohorts around 1908 and 1878. taken together with data from previous studies, there is strong evidence that those born before 1878 or after 1908 were not at increased risk of dying of 1918 pandemic influenza. although the peak death risk coincided with the 1889–1892 pandemic, the 1908 and 1878 break points do not correspond with known pandemics. an increasing number of interdisciplinary studies covering fields such as virology, phylogenetics, death, and serology offer exciting insights into patterns and reasons for the unusual extreme 1918 pandemic mortality risk in young adults. initially submitted april 30, 2018 ; accepted for publication july 13, 2018. in the century since the 1918 influenza pandemic, insights have been sought to explain the pandemic's signature pattern of high death rates in young adults and low death rates in the elderly and infants. our understanding of the origin and evolution of the pandemic has shifted considerably. we review evidence of the characteristic agerelated pattern of death during the 1918 pandemic relative to the "original antigenic sin" hypothesis. we analyze age-stratified mortality data from copenhagen around 1918 to identify break points associated with unusual death risk. whereas infants had no meaningful risk elevation, death risk gradually increased, peaking for young adults 20-34 years of age before dropping sharply for adults ages 35-44 years, suggesting break points for birth cohorts around 1908 and 1878. taken together with data from previous studies, there is strong evidence that those born before 1878 or after 1908 were not at increased risk of dying of 1918 pandemic influenza. although the peak death risk coincided with the 1889-1892 pandemic, the 1908 and 1878 break points do not correspond with known pandemics. an increasing number of interdisciplinary studies covering fields such as virology, phylogenetics, death, and serology offer exciting insights into patterns and reasons for the unusual extreme 1918 pandemic mortality risk in young adults. 1918 spanish flu; age patterns; antigenic sin; excess mortality; pandemic influenza abbreviation: wwi, world war i. this year marks the 100th anniversary of the iconic 1918 influenza pandemic. over the years, the impact of the pandemic on death, demography, society, and its general characteristic features have been studied in depth. although many questions have already been resolved, answers to some key questions continue to elude us, such as the origin of the virus, the role of world war i (wwi), the economic and societal impacts, and, most importantly, the unusual death-rate pattern in young adults. in this article, we review some of these outstanding questions, focusing on the origin of the pandemic as well as its "signature" age pattern of an extremely high death rate among young adults, whereas the elderly tended to be spared. we address the hypothesis of "original antigenic sin" (1)-that early childhood exposure may determine death risk during influenza pandemics encountered later in life-which may explain why some age cohorts fared differently in this pandemic. this hypothesis has brewed for some time (2) , and detailed analyses of 1918 data from kentucky (3) as well as analysis of the dramatically different age patterns among victims of avian h5n1 and h7n9 influenza who were born before and after the 1968 pandemic (4-6) have brought new steam to this old question. to further investigate the age-related patterns of death rates and risk change points in 1918, we analyzed monthly all-cause and age-stratified mortality data from copenhagen to address the antigenic sin hypothesis. specifically, we reviewed data on 12 age groups from the 1918 pandemic and sought to pinpoint change points in relative risk elevation. we also sought to link these change points to particular years when the so-called original sin would have occurred. for this purpose, we used weekly surveillance for outpatient influenzalike illness in copenhagen and looked for unusual influenza activity. it has been argued that the 1918 h1n1 virus originated in the context of wwi efforts in the trenches and army camps in england and france in 1916; affected persons received a diagnosis of "purulent bronchitis." the reports of an unusual clinical picture of young men with respiratory febrile illness, heliotrope cyanosis, and bloody coughing are strong support for this hypothesis (7) (8) (9) . others have argued that it all started in a military camp in kansas in early march 1918; again, military doctors saw a similar unusual picture of hemorrhage and edematous lungs on autopsy among enlisted men who had clinical symptoms of influenza (10, 11) . according to a third theory, the pandemic originated in inner northern china, where in 1917-1918, an epidemic of "pneumonic plague" (12) may have been pandemic influenza that then spread to europe via chinese migrant workers (13) . although we cannot resolve these different views, the evidence of unusual cyanotic respiratory illness in young men that was later a signature clinical feature in the severe autumn 1918 pandemic supports the idea that the emerging h1n1 pandemic virus had festered in immunesuppressed wwi army populations for some time before it gained effective transmissibility. indeed, it may have been wwi troop movements that brought the emerging virus to the united states and it was there that the first documented epidemics took place. the central role of wwi troop movements has previously been documented in a study of brazilian naval ships whose personnel became infected with pandemic influenza after an encounter with the british fleet along the african coast in the early summer of 1918 (14) . meanwhile, the genetic origins of the h1n1 pandemic virus have been studied through phylogenetic analysis of fully sequenced viral rna isolated from lung specimens of victims of the 1918 pandemic. taubenberger et al. (15) concluded that the pandemic virus emerged as an all-avian virus crossing over to human populations in 1918. however, evidence from smith et al. (16) suggests the virus arose through multiple reassortment events among circulating swine, avian, and human strains in the decade before the pandemic. in 2014, contradictory evidence was brought forth by worobey et al. (6) , who argued that the h1n1 virus was not all avian but rather was assembled by reassortment of a human h1 hemagglutinin and avian viral segments shortly before 1918. they concluded that the hemagglutinin segment had already emerged in human strains around 1907 and that about a decade later, the h1n1 pandemic virus fully formed in a single event when the human h1 strain reassorted with an avian source. taken together with clinical evidence, it is not easy to reconstruct the actual reassortment timeline. it is possible that the unusual occurrence of cyanotic respiratory illness in wwi army camps was, in fact, a manifestation of the h1 reassortant circulating in the years before the pandemic virus had fully formed and gained the ability to spread effectively. epidemiologists have long analyzed death time series to study the signature age patterns of the 1918 pandemic influenza, characterized by extreme death rates in young adults while seniors were spared (17) (18) (19) . using unique influenza outpatient and death time-series data from copenhagen, we demonstrated the existence and the mild nature of the first pandemic wave in the summer of 1918 (18, 20) and we recently reviewed all evidence of herald waves in 1918 (21) . why this first summer wave was milder than the following fall and winter waves remains unclear. it is possible that the virus had not yet acquired the virulence mutations before autumn, or that important bacterial cofactors were not present during the summer wave. the devastating impact of deaths resulting from the 1918 pandemic was due to a combination of high attack rates (50%-70%), high case-fatality rates (2%-4%), and the unusual age distribution: an estimated 95% of pandemic deaths occurred in young adults (22) . the unique 1918 pandemic age pattern holds important clues about the meeting of the pandemic virus with the immune landscape of the human population that was shaped by decades of experience with influenza. so far, the observation that adults older than 45 years suffered no excess mortality in cities like new york city and in copenhagen has been interpreted as evidence of "recycling" of the h1 antigen that age group had encountered during their childhood some 50 years earlier (18, 20) . meanwhile, the extreme death rate in young adults suggests that having been born between the 1900 (pseudo) pandemic and the 1889 russian pandemic resulted in that age group's "antigenic sin" (2) . the exact break points on the age-risk pattern have been elegantly studied using individual-level 1918 death records from kentucky (3). viboud et al. (3) found several change points in age-specific excess death rates: a minimum at approximately 10 years of age, followed by a steep increase that peaked at ages 24-26 years and another minimum at ages 56-59 years. viboud et al. hypothesized that these peaks and valleys in the corresponding birth years (cohorts born during 1859-1862, 1892-1894, and 1908-1909) should correspond with known dates of historic pandemics. however, they found that this was not the case. the large geographical discrepancies in age patterns provide additional clues. it was found in studies of death patterns in south american populations that elderly people were not spared from pandemic influenza; in fact, all age groups seemed to be at highly elevated risk (23, 24) . similarly, observations of high pandemic impact in remote populations such as inuits in newfoundland and the maoris in new zealand can be interpreted in the same way (25, 26) . for example, the maoris were 7-fold more likely to die during the 1918 influenza pandemic than were the new zealand population who were of european descent. although far higher death risk in ethnic populations could also be interpreted as a consequence of genetic risk factors, we think a more parsimonious explanation is the remoteness of these ethnic populations in their childhood some 20-50 years earlier. these findings of high risk for death associated with pandemic influenza in elderly adults living in remote settings would then support the recycling hypothesis. gostic et al. (4) brought new evidence to this immunity agesignature puzzle. they demonstrated that victims of h5n1 and h7n9 avian influenza had very different age distributions, such that birth cohorts born before and after the 1968 pandemic had completely opposite risk profiles for these 2 zoonotic viruses (4) . their findings expand on the fascinating possibility proposed earlier by worobey et al. (6) that it is the phylogenetic group of influenza a hemagglutinin segment (group 1 or group 2) that may determine death risk of a novel influenza infection. members of these groups may elicit crossimmunity because their subtypes (h1, h2 and h5, belonging to group 1, and h3 and h7 belonging to group 2) are from the same major hemagglutinin phylogenetic clade. thus, the recycling hypotheses can be relaxed to having experienced an original sin of group 1 versus group 2 influenza a hemagglutinin in childhood, rather than requiring that the original exposure had to be the exact same hemagglutinin subtype. the high young adult death rate during the 1918 pandemic may be due to different imprinting between age groups. all-cause, age-specific death patterns can help us determine which age groups were more affected by the 1918 pandemic and thus help uncover clues about the evolutionary history of the virus. therefore, we looked at the death patterns of different age cohorts in copenhagen in 1918 and sought to place these patterns in the context of long time series of outpatient records of influenza-like illness. we used detailed, long time series of age-stratified monthly death records from copenhagen along with population census statistics (see andreasen et al. (20) for more information on the data sources) to look for break points in the age profile of cases during the various seasons of the 1918 pandemic ( figure 1) . we took the same general approach as viboud et al. (3) , but rather than look at excess death rates, we studied the relative death risk over the baseline level for each age group. all-cause death data with a 5-to 10-year age resolution were available from 1879 through 1923 through annual reports from the medical officers of copenhagen. as a baseline for 1918 and 1919 data, we interpolated the death rates for each month between 1917 and 1921 and calculated incidence rates (the baseline) during each of the 4 pandemic waves. all rates were expressed as all-cause deaths per 10,000 individuals (linearly interpolating between successive census data). we then computed the incidence ratio as the ratio of incidence rates for each wave over the baseline. there were 4 pandemic waves in copenhagen during 1918-1920: a milder first (herald) wave in july to august 1918, followed by the main wave peaking in october to november. this was followed by a winter wave peaking in january to february 1919, and a fourth recrudescent wave in january to february 1920. already in june 1918, the danish national newspapers began reporting on the "spanish sickness" (27) (28) (29) . in the second week of july, the pandemic broke out in both copenhagen and the town of roskilde, 30 km to the west of copenhagen. although the source of the outbreak in copenhagen is impossible to track, in roskilde, it was likely introduced by a circus artist arriving from oslo, norway, where the first wave had already reached epidemic levels a week earlier (table 1) . interestingly, before the outbreak in copenhagen and roskilde, an outbreak occurred in the town of christiansfeld just south of the danish border in jutland, apparently introduced by a postal clerk returning from germany (30, 31) . it was widely accepted among physicians in copenhagen at the time that the outbreak was a novel, atypical form of influenza and they likened it to the influenza that had caused substantial impact in madrid, spain, in june (19, 29) . also, they commented on the outbreak's mildness and suggested that people should not worry-unfortunate advice, in hindsight, because the subsequent autumn wave killed 0.2% of the danish population and 1%-2% of the entire world population (20, 22) . across the study period, the 1918 pandemic death rate in young adults stood out dramatically in the severe autumn 1918 wave (the second wave) and the recrudescent (fourth) wave during winter 1919-1920. in contrast, young children as well as older adults did not have unusual excess death in the 1918-1920 period (figure 1 ; web figure 1 , available at https:// academic.oup.com/aje). during the severe autumn 1918 pandemic wave, the risk ratio was highest for persons in age groups between approximately 10 and 44 years, reiterating earlier findings in other studies (figure 2 ). the risk ratio steadily increased to more than 11-fold of baseline for the 25-34-years age group during the autumn of 1918. the incidence ratio then dropped sharply for the 35-44-years age group and reached a risk ratio of approximately 1 for older age groups, consistent with no risk elevation during the pandemic. the exact risk break point is likely approximately 40 years of age, judging by the steep decline in this group compared with the surrounding age groups. a similar risk-ratio pattern was seen in all 4 pandemic waves, in particular in the second and fourth waves. we next looked for evidence of pandemic-level activity in the decade before 1918 in long time series of weekly influenza outpatient morbidity data available since 1889 when influenza was added to the list of notifications (figure 3 ). we could not identify any standout epidemics between the 2 pandemics of 1889 and 1918. unfortunately, these morbidity data did not allow us to go back further to check for epidemics around 1873. we next perused annual medical reports for denmark and found that influenza, in fact, was noted and discussed during 1876-1883 but without mention of pandemic or severe activity before 1889. one physician commented that there had been no notable influenza in the years between the pandemic in the 1830s and 1889 (32) . reviewing the evidence that has accumulated from various disciplines, including medical history, quantitative epidemiological analyses, seroepidemiology, virology, and phylogenetics, answers to key questions about the 1918 pandemic still elude us. however, we are moving closer, in particular regarding the unusual age pattern of deaths. the recycling hypothesis was first put forward to explain the 1918 patterns and has since been investigated for more recent pandemics. age-specific excess death rates were used in a study to review age-groups data to identify points at which pandemic protection begins. such change points have been found for persons older than 77 years during the 1968 pandemic, corresponding to those born before the pandemic in 1889 (2) . for the recent 2009 pandemic, middle-aged and older adults born before the 1957 pandemic were nearly completely spared and showed evidence of preexisting cross-reactive antibodies (33, 34) . thus, there is good reason to believe that adult protection relates to exposure to pandemics in childhood. for the 1918 pandemic, such an inquiry can only be done with epidemiologic excess death data, because of the absence of seroepidemiology from blood sampled before that pandemic and the absence of virologic evidence from the 19th century. 1913-1918, 1908-1913, 1903-1908, 1898-1903, 1893-1898, 1883-1893, 1873-1883, 1863-1873, 1853-1863, 1843-1853, 1833-1843 , and 1833 or earlier. in our analysis of copenhagen data, we managed to pinpoint the break point from high to low death risk at approximately 40 years of age (in the middle of the 35-44-years age group). this corresponds to having been born around 1878 (range, 1873-1882), which is curiously a pandemic-free period in humans as far as we know (there was a severe pandemic in horses (35) ). in denmark, certainly, there is no mention in the medical literature of a human pandemic event in that period. however, our finding that the maximum death risk occurred in the 25-34-years age group (probably at approximately 29 years of age) is consistent with having been born around the time of the 1889-1892 pandemic, as if exposure to the 1889 emerging pandemic virus led to enhanced risk later in life. unfortunately, we could not pinpoint with accuracy a particular break point in terms of risk among the pediatric age groups; rather, we found a gradual increase in the incidence ratios, starting with the 5-9-years age group. explanations for the lack of a steep cutoff could be the limited total number of deaths in the toddler and schoolchildren age groups, or the resolution of the age groups. infants were at no particular 1902 1905 1908 1911 1914 1917 1920 1923 year no. of influenza cases increased risk relative to the baseline level. this is not to say that there were no infant deaths due to the pandemic-rather, those would be few compared with the overall background number of deaths. our results are remarkably similar to those of viboud et al. (3) , given the lower resolution of our data (table 2) . whereas in kentucky it was clear that those approximately 10 years of age were at the lowest risk for death associated with the pandemic, our analysis points to children under 5 being at lower risk than those in other pediatric age groups. however, it is also clear that those 5-9 years of age were at a relatively low risk compared with those 10-44 years old. the highest risk in kentucky was in people between 20 and 30 years old-similar to data from copenhagen. in kentucky, the excess death declined steadily after 25 years; in copenhagen, this decline was sharper and mainly evident in the age groups older than 35 years. these results suggest diverging antigenic sins between birth cohorts. birth cohorts born before 1873 may have been exposed predominantly to group 1 influenza a hemagglutinin, whereas those born between 1873 and 1908 may be been exposed to group 2 influenza a hemagglutinin, and those born after 1908 may have an antigenic sin related to reemerging group 1 influenza a hemagglutinin, very likely of the h1 subtype. it is possible that h1 was also circulating around 1873, which would explain the low risk among the elderly. seroepidemiology, epidemiologic, and phylogenetic evidence seem to point to the same time period around 1907 (table 2) (3, 6, 36). the possibility of a 2-step assembly raised by worobey et al. (37) in a newer phylogenetic analysis is in disagreement with earlier phylogenetic analyses by taubenberger et al. (15) and reid et al. (38) , who concluded that the 1918 pandemic was an all-avian zoonosis. although these hypotheses disagree on the origin of the pandemic and its reassortment history, they are consistent with the idea that h1 (of avian or human origin) was already circulating well before the 1918 pandemic arose and was likely introduced in humans between 1900 and 1907. it is possible that the pandemic virus was assembled in multiple steps: around 1907, the virus acquired h1 by recombination (6). this precursor virus may have been circulating for a decade or more before the 1918 pandemic, along with the previous group 2 influenza virus, thereby explaining the intermediate risk profile for those born between 1908 and 1913 (some would have experienced a group 1 infection and others a group 2 infection as their first influenza illness). certainly, a consolidating view on this issue, and of the possible contribution of other segments, like neuraminidase, would be most helpful to elucidate the likely human immunity landscape at the time. the nature, origin, and timing of a future pandemic may be unknown; however, it is clear from historical accounts that one will occur again. pandemic preparedness relies on our understanding of what might happen given our pandemic experiences; in particular, our understanding of patterns of severity and highrisk age groups. studying historical influenza pandemics is only natural, therefore, and may resolve important conundrums about the interaction between population immunity and pathogen evolution. although some aspects of events such as the h1n1 1918 outbreak and other pandemics of the 20th and 21th centuries still elude us, they provide invaluable insights for informing pandemic planning whatever the next threat may be. a serological recapitulation of human infection with different strains of influenza virus the virtues of antigenic sin: consequences of pandemic recycling on influenzaassociated mortality age-and sex-specific mortality associated with 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emerg infect dis regional patterns of mortality during the 1918 influenza pandemic in newfoundland black flu 1918 -the story of new zealand's worst public health disaster det drejer sig om influenza, ledsaget af staerk feber kliniske og aetiologiske bemaerkninger om influenzaepidemien den spanske syge i sønderjylland. nationaltidende bidrag til oplysning om influenza, med hensyn til dens forhold til denguefeber cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus incidence of 2009 pandemic influenza a h1n1 infection in england: a crosssectional serological study an avian outbreak associated with panzootic equine influenza in 1872: an early example of highly pathogenic avian influenza? influenza other respir viruses recycling of h1n1 influenza a virus in man-a haemagglutinin antibody study a synchronized global sweep of the internal genes of modern avian influenza virus origin and evolution of the 1918 "spanish" influenza virus hemagglutinin gene kommer farsoten hertil? -en lille samtale med stadslaegen. nationaltidende en influenzaepidemi i roskilde? roskilde avis den spanske syge i flaaden? 20 mariner indlagt paa epidemihospitalet. politiken den spanske syge i taarbaek. politiken the next influenza pandemic: can it be predicted? conflict of interest: none declared. key: cord-278508-h145cxlp authors: streng, andrea; prifert, christiane; weissbrich, benedikt; liese, johannes g. title: continued high incidence of children with severe influenza a(h1n1)pdm09 admitted to paediatric intensive care units in germany during the first three post-pandemic influenza seasons, 2010/11–2012/13 date: 2015-12-18 journal: bmc infect dis doi: 10.1186/s12879-015-1293-1 sha: doc_id: 278508 cord_uid: h145cxlp background: previous influenza surveillance at paediatric intensive care units (picus) in germany indicated increased incidence of picu admissions for the pandemic influenza subtype a(h1n1)pdm09. we investigated incidence and clinical characteristics of influenza in children admitted to picus during the first three post-pandemic influenza seasons, using active screening. methods: we conducted a prospective surveillance study in 24 picus in bavaria (germany) from october 2010 to september 2013. influenza cases among children between 1 month and 16 years of age admitted to these picus with acute respiratory infection were confirmed by pcr analysis of respiratory secretions. results: a total of 24/7/20 influenza-associated picu admissions were recorded in the post-pandemic seasons 1/2/3; incidence estimates per 100,000 children were 1.72/0.76/1.80, respectively. of all 51 patients, 80 % had influenza a, including 65 % with a(h1n1)pdm09. influenza a(h1n1)pdm09 was almost absent in season 2 (incidence 0.11), but dominated picu admissions in seasons 1 (incidence 1.35) and 3 (incidence 1.17). clinical data was available for 47 influenza patients; median age was 4.8 years (iqr 1.6–11.0). the most frequent diagnoses were influenza-associated pneumonia (62 %), bronchitis/bronchiolitis (32 %), secondary bacterial pneumonia (26 %), and ards (21 %). thirty-six patients (77 %) had underlying medical conditions. median duration of picu stay was 3 days (iqr 1–11). forty-seven per cent of patients received mechanical ventilation, and one patient (2 %) extracorporeal membrane oxygenation; 19 % were treated with oseltamivir. five children (11 %) had pulmonary sequelae. five children (11 %) died; all had underlying chronic conditions and were infected with a(h1n1)pdm09. in season 3, patients with a(h1n1)pdm09 were younger than in season 1 (p = 0.020), were diagnosed more often with bronchitis/bronchiolitis (p = 0.004), and were admitted to a picu later after the onset of influenza symptoms (p = 0.041). conclusions: active screening showed a continued high incidence of a(h1n1)pdm09-associated picu admissions in the post-pandemic seasons 1 and 3, and indicated possible underestimation of incidence in previous german studies. the age shift of severe a(h1n1)pdm09 towards younger children may be explained by increasing immunity in the older paediatric population. the high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in germany. influenza is one of the most common vaccine-preventable viral diseases, with the highest morbidity reported for children and elderly patients [1, 2] . influenza infections during childhood usually present as mild respiratory upper airway disease, but severe complications and fatalities also occur, especially in children less than 2 years of age and in children with underlying chronic conditions [2] [3] [4] [5] [6] [7] . however, 40-50 % of influenza-associated fatalities occur in previously healthy children [4, 8] . before the influenza a(h1n1)pdm09 pandemic in 2009/ 2010, comparisons of clinical characteristics between patients infected with different influenza types (a vs. b) and between patients infected with different influenza a subtypes showed only small differences when controlling for age [9, 10] . during the pandemic, however, some studies observed increased morbidity and mortality among children compared to previous seasonal influenza [6, [11] [12] [13] , while other studies described the clinical features of a(h1n1)pdm09 as being similar or even milder [14, 15] . acute respiratory distress syndrome (ards) and fatal viral pneumonia was observed more frequently during the pandemic [16] . post-pandemic surveillance was recommended, as circulation of a(h1n1)pdm09 was expected to continue for several years, gradually assuming a seasonal influenza pattern [16] . in germany, influenza sentinel surveillance on outpatients of all ages [17] confirmed that the first postpandemic season 2010/11 was dominated by influenza a(h1n1)pdm09 (62 %), co-circulating with influenza b (37 %) whereas a(h3n2) was rare (<1 %). during the second season 2011/12, a(h1n1)pdm09 was rare (1 %), whereas a(h3n2) was diagnosed in 75 % of cases and co-circulated with influenza b (24 %). in the third season 2012/13, all three types/subtypes co-circulated in similar proportions (34 % a(h1n1)pdm2009, 31 % a(h3n2), and 35 % b). information on the incidence and clinical characteristics of severe paediatric influenza resulting in intensive care treatment and/or fatal outcome is still limited in germany, and post-pandemic data is thus far available only for the season 2010/11 [18] [19] [20] . based on cases recorded by a nation-wide paediatric intensive care unit (picu) reporting system, the pre-pandemic (2005/06-2007/08), pandemic (2009/10) and post-pandemic (2010/ 11) annual incidence of severe influenza cases per 100,000 children below 15 or 17 years of age was estimated as 0.05, 0.8-1.0, and 0.4, respectively [18] [19] [20] . the data so far available indicated a shift towards younger children in a(h1n1)pdm09 cases from the pandemic to the first post-pandemic season [20] . in these studies, it remained unclear whether the higher pandemic and post-pandemic incidence in children was caused by higher influenza activity, heightened physician awareness, more frequent or more sensitive influenza testing, or a more severe course of disease of a(h1n1)pdm09 [18] . furthermore, all these previous studies may have been affected by underreporting, as influenza cases were reported at the discretion of the picu physician without systematic screening for influenza in patients with severe acute respiratory infection. in the study presented here, we used active screening to estimate the incidence of laboratory-confirmed influenzaassociated picu admissions in one of germany's largest federal states during the first three post-pandemic seasons. furthermore, we described the clinical characteristics of influenza picu patients and compared patients with severe a(h1n1)pdm09 disease between the post-pandemic seasons. prospective, active surveillance was conducted in picus of paediatric hospitals in bavaria, germany. on december 31 st , 2010 roughly 2,001,700 children <17 years of age were registered in bavaria [21] , representing 16 % of the german population in this age group [22] . the annual study population was defined as the sub-group of all children in bavaria at least 1 month and <17 years of age. all 30 paediatric hospitals in bavaria equipped for paediatric intensive care treatment of children older than 1 month of age were invited to participate. these picus reported a total of 432 intensive care beds (median 14, iqr [11] [12] [13] [14] [15] [16] , including 207 beds (median 9, iqr 6-12) equipped with ventilation facilities. , all patients who fulfilled the following inclusion criteria were enrolled: i) admission to a participating picu with suspected acute respiratory infection (ari) of the upper or lower respiratory tract, with arirelated symptoms (for example, coryza, cough, or sore throat); ii) age at picu admission due to ari at least 1 month and below 17 years of age; iii) parental written informed consent. enrolled children with pcr-confirmed influenza were classed as influenza-associated ari. the picu physician documented demographic characteristics, underlying chronic medical conditions, influenza vaccination status, diagnostic findings, ari-associated diagnoses and complications, treatment, duration of hospital and picu stay, and outcome in a structured questionnaire. a respiratory sample, usually a flocked nasopharyngeal or pharyngeal swab, was collected on the day of picu admission for pcr-confirmation of influenza. microbiological testing for bacteria or fungi was at the discretion of the picu physician; pathogens detected at usually sterile sites or in tracheal aspirates were classified as bacterial or fungal co-infection. pcr confirmation of influenza was performed either at the local laboratories of the participating picus using influenza-specific pcr, or (in the majority of cases) at the central laboratory at the institute of virology and immunobiology of the university of würzburg using multiplex pcr for respiratory viruses. for the latter, respiratory samples were placed in a viral transport medium (mast diagnostica gmbh, reinfeld, germany). at the central laboratory, they were tested using the commercial multiplex pcr 'ftd® respiratory pathogens 21' (fast track diagnostics, luxembourg) to screen for respiratory viruses (sensitivity and specificity of 99-100 % compared to singleplex pcr assays for all included viruses in clinical samples). pathogens detected by the test kit included influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza virus (piv) 1-4, coronavirus (cov) nl63, oc43, hku1, and 229e, human metapneumovirus (hmpv), human bocavirus (hbov), adenovirus (adv), rhinovirus (rhv), enterovirus (ev), parechovirus (pv), and additionally mykoplasma pneumoniae. samples positive for influenza a and b virus rna in the multiplex pcr were further analysed to determine the subtype and lineage, respectively. primers and probes specific for influenza a(h1n1)pdm09 were included in the 'ftd respiratory pathogens 21' kit. all samples positive for influenza a virus rna but negative for influenza a virus (h1n1)pdm09 rna were tested by a pcr specific for influenza a virus h3. all data was entered into a microsoft access database and transferred to ibm spss 21.0 for statistical analysis. data was analysed descriptively (percentages, or median with inter-quartile range, iqr). comparisons between groups were assessed for significance (p < 0.05, twosided) using pearson's chi 2 -test or fisher's exact test for categorical data, and the mann-whitney u-test for continuous data. the minimum incidence of influenza-associated picu admissions per 100,000 children <17 years of age was calculated for each season based on the observed number of influenza picu patients with a residential address in bavaria. to correct for non-participating picus, the estimates of the total number of picu influenza cases treated in all eligible picus in bavaria per season were derived taking into account the annual percentage of participating picus. the annual study population was used as denominator. a similar questionnaire and case definition had been used in previous studies on influenza-related picu admission [18, 19] . key variables were extracted from these publications for comparison purposes. data from streng et al. [18] and the present study were pooled for statistical comparison of pre-and post-pandemic seasons. the study was approved by the ethical committee of the medical faculty, university of würzburg, germany. based on the observed cases, the minimum incidence for pcr-confirmed influenza-associated picu admission per 100,000 children <17 years of age in bavaria was calculated as 1.15/0.36/1.03 for seasons 1/2/3, respectively. taking into account that the observed cases were based on data from 67 %/47 %/57 % of all eligible picus, the total number of influenza-associated picu admissions in bavaria was estimated and corrected incidences were calculated as 1.72/0.76/1.80 per 100,000 children <17 years. subtype-specific corrected incidences were 1. 35 (table 2) . after onset of ari symptoms, children were admitted to hospital after a median interval of 3.0 days; 83 % were transferred to the picu on the day of hospital admission or the following day (table 2) . two long-term hospitalized children (4.2 %) required picu treatment due to ari and were diagnosed with suspected nosocomial influenza a(h1n1)pdm09 infection. median length of picu stay was 3.0 days and median length of total hospital stay was 7.5 days (table 2) . underlying chronic medical conditions were reported for a total of 36 influenza picu patients (76.6 %) ( table 3) . chronic neurological diseases were most frequent (34.0 %), followed by chronic lung disease (25.5 %), preterm birth (21.3 %), cardiac malformations (17.0 %), obesity (10.6 %), genetic disorders (8.5 %), and immunocompromising conditions (8.5 %). of 36 influenza picu patients with underlying chronic conditions, four (11.1 %) were too young (<6 months of age) to have been immunized against influenza, and for two patients (5.6 %), data on their influenza vaccination status was unavailable. twenty-nine (80.6 %) patients from this risk group had not been vaccinated against influenza although they would have been eligible. one immunocompromised child (2.8 %) had been vaccinated in october 2012, but was diagnosed with a(h3n2) in january 2013. one or more specific influenza-associated diagnoses/complications were reported for 42 (89.4 %) of the 47 children ( table 4 ). the most frequent diagnosis was influenzaassociated pneumonia (61.7 %), followed by bronchitis/ bronchiolitis (31.9 %), and secondary bacterial pneumonia (25.5 %). ards was reported for 10 (21.3 %) and sepsis for six children (12.8 %); other complications were rare. thirty-nine of the 47 patients (83.0 %) underwent a chest radiograph. in addition to influenza, laboratory-confirmed co-infections were reported for 16 children (34.0 % out of 47 (1)). the majority of the 47 picu patients were treated intravenously with antibiotics (72.3 %), and with antipyretics (70.2 %) ( five children (10.6 %), infected with a(h1n1)pdm09, died at an age of 4 to 11 years; four were male patients ( table 6 ). four of these children suffered both from severe neurological conditions (two children with previous peripartal asphyxia and spastic tetraparesis; one child with cerebral paresis and tetraspasticity; one child with congenital cerebral disorder), and from chronic pulmonary conditions; two of these four children were also born pre-term. influenza-associated pneumonia was diagnosed in all four of these children; three additionally had secondary bacterial pneumonia, and one child also developed sepsis. for the fifth child, obesity was reported as the only risk factor; and sepsis and suspected encephalitis as complications. bacterial co-pathogens were detected in three of these five children and suspected in one child; two viral and two fungal co-infections were also reported. all five children received intratracheal ventilation, antibiotics and catecholamines; two were additionally treated with antiviral medication. death occurred 1, 2, 4, 19, and 26 days after picu admission, with ards reported as cause of death in three children. sequelae were reported for five patients (10.6 %): state after surgery due to pleural effusion/empyema in two children; increased oxygen requirements in two children who had previously already received oxygen therapy at home; damage of the lung after high-pressure ventilation in one child. table 2 ). figure 1 shows the difference in age distribution between both seasons, and the high proportion of children below 2 years of age as opposed to low proportions in all other age groups in season 3. after onset of symptoms, children were admitted to a picu after a significantly shorter period, with a median of 3 days (iqr 1-4) in season 1 compared to 6 days (iqr 2.0-7.5) in season 3 ( table 2 ). in season 1, significantly fewer children were diagnosed with bronchitis/bronchiolitis (table 4 ), and they tended to require cpap treatment less frequently than in season 3 (11.1 % vs. 41.7 %, p = 0.084, table 5 ). in the pre-pandemic period, median duration of picu stay was longer (19 days) , and children were more often diagnosed with encephalitis/encephalopathy (25 %) and co-infections (65 %) than in later periods ( table 7) . the proportion of children with influenza-associated pneumonia was highest (74 %) during the pandemic, whereas secondary bacterial pneumonia (17 %), bronchitis/ bronchiolitis (12 %) and sepsis (6 %) were reported less frequently during the pandemic than in the pre-and post-pandemic seasons. oseltamivir treatment decreased significantly in the post-pandemic period (table 7) . during the first three post-pandemic seasons 2010/11, 2011/12 and 2012/13, active screening of children with acute respiratory infection admitted to 24 paediatric intensive care units in bavaria identified a total of 51 pcr-confirmed influenza cases, resulting in annual incidence estimates of 1.7, 0.7, and 1.8 influenza-associated picu admissions per 100,000 children, respectively. these figures would, by extrapolation, correspond to a total number of 559 children with influenza-associated picu admission in germany within the 3-year post-pandemic period, with an annual average of 186 children. this is almost 28 times as high as the annual average of six to seven influenza-associated picu admissions detected by nation-wide picu surveillance in germany during three pre-pandemic years without active screening [18] . furthermore, the incidence estimates for the subtype a(h1n1)pdm09 derived from our active screening study were higher in the first and third post-pandemic seasons (1.35 and 1.17, respectively) than previous incidence estimates for picu patients in the pandemic (0.8-1.0) and the first post-pandemic (approximately 0.4) season in germany [19, 20] . thus, our results indicate possible underreporting in previous studies, and show a continued high level of a(h1n1)pdm09-associated picu admissions even 3 years after the pandemic. in our study, the proportions of children with bacteriaassociated complications (secondary bacterial pneumonia, sepsis) were similar to the proportions observed during the pre-pandemic period, but appeared higher than those observed during the pandemic 2009/10 [19] . the lower proportions observed during the pandemic might be explained by the time shift of the peak of influenza cases, which was observed as early as november 2009 in germany [19] . thus, the pandemic influenza peak did not coincide with the seasonal peak of streptococcus pneumoniae, the bacterial pathogen most frequently associated with community-acquired influenza [23] . antiviral treatment patterns changed considerably during the post-pandemic period, with a decrease in the proportion of paediatric influenza cases receiving oseltamivir from previously 50 % [18] and 61 % [19] to 19 %. oseltamivir is considered to be most advantageous when administered within the first 48 h of influenza disease. the reduced use in the post-pandemic period may therefore be partly due to the fact that median time between onset of influenza symptoms and picu admission was longer than during the pandemic (3 vs. 2 days [19] ). increasing uncertainty regarding the effectiveness of oseltamivir in the treatment of paediatric influenza may also have played a role [24, 25] . post-pandemic oseltamivir treatment was associated with co-infections and longer picu stay, suggesting that it were mainly children with severe complications or with serious underlying conditions who received this medication. in our study, about two-thirds of influenza cases and all fatalities were a(h1n1)pdm09-associated. during the postpandemic seasons 1/2/3, the proportion of a(h1n1)pdm09 cases among the picu patients was 79 %/14 %/65 % and, thus, considerably higher than the proportion of this subtype reported among outpatients by national influenza surveillance (65 %/1 %/34 %) [17] . this observation suggests that a(h1n1)pdm09 may be associated more often with a severe course of influenza requiring picu treatment than other influenza types/ subtypes. similar observations on the proportion of a(h1n1)pdm09-associated picu admissions have been reported in the united states [9] . comparison of picu patients with a(h1n1)pdm09 between the post-pandemic seasons showed that median age was 1.7 years in the third season and, thus, significantly lower than in the first season. a significant age shift towards younger children, from a median age of 5 to 3 years, had already been observed in a comparison of the pandemic and the first post-pandemic season in germany [20] . the continued shift towards younger patients in the third season is likely to be due to increasing immunity in the older paediatric population, after previous contact with a(h1n1)pdm09. seroprevalence data from germany had already shown evidence for a(h1n1)pdm09 infection in as many as 25 % of children aged 1-4 years and 48 % of 5-17 year-old children for the pandemic season 2009/10 [26] . a similar shift towards younger hospitalized children [27, 28] and towards younger children with severe paediatric a(h1n1)pdm09-associated influenza from the pandemic season to the first post-pandemic season had also been detected in other european countries [28] [29] [30] [31] . in germany, paediatric influenza vaccination for pandemic influenza a(h1n1)pdm09 was recommended and funded for all children as monovalent vaccination from october 2009 to july 2010 [32] . for seasonal influenza, however, paediatric influenza vaccination was and is currently recommended only for specific risk groups with underlying chronic conditions [33] . vaccination uptake was low, even in this target group. pre-pandemic vaccination rates were 5 % for all children and about 15 % for children with chronic underlying conditions in 2007/2008 [34] . for the pandemic and post-pandemic seasons, no data on vaccination rates is available for children, but vaccination rates as low as 14 % (2009/10) and 11 % (2010/11) were reported for adults, with a vaccination rate of only 17 % even for risk group adults [35] . in our study, more than 75 % of influenzaassociated picu patients were children with underlying chronic conditions. analysis of their reported influenza [20] ). data are given in %, by age group and season. season 1: oct10-sep11 (n = 18), season 3: oct12-sep13 (n = 13); season 2: oct11-sep12 (n = 1) is not shown vaccination status showed that among these were a high proportion of vaccine-eligible but unvaccinated children. patients with chronic conditions too young to be vaccinated and other paediatric risk groups, such as otherwise healthy children below 2 years of age, are not covered by the current german recommendation. all these groups could profit considerably from universal influenza vaccination for children, either directly or by herd protection. in contrast to the situation in germany, in the united states universal influenza vaccination for all children older than 6 months of age has been established, and vaccination coverage reached a level of approximately 41 % in 2013 [36] . compared to 348 influenza-associated paediatric deaths observed in the united states during the pandemic 2009/10, only 79 were observed in the strong a(h1n1)pdm09 season 2013/14 [37] . this might partly be explained by increasing immunity in children after previous a(h1n1)pdm09 infection, but may in part also be a result of the influenza vaccination program [37] . in england, a universal childhood vaccination programme with a new live attenuated influenza vaccine (laiv) with intra-nasal application was started in the 2013/14 influenza season [38] . first results showed an overall uptake of 53 % in primary school aged children, indicating a good acceptance of laiv, and suggesting direct and indirect impacts on disease incidence, including reduction of paediatric influenza-associated hospitalisations. to our knowledge, our study is the first in europe to investigate paediatric influenza in picu patients during the first three post-pandemic seasons after the 2009/10 pandemic. the strengths of our study include the multicentre design covering the majority of picus in bavaria, the active screening for influenza in patients admitted to picus, and pcr-confirmation of all influenza cases. a limitation is that the corrected incidence estimates were based on the assumption that participating and nonparticipating picus treated a similar number of severe paediatric influenza patients. although picus of both groups were of similar size, some of the non-participating picus, where paediatricians indicated lack of time as reason for non-participation, may have treated a higher number of patients, or patients with higher acuity. further limitations include potential over-and underreporting in participating picus. on the one hand, due to different hospitalization rules some children may have been admitted to picus mainly for the purpose of monitoring their course of influenza disease more closely, thus resulting in an overestimate of severe cases. on the other hand, some parents of children with severe influenza may have refused study participation, or children with a fulminant course of influenza disease may have died before they were admitted to a picu [4] . thus, the high incidence estimates derived in this study may still underestimate the true burden of severe influenza. [19] b key data from this study were pooled with data from streng et al. 2011 [18] and compared using fisher's exact test or mann-whitney u-test, respectively the incidence estimates of influenza a(h1n1)pdm09associated picu admissions, derived from active screening of picu patients with acute respiratory infections, reached similarly high levels in the first and third postpandemic seasons. both incidence estimates were higher than those previously reported by nation-wide picu surveillance for the pandemic and the first post-pandemic season, suggesting possible underreporting in previous studies without active screening. comparison of the first and third post-pandemic seasons indicated an age shift of severe a(h1n1)pdm09 towards younger children, which might be explained by increasing immunity in the older paediatric population. the large proportion of children with underlying chronic conditions indicates the need for a more consistent implementation of the current recommendations for influenza vaccination of specific risk groups in germany. these children could also profit from herd protection, if universal influenza vaccination was successfully introduced in germany. authors' contributions as designed the study, coordinated data collection, performed the analysis, interpreted the data, and drafted the manuscript. bw and cp performed multiplex pcr and subtyping on laboratory specimens, interpreted virological data, and revised the manuscript. jgl supervised the study, supported data interpretation, and revised the manuscript. the clinical data were collected by the bavarian picu study group on influenza and other viral ari, from oct 2010 to september 2013. all authors read and approved the final manuscript. group on influenza and other viral ari participants and their affiliations while participating during the study period städtisches klinikum münchen gmbh, klinikum harlaching städtisches klinikum münchen gmbh, klinikum schwabing christoph schmidtlein (kinderklinik dritter orden tobias trips (kliniken südostbayern ag missionsärztliche klinik ggmbh, kinderklinik; würzburg) references 1. heikkinen t. influenza in children the underrecognized burden of influenza in young children risk factors associated with severe influenza infections in childhood: implication for vaccine strategy influenzaassociated pediatric deaths in the united states identification of children at risk of influenza-related complications in primary and ambulatory care: a systematic review and meta-analysis the burden of seasonal and pandemic influenza in infants and children the burden of influenza illness in children with asthma and other chronic medical conditions implications for immunization recommendations patients hospitalized with laboratory-confirmed influenza during the 2010-2011 influenza season: exploring disease severity by virus type and subtype the burden of influenza b: a structured literature review hospitalized children with 2009 pandemic influenza a (h1n1): comparison to seasonal influenza and risk factors for admission to the icu influenza a (ph1n1) infection in children admitted to a pediatric intensive care unit: differences with other respiratory viruses clinical characteristics of pediatric hospitalizations associated with influenza a (h1n1) in northern bavaria the role of infections and coinfections with newly identified and emerging respiratory viruses in children comparative analysis of clinical characteristics of pandemic influenza a/h1n1 and seasonal influenza a infections in hospitalized children forward look risk assessment -likely scenarios and uncertainties in the 2010/2011 influenza season in europe and beyond bericht zur epidemiologie der influenza in deutschland saison 2012/13 severe influenza cases in paediatric intensive care units in germany during the pre-pandemic seasons severe cases of pandemic (h1n1)pdm2009 in children unchanged severity of influenza a(h1n1)pdm09 infection in children during first postpandemic season bavarian state office for statistics and data processing bacterial and viral infections associated with influenza. influenza other respir viruses neuraminidase inhibitors for preventing and treating influenza in healthy adults and children effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1pdm09 virus infection: a meta-analysis of individual participant data post-pandemic seroprevalence of pandemic influenza a (h1n1)pdm2009 infection (swine flu) among children <18 years in germany clinical features of influenza disease in admitted children during the first postpandemic season and risk factors for hospitalization: a multicentre spanish experience influenza in hospitalized children in ireland in the pandemic period and the 2010/2011 season: risk factors for paediatric intensive-care-unit admission critical care surveillance: insights into the impact of the 2010/11 influenza season relative to the 2009/10 pandemic season in england burden and characteristics of influenza a and b in danish intensive care units during the 2009/10 and 2010/11 influenza seasons first influenza season after the 2009 pandemic influenza: characteristics of intensive care unit admissions in adults and children in vall d'hebron hospital änderung der empfehlung zur impfung gegen influenza analyse regionaler unterschiede der influenza-impfraten in der impfsaison influenza a(h1n1)pdm09 antibodies after pandemic and trivalent seasonal influenza vaccination as well as natural infection in update: influenza activity -united states influenza activity -united states, 2013-14 season and composition of the 2014-15 influenza vaccines uptake and impact of a new live attenuated influenza vaccine programme in england: early results of a pilot in primary school-age children, 2013/14 influenza season the authors thank all participating hospitals, and picu and university staff involved in data collection and virological testing. karin seeger we thank for helpful comments on the manuscript. the study was supported by an unrestricted grant from glaxosmithkline gmbh & co. kg, munich, germany. apart from financial support, the company was not involved in any part of the study. the publication was funded by the german research foundation (dfg) and the university of wuerzburg in the funding programme open access publishing. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-290004-v3ruj5bq authors: madsen, anders; cox, rebecca jane title: prospects and challenges in the development of universal influenza vaccines date: 2020-07-06 journal: vaccines (basel) doi: 10.3390/vaccines8030361 sha: doc_id: 290004 cord_uid: v3ruj5bq current influenza vaccines offer suboptimal protection and depend on annual reformulation and yearly administration. vaccine technology has rapidly advanced during the last decade, facilitating development of next-generation influenza vaccines that can target a broader range of influenza viruses. the development and licensure of a universal influenza vaccine could provide a game changing option for the control of influenza by protecting against all influenza a and b viruses. here we review important findings and considerations regarding the development of universal influenza vaccines and what we can learn from this moving forward with a sars-cov-2 vaccine design. an influenza virus infection typically manifests as a fever, sore throat, cough, runny nose, myalgia, and headaches. people in certain risk groups (the elderly, children <59 months of age, pregnant women, and individuals with chronic conditions) have a higher chance of developing more severe illness with complications such as fulminant pneumonia. seasonal influenza causes around 290,000 to 650,000 respiratory deaths each year [1] . however, this number can increase dramatically when, at unpredictable intervals, influenza pandemics occur. these global epidemics are characterized by the emergence of a novel influenza virus to which most humans are immunologically naïve [2] . the emergence of the a/h1n1pdm09 virus ("swine flu") was the first influenza pandemic of the 21st century. this h1n1 virus has subsequently continued to circulate in the human population as a seasonal virus, together with influenza a/h3n2 and influenza b (yamagata-like and victoria-like) viruses. the current seasonal influenza vaccines contain strains from each of these viruses. the world health organization (who) decides biannually which strains should be included in the vaccine based on which viruses are most likely to circulate in the upcoming season. annual seasonal influenza vaccination remains the most cost-effective measure to reduce burden of the disease. however, the currently licensed influenza vaccines lack two essential attributes: firstly, they do not produce durable protective immunity. secondly, they do not produce a cross-reactive immune response that can neutralize diverse influenza virus strains. cross-reactivity is necessary because of the antigenic plasticity of the viral membrane protein hemagglutinin (ha), which leads to antigenically drifted viruses and potential mismatches with the vaccine strains [3] . this shortcoming can be addressed by developing new vaccines targeting the more antigenically conserved regions of the influenza virus [4] . in order to overcome these barriers, efforts have been made to develop next-generation influenza vaccines that provide robust, long-lasting protection against diverse influenza a subtypes (table 1 ) [5] . the ultimate goal is to develop a "universal" influenza vaccine that covers all influenza a and b viruses. [6] . provide protection against all ha subtypes and at a minimum protection against h1, h2, h3, h5, h7 and h9 subtypes. provide a decade or more of protection. use manufacturing technology that can be readily transferred to developing-world countries. provide immunologic protection for those populations most at risk for severe disease and increased mortality use inexpensive manufacturing technology that permits rapid and highly scalable production, particularly to address emergence of a pandemic virus. offer heat stability, thereby eliminating the need to maintain a cold chain. rarely cause adverse events, and any adverse events are mild and temporary. do not require injection for administration. do not require injection for administration. the influenza virus is an enveloped virus with a segmented, negative-sense, single-strand rna genome ( figure 1 ). there are four types of influenza: a, b, c, and d. influenza a and b are the only types that cause seasonal epidemics in humans. influenza a viruses are further subdivided into subtypes and strains depending on the characteristics of the most abundant surface proteins, ha and neuraminidase (na). ha is a trimeric glycoprotein responsible for the attachment of the virus to the surface of the host cell by binding to sialic acid receptors. there are 18 subtypes of ha within the influenza a viruses. these can be divided into two distinct phylogenetic groups: group 1 ha (h1, h2, h5, h6, h8, h9, h11, h12, h13, h16, h17, and h18) and group 2 ha (h3, h4, h7, h10, h14, and h15). the ha protein comprises a head domain and a stalk domain, the latter being more antigenically conserved. antibodies targeting the ha head domain are usually strain-specific and neutralize influenza viruses by inhibiting the binding of ha to sialic acid receptors on the surface of the host cell. ha stalk-specific antibodies can provide heterosubtypic protection by blocking viral fusion with the host cell and by eliminating infected host cells through antibody-dependent cellular cytotoxicity (adcc) [7, 8] . the stalk-specific antibodies are broadly cross-reactive within an ha group [9, 10] . while ha remains the main target of current inactivated influenza vaccines (iivs), na has recently been recognized as a potential target for universal vaccines [11, 12] . na functions by catalyzing the cleavage of sialic acids on the surface of an infected cell, leading to the release of newly formed viruses. several studies have shown that na antibody titers correlate with protection in humans [13-15]. na-specific antibodies function by inhibiting the enzymatic activity of na and thus preventing the spread of virions from infected cells. fc receptor-mediated effector functions such as adcc have also been reported for na antibodies [16, 17] . the matrix protein 2 ectodomain (m2e) protein functions by transferring h + ions through the viral membrane as the virus is taken up by the endosomes. this leads to conformational changes of the ha molecule and fusion between the viral membrane and the endosome membrane. as a result, the viral rna is released into the cytosol. m2e-specifc antibodies have been shown to be protective in animal studies, although the exact defense mechanisms are not fully understood. t-cell activation also plays an important role in targeting other conserved proteins that are generally not exposed on the outside of virus particles, such as the nucleoprotein (np) and matrix protein 1 (m1) [25] . these proteins are highly conserved between influenza a viruses. influenza-specific interferon-secreting t cells, cd4+ t cells, and cd8+ t cells play an important role in recovery from influenza in humans [26] [27] [28] [29] . there are currently three types of licensed influenza vaccines: inactivated, live attenuated, and recombinant ha ( figure 2 ). inactivated influenza vaccines (iivs) are the most commonly used, partly because of their well-documented safety and low production costs. the iivs come in three variants: whole-virion vaccines, split-virion vaccines, and subunit vaccines, which contain purified ha and na. iivs are traditionally produced in embryonated chicken eggs. this egg-based approach has several drawbacks: firstly, the production time is relatively long. consequently, the producers have to start production months in advance of the flu season, which can lead to mismatches between the vaccine strain and the circulating strain [30] . in addition, the prolonged production time becomes a crucial problem during influenza pandemics, where rapid production and distribution of vaccines are essential. another issue with the egg-based approach is the occurrence of egg-adapted mutations during the production process, which can lower vaccine effectiveness [31, 32] . cell-based iivs have been licensed in europe and the united states as an alternative to the egg-based approach. another approach for overcoming egg-based vaccine production is the use of recombinant ha vaccines, which are based on a protein-expression system using baculoviruses and insect cells [33] . these vaccines have been licensed in the united states since 2013. however, as with the iivs, the protection appears to be strain-specific, with limited immunogenicity. some vaccine formulations include adjuvants in order to improve immunogenicity and vaccine effectiveness [34] . adjuvants function by increasing antigen uptake and presentation in the local draining lymph nodes. during the 2009 h1n1 pandemic, the use of the oil-in-water emulsion (as03) led to higher b and cd4 t-cell responses than vaccination with nonadjuvanted pandemic vaccines [35, 36] . in contrast to iivs and recombinant ha vaccines, the live-attenuated influenza vaccines (laiv) contain live, cold-adapted viruses that are administered as a nasal spray, leading to a restricted viral replication in the upper respiratory tract of the recipient [37, 38] . the laiv induces broader immune response, including t-cell and mucosal immunity, by mimicking natural infection [39] [40] [41] . laiv is currently licensed in north america, europe, and india. it is generally not recommended for people younger than 2 years of age due to increased risk of wheezing [42] or for immunosuppressed individuals who are at greater risk of severe influenza illness as the vaccine may produce higher virus titers in these individuals, leading to severe side effects. recent scientific advances have made way for novel vaccine approaches, enabling a more targeted delivery of conserved antigens that can stimulate the innate and adaptive immune systems. here, we highlight three vaccine platforms that are being used in the development of next-generation influenza vaccines (figure 2 ). virus-like particles (vlps) have similar morphological and structural features to viruses but lack the viral genome [43] . they are a useful vaccine component as the immune system recognizes vlps similarly to viruses but without the risk of replication and recombination. a variety of immunogens have been tested on vlps, including ha, matrix protein 2 (m2), and na [44] [45] [46] . one of the main barriers to the use of this vaccine construct is the challenge of generating sufficient immunogenicity. however, the inclusion of adjuvants such as toll-like receptor ligands has led to improved vaccine effectiveness [47] . in contrast to vlps, peptide-based vaccines focus on minimal epitopes of the influenza virus, such as t-cell-inducing np and m1 peptides [48, 49] . however, as with vlp-based vaccines, they often require the use of adjuvants or particulate carriers for delivery in order to be sufficiently immunogenic. nucleic-acid-based vaccines use rna-or dna-sequences for the vaccines antigens. this technology is especially useful in disease outbreaks where rapid vaccine development is needed. using self-amplifying mrna, researchers were able to develop a vaccine candidate for the influenza a h7n9 outbreak in china just 8 days after a/shanghai/2/2013 (h7n9) sequences were released [50] . this vaccine platform could be a viable option in the ongoing efforts to develop a sars-cov-2 vaccine. a phase 1 clinical trial of a dna vaccine targeting the spike protein of sars-cov-1 found that the vaccine was safe and capable of eliciting neutralizing antibodies and cellular immune responses [51] . viral vector vaccines use carrier viruses such as modified vaccinia virus ankara (mva) or adenovirus containing genes expressing influenza-proteins of interest [52] . these vaccines allow for any influenza antigens to be expressed in their native conformation or with modifications, inducing both humoral and cellular immune responses [53] [54] [55] . most viral vectors are replication-deficient in mammalian host cells and are therefore safe to use. issues with pre-existing immunity to some viral vectors have been reported [56] , although other vectors, such as mva, remain immunogenic despite the presence of pre-existing immunity. influenza vaccines can be divided into two categories based on their immunological features: (1) vaccines providing sterile immunity by eliciting ha head antibodies that prevent viral attachment to sialic acid receptors and (2) vaccines that provide infection-permissive immunity by inducing cellular immunity or broadly reactive antibodies that function in the later stages of the viral life cycle (figure 3) . many of the infection-permissive vaccines that are in development will probably not be universal vaccine candidates as stand-alone vaccines but could be combined with other approaches to increase immunogenicity. ha stalk antibodies have emerged as one of the leading strategies in the development of universal vaccine candidates [57] . several approaches for avoiding the immunodominant ha head domain are under development. one strategy is to remove the globular head domain while maintaining the correct immunogenic conformation of the ha stalk domain [58] . animal models have shown that vaccination with these "headless" ha proteins can induce heterosubtypic protection within group 1 ha proteins [59] [60] [61] [62] . other strategies for refocusing the antibody response toward the ha stalk involve maintaining the full-length ha. krammer and colleagues have developed a method for directing the antibody response to the conserved ha stalk domain by sequential vaccination with chimeric ha proteins consisting of exotic ha head domains to which the recipient is immunologically naïve. the results of a phase 1 clinical trial (nct03300050) provide evidence that group 1 chimeric ha-based vaccines induce high titers of stalk-reactive immunoglobulin g (igg) that cross-react to other ha proteins within the same group [63] . a trivalent vaccine comprising group 1, group 2, and influenza b virus chimeric constructs could potentially lead to a universal influenza vaccine. advances in the development of monoclonal antibodies (mabs) have shown that there are cross-reactive epitopes on the ha head domain that can be targeted to give heterosubtypic protection [64] [65] [66] . a new vaccine approach called mosaic ha has been developed to combine the immunity of conserved epitopes of the ha stalk and head domains. mosaic ha vaccines are developed by replacing the immunodominant and strain-specific antigenic sites with sequences from exotic ha subtypes. through sequential immunization, the immune response is refocused toward more cross-reactive immunosubdominant epitopes of the ha stalk and head domains. cross-reactive ha stalk-and head-specific antibodies can also be induced by computationally optimized, broadly reactive antigens (cobras). this approach is based on developing novel ha proteins after multiple rounds of consensus sequence generation from a variety of different ha isolates. preclinical trials have demonstrated that cobra-based vaccines provide protection against multiple influenza viruses within a subtype in murine and ferret models [67] [68] [69] . there has been increased recognition of na as a target for improved vaccines following the development of practical assays for measuring antibody responses to na [11] . although antibodies targeting na can bind to conserved epitopes and provide heterologous protection [16], they are not really induced by seasonal iivs. this is mostly due to the amount and stability of the na in the vaccines. enhanced na-antibody responses can be achieved by delivering na as a supplement to current iivs or vaccinating with recombinant or viral vector na in addition to the seasonal iivs [70] [71] [72] [73] [74] . m2e has been an attractive target in the development of the future universal influenza vaccine, as it is highly conserved between influenza viruses [75] . because of the relatively small size of the m2e, vaccines usually depend on carrier constructs such as vlps. preclinical trials have demonstrated that m2e antibodies are protective in animal models, and some m2e vaccines have advanced into clinical trials [76, 77] . while the effectiveness of a stand-alone m2e-based vaccine remains limited, recent studies have found promising results by combining an m2e-based vaccine (m2e5x vlp) with other vaccine constructs such as laivs [78] and ha vlps [79] . most vaccine approaches targeting internal proteins such as m1 and np involve t-cell activation. t cells are important during an influenza infection because they limit viral replication and shedding by clearing influenza-infected cells. an ongoing phase 3 clinical trial (clinicaltrials.gov: nct03450915) is testing a recombinant-peptide based vaccine consisting of conserved epitopes from ha, np, and m1 [80] . other vaccine constructs use viral vectors expressing m1 and np [54, 81] . these have been shown to induce cd4+ and cd8+ t cells in clinical trials. selecting the best approach for inducing t-cell-mediated immunity can be challenging due to the diverse cd4 t-cell repertoire in humans with different degrees of pre-existing immunity. however, with increasing insights into the quantity, functionality, and specificity of cd4 t-cell subsets, there is hope that new correlates of protection can be established in the near future, which would help in selecting the best vaccine candidates for clinical trials [29, 82, 83] . some studies evaluating the t-cell response in various infection models have reported immunopathology with excessively large cd8 t-cell responses [84, 85] . other studies have reported a narrowing of t-cell receptors during heterosubtypic challenges [86] . additional studies that reflect pathogen encounters in humans are needed in order to select the best candidates for next-generation t-cell-based vaccines [87] . there has been significant progress in the development of novel approaches for universal influenza vaccines. however, additional efforts are needed in order to translate more of these novel approaches into products ready for late-stage clinical trials. selecting the best candidates for clinical trials is a challenging process for several reasons: (1) there is no uniform evaluation of novel influenza vaccines in preclinical trials. this is partly due to the variety of vaccine platforms and targets. in addition, there is no consensus on which animal model is best suited to accurately reflect human infection [3, 88] . this is partly due to the existence of pre-existing immunity in humans, which is difficult to translate into animal models. other host factors to be considered include age, gender, and chronic diseases, which could affect vaccine efficacy. (2) there are no established correlates of protection for broadly cross-reactive immune responses to influenza. assays measuring non-ha-head immune responses, such as ha stalk antibodies, na antibodies, and cellular immunity, are inadequately standardized. optimization and standardization of such assays are needed in order to assess the protective immunity contributed by broadly reactive or universal vaccines. (3) the funding for universal vaccine development is limited. this leads to down selection of promising candidates before they advance to clinical trials. regulatory initiatives are needed to enhance advocacy for moving from an annually reformulated vaccine to a universal vaccine, despite the financial risks and inadequate incentives [6] . as the search for an appropriate sars-cov-2 vaccine continues, comparisons can be drawn to influenza vaccine development. some of the next-generation influenza vaccines use vaccine platforms similar to those used by the sars-cov-2 vaccine candidates [89] . given the urgency of moving forward with a sars-cov-2 vaccine, a nucleic-acid-based vaccine would be a convenient approach in order to facilitate rapid production. although this method has been tested for several influenza vaccine candidates, no dna or rna vaccines have yet been licensed for humans. the disadvantages of such vaccines include limited immunogenicity and reports of increased reactogenicity. another option is the use of viral-vector-based vaccines, which have shown promising results against mers-cov and other emerging viruses [90] [91] [92] . some viral-vector-based vaccines focusing on the s protein of the sars-cov-2 virus are in the preclinical phase. however, it might be years until a nucleic-acidor viral-vector-based vaccines are licensed for human use since both of these vaccine platforms are novel and have not been extensively tested. other sars-cov-2 vaccines that are currently being tested include recombinant protein vaccines, live-attenuated vaccines, and inactivated vaccines. all of these platforms can be found among the licensed influenza vaccines, each with their own advantages and disadvantages. in the end, a sars-cov-2 vaccine or a universal influenza vaccine can only be successful if implemented effectively [93] . efforts are needed to improve vaccine uptake by restoring widespread public confidence in vaccines. this is especially important in a pandemic setting. in addition, as the 2009 influenza a/h1n1 pandemic demonstrated, there will likely be an issue of vaccine availability in low-and middle-income countries. therefore, strategies are needed to ensure sufficient and equitable vaccine distribution to these countries. encouraging progress has been made in the development of universal influenza vaccines. future universal vaccines will likely consist of a combination of different approaches to induce broad immunity to influenza. ultimately, the path forward depends on combined action from governments, industries, and the scientific community. estimates of global seasonal influenza-associated respiratory mortality: a modelling study in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses the future of influenza vaccines: a historical and clinical perspective development of a universal influenza vaccine a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases the compelling need for game-changing influenza vaccines an analysis of the influenza vaccine enterpris the role of fc-fcγr interactions in igg-mediated microbial neutralization broadly neutralizing anti-influenza antibodies require fc receptor engagement for in vivo protection highly conserved protective epitopes on influenza b viruses natural t cell-mediated protection against seasonal and pandemic influenza. results of the flu watch cohort study cytotoxic t-cell immunity to influenza preexisting influenza-specific cd4+ t cells correlate with disease protection against influenza challenge in humans influenza virus: dealing with a drifting and shifting pathogen h3n2 influenza viruses in humans: viral mechanisms, evolution, and evaluation. hum. vaccines immunother efforts to improve the seasonal influenza vaccine safety, efficacy, and immunogenicity of flublok in the prevention of seasonal influenza in adults adjuvanted influenza vaccines. hum. vaccines immunother van der most, r. investigating the effect of as03 adjuvant on the plasma cell repertoire following ph1n1 influenza vaccination adjuvant system as03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity recombinant cold-adapted attenuated influenza a vaccines for use in children: molecular genetic analysis of the cold-adapted donor and recombinants multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60 the role of nasal iga in children vaccinated with live attenuated influenza vaccine comparisons of the humoral and cellular immune responses induced by live attenuated influenza vaccine and inactivated influenza vaccine in adults immune responses after live attenuated influenza vaccination live attenuated versus inactivated influenza vaccine in infants and young children novel platforms for the development of a universal influenza vaccine multi-antigen avian influenza a (h7n9) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models virus-like particles containing multiple m2 extracellular domains confer improved cross-protection against various subtypes of influenza virus an intranasal virus-like particle vaccine broadly protects mice from multiple subtypes of influenza a virus tlr-based immune adjuvants heterosubtypic influenza protection elicited by double-layered polypeptide nanoparticles in mice synthetic multi-epitope peptides identified in silico induce protective immunity against multiple influenza serotypes rapidly produced sam ® vaccine against h7n9 influenza is immunogenic in mice a sars dna vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a phase i clinical trial viral vector-based influenza vaccines. hum. vaccines immunother safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for h5n1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study preliminary assessment of the efficacy of a t-cell-based influenza vaccine, mva-np+ m1, in humans safety and immunogenicity of adenovirus-vectored nasal and epicutaneous influenza vaccines in humans failure of a recombinant fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to provide consistent protection against influenza in chickens preimmunized with a fowl pox vaccine universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines a stable trimeric influenza hemagglutinin stem as a broadly protective immunogen influenza virus vaccine based on the conserved hemagglutinin stalk domain mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed nhp that protect mice against lethal influenza challenge hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection immunogenicity of chimeric haemagglutinin-based, universal influenza virus vaccine candidates: interim results of a randomised, placebo-controlled, phase 1 clinical trial cross-neutralization of influenza a viruses mediated by a single antibody loop receptor mimicry by antibody f045-092 facilitates universal binding to the h3 subtype of influenza virus heterosubtypic antibody recognition of the influenza virus hemagglutinin receptor binding site enhanced by avidity design and characterization of a computationally optimized broadly reactive hemagglutinin vaccine for h1n1 influenza viruses a computationally optimized broadly reactive antigen (cobra) based h5n1 vlp vaccine elicits broadly reactive antibodies in mice and ferrets a computationally optimized broadly reactive antigen subtype-specific influenza vaccine strategy elicits unique potent broadly neutralizing antibodies against hemagglutinin purified influenza a virus n2 neuraminidase vaccine is immunogenic and non-toxic in humans vaccination with adjuvanted recombinant neuraminidase induces broad heterologous, but not heterosubtypic, cross-protection against influenza virus infection in mice vaccination with recombinant parainfluenza virus 5 expressing neuraminidase protects against homologous and heterologous influenza virus challenge immunogenicity and protection against influenza h7n3 in mice by modified vaccinia virus ankara vectors expressing influenza virus hemagglutinin or neuraminidase neuraminidase expressing virus-like particle vaccine provides effective cross protection against influenza virus m2e-based universal influenza vaccines: a historical overview and new approaches to development immunopotentiation of trivalent influenza vaccine when given with vax102, a recombinant influenza m2e vaccine fused to the tlr5 ligand flagellin safety and immunogenicity of a recombinant m2e-flagellin influenza vaccine (stf2. 4xm2e) in healthy adults enhancing the cross protective efficacy of live attenuated influenza virus vaccine by supplemented vaccination with m2 ectodomain virus-like particles influenza m2 virus-like particle vaccination enhances protection in combination with avian influenza ha vlps safety and immunogenicity of multimeric-001-a novel universal influenza vaccine at cell-inducing influenza vaccine for the elderly: safety and immunogenicity of mva-np+ m1 in adults aged over 50 years crtam determines the cd4+ cytotoxic t lymphocyte lineage nkg2c/e marks the unique cytotoxic cd4 t cell subset, thctl, generated by influenza infection protective heterologous antiviral immunity and enhanced immunopathogenesis mediated by memory t cell populations memory cd8 t cells mediate severe immunopathology following respiratory syncytial virus infection t cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens past life and future effects-how heterologous infections alter immunity to influenza viruses workshop report: experimental animal models for universal influenza vaccines vaccines: status report. immunity immunogenicity of an adenoviral-based middle east respiratory syndrome coronavirus vaccine in balb/c mice systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of middle east respiratory syndrome coronavirus recent advances in the vaccine development against middle east respiratory syndrome-coronavirus improving influenza vaccines: challenges to effective implementation the authors declare no conflict of interest. key: cord-281228-8kqohdcr authors: li, xin; leng, sean x. title: influenza immunization among chinese seniors: urgent calling for improving vaccination coverage, education, and research date: 2020-03-27 journal: aging med (milton) doi: 10.1002/agm2.12103 sha: doc_id: 281228 cord_uid: 8kqohdcr nan it is with great pleasure that we provide this commentary with a focus on influenza vaccination for an expert consensus entitled "recommendations for influenza and streptococcus pneumoniae vaccination in elderly people in china" to be published in this issue of aging medicine. 1 influenza is a major global public health burden with pandemic threat. seasonal influenza infection is responsible for 3-5 million severe illness cases and 290 000-650 000 respiratory deaths annually worldwide. 2, 3 according to the centers for disease control and prevention (cdc), influenza affects 5%-20% of the population each year in the united states. 4 it is estimated that influenza causes 226 000 excess hospitalizations, 25 000-69 000 deaths, and us $87 billion excess health-care cost with over 600 000 life-years lost annually. 5, 6 among all infectious diseases, influenza is foremost in its age-related increase in serious complications, leading to hospitalization, catastrophic disability, and death in older adults. 7, 8 moreover, influenza frequently causes exacerbation of many chronic conditions that are common in older adults, including cardiovascular diseases, 9, 10 further indirectly impacting senior health and mortality. in fact, over 90% of influenza-related mortality occurs in persons aged over 65 years. 11 in the united states, influenza and its secondary pneumonia are the fourth leading cause of death in this population. 12 therefore, prevention and treatment of influenza in older adults have become a major public health priority. like many preventable infectious diseases, immunization is the main strategy for influenza prevention in addition to the universal contact-and airborne-precaution measures. in the united states, annual influenza vaccination is recommended for all adults aged 50 years and older. the annual vaccination is overall efficacious, with estimated risk reduction of 50%-70% for influenza infection in young adults and less robust risk reduction in older adults. 13, 14 this reduced efficacy in older adults is thought to be due to immunosenescence and common health conditions, such as frailty. 15 in recent years, a new generation of influenza vaccines, including high-dose (hd) and adjuvanted ones, have been approved by the us food and drug administration (fda) for older adults in the united states and elsewhere, in addition to the standard dose trivalent inactivated influenza vaccine (iiv3). as chen et al 1 point out in the expert consensus paper, influenza vaccination coverage for older adults in china is extremely low. this is in the context of the largest and fastest growing older adult population, 16 leaving the majority of chinese seniors unprotected by vaccination against influenza. historically, china has classified vaccines into two categories. the first-category vaccines are mostly for childhood immunization, such as measles, mumps, and rubella; diphtheria, tetanus, and pertussis; varicella; and bacillus calmette-guerin. the chinese authorities have mandated that these first-category vaccines be given to every child born in china, free of charge. the second-category vaccines, influenza vaccine included, are mostly for adult immunization. immunization with the second-category vaccines is not mandated and immunization cost is not covered by government-sponsored health insurance. as such, childhood imshould also be considered before these vaccines can be produced domestically with quality assurance. this pillar is particularly important to reestablish public confidence since the recent vaccine scandals stated above. last but not least is the pillar of innovation and research. after all, it is the effort of innovation and research that has led to the development and fda-approved clinical application of the new generation of influenza vaccines described above. in china, the pillar of innovation and research will need to be built from the ground up with special consideration of the following three aspects. in-depth studies of vaccine-induced immunity agaist influenza as well as age-related immunosenescence. 3. host factors are the third aspect that is equally critical and deserves more attention. if the host fails to mount an adequate immune response or refuses to get vaccinated, it is irrelevant no matter how good the influenza vaccine is. immunosenescence that occurs during aging is believed to be a major host factor responsible for the impaired immune protection of influenza vaccination in older adults, further review of which is beyond the scope of this article. [21] [22] [23] age-related conditions, such as frailty, as well as sex and gender are other important clinical, biological, and social host factors that have significant impact on influenza vaccine immune response and clinical protection. 15, 24, 25 emerging evidence suggests that stem cell therapy may enhance immune response to influenza immunization in aging frailty. 26, 27 innovation and research will help develop strategies to address these and other host factors. cutting-edge geroscience research may pave the way for the development of such interventional strategies. 28, 29 government agencies, health-care providers, academic institutions, and the vaccine manufacturing industry are key players in this proposed roadmap. other entities, including philanthropic foundations, international organizations, and venture capitalists, may also play important roles. government agencies are responsible for policies and regulations, such as those on health insurance coverage of influenza vaccination cost and vaccine quality control and supply (red solid arrows, figure 1 ). they also provide funding for education as well as innovation and research (red dotted arrows). academic institutions and health-care providers play a direct role in education (educating primary-care physicians and other health-care workers as well as the public) as well as innovation and research (blue solid arrows). with their expertise and leading roles in innovation and research, they may also influence policy-making and vaccine supply (blue dotted arrows). likewise, the vaccine manufacturing industry plays a role directly in vaccine supply as well as innovation and research (black solid arrows), and perhaps also indirectly in providing information on the influenza vaccines they manufacture to healthcare providers as well as the public (education, black dotted line). other entities can directly play a role in education and innovation and research (green solid lines) and vaccine development and supply (green dotted arrow). working together, it is hoped that these key players along with the public will improve policy, education, vaccine supply, as well as innovation and research, ultimately leading to universal influenza vaccination coverage for seniors and those who need vaccine protection in china. nothing to disclose. recommendations for influenza and streptococcus pneumoniae vaccination in elderly people in china influenza (seasonal): fact sheet. world health organization website estimates of global seasonal influenza-associated respiratory mortality: a modelling study influenza (flu) influenzaassociated hospitalizations in the united states the annual impact of seasonal influenza in the us: measuring disease burden and costs estimating influenza vaccine effectiveness: evolution of methods to better understand effects of confounding in older adults hospital diagnoses, medicare charges, and nursing home admissions in the year when older persons become severely disabled risk of myocardial infarction and stroke after acute infection or vaccination effect of influenza vaccine on tumor necrosis factor-like weak inducer of apoptosis (tweak) in older adults impact of influenza vaccination on seasonal mortality in the us elderly population mortality associated with influenza and respiratory syncytial virus in the united states efficacy and effectiveness of influenza vaccines in elderly people: a systematic review effectiveness of influenza vaccine in the community-dwelling elderly frailty is associated with impairment of vaccine-induced antibody response and increase in post-vaccination influenza infection in community-dwelling older adults the aging tsunami and senior healthcare development in china using the immunization information system to determine vaccination coverage rates among children aged 1-7 years: a report from zhejiang province, china an overview of coverage of bcg vaccination and its determinants based on data from the coverage survey in zhejiang province overview of influenza vaccination policy in beijing, china: current status and future prospects meeting report: the pathway to a universal influenza vaccine telomere length as an indicator of the robustness of b and t cell response to influenza immunization in older adults understanding immunosenescence to improve responses to vaccines chronic low-grade inflammatory phenotype (clip) and senescent immune dysregulation aging, sex, inflammation, frailty, and cmv and hiv infections sex and gender differences in the outcomes of vaccination over the life course allogeneic mesenchymal stem cell therapy: a regenerative medicine approach to geroscience allogeneic mesenchymal stem cell therapy improves immune response to influenza vaccination in aging frailty: preliminary results from hera trial. paper presented at: maryland stem cell fund and tedco scientific symposium aging mechanisms and interventions that impact senior health: introduction to a special issue on geroscience geroscience and the trans-nih geroscience interest group key: cord-288238-36hiiw91 authors: keshavarz, mohsen; solaymani-mohammadi, farid; namdari, haideh; arjeini, yaser; mousavi, mohammad javad; rezaei, farhad title: metabolic host response and therapeutic approaches to influenza infection date: 2020-03-05 journal: cell mol biol lett doi: 10.1186/s11658-020-00211-2 sha: doc_id: 288238 cord_uid: 36hiiw91 based on available metabolomic studies, influenza infection affects a variety of cellular metabolic pathways to ensure an optimal environment for its replication and production of viral particles. following infection, glucose uptake and aerobic glycolysis increase in infected cells continually, which results in higher glucose consumption. the pentose phosphate shunt, as another glucose-consuming pathway, is enhanced by influenza infection to help produce more nucleotides, especially atp. regarding lipid species, following infection, levels of triglycerides, phospholipids, and several lipid derivatives undergo perturbations, some of which are associated with inflammatory responses. also, mitochondrial fatty acid β-oxidation decreases significantly simultaneously with an increase in biosynthesis of fatty acids and membrane lipids. moreover, essential amino acids are demonstrated to decline in infected tissues due to the production of large amounts of viral and cellular proteins. immune responses against influenza infection, on the other hand, could significantly affect metabolic pathways. mainly, interferon (ifn) production following viral infection affects cell function via alteration in amino acid synthesis, membrane composition, and lipid metabolism. understanding metabolic alterations required for influenza virus replication has revealed novel therapeutic methods based on targeted inhibition of these cellular metabolic pathways. influenza virus infection (ivi) is one of the most common infectious agents, capable of infecting a variety of avian and mammalian species. the virus is responsible for seasonal epidemics, leading to 3-5 million severe infections and 250,000-500,000 deaths annually [1, 2] . despite the annual vaccination program, the high mortality rate caused by influenza infection and its various complications, including chronic lung disease, cardiac disease, asthma, and metabolic disorders, is yet to be adequately addressed [3] [4] [5] . in 1956, eagle et al. first indicated that the addition of glucose to hela cell medium could promote the generation of poliovirus progeny [6] . results of a published study showed that the replication of the influenza virus depends on host cellular metabolism such that metabolites including nucleic acids, proteins, glycoproteins, and lipids are crucially required for the life cycle of the influenza virus [7] . recent research on a mouse model showed that influenza infection could affect more than 100 metabolite markers in serum, lung, and bronchoalveolar lavage fluid [8] . acquiring the required materials from the host cell to self-replicate, the virus can disrupt biochemical processes such as glycolysis, fatty acid (fa) synthesis, and glutamine pathways [9] . it is of particular importance for scientists to broaden their horizon on the metabolic changes during influenza infection, which in turn paves the way for preventing life-threatening consequences. influenza infection actively provokes the pro-oxidant condition in the host cell to facilitate viral proliferation and pathogenesis. increased expression of influenza m2 protein can activate protein kinase c and increase reactive oxygen species (ros) production [10] . on the other hand, pb1-f2 decreases superoxide anion dismutase 1 (sod1) expression and consequently disrupts the ros scavenging process [11] . in people with influenza infection, increased levels of dna, lipid, and protein oxidation products are found in plasma and urine [12] [13] [14] . also, increased levels of ros and inducible nitric oxide synthase (inos) have been observed as markers of oxidative stress in the lungs of people who died due to pandemic influenza infection [15] . ros-producing enzymes induced by influenza infection mainly include nadph oxidase (nox) and xanthine oxidase, upregulation of which causes the impaired defensive function of antioxidants [16] . an increase in ros production, along with impaired antioxidant function, ultimately leads to a profound change in redox homeostasis of the cell [16] [17] [18] [19] . nox2 is a phagocytic enzyme that is involved in the production of ros induced by influenza virus [19] [20] [21] [22] , and impaired nox2 expression results in a lack of increased rns and ros production following influenza infection [20] . xanthine oxidase is also an ros-producing enzyme that is induced by influenza infection [23, 24] , and its inhibition can hinder ros increase in the cell. on the other hand, increased expression of sod1 reduces influenza virus titers within the cell [25] . it is also reported that influenza infection significantly increases ros production by inducing nox4, and the proliferation of this virus in lung epithelial cells is dependent on redox-sensitive pathways activated by nox4-derived ros [16] . glutathione (gsh) is a vital antioxidant in the cell, and its cellular content is inversely related to influenza virus replication in the cell [26, 27] . it is indicated that higher levels of gsh, antioxidant enzymes such as glutathione peroxidase, and the anti-apoptotic protein bcl-2 in the lungs of female mice result in superior resistance of these mice to influenza infection. in contrast, male mice are more susceptible to this infection due to higher expression of nox4. this difference is due primarily to the higher ability of female mice to maintain cellular redox homeostasis [28] . amatore et al. also showed that an increase in gsh content in organs by affecting gsh-dependent antiviral pathways strengthens the immune system, in particular th1 cell response, and decreases viral replication [29] . however, gsh depletion results in a deviation of the response towards th2 cells [30] . furthermore, oxidative stress following infection can induce the transcription factor nf-kb, which subsequently leads to increased levels of inflammatory cytokines, including interleukin (il)-1β, il-6, ifn, and tnf [31, 32] . ifns are one of these cytokines that trigger and affect t cell metabolism via mediating glucose uptake, glycolysis, and lipid synthesis. ifn can also exert its function on metabolic changes by producing several mediators including indoleamine-2,3-dioxygenase (ido) and nitric oxide (no), both of which appear to have either an inducible or an inhibitory role in viral replication [33] . since tryptophan is critical for t cell proliferation, depletion of this amino acid by ido suppresses the immune system through the stimulation of t regulatory cells. no, on the other hand, inhibits viral replication via changes in the structure of viral proteins [34] . in this review, we first discuss the metabolic abnormalities during influenza infection and then shed light on the role of immunometabolites that regulate cellular metabolism. the following sections summarize recent evidence about the novel therapeutic approaches that target metabolic pathways in influenza infection. viruses take advantage of various cellular mechanisms to replicate efficiently. vital metabolic pathways of host cells are one of the most widely used mechanisms targeted by viruses, resulting in considerable changes. in this context, studies have revealed that various human viruses, such as cytomegalovirus [35] [36] [37] , rubella [38, 39] , dengue [40] , mumps [41] , poliovirus [42, 43] and reovirus [44] can strongly affect host cell glycolysis, lipid metabolism, and glutaminolysis. furthermore, a review by sanchez and lagunoff delineated the activation of these metabolic processes by several viruses [45] . as will be discussed in this section, influenza as a highly pathogenic human virus interferes tremendously with the host metabolic cycles and thereby forces them to produce viral particles more efficiently. the metabolism and concentration of glucose in the cell play a cardinal role in the homeostasis of cellular metabolic procedures [46] . shortly after the onset of ivi, the rate of glucose uptake by infected cells increases continually, and the subsequently enhanced glycolysis results in higher glucose consumption, production of viral particles [47, 48] , and extracellular concentration of lactate. the relationship between glycolysis and ivi has shown that influenza infection at a higher multiplicity of infection (moi) raises the glycolytic activity of the cells [49] . in a study by kohio and adamson, a dose-specific increase in influenza infection was associated with higher glucose levels, whereas the treatment of cells with glycolysis inhibitors remarkably suppressed the viral replication. however, the viral infection could be retriggered by adding atp to the cell environment. this study revealed that enhancing vacuolar-type atpase (a proton pump essential for influenza uncoating) via increasing glucose metabolism and, as a result, higher available atp resources, augments the virus infection [50] . the observations, as mentioned above, reveal a significant increase in atp and glucose consumption within cells following influenza infection and also highlight the dependence of the influenza virus on the glycolysis pathway for energy production. the viral replication has the highest use of atp during influenza infection, releasing large quantities of energy in the form of heat. this process can increase the temperature of infected cells by 4-5°c. as viral proliferation increases, the cellular atp level drops sharply, resulting in reduced potential and stability of the mitochondrial membrane [51] . based on available results, patients with metabolic disorders can develop more severe influenza infection compared to healthy hosts. there have been several studies showing that diabetes can increase the risk of influenza infection, the severity of the disease, and the fatal consequences of this infection [52] [53] [54] [55] . about 90% of patients with type 2 diabetes are overweight, and obesity is a significant risk factor for severe influenza infection [56] . this reinforcing effect of diabetes on influenza may be due to the inhibitory effect of hyperglycemia on the immune system [57] [58] [59] . it has been shown that this hyperglycemia-associated immunosuppression and susceptibility to influenza infection can be alleviated by insulin administration and diabetes control [60] . on the other hand, the pentose phosphate pathway (ppp), as another glucose-consuming pathway reported to be enhanced by ivi [61] , contributes to a higher yield of nucleotides and atp for viral replication [62] . significant up-regulation of the ppp key enzymes in influenza-infected cells, including glucose 6-phosphate dehydrogenase (g6pd) and 6-phosphogluconate dehydrogenase (6pgd), was reported by janke et al. [63] . g6pd enzyme is also responsible for the generation of nadph [64] , a critical component of fatty acid biosynthesis. the level of g6pd activity specifies the ability of the cell to clear the accumulated ros. cells with an average g6pd level can retain the appropriate gsh/gssg ratio and keep the ros production at a tolerably low level, indicating that g6pd activity has an inverse correlation with cellular ros level [65] . disruption of redox balance has been shown to contribute to replication and virulence of several viruses [66] [67] [68] , and g6pd deficiency can cause this disruption. despite the results reported by janke et al., g6pd activity seems to have an inverse relation with some other respiratory viral infections. for example, in an in vitro study, after infection with human coronavirus (hcov) 229e, the production of viral particles in g6pd-deficient or g6pd-knockdown cells was higher than in healthy cells, and this was correlated with increased oxidant production [67] . molecular mechanisms through which the virus can control the metabolic pathways have been thoroughly identified. smallwood et al. have shown that an increase in glucose uptake, glycolysis, and glutaminolysis following influenza infection may be related to the loss of pi3k/akt/mtor pathway homeostasis and subsequent increase in c-myc expression in the infected cells [9] . regarding the available results, the mechanistic target of rapamycin complex 1 (mtorc1) and mtorc2 signaling can be activated by a variety of influenza virus proteins. the viral hemagglutinin (ha) protein, along with virus replication, can upregulate pdpk1-mediated phosphorylation and activate akt, which is required for induction of the mtorc1 signaling pathway by the influenza virus. on the other hand, influenza m2 protein is capable of down-regulation of the mtorc1 inhibitor redd1, thereby enhancing the mtorc1 activation [69] . mtorc1 signaling, in turn, promotes c-myc expression at the translational level [70] . additionally, the ns1 protein can effectively promote the activity of mtorc2, which, in turn, upregulates c-myc through foxo inhibition [71] . moreover, akt-dependent inactivation of foxos can increase glycolysis [72, 73] by removing the suppressive force of c-myc [74] [75] [76] . myc enhances glycolysis by upregulating expression of the glucose transporter glut1, glycolytic genes, and lactate dehydrogenase (ldh), as the converter of pyruvate to lactate [77, 78] . mtorc1 also mediates upregulation of hypoxia-inducible factor-1α (hif-1α), a factor that increases the expression of various genes [79] , including several glycolytic enzymes, glucose transporters, and ldh [80] [81] [82] . akt is able to promote the expression and membrane localization of glut1 as well as the function of phosphofructokinase [83, 84] . furthermore, akt is demonstrated to activate srebp in an mtorc1-dependent manner [85] and to upregulate srebp by enhancing the stability of its processed form [86] . srebp1 is shown to be required for the mtorc1induced increase in the expression of g6pd, which is the rate-limiting enzyme of the ppp oxidative branch [79] . in addition to the above-mentioned metabolic pathways, the influenza virus exhibits disruptive effects on some other metabolic processes, which gives rise to metabolic disorders and atp crisis. previous studies have found that the influenza infection increases the cellular synthesis of fatty acids [87] , with some of their derivatives, including eicosanoids [88] , and these molecules are natural endogenous ligands and stimulators of peroxisome proliferation-activated receptors (ppars) [88] [89] [90] . ppars are a group of nuclear receptor proteins that act as transcription factors and regulate the expression of different genes [91] involved in cellular differentiation and metabolism of carbohydrates, lipids, and proteins [92] . furthermore, all types of ppars discovered so far are able to suppress the activity of the pyruvate dehydrogenase (pdh) enzyme (known as a catalyzer of the oxidative decarboxylation of pyruvate leading to acetyl-coa production) in various organs through the upregulation of pyruvate dehydrogenase kinase (pdk)-4 [93] . in this respect, extremely low pdh enzyme activity has been found after influenza infection in vitro. enhanced pdk4-mediated inhibition of pdh has been found in the lung tissue of influenza-infected mice. this enzyme inhibition contributes to an erroneous process, which causes significant disruption of glucose oxidation, cellular respiration, and lipid metabolism ( fig. 1) [7, 94] . following infection, levels of phospholipids and several lipid derivatives undergo perturbations. several lines of evidence have shown that in obese mice due to abnormalities in the fig. 1 metabolic changes caused by influenza infection and related mechanisms. several anabolic and catabolic processes can be affected: higher glucose uptake and metabolism in glycolysis and pentose phosphate pathways, higher nucleotide catabolism, increase in biosynthesis of fatty acids including arachidonic acid, the precursor of proinflammatory lipids, and also enhanced glutaminolysis and protein synthesis. activation of mtorc1&2 signaling and downstream factors by influenza infection may have an essential role in the upregulation of these metabolic processes. in addition, high atp consumption and reduced β-oxidation, as well as glucose oxidation by influenza infection, contribute to the atp crisis and hence influenza-related multi-organ failure [96] . this influenza-mediated elevation of aa, consistent with inflammation, has also been reported in the same study [97] . the accumulation of the above-mentioned proinflammatory lipids in the cell leads to the promoted synthesis of eicosanoids and inflammatory mediators, thus exacerbating post-infection necrosis, inflammation, and tissue damage in the lungs [96] . in a prospective cohort study on influenza-infected subjects, lipid inflammatory mediators in serum samples of patients were mainly aa-derived oxylipins, including txb2, 15-deoxy-12,14-pgd2, 20-hete, 5,6-dhet, 5-oxoete, lte4, and 12-hpete. although all of these metabolites were shown to be elevated shortly after the infection, 5,6-dhet and 5-oxoete levels remained considerably high up to 4 weeks postinfection, indicating a constant pulmonary inflammation [98] . the pulmonary surfactant system, which is involved in suppressing influenza infection in the respiratory tracts [99] , can be disrupted due to significant influenza-induced changes in the abundance of different types of pc and pe species as the major components of surfactants [96, 100] . tanner et al. proposed a principal correlation between influenza replication and choline lipids metabolism. they found an ivi-mediated reduction in ester-linked pc species as well as an increased level of sphingomyelin (sm) [101] , probably connected with expending cellular choline stores for sm synthesis. this led to an increase in sm species within the infected cell. sm and short-chain fatty acid-containing ether-linked pc (epc) species were found in higher amounts in both infected cells and virions and therefore appeared to be involved in viral morphogenesis. on the other hand, long-chain fatty acidcontaining epc was increased in infected cells while having low levels in the structure of the virion, highlighting the role of this phospholipid in replication of the virus [101] . evidently, higher production of these complex lipids in the cell will require increased biosynthesis of fatty acids. in this regard, the results of a study revealed that influenza infection could induce fatty acid biosynthesis and cholesterol metabolism in human lung basal epithelial tumor cells [87] . since srebps are thoroughly identified as stimulators of expression of many genes involved in lipid and sterol biosynthesis, including fatty acid synthase [102] [103] [104] , their upregulation by the influenza virus (through induction of mtorc1 signaling, as discussed earlier) may logically explain the increased rate of lipogenesis [105] . a coincidence between increased fatty acid synthesis and a decline in fatty acid β-oxidation has been found during influenza infection, which is attributed to a variety of mechanisms directly or indirectly related to viral replication. for instance, the sharp increase of proinflammatory cytokines during influenza infection [106] causes decreased hepatic fatty acid β-oxidation both in vitro and in vivo [107, 108] , most likely through excessive nitric oxide and other related free radicals [109] . in addition, increased temperature of cells during infection (which could be the result of virus replication and fever) causes heat stress which in turn can considerably downregulate carnitine palmitoyltransferase ii (cpt ii) activity and reduce the β-oxidation and atp levels in fibroblasts of influenza-associated encephalopathy patients and healthy volunteers [110] . a study on influenza-infected mice demonstrated a significant depression in long hepatic chain fatty acid β-oxidation at both the mrna and protein level, as several β-oxidation essential enzymes were reduced by > 50% [97] . a significant decrease in mitochondrial fatty acid β-oxidation simultaneously with increased biosynthesis of fatty acids and membrane lipids may reflect the fact that the virus stores structural lipids to produce more infectious particles. in addition to impaired metabolism in mitochondria, influenza infection induces severe peroxisomal lipid metabolism disorders, which can be inferred from abnormal levels of several specific long-chain fatty acids [101] (fig. 1) . the aforementioned metabolic processes are not the only pathways affected by influenza virus infection. this virus has the ability to induce higher consumption rates of glutamine during glutaminolysis, which can be attributed to transient c-myc overexpression [9] . myc acts to regulate glutamine uptake and its utilization in the cell [111] . it has been demonstrated that catalytic activity of glutaminase, as the key enzyme in glutaminolysis, greatly increases following the infection [63] . moreover, essential amino acids, especially tryptophan, are other materials whose quantities have been shown to decline in infected tissues [112] . mtorc1 can up-regulate protein synthesis through several downstream factors [113] . thus, induction of mtorc1 signaling by the influenza virus leads to higher usage of essential amino acid storages for concurrent production of large amounts of viral and cellular proteins. infection of influenza virus can also alter the cellular level and metabolism of purines and pyrimidines [8, 98, 100] , and is associated with both increased activities of nucleotide catabolism core enzymes including adenosine deaminase (ada) and xanthine oxidase (xo) and elevated levels of inosine, hypoxanthine, xanthine, and uric acid in serum and bronchoalveolar lavage fluid. enhanced catabolic degradation of nucleotides and their metabolites can facilitate the production of superoxide and contribute to the pathogenesis of influenza infection [23] . interferons are well-known cytokines with a powerful capability of altering the cellular functions following viral infections. these alterations affect protein synthesis, composition of the membrane, cellular proliferation, and nutritional status [34] . interferon stimulated genes (isgs) are the effector components whose transcription could be induced by type i ifns and ifnγ [114, 115] . studies have recently underscored the general effect of ifns on the energy metabolism of cells, mostly by promoting glycolysis. for instance, ifnβ has been shown to induce the glucose uptake of embryonic fibroblasts in a pi3/akt-dependent manner, thereby increasing atp production [116] . it has also been demonstrated that type i ifn can stimulate oxygen consumption in a range of cells, including conventional dendritic cells (dcs), keratinocytes, and memory t cells [117] . indeed, the high yield of atp and mitochondrial fitness guarantee the host cell's need for energy in plasmacytoid dcs (pdcs) and non-hematopoietic cells following challenges with viral pathogens [118] . these studies emphasized the mediatory effect of type i ifn on glycolysis induction via ifnar1, tyk2, and stat1. it has also been shown that influenza infection stimulates pdcs to enhance their glycolysis and develop a warburg-like remodeling of energy metabolism. this enhanced glycolysis leads to higher ifn production and, consequently, more potent antiviral activity [118] . ifnγ induces metabolic reprogramming of m1 macrophages as a rapid increase in aerobic glycolysis, followed by a reduction in oxidative phosphorylation. this metabolic reprogramming maintains cell viability and the inflammatory response while reducing dependence on mitochondrial oxidative metabolism. excessive production of pro-inflammatory cytokines and chemokines in human monocytes/macrophages can be blocked by inhibition of aerobic glycolysis [119] . also, activation of macrophages by ifnγ induces expression of the atp-citrate lyase enzyme (acly), and blockage of acly activity reduces the production of ros and nitric oxide [120] . there is a strong consensus that influenza replication is crucially dependent on fatty acids [97] , which makes it a fascinating target for therapeutic modalities [45] . thus, the ability of ifn to channel the fas from biosynthesis to catabolism via fatty acid oxidation (fao) is currently known as a promising antiviral strategy in pdcs [117] , which requires further research for more elucidation. several lines of current evidence have revealed the antiviral activity of type i ifn to be exerted through hampering glucosederived cholesterol and fatty acid synthesis [121, 122] . sterol regulatory binding protein 2 (srebp2), along with srebp1, is known as the leading transcription factor which orchestrates the biosynthesis pathway of sterol, whose inhibited transcription and expression can be strongly mediated by ifns via ifnar1 [123] (fig. 2) . [124] . this recognition can lead to the induction of an inflammatory response that, in turn, controls the replication and spread of the virus [31] . h5n1, h7n7, and h7n9 were correlated with increased transcription of the cytokine response in mice. severe infection with h7n9, h7n7, h5n1, or 1918 virus can lead to upregulation of inflammatory cytokine genes along with downregulation of lipid metabolism and coagulation genes [125] . this uncontrolled proinflammatory response accompanied by an inadequate anti-inflammatory response is referred to as the cytokine storm [31] . monocytes/macrophages, neutrophils, and lung epithelial cells have useful roles in the cytokine storm developed by influenza infection [126] . severe cytokine storm, with greater levels of interferons and tumor necrosis factors, has been recognized in patients hospitalized due to influenza infection [127] . such influenza-induced cytokine storms, together with viral virulence, can develop severe lung injury in patients [128, 129] . it is believed that the level of the cytokine storm is directly associated with the severity of the disease caused by influenza infection [130, 131] . some specific polymorphisms in immune system genes have determinative roles in the outcome of influenza infection. our previous studies have shown a relationship between cytokine gene polymorphisms and severity of the influenza disease. several cytokines were evaluated after influenza a/h3n2 virus infection, among which il-17 rs2275913 gg and ag, gg and gt of il-10 (rs1800872) and il-28 (rs8099917) genotype tt polymorphisms were associated with increased risk of influenza infection. in contrast, il-1β (rs16944) (gg) and il-28 (rs8099917) gg and tg genotypes were associated with reduced risk of infection [132] . in another study, an association between il-1β rs16944 and il-17 rs2275913 genotypes and severe influenza disease was found while il-10 rs1800872 and il-28 rs8099917 polymorphisms were not associated with influenza disease. also, lacking an a allele in il-17 rs2275913 could increase the risk of influenza a (h1n1) infection [2] . such polymorphisms in immune system genes may be associated with some metabolic changes and, in turn, may reinforce the metabolic disorders following influenza infection. however, additional studies are needed in this field to confirm or reject this opinion. ifns are known to be capable of depletion of polyamines to limit virus replication. polyamines are small ornithine-derived polycationic molecules which encompass three molecules: putrescine, spermidine, and spermine. spermidine and spermine depletion is one of the compelling mechanisms through which ifns produce their antiviral effects on the replication of rna viruses. mechanistically speaking, polyamines appear to play a pivotal role in the processes of rna transcription and protein translation of viruses, making them a promising target to combat viral infections [133] . l-tryptophan is one of the nine essential amino acids with a remarkable role in immunosuppression and tolerance and is also essential in protein, kynurenine, and serotonin synthesis [122] . ido is an intracellular enzyme that induces production of kynurenine from l-tryptophan, thereby acting to deplete tryptophan and modulate the immune system following viral infections [134] . having two ifn-stimulated response elements and three ifnγ-activated sites in the promoter of ido, ifnγ acts as the most powerful inducer of ido1 expression [135] . ido has also been shown to be expressed during influenza infection [136] . dendritic cells, macrophages, and epithelial cells can express ido [137, 138] , and since the primary target for replication of influenza is primarily found to be respiratory epithelial cells, understanding the role of ido during influenza infection is of particular importance. there exists a coincidence of peak ido1 and ifn-k expression during influenza infection. also, mouse lung airways considerably express ifn type i and iii following infection with influenza [139] . these findings emphasize that there is upregulated expression and enhanced function of ido during influenza infection, which is found to be induced by ifn-i. moreover, ifn-i is thought to signal the adjacent cells via ifn-ir and stimulate them to produce ido [140] . nonetheless, the ifn-mediated ido induction during influenza infection generally has undesirable consequences and establishes immune tolerance [136] . indeed, an inhibitory effect of tryptophan depletion on t cell responses has been confirmed. also, ido induces kynurenine derivation from tryptophan, leading to stimulation of regulatory t cells [141] . nowadays, ido is hypothesized to be part of the "metabolic, immune regulation," which plays a protective role in immune responses and inhibits the overreaction of these responses against influenza infection. a pleiotropic role has been attributed to ido during infections, which gives rise to the opposing outcomes (fig. 3) [142] . research on the role of ido in influenza infection has been mainly focused on the murine models of influenza infection, emphasizing the increased ido activity and its maximum expression correlated with increased lymphocyte numbers in the respiratory tract [143] . nitric oxide (no) is a gaseous free radical with accessible vasodilatory and microbicidal functions [144] . however, the antiviral effect of no has also been documented, leading to reduced viral load and more efficient clearance of infection [145] . nitrosylation of viral molecules has been offered as an antiviral mechanism employed by no [146] . also, no synthase-mediated generation of no leads to the depletion of l-arginine, thereby reducing the level of polyamines. thus, ifn-induced nos2 represents antiviral activities, and this, in turn, may exhibit another mechanism through which ifns hinder viral infections such as influenza [34] . despite existing data regarding the antiviral activity of no, many studies have considered no as a double-edged sword with both pathogenic and viricidal effects. the role of no in the pathogenesis of pneumonia caused by influenza virus infection has been described in mice. the ifnγ response induces greatly increased levels of inos in the lungs of infected mice, leading to the production of a significant amount of no and peroxynitrite species, which are among the most important pathogenic factors in influenza virus-induced pneumonia in mice [147] . uetani et al. also observed overexpression of the inos gene in human airway epithelial cells induced by influenza a virus infection [148] . in addition, no produced by phagocytic cells has antiviral activity that is simultaneous with nonspecific damage of host cells and viral pathogenesis [149] . in a survey by nin et al. on pandemic a/h1n1 influenza infection, all cases showed increased levels of inos protein, tyrosine nitration, and oxygen free radicals, indicating the production of peroxynitrite. their results revealed the involvement of oxidative and nitrative stress in the pathogenesis of h1n1 influenza virus-induced acute respiratory distress syndrome (ards) [15] . influenza-induced cytokines such as ifnγ stimulate no release from human airway epithelial cells [150] [151] [152] . as mentioned previously, the influenza infection induces upregulation of hif-1α. interestingly hif-1α-knockout macrophages show decreased expression of inos after ifnγ stimulation [153] , indicating the possible involvement of hif-1α in influenza pathogenesis. it has been shown that infection of h5n1 and 1918 viruses induces higher levels of no in mice compared to the seasonal h1n1 virus and, as a result, they develop more intense pathogenic outcomes, while mice with inos deficiency showed reduced morbidity, mortality, and cytokine production in the lungs following h5n1 and 1918 virus infection. also, systemic administration of nos inhibitor could postpone weight loss and death among 1918 virus-infected mice [154] . in another survey, the delivery of no to influenza-infected mice could not improve the lung infection and survival of mice, indicating that no administration was not a suitable treatment strategy for influenza although this was probably due to the difficulty of determining concentrations of no that are both viricidal and safe in host airways [155] . also, it is reported that no released from s-nitroso-n-acetylpenicillamine (snap) reduces the replication of influenza virus in a dose-dependent manner. the production of no in airway epithelial cells can lead to antiviral rather than harmful effects following influenza infection provided that its production is precisely controlled [156] . since the influenza virus affects about 20% of the world population annually, preventive and therapeutic approaches require much closer attention. therapeutic drugs for influenza infection fall into three groups: 1) neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir), 2) m2 inhibitors (rimantadine and amantadine), and 3) polymerase inhibitors (favipiravir) [157, 158] . antiviral drug resistance has recently emerged as a global problem which can bring about a remarkable financial and social burden [159] . therefore, further research is urgently needed to develop novel and promising antiviral drugs. many of the metabolic pathways in influenza infections are increasingly changing, dampening of which appears to hamper the virus replication. one of the newly developed strategies aiming to hinder influenza infection is targeting metabolic pathways and restoration of hemostasis in cells ( table 1 ). the pi3k/mtor signaling pathway has been shown to play a pivotal role in a variety of cellular pathways, including proliferation and nutrient uptake, and its activation increases the glucose uptake through the up-regulation of cell surface glucose transporter [163] . bez235 alters glucose metabolism via blockage of the pi3k/mtor pathway, and some clinical trials are underway to assess this strategy in cancer therapy (smith et al., 2012). on the other hand, several lines of evidence have demonstrated that sirna targets the pi3k-akt-mtor pathway, thereby warding off influenza infection [164] . in a new study by smallwood et al., it was found that although bez235 did not interfere with the early stages of the infection, it could finally reduce the viral progeny and result in prolonged survival in mice challenged by the influenza virus. indeed, bez235 induced hemostasis in the pi3k/mtor pathway via phosphorylation of p85 and 4e-bp1 and through reconstitution of metabolic status, which was already altered by the virus [9] . it has been found that there is an elevated level of pdk4 in lung, liver, and heart during influenza infection, while the levels of atp and pdh, a key enzyme in the regulation of glucose, lipid and atp levels in human cells, are shown to be reduced [156] . furthermore, dichloroacetate (dca) is a pyruvate dehydrogenase kinase inhibitor with anti-tumor activity in a variety of carcinomas. studies have also indicated that diisopropylamine dichloroacetate (dada) could ameliorate the metabolism of hepatocytes in chronic liver disease [165] . in a study by yamane et al. attempting to evaluate the effect of dada on influenza-infected mice (pr8), oral administration of dada was found to not only restore the activity of pdh and atp in affected organs but also suppress cytokine storm and viral replication [94] . sterols are intermediate metabolites that play an essential role in a broad spectrum of biological pathways, including inflammation. research has shown that interferon production following the immune response in viral infection regulates sterol production paths. blanc et al. revealed that sterol metabolism pathway regulators such as simvastatin, zometa (zoledronic acid), and fpt inhibitor iii could effectively hinder h5n1 influenza replication and cytokine production, which makes them promising therapeutic candidates in acute patients [121, 161] . on the other hand, as mentioned earlier, srebps are transcription factors that have a critical role in the process of lipogenesis. studies have shown that these factors can play a variety of roles, such as energy supply and post-translational protein modification, as well as in the propagation of various groups of viruses such as influenza viruses. a study has shown that the am580 compound, which is a retinoid derivative, inhibits srebp-linked pathways, and it has antiviral activity against influenza a and coronavirus in vitro and in vivo [105] . concerning the fact that mitochondria and glycolysis are two sources of energy production, they play vital roles in the regulation of innate immunity responses. during the immune system response, and especially the cytokine storm, following influenza infection, atp synthesis in the mitochondria decreases, leading to weakened innate immune responses (dengbing yao). studies have revealed that traditional herbal medicines have an important role in improving influenza-like symptoms in infected patients. results of a study demonstrated that pre-treatment of infected cells with hochuekkito (a traditional japanese herbal medicine) could activate both mitochondrial and glycolytic energy metabolism and thereby intensify symptoms [160] . also, the effects of traditional chinese medicine (modified jiu wei qiang huo) on h1n1 infected mice were evaluated in another study. the results showed that this herbal medicine could ameliorate weight loss and inflammatory mediators in infected mice through the regulation of amino acid, fatty acid, and arachidonic acid pathways [162] . based on recent studies, influenza virus infection can interfere with cellular metabolic pathways either directly or indirectly via stimulation of immune system mediators. through enhancing the activity of the mtorc1 complex, the influenza virus strengthens several metabolic pathways, including glycolysis, glutaminolysis, pentose phosphate, and fatty acid synthesis, to provide more atp and structural materials for viral replication. on the other hand, β-oxidation suppression following viral infection can help to supply essential fatty acids for the synthesis of structural lipids. however, exhausting cellular atp resources due to virus replication, as well as an increase in pro-inflammatory lipid synthesis, will ultimately lead to irreversible cell damage. innate immune responses following influenza infection play a crucial role in metabolic alterations. ifn is one of these mediators that acts on several metabolites such as ido and no and thereby affects lipid and amino acid pathways. since the drug resistance in influenza infection is a global concern, research on designing novel therapeutic modalities to tackle pandemics is of particular importance. thus, a clear understanding of the metabolic alterations during influenza infection would be tremendously helpful for 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diabetes mellitus: abnormalities in metabolism and function increased susceptibility of diabetic mice to influenza virus infection: compromise of collectin-mediated host defense of the lung by glucose? glucose breakdown in chick embryo cells infected with influenza virus adenovirus e4orf1-induced myc activation promotes host cell anabolic glucose metabolism and virus replication effect of influenza virus infection on key metabolic enzyme activities in mdck cells identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. inactivation leads to a nutritional requirement for organic sulfur impact of glucose-6-phosphate dehydrogenase deficiency on the pathophysiology of cardiovascular disease glucose-6-phosphate dehydrogenase deficiency enhances enterovirus 71 infection glucose-6-phosphate dehydrogenase deficiency enhances human coronavirus 229e infection a nutritional supplement formula for influenza a (h5n1) infection in humans influenza virus differentially activates mtorc1 and mtorc2 signaling to maximize late stage replication translational control of c-myc by rapamycin promotes terminal myeloid differentiation mtor complex 2 controls glycolytic metabolism in glioblastoma through foxo acetylation and upregulation of c-myc protein kinase b/akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor fkhr1 links between metabolism and cancer myc on the path to cancer foxo3a regulates reactive oxygen metabolism by inhibiting mitochondrial gene expression antagonism between foxo and myc regulates cellular powerhouse hif and c-myc: sibling rivals for control of cancer cell metabolism and proliferation myc-induced cancer cell energy metabolism and therapeutic opportunities activation of a metabolic gene regulatory network downstream of mtor complex 1 mammalian target of rapamycin upregulation of pyruvate kinase isoenzyme type m2 is critical for aerobic glycolysis and tumor growth defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics frataxin participates to the hypoxia-induced response in tumors regulation of glut1 gene transcription by the serine/ threonine kinase akt1 akt-directed glucose metabolism can prevent bax conformation change and promote growth factor-independent survival srebp activity is regulated by mtorc1 and contributes to akt-dependent cell growth control of lipid metabolism by phosphorylation-dependent degradation of the srebp family of transcription factors by scffbw7 gc/ms-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza a virus fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors α and γ differential activation of peroxisome proliferator-activated receptors by eicosanoids fatty acids, eicosanoids and ppar gamma international union of pharmacology. lxi. peroxisome proliferator-activated receptors regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by ppar agonists the pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility diisopropylamine dichloroacetate, a novel pyruvate dehydrogenase kinase 4 inhibitor, as a potential therapeutic agent for metabolic disorders and multiorgan failure in severe influenza obesity increases mortality and modulates the lung metabolome during pandemic h1n1 influenza virus infection in mice integrated omics analysis of pathogenic host responses during pandemic h1n1 influenza virus infection: the crucial role of lipid metabolism kupffer cells modulate hepatic fatty acid oxidation during infection with pr8 influenza serial metabolome changes in a prospective cohort of subjects with influenza viral infection and comparison with dengue fever phosphatidylglycerol suppresses influenza a virus infection metabolomics investigation reveals metabolite mediators associated with acute lung injury and repair in a murine model of influenza pneumonia lipidomics identifies a requirement for peroxisomal function during influenza virus replication regulation of sterol synthesis in eukaryotes the akt-srebp nexus: cell signaling meets lipid metabolism activation of fatty acid synthesis during neoplastic transformation: role of mitogen-activated protein kinase and phosphatidylinositol 3-kinase srebp-dependent lipidomic reprogramming as a broad-spectrum antiviral target cytokine responses in patients with mild or severe influenza a (h1n1) pdm09 inhibition of hepatic ketogenesis by tumor necrosis factor-alpha in rats tnf-α and il-6 synergistically inhibit ketogenesis from fatty acids and α-ketoisocaproate in isolated rat hepatocytes regulation of energy metabolism by interleukin-1beta, but not by interleukin-6, is mediated by nitric oxide in primary cultured rat hepatocytes bezafibrate upregulates carnitine palmitoyltransferase ii expression and promotes mitochondrial energy crisis dissipation in fibroblasts of patients with influenza-associated encephalopathy myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads to glutamine addiction effect of feed restriction on performance and postprandial nutrient metabolism in pigs co-infected with mycoplasma hyopneumoniae and swine influenza virus mtor signaling at a glance influenza virus drug resistance: a time-sampled population genetics perspective interferons and viral infections beta interferon regulation of glucose metabolism is pi3k/akt dependent and important for antiviral activity against coxsackievirus b3 type 1 interferons induce changes in core metabolism that are critical for immune function cutting edge: critical role of glycolysis in human plasmacytoid dendritic cell antiviral responses interferon gamma induces reversible metabolic reprogramming of m1 macrophages to sustain cell viability and pro-inflammatory activity atp-citrate lyase is essential for macrophage inflammatory response host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis limiting cholesterol biosynthetic flux spontaneously engages type i ifn signaling the srebp pathway: regulation of cholesterol metabolism by proteolysis of a membranebound transcription factor pattern recognition receptor immunomodulation of innate immunity as a strategy to limit the impact of influenza virus h7n9 and other pathogenic avian influenza viruses elicit a three-pronged transcriptomic signature that is reminiscent of 1918 influenza virus and is associated with lethal outcome in mice pathogenesis of influenza-induced acute respiratory distress syndrome writing committee of the world health organization consultation on human influenza a/h5: avian influenza a (h5n1) infection in humans pathogenesis of influenza virus infections: the good, the bad and the ugly. current opinion in virology chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza a virus infection in mice molecular analysis of serum and bronchoalveolar lavage in a mouse model of influenza reveals markers of disease severity that can be clinically useful in humans respiratory mucosal proteome quantification in human influenza infections seasonal influenza a/h3n2 virus infection and il-1β, il-10, il-17, and il-28 polymorphisms in iranian population interferon-induced spermidine-spermine acetyltransferase and polyamine depletion restrict zika and chikungunya viruses role of indoleamine 2,3-dioxygenase in health and disease targeting a metabolic pathway to fight the flu new insights into ido biology in bacterial and viral infections functional expression of indoleamine 2,3-dioxygenase by murine cd8 alpha(+) dendritic cells ifn-gamma amplifies il-6 and il-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase lambda interferon is the predominant interferon induced by influenza a virus infection in vivo characterization of an indoleamine 2,3-dioxygenase-like protein found in humans and mice ido expression by dendritic cells: tolerance and tryptophan catabolism indoleamine 2,3 dioxygenase and metabolic control of immune responses induction and role of indoleamine 2,3 dioxygenase in mouse models of influenza a virus infection nitric oxide synthase in innate and adaptive immunity: an update does nitric oxide play a critical role in viral infections? role of nitric oxide in immune responses against viruses: beyond microbicidal activity pathogenesis of influenza virusinduced pneumonia: involvement of both nitric oxide and oxygen radicals central role of doublestranded rna-activated protein kinase in microbial induction of nitric oxide synthase nitric oxide-induced nitrative stress involved in microbial pathogenesis constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice with influenza pneumonia differential activation and antagonistic function of hif-α isoforms in macrophages are essential for no homeostasis inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza inhibition of influenza virus replication by nitric oxide emergence of multidrug-resistant influenza a(h1n1)pdm09 virus variants in an immunocompromised child treated with oseltamivir and zanamivir affinity of rimantadine enantiomers against influenza a/m2 protein revisited influenza virus drug resistance: a time-sampled population genetics perspective japanese herbal medicine, restores metabolic homeostasis between mitochondrial and glycolytic pathways impaired by influenza a virus infection modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza a virus infected human alveolar epithelial cells modified jiu wei qiang huo decoction improves dysfunctional metabolomics in influenza a pneumonia-infected mice proliferation, survival and metabolism: the role of pi3k/akt/mtor signalling in pluripotency and cell fate determination inhibition of influenza a virus replication by antagonism of a pi3k-akt-mtor pathway member identified by gene-trap insertional mutagenesis superior anti-tumor efficacy of diisopropylamine dichloroacetate compared with dichloroacetate in a subcutaneous transplantation breast tumor model key: cord-023859-3v9cmok0 authors: pinsky, benjamin a. title: influenza a (h1n1) date: 2011-03-08 journal: diagnostic molecular pathology in practice doi: 10.1007/978-3-642-19677-5_36 sha: doc_id: 23859 cord_uid: 3v9cmok0 in the summer of 2009 a 12-year-old boy with a history of multiply relapsed acute lymphoblastic leukemia now in his fourth remission on an individualized chemotherapy protocol, presented to his local hospital’s emergency room with a two day history of fever to 102.5°c (39.2°c) and upper respiratory symptoms including cough, sore throat, and runny nose. his mother developed similar symptoms approximately one week ago. in addition, he complained of abdominal pain with persistent diarrhea and one episode of emesis. the patient had a history of obstructive lung disease of uncertain etiology for which he used an albuterol inhaler on an “as needed” basis. since the onset of this acute illness he had been using his inhaler every four hours. in the summer of 2009 a 12-year-old boy with a history of multiply relapsed acute lymphoblastic leukemia now in his fourth remission on an individualized chemotherapy protocol, presented to his local hospital's emergency room with a two day history of fever to 102.5°c (39.2°c) and upper respiratory symptoms including cough, sore throat, and runny nose. his mother developed similar symptoms approximately one week ago. in addition, he complained of abdominal pain with persistent diarrhea and one episode of emesis. the patient had a history of obstructive lung disease of uncertain etiology for which he used an albuterol inhaler on an "as needed" basis. since the onset of this acute illness he had been using his inhaler every four hours. the patient had a central line for the administration of chemotherapy and previously had numerous positive blood cultures with a variety of bacterial organisms. however, current blood cultures were negative and his chest x-ray was unremarkable. consistent with his presentation, a rapid influenza a antigen test was positive. the patient had not been vaccinated for influenza as neither the seasonal vaccine nor the 2009 a (h1n1) pandemic vaccine was available at the time. he was started on the standard pediatric dose of oseltamivir (tamiflu) and arrangements were made for the child to be seen by his hematologist early the following morning. given the potential significant adverse morbidity and mortality associated with influenza infection in the immunocompromised, including patients being treated for hematologic malignancies, the hematologist admitted the patient to a quaternary medical center for further treatment and supportive care. the hospital stay was uneventful and after completion of the five day course of oseltamivir, the patient's upper respiratory illness resolved. upon discharge, a direct fluorescent antibody (dfa) test for the presence of influenza a antigen was negative. further antigen testing was negative two days later, when the patient resumed his chemotherapy regimen. however, nucleic acid testing demonstrated the presence of pandemic, 2009 influenza a (h1n1) rna. after three days, the patient again developed fever and upper respiratory symptoms. a nasopharyngeal specimen was positive for influenza a antigen and oseltamivir was restarted. the clinical team considered the possibility that the patient had been reinfected with a different influenza a subtype. however, they were most concerned about persistent infection and the development of oseltamivir resistance. oseltamivir resistance. both of these questions are critical to optimize treatment. at the time of this case there were two common classes of influenza antivirals; the neuraminidase (na or n) inhibitors [oseltamivir (tamiflu) and zanamivir (relenza)] and the m2 ion channel inhibitors (the adamantanes: amantidine and rimantidine) [1] . in 2009, essentially all of the seasonal h1n1 strains carried the histidine 275 to tyrosine (h275y) oseltamivir-resistance mutation. these strains were sensitive to both the adamantanes and zanamivir. in contrast, essentially all seasonal h3n2 strains carried the s31n adamantaneresistance mutation and were sensitive to the na inhibitors. interestingly, the 2009 a (h1n1) pandemic strain resembled the h3n2 subtype in terms of its resistance profile, and was generally sensitive to the na inhibitors and resistant to the adamantanes (table 36.1) . immunocompromised patients are particularly at risk for the development of resistance to influenza antivirals [2] . though 2009 a (h1n1) oseltamivir resistance was uncommon at the time of this case (less than 50 reports worldwide), the oseltamivir exposure and clinical course were suggestive of the emergence of a resistant virus. importantly, the patient's underlying obstructive pulmonary disease contraindicated the use of zanamivir, which requires inhaled dosing and has been associated with a decrease in respiratory function in these patients. 2009 influenza a (h1n1) subtyping and h275y oseltamivir resistance mutation analysis by real time, reversetranscriptase polymerase chain reaction (rrt-pcr). the laboratory-developed, duplex rrt-pcr assay targeted the 2009 influenza a (h1n1) na gene. the probes were specific for either the wild-type 2009 a (h1n1) na sequence or the 823c > t point mutation encoding the h275y change responsible for oseltamivir resistance. the probes utilized hydrolysis chemistry and were differentially fluorescently labeled on the 5¢ end with either fam or calfluor560 (hexequivalent). on the 3¢ ends were black-hole quencher moieties (bhq). these probes were further modified with a proprietary dna duplex stabilizing technology (biosearch technologies, novato, ca) that improves specificity and allows single base-pair discrimination. numerous rrt-pcr methods, both commercially available and laboratory-developed, are available for the diagnosis of influenza a infections [3] . these assays typically target the highly conserved influenza a matrix (m) gene and are generally ~5-10% more sensitive than viral culture [3, 4] . because the target is so well conserved, the matrix rrt-pcr tests are able to detect most influenza a subtypes but are unable to distinguish between them. in order to subtype influenza a, a majority of assays target unique sequences in the influenza a hemagglutinin (ha or h) gene [4] . for example, during the 2009 pandemic in the state of california, county public health laboratories performed individual rrt-pcr reactions to amplify matrix, seasonal h1, and seasonal h3 sequences from respiratory specimens. if positive only for influenza a matrix rna, the subtype was presumed to be 2009 a (h1n1) and confirmatory rrt-pcr testing targeting the swine-origin h1 was performed at the state public health laboratory. another methodology used for the diagnosis of influenza a is traditional rt-pcr, followed by array hybridization, most often to liquid-phase, bead-based arrays. these tests utilize the same nucleic acid targets as the rrt-pcr assays for the identification and subtyping of influenza a, though they may be slightly less sensitive [5] . however, arrays have increased multiplexing capabilities compared to rrt-pcr and therefore allow the simultaneous detection of a large panel of respiratory viral pathogens, most of which have very similar clinical presentations. the standard molecular approach for tamiflu resistance testing is pyrosequencing of the na gene [6, 7] . while sequencing allows the identification of na mutations other than the 823c > t change, rrt-pcrbased testing may be more sensitive for the detection of specific resistance mutations. at the time this chapter was written, all reported oseltamivir-resistant 2009 a (h1n1) strains carried the h275y mutation. yes. the blank is blank and the negative control is negative in both channels. the h275y control amplifies only in the green channel and the wild-type control amplifies only in the yellow channel. a report for this patient specimen might read: yes. the clinical course is consistent with the development of oseltamivir-resistant influenza a virus following treatment with oseltamivir. there is no evidence for infection with previous seasonal influenza a subtypes and it is very unlikely that the patient was subsequently infected with a second, independent oseltamivir-resistant 2009 a (h1n1) strain. after receiving this result, the clinical team treated the patient with intravenous zanamivir (at the time an investigational drug) [8] . other strategies were considered, including an increased dose of oseltamivir and the administration of ivig (intravenous immunoglobulin), which contains some 2009 a (h1n1) neutralizing antibodies and may provide limited passive immunity. the patient's symptoms resolved and after two weeks no influenza a rna was detected in the patient's nasopharyngeal specimens. for epidemiological purposes, the specimen containing the resistant virus was sent to the state public health laboratory for confirmatory testing. interestingly, their pyrosequencing approach was unable to detect the resistance mutation. this specimen contained a mixture of wild-type and mutant virus that was below the lower limit of detection for the sequencing assay but above the detection limit for the rrt-pcr test. to resolve this discrepant result, our laboratory used a very sensitive low-copy-number, high resolution melting approach to determine that indeed, the sample in question contained the mutant virus [9] . in addition, the public health laboratory was able to detect the resistance mutation by sequencing a subsequent sample from this patient that contained predominantly mutant virus by rrt-pcr. influenza a is a member of the family orthomyxoviridae [10] . the virus contains a single-stranded, negativesense, segmented rna genome, and is subtyped based on its hemaggluttinin and neuraminidase genes. the year 2009 saw the emergence of a novel a (h1n1) subtype derived, in part, from an influenza a virus known to infect swine [11] . this novel a (h1n1) virus spread rapidly through the human population worldwide and represents the first influenza pandemic of the twenty-first century [12] . influenza infections are transmitted from person-toperson via contact and large particle respiratory droplets [13] . there is a one to four day incubation period and the virus is shed the day before symptoms begin through five to 10 days after illness onset. the signs and symptoms of influenza infection may include fever, myalgia, headache, malaise, nonproductive cough, sore throat, rhinitis, otitis media, nausea, and vomiting. uncomplicated illness typically resolves in three to seven days, though cough and malaise can persist for more than two weeks. complications include primary influenza pneumonia, exacerbation of underlying medical conditions, and secondary bacterial pneumonia. the complications are typically highest in those over the age of 65, young children, and patients with underlying disease. seasonal influenza infections cause significant morbidity and mortality, on average 225,000 hospitalizations and 36,000 deaths per respiratory virus season in the united states. pandemic influenza has the potential to cause an even greater burden of disease. influenza a nucleic acid testing is indicated in patients demonstrating signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors. influenza a subtyping is indicated to track the local influenza a epidemiology and, as demonstrated in this case, to adjust empiric antiviral therapy (e.g., to discontinue adamantanes if the strain is 2009 h1n1, or to discontinue oseltamivir if the strain is a previous seasonal, circulating h1n1). furthermore, oseltamivir-resistance testing is indicated in patients who do not show clinical improvement and/or viral clearance after completion of an oseltamivir-containing therapeutic regimen. developing new antiviral agents for influenza treatment: what does the future hold? how i treat influenza in patients with hematologic malignancies detection of respiratory viruses by molecular methods who/cdc protocol of realtime rtpcr for influenza a(h1n1) comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis detection of molecular markers of drug resistance in 2009 pandemic influenza a (h1n1) viruses by pyrosequencing influenza a(h1n1) na-h274 detailed pyrosequencing protocol for antiviral susceptibility testing intravenous zanamivir for oseltamivir-resistant 2009 h1n1 influenza high-resolution melting analysis (hrma): more than just sequence variant screening wolters kluwer health/lippincott williams & wilkins, philadelphia 11. novel swine-origin influenza a virus investigation team historical perspective-emergence of influenza a (h1n1) viruses h1n1 influenza preferential lower respiratory tract infection in swine-origin 2009 a(h1n1) influenza answers to multiple choice questions 1 . the correct answer is c. 2 . the correct answer is c. 3 . the correct answer is e.influenza a virus has a single-stranded, negativesense, rna genome. the use of single-tube rt-pcr containing both forward and reverse primers allows amplification of genomic rna (negative-stranded) as well as viral complementary and messenger rna (both positive-stranded). in contrast, the use of a separate rt reaction with a single primer would allow subsequent amplification of only the negative-or positive-stranded rna species. for example, an rt reaction with only the forward matrix primer would generate complementary dna (cdna) only from the genomic, negative-stranded matrix rna. the signs and symptoms of respiratory viral illnesses are not specific enough to make a definitive diagnosis. respiratory virus panels often cover influenza a and b, respiratory syncytial virus, metapneumovirus, adenovirus, and parainfluenza 1, 2, and 3. some tests also detect rhinoviruses and coronaviruses. these panels are particularly important for hospitalized patients where results not only guide patient care but also isolation and infection control. viral pneumonia is a serious complication of influenza a viral infections but may be difficult to distinguish from a secondary bacterial pneumonia. appropriate diagnosis requires testing a lower respiratory tract specimen, such as bronchoalveolar lavage (bal) fluid, for viral and bacterial pathogens. interestingly, 2009 a (h1n1) can present with predominantly lower-tract disease, so testing bal or endotracheal (et) aspirate specimens should be considered in patients with severe respiratory illness even in the absence of viral detection in nasopharyngeal swabs [14] . key: cord-292528-8kdhf123 authors: lau, yuk-fai; tang, lay-hoon; ooi, eng-eong title: a tlr3 ligand that exhibits potent inhibition of influenza virus replication and has strong adjuvant activity has the potential for dual applications in an influenza pandemic date: 2009-02-25 journal: vaccine doi: 10.1016/j.vaccine.2008.12.048 sha: doc_id: 292528 cord_uid: 8kdhf123 the appearance and spread of the h5n1 highly pathogenic avian influenza (hpai) raise concern of a possible pandemic. current preventive measures include the development of a pre-pandemic influenza vaccine and stockpiling of neuraminidase inhibitors. however, their benefits can be significantly reduced by mutations in the hemagglutinin or neuraminidase resulting in antigenic changes and the appearance of drug-resistance, respectively. drugs that target the innate immune system to achieve a ‘heightened antiviral’ state represent another class of antiviral agents that could contribute to the control and treatment of influenza infection. in this study, pika (a stabilized dsrna) provides broad-spectrum prophylaxis against a number of influenza a viruses. in addition, when pika was admixed with influenza vaccine preparations, including a formalin-inactivated whole-virion h5 vaccine, significant adjuvanting effect leading to accelerated viral clearance was observed in a murine model. these biological effects appear to be mediated by the ability of pika to promote the maturation of dendritic cells, including up-regulation of co-stimulatory molecules, such as cd80 and cd86, and the induction of various cytokines and chemokines. toll-like receptor 3 (tlr3) was shown to recognize pika in a concentration-dependent manner. the potency and versatility in its activities make pika an attractive candidate for use in an influenza pandemic. the most effective tool for controlling an influenza pandemic is a suitable vaccine. for optimal effectiveness, the pandemic vaccine needs to be based on the causative strain and therefore, vaccine cannot be produced until the pandemic strain is identified. this criterion poses serious constraints in the capability of vaccine manufacturers to prepare pandemic vaccines in advance for stockpiles. in addition, a number of studies have shown that the hemagglutinin (ha) molecules of avian h5 viruses are poorly immunogenic [1, 2] , where up to 90 g of antigen (6 times the normal dose of human influenza virus ha) was required to elicit potentially protective responses in a substantial number of subjects [2] . this need for a larger antigen dose further reduces the availability of the 'limited' pandemic vaccines. in an attempt to increase the immunogenicity of the antigen, adjuvants like alum and mf-59 have been incorporated into vaccine formulations. although alum may enhance the magnitude of the antibody response to a high dose of antigen, it does not have significant antigen-sparing effect [3, 4] . other adju-vants, such as mf59 or a proprietary oil emulsion adjuvant, have been tested and data indicate that the inclusion of these adjuvants in the vaccine preparation would significantly increase antibody titers [5, 6] . given the likelihood that a suitable vaccine will not be available before the onset of a pandemic, initial protection in a pandemic may have to rely on antiviral treatment and prophylaxis. currently available anti-influenza drugs include neuraminidase inhibitors (oseltamivir and zanamivir) or ion-channel blockers (adamantanes). the success of these drugs, both for prophylaxis or therapeutics, is based on the susceptibility of the viruses to these drugs. recently reported studies, however, have shown that a number of h5 isolates are resistant to these drugs [7] [8] [9] . these drugs or classes of drugs alone may thus be insufficient in protecting the human population from an influenza pandemic. an alternative, antigen-independent approach that could slow down the transmission of the virus and provide more time for vaccine production is thus highly desirable. the toll-like receptors (tlrs) are a collection of receptors that detect conserved molecular components encoded by microorganisms (reviewed in refs. [10, 11] ) and stimulation of these receptors by specific ligands leads to the activation of the innate immune cells, such as dendritic cells (dc), macrophages and nk cells [12, 13] , through a variety of signaling pathways, such as nf-b transcription factors, c-jun nh2 terminal kinase (jnk), mitogen-activated protein kinases (mapks), resulting in quantitative and qualitative changes in immunological functions, including antigen presentation. in addition, cytokines, such as ifn-␣, tnf-␣ and il-12p40, are produced in vivo shortly after tlr activation [13, 14] . the production of these cytokines results in antiviral effects observed in a number of infection models. for example, administration of synthetic lipid a analogs (tlr4 agonists) protects mice against an influenza challenge [15] . a possible explanation for this anti-influenza effect could be related to the production of type 1 interferons, which induce the activation of ifn-stimulated genes including mx proteins, protein kinase r and 2 5' oligoadenylate synthetase [16] . the induction of these proteins increases the resistance to the replication of the influenza virus [17] [18] [19] . the present study was designed to investigate the efficacy of pika, which is a synthetic stabilized form of double stranded rna, as a stand-alone agent for its antiviral effect and to evaluate the efficacy of pika as an adjuvant when co-administered with inactivated influenza vaccines, including an h5n1 vaccine. our results show that pika has potent anti-influenza effect when administered 6 h prior to or immediately after influenza virus challenge in mice. the antiviral effect is not strain-specific. when used as an immuno-adjuvant, pika was able to achieve significant antigen-sparing effect. when incubated with immature dc, pika altered the expression pattern of a number of immune genes. in addition, pika was capable of interacting with both human and mouse tlr3, leading to the production of nf-b. thus, in the face of a potential influenza pandemic, pika could be a candidate as a prophylactic drug or could be used as an adjuvant to increase the population coverage when pandemic vaccines become available. specific pathogen-free (spf) male balb/c mice (6-8 weeks old) were purchased from the centre for animal resources (care), national university of singapore. the animal protocol used in this study was reviewed and approved by the institutional animal care and use committee (iacuc) of dso national laboratories, singapore. a/puerto rico/8/34 (h1n1) and the reassortant virus mem71, which bears the ha of a/memphis/1/71 (h3n2) and the neuraminidase (na) of a/bellamy/42 (h1n1) are gifts from professor lorena brown, the university of melbourne. a/ws/33 (vr-1520) is from the american type culture collection (atcc). the reverse-engineered h5n1 influenza virus was made based on the method described by hoffmann et al. [20] , using an expression plasmid provided by dr brendon hanson (dso national laboratories, singapore). the ha sequence of a/indonesia/cdc625l/2006 (cy014465, a clade 2, subclade 1 virus which is closely related to a/indonesia/5/05) was downloaded from genbank and was synthesized by geneart ag (regensburg, germany). the viruses were grown in the allantoic cavity of 10-day-old embryonated chicken eggs for 2 days at 37 • c. allantoic fluids were pooled and divided into aliquots, and stored at −80 • c until use. the 2006-2007 south hemisphere seasonal influenza vaccine, fluvax, was from csl ptd ltd. (victoria, australia). virions of the reverse-engineered h5n1 virus were concentrated by ultra-centrifugation and resuspended in pbs. formalin-inactivated purified whole-virion vaccine was prepared by inactivating the virions with 0.5% formalin for 3 days at 37 • c followed by dialysis in pbs. the total protein concentration in the vaccine was measured by coomassie plus (pierce biotechnology, inc., il, usa). the amount of ha content was estimated to be 30% of the total protein content. complete and incomplete freund's adjuvant (cfa and ifa) were obtained from sigma-aldrich (st louis, usa). pika was obtained from newbiomed pika pte ltd. (singapore). for intranasal (i.n.) immunization, groups of mice were anesthetized with an intraperitoneal (i.p.) injection of ketamine and xylaxine followed by i.n. administration of immunogens in 50 l. for immunization by the subcutaneous (s.c.) route, the immunogens were administered in 100 l at the base of the tail. in selected experiments, 100 g of pika was given to anesthetized mice i.n. in a volume of 50 l. when given by the s.c. or i.p. route, 100 g of pika was given in 100 l volume per dose. pika contains dsrna that is greater than 100 base pairs in length. three weeks after final vaccination, mice were challenged i.n. with 10 1.7 -10 4.5 pfu of various strains of influenza virus in a volume of 50 l. mice were sacrificed at various time-points and pulmonary viral titers were determined using previously described methods [21] . mice were anesthetized by ketamine/xylaxine injection and blood samples were collected from the orbital plexus. sera were collected and stored at −20 • c until analysis. the titer of immunogen-specific antibodies were quantified by elisa, as previously described [22] . avidity elisa was carried out using previously described method [23] . hek293 cells were maintained in optimem (invitrogen) with 5% fetal calf serum and were transfected in flat-bottomed 96-well plates as previously described [24] . each sample was tested in triplicate. six hours after the stimulation, luciferase and ␤-galactosidase activity in each sample was measured by commercial kits (promega corporation, wi, usa). the relative stimulation of nf-b was calculated by normalizing luciferase activity with ␤-galactosidase activity and was expressed using the readings from unstimulated wells as a baseline. the level of expression of various cytokine genes was determined using a lightcycler (roche, in, usa) using primers described in giulietti et al. [25] . samples containing 1 g of total rna from the abovementioned dc experiment were used for microarray analysis. piqor immunology microarrays (miltenyi biotech, gladbach, germany) were used. samples were amplified and labeled with different fluorochromes according to manufacturer's instructions. samples from pika-stimulated dc and unstimulated dc were labeled with cy5 and cy3, respectively. after hybridization, the slides were scanned using the imagene software (biodiscovery). each gene was printed in quadruplicate on the array. net signal intensity, data normalization and calculation of the cy5/cy3 ratios were performed by miltenyi biotech using the piqor analyzer software. one million d1 cells were stimulated with 100 g of pika or 1 g of lps (0111:b4, sigma-aldrich) overnight. the supernatants were collected and the cytokine protein levels measured using the bioplex protein array system (bio-rad, hercules, ca), in duplicates and against a standard curve according to manufacturer's instructions. the significance of any difference between any two different groups was assessed by the mann-whitney test using prism 4 (graphpad software, ca, usa). reported p values < 0.05 are considered significantly different. in this study, the murine influenza model was employed to demonstrate the antiviral properties of pika. in this model, anesthetized mice were challenged with influenza virus i.n. this mimics a total respiratory tract infection and the pulmonary viral titer reaches its peak on day 2 post-infection (pi) and remains at a high level till day 6 pi (data not shown). using this model, groups of five mice were given 100 g of pika i.n. 6 h prior to challenge with 50 plaque forming unit (pfu) of influenza/a/pr/8/34. the pulmonary viral titer in the lungs was determined on day 3 pi. as shown in fig. 1a , mice that received pika treatment at 24 and 48 h pi had the lowest pulmonary viral titer at day 3 and this was significantly lower than the titer in control mice that received pbs (p = 0.0079). in addition, significant reductions in pulmonary viral titers were also observed in mice that received only one dose of pika at 6 h prior to infection. the difference in the pulmonary viral titer between the group that received daily treatment and the group that received only one dose of pika was not statistically significant (p = 0.75). after demonstrating that pika has an inhibitory effect on influenza viral replication in vivo, it was of interest to determine the optimal conditions for the treatment. to examine this, mice were given different concentrations of pika i.n. and were challenged with 50 pfu of pr8 virus i.n. the mice were treated at 24 and 48 h and were sacrificed on day 3. a dose-dependent response was observed (fig. 1b) , in which mice given 100 g of pika daily had the lowest mean viral titer among all the groups. an inhibitory effect of pika on viral replication was detected at 10 g per dose but not at 1 g per dose (p = 0.0556). to determine whether pika could be used as a treatment option at the time of infection, mice were given pika immediately after infection with 50 pfu of pr8 virus. as shown in fig. 1c , this treatment protocol was effective in reducing the pulmonary viral titer and the titer was not significantly different from the prophylaxis group that received pika 6 h prior to infection (p = 0.1508). however, it is apparent that daily treatment is preferable because the group that received only a single dose had a significantly higher titer of virus in the lungs compared to those that received daily doses (p = 0.0159). intranasal administration of pika produced the most significant anti-influenza effect compared to s.c. or i.p. administration of the drug (fig. 1d ) though the pul-monary viral titers in the treated mice were still significantly lower than the titers of the pbs control group (p = 0.0079). after demonstrating the effectiveness of pika in inhibiting the replication of pr8 virus in vivo, we sought to determine whether similar inhibition would be observed with different influenza viruses. groups of five mice were given pika treatment and were challenged with 50 pfu of pr8 (h1n1), mem71 (h3n1) and a/ws/33 (h1n1), as previously described. on day 3 pi, the viral titer of the pika-treated groups were significantly lower than the titers of the pbs group, regardless of the subtype of the challenge viruses ( fig. 2a -c, p = 0.0079, 0.0159, 0.0119) respectively. in addition, mice were also challenged with higher doses of virus, 500 or 5000 pfu (5 and 50 ld 50 respectively) of pr8. as shown in fig. 3a and b, the pika-treated mice had lower pulmonary titers than the pbs control group at these higher challenge doses (p = 0.0079). in addition, when challenged with 10 4.5 pfu of mem 71, there was a 99% reduction in viral titer in pika-treated mice compared with the pbs control group (fig. 3c) . to demonstrate the therapeutic potential of the treatment, groups of five mice were challenged with 50 pfu of pr8 i.n. and received pika treatment 24 or 48 h pi. on day 5 pi, as shown in fig. 3d , significant reduction in pulmonary viral titers was observed in pika-treated mice (p = 0.0079, 0.0273) compared with the pbs-treated group. therefore, in summary, we demonstrated that pika has the ability to inhibit the replication of several strains of influenza in vivo. although intranasal administration of the drug before the establishment of an infection provided the best protection, substantial viral reduction could still be achieved if the drug were given in the course of an established infection. since pika is an effective adjuvant for hepatitis b vaccine [26] , we examined whether pika could enhance the immunogenicity of influenza vaccine. we sought to demonstrate that the enhancement of the humoral responses would lead to significant viral reduction in replication of challenge virus. previous studies have showed that when administrated at between 1.5 and 5 g per dose, different forms of influenza vaccine could induce robust antibody responses without any external adjuvant [27] [28] [29] with protection against viral challenge [27] . as shown in fig. 4a , mice immunized with a single dose of the trivalent vaccine at 1.5 g s.c. had robust antibody responses which were comparable to those induced when the vaccine was admixed with cfa. when the vaccine dose was reduced by 10fold (0.15 g) , there was about 10-fold reduction in the mag-nitude of the antibody responses. to mimic a vaccine shortage situation, a dose of 0.015 g, (∼100-fold reduction from the optimal dose) was selected and we evaluated whether the inclusion of pika could compensate for the reduction in antigen concentration. groups of five mice were vaccinated with a suboptimal dose of fluvax vaccine by the s.c. or i.n. route. three weeks after priming, sera were collected from the mice before they were boosted. sera were collected from the mice 3 weeks after boost. as shown fig. 4 . pika acts as a potent adjuvant by enhancing the immunogenicity of the seasonal influenza vaccine. (a) groups of four mice were vaccinated with either 50 or 5 l of fluvax influenza vaccine (containing 1.5 or 0.15 g of ha from each subtype) in pbs or in cfa by the subcutaneous route. on day 21, sera were collected and the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. (b) groups of five mice were vaccinated with 0.5 l of fluvax influenza vaccine (containing 15 ng of ha from each subtype) in pbs, either by the subcutaneous route at the base of the tail or by the intranasal route. some groups received the vaccine with additional adjuvant as indicated in the x-axis. for those that received pika as an adjuvant, 100 g of pika was admixed with the vaccine prior to administration. on day 21 post-vaccination, sera were collected and the mice received another boost by the same route and sera were collected on day 42. the antibody titer of the sera was determined by an elisa assay, using fluvax as the coating antigen. each dot represents the antibody titer of an individual mouse. the primary and secondary antibody titers are represented by the black and red dots respectively and the lines represent the geometric mean antibody titer of each group. the '*' symbol indicates that the difference between two groups is statistically significant (p < 0.05). (c) to determine the level of protection mediated by the antibody response, the mice were challenged with 50 pfu of pr8 intranasally 3 weeks after the boosting and the pulmonary viral titer was determined on day 5 post-infection. closed circles represent the lung virus titer of an individual mouse and the line represents the geometric mean titer of the group of mice. the percent reduction in mean viral titer relative to the pbs control group is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < 0.05). (d) serum samples were diluted 1:2000 and the avidity of the antibodies was tested in a urea-displacement elisa. the o.d. of the urea-treated plates was expressed as a percent of the untreated plates. data are represented as the mean and standard derivation of five mice. in fig. 4b , only two out of the five mice that received the vaccine without an adjuvant by the s.c. route had detectable amounts of anti-ha antibody in the primary response. for the cfa group, four mice had anti-ha antibody whereas all animals in the vaccine with pika group had detectable levels of anti-ha antibody. in the secondary responses, although all mice given vaccine alone developed anti-ha antibody, the antibody titers were significantly lower than those that were given vaccine with pika (p = 0.0119). as for intranasal delivery of vaccine, none of the vaccine protocols was able to elicit detectable anti-ha responses after one dose. when two doses of vaccine were given with pika, the vaccine recipient group showed a low titer of anti-ha response. the response was substantially improved by the addition of pika to the vaccine (p = 0.0079). interestingly, the titer of the sera attained by two doses administered i.n. was comparable to titers achieved by two doses given by s.c. injection (p = 0.5762). in order to demonstrate that the enhanced antibody responses can lead to improved efficacy, vaccinated mice were challenged with 50 pfu of pr8 i.n. and the pulmonary viral titers were determined 5 days after challenge. as shown in fig. 4c , mice given two doses of the vaccine showed a 83% reduction in viral titer compared to the pbs control group (p = 0.0556). although the cfa group had the highest titer of anti-ha responses (fig. 4a) , the group only showed a 91% reduction in viral titer, which was not statistically different from the group that received unadjuvanted vaccine (p = 0.4206). on the other hand, the group given vaccine with pika the pulmonary viral titer was determined as previously described. the percent reduction in mean viral titer relative to the group treated with pbs is shown above each column of data. the '*' symbol indicates that the difference between the two groups was statistically significant (p < 0.05). showed the most potent viral reduction, with 99.8% reduction in viral titer compared to the pbs control group, and this difference was statistically significant (p = 0.0079). the group that received pika alone served as control to ensure that the observed reduction in pulmonary viral titer was due to the anti-ha antibody responses, instead of the direct inhibitory effects of pika on viral replication shown in the fig. 1 . the enhancement in viral clearance was also observed in the group that received the vaccine by the i.n. route (fig. 4c) . mice that received either vaccine alone or vaccine with pika showed a 66.4% and 99.8% reduction, respectively, and the difference between the 2 groups was statistically significant (p = 0.0079). it is interesting to note that, although the group received the vaccine admixed with cfa had the highest antibody titers (fig. 4b) , however, the mice had higher pulmonary viral titers compared with mice received the vaccine administrated with pika (fig. 4c ). to investigate this discordance between the two parameters, the relative avidity of the antibodies of the two groups against the vaccine antigen was examined in a urea-displacement elisa. as shown in fig. 4d , the group received the vaccine with pika had antibodies with higher avidity to the surface glycoprotein of the virus as significantly more antibodies remained bound to the ha when exposed to 6 m urea as compared to those induced with cfa as an adjuvant (p = 0.0317). there is an accumulating body of data showing that h5n1 virus vaccines are generally poorly immunogenic. we therefore proceeded to examine whether pika was an effective adjuvant for an h5n1 vaccine. groups of five mice were inoculated by the s.c. route with a formalin-inactivated h5 vaccine, with or without an adjuvant, and 3 weeks later, sera were collected to determine the antibody titer against the virus. as shown in fig. 5a , the whole-virion vaccine was capable of inducing a measurable titer of antibody after one dose. however, with the inclusion of pika, there was a ∼3-fold increase in antibody titer (p = 0.0157), and this was comparable to the titer achieved in the group that received the vaccine with cfa. when challenged with the homologous virus i.n., however, this group of mice was the only group which showed significant reduction in pulmonary titer on day 5 pi (fig. 5b, p = 0.0079) . the efficacy was further boosted by a second dose of h5 vaccine with pika. mice in this group showed a further 1000-fold reduction in viral titer (fig. 5c ). in addition, the vaccine-pika formulation was efficacious in reducing viral replication even when the vaccine was delivered intranasally. in contrast, in the absence of pika, the vaccine administered by the i.n. route failed to induce a significant reduction in viral titer compared with the pbs control (p = 0.2492). in summary, we have demonstrated that the inclusion of pika in two different formations of influenza vaccine can achieve substantial antigen-sparing with robust humoral immune responses, leading to potent pulmonary viral titer reduction in vivo. in an attempt to elucidate the biological basis for the inhibition of viral replication and the adjuvant effects of pika, we sought to identify the receptor(s) that might be involved in these processes and to examine how cells of the innate immune system respond to pika stimulation. using a human tlr3-expressing plasmid, we showed that pika is capable of interacting specifically with tlr3 in a dose-dependent manner (fig. 6) . similar results were obtained when the experiment was carried out with mouse-tlr3-expressing plasmid (data not shown). next, we investigated the outcome of stimulating dc in vitro with pika given that dcs are the most potent antigen presenting cells (apc), responsible for antigen capture and presentation to naïve t lymphocytes. using primary immature dc culture, d1 cells, we examined how dc responded to pika stimulation in vitro. pika was added into dc cultures for 16 h following which total rna was extracted from the cells. the expression level of more than 1000 immunologically related genes between pika-stimulated and unstimulated samples were compared using microarray technology. as shown in fig. 7a , pika-stimulated dcs up-regulated expression of a number of cellular activation markers, such as cd80 and cd86 compared to unstimulated cells. a number of genes that have important immunological functions were also found to be up-regulated after pika treatment, including irf-7 (interferon regulatory factor 7), il-1, il-15, il-6 and il-12 receptor. many chemokines, such as mip (macrophage inflammatory protein), rantes, mcp-2 (macrophage chemoattractant protein) and ip-10 (interferon gamma inducible protein) were also found to have increased expression. quantitative real-time pcr was used to confirm these findings. using cdna from unstimulated dc as the baseline, many genes were up-regulated in a dose-dependent manner (fig. 7b) . while certain genes, such as inos and il-12, were up-regulated to a comparable level in lpsstimulated dc, other genes, such as cd40l, mip3 and ifn-␥, were up-regulated more readily by pika stimulation. to demonstrate that the observed changes in gene expression lead to changes in these cytokines/chemokines, the level of these proteins in the culture supernatants was measured. consistent with the up-regulation of cytokine/chemokines genes, there was an increase in the production of these cytokines/chemokines in the supernatant from pika-stimulated dc compared with those from the unstimulated dc (fig. 7c ). it has been 11 years since the first case of human infection with a highly pathogenic h5n1 virus was identified in hong kong [30] and the virus has now spread to many regions, including southeast asia, western china [31] , africa [32] , turkey [33] and siberia [34] . most human cases to date involve close contact with infected poultry and human-to-human transmission remains limited [35] . nonetheless, the lack of anti-h5 immunity in humans, together with the continuous evolution of the virus in close proximity with the human population, threatens a pandemic. should the virus acquire the property of efficient transmissibility between humans, in combination with the high case-fatality rate resulting from infection, this virus has the potential of causing an influenza pandemic with damage on a scale similar to that achieved by the 1918 'spanish' flu pandemic. therefore, there is an urgent need to develop effective prophylaxis and therapeutic strategies in preparation for a possible pandemic. in the current study, using a murine influenza model, we have evaluated the potential of pika for prophylaxis and treatment of influenza infection as well as an adjuvant for influenza vaccine. our data demonstrate that administration of pika intranasally prior to or shortly after an influenza infection can inhibit influenza replication, leading to a significant reduction in pulmonary viral titers. this effect is unlikely to be due to a direct antiviral activity, because pre-incubating influenza virus with pika did not inhibit virus infectivity for embryonated chicken eggs (lynn tang, unpublished data). instead, pika appears to act by stimulating the innate immune system (fig. 7) , resulting in the production of several chemokines, cytokines and interferons and these, in turn, mediate the observed antiviral activity. myxovirus resistance (mx) proteins, protein kinase r and 2'5' oligoadenylate synthetase are some of the products that have been reported to be up-regulated after type 1 interferon production (reviewed in ref. [16] ), achieving an antiviral state in the host [36] . the fact that pika administration results in the stimulation of several antiviral proteins in the host, it decreases the likelihood that viruses will develop resistance through mutation. furthermore, as pika does not target a specific component of the virion for its antiviral activity, it is likely to be effective against multiple strains and subtypes of influenza viruses as demonstrated in this study. this broad-spectrum activity is not limited to influenza a viruses only. a number of studies have shown that tlr-ligands are capable of inhibiting a wide range of viruses, including herpes simplex virus-2 [37] , cytomegalovirus [38] , parainfluenza [39] , west nile virus [40] , severe acute respiratory syndrome coronavirus (sars-cov) [41] and influenza virus [42, 43] . the abovementioned advantages make this novel antiviral approach a compelling alternative to traditional antiviral drugs and vaccines especially since avian influenza viruses have rapidly developed resistance to antiviral drugs [44] and some mutant viruses can maintain drug-resistance without losing virulence [45] . in addition, thitithanyanont et al. showed that stimulating dc with poly(i:c) conferred the cells with resistance to the cytopathic effects of h5n1 virus which might be important for the induction of virus-specific immune responses during the infection [46] . apart from being an effective agent for prophylaxis and treatment, pika can also act as an effective adjuvant. using two different forms of influenza vaccine (split subunit vaccine and whole inactivated vaccine), the magnitude of the humoral responses elicited by the combination with pika was comparable to those formulated in freund's adjuvant. our observation is consistent with the report by shen et al. [26] which showed that administering fig. 7 . pika induces maturation of dendritic cells, with expression of a wide range of immunological genes. immature murine dendritic cells, d1 cells, were incubated with 500 g per ml of pika or were unstimulated overnight. (a) total rna was harvested from the cells and converted into cdna. the cdnas derived from pika-stimulated dc and unstimulated dc were amplified and labeled with cy5 and cy3 dye respectively. the samples were hybridized overnight to a microarray chip and fluorescence signals were measured by an array scanner. each gene target was printed 4 times on the array and the normalized mean cy5/cy3 ratio and coefficient of variation (cv) of the four replicates were determined by the software. (b) rna was harvested from the dcs and converted into cdna. the expression level of each cytokine gene was determined by quantitative real-time pcr. the expression level was normalized with beta-actin, a house-keeping gene, and data were expressed relative to unstimulated samples. the bars and error bars represent the mean and standard deviation of triplicate samples and are representative of two independent experiments. (c) supernatants were harvested from dcs stimulated overnight with either 100 g of pika, 1 g of lps or unstimulated. fifty microliters of the supernatant was used to test for the presence of various cytokines/chemokines in the supernatants using the bioplex protein array system and were measured in duplicate. the bars and error bars represent the mean and standard error. pika with hepatitis b vaccine boosted the humoral response. we explored the concept further by demonstrating that a substantial antigen-sparing effect could be achieved by incorporating pika into vaccines. using the commercial split vaccine, the antigensparing effect was about 100-fold ( fig. 4a and b) . furthermore, we demonstrated that the augmented humoral response led to a func-tional reduction in viral titer (fig. 4c ). our data also showed that successful immunization could be achieved by a non-parenteral route. with two doses, the magnitude of the response achieved by intranasal immunization was comparable to that achieved by subcutaneous immunization and was associated with a similar level of viral clearance. our finding is consistent with the report from ichinohe et al. [29] in which they showed that another tlr3 ligand, ampligen, was capable of enhancing the immunogenicity of a trivalent inactivated influenza vaccine when administrated intranasally. in their study, the enhancement effect was demonstrated using a higher dose of the trivalent vaccine (1 g) and in this current study, we extended the observation further by showing that similar enhancement effect can be achieved even when the concentration of the immunogen was reduced to 0.045 g (22-fold lower). the antigen-sparing effect of pika might be an important strategy for maximizing vaccine coverage when vaccine supplies are limited. it is of interest to note that although the group that received the influenza vaccine with pika did not have the highest antibody titer, it had the lowest pulmonary viral titer (a 10-fold reduction compared with the cfa adjuvant vaccine group). the higher avidity of the antibodies induced by the vaccine with pika might allow more effective inhibition of viral replication in vivo which translated into lower pulmonary viral titers as observed in this study. this suggests that in addition to augmenting the amount of antibody produced, pika may also have an impact on the quality of the humoral responses, such as affinity maturation [47, 48] . because pika is a stabilized form of double stranded (ds) rna that lacks direct anti-influenza activity, we evaluated its effect on the innate immune system in search of the mechanism underlying the observed activity. dsrna was shown to be a ligand of tlr3 [49] and using a transient transfection system, we showed that pika maintained its tlr3 binding property. incubation of pika with primary immature murine dc in vitro resulted in maturation of dendritic cells, with marked up-regulation of co-stimulatory molecules, such as cd80 and cd86 and cytokines with potent immunostimulatory functions such as il-12 and il-15. our observation is consistent with those observed by shen et al. with bmdc [26] . the maturation process enables dc to act as a potent antigen presenting cells for effective activation of naïve lymphocytes [13, 50] , which in turn, leads to a more robust immune response. in addition, a study conducted by perera et al. [51] showed that a vaccinia virus expressing il-15 induced superior cellular and humoral immune responses compared to the parental strain. therefore, the induction of il-15 by pika could also have resulted in a more efficient induction of cd4 + t cell responses, translating to more robust humoral responses. lung tissue has recently been shown to constitutively express tlr3 in vivo in mice [52] and intranasal administration of pika could directly stimulate these tlr-3 expressing cells, which is perhaps why pika worked most effectively when administrated intranasally (fig. 1d) . we believe that pika will have similar antiviral activity in humans, because we showed that human tlr-3 can recognize pika (fig. 6) and a number of studies have demonstrated the expression of tlr-3 in various respiratory cell types, such as nasal epithelial cells [53] , airway smooth muscle cells [54] and airway epithelial cells [55] . in addition, ds rna was found to be the most effective tlr ligand in activating airway epithelial cells in producing il-8 [55] . although our experimental data are limited to tlr-3, dsrna can also be recognized by rna helicase, rig-1 and mda5, and can induce nf-b activation with type-1 ifns [56] . it is possible that pika stimulates several pathways in vivo to achieve its antiviral and adjuvant activities. the knockout-mice, such as tlr3 k/o, mda5 k/o and rig-1 k/o mice, will be useful in shedding more light on the contribution of these receptors to the activity of pika. in summary, although poly(i:c) has been shown to be an effective mucosal adjuvant, its use in clinical trials was associated with side-effects that has limited its usage [57] . in a recent toxicity trial, pika showed no significant toxicity in mice and a phase 1 clinical study showed that the use of pika as an adjuvant was well-tolerated in humans without significant side-effects (peter brazier, unpublished data). the safety profile, together with the long shelf-life, makes pika an attractive complement to currently licensed antivi-ral drugs. in the early phase of an influenza pandemic, when a well-matched vaccine is unlikely to be available, pika can be used as an antiviral drug for temporary protection of the susceptible population, slowing down the rate of viral transmission and allowing more time for vaccine production. furthermore, global influenza vaccine production is likely to lag well behind the demand for pandemic influenza vaccine, so the inclusion of an effective antigensparing adjuvant such as pika can ensure that limited supplies of vaccine achieve maximum coverage. safety and antigenicity of non-adjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a randomised trial of two potential vaccines against h5n1 influenza safety and immunogenicity of an inactivated subvirion influenza a (h5n1) vaccine safety and immunogenicity of an inactivated split-virion influenza a/vietnam/ 1194/2004 (h5n1) vaccine: phase i randomised trial 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humoral and cellular immune responses following the vaccination of mice with hbsag plus pika immune responses of mice to influenza subunit vaccine in combination with cia07 as an adjuvant intranasal immunisation with influenza-iscom induces strong mucosal as well as systemic antibody and cytotoxic t-lymphocyte responses crossprotection against h5n1 influenza virus infection is afforded by intranasal inoculation with seasonal trivalent inactivated influenza vaccine human influenza a h5n1 virus related to a highly pathogenic avian influenza virus properties and dissemination of h5n1 viruses isolated during an influenza outbreak in migratory waterfowl in western china confirmation of h5n1 avian influenza in africa h5n1 avian influenza: the turkish dimension h5n1 influenza virus avian influenza a (h5n1) infection in humans type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors posttherapy suppression of genital herpes simplex virus (hsv) recurrences and enhancement of hsv-specific t-cell memory by imiquimod in guinea pigs efficacy of s26308 against guinea pig cytomegalovirus infection attenuation of virus-induced airway dysfunction in rats treated with imiquimod effect of interferon-alpha and interferon-inducers on west nile virus in mouse and hamster animal models evaluation of immunomodulators, interferons and known in vitro sars-cov inhibitors for inhibition of sars-cov replication in balb/c mice prophylaxis of acute respiratory virus infections using nucleic acid-based drugs nucleic acidbased antiviral drugs against seasonal and avian influenza viruses avian flu: isolation of drug-resistant h5n1 virus neuraminidase inhibitor-resistant recombinant a/vietnam/1203/04 (h5n1) influenza viruses retain their replication efficiency and pathogenicity in vitro and in vivo high susceptibility of human dendritic cells to avian influenza h5n1 virus infection and protection by ifn-alpha and tlr ligands coadministration of cpg oligonucleotides enhances the late affinity maturation process of human anti-hepatitis b vaccine response enhancement of antibodies to the human immunodeficiency virus type 1 envelope by using the molecular adjuvant c3d recognition of double-stranded rna and activation of nf-kappab by toll-like receptor 3 dendritic cell maturation is required for initiation of the immune response development of smallpox vaccine candidates with integrated interleukin-15 that demonstrate superior immunogenicity, efficacy, and safety in mice detrimental contribution of the toll-like receptor (tlr)3 to influenza a virusinduced acute pneumonia expression of toll-like receptors in cultured nasal epithelial cells toll-like receptor 2, 3, and 4 expression and function in human airway smooth muscle activation of airway epithelial cells by toll-like receptor agonists the rna helicase rig-i has an essential function in double-stranded rnainduced innate antiviral responses a phase i-ii trial of multiple-dose polyriboinosic-polyribocytidylic acid in patieonts with leukemia or solid tumors we gratefully acknowledge kanta subbarao (niaid, nih) for helpful discussions and critical reading of the manuscript. this study was supported by future systems directorate, ministry of defence, the republic of singapore. key: cord-268369-yj7m0n0f authors: wang, keyang; holtz, kathleen m.; anderson, karl; chubet, richard; mahmoud, wafaa; cox, manon m.j. title: expression and purification of an influenza hemagglutinin—one step closer to a recombinant protein-based influenza vaccine date: 2006-03-15 journal: vaccine doi: 10.1016/j.vaccine.2005.11.005 sha: doc_id: 268369 cord_uid: yj7m0n0f numerous human infections with avian influenza viruses in asia in recent years have raised the concern that the next influenza pandemic is imminent. the most effective way to combat influenza is through the vaccination of the public. however, a minimum of 3–6 months is needed to develop an influenza vaccine using the traditional egg-based vaccine approach. the influenza hemagglutinin protein (ha), the active ingredient in the current vaccine, can be expressed in insect cells using the baculovirus expression vector system and purified rapidly. an influenza vaccine based on such a recombinant antigen allows a more timely response to a potential influenza pandemic. here, we report an innovative monitoring assay for recombinant ha (rha) expression and a rapid purification process. various biochemical analyses indicate that the purified rha is properly folded and biologically active. influenza is a highly contagious, acute viral respiratory disease, which causes significant morbidity and mortality worldwide each year [1] [2] [3] . influenza viruses are singlestranded ribonucleic acid (rna) viruses surrounded by a lipid containing envelope spiked with two glycoproteins: hemagglutinin (ha) and neuraminidase (na). these glycoproteins, and ha, in particular, have been recognized as key antigens in the host response to influenza virus in both natural infection and vaccination [4, 5] . the viruses are well known for their ability to mutate to circumvent immunity and re-infect the host. an antigenic shift, a major antigenic change of the virus due to the genetic re-assortment of two subtype strains that co-infected a host, can cause an influenza pandemic since the population may have no inherent immunity against the new strain [6, 7] . the avian influenza a (h5n1) epizootic outbreak and numerous human infections with h5n1 in asia in recent years, and events such as the infection of two nurses attending to avian influenza patients in vietnam (who, march 14, 2005 ) and a possible personto-person transmission in a family cluster of the disease in thailand [8] , continue to raise concern that the next influenza pandemic is imminent. a proven, effective way to combat influenza is through vaccination of the public using the trivalent vaccine produced in embryonated chicken eggs. in the current process, three influenza strains selected by who/cdc are propagated in chicken eggs, chemically inactivated, and semi-purified. the egg-based technology, however, is unable to respond to a pandemic crisis. vaccine development and production take several months following the identification of potential strains and typically requires the re-assortment with a high yield strain to obtained adequate growth properties [9] [10] [11] [12] . a minimum of 3-6 months is needed to develop an influenza vaccine using this approach. more importantly, the h5 avian influenza strains responsible for recent epizootic outbreak involving numerous human infections are lethal to chicken eggs used for vaccine production and to the chickens that lay the eggs. if the 2003 severe acute respiratory syndrome (sars)-coronavirus outbreak serves as a guide, the next influenza pandemic will likely have global consequences spreading within weeks, if not within days. thus, a system that can rapidly produce new influenza vaccine is needed to prevent or to effectively reduce the impact of pandemic influenza. two new approaches have shown great promise to replace the egg-based technique [9] [10] [11] . one is cell culture-based, and the other is recombinant protein (antigen)-based. the cell culture-based approach involves production of influenza viruses in cell culture followed by the current (egg-based) virus inactivation and purification for the down stream processing. the advantages are: cell cultures are easier to handle and can be scaled up in a short period of time, and the influenza vaccines produced with this approach have been tested in phase i and phase ii clinical trials and were found to be safe and at least as effective as the vaccines produced in embryonated chicken eggs [13] [14] [15] . a limitation of the cell culture-based approach is that the process still requires the production of a high-yielding re-assorted virus. this process also may introduce cell line specific mutations in the genes that can lead to the selection of variants characterized by antigenic and structural changes in the ha protein, potentially resulting in less-efficacious vaccines [16] [17] [18] . additional hurdles include: the production and handling of a dangerous virus requires the availability of a high containment facility; mammalian cells can harbor animal viruses that may lead to safety concerns; the residues from the expressing cells may cause some unknown side-effects since no thorough purification process has been introduced into the manufacturing process. on the other hand, the recombinant protein-based approach involves production of viral antigens such as ha and na in cell culture with recombinant dna technology and utilization of the purified antigens as the active ingredients in the vaccine. the rha influenza vaccines developed using the baculovirus-insect cell expression system has been tested in several phase i and phase ii human clinical trials involving over 1200 subjects that demonstrated safety, immunogenicity and efficacy [19] [20] [21] [22] [23] . in elderly adults, rha vaccine is equally or more immunogenic than the egg-based vaccine (treanor jj, et al. dose-related safety and immunogenicity of a trivalent baculovirus-expressed influenza virus ha vaccine in elderly adults, manuscript in preparation). interestingly, two h1n1 rha vaccines (derived from two strains of a/new caledonia/20/99 or a/texas/36/91) provided partial protection against the lethal challenge of a reconstructed highly lethal 1918 pandemic influenza virus (also a h1n1 strain) in mice, suggesting that cross protection against drifted strains is definitely feasible [24] . to meet the challenge of a potential influenza pandemic, however, a reliable expression system and a quick, efficient downstream purification process are needed. in this communication, we reported a rapid process capable to purify rha (h1n1, a/new caledonia/20/99) from the fermentation to 95% purity within 6 h with a 57% overall yield. since all chromatographic media used here are chemically stable and commercially available, the process can be easily scaled up in a gmp facility. various biochemical analyses indicated that the purified rha is properly folded and biologically active. in addition, we also developed a quick, simple analytical assay to monitor the expression of rha in the insect cell fermentation to ensure the rha production. the influenza vaccine strain-a/new caledonia/20/99 (h1n1)-was obtained from the cdc. the full-length ha gene (containing the ha1 and ha2 genes) from the influenza viruses was cloned using rt-pcr and inserted into a baculovirus transfer vector developed by protein sciences corporation. this specialized vector contained the promoter from the baculovirus polyhedrin gene flanked by sequences naturally surrounding the polyhedrin locus. next, the transfer vector was co-transfected into insect cells with the linearized baculovirus genomic dna (autographa californica nuclear polyhedrosis virus) depleted of the polyhedrin gene and part of an essential gene downstream of the polyhedrin locus. the homologous recombination between the transfer plasmid and the linearized viral dna rescued the virus, resulting in recovery efficiencies of recombinant virus of nearly 100%. recombinant viruses were then selected by plaque assay. the plaque-derived recombinant baculovirus was then used to create a virus stock by infecting increasingly larger cultures of the proprietary insect cells (expressf+ ® , derived from sf9 cells) in serum-free culture medium (protein sciences fortified medium). the virus stock was then used to infect insect cells (2.0 × 10 6 cells/ml) to produce rha in a 15-liter applikon bioreactor. the multiplicity of infection (moi) of the virus stock was 1 for the experiment. to monitor the infection process and the expression of ha, 4 ml samples were taken from the bioreactor at various times. one millilitre was used for analyzing the changes in cell density, cell viability and cell size distribution. two millilitres were centrifuged at 1600 rpm. the supernatant and pellet were stored separately at −80 • c to be used for srid, gel and blot analysis. one millilitre was used for hemadsorption analysis. the rest was used for protein purification. to 0.5 ml fermentation samples (insect cells uninfected, infected with recombinant baculovirus containing ha genes, and infected with recombinant baculovirus containing a non-ha gene) in a 1.5 ml eppendorf tube, 0.10 ml of 5% chicken red blood cells (charles river-spafas, north franklin, ct) in pbs was added and shaken gently for 10 min at room temperature. at the end of incubation, the tube was flipped gently for five times to get a homogenous suspension. then, 10 l of the suspension was pipetted on a glass plate and observed under a microscope (ck2, olympus optical co., japan) in three representative view fields. on average, about 20-70 insect cells were counted in each field. to reduce the chance of false positives, only the insect cells attached by three or more rbcs were counted as the rbc-bound insect cell. the percentage of rbc-bound insect cells against the total insect cells in each time point was calculated from three fields. at each time point, 1.0 ml of fermentation sample was analyzed with an automated cell analyzer (cedex as20, innovatis gmbh, germany) for cell density, cell viability and cell size distribution using the procedure described by the manufacture. buffer a: 20 mm sodium phosphate, 1.0 mm edta, 0.01 % tergitol-np9, 5% glycerol, ph 5.89. buffer b: 20 mm sodium phosphate, 0.03 % tergitol, 5% glycerol, ph 7.02. buffer c: 20 mm sodium phosphate, 150 mm nacl, 0.03% tergitol, 5% glycerol, ph 7.02. buffer d: 40 mm sodium phosphate, 0.05% tween-20, 5% glycerol, ph 7.20. buffer e: 100 mm sodium phosphate, 0.05% tween-20, 5% glycerol, ph 7.20. buffer f: 500 mm sodium phosphate, 0.05% tween-20, 5% glycerol, ph 7.20. buffer g: 10 mm sodium phosphate, 150 mm nacl, 0.01% tween-20, ph 7.22. sanitation buffer: 1.0 m nacl, 0.5 m naoh. unosphere-q (bio-rad, hercules, ca) column, ø 1.6 cm × 10 cm, 20 ml; sp-sepharose fast flow (ge/amersham/pharmacia, piscataway, nj) column, ø 1.6 cm × 10 cm, 20 ml; hydroxyapatite type i column (hx-i, bio-rad, hercules, ca), ø 1.0 cm × 4.6 cm, 3.6 ml. the fermentations producing rha were harvested by centrifugation at 56-65 h post-infection. the cell pellet (6.4 g) was extracted with 225 ml of 1% tergitol np-9 in buffer a by stirring on a magnetic stirrer at 4 • c for 30 min. the extract was clarified by centrifugation at 10,000 × g for 25 min. the supernatant was loaded on q/sp columns (equilibrated with buffer a) in tandem at 5 ml/min. after loading, the columns were washed with 140 ml of buffer a. then, the columns were disconnected. ha was eluted from the sp column with 140 ml of buffer b and 80 ml of buffer c, consecutively. the q/sp columns can be regenerated by washing with 5 column volumes (cv) of sanitation buffer and 5 cv of water and equilibrated with 5 cv of buffer a. the ha fraction in buffer b (40 ml) was loaded on a hx-i column at 2 ml/min. the column was washed with 18 ml of buffer b. the ha was eluted from the hx-i column with increasing phosphate concentration (buffers d, e and f). the ha preparation in buffer d was further purified and concentrated by ultrafiltration with a stir cell using a 100 kda mwco regenerated cellulose membrane with buffer g. the hx-i column can be regenerated by washing with 10 cv of water, and equilibrated with 10 cv of buffer c. the rha contents in all preparations were determined with srid assay as described by williams [25] and manchini et al. [26] . the assay is based on the diffusion of rha into a 1% agarose gel containing antibodies against the ha. the interaction between antigen and antibody produced a precipitation ring of which the size was directly proportional to the amount of antigen applied. the diameters of the rings in the srid assays were determined with a measuring magnifier (baush/lomb, 81-34-38). the diameters of the precipitate ring were used to determine the actual concentrations based on standards provided by the center for biologics evaluation and research of fda. for complete deglycosylation, 20 g of purified rha was deglycosylated with 5000 units of peptide-n-glycosidase f (pngase f, new england biolabs, beverly, ma, usa) or endoglycosidase h (endo h, new england biolabs) at 37 • c for 60 min as described previously [27] . for limited deglycosylation, 20 g of rha was treated with 0.2 g of trypsin on ice for 30 min. the digestion was stopped by adding 10× denaturing buffer and boiling for 5 min. then the trypsin treated rhas were deglycosylated with 2, 20 or 200 units of pngase f or endo h on ice, at 25 • c or at 37 • c for 2-60 min. the reactions were stopped by adding 2× sds sample buffer and boiling for 5 min. the protein species at various deglycosylation stages were resolved on sds-page. vaxigrip influenza vaccine 2004-2005 was purchased from canada drug delivery (nanaimo bc, canada). the purity of rha was measured on sds-polyacrylamide gels stained with coomassie blue using scanning laser densitometry (model 710, bio-rad, hercules, ca, usa) and peak integration analysis. the total amino acid analysis was carried out with a beckman amino acid analyzer at keck facility of yale university. the n-terminal amino acid sequence analysis was executed at the protein core facility of columbia university. the molecular size of the purified rha was analyzed on a size-exclusion columns (tsk-4000, 7.5 × 300 mm, tosa-haas, japan) at a constant flow rate of 0.8 ml/min, using the protein molecular weight markers as the reference (sigma, st. louis, usa) as previously described [28] . elution buffer: 50 mm sodium phosphate, 50 mm nacl and 0.001% nan 3 . a trypsin resistance assay was carried out by incubating rha for 30 min at 0 • c without or with 50 g/ml tpck-treated trypsin as described by copeland et al. [29] . for this assay, the denatured ha was produced by boiling rha for 5 min. hemagglutination activity assays were done essentially as described by barrett and inglis [30] with a 0.5% solution of fresh chicken red blood cells in a u-bottom 96-well microtiter plate. a critical issue in production of a therapeutic protein using recombinant dna technology is determining when to harvest the fermentation [31, 32] . too early, the yield may be suboptimal. too late, the expressed protein may be degraded by a variety of proteases released during the lytic process of infected cells. thus, a rapid and sensitive assay is needed to monitor protein expression and to choose the right harvest time. ha is well known for its ability to bind the sialic acid on the surfaces of red blood cells (rbcs) and agglutinate these cells [30] . this phenomenon has been success-fully used to detect cells and tissues infected with influenza viruses. to determine whether the insect cells infected with the recombinant baculoviruses containing the ha gene can also agglutinate rbcs, both uninfected and infected insect cells were incubated with rbcs for 10 min and observed under a microscope. in the uninfected insect cell sample, the rbcs (the smaller cells) were scattered around on the slide, and no specific binding of rbcs to the insect cells (the larger cells, about 16 m in diameter) was observed as shown in fig. 1a . on the other hand, most of rbcs were bound to the insect cells infected with baculovirus containing the ha gene derived from influenza strain a/new caledonia/20/99 (h1n1) (fig. 1b) . this method also works for expression of the has derived from other strains such as b/jiangsu/10/2003 (fig. 1c) and a/new york/55/2004 (h3n2) (data not shown). to verify that the observed hemadsorption is due to the ha genes and not to the other genes in the baculovirus, insect cells infected with recombinant baculovirus containing a non-ha gene were also incubated with rbcs. no binding of rbcs on insect cells was observed (fig. 1d) . these observations clearly demonstrate that rbc's binding to the surface of insect cells is ha expression dependent, not infection dependent. the data also support the conclusion that the has table 1 . at the peak of hemadsorption, clusters of 20-200 insect cells agglutinated by rbcs have also been observed. later, the binding gradually reduces to 30-40% of insect cells, most likely caused by the breakdown of some infected cells and the loss of cell membrane, as evidenced by the rapid decrease of cell viability. to determine the rha levels in the fermentation at each sampling time, the single radial immunodiffusion (srid) assay has been used, since it is a simple, reproducible technique, and relatively unaffected by other proteins in the crude extract [25, 26] . consistently, the rbc binding to insect cells correlated well with the ha levels determined by srid. late ha expression is expected because the ha gene is regulated by the polyhedrin promoter, which is a late stage promoter in baculovirus infection. similar results were also obtained for the expression of other has such as b/jiangsu/10/2003 and a/new york/55/2004 (data not shown). a major challenge in the biotechnology industry is purification of biologically active recombinant proteins [33, 34] . an ideal purification process should be mild, efficient and capable of achieving high purity in a short period of time. accordingly, each purification step has been carefully designed to optimize the whole process. as demonstrated in the hemadsorption studies, rhas are expressed, folded and transported to the cell membrane at a late stage. to extract rha from the cell membranes, several non-ionic detergents at various concentrations were tested for their efficiency. the best result was obtained using 1% tergitol np-9. to get a relatively clean extraction, a magnetic stirrer was used to avoid the disruption of cell nuclei and other organelles. the extract was clarified by centrifugation to remove cell nuclei and other debris. the ha monomer of a/new caledonia/20/99 influenza virus strain consists of 547 amino acids with a theoretical molecular weight of 63,156.43 and a pi of 6.30. therefore, this rha can be bound on a cation-exchange media like sp using a lower ph buffer and eluted with a higher ph buffer to achieve the primary purification and concentration. the supernatant of the extract was loaded on uno sphere q/sp columns in tandem. the anion-exchange q column acts as a scavenger by binding the negative-charged impurities that may foul the sp column. after loading and washing, the columns were detached and eluted separately. as shown in the chromatogram (fig. 2a) , about 50% of the proteins flow through q/sp columns, a small amount of protein elutes from the sp with the ph 7 buffer and the rest bind tightly to either the sp or the q column. as shown on the sds-page (fig. 2b) , the rha captured on the sp at ph 6 was selectively eluted by a shift to ph 7. the ph shift resulted in a 14-fold increase in purity based on densitometry of the sds-page gel. to further purify rha, use of hydroxyapatite type i (hx-i) media was explored. the binding preference of hx-i media is significantly different from the ion-exchange media, and yet the binding and eluting conditions are relatively mild so as to preserve the biological activity of the target protein [35, 36] . thus, the ph 7 sp column eluate was loaded on a hx-i column. after washing, rha was eluted from the column with increased phosphate concentration. as shown in fig. 2c , most rha was eluted in 40 mm phosphate, and there was a small loss in the wash and in the 100 mm phosphate elution. the purity was increased from 52% to 91% according to the densitometry of the sds-page. however, there was still a protein contaminant of about 36 kda in the preparation as revealed on lane 5 of fig. 2c . since the size of rha trimer is around 210 kda, the difference between rha size and this impurity could be explored to remove this impurity. thus, the 40 mm phosphate eluate was further purified with ultrafiltration using a stir cell (100 kda mwco). as demonstrated in fig. 2d , the 36 kda band (lane 5) was selectively removed from the retentate. the purified rha migrated on sds-page gel as a single polypeptide (rha) with an apparent molecular weight of approximately 70 kda. on the blot, a small amount of rha1 and rha2 (the cleavage products of rha) were also observed, with apparent molecular weights of ≈50 and ≈28 kda, respectively (data not shown). trace amounts of rha dimers and trimers were also detectable, with apparent molecular weights of ≈140 and ≈220 kda, respectively. as summarized in table 2 , the process described here can purify rha from the fermentation to 95% purity within 6 h with a 57% overall yield. the largest single step loss (27%) is on hx-1 column. to confirm the authenticity of rha, the purified protein was examined by n-terminal amino acid sequencing and total amino acid analysis (aaa). the n-terminal amino acid sequence matched the predicted one (10 cycles were used) and the signal sequence peptide (the first 17 amino acids) of the full-length ha gene was absent in rha. the measured amino acid composition of the purified rha was consistent with the theoretical one (data not shown). the authenticity of rha was further verified by the western blot using a/new caledonia/20/99 antibody provided by the food and drug administration (fda). the purified rha in 0.005% tween/pbs solution was analyzed using size-exclusion chromatography. it was eluted as a single peak at 9.1 min as shown in fig. 3a , corresponding to a molecular weight around 800-1000 kda, likely a complex of four to five ha trimers ((4-5) × 3 × 70 kda). to test whether the purified rha still retained its native structure, the purified protein was treated with trypsin on ice. as shown in the lane 3 of fig. 3b , rha was cleaved into only two bands, ha1-50.9 kda and ha2-27.5 kda. on the other hand, the heat-denatured rha was digested into numerous small fragments. the trypsin-resistance data demonstrate that the rha expressed in insect cells folded properly and retained its native structure after purification. table 2 stepwise mass balance of ha-nc purification by srid assay ha (g/ml) volume (ml) ha (mg) purity a (%) step recovery (%) total recovery (%) since glycosylation may play an important role in the biological function of ha [37] [38] [39] , it is of interest to explore whether the rha produced in insect cells is properly glycosylated. thus, rha was deglycosylated with peptide-nglycosidase f (pngase f) or endoglycosidase h (endo h) and resolved on sds-page. as shown in fig. 3c , the untreated ha migrated at 70.4 kda, pngase f deglycosylated ha at 57.9 kda, and endo h treated ha at 64.8 kda, respectively. these data indicate that the rha produced in insect cells is indeed glycosylated with n-linked oligosaccharide side chains. about 5.6 kda of oligosaccharide chains have high mannose content susceptible to endo h, and 6.9 kda of oligosaccharide chains have low mannose residues resistant to endo h. to assess the number of n-linked oligosaccharide chains, the trypsin treated rha was subjected to limited deglycosylation with pngase f or endo h under a variety of conditions. on the sds-page of pngase f treated samples (fig. 3d) (fig. 3e) , there are three distinguishable ha1 bands, 51.2, 49.4 and 46.5 kda, and one ha2 band, 27.1 kda. the data suggest that there are six n-linked oligosaccharide chains in the ha1 region, and two of them have high mannose content. there is only one n-linked oligosaccharide chain in the ha2 region, which is to directly compare with the a/new caledonia antigen present in the egg-based vaccine, the purified rha/ a/new caledonia was formulated either alone into a 15 g/0.5 ml solution or with 15 g rha/b/jiangsu and 45 g rha/a/wyoming in a 0.5 ml dosage (flubløk tm , the expected trade name of protein sciences' rha vaccine). as judged by the hemagglutination assay, flubløk is as active as vaxigrip (a licensed egg-based vaccine manufactured by sanofi-pasteur-aventis) in agglutinating rbcs and preventing them from forming a tight pellet as shown in fig. 4a . antigen specific srid assay is widely used to determine the concentration of active ingredients in a vaccine. on the srid gel prepared with a/new caledonia/20/99 antibody (fig. 4b) , the diffusion ring of flubløk is slightly larger than that of vaxigrip, suggesting that flubløk is equivalent to vaxigrip for the active ingredient of a/new caledonia/20/99 strain. on the sds-page (fig. 4c) , the flubløk lane is clean, and only three broad bands are visible, representing the ha, ha1 and ha2 of the three strains, respectively. the vaxigrip lane is complicated with the major ha band around 59 kda along with many minor bands. similarly, numerous impurities have also been found in other egg-based vaccines [40] . all these data demonstrate that the purified rha expressed in insect cells is correctly translated, properly glycosylated and folded and biologically active. the traditional egg-based vaccines have been successfully used for more than 50 years to prevent influenza. they are reliable, effective (if there is a good match), and affordable. however, the production cycle of the egg-based vaccines is lengthy and heavily dependent on egg supply and unable to be developed quickly in response to the urgent need in an influenza pandemic [9] [10] [11] [12] . to replace or supplement the egg-based vaccines, the new vaccine has to be equally effective, reliable, economical, and capable of being developed and delivered in a short period of time. the work reported here is some important progress toward an alternative influenza vaccine-the recombinant protein-based vaccine. a new analytical method based on hemadsorption has been developed to closely monitor the expression of ha in insect cells. this method plays a critical role in ensuring optimal ha production and in determining the right harvest time, in addition to other harvest parameters such as hpi, cell's morphology and viability. it has also been successfully used at protein sciences corporation to accelerate the screening process for the recombinant baculoviruses used for ha manufacturing. a purification process has been developed to quickly purify the recombinant ha from the bulk harvested from the bioreactor while retaining its biological activity. previously, ha purifications were heavily dependent on affinity chromatography using specific monoclonal antibodies or various lectins [41, 42] . such methods are highly selective, but difficult to scale up to commercial levels due to a number of limiting factors: (1) some of the ligands will leach off the column during the purification and they must be removed from the final product; (2) it is difficult to regenerate an affinity column after use, and thus the performance declines after each use; (3) the batch to batch variations in the quality of affinity media make it almost impossible to have a robust purification process from time to time; (4) most affinity media are too expensive to be used at large scale. on the other hand, all chromatographic media used in the present process are chemically stable (can be regenerated repeatedly), commercially available and relatively inexpensive; thus more suitable to scale up in a gmp facility. if the reagents, columns and ancillary equipment are well prepared in advance, the whole purification can be completed in one full working day, avoiding the overnight storage of intermediate rha preparation and possible inactivation of rha. several factors have made this rapid process possible. first, there is no sample manipulation between purification steps, which makes a quick, continuous purification process possible. second, q/sp is connected in tandem, combining two chromatographic processes into one. third, a chemically different chromatographic media-hx-i is used to differentiate rha from the remaining impurities. unlike ion-exchange media, the adsorption of proteins to hx-i involves both anionic and cationic interaction. the calcium group can interact with carboxylate residues, whereas the phosphate group can bind the basic residues on the surface of the protein. the bound proteins can be eluted by an increasing phosphate gradient or a gradient of calcium, magnesium ions. it is worth to point out that the purification process described here needs to be further optimized for large-scale production, for example, a tangential flow filter should be used to replace a stir cell at the polish step. nonetheless, we believe the strategies described here can also be used to develop a rapid purification process for other recombinant proteins. biologically, flubløk is as active as the egg-based vaccine-vaxigrip as determined by the hemagglutination assay. based on the srid assay, flubløk is equivalent to vaxigrip for the active ingredient of a/new caledonia/20/99 strain of influenza. in challenge studies, chickens were effectively protected against the h5n1 virus infection after inoculation with the rha of the virus [43] . moreover, two distantly related h1n1 rha influenza vaccines using the baculovirusinsect cell expression system have also been demonstrated to partially protect mice against the lethal challenge of a recombinant 1918 pandemic influenza virus [24] . in clinical trials, the trivalent rha vaccine (flubløk) stimulates anti-ha antibody production at least as well as, and in the case of h3 rha, superior to the traditional egg-based vaccine [19] [20] [21] [22] [23] . the 2004/05 influenza season phase iib field trial of flubløk that enrolled 460 healthy subjects aged 18-49 further showed that the 45/45/45 dose was 100% efficacious in preventing culture positive influenza illness compared to placebo (press release, protein sciences corp., june 14, 2005) . furthermore, a recombinant protein-based vaccine, such as the flubløk described here, also has other advantages over the traditional egg-based vaccines. it consists solely of three antigens (proteins) stored in sterile phosphate buffered-saline and without preservatives such as thimerosal (a mercury derivative currently used in the egg-based vaccine), antibiotics or adjuvants. unlike the egg-based vaccines, no live influenza viruses, biocontainment facilities or harsh chemicals such as formaldehyde are used in manufacturing. this may explain why flubløk has shown lower side effects than the licensed vaccines in clinical trials [19] [20] [21] [22] [23] . therefore, a reliable, effective, and affordable recombinant protein-based influenza vaccine can be and should be developed to meet the challenge of a potential influenza pandemic. for pandemic preparedness, developing and stockpiling rha influenza vaccines against the present h5n1 strain may be a good option to provide some protection for the first response personnel and the 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proteins recombinant protein expression for therapeutic applications hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of ph separation between the alpha and beta forms of human antithrombin by hydroxyapatite high-performance liquid chromatography importance of hemagglutinin glycosylation for the biological functions of influenza virus effects of glycosylation on the properties and functions of influenza virus hemagglutinin roles of n-linked glycans in the endoplasmic reticulum morphological and biochemical characterization of influenza vaccines commercially available in the united kingdom production of a recombinant influenza vaccine using the baculovirus expression vector system the glycosylation of the influenza a virus hemagglutinin by mammalian cells. a site-specific study baculovirus-derived rha vaccines protect against lethal influenza infections by avian h5 & h7 subtypes key: cord-015764-ly68q5z0 authors: poissy, j.; terrier, o.; lina, b.; textoris, j.; rosa-calatrava, m. title: la modulation de la signature transcriptomique de l’hôte infecté : une nouvelle stratégie thérapeutique dans les viroses graves ? exemple de la grippe date: 2016-04-07 journal: reanimation doi: 10.1007/s13546-016-1188-1 sha: doc_id: 15764 cord_uid: ly68q5z0 during the last decades, emergence and reemergence of viruses were responsible for epidemic and pandemic infectious diseases, with variable degrees of severity. current preventive strategies are not sufficient at all, and available therapeutic drugs are very limited. indeed, genetic variations of viruses can impair the efficacy of antiviral compounds by the apparition of resistance. moreover, current delay needed for de novo development of drugs does not allow a rapid response in case of important epidemic or pandemic events. in this context, new therapeutic approaches are necessary. an innovative concept is to repurpose already marketed compounds that can reverse the host cellular transcriptomic response to the infection. by targeting the host, these molecules exhibit a broad-spectrum activity and are potentially effective even against new emergent strains. this strategy implements the characterization of specific host gene expression profiles, the in silico screening of drugs, and their validation in in vitro and in vivo models, until their evaluation in clinical trials. here, we will present this approach, with the example of the flu. résumé ces dernières décennies ont été marquées par l'émergence ou la réémergence de virus responsables d'épidémies ou de pandémies plus ou moins sévères. les stratégies préventives sont prises à défaut, et l'arsenal antiviral curatif est limité d'autant plus que les résistances virales peuvent apparaître rapidement. par ailleurs, le développement de nouvelles molécules nécessite un délai incompatible avec la réponse rapide nécessaire lors d'une épidémie d'envergure ou d'une pandémie. c'est la raison pour laquelle de nouvelles approches thérapeutiques sont nécessaires. un concept novateur est le repositionnement de molécules déjà sur le marché en exploitant leur capacité à inverser la réponse transcriptomique cellulaire de l'hôte infecté. en identifiant des molécules qui visent l'hôte et non le virus, cette stratégie permet d'avoir un large spectre d'action et d'être potentiellement actif sur de nouveaux variants. la mise en place de cette stratégie nécessite de caractériser les réponses cellulaires spécifiques de l'infection virale d'intérêt, de cribler in silico des molécules candidates, de les tester sur modèles cellulaires et animaux, avant d'envisager des essais cliniques chez l'homme. nous présenterons cette démarche en prenant pour exemple l'infection grippale. mots clés viroses · grippe · transcriptome · repositionnement de médicaments abstract during the last decades, emergence and reemergence of viruses were responsible for epidemic and pandemic infectious diseases, with variable degrees of severity. current preventive strategies are not sufficient at all, and available therapeutic drugs are very limited. indeed, genetic variations of viruses can impair the efficacy of antiviral compounds by the apparition of resistance. moreover, current delay needed for de novo development of drugs does not allow a rapid response in case of important epidemic or pandemic events. in this context, new therapeutic approaches are necessary. an innovative concept is to repurpose already marketed compounds that can reverse the host cellular transcriptomic response to the infection. by targeting the host, these molecules exhibit a broad-spectrum activity and are potentially effective even against new emergent strains. this les virus ont une place prépondérante en pathologie humaine. en effet, environ 15 % des 1 400 pathogènes humains sont des virus, et 40 % de ces virus sont émergents ou réémergents [1] . l'émergence est définie par l'apparition d'une maladie liée à un agent infectieux jusque-là inconnu ou dont l'imputabilité dans la maladie n'avait pas encore été mise en évidence, ou d'un nouveau variant d'un agent connu. la réémergence est définie par la réapparition de maladie(s) qui avai(en)t disparu ou l'augmentation de son (leur) incidence. depuis les années 1980 et à raison de deux par an en moyenne, 87 virus ont été responsables de pathologies émergentes de gravité variable [2] . les projections prévoient l'émergence d'une quarantaine de nouveaux virus dans les dix ans qui viennent [3] . ces virus sont principalement des virus à arn à tropisme respiratoire et dont le réservoir est animal [2] . les points d'orgue de ces infections virales ont été l'épidémie de sras en 2003 [4] , l'apparition de la souche h1n1pdm09 en 2009 et le mers coronavirus en 2012 [5] , du fait de leur propagation à l'échelle mondiale, de leur mortalité importante, et atteignant spécifiquement des populations inattendues. de manière plus dramatique, l'épidémie récente d'ebola souligne davantage la nécessité d'avoir à disposition des molécules à action antivirale et de pouvoir les utiliser rapidement. en effet, en dehors de l'infection par le vih et des hépatites virales b et c, l'utilisation d'antiviraux reste très limitée et se heurte à la problématique générale des résistances. une des approches possibles pour le développement de nouveaux antiviraux à plus large spectre d'action est de cibler non pas le virus, mais la cellule de l'hôte infecté, en utilisant la signature transcriptomique cellulaire de l'infection pour sélectionner et repositionner des molécules déjà sur le marché qui vont moduler ce profil transcriptomique, supposé comme étant favorable à l'infection. alors que cette approche est aujourd'hui développée en cancérologie, elle est d'utilisation très récente en infectiologie. nous proposons ici de présenter les concepts sur lesquels elle repose, en illustrant notre propos par les travaux que nous avons menés, essentiellement sur les virus influenza. les infections respiratoires aiguës (ira) représentent une des principales causes de consultations, d'hospitalisations et de décès dans le monde, étant notamment la première cause de mortalité chez les jeunes enfants avec près de deux millions de décès par an. chaque année, le coût de leur prise en charge est estimé entre 1,5 et 2 milliards d'euros [6] . les progrès en termes de diagnostic ont permis de montrer qu'une large part de ces infections est due à des virus. ainsi, dans une étude épidémiologique récente sur les pneumonies communautaires aux états-unis nécessitant une hospitalisation des patients, les virus représentaient deux tiers des pathogènes identifiés. les virus influenza de type a représentaient la deuxième étiologie, après les rhinovirus [7] . par ailleurs, ces virus pathogènes respiratoires sont un facteur prédisposant des pneumonies bactériennes chez l'adulte [8] . les virus influenza, responsables de la grippe, occupent donc une place prépondérante parmi les agents pathogènes responsables des ira. ces virus enveloppés à génome arn segmenté font partie de la famille des orthomyxoviridae et sont divisés en trois types distincts, a, b et c, sur la base de leurs différences majeures antigéniques, leur organisation génomique, leur spécificité d'hôte et leur épidémiologie [9] . les virus influenza a et b sont les responsables de la grippe saisonnière. à leur surface se trouvent deux glycoprotéines, l'hémagglutinine (ha) qui se fixe aux récepteurs sialylés cellulaires et la neuraminidase (na) qui permet la libération des nouveaux virions. il existe différents sous-types de virus influenza a selon la nature de ces glycoprotéines [9] . chez l'homme, les virus circulant depuis plusieurs décennies sont les sous-types h1n1 et h3n2. des transmissions interespèces occasionnelles, notamment de l'animal à l'homme, sont aussi décrites pour les virus aviaires de type h5, h7 ou h9 [10] . ces transmissions interespèces à l'origine de pandémie résultent notamment de cassures antigéniques qui correspondent à des échanges de matériels génétiques entre soustypes viraux, conduisant à l'apparition de virus dotés de nouveaux antigènes ha et na, vis-à-vis desquels la population humaine est immunologiquement naïve. ce phénomène fut à l'origine de l'émergence en 2009 d'un nouveau virus grippal pandémique h1n1, d'origine porcine, aviaire et humaine [11] , ou encore des épidémies de h5n1 aviaires hautement pathogènes depuis 2003 en asie, ou des épisodes de grippe h7n7 aux pays-bas en 2003 et h7n9 dans le sud-est asiatique en 2013. les épidémies de grippe saisonnière sont le résultat d'une dérive génétique permanente des virus influenza a et b (apparition de mutations spontanées, notamment dans les gènes qui codent pour les glycoprotéines de surface) [11] et constituent une cause majeure de morbidité et de mortalité accrue, notamment chez les individus très jeunes, les personnes âgées, les individus immunodéprimés et ceux souffrant de maladies cardiopulmonaires chroniques. la grippe est une infection virale saisonnière qui touche chaque hiver d'un à huit millions de personnes en france, ayant un impact important non seulement en termes de santé publique, mais également d'un point de vue économique, en occasionnant un absentéisme (deux à quatre millions de journées) et une perte de productivité importants. l'incidence des virus influenza dans la population est estimée de 5 à 10 % chez les adultes et de 20 à 30 % chez les enfants. leur taux de mortalité globale varie de 0,1 à 1 % selon le type de souches circulantes et la couverture vaccinale annuelle. en france, la grippe est responsable en saison hivernale de 500 à 900 consultations par semaine, avec un pic de 50 à 100 nouveaux cas graves par semaine [12] . les admissions en réanimation pour détresse respiratoire associée à une infection par influenza de type a sont estimées à 12/100 000 personnes.an chez l'adulte [13] . outre les personnes âgées avec des comorbidités, les jeunes enfants et les femmes enceintes sont des populations particulièrement à risque. streng et al. ont ainsi récemment rapporté dans une cohorte de cas de grippe grave hospitalisés en réanimation pédiatrique 11 % de séquelles pulmonaires et 11 % de décès [14] . par ailleurs, dans une série épidémiologique américaine ayant analysé 915 décès survenus durant la saison hivernale 2009-2010 au cours d'une grossesse, il a été démontré que l'infection par influenza de type a était la cause certaine du décès dans 8,2 % des cas et suspectée dans 3,7 % [15] . la vaccination constitue la mesure préventive la plus efficace contre la grippe. cependant, le délai incompressible de six à neuf mois entre le choix par l'oms des sous-types entrant dans la composition vaccinale et la délivrance des lots de vaccins ainsi que le manque d'adhésion de plus en plus important de la population, y compris chez les personnels de santé (en france, 53 % des personnes à risque n'étaient pas vaccinées en 2014) limitent grandement son efficacité [16, 17] . cela n'est pas sans conséquence à l'échelle de la population, puisqu'un faible taux de couverture vaccinale est associé à un risque augmenté d'admission en réanimation et de ventilation mécanique [18] . l'émergence de nouveaux variants viraux par dérive antigénique (comme observé en une stratégie innovante d'identification de nouveaux antiviraux ciblant la cellule infectée transcriptome le succès de telles stratégies repose en grande partie sur notre capacité à caractériser davantage la biologie cellulaire de ces virus respiratoires et leurs interactions moléculaires et fonctionnelles avec leur hôte. ces dernières années, de nouvelles approches de biologie cellulaire de l'infection [38] [39] [40] [41] [42] , et en particulier le développement de la biologie des systèmes, ont considérablement enrichi les connaissances concernant les interactions complexes et multiples entre ces virus et la « machinerie » cellulaire. parmi elles, les études de la réponse transcriptomique de l'hôte ont permis de mettre en évidence les voies de signalisation sollicitées et/ou détournées lors de l'infection [43, 44] . le transcriptome correspond à l'ensemble des arn transcrits à partir du génome cellulaire, codé par l'adn [45] . ces arn transcrits sont composés d'arn messager (arnm), d'arn de transfert, d'arn ribosomal et d'arn non codant [46] . déterminer le transcriptome revient donc à « photographier » l'état d'expression du génome, à un moment donné. il existe différentes techniques de biologie moléculaire permettant d'analyser le transcriptome cellulaire. cette analyse peut être qualitative et comparative, mais aussi quantitative. les techniques historiques (banques soustractives) sont maintenant remplacées par des techniques dites de puces à adn (microarrays) ou de séquençage (rnaseq). ces techniques permettent de déterminer en une seule expérience l'expression transcriptomique [47] et nécessitent l'utilisation d'outils informatiques adaptés. dans les réactions inflammatoires, le profil de réponse transcriptomique est différent selon que la cause est infectieuse ou non. dans les infections, il a été démontré, sur modèle animal, qu'il était possible d'établir des réponses spécifiques du microorganisme responsable [48, 49] , faisant ainsi du transcriptome un éventuel outil diagnostique. cette réponse transcriptomique a également été un succès en termes de médecine personnalisée dans le domaine de l'oncologie et de la prise en charge du cancer [50] . l'ensemble de ces données permet d'envisager la détermination d'une signature cellulaire spécifique d'une infection et d'imaginer moduler sa réponse. utilisation du transcriptome comme outil de développement de nouvelles approches thérapeutiques une des approches visant à minimiser les résistances aux antiviraux tout en permettant des activités antivirales à large spectre consiste à cibler les réponses cellulaires de l'hôte pour contrecarrer la réplication virale et/ou atténuer la pathogenèse associée [51] . cette stratégie a déjà été utilisée avec succès dans les thérapies antirétrovirales avec notamment le développement d'antagoniste de ccr5, corécepteur du vih [52] . en ce qui concerne l'application de ce concept aux viroses respiratoires, cameron et al. ont par exemple montré par analyse transcriptomique que l'expression du gène cxcl10 était fortement induite dans les poumons de furets infectés par une souche hautement pathogène h5n1. le traitement par un antagoniste de la voie de signalisation associée à cxcl10 a permis une réduction de la mortalité et de la sévérité des symptômes dans ce modèle animal [43] . par ailleurs, plusieurs méthodes de criblage à haut débit (petits arn interférents [sirna], interaction protéine-protéine) ont été menées ces dernières années et ont identifié plusieurs centaines de facteurs cellulaires impliqués dans la réplication des virus influenza et/ou la pathogenèse associée à l'infection, proposant ainsi autant de cibles thérapeutiques potentielles [43, 44] . cependant, malgré des résultats prometteurs sur le plan expérimental, ce type d'approche focalisée sur une voie précise de signalisation cellulaire et un de ses effecteurs s'est pour l'instant toujours soldée par un échec lors de son application en clinique humaine [53, 54] , probablement du fait de la grande redondance des voies de signalisation de la cellule et de leurs interconnexions multiples. fondée sur plusieurs études assez récentes suggérant que le mode d'action des médicaments est davantage dépendant de leur effet transcriptionnel sur la cellule hôte que de leur interaction directe avec leur(s) cible(s) cellulaire(s) [55, 56] , nous avons mis en place un programme de recherche translationnelle visant à exploiter la réponse transcriptomique de l'hôte infecté en considérant le profil d'expression des gènes cellulaires comme une empreinte globale de l'infection. nous avons ainsi postulé que les signatures transcriptomiques cellulaires des virus influenza reflètent un état cellulaire globalement favorable à leur réplication, et nous avons émis l'hypothèse que des molécules associées à une signature cellulaire transcriptomique inverse pourraient induire un état cellulaire globalement défavorable à l'infection. l'ensemble de cette stratégie de repositionnement moléculaire est schématisé sur la figure 1 . il faut préciser que dans cette approche nous ne cherchons pas à distinguer la signature cellulaire favorisant la réplication virale des éléments de la réponse antivirale eux aussi stimulés lors de l'infection, car nous avons choisi d'adopter une approche globale de la cellule en sachant que les voies de signalisation, biogenèse et métabolique cellulaires, sont ambivalentes (à la fois pro-et antivirales en fonction de l'étape, du cycle infectieux et de l'état cellulaire). par exemple, l'induction des voies apoptotiques par les virus influenza est à la fois provirale dans les étapes amont (le clivage de caspase est nécessaire au trafic nucléocytoplasmique des génomes viraux néosynthétisés) et antivirale dans les étapes d'aval (qui sont pour le coup inhibées par des facteurs viraux). c'est donc bien l'état cellulaire global auquel nous nous sommes intéressés. nos premiers travaux ont permis de caractériser des signatures transcriptomiques in vitro spécifiques de l'infection par différents virus influenza humains et aviaires [57] [58] [59] . il existe bien sûr des variations dans les signatures transcriptomiques entre les différents sous-types viraux, mais nous nous sommes focalisés sur les similarités, permettant ainsi de dégager un profil transcriptomique commun à tous les virus. nous avons également développé une méthodologie d'analyse bio-informatique adaptée à un criblage informatique in silico permettant la sélection de molécules sur la base de leur signature cellulaire anticorrélée à celle de l'infection (fig. 2) . cette analyse repose notamment sur l'exploitation de la base de données connectivity map [60] (broad institute, mit) qui recense plus de 7 000 profils transcriptomiques obtenus après traitement de cellules par 1 309 molécules différentes, la plupart possédant une autorisation de mise sur le marché pour des indications thérapeutiques très diverses (oncologie, rhumatologie, etc.). parmi les dix molécules que nous avons sélectionnées, la présence de la ribavirine, connue pour son activité antivi-rale [61] , a ainsi constitué une première validation de notre stratégie de criblage non conventionnelle. de manière très intéressante, les neuf autres molécules identifiées dans le criblage n'étaient pas connues pour une indication thérapeutique anti-infectieuse supposée. à l'issue de leur évaluation dans différents tests in vitro cellulaires spécifiques, nous avons pu caractériser quatre de ces molécules pour leur propriété antivirale à large spectre contre plusieurs virus influenza humains et aviaires. ces résultats d'efficacité constituaient ainsi une seconde validation de notre stratégie de criblage et de repositionnement de médicaments. ces molécules ont fait l'objet de deux dépôts de brevet d'invention (fr n o 0958810-pct/ep2010/069023-wo 2011069990, us n o 61/279 997) pour une seconde indication thérapeutique anti-infectieuse et ont été décrites dans une publication [57] . parmi les molécules identifiées, la midodrine, un agoniste des récepteurs alpha-1 adrénergique, usuellement utilisée comme antihypotenseur (gutron ® ) [62] , présentait la meilleure activité antivirale à dose non cytotoxique contre tous les virus testés in vitro. nous avons mené un essai clinique multicentrique et randomisé (essai flumed eudract n o 011-004552-19, promoteur : hospices civils de lyon, investigateur : pr bruno lina) pour évaluer l'activité antivirale in vivo de cette molécule administrée à une dose usuelle de 7,5 mg/j dans la prise en charge des grippes ambulatoires, donc non sévères cette étude prospective multicentrique randomisée en double insu comptera trois bras parallèles, afin de comparer l'efficacité de l'association de chacune des molécules à l'oseltamivir, contre l'oseltamivir seul. le bénéfice attendu est une clairance virale plus rapide et plus importante que dans le groupe oseltamivir seul, avec un bénéfice potentiel sur la durée de ventilation mécanique, la mortalité, les séquelles, notamment respiratoires. l'efficacité (objectif primaire) sera évaluée sur le pourcentage de patients vivant avec une négativation de la charge virale influenza a (par rt-pcr) au niveau d'un écouvillon nasopharyngé à j7 de l'inclusion. les objectifs secondaires seront : • de vérifier si l'administration de ces molécules restaure bien un profil transcriptionnel physiologique (inversion in vivo de la signature induite par l'infection par influenza) ; • d'évaluer l'action de ces molécules sur l'évolution de la charge virale (suivi pendant les dix jours du traitement) ; • d'évaluer l'impact de ces associations sur d'autres critères pronostiques tels que la durée de ventilation mécanique, la durée de séjour en réanimation et la mortalité. cette stratégie innovante de criblage de molécules antivirales ciblant la cellule plutôt que le virus est bien adaptée aux viroses respiratoires. celle-ci s'inscrit dans une démarche de repositionnement thérapeutique. outre les perspectives de limiter les possibilités d'émergence de résistances, les avantages réglementaires et financiers de cette stratégie sont évidents par rapport au processus long et coûteux du développement de molécules de novo. en perspective, il est tout à fait envisageable que cette stratégie rationnelle et rationalisée que nous proposons pour cribler, sélectionner et repositionner des médicaments puisse non seulement permettre de puiser dans la pharmacopée existante et constituer ainsi une force de réaction rapide en cas d'épidémie ou de pandémie, ou d'émergence de nouveaux virus, mais également être déclinée pour d'autres pathogènes que ceux respiratoires 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validation of drug indications using compendia of public gene expression data gene expression signature-based screening identifies new broadly effective influenza a antivirals cellular transcriptional profiling in human lung epithelial cells infected by different subtypes of influenza a viruses reveals an overall down-regulation of the host p53 pathway host microrna molecular signatures associated with human h1n1 and h3n2 influenza a viruses reveal an unanticipated antiviral activity for mir-146a the connectivity map: a new tool for biomedical research megaribavirin aerosol for the treatment of influenza a virus infections in mice midodrine. a review of its therapeutic use in the management of orthostatic hypotension key: cord-285856-0sw3wt1i authors: naesens, lieve; vanderlinden, evelien; rőth, erzsébet; jekő, józsef; andrei, graciela; snoeck, robert; pannecouque, christophe; illyés, eszter; batta, gyula; herczegh, pál; sztaricskai, ferenc title: anti-influenza virus activity and structure–activity relationship of aglycoristocetin derivatives with cyclobutenedione carrying hydrophobic chains date: 2009-02-05 journal: antiviral res doi: 10.1016/j.antiviral.2009.01.003 sha: doc_id: 285856 cord_uid: 0sw3wt1i previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chain-substituted cyclobutenedione. the lead compound 8e displayed an antivirally effective concentration of 0.4 μm, which was consistent amongst influenza a/h1n1, a/h3n2 and b viruses, and a selectivity index ≥50. structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure–activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound 8e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. previous studies have demonstrated that glycopeptide compounds carrying hydrophobic substituents can have favorable pharmacological (i.e. antibacterial and antiviral) properties. we here report on the in vitro anti-influenza virus activity of aglycoristocetin derivatives containing hydrophobic side chainsubstituted cyclobutenedione. the lead compound 8e displayed an antivirally effective concentration of 0.4 m, which was consistent amongst influenza a/h1n1, a/h3n2 and b viruses, and a selectivity index ≥50. structural analogues derived from aglycovancomycin were found to be inactive. the hydrophobic side chain was shown to be an important determinant of activity. the narrow structure-activity relationship and broad activity against several human influenza viruses suggest a highly conserved interaction site, which is presumably related to the influenza virus entry process. compound 8e proved to be inactive against several unrelated rna and dna viruses, except for varicella-zoster virus, against which a favorable activity was noted. © 2009 elsevier b.v. all rights reserved. currently available drugs for the treatment of influenza virus infections comprise the m2 ion channel blockers amantadine and rimantadine, and the neuraminidase inhibitors oseltamivir and zanamivir (de clercq, 2006; moscona, 2008) . stockpiling of oseltamivir and, to a lesser extent, zanamivir has been advocated in the context of pandemic preparedness (schünemann et al., 2007) , yet the recent isolation of oseltamivir-resistant seasonal influenza virus mutants, even from untreated patients, warrants for continued caution (lackenby et al., 2008; van der vries et al., 2008) . additional anti-influenza virus compounds should be urgently developed, having a novel antiviral target that is highly conserved amongst influenza virus (sub)types and, hence, less prone to genetic variation and resistance selection. one of the attractive therapeutic strategies would be a blockade of the viral entry into the host cell. the cellular entry process of influenza viruses has been unraveled since many years (reviewed in skehel and wiley, 2000) . a key role is being played by the viral envelope glycoprotein hemagglutinin (ha), which contains the receptor-binding site for initial attachment to the sialylated cellular receptors, and governs the receptor specificity of human versus avian influenza virus subtypes (chandrasekaran et al., 2008; nicholls et al., 2008) . in addition, after cellular uptake of the virus by endocytosis, the ha mediates the low ph-induced fusion of the viral envelope with the endosomal membrane, leading to release of the viral ribonucleoprotein in the cytosol. although the ha has been extensively studied from a biochemical and epidemiological perspective, specific antiviral drugs blocking the ha-receptor interaction remain to be clinically developed. several reports are available on the in vitro activity of small-molecule inhibitors of influenza virus fusion, which act by preventing the conformational change of the ha at low ph deshpande et al., 2001; plotch et al., 1999) . unfortunately, their development has been slow due to their inferior activity against some human influenza virus (sub)types, rapid selection for resistance and/or unsatisfactory outcome in animal models (yagi et al., 1999) . glycopeptide compounds represent a large series of natural, semisynthetic or fully synthetic compounds, which are widely recognized for their potent activity against gram-positive bacteria (nicolaou et al., 1999) . several studies have demonstrated that hydrophobic derivatives of glycopeptide antibiotics (e.g. vancomycin and eremomycin), and/or their aglycones exert antibacterial activity against glycopeptide-resistant enterococci. (cooper et al., 1996; printsevskaya et al., 2002; pace and yang, 2006) . remarkably, some of these lipophilic glycopeptide compounds were found to have inhibitory activity against coronaviruses and hiv, the latter being ascribed to inhibition of the hiv entry process (balzarini et al., 2003 (balzarini et al., , 2006 preobrazhenskaya and olsufyeva, 2006) . we here report on the chemical synthesis, anti-influenza virus activity and structure-activity relationship of novel glycopeptide compounds carrying a hydrophobic side chain on an aglycoristocetin backbone ( fig. 1) . high-yield synthesis of aglycovancomycin (1) and aglycoristocetin (2) (fig. 1 ) was performed as originally described by wanner et al. (2003) and consisted of deglycosidation of the parent antibiotics with hydrogen fluoride in anisole at neutral ph ( fig. 2) (sztaricskai et al., 2006) . the aglycones were converted into the squaric acid amide esters (4 and 5) by coupling with dimethyl squarate (3). this was followed by reaction with primary amines (6a-j) to yield the corresponding asymmetric squaric diamides (7a-g, 8a-j), using a regioselective procedure without the requirement for a protecting group strategy (tietze et al., 1991; sztaricskai et al., 2006) . the primary amines were: 6aminohexanol (6a); 6-aminohexanecarboxylic acid (6b); triglycine (6b1); dopamine (6c); n-(4-aminophenyl)piperidine (6d) and 4phenyl-benzylamine (6e). based on the observation that compound 8e displayed favorable anti-influenza virus activity (see below), subsequent modifications were performed to study the impact of an increasing steric bulk in the rigid aromatic side chain of 7f-g and 8f-h, which were prepared with 1-naphthylamine (6f), 4-aminoterphenyl (6g) or 2-aminoanthracene (6h). it has been shown that introduction of hydrophobic substituents into glycopeptide antibiotics enhances their activity against glycopeptide-resistant bacteria (cooper et al., 1996; printsevskaya et al., 2002; pace and yang, 2006) , but, unfortunately, these products have lower water-solubility. to improve solubility, the squaric acid amide ester 5 was reacted (sztaricskai et al., 2007b) with d-glucosamine (6i) or d-galactosamine (6j), resulting in the asymmetric squaric diamides 8i and 8j, respectively. in these products, the carbohydrate moiety is linked to the aglycone in an unusual manner, i.e. through a cyclobutandione moiety, and not directly through one of the hydroxyl groups, as in the parent glycopeptide antibiotics. the structure, reaction yield and physico-chemical data for the aglycones, their squaric acid amide esters and corresponding asymmetric diamides, are summarized in table 1 . the homogeneity of all compounds was checked by tlc and hplc, and the structures were confirmed by mass spectrometry. (2) were first converted into their squaric acid amide esters (4 and 5, respectively), followed by conversion to the asymmetric squaric diamides (7a-g and 8a-j, respectively). see table 1 for the structures of the r1 group, as present in the primary amines 6a-j and the final products 7a-g and 8a-j. (2) into their squaric acid amide esters (4 and 5, respectively), these were converted to the asymmetric squaric diamides (7a-g, derived from aglycovancomycin, and 8a-j, derived from aglycoristocetin). b hplc conditions: instrument: waters 600 with uv230nm detection; column: lichrospher rp-8 (4 mm × 250 mm; 10 m); injection volume: 20 l (corresponding to 2 g compound); solvents: table 2 , several asymmetric squaric diamides derived from aglycoristocetin exerted marked activity against influenza virus, the most potent compounds being the phenylbenzyl derivative 8e [average antiviral ec 50 : 0.4 m; selectivity index (si), defined as the ratio of mcc to ec 50 : 50]; the hexanol deriva-tive 8a (ec 50 : 1 m; si: 14) and the naphthyl derivative 8f (ec 50 : 1.4 m; si: 10). their activity was 2-to 5-fold higher than that of the squaric acid amide ester of aglycoristocetin 5 (ec 50 : 2.4 m; si: 42). an intermediate activity (ec 50 : 5 m) was observed for the triglycyl derivative 8b1 which, surprisingly, was comparably active as unsubstituted aglycoristocetin 2. the 3,4-dihydroxybenzyl derivative 8c and the d-galactosamine derivative 8j were active against two of the three influenza virus strains tested, and the derivatives containing carboxypentyl (8b) and d-glucosamine (8i) substituents had activity against only one virus strain. the compounds carrying 4-aminophenylpiperidine (8d), terphenyl (8g) and anthracene (8h) substituents were completely inactive. thus, the intrinsic antiinfluenza virus activity of aglycoristocetin is markedly increased by squaric acid amide coupling and addition of a hydrophobic side chain, with the phenylbenzyl group being the optimal substituent. the favorable effect of this side chain appears to depend on different factors, namely: neutral charge (the alcoholic aliphatic derivative 8a is clearly more active than the corresponding carboxypentyl compound 8b) and steric bulkiness (8e and 8f are active while the more bulky compounds 8g and 8h are not). the aglycoristocetin backbone structure was shown to be critical for inhibition of influenza virus, since no activity was observed for compound 7e, which represents the aglycovancomycin analogue of 8e. aglycoristocetin and aglycovancomycin both contain a central heptapeptide core and nonproteinogenic phenolic amino acids, i.e. ␤-hydroxytyrosine (c and e units), 4-hydroxyphenylglycine (b and d units) and 3,5-dihydroxyphenylglycine (a unit) (fig. 1) . the main structural differences between both glycopeptide aglycones are as follows (fig. 1) : (i) whereas aglycovancomycin has five aromatic rings (a-e) and two known amino acids (l-aspartic acid and n-methyl-d-leucine), aglycoristocetin has seven aromatic moitable 3 cytotoxicity of selected aglycoristocetin derivatives in human and animal cell lines. a . cytotoxic concentration ≥100 >100 100 >100 a human embryonic lung (hel) fibroblasts; human cervix epithelial (hela); african green monkey kidney (vero); and crandell feline kidney (crfk) cells; nd: not done. b the cytotoxic concentration was defined as the minimum cytotoxic concentration (mcc) or 50% cytotoxic concentration (cc50); cf. legend to table 2. eties (a-g); (ii) the additional f and g rings of aglycoristocetin are interconnected via a diphenylether linkage (constituting the ristomycinic acid moiety); (iii) aglycoristocetin lacks the chloro substituents on the c and e rings, present in aglycovancomycin; (iv) the c-terminal carboxyl function is free in aglycovancomycin, but contains a methyl ester in aglycoristocetin and (v) aglycoristocetin has a primary amine function, whereas aglycovancomycin contains a secondary amine group (crowley et al., 2004) . at present, we cannot speculate on which of these structural components explain the antiviral specificity of the aglycoristocetin derivatives. our basic test panel of influenza viruses contained one chimeric virus (a/x-31; h3n2 subtype), one a/h3n2 virus and one b virus strain (table 2) . when the lead compound 8e was further evaluated for activity against a/h1n1 (strain a/puerto rico/8/34), its antiviral ec 50 value was 0.12 ± 0.04 m, which is in the same range as that for the a/h3n2 and b strains. determination of its cytostatic activity in mdck cells, using a cell counting assay, revealed an ic 50 value of 67 ± 19 m. thus, 8e emerged as a potent inhibitor of influenza virus replication, with broad activity against different (sub)types and favorable selectivity. the glycopeptide compounds were evaluated for activity against other viruses besides influenza virus. none of the following rna viruses was found to be significantly inhibited by any of these glycopeptides, as evaluated by cpe or plaque reduction assay: feline coronavirus [examined in crandell-rees feline kidney cells]; vesicular stomatitis virus, coxsackie b4 virus and respiratory syncytial virus [performed in human epithelial hela cells]; parainfluenza-3 virus, reovirus-1, sindbis virus and punta toro virus [tested in african green monkey vero cells]. in human embryonic lung fibroblast cells infected with various dna viruses, the only glycopeptide displaying some activity was compound 8e, with antiviral ec 50 values of 10 m (herpes simplex virus type 1; wild-type or thymidine kinase-deficient); 6 m (herpes simplex virus type 2); >100 m (cytomegalovirus) and 12 m (vaccinia virus), and a selectivity index of 10-17. of note, 8e was found to be highly active against varicella-zoster virus (vzv; wild-type or thymidine kinasedeficient), with an antiviral ec 50 value of 0.55 m and a selectivity index of 180. the diverse antiviral assays performed in human or animal cell lines permitted to obtain a more detailed insight into the cytotoxicity of the aglycoristocetin derivatives selected from the influenza virus experiments (table 3 ). all compounds were either not or minimally cytotoxic at a concentration of 70-100 m. the cytotoxicity values for the lead compound 8e were in the same range as previously obtained in mdck cells. some glycopeptides in this study were previously evaluated for antibacterial activity (sztaricskai et al., 2006) , with 8e emerging as a highly active compound. our present anti-influenza virus data thus agree with the view that hydrophobic substitution has a positive impact on the pharmacological (i.e. antibacterial and antiviral) activities of glycopeptide compounds (printsevskaya et al., 2002; balzarini et al., 2003 balzarini et al., , 2006 . with regard to the antiviral mode of action, time-of-addition studies suggested that 8e blocks the viral entry process, since optimal anti-influenza virus activity was obtained when the compound was added to mdck cells 30 min prior to or simultaneously with virus infection. a detailed analysis is currently ongoing to determine the effect of 8e on the virus-receptor interaction (i.e. binding of the viral ha to the sialic acid terminus of cell surface glycans), endocytosis or membrane fusion (i.e. fusion of the viral envelope with the endosomal membrane). whatever the precise mode of action, the subtype-independent activity of 8e provides strong support that the interaction site of 8e is highly conserved amongst human influenza virus strains. this is consistent with our observation that influenza virus fully retained its sensitivity to 8e after eleven sequential virus passages in mdck cells in the presence of 8e (at concentrations up to 25 m). within human influenza virus ha sequences, only few residues are fully conserved, in particular in the receptor-binding site and fusion peptide (skehel and wiley, 2000) . the aglycoristocetin compounds described here and represented by the lead compound 8e are not the first glycopeptides reported to have activity against influenza virus. in 1993, naruse et al. reported on the isolation, characterization and anti-influenza virus activity of two kistamicin antibiotics (naruse et al., 1993) . similarly to our compounds, both kistamicins showed strong activity against influenza virus and low activity against herpes simplex virus type 1. the kistamicins were reported to be inactive in hiv syncytium assays (naruse et al., 1993) . the observation that the anti-influenza virus activity of the kistamicins was higher when a lipophilic substituent was present at the terminal amine function, is reminiscent of our findings with the aglycoristocetin derivatives. in conclusion, the broad and robust anti-influenza virus activity of the aglycoristocetin derivatives described in this study creates a new avenue for the development of anti-influenza virus agents with a novel mode of action. the relatively narrow structure-activity relationship points to a highly specific interaction with the antiviral target, which is probably related to interaction of the influenza virus hemagglutinin with its cellular receptor. further studies to unravel the structure-activity relationship and precise mode of action are underway in our laboratories. antiretroviral activity of semisynthetic derivatives of glycopeptide antibiotics inhibition of feline (fipv) and human (sars) coronavirus by semisynthetic derivatives of glycopeptide antibiotics glycan topology determines human adaptation of avian h5n1 virus hemagglutinin reductive alkylation of glycopeptide antibiotics: synthesis and antibacterial activity total synthesis of the ristocetin aglycon antiviral agents active against influenza a viruses an approach to the identification of potent inhibitors of influenza virus fusion using parallel synthesis methodology emergence of resistance to oseltamivir among influenza a (h1n1) viruses in europe medical management of influenza infection new antiviral antibiotics, kistamicins a and b. i. taxonomy, production, isolation, physico-chemical properties and biological activities chemistry, biology, and medicine of the glycopeptide antibiotics evolving complexities of influenza virus and its receptors glycopeptides: update on an old successful antibiotic class inhibition of influenza a virus replication by compounds interfering with the fusogenic function of the viral hemagglutinin polycyclic peptide and glycopeptide antibiotics and their derivatives as inhibitors of hiv entry synthesis and mode of action of hydrophobic derivatives of the glycopeptide antibiotic eremomycin and des-(n-methyl-d-leucyl)eremomycin against glycopeptide-sensitive andresistant bacteria structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion who rapid advice guideline panel on avian influenza who rapid advice guidelines for pharmacological management of sporadic human infection with avian influenza a (h5n1) virus receptor binding and membrane fusion in virus entry: the influenza hemagglutinin a new series of glycopeptide antibiotics incorporating a squaric acid moiety. synthesis, structural and antibacterial studies application of squaric acid esters in aminodeoxy sugar chemistry squaric acid diethyl ester: a new coupling reagent for the formation of drug biopolymer conjugates synthesis of squaric acid ester amides and diamides fatal oseltamivir-resistant influenza virus infection a new and improved method for deglycosidation of glycopeptide antibiotics exemplified with vancomycin, ristocetin, and ramoplanin development of anti-influenza virus drugs. i. improvement of oral absorption and in vivo anti-influenza activity of stachyflin and its derivatives this study was supported by grants from the fonds voor wetenschappelijk onderzoek vlaanderen (fwo no. 9.0188.07), the international consortium for anti-virals (icav) and the hungarian national scientific research foundation (no. otka t 46744). we thank leentje persoons, frieda de meyer and vicky broeckx for dedicated assistance, and dr. sándor kéki (department of applied chemistry, university of debrecen) for recording the mass spectra. we thank dr. t. cihlar (gilead sciences, usa) for the generous gift of oseltamivir carboxylate. key: cord-020789-slsfhrkx authors: kleines, michael title: virale atemwegserkrankungen – influenza, rsv und neue viren date: 2017-10-27 journal: nan doi: 10.1055/s-0043-114856 sha: doc_id: 20789 cord_uid: slsfhrkx nan atemwegsinfektionen sind häufig und werden meist durch viren ausgelöst. bei den meisten fällen ist der obere respirationstrakt betroffen, aber bei bis zu einem drittel der fälle werden auch erkrankungen des unteren respirationstrakts beobachtet. dabei kann es u. a. zur bronchitis, zu exazerbationen chronisch obstruktiver atemwegserkrankungen oder zu ambulant erworbenen pneumonien kommen. die hauptlast der respirationstraktinfektionen tragen kinder. kinder machen etwa 5-6 akute atemwegserkrankungen im jahr durch, die mit einer obstruktiven klinik einhergehen. es gibt hinweise, dass genetische faktoren des wirts symptomatische infektionen mit respirationstraktpathogenen begünstigen, z. b. ein einzelner nukleotidaustausch im interleukin-4-gen [1] . die große mehrheit der atemwegsinfektionen wird in mitteleuropa von viren ausgelöst. trotzdem werden viele patienten mit antibiotika behandelt. virusinfektionen des respirationstrakts verändern die zusammensetzung des bakterioms des oberen teils und begünstigen die bakterielle kolonisierung des unteren respirationstrakts. beides erleichtert die entwicklung einer pneumonie im rahmen einer bakteriellen superinfektion [2] . infektionen mit viralen respirationstraktpathogenen können auch erkrankungen beeinflussen, die sich außerhalb des respirationstrakts manifestieren. so wurde eine assoziation von infektionen des oberen respirationstrakts mit schüben der multiplen sklerose postuliert [3] . für andere, bereits bekannte viren wurde ein größeres pathogenes potenzial festgestellt (rhinoviren, enteroviren). zur orientierung über die jeweils aktuell in deutschland zirkulierenden respiratorischen viren und deren typische saisonalität steht seit einigen jahren der internetauftritt des netzwerks für respiratorische viren zur verfügung (http://rvdev.medical-dpc.com/ inhalte/start-viren.html; ▶ abb. 1). früher bewährte verfahren (z. b. virusanzucht oder antikörpernachweis) sind nicht mehr methode der wahl, weil sie zeitaufwendig und nicht für alle erreger im routinelabor verfügbar sind (virusanzucht) oder weil sensitivität und spezifität für den nachweis akuter infektionen nicht ausreichen (antikörpernachweis). heutzutage sind virusdirektnachweise durch nukleinsäureamplifikationsverfahren, z. b. polymerasekettenreaktion (pcr) oder isothermale amplifikationsverfahren, die diagnostischen methoden der wahl für den nachweis respiratorischer viren. die bedeutung der entsprechenden viren ließ sich nur durch die anwendung moderner molekularer methoden erkennen, und sie werden auch am besten durch molekulare verfahren diagnostisch erfasst. in den letzten jahren hat es eine weiterentwicklung der verfügbaren technologien (ausgehend von der für ein einzelnes virus spezifischen standard-pcr mit einem zeitbedarf von 2-3 h) in 2 richtungen gegeben: zum einen sind nun multiplexverfahren verfügbar, die organsystemorientiert alle relevanten erreger für ein definiertes krankheitsbild in einem reaktionsansatz nachweisen können, z. b. für bis zu 19 verschiedene respiratorische viren. zum anderen sind point-of-care-systeme entwickelt worden, die in wenig störanfälligen geschlossenen systemen den nachweis von viren in sehr kurzer zeit ermöglichen und geeignet sind für nicht speziell in molekularen verfahren geschultes personal. der nachweis von influenzaviren durch nukleinsäureamplifikation ist mittlerweile in einer reaktionszeit von 15 min möglich. die bedeutung, die next-generation-sequencing-verfahren für das routinelabor gewinnen werden, lässt sich noch nicht abschließend beurteilen [4, 5] . parallel zur entdeckung weiterer viraler respirationstraktpathogene hat es auch fortschritte im bereich der entwicklung antiviraler wirkstoffe sowie impfstoffen gegeben. davon hat die möglichkeit zum management von influenzavirusinfektionen am meisten profitiert. aber auch für das management von rsv-infektionen und infektio-nen mit anderen respiratorischen viren deuten sich substanzielle fortschritte an. so sind mittlerweile 16 impfstoffkandidaten für rsv in klinischer entwicklung. zusätzlich sind vielversprechende spezifisch antiviral wirkende substanzen in der entwicklung, u. a. monoklonale antikörper [6, 7] . biologie virustypen influenzavirusinfektionen lassen sich weltweit nachweisen. sie werden ausgelöst durch den influenza-a-, influenza-b-und influenza-c-virus aus der familie der orthomyxoviren. die genannten virustypen werden aufgrund der sequenzinformation des viralen ribonukleoproteins unterschieden. influenza-c-viren sind überwiegend humanspezifisch, wurden aber auch aus schweinen isoliert [8] . sie haben im vergleich zu influenza-a-und influenza-b-viren ein reduziertes pathogenes potenzial. auch influenza-b-viren lassen sich überwiegend in menschen nachweisen, aber auch in seehunden und pferden [8] . influenza-a-viren dominieren in verschiedenen wasservogelarten, können aber auch säugetierspezies infizieren. dazu gehören neben dem menschen u. a. pferde, schweine und seehunde. influenza-a-viren teilt man anhand der sequenz ihrer oberflächenantigene hämagglutinin (h) und neuraminidase (n) in subtypen (z. b. h1n1) ein: man unterscheidet 18 h-varianten und 11 n-varianten [9] . die meisten der denkbaren kombinationsmöglichkeiten von h und n wurden in der natur bereits beobachtet. menschliche infektionen wurden in der vergangenheit überwiegend durch die subtypen h1n1, h2n2 und h3n2 ausgelöst. es ließen sich aber auch humane infektionen mit h5, h6, h7, h9 und h10 beobachten (▶ tab. 1). merke sowohl antigenic drift als auch antigenic shift führen zu neuen influenzavirusvarianten, die den durch den wirtsorganismus aufgebauten immunschutz unterlaufen und so zu neuen ausbrüchen führen können. influenzavirusinfektionen treten saisonal in der kalten jahreszeit auf, sodass es auf jeder erdhalbkugel alljährlich eine eigene grippewelle gibt. im rahmen dieser grippewellen werden schätzungsweise 5-20 % der bevölkerung im ausbruchsgebiet durch influenzaviren infiziert. auch außerhalb der klassischen influenzasaison werden influenzavirusinfektionen labortechnisch nachgewiesen. diese können u. u. zu örtlich und zeitlich limitierten kleinstausbrüchen führen [10] . grundlage der alljährlichen grippewelle ist die antigenic drift, durch die virusvarianten immer wieder auf eine durch vorjahresinfektionen nicht optimal geschützte bevölkerung treffen. influenza-b-viren sindje nach saisonfür 1-60 % aller influenzavirusinfektionen verantwortlich. meist schwankt der anteil der influenza-b-viren zwischen 15 % und 35 %. der anteil der influenza-b-virus-infektionen steigt zum ende der influenzasaison an. seit mehr als 20 jahren zirkulieren 2 durch antigenic drift auseinander hervorgegangene influenza-b-virus-linien. jeweils einer davon dominiert in einer saison stark. für die beiden influenza-b-virus-linien besteht keine kreuzprotektion [11] . häufig sind superinfektionen durch bakterien oder pilze, die 5-10 tage nach der influenzavirusinfektion auftreten [10] . eine wichtige erkrankung aus der gruppe der grippalen infekte des unteren respirationstrakts ist die bronchitis. sie ist die zweithäufigste grundlage für das ausstellen von arbeitsunfähigkeitsbescheinigungen. die ursache für bronchitis ist bei patienten ohne schwere grunderkrankungen häufig nicht bakteriell. trotzdem werden oft antibiotika verschrieben. eine auswertung randomisierter studien ergab nur marginale vorteile (eine um einen halben tag verkürzte hustendauer, aber keine signifikante verkürzung der krankheitsdauer) bei mit antibiotika behandelten patienten gegenüber placebogruppen [12] . es konnte abgeleitet werden, dass in deutschland influenzaviren während der influenzasaison für bis zu 25 % der akuten bronchitisfälle verantwortlich sind [10] . auch andere respiratorische viren spielen hier eine wichtige rolle. aufgrund des hohen änderungspotenzials von influenzaviren durch antigenic drift und antigenic shift ist das auftreten neuer humanpathogener influenzaviren ein regelmäßiges phänomen. in der regel handelt es sich nur um geringe veränderungen durch punktmutationen von saison zu saison. aber auch diese haben medizinisch relevante konsequenzen, da eine jährliche überprüfung und ggf. anpassung der impfstoffzusammensetzung nötig ist. die who empfiehlt, den etablierten trivalenten durch einen quadrivalenten impfstoff zu ersetzen. grund ist das neuauftreten der zweiten, nicht kreuzprotektiven linie des influenza-b-virus durch die mangelnde vorhersehbarkeit des in der nächsten saison dominierenden stamms. die saison 2015/2016 hat wieder die notwendigkeit des quadrivalenten impfstoffs unterstrichen, da auch in dieser saison im impfstoff keine schützende komponente gegen das influenza-b-virus vorhanden war. möglicherweise ist dies die grundlage für die beobachtete hohe zahl an influenza-b-virus-infektionen. influenza-a-viren haben aufgrund des hier möglichen antigenic shift ein noch viel größeres änderungspotenzial. seit 1997 (h5n1, ▶ tab. 1) kommt es in kurzer folge immer wieder zum auftreten von influenza-a-virus-subtypen, die den speziessprung z. b. vom vogel in den menschen schaffen und dort zu infektionen mit hoher letalität führen können. letzter wichtiger vertreter dieser reihe, die von subtypen mit den hämagglutininkomponenten h5, h7 und h9 dominiert wird, ist ein influenza-a-virus vom subtyp h7n9, das 2013 in china nachgewiesen wurde und eine größere zahl humaner infektionen verursacht hat (▶ tab. 1). dieses virus hat eine letalität von bis zu 39 %. mehr als 60 % der todesfälle hatten eine grunderkrankung. nach 2010 ist es zusätzlich zu vereinzelten humanen fällen von h5n6, h6n1 und h10n8 gekommen, sowie einer anzahl von humanen infektionen mit h3n2v, einem schweineinfluenzavirus, das sehr verschieden vom humanen h3n2-subtyp ist [13] . bisher hat keiner der im vorhergehenden abschnitt genannten virusstämme das potenzial entwickelt, sich effizient von mensch zu mensch auszubreiten. aus diesem grund ist es bisher noch nicht zu einer pandemie mit humanen infektionen mit einem dieser viren gekommen. bei fledermäusen wurden in den letzten jahren die influenza-a-virus-subtypen h17n10 und h18n11 neu entdeckt. zurzeit gibt es keinen hinweis, dass menschliche infektionen mit diesen subtypen möglich sind. 2009 kam es zu einer humanen influenzapandemie mit dem varianten influenza-a-virus-subtyp h1n1 (schweinegrippe). in deutschland wurden mehr als 226 000 fälle gemeldet. morbidität und letalität waren geringer als in den jahren, die von den bekannten saisonalen stämmen dominiert waren. zu letalen verläufen kam es in deutschland weit überwiegend bei patienten mit grunderkrankungen. auffällig war allerdings die weltweit hohe gefährdung von kleinstkindern und schwangeren. nur in japan gab es keine signifikante erhöhung der letalität bei schwangeren. hier wurde eine durchimpfungsrate der schwangeren von 67 % erreicht. 95 % der infizierten schwangeren wurden mit oseltamivir behandelt, 88 % davon innerhalb von 2 tagen nach symptombeginn. als ursache der besonders guten epidemiologischen situation bei schwangeren während der pandemie 2009 in japan ist das effiziente management von prävention und therapie der influenzavirusinfektion anzunehmen [14] . während der influenzasaison lässt sich eine influenza i. d. r. anhand von klinischen symptomen diagnostizieren. auf eine labordiagnostische abklärung kann man im ambulanten bereich dann verzichten. bei sporadischen fällen oder zu beginn der influenzasaison oder bei schweren verläufen sollte die labordiagnostik auf jeden fall erfolgen. gute gründe für die durchführung der diagnostik sind ebenso die möglichkeit zur durchführung von kohortierungsmaßnahmen oder die erfassung der infektionsepidemiologie (wirksamkeit der impfung, zuordnung von krankheitslast zu virustypen und -subtypen). goldstandard für die diagnostik von influenzavirusinfektionen war lange zeit die virusisolation aus patientenmaterial durch anzucht auf geeigneten zellkulturen. aufgrund der heute zur verfügung stehenden schnelleren, sensitiveren und automatisierbaren verfahren ist die virusanzucht in der täglichen routine aber in den hintergrund getreten. serologische antikörpernachweisverfahren sind ebenfalls seit langer zeit verfügbar und werden nach wie vor häufig vom klinisch tätigen arzt angefordert. aufgrund von eingeschränkter sensitivität und spezifität der antikörpernachweisverfahren ist der positive vorhersagewert positiver antikörpernachweise für eine frische influenzavirusinfektion gering. somit ist der antikörpernachweis im wesentlichen für epidemiologische fragestellungen oder für infektionsnachweise in einer späten erkrankungsphase von bedeutung, wenn direktnachweise nicht mehr zum ziel führen. in der frühen phase der influenzavirusinfektion ist der virusdirektnachweis heute die methode der wahl. zur verfügung stehen: in der frühphase der impfstoffentwicklung wurden trivalente (2 influenza-a-virus-stämme, 1 influenza-b-virus-stamm) ganzvirus-impfstoffe der 1. generation zugelassen, die eine optimale immunantwort auslösten, aber ein großes nebenwirkungsspektrum zeigten. die spaltvirus-impfstoffe der 2. generation, die auch heute noch verfügbar sind, zeigen nur noch ein mäßiges nebenwirkungsspektrum bei suboptimaler immunantwort. die subunit-impfstoffe der 3. generation zeigen ein geringes nebenwirkungsspektrum bei einer zur 2. generation der impfstoffe vergleichbaren immunantwort bzw. schutzwirkung. die adjuvantierten impfstoffe der 4. generation verbinden starke immunantwort mit geringem nebenwirkungsspektrum. in der vergangenheit wurden auch impfstoffe gegen neue virusvarianten zugelassen, z. b. den influenza-a-virus-subtyp h5n1 (vogelgrippe), sowie pandemieimpfstoffe, die eine schnelle adaptation eines basisimpfstoffs an einen pandemischen influenza-a-virus erlauben sollen. bocaviren werden durch nukleinsäureamplifikation nachgewiesen. andere verfahren spielen in der routinediagnostik heute keine rolle (▶ tab. 4). die viren sind sowohl durch singuläre pcrs erfassbar als auch in gängigen multiplex-pcrs für respiratorische viren enthalten. eine frische bocavirusinfektion lässt sich nur auf basis einer mittleren bis hohen viruslast (> 10 000 kopien/ml) diagnostizieren. die diagnose wird durch die monatelange persistenz von bocavirus-dna erschwert. im fall von persistenz ist die viruslast i. d. r. niedrig. es gibt keine arznei-oder impfstoffe, spezifische therapeutika oder vakzine sind entweder nicht zugelassen oder in keinem fortgeschrittenen entwicklungsstadium [27] . enteroviren gehören zur gattung enterovirus in der familie der picornaviren. die gattung umfasst 4 enterovirusspezies (enterovirus a-d) und 3 rhinovirusspezies (rhinovirus a-c). die unterschiedlichen enterovirustypen können ursache eines großen erkrankungsspektrums sein, das von exanthematischen erkrankungen und fieber über myokarditis/perikarditis und lähmungen bis hin zur enzephalitis reichen kann. enteroviren sind die häufigste ursache viraler meningitiden. einige enterovirustypen werden nur im respirationstrakt nachgewiesen. diese gehören überwiegend zu den enterovirusspezies c und d. diese virustypen sind weltweit verbreitet und können symptome von der banalen infektion des oberen respirationstrakts bis hin zur pneumonie auslösen [28] . schwere verlaufsformen treten überwiegend bei kindern und patienten mit grunderkrankungen aus. aufgrund der großen zahl an respiratorischen enterovirustypen ist die seroprävalenz hoch. es gibt keine spezifischen arznei-oder impfstoffe, spezifische therapeutika oder vakzine sind für respiratorische enteroviren nicht zugelassen. der wirkstoff pleconaril ist in vitro gut wirksam, führte in klinischen studien aber häufig zur aggravierung der symptome und hat daher keine zulassung zur therapie von patienten mit respiratorischen infektionen erhalten. der wirkstoff wurde in einzelfällen erfolgreich bei zns-infektionen durch enteroviren eingesetzt [10] . die neue systematische einordnung der humanen rhinoviren fasst sie zu 3 spezies (rhinovirus a-c) zusammen und ordnet sie der gattung enterovirus in der familie der picornaviren zu. man kann über 100 rhinovirustypen unterscheiden. bis 2006 wurden durch molekularbiologische untersuchungen mehr als 50 nicht anzüchtbare rhinoviren neu entdeckt. diese fasste man in der dafür neu geschaffenen spezies rhinovirus c zusammen. merke bei bis zu 20 % der kinder ist eine apnoeepisode das erste symptom einer rsv-infektion [32] . die infektion der unteren atemwege durch rsv bei kleinkindern ist ein signifikanter risikofaktor für giemen und asthma in der ersten lebensdekade, aber möglicherweise auch in der adoleszenz und bei erwachsenen. die lungenfunktion kann bei diesen kindern langfristig gestört sein und zu eingeschränkter lebensqualität und überdurchschnittlichem arztkontakt führen [33] . goldstandard für die diagnostik von rsv war lange zeit die virusisolation aus patientenmaterial durch anzucht auf geeigneten zellkulturen. aufgrund der heute zur verfügung stehenden schnelleren, sensitiveren und automatisierbaren verfahren ist die virusanzucht in der täglichen routine aber in den hintergrund getreten. serologische antikörpernachweisverfahren sind ebenfalls seit langer zeit verfügbar und werden nach wie vor häufig vom klinisch tätigen arzt angefordert. aufgrund von eingeschränkter sensitivität und spezifität der antikörpernachweisverfahren ist der positive vorhersagewert positiver antikörpernachweise für eine frische rsv-infektion gering. somit ist der antikörpernachweis im wesentlichen für epidemiologische fragestellungen oder für infektionsnachweise in einer späten erkrankungsphase von bedeutung, wenn direktnachweise nicht mehr zum ziel führen. in der frühen phase der rsv-infektion ist der virusdirektnachweis heute die methode der wahl. es stehen schnelltests (immunchromatografie), antigennachweise durch elisa, antigennachweise durch immunfluoreszenztest (ift) und nukleinsäureamplifikationsverfahren zur verfügung (pcr, isothermale amplifikationsverfahren). die sensitivität der schnelltests liegt bei 75,3 % und die spezifität bei 98,7 % im vergleich zur pcr-diagnostik [34] . die molekulare diagnostik ist also deutlich überlegen. mittlerweile sind auch molekulare verfahren verfügbar, die bezüglich der geschwindigkeit mit den schnelltests konkurrieren können (isothermale amplifikation). therapie der wahl ist heute die symptomatische therapie mit angemessener flüssigkeits-und sauerstoffversorgung. eine therapie mit ribavirin kann erwogen werden, ist aber aufgrund der schwierigkeiten und gefahren bei der administration sowie der im normalfall schon absinkenden viruslast bei symptombeginn nur für immunkompromittierte patienten empfohlen. in entwicklung befinden sich die wirkstoffe gs-5806 (presatovir) und als-008176, die beide phase-ii-studien erfolgreich abgeschlossen haben. bei gs-5806 handelt es sich um einen kleinen, oral administrierbaren fusionsinhibitor, der die viruslast und die mukusproduktion reduziert sowie die gesamtsymptomatik verbessert. als-008176 ist ein cytosin-nukleosid-analogon, das als prodrug oral administrierbar ist. der wirkstoff reduziert die viruslast [32] . weitere wirkstoffkandidaten befinden sich in weniger weit fortgeschrittenen entwicklungsstadien. zurzeit steht nur ein einziger wirkstoff zur immunprophylaxe zur verfügung. es handelt sich dabei um den gegen das f-protein von rsv gerichteten monoklonalen antikörper palivizumab. der wirkstoff wird während der rsv-saison monatlich administriert. er führt zu einer reduktion der hospitalisierungen um 50 %. aufgrund der hohen kosten wird palivizumab nur bei definierten risikopatienten wie ehemaligen frühgeborenen unter 32 ssw oder säuglingen mit herzfehlern eingesetzt. die verwendung aktueller epidemiologischer daten zu rsv, wie sie das respvir-netzwerk zu verfügung stellt, könnte die rsv-saison präziser definieren und so zu einem ökonomischeren einsatz von palivizumab führen. alx-0171, ein nanoantikörper (monomere variable domäne eines klassischen antikörpers), reduziert viruslast und virusreplikation. der wirkstoff hat gerade für kinder unter 2 jahren eine phase-i/ii a-studie erfolgreich durchlaufen, eine phase-ii-studie beginnt in diesem jahr. weiter fortgeschritten ist die klinische testung von regn2222, einem humanen monoklonalen igg-antikörper, der gegen das f-protein gerichtet ist. hier wird im jahr 2017 eine phase-ii-studie bei frühgeborenen abgeschlossen [32] . zurzeit ist kein aktiver impfstoff gegen rsv verfügbar. dies ist auf die schwierigkeit zurückzuführen, gleichzeitig eine hinreichend lang protektive immunantwort zu induzieren, aber ved (vaccine induced disease) zu vermeiden. ved wurde in den 1960er-jahren beim ersten versuch der impfstoffentwicklung mit einem formalininaktivierten rsv-stamm beobachtet. welche aussage zu bocaviren ist richtig? a auslöser der meisten respiratorischen bocavirusinfektionen ist die spezies hbov3. b bei der diagnostik von bocavirusinfektionen steht die serologie im vordergrund. c ein impfstoff gegen bocaviren befindet sich in einem fortgeschrittenen entwicklungsstadium. d bocaviren verursachen ausschließlich banale infektionen des oberen respirationstrakts. e nur eine viruslast > 10 000 kopien/ml zeigt eine frische bocavirusinfektion an. welche aussage zu entero-und rhinoviren ist falsch? a es wurden mehr als 100 rhinovirustypen identifiziert. b entero-und rhinoviren gehören beide zur familie der picornaviren. c enterovirus d68 ruft leichte bis schwere respirationstrakterkrankungen hervor. d entero-und rhinoviren lassen sich ausschließlich durch serologische verfahren nachweisen. e es gibt keine antiviralen impfstoffe gegen rhino-und enteroviren. entwicklung befinden sich mehrere lebend attenuierte impfstoffkandidaten in phase-i-und phase-ii-studien, mehrere vektorbasierte impfstoffkandidaten in phase-iund phase-ii-studien und proteinbasierende impfstoffkandidaten in phase-i-bis phase-iii-studien der autor gibt an, dass kein interessenkonflikt vorliegt studium der biologie in aachen. 1993-1997 promotion am max-planck-institut für züchtungsforschung in köln. 1997-2010 institut für medizinische mikrobiologie in aachen leiter der virologie am labordiagnostischen zentrum, aachen. korrespondenzadresse pd dr. michael kleines bereichsleiter virologie/serologie labordiagnostisches zentrum universitätsklinikum aachen pauwelsstr. 30 52074 aachen mkleines@ukaachen.de wissenschaftlich verantwortlich gemäß zertifizierungsbestimmungen wissenschaftlich verantwortlich gemäß zertifizierungsbestimmungen für diesen beitrag ist dr erstveröffentlichung dieser beitrag ist eine aktualisierte version des artikels: kleines m. virale atemwegserkrankungen -neue viren the role of host genetic factors in respiratory tract infectious diseases: systematic review, meta-analyses and field synopsis the role of respiratory viruses in the etiology of bacterial pneumonia factors associated with onset, relapses or progression in multiple sclerosis: a systematic review molecular diagnosis of respiratory virus infections molecular diagnosis of respiratory viruses advances in rsv vaccine research and development -a global agenda new options in the treatment of respiratory syncytial virus disease zoonoses and communicable diseases common to man and animals. 3. aufl. washington dc: pan american health organization new world bats harbor diverse influenza a viruses die rolle von viren bei tiefen atemwegsinfektionen des erwachsenen -teil 1: erreger, pathogenese und diagnostik live attenuated intranasal influenza vaccine antibiotics for acute bronchitis overview of the 3rd isirv-antiviral group conference -advances in clinical management review of the pandemic (h1n1) 2009 among pregnant japanese women emerging respiratory tract viral infections synergistic combinations of favipiravir and oseltamivir against wild-type pandemic and oseltamivir-resistant influenza a virus infections in mice influenza vaccination coverage rates in five european countries during season 2006/07 and trends over six consecutive seasons new approaches for immunization and therapy against human metapneumovirus the role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience treatment of severe human metapneumovirus (hmpv) pneumonia in an immunocompromised child with oral ribavirin and ivig catalytic function and substrate specificity of the papain-like protease domain of nsp3 from the middle east respiratory syndrome coronavirus the emergence of the middle east respiratory syndrome coronavirus middle east respiratory syndrome coronavirus: transmission, virology and therapeutic targeting to aid in outbreak control coronaviruses: an overview of their replication and pathogenesis current advancements and potential strategies in the development of mers-cov vaccines human bocaviruses: possible etiologic role in respiratory infection a novel human enterovirus c (ev-c118) identified in two children hospitalised because of acute otitis media and community-acquired pneumonia in israel a novel outbreak enterovirus d68 strain associated with acute flaccid myelitis cases in the usa (2012-14): a retrospective cohort study human rhinoviruses rhinoviruses and respiratory enteroviruses: not as simple as abc rsv infection: state of the art ongoing developments in rsv prophylaxis: a clinicianʼs analysis the burden and longterm respiratory morbidity associated with respiratory syncytial virus infection in early childhood rapid tests for influenza, respiratory syncytial virus, and other respiratory viruses: a systematic review and meta-analysis key: cord-013073-siy7dvlo authors: pfäfflin, albrecht title: influenza virus-flow from insects to humans as causative for influenza seasonality date: 2020-10-09 journal: biol direct doi: 10.1186/s13062-020-00272-5 sha: doc_id: 13073 cord_uid: siy7dvlo virus biomass outweighs human biomass, and insects biomass outweighs human biomass. insects are regularly habited by viruses as well as humans, humans are further inhabited via insects. a model of viral flow is described and specified to explain influenza virus seasonality, which, in temperate climate, usually evolves when insects have mostly disappeared. with this hypothesis a coherent description of regular seasonal influenza and other seasonal respiratory virus infections in temperate climates is possible. the incidence of influenza under different circumstances e.g. temperature, humidity, or tropical conditions and different aspects like synchronicity of infections or in respect to evolutionary conditions do sustain this hypothesis if the behaviour of insects is considered. seasonality of influenza is not understood. the complex transmission behaviour of influenza is enigmatic [1] . the clock-like consistency of the winter incidence peaks of influenza virus in temperate climatic regions represents a strong example of seasonality in infectious disease [2] . approaching this phenomenon via biomass of involved organisms, a flow may explain this phenomena. influenza is considered a zoonosis. the reservoir of influenza virus are aquatic birds [3] which have usually enteral infections often without clinical signs. influenza virus crosses species barriers from time to time and persists in the species (e.g. humans, dogs, horses) for a certain time in a seasonal manner, and is then eventually lost. crossing species barriers leads eventually to pandemics [4] which are followed by seasonal epidemics. the question and focus of this paper is laid on the consecutive regular epidemics. when influenza virus has reached humans and persists there, it disappears during off-season but re-emerges regularly. the question is, where does the virus persist during the off-season time of more than 6 months? several molecular studies suggesting a lack of influenza virus persistence in the offseason in temperate areas [5, 6] . the reintroduction of influenza virus is thought to involve the importation from a locality either in the alternate hemisphere where the influenza season is current, or from the tropics where low levels of virus may circulate year-round, particularly the densely populated regions of east and south-east asia [7, 8] . however, in this paper, an alternative view is proposed. in this model, insects serve as a buffer for influenza virus. if insects are intact, they enclose virus particles preventing these particles to reach humans. if insects deteriorate, virus particles are set free, and humans are infected. so, according to season and insect biomass, a flow of virus particles is ping-ponged between humans and insects. seasonality of influenza is explainable using this insect-compartment model in temperate climate conditions. if this scenario is truth, it may be speculated, that virus is retransmitted from humans to insects (where virus eventually proliferates), and the circle would be closed. in this model, insects serve as a connecting transmitter from and to humans to coordinate regular epidemics. they connect birds to humans or other animals and come close to these species. insects serve as a reservoir for viruses and are able to transport different viral species. when the cold season starts, insects may have, because of the flyaway of many birds, eventually an interest to come in proximity to humans. humans provide them warmness and nutrition, otherwise this proximity fosters viral transmission from insects to humans. it is not possible, to proof this hypothesis, but it is possible to describe circumstances which fit to this hypothesis. such circumstances are discussed in the following. biomass composition of the earth [9] is such: half of animals are arthropods. there are 17fold more arthropod biomass compared to humans. there is a 3fold biomass of virus compared to humans. insects are living closer to humans as expected [10] . arthropods and viruses predate humans, and held and hold manifold interactions. viruses predate any life [11] . only because of these mass relations, an interaction of viruses with arthropods seems plausible and is found in numerous examples e.g. zika-virus and aedes albopictus. indeed, insects are able to transmit influenza virus, experimentally proven at least in birds [12] , and, insects e.g. musca indeed act as vectors [13, 14] at least for avian influenza. life of vertebrates does not occur without insects even under current conditions. some centuries ago, the interactions of humans with insects were, compared to now, because of a more simple life style, surely more intense. so, an exchange of insect contaminants is unavoidable. in analogy, transmission of virus from plants to plants takes place mostly via insects [15] . many details of these interactions have been elucidated revealing a high-level of complexity between plant-viruses and insects. in this context, it is astonishing that the transmission of human influenza virus via insects is yet neglected. an argument for insects as an influenza vector are their presence during off-season of influenza and vice versa. isolated insect-free regions without virus do not show seasonality of influenza [16] . human immune response is not obligatory discriminative between influenza and insect structures indicating eventually that insect and influenza structures are linked [17] . a synergy between insects and influenza may indicate an ancient coupling between these organisms [18] . an ecological niche that is currently underrepresented in global surveillance efforts was postulated, namely the rare influenza subtype h14 was found only after several decades [19] . such an ecological niche in the environment would perfectly fit to insects. these arguments have been at least partly discussed as an insect-compartment model contributory to explain seasonality of influenza [20] . influenza epidemics occur usually in synchronicity [21] . seasons in temperate climates begin when temperatures are permanently low. direct human to human transmission does not cause synchronicity because of a lag-time during the incubation period. synchronicity is to be triggered to happen regularly, therefore natural processes may be responsible for. insects emerge and cease in a synchronized manner. humans may act, in this context, as a dead-end host for influenza according to a source-sink model for the ecology of influenza [22] . synchronicity of influenza may be triggered via environmental conditions. temperature and absolute humidity are revealed to be important for transmission [23] as well as low temperature [24] . cold and dry air facilitates virus stability [25] . however, such observations do not explain why influenza disappears and where it stays in the meantime, but such factors will influence survival of insects as well. global weather conditions are connected to pandemic influenza [26] . associations of global weather conditions to the dynamics of seasonal influenza are found regularly, but the biological mechanism between climate variations and influenza epidemics is dubious [27] . insects might be this biological mechanism. weather conditions imperatively will affect the biomass of insects and migration birds. general weather phenomenon e.g. el nino southern oscillation probably may lead also to an increase or decrease of insect biomass providing a biological reason for observed associations [28] viewed from the viral-flow model. seasonality of influenza in the tropics is also not well understood because accepted influence factors like temperature or absolute humidity show weak or absent associations in tropical countries. epidemics occur there during the rainy season [27] . eventually, insect biomass is different during and out of rainy season fitting to the model proposed here. accepting this approach may imply eventually new proceedings for prevention of influenza e.g. distribution of insects or insect particles to neutralize virus. a kind of therapeutic use of insect particles [29] was already described. here, an viral-flow model contributory to explain seasonality of influenza is applied to elucidate questions and circumstances concerning influenza infections like synchronicity, environmental factors. with this approach, an insect-compartment relevant for the transmission of influenza is not proven, and other transmission routes e.g. direct transmission or transmission via aerosols are active as well, but this approach provides answers to question where influenza virus persists between epidemics, and how it is reintroduced regularly. of note, many other seasonal viruses show an enigmatic seasonal behaviour like parainfluenza, respiratory syncytial, human metapneumovirus and others. they may act in a similar way as described here. the human-centred view on virus transmission does not allow recognizing a viral flow. this human-centred view is the perception that viral infections are rare and viruses proliferate preferentially or exclusively in humans. viruses are invisible and difficult to detect. therefore, the notification of viral-flow is an abstraction and unusual as a concept, but, from an evolutionary point-of-view, at least debatable. influenza seasonality: underlying causes and modeling theories the evolution of epidemic influenza fouchier ra global patterns of influenza a virus in wild birds kash jc influenza virus evolution, host adaptation, and pandemic formation contrasting the epidemiological and evolutionary dynamics of influenza spatial transmission phylogenetic analysis reveals the global migration of seasonal influenza a viruses the global circulation of seasonal influenza a (h3n2) viruses inter-seasonal influenza is characterized by extended virus transmission and persistence the biomass distribution on earth ubiquity and diversity of human-associated demodex mites the ancient virus world and evolution of cells pakpinyo s experimental assessment of houseflies as vectors in avian influenza subtype h5n1 transmission in chickens the potential of house flies to act as a vector of avian influenza subtype h5n1 under experimental conditions kittayapong p detection of h5n1 avian influenza virus from mosquitoes collected in an infected poultry farm in thailand insect vector-mediated transmission of plant viruses influenza epidemics in iceland over 9 decades: changes in timing and synchrony with the united states and europe shifting of immune responsiveness to house dust mite by influenza a infection: genomic insights potentiation of infectivity and pathogenesis of influenza a virus by a house dust mite protease evidence for the circulation and inter-hemispheric movement of the h14 subtype influenza a virus an insect-compartment model as contributory to explain the seasonality of influenza analysis of multi-level spatial data reveals strong synchrony in seasonal influenza epidemics across norway the genomic and epidemiological dynamics of human influenza a virus influenza virus transmission is dependent on relative humidity and temperature global environmental drivers of influenza low temperature and low uv indexes correlated with peaks of influenza virus activity in northern europe during association of influenza epidemics with global climate variability global influenza seasonality: reconciling patterns across temperate and tropical regions the el nino-southern oscillation (enso)-pandemic influenza connection: coincident or causal? house dust mite exposure attenuates influenza a infection in a mouse model of pulmonary allergic inflammation author's contributions ap designed the concept and wrote the paper. the author(s) read and approved the final manuscript. not applicable. availability of data and materials not applicable.ethics approval and consent to participate not applicable. the author transfers the non-exclusive publication rights and he warrants that his contribution is original and that he has full power to make this grant. the author signs for and accepts responsibility for releasing this material on behalf all authors. none.received: 18 june 2020 accepted: 25 september 2020 key: cord-253049-vm46wq1m authors: rößler, steve; ankert, juliane; baier, michael; pletz, mathias w.; hagel, stefan title: influenza-associated in-hospital mortality during the 2017/2018 influenza season: a retrospective multicentre cohort study in central germany date: 2020-09-27 journal: infection doi: 10.1007/s15010-020-01529-x sha: doc_id: 253049 cord_uid: vm46wq1m the aim of this retrospective cohort study at eight hospitals in germany was to specify influenza-associated in-hospital mortality during the 2017/2018 flu season, which was the strongest in germany in the past 30 years. a total of 1560 patients were included in the study. overall, in-hospital mortality was 6.7% (n = 103), in patients treated in the intensive care unit (n = 161) mortality was 22.4%. the proportion of deceased patients per hospital was between 0% and 7.0%. influenza was the immediate cause of death in 82.8% (n = 82) of the decedents. it is estimated that 5-20% of the population contracts influenza in an average flu season [1] . among the 30 most relevant infectious diseases in europe, influenza, with 5.9 infections per 100,000 inhabitants annually, has the highest incidence and the highest mortality (5.89 deaths per 100,000 inhabitants per year) [2] . approximately one-third of the total burden of all infectious diseases, measured in disability-adjusted life years, is attributable to influenza [2] . however, it has been recognized for many years that influenza is underreported on death certificates. it is, therefore, common practice to estimate mortality attributed to influenza with statistical methods [3] . this so-called excess mortality is obtained by subtracting the expected mortality (i.e. background mortality) from the observed mortality during an influenza season. if an increase in mortality is observed which is significantly higher than background mortality, this is attributed to influenza. for example, according to the robert koch institute (rki), an estimated 25,100 people in germany died due to influenza during the 2017/2018 flu season, but only 1674 influenza-associated deaths were officially reported [3] . as in-hospital mortality is not collected as part of rki influenza surveillance, no data are available on this. however, accurate data on the burden of disease are important for making comparisons with other diseases, e.g., coronavirus disease 2019 (covid-19) [4] . therefore, the primary aim of this study was to specify influenza-associated in-hospital mortality during the 2017/2018 influenza season, the strongest influenza season in the past 30 years. in addition, the need for intensive care support was assessed, an important number to describe severity of disease. this retrospective cohort study was performed at eight hospitals with 4976 beds (median 366 (range 80-1735) beds) in central germany (table 1) . six hospitals were small regional hospitals providing basic and standard care, and the two largest hospitals were hospitals providing maximum care. all hospitalized patients with a discharge diagnosis of influenza infection (j.10.-) between december 2017 and april 2018 were included in the analysis. in addition to demographics, length of hospital stay, intensive care unit (icu) stay and hospital discharge data were collected from all patients. in patients who died during steve rößler and juliane ankert contributed equaly. their hospital stay, chart review was performed to document information about comorbidities, the cause of death and therapy. immunosuppression was defined as the presence of congenital or acquired immunosuppression (e.g., chronic therapy with corticosteroids > 10 mg/day, radio/ chemotherapy, transplantation, hiv/aids). to summarize characteristics of deceased patients, we provide absolute and relative frequencies and the median complemented by the first and third quartiles (q1, q3). we applied the χ 2 test or, if indicated, fisher's exact test and the mann-whitney u test to assess differences between deceased patients with icu care and without icu care. ethical approval for the study was provided by the ethics committee of the jena university hospital, with a waiver for informed consent from the patients (5532-05/18). a total of 1560 patients were diagnosed with influenza infection during the study period. in 21 patients, data were incomplete; therefore, 1539 patients were included in the subsequent analysis. table 1 shows an overview of the number of influenza cases per hospital. the median age of the patients was 72 (range 0-102) years, and 769 (50%) patients were male. the average length of hospital stay was 11.4 ± 13.4 days (median 7 days, range 0-130 days). overall, 103 [6.7%, 95% confidence interval (ci) 5.5-8.1%] out of 1539 patients died during their hospital stay. the proportion of deceased patients per hospital was between 0% and 7.0% (table 1 ). in 82.8% (n = 82) of the cases, influenza infection was considered to be the direct cause of death; in 15.2% (n = 15) of the cases, it was an indirect cause of death; and in 2% (n = 2) of the cases, it did not affect patient death. in total, 161 patients (10.3%) were treated in an icu during their hospital stay. the median age of these patients was 72 (range 32-96) years, and 60% (n = 96) were male. detailed information on the course of the disease was obtained from 111 patients receiving intensive medical care. in 44.1% (n = 49) of the patients, influenza infection was the immediate and primary reason for admission to the icu; in 13.5% (n = 15) of patients, infection was acquired in the icu. influenza infection was present in 20.7% (n = 23) of patients, but it had no influence on admission to the icu. in another 21.6% (n = 24) of patients, influenza infection worsened the underlying illness, leading to icu admission. extracorporeal membrane oxygenation was successfully performed in one patient. a total of 36 patients (22.4%, 95% ci 16.1-29.6) died during their stay in the icu. influenza infection was the immediate cause of death in 29 patients (80.6%) and an indirect cause in 7 (19.4%) patients. influenza subtyping was performed in 97 patients in the icu. sixty-seven patients (69.1%) had influenza b infection, and 30 patients (30.9%) had influenza a infection (p < 0.001) ( table 2 ). during the 2017/18 influenza season, approximately 334,000 laboratory-confirmed influenza cases were reported to the rki. approximately 60,000 (17%) patients were reported to be hospitalized. in the present study involving eight hospitals in central germany, influenza-related in-hospital mortality in the examined patient population was 6.7%, and every tenth patient was cared for in an icu during their hospital stay. the in-hospital mortality in this group of patients was significantly higher (22.4%) than that in those who did not require admission to an icu. our observed in-hospital mortality rate of 6.7% was comparable to the in-hospital mortality rate of 5.8% reported by another german university hospital [5] but lower than the in-hospital mortality (8.3%) rate reported by a tertiary hospital in austria [6] . interestingly, the in-hospital mortality observed in this season was significantly lower compared to the in-hospital mortality in the 2014/2015 season (7.0% vs. 10.4%) in study hospital no. 7 [7] . applying our observed in-hospital mortality of 6.7% to the 60,000 hospitalized patients with a laboratoryconfirmed influenza infection reported to the rki would correspond to approximately 4000 deceased hospitalized patients during the 2017/2018 flu season in germany. this in contrast to the 1674 influenza-associated deaths officially reported to the rki, for both, hospitalized and non-hospitalized patients, clearly underlying the notion that influenza-associated deaths are underreported. however, our observed in-hospital mortality does not allow to draw conclusions about the overall influenza-associated mortality and burden of disease. for one thing, the mortality in non-hospitalized patients with influenza most probably differs from hospitalized patients with influenza. on the other hand, the overall number of patients infected with influenza each season is unknown. not every person who truly has influenza will seek medical care, will be tested for influenza, have a positive test, and, therefore, be reported through influenza surveillance [8] . routinely available influenza diagnostic tests also vary in sensitivity. thus, data collected through influenza surveillance and case finding represent only a fraction of persons infected with influenza. for example, a recently published study calculated that in the us, only between 0.08-0.61% and 0.07-0.33% of symptomatic influenza illnesses were laboratory-confirmed in the 2011-2012 and 2012-2013 seasons, respectively [8] . in 1129 (67.4%) influenza-associated deaths reported to the rki, it was stated that the patient died from influenza disease and its consequences [3] . this proportion was lower than that in our study, in which influenza infection was considered to be the immediate cause of death in 82.8% of the deceased patients. this discrepancy could be because, unlike in the current study in which a doctor made a decision regarding whether death was associated with influenza infection based on a medical chart review, the health authorities made the decision solely on the basis of the information available to them. these decisions considered assessments by the supervising doctor or information on the death certificate. however, these types of assessments are problematic because, in contrast to other diseases, influenza is often not recorded on the death certificate as the cause of death, even if influenza had been confirmed by laboratory testing in the course of the disease. there are several limitations in the current study. patients with influenza infection were identified retrospectively on the basis of the diagnosis code at discharge. since it is possible that not every microbiologically confirmed influenza infection was coded at discharge and that microbiological diagnostics were not always performed to ensure the clinically suspected diagnosis, the true number of hospitalized influenza patients could be correspondingly higher than that reported here. in addition, as the assessment of whether the death of a patient was related to influenza infection was made by a study physician, the results may be subject to bias. nevertheless, the results of the present study underline the high burden of disease in hospitalized patients with influenza and allow a comparisons with other diseases, e.g., coronavirus disease 2019 (covid-19). funding open access funding enabled and organized by projekt deal. conflict of interest on behalf of all authors, the corresponding author states that there is no conflict of interest. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. erkrankungen durch saisonale influenzaviren impact of infectious diseases on population health using incidence-based disability-adjusted life years (dalys): results from the burden of communicable diseases in europe study, european union and european economic area countries bericht zur epidemiologie der influenza in deutschland assessment of deaths from covid-19 and from seasonal influenza clinical characteristics of influenza in season 2017/2018 in a german emergency department: a retrospective analysis intrahospital mortality of influenza patients during the 2017-2018 influenza season: report from a tertiary care hospital in austria characteristics and management of patients with influenza in a german hospital during the 2014/2015 influenza season quantifying the annual incidence and underestimation of seasonal influenza: a modelling approach key: cord-318753-ribybqfo authors: kwok, c. s.; aslam, s.; kontopantelis, e.; myint, p. k.; zaman, m. j. s.; buchan, i.; loke, y. k.; mamas, m. a. title: influenza, influenza‐like symptoms and their association with cardiovascular risks: a systematic review and meta‐analysis of observational studies date: 2015-05-04 journal: int j clin pract doi: 10.1111/ijcp.12646 sha: doc_id: 318753 cord_uid: ribybqfo aims: to synthesise the evidence relating influenza and influenza‐like symptoms to the risks of myocardial infarction (mi), heart failure (hf) and stroke. methods: we conducted a systematic review and meta‐analysis of the evidence relating influenza and influenza‐like symptoms to the risks of mi, hf and stroke. we systematically searched all medline and embase entries up to august 2014 for studies of influenza vs. the cardiovascular outcomes above. we conducted random effects meta‐analysis using inverse variance method for pooled odds ratios (or) and evaluated statistical heterogeneity using the i (2) statistic. results: we identified 12 studies with a total of 84,003 participants. the pooled or for risk of mi vs. influenza (serologically confirmed) was 1.27 (95% ci, confidence interval 0.54–2.95), i (2) = 47%, which was significant for the only study that adjusted for confounders (or 5.50, 95% ci 1.31–23.13). the pooled or for risk of mi vs. influenza‐like symptoms was 2.17 (95% ci 1.68–2.80), i (2) = 0%, which was significant for both unadjusted (or 2.23, 95% ci 1.65–3.01, five studies) and adjusted studies (or 2.01, 95% ci 1.24–3.27, two studies). we found one study that evaluated stroke risk, one study in patients with hf, and one that evaluated mortality from mi – all of these studies suggested increased risks of events with influenza‐like symptoms. conclusions: there is an association between influenza‐like illness and cardiovascular events, but the relationship is less clear with serologically diagnosed influenza. we recommend renewed efforts to apply current clinical guidelines and maximise the uptake of annual influenza immunisation among patients with cardiovascular diseases, to decrease their risks of mi and stroke. • we conducted random effects meta-analysis using inverse variance method for pooled odds ratios (or) and evaluated statistical heterogeneity using the i 2 statistic. message for the clinic • there is an apparent association between influenza-like illness and adverse cardiovascular events. during a typical flu season in england and wales, there are around 1.1 million extra consultations for acute respiratory infections, over 3000 excess hospital admissions, and around 12,500 deaths (1) . in the h1n1 influenza pandemic, around 540,000 people in england had symptomatic h1n1 infection, with a case fatality rate of 26 deaths per 100,000 cases (2) . there is some observational evidence that major cardiovascular events are a prominent mechanism in deaths linked to influenza (3, 4) . excess mortality during influenza epidemics in europe and the usa in the early 1900s was indeed attributed to causes other than influenza, such as heart disease (5), a finding replicated in more contemporary studies (6) (7) (8) . stroke also appears to be more common after a respiratory infection (9) (10) (11) . while some studies suggests that influenza may be a precursor to incident cardiovascular events, there remain inconsistencies in the literature. several reviews have evaluated the potential for influenza to trigger cardiovascular events (3, 8, (12) (13) (14) . these articles have summarised cardiovascular manifestations of influenza and described the direct effects of the virus on the myocardium (3, 14) , as well as the potential mechanisms of acute coronary syndrome with infection (13) . while existing studies have shown some evidence that influenza may be associated with cardiovascular disease (cvd) there has been no published metaanalysis of the association. in addition, these studies focused on coronary heart disease, while less is known about stroke and heart failure (hf). in view of the uncertainty regarding the association between influenza and influenza-like symptoms and risk of cvd, we conducted a systematic review and meta-analysis to quantify the risk of myocardial infarction (mi), hf and stroke among patients recorded as having influenza and influenza-like symptoms. we selected studies that evaluated the association between influenza and adverse cardiovascular outcomes. we included all study designs and placed no restriction on the definition of influenza that could be based on recording flu-like symptoms or serologically confirmed cases. the end-points were considered were mi, hf, stroke and cardiovascular death. we searched (via ovid) medline and embase from inception up to the end of august 2014 with no language limitations and using the broad free-text and indexing search terms [(influenza or flu) and ((myocardial infarction or acute coronary syndrome or ischemic heart disease or ischaemic heart disease) or (heart failure or cardiac failure or left ventricular impairment) or (stroke or cerebrovascular disease or cerebrovascular accident) or (cardiac death or cardiovascular death or cardiovascular mortality)]. additional relevant studies were identified by checking the bibliographies of included articles. two reviewers (csk and su) evaluated all titles and abstracts for studies that met the inclusion criteria and studies that did not clearly meet the selection criteria were excluded. one senior author (ykl) checked the potential inclusions and full reports (where available) of potentially relevant studies were retrieved and independently checked for eligibility. two reviewers independently extracted data from included studies into a proforma spreadsheet covering study design; study location; characteristics of participants; definition of influenza; outcome ascertainment and results. the spreadsheet data were then checked for completeness and accuracy by a senior reviewer (ykl). two reviewers (csk and su) then independently extracted the number of events and denominators from the sources papers, thereby enabling calculations of crude (unadjusted) estimates of the risk of adverse events among patients with influenza. where raw results were not available, we collected results that were the most adjusted risk estimates [adjusted relative risk, odds ratio (or), or hazard ratios] for cardiovascular events with influenza. any differences were reconciled through the arbitration and further investigation of a senior clinical researcher (ykl). authors of manuscripts were contacted to seek further information where necessary. the quality of included studies was evaluated using a risk of bias assessment including: ascertainment of exposure to influenza, ascertainment of selected cardiovascular outcomes and adjustments for potential confounders. overall risk of bias of studies was deemed to be low if all three categories were satisfied and deemed to be moderate risk of bias if two categories were satisfied and high risk of bias if zero or one categories were satisfied. where there was no evidence of substantial heterogeneity and more than 10 studies available for meta-analysis, we used funnel plots to assess publication bias (15) . we used revman 5.2. (nordic cochrane centre, copenhagen, denmark) to conduct a dersimonian-laird random effects meta-analysis using an inverse variance weighting approach, to calculate a pooled or. we assumed asymptotic convergence of risk ratio and or as the proportion of adverse outcomes was low (16) . we evaluated both adjusted and unadjusted data from primary studies, although we preferentially used adjusted data where available. we stratified the main analysis based on the measures of ascertaining influenza (e.g. laboratory serology tests or based on clinical presentation suggesting influenza-like illness) and use of adjustments to account for potential confounders. if different types of influenza were reported, we used the category with the largest number of patients. statistical heterogeneity was assessed using i 2 statistic (17) , with i 2 values of 30-60% representing a moderate level of heterogeneity. statistical results are presented as the main effect with 95% confidence intervals (cis) in braces unless otherwise specified. we identified 12 studies that met the inclusion criteria (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) . details of the study selection are shown in figure s1 . there were seven case-control studies, two case-cross-over studies, two cohort studies and one self-controlled case-series study. there were a total of 84,003 participants. the mean age from four studies ranged from 42 to 65 years and median age ranged from 64 to 75 years from four other studies. the percentage of male patients ranged from 42% to 100%. details are shown in table 1 . the study quality assessment is shown in table s1 . the methods for ascertaining exposure to influenza included the use of questionnaires, clinical assessments, review of patient records and laboratory testing. seven studies used well-characterised methods of ascertaining influenza exposure blood tests (18, 20, (22) (23) (24) , lung tissue testing (26) and a validated algorithm (19) . apart from one study where ascertainment of outcome was unclear (29) , all studies reported methods that could reliably identify cardiovascular events. three studies reported use of adjustments for more than two potential confounders (18, 27, 28) . in terms of risk of bias, one study was classified as low risk of bias (18) and three studies were classified as high risk of bias (21, 25, 29) . influenza exposure and cardiovascular outcome evaluation results are shown in table 2 . influenza exposure was defined by serology or laboratory tests in four studies while in the other five studies it was based on symptoms and questionnaires. in madjid et al., influenza epidemics were defined as weekly acute respiratory disease morbidity exceeding the predefined epidemic thresholds (21) . the reporting of the follow-up or timing of influenza exposure was variable. three studies did not report any information on this (18, 20, 26) , but among the other studies, influenza exposure ranged from the preceding week to preceding 3 months. finally, nine studies evaluated mi as the cardiovascular outcome while one study evaluated death from mi (21), one study evaluated ischaemic stroke (19) and one study evaluated hf (24) . of the nine studies with a mi outcome, we excluded the study by pesonen et al. since it compared the risk of mi at different influenza symptom levels, rather than symptoms vs. no symptoms (25) . the remaining eight studies were then stratified into four studies, which used serology or laboratory tests to confirm influenza and seven studies that diagnosed influenza based on symptoms or clinical presentation. the pooled or for the risk of mi with serologically diagnosed influenza was or 1.27 (95% ci, 0.54-2.95), i 2 = 47% (956 participants), which was significant for the only study that adjusted for confounders (or 5.50, 95% ci 1.31-23.13; figure 1 ). the pooled or for risk of mi with influenza-like symptoms was 2.17 (95% ci 1.68-2.80), i 2 = 0%, 6658 participants, which was significant for both unadjusted or 2.23 (95% ci 1.65-3.01, five studies, 2597 participants) and adjusted (or 2.01, 95% ci 1.24-3.27, two studies, 4061 participants) studiessee figure 2 . pesonen et al. found that two to three vs. one or no symptoms of influenza-like illness was associated with increased risk of mi (or 3.8, 95% ci 1.4-10.8) (25) . the study by nicholls et al. was the only study that evaluated the risk of hf with influenza (24) . this study found that three of eight participants who tested positive for influenza a had hf (left ventricular failure or congestive cardiac failure) while four of the other 51 participants had hf. the crude or for risk of hf from this study was 7.05 (95% ci 1. 22-40.90 ). the study by luna et al. evaluated the timing of flulike illness and risk of ischaemic stroke (19) . they found that the greatest risk was present within the first 15 days (adjusted or 6.5, 95% ci 2.2-19.7) and decreased in magnitude with prolonged duration to adjusted or 3.3 (95% ci 1.9-5.8) at 90 days. madjid et al. conducted a study that was not included in the meta-analysis because it evaluated outcomes indirectly, namely autopsy rates of cvd vs. influenza epidemics (21) . this was the only study that evaluated risk of cardiovascular mortality and found that that influenza epidemics were associated with increased odds of death from acute mi (or 1.30, 95% ci 1.08-1.56). our meta-analysis suggests that influenza-like illness is associated with a twofold increase in mi. the risk of mi with serologically defined influenza is less evident, however, the only study that adjusted for potential confounders showed an increased risk of mi with positive influenza tests. in addition, there is limited evidence that flu-like illness precipitates both stroke and hf events, although these observations are based on just single publications, thus warranting further study. a major challenge of studies which associate flulike symptoms and cvd is the spectrum of viral infections that can initiate flu-like symptoms. a review of influenza-like illness suggests that respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza viruses and human coronaviruses can present similar to influenza (30) . a study of influenza-like illnesses over successive winters in the uk found that 480 of 2226 swabs (21.6%) were positive for respiratory syncytial virus (31) . a smaller study in scotland found that picornavirus was also an important cause of influenza-like illness (32) . other studies have also identified rhinovirus (33), adenovirus (33), human corona virus (33) and parainfluenza virus (34) as important causes of influenza-like illness. our findings suggest that exposure to influenzalike illnesses may be associated with increased risk of cardiovascular events, hence measures such as influenza vaccination should be supported in line with current recommendations, particularly among patients who are at risk of cvds. the definition of influenza varied among studies in the current analysis since both serological tests and clinical assessments were used. we have observed that influenza-like illness defined by clinical features, which may be caused by both influenza and other viral infections such as those highlighted above are associated with an increased risk of mi. this association appears to be absent in influenza cases defined by serology. there are a number of possible explanations that might account for this. the clinical features of influenza may be secondary to other viral infections so using clinical features may include other non-influenza infections. participants in whom influenza was diagnosed by serology may represent a different biological cohort than those patients in whom influenza diagnosis is based on clinical features. this may be because participants identified from serology may be asymptomatic, have atypical or milder symptoms because of infection with less virulent genotypes of influenza virus while participants who are diagnosed with influenza on clinical grounds might have more severe influenza infections which could manifest cardiovascular complications. inclusion of asymptomatic cases may lead to a cohort with fewer cardiovascular events and reduced likelihood of detecting differences when compared with control group. second, the timing of the serology test is important to determine if it is associated with cardiovascular event. serology tests are able to measure the antibodies that develop in response to influenza virus but this response develops within 2-3 weeks of infection (35) (36) (37) . once developed, antibodies remain at detectable levels for months (38) , thus making the test reliable in confirming influenza infection exposure. consequently, if serological testing took place in the acute phase infection, the test result may represent a false negative. the quality of the serology test for influenza may also present a problem as high quality and standardised methods should be used to minimise variability in results. it is notable that two of the studies of serology took place in 1977 and 1981 and it is unclear, if the laboratory tests are as reliable and robust as those having been conducted more recently. serological tests may detect cross-reactive antibodies generated by previous exposure to antigenically similar viruses leading to false positive results. the current gold standard for laboratory confirmation of influenza virus infection is reverse transcription-polymerase chain reaction or viral culture although this methodology was not used in studies included in the current analysis. it is notable that there are differences between influenza and influenza-like illness. true influenza, which is confirmed by serology, will not include other flu-like mimics unless there is dual infection. however, serologically defined influenza infection will also include asymptomatic cases that may not be clinically relevant and may be missed unless a study defines a cohort and tests all participants regardless of whether they are symptomatic. influenza-like illness on the other hand may include other viral illnesses as previously described. this population will represent the clinically relevant cohort and may be larger than the true influenza cohort. however, it may be more heterogeneous in clinical features than the influenza cohort because it may include infections with other viruses. the ideal study should actually consider both in parallel. this can be conducted by taking a defined population and testing all participants for influenza and then prospectively collect data on whether participants develop symptoms of influenza. this study will allow understanding of the specificity of the influenza-like illness that is unclear. while we identified 12 studies of cvds and patients with influenza and influenza-like illness, and built on existing reviews. this study was the first to quantify the risk, to consider serological diagnosis separately from symptomatic diagnoses and to review the limited evidence for risk of stroke and hf with influenza-like illness. however, many of the studies were of low quality and more than 10-years old, so their findings may not generalise well to current clinical practice and settings. while we found some evidence to support the role of influenza-like illnesses as a trigger for cardiovascular events the precise mechanism is unclear. it has been suggested that influenza and influenza-like illnesses may increase the risk of acute mi by mechanisms such as: antigenic cross-reactivity; increased proinflammatory and pro-thrombotic cytokines; loss of anti-inflammatory properties of hdl particles; increased trafficking of macrophages into the arterial wall; pronounced expression of inflammatory cytokines by infected monocytes; reduced clotting times and induction of pro-coagulant activity by infected endothelial cells and increased expression of tissue factor (18) . coagulopathy and inflammation are thought to be key factors (39) . inflammation is also an important part of the atherosclerotic process and inflammatory cytokines such as tnf-a and il-6 are increased in the context of studies of influenza in mice (40) . repeated influenza infection may injure the vascular endothelial cells and initiate the inflammatory response that is required to accelerate and enhance atherosclerosis development (18) . furthermore, viral infections can trigger the production of inflammatory cytokines, which could destabilise existing vulnerable plaques leading to mi (41) . it is worth noting that included studies were of high risk of bias because of their retrospective nature and study designs, which were case-control or case cross-over. in addition, bias may arise from the variation in the way influenza was diagnosed which ranged from symptoms associated with respiratory infection to diagnoses based on serology tests. the most reliable diagnostic methodology is laboratory serological tests, as other chest infections or atypical respiratory infections may have clinical features consistent with influenza infection. however, even when serological tests were used to ascertain influenza infection, it is possible that patients who did not have serology positive influenza did not have flu and this may account for the dissimilarity between the two large cohorts evaluating public health records and the remaining studies. furthermore, adjustments for potential confounders are important to reduce risk of bias. only a limited number of studies in the current review adjusted for baseline variables and there is always the risk of unmeasured confounders. reporting bias represents a limitation in some of the studies included in the current review. data from large databases may fail to capture asymptomatic cases of influenza, which do not present the health services or patients who self medicate and do not seek medical attention. another limitation relates to the inclusion of older studies that used different diagnostic criteria for mi. the diagnostic criteria for mi in older studies were based mainly on clinical criteria, echocardiography, electrocardiographical changes and possible enzyme rises of creatinine kinase that are not as sensitive as high sensitivity troponin assays used in contemporary practice to detect myonecrosis, which may have resulted in underreporting of incident mi in older studies further studies should be conducted to evaluate the risk of cvd with influenza. these observational studies should be prospective in design with outcomes linked to a cvd register. once potential cases of influenza are identified, further laboratory tests should be performed to confirm diagnosis of influenza. in addition, important potential confounders such as smoking, socioeconomic status, underlying chronic respiratory disease and other cardiovascular risk factors need to be accounted for in risk-adjusted models. in conclusion, there is an apparent association between influenza-like illness and adverse cardiovas-cular events. this association is less clear for serologically defined influenza, which may be a limitation of published data and study designsthere are many biologically plausible mechanisms to explain the relationship. we recommend renewed efforts to apply current clinical guidelines and maximise the uptake of annual influenza immunisation among patients with cvds, to decrease their risks of mi and stroke. mam conceptualised the review. csk and ykl performed the literature search. csk and su screened the search results for relevant studies and extracted the data. csk, ek and ykl were involved in the data-analysis. csk drafted the manuscript and all authors contributed in writing of the paper. additional supporting information may be found in the online version of this article: table s1 . risk of bias assessment of included studies. figure s1 . flow diagram of study selection. the contribution of influenza to combined acute respiratory infections, hospital 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antibodies in patients with myocardial infarction coxsackie virus infection in acute myocardial infarction elevated infection parameters and infection symptoms predict an acute coronary events respiratory viral antigens in autopsy lung tissue specimens from patients with cancer of myocardial infarction influenza infection and risk of acute myocardial infarction in england and wales: a caliber self-controlled case series study influenza-like illness in acute myocardial infarction patients during the winter wave of the influenza a h1n1 pandemic in london: a case-control study triggers of myocardial infarction and sudden death the causes and diagnosis of influenza-like illness contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study virological surveillance of influenza-like illness in the community using pcr and serology surveillance of 16 respiratory viruses in patients with influenza-like illness in nanjing china population-based surveillance of hospitalizations associated with respiratory syncytial virus, influenza virual and parainfluenza viruses among young children incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic h1n1 virus in u.s. populations serological response in rt-pcr confirmed h1n1-2009 influenza a by hemagglutination inhibition and virus neutralization assays: an observational study influenza serological studies to inform public health action: best practices to optimise timing, quality and reporting inflammation and coagulation in the cardiovascular system: the contribution of influenza role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice association of influenza vaccination and reduced risk of recurrent myocardial infarction key: cord-258366-fu9b446y authors: couto, carla r.; pannuti, cláudio s.; paz, josé p.; fink, maria c. d.; machado, alessandra a.; de marchi, michela; machado, clarisse m. title: fighting misconceptions to improve compliance with influenza vaccination among health care workers: an educational project date: 2012-02-06 journal: plos one doi: 10.1371/journal.pone.0030670 sha: doc_id: 258366 cord_uid: fu9b446y the compliance with influenza vaccination is poor among health care workers (hcws) due to misconceptions about safety and effectiveness of influenza vaccine. we proposed an educational prospective study to demonstrate to hcws that influenza vaccine is safe and that other respiratory viruses (rv) are the cause of respiratory symptoms in the months following influenza vaccination. 398 hcws were surveyed for adverse events (ae) occurring within 48 h of vaccination. ae were reported by 30% of the hcws. no severe ae was observed. a subset of 337 hcws was followed up during four months, twice a week, for the detection of respiratory symptoms. rv was diagnosed by direct immunofluorescent assay (dfa) and real time pcr in symptomatic hcws. influenza a was detected in five episodes of respiratory symptoms (5.3%) and other rv in 26 (27.9%) episodes. the incidence density of influenza and other rv was 4.3 and 10.8 episodes per 100 hcw-month, respectively. the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. the compliance with influenza vaccination has been historically poor among health care workers (hcws) varying from 2 to 36% around the world [1] [2] [3] [4] . a recent review of relevant predictor studies of self-reported reasons for accepting or rejecting influenza vaccination showed that the two major reasons for rejecting are misconceptions or lack of knowledge about influenza infection; and a lack of convenient access to vaccine. on the other hand, hcws compliant to seasonal vaccination are generally older, believe in vaccine efficacy, take the vaccine for self-protection, and have been previously vaccinated [5] . at hospital das clinicas, university of sã o paulo school of medical sciences, a previous study showed a 34% compliance with influenza vaccination among hcws. in the mentioned study, the main reasons for non-compliance were the perception of vaccine inefficacy and the fear of adverse reactions [4] . respiratory symptoms occurring after vaccination are frequently misinterpreted as vaccine failure which reinforces the hcw's skepticism on vaccine efficacy. to overcome these false beliefs, we proposed a prospective study in a cohort of hcws to demonstrate that influenza vaccine is safe and other respiratory viruses (but not influenza) are generally the cause of respiratory symptoms in the months following influenza vaccination. this study was conducted at hospital das clinicas, university of são paulo school of medical sciences (hc-fmusp) from may to october 2006. the hospital das clinicas is a 2,000-bed tertiary teaching hospital consisting of 5 buildings attached to the university of são paulo. the main building has approximately 900 beds and contains most of the surgical and clinical wards and 12 intensive care units. hospital das clinicas has an estimated 15,000 hcws, including permanent and casual staff, employees, students, and volunteers. since 1999, annual influenza vaccination has been offered free of charge to all hcws. vaccination usually takes place at the hospital's immunization center during working hours, from monday to friday. in 2006, as a strategy to increase compliance with influenza vaccination, vaccine was offered at places of easy access during expanded hours, as suggested by 61% of the interviewed in previous survey [4] . in addition, an educational campaign was carried out emphasizing the safety and importance of influenza vaccination. detailed information about the 2006 educational and vaccination campaign have been published elsewhere [6] . during vaccination campaign, hcws were invited to participate in the present study which had two steps: 1) evaluation of vaccine safety and, 2) cohort study to evaluate which respiratory viruses were more frequently detected in hcws presenting respiratory symptoms in the four-month period following vaccination. sample size was estimated taking into account an expected frequency of 10% of adverse events in adult population. considering an acceptable frequency rate of up to 13%, we estimated to enroll at least 377 hcws (epiinfo version 6). three hundred and ninety eight vaccinated hcws were surveyed for adverse events occurring within the first 48 h after influenza vaccination. a subset of 337 hcws participated in the follow-up phase of the study. to assure that all hospital sectors were represented, the cohort was defined during the assessment of adverse events which was performed by a hospital epidemiologist nurse, through visits in all hospital floors and sectors. afterward, these hcws were actively surveyed twice a week, at work place, during four months, to check for the occurrence of respiratory symptoms and nasal wash sampling which was done in 93 of the participants. figure 1 shows the algorithm of the study. a followup time of four months was proposed taking into account the period when serum antibodies elicited by influenza vaccine are expected to maintain protective levels. the following adverse events were actively surveyed: fever, headache, malaise, myalgia, local pain, local edema and allergic reaction. other adverse events spontaneously reported were also registered. during follow-up visits, participants were asked about the presence of the following symptoms: fever, coryza, blocked nose, sneeze, cough, watery eyes, headache, myalgia, sore throat, hoarseness, sibilance, and dyspnea. allergy was ruled out in those with sneezing as the only symptom. influenza like illness (ili) was defined by the presence of fever and cough and/or sore throat according to the cdc definition [7] . in the presence of any of the above mentioned symptoms, a nasal wash sample was taken according to englund et al, kept at 4uc to 8uc and processed at the virology laboratory within four hours from sampling [8] . nasal washes were taken from hcws who consent with sampling and whose duration of symptoms did not exceed three days. respiratory syncytial virus (rsv), influenza (inf) a and b, adenovirus (adv) and parainfluenza virus (piv) were diagnosed by direct immunofluorescent assay (dfa) according to the manufacturer's instructions (imagenh dako, cambridgeshire, uk). aliquots of nw samples were stored at 280uc for later pcr and real time pcr processing. pcr was used to detect coronavirus and picornavirus. rt-pcr products for picornavirus were subsequently sequenced to differentiate rhinovirus from enteroviruses. real time pcr (taqman assay) was used to diagnose human metapneumovirus (hmpv). to increase the sensitivity of influenza diagnosis, a real time pcr (taqman assay), was added to the diagnostic tools. similarly, a nested adenovirus pcr was used along with dfa due to the low sensitivity of the latter in diagnosing adv. the pcr protocols used in the present study have been published elsewhere [9] [10] [11] [12] . hcws were informed about the results of the dfa up to 48 h after sampling. spss 15 .0 (spss inc., chicago, il) was used with the x 2 test or fisher's exact test for discrete variables, and student's t test or the mann-whitney u test for continuous variables. tests of significance were two sided, and p,0.05 was considered to be statistically significant. incidence density (id) of respiratory symptoms, influenza virus infections and other respiratory virus infections were calculated by the formula described below. id results were expressed per 100 hcw-month [13] . the interviews for adverse events occurring within 48 h of vaccination were made from one to 10 days after vaccination. the majority of the participants (81.4%) were surveyed within the first week of vaccination. all hospital sectors were represented (table 1) . one hundred and twenty of the 398 hcws (30.2%) reported at least one adverse event (ae). table 1 shows the occurrence of adverse events according to the demographic characteristics of the vaccinees. sector of work was the only variable associated with the presence of adverse events (p = 0.017). the sectors with highest frequency of adverse events were the virology laboratory (87.5% of the subjects), burn unit (54.5%), nephrology (52.4%) and pneumology (45.5%). in the remaining sectors, ae were reported by less than 40% of the subjects. those surveyed in the first five days after vaccination were more likely to report such events [95 (79.2%) versus 25 (20.8%); p,0.0001]. local ae were reported by 18.3% of the participants, systemic ae by 71.6%, and 10% of the participants reported both local and systemic aes. headache, myalgia and malaise were more frequently reported (50%, 45.8% and 45%, respectively). local pain and local edema was reported by 17.5% and 5% of the hcws. no severe adverse event was observed. during the 4-month period following influenza vaccination, respiratory symptoms were evaluated in 337 hcws. a total of 4,182 follow-up visits were performed (median 12 per hcw, ranging from one to 25 visits). one hundred and twenty-one hcws (36%) developed 192 episodes of respiratory symptoms. coryza, cough, sore throat and myalgia were reported by 36.3%, 25%, 17.4% and 13.7% of the participants, respectively. seventy-one of them (58.7%) presented more than one episode suggestive of upper respiratory infection (uri). ili was observed in 17 of the 192 episodes (8.8%). mean time to the occurrence of respiratory symptoms was 2.9 (0.7 to 5.2) months. the incidence density of respiratory symptoms was 12.4 episodes per 100 hcw-month. nasal washes were taken in 93 of the 192 episodes of uri. in 61 episodes (66.3%) no respiratory virus was found, even though 82% had coryza, 53% had cough and 49% had sore throat. the frequency of ili was similar among hcws who agreed with sampling and those who did not agree (58.8% versus 41.2%, p = 0.37) ( misconceptions about influenza vaccine, be it about its safety or its effectiveness, have been identified in all studies included in a recent review of attitudes and predictors of influenza vaccination among hcws, highlighting the importance of education efforts [5] . initial symptoms of rv infections are often unspecific such as fever, malaise, or myalgia. as influenza vaccine is offered when other rv are circulating (e.g., rsv), vaccinated hcws developing symptoms within 48 h of vaccination misinterpret those signs as vaccine adverse events. in addition, the occurrence of respiratory symptoms in the months following vaccination is mistaken as vaccine failure. other respiratory infections as the cause of such symptoms are hardly ever considered. to diminish the arguments of fear of adverse events or perception of vaccine inefficacy, this prospective study was conducted to demonstrate to a subset of hcws from our hospital, that severe adverse events following influenza vaccination are rare and the episodes of respiratory symptoms occurring in the first four months after vaccination are generally caused by other respiratory viruses and not by influenza virus. as expected, no severe adverse event was observed in the present study, and the events more frequently reported, such as headache, myalgia and malaise could be related to influenza vaccine itself as well as to other causes, given their unspecificity. in adults, the adverse event more frequently reported after intramuscular administration of inactivate vaccines is local pain, affecting 10% to 64% of the vaccinated [14] [15] [16] . in the present study, 17.5% of the participants reported local pain. systemic reactions like fever, malaise and myalgia can also occur after inactivate vaccines. in the present study, the frequency of systemic aes (over 70%) was higher than reported in previous studies. recent publications have shown rates of systemic adverse events ranging from 30% to 59% in hwcs [17, 18] . ideally, the subjects should have been surveyed within the first four days of vaccination. as we preferred to apply the questionnaire personally, rather than by mail or phone calls, only 49.7% of the participants were interrogated up to the fourth day, due to the great number of interviews. thus, the high rate of systemic adverse events observed in the present series may be either an overestimation by the subjects or a consequence of the survey method applied. a recent study evaluating vaccine coverage in korea has demonstrated that interview surveys provide more reliable information than telephone surveys, showing lower missing rates and 100% of agreement with the immunization registry record [18] . anaphylaxis and neurological reactions are rare [15, 19] . the frequency of adverse events observed in the present study may be overestimated taking into account the subjectiveness of selfreported unspecific symptoms. as hcws are aware of vaccine adverse events and fear its consequences, it is comprehensible that these events will be more frequently reported by them than by general population. another study conducted in the same hospital demonstrated that hcws reported significantly more adverse events (52.9%) than the elderly (25.3%) [20] . the higher frequency of adverse events reported by hcws surveyed in the first five days of vaccination, as compared with those surveyed after the fifth day, may suggest that people may be more predisposed to remember any symptom possibly associated with the vaccine if inquired within the first days of vaccination. on the other hand, we believe that if the adverse events were severe or important, they would not be missed if inquired after 6 to 10 days. interestingly, we observed that some sectors showed significantly higher rates of ae than others, supporting the subjectivity of the information. also, this data may suggest a mouth to mouth effect among sector coworkers influencing the self-report of ae. among hcws, the belief that coworkers take influenza vaccine influences the vaccine uptake. thus, it is possible that the same occurs concerning to adverse events. continued education of health professionals is essential to highlight not only the epidemiological importance of the vaccine, but also its safety and the low risk of severe adverse events. our study also demonstrated that the respiratory symptoms occurring in the months following influenza vaccination were more frequently caused by other respiratory viruses and generally do not mean vaccine failures. one limitation of our study is that in only 93 of the 192 episodes of respiratory symptoms (48.4%) the subjects agreed with nw sampling. nw sampling is a simple but uncomfortable procedure and this fact may explain why some hcws preferred not to get tested during working hours. one could argue that influenza cases could be missed among those not tested. however, we believe that this loss has not affected our results as the frequency of ili was similar between those who agreed with sampling and those who did not ( table 2 , p = 0.37). the incidence density of other respiratory viruses was 2.4 times greater than incidence density of influenza. probably, this difference would be even greater if real time pcr was also performed to increase the sensitivity of the diagnosis of other respiratory viruses as well. in addition, more cases of other rv infections would be diagnosed if a larger number of professionals were tested, increasing the difference between the incidence density of influenza and other rv. influenza infection is characterized by the abrupt occurrence of fever, headache, myalgia, and dry cough. during influenza season, the presence of these symptoms is highly predictive of influenza infection and summarizes the case definition of influenza-like illness (ili), which has been used worldwide for influenza surveillance purposes. however, the sensitivity and positive predictive value of such definition can vary greatly depending on the co-circulation of other respiratory viruses in the community [21] . indeed, bellei et al. have recently reported that 70% of ili cases in the city of são paulo were caused by other agents, mainly rhinovirus, which peaks along with influenza [22] . similar results have been previously published by other authors [21] . in our series, influenza cases in vaccinated hcws were mild and occurred significantly earlier following vaccination in comparison to other respiratory viruses. this finding may be explained by the marked seasonality of influenza in são paulo city as reported previously [23, 24] , peaking in early winter and coinciding with the initial period of the study. the effectiveness of influenza vaccines is related predominantly to the age and immune competence of the vaccinee and the degree of similarity between the viruses in the vaccine and those in circulation. vaccine effectiveness in preventing laboratory-confirmed influenza illness when the vaccine strains are well matched to circulating strains is 70-90% in randomized, placebo-controlled trials conducted among children and young healthy adults, but is lower among elderly or immunocompromised persons [25] . in adults $65 years old, the efficacy of influenza inactivate vaccine varies from 30% to 40% [26] . trials that measure laboratory-confirmed influenza virus infections as the outcome are the most persuasive evidence of vaccine efficacy [25] . in the present study, only five of the 337 vaccinated hcws (1.5%) acquired influenza. in accordance with the educational nature of our study, we considered all cases of influenza as vaccine failures, since vaccinated health personnel look forward to be protected against influenza. molecular characterization of influenza cases was not performed to check for possible mismatches between circulating viruses and vaccine strains, which could possibly justify those failures. our study demonstrated that the fear of severe adverse events seems unjustified as well as the perception of vaccine inefficacy. uri following influenza vaccination were generally caused by other respiratory viruses and not by influenza. in times of pandemic influenza a h1n1 and widespread vaccination, healthcare and emergency medical services personnel are among the priority groups recommended to receive the h1n1 influenza vaccine. it is time to overcome definitively the misconceptions about the vaccine as well as the fear of adverse events. so far, the vast majority (93%) of adverse events reported to vaers after receiving the trivalent 2010-2011 influenza vaccine, were classified as ''non serious'', e.g., soreness at the vaccine injection site [27] . we believe that the educational nature of the present study may persuade hcws to develop a more positive attitude to influenza vaccination. influenza vaccination among medical residents in a teaching 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vaccination of healthy working adults: a randomized controlled trial frequency of adverse reactions after influenza vaccination the effectiveness of vaccination against influenza in healthy, working adults immunogenicity of a monovalent pandemic influenza a h1n1 vaccine in health-care workers of a university hospital in japan adverse events associated with the 2009 h1n1 influenza vaccination and the vaccination coverage rate in health care workers adverse reactions to influenza vaccine in the elderly occurrence of early adverse events after vaccination against influenza at a brazilian reference center predicting influenza infections during epidemics with use of a clinical case definition acute respiratory infection and influenza-like illness viral etiologies in brazilian adults low mortality rates related to respiratory virus infections after bone marrow transplantation use of oseltamivir to control influenza complications after bone marrow transplantation prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) the efficacy of influenza vaccine in elderly persons. a meta-analysis and review of the literature summary of 2010-2011 trivalent influenza vaccine data from the u.s. vaccine adverse event reporting system the authors thank the infection control group of hospital das clínicas, school of medical sciences, university of são paulo. key: cord-013022-c8a8ocge authors: vázquez-espinosa, emma; laganà, claudio; vazquez, fernando title: the spanish flu and the fiction literature date: 2020-07-07 journal: rev esp quimioter doi: 10.37201/req/049.2020 sha: doc_id: 13022 cord_uid: c8a8ocge this review focuses on the fictional literature in which the spanish flu is represented either as an anecdotal or as a historical aspect and the effect on the author or fictional character. we examine this sociocultural period in the press and mainly in anglo-saxon literary works and from other countries, including spanish and latin american literature that is not very represented in some international reviews on the subject. also, we include books about the previous and subsequent influenza pandemics to the spanish flu. flu has caused global pandemics over the centuries. in the 18th century, the influenza pandemic between 1708-1709 was not fairly assessed [1] . during epidemics and pandemics in 1847-1848 and 1889-1893, it was recognized that the respiratory complications of flu could greatly elevate the death rate [2] . another pandemic has been in 2005, the avian flu, with the emerging cultural patterns and interpretative repertoires and metaphors [3] . the spanish flu, in 1918, killed 50-100 million people in the world and, in spain, caused as many deaths as in the spanish civil war. about the spanish flu, there are different studies, this is not an exhaustive list, in the world [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] and in spain [14] [15] [16] [17] with its spatial-temporal patterns [18] . this pandemic has been reviewed from different points of view: sociological or historical and the origin of the flu [19] [20] [21] . this review focuses on the fictional literature in which influenza is represented either as an anecdotal or as a historical aspect and the effect on the author or fictional character. in neither case it is an exhaustive review, but it focuses mainly on anglo-saxon literary work and from other countries, including spanish and latin american literature that is not very represented in some international reviews on the subject. all literature books are cited in the tables with authors and their books in english and spanish and we include books about the previous and subsequent influenza pandemics to the spanish flu. the spanish flu and the fiction literature 1 servicio de neumología, hospital universitario la princesa, madrid, españa 2 servicio de radiodiagnóstico, hospital universitario la princesa, madrid, españa 3 servicio de microbiología. hospital universitario central de asturias, oviedo, españa. 4 departamento de biología funcional, área de microbiología, facultad de medicina. universidad de oviedo, oviedo, españa. 5 instituto oftalmológico fernández-vega, fundación de investigación oftalmológica, universidad de oviedo, oviedo, españa. 6 grupo de microbiología translacional, instituto de investigación sanitaria del principado de asturias (ispa), oviedo, spain. fonso xiii had to interrupt this work and his conversations with germany to stop sinking spanish ships in the atlantic. the "abc" newspaper reported on september 30, 1918 that «h. m. the king is sick with the flu. the attack is mild, and although his majesty has a fever, so far the ailment is of no importance". on october 4, however, the official party pointed out a fact that indicates that the king did not suffer from the flu: a series of "scarlet fever eruptions of normal evolution" on his body. skin rashes that do not fit with the usual symptoms of the spanish flu, in the same way that the fact that ten days later the press continued to report mild fever and more skin problems does not coincide with the picture of this disease for cervera c [24] . the first signs of scarlet fever can be flu-like symptoms, including a high temperature of 38 ºc or above, a sore throat and swollen neck glands. it could be probable that these two theories may be both true, the flu can later in the course of the disease be complicated by scarlet fever in a percentage of patients. scarlet fever circulating with chickenpox or influenza can be particularly dangerous. another explanation is, although infrequently, the flu can take with an exanthematous skin rash. the treatment and vaccines of the spanish flu pandemic. the treatment was based in several substances and bacterial vaccines in relationship with the belief in the bacterial theory of disease: "bacterial vaccines, some were derived exclusively from the pfeiffer's bacillus, the presumed cause of influenza, were widely used, while others contained one or more other organisms found in the lungs of victims" [25] . the treatment included "symptomatic therapy with salicylates and quinine and codeine, for pneumoniae intramuscular or intravenous silver or platinum colloid, digitalis, alcamphor oil, or adrenaline, and bleeding" [26] . in the espasa encyclopedia (popular spanish encyclopedia), it was cited that "the serums and vaccines inspired by bacterial associations are now abandoned"; and that "strychnine, oxygen inhalations, arsenicals, salicylates and bleeding are prescribed" [27] . other treatments were vapors from aromatic plants, purgatives, sweats, medicinal plants, and hydrotherapy, strong showers with alternating hot and cold water, iodine, leeches, cardenal brand water filter to trap all microbes. marañón advocated to use a light antiseptic nasal douches twice a day [19] . loeb l [28] found "striking similarities between orthodox and commercial suggestions for treating influenza" in the lancet and the british medical journal between 1889 and 1919. the first reference in the spanish press to an epidemic the name of influenza. the name of influenza is believed to have been used in the city of florence in the 14th century (by villani in 1358), considering that the disease was due to the «influenza di freddo» (to the cold) or «di stelle» (to the stars, by the astrological theories in those times) [22] [23] . in 1742, sauvage use the name «grippe». the terms «gripper» (french), «to grip» (english) or «greifen» (german) mean in spanish «agarrar, atrapar». perhaps the abrupt way of presenting this disease on many occasions has justified the name «grippe», that in spanish it was written «grippe» until at least 1925, and then with a single p. at the beginning of the tolstoi´s book, "war and peace" (1869), a novel that is the chronicles of the french invasion of russia and the impact of the napoleonic era on tsarist society, tolstoi writes that: "on a july day in 1805… anna pávlovna had been coughing for a few days; it was a "grippe", as she said ("grippe" was a new word then, that very few used"). another word in spanish was "trancazo" ("strike with a bar"), "tranca" means "iron or wood bar" and from this word derives "trancazo" that is a colloquial name for flu. other nicknames were spanish flu or spanish lady, also french flu. it appears that french journalists had, initially, called it the "american flu"; but the fact that the american soldiers were his allies in the warlike conflict advised not to assign such a link to them; and as there were also cases of influenza in spain, it was decided to generalize the use of this expression, which was later assumed by germans and others [22] . another most popular name in madrid, was the "soldado de nápoles" ("naples soldier"), a popular song in the zarzuela (popular musical genre or "género chico" in spain) called la canción del olvido (the forgotten song) due both, were "highly contagious". today, there are many authors who avoid such a name (the spanish flu) and they aptly refer to it as the "1918-1819 influenza pandemic". the origin of the spanish flu pandemic. there are several theories about the origin: a) the origin could be in china and after in philippines and the usa and the army in europe. b) english soldiers in france in 1916, the disease soon spread to other neighboring countries (england, italy, spain) and to more distant ones (the usa) as a consequence of the displacement of the troops [22] . c) the regular arrival of chinese workers to africa and europe, throughout those years, could have been the origin of an earlier introduction (coinciding with the war). and this is a very plausible interpretation due to the circumstance that the spanish royal family and the spanish ministers suffered the flu, in the month of may 1918, and could contribute to this unjustified name [21] . the flu in the spanish king is debated, the majority of scholars think that it was a flu. for cervera c [24] was scarlet fever: between september and october 1918, al-war could represent the newness material to build your novels, whereas the pandemic represented historical continuity of the past plagues and this matter was not modern for their literature [32] . in the essay "on being ill" (1926), virginia woolf lamented that flu hadn't become a central theme in literature [33] . susan sontag pointed out, "novelists tend to focus on illnesses that can be "used" as metaphors, plague with its medieval aura, cancer with its mysterious provenance, tuberculosis with its rosy-cheeked energy and dickensian associations. these illnesses, unlike influenzae, carry built-in mythologies primed for literary appropriation" [33] . for hovanec [34a] . "the flu acts as metaphor for the dehumanizing and denaturalizing aspects of modern life, which take on many forms". belling [31] divides fiction representing the pandemic in two groups: the authors with "experienced" disease, or autobiographical works, and those with "registered rather than experienced" motifs. the first group: authors who were alive at the time ("experienced") (table 1) the best known is katherine anne porter's novella pale horse, pale rider. a) at the beginning, was considered not a serious infection. on 24 june 1918, the war poet wilfred owen composed an ironic letter to his mother and considered the flu something of a joke: "stand back from the page! and disinfect yourself" [35] . b) t. s. eliot makes a possible reference to the spanish flu in his poem sweeney among the nightingale: "the person in the spanish cape". elliot and vivien (his wife) caught the disease in november 1918 and he was working in his masterpiece poem "the waste land". "-he represented beatrice's immortality, also outbreak in the spring of 1918 can be found in the madrid newspapers "abc" and "el sol" (the sun), the latter one published its first headline about the subject on 22 may 1918 [18] : "what is the cause? an epidemic in madrid. in june 2 of 1918, "the times" in madrid, talked about an epidemic with the name the spanish flu and this name began to circulate and in august the 'journal of the american medical association' dedicated its number to the "spanish flu" [18] . an important book about the spanish flu and the press is that of davis ra [19] . in the spanish press, it was the subject of attention with different comic strips that are not included in this work but that can be found in the digital newspaper archives [29a] . these comic strips remember the previous coloured engraved satires, in the wellcome collection, such as "an address of thanks from the faculty to the right hon.ble mr. influenzy for his kind visit to this country" (by temple west) (https://wellcomecollection.org/ works/kn2xshu9) [29b] . the god punishes is a typical approach to the plagues and pandemics. this is just one example: in león, spain, during the spanish flu in his prayer "pro tempore pestilentiae" ("for the times of pestilence"), the bishop: "exhorts their parishioners to repent of their guilt because sins are the cause of scourging with that god punishes us". among the reasons cited by the bishop to explain the incidence of influenza are the desecration of holidays, blasphemy, obscene and immoral amusements and debauchery [29a] . and in zamora (city in castilla), with one of the highest mortality, there were a lots of mass and the consequent spreading of the flu. instead the literature of the plague (bocaccio´s decamerone, camus´s the plague), or tuberculosis (such as thomas mann´s the magic mountain), the 1918-1919 pandemic have hardly been the subject of novel or realistic descriptions by writers. could it be the coincidence in the time of the first world war with the most fatal stages of the pandemic that contributed to the desire not to insist more on the evocation of so many sufferings, and thus favor a deliberate forgetfulness? stalin said: "a single death is a tragedy; a million deaths is a statistic", the little literature in the 1918 pandemic, perhaps was due to the "flu overwhelmed language in ways that world war i did not" [31] . the spanish flu is called a "forgotten pandemic" [31] , that's the difference for example, between literature of the spanish flu and the literature of the world war i and the poets of the war. or for f. scott fitzgerald, gertrude stein, ernest hemingway and john dos passos, the flu did not represent a topic in their novels, the great g) thomas wolfe, look homeward, angel; a story of the buried. this is his first novel, a semi autographic story, covers the span of time from eugene's birth in 1900 to his definitive departure from home at the age of 19. the setting is a fictionalization of his home town of asheville, north carolina, called altamont, catawba in the novel. brother´s writer died with influenza. h) john o´hara, in a short story the doctor´s son. his father worked during the flu outbreak in the pennsylvania mining and o´hara accompanied his father on house calls. here, o´hara, as the narrator, adopts a point of view of observer not such as a personal or familiar victim also dramatizes another major public health risk: the gathering of crowds [34] . i) while pale horse, pale rider represents the best literature of the flu and perhaps the paradigm of the spanish flu, there was a lack the interest on it, maybe the reason is the traumatic experience just of a person [36] and without importance in comparison to the world war i. katherine anne porter, the author, suffered influenza at twenty eight years old in 1918, and her father had planned in advance her funeral. twenty years later, she published pale horse, pale rider, a novella in which her autobiographical protagonist, miranda, almost dies of the flu. the story closely follows an account of porter's own illness and recovery during the pandemic, when she was working as a reporter in denver. j) william keepers maxwell wrote they came like swallows. a novel about a midwestern family that falls ill love-affairs of numerous dead men who surely had never thought of him... if it wasn't appendicitis, influenza maybe". e) michael arlen, in the green hat, the protagonist was inspired in the heiress nancy cunard, who caught the flu in 1919 with pneumonia and depression. f) virginia woolf wrote an essay on flu, on being ill, and she describes the mental effects of disease. her mother had died of influenza in 1895. in her diary writes: "influenza, which rages all over the place, has come next door." "rain for the first time for weeks today and a funeral next door; dead of influenza". she had several bouts of influenza: in 1918 was kept in bed 8 days, in 1920, 1922, 1923 and 1925. in her book, mrs. dalloway, there are two flu quotations: "for having lived in westminster-how many years now? over twenty, -one feels even in the midst of the traffic, or waking at night, clarissa was positive, a particular hush, or solemnity; an indescribable pause; a suspense (but that might be her heart, affected, they said, by influenza) before big ben strikes". "thus, when she said in her offhand way "how's clarissa?" husbands had difficulty in persuading their wives and indeed, however devoted, were secretly doubtful themselves, of her interest in women who often got in their husbands' way, prevented them from accepting posts abroad, and had to be taken to the seaside in the middle of the session to recover from influenza". a chaotic city and many did not want to listen to the rational explanations for the reason for that epidemic, so on october 1 was celebrated the first black wedding (shvartze khasene in yiddish). sforim recounts the first black wedding in his book: it was a jewish ritual to protect themselves from deadly epidemics, consisting of looking for a boyfriend and girlfriend among the most disadvantaged in the city (crippled or destitute) and marrying them in a cemetery [20] . d) géza csáth, hungary writer, in the short story the redhaired girl: "i had taken to my bed. i had contracted influenza. in the evening, fever developed. at such times it is as though the air has become as dense as oil, and everything seems to be swimming in a soft warm fluid…. i saw my father hurrying to with the ongoing russian civil war, it had a major problem of food shortages and the existence of gangster gangs. due to its strategic position and suffering from different infections throughout history with quarantines since the time of catherine the great, iliá mechnikov chose odessa in 1886 as the first center of disease control (bacteriological institute) of russia with vaccinations against the rage. his assistant yakóv bardakh continued his work investigating anthrax, typhoid, cholera, malaria and tuberculosis. bardakh's jewish origin led to his dismissal and one of his students, stefansky, was put in his place, but his fame was so great that he was the most famous doctor in southern russia. the arrival of the spanish flu (ispanka) and other infections such as cholera and typhus made odesa felice (1912 felice ( -1917 . he contracted the flu in prague on october 14, 1918 and while in his sickbed he witnessed the fall of the austro-hungarian empire from his window. "getting the fever as a subject of the habsburg monarchy and recovering from it as a citizen of a czech democracy was certainly overwhelming, but also a little comical" wrote his biographer. in: letters to felice (18) (19) xii, 12) [20] . i) saneatsu mushanokōji, member of the japanese avant-garde shirakaba, in love and death, described the death by flu of a young´s girlfriend. j) suryakant tripathi (nirala) (first modern hindi poet of india). in 1918 many members of nirala´s family died of flu and there was not enough wood to cremate them. "my family disappeared in the blink of an eye" [20] . the authors talk about the facts: "i caught the flu" in their diaries and letters, while mainly about the consequences of the flu in the novels and poems. the most important spanish writers in this period and the spanish flu are josep plá, rosa chacel and miguel delibes the first group: authors who were alive at the time ("experienced") a) ramón de valle-inclán, an spanish playwright, poet and novelist, letters: "letter to don julio romero de torres (a famous spanish painter): ... i beg your pardon that i did not write to you before thanking you. the cause has been not having josefina, in all this time, with a health day. all this as a result of a "flu" that left her very delicate" [39] . b) josep plá, the gray notebook. josep plá´s dietary wrote between march 1918 and november 1919. plá caught the flu that year. spanish flu caused the faculties to close and plá returns to his town, palafrugell, to the family home: "since there is so much flu they have had to close the college… the flu continues to relentlessly kill people. in these last days i have had to attend various burials". there are extensive references in the text. c) juan pérez zúñiga, the fashionable illness (poem). he was a writer, journalist and humorist. he wrote this potable 5 spanish epistolary (years 1957, 1961, 1964, 1965, 1966 (years 1957, 1961, 1964, 1965, 1966 and 1968) j) rosa chacel, her novel acropolis is a look at women and their stage of growth in a generation that lived the spanish flu. it covers the period of the spanish flu until the proclamation of the second republic ("segunda república") in 1931: "it was not enough with the date, from 15 24, 1918) , he echoes the song of the "zarzuela" of barbieri "gloria and wig" (gloria y peluca): "do not cover your face/ pretty girl,/ that whoever hides the good/ god takes it away" and he changed it: "if you cover your face,/ pretty girl/ you will get rid of flu/ and scarlet fever". he was following professor marchoux´s advice, in the pasteur institute, advocated wearing mask for the flu [19] . d) ramón gómez de la serna, a spanish writer, dramatist and avant-garde agitator especially known for "greguerías" (a short form of poetry that roughly corresponds to the one-liner in comedy): "the flu is the fog of death, that little smoke that she also throws on the harsh days of winter" "some guys on the fringes of life, who seem to go down unspecified sidewalks, are household disinfectants. they pass with their big appliances on their backs talking about indifferent things to avoid the contagion of the epidemics that have just died out. they know that their path had to be secret so as not to startle the life of the street, which is neglected of all the problems and on which they are cast as shadows. they leave a wake of fallen microbes, but they are looked upon with benevolence, since they are heroic soldiers who enter the house where there has been a flu, and close the doors of the fateful rooms, and are inexorable executioners of evil in the hermetic rooms". in the bullfighter caracho (1926): "the sensible man maintained that if those misfortunes were exorbitant it is because he had been focused with a telescope in agony and that of a tuberculosis patient or that of a carpentry officer who dies of the flu calling his mother cannot be focused in this way"). in social gatherings in the café (pombo): "salvador sometimes has long absences. pombo misses the little satan who sniffs everything and is distracted from ideas by sniffing things, sensual and dissolute. it is that salvador always has the flu, his grip is temporary, so bartolozzi and flu are also savior". "crespo: "if i had not said that the great savior is lucifer, i would say that this man is mephistopheles, although of course salvador is that in a very serious way with a black coat". he also alludes to his chronic flu in one of his characteristic humorous notes (in the pombo´s holy crypt). e) carlos arniches, a spanish playwright. his prolific work, drawing on the traditions of the "género chico", the zarzuela and the grotesque, came to dominate the spanish comic theatre in the early twentieth century. in this theater comedy, the caciques, released in madrid on february 13, 1920 abundance of the elderly with the flu; but "ou" will see later the best i have found. and the boys are picking them up for me, my wife. i have told him to pay them six pesetas for half a dozen ... i was already nine when i came; but the nine of both sexes, as "ou" wanted". f) ramón pérez de ayala, in his epistolary between pérez de ayala and jesús pabón: "i did not answer immediately because a whole month i suffered from the flu, which had me perfectly asthenic. the south american flus are very impertinent. i have not recovered yet, but i am improving". table 6 the finally, in table 6 we correlate the spanish flu topics and the authors. in conclusion, we show the different literary works about the spanish flu and an extensive list of them. all these works show the wrong idea of virginia woolf complained about the lack of novels devoted to influenza and we have contextualized the works with the historical situation of the spanish flu. none to declare la epidemia europea de gripe de 1708-1709. difusión témporo epidemic influenza: a study in comparative statistics avian flu: the creation of expectations in the interplay between science and the media the influenza pandemic of 1918-1919 in the british caribbean a historical note on influenza in ethiopia the age pattern of mortality in the 1918-19 influenza pandemic: an attempted explanation based on data for england and wales the effects of the 1918-1919 influenza pandemic on infant and child health in derbyshire influenza: the mother of all pandemics a most protean disease': aligning medical knowledge of modern influenza waiting for the flu: cognitive inertia and the spanish influenza pandemic of 1918-19 the ghost of pandemics past: revisiting two centuries of influenza in sweden stacking the coffins: influenza, war and revolution in ireland we all expected to die: spanish influenza in labrador la gripe española. la pandemia de 1918-1919 una ciudad en crisis: la epidemia de gripe de 1918-19 en madrid (thesis) la pandemia de gripe española en el país vasco (1918-1919). ed. fundación museo vasco hª medicina y de la ciencia la gripe española spatial-temporal excess mortality patterns of the 1918-1919 influenza pandemic in spain the spanish flu: narrative and cultural identity in spain the spanish flu of 1918 and how it changed the world. pale rider. publicaffairs la gripe de 1918. centenario de una crisis sanitaria devastadora la grippe et ses virus. presses universitatires de france. parís datos sobre las pandemias de gripe de 1889-90 y 1918-19 en madrid y salamanca, y estudios sobre la sialidasa de los virus de la gripe a y b y la esterasa del virus c. discurso de recepción. real academia de farmacia madrid la mentira sobre que el rey alfonso xiii contrajo la gripe española en 1918 the fog of research: influenza vaccine trials during the 1918-19 pandemic sueros y vacunas en la lucha contra la pandemia de gripe de 1918-1919 en españa beating the flu: orthodox and commercial responses to influenza in britain hemeroteca digital. biblioteca nacional hispánica. available in a satire on the influenza of 1803 el asesino que diezmó león. diario de león 18/12/2017 overwhelming the medium: fiction and the trauma of pandemic influenza in 1918 waste in a great enterprise": influenza, modernism, and one of ours anxiety in the time of influenza: a flu literary review the 1918 influenza pandemic in literature and memory. (thesis) regulating the 1918-19 pandemic: flu, stoicism and the northcliffe america's forgotten pandemic somehow a word must be found: william carlos williams, the legacies of duchamp, and the troping of the found psychology of the pandemic pp the authors declare that they have no conflicts of interest key: cord-282140-teplpmi6 authors: horm, srey viseth; tarantola, arnaud; rith, sareth; ly, sowath; gambaretti, juliette; duong, veasna; y, phalla; sorn, san; holl, davun; allal, lotfi; kalpravidh, wantanee; dussart, philippe; horwood, paul f; buchy, philippe title: intense circulation of a/h5n1 and other avian influenza viruses in cambodian live-bird markets with serological evidence of sub-clinical human infections date: 2016-07-20 journal: emerg microbes infect doi: 10.1038/emi.2016.69 sha: doc_id: 282140 cord_uid: teplpmi6 surveillance for avian influenza viruses (aivs) in poultry and environmental samples was conducted in four live-bird markets in cambodia from january through november 2013. through real-time rt-pcr testing, aivs were detected in 45% of 1048 samples collected throughout the year. detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. influenza a/h5n1 virus was detected in 79% of samples positive for influenza a virus and 35% of all samples collected. sequence analysis of full-length haemagglutinin (ha) and neuraminidase (na) genes from a/h5n1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with cambodian human cases during 2013 was the only a/h5n1 virus detected during the year. however, multiplex reverse transcriptase-polymerase chain reaction (rt-pcr) analysis of ha and na genes revealed co-circulation of at least nine low pathogenic aivs from ha1, ha2, ha3, ha4, ha6, ha7, ha9, ha10 and ha11 subtypes. four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to a/h5n1 and a/h9n2, respectively. seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for a/h5n1 and a/h9n2, respectively. peak aiv circulation was associated with the lunar new year festival. knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel aivs. avian influenza viruses (aivs) naturally infect the gastrointestinal tracts of wild birds from the orders anseriformes (ducks, geese and swans) and charadriformes (waders and gulls). within these hosts, 16 haemagglutinin (ha) and nine neuraminidase (na) surface glycoprotein types have been described. the ha and na types carried by the virus are used for classification and can be found in various combinations, such as a/h5n1 and a/h3n2. wild birds have been implicated in the global dissemination of aivs and, in some instances, the introduction of aivs into domestic poultry populations. 1 although low pathogenic avian influenza (lpai) viruses (which have limited impact on poultry mortalities) constitute the majority of these viruses, highly pathogenic avian influenza (hpai) viruses such as a/h5n1 can result in mortalities approaching 100% when introduced into domestic flocks. 2 influenza a viruses, which constitute all of the known aivs, have a segmented genome with eight separate rna strands enclosed in the virus. when an animal is co-infected with two different influenza viruses, reassortment of these rna segments can occur, resulting in a new virus with characteristics distinct from the parent viruses. 3 the 2009 emergence and resulting pandemic from a/h1n1pdm09 virus was caused by a virus that underwent multiple reassortment events with strains from pigs, birds and humans before suddenly gaining the ability to efficiently transmit between humans. 4 pandemic events resulting from reassortment of influenza viruses have occurred at least three times in the last 100 years, 5, 6 resulting in high human morbidity and mortality worldwide. the most severe influenza pandemic in recorded history, the 1918 'spanish flu', is thought to have arose from an avian influenza strain that directly adapted to the human host. 7 clearly it is imperative that close monitoring of reassortments and mammalian-adapted mutations in avian influenza strains is needed. since the emergence of hpai a/h5n1 in southern china in 1996 and 1997, 8 descendants of this virus have caused considerable economic losses in poultry populations, primarily in east and southeast asia, but also in africa and europe. influenza a/h5n1 has been identified as a significant threat for pandemic emergence. to date, at least 844 people have been infected worldwide, resulting in 449 deaths (53% case fatality rate). the virus was first detected in cambodian poultry in early 2004 and the first human cases were detected in 2005. 9 as of december 2015, 56 human cases (including 37 deaths) and 43 poultry outbreaks of influenza a/h5n1 have been recorded in the country. 10, 11 live-bird markets (lbms) have been implicated as an important source of human sporadic cases and dissemination of avian influenza since the emergence of influenza a/h5n1. 12 previous studies have established that lbms serve as hubs for the circulation and persistence of aivs through the presence of multiple avian species, the constant introduction of immunologically naive hosts and the frequent lack of biosecurity measures. 1, 13, 14 recent surveillance studies have documented that a/h5n1 and a/h7n9 commonly co-circulate with other subtypes of avian influenza in lbms, [15] [16] [17] thus increasing the likelihood of reassortment events. in the present study we initiated poultry and environmental surveillance for a/h5n1 and other aivs in four lbms in cambodia to better ascertain virus circulation in this setting. a longitudinal human serosurvey was also conducted to investigate the risk of human avian influenza infections in exposed lbm workers. commencing in january 2013, through november 2013, environmental and poultry samples were collected at four lbms in cambodia: an lbm in central phnom penh (m1), a wholesaling farm/slaughter house in phnom penh (m2), an lbm in kampong cham province (m3) and an lbm in takeo province (m4) (figure 1 ). these lbms were selected for the study as they represent the largest poultry collection sites in the most densely populated region of the country. samples were collected from each market every 1-2 weeks (figure 1 ). the lbms in cambodia typically have poor biosecurity, with slaughtering of animals onsite occurring for a range of domestic animals ( figure 2 ). birds are typically sourced from backyard flocks by middlemen who transport the animals through a convoluted system of semi-commercial farms and stock houses before being transported to the main lbms. 18 thus, tracking the original source of poultry is usually not possible. during each market investigation, tracheal and cloacal swabs were collected (both samples pooled in one tube of viral transport medium for each animal) from four randomly selected poultry (three ducks and one chicken). environmental samples were also collected in the same cage/site where the poultry swabs were collected to investigate contamination of the lbms with aivs. during each mission, 50 ml of carcass wash water (water used to wash the carcasses once the poultry has been slaughtered and defeathered), 50 ml of poultry drinking water (small bowl of water placed in cages), soil/mud from an area within 50 cm around the poultry cages/poultry resting area and samples of discarded feathers were collected. the water, soil/mud and feather samples were processed and nucleic acids extracted following techniques described previously. 19, 20 extracts were then tested by quantitative real-time reverse-transcriptase polymerase chain reaction (qrt-pcr) for the detection of m, h5 and n1 genes. m-gene-positive samples, for which there was sufficient sample and high virus concentration (cto30; n = 78), were inoculated into specific pathogen-free embryonated chicken eggs for virus isolation. 21 isolates were then tested using influenza universal multiplex rt-pcr assays to test for all known subtypes of influenza. 22 universal multiplex rt-pcr typing assays were also applied to the original samples where isolation could not be achieved but discordance between m-gene and h5 qrt-pcr testing suggested the presence of a non-h5 aiv. the amplified rt-pcr products from isolates and original material were submitted to a commercial sequencing facility (macrogen, seoul, south korea) for sequencing by the sanger method. full-genome sequences were generated from representative a/h5n1 isolates using methods previously described. 9, 23 contiguous sequences were assembled using clc workbench (clc bio) and compared to representative influenza virus sequences downloaded from the ncbi genbank database. neighbour-joining trees were constructed with mega5 24 and bootstrap values were calculated and expressed as a percentage from 1000 replicates. the longitudinal human serological study was approved by the national ethics committee for human research (approval no. 267, 24 december 2012). serum samples were collected, after obtaining informed consent, from lbm workers at the start of the study (january 2013) to form a baseline, and 8 weeks after the three major national festivals shown by previous work to be associated with increased a/h5n1 circulation in markets: 25 lunar new year, week 6; khmer new year, week 15; pchum ben, week 40. all adult-age lbm sellers or workers were exhaustively recruited in the four targeted livepoultry markets. the sample size could not be calculated as transmission to humans was unlikely and its probability in cambodia is unknown. participants were informed to report any acute febrile, respiratory or digestive signs, and were provided with a toll-free phone number. serum samples were tested for avian influenza a/h5n1, a/h9n2 and a/h7n9 antibodies using the haemagglutination inhibition assay (hia) and microneutralization assay (mn). the hia and mn testing were performed using influenza a/h5n1 clade 1.1.2 reassortant viruses (a/cambodia/x0121311/2013 and a/cambodia/ x0125302-/2013), which were isolated from human cases during 2013, and influenza a/h9n2 virus (a/environment/cambodia/e265/2013), which was isolated from the lbms during 2013. the hia and mn testing for a/h7n9 were performed using the strain a/anhui/01/2013, supplied through the world health organization (who) global influenza surveillance and response system (dr sylvie van der werf, department of virology, institut pasteur, paris, france). exposure to these viruses was considered 'confirmed' with a hia titre of ≥ 80 and a mn titre of ≥ 40. a seroconversion was defined as the detection of antibodies equal to or above the thresholds defined above following no detection of antibodies in the serum sample from the previous period. laboratory data were entered using an excel spreadsheet (microsoft excel, microsoft, redmond, wa, usa). a baseline assessment was documented using point prevalence for influenza antibodies, and incidence rates during follow-up were then computed using data on laboratory-confirmed seroconversions in lbm workers (numerator), and the number of days elapsed during the last serosurvey (denominator). poisson confidence intervals for the incidence rates were computed using stata 11 (stata corp., college station, tx, usa) with the function cii for the binomial ci, and the functions cii and the option 'poisson' for the poisson ci. during the study, a total of 1048 samples were collected, with 45% of all samples positive for influenza a rna by qrt-pcr (table 1) . influenza a viruses were detected in at least one sample during 93% of 120 collection missions, with influenza virus detected in three of the markets (m1, m2 and m3) on all but one sampling mission and detected in 83% of sampling missions from the remaining market (m4). influenza a (m-gene) ribonucleic acid (rna) was detected most frequently in carcass wash water samples (75%, n = 146), followed by feathers (61%, n = 138), poultry drinking water (50%, n = 138), soil/mud (48%, n = 146), duck swabs (32%, n = 358) and chicken swabs (18%, n = 122). h5 and n1 genes were detected in 79% (n = 372) and 58% (n = 270), respectively, of the 468 samples that tested positive for influenza a (m-gene), which accounted for 35% and 26% of all samples collected, respectively. peak aiv circulation was detected from january through march, with particularly high detection rates during the lunar new year festival period (figure 3) . influenza a/h5n1 virus was isolated from 71% (55/78) of samples from which h5 was detected and isolation was attempted. isolation was only attempted on high viral load samples (m-gene qrt-pcr ct full-gene sequences were generated for h5 (n = 22) and n1 (n = 21) for all viruses detected in the study where sufficient virus concentration allowed for successful sequencing (figure 4) . 10, 23 fullgenome sequences (eight fragments) were generated for six influenza a/h5n1 viruses detected during the study (supplementary figure s1) . for other viruses full-gene sequences could not be generated for all fragments (supplementary table s1 ). all a/h5n1 viruses detected in the study clustered with the clade 1.1.2 reassortant strains associated with human cases and poultry outbreaks of a/h5n1 during 2013. 10 non-h5 avian influenza detection a large number and variety of non-h5 avian influenza strains were detected during the study. full-ha sequences were generated from two h1, two h2, three h3, nine h4, 15 h6, one h7, 27 h9, one h10 and twelve h11 viruses (supplementary figure s2) . full na sequences from twenty-two n2, one n3, one n5, two n6, one n8 and two n9 subtypes were also generated from the same samples (supplementary figure s3) . however, as many of the environmental samples contained evidence of multiple avian influenza strains, it was difficult to ascertain that ha and na sequences derived from the same sample actually belonged to the same virus strain. co-infections between aivs were not detected in any poultry samples. many other partial ha and na sequences were also generated, but not included in these results due to difficulties in determining close phylogenetic relationships from incomplete gene sequences. aivs were detected throughout the year ( figure 5) , with a distinct peak of activity during january − march, perhaps mostly due to the increased circulation of a/h5n1 during major cambodian festivals, as previously reported. 25 longitudinal human serosurvey successive serological surveys in the poultry worker cohort provided evidence of seroconversions and some prior exposure (table 2) . at baseline sampling (january 2013), 125 participants were enrolled in the study, with one person testing positive to a/h5n1 antibodies and another testing positive to a/h9n2 antibodies. participant retention was high throughout the study, with 117, 105 and 106 people resampled at the second (march 2013), third (june 2013) and fourth (november 2013) sampling missions, respectively. seroconversions to a/h5n1 were detected for two participants at the third sampling and two participants at the fourth sampling. seroconversions to a/h9n2 were detected for one participant at the third sampling. overall seroprevalence was 4.5% for a/h5n1 and 1.8% for a/h9n2. rates of seroconversion were 3.7 infections per 1000 person-months for a/h5n1 and 0.9 infections per 1000 person-months for a/h9n2. there was no serological evidence of exposure or molecular detection of a/h7n9 before or during the study. there were no calls to the toll-free number reporting an incident with clinical signs or symptoms compatible with influenza infection. lbms have been established as important foci for the transmission of aivs and the potential emergence of reassortant strains. 13 the presence of multiple host species and the continued introduction of naive birds create an ideal environment for the persistence and emergence of aivs. exposure to live poultry has been associated with fatal human a/h5n1 infections, which for instance led to the government of hong kong rapidly closing lbms in 1997 and slaughtering large numbers of poultry. 26 the lbms surrounding phnom penh have been established as foci for poultry movement in the country 18, 27 and a previous market surveillance study in 2011 revealed a high rate of influenza a/h5n1 circulation in this setting. 25 to our knowledge, aivs were detected in cambodian lbms during 2013 at a higher frequency than any other study published previously, 25 5 6 7 9 11 12 13 14 15 16 17 19 21 23 25 27 29 31 33 35 37 38 39 40 41 42 avian influenza viruses in cambodian live-bird markets sv horm et al chicken swabs positive for influenza a rna. the majority of the aivs detected were likely a/h5n1, with 35% of samples positive for h5 qrt-pcr. although there was clearly high co-circulation of lpai viruses, much of the difference between the detection rates of m-gene, h5 and n1 was probably due to the differing qrt-pcr assay sensitivities (m4h54n1). we previously detected high circulation of a/h5n1 in these same markets in 2011. 25 however, in 2013 there was a 42.5-fold increase in the frequency with which the virus was detected despite the same sample processing, nucleic acid extraction and rt-qpcr methods being used. in 2013, cambodia had the highest confirmed human a/h5n1 caseload per capita in the world. during 2013 alone, 26 human a/h5n1 cases (14 deaths) were detected in cambodia, a dramatic increase in the 21 total cases that had been detected in the preceding 8 years, 2005-2012. this rise in reported human cases of a/h5n1 coincided with the emergence of a reassortant virus that contained the ha and na genes from the previously circulating clade 1.1.2 genotype z virus, and the matrix and internal genes from a clade 2.3.2.1 virus previously circulating in southern vietnam. 10 sequence and phylogenetic analyses of the ha and na genes ( figure 4 ) and the matrix and internal genes (supplementary figure s1 ) from the a/h5n1 market strains revealed that all of the viruses clustered closely with other clade 1.1.2 reassortant strains associated with poultry outbreaks and human cases in 2013. 10 questions remain regarding the causes of the dramatic increase in human cases in 2013 and whether the reassortant strain is more transmissible in poultry, resulting in the intense circulation that we observed in this report. alternatively, increased surveillance and education of clinicians may have resulted in improved detection of human a/h5n1 cases. presently we do not know the exact role played by lbms in the infection of poultry workers and market clients. similarly to our previous lbm surveillance in 2011, 25 increased circulation of aivs was detected before and during the major cambodian festivals (figure 3 ). in particular, increased circulation of influenza a viruses was detected during the period between the lunar new year and khmer new year festivals. a previous study on the poultry trade links in cambodia established that there was a significant increase in the trade volume of poultry prior to these festivals, with an assumed rise in cross-border poultry trade in response to increased demand. 18 the greater volume of poultry trade and the expansion of poultry trading networks during these festival periods present opportunities for virus spread throughout domestic flocks and the introduction of new strains of aivs. peaks in aiv levels were also observed during times that were not associated with known festivals (e.g. week 25 and 35). climatic factors, which were not analysed in this study, are also likely to have an influence on levels of aiv circulation. knowledge of these periods of intense circulation should inform future control policies such as targeted poultry vaccination, quarantining and improved market cleaning. such measures have proven effective in reducing avian influenza viral isolation rates in lbms [35] [36] [37] and may reduce the spread of aivs back to farms through fomites and personnel. 38 environmental sample testing showed that the lbm environment is highly contaminated with aivs (table 1) . influenza a detection rates were highest in carcass wash water samples, which serve as 'pooled' samples for multiple slaughtered birds. during the slaughtering process at the markets, birds are defeathered and eviscerated before carcasses are washed in a large bucket of water. we observed that 20-30 carcasses were often washed in the same container of water before it was refreshed. these samples were positive for influenza a rna in 75% of samples from all four markets (ranging from 63 to 86%). poultry drinking water was also a useful surveillance sample, with 50% of samples testing positive for influenza a rna; a/h5n1 isolation rates were higher when compared to carcass wash water. detection rates and isolation rates were also high with soil/mud and discarded feather samples, but these specimens required more complex processing before testing. high contamination of the lbm environment, including relatively high rates of virus isolation, is evidence that the risk of human exposure is very high. carcass wash water and poultry drinking water samples proved a useful adjunct to poultry swabs for monitoring purposes in the lbms. based solely on the ha typing, analysis of lbm samples collected in 2013 revealed the presence of at least nine other subtypes of aivs co-circulating with a/h5n1 ( figure 5 ). this situation increases the likelihood of reassortment events occurring, which may result in the emergence of new influenza subtypes. the recent emergence of reassortant hpai subtypes of h5, such as a/h5n2, a/h5n5, a/h5n6 and a/h5n8, [39] [40] [41] [42] [43] is evidence of the risk of new viruses arising through reassortment events with a/h5n1. the sudden emergence of multiple h5 subtypes has not been adequately explained but could be related to the diversity of aivs currently circulating in domestic poultry populations. the emergence of a/h7n9 virus in china in early 2013 44 has also prompted increased concerns about the possibility of a pandemic virus emerging in the asia-pacific region. although most of the non-h5 aivs detected in this study likely pose little or no risk to humans, the high prevalence of multiple avian influenza strains is an indictment on the poor biosecurity associated with poultry rearing and selling in the region. previous studies and surveillance have also established that there is intense circulation exposure to these viruses was considered positive with a haemagglutination inhibition assay titre of ≥ 80 and a microneutralization assay titre of ≥ 40. c only participants who provided samples for at least two time points were included in these analyses. d participants were removed from further calculations once they were recorded as 'positive' in the assumption that antibodies are protective against further infections. e one-sided, 97.5% ci. of poultry between regions and across borders, which facilitates the regional spread of aivs. 9, 23 in our study, the detection of a/h5n1 was achieved using qrt-pcr, whereas the detection of other aivs was done using universal multiplex rt-pcr assays with comparatively reduced sensitivity. in addition, multiplex testing was only conducted where there was disparity between m-gene and h5 qrt-pcr assays. furthermore, due to the difficulties with classifying influenza viruses using incomplete gene sequences we have only reported lpai detection where we have been able to generate full ha or na sequences. it is therefore likely that the true prevalence of non-h5 aivs was far greater than what we have reported in these market samples. the potential for reassortment between aivs, including a/h5n1, could result in the emergence of viral strains with considerable impacts on poultry and/or human health. this study, coupled with other recent studies in lbms from other asian countries, [15] [16] [17] confirms that the environment of lbms provides a pool of genes for potential emergence of new pandemic viruses. the silent circulation of a multitude of lpai viruses, which remain undetected and unmonitored in most asian countries, heightens the threat when they co-circulate with pandemic candidates such as a/h5n1 and a/h7n9. as no poultry workers reported any symptoms in relation to acute avian influenza infection, seroconversions in this study would most likely be related to sub-clinical or very mild cases. it has been reported that asymptomatic and mild avian influenza infections lead to seroconversions with low antibody titres that quickly decrease below the threshold recommended by who for a confirmed a/h5n1 case (hia ≥ 160; mn ≥ 80). 45 for this reason, we considered as positive all individuals with a hia titre of ≥ 80 and a mn titre of ≥ 40, which is consistent 46 or more stringent 47-52 than the cut-off levels suggested by other authors. however, it is difficult to directly compare the results between studies as there is no consensus on the antibody titre that results from a mild or asymptomatic infection, and consistent cut-off levels have not been used in past studies. according to our classification, 4.5% of workers who participated in the study had antibodies against a/h5n1. this prevalence is much higher than that reported among lbm workers in egypt, 53 thailand 47 or china, 54 and much higher than in villagers in thailand 51 or cambodia. 48 the incidence was 3.7 per 1000 person-months in our study, a figure twice that described in bangladesh in 2009-2010. 46 there were no reported clinical signs in our cohort, confirming that a large proportion of human a/h5n1 infections may go undetected. poultry workers in cambodia and other asian countries, where there is endemic circulation of a/h5n1, are constantly exposed to high levels of virus. although this may lead to mild or sub-clinical infections with seroconversions, it seems that transmission to humans resulting in acute infections is still rare. influenza a/h9 viruses, predicted to be a/h9n2 based on the phylogenetic analysis of the ha and na sequences repeatedly detected in the same samples, were detected in 2.6% of samples analysed in this study. although a/h9n2 viruses have low pathogenicity for poultry, novel hpai and lpai viruses affecting humans, such as a/h5n1, a/h7n9 and a/h10n8, contain internal genes originating from influenza a/h9n2. [55] [56] [57] this may have facilitated the ability of these viruses to cause infection and deaths in humans. human infections with a/h9n2 have been reported in the literature, and seroprevalence studies have suggested that asymptomatic or mild infections commonly occur in high-risk locations such as lbms and slaughterhouses. 50, 53, 58, 59 the detection of a/h9n2 antibodies in 1.8% of lbm workers participating in our study and an incidence of 0.9 infections per 1000 person-months confirm other data on poultry-tohuman transmission of a/h9n2, 53,59 including among rural villagers in cambodia. 48 in addition, a/h9n2 infections have also been detected in other mammals such as guinea pigs, dogs, horses and pigs. 59, 60 the ability of a/h9n2 to cross the species barrier and the relatively high frequency in which it has been implicated in reassortment events suggest that this virus significantly contributes to the emergence of viruses that pose an important public health risk and should therefore be very closely monitored. in this study we document intense co-circulation of influenza a/h5n1 and lpai viruses in cambodian lbms during 2013. in addition, serological surveys provided evidence of sub-clinical a/h5n1 and a/h9n2 infections. interventions such as regular cleaning/disinfection, bans on overnight poultry storage, targeted closure during periods of peak circulation and segregation of poultry slaughtering areas should be considered in lbms to reduce the threat of the emergence of aivs with public health or animal health impacts. further monitoring of the circulation of influenza a/h5n1 in cambodian lbms and research into the mechanisms associated with human cases is warranted. ); and the world health organization country office in cambodia (grant no ecology of avian influenza viruses in a changing world ecology of avian influenza virus in birds a new avian influenza virus from feral birds in the ussr: recombination in nature? origins and evolutionary genomics of 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of low pathogenic avian influenza viruses of domestic poultry in vietnam and their genetic relevance with asian isolates the diversity of avian influenza virus subtypes in live poultry markets before and during the second wave of a(h7n9) infections in hangzhou genetic and molecular characterization of h9n2 and h5 avian influenza viruses from live poultry markets in zhejiang province, eastern china poultry movement networks in cambodia: implications for surveillance and control of highly pathogenic avian influenza (hpai/h5n1) development and validation of a concentration method for the detection of influenza a viruses from large volumes of surface water direct detection of highly pathogenic avian influenza a/h5n1 virus from mud specimens environment: a potential source of animal and human infection with influenza a (h5n1) virus. influenza other respir a high diversity of eurasian lineage low pathogenicity avian influenza a viruses circulate among wild birds sampled in egypt dynamic of h5n1 virus in cambodia and emergence of a novel endemic sub-clade mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods influenza a (h5n1) virus surveillance at live poultry markets, cambodia case-control study of risk factors for avian influenza a (h5n1) disease, hong kong identifying live bird markets with the potential to act as reservoirs of avian influenza a (h5n1) virus: a survey in northern viet nam and cambodia isolation and characterization of avian influenza viruses, including highly pathogenic h5n1, from poultry in live bird markets in hanoi influenza virus (h5n1) in live bird markets and food markets circulation of avian influenza h5n1 in live bird markets in egypt environmental sampling for avian influenza virus a (h5n1) in live-bird markets detection of influenza a virus in live bird markets in kenya detection of hpai h5n1 viruses in ducks sampled from live bird markets in vietnam avian influenza h5n1 surveillance and its dynamics in poultry in live bird markets the impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in hong kong effect of interventions on influenza a (h9n2) isolation in hong kong's live poultry markets avian influenza and ban on overnight poultry storage in live poultry markets risk for infection with highly pathogenic influenza a virus (h5n1) in chickens emerging multiple reassortant h5n5 avian influenza viruses in ducks, china emergence and evolution of avian h5n2 influenza viruses in chickens in taiwan novel reassortant influenza a(h5n8) viruses, south korea novel eurasian highly pathogenic avian influenza a h5 viruses in wild birds reassortant highly pathogenic influenza a(h5n6) virus in laos human infection with a novel avian-origin influenza a (h7n9) virus kinetics of neutralizing antibodies in patients naturally infected by h5n1 virus highly pathogenic avian influenza a(h5n1) virus infection among workers at live bird markets evidence for subclinical avian influenza virus infections among rural thai villagers evidence for avian h9n2 influenza virus infections among rural villagers in cambodia serological evidence for avian h9n2 influenza virus infections among romanian agriculture workers seroprevalence of avian influenza h9n2 among poultry workers in shandong province, china prospective study of avian influenza virus infections among rural thai villagers seroprevalence survey of avian influenza a(h5n1) among live poultry market workers in northern viet nam avian influenza a(h5n1) and a(h9n2) seroprevalence and risk factors for infection among egyptians: a prospective, controlled seroepidemiological study seroprevalence of avian influenza a (h5n1) virus among poultry workers in jiangsu province, china: an observational study molecular characterization of h9n2 influenza viruses: were they the donors of the 'internal' genes of h5n1 viruses in hong kong? clinical and epidemiological characteristics of a fatal case of avian influenza a h10n8 virus infection: a descriptive study evolution of the h9n2 influenza genotype that facilitated the genesis of the novel h7n9 virus avian influenza h9n2 seroprevalence among poultry workers in pune a systematic review and meta-analysis of the seroprevalence of influenza a (h9n2) infection among humans a global phylogenetic analysis in order to determine the host species and geography dependent features present in the evolution of avian h9n2 influenza hemagglutinin the authors would like to thank poultry workers at the live-bird markets for their cooperation with the study team. we thank the technical staff at the virology unit and investigation team at the epidemiology and public health unit, institut pasteur in cambodia. the authors would also like to thank the investigation team from the national veterinary research institute, ministry of agriculture, forestry and fisheries. this work was funded by the key: cord-252293-8286lsof authors: suzuki, motoi; katsurada, naoko; le, minh nhat; kaneko, norihiro; yaegashi, makito; hosokawa, naoto; otsuka, yoshihito; aoshima, masahiro; yoshida, lay myint; morimoto, konosuke title: effectiveness of inactivated influenza vaccine against laboratory-confirmed influenza pneumonia among adults aged ≥65 years in japan date: 2018-05-17 journal: vaccine doi: 10.1016/j.vaccine.2018.04.037 sha: doc_id: 252293 cord_uid: 8286lsof background: the effectiveness of inactivated influenza vaccine (iiv) against laboratory-confirmed influenza pneumonia in older adults remains to be established. methods: pneumonia patients aged ≥65 years who visited a study hospital in chiba, japan, were prospectively enrolled from february 2012 to january 2014. sputum samples were collected from participants and tested for influenza virus by polymerase chain reaction assays. influenza vaccine effectiveness (ive) against laboratory-confirmed influenza pneumonia was estimated by a test-negative design. results: among a total of 814 pneumonia patients, 42 (5.2%) tested positive for influenza: 40 were positive for influenza a virus, and two were positive for influenza b virus. the ive against laboratory-confirmed influenza pneumonia was 58.3% (95% confidence interval, 28.8–75.6%). the ive against influenza pneumonia hospital admission, severe pneumonia, and death was 60.2% (95% ci, 22.8–79.4%), 65.5% (95% ci, 44.3–78.7%), and 71% (95% ci, −62.9% to 94.8%), respectively. in the subgroup analyses, the ive against influenza pneumonia was higher for patients with immunosuppressive conditions (85.9%; 95% ci, 67.4–93.9%) than for those without (48.7%; 95% ci, 2.7–73%) but did not differ by patients’ statin use status. conclusion: iiv effectively reduces the risk of laboratory-confirmed influenza pneumonia in older adults. influenza is a major public health concern for older adults. influenza infections generally cause self-limited illnesses but can result in severe disease such as pneumonia in older adults with and without underlying conditions. older age is associated with a higher risk of pneumonia and mortality in influenza patients [1] . based on our recent estimates, the incidence of influenza pneumonia and its related mortality among people aged !65 years in japan were 210 and 24 per 100,000 persons/year, respectively [2] . cumulative evidence has suggested that influenza vaccines are effective at reducing the risk of medically attended influenza in children and adults [3, 4] . currently, seasonal influenza vaccination is recommended for older adults in more than 90 countries [5] . however, its clinical benefit has long been discussed because vaccine responses are reduced by an age-related decline in adaptive immunity [6, 7] . positive results have been reported from recent meta-analyses: influenza vaccines reduce medically attended influenza by 20-44% [8] and influenza-associated hospitalization by 37% in older adults [9] . however, evidence is lacking for the protective effect of influenza vaccination on influenza pneumonia, including primary influenza pneumonia and secondary bacterial pneumonia. in a study by grijalva influenza pneumonia by 56.7%, although the majority of their patients were people aged <65 years [10] . therefore, the influenza vaccine effectiveness (ive) against laboratory-confirmed influenza pneumonia in older adults remains to be established. we conducted this study to investigate the effectiveness of the trivalent inactivated influenza vaccine (iiv) against laboratoryconfirmed influenza pneumonia and its related outcomes in adults aged !65 years. we also conducted subgroup analyses to explore differences in ive by patient characteristics, particularly those related to immunosuppressive status. this single-center prospective study was conducted at kameda medical center (kmc), kamogawa, chiba, japan, as part of the adult pneumonia study group-japan (apsg-j) study [2, [11] [12] [13] . the apsg-j study was a multicenter prospective study of adult pneumonia conducted at four community-based hospitals in japan from september 2011 to august 2014. to investigate ive, influenza vaccination history was systematically collected at kmc. in this study, pneumonia patients aged !65 years who visited kmc from february 2012 to january 2014 were included. the diagnosis of pneumonia was made by staff physicians according to clinical signs, symptoms, and radiological findings. demographic and clinical information was collected from patients and medical charts. sputum samples were collected from patients at the time of enrollment. if the patient was unable to cough up sputum, sputum was induced with the inhalation of hypertonic saline solution. details of study settings and designs have been described previously [2, 13] . gram staining and sputum culture were performed on site. sputum samples were transferred to the institute of tropical medicine, nagasaki university, and tested by in-house multiplex polymerase chain reaction (pcr) assays to identify the influenza virus (a and b) and 11 other viral pathogens (respiratory syncytial virus [rsv], human metapneumovirus, human parainfluenza virus types 1-4, human rhinovirus [hrv], human coronavirus 229e/oc43, human adenovirus, and human bocavirus) [14] . the detection limits of the multiplex pcr assays were 10 -100 copies per reaction as reported previously [14] . influenza virus subtyping was performed for influenza a-positive samples via rt-pcr of the influenza ha genes using previously published methods [15, 16] . patients were defined as having laboratory-confirmed influenza pneumonia if their sputum sample tested positive for influenza a or b virus by pcr. influenza pneumonia patients were classified as having influenza-associated bacterial pneumonia if their sputum samples were microscopically purulent (i.e., geckler's classification groups 4 and 5) and tested positive for bacterial pathogens by culture or pcr; otherwise, they were classified as having primary influenza pneumonia. a test-negative design (tnd) case-control study was applied to estimate ive [17] . unlike the conventional case-control design, the tnd does not require non-disease controls; instead, in this study design, researchers collect clinical samples from patients with a specific condition (eg, influenza like illnesses) and classify the patients into cases (i.e., influenza tested positive patients) and controls (i.e., influenza tested negative patients) according to the influ-enza test results. the tnd is less susceptible to bias due to differences in health care-seeking behavior among cases and controls and provides reliable ive estimates [18, 19] . recently, tnd studies have been widely used to estimate ive against medically attended influenza and influenza-associated hospitalization [8, 9] . in the current study, our primary outcome was laboratoryconfirmed influenza pneumonia. cases were pneumonia patients who tested positive for influenza a or b, and controls were pneumonia patients who tested negative for both influenza a and b. the odds of vaccination were compared between cases and controls, and ive was expressed as (1-odds ratio) â 100%. our secondary outcomes were (1) primary influenza pneumonia, (2) influenza-associated bacterial pneumonia, influenza pneumonia-related hospital admission, (4) severe influenza pneumonia, and (5) influenza pneumonia death. in japan, all adults aged !65 years are recommended by the ministry of health, labor and welfare to receive one dose of the seasonal influenza vaccine [20] . the trivalent iiv vaccine was used during the study period (2011-12, 2012-13, and 2013-14 seasons); the quadrivalent iiv vaccine was introduced in the 2015-16 season. high-dose or adjuvanted iivs have not been licensed in japan. the compositions of the trivalent iiv vaccines used during the study seasons and their antigenic match status are summarized in supplementary table 1 . influenza vaccination histories were collected from medical records and confirmed by patients and/or their guardians. patients were considered vaccinated for influenza if they had received at least one dose of influenza vaccine in the 12 months before the hospital visit. because the duration from influenza vaccination to the hospital visit was recorded as a monthly data, all patients who had been vaccinated within a month were considered vaccinated in our primary analysis. patients were considered as having unknown influenza vaccination statuses if their influenza vaccination histories were not recorded in medical charts and could not be confirmed by the patients or their guardians; this group was excluded from our primary analysis. patients were categorized into three age groups: 65-74 years, 75-84 years, and 85 years or older. patient disability status was evaluated using the eastern cooperative oncology group performance status score [21] . body mass index (bmi, kg/m 2 ) was classified as underweight (<18.5), normal (18.5-24.9), or overweight (!25.0). chronic conditions included diabetes mellitus, heart failure, ischemic heart disease, cerebrovascular disease, liver disease, renal disease, neurological disease, cancer, chronic obstructive pulmonary disease, bronchial asthma, and previous tuberculosis disease. immunosuppressive status included cancer, oral steroid use, and immunosuppressive drug use. patients were considered to have severe pneumonia if they required oxygen therapy, mechanical ventilation, or a vasopressor after admission. the period from november to april was considered the influenza season. the characteristics of patients were compared according to influenza infection status (i.e., influenza pneumonia vs. noninfluenza pneumonia) and influenza vaccination status (i.e., vaccinated vs. unvaccinated) using chi-square tests and fisher's exact tests for categorical variables and wilcoxon rank sum tests for numerical variables. ive was estimated using logistic regression models. pre-specified confounding factors were sex, age, the pres-ence of chronic conditions, the presence of immunosuppression, smoking status, the duration from onset to hospital visit, and the period of the study, and all these variables were included in the final multivariable logistic regression models. we also considered the performance status score and bmi category as potential confounders and examined if ive estimates changed after adjusting for these variables. confidence intervals (cis) were adjusted for the residential area level clustering of patients using robust standard errors. we conducted sensitivity analyses as follows: (1) restricting the analysis to patients who visited during influenza seasons; (2) excluding patients vaccinated <1 month prior to hospital visit; (3) excluding patients vaccinated >6 months prior to hospital visit; (4) using patients who were negative for influenza virus but positive for non-influenza respiratory viruses as controls; (5) using patients who were negative for all viruses as controls [22] ; (6) using propensity scores for adjustment; and (7) including patients with unknown vaccination status using multiple imputation. stratified analyses were conducted to investigate the potential effect modifications by patient characteristics (i.e., sex, age group, underlying condition, immunosuppressive status, and statin use status). stratum-specific ive estimates were compared using a likelihood ratio test (test for interaction). this study was approved by the institutional review board (irb) of the institute of tropical medicine, nagasaki university, nagasaki, japan and the irb of kameda medical center, chiba, japan. anonymized data were used in this study. during the study period, a total of 1494 pneumonia patients aged !65 years were enrolled in the study. among them, sputum samples were obtained from 1044 patients (70%). after excluding 230 patients whose influenza vaccination history were unavailable (22% of patients with sputum samples), a total of 814 patients were eligible for our analyses (fig. 1) . among them, 42 (5%) tested positive for influenza virus by pcr: 40 were positive for the influenza a virus, and the other two were positive for the influenza b virus. among the 26 influenza a-positive samples that were subtyped (65% of all influenza a-positive samples), all were positive for the h3n2 strain. non-influenza viruses were detected in 178 patients: hrv was the leading virus detected (n = 77, 9%), followed by rsv (n = 36, 4%). non-influenza viruses were co-detected in 6 of the 42 influenza-positive patients (14%) and detected in 172 of the 772 influenza-negative patients (22%). bacterial pathogens were co-detected in 26 of the 42 influenza-positive patients (62%). demographic and clinical characteristics were compared between influenza pneumonia patients (i.e., cases) and noninfluenza pneumonia patients (i.e., controls) (tables 1 and 2 ). cases were more frequently found in winter seasons than controls, but other characteristics were similar between cases and controls. among 814 patients, 525 (65%) had been vaccinated for influenza. vaccinated patients more frequently had received home oxygen therapy and had been diagnosed with chronic respiratory obstructive disease than unvaccinated patients, while other characteristics were similar between two groups (tables 1 and 2) . after adjusting for confounders, the ive against laboratoryconfirmed influenza pneumonia was 58.3% (95% ci, 28. ( table 3 ). the change in ive estimates was marginal after additional adjustment for performance status (58.9%; 95% ci, 30.6-75.7%) or bmi category (58.0%; 27.6-75.6%); therefore, these variables were not included in the final models. the sensitivity analyses showed similar results. ive was relatively higher (68.9%; 95% ci, 46.4-81.9%) when we used patients who were negative for influenza but positive for non-influenza viruses as controls, but the value was almost identical to the primary analysis when we used patients who were negative for all viruses (57.8%; 95% ci, 26.9-75.7%). for the secondary outcomes, the ive against primary influenza pneumonia (70.1%; 95% ci, 19.8-88.9%) was higher than that table 4 . the ive against influenza pneumonia was higher in patients with immunosuppressive conditions (85.9%; 95% ci, 67.4-93.9%) than in those without these conditions (48.7%; 95% ci, 2.7-73%; test for interaction, p = 0.001). ive did not differ by sex. the point estimate of ive decreased with increased age, but the difference did not reach a statistically significant level (test for interaction, p = 0.17). patients' chronic conditions and statin use status did not modify ive. iiv effectively reduced the risk of laboratory-confirmed influenza pneumonia in adults aged !65 years. ive was higher among patients with immunosuppressive conditions, while statins did not modify ive. to our knowledge, this is the first study that confirmed the beneficial effect of seasonal influenza vaccination against laboratory-confirmed influenza pneumonia in older adults. the benefit of seasonal influenza vaccination in older adults is still debated [3, 23] . in this age group, the age-related decline in adaptive immunity results in reduced responses to influenza vaccination [6, 7] ; moreover, multiple chronic conditions and frailty may also contribute to weak immune responses [24] . however, despite an observed lower antibody response compared with that of younger adults [25] , recent evidence supports the protective effect of influenza vaccination against medically attended influenza in older adults. according to a systematic review by belongia et al, the pooled ive was 24% (95% ci, à6% to 45%) for the h3n2 strain, 63% (95% ci, 33-79%) for type b, and 62% (95% ci, 36-78%) for the h1n1pdm09 strain among adults aged >60 years [4] . darvishian et al conducted an individual participant data meta-analysis of tnd studies and demonstrated that influenza vaccination is moderately effective against laboratory-confirmed influenza in this age group during epidemic seasons but not during non-epidemic seasons [8] . on the other hand, evidence is still limited for the beneficial effect of influenza vaccination against influenza-related severe outcomes such as pneumonia. previous studies have estimated the ive against all-cause pneumonia or influenza-related hospitalization in older adults [9, [26] [27] [28] ; however, these studies used less specific outcomes and may have underestimated the true ive [17] . the tnd study by grijalva et al demonstrated that the overall estimate of ive against hospitalization with laboratory-confirmed influenza pneumonia was 56.7% (95% ci, 31.9-72.5%) [10] . however, their study included all age groups, and only 16% of their patients were aged !65 years. in their analysis restricted to patients aged !65 years, ive showed a positive effect but did not reach a statistically significant level (48.4%; 95% ci, à33.3% to 80%). therefore, the authors concluded that additional studies were needed to establish the ive against pneumonia in older adults. our study targeted this age group and demonstrated that the vaccine effectively reduces the risk of laboratory-confirmed influenza pneumonia by 58.3% (95% ci, 28.8-75.6%). our ive estimates against influenza pneumonia in older adults may be higher than generally expected values. ive is commonly lower for severe outcomes than for medically attended influenza and is lower in older adults than in children [3, 9] . in addition, ive is usually lower for the h3n2 stain than for the h1n1pdm09 strain [4] . however, our estimates are not dissimilar to those of previous reports: in the study by grijalva et al, the ive against influenza pneumonia related to the h3n2 strain in all age groups was 45.1% (95% ci, à9.3% to 72.4%) [10] , and in another study conducted during the 2011-12 influenza season when h3n2 was the dominant circulating strain, the ive against influenza hospitaliza[29] . the use of sputum samples in our study may also explain our high ive estimate. identification of influenza from sputum samples may be more sensitive and specific than that from upper respiratory tract samples in diagnosing influenza pneumonia and may provide less biased ive estimates [17, 30, 31] . consistent findings in our sensitivity analyses also support the robustness of our ive estimates. although a higher ive estimate was observed for primary influenza pneumonia, iiv was also effective at preventing influenzaassociated bacterial pneumonia (49.1%; 95% ci, 17.1-68.7%). this finding is important because influenza-bacterial co-infection increases the risk of severe outcomes [32] . our finding also suggests that iiv may be effective at preventing influenza pneumonia death; however, the association did not reach a statistically significant level because of the limited sample size. it was unexpected that the ive was significantly higher among people with immunosuppressive conditions (85.9%; 95% ci, 67.4-93.9%) than among those without (48.7%; 95% ci, 2.7-73%). the opposite finding was observed in the study by grijalva et al, which included children and adults (à21.9% vs. 73.4%) [10] . this difference might be, at least partially, explained by a lower hiv prevalence in our patients. although seasonal influenza vaccinations have been recommended for adults with immunosuppressive conditions [33] , only a few studies have evaluated the ive against clinical outcomes among this population [34] . our finding provides supporting evidence for the current recommendations but needs to be confirmed in future studies. recent studies have suggested that statins may reduce the ive against medically attended influenza among older adults by their immunomodulatory effects [35] [36] [37] [38] . however, such an effect has not been observed in our study. although the degree of its effect remains controversial, statins are also known to modify the risk of pneumonia and pneumonia-related outcomes [39] [40] [41] . the impact of statin use on the ive may be different according to influenza outcomes. influenza infection is a threat to older adults because of its potential to cause pneumonia and secondary bacterial infections [13] . the burden of pneumonia is rapidly increasing in highincome countries such as japan because of the aging population [2] . therefore, the prevention of influenza pneumonia is an important public health measure in controlling pneumonia. the moderate effectiveness observed in our study supports the current seasonal influenza vaccination policy. in japan, the proportion of people vaccinated against influenza among adults aged !65 years has been increasing but still remains approximately 60% [42] . in addition to improving vaccination coverage, an introduction of newer vaccines such as the more immunogenic high-dose influenza vaccine must be considered [43, 44] . on the other hand, it must be noted that only 5% of pneumonia cases have influenza pneumonia, and thus, the impact of influenza vaccination on allcause pneumonia is limited [45] . newer multidimensional approaches are needed to reduce the pneumonia burden in the aging population. our study has limitations. influenza vaccination history was not documented for 22% of our patients. however, our sensitivity analysis using multiple imputations showed very robust estimates. we believe that the exclusion of this patient group did not affect our ive estimates. although all potential confounders were considered, unmeasured confounders may have remained. recently, andrew et al argued that frailty must be considered in estimating ive for older adults [29] . we have not measured the frailty of our patients but measured their performance status and bmi. we confirmed that the inclusion of performance status or bmi category in the final model did not change the ive estimates. our observation is based on the analyses of older patients aged !65 years and therefore may not be generalizable to younger adults. finally, our sample size was too small to estimate subtype-specific ive. seasonal influenza vaccination is moderately effective against laboratory-confirmed influenza pneumonia in adults aged !65 years. considering the increasing burden of pneumonia in an aging population, we must improve influenza vaccination coverage and establish newer approaches. konosuke morimoto reports speaker fees from taisho toyama pharmaceutical, pfizer, and asahi kasei pharma. all other authors declare no competing interests. populations at risk for severe or complicated influenza illness: systematic review and meta-analysis the burden and etiology of community-onset pneumonia in the aging japanese population: a multicenter prospective study efficacy and effectiveness of influenza vaccines: a systematic review and meta-analysis variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies who. who vaccine-preventable diseases: monitoring system innate immunity in aging: impact on macrophage function the effect of ageing of the immune system on vaccination responses effectiveness of seasonal influenza vaccination in community-dwelling elderly people: an individual participant data meta-analysis of test-negative design case-control studies effectiveness of influenza vaccines in preventing severe influenza illness among adults: a systematic review and meta-analysis of test-negative design case-control 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accuracy and precision of vaccine efficacy estimates compared to the gold standard of randomised placebo-controlled clinical trials. euro surveillance: bulletin europeen sur les maladies transmissibles = theoretical basis of the testnegative study design for assessment of influenza vaccine effectiveness negative impact of prior influenza vaccination on current influenza vaccination among people infected and not infected in prior season: a testnegative case-control study in japan toxicity and response criteria of the eastern cooperative oncology group potential effect of virus interference on influenza vaccine effectiveness estimates in test-negative designs vaccines for preventing influenza in the elderly influenza vaccine response in community-dwelling german prefrail and frail individuals antibody response to influenza vaccination in the elderly: a quantitative review influenza vaccine effectiveness in preventing hospitalization for pneumonia in the elderly influenza vaccine effectiveness among elderly nursing home residents: a cohort study effectiveness of influenza vaccine in the community-dwelling elderly the importance of frailty in the assessment of influenza vaccine effectiveness against influenza-related hospitalization in elderly people yield of sputum for viral detection by reverse transcriptase pcr in adults hospitalized with respiratory illness comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses mechanisms of severe mortalityassociated bacterial co-infections following influenza virus infection vaccination recommendations for the adult immunosuppressed patient: a systematic review and comprehensive field synopsis influenza vaccines in immunosuppressed adults with cancer. cochrane database syst rev influence of statins on influenza vaccine response in elderly individuals impact of statins on influenza vaccine effectiveness against medically attended acute respiratory illness effect of statin use on influenza vaccine effectiveness how regulatory t-cell induction by statins may impair influenza vaccine immunogenicity and effectiveness the effect of statins on mortality from severe infections and sepsis: a systematic review and metaanalysis is statin use associated with reduced mortality after pneumonia? a systematic review and meta-analysis statin use and hospital length of stay among adults hospitalized with community-acquired pneumonia estimated influenza vaccination rates in japan comparative effectiveness of high-dose versus standard-dose influenza vaccines among us medicare beneficiaries in preventing postinfluenza deaths during comparative effectiveness of high-dose versus standard-dose influenza vaccination on numbers of us nursing home residents admitted to hospital: a cluster-randomised trial inactivated influenza vaccines for prevention of community-acquired pneumonia: the limits of using nonspecific outcomes in vaccine effectiveness studies we are grateful to all the adult pneumonia study group-japan contributors. we would like to thank professor koya ariyoshi, dr. eiichiro sando, and dr. tomoko ishifuji for their contribution to the study. we also thank rina shiramizu, kyoko uchibori for performing the pcr and yumi araki for administrative work. this study was supported by nagasaki university and pfizer japan, inc. supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.vaccine.2018.04. 037. key: cord-017291-bhe34dky authors: cohen, cheryl; reubenson, gary title: influenza date: 2017-05-05 journal: viral infections in children, volume i doi: 10.1007/978-3-319-54033-7_2 sha: doc_id: 17291 cord_uid: bhe34dky influenza is one of the commonest infections in human populations, and causing substantial morbidity and mortality globally. the influenza virus is divided into different types and subtypes, three of which are currently circulating widely in humans: influenza a(h3n2) and influenza b. the virus undergoes constant evolution, leading to annual seasonal winter epidemics in temperate countries and necessitating annual updates to the vaccine. rarely, completely new influenza viruses can emerge in human populations, giving rise to influenza pandemics. children aged <5 years (especially those <2 years) and those with underlying illness such as cardiac, respiratory and severe neurologic disease have an increased risk of severe outcomes associated with influenza. pregnant women have an increased risk of severe influenza. complications may involve the respiratory tract (e.g. otitis media or pneumonia) or, less commonly, other organ systems (e.g. encephalitis or myocarditis). specific antiviral treatment should be offered as soon as possible for hospitalized children with presumed or confirmed influenza and for influenza of any severity for children at high risk of severe complications of influenza without waiting for laboratory confirmation. antiviral treatment is usually not warranted for uncomplicated influenza as this is usually self-limiting. annual influenza vaccination should be offered to all individuals at increased risk for complications of influenza. vaccine cannot be given to children aged <6 months but maternal influenza immunization during pregnancy is recommended and can confer protection to the young infant. influenza is one of the commonest infections in human populations, infecting a significant percentage of the population each year and causing substantial morbidity and mortality globally. while seasonal influenza is an important cause of morbidity and mortality, novel influenza virus strains can emerge with the potential to cause global pandemics. children are a common source of inluenza transmission in the community and form an important risk group for severe influenza illness (particularly infants and children with underlying chronic illnesses). influenza viruses are enveloped viruses from the family orthomyxoviridae [1] . influenza has a negative sense rna genome divided into eight segments. the segmented genome allows for exchange of genes between influenza viruses of the same type through genetic reassortment. two types of influenza viruses, influenza a and b, cause epidemic disease in humans. the influenza a viruses are divided into subtypes and influenza b viruses into lineages based on their antigenic structure. the influenza a subtypes are differentiated based on characteristics of the haemagglutinin (ha) and neuraminidase (na) surface antigens. haemaglutinin is responsible for virus attachment during the early stages of infection and is the main antigen against which the host immune response is directed. neuraminidase facilitates the release of mature virus from the cell surface. currently there are two influenza a subtypes (influenza a(h3n2) and influenza a(h1n1)pdm09 and two influenza b lineages (yamagata and victoria) co-circulating globally in human populations. influenza a viruses of at least 17 ha and 9 na subtypes have been isolated from animals such as birds, pigs, horses and dogs. because many different ha and na subtypes can circulate in animals, animals such as birds and pigs may be the reservoir of emerging influenza virus subtypes which can infect humans [1] . several of these subtypes such as influenza a(h7n1) or influenza a(h5n2) can cause severe illness and even death in individuals in close contact with animals but are not able to be efficiently transmitted from person-to-person [2] . antigenic drift is the emergence of new influenza virus antigenic variants as a result of point mutations and recombination which occurs during viral replication [1] . this frequent emergence of antigenic variants contributes to seasonal influenza epidemics and leads to the requirement for annual assessment of the need to update the viruses included in the influenza vaccine. antigenic shift is a term for larger genetic changes which occur infrequently in influenza a viruses. new, or substantially different, influenza a virus subtypes which emerge in humans, have the potential to cause pandemics if they are efficiently transmitted between humans in the presence of little or no pre-existing population immunity [2] (fig. 2.1 ). epidemiology including pandemics it is estimated that 5-20% of the population become infected with influenza each year and about 20% of these develop symptomatic illness. rates of influenza infection are highest in children aged 5-15 years [3] . annually in children aged <5 years there are approximately 90 million new cases of influenza, 20 million cases of influenza-associated acute lower respiratory tract infection (alri) (13% of all cases of paediatric alri) and one million cases of influenza-associated severe alri (7% of all severe alri) globally [4] . between 28,000 and 111,500 influenza-associated deaths in children <5 years are estimated to occur each year, with 99% of these occurring in developing countries. influenza is associated with approximately 10% of respiratory hospitalizations in children <18 years worldwide ranging from 5% in children aged <6 months to 16% in children aged 5-17 years [5] . influenza-associated hospitalization rates are more than three times higher in developing than industrialised countries. the incidence and mortality associated with influenza can vary substantially from year-to-year as a result of different circulating types and subtypes with differing propensity to cause severe illness. years in which influenza a(h3n2) predominates may typically be associated with increased risk of severe disease [6] . people of all ages may develop symptomatic influenza infection but the highest rates of influenza-positive influenza-like illness (ili) are seen in children aged 2-17 years [7] . school-age children are an important source of infection in the community and influenza outbreaks can occur in schools during the influenza season [8] . during the influenza season, influenza is an important cause of school absenteeism. illness in children can cause a substantial economic burden as a result of caregiver absenteeism from work to care for ill children as well as outpatient visits in children and can lead to additional antibiotic courses being prescribed. hospitalizations and mortality during the influenza season can be substantial. in severe influenza seasons, the large number of medical care visits as a result of influenza can overwhelm health systems. the highest rates of influenza-associated hospitalizations and deaths are typically seen in individuals aged ≥65 years, <5 years and those with underlying medical conditions that confer an increased risk for severe influenza [9] . children aged <2 years and, to a lesser extent, those aged 2-5 years have increased rates of influenzaassociated hospitalization and mortality compared to older children. children with underlying illnesses, particularly cardiac, respiratory and severe neurologic disease have an increased risk of severe outcomes associated with influenza. a study from south africa, found that amongst children aged <5 years, malnutrition, prematurity and hiv infection were associated with increased odds of influenza-associated hospitalization [10] . hiv-infected children have an approximately two times elevated risk of influenza hospitalization and are more likely to die of influenza once hospitalized compared to hiv-uninfected children [11, 12] . pregnant women have an increased risk of severe influenza. some studies suggest that influenza in pregnancy may be associated with adverse outcomes in infants born to these women (such as low birth weight, pre-term birth and stillbirth), but others have disputed this [13] . in temperate climates influenza typically causes annual seasonal epidemics in the winter months, between april and september in the southern hemisphere and between october and april in the northern hemisphere [9, 14] . in more tropical climates influenza commonly circulates year-round with two or more peaks which may coincide with climatic events such as the rainy season [15] . this may present challenges for decision-making around the best time to vaccinate and which vaccine formulation (the northern or southern hemisphere) should be used (see section on vaccines) [16] . the start, peak, size and duration of the influenza season may vary substantially from year-to-year. seasonal influenza can give rise to outbreaks in closed settings such as schools, these can occur at any time of year but are more common during the influenza season [9] . influenza pandemics are caused by the emergence and spread in human populations of a new influenza a virus with either a new or substantially altered ha or na combination against which there is little or no immunity in humans, which is easily transmitted between humans and causes clinical illness in humans [1] . the emergence of a pandemic influenza strain is unpredictable and can occur through two mechanisms. firstly, a host could be simultaneously infected with two different influenza virus subtypes which could allow for exchange of genetic material or reassortment and the emergence of a new subtype. for example this could occur if a pig were infected by both a human and avian origin influenza subtype simultaneously with genetic exchange leading to the emergence of a virus adapted to spread in humans, but with ha and/or na not currently circulating in humans. the second way that novel subtypes can emerge is if avian or other animal adapted subtypes are directly transmitted to humans and then undergo adaptation to allow transmission between humans. currently some avian influenza virus subtypes such as influenza a(h5n2) can be transmitted to humans, usually following close contact with poultry, and cause severe infections. however, these viruses are not adapted for efficient transmission from person to person and therefore have not given rise to a new pandemic strain [2] . global surveillance for new influenza virus strains is essential for early identification of novel strains to allow a global public health response. the 1918 pandemic of influenza a(h1n1) is widely acknowledged as the most severe in recent times with an estimated >20 million deaths worldwide. other recent pandemics (1957, asian flu h2n2 and 1968 hong kong flu h3n2) have been associated with a lower death toll [17] . a characteristic of pandemic influenza strains is the shift in the age distribution of deaths from predominantly affecting the extremes of age (young infants and the elderly) to mortality in young adults aged 20-40 years [17] . although influenza pandemics can cause substantial mortality, the annual cumulative deaths each year, associated with seasonal mortality, far outweigh this burden. in 2009, a novel influenza a virus, influenza a(h1n1)pdm09 emerged in the human population and caused a global pandemic. this virus, was antigenically distinct from the h1n1 virus which had been circulating in human populations from 1997 to early 2009 and was thought to have entered the human population from pigs (hence the colloquial name "swine flu"). the overall mortality burden of this strain was estimated at between 123,000 and 203,000 deaths globally, similar to the annual mortality burden from seasonal influenza, although this strain did exhibit the characteristic pandemic age shift, disproportionately affecting individuals aged 20-40 years [18] . subsequently, influenza a(h1n1)pdm09 has been circulating in human populations and immunity in the population has built up. influenza a(h1n1)pdm09 has become the predominant h1n1 seasonal train, replacing those that previously circulated and behaves like any other seasonal influenza virus [9] . influenza is predominantly spread person-to-person by large droplets and through direct contact with respiratory secretions [19] . the contribution of airborne transmission is unclear. the incubation period for influenza typically ranges from 1 to 4 days (median 2 days) [20] . influenza virus is typically shed from the nasopharynx for up to 5 days after illness but viral shedding may be longer in severely ill individuals, young children and immunocompromised individuals. the reproductive number for influenza is between 1 and 2 and the serial interval usually estimated at 2-3 days. an individual's susceptibility to infection and disease will depend on host characteristics including preexisting cellular or humoral immunity to influenza [20] . young children may have no pre-existing immunity to influenza, but older children and adults have often been exposed to circulating influenza several times before and may also have pre existing immunity from vaccination. natural immunity is not fully protective, largely because of the variability of influenza ha and na. influenza virus replication predominantly occurs in the respiratory tract columnar epithelial cells, with infection leading to loss of cilia and cell death [20] . damage to the respiratory tract as well as immunologic changes can lead to increased susceptibility to bacterial superinfection. viremia with influenza is relatively uncommon although constitutional symptoms are a prominent feature of clinical disease. in most children influenza infection results in acute self-limiting upper respiratory tract (urt) symptoms, however, systemic manifestations are not uncommon [21] . factors that influence clinical presentation include: age of the child, previous influenza exposure, vaccination status, underlying disease states or co-morbidities, as well as viral factors. children are considered important influenza "vectors" and are often responsible for introducing the virus into their homes and broader social settings [22] . influenza classically presents with the sudden onset of systemic (fever, myalgia, headache, and malaise) and urt symptoms (sore throat, cough, rhinitis). since many patients do not have all these typical symptoms, accurate clinical diagnosis is challenging particularly in the younger pre-verbal child and outside of the influenza season [23] . a large study evaluating the clinical presentation of influenza in children found that almost all (95%) had fever; cough (77%) and rhinitis (78%) were also very common, but much lower proportions experienced headache (26%) or myalgia (7%) [24] . younger children have not yet been exposed to influenza very often and so have yet to acquire immunity to a substantial repertoire of circulating seasonal influenza strains. they, therefore, are more likely to develop severe or complicated disease [25] . further, they are less likely to manifest with classic symptoms, experience higher fevers (not uncommonly associated with febrile convulsions), less prominent urt involvement and more gastro-intestinal symptoms (vomiting, diarrhea, abdominal pain, loss of appetite). examination may be completely normal in some children, others may manifest with tachypnea, conjunctival injection, nasal inflammation and discharge, or cervical lymphadenopathy. oropharyngeal findings are often limited, even in those children complaining of a sore throat [24] . symptoms of uncomplicated influenza usually start improving within a few days, but symptoms lasting more than a week are not uncommon. cough, in particular, may persist for a number of weeks, but steady improvement can be expected [26] . the differential diagnosis of influenza largely depends on the presenting symptoms and clinical findings, but includes other respiratory viruses (rhinovirus, coronavirus, respiratory syncitial virus, human metapneumovirus, adenovirus, parainfluenza) and some bacterial urt infections (streptococcus pyogenes, mycoplasma). the clinical manifestations of these conditions are very similar, regardless of the implicated pathogen [27] . all influenza strains may result in severe illness and knowing which infection a particular child has, is not helpful in predicting their disease course. complications may involve the respiratory tract (e.g. otitis media, pneumonia) or, less commonly, other organ systems (e.g. encephalitis, myocarditis). otitis media may occur in as many as 50% of cases; this may be related to the influenza virus itself or secondary infection with bacteria or other viruses [24, 28] . symptoms of acute otitis media generally present a few days after onset of influenza symptoms. lower respiratory tract complications may include the following [21, 29] : • laryngo-tracheo-bronchitis ("croup") • bronchiolitis • pneumonia-especially in children <2 years of age, often mild but may be severe, rapidly-progressive and occasionally fatal, particularly if associated with secondary bacterial infection (usually streptococcus pneumoniae or staphylococcus aureus). a variety of radiographic appearances have been described • acute exacerbation of asthma-this is the most common respiratory tract complication of influenza. central nervous system involvement can include the following [30] [31] [32] : • aseptic meningitis • acute cerebellitis • transverse myelitis • guillain-barré syndrome • febrile seizure • necrotizing encephalitis • postinfectious encephalitis (also referred to as acute disseminated encephalomyelitis). neurologic complications appear to be more common in younger children and in those with underlying neurologic and neuromuscular disease. following the rapid decline in aspirin use over the last few decades, influenza-associated reye syndrome is now rare. mild transient myositis is common with influenza infection; it is more likely with influenza b and is associated with moderate elevations in creatine kinase levels [24] . acute myositis is an important, severe, but rare, complication of influenza infection [33] . it presents with extreme muscle tenderness, often involving the calf muscles, extreme elevations in creatine kinase as well as significant myoglobinuria. during influenza season, influenza should be considered in all children presenting with suggestive clinical features-this includes those already admitted to hospital, as nosocomial transmission of influenza is well described. influenza should still be considered outside of the influenza season, particularly in travelers and children residing in tropical and sub-tropical climates where year-round influenza transmission occurs. accurate clinical diagnosis of influenza is challenging, particularly in younger children. the lack of specific signs or symptoms results in patients receiving diagnoses of "influenza-like illness" or "viral upper respiratory tract illness" unless further diagnostic testing is undertaken. this degree of diagnostic uncertainty should be acknowledged, however, since most such cases are self-limiting and management is largely supportive, there is usually no need to obtain a precise microbiological diagnosis. currently available diagnostic tests for influenza include the following [23, 34] : • rapid, point-of-care, antigen detection tests a number of different tests are currently available, although they remain unavailable in many settings. they provide results within 30 min, and, when used appropriately, are helpful in confirming influenza infection. in general, they are insufficiently sensitive to reliably exclude the disease. further, their performance will depend on which antigens are expressed by currently circulating strains. when influenza activity is low, positive results are likely to be false-positives, however, their positive predictive value improves as influenza activity increases. conversely, during periods of high influenza activity, false-negatives are more likely and may warrant additional testing in some patients. since diagnostic confirmation seldom affects management of such children, the use of these tests is generally not recommended in low-resource settings and should be used judiciously in better-resourced areas. when used, it should be clear in the clinician's mind as to how the result is going to alter treatment: positive results can potentially reduce antibiotic usage and allow for early use of antivirals in those at high risk of complications or severe disease. however, it is important to recognize that the identification of influenza does not exclude the presence of bacterial co-infections. these tests are currently considered to be the most reliable for the diagnosis of influenza in children. amongst the available options, they are the most sensitive and specific. they can be performed on most respiratory samples, most commonly nasopharyngeal aspirates or swabs. they are also able to differentiate influenza a and b, as well as subtypes of influenza a. more recently, point-of-care pcr assays have becomes available in some developed world settings. they are performed on nasal swabs and can supply a reliable result in as little as 15 min. while, not currently available in most developing world settings, if more affordable they could become an important new influenza diagnostic. since the live attenuated influenza vaccine contains influenza genetic material, recent receipt of this vaccine will also result in a positive pcr test. these are also performed on nasal or nasopharyngeal swabs and allow for the direct or indirect detection of influenza antigens. influenza a and b can be differentiated, however, the sensitivity of these tests is moderate and, particularly during periods of high transmission, negative tests may need to be repeated using a more sensitive methodology (pcr or culture). • viral culture viral culture takes at least 48-72 hours and so has limited utility in the routine diagnosis of influenza. however, they are helpful as part of surveillance activities and isolates can be used to inform annual vaccine planning. a variety (hemagglutination-inhibition, enzyme-linked immunosorbent assay (elisa), and complement fixation assays) of serological assays can be performed but are of limited diagnostic value as they require acute and convalescent sampling. a fourfold increase in titre allows for a retrospective influenza diagnosis to be made. their primary role is as a research tool. antivirals are available for the specific treatment of influenza. two classes of antiinfluenza drugs are available: neuraminidase inhibitors (e.g. oseltamivir, zanamivir) and m2 inhibitors (e.g. amantadine, rimantadine). both classes are inactive against all other respiratory viruses. resistance can emerge in circulating influenza virus strains and for this reason antiviral resistance should be constantly monitored through surveillance and recent guidance consulted for the latest antiviral resistance profiles. • neuraminidase inhibitors [35] neuraminidase inhibitors prevent the release of new virions from influenza infected cells and are active against both influenza a and b. oseltamivir is the most widely available member of this drug class and can be used for children and adults. it is dosed orally and is approved for both treatment and prevention of influenza. it is available both in capsule form and as a powder for suspension, although the suspension has a relatively short shelf life and is often substantially more expensive than the capsule. as a result, the capsule is the most widely used formulation. when the oral suspension is unavailable the capsule can be opened and diluted with sweetened liquids to provide the appropriate dose. zanamivir is predominantly available as an inhaled formulation, although intravenous zanamivir is available for investigational use, particularly for severely ill patients or those with suspected or confirmed oseltamivir-resistant virus. the inhaled preparation is contra-indicated in children with a history of wheezing or other chronic respiratory condition. its use is not recommended for children younger than 5 years of age. peramivir is an intravenous neuraminidase inhibitor that is not approved for use in children and is not widely available. neuraminidase inhibitors are generally well tolerated. common side effects include nausea, vomiting and rash, as well as bronchospasm with zanamivir. neuropsychiatric symptoms have been linked to oseltamivir use, particularly in japan, however, more recent evidence suggests no such causative link [36, 37] . severe adverse reactions have been reported but are considered rare [38] . the mainstay of influenza prevention remains immunization, but in high risk situations, amongst partially or unimmunized children, chemoprophylaxis can be considered. both pre-and post-exposure chemoprophylaxis have been advocated, however, concerns regarding induction of oseltamivir-resistance have tempered enthusiasm for this approach and their prophylactic use is increasingly discouraged. antiviral treatment should be offered as soon as possible for hospitalized children with presumed or confirmed influenza and for influenza of any severity for children at high risk of severe complications of influenza (table 2 .1) [39] . timely oseltamivir treatment can reduce the duration of fever and symptoms. there are no prospective randomized controlled trials of treatment efficacy of neuraminidase inhibitors in hospitalized children and for severe outcomes but observational data suggest that treatment does reduce the risk of hospitalization and death, although there is some controversy in the literature (table 2. 2) [40] . treatment should be initiated as early as possible. the benefit of any treatment is maximal early in the course of the disease and should be started within 48-72 hours of symptom onset. influenza diagnosis may not have been confirmed within this timeframe and so treatment will often be initiated empirically prior to microbiological confirmation. some evidence suggests benefit to those with very severe illness even when treatment is initiated later than 72 hours into the disease course. therefore, in table 2 .1 children at high risk of severe influenza in whom influenza antiviral treatment is recommended by the centers for disease control and prevention (cdc) and american academy of pediatrics (aap) current guidance [9, 39] 1. children aged <2 years 2. persons with chronic pulmonary (including asthma), cardiovascular (except hypertension alone), renal, hepatic, hematologic (including sickle cell disease), or metabolic disorders (including diabetes mellitus) or neurologic and neurodevelopment conditions (including disorders of the brain, spinal cord, peripheral nerve, and muscle such as cerebral palsy, epilepsy [seizure disorders], stroke, intellectual disability, moderate to severe developmental delay, muscular dystrophy, or spinal cord injury) 3. persons with immunosuppression, including that caused by medications or by hiv infection 4. persons who are receiving long-term aspirin therapy 5. american indian/alaska native persons 6. residents of chronic care facilities patients with severe or complicated disease, treatment should be initiated even if >48 hours after illness onset. children meeting the clinical criteria for treatment should be treated irrespective of whether they have been vaccinated. since influenza is generally a mild, self-limiting disease in previously well children, most such children will not require antiviral treatment even when they present soon after symptom onset. treatment of such children increases the risk of adverse events, potentially increases the risk of resistance developing and may deplete medicine supply for those in greater need. • adamantanes [9, 39] these agents target the influenza a m2 protein, which is essential for efficient viral replication. they have no activity against influenza b and are not active 10 mg (two 5-mg inhalations) twice daily 10 mg (two 5-mg inhalations) once daily against currently circulating influenza a strains. as such, there use is not currently recommended for the treatment or prevention of influenza. it has been suggested that they may have a role, in combination with oseltamivir, for the treatment of oseltamivir-resistant influenza a. vaccines and guidelines for vaccination influenza vaccination can be given to any person who wishes to reduce the risk of becoming ill during the influenza season. some countries such as the united states of america (usa) and united kingdom (uk) recommend influenza vaccination for all children, or all individuals. in addition, special effort should be made to vaccinate children at risk of severe influenza listed in table 2 .3. individuals such as healthcare personnel and childcare providers (especially those in contact with infants aged <6 months and children with underlying risk conditions) should be vaccinated to reduce the risk of transmission to high risk children. lastly, pregnant women are recommended to receive influenza vaccination, to reduce the risk of severe illness in the mother, to provide direct protection to the young infant through trans-placental transfer of maternal antibodies and to reduce the risk of transmission of influenza from the mother to the young infant [41] . iiv3 has been available for many years and includes inactivated components of two influenza a (one each of influenza a(h1n1)pdm and influenza a(h3n2)) and one influenza b strains. since the 1980s, two antigenically distinct influenza b lineages (victoria and yamagata) have been circulating globally. this is a limitation of iiv3, as protection may be reduced when the circulating influenza b strain is of the lineage which is not included in iiv3. the iiv4 includes an additional strain of the other influenza b lineage not included in tiv to make a total of four strains and thus potentially offers additional benefit of protection against both circulating influenza b lineages. inactivated influenza vaccines (iiv3 and iiv4) contain no live virus. standarddose iiv should contain 15 μg of each haemagglutinin antigen in each 0.5 ml dose. iiv3 and iiv4 are available in formulations for both intramuscular (im) and intradermal (id) use but the id formulation is only licensed for use in individuals aged 18 years and older. two formulations of iiv3 manufactured using technologies that do not include eggs have become available in recent years, but neither is licensed for individuals aged <18 years. these are cell-culture based inactivated influenza vaccine and recombinant influenza vaccine. d. hiv infection e. sickle cell anemia and other hemoglobinopathies f. diseases that necessitate long-term aspirin therapy, including juvenile idiopathic arthritis or kawasaki disease g. chronic renal dysfunction h. chronic metabolic disease, including diabetes mellitus i. any condition that can compromise respiratory function or handling of secretions or can increase the risk of aspiration, such as neurodevelopmental disorders, spinal cord injuries, seizure disorders, or neuromuscular abnormalities j. morbid obesity k. pregnancy 2. children aged 6 months-59 months as this group (particularly those aged <24 months) has an increased risk of influenza-associated hospitalization and mortality b. individuals who come into contact with children at risk of severe influenza 1. all household contacts and out-of-home care providers of the following: children with high-risk conditions; and children younger than 5 years, especially infants younger than 6 months 2. all health care personnel (hcp) 3. all child care providers and staff; and 4. all women who are pregnant, are considering pregnancy, are in the postpartum period, or are breastfeeding during the influenza season laiv is a live attenuated influenza vaccine which is administered intranasally and licensed for use in individuals aged 2-49 years of age. since 2013 the laiv has only been available in a quadrivalent formulation. adjuvanted formulations of influenza vaccine are licensed for use in individuals aged ≥65 years in the usa but not currently in children [39] . they have been shown to have a higher efficacy then iiv in a randomized controlled trial in children [42] . adjuvants have several potential advantages over more traditional vaccine formulations including increased immunogenicity, potentially reducing the amount of antigen required. they elicit a more robust immune response and could potentially reduce the number of doses needed in children. children aged 6 months through 8 years should receive two influenza doses administered ≥4 weeks apart the first time influenza vaccine is administered. for young children who require two doses of influenza vaccine, vaccination should not be delayed to ensure that both doses are given with the same product. any licensed, effective influenza vaccine product may be used for each dose. it is important to document all doses of influenza vaccine administered in the child's medical records. in temperate countries, influenza vaccine should be administered as soon as possible after the influenza vaccine becomes available. the recommended dosage of influenza vaccine for patients of different age groups is described in table 2 .5 [9] . influenza vaccine effectiveness depends on characteristics of those being vaccinated (age and health), whether there is a good match between the circulating viruses and the viruses contained in the vaccine, and on influenza types and subtypes circulating each year. in general, influenza vaccines work best among children ≥2 years and healthy adults. older people (≥65 years), children <2 years and severely immunocompromised individuals often have poorer immune responses to inactivated influenza vaccine (iiv) compared with healthy adults. however, even for these people influenza vaccine still provides some protection. other products, e.g. high-dose influenza vaccine and adjuvanted vaccines, have been shown to be more effective in certain groups [43] but these vaccines may not be available in all settings and are not licensed for use in all age groups. there have been a number of studies of iiv effectiveness in children aged 6-59 months. for seasonal iiv in young children, two doses of influenza vaccine provides better protection than one dose in the first season a child is vaccinated. estimates of iiv efficacy in young children are limited and vary by season and study design. efficacy is lower in children aged 6-23 months. data are unclear as to the effectiveness in hiv-infected children aged <5 years [44] . a randomized controlled trial of laiv3 in healthy children aged 15-71 found a vaccine effectiveness of 92% (95% ci 65-96%). several other randomized controlled trials and observational studies have demonstrated high efficacy of laiv3 against laboratoryconfirmed influenza. studies comparing efficacy of iiv and laiv have generally found that laiv has similar, or better efficacy, than iiv. since 2013, laiv4 has replaced laiv3 as the available laiv. licensure was on the basis of immunogenicity studies. in 2016, conflicting findings on the effectiveness of laiv emerged from different settings, with the usa withdrawing its recommendation for laiv use in the 2016-2017 influenza season based on concerns of reduced effectiveness, particularly against the a(h1n1)pdm component of the vaccine [9] . studies from europe in the same seasons found moderate laiv effectiveness, similar to that of iiv for the same year [45, 46] with recommendations for laiv remaining unchanged in england and europe. vaccinating individuals at risk of severe influenza may provide direct protection for these individuals. in addition, vaccinating individuals in close contact with people at risk for severe influenza may provide indirect protection through preventing transmission to high-risk individuals. vaccinating children can protect children directly and the general population indirectly. this strategy is especially important for individuals in whom influenza vaccine is not indicated, such as children aged <6 months (who may be protected through maternal immunization) [47] [48] [49] . a randomized controlled trial conducted in south africa has shown that when pregnant women receive the influenza vaccine, their risk of developing influenza is halved, as is the risk to their infants in the first 24 weeks of life [47] . the vaccine has been shown not only to be efficacious for prevention of influenza in both mothers and their infants, but also safe [47] [48] [49] . vaccination of healthcare workers may decrease the risk of spreading influenza to their patients. recent influenza vaccination does not preclude a diagnosis of influenza as the vaccine is not 100% effective. because of the large year-to-year variability in influenza vaccine effectiveness depending on the circulating influenza strains, many countries publish annual estimates of influenza vaccine effectiveness using a test-negative case-control study design. a systematic review of test-negative case-control studies found that the pooled ve was 33% (95% ci 26-39) for h3n2, 54% (46-61) for type b, 61% (57-65) for h1n1pdm09, suggesting reduced protection of available vaccines against influenza a(h3n2) in recent years [50] . [35, 39] the iiv is an inactivated vaccine, and has a well-established safety record. it is safe for use in pregnancy and in children ≥6 months of age. minor illness, with or without fever, is not a contraindication to influenza vaccine administration. clinicians should always consult the manufacturer's package insert for current contraindications and precautions for particular products. contraindications to the administration of iiv include: • a history of severe (anaphylactic) hypersensitivity to any components of the vaccine including, egg protein, or after previous dose of any influenza vaccine. anaphylaxis is rare and a careful history will distinguish between anaphylactic reactions and allergic reactions like rashes. mild egg protein allergy is not a contraindication for influenza vaccine • infants <6 months of age. precautions to iiv administration include: • persons with moderate to severe illness with or without fever should preferably be immunized after symptoms have resolved • person who developed guillian-barrè syndrome within 6 weeks of receiving an influenza vaccine. contraindications to the administration of laiv include the following: • children 2-4 years of age with a history of recurrent wheezing or a medically attended wheezing episode in the previous 12 months because of the potential for increased wheezing after immunization • children with the diagnosis of asthma • children with a history of egg allergy • children who have received other live virus vaccines within the past 4 weeks; however, other live virus vaccines can be given on the same day as laiv • children who have known or suspected immunodeficiency disease or who are receiving immunosuppressive or immunomodulatory therapies • children who are receiving aspirin or other salicylates • any female who is pregnant or considering pregnancy • children with any condition that can compromise respiratory function or handling of secretions or can increase the risk for aspiration • children taking an influenza antiviral medication (oseltamivir or zanamivir), until 48 hours after stopping the influenza antiviral therapy • children with chronic underlying medical conditions that may predispose to complications after wild-type influenza infection. as for all vaccines, influenza vaccine should be administered in a setting where there is the ability to respond to acute hypersensitivity reactions. the most common adverse events following intramuscular iiv administration are pain and tenderness at the injection site. fever can occur in 10-35% of children aged <2 years within 24 hours of vaccination, but is much less common in older children. in trials, when iiv are administered, 16-20% of those vaccinated experience local reactions in the arm, lasting for 1 or 2 days. short-term reactions (mild fever, malaise and muscle pains) have been reported in a much smaller proportion in the first few hours following vaccination. trials of the split and subunit vaccines show even fewer systemic reactions. there have been no strong temporal or causal associations of the current vaccines with more severe reactions. anaphylaxis is very rare but does occur as with all vaccines. more severe adverse events, like guillain-barré syndrome have been reported with a particular vaccine in the 1970s but they are extremely rare. with the modern influenza vaccines, the causative risk is either found to be very rare (0.8 per million doses) [51] or there is no causal link found at all [52] [53] [54] and more association is found with influenza infection than vaccination [55] . an increased risk of fever and febrile seizures was reported from australia in 2010 associated with the southern hemisphere iiv3 produced by csl biotherapeutics (now sequirus) [39] . following this, many countries do not recommend the csl iiv3 for children aged <9 years. influenza vaccination during pregnancy has been shown to protect both the mother and her baby (up to 6 months old) against influenza [47, 48, 56, 57] . influenza vaccination is safe in pregnancy and influenza vaccines have been administered to millions of pregnant women over many years and have not been shown to cause harm to pregnant women or their babies [58] . the most common adverse events associated with laiv include runny nose or nasal congestion, headache, lethargy and sore throat. laiv should not be administered to children with marked nasal congestion as this can reduce vaccine delivery. the safety of laiv is not established in people with a history of asthma, diabetes mellitus, or other high-risk medical conditions associated with an elevated risk of complications from influenza. the use of laiv in young children with chronic medical conditions is not recommended in the usa but is in some other countries. appropriate hand and respiratory hygiene (cough etiquette) has been shown to reduce influenza transmission in children in day-care or school [59] . sick children and adults should remain at home and not attend school or 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presentation of influenza in unselected children treated as outpatients clinical and epidemiologic characteristics of children hospitalized with 2009 pandemic h1n1 influenza a infection duration of cough in acute upper respiratory tract infections understanding the symptoms of the common cold and influenza a longitudinal study of respiratory viruses and bacteria in the etiology of acute otitis media with effusion respiratory viruses in laryngeal croup of young children neurologic complications in children hospitalized with influenza: characteristics, incidence, and risk factors acute childhood encephalitis and encephalopathy associated with influenza: a prospective 11-year review neurological manifestations of influenza infection in children and adults: results of a national british surveillance study influenza-associated myositis in children seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america healthcare workers handbook on influenza. the national institute for communicable diseases (nicd) the association between oseltamivir use and adverse neuropsychiatric outcomes among tricare beneficiaries, ages 1 through 21 years diagnosed with influenza assessment of neuropsychiatric adverse events in influenza patients treated with oseltamivir: a comprehensive review neuropsychiatric adverse effects of oseltamivir in the fda adverse event reporting system committee on infectious diseases. recommendations for prevention and control of influenza in children neuraminidase inhibitors for preventing and treating influenza in healthy adults and children vaccine against influenza who position paper oil-inwater emulsion adjuvant with influenza vaccine in young children efficacy of high-dose versus standard-dose influenza vaccine in older adults efficacy and immunogenicity of influenza vaccine in hiv-infected children: a randomized, double-blind, placebo controlled trial effectiveness of the live attenuated and the inactivated influenza vaccine in two-year-olds-a nationwide cohort study finland, influenza season 2015/16 effectiveness of seasonal influenza vaccine for adults and children in preventing laboratory-confirmed influenza in primary care in the united kingdom: 2015/16 end-of-season results influenza vaccination of pregnant women and protection of their infants effectiveness of maternal influenza immunization in mothers and infants maternal influenza vaccination and effect on influenza virus infection in young infants variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of testnegative design studies the guillain-barre syndrome and the 1992-1993 and 1993-1994 influenza vaccines guillain-barre syndrome and the 1978-1979 influenza vaccine guillain-barre syndrome and influenza vaccination in the us army, 1980-1988 guillain-barre syndrome in the united states, 1979-1980 and 1980-1981. lack of an association with influenza vaccination investigation of the temporal association of guillain-barre syndrome with influenza vaccine and influenza like illness using the united kingdom general practice research database maternal influenza immunization and reduced likelihood of prematurity and small for gestational age births: a retrospective cohort study influenza vaccine given to pregnant women reduces hospitalization due to influenza in their infants safety of influenza vaccination during pregnancy effectiveness of hand hygiene interventions in reducing illness absence among children in educational settings: a systematic review and meta-analysis key: cord-011754-lumzp1ca authors: jackson, michael l.; yu, onchee; nelson, jennifer c.; naleway, allison; belongia, edward a.; baxter, roger; narwaney, komal; jacobsen, steven j.; shay, david k.; jackson, lisa a. title: further evidence for bias in observational studies of influenza vaccine effectiveness: the 2009 influenza a(h1n1) pandemic date: 2013-10-15 journal: am j epidemiol doi: 10.1093/aje/kwt124 sha: doc_id: 11754 cord_uid: lumzp1ca preinfluenza periods have been used to test for uncontrolled confounding in studies of influenza vaccine effectiveness, but some authors have claimed that confounding differs in preinfluenza and influenza periods. we tested this claim by comparing estimates of the vaccine-mortality association during the 2009/2010 influenza year, when there was essentially no circulation of seasonal influenza in the united states, and 2007/2008, a typical influenza year. we pooled data on seniors (adults aged ≥65 years) from 7 us managed care organizations that participated in the vaccine safety datalink project. we defined influenza vaccination, all-cause mortality, and potential confounders from administrative databases. we quantified the vaccine-mortality association using cox regression. during 2007/2008, the adjusted hazard ratio was 0.44 prior to influenza season, 0.62 during influenza season, and 0.71 after influenza season. a similar pattern was observed during 2009/2010, when any effect of seasonal influenza vaccine observed during all time periods must have resulted from confounding: 0.65 during the autumn, 0.80 during the winter, and 0.84 during the summer. in a year with minimal seasonal influenza, we found no evidence that confounding in autumn preinfluenza periods is qualitatively different from confounding in winter. this supports the use of preinfluenza periods as control time periods in studies of influenza vaccine effectiveness. influenza vaccine effectiveness (ve) in seniors (adults aged ≥65 years) remains controversial (1) (2) (3) (4) (5) . numerous observational studies have estimated that influenza vaccine reduces the risk of all-cause mortality among seniors during the winter months by 40% or more and have concluded that influenza vaccine is highly effective (4, (6) (7) (8) . in recent years, other researchers have noted that these estimates are implausibly large given the small proportion of winter deaths attributable to influenza (9) and that the apparent ve is perhaps entirely the result of uncontrolled confounding (1, 10) . strong evidence for confounding comes from studies that have estimated the association between influenza vaccination and risk of death during time periods before, during, and after the seasonal circulation of influenza. time periods before and after influenza circulates are designated control time periods, during which no true vaccine benefit is expected. studies including these control time periods have consistently found that vaccinated seniors have a significantly lower risk of death than unvaccinated seniors during all time periods, with the strongest association being observed prior to circulation of influenza viruses (1, (11) (12) (13) (14) . a highly plausible explanation for this trend is that seniors at high risk of dying are less likely to receive influenza vaccine, leading to a strong but spurious association between vaccination and reduced mortality during the months immediately following vaccination. over time, the seniors at high risk of mortality (who are predominantly unvaccinated) die. this reduces the underlying differences in mortality risk between vaccinated and unvaccinated seniors, and the spurious association between vaccination and mortality risk attenuates towards the null. notably, the apparent ve during influenza seasons can be fully explained by the confounding observed in the preinfluenza control time period. although incorporation of the preinfluenza control periods is effective and innovative, their use has been criticized by some (3, 15) . these authors have claimed that the confounding observed in autumn preinfluenza seasons is qualitatively different from confounding in winter influenza seasons. specifically, they claim that persons who die prior to influenza season may be less likely to be offered vaccine, or may have fewer opportunities to receive vaccine, than persons who survive until influenza season. on this basis, they infer that confounding observed during preinfluenza seasons is not present when influenza circulates and that preinfluenza periods are not appropriate control time periods for studies of influenza ve. this claim could be tested by estimating the association between seasonal influenza vaccination and risk of mortality over time in a year with no seasonal influenza. if the same trends in apparent associations between vaccination and mortality were observed during a year when there could be no true vaccine effect, the claim that confounding differs between time periods would be refuted. circulation (16) . thus, we hypothesized that during the entire 2009/2010 influenza year, no true association between seasonal influenza vaccine and disease outcomes should be found if confounding factors were appropriately adjusted for in multivariate analyses. we estimated the association between influenza vaccination and all-cause mortality among seniors during the 2009/2010 influenza year to see whether the confounding trend seen in prior years with typical influenza circulation remained in a year with no seasonal influenza. we conducted a retrospective cohort study among seniors enrolled in managed care organizations (mcos) participating in the vaccine safety datalink project, which has been described elsewhere (17 (4, 7, 18) . this set consisted of binary indicators for the presence or absence of diagnosis codes for chronic conditions in broadly defined categories (appendix table 1 ). adjusting for this "traditional" set of variables has been shown to have a negligible impact on confounding in observational ve studies (1, 19) , in part because the conditions defined within each binary variable are overly broad (10) . we also explored whether confounding could be reduced by adjusting for a second "alternate" set of more precise covariates. in addition to covariates based on icd-9-cm codes, the alternate comorbid conditions included indicators for filled prescriptions for bronchodilators, corticosteroids, and statins (appendix table 1 ), based on pharmacy data from each mco, since adjusting for prescription data has been shown to attenuate confounding in studies of influenza ve against all-cause pneumonia (19) . subjects were followed for 1 year from the date of study enrollment (september 1 of 2007 or 2009). the outcome of interest was all-cause mortality. subjects were followed until death, disenrollment from the mco, or august 31 of 2008 or 2010 (respectively), whichever was earliest. we used cox proportional hazards regression to estimate the hazard ratio for the association between receipt of seasonal influenza vaccine and incidence of all-cause mortality. influenza vaccination was treated as a time-varying covariate, with individuals being classified as vaccinated 14 days after receipt of vaccine. we fitted 3 separate models for each year. in the first model, only age (as a set of categorical variables), sex, and mco were included as model covariates. in ratio estimates were stratified by time by including an interaction term between vaccination and time period. in 2007/ 2008, we divided time into preinfluenza, influenza, and postinfluenza periods. these time periods were defined at each mco based on regional influenza surveillance (20) . in 2009/ 2010, we divided time into pandemic (september 1-december 15), winter (december 16-march 31), and summer (april 1-august 31) periods. to further explore the change in hazard ratios over time, we estimated the vaccine-mortality hazard ratio separately for each 2-week period during each study year, adjusting for age, sex, and mco. for the 2009/2010 influenza year, we repeated all analyses while adjusting for receipt of monovalent 2009 h1n1pdm vaccine as a time-varying covariate. the proportion of cohort members from each mco was consistent between the two study years. vaccinated seniors in both cohorts tended to be older than unvaccinated seniors and were more likely to have icd-9-cm codes for heart disease, cancer, lung disease, and other chronic illnesses (table 1) association between vaccine and all-cause mortality during the 2007/2008 year, a year with typical influenza circulation, receipt of seasonal influenza vaccine was associated with a decreased risk of all-cause mortality after adjusting for age, sex, and mco ( figure 1a) . consistent with prior studies (1, (11) (12) (13) (14) , the apparent association was strongest in the preinfluenza time period (hazard ratio = 0.44, 95% confidence interval (ci): 0.42, 0.46), decreasing to 0.62 (95% ci: 0.60, 0.65) during influenza season and to 0.71 (95% ci: 0.69, 0.74) in the summer time period. also consistent with prior studies, adjusting for the traditional set of comorbidity indicators did not appreciably alter the estimated associations. adjusting for the alternate comorbidity indicators also did not appreciably alter the estimated associations (figure 1 ). during the 2009/2010 pandemic year, receipt of seasonal influenza vaccine was also associated with a decreased risk of all-cause mortality after adjusting for age, sex, and mco ( figure 1b) . the apparent association was strongest during the pandemic (fall) period (hazard ratio = 0.65, 95% ci: 0.62, 0.68), decreasing to 0.80 (95% ci: 0.77, 0.83) during the winter and 0.84 (95% ci: 0.81, 0.87) during the summer. adjusting for the traditional or alternate comorbid conditions did not appreciably change the vaccine-mortality association. adjusting for monovalent 2009 h1n1pdm vaccine also did not appreciably affect the results. when hazard ratios were estimated biweekly, both study years showed similar patterns over time (figure 2 , parts a and b). after initial instability during weeks when few doses of seasonal influenza vaccine had been given, the hazard ratio trended steadily toward the null over time. our findings refute the claim that confounding in preinfluenza seasons does not operate in influenza or summer seasons (3, 15) . if this claim were true, we would have observed a non-null association between influenza vaccination and mortality during the fall of 2009, followed by a null association during the winter and summer of 2009/2010. instead, we observed a pattern in the vaccine-mortality association over time during the 2009/2010 pandemic year that was similar to the pattern observed in typical influenza seasons (1, 11, 14) , including the 2007/2008 season, which we examined in the current study. specifically, the estimated association was highest during the preinfluenza season and trended steadily toward the null over time, but it remaining significantly protective in all time periods. our findings are consistent with our hypothesis about confounding in observational ve studies among seniors: differences between seniors who choose to receive vaccine and those who do not are greatest when vaccine is first made available, and the differences between vaccinated and unvaccinated groups decline slowly over time as seniors at high risk of mortality (who are predominantly unvaccinated) die, making vaccinated and unvaccinated groups more similar with respect to mortality risk over time. this study supports the use of preinfluenza periods for identifying uncontrolled confounding in observational influenza ve studies. furthermore, this study affirms that the magnitude of uncontrolled confounding in observational ve studies is strong, as the observed confounding during preinfluenza periods is sufficient to entirely account for the apparent ve during influenza seasons. finally, this study affirms that adjustment for the traditional comorbidity indicators does not remove or reduce the confounding in observational studies of influenza ve against nonspecific outcomes in seniors (1, 11, 21) . prior studies have shown that the prevalence of comorbid chronic illnesses (based on icd-9-cm codes) is higher in vaccinated seniors than in unvaccinated seniors (1, 4, 11) . this could contribute to confounding by indication and underestimation of ve (i.e., bias toward the null) if these chronic illnesses are associated with an increased risk of death (22) . however, we have previously shown that the use of icd-9-cm codes to define chronic illnesses can lead to substantial misclassification of health status and frailty (10) . importantly, this misclassification is differential by influenza vaccination, and adjustment for the presence or absence of chronic illness (defined by icd-9-cm codes) can actually increase bias in influenza ve estimates. in the present study, we found that this bias was not reduced even when we used the expanded set of icd-9-cm comorbidity indicators. this finding suggests that increasing the specificity of potential confounders defined from icd-9-cm codes does not capture relevant information on the difference between vaccinated and unvaccinated seniors. available evidence suggests that detailed data on cognitive impairment, functional status, and severity of comorbid illnesses (rather than presence or absence) is necessary to substantially reduce or eliminate confounding of influenza ve estimates in seniors (19) . this study provides further evidence that outcomes such as all-cause mortality that are highly nonspecific to influenza are generally not appropriate endpoints for influenza ve estimation in seniors. we note that the hazard ratio estimates were closer to the null during the 2009/2010 pandemic year than during the 2007/2008 influenza year. we do not believe there was a true difference between associations in these two years, as the hazard ratio estimates were further from the null during the 2007/2008 control time periods ( preinfluenza and summer, when any apparent association is the result of uncontrolled confounding) as well as during influenza season. rather, our findings suggest that the availability of the pandemic vaccine and expected lack of benefit of seasonal influenza vaccine (table 1) . although the pharmacy-and icd-9-cm-based comorbidity indicators do not capture informa-tion on the true confounders, they may indicate differences in the seniors receiving seasonal influenza vaccine between the two study years. a limitation of this study is that, in contrast to typical years, influenza viruses were circulating intensely during the fall of 2009, which could make the 2009 fall atypical with regard to death rates as compared with other preinfluenza caused little morbidity and mortality among seniors (16) , as many of these seniors had cross-protective antibodies from exposure to influenza a(h1n1) strains that circulated before 1957 (23) . thus, the impact of the fall pandemic wave on deaths in seniors was small and should not have affected the presence of confounding in our study. another potential limitation is that seniors could have received influenza vaccine outside of the mco in which they were enrolled; if vaccination records were not subsequently transferred to the mco, these seniors might have been falsely classified as unvaccinated. while previous studies conducted with the group health cooperative and northern california kaiser populations have found that vaccine misclassification makes a negligible contribution to the bias in ve estimates (1, 24) , pandemic influenza vaccination may have been less likely to be captured in mco electronic records than seasonal vaccination, as pandemic vaccines were more likely to be administered in non-mco settings in some of the geographical areas in which the participating mco provided care. when evaluated in a year with essentially no seasonal influenza circulation, estimates of the association between influenza vaccination and all-cause mortality showed a pattern of steady trend towards the null from fall through summer. our findings are consistent with strong confounding by unmeasured factors, the strength of which declines over time. they also illustrate the usefulness of the preinfluenza time period as a control period for identifying and removing confounding in studies of influenza ve. investigators conducting observational studies of influenza ve against nonspecific outcomes should report adjusted ve estimates during preinfluenza periods, to demonstrate the presence and strength of residual confounding. evidence of bias in estimates of influenza vaccine effectiveness in seniors to rule out confounding, observational studies of influenza vaccine need to include analyses during the "preinfluenza period challenges in evaluating influenza vaccine effectiveness and the mortality benefits controversy effectiveness of influenza vaccine in the community-dwelling elderly mortality benefits of influenza vaccination in elderly people: an ongoing controversy clinical effectiveness of influenza vaccination in manitoba influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly influenza vaccination: reduction in hospitalizations and death rates among members of "maccabi healthcare services" during the 2000-2001 influenza season impact of influenza vaccination on seasonal mortality in the us elderly population functional status is a confounder of the association of influenza vaccine and risk of all cause mortality in seniors influenza vaccination and all-cause mortality in community-dwelling elderly in ontario, canada, a cohort study influenza vaccination and mortality: differentiating vaccine effects from bias influenza vaccination and mortality: prospective cohort study of the elderly in a large geographical area influenza vaccine effectiveness in the elderly based on administrative databases: change in immunization habit as a marker for bias benefits of influenza vaccine in us elderly-appreciating issues of confounding bias and precision and composition of the 2010-11 influenza vaccine the vaccine safety datalink: a model for monitoring immunization safety influenza vaccine effectiveness in preventing hospitalizations and deaths in persons 65 years or older in minnesota influenza vaccination and risk of community-acquired pneumonia in immunocompetent elderly people: a population-based, nested casecontrol study us department of health and human services why do covariates defined by international classification of diseases codes fail to remove confounding in pharmacoepidemiologic studies among seniors? confounding by indication in non-experimental evaluation of vaccine effectiveness: the example of prevention of influenza complications cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus evidence of bias in studies of influenza vaccine effectiveness in elderly patients evidence of bias in influenza vaccine studies 1335 key: cord-261241-eqf6ame6 authors: van beek, josine; veenhoven, reinier h; bruin, jacob p; van boxtel, renée a j; de lange, marit m a; meijer, adam; sanders, elisabeth a m; rots, nynke y; luytjes, willem title: influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections date: 2017-08-15 journal: j infect dis doi: 10.1093/infdis/jix268 sha: doc_id: 261241 cord_uid: eqf6ame6 background: data on the relative contribution of influenza virus and other respiratory pathogens to respiratory infections in community-dwelling older adults (≥60 years) are needed. methods: a prospective observational cohort study was performed in the netherlands during 2 winters. nasopharyngeal and oropharyngeal swabs were collected during influenza-like illness (ili) episodes and from controls. viruses and bacteria were identified by multiplex ligation–dependent probe amplification assay and conventional bacterial culture. results: the ili incidence in the consecutive seasons was 7.2% and 11.6%, and influenza virus caused 18.9% and 34.2% of ili episodes. potential pathogen were detected in 80% of the ili events with influenza virus, coronaviruses, rhinoviruses, human metapneumovirus, respiratory syncytial virus, parainfluenza viruses, and haemophilus influenzae being the most common. influenza vaccination reduced influenza virus infection by 73% (95% confidence interval [ci], 26%–90%) and 51% (95% ci, 7%–74%) in ili patients. however, ili incidence was similar between vaccinated (7.6% and 10.8%) and nonvaccinated (4.2% and 11.4%) participants in 2011–2012 and 2012–2013, respectively (p > .05). conclusions: influenza virus is a frequent pathogen in older adults with ili. vaccination reduces the number of influenza virus infections but not the overall number of ili episodes: other pathogens fill the gap. we suggest the existence of a pool of individuals with high susceptibility to respiratory infections. clinical trials registration: ntr3386. influenza virus causes seasonal epidemics, resulting in 3-5 million severe cases and 250 000-500 000 deaths globally each year [1] . elderly persons, individuals with certain medical conditions and children aged <2 years have the highest risk for complications. vaccination is an important tool to prevent infection and to reduce morbidity and mortality [1] . in the netherlands, individuals aged ≥60 years are offered the annual influenza vaccination. however, public acceptance of vaccination is moderate [2] . vaccine effectiveness (ve) varies per season and depends on age and health of the recipients, and the antigenic match of vaccine strains with circulating strains [3] [4] [5] . furthermore, there is scientific debate about the methodology of determining ve [5, 6] . these discussions reach the media and influence the general opinion on influenza vaccine benefit. moreover, to the public, flu as caused by influenza virus is the same as influenza-like illness (ili) caused by respiratory pathogens, against which influenza vaccination will not protect. consequently, influenza vaccination is perceived to be ineffective and vaccine uptake is reduced. to counter this trend, data on the relative contribution of influenza virus and other respiratory infections to ili in older community-dwelling adults are lacking and needed [7] [8] [9] [10] . this group is underrepresented in the dutch primary care sentinel surveillance system [10, 11] and by definition absent in the dutch sentinel nursing home surveillance network [12] . this is also the case in many other studies worldwide [7] [8] [9] . the aim of this prospective observational study was to determine the relative contribution of influenza virus and other respiratory pathogens to ili in older adults (aged ≥60 years) in 2 consecutive seasons in the netherlands. in addition, influenza ve was estimated in both seasons. upon ili, we determined the presence of potential pathogens in the pharynx of the participant within 72 hours of symptom onset. as a control, we analyzed samples of the same individuals taken after recovery (8 weeks) and in a subset of asymptomatic controls for the same potential pathogens. this prospective observational study was conducted during 2 consecutive influenza seasons (from december 2011 to april 2012 [2011] [2012] and from october 2012 to may 2013 [2012] [2013] ) in the netherlands. adults aged ≥60 years were recruited through their general practitioner or through the civil registry. for the second season, participants were reinvited and additional participants were recruited through the civil registry. we also started earlier to use the same monitoring period as in the dutch sentinel surveillance system [11] . there were no exclusion criteria for the study. influenza vaccination status was recorded (2009) (2010) (2011) (2012) . participants were part of the study for the entire duration of each season and contacted at the end of season to verify participation. written informed consent was obtained from all participants. all trial-related activities were conducted according to good clinical practice, which includes the provisions of the declaration of helsinki. the study was approved by the acknowledged ethical committee metc noord holland (http://www.trialregister.nl; ntr3386). participants were instructed about ili symptoms according to the dutch pel criteria, defined by fever (≥37.8°c) with at least 1 other symptom of headache, myalgia, sore throat, coughing, rhinitis, or chest pain [13] and to report ili as soon as possible after onset. a research nurse performed a home visit within 72 hours of fever onset. during this acute phase, nasopharyngeal and oropharyngeal swabs were obtained and additional information on demographics and comorbidities was recorded. a second visit (recovery phase) was performed 8 weeks (±1 week) later, during which the same samples were collected. if a new ili episode was reported, participants were visited again. in the second season, a control group of asymptomatic participants, equally distributed over the different age groups and season, was sampled and questioned. nasopharyngeal and oropharyngeal samples were obtained with a sterile swab with a flocked nylon tip and stored separately in 1 ml modified liquid amies transport medium (eswab, copan, brescia, italy). samples were transported at room temperature to the laboratory and processed and stored at -80°c within 8 hours after sampling. dna and rna were isolated from 200 µl of both swabs by easy-mag isolation and eluted in 25 µl of buffer (biomérieux, the netherlands). five microliters was used for the detection of a panel of respiratory viruses and bacteria by a real-time polymerase chain reaction (pcr)-based multiplex ligation-dependent probe amplification (mlpa) assay (respifinder smart 22 kit; pathofinder, the netherlands). all analyses were performed on a roche lightcycler 480. mlpa analysis was performed on both swabs separately. a participant was excluded from the analysis and considered missing if either of the swabs or data for a swab were missing. for mlpa data analysis, a participant was considered positive for a target if at least 1 swab of the participant was positive. influenza virus-positive samples were subtyped by real-time reverse-transcription pcr using the roche lightcycler 480 system with slightly modified protocols as described previously [14, 15] . conventional culture of oropharyngeal swabs was performed for streptococcus pneumoniae and haemophilus influenzae according to standard procedures [16] . discrimination of h. influenzae and haemophilus haemolyticus was performed by matrix-assisted laser desorption/ionization-time of flight (maldi-tof) [17] . participants without available oropharyngeal swabs were excluded from analysis and designated as missing. for analysis of the contribution of influenza virus, a sample size of 200 ili cases was estimated to be required. based on an expected ili incidence of 7.5% and a drop-out rate of 5%, a cohort size of 2100 participants was calculated. based on results of the first season, 2500 participants were included in the second season. pearson χ 2 testing and independent samples t test of the means was applied to analyze participant characteristics with spss 19.0 for windows software. a p value ≤.05 was considered significant. the incidence of different viruses or bacteria was calculated as the percentage of swabs positive with the potential pathogen of the number of ili events during the season. attack rates were calculated as percentage of detected pathogens per number of monitored participants. vaccine effectiveness was determined by test-negative design analysis of ili positive participants in the influenza-active period [18] . the analysis is restricted to the period that influenza virus was circulating in the netherlands for that particular season, defined by the national influenza surveillance weekly reports. participants with <14 days between the date of vaccination and the date of home visit were excluded from the ve analysis, as it is uncertain whether the vaccine already had any effect in this period. second and third ili periods were included in the analysis only if an earlier ili period was influenza virus negative. for ili participants, influenza virus positive was considered "case" and influenza negative "control. " the ve is calculated as (1 -odds ratio [or]) × 100% with 95% confidence interval (ci) and is calculated per influenza virus subtype or lineage. the following factors were regarded as potential confounders: period in the season (early and late season), sex, smoking, comorbidity, and age (natural smoothing spline, 4 degrees of freedom). the association between the potential confounders and influenza virus positivity (any subtype) was analyzed with univariate logistic regression. variables with p < .20 were considered in the multivariable analysis. variables that changed the or by at least 5% are included in the final multivariable logistic regression model for any influenza subtype (backward selection). all analyses were performed with sas version 9.4 software. sensitivity analyses were performed in the ve analyses for other control groups (supplementary materials). in this prospective study, we observed an ili incidence of 7.2% (143/1992) and 11.6% (275/2368) in 2 consecutive seasons (2011-2012 and 2012-2013, respectively) ( figure 1a ; table 1 ). the average age of vaccinated individuals was significantly higher than that of unvaccinated individuals (respectively, 70.4 vs 66.9 years in 2011-2012 and 71.9 vs 67.9 years in 2012-2013; table 2 ). the asymptomatic controls from the second season were older and more often vaccinated compared with the overall cohort ( figure 1b ; table 2 ). furthermore, participants who reported comorbidities were vaccinated significantly more often than participants who did not report comorbidities (table 3) . no significant differences were found between individuals with or without ili with respect to sex, age, and chronic illnesses (tables 1 and 3) . importantly, no differences were found in the incidence of ili episodes between vaccinated and unvaccinated participants (table 1) . in 79.1% and 78.0% of the acute ili samples from the 2 seasons, at least 1 potential pathogen could be identified by mlpa or bacterial culture ( in 16.2% and 17.0% of the samples from the acute phase, >1 potential pathogen was detected, but no specific combinations of viruses and/or bacteria were observed (data not shown). in recovery samples, 8 weeks after acute ili, potential pathogens were detected in 27.0% and 24.8% of cases in 2011-2012 and 2012-2013, respectively. in asymptomatic control samples, similar potential pathogens were observed as in recovery samples (21.5%) ( figure 2c ). in 2011-2012, influenza virus was detected in 18.9% of the acute ili samples, predominantly of the a(h3n2) subtype (96.3%) ( figure 3 ; supplementary table 1a) . influenza virus was not detected in the corresponding recovery samples in this season, suggesting that influenza virus was the actual cause of ili. in 2012-2013, influenza virus was detected in 34.2% of the acute ili samples, and all 4 circulating subtypes were detected: 43.6% a(h3n2), 25.5% a(h1n1)pdm09, 25.5% b/yamagata lineage, and 5.3% b/victoria lineage. in addition, influenza virus was detected at a very low level in both the recovery samples and in samples of asymptomatic controls (1.1% and 0.9%, respectively). we investigated which other viruses and bacteria were detectable during ili episodes. in 60.8% (2011-2012) and 44.7% (2012-2013) of ili samples, potential pathogens other than influenza virus were detected (figure 3 ; supplementary table 1 ). coronaviruses of all 4 common human subtypes (18.2% in 2011-2012 and 11.3% in 2012-2013), human metapneumovirus (hmpv) (20.3% and 3.6%), rhinoviruses (8.4% and 21.1%), respiratory syncytial virus (rsv) (4.9% and 6.5%), and parainfluenza viruses (2.8% and 5.1%) were detected in >5% of the ili samples in at least 1 season. hmpv and rhinovirus varied most between the 2 seasons. all viruses were detectable at low levels in recovery and control samples. rarely, the same virus was observed during both the ili event and the recovery sampling, except for rhinoviruses, which were detected frequently at both visits (8.3% in 2011-2012 and 17.2% in 2012-2013), but as our test only detected rhinovirus in general, we cannot exclude that these are different serotypes. the attack rate of influenza virus was significantly higher in the second season compared to the first season (p < .0001; table 4 ). interestingly, the increased attack rate could be attributed to significant increases in the attack rates of h1n1, the b/victoria-like subtype, and the b/yamagata-like subtype (p < .0001, p = .04, and p < .0001, respectively), whereas the attack rate of the h3n2 subtype was not significantly different between these seasons. for the other viruses, we observed significant increased attack rates in the second season for rhinovirus (p < .0001) and parainfluenza viruses (p = .04), whereas hmpv attack rates were significantly lower in the second season (p = .0004). the other viruses had similar attack rates during the 2 seasons. the only bacterial species detected by conventional culture in a significant number of acute ili cases was h. influenzae (15.4% in 2011-2012 and 11.3% in 2012-2013), frequently as the sole pathogen, while presence of other bacteria such as h. haemolyticus and s. pneumoniae was low. haemophilus influenzae was also detected frequently in recovery and control samples. we evaluated whether influenza vaccination reduced the overall influenza virus infection incidence and whether this influenced the incidence of ili. although influenza virus lost to follow-up n=20 no ili reported n=2240 per protocol no ili reported n=2108 a subject could have multiple ili episodes per season. an ili visit (v1) was considered "out of window" if the sample was taken >72 hours after start of fever. for the recovery visit (v2), the window was 7-9 weeks after ili onset. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (a). after the baseline visit had been performed, a subject could have an ili event. subjects were considered lost to follow-up if they did not respond to the end of study mailing and had no ili visit (b). infection incidence was significantly lower in influenza vaccinated than in unvaccinated individuals (table 5) , the incidence of ili cases was not reduced by vaccination (table 1) . among participants with ili, we observed a high ve of 73% (95% ci, 26%-90%) in 2011-2012 and a moderate ve of 51% (95% ci, 7%-74%) in 2012-2013 against influenza virus during the influenza-active period (table 6 ). furthermore, the ve for the predominant influenza virus subtype a(h3n2) in 2011-2012 was 71% (95% ci, 19%-90%). in 2012-2013, the ve against influenza virus type a(h3n2) was 67% (95% ci, 20%-86%). additional sensitivity analyses for multiple ilis, households with ili, and the presence or absence of other virus infections did not affect this conclusion (supplementary table 2 ). in this study in a cohort of community-dwelling older adults in the netherlands, we show that influenza virus was present in 18.9% and 34.2% of ili cases in 2 consecutive seasons and that influenza vaccination significantly reduced laboratory-confirmed influenza virus infection. in 60.8% and 44.7% of the acute ili cases, potential pathogens other than influenza virus were detected. in addition, these pathogens were more often present during ili than after recovery or in asymptomatic elderly persons. in 20% of the ili cases, no potential pathogen was detected. the incidence of ili cases was expected to decrease by a reduction in influenza virus-caused ili through vaccination; however, this effect was not observed. instead, the incidence of c individuals in the asymptomatic subset were selected to be evenly distributed over the different age groups; therefore, the overall vaccination level was higher in the asymptomatic subset compared to the ili and non-ili groups. ili remained the same between the vaccinated and nonvaccinated individuals. influenza vaccination is offered to individuals 60 years and older in the netherlands, as older adults are at increased risk for morbidity and mortality from influenza virus infections due to an aging immune system and age-related chronic illnesses [19, 20] . however, this policy is based on studies mostly performed in elderly persons in nursing homes, who are substantially older and more frail compared with community-dwelling elderly individuals. studies in community-dwelling older adults are scarce [7] [8] [9] [10] , and only a restricted number of pathogens have been analyzed in these surveillance studies. to fill the gap in knowledge, we recruited participants through general practitioners and the civil registry, resulting in >99% of the participants living in the community. in line with the risk-based vaccination strategy in the western world, older participants and participants with chronic conditions were more likely to be vaccinated [2] . the age distribution in the cohort was similar to that observed for those ≥60 years in the general dutch population; only the oldest age group (>80 years) was underrepresented (table 2) . these elderly persons are often less likely to participate in studies. "healthy user effect" or "frailty selection bias" is a well-known issue in observational influenza vaccine studies, as the very frail elderly persons are difficult to reach [21, 22] . we found that influenza virus is involved in 18.9%-34.2% of ili cases in the 2 seasons studied. in addition, coronaviruses, rhinoviruses, hmpv, rsv, parainfluenza viruses, and h. influenzae were frequently observed as the sole detected pathogen in acute ili cases, whereas they were low to absent in most of the recovery samples. however, rhinoviruses and the bacterium h. influenzae were also commonly detected in asymptomatic controls. the rhinoviruses may be of different subtypes, as we did not type these viruses. viruses can often be detected in individuals without clinical manifestations [7] , but when they are found as the sole agent in the context of disease, they are commonly considered to be the cause of the disease. for bacteria, this is less clear as they are commonly carried in asymptomatic persons but may expand during respiratory viral infection or new acquisition. haemophilus influenzae may induce an enhanced inflammatory state in the presence of other pathogens, as was shown in children [23] . this may lead to ili. however, in most ili cases where we found h. influenzae, we did not find a second pathogen that could also explain the ili symptoms. influenza virus incidence in our cohort broadly matched the incidence reported in the dutch sentinel surveillance system [11] , reported a high ili incidence and mortality in the 2011-2012 season [24, 25] . this underlines a potential difference between the generally healthy community-dwelling elderly persons and that of the generally more frail institutionalized elderly persons, who may overlap in age. it also shows that data acquired in one group do not necessarily apply to the other, including exposure and susceptibility to infection, as well as influenza ve. we show that the incidence of influenza virus infection was reduced in individuals who received influenza vaccination and that ve to laboratory-confirmed influenza virus infection was high to moderate in the 2 seasons. the 95% cis of the data are wide, probably due to the relatively small sample size, a common problem in similar studies. ve estimates are notoriously variable between studies [18], but the ve data from this study are in line with those reported by van der hoek et al [18] . the most striking finding in this study was the similar incidence in ili cases observed between vaccinated and the nonvaccinated individuals. this may be explained by assuming that a pool of people exists that is highly susceptible to respiratory infections. the reduction by vaccination in the number of cases caused by influenza virus infections is offset by a rise in the number of cases caused by infections by other pathogens. cowling et al have described a similar increased risk of noninfluenza respiratory virus infection in influenza-vaccinated children [26] . however, we cannot attribute this effect to a specific pathogen. influenza virus may take preference over other viruses and prevent them from filling the niche, possibly by inducing a prolonged antiviral state, as has been described for other viruses [23, 27] . when vaccination reduces influenza virus infections, the other pathogens can fill the gap. it needs to be confirmed in other studies whether influenza virus vaccination has no effect on the total number of ili cases. it would have important consequences for decisions on implementation of vaccination for community-dwelling older adults when looking at the overall disease burden and cost-effectiveness. however, a limitation of our study is that we did not directly monitor the duration and severity of disease in the ili cases: difference in disease or hospitalization was only registered in post-ili questionnaires, and no significant events were reported. more detailed data on severity and duration of symptoms would allow assessing the relative risk posed by different pathogens, taking into account that influenza virus, unlike most other potential pathogens, can vary in pathogenicity between seasons. in combination with other medical information, it may then be possible to assemble a profile of individuals potentially at risk for ili or worse. such a profile would be of great value to public health professionals. a limitation of this study is the definition of ili used, which differs between the world health organization, the european centre for disease prevention and control, and different countries, although most include fever and cough [28] [29] [30] . in this study we used the dutch pel criteria [13] , which includes fever. it has been described that this specific ili definition can affect the number and type of pathogens detected. falsey et al showed that fever is more frequently associated with influenza virus infection in the elderly persons compared with other respiratory infections [7] . cough is not a prerequisite in the pel criteria, but as >80% of the participants with ili and >90% of the participants with influenza virus-positive ili reported coughing (data not shown), it is unlikely that cases were missed due to this difference in definition. in summary, we show that influenza virus caused between 18.9% and 34.2% of ili cases in community-dwelling older adults aged ≥60 years in 2 influenza seasons in the netherlands, leaving the remainder caused by other pathogens. we also show that influenza vaccination was effective in reducing the incidence of influenza virus infections but did not reduce the ili incidence, which may have important public health and healthcare consequences. our data will also help to better for more details, see supplementary table 3 . abbreviations: ci, confidence interval; ve, vaccine effectiveness. a corrected for the possible confounders age group, comorbidity, sex, and smoking. data were calculated for the influenza-active period in the netherlands as defined by the netherlands institute for health services research [11] . inform the public what to expect from influenza vaccination and how it will not protect against all cases of ili, popularly seen as "flu. " supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. notes influenza (seasonal) social determinants of health and seasonal influenza vaccination in adults ≥65 years: a systematic review of qualitative and quantitative data effectiveness of seasonal influenza vaccine in community-dwelling elderly people: a meta-analysis of test-negative design case-control studies the efficacy of influenza vaccination in elderly individuals. a randomized double-blind placebo-controlled trial vaccines for preventing influenza in the elderly cochrane re-arranged: support for policies to vaccinate elderly people against influenza respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza vaccine effectiveness among elderly persons living in the community during the 2003-2004 season a case-control study of acute respiratory tract infection in general practice patients in the netherlands nivel influenza surveillance sentinel surveillance network on infectious diseases in nursing homes study group. absence of influenza a(h1n1) during seasonal and pandemic seasons in a sentinel nursing home surveillance network in the netherlands proefonderzoek naar de frequentie en de aetiologie van griepachtige ziekten in de winter 1963-1964 preparing the outbreak assistance laboratory network in the netherlands for the detection of the influenza virus a(h1n1) variant differentiation of influenza b virus lineages yamagata and victoria by real-time pcr effect of 7-valent pneumococcal conjugate vaccine on nasopharyngeal carriage with haemophilus influenzae and moraxella catarrhalis in a randomized controlled trial identification of haemophilus influenzae and haemophilus haemolyticus by matrix-assisted laser desorption ionization-time of flight mass spectrometry letter to the editor: influenza vaccine effectiveness: heterogeneity in estimates for the 2012/13 season frailty, inflammation, and immunity vaccines for the twenty-first century society mortality benefits of influenza vaccination in elderly people: an ongoing controversy effectiveness of influenza vaccine in aging and older adults: comprehensive analysis of the evidence dutch rsv neonatal network. respiratory syncytial virus and recurrent wheeze in healthy preterm infants excess mortality among the elderly in 12 european countries increased risk of noninfluenza respiratory virus infections associated with receipt of inactivated influenza vaccine does viral interference affect spread of influenza? world health organization. ili sari surveillance case definition overview of influenza surveillance in the united states european centre for disease prevention and control. influenza case definitions we gratefully acknowledge all participants for their time and commitment to the study. we thank the study staff at the spaarne hospital and the laboratory staff members at the regional laboratory kennermerland and the centre for infectious disease control at the national institute for public health and the environment.financial support. this work was supported by the dutch ministry of health, welfare and sport.potential conflicts of interest. e. a. s. has received research grants from pfizer and gsk, in addition to fees paid to wilhemina children's hospital/university medical center for advisory boards and participation in independent data monitoring committees for pfizer and gsk. r. v. has received research support from gsk and pfizer for vaccine studies and consulting fees for gsk. all other authors report no potential conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-254117-2ttwaegh authors: priest, patricia c.; duncan, alasdair r.; jennings, lance c.; baker, michael g. title: thermal image scanning for influenza border screening: results of an airport screening study date: 2011-01-05 journal: plos one doi: 10.1371/journal.pone.0014490 sha: doc_id: 254117 cord_uid: 2ttwaegh background: infrared thermal image scanners (itis) appear an attractive option for the mass screening of travellers for influenza, but there are no published data on their performance in airports. methods: itis was used to measure cutaneous temperature in 1275 airline travellers who had agreed to tympanic temperature measurement and respiratory sampling. the prediction by itis of tympanic temperature (37.8°c and 37.5°c) and of influenza infection was assessed using receiver operating characteristic (roc) curves and estimated sensitivity, specificity and positive predictive value (ppv). findings: using front of face itis for prediction of tympanic temperature ≥37.8°c, the area under the roc curve was 0.86 (95%ci 0.75–0.97) and setting sensitivity at 86% gave specificity of 71%. the ppv in this population of travellers, of whom 0.5% were febrile using this definition, was 1.5%. we identified influenza virus infection in 30 travellers (3 type a and 27 type b). for itis prediction of influenza infection the area under the roc curve was 0.66 (0.56–0.75), a sensitivity of 87% gave specificity of 39%, and ppv of 2.8%. none of the 30 influenza-positive travellers had tympanic temperature ≥37.8°c at screening (95%ci 0% to 12%); three had no influenza symptoms. conclusion: itis performed moderately well in detecting fever but in this study, during a seasonal epidemic of predominantly influenza type b, the proportion of influenza-infected travellers who were febrile was low and itis were not much better than chance at identifying travellers likely to be influenza-infected. although febrile illness is more common in influenza a infections than influenza b infections, many influenza a infections are afebrile. our findings therefore suggest that itis is unlikely to be effective for entry screening of travellers to detect influenza infection with the intention of preventing entry of the virus into a country. rising concerns regarding influenza a (h5n1) and the pandemic of influenza a (h1n1) 2009 have led to the use of infrared thermal image scanners (itis) at some borders for the mass screening of travellers to detect those who might be infected with influenza [1] . itis measure body surface temperature rapidly, non-invasively, and with no contact, minimising the risk of contagion. they therefore have the potential to comply with the international health regulations' emphasis on containing the spread of disease in ways that avoid unnecessary interference with international traffic and trade [2] . evaluations of the use of itis in clinical settings have been conducted, and have reported sensitivities of 15% to 90% for confirmed fever depending on the cut-off used to define fever [3, 4, 5, 6] . however, these findings may not be applicable to border screening. itis measure body surface temperature, not body core temperature, and so itis temperature measurements are subject to the influence of a range of human and environmental factors. these include whether a person is sunburnt, has taken antipyretics or has circulatory problems, and also the ambient temperature and humidity. consequently it is important that the relationship between body surface temperature and body core temperature be evaluated within the environment in which itis are to be operated. in the airport setting, thermal scanning of arriving travellers has been used to screen for several different infectious diseases. during the outbreak of severe acute respiratory syndrome (sars) itis use was documented, however only the numbers of travellers triggering the scanner were reported, without stating the cut-off threshold used for fever or reporting on any subsequent method used to confirm febrile status [7, 8, 9] . a trial dengue fever screening programme found that among travellers arriving into cairns airport [10] 12% (118/963) of travellers who triggered the pre-set alarm threshold were confirmed to be febrile on tympanic temperature measurement. influenza screening in singapore found that only 12% of cases of pandemic (h1n1) 2009 infection with onset within 10 days of arrival were detected by itis on entry [11] . proper evaluation of a screening test requires that the 'gold standard' test is applied to both test positive and test negative participants in the study. to evaluate the use of itis in border screening for influenza, its performance in predicting both fever and also influenza infection is necessary. however to date no studies have been reported that tested itis negative travellers for either fever or influenza infection [12, 13] . we undertook both itis and tympanic temperature measurement on, and collected specimens for testing for influenza from, symptomatic and asymptomatic air travellers arriving into christchurch, new zealand during the southern hemisphere winter in 2008. this paper assesses the performance of itis in detection of fever and infection with seasonal influenza in these airline travellers. this evaluation of thermal image scanning was carried out as part of a larger study to measure the prevalence of seasonal influenza infection in arriving airline travellers and the effectiveness of a screening questionnaire for detecting those with influenza infection. the design followed closely a pilot study carried out in 2007 [14] . three airlines agreed to have their staff distribute a screening questionnaire to travellers (passengers and crew) during flights travelling from australian airports to christchurch, new zealand. the questionnaires were collected by research assistants following immigration processing on arrival in christchurch. 'symptomatic' travellers were defined as those who reported one or more of the following symptoms: cough, sore throat, sneezing, fever or chills, runny or blocked nose, muscle aches or pains, feeling generally unwell, chest discomfort or breathing difficulties. symptomatic travellers were all invited to have throat and nose swabs (copan italia spa, brescia, italy) taken and their temperature measured. in addition, half the questionnaires were marked and were randomly placed into the sets of questionnaires delivered to the flight crew (the sequence was determined by the rand function of microsoft excelß). arriving travellers carrying a marked questionnaire were also invited to have swabs and temperature taken. the nurse taking the swabs noted on the request form whether the traveller was symptomatic or asymptomatic. for the 23 working days from 21 august to 12 september 2008, cutaneous temperature from those travellers invited to participate who had given consent was measured using itis (therma-cam tm e45, flir systems, sweden) prior to swabs being taken. a focal plane array (1606120 pixels) was used on the front of the face and the side of the face (see figure 1 ) and the maximum temperature reading for each was recorded. after the swabs were taken, each participant's tympanic temperature was measured using an infrared tympanic thermometer (thermascan pro4000, braun, germany). the ambient temperature in the arrivals hall was a consistent 20.5uc at all times during data collection. all nasal and throat swab samples were analysed at canterbury health laboratories, christchurch. a multiplexed tandem polymerase chain reaction (mt-pcr) assay was employed to detect the presence of influenza a and b virus infection, as described by the manufacturer (easy-plex influenza a+b kit, cat. no. 3005.01, ausdiagnostics pty ltd, sydney, australia). stataß 10 was used to analyse the data. the cii command was used to calculate poisson exact confidence intervals around the proportion of influenza-infected travellers who were febrile. information about temperature measurements was collected on the swab consent form and linked to the symptom information on the questionnaire using a unique swab identifier. nine swab results were unable to be linked as their identifier had not been attached to any questionnaire. for these individuals the nurse's note of whether or not they were symptomatic was used to define their symptom status. analyses were performed to assess the accuracy of itis measurements in predicting two different tympanic temperature thresholds: 1. tympanic temperature $37.8uc (.100uf -the level used by the centers for disease control in defining 'influenza-like illness') [15] 2. tympanic temperature $37.5uc (the threshold used in the majority of reports) [12] . firstly, a receiver operating characteristic (roc) curve [16] was constructed. roc curves assess the ability of a test (in this case the itis measure) to discriminate between people who have, and who do not have, a condition (fever). the area under the roc curve for an uninformative test is 0.5. secondly, a level of itis temperature with sensitivity closest to 85% was chosen and the specificity calculated. finally, the positive predictive value (ppv) of the chosen level of itis temperature was estimated. the positive predictive value is the proportion of people who test positive (i.e. are 'positive' on itis) who actually have the condition of interest. it is not appropriate to calculate the ppv of itis measures directly in this sample since it was not a random sample of the population of travellers but was instead 'enriched' by including as many symptomatic travellers as were prepared to provide respiratory samples. therefore, the prevalence of fever (by each definition) in the holders of marked questionnaires was combined with the sensitivity and specificity of itis for detecting fever to estimate the ppv in the population of all travellers who arrived on the flights that took part in the study. to assess the utility of fever as a screening test for influenza infection (mt-pcr result), sensitivity, specificity, and population ppv for influenza were estimated for each tympanic temperature threshold, and the itis threshold used above. this study was approved by the new zealand health and disability multiregion ethics committee. written informed consent was obtained from all participants. in total, 5274 travellers returned a questionnaire during the study period, of whom 823 (15.6%) were symptomatic by our definition. figure 2 shows the pathway of potential participants through the study. accuracy of thermal scanning in predicting core temperature seven participants had a tympanic temperature of $37.8uc (2 reported no symptoms and 5 were symptomatic). five held marked questionnaires, giving a prevalence of fever by this definition of 0.5% (5/1063). half of the 38 participants with a tympanic temperature of $37.5uc were symptomatic. thirty-two of them held a marked questionnaire, so the prevalence of fever by this definition was 3.0% (32/1063). figure 3 is a roc curve showing the ability of itis front of face measurement to predict a tympanic temperature of $37.8uc. table 1 shows the test characteristics of itis as a predictor of tympanic temperature. for each definition of 'fever' (determined by tympanic temperature measurement), and for each site of itis measurement (front and side of face), the table shows: the itis threshold that gave a sensitivity closest to 85% in our data; the proportion of travellers with an itis measure above that threshold (i.e. who would have 'triggered' the itis during screening); the area under the roc curve; the actual sensitivity of that threshold; and its specificity. the prevalence of fever at each threshold in holders of marked questionnaires (as an estimate of the prevalence in this population of arriving travellers) is also shown, as well as the estimated ppv of itis for fever in this population. most (90%; 27/30) influenza-positive participants were symptomatic, but none (0%) had a measured tympanic temperature of $37.8uc (99%ci 0% to 18%), and only two (7%) had a measured tympanic temperature of $37.5uc(99%ci 0.3% to 31%). table 2 shows the ability of tympanic and itis temperatures to predict influenza infection in a population where the prevalence is 2% (the estimate of the prevalence of infection in this population of arriving travellers). with high sensitivity, specificity is very low. combined with the low prevalence of influenza infection in this population, ppv is also very low. influenza-positive participants reported that the first of their symptoms started between 12 hours and 24 days prior to answering the questionnaire, with symptom duration of 2 days or less in 11 participants, more than 2 and up to 5 days in 7 participants, and more than 5 days in 8 participants (3 were asymptomatic and 1 did not respond to this question). the greatest potential for the use of itis to screen incoming or departing travellers for infectious diseases such as a pandemic strain of influenza would be as the first stage of screening; that is, to identify and select out a high risk group for further assessment, for example by questionnaire, body core temperature measurement, and/or respiratory sample collection. this would require very high sensitivity for raised body temperature, as any travellers who 'slipped through' the screening process would enter the community and potentially spread infection. in addition, core temperature would need to be a good predictor of infection. can thermal scanning predict core temperature? this study shows that, among a group comprising both asymptomatic and symptomatic arriving international airline travellers, itis can have moderately high sensitivity and specificity for a high body core temperature of $37.8uc. however, the low prevalence of fever in arriving travellers means that the ppv is very low. measurement of the sensitivity of fever for influenza infection requires that afebrile as well as febrile people, from the same population, are tested for influenza infection. there are few studies that have done this, as symptoms of 'influenza-like illness', which include fever, are usually criteria for entry to studies of influenza [17, 18, 19] . such studies, with selected participants with a high prevalence of influenza infection, overestimate the sensitivity and dramatically overestimate the ppv of fever for influenza infection in unselected populations, such as airline travellers. a review of volunteer challenge studies [20] showed that not only were approximately 30% of influenza infections asymptomatic, but only 35% of those with symptoms had a measured fever .37.8uc. this study found a lower prevalence of fever among the participants infected with influenza b (7/101) than with influenza a h1n1(88/285; 31%) [20] . in this study, none of the 30 travellers subsequently identified as infected with influenza (most of whom had influenza b) had a temperature $37.8uc, and only two had a temperature $37.5uc. these results emphasise what is already known about fever as a symptom of influenza -while it clearly is one of the symptoms that can be experienced by people with influenza infection, it does not occur in all infected people [20] . the prevalence of fever is high in case series of patients with confirmed influenza infection [11, 21] , since often one of the criteria that is often used to determine whether testing takes place is the presence of fever. however, where fever is not used as a criterion for influenza testing, the prevalence of fever is by no means 100%, even among people with severe symptoms. for example, among 106 patients hospitalised with respiratory disease [22] , 39% of those with confirmed pandemic (h1n1) 2009 infection did not have a temperature of $37.8uc at any time during admission. in this study, the predominance of influenza b infection may partly explain the low prevalence of fever among infected participants (although the three with influenza a all had tympanic temperatures ,37.2uc ).even with more pyrexigenic strains, among travellers, who by definition are not severely unwell and in fact who are mostly not unwell at all, the proportion of influenza infected people who are afebrile can be expected to be much higher than among hospitalised patients [22] (because the sicker infected people don't travel), as shown in this study. it was a condition of conducting this study that we did not delay the transit of passengers through the airport by more than a few minutes and, therefore, measurements had to be made efficiently. we used a single measurement by an infrared tympanic thermometer as our 'gold standard' measure of core temperature. this approach may have introduced some random error into our results, but is unlikely to have caused systematic bias and is likely to be similar to the way that temperature would be confirmed in practice. in addition, our participants sat still at approximately 1m from the scanner for the itis measure and those who were wearing glasses were asked to remove them, steps likely to have provided greater accuracy than itis measures that are taken as numerous people walk past a fixed scanner in an arrivals hall. therefore our study provides an assessment of the best results that could be expected from the use of itis in border screening for influenza. in this study, no influenza-infected travellers had a measured tympanic temperature $37.8uc. we do not believe that this was because of systematic errors in tympanic temperature measurements, as these were measured by trained nurses using standard thermometers. we acknowledge that the number of infected travellers was relatively small at 30 but the probability is only 0.005 (0.5%) that the prevalence of fever among the population of infected travellers arriving from australia into christchurch at this time was greater than 18%; in other words the vast majority of infected travellers in this population were afebrile. among travellers, the proportion of influenza cases who are febrile may be low because those infected with influenza that is causing fever may feel too unwell to travel; 25% of travelassociated cases of pandemic (h1n1) 2009 infection with onset in singapore were symptomatic on embarkation but the proportion who were febrile was not reported [11] . in addition, it is possible that unwell infected travellers had used anti-pyretics prior to or during the flight, but this is a limitation of itis rather than of our study. the study assessed the performance of itis in the real world, which includes the fact that some unwell people take antipyretics. also, the flights that were part of this study were relatively short -3 to 4 hours -and it is possible that on longer flights some of the infected travellers might have become febrile. however, it remains unlikely that fever would occur in all, or even most, infected travellers arriving at any international airport [23] . good evidence on influenza virus transmissibility during the various phases of viral infection, (including afebrile infection and asymptomatic infection) is not available, but detection of viral rna on a respiratory sample does not necessarily mean that the infected person is, or will be, infectious. we were not able to perform culture for influenza virus in this study, so it is possible that some of the infected travellers were not shedding viable virus. although the approximately one third of participants whose symptoms were of 2 days' duration or less were likely to be in the early stages of their infection, those with longer duration of symptoms may not have been. unfortunately the symptoms of influenza are so non-specific that it is difficult to estimate the stage of influenza infection in a traveller with, for example, a cough that has been present for several weeks. nonetheless, it seems reasonable to conclude that at least a third, and probably more, of the infected (and afebrile) participants in this study were infectious on or after arrival into new zealand. influenza-infected arriving travellers include those who are symptomatic (with or without fever), those who become symptomatic during the flight, those who will develop symptoms following arrival, and those who will never have symptoms. it is not known whether the latter group are infectious, but clearly only the first two categories could potentially be detected by entry screening. most people who were infected but asymptomatic on boarding will still be asymptomatic on arrival at their destination [23] . however, in the absence of effective exit screening during the h1n1 2009 pandemic, some countries decided to use itis in entry screening with the hope that detecting travellers who were febrile on arrival would be worthwhile to reduce the probability of infected travellers entering the country, and that itis could detect them [1] . this study provides evidence to the contrary. the low ppv of itis measures for fever in this population means that the number of false positives who would require further investigation, presumably by taking a tympanic temperature, would be very high. in this study, using a front of face itis threshold of 35.4uc identifies 69% of travellers as requiring further investigation, of whom only 4.1% had a tympanic temperature $37.5uc. the ppv of any of the measures of temperature for influenza infection itself was lower, at less than 3%. however, the prevalence of disease is an important determinant of ppv, and the prevalence of influenza infection in this study, performed during the 'influenza season', was low at 1.9%. there are no other published estimates of the prevalence of influenza in arriving travellers, but it could be argued that the prevalence of infection would be higher during a pandemic, which typically infects a higher proportion of the population than seasonal influenza, than in this study. on the other hand, particularly if local containment strategies were in place in originating countries, the prevalence of infection in travellers might be lower during a pandemic. at the beginning of a pandemic, when effective entry screening would be most useful, the prevalence of infection among travellers and therefore the ppv will likely be much lower than the prevalence of seasonal influenza in this study. more importantly, raised temperature itself by any measurement technology is insufficiently sensitive for influenza infection for its measurement to be effective for mass screening in a pandemic situation. use of itis to identify travellers at high risk of fever, measuring the core temperature of itis-positive travellers, and then taking specimens from those with high core temperatures would have failed to identify all the influenza-infected travellers in this study. using a lower temperature threshold (however measured) for taking specimens could detect a high proportion of influenza-infected travellers only by taking specimens from what is likely to be an unfeasibly high proportion of travellers. governments may decide to implement entry screening, including itis, for reasons other than to actually detect most influenza-infected arrivals, for example to deter unwell people from travelling, or to demonstrate to their citizens that they are doing everything they can to protect population health. the risks associated with this approach include the potentially very large opportunity cost of further investigating itis 'positive' travellers, including quarantine of those febrile on tympanic temperature measurement pending specimen processing, and the potential for the loss of public confidence in the pandemic response when it becomes clear that many infected travellers were not detected by the screening and entered the country. in this study, during a seasonal epidemic of predominantly influenza type b, influenza-infected arriving travellers had a very low prevalence of fever. consequently, itis would not have identified influenza-infected travellers even though it performed moderately well at detecting febrile travellers. some aspects of this study may not generalise to a pandemic of influenza a. although febrile illness is more common in influenza a infections than influenza b infections, many influenza a infections are afebrile. our findings therefore suggest that itis is unlikely to be effective for entry screening of travellers to detect influenza infection with the intention of preventing entry of the virus into a country. entry screening to delay local transmission of 2009 pandemic influenza a (h1n1) revision of the international health regulations world health organisation screening for fever by remote-sensing infrared thermographic camera limitations of forehead infrared body temperature detection for fever screening for severe acute respiratory syndrome analysis of ir thermal imager for mass blind fever screening cutaneous infrared thermometry for detecting febrile patients world health organization working group on international and community transmission of sars (2004) public health interventions and sars spread thermal image scanners to detect fever in airline passengers, vancouver and toronto border screening for sars investigation of the optimal assessment of febrile passengers detected by infrared thermal scanning at an international airport epidemiology of travel-associated pandemic (h1n1) 2009 infection in 116 patients international travels and fever screening during epidemics: a literature review on the effectiveness and potential use of non-contact infrared thermometers physical interventions to interrupt or reduce the spread of respiratory viruses: systematic review screening for influenza infection in international airline travellers manual for the surveillance of vaccine-preventable diseases diagnostic tests 3: receiver operating characteristic plots clinical signs and symptoms predicting influenza infection predicting influenza infections during epidemics with use of a clinical case definition diagnosis of influenza in the community: relationship of clinical diagnosis to confirmed virological, serologic, or molecular detection of influenza time lines of infection and disease in human influenza: a review of volunteer challenge studies clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china clinical diagnostic criteria for isolating patients admitted to hospital with suspected pandemic influenza entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation we thank the health emergency management branch, department of health and ageing, australia for lending us the scanner; christchurch international airport limited, new zealand customs service, and the participating airlines for their cooperation and assistance; and andrew strathdee for the laboratory testing.we are grateful to elisabeth wells, heath kelly, jonathan van-tam, and rebecca psutka for their helpful comments on drafts of this article, and to the anonymous reviewers for their helpful comments on the originally submitted version. key: cord-253083-4mk5u0wg authors: lazarus, rajeka; lim, poh lian title: avian influenza: recent epidemiology, travel-related risk, and management date: 2014-12-05 journal: curr infect dis rep doi: 10.1007/s11908-014-0456-3 sha: doc_id: 253083 cord_uid: 4mk5u0wg h5n1 influenza continues to smolder in southeast asia over the past 5 years, but the emergence of h7n9 in china in 2012 raised concerns for a new avian influenza threat. in contrast with h5n1 with over 650 confirmed cases over 11 years, h7n9 has infected over 450 persons within 2 years. the case fatality rate for h7n9 (35 %) is lower than for h5n1 (60 %) or h10n8 (67 %) but is comparable to that for the middle east respiratory syndrome coronavirus (mers cov), another emerging zoonosis with travel-associated importations. exposure to poultry and fomites are considered the likely sources of infection for h7n9, h5n1, and h10n8, with limited human-to-human transmission in close contacts. most cases have occurred in local populations of affected countries, and travel-related risk can be mitigated by avoiding exposure. vaccines, antivirals, and other therapeutics remain in development stage or of modest benefit for dangerous infections carrying high morbidity and mortality. for decades, influenza a has been on the watch list as a potential pandemic pathogen. in the past 15 years, two avian influenza strains, h5n1 and h7n9, have successfully jumped the species barrier, resulting in outbreaks of human infection. although there has been no evidence of sustained human-tohuman transmission to date, if this occurs, we would be closer to fulfilling all the necessary conditions for a devastating human pandemic. h5n1 influenza continues to smolder in southeast asia since 1997 [1], but the emergence of h7n9 in china in 2013 raised concerns for a new avian influenza threat [2] . in contrast with h5n1 with 650 confirmed cases since 2003, h7n9 has infected over 400 persons in just over 1 year [3, 4] . how this situation evolves over the next few seasons may have far-reaching implications for our cumulative pandemic risk globally (fig. 1) . the h5n1 virus was first isolated from a farmed goose in the guangdong province of china in 1996, with the first human cases reported a year later in hong kong [5] . after this first cluster of 18 cases, h5n1 reemerged in 2003 in china. subsequently, h5n1 spread through southeast asia. turkey marked the first occurrence outside of asia, followed by iraq, azerbaijan, and egypt between january and april 2006 [1] . a total of 650 laboratory-confirmed human infections have been officially reported to the world health organization (who) from 15 countries in asia, africa, the middle east, and europe [3] . sporadic importations have occurred rarely, most recently in canada from a traveler returning from beijing [6] . h5n1 cases peaked in january 2006, when the outbreak was brought under control through expensive culling campaigns in affected countries like thailand, vietnam, and indonesia. yearly peaks around january have continued since then, but these have gradually stabilized [3] . in the aftermath of sars, china established a national surveillance system to detect novel pathogens using cases of pneumonia of unknown etiology (pue) as beacons. health care facilities in china are mandated to report details to local public health officials of all patients who have fever >38°c, radiographic features of pneumonia, low or normal leukocyte count, and no response or deterioration after 3 to 5 days of standard antimicrobial therapy. in such cases, respiratory tract specimens are tested for influenza h5n1 and for sars coronavirus. clusters, defined as two or more cases of pue with an epidemiological link that cannot be explained by local investigators, are escalated to the national centers for disease control (cdc) for further investigation [7] . in march 2013, a case of pue was tested for new influenza subtypes; this became the first documented case of h7n9 and led to the first three cases being diagnosed in shanghai and anhui, although no epidemiological link was established between the three cases [8] . after these cases were described, the definition of pue was broadened and more discretion given to individual physicians to test when clinically appropriate in order to increase the sensitivity of the program. by december 2013, 139 cases of h7n9 had been confirmed, and reporting continued until may 2013 [4] . this outbreak resurged in december 2013, peaking around the chinese new year celebration in february 2014. sixty-eight percent of individuals with h7n9 identified through the pue surveillance system were from the shanghai municipality and zhejiang province. these two regions, however, only contain about 6 % of the total chinese population; conversely, 10 % of h7n9 cases were reported from the remaining 21 provinces where approximately 57 % of the populations reside. these numbers may represent concentrated foci of infection; however, variable reporting among regions may yield inaccurate estimates of the true disease burden and distribution. such data may provide a false and potentially dangerous reassurance to travelers [7] . pue surveillance only provides numbers of ill cases admitted to a hospital. to establish the true spectrum of clinical cases, a sentinel survey was conducted among 541 outpatient clinics and emergency departments spread across 31 provinces. this yielded 5 of the total 130 lab-confirmed cases by may 27 2013 . three of the cases were in young children; four had exposure to live animals. three had mild to moderate illness while two were admitted to a hospital, one with pneumonia; subsequently, all recovered [9] . seroepidemiology investigations have been conducted to determine the extent of virus circulation and spectrum of disease. most studies use standard hemagglutinin inhibition assays or microneutralization assays according to who criteria [10] . a meta-analysis of h5n1 studies which included seroepidemiology studies that met who criteria and excluded diagnosed h5n1 cases found a seroprevalence of 1.2 % (95 % ci 0.6 to 2.1) in 7304 participants, with higher seropositivity (1.4 %) in poultry workers [11] . given the higher h5n1 seropositivity rate among poultry workers, this occupational group would likely have a higher risk of early exposure to emerging avian influenza viruses. a retrospective study using stored serum samples from 1544 occupational groups including workers in live poultry markets, farms, slaughterhouses, and wild bird habitats in eastern china (shanghai, zhejiang, jiangsu, and anhui) was analyzed to look for evidence of circulating h7n9 prior to the onset of the outbreak. surprisingly, samples taken between january and november 2012 showed no evidence of immune reactivity to h7n9 [12] . h5n1 human infections have occurred mostly in young persons, with children under age 12 years comprising 50 % of the 1997 outbreak cases [13] . by contrast, h7n9 infections have affected predominantly older men. in the first large series of 139 laboratory-confirmed h7n9 cases, 71 % were male with a median age of 60 and 73 % lived in urban areas, while those who were diagnosed as part of the outpatient sentinel survey who tended to have less severe disease were predominantly children [9, 14] . several investigators have explained this finding as reflecting social patterns: grandfathers are exposed shopping at live poultry markets, while grandmothers stay at home and younger adults are at work. the evidence for avian-to-human transmission comes from epidemiological and molecular investigations. among the first 139 h7n9 cases, 82 % reported a history of exposure to live animals including chickens [14] . similarly, a case-control study of the first 15 h5n1 cases demonstrated that 64 % of cases compared to 29 % of controls had visited live poultry markets. however, eating or preparing poultry products was not a risk factor [15] . another case-control study of 25 h7n9 cases with 93 matched controls found that direct exposure to poultry in the 2 weeks prior to illness onset was a risk factor for acquiring infection [16] . although transmission is predominantly after poultry contact, limited human-to-human transmission is possible. in the first h7n9 outbreak, four household clusters were reported, yet in three of these households, only the index case had exposure to poultry. infected household members had prolonged close contact to the index cases [14] . similarly for h5n1, investigations indicate at least three clusters with a likely human-to-human transmission in thailand and indonesia [17, 18] . transmission is not efficient; in one study, throat swabs were collected from symptomatic contacts, and paired sera from asymptomatic contacts showed no evidence of infection. spread may occur outside households, if there is close contact. seroepidemiology studies showed seropositivity in 1 out of 26 in a tour group, and exposed health care workers had 3.7 % seropositivity compared to 0.7 % among unexposed health care workers [18] . as the number of older travelers increase, with their propensity for tour groups, this may become an important consideration [19] . the most recent avian influenza jumping the species barrier into humans is h10n8. in november 2013, a 74-year-old woman visited a live poultry market 4 days before becoming ill; she died 9 days later. genetic analysis confirmed that all viral genes were of avian origin, prompting speculation whether this could start the next epidemic. there was concern that h10n8 could be even more widespread than h7n9 because h10n8 has primarily been found in migratory birds, rather than poultry. thankfully, only two cases have officially been reported to date (table 1) [20] . influenza viruses belong to the orthomyxovirus family, with human infections caused by both influenza a and b. novel subtypes result from the reassortment of gene segments when two viruses are circulating in a single host. phylogenetic analysis has indicated that h5n1 is a reassortment of at least four viruses, including goose h5n1 hemagglutinin and teal h6n1 neuraminidase, with internal genes from both quail h9n2 and teal h6n1 [21] . avian sampling data suggests that the h7 component of the current h7n9 human outbreak comes from the h7n3 domestic duck virus in china, while the closest n9 probably originated from wild birds in south korea. screening of virus archives with subsequent computation of the evolutionary pathways indicates that reassortments have happened on at least two independent occasions, firstly, h7 with h9n2 to generate h7n2 followed by a second reassortment event with bird h7n9 [22] . the current low transmissibility of h5n1 and h7n9 is believed to be related to the site at which the avian influenza viruses bind to human respiratory tract. influenza viruses bind with sialic acid receptors of which there are two subtypes relevant for avian influenza strains: α-2,3 sialic acid receptors found in the gastrointestinal tract of birds and also in the lower respiratory tract of humans and α-2,6 sialic acid receptors which are found in abundance in the human upper respiratory tract. influenza viruses adapted to avian hosts selectively bind to α-2,3 sialic acid receptors, whereas those adapted to humans bind to the α-2,6 subtype. genetic sequencing of binding sites and x-ray crystallography reveal that h7n9 binds strongly to α-2,3 receptors and weakly to α-2,6, unlike pandemic h1n1 which preferentially binds to α-2,6 receptors [23, 24] . although h7n9 and h5n1 are poorly adapted for human transmissibility at the present time, changes in receptor binding may occur with accumulated mutations arising from host pressure and faulty viral rna replication. some of the necessary mutations have already been identified in h5n1; passage through ferrets has produced isolates capable of preferential binding to α-2,6 receptors, making this situation theoretically possible [25] . case series are useful in reporting clinical features of avian influenza. however, these have the inherent limitation of small series and may not describe the full spectrum of disease. for h7n9, there is a detailed description of 113 out of 132 of the first reported cases [26] . the largest h5n1 report contains information for 79 cases in 5 different countries over a period of 8 years, with variable amounts of information from each country's dataset [27, 28] . fever and cough were described in all the h7n9 cases, whereas one case of h5n1 did not have fever, 67 to 100 % had cough, and 3 h5n1 cases had no respiratory symptoms at all. sore throat and rhinorrhea are reported for h5n1 but is not mentioned in h7n9 reports, while dyspnea was present in most cases of h7n9 (62 %) and h5n1 (85 %). gastrointestinal symptoms are more common with h5n1 (98 %) compared to h7n9 (13 %) infections [26] [27] [28] . h7 infections had previously been associated with conjunctivitis. two small outbreaks occurred previously, in the netherlands (2003) and canada (2004), both originated in poultry and resulted in a single fatal case as a result of pneumonia in each outbreak [29] . key laboratory features in both h5n1 and h7n9 were lymphopenia, thrombocytopenia, and raised alanine transaminase, with all three features reported more frequently in h7n9 [27] [28] [29] . both h5n1 and h7n9 had radiological changes consistent with pneumonia, including air bronchograms, diffuse ground glass shadowing, and pleural effusions [30] . severe disease was associated with more extensive lung pathology [31] . the most common complication was ards with 76 % of h7n9 patients admitted to an intensive care unit at a median of 7 days after illness onset. patients requiring mechanical ventilation experienced complications including ventilatorassociated pneumonia, pulmonary hemorrhage, and pneumothorax. organ dysfunctions included acute kidney injury, cardiac dilatation, arrhythmias, myocarditis, pericarditis, gastrointestinal hemorrhage, encephalitis, rhabdomyolysis, and sepsis of unknown source. most deaths have resulted from rapidly progressive pneumonia, typically primary influenza infection rather than secondary bacterial infection. mortality with h7n9 is 30-35 %, with death occurring 6-48 days after onset. by contrast, h5n1 mortality is 60-70 %, with patients dying sooner at 4 to 29 days. mortality rates are based on the outcomes of hospitalized patients and may overestimate the true mortality rate [26] [27] [28] . yu's modeling study, using assumptions about sentinel surveillance coverage and healthseeking behavior, suggests that h7n9 mortality could be as low as 16 %, which is nevertheless 100-fold higher than that of seasonal influenza [32] . risk factors of mortality are different between h5n1 and h7n9, particularly for age. h7n9 patients older than 60 had higher mortality (49 %) compared to those under 16 (18 %), whereas the opposite is true for h5n1, which saw 89 % of patients under 15 succumbing. in a multivariate analysis, coexisting medical conditions was the only other predictor for death from h7n9, despite this being a confounder with older age [26] [27] [28] . severity of disease is believed to be driven by the release of pro-inflammatory cytokines. although studied in a few cases, levels of il-2, il-6, and γ-ifn measured post-mortem were found to be higher in patients infected with h5n1 compared to uninfected individuals [33, 34] . testing of acute sera from h7n9 patients also indicated that levels of particular cytokines were predictive of more severe outcomes, which may suggest possible therapeutic approaches [35] . diagnosis should be considered for individuals presenting with travel exposure in h5n1-or h7n9-affected areas, with or without poultry exposure. for residents in avian influenzaaffected areas, influenza-like illness with fever and cough should prompt consideration of possible avian influenza, especially if disease is severe and rapidly progressive, or if patient has history of exposure to poultry within the previous 2 weeks, or contact with an infected person. for both h5n1 and h7n9, definitive diagnosis can be confirmed by pcr or viral culture of respiratory samples including nasal/throat swabs, sputum, or bronchoalveolar lavage samples. paired sera that meet the who criteria may also be useful for detecting asymptomatic cases, for retrospective diagnosis, and for epidemiological studies [10] . specific antivirals for these influenza strains are primarily the neuraminidase inhibitors oseltamivir and zanamivir, with newer agents such as peramivir available for compassionate or emergency use in different countries. agents that block the influenza matrix protein 2 ion channel such as amantadine are not recommended due to resistance. neuraminidase inhibitors stop the virus from exiting the host cell, thereby acting to reduce viral loads in the blood. h5n1 survivors have demonstrated lower levels of the virus in their blood than non-survivors [36] . in a subgroup of 38 patients treated with oseltamivir, it took an average of 11 days for viremia to drop to undetectable levels [27, 28] . timing of oseltamivir initiation (less than or more than 5 days) was not a significant risk factor for death [26] . in seasonal influenza, starting treatment within 72 h of the onset can reduce the duration of symptoms [36] . observational studies of hospitalized patients show that the use of oseltamivir can reduce mortality, and there is clinical benefit even when started after 72 h [37••] . although the effectiveness of oseltamivir in treating h7n9 is not proven, there are data to support its effectiveness against h5n1. a retrospective, observational study of 308 cases from 12 countries looking at oseltamivir efficacy across several h5n1 clades demonstrated 49 % reduction in mortality in those who received oseltamivir compared to those who did not [38] . oseltamivir is currently recommended for treatment of confirmed and probable cases of avian influenza. preemptive treatment may be considered in close contacts and in suspect cases requiring hospitalization [39] . although early treatment likely confers greater benefit, confirmed and probable cases should receive neuraminidase inhibitor treatment even if they present more than 48 h after illness onset. longer courses of treatment are recommended for those with severe illness. animal data suggests that h5n1 clades circulating more recently have required higher doses of neuraminidase inhibitor for the same antiviral effects. using higher doses of oseltamivir for longer durations may therefore be considered in those with severe infection or immunocompromise, although the clinical benefit remains unproven [40] . unfortunately, oseltamivir resistance has been reported with h5n1 as early as 2005 [41] . a single mutation at position 274 (h274y) confers resistance to neuraminidase inhibitors, resulting in reduced efficacy and poor clinical outcomes. to date, most strains of h7n9 remain susceptible to neuraminidase inhibitors. plasma from convalescent patients has been used in several emerging infection outbreaks, including ebola, sars, and most recently h1n1 [42] [43] [44] . phase 1 studies have been conducted using polyclonal equine immunoglobulin against h5n1 by fab'entech, a french biopharmaceutical company specializing in developing immunotherapies. purification for the immunoglobulin f(ab′) fragments permits reduced reactogenicity [45] . used early, this passive protection may prevent descent into the cytokine cascade, which is thought to cause severe disease. close to 60 % of patients in the h7n9 case series were given intravenous immunoglobulin, but there is no analysis as to effectiveness [26] . adjunctive therapy for h5n1 and h7n9 remains in the experimental stage. high doses of corticosteroids are generally not recommended and remain controversial. in a randomized trial of vietnamese patients with h5n1, all four patients given dexamethasone died. two patients treated with steroids in the 1997 hong kong outbreak survived [46] . in the first h7n9 outbreak, 62 % of patients received corticosteroids [26] . in summary, there is sparse evidence that adjunctive therapies significantly improve outcomes for avian influenza. the mainstay of treatment is supportive care including mechanical ventilation, and access to intensive care may be an important factor for survival. sporadic human cases of h5n1 continue to occur, primarily in southeast asia. imported h7n9 cases have been reported in several asian countries in travelers from chinese provinces with ongoing transmission. the first such case turned up in taiwan; a traveler returned from jiangsu, china, on april 9 2013, and developed fever 3 days later [47] . travel-associated h7n9 importations have also been reported in malaysia and hong kong [48, 49] . currently, the risk level does not warrant travel restrictions to particular countries [50] . the us centers for disease control and prevention (cdc) advises individuals traveling in avian influenza-affected regions to avoid touching dead or live birds, eat only food that is fully cooked, practice good hygiene, and seek medical advice early if ill [51, 52] . some countries have adopted thermal screening at airports. those with fever who have been in areas with ongoing transmission may be investigated and quarantined until confirmatory tests are completed. these suspected or confirmed cases of avian influenza should be admitted to facilities that permit airborne isolation precautions, if available. although influenza is generally transmitted via droplet spread, some airborne transmission is possible with medical procedures. given the high morbidity and mortality with h5n1 and h7n9 and lack of highly effective therapy, an abundance of caution would lead to recommending the use of n95 respirators, gowns, goggles, and eye protection in order to minimize risk to health care workers and other hospitalized patients. control remains a challenge because avian influenza can spread through poultry and wild bird populations, causing both symptomatic and asymptomatic infection. who has released guidance for countries undertaking poultry control activities [53] . the 1997 hong kong h5n1 outbreak was brought to an end by culling over a million poultry, however came a cost of many lost livelihoods and over us$10 billion [54] . according to food and agriculture organisation (fao) estimates, 20 % of indonesian workers on industrial farms lost their jobs as a result of poultry culling in 2004 [53] . however, the demand for meat and poultry in china continues to rise; from 1985 to 2011, annual per capita poultry consumption increased threefold from 3.2 to 10.6 kg [55] . after the 1997 outbreak, new regulations imposed on wet markets included regular cleaning of transport cages, banning overnight poultry storage, and screening chinese farms exporting poultry to hong kong. these measures reduced avian carriage of h9n2 from 10 to 1 % [55] . live bird market closures in shanghai, hangzhou, huzhou, and nanjing areas appear to have reduced h7n9 infections by 99 %. the rapid drop in cases after closuring markets indicates that the mean h7n9 incubation period is 3.3 days [14] . who also advises that poultry should be slaughtered away from other food products to avoid contamination, different species should be kept apart, and there should be surveillance for sick poultry [53] . in countries where most poultry are "backyard" chickens rather than farms, poultry vaccination may be more useful, as was the experience in vietnam in 2004 [53] . pre-pandemic vaccination can potentially prevent millions of people from succumbing to novel influenza virus. challenges for pandemic vaccine manufacturers include short timelines and large amounts of vaccine to protect entire populations. ideally, the product should facilitate mass vaccination and provide cross clade protection in anticipation of subsequent waves of the pandemic. influenza vaccine production is dependent on embryonated chicken eggs for culture, which is the rate-limiting production factor currently. egg-independent vaccine production strategies are undergoing active investigation. examples include cell-derived whole-virus inactivated vaccines which can take 1-3 months to manufacture, compared to 5-6 months for egg-based vaccines which have been tested in phase 3 trials. other vaccine types that are still in trial stage include recombinant protein-based vaccines, virus-like particle-based vaccines, dna vaccines, and viral vector-based vaccines [reviewed in 56] . novel influenza strains are less immunogenic than viruses that are genetically closer to strains that we may have encountered previously either through vaccine or disease. much of the focus on pandemic vaccine production has been on dosesparing formulations used alongside adjuvants. the most immunogenic of these preparations has been the squalene adjuvants (mf59, aso3). an mf59-adjuvanted inactivated subunit h5n1 vaccine demonstrated seroprotection at doses as low as 7.5 μg over a two-dose schedule. this vaccine derived from a clade 1 virus also demonstrated cross protection against the clade 2 virus [57] . similar results were obtained with as03-adjuvanted h1n1 vaccine in children; however, post marketing surveillance of the pandemic h1n1adjuvanted vaccine detected an increase in narcolepsy in children aged up to 18 with symptoms most likely to start in the first 6 months post vaccination [58, 59] a us survey of 1301 travelers due to visit asia found that although the majority of travelers were aware of general influenza prevention, there was less awareness of h5n1 transmission risks, and this was more marked among asians, foreign-born travelers, and those not working in animal or health care [60] . to underscore this issue, the first case of h5n1 was imported into canada by a returning traveler. the patient was ethnically chinese, resided in canada, and had spent a month in beijing. she developed symptoms on her way home and died soon after [6] . a survey of chinese communities living in europe demonstrated that the perceived risks and severity of avian influenza were lower compared to the general population [59] . travelers visiting friends and family (vfr) in avian influenza-affected countries may therefore be at increased risk compared to other travelers to the region ( table 2) . for the coming year, h5n1 caseloads are expected to continue at present levels, but we anticipate that h7n9 cases may increase as we enter the northern hemispheric winter of 2014-2015, especially as intensive surveillance in china continues. individuals may continue to be at risk due to poultry exposures. the risk of a global pandemic remains low currently, in the absence of sustained, efficient transmission between humans. however, with reservoirs that cannot be completely eradicated, these pre-pandemic threats will remain with us for the foreseeable future. appropriate preventive measures will protect most travelers to areas affected by avian influenza, but those at higher risk may include vfr travelers and immigrants who are less likely to be reached by pre-travel advice. all three avian influenza strains still carry high morbidity and mortality, with proven treatment options limited to antivirals and supportive care. conflict of interest poh lian lim has no disclosures relevant to this work. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by the authors. particular interest, published recently, have been highlighted as: •• of major importance 1. h5n1 avian influenza: timeline of major events 13 emergence of avian influenza a(h7n9) virus causing severe human illness-china world health organisation. influenza monthly risk assessment who risk assessment of human infection with avian influenza a(h7n9) virus isolation of avian influenza a(h5n1) viruses from humans-hong kong american h5n1 bird flu death confirmed in canada use of national pneumonia surveillance to describe influenza a(h7n9) virus epidemiology, china human infection with a novel influenza virus detection of mild to moderate influenza a/h7n9 infection by china's national sentinel surveillance system for influenza-like illness: case series who case definitions for human infections with influenza a(h5n1) virus 26 seroevidence for h5n1 influenza in human: meta-analysis serologic study for influenza a (h7n9) among high-risk groups in china clinical features and rapid viral diagnosis of human disease associated with avian influenza a/h5n1 virus epidemiology of human infections with avian influenza a(h7n9) virus in china 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potential for respiratory droplet-transmissible a/h5n1 influenza virus to evolve in a mammalian host clinical findings in 111 cases of influenza a (h7n9) virus infection consultation on human influenza a/h5n1 avian influenza a (h5n1) infection in humans past, present, and possible future human infection with influenza virus a subtype h7 emerging h7n9 influenza a (novel reassortant avian-origin) association of radiologic findings with mortality in patients with avian influenza h7n9 pneumonia human infection with avian influenza a h7n9 virus: an assessment of clinical severity pathology of fatal human infection associated with avian influenza a h5n1 virus fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia biological features of novel avian influenza a (h7n9) virus neuraminidase inhibitors for preventing influenza in healthy adults and children effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h1n1 virus infection: a meta-analysis of individual participant data effectiveness of antiviral treatment in human influenza a(h5n1) infections: analysis of a global patient registry centres for disease control and prevention. interim guidance on the use of antiviral agents for treatment of human infections with avian influenza a (h7n9 virus efficacy of oseltamivir therapy in ferrets inoculated with different clades of h5n1 influenza virus oseltamivir resistance during treatment of influenza a (h5n1) infection experience of using convalescent plasma for severe acute respiratory syndrome among healthcare workers in a taiwan hospital treatment of ebola hemorrhagic fever with blood transfusions from convalescent patients. international scientific and technical committee convalescent plasma treatment reduced mortality in patients with severe pandemic influenza a (h1n1) 2009 virus infection specific polyclonal f(ab′)2 neutralize a large panel of highly pathogenic avian influenza a viruses (h5n1) and control infection in mice human infection with highly pathogenic avian influenza virus (h5n1) in northern vietnam surveillance of avian influenza a(h7n9) virus infection in humans and detection of the first imported human case in taiwan centres for disease control. h7n9 case detected in malaysia hong kong on high alert after first human case of h7n9 bird flu world health organisation global alert and response avian flu travel information stop the spread measures to stop the spread of highly pathogenic bird flu at its source outbreak of avian influenza a(h5n1) virus infection in hong kong in 1997 the emergence of influenza a h7n9 in human beings 16 years after influenza a h5n1: a tale of two cities avian influenza pandemic preparedness developing prepandemic and pandemic vaccines against a moving target antigen sparing and cross-reactivity immunity with an adjuvanted rh5n1 prototype pandemic influenza vaccine: a randomised controlled trial safety and immunogenicity of as03b adjuvanted split virion versus non-adjuvanted whole virion versus whole virion h1n1 influenza vaccine in uk children aged 6 months-12 years: open label, randomised, parallel group, multicentre study risk of narcolepsy in children and young people receiving as03 adjuvanted pandemic a/h1n1 2009 influenza vaccine: retrospective analysis knowledge, attitudes, and practices of us travelers to asia regarding seasonal influenza and h5n1 avian influenza prevention measures sources of information and health beliefs related to sars and avian influenza among chinese communities in the united kingdom and the netherlands, compared to the general population in these countries h10n8 case list key: cord-007583-owxcokge authors: kohn, william g. title: emerging and re-emerging infectious diseases: be prepared date: 2014-12-26 journal: j am dent assoc doi: 10.14219/jada.archive.2010.0002 sha: doc_id: 7583 cord_uid: owxcokge nan be prepared e ach time a new infectious agent or variant emerges, the public and health care providers struggle to understand how to stay safe. in the case of health care providers, we have the added responsibility for the safety of our patients, staff members and families. in the past 30 years, we have responded to a litany of diseases and organisms with exotic names, such as hepatitis b virus (hbv); human immunodeficiency virus (hiv); hantavirus; lyme disease; variant creutzfeldt-jakob disease and prion diseases; severe acute respiratory syndrome/coronavirus; avian influenza (bird flu); west nile virus; human papilloma virus; escherichia coli o157:h7; methicillin-resistant staphylococcus aureus; and, of most recent concern, 2009 h1n1 influenza. there also are potential threats from deliberate exposures with uncommonly seen yet virulent agents such as anthrax, plague, tularemia and viral hemorrhagic fevers. in these circumstances, it is almost impossible for a clinician to stay fully informed about the variety of signs, symptoms, routes of transmission, virulence and other important factors related to risk of transmission. when considering how to apply infection control practices to emerging infectious diseases, it might be best to consider the boy/girl scout motto: "be prepared." "be prepared for what?" someone once asked robert baden-powell, the founder of scouting. "why, for any old thing," he replied. the most important thing we, as clinicians, can do is to be informed and prepared-for any old or new thing that comes our way. preparation would be a daunting task if each agent required a special set of infection control practices, particularly when infected patients can be asymptomatic or unaware they are infected. fortunately, dental health care workers follow a set of standard infection control precautions. in 1985, the centers for disease control and prevention (cdc), atlanta, established "universal precautions," a set of infection control practices based on the concept that all blood and bodily fluids that might be contaminated with blood the most important part of being prepared is to have a written plan and to train staff members in executing the plan. viruses. dpost signs in languages appropriate to your patient population with instructions to patients and accompanying family members or friends to report immediately symptoms of a respiratory infection as directed. dapply source-control measures such as covering the mouth and nose with a tissue when coughing and disposing of used tissues. use a mask on a coughing person when it can be tolerated and is appropriate. dconduct hand hygiene after every contact with respiratory secretions. duse spatial separation, ideally three feet or more, of people with respiratory infections in common waiting areas when possible. it is worth noting that the occupational safety and health administration (osha) recently posted regulatory controls that health care employers should be using to protect workers from exposure to the 2009 h1n1 influenza virus. 2 these general principles are intended to follow cdc guidance. in addition, cdc 3 recently posted specific guidance for dental health care: dencourage all dental health care personnel to receive vaccinations for seasonal influenza and 2009 h1n1 influenza. duse patient-reminder calls to identify patients reporting influenzalike illness and resched-ule nonurgent visits until after the patient is free of fever for 24 hours without the use of feverreducing medicine. didentify patients with influenzalike illness at check-in; offer a face mask or tissues to symptomatic patients; follow respiratory hygiene and cough etiquette 4 and reschedule appointments for patients needing nonurgent care; separate ill patients from others whenever possible if evaluating for urgent care. durgent dental treatment can be performed without the use of an airborne infection isolation room because transmission of 2009 h1n1 influenza is thought not to occur across longer distances through the air, such as from one patient room to another. duse a treatment room with a closed door, if available. if not, use one that is farthest from other patients and personnel. dwear recommended personal protective equipment before entering the treatment room. ddental health care personnel should wear a disposable n95 respirator fit-tested by the national institute for occupational safety and health when entering the operatory and when performing dental procedures in patients with suspected or confirmed 2009 h1n1 influenza. dif n95 respirators and/or fit testing is not available despite reasonable attempts to obtain them, non-fit-tested disposable n95 respirators or surgical face masks can be considered as a lower level of protection for personnel at lower risk of exposure or lower risk of experiencing complications resulting from influenza until fit-tested n95 respirators are available. dminimize spray and spatter should be treated as infectious. these were designed to prevent transmission of hiv, hbv and other bloodborne pathogens in all health care settings. in 1996, cdc expanded this guidance into "standard precautions," a standard of care designed to protect health care personnel and patients from pathogens that can be spread by blood or any other bodily fluid, excretion or secretion. standard precautions are the foundation of a comprehensive infection control program and include a group of infection control practices that apply to all patients, regardless of suspected or confirmed infection status, in any setting in which health care is delivered, including dental settings. at press time, we are in the middle of the 2009 h1n1 influenza pandemic. exposure to 2009 h1n1 influenza virus occurs in household, community and occupational settings, and transmission is thought to occur through droplet exposure of mucosal surfaces; through indirect contact, usually via the hands, with respiratory secretions from an infectious patient or contaminated surface; and through inhalation of small-particle aerosols in the vicinity of the infectious person. in theory, any measure that limits the dispersal of respiratory droplets should reduce the opportunity for transmission. an important element added to standard precautions in 2007 was respiratory hygiene/cough etiquette. 1 basic principles of this etiquette include the following: deducate staff members, patients and visitors regarding the importance of containing respiratory secretions to help prevent the transmission of influenza and other respiratory in theory, any measure that limits the dispersal of respiratory droplets should reduce the opportunity for transmission of infection. when possible (for example, use a dental dam and a high-volume evacuator). despite the economic and ethical pressure to keep working despite illness, dental personnel should take action to prevent the transmission of 2009 h1n1 influenza in their practices. among these actions are the following: dself-assess daily for symptoms of febrile respiratory illness (fever plus one or more of the following: nasal congestion/runny nose, sore throat or cough). ddo not report to work with fever and respiratory symptoms. dremain at home until at least 24 hours after being free of fever (100°f/37.8°c), or signs of fever without the use of feverreducing medications. dif a family member is diagnosed with 2009 h1n1 influenza, you still can go to work but should monitor yourself for symptoms. all aspects of preparedness planning for pandemic influenza must allow for flexibility and real-time decision making that take new information into account as the situation unfolds. cdc provides updated infection control guidance as necessary. the most important part of being prepared is to have a written plan and to train staff members in executing the plan. excellent sources of information exist to help in developing an influenza plan, including a cdc checklist and action steps for 2009 h1n1 influenza planning and response for outpatient facilities. [4] [5] [6] [7] when considering infection control guidelines and infectious disease transmission in caring for our patients, we also might do well to remember another scout guideline-"leave no trace" (after camping or other outdoor activities)-or, in our own hippocratic terms, "first, do no harm." ■ centers for disease control and prevention. respiratory hygiene/cough etiquette in healthcare settings osha instruction: enforcement procedures for high to very high occupational exposure risk to 2009 h1n1 influenza infection control in dental settings: prevention of 2009 h1n1 influenza transmission in dental health care settings department of health & human services department of health and human services, medical offices and clinics pandemic influenza planning checklist centers for disease control and prevention. 10 steps you can take: actions for novel h1n1 influenza planning and response for medical offices and outpatient facilities department of health and human services. hhs pandemic influenza plan: supplement 4 infection control key: cord-318556-a28bowqy authors: kuliese, monika; jancoriene, ligita; grimalauskaite, rita; zablockiene, birute; damuleviciene, gyte; velyvyte, daiva; lesauskaite, vita; ambrozaitis, arvydas; mickiene, aukse; gefenaite, giedre title: seasonal influenza vaccine effectiveness against laboratory-confirmed influenza in 2015–2016: a hospital-based test-negative case–control study in lithuania date: 2017-10-10 journal: bmj open doi: 10.1136/bmjopen-2017-017835 sha: doc_id: 318556 cord_uid: a28bowqy objective: a case–control study was conducted to assess seasonal influenza vaccine effectiveness (sive) during the 2015–2016 influenza season. methods: a study was performed in three departments in lithuania between 1 december 2015 and 1 may 2016. data on demographic and clinical characteristics including influenza vaccination status were collected from the patients recommended to receive the seasonal influenza vaccine. influenza virus infection was confirmed by multiplex reverse transcription polymerase chain reaction (rt-pcr). results: ninety-one (56.4%) of the 163 included subjects were ≥65 years old. fifteen (9.2%) subjects were vaccinated against influenza at least 2 weeks before the onset of influenza symptoms, 12 of them were ≥65 years old. of the 72 (44.2%) influenza virus positive cases, 65 (39.9%) were confirmed with influenza a (including 50 cases of influenza a(h1n1)pdm09), eight (4.9%) were confirmed with influenza b and one was a co-infection. unadjusted sive against any influenza, influenza type a and influenza a(h1n1)pdm09 was 57% (95% ci −41% to 87%), 52% (95% ci −57% to 85%) and 70% (95% ci −43% to 94%) respectively. conclusion: although sive estimates were not statistically significant the point estimates suggest moderate effectiveness against influenza type a. objective a case-control study was conducted to assess seasonal influenza vaccine effectiveness (sive) during the 2015-2016 influenza season. methods a study was performed in three departments in lithuania between 1 december 2015 and 1 may 2016. data on demographic and clinical characteristics including influenza vaccination status were collected from the patients recommended to receive the seasonal influenza vaccine. influenza virus infection was confirmed by multiplex reverse transcription polymerase chain reaction (rt-pcr) . results ninety-one (56.4%) of the 163 included subjects were ≥65 years old. fifteen (9.2%) subjects were vaccinated against influenza at least 2 weeks before the onset of influenza symptoms, 12 of them were ≥65 years old. of the 72 (44.2%) influenza virus positive cases, 65 (39.9%) were confirmed with influenza a (including 50 cases of influenza a(h1n1)pdm09), eight (4.9%) were confirmed with influenza b and one was a co-infection. unadjusted sive against any influenza, influenza type a and influenza a(h1n1)pdm09 was 57% (95% ci −41% to 87%), 52% (95% ci −57% to 85%) and 70% (95% ci −43% to 94%) respectively. conclusion although sive estimates were not statistically significant the point estimates suggest moderate effectiveness against influenza type a. influenza is a highly contagious viral airborne disease that in the northern hemisphere typically occurs during the winter months. 1 annual influenza epidemics result in high morbidity (three to five million cases of severe illness) and significant mortality rates (250 000-500 000 deaths) worldwide. 2 in addition, for people with chronic underlying medical conditions and those of 65 years old and older influenza is associated with significant adverse health outcomes, [3] [4] [5] such as influenza-related hospitalisations and deaths. 6 7 the most effective way to prevent potentially severe influenza complications is vaccination. 8 9 annual influenza immunisation is recommended for the most vulnerable groups, such as adults aged ≥65 years and people with co-morbidities. 10 however, the evidence base for this recommendation is weak, and except for a few randomised control trials, is based on the results of observational studies. 11 12 the lack of laboratory confirmation of influenza infection and the assessment of influenza vaccination effectiveness against non-specific outcomes are the major limitations of the majority of the existing observational studies. relatively recently, the test-negative case-control studies have been introduced to assess seasonal influenza vaccine effectiveness (sive), and despite some limitations, they are currently considered to be the most accurate and efficient way to monitor sive. 13 due to frequent change in the circulating influenza strains, sive should be monitored on a routine basis. 14 this is useful in guiding the influenza prevention and informing the treatment strategies during the influenza epidemics and it could also help when making a decision on the next season's influenza vaccine content. 11 furthermore, due to different timing and spread of the influenza viruses across europe, 15 sive estimates derived from different geographical areas are of particular interest and of great relevance as well. in lithuania, adults≥65 years of age, pregnant women, people with underlying medical conditions and healthcare workers are eligible to receive influenza vaccination free of charge. the vaccines used for the immunisation of the risk groups in lithuania are trivalent influenza vaccines. for the 2015-2016 influenza season, one dose of a subunit influenza vaccine influvac (bgp products, hoofd-dorp, the netherlands) was used. 16 according to the recommendations of the who, 17 the objective of this study was to measure sive against laboratory-confirmed influenza in patients admitted to hospital due to severe acute respiratory infection (sari) in lithuania in 2015-2016. a test-negative case-control study was conducted between 1 december 2015 and 1 may 2016. the subjects were recruited from three participating sites: centre of infectious diseases, vilnius university hospital santaros klinikos, vilnius, lithuania; the department of infectious diseases and the department of geriatrics of lithuanian university of health sciences, kaunas. the study population consisted of 18 years and older individuals with underlying medical conditions, healthy ≥65 years old individuals and pregnant women living in the community, who were admitted to one of the participating sites due to sari with no contraindication for influenza vaccination, that is, allergies to influenza vaccine and other adverse events to vaccinations in the past. patients were eligible to be included in the study when they were hospitalised for at least 24 hours, but not longer than 48 hours, had a swab taken ≤7 days after self-reported disease onset, did not test positive and were not hospitalised for any influenza virus in the current season before the inclusion, and were suffering from sari with at least one of the systemic symptoms (fever, malaise, headache and myalgia) or deterioration of general condition or deterioration of functional status, and at least one of the respiratory symptoms (cough, sore throat and shortness of breath). patients were not eligible to be included in the study when they were institutionalised, unwilling to participate, not able to communicate, not able and/or willing to give written informed consent (ic). eligible patients were asked to provide one throat and one nose swab specimen for influenza testing by the multiplex rt-pcr. as data from shedding studies propose that influenza virus detection decreases after 7 days, recruitment was limited to patients who were swabbed not more than 7 days after onset of the symptoms. 18 swabbing was done after the information on demographic and clinical characteristics was collected from the medical history and patient self-report. the outcome was laboratory-confirmed influenza virus infection in patients admitted to hospital for sari. sari patients positive for influenza a(h1n1)pdm09, a(h3n2), non-typed a or influenza b viruses were considered as cases. the control group consisted of patients who were negative for any influenza virus infection. the exposure of interest was vaccination with trivalent seasonal influenza vaccine, available in lithuania during 2015-2016 influenza season. 16 subjects were considered as vaccinated if their vaccination status was confirmed by their general practitioner (gp) records, and the vaccination occurred more than 14 days before disease onset or more than 14 days before being selected as controls, who also had sari onset. otherwise, they were considered as unvaccinated. covariates information about age, sex, antiviral drug use during current hospitalisation, transfer to the intensive care unit, hospitalisations due to disease exacerbation in the last 12 months, length of hospitalisation and occurrence of underlying medical conditions (cardiovascular, respiratory, renal, rheumatological, endocrine diseases and diabetes, haematological and non-haematological cancer, immunodeficiency and transplantation, dementia, stroke, anaemia (according to the international classification of diseases 10) were collected from the medical records (online supplementary appendix 1). socioeconomic status (education, occupation, income per household member), living in urban or rural areas, smoking status, body mass index (bmi), obesity (bmi≥30), number of hospitalisations and number of visits to gp due to the underlying medical conditions (but not repeated prescriptions) in the preceding year, and barthel scores to assess dependence in activities of daily living before the hospitalisation were collected from the self-reports. the nasal and throat swabs were kept in the fridge at the ward at +4°c up to 72 hours. during this time, the samples were transported to the national public health laboratory (nphl). if the samples were not transferred to the nphl within 72 hours, they were frozen at −70°c. when transported to the nphl the samples were kept in −70°c while analysed. viral rna from the samples was isolated using an automatic magnetic particle method based on the centre for disease control and prevention (cdc) recommendations, 19 20 and influenza vaccination rates among the risk groups in lithuania, we assumed the vaccination rates among the cases and controls of 2% and 15% respectively. to achieve the statistical power of 80% with a confidence level of 95%, the required sample size was 170 subjects. the sample size was estimated for the total sample only. the demographic and clinical characteristic of cases and controls were compared by using fisher's chi-square test and student's t-test. the analysis was adjusted for confounding when the variables were associated with both the outcome and the vaccination at alpha level of 10%. sive and its 95% confidence interval (95% ci) was estimated by using the formula (1-or)*100%. the study was conducted in accordance with the lithuania legislation and the declaration of helsinki. kaunas regional biomedical research ethics committee (kaunas, lithuania) and state data protection inspectorate approvals p2-158200-04-476-138/2012, dated 23 february 2016 and 2 r-372(2.6-1.) dated 21 january 2016 were received respectively. the written ic was obtained from the study subjects. overall, 1003 patients were screened and 180 met the inclusion criteria, of which 163 (91%) subjects gave an ic and were included into the study. ninety-one (55.8%) subjects were negative for any influenza virus infection, 72 (44.2%) subjects tested positive for influenza virus infection. sixty-five subjects (39.9%) were confirmed with influenza virus a infection, including 50 cases of influenza a(h1n1)pdm09. the subtyping was inconclusive for 15 specimens of influenza type a. there were eight (4.9%) cases with influenza b virus infection (seven b/victoria; one unsubtyped) and one (0.6%) had a co-infection with unsubtyped influenza virus a and b/victoria. eighty-four (91.3%) patients were swabbed within 4 days of antiviral administration, of which 32 (34.8%) were administered antivirals before or on the day of swabbing and 39 (42.4%) patients were swabbed 1 day after the administration of antivirals. in addition to influenza, seven subjects were co-infected: two with adenovirus (2.8%), one with coronavirus (1.4%), two with metapneumovirus (2.8%) and two with rsv (2.8%). other respiratory pathogens isolated from the study participants (n=25) were rsv (6, 3.7%), adenovirus (5, 3.1%), metapneumovirus (5, 3.1%), rhinovirus (6, 3.7%), coronavirus (2, 2.3%) and parainfluenza (1, 0.6%). the observed influenza peak in our study occurred in week 6 (figure 1), which overlapped with the nationally detected influenza season's peak in lithuania in 2015-2016 (figure 2). the average age of the influenza cases was significantly lower than the controls (59 vs 67 years old). influenza cases had significantly less underlying medical conditions, such as cardiovascular and lung diseases, were less often hospitalised during the last 12 months due to the exacerbations of the underlying illnesses, and were prescribed oseltamivir twice more often (table 1) . fifteen (9.1%) subjects were vaccinated against influenza in the 2015-2016 season, of which 12 were ≥65 years old (table 1). all vaccinations occurred more than 14 days before the onset of sari. vaccinated individuals were older, more likely to have received the seasonal influenza vaccine during the previous season and had slightly shorter length of hospitalisation (table 1) . influenza cases appeared to have more cough and less shortness of breath and deterioration of general condition than the controls (table 2) . in the total sample, five out of 163 patients died during the hospitalisation, of which 4/72 (5.6%) within the influenza confirmed cases (including one vaccinated death), and 1/91 (1.1%) within the controls (ceased subject not vaccinated). unadjusted sive point estimates against any influenza, influenza type a and influenza a(h1n1)pdm09 were 57% (95% ci −41% to 87%), 52% (95% ci −57% to 85%) and 70% (95% ci −43% to 94%) respectively, however they were not statistically significant (table 3) . as age was associated with both vaccination status and the outcome, we performed sive analysis by age group (18-64 and ≥65 years old). in the stratified by age analysis, the sive point estimate for any influenza and influenza type a for the subjects of 18-64 years old increased, while it dropped in the ≥65 years old subjects (table 3) . this was not the case for the sive against a(h1n1)pdm09. none of the results in the stratified analysis were statistically significant. due to low statistical power, the analysis to estimate sive against influenza type b/victoria was not performed. the sensitivity analysis with a scenario when 0 vaccinated influenza b cases were replaced with one was used. 21 however, the unadjusted or resulted in 1.04, indicating no sive (95% ci 0.02 to 9.47). this study aimed to estimate sive against laboratory-confirmed influenza virus infection in patients admitted to the hospital due to sari in lithuania during the 2015-2016 season. our estimates indicated vaccine effectiveness against any influenza, influenza type a and influenza a(h1n1) pdm09 of 57%, 52% and 70% respectively, but none of them were statistically significant. after stratifying by age sive point estimates against any influenza and influenza type a slightly increased in the 18-64 years old individuals, and decreased in those ≥65 years old. due to the lack open access of precision in the subgroup analysis, these results can serve only as indicatory. in our study, sive was higher than the mid-season estimates against influenza a(h1n1)pdm09 reported in the primary and hospital patient populations in denmark (35% in ≥65 years old subjects) and in the gp patient population in the uk (49% in all age groups). 12 22 lower point estimates (crude 26.2% and adjusted 6.2%) against the predominant a(h1n1)pdm09 were reported from multicentre european network of hospitals during the 2012-2013 influenza season. 11 in the individual participant data meta-analysis in the community dwelling elderly confounder-adjusted vaccine effectiveness estimate against a(h1n1)pdm09 resulted in 53.2%. 23 during the 2013-2014 influenza season with predominant influenza a(h1n1)pdm09 in canada an adjusted sive in elderly of 67% was found. 24 due to the lack of published data reporting vaccine effectiveness in 2015-2016, sive comparison across countries and/or regions is still missing. in addition, in the previous study conducted by our group in a similar setting in the 2012-2013 we found 30% higher sive than during the 2015-2016 season. 20 it is necessary to point out that influenza a(h1n1), influenza a(h3) and influenza b cases were predominant open access during the 2012-2013 season, while influenza a(h1n1) pdm09 was predominant during the season 2015-2016. we used influenza virus negative patients as controls in our study. it has been shown that the use of different control groups (ie, negative for influenza viruses; negative for influenza viruses, but positive for other respiratory viruses; negative for both influenza and other respiratory viruses) in test-negative design case-control studies resulted in different sive estimates. [25] [26] [27] [28] [29] [30] however, the results so far have not been consistent, and the discussion about the study designs and methodological challenges using each of the control groups is still ongoing. 26 30 31 some differences in point estimates of sive when using different control groups have recently been reported, but the confidence intervals of the estimates overlapped. 30 31 given these inconsistencies and even lower sample size when restricting the controls to only influenza negative or all respiratory viruses negative in our study, the control group of influenza virus negative patients remains a valid choice. seven influenza b/victoria cases were detected in our study. although we were not able to perform sive due to extremely low numbers of influenza b cases, our findings show that the circulating strain mismatched the b/yamagata lineage virus, which was included in the 2015/2016 season's vaccine. this was confirmed by the or of 1 when 0 vaccinated influenza b cases were replaced with one case, as well as not significant mid-season estimate of 4% found in denmark. 12 five deaths occurred in our study during the 2015-2016 influenza season, of which four subjects were positive for influenza a(h1n1)pdm09. all five subjects had at least one underlying medical condition. three of the four influenza cases were younger than 65 years. only one (influenza positive) patient was vaccinated against influenza this season. during hospitalisation, all patients developed the unilateral or bilateral pneumonia. the main cause of death among the influenza cases was the respiratory insufficiency due to the acute respiratory distress syndrome. such high mortality in our study sample, and especially among the influenza confirmed cases (5.6%) as compared with controls (1.1%) reflects the severity of the condition of the hospitalised population and the increased risk for severe influenza-related outcomes and calls for better influenza prevention measures for the risk groups. the response rate in this study was very high and exceeded 90%. the selection bias was reduced by including the patients into the study before the laboratory result of influenza status was known; cases and controls were similar with regard to the demographic and clinical characteristics. the outcome bias was further reduced by using a very sensitive influenza detection method. 32 the exposure status was verified with the gp, and therefore exposure misclassification was very unlikely. that together gives us confidence that despite low sample size and low precision of the sive estimates, the bias in this test-negative case-control study, except for unmeasured confounding, which we were not able to assess, is limited. 33 34 this study has several limitations. first, the two participating university hospitals are located in two of the 10 districts in the country. however, the infectious diseases units in these hospitals are the main centres where the majority of patients with clinically suspected influenza are admitted in the vilnius and kaunas regions and they cover about 35% of the lithuanian population. 35 the low precision of the sive estimates was partly due to low number of vaccinated patients, which would require a much higher sample size to be able to provide precise sive estimates. nevertheless, we addressed the potential biases in the design and the analysis stages of the study, and at least for influenza type a the obtained estimates are likely to be a reasonable indication of sive during the 2015-2016 season. in lithuania, the vaccination coverage among the risk groups is very low. according to the centre for communicable diseases and aids, 19.5% of the elderly population were vaccinated during the 2015-2016 influenza season, while in several other european countries the vaccination coverage among the risk groups varies from 28% in portugal to 80.2% in northern ireland. 36 different vaccination rates in the european countries that generally adopt the same recommendations might be explained by different communication activities, differences in vaccination provision systems, funding schemes, attitudes and trust in seasonal influenza vaccination recommendations. in england, for example, gps and other providers are encouraged to contact eligible patients in september and actively invite them to attend the clinics and get vaccinated against influenza. 17 37 in lithuania, personal invitations are usually not sent and the vaccination coverage among the healthcare workers themselves is quite low, 36 which likely influences the patients' decisions as well. the patients eligible for influenza vaccination (ie, those suffering from underlying condition, ≥65 years old, pregnant women) are usually offered and administered the vaccine if they are visiting their gp during the weeks when the vaccine is available (october-december), which limits the number of patients who would potentially be willing to get vaccinated if they were actively invited. in addition to the routine sive assessments to inform influenza prevention and treatment strategies, multiple questions remain to be tackled by future studies. multiple years are needed to investigate the role of the previous influenza infection as a potential effect modifier for the vaccine effectiveness estimates. 38 due to absence of electronic record system, we do not have information about (laboratory-confirmed) influenza illness in the previous seasons for the same subset of patients. we are therefore not able to determine the effect of previous laboratory-confirmed influenza on sive. also, bigger sample size is needed to monitor within-season waning immunity and to determine whether the timing of the influenza immunisation campaigns needs to be revised. 39 open access conclusions although sive estimates confidence intervals are broad, this study suggests moderate effectiveness against influenza type a. even with moderate vaccine effectiveness estimates, given such high prevalence of influenza in hospitalised cases and relatively high number of deaths, there is an urgent need for adopting more effective vaccination campaign strategies in countries with low vaccination uptake rates. ethics approval kaunas regional biomedical research ethics committee (kaunas, lithuania). provenance and peer review not commissioned; externally peer reviewed. data sharing statement no additional unpublished data are available for sharing. open access this is an open access article distributed in accordance with the creative commons attribution non commercial (cc by-nc 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. see: http:// creativecommons. org/ licenses/ by-nc/ 4. 0/ on seasonal influenza vaccination 2011-12 seasonal influenza vaccines effectiveness against confirmed a(h3n2) influenza 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effectiveness in adults 65 years and older, denmark, 2015/16 -a rapid epidemiological and virological assessment potential of the test-negative design for measuring influenza vaccine effectiveness: a systematic review first steps in the design of a system to monitor vaccine effectiveness during seasonal and pandemic influenza in eu/eea member states the community impact of the 2009 influenza pandemic in the who european region: a comparison with historical seasonal data from 28 countries vinfluvac sub-unit suspension for injection influenza vaccine surface antigen (inactivated) season recommended composition of influenza virus vaccines for use in the 2015-2016 northern hemisphere influenza season ? dbfrom= pubmed& id= 25771542& retmode= ref& cmd= prlinks% 5cnpapers3:// publication/ uuid/ d5a5ff30-19e1-45f0-a209-809291d78f8f comparison of shedding characteristics of seasonal influenza virus (sub)types and influenza a(h1n1)pdm09 protocol of realtime rt-pcr for influenza a (h1n1) seasonal influenza vaccine effectiveness against influenza in 2012-2013: a hospital-based case-control study in lithuania computation of exact confidence intervals for the odds ratio effectiveness of seasonal influenza vaccine for adults and children in preventing laboratoryconfirmed influenza in primary care in the united kingdom: 2015/16 end-of-season results effectiveness of seasonal influenza vaccination in community-dwelling elderly people: an individual participant data meta-analysis of test-negative design case-control studies integrated sentinel surveillance linking genetic, antigenic, and epidemiologic monitoring of influenza vaccine-virus relatedness and effectiveness during the 2013-2014 influenza season the importance of circulating influenza virus types and subtypes estimates of 2012/13 influenza vaccine effectiveness using the case test-negative control design with different influenza negative control groups vaccine effectiveness against laboratory-confirmed influenza in healthy young children effectiveness of trivalent flu vaccine in healthy young children effectiveness of the 2010-2011 seasonal influenza vaccine in preventing confirmed influenza hospitalizations in adults: a case-case comparison, casecontrol study influenza vaccination is not associated with detection of noninfluenza respiratory viruses in seasonal studies of influenza vaccine effectiveness influenza vaccine effectiveness estimates in the dutch population from 2003 to 2014: the test-negative design case-control study with different control groups role of the laboratory in diagnosis of influenza during seasonal epidemics and potential pandemics effectiveness of seasonal influenza vaccine in community-dwelling elderly people: a metaanalysis of test-negative design case-control studies a probability model for evaluating the bias and precision of influenza vaccine effectiveness estimates from case-control studies teritory and population st& rvar0=& rvar1=& rvar2=& rvar3=& rvar4=& rvar5=& rvar6=& rvar7=& rvar8=& rvar9=& rvar10=& rvar11=& rvar12=& rvar13=& rvar14= national seasonal influenza vaccination survey for 2012-13 influenza season in eu/eea (provisional data importance of timely monitoring of seasonal influenza vaccine effectiveness i-move multicentre casecontrol study 2010/11 to 2014/15: is there within-season waning of influenza type/subtype vaccine effectiveness with increasing time since vaccination? key: cord-275462-7a55odok authors: journeay, w shane; burnstein, matthew d title: pandemic influenza: implications for occupational medicine date: 2009-06-23 journal: j occup med toxicol doi: 10.1186/1745-6673-4-15 sha: doc_id: 275462 cord_uid: 7a55odok this article reviews the biological and occupational medicine literature related to h5n1 pandemic influenza and its impact on infection control, cost and business continuity in settings outside the health care community. the literature on h5n1 biology is reviewed including the treatment and infection control mechanisms as they pertain to occupational medicine. planning activity for the potential arrival of pandemic avian influenza is growing rapidly. much has been published on the molecular biology of h5n1 but there remains a paucity of literature on the occupational medicine impacts to organizations. this review summarizes some of the basic science surrounding h5n1 influenza and raises some key concerns in pandemic planning for the occupational medicine professional. workplaces other than health care settings will be impacted greatly by an h5n1 pandemic and the occupational physician will play an essential role in corporate preparation, response, and business continuity strategies. the occupational medicine community has been adressing occupational diseases of epidemic proportions since ramazzini first studied injured workers. traditionally, these diseases have been musculoskeletal, psychiatric or toxicologic in nature. when the etiology of these conditions has been identified, appropriate measures have been taken to mitigate the risk of becoming ill or injured. occupational health specialists are therefore quite adept at looking at prevention when the causative factors are known and their mechanism of action understood. however, when the process is poorly understood, as is the case with pandemic influenza, determining the most appropriate prevention and mitigation strategy is more complex. despite this uncertainty, government agencies and businesses are taking measures to address the impact of a potential pandemic influenza on their workforce [1, 2] . the field of occupational medicine is being consulted to assist in mitigating the impact of an avian influenza pandemic on their human resources, business continuity and also the societal impact associated with essential services and disease transmission. this article will outline the nature of pandemic avian influenza and some of the unique considerations related to the occupational environment outside the health care setting. occupational medicine professionals are uniquely positioned to provide information on the potential impact of a pandemic influenza. indeed, infectious disease may disproportionately impact the occupational environment. this is due to factors associated with transmission such as the proximity of co-workers to one another in the workplace, during the daily commute to work, or simply dealing face to face with customers. of particular concern is the health and safety of those health care professionals caring for infected patients. the recent experience with severe acute respiratory syndrome (sars) provides some useful insight into the consequences of a novel infection on a modern society and more specifically on the health care community. there are many similarities between the sars epidemic and the anticipated experience with avian influenza. both have been associated with food and animals. in the early stages of sars, more than a third of infected humans were food handlers [3] , and it was later inferred that the sars coronavirus had originated in civet cats, and that the first transmission of infection to humans may have occurred in those workers handling civet cats [4] . however, the greatest impact of sars was subsequently felt in health care workers where they were estimated to have accounted for over 20% of total sars cases in singapore and 40% in canada [5] . thus, not only are individuals working closely with infected animal hosts at risk for first line crossover transmission of an emerging virus but they are also at risk of acquiring the virus from coworkers, or in the case of health care professionals, from patients. influenza are single stranded rna viruses and are part of the orthomyxoviradae family [6] . influenza a and b can recur in individuals because of their ongoing mutation. antigenic drifts can occur in seasonal influenza and if sufficient mutations arise in the surface proteins hemagluttinin or neuraminidase, it can result in a novel strain. thus 'h' and 'n' components determine the different potential subtypes of a given influenza virus, and at present a total of 15 h variants exist while 9 n subtypes have been identified. the ongoing emergence of small but significant mutations can lead to epidemics which we experience as seasonal influenza. the yearly influenza vaccination program is based on correctly determining which of these subtle changes (drift) will become predominant. pandemic influenza, such as the suspected h5n1avian influenza, occurs as a result of major changes in surface proteins of influenza viruses known as an antigenic shift. this novel strain which is present in animals still requires further modification before it can effectively spread among the human population. this situation can be created via transmission from a different species with frequent exposure leading to adaptation, or from genetic reassortment [6] . the process of reassortment happens when an individual simultaneously has both human and avian influenza subtypes. this allows for a recombination of viral components, leading to a new viral form with the potential for efficient transmission between humans. this form of the virus would still contain avian viral surface proteins. when this occurs humans have minimal or no immunity against the virus, enabling a large geographic spread of disease with high attack rates [6, 7] . it should be noted that h5n1 is not the only avian influenza that has the capacity to affect humans. h7n2 is slowly progressing globally, and while less pathogenic than h5n1, has caused illness in poultry workers. to date, neither of these avian influenzas has gained the capacity to spread efficiently from human to human. pandemic influenza occurs when a new strain of human influenza arises that humans have minimal or absent preexisting natural immunity, which causes disease, can be easily transmitted from person to person, and is globally widespread (on 3 continents at one time) [7] or exhibits community level outbreaks in two who regions. in today's globalized economy and interdependent supply chain, the work force is particularly sensitive to pandemic infections and it is also a key mechanism for the geographic spread of a pandemic. on average, we experience a pandemic about every thirty years. indeed, in the 20 th century, there were three pandemic influenza outbreaks which included: the spanish influenza (1918) (1919) (1920) , asian influenza (1957) (1958) and the hong kong influenza (1968) (1969) [8] . this is not to suggest that simply because 30 years have passed since the last pandemic, we are overdue; it is simply meant to point out that pandemics are relatively common events given the right conditions. the current strain of influenza considered to have pandemic potential is the highly pathogenic h5n1 strain of avian influenza which has spread from asia to europe. moreover, its transmission to humans has intensified concerns that a novel strain will emerge leading to human infections of pandemic proportions [7] . the three criteria that are required to enable a pandemic include: 1) the presence of a new viral strain that is capable of infecting humans, 2) ability to be transmitted from person to person, and 3) availability of a susceptible global population [6] . thus, should a new viral strain emerge, the global workforce provides and ideal vehicle in which transmission from person to person can occur within a susceptible global population. the ability of h5n1 to propagate between humans after an initial infection has not been established and its probability is unknown. thus avian influenza has currently not developed into a pandemic [6, 9] . however, it is generally accepted that this will occur; it is a matter of "when, not if". when this occurs, the health care system will be particularly susceptible to pandemic influenza events. this is because patients with influenza will place an enormous burden on already fully taxed health care services and because health care professionals will come into direct contact with infected patients rendering them susceptible to acquiring the virus. however, there are no industries that would be left unaffected by an avian influenza pandemic, and therefore public health agencies, government, and industry will need to consider the level of interdependence they share. it is generally accepted that transmission of the influenza virus occurs by host inhalation of viral droplets usually greater than 5 μm in size [7, 10] . a recent review of the mechanism of influenza transmission concluded that the virus is primarily transmitted at close quarters [11] . it can also be transmitted by coming into contact with viral laden fomites. both of these methods are of great concern in the workplace, due to use of communal equipment and also in areas where employees work in close proximity. therefore, infection control measures will need to vary between industries. for example, staff that work in isolation or even outdoors could be at far less risk of transmission than having many employees in a single room such as a telecommunications call center where individuals are separated by small distances. moreover, unlike seasonal influenza which has an incubation period of one to four days (average two), avian influenza has an incubation period ranging from two to eight days [12] . this has implications for staffing schedules and return to work policy when developing guidelines for pandemic influenza in the workplace. once again the nature of the control measures and advisement to employees may vary considerably depending on the physical layout of the worksite. it is well established that occupational disease is already an enormous contributor to the economic and human resource strain on our health care systems. many mechanisms are in place to prevent or manage such disease which may include ergonomic initiatives, exposure limits, and corporate health and wellness programs. at the same time, the workplace is one of the key pillars of societal function, such that the health of a workplace is vital to the health and functioning of our interdependent society. this is particularly true when one considers such essential services as health care, energy, communications, and food supply sectors. in the event of a pandemic influenza absenteeism will be an enormous challenge. employees will not be present due to reasons such as: infection and illness from the pandemic influenza strain, exclusion from work while suffering an illness that is mistaken for or treated empirically as influenza, caring for sick relatives, caring for children in the event of day care and school closures by governments, loss of public transportation and based on the fear of real or perceived risk of infection at work or during travel [13] . the public health agency of canada is predicting total work absenteeism of 35 to 50% during the whole disease wave with the peak work absence ranging from 15 to 27%. while it is tempting to look at absenteeism from within a single organization, the functioning of a company is almost always dependent on external clients, supply chains, or multi-national locations. thus, a large manufacturing plant in united states may require final product detailing in another region of the country, which in turn receives its raw materials from asia or south america. "just on time" delivery processes have created a society in which most companies (including health care institutions) have less than a few weeks supply of essential goods (including medications). little is known about the global timing and progression of h5n1 avian influenza at present but it is entirely possible that while an organization in north america is healthy, its supplier abroad is experiencing a disease wave leading to uncoordinated business efforts. each company has an obligation to ensure that occupational transmission is attenuated and planned for, but this will also require cooperation with governments that may impose social and travel restrictions to suppress the spread of the disease while still maintaining business continuity and societal function. pandemic influenza, will have the capacity to disrupt services and supply chains and thus requires significant planning and foresight from occupational medicine professionals to help mitigate the health and economic impacts to their organizations and to the functioning of society [14] . as with any occupational disease, the interventions available to health professionals can be considered as engineering or administrative controls. as well, pharmaceutical controls (prophylaxis) for avian influenza may provide an important role in prevention. however, there is limited clinical evidence for the effectiveness of currently available medications or vaccines. vaccination strategies, such as the annual influenza vaccine programs, have been the traditional first line of defense against viral infections. research is currently being devoted to the development of vaccines as a possible intervention for pandemic influenza. the need for a rapidly deliverable vaccine for pandemic influenza has become more urgent since de jong et al. [15] reported the emergence of oseltamivir resistance to h5n1. given the current 4 to 6 month development time, it is unlikely that a vaccination will be available during the first wave of a pandemic. the impact of antigenic drift on vaccination for influenza is an on ongoing challenge and is the reason vaccination for seasonal influenza must be administered annually to protect against the new antigenic strain. increased demand for vaccine during a pandemic influenza may be tempered by the supply. specifically, the substrate used for vaccine manufacturing for all major suppliers worldwide is chicken eggs [16] . during a pandemic several times the current supply of eggs would be required. what is even more challenging is that h5n1, which is the current predicted pandemic strain, is lethal in eggs and is also a biosafety level 3 pathogen which decreases the potential of scaling up the manufacture of vaccine for international deployment [16] . one must also consider that poultry workers may be at increased risk of exposure to pandemic influenza zoonotically or may also be stretched from a human resource perspective when measures need to be taken to curb a poultry influenza outbreak [17] . acambis labs, and others, are working on the development of a universal influenza vaccination that is based on more stable surface proteins such as m2e, which is found on the surface of all influenza a strains. the first vaccine approved by the us food and drug administration for pandemic influenza is a reverse genetics vaccine and demonstrated low immunogenicity except for high doses with an adjuvant [18] . when this was approved by the fda it was noted that the vaccine would not be marketed to the general public but rather stockpiled by governments [16] . it has previously been suggested that an appropriate vaccine will likely not be determined until the initial phase of a pandemic [19] . furthermore, once a vaccine is developed a mechanism needs to be put in place that can provide an adequate supply at an affordable cost globally in lock step with the progression of the pandemic. a unique challenge for the occupational medicine physician in the event of a pandemic outbreak is to determine who gets priority for receiving vaccination. maintenance of essential services will be central to the continuity of a functioning society. health care workers and workers in critical occupations will be a priority for vaccination programs, once available. decisions on vaccination programs are complicated by the eventual timing of the disease wave, number of employees, nature of the work environment, and the availability of vaccine. for example, should employees who are in close proximity to one another be given priority or only those critical to maintaining business continuity? the public health agency of canada has created priority lists for receipt of vaccinations [20] . not surprisingly health care workers are part of group 1, followed by key societal decision makers and critical protection and utility workers (police, fire fighters, sewage workers, public transportation and communications). another treatment option is the use of anti-viral medications. the two main classes of antivirals available at present are the neuraminidase inhibitors and the adamantanes. there has been an emergence of resistance to adamantanes for seasonal influenza [21] leading many to reconsider them as agents in the treatment of pandemic avian influenza [22] . in preliminary studies using oseltamivir [23] or zanamivir [24] , patients showed a reduc-tion in the duration of symptoms ranging from 1-2 days. whether a 1-2 day reduction in symptoms will translate into reduced absenteeism, cost-savings and disease transmission is unknown. additionally, the cost-benefit of stockpiling anti-virals for treatment of pandemic influenza remains unknown. as noted previously, oseltamivir has also demonstrated resistance [15] . adding to the complexity of managing h5n1 treatment, is once again the manner in which one decides who receives the medication and the fact that the modest reduction in influenza symptoms will depend on timing of administration of the drug. in individuals with confirmed h5n1 influenza that were treated with oseltamivir, mortality was still close to 80% [25] . it has also been noted by tambyah [22] , that despite guidelines from the world health organization concerning the use of anti-virals in pandemic avian influenza, there remains little 'level 1' clinical evidence to support such guidelines. more recently, a group in singapore has gathered a set of practical guidelines for clinicians encountering h5n1 avian influenza in humans [26] . despite the lack of scientific evidence for their effectiveness in a pandemic situation, governments and many employers are stockpiling anti-virals to be used not only as therapy for ill individuals, but also as prophylaxis for critical staff. this may be driven by the recognition that once the pandemic is recognized, it will be nearly impossible to purchase these products. it reflects a significant investment: at approximately $3/pill, an eight week course would cost over $200 per employee. a company of 1000 employees would need to invest $200,000 on a product which they hope they will never use, is unproven, and has a limited shelf life. again, one is faced with decisions regarding dispensing medication -to all workers, critical workers, families? non-pharmaceutical controls while the world waits for an effective pharmaceutical intervention, non-pharmaceutical controls will need to be considered to combat the spread of illness in the community and the workplace. low [7] has outlined and adapted [27] five non-pharmaceutical public health interventions that would aid in the mitigation of pandemic influenza. they include: hand hygiene and respiratory etiquette, human surveillance, rapid viral diagnosis, provider and patient use of masks and other personal protective equipment and isolation of the sick. all of these interventions will need to be coordinated at organizational and government levels due to the tremendous interrelationships affected by a pandemic. some of the above interventions have some unique implications from an occupational medicine perspective. hygiene and respiratory etiquette are particularly effective in reducing the spread of infectious disease and represent a key defense against nosocomial infection in hospitals. this also applies to a workplace where people are in close proximity to one another where viral droplets may exist in the air and on equipment or surfaces used by multiple people each day. the spread of infection between employees is one possible transmission pathway, however the occupational medicine professionals of large and complex organizations must also consider the families of the employees and the consumers of products where interaction occurs with the public. protection of the consumer raises the issue of due diligence which can be complex for service oriented organizations. hand washing, social distancing and respiratory etiquette, if normalized and rigorously adopted, may provide the most effective (certainly most cost effective) means of protection. the role of personal protective equipment in reducing the spread of pandemic influenza is one of considerable debate. both the perceived and/or real efficacy of such measures and the cost associated with the provision of such materials are legitimate concerns for those coordinating pandemic plans in the workplace. the gold standard for particulate inhalation in most cases is the use of the n95 respirator. droplet transmission is thought to be the primary mode of transmission and is the basis of guidelines for health professionals coming within 3 feet of patients during seasonal influenza [7, 28] . therefore, because n95 respirators can trap more than 95% of airborne particles [28, 29] , experience from their use in seasonal influenza supports some effectiveness of their application to pandemic avian influenza. regardless of the real or perceived protection that n95 respirators provide to employees from transmitting or contracting h5n1 influenza via inhalation, many challenges exist with the use of such protective equipment. n95 respirators require fit testing, need to be replaced, and tend to be uncomfortable which create opportunities for their improper and therefore ineffective use. moreover, the n95 respirators would impose a large cost to an organization who decides they will outfit their employees with them in the event of a pandemic. this cost is imposed by buying a stockpile of the respirators, and the provision of fit-testing for each and every employee issued a respirator. consider an organization that decides that during a two week pandemic disease wave they will issue n95 masks to 1000 employees. each respirator unit has a cost of $1, and because the respirators need to be changed every 2-3 hours, each employee working an 8-hour day will require 3 masks per day. therefore, each employee would require 30 masks over 2-weeks (10 working days), leading to a cost of $30 per employee for a total of $30k for 1000 employees for two weeks. this does not include the cost associated with fit-testing which takes approximately 20-minutes per person, which would therefore require 333 hours of time to fit test 1000 employees. furthermore, a trained professional is required to perform the fit testing procedure. finally, does the employer provide n95 masks for the families of the employees such that protection is afforded to the family and the employee at home? all of these measures will vary as the risk of transmission will depend upon the nature of the worksite and the controls put in place. for example, teleworking would greatly reduce the number of employees that congregate at the worksite. not all industries will have this luxury. creating an environment in which employees are comfortable and confident of their safety in the workplace is critical in enhancing their work attendance. fear will be rampant, and employee education well in advance of the event will be vital in reducing the spread of disease, myths, and ensuring corporate and social stability. indeed addressing both real and perceived risk of infection may be the most crucial factor in maintaining business continuity in the face of a pandemic. the scientific community is devoting a great deal of effort and research funding towards what is considered by many to be an inevitable pandemic. it has also been suggested that even the most stringent non-pharmaceutical interventions are unlikely to prevent the pandemic or alter the underlying biological susceptibility of a population to a pandemic virus [7] . however, the prevention and management of disease transmission in the occupational environment will play a central role in the health and economic burden of pandemic influenza. with a longstanding record of applying the latest science to appropriate engineering and administrative disease controls, the occupational medicine community can utilize these concepts to prepare for and mitigate the potential impact on industry and society. pandemic influenza preparedness: a survey of businesses planning for avian influenza consensus document on the epidemiology of severe acute respiratory syndrome (sars) molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms emerging infections among health care workers: the severe acute respiratory syndrome (sars) experience influenza and the pandemic threat pandemic planning: non-pharmaceutical interventions updating the accounts: global mortality of the 1918-1920 'spanish' influenza pandemic avian flu from an occupational health perspective clinical manifestations and consequences of influenza transmission of influenza a in human beings avian influenza a (h5n1) infection in humans. the writing committee of the world health organization (who) consultation on human influenza a/h5 business continuity management and pandemic influenza roundtable discussion: corporate pandemic preparedness oseltamivir resistance during treatment of influenza a (h5n1) infection update on influenza vaccines pandemic influenza planning: shouldn't swine and poultry workers be included? vaccine safety and immunogenicity of an inactivated split-viron influenza a/vietnam/1194/2004 (h5n1) vaccine: phase i randomised trial are we ready for pandemic influenza? public health agency of canada: preparing for the pandemic vaccine response -annex d. ottawa: public health agency of canada the emergence of adamantane resistance in influenza a (h1) viruses in australia and regionally in 2006 update on influenza anti-virals efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial zanamivir for treatment of influenza a and b infection in high-risk patients: a pooled analysis of randomized controlled trials the working committee of the who: consultation on human influenza a/h5 avian influenza a (h5n1) infection in humans practical management of avian influenza in humans non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base transmission of influenza: implications for control in health care settings influenza transmission and the role of personal protective respiratory equipment: an assessment of evidence. ottawa: council of canadian academies at the time this paper was submitted to this journal the who and many governments are monitoring an outbreak of h1n1 swine influenza which has recently been declared a pandemic. cases have been confirmed here in nova scotia, united states, uk, spain and israel with the epicenter in mexico where over 100 people have died. while much of the literature focused on the future possibility of h5n1 avian influenza pandemic, the h1n1 swine influenza strain was not of immediate concern to the international community until the current outbreak in mexico. the authors declare that they have no competing interests. wsj conceived, researched, wrote and edited the manuscript. mdb provided background information, guidance and editing. both authors reviewed and approved the final submitted manuscript. key: cord-028564-sltofaox authors: gutiérrez-spillari, lucia; palma m., geovani; aceituno-melgar, jorge title: obesity, cardiovascular disease, and influenza: how are they connected? date: 2020-07-06 journal: curr trop med rep doi: 10.1007/s40475-020-00207-0 sha: doc_id: 28564 cord_uid: sltofaox purpose of review: to better understand the impact of obesity and cardiovascular diseases on influenza a infection. recent findings: this infection could have detrimental outcomes in obese patients with cardiovascular diseases, such as an increased risk, length of hospitalization, disease severity, morbidity, and mortality. nevertheless, there also might be some cardioprotective benefits associated with influenza vaccination, such as a reduced mortality, hospitalization, and acute coronary syndromes, in patients with coronary heart disease and/or heart failure. summary: obesity negatively impacts immune function and host defense. recent studies report obesity to be an independent risk factor for increased morbidity and mortality following infection. obese patients might need special considerations in the treatment; however, there is not enough evidence to fully comprehend the mechanisms behind the reduced immunocompetence when influenza a infection occurs. future studies should focus on special consideration treatments when the patients have not been vaccinated and have cardiovascular diseases. this review focuses on how obesity and cardiovascular disease impact influenza response. retrospective studies demonstrate that during the 2009 h1n1 pandemic, obesity was identified as a risk factor for hospitalization, mechanical ventilation, and mortality upon infection. these data must be highlighted since it is projected that nearly 50% of the worldwide population is going to be obese by 2050. several case studies have identified possible effects of obesity on viral replication in the deep lung, progression to viral pneumonia, and prolonged viral shedding [1] . therefore, management of the influenza infection in this at-risk population has to have special consideration given that they may not respond optimally to vaccination [2] . excessive fat accumulation that results in obesity impairs health for adults [3] . its low-grade chronic inflammatoryinduced state negatively impacts immune function and host defense [2] , as shown during the 2009 influenza a virus h1n1 pandemic, where obesity resulted to be an independent risk factor for severe disease, hospitalization, mechanical ventilation, and mortality upon infection [1] . it is well known that influenza a virus infection is characterized by fever, myalgia, rhinorrhea, sore throat, and sneezing. these symptoms peak 3-5 days post-infection, with viral shedding peaking at days 2-3. usually, it is limited to the upper respiratory tract; however, in severe cases, the lower respiratory tract, including the lungs, can be affected and often requires hospitalization. this progression is more common in obese patients, leading to diminish infection resolution when compared with non-obese patients [1] . obesity also plays a role in the outcome of critical complications from influenza this article is part of the topical collection on metabolism in tropical medicine a/pdmh1n1 infection and is associated with longer mechanical ventilation for severe acute respiratory distress syndrome and shock [4] . a higher body mass index (bmi) and metabolic syndrome in patients with influenza have shown an increased risk and length of hospitalization [4] [5] [6] , increased disease severity, morbidity, and mortality during lower respiratory tract infections. this might be explained in part by increased lung permeability during infection, found in mice studies. obese mice have an increased protein leakage from the lung into the bronchoalveolar lavage fluid when compared to lean mice. additionally, lung edema and oxidative stress are also increased, which emphasizes the multiple etiologies of increased lung pathology in the obese host and the impairment in wound repair [1, 4] . the obesogenic state can also affect the influenza a virus evolution. it is well known that obese individuals are malnourished besides their excess in fat; they might also present nutrient deficiencies, such as vitamins [7] , minerals, and trace elements [8] . there are a variety of mechanisms by which nutritional imbalances could alter within-host viral evolution [8] . studies have shown that such imbalances prolong infections, delay clearance, and increase shedding (42% longer than non-obese) [9] , all of which potentially increase viral transmission [1] . in addition to the decreased immunocompetence mechanisms, other potential factors might contribute to its increased susceptibility to infection in the hospital setting. some examples, which are underlying diseases that affect mobility, can also increase risk for skin problems, prolonged visits at hospitals and nosocomial infections, alteration in the pharmacokinetics of some drugs, and an increased susceptibility to postsurgery infections [2] . thus, it is a complex problem that needs further evidence to develop better treatments for this increasing population. obesity causes a chronic state of inflammation in a generalized and constant way with negative effects on immunity. obese people have delayed immune responses to influenza virus infection and experience slower recovery from the disease. in addition, the efficacy of the treatment and the vaccine is reduced in this population causing an alteration of the viral life cycle and, coupled with an already weakened and delayed immune response, leading to a more serious condition. poor initial and adaptive responses to infection and vaccination create an impaired ability to respond appropriately to infection. the efficacy of the vaccine may decrease in obese humans; however, more studies are needed to better understand how the obese state affects infection control [1] . previous studies suggest that the severity of influenza virus infection is multifactorial and may be related to lung spread and repair, the formation of extracellular concentrates of neutrophils at the lung level; however, this mechanism in individuals is unknown [10] . the efficacy of the vaccine in human groups has shown that initial seroconversion rates are high in the obese population, but that over time there is a greater decrease in efficacy than that observed in non-obese populations [11] . influenza vaccine as a method of prevention is formulated each year, typically containing both influenza a and b. a study conducted in 2013-2015 aimed at evaluating whether obesity was associated with an increased risk of influenza for influenza and influenza-like illness among vaccinated obese and nonobese adults, finding that, among the obese, 9.8% had confirmed influenza or influenza-like illness compared with 5.1% of healthy weight participants. compared with the vaccinated healthy weight, obese participants had twice the risk of developing influenza or influenza-like illness (relative risk = 2.01, 95% ci 1.12, 3.60, p = 0.020); therefore, in this risk group, in the same way, vaccination is very important [12] . although it appears that in high-risk groups, such as the obese and overweight population, vaccination may not provide optimal protection, and because of the growing trend of obesity worldwide, the efficacy of the vaccine should be improved [10] . among cardiovascular disease patients, there is compelling evidence that shows lower risk of major adverse cardiovascular events, reduced hospitalization, and mortality [13] [14] [15] , being the greatest treatment effect was seen among the highestrisk patients with more active coronary disease [15] . the recent recommendation advocates the priority of vaccination against influenza in obese patients; a vaccination program should be fully evaluated in obese adults. high-dose vaccines designed to vaccinate people over 65 can also be used in the obese population [9, 10] . in the twentieth century during influenza epidemics, there has been an excess of mortality from cardiovascular disease [16] . a recent study that included 364 hospitalizations for acute myocardial infarction demonstrated an increased risk of acute myocardial infarction within one week after influenza virus infection to a risk that was six times higher than the risk during the year before or after the onset of infection [17] . cardiovascular complications associated with influenza infection include myocarditis, pericardial effusion, myopericarditis, right and left ventricle dysfunction, myocardial infarction, heart failure, stroke, and circulatory failure due to septic shock [13, [18] [19] [20] . the risk of myocardial infarction after mild respiratory infection returns to baseline within approximately 5 weeks, but in the case of pneumonia complicated by sepsis the risk persists up to 10 years after the infection [16] [21] . infectious agents (including influenza virus) have been implicated in the etiology of atherosclerosis [22] . there have been described several mechanisms by which influenza increases the risk of cardiovascular events; they may be related to pro-inflammatory mediators, sympathetic stimulation, and the activation of the coagulation cascade [19] . according to the fourth universal definition of myocardial infarction, there are 5 types of myocardial infarction based on clinical, electrocardiographic, and laboratory evaluation [23] . influenza infection can trigger type 1 and type 2 myocardial infarctions [16] . type 1 myocardial infarction is defined as myocardial ischemia caused by atherothrombotic coronary artery disease, and it is usually precipitated by atherosclerotic plaque disruption that can be rupture or erosion [23] . it is important to remember that atherosclerotic plaques also contain inflammatory cells, and pro-inflammatory cytokines, such as interleukins 1, 6, and 8, and tumor necrosis factor α are generated as a response of infection. these inflammatory cytokines can activate inflammatory cells in atherosclerotic plaques [16] [24] . acute influenza infection is associated to a procoagulant state that increases the risk of coronary thrombosis at sites of plaque disruption [16] [25, 26] . infection with influenza virus is associated with expression of genes that have been linked to platelet activation: h1n1 exposure increases platelet gene expression signature, which is associated with myocardial infarction [25] . type 2 myocardial infarction might be considered with a rise and/or fall of cardiac troponin values and evidence of an imbalance between myocardial oxygen supply and demand unrelated to coronary thrombosis. [23] . influenza infection produces a systemic inflammatory response with a resulting increase in heart rate and shortens the filling time during diastole compromising in that way, the coronary blood supply. if septic shock occurs, it may have a substantial adverse effect on coronary perfusion. in older patients with chronic coronary plaques, systemic inflammation causes cardiac metabolic mismatch increasing the risk of myocardial infarction [16, 18] . influenza infection is also associated with increased mortality in patients with heart failure [27] and is vulnerable to influenza-associated complications [28] [29] . this type of patients has limited cardiac and respiratory reserves and may not tolerate the metabolic demand and hypoxemia, exacerbating underlying cardiac disease probably due to an increased sympathetic nervous system activity, hypoxemia, and renal dysfunction that can lead to volume overload [28] . in a healthy heart, severe acute influenza infection produces pro-inflammatory cytokine level elevations that can cause acute myocarditis [28] [18] , characterized by a broad spectrum of symptoms that go from asymptomatic courses to signs of myocardial infarction to devastating illness with cardiogenic shock [30] [31] . myocarditis often results from common viral infections and post-viral immune-mediated responses [30] . in acute myocarditis, there is a high incidence of wall motion abnormalities. during influenza epidemics, 15% of the patients admitted to a military hospital with influenza infection had wall motion abnormalities on echocardiogram [18] . this becomes important, due to the fact that the myocardium inflammatory disease is regarded as a precursor of dilated cardiomyopathy [30] [32] . the electrocardiographic findings in patients with myocarditis range from nonspecific t-wave and st segment changes to st segment elevation resembling an acute myocardial infarction; supraventricular and ventricular arrhythmias can also be present. electrocardiographic findings that are related to poor clinical outcome include a qtc prolongation at 440 ms, an abnormal qrs axis, and ventricular ectopic beats [30] . a recent study developed to evaluate the incidence and hemodynamic consequences of right ventricular and left ventricular dysfunction in patients with h1n1 infection demonstrated that on admission 72% had abnormal ventricular function (46% had isolated left ventricular abnormalities and 39% had isolated right ventricular dysfunction) and 14% had biventricular dysfunction. on the follow-up, right ventricular function tended to worsen during hospitalization, but left ventricular function tended to normalize. however, patients with ventricular dysfunction needed more aggressive therapy and of rescue ventilatory strategies, such as inhaled nitric oxide, prone positioning, and extracorporeal membrane oxygenation [18] . during influenza epidemics, hospitalizations for cerebrovascular diseases increase [33] . an increase in incidence of ischemic stroke within 2 weeks after influenza infection has been suggested [34, 35] . protein c pathway and endogenous fibrinolysis are mechanisms associated with cerebrovascular ischemia and influenza infection [19] . influenza infection develops a prothrombotic state by increasing tissue factor expression and decreases fibrinolytic capacity by increased plasminogen-activator inhibitor-1 (pai-1) expression. this results in an unbalance between coagulation and anticoagulant pathways [36] . as in myocardial infarction, the relationship between systemic inflammation and stroke pathophysiology has shown that stroke often occurs in a pre-existing state of inflammation due to atherosclerosis, obesity, or infection [37] . the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice recommend that annual influenza vaccination can be considered in patients with established cardiovascular disease (class iib, level c) [38] , based on the fact that the risk of a cardiovascular event (myocardial infarction or stroke) is more than four times higher after a respiratory tract infection, with the highest risk in the first 3 days after infection [39] . there have been developed several studies that demonstrate that influenza vaccination reduces mortality, hospitalization, and acute coronary syndromes in patients with coronary heart disease and/or heart failure [13] . the mechanisms by which acute inflammation affect the risk of vascular events include the following: endothelial dysfunction, procoagulant state, and inflammatory changes in atherosclerotic plaques [39, 40] . it is also known that persistent systemic inflammatory activity is a risk of factor for cardiovascular disease, and higher interleukin-6 blood levels increase cardiovascular mortality at one year after pneumonia infection [41] . this systemic inflammatory response can be reduced by vaccination [13] . when estimating the cost and benefit of interventions to prevent pneumonia, the association of pneumonia with cardiovascular disease risk should also be considered [40] . patients with chronic heart failure are vulnerable to influenza-related complications (including secondary infections, such as pneumonia and acute heart failure exacerbations). recently, the paradigm-hf trial assessed associations between receiving influenza vaccine and cardiovascular death or heart failure hospitalizations, all-cause hospitalizations, and cardiopulmonary or influenza-related hospitalizations, concluding that vaccination was associated with reduced risk for death [42] . there have been described two possible mechanisms by which influenza vaccination may reduce cardiovascular events: unspecific and specific effects [13] . the unspecific mechanism is based on the fact that influenza infection causes a systemic inflammatory response, endothelial dysfunction, and a procoagulant state. these factors have negative effects on patients with previous cardiovascular diseases, such as ischemic heart disease and heart failure, causing acute heart failure, pulmonary edema, or destabilization of chronic ischemic heart disease, leading to myocardial infarction or sudden cardiac death [13] [15] . influenza vaccination reduces the risk fig. 1 possible cardioprotective mechanisms of influenza vaccination of infection and inflammation by decreasing the secretion of pro-inflammatory mediators, such as cytokines (that cause reduced myocardial contractility) and metalloproteinases (that cause adverse cardiac remodeling and plaque rupture); influenza vaccination also causes inhibition of platelet activation and cloth formation [13] [43] . the specific mechanism takes into account the immunological properties of the vaccine. the protective effect of the influenza vaccine has been demonstrated in multiple studies. to explain the pleiotropic effect of the influenza vaccine, the "antigen mimicry" between atherothrombotic plaque and influenza virus has been proposed [22] . it has also been proposed that there is an autoimmune "cross-reaction" between influenza and atherosclerosis [13] [44] . figure 1 summarizes the possible cardioprotective mechanism of influenza vaccination [13] . it is well studied that obese patients can develop cardiovascular diseases; however, it is less known that the lowinflammatory chronic state might affect host defense and immune cell dysfunction and infections, such as influenza a, could have detrimental outcomes in such patients, such as an increased risk, length of hospitalization, disease severity, morbidity, and mortality. cardiovascular diseases, such as ischemic heart disease and heart failure combined with influenza a infection, can trigger acute heart failure exacerbations that increase the overall mortality in a hospitalized setting. cardiovascular complications associated with influenza infection include myocarditis, pericardial effusion, myopericarditis, right and left ventricle dysfunction, myocardial infarction, heart failure, stroke, and circulatory failure due to septic shock. there have been described several mechanisms by which influenza increases the risk of cardiovascular events; they might be related to pro-inflammatory mediators, sympathetic stimulation, and activation of the coagulation cascade. while influenza vaccination is associated with a significant reduction in all-cause mortality risk in patients with heart failure, this cardioprotective mechanism may not function as intended in the obese population since they do not always respond optimally to vaccination. therefore, in an effort to prevent these complications and in the absence of special consideration treatments for this population, we strongly suggest a weight-loss approach. future studies should focus on developing targeted treatments that can combat the reduced immunocompetence that excess adiposity causes to the patient. particular interest, published recently, have been highlighted as: • of importance impact of obesity on influenza a virus pathogenesis, immune response, and evolution the impact of obesity on the immune response to infection immunity to influenza: impact of obesity high body mass index as a risk factor for hospitalization due to influenza: a case-control study epidemiology of severe influenza outcomes among adult patients with obesity in detroit association between vitamin deficiency and metabolic disorders related to obesity rna virus evolution, population dynamics, and nutritional status obesity increases the duration of influenza a virus shedding in adults obesity outweighs protection conferred by adjuvanted influenza vaccination obesity is associated with impaired immune response to influenza vaccination in humans increased risk of influenza among vaccinated adults who are obese cardioprotective effect of influenza and pneumococcal vaccination in patients with cardiovascular diseases influenza vaccines for preventing cardiovascular disease association between influenza vaccination and cardiovascular outcomes in high-risk patients acute infection and myocardial infarction acute myocardial infarction after laboratoryconfirmed influenza infection myocardial dysfunction during h1n1 influenza infection beneficial effects of vaccination on cardiovascular events: myocardial infarction, stroke, heart failure. cardiol cardiovascular manifestations associated with influenza virus infection severe infections and subsequent delayed cardiovascular disease influenza and cardiovascular disease: a new opportunity for prevention and the need for further studies fourth universal definition of myocardial infarction diffuse and active inflammation occurs in both vulnerable and stable plaques of the entire coronary tree: a histopathologic study of patients dying of acute myocardial infarction gene expression profiles link respiratory viral infection, platelet response to aspirin, and acute myocardial infarction. schulz c, editor sepsis, thrombosis and organ dysfunction relation of concomitant heart failure to outcomes in patients hospitalized with influenza association of influenza-like illness activity with hospitalizations for heart failure: the atherosclerosis risk in communities study decreased immune responses to influenza vaccination in patients with heart failure update on myocarditis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases management of myocarditis-related cardiomyopathy in adults influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly temporal relationship between influenza infections and subsequent first-ever stroke incidence influenza-like illness as a trigger for ischemic stroke influenza and stroke risk: a key target not to be missed? influenza virus infection aggravates stroke outcome european guidelines on cardiovascular disease prevention in clinical practice. the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of 10 societies and by invited experts. developed with the special contribution of the european association for cardiovascular prevention & rehabilitationg ital cardiol (rome) risk of myocardial infarction and stroke after acute infection or vaccination association between hospitalization for pneumonia and subsequent risk of cardiovascular disease inflammatory markers at hospital discharge predict subsequent mortality after pneumonia and sepsis influenza vaccination in patients with chronic heart failure: the paradigm-hf trial from vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: part ii influenza vaccine as prevention for cardiovascular diseases: possible molecular mechanism publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-269623-9pxdeva3 authors: nicholson, karl g; wood, john m; zambon, maria title: influenza date: 2003-11-22 journal: lancet doi: 10.1016/s0140-6736(03)14854-4 sha: doc_id: 269623 cord_uid: 9pxdeva3 although most influenza infections are self-limited, few other diseases exert such a huge toll of suffering and economic loss. despite the importance of influenza, there had been, until recently, little advance in its control since amantadine was licensed almost 40 years ago. during the past decade, evidence has accrued on the protection afforded by inactivated vaccines and the safety and efficacy in children of live influenza-virus vaccines. there have been many new developments in vaccine technology. moreover, work on viral neuraminidase has led to the licensing of potent selective antiviral drugs, and economic decision modelling provides further justification for annual vaccination and a framework for the use of neuraminidase inhibitors. progress has also been made on developing near-patient testing for influenza that may assist individual diagnosis or the recognition of widespread virus circulation, and so optimise clinical management. despite these advances, the occurrence of avian h5n1, h9n2, and h7n7 influenza in human beings and the rapid global spread of severe acute respiratory syndrome are reminders of our vulnerability to an emerging pandemic. the contrast between recent cases of h5n1 infection, associated with high mortality, and the typically mild, self-limiting nature of human infections with avian h7n7 and h9n2 influenza shows the gaps in our understanding of molecular correlates of pathogenicity and underlines the need for continuing international research into pandemic influenza. improvements in animal and human surveillance, new approaches to vaccination, and increasing use of vaccines and antiviral drugs to combat annual influenza outbreaks are essential to reduce the global toll of pandemic and interpandemic influenza. influenza viruses have segmented genomes and show great antigenic diversity. of the three types of influenza viruses-a, b, and c-only types a and b cause widespread outbreaks. influenza a viruses are classified into subtypes based on antigenic differences between their two surface glycoproteins, haemagglutinin and neuraminidase. 15 haemagglutinin subtypes (h1-h15) and nine neuraminidase subtypes (n1-n9) have been identified for influenza a viruses (figure 1). viruses of all haemagglutinin and neuraminidase subtypes have been recovered from aquatic birds, but only three haemagglutinin subtypes (h1, h2, and h3) and two neuraminidase subtypes (n1 and n2) have established stable lineages in the human population since 1918. only one subtype of haemagglutinin and one of neuraminidase are recognised for influenza b viruses. 1733 although most influenza infections are self-limited, few other diseases exert such a huge toll of suffering and economic loss. despite the importance of influenza, there had been, until recently, little advance in its control since amantadine was licensed almost 40 years ago. during the past decade, evidence has accrued on the protection afforded by inactivated vaccines and the safety and efficacy in children of live influenza-virus vaccines. there have been many new developments in vaccine technology. moreover, work on viral neuraminidase has led to the licensing of potent selective antiviral drugs, and economic decision modelling provides further justification for annual vaccination and a framework for the use of neuraminidase inhibitors. progress has also been made on developing near-patient testing for influenza that may assist individual diagnosis or the recognition of widespread virus circulation, and so optimise clinical management. despite these advances, the occurrence of avian h5n1, h9n2, and h7n7 influenza in human beings and the rapid global spread of severe acute respiratory syndrome are reminders of our vulnerability to an emerging pandemic. the contrast between recent cases of h5n1 infection, associated with high mortality, and the typically mild, self-limiting nature of human infections with avian h7n7 and h9n2 influenza shows the gaps in our understanding of molecular correlates of pathogenicity and underlines the need for continuing international research into pandemic influenza. improvements in animal and human surveillance, new approaches to vaccination, and increasing use of vaccines and antiviral drugs to combat annual influenza outbreaks are essential to reduce the global toll of pandemic and interpandemic influenza. we reviewed international reports published in english before december, 2002 . the data for this non-systematic review of articles were identified by searches of medline, embase, integrated science citation index, pubmed, and the cochrane library electronic databases with relevant keywords. we also searched cited references in retrieved articles, reviewed articles we have collected over many years, referred to the textbook of influenza, 2 and used knowledge of new data presented at international scientific meetings. because of the large number of articles that are published every year and limitations on the number of citations, we gave emphasis to clinically relevant issues, particularly disease burden, the emergence of new subtypes, vaccines, and antivirals, and diagnosis. we gave priority to randomised controlled trials when available, to larger studies, articles published in high-impact journals that have a wide readership, and the systematic review and economic decision modelling, for the prevention and treatment of influenza, commissioned by the health technology assessment programme on behalf of the national institute of clinical excellence. 1 we also drew on our own knowledge when it seemed appropriate to fill in the gaps in the published work and included several recent pertinent articles. haemagglutinin facilitates entry of the virus into host cells through its attachment to sialic-acid receptors. it is the major antigenic determinant of type a and b viruses to which neutralising antibodies are directed and the crucial component of current influenza vaccines. an important function of neuraminidase, the second major antigenic determinant, is to catalyse the cleavage of glycosidic linkages to sialic acid, thereby assisting in the release of progeny virions from infected cells. accordingly, neuraminidase has become an important target for antiviral activity. the m2 ion channel of influenza a, which is blocked by the antiviral drug amantadine, regulates the internal ph of the virus, which is crucial during early viral replication. the epidemiological behaviour of influenza in people is related to the two types of antigenic variation of its envelope glycoproteins-antigenic drift and antigenic shift. during antigenic drift, new strains of virus evolve by accumulation of point mutations in the surface glycoproteins. the new strains are antigenic variants but are related to those circulating during preceding epidemics. this feature enables the virus to evade immune recognition, leading to repeated outbreaks during interpandemic years. antigenic shift occurs with the emergence of a "new", potentially pandemic, influenza a virus that possesses a novel haemagglutinin alone or with a novel neuraminidase. the new virus is antigenically distinct from earlier human viruses and could not have arisen from them by mutation (figure 2). four or five pandemics of influenza occurred during the 20th century with intervals of 9-39 years. the h1n1 pandemic of 1918-19 was the most devastating, with 40-50 million deaths; an estimated 4·9 million excess deaths, representing 2% of the population, occurred in india alone. however, the cumulative mortality from influenza during the intervening years is generally many times greater than that associated with pandemics. 3 although influenza a or b viruses circulate virtually every winter in temperate zones of the northern and southern hemispheres, quantification of the burden of influenza on consultations, emergency-department examinations, hospital admissions, and mortality has been difficult because influenza lacks pathognomonic features, it cocirculates with other respiratory pathogens, and it causes a range of nonspecific complications, such as exacerbations of chronic cardiopulmonary disease. nevertheless, there is much evidence that the h3n2 subtype of influenza a virus causes more severe illness than h1n1 or influenza b, 4-6 more hospital admissions for pneumonia and influenza, 7 and higher numbers of excess deaths. 3 during outbreaks, sentinel schemes, such as the royal college of general practitioners' network in england, report increased consultation rates for influenza-like illness and other respiratory syndromes that are strongly associated with excess mortality. 8 in england and wales, an estimated 6200-29 600 people died during each of the epidemics between 1975-76 and 1989-90. 8 these estimates are about ten times the number of death certifications for influenza, because the disease is the cause of many "hidden deaths". in the usa, during the 9 about 90% of these influenzaassociated excess deaths are among people aged 65 years and older. although there are age-related increases in deaths from influenzal illness in both at-risk and low-risk groups, 10 most deaths and hospital admissions occur in elderly people with chronic cardiopulmonary disorders. among toddlers, rates of influenza-associated hospital admission in the usa have ranged from about 500 per 10 5 population for those with high-risk conditions to 100 per 10 5 for those without high-risk conditions. [11] [12] [13] [14] admission rates are highest among children younger than 1 year and are similar to rates found among people aged 65 years and older. 13, 14 among children in hong kong, china, the numbers of excess hospital admissions attributed to influenza are very high in children younger than 12 months (2785 and 2882 per 10 5 in 1998 and 1999, respectively) and decrease with age (2184 and 2093 per 10 5 children aged 12-23 months; 1256 and 773 per 10 5 children aged 2-4 years; 573 and 209 per 10 5 children aged 5-9 years; and 164 and 81 per 10 5 children aged 10-15 years). 15 in the tropics and subtropics, influenza occurs either throughout the year with no distinct seasonality or visible excess mortality, or twice a year, with the more intense activity during the rainy season. consequently, the morbidity and mortality from influenza are probably greatly underestimated in these regions. during summer, 2002, an epidemic of respiratory illness with 22 646 cases and 3% case-mortality affected madagascar; it was attributable to influenza a/panama/ 2007/97-like (h3n2) virus. the loss of life was greatest in young children and was ascribed to malnutrition and poor access to health care. 16 another outbreak attributable to influenza a/panama/2007/97-like (h3n2) virus occurred during november and december, 2002, in the district of bosobolo, democratic republic of congo. the casefatality rate was 3·5% in children younger than 5 years and 3·2% in people over 65. these rates illustrate the seriousness of such outbreaks and are one of the reasons why improved linkage of morbidity and mortality analysis with virological surveillance is one of the key objectives of the who global agenda on influenza, formulated in 2002. in southern china, influenza viruses circulate throughout the year. there is evidence for the origin in china of the viruses that caused the pandemics of h2n2 influenza in 1957, h3n2 influenza in 1968, and the re-emergence of h1n1 influenza in 1977. recent outbreaks of avian influenza a h5n1 and h9n2 in people in hong kong show the importance of virological surveillance in this region for the early detection of potentially pandemic viruses. there is also evidence that some drift variants circulate in china for up to 2 years before causing epidemics in europe and north america. 17, 18 this region is thought to provide an appropriate ecological niche for the emergence of new influenza viruses with pandemic potential, owing to the proximity of dense populations of people, pigs, and wild and domestic birds, thereby facilitating genetic reassortment of viruses from different species (figure 2), or for the emergence of drift variants, given the high human population density and year-round virus circulation. these observations provided the impetus for improving the who global influenza surveillance programme in china that has provided many of the vaccine strains recommended by who in the past decade. the examples cited below indicate the unpredictability of influenza-virus variation and the great capacity for evolution, but they also show that novelty alone is insufficient for the emergence of pandemic influenza. adaptation to replication in human beings, the ability to spread from person to person, and a susceptible population are also prerequisites. thus, the emergence of new influenza-virus variants in the human population does not necessarily herald pandemic influenza. influenza a/hong kong/97 (h5n1) in may and november-december, 1997, 18 cases of influenza h5n1 infection were identified in people in hong kong. this outbreak, which followed serious outbreaks of avian h5n1 influenza in chicken farms, signalled the possibility of an incipient pandemic. the human influenza isolates were of avian origin and were not derived by reassortment. 19 the high mortality (six of 18 patients died from acute respiratory distress syndrome or multiple organ failure, most previously healthy young adults 20 ) suggested an unusually aggressive clinical course. deterioration was rapid, with pneumonia necessitating ventilatory support developing within a few days of illness onset. striking features of severe cases were the early onset of lymphopenia and high concentrations of serum transaminases. fortunately, there were few if any secondary infections, and the h5n1 outbreak ceased when all chickens in hong kong (about 1·5 million) were slaughtered. the territory's poultry stocks were again depopulated when highly pathogenic a/hong kong/97 (h5n1) virus reemerged in flocks in may, 2001, and february and april, 2002. however, no further human cases of h5n1 influenza were identified until february, 2003, when two cases were confirmed in a family of hong kong residents. the first patient, a 9-year-old boy who was admitted to hospital in hong kong and recovered, became unwell during travel to fujian province, mainland china. the boy's 33-year-old father died in a hong kong hospital and his 8-year-old sister died in a hospital while the family was in china; the cause of her death is not known. genetic analysis of the two h5n1 isolates showed that the virus genes were purely avian in origin, but differed from the 1997 strains that infected human beings. after the h5n1 outbreak in hong kong, heightened surveillance in the adjoining guandong province led to recovery of nine human isolates of h9n2 virus during july-september, 1998. 21 in march, 1999, influenza h9n2 viruses were isolated from two children in hong kong. the illness in both was mild and self-limited. 22 no serological evidence of h9n2 infection was found in family members or health-care workers who had close contact with the children; thus, h9n2 viruses, like h5n1 viruses, seem not to be easily transmitted from person to person. 23 three lineages of h9 virus have been defined, with the prototype viruses being g1, g9, and y439. 24 the g1 "avian" h9n2 viruses isolated from human beings have some receptor properties similar to those of other human viruses-ie, binding to ␣2,6 sialic acid linkages, in contrast to the binding preference to the ␣2,3 linkages normally found with avian influenza viruses. in hong kong, antibody to h9 viruses was found in about 4% of blood donors, 22 which suggests that human infection with h9n2 may occur in this locality. surveillance of pigs in southern china has shown that h9n2 viruses are cocirculating with human a/sydney/97-like h3n2 viruses and other porcine h1n1 and h3n2 viruses. together, these observations indicate that all the precursors of potentially pandemic h9 human-avian reassortants are in place. during february, 2002, a new influenza h1n2 virus was isolated from patients with influenza-like illness in england and the middle east. 25 in the uk it affected mainly young children. 26 these h1n2 viruses arose after reassortment of the segments of the currently circulating influenza a (h1n1) and a (h3n2) subtypes. 25 although influenza a (h1n2) viruses have been identified previously, during 1988-89, when 19 influenza a (h1n2) viruses were isolated in six cities in china, the virus did not spread further. 27, 28 the limited effect of h1n2 in 1988 and during the 2001-02 and 2002-03 seasons is attributable to the good pre-existing immunity in the population. in 1980, four people contracted purulent conjunctivitis within 2 days of post-mortem examination of harbour seals that died during an outbreak of influenza a/seal/mass/1/80 (h7n7), an a/fowl plague/dutch27 (h7n7)-like virus, in cape cod, ma, usa. 29 subsequently, a/seal/mass/1/80 (h7n7) was recovered from the conjunctiva of an investigator who developed conjunctivitis when an infected animal sneezed into his face. 29 in 1996, avian h7n7 virus was isolated in the uk from a woman with conjunctivitis who kept ducks. 30 although none of these six patients had respiratory symptoms, an outbreak of highly pathogenic avian h7n7 influenza in poultry farms in the netherlands, which began at the end of february, 2003, was associated with fatal respiratory illness in one of 82 human cases by april 21. the person who died was a previously healthy 57-year old veterinary surgeon who developed severe headache, renal impairment, interstitial pneumonia, and acute respiratory distress after visiting an affected poultry farm. 31 most patients presented with conjunctivitis (n=79), and only seven (<10%) had respiratory illness. transmission of h7n7 influenza from poultry workers to family members was found on three occasions. 31 most virus isolates obtained from human beings had not accumulated significant genetic changes, including those from cases of human-to-human transmission. however, the virus isolated from the person who died had 14 aminoacid substitutions, which suggests a role in pathogenicity. rapid, near-patient tests for influenza can aid clinical management, but the usefulness of existing tests for decisions on whether to start antiviral drug treatment is limited because they are complex or have low sensitivities (table 1) . [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] however, rapid influenza tests can show whether virus is circulating in specific populations or localities, and they may become a useful adjunct to surveillance programmes. 42 thus, treatment with anti-influenza drugs commonly depends on patients' symptoms. although influenza has no pathognomonic features, it was diagnosed correctly in clinical trials in about two-thirds of adults when the clinical entry criteria were met. 43 cough and fever (temperature 37·8°c or above) are the most predictive symptoms of influenza, 43 but fever may not be present in elderly people. 44 in primary care, about 25-50% of patients with influenza-like illness have the disease during outbreaks. [45] [46] [47] thus, optimum implementation of guidelines for antiviral treatment depends on continuous community-based clinical and virological surveillance and awareness by general practitioners that influenza is circulating. most of what we know about highly pathogenic influenza viruses derives from studies with avian influenza viruses in birds. this situation is potentially relevant to disease in human beings because some mechanisms of pathogenicity in birds may operate in mammals and new human influenza a strains may come ultimately from the avian reservoir. 48 tissue tropism and the capacity for systemic spread are the most important determinants of pathogenicity in birds. the molecular correlates of these pathogenic properties reside in the viral haemagglutinin and have been well studied. [49] [50] [51] however, in mammals, factors other than viral haemagglutinin are involved in determining pathogenicity, including viral non-structural protein 1, pb2, and neuraminidase. recovery of nucleic acid of the 1918 pandemic virus from post-mortem tissue or preserved human remains has shown that this highly pathogenic virus did not have the molecular motifs in haemagglutinin associated with virulence in avian strains. 52, 53 the use of reverse genetics techniques has allowed the direct manipulation of influenza-virus gene products and creation of new recombinant viruses. selective testing of specific mutations engineered into recombinant viruses in a mouse model has shown that greater virulence is obtained by specific molecular properties of haemagglutinin, but these do not fully explain pathogenicity. 54 the balance between viral replication and host immune response determines the outcome of viral infection. highly virulent viruses, such as h5n1, have a remarkable capacity to resist the antiviral effects of host cytokines. 55 infection of human macrophages also results in induction of high cytokine expression, suggesting that severe outcome of infection is due both to lack of inhibition of viral replication by cytokines and to excess induction of cytokines leading to tissue damage in the infected host. 56 the key genetic component determining replication of highly pathogenic virus may be the nonstructural protein 1 of the virus, which has been identified as the major immune modulator. 57 factors contributing to the pathogenesis of influenza in people are incompletely understood. these include understanding of the nature of tissue restriction of proteases, differences in reactivity of the innate immune system at different stages of life, and varying susceptibilities in different human populations, leading to different ranges of disease in certain populations; for example, encephalopathy is well recognised in japan, but less so in other populations. 58, 59 study of mechanisms of virulence should provide more than simply elucidation of pathogenesis, notably development of alternative means of attenuating influenza viruses (eg, by deletion of non-structural protein or m2 [matrix protein 2] genes) to make safer vaccine strains. current vaccines are produced from virus grown in fertile hens' eggs and inactivated by either formaldehyde or ␤-propiolactone. they consist of whole virus, detergent-treated split product, or purified haemagglutinin and neuraminidase surface antigen formulations of the three virus strains currently recommended by who. about 50 countries have government-funded national immunisation programmes, and influenza vaccine is available in many others. about 234 million of the world's population of 6 billion were vaccinated during 2000. specific vaccine recommendations vary, but most involve annual vaccination of elderly people and those with certain chronic medical disorders. these recommendations were founded on proven morbidity and mortality in the at-risk groups, consistent demonstrations of vaccine efficacy in military recruits, recognition of the relation between antibody and protection, and proof of vaccine antigenicity. whole-virus vaccines are not widely available because they cause adverse reactions in young children, whereas split-product formulations and those containing purified surface antigen are well tolerated and extremely safe. the virtual absence of published reports suggests that hypersensitivity reactions are rare. recent randomised controlled trials 60 efficacy and effectiveness of current vaccines studies on the efficacy and effectiveness of inactivated influenza vaccines reveal substantial benefits. in children, meta-analysis 1 of double-blind 64-66 and singleblind 67,68 randomised controlled trials estimated the efficacy of vaccine in preventing symptomatic laboratory-confirmed influenza at 80% (table 2) . other benefits include reductions in school absenteeism, otitis media, asthma exacerbations, and febrile respiratory illness in unvaccinated household contacts. 67, [75] [76] [77] [78] [79] in adults of working age, meta-analysis 1 of two randomised controlled trials of split influenza vaccines 69,70 estimated the efficacy in preventing laboratory-confirmed influenza at 77% (table 2) . associated benefits include reductions in absenteeism, consultations, antibiotic use, and use of over-thecounter medication. [80] [81] [82] the frequency of laboratory-confirmed influenza fell by 52% in vaccinees in a randomised controlled trial (table 2) 71 and by 94% in a prospective cohort study of elderly people living in the community. 83 many cohort and case-control studies have shown lower rates of hospital admissions for pneumonia and influenza, 84-92 all respiratory disorders, 85,87 respiratory disorders and heart failure, 93 deaths from pneumonia and influenza, 84, 87 and all-cause mortality 85, 86, 94 in vaccinees than in controls (table 3) . a meta-analysis of reports published before 2001 showed that vaccination reduces numbers of cases of influenza-like illness by 35%, hospital admissions for pneumonia and influenza by 47%, and all-cause mortality by 50%. 95 a prospective observational study of 22 462 nursinghome residents in japan showed a 60% reduction in laboratory-confirmed influenzal illness among vaccinees. 72 vaccination also reduced numbers of hospital admissions among vaccine failures and thus appears to ameliorate illness severity. gross and colleagues 96 did a meta-analysis of 20 cohort studies. the odds ratios for development of respiratory illness (0·44) or pneumonia (0·47), hospital admission (0·50), and mortality (0·32) indicate substantial protection. vaccination of elderly patients with chronic lung disease reduces hospital admissions for pneumonia and influenza by 52%, 97 all-cause mortality by 70%, 97 and complications (death, exacerbations of lung disease, pneumonia, heart failure, angina, and myocardial infarction) by 50%. 98 influenza vaccination also prevents heart failure, 85 brain infarction, 99 recurrent myocardial infarction, 100 and primary cardiac arrest, 101 indicating important benefits in patients with cardiovascular disease. vaccination of patients with diabetes mellitus is associated with an estimated 79% reduction in hospital admissions, mostly (86%) for reasons of diabetic control. 102 thus, influenza vaccine protects against several potentially fatal events that explain the many hidden deaths that accompany epidemics. staff have been implicated as the source of influenza in several outbreaks in nursing homes. accumulating that influenza vaccination of staff caring for elderly people in long-stay facilities provides benefits to residents. [103] [104] [105] one study showed substantial herd immunity (68-87% protection) among patients exposed to staff with high vaccine coverage. 105 during the past decade, there have been many new developments in vaccine technology that have aided vaccine production or aimed to improve vaccine immunogenicity and acceptability. subunit influenza vaccine with adjuvant mf59, an emulsion of squalene in water for parenteral use, is licensed in some european countries but not the uk. mf59 significantly increases haemagglutinationinhibition antibody responses to interpandemic influenza a h3n2 and influenza b antigens, particularly in older people with chronic diseases, and is well tolerated despite slightly higher rates of transient mild local reactions than with other vaccines. 106 virosomes consist of bilayers of phospholipids (liposomes) containing virus surface proteins embedded in the bilayer. virosomes have been extensively evaluated in various human populations. 107 typically, virosomes induce higher concentrations of antibody after vaccination, higher rates of seroconversion, and a greater proportion of individuals with "protective" antibody titres than conventional inactivated vaccines. virosomal influenza vaccine for parenteral use became available in the uk during the 2002-03 season. cell-culture vaccines offer the potential of being able to respond quickly to epidemics or a pandemic at any time of the year and avoid the risk of contaminated eggs, which could affect the bioburden and endotoxin content of vaccines. moreover, influenza viruses grown in mammalian cells more closely resemble those present in clinical samples than viruses isolated and grown in eggs, hence offering the potential for more effective vaccine. influenza vaccines prepared with madin darby canine kidney (mdck) and african green monkey (vero) cells as substrate have been licensed in the netherlands but are not yet available commercially. live attenuated influenza vaccines intranasal delivery of live influenza vaccines offers the advantage of mimicking natural infection, thereby providing a broader immunological response and more durable protection than with inactivated vaccines. strategies for use of live influenza vaccines based on the transfer of genes coding for cold adaptation (ca) and temperature sensitivity from an attenuated parental virus donor have been used in russia for many years and were approved in june, 2003, by the us food and drug administration for use in healthy children and adolescents aged 5-17 years, and in healthy adults aged 18-49 years after three decades of clinical evaluation. vaccines based on ca replicate well at the temperatures found in the nasopharynx but not at temperatures in the lower airways. in young children, a recent study of us ca vaccine showed very high protection with benefits in terms of both influenzal illness and otitis media. 108 during the second year of this study, ca vaccine afforded a high degree of protection against a variant not closely matched to the vaccine antigen. 109 studies in nursing-home residents have suggested that a combination of live and inactivated influenza vaccines may improve protection in these communities. immunostimulating complexes are cage-like structures that were originally formed as a complex between cholesterol and saponin derived from the tree quillaia saponaria. vaccines containing a defined saponin called iscoprep 703 stimulated an accelerated serum antibody response in human beings compared with conventional inactivated vaccines, an improved proliferative t-cell response, and a cytotoxic t-cell response. although nasally delivered influenza vaccine could greatly increase vaccine coverage and provide mucosal immunity, intranasal administration of conventional inactivated influenza vaccines has typically been unsuccessful. in animals, incorporation of a mucosal adjuvant derived from bacteria has been necessary to improve immunogenicity, and several such vaccines administered intranasally have been evaluated clinically, with promising results. an intranasal spray formulation containing trivalent subunit influenza vaccine prepared from virosomes and wild-type escherichia coli enterotoxin was licensed briefly in switzerland. although it met the immunogenicity criteria set for yearly relicensing of conventional influenza vaccines, it was withdrawn after a possible association with bell's palsy could not be discounted. various microparticles are being investigated as adjuvants and delivery systems, for parenteral delivery of influenza-virus antigens or delivery to mucosal sites including the gut. subunit influenza vaccines have been prepared from recombinant haemagglutinin and neuraminidase proteins expressed in insect cells by baculoviruses. the recombinant haemagglutinins are well tolerated by young adults and elderly people, and there are significant doseresponse effects for both h1 and h3 haemagglutinin vaccines. phase i and virus-challenge studies of baculovirus-expressed recombinant neuraminidase in healthy volunteers have had promising results. the development of reverse-genetics techniques for negative-sense rna viruses has allowed the direct manipulation of influenza-virus gene products and creation of new recombinant viruses. this approach offers enormous potential for preparing interpandemic vaccines. nucleic-acid vaccines dna vaccines present a promising new approach to vaccination, evoking a full range of immune responses, including antibody, cytotoxic, and helper-t-cell responses. dna vaccines with constructs encoding the nucleoprotein (np), haemagglutinin, neuraminidase, matrix protein 1 (m1), and non-structural protein 1 of influenza virus have been studied extensively, either singly, in combination with one another, or together with dna encoding various cytokines. currently, two drug classes are available to manage influenza: the inhibitors of m2, amantadine and rimantadine, and the neuraminidase inhibitors, zanamivir seminar the lancet • vol 362 • november 22, 2003 • www.thelancet.com and oseltamivir. rimantadine causes less neurotoxicity than amantadine but is not available in most parts of the world and is not discussed further. amantadine inhibits the m2 membrane protein ionchannel activity of the influenza a virus but has no effect on influenza b. amantadine has three important limitations: its range of activity excludes influenza b; it has adverse side-effects, including insomnia, lightheadedness, hallucinations, dizziness, headache, and falls, which are particularly troublesome in elderly people; and drug resistance emerges rapidly during treatment. the genetic basis of resistance is a single nucleotide change, resulting in an aminoacid substitution at position 26, 27, 30, 31, or 34 in the membrane-spanning region of m2. estimates of amantadine's therapeutic effectiveness are uncertain owing to the clinical and methodological heterogeneity of clinical trials, a paucity of data by dose, the small number of trials in children and elderly people, and low trial-quality scores. 1 treatment of healthy adults with 100-300 mg daily of amantadine cuts the duration of fever compared with placebo by 1 day. 110 there are few data on use of the currently licensed dose in the uk, 100 mg daily. 1 at this dose, amantadine reduced the duration of fever compared with placebo by 1 day, but in a meta-analysis of data from six trials involving a total of 232 patients the effect did not attain statistical significance. 111 there is no high-quality evidence from randomised controlled trials of the effectiveness of amantadine 100 mg daily for the treatment of influenza in at-risk individuals, and illness was significantly shortened with treatment by 1·2 days in only one of two small randomised controlled trials in children. 1, 111 no randomised trial has tested amantadine during outbreaks in nursing homes. moreover, its use in this setting is complicated by toxicity, treatment failures, and frequent recovery of drug-resistant virus (about 32%). amantadine prophylaxis of other populations during interpandemic outbreaks is precluded by the lack of high-quality evidence from randomised controlled trials at the licensed dose and the high incremental cost per quality-adjusted life-year gained. 1 this second-generation neuraminidase inhibitor is a potent and specific inhibitor of a wide range of influenza virus types a and b. it has poor oral bioavailability and is delivered through an inhaler. zanamivir is licensed for the treatment of influenza a and b in people aged 12 years and over. it is well tolerated; the number, type, and severity of adverse events in healthy adults or people with stable chronic underlying medical disorders differ little from those with placebo. 112 the main safety concern is that inhaled zanamivir may cause bronchospasm. 113 however, respiratory viruses including influenza regularly exacerbate asthma and chronic obstructive pulmonary disease, so the role of zanamivir in bronchospasm is unclear. zanamivir was administered with apparent safety in two studies involving patients with asthma or chronic obstructive pulmonary disease. 114, 115 difficulty in using the inhaler may limit use of zanamivir. in one study, half of a very elderly group were unable to use the inhaler after training, and two-thirds zanamivir healthy individuals, aged 12-65 years 0·78 (0·26 to 1·31) 1·26 (0·59 to -1·93) 0·51 (-0·02 to 1·04) 0·46 (0·02 to 0·90) at-risk individuals, including older than 65 years 0·93 (-0·05 to 1·90) 1·99 (0·90 to 3·08) 0·09 (-0·78 to 0·95) 0·2 (-0·79 to 1·19) healthy children 1·0 (0·50 to 1·50) 1·0 (0·40 to 1·60) 0·5 (-0·30 to 1·30) 0·5 (-0·40 to 1·40) "all" individuals 0·94 (0·65 to 1·23) 1·26 (0·90 to 1·61) 0·37 (0·01 to 0·74) 0·37 (0·02 to 0·72) healthy individuals, aged 12-65 years 0·86 (0·31 to 1·42) 1·38 (0·79 to 1·96) 1·33 (0·70 to 1·96) 1·64 (0·69 to 2·58) at-risk individuals, including older than 65 years ϫ0·34 (-0·71 to 1·40) 0·45 (-0·97 to 1·88) 2·45 (0·05 to 4·86) 3·0 (0·13 to 5·88) healthy children 0·87 (0·25 to 1·49) 1·49 (0·76 to 2·20) 1·25 (0·70 to 1·80) 1·86 (1·06 to 2·65) "all" individuals 0·80 (0·41 to 1·18) 1·33 (0·90 to 1·77) 1·32 (0·91 to 1·73) 1·64 (1·17 to 2·10) itt=intention-to-treat. were unable to use it the next day. 116 however, in three other studies, it was used successfully by about 80% of more than 400 elderly people. 1 summary results, 1 which draw from published [117] [118] [119] [120] [121] [122] and unpublished treatment studies with zanamivir, are shown in table 4. overall, symptoms were alleviated sooner with zanamivir than with placebo-a median of 0·9 days on an intention-to-treat basis and 1·3 days for the influenzapositive subgroup. with zanamivir, the median time to return to normal activities was 0·4 days shorter for the treatment group, for both the intention-to-treat and influenza-positive populations. in a pooled analysis of intention-to-treat data from trials including both otherwise healthy and at-risk individuals, antibiotics were given to a smaller proportion of patients receiving zanamivir than of those assigned placebo (table 5). 123 similar, but nonsignificant, reductions in need for antibiotics in high-risk individuals and in pneumonia were seen with treatment in published 124 and unpublished 1 marginal analyses. seasonal prophylaxis with zanamivir 10 mg daily of mostly unvaccinated healthy adults provided an estimated 69% reduction in the incidence of laboratory-confirmed clinical influenza compared with placebo, 125 and metaanalysis of two randomised controlled trials of postexposure prophylaxis, with treatment given for 5 days 126 or 10 days, 120 suggested 81% protection against symptomatic laboratory-confirmed influenza. 1 oseltamivir this third-generation neuraminidase inhibitor is an orally active prodrug of oseltamivir carboxylate. it is licensed for the treatment of influenza a and b in people aged 1 year or older and for the prophylaxis of influenza a and b in people aged 13 years or older. the frequency of nausea is 3-7% higher and of vomiting up to 2% higher than with placebo; 127,128 these gastrointestinal side-effects can be ameliorated if the drug is taken shortly after food. summary results, 1 which draw from published [129] [130] [131] and unpublished treatment studies with oseltamivir (table 4) show that for all treatment groups combined, symptoms were alleviated sooner with oseltamivir than with placebo, by 0·8 days on the basis of intention to treat and 1·3 days for the influenza-positive subgroup. similarly, normal activities were resumed 1·3 days and 1·6 days sooner with oseltamivir for the intention-to-treat and influenzainfected group, respectively. treatment with oseltamivir reduces the frequencies of otitis media, antibiotic use, pneumonia, and hospital admissions. in children with influenza, the frequency of otitis media was 21% with placebo and 12% with oseltamivir. 131 the rate of antibiotic use in the intention-totreat population in one study was 3·4% (eight of 235) with placebo and 0·4% (one of 241) with treatment. 129 pooled marginal analyses showed lower rates of antibiotic use for lower-respiratory-tract complications in "healthy" and "high-risk" people with influenza with oseltamivir than with placebo; 1 a lower frequency of pneumonia in the influenza-positive group of ten studies (2% among placebo recipients vs <1% with oseltamivir; table 5); 1 and a significant reduction in the occurrence of hospital admissions in influenza-positive populations of ten trials (1·7% vs 0·7%). 1 three different strategies in preventing laboratoryconfirmed symptomatic influenza with oseltamivir have been investigated in randomised controlled trials. metaanalysis of data from two trials of seasonal prophylaxis in non-vaccinated healthy adults with oseltamivir, 75 mg once daily, 127 gave an estimate of 74% protection. 1 in households, postexposure prophylaxis with oseltamivir, 75 mg once daily for 7 days, gave 89% protection. 128 similarly, seasonal prophylaxis of mostly vaccinated elderly people receiving residential care with oseltamivir, 75 mg daily for 6 weeks, provided 91% protection. 132 resistance to neuraminidase inhibitors influenza viruses with low susceptibility to the neuraminidase inhibitors have been isolated in vitro and in vivo. resistance involves either a mutation in the active site of the neuraminidase, altering its sensitivity to inhibition, or a mutation in the haemagglutinin. mutations in haemagglutinin that confer drug resistance decrease the affinity of the protein for the cellular receptor, thus enabling virus to escape from infected cells without the need for viral neuraminidase. to date, few viruses with altered susceptibility to neuraminidase inhibitors have been recovered from patients. the first report of emergence of neuraminidaseinhibitor resistance (r152k) during treatment with zanamivir involved a recipient of a bone-marrow transplant. 133 during clinical trials with oseltamivir, 1·3% (four of 301) of post-treatment isolates from adults and adolescents and 8·6% (nine of 105) from children had low neuraminidase-inhibitor susceptibility, 134 indicating that such viruses are likely to emerge in clinical practice. three resistant variants with neuraminidase mutations (e119v, h274y, and r292k) that have emerged in clinical trials show low infectivity and virulence in animal models, thus the relevance of these mutations in clinical practice remains uncertain. in 1999, an international neuraminidase susceptibility network was established to oversee global surveillance of neuraminidase-inhibitor resistance. the uk national institute for clinical excellence (nice) has recently issued new guidance on the interpandemic use of antivirals for the treatment of influenza. 111 amantadine is not recommended. neither zanamivir nor oseltamivir is recommended for the treatment of influenza in children or adults unless they are at risk. within their licensed indications, zanamivir and oseltamivir are both recommended for the treatment of atrisk adults, and oseltamivir for the treatment of at-risk children, who present with influenza-like illness and can start therapy within 48 h of the onset of symptoms, when it is known that influenza a or b is circulating in the community. nice guidance on the use of antiviral drugs for the prevention of influenza was also issued lately. 135 oseltamivir is recommended for postexposure prophylaxis of influenza in at-risk people aged 13 years and older, who can begin prophylaxis within 48 h, if they live in a residential care establishment, whether or not they have been vaccinated, and a resident or staff member has influenza-like illness; or if they are not effectively protected by vaccination and can begin prophylaxis within 48 h of exposure. oseltamivir is not recommended for postexposure prophylaxis of healthy people up to age 65 years or for seasonal prophylaxis. amantadine is not recommended for either postexposure or seasonal prophylaxis. this guidance does not cover the circumstances of a pandemic. the hong kong "chicken flu" situation in 1997 and the rapid global spread of severe acute respiratory syndrome highlighted how ill prepared we are to introduce preventive measures for pandemic influenza. the problems encountered in 1997 were due mainly to the dangers of working with the chicken h5n1 virus and the need to produce a safe vaccine strain. conventional technology was unable to produce a safe productive vaccine strain. however li and colleagues 136 were able to modify the haemagglutinin gene of the a/hong kong/97 virus. they deleted the series of basic aminoacid residues at the cleavage site associated with virulence, then by use of reverse genetics rescued the modified haemagglutinin gene and the neuraminidase gene from the wild-type a/hong kong/97 virus into ca a/ann arbor/6/60 virus. the resultant ca virus was non-pathogenic in animal models of infection, grew well in eggs, and protected chickens from challenge with lethal virus; it could be a suitable candidate vaccine strain. these experiments show the potential for using reverse genetics technology to develop live and inactivated vaccines for both pandemic and interpandemic use. a second strategy for pandemic vaccine development is the use of recombinant haemagglutinin. however, the disappointing results from clinical trials of baculovirusexpressed haemagglutinin from the a/hong kong h5n1 virus, even after two doses of up to 90 µg, 137 question the role of this strategy alone. clinical trials of conventional inactivated-surface-antigen vaccine produced from an h5n3 virus showed that extremely poor antibody responses were stimulated, even after two doses, whereas an h5n3 subunit vaccine with mf59 adjuvant was much more immunogenic. 138 the benefit of adjuvants for use in naïve populations has also been shown with a whole-virus h2n2 vaccine with aluminium salts adjuvant. 139 thus, like mf59, aluminium salts have promise in increasing vaccine coverage in response to pandemic influenza by allowing scarce antigen to be used more efficiently. from the limited information available, conventional influenza vaccines seem not to be sufficiently immunogenic in a pandemic situation and two doses in conjunction with an adjuvant may be needed. different dosing strategies with various influenza-virus subtypes should be investigated so a robust strategy can be developed. vaccines will be in very short supply during the first stages of a pandemic, and antiviral drugs could have an important role in prevention. who has recently prepared draft guidelines for use of both vaccines and antiviral drugs during a pandemic; 140 they emphasise the need to stockpile drugs and to develop plans for their distribution and use. as with vaccines, there are gaps in our knowledge as to how antiviral drugs should be used. research is urgently required to ensure effective use of both vaccines and drugs in response to an emerging pandemic. kgn has received fees or honoraria from roche pharmaceuticals, wyeth, and berna biotech for speaking at meetings on influenza; research has been supported by chiron vaccines, aventis pasteur, wyeth, and berna biotech. solvay and berna biotech have provided h9 avian influenza viruses free of charge for a project on candidate pandemic vaccines. mz has received honoraria from berna biotech to speak at pharmaceutical-industry conferences on influenza and has been supported in attendance at an international who workshop on virus neutralisation sponsored by glaxosmithkline. the health protection agency has received funding from chiron vaccines, wyeth vaccines, aventis pasteur, roche, and glaxosmithkline, to carry out analytical work on a contractual basis in mz's laboratory. jmw has no conflicts of interest to declare in relation to this paper. none of these sources of funding had any role in the writing of this seminar. we received no funding in relation to this seminar other than a small payment from the lancet. learning from experience? uses of error reflecting on my medical errors over 35 years of clinical experience, i was disturbed to note that most errors that came to my mind were from the early part of my career. is this a sign that i really did become a better doctor or is it a symptom of ageing? were my early errors more influential on my subsequent practice or are my current errors not recognised or not made known to me by sympathetic colleagues? during my first house job, a middle-aged man was under my care for several months with an infected pleural cavity following a plombage operation for tuberculosis some years previously. he was memorable not only because of his sickness, but also because he was the first patient to give me a present, which i have to this day. 18 months later, i was a casualty officer at a teaching hospital. an intern showed me the chest radiograph of a man with a febrile illness and an opaque hemithorax. i lectured the intern on my patient with the infected plombage, not thinking for a moment that this story was directly relevant to the radiograph i was shown. the patient died of a cardiac arrest in the casualty department. the autopsy showed an infected plombage site and death from untreated septicaemia. 3 years after qualifying, while a locum physician in the northern parts of canada, i was required to do a coroner's post-mortem on a middle-aged man found dead in his cabin. fortunately, the policeman knew what to do and this overcame my anxiety. 2 hours later, i had no doubt that the man was dead, but knew neither why nor how. i dissected the heart and coronary vessels and decided they were atheromatous and that death was from natural causes, probably from myocardial ischaemia. in the coroner's court, i presented my conclusions and the case was closed. i received the pathologist's report as i was due to finish the locum . . . normal heart! being on-call for a unit and not just one's own patients can be an onerous task, particularly as the staff become more numerous and anonymous. in the past 5 years, i have been required to be on-call for a unit of ten consultants at two hospitals, one of which i visit only when on call. i was telephoned in the middle of the night about a patient who had had a cardiac arrest. i did not recognise the patient's name. i was told the arrest team wanted me to advise on their "enthusiasm" for resuscitation. i said that if she was not readily resuscitated, they should not resort to extreme measures, presuming she was a cancer patient admitted under the care of one of my oncology colleagues. on visiting the ward the next morning, i learnt that the patient who had died was a young woman under my care. (i had been given her first name and not her surname). she had been successfully treated for ovarian cancer 10 years previously and had been diagnosed with metastatic disease a couple of weeks earlier. she was neutropenic after the first chemotherapy treatment and a fatal outcome was not anticipated, particularly since she was being treated with curative intent. systematic review and economic decision modeling for the prevention and treatment of influenza a and b textbook of influenza the impact of influenza epidemics on mortality: introducing a severity index differing virulence of h1n1 and h3n2 influenza strains comparison of infection rates and severity of illness for influenza a subtypes h1n1 and h3n2 the tecumseh study of illness: xiii, influenza infection and disease, 1976-1981 the impact of influenza epidemics on hospitalizations impact of influenza and respiratory syncytial virus on mortality in england and wales from mortality associated with influenza and respiratory 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a/duck/singapore/97 (h5n3) vaccine: a randomized trial of two potential vaccines against h5n1 influenza pandemic preparedness: lessons learnt from h2n2 and h9n2 candidate vaccines key: cord-251979-j3mme15e authors: kandeel, amr; dawson, patrick; labib, manal; said, mayar; el-refai, samir; el-gohari, amani; talaat, maha title: morbidity, mortality, and seasonality of influenza hospitalizations in egypt, november 2007-november 2014 date: 2016-09-08 journal: plos one doi: 10.1371/journal.pone.0161301 sha: doc_id: 251979 cord_uid: j3mme15e background: influenza typically comprises a substantial portion of acute respiratory infections, a leading cause of mortality worldwide. however, influenza epidemiology data are lacking in egypt. we describe seven years of egypt’s influenza hospitalizations from a multi-site influenza surveillance system. methods: syndromic case definitions identified individuals with severe acute respiratory infection (sari) admitted to eight hospitals in egypt. standardized demographic and clinical data were collected. nasopharyngeal and oropharyngeal swabs were tested for influenza using real-time reverse transcription polymerase chain reaction and typed as influenza a or b, and influenza a specimens subtyped. results: from november 2007–november 2014, 2,936/17,441 (17%) sari cases were influenza-positive. influenza-positive patients were more likely to be older, female, pregnant, and have chronic condition(s) (all p<0.05). among them, 53 (2%) died, and death was associated with older age, five or more days from symptom onset to hospitalization, chronic condition(s), and influenza a (all p<0.05). an annual seasonal influenza pattern occurred from july–june. each season, the proportion of the season’s influenza-positive cases peaked during november–may (19–41%). conclusions: in egypt, influenza causes considerable morbidity and mortality and influenza sari hospitalization patterns mirror those of the northern hemisphere. additional assessment of influenza epidemiology in egypt may better guide disease control activities and vaccine policy. from november 2007-november 2014, 2,936/17,441 (17%) sari cases were influenzapositive. influenza-positive patients were more likely to be older, female, pregnant, and have chronic condition(s) (all p<0.05). among them, 53 (2%) died, and death was associated with older age, five or more days from symptom onset to hospitalization, chronic condition(s), and influenza a (all p<0.05). an annual seasonal influenza pattern occurred from july-june. each season, the proportion of the season's influenza-positive cases peaked during november-may (19-41%) . in egypt, influenza causes considerable morbidity and mortality and influenza sari hospitalization patterns mirror those of the northern hemisphere. additional assessment of influenza epidemiology in egypt may better guide disease control activities and vaccine policy. globally, acute respiratory infections (ari) are the fourth leading cause of death [1] and second highest cause of years of life lost [2] , and cause over 4.25 million deaths each year [3] . previous studies have indicated that influenza comprises a substantial portion of ari morbidity and mortality [4] [5] , with one estimate that 18% of global ari deaths were due to influenza infection in 2010 [6] and another that influenza causes 250,000-500,000 deaths each year [7] . however, little is known about influenza epidemiology in many parts of the developing world, particularly in the eastern mediterranean region (emr). the arab republic of egypt, the second most populous country of the emr, is a medium human-development country [8] located at the junction of northeastern africa and southwestern asia. egypt has a desert climate with hot, arid summers and moderate winters. the population is over 86.8 million, and 32% are under 15 years old [9] . in egypt, studies have evaluated limited aspects of human infection with avian influenza and the advent of the 2009 influenza pandemic [10] [11] [12] [13] [14] ; however, nationwide data on influenza morbidity, mortality, and seasonality are limited. resolving knowledge gaps in influenza epidemiology in egypt serves several purposes. first, a seasonal influenza vaccine is available each year that can prevent infections. recent u.s. estimates of the effectiveness of northern hemisphere influenza vaccines ranged from 19-60% [15] . even a moderately effective vaccine could reduce influenza-associated hospitalizations and lost worker productivity. understanding whether influenza strains match those in either northern or southern hemisphere circulation will elucidate whether northern or southern hemisphere seasonal influenza vaccine is a viable policy. additionally, data on whether demographic subpopulations are disproportionately affected by influenza may lead to designation of priority groups if vaccine availability is low. second, improved understanding of the timing of influenza circulation may result in a more cost-efficient timing of resource allocation (e.g., antivirals) for hospitals and other clinical settings. this could reduce financial burdens on hospitals during times of elevated influenza transmission. finally, enhanced data on annual influenza activity may provide a baseline metric to evaluate future activity. therefore, influenza epidemics may be detected earlier allowing public health officials to respond faster and have more opportunity to interrupt transmission. in 2007, the eastern mediterranean acute respiratory infection surveillance (emaris) network was established to initiate sentinel surveillance for severe acute respiratory infections (sari) to provide a better understanding of the epidemiology and viral etiologies of sari in the emr. the emaris network was formed through a collaboration of participating countries' ministries of health, u.s. centers for disease control and prevention (cdc), u.s. naval medical research unit no. 3 (namru-3), and world health organization (who) eastern mediterranean regional office. we describe seven years of influenza epidemiology from the emaris-egypt multi-site sentinel sari surveillance system. the aims of this study were to (1) assess the proportion of sari cases having influenza infection in egypt; (2) examine the types and subtypes of detected influenza viruses in egypt; (3) compare demographic and clinical characteristics of influenza-positive sari cases to those of influenza-negative sari cases in egypt; (4) quantify influenza deaths and assess influenza mortality risk factors in egypt; and (5) establish a defined period of influenza seasonality in egypt. a hospital surveillance team conducted sari surveillance at each site. these teams included a surveillance coordinator, internist, pediatrician, and laboratory focal person, each of whom was trained on case definitions, enrollment procedures, specimen collection, and data recording. teams screened all hospitalized patients with the sari case definition. all hospitalized patients (including weekend admissions) who met the case definition and were admitted between november 1, 2007 and november 30, 2014 were eligible for enrollment, and those who provided informed consent were enrolled and assigned a unique study identification (id) number. the case definition for identifying eligible patients was standardized across the emaris network. emaris participants reviewed the case definition each year, and it changed several times due to partner input and updated who guidance on sari: from 2007-2009, the 2006 who sari case definition was used [16] ; from 2010-2011, the case definition became any hospitalized patient 31 days or older meeting the 2006 who definition, or any patient meeting the cdc international emerging infection program pneumonia case definition [17] , or any patient suspected to have sari; from 2012-2014, the 2011 revised who sari case definition was used (any hospitalized patient with a history of fever (38°c) and cough in the past seven days) [18] , although clinicians could still enroll patients suspected to have sari; and finally, in 2014, the symptom history was extended to the past ten days to align with the 2014 revised who sari case definition [19] . each enrolled patient had an oropharyngeal and nasopharyngeal swab taken with a sterile tip flocked with nylon fiber swab applicator within 24 hours of hospital admission. all collected swabs from a patient were placed in a 15 ml tube with 2 ml viral transport medium and agitated vigorously for 10 seconds in a vortex mixer. the resulting supernatant was split into two cryovials pre-labeled with the patient's study id number and stored in -70°c freezers before testing. real-time reverse transcription polymerase chain reaction (rtrt-pcr) was conducted to detect influenza virus, types, and influenza a subtypes (determination of influenza b lineage was not done) [20] . specimens were first tested at the hospital or central public health laboratory (cphl) of the egypt ministry of health (moh), a recognized who national influenza center, and then at namru-3, a who regional reference laboratory, for quality assurance. namru-3 results were recorded as the gold standard final result by study id into microsoft excel (redmond, wa, usa). laboratory results were recorded as influenza negative, a/h1n1, a/h1n1pdm09, a/h3n2, a/h5n1, a/unsubtyped, or b. for analytical purposes, specimens positive for two or more influenza types were coded according to the first specimen listed of: a/h5n1, a/h1n1pdm09, a/h3n2, a/h1n1, b, a/unsubtyped. hospital surveillance teams completed a case report form including the unique study id and demographic and clinical data for each enrolled patient. no other identifying information was recorded. the form was standardized across the emaris network. teams followed each patient prospectively until discharge, transfer to another hospital, or death, and collected data including age, sex, reported history of fever and cough, date of symptom onset, date of hospitalization, and date of last follow-up (i.e., the date of discharge, transfer to another hospital, or death). sari cases and associated laboratory results were dated as the patient's hospital admission date. teams also abstracted data from medical records, such as chronic medical conditions and pregnancy status. each form was entered into microsoft access at moh and sent to a central database at namru-3. data managers performed double data entry and resolved any identified discrepancies amongst each other. the surveillance case report form database was merged with the laboratory result database, and the resulting database was cleaned and imported into sas 9.4 (sas institute, cary, nc, usa) for analysis. bivariate analyses of demographic and clinical data were conducted with the influenza laboratory result (positive versus negative) to generate counts and percentages and compare categorical levels by pearson's chi squared test. the odds of death among influenza-positive cases were modeled with logistic regression using different explanatory variables (age group: pediatric <15 years old versus adult 15 years old; sex: male versus female; days from symptom onset to hospitalization: 0-2 versus 3-4 versus 5; chronic conditions: at least one versus none; and influenza type: a versus b). statistical significance was set at an alpha level of 0.05. continuous variables were assessed individually to generate the median, range, and interquartile range (iqr). monthly influenza positivity rate was calculated as the proportion of specimens positive for influenza out of the total specimens tested for influenza each month. influenza seasonality was evaluated by examining the monthly average number and proportion of influenza-positive specimens. the influenza season was defined as beginning on the month having the lowest average influenza activity over the entire period and ending one year later. the month having the highest proportion of a season's influenza-positive specimens was considered that season's peak while the month having the lowest proportion was considered that season's nadir. namru-3 institutional review board (irb), cdc institutional review board, and egypt moh institutional review board approved the sari surveillance protocol in 2007 and maintained approval for the duration of the surveillance period. written informed consent was not required by the irbs due to the minimal risk faced by patients and because sentinel sari surveillance is part of egypt's national routine respiratory infection surveillance system of the egypt moh (the requirement was waived), but verbal informed consent was required. hospital surveillance teams provided consent information to patients both written and verbally in arabic, allowing adequate time for consideration. verbal informed consent was also obtained from the parents, caretakers, or guardians on behalf of all children/minors enrolled in the study. verbal informed consent was documented by the enrolling physician signing the consent form if the patient approved. the informed consent process for this study was approved by the three aforementioned institutional ethical review boards. from november 2007-november 2014, 17,441 patients met the sari case definition and had a specimen tested for influenza by rtrt-pcr (s1 file). of these, 2,936 (17%) were influenzapositive. a total of 2,013 (12%) were influenza a-positive and 923 (5%) were influenza b-positive. of the 2,013 influenza a-positive specimens, 115 (6%) were subtyped as seasonal a/ h1n1, 1,200 (60%) as a/h1n1pdm09, 674 (33%) as a/h3n2, 16 (1%) as a/h5n1, and 8 (<1%) as a/unsubtyped. demographic and clinical characteristics of sari patients by viral influenza result are shown in table 1 . influenza-positive patients differed from influenza-negative patients by age group (p<0.0001). among those with available data on age, 30% of influenza-negative patients were under five years old compared to 16% of influenza-positive patients. the median age of influenza-positive patients was higher than that of influenza-negative patients: 30 years (range 1 month-90 years, iqr 12-50 years) compared to 27 years (range 1 month-95 years, iqr 3-50 years). including missing data, influenza-positive cases were more likely to be female (and if female, be pregnant), have a preexisting chronic condition, require ventilation, be admitted to an intensive care unit (icu), and die (all p<0.0001). when missing data were excluded, statistically significant (p<0.05) differences persisted except for ventilation, icu admission, and death. the median duration from symptom onset to hospitalization among all patients was 3 days (iqr 2-4 among influenza-positive and 2-5 among influenza-negative), and this did not change by sex, age group, or both sex and age group. the median duration from hospitalization to end of follow-up among all patients was 5 days (iqr 4-6 among influenza-positive and 4-7 among influenza-negative), and this did not change by sex, age group, or both sex and age group. of the 2,936 influenza-positive cases, 53 (2%) died. influenza-positive deaths comprised 19% of sari deaths. among the 53 influenza deaths, 83% were 15-64 years old, 53% were male, and 55% had a preexisting chronic condition. twelve percent (3/25) of females who had influenza and died were pregnant. influenza a accounted for 83% of influenza-positive deaths, and of the 44 influenza a deaths, 70% were subtyped as a/h1n1pdm09, 18% as a/h5n1, 9% as a/ h3n2, and 2% as a/unsubtyped. the sari case fatality rates by influenza virus type were: 50% (8/16) a/h5n1, 13% (1/8) a/unsubtyped, 3% (31/1,200) a/h1n1pdm09, 1% (9/923) b, 1% (4/ 674) a/h3n2, and 0% (0/115) seasonal a/h1n1. the median duration from symptom onset to hospitalization and the median duration from hospitalization to end of follow-up were both 4 days among influenza-positive cases who died. factors associated with death among influenza-positive cases are displayed in table 2 . adjusting for all covariates, the odds of death among influenza-positive cases were significantly greater for adult versus pediatric patients, patients having symptoms for five or more days before hospitalization versus two or less days, patients with a preexisting chronic condition, and patients having influenza a versus b (all p<0.05). the calendar month with the lowest average number and proportion of influenza-positive cases was july, making july the influenza activity nadir. the influenza season in egypt was therefore defined as beginning in july and concluding in june. the calendar month having the highest average number and proportion of influenza-positive cases was december, making december the influenza activity peak. seasonal influenza activity peaks (fig 1) discussion this is the first study to examine national influenza epidemiology and seasonality in egypt. data from egypt's sari surveillance system were used to describe influenza hospitalizations at eight geographically-representative egyptian hospitals across a seven-year period. we found that approximately one of every six sari patients had an influenza infection, and during months of peak influenza activity, this was as high as one of every two. influenza-positive patients were different from influenza-negative patients in a number of ways, though differences regarding ventilation, icu admission, and death were inconclusive. death among influenza-positive patients was associated with four characteristics. we also found marked influenza seasonality beginning in july each year and ending in june. seasonal peaks occurred from november to may, and the peak month contained an average of 27% of the entire season's influenza infections. our findings indicate that influenza leads to substantial morbidity and mortality in egypt among people of all ages and both sexes. from 2007-2014, nearly 3,000 influenza hospitalizations were detected by the eight sentinel hospitals, approximately 17% of sari hospitalizations. this figure is nearly double those recently found in other emaris network countries: 8% in oman and 9% in jordan [21] [22] , and larger than estimates obtained in multiple african [5, 23] and asian countries [24] [25] . furthermore, influenza accounted for 19% of sari deaths in egypt. this mirrors the recent worldwide estimate of 18% [6] . comparison of demographic characteristics revealed that some individuals have increased odds of severe influenza infection in egypt. among the age groups encompassing 5-65 years, patients were more likely to have influenza. more influenza-negative patients were under five years old, possibly due to pediatric respiratory syncytial virus (rsv) infection which has been shown to be more prevalent among infants and young children in egypt [13, [26] [27] . a similar proportion of influenza-positive and influenza-negative patients were 65 years or older, possibly reflecting that elderly adults are simply predisposed to aris of various etiologies. other factors associated with influenza among those hospitalized with sari from this study include pregnancy and chronic conditions. influenza-positive women were more likely to be pregnant, and numerous other studies have identified pregnancy as a risk factor for influenza and influenza complications [28] [29] [30] [31] [32] . chronic conditions were also associated with influenza hospitalization, as 27% of influenza-positive patients had at least one. previous studies have shown that chronic disease is a risk factor for influenza and complications due to influenza [33] [34] [35] [36] . although 2% of patients with influenza died, there was no apparent difference in mortality between influenza-positive and negative patients. regardless, patients with influenza infection comprised 19% of total sari deaths. analysis of influenza mortality among hospitalized patients with influenza found four potential risk factors: older age, chronic conditions, a longer duration from symptom onset to hospitalization, and influenza a virus infection. overall, the number of deaths was greater for those with influenza a than influenza b, which aligns with previous research [37] [38] . the influenza subtype with the highest mortality (50%) was a/ h5n1 avian influenza. as of march 3, 2015, the worldwide a/h5n1 case fatality rate was 55% [39] , similar to our finding. furthermore, though 13% of patients with influenza a/unsubtyped died, there were only eight such infections. additionally, a longer duration from symptom onset to hospitalization increased the odds of death among influenza-positive patients. this supports previous research that found a longer duration from influenza symptom onset to hospitalization increased the odds of death [40] [41] , which may be due to individuals' healthcareseeking behavior, i.e., avoiding a hospital visit until it is too late for medical intervention. in addition to influenza hospitalization morbidity and mortality in egypt, we were able to assess seasonality over seven years. we observed an annual seasonal pattern beginning in july and ending the following june. overall influenza activity peaked in november-may, with the peak month having 19-41% of that entire season's influenza infections and accounting for 30-50% of all sari cases that month. these seasonal peaks were largely driven by influenza a, and in some seasons, there were bimodal influenza peaks corresponding to an earlier influenza a peak followed by a later influenza b peak. climate has been shown to correlate with influenza activity worldwide, particularly lower temperatures and lower relative humidity [42] [43] [44] . although egypt has a desert climate with year-round high temperatures [9], we observed annual northern hemisphere influenza seasonality with activity peaks in the winter and spring, perhaps due to lower temperatures during those months. based on these data, seasonal influenza vaccination in egypt may be a useful recommendation. the system identified predominant influenza strains each season [45] , indicating some seasonal influenza transmission could have been interrupted by vaccination. however, future studies are needed to antigenically characterize the predominant influenza strains in egypt to assess their similarity to strains included in the corresponding season's vaccine. possible priority groups for vaccination in egypt include pregnant women, individuals with chronic conditions, and older adults based on aforementioned findings. influenza seasonality in egypt suggests an ideal timing for resource allocation and provides a baseline metric on which to compare future activity. these data indicate that egyptian hospitals should prepare for peak influenza activity from november-may. stockpiling antiviral medication, increasing staff capacity, and reserving hospital beds could begin in october and be scaled back in may. also, moh and partners could use these data to determine average weekly influenza hospitalization activity (smoothed with moving averages) for evaluation of future activity. this will allow for earlier discovery of unusual influenza activity, including crossing of epidemic thresholds, allowing for a faster public health intervention. there were limitations to these data. first, the system's detection of influenza relied on the case definition's sensitivity and specificity, and the definition changed over time based on who guidance. it is possible the system missed some hospitalized individuals with influenza. however, the case definition was standardized across the network and the system detected a higher proportion of influenza sari hospitalizations than other countries. second, more comprehensive demographic and clinical data were unavailable, and thus more nuanced analyses of risk factors were not possible. this is a reality of surveillance as only basic data are collected to reduce staff burden. third, data were missing for a sizeable number of individuals for some variables. therefore, statistical comparisons were presented both including and excluding missing data, and some comparisons yielded divergent results (implying statistical significance was driven by the extent of missing data for some variables). work to maintain the quality and integrity of surveillance data through the emaris network is ongoing, including improving motivation among hospital surveillance teams. strengths of this study stem from its scale, length of observation, and capabilities to detect influenza virus. the use of eight sentinel hospitals representatively distributed throughout egypt allowed for adequate generalizability of findings to the national level. furthermore, with over 17,000 patients enrolled, there was ample power to detect meaningful differences in demographic, clinical, and virological characteristics. the seven-year period of surveillance afforded the opportunity to examine influenza seasonality over time, which is only possible with several years of data. finally, egypt's cphl and namru-3 enabled sensitive and specific detection of influenza viruses and types via rtrt-pcr for all 17,441 sari patients. this is the first documented examination of national influenza epidemiology in egypt. given the dearth of influenza knowledge in the emr, our study helps fill a critical epidemiologic gap for the second most populous country in the region. our findings indicate that influenza is an important cause of morbidity and mortality. furthermore, it appears to have northern hemisphere seasonality, though not as pronounced as in temperate climate countries. therefore, seasonal influenza vaccination may be a viable policy. ultimately, the emaris network provided numerous benefits, namely increased epidemiologic and laboratory capacity, enhanced international health regulations (2005) compliance, and a sustainable sari surveillance system. such a system is a crucial component of modern disease control, as evidenced by the recent appearance of middle east respiratory syndrome coronavirus [46] . it may also be adapted for other novel aris, which is imperative given growing concern over the emergence of new influenza viruses with pandemic potential [47] [48] . continued surveillance for influenza and further assessment of its molecular epidemiology will better inform decision-makers about disease control activities in egypt and the greater emr. supporting information s1 file. raw data. data file (sas format) for the severe acute respiratory infection (sari) sentinel surveillance system in egypt. (sas7bdat) the findings and conclusions in this reports are those of the authors and do not necessarily reflect the official policy or position of the department of the navy, department of defense, the centers for disease control and prevention, u.s. government, nor egypt ministry of health. patrick dawson and maha talaat are contractors of the u.s. government. mayar said is an employee of the u.s. government. this work was prepared as part of their official duties. title 17 uscx105 provides that "copyright protection under this title is not available for any work of the united states government." title 17 usc x 101 defines u.s. government work as work prepared by a military service member or employee of the u.s. government as part of that person's official duties. the study protocol was approved by the naval medical research center institutional review board in compliance with all applicable federal regulations governing the protection of human subjects. conceptualization: ak mt. data curation: pd. world health organization. global health observatory data repository: causes of death acute respiratory infections: making inroads against a forgotten pandemic global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis influenza surveillance in 15 countries in africa global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study united nations development programme age-based human influenza a virus (h5n1) infection patterns burden of pediatric influenza a virus infection post swine-flu h1n1 pandemic in egypt severe atypical pneumonia outbreak associated with influenza a(h1n1) pdm09 in egypt, 2013-2014 season risk factors of prolonged hospital stay in children with viral severe acute respiratory infections avian influenza a(h5n1) and a (h9n2) seroprevalence and risk factors for infection among egyptians: a prospective, controlled seroepidemiological study centers for disease control and prevention. seasonal influenza vaccine effectiveness world health organization. paho-cdc generic protocol for influenza surveillance centers for disease control and prevention international emerging infections program (ieip): thailand. description of clinical pneumonia world health organization. who global technical consultation: global standards and tools for influenza surveillance world health organization. global epidemiologic surveillance standards for influenza rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4 the burden of influenza-associated hospitalizations in oman influenza hospitalization epidemiology from a severe acute respiratory infection surveillance system in jordan sentinel surveillance for influenza-like illness, severe acute respiratory illness, and laboratory-confirmed influenza in kinshasa, democratic republic of congo incidence of influenza-like illness and severe acute respiratory infection during three influenza seasons in bangladesh estimating influenza outpatients' and inpatients' incidences from 2009 to 2011 in a tropical urban setting in the philippines viral etiologies of lower respiratory tract infections among egyptian children under five years of age incidence and clinical features of respiratory syncytial virus infections in a population-based surveillance site in the nile delta region risk of acute respiratory disease among pregnant women during influenza a epidemics fatal swine influenza pneumonia during late pregnancy hospitalizations with respiratory illness among pregnant women during influenza season severity of 2009 pandemic influenza a (h1n1) virus infection in pregnant women pregnant women infected with pandemic influenza a(h1n1)pdm09 virus showed differential immune response correlated with disease severity impact of epidemic type a influenza in a defined adult population survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston impact of respiratory virus infections on persons with chronic underlying conditions the burden of influenza illness in children with asthma and other chronic medical conditions the impact of influenza epidemics on mortality: introducing a severity index mortality associated with influenza and respiratory syncytial virus in the united states world health ogranization. cumulative number of confirmed human cases for avian influenza a(h5n1) reported to who infection and death from influenza a h1n1 virus in mexico: a retrospective analysis outcomes of adults hospitalised with severe influenza association of influenza epidemics with global climate variability influenza virus transmission is dependent on relative humidity and temperature seasonality, timing, and climate drivers of influenza activity worldwide available candidate vaccine viruses and potency testing reagents world health organization. interim surveillance recommendations for human infection with middle east respiratory syndrome coronavirus pandemic threat posed by avian influenza a viruses epidemiology, ecology and gene pool of influenza a virus in egypt: will egypt be the epicentre of the next influenza pandemic? the authors wish to thank all egypt ministry of health and central public health laboratory staff and hospital surveillance coordinators for their participation and continued excellence in sari surveillance. key: cord-007294-qeb2r08t authors: edmondson, william p.; rothenberg, richard; white, paul w.; gwaltney, jack m. title: a comparison of subcutaneous, nasal, and combined influenza vaccination. ii. protection against natural challenge(1)(2) date: 1971-06-17 journal: am j epidemiol doi: 10.1093/oxfordjournals.aje.a121282 sha: doc_id: 7294 cord_uid: qeb2r08t edmondson, w. p., jr., r. rothenberg, p. w. white and j. m. gwaltney, jr. (univ. of virginia school of. medicine, charlottesville, va. 22901). a comparison of subcutaneous, nasal, and combined influenza vaccination. ii. protection against natural challenge. amer j epidem 93: 480–486, 1971.—monovalent killed influenza a(2) hong kong vaccine in doses (400 cca units) recommended for civilian use was given to insurance company employees and elderly psychiatric patients by injection, nasal spray, or a combination of both methods. vaccinees and controls were then studied for evidence of immunity to influenza during the 1968–1969 epidemic parenteral vaccination was well tolerated and effective in reducing influenza infection and illness rates in both groups. vaccine had no effect on total respiratory illness in the insurance group, although total absenteeism was lowered because of the greater effect of influenza over that of colds in causing time lost from work. vaccine given by spray into the respiratory tract was ineffective. the addition of spray to parenteral vaccination provided no additional advantage over parenteral vaccination alone. and pharynx, or a combination of these methods. during the epidemic, vaccinees and controls were studied for clinical, virological, and serologic responses to natural influenza infection. parenteral vaccination was associated with protection from influenza infection and illness and with a reduction in industrial absenteeism. persons vaccinated by spray alone were not protected and had influenza infection and illness rates which were similar to rates in controls given type b influenza vaccine or to unvaccinated persons. the details of these findings are described below. details on the characteristics of the study populations, vaccine composition, and techniques of vaccine administration are reported in the accompanying paper (1) . measurements of vaccine efficacy were made only among members of the insurance company and psychiatric patient populations. as part of an ongoing respiratory virus study, each insurance company employee recorded symptoms daily on an ibm card. questionnaires filled out at the time of sampling were compared with the daily symptom records as a check on the accuracy of reporting. employees with new complaints were seen by a nurse-epidemiologist who collected specimens and personally contacted each employee weekly to encourage accurate symptom reporting. home visits were made to employees who were absent from work. surveillance of the elderly psychiatric patients was done by monitoring illness reports from the regular hospital staff combined with ward visits by a member of the research team. in addition to seeing patients with reported illnesses, an investigator visited each of the 47 wards weekly and recorded and sampled minor unreported illnesses. the following criteria were established to define the types of illnesses studied. febrile illness: feverishness and/or recorded body temperature above 99.9 f together with one or more respiratory symptoms or two or more systemic complaints lasting longer than one day. a febrile illness: three respiratory symptoms or two respiratory symptoms with two systemic complaints of two days or longer in the absence of feverishness or body temperature above 99.9 f. influenza specific illness: any febrile or afebrile illness in which influenza virus was recovered within six days of the onset or in which a fourfold or greater hemagglutination inhibition, neutralization, or complement fixing antibody rise was measured. minor differences in sampling rates of the study groups were adjusted by the formula: corrected number infected = (number infected/number tested) x number reported. the corrected number infected was used to derive attack rates. collection of specimens for virus isolations: specimens of respiratory secretions from insurance employees and elderly patients were tested fresh or were frozen rapidly and stored at ~70 c for later testing. all specimens were collected within six days of the initial complaint and refrigerated from the time of collection to processing in the laboratory (0-6 hours). influenza virus cultures were done on specimens collected from november 12, 1968, through february 14 (employees), or march 30, 1969 (patients). specimens were inoculated into three 9-10 days old embryonated eggs and three culture tubes of primary rhesus monkey kidney (mk) cells. negative specimens received two further passages. specimens from the insurance company employees were also inoculated into three tubes containing human diploid fibroblast (wi-38) cells as part of the ongoing study. influenza virus isolates from egg and monkey kidney cells were identified by a standard hemagglutination inhibition test (2) . rhinoviruses were identified by characteristic cpe and acid lability. the methods of performing serologic tests are reported in the previous paper (1). local reactions of three cm or greater occurred in less than 1 per cent of 263 elderly patients evaluated at 24 or 48 hours after vaccination. no vaccine-associated systemic complaints were reported by the elderly patients or the insurance employees. vaccine: insurance company employees occurrence of influenza: during december of 1968, a peak of respiratory illness occurred which exceeded by over 1.7 stand-ard deviations the six-year cumulative mean respiratory illness rate for the population at that season of the year (figure 1). this large excess of respiratory illness was associated with a high isolation rate of aa/ hk influenza virus. influenza infections were documented during the period november 27,1968 through january 7,1969. there was no laboratory evidence of influenza infections in the three weeks before or after this interval. the period of time chosen for the analysis of vaccine efficacy was the eight weeks from november 20 to january 17. infection and illness rates by vaccine status: the total number of illnesses (influenza and non-influenza) reported per group (table 1) were used to derive overall respiratory illness rates. there were 63 illnesses among the 190 unvaccinated employees, 24 among the 55 spray vaccinees, 26 among the 76 gun vaccinees, and 18 among the 55 employees receiving combined vaccination. this gave similar overall respiratory illness rates of 33.2, 43.6, 34.2, and 32.7/100 for these groups, respectively (figure 2). infections due to agents other than influenza virus thus accounted for a larger proportion of illness in the protected than in the unprotected groups. febrile and afebrile influenza illnesses were markedly reduced in employees receiving vaccine by gun and by gun and spray compared to those receiving no vaccine or spray alone (table 1). the total influenza illness rate for the unvaccinated group (17.9/100) was almost identical with that for spray vaccinees (18.0/100). these high rates contrast with total influenza illness rates of 1.9/100 and 4.7/100 for the gun and combined groups, respectively. febrile illnesses constituted the major portion of all influenza cases. among spray vaccinees and nonvaccinated employees, febrile influenza illness rates averaged approximately 12.5/100. other viral infections in insurance employees: non-influenza virus specific infection rates were 14, 10, 10, and 12 per 100 persons for the combined, gun, spray, and control groups, respectively. eighteen rhinoviruses, two herpes simplex, one mumps, one parainfluenza type 2, and five unidentified acid sensitive viruses were isolated during the study. twelve persons with documented influenza had one or more other illnesses, three of which yielded other viruses. seventy-four (55 per cent) of all illnesses were not associated with any agent and were presumably due to coronaviruses and/or as yet undiscovered viruses. reduction of industrial absenteeism: between november 26, 1968 and january 10, 1969, there were 27 working days. fortyone of the 245 (16.7 per cent) unvaccinated employees and spray vaccinees lost time from work due to influenza compared to six of 131 (4.6 per cent) gun plus gun and spray vaccinees (table 2) . also, vaccination resulted in a reduction in the total number of days lost from work due to any illness (respiratory and nonrespiratory). parenteral and combined vaccinees averaged 0.6 absentee days per person; unvaccinated employees and spray vaccinees averaged 0.9 absentee days per person. based on a net influenza rates: influenza illness rates in the control and spray vaccine groups were 14.7 and 12.2/100, respectively (table 3) . these rates were lower than those observed in the susceptible insurance employees but still considerably higher than the rates for elderly patients receiving parenteral or combined vaccination. these latter two groups had rates at or near 1.5/100. thus, parenteral administration of the a2/hk vaccine also provided-protection to elderly adults while spray vaccination was ineffective. unlike the young adults, the elderly patients had few minor non-influenzal respiratory illnesses during the time of the trial. sixty-six of 283 (23 per cent) persons had serologic evidence of influenza virus infection. (these numbers include 24 of 73 insurance employees, 33 of 104 psychiatric patients, and 9 of 106 medical students.) fifty-seven serology positive persons were cultured within six days of the onset of respiratory symptoms and 34 (60 per cent) were virus positive. sixteen parenterally vaccinated persons had serologic evidence of natural infection. they yielded virus at a similar rate (66 per cent) to those not receiving parenteral vaccine (62 per cent). while yearly immunization against influenza has been recommended for persons who are aged and chronically ill (3), little information has been accumulated which documents that these persons are significantly protected when such programs are undertaken (4). in the united states, evidence for the effectiveness of inactivated influenza vaccines has come largely from field trials in military populations (5), and questions have been raised about the justification for continued vaccine use in civilians (4) . several conditions appear to be necessary for successful testing of influenza vaccine. these include close and accurate surveillance, high sampling rates, and influenza attack rates which are sufficient to allow valid comparisons between control and vaccinated populations (6) . these conditions are often difficult to achieve in civilian groups where living conditions are less uniform than in the military. in the current work, advantage was taken of an efficient program of surveillance and sampling which was already in progress in one group. the trial also coincided with a sharp outbreak of influenza in which up to 20 per cent of unvaccinated controls experienced infection. under these conditions, parenterally administered vaccine gave substantial pro-tection from influenza to young, healthy adults and to elderly, debilitated psychiatric patients. total respiratory illness rates were not reduced in the industrial population although total illness absenteeism was, reflecting the greater morbidity of influenza over that of common endemic respiratory disease. immunization failures after parenteral vaccination were observed primarily in persons with limited antibody responses. the majority (67 per cent) of parenteral vaccinees developed hi antibody titers of 80 or greater. eight of the 11 vaccine failures occurred in insurance employees and elderly patients with post-vaccination titers of less than 80. at least three possibilities could explain the failure of the vaccine to reduce total respiratory illness in the insurance group. one is that influenza was prevented by the vaccine but was replaced by illnesses due to other prevalent respiratory viruses. a second possibility is that all groups suffered equally from colds, but the nonprotected employees had superimposed influenza which obscured these relatively minor illnesses. some support for this idea comes from the finding of three influenza infections in persons shedding a second respiratory virus. a third hypothesis is that vaccination interfered with the laboratory diagnosis of influenza. evidence against this was the finding that influenza virus shedding rates were not reduced in vaccinees with serologic evidence of natural infections. also, total respiratory illness rates were reduced for the protected elderly patients among whom colds were at a minimum. the causes of most non-influenzal infections in the protected groups were not discovered because of technical limitations of testing which prevented making an etiologic diagnosis in 65 per cent of cases. the undiagnosed illnesses were presumably due to "winter cold viruses" such as coronaviruses. a final answer to the question of why the vaccine did not affect total illness rates will have to await the time when the diagnosis of acute respiratory diseases is more complete. another goal of this trial was to extend observations on the effectiveness of influenza vaccine given by spray into the respiratory tract. a recent report of successful vaccination by this method (7) has stirred interest in its possible adoption for general use. in the current work, a standard dose of vaccine sprayed into the respiratory tract gave no protection when given alone and did not augment the effectiveness of simultaneously administered parenteral vaccine. the different results of this and the previous trial may be explained by the partial immunity which was present in the group studied earlier and by differences in methods of surveillance and sampling. nevertheless, respiratory tract antibody may be of primary importance in host defenses against influenza. if this is true, then the current resulte suggest that parenteral vaccination evokes secretory antibody more effectively than vaccination by spray when standard doses of vaccine are used. in conclusion, it is suggested that while vigorous efforts to improve influenza vaccines should be continued, the value of currently available products given parenterally should not be lost due to lack of enthusiasm for their use. addendum more recently, testing of serum pairs obtained during the trial has been done by means of a hemagglutination inhibition test using coronavirus strain oc43 antigen. nine coronavirus infections were documented among the participants in the study. a comparison of subcutaneous, nasal, and combined influenxa vaccination influenza virus the epidemiological basis for the control of influenza inactivated influenza virus vaccines, past, present, and future immunization against influenia prevention of illness in man by aerosolized inactivated vaccine key: cord-258270-67f5z8et authors: he, biao; zheng, bo-jian; wang, qian; du, lanying; jiang, shibo; lu, lu title: adenovirus-based vaccines against avian-origin h5n1 influenza viruses date: 2014-12-03 journal: microbes infect doi: 10.1016/j.micinf.2014.11.003 sha: doc_id: 258270 cord_uid: 67f5z8et since 1997, human infection with avian h5n1, having about 60% mortality, has posed a threat to public health. in this review, we describe the epidemiology of h5n1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based h5n1 systemic and mucosal vaccines. influenza a viruses are notorious for antigen drift and shift for segmented negative-sense rna genomes, and they are named according to their two surface proteins: hemagglutinin (ha) and neuraminidase (na), with 18 and 11 subtypes, respectively [1] . before 1997, human infection by influenza virus was confined to h1, h2 and h3 subtypes, according to records. however, the first recorded human infection of avian influenza virus occurred in 1997 in hong kong, which was caused by h5n1 virus. the causal h5n1 was a reassortant with hemagglutinin (ha) from goose h5n1 and seven viral genes from other avian influenza viruses [2] . pathogenetic and sequence analysis revealed that h5n1 belonged to the group of highly pathogenic avian influenza viruses (hpai) based on its lethality to chickens and polybasic amino acids at the cleavage site of ha, which allowed ha processing by ubiquitously expressed proteases [3, 4] . since then, the hpai h5n1 subtype has caused numerous outbreaks in poultry and more than 700 human h5n1 infection cases with about 60% mortality (http://www.who.int/influenza/human_animal_interface/ en_gip_20140727cumulativenumberh5n1cases.pdf? ua¼1). moreover, similar to seasonal influenza viruses circulating in humans [5] , the hpai h5n1 subtype is genetically highly variable, and it has diversified into multiple phylogenetic clades over the past decade [6] . hence, the development of vaccines against h5n1 influenza viruses is challenging. the asian h5n1 virus, which killed some geese, was detected in guangdong province, china, in 1996 [7] . in 1997, the hpai virus subtype h5n1 caused disease in 18 patients with six deaths in hong kong [3] . exposure to live poultry a week before the onset of illness was associated with disease in humans. the culling of all poultry in hong kong ended the first wave of h5n1, but the virus continued to circulate among avians. no further human cases occurred until 2003, when two hong kong residents contracted the disease, one of whom died [8] . the second wave of the asian h5n1epidemic occurred in the winter of 2003/04 in eight asian countries, including china, cambodia, indonesia, japan, korea, lao people's democratic republic, thailand and vietnam. the impact was particularly severe in thailand and vietnam, where widespread disease was reported in poultry, as well as human case fatalities [9] . from 1997 to may 2005, h5n1 viruses were largely confined to southeast asia, but after they had infected wild birds in qinghai lake, china, they rapidly spread worldwide [10, 11] . even though sustained human-to-human transmission of influenza a(h5n1) has not yet been described, it has been suggested in several household clusters [12e14]. in addition, several studies showed that airborne transmission of h5n1 can be achieved through adaptation in experimental tests [15e18] . therefore, h5n1 remains a critical public health concern. vaccines are the most effective way to fight against influenza pandemic. however, h5n1 poses a particular challenge to conventional inactivated influenza virus-based vaccine as a result of its poor immunogenicity [19, 20] . even though live attenuated influenza vaccines (laiv) are attractive for their ability to mimic the natural route of infection by which both strong systemic and mucosal immunity can be induced, laiv is only licensed for healthy people from 2 to 49 years of age, excluding high-risk human groups. furthermore, reversion to virulence during production may occur, and reassortment with circulating influenza viruses may happen [21] . dnavaccine can be made rapidly and can be produced in large quantities; however, dna vaccine alone is poorly immunogenic, and specific instrumentation is needed during immunization. virus-like particles (vlps)-based vaccines antigenically mimic native virions with intact biochemical activities. khurana et al. showed that two doses of h5n1 vlps at 15, 45, or 90 mg ha/dose induced hemagglutination inhibition (hai), seroconversion rates of 40%, 57%, and 61%, and microneutralization (mn) of 39%, 52%, and 76%, respectively, in human [22] , indicating that h5n1 vlp is, like h5n1 inactivated vaccine, poorly immunogenic. even though subunit vaccines serve as a convenient method of generating large amounts of influenza vaccines for a potential pandemic, properties such as antigenicity and immunogenicity are indeed challenging [23] . however, adenovirusbased h5n1 vaccines are attractive based on selfadjuvanticity, rapid production by the egg-independent method, and administration by various routes. 3. the production of adenovirus-based vaccine and the relationship between adenovirus and the immune system adenoviruses are non-enveloped icosahedral, doublestranded dna viruses. a targeted gene can be inserted into an adenovirus genome and expressed after recombinant adenovirus (rad) infection (fig. 1) . based on the deletion of adenovirus genome sequence, three generations of rad are established. in the first generation, replacement of the e1 or e1/e3 gene of the adenovirus genome with a gene encoding an antigen of interest renders virus replication incompetent, but able to be produced and propagated in hek 293 or per.c6 cells, allowing for expression of the gene of interest by the recombinant virus upon infection [24] . in order to increase cloning capacity, prolong gene expression, and reduce toxicity associated with adenovirus backbone gene expression, the second generation of rad was developed by further deletion of e2 or e4 gene stably expressed in the production cells [25] . the third generation of rad is helper virus-dependent and lacks nearly all viral encoding sequences, except packaging signal and two inverted terminal repeats (itrs), enlarging cloning capacity, but reducing immunogenicity for gene therapy [26] . however, the first generation of rad is widely used for its ease of manufacture, including production and purification, compared to the other two generations. an important advantage for adenovirus-based vaccine is its adjuvanticity by stimulating innate immune response. adenoviruses can infect various types of cells, such as epithelial cells, macrophage cells and dendritic cells. as shown in fig. 2 , upon infection, adenoviruses activate innate immunity in a toll-like receptor (tlr)-dependent manner, as well as tlrindependent pathways, causing upregulation of type i interferons (ifns) and inflammatory cytokines [27] . it was reported that neutralizing antibodies to type i ifns could block innate and adaptive immune responses to adenoviral vectors, which showed that the induction of type i ifns plays an important role in the adjuvanticity of adenoviruses [28] . after endocytosis of adenovirus, the genome can be recognized by tlr9, which leads to the activation of interferon regulatory factor 7 (irf7) that promotes the induction of type i ifns [29] . cytosolic adenovirus genome can be recognized by dna sensor cyclic guanosine monophosphateeadenosine monophosphate (cgamp) synthase (cgas) and another unknown dna sensor that leads to activation of irf3 and irf7 [30, 31] , respectively, adding to the induction of type i ifns to promote both cellular and humoral immune response. since revealing the low immunogenicity of h5n1 inactivated vaccine, adenovirus-based h5n1 vaccines have been widely investigated (table 1 ). in 2006, gao et al. first reported that adenovirus serotype 5-based vectors, which expressed codon-optimized hemagglutinin (ha) gene from a/vietnam/ 1203/2004(h5n1) influenza virus were prepared within 36 days after acquiring the virus sequence. intramuscularly vaccinated mice were fully protected from challenge with the homologous virus. in addition, chickens, which had played a critical role as mediator in transmitting h5n1 to human, were completely protected from lethal challenge after a single subcutaneous immunization of the adenovirus-based h5n1 vaccine [32] . later, it was reported that a single-dose in ovo vaccination with a human adenovirus vector encoding an h5n9 avian influenza virus ha not only fully protected against homologous virus, but also provided 68% protection against h5n1, demonstrating that adenovirus-based h5n1 vaccine could be administered en masse, using available robotic in ovo injectors [33] . the combination of different has in adenovirus-based h5n1 vaccine was also studied. hoelscher et al. revealed that mice vaccinated with two adenovirus vaccines encoding hemagglutinins from clade 1 and clade 2 of h5n1 viruses were protected against both viruses with minimal interference against each other [34] . in addition, adenovirus-based multivalent vaccines encoding hemagglutinins from h5, h7 and h9 were broadly protective, and significantly high levels of ha stalk-specific antibodies were induced following immunization with the multivalent vaccines [35] . scallan et al. reported that vaccination with h5n1 adenovirus-based vaccine by the oral route protected mice and ferret upon lethal h5n1 challenge [36] . in addition, cross-clade neutralizing antibodies were induced in ferrets immunized by the peroral route. it was also reported that dna prime-adenovirus boost vaccinations based on np and m2 from h1n1 were superior to trivalent live attenuated influenza vaccines in protection against heterosubtypic h5n1 influenza challenge [37] . these cited studies show that adenovirus-based h5n1 vaccines are effective in preclinical research. clinical application of adenovirus-based h5n1 vaccine was also investigated. a phase i clinical study of h5n1 vaccine based on recombinant adenovirus serotype 4 showed that orally administered vaccine induced significant specific t cell response. after one boost with inactivated h5n1 vaccine, 80% for hai and 67% for mn were induced in those vaccinated [38] . antrobus et al. reported that a replication-deficient chimpanzee adenovirus-vectored vaccine expressing the conserved influenza antigens np and m1 was safe and immunogenic in human [39] . even though adenovirus-based h5n1 vaccines can induce systemic immunity, most human populations have pre-existing immunity to human adenovirus, and it is believed that preexisting adenovirus neutralizing antibodies (vector immunity) may negatively impact the immune response to vaccine antigens when delivered by human adenovirus vectors [40] . in naive mice immunized with human adenovirus vector expressing h5n1 ha and boosted with bovine adenovirus vector expressing h5n1 ha, singh et al. reported that the humoral responses were significantly higher than those with either alone [41] . however, immunity against non-human adenovirus will be induced after immunization with nonhuman adenovirus, a fact which should be taken into consideration for further immunization. pandey et al. reported that pre-existing immunity to adenovirus can be overcome by either increasing the vaccine dose or using alternate routes of fig. 2 . three signal pathways of ifn-a and -b production induced by adenovirus stimulation. in the endosome, the genome of adenovirus can be recognized by tlr9 which leads to irf7 activation in myd88-and ikka-dependent pathways. in addition, the genome of adenovirus in cytoplasm can be recognized by cgas and a suggested unknown dna sensor which leads to the activation of irf3 and irf7, respectively, both leading to the production of ifn-a and b. [38] vaccination in mice [42] . however, whether these approaches will work in human needs further investigation. mucosal immunization via nasal route could induce both cellular and humoral immune response generating not only mucosal cytotoxic lymphocytes and igg, but also siga (secretory iga), which effectively prevented penetration of pathogen inside the cells and its dissemination throughout the human body [43e45]. however, mucous membranes of the upper respiratory tract exhibited low epithelium permeability [46] , thus, free antigens, such as proteins and peptides, are unable to induce strong immune response. adenovirus-based vaccines are indeed satisfactory in mounting mucosal immune response for high transduction efficiency in various cell types, such as epithelial cells or dendritic cells, and the activation of innate immune response [47] . adenovirus-based mucosal vaccines have achieved promising results. hoelscher et al. showed that intranasal immunization with adenovirus serotype 5 expressing h5ha induced both humoral and cell-mediated immunity against h5ha, especially interferon-gamma-secreting cd8 þ t cell response [48] . in order to elicit high levels of potent and durable humoral and cellular responses in the lower airways, song et al. demonstrated that aerosolized recombinant adenovirus encoding h5ha completely protected ferrets against h5n1 [49] . the results of adenovirus vaccine-based influenza conserved protein are encouraging. park et al. reported that mucosal immunity induced by adenovirus-based h5n1 vaccine, including h5ha and two conserved influenza proteins, m1 and m2, confers protection against a lethal h5n2 avian influenza virus challenge [50] . importantly, a single intranasal, but not sublingual, immunization of adenovirus expressing np from h1n1 provided 100% protection against h1n1, 100% protection against h3n2, and 40% protection against h5n1 [51] . also, a single-dose mucosal immunization with adenovirus vaccine encoding np and m2 provided rapid and full protection against virulent h5n1, h3n2 and h1n1 viruses [52] . these results suggest that adenovirus-based vaccines can serve as a platform for ha and influenza conserved proteins through mucosal immunization [53] . adenovirus-vectored nasal vaccines can also bypass preexisting immunity against adenovirus, a weakness of all viral vectors [42] . in addition, intranasal immunization of adenovirus-vectored vaccines through the use of a nasal spray would be suitable for mass vaccination by its simplicity and practicality. however, clinical data of mucosal adenovirus h5n1 vaccine in human is not yet available. since 1997, human infection of avian influenza virus other than h5n1 has also been reported [54] . however, h5n1 continues to play a major role in human infection of avian influenza viruses for its high mortality rate. in addition, given its widespread presence among avians, the potential of human-to-human transmission cannot be ignored. vaccination is the most effective way to prevent a potential h5n1 pandemic. by its avian origin, high mortality rate, and poor immunogenicity, h5n1 vaccines based on traditional methods, such as those used against seasonal influenza, are unsatisfactory. therefore, new vaccination methods are needed to compensate for the weaknesses of inactivated h5n1 vaccine. adenovirus-based h5n1 vaccines are an attractive alternative since they (i) serve as an adjuvant for h5n1 by activating innate immunity, (ii) are egg-independent, and (iii) can be administered by various routes, especially the nasal route, which endows the vaccine with strong mucosal immunity and the convenience of application for mass vaccination. therefore, adenovirus-based h5n1 vaccines, especially mucosal vaccine, may be an effective weapon against a new outbreak of h5n1. however, problems, such as pre-existing immunity to human adenovirus and the question of whether immunity against non-human adenovirus after immunization with nonhuman adenovirus in human will impede further immunization, should be taken into account, and other strategies should be developed. we have previously reported that a recombinant adeno-associated virus (aav) encoding receptor binding domain of sars-cov spike protein induced sars-covspecific igg antibody with neutralizing activity [55, 56] . other studies suggested that activation of the immune system by the transgene product following aav-mediated gene transfer might be easier to control than that following adenovirus-mediated gene transfer, indicating that aav-based vaccines lead to mild immune response [57] . therefore, the combination of adenovirus and aav in h5n1 vaccine may result in a synergy that achieves an optimal immune response. adenovirus-based vaccines may also apply to the new emerging viruses, such as h7n9 and mers-cov [58, 59] . like h5n1, h7n9 is of avian origin [60, 61] , which indicates that an egg-independent vaccine should be developed. in addition, h7n9 causes more serious infection in older people who respond less effectively to vaccines; thus, immunization with adenovirus-based h7n9 may be more efficacious in persons above the age of 65. an adenovirus-based mers vaccine, especially the rbd region [62] , may be effective and, most importantly, can be administered through both intramuscular and intranasal immunization. in conclusion, adenovirus-based vaccines are promising for both new emerging and reemerging viruses. collaborative programs (201200007673) and national grand program on key infectious disease control (2012zx10001008-002). the funders have no role in the design, implementation, interpretation, or publication of study. bat-derived influenza-like viruses h17n10 and h18n11 characterization of the influenza a virus gene pool in avian species in southern china: was h6n1 a derivative or a precursor of h5n1? characterization of avian h5n1 influenza viruses from poultry in hong kong receptor binding and transmission studies of h5n1 influenza virus in mammals 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viruses research and development of universal influenza vaccines influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than a(h5n1) recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus s protein elicits neutralizing antibodies: implication for developing sars vaccines priming with raav encoding rbd of sars-cov s protein and boosting with rbd-specific peptides for t cell epitopes elevated humoral and cellular immune responses against sars-cov infection successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors the outbreak of avian influenza a (h7n9) in china: current status and future prospects middle east respiratory syndrome coronavirus (mers-cov): challenges in identifying its source and controlling its spread genomic signature and protein sequence analysis of a novel influenza a (h7n9) virus that causes an outbreak in humans in china genetic tuning of avian influenza a (h7n9) virus promotes viral fitness within different species receptor-binding domains of spike proteins of emerging or re-emerging viruses as targets for development of antiviral vaccines comparative efficacy of hemagglutinin, nucleoprotein, and matrix 2 protein gene-based vaccination against h5n1 influenza in mouse and ferret matrix protein 2 vaccination and protection against influenza viruses, including subtype h5n1 partial protection against h5n1 influenza in mice with a single dose of a chimpanzee adenovirus vector expressing nucleoprotein new pre-pandemic influenza vaccines: an egg-and adjuvant-independent human adenoviral vector strategy induces long-lasting protective immune responses in mice a porcine adenovirus with low human seroprevalence is a promising alternative vaccine vector to human adenovirus 5 in an h5n1 virus disease model vaccination focusing immunity on conserved antigens protects mice and ferrets against virulent h1n1 and h5n1 influenza a viruses pre-clinical evaluation of a replication-competent recombinant adenovirus serotype 4 vaccine expressing influenza h5 hemagglutinin multi-antigen vaccines based on complex adenovirus vectors induce protective immune responses against h5n1 avian influenza viruses this study was supported by the national natural science the authors have declared that no conflict of interests exist. key: cord-011438-imbpgsub authors: zhang, yun; xu, zhichao; cao, yongchang title: host–virus interaction: how host cells defend against influenza a virus infection date: 2020-03-29 journal: viruses doi: 10.3390/v12040376 sha: doc_id: 11438 cord_uid: imbpgsub influenza a viruses (iavs) are highly contagious pathogens infecting human and numerous animals. the viruses cause millions of infection cases and thousands of deaths every year, thus making iavs a continual threat to global health. upon iav infection, host innate immune system is triggered and activated to restrict virus replication and clear pathogens. subsequently, host adaptive immunity is involved in specific virus clearance. on the other hand, to achieve a successful infection, iavs also apply multiple strategies to avoid be detected and eliminated by the host immunity. in the current review, we present a general description on recent work regarding different host cells and molecules facilitating antiviral defenses against iav infection and how iavs antagonize host immune responses. influenza a virus (iav) can infect a wide range of warm-blooded animals, including birds, pigs, horses, and humans. in humans, the viruses cause respiratory disease and be transmitted by inhalation of virus-containing dust particles or aerosols [1] . severe iav infection can cause lung inflammation and acute respiratory distress syndrome (ards), which may lead to mortality. thus, causing many influenza epidemics and pandemics, iav has been a threat to public health for decades [2] . the virus is an enveloped, segmented, negative-strand rna virus, belonging to the orthomyxoviriae family. the eight viral gene segments encode as many as 18 proteins. besides polymerase basic 1 (pb1), pb1-n40, pb1-f2, pb2, polymerase acid (pa), hemagglutinin (ha), nucleoprotein (np), neuraminidase (na), matrix 1 (m1), matrix 2 (m2), nonstructural protein 1 (ns1) and ns2 (also known as nuclear export protein, nep), new viral proteins were recently uncovered, such as pb2-s1 [3] , pa-x (product of ribosomal frameshifting) [4] , pa-related proteins pa-n155 and pa-n182 [5] , m42 [6] , and ns3 [7] . ha, na, and m2 proteins constitute surface of the iav virion, where ha is the most abundant surface protein. according to the genetic and antigenic diversity of the ha and na proteins, iavs were divided into 18 ha and 11 na subtypes. h17n10 and h18n11 subtypes were recently identified in bats [8, 9] . ha is a type i glycosylated protein, which is responsible for virus entry to host cell. functional ha protein is a homotrimer structurally composed of a stem region and a globular head region in each monomer. the head region bearing n-acetylneuraminic acid (sialic acid, sa) binding pocket is critical for receptor attachment, and contains most antigenic determinants. the stem region undergoing conformational changes is responsible for low ph-triggered membrane fusion [10] , and plays an important role in cross protection against heterosubtypic iav infection [11] . n38 glycan at this region iavs can infect a broad spectrum of host species, including both wild and domestic birds, as well as many mammalian species. the virus is capable of interspecies transmission to new species. however, no interspecies transmission of the bat iavs has been reported so far [54] . furthermore, the high frequency of mutations and recombination increases the risk of iav adaptation in humans. besides three pandemic subtypes (h1n1, h2n2, and h3n2), other subtypes, including h5n1, h5n6, h7n7, h7n9, n9n2, and h10n8 could cross the species barrier and cause human infections [55] [56] [57] [58] . several effect factors are essential in iav host switch events, including the receptor-binding properties of ha [16] , as well as cellular receptors [59] [60] [61] [62] . long and his colleagues summarized the role of host factors in iavs adaption to humans, and the review is recommended here for further reading [63] . noteworthy is the fact that most phylogenetically diverse iavs with different origins could successfully replicate in swine [64] . since pigs have both sa α-2,6 and sa α-2,3 galactose receptors [65] , they can serve as a suitable mixing reservoir for both human and avian iavs, thus raising global concern on periodic zoonotic infections. take the emergence of influenza a (h1n1) pdm09 (ph1n1) and influenza a (h3n2) a/canada/1158/2006 strains for instance, both strains are swine-origin iavs and were the consequence of adaption and reassortment of several swine lineages [66, 67] . furthermore, some genes of these strains originated from avian iavs [68] . with the development of gene sequencing technology, machine learning (ml) facilitated with large genomic datasets are used in prediction about sequence changes in newly invaded viruses from other animal hosts, take the "batch-learning self-organizing map (blsom)" method for instance [69] . ml is also applied in characterization of distinct host tropism protein signatures [70] , and prediction of amino acid changes for interspecies transmission [71] . these studies provided measures in identification of potential high-risk strains. in addition, the nucleotides and dinucleotide compositions of viruses play important roles in prediction of viral host species [72] . combining gene sequencing technology viruses 2020, 12, 376 4 of 23 and ml methods, researchers applied large iav genomic datasets to analyze species selection bias of iav mono-/dinucleotide composition and predict human-adaptive swine or avian iavs [73, 74] . the application of multi-disciplinary subjects would provide useful information for prediction of pandemic influenza. in general, the life cycle of the iav is generally divided into four steps: virus entry into the host cell, transcription and replication of the viral genome, assembly, and virus budding. though alveolar epithelial cell is the primary target cell for iavs, different iav subtypes have different patterns of viral attachment (pva). for human iavs, alveolar type ii epithelial cells, as well as immune cells such as alveolar macrophages and dendritic cells, are major target cells for an established infection [75, 76] . two seasonal iavs and pandemic h1n1 virus, preferred to attach to ciliated epithelial cells and goblet cells in the upper respiratory tract (urt), and avian iavs, take h5n1 for instance, attached seldom to these cells [77] . in the lower respiratory tract (lrt), human iav h1n1 and h3n2 attached to more cell types than avian iav h5n1, a highly pathogenic avian iav (hpaiv) strain. however, h5n1 could bind to type ii pneumocytes [78] . considering the fact that metabolism in the type ii pneumocytes is quite active, infection of hpaivs is more likely to cause severe pneumonia [78] . other research on low pathogenic avian iavs (lpaivs), which generally do not cause severe pneumonia, showed that these viruses usually attach to human submucosal gland cells, thus can be cleared by the mucus [79] . iav infection starts from recognition of sa by ha protein, though in vitro research claimed that these n-linked glycans were not essential for virus entry [80] . the cleavage of ha precursor protein ha0 into ha1 (containing receptor binding domain) and ha2 (containing fusion peptide) in low ph environment during ha transport is critical for virion internalization [81] . some research showed that type ii transmembrane serine protease such as transmembrane protease serine 2 (tmprss2), human airway trypsin-like protease (hat), transmembrane protease serine 4 (tmprss4), homo sapiens serine protease desc1 and homo sapiens transmembrane protease, serine 13 (mspl) can cleave human and avian iav ha proteins at an arginine residue [82] . in addition, for avian iavs, ha0 of hpaivs can be cleaved by subtilisin-like protease, while that of lpaivs is cleaved by trypsin-like proteases [83] or thrombin [84] . therefore, in avian iavs, the cleavage sites are considered to be the major determinants for virus virulence [85] , and rna folding in the cleavage region could be an important factor for virulence determination [86, 87] . proteins in the vrnp complex contain different nuclear localization signals (nlss), thus helping the vrnp complex to enter the host cell nucleus via active transport, take the crm1-dependent pathway for instance [88] . the acidic environment of the endosome also activates m2 ion channel, hence acidifies the viral core, resulting in entrance of vrnp complex into the host cell [34] . replication of viral genome does not require a primer but a full-length complementary rna (crna), which is essential for the newly formed vrnp complex. the viral rna polymerases first bind to the 3 end and the 5 end of the segmented viral rna and crna, respectively, then start replication with the help of the 5 cap of host pre-mrnas via a pb1-pb2-mediated "cap snatching" mechanism [27, 89] . the conserved segment-specific nucleotides at the 3 and 5 ends of the viral genome could modulate genome expression and replication during infection [90] . in addition, dephosphorylation at a specific position of the h1n1 ns1 protein results in attenuated virus replication [91] . mature viral mrnas are transported to the cytoplasm by a "daisy-chain" complex and translated subsequently [88, 92] . new synthesis of ha occurs on the rough endoplasmic reticulum (er). glycosylation and palmitoylation of the protein are completed later in the golgi [93, 94] . after synthesis and maturation of na and m2 proteins, the trans-golgi network (tgn), together with coat protein i (copi) complex and gtpase rab proteins, transport the newly synthesized ha, na, and m2 proteins to the apical plasma membrane (pm). these proteins then assemble with viral genomic segments. the virions are finally closed and m1 and m2 proteins mediate virion budding from the apical side of the viruses 2020, 12, 376 5 of 23 cells [28, [95] [96] [97] [98] . na protein cleavages the sa residues, which allows the virions to be released from the plasma membrane [99] . since iav has a relatively small genome, host machinery is required in order to accomplish the viral life cycle. to uncover host dependency factors that are necessary for iav replication, numerous large-scale rna interference (rnai) screens and genome-wide crispr/cas 9 screen were performed [29, [100] [101] [102] [103] . for instance, son dna binding protein was important for iav virion trafficking in an early infection stage and cdc-like kinase 1 facilitated aiv replication [29] . usp47 facilitated viral entry, whereas tnfsf12 (april) and tnfsf12-tnfsf13 (twepril) helped with viral replication [101] . using genome-wide crispr/cas9 screen, several genes of sialic acid biosynthesis and related glycosylation pathways were involved with h5n1 infection [102] , and wdr7, ccdc115, and tmem199 were essential for viral entry and regulation of v-type atpase assembly [103] . furthermore, single-cell transcriptome sequencing (rna-seq) was applied to explore host-virus interactions, revealing a correlation between defective viral genomes and virus-induced host transcriptional programs [104] . these data provide valuable information for developing host-targeted therapeutics. host immune system functions immediately after detection of the virus. host mucosal immune system (mis), induced after virus invasion, serves as the first line to prevent iav from adhering to the susceptible cells. in the urt, mucosal response is induced in the naso-associated lymphoid tissues (nalt), while in the lrt, it occurs in bronchus-associated lymphoid tissues (balt). host innate immunity, including phagocytic cells, interferons (ifns), proinflammatory cytokines, etc., applies multiple mechanisms in defending iav infection [105] . host adaptive immunity, mediated by b lymphocytes and t lymphocytes, together with other immune mechanisms, reacts specifically to neutralize and eliminate the virus. on the other hand, to establish a successful infection, iavs also employ a plethora of strategies to avoid being detected or being cleared by the host immunity. notable strategies include regulation of ifn signaling [106] , inhibition of cytokine expressions [107, 108] , modulation of apoptosis [109] [110] [111] , interference of autophagy [112] , and effects on antibody production [113] . the iav-host immunity interaction was summarized by several reviews [114, 115] . upon detection of infection, innate effector cells, including natural killer (nk) cells, neutrophils, and dendritic cells (dcs), etc., are recruited to the infected sites. nk cells are large granular lymphocytes, making up 10% of the resident lymphocytes in the lung. after recruitment from the blood, nk cells interact with dcs and macrophages to secret various cytokines and restrict infection via lysis of the iav-infected cells. the lysis process is mediated by interaction between nk receptors p46 (most nkp46) and iav ha protein expressed by the infected cell [116, 117] . interestingly, liver nk cells other than lung nk cells possessed a memory phenotype to protect mice against subsequent iav infection, though the lung nk cells are important in control of primary iav infection [118] . however, nk cells are also shown to exacerbate iav pathology, since depletion of nk cells led to increased resistance to high dose h1n1 infection in mice [119, 120] . the contribution of nk cells to anti-iav defense in mouse models was later shown to be strain and dose dependent. in addition, the host genetic background also played an important role [121] . neutrophils are key innate immune cells recruited to infection sites by cellular migration through vascular endothelium. they function in clearance of pathogens via phagocytosis, producing extracellular traps, and degranulation [122] . in addition, they also regulate adaptive immunity via guiding influenza specific cd8 + t cells to the infection sites [123] . the function of dendritic cells (dcs) is to monitor invading pathogens. after iav infection, the conventional dcs migrate from lung to lymph nodes through interaction between ccr7 and its ligand, and present antigens to t cells [124, 125] . one study based on a mouse model showed that, during iav infection, immature and mature dcs were specialized in iav ha processing, since both types of dcs could present one epitope of h1n1 ha (ha amino acids 107-119), whereas another epitope (ha amino acids 302-313) could only be processed by mature dcs [126] . the complex role of dcs in initiation of robust immunity against iav infection is reviewed by waithman and mintern [127] . t cells and b cells are critical components in adaptive immunity against iav infection. cd8 + t cells differentiate into cytotoxic t lymphocytes (ctls) and defend iav infection via producing cytokines and effector molecules, and cytotoxic effects (i.e., lysis) of infected cells mediated by mhc class i. cd4 + t cells target iav-infected epithelial cells through binding with mhc class ii molecules and contribute to b cell activation thus consequently promote antibody production. the activation of t cells and b cells in iav infection will be exposited in section 3.5. the reaction of innate immunity is nonspecific. it is triggered by recognition of pathogen associated molecular patterns (pamps) via host pathogen recognition receptors (prrs). toll-like receptors (tlrs), retinoic acid-inducible gene-i proteins (rig-i), and nod-like receptors are common prrs, the activation of which leads to activation of innate immune signaling and further production of cytokines as well as other antiviral molecules. toll-like receptors are responsible for sensing pathogens at cell membranes, endosomes, and lysosome [128] . tlr3 and tlr7 are shown to be involved in iav detection at endosomes [105] . tlr3 recognizes double stranded rna (dsrna) which may be released by cellular stress and cell death [105] and unidentified rna structures in phagocytosed cells infected with iavs [129] . in macrophages and dendritic cells, tlr3 interacted with tir-domain-containing adapter, then activated the serine-threonine kinase iκkε (ikkε) and tank binding kinase 1 (tbk1) to phosphorylate interferon regulatory factor 3 (irf3), the process of which further led to expression of ifn-β [130] . in addition, an over-reacting tlr3 activation promoted iav pathogenesis, which could be reduced by a single-stranded oligonucleotide (sson) functioning as a tlr3 inhibitor, resulting in restrained viral loads both in vitro and in vivo [131] . tlr7 recognizes single stranded rna (ssrna). in plasmacytoid dendritic cells (pdcs), after activation of tlr7 during iav infection, irf7 or nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) were activated via myeloid differentiation factor 88 (myd88) to induce type i ifns [132] . in avian macrophages, activation of tlr7 produced pro-inflammatory molecules such as interleukin (il)-1β [133] . in addition, in mouse models, tlr7 played an important role in activation of nk cells [134] . it was also shown to be involved in development of adaptive immunity to prevent iav infection [135, 136] . rig-i recognizes ssrnas and transcriptional products of iavs, which triggers activation of the caspase activation and recruitment domains (cards) via dephosphorylation or ubiquitination by e3 ligases, resulting in activation of transcription factors including irfs and nf-κb [137] . otub1 played an essential role in regulation of rig-i [138] . in addition, melanoma differentiation-associated gene 5 (mda5) was also involved in sensing transcriptional products of iavs in the cytoplasm [139] . for nod-like receptor family, pyrin domain containing 3 (nlrp3) and nlr apoptosis inhibitory protein 5 were activated after iav infection [140] . iav m2 ion channel and pb1-f2 were involved in activation of nlrp3 inflammasome and stimulate il-1β secretion subsequently [141, 142] . the role of the nlrp3 inflammasome in regulation of anti-iav responses is discussed in detail by sarvestani and his colleagues [143] . delayed oseltamivir and sirolimus combined treatment could suppress nlrp3 inflammasome mediated secretion of il-1β and il-18, resulting in attenuation of h1n1-induced lung injury [144] . after detecting viral components, transcription factors including nf-κb and irfs are activated, leading to transcription of ifns and pro-inflammatory cytokines. ifns bind to receptors, resulting in upregulation of multiple interferon-stimulated genes (isgs) [145] . it is well known that type i ifns viruses 2020, 12, 376 7 of 23 (ifn-α and ifn-β) and type iii ifns (ifn-λ 1-4) play critical roles in antiviral responses. mice failed to restrict non-pathogenic iav when both type i and type iii ifn receptors were knocked out [146] . the expressed ifns consequentially bind to different receptors. type i ifns interact with ifn-α/β receptors (ifnar), whereas type iii ifns interact with ifn-λ receptors (ifnlr). janus kinase-signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifn-λs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim41 targeted np for ubiquitination and degradation in vitro [159] . for further reading on other isgs, several reviews regarding ifn responses during iav infection are recommended here [160, 161] . a general description of activation of innate immunity and ifn signaling pathway after iav infection is illustrated in figure 1 . signal transducer and activator of transcription (jak-stat) signaling pathway is then activated, resulting in transcription of numerous ifn-stimulated genes (isgs) [147, 148] . though ifn-λs share many characteristics such as expression patterns, signaling pathways, etc. with type i ifns, they are the first ifns produced at the infected epithelial sites to block virus spread [149] . furthermore, ifnλs served an important role in programming dcs to direct effective t cell immunity against iav infection [150] . isgs encode various antiviral proteins functioning in different ways to defend iav infection. for instance, mxa gtpase from the mx family could retain viral genome from entry to the cytoplasm via blocking the function of iav np. in addition, in vitro research found that avian iavs were more sensitive to mxa than human iavs [151, 152] . cholesterol 25-hydroxylase (ch25h) were identified to block iav entry via altering the cellular membrane properties to interfere with viral fusion, and amplified the activation of immune cells [153] . guanylate-binding protein 3 (gbp3) of ifn-inducible gtpases inhibited iav replication via binding to the viral polymerase complex [154] . members of the tripartite motif-containing (trim) family are also involved in cellular anti-iav processes. for instance, trim14 could interact with iav np for ubiquitination and proteasomal degradation, thus restricting iav replication in a type i ifn and nf-κb independent manner [155] . trim22 degraded iav np via polyubiquitination, thus resulting in inhibition of iav infection [156] . trim 25 regulated the re-localization of rig-i and was responsible for rig-i ubiquitination as well as rig-i-mediated ifn production [157] . trim32 recognized iav pb1 protein and reduced its polymerase activity [158] . trim in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), in order to counter ifn-stimulated antiviral proteins, iav viral proteins apply multiple strategies. for instance, ha protein was shown to trigger ubiquitination of ifnar to attenuate the type i ifn signaling pathway [162] . the follow-up work showed that poly (adp-ribose) polymerase 1 (parp1) functions as an interacting partner of ha protein to mediate the ha-induced ifnar degradation [163] . ns1 is the most important ifns antagonist protein via mechanisms including inhibition of the trim25-mediated rig-i ubiquitination, suppression of protein kinase r (pkr), phosphorylation of iκb kinases (ikk) α and β in the nf-κb pathway, interruption of the phosphorylation of stat1, stat2, and stat3 [39, 115] , and degradation of otub1 [138] . phosphorylation of ns1 is crucial for its function of antagonizing ifn-β expression, since dephosphorylation at position 73 and 83 of the protein induced a high level of ifn-β [91] . non-structural protein pb1-f2, identified from a+1 open reading frame (orf) of pb1 gene segment [164] , is multifunctional in deregulation of type i interferon [165, 166] . it counteracted rlr-mediated activation of ifn pathway not only by targeting mitochondrial mavs [115, 165, 167] , but also by binding to the dead-box helicase ddx3 to induce proteasome-dependent degradation [166] . furthermore, pb1-f2 interacted with mitochondrial tu translation elongation factor (tufm) to mediate formation of autophagosome, thus inducing complete mitophagy, which is critical for mavs degradation [167] . novel pa-x protein could also modulate innate immune responses. a review regarding the function of ns1 and pa-x proteins in antagonizing host innate immunity is recommended here [114] . though autophagy is essential for cellular metabolism and homeostasis, it also plays important roles in innate immune responses against pathogen infection. for cellular homeostasis, the mtor pathway is one of the most conserved autophagic pathways. the mtor complex 1 (mtorc1) negatively regulates the ulk1 kinase activity, thus affecting the autophagy induction [168] . c-jun n-terminal protein kinase 1 (jnk1) disrupts the bcl-2/beclin-1 complex through phosphorylation, thus regulating the autophagy induction [169, 170] . jnk1 is also reported to upregulate beclin-1 expression through phosphorylation of transcription factor c-jun in vitro [171] . in contrast to the autophagic pathways for cellular metabolism and homeostasis, less is known about autophagosome formation after iav infection [172] . to restrict infection of multiple viruses including iavs, trim23 is essential to mediate autophagy via its ring e3 ligase and adp-ribosylation factor (arf) gtpase activity [173] . beclin-1 and tufm-regulated autophagy also inhibited iav replication [112] . in hela cells and a549 cells, iav infection activated jnk1 to induce autophagosome formation and tgf-β-activated kinase 1 might contribute to the process [174, 175] . furthermore, autophagy was involved in maintaining memory b cells to counteract iav infection [176] . iav also utilizes autophagy to complete its life cycle. ns1 protein is proposed to suppress jnk1-mediated autophagy induction [174] . m2 could also block autophagosome maturation and mediate microtubule-associated protein 1 light chain 3 (lc3)-bound membrane redistribution, thus allowing filamentous budding of iav [177] [178] [179] . circ-gatad2a (gata zinc finger domain containing 2a), induced by iav infection, could inhibit autophagy and promote iav replication [180] . for a comprehensive reading on iav-induced apoptosis, a review is recommended here [181] . upon detection of iavs, dcs trigger production of ifns and cytokines, which in turns assist maturation of the dcs into antigen presenting cells (apcs), and initiate t cell immune responses. through the activation of ag-bearing dcs, naïve cd4 + t cells differentiate into th1, th2, th7, regulatory t cells (treg cells), follicular helper t cells, and killer cells. th1 and follicular helper t cells are the most abundant cd4 + t helper cells. they can secret antiviral cytokines, regulate cd8 + t cell differentiation, promote b cell activation, and maintain immunological memory [182, 183] . th17 cells induced pulmonary pathogenesis and could decrease mortality of iav-infected mice [184, 185] . in addition, γδ t cells, expanding in the late stage of iav infection with a t cell receptor (tcr)-independent viruses 2020, 12, 376 9 of 23 manner, could efficient eliminate iav-infected airway epithelial cells, resulting in lower viral titers [186] . new surrogate markers cd49d and cd11a were used to explore the kinetics of iav-specific cd4 t cells responses, revealing endogenous cd4 t cell response to primary iav infection is predominantly composed of t-bet+ cells [187] . cd8 + t cells are major components for virus clearance in adaptive immunity. after activated by dcs, cd8 + t cells undergo rapid expansion, differentiation, and migration to the infected sites. in general, to establish effective primary cytotoxic t lymphocyte (ctl) responses, cd4 + t cells play an essential role, with a mouse model as an exception [188] . ctls produce cytotoxic granules containing perforin and granzymes (gra and grb) to induce apoptosis and interrupt iav replication [189] . in addition, ctls produce cytokines, such as tnf, fasl, and trail, which recruit death receptors to induce apoptosis [190] . in addition, il16 deficiency enhanced the th1 and ctl responses upon iav infection [191] . furthermore, as cd8 + cells could last for two years in murine models, iav-specific memory ctls reacted specific to epitopes in conserved iav proteins [192] . in the nasal epithelia, they could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blocking iav transmission [197] . in addition, iav-specific antibody-dependent cell-mediated cytotoxicity (cdcc) also plays a role in cross-protection against iav infection. a general description of adaptive immunity against primary iav infection is illustrated in figure 2 . antigenic shift and drift, resulting in reassorted and mutated ha and/or na, are responsible for aiv escaping from host immunity [50] [51] [52] . furthermore, additional glycosylation on h5 ha could also induce virus escape from neutralizing antibodies [198] . apoptosis represents programmed single cell death that occurs in cell physiological remodeling, cell proliferation, or immune response to invading pathogens [199] . besides prototypical changes, cells undergoing apoptosis can be detected through dna and biochemical assays, take the tunel and in situ end-labeling (isel) techniques for instance. two primary pathways are involved in activation of apoptosis: the intrinsic or mitochondrial pathway, and the extrinsic or death receptor pathway. the intrinsic pathway is also known as "the mitochondrial pathway", which operates in response to various intracellular stress. several factors such as nitric oxide (no), cytochrome c, and second mitochondria-derived activator of caspases (smac) can activate this pathway, and the key player of this pathway is proteins in the bcl-2 family, which are activated by stress signals and then release apoptotic factors via destabilizing the mitochondrial membrane [200, 201] , resulting in release of mitochondrial cytochrome c. cytochrome c then binds to apoptosis protease activating factor-1 (apaf-1) and forms a complex with pro-caspase 9 (then cleaved into caspase 9), the function of which is to cleave its effector pro-caspase 3 [202] . in addition, smac, localizing in the cytosol, could initiate activation of caspase 9 via blocking the activity of iap [203] . the extrinsic pathway is regulated by extracellular ligands acting on transmembrane "death receptors": the first apoptosis signal (fas) receptor-fas ligand (fasr/fasl) and the tnf-αtnf receptor 1 (tnfα/tnfr1) [199] . in the fasr/fasl model, fas ligand binds to its receptor fasr [204] , forming the death-inducing signaling complex (disc) with pro-caspase 8, resulting in activation of caspase 8 and downstream activation of other caspases (caspase-3, caspase-6, and caspase-7) [199] . in the tnfα/tnfr1 pathway, tnfr1-associated death domain protein (tradd) is activated after binding of tnfα to tnfr1, leading to recruitment of fadd and receptor interacting protein (rip) [205] . fadd then associates with pro-caspase 8 to form the disc, resulting in activation of caspase 8 and apoptosis. could prevent the spread of the virus from the urt to the lung [193] . to establish memory cd8 + t cells, autophagy plays an important role [194] , while the function of cd4 + t cells in memory ctl responses is "context-dependent". a recent study showed that cd4+ t cells promoted iav-specific ctl memory at the initial priming stage of viral infection [195] . grant and her colleagues summarized and discussed the importance of cd8 + t cell immunity against iavs [192] , and this review is recommended for further reading. with the help of cd40 ligand (cd40l), cd4 + cells contribute to b cell activation [183] . with the help of memory t cells, naïve b cells could reduce morbidity and promote recovery on heterosubtypic infection [196] . for different types of antibodies, igg could inhibit pathogenesis, while iga functions in blockin during iav infection, viruses modulate host apoptotic responses in a time-dependent manner [206] . for instance, in order to earn enough time for replication and virion formation, iav inhibited apoptosis via upregulating the anti-apoptotic phophoinositide-3-kinase-protein kinase b (pi3k-akt) pathway at the beginning of infection. however, in the later phase of infection, the virus suppressed this pathway to upregulate the pro-apoptotic p53 pathway, thus allowing successful release of virions [207] . several viral proteins are involved in regulation of host apoptosis. np protein induces host apoptosis to favor viral replication through interaction with ring finger 43 (rnf43) [208] , apoptotic inhibitor 5 (api5) [209] , or clusterin [111] . pb1-f2 also induced apoptosis and promoted viral replication through dysregulating mitochondrial potential [206] . furthermore, m1 promoted apoptosis by binding to heat shock protein 70, thus activating caspase and the subsequent apoptosis [210] . in addition, ns1 expression was reported to induce apoptosis in mdck and hela cells [211] . however, mutant iav lacking the ns1 gene could induce apoptosis in cultured cells [212] . the function of ns1 in inhibiting apoptosis may be explained by its ability to inhibit type i ifn [213, 214] . these data demonstrate sophisticated mechanisms of iav in regulating host apoptosis. furthermore, the role of these viral proteins in apoptosis suggests that these proteins may present suitable targets for anti-iav therapies. a comprehensive review on influenza a virus-induced apoptosis discussed by ampomah and lim is recommended here [181] . in addition, recent in vitro research found that apoptosis was induced at early iav infection stage, while later the cell death pathway was shifted to pyroptosis. the switch process was promoted by the type i ifn-mediated jak-stat signaling pathway through expression of the bcl-xl gene [215] . during iav infection, multiple immune systems coordinate together to protect the host. accordingly, viruses antagonize the immune system through multiple measures to establish a successful infection. considering the high frequencies in genome mutations and recombination, vaccination is the most effective way to defend against the viruses via inducing cross-protective antibodies and/or enhancing immune responses. several studies in vaccine development have tried to enhance host immune responses. for instance, vaccine candidate containing ha targeted to chemokine receptor (porcine mip1α) was shown to enhance t cell responses, resulting in a strong and cross-reactive cellular immunity in vaccinated pigs [216] . another example is an attempt to intranasally administer a polyanhydride nano vaccine (iav-nanovax), which could promote robust lung-resident germinal center (gc) b cells with lung-localized iav-specific antibody responses as well as lung-resident memory cd4 + and cd8 + t cell responses [217] . for anti-iav drugs, currently, na inhibitors (relenza tm and tamiflu tm ) are applied clinically as anti-influenza drugs [218] . these drugs inhibit the activity of na by preventing viral budding [21] . in addition, cap-dependent endonuclease inhibitor (baloxavir marboxil) targeting pa is also applied against influenza a and b virus infection [219] . our progressing understanding of the iav life cycle of the virus and iav-host interaction could contribute to anti-influenza drug design. since the recognition of ha protein to sa linked glycoproteins is the first step in iav infection, effective blocking of the interaction between viral ha and sa receptor serves as a favorable target in drug design [220, 221] . favipiravir, a nucleotide analogue that selectively inhibits the rna-dependent rna polymerase, is licensed in japan to be applied against emerging influenza viruses resistant to other antivirals [222, 223] . oleanolic acid (oa), a kind of pentacyclic triterpene natural product, and its analogues, as well as its derivatives, were shown to bind to ha, thus blocking the attachment of iavs to mdck cells [224] [225] [226] . pvf-tet is a peptide-based ha inhibitor, which was shown to sequester ha into amphisome (fusion of late endosome with autophagosome) and protected mice from the lethal iav infection [227] . new effective drugs targeting the polymerase would be a promising strategy against iav infection, since they would directly reduce or eliminate viral replication. numerous sites, including the cap-binding site [228] , the endonuclease [229, 230] , and pa-pb1 inter-subunit interface [231] can serve as potential targeting sites for new drug design. coumarin compounds, including eleutheroside b 1 , isofraxidin, fraxin, esculetin, fraxetin, and scoparone, were investigated for their antiviral and anti-inflammatory activities against influenza virus in vitro [31] . other candidates, such as naproxen, a non-steroidal anti-inflammatory drug, was shown to target np protein at residues f209 and y148, thus antagonizes the crm1-mediated nuclear export of np. it is suggested to have a broad-spectrum anti-influenza activity [232] . verdinexor (kpt-335), a novel orally bioavailable drug, blocks crm1-mediated nuclear export of np and repress nf-κb activation, thus reducing cytokine production and eliminating virus-associated immunopathology [233] . for further reading on candidate anti-iv therapeutics, a review summarized by davidson is recommended here [234] . with the increasing knowledge obtained through massive investigations on host immunity against iav infection, promoting host immune responses not limited to antibody enhancement would have good prospects not only for vaccine design, but also for development of novel antiviral agents. author contributions: manuscript preparation, y.z.; revision, z.x.; supervision, y.c.; funding acquisition, y.z. all authors read and approved the final version of the manuscript. funding: this study was supported by the "zhujiang talent program" overseas youth talent introduction program (post-doctoral program) and doctoral initiative project of natural science foundation of guangdong province (18zxxt49). the authors declare that they have no financial and personal relationships with other people or organizations that can influence the work. there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in this review. they do not have any commercial or associative interest that represents conflicts of interest in connection with the work submitted. influenza virus aerosols in the air and their infectiousness influenza: the once and future pandemic identification of a novel viral protein expressed from the pb2 segment of influenza a virus an overlapping protein-coding region in influenza a virus segment 3 modulates the host response 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cell responses resident memory cd8+t cells in the upper respiratory tract prevent pulmonary influenza virus infection autophagy is a critical regulator of memory cd8+ t cell formation cd4+t help promotes influenza virus-specific cd8+t cell memory by limiting metabolic dysfunction b cells promote resistance to heterosubtypic strains of influenza via multiple mechanisms1 recombinant iga is sufficient to prevent influenza virus transmission in guinea pigs addition of n-glycosylation sites on the globular head of the h5 hemagglutinin induces the escape of highly pathogenic avian influenza a h5n1 viruses from vaccine-induced immunity apoptosis: a review of programmed cell death bcl-2 gene family in endocrine pathology: a review nitric oxide: no apoptosis or turning it on? promotion of caspase activation by caspase-9-mediated feedback amplification of mitochondrial damage smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition the many roles of fas receptor signaling in the immune system the fas signaling pathway: more than a paradigm viral control of mitochondrial apoptosis control of apoptosis in influenza virus-infected cells by up-regulation of akt and p53 signaling the nucleoprotein of influenza a virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase rnf43 nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api5 to enhance e2f1-dependent apoptosis and virus replication cell death regulation during influenza a virus infection by matrix (m1) protein: a model of viral control over the cellular survival pathway influenza virus ns1 protein induces apoptosis in cultured cells loss of function of the influenza a virus ns1 protein promotes apoptosis but this is not due to a failure to activate phosphatidylinositol 3-kinase (pi3k) pathogenic potential of interferon αβ in acute influenza infection ifnλ is a potent anti-influenza therapeutic without the inflammatory side effects of ifnα treatment influenza a virus infection triggers pyroptosis and apoptosis of respiratory epithelial cells through the type i interferon signaling pathway in a mutually exclusive manner targeting of ha to chemokine receptors induces strong and cross-reactive t cell responses after dna vaccination in pigs polyanhydride nanovaccine induces robust pulmonary b and t cell immunity and confers protection against homologous and heterologous influenza a virus infections influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir. viruses baloxavir marboxil: the new influenza drug on the market emerging antiviral strategies to interfere with influenza virus entry discovery of the first series of small molecule h5n1 entry inhibitors favipiravir (t-705), a novel viral rna polymerase inhibitor favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase synthesis, structure activity relationship and anti-influenza a virus evaluation of oleanolic acid-linear amino derivatives design, synthesis of oleanolic acid-saccharide conjugates using click chemistry methodology and study of their anti-influenza activity design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors the inducible amphisome isolates viral hemagglutinin and defends against influenza a virus infection discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (vx-787) of influenza pb2 a novel endonuclease inhibitor exhibits broad-spectrum anti-influenza virus activityin vitro identification and characterization of influenza variants resistant to a viral endonuclease inhibitor polymerase acidic protein-basic protein 1 (pa-pb1) protein-protein interaction as a target for next-generation anti-influenza therapeutics naproxen exhibits broad anti-influenza virus activity in mice by impeding viral nucleoprotein nuclear export verdinexor targeting of crm1 is a promising therapeutic approach against rsv and influenza viruses treating influenza infection, from now and into the future key: cord-011251-rjyipcfv authors: chernyshov, vladimir v.; yarovaya, olga i.; fadeev, dmitry s.; gatilov, yuriy v.; esaulkova, yana l.; muryleva, anna s.; sinegubova, katherina o.; zarubaev, vladimir v.; salakhutdinov, nariman f. title: single-stage synthesis of heterocyclic alkaloid-like compounds from (+)-camphoric acid and their antiviral activity date: 2019-02-28 journal: mol divers doi: 10.1007/s11030-019-09932-9 sha: doc_id: 11251 cord_uid: rjyipcfv abstract: an effective technique for one-stage synthesis of new polycyclic nitrogen-containing compounds has been developed. the procedure involves refluxing mixtures of camphoric acid with aliphatic or aromatic diamine without catalysts. in cases where the starting amine has a low boiling point (less than 200 °c), phenol is used as a solvent, as it is the most optimal one for obtaining products with good yields. it has been shown that the use of lewis acids as catalysts reduces the yield of the reaction products. a set of compounds have been synthesized, which can be attributed to synthetic analogues of alkaloids. in vitro screening for activity influenza virus a was carried out for the obtained compounds. the synthesized quinazoline-like agent 14 has inhibitory activity against different strains of influenza viruses. graphical abstract: [image: see text] electronic supplementary material: the online version of this article (10.1007/s11030-019-09932-9) contains supplementary material, which is available to authorized users. influenza represents one of the most serious challenges to medical science and health care all over the world. it causes annual epidemics and from time to time pandemics, both resulting in significant increase in morbidity and mortality [1] . due to fast replicative cycle, ability to reassort the fragments of segmented genome and lack of correcting activity of viral polymerase influenza virus can quickly select mutants that do not match the virus-inhibiting antibodies and can therefore escape from the immune response [2] . several classes of chemically distinct compounds are currently used for treatment of influenza: amantadine and rimantadine (two blockers of m2 proton channel) [3, 4] , four neuraminidase inhibitors (oseltamivir, zanamivir, peramivir and laninamivir) prevent budding of viral progeny [5] , umifenovir (arbidol) is used against influenza in russia and china [6, 7] , pyrazinecarboxamide derivative favipiravir (t-705) was approved for stockpiling for potential treatment of pandemic influenza [8, 9] , and baloxavir marboxil (xofluza ® ) that has been recently approved by fda interferes with endonuclease activity of viral pa subunit of polymerase complex [10] . the same features of influenza virus that cause the emergence of antibody-escape mutants lead also to the selection of drug resistance to direct-acting antivirals. indeed, all current isolates of influenza virus are resistant to adamantane derivatives so that who does not recommend their use for treatment of influenza anymore [10] [11] [12] . in 2007-2009 almost total resistance of seasonal influenza a(h1n1) viruses to oseltamivir was achieved. these strains were further replaced with oseltamivir-susceptible pandemic influenza viruses a(h1n1)pdm09 [13, 14] . taken together, these facts suggest that novel anti-influenza drugs of alternative mechanism(s) of activity and viral target(s) are therefore of high priority for medicinal science and health care. the abundance, crystallinity and variety of transformations of (+)-camphor were interesting throughout the history of organic chemistry. our work is devoted to the investigation of chemical properties of (+)-camphoric acid (product of oxidation of (+)-camphor). camphoric acid is a cyclopentane derivative containing two carboxylic acid functional groups on the first and third carbon atoms. the c-1 atom has an additional methyl group, thereby converting camphoric acid into an enantiomeric ditopic organic linker with different coordination regimes. moreover, the coordination chemistry of camphoric acid specifically gives rise to many interesting chiral features applicable to both materials and life sciences, such as asymmetrical synthesis or crystallization, homochiral structural design, chiral induction, absolute helical control and ligand handedness [15] . there are examples of the use of camphoric acid derivatives as ligands [16] [17] [18] . the condensation of carboxylic acids with amines and anilines is known to be a classical way of preparing amides. this interaction is shown for a wide spectrum of various substrates [19] . the methods for preparing 2-substituted benzimidazoles based on direct cyclocondensations of carboxylic acids with benzene-1,2-diamine are also well known, but almost all of them involve rigid conditions, high temperatures or acidic conditions [20] [21] [22] . for example, many carboxylic acids are condensed with benzene-1,2-diamine at temperatures of about 200° [23] [24] [25] . thus, we consider it relevant to study the interaction of camphoric acid with various aliphatic and aromatic diamines. this study is important, because all nitrogen-containing heterocyclic compounds play a great role in medicinal and organic chemistry in whole. the present work is devoted to the synthesis of new polycyclic nitrogen-containing compounds from (+)-camphoric acid and aliphatic or aromatic diamines. the first investigated reaction was interaction between ethylenediamine 2 and (+)-camphoric acid 1. as a result of refluxing 1 eq. camphoric acid with 2 eq. ethylenediamine in phenol, product 3 was obtained (scheme 1). this technique led to an almost quantitative conversion to 3 after 2 h with 80% yield. product 3 was not observed, if refluxing the starting reagents was carried out without a solvent, apparently due to the low boiling point of the starting amine 2. also we used toluene, dmso and o-xylene as a solvent for this interaction. it should be noted that using toluene or xylene significantly increased the conversion time to 24 h. the use of dmso as a solvent resulted in a double decrease in the desired product. thus, using phenol as a solvent proved to be the most optimal for the production of the tricyclic product 3. refluxing a tenfold excess of camphoric acid with diamine 2 in i-proh gives us a mixture of cyclic amide of symmetrical structure 4 and product 3 (1:1). product 4 was purified by column chromatography and isolated with 9% yield. further, the interaction of 1,3-diaminopropane 5 with camphoric acid was investigated. refluxing 5 with 1 in phenol for 3 h led to compound 6 with 90% yield (scheme 1). unfortunately, we were unable to isolate the cyclic amide of a symmetric structure based on 1,3-diaminopropane. product 8 was obtained by refluxing 1 with 7 in an i-proh with 25% yield. this interaction allows us to obtain compound 8 and presumably mixture of polycyclic compounds with a similar structure to the compound 6, which, unfortunately, are not received in a pure form. for compounds 3, 8 and perchlorate of the compound 6 (6·hclo 4 ) x-ray crystallographic analysis was carried out (fig. 1) . then, we decided to investigate the possibility of obtaining compounds of a similar structure from aromatic amines. thus, o-phenylenediamine 9 was chosen as the next object of our research. we show that refluxing of mixture of 9 with 1 without a solvent for 3 h leads to the formation of a mixture of two benzimidazole derivatives 10a and 10b with a total yield of 70% (scheme 2). in this case, we carried out a study of the conversion rate and the ratio of the final products, by replacing camphoric acid 1 with its anhydride, and also by adding 5 mol% of lewis acid to the reaction mixture, for example, anhydrous zinc chloride. it is shown that the use of the lewis acid increases the conversion time from 3 to 5 h and decreases the total yield of the reaction products to 55% and the quantity of the major isomer 10a in the final mixture. using camphoric acid anhydride as a starting reagent slightly reduces the overall yield of the reaction products. compounds 10a and 10b have been isolated after the column chromatography with a yield of 40% and 2%, scheme 1 interaction of camphoric acid with aliphatic diamines respectively. the structure of compound 10a has been confirmed by x-ray crystallographic analysis (fig. 1) . the benzimidazole derivative 10a was previously described [19] . the procedure involved refluxing the mixture of reagents with 10 mol% of boric acid in toluene for 48 h, and a dean-stark trap was used for the azeotropic removal of h 2 o. we have also shown the possibility of synthesizing compound 10a by single-stage synthesis without solvent and catalysts, which significantly reduced the reaction time. then, we chose naphthalene-1,8-diamine 11 as parent aromatic diamine. refluxing double excess of 11 with 1 without a solvent for 6 h led to the mixture of compounds 12a and 12b with a total yield of 50% (scheme 2). compounds 12a and 12b have been isolated by column chromatography with a yield of 30% and 2%, respectively. we consider that the minor product in all the transformations above is obtained by the primary nucleophilic attack to a more sterically hindered carboxylic group. this assumption is confirmed by 1 h, 13 c and 2d nmr spectra (see supporting information). also, the interaction of o-aminobenzylamine 13 with 1 was studied. refluxing the mixture of 1 and excess of 13 without a solvent for 4 h resulted in compound 14 with 45% yield (scheme 2). x-ray crystallographic analysis shows that, in this case, the main product is the compound formed as a result of the primary nucleophilic attack to a more sterically hindered carboxyl group (fig. 1 ). synthesized polycyclic nitrogen-containing compounds 3, 6, 10a, 10b, 12a, 12b, 14 can be referred to as synthetic analogues of natural quinazoline alkaloids. nowadays, natural and synthetic quinazolines attract considerable attention due to their diverse and sometimes very high biological activity [26] . for example, the most common active metabolites of plants of the genus peganum are quinazoline alkaloids (such as vasicine 15a, desoxyvasicine 15b, vasicinone 16a, deoxyvasicinone 16b [27] ). these natural compounds have a broad spectrum of native biological activity, in particular: anti-ad for 15a-b [28] , anti-parasitic for 16a-b [29] , insecticidal for 15a [30] . at the same time, synthetic derivative of quinazoline diproqualone 17 was previously used as an analgesic for osteoarthritis and rheumatoid arthritis [31] (fig. 2) . along with the pronounced biological activity, the compounds of the quinazoline series can be successfully used as chiral catalysts [32, 33] . thus, the use of vasicine 15a as an organic catalyst for direct c-h arylation of unactivated arenes with aryl iodides/bromides without assistance of any transition metal catalyst has been described [34] . vasicine 15a, a quinazoline alkaloid, from the leaves of adhatoda vasica, has been utilized as an efficient catalyst for metal-and base-free henry reaction of various aldehydes with nitro alkanes [35] . the quinazoline structure possibly imparts rigidity to the ligand and hence consistently high scheme 2 interaction of camphoric acid with aromatic diamines enantioselectivity [36] . at present time, attention of numerous groups is being paid to the synthesis of analogues of natural alkaloids. synthetic and natural quinazoline alkaloids can exhibit pronounced antiviral properties [37, 38] . the compounds synthesized in this work contain both the monoterpenic fragment and the n-heterocycle. we have previously shown that various derivatives of monoterpenoids, in particular compounds including a 1,7,7-trimethylbicyclo[2.2.1]heptane scaffold and n-heterocyclic fragment, exhibit antiviral properties against the influenza virus [39, 40] . in this regard, the obtained derivatives were screened for their inhibitory activity against influenza virus a h1n1. for each compound, the values of 50% cytotoxic dose (cc 50 ), 50% virus-inhibiting dose (ic 50 ) and selectivity index (si) were calculated. the results are shown in table 1 . adamantane-and norbornanebased derivatives were used as reference compounds due to their close similarity to the compounds under investigation in having rigid cage fragments in their structures. it is worth noting that compounds 3, 6, 10b, 12b, 14 are less cytotoxic than reference compounds. compounds 3, 6, 10b, 14 are most effective in inhibiting the influenza virus a (h1n1) and can be used for further studies this type of activity. we believe that aliphatic polycyclic compounds (with a similar structure to compounds 3, 6) or compounds containing an additional aromatic cycle may exhibit potentially high antiviral activity, but in this case, we are particularly interested in the isomers with the hem-dimethyl bridge directed upwards. for compound 14, which showed the highest activity, we studied the antiviral activity against different strains of influenza virus (table 2) . it has been shown that compound 14 has inhibitory activity against different strains of influenza virus a. the compound synthesized has inhibitory activity against strain h5n2 (comparable to reference compounds) and strain h1n1 (exceeding that of reference compounds). unfortunately, the inhibitory activity of compound 14 against strain h3n2 is lower than that of the reference compounds. activity of compounds 3, 4, 6, 10a, 12a in conclusion, a simple and effective method of singlestage synthesis of polycyclic nitrogen-containing heterocyclic compounds, synthetic analogues of natural alkaloids, is suggested for the first time. it was shown that the optimal technique of synthesis is refluxing mixtures of parent compounds in phenol or without solvent and catalysts. using this method, we synthesized a number of polycyclic amides containing in their structure both a heterocyclic fragment and a bicyclic fragment (3, 6, 10a, 10b, 12a, 12b, 14) and two cyclic symmetrical amides (4, 8) . the structures of all synthesized compounds were confirmed by a complete set of spectral data, including x-ray crystallographic analyses of crystalline products 3, 6, 8, 10a and 14. in vitro screening for inhibitory activity against influenza virus was carried out for the obtained compounds and compound 14, was shown, exhibits inhibitory activity against different strains of influenza virus a (h1n1, h3n2, h5n2). compounds 3, 6, 10b and their derivatives, in turn, can be used in the further study of this type of activity. media centre influenza (seasonal) fact sheet constraints, drivers, and implications of influenza a virus reassortment multiscale simulation reveals a multifaceted mechanism of proton permeation through the influenza a m2 proton channel structural basis for proton conduction and inhibition by the influenza m2 protein antiviral treatments arbidol as a broad-spectrum antiviral: an update arbidol: a broad-spectrum antiviral compound that blocks viral fusion favipiravir as a potential countermeasure against neglected and emerging rna viruses favipiravir (t-705), a broad spectrum inhibitor of viral rna polymerase baloxavir marboxil investigators group baloxavir marboxil for uncomplicated influenza in adults and adolescents adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states the origin and global emergence of adamantane resistant a/h3n2 influenza viruses comparison of antiviral resistance across acute and chronic viral infections drug resistance in influenza a virus: the epidemiology and management chiral chemistry of metalcamphorate frameworks novel tridentate ligands derived from (+)-camphoric acid for enantioselective ethylation of aromatic aldehydes enantioselective alkylation of aromatic aldehydes with (+)-camphoric acid derived chiral 1,3-diamine ligands synthesis of some new chiral bifunctional o-hydroxyarylphosphonodiamides and their application as ligands in ti(iv) complex catalyzed asymmetric silylcyanation of aromatic aldehydes boric acid-catalyzed direct condensation of carboxylic acids with benzene-1,2-diamine into benzimidazoles synthesis, reactivity and biological activity of benzimidazoles synthesis and biological evaluation of 4′-[(benzimidazole-1-yl)methyl]biphenyl-2-sulfonamide derivatives as dual angiotensin ii/endothelin a receptor antagonists novel pyrazolo[3,4-d]pyrimidine with 4-(1h-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer agents: synthesis, cell based assay, topoisomerase inhibition, dna intercalation and bovine serum albumin studies efficient propylphosphonic anhydride ( ® t3p) mediated synthesis of benzothiazoles, benzoxazoles and benzimidazoles simulating microwave chemistry in a resistance-heated autoclave made of semiconducting silicon carbide ceramic synthesis and tuberculostatic activity evaluation of novel benzazoles with alkyl quinazoline derivatives: synthesis and bioactivities chemistry, pharmacology and medicinal properties of peganum harmala l rapid and sensitive detection of the inhibitive activities of acetyl-and butyryl-cholinesterases inhibitors by uplc-esi-ms/ms alkaloids from the seeds of peganum harmala showing antiplasmodial and vasorelaxant activities toxicity and growth inhibitory activities of methanol extract and the β-carboline alkaloids of peganum harmala l. against two coleopteran stored-grain pests quinazolinone: an overview evidence for involvement of cationic intermediate in epoxidation of chiral allylic alcohols and unfunctionalised alkenes catalysed by mn iii (quinazolinone) complexes 3-aminoquinazolinones as chiral ligands in catalytic enantioselective diethylzinc and phenylacetylene addition to aldehydes vasicine catalyzed direct c-h arylation of unactivated arenes: organocatalytic application of an abundant alkaloid vasicine from adhatoda vasica as an organocatalyst for metal-free henry reaction and reductive heterocyclization of o-nitroacylbenzenes vasicine as tridentate ligand for enantioselective addition of diethylzinc to aldehydes synthesis, antiviral activity and cytotoxicity evaluation of schiff bases of some 2-phenyl quinazoline-4(3)h-ones antiviral alkaloids produced by the mangrove-derived fungus cladosporium sp. pjx-41 synthesis and in vitro study of novel borneol derivatives as potent inhibitors of the influenza a virus synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity acknowledgements this work was supported by the russian science foundation (18-03-00271 a). the authors confirm that this article content has no conflict of interest. key: cord-278554-rg92gcc6 authors: aoyagi, yumiko; beck, charles r; dingwall, robert; nguyen-van-tam, jonathan s title: healthcare workers' willingness to work during an influenza pandemic: a systematic review and meta-analysis date: 2015-04-23 journal: influenza other respir viruses doi: 10.1111/irv.12310 sha: doc_id: 278554 cord_uid: rg92gcc6 to estimate the proportion of healthcare workers (hcws) willing to work during an influenza pandemic and identify associated risk factors, we undertook a systematic review and meta-analysis compliant with prisma guidance. databases and grey literature were searched to april 2013, and records were screened against protocol eligibility criteria. data extraction and risk of bias assessments were undertaken using a piloted form. random-effects meta-analyses estimated (i) pooled proportion of hcws willing to work and (ii) pooled odds ratios of risk factors associated with willingness to work. heterogeneity was quantified using the i(2) statistic, and publication bias was assessed using funnel plots and egger's test. data were synthesized narratively where meta-analyses were not possible. forty-three studies met our inclusion criteria. meta-analysis of the proportion of hcws willing to work was abandoned due to excessive heterogeneity (i(2) = 99·2%). narrative synthesis showed study estimates ranged from 23·1% to 95·8% willingness to work, depending on context. meta-analyses of specific factors showed that male hcws, physicians and nurses, full-time employment, perceived personal safety, awareness of pandemic risk and clinical knowledge of influenza pandemics, role-specific knowledge, pandemic response training, and confidence in personal skills were statistically significantly associated with increased willingness. childcare obligations were significantly associated with decreased willingness. hcws' willingness to work during an influenza pandemic was moderately high, albeit highly variable. numerous risk factors showed a statistically significant association with willingness to work despite significant heterogeneity between studies. none of the included studies were based on appropriate theoretical constructs of population behaviour. although variable in severity, 1,2 one consistent feature of pandemic influenza is a surge in demand for health care. 3, 4 hospitalization due to influenza a(h1n1)pdm09 in the usa was estimated at approximately 274 000 cases between april 2009 and april 2010 5 contrasting with 95 000 annual influenza-associated primary hospitalizations from 1979 to 2001. 6 in 2009-10, the availability of intensive care unit beds came under pressure in most national health systems. 1, 7 healthcare workers (hcws) play key roles during an influenza pandemic, but a serious shortage of personnel may occur at peak times or in severe pandemics because of absenteeism due to illness, caring for family members who are ill, or refusal to work. 8 effective preparation for the next pandemic requires estimates of hcws' willingness to work and an understanding of influencing factors. the available data are highly variable. one nigerian study found only one quarter of hcws stating they would be willing to work in a unit treating patients with influenza a(h1n1)pdm09, 9 whilst an australian qualitative study of family physicians found 100% of participants willing to work. 10 chaffee 11 first reviewed willingness to work during disasters and reported that the following factors would be influential: type of disaster, concern for close family, friends and pets, responsibility for dependants, the perceived value of one's response, belief in a duty of care, access to personal protective equipment (ppe), provision of basic needs (water, food, rest, shelter and communication tools) and prolonged working hours. three published reviews reported that similar factors would be associated with willingness to work during an influenza pandemic, [12] [13] [14] but the data were not summarized quantitatively. we addressed this evidence gap by conducting a systematic review and meta-analysis in accordance with the preferred reporting items for systematic review and meta-analyses (prisma) statement. the review questions sought to elucidate the proportion of hcws willing to work during an influenza pandemic, and to identify risk factors associated with willingness to work. our findings are interpreted with reference to sociological understandings of population behaviour, which have to date largely been absent from the peer-reviewed literature, but are highly relevant to the development of appropriate interventions to minimize refusal to work. the study protocol was registered with the national institute for health research international prospective register of scientific reviews (prospero; #crd42013004865) prior to executing the literature search strategy. 15 the prisma checklist is available as supporting information. we sought to analyse data collected exclusively from hcws including doctors, nurses, hospital workers, emergency healthcare service workers, public health workers, medical and nursing students, non-clinical support staff and retirees. the outcome measures of interest were the proportion of hcws reporting willingness to work during an influenza pandemic, and odds ratios or case counts allowing the derivation of odds ratios pertaining to factors associated with willingness to work. we included study manuscripts written in english reporting original quantitative research derived from a cross-sectional design, studies pertaining to a prior or hypothetical influenza pandemic, and studies reporting data pertaining to the aforementioned outcome measures, with no limitations on the time and place of publication. the following databases were searched from their inception to april 2013: medline, embase, web of knowledge, scopus, amed, assia, bioethicsweb, cinahl, cochrane library and psycinfo. google scholar and opengrey were also searched. search terms were 'pandemic + influenza + willingness to work/report to work' to avoid including studies on willingness to accept vaccination. these terms were used in both keyword and mesh searches as appropriate for each database as follows: #1. pandemics (mesh); #02. influenza, human (mesh); #03. 'attitude of health personnel' (mesh) or willingness (keyword); #04. hospital administration (mesh) or report to work (keyword); #05. willing* adj5 work (keyword); #06. respon* adj5 work (keyword); #07. would come (keyword); #08. #03 or #04 or #05 or #06 or #07; #09. #01 and #02 and #08 (see also table s1 ). reference lists in eligible articles were also searched. all identified records were imported to endnote software x4 (thomson reuters, toronto, ca, usa) and duplicate entries removed. the remaining records were screened by a single researcher (ya) against the protocol eligibility criteria following a sequential assessment of the study title, abstract and full-text article. where this was unclear, agreement on eligibility of each study was achieved through discussion with a second researcher (rd or jsn-v-t). data extraction was performed by a single researcher (ya) using a piloted form collecting details of study characteristics {title, author, publication year, place, study period, study design, participants, subject [pandemic of avian influenza origin/influenza a(h1n1)pdm09/non-specified, hypothetical influenza pandemic]}; definition of outcome measures; questionnaire type; validation; statistical analysis and any stated limitations; percentage of willingness to work; and risk factors association with willingness. odds ratios (ors) of factors both unadjusted and adjusted were extracted to estimate the association with willingness to work. crude case counts and the percentage of people in each risk factor stratum were extracted where available. risk of bias was assessed for each study using a newcastle-ottawa assessment scale modified for crosssectional studies by herzog et al. 16 descriptive statistics were calculated using microsoft â office excel â 2010 (microsoft corporation, richmond, va, usa). random-effects meta-analysis estimated the proportion of hcws (including 95% confidence intervals [cis]) who reported willingness to work during an influenza pandemic. random-effect meta-analysis of pooled odds ratios (including 95% cis) estimated the association of factors with willingness to work. 17 heterogeneity between studies was assessed using the i 2 statistic. 18 we considered it statistically inappropriate to perform meta-analysis where i 2 exceeded 85%. 19 to explore sources of heterogeneity, we planned to conduct subgroup analyses according to the type of influenza pandemic; geographical region; survey time period; type of questionnaire; type of participants; sex of participants; and newcastle-ottawa assessment scale score. we used galbraith plots to detect those studies that contributed substantial heterogeneity and conducted sensitivity analyses excluding them from our pooled estimates. 20 for each meta-analysis, publication bias was assessed graphically using a funnel plot of effect size versus standard error and statistically using egger's regression test. 21 meta-analysis of pooled proportions was conducted using statsdirect version 2.7.9 (statsdirect ltd., cheshire, uk), and meta-analysis of pooled odds ratios was conducted using we identified a total of 1133 unique records of which 43 studies met protocol eligibility criteria (see figure 1 ). two the included studies comprised entirely of cross-sectional surveys including two pre-/post-intervention studies and are summarized in table 1 . the participant population sizes ranged from 60 to 4306 with a median of 725 (interquartile range [iqr] 308-1711). the earliest publication was in 2006, and the majority of articles were published in 2009 (11; 25á6%) and 2010 (13; 30á2%). 28 of 43 (67á4%) studies used a hypothetical influenza pandemic as the subject, 21 (48á8%) were conducted in the usa, and 21 (48á9%) investigated both clinical and non-clinical staff within hospital settings. assessments using the modified newcastle-ottawa scale showed that 23 of 43 studies were at moderate risk of bias (2-3 of five stars) for the selection domain, whilst 10 studies were at low risk (4-5 stars) and ten studies were at high risk (0-1 stars); many studies used convenience sampling and few justified the study sample size, appropriately considered nonresponders and used a validated measurement tool. for the comparability domain, 24 were at high risk (0 of two stars), eight at moderate risk (one star) and 11 at low risk of bias (two stars). many studies did not clarify how statistical adjustment for confounding variables was carried out, or reported unadjusted estimates only. for the outcome domain, 39 studies were at moderate risk of bias (two of three stars) and four were at high risk (one star). willingness to work was self-reported in all 43 studies although the statistical test used was clearly described in only 39 studies (see figure s1 ). the percentage of participants who expressed a willingness to work ranged from 23á1% (community nurses during the influenza a(h1n1)pdm09 pandemic in hong kong in 2009) 22 to 95á8% (a study of us medical students targeting a hypothetical influenza pandemic). 23 we abandoned metaanalysis to estimate a pooled mean proportion of hcws willing to work due to very high statistical heterogeneity between studies (i 2 = 99á2%). our planned subgroup analyses were unable to adequately explain the sources of heterogeneity between studies as this remained above our threshold of 85% in each analysis. the percentage of willingness to work seemed to depend on the particular context of the study. studies of hypothetical influenza pandemics, which did not include detailed conditions such as virulence of the strain and availability of protective equipment, tended to show a high level of willingness to work. however, studies of precise scenarios or those which investigated willingness during the relatively mild influenza a (h1n1)pdm09 pandemic tended to present relatively low levels of willingness. this finding may correspond with earlier work by syrett et al. 24 which showed that willingness factors associated with willingness to work data were extracted from 33 studies. pooled estimates from meta-analyses of individual factors associated with willingness to work are summarized in table 2 . overall, females were one-third less likely to be willing to work compared with males. by occupational group, physicians were most likely to be willing to work, followed by nurses, then other health workers. urban or metropolitan area workers were less likely to be willing to work than rural area workers. full-time workers were more likely to be willing to work than parttime employees. respondents living with children or having childcare obligations were one-third less likely to be willing to work compared with those without these obligations. one study identified that pregnancy in a family member reduced willingness to work. 25 marital status (not meta-analysed) did not influence willingness to work. perceived personal safety at work and perception of pandemic risk (aware that a pandemic was likely) were both associated with increased willingness to work. likewise, the provision of protective measures (mainly personal protective equipment) increased willingness to work, although metaanalysis was abandoned due to high heterogeneity (i 2 = 97á8%). training in pandemic preparedness, general and specific role knowledge, confidence in personal skills, good communication skills and perception of role importance all had positive effects on willingness to work. confidence in employers as judged by 'belief that the employer can provide timely information' also positively influenced willingness to work, although meta-analysis was abandoned due to high heterogeneity. the funnel plot of the percentage of hcws willing to work did not present a clear funnel shape, appeared to scatter widely without any detectable association with the standard error and overflowed the false 95% ci range. egger's regression test reached statistical significance and showed that studies reporting a lower percentage were more likely to be published (p = 0á004). funnel plots and egger's regressions tests pertaining to meta-analyses of factors associated with willingness to work revealed no evidence of publication bias except for previous training and comparison of physicians and nurses (see table 2 ), which suggested possible underreporting of studies with an adverse result. this study advances knowledge from previous reviews on willingness to work during influenza pandemics by adding further new studies and subjecting the findings to statistical evaluation where possible. the search was conducted comprehensively and yielded 43 studies from 11 countries. however, quality of the included studies was not uniformly high and excessive statistical heterogeneity prevented metaanalysis of the primary outcome measure. whilst it was not possible to identify a single clear source of the heterogeneity encountered, almost certainly the wide variation in settings, scenarios and respondents contributed significantly. metaanalysis suggested that sex and job category would affect willingness to work although studies varied greatly in the composition of their samples. hypothetical scenarios varied in virulence, stage and the amount of information provided to respondents. studies of influenza a(h1n1)pdm09 were conducted at different junctures during the evolution of the 2009-10 pandemic. there was no consistency in terms of how respondents were asked about their willingness to work, and the design of questionnaires used to collect outcome data from respondents varied between studies. remarkably, despite such high heterogeneity, some factors emerged showing a consistent association with willingness to work. whilst previous reviews suggested these from a narrative approach, this study has confirmed them statistically. being male, a physician or nurse (especially the former), and a full-time worker were all positively associated with willingness to work. these factors are essentially nonmodifiable; without access to the raw data, we could not disentangle any potential confounding between being male and the likelihood of being a physician or full-time worker in studies providing only unadjusted ors. nevertheless these were consistent findings across most studies and firm knowledge that these are reliable and statistically proven influencers of willingness to work is important information for both policy makers and healthcare service managers, even though they are difficult factors to influence. childcare obligation was a consistent barrier to hcws' willingness to work. the importance of this factor may be an artefact of the high participation of women in the hcw workforce in most countries, combined with traditional cultural expectations that they will take primary responsibility for childcare. it is, nevertheless, an important finding for managers. it is not clear whether this is driven mainly by practicality, that is the need to provide childcare at home, or by concerns about whether the safety of children might be compromised by infection brought in from the parental workplace. paradoxically, the evidence that hcws are at increased risk of influenza infection is rather mixed and somewhat inconsistent, 26 whereas the evidence that children (rather than adults) are usually the introducers of influenza infection into households is firmly established. 27 this question should be further investigated because it has implications for appropriate organizational responses. if it is simply a practical matter, then managers need to consider what help could be given in emergencies through the expansion of onsite or community childcare provision. if it is a concern about cross-infection, then appropriate education and information programmes may resolve the problem. in either case, it is unlikely that simple disciplinary sanctions will be effective, because of the social force of parental obligations. indeed, these may well be counterproductive, if other workers perceive them to have been unreasonably applied by managers unsympathetic to real personal dilemmas. confidence in safety, risk perception, prior training, general and role knowledge and confidence in skills were statistically proven facilitators for willingness to work. these are all addressable by detailed pandemic preparedness educational activities at healthcare unit level. importantly, one message arising from assessments of pandemic planning activities prior to the 2009-10 pandemic was that whilst national level pandemic planning was generally successful, the level of planning at local level was insufficient, including training on pandemic influenza for hcws. 28 a particular feature of pandemics is the level of anxiety provoked by the disruption of 'business as usual' and the destabilization of usually stable organizational environments. 29 whilst it is not necessary to retrain hcws frequently, this is a topic that should be addressed in their basic education and managers should ensure that updating materials are readily available, and regularly revised, so that programmes can rapidly be rolled out when a pandemic is identified. evidence of organizational preparedness will contribute to the confidence of hcws that they will not be placed at undue risk by being asked to work in different ways or in different environments from those that they are accustomed to. a number of limitations with the present study warrant discussion. our literature search was limited to records published in english. therefore, we cannot exclude the possibility of having omitted outcome data published in other languages. many of the included studies were at moderate or high risk of bias. moreover, only a small number were available for analysis in relation to some risk factors; these results should be interpreted cautiously. the possibility of publication bias might also be a limitation. however, considering that the percentage of willingness was relatively high in most studies, this suggests that unpublished data may not have found statistically significantly higher percentages of willingness to work. whilst some studies used questionnaires based on recognized psychological theories, these were commonly 'fear-appeal' theories. unfortunately, this may not be appropriate as the preferable behaviour (working during an influenza pandemic) would not result in release from personal fear. 30 we did not identify any studies that investigated the interaction between individual and organizational responses, which biased the findings towards individual fears rather than the social conditions that might provoke or alleviate these. as important as our specific results themselves, is the fact that we identified a multiplicity of approaches to studying the issue of hcw willingness to work during a pandemic; mainly small, ad hoc enquiries, not based on any consistent scenarios or theoretical approaches. to solve this, a consistent methodological framework is needed before any further studies are undertaken. the outbreaks of ebola virus disease in west africa and mers-cov in the middle east offer two very different settings in which to improve study designs and understanding of hcws' willingness to work where infectious disease creates appreciable personal risk. in the meantime, policy makers should recognize that hcw willingness to work during an influenza pandemic is likely to be improved by practical measures to support childcare responsibilities and by the timely provision of relevant and high-quality training and information as a pandemic develops. whilst the above would hold true for influenza, the actual risks and perceptions are not consistent across all novel respiratory viruses. for example, 5% of nurses in ontario refused to work during the sars crisis when the risk to hcws was almost exclusively nosocomial (compared with pandemic influenza where the risk is community-wide). 31 similarly, in the ongoing mers-cov epidemic, the risk of nosocomial infection is presently greater than in wider community settings. 32, 33 conclusions hcws' willingness to work during an influenza pandemic is moderately high although highly variable, and substantial statistical heterogeneity precluded formal meta-analysis. numerous risk factors are associated with willingness of hcws to work during an influenza pandemic, revealing potential points of intervention to increase willingness to work. we identified a wide variety of approaches to the study of willingness to work. for improved future understanding, we advocate a coordinated global approach with standardized protocols and based on appropriate theoretical constructs; and the evaluation of packages of intervention through controlled studies. additional supporting information may be found in the online version of this article: table s1 . full electronic search strategy (medline). figure s1 . summary of risk of bias of included studies using the modified newcastle-ottawa scale (n = 43). data s1. prisma 2009 checklist. pandemic influenza preliminary estimates of mortality and years of life lost associated with the a/h1n1 pandemic in the us and comparison with past influenza seasons world health organization. pandemic influenza preparedness and response: a who guidance document. geneva: world health organization america's forgotten pandemic estimating the burden of 2009 pandemic influenza a (h1n1) in the united states influenza-associated hospitalizations in the united states complications among adults hospitalized with influenza: a comparison of seasonal influenza and the 2009 h1n1 pandemic pandemic influenza and hospital resources pandemic 2009): how prepared are healthcare providers in calabar, nigeria? the gp's response to pandemic influenza: a qualitative study willingness of health care personnel to work in a disaster: an integrative review of the literature healthcare workers willingness to work during a pandemic factors associated with the willingness of health care personnel to work during an influenza public health emergency: an integrative review healthcare workers' willingness to report to work during an influenza pandemic: a systematic literature review an international registry of systematic-review protocols are healthcare workers' intentions to vaccinate related to their knowledge, beliefs and attitudes? a systematic review meta-analysis in clinical trials measuring inconsistency in meta-analyses influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective graphical display of estimates having differing standard errors bias in meta-analysis detected by a simple, graphical test will the community nurse continue to function during h1n1 influenza pandemic: a cross-sectional study of hong kong community nurses? perspectives of future physicians on disaster medicine and public health preparedness: challenges of building a capable and sustainable auxiliary medical workforce will emergency health care providers respond to mass casualty incidents? how would australian hospital staff react to an avian influenza admission, or an influenza pandemic incidence of influenza in healthy adults and healthcare workers: a systematic review and metaanalysis estimating household and community transmission parameters for influenza who regional office for europe. recommendations for good practice in pandemic preparedness: identified through evaluation of the response to pandemic (h1n1) 2009. copenhagen: who regional office for europe introduction: why a sociology of pandemics? putting the fear back into fear appeals: the extended parallel process model spring of fear, chapter 6: the nurses' survey hospital-associated outbreak of middle east respiratory syndrome coronavirus: a serologic, epidemiologic, and clinical description hospital outbreak of middle east respiratory syndrome coronavirus gauging us emergency medical services workers' willingness to respond to pandemic influenza using a threat-and efficacy-based assessment framework assessment of local public health workers' willingness to respond to pandemic influenza through application of the extended parallel process model assessment of medical reserve corps volunteers' emergency response willingness using a threat-and efficacy-based model determinants of emergency response willingness in the local public health workforce by jurisdictional and scenario patterns: a cross-sectional survey nurses' ability and willingness to work during pandemic flu health care workers' ability and willingness to report to work during public health emergencies hospital health care workers' understanding of and attitudes toward pandemic influenza in beijing who will show up? estimating ability and willingness of essential hospital personnel to report to work in response to a disaster will they just pack up and leave?"-attitudes and intended behaviour of hospital health care workers during an influenza pandemic characterizing hospital workers' willingness to report to duty in an influenza pandemic through threat-and efficacy-based assessment ethical planning for an influenza pandemic survey study of the knowledge, attitudes, and expected behaviors of critical care clinicians regarding an influenza pandemic hospital disaster staffing: if you call, will they come? survey of alberta family physicians' reactions to the spring and summer phase of ph1n1 (pandemic swine flu) outbreak willingness of university nursing students to volunteer during a pandemic preparing for an influenza pandemic: healthcare workers' opinions on working during a pandemic willingness of frontline health care workers to work during a public health emergency factors associated with the ability and willingness of essential workers to report to duty during a pandemic will the nhs continue to function in an influenza pandemic? a survey of healthcare workers in the west midlands healthcare workers' perceptions of the duty to work during an influenza pandemic evaluation of a pandemic preparedness training intervention of emergency medical services personnel pre-pandemic planning survey of healthcare workers at a tertiary care children's hospital: ethical and workforce issues anticipated behaviors of emergency prehospital medical care providers during an influenza pandemic assessing public health department employees' willingness to report to work during an influenza pandemic local public health workers' perceptions toward responding to an influenza pandemic senior clinical nurses effectively contribute to the pandemic influenza public health response pandemic-related ability and willingness in home healthcare workers mitigating absenteeism in hospital workers during a pandemic knowledge and anticipated behavior of health care workers in response to an outbreak of pandemic influenza in georgia a national survey of emergency nurses and avian influenza threat are belgian senior medical students ready to deliver basic medical care in case of a h5n1 pandemic ensuring adequate human medical resources during an avian influenza a/h5n1 pandemic nurses' fears and professional obligations concerning possible human-to-human avian flu survey of hospital healthcare personnel response during a potential avian influenza pandemic: will they come to work knowledge and attitudes of healthcare workers in chinese intensive care units regarding 2009 h1n1 influenza pandemic perception, attitudes and knowledge regarding the 2009 swine-origin influenza a (h1n1) virus pandemic among health-care workers in australia influenza vaccination and intention to receive the pandemic h1n1 influenza vaccine among healthcare workers of british columbia, canada: a cross-sectional study nurses' perspectives and concerns towards an infectious disease epidemic in egypt influenza a h1ni (pandemic 2009): how prepared are healthcare providers in calabar, nigeria? factors associated with motivation and hesitation to work among health professionals during a public crisis: a cross sectional study of hospital workers in japan during the pandemic (h1n1) we thank the authors of the articles cited in this paper. we also thank nicola darlington (university of nottingham) for assistance with developing the search terms and john mair jenkins (health education east midlands) and roshni joshi (university of nottingham) for help with manuscript preparation. this research was supported by the university of nottingham as a master of public health dissertation project. jsn-v-t and crb are respectively editor-in-chief and associate editor for influenza and other respiratory viruses; however they played no role whatsoever in the editorial process for this paper, including decisions to send the manuscript for independent peer-review or about final acceptance of a revised version. all of the above functions were handled alone by dr john wood, senior editor (reviews). key: cord-010416-u0yo0lk6 authors: tejada, sofia; campogiani, laura; solé-lleonart, candela; rello, jordi title: alternative regimens of neuraminidase inhibitors for therapy of hospitalized adults with influenza: a systematic review of randomized controlled trials date: 2020-04-28 journal: adv ther doi: 10.1007/s12325-020-01347-5 sha: doc_id: 10416 cord_uid: u0yo0lk6 introduction: influenza in hospitalized intensive care unit (icu) patients with respiratory failure is associated with 25% mortality, despite timely oseltamivir treatment. a systematic review of randomized controlled trials (rcts) was conducted to evaluate the efficacy and safety of alternative neuraminidase inhibitor (nai) regimens compared to standard of care in patients hospitalized for h1n1, h3n2, or b influenza. methods: the cochrane collaboration searching methods were followed in cochrane library, pubmed, and web of science databases (2009–2019). eligibility criteria were rcts comparing different regimens of nais in hospitalized patients (at least 1 year old) for clinically diagnosed influenza (h1n1, h3n2, or b). pre-defined endpoints were time to clinical resolution (ttcr), overall mortality, hospital discharge, viral clearance, drug-related adverse events (aes), and serious adverse events. results: seven trials (1579 patients) were included. two trials compared two regimens of oral oseltamivir therapy, and one trial compared two regimens of intravenous zanamivir therapy vs oral oseltamivir therapy. four trials focused on intravenous peramivir therapy: two trials compared two different regimens and two trials compared two different regimens vs oral oseltamivir therapy. overall, the different regimens were well tolerated, with no significant differences in aes; nonetheless non-significant differences were reported among different regimens regarding ttcr, mortality, and viral clearance. conclusion: higher compared to standard doses of nais or systemic peramivir therapy compared to oral oseltamivir therapy did not demonstrate benefit. electronic supplementary material: the online version of this article (10.1007/s12325-020-01347-5) contains supplementary material, which is available to authorized users. influenza virus infection is a worldwide problem and it is the leading cause of respiratory viral disease in hospitalized patients [1] [2] [3] . both the world health organization (who) and the european centre for disease control (ecdc) recommend the use of neuraminidase inhibitors (nai) for hospitalized adults with influenza [4] . the infectious diseases society of america (idsa) released guidelines on influenza management in 2018, identifying nais as first-line therapy in hospitalized patients regardless of illness duration prior to hospitalization, with no differences between oral oseltamivir therapy, intravenous peramivir therapy, or inhaled zanamivir therapy [5] . while there is a consensus on dosing and duration of treatment for outpatients and high-risk population, management of influenza treatment in hospitalized and severely ill patients is suboptimal. in spite of early initiation of nais, mortality rates exceed 25% in primary influenza pneumonia with acute respiratory failure (arf). critically ill patients are characterized by a variety of conditions that may alter drug absorption, like altered gastrointestinal motility, and pharmacokinetics, such as the need for renal replacement therapy or extracorporeal membrane oxygenation. furthermore, in mechanically ventilated patients, administration of inhaled zanamivir is contraindicated because of reported fatal complications, and oseltamivir has to be administered via nasogastric tube [6] . the idsa recommends against the routine use of higher doses of food and drug administration (fda)-approved nai drugs for therapy of seasonal influenza [5] . double oseltamivir dose has been used as salvage therapy in presence of arf in some settings, but robust data are lacking [7] . peramivir is the only fda-approved intravenously administered drug for influenza, but optimal dosing regimen remains controversial [5] . to date, there are no unanimous data on nai use for the treatment of hospitalized patients because treatment dosing, administration route, and duration are still debated in these patients, who require intensive care admission, and effect on outcomes and safety of different therapies is not clear. the hypothesis was that in patients admitted to hospital with influenza infection, the optimization of nai administration may improve outcomes. thus, the study's aim was to perform a systematic review (sr) of randomized controlled trials (rct) to evaluate the efficacy and safety of alternative nai regimens compared with 75 mg orally administered oseltamivir twice/daily or 600 mg intravenously administered peramivir once/daily in patients hospitalized for h1n1, h3n2, or b influenza. this report describes the results of the sr following the guidance of the preferred reporting items for systematic reviews and meta-analyses (prisma) statement [8] . pico (population, intervention, comparator, outcome) questions are detailed in the supplementary material 1. the protocol was published in the national institute for health research international prospective register of systematic reviews (prospero), registration number crd42018110060. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. a global search strategy was systematically performed in three databases: medline database through the pubmed search engine, the cochrane library database, and web of science database. websites from clinicaltrials.gov and clinicaltrialsregister.eu were consulted for other ongoing trials. search terms were detailed in the supplementary material 2. restrictions in the search were applied regarding the language: only studies published in english, spanish, french, italian, and portuguese were considered. abstracts presented at scientific conferences, unpublished studies, and other unpublished data deriving from industry sites were excluded. a restriction was also applied to the publication period of time, between 2009 and 2019, partially because before 2009 there were no diagnostic tests of influenza and also since the outbreak of a/h1n1 in 2009 [9] , the use of nais has increased. the first search was performed in january 2019 and repeated in november 2019. two authors (st and lc) independently evaluated all the studies identified in the literature search by screening their titles, abstracts, and full text. in case of disagreement, a third author (csl) independently determined eligibility. a predesigned spreadsheet was used to collect study data in a standardized way. data extracted from each trial included were the study design, quality assessment, characteristics of the study populations, method used for confirmation of the influenza infection, characteristics of compared treatment arms, the intention to treat (itt) population and the subgroup of patients with laboratory-confirmed influenza infection, as well as data regarding the effectiveness and safety outcomes. studies were considered eligible for inclusion in the sr if they were a rct that enrolled patients older than 1 year of age, requiring hospitalization with clinically diagnosed influenza (with h1n1, h3n2, or b) or influenza-like syndrome, with or without laboratory confirmation. pre-defined treatments for inclusion were oseltamivir (oral administration), zanamivir (oral, intravenous, or inhaled administration), peramivir (oral or intravenous administration), and laninamivir (inhaled administration). studies involving children less than 1 year old, nais against other serotypes of influenza such as h5n1, pregnant women, immunocompromised patients (more than 30% of the overall population), or outpatients were excluded. also, observational cohort studies or studies with different intervention of nais such as polymerase inhibitors (baloxavir marboxil) treatment were excluded. clinically suspected influenza was defined by the presence of respiratory symptoms (sore throat, cough, nasal congestion) and fever (c 37.7°c) within 48 h of study enrollment, regardless of prior symptoms duration. influenza infection was defined by the presence of a positive polymerase chain reaction (pcr), immunofluorescence assay, or rapid antigen test (rat) for influenza virus. the itt population included all patients randomized to receive the respective study regimens. the influenza-positive population included only patients with confirmed influenza. time to clinical resolution (ttcr), defined by the individual study protocol as the time from initiation of the study treatment until resolution of vital sign abnormalities (the supplementary material 3), and overall mortality were considered as the primary effectiveness outcomes of this sr. secondary effectiveness outcome was viral clearance, defined as the proportion of influenza virus-negative patients detected by pcr on nasopharyngeal samples at 5 day. samples analyzed with different methods (e.g., viral culture) or at different time frames were excluded from the comparison. safety was evaluated in terms of occurrence of respiratory and/or systemic drug-related adverse events (aes) and serious adverse events (saes). risk of bias was assessed for each included study independently by st on the basis of the cochrane handbook for systematic reviews of interventions [10] and using the cochrane review manager 5.3 risk of bias tool which takes account of allocation sequence generation, concealment of allocation, masking of participants and investigators, incomplete outcome reporting, selective outcome reporting, and other sources of bias. each potential source of bias was graded to determine whether studies were considered at high, low, or moderate risk of bias. in case of disagreement, a second author (csl) independently determined the quality assessments. for categorical outcomes, the numbers of patients who had each outcome and denominator were extracted, and for continuous outcomes, sample size, mean [standard deviation (sd)], or median [interquartile range (iqr)] were extracted on the basis of the information provided within studies. where results were not reported in the same format for analysis, we used recommended methods from the cochrane collaboration to extract or estimate effects including contacting study authors and using formulae for conversion of medians (iqr) to estimated mean (sd) as previously described [11] . a total of 6692 studies were identified: 5732 studies in the medline (pubmed), 563 in web of science, and 397 in the cochrane library databases. seven trials and 1579 itt patients were included. the prisma flow diagram of the studies' selection is presented in fig. 1 . a summary of the risk of bias of the included rct is detailed in fig. 2 . a total of seven trials were included, analyzing different nai regimens. main characteristics of the included studies are described in table 1 . two trials focused on oral oseltamivir therapy [12, 13] , comparing high dose (150 mg twice/daily) vs standard dose (75 mg twice/daily). one trial compared two regimens of intravenous zanamivir therapy [14] (300 mg vs 600 mg twice/daily) vs standard dose of orally administered oseltamivir (75 mg twice/daily). two trials compared two different regimens of intravenous peramivir therapy [15, 16] (300 mg vs 600 mg once daily; or 200 mg vs 400 mg once daily) vs standard dose of orally administered oseltamivir (75 mg twice/daily), and two trials of intravenous peramivir therapy [17, 18] compared high dose (600 mg once/daily) vs standard dose (300 mg twice/daily or once/daily). no study analyzed inhaled zanamivir, given the contraindication of its use in severely ill patients on mechanical ventilation [5] . no laninamivir trial respecting all the inclusion criteria was found; hence laninamivir was not included in the sr. a total of 1579 patients were included in the seven analyzed trials. of these, 1312 (83.0%) had confirmed influenza infection, and 205 (12.9%) were vaccinated against influenza. baseline characteristics of the population included are described in table 2 . five trials involved only adult patients (c 16 years of age), whereas the remaining two trials involved children and adults (c 6 years or c 1 year old). a total of 545 (34.5%) patients received other antiviral treatment prior to study drug initiation and 342 (21.6%) patients needed admission to the icu. the most common underlying diseases were chronic obstructive pulmonary disease (copd) (15.0%), diabetes (11.0%), and asthma (7.2%). all data on outcomes extracted from each trial included are presented in table 3 . the median days of clinical resolution was assessed in five studies. the study by lee et al. [12] , focused on oral oseltamivir therapy, reported a non-significant ttcr decrease in the group of patients treated with standard dose twice/daily ( the drug-related aes were assessed in six studies. two studies focused on oral oseltamivir therapy [12, 13] reported a significant decrease of aes incidence in the group of patients treated with low dose twice/daily in one study [12] ( this is the first sr of rcts that have evaluated the efficacy and safety of different dosage and/ or regimens of systemic nais in an important clinical and public health challenge, such as hospitalized patients with seasonal or pandemic influenza. our data suggest that alternative regimens are safe to use in a hospitalized population but do not significantly change mortality in the efficacy analyses. also, the evidence is inconclusive for other meaningful outcomes, such as ttcr or viral clearance. our findings confirm the variability of efficacy of antiviral treatment regimen for severe hospitalized patients with influenza infection. several sr, including both rct and observational studies, conducted in the past years have addressed the efficacy and safety of nai treatment, demonstrating the effectiveness of nai treatment to reduce severity of influenza in outpatients, and mortality in hospitalized patients, compared without treatment [19] [20] [21] [22] [23] . furthermore, it is widely accepted that the efficacy of nai treatment is higher if administered within 48 h from symptoms onset [5] . nonetheless, given the variety of the population enrolled in the published studies, involving both in-and outpatients, treated with different nai regimens, no consensus exists on which nai represents the best option in hospitalized patients with influenza. in a meta-analysis [23] of individual participant data in 29,234 hospitalized patients from 78 studies with influenza a (h1n1)pdm09 with infection, nai therapy was associated with a reduction of mortality in the subgroup of icu patients, compared with no treatment. moreover, treatment within 2 days of symptoms onset was associated with a reduction in mortality compared with the late administration. in our sr, time from symptoms onset to nai treatment was heterogeneous among the included studies, from 48 h to 10 days, with median duration of illness from 2 to 5 days, adding a confounding factor. standardized rct protocols might help in reducing controllable variables, to equalize studies conducted in different settings, and further investigate nai time-efficacy. oseltamivir, zanamivir, peramivir, and laninamivir are the nais currently available, approved for a variety of indications and formulations by the different regulatory agencies (table 4 ). oral oseltamivir therapy is approved to treat patients with uncomplicated influenza by both the fda and european medicines agency (ema); no information is available on safety and efficacy in hospitalized patients [24] . for severely ill patients, double dose oseltamivir has been used in some settings but robust data on its efficacy are lacking, and guidelines recommend against its use [7] . a recent study [25] among adult patients with pandemic influenza requiring ventilator support concluded that oseltamivir had a good enteric absorption, and the dosage of 75 mg twice daily achieved adequate plasma concentrations, far in excess of those required to inhibit viral neuraminidase activity. accumulation of oseltamivir in patients with extracorporeal membrane oxygenation and continuous venovenous hemodiafiltration lead to 4-to 5-fold increase in plasma levels [26] . if oral or enteral administration of oseltamivir is impossible or its absorption is altered, intravenously administered nais might be used. zanamivir is typically used as inhaled drug in outpatients, but lack of safety in subjects with airway diseases limits its use in hospitalized and mechanically ventilated patients [5] . on the basis of the trial from marty et al. [14] , the ema approved the use of intravenously administered zanamivir 600 mg twice/daily in complicated influenza [5] ; this formulation is not fda approved and not included in the latest idsa guidelines. given the recent introduction, to date there are only a few anecdotical case reports and a small case series of four icu patients treated with intravenous zanamivir therapy, but they showed a high efficacy and tolerability [27, 28] . intravenously administered zanamivir could represent a good therapeutic option in severely ill patients with influenza infection, not only when oral or aerosolized antiviral medication cannot be administered but also in the unlikely event of oseltamivir resistance. in these cases, intravenous peramivir therapy might also be considered, but it is only approved for uncomplicated influenza and no consensus has been reached on the appropriate dosing and duration of treatment [5] . a recent sr [19] confronted intravenous peramivir therapy vs oral oseltamivir therapy demonstrating peramivir efficacy in reducing ttcr only in outpatients, with no differences in mortality and length of hospital stay for both in-and outpatients. to date, european guidelines do not include indication for intravenously administered peramivir at dosages different from 600 mg single administration in outpatient settings, while idsa guidelines suggest to consider administering a multiday dosing regimen, although the optimal regimen is unknown. finally, laninamivir is approved only in japan (2010), used as a single dose aerosol in outpatients, with no data available in inpatients [29] . among the different nais available for treating patients with influenza, no consensus has been reached about which regimen should be recommended to treat hospitalized patients. comorbidities, clinical conditions, and clinical setting might play an important role in guiding nai choice. new drugs are being developed, and studied in severe hospitalized patients: baloxavir marboxil is a novel polymerase inhibitor approved in japan, the usa, and other countries. two phase iii trials [30, 31] in non-hospitalized patients with placebo found that single dose was superior to placebo in alleviating influenza symptoms, and was superior to both oseltamivir and placebo in reducing viral replication. a double-blind rct (nct03684044) comparing the combination of oseltamivir and baloxavir marboxil to oseltamivir alone is currently in progress in hospitalized patients. limitations should be considered when interpreting the results of this systematic review. we judged that the included studies were generally of low quality based upon the selection bias. the main limitation is the heterogeneity in dosage and comparators that precluded a meta-analysis, as well as and the size of the study population (large rcts are needed) and the inclusion of clinically diagnosed influenza in two studies. despite identifying many studies (e.g., trials with outpatients or observational studies), there were few rcts about hospitalized patients with influenza treated with nais. none of the included studies assessed the penetration of antivirals into the lung tissue or analyzed the effect of antiviral concentrations on alveolar viral load. no study involving laninamivir met the inclusion criteria. finally, only a small percentage of mechanically ventilated (mv) patients with acute respiratory distress syndrome (ards) or pneumonia were enrolled, and the impact of viral susceptibility on treatment could not be analyzed because of the scarcity of data. even if rare, nai resistance might influence the outcomes of different treatment regimens. only four out of seven studies analyzed viral strain susceptibility pre-treatment, and six studies conducted a post-treatment analysis, with overall only four new resistances identified. the small numbers did not allow a correlation with clinical outcomes; furthermore, different analysis methods were used, not allowing a standardized comparison. despite these limitations, our study provides information that is not available in the published literature, being an important strength and having implications for further research. furthermore, the results were based on rcts, rather than observational cohort studies, so that it illustrates the need for research in the form of rcts in the subset of patients with respiratory failure requiring hospitalization or icu admission, focusing on meaningful pre-defined outcome criteria. the evidence evaluated in this sr indicates that the alternative nai regimens to orally administered oseltamivir 75 mg twice/daily or intravenously administered peramivir 600 mg once/daily to treat hospitalized patients with influenza infection are equally safe but do not modify meaningful clinical outcomes when compared with the standard dose. disclosures. jordi rello served as a consultant and received grant support from genentech and roche. jordi rello is a member of the journal's editorial board. sofia tejada, laura campogiani and candela solé-lleonart have nothing to declare. compliance with ethics guidelines. this article is based on previously conducted studies and does not contain any studies with human participants or animals performed by any of the authors. data availability. all data and materials used in this research are freely available. references have been provided. open access. this article is licensed under a creative commons attribution-noncommercial 4.0 international license, which permits any noncommercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/ licenses/by-nc/4.0/. disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (daly) model influenza and other respiratory viruses: standardizing disease severity in surveillance and clinical trials reviewing the history of pandemic influenza: understanding patterns of emergence and transmission. pathogens public health use of influenza antivirals during influenza pandemics. ecdc interim guidance clinical practice guidelines by the infectious diseases society of america: 2018 update on diagnosis, treatment, chemoprophylaxis, and institutional outbreak management of seasonal influenzaa the emerging influenza virus threat: status and new prospects for its 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neuraminidase inhibitors for the treatment of influenza influenza virus polymerase inhibitors in clinical development phase 3 trial of baloxavir marboxil in high-risk influenza patients (cap-stone-2 study) this study was part of the doctoral thesis from st at the medicine department, university of barcelona. funding. this work was funded by ciberes, instituto salud carlos iii, madrid, spain (fondos feder) (cb06-06-036). no funding or sponsorship was received for the publication of this article.authorship. all named authors meet the international committee of medical journal editors (icmje) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. key: cord-006345-03kqeed3 authors: takayama, koji; kuramochi, jin; oinuma, takeshi; kaneko, hiromi; kurasawa, satoshi; yasui, makito; okayasu, kaori; ono, hiroshi; inase, naohiko title: clinical features of the 2009 swine-origin influenza a (h1n1) outbreak in japan date: 2010-12-21 journal: j infect chemother doi: 10.1007/s10156-010-0187-9 sha: doc_id: 6345 cord_uid: 03kqeed3 to clarify the clinical symptoms of the influenza a virus during the 2009 pandemic influenza outbreak, we describe the clinical features of outpatients diagnosed with type a influenza by use of the rapid influenza diagnostic test (ridt) from september to december 2009. questionnaires were used to collect prospective data on 1,122 cases with influenza-like illness at our medical institutions. the independent predictors of influenza a virus were identified on the basis of demographic features and the clinical symptoms of the patients who tested positive for influenza a virus in the ridt test. of the 1,122 cases tested, 389 (34.7%) were positive for the influenza a virus. the median age of the influenza-positive patients was 14, and 58.9% of the patients were male. the symptoms fever, cough, rhinorrhea, and headache were statistically dominant. a history of recent contact with persons suffering from influenza or influenza-like illness at home, school, or in the workplace was significantly more common in the positive group than in the negative group. pneumonia was observed in 2 (0.5%) of the positive patients, but the symptoms were only severe enough to require hospitalization in 1 of the 2. no deaths were observed among the 389 ridt-positive patients. although the spread of influenza a virus was both rapid and extensive, mainly among children under the age of 18, it seemed to be mild. appropriate interpretation of the ridt on the basis of recent clinical information, and early treatment with antiviral drugs might help to prevent severe illness from influenza pandemics in the future. the first three cases of swine-origin influenza a (h1n1) virus in japan were reported by the ministry of health, labour and welfare on may 9, 2009 [1] . all three of the cases were travelers who had recently returned from ontario, canada, via detroit, usa. following this initial report, the number of confirmed cases steadily rose. about a month later, on june 11, 2009 , the world health organization (who) [2] raised the level of influenza pandemic alert from phase 5 to phase 6, the highest level. for a period of several months, many articles on the influenza pandemic were published in different parts of the world. according to these reports, the symptoms, disease severity, and mortality of the pandemic influenza virus seem to have differed from nation to nation. available findings highlight the importance of early use of antiviral drugs. in japan, antiviral drugs for influenza, including both oseltamivir and zanamivir, are available at all medical institutions. our group prospectively identified the demographic features and clinical symptoms of patients who tested positive for influenza a virus in the rapid influenza diagnostic test (ridt) in japan from september to december 2009. according to the japanese infectious agents surveillance report from the infectious disease surveillance center (idsc), isolation and detection of pandemic influenza from the influenza sentinel clinics started to increase from august 2009, attained its peak in late october 2009 and then gradually declined [3] . the proportion of pandemic influenza detected and isolated during our 4-month investigation period made up approximately 60% of the total cases reported between may 2009 and may 2010. then we determined the independent predictors of positive testing in the ridt, which has been in wide commercial use in clinical practice worldwide. a questionnaire was used to collect data prospectively from patients who visited three medical institutions (two clinics and one hospital) because of influenza-like illness (self-reported fever with cough, sore throat, or both) in utsunomiya, which is approximately 100 km north of tokyo and the capital of tochigi prefecture, japan. in 2009, the city had an estimated population of 510,000. in total, 1,122 tests were performed after informed consent was obtained and 190 patients were examined more than twice during our investigation period. out of the 1,122 cases tested, 608 (54.2%) were male. the age of the patients ranged from 10 months to 94 years (median 18, interquartile range 10-34). the clinical features were compared between the patients who tested positive and negative for the influenza a virus in the ridt. after diagnosis, all of the patients were given warning leaflets with the emergency phone numbers of our institutions. the study conformed to the declaration of helsinki and was approved by the internal review boards of our institutions. informed written consent was obtained from every subject. a patient questionnaire was used to obtain detailed information in advance. the information covered in the questionnaire was: name, sex, age, date of birth, time from the onset of symptoms, maximum body temperature, occupation or school, family structure, and symptoms (headache, nausea, vomiting, diarrhea, abdominal pain, cough, sputum, sore throat, rhinorrhea, muscle pain, arthralgia, dyspnea, general fatigue, and anorexia). the patients were asked to indicate whether they had come into contact with other persons with influenza-like illness around them, and if so, to indicate whether or not those persons had been diagnosed with influenza. clearview exact influenza a and b (inverness medical japan, tokyo, japan) or capilia flu a ? b (alfresa pharma corporation, osaka, japan) was used according to the manufacturers' instructions. all reported values are medians with interquartile ranges. we compared clinical features between cases positive for influenza a virus and cases negative for influenza a virus by using the chi-squared test or fisher's exact test for dichotomous categorical variables, as appropriate. continuous data were tested by means of the mann-whitney u test. multiple logistic-regression analysis was used to identify independent predictors of positive testing for the influenza a virus in the ridt. the outcome was predicted on the basis of factors such as age, sex, maximum body temperature, presence or absence of symptoms, and contact with persons with influenza-like illness or influenza. we first included factors which were selected if the p value in the univariate analysis was.20. we then used backwardelimination techniques. a p value of less than 0.05 was considered to indicate statistical significance. all statistical analyses were performed using excel statistics 2008 (ssri, japan). over the period from september to december 2009, a total of 1,122 cases with influenza-like illness were seen at our medical institutions and tested by use of the ridt. positive results were obtained from 34.8% (390 cases) of the patients tested. only one of 390 patients was positive for influenza b antigen, and this patient was excluded from the analysis. none of the patients tested positive in the ridt more than once. the median age of the positive patients was 14 years (range 1-68 years). among the 389 positive patients, 72.2% (281 patients) were younger than 18 years and only 3.1% were 51 years of age or older. in total, 229 (58.9%) of the positive patients were male, and the difference between the percentages of males and females was significant. the median time from the onset of symptoms to the positive result in the ridt was 24 h (interquartile range 14.5-24). the maximum body temperature was significantly higher in the positive group (median 38.5°c, interquartile range 38-39) than in the negative group (p \ 0.001) ( table 1 ). as shown in table 2 , symptoms of cough, headache, and rhinorrhea were present in 81.7, 51.4, and 48.8% of the patients in the positive group, respectively. the incidence of diarrhea was quite low. incidence of muscle pain and arthralgia did not differ significantly between the two groups. impaired consciousness was not observed in any of the patients in our series. a history of recent contact with persons suffering from influenza-like illness or influenza at home, school, or in the workplace was significantly more frequent in the positive group than in the negative group, (influenza-like illness 74.3 vs. 47.1%, p \ 0.001; influenza 62.2 vs. 39.9%, p \ 0.001). pneumonia were recognized in only two patients from the positive group. one of these patients was admitted to a university hospital and definitively diagnosed with pandemic influenza by a reverse-transcriptase-polymerasechain-reaction (rt-pcr) assay. oseltamivir was administered to 284 (73.6%) of the positive patients and zanamivir was administered to 98 (25.4%) of the positive patients. four of the patients received neither oseltamivir nor zanamivir. of the 389 positive patients, 94.9% visited one of our institutions within 48 h of symptom onset and received either oseltamivir or zanamivir immediately. no deaths were observed among the 389 ridt-positive patients. in addition, more than 20% of the patients who tested negative in the ridt also received antiviral drugs. nausea and vomiting occurred in 3 patients, all of whom were taking oseltamivir. no severe adverse effects were reported. multivariable logistic-regression analysis identified the following as independent risk factors for positive ridt findings for the influenza a virus: age less than 30 years (odds ratio (or) 2. 16 two cases of influenza-associated pneumonia a 7-year-old, previously healthy girl, developed a temperature of 39°c in association with a headache 2 days before visiting our clinic. the oxygen saturation was 94% in ambient air when she visited our clinic. rhonchi were audible in the right upper lung field. chest computed tomography (ct) scans revealed ground-glass opacity and thickened bronchial walls in the right upper lobe, and scattered ground-glass opacities in both lung fields (fig. 1a, b ). an ridt was positive for influenza a virus. she was admitted to a university hospital near our institutions and treated with oseltamivir, broad-spectrum antibiotics, and corticosteroids. she was discharged, in improved condition, on the 7th hospital day. pandemic influenza a virus was confirmed by a rt-pcr assay. a 35-year-old woman with allergic rhinitis visited our clinic after a 2-day history of fever, productive cough, and sore throat. an ridt was positive for the influenza a virus. ct scans revealed localized ground-glass opacity in the left lower lobe. she was treated with oseltamivir and recovered fully in 3 days. according to the japanese infectious agents surveillance report from the idsc, 96% of the influenza viruses detected and isolated from specimens collected from influenza cases by may 2010 were pandemic influenza [3] . moreover the proportion of pandemic influenza to all influenza virus is 99% only during our investigation period. therefore almost all of the patients who tested positive for the influenza a virus in the ridt were thought to have been infected with pandemic influenza in japan. reports from other countries for example mexico [4, 5] , the united states [6] , and australia [7] have described icu admissions for pneumonia, ards, and other severe conditions caused by the recent pandemic influenza virus. most of the affected patients have been young. during our investigation period, more than 70% of the ridt-positive outpatients were under the age of 18 years, and only 3.1% were 51 years of age or older. influenza morbidity was much more common in the young than in the old in our investigation. this was identical to the trend observed for the morbidity of seasonal influenza. one reason proposed for this young age distribution has been the higher risk of fisher's exact test c influenza-like illness is defined as self-reported fever with cough, sore throat, or both influenza exposure in school environments. although seasonal influenza-associated hospitalization rates and mortality are higher among persons 65 years of age or older and young children than among healthy older children and younger adults [8, 9] , none of the patients in our series, including the older adults, deteriorated to serious conditions. chowell et al. [4] reported that low rates of morbidity and mortality from the pandemic influenza among the elderly reflect relatively stronger protection for persons who had been exposed to h1n1 strains during childhood, before the 1957 pandemic. there is reported evidence that exposure to a 1918-like h1n1 virus contributed to the induction of a cross-reactive antibody response to the pandemic influenza virus [10] . similarly, both studies in england and in japan have revealed that serum donors from older adults who had probably been exposed to the 1918 virus or a closely related h1n1 virus carried high levels of neutralizing antibodies against the 2009 h1n1 [11, 12] . the most common symptoms in our patients were fever, cough, and rhinorrhea. however, somewhat fewer patients in our series presented with gastrointestinal disturbances including diarrhea, nausea, and vomiting, compared with the patients reported in the united states [13] and mexico [5] . these previous reports included a number of hospitalized patients who deteriorated into severe conditions as a result of the difficulty in rapid identification of the novel pandemic influenza and delays in the initiation of antiviral drugs during the early phase of the epidemic. therefore, differences in seasonal effects, the length of time from onset to treatment, and the severity of the illness might have affected the incidence of gastrointestinal disturbances. positive findings in the ridt were significantly more frequent in our male patients than in our female patients. among patients with pandemic influenza in china, multivariable logistic-regression analysis identified male gender as an independent risk factors for prolonged infection with the pandemic influenza virus on the basis of real-time rt-pcr test [14] . however, who reports on whether or not the incidence of pandemic influenza infection differs between the sexes remains unresolved [15] . pneumonia was identified in two patients (0.5%) from our ridt-positive group. in contrast with the experience in mexico [5] , the pneumonia was somewhat mild in our two our study has two important limitations. first, most of our cases were not confirmed by rt-pcr. without this diagnostic confirmation, we were unable to determine whether there were any cases with influenza a subtypes other than pandemic influenza. however, the japanese idsc reports that the proportion of pandemic influenza to all influenza virus was 99% during our investigation period [3] . only a very small number of positive patients were thought to suffer from seasonal influenza virus. second, the sensitivity of the ridts is still poor [16] [17] [18] . according to the report and to guidance from the cdc, the specificity of ridts is generally high [16, 17] . when influenza viruses are circulating in a community, a positive test indicates that the specimen is likely to be infected with the influenza virus. on the other hand, the cdc guidance also reports that the sensitivity of ridts for detecting pandemic influenza a virus infections ranges from 10 to 70% compared with rt-pcr. for this reason, false negatives may have led to underestimation of the true burden of the influenza a virus. in conclusion, the spread of influenza a virus during our investigation period was extensive and rapid among children under the age of 18 years. only one patient with viral pneumonia in association with pandemic influenza was hospitalized. the low rate of hospitalization seemed to be attributable to prompt hospital visits, prompt diagnosis, and prompt treatment with antiviral drugs. understanding the limitations of ridt and appropriate interpretation of the results based on the recent clinical information seem to be important in the management of influenza pandemics in the future. world health organization. world now at the start of 2009 influenza pandemic flash report of influenza virus in japan, 2009/10 season (seasonal ? ah1pdm) severe respiratory disease concurrent with the circulation of h1n1 influenza pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico hospitalized patients with 2009 h1n1 influenza in the united states critical care services and 2009 h1n1 influenza in australia and new zealand influenza-associated hospitalizations in the united states cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses household transmission of 2009 pandemic influenza a (h1n1) virus in the united states clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china world health organization. sex, gender and influenza evaluation of rapid influenza diagnostic tests for detection of novel influenza a (h1n1) virus-united states interim guidance for the detection of novel influenza a virus using rapid influenza diagnosis tests rapid-test sensitivity for novel swine-origin influenza a (h1n1) virus in humans key: cord-048448-kfwbqp4p authors: sandrock, christian; kelly, terra title: clinical review: update of avian influenza a infections in humans date: 2007-03-22 journal: crit care doi: 10.1186/cc5675 sha: doc_id: 48448 cord_uid: kfwbqp4p influenza a viruses have a wide host range for infection, from wild waterfowl to poultry to humans. recently, the cross-species transmission of avian influenza a, particularly subtype h5n1, has highlighted the importance of the non-human subtypes and their incidence in the human population has increased over the past decade. during cross-species transmission, human disease can range from the asymptomatic to mild conjunctivitis to fulminant pneumonia and death. with these cases, however, the risk for genetic change and development of a novel virus increases, heightening the need for public health and hospital measures. this review discusses the epidemiology, host range, human disease, outcome, treatment, and prevention of cross-transmission of avian influenza a into humans. human influenza pandemics over the last 100 years have been caused by h1, h2, and h3 subtypes of influenza a viruses. more recently, avian influenza virus subtypes (that is, h5, h7) have been found to directly infect humans from their avian hosts. the recent emergence, host expansion, and spread of a highly pathogenic avian influenza (hpai) h5n1 subtype in asia have heightened concerns globally, both in regards to mortality from hpai h5n1 infection in humans and the potential of a new pandemic. this paper will review the current human infections with avian influenza and their public health and medical implications. influenza a, b and c are the most important genera of the orthomyxoviridae family, casusing both pandemic and seasonal disease in humans. influenza a viruses are enveloped, single-stranded rna viruses with a segmented genome (table 1 ) [1] . they are classified into subtypes on the basis of the antigenic properties of the hemagglutinin (ha) and neuraminidase (na) glycoproteins expressed on the surface of the virus [1, 2] . influenza a viruses are characterized by their pathogenicity, with highly pathogenic avian influenza (hpai) causing severe disease or death in domestic poultry [3] . molecular changes in the rna genome occur through two main mechanisms: point mutation (antigenic drift) and rna segment reassortment (antigenic shift) [4, 5] . point mutations cause minor changes in the antigenic character of viruses and are the primary reason a vaccination for influenza a is given yearly. reassortment occurs when a host cell is infected with two or more influenza a viruses, leading to the creation of a novel subtype. the influenza subtypes of the 1957 (h2n2) and 1968 (h3n2) pandemics occurred through reassortment, while the origins of the 1918 (h1n1) pandemic are unclear. the ha glycoprotein mediates attachment and entry of the virus by binding to sialic acid receptors on the cell surface. the binding affinity of the ha to the host sialic acid allows for the host specificity of influenza a [6, 7] . avian influenza subtypes prefer to bind to sialic acid linked to galactose by α-2,3 linkages, which are found in avian intestinal and respiratory epithelium ( table 2 ) [8] . human virus subtypes bind to α-2,6 linkages found in human respiratory epithelium [8, 9] . swine contain both α-2,3 and α-2,6 linkages in their respiratory epithelium, allowing for easy co-infection with both human and avian subtypes (thus acting as a 'mixing vessel' for new strains) [10] . humans have been found to contain both α-2,3 and α-2,6 linkages in their lower respiratory tract and conjunctivae, which allows for human infections by avian subtypes [9, 11, 12] . the ha glycoprotein is the main target for immunity by neutralizing antibodies. the na glycoprotein allows the spread of the virus by cleaving the glycosidic linkages to sialic acid on host cells and the surface of the virus. the virus is then spread in secretions or other bodily fluids. the na glycoprotein is not the major target site for neutralization of the virus by antibodies. influenza a viruses infect a wide range of hosts, including many avian species, and various mammalian species, such as swine, ferrets, felids, mink, whales, horses, seals, dogs, civets, and humans [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] . wild birds (ducks, geese, swans, and shorebirds) are important natural reservoirs of these viruses, and all of the known 16 ha and 9 na subtypes have been found in these birds [32] [33] [34] [35] . in most cases, these subtypes are found within the gastrointestinal tract of the birds, are shed in their feces, and rarely cause disease [32] . since 2002, however, hpai h5n1 viruses originating in asia have been reported from approximately 960 wild bird species, causing disease in some instances and asymptomatic shedding in others [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] . the virus has now spread across asia, europe, the middle east, and some african countries. additional species, such as tigers, leopards, cats, stone martens, and humans have also become infected with hpai h5n1 [49] . this spread of h5n1 into a wide range of animal and avian species may enhance the spread of the virus into the human population as it interacts with animals in a number of ways (increased land use, markets, consumption) [44] . thus, the potential contact, transmission, and mutability of hpai h5n1 worldwide will increase as the number of species and their interactions increase, complicating prevention, surveillance and treatment possibilities. the incidence of avian influenza infections in humans has increased over the past decade (table 3) . initially, cases of avian influenza (h7n7) in humans occurred in association with poultry outbreaks, manifesting as self-limiting conjunctivitis [30, [50] [51] [52] [53] . then, in 1997, a large scale hpai h5n1 outbreak occurred among poultry in hong kong, with 18 documented human cases [29, 31, 54, 55] . two subsequent poultry outbreaks in hong kong in 1999 and 2003 with hpai h5n1 occurred without human cases until 2003 when two members of a family in hong kong contracted hpai h5n1 [56] . in december of 2003, hpai h5n1 surfaced in poultry in korea and china, and from 2003 to 2006 the outbreak stretched worldwide in the largest outbreak in poultry history. human cases of hpai h5n1 followed the poultry outbreak, with a total of 256 cases and 151 fatalities thus far [57] . other limited outbreaks have occurred, causing variable human disease (table 3 ) [52, 58] . however, hpai h5n1 remains the largest and most significant poultry and human avian influenza outbreak. epidemiological investigations of human cases of avian influenza show that the virus was acquired by direct contact with infected birds [29] [30] [31] [50] [51] [52] [53] [54] [55] [56] . influenza a is transmitted through the fecal-oral and respiratory routes among wild birds and poultry [32] . human interaction with these infected secretions and birds was the major mode of transmission, with contact including consumption of undercooked or raw poultry products, handling of sick or dead birds without protection, or food processing at bird cleaning sites. all birds were domesticated (chicken, duck, goose) and no transmission from birds in the wild (migrating) or contaminated waterways has been documented. in a few cases, limited human to human transmission has been reported among health care workers and family members (table 4 ) [59] [60] [61] [62] [63] . in each of these cases, no personal protective equipment was used, which is the major factor in transmission between humans [60] . the clinical manifestations of avian influenza in humans has ranged from mild conjunctivitis to severe pneumonia with multi-organ system failure ( [53] . however, with hpai in hong kong in 1997 and in southeast asia currently, pneumonia progressing to multiorgan failure, acute respiratory distress syndrome (ards), and death are the predominant findings [17, 55, [65] [66] [67] [68] . rye syndrome, pulmonary hemorrhage, and predominant nausea, vomiting, and diarrhea complicate these cases [68] . laboratory findings include both thrombocytopenia and lymphopenia [65, 66] . chest radiographic findings include interstitial infiltrates, lobar consolidation, and air bronchograms. the clinical course of patients with hpai h5n1 is rapid, with 68% percent of patients developing ards and multiorgan failure within 6 days of disease onset [69] . the case fatality rate ranges form 67% to 80%, depending on the case series [17, 55, 65, 66] . once the patients reached the critical care unit, however, the mortality rate was 90% [69] . the average time of death from disease onset was nine to ten days. avian influenza a infections in humans differ from seasonal influenza in several ways. the presence of conjunctivitis is available online http://ccforum.com/content/11/2/209 number of fatalities (percent) 0 (0) 6 (33) 0 (0) 1 (1) 0 (0) 151 (59) h, hemagglutinin; ili, influenza like illness; n, neuroaminidase. more common with avian influenza a infections than with seasonal influenza. gastrointestinal symptoms, as seen with hpai h5n1, and reports of primary influenza pneumonia and development of ards are also more common with avian influenza a infections [65, 67, 69] . finally, the rapid progression to multi-organ failure and eventually death occurs at a much higher rate with avian influenza a infections [69] . post-mortem studies have illustrated findings consistent with an overwhelming systemic inflammatory response syndrome, including diffuse alveolar damage, acute tubular necrosis and atrophy, disseminated intravascular coagulation, and multiorgan damage [70, 71] . interestingly, the virus has been isolated from the lungs, intestine, spleen, and brain, suggesting viremia, but active replication of the virus has been limited to the lungs [71] . this overwhelming inflammatory response, with acute lung injury and ards as the predominant features, coincides with the findings of preferential binding of the avian influenza a viruses to α-2,3 linkages in type ii pneumocytes of the lower respiratory tract of humans and a vigorous cytokine response, including increased interleukin-6, interleukin-10, and interferon beta release [11, 12, 70, 71] . the clinical diagnosis of avian influenza infection in humans is difficult and relies on the epidemiological link to endemic areas, contact with sick or dead poultry, or contact with a confirmed case of avian influenza (table 6 ). since many infectious diseases present with similar symptoms, the only feature significant to the clinician may be contact in an endemic area, through travel or infected poultry, and the clinician should always elicit a detailed patient history. the definitive diagnosis is made from isolation of the virus in culture from clinical specimens. this method not only provides the definitive diagnosis, but the viral isolate is now available for further testing, including pathogenicity, antiviral resistance, and dna sequencing and analysis. alternatively, antibody testing can be performed, with a standard four-fold titer increase to the specific subtype of avian influenza virus. neutralizing antibody titer assays for h5, h7 and h9 are performed by the micorneutralization technique [72] . western blot analysis with recombinant h5 is the confirmatory test for any positive microneutralization assay [59, 60, 72] . more recently, rapid diagnosis can be performed with reverse transcription-pcr on clinical samples with primers specific for the viral subtype [73] [74] [75] . this test should be performed only on patients meeting the case definition of possible avian influenza a infection. any suspected case of avian influenza in a human should be investigated by the public health officials in the province or country of origin [39, 76] . additionally, governmental labs are often equipped with the appropriate biolevel safety 3 laboratories, primer libraries, and associated expertise to confirm the diagnosis quickly and efficiently. any clinical specimens should be submitted with the assistance of the public health experts. treatment of avian influenza infections in humans includes antiviral therapy and supportive care. controlled clinical trials on the efficacy of antivirals (na inhibitors), supportive therapy, or adjuvant care have never been performed, so current recommendations stem from the experiences of past avian influenza outbreaks and animal models. the adamantanes (rimantadine and amantadine) and na inhibitors (oseltamivir and zanamivir) are the antivirals used for treatment and prophylaxis of influenza infections in humans. in avian influenza virus infections, adamantanes have no role due to widespread resistance through a m2 protein alteration. in addition, over 90% of isolates of h1 and h3 human subtypes during seasonal influenza have had resistance to the adamantanes [77] . their role has now been limited to prophylaxis in the community when the circulation strain is know to be susceptible to the adamantanes [78] [79] [80] . na inhibitors (oseltamivir and zanamivir) have been studied for both treatment and prophylaxis with the human influenza a subtypes h1, h2, and h3 as well as influenza b (table 7 ) [80] [81] [82] . in animal models with hpai h5n1, their efficacy has been well documented, with improved survival rates seen after infection [83] [84] [85] . oseltamivir has been used in avian influenza outbreaks involving h7n7 and hpai h5n1, and therapy with oseltamivir has been shown to decrease the viral load in nasal secretions in patients infected with hpai h5n1 [11, 86, 87] . resistance to oseltamivir has been documented in a hpai h5n1 subtype in a vietnamese girl treated with 75 mg daily for 4 days as post-exposure prophylaxis [68] . the na glycoprotein had a histidine to tyrosine substitution at position 274, conveying a markedly higher ic50 for oseltamivir [68, 88] . in one study, the viral count of hpai h5n1 in nasal secretions did not decrease with the administration of oseltamivir when the h5n1 isolate carried this resistance mutation [68] . however, resistance produced by this change may be overcome with higher doses of oseltamivir in vitro, and this change has not been documented to confer resistance to zanamivir [88] . the timing of treatment with na inhibitors is paramount, as early therapy is directly related to improved survival [66, [83] [84] [85] . the greatest level of protection was seen if the na inhibitors were started within 48 hours of infection, and protection rapidly dropped after 60 hours [78, 79] . these initial studies, however, were performed with seasonal human influenza a and b, where the period of viral shedding is approximately 48 to 72 hours. in hpai h5n1 cases from southeast asia, survival appeared to be improved in patients who received oseltamavir earlier (4.5 days versus 9 days after onset of symptoms) [66] . both of these time periods are much longer than documented in animal models, so the window of optimal therapy is still unknown, particularly if viral shedding exceeds the average 48 to 72 hour period seen in seasonal influenza a and b infections. combination therapy with influenza a viruses has not been studied [84] . ribaviron by inhalation has been evaluated in vitro with some avian influenza a subtypes and has been found to reduce mortality from influenza b in a mouse model [89] . further animal model studies are indicated to determine if there is a role for ribaviron or combination therapy with avian influenza a viruses. supportive care with intravenous rehydration, mechanical ventilation, vasopressor therapy, and renal replacement therapy are required if multiorgan failure and ards are a feature of disease [69, 90] . due to the progression of pneumonia to ards, non-invasive ventilation is not recommended, and early intubation may be beneficial before overt respiratory failure ensues. corticosteroids have been used in some patients with hpai h5n1, but no definitive role for steroids has been determined. other immunomodulatory therapy has not been reported [91] . human vaccination for avian influenza viruses has not been widely used, although multiple vaccination trials are underway. prior avian vaccines in humans have been poorly immunogenic and thus have limited use. an inactivated h5n3 has been tested and was tolerated but with limited immunogenicity [91, 92] . other h5 vaccines have resulted in the development of neutralizing antibodies, but to a limited degree [93, 94] . recently, a large randomized trial looked at an h5n1 attenuated vaccine from the vietnam strain [95] . only a modest immune response was seen, with microneutralization antibodies being developed at 12 times the dose used in the seasonal influenza vaccine. the side effects were minimal. a number of other industry trials with adjuvant vaccines are currently ongoing. although promising, human vaccination against avian influenza viruses is still under development. underscoring this development is the uncertainty of a pandemic strain, which may have vastly different antigenic properties from any developed h5 vaccine. health care infection control is a crucial component in the management of avian influenza infection or a new pandemic strain. experience from the severe ards outbreak in 2002 has illustrated that appropriate infection control measures are paramount to reduce spread to health care workers and, possibly, the community [96] [97] [98] . therefore, the world health organization (who) and centers for disease control and prevention (cdc) recommend contact and airborne precautions for any initial suspected case of avian influenza in a human [99] . in late october 2006, the cdc released updated interim guidance on the use of masks and respirators in the health care setting (table 8 ) [99] . in certain high risk procedures, additional protection may be considered given the likelihood of generating aerosol particles that may enhance transmission (table 9 ) [99] . respiratory protection should be worn along with an impermeable gown, face shield, and gloves. initial cases should be placed in a negative pressure isolation room with 6 to 12 air changes per hour. hand hygiene with antibacterial soap or alcohol based washless gel should be standard, with appropriate basins at each patient room. seasonal vaccination of all health care workers should be preformed and further emphasized in order to reduce the likelihood of co-infection with two stains of influenza. visitors and family members should be strictly monitored and their access to the patient limited to reduce the likelihood of spread. finally, antiviral chemoprophylaxis should be available to any health care workers exposed to an infected individual. any symptomatic worker should be taken off duty and workplace surveillance should occur. with these aggressive measures, risk to health care workers, patients, and family members will be reduced. avian influenza viruses have occurred with increased incidence within the human population, reflecting the delicate and tangled interaction between wildlife, domesticated animals, and humans. disease in humans can be limited to conjunctivitis or an influenza-like illness, but hpai h5n1 causes mainly severe pneumonia, respiratory failure, and death. most cases have occurred through direct transmission from infected poultry or waterfowl, with only a few limited cases of human to human transmission. treatment has been successful with the na inhibitors if started early, and vaccine development is underway with a more immunogenic attenuated h5n1 virus preparation. infection control measures are the mainstay for prevention and disease reduction. avian influenza viruses may constitute part of the next pandemic, so appropriate knowledge, prevention, and treatment will reduce the likelihood of this occurrence. table 9 high risk aerosol procedures in avian influenza non-invasive mechanical ventilation bronchoscopy humidified oxygen delivery non-rebreather mask without expiratory filter this article is part of a thematic series on disaster management edited by j christopher farmer. other articles in this series can be found online at http://ccforum.com/articles/ theme-series.asp?series=cc_disaster world health organization expert committee: a revision of the system of nomenclature for influenza viruses: a who memorandum characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained from black-headed gulls highly pathogenic avian influenza: manual of diagnostic tests and vaccines for terrestrial animals molecular mechanism of acquisition of virulence in influenza virus in nature epidemiologic implications of changes in the influenza virus genome differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 h5n1 chicken and human influenza viruses from hong kong molecular basis for the generation in pigs of influenza a viruses with pandemic potential influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium: the role of the host cell in selection of hemagglutinin receptor specificity human and avian influenza (ai) viruses target different cell types in cultures of human airway epithelium the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties avian flu: influenza virus receptors in the human airway h5n1 virus attachment to lower respiratory tract evidence for the natural transmission of influenza a virus from wild ducks to swine and its potential importance for man genetic relatedness of hemagglutinins of the h1 subtype of influenza a viruses isolated from swine and birds replication of avian influenza a viruses in mammals influenza a virus (h5n1) infection in cats causes systemic disease with potential novel routes of virus spread within and between hosts avian influenza h5n1 in tigers and leopards avian influenza a virus causing an outbreak of contagious interstitial pneumonia in mink an avian influenza a virus killing a mammalian species -the mink characterization of two influenza a viruses from a pilot whale seroepidemiological and molecular evidence for the presence of two h3n8 equine influenza viruses in china in 1993-94 characterization of a new avian-like influenza a virus from horses in china the appearance of h3 influenza viruses in seals are seals frequently infected with avian influenza viruses? characterization of an influenza a virus from seals transmission of equine influenza virus to dogs avian influenza h5n1 in viverrids: implications for wildlife health and conservation characterization of an avian influenza a virus isolated from a human -is an intermediate host necessary for the emergence of pandemic influenza viruses human influenza a h5n1 virus related to a highly pathogenic avian influenza virus avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness evolution and ecology of influenza a viruses antigenic and genetic characterization of a novel hemagglutinin subtype of influenza a viruses from gulls molecular characterization of a new hemagglutinin, subtype h14, of influenza a virus characterization of a novel influenza hemagglutinin, h15: criteria for determination of influenza a subtypes investigation of outbreaks of highly pathogenic h5n1 avian influenza in waterfowl and wild birds in hong kong in late highly pathogenic h5n1 influenza virus infection in migratory birds reemerging h5n1 viruses in hong kong in 2002 are highly pathogenic to ducks genesis of a highly pathogenic and potentially pandemic h5n1 virus in eastern asia new genotype of avian influenza h5n1 viruses isolated from tree sparrows in china characterization of h5n1 influenza a viruses isolated during the 2003-2004 influenza outbreaks in japan. virol highly pathogenic h5n1 influenza virus in smuggled thai eagles establishment of multiple sublineages of h5n1 influenza virus in asia: implications for pandemic control role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia intestinal influenza: replication and characterization of influenza viruses in ducks perpetuation of influenza a viruses in alaskan waterfowl reservoirs factors in the emergence of infectious diseases mallards and highly pathogenic avian influenza ancestral viruses host range and emerging and reemerging pathogens conjunctivitis in human beings caused by influenza a virus of seals avian influenza virus isolated from a woman with conjunctivitis human infection with influenza h9n2 transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus outbreak of avian influenza a (h5n1) virus infection in hong kong in 1997 re-emergence of fatal human influenza a subtype h5n1 disease world health organization: cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who human illness from avian influenza h7n3, british columbia antibody response in individuals infected with avian influenza a (h5n1) viruses and detection of anti-h5 antibody among household and social contacts risk of influenza a (h5n1) infection among health care workers exposed to patients with influenza a (h5n1), hong kong probable person-to-person transmission of avian influenza a (h5n1) world health organization international avian influenza investigation team, vietnam: lack of h5n1 avian influenza transmission to hospital employees avian influenza h5n1 and healthcare workers avian influenza, human (09): indonesia avian influenza a (h5n1) in 10 patients in vietnam human disease from influenza a (h5n1) the writing committee of the world health organization (who): consultation on human influenza a/h5. avian influenza a (h5n1) infection in humans fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma avian influenza (h5n1): implications for intensive care induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? pathology of fatal human infection associated with avian influenza a h5n1 virus risk of influenza a (h5n1) infection among poultry workers development of a realtime reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes application of real-time rt-pcr for the quantitation and competitive replication study of h5 and h7 subtype avian influenza virus single-step multiplex reverse transcription-polymerase chain reaction (rt-pcr) for influenza a virus subtype h5n1 detection the world health organization global influenza program surveillance network: evolution of h5n1 avian influenza viruses in asia emergence of drug-resistant influenza virus: population dynamical considerations antiviral agents. clinical virology antiviral agents active against influenza a viruses neuroaminidase inhibitors for preventing and treating influenza in healthy adults pharmacokinetics of zanamivir after intravenous, oral, inhaled or intranasal administration to healthy volunteers the pharmacokinetics and tolerability of the oral neuraminidase inhibitor oseltamivir (ro 64-0796/gs4104) in healthy adult and elderly volunteers the neuraminidase inhibitor gs4104 (oseltamivir phosphate) is efficacious against a/hong kong/156/97 (h5n1) and a/hong kong/1074/99 (h9n2) influenza viruses neuraminidase inhibitor-rimantadine combinations exert additive and synergistic anti-influenza virus effects in mdck cells efficacy of zanamivir against avian influenza a viruses that possess genes encoding h5n1 internal proteins and are pathogenic in mammals resistant influenza a viruses in children treated with oseltamivir: descriptive study oseltamivir resistance during treatment of influenza a (h5n1) infection avian flu: isolation of drug-resistant h5n1 virus in vitro and in vivo influenza virus inhibitory effects of viramidine medical treatment of viral pneumonia including sars in immunocompetent adult safety and antigenicity of non-adjuvanted and f59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a randomized trial of two potential vaccines against h5n1 influenza cross-reactivity to highly pathogenic avian influenza h5n1 viruses after vaccination with nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a potential priming strategy safety and immunogenicity of a recombinant hemagglutinin vaccine for h5 influenza in humans efficacy of h5 influenza vaccines produced by reverse genetics in a lethal mouse model saftey and immunogenicity of an inactivated subviron influenza a (h5n1) vaccine world health organization: summary of probable sars cases with onset of illness from 1 evidence of airborne transmission of the severe acute respiratory syndrome virus toronto sars critical care group: critically ill patients with severe acute respiratory distress syndrome interim guidance on the planning for the use of surgical masks and respirators in health care settings during an influenza pandemic the authors declare that they have no competing interests. key: cord-017748-xy26tk0t authors: georgiev, vassil st. title: influenza date: 2009 journal: national institute of allergy and infectious diseases, nih doi: 10.1007/978-1-60327-297-1_13 sha: doc_id: 17748 cord_uid: xy26tk0t influenza is a highly contagious, acute respiratory illness afflicting humans. although influenza epidemics occur frequently, their severity varies (1). not until 1933, when the first human influenza virus was isolated, was it possible to define with certainty which pandemics were caused by influenza viruses. in general, influenza a viruses are more pathogenic than are influenza b viruses. influenza a virus is a zoonotic infection, and more than 100 types of influenza a viruses infect most species of birds, pigs, horses, dogs, and seals. it is believed that the 1918–1919 pandemic originated from a virulent strain of h1n1 from pigs and birds. influenza is a highly contagious, acute respiratory illness afflicting humans. although influenza epidemics occur frequently, their severity varies (1) . not until 1933, when the first human influenza virus was isolated, was it possible to define with certainty which pandemics were caused by influenza viruses. in general, influenza a viruses are more pathogenic than are influenza b viruses. influenza a virus is a zoonotic infection, and more than 100 types of influenza a viruses infect most species of birds, pigs, horses, dogs, and seals. it is believed that the 1918-1919 pandemic originated from a virulent strain of h1n1 from pigs and birds. the natural reservoir of influenza viruses was identified as wild aquatic birds, from whose populations viruses with new surface proteins could emerge through reassortment. however, it is still not possible to predict how and when new influenza strains will emerge or how virulent a new strain will prove (2) . currently, influenza a viruses of subtypes h1n1 and h3n2 and influenza b viruses of two antigenically distinct hemagglutinin lineages are present in human populations. the occurrence of sporadic avian influenza subtypes (e.g., h9, h7, and particularly h5) in human populations have led to widespread concerns about the possibility of an influenza pandemic. since 1889, at least five major pandemics have been recorded, when new hemagglutinin and/or neuraminidase subtypes have been introduced into human populations. the pandemic of 1918-1919 was by far the worst of its kind. it was followed by pandemics of decreasing severity in 1957 (asian flu; subtype h2n2), 1968 (hong kong flu; subtype h3n2), and 1977 (russian flu; subtype h1n1). based on the antigenic differences between their nucleoprotein (np) and matrix (m) protein antigens, the influenza viruses are divided into types a, b, and c (1). the influenza a viruses are further divided into subtypes. the influenza viruses are single-stranded rna viruses and share structural and biological similarities. the viral rna core consists of 8 gene segments surrounded by a coat of 10 (influenza a) or 11 (influenza b) proteins. immunologically, the most important surface proteins are hemagglutinin and neuraminidase, as the influenza viruses are typed on the basis of these proteins. for example, influenza a subtype h3n2 expresses hemagglutinin 3 and neuraminidase 2. the most prevalent human influenza a strains are h1n1 and h3n2. although the morphologic characteristics of the influenza viruses are a genetic trait, the spherical morphology appears to be dominant on passage in chicken embryos or tissue culture systems. the most distinct feature of the influenza virions is the presence of a layer of spikes projecting radially outward over the surface. the surface spikes are of two distinct types, corresponding with the hemagglutinin and neuraminidase components of the virus. the hemagglutinin (ha) is the surface glycoprotein, which accounts for approximately 25% of viral protein and is distributed evenly on the virion surface. it is responsible for the virus's binding to the host receptor, internalization of the virus, and subsequent membrane-fusion events within the endosomal pathway in the infected cell. furthermore, ha is also the most abundant antigen on the viral surface and harbors the primary neutralizing epitopes for antibodies (1) . structurally, ha is a glycoprotein consisting of two polypeptide chains, ha1 and ha2. ha1 and ha2 are linked by a single disulfide bond, and each ha "spike" contains three of these ha1 and ha2 chains. furthermore, ha contains up to seven oligosaccharide chains (six in ha1 and one in ha2) linked to asparagine. the majority of these carbohydrate chains are on the lateral surface of the trimer. no obligatory function has been assigned to these side chains (1). the neuraminidase (na) exists as a mushroom-shaped spike containing a hydrophobic region by which it is embedded in the viral membrane in the opposite way to the ha. na is the second subtype-specific glycoprotein on the influenza virion and is composed of a single polypeptide chain (1). it is not evenly distributed on the virion surface but rather found in patches. the principal biologic role of na protein of the influenza a virus is the cleavage of the terminal sialic acid residues that are receptors of the virus's ha protein. removal of these residues from the surface of infected cells and from newly formed viruses will prevent the budding viruses from clumping to each other or to the cell surfaces. in addition, the ability to cleave sialic acid is also thought to help the virus to penetrate mucus (3) . because na functions largely in the release of newly formed viral particles, antibodies against it do not prevent initial infection. however, they sharply limit its spread and therefore, in humans, selection favors na variants with mutations that hinder antibody recognition (antigenic shift) (3). the nucleoprotein (np) is one of the type-specific antigens of influenza viruses that distinguish among types a, b, and c viruses (1) . the nucleoproteins are basic proteins that constitute the backbone of the helical internal complex and have a putative role in transcription and replication. during infection, the np accumulates in the nucleus, and karyophilic sequences have been identified that are partially conserved among the influenza a, b, and c viruses. the rna segment 7 of influenza a viruses codes for two proteins m1 and m2 (1) . the basic organization of rna segment 7 is present in all influenza a and b viruses sequenced. the m1 protein is a virion structural protein that is intimately associated with the lipid bilayer. it is believed to be a multifunction protein having a role in the downregulation of the activity of the virion-associated transcriptase. it is located in the nucleus, cytoplasm, and plasma membrane of infected cells. passively transferred monoclonal antibodies to the m1 protein did not confer resistance to infection. the m2 protein of influenza a virus is an integral membrane protein that is expressed on the surface of infected cells with an extracellular domain (1) . the m2 protein is present in high copy number in infected cells and thus appears to be actively excluded from the virions. in just 8 months, the 1918-1919 pandemic known as the "spanish influenza" killed between 20 million and 40 million people worldwide (2) . the virus causing this influenza pandemic was not isolated at the time. however, its enhanced severity, multiple waves in just 1 year, and its predilection for the young and healthy all suggested that this influenza pandemic was unique. as the most deadly influenza virus ever experienced, the 1918 strain offers a unique opportunity to understand the connection between genotype and virulence (2) . from preserved autopsy samples of two u.s. soldiers and from the frozen lungs of an inuit woman, fragments of the deadly virus have been isolated, copied, and analyzed. the crystal structure of the 1918 influenza ha has been elucidated from a second human subtype (h1) derived from reassembling the extinct 1918 influenza virus (8, 9) . in addition, two closely related has in complex with receptor analogues have also been determined (9) . these two related has may explain how the 1918 ha, while retaining receptor binding site amino acids characteristic of an avian precursor ha, was able to bind human receptors and how, as a consequence, the virus was able to spread in human populations (9) . the primary event in influenza infection is the binding of the virus to the host receptor. the crystal structure of ha has shown that its receptor-binding site is situated in a shallow pocket in the membrane-distal ha1 domain in each subunit of the ha trimer (8, 9) . the nature of the receptor's sialic acid linkage to the vicinal galactose is the primary determinant in lung epithelial cells that differentiates avian viruses from mammalian. sialic acids are usually found in either α2,3or α2,6-linkages to galactose, which is the predominant penultimate sugar of the n-linked carbohydrate side chains. the binding preference of a given ha for one or other of these linkage types correlates with the species specificity for infection (species barrier). thus, avian viruses preferentially bind to receptors with an α2,3-linkage to galactose, whereas human-adapted viruses are specific for the α2,6-linkage (9) . for example, the has of all 15 antigenic subtypes found in avian influenza viruses bind preferentially to sialic acid in α2,3-linkage, and it is this form of the sialosaccharide that predominates in the avian enteric tract where these viruses replicate. human viruses of the h1, h2, and h3 subtypes that are known to have caused pandemics in 1918, 1957, and 1968, respectively, recognize α2,6-linked sialic acid, the major form found on cells of the human respiratory tract (9) . because an avian origin has been proposed for the has of swine and human viruses, changes in the binding specificity of has will be required for cross-species transfer. the the influenza a viral heterotrimeric polymerase complex (pa, pb1, pb2) is known to be involved in many aspects of viral replication and to interact with host factors, thereby having a role in host specificity. recently, an additional small open reading frame has been identified that codes for a peptide (pb1-f2) that is thought to play a role in the virusinduced cell death. it is not yet clear how the polymerase complex must change to adapt to a new host. however, a single amino acid change in pb2, e627k, was shown to be important for adaptation in mammals. the polymerase protein sequences from the 1918 human influenza virus differ from avian consensus sequences at only a small number of amino acids; that is consistent with the hypothesis that they derived from an avian source shortly before the 1918 pandemic. however, when compared with avian sequences, the nucleotide sequences of the 1918 polymerase genes have more synonymous differences than expected, suggesting evolutionary distance from known avian strains (10) . in 2005, the sequence and phylogenetic analyses of the complete genome of the 1918 influenza virus were determined (10) . the data suggested that the 1918 virus was not a reassortant virus like those of the 1957 and 1968 pandemics. more likely, the 1918 virus was an entirely avian-like virus that had adapted to humans. one interesting feature found in the polymerase complex of the 1918 virus and subsequent human isolates as well was the presence of a lys residue at position 627. this residue has been implicated in host adaptation and has been previously shown to be critical for high pathogenicity in mice infected with the 1997 h5n1 virus (11). one of the characteristics of the 1918 influenza pandemic was its unusual virulence, reflected in the dramatic increase in the severity of the illness and the prevalence of pneumonic complications (2, 3) . the virulence of the influenza viruses is a complicated function of the genetic characteristics of the virus itself, the immune status of the infected person, and the dose and route of transmission. the severity of the 1918 pandemic suggested that both the ha and na were antigenically novel as supported by sequence and phylogenetic analyses of both 1918 ha and na proteins (5), as well as that the virus had not circulated widely in the human population before spring 1918 (3) . the relationship between virulence and the genetic structure of the influenza virus is complex. there have been several examples where simple changes in a single gene resulted in dramatic changes in virulence. thus, one of these changes is the insertion of basic amino acids in the ha cleavage site, which will allow the virus to grow in many tissues outside its normal host cells. although this change has been found in the h5 and h7 subtypes in birds, it was not found in the 1918 ha (5) . in another change observed in the na gene, the loss of a glycosylation site at amino acid 146 in wsn/33 contributed to making the virus exceptionally virulent as well as neurotropic in mice. this change was also not observed in the 1918 virus strain (3). although milder than the 1918 spanish h1n1 influenza pandemic, both the 1957 asian influenza h2n2 pandemic and the 1968 hong kong influenza h3n2 pandemic caused significant morbidity and mortality worldwide (1, 2) . the 1957 influenza pandemic was caused by a reassortant virus that was derived from the ha (h2), na (n2), and pb1 (polymerase basic protein 1) genes from an avian influenza virus infecting ducks and the remaining gene segment from the previously circulating human h1n1 virus (12) (13) (14) . the h3n2 virus that caused the 1968 pandemic consisted of avian ha (h3) and pb1 genes in a background of other internal protein genes of the human h2n2 virus that was circulating at the time (12) (13) (14) . the presence of an avian ha h3 glycoprotein made the reassortant virus antigenically novel to humans, and it spread in the susceptible human population causing a pandemic (12, 13) . studies on the origin and evolutionary pathways of the pb1 genes of influenza a viruses responsible for the 1957 and 1968 human pandemics and the variable or conserved region of the pb1 protein have shown that the evolutionary tree constructed from nucleotide sequences suggested that (i) the pb1 gene of the 1957 human pandemic strain, a/singapore/1/57 (h2n2), was probably introduced from avian species and was maintained in humans until 1968; (ii) in the 1968 pandemic strain, a/nt/60/68 (h3n2), the pb1 gene was not derived from the previously circulating virus in humans but probably from another avian virus; and (iii) a current human h3n2 virus inherited the pb1 gene from an a/nt/60/68-like virus (12) . nucleotide sequence analysis also showed that the avian pb1 gene was introduced into pigs. hence, transmission of the pb1 gene from avian to mammalian species is a relatively frequent event. comparative analysis of deduced amino acid sequences disclosed highly conserved regions in pb1 proteins, which may be key structures required for pb1 activities (12) . the rna of the human influenza virus singapore (h2n2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins (2) . the base sequence homology between the individual genes (segments) of the h2n2 virus and those of different influenza a strains has been determined by molecular hybridization. segments 1, 5, 7, and 8 of the singapore strain exhibit a base sequence homology of almost 100% compared with those of the fm1 strain (hlnl), whereas the homology between the other segments was significantly lower (24% to 76%). for the singapore and hong kong (h3n2) strains, all segments except that coding for the ha (24%) exhibit a homology close to 100%. the 32 p-labeled segment 4 (ha gene) of the avian influenza a strain duck ukraine (hav7neg2) showed a homology of 92% to hong kong, whereas the homology of at least two other segments was significantly lower. these results were interpreted as an indication that the h2n2 subtype is derived from the hln1 subtype by a recombination event retaining four h1n1 segments, whereas the other four segments were gained from another yet unknown strain. the h3n2 subtype is presumably derived from a h2n2 subtype, retaining seven segments of the h2n2 subtype, whereas the gene coding for the ha is obtained from the duck ukraine or another highly related strain (13). an unprecedented epizootic avian influenza a (h5n1) virus that is highly pathogenic has crossed the species barrier in asia to cause human fatalities and thus poses an increased threat of pandemic (15) . in 1997, an avian subtype, h5n1, was first described in hong kong. the infection was confirmed in only 18 people, but 6 of them died. subsequent, although sporadic, cases of avian (bird flu) influenza continued to be recorded, mainly in southern china, but also in other regions of southeast asia. the h5n1 influenza, in nearly all cases, has been transmitted to humans from birds. other routes of transmission include possibly environment-to-humans, and limited, nonsustained, human-to-human transmission. transmission to felids has been observed by feeding raw infected chicken to tigers and leopards in zoos in thailand and to domestic cats under experimental conditions. transmission between felids has been found under such conditions. because of the poultry outbreaks and bird-to-human transmission, hundreds of new cases of avian influenza have been reported, stretching from southeast asia (mainly china, vietnam, thailand, and indonesia) through mongolia, kazakhstan, and russia to turkey, raising the concern that a slight mutation may convert subtype h5n1 into a strain that would be easily transmitted from human to human. to date, human-to-human transmission of influenza a virus (h5n1) has been suggested in several household clusters (11) and in one case of apparent child-to-mother transmission (16) . there has been a who report suggesting that local virus h5n1 strains (northern vietnam) may be adapting to humans (17) (http://www.who.int/csr/resources/publications/influenza/w-ho cds csr gip 2005 7/en/). however, epidemiologic and virologic studies will be needed to confirm these findings. studies of isolates of avian influenza a (h5n1) from patients in 1997 have shown that the virulence factors included (i) the highly cleavable hemagglutinin that can be activated by multiple cellular proteases; (ii) a specific substitution in the polymerase basic protein 2 (glu627lys) that enhances replication; and (iii) a substitution in nonstructural protein 1 (asp92glu) that confers increased resistance to inhibition by interferons and tumor necrosis factor α (tnf-α) in vitro and prolonged replication in swine, as well as greater elaboration of cytokines, particularly tnf-α, in human macrophages exposed to the virus (15) . since 1997, studies of influenza a (h5n1) have indicated that these viruses continue to evolve, including (i) changes in antigenicity and internal gene constellations; (ii) an expended host range of avian species and the ability to infect felids; (iii) enhanced pathogenicity in experimentally infected mice and felids, in which they caused systemic infections; and (iv) increased environmental stability (15) . phylogenetic analyses have demonstrated that the z genotype has become dominant (18) and that the virus has evolved into two distinct clades, one encompassing isolates from cambodia, laos, malaysia, and vietnam, and the other isolates from china, indonesia, japan, and south korea (17) . recently, a separate cluster of isolates has appeared in northern vietnam and in thailand, which included variable changes near the receptor-binding site and one fewer arginine residue in the polybasic cleavage site of the hemagglutinin (12) . however, the importance of these genetic and biologic changes with respect to human epidemiology or virulence is uncertain. the relatively low frequency of influenza a (h5n1) illness in humans despite widespread exposure to infected poultry has suggested that the species barrier to acquisition of this avian virus is substantial. clusters of cases in family members may be caused by common exposures, although the genetic factors that may affect a host's susceptibility to disease will require more studies (15) . the innate immune responses to influenza a (h5n1) may contribute to disease pathogenesis. in the 1997 outbreaks, elevated blood levels of interleukin-6, tnf-α, interferon-γ, and soluble interleukin-2 receptor were observed in individual patients (19) , and in patients in 2003, elevated levels of the chemokines interferon-inducible protein 10, mono-cyte chemoattractant protein 1, and monokine induced by interferon-γ were found 3 to 8 days after the onset of illness (20) . recently, plasma levels of inflammatory mediators (interleukin-6, interleukin-8, interleukin-1β, and monocyte chemoattractant protein 1) were found to be higher among patients who died than among those who survived, and the average levels of plasma interferon-α were about three times as high among patients with avian influenza a who died as among healthy controls (15) . among survivors, specific humoral immune responses to influenza a (h5n1) are detectable by microneutralization assay 10 to14 days after the onset of illness. the clinical spectrum of influenza a (h5n1) in humans has been based on descriptions of hospitalized patients (15) . the frequencies of milder illness, subclinical infections, and atypical presentations (e.g., encephalopathy, gastroenteritis) have not been determined, but case reports indicated that each have occurred. most of the hospitalized patients were previously healthy young children or adults. incubation. the incubation period of the avian influenza a (h5n1) may be longer compared with those of other known human influenzas-in most cases it is within 2 to 4 days after exposure but occasionally up to 8 days (15) . the case-to-case intervals in household clusters have generally been 2 to 5 days with a upper limit between 8 and 17 days (possibly resulting from unrecognized exposure to infected animals or environmental sources). initial symptoms. most patients have initial symptoms of high fever (38 o c or higher) and an influenza-like illness with lower respiratory tract symptoms (21) (http://www.who.int/ csr/disease/avian influenza/guidelines/guidelines clinical% 20management h5n1 rev.pdf). upper respiratory tract symptoms are manifested only occasionally. also, unlike patients with infections caused by avian influenza a (h7n7) viruses, patients with avian influenza a (h5n1) rarely have developed conjunctivitis (22) . diarrhea, vomiting, abdominal pain, pleuritic pain, and bleeding from the nose and gums have also been reported early in the course of illness. watery diarrhea may precede the respiratory manifestations by up to 1 week (15) . clinical course and management. lower respiratory tract manifestations develop early in the course of the diseaserespiratory distress, tachypnea, and inspiratory crackles are common. sputum production is variable and occasionally bloody. nearly all patients have clinically apparent pneumonia. progression to respiratory failure has been associated with diffuse, bilateral, ground-glass infiltrates and manifestations of acute respiratory distress syndrome (ards). multiorgan failure with signs of renal dysfunction, and sometimes cardiac compromise including cardiac dilatation and supraventricular tachyarrhythmias, have been common (15) . most hospitalized patients with avian influenza a (h5n1) have required ventilatory support within 48 hours after admission, as well as intensive care for multiorgan failure and sometimes hypotension. empirical treatment with broad-spectrum antibiotics, antiviral agents-alone or with corticosteroids-has been tried in most patients, although their effects have not been rigorously assessed (15) . early initiation of antiviral drugs appears to be beneficial (11) . mortality. death has occurred an average of 9 or 10 days after the onset of illness (range, 6 to 30 days), and most patients have died of progressive respiratory failure (15) . the mortality rate among hospitalized patients has been high although the overall rate has probably been much lower (17) . in contrast with 1997, when most deaths occurred among patients older than 13 years of age, recent infections have caused high rates of death among infants and young children (89% among children younger than 15 years of age in thailand). since the late 1990s, several cases of human infections with the avian influenza a (h9n2) virus have been reported (23) . however, despite concerns after the initial cases of human infections with the h9n2 strain occurred, no virulent outbreak of human h9n2 infection did occur. as with influenza a (h5n1) virus outbreaks, there has been considerable apprehension that a virulent h9n2 strain might still mutate to allow human-to-human infection and that such a strain might also possess the triad of infectivity, lethality, and transmissibility. the avian h5n1 influenza virus that was transmitted from poultry to humans in 1997 and caused high mortality in both species is unusual in having a large proportion of amino acid substitutions in all gene products except in the surface antigen, thus suggesting that the h5n1 virus may be a reassortant (24) . phylogenetic and antigenic analyses of the h9n2 and h5n1 viruses and a quail h9n2 virus, all isolated from hong kong, provided evidence that the h5n1 and h9n2 influenza viruses were indeed reassortants, and that the quail h9n2 virus may have been the internal gene donor (25, 26) . the reassortment between n5n1 and n9n2 had occurred prior to the human infection by h5n1 in 1997. further results have indicated the presence of multiple lineages of h9n2 viruses in asia and at least three distinguishable subgroups in hong kong poultry (25), as well as that h9n2 influenza viruses possessing h5n1-like internal genomes continue to circulate in poultry in southeastern china (27) . in another study (28) , the h9n2 influenza viruses were found to have receptor specificity similar to that of human h3n2 viruses. in addition, the neuraminidase of poultry h9n2 viruses has mutations in its hemadsorbing site, a characteristic resembling that of human h2n2 and n3n2 viruses, but differing from that of other avian viruses. these peculiar features of the surface glycoproteins of h9n2 viruses from hong kong suggest an enhanced propensity for introduction into humans and emphasize the importance of poultry in the zoonotic transmission of influenza viruses. the influenza infection (flu) is a contagious respiratory illness that can cause mild to severe illness, which at times can lead to death. the best way to prevent influenza is by getting a flu vaccination each year. according to statistics by the cdc, every year in the united states, on average: (i) 5% to 20% of the population will contract flu; (ii) more than 200,000 people are hospitalized from flu complications; and (iii) about 36,000 people die from flu (29) (http://www. cdc.gov/flu/keyfacts.htm). recent studies have revealed that children 6 to 23 months of age have a substantially higher risk for influenza-associated morbidity (30, 31) . despite the high annual rates of influenza in children, preventive vaccines are given infrequently (27 r basic biology. niaid supports many basic research projects aimed at understanding how the influenza virus replicates, interacts with the host, stimulates an immune response, and evolves into new strains. results from these studies lay the foundation for the design of new antiviral drugs, diagnostics, and vaccines. r antiviral drugs. niaid currently supports the identification, development, and evaluation of new antiviral drugs against influenza, including the screening of new drug candidates to see if they have activity against the virus both in laboratory cells and in animals. niaid is also focused on developing novel broad-spectrum therapeutics intended to work against many influenza virus strains; some of these target viral entry into human cells, whereas others specifically attack and degrade the viral genome. development and evaluation of a combination antiviral regimen against potential pandemic influenza strains is also now under way. r diagnostics. niaid supports the development of rapid, ultrasensitive devices to detect influenza virus infection. although early in development, these devices will allow detection of newly emerging viral mutants and discrimination between different antigenic subtypes. r surveillance and epidemiology. the threat from influenza, like virtually all emerging and re-emerging infectious disease threats, is global in scope. for this reason, in recent years niaid has expanded its activities in other countries. through a contract for pandemic influenza preparedness, niaid supports a long-standing program in hong kong to detect the emergence of influenza viruses with pandemic potential in animals. under this program, scientists had detected the re-emergence of highly pathogenic h5n1 avian strains in this area in 2002 and 2003 and were instrumental in the early detection and characterization of the sars coronavirus in 2003. this approach has underscored the concept that research on one type of infectious disease often supports or can be applied to research on other types of infectious diseases, whether newly emerging, re-emerging, or deliberately introduced. r vaccine development and evaluation. because influenza is so easily transmitted, effective vaccines are essential to controlling annual influenza epidemics. the current egg-based system used to produce licensed influenza vaccines-despite being reliable for more than 40 yearscan still be improved. limitations of the current system include (i) a lengthy manufacturing process; (ii) the need to select which virus strains will be in the vaccine at least 6 months in advance of the influenza season; (iii) the need to produce nearly 90 million doses of a new influenza vaccine each year; and (iv) the need for hundreds of millions of fertilized chicken eggs to manufacture the vaccine. the early decision about which strains to include in the influenza vaccine will not always be correct, and the long lead time required to produce the vaccine makes midstream corrective action impossible. additional limitations may also include allergenicity of eggs in some individuals, and the inability to use eggs for propagating viruses lethal to chickens. niaid is currently supporting several research projects aimed at developing vaccines that can be manufactured more rapidly, are more broadly cross-protective, and are more effective. the use of reverse genetics-a genetic tool developed by niaid-supported scientists-holds the promise of more rapid generation of high-yielding vaccine candidates that match the anticipated epidemic strain. reverse genetics can also be used to turn highly pathogenic influenza viruses into vaccine candidates more suitable for manufacturing of vaccine by removing or modifying certain virulence genes; laboratories around the world are using the technique to prepare vaccine candidates against the h5n1 viruses emerging in asia. niaid also is funding the development of new influenza vaccine technologies. recently, niaid supported a phase ii clinical trial of a new influenza vaccine produced in a cell culture system as an alternative to manufacturing the vaccine in eggs. another approach has focused on improving the effectiveness of current inactivated virus vaccines by giving increasing doses of influenza vaccine to elderly individuals, the population that frequently accounts for up to 90% of influenza deaths each year in the united states. niaid is also funding the development of new technologies for the production of influenza vaccines; these involve dnabased approaches and broadly protective vaccines based on influenza virus proteins that are shared by multiple strains of the influenza virus. (i) niaid's continuing support for the development of novel vaccination strategies for the elderly. thus, a study conducted to assess the immunogenicity and reactogenicity of a current u.s. vaccine formulation at increased doses in the elderly population (65 years and older; n=202) has shown that at the increased dose (60 μg), higher levels were observed for the mean serum hemagglutination inhibition and neutralizing antibody levels (44% to 71% and 54% to 79%, respectively) compared with the standard dose (15 μg) of the vaccine (33) . increasing the antigen content of inactivated vaccines may provide a straightforward approach to improving protection in the elderly. undertaken an initiative to develop a protective vaccine that is effective against multiple influenza strains (see section 13.8). the proposed approach is to incorporate both conserved and variable genes into dna and adenoviral (adv) vectors that can be readily produced by existing methodologies. in particular, the vrc has been involved in developing three new vaccines, each comprising a single plasmid dna encoding hemagglutinin protein from h1n1, h3n2, and h5n1 subtypes isolated from recent human outbreaks of influenza. adenoviral construct expressing the same inserts are also being constructed. in addition, protein subunit vaccines based on production from insect and mammalian cells are being developed and tested. r immunity to influenza. niaid is currently involved in supporting a robust program that will further broaden the knowledge regarding immunity to influenza, as follows: (i) nih tetramer facility at emory university. since 1999, the tetramer facility has prepared nearly 2,400 unique tetramers, of which 459 have been directly related to monitoring t-cell functions against infectious diseases; of those, 45 tetramers were produced specifically for influenza-related studies. (iedb) at la jolla institute of allergy and immunology. in 2005, iedb became publicly available (http://www.immuneepitope.org). the iedb contains extensively curated information from the published literature on antibody and t-cell epitopes, as well as tools to predict antibody and t-cell epitopes or visualization/mapping of epitopes onto known protein structures. there are currently 17,868 unique epitopes within the database, including all published influenza antibody and t-cell epitopes. (iii) modeling pulmonary immunity. this program, conducted at the university of pittsburgh, involves analysis of innate and adaptive immunity in the lungs and draining lymph nodes of mice (various ages) either infected with or vaccinated against influenza viruses. the computational models to be developed may be used to simulate human innate responses to adjuvants or immune modulators, as a method for screening novel compounds against influenza. (iv) biodefense immune modeling. scientists from the university of rochester will conduct a comprehensive examination of b-and t-cell-mediated immunity to influenza vaccination in healthy adults and influenza a infection or vaccination in mouse model systems. the major goal of these studies is to produce computational models capable of predicting human immune responses to natural variants or genetically engineered influenza viruses and can be used to test novel vaccine strategies or immune modulators in silico, prior to further testing in animal models and humans. (v) pathways in the interferon signaling cascade. this niaid-supported program is aimed at creating computational models to decipher the type 1 interferon signaling networks in primary human dendritic cells that are modulated by viral proteins. niaid has had a long history of supporting research activities to provide more effective approaches to controlling influenza virus infections. these activities include both basic and applied research on the influenza virus basic biology and replication, pathogenesis, immunology, epidemiology, and clinical research to develop new and improved diagnostics, antiviral drugs, and vaccines. because of the ever-present threat of an influenza pandemic, niaid has initiated a program to establish niaid centers of excellence for influenza research and surveillance (ceirs) to support the research agenda of the hhs pandemic influenza plan. the overall goal of this program is to provide the government with the information and public health tools and strategies needed to control and lessen the impact of epidemic influenza and the increasing threat of pandemic influenza. the activities undertaken by the niaid centers of excellence for influenza research and surveillance will lay the groundwork for developing new and improved control measures for emerging and re-emerging influenza viruses, including determining the prevalence of avian influenza viruses in animals in close contact with humans, understanding how influenza viruses evolve, adapt, and transmit, and identifying immunologic factors that determine disease outcome. in the event of an urgent public health emergency involving the emergence and rapid spread of an influenza pandemic in humans, the network of centers will also develop and implement a niaid pandemic public health research response plan. each of the niaid centers of excellence for influenza research and surveillance has a focus on one or both of the following research areas: r research area 1: animal influenza surveillance. this is designed to conduct prospective international and/or domestic surveillance of animal influenza for the rapid detection and characterization of influenza viruses with pandemic potential. research. this is designed to enhance understanding of the molecular, ecologic, and environmental factors that influence pathogenesis, transmission, and evolution of influenza viruses, as well as to characterize the protective immune response. (i) niaid has awarded two contracts for the production of inactivated h5n1 vaccine to aventis pasteur (swiftwater, pennsylvania) and chiron (liverpool, united kingdom). (ii) niaid is also supporting the production of inactivated h9n2 vaccine manufactured with and without adjuvant by chiron (sienna, italy). (iii) clinical trials of pandemic influenza vaccine. in 2003, niaid conducted a phase i/ii clinical trial to evaluate increasing doses of inactivated influenza vaccine made by using the h9n2 virus isolated in 1999 from two infected children in hong kong. immunogenicity assays have been generated and data analysis is ongoing. the preliminary data showed that the vaccine was well tolerated. in 2004, niaid expanded its "pandemic preparedness in asia" contract to st. jude children's research hospital (n01-ai-95357). activities conducted under this expansion include (i) establishing animal influenza surveillance sites in asia; (ii) generating high-yielding vaccine candidates against influenza strains with pandemic potential accompanying reagents; (iii) supporting an international animal surveillance training course in hong kong; and (iv) studying newly emerging influenza strains infecting swine in the united states. in 2004, niaid launched the influenza genome sequencing project, which will rapidly provide influenza sequence data to the scientific community to enable further studies of how the influenza viruses evolve, spread, and cause disease, and which may ultimately lead to improved methods of treatment and prevention. during the past several years, there have been increasing reports of direct transmission of avian influenza viruses to humans (36) (37) (38) . furthermore, the continuing outbreaks of h5n1 influenza virus infections in avian species and humans in several countries (36, 39, 40) has emphasized the considerable threat posed by highly pathogenic avian influenza (hpai) and low pathogenic avian influenza lpai viruses to human health (14) . this, coupled with the difficulty to predict which subtype of avian influenza virus will cause the next human pandemic means that an ideal vaccine would elicit an immune response that protects the host from infection with a broad range of influenza viruses from the same or different subtypes (14) . the ha and na glycoproteins of influenza viruses undergo genetic and antigenic variation to escape the immune response (14, 41, 42) . whereas the presence of neutralizing antibodies specific for the ha glycoprotein at systemic or mucosal sites of infection would provide immediate protection against infection with influenza viruses, the clearance of human influenza viruses depends mainly on cellmediated immunity (43) . although antibodies specific for the na glycoprotein do not neutralize infectivity, they restrict virus replication by preventing the release of new virus particles, a process that requires viral na proteins. therefore, antibodies specific for na can decrease the severity of the disease (44, 45) . epitopes recognized by cytotoxic t lymphocytes (ctls) are present on np, pb2, and pa proteins of human influenza viruses. therefore, if a virus with a new ha and/or na glycoprotein emerges in the human population, cell-mediated immunity directed against the highly conserved internal proteins could have a role in protection at the time of a pandemic (14) . the principle underlying the currently licensed vaccines against human influenza viruses is the induction of protective antibodies specific for the ha glycoprotein of the predicted epidemic strain. the concentration of ha glycoprotein in licensed, inactivated virus vaccines for seasonal influenza is standardized, but the concentration of na glycoprotein is not standardized (14) . although most influenza vaccines are designed to induce ha-specific antibody responses to protect the host from infection, the biology of avian influenza viruses presents several unique challenges compared with human influenza viruses. these challenges include the presence of different subtypes of ha and na glycoproteins and the genetic and antigenic diversity within each subtype (14) . whereas the antigenic diversity has consequences for pandemic vaccines that must be considered in the design of a protective vaccine, not all of the 16 ha and 9 na subtypes of avian influenza viruses have similar pandemic potential. although hpai h5n1 viruses are the main focus of global attention, lpai h9n2 viruses are also widespread in poultry in asia (46) and hpai h7 viruses have caused large outbreaks in poultry in europe (47) , north america (48) , and south america (49) . although hpai viruses cause morbidity and mortality in poultry, hpai viruses might not be intrinsically more likely to cause a human pandemic than would lpai viruses. to this end, there are no known examples of a pandemic caused by an h5 or h7 hpai virus, although virologic data are limited to those from the three influenza pandemics that occurred in the past century (38) . because of this uncertainty, it would be highly desirable to develop vaccines against each of the subtypes of avian influenza virus, although the order of development can be prioritized on the basis of epidemiologic data (14) . a comparison of the predicted protein structures of ha glycoprotein subtypes 1 to 15 has led to the classification of these subtypes into four different clades: clade 1 (h1, h2, h5, h6, h11 and h13), clade 2 (h8, h9 and h12), clade 3 (h3, h4 and h14), and clade 4 (h7, h10 and h15) (51) . phylogenetic analysis of the genes encoding certain subtypes of ha glycoprotein reveals a separation into lineages that correspond with the geographic separation of the birds that they infect. these genetic lineages are referred to as the eurasian and north american lineages, and they generally correspond with the flight paths of migratory birds (51) (52) (53) . viruses from these two lineages might also be antigenically distinguishable, but the consequences of these genetic and antigenic differences for vaccine development are not known (14) . circulating human influenza viruses undergo rapid mutation owing to the low fidelity of the viral rna-dependent rna polymerase (54) . antigenic drift occurs when the genes encoding the ha and/or na glycoproteins undergo stepwise mutations, resulting in variant viruses with amino acid changes at one or more antibody-binding sites of ha and/or na (55) that allow the viruses to evade neutralization by antibodies generated as a result of previous natural infection or vaccination. the internal protein genes of avian influenza viruses are not under positive immune selection in waterfowl and shorebirds. however, the use of veterinary vaccines to protect poultry from infection with avian influenza viruses might drive evolution of the ha glycoprotein if such vaccines do not induce sterilizing immunity (14) . the viral determinants of pathogenicity of avian influenza viruses in humans are multigenic. further studies are required to understand how the pathogenicity of avian influenza viruses affects the infectivity and transmissibility of these viruses in humans and to establish whether these factors have implications for vaccine design (14) . inactivated virus vaccines and live attenuated virus vaccines that are being developed for pandemic influenza are based on technologies that are licensed for the existing seasonal human influenza vaccines (14) . vaccines based on various other platforms, such as live virus vectors expressing influenza virus proteins and dna vaccines, are also being developed and have shown promise in preclinical studies (see table 2 in ref. 14) . the currently licensed vaccines against human influenza viruses are produced in embryonated chicken eggs, and the manufacturing process can take 6 to 9 months (14) . consequently, for vaccines that are based on the currently licensed technologies, the availability of embryonated eggs is a crucial factor, and if the pandemic virus causes widespread morbidity and mortality in poultry, the supply of embryonated eggs might be compromised. therefore, alternative substrates, including mammalian cell lines, such as madin-darby canine kidney (mdck) cells and vero cells, have been developed for the production of influenza viruses for use in vaccines. to this end, considerable progress was made in the development of vaccines based on inactivated influenza viruses and live cold-adapted influenza viruses grown in these cell lines in microcarrier fermentors (56) (57) (58) . influenza a viruses replicate in several experimental animals, including chickens, mice, cotton rats, ferrets, hamsters, guinea pigs, and non-human primates. the use of mouse models for the study of influenza is limited because intranasally administered influenza a viruses do not cause symptoms of respiratory tract disease in mice, although some influenza a viruses are lethal in some other animal models (14) . ferrets are generally thought to be the best available model for influenza research. unlike mice, ferrets develop fever, rhinorrhea, and sneezing after infection with intranasally administered human influenza viruses and the virus replicates in the respiratory tract of these animals. seronegative ferrets develop a strain-specific immune response to human influenza viruses. currently, preclinical studies of pandemic influenza vaccines are carried out in mice and ferrets (14) . in preclinical studies, parenterally administered, inactivated whole-virus h9 and h5 subtype vaccines were shown to be effective in mice against challenge with homologous and heterologous viruses (14, (59) (60) (61) (62) (63) . recombinant h5 influenza viruses-which contain a modified ha glycoprotein, a wildtype na glycoprotein from the 1997 or 2003 h5n1 viruses or from an lpai h5n3 virus, and internal protein genes from the pr8 h1n1 influenza virus (a/puerto rico/8/34) that confer high yield in eggs-have been generated by reverse genetics (64) (65) (66) (67) (68) . the removal of the multibasic amino acid motif in ha that makes the ha0 precursor of hpai viruses highly cleavable attenuated the virus for infection of chickens, mice, and ferrets without altering the antigenicity of the ha glycoprotein (38) . two doses of these inactivated virus vaccines provided complete protection from lethal challenge with homologous and heterologous h5n1 viruses in mice and ferrets (64) (65) (66) (67) (68) . data from phase i clinical trials of inactivated virus vaccines against h9n2, h5n3, h5n1, and h2n2 viruses have been reported and other vaccines are still under evaluation (see table 2 in ref. 14) . studies that were carried out to date indicated that inactivated split-virion vaccines against avian influenza viruses-in which the virions were disrupted or split by detergent treatment and the surface glycoproteins were then partially purified-were not optimally immunogenic (69) and required multiple doses (70) or the inclusion of an adjuvant (71) (72) (73) (74) to induce a protective immune response (14) . whole-virus vaccines are more immunogenic than are split-virion vaccines, but they are likely to be more reactogenic (75) . adjuvants are required to increase the immunogenicity of inactivated virus vaccines and to decrease the concentration of viral proteins that is required to induce protective immunity, and several adjuvants for this purpose are under investigation, including aluminum salts, the squalene-oil-water emulsion (mf59), and other proprietary compounds (14) . an inactivated whole-virus h9n2 vaccine was found to be immunogenic in individuals who had circulating antibodies induced by prior exposure to h2n2 viruses that cross-reacted with h9n2 viruses, but the vaccine was not immunogenic in individuals who were born after 1968, when h2n2 viruses stopped circulating in humans (76) . this observation is consistent with findings from studies of an h1n1 vaccine in 1976-1977, when prior exposure to h1n1 viruses that had circulated in the population earlier ("priming") was found to be a determinant of the response to vaccination (75, 77) . these studies also emphasized the need for two doses of vaccine in "unprimed" individuals. in other studies of vaccines against h9n2 viruses, aluminum hydroxide and mf59 adjuvants improved immunogenicity (71, 73) . inactivated virus vaccines prepared from recombinant pr8 viruses that consisted of a modified ha glycoprotein and wild-type na glycoproteins from h5n1 viruses isolated in 2004 were evaluated as subvirion vaccines or wholevirus vaccines, with or without adjuvants (68, 72, 78, 79) . the subvirion vaccines were safe and well-tolerated in healthy adults, and the antibody response that was induced could be enhanced by increasing the dose of antigen used or by the addition of an adjuvant (72, 79) . a whole-virus vaccine was also well-tolerated by humans, and when administered with an adjuvant, this vaccine was immunogenic at a lower dose than that of the subvirion vaccines (74) . however, the available data indicate that inactivated h5 influenza virus vaccines are poorly immunogenic and require a large concentration of ha glycoprotein or co-administration with an adjuvant to achieve the desired antibody response (14) . live attenuated, cold-adapted influenza virus vaccines against human influenza viruses elicit both systemic immunity and mucosal immunity at the primary portal of infection (14) . these vaccine strains are generated by the reassortment of a wild-type influenza virus carrying the ha and na genes of interest with a cold-adapted donor aa (h2n2) influenza virus (a/ann arbor/6/60), which was generated by serial passage of the wild-type aa virus at successively lower temperatures (80) . the temperature-sensitive, attenuated, coldadapted donor aa virus has five mutations in three gene segments that contribute to the temperature-sensitive or attenuation phenotype (81) , and the virus has a high degree of phenotypic and genotypic stability (82) . candidate live attenuated virus vaccines against h9n2 and h5n1 avian influenza viruses generated on this cold-adapted donor backbone using reassortment and plasmid-based reverse genetics, respectively (see fig. 3 in ref. 14) , were safe and effective in mice and ferrets (83) (84) (85) . phase i clinical evaluation of these vaccines is currently in progress (14) . generally, live attenuated virus vaccines must retain some infectivity to be immunogenic. hence, virus shedding during clinical testing of these vaccines must be closely monitored (14) . there are potential challenges in the development of live attenuated virus vaccines for pandemic influenza, namely, (i) to generate reassortant viruses that are sufficiently infectious when the ha glycoprotein is derived from an avian influenza virus, in particular if the ha used has a preference for α2,3-linked oligosaccharides; (ii) to reproducibly achieve the desired level of viral attenuation with different combinations of ha and na genes; and (iii) to minimize the risk of reassortment with circulating human influenza viruses. the evaluation of live attenuated virus vaccines of different subtypes in preclinical studies in appropriate animal models and in clinical studies will address the first two challenges. the standard approach of preclinical evaluation that is applied to vaccines against human influenza viruses might not be uniformly applicable to avian influenza viruses, because the infectivity, immunogenicity, and protective efficacy of avian influenza viruses of different subtypes have not been studied extensively (86) . the risk of reassortment of the live attenuated vaccine virus with human influenza viruses during clinical trials can be minimized by conducting vaccine studies in isolation units when human influenza viruses are not circulating in the community. in the event of an influenza pandemic, the potential benefits of a live attenuated virus vaccine will have to be balanced against the risks associated with it, and this type of vaccine will only be introduced judiciously when a pandemic is imminent (14) . the use of recombinant or expressed proteins of the influenza virus in a vaccine is an attractive option for vaccine development because these approaches do not require handling of hpai or infectious viruses for vaccine production (6) . preclinical studies of recombinant ha, na, and m2 proteins as vaccine antigens (see table 2 in ref. 14) showed that the proteins were poorly immunogenic and required multiple doses (87) or the inclusion of adjuvants (88, 89) for improved immunogenicity and efficacy. dna vaccines encoding the ha and na glycoproteins of avian influenza viruses or conserved internal virus proteins, such as matrix proteins and nucleoproteins, induced protective immunity in mice and chickens (90) (91) (92) (93) . the protective efficacy of a nucleoprotein-encoding dna vaccine was increased by a booster vaccination in the form of a recombinant replicationdefective adenovirus (radv) expressing the nucleoprotein (94) . in two recent studies, intramuscular or intranasal immunization of mice with a human radv vaccine expressing the influenza virus ha glycoprotein induced both humoral and cell-mediated immune responses and conferred protection against challenge with the wild-type virus in mice and chickens (95, 96) . a recombinant baculovirus-expressed h5 glycoprotein subunit vaccine was well tolerated but was poorly immunogenic in humans, indicating the need for an adjuvant (97) . the production of recombinant proteins and dna vaccines is safe and economical, but clinical studies of their safety and immunogenicity in humans are awaited (14) . an ideal influenza vaccine would be effective against a range of virus subtypes and could be useful during pandemic and interpandemic periods. one approach to creating a universal vaccine would be to target an antigenically stable protein or an antigenically stable part of a variable protein that is essential for virus replication (14) . the high degree of conservation of the m2 protein makes it a prime candidate for a universal influenza vaccine. the m2 protein induced crossreactive immunity that decreased the severity of disease in animal models after challenge with wild-type virus (98, 99) . however, the emergence of immune-escape mutants of the m2 protein in mice in the presence of specific antibodies raised concerns regarding the usefulness of the m2 protein as a target for a universal vaccine (100) . clinical studies would be required to evaluate the immunogenicity of the m2 protein in humans (14) . it has been suggested that the use of the na glycoprotein, which is less variable than the ha glycoprotein, to induce cross-protective immunity should be explored (101) . na-specific immunity in mice provides significant crossprotection against antigenically distinct viruses of the same subtype (102, 103) . although na-specific antibodies do not prevent infection with influenza viruses, they decrease the severity and duration of illness in humans by limiting the release and spread of the virus (44, 45) . furthermore, if common immunogenic epitopes are identified within the four clades of ha glycoprotein subtypes, ha-based immunogens could induce widely crossreactive immunity (104) . alternatively, genetically engineered viruses that have several conserved immunogenic epitopes on the viral envelope could be developed and evaluated for use as a universal influenza vaccine (101) . recombinant viruses expressing chimeric ha glycoproteins have also been described recently (105) . although universal influenza vaccines are still in preclinical development, the potential benefits of such vaccines are so great that strategies to develop them must be encouraged (14) . the evaluation of vaccines against potential pandemic strains of avian influenza viruses presents a unique challenge because vaccines developed against these viruses can only be evaluated for safety and immunogenicity, and not for protection, in clinical trials because challenge studies to assess the efficacy of the vaccines cannot be undertaken in humans (14) . in addition, when the immunogenicity of candidate pandemic vaccines is assessed, the data can be difficult to interpret because specific information on the nature and magnitude of the antibody response that correlates with protection is lacking. if a vaccine is immunogenic, it might be possible to assess its efficacy by testing the vaccine in a large group of people who are at high risk from infection with avian influenza virus, such as poultry farmers in areas with severe epizootics (14) . serum and mucosal antibodies can independently mediate immunity to influenza viruses. live viruses and inactivated virus vaccines differ in the induction of protective antibodies, but there are no standardized methods for evaluating the mucosal antibody response. the conventional assay for assessing the immunogenicity of a human influenza vaccine is the hemagglutination-inhibition assay. although the standard hemagglutination-inhibition assay, which uses chicken or turkey erythrocytes, is relatively insensitive for the detection of antibodies specific for h5n1 viruses, there is a modified assay using horse erythrocytes that was found to be more sensitive because horse erythrocytes exclusively express the α2,3-linked oligosaccharide side chains that are preferred for binding by avian influenza viruses. however, the horse erythrocyte hemagglutination-inhibition assay has not been well standardized, and the antibody titers determined by this assay that correlate with protection are not known. therefore, the choice of assays by which the immune response is assessed poses a practical challenge for the evaluation of pandemic influenza vaccines (14) . an alternative to the hemagglutination-inhibition assay that might be more biologically relevant is a neutralization assay, in which the ability of antibodies to neutralize the infectivity of the avian influenza virus is assessed (14) . using paired sera from individuals infected with h5n1 influenza virus in 1997 in hong kong collected at the acute and convalescent stages of infection, a neutralizing antibody titer of 1:80 was shown to be indicative of infection with an h5n1 virus (106) . however, it is not known whether this antibody titer correlates with protection from re-infection. other barriers to the use of the neutralization assay are the requirement for appropriate biosafety containment measures, as the assay requires handling of the infectious virus, and the fact that the test has not yet been standardized (107). virus infection. a recombinant fusion protein (das181) composed of a sialidase catalytic domain derived from actinomyces viscosus was fused with a cell surfaceanchoring sequence. when applied topically via inhalation, das181 effectively removed the influenza viral receptors, sialic acids, from the airway epithelium (108). by effectively cleaving the sialic acid receptors used by both the human and avian influenza viruses, das181 prevented the virus from binding to and entering the host cells. this is an innovative antiviral strategy because da181 acts on the receptors used by the influenza virus rather than targeting the virus itself. r large-scale sequence analysis of avian influenza isolates. avian influenza is a significant human health threat globally because of its potential to infect humans and result in a global influenza pandemic; however, very little sequence information for avian influenza virus (aiv) has been publicly available. a more comprehensive collection of publicly available sequence data for aiv is necessary for research in influenza to understand how flu evolves, spreads, and causes disease to shed light on the emergence of influenza epidemics and pandemics and to uncover new targets for drugs, vaccines, and diagnostics. a team of niaid-supported scientists has released genomic data from the first large-scale sequencing of aiv isolates, doubling the amount of aiv sequence data in the public domain (109) . these sequence data include 2,196 aiv genes and 169 complete genomes from a diverse sample of birds. the preliminary analysis of these sequences, along with other aiv data from the public domain, revealed new information about aiv, including the identification of a genome sequence that may be a determinant of virulence. this study provides valuable sequencing data to the scientific community and demonstrates how informative large-scale sequence analysis can be in identifying potential markers of disease. r architecture of ribonucleoprotein complexes in influenza a virus particles. data from transmission electron microscopy of serially sectioned virions of influenza a viruses have shown that the ribonucleoprotein complexes (rnps) of the virus are organized in a distinct pattern: seven segments of different lengths surrounding a central segment (110) . furthermore, the individual rnps are suspended from the interior of the viral envelope at the distal end of the budding virion and are oriented perpendicular to the budding tip. these findings have argued against a random incorporation of rnps into virions, supporting instead a model in which each segment contains specific incorporation signals that would enable the rnps to be recruited and packaged as a complete set. the selective mechanism of rnp incorporation into virions and the unique organization of the eight rnp segments may be crucial to maintaining the integrity of the viral genome during repeated cycles of replication (110) . r structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus. the hemagglutinin structure at 2.9å resolution from a highly pathogenic vietnamese h5n1 influenza virus (viet04) has been elucidated (111) . its structure was found to be more related to that of the 1918 (8) and other human h1 hemagglutinins than to a 1997 duck h5 hemagglutinin. glycan microarray analysis of the viet04 virus revealed an avian α2,3 sialic acid receptor binding preference. introduction of mutations that can convert h1 serotype hemagglutinins to human α2,6 receptor specificity only enhanced or reduced affinity for the avian-type receptors. however, mutations that can convert avian h2 and h3 hemagglutinins to human receptor specificity, when inserted onto the viet04 hemagglutinin framework, permitted binding to a natural α2,6 glycan, thereby suggesting a path for this h5n1 virus to gain a foothold in the human population (111) . r ns1-truncated modified live-virus vaccine. swine influenza viruses (sivs) naturally infect pigs and can be transmitted to humans. furthermore, in the pig, genetic reassortment to create novel influenza subtypes by mixing avian and human influenza viruses is possible. therefore, a vaccine against siv and inducing cross-protective immunity between different subtypes and strains circulating in pigs will be highly advantageous. to this end, an h3n2 siv (a/swine/texas/4199-2/98) (labeled tx98) containing a deleted ns1 gene expressing a truncated ns1 protein of 126 amino acids (ns1/126) was attenuated in swine. subsequently, 4-week-old pigs were vaccinated with the tx98 ns1/126 modified live-virus (mlv) vaccine. the highly attenuated mvl completely protected against challenge with the homologous siv (112) . vaccinated pigs challenged with the heterosubtypic n1n1 virus demonstrated macroscopic lung lesions similar to those of the unvaccinated h1n1 control pigs. remarkably, vaccinated pigs challenged with the h1n1 siv had significantly less microscopic lung lesions and less virus shedding from the respiratory tract than did unvaccinated, h1n1-challenged pigs. furthermore, all vaccinated pigs developed significant levels of hemagglutination inhibition and enzyme-linked immunosorbent assay titers in serum and mucosal immunoglobulin a antibodies against h3n2 siv antigens (112). r immunization by h5 avian influenza hemagglutinin scientists from niaid's vrc have developed a strategy to generate vaccines and therapeutic antibodies that could target predicted h5n1 mutants before these viruses evolve naturally. this advance was made possible by creating mutations in the region of the h5n1 hemagglutinin protein that directs the virus to bird or human cells and eliciting antibodies to it (113) . r protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. using the genetic sequence information for the 1918 flu virus, vrc scientists have created plasmidssmall strands of dna designed to express specific characteristics-carrying genes for the virus's hemagglutinin protein, the surface protein found in all flu viruses that allows the virus to stick to a host cell and cause infection (114) . the researchers created two types of plasmids: one to reflect the ha found in the original 1918 flu virus; the other an altered ha protein designed to attenuate the virus. mice were then injected with a dna vaccine containing both types of plasmids to determine whether they would generate immune responses to the 1918 virus. the researchers found significant responses both in terms of the production of t cells and the production of neutralizing 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sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection large-scale sequence analysis of avian influenza isolates architecture of ribonucleoprotein complexes in influenza a virus particles structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus vaccination of pigs against swine influenza viruses by using an ns1-truncated modified live-virus gene immunization by avian h5 influenza hemagglutinin mutants with altered receptor binding specificity protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination key: cord-016995-5izyl234 authors: auewarakul, prasert title: the past and present threat of avian influenza in thailand date: 2008 journal: emerging infections in asia doi: 10.1007/978-0-387-75722-3_2 sha: doc_id: 16995 cord_uid: 5izyl234 avian influenza h5n1 infection was first identified in thailand in january 2004. since then, there have been three major outbreaks in the cold season of 2003–2004 and in the rainy and cold seasons of 2004–2005 and 2005–2006. more than 62 million birds died or were culled. the burden shifted from large industrial farming in the first outbreak to small farms, backyard chickens, and free-grazing ducks. up to november 2005, there were 20 confirmed cases of human h5n1 infection. thirteen of these died. most of the confirmed cases were solitary ones except for three persons in a single family, and epidemiological evidence indicated that person-to-person transmission may have been involved in this cluster. however, sequence analysis of the virus in the cluster did not suggest any changes that might enhance the viral ability to get transmitted among humans. h5n1 viruses in thailand and vietnam belong to a single lineage genetically and are antigenically distinguishable from the viruses of the same genotype z from southern china and indonesia. despite the seemingly subsiding epidemic in thailand, the problem is far from resolved. h5n1 viruses are still sporadically isolated from domestic poultry as well as from wildlife. more important, isolates were also found in asymptomatic animals. natural selection may have adapted the virus to a less aggressive form. this would make the virus more elusive and difficult to control. a threat of a pandemic strain emerging from the h5n1 virus is still imminent. a national strategic plan for avian influenza control and influenza pandemic preparedness has been implemented. the plan aims at effective control of avian influenza spread in animals as well as in humans for a three-year period and at efficient pandemic preparedness within one year. nevertheless, more regional and international collaboration is needed. with proper collective preparedness, there is a hope that the threatening influenza pandemic can be prevented by confining and eliminating a potential pandemic strain at its origin. in december 2003, poultry farms in the eastern, central, and northern regions of thailand experienced large-scale die-offs. the outbreak started from the eastern region of the country. the disease caused rapid death, with a very high attack rate. at that time, h5n1 outbreaks had been reported in south korea, vietnam, and japan (oie, 2005). a few humans with pneumonia were suspected to originate from contact with sick or dead poultry. final diagnosis in these patients was not done as clinical samples were not available at the time when proper diagnostic testing became available. on 23 january 2004, the first case of human h5n1 infection in thailand was reported. it was a boy from kanchanaburi, a province about 100 km west of bangkok. he was admitted to siriraj hospital in bangkok and was diagnosed to have severe progressive pneumonia. the patient was initially treated with broad spectrum antibiotics, and respiratory samples were tested for influenza virus. the laboratory result showed that the patient harbored influenza virus, and sequencing of the viral rna indicated that the virus belonged to the h5 subtype (chokephaibulkit et al., 2005; puthavathana et al., 2005). when this result was reported to the ministry of public health, the government announced that there was a highly pathogenic avian influenza (ai) outbreak in thailand. the department of livestock development (dld) confirmed the presence of h5n1 viruses in poultry on the same day. subsequent analysis of the virus from patients and animals confirmed that it was h5n1 ai virus of genotype z and was closely related to the virus from vietnam (viseshakul et al., 2004; puthavathana et al., 2005). done as clinical samples were not available at the time when proper diagnostic testing became available. on 23 january 2004, the first case of human h5n1 infection in thailand was reported. it was a boy from kanchanaburi, a province about 100 km west of bangkok. he was admitted to siriraj hospital in bangkok and was diagnosed to have severe progressive pneumonia. the patient was initially treated with broad spectrum antibiotics, and respiratory samples were tested for influenza virus. the laboratory result showed that the patient harbored influenza virus, and sequencing of the viral rna indicated that the virus belonged to the h5 subtype puthavathana et al., 2005) . when this result was reported to the ministry of public health, the government announced that there was a highly pathogenic avian influenza (ai) outbreak in thailand. the department of livestock development (dld) confirmed the presence of h5n1 viruses in poultry on the same day. subsequent analysis of the virus from patients and animals confirmed that it was h5n1 ai virus of genotype z and was closely related to the virus from vietnam (viseshakul et al., 2004; puthavathana et al., 2005) . figure 1 shows the time distribution of the outbreak in humans and poultry in thailand (dld, 2005a) . the outbreak activity has a clear seasonal variation. the disease activity starts at the beginning of the rainy season (july), peaks in october at the transition from the rainy season to winter, and subsides in march when summer starts. the first round of outbreaks in early 2004 was widely spread. most of the outbreaks were in the central and lower northern regions of the country, along the major river basin, where poultry density is the highest, especially free-grazing ducks (fig. 2) . outbreaks in humans and poultry had similar time and geographical distributions, indicating poultry as the source of infection in humans. repetitive outbreaks in same areas suggested that even though there was no apparent disease between the outbreaks, the virus remained resident either in domestic animals or wildlife in that area. the similarity of the viruses between the outbreaks further proved that latter outbreaks were caused by the remnant viruses from previous outbreaks and not by a reintroduced virus . up to the end of october 2005, there have been a total of 20 confirmed cases in humans, of which 13 died. of the 20 cases, 12 were in the first round of the outbreak in the winter of [2003] [2004] 5 were in the rainy season and winter of 2004, and 3 were in 2005 (centers for disease control and prevention, 2004; beigel et al., 2005; chotpitayasunondh et al., 2005) . besides these 20 cases, there were 23 suspected cases with comparable clinical and epidemiological features but lack laboratory confirmation. most of the suspected cases were in the early part of the first outbreak when the laboratory test was not readily available and surveillance and specimen referral system had not been well established. they also had direct contact with dying poultry except for one cluster that probably resulted from person-to-person transmission . all h5n1 ai viruses since the first outbreak in 1997 carry h5 and n1 genes that originated from a common ancestor closely related to a goose virus isolated from guangdong in 1996 (a/goose/gaungdong/1/96) . although the virus responsible for the 1997 outbreak in hong kong was completely eliminated by a total depopulation of poultry on the island, the ancestral virus still circulated in southern china and gave rise to several genotypes by reassortment with other ai viruses. since 1997, many of these genotypes have emerged, disappeared, and been replaced by other genotypes (guan et al., 2002 . all the viruses in the outbreaks since 2004 belong to the genotype z. this suggested that the genotype z has the optimal genetic makeup for efficient spread among poultry. all thailand isolates contain multiple basic amino acid substitutions at the protease cleavage site in the ha protein, a 20-codon deletion in the na gene, and a 5-codon deletion in the ns gene, which are characteristics of the genotype z viruses. amino acid residues at the receptor-binding site of ha of human viruses were similar to those of chicken viruses. the presence of amantadine resistance in the thailand viruses was indicated by a mutation in the m2 transmembrane protein and was phenotypically confirmed. the thailand viruses contained more avianspecific residues than did the 1997 hong kong h5n1 viruses, suggesting that the virus may have adapted to allow a more efficient spread in avian species (viseshakul et al., 2004; puthavathana et al., 2005) . it was initially hypothesized that the thailand-vietnam clade might be more efficient in causing disease in humans, because there was no human infection in indonesia earlier in the outbreak, despite extensive spread of the disease in poultry. however, the more recent outbreak of human infection in indonesia indicated that the indonesian clade is also pathogenic in man (kandun et al., 2006) . whether this is a result of viral adaptation or a property of the original virus is unclear. influenza is a rapidly evolving virus. experiments in ducks demonstrated that only a single round of nonlethal infection in one host could exert enough selection pressure to cause an antigenic drift and reduce virulence . it is therefore likely that the endemic virus will gradually become less pathogenic in ducks, which is a natural host of influenza virus. observation of the outbreak pattern in the third round (rainy season 2005) suggested that pathogenicity in other avian species may not be similar to what had been observed in the first round of outbreaks. in the latest outbreak, a smaller fraction of sick and dying animals was observed and more viruses were isolated from apparently healthy animals. experimental data have shown that the viruses in 2005 were indeed less pathogenic in ducks sturm-ramirez et al., 2005) . interestingly, the h5n1 viruses of 1997 were also nonpathogenic in ducks. the virus became pathogenic in ducks in 2002-2003 and reverted to a nonpathogenic strain in 2005 sturm-ramirez et al., 2005) . this probably indicated a temporary loss of the equilibrium between the virus and its natural host by the adaptation into a new host, that is, domestic poultry. the reduction of pathogenicity in 2005 suggested that the virus is setting a new equilibrium with its natural host. the impact on the poultry industry during the first outbreak was devastating. because thailand is a major poultry meat exporting country, direct loss in production caused by the outbreak and culling, the trade ban imposed by importing countries, and reduced domestic consumption caused severe economic loss in the industry. more than 62 million birds were either killed by the disease or culled for outbreak control (tiensin et al., 2005) . the government compensated farmers for their losses. farmers were entitled to compensation of 75% of the value of animals that were destroyed. different sectors of the poultry industry vary in their practice of biosecurity and outbreak prevention. after the first outbreak, most large-scale industries improved biosecurity measures. consequently, little problem remained in the large-scale industry sector in the second and third outbreaks. the major concern remains in the smallscale backyard farms of chickens and ducks, where it is very difficult to employ proper biosecurity. another area of concern is the farms in the central part of thailand, where paddy fields are plentiful. it is a common practice of free-grazing duck raising in thailand to move flocks of ducks from field to field by trucks to let them feed on the dropped grains after the harvest. flocks can move over a very long distance in search of a suitable feeding area. because ducks can be infected and can shed virus without clinical sign, migrating flocks are ideal for spreading the disease sturm-ramirez et al., 2005) . the geographical distribution of the outbreaks coincided with the area with the highest density of free-grazing duck raising (gilbert et al., 2006) . this further supports the belief that free-grazing ducks may have been a major source of the spread of disease. another activity that the government has been trying to control is cock fighting. it involves not only the movement of fighting cocks from place to place for fighting, but also close contact between cocks and people, which can increase the chance of transmission to humans. the control measures now in place are registration and the obligatory screening of fighting cocks and temporary prohibition of cock fighting during the peak of influenza outbreaks (dld, 2005b). open-bill storks are probably the most affected species in thailand (keawcharoen et al., 2005) . they are migratory birds that migrate to central thailand in winter. some of the birds remain in thailand all year round. open-bill storks are vulnerable to infection, probably because they live in big flocks in the wetlands of central thailand. continuous surveillance confirmed the presence of virus in these birds. evidence of h5n1 infection was also found in other birds, such as sparrows and pigeons, which share natural habitats with humans and domestic poultry. although free-living birds are not likely to play a major role in the introduction of new outbreaks, it is possible that these birds may spread the infection locally between farms and maintain the viral reservoir locally between outbreaks. of the 20 confirmed cases, 11 were children under the age of 14 years and 13 died, leading to a mortality of 65% (chotpitayasunondh et al., 2005) . in most of the confirmed cases, the source of infection was backyard chickens. most of the patients had a fever as the starting symptom followed by coughing, dyspnea, and pneumonia in a median time of 4 days. about half of the patients (53%) had rhinorrhea, 71% had sore throat, 53% had myalgia, and 41% had diarrhea (chotpitayasunondh et al., 2005) . abnormal chest radiographs in these patients included interstitial infiltration and patchy lobar infiltrates in a variety of patterns (single lobe, multiple lobes, unilateral, or bilateral distributions). in patients who developed ards (acute respiratory distress syndrome), the radiographic pattern progressed to a diffuse bilateral ground-glass appearance (beigel et al., 2005; chotpitayasunondh et al., 2005) . an autopsy study showed that the lung, but not upper airway epithelium, was the site of viral replication, and the cellular target of the virus was the alveolar epithelial cell . the most remarkable laboratory findings in these patients were lymphopenia, leucopenia, and thrombocytopenia. more importantly, the lymphocyte counts were significantly different between fatal cases and survivors and between patients with and without ards (chotpitayasunondh et al., 2005) . this suggested that a simple blood count can not only help in the preliminary diagnosis of bird flu, but can also be used as a prognostic marker. most of these patients were treated with oseltamivir, but it is not clear whether the treatment changed the course of the illness. although most confirmed ai infections in humans caused respiratory infection and pneumonia, there were some cases with other manifestations. a case presenting with acute diarrhea and later developing fatal respiratory failure was reported . viral replication could be detected in an autopsy sample from the intestine of a patient even in the absence of diarrhea . this suggested that the virus may have a tropism for the intestinal tract similar to the infection in avian species. reports from vietnam also showed other unusual manifestations of h5n1 infection in humans, such as encephalitis . this will make the disease surveillance more difficult, and monitoring of only pneumonia and respiratory failure may not be adequate to cover all cases of human h5n1 infection. avian and human influenza viruses were thought to be separated by the receptor preference: avian viruses use 2,3-α-linked sialic acid while human viruses use 2,6α-linked sialic acid (suzuki, 2005) . h5n1 ai viruses showed avian-type receptor specificity (matrosovich et al., 1999; gambaryan et al., 2004) . nevertheless, they can infect humans. infection by the h5n1 virus in humans is not efficient, and person-to-person transmission cannot readily occur. the inability of the virus to transmit from person to person is the only barrier preventing the virus from becoming a pandemic strain. experimental data showed that only two substitutions in the receptor-binding site of the hemagglutinin gene are needed to change the receptor-binding preference of h5n1 virus from 2,3-to 2,6-α-linked sialic acid (harvey et al., 2004) . it is not known whether this receptor preference is the only barrier the virus needs to cross to infect humans efficiently. although several clusters of h5n1 infections have been observed in thailand, vietnam, and indonesia, it is difficult to prove human-to-human transmission, as most of these patients had exposure to poultry and it is not possible to prove whether they contracted the disease from animals or humans. the very low sequence variability among the viruses in the outbreak made it impossible to infer chain of transmission from sequence data. it was a unique incidence in thailand that allowed an inference of probable person-to-person transmission . it was a cluster of three patients: a young girl, her mother, and her aunt. the girl and her aunt lived in kamphaengphet where there was an ai outbreak in poultry. the mother lived in nonthaburi, a province near bangkok where there was no ai outbreak and did not have any contact with poultry. she went to take care of her sick daughter 1 day before the girl died. the mother had an onset of fever 4 days later, went back to nonthaburi, had pneumonia, and died 14 days later. the aunt who also took care of the sick girl also developed pneumonia 8 days after the girl's death. although she had contact with a dead chicken, the last exposure was 17 days before the onset of fever, which was too long for an incubation period of influenza. because the mother and the aunt had no contact with poultry within a time interval compatible with an incubation period of influenza and the time of onset after exposure to the index case was compatible with the incubation period of ai, it was concluded that the two cases were likely to contract the disease from the index case . as soon as this cluster was recognized, effort was made to contain possible further person-to-person transmission. all household members, other family contacts, exposed neighbors, and exposed health care workers were placed under active surveillance for fever and respiratory symptoms for 14 days. fortunately, there was no further transmission or evidence that the virus in this cluster facilitated more efficient human-to-human transmission. however, if the virus is allowed to transmit from humans to humans without interruption, it is likely that it will eventually evolve to become more transmissible in humans. it is therefore crucial that every effort has to be made to prevent human-to-human transmission. ai virus h5n1 infection was observed in several mammalian species, including cat, tiger, leopard, and dog (keawcharoen et al., 2004; thanawongnuwech et al., 2005; songsermn et al., 2006; butler, 2006) . the exposure that led to the infection was mostly from feeding on the carcasses of infected poultry. the infection caused severe disease, with high mortality in these animals. experimental infections were also reported in cats, mice, ferrets, monkey, and pigs (rimmelzwaan et al., 2001 kuiken et al., 2003 kuiken et al., , 2004 govorkova et al., 2005; maines et al., 2005) . most of these animals presented severely fatal disease manifestations except for pigs, in which the infection caused only mild disease (choi et al., 2005) . nevertheless, the virulence of h5n1 virus in mammals is probably heterogeneous among strains and is continuously evolving. experimental data suggested that the virus is evolving to become more pathogenic in mammals and that the genetic determinants of virulence lie in the polymerase genes, resulting in a high-replication phenotype li et al., 2005) . this further emphasized the danger of this virus and suggested that it may also become more pathogenic and transmissible in humans. active surveillance of suspected cases is continuously employed by the public health authority in thailand. cases of pneumonia in those who have history of exposure to poultry are reported to the department of disease control and investigated for the presence of h5n1 virus by viral culture and rt-pcr. clusters of pneumonia and pneumonia in hospital personnel are also the targets of surveillance and disease control in order to detect human-to-human transmission of severe influenza of pandemic potential. emphasis has been made to detect any potential pandemic strain as early as possible. recent studies using computer simulation predicted that early detection and proper outbreak control by social distancing measures and antiviral drugs may be effective in containing the outbreak and eliminating the potential pandemic virus provided the virus does not have a greater ability to get transmitted than does the previous pandemic strains, that is, it has a basic reproductive number below 1.6-1.8 (ferguson et al., 2005; longini et al., 2005) . the amount of the antiviral drug oseltamivir that should be enough for the elimination of a potential pandemic virus has been predicted differently between the two studies: 100,000 to 1 million and 3 million courses (ferguson et al., 2005; longini et al., 2005) . having that in mind, the ministry of public health has started to stockpile oseltamivir, and as of october 2005, the amount stockpiled is 72,500 courses (725,000 tablets). outbreaks in poultry are monitored and controlled by the dld. specifically, if the poultry death rate in any facility was greater than 10% within a single day, all birds, their products, and other potentially contaminated materials have to be destroyed without delay. cloacal swabs of affected flocks would then be collected for laboratory confirmation. subsequently, neighboring flocks would be destroyed immediately or quarantined and destroyed when h5n1 laboratory diagnosis was confirmed. movement of poultry and their products would be restricted to a 1-5-km radius. in the first round of outbreaks, neighboring flocks within a 5-km radius were preemptively culled as quickly as possible. after july 2004, preemptive culling was implemented only within a village, within an area of 1 km around an outbreak, or on suspected farms. dld has launched a nationwide surveillance program (known as "x-ray survey") in january 2004 , october 2004 , and july 2005 . the program was conducted in close collaboration with the ministry of agriculture and cooperatives, ministry of public health, provincial governors, volunteer public health workers, and dld livestock workers (dld, 2005b) . the program is planned to be launched biannually, in the winter before chinese new year and in the rainy season. these two periods are considered the riskiest times of the year because there is an unusually high volume of movement of poultry for the chinese new year festivities, which can promote the spreading of the virus. furthermore, the high humidity and low temperature in the rainy season may be optimal for the viral spread. other specific measures include the control of free-grazing ducks and fighting cocks. there have been serious debates over the continuation of raising free-grazing ducks. the practice is favored by environmentalists because it makes efficient use of the paddy field after harvesting and provides biological pest control, as they prey on the golden apple snail, a major pest for rice cultivation. although the debates are not yet totally resolved, long distance moving of flocks by trucks is temporarily prohibited, especially moving from or into the central region of the country where the disease is not yet fully eliminated. the flocks are registered and allowed to migrate only in a limited zone. the limitation of grazing area by zoning was designed so that there will be no extensive movement, so as to limit the area of potential contamination. the zoning strategy is also implemented to other poultry, dividing the country into five zones: central, north, northeast, east, and south, in order to limit movement of poultry from the central plan to the other regions that are disease-free, especially the eastern region where most exporting poultry industry farms are located. in contrast to other countries in the region, thailand does not use the ai vaccine in poultry. although the illegal vaccine may have been used to some extent, the official policy is still against the vaccine (fao newsroom, 2006) . although the decision was probably influenced mainly by the international trade barrier, the major scientific concern against vaccination in poultry is the risk of having undetectable asymptomatic infection that may shed the virus and spread the infection (swayne et al., 2001; liu et al., 2003) . with the changing phenotype of the virus towards lower virulence in ducks, the vaccination policy may need to be reconsidered, because without vaccination the ducks will be asymptomatically infected as well and vaccination is effective in reducing the level of viral shedding . a previous report showing the ability of vaccination to abort an outbreak in a chicken farm strongly supports the use of the vaccine in adjunction to biosecurity measures . sufficient facility for viral testing is crucial for the success of outbreak control and surveillance. both the national institute of health and the national institute of animal health have strengthened their diagnostic capability and extended the service to their regional laboratories. two national plans, national strategic plan for avian influenza control and national strategic plan for influenza pandemic preparedness for a 3-year period (2005) (2006) (2007) , have been set and endorsed by the cabinet since january 2005 (wibulpolprasert et al., 2005) . the national strategic plan for avian influenza control has targets within the 3-year period as follows: 1. no outbreak of ai in economic poultry in 2 years. 2. reduce outbreak in domestic poultry, fighting cocks, exotic birds, and migratory birds to a level that is not considered a problem in 3 years. 3. no outbreak in other animals in 3 years. 4. no disease contract from animals to humans in 2 years. 5. thailand is efficiently prepared to handle an influenza pandemic in 1 year. the national strategic plan for influenza pandemic preparedness has targets within the 3-year period as follows: 1. to strengthen an effective influenza surveillance system, including clinical surveillance in the communities, work places, educational institutions, and every public health facilities, as well as establishing 12 centers for laboratory surveillance of the viruses throughout the country within 3 years. 2. to enable thailand to be ready for efficient management of the emergency situations during the influenza pandemic within 2 years. 3. to stockpile antiviral drugs (oseltamivir) so as to treat 325,000 patients (3,250,000 tablets) and to stockpile the raw materials for manufacturing antiviral drugs (oseltamivir) so as to treat 1,625,000 patients within 5 years. 4. to develop the capacity to manufacture or stockpile influenza vaccines within 5 years. 5. in case of an influenza pandemic, hospitals throughout the country have the capacity of up to 100,000 beds for taking care of influenza patients in critical conditions. in case of outbreaks in specific areas, field hospitals with a capacity of 5,000 beds will be ready for services. the threat of influenza pandemic is eminent. while any ai virus has a potential to evolve and eventually become a pandemic strain, the present danger is the h5n1 virus, which has already crossed the interspecies barrier from avian to human. without proper intervening measures, it is just a matter of time before the virus adapts to transmit efficiently from person to person and become the next pandemic virus. effective control of the outbreak in animals, prevention of exposure in humans, and early detection of a potential pandemic strain are essential to the success of preventing the pandemic. preparedness in case of a pandemic is also crucial to minimize the loss of human life. strong international collective effort is essential for the success. genetic characterization of h5n1 influenza a viruses isolated from zoo tigers in thailand atypical avian influenza (h5n1). emerg infect dis avian influenza a (h5n1) infection in humans thai dogs carry bird-flu virus, but will they spread it? the evolution of h5n1 influenza viruses in ducks in southern china studies of h5n1 influenza virus infection of pigs by using viruses isolated in vietnam and thailand in 2004 a child with avian influenza a (h5n1) infection human disease from influenza a (h5n1) fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma department of livestock development. date of access vaccination of chickens against h5n1 avian influenza in the face of an outbreak interrupts virus transmission thailand shares secrets of success strategies for containing an emerging influenza pandemic in southeast asia h5n1 chicken influenza viruses display a high binding affinity for neu5acalpha2-3galbeta1-4(6-hso(3) )glcnac-containing receptors free-grazing ducks and highly pathogenic avian influenza lethality to ferrets of h5n1 influenza viruses isolated from humans and poultry in 2004 emergence of multiple genotypes of h5n1 avian influenza viruses in hong kong sar h5n1 influenza: a protean pandemic threat restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia three indonesian clusters of h5n1 virus infection in 2005 avian influenza h5n1 in tigers and leopards characterization of the hemagglutinin and neuraminidase genes of recent influenza virus isolates from different avian species in thailand pathology of human influenza a (h5n1) virus infection in cynomolgus macaques (macaca fascicularis) avian h5n1 influenza in cats genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia molecular basis of replication of duck h5n1 influenza viruses in a mammalian mouse model preparation of a standardized, efficacious agricultural h5n3 vaccine by reverse genetics containing pandemic influenza at the source avian influenza (h5n1) viruses isolated from humans in asia in 2004 exhibit increased virulence in mammals the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties world organization for animal health. date of access molecular characterization of the complete genome of human influenza h5n1 virus isolates from thailand pathogenesis of influenza a (h5n1) virus infection in a primate model a primate model to study the pathogenesis of influenza a (h5n1) virus infection avian influenza h5n1 in naturally infected domestic cat are ducks contributing to the endemicity of highly pathogenic h5n1 influenza virus in asia sialobiology of influenza: molecular mechanism of host range variation of influenza viruses efficacy of vaccines in chickens against highly pathogenic hong kong h5n1 avian influenza probable tiger-to-tiger transmission of avian influenza h5n1 protective efficacy in chickens, geese and ducks of an h5n1-inactivated vaccine developed by reverse genetics highly pathogenic avian influenza h5n1 influenza a h5n1 replication sites in humans probable person-to-person transmission of avian influenza a (h5n1) the genome sequence analysis of h5n1 avian influenza a virus isolated from the outbreak among poultry populations in thailand national strategic plan for avian influenza control and influenza pandemic preparedness in thailand key: cord-019010-9xgwjvsv authors: luna, c. m.; valentini, r.; rizzo, o. title: life-threatening respiratory failure from h1n1 influenza: lessons from the southern cone outbreak date: 2010-06-23 journal: yearbook of intensive care and emergency medicine 2010 doi: 10.1007/978-3-642-10286-8_20 sha: doc_id: 19010 cord_uid: 9xgwjvsv a sharp increase in the hospitalization rate for pneumonia, particularly among adults between 20 and 40 years old, and an unusual series of deaths, coincident with an increase in laboratory-confirmed influenza cases, were reported in the spring of 2009 in mexico. this outbreak appeared after the end of influenza season, and was associated with mortality in a younger age-group than the pattern observed in temperate areas in the northern hemisphere [1]. the concurrent finding of a novel, swine-origin influenza a virus (so called pandemic influenza [h1n1] 2009) from infected children in the united states [2] completed the picture. . sharp increase in the number of cases and rapidly argentina and brazil became the countries with the highest numbers of deaths due to microbiologically confirmed pandemic influenza (h1n1) 2009. consistent with this particular situation, the health system in the metropolitan area of buenos aires began to show evidences of collapse, use of ventilators increased critically, achieving an extremely unusual level; about a quarter of the available icu beds were occupied by young and previously healthy patients with ards associated with severe bilateral pneumonia due to 'swine flu' who needed mechanical ventilation. by the time of writing this chapter, during the end of the winter in the southern hemisphere, it is evident that pandemic h1n1 influenza is highly prevalent in south america. in september 2009, the world health organization (who) director general, margaret chan, estimated that up to 30 % of people in densely populated countries risk being infected with h1n1 pandemic 2009 influenza, while dr. thomas frieden, head of the us centers for disease control and prevention, predicted that about 800,000 people may potentially have been infected in new york city by the spring. these figures are difficult to extrapolate globally and to confirm, as epidemiological studies looking at the population at risk in different world areas are lacking, but the huge number of severely ill patients with ards due to primary influenza pneumonia (an extremely unusual complication) observed in the southern cone, suggest that these estimations could be realistic. calculating the population-corrected mortality rate from estimations made in new zealand [5] , it can be inferred that by the end of winter in the southern hemisphere, up to about 40 % of the population in argentina could be infected by this novel agent. influenza complications during seasonal influenza occur most frequently in patients older than 64 years old, in those with chronic disorders, including cardiac or pulmonary diseases, diabetes mellitus, hemoglobinopathies, renal dysfunction, and immunosuppression. pregnant women in the second or third trimester, particularly in the 1918 and 1957 pandemics, had a higher risk of complications, especially of primary influenza pneumonia, and higher hospitalization rates. pneumonia is the most significant complication of influenza. the presentation of pneumonia includes: 'primary' influenza viral pneumonia secondary bacterial pneumonia and mixed viral and bacterial pneumonia. primary influenza viral pneumonia may be the least common of the pneumonic complications but it is also the most severe. it presents as acute influenza that does not resolve but instead progresses relentlessly, with persistent fever, dyspnea, and eventual cyanosis. sputum production is generally scanty, but the sputum can contain blood. few physical signs may be evident early in the illness. in more advanced cases, diffuse rales may be noted, and chest x-ray findings consistent with diffuse interstitial infiltrates and/or ards may be present (fig. 1) . viral cultures of respiratory specimens, especially if a 29 year-old obese male with arterial hypertension secondary to cushing's disease (hypophyseal adenoma) developed bilateral pneumonia and died from respiratory failure secondary to acute respiratory distress syndrome (ards) after 13 days on mechanical ventilation, with multiple organ failure, including renal and hemodynamic compromise requiring high doses of vasopressors. his disease began as an influenza-like illness 5 days before admission; influenza a h1n1 was confirmed with rt-pcr performed on pharyngeal swab. post-mortem microscopic histopathologic findings in the lung included extensive alveolar edema (small arrow) replacing up to 90 % of the effective alveolar space, with hyaline membrane development (big arrow); alveolar cellular infiltrate and bacterial superinfection (arrowhead) were also observed (diffuse alveolar damage pattern) (panel a). there was also mild evidence of a fibroproliferative stage, microthrombi (gray arrow), small areas with well preserved pulmonary parenchyma (blue arrow), and hemorrhagic infarcts (panel b). suprarrenal hyperplasia and acute tubular necrosis were found. samples are taken early in illness, yield high titers of virus. in fatal cases of primary viral pneumonia, histopathologic examination reveals a marked inflammatory reaction in the alveolar septa, with edema and infiltration by lymphocytes, macrophages, occasional plasma cells, and variable numbers of neutrophils (fig. 2) . fibrin thrombi in alveolar capillaries, along with necrosis and hemorrhage, have also been noted. hyaline membranes can be found lining alveoli and alveolar ducts. primary influenza viral pneumonia has a predilection for individuals with cardiac disease, particularly those with mitral stenosis, but has also been reported in otherwisehealthy young adults as well as in older individuals with chronic pulmonary disorders. secondary bacterial pneumonia follows acute influenza; in these cases typically improvement in the patient's condition over 2 to 3 days is followed by a reappearance of fever along with clinical signs and symptoms of pneumonia, including cough, purulent sputum, and physical and x-ray signs of consolidation. the most common bacterial pathogens in this setting are streptococcus pneumoniae, staphylococcus aureus, and haemophilus influenzae -usual nasopharynx colonizers. secondary bacterial pneumonia occurs most frequently in high-risk individuals with 206 c.m. luna, r. valentini, and o. rizzo chronic pulmonary and cardiac disease and in elderly individuals. patients with secondary bacterial pneumonia often respond to antibiotic therapy when it is instituted promptly. the risk factors for acquiring severe h1n1 2009 primary influenza pneumonia include age (particularly young children) and comorbidities; some series have observed a particular prevalence of overweight individuals in this group of patients [1, 6, 7] . obesity has not previously been mentioned among the risk factors for complications in patients with influenza. being overweight is associated with a chronic increase in pro-inflammatory cytokines, such as interleukin (il)-6 and tumor necrosis factor (tnf)-α. in an experimental model of influenza a, smith et al. described higher mortality rates in overweight patients than in lean controls related to minimally expressed interferon (ifn)-α and -β and a delay in expression of the proinflammatory cytokines, il-6 and tnf-α, which may lead to increased morbidity and mortality from viral infections [8] . in contrast to what happens with the usual annual seasonal influenza outbreak, in this outbreak of pandemic influenza h1n1 2009, young adults are dying and between one quarter and one half of the deaths around the world have happened in patients who were previously in good health and without any specific risk factors. in one of the earlier case report publications during the beginning of the pandemic in mexico, the authors observed that 87 % of deaths and 71 % of cases of severe pneumonia involved patients between the ages of 5 and 59 years, compared with average rates of 17 % and 32 %, respectively, in that age group during the reference periods [1] . features of this epidemic were similar to those of past influenza pandemics in that circulation of the new influenza virus was associated with an off-season wave of disease affecting a younger population [1] . in the setting of a disease with very high mortality, with no available controlled human clinical data to guide clinicians, in which most patients present with severe disease, a number of combined strategies should be considered for therapy. these include pharmacological strategies (antiviral treatment) and non-pharmacological strategies (standardization of optimal ventilator and fluid management, especially for ards, and management of other complications) necessarily given empirically, as diagnostic confirmation using real time reverse-transcriptase polymerase chain reaction (rt-pcr), can take from several hours to days. p plat eetp c stat pulm drive pr eitp v d /v t most of these patients have ards, and in these patients, ventilatory support should follow the concepts of protective ventilation, with a tidal volume (v t ) of 6 ml/kg of predicted body weight [9] . ards is usually severe, with pao 2 /fio 2 < than 150 and positive end-expiratory pressure (peep) should be high and optimized according to a mechanical basis. in our experience, we initially select peep according to the methods used in the express trial where peep was adjusted based on airway pressure and was kept as high as possible without increasing the maximal inspiratory plateau pressure above 28 to 30 cmh 2 o [10] . in more severe respiratory failure, we also set peep according to the transpulmonary pressure, by using esophageal-pressure measurements. in secondary, but also in primary ards the lungs can suffer substantial effects of chest wall elastance and may be effectively compressed by high pleural pressures with their alveoli collapsed at the end of expiration, even though moderate or high peep levels are applied. therefore, peep is set at a level necessary to obtain a positive end-expiratory transpulmonary-pressure to improve the oxygenation, an end-inspiratory transpulmonary-presure less than 20 cmh 2 o to minimize stress-inducing ventilator lung injury, and a pulmonary driving pressure (end-inspiratory transpulmonary pressure less end-expiratory transpulmonary pressure) e 10 cmh 2 o to avoid straininducing ventilator lung injury (fig. 3) . using these premises, the mean peep applied in patients with severe influenza h1n1 2009 and ards was 20 cmh 2 o. interestingly, in contrast to other etiologies of ards, in primary influenza pneumonia, high peep levels were necessary for many days. in a group of 23 patients variation in gas exchange and respiratory system mechanics from the onset of mechanical ventilation in a group of mechanically ventilated ards patients with h1n1 pneumonia observed in one of our icus [11] . all variables improved from day 0 to day 3 of mechanical ventilation; however, in the majority of the patients the pao 2 /fio 2 ratio remained low for many days, inducing us to maintain high levels of peep. cstat: static lung compliance; peep: positive end-expiratory pressure managed by one of us (rv) in cemic medical center, the mean peep after 10 days on mechanical ventilation was 18 cmh 2 o [11] . at the beginning of this outbreak, we decreased the peep level after a few days of mechanical ventilation, based on improvement in oxygenation levels; however, this produced a dramatic worsening of the pao 2 /fio 2 ratio. because of this observation, it was decided that, in patients with severe ards, high peep levels should be maintained for at least two weeks regardless of the oxygenation levels (fig. 4) . most of the patients with severe influenza pneumonia responded to recruitment maneuvers. a recruitment maneuver in pressure controlled ventilation (pcv) with a peep of 25-30 cmh 2 o and an inspiratory pressure of 25 cmh 2 o (peak pressure 50 -55 cmh 2 o) was performed in patients with a pao 2 /fio 2 < 200 mmhg. many of these patients were young, healthy and had good cardiac performance and tolerance of high ventilatory pressures during the recruitment maneuver with adequate intravascular volume repletion. several trials have demonstrated no survival benefit in ards patients managed in the prone position. however, these trials did not select the most severe patients. many of our patients had severe ards with pao 2 /fio 2 < 100 mmhg despite peep optimization and recruitment maneuvers. in this setting, prone ventilation was used and, if pao 2 /fio 2 did not reach > 200 mmhg, a recruitment maneuver was applied in the prone position. prone ventilation was used in 22 % of the patients with ards and in 50 % of patients with severe ards, and was associated with improved oxygenation and reduced distending pressures. we suggest the use of adjunctive therapies when plateau pressure is higher than 35 cmh 2 o, despite a v t of 6 -8 ml/kg predicted weight, severe hypercapnic acidosis, and refractory hypoxemia (defined as a pao 2 /fio 2 ratio < 100 mmhg after optimization of peep, recruitment maneuvers, prone position, and recruitment maneuvers in the prone ventilatory position). the adjunctive therapies developed to reduce the stress of mechanical ventilation on the already damaged lungs include: nitric oxide (no), extracorporeal membrane oxygenation (ecmo), arterial venous carbon dioxide removal, high-frequency oscillatory ventilation, and liquid ventilation. we prefer to use no because of its availability and easy implementation and we have observed better improvement in oxygenation combining this therapy with prone ventilation, as previously described [12] . non-invasive positive pressure ventilation (nppv): nppv has been used in respiratory failure due to viral pneumonia, even in cases of high transmission risk like in the epidemic of severe acute respiratory syndrome (sars) in hong kong [13] . in one study, the efficacy in sars pneumonia with mild acute lung injury (ali) was high and no cases of healthworker infection were observed. however, application of nppv to patients with h1n1 influenza has not been well evaluated and it is not indicated for impending respiratory failure. in mild cases or in patients with chronic obstructive pulmonary disease (copd) or chronic respiratory restriction, nppv could be useful to support the respiratory system, but it should be applied in healthcare facilities where staff have been adequately trained and with strict enforcement of personal protection measures; use of expiratory viral and bacterial filters are necessary to provide safer ventilation. most of the patients with influenza h1n1 2009 will recover without any antiviral therapy. antivirals are indicated to prevent the rapid spread of the disease in a specific population, to prevent the pneumonia syndrome in susceptible patients, or to treat patients with influenza pneumonia. for critically ill influenza patients, antiviral treatment options are limited because no parenteral drug is available and no drug has been proved to be effective once life-threatening disease occurs. currently, four antiviral drugs are available for the treatment of influenza: amantadine, rimantadine (both cannot be used for the treatment of h1n1 influenza due to resistance), oseltamivir, available only for oral administration, and zanamivir, available as an inhalation agent; the two latter drugs are both sialic acid analogs that inhibit viral neuraminidases by competitively binding with the active enzyme site of influenza a and b viruses. the neuraminidase is critical for viral release from infected cells after replication. the earlier the administration of these agents, and the shorter the duration of fever, the greater the benefit of drug intervention [14, 15] . oseltamivir has also been shown to reduce lower respiratory tract complications such as bronchitis and pneumonia [16] . in a prospective case control study, multivariate analysis suggested that treatment with oseltamivir decreased the likelihood of death (odds ratio 0.21 [confidence interval 0.06 -0.80, p = 0.02]) [17] . immunosuppressed patients (leukemia, organ transplantation, and hematopoietic stem cell transplantation) have a higher rate of viral pneumonia and higher attributable mortality [18] ; viral shedding is also prolonged in these patients to an average of 11 days [19] , which is associated with the development of resistance [20] . a standard dose and duration of antivirals may not be adequate in this population; for these reasons, some authors have advocated a higher dose of oseltamivir (300 mg daily) in these patients [18] . during the pandemic, the therapeutic strategy proposed by the argentinean health authority for mechanically ventilated patients with presumptive primary influenza pneumonia was to use oseltamivir at a dose of 300 mg daily during an extended period of time, typically until the patient was weaned from mechanical ventilation. the most frequent reported adverse effect seen with oseltamivir is nausea and vomiting, but this leads to medication interruption in only a small number of cases. neuropsychiatric disorders (seizure, confusion or hyper-excitation of the nervous system) and severe skin reactions (e.g., toxic epidermal necrolysis) are more severe adverse events that have been observed in some cases during the pandemic. these unusual events have been related to a single nucleotide polymorphism in a gene located near the enzymatic active site of human cytosolic sialidase, a homolog of the virus neuraminidase that is the target of oseltamivir. this polymorphism has been found to occur in 9.3 % of the asian population [21] . because of the high frequency of bacterial co-infection, antibiotic administration is recommended for all patients with pandemic h1n1 2009 influenza infection who require admission to a critical care unit. in immunocompetent patients, without recent antibiotic exposure, combination therapy with a beta-lactam plus a macrolide or a respiratory fluorquinolone, is recommended [22] . corticosteroids may be used to treat airflow obstruction due to asthma or copd, to maintain immunosuppression in transplant patients, and when adrenal dysfunction is suspected because of refractory vasodilatory shock. corticosteroids are not indicated for ali; prolonged or high-dose corticosteroid therapy can result in serious adverse events, including opportunistic infections. in patients with h5n1 pulmonary infection, corticosteroids were not effective and in one series mortality was 59 % in 29 recipients of corticosteroids, compared with 24 % in 38 patients who did not receive corticosteroids [23] . one exception to this is cryptogenic organizing pneumonia (cop) described below under 'complications'. in addition to primary viral pneumonia, viral and bacterial co-infection and secondary bacterial pneumonia are frequent. co-infection with s. pneumoniae, s. aureus, and mycoplasma pnemoniae has been detected in some of the reported series from argentina; this co-infection occurs after several days of influenza infection and occurs more frequently in the elderly and in patients with chronic pulmonary diseases [24] . it has been observed in one series that 9 % of hospitalized patients with communityacquired pneumonia had dual infection with a respiratory virus and a bacterial pathogen, influenza being the most common viral agent [25] . proposed theories for the high incidence of superimposed bacterial infections in influenza pneumonia emphasize the synergistic effects of viral and bacterial pathogens in producing lung injury. studies suggested that influenza virus can directly damage the respiratory epithelium, allowing free access to invading bacteria. it has also been demonstrated that some life-threatening respiratory failure from h1n1 influenza 211 vi staphylococcus and streptococcus strains may increase viral replication and pathogenicity, contributing to influenza viral pneumonia [26] . pulmonary embolism has not been recognized as a common complication of severe influenza with ards. however, in a series of 10 patients with pandemic influenza h1n1 2009 infection and ards at a tertiary-care icu in michigan, five had pulmonary emboli [6] . influenza infections have been associated with procoagulant changes [27] . pathologic fibrin deposition also occurs in the vasculature in ards and pulmonary artery thrombi are found, implying an anatomic mechanism for the occurrence of increased pulmonary vascular resistance in ards [28] . it remains unknown whether these cases were secondary to some of the several risk factors that these bed-ridden severely ill patients had, or whether it was a direct consequence of a particular risk in influenza patients. meanwhile, clinicians should periodically search for thrombosis and if necessary use chest multislice spiral computed tomography (ct) to confirm pulmonary embolism. influenza virus does not replicate in the alveoli or tissues beyond the respiratory tract. histopathological analyses revealed that no virus was detected in the liver, spleen, kidney, or brain of animals inoculated with influenza h1n1 2009 virus at 3 or 7 days after inoculation [29] . however, myocarditis and pericarditis have been described in association with influenza infection and it has been suggested that influenza-associated myocarditis can take two forms: immediate, associated with fulminating disease, and delayed, occurring during late convalescence [30] . renal failure has been described in a number of influenza patients [7, 31] . it is usually the consequence of shock and multiorgan dysfunction. we recommend adequate fluid replacement and, in patients with severe ards, fluid infusion should not be restrictive and diuretic use should be avoided to prevent the progression of renal dysfunction [11] . using this strategy in our patients, the positive fluid balance at the 7 th day was 10,000 ml and hemodialysis was necessary in only 18 % of patients [11] . occasionally, rhabdomyolysis may facilitate the development of renal failure; in fact, high levels of serum creatine phosphokinase have been described in reports of h1n1 infection [31] . this condition, occasionally associated with influenza, is characterized by progressive respiratory failure after 1 week of influenza symptoms with chest computerized axial tomography demonstrating multiple, bilateral, patchy alveolar opacities [11] . if identified, this complication must be treated with high doses of corticosteroids [32] . ho et al. performed a study to define the prognostic factors for fatal adult influenza pneumonia [33] . univariate analysis demonstrated that, compared with survivors of septic shock, a respiratory rate & 25 breaths per min, an arterial ph < 7.35, a pao 2 / fio 2 ratio < 150 mmhg, a creatinine value & 2 mg/dl, a pneumonia severity index (psi) of iv or v, and an apache ii score & 20 were all associated with decreased survival. adjustments were made for septic shock, respiratory rate, arterial ph, creatinine and psi in the cox proportional hazard model. the multivariate analysis demonstrate that only the pao 2 /fio 2 ratio < 150 mmhg (p = 0.024) and an apache ii score & 20 (p = 0.017) remained associated with death. in another study, the development of ards and a history of immunosuppression were independent risk factors for hospital mortality in critically ill patients with confirmed influenza virus infection [34] . the emergence of an antigenically novel influenza virus to which little or no antibody was present in a community, resulted in an extensive outbreak; the absence of antibody is worldwide, and for that reason there has been a pandemic. independent of this antigenically new virus, questions regarding the potential effectiveness of vaccination for seasonal influenza arises. in one interim analysis of the pandemic in australia, the authors found that there was no evidence of significant protection from seasonal vaccine against pandemic influenza virus infection in any age group [35] . a new vaccine has been developed, but there have been concerns based on the experience during the 1976 -77 flu season, during which a swine flu outbreak at fort dix, new jersey led the federal government to expedite vaccine production. some 40 million people had been vaccinated by the time guillain-barré syndrome was identified as a side effect. however, with the pandemic as a reality, it is considered that the benefit of the vaccine far outweighs the risks. pandemics provide the most dramatic evidence of the impact of influenza. the morbidity and mortality caused by this first influenza pandemic in the 21 st century, characterized by an unusual increase in the number of cases of primary viral severe community-acquired pneumonia requiring mechanical ventilation, has been substantial. interestingly this higher incidence of severe cases appeared in a younger age group than that usually involved in the annual seasonal flu outbreak. the percentage of the population that acquired influenza during this pandemic has not yet been estimated but certainly it was much higher than during seasonal influenza; this higher incidence may explain the high number of cases of severe primary pneumonia observed in the southern cone. the apparently less aggressive nature of the infection and the younger population affected may explain an estimated mortality rate of 0.05 -0.1 %, lower than that observed in seasonal influenza, as complications and mortality in seasonal flu are more frequent among patients & 65 years old and in those with chronic disorders, including cardiac or pulmonary diseases, diabetes mellitus, hemoglobinopathies, renal dysfunction, and immunosuppression, also usually associated with older age. improved and standardized optimal icu care for patients with influenza h1n1 2009, including young and immunocompetent patients, with or without comorbidities, should lead to lower mortality than that previously observed for influenza pneumonia when mechanical support is required. the pandemic h1n1 2009 influenza has resulted in tremendous pressures on the critical care system. the unexpected and rapid influx of such a large number of patients to emergency room and critical care services has highlighted not only a shortage of critical care capacity but also an inadequate supply of critical care resources. the extreme severity of ards in these patients has necessitated a change in the usual approach to the management of these patients to improve success rates. the health system must be prepared to reallocate resources in response to demand. therefore, early recognition of probable viral pneumonia is crucial in order to implement early infection-control strategies and to reduce transmission to health-care workers who are at high risk for 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reactions to oseltamivir infectious diseases society of america/ american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus 2008 update on avian influenza a (h5n1) virus infection in humans mandell, douglas, and bennett's principles and practice of infectious disease viral community-acquired pneumonia in nonimmunocompromised adults influenza pneumonia: a descriptive study influenza infection and risk of acute pulmonary embolism anticoagulants for acute respiratory distress syndrome: can they work? pathogenesis and transmission of swine-origin 2009 a(h1n1) influenza virus in ferrets the prevalence of myocarditis and skeletal muscle injury during acute viral infection in adults: measurement of cardiac troponins i and t in 152 patients with acute influenza infection pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico case records of the massachusetts general hospital. weekly clinicopathological exercises. case 36 -2001. acute febrile respiratory illness in a 57-year-old man with recurrent pulmonary disorders prognostic factors for fatal adult influenza pneumonia outcome of critically ill patients with influenza virus infection interim analysis of pandemic influenza (h1n1) 2009 in australia: surveillance trends, age of infection and effectiveness of seasonal vaccination severe pneumonia requiring mechanical ventilation, coincident with the influenza h1n1 2009 pandemic key: cord-011712-fyrbe8tw authors: venkatesan, sudhir; myles, puja r; bolton, kirsty j; muthuri, stella g; al khuwaitir, tarig; anovadiya, ashish p; azziz-baumgartner, eduardo; bajjou, tahar; bassetti, matteo; beovic, bojana; bertisch, barbara; bonmarin, isabelle; booy, robert; borja-aburto, victor h; burgmann, heinz; cao, bin; carratala, jordi; chinbayar, tserendorj; cilloniz, catia; denholm, justin t; dominguez, samuel r; duarte, pericles a d; dubnov-raz, gal; fanella, sergio; gao, zhancheng; gérardin, patrick; giannella, maddalena; gubbels, sophie; herberg, jethro; higuera iglesias, anjarath lorena; hoeger, peter h; hu, xiao yun; islam, quazi t; jiménez, mirela f; keijzers, gerben; khalili, hossein; kusznierz, gabriela; kuzman, ilija; langenegger, eduard; lankarani, kamran b; leo, yee-sin; libster, romina p; linko, rita; madanat, faris; maltezos, efstratios; mamun, abdullah; manabe, toshie; metan, gokhan; mickiene, auksė; mikić, dragan; mohn, kristin g i; oliva, maria e; ozkan, mehpare; parekh, dhruv; paul, mical; rath, barbara a; refaey, samir; rodríguez, alejandro h; sertogullarindan, bunyamin; skręt-magierło, joanna; somer, ayper; talarek, ewa; tang, julian w; to, kelvin; tran, dat; uyeki, timothy m; vaudry, wendy; vidmar, tjasa; zarogoulidis, paul; nguyen-van-tam, jonathan s title: neuraminidase inhibitors and hospital length of stay: a meta-analysis of individual participant data to determine treatment effectiveness among patients hospitalized with nonfatal 2009 pandemic influenza a(h1n1) virus infection date: 2020-02-01 journal: j infect dis doi: 10.1093/infdis/jiz152 sha: doc_id: 11712 cord_uid: fyrbe8tw background: the effect of neuraminidase inhibitor (nai) treatment on length of stay (los) in patients hospitalized with influenza is unclear. methods: we conducted a one-stage individual participant data (ipd) meta-analysis exploring the association between nai treatment and los in patients hospitalized with 2009 influenza a(h1n1) virus (a[h1n1]pdm09) infection. using mixed-effects negative binomial regression and adjusting for the propensity to receive nai, antibiotic, and corticosteroid treatment, we calculated incidence rate ratios (irrs) and 95% confidence intervals (cis). patients with a los of <1 day and those who died while hospitalized were excluded. results: we analyzed data on 18 309 patients from 70 clinical centers. after adjustment, nai treatment initiated at hospitalization was associated with a 19% reduction in the los among patients with clinically suspected or laboratory-confirmed influenza a(h1n1)pdm09 infection (irr, 0.81; 95% ci, .78–.85), compared with later or no initiation of nai treatment. similar statistically significant associations were seen in all clinical subgroups. nai treatment (at any time), compared with no nai treatment, and nai treatment initiated <2 days after symptom onset, compared with later or no initiation of nai treatment, showed mixed patterns of association with the los. conclusions: when patients hospitalized with influenza are treated with nais, treatment initiated on admission, regardless of time since symptom onset, is associated with a reduced los, compared with later or no initiation of treatment. seasonal influenza epidemics and pandemics increase pressure on hospital bed capacity. early initiation of monotherapy with neuraminidase inhibitors (nais) reduces illness duration in patients with uncomplicated influenza [1] [2] [3] ; associated reductions in complications, hospitalizations, and mortality are supported by systematic reviews of observational data [4] [5] [6] [7] [8] . the evidence is less clear that nai treatment reduces length of stay (los) in hospitalized patients with influenza, compared with supportive care without antiviral treatment [9] [10] [11] [12] [13] [14] [15] . minimizing the los is important in managing hospital surge and limiting healthcare costs due to seasonal influenza epidemics and pandemics. we undertook a one-stage individual participant data (ipd) [16] meta-analysis to explore the association between nai treatment of patients hospitalized with 2009 pandemic influenza a(h1n1) virus (a[h1n1]pdm09) infection and the los during the 2009-2010 influenza pandemic. details regarding identification of study centers and inclusion of patients have been published previously [6] . briefly, we requested data from multiple clinical centers worldwide on patients admitted to hospital with laboratory-confirmed or clinically diagnosed a(h1n1)pdm09 infection for whom a minimum data set was available. of the ipd that we received, we excluded patients who had laboratory-confirmed absence of a(h1n1) pdm09 infection, retaining only patients who had laboratoryconfirmed a(h1n1)pdm09 infection and patients with clinically diagnosed pandemic influenza (ie, those for whom the clinical suspicion and working diagnosis was one of pandemic influenza but laboratory confirmation was not performed) [6] . the pride study protocol was registered with the prospero register of systematic reviews (crd42011001273) prior to data collection [17] . this states that the study will investigate the impact of nai treatment on multiple outcomes of public health interest in a(h1n1)pdm09-infected patients, using mixedeffects models. after collection and standardization of the data, sufficient data existed to assess 2 indicators of "severe hospital outcomes"-requirement for ventilatory support (ie, intensive care unit [icu] admission) and los. in this article, we present the findings relating to the los. we standardized data from individual data sets before we pooled the data (supplementary table 1 ). the primary outcome was the los (in whole days). we excluded patients with known receipt of nai treatment before admission, to ensure uniform potential for treatment to influence the los. we excluded patients with continuing postdischarge nai treatment; patients with a los <1 day, on the grounds that they would have received a maximum of 2 doses of nai inpatient treatment and that their admission may have been precautionary; and patients with nosocomial influenza (defined as influenza with symptom onset after the hospital admission date; figure 1 ). finally, since rapid deterioration and early death during hospitalization would be an adverse outcome associated with a paradoxically short los, those who died in the hospital were excluded from analysis. the primary exposure variable was in-hospital nai treatment received on the day of hospital admission, compared with later or no nai treatment. additionally, where data were available, we defined 3 further exposure variables: nai treatment (at any time) versus no nai treatment, early nai treatment (initiated within ≤2 days after symptom onset) versus no nai treatment, and early nai treatment versus later treatment (initiated >2 days after symptom onset). we derived propensity scores via multivariable logistic regression for each exposure variable, as described by hirano and imbens [18] , separately for individual study centers, based on patient characteristics recorded on admission. propensity score derivation models included, a priori, the following variables: age, sex, comorbidity (yes/no), and an indicator of disease severity, plus any additional covariates (ie, obesity, smoking, pregnancy, asthma, chronic obstructive pulmonary disease, lung disease, heart disease, immunosuppression, neurological disease, renal disease, and/or diabetes) that remained statistically significant in a regression model. variables for which data from >25% of participants were missing were excluded from propensity score derivation. to investigate the impact of nai treatment on the los, we performed a one-stage ipd meta-analysis using a mixed-effects negative binomial regression model, including study center as a random intercept to account for clustering. a negative binomial model was chosen to account for overdispersion in the los data (as represented in supplementary figure 1 ). we tested a zeroinflated negative binomial regression model on a subgroup of the data and found that the model fit was inferior to that of a negative binomial regression model. in our primary analysis, we aimed to quantify the potential benefits of a pragmatic treat-on-admission policy (irrespective of the time elapsed since symptom onset), compared with patients who received no nai treatment and those whose treatment was delayed until after the day of admission. by way of sensitivity analysis, we restricted the comparator group to patients who did not receive nai treatment at any point. for both analyses, we adjusted for propensity score quintile, in-hospital antibiotic treatment, in-hospital corticosteroid treatment, and the delay between symptom onset and hospital admission. in addition, we performed secondary analyses for the following exposures: nai treatment (at any time) versus no nai treatment, early nai treatment (≤48 hours after symptom onset) versus later nai treatment (>48 hours after symptom onset), and early nai treatment versus no nai treatment, adjusting for propensity score, in-hospital antibiotic treatment, and corticosteroid treatment. we performed a priori-specified analyses for the following subgroups: patients with laboratory-confirmed a(h1n1) pdm09 infection, children (age, <16 years), elderly patients (age, ≥65 years), patients with chest radiography-confirmed influenza-related pneumonia (irp), and patients with confirmed absence of irp. we looked at pregnant women and obese patients as post hoc subgroups. furthermore, we investigated, by stratification, the impact of nai treatment on the total los in patients admitted to critical care facilities (ie, icus) at any point and patients treated exclusively by using standard ward-based care. both unadjusted and adjusted models were run, and results are presented as unadjusted incidence rate ratios (irrs) or adjusted irrs (airrs) with 95% confidence intervals (cis). missing data in the covariates were included in the analysis as dummy variable categories. using airr point estimates, we determined the difference in the los (in days) between a treated patient and an untreated patient with similar characteristics by scaling the model prediction for los without treatment by (airr-1). repeating this for all patients in our data set gave us a distribution of expected changes in the los due to treatment (with timing as defined for each regression analysis). this does not account for error in the estimates of model covariates, which would require a bayesian approach; however, it offers a clinically relevant interpretation of airrs. the statistical analyses were performed using stata (version 14.2; statacorp, college station, tx). we identified 29 234 patients admitted to the hospital between 2 january 2009 and 14 march 2011 with laboratory-confirmed or clinically diagnosed a(h1n1)pdm09 infection [4] . the analysis population included 18 309 patients (62.6%; figure 1 ). the included patients came from 70 clinical centers in 36 countries across all 6 world health organization regions. the americas contributed the most data (46.2% of patients), followed by europe (for 33.3%). the country that contributed the most to the pooled data set was mexico (28.8% of patients), followed by spain (8.6%), the united states (7.6%), and the united kingdom (7.5%). among patients in the final study population, 67.4% were adults, and 81.1% had laboratoryconfirmed a(h1n1)pdm09 infection; general characteristics of the included population are further described in table 1 . among the 8621 patients (47.1%) for whom data on the timing of nai treatment were available, 3678 (42.7%) received early nai treatment, and 4816 (55.9%) initiated treatment on the day of admission. the median delay from illness onset to hospital admission was 2 days (interquartile range [iqr], 1-5 days), and among patients with data on the timing of treatment, 42.7% presented ≤48 hours after symptom onset; the median los was 5 days (iqr, 3-9 days; supplementary figure 1 ). in patients whose nai treatment was initiated on the day of hospital admission, the median interval between symptom onset and admission was 2 days (iqr, 1-4 days). in our primary analysis, we observed that nai treatment started on the day of admission was associated with a 19% overall reduction in the los (airr, 0.81 [95% ci, .78-.85]; median decrease, 1.19 days [iqr, 0.85-1.55 days]), compared with no or later initiation of nai treatment. this association was of similar magnitude and remained significant in all subgroups (table 2 and supplementary table 3 ). in the sensitivity analysis, we observed that nai treatment on the day of hospital admission was associated with an 8% reduction in the los among patients not admitted to the icu ( after adjustment, nai treatment at any time was associated with an 11% overall increase in the los (airr, 1.11 [95% ci, 1.07-1.16]; median increase, 0.74 days [iqr, 0.60-1.05 days]), compared with no nai treatment. by exploring subgroups, we identified corresponding statistically significant findings in patients with laboratory-confirmed a(h1n1)pdm09 infection, children, patients admitted to the icu, and patients with confirmed irp but not in the elderly, patients requiring non-icu care, or patients with confirmed absence of irp (table 2) . we did not find any evidence of effect modification by pandemic influenza vaccination (p = .68) or by in-hospital antibiotic treatment (p = .20); however, a borderline significant effect modification was observed for in-hospital corticosteroid treatment (p = .05), the irr was further adjusted for time from onset to admission. c statistically significant (p < .05). d data are for patients admitted to the icu at any point. the irr was calculated for the total length of hospital stay, not time in the icu. our sensitivity analyses and secondary analyses must be interpreted with caution because they may be affected by various time-dependent biases and patients with confirmed irp ( table 3 ). our study extends the existing literature by offering data on the association between nai treatment and the los in >18 000 adult and pediatric patients, of whom >80% had a laboratoryconfirmed diagnosis of a(h1n1)pdm09 infection. we found a mixed pattern of association between nai treatment and los, depending on the delay to initiation of treatment, age, and case severity. the most pragmatic and important question is whether nai treatment, started on admission, irrespective of delay since symptom onset, reduces the los in hospitalized patients with influenza. clinically, this is important because there can be significant uncertainty in ascertaining symptom onset, even by the attending physician. the uncertainty in ascertaining symptom onset could mean prescribing nai treatment outside the recommended (licensed) window of ≤48 hours after symptom onset. however, there is evidence pointing to the effectiveness of nai therapy, albeit reduced, even when given >48 hours after symptom onset [6] . statistically, by defining our exposure variable on the basis of treatment decisions made on admission, we avoided introducing correlations between exposure and los that can lead to survivorship bias in linear regression models of time-to-event data [19, 20] . additionally, this approach ensures that the propensity scores, modeled on symptom severity at admission, should appropriately correct for treatment bias [21] . however, this choice of exposure variable also reflects the clinical reality that patients present to the hospital at varying intervals after symptom onset (ranging from 0-20 days in our study) and that clinicians and policy makers want to know whether a so-called treat-at-the-door policy applied to patients admitted to the hospital with clinically recognized influenza will be beneficial, compared with no nai treatment or a watch-and-see approach. this was addressed by our primary analysis, which revealed that initiation of nai treatment on the day of admission was associated with a 19% reduction in the los (median decrease, 1.19 days), compared with later or no treatment, with similar statistically significant findings across all patient subgroups including children, pregnant women, and obese patients. these findings emphasize the importance of presumptive nai treatment in patients admitted to the hospital with suspected influenza, coupled with early diagnosis using standard laboratory or rapid diagnostic tests. in our sensitivity analysis, we found a significant reduction of 19% in the los (median decrease, 1.24 days) among patients with confirmed absence of irp and a reduction of 8% (median decrease, 0.5 days) among patients who required supportive ward-based care. in contrast, nai treatment (compared with no treatment) was associated with a 28% increase in the los (median increase, 1.73 days) among patients with irp. these data suggest that nais may be more effective in reducing the los when patients do not have irp and are consistent with the fact that nais have no known antibacterial properties. in secondary analyses, we observed an 11% overall increase in the los associated with nai treatment, equivalent to a median increase of about 0.74 days and irrespective of the time between symptom onset and initiation of therapy. compared with no treatment, nai treatment initiated within 48 hours after symptom onset was associated with a 7% overall reduction in the los, equivalent to a median decrease of 0.40 days; this effect was not observed in children and patients requiring icu care. this finding is clinically important because it suggests that rapid access to antiviral treatment after symptom onset may influence the los in adults and elderly individuals; nevertheless, we did not observe the same result among patients requiring icu care. our results in children may be influenced by a higher a(h1n1) pdm09 viral load in children [22] than in adults, leading to prolonged hospital stay, suboptimal dosing in very young children [23] , increased likelihood of antiviral resistance emergence in children [24] , secondary bacterial infections, confounding by indication related to baseline illness severity [25] , or a combination of these factors. although we attempted to adjust for influenza severity by using propensity scores, we found icu care to be very strongly associated with a prolonged los (irr, 2.96; 95% ci, 2.84-3.09) and nai treatment to be associated with a higher likelihood of requiring icu care (adjusted odds ratio, 3.11; 95% ci, 2.42-3.98). furthermore, we found that patients who presented to the hospital >2 days after symptom onset were 73% more likely to eventually require icu care than patients who presented earlier (odds ratio, 1.73; 95% ci, 1.53-1.95). in addition, patients requiring icu care have frequently developed extrapulmonary manifestations of influenza and multiorgan decompensation; therefore, inhibition of virus replication may not correspond with rapid clinical recovery. we noted no association between nai treatment and los among hospitalized children with influenza when considering early treatment versus no treatment. the study may have been underpowered in children, but other factors might have contributed to our findings. the los is typically shorter among children, compared with adults; mortality and serious outcomes are less common among hospitalized children with influenza, compared with adults; and different discharge policies and thresholds for children could also influence the findings. in addition, vomiting is a recognized side effect of oseltamivir in children [3] , and this may have prevented discharge in some cases. previous studies examining whether use of nais in patients hospitalized with influenza affects the los have generally been of smaller size (<1300 individuals) as compared to our study and reached variable conclusions. of note, 8 studies [11] [12] [13] [14] [15] [26] [27] [28] (of which one [12] was a randomized trial) assessed nai treatment of hospitalized children, but only 2 (both with an observational design) concluded that the total number of hospital days in the nai-treated hospital cohort was reduced (by 18% [8.3 days]) [11, 28] , with the other 6 reporting no differences [12-15, 26, 27] . only 4 studies have addressed the same question in adults. in hong kong, a study of 356 adult patients hospitalized with laboratory-confirmed seasonal influenza showed that early oseltamivir treatment was associated with a reduced los in both unadjusted and multivariable analyses [9] , compared with no or later treatment, with the median los decreasing from 6 to 4 days; this accords with our primary analysis. a canadian study of adult patients with seasonal influenza found that oseltamivir treatment was not associated with the los among surviving patients [29] . a further study in 13 spanish hospitals involving 538 patients with laboratory-confirmed a(h1n1)pdm09 infection noted that the los increased by 7% (odds ratio, 1.07), after adjustment for confounders, if nai treatment was instigated <48 hours after symptom onset; however, this was of borderline statistical significance [10] . a recent american study analyzed data on 201 adult patients with laboratory-confirmed seasonal influenza, reporting that nai treatment was not associated with the los overall but was associated with a reduced los among vaccinated individuals (hazard ratio of discharge, 1.6; 95% ci, 1.0-2.4; p = .04) [30] . finally, 2 studies included patients of all age groups. one of them, performed in 813 hospitalized patients with a(h1n1)pdm09 infection in spain, found that early nai treatment reduced the los by 1.9 days (p = <.001) [31] . the other, an american study using insurance claims data from patients with seasonal influenza, reported that patients treated with nais spent fewer days in the hospital (p = <.0001) [32] . this study has a number of strengths and weaknesses. we combined data from geographically diverse centers, offering broad generalizability of our findings. we used propensity scores to adjust for major confounders. by excluding patients who died (10%), we removed the paradoxical possibility that a short los (a positive outcome in our analyses) was associated with an extremely unfavorable clinical outcome. however, a limitation of this approach is that it does not explain the impact of nai treatment on the relationship between los and in-hospital mortality. in our primary analysis, we adjusted for the delay between illness onset and admission, to address length bias [20] , and chose our exposure variable to avoid time-dependent/survivorship bias [19, 21] . however, our secondary analyses, which used time since onset to define the exposure variable, are subject to time-dependent biases and must therefore be interpreted with caution. indeed, the benefit of early versus late treatment ( table 2 ) will be partially driven by this bias [19] . all of our analyses may be subject to residual competing risk bias, which was not removed through adjustment; for example, we found a significant difference between propensity scores to receive nais in the hospital for surviving and nonsurviving patients in the data set (p < .05, by the kruskal-wallis test), signaling that our removal of nonsurviving patients altered the aggregate presenting patient characteristics for which our results hold. our data, generated during the 2009-2010 influenza pandemic, contained relatively few elderly patients and children, consistent with patterns of a(h1n1)pdm09 infection [33] , and differs in profile from seasonal influenza a(h3n2) virus infection, for which patients admitted to the hospital tend to be much older and to have a median los higher than the los of 5 days we observed [34, 35] . in addition, the prevalence proportions of clinically recorded obesity (12%) and pregnancy (23%) were both comparatively high. optimally, clinicians wish to treat patients with influenza within 48 hours after symptom onset, yet in many cases patients with influenza do not seek medical care during this therapeutic window. our data show that 57.3% of included patients were hospitalized >48 hours after symptom onset. what then matters is whether initiation of treatment upon hospitalization (on the day of admission), irrespective of the time elapsed since symptom onset, is effective and whether this is preferable to nontreatment or further delays in treatment. we revealed a 19% reduction in the los (median decrease, 1.19 days) among patients who were treated with an nai upon admission, compared with those who received no or later treatment; the trend was observed across all subgroups, including children. this treatment approach would avoid the uncertainties associated with ascertaining the symptom onset date. our data support current recommendations to treat adults hospitalized with clinically suspected influenza with nais as soon as possible upon admission; furthermore, this approach appears to be superior to no treatment or delayed treatment in terms of a reduced los. if used consistently, this strategy would contribute to the management of surge pressures and healthcare costs during seasonal influenza epidemics and pandemics. the pride consortium investigators are as follows (affiliations are listed in supplementary 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opportunity to preview results/findings before entry into the public domain; and no opportunity to contribute to, preview, or comment on manuscripts and presentations arising from this work. the research contract between the university of nottingham and the funder is freely available for inspection (with commercial details redacted) at: http://www.nottingham.ac.uk/research/groups/healthprotection/projects/pride.aspx. no data were provided or funded for collection by pharmaceutical companies.financial support. this work was supported by f. hoffmann-la roche (unrestricted educational grant to the pride study).potential supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. key: cord-017354-cndb031c authors: janies, d.; pol, d. title: large-scale phylogenetic analysis of emerging infectious diseases date: 2008 journal: tutorials in mathematical biosciences iv doi: 10.1007/978-3-540-74331-6_2 sha: doc_id: 17354 cord_uid: cndb031c microorganisms that cause infectious diseases present critical issues of national security, public health, and economic welfare. for example, in recent years, highly pathogenic strains of avian influenza have emerged in asia, spread through eastern europe, and threaten to become pandemic. as demonstrated by the coordinated response to severe acute respiratory syndrome (sars) and influenza, agents of infectious disease are being addressed via large-scale genomic sequencing. the goal of genomic sequencing projects are to rapidly put large amounts of data in the public domain to accelerate research on disease surveillance, treatment, and prevention. however, our ability to derive information from large comparative genomic datasets lags far behind acquisition. here we review the computational challenges of comparative genomic analyses, specifically sequence alignment and reconstruction of phylogenetic trees. we present novel analytical results on two important infectious diseases, severe acute respiratory syndrome (sars) and influenza. sars and influenza have similarities and important differences both as biological and comparative genomic analysis problems. influenza viruses (orthymxyoviridae) are rna based. current evidence indicates that influenza viruses originate in aquatic birds from wild populations. influenza has been studied for decades via well-coordinated international efforts. these efforts center on surveillance via antibody characterization of the hemagglutinin (ha) and neuraminidase (n) proteins of the circulating strains to inform vaccine design. however, we still do not have a clear understanding of (1) various transmission pathways such as the role of intermediate hosts like swine and domestic birds and (2) the key mutation and genomic recombination events that underlie periodic pandemics of influenza. in the past 30 years, sequence data from ha and n loci has become an important data type. in the past year, full genomic data has become prominent. these data present exciting opportunities to address unanswered questions in influenza pandemics. sars is caused by a previously unrecognized lineage of coronavirus, sars-cov, which like influenza has an rna based genome. although sars-cov is widely believed to have originated in animals, there remains disagreement over the candidate animal source that lead to the original outbreak of sars. in contrast to the long history of the study of influenza, sars was only recognized in late 2002 and the virus that causes sars has been documented primarily by genomic sequencing. in the past, most studies of influenza were performed on a limited number of isolates and genes suited to a particular problem. major goals in science today are to understand emerging diseases in broad geographic, environmental, societal, biological, and genomic contexts. synthesizing diverse information brought together by various researchers is important to find out what can be done to prevent future outbreaks [jon03]. thus comprehensive means to organize and analyze large amounts of diverse information are critical. for example, the relationships of isolates and patterns of genomic change observed in large datasets might not be consistent with hypotheses formed on partial data. moreover when researchers rely on partial datasets, they restrict the range of possible discoveries. phylogenetics is well suited to the complex task of understanding emerging infectious disease. phylogenetic analyses can test many hypotheses by comparing diverse isolates collected from various hosts, environments, and points in time and organizing these data into various evolutionary scenarios. the products of a phylogenetic analysis are a graphical tree of ancestor–descendent relationships and an inferred summary of mutations, recombination events, host shifts, geographic, and temporal spread of the viruses. however, this synthesis comes at a price. the cost of computation of phylogenetic analysis expands combinatorially as the number of isolates considered increases. thus, large datasets like those currently produced are commonly considered intractable. we address this problem with synergistic development of heuristics tree search strategies and parallel computing. in the past, most studies of influenza were performed on a limited number of isolates and genes suited to a particular problem. major goals in science today are to understand emerging diseases in broad geographic, environmental, societal, biological, and genomic contexts. synthesizing diverse information brought together by various researchers is important to find out what can be done to prevent future outbreaks [jon03] . thus comprehensive means to organize and analyze large amounts of diverse information are critical. for example, the relationships of isolates and patterns of genomic change observed in large datasets might not be consistent with hypotheses formed on partial data. moreover when researchers rely on partial datasets, they restrict the range of possible discoveries. phylogenetics is well suited to the complex task of understanding emerging infectious disease. phylogenetic analyses can test many hypotheses by comparing diverse isolates collected from various hosts, environments, and points in time and organizing these data into various evolutionary scenarios. the products of a phylogenetic analysis are a graphical tree of ancestor-descendent relationships and an inferred summary of mutations, recombination events, host shifts, geographic, and temporal spread of the viruses. however, this synthesis comes at a price. the cost of computation of phylogenetic analysis expands combinatorially as the number of isolates considered increases. thus, large datasets like those currently produced are commonly considered intractable. we address this problem with synergistic development of heuristics tree search strategies and parallel computing. phylogenetics is the study of the evolutionary relationships of genes and organisms, thus providing a retrospective analysis of biological change and adaptation over time. phylogenetic trees are represented by acyclic graphs in which the leaves of these graphs represent the observed biological entities (taxa) being compared (e.g., sequences of genes, genomes, and/or anatomy of individuals, isolates or cultivars, species, or any higher level taxonomic unit). the internal nodes of the tree are interpreted as a nested set of hypothetical evolutionary ancestors of the entities under consideration as depicted in fig. 2 .1. once a tree is complete, changes such as mutations and host shift can be traced along branches of the tree that contain important disease causing strains. this retrospective analysis of features provides means of finding mutations that are diagnostic of pathogens, correlating phenotypes and genotypes, and predicting strains that are important for vaccine design. the classification of organisms dates back to aristotle [ari343] . however, it was only a few decades ago that the theoretical foundations of the field of phylogenetics as it is practiced today were established. the modern school of phylogenetics arose from the application of the ideas, termed cladistics, originally proposed by hennig [hen66] . cladistics lead v x w y p q fig. 2.1 . phylogenetic tree of four taxa labeled v, w, x, and y and two hypothetical ancestors labeled p and q biologists to use shared derived similarities (termed synapomorphies) that distinguish various natural groups of organisms. nested sets of natural groups of organisms based on synapomorphies are then used to discover the evolutionary relationships between organisms and reconstruct patterns of modification in the features of organisms. subsequently, these principles have been used to develop optimization techniques to find the most justifiable sets of synapomorphies in large datasets. optimization techniques are necessary with most large and real world datasets as they often contain several, often conflicting, evolutionary signals (treated below). in contrast, advocates of another way of thinking, termed phenetics [sne73] , group organisms based on gross measures of similarity. groups are based on measures evolutionary distance rather than the concept of shared derived characters. in modern practice, similarity methods espousing phenetic concepts are used in searches of nucleotide databases and some multiple alignment methods. clustering algorithms in which least distant groups are clustered first and then distant clusters are connected are termed distance methods, in phylogenetics. distance methods typically produce a single tree and cannot, on their own, trace patterns of change in the features of organisms as they convert raw data to distances. next we discuss how various viewpoints have influenced methods, algorithms, and implementations in phylogenetics. a wide variety of methods have been proposed in order to infer the phylogenetic relationships of organisms. most methods are based on minimizing edit cost (such as a hamming distance) to transform one string of nucleotides or organismal characters into another. phylogenetic methods can be further classified in two different categories: distance-based and character-based. in this paper, we compare and contrast the applications of distance and characterbased methods used in infectious disease research. we illustrate applications of these technique to study the evolutionary relationships of groups of rna viruses and the patterns of mutations and phenotypic changes that can be reconstructed. among the distance-based methods, the most commonly used is neighbor-joining [sai87] . distance based methods require a precomputed multiple alignment of dna or amino acid sequences drawn from homologous genes. the most similar pair of taxa (as represented by sequences) are clustered. the clustered pair is then considered as a single taxon and the next most similar pair of taxa is clustered until only the last taxon is joined and the tree is completed. although the use of distance-based methods is relatively common in analysis of organisms that cause infectious disease, several authors have criticized the performance of this method for phylogenetic reconstruction (see [far96] ). one strategic flaw of the method is that it is computationally greedy. distance methods form the most similar clusters instantaneously without considering locally suboptimal paths that may lead to a better global optimum. other methods of phylogenetic analysis focus on characters, which are typically polymorphisms, recognized in columns of aligned nucleotides or amino acids from sequences of interest or investigator encoded characters of polymorphic phenotypes. character-based methods seek to find the phylogenetic trees that optimize a particular criterion. major optimality criteria include parsimony [far83] and maximum likelihood [fel73, fel81] . bayesian analysis [ran96, li00, hue02] uses a maximum likelihood optimality criterion but incorporates the probability, termed the prior, that a hypothesis is correct in the absence of data. the unifying feature of character-based methods is that they examine many randomly generated trees each representing an evolutionary hypothesis of character transformations and organismal relationships. as a character based analysis progresses, edit costs are calculated for transformations that candidate tree imply and optimal trees are stored for further consideration and refinement. the concept of optimality can be associated with cladistics or maximum likelihood but not distance methods. distance techniques lack a measure of tree quality and means to compare trees. cladistics employs parsimony as an optimality criterion. the core concept of cladistics is that the least number of transformations in the data implies the most defensible hypothesis. in cladistics, various edit costs can be applied to different genomic and phenotypic transformations. in the case of weighted parsimony the goal of tree search is to minimize weighted costs. under the maximum likelihood criterion the probability of the data, given the tree, calculated with a model for nucleotide or amino acid substitution is optimized. the related technique of bayesian phylogenetic inference uses maximum likelihood to evaluate trees. bayesian analysis aims to capture a posterior probability distribution of trees. typically the results of a bayesian analysis are displayed not as an optimal tree but rather as the probability that a set of evolutionary relationships is "true," given the prior probabilities, the substitution model, and data. all character-based methods of molecular phylogenetics [cladistics, maximum likelihood (and related bayesian methods)] rely on explicit assumptions about ancestral character states to polarize transformations of phenotypes and genotypes that can be reconstructed from data. as an example of such assumptions, in character based analyses is the outgroup criterion (treated below). in contrast, distance based methods do not use an outgroup criterion. distance based methods do not use the outgroup criterion. parsimony is a widely used optimality criterion. this criterion is associated with the concept that simpler explanations provide for more supportable hypotheses. in phylogenetics, the most parsimonious tree(s) is that which implies the minimum number of transformations in sequence and/or phenotypic character states among organisms of interest. the biological justification of this use of parsimony is that descent with modification from a common ancestor is a primary pattern of organismal diversification and the record of transformations can be used to reconstruct that pattern. as such, the tree(s) that minimizes the overall number of independent transformations (convergences or reversals in character state) that are needed to explain the observed data are to be preferred [far83] . recombination and horizontal gene transfer as seen among rna viruses are violation of the assumption of ancestor to descendent evolution, not parsimony per se. some novel techniques for discovery and understanding of reassortment and horizontal gene transfer have been developed under the parsimony criterion [wan05, whe05] . the parsimony score for a tree is measured based on the number of transformations implied by the tree, known as the tree length [far70] . the tree length is the sum, over all edges, of the hamming distances between the labels at the endpoints of the edge [ric97] . the labels located at the leaves of the tree are the observed characteristics (either genotypic of phenotypic) of the organisms being analyzed. the internal nodes are labeled in order to minimize the tree length of each tree being evaluated. given a tree and a matrix of features or aligned sequences for each taxon, the tree length is calculated using the fitch algorithm [fit71] . this algorithm works in polynomial time with the amount of data being analyzed (both in the number of characters and taxa). thus for a sequence alignment of thousands of taxa, each of which is labeled with thousands of nucleotides, the tree length of a particular tree can be computed using modern implementations of the fitch algorithm [gol03] in fractions of a second. given a tree and a data matrix of sequences and features, the parsimony method can pinpoint the branches on which certain evolutionary events are inferred to occur between ancestor or descendent. in an infectious disease context, these events can be a shift by a viral lineage from animal to human host. in the case of standard nucleotide sequence analysis, transformation events include substitution mutations (replacement of a given nucleotide by other) and nucleotide insertions and deletion mutations. some analyses invoke more complex parsimony models with weighted recombination and horizontal transfer events, as well as differentially weighting certain classes of mutation such as transversions (pyrimidine-purine shifts), transitions (pyrimidine-pyrimidine or purine-purine shifts), or insertion-deletion events [whe05] . note that in using the fitch algorithm to optimize a phylogenetic tree, both the tree length and the branch in which a particular transformation event is inferred to occur can be calculated in unrooted or rooted trees. the results of these calculations are independent of the root chosen for the tree. for example, in an unrooted tree relating four taxa known from their nucleotide sequences (see fig. 2 .2), the fitch algorithm can be used to identify a specific branch of a tree in which a transformation occurs (e.g., a mutation between nucleotides c and t of the third sequence position occurring in the only internal edge of the tree in fig. 2.3) . however, the polarity of a transformation event is dependent on how the tree is rooted. inferring polarity of change requires an external criterion, termed the outgroup. in character-based methods explicit assumptions of ancestral character states are set up by the investigator via the designation of at least one taxon as the outgroup. a good outgroup is known to be closely related to the taxa of interest (termed the ingroup). however, the outgroup must be clearly not a member of the ingroup. the underlying logic of the outgroup criterion is that the transformation events that occurred at evolutionary origin of the ingroup can be identified by comparison to modern organisms of another clade but with which the ingroup shares a common ancestor. the common ancestor is a hypothetical organism that provides a baseline set of character states from which polarity determinations can be made. thus the outgroup method, like bayesian inference, incorporates some previous knowledge of the relationships of the organisms. if the phylogenetic results show that the ingroup includes some members of the outgroup the previous knowledge must be reevaluated. the outgroup taxon is included in the data matrix of the phylogenetic analysis and the entire data set is analyzed simultaneously. the phylogenetic position and relationships of the outgroup are determined by the optimality criterion. in the case of the parsimony method, the outgroup is treated as any other taxon and is positioned in the tree in the position that minimized tree length. once the phylogenetic affinities outgroup are established, the outgroup can be used to root the tree, and the polarities of the transformations can be established (note the unidirectional arrows in fig. 2.4) . if chosen carefully, the outgroup will not be clustered with any of the ingroup. model based methods can also be used in reconstruction of ancestral character states (e.g., [cha00, thr04] to communicate the choice of outgroup taxon or taxa and clarify the relationships of the taxa, character based trees are often drawn as directed acyclic graphs with the root positioned on the branch between the outgroup and ingroup. in the example diagrammed in fig. 2 .4, a mutation is inferred to occur in the third sequence position from the ancestral state of c to the derived state of t. the presence of a t in the third sequence position is a synapomorphy, a derived character state that can be used to distinguish the members of the group formed by the taxa w and y. in contrast, the presence of a c in the third sequence position in taxa x and v cannot be used to distinguish these taxa since a c is also present in the third position in the outgroup. in this case the third position c is a primitive similarity of x and y or a symplesiomorphy. the other mutations occurring in sequence positions 2, 5, and 6 are found only in one taxon and thus cannot be used to infer relationships. these are termed autapomorphies. sequence position 4 is inferred to have not changed in this example and is thus of no value in discovering groups. in cladistics only the shared derived characteristics, synapomorphies, are used to diagnose a group. although other criteria have been proposed to root phylogenetic trees, the outgroup criterion is the least arbitrary. as a result, outgroup rooting is widely used for character-based phylogenetic analyses [nix94] . problems of the outgroup criterion. as seen, the use of the outgroup taxon provides an informative way test hypotheses on the content of natural groups and to root the phylogenetic tree in a way that allows interpretation of the polarity of change of evolutionary events. however, the choice of an outgroup taxon is key to the success of this method. if the nucleotide sequences of a candidate outgroup are divergent from the sequences of the ingroup taxa, the phylogenetic position of the outgroup might be hard to establish [whe90] . therefore, the choice of the outgroup requires judicious selection and searches for organisms that (1) are safely outside the ingroup but (2) that have comparable data [whe90] . the cases shown in figs. 2.1-2.4 are based on the simplifying assumption that the genes sequenced for the taxa of interest have equal number of residues (i.e., amino acid or nucleotide sequences of the same length). frequently, in empirical studies of related organisms, homologous genes have sequences with different number of residues. sequence length variation occurs in both coding and noncoding loci. the causes can be genetic drift, mutation, recombination, or horizontal transfer events. the phylogenetic analysis of molecular sequences, like that of all other comparative data, is based on schemes of putative homology that are then tested via phylogenetic analysis. unlike some other data types, however, putative homologies in molecular data are not directly observable. sequences from various organisms are often unequal in length. hence, the correspondences among sequence positions are not evident and some sort of procedure is required to determine which regions are homologous. this procedure is typically multiple sequence alignment. alignment inserts gaps to make the putatively corresponding residue line up into columns. these columns (characters) comprise the matrix used to reconstruct cladograms. the matrix is then submitted to phylogenetic analysis in the same manner as other forms of data such as morphological characters scored by an investigator. thus the primary reason in phylogenetics to create an alignment has a strongly operational basis -to make it possible to submit these data to standard phylogeny programs that were designed to handle column vectors of morphological characters. nevertheless, alignment followed by tree search is the standard procedure. two major options are currently available to analyze sequence data in a phylogenetic framework: a twostep analysis or a one-step analysis. phylogenetic analysis of large genomic datasets can present several nested npcomplete problems: multiple alignment, tree-search, and in some cases, gene order and complement differences among organisms. just as in distance methods, in most character-based methods, alignments are precomputed before any phylogenetic analysis. the alignment procedure is usually done through algorithms that produce a matrix from the raw dna sequences of the organisms being analyzed (fig. 2 .5). this data set is then analyzed (second step) in order to find the optimal tree (see sect. 2.1.4). the multiple alignment procedure ranges from easy in many coding loci to very difficult in noncoding loci such as functional rnas and genes containing introns [mor97] . in the case of some protein coding loci the alignment may be a nonissue if there are no significant length differences in sequences. however, various investigators who employ different primer sets and editing styles often produce various length sequences. leading and trailing gaps produced by experimental artifact should not be counted in tree length calculations. results of multiple alignment of functional rnas and genes containing introns can be sensitive to parameter choices [fit83] . important parameters include the addition order of taxa, relative costs of various classes of mutations (transversions, transitions, insertion-deletion), and differential costs applied to opening or extending regions of insertion-deletions. analyses of different alignments of the same raw sequences can lead to different trees irrespective of tree search procedures [mor97] . in such cases investigators must search parameter space [whe95, phi00] or otherwise justify their assumptions during alignment [gra03] just as they are required to justify optimality criteria used during tree search. several researchers have noted that performing phylogenetic analysis into two steps is not consistent with the goals of finding the most parsimonious solutions due to the interdependence of multiple alignment and tree estimation [phi00, jan02] . in fact, popular multiple alignment programs such as clustal [tho94] use a guide tree used to construct the alignment. therefore, methods have been proposed to make a simultaneous estimation of the optimal sequence alignment and the optimal phylogenetic tree [san83] . a modern implementation of the one-step concept in poy [whe05] , termed direct optimization, allows unaligned sequence data to be analyzed without precomputing an alignment. in direct optimization, sequence data are aligned as various trees are built and their optimality is assessed. thus for each tree considered in a search, various sets of homology statements for the sequence data are considered. one advantage of direct optimization is that the outgroup need not be designated by the investigator. poy allows for randomization of the outgroup taxon and thus adds rigor to the search for optimal trees and homology statements. in some implementations of character-based methods where prealignment is necessary the outgroup can be randomized by scripting a series of analyses, e.g., tnt [gol03] . one important difference is that in molecular data a rigorous tree search with on a prealigned dataset with unordered characters should lead to the same tree length irrespective of outgroup choice; whereas in direct optimization the homology statements and hence tree length can be dependent on outgroup choice. several groups are developing algorithms for simultaneous estimation of alignment and phylogenetic trees. methods for a one-step phylogenetic analysis have been developed using maximum likelihood [tkf92, fle05] and parsimony optimality criteria [whe96] , as well as for bayesian analysis [red05] . although the one-step approach has the appeal of using a unified and epistemologically consistent method of alignment and tree estimation, the time and space requirements for computation are considerable. this problem of tree-based alignment is known to be np-complete [wan94] . in this situation, genes that vary in length (as most noncoding and intronic containing genes do) present a huge number of possible hypothetical ancestral sequences even for a single binary tree. during a phylogenetic analysis many trees will be examined and compared. for s taxa and l nucleotides per taxon, the cost of computation per tree ranges from (s−l)l 2 to (s−2)l 3 , depending on the heuristics applied. the memory requirements scale proportional to l 3 . fortunately, procedures such as the optimized diagonal transition algorithms described by ukkonen [ukk85] abate the space and time dependence on l, the number of nucleotides [whe05] . phylogenetic analysis under the parsimony criterion is based on an objective function (tree length). tree length is used to evaluate the optimality of each phylogenetic tree considered. however, finding the optimal phylogenetic tree (among all possible topologies) is an np-hard problem [fg82] , that resembles the steiner tree problem. the combinatorial optimization problem of phylogenetic analysis consists of finding the optimal solution from a very large number of possible trees. the number of possible trees increases dramatically with the number of organisms being analyzed [fel78] . the number of possible (unrooted) phylogenetic trees (t ) for a given set of organisms increases following where s is the number of organisms (leaves) of the phylogenetic tree. therefore, the number of possible phylogenetic trees is extremely large even for trees with moderate number of organisms. as stated above, a phylogenetic analysis consists evaluating topologies in order to find the optimal solution [i.e., the tree(s) with the minimum length]. it is interesting to note that the computing time of an exhaustive evaluation of all possible trees for a fixed number of taxa will increase nearly linearly with the number of characters (e.g. length of dna sequences) because the fitch algorithm [fit71] for evaluating the tree length of a particular topology works in polynomial time. however, the excessively large number of possible phylogenetic trees of 20 or more organisms (see table 2 .1) makes exhaustive evaluation of all phylogenetic trees intractable. note on multiple optimal trees. frequently, in phylogenetic analysis based on an optimality criterion, there are multiple trees that score the same minimum order of magnitude is given for the number of trees with more than 10 organisms for tree length or likelihood. in the set of known optimal trees, the transformations may be differentially distributed and different organismal groups may be implied. therefore, this set of known optimal trees must be considered equally valuable. these cases represent alternative hypotheses (i.e., phylogenetic trees) that are equally supported by the available data and can be summarized through a consensus tree. several kinds of techniques for consensus estimation exist. the strict consensus tree is one of the most frequently used. a strict consensus calculation represents a tree that has all the edges shared by all the known optimal trees. see [swo91] for further information on various consensus trees. two algorithms can be applied to perform exhaustive searches that evaluate (explicitly or implicitly) all possible phylogenetic trees in order to find the optimal tree. the first of these is exhaustive enumeration, which computes the optimality value (e.g., tree length) of every possible phylogenetic tree and select the tree (or trees) with the minimal value. the second method is the branch and bound algorithm [hen82] that implicitly evaluates all possible trees but avoids, in practice, computing all possible trees (see [sea96] ). in current phylogenetic software packages (e.g., [swo02, gol03] ) this algorithm can be applied to data sets of up to 20 (or 25) organisms and guarantees to find the optimal trees (or trees) for a given phylogenetic data matrix. for analysis of data sets with larger number of organisms, the number of trees is prohibitively large for conducting an exhaustive search. in modern biology, interesting data sets consider hundreds to thousands organisms. thus the problem of phylogenetic tree search is compute bound and must be approached through heuristic searches. in these tree searches, a large number of phylogenetic trees are evaluated and the best solution is kept as known estimate of minimum tree length. some early examples of heuristic tree searches include the algorithm to compute wagner trees [far70] . the wagner algorithm creates a phylogenetic tree of three taxa and progressively adds organisms, attaching them to the branch that generates the minimal increase in tree length at that step. this stepwise procedure is conducted until the last organism is added to the tree. although this procedure usually results in a tree that has a suboptimal tree length, in most cases this score is significantly better than that obtained with a random choice among all possible topologies. as various starting points are used for building wagner trees, this aspect of phylogenetic analysis can be considered a type of monte carlo randomization. given one or many wagner trees, the next standard heuristic refinement techniques that would be typically applied in tree search are known as branch swapping or hill-climbing procedures (see [sea96] ). this class of refinement procedures consists of performing minor rearrangements of branches in the starting tree. each wagner tree is modified by pruning a subtree and reattaching it to a different branch of the remaining tree. the tree length of the modified tree is then calculated. if the modified tree has a shorter tree length it is kept in a buffer of new candidate trees. branch swapping is applied to all wagner and candidate trees until the algorithm converges. when no further rearrangements can improve the current topology the branch swapping is finished. the results of the branch-swapping algorithm depend on the quality of the starting point (i.e., wagner tree). in many cases, the tree resulting from the application of branch swapping to a wagner tree is a local optimum that cannot be further improved by swapping. therefore, multiple replicates (100s to 1,000s) of independent wagner trees followed by swapping are typically preformed. at the end of these stages of analysis, the best trees found in all the replicates are kept as a set representing topologies at the known minimum length. replication of wagner builds plus swapping (or random-restart hill climbing) is the most widely used routine implemented in most software packages (e.g., [swo02, gol03] ). one major drawback of wagner builds plus swapping is that this procedure is subject to finding only local optima. finding the globally optimal tree(s) for a dataset of >20 taxa is a np-hard problem [fg82] . however, performing multiple replicates of this procedure can provide a relative degree of confidence if the minimum length tree(s) converge at the same tree length from numerous independent starting points [gol99] . replication of wagner builds plus swapping is usually efficient for data sets smaller than a hundred taxa. because of advances in automated dna sequencing technology, the size of modern comparative data sets far exceed the limits for which these techniques are efficient analytical tools for phylogenetic analysis. large phylogenetic problems are becoming increasingly common across the life sciences due to the prevalence of high throughput nucleotide sequencing technology. large data sets are of interest to biologists because they provide a rich context of phenotypes and genotypes and permit worldwide and longitudinal sampling of genomes. these large phylogenetic problems will become increasingly common in the years ahead. thus, phylogenetic methods suited to large datasets will have important consequences not only for the study of organismal classification and evolution, but also for many aspects of public health (see sect. 2.2). furthermore, in a operational context, strong organismal sampling has been shown to correlate with improved performance of phylogenetic methods [hil96, poe98, ran98, zwi02, hil03]. the dauntingly high cost of computation of large-scale phylogenetic analysis stunted this line of research. however, in recent years two main lines of research have provided efficient tools to analyze large phylogenetic datasets: the development of new algorithms and the use of parallel computing. several researchers have combined groups of algorithms into heuristic tree search strategies that have proven to be efficient for phylogenetic analyses of hundreds to thousands of organisms under the parsimony criterion. these heuristic search strategies are based on basic monte carlo and hill climbing techniques with the addition of other classes of algorithms including simulated annealing [gol99] , data perturbation [nix99] , divide-and-conquer [gol99, ros04] , and genetic algorithms [moi99, gol99] . similar search strategies that combine several layers of algorithms have been employed using other optimality criteria such as maximum likelihood [lew98, sal01, lem02, bra02] . the judicious application of various algorithms has provided efficient solutions for the analysis of datasets of several hundreds organisms [gol99] in a single cpu [teh03] . in particular, the successive combination hill-climbing, genetic, and simulated annealing algorithms of tree search have produced a drastic speed up in comparison to other strategies [gol99] . efficient implementations of these algorithms have become recently available in software packages [gol03] . the need of phylogenies depicting the evolutionary relationships of datasets consisting of thousands of taxa has prompted the synergistic implementation of efficient heuristic tree search strategies and parallel computing hardware. an increasing number of researchers are developing software suited for parallel computing using beowulf class clusters [ste00] . beowulf clusters are simply arrays of commodity pcs and switches enabled by scalable, open source operating systems (e.g. linux) and message passing software (e.g. pvm or mpi). although the advantages of parallel computing in phylogenetics and multiple alignment have been clear for some time [whe94] , the means to exploit this potential for research gain have not been broadly and economically available until the beowulf concept was developed by the end of the 1990s. alignment and tree search problems are naturally suitable for parallel computing. phylogenetic researchers quickly realized the opportunity presented by beowulf computing [cer98, jan01] . finding an optimal phylogeny requires the evaluation of the same objective function on a large number of alternative trees. because many trees can be examined concurrently and independently, this has led several authors to implement phylogenetic tree searches in parallel [jon95, sne00, cha01, gol02, bra02, sta02]. these implementations use the parsimony and maximum likelihood optimality criteria as well as bayesian analysis. researchers have also used parallelism to speedup one-step phylogenetic analysis [jan01] and multiple alignment [whe94, li03] . originally, phylogenetics was considered relevant only to taxonomic and evolutionary studies. however, the ability to identify conserved and divergent regions of genomes is becoming critical data for numerous disciplines in biology and medicine. these fields include vascular genomics [rub03] , ecology [sil97] , physiology [car94] , pharmacology [sea03] , epidemiology [ros02] , developmental biology [whi03] , and forensics [bud03] . phylogenetics has even been used in successful criminal prosecution of a doctor who attempted to cause hiv infection in his former girlfriend via blood products taken from a hiv patient under his care [met02] . here we focus on cases in which phylogenetic analyses have helped researchers to understand the evolution and spread of infectious diseases. we provide exemplar cases in which phylogenetic analyses of viral genomes have been crucial to understand complex patterns of transmission among animal and human hosts: severe acute respiratory syndrome (sars) [ksi03] and influenza [web92] . emergent infectious diseases often evolve via zoonosis; shifts of an animal pathogen to human host. in fact, most category a pathogens and potential agents of bioterrorism and more than 75% of emergent diseases have zoonotic origins [tay01, fra02] . a typical set of tests for the hypothesis of animal host of a disease might be (1) experimentally exposing the candidate host animals with isolated viruses and ascertaining whether infection and viral shedding occurs [mar04] ; or (2) survey populations of animals with antibodies for exposure to the virus [gua03] . these activities often provide model organisms for vaccine and drug development, data on seroprevalance, and sequence data for viruses isolated from various candidate hosts. phylogenetic analysis of genomes is a complement to laboratory and survey studies with a distinct advantage. with phylogenetics, the researcher is not restricted to testing a single hypothesis for a specific candidate host in each experiment. provided with sequence data for a diverse set of candidate hosts, a researcher performing a single phylogenetic analysis makes a vast number of comparisons, thus evaluating simultaneously many alternative hypotheses. these hypotheses include the evaluation of pathways of transmission among several hosts and the polarity of the transmission events. for example, experimentalists report that small carnivores in chinese markets have been exposed to sars-cov [gua03] and the virus can infect domestic cats [mar04] . on their own these data do not necessarily reconstruct the history of the zoonotic and genomic events that underlie the sars epidemic. furthermore, whether or not interspecies transmission is observed or enhanced under controlled laboratory conditions, phylogenetic research is distinct as it can address whether the genomic record has evidence to support a hypothesis for a particular transmission pathway. for example, if phylogenetic analysis reveals multiple independent events of human to avian transmission of influenza viruses without intermediate hosts such as swine that provides a strong argument to reevaluate the hypothesis that pigs serve as "mixing vessels" for avian and human viruses leading to influenza epidemics [sch90] . in many cases, host shifts occur via recombination between two ancestral pathogen genomes to produce a chimeric descendent. epidemics can occur when, subsequent to recombination, a lineage of pathogens establishes itself in a new population of hosts, vectors, or reservoir species that can amplify and distribute the pathogen [mor95] . a host shift can require key mutations and rearrangement of the pathogen genome to infect cells of new hosts followed by adaptation to novel regulatory machinery. phylogenetics can reconstruct genomic changes at the level of each nucleotide and unravel parental and descendent strains in recombination mediated host shifts [wan05] . influenza is a widespread respiratory disease caused by an rna virus (orthomyxoviridae). the influenza virus has been traditionally divided in three major types: a, b, and c. influenza viruses of type a are known from many strains that infect both mammal and avian hosts, whereas the other two type are primarily known from humans. influenza a is characterized by antigenic subtypes (see sect. 2.3). influenza is interesting from both epidemiological and evolutionary points of view due to the interplay between genetic changes in the viral population and the immune system of hosts [ear02] . there are two basic hypotheses on how influenza a viruses escape the immune response in host population to cause epidemics: (1) antigenic drift, meaning that random point mutations produces novel influenza strains that succeed and persist if they can infect and spread among hosts; (2) antigenic shift, meaning that genes derived from two or more influenza strains reassort thus creating a novel descendent genome with a constellation of genes that can infect and spread among hosts. in both scenarios zoonosis is often involved. in case of antigenic shift the ancestry of only a fraction of the influenza genes may be zoonotic. two major classes of influenza epidemics are recognized in humans: seasonal outbreaks and large-scale epidemics known as pandemics [web92] . seasonal influenza is a significant public health concern causing 36,000 deaths and 200,000 hospitalizations in the united states in an average year [ger05] . elderly and children account for many of these severe cases of seasonal influenza. much of the population has partial immunity to seasonal influenza strains that are typically descendents of strains circulating in previous years. pandemics are often caused by infection, replication, and transmission among the human population with influenza strains of zoonotic origin to which few people have prior immunity. pandemics are rare but can affect the entire human population, irrespective of an individual's predisposition to respiratory diseases. in fact, the 1918 pandemic disproportionately affected young adults [tau06] , suggesting that older adults may have had some immunity. there have been three major influenza a pandemics, 1918 (h1n1), 1957 (h2n2), and 1968 (h3n2). the pandemic of 1918 is estimated to have killed tens to a hundred million people worldwide and 675,000 in the united states [tau01] . the asian flu pandemic of 1957 and the hong kong flu pandemic of 1968 were less severe, but caused tens of thousands of deaths in the united states [hhs04] all of these pandemic strains are thought to have originated in wild birds [web92] . the 1957 and 1968 strains are believed to be the results of antigenic shift. however, recent studies suggest that the h1n1 influenza virus that caused the pandemic of 1918 was entirely of avian origin rather than a humanavian reassortant [tau05] . other researchers have countered that the 1918 h1n1 strains had a more commonly accepted route to infection of human populations by reassortment in mammals [gib06; ant06]. pandemics can theoretically occur with any strain of influenza. most influenza infections since 1968 have been attributed to influenza a h3n2 or h1n1 strains. however, there have been several recent reports of novel human infections from avian strains of influenza with subtypes thought to occur rarely in humans. several cases of human infection of viruses of subtype h7 of avian origin have recently occurred in canada [twe04] and the netherlands [koo04] . avian influenza of antigenic subtype h5 and h7 viruses can be found as low or high pathogenic forms depending on the severity of the illness they cause in poultry. thus far, influenza h9 virus has only been identified as strains with low pathogenicity [lin00] . alarmingly, highly pathogenic strains of influenza a with an h5n1 subtype have spread rapidly among various species of birds in china, southeast asia, russia, india, the middle east, africa, eastern, and western europe [who07a] . these h5n1 influenza a strains share common ancestry with the outbreak of h5n1 that lead to a massive chicken cull and six human deaths in hong kong in 1997 [li04] . between 2003 and september 10, 2007, there have been 328 cases and 200 deaths among humans [who07b] . there are several instances of h5n1 infection of felids and swine in asia. there is scant evidence of human-to-human transmission in thailand [ung05] and indonesia [yan 2007 ]. if lethality to human cases of h5n1 drops, the virus might spread rapidly and without being detected. many predict an upcoming avian influenza pandemic of devastating human and economic costs. in the united states alone, it is projected that 15-35% of the population will be affected and the costs could range from 71.6 to 166.5 billion united states (us) dollars [ger05] . although vaccine production can in theory be modified to include h5n1 strains [dut05] , the genomes of interest are moving targets. it remains unknown whether the descendents of the contemporary h5n1 virus will achieve efficient human-to-human transmission and if this will occur via incremental mutations or a more punctuated reassortment mediated change. thus phylogenetics is a key technology to track the evolution of h5n1 and compare those changes to genomic and zoonotic events that underlie pandemics. the viruses of influenza type a are classified as various subtypes that represent differences in the antigenic reaction of two key glycoproteins: hemagglutinin (ha) and neuraminidase (na). these proteins reside on the surface of the virion. these proteins play key roles in recognition and infection of susceptible hosts (ha) and viral replication (na). these surface proteins are primary antigens recognized by the host immune system [web92] . the subtypes of influenza a are labeled according to the reaction of standard monoclonal antibodies to these ha and na proteins provided by the us centers for disease control to laboratories participating in the world health organization's (who) surveillance program [hhsb] . although this number will soon expand, there are currently 16 different antigenic subtypes recognized for ha (labeled from h1 to h16) and 9 different antigenic subtypes of na (from n1 to n9). thus, a subtype of influenza virus type a is labeled with the number associated with ha and na proteins (e.g., the most common subtype found in humans h3n2). since 1948, influenza viruses have been the focus of a coordinated surveillance program organized by the who [who05] . the hemagglutinin gene (ha) is the major target of the influenza surveillance. this program helps track predominant strains to inform the development of new vaccines. influenza viruses are sampled worldwide through the national influenza centers located in 54 countries [who05] . many of the viral isolates sampled by these programs are sequenced for the hemagglutinin gene, although there has been an increasing interest in sampling complete influenza genomes [ghe05, obe06] . an extensive record of hemagglutinin sequences of the influenza viruses type a isolated since 1902 are publicly available. these data provide a unique set of challenges and opportunities for phylogenetics. the geographically wide and temporally long sampling of viral isolates provides an unprecedented opportunity to study evolutionary patterns underlying the spread and host range of an infectious disease. however, as described earlier, large datasets present an enormous search space of possible evolutionary scenarios to be evaluated. the availability of nucleotide sequences of influenza viruses has triggered numerous research groups to attempt reconstruction of the phylogenetic history of these viruses (e.g., [bus99, yua02, fer03, bus04] ). these groups draw on data from currently circulating strains as well as from historically important strains gathered from archival tissue samples. examples of archival tissues that have provided date of interest to the 1918 epidemic include lung biopsies of deceased soldiers, victims frozen in alaskan permafrost [tau97] , and waterfowl collected for the smithsonian in 1916-1917 [fan02] . phylogenetic analysis of seasonal influenza sequence data has been used to classify nucleotide substitution mutations. in many codons of the ha gene mutations that produce a change in protein sequence are more frequent than those that do not [bu99] . this finding indicates that selective pressures imposed by the immune system of the hosts can drive the evolution of some codons of ha. thus an evolutionary perspective can illuminate functional studies of infectious disease [ear02] . as noted, phylogenetics have been widely used to understand history of influenza epidemics, host shifts, as well as evolutionary interactions with the hosts immune system (see sect. 2.3.1). however, most phylogenetic analyses of influenza thus far have used only fractions of the dataset of influenza nucleotide sequences in the public domain. the sequences in the public domain are largely ha, but recently whole genomes have been produced. the institute for genomic research (tigr) is rapidly sequencing and releasing into the public domain thousands of influenza genomes under the microbial sequencing center (msc) program sponsored by the national institute of allergy and infectious disease (niaid) [ghe05] . st. jude children's research hospital in memphis has contributed a significant increase in the number of avian influenza genomes sequences [obe06] . most existing phylogenetic analyses of influenza have focused on the phylogenetic relationships of particular subgroups of influenza type a, such as the h5n1 subtype (e.g., [li04] ) or the h3n2 subtype (e.g., [bus99] ). these analysis have provided useful information but have depicted a disjoint picture of the evolution of the major lineages of influenza. in contrast, other studies have attempted broader subtype scope; however, they included a single viral isolate as an exemplar of each subtype [suz02] . this study failed to include an extensive sampling of strains. poor strain sampling can have a negative impact on the performance of phylogenetic methods (see sect. 2.1.5) and does not test whether the subtypes are natural groups (i.e. monophyletic). a very recent study has used whole genomes of 136 isolates drawn from a variety of avian influenza subtypes [obe06] . here we show results of a comprehensive phylogenetic analysis based on hemagglutinin dna sequences of 2,359 viral isolates. these sequences include representatives of the 16 different subtypes of the hemagglutinin protein of influenza type a, recorded worldwide by the world health organization surveillance program. the analyzed viruses were isolated as early as 1902, from tissues of patients who died during the 1918 spanish flu epidemic, to recently sequenced isolates from the 2004 seasonal flu and h5n1 outbreak. the analyzed dna sequences also implies a broad range of host organisms, including multiple species of wild and domestic birds, humans, swine, horses, felids, and whales. an inclusive phylogenetic analysis with a large number of taxa require the use of efficient tree search strategies (see sect. 2.1.5) and the use of multiple computers dedicated to the phylogenetic analysis. the cost of computation is tied primarily to the number of strains, not nucleotides. thus the inclusion of whole genomes does not contribute significantly to the compute bound nature of phylogenetic analysis. however, the inclusion of whole genome data does increase memory demands. this 2,359 isolate dataset was analyzed with a parallelization of the tree search strategy implemented in a recently developed software for parsimony analysis [gol03] . the results of this analysis are used here to illustrate two new uses, longitudinal analyses of patterns of zoonotic transmission and assessment of surveillance quality. our results on the relationships of ha subtypes shown in fig. (2.6) has similarities with the results of suzuki and nei [suz02] , including the clades ((h8 h12) h9), ((h15 h7) h10), ((h4 h14) h3), and (((h2 h5) h1) h6). however, the position of h13 and h11 differ in our trees due to our inclusion of h16. moreover, the relationship of these clades to one another differs in our assessments. our tree has a staircase shape with ((h8 h12) h9) basal most, whereas suzuki and nei's [suz02] tree has a symmetrical shape with no clear basal group. influenza a viruses from wild aquatic birds have been identified as the source of influenza viruses isolated from birds of the order galliformes (e.g., turkeys, grouse, quails, pheasants, domestic chickens, and their ancestral stock the jungle fowl) [web92] . direct human infection by avian strains of influenza a is considered rare [lip04] . after the discovery of receptors for both avian fig. 2.6 . phylogeny of hemagglutinin (ha) sequences representing 2,358 isolates of influenza a, with a single sequence of influenza b as outgroup. to summarize the source tree we have condensed each subtype clade into a single branch. the numbers of isolates included in the full tree are presented as the numerals above each branch. the numerals below each branch represent jackknife support values (0 worst to 100 best). sequence and character data was drawn from genbank (www.ncbi.nlm.nih.gov) and the influenza sequence database (www.flu.lanl.gov) and mammalian strains of influenza in the trachea of pigs, it has been hypothesized that domestic swine act as intermediate hosts in which human and avian viruses can recombine [sch90] . this mechanistic hypothesis of viral transmission is widespread. however, as discussed above, a number of events of suspected direct transmission of avian influenza viruses to humans have been reported [lip04, ung05] . hypotheses on the relative frequency of host shifts can be made on a phylogenetic tree through the optimization [fit71] of a character with states representing various hosts of the viral isolates under consideration (see sect. 2.2.1). we performed this analysis on our tree of 2,359 ha sequences and found that most of the internal nodes close to the root are optimized as having an avian origin (fig. 2.7) . thus the results of this analysis are consistent with the hypothesis of an avian origin of all influenza type a viruses [web92] . these results also show that most major lineages of influenza a that infect domestic birds originated in aquatic birds. this is compatible with the hypothesis that wild aquatic birds as the natural reservoir of influenza viruses of type a [web92] . however, the pattern of host shifts resulting from our study of 2,359 ha sequences seems to be much more complex than previously thought [gam90, lip04] . for instance, in many cases, after the spread of influenza type a viruses into domestic bird and mammal populations (including humans), some derived lineages are later spread again to aquatic birds. furthermore, the results indicate that direct shifts from avian to human hosts have occurred 18-27 times independently in different lineages (without observed intermediate hosts). it must be noted that the possibility of an intermediate host in avian-to-human transmission events cannot be completely rejected. it is possible that an intermediate host existed in nature but it was not sampled by the surveillance program and therefore not included in the analysis. however, based on the available evidence, it seems that host shifts from birds to humans have been frequent in the evolutionary history of influenza type a. moreover, avian-to-human shifts are more common than swine to human shifts in the history of influenza. multiple direct avian-to-human shifts appear to occur in the case of the putative pandemic strains of influenza a (subtype h5n1) that have spread across eurasia since 1997 [who05] . in addition to being highly pathogenic, these h5n1 strains have independently infected other hosts such as felids and pigs in several instances. phylogenetics is practiced by most as a historical science; however, several researchers noted that aspects of the tree shape may be used in predicting future genetic lineages of influenza against which it is important to design vaccines [gre04] . notable among these assertions are the studies in the shape of influenza a phylogeny as viewed through the hemagglutinin (ha) gene fig. 2.7 . two character optimizations on the for hemagglutinin (ha) sequences representing 2,358 isolates of influenza a, with an influenza b outgroup at the root. the top tree has an optimization of the character "ha antigenic subtype". the lower tree depicts optimization of the character "host". character data was drawn from genbank (www.ncbi.nlm.nih.gov) and the influenza sequence database (www.flu.lanl.gov). optimizations and tree graphics were made with mesquite (www.mesquiteproject.org). for better visualization contact the authors for files in scalable pdf format [bus99, fer02] . the ha gene codes for a surface glycoprotein of the virion responsible for binding to sialic acid on host cell surface receptors. at a genomic level, lineages of influenza are constantly changing due to mutation that occurs at high rates in rna viruses. extinction of evolutionary lineages of viruses to which hosts have become immune or when susceptible hosts are in short supply is common [gre04] . this process of constant replacement of influenza lineages produces a characteristic coniferous shape to a phylogeny reconstructed from ha sequences [bus99] . the "conifer" metaphor refers to the hypothesis that influenza ha is constantly changing but there is limited diversity at any time [fer02] . thus an influenza ha tree appears to be formed by addition of strains to the apex of the tree's trunk that contains the contemporary "infectious" viruses rather than more basal presumably "extinct" lineages to which hosts are immune. other groups of researchers have used the assumption that there is limited influenza a diversity at any one time to downplay the utility of phylogenetic approaches [plo02] . as an alternative to phylogenetics, which they consider difficult, these groups make predictions based on size of various clusters of related isolates, termed "swarms" [plo02] . several groups, whether using trees or swarms, have identified putatively dominant strains of influenza to predict the genetic makeup of future viral populations [plo02] [bus99] . if these assumptions were never violated, the diversity of a previous year's flu season could be assessed, forthcoming strains predicted, and thus used to inform vaccine design. in practice, the cdc uses a mixture of viral strains comprised of h1n1 and h3n2 of influenza a and an influenza b virus. [pal06] . notably the h5n1 strain (or any of the other avian strains with potential to infect humans) is currently not considered in the vaccine that is seasonally administered to civilians in the united states. the ability to predict influenza viral strains that will affect human and animal populations is important. however, prediction methods and experimental designs that are relevant to those methods are in their infancy. current surveillance programs are focused on detection of antigenically novel strains. as such, surveillance programs are not designed as ecological experiments to quantitatively measure strain-specific incidence and cluster size. furthermore, the current sample of influenza diversity may be biased by partial genomic sequencing, differences in effort within various geographic and political boundaries, focus on certain subtypes of interest, and differential efforts over time due to variable public concern. recent papers using whole genome data have indicated that the conifer like growth assumption of habased phylogenies that has been central to predictive models of h3n2 seasonal influenza [bus99, fer02] may be violated. full genome analysis of h3n2 has shown that there are multiple co-circulating lineages; some of which may be overlooked by vaccine designs [hol05, ghe05] . similarly, our large scaleanalysis of 2,359 ha sequences depicts that many subtypes and lineages within subtypes of influenza are circulating and being exchanged among human and animal populations at any one time fig. 2. 7. in addition to providing hypotheses on the relationships of a group of organisms, phylogenetic trees imply a temporal order of the successive internal nodes (i.e., the time at which a single evolutionary lineage splits producing two independent descendent lineages). minimal estimates on the date at which these evolutionary splits occur can be obtained through the analysis of the time at which the descendant organisms (leaves) are known to occur. these estimates can be computed with the implementation of an irreversible sankoff character in which the cost of transformation between two character states represents the amount of time elapsed between the time of appearance of two terminal taxa [pn01] . influenza a viral sequences are named with the host, locale, and year in which each isolate was sampled by the surveillance program. several methods exist to measure the correlation between the temporal dates of sampled organisms and the relative order they show in the phylogenetic tree. here we adapt the manhattan stratigraphic metric (msm*) to influenza surveillance. the msm* was originally developed to assess the quality of the fossil record [pn01] (table 2. 2). however, the msm* is simply a quantitative measure of how well the available data reflects the diversification pattern of the taxa present in the optimal phylogenetic trees and is thus of general utility. an extensive sampling of sequences, such as the one gathered for the study of 2,359 isolates, is critical to comparatively assess quality of surveillance in various regions, among various strains, and over periods of time. our results show that this correlation between branching pattern and dates of viral isolation is good in that it significantly differs from a random expectation. this is true over the entire tree as well as when some individual lineages are measured. however, the relative quality of surveillance differs markedly between lineages. one example of differential surveillance quality occurs in two closely related groups of avian influenza of h5 hemagglutinin subtype. one group in this example contains the highly pathogenic h5n1 strains that currently circulate in eurasia, the middle east, and africa. this large clade has been the focus of intense surveillance since the discovery of widespread infection among wild and domestic birds and some avian-to-human transmission [yua02, ung05] . the h5n1 viral isolates form a sister clade to h5n2 known from domestic and wild bird in the americas (h5n2). the number of available hemagglutinin sequences of h5n2 comprise less than one fifth of the number of ha sequences for h5n1. this in itself represents a measure of the surveillance intensity devoted to these two groups of avian influenza. however, even if the number of sequences is normalized at 100 sequences to perform the msm test, the surveillance quality of the h5n1 clade is far superior to the h5n2 clade. we can also use visualization techniques to assess surveillance quality. typically branches of a phylogenetic tree are scaled used to depict the number of mutations or other character changes assigned to each branch. however, we have adapted this use of branch scaling to reflect the number of years that have passed between sampling of related isolates rather than mutations or characters. compare fig. 2 .8 which has short branch lengths reflecting good surveillance quality with fig. 2 .9 which has long branch lengths implying poor surveillance quality. cases in which there is poor correlation between the date of sampling of a given isolate and its inferred date of origin would indicate that the surveillance program is failing to closely monitor the persistence of diverse lineages of influenza (2.9). no matter the type of phylogenetic perspective they may espouse, most virologists produce the same basic data by surveying putative host animals and patients with antibodies, then isolating and sequencing partial or whole genomes of various viruses detected in hosts. molecular phylogenetic analyses of the nucleotide or inferred amino acid sequence data from various viral isolates can then be used to reconstruct the history of the transmission events the virus among hosts. the fundamental belief associated with this research program is that the branching pattern of the phylogeny will reveal a temporal series of transformations when character of interest such as the host is optimized on the viral phylogeny. most virology researchers rely on distance methods. the most popular distance method among virologists is neighbor-joining (nj) [sai87] . distance methods require a precomputed multiple alignment of dna or amino acid sequences drawn from homologous genes of the viral strains of interest. then in nj, the most grossly similar pair of isolates (as represented by sequences) are clustered. the clustered pair is then considered as a single taxon and the next most similar pair of taxa is to cluster until only two taxa remain and are joined. in distance methods no outgroups are proposed and no assumptions of ancestral character states are considered. as a result, polarity of transformations can only be inferred as from dissimilar to similar. distance methods output a single unrooted, star-shaped graph. nevertheless preparing figures, some investigators who use distance methods choose to impart directionality by selecting an edge of the graph to serve as a root of the tree. the choice of root is crucial in depicting the polarity of host shifts and depicting clades. the rooting step has been executed variably by researchers comparing sequence data from covs isolated from humans with sequence data covs isolated from small carnivores. in the case of guan et al. fig. 1a) , rooted on a clade comprised of two sars-cov isolates, one from human and the other from a carnivor ( [son05] their fig. 1b) , and in a regression analysis a date for a common ancestor of sars-cov isolated from humans is calculated using a human basal group ([son05] their fig. 3) . in papers comparing sequence data from sars-like cov recently isolated from bats to that from humans and small carnivores lau et al., [lau05] do not root their trees (their fig. 2) and li et al., [li05] and force the root position on their drawing such that one of the bat sequences is ancestral (their fig. s4 of the supplemental material). thus, although all these studies employ distance methods, the researchers use various, often facultative means to infer the animal origins of sars-cov. other methods of phylogenetic analysis focus on characters, states, edit costs for changes among states, outgroup assumptions, and polarity of change among states -rather than gross similarity in the case of distance methods. characters can be polymorphisms recognized in columns of aligned nucleotides or amino acids from sequences of interest or phenotypic states such as host, date of isolation, or antigenic subtype. another feature of most character based methods that differs from nj is that character based methods examine many randomly generated trees (each representing an evolutionary hypothesis of character transformations and organismal relationships). thus the concepts of optimality and hypothesis testing are tightly associated with cladistic and maximum likelihood inference. optimal trees represent more defensible hypotheses. moreover, character based methods of molecular phylogenetics rely on explicit choices of outgroup to make assumptions about ancestral character states and thus polarize transformations of phenotypes and genotypes that can be reconstructed from data. in order to make an explicit assumption of ancestral character states the investigator designates at least one taxon as the outgroup. the outgroup method originated in cladistics [wat81] and has become central to the phylogenetic inference [nix94] . if chosen carefully, the outgroup estimates baseline character states in sequence and phenotypes such that transformations (such as host shifts) can be reliably inferred. to illustrate the choice of outgroup taxon or taxa and clarify the relationships of the organisms, character based trees are often rooted from the outgroup and ingroup. just as in distance methods, in most character-based methods, sequence data is aligned before the phylogenetic analysis. novel implementations, termed direct optimization, allow unaligned sequence data to be analyzed without precomputing an alignment, wheeler [whe96] . in direct optimization, sequence data are aligned as various trees are built and their optimality is assessed (using maximum likelihood and cladistic optimality criteria as specified by the investigator). thus for each tree a specific alignment that is optimal for that tree is constructed. one additional advantage of direct optimization is that the outgroup need not be designated by the investigator but rather randomized during the search for optimal trees and alignments. in some implementations of character based methods where prealignment is necessary, the outgroup can be randomized by scripting a series of analyses. outgroup randomization enables analyses of taxa where previous knowledge of ingroup/outgroup relationships is lacking or is among the hypotheses the investigator wants to test via tree search. large-scale phylogenetic analyses are particularly useful to study global problems of infectious disease. however, phylogenetic analysis of large number of organisms and whole genomes is an extremely challenging computational problem. recent advances in heuristic tree search algorithms, alignment methods, and parallel computing strategies have been successful. these advances have pushed upward the limits of taxon sampling considered tractable. large data sets analysis is interesting not only because it presents interesting computational challenges, moreover large dataset analysis is leading to new knowledge about natural phenomena. for example, in the recent past, researchers working on small datasets argued that influenza had limited diversity at any one time and that this should allow us to predict which strains are important for vaccine design. on the contrary, with large datasets, we find that there are multiple co-circulating lineages at any one time. thus, large datasets and means to analyze them are important for future vaccine design. character-based approaches to phylogenetics provide a wide variety of tools that can be 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the study of development, variation and evolution h5n1 avian influenza: timeline of major events cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who detecting human-to-human transmission of avian influenza a (h5n1) emergence of multiple genotypes of h5n1 avian influenza viruses in hong kong sar increased taxon sampling greatly reduces phylogenetic error mr. farhat habib m.s. was instrumental to build and maintain cluster computers. rebecca allen and jiarui lian helped to organize phenotype and genetic data. the authors have no competing interests, financial or otherwise. dp acknowledges the national science foundations (nsf) support of the mathematical biosciences institute (mbi) and the mbi. dp and dj acknowledge the support of the department of biomedical informatics and the ohio state university medical center. dj acknowledges that this material is based upon work supported by, or in part by, the us army research laboratory and the us army research office under contract/grant number w911nf-05-1-0271 and nsf 0531763. key: cord-255181-du6rqc6i authors: louz, derrick; bergmans, hans e.; loos, birgit p.; hoeben, rob c. title: cross‐species transfer of viruses: implications for the use of viral vectors in biomedical research, gene therapy and as live‐virus vaccines date: 2005-06-29 journal: j gene med doi: 10.1002/jgm.794 sha: doc_id: 255181 cord_uid: du6rqc6i summary all living organisms are continuously exposed to a plethora of viruses. in general, viruses tend to be restricted to the natural host species which they infect. from time to time viruses cross the host‐range barrier expanding their host range. however, in very rare cases cross‐species transfer is followed by the establishment and persistence of a virus in the new host species, which may result in disease. recent examples of viruses that have crossed the species barrier from animal reservoirs to humans are hantavirus, haemorrhagic fever viruses, arboviruses, nipah and hendra viruses, avian influenza virus (ai), monkeypox virus, and the sars‐associated coronavirus (sars‐cov). the opportunities for cross‐species transfer of mammalian viruses have increased in recent years due to increased contact between humans and animal reservoirs. however, it is difficult to predict when such events will take place since the viral adaptation that is needed to accomplish this is multifactorial and stochastic. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live‐vector vaccines is a cause for concern. this review addresses a number of potential risk factors and their implications for activities with viral vectors from the perspective of cross‐species transfer of viruses in nature, with emphasis on the occurrence of host‐range mutants resulting from either cell culture or tropism engineering. the issues are raised with the intention to assist in risk assessments for activities with vector viruses. copyright © 2005 john wiley & sons, ltd. is known as a zoonosis in the case where the virus is transmitted from non-human hosts to humans and causes disease [6] . crossing the species barrier is an unpredictable event that involves complex interactions between the virus and the newly adopted host [7] . the hiv virus and contemporary human influenza viruses are prominent examples of viruses that have crossed the species barrier and established themselves permanently in the human population without further dependence on the presence of the original animal host reservoir. fortunately, natural adaptation of a new virus leading to permanent establishment and dissemination within the human population is a rare event [8] . often, the virus is not readily adapted to infect and spread efficiently from human to human. the emergence of new viral infections often follows environmental, ecological and technological changes caused by human activities [9] . these activities may lead to an increased contact between humans and animal hosts acting as reservoirs of zoonotic viruses. agricultural development, an increased exploitation of environmental resources, growth and increase in the mobility of the human population and trade and transportation of food and livestock, have been identified as important factors contributing to the introduction and spread of a number of new viruses in the human population [10] [11] [12] [13] . the road map for cross-species transfer may differ for individual viruses. however, some common underlying factors that affect the probability of zoonotic events can be identified ( table 1 ). the potential of viruses to adapt to new or changing cellular environments or ecological niches via genetic variation appears to be a key feature [8, 14] . the advent of novel technologies for genetic modification of viruses offers new opportunities for biomedical research. often, these activities involve handling of viruses and their genetically modified variants in large quantities. the use of viral vectors in experimental gene therapy or as live vaccines may be also a cause for concern. indeed, such activities meet the primary requirements for cross-species mutations that facilitate the use of alternative receptors in the newly adopted host species feline parvovirus [29] transmission to humans after occupational exposure hendravirus [135] intensified contact between natural and new host species due to climatic changes hantavirus [136] immune evasion by genetic variation (e.g. antigenic drift and shift) influenza virus [55] introduction in new geographic areas by migrating birds west nile virus [137] initial close contact between natural and newly adopted host due to changes in natural infrastructures nipah virus [135] transfer, i.e. contact between infectious viruses and a potential new host species. therefore, it is important to identify those activities that have a finite risk of leading to new viral infections and to practice appropriate biosafety measurements. in this review risk factors associated with activities with viral vectors will be addressed from the perspective of emerging viruses that have crossed the host barrier in nature. the processes that underlie cross-species transfer through host-range expansion and establishment of viruses in new host species depend on the accumulation of genetic changes [7, 15] . these are likely to differ for various viruses and may affect virtually every aspect of the viral life cycle. this process of adaptation can occur by a variety of mechanisms including mutation, recombination and reassortment. mutations occur in the genomes of dna as well as rna viruses. in general, mutations occur slower in dna viruses than in rna viruses because of the proofreading function of many dna polymerases. this corrects mistakes made by the polymerase during replication. rna genomes are replicated by rna polymerases that lack proofreading. therefore, mutations in rna viruses can occur up to a million-fold more frequently than in dna viruses [16, 17] . as a consequence, rna viruses generally evolve more rapidly, and lead to genetic heterogeneity and the presence of so-called viral quasispecies [18] . the concept of quasispecies states that in an infected host, the virus exists as a population of genetically related but divergent variants defined by a master sequence and a complex and dynamic series of mutant sequences. while the master sequence remains the predominant sequence present within the population, the spectrum of mutants may shift in response to selective pressures. from the quasispecies population a variant may be selectively expanded [19] [20] [21] . although the major mechanism that drives adaptation is based on accumulation of point mutations, evolution of viruses also occurs through recombination. recombination occurs in both dna and rna viruses leading to the exchange of parts of genomes. this may result in the emergence of new virus variants. for recombination to take place, at least co-infection of a cell by two different virus variants is required. recombination plays an important role in evolutionary changes of dna viruses. in some cases, recombination between viruses and cellular nucleic acid can lead to the capture of cellular coding sequences [22] . reassortment is another important evolutionary mechanism in rna viruses with a segmented genome, such as influenza viruses and reoviruses. after co-infection of a cell with different strains or subtypes, genomic segments may be shuffled and rearranged in progeny virus particles resulting in the generation of new viruses with different biological properties. this offers viruses a large adaptive potential by facilitating evolutionary leaps in response to changing cellular environments without the need for gradual accumulation of favourable mutations [23] . in this section a number of examples of animal viruses that have crossed the species barrier are chosen to illustrate how they have evolved in nature through genetic changes. the examples illustrate that under the right environmental conditions host-range variants evolve that may establish themselves in the newly recruited host as new viruses causing disease. after the initial cross-transfer to the new host species, a period of further adaptation may be required. the examples also show how the evolutionary processes continue while an epidemic evolves. in the late 1970s, a new syndrome of viral enteritis and myocarditis emerged in dogs and subsequently swept rapidly across the world, killing thousands of dogs within a few years after its initial appearance. the virus was named canine parvovirus type 2 (cpv-2). phylogenetic analysis revealed that this virus was remarkably similar to feline parvovirus-like viruses such as feline panleukemia virus (fpv) which infects cats, mink, and raccoons, but not dogs [24] . therefore, cpv-2 presumably emerged as a natural host-range mutant of a feline parvovirus [25, 26] . host-range properties are determined by the capsid protein for both cpv-2 and fpv. although cpv-2 is capable of infecting feline cells in culture, the virus does not replicate in cats. fpv, on the other hand, is able to replicate in dogs in a restricted fashion, i.e. in bone marrow and thymus, but not in cultured dog cells [27] . cpv-2 differs from fpv by only two nucleotide substitutions within the capsid gene resulting in two amino acid substitutions [28] . these changes are associated with the ability of cpv-2 to bind to the canine transferrin receptor with high affinity [29] . as a result, cpv-2 acquired the capacity to infect canine intestinal tissue. it is thought that subsequently this virus acquired transmissibility between dogs and further adapted to replicate more efficiently in dogs as it became pandemic. interestingly, within three years after its initial appearance in 1978, cpv-2 was replaced worldwide by an antigenically and genetically variant virus cpv-2a [25] . this indicates that cpv-2a had a strong selective advantage over cpv-2. another antigenic variant derived from cpv-2a, cpv-2b which differs by only two amino acids, arose in 1984. this implies that variants of cpv gradually arose due to further adaptation and selection in dogs, in reaction to selective, most likely immunological, pressure. the two new variants (types 2a and 2b) differ at 5 or 6 amino acid from cpv-2 isolates [30] . at present, the two variants are still endemic in the canine population. these two variant viruses have an expanded host range compared to the original cpv-2 since they replicate in cats in both experimental settings and in the wild although with no or relatively low pathogenicity [31, 32] . remarkably, the prevalence of cpv-2a and cpv-2b and new antigenic variants (cpv-2c) has now been demonstrated in a wide range of feline populations worldwide (reviewed in [33] ). the emergence of cpv-2 serves as an example of rapid global distribution and establishment of host-range mutants in an immunologically naï ve new host. whereas cpv-2 most likely arose as a natural host-range mutant derived from cats, some other scenarios on its emergence have been suggested [34] . it was suggested that cpv-2 may have emerged after cross-transfer from a yet unidentified animal host to dogs, or cpv-2 may have arisen under selective growth conditions during fpv livevirus vaccine production in canine cells and subsequently spread via vaccination. the high titers of the virus shed in faeces and its resistance to inactivation may explain its initial rapid dissemination, also into countries with strict quarantine regulations for dogs. human activity may have stimulated the spread through mechanical transport, presumably aided by long-distance air travel [25, 35] . the aids pandemic is now generally accepted to stem from a viral zoonosis. human immunodeficiency viruses 1 and 2 (hiv-1 and hiv-2) emerged separately around the same time in distinct geographically areas as the result of multiple zoonotic transmissions from simian immunodeficiency virus (siv)-infected non-human primates to humans [36] . based on their genomic organization and phylogenetic analyses it is clear that hiv-1 and hiv-2 fall into two different siv lineages [37] . this implicates that both viruses must have had distinct origins. both phylogenetic and epidemiologic evidence indicate that hiv-1 evolved as a consequence of sivcpz transmission from chimpanzees to humans in central africa [38, 39] . however, to date, no serological or genetic evidence of widespread prevalence of hiv-1-related strains exists in chimpanzees in the wild in africa. transmission of siv from sooty mangabeys in west africa most probably caused the emergence of hiv-2 since siv strains derived from sooty mangabeys are phylogenetically closely related to hiv-2. hiv-2 is found at a high prevalence in sooty mangabeys [40, 41] . both hiv-1 and hiv-2 show enormous genetic diversity. hiv-1 comprises three genetically distinct virus groups (m, n, and o) of which the predominant group m consists of 11 subtypes or clades of which all but two have spread throughout the world. in contrast, hiv-2 is mainly confined to the african continent and comprises seven distinct phylogenetic lineages, subtype groups a through g, which can be categorized in epidemic subtypes a and b and non-epidemic subtypes c through g [37, 42] . it has been estimated from phylogenetic and epidemiological data that initial cross-species transfers of both the m group of strains of hiv-1, and the progenitor group of subtypes of hiv-2, may have taken place around 1930 in west africa [43, 44] . hiv-1 may have initially started to spread in africa at the beginning of the 1960s [45, 46] . therefore, more than two decades of 'silent' human-tohuman transmission may have occurred in africa before aids became apparent and hiv was identified as its causative agent in the early 1980s. in the advent of the hiv epidemic early siv strains initially may have crossed the species barrier as a result of an increase of contact between humans and siv-infected simian species. apparently, activities such as hunting, handling and consumption of contaminated uncooked simian meat led to direct exposure to animal blood and body fluids [47] . the following years passaging between infected humans of partially adapted siv strains may have resulted in series of cumulative mutations and genetic changes. the large genetic differences that exist between siv and hiv indicate that the initial siv strains that crossed the species barrier must have undergone adaptation in humans in a relatively short period of time. this suggests the involvement of some modern iatrogenic event. massive vaccination programs carried out at that time using non-sterile injection needles may have provided opportunities for transmission and further adaptation of the virus to humans in africa [48, 49] . the genetic and phenotypic evolution of the hiv virus still proceeds at a high pace not only between individuals worldwide, but also within infected individuals. during the time-course of infection the extensive genetic diversity originates from the rapid viral turnover and replication errors caused by reverse transcriptase [50, 51] . in addition, among the globally pandemic hiv-1 m group, several circulating recombinant forms (crfs) exist. these crfs result from recombination events between two different strains within the same individual and now constitute 10-20% of newly characterized circulating strains [52] . this diversity allows the hiv virus to rapidly adapt under selective pressure generated by antiretroviral drugs and host immune responses. selection of hiv variants has been implicated in the use of different co-receptor molecules and selection for different cell types and tissues and body compartments such as lymph nodes and the brain during late stages of infection and manifestation of different disease patterns [53, 54] . the emergence of hiv exemplifies how multiple independent cross-species transmissions of simian viruses that are not associated with disease in their natural hosts eventually resulted in the establishment of two types of hiv in the human population. while adapting to its new host the virus underwent a myriad of molecular changes. changes in social behaviour of humans may well have offered opportunities for newly evolved hiv strains to become pandemic. pandemic influenza a is a zoonotic disease caused by cross-species transfer of influenza a viruses from animal reservoirs. the twentieth century has witnessed three influenza pandemics, spanish influenza (1918), asian influenza (1957), and hong kong influenza (1968), that killed millions of people worldwide. although influenza a viruses have been isolated from a variety of vertebrates, including pigs, horses, seals, and whales, birds serve as the main reservoir and are a potential source for new pandemic strains [55] . influenza a viruses contain eight negative-sense rna segments that code for at least ten polypeptides of which eight are structural viral proteins and two nonstructural proteins. influenza a viruses are divided into subtypes based on both serological and genetic differences between the surface proteins and their encoding genes, respectively. to date 15 hemagglutinin (ha) subtypes (h1-h15) and nine subtypes (n1-n9) of the neuraminidase (na) proteins have been identified. influenza a viruses containing all different combinations of the ha and na subtypes have been identified in aquatic birds. in humans only influenza a viruses of hemagglutinin subtypes h1 through 3 and neuramidase subtypes n1 and n2 have established permanent lineages. these viruses are considered human influenza a viruses [56] . in nature new influenza a viruses emerge via two mechanisms of antigenic variation. the first, antigenic drift, is caused by accumulation of point mutations in both the na and ha surface proteins enabling new antigenic variant viruses to evade the human immune system and emerge via selection. influenza a viruses that emerge via antigenic drift are responsible for the yearly epidemics in the human population. antigenic shift occurs when a variant influenza a virus arises that is antigenitically completely distinct from former circulating influenza a viruses. the new virus is a reassortant that is characterized by the presence of a novel hemagglutinin gene segment alone or in combination with a complete novel neuraminidase gene segment. influenza a variants that emerge through antigenic shift are potentially capable of causing novel pandemics in an immunologically naï ve human population [55] . the avirulent nature of avian influenza virus infections in ducks and waterfowl results from adequate adaptation to their hosts [55] . avian influenza viruses do not replicate efficiently in humans. similarly, human influenza a viruses do not replicate efficiently in birds [57, 58] . the trachea tissue of the pig contains receptors for both avian and human influenza a viruses. therefore, they are permissive to both human and avian viruses and thought to function as a 'mixing vessel' for reassortment of not only human and avian, but also swine influenza a viruses [59] . pigs are therefore also considered to be ecological niches important for the emergence of new influenza a viruses in humans. in pigs, newly reassorted viruses may further evolve by accumulation of additional mutations, further adapt to a mammalian host and eventually be transmitted to humans. ample data indicate that further adaptation to the human cellular environment is necessary for replication and efficient transmission in humans. adaptation has a polygenic basis and may involve multiple viral gene segments. it should be noted, however, that only in rare cases will cross-species transmission lead to permanent establishment of new lineages of influenza a viruses in humans [56, 60] . analyses of the viruses that caused the asian and hong kong pandemics revealed that these were caused by reassortants that contained a mixture of avian and human genome segments [61, 62] . genetic analysis of the 1918 spanish influenza virus initially suggested that the epidemic originated from a whole avian influenza virus that had been transmitted from infected pigs to humans [63] . however, the origin of the 1918 pandemic strain still remains an enigma since the presence of its ha molecule did not originate from any known avian strain. in addition, there was no evidence of adaptation to a mammalian host [64] . it was not until the 1997 hong kong epidemic that direct transmission of whole avian viruses to humans was observed. analysis of the virus that caused this epidemic revealed that a reassorted influenza a virus (h5n1) of entirely avian origin had crossed the species barrier, apparently without adaptation to a mammalian host. interestingly, the virus was able to replicate in humans but had not acquired human-tohuman transmissibility, preventing efficient spread and, potentially, a global epidemic (reviewed in [65] ). receptor specificity is considered to be a major determinant of the host range of influenza a viruses. the ha protein plays a pivotal role in host-cell receptor recognition and attachment. it binds sialic acid (sa) on the host cells. avian influenza a viruses preferentially bind to terminal sa which is joined by an alpha2,3-linkage to the sugar chain of the glycoprotein or glycolipid in the gut. however, human influenza a strains bind to terminal sa through an alpha2,6-bond to cells in the respiratory tract as a result of acquired mutations [66] [67] [68] . the 1997 hong kong avian h5n1 strain, however, possessed avian binding properties [69] . this indicates that receptor specificity alone is not an absolute requirement for birdto-human transmission. the host range of influenza a viruses is determined by a complex interplay of multiple factors [70] . the influenza a virus illustrates the unpredictability of virus variation as well as the virus's great potential for adaptation. regular close contact between birds, pigs and humans offers opportunities for reassortment and crossspecies transfer. hence, the live-bird markets in south-east asia are considered a risk [71] . a variety of experimental conditions are applied in the laboratory for propagation and isolation of viruses and their genetically modified derivatives. as a result, these viruses are subject to selective forces that are likely to differ from those experienced in nature. although conditions may be well defined and controlled, various selective pressures are generated in culture due to, e.g., changes of concentrations of nucleotide substrates, the addition of mutagenic substances, the use of different incubation temperatures, incubation with antibodies, or a change of host cells. these different selective forces have unpredictable influences on the virus. since cell culture conditions can have profound effects on the composition of viral populations, viral stocks consist of genetically heterogeneous populations (reviewed in [20, 72] the following examples demonstrate that upon persistent infection and passage in cell culture, cross-species transmissibility may be promoted by selection of virus variants with an altered host range. adaptation in cell culture may result in changes in receptor specificity and tropism, and leads to the emergence of host-range mutant viruses. the mouse hepatitis virus (mhv) is characterized by a narrow host-range and tissue specificity, both in vivo and in vitro. this specificity is primarily determined by the virus's surface spike (s) glycoprotein, which is responsible for attachment to specific host-cell receptors [73] . mhv virus variants with both an altered receptor specificity and a broadened host range were selected during continued passaging in murine or mixed cultures consisting of murine and non-permissive hamster cells. here mhv acquired the ability to infect human, hamster and monkey cells. mhv host-range expansion has been attributed to the presence of virus variants recognizing homologues of the normal receptor. adaptation required mutations in the surface s protein and selection of host-range mutants for the use of the alternative cellular receptor [74] [75] [76] [77] . selection of variants with a changed receptor specificity resulting from passage in cell culture has also been demonstrated for foot-and-mouth disease virus (fmdv). host-cell specificity of the parental virus is based on an rgd motif-dependent integrin-mediated entry pathway [78] . the rgd motif is an arginine-glycine-aspartic protein sequence within the virus capsid that recognizes and binds to some integrins on the cell surface of the host cell [79] . yet, upon multiple passages in bhk-21 cells, fmdv variants emerged that acquired the ability to infect several initially non-permissive human and animal cell lines via an alternative entry pathway. analysis of these variants revealed that adaptation of fmdv in cell culture led to an enhanced affinity for heparan sulfate as a receptor, independent of the rgd motif. interestingly, these host-range variants were able to maintain infectivity in cell culture not only independent of an rgd motif, but also without the requirement to bind to heparin sulfate [80, 81] . this implies the use of alternative receptors. selection of the fmdv host-range mutants was associated with amino acid substitutions in or near the capsid rgd motif [82] [83] [84] . recently it was demonstrated that passaging of fmdv in bhk-21 cells led to an expansion of the host-cell tropism to non-human primate and human cell lines. selection of these host-range variants was also associated with amino acid substitutions in the viral capsid proteins [85] . there is less data on mutation frequencies and adaptation of dna viruses in cell cultures compared to rna viruses. however, there are a number of illustrative examples. adaptation of sv40 and polyomavirus to different cellular environments resulted in the emergence of host-range mutants in cell cultures (reviewed in [86] ). random mutagenesis of human adenovirus followed by repeated passaging in certain cell lines allowed isolation of adenovirus host-range mutants [87, 88] . human adenovirus 2 (hadv2) mutants with altered specificity resulting from reduced binding affinity of the adenovirus penton-base protein for the integrins on the cells were selected in persistently infected cell lines [89] . more recently, upon passaging in cell culture, adaptation led to the emergence of herpesvirus host-range mutants. these viruses use alternative receptors and replicate in different cell types in the natural host, and in cells from different species that were previously nonpermissive. several glycoproteins are essential for the entry of alphaherpesviruses such as pseudorabies virus (prv), herpes simplex virus (hsv) and bovine herpesvirus 1 (bhv-1) (reviewed in [90] ). interaction between, e.g., the viral glycoprotein c (gc) and heparan sulfate mediates primary attachment. for infection, however, a secondary interaction between glycoprotein d (gd) and one of several entry receptors is required. single amino acid substitutions in the gd glycoprotein of herpes simplex 1 (hsv-1) as well as complete ablation of this glycoprotein in the swine pseudorabies virus (prv) led to a gd glycoprotein-independent entry mode using alternate receptors [91] [92] [93] : at least three classes of cell-surface proteins are now thought to be involved in alphaherpesvirus entry [94] . also, cell culture adaptation of human cytomegalovirus (hcmv), a betaherpesvirus, resulted in the selection of phenotypic variants that had lost their endothelial tropism [95] . in cell culture viruses may readily adapt by mutation, selection and competition. these processes are stochastic in nature. adaptation is therefore an unpredictable process, strongly influenced by the experimental setting, e.g. the multiplicity of infection used, the number of passages employed, and the type of selection employed. one should be aware of the potential adaptation when working with virus-infected cell cultures. recombinant dna technology, including 'reverse genetics' and the availability of complete (infectious) clones for a large number of rna and dna viruses, allows genetic modification of viral genomes and generation of recombinant [90, 90] viruses in vitro. these technologies offer the possibility to deliberately change the tropism or host range of the viruses. some of the latest technologies are discussed in the context of two important virus groups, i.e. influenza a viruses and coronaviruses. the advent of reverse genetics systems now allows the generation of recombinant infectious influenza a viruses entirely from cloned cdnas in cell culture. these systems are based on transfection of at least eight plasmids, each containing a copy of one of the eight influenza a virus genomic segments [96, 97] . the technology permits the generation of custom-made recombinant influenza a viruses (reassortants) containing specific (heterologous) gene segments of interest and offers the possibility to study their biological properties in cellular and animal model systems. in addition, reverse genetics can be used in the development of vaccine strategies. the use of reverse genetics allows the deliberate introduction of specific mutations in viral genes allowing selective evaluation of the contribution of individual genes or segments to, e.g., the virus's virulence/pathogenicity, transmissibility and host range. this approach has already been shown to be pivotal for the generation and characterization of reassortants containing heterologous influenza a segments from, e.g., highly pathogenic avian influenza (hpai) h5n1 strains [98] or the 1918 pandemic strain [99, 100] . in addition, this technology allowed the generation from cloned segments of the hpai h5n1 strain that caused the deadly 1997 hong kong outbreak [101] . reverse genetics systems can also be used as an alternative for the production of both live attenuated and inactivated vaccines in preparing for pandemic influenza a virus threats. conventional annual (inactivated) vaccine production is based on simultaneous infection of chicken eggs with two different influenza a strains followed by selection of the desired vaccine virus. this reassorted virus then contains the na and ha segments of the relevant circulating influenza a virus against the background of six complementary segments derived from an attenuated reference strain (e.g. a/puerto rico/8/34 h1n1), which is safe for humans [102] . to overcome the difficulties of selecting such reassortants and subsequent laborious time-consuming passaging of these viruses, plasmidbased reverse genetics can be used for fast and directed generation of vaccine strains [103] . for vaccines based on hpai viruses the use of reverse genetics has another important advantage. such highly pathogenic viruses are lethal to chicken embryos and cannot be grown in large quantities in this way. the virus's high pathogenicity is associated with the presence of basic amino acids adjacent to the cleavage site within the ha molecule [104] . by using recombinant dna technology this sequence can be eliminated. plasmid-based reverse genetics can then be used to generate the desired vaccine strain containing the attenuated ha molecule [105, 106] . in conclusion, plasmid-based reverse genetics enables the generation of defined reassorted influenza a viruses consisting of, e.g., human and avian viral gene segments of interest. however, it is usually not possible to predict the biological properties from the gene constellation of such variant viruses. such activities therefore pose potential risks, in particular when the gene constellation is not based on characterized isolates. recombinant dna technology has allowed the construction of infectious cdna clones of large rna viruses such as coronaviruses, including the sars-associated coronavirus (sars-cov) [107] [108] [109] . these reverse genetics systems can now be used as tools for the production of defined genetically modified coronaviruses [110] . this allows the introduction of specific mutations into the genome of coronaviruses, and, e.g., the exchange of specific viral genes between different coronaviruses to study their pathogenesis, replication strategy, and cross-species transmissibility. the possibility to engineer tissue and host tropism using these technologies makes coronaviruses potential vectors for vaccine development and possibly for gene therapy [111] [112] [113] . host-range specificity of coronaviruses is primarily determined at the virus entry level. several studies have demonstrated that sequence changes in the gene encoding the coronavirus surface spike (s) glycoprotein can lead to a change of tropism and host-range specificity [73] . this is illustrated by the generation of a chimeric coronavirus by targeted recombination, in which the ectodomain of the s glycoprotein of mouse hepatitis virus (mhv) was replaced by the ectodomain of the s glycoprotein of feline infectious peritonitis virus (fipv). this substitution conferred specific tropism for feline cells, while the ability to infect murine cells was lost [114] . vice versa, a reverse genetics strategy for fipv was developed conferring the ability to infect murine cells [115] . similar techniques may aid the studies on the pathogenicity of the sars-cov. the examples mentioned above indicate that the use of recombinant dna technology now provides for powerful systems to generate and modify, e.g., highly pathogenic viruses such as pandemic influenza a viruses and sars-cov. live attenuated virus vaccines are among the most successful viral vaccines known to date. traditionally, attenuation is achieved by the 'jennerian approach', i.e. serial passaging in cell culture [116] . by this method a number of useful vaccines currently in use have been generated. still, the mechanism by which the attenuated phenotype evolves is largely unknown. for instance, the nature and degree of genetic variation present at different stages of the attenuation process is usually not known. the presumed mechanism of attenuation is based on host-range restriction due to accumulation of changes in surface (glyco)proteins [117] . thus selection of variants seems inherent to the process of generating the desired level of attenuation and genetic stability to prevent reversion to the wild-type virus therefore, dependent on the passage history, diversity within the virus population is likely to represent adaptations to growth in cell culture. as a result, genetic variants with different host-range phenotypes may be present in the vaccine strain of the virus. examination of substrains of live attenuated vaccine lots based on the yellow virus 17d strain and measles virus edmonston strain demonstrate that these virus stocks indeed consist of a heterogeneous population of variants [118] [119] [120] [121] . a number of adverse consequences of the use of such virus stocks have been reported and associated with possible selective growth advantage of host-range variants in the recipient [122, 123] . therefore, caution should be taken before releasing live attenuated viral vaccines based on non-human animal viruses. significant progress has been made in approaches to genetically modify the tropism of vector viruses. such strategies have been used in the development of cancer gene therapy. initially, replication-deficient vectors were used for this purpose. however, to improve efficacy, tumor-targeted replication-competent viruses have been developed for the use of viral therapy of cancer (virotherapy) [124, 125] . here we discuss some of the developments with human adenovirus type 5 (hadv5). hadv5 has been widely used for a number of vector applications [126] . however, the use of genetically modified adenoviral vectors has some limitations [127] . their efficacy relies on the presence of its receptor, car (coxackievirus and adenovirus receptor for hadv5), on target cells. primary binding of the virus to car is mediated by the knob domain of the adenoviral fiber protein. subsequent internalization is mediated by the interaction between the rgd motif in the penton base of the virus and secondary host-cell integrin receptor molecules [128, 129] . to achieve cell-type specificity and a high efficacy in the absence of the car receptor different strategies have been developed. these include redirecting adenoviral binding to alternative cellular receptors by genetic modification of genes coding for the capsid proteins fiber, hexon and penton base [130] . this may result in either an expanded tropism or in abolishment of the adenoviral native tropism. table 2 summarizes a number of properties contributing to the relative risk for the use of vector viruses. for this purpose a numerical hazard score was assigned to each property. table 3 summarizes a number of adenoviral vectors with altered properties and their relative risks. such modified viruses are now being evaluated in a clinical setting for experimental gene therapy. table 3 illustrates that a change of cell tropism, tissue tropism, or host range of a viral vector should be considered as factors in risk assessment for activities with genetically altered vector viruses. in general, the use of replicationcompetent viral vectors poses special concerns with regard to unintended spread to new and undesired cell types, as well as horizontal transmission of the vector [131, 132] . a replication-competent vector virus with an altered tropism or host range virtually constitutes a new viral pathogen with the potential of a new disease manifestation. in nature many factors may contribute to the emergence of a new zoonotic viral disease. these factors consist of viral evolutionary processes such as mutation, natural selection and competition, host determinants, e.g., immune status and physiological factors, and environmental determinants such as ecological and climatological circumstances. as highlighted by the emergence of new viral diseases in the last two decades, the process of adaptation often involves the acquisition of an altered cell tropism or host range. against this background the intensified use of viruses and their genetically modified variants as viral gene transfer vectors for biomedical research, experimental gene therapy and for live-vector vaccines is a cause for concern. this review highlights the importance of identifying and evaluating the risks and consequences of activities that may generate host-range mutants with the capacity of cross-species transmission. the use of such viruses may lead to inadvertent introduction of vector viruses with a changed cell or tissue tropism and/or host range through an immunologically naï ve and non-adapted hosts. interactions between the virus and the cellular receptor often determine the host range of the virus and therefore constitute a species barrier [133] . minor mutations in the viral capsid or surface glycoproteins may already result in profound changes in cell tropism or host range of a virus. in this review we have therefore focused on the level of virus entry to address some implications for activities with viral vectors, and in particular with hostrange mutants. this could contribute to a rational and reasoned inventory of factors that should be considered in risk assessments of activities with viral vectors. in considering possible risks involved in handling replication-competent vector viruses in the laboratory, [134] . this implies that concepts such as host-range barrier and hostcell specificity may be rather flexible than rigid. if the host range and completion of the viral life cycle is exclusively restricted to the level of entry, forced entry may bypass important discriminatory host-cell restriction steps. this may result in distinct pathological phenotypes and new disease manifestations. therefore, precaution should be taken to avoid inadvertent release and spread of such potential harmful vector viruses. it goes without saying that live-virus vaccines are among the most effective modalities to control viral pathogens. it is evident that for such vaccines the benefit is higher than the possible adverse effects. nevertheless, the risks associated with handling vaccine viruses, viral vectors, and exotic viruses are small but finite. we are just beginning to understand the mechanisms that drive the emergence of new viruses and viral diseases in nature. there are parallels between the patterns that are seen in emerging viral diseases and certain virus modifications that are generated, either deliberately or inadvertently, when handling viruses in the 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that replicates selectively in p53-deficient human tumor cells a novel threepronged approach to kill cancer cells selectively: concomitant viral, double suicide gene, and radiotherapy infectivity enhanced, cyclooxygenase-2 promoter-based conditionally replicative adenovirus for pancreatic cancer the authors wish to thank prof. alex j van der eb and dr. huub schellekens for critical review of the manuscript. in addition the authors wish to acknowledge the useful commentaries of the anonymous reviewers. key: cord-280218-zwjrcaab authors: he, xiao-song; holmes, tyson h.; sanyal, mrinmoy; albrecht, randy a.; garcía-sastre, adolfo; dekker, cornelia l.; davis, mark m.; greenberg, harry b. title: distinct patterns of b-cell activation and priming by natural influenza virus infection versus inactivated influenza vaccination date: 2014-10-21 journal: journal of infectious diseases doi: 10.1093/infdis/jiu580 sha: doc_id: 280218 cord_uid: zwjrcaab background. the human b-cell response to natural influenza virus infection has not been extensively investigated at the polyclonal level. methods. the overall b-cell response of patients acutely infected with the 2009 pandemic influenza a(h1n1)pdm09 virus (a[h1n1]pdm09) was analyzed by determining the reactivity of plasmablast-derived polyclonal antibodies (ppabs) to influenza proteins. recipients of inactivated influenza vaccine containing the same a(h1n1)pdm09 strain were studied for comparison. results. during acute infection, robust plasmablast responses to the infecting virus were detected, characterized by a greater ppab reactivity to the conserved influenza virus nuclear protein and to heterovariant and heterosubtypic hemagglutinins, in comparison to responses to the inactivated a(h1n1)pdm09 vaccine. in a(h1n1)pdm09 vaccinees, the presence of baseline serum neutralizing antibodies against a(h1n1)pdm09, suggesting previous exposure to natural a(h1n1)pdm09 infection, did not affect the plasmablast response to vaccination, whereas repeated immunization with inactivated a(h1n1)pdm09 vaccine resulted in significantly reduced vaccine-specific and cross-reactive ppab responses. conclusions. natural a(h1n1)pdm09 infection and inactivated a(h1n1)pdm09 vaccination result in very distinct patterns of b-cell activation and priming. these differences are likely to be associated with differences in protective immunity, especially cross-protection against heterovariant and heterosubtypic influenza virus strains. the 2009 pandemic due to influenza a(h1n1) virus (a[h1n1]pdm09) and recent human cases of infection due to highly pathogenic avian influenza a(h5n1) and a(h7n9) viruses emphasize the urgent need to adequately prepare for influenza pandemics. vaccination is considered the most effective means to protect against influenza. two types of seasonal influenza vaccines are currently available in the united states: inactivated influenza vaccine (iiv) and live, attenuated influenza vaccine (laiv). in children 6 months to 18 years of age, laiv has been consistently more efficacious than iiv against both antigenically matched and drifted strains [1] [2] [3] . laiv was recently recommended as the preferred vaccine for healthy children 2-8 years of age by the centers for disease control and prevention's advisory committee on immunization practices [4] . in adults <50 years of age, laiv is either equally effective or, in some reports, somewhat less effective than iiv [1, 5, 6] . both wild-type infection and iiv immunization, but not laiv immunization, efficiently induce serum antibody responses against influenza virus antigens. influenza hemagglutinin (ha)-specific serum antibody levels after wild-type influenza virus infection or iiv immunization are good predictors of substantial protection against infection with antigenically matched strains, but not antigenically drifted variant strains [7] [8] [9] [10] [11] [12] . with the exception of studies involving the generation of recombinant monoclonal antibodies with broad neutralizing activity from patients with acute influenza [13, 14] , the b-cell responses to natural influenza virus infection have not been well characterized. since the 2009 influenza pandemic, a(h1n1)pdm09 has been the annual h1n1 component of seasonal influenza vaccines, and most vaccine recipients have received iiv. the influenza vaccine coverage rate in the united states was 60%, and vaccine effectiveness was estimated as 62% for the 2013-2014 influenza season [15] . despite the consistent inclusion of a(h1n1)pdm09 in the vaccine formulations since 2010, a(h1n1)pdm09 became the dominant strain isolated from patients with influenza in the 2013-2014 influenza season for the first time since 2010. moreover, changes in the antigenicity of the circulating a(h1n1)pdm09 were not detected in 2013-2014 [15] , suggesting a lack of immunological selective pressure on the circulating virus. in the current study, we collected blood samples from patients with polymerase chain reaction (pcr)-confirmed acute a(h1n1)pdm09 infection during the 2013-2014 influenza season and generated plasmablast-derived polyclonal antibodies (ppabs) [16] . ppabs are representative of the overall antibody repertoire of infection-or vaccination-activated b cells; analysis of these antibodies avoids interference by preexisting cross-reactive serum antibodies, a pitfall difficult to avoid when using serum-based assays. reactivities of ppabs from these infected patients and from recipients of iiv to proteins from the infecting virus and from heterovariant and heterosubtypic influenza virus strains were examined. in addition, priming of the immune system by natural infection versus iiv immunization was evaluated by examining the ppab response to a subsequent immunization with iiv. to our knowledge, this is the first comparison of the characteristics of b-cell responses in natural influenza virus infection versus responses to iiv immunization at the polyclonal level. patients with acute influenza-like illness (ili) were enrolled at stanford hospital and clinics during the 2013-2014 influenza season. a nasopharyngeal swab was tested by the stanford hospital virology laboratory with the esensor respiratory viral panel (genmark) to identify infecting virus. a blood sample was collected at the time of enrollment. after each vaccination to measure levels of neutralizing antibodies against a(h1n1)pdm09 by influenza virus neutralization assay as previously described [17] . a blood sample was collected at day 6-8 after vaccination for ppab studies. all studies were approved by the stanford institutional review board, and written informed consent was obtained from all participants. b cells were isolated from blood samples by using the rosettesep human b-cell enrichment cocktail (stemcell technologies) and cultured to collect ppabs [16] . enzyme-linked immunosorbent assays (elisas) were performed as described previously [17] . in brief, 96-well plates (greiner) were coated with purified, coldadapted influenza a(h1n1)pdm09 (kindly provided by dr h. jin of medimmune vaccines) at 10 6 fluorescent focus forming units per well or with recombinant ha from 293 cells (immune technology), matrix protein 1 (m1) from escherichia coli (immune technology), or nucleoprotein (np) from insect cells (imgenex) at 5 µg/ml. plates were blocked and then incubated with 10-fold serially diluted ppabs starting at 100-fold dilution. wells incubated with complete medium without human immunoglobulin were used to determine background. plates were washed and incubated with peroxidase-conjugated goat anti-immunoglobulin g (igg) γ antibody or goat anti-immunoglobulin a (iga) α antibody (kpl) and developed with tmb substrate (kpl). the od 450nm was measured. background was subtracted, and the area under curve (auc) of each serially diluted sample was calculated as described elsewhere [17] . hypothesis testing used 2-sample and paired-sample t tests, as indicated. for comparison of immune priming by influenza virus infection versus iiv immunization, because the 2 data sets to be compared had some participants in common, comparisons of means used perturbation resampling [18] with bias correction [19] to estimate p values. sequential bonferroni adjustment [20] was used to adjust for multiple comparisons across all hypothesis tests in each figure. analyses were performed in sas v. 9.4 (sas institute). code is available upon request. hypothesis tests were declared statistically significant for p < .05. during the 2013-2014 influenza season, we enrolled 12 patients with acute ili (table 1) , including 9 patients infected with a(h1n1)pdm09, 1 infected with influenza b virus, 1 infected with metapneumovirus, and 1 infected with coronavirus. blood samples were collected on the enrollment day, from 2 to 8 days after the onset of illness. ppabs were derived from samples [16] and tested by elisa for binding reactivity to a(h1n1)pdm09 ( figure 1a and 1b). for control, we used a pool of ppabs derived from blood samples collected on days 6-8 after vaccination from a group of recipients of the 2011 iiv, which contained a(h1n1)pdm09. a(h1n1)pdm09specific binding was detected in the ppab pool from the iiv recipients and from individual ppab samples from 6 of the 7 a(h1n1)pdm09-infected patients who presented at day 4 or later after symptom onset ( figure 1b) . binding was not detected in the ppab samples collected from a(h1n1)pdm09-infected patients on days 2 or 3 or from patients infected with influenza b virus, metapneumovirus, or coronavirus. in a(h1n1)pdm09reactive ppab samples from a(h1n1)pdm09-infected patients, the binding activity of igg was significantly higher than that of iga (p = .01, by the paired t test). these results suggest that, in patients infected with a(h1n1)pdm09, substantial virus-specific plasmablast responses are detectable in the blood 4 days after symptom onset and that the igg response is dominant. next, we examined the igg binding of ppab to 3 individual influenza virus proteins: ha of a(h1n1)pdm09 and np and m1 of influenza a(h1n1). substantial binding to ha was detected in 5 of 6 ppab samples from a(h1n1)pdm09-infected patients ( figure 2a ). np-specific binding was detected in all 6 patient samples, whereas m1-specific binding was detected in only 3. in contrast, the iiv ppab pool only bound to the ha antigen but not to np or m1. the ppab from the influenza b virus-infected patient did not bind any of the influenza a virus proteins ( figure 2a ). we then compared ppab reactivity to the 3 influenza virus proteins between the a(h1n1)pdm09-infected patients and a group of 15 iiv recipients. since the precise kinetics of the peripheral plasmablast response in influenza virus infection are not known, the observed reactivity of ppab samples collected on different days after disease onset might not represent the peak plasmablast response. therefore, instead of comparing the reactivity to each influenza virus protein directly, we normalized the np and m1 binding activity to ha reactivity, since ha is the primary antigenic target of iiv. these normalized reactivities provide information about the relative pattern of ppab responses to different influenza virus proteins. as shown in figure 2b , the normalized np reactivity was significantly higher in the a(h1n1)pdm09-infected patients than in the iiv recipients. the mean of normalized binding activity for m1 was also higher in the infected patients than in the iiv recipients, but the difference was not statistically significant, perhaps because of the small sample size and large variability among the infected patients. taken together, these results indicate that, in addition to a b-cell response to the variable ha, patients infected with a(h1n1)pdm09 developed b-cell responses to the conserved np that were significantly higher than those mounted by iiv recipients despite the fact that iiv preparations contain large amounts of np [21, 22] . we tested the ppab samples from a(h1n1)pdm09-infected patients and iiv recipients for their ability to bind full-length ha proteins of 3 influenza a virus strains: the homotypic infecting a(h1n1)pdm09 strain ( ph1), the heterovariant a/ brisbane/59/2007(h1n1) strain (sh1), and the heterosubtypic avian a/vietnam/1203/2004(h5n1) strain (h5) and to the ha2 peptide of h5. each a(h1n1)pdm09-infected ppab sample with detectable ph1 binding also bound to sh1 and h5 ( figure 3a ). in addition, these ppabs bound to the ha2 portion of h5, which contains the conserved major antigenic site of the stalk domain. in agreement with our previous reports [17, 23] , the iiv ppab pool also bound the heterovariant sh1. these results indicate that a cross-reactive ppab response to heterovariant and heterosubtypic has was induced in patients with acute a(h1n1)pdm09 infection and that cross-reactivity was in part mediated by the binding to the conserved ha stalk domain. next we normalized the sh1 and h5 reactivity to the homotypic ph1 reactivity and compared these normalized crossreactivities of ppabs from infected and iiv immunized groups. as shown in figure 3b , the normalized sh1 and h5 reactivities were both significantly higher in the infected patients than in the iiv recipients, indicating that plasmablast responses to influenza virus infection had greater relative cross-reactivity against heterovariant and heterosubtypic has than those induced by iiv. for this analysis, we identified a subset of 43 individuals aged 18-30 years who did not receive a monovalent a(h1n1)pdm09 vaccine in 2009 (by self-report) in our cohort of healthy iiv recipients. before the a(h1n1)pdm09 pandemic, individuals younger than 30 years had little a(h1n1)pdm09-reactive serum antibodies [24] . at the time of enrollment, 26 of 43 individuals had detectable serum neutralizing antibodies against a(h1n1)pdm09 with a geometric mean titer of 84 (range , suggesting that they had been previously infected with wild-type a(h1n1)pdm09. seventeen subjects were seronegative (titer lower than 10) for a(h1n1)pdm09. all 43 individuals were immunized for the first time with a(h1n1)pdm09-containing trivalent iiv in 2010/2011 or 2011/2012 by either intramuscular or intradermal injection. of note, no detectable differences in the frequency of vaccinespecific antibody secreting cells or in vaccine-specific ppab reactivity were observed in samples from patients vaccinated intramuscularly versus those vaccinated intradermally (data not shown). the iiv from both years had identical strain composition and contained a(h1n1)pdm09 as their h1n1 component. we compared the b-cell responses after iiv immunization in baseline a(h1n1)pdm09-seronegative and a(h1n1)pdm09seropositive individuals by measuring the ppab reactivity to the homotypic ph1, heterovariant sh1, and heterosubtypic h5 proteins. significant differences were not detected in the binding to ph1, sh1, or h5 between the seronegative and seropositive individuals ( figure 4a ), suggesting that previous natural infection with a(h1n1)pdm09 did not result in detectable differences in the plasmablast response to homotypic or heterovariant has after iiv immunization. of note, the mean ppab binding to the homotypic ph1 was higher in the seropositive individuals than the seronegative individuals, although the difference was no longer significant after adjustment for multiple comparisons. in the subsequent 2012-2013 influenza season, 18 of 43 2010 or 2011 iiv recipients received the 2012 iiv, which contained the same a(h1n1)pdm09 component. comparison of responses to the first versus the second iiv immunization as assessed by ppab reactivity to the 3 ha proteins reflects the priming effect of inactivated a(h1n1)pdm09 vaccine (the first immunization) on the b-cell response to subsequent vaccination with the same vaccine (the second immunization). ppab reactivities to ph1, sh1, and h5 were all significantly lower after the second iiv immunization than after the first immunization ( figure 4b ). in agreement with these results, the levels of a(h1n1)pdm09specific serum neutralizing antibodies increased significantly after the first and second iiv immunization, but the foldincrease of titers after the second immunization was significantly lower than that after the first (supplementary 1). therefore, priming with inactivated a(h1n1)pdm09 vaccine reduced the plasmablast response to a subsequent immunization with the same vaccine. finally, we compared the fold difference in ppab reactivity between the seronegative and seropositive subjects ( figure 4a ) and the fold difference between the first and second iiv immunizations ( figure 4b ). these differences represent the priming effects of a(h1n1)pdm09 infection versus inactivated a(h1n1)pdm09 immunization, respectively. as shown in figure 4c , the priming effects were significantly different for ppab reactivity to ph1 and sh1 but not to h5. taken together, these results show that priming with inactivated a(h1n1)pdm09, but not with a(h1n1)pdm09 infection, results in significantly reduced plasmablast responses to a subsequent immunization with the same inactivated a(h1n1)pdm09 vaccine. we observed a vigorous b-cell response in patients acutely infected with a(h1n1)pdm09, as demonstrated by broad ppab reactivity against select a(h1n1)pdm09 structural proteins h5 (h5-ha2) . b, igg binding activity for sh1 and h5 were normalized to that for ph1. the a(h1n1)pdm09-infected patient ppabs with detectable binding to ph1 (n = 5) and ppabs from the 15 randomly selected individual iiv recipients were included for this analysis. horizontal bars indicate geometric mean of the area under the curve (auc) ratio. the p values were determined by unpaired t tests and adjusted by sequential bonferroni adjustment for multiple comparisons. the asterisks indicate a statistically significant difference after the adjustment. and against has derived from a heterovariant h1n1 strain and a heterosubtypic avian h5n1 strain. compared with the b-cell response in iiv recipients, influenza virus infection elicited greater b-cell responses to the conserved np, as well as to the heterovariant and heterosubtypic has. we also identified distinct priming effects after infection versus iiv immunization: wild-type a(h1n1)pdm09 infection did not affect the antigen recall response to a subsequent immunization with an iiv containing the same strain-specific ha, whereas iiv immunization resulted in significantly reduced homotypic and cross-reactive ppab responses after a subsequent immunization with the same iiv strain. on day 7 after vaccination with either iiv [25] [26] [27] or laiv [28, 29] (and unpublished data), specific plasmablasts reach their peak concentration in the circulation. in a recent human challenge study with wild-type influenza virus, virus-specific plasmablasts were detected at day 7 after infection [30] . this study did not, however, examine the kinetics of the infectioninduced plasmablast response or the antibody specificities of these plasmablasts. the kinetics of the b-cell response in naturally infected patients is likely to be more variable than the kinetics in volunteers who receive standardized vaccines or who are experimentally challenged with wild-type influenza virus preparations at a prespecified time and dose. this supposition is supported by the report that plasmablast-derived influenza virus-specific monoclonal antibodies were isolated from influenza virus-infected patients 9-31 days after the onset of symptoms [13] . in our current study, we detected substantial ppab responses in infected patients for several days after the onset of symptoms, although multiple sampling was not performed to identify the peak day. other limitations of the current study are the lack of randomization in the comparisons of infected versus vaccinated individuals and the seronegative versus seropositive individuals, and the small number and wide age range and underlying diseases of the infected patients examined. the only elderly patient ( patient 1) had substantial ppab reactivity to np but not ha; this differed from the response of all the younger patients. we previously reported a weaker plasmablast response in elderly subjects than in younger individuals after immunization with iiv, and ha is the primary antigenic component in this vaccine [31] . it is not clear whether the lack of a detectable ha-specific ppab reactivity in this elderly patient was due to off-peak sample collection or to a generally reduced ha-specific response. thus, we suggest that the kinetics of the plasmablast response, including its quantitative because the 2 data sets (in panels a and b) had some subjects in common, analysis used perturbation resampling [18] , which is essentially a smoothed bootstrap. the p values were adjusted by sequential bonferroni adjustment for multiple comparisons across all 9 tests in the figure. the asterisk indicates a statistically significant difference after the adjustment. and qualitative characteristics over the disease course, should be addressed in future studies of defined subject populations experimentally challenged with wild-type virus. in addition to plasmablasts specific for the ha antigen of the infecting virus, natural a(h1n1)pdm09 infection elicited a greater plasmablast response to the conserved np and to has from a heterovariant h1n1 strain and an avian h5n1 strain than iiv immunization. recently, we reported that laiv also induces proportionally greater plasmablast responses to np and variant ha proteins than iiv [17] . our findings suggest that laiv immunization recapitulates some distinct qualitative characteristics of wild-type influenza virus infection in terms of b-cell response, but at a much lower magnitude. thus, wildtype influenza virus and laiv may induce protective immunity through similar mechanisms that are distinct from those of iiv. of note, it has been proposed that antibodies targeting conserved virus components, including the stalk domain of ha from the group 1 influenza a viruses [32, 33] and the np [22] , have potential as the basis of universal influenza vaccines that would offer protection against a broad range of seasonal and pandemic influenza viruses. this is consistent with the data showing that laiv is superior to iiv in inducing crossprotection [3] . the basis for differences in b-cell responses to influenza virus infection and iiv immunization is currently unknown and should be addressed in future studies that include analyses of immunoglobulin gene sequences and antibody functions of the activated b-cell repertoire at the clonal level [34] and also, of course, t-cell responses. elucidating different aspects of immune responses to natural influenza virus infection and identifying the immune correlates with protective immunity will be important for development of next-generation influenza vaccines. a recent influenza cohort study revealed high rates of asymptomatic natural influenza virus infection in the community; infection was only detected by strain-specific seroconversion [35] . it will be interesting to determine whether such asymptomatic influenza virus infection differs from symptomatic cases or from immunization with laiv in terms of protective immune responses and to determine the factors that affect the outcome (symptomatic vs asymptomatic) of natural infection. this information could lead to a new approach for influenza vaccine development that is based on reducing influenza pathogenicity while preserving immunogenicity. because of the repeated exposure to influenza virus infection and vaccination, the b-cell response to influenza vaccination is an antigen recall response in almost all individuals [25] , except for very young children who are naive to influenza virus infection and vaccination. activation of antigen-specific b cells occurs in germinal centers of lymphoid tissues with the help of antigen-specific follicular helper t-cells and results in generation of plasmablasts and switched memory b cells that express high-affinity antibodies [36, 37] . exposure to a newly emerged strain, such as a(h1n1)pdm09, primes strain-specific b cells and t-cells to generate memory cells that in turn alter the b-cell response to a subsequent vaccination or infection. in the current study, we showed that priming with iiv resulted in diminished strain-specific recall responses to a subsequent iiv immunization, whereas such suppression was not observed in individuals with prior natural a(h1n1)pdm09 infection. laiv immunization also does not result in detectable differences in the serum antibody and plasmablast responses to iiv or laiv immunization in the subsequent year [29] . a recent study found that immunization with a live, attenuated avian h5n1 influenza vaccine, which stimulated virtually no serum antibodies, effectively primed the immune system, resulting in a robust serum antibody response to a subsequent immunization with an inactivated h5n1 vaccine including broader cross-reactivity, compared with repeated immunization with the inactivated vaccine alone [38] . in mice primed with a t-cell-dependent antigen, a second immunization with an epitope-matched t-cell independent antigen results in induction of tolerant memory b cells rather than a recall response [39] . a substantial cd4 + t-cell response was detected in volunteers experimentally infected with wild-type influenza viruses [40] . in another human study a specific subset of icos + cxcr3 + cxcr5 + cd4 + follicular helper t-cells correlated with the induction of antibodies to iiv in previously primed individuals but not in naive subjects [41] . thus, deficiencies in the magnitude or functional properties of the cd4 + t-cell repertoire specific for iiv are likely a contributing factor to the reduced b-cell response in repeated iiv immunization. the efficacy of repeated iiv immunization has been an issue of debate for some time [42] [43] [44] [45] [46] . however, this controversy has not previously focused specifically on the generation of humoral immunity in recipients of repeated iiv immunization in comparison to those previously primed by natural infection. since annual immunization with iiv is common practice, the implication of our findings to the long-term effectiveness of influenza vaccination needs to be carefully evaluated, especially in the context of maximizing protective immunity in vulnerable populations, such as elderly individuals. supplementary materials are available at the journal of infectious diseases online (http://jid.oxfordjournals.org). supplementary materials consist of data provided by the author that are published to benefit the reader. the posted materials are not copyedited. the contents of all supplementary data are the sole responsibility of the authors. questions or messages regarding errors should be addressed to the 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'the hoskins' paradox' revisited variable efficacy of repeated annual influenza vaccination impact of repeated vaccination on vaccine effectiveness against influenza a(h3n2) and b during 8 seasons acknowledgments. we thank our study subjects, for their participation; c. zhang, for technical assistance; b. pinsky, for performing the diagnostic pcr assay; h. jin, for providing purified a(h1n1)pdm09; s. mackey, for coordinating the clinical study; s. swope, n. mastman, t. trela, and m. ugur, for enrolling subjects, administering vaccine, and collecting b-cell response to acute influenza • jid 2015:211 (1 april) • 1057 samples and clinical data; and a. goel, t. quan, r. fleischman, s. batra, and i. chang, for screening and scheduling subjects and providing regulatory and clinical data management support.financial support. this work was supported by the national institutes of health (nih; grants ai090019, ai057229, ai089987, p01ai097092, and u19ai089987); the national center for research resources, nih (clinical and translational science award ul1rr025744); and the center for research on influenza pathogenesis (a national institute of allergy and infectious diseases-funded center of excellence for influenza research and surveillance; contract hhsn272201400008c).potential conflicts of interest. h. b. g. is on the scientific advisory board of novartis vaccines, a major producer of influenza vaccines, and a consultant for vaxart and paxvax, both of which are developers of novel influenza vaccines. the icahn school of medicine owns intellectual property in the area of influenza vaccines for which a. g.-s. is an inventor. all other authors report no potential conflicts.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-000161-hxjxczyr authors: rello, jordi; pop-vicas, aurora title: clinical review: primary influenza viral pneumonia date: 2009-12-21 journal: crit care doi: 10.1186/cc8183 sha: doc_id: 161 cord_uid: hxjxczyr primary influenza pneumonia has a high mortality rate during pandemics, not only in immunocompromised individuals and patients with underlying comorbid conditions, but also in young healthy adults. clinicians should maintain a high index of suspicion for this diagnosis in patients presenting with influenza-like symptoms that progress quickly (2 to 5 days) to respiratory distress and extensive pulmonary involvement. the sensitivity of rapid diagnostic techniques in identifying infections with the pandemic 2009 h1n1v influenza strain is currently suboptimal. the most reliable real-time reverse transcriptase-polymerase chain reaction molecular testing is available in limited clinical settings. despite 6 months of pandemic circulation, most novel h1n1v pandemic strains remain susceptible to oseltamivir. ensuring an appropriate oxygenation and ventilation strategy, as well as prompt initiation of antiviral therapy, is essential in management. as the novel swine-origin influenza a (h1n1)v global pandemic is under way, the medical community has already experienced an increase in hospitalizations from influenzarelated complications in many geographic regions. primary viral pneumonia is recognized as the most severe pulmonary manifestation of influenza. while uncommon during seasonal epidemics, the syndrome has been well documented during the h2n2 pandemic of 1957-1958 and is thought to be responsible for much of the mortality associated with the young healthy adult population during the 1918 h1n1 pandemic [1] . this paper reviews the clinical aspects of influenza and primary influenza pneumonia that may be of most interest to the practicing physician in the 2009 pandemic environment. seasonal influenza epidemics occur each year as a result of minor changes in the antigenic characteristics of the hemagglutinin and neuraminidase glycoproteins of the influenza viruses (antigenic drift) [2] . the morbidity and mortality associated with seasonal influenza outbreaks are significant, especially in older patients, who incur more than 90% of the influenza-related mortality each year [3] . factors contributing to their increased vulnerability include a decline in cellmediated and humoral immune responses, a reduction in lung compliance and respiratory muscle strength, a diminished cough reflex associated with normal aging, the frequent presence of multiple comorbid conditions, nutritional deficiencies, and in the case of residents of long-term care facilities, greater exposure risk due to close living quarters and shared caregivers [4, 5] . influenza pandemics occur less frequently, as a result of major changes in the surface glycoproteins of the virus (antigenic shift). the emerging novel influenza strain then easily spreads into an immunologically susceptible population. consequently, pandemics are characterized by a shift in mortality toward the otherwise young and healthy 18to 35-year-old adults, with relative sparing of older patients, as evidenced by epidemiological analyses of the 1918 influenza a pandemic [6] . this is likely due to the persistence of immunological memory in older patients after previous exposures to h1-type viruses similar to the pandemic strain [7, 8] . the virulence of the pandemic strain may also play a role, as demonstrated by recent experiments with the highly fatal 1918 influenza strain [9] . preliminary data from the 2009 h1n1 pandemic suggest a similar shift in age-related mortality. an analysis of 532 cases of 2009 pandemic h1n1 influenza a in the us, for example, has revealed that 60% of the cases occurred in patients not older than 18 years of age and that only 5% occurred in patients older than 50 years [10] . in the cohorts recently tested, the modest extent of immunological memory in older patients was confirmed by the presence of serum crossreactive antibodies to the pandemic h1n1 influenza a strain found in 33% of the adults older than 60 years of age versus 6% to 9% of the adults 18 to 64 years of age and none of the children [11] . influenza attack rates during seasonal epidemics vary between 10% and 20% but can be much higher during pandemics. for example, an analysis of the pandemic 2009 h1n1 influenza a outbreak in la gloria, veracruz, found clinical attack rates of 29% in adults older than 15 years and 61% in children younger than 15 years of age [12] . however, these rates may be different in geographic areas of low population density. groups at high risk for severe disease and complications secondary to 2009 pandemic h1n1 influenza a include patients with underlying pulmonary (asthma) and cardiac comorbid conditions, some immunosuppressive states, pregnancy and post-partum states, diabetes mellitus, obesity [13, 14] , and, in children, prior neurological disabilities [15] . severe primary h1n1 influenza pneumonia can also affect young adults without any underlying comorbidities [14] . person-to-person transmission occurs primarily through droplet spread via small particle-sized aerosols generated by coughing, sneezing, or talking [16] . airborne transmission should be considered in those patients exposed to aerosolgenerating techniques, such as intubation or mechanical ventilation. the incubation period is usually 24 to 48 hours. in the absence of antiviral treatment, viral shedding starts within 24 hours before the onset of symptoms and continues for approximately 5 days in healthy adults [17] . viral shedding can last longer in children, patients with extensive comorbidities, older patients, patients who undergo mechanical ventilation, and immunocompromised hosts [18] [19] [20] . the infectious period can be significantly reduced by the use of antiviral medications within the first 48 to 96 hours of illness [20] . after inhalation, the virus is deposited onto the respiratory tract epithelium, where it attaches to ciliated columnar epithelial cells via its surface hemagglutinin. local host defenses, such as mucociliary clearance, or secretion of specific secretory iga antibodies can remove some of the virus particles. however, if mucociliary clearance is impaired (as in smokers [21] or older patients [22] ) or secretory antiinfluenza iga antibodies are absent (as in no antecedent exposure to the virus), infection continues unabated [23] . respiratory epithelial cells are invaded, and viral replication occurs. newer viruses then infect larger numbers of epithelial cells, shut off the synthesis of critical proteins, and ultimately lead to host cell death [24] . in patients with uncomplicated influenza, bronchoscopy typically reveals diffuse inflammation and edema of the larynx, trachea, and bronchi, and biopsy may show cellular infiltration with lymphocytes and histocytes and desquamation of the ciliated columnar epithelium [25] . in patients with severe influenza infections that progress to primary viral pneumonia, the involvement of the respiratory tree is extensive, with necrotizing tracheobronchitis, ulceration and sloughing of the bronchial mucosa [26] , hyperemic alveolar capillaries with intra-alveolar hemorrhage, infiltration of alveolar spaces with fluid, fibrin, and cellular exudates, and lining of the alveoli with acellular hyaline membranes [1] . autopsies from patients with primary influenza pneumonia confirmed bilateral severe hemorrhagic pneumonitis with interstitial inflammation, diffuse alveolar damage, and heavy viral loads observed in the periphery of the lungs. the clinical features of uncomplicated influenza are virtually indistinguishable from those of other respiratory viral infections. influenza is classically characterized by an abrupt onset of headache, high-grade fever, chills, dry cough, pharyngeal irritation, myalgias, malaise, and anorexia. the fever lasts an average of 3 days (range of 2 to 8 days). the cough, initially nonproductive and nonpurulent, may persist for weeks. bronchial hyper-reactivity and small-airway dysfunction are often present in influenza virus infection. in the presence of asthma or structural lung disease, wheezing may be a prominent manifestation [24] . vomiting and diarrhea, while rare in seasonal influenza, have been frequently reported in infections with the 2009 pandemic influenza a h1n1v strain [10] , particularly in children. the clinical presentation of influenza in the immunocompromised host may be more subtle and manifest only as coryza; similarly, the classic fever symptom may be absent in the older patient, who may present only with lethargy, confusion, anorexia, and cough [27] . influenza pneumonia and respiratory complications in patients with th1 defects, such as hiv infection, are uncommon. pneumonia and the acute respiratory distress syndrome (ards) account for the majority of severe morbidity and mortality that accompany pandemic influenza infection [14] . pneumonia may occur as a continuum of the acute influenza syndrome when caused by the virus alone (primary pneumonia) or as a mixed viral and bacterial infection after a delay of a few days (secondary pneumonia) [28] . identifying patients who are more likely to develop severe complications from influenza pneumonia requires a high clinical vigilance. commonly used pneumonia severity assessment tools, such as the pneumonia severity index [29] or curb65 [30] , are not useful in deciding which patients to hospitalize in the context of primary influenza pneumonia since these tools have not been developed and validated during a pandemic scenario. thus, careful triage in the emergency department and early identification of young patients with decreased oxygen saturation, respiratory rate above 25, concomitant diarrhea, or hypotension are crucial. elevated lactate dehydrogenase, creatine phosphokinase, and creatinine at hospital admission may also serve as prognostic indicators of severe disease [14] . c-reactive protein and procalcitonin are increased during this acute lung injury stage of early fibroproliferation. the most ominous cases are those infections that progress rapidly to ards and multilobar alveolar opacification. these patients usually present with gradually increasing dyspnea and severe hypoxemia after an antecedent of 2 to 5 days of typical influenza symptoms [14] . the cough is usually productive of thin, often bloody, sputum with few cells. hypoxemia increases progressively to the point of respiratory failure requiring intubation and mechanical ventilation, often after only one day of hospitalization [14] . the radiological appearance of primary influenza pneumonia can be difficult to distinguish on chest x-ray from pulmonary edema, given the presence of perihiliar congestion and hazy opacification, at least in the lower lobes (figure 1a,b) . pleural effusions may also be present. computed tomography scans ( figure 2) can add further diagnostic insight and may be useful to differentiate primary viral pneumonia from bronchiolitis and interstitial pneumonias, which occur frequently in children and young adults but have a benign outcome. concomitant myopericarditis should be excluded by echocardiography. concurrent pulmonary emboli, as suggested by early case reports from hospitalized patients with pandemic influenza a h1n1v 2009 in the us [13] , may further contribute to clinical deterioration in some patients. however, the occurrence of concomitant pulmonary emboli has not been reproduced in other geographic regions so far. bacterial co-infection, though uncommonly reported in the early stages of the 2009 h1n1 pandemic, may be more prevalent than initially thought. a recent analysis of lung specimens from 77 fatal cases of pandemic h1n1v 2009 infection found a prevalence of concurrent bacterial pneumonia in 29% of these patients [31] . the most common coinfecting bacterial pathogens were pneumococcus, staphylococcus aureus, and streptococcus pyogenes, with a median duration of illness of 6 days [31] . the real-time reverse transcriptase-polymerase chain reaction (rrt-pcr) swine flu panel for detection of pandemic h1n1 influenza, developed by the centers for disease control and prevention (atlanta, ga, usa) and distributed to many laboratories in us and worldwide, is a reliable and timely method of diagnosing the pandemic strain [32, 33] . the viral culture, while the gold standard in influenza diagnostics, takes several days before the results are known [24] . the direct fluorescent antigen influenza test was recently reported to have a sensitivity of 93% compared with the rrt-pcr [34] , but the test requires considerable technical expertise in addition to a fluorescent microscope. the commonly used point-of-care rapid influenza tests provide results in less than 1 hour but are of only modest sensitivity for seasonal influenza viruses (63%) [35] and unacceptably insensitive for the detection of pandemic h1n1 influenza [35, 36] . thus, for the majority of clinicians practicing during the 2009-2010 influenza pandemic, the access to a reliable and timely diagnostic modality may still be limited. as such, it is chest x-rays of a patient with primary h1n1 (swine-origin influenza a) influenza pneumonia on day 1 (a) and day 6 (b) of hospitalization. reassuring to know that the patients presenting during influenza epidemics with both cough and fever within the first 48 hours of symptom onset are very likely to have actual influenza (79% positive predictive value) [37] . the majority of patients with primary influenza pneumonia require ventilatory support. mortality is high but can be decreased with an optimal protective ventilatory strategy (tidal volume of not more than 6 ml per kilogram of predicted body weight, with a plateau airway pressure goal of not more than 30 cm h 2 o), as shown in acute respiratory distress syndrome network clinical trials; this strategy is therefore recommended in acute lung injury [38, 39] . maintaining an adequate fluid balance is also important for survival in acute lung injury. the hemodynamic status should be optimized by appropriate repletion of intravascular volume deficits during the early systemic inflammatory stage [40] . once acute lung injury has become established, a conservative fluid management protocol, which was associated with beneficial effects in clinical trials, should be considered [41, 42] . in severe refractory cases of primary influenza pneumonia, some patients require venovenous extracorporeal membrane oxygenation support and continuous renal replacement for acute renal failure. antiviral treatment should be initiated as soon as possible, particularly in patients at high risk of complications. the majority of treatment benefits are derived when antivirals are initiated within the first 48 hours from onset of symptoms. unfortunately, most patients with primary viral pneumonia receive oseltamivir after 3 to 8 days of influenza onset [14] . however, the experience with seasonal influenza suggests that a reduction in mortality for hospitalized patients has been documented even when oseltamivir was initiated after the first 48 hours following illness onset [43] . thus, being out of the ideal therapeutic window should not be a reason to withhold antiviral treatment at any stage of active disease. both neuraminidase inhibitors (oseltamivir and zanamivir) are active against the novel h1n1v 2009 pandemic influenza a strain. the recommended adult dose for oseltamivir, considered the first-line therapy for h1n1 influenza infection, is 75 mg orally twice a day for a total of 5 days [44] . dose adjustment may be required in the presence of reduced creatinine clearance, but the dosage should be maintained for patients undergoing continuous venovenous hemodialysis. a recent world health organization treatment guideline for pharmacological management of 2009 pandemic h1n1v influenza a recommends the consideration of higher doses of oseltamivir (150 mg twice a day) and longer duration of treatment for patients with severe influenza pneumonia or clinical deterioration [44] . since hospitalized patients can shed influenza virus for prolonged periods of time, extending antiviral treatment beyond the first 5 days of treatment in cases of persistent influenza symptoms may be necessary. however, clear guidelines for these circumstances have not been established, and clinical trials examining the appropriate treatment dose and duration for severe h1n1 influenza in various patient populations are acutely needed. development of oseltamivir resistance in novel h1n1 influenza, though still exceedingly rare, has been reported from several countries [45] . it should be suspected in patients who remain symptomatic or have evidence of viral shedding despite a full treatment course of oseltamivir. immunosuppression and prior exposure to oseltamivir, such as receipt of prolonged post-exposure prophylaxis, increase the risk for oseltamivir resistance [45] . zanamivir remains an effective therapeutic option for these cases. zanamavir is also indicated in the rare circumstance when an oral route for oseltamivir administration is not available for critically ill patients in the intensive care unit. the risk of bronchospam rarely associated with zanamivir, particularly in patients with underlying reactive airway disease, can be minimized by concurrent bronchodilator administration. adamantanes (amantadine and rimantadine) have no activity against the 2009 influenza a h1n1v pandemic strain. they are effective for seasonal h1n1 influenza strains, which are 100% resistant to oseltamivir. therefore, for patients presenting with primary influenza pneumonia in geographic regions where seasonal h1n1 strains are circulating in addition to the novel h1n1 pandemic strain, amantadine or computed tomography scan of the patient with primary h1n1 (swineorigin influenza a) influenza pneumonia whose chest x-rays appear in figure 1 . rimantadine should be added to oseltamivir [46] . rimantadine is also associated with immunomodulatory effects. patients presenting with severe influenza pneumonia who may have concurrent bacterial superinfection should also receive antibacterial agents effective against the most common etiologic pathogens, such as streptococcus pneumoniae, streptococcus pyogenes, and staphylococcus aureus, including methicillin-resistant staphylococcus aureus, according to published guidelines in the management of communityacquired pneumonia [47] . corticosteroids remain controversial in persistent ards and are not routinely recommended [48] . further research is required to clarify their impact on outcome. whether other adjunctive immunomodulatory therapies such as statins, chloroquine, and fibrates could prove useful in the context of an influenza pandemic [49] remains to be determined. primary influenza pneumonia caused by the 2009 pandemic influenza a h1n1v strain, though rare, carries a high mortality. the rapid progression from initial typical influenza symptoms to extensive pulmonary involvement, with acute lung injury, can occur both in patients with underlying respiratory or cardiac morbidities and in young healthy adults, especially if obese or pregnant. prompt initiation of effective antiviral treatment, appropriate oxygenation and ventilation support, and antibacterial treatment in the case of concurrent bacterial pneumonia are critical for survival. the most reliable and timely diagnostic method for 2009 pandemic influenza a h1n1v infection is the rrt-pcr developed by the centers for disease control and prevention. common 'point-of-care' rapid influenza tests are very insensitive. a negative test result in a patient with clinical symptoms compatible with influenza pneumonia does not accurately rule out influenza and should not be a deterrent to prompt oseltamivir treatment during this current pandemic. further research is needed in order to identify the 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it be done? the patient whose radiological images appear in figures 1 and 2 has given written consent for their publication. the authors declare that they have no competing interests.this article is part of a review series on influenza, edited by steven opal.other articles in the series can be found online at http://ccforum.com/series/influenza key: cord-000724-lzhobnch authors: zhang, j.; while, a. e.; norman, i. j. title: seasonal influenza vaccination knowledge, risk perception, health beliefs and vaccination behaviours of nurses date: 2011-11-18 journal: epidemiol infect doi: 10.1017/s0950268811002214 sha: doc_id: 724 cord_uid: lzhobnch the relationship between knowledge, risk perceptions, health belief towards seasonal influenza and vaccination and the vaccination behaviours of nurses was explored. qualified nurses attending continuing professional education courses at a large london university between 18 april and 18 october 2010 were surveyed (522/672; response rate 77·7%). of these, 82·6% worked in hospitals; 37·0% reported receiving seasonal influenza vaccination in the previous season and 44·9% reported never being vaccinated during the last 5 years. all respondents were categorized using two-step cluster analyses into never, occasionally, and continuously vaccinated groups. nurses vaccinated the season before had higher scores of knowledge and risk perception compared to the unvaccinated (p<0·001). nurses never vaccinated had the lowest scores of knowledge and risk perception compared to other groups (p<0·001). nurses' seasonal influenza vaccination behaviours are complex. knowledge and risk perception predict uptake of vaccination in nurses. annual epidemics of seasonal influenza result in about 3-5 million cases of severe illness and 250 000-500 000 deaths worldwide [1] . healthcare workers (hcws) can be a key source for influenza transmission in communities and hospitals as they are exposed to both infected patients and high-risk groups [2, 3] . vaccination is the most effective way to prevent infection and severe outcomes [1] and the principal measure to reduce the impact of epidemics, such as hospitalization, mortality and morbidity [2, [3] [4] [5] . moreover, studies suggest that the vaccination of hcws has substantial economic benefits as well as health-related benefits, including reduced absenteeism from work and the extra costs of sick leave and staff replacement [4, 6, 7] . for the above reasons, the world health organization (who), united kingdom department of health (doh) [8] , united states centers for disease control and prevention (cdc), other healthcare professional organizations and many countries' government agencies [1, 9, 10] strongly recommend the annual seasonal influenza vaccination of hcws. however, studies suggest that influenza vaccine uptake in hcws is often low worldwide [11] [12] [13] [14] . for example, the overall seasonal vaccination rate in england for hcws was 26 . 4% for the 2009/2010 season [15] . nurses, as the group having the most patient contact, are more reluctant to be vaccinated than other hcws [16] [17] [18] [19] [20] [21] [22] [23] . although predictors influencing nurses' vaccination practices have been identified to some extent regarding knowledge and risk perception [16] [17] [18] [19] [23] [24] [25] [26] [27] , further studies are needed to explore the influences on nurses' attitudes and practices regarding influenza vaccination and to identify the major influencing factors for their vaccination behaviours. this study aimed to examine the relationship between knowledge, risk perceptions, health beliefs towards seasonal influenza and vaccination and the vaccination behaviours of nurses. a cross-sectional survey was conducted of qualified nurses between 18 april and 18 october, 2010. qualified nurses attending continuing professional education courses at a large university in central london were invited to participate in the study. potential respondents were given a study information sheet and a questionnaire by the investigator. completed questionnaires were collected immediately by the investigator or returned by mail to the research team using freepost addressed envelopes. questionnaire completion was anonymous so that it was not possible to follow up non-response. ethical approval was obtained from the university ethics committee. the questionnaire collected the following data : (1) knowledge about seasonal influenza and vaccination (22 items requiring true, false or unsure responses) included five dimensions to assess general information, severity of influenza, influenza vaccination, high-risk groups and vaccination-recommended groups; (2) risk perception (12 items with a 4-point likert scale) towards influenza and pandemic with three dimensions (i.e. personal vulnerability to illness, negative consequences of contracting influenza and severity of influenza) ; (3) health locus of control including internal, chance and powerful others dimensions assessed by the multidimensional health locus of control (mhlc) scales [28] (18 items) ; (4) vaccination behaviours (nine items) including vaccination status (whether respondents had been vaccinated in the previous season), vaccination intent (whether respondents intended to be vaccinated next season) and vaccination history (how many times respondents had been vaccinated in the last 5 years) ; (5) reasons for accepting or refusing vaccination using two open questions; and (6) demographic characteristics (10 items) including gender, age group, highest educational qualification, place of work, clinical speciality, year of qualification as a nurse and whether or not respondents had direct patient contact. the cronbach's a-coefficients for the three newly developed scales (sections 1, 2, 4) ranged from 0 . 701 to 0 . 763 and principal components analysis produced a good fit and confirmed the internal design of the instrument. statistical analysis was performed using spss version 15.0 (spss inc., usa). the x 2 test or fisher's exact test was used to explore the statistical differences between categorical variables. the independentsamples t test was used to compare statistical difference between continuous variables in two groups. the one-way between-groups analysis of variance (anova) was used to explore the differences between more than two groups. logistic regression was performed to explore the impact of the variables on vaccination status. the two-step cluster analysis procedure was performed to explore the natural groupings (i.e. clusters) within the respondents. the clustering criterion was that the solution had smaller values of schwarz's bayesian information criterion (bic), a reasonably large ratio of bic changes and a large ratio of distance measures. a p value <0 . 05 was considered to denote statistical significance. in total, 672 questionnaires were distributed and 522 were returned representing a response rate of 77 . 7%. the characteristics of the respondents are summarized in table 1 . overall 188/508 respondents (37 . 0%) reported receiving a vaccination in the previous season with 44 . 9% never receiving a vaccination during the last 5 years. there was no difference in the demographic characteristics of the vaccinated or unvaccinated respondents in the previous season. the number of years qualified as a nurse for the two groups were 11 . 99¡9 . 085 years and 11 . 89¡8 . 624 years (p=0 . 898), respectively. comparison of knowledge and risk perception scores and sub-scores of mhlc are summarized in table 2 . there were significant differences in knowledge scores and risk perception between the vaccinated and unvaccinated nurses and between those with vaccination intent, no intent or unsure. there was no significant difference in the sub-scores of mhlc between the vaccinated and unvaccinated (data not shown in table) but there was a significant difference for the sub-score of powerful others between those groups with different vaccination intent. direct logistic regression was performed to assess the impact of a number of factors on the likelihood that respondents had been vaccinated in the previous season. the model contained five independent table 3 , only two of the independent variables made a unique statistically significant contribution to the model (knowledge score and risk perception score). the strongest predictor of vaccination status was the risk perception score, recording an odds ratio of 1 . 76, indicating that respondents who had higher risk perception scores were >1 . 76 times more likely to have been vaccinated in the last 12 months than those with lower scores, controlling for all other factors in the model. knowledge score with an odds ratio of 1 . 05 indicated that knowledgeable respondents were more likely to be vaccinated than the unknowledgeable, controlling for other factors in the model. the two-step cluster analysis procedure was used to explore the natural groupings within the respondents. first, the auto-clustering exploratory analysis was performed using the categorical variables of vaccination status, vaccination intent, vaccination history and the continuous variables of knowledge score and risk perception score. of the 522 respondents, 64 were automatically excluded from the analysis due to missing values on one or more of the variables. of the 458 respondents assigned to clusters, 195 (42 . 6%) were assigned to the first cluster, 143 (31 . subsequently the analysis was performed using the combined categorical variables of vaccination status in the previous season (=yes) and vaccination history and the continuous variables of knowledge and risk perception scores. the results were auto-clustered into four groups but not explainable. the procedure was repeated with the cluster number fixed to 2 due to the values of bic, ratio of bic changes and ratio of distance measures. of the total 188 vaccinated respondents, 12 were excluded due to missing values. of the remaining 176 respondents, 107 (60 . 8%) were assigned to cluster 1 and 69 (39 . 2 %) to cluster 2. vaccinated cluster 1 comprised those vaccinated only in the previous season, i.e. the newly vaccinated group and vaccinated cluster 2 contained those vaccinated in the previous season who had more than one previous vaccination, i.e. the continuously vaccinated group. then, the same analysis was repeated for the unvaccinated respondents and two clusters emerged, i.e. unvaccinated cluster 1 (never vaccinated) and unvaccinated cluster 2 (used to be vaccinated). the analysis had therefore separated the respondents into reasonable categories. a comparison of variables across all clusters revealed that the never vaccinated had the lowest knowledge score, risk perception score and powerful others sub-score of mhlc compared to the other clusters (p<0 . 001, p<0 . 001, p=0 . 020, respectively) and this difference was statistically significant. for the vaccinated, there were no significant differences across any variable for the newly vaccinated and continuously vaccinated clusters although there was a trend of higher average scores for knowledge and risk perception in the newly vaccinated cluster compared to those of the other clusters (p=0 . 652, p=0 . 288, respectively). for the unvaccinated, there were no statistically significant differences across the variables except for the mhlc 'powerful others ' sub-score (p=0 . 008). further comparisons were performed to explore whether there were differences across the different items of knowledge and risk perception in the clusters. in the clusters of never vaccinated, other vaccination history and vaccinated with intent, there were significant differences in knowledge related to general information, high-risk groups and vaccination of recommended groups with p values of <0 . 001, <0 . 003 and <0 . 006, respectively. on average those never vaccinated had the lowest score while those vaccinated with intent had the highest scores across all knowledge items. for only one item of risk perception, i.e. personal vulnerability to illness, was there a significant difference between the clusters of never vaccinated and other vaccination history and between never vaccinated and vaccinated with intent (p<0 . 000 respectively). those never vaccinated had the lowest average score. there was no statistically significant difference in the knowledge and risk perception item scores between the two vaccinated clusters. however, the newly vaccinated usually had higher scores than those of the continuously vaccinated except for one item, i.e. the vaccination of recommended groups. similarly, for the two unvaccinated clusters there was no difference for knowledge scores, but there was a significant difference in one risk perception item, i.e. personal vulnerability to illness (p=0 . 001). those never vaccinated had a lower score for this item than those who used to be vaccinated and they were also less knowledgeable compared to the other group. tables 4 and 5 . in this study, the seasonal influenza vaccination rate in nurses was 37 . 0 % which is higher than previous reports of vaccination coverage ranging from 14 . 3-26 . 4% in hcws in uk [12, 29, 30] and 16% in nurses reported by chalmers [27] and similar to o'reilly et al.'s reported vaccination coverage of nurses in elderly care units [19] . this higher vaccination rate might be explained to some extent by the uk media reports of the risk of seasonal influenza and h1n1 pandemics in 2009 which may have increased the sample nurses' risk perception towards influenza and consequently changed their vaccination decisions as noted in a previous study [31] . this study found that vaccination behaviours in nurses were more complex requiring an analysis of both vaccinated and unvaccinated nurses' behaviours. more levels of vaccination behaviours existed in the sample with the two-step cluster analysis revealing three whole population clusters, i.e. those never vaccinated, those vaccinated this season with intent next year, and those with other vaccination history. two clusters, the newly vaccinated and continuously vaccinated, were identified for the vaccinated group and another two clusters, never vaccinated and used to be vaccinated, were identified in the unvaccinated group. to improve the influenza vaccination rates in nurses, it may be helpful to develop different strategies which target the nurse groups of the never vaccinated and the occasionally vaccinated. we found that a lack of knowledge about influenza and vaccination was a strong predictor of nurses' vaccination behaviours, especially for those never vaccinated. this cluster had the lowest knowledge score, suggesting that increasing their knowledge might improve their vaccination behaviours. however, it seems there are 'persistent decliners ' who are in the 'habit ' of not having a vaccination. this suggests that future educational campaigns need to be persistent, durative, and intensive if their vaccination behaviours are to be modified. for those who had been vaccinated in the past but not in the current season, knowledge was also a predictor for their vaccination behaviours, which suggests that current vaccination campaigns have failed to address their misgivings about vaccination to maintain their compliance with the annual vaccination recommendation for hcws. between those occasionally vaccinated and continuously vaccinated, knowledge levels were not significantly different but the newly vaccinated in 2009 had on average higher knowledge scores than those continuously vaccinated. this may reflect an increase in their risk perceptions towards influenza due to widespread reporting of the risks in the media encouraging them to be vaccinated for the first time in their lives. this suggests that timing may be crucial to the success of vaccination campaigns making behaviour modification easier. future studies are required to explore the relationship between the content and timings of vaccination campaigns and nurses' first vaccination uptake. this study showed that the perception of personal vulnerability to illness was important in nurses making vaccination decisions. but perceptions of the negative consequences of contracting influenza and severity of influenza were not major factors, a finding which is consistent with findings of previous studies [16] . this suggests that future educational campaigns might be more effective if they focus on the negative personal consequences of contracting influenza and its sequelae rather than nurses' professional duty to protect patients or other vulnerable groups. additionally, the reasons which nurses gave for having vaccination focused upon their personal health motivation rather than a professional responsibility regardless of whether they were vaccinated or unvaccinated. concerns about the vaccine's side-effects and effectiveness or safety were the two most frequent reasons for not having a vaccination indicating continuing misconceptions about influenza vaccine in nurses. future educational campaigns may wish to consider providing targeted information to change these widespread myths in nurses. however, these concerns did not seem to influence vaccination decisions because both vaccinated as well as unvaccinated nurses noted these reasons against vaccination. it may be the case that 2 days of minor discomfort postvaccination is tolerable when set against a year's influenza protection. unvaccinated nurses reported 'no need ' as their reason not having a vaccination which is consistent with their low-risk perception of contracting influenza. the convenience of the vaccination programme was identified as an organizational reason highlighting the importance of easy access to vaccination to increase its coverage in nurses. our analysis of health locus of control data found that those never vaccinated had a lowest 'powerful others ' locus of control for their vaccination behaviours, indicating that they did not believe their health was something over which they had no control [32] . this pattern of health beliefs towards influenza vaccination is consistent with their low-risk perception of personal vulnerability to illness and 'no need ' as their reason refusing vaccination and may be an important factor for never vaccinated nurses. further studies are needed to explore what may influence this pattern of health locus of control in order to modify nurses' vaccination behaviours. some organizations have recently required mandatory seasonal influenza vaccination for hcws as a professional and ethical obligation to protect their patients' health [33, 34] . however, ethical issues have been raised with mandatory vaccination because, while promoting the interests of patients and employers, it challenges hcws' personal autonomy and freedom of choice [35, 36] . moreover, it has been suggested that vaccination is not the only avenue of influenza prevention and there are several other important measures that healthcare organizations may take to protect both patients and hcws [37] . further previous studies have also suggested that not all hcws support mandatory vaccination [38] . until mandatory influenza vaccination for hcws is accepted worldwide, continued efforts to improve nurses' vaccination behaviours will be required. this study has some limitations. first, there is possible selection bias of a convenience sample ; however, the broad range of qualified nurses together with a high response rate strengthen the results. the extent of bias is unknown especially regarding nurses not working in london or in different care settings. second, the survey relied on self-report vaccination data ; however, zimmerman et al. [39] found that selfreport data were reliable in comparison with medical records. third, the three factors explored relating to nurses' vaccination behaviours explained only 8 . 7-11 . 9% of the variance according to the logistic regression analysis (although it was statistically significant) and therefore our results cannot fully explain nurses' vaccination behaviours. additional predictors will need to be introduced into the model in future studies to fully explain nurses' vaccination behaviours. in conclusion, this study revealed that nurses' influenza vaccination behaviours are complex. knowledge and risk perception were identified as two predictors influencing nurses' vaccination decisions with the health belief pattern of 'less powerful others ' being an important predictor in the never vaccinated ; however, there are other influential factors which need to be identified in future studies. world health organization. world health organization (who) influenza (seasonal) factsheet n211 preventing nosacomial influenza by improving the vaccine acceptance rate of clinicians assessing the role of basic control measures, antivirals and vaccine in curtailing pandemic influenza : scenarios for the us, uk and the netherlands effects of influenza vaccination of health-care workers on mortality of elderly people in long-term care : a randomised controlled trial effectiveness of an influenza vaccine programme for care home staff to prevent death, morbidity, and health service use among residents : cluster randomised controlled trial organizational and environmental factors that affect worker health and safety and patient outcomes effectiveness of influenza vaccine in health care professionals : a randomized trial summary of flu immunisation policy centres for disease control and prevention. prevention and control of influenza. recommendations of the advisory committee on immunization practices (acip) prioritization strategies for pandemic influenza vaccine in 27 countries of the european union and the global health security action group : a review national seasonal influenza vaccination survey in europe influenza vaccination coverage rates in five european countries during season 2006/07 and trends over six consecutive seasons influenza vaccination among primary healthcare workers influenza vaccination acceptance among health-care workers : a nationwide survey influenza vaccination uptake monitoring on behalf of the department of health attitudes, knowledge and factors related to acceptance of influenza vaccine by pediatric healthcare workers correlation between healthcare workers' knowledge of influenza vaccine and vaccine receipt factors affecting nurses' decision to get the flu vaccine factors affecting influenza vaccine uptake among health care workers knowledge and attitudes about influenza vaccination amongst general practitioners, practice nurses, and people aged 65 and over influenza vaccination coverage among hospital personnel over three consecutive vaccination campaigns influenza vaccination rates and motivators among healthcare worker groups influenza vaccination in paediatric nurses : cross-sectional study of coverage, refusal, and factors in acceptance predictors of influenza vaccination amongst australian nurses impact of severe acute respiratory syndrome and the perceived avian influenza epidemic on the increased rate of influenza vaccination among nurses in hong kong influenza vaccination among registered nurses : information receipt, knowledge, and decision-making at an institution with a multifaceted educational program understanding healthcare worker uptake of influenza vaccination : a survey accessed trends in influenza vaccination coverage rates in the united kingdom over six seasons from 2001-2 to 2006-7 pandemic h1n1 (swine flu) and seasonal influenza vaccine uptake amongst frontline healthcare workers in england avian flu : the creation of expectations in the interplay between science and the media development of the multidimensional health locus of control (mhlc) scales policy statementrecommendation for mandatory influenza immunization of all health care personnel revised shea position paper : influenza vaccination of healthcare personnel mandatory vaccination of health care workers the ethics of mandatory vaccination against influenza for health care workers point counterpoint : mandatory flu vaccination for health care workers beliefs on mandatory influenza vaccination of health care workers in nursing homes : a questionnaire study from the netherlands sensitivity and specificity of patient self-report of influenza and pneumococcal polysaccharide vaccinations among elderly outpatients in diverse patient care strata we are grateful for the statistical advice of peter milligan. none. key: cord-000891-5r2in1gw authors: giannella, maddalena; rodríguez-sánchez, belen; roa, paula lópez; catalán, pilar; muñoz, patricia; de viedma, darío garcía; bouza, emilio title: should lower respiratory tract secretions from intensive care patients be systematically screened for influenza virus during the influenza season? date: 2012-06-14 journal: crit care doi: 10.1186/cc11387 sha: doc_id: 891 cord_uid: 5r2in1gw introduction: influenza is easily overlooked in intensive care units (icus), particularly in patients with alternative causes of respiratory failure or in those who acquire influenza during their icu stay. methods: we performed a prospective study of patients admitted to three adult icus of our hospital from december 2010 to february 2011. all tracheal aspirate (ta) samples sent to the microbiology department were systematically screened for influenza. we defined influenza as unsuspected if testing was not requested and the patient was not receiving empirical antiviral therapy after sample collection. results: we received ta samples from 105 patients. influenza was detected in 31 patients and was classified as unsuspected in 15 (48.4%) patients, and as hospital acquired in 13 (42%) patients. suspected and unsuspected cases were compared, and significant differences were found for age (53 versus 69 median years), severe respiratory failure (68.8% versus 20%), surgery (6.3% versus 60%), median days of icu stay before diagnosis (1 versus 4), nosocomial infection (18.8% versus 66.7%), cough (93.8% versus 53.3%), localized infiltrate on chest radiograph (6.3% versus 40%), median days to antiviral treatment (2 versus 9), pneumonia (93.8% versus 53.3%), and acute respiratory distress syndrome (75% versus 26.7%). multivariate analysis showed admission to the surgical icu (odds ratio (or), 37.1; 95% confidence interval (ci), 2.1 to 666.6; p = 0.01) and localized infiltrate on chest radiograph (or, 27.8; 95% ci, 1.3 to 584.1; p = 0.03) to be independent risk factors for unsuspected influenza. overall mortality at 30 days was 29%. icu admission for severe respiratory failure was an independent risk factor for poor outcome. conclusion: during the influenza season, almost one third of critical patients with suspected lower respiratory tract infection had influenza, and in 48.4%, the influenza was unsuspected. lower respiratory samples from adult icus should be systematically screened for influenza during seasonal epidemics. influenza is a common cause of admission to the intensive care unit (icu) during the influenza season and influenza pandemics [1] [2] [3] [4] . however, it may be overlooked, particularly in patients with clinical manifestations that can be explained by alternative infectious or noninfectious causes [5] . furthermore, influenza may not be suspected when respiratory function deteriorates or fails in patients already admitted to the icu. at present, information on influenza acquired during icu stay is scarce and incomplete [5] . timely knowledge of the presence of influenza virus in patients admitted to the icu has obvious epidemiologic, diagnostic, and therapeutic advantages [4] . we assessed the burden of influenza in adult icus and the number of overlooked cases when the routine diagnostic workup was applied during the influenza season. we screened all tracheal aspirates sent to the microbiology department for the diagnosis of lower respiratory tract infection, even when not requested by the attending physician. our hospital is a 1,550-bed tertiary referral teaching institution caring for a population of approximately 750,000 inhabitants. it has three different adult icus (medical, surgical, and cardiac surgery) with a total of 42 beds. from december 15, 2010, through february 28, 2011, all tracheal aspirate (ta) samples obtained from adult patients (≥18 years) admitted to our icus and sent to the microbiology department were systematically screened for influenza virus. icu admission criteria and management for all patients, including the need for intubation and for obtaining ta samples, were not standardized, and decisions were made at the discretion of the attending physician. patients with laboratory-confirmed influenza, by realtime reverse transcriptase polymerase chain reaction (rt-pcr) on ta and nasopharyngeal samples, were prospectively followed up by an infectious diseases specialist and treated with oseltamivir, 150 mg/day, for 5 to 10 days. clinical and microbiology data were recorded in a preestablished protocol and entered into a database. the study was approved by the ethics committee of the "fundación para la investigación biomédica del hospital gregorio marañón." the requirement for informed consent was waived because we applied an excellent diagnostic technique to improve the quality of patient care without any negative impact. our objectives were to determine the incidence of influenza among adult icu patients with a ta sample obtained during the influenza season, and to demonstrate the frequency of unsuspected cases and the rate of hospital-acquired episodes. the variables recorded were age, sex, classification of the severity of underlying conditions according to the charlson comorbidity index [6] , type of icu, date and cause of icu admission, apache ii score [7] on admission to the icu, date of onset of influenza symptoms, clinical manifestations and radiologic findings at diagnosis, date of ta sample collection, other samples tested for influenza and result, date of initiation of antiviral treatment, complications (septic shock, acute respiratory distress syndrome (ards)), outcome including mortality within 30 days after influenza diagnosis, and length of icu and hospital stay. we defined the diagnosis of influenza as unsuspected when influenza testing was not explicitly requested or had not been previously requested in other samples, such as nasopharyngeal swabs, and the patient was not receiving empirical antiviral treatment immediately after sample collection. influenza was classified as community acquired if the flu syndrome (fever, chills, malaise, sore throat, rhinorrhea, cough, dyspnea, myalgia, nausea, and diarrhea) began before or during the first 72 hours of hospital admission. the infection was classified as hospitalacquired, if symptoms started after the first 72 hours [8] . as for causes of icu admissions, severe respiratory failure was defined as severe hypoxemia (pao 2 < 60 mm hg) refractory to high-flow oxygen therapy (fio 2 , 50%) with a venturi mask. as for underlying conditions, chronic obstructive pulmonary disease was defined according to the criteria of the 2007 global initiative for chronic obstructive lung disease [9] . immunosuppressed patients were those with hematologic malignancy (with or without bone marrow transplantation), hiv infection, inflammatory diseases under biologic or immunosuppressive treatment and solid organ transplant. as for influenza vaccination, we considered patients who had been vaccinated against influenza within 6 months before admission. pneumonia was defined according to the current idsa/ats guidelines [10] . ards and septic shock were defined by using standard criteria [11, 12] . samples for microbiologic diagnosis were taken by endotracheal aspiration with a 14f sterile probe to a depth of 2 cm from the distal end of the endotracheal tube. the secretions obtained were collected in a sterile container (lukens specimen container; sherwood medical, tullamore, ireland) and transported in sterile packages to the microbiology laboratory for gram staining and bacterial and viral procedures. standard bacterial procedures included quantitative culture performed on blood agar, chocolate agar, mcconkey agar, and, when required, legionella agar (bcye) [13] . positive samples were defined as those with bacterial counts ≥10 5 cfu/ml of each significant microorganism. the microorganisms were identified and antimicrobial susceptibility testing performed by using an automatic system (microscan; dade behring, sacramento, ca, usa). breakpoints were determined after the clinical and laboratory standards institute (clsi) guidelines [14] . unless proven otherwise, we considered as nonpathogenic the isolation (at any concentration) of the following microorganisms: viridans-group streptococci, enterococcus spp., coagulase-negative staphylococcus, neisseria spp., corynebacterium spp., and candida spp. samples were collected in viral-transport medium (copan 305c; copan innovation, brescia, italy). a 200μl aliquot was stored at 4°c for no longer than 48 hours until analysis. the rest of the sample was stored at -80°c for further amplification and sequencing. rna was extracted in a nuclisens easymag system (biomérieux, boxtel, the netherlands) by following the manufacturer's instructions. pandemic influenza a ph1n1 was detected by real-time reverse transcriptase polymerase chain reaction (rt-pcr) by following the who/cdc protocol in a stratagene mx3000 thermocycler (stratagene, la jolla, ca, usa). those samples rendering indeterminate results (low-fluorescence signal or high ct values) were tested again with the realtime ready inf a/h1n1 detection set (roche diagnostics, mannheim, germany). influenza b was detected by using the realtime ready influenza b detection set (roche diagnostics). h3n2 and seasonal h1n1 strains were detected as described elsewhere [15] . relative dna was quantified by combining the rt-pcr methods described with the detection of a housekeeping gene with real-time rt-pcr, as described by the cdc. this method allowed normalization of the initial amount of rna present in each sample [16] . categoric variables appear with their frequency distribution. nonnormally distributed continuous variables are expressed as the median and interquartile range (iqr). the association between categoric variables was evaluated by using the χ 2 test or fisher exact test; the association between continuous variables was evaluated by using the mann-whitney u test. a logistic binary model was used to analyze the independent risk factors for unsuspected influenza and 30-day mortality. variables with p ≤ 0.1 in the univariate analysis were entered into the multivariate model. the level of significance was set at p < 0.05 for all the tests. the statistical analysis was performed by using spss 13.0. during the study period, 618 patients were admitted to our adult icus. overall, one or more ta samples were obtained from 105 patients, and a microbiologic diagnosis was made in 65 of them (see figure 1 ). bacterial infection was diagnosed in 29 patients, and the frequencies of the pathogens isolated were as follows: staphylococcus aureus, 37.9%; enterobacteriaceae, 24.1%; pseudomonas aeruginosa, 17.2%; streptococcus pneumoniae, 13.7%; and acinetobacter baumannii, 6.8%. a diagnosis of viral infection only was made in 25 patients: 23 with influenza virus, one with adenovirus, and one with herpes simplex virus. aspergillus fumigatus was the only microorganism isolated in three patients. the remaining eight patients initially had coinfection with influenza virus and the following microorganisms: s. aureus, three; s. pyogenes, one; s. pneumoniae, one; a. baumannii, one; p. aeruginosa, one; and aspergillus fumigatus, one. during the study period, the overall incidence of influenza in the adult icus of our hospital was 5.3 cases per 100 icu admissions. the incidence of influenza among the patients with at least one ta sample sent to the microbiology department was 29.5 cases per 100 icu patients. the reasons for admission to the icu and the characteristics and outcome of the 31 patients with influenza are shown in table 1 . influenza was unsuspected in 15 (48.4%) patients and hospital-acquired in 13 (42%) patients. at the time of influenza diagnosis, all patients but one were intubated. among patients with co-infection, the reasons for admission to the icu were as follows: surgery, five; respiratory failure, one; cardiac arrest, one; and decompensated cirrhosis, one. influenza was classified as hospital acquired in five (62.5%) of them, and pneumonia was diagnosed in seven (87.5%) patients. overall, viral infection was diagnosed in 33 patients, and in 31 (93.9%) of them, influenza was detected. influenza was due to the 2009 pandemic influenza a h1n1 strain in 27 (87%) patients, influenza b in three (9.7%) patients, and influenza a h3n2 in one (3.2%) patient. in 17 of the 31 patients, influenza testing was performed simultaneously in the ta and nasopharyngeal samples. the upper respiratory tract sample failed to detect influenza in 17.6% of cases. overall, the median relative viral load at diagnosis was 1.55 (iqr, 0.68 to 3.16) . this tended to be higher in patients with suspected influenza ( table 2) . patients with suspected influenza were compared with those with unsuspected influenza ( table 2 ). the univariate analysis revealed significant differences for age (53 versus 69 years; p = 0.008), medical icu (93.8% versus 40%; p = 0.002), admission to the icu for severe respiratory failure (68.8% versus 20%; p = 0.002), length of icu stay before the influenza diagnosis (1 (iqr, 0 to 1) versus 4 (iqr, 1 to 17) days; p = 0.01), classification as having hospital-acquired influenza (18.8% versus 66.7%; p = 0.01), cough (93.8% versus 53.3%; p = 0.01), localized pulmonary infiltrate on radiograph (6.3% versus 40%; p = 0.04), median days to initiation of antiviral therapy after onset of symptoms (2 (iqr, 2 to 6) versus 9 (iqr, 4.5 to 18) days; p = 0.02), pneumonia (93.8% versus 53.3%; p = 0.01), and development of ards (75% versus 26.7%; p = 0.01). mortality at 30 days after the influenza diagnosis was 37.5% and 20% (p = 0.43) in patients with suspected and unsuspected influenza, respectively. multivariate analysis showed the independent risk factors associated with unsuspected influenza to be admission to the surgical icu (or, 37.13; 95%ci, 2.06 to 666.60; p = 0.01) and localized pulmonary infiltrate on radiograph (or, 27.78; 95%ci, 1.32 to 584.06; p = 0.03). longer icu stay before the diagnosis of influenza was also associated with unsuspected influenza but was not significant (table 3) . overall mortality at 30 days after influenza diagnosis was 29%. the univariate analysis of the risk factors for mortality is shown in table 4 . nosocomial acquisition of influenza was associated with better outcome (54.5% versus 11.1%; p = 0.04). the only independent risk factor for 30-day mortality in the multivariate analysis was severe respiratory failure as the reason for admission to the icu (or, 7.5; 95%ci, 1.23 to 45.8; p = 0.03). during the influenza season, almost one third of patients hospitalized in our adult icus and with suggestion of lower respiratory tract infection had influenza. influenza was unsuspected in 48.4% and hospital acquired in 42%. patients with unsuspected influenza were more frequently admitted to the icu for surgery, had a localized infiltrate on chest radiograph, and stayed longer in the icu before being diagnosed with influenza. antiviral treatment was initiated later in patients with unsuspected influenza, although mortality was similar in both groups. overall mortality at 30 days after the influenza diagnosis was 29%; however, it was lower in patients with nosocomial influenza. severe respiratory failure as the cause of admission to the icu was the only independent factor associated with poor outcome. acute febrile respiratory illness is a common cause of respiratory failure and admission to the icu [2] [3] [4] . in most cases, the etiology is bacterial, although viruses have been implicated in almost 9% of cases [17] . during the 2009 pandemic, the rate of icu admission for respiratory failure among hospitalized patients with a confirmed diagnosis of influenza a (h1n1v) ranged from 15% to 34% [18] [19] [20] [21] [22] . however, no studies have investigated the rates of bacterial and viral etiologies among patients admitted to the icu with suggestion of lower respiratory tract infection during the 2009 pandemic. here, we demonstrated that, after the pandemic influenza season, the etiology was viral in 31.4% of patients admitted to the icu with suggestion of lower the etiology of acute febrile respiratory illness causing respiratory failure is often unknown at admission to the icu [17] . about half of the cases are diagnosed as bacterial pneumonia shortly after admission, with a small number of cases found to be viral pneumonia when the initial bacterial studies are negative [10] . detection of influenza virus often depends on specific epidemiologic risk factors and clinical suspicion. the combination of fever, malaise, and cough was shown to have a 79% positive predictive value during the pandemic and seasonal epidemics [23, 24] ; however, these criteria may be not accurate in icu patients, because other etiologies, or conditions like as postsurgery sedation, may confound the diagnosis [25] . in our study, influenza was unsuspected in 48.4% of cases. suspicion of influenza was lower in older patients, in those admitted to the icu for surgical conditions, in those who stayed for a longer time in hospital and icu, and in those who did not have a cough and diffuse pulmonary infiltrates. the direct consequence of overlooked influenza was a significant delay in the initiation of antiviral treatment. definitive diagnosis of influenza is by detection of the virus in culture or rt-pcr with a nasopharyngeal aspirate/swab or lower respiratory tract sample [23, 24] . because viral shedding peaks at 48 hours after the onset of illness and declines thereafter, testing of lower respiratory tract samples in patients with compromised lung parenchyma may be more beneficial [23, 26, 27] . accordingly, we found that the upper respiratory tract sample did not reveal influenza in 17.6% of cases. diagnostic viral load tended to be higher in patients with suspected influenza, possibly as a result of the earlier diagnosis of influenza after onset of symptoms in this group compared with patients with unsuspected influenza. hospital-acquired influenza is a well-recognized problem [28, 29] . nosocomial outbreaks of pandemic and seasonal influenza have been documented in various settings, including icus, pediatric wards, transplant units, medical wards, and surgical wards [28] [29] [30] [31] [32] . however, few sporadic cases of hospital-acquired influenza have been reported during surveillance activities [33] . in a study including 1,520 patients hospitalized with the pandemic 2009 influenza a in 75 hospitals in the united kingdom, the authors identified 30 (2%) cases of sporadic nosocomial influenza [33] . these comprised 15 adults and 15 children. most had serious underlying illnesses and were admitted to nonmedical areas, as in our study. unexpectedly, we found that the 30-day mortality rate was lower in patients with hospital-acquired influenza. this figure can be associated with viral factors, such as lower virulence of the influenza strains circulating in the hospital, or with host factors, such as older age and surgical conditions. overall, 30-day mortality was high (29%), and admission to the icu for severe respiratory failure was an independent risk factor for death. these data are consistent with those of martin-loeches et al. [34] , who showed that patients from the postpandemic influenza ph1n1 period had an unexpectedly high mortality rate. early administration of antiviral therapy has been associated with better outcome in critically ill patients [35] . in our study, although the timing to initiation of antiviral treatment was longer among patients with unsuspected influenza, a trend to lower mortality was seen in this group compared with patients with suspected influenza. a possible explanation of this finding could be that: suspected and unsuspected groups were epidemiologically very different, and the median relative viral load was lower in the unsuspected group; thus, epidemiologic and viral factors could influence the outcome in the two groups independently of the timing of antiviral treatment. conversely, the benefit of testing will not be necessarily to the patient in terms of improved outcome due to early therapy, but more likely to preventing the nosocomial transmission of influenza. our study is limited in that the small number and heterogeneity of patients diminishes the power of our data analysis. we performed the study during the postpandemic period (2010 to 2011), when the prevalence of the pandemic influenza a h1n1 strain was still high. findings could vary between one influenza season and another, depending on the characteristics of the prevalent influenza virus stain. we did not perform a costeffectiveness analysis, although the finding of a longer icu and hospital stay in patients with unsuspected influenza suggests a potential favorable impact on care management. we could not perform an analysis of the possible routes of transmission of the nosocomial cases. however, we can exclude with sufficient certainty the occurrence of an outbreak for the following reasons: (a) the cases of hospital-acquired influenza were distributed uniformly between the three icus (postsurgery icu, six; medical icu, five; and postcardiosurgery icu, two); (b) no case of influenza was recognized among the healthcare staff during the study period; (c) the preventive measures included vaccination of staff, respiratory isolation, and droplet-contact precautions, as recommended by the centers for disease control and prevention [36] . we showed that influenza is a common cause of acute respiratory illness among patients admitted to the icu during seasonal epidemics, and that it is often overlooked, and it could lead to a delay in the initiation of antiviral treatment and possible nosocomial transmission of influenza. microbiology departments should systematically investigate the presence of influenza in respiratory samples obtained from icu patients during the seasonal epidemic. • the incidence of influenza in the adult icu during the influenza season is high. • the diagnosis of influenza is often overlooked in icu patients. among patients with unsuspected influenza, the timing to initiation of antiviral treatment was longer, and the rate of hospital-acquired influenza was higher compared with that of patients with suspected influenza. • microbiology departments should systematically investigate the presence of influenza in respiratory samples obtained from icu patients during the seasonal epidemic. abbreviations apache: acute physiology and chronic health evaluation; ards: acute respiratory distress syndrome; ats: american thoracic society; cdc: centers for disease control; copd: chronic obstructive pulmonary disease; icu: intensive care unit; idsa: infectious diseases society of america; iqr: interquartile range; rt-pcr: reverse transcriptase-polymerase chain reaction; ta: tracheal aspirate; who: world health organization. case mix, outcome and length of stay for admissions to adult, general critical care units in england, wales and northern ireland: the intensive care national audit & research centre case mix programme database febrile respiratory illness in the intensive care unit setting: an infection control perspective acute febrile respiratory illness in the icu: reducing disease transmission severe febrile respiratory illnesses as a cause of mass critical care nosocomial influenza: new concepts and practice morbidity during hospitalization: can we predict it? apache ii: a severity of disease classification system incubation periods of acute respiratory viral infections: a systematic review the 2007 gold guidelines: a comprehensive care framework infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults the american-european consensus conference on ards: definitions, mechanisms, relevant outcomes, and clinical trial coordination surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock guidelines for performance of respiratory tract cultures performance standards for antimicrobial susceptibility testing: fifteenth informational supplement typing (a/b) and subtyping (h1/h3/h5) of influenza a viruses by multiplex real-time rt-pcr assays prolonged viral shedding in pandemic influenza a h1n1: clinical significance and viral load analysis in hospitalized patients acute respiratory distress syndrome and pneumonia: a comprehensive review of clinical data critically ill children with pandemic influenza (h1n1) in pediatric intensive care units in turkey outcomes from pandemic influenza a h1n1 infection in recipients of solid-organ transplants: a multicentre cohort study hospitalized children with 2009 pandemic influenza a (h1n1): comparison to seasonal influenza and risk factors for admission to the icu pandemic influenza a (h1n1) virus hospitalizations investigation team: hospitalized patients with 2009 h1n1 influenza in the united states hospitalized patients with 2009 pandemic influenza a (h1n1) virus infection in the united states efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial neuraminidase inhibitor flu treatment investigator group does this patient have influenza? when should a diagnosis of influenza be considered in adults requiring intensive care unit admission? results of population-based active surveillance in toronto implications of antiviral resistance of influenza viruses intensive care adult patients with severe respiratory failure caused by influenza a (h1n1)v in spain nosocomial influenza in children influenza in the acute hospital setting nosocomial outbreak of influenza virus a (h3n2) infection in a solid organ transplant department a nosocomial outbreak of 2009 pandemic influenza a(h1n1) in a paediatric oncology ward in italy outbreak of novel influenza a (h1n1) in an adult haematology department and haematopoietic cell transplantation unit: clinical presentation and outcome nosocomial pandemic (h1n1) 2009 pandemic and post-pandemic influenza a (h1n1) infection in critically ill patients impact of early oseltamivir treatment on outcome in critically ill patients with 2009 pandemic influenza a should lower respiratory tract secretions from intensive care patients be systematically screened for influenza virus during the influenza season? critical care 2012 16:r104. submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank thomas o'boyle for his help with the preparation of the manuscript. this study was partially financed by the programa de centros de investigación biomédica en red (ciber) de enfermedades respiratorias cb06/06/0058. maddalena giannella (cm08/00279) is contracted by the fis. all the authors made a substantial contribution. eb, dgdv, pc, and pm assisted in the conception and design of the study, revised the manuscript critically, and gave the final approval of the version to be published. mg, br, and plr were responsible for data acquisition, analysis, and interpretation. mg drafted the manuscript. members of gang study group revised and approved the study design and assisted in the data acquisition. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-253143-73dsc6q3 authors: tang, julian w.; shetty, nandini; lam, tommy t.y.; hon, k.l. ellis title: emerging, novel, and known influenza virus infections in humans date: 2010-08-02 journal: infect dis clin north am doi: 10.1016/j.idc.2010.04.001 sha: doc_id: 253143 cord_uid: 73dsc6q3 influenza viruses continue to cause yearly epidemics and occasional pandemics in humans. in recent years, the threat of a possible influenza pandemic arising from the avian influenza a(h5n1) virus has prompted the development of comprehensive pandemic preparedness programs in many countries. the recent emergence of the pandemic influenza a(h1n1) 2009 virus from the americas in early 2009, although surprising in its geographic and zoonotic origins, has tested these preparedness programs and revealed areas in which further work is necessary. nevertheless, the plethora of epidemiologic, diagnostic, mathematical and phylogenetic modeling, and investigative methodologies developed since the severe acute respiratory syndrome outbreak of 2003 and the subsequent sporadic human cases of avian influenza have been applied effectively and rapidly to the emergence of this novel pandemic virus. this article summarizes some of the findings from such investigations, including recommendations for the management of patients infected with this newly emerged pathogen. virus, which is why the seasonal influenza vaccine is mainly characterized by its ha (rather than its na) composition for influenza a. so far, only 3 subtypes of ha (h1, h2, h3) and 2 subtypes of na (n1, n2) have caused pandemics in humans. traditional pandemic surveillance has focused on monitoring the antigenic shift, that is, the reassortment of ha and/or na genes between human and zoonotic influenza a viruses during rare events of dual infections in a human or an intermediate host. for the surveillance of currently circulating seasonal influenza viruses, most recently the a (h3n2) and a (h1n1) viruses, viral isolates are collected throughout the year to determine the most appropriate seasonal influenza vaccine composition for the coming influenza seasons in the northern and southern hemispheres. a zoonotic virus (ie, originating from animals and spreading to humans), avian influenza a (h5n1) emerged for the first time in hong kong in 1997 from chickens to infect humans, infecting 18 people and killing 6 of them-a high mortality rate of more than 30%. 1 originally discovered to be circulating on geese farms in 1996 in guangdong, china, this highly pathogenic avian influenza a (h5n1) virus soon spread to hong kong, causing outbreaks among poultry in 1997. it was eventually eradicated from hong kong after a mass cull of all poultry, but it apparently continued to circulate asymptomatically amongst birds in southern china, 2 from where it eventually reemerged in the human population in 2002 to 2003 and has been causing ongoing sporadic human infection and disease, with a high mortality (close to 60%) till present. as of august 31, 2009 , the world health organization has reported 440 cases of sporadic h5n1 human infection, of which 262 were lethal (a 60% case-fatality rate). 3 in addition, sporadic, generally mild (although there has been at least 1 recorded death because of h7n7) human infections resulting from occasional bird-to-human transmissions, with low pathogenic avian influenza strains (eg, subtypes h9n2, h7n7, h7n2, and h7n3) have been ongoing since 1997, when heightened surveillance for avian influenza viruses began (fig. 2) . 4 so far, all these low pathogenic avian influenza viruses isolated from these sporadic human infections have been genetically similar to the corresponding avian influenza viruses circulating in birds. thus, so far and within the limits of current surveillance, there seems to have been no further reassortment of these viruses with either human or swine influenza viruses. until recently, avian influenza a (h5n1) was considered the prime virus subtype candidate for causing the next influenza pandemic. however, the recent unexpected emergence of the pandemic influenza a (h1n1) 2009 virus (also referred to as h1n1v) of swine origin from mexico has demonstrated that even influenza subtypes that have been encountered in previous influenza pandemics may constitute new pandemic threats. 5 before the emergence of the recent pandemic influenza a (h1n1) 2009 virus, there were 3 pandemics during the twentieth century: in 1918 (the spanish flu), 1957 (the asian flu), and 1968 (the hong kong flu). these incidents have been widely studied with the help of available (and sometimes extensive) epidemiologic records and any preserved, archived viral isolates or infected tissue specimens. the first of these pandemics in 1918 coincided with world war i and infected an estimated one-third of the world's population (approximately 500 million people), with approximately 50 million deaths. in contrast, the subsequent 1957 and 1968 pandemics (now shown to have originated in asia) resulted in a lower morbidity and mortality but still had a significant global effect. perhaps most importantly, the occurrence of these subsequent pandemics gave rise to the concept that such pandemics could and would recur. pandemic influenza viruses are thought to arise when there is frequent human contact with certain animal species that can be infected with their own specific influenza viruses and when these viruses develop the ability to jump the species barrier to infect humans. this crossing is made possible in the presence of certain gene mutations permitting the binding of such animal influenza viruses to surface proteins on human respiratory epithelial cell receptors. 6 birds are the natural reservoir for influenza a viruses, although other animals such as pigs and horses have also acquired and maintained their own separate genetic lineages of influenza. 7 the origin of the 1918 a (h1n1) pandemic influenza virus has become more controversial recently, and there is a debate over whether it was derived from a human influenza strain existing before 1918, 8 or directly from a purely avian influenza strain from around 1918, 9 or whether it was generated by the reassortment or recombination between human and avian influenza viruses cocirculating around that time. the reason for this controversy is that there are very few viral isolates available for analysis from around this time (before 1918); therefore, the complete diversity of avian influenza viruses circulating then cannot be known. in contrast, the origins of the 1957 and 1968 influenza pandemics have been more clearly defined (see fig. 2 ). the 1957 pandemic was caused by an a (h2n2) reassortant strain admixing ha, na, and polymerase basic protein 1 (pb1) gene segments from avian influenza strains, with the remaining gene segments from the a (h1n1) human pandemic influenza virus subtype that had been circulating since its emergence in 1918. 10 the strain a (h2n2) eventually replaced a (h1n1); then a (h2n2) was itself replaced by the 1968 a (h3n2) pandemic subtype. the 1968 a (h3n2) virus was also a reassortant strain in which the ha and pb1 gene segments from an avian influenza strain reassorted with the then currently circulating a (h2n2) virus. 10 since 1977, this a (h3n2) virus has been cocirculating with an a (h1n1) strain similar to the 1918 a (h1n1) pandemic virus, which was accidentally released from a laboratory. 11 thus, these 2 viruses have now become familiar to us as the seasonal influenza a subtypes for more than 30 years. analyses of the viruses that caused the 1957 and 1968 influenza pandemics therefore proved that zoonotic transmissions of influenza viruses (ie, from animals to man) with gene reassortment were capable of generating antigenically new influenza strains, novel to human immunity, with significant effects on the public health. the emergence of the first influenza pandemic virus in april 2009 in more than 40 years caught the world by surprise. it was a surprise not just because of the zoonotic origin of the virus (ie, swine rather than avian) but also because of the geographic origins (ie, the americas rather than southeast asia). 12 however, the pandemic preparedness that was already in place to combat the more expected avian influenza pandemic has been used to good effect. the stockpiling of antivirals and a lot of basic and applied research into developing vaccines against novel influenza viruses had already commenced. apart from the clinical preparedness, a lot of groundwork has also already been done on the development and application of mathematical models for describing and predicting how the pandemic will evolve, 13,14 as well as identifying and prioritizing public health interventions. 15 this approach has been stimulated greatly by the severe acute respiratory syndrome outbreaks of 2003, and it was easy to apply these techniques to influenza. these mathematical models included not just the traditional epidemiologic models but also the newer approach of phylogenetic analysis applied to partial or whole viral genomes. in some recent analyses of the novel pandemic influenza a (h1n1) virus, this latter approach has been used to give unique insights into the evolution of this new virus. 16 case definitions form the cornerstone of the investigation and management of individual patients and outbreaks, although the different influenza subtypes may present in slightly different ways. however, these differences, although statistically noticeable in comparative case series, may not be necessarily useful when faced with individual patients. ultimately, laboratory diagnosis will always be required to distinguish among the different infecting subtypes. clinically, influenza is usually a self-limiting disease. after an average incubation period of around 1 to 2 days, onset of illness is characterized by an abrupt onset of fever and chills accompanied by headache, generalized myalgia, rhinorrhea, sore throat, and cough. gastrointestinal symptoms such as vomiting, abdominal pain, and diarrhea are often reported. the most common cause of hospitalization is lower respiratory tract infection, including croup, bronchitis, bronchiolitis, and pneumonia. manifestations involving the central nervous system may be observed, including encephalopathy, postinfluenza encephalitis, transverse myelitis, guillain-barr e influenza virus infections in humans syndrome, and acute necrotizing encephalitis. myositis often occurs 3 days (range, 0-18 days) after onset of illness. in young infants, influenza can mimic generalized sepsis. myocarditis is a rare complication. epidemiologically, most deaths occur in infants and the elderly (>65 years old) during the annual influenza epidemics as a result of decreased immunity against influenza virus infection. the mortality curve typically presents with a u shape when age-specific excess mortality caused by pneumonia and seasonal influenza is plotted. 17 the incubation period for this virus has been estimated to be up to 7 days, but it is more commonly 2 to 5 days after the last known exposure to sick or dead poultry. in cases where limited human-to-human transmission likely occurred, the incubation period was estimated to be between 2 and 10 days. 18 analyses of the human a (h5n1) infections in hong kong, vietnam, thailand, and cambodia revealed that fever and cough were the most common initial symptoms. gastrointestinal symptoms including vomiting, diarrhea, and abdominal pain were reported early in the course of illness in some cases. 19 others reported pleuritic pain and bleeding from the nose and gums. generally, patients with h5n1 virus infection were hospitalized 4 to 6 days after onset of illness. 18 common laboratory findings in patients with a (h5n1) infection at the time of hospital admission include leukopenia, lymphopenia, and mild-to-moderate thrombocytopenia. 19 however, for patients with a clinically mild illness, there was no decrease in the white cell count. chest radiographic findings included patchy, interstitial, lobar, and/or diffuse infiltrates; consolidation; pleural effusion; and pneumothorax. in fatal a (h5n1) cases, the median time from onset to death was 9 days. 18 the fatality rate among hospitalized patients has been high and varies considerably between countries (33%-100%), although the true rate maybe much lower because of an unknown number of milder nonfatal infections in the community. 20 acute respiratory distress syndrome complicated 76.5% (13 in 17) of cases in thailand and 44.4% (8 in 18) of cases in hong kong. multiple organ failure, with signs of renal dysfunction and sometimes cardiac compromise, was often noted. in the severe human a (h5n1) infections in hong kong, reactive hemophagocytic syndrome was a unique pathologic feature in 3 fatal cases, as were increased blood levels of interferon-a, tumor necrosis factor a, and other cytokines, providing evidence that cytokine responses contributed to the pathogenesis of human h5n1 infections. 21 exactly how the severity of illness varies by clade or subclade of h5n1 virus infection, by age, or by immunologic, genetic, or other factors is unknown. 18 most patients who died did not have a preexisting disease, in contrast to situations where other subtypes of human influenza virus infections caused epidemics during interpandemic periods. however, patients with underlying cardiovascular, pulmonary, or renal diseases were, as expected, still more susceptible to severe influenza infection. 20 an analysis of 18 cases of pneumonia with confirmed a (h1n1) 2009 infection among 98 patients hospitalized for acute respiratory illness in mexico city, mexico, showed that more than 50% of them were between ages 13 and 47 years and only 8 had preexisting medical conditions. all patients had fever, cough, dyspnea or respiratory distress, increased serum lactate dehydrogenase levels, and bilateral patchy pneumonia (fig. 3) . other common findings were an increased creatine kinase level (in 62% of the patients) and lymphopenia (in 61%). twelve patients required mechanical ventilation, and 7 died. within 7 days after contact with the initial case patients, a mild tang et al or moderate influenzalike illness developed in 22 health care workers, none of whom required hospitalization. 22 in a study of 642 confirmed cases of human a (h1n1) 2009 infection identified from the rapidly evolving us outbreak in april 2009, the age of patients ranged from 3 months to 81 years; 60% of patients were 18 years old or younger. of patients with available data, 18% had recently traveled to mexico and 16% were identified from 2009. the initial appearance is suggestive of a developing viral pneumonitis, which then seems to resolve (a-c), but then the patient probably developed a secondary bacterial infection (although not proven conclusively) (d) that necessitated a transfer to the intensive care unit (e) before finally resolving (f). the patient was finally discharged feeling well, with no long-term sequelae. (courtesy of university college london hospitals nhs trust, london, uk.) school outbreaks of a (h1n1) 2009 infection. the most common presenting symptoms were fever (94%), cough (92%), and sore throat (66%); 25% of patients had diarrhea, and 25% had vomiting. of the 399 patients for whom hospitalization status was known, 36 (9%) required hospitalization and 2 died. 12 a canadian study also reported cough in 90% of patients but fever in only 59% of confirmed and probable cases. other common symptoms included headache (83%), sore throat (76%), and nasal congestion (76%). none of the cases was admitted to hospital. no deaths were associated with the cluster. 23 it is now becoming clear that most cases of a (h1n1) 2009 infection are mild and self-limiting and present in a manner that is indistinguishable from seasonal influenza. as for seasonal influenza, those with preexisting medical conditions such as the traditional chronic diseases (eg, diabetes, asthma, renal or cardiac failure, and any form of immunosuppression) seem to be at greater risk of severe disease and death (and are therefore routinely targeted for the annual seasonal influenza immunization). even so, with this virus, it has been suggested that obesity may be an additional risk factor for serious disease, 24 as is pregnancy, 25 which is also a recognized risk in seasonal influenza infection. the age distribution of infection with this novel virus also differs from seasonal influenza. for example, the older age groups (>65 years) have always been considered to be vulnerable to seasonal influenza infection, but they seem to be less frequently infected by this novel virus. this trend is now thought to be caused by some preexisting cross-reacting immunity to this virus as a result of their past exposure to the older circulating seasonal influenza a (h1n1) strains that have been more similar to the current pandemic a (h1n1) 2009 virus. the current circulating seasonal influenza a (h1n1) virus and its corresponding seasonal influenza vaccine antigen components seem to not provide any cross-immunity to the pandemic strain. 26 in the more frequently targeted younger adult age groups, an unusual feature has been observed; more patients in this group progress to more serious respiratory disease, whereas there is also a significant gastrointestinal component (nausea, vomiting, and diarrhea in 10%-50% of cases) involved. 12 in addition, in children infected with a (h1n1) 2009, the incidence of seizures seems to be prominent. although this is also seen with seasonal influenza, 27 the few recent case reports available so far suggest that outcomes are better with the pandemic a (h1n1) 2009 infection. 28 antivirals for all these influenza subtypes, apart from the usual respiratory support and monitoring, there are only a few specific antiviral drugs for treatment. in the cases of seasonal influenza a (h3n2), avian influenza a (h5n1), and pandemic influenza a (h1n1) 2009, virtually all these viruses are resistant to treatment with the adamantane drugs (amantadine and rimantadine) but still susceptible to the na inhibitors (nais) such as oseltamivir and zanamivir. in the case of seasonal influenza a (h1n1), most viruses are resistant to oseltamivir (although zanamivir is still effective in most cases) but still susceptible to the adamantane drugs, although resistance seems to be increasing. another member of the nai group, peramivir, is still in clinical trials. peramivir has an advantage over oseltamivir (taken orally) and zanamivir (taken by inhalation) in that it can be given intravenously. combination therapy with oseltamivir and rimantadine can be given empirically if the influenza subtype is unknown, and some patients infected with a (h1n1) 2009 have been given this combination as initial empiric therapy. 28 according to the manufacturer's information, oseltamivir is generally well tolerated and its adverse effects are mild and mainly gastrointestinal (ie, nausea, vomiting, tang et al diarrhea). however, reports from the use of oseltamivir as postexposure prophylaxis (75 mg once daily for 10 days) in primary and secondary school children (age, 4-12 years) have described additional symptoms such as feeling sick, headaches, stomach aches, difficulty sleeping, nightmares, and poor concentration. 29, 30 table 1 shows the currently recommended nai doses for treatment and postexposure prophylaxis for patients of different ages. treatment is recommended for 5 days, whereas prophylaxis is recommended for at least 2 weeks or a minimum of 7 days (eg, at least 10 days as per cdc recommendations in table 1 ) after contact with the last infected individual and onset of illness. pediatric dosing is based on weight for those weighing less than 40 kg and older than 1 year. for children younger than 1 year, oseltamivir is not licensed to be administered; however, it can be used on an ''emergency authorization use'' basis, for which the recommended dosing regimen is also shown in table 1 . there should be careful monitoring for adverse effects when the drug is used in this younger age group outside its licensure. the treatment of pregnant women infected with a (h1n1) 2009 is considered a priority, because there seems to be an increased risk (although this is relative) of complications in this population. 25 a recent study suggests that, so far, treatment with oseltamivir appears to be safe in pregnancy 31 and that, on this basis, it should be commenced as soon as possible after the onset of symptoms, 32 as well as being offered for postexposure prophylaxis. 33 however, as always, the actual application of these recommendations is left to the individual decision and risk assessment of the patient and the doctor. resistance to oseltamivir arising in patients treated with it has been reported in a few cases so far. the most commonly reported resistance mutation, h275y, occurs in the na gene. 34, 35 the incidence and prevalence of oseltamivir-resistant a (h1n1) 2009 viruses is likely to increase, given the continued widespread use of the drug for treatment. however, oseltamivir should be used for treating only severely ill cases of a (h1n1) 2009 infection (as per seasonal influenza use recommendations) and not for postexposure prophylaxis unless vulnerable groups have been exposed. 32 there has been one case of oseltamivir resistance reported in an individual with no history of oseltamivir use. 36 the issue of worry then is whether such resistant viruses will eventually become fit enough to transmit efficiently in the population (perhaps displacing the wild-type susceptible virus), making the worldwide stockpiles of oseltamivir effectively useless. there is increasing evidence and conviction that a significant proportion (20%-30%) of deaths caused by past influenza pandemics may have been a result of secondary bacterial infections. recent analyses of the 1918 pandemic mortality figures suggest that a significant number of deaths were caused by secondary infections with haemophilus influenzae, streptococcus pneumoniae, streptococcus pyogenes, and/or staphylococcus aureus. 37 these findings suggest that antibiotics and antibacterial vaccines may be important in the management of influenza infections. 38 there are several approaches to prevent infection by the pandemic influenza a (h1n1) 2009 virus, although not all of them are currently available and the evidence supporting the use of some approaches is limited or controversial. at the individual and population level (ie, in terms of increasing overall herd immunity), immunization with a specific a (h1n1) 2009 vaccine is one of the most effective ways to prevent infection. there are multiple ongoing vaccine trials, 39, 40 and recently, a specific live attenuated vaccine (indicated for children and adults, aged 2 to 49 years) has been approved by the us food and drug administration, as of september 15, 2009 . other countries are in the process of approving the vaccine, and it is likely that vaccines against the pandemic influenza virus will be widely available in most countries by the time this article is published. the rapid licensing of this new vaccine has only been made possible by the urgency of the pandemic situation and the application of existing seasonal influenza vaccine manufacturing regulations that do not require annual changes in influenza antigen composition of the seasonal influenza vaccine to undergo relicensing each year. to make the limited supply of these vaccines go further, lower antigenic doses and different adjuvants are being tested to immunize as much of the population as required. 41 in addition, individual vaccination benefits the population as a whole by reducing the number of susceptible individuals who can be infected by the virus. given the currently accepted relatively low value for the reproductive number (r o ) of 1 to 2, 13,14 less than 60% (where required vaccine coverage, v c , is estimated by the formula v c 5 1 ã� 1/r o ) of the population requires vaccination to curtail the onward transmission of influenza. however, there are significant technical (ie, how to make and deliver such a huge amount of vaccine in a short time), moral, and economic (ie, who gets vaccinated first and for what price) challenges that will be faced by the planned global immunization program against the pandemic a (h1n1) 2009 influenza virus. 42, 43 with such large populations being vaccinated, there needs to be careful monitoring for common and rarer (eg, guillan-barr e syndrome) adverse effects. 44 postexposure prophylaxis with oseltamivir for those in close contact with confirmed cases has already been discussed. the disadvantage of mass prophylaxis has been seen in school children when the incidence of adverse effects may be greater than the incidence of secondary infections. 29, 30 this situation becomes more relevant with lower estimates of influenza transmissibility (ie, the lower the value of r o ), although this may be higher in certain situations, such as the dense crowding seen in schools and other public entertainment venues. simple surgical and n95 masks are probably effective to a certain extent either in preventing the noninfected wearer (eg, health care workers) from inhaling influenza-containing droplets (from either a close or more distant source) or in containing the infectious exhaled air from an infected wearer (eg, patients). the problem with wearing masks for either purpose tends to mainly be that of box maintaining an effective mask position on the face for long periods of time. 45 sweating and contact irritation can combine to cause mask displacement or removal (noncompliance). it may be particularly difficult when patients suffering from coughing or sneezing are made to wear masks to contain their infection as a form of infection control. social distancing has received much attention because of its potential to mitigate and perhaps even curtail the widespread transmission of pandemic influenza. one well-investigated example of this has been the effects of school closure on the subsequent progression of an influenza pandemic. various mathematical modeling studies have suggested that, although there may be some delay in the spread of the pandemic from closing schools, this measure will only be effective if the subsequent behavior of children outside the school does not result in a similar number of contacts and that such relative isolation is prolonged during the pandemic period. however, the social and economic disruption for the working parents with such a strategy may be difficult to overcome because at least one parent will have to take time off work to look after young children at home. in particular, school closure has to be part of an overall mitigation strategy, including the treatment and home isolation of infected individuals to reduce further contacts. 15 air travel can rapidly transport infections between different destinations around the world and also act as a source for generating new infections within the crowded confines of modern passenger planes. various mathematical modeling tools have been used to assess the effect of restricting air travel on the spread of pandemic influenza. however, the benefits may be fairly minor and may not be worth the inevitable and serious social and economic disruption this will cause. 46 the possible nonpharmaceutical public health interventions are summarized in box 1. 47 the understanding of how novel influenza viruses arise (usually from animal reservoirs) has increased at an incredible rate assisted by the rapid advances in sequencing technologies and phylogenetic methods. such understanding allows more effective public health surveillance of seasonal human influenza viruses, as well as candidate pandemic viruses that may cross the species barrier from animals to humans. development in antiviral drugs for influenza is still slow (compared with rapid advances and the variety in the case of anti-human immunodeficiency virus drugs), but this is counterbalanced by the effective and highly organized and regulated vaccinemanufacturing base that is already in existence for the seasonal influenza vaccines. unlike infectious agents that infect humans only (such as smallpox and measles), influenza viruses, being zoonotic (with animal and human reservoirs), will continue to pose a persistent and variable threat to human health for the foreseeable future. it is therefore important that systems are in place, in health care institutions and in the general community, to react and adapt quickly to limit human morbidity and mortality caused by this ever-changing pathogen. characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness characterization of h5n1 influenza viruses that continue to circulate in geese in southeastern china confirmed human cases of avian influenza a(h5n1) avian influenza virus infections in humans the 2009 h1n1 influenza outbreak in its historical context haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to human-type receptors evolution and ecology of influenza a viruses influenza: the mother of all pandemics 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics antigenic similarity of influenza a (h1n1) viruses from epidemics in 1977-1978 to ''scandinavian'' strains isolated in epidemics of 1950-1951 emergence of a novel swine-origin influenza a (h1n1) virus in humans initial human transmission dynamics of the pandemic (h1n1) 2009 virus in north america pandemic potential of a strain of influenza a (h1n1): early findings closure of schools during an influenza pandemic origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic influenza pandemics: past, present and future human infection with highly pathogenic avian influenza a (h5n1) virus: review of clinical issues avian influenza a (h5n1) in 10 patients in vietnam avian influenza a (h5n1) infection in humans re-emergence of fatal human influenza a subtype h5n1 disease pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico investigation of the first cases of human-to-human infection with the new swine-origin influenza a (h1n1) virus in canada epidemiology of fatal cases associated with pandemic h1n1 influenza h1n1 2009 influenza virus infection during pregnancy in the usa cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus influenza a virus-associated acute necrotizing encephalopathy in the united states neurologic complications associated with novel influenza a (h1n1) virus infection in children oseltamivir adherence and side effects among children in three london schools affected by influenza a(h1n1)v compliance and side effects of prophylactic oseltamivir treatment in a school in south west england safety of neuraminidase inhibitors against novel influenza a (h1n1) in pregnant and breastfeeding women novel influenza a (h1n1) virus infections in three pregnant women-united states oseltamivir-resistant 2009 pandemic influenza a (h1n1) virus infection in two summer campers receiving prophylaxis-north carolina oseltamivir-resistant novel influenza a (h1n1) virus infection in two immunosuppressed patients center of disease control and prevention. hong kong oseltamivir resistance deaths from bacterial pneumonia during 1918-19 influenza pandemic bacterial pneumonia and pandemic influenza planning trial of influenza a (h1n1) 2009 monovalent mf59-adjuvanted vaccine -preliminary report response after one dose of a monovalent influenza a (h1n1) 2009 vaccine-preliminary report pandemic influenza vaccine policy-considering the early evidence influenza vaccine manufacture: keeping up with change poverty, wealth, and access to pandemic influenza vaccines prepandemic'' immunization for novel influenza viruses, ''swine flu'' vaccine, guillain-barre syndrome, and the detection of rare severe adverse events face mask use and control of respiratory virus transmission in households frequent travelers and rate of spread of epidemics non-pharmaceutical public health interventions for pandemic influenza: an evaluation of the evidence base key: cord-103560-28o0bauv authors: yechezkel, m.; ndeffo-mba, m.; yamin, d. title: optimizing antiviral treatment for seasonal influenza in the united states: a mathematical modeling analysis date: 2020-07-30 journal: nan doi: 10.1101/2020.07.28.20163741 sha: doc_id: 103560 cord_uid: 28o0bauv seasonal influenza remains a major health burden in the united states. despite recommendations of early antiviral treatment of high-risk patients, the effective treatment coverage remains very low. we developed an influenza transmission model that incorporates data on infectious viral load, social contact, and healthcare-seeking behavior, to evaluate the population-level impact of increasing antiviral treatment timeliness and coverage among high-risk patients in the us. we found that increasing the rate of early treatment among high-risk patients who received treatment more than 48 hours after symptoms onset, would substantially avert infections and influenza-induced hospitalizations. we found that treatment of the elderly has the highest impact on reducing hospitalizations, whereas treating high-risk individuals aged 5-19 years old has the highest impact on transmission. the population-level impact of increased timeliness and coverage of treatment among high-risk patients was observed regardless of seasonal influenza vaccination coverage and the severity of the influenza season. seasonal influenza continues to be a major cause of health and economic burden (molinari et al., 2007) . although influenza infection is generally a self-limiting disease, it can result in severe illness and death. in particular, the disease carries substantial health burden among young children, the elderly, and people with certain health conditions (fiore et al., 2010; monto, 2008) . in the united states, seasonal influenza results in an estimated incidence of 9.3-49.0 million illnesses, 140,000-710,000 hospitalizations, and 12,000-56,000 deaths annually (molinari et al., 2007; tokars et al., 2018a) . treatment of patients at risk of severe complications (ison, 2018; o'halloran et al., 2016) . neuraminidase inhibitors (nais) are a class of antiviral medications recommended for the pharmacologic treatment of influenza (fiore et al., 2011) . early therapy of influenza patients with nais reduces the duration and intensity of viral shedding, the duration of symptoms, and disease-associated complications, hospitalizations, and mortality (hayden and pavia, 2006) . despite the high burden of influenza-induced complications among high-risk individuals, their rate of treatment for influenza infection has remained low (stewart et al., 2018) . approximately 40% of high-risk patients with laboratory-confirmed influenza seek care within two days of symptoms onset (stewart et al., 2018) . among these patients, on average, 37% are prescribed an antiviral medication (biggerstaff et al., 2014; stewart et al., 2018) . the acip guidelines recommend that antiviral treatment be given to high-risk patients with clinically suspected influenza infection, even with deferred laboratory confirmation, when influenza is known to be circulating in the population (fiore et al., 2011) . however, current clinical practice is far from keeping with these guidelines. antiviral treatment not only provides direct benefits to treated patients by reducing their risk of influenza-induced hospitalization and/or mortality but may also provide indirect protection to noninfected individuals by reducing their risk of infection. this indirect benefit is achieved by decreasing the contribution of treated patients to disease transmission by reducing their viral shedding and duration of infectiousness. household-based trials have shown that early treatment of infected individuals with nais may reduce their contribution to disease transmission by 50 -80% (e. et al., 2009; halloran et al., 2007) . to evaluate the population-level impact of increased antiviral treatment coverage and timeliness of influenza-infected high-risk individuals during influenza seasons, we developed a data-driven influenza transmission model that incorporates data on infectious viral load, social contact, healthcare-seeking behavior, time to seek healthcare, and antiviral treatment. we developed a dynamic model for influenza infection progression and transmission in texas, california, connecticut, and virginia. our model is a modified susceptible-infected-recovered compartmental framework (vynnycky and white, 2010) , in which transitions between the health-related compartments occur over time (figure 1 ). to model age-dependent transmission, we stratified the population into five age groups: 0-4 y, 5-19 y, 20-49 y, 50-64 y, and ≥65 y. we also distinguished between high-risk and low-risk individuals for each age group based on the acip case definition (fiore et al., 2011) . susceptible individuals in the model may interact with infectious individuals and become either asymptomatically or symptomatically infected (furuya-kanamori et al., 2016; leung et al., n.d.) , where they can transmit the disease to others until recovery. consistent with previous models (medlock and galvani, 2009; ndeffo mbah et al., 2013; yamin et al., 2014) , we assumed that upon recovery, individuals are fully protected for the entire season. this assumption is supported by prospective studies demonstrating that reinfection in the same season is rare (möst et al., 2019; möst and weiss, 2016) . force of infection. the rate at which infectious individuals transmit depends on 1) agespecific contact rates (table supplementary s1 ) between an infected individual and his or her contacts, 2) age-specific susceptibility to infection, and 3) infectiousness of the infected individual based on her/his daily viral loads and time in the season (figure supplementary s1, and si appendix for details). in the us, influenza incidence is seasonal, with a peak typically striking in the winter, yet the driver for this seasonality remains uncertain (lipsitch and viboud, 2009 ). thus, we included general seasonal variation in the susceptibility rate of the model as ( ) = (1 + [ 2 ( − ) 365 ]), where is a seasonal offset. this formulation was previously shown to accurately capture the seasonal variation in the incidence of respiratory diseases by us state (pitzer et al., 2015; yamin et al., 2016) . hospitalizations. hospitalization was not model explicitly. however, we computer the number of hospitalizations for each age and riskgroup by multiplying the number of symptomatic infected individuals by the rate of hospitalization given influenza infection. these age-and risk-specific rates were obtained from epidemiological studies (pitzer et al., 2015; yamin et al., 2016) . for each year, we parameterized vaccination uptake from state-specific influenza vaccine coverage data for different age groups, as observed from 2013 to 2018 (table supplementary s2) ("2010-11 through 2018-19 influenza seasons vaccination coverage trend report | fluvaxview | seasonal influenza (flu) | cdc," n.d.). we estimated vaccine efficacy using the cdc estimates for influenza vaccine efficacy between 2013 and 2018 ("cdc seasonal flu vaccine effectiveness studies | cdc," n.d.). antiviral treatment is provided to high-risk individuals who seek care in health clinics and hospitals. we parameterized our model using data from recent large-scale studies on the time to seek care and antiviral prescription among laboratory-confirmed high-risk influenza patients in the us (biggerstaff et al., 2014) . these data were used to inform our baseline treated scenario for each state. high-risk individuals receiving antiviral treatment have a reduced rate of hospitalization compared to nontreated high-risk patients. the age-specific reduction in hospitalization rate among treated high-risk individuals was obtained from previous retrospective and prospective studies (kaiser et al., 2003; piedra et al., 2009) . model calibration. we calibrated our model to weekly cases of influenza (confirmed by viral isolation, antigen detection, or pcr) and influenza-like illnesses (ili) cases reported ("fluview interactive | cdc," n.d.) to estimate empirically unknown epidemiological parameters (si appendix, tables supplementary s3 and s4). these data were collected by the cdc's national respiratory and enteric virus surveillance system and state health departments from 2014 to 2019. we used data from a recent meta-analysis (tokars et al., 2018a) of seasonal influenza in the us between 2011 and 2016 to obtain the median annual attack rate per age group. interventions. we evaluated two interventions for increasing the number of high-risk patients seeking care and being treated within the first two days. in the first intervention, we increased the number of individuals who are treated within the first two days of symptoms onset by assuming that a proportion of those who received treatment after the first two days of symptoms onset would receive treatment within the first two days of symptoms onset. in the second intervention, we increased the total number of infected high-risk individuals who received treatment while assuming that they all received treated within the first two days of symptoms onset. we evaluated the population-and individual-level benefits of these interventions in terms of infections and hospitalizations averted in during a single influenza season. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 sensitivity analysis. we conducted sensitivity analyses to examine the robustness of our results. in the first analysis, we investigated the impact of effective vaccination coverage on the effectiveness of antiviral treatment. effective vaccination coverage is defined as the product of vaccine efficacy times vaccine coverage, and represents the level of vaccine induced immunity in the population. in the second analysis, we conducted a two-way sensitivity analysis to investigate the joint impact of changing both the attack rate and the effective vaccination coverage. the transmission model was calibrated to age-stratified weekly incidence data for the 2014all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint a figure 1. structure and fit of the model. (a) compartmental diagram of the transmission model. following exposure, susceptible individuals s move to the symptomatic or asymptomatic compartment, i τ=0 , a τ=0 , representing the first day of infection. then, on each following day, they transition to a matching compartment, where they may transmit the disease to others based on 1) their contact mixing patterns, 2) the per-day viral load, 3) the seasonal forcing, and 4) their antiviral treatment regimen. recovered individuals move to compartment r, where they are fully protected for the entire season. for clarity, age and risk stratification are not displayed (si appendix). (b, d) time series of recorded weekly influenza cases and model fit to california and texas (the model fit to connecticut and virginia is provided in si appendix, figure supplementary s2). (c, e) data and model fit to the age distribution among influenza infections. we simulated five influenza seasons and projected the number of cases and influenzainduced hospitalization that would have been averted by early treatment. specifically, we evaluated the population-level benefit of increasing the proportion of high-risk individuals who initiate treatment within 48 hours of symptoms onset without increasing the baseline all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 given that the benefit of treatment depends on the underlying vaccination coverage within the population, we also examined how variation in vaccination coverage affects the benefit of treating high-risk individuals. we found that increasing effective vaccination coverage would decrease both influenza burden and the benefit of treatment ( figure 3) . nevertheless, for a 20% increase in influenza effective vaccination coverage among all age groups, our results suggest that the benefit of treatment remains substantial ( all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 to estimate the benefits of a policy that targets specific age groups for early treatment, we evaluated the effectiveness of age-targeted treatment strategies for averting influenza cases and influenza-induced hospitalizations ( figure 4 and figure supplementary s5). we found that treatment of the elderly (>65 years old) has the highest impact on reducing hospitalizations (figure 4 b and d). this result was driven mainly by the fact that this age group has the highest risk for influenza complications, which leads to a higher rate of hospitalization. the highest impact on reducing transmission was achieved by targeting highrisk individuals aged 5-19 years old. for example, in texas, early treatment of the 5-19 years old age group will avert 2.31 cases per treatment, and early treatment of the >65 years old age group will avert 0.04 hospitalizations per treatment. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint the yearly attack rate of influenza varies considerably between seasons. thus, we explored the benefit of treating high-risk patients under different attack rates and effective vaccination coverage. we found that the benefit conferred by treatment decreases with increasing attack rate and increases with increasing effective vaccination coverage ( figure 5 and figure supplementary s6). with a medium attack rate of 12.03% and 11.86% effective vaccination coverage, which is 60% from the yearly mean effective vaccination coverage, 0.69-0.78 cases are averted per treatment in california. with an attack rate of 5.70% and the same effective vaccination coverage, 0.96-1.11 cases are averted per treatment. for the same transmission settings in texas, for a yearly attack rate of 15% and 12.11% effective vaccination coverage, 0.49-0.55 cases are averted per treatment. with an attack rate of 2.81% and the same effective vaccination coverage, 1.65-1.81 cases are averted per treatment. in texas, the benefit conferred by treatment was found to decrease when the effective vaccination coverage exceeds 22.50% ( figure 5c ). in all settings considered, the marginal benefit of treatment per dose was found to decrease with increasing treatment coverage and vaccination coverage ( figure 5 ). this saturation in the benefit of treatment is driven by the increased herd immunity resulting in a decrease in the indirect benefit of treatment. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint our key finding shows that increasing the timeliness of treatment of high-risk patients, even without increasing the current treatment coverage, is highly effective in reducing morbidity and mortality associated with influenza at the population level. the reason behind this finding is that the viral load of influenza is the highest during the first three days from symptoms onset. earlier treatment reduces the viral loads and thus has a pivotal nonlinear effect of reducing transmission. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 interventions that could improve the timeliness of high-risk patients seeking care and their access to timely antiviral prescriptions and potentially reduce influenza-associated morbidity and mortality are urgently needed. these interventions could include education of high-risk patients, education of physicians about the benefits of early antiviral treatment among highrisk patients, and innovative tools to enhance early detection of influenza infection and treatment. these tools include providing phone consultations or remote electronic visits (virtual visits). vaccination remains the main tool for controlling seasonal influenza. however, vaccine efficacy varies widely between influenza seasons, and vaccination coverage remains suboptimal ("cdc seasonal flu vaccine effectiveness studies | cdc," n.d.; . our study shows that the benefit of treating early and increasing the treatment coverage is substantial, regardless of vaccine efficacy and coverage. counterintuitively, we found that the higher the effective vaccine coverage is, the higher the marginal outcome of treatment. this phenomenon is driven by the fact that high effective vaccination coverage results in low disease transmission, which in turn increases the indirect benefit of treatment. this finding emphasizes the importance of antiviral treatment as a complementary effort to vaccination. despite the effectiveness of antivirals in reducing influenza-related morbidity and mortality, the emergence of drug resistance poses a critical limitation on their application. therefore, parsimonious use of antivirals is needed to mitigate the emergence of influenza antiviralresistant strains. studies have suggested that to reduce the risk of antiviral overuse while maximizing their use to mitigate the burden of influenza, low-risk patients should be tested before treatment with antivirals and high-risk patients with clinically diagnosed influenza all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint infection should receive prompt treatment pending results of a laboratory-confirmed test (sintchenko et al., 2002) . our study shows that increasing the timeliness of treatment without increasing the number of treated individuals would substantially increase the population-level benefit of antiviral treatment. for example, if the proportion of high-risk patients who receive treatment within the first 48 hours of symptoms onset were to increase from its baseline value of 8.1% to 14.85% (the total number of high-risk patients who receive treatment: both within and after 48 hours), it could avert an additional 65,210 (50,206-83,713) cases annually in texas, 90, 842 (74, 531) cases in california, 7,012 (6,297-10,187) cases in connecticut, and 18, 230 (15, 471 ) cases in virginia. the ongoing coronavirus (covid-19) pandemic has already put unprecedented strain on the health system of many countries. as the disease continues to unfold across the world, its impact on national health systems is yet to be fully understood. in the us, the possibility of covid-19 transmission during the next influenza season is raising a substantial concern about the health system being overwhelmed by visits from both covid-19 and influenzarelated complications among high-risk patients. the specter of this challenging scenario emphasizes the importance of the results of this study and the urgent need for increase influenza vaccine uptake and timeliness of antiviral treatment among high-risk patients in the us our study includes several limitations that should be addressed by future studies. although several studies have attempted to estimate the annual attack rate of influenza in the us (jayasundara et al., 2014; molinari et al., 2007; tokars et al., 2018b) , the state-level rates remain unknown. therefore, we used the nationwide attack rate to normalize the statespecific influenza cases. moreover, we used nationwide data to estimate the treatment all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint coverage and timeliness for each state, as state-specific data are not available. under these assumptions, our results were qualitatively similar across all states, with quantitative differences being driven by state-specific information on population size and demography, vaccination coverage, and influenza seasonality. in conclusion, increasing the timeliness and coverage of antiviral treatment among high-risk individuals has the potential to substantially reduce the burden of seasonal influenza in the us. timely treatment not only reduces the risk of influenza-induced hospitalization for the treated individual but may also reduce disease transmission. public health decision makers should invest continuous efforts to follow the cdc guidelines by treating influenza patients at high risk as soon as possible. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint 1. model we developed a dynamic model for age-stratified influenza infection progression and transmission in four states in the united states. our model is a modified susceptible-exposed-infected-recovered (seir) compartmental framework (vynnycky and white, 2010) , whereby the population is stratified into health-related compartments, and transitions between the compartments occurs over time (main text, figure 1 ). to model age-dependent transmission, we stratified the population into n = 5 age groups: 0-4 years, 5-19 years, 20-49 years, 50-64 years, and ≥65 years. consistent with immunological observations ("antibodies crossvaccine on cross-reactive antibodies -united states," n.d.; branch et al., 2012; hancock et al., 2009; mandelboim et al., 2014; ranjeva et al., 2019; sharabi et al., 2016) , we assumed age-dependent susceptible reduction due to preexisting serum influenza neutralizing antibodies from previous exposure which reduce susceptibility to infection. consistent with previous models (medlock and galvani, 2009; ndeffo mbah et al., 2013; yamin et al., 2014) , we assumed that upon recovery individuals are fully protected for the entire season. this assumption is also supported by prospective studies demonstrating that reinfection in the same season is rare, yet possible (möst et al., 2019; möst and weiss, 2016) . accordingly, we stratified the population into four health-related compartments: where the index ∈ {1,2, . . . , } represents the age-group of each individual, and the index ∈ { , } specifies the risk-group of each individual (i.e. high-risk, or low-risk). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint before the beginning of each influenza season, individuals start from susceptible compartment , (0). individuals who are immune, due to preexisting serum influenza neutralizing antibodies from previous exposure (proportion of of each age-group), are not included in the susceptible compartment. thus, they are transitioned to the recovered compartment , (0) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. the rate at which individuals transmit influenza at time t is ( ). this rate depends on the combination of 1) age-specific contact rates between an infected individual and his or her contacts, 2) infectiousness of the infected individual based on his or her daily viral loads, and 3) age-specific susceptibility to infection. in the us, influenza incidence is seasonal, with a peak typically striking in the winter, yet the driver for this seasonality remains uncertain (lipsitch and viboud, 2009 ). thus, we included general seasonal variation in the susceptibility rate of the model as the seasonal offset will be calibrated, for each season separately, in order to fit the influenza case data. we set the boundaries of the search between the fourth week and the twenty-seventh week of the season (according to the data). this formulation was previously shown to accurately capture the seasonal variation in the incidence of respiratory diseases by us state (pitzer et al., 2015; yamin et al., 2016) . we incorporate age-specific contact patterns between individuals, represented by contact rate between an infected individual in age-group and each of their contacts with susceptible in age-group , denote by , . i.e. the contact matrix will be detailed in the next section. for high-risk individuals from age-group , we parameterize the timing of the antiviral treatment uptake (see si appendix 2.1 data set and parameters). for each day during the all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint exposed and infection periods ( ), we incorporate the proportion of untreated high-risk individuals , =0 , the proportion of high-risk individuals who got treated on the second day since symptoms onset , =2 , and the proportion of high-risk individuals who got treated on the third day since symptoms onset , =3 . high-risk individuals who got treated after the third day since symptoms onset are considered as treated ineffectively, with treatment having no impact on disease progression and severity (aoki et al., 2003; heinonen et al., 2010 ; "use of antivirals | cdc," n.d.). therefore, we include them in , =0 . given a contact with an infected host, the logarithm of the infectious viral load has been shown to be correlated with the transmissibility of several respiratory viruses (couch et al., 1969; tellier, 2009) . the logarithm of the viral load depends on the risk-group of the infected individual ∈ { , }, the timing of the antiviral treatment for the high-risk individuals , the day of infection which includes the exposed and infection periods, and on the type of infection. (see si appendix 2.1 data set and parameters). in addition, we consider the agespecific susceptibility rate of and individual , was parametrized by calibrating our model with weekly influenza records (see section 2.2. calibrated parameters). taken together, the force of infection ( ) is given by: (4) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 we parameterized the age-specific contact rates between an infected individual and their contact , , based on the contact matrix parameterized by a previous study (yamin et al., 2016) . we adjusted the contact mixing matrix to our model age-groups. for age-groups, 5 − 19 , 20 − 49 we analyzed the data used to build the contact matrix using the same methods described in that study. this contact data exhibits frequent mixing between similar age-groups, moderate mixing between children and adults in their thirties (likely their parents), and infrequent mixing between other groups. table s1 . age-specific rates , between an infected individual and their contact . using recent prospective studies of the course of influenza infections in young children and adults, we estimated the viral load for asymptomatic, symptomatic high-and low-risk and treated high-risk who got treated on the first three days since symptoms onset (ip et al., 2017; lee et al., 2013) . viral load for asymptomatic estimated using the results of a prospective study that tracked after households in hong kong with an influenza confirmed patient (ip et al., 2017) . to estimate the viral load for the asymptomatic infected individuals, we smoothed the data of paucisymptomatic. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 for symptomatic high-and low-risk and treated high-risk who got treated on the first three days from symptoms onset, we estimated the viral load using the data of a prospective study monitored patients with laboratory-confirmed influenza who admitted to the medical department of the prince of whales hospital (lee et al., 2013) . day zero and days eight to fourteen since symptoms was estimated by fitting an exponential curve to the data. to estimate the viral load during the exposure period we scaled the data using the data of asymptomatic. for treated high-risk, the viral load prior to initiating treatment was set to be the same as for not treated high-risk individuals ( figure s1 ). figure s1 . daily log viral load following influenza infection for asymptomatic, symptomatic high and low-risk and treated high risk who got treated on the first three days since symptoms onset. the number of hospitalizations for each age-and risk-group was calculated by multiplying the number of symptomatic infected individuals by the probability of hospitalization given influenza infection (see table s3 ). using data from epidemiological study, we calculated the fraction of symptomatically infected individuals that will be hospitalized based on the ratio between hospitalizations and infection cases stratified by age (rolfes et al., 2018) . for agegroups 5 − 19 , 20 − 49y, 50 − 64y, ≥ 65 , the proportion of hospitalizations related to each risk-group estimated based on the ratio between hospitalizations of high-risk and low-risk as suggested by previous epidemiological study (mullooly et al., 2007) . for age-group 0 − 4 a previous retrospective study suggests that 37% of hospitalizations related to high-risk children all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint (ampofo et al., 2006) . these data are consistent with the us influenza hospitalization surveillance network data (chaves et al., 2015) . infected high-risk individuals who treated within three days from symptoms onset have a lower probability to be hospitalized (kaiser et al., 2003; piedra et al., 2009) . for individuals who younger than 19 years old, we reduced the probability of hospitalization given an infection by 75% (piedra et al., 2009) . for adults (older than 19 years old) we reduced the probability by 59% (kaiser et al., 2003) . we estimated the vaccination coverage based on data obtained from center for disease coverage trend report | fluvaxview | seasonal influenza (flu) | cdc," n.d.). the data is stratified by state, age, season, and risk-group. to adjust the age stratification in the data to the age-groups used in our model, we assumed uniform distribution for each age-group in the data. for age-groups without risk-group stratification we assumed equal vaccination coverage for both low-and high-risk individuals. we parametrize the vaccination coverage for each year at both national and state-level as observed from 2013-2018 (see table s2 ). for the national coverage we used the median vaccination coverage for each year. as some of the states do not have data for season 2013-14, we used the average coverage of seasons 2012-13,2014-15 as the coverage for season 2013-14. in both levels, we used the mean coverage over the five seasons as our baseline. as mentioned in the equation (2), the vaccination coverage , is multiplied by the vaccination efficacy to calculate the effective vaccination coverage. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint antiviral treatment is only provided to high-risk individuals that seek care in health clinics and hospitals. we assumed that following clinic or hospital visit and treatment prescription, it takes at least one day to a patient to initiate course of treatment. therefore, the earliest treatment initiation time is two days from symptoms onset. moreover, people who sought care on the third and fourth day since symptoms onset are considered as treated ineffectively, with treatment having no impact on disease progression and severity (aoki et al., 2003; heinonen et al., 2010 ; "use of antivirals | cdc," n.d.) . thus, we included them with the untreated individuals. hence, the probability of infected high-risk to seek care and get treated effectively (getting treated within 3 days from symptoms onset) is given by equation (5): the probability of seeking care given a high-risk infection stratified by age was calculated using data from an epidemiological study (molinari et al., 2007) (see table s3 ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 using data from recent large scale studies on the time to seek for care and antiviral prescription among laboratory-confirmed high-risk influenza patients in the us (biggerstaff et al., 2014; stewart et al., 2018) , we estimated the probability of seeking care withing two days from symptoms onset given an infected high-risk who sought for care and the probability of getting treated given an infected high-risk who sought for care withing two days since symptoms onset. those studies provided age-stratified data on the proportion of treated individuals who sought care during the first two days, 3 to 7 days, and 7 days since symptoms onset. further to our assumptions, we assumed high-risk individuals who sought care during the first two days since symptoms onset, have equal probability of treatment initiation time on the second and third day since symptoms onset. high-risk patients treated effectively are accounted for 54.5% of the treated high-risk patients (biggerstaff et al., 2014) . (sharabi et al., 2016) , population size of risk-group k age-group . varies between states (molinari et al., 2007) ("population distribution by age | the henry j. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 kaiser family probability of becoming asymptomatic given an infection. 0.191 (leung et al., n.d.) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 .07.28.20163741 doi: medrxiv preprint (furuya-kanamori et al., 2016 length of infection since exposure. 14 days (bell et al., 2006) , proportion of treated high-risk in age-group on day , initiate treatment on day ∈ {0,2,3} from symptoms onset. = 0 means either no treatment provided or treatment ineffectively. to estimate empirically unknown epidemiological parameters, we calibrated our model to the weekly number of influenza incidence according to the cdc data. we obtained the weekly ili cases for each state and season. we also obtained the weekly proportion of positive specimen to influenza (confirmed by viral isolation, antigen detection, or pcr). these data were collected by the cdc's national respiratory and enteric virus surveillance system and state health departments during 2014 to 2019 ("fluview interactive | cdc," n.d.). to account for all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted . . https://doi.org/10.1101 the influenza cases, we multiply the weekly ili cases by the proportion of positive specimens to influenza. moreover, to derive the weekly cases for each age-group, we assumed that the proportion of weekly cases for each age-group is the same as the state's population age structure. the age-group distribution for each state was obtained from the henry j. kaiser foundation's database ("population distribution by age | the henry j. kaiser family foundation," n.d.) . to account for unreported cases, the influenza weekly cases data described above was adjusted to fit the median attack rate of the age-group according to yamin et. al (yamin et al., 2016) . we used data from a recent meta-analysis study, of seasonal influenza in the us between 2011-2016, to obtain the median annual attack rate per age-group (tokars et al., 2018) . to derive the influenza weekly incidence, we divided the weekly influenza cases by the mean of the seasonal cases such that the yearly average attack rate is equal to the median attack rate. to calibrate the model to the incidence data we minimized the squared error between model predictions and incidence data. this is equivalent to maximum likelihood estimation assuming a normal distribution of the error. we conducted this calibration for each of the five seasons (2014-2019) separately. for the calibration, we assumed the median us vaccination coverage by age to account for the variation in attack rates due to vaccination uptake. also, we assumed the same susceptibility rate for both low-and high-risk in each age-group. the final transmission model (main text figure 1a ) included five parameters to be estimated through model calibration: seasonal offset ; seasonal susceptibility rate for age-group : 0-4y, 5-49y, 50-64y, 65y. table s4 . calibrated parameters. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint main text figure 2 to evaluate the number of cases and hospitalizations averted by early treatment (within 48 hours since symptoms onset. for conservative purposes, we assumed treating on the second day) of infected high-risk individuals who are currently receiving treatment on the third and more than three days after symptoms onset (main text, figure 2) , we ran the model for each of the five seasons with an increased proportion of the infected high-risk treated on the second day since symptoms onset. for each season, we ran the model using the average state's vaccination coverage. the addition to the proportion of the infected high-risk treated on the second day was done by shifting first the individuals who treated ineffectively (more than 72 hours since symptoms onset), followed by the individuals who treated on the third day. the number of additional cases and hospitalizations averted was computed as the average over the model projections of 5 seasons compared to the baseline case. for the range, we did the same analysis described above but we used the vaccination coverage of the season with the highest (lower bond) and the lowest (higher bond) vaccination coverage in the seasons between (2013 -2018) for each state. figure 3 we estimated the number of additional cases and hospitalizations averted due to increasing the treatment coverage of high-risk patients assuming that all receive treatment within 48 hours from symptoms onset. to assess the benefit in terms of cases and hospitalizations averted we ran the model with treatment coverage varying from 10%-30% for each season and state using the state's average vaccination coverage. we also assumed that there are no infected high-risk individuals who getting treatment three days since symptoms onset. for each fixed portion we compared the mean model projections to the baseline scenario. we also conducted a sensitivity analysis to examine the robustness of our results. in this analysis, we examined the effect of the vaccination coverage on the number of additional cases and hospitalizations averted by changing vaccination coverage for both model's projections of the new treatment policy and the baseline treatment scenario. we increased and decrease the state's mean vaccination coverage by 10%, 20%. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 main text figure 4 to estimate cases and hospitalizations averted per treatment for each age group we analyzed the scenario in which all the infected high-risk individuals in the examined age-group are seeking care and getting treatment within 48 hours since symptoms onset. for the rest of the age-groups we change the timeliness of initiating treatment to 48 hours since symptoms onset without changing the baseline treatment coverage. the number of additional cases and hospitalizations averted per treatment was computed by comparing the model's projections of the new scenario with the baseline scenario, while considering the increase in the number of treated high-risk patients. figure 5 we conducted a two-way sensitivity analysis to investigate the joint impact of seasonal attack rate and vaccination coverage in each state on the number of cases averted per treatment. we ran the model with varying proportion of infected high-risk who seeks care and gets treated within 48 hours from symptoms onset from 10%-30% while increasing and decreasing the state's average vaccination coverage by 10%-40%. we conducted the analysis for median, high, and low seasonal attack rate for each state. the high and low attack rate were informed by the year with the lowest and the year with the highest attack rate for each state between 2013-2018. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10.1101/2020.07.28.20163741 doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july 30, 2020. . https://doi.org/10. 1101 update: influenza activity in the united states during the 2016-17 season and composition of the 2017-18 influenza vaccine patterns of influenza vaccination coverage in the united states from 2009 to 2015 influenza activity -united states, 2015-16 season and composition of the 2016-17 influenza vaccine interim estimates of 2018-19 seasonal influenza vaccine effectiveness -united states oseltamivir for treatment and prevention of pandemic influenza a/h1n1 virus infection in households antiviral agents for the treatment and chemoprophylaxis of influenza ---recommendations of the advisory committee on immunization practices (acip) prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) heterogeneous and dynamic prevalence of asymptomatic influenza virus infections update: influenza activity in the united states during the 2017-18 season and composition of the 2018-19 influenza vaccine antiviral effects on influenza viral transmission and pathogenicity: observations from household-based trials antiviral management of seasonal and pandemic influenza improving delivery of early treatment to influenza-infected patients natural attack rate of influenza in unvaccinated children and adults: a meta-regression analysis epidemiology, complications, and cost of hospitalization in children with laboratoryconfirmed influenza infection antibodies cross-reactive to influenza a (h3n2) variant virus and impact of 2010-11 seasonal influenza vaccine on cross-reactive antibodies -united states early administration of oral oseltamivir increases the benefits of influenza treatment nonpharmaceutical interventions for pandemic influenza, international measures influenza-like illness, the time to seek healthcare, and influenza antiviral receipt during the 2010-2011 influenza season -united states antibodies cross-reactive to influenza a (h3n2) variant virus and impact of 2010-11 seasonal influenza vaccine on cross-reactive antibodies -united states cdc seasonal flu vaccine effectiveness studies | cdc the us influenza hospitalization surveillance network 21 the minimal infectious dose of adenovirus type 4; the case for natural transmission by viral aerosol berman all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity heterogeneous and dynamic prevalence of asymptomatic influenza virus infections cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus early oseltamivir treatment of influenza in children 1-3 years of age: a randomized controlled trial viral shedding and transmission potential of asymptomatic and paucisymptomatic influenza virus infections in the community impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations influenza virus load in hospitalised patients the fraction of influenza virus infections that are asymptomatic: a systematic review and meta-analysis influenza seasonality: lifting the fog significant cross reactive antibodies to influenza virus in adults and children during a period of marked antigenic drift optimizing influenza vaccine distribution. science (80-) all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted the annual impact of seasonal influenza in the us: measuring disease burden and costs multiple influenza virus infections in 4 consecutive epidemiological seasons: a retrospective study in children and adolescents consecutive infections with influenza a and b virus in children during the 2014-2015 seasonal influenza epidemic influenza-and rsv-associated hospitalizations among adults optimal targeting of seasonal influenza vaccination toward younger ages is robust to parameter uncertainty effects of oseltamivir on influenza-related complications in children with chronic medical conditions environmental drivers of the spatiotemporal dynamics of respiratory syncytial virus in the united states population distribution by age | the henry j. kaiser family foundation age-specific differences in the dynamics of protective immunity to influenza annual estimates of the burden of seasonal influenza in the united states: a tool for strengthening influenza surveillance and preparedness epidemiological and virological characterization of influenza b virus infections influenza antiviral prescribing for outpatients with an acute respiratory illness and at high risk for influenza-associated complications during 5 influenza seasons-united states aerosol transmission of influenza a virus: a review of new studies seasonal incidence of symptomatic influenza in the united states introduction. the basics: infections, transmission and modelsan introduction to infectious disease modelling an innovative influenza vaccination policy: targeting last season's patients vaccination strategies against respiratory syncytial virus no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted key: cord-006517-845w9r6l authors: lalueza, a.; trujillo, h.; laureiro, j.; ayuso, b.; hernández-jiménez, p.; castillo, c.; torres, m.; folgueira, d.; madrid, o.; díaz-pedroche, c.; arrieta, e.; arévalo, c.; lumbreras, c. title: impact of severe hematological abnormalities in the outcome of hospitalized patients with influenza virus infection date: 2017-05-13 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-017-2998-4 sha: doc_id: 6517 cord_uid: 845w9r6l although hematological abnormalities have been described among patients with influenza virus infection, little is known about their impact on the outcome of the patients. the aim of this study was to assess the frequency and clinical impact of severe hematological abnormalities in patients with confirmed influenza virus infection. this was an observational retrospective study including all adult patients with diagnosis of influenza virus infection hospitalized from january to may 2016 in our institution. influenza virus infection was diagnosed by means of rrt-pcr assay performed on respiratory samples. poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) sofa ≥2, or (c) death. two hundred thirty-nine patients were included. applying the hlh-04 criteria for the diagnosis of hemophagocytic syndrome, cytopenias (hemoglobin ≤9 g/dl, platelets <100,000/μl or neutrophils <1,000/μl) were present in 51 patients (21%). patients with hematological abnormalities showed higher sofa scores, respiratory failure, septic shock and in-hospital mortality than the remaining patients. the composite endpoint was present in 33.3% in the cytopenias group vs. 13.3% in the group without cytopenias (p=0.001). in a multivariate analysis, variables associated with the composite endpoint were: use of steroids prior to present admission (or: 0.12; 95% ci: 0.015–0.96, p=0.046), presence of any hematological abnormality (or: 3.54; 95% ci:1.66–7.51, p= 0.001), and ldh>225 u/l (or:4.45; ci:1–19.71, p=0.049). hematological abnormalities are not uncommon among hospitalized patients with influenza virus infection, and they are associated with a poorer outcome. hematological abnormalities have been previously described among patients with influenza virus infection [1, 2] . moderate thrombocytopenia [1, 3, 4] and lymphopenia [1] [2] [3] [4] [5] [6] are the most common findings, whilst anemia and neutropenia are rarely described [4] . as an example, the incidence of leukopenia and thrombocytopenia in patients who were hospitalized for the treatment of influenza a (h1n1) pdm09 early in the u.s. epidemic was 20% and 14%, respectively [7] . most studies directed toward identifying mortality risk factors in influenza virus infection mainly take into consideration clinical variables, especially those related to comorbidities [2, 3] . little is known about the impact of hematological abnormalities in the prognosis of this infection [1, 2] . lymphopenia and thrombocytopenia are more frequently seen in patients with respiratory failure and shock [7, 8] . additionally, a tendency toward increased mortality has been observed in the presence of lymphopenia or thrombocytopenia. however, these findings have not been confirmed in large multivariate analyses [3] . moreover, hemophagocytic syndrome (hps) secondary to influenza virus infection has been rarely described in immunocompetent and immunocompromised patients, with a high mortality rate [1, [9] [10] [11] [12] [13] [14] [15] [16] [17] . nevertheless, the real incidence of this severe complication in patients with influenza is not known. to the best of our knowledge, there is only one prospective observational study carried out in 25 critically ill patients with respiratory failure and hps secondary to influenza a (h1n1) pdm09 infection. hps was found in 9 patients (36%), with a high mortality rate (89%) compared to those without hps (25%) [18] . the aim of the present study was to assess the frequency and clinical impact of hematological abnormalities in the range of those accepted by the histyocite society for the suspicion of hps [19] in patients who were admitted to the hospital with a confirmed influenza virus infection. we conducted an observational retrospective study including all adult patients with a diagnosis of influenza virus infection hospitalized from january to may 2016 in a 1300-bed tertiary teaching hospital in madrid, spain. the study protocol was approved by the university hospital 12 de octubre review board. a confirmed case was defined by a positive result of a real-time reverse-transcriptase-polymerase-chain-reaction (rrt-pcr) assay performed at the local laboratory performed on respiratory samples [nasopharyngeal swabs (flocked swabs in utm™ viral transport medium, copan, brescia, italy)] from adult patients with respiratory tract symptoms. for the molecular diagnosis, rna was extracted from 200 μl of the specimen using nuclisens®easymag instrument (biomérieux diagnostics, marcy l'etoile, france) and eluted in 50 μl. five μl of the elution were employed to perform each rt-pcr reaction. the modular duplex rrt-pcr for influenza a/influenza b detection (influenza a/b r-gene ™, biomérieux) was run in the lightcycler 480 instrument (roche) [20] . all samples testing positive for influenza awere subtyped using rrt-pcr previously described [21] to detect specific regions of subtypes h1 and h3 hemagglutinin. for the detection of influenza a (h1n1) pdm09 subtype commercially available primers and probe (realtime ready infa/h1n1 detection set, roche) [22] were used. hematological abnormalities secondary to influenza virus infection were only considered when they are in the range of the hlh-04 updated criteria proposed by the histiocyte society [19] for the diagnosis of hematophagocytic syndrome (hemoglobin ≤9 g/dl, platelets <100,000/μl, neutrophils <1000/μl). a diagnosis of hemophagocytic syndrome needed to fulfill at least five of the clinical and non-clinical findings included in the previously mentioned hlh-04 updated criteria [19] : (a) fever of 38.5°c or more, (b) splenomegaly, (c) cytopenias affecting at least two of three cell lineages in peripheral blood (hemoglobin ≤9 g/dl, platelets <100,000/μl, neutrophils <1000/μl), (d) hypertriglyceridemia (≥265 mg/dl) and/or hypofibrinogenemia (≤150 mg/dl), (e) hyperferritinemia (≥500 ng/ml), (f) hemophagocytosis in bone marrow, spleen, lymph nodes, or liver, (g) low or absent nk cell activity or (h) increased soluble il-2 receptor concentration. comorbidity was defined using the charlson index. immunosuppression was defined as the presence of any the following: active malignant neoplasia, autoimmune disease, solid organ transplantation, hiv infection, use of steroids or chemotherapy. use of steroids was defined as: (1) more than 20 mg/day of oral prednisone for 7 days or longer or (2) less than 20 mg/day over a minimum of 3 months [23] . respiratory failure was defined as the need for mechanical ventilation, either non-invasive positive pressure ventilation or invasive mechanical ventilation, including those patients who had a clinical indication for ventilatory support but were finally not ventilated. sepsis, septic shock and organ dysfunction were defined according to the terms proposed recently by the third international consensus definitions for sepsis and septic shock [24] using for this purpose the sofa score and the qsofa score. acute respiratory distress syndrome (ards) was defined according to the american-european consensus conference on ards [25, 26] . poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) sofa ≥2, or (c) death (related or not related to influenza infection). a descriptive analysis of patients was initially performed, comparing those who had cytopenias with those who did not have cytopenias. descriptive analysis was performed using means (±sd) or medians with interquartile ranges (iqr). student's t-test for independent samples was used to compare continuous variables. mann-whitney u test was used to compare continuous variables with a non-normal distribution, and the fisher exact test to compare proportions. we further analyzed the risk factors associated with poor outcome by means of a logistic regression model that included those variables found to be statistically significant at the univariate level or those deemed clinically relevant. associations were expressed as odds ratios (ors) with 95% confidence intervals (95% ci). seventeen independent variables were initially selected based on clinical judgment and published literature. prior to model fitting, cluster analysis was used to reduce the number of candidate variables. backward selection with a type i error rate of 0.05 was used to reach a final reduced model containing three predictor variables. discrimination of the final model was quantified via a c index (roc area). all statistical tests were 2-tailed and the threshold of statistical significance was p < 0.05. statistical analysis was performed with computer software ( during the study period 401 cases of influenza virus infection were confirmed by rt-pcr at our institution, of which 239 were included in the present study (fig. 1 ). all cases were diagnosed between january and may 2016. patient's demographic and clinical characteristics are shown in table 1 . only 4 (1.7%) patients were living in a nursing home at the time of admission, and three patients were hiv positive. only 40% of the cases were vaccinated against influenza with a mean time between vaccination and confirmed infection of 117.8 ± 26.3 days. symptoms initiated a median of 3 days (range 2-5 days) before influenza diagnosis. dyspnea was present in 59.4% of the patients, and bronchospasm was described in 43% of them. laboratory parameters of the study population are presented in table 2 . both baseline and nadir values were significantly lower in all cell lineages in patients who developed cytopenias during admission (fig. 2 ). the median time to nadir of leukocytes, neutrophils, lymphocytes, platelets and hemoglobin was 1 day (range, 0-4), 2 days (range, 0-4), 0 days (range, −1 to 0), 0 days (range, −1 to 1), and 1 day (range, 0-3), respectively. when applying the hlh-04 criteria, we observed that 21% (n = 51) of the patients had at least one hematological abnormality, while 7.5% (n = 18) had cytopenias affecting at least two of three cell lineages. as is shown in table 2 , neutropenia (<1000 neutrophils/μl) was present in 5.9% of the cases (n = 14), thrombocytopenia (<100,000 platelets/μl) in 15.1% of the cases (n = 36), and anemia (hb <9 g/dl) in 10.5% (n = 25) of the cases. in the group of patients with at least one hematological abnormality, neutropenia was present in 27.5%, thrombocytopenia in 70.6%, and anemia in 49%, respectively. only one patient fulfilled the five criteria required for the diagnosis of hps. one patient met three criteria for the diagnosis of hps, six patients met two criteria, and 23 cases met one criterion. overall, 20 of the 51 patients with at least one hematological abnormality did not meet any of the hlh-04 criteria. however, it is noteworthy that fibrinogen levels were only available in eight patients (>150 mg/dl in all cases), triglyceride levels in 20 patients (≥265 mg/dl in one case), and ferritin levels in 20 patients (>500 ng/dl in 8 cases). nk cell activity was not available in any case, while increased soluble il-2 receptor concentration was observed in one of the two patients from whom it was available. bone marrow biopsy was performed in one patient, which showed 26% of hemophagocytosis. splenomegaly was not reported in any patient. of 225 patients in which chest x-ray was performed, 50.2% (n = 113) had abnormal findings (56.8% in the cytopenias group vs. 48.6% in the group without cytopenias, p = 0.32). the most common radiologic findings were patchy infiltrates (22.2%) and interstitial infiltrates (8.4%). thoracic computed tomography was performed in 18 cases, of which 83.3% (n = 15) had abnormal findings (66.7% in the cytopenias group vs. 86.7% in the group without cytopenias, p = 0.44). the most common abnormalities were patchy infiltrates (39.8%) and ground-glass opacities (16.7%). as is shown in table 3 , 28 patients (11.7%) developed an associated bacterial pneumonia in the course of admission (p = ns between groups) with staphylococcus aureus being the causative microorganism in two cases (0.8%), both in the cytopenias group. antiviral treatment with oseltamivir was started in 96.7% (n = 231) of the patients and it was always initiated early upon confirmation of influenza diagnosis. empiric antibiotic treatment was started in 72.8% (n = 174) of the cases, with a median duration of treatment of 6 days (range 4.7-9). in [4] [5] [6] [7] [8] [9] [10] [11] in the group without cytopenias, p = 0.15). overall in-hospital mortality was 5.2%, with 4.6% influenza related mortality, and a 30-day in-hospital mortality of 4.6%. as is shown in table 3 , patients with hematological abnormalities presented with higher sofa and qsofa scores, had longer hospital and icu stays, and more frequently presented respiratory failure and septic shock that required results are expressed as mean ± standard deviation or median with interquartile range (iqr) or as absolute value (percentage). values in bold text are considered statistically significant a upper limit of ldh in the local laboratory is 225 mg/dl. b nk cell activity was not determined in any case, splenomegaly was not reported in any patient, bone marrow biopsy was performed in only one patient, and soluble il-2 receptor concentration was determined in only two patients. c dexamethasone (10 mg/m 2 ), observing a prompt clinical improvement icu admission. as a consequence, we did notice significant differences between the two groups in the poor outcome composite endpoint (33.3% vs. 13.3%, p = 0.001). patient characteristics with respect to the poor outcome composite endpoint are presented in table 4 . no statistically significant differences between active smokers (18.8% in the good outcome group vs. 26.2% in the poor outcome group) or ex-smokers (23.4% in the good outcome group vs. 23.8% in the poor outcome group) were observed. in the favorable outcome group, 181 of the cases (95.3%) presented complete recovery of the episode and nine patients (4.7%) presented sequelae, while in the poor outcome group only 25 of the cases (59.5%) presented complete recovery and five cases (11.9%) presented sequelae. twelve patients died during hospitalization. thirty-day related mortality was 0.5% in the good outcome group vs. 23.8% in the poor outcome group (p < 0.0001). a reduced model to identify factors associated with poor outcome was generated. the three following variables remained significantly associated with the presence of the composite endpoint: use of steroids prior to present admission (or: 0.12; 95% ci: 0.015-0.96, p = 0.046), presence of any hematological abnormality (or: 3.54; 95% ci: 1.66-7.51, p = 0.001), and ldh > 225 u/l (or: 4.45; ci:1-19.71, p = 0.049) ( table 5 ). this observational retrospective study seeks to address mainly two issues. on the one hand, we seek to identify the frequency of hematological abnormalities (cytopenias) secondary to influenza virus infection in patients who required in-hospital treatment. on the other hand, we seek to determine if these hematological abnormalities are associated with a poor outcome defined as a composite endpoint that included the presence of respiratory failure, sofa score ≥ 2, and mortality. values are expressed as the mean percentage difference (or median) in both groups (cytopenias vs no cytopenias) between initial laboratory findings and nadir. the mean had been used for the hemoglobin and the median for the rest of the variables. (1) group with cytopenias, (2) group without cytopenias) in the present study, hematological abnormalities were defined according to the criteria proposed by the hlh-04 for the diagnosis of hps [19] . one theoretical disadvantage of these criteria is that lymphocyte count is not considered as part of the diagnosis and lymphopenia is frequently seen in patients with influenza virus infection. additionally, more severe cytopenias are considered in the hlh-04 definition than those frequently reported in patients with influenza virus infection [1] [2] [3] [4] [5] [6] . however, it is noticeable that in the present study we observed that 21.3% of the patients presented with at least one cytopenia in the hlh-04 range, while 7.5% of the cases presented with cytopenias affecting at least two of three cell lineages. thrombocytopenia (15% of all patients) was the most common hematologic abnormality. as is shown in table 2 , these hematological abnormalities were already present in baseline laboratory data at admission. additionally, the median time to nadir was between 0 and 1 day depending on the affected cell lineage. these observations suggest that cytopenias present early in the course of the infection. we therefore believe that the presence of hematological abnormalities is an early predictive marker of poor outcome in patients with influenza virus infection who required hospitalization. interestingly, we observed a tendency of a decrease in peripheral blood cell values even in patients without cytopenias, with a median time to nadir similar to those patients with cytopenias. we did not randomly choose the hlh-04 criteria for defining the hematological abnormalities in the present study. we suspect that hemophagocytic syndrome triggered by influenza virus infection was more frequent than previously reported. unfortunately however, no specific disease markers were ordered in most cases, making it impossible for us to retrospectively know the exact incidence of this disorder in our present cohort. in fact, albeit 51 patients presented with at least one cytopenia according to the hlh-04 criteria [19] , fibrinogen levels were available in only eight cases, triglycerides and ferritin levels in 20 cases, and soluble il-2 receptor concentration in only two cases. although rarely, hemophagocytic syndrome has been described in patients with severe influenza virus infection, usually associated with a poor outcome and a high mortality rate [3, 27] . in beutel's study of 25 critically ill patients with influenza a (h1n1) pdm09 virus associated hemophagocytic syndrome, the absence of steroid therapy in the early phase of the infection might have contributed to the high incidence of hps (9 out of 25 patients) and the rather poor outcomes [18] . we consider that a low clinical index of suspicion for influenza associated hemophagocytic syndrome is the main factor for delayed initiation of immunomodulatory therapy, which could have improved the outcome in these patients [28] . in the present study, a multivariate model was conducted to identify risk factors associated with a poor outcome. presence of hematological abnormalities and ldh levels >225 u/l remained significantly associated with a worse outcome in patients with influenza virus infection, while previous use of steroids was identified as a favorable prognostic factor. although no conclusive evidence has been reached, thrombocytopenia has been associated with a worse outcome in previous studies [3, 4] . in our univariate analysis, both thrombocytopenia and anemia were significantly associated with poor outcome. to our knowledge, anemia as a poor prognostic marker in patients with influenza virus infection had not been previously described. indeed, the two most notable aspects of our model are the presence of any hematological abnormality as a marker of poor outcome and the potential beneficial role of steroids in these patients. an interesting finding in our study was the better outcome observed in patients who were already using steroids before admission, while the use of steroids during admission was associated with poor outcome. this paradox could be explained by two facts. first, as in other series [4, 18] , steroids were mostly prescribed for copd exacerbations, asthma exacerbations or bronchospasms, but not as immunomodulatory therapy; therefore, time to treatment initiation, duration of treatment, and prescribed doses, were not always optimal. in fact, only one patient in our series received high.dose steroids (dexamethasone 10 mg/m 2 ) and that was for the treatment of hemophagocytic syndrome triggered by influenza virus infection with marked hemophagocytosis in bone marrow. second, it is known that onset of influenza a viruses infections is very acute by triggering a cascade of immune responses and switching on almost all parts of the immune defense system [29, 30] . in fact, in the present study, hematological abnormalities presented early in the course of the infection in a large number of patients. we believe that steroid therapy before admission could have modulated the acute inflammatory response triggered by influenza virus infection in the early course of the disease, and with this, prevented an uncontrolled immune response [28] . this finding, if confirmed in properly prospective designed studies, may establish the utility of early use of steroids in some subgroup of patients with influenza virus who required hospitalization. by using more restrictive criteria for the definition of cytopenias as compared to other series, our study might have underestimated the percentage of patients who presented with hematological abnormalities. additionally, the observational retrospective nature of the study complicates the accurate assessment of the role of steroid therapy during admission, especially since it was mostly prescribed for other indications rather than the infection itself. this wide range of indications, although similar to other series, precludes a correct assessment of steroid therapy as a poor prognostic factor. the potential benefit of steroid therapy in patients with influenza virus infection with risk factors for poor outcome should be further studied. unfortunately, an active pursuit of hemophagocytic syndrome secondary to influenza virus infection was conducted only in a few cases, which precluded us to confirm the real incidence of this severe complication in the subgroup of patients with hematological abnormalities. since a complete blood count is an easily accessible test, we believe that it should be the cornerstone in the screening of a possible underlying hps secondary to influenza virus infection. if severe cytopenias were observed, we consider that a systematic determination of ferritin, triglycerides and fibrinogen levels would be beneficial. in the case of abnormal levels of any of these three hps markers, determination of nk cell activity and soluble il-2 receptor concentration should be ordered to confirm the diagnosis. significant hematological abnormalities are frequently seen in patients with influenza virus infection who required hospital admission and are associated with a poor outcome. the ongoing use of steroids upon the start of influenza was associated with a better prognosis suggesting that an early immunomodulatory therapy could improve the outcome of these patients. funding information this study has not received any funding. predictors and outcomes of respiratory failure among hospitalized pneumonia patients with 2009 h1n1 influenza in taiwan severe influenza in 33 us hospitals, 2013-2014: complications and risk factors for death in 507 patients risk factors for death from influenza a(h1n1)pdm09, state of sao paulo, brazil hospitalized patients with 2009 pandemic influenza a (h1n1) virus infection in the united states clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china diagnostic importance of relative lymphopenia as a marker of swine influenza (h1n1) in adults hospitalized patients with 2009 h1n1 influenza in the united states swine-origin influenza virus h1n1, seasonal influenza virus, and critical illness in children virus associated hemophagocytic syndrome accompanied by acute respiratory failure caused by influenza a (h3n2) a case of novel swine influenza a (h1n1) pneumonia complicated with virus-associated hemophagocytic syndrome virus-associated hemophagocytic syndrome caused by pandemic swine-origin influenza a (h1n1) in a patient after unrelated bone marrow transplantation fatal case of swine influenza virus in an immunocompetent host hemophagocytic lymphohistiocytosis associated with influenza a (h1n1) infection in a patient with chronic lymphocytic leukemia: an autopsy case report and review of the literature viral infections associated with haemophagocytic syndrome whole-exome sequencing reveals mutations in genes linked to hemophagocytic lymphohistiocytosis and macrophage activation syndrome in fatal cases of h1n1 influenza novel influenza a (h1n1) virus-induced hemophagocytosis: first case reported in saudi arabia fulminant hemophagocytic lymphohistiocytosis induced by pandemic a (h1n1) influenza: a case report virus-associated hemophagocytic syndrome as a major contributor to death in patients with 2009 influenza a (h1n1) infection hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections typing (a/b) and subtyping (h1/h3/h5) of influenza a viruses by multiplex real-time rt-pcr assays comparison of the roche realtime ready influenza a/h1n1 detection set with cdc a/h1n1pdm09 rt-pcr on samples from three hospitals in ho chi minh city risk of infectious complications in patients taking glucocorticosteroids the third international consensus definitions for sepsis and septic shock (sepsis-3) the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination the distinct clinical profile of chronically critically ill patients: a cohort study infections associated with haemophagocytic syndrome cytotoxic therapy for severe avian influenza a (h5n1) infection induction of innate immunity and its perturbation by influenza viruses the host immune response in respiratory virus infection: balancing virus clearance and immunopathology conflict of interest the authors declare no conflicts of interest.ethical approval the study protocol was approved by the university hospital 12 de octubre review board. key: cord-017893-ck0m3h7u authors: sandrock, c. title: update on avian influenza for critical care physicians date: 2007 journal: intensive care medicine doi: 10.1007/978-0-387-49518-7_90 sha: doc_id: 17893 cord_uid: ck0m3h7u human influenza pandemics over the last 100 years have been caused by h1, h2, and h3 subtypes of influenza a viruses. more recently, avian influenza viruses have been found to directly infect humans from their avian hosts. the recent emergence, host expansion, and spread of a highly pathogenic avian influenza (hpai) h5n1 subtype in asia has heightened concerns globally, both in regards to mortality of hpai h5n1 in humans and the potential of a new pandemic. in response, many agencies and organizations have been working collaboratively to develop early detection systems, preparedness plans, and objectives for further research. as a result, there has been a large influx of published information regarding potential risk, surveillance, prevention and control of highly pathogenic avian influenza, particularly in regards to animal to human and subsequent human to human transmission. this chapter will review the current human infections with avian influenza and its public health and medical implications. human influenza pandemics over the last 100 years have been caused by hi, h2, and h3 subtypes of influenza a viruses. more recently, avian influenza viruses have been found to directly infect humans from their avian hosts. the recent emergence, host expansion, and spread of a highly pathogenic avian influenza (hpai) h5n1 subtype in asia has heightened concerns globally, both in regards to mortality of hpai h5n1 in humans and the potential of a new pandemic. in response, many agencies and organizations have been working collaboratively to develop early detection systems, preparedness plans, and objectives for further research. as a result, there has been a large influx of published information regarding potential risk, surveillance, prevention and control of highly pathogenic avian influenza, particularly in regards to animal to human and subsequent human to human transmission. this chapter will review the current human infections with avian influenza and its public health and medical implications. influenza a, b and c are the most important genera of the or thorny xoviridae family. influenza a is responsible for human pandemic outbreaks and seasonal epidemics and influenza b is responsible for increasing cases of seasonal disease. influenza a viruses are enveloped, single-stranded rna viruses with a segmented genome. the eight rna segments of the genome encode for 11 viral proteins, including the polymerase proteins (fbi, pb2), matrix proteins (ml, m2) and the surface glycoprotiens hemagglutinin (ha) and neuraminidase (na). influenza a viruses are classified into subtypes on the basis of the antigenic properties of the hemagglutinin and neuraminidase glycoproteins expressed on the surface of the virion [1] . to date, 16 hemagglutinin and 9 neuraminidase subtypes have been identified and are found in 144 different combinations (e.g., hlnl, h3n2, h5n1, etc.) [2] . the hemagglutinin glycoprotein mediates attachment and entry of the virus by binding to sialic acid receptors on the cell surface [3, 4] . the binding affinity of hemagglutinin to the host sialic acid allows for the host specificity of influenza a. more specifically, avian influenza subtypes prefer to bind to sialic acid linked to galactose by a-2,3 linkages, which are found on duck intestinal epithelium and poultry and duck respiratory epithelium [5] . human virus subtypes, hi, h2, and h3, bind to a-2,6 linkages found in human respiratory epithelium. swine contain both a-2,3 and a-2,6 linkages in their respiratory epithelium allowing for easy co-infec-tion with both human and avian subtypes [6] . this reason has been cited as the hkely genesis of novel strains, as in the 1968 h3n2 human pandemic, and has given pigs the designation of a ^mixing vessel' for new strains [7, 8] . to a lesser degree, humans have been found to contain both a-2,3 and a-2,6 unkages in their lower respiratory tract and conjunctivae which allows for human infections of avian strains [9] . the hemagglutinin glycoprotein is the main target for immunity by neutralizing antibody. the neuraminidase glycoprotein allows the spread of the virus by cleaving the glycosidic linkages to sialic acid on host cells and the surface of the virus [10] . the virus is then spread in secretions or other bodily fluids. the neuraminidase glycoprotein is a lesser target for immunity by neutrahzing antibodies, but is the target site for the antiviral neuraminidase inhibitors. in addition to hemagglutinin and neuraminidase classification, influenza a viruses are characterized by their pathogenicity. highly pathogenic avian influenza (hpai) is defined by the world organization for animal health (oie) as any influenza that causes severe disease or death in domestic poultry. hpai viruses, with very few exceptions, are of the h5 or h7 subtype, but not all h5 and h7 subtypes are hpai viruses. the potential pathogenicity of h5 and h7 subtypes can be evaluated by sequencing the hemagglutinin gene, since pathogenicity is associated with the presence of multiple basic amino acids at the hemagglutinin cleavage site. a change from a low pathogenic h5 or h7 subtype to a highly pathogenic form may occur upon introduction into poultry and is thought to occur primarily as a result of insertion of basic amino acids in the hemagglutinin cleavage site. molecular studies have shown that the 1918 human pandemic hlnl subtype originated as a low pathogenic avian virus in contrast with current human cases of h5n1 worldwide, which are the result of a highly pathogenic avian influenza virus. influenza a viruses are highly variable as a result of molecular changes in the rna segments that occur through a number of mechanisms; the most important of which are point mutation (antigenic drift) and rna segment reassortment (antigenic shift) [10] . like other rna viruses, the influenza a viruses lack proofreading ability, and are, therefore, subject to point mutations [10] . these individual mutations in the viral genome cause minor changes in the antigenic character of virus, with amino acid changes in hemagglutinin and neuraminidase of principal importance. reassortment occurs when a host cell is infected with two or more influenza a viruses and leads to the creation of a novel subtype containing a new hemagglutinin or neuraminidase that is immunologically distinct from those of the previous circulating strains, as can be seen in pigs, which possess receptors for both the human and avian subtypes [8] . three major pandemics have occurred in the last century (1918 hlnl, 1957 h2n2, and 1968 h3n2) through reassortment. however, point mutations leading to viral adaptation to a human host can occur with any avian influenza subtype. influenza a viruses infect a wide range of hosts including many avian species, and various mammalian species such as swine, ferrets, felids, mink, whales, horses, seals, dogs, civets, and humans [11] [12] [13] . wild birds (ducks, geese, swans, and shorebirds) are important natural reservoirs of these viruses, and all of the known 16 hemagglutinin and 9 neuraminidase subtypes have been found in these birds. in most cases. these subtypes are found within the gastrointestinal tract of the birds, shed in their feces, and rarely cause disease. since 2002, however, hpai h5n1 viruses originating in asia have been reported from approximately 960 wild bird species, causing disease in some instances and asymptomatic shedding in others [14] . the virus has now spread across asia, europe, the middle east, and some african countries. additional species, such as tigers, leopards, cats, stone martens, and humans have also become infected with hpai h5n1. the wide host range of many of these bird species may be one potential mechanism of spread of hpai h5n1 worldwide, thus complicating the potential contact, transmission, and mutability of hpai h5n1 in animal and human populations. the incidence of avian influenza infections in humans has increased over the last decade (table 1) [ii, 12, [15] [16] [17] [18] [19] [20] . initially, cases of avian influenza (h7n7) in humans occurred in association with poultry outbreaks, manifesting as self-limiting conjunctivitis [11] . then, in 1997, a large scale hpai h5n1 outbreak occurred among poultry in hong kong, with 18 documented human cases [17] . through the fecal-oral and respiratory routes among wild birds and poultry [13, 14] . human interaction with these infected secretions and birds was the major mode of transmission, with contact including consumption of undercooked or raw poultry products, handling of sick or dead birds without protection, or food processing at bird cleaning sites [11, 12, 15-17, 19, 22-24] , all birds were domesticated poultry or waterfowl, and no transmission from wild birds or contaminated water has been reported. in a few cases, limited human to human transmission was reported among health care workers and family members ( table 2 ) [24] [25] [26] [27] [28] . in each of these cases, no personal protective equipment was utilized and was the major factor in transmission between humans. the clinical manifestations of avian influenza in humans have ranged from mild conjunctivitis to severe pneumonia with multiple organ failure (mof) [11, 12, 15-20, 22, 23, 29] . while the ages of the patients have varied, the majority of cases in both the 1997 and 2003 hpai outbreaks were young. in 1997, the median age of the cases was 17.2 years, while the cases from 2003-2004 in southeast asia had a median age of 16 (range 2 months to 90 years). nearly all cases were linked with sick and infected poultry, and the incubation period ranged from 2 to 8 days from contact to symptoms. the symptoms in each outbreak have varied with the avian influenza a subtype. in 2003 during the netherlands outbreak with subtype h7n7, 92 % (82 of 89) presented with conjunctivitis [11, 29] . the other cases in canada and the uk also presented with conjunctivitis [22] . however, with hpai in hong kong in 1997, 18 of the cases had an influenza-like illness [17, 19] . in 11 cases, pneumonia developed with 6 of these progressing to mof, acute respiratory distress syndrome (ards), and death [17] . reye syndrome, pulmonary hemorrhage, and predominant nausea, vomiting, and diarrhea complicated cases. cases from the worldwide outbreak originating in southeast asia had similar presentations to the 1997 hpai h5n1 cases [30] [31] [32] [33] . the main presenting symptom was pneumonia with fever and an influenza-like illness. diarrhea was present in up to 70% of the cases. many cases had both thrombocytopenia and lymphopenia. chest radiographic findings included interstitial infiltrates, lobar consolidation, and air bronchograms. sixty-eight percent of patients developed ards and mof within 6 days of disease onset. the case fatahty rate has ranged from 67 -80 %, depending on the case series [34] . once the cases reached the critical care unit, however, the mortality was 90%. the average time of death from disease onset was 9-10 days. post-mortem studies have illustrated findings consistent with mof and overwhelming systemic inflammatory response syndrome (sirs), including diffuse alveolar damage, acute tubular necrosis and atrophy, disseminated intravascular coagulation (dig), and multi-organ damage [35] . interestingly, the virus has been isolated from the lungs, intestine, spleen, and brain, suggesting viremia. however, active replication of the virus was limited to the lungs. this overwhelming inflammatory response, with acute lung injury (ali) and ards as the predominant feature, coincides with the findings of a preferential binding of the avian influenza a viruses to a-2,3 linkages in type ii pneumocytes of the lower respiratory tract of humans [36] . subsequent viral replication, cytokine release, overwhelming host immune response, and the subsequent systemic manifestations then occur. the clinical diagnosis of avian influenza infection in humans is difficult and relies on the epidemiological link to endemic areas, contact with sick or dead poultry, or contact with a confirmed case of avian influenza. since many infectious diseases present with these findings, the only feature significant to the clinician may be contact in an endemic area, through travel or infected poultry, and the clinician should always elicit this detailed history. the definitive diagnosis is made from isolation of the virus in culture from clinical specimens. this method not only provides the definitive diagnosis, but the viral isolate is now available for further testing, including pathogenicity, antiviral resistance, and dna sequencing and analysis. alternatively, antibody testing can be performed, with a standard four-fold titer increase to the specific subtype of avian influenza virus. neutralizing antibody titer assays for h5, h7 and h9 are performed by a microneutralizaiton technique. western blot analysis with recombinant h5 is the confirmatory test for any positive microneutralization assay. more recently, rapid diagnosis can be performed with reverse transcription-polymerase chain reaction on clinical samples with primers specific for the viral subtype [37] . this test should only be performed on patients meeting the case definition and with an indirect immunoflorescence or enzyme immunoassay test confirming influenza a. any suspected case of avian influenza in a human should be investigated by the public health officials in the province or country of origin [38] . additionally, governmental laboratories are often equipped with the appropriate biolevel safety 3 laboratories, primer libraries, and associated expertise to confirm the diagnosis quickly and efficiently. any clinical specimens should be submitted with the assistance of the public health experts treatment of avian influenza infections in humans includes antiviral therapy, supportive care, and adjunctive therapies [39] [40] [41] [42] [43] . controlled chnical trials on the efficacy of antivirals (neuraminidase inhibitors), supportive therapy, or adjuvant care have never been performed, so current recommendations stem from the experiences of past avian influenza outbreaks and animal models. the adamantanes (rimantadine and amantadine) and neuraminidase inhibitors (oseltamivir and zanamivir) are the antivirals used for treatment and prophylaxis of influenza infections in humans [42] . adamantanes bind to the m2 protein on the viral capsule, inhibiting dissociation of the matrix proteins from the nucleocapsid during viral uncoating. in avian influenza virus infections, adamantanes have no role due to widespread resistance. hpai h5n1 isolated from southeast asia carried the mutation in m2 that conferred resistance to this group of antivirals. in fact, over 90% of isolates of hi and h3 human subtypes during seasonal influenza have had resistance to the adamantanes, thereby limiting their use in seasonal epidemics with human subtypes. their role has been limited now to prophylaxis in the community when the circulating strain is known to be susceptible to the adamantanes. neuraminidase inhibitors (oseltamivir and zanamivir) have been extensively studied for both treatment and prophylaxis in the human influenza a subtypes, hi, h2, and h3, as well as influenza b [40, 42] . in avian influenza, the efficacy has been well documented in animal models where improved survival has been seen after infection with hpai h5n1. the timing of treatment is paramount, as earlier therapy is directly related to improved survival. the greatest level of protection was seen if the neuraminidase inhibitors were started within 48 hours of infection, and protection rapidly dropped after 60 hours. in hpai h5n1 cases from southeast asia, survival appeared to be improved in patients who received oseltamavir earlier at 4.5 days compared to 9 days. both of these times are much longer than documented in the animal models, so the window of optimal therapy is largely unknown. additionally, therapy with oseltamivir has been shown to decrease the viral level in nasal secretion in patients infected with hpai h5n1. for oseltamavir, therapy has been at 75 mg twice daily, with 75 mg once daily reserved for prophylaxis. the drug has a 90 % oral bioavaflability and reaches significant plasma and broncho alveolar lining fluid levels. zanamivir is available in a dry powder inhalation at 10 mg twice daily for treatment and 10 mg daily for prophylaxis. zanamivir has not been used in human avian influenza cases, and some concern exists over treatment with an inhalation powder as plasma levels are significantly lower than with oseltamivir. neuraminidase inhibitor resistance has been documented in hpai h5n1 subtype in a vietnamese girl treated with 75 mg daily for 4 days for post-exposure prophylaxis [44] . the na glycoprotein had a histadine to tyrosine substitution at position 274, conveying markedly higher ic 50 for oseltamivir. zanamivir resistance was not found with this change [39, 41] , neuraminidase resistance has not been documented in other hpai h5n1 cases to date. combination therapy has not been studied in influenza a viruses. ribaviron by inhalation has been evaluated in vitro with some avian influenza a subtypes and has been found to reduce mortality from influenza b in a mouse model. further animal model studies are indicated to determine if there is a role for ribaviron or combination therapy with avian influenza a viruses [42] . supportive care with i.v. rehydration, mechanical ventilation, vasopressor therapy, and renal replacement therapy are required if mof and ards are a feature of disease [43] . due to the progression of pneumonia to ards, non-invasive ventilation is not recommended, and early intubation may be beneficial before overt respiratory failure ensues. corticosteroids have been used in some patients with hpai h5n1, but no definitive role for steroids has been determined. other immunomodulatory therapy has not been reported. human vaccination for avian influenza viruses has not been widely used, although multiple vaccination trials are underway. prior avian vaccines in humans have been poorly immunogenic and thus have limited use [45] [46] [47] [48] . an inactivated h5n3 has been tested and was tolerated but with hmited immunogenicity [45] , other h5 vaccines have developed neutralizing antibodies, but to a limited degree. recently, a large randomized trial looked at an h5n1 attenuated vaccine from the vietnam strain. only a modest immunologic response was seen, with microneutralization antibodies being developed at 12 times the dose for seasonal influenza. the side effects were minimal. a number of other industry trials with adjuvent vaccines are currently ongoing. sandofi-adventis has recently reported success with a h5n1 attenuated vaccine at low doses, but the results have not been released thus far. although promising, human vaccination against avian influenza viruses is still under development. underscoring this development is the uncertainty of a pandemic strain, which may have vastly different antigenic properties of any developed h5 vaccine. health care infection control is a crucial component in the management of avian influenza infection or a new pandemic strain. experience with the severe acute respiratory syndrome (sars) outbreak in 2002 has illustrated that appropriate infection control measures are paramount to reducing spread to health care workers and possibly the community [49] . therefore, the world health organization (who) and centers for disease control and prevention (cdc) recommend contact and airborne precautions for any initial suspected case of avian influenza in a human. in late october 2006, the cdc released an updated interim guidance on the use of masks and respirators in the health care setting (table 3 ). in certain high risk procedures, additional protection with an n-95 particulate respirator may be considered given the likelihood of generating aerosol particles that may enhance transmission (table 4 ). respiratory protection should be worn along with an impermeable gown, face shield, and gloves. initial cases should be placed in a negative pressure isolation room with 6-12 air changes per hour. hand hygiene with antibacterial soap or alcohol based washless gel should be standard, with appropriate basins at each patient room. seasonal vaccination of all health care workers should be preformed and emphasized to reduce spread. visitors and family members should be strictly monitored and limited to reduce the likelihood of spread. finally, antiviral chemoprophylaxis should be available to any health care workers with exposure to an infected individual. any symptomatic worker should be taken off duty and workplace surveillance should occur. with these aggressive measures, risk to the health care worker, patients, and family members will be reduced. avian influenza viruses have occurred with increased incidence within the human population, reflecting the delicate and tangled interaction between wildlife, domesticated animals, and humans. disease in humans can be limited to conjunctivitis or an influenza-like illness, but hpai h5n1 causes mainly severe pneumonia, respiratory failure, and death. most cases have occurred with direct transmission from infected poultry or waterfowl, with only a few limited cases of human to human transmission. treatment has been successful with the neuraminidase inhibitors if started early, and vaccine development is underway with a more immunogenic attenuated h5n1 virus preparation. infection control measures are the mainstay for prevention and disease reduction. avian influenza viruses may constitute part of the next pandemic, so appropriate knowledge, prevention, and treatment will reduce the likelihood of this occurrence. world health organization expert committee (1980) a revision of the system of nomenclature for influenza viruses: a who memorandum characterization of a novel influenza a virus hemagglutinin subtype (h16) obtained 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the severe acute respiratory syndrome virus single-step multiplex reverse transcription-polymerase chain reaction (rt-pcr) for influenza a virus subtype h5n1 detection genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia avian flu: isolation of drug-resistant h5n1 virus the neuraminidase inhibitor gs4104 (oseltamivir phosphate) is efficacious against a/hong kong/156/97 (h5n1) and a/hong kong/ 1074/99 (h9n2) influenza viruses resistant influenza a viruses in children treated with oseltamivir: descriptive study antiviral agents medical treatment of viral pneumonia including sars in immunocompetent adult oseltamivir resistance during treatment of influenza a (h5n1) infection safety and antigenicity of non-adjuvanted and f59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a randomized trial of two potential vaccines against h5n1 influenza safety and antigenicity of nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a randomised trial of two potential vaccines against h5n1 influenza cross-reactivity to highly pathogenic avian influenza h5n1 viruses after vaccination with nonadjuvanted and mf59-adjuvanted influenza a/duck/singapore/97 (h5n3) vaccine: a potential priming strategy efficacy of h5 influenza vaccines produced by reverse genetics in a lethal mouse model h5n1 virus attachment to lower respiratory tract key: cord-026641-eemp6b5j authors: kabiljo, julijan; laengle, johannes; bergmann, michael title: from threat to cure: understanding of virus-induced cell death leads to highly immunogenic oncolytic influenza viruses date: 2020-06-11 journal: cell death discov doi: 10.1038/s41420-020-0284-1 sha: doc_id: 26641 cord_uid: eemp6b5j oncolytic viruses constitute an emerging strategy in immunomodulatory cancer treatment. the first oncolytic virus, talimogene laherparepvec (t-vec), based on herpes simplex virus 1 (hsv-1), was approved by the food and drug administration (fda) and european medicines agency (ema) in 2015. the field of oncolytic virotherapy is still in its beginnings, since many promising viruses remain only superficially explored. influenza a virus causes a highly immunogenic acute infection but never leads to a chronic disease. while oncolytic influenza a viruses are in preclinical development, they have not made the transition into clinical practice yet. recent insights into different types of cell death caused by influenza a virus infection illuminate novel possibilities of enhancing its therapeutic effect. genetic engineering and experience in influenza a virus vaccine development allow safe application of the virus in patients. in this review we give a summary of efforts undertaken to develop oncolytic influenza a viruses. we discuss strategies for targeting viral replication to cancerous lesions and arming them with immunogenic transgenes. we furthermore describe which modes of cell death are induced by influenza a virus infection and how these insights may be utilized to optimize influenza a virus-based oncolytic virus design. • influenza a virus can be targeted to tumors and armed with cytokines. • growth optimized influenza a virus for a phase i application can be generated. • oncolytic influenza a virus prototypes have been tested in humans as vaccine candidates and proven to be safe. • optimal viral subtypes as well, as the impact of preexisting anti-viral immunity may still be determined. the observation of viral infections leading to the reduction of cancerous tumors was reported throughout the 20th century 1,2 . the first report of a complete tumor remission in the context of an influenza a infection dates back to 1904 3 . preclinical models solidified the assertion of oncolytic effects of influenza a viruses 4, 5 . however, the lack of understanding of virus biology made a safe and effective development of oncolytic a viruses impossible at that time. in the last decades advancements in molecular virology and viral engineering led to the clinical development of a variety of oncolytic viruses, including herpes simplex virus (hsv) 6 , reovirus 7 , vaccinia virus (vv) 8 , vesicular stomatitis virus (vsv) 9 , adenovirus 10 , newcastle disease virus (ndv) 11 , measles virus (mev) 12 and picornaviridae 13 . this renewed interest in oncolytic viruses resulted in the food and drug administration (fda) and european medicines agency (ema) approval of the first oncolytic virus, talimogene laherparepvec (t-vec), in 2015 6 . t-vec is an hsv, which is modified to grow selectively in tumor cells and express the immunostimulatory transgene granulocyte-macrophage colony-stimulating factor (gm-csf). the development of oncolytic viruses was based on conditional replication of prototype viruses in malignant tissue, while being attenuated in normal tissue 14 . the lytic effect of the virus was initially thought to be the main principle of their anti-cancer activity. later it turned out that the therapeutic effect was mainly promoted by a pro-inflammatory stimulation of the tumor immune microenvironment (time) counteracting tumor-associated immunosuppression. thus, the concept of virotherapy largely overlapped with the rational principles of immune checkpoint inhibiting antibodies [15] [16] [17] [18] [19] [20] [21] [22] . importantly, the combination of oncolytic viruses with checkpoint inhibitors appears to be highly beneficial in a number of preclinical models [23] [24] [25] . in this line, a seminal paper by zamarin et al. indicated , that an oncolytic ndv sensitized an immunologically "cold" murine tumor to systemic checkpoint inhibitors, which increased the rate of tumor remission 23 . similarly, combining t-vec with immune checkpoint inhibitors was associated with complete remission in 22% of stage iiib/iv melanoma patients in a small phase i study strongly supporting this concept 26 . those observations prompted a high level of interest in the field of virotherapy. with respect to oncolytic influenza a viruses, only preclinical studies have been accomplished yet, in spite of the fact, that this virus family is well studied and known to be highly immunogenic. in this review we summarize major milestones in the development of oncolytic influenza a viruses. we delineate in which manner targeted replication in cancer cells was achieved. furthermore, we discuss strategies to arm influenza a viruses with immuno-stimulatory transgenes. finally, we discuss which types of cell death influenza a virus-infected cancer cells succumb to and their implications for future design of oncolytic a viruses. there are several hallmarks viral candidates should possess in order to be considered for development into oncolytic agents: their genetics and biology should be well known and targeting to cancerous tissue needs to be feasible. they should be highly immunogenic and exert lytic activity leading to an immunogenic cell death (icd) of malignant cells, while sparing normal tissue 27 . they should not lead to a chronic disease or retain capability of integrating into the human genome. moreover, the use of the viruses as an oncolytic agent must be safe, in terms of excluding the possibility of the induction a pathogenic virus, which causes a disease the human population. it should also be feasible to genetically modify the virus and arm it with recombinant transgenes to enhance its immunogenicity or stimulate targeted anti-cancer mechanisms. utilizing an influenza a virus as an oncolytic agent has several advantages. the influenza virus is a small virus of the orthomyxoviridae family, commonly known for causing the flu 28 . it comprises 4 genera, influenza a, b, c, and d viruses, type a being the most extensively studied one 28, 29 . while the influenza virus can induce strong immunogenic reactions and intense pathology in humans, it never leads to chronic disease and attenuated forms have been described 30, 31 . influenza virus is an enveloped, negative-strand rna virus with no reverse transcriptase or dna integration activity 28 . these factors predispose it as an ideal vector for oncolytic therapy. oncolytic virus development focused on influenza a virus. this virus subtype contains 8 separate rna fragments, kept in cyclical conformation within the 80-120 nm large virion 28 . these segments encode 11 viral proteins necessary for viral structure and replication ( fig. 1) , as well as the nonstructural protein 1 (ns1) 28 , which antagonizes the anti-viral reaction of the host 32 . the extensive knowledge and infrastructure that has previously been established for the production of seasonal influenza vaccinations reduces the amount of novel biotechnological engineering and regulatory issues, which are necessary for clinical development of the virus in the field of oncology 33 . the rational development of a conditionally replicating phenotype of a virus in tumor tissue requires the understanding of virus-host interactions, particularly how viruses lyse infected cells and how cells protect themselves from the lytic infection. influenza a viruses have been shown to induce multiple distinct modes of cell death 34 . in the early phase of infection, the virally encoded protein ns1 inhibits apoptosis 35, 36 , suggesting that apoptosis plays a role in anti-viral defense 37, 38 . in the absence of ns1 apoptosis appears to be induced through the viral-rna-mediated induction of retinoic acidinducible gene i (rig-i) and interferon (ifn) signaling including protein kinase r (pkr) and eukaryotic initiation factor 2 alpha (eif2α) activation and subsequent block of translation [39] [40] [41] . ns1 has also been shown to inhibit apoptosis though interaction with the pro-apoptotic scribbled planar cell polarity protein (scribble) 42 . however, influenza a viruses have a two-sided relationship to apoptosis 37 . there is evidence, that growth of influenza viruses is dependent on apoptosis 43 . specifically, caspase 3 appears important for viral replication 44 . in this line, the influenza a virus can actively induce apoptosis. apoptotic signaling may be initiated intrinsically through the viral protein pb1-f2 45 . a further major inducer of apoptosis during influenza a virus infections is the viral nucleoprotein (np), interacting with the host's bcl-2associated x protein (bax) inhibitor clusterin, leading to bax induced apoptosis 46 . extrinsic induction of cell death, which inhibits viral replication at a late stage of viral life cycles, has been reported to occur through the release of tumor necrosis factor (tnf) receptor ligands, depending on nuclear factor kappa-light-chain-enhancer of activated b-cells (nf-κb) activation 47 . this process is counterbalanced by nf-κb inactivation through ns1 48 . the viral surface glycoprotein neuraminidase (na) can also be involved in induction of cell death, as it enhances apoptosis through activation of transforming growth factor beta (tgf-β) 49 . there are multiple theories, why influenza a virus may actively induce apoptosis. overall, there seems to be a fine, time-dependent balance of pro-and anti-apoptotic stimuli, which are tightly controlled by the virus. upon overexpression of anti-apoptotic molecules influenza a virus titers are reduced due to viral rna-protein complexes being retained in the nucleus 43, 50 . interestingly, caspase activation has been shown to enable diffusion of nuclear proteins into the cytoplasm 51 . this suggests that inhibition of both apoptosis and innate anti-viral responses through ns1 is necessary for viral propagation, especially in the initial phases of infection. in the late phase, activated caspases are needed to release viral rna from the nucleus (fig. 2 ). this theory is further reinforced by the observation, that the anti-mycotic amphotericin b enhances influenza virus growth 52 . amphotericin b stabilizes pores within cellular membranes. this mechanism has been shown to aid rna particles in passing through different cellular compartments 53 and might also enable viral rna release from the nucleus. influenza viral matrix-protein 2 (m2) and ns1 have been shown to induce autophagy 54 . this process leads to active transport of the danger associated molecular pattern (damp) adenosine triphosphate (atp) out of the cell, resulting in immunogenic reactions 55, 56 . in conclusion, influenza a virus induces an apoptotic cell death through a number of pathways. it should be taken into account, that virally encoded danger molecules convert the apoptotic cell death into an icd, leading to the stimulation of cytokines. in order to create a virus that initiates potent anticancer-immune responses without causing an infectious disease, it is necessary to target them to cancer cells. mammalian cells react to viral infections by secreting type i ifn. most viruses develop strategies to circumvent these immunogenic effects. it was shown, that the influenza a virus lacking ns1 (delns1) generated by egorov et al. 32 was unable to replicate in mice with a functioning ifn signaling pathway 57 . however, it retained lethality in signal transducer and activator of transcription 1 (stat1) knockout mice, which are unable to react to ifn 57 . since many cancers downregulate components of the ifn signaling pathway [58] [59] [60] , attenuation of the ns1 protein appeared to be an attractive strategy for targeting growth of influenza a viruses to malignant tissue. we were able to show that deletions of various lengths within the ns1 gene of an influenza a/puerto rico/8/34 (pr8) based h1n1 virus yielded potent anti-cancer effects in ifn resistant human melanoma (sk-mel1) xenografts 61 . another mechanism through which ns1 counteracts cellular responses to influenza a virus infection is the inhibition of the double-stranded rna sensor pkr 36, 62, 63 . we demonstrated that ns1 deleted influenza a virus was lethal to pkr knockout mice but was not lethal in wildtype mice 36 . oncogenic rat sarcoma (ras) gene mutation, present in approximately a third of all cancer subtypes, results in pkr inhibition 64 . we showed that ns1 deletion targets a pr8 influenza virus towards ras mutated tumors in a pkr dependent manner, using mouse xenografts of human 518 melanoma cells, transfected with oncogenic ras 65 . there are also developments of oncolytic influenza a viruses, which are based on attenuation markers other than the ns1 deletion. one preclinical study in murine non-small cell lung cancer (nsclc) xenografts showed efficacy and safety of wild-type ns1, laboratory adapted, pr8 h1n1 virus 66 . another study screened a variety of wild-type influenza a viruses for their infectivity in pancreatic carcinoma cell lines and showed oncolytic effectiveness in a mouse model of human pancreatic cancer 67 . similarly, the seasonal flu vaccination has recently been assessed for sensitizing murine b16 melanoma models to immune check point inhibitor therapy, which showed promising results 24 . ns1-deleted (delns1) viruses are potent stimulators of the immune system apart from the conditionally replicating phenotype in malignant cells influenza a viruses with ns1 deletions are associated with a more potent stimulation of the innate immune system than wild-type viruses. this is due to the fact that the attenuation is linked to deletions in the viral inhibitor of the innate immune system. the inhibitory effect of ns1 on the immune system is based on its polyfunctional nature (fig. 3a) . with respect to cellular innate immune mediators, ns1 was shown to inhibit interferon regulatory factor 3 (irf3) and nf-κb 48, 68 . since ns1 deleted influenza a viruses are capable of inducing strong pkr pathway upregulation, it seems plausible that known downstream signaling might lead to calreticulin (calr) exposure on the cell membrane 69 . here calr acts as a damp, causing enhanced immunogenicity of apoptotic bodies after influenza a virus infection in pkr sensitive cancers 70 . a transcriptional profiling confirmed that a delns1 virus infection caused a much more potent induction of an immuno-stimulatory gene pattern compared with wild-type virus 71 . correspondingly, infection of macrophages with the delns1 virus is not only associated with an icd, but also with a more potent stimulation of innate cytokines as compared with wild-type viruses 72 . the removal of c-terminal domains of ns1 provoked release of type i ifn, interleukin 6 (il-6), tumor necrosis factor α (tnf-α), and chemokine (c-c motif) ligand 3 (ccl3) 72 . removal of n-terminal domains induced release of interleukin 1β (il-1β) and interleukin 18 (il-18) 72 . importantly, whereas partial truncation of ns1 increased upregulation of pro-inflammatory genes, the full ns1 deletion leads to further increased expression 71 . the length of the ns1 deletion correlated with the potency of its immune stimulation and the level of its attenuation 31, 32 . full ns1 deletion results in viruses characterized by an abortive infection. thus, the level of pro-inflammatory immune stimulation and the level of attenuation can be titrated by the length of the deletion. correspondingly, we observed that partial ns1 deletion to 80 or 116 remaining amino acids (aa; removing the effector domain, but leaving the rna binding domain and one nuclear locating sequence) induced an optimal balance between attenuation and the oncolytic effect 61,73,74 . this truncation should reduce pkr pathway inhibition while retaining inhibition of immunogenic signaling through the rig-i pathway 62, 63, 75 (fig. 3a) . both pkr and rig-i have been implicated in type i ifn production 76, 77 . they both recognize the short double-stranded rna "panhandle", which holds the single stranded rna segments of the influenza a virus genome in a circular conformation [78] [79] [80] [81] . the immunogenic character of the delns1 virus had also been explored in the context of potential vaccination agents. in a preclinical study the use of complete or partial ns1 deleted h1n1, h3n2, and h5n1 subtypes as vaccines induced potent t cell and b cell responses, protecting mice and ferrets against influenza a virus challenge 31, 82 . importantly, the virus did not cause any disease in mice or the primate macaca mulatta when applied intranasally, corresponding to systemic application 82 . in a phase i clinical trial we evaluated a ns1deleted h1n1 virus as an intranasal vaccination vector 83 . another clinical trial conducted a similar study using the h5n1 virus 84 . these vaccine studies are relevant for the development of ns1-deletion viruses as oncolytic agents since they indicated that ns1-deletion viruses are safe in humans and are able to stimulate a potent adaptive immune response at the same time. certainly, the major requirement of oncolytic viruses is the stimulation of an adaptive t cell response against tumor-associated antigens (taa). in this line, we and others have demonstrated, that ns1-deletion viruses can be employed to stimulate dendritic cells (dc) to mount a t cell response against malignant cells via cross presentation 85, 86 . stimulation of t cells can either be induced by dcs exposed to delns1 virus generated virolysates or by exposing dcs infected with delns1 viruses to tumor cell lysates. again, partial ns1-deletion viruses were more effective than full deletion of the ns1 protein. those ex vivo assays might somewhat model the in vivo tumor microenvironment during oncolytic therapy. exposure of immune cells to oncolytic ns1 deletion viruses was also shown to induce direct ifn dependent cytotoxic effects of peripheral blood mononuclear cells (pmbcs), including t cells, b cells, monocytes and natural killer (nk) cells against various cancer cell lines 87 . these cytotoxic effects might contribute to the viruses' therapeutic effect in the tumor microenvironment. influenza a virus entry depends on a protease to cleave the hemagglutinin (ha), a protein mediating viral entry. thus, the presence of trypsin or equivalent proteases in the tissue is substantial for host restriction. it restricts the influenza a virus infection to the lung, the enteric system and for some viral isolates to the brain. we discovered that various colon cancer cell lines express trypsin and allow oncolytic influenza a virus growth in the absence of exogenous protease 73 . to further target the influenza a virus to tumor tissue we generated a virus in which the conformational change of ha, which enables viral entry, relies on the protease elastase 74 . elastase has been shown to be present in tumor tissue due to expression by neutrophils. elastase is also strongly expressed by malignant cells of pancreatic origin. replacing the trypsin cleavage site with an elastase cleavage site has been shown to attenuate influenza a virus replication in swine and mice [88] [89] [90] . we therefore exchanged the trypsin cleavage site within the partially ns1 deleted (116aa) pr8 influenza virus to elastase. elastase-dependent viruses yielded a potent therapeutic efficacy in murine melanoma (b16) and pancreatic ductal adenocarcinoma (panc-1) xenograft models 74 . thus, this attenuation marker present in ha can be used to target virus to tumor tissue and might allow for the use of ha which is currently not present in the human population, including the h7 or h9 subtypes. many clinical and preclinical studies have shown effectiveness of "non-armed" oncolytic viruses in cancer treatment. still, their immuno-stimulatory properties do not always result in the expected potent anti-cancer effect. in order to optimize anti-cancer activity viruses are engineered to express various immuno-stimulatory (see figure on previous page) fig. 3 functions of the influenza a virus ns1 protein and generation of an armed oncolytic influenza a virus. a schematic representation of the ns1 protein. major domains are represented and their aa positions indicated above. the yellow line represents parts of ns1 expressed after truncation to 116 aa. inhibitory functions of ns1 relevant to oncolytic virus development and the domains they have been attributed to are indicated 42, 48, 62, 63, 68, 72, 75 . b example of genetic modifications in an influenza virus designed to be used as oncolytic agent 94 . modifications are carried out on the ns segment. in the first step ns1 is truncated to a length of 116 aa, leaving nep intact. in a second step the armed transgene, in this example il-15, is encoded in the reading frame of ns1. it is connected to an igk and separated from ns1 with the h2 fmdv. reading frames for ns1 and nep have a common beginning. ns1 continues on, while an alternative reading frame is created for nep through splicing. the short, frame shifted overlap of the end of ns1 and middle part of nep is indicated by the diagonal hatching pattern. the complete nep reading frame is generated through splicing in the attenuated and armed examples as well. aa amino acids, il-15 interleukin-15, ns1 nonstructural 1 protein, nep nuclear export protein, igk mouse-derived igkappa signal peptide, 2a fmdv 2a cleavage site of the foot and mouth disease virus. transgenes, most prominently t cell and dc activating cytokines like interleukin-2 (il-2), interleukin-15 (il-15) or gm-csf 91 . within the influenza a virus background we were able to establish potent expression of various transgenes from a deleted or truncated ns1 reading frame, including the reporter gene green fluorescing protein (gfp), the cytokine il-2, or cc-chemokine ligand 20 (ccl20) 92, 93 . using the viruses' inherent property of a high mutational frequency we were able to adapt viruses to stably express transgenes up to 441 aa from the ns segment, together with nuclear export protein (nep) and ns1 truncated to 116 aa 92 using initial selection pressure. it seems likely that very long transgenes might be deleted, but the maximum transgene loading capacity has not yet been tested in influenza viruses. we demonstrated superior therapeutic efficacy and enhancement of nk cell and t cell activation and proliferation when the partially ns1 deleted (116) influenza virus was armed with il-15 94 (fig. 3b) in murine models. subsequently, penghui et al. were able to show anticancer activity of an ns1-deleted influenza a virus armed with gm-csf in a human hep-g2 liver cancer cell line xenograft model 95 . hamilton et al. expressed a recombinant humanized cytotoxic t-lymphocyte-associated protein 4 (ctla4) immune checkpoint inhibiting antibody from two different rna fragments of the influenza a virus genome in order to enhance its anti-cancer effectiveness in a murine b16 melanoma model 96 . in order to elicit specific immunological memory against known cancer epitopes, efferson et al. established a combined strategy of oncolytic influenza a viruses armed with a vaccination peptide against the human epidermal growth factor receptor 2 (her2) 97 . this approach led to potent initiation of effector and memory t cells in an in vitro dc-based assay. overall, multiple promising transgenes that enhance therapeutic outcomes in preclinical models have been postulated for oncolytic influenza a viruses. influenza viruses remain a major health concern as they cause epidemics and pandemics. this happens due to the emergence novel reassortments as well, as novel mutants, generated by genetic changes caused by viral polymerases characterized by a low fidelity. therefore, the use of the virus as a therapeutic agent requires specific safety attention. any oncolytic virus candidate needs to be tested for its stable attenuation. the existence of licensed live influenza a virus vaccines clearly indicates, that the genetic stability of attenuated live influenza viruses is feasible. we could demonstrate that delns1 viruses, including armed versions, can be safely passaged for more than 5 times, without losing their transgene or phenotype 83, 92 . moreover, expressing a transgene from a truncated ns1 reading frame within the ns segment, which codes for the attenuation marker at the same time, prevents the unwanted transmission of the transgene to a wild-type virus (fig. 4) . this is particularly desirable, since reassortment of the chimeric segment with wild-type segments of other influenza virus subtypes might lead to an unexpected pathogenic phenotype. thus, in case transgenes are expressed within a wild-type segment, the apathogenic character of such chimeric viruses may need to be proven within the background of various subtypes. enhancing oncolytic activity of the influenza a virus by optimizing immunogenic cell deathpotential strategies one strategy to optimize oncolytic virus-induced cell death is to enhance induction of apoptosis of neighboring cells. this can be achieved by arming the virus with a proapoptotic cytokine such as interleukin-24 (il-24). we have shown that il-24 sensitizes cancer cells to apoptosis within the background of an influenza a virus infection 98 . importantly the apoptotic effect of il-24 was strongly dependent on a second signal, namely the activation of the toll-like receptor 3 (tlr3) by viral rna. this dependency targets the strong induction of apoptosis achieved by il-24 to the tumor microenvironment, as the attenuated influenza virus used in this experiment cannot replicate otherwise. however, an influenza virus armed with il-24 might be difficult to translate to clinical studies, due to il-24 inhibiting viral growth and leading to low production titers 99 . another strategy to optimize oncolysis is the induction of a more immunogenic form of cell death such as necrosis or necroptosis 100 . necroptosis is a form of programmed death a cell may engage in as an alternative to apoptosis depending on abundance of caspases and various signaling proteins within the cell. it is dependent on receptor-interacting serine/threonine-protein kinase 3 (ripk3) activation and subsequent membrane lysis through mixed lineage kinase domain-like pseudokinase (mlkl) activation. this leads to a phenotype resembling necrosis. necroptosis is considered to be highly immunogenic due to passive release of a large variety of damps and cancer neo-antigen 101 . a recent report implicates necroptosis signaling through ripk3 in inducing potent anti-tumor immune responses independent of the subsequent necrotic phenotype induced by mlkl and damp release 102 . influenza a virus infection can be sensed by the cell through z-dna binding protein 1 (zbp1). this can lead to either necroptosis or apoptosis 103 . conversely, the cellular inhibitor of apoptosis protein 2 (ciap2) protein has been shown to protect against influenza a virus-induced necroptosis 104 . necroptosis but also necrosis might be induced by specific influenza subtypes. examples are the avian h5n1 and a reconstructed 1908 pandemic h1n1 105, 106 . similarly, h5n1 has been shown to inhibit apoptosis, which possibly enables the cell to undergo necroptosis 107 . h5n1 was also more potent than h1n1 in initiating cytokine responses 108 . specifically, h5n1 seems to induce stronger chemotactic signals and thus stronger modulation of chemoattraction of immune cells 109 102 . on the other hand, ripk3 mediated effects seemed to depend on nf-κb activation in the same study. infection with delns1 virus may lead to similar effects, as it strongly induces nf-κb activation 48 . using in vitro models, we were not able to show necroptotic cell death after infection with delns1 or wildtype viruses 98 . this may have been due to the lack of ripk3 expression in the cancer cell lines examined. it is well established, that ripk3 is downregulated in a variety of cancers 112 . this is usually correlated with worsened prognosis 112 . interestingly, murine models indicate that necroptotic non-cancerous cells within the tumor microenvironment contribute to positive outcomes at least as much as necroptotic cancer cells 102 . therefore, the potential of candidate oncolytic viruses to induce necroptosis in stroma cells should be examined. a novel pathway of regulating immunogenicity in viral infections is oxeiptosis 113 . viruses like influenza a virus generate radical oxidative species (ros). cells can sense ros through kelch-like ech-associated protein 1 (keap1) which produces a dose-dependent effect 113 . at low doses of ros the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2) is activated by keap1 and promotes cell survival 113 . also, keap1 binds to pgam family member 5 (pgam5), and consequently inactivates it. at higher doses, keap1 dissociates from pgam5, leading to induction of oxeiptosis, a caspaseindependent, immune-silent form of cell death 113 . engaging in this type of cell death protects the cell from undergoing more immunogenic forms of death like necroptosis. pgam5 knockout mice were shown to react to influenza a virus infection with enhanced necrotic histology and rapid death 113 . a malignant tumor might protect itself from ros induced immunogenic forms of cell death through intact oxeiptotic signaling, which potentially reduces the effectiveness of oncolytic viruses. downregulation of oxeiptotic cell death in the tumor microenvironment may be a promising strategy to enhance oncolytic virotherapy. further effects of oncolytic influenza a viruses on the cancer-immune microenvironment shown in murine models include activation of nk-cells and macrophage polarization towards immuno-stimulatory m1 phenotypes 66, 114 . recent evidence suggests ns1 deleted influenza virus to exhibit enhanced growth when combined with an ifn blocking agent in vitro 115 . therefore, coinjection with such an agent may lead to intensified initial growth and enhanced subsequent reactions against the tumor. oncolytic viruses have been combined with multiple agents in preclinical studies, like histone deacetylase inhibitors or the her2 antibody trastuzumab 116, 117 . such novel combinations may prove to activate immunological anti-cancer mechanisms like antibody-dependent, cellmediated cytotoxicity (adcc), which is reduced in breast cancer patients 118, 119 . the clinically approved oncolytic virus t-vec is experimentally combined with other therapeutic modalities, such as radiotherapy 120 or immune checkpoint blockade 26, 121 . similar methods may prove to be successful in influenza virus-based oncolytic virotherapy. influenza viruses attenuated by means of truncated or deleted ns1 or by other attenuation markers have proven to be safe vaccines in clinical studies and have promising characteristics for use as oncolytic agents. various factors governing their immunogenicity have been described, and strategies for optimizing their oncolytic effects established. arming viruses with cytokines or checkpoint inhibitors appears to enhance their therapeutic effect. these insights into the immunomodulatory properties of such viruses may help further refine choices and design of viruses to be used in clinical trials. the future challenge is bringing this promising novel immunotherapeutic agent into clinical routine. open questions remain the impact of preexisting immunity and combination with other anticancer drugs. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. received: 19 january 2020 revised: 18 february 2020 accepted: 20 february 2020 regression of burkitt's lymphoma in association with measles infection regression of hodgkin's disease after measles the influence of complicating diseases upon leukaemia viral oncolysis: increased immunogenicity of host cell antigen associated with influenza virus oncolytic effect of influenza virus upon ehrlich carcinoma and yoshida ascites hepatoma clinical development of talimogene laherparepvec (t-vec): a modified herpes simplex virus type-1-derived oncolytic immunotherapy intravenous delivery of oncolytic reovirus to brain tumor patients immunologically 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nucleoprotein and host protein clusterin nf-kappab-dependent induction of tumor necrosis factorrelated apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation influenza a virus ns1 protein prevents activation of nf-kappab and induction of alpha/beta interferon influenza virus neuraminidase activates latent transforming growth factor beta apoptosis: a mechanism of cell killing by influenza a and b viruses caspases disrupt the nuclear-cytoplasmic barrier antimycotic-antibiotic amphotericin b promotes influenza virus replication in cell culture overcoming endosomal barrier by amphotericin b-loaded dual ph-responsive pdma-b-pdpa micelleplexes for sirna delivery influenza a virus proteins ns1 and hemagglutinin along with m2 are involved in stimulation of autophagy in infected cells an autophagy-driven pathway of atp secretion supports the aggressive phenotype of braf(v600e) inhibitor-resistant metastatic melanoma cells the p2x7 receptor in infection and inflammation influenza a virus lacking the ns1 gene replicates in interferon-deficient systems interferon signaling is frequently downregulated in melanoma inactivation of interferon receptor promotes the establishment of immune privileged tumor microenvironment progesterone receptor attenuates stat1-mediated ifn signaling in breast cancer interferon resistance promotes oncolysis by influenza virus ns1-deletion mutants a site on the influenza a virus ns1 protein mediates both inhibition of pkr activation and temporal regulation of viral rna synthesis mutant influenza viruses with a defective ns1 protein cannot block the activation of pkr in infected cells oncogenic ras induces an inhibitor of doublestranded rna-dependent eukaryotic initiation factor 2 alpha-kinase activation a genetically engineered influenza a virus with rasdependent oncolytic properties oncolytic influenza virus infection restores immunocompetence of lung tumor-associated alveolar macrophages oncolytic activity of avian influenza virus in human pancreatic ductal adenocarcinoma cell lines activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein pkr and gcn2 stress kinases promote an er stressindependent eif2α phosphorylation responsible for calreticulin exposure in melanoma cells calreticulin exposure dictates the immunogenicity of cancer cell death cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza influenza a mutant viruses with altered ns1 protein function provoke caspase-1 activation in primary human macrophages, resulting in fast apoptosis and release of high levels of interleukins 1β and 18 endogenous expression of proteases in colon cancer cells facilitate influenza a viruses mediated oncolysis targeting an oncolytic influenza a virus to tumor tissue by elastase influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i protein kinase pkr amplification of interferon β induction occurs through initiation factor eif-2α-mediated translational control rig-i detects viral genomic rna during negative-strand rna virus infection genomic rnas of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing the relative amount of an influenza a virus segment present in the viral particle is not affected by a reduction in replication of that segment rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates preclinical evaluation of a replication-deficient intranasal deltans1 h5n1 influenza vaccine a novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist ns1 safety and immunogenicity of a replication-deficient h5n1 influenza virus vaccine lacking ns1 improvement of a dendritic cell-based tumour vaccine by an influenza virus prostate tumor cells infected with a recombinant influenza virus expressing a truncated ns1 protein activate cytolytic cd8+ cells to recognize noninfected tumor cells influenza a virus induces an immediate cytotoxic activity in all major subsets of peripheral blood mononuclear cells reverse genetics-generated elastasedependent swine influenza viruses are attenuated in pigs a new approach to an influenza live vaccine: modification of the cleavage site of hemagglutinin immunogenicity and protective efficacy of an elastasedependent live attenuated swine influenza virus vaccine administered intranasally in pigs armed oncolytic viruses: a kick-start for anti-tumor immunity adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza a virus vector establishment of a chimeric, replication-deficient influenza a virus vector by modulation of splicing efficiency oncolytic influenza a virus expressing interleukin-15 decreases tumor growth in vivo oncolytic activity of a novel influenza a virus carrying granulocyte-macrophage colony-stimulating factor in hepatocellular carcinoma. hum a recombinant antibodyexpressing influenza virus delays tumor growth in a mouse model activation of tumor antigen-specific cytotoxic t lymphocytes (ctls) by human dendritic cells infected with an attenuated influenza a virus expressing a ctl epitope derived from the her-2/neu proto-oncogene il-24 sensitizes tumor cells to tlr3-mediated apoptosis interleukin-24 inhibits influenza a virus replication in vitro through induction of toll-like receptor 3 dependent apoptosis regulated necrosis: the expanding network of non-apoptotic cell death pathways radiotherapy as a backbone for novel concepts in cancer immunotherapy intratumoral activation of the necroptotic pathway components ripk1 and ripk3 potentiates antitumor immunity zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways cellular inhibitor of apoptosis protein ciap2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival pathology of natural infections by h5n1 highly pathogenic avian influenza virus in mute (cygnus olor) and whooper (cygnus cygnus) swans genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus highly pathogenic avian influenza h5n1 virus delays apoptotic responses via activation of stat3 h5n1 influenza virus-induced mediators upregulate rig-i in uninfected cells by paracrine effects contributing to amplified cytokine cascades h5n1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response pandemic h1n1 influenza a viruses suppress immunogenic ripk3-driven dendritic cell death the ns1 protein of influenza a virus participates in necroptosis by interacting with mlkl and increasing its oligomerization and membrane translocation the role of necroptosis in cancer biology and therapy oxeiptosis, a ros-induced caspase-independent apoptosis-like cell-death pathway tumor cells infected with oncolytic influenza a virus prime natural killer cells for lysis of resistant tumor cells inhibitors of the interferon response enhance virus replication in vitro combining hdac inhibitors with oncolytic virotherapy for cancer therapy oncolytic reovirus combined with trastuzumab enhances antitumor efficacy through trail signaling in human her2-positive gastric cancer cells histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis trastuzumab mediates antibody-dependent cell-mediated cytotoxicity and phagocytosis to the same extent in both adjuvant and metastatic her2/neu breast cancer patients radiation therapy and talimogene laherparepvec (tvec) synergize in the treatment of melanoma complete intracranial response to talimogene laherparepvec (t-vec), pembrolizumab and whole brain radiotherapy in a patient with melanoma brain metastases refractory to dual checkpoint-inhibition key: cord-258021-xhx74vr6 authors: waterer, grant w. title: diagnosing viral and atypical pathogens in the setting of community-acquired pneumonia date: 2016-12-21 journal: clin chest med doi: 10.1016/j.ccm.2016.11.004 sha: doc_id: 258021 cord_uid: xhx74vr6 the ‘atypical’ pathogens causing pneumonia have long been problematic for physicians because we have had to rely on serologic tests to make a diagnosis. the introduction of polymerase chain reaction techniques revolutionized the diagnosis of respiratory infections and now a new wave of technologies promising faster, cheaper, and more comprehensive testing are becoming available. this review focuses principally on the diagnosis of legionella, mycoplasma, and influenza infections, but also covers recent publications on the cutting edge of diagnostic tools likely to transform the field of infectious diseases over the coming decade. despite many promises that molecular diagnostics would transform the management of infection, empiric therapy remains the standard of care in community-acquired pneumonia (cap). outside of etiologic studies, the vast majority of patients never have a pathogen diagnosed as the cause of their pneumonia. although physicians are generally quite comfortable with empiric therapy, the need to guess and fear of missing an important pathogen inevitably leads to a broader than necessary spectrum of coverage, particularly in the setting of more severe illness. that viruses are an important cause of pneumonia has been known since the identification of influenza in the early 1930s. 1 despite an awareness that viruses can cause cap, it is only recently that they have appeared as more than a footnote on the list of common pathogens. however, with modern generations of diagnostic panels, and particularly nucleic acid amplification tests, viral pathogens are being identified increasingly as not only common causes of cap, but possibly as being overall more common that bacteria. 2, 3 with more sensitive tests has also come confirmation that patients with cap frequently have multiple pathogens present, particularly the combination of bacterial and viral infection. the term "atypical pneumonia" was coined in first half of the 20th century and used to describe pneumonia owing to pathogens that were not detectable by standard gram staining or traditional culture methods and typically associated with headache, low-grade fever, cough, and malaise. the predominant pathogens that have become associated with atypical pneumonia are mycoplasma pneumoniae (first identified in human lung in 1944), 4 legionella pneumophila (first identified as a significant pneumonia pathogen in 1977 after the outbreak at a convention in philadelphia in 1976) 5 and chlamydophila pneumoniae (first identified in the respiratory tract in 1984). 6 a variety of different species of these genera are now recognized as pneumonia pathogens. this review covers the main approaches to the diagnosis of atypical and viral infections in the setting of pneumonia. the most common approach has been the use of pathogen-specific assays for use in urine, blood, or sputum. although serologic tests based on detecting antibodies to specific pathogens were the predominant technique for decades, they all have limitations in early disease before an adaptive immune response being constituted as well as issues of cross-reactivity reducing specificity. polymerase chain reaction (pcr)-based techniques are now the primary modality for the detection of atypical pathogens in most settings. more recently, there has been the development of multipathogen detection platforms that have become used increasingly in the setting of pneumonia. before moving to laboratory tests, it is worth briefly looking at the evidence of whether there are any specific clinical or radiological features in cap that help to deduce reliably the pathogen. there are definitely clinical features that are seen more commonly in some of the atypical pathogens than with disease owing to streptococcus pneumoniae. examples include erythema multiforme with m pneumoniae, diarrhea with l pneumophila, and rhinorrhea with influenza. however, there is ample evidence that no set of clinical symptoms or signs has sufficient predictive ability to rule in or out any atypical or viral pathogens, especially m pneumoniae 7 and legionella. [8] [9] [10] a number of nonmicrobiological tests have also been proposed as being able to discriminate between "atypical" and "typical" pathogens, including the peripheral white cell count and procalcitonin. although peripheral white cell counts do tend to be lower in viral infections compared with bacterial infections, this is not particularly discriminating at an individual patient level and certainly not accurate enough to use to determine empiric therapy. 11 procalcitonin seems to be more accurate than white cell count, 11 but does not discriminate between atypical bacterial infection and viral infection 12 and may be misleading, particularly in critically ill patients or in patients with bacterial and viral coinfection. 13 a definitive diagnosis based on detecting the infection pathogen(s), therefore, remains critical if we are to improve the accuracy of empiric therapy. very little has changed in the diagnosis of legionella infection since we reviewed this topic comprehensively 15 years ago. 14 in most settings, legionella is underdiagnosed and therefore underrecognized owing to routine testing not being performed. 15 legionella infections seem to be increasing in the united states, 16, 17 possibly owing to recent climate change, including a number of severe outbreaks with multiple fatalities, 18 which has led to increased interest in its diagnosis. because legionellae will not grow on standard culture media, the diagnosis has traditionally rested on either positive serology or a positive urinary antigen test. both of these tests have significant limitations. in the case of serology, 20% or more of patients with culture-proven legionella infection do not ever seroconvert, 19, 20 and seroconversion may take months, requiring testing out to at least 2 months if not longer. 21 urinary antigen testing is quite specific, but will only reliably detection l pneumophila serogroup 1, and usually serogroup 6, but in many areas other species (particularly legionella longbeachae and legionella micdadei) are more predominant. despite these limitations, urinary antigen testing for legionella is recommended in all patients with severe cap (ie, admitted to the intensive care unit) for both diagnostic and public health reasons. 22 the mainstay of diagnosis of legionella infection has been from one or more of direct antigen detection or nucleic acid detection in respiratory secretions. direct fluorescent antigen detection was developed in the pre-pcr era but have now largely been replaced by pcr because the latter is more sensitive, less technician dependent, and easier to automate. pcr tests for legionella are a mix of "home-grown" assays and commercially available products, with reported sensitivity and specificity (using all other tests as the gold standard) in the range of 91% to 99% and 94% to 99%, respectively. 23 because pcr tests for legionella are generally able to detect all species, 24 not surprisingly they have a greater degree of sensitivity than urinary antigen testing. 23, 25 there is, however, a reasonable argument for performing both urinary antigen testing and pcr on respiratory secretions because there is an increased diagnostic yield from this approach. 26 it is worth noting that both nasopharyngeal aspirates [27] [28] [29] and throat swabs 15 have substantially lower yields for the detection of legionella by pcr, but may be of use in patients in whom it is not possible to get spontaneous or induced sputum samples. traditionally, mycoplasma infections have most often been diagnosed on the basis of serology; however, as with serologic tests for many pathogens, this has significant limitations early in disease when false-negative results are common. difficulties in making the diagnosis as well as the marked season to season variation in its prevalence probably explain the enormous variation in the estimated proportion of cases of cap owing to m pneumoniae, which range from less than 1% to greater than 50%. pcr does overcome some of the limitations of serology for the diagnosis of mycoplasma infection and the nuances of assay development and relative performance characteristics has been reviewed comprehensively elsewhere. 30 the performance characteristics for pcr assays for m pneumoniae seem to be at least as good as those for legionella infections and possibly better. 31, 32 as with other pathogens, the detection rate of m pneumoniae using pcr on nasopharyngeal aspirates is lower than in sputum samples. 28 recently, there has been interest in antigen detection assays for the diagnosis of m pneumoniae because these offer the potential for point-of-care testing, but so far these have yet to enter the clinical mainstream. 33, 34 chlamydophila the nomenclature for the chlamydia has changed recently with chlamydia and chlamydophila being combined back into a single genus. 35 both chlamydia psittaci and c pneumoniae are wellaccepted as causes of cap, although almost always being identified as much less common than either mycoplasma or legionella infections. a number of other chlamydophila-like pathogens (such as parachlamydia acanthamoebae and simkania negevensis) also been suggested as potential causes of the 50% or more of cases of cap where no pathogen is identified. 36, 37 the specificity of positive serology for c pneumoniae has also been questioned, because studies using pcr-based diagnosis typically find much lower rates of infection than earlier serology-based studies and a large variety of assays with different performance characteristics have been used. 38 as with mycoplasma, in early disease chlamydophila serology is often negative making pcr a superior diagnostic test. 39 unlike legionella and mycoplasma, chlamydophila cannot be detected by 16s-based pcr assays. because culture of chlamydophila is difficult and has a low yield, it is rarely done 40 ; therefore, pcr assays that have been developed are generally compared with serologic tests, with their known limitations as discussed. the true sensitivity of pcr for chlamydophila species is, therefore, unknown. however the reported specificity of most assays is well over 95% 41 and, therefore, a positive result in the right clinical context should be acted on. all major etiologic studies of cap have identified influenza as a significant cause of cap, particularly in hospitalized patients. since the recent h1n1 09 influenza pandemic, there is evidence that the use of empiric antiinfluenza therapy in the setting of cap has increased significantly, with an unclear impact on outcome. 42 a fast and reliable diagnostic test for influenza is, therefore, attractive not only to prescribe antivirals appropriately (for treatment and prophylaxis), but also to aid in the allocation of respiratory isolation beds, which are often in limited supply, especially in influenza season. for this reason, the diagnostic tools available for influenza have significantly outpaced those for the other causes of atypical pneumonia. in the united states, there are more than a dozen approved rapid influenza tests primarily based on the detection of influenza antigens in respiratory samples. most available assays have been compared with a gold standard of real-time reverse transcription pcr in the same sample. the sensitivity of these assays varies between 10% and 75% depending on age, quality of the sample, and duration of symptoms. complicating the assessment of the usefulness of these assays is that the performance seems to vary between influenza strains and, unfortunately, during the h1n1 09 pandemic they were less than optimal. 43-47 a recent metaanalysis of 159 published studies of rapid influenza tests found the pooled sensitivity, sensitivity, specificity, and positive and negative predictive values to be 62%, 98%, 34%, and 38%, respectively. 47 not surprisingly, given these data, there is little evidence that rapid influenza tests are currently used by clinicians to alter patient management. 48 however, this is a rapidly changing field and more recent publications suggest that there are incremental improvements with a range of sensitivity from 68% to 79% and specificity of 99% to 100%. [49] [50] [51] this is clearly an area where we can expect to see significant advances over the next few years. in the absence of rapid diagnostic tests, existing commercial pcr assays for influenza have well-documented good performance characteristics for influenza a and b, and these data are well-reviewed elsewhere. 52 what is interesting from etiologic studies is the high degree of copathogen involvement with influenza, particularly the codetection of bacterial infection with s pneumoniae. 2, 3 whether this is genuine coinfection or sequential infection is a current controversy and major area of research interest. unlike bacterial pathogens, the constant genomic shifts in influenza a do affect the performance of assays and they need to be revalidated constantly as new strains appear. 53 a variety of point-of-care platforms have been developed for detecting influenza, of which the genexpert system (cepheid, sunnyvale, ca) is perhaps so far the best studied. 54 genexpert is an "all-in-one" platform requiring minimal technical expertise, and is a potential point-of-care platform for diagnosing influenza. a sputum sample is placed in a cartridge that plugs into the platform without the need for further processing or expert microbiological assistance. with a turnaround time of less than 2 hours, results can be available fast enough to impact on empiric therapy. this system has been evaluated extensively for the diagnosis of tuberculosis, including multidrug-resistant tuberculosis, where it has been proven to have excellent sensitivity and specificity. 55 the influenza a and b genexpert assay has been evaluated in comparison to a number of commercially available rapid antigen tests and pcr tests and found to have excellent sensitivity (97% à100%) and specificity (99%-100%). [56] [57] [58] [59] [60] the potential clinical usefulness has been studied in the emergency department setting, again with good performance and efficiency. 61, 62 point-of-care testing for influenza is a highly competitive area with potential new products regularly entering the market offering greater speed, lower cost, and/or greater accuracy (for example [63] [64] [65] [66] ). a large number of other viruses are well-known to cause pneumonia, with the most common being adenovirus, respiratory syncytial virus, metapneumonvirus, parainfluenza, and coronaviruses. in the absence of specific treatments for any of these viruses, discussion of specific diagnostic tests is relatively superfluous; however, many of the multipathogen approaches are discussed herein and include 1 or more of these viruses in their "panels." with an ever-expanding list of pneumonia-causing pathogens, it is both time consuming and expensive to test for each organism individually. the ability to detect multiple pathogens in a single test is, therefore, highly appealing and has been the subject of significant research, development, and validation in the setting of respiratory tract infection. starting with "home-grown" multiplex pcr assays, a variety of new platforms have been developed to speed up pathogen identification, and in some cases combining this with antibiotic sensitivity testing. because the focus of multipathogen detection tools is to find the cause of the pneumonia, they all combine assays for "typical" pathogens such as s pneumoniae with the "atypical" pathogens. multipathogen detection systems can in general these can be categorized into those specifically designed to speed up pathogen recognition from positive blood cultures (eg, including systems such as the verigene grampositive blood culture nucleic acid test; nanosphere, northbrook, il), prove-it sepsis striparray technology (mobidiag, espoo, finland), and filmarray (biofire diagnostics, salt lake city, ut), and those designed for clinical samples such as sputum, blood, or urine the genexpert system has already been discussed, but it is worth noting that the range of pathogen assays is steadily increasing and now includes respiratory syncytial virus and methicillinresistant staphylococcus aureus, which are clearly relevant to pneumonia. filmarray is another novel "all-in-one" multiplex pcr platform with minimal technical expertise required and a turnaround time of approximately 1 hour. manual handling is very limited, as with genexpert, and a variety of panels are available. the commercially available respiratory panel detects 17 viral and 3 bacterial pathogens. the performance of the respiratory panel has been compared with "in-house" pcr tests with favorable results 67,68 and the system seems to be robust enough to be useful in routine clinical practice. 69, 70 curetis unyvero the curetis unyvero p50 pneumonia cartridge can detect 17 bacterial and fungal pathogens and 22 antibiotic resistance markers from respiratory samples in a single run in approximately 4 hours. 71 the panel includes l pneumophila and m pneumoniae, but specific performance data on these pathogens from clinical studies has not been reported. a preliminary study in critically ill patients found the performance of the curetis unyvero to be questionable, but noted the system was still under development. 72 mass spectrometry has been available for decades, but improvements in size, speed, and cost have brought this technology to a point where it can be used for both broadrange and target-specific identification of pathogens. pcrelectrospray ionization mass spectrometry holds particular promise given that it can identify minute quantities and mixtures of nucleic acids from microbial isolates or directly from clinical specimens. the performance of pcr-electrospray ionization mass spectrometry for detecting influenza in clinical samples seems at least as good as conventional pcr assays. 73 a single study from taiwan indicates that pcr-electrospray ionization mass spectrometry has promise for the detection of multiple viruses in the setting of respiratory tract infection but this was done retrospectively rather than in real time. 74 a different use of mass spectrometry, matrixassisted laser desorption/ionization time of flight mass spectrometry (maldi-tof-ms) is also a protein/peptide diagnostic tool that has been shown to have usefulness in identifying microorganisms at a species level. maldi-tof-ms has been assessed predominantly as a means of rapidly identifying the identity of both bacteria and their bacterial products from positive blood cultures, up to 24 hours faster than conventional methods. a comparison of the diagnostic accuracy of maldi-tof-ms with liquid chromatography ms for influenza a, metapneumovirus, and respiratory syncytial virus suggested the latter may be superior. 75 a potential and significant limitation of current maldi-tof-ms is that when a large mixture of bacteria are present, as occurs more commonly in hospital-acquired pneumonia and ventilator-acquired pneumonia, the sensitivity and specificity become suboptimal. 76 next-generation sequencing, also known as highthroughput sequencing, is a generic term used to describe a group of different modern sequencing technologies including illumina (solexa, san diego, ca) sequencing, roche 454 sequencing, ion torrent: proton/pgm sequencing (thermo-fisher scientific, waltham, ma), and solid sequencing (thermofisher scientific, waltham, ma). these recent technologies allow sequencing of dna and rna much more quickly and cheaply than the previously used sanger sequencing (thermofisher scientific, waltham, ma). to date, there are few data on the applicability of nextgeneration sequencing to immediate clinical care, but it has been particularly useful in diagnosing new and/or novel pathogens for which there are no available assays. as technology has improved, we have moved from relying on serologic tests to 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influenza a, influenza b, and respiratory syncytial virus performance characteristics of xpert flu/rsv xc assay prospective and retrospective evaluation of the cepheid xpert(r) flu/rsv xc assay for rapid detection of influenza a, influenza b, and respiratory syncytial virus comparison of xpert flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza a and b evaluation of the xpert flu rapid pcr assay in high-risk emergency department patients economic impact of a new rapid pcr assay for detecting influenza virus in an emergency department and hospitalized patients performance of the alere i influenza a&b assay and maripoc test for the rapid detection of influenza a and b viruses integrated microfluidic device using a single universal aptamer to detect multiple types of influenza viruses sensitive detection of influenza viruses with europium nanoparticles on an epoxy silica sol-gel functionalized polycarbonate-polydimethylsiloxane hybrid microchip hira-tan: a real-time pcr-based system for the rapid identification of causative agents in pneumonia comparison of the idaho technology filmarray system to real-time pcr for detection of respiratory pathogens in children comparison of the filmarray assay and in-house real-time pcr for detection of respiratory infection impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients the potential of molecular diagnostics and serum procalcitonin levels to change the antibiotic management of community-acquired pneumonia evaluation of curetis unyvero, a multiplex pcr-based testing system, for rapid detection of bacteria and antibiotic resistance and impact of the assay on management of severe nosocomial pneumonia point-of-care multiplex pcr promises short turnaround times for microbial testing in hospital-acquired pneumoniaan observational pilot study in critical ill patients evaluation of a polymerase chain reaction-electrospray ionization time-of-flight mass spectrometry for the detection and subtyping of influenza viruses in respiratory specimens viral respiratory tract infections in adult patients attending outpatient and emergency departments rapid and generic identification of influenza a and other respiratory viruses with mass spectrometry direct identification of bacteria in positive blood culture bottles by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry key: cord-001746-pbahviaz authors: garg, shikha; jain, seema; dawood, fatimah s.; jhung, michael; pérez, alejandro; d’mello, tiffany; reingold, arthur; gershman, ken; meek, james; arnold, kathryn e.; farley, monica m.; ryan, patricia; lynfield, ruth; morin, craig; baumbach, joan; hancock, emily b.; zansky, shelley; bennett, nancy; thomas, ann; schaffner, william; finelli, lyn title: pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection—united states, 2005–2008 date: 2015-08-26 journal: bmc infect dis doi: 10.1186/s12879-015-1004-y sha: doc_id: 1746 cord_uid: pbahviaz background: influenza and pneumonia combined are the leading causes of death due to infectious diseases in the united states. we describe factors associated with pneumonia among adults hospitalized with influenza. methods: through the emerging infections program, we identified adults ≥ 18 years, who were hospitalized with laboratory-confirmed influenza during october 2005 through april 2008, and had a chest radiograph (cxr) performed. pneumonia was defined as the presence of a cxr infiltrate and either an icd-9-cm code or discharge summary diagnosis of pneumonia. results: among 4,765 adults hospitalized with influenza, 1392 (29 %) had pneumonia. in multivariable analysis, factors associated with pneumonia included: age ≥ 75 years, adjusted odds ratio (aor) 1.27 (95 % confidence interval 1.10–1.46), white race aor 1.24 (1.03–1.49), nursing home residence aor 1.37 (1.14–1.66), chronic lung disease aor 1.37 (1.18–1.59), immunosuppression aor 1.45 (1.19–1.78), and asthma aor 0.76 (0.62–0.92). patients with pneumonia were significantly more likely to require intensive care unit (icu) admission (27 % vs. 10 %), mechanical ventilation (18 % vs. 5 %), and to die (9 % vs. 2 %). conclusions: pneumonia was present in nearly one-third of adults hospitalized with influenza and was associated with icu admission and death. among patients hospitalized with influenza, older patients and those with certain underlying conditions are more likely to have pneumonia. pneumonia is common among adults hospitalized with influenza and should be evaluated and treated promptly. electronic supplementary material: the online version of this article (doi:10.1186/s12879-015-1004-y) contains supplementary material, which is available to authorized users. influenza illness is generally characterized by acute onset of fever, mylagias, and respiratory symptoms, and while disease usually resolves without complications in healthy indiviudals, influenza is associated with an annual increase in hospital admissions for pulmonary, cardiovascular and neuromuscular compliations [1] [2] [3] . the etiology of influenza-associated pneumonia may include primary influenza pneumonia, secondary bacterial pneumonia, or concomitant viral and bacterial pneumonia [1, 4, 5] . pulmonary complications of influenza, including pneumonia and exacerbations of chronic pulmonary disease, are common and result in significant morbidity and mortality. oliveira and colleagues found that among all patients admitted to a large metropolitan hospital with influenza during the 1999-2000 season, 49 % had pneumonia [6] . further, in a study conducted over 4 influenza seasons (1999) (2000) (2001) (2002) (2003) , murata and colleagues found that among 193 patients hospitalized with influenza a, 52 % had some type of acute findings on chest radiograph and 17 % had definitive pneumonic infiltrates [7] . although there is evidence that adult patients with underlying cardiac or pulmonary disease are more likely to develop influenza-associated pneumonia than those without underlying medical conditions [6, 7] , much of the data describing factors associated with influenzaassociated pneumonia among adults comes from case series conducted at single sites and during a limited number of seasons. using data from a large multi-center, geographically diverse, population-based surveillance system, we describe factors associated with pneumonia among adults hospitalized with influenza over three consecutive years in which seasonal influenza viruses circulated. the emerging infections program (eip) network conducts active population-based surveillance for laboratory-confirmed influenza-associated hospitalizations. the network began adult surveillance in 2005 and covers over 80 counties in 10 states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, new york, oregon, and tennessee), representing approximately 7 % of the adult u.s. population [8] . patients were included in eip influenza surveillance if they resided and were hospitalized in an eip catchment area and were hospitalized within 14 days of a positive influenza diagnostic test result. patients were excluded if the first positive influenza specimen was obtained >3 days after hospital admission because these patients might have had healthcare-associated influenza infection. influenza testing was performed at the discretion of health care providers. medical charts of hospitalized patients with laboratory-confirmed influenza were retrospectively reviewed [8, 9] . the study period comprised 3 influenza seasons, 2005-2006 to 2007-2008. patients were included in this analysis if they were ≥ 18 years of age, were hospitalized with laboratory-confirmed influenza during the 2005-2006 through 2007-2008 influenza seasons, and had a chest radiograph (cxr) performed during hospitalization. the following data were collected on patients: demographics, results of laboratory tests for influenza, influenza vaccination status for the current season, underlying medical conditions, bacterial coinfections, cxr data, antiviral treatment, clinical outcomes, and discharge diagnoses. laboratory confirmation of influenza was based on viral culture, direct or indirect immunoflourescence antibody staining, reverse-transcription polymerase chain reaction, or a rapid antigen test. surveillance staff completed medical record abstractions using check boxes to indicate whether or not a new infiltrate or consolidation was recorded on the official cxr transcript. discharge diagnoses were captured in two ways: 1) the first nine international classification of diseases (icd-9-cm) codes for each case were abstracted from the medical record; 2) check boxes were marked for certain diagnoses, including pneumonia, if they were recorded by clinicians on the discharge summary. pneumonia was defined as the presence of a new infiltrate on cxr and either an icd-9-cm discharge diagnosis code for pneumonia (480-487.0) or a diagnosis of pneumonia recorded on discharge summary. information on the presence of selected bacterial infections was available only for patients who had a positive culture. a bacterial infection was recorded if bacteria other than those that are commonly considered to be contaminants grew from a sterile body site or a non-sterile respiratory site culture obtained within 3 calendar days of hospital admission. sterile body sites for bacterial infections included blood, pleural fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, and deep tissue biopsy. non-sterile respiratory sites included sputum and endotracheal aspirates. use of influenza antiviral therapy was examined for all individuals. among those who were treated with antiviral agents, timing of treatment was assessed in relation to hospitalization date. early antiviral treatment was defined as initiation of antiviral treatment within 2 days of hospital admission. we used bivariate analysis to compare adults hospitalized with influenza with and without pneumonia. we used χ 2 and fisher exact tests for categorical variables and t-tests and wilcoxon-rank sum tests for continuous and ordinal variables. all variables significant in bivariate analysis, as well as biologically plausible variables, and potential confounders were included in a multivariable logistic regression model to identify factors independently associated with influenza-associated pneumonia. we used the breslow-day test for homogeneity to assess for effect modification of select variables. all tests were two-tailed and a p-value of 0.05 was considered significant. analyses were conducted using sas version 9.2 (sas institute inc., cary, nc). ethics statement eip adult influenza hospitalization surveillance activities during the 2005-2007 influenza seasons were determined by the centers for disease control and prevention (cdc) institutional review board (irb) not to involve research in accordance with the federal regulations for the protection of human subjects in research. starting with the 2007-2008 season, research questions were added to evaluate factors associated with severe outcomes during hospitalizations, and irb review was conducted at all surveillance sites and the cdc. the protocol was approved by the cdc irb and was either approved or received exempt status by all surveillance site irbs. because all surveillance data was analyzed anonymously, neither verbal nor written informed consent was obtained from participants. during the study period, of 5055 adults hospitalized with laboratory-confirmed influenza, 4765 (94.3 %) had an available cxr report and discharge diagnosis information and were therefore included in our study. of the 4765 adults, 1392 (29 %) had pneumonia. the prevalence of pneumonia did not vary significantly over the 3 influenza seasons included in the analysis. adults ≥75 years of age represented the age group with the highest proportion of patients hospitalized with and without influenza-associated pneumonia (fig. 1) . the median age of patients with pneumonia compared with patients without pneumonia was 74 years versus 69 years (p <0.01) ( table 1 ). the majority of patients hospitalized with and without influenza-associated pneumonia were white. white patients were older (median age 74 years) than black patients (53 years), hispanic patients (56 years), and patients of other races including asian, pacific islander, american indian, alaskan native, and multi-race (69 years) (p <0.01). patients aged 65 years and above had a higher proportion of underlying conditions (90 %) compared to patients aged < 65 years (79 %) (p < 0.01). influenza was diagnosed by rapid test only in 1048/1390 (75 %) patients with pneumonia and in 2396/3368 (71 %) patients without pneumonia (p < 0.01). the median number of days from symptom onset to hospital admission was 2 days for patients with and without pneumonia (table 1) . patients with pneumonia were significantly more likely than patients without pneumonia to reside in a nursing home prior to hospital admission, to have received influenza vaccine, and to have the following underlying medical conditions: chronic lung disease, cardiovascular disease, and immunosuppression. patients with pneumonia were significantly less likely than patients without pneumonia to have asthma (table 1) . a description of the most frequent discharge diagnoses (based on first listed icd-9 diagnosis code) among patients with and without pneumonia can be found in additional file 1: table s1 . except for influenza vaccination and cardiovascular disease, all factors included in a multivariable model remained independently associated with pneumonia including age ≥75 years [adjusted odds ration (aor) 1.27], white race (aor 1.24), nursing home residence (aor 1.37) chronic lung disease (aor 1.37), immunosuppression (aor 1.45) and asthma (aor 0.76) ( table 1) . sixty-one patients with pneumonia and 68 patients without pneumonia had sterile site bacterial infections, 90 % of which were cultured from the blood ( table 2 ). the most common pathogens cultured from sterile sites in patients with pneumonia were staphylococcous aureus (s. aureus) and streptococcus pneumonia (s. pneumonia). patients with pneumonia had a longer median length of hospital stay than patients without pneumonia (5 days versus 3 days; p <0.01). patients with pneumonia were also significantly more likely to have a hospital length of stay greater than one week (aor 2.99), require intensive care unit (icu) admission (aor 3.62), require mechanical ventilation (aor 4.79), and die (aor 6.06) ( table 3) . among patients with pneumonia, factors independently associated with a poor outcome, defined as icu admission, need for mechanical ventilation or death, included nursing home residence (aor 1.6), chronic lung disease (aor 1.6), cardiovascular disease (aor 1.4), (table 4 ). of note, older age was inversely associated with a poor outcome (aor 0.7) among patients hospitalized with pneumonia (table 4) . patients with pneumonia [823/1392 (59 %)] were significantly more likely to receive influenza antiviral therapy than patients without pneumonia [1815/3373 (54 %); p <0.01]. through this large, population-based surveillance system, we found that pneumonia was present in almost one-third of u.s. adults hospitalized with laboratoryconfirmed influenza over three consecutive years in which seasonal influenza viruses circulated. patients with pneumonia were older and were more likely to have certain underlying medical conditions than patients without pneumonia. patients with pneumonia were also more likely to have a prolonged hospital stay, be admitted to an icu, require mechanical ventilation for respiratory failure, and die. while patients with pneumonia were more likely to receive antiviral therapy than those without pneumonia, treatment was more often delayed among patients with pneumonia. similar to findings from smaller inter-pandemic studies [6, 7] pneumonia was common among adults hospitalized with influenza in this study. among those hospitalized with influenza, older adults and nursing home residents were at significantly increased risk for having influenza-associated pneumonia. respiratory viruses including influenza are a common etiology of pneumonia in older adults, and several factors may contribute to the development of severe lower respiratory tract disease in these individuals, including decreased respiratory muscle strength and lung compliance, and waning humoral and cell-mediated immunity [10] [11] [12] . additional risk factors for lower respiratory tract disease among older nursing home residents include immobility and swallowing difficulties leading to aspiration [13] . within closed settings such as nursing homes, large outbreaks of influenza and its subsequent complications, including severe pneumonia, may rapidly evolve and lead to significant morbidity and mortality [10, 14] . influenza virus infection should thus be considered a potential cause of pneumonia in older individuals and nursing home residents during fall and winter months (15) other streptococci c 4 (7) 8 (12) other pathogens d 2 (3) 5 (7) unknown pathogens 1 (2) 6 (9) [2] and should be diagnosed and treated promptly. influenza vaccination is the most effective method to prevent influenza and its complications, and older adults, residents of nursing homes and other long-term-care facilities, and adults with underlying medical conditions should be considered high priority groups for receipt of annual influenza vaccination [15] . similar to earlier studies conducted during periods of seasonal influenza virus circulation, patients with pneumonia in this study were more likely to have underlying medical conditions including chronic lung disease and heart disease [6, 7] . an unexpected finding was that patients with asthma in our analysis were less likely to have a diagnosis of pneumonia than patients without pneumonia. our study results contrast with eip surveillance data in hospitalized children <18 years of age which has shown that children with influenza-associated pneumonia were more likely to have asthma than those without pneumonia [16] . studies of the association between asthma and seasonal influenza-associated pneumonia among adults are lacking. a possible explanation for our finding is that respiratory distress caused by influenza-associated asthma exacerbation provided an alternate reason for hospitalization in adult patients in the absence of pneumonia. biases in hospital admission practices based on the presence of underlying conditions may have also contributed to admission of asthmatic patients with a less severe respiratory presentation compared to patients without underlying medical conditions. invasive bacterial infections, especially due to s. aureus and s. pneumoniae, were observed among patients with influenza-associated pneumonia in this study as well as other studies conducted during inter-pandemic [7] and pandemic periods [17] . among patients with pneumonia, s. aureus was the most common organism cultured from specimens collected from sterile sites. influenza virus and s. aureus co-infections are increasing [18] [19] [20] and have been associated with particularly severe cases of community-acquired pneumonia during periods of seasonal influenza virus circulation [21] . in patients hospitalized with influenza, sterile site cultures should be collected as early as possible for detection of bacterial infection and empiric antimicrobial coverage of the most likely bacterial organisms should be considered [22, 23] . in our study, s. pneumoniae was the only organism to be cultured from a sterile site more frequently in patients with pneumonia that in patients without pneumonia. in addition to annual influenza vaccination, pneumococcal vaccine should be administered to adults aged 18-64 years with certain health conditions and to all persons aged ≥65 years [24] . patients with influenza-associated pneumonia had a significantly increased risk of icu admission, respiratory failure requiring mechanical ventilation, and death compared with patients without pneumonia. while case series conducted during the 2009 h1n1 pandemic demonstrated elevated frequencies of icu admission (36-58 %) [25, 26] , respiratory failure (10-67 %) [25, 27] and death (7-39 %) [25] [26] [27] [28] among patients hospitalized with pandemic h1n1 influenza-associated pneumonia, limited data is available on the association between seasonal influenzaassociated pneumonia and severe outcomes. in a small case series of patients hospitalized with influenza during the 1999-2000 season, 10 (58 %) of 17 patients with pneumonia were admitted to the icu and 5 (29 %) patients died [7] . in another observational study of patients hospitalized with influenza during 1999-2003, 16 (16 %) of 101 patients with acute pulmonary disease were admitted to the icu, 10 (10 %) required mechanical ventilation, and 6 (6 %) died [6] . while pneumonia and acute respiratory distress syndrome (ards) have been shown to account for a majority of deaths associated with influenza virus infection during pandemics [28] , data is limited on the association between seasonal influenza virus infection and death from pneumonia or ards. in our analysis, only 55 % of patients hospitalized with laboratory-confirmed influenza received influenza antiviral treatment. when limiting the analysis to patients who presented to the hospital within 2 days of symptom onset, only 66 % of all patients received antiviral treatment; the majority received antiviral treatment within 1 day of hospital admission. multiple studies have found early antiviral treatment to be associated with a reduction in serious influenza-associated outcomes including the development of lower respiratory tract infections [29] [30] [31] . the advisory committee on immunization practices recommends empiric influenza antiviral treatment for all adults with suspected or confirmed influenza who are hospitalized, have severe, complicated, or progressive illness, or are at high risk for influenza-associated complications [32] . several limitations to this study should be noted. influenza diagnostic testing was performed at the discretion of treating clinicians at the various eip hospital sites. while all hospitalized patients who tested positive for influenza were included in surveillance, data is unavailable for hospitalized patients who tested negative for influenza or who were not tested. thus, these data may not be representative of all individuals hospitalized with influenza who may not have been tested or have laboratory confirmation of influenza virus infection. it is possible that patients included in surveillance were more likely to be tested for influenza because they were more severely ill; thus a higher proportion of patients exhibiting pneumonia-like symptoms may have been tested for influenza than patients presenting with other symptoms. furthermore, in our analysis, patients with pneumonia were compared to patients without pneumonia but with a wide array of other diagnoses. clinican influenza testing practices based on patient diagnoses at presentation may have biased our findings. in one study conducted in an emergency department in australia, patients presenting with fever and respiratory diagnoses were more likely to be tested for influenza than patients presenting with cardiac or other diagnoses [33] . this study assessed pneumonia specifically among adults hospitalized with laboratory-confirmed influenza, including those whose influenza virus infection preceded hospitalization by more than a few days, and findings are not generalizable to all hospitalized individuals with pneumonia of other etiologies or to non-hospitalized individuals. several of the findings in this study may have been biased by hospital admission practices. for example, the finding of an inverse association between asthma and pneumonia may have been due to more aggressive admission of asthmatic patients presenting with respiratory distress despite the absence of pneumonia, compared with patients without asthma. biases related to hospital admission practices were likely reduced by including patients from multiple hospital sites in geographically diverse settings. for certain underlying conditions such as chronic lung disease and cardiovascular disease, disease type and severity were not captured by the case report form. availability of detailed data on type and severity of underlying conditions may have helped to better identify factors more strongly associated with development of influenza-associated pneumonia. radiographic data were based on review of cxr reports by surveillance officers and not by actual review of radiographs by a designated study radiologist. as a result, some individuals may have been misclassified as having pneumonia based upon a report of infiltrates or opacities, when in fact they had a more chronic pulmonary condition or a transient episode of pulmonary edema or effusion. there was no requirement regarding timing of identification of radiologic abnormalities during the hospitalization, and the timing of chest radiographs during the hospitalization was not collected as part of eip surveillance; thus, some misclassification of communityacquired pneumonia versus nosocomial pneumonia may have occurred. using icd-9-cm code data may also have led to misclassification if a diagnosis code was listed incorrectly or not listed at all. a joint case definition for pneumonia which used both radiographic data and discharge diagnosis data from icd-9-cm codes or discharge summaries was utilized to minimize some of these biases. bacterial culture data was only available for patients with a positive culture result rather than for all specimens spent, thus limiting the interpretation of the culture data. pneumonia is common among adults hospitalized with seasonal influenza virus infection. among patients hospitalized with influenza, older adults and those with underlying medical conditions may be more likely to have pneumonia. further studies are needed to explore the association between influenza-associated pneumonia and asthma in adults. influenza-associated pneumonia can lead to severe outcomes including icu admission and death. adults hospitalized with suspected or confirmed influenza should receive early antiviral therapy, prompt evaluation for pneumonia, and appropriate management upon diagnosis of pneumonia. additional file 1: table s1 . the 10 most frequent icd-9 diagnosis categories based on first icd-9 code listed among adults hospitalized with laboratory-confirmed influenza with and without pneumonia (n=4177). abbreviations cxr: chest radiograph; aor: adjusted odds ratio; eip: emerging infections program; icd-9-cm: international classification of diseases; cdc: centers for disease control and prevention; irb: institutional review board; icu: intensive care unit; s. aureus: staphylococcus aureus; s. pneumonia: streptococcus pneumonia. the authors declare that they have no competing interests. authors' contributions sg: contributed to conception and design of study, analysis of data and interpretation of results, and drafting of the manuscript; sj: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; fd: contributed to conception and design of study, analysis of data, and critical review of manuscript; mj: contributed to conception and design of study, interpretation of data, and criticial review of the manuscript; ap: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; td: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; ar: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; kg: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ke: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; mf: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; pr: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; rl: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; cm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; eh: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; sz: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; nb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; at: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ws: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; lf: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; all authors read and approved the final manuscript. complications of viral influenza influenza circulation and the burden of invasive pneumococcal pneumonia during a non-pandemic period in the united states interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza rates of pneumonia during influenza epidemics in seattle influenza pneumonia: a descriptive study pulmonary complications of interpandemic influenza a in hospitalized adults adult hospitalizations for laboratory-positive influenza during the influenzaassociated pneumonia in children hospitalized with laboratory-confirmed influenza viral pneumonia in older adults immunology of the aging lung physiological changes in respiratory function associated with ageing risk factors for pneumonia and other lower respiratory tract infections in 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the treatment of methicillin-resistant staphylococcus aureus infections in adults and children updated recommendations for prevention of invasive pneumococcal disease among adults using the 23-valent pneumococcal polysaccharide vaccine (ppsv23) clinical characteristics and outcomes of h1n1-associated pneumonia among adults in south korea factors associated with death in hospitalized pneumonia patients with 2009 h1n1 influenza in shenyang, china pneumonia and respiratory failure from swineorigin influenza a (h1n1) in mexico factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california oseltamivir and risk of lower respiratory tract complications in patients with flu symptoms: a meta-analysis of eleven randomized clinical trials benefit of early treatment with oseltamivir in hospitalized patients with documented 2009 influenza a (h1n1): retrospective cohort study impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) van hal sj. influenza a testing and detection in patients admitted through emergency departments in sydney during winter 2009; implications for rational testing we wish to thank the following individuals for their help with surveillance efforts: deborah submit your next manuscript to biomed central and take full advantage of: key: cord-000757-bz66g9a0 authors: davis, kailah; staes, catherine; duncan, jeff; igo, sean; facelli, julio c title: identification of pneumonia and influenza deaths using the death certificate pipeline date: 2012-05-08 journal: bmc med inform decis mak doi: 10.1186/1472-6947-12-37 sha: doc_id: 757 cord_uid: bz66g9a0 background: death records are a rich source of data, which can be used to assist with public surveillance and/or decision support. however, to use this type of data for such purposes it has to be transformed into a coded format to make it computable. because the cause of death in the certificates is reported as free text, encoding the data is currently the single largest barrier of using death certificates for surveillance. therefore, the purpose of this study was to demonstrate the feasibility of using a pipeline, composed of a detection rule and a natural language processor, for the real time encoding of death certificates using the identification of pneumonia and influenza cases as an example and demonstrating that its accuracy is comparable to existing methods. results: a death certificates pipeline (dcp) was developed to automatically code death certificates and identify pneumonia and influenza cases. the pipeline used metamap to code death certificates from the utah department of health for the year 2008. the output of metamap was then accessed by detection rules which flagged pneumonia and influenza cases based on the centers of disease and control and prevention (cdc) case definition. the output from the dcp was compared with the current method used by the cdc and with a keyword search. recall, precision, positive predictive value and f-measure with respect to the cdc method were calculated for the two other methods considered here. the two different techniques compared here with the cdc method showed the following recall/ precision results: dcp: 0.998/0.98 and keyword searching: 0.96/0.96. the f-measure were 0.99 and 0.96 respectively (dcp and keyword searching). both the keyword and the dcp can run in interactive form with modest computer resources, but dcp showed superior performance. conclusion: the pipeline proposed here for coding death certificates and the detection of cases is feasible and can be extended to other conditions. this method provides an alternative that allows for coding free-text death certificates in real time that may increase its utilization not only in the public health domain but also for biomedical researchers and developers. trial registration: this study did not involved any clinical trials. the ongoing monitoring of mortality is crucial to detect and estimate the magnitude of deaths during epidemics, emergence of new diseases (for example, seasonal or pandemic influenza, aids, sars), and the impact of extreme environmental conditions on a population such as heat waves or other relevant public health events or threats [1, 2] . the surveillance of vital statistics is not a novel idea; mortality surveillance has played an integral part in public health since the london bills of mortality were devised in the seventeenth century [3] . the bills served as an early warning tool against bubonic plague by monitoring deaths from the 1635 to the 1830s. today, mortality surveillance continues to be a critical activity for public health agencies throughout the world [4] [5] [6] [7] . pneumonia and influenza are serious public health threats and are a cause of substantial morbidity and mortality worldwide; for instance, the world health organization (who) estimates seasonal influenza causes between 250,000 to 500,000 deaths worldwide each year [8] while pneumonia kills more than 4 million people worldwide every year [9] . worldwide, the morbidity and mortality of influenza and pneumonia have a considerable economic impact in the form of hospital and other health care costs. each year in the united states approximately 3 million persons acquire pneumonia and, depending on the severity of the influenza season, 15 to 61 million people in the us contract influenza [9] . these numbers contribute to approximately 1.3 million hospitalizations, of which 1.1 million are pneumonia cases [10] and the remainder for influenza [11] . moreover, pneumonia cases and influenza together cost the american economy 40.2 billion dollars in 2005 [12] . in the netherlands it has been estimated that influenza accounts for 3713 and 744 days of hospitalization per 100,000 highrisk and low-risk elderly, respectively [13] . due to the public health burden and the unpredictability of an influenza season, strong pneumonia and influenza surveillance systems are a priority for health authorities. mortality monitoring is an important tool for the surveillance of pneumonia and influenza which can aid in the rapid detection and estimates of excess deaths and inform and evaluate the effect of vaccination and control programs. traditionally, influenza mortality surveillance often uses the category of "pneumonia and influenza" (p-i) on death certificates as an indicator of the severity of an influenza season or to identify trends within a season; however, only a small proportion of these deaths are influenza related. it has been reported that only 8. [5] [6] [7] [8] [9] .8% of all pneumonia and influenza deaths are influenza related [14, 15] . the non-influenza-related pneumonia deaths tend to be stable from year to year and fluctuations in this category are largely driven by the prevalence and severity of seasonal influenza. as a result, the p-i category is an important sentinel indicator. in the us, death certificates are the primary data source for mortality surveillance whose findings are widely used to exemplify epidemics and measure the severity of influenza seasons [16] . currently, there are three systems to monitor influenza-related mortality; one system in particular, the 122 cities mortality reporting system, provides a rapid assessment of pneumonia and influenza mortality [6] . each week, this system summarizes the total number of death certificates filed in 122 us cities, as well as the number of deaths due to pneumonia and influenza. however, even these data can be delayed by approximately 2-3 weeks from the times of death. this delay can be attributed to one of the following reasons: 1) timeliness of death registration and 2) reviewing of the death certificates to identify pneumonia and influenza deaths [6, 16, 17] . the registration and reviewing of death certificates varies by states and, as a result, there is variability in length of time to report a death to cdc. for instance, states with paper-based death registration system typically perform manual reviews of the death certificates which can take up to 3 weeks; however states with electronic death registration systems (edrs) may perform automatic reviews which can decrease this time significantly. the current 122 cities mortality reporting system surveillance system also lacks flexibility for expanding the number of conditions and/or the geographic distribution. moreover, the unavailability of coded death records due to the complexity of the national center of health statistics (nchs) coding process results in multiple strategies to identify common outbreaks such as pneumonia and influenza deaths, which greatly vary by jurisdiction. to bypass the lengthy nchs process, a variety of approaches have been attempted that are close to 'realtime' but less than optimal. for instance, in utah keyword searching is used to identify pneumonia and influenza deaths; although this method is fast and easy to implement, it can easily result in the over or under estimation of cases. this can occur by missing cases due to misspelled terms, synonyms, variations, or the selection of strings containing the search term. other research groups [18, 19] have demonstrated the feasibility of using mortality data for real time surveillance but all used "free text" search for the string "pneumonia", "flu" or "influenza." as noted earlier, although this method can provide the semi quantitative measurements for disease surveillance purposes, keyword searches can also result in an array of problems that result from complexities of human language such as causal relationships and synonyms [20] . therefore, the lack of coded death data that may not be available for months [21] seriously limits the use of death records in automated systems. at this time, there is little published on the automatic assignment of codes to death certificates for automatic case detection. currently the coding of death certificates is a complex process which involves many entities. in the us, where we are focusing this study, the codes on death certificates that are generated by the national center for health statistics (nchs) depend on information reported on the death certificate by the medical examiner, coroner, or another certifier, and there is substantial variation in how certifiers interpret and adhere to causeof-death definitions [22] . the cause of death literals are coded into international classification of diseases tenth revision (icd-10) [23] and the underlying and multiplecause-of-death codes are selected based on the world health organization coding rules. these coding rules have been automated by cdc with the development the mortality medical data system (mmds) which consists of four programs: super mortality medical indexing classification and retrieval (supermicar) data entry; mortality medical indexing classification and retrieval (micar); automated classification of medical entities (acme) and transax (translation axes). supermi-car was designed to facilitate the entry of literal text of causes of death in death certificates and convert them into standardized expressions acceptable by micar. it contains a dictionary which assigns an entity reference number (ern) to statements on the death certificate. these erns are fed into micar200 which transforms the erns into icd-10 codes by using specific mortality coding rules; the rules require look-up files and a dictionary. acme and transax then selects the underlying and multiple causes of death respectively. icd-10 codes from micar200 are fed into acme which assigns the underlying cause of death using decision tables. the decision table contains all possible pairings of diseases for which the first disease can cause the second. in the latest version of the system, acme is comprised of eight decision tables including three tables of valid and invalid codes, causal relationships (general principle and rule 1), and direct sequel (rule 3), and three other tables needed by modification rules. figure 1 provides the workflow for the mmds system. of the 2.3 million deaths that occur each year 80-85 percent are automatically coded through super-micar, and the remaining records are then manually coded by nosologists, a medical classification specialist [24] ; this is a tedious and lengthy process lasting up to 3 months. although the automation process has decreased the time required for coding death data to 1-2 weeks, the national vital statistics data is not available for at least two years. therefore, local health department still manually code records or perform basic process techniques to quickly characterize disease patterns [25] . records that were processed through super-micar or were manually coded are then processed through the remaining components (micar200, acme and transax) of mmds. in 1999, micar200 had a throughput rate of 95-97%, while acme rate was 98 percent. moreover, based on a reliability study, acme error rate for selecting the underlying cause is at onehalf percent, while transax, the multiple cause codes had a one-half percent error rate [26] . due to the high processing rates and low error rates, mmds is considered by practitioners as the gold standard for the processing and coding of death certificates in the us and other countries (such as canada, the united kingdom (uk) and australia). therefore, we used the codes produced by this system as the "gold standard" when comparing with the methods developed here. in 1997, the us steering committee to reengineer the death registration process (a task force representing federal agencies, the national center for health statistics and the social security administration, and professional organizations representing funeral directors, physicians, medical examiners, coroners, hospitals, medical records professionals, and vital records and statistics officials (naphsis) published the report "toward an electronic death registration system in the united states: report of the steering committee to reengineer the death registration process." this report explained the feasibility of developing electronic death registration in the united states [27] and argued that these electronic death records have the potential to be an effective source of information for nation-wide tracking and detecting of disease outbreaks. however, little actions have been taken to implement such recommendations in a comprehensive manner. as of july 2011, electronic death registration systems were operating in 36 states, the district of colombia, and in development or planning stage in a dozen others [28] . information representing the 'cause of death' field on the death certificates is free text. one major goal of natural language processing (nlp) is to extract and encode data from free-texts. there have been many research groups developing nlp systems to aid in clinical research, decision support, quality assurance, the automation of encoding free text data and disease surveillance [29] [30] [31] . although, there have been a few nlp applications to the public health domain [32, 33] , little is known about its capability to automatically code death certificates for outbreak and disease surveillance. recently, medical match master (mmm) [25] , developed by riedl et al at the university of california davis, was used to match unstructured cause of death phrases to concepts and semantic types within the unified medical language system (umls). the system annotates each death phrase input with two types of information, the concept unique identifier, cui, and a semantic type both assigned by the umls. mmm was able to identify an exact concept identifier (cui) from the umls for over 50% of 'cause of death' phrases. although, the focus of this study was to use nlp techniques to process death certificates, the description of this system reported in the literature did not show how well coded data from an nlp tool along with predefined rules can detect countable cases for a specific disease or condition. the purpose of our project is to create a pipeline which automatically encodes death certificates using a nlp tool and identify deaths related to pneumonia and influenza which provides daily and/or weekly counts. we compared the new technique developed here with keyword searching and mmds as exemplars of the easiest possible approach and the current "gold standard", respectively. the comparison of the techniques was done by calculating recall, precision, f-measure, positive predictive value and agreement (cohen's kappa). we obtained 14,440 de-identified electronic death records all with multiple-cause-of-death from the utah department of health (udoh) for the period 1 january 2008 to 31 december 2008. the records included a section describing the disease or condition directly leading to death, and any antecedent causes, co-morbid conditions and other significant contributing conditions. an example of a paper and electronic death certificate are shown in figures 2 and 3 respectively. all death certificates used in this study have been processed using the mortality medical data system (mmds) and the record axis codes were received from udoh. for our study we randomly selected 6,450 (45%) records. all death records included in the study were previously also coded by nchs into icd-10, but this information was not used for our coding, it was only used as posteriori to assess to quality of the automatic coding. we chose to apply the centers of disease control and prevention case definition of pneumonia and influenza deaths defined by cdc's epidemiologist staff through personal communication. therefore, the operational definition for deaths from influenza includes deaths from all types of influenza with the exception of deaths from haemophilus influenzae infection and deaths from parainfluenzae virus infection. pneumonia deaths include deaths from all types of pneumonia including pneumonia due to h. influenza and pneumonia due to parainfluenzae virus. the exceptions include aspiration pneumonia (o74.0, o29, o89.0, j69.-and p24.-)1, pneumonitis (j84.1, j67-j70), and pneumonia due to pneumococcal meningitis (j13, g00.1) 1. pneumonia and influenza related deaths were defined as one of the diagnoses listed in table 1 which were reported in any cause of death field. these codes were selected through manual review of the icd-10 version 2007 manual [23] . the death certificates pipeline, dcp, was developed to identify pneumonia and influenza cases. the pipeline consisted of two components. the first component of the system was the natural language processor, for which we used metamap [34] , and the second component was the definitional rules that were applied to the output generated by metamap. the study procedures for this pipeline included: preprocessing, nlp, extraction of coded data and the detection of pneumonia and influenza cases (figure 4 ). spelling errors are common on death certificates; therefore, the death records were first processed through a spell checker to identify misspellings. although the umls sl has a spell suggestion tool called gspell [35] [36] [37] , we decided not to use it and chose to utilize aspell [38] . our motivation for this decision was based upon an evaluation which showed aspell outperforming gspell; aspell performed better on three areas of performance which were (2) whether the correct word was ranked in the top ten; and (3) whether the correct word was found at all [35] . perl (www.perl.org), a high-level computer programming language that aids in the manipulation and processing of large volume of text data was then used to prepare the cause of death free text for nlp. the preprocessing also involved the removal of non-ascii characters; this was a required technical step for metamap processing. step 2: natural language processing metamap was used to convert the electronic death records to coded descriptions appropriate for the rule based system. metamap [34] , developed by the national library of medicine (nlm), is useful in identifying biomedical concepts from free-form textual input and maps them into concepts from the unified medical language system (umls) metathesaurus [34, 39] . metamap works by breaking the inputted text into words or phrases, map them to standard terms, and then match the terms to concepts in the unified medical language system (umls) [40] . for each matched phrase, metamap classifies it into a semantic type then returns the concept unique identifier (cui) and the mapping options which are ranked according to the strength of the mapping. output from metamap. text bolded in the output from nlp represent the code and its corresponding phrase. step 3: extraction of coded data the data produced by metamap (xml format) was processed through a perl script to extract the inputted text and its corresponding meta-mapped cuis. this extracted data was outputted to a text document. step 4: identification of p-i deaths the identification of pneumonia and influenza cases involved two steps: 1) identifying cuis relating to pneumonia and influenza and 2) use of the cuis to create a rules based algorithm to identify cases. details of each step are explained in the following paragraphs. to determine which cui codes were relevant for identifying pneumonia and influenza deaths it was necessary to create a "cui code list" that represents all the icd-10 codes of interest (see table 1 ). to create this list, we generated a subset of the umls 2010 ab database [41] using the metamorphosys [40] tool provided by the national library of medicine, nlm. the umls database includes many vocabularies, therefore, to determine which vocabularies are relevant to our aims we used the procedure used by riedl three queries were performed on the subset described above to map pneumonia and influenza icd-10 codes to cuis and identify related pneumonia and influenza concepts. each query was then placed in a separate database, all duplicates were removed and a sub-query was run to ensure that only the icd-10 codes in table 1 were included in this list. this produced 241 distinct concept identifiers (cuis) relating to pneumonia or influenza. these codes were used to develop the rules to identify the cases of interest. the coded data produced by metamap was accessed by rules, aimed at identifying the presence of pneumonia and influenza based on the coded data. the rules for identifying these deaths used the cui code list described above. the rule looks at each cause of death field (underlying cause, additional causes, etc.) to flag records with relevant codes. these rules used boolean operators (and, or, not) and if-then statements to create a chain of rules ( figure 5 ). the list of cases identified by our automated detection system was compared with those identified by two other methods: a) keyword searching and b) the reference standard: the icd-10 codes given by the cdc mmds method. for key-word searching we followed the process to evaluate the performance of both techniques against the reference standard, we needed to specify what constituted a match. each death record is associated to a unique number; therefore, we considered a match if the unique identifier was identified by the comparator and also found by the reference standard. three standard measures were used to evaluate the performance of one method in relation to the reference standard used in this study: precision (equivalent to positive predictive value; recall (equivalent to sensitivity or true positive rate), and f-measure. kappa statistics were used to assess agreement and mcnemar's test was used to analyze the significance between the two methods. all calculations were performed in r [42] . to calculate these values, pneumonia and influenza related deaths were examined by comparing the reference standard output vs. the two comparators: dcp and keyword search. for both comparators, the deaths were counted and categorized as true posi-tives (cases found by the comparator-pneumonia deaths being correctly classified); false positives (incorrect cases found by the comparator-the number of pneumonia and influenza deaths incorrectly identified by the comparator); false negatives (correct cases not found by the comparator-the number of pneumonia deaths not identified by the comparator). precision, recall and f-score were calculated as follows: precision = true positives/(true positives + false positives) (1) recall = true positives/(true positives + false negatives) (2) f-measure = 2 *(p r/ p + r) (3) mcnemar's test was also calculated to evaluate the significance of the difference between the two comparators. to calculate this value a confusion matrix was created where a is the number of times both methods have correct predictions; b is the number of times method 1 has a correct prediction and method 2 has a wrong prediction; c is the number of times method 2 has a correct prediction and method 1 has a wrong prediction; d is the number of times both methods have incorrect predictions. ethics approval was not required for this study. identifying variables that could be used for re-identifying individuals were excluded from the study data. the records were processed and analyzed on a server with two opteron dual-core 2.8 ghz processors and 16 gb ram at the center of high performance computing at the university of utah. using keyword searching the cpu processing time to identify pneumonia and influenza cases was 0.21 seconds and the wall time was 0.37 seconds. for the dcp, the total cpu processing time was 881.83 seconds. the nlp portion of the pipeline attributed to 99.4 percent of the processing time (nlp-877 seconds). while the dcp execution time is much longer, still it is well within the "in real time" realm. for instance, it would take 6,364.3 seconds cpu time seconds for dcp to code and flag all the weekly death records of the us ( 46,523). recall and precision were calculated at a 0.95 confidence intervals; the f-measure was also calculated. the performance of each method is described below. of the 6,450 records analyzed keyword search identified 473 records as pneumonia and influenza deaths, 21 being identified as false positives. precision for keyword searching was calculated at 96%. of the 21 false positives, 6 records correctly mentioned pneumonia in the cause of death text but their corresponding icd-10 codes failed to provide any code related to pneumonia, while 2 records were flagged because it included the sub-string "pneumonia" in the additional cause of death field. the death literal for these two records were "bacteremia due to streptococcus pneumonia" and "streptococcal pneumoniae septicemia", the remaining 13 errors were due to the entry of the death literals; in all cases the negation of 'aspiration pneumonia' either due to: 1) 'pneumonia' being in a separate cause of death field to 'aspiration' or 2) 'pneumonia' not being directly followed by 'aspiration' in the death text (example "pneumonia due to secondary aspiration"). a total of 20 false negatives were recorded, yielding a recall of 96%. the false negatives could be generalized into two categories: 1) misspellings of pneumonia on the death certificated (n = 8) and 2) appropriate pneumonia or influenza icd-10 code was coded but the death literals did not mention an appropriate scanned phrase (n = 12). f-measure was also calculated at 96%. a high level of agreement was seen among keyword searching and the reference standard (kappa 0.95). utilizing the death certificates pipeline (dcp), we identified 481 records as pneumonia and influenza deaths, 9 of which were false positives. the precision for this method was calculated at 98%. like the keyword searching method, of the 9 false positives, 6 records mentioned pneumonia in the cause of death field but their corresponding icd-10 codes failed to provide any code related to pneumonia and the remaining errors were due to the reporting of aspiration pneumonia on the death certificate. this method had only 1 false negative for the death literal stating "recurrent aspiration with pneumonia", thus yielding a recall at 99.8%, being less than keyword searching. f-measure was calculated at 99%. the level of agreement between the pipeline and the gold standard was almost perfect with a cohen's kappa of 0.988. the precision and recall scores that are reported above suggest that the dcp is a better method for identifying pneumonia and influenza deaths than keywordsearching. therefore, we investigated if this observation is supported by statistical analysis. performing a fisher's exact test at î± = 0.05, significant difference was seen for both recall (p = 1.742e-05) and precision (p = 0.026). the mcnemar's test result also showed dcp to be a better method with a p-value = 2.152e-05. for the 472 pneumonia and influenza cases found by the reference standard, dcp correctly identified 471 cases, missed one case and incorrectly flagged nine cases. most failures were due to discrepancies between the death literal and its respective icd-10 code. for the only case which the pipeline did not match, the phrase 'recurrent aspiration with pneumonia' was present in the death literal. metamap coded this literal as aspiration pneumonia which was excluded from the cui code list, but its respective icd-10 included j189. for the 9 additional cases which were not present in the reference standard, we noticed two categories of errors: 1)cases where the string 'pneumonia' is present in the death literal but not coded into icd-10 and 2) the reporting of aspiration pneumonia on the death certificate. the first category of errors was not due to metamap or the rule algorithm, but perhaps due to the coding process. as described earlier, mmds produces entity axis and record axis codes. the entity axis codes would be a more appropriate reference standard for they provide the icd 10 codes for the conditions or events reported as listed by the death certifier and maintains the order as written on the death certificate [43] ; but as noted earlier only the record axis codes were made available for this study. the algorithm used to produce record axis codes from the entity axis data removes duplicate codes and contradictory diagnoses within the entity axis data to produce the more standardized record axis [44] . for example, if a medical examiner reports pneumonia with chronic obstructive pulmonary disease both conditions will be shown in entity axis code data. however, in record axis code data, they will be replaced with a single condition: chronic obstructive pulmonary disease with acute lower respiratory infection (j44.0). we were unable to verify that codes related to pneumonia were present in the entity axis codes for the six cases; therefore, we can only speculate the reason for this failure. the second category of errors was due to the reporting of aspiration pneumonia on the death certificate. in cases where the string "aspiration" and pneumonia" were not reported in the same text field metamap processed the string separately thus yielding two codes: one for aspiration and the other pneumonia, instead of one code for "aspiration pneumonia" [c0032290]. in an initial review of metamap we found metamap had difficulties processing the phrase "pneumonia secondary to acute aspiration", therefore, our rule detection algorithm excluded cases where the code for pneumonia and aspiration were present in the same text field. to our knowledge, this is the first published report on using a natural language processing tool and the umls to identify pneumonia and influenza deaths from death certificates. we found that automated coding and identification of pneumonia and influenza deaths is possible and computationally efficient. the death certificates pipeline developed here was statistically different to keyword searching and has higher recall and precision when compared to the current semi-automatic methods in use by the cdc. a good recall is required to help capture the 'true' p-i deaths and a good precision is needed to avoidoverestimating the number of p-i deaths. this study also indicated that keyword searching underestimated pneumonia and influenza deaths in utah. the simple keyword search method not only decreased recall and precision but also reduced the level of agreement. when reporting counts for surveillance purposes it's best to be as accurate as possible; however, there's a trade-off between recall and precision. for disease surveillance, increased precision enables public health officials to more accurately focus resources for control and prevention, therefore, although both methods had good precision the pipeline developed would be more advantageous to utilize. metamap did an excellent job at extracting cause of deaths from free-form text which is consistent with the results of reid et al [25] . most of the concepts were present in the umls which attributed good recall. both recall and precision depended on the comprehensiveness of the cui code list. the performance of this system is determined largely by the coverage of terms and sources in the umls. both keyword searching and the system's weakest point is its lack of precision. most of the concepts the system did not identify had either the aspiration text in another field or pneumonia was mentioned in the cause of death text but not coded (9 cases fit these criteria). the sample size was sufficient to show difference between the two methods. it is important to note that utilizing trained nosologists, who would manually code the death certificates, would have developed an absolute gold standard which may or may not be a better reference standard than icd-10 codes. however, our motivation for utilizing icd codes was influenced due to the fact that the use of icd codes to identify all-cause pneumonia has been examined and has showed to be a valid tool for the identification of these cases [45, 46] . in terms of timing, while keyword searching is faster than the dcp, our method is also sub 1/10 second range, which implies that it is possible to process the daily utah deaths (~40) in approximately 5.47 seconds and all deaths in the us (~6646) in approximately 909.17 seconds using current hardware. this timing would be much faster than the minimum of two weeks to receive the coded data from the current cdc process. moreover, these timings make it apparent that this system can be integrated in a real time surveillance system without introducing any additional bottlenecks. there are several potential limitations with this analysis. first, the generalizability of the findings is limited because the death records were only from one institution. although death certificates have a standardized format, the death registration process and the reviewing of death records differ by institutions. udoh utilizes keyword searching to identify pneumonia and influenza cases, other institutions may use more accurate (manual review) or less accurate methods for finding cases. second, a separate evaluation of the nlp component of the dcp was not performed. further research is needed to examine the use of nlp on electronic death records across institutions and countries which may have different documentation procedures. this study shows that it is feasible to achieve high levels of accuracy when using nlp tools to identify cases of pneumonia and influenza cases from electronic death records while still providing a system that can be used for real time coding of death certificates. identification of concept identifiers related to the cdc's case definition of pneumonia and influenza was very important in producing a highly accurate rule for the identification of these cases. future work will aim to improve the preprocessing phase of the pipeline by providing the inclusion of the spellchecker used by the cdc's mortality medical data system. future work will also involve evaluating the flexibility (e.g. identification of different diseases) of the system to deploy the pipeline tool, along with other public health related analytical tools, as a grid service to provide to real time public health surveillance tool that uses data and services under the control of different administrative domains. we have shown that it is feasible to automate the coding of electronic death records for real-time surveillance of deaths of public health concern. the performance of the pipeline outperformed the performance of current methods, keyword searching, in the identification of pneumonia and influenza related deaths from death certificates. therefore, the pipeline has the potential to aid in the encoding of death certificates and is flexible to identify deaths due to other conditions of interest as the need arises. participants of a workshop on mortality monitoring in europe: monitoring excess mortality for public health action: potential for a future european network public health surveillance: historical origins, methods and evaluation communicable disease surveillance the new automated daily mortality surveillance system in portugal description of a new all cause mortality surveillance system in sweden as a warning system using threshold detection algorithms a method for timely assessment of influenza-associated mortality in the united states mortality surveillance 1968-1976, england and wales. deaths and rates by sex and age group for 8th revision causes, a-list and chapters. london: great britain office of population census and surveys 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medical records to facilitate epidemiologic case investigations evaluating natural language processing applications applied to outbreak and disease surveillance a: effective mapping of biomedical text to the umls metathesaurus: the metamap program a frequency-based technique to improve the spelling suggestion rank in medical queries lexical systems: a report to the board of scientific counselors umls language and vocabulary tools metamap: mapping text to the umls metathesaurus the unified language system (umls): integrating biomedical terminology umls distribution team: r: a language and environment for statistical computing. vienna: r foundation for statistical computing entity axis codes documentation of the mortality tape file for community-acquired pneumonia: can it be defined with claims data? icd-10 codes are a valid tool for identification of pneumonia in hospitalized patients aged > or = 65 years identification of pneumonia and influenza deaths using the death certificate pipeline this study has been supported in part by the grants from the national library of medicine (lm007124) and from the centers of disease control and prevention center of excellence (ip01hk000069-10). the authors declare that they have no competing interests. all the authors contributed equally to this research. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord-103972-kbv9kh6z authors: singer, gregor; graff zivin, joshua; neidell, matthew; sanders, nicholas title: air pollution increases influenza hospitalizations date: 2020-04-10 journal: nan doi: 10.1101/2020.04.07.20057216 sha: doc_id: 103972 cord_uid: kbv9kh6z seasonal influenza is a recurring health burden shared widely across the globe. we study whether air quality affects the occurrence of severe influenza cases that require inpatient hospitalization. using longitudinal information on local air quality and hospital admissions across the united states, we find that poor air quality increases the incidence of significant influenza hospital admissions. effects diminish in years with greater influenza vaccine effectiveness. apart from increasing vaccination rates, improving air quality may help reduce the spread and severity of influenza. seasonal influenza is a global health threat, with an average of 3-5 million severe cases per year and 290,000 to 650,000 respiratory deaths (1, 2) . the disease exhibits variability in spread and severity across individuals, regions, and over time. prior research has produced two broad sets of findings to explain this variation: a) meteorological factors that affect the spread of the virus, such as temperature, sunlight and humidity (3, 4, 5, 6, 7, 8) ; and b) individual level host factors, such as age, sex, underlying health and smoking that affect the intensity of symptoms (9, 10). we know considerably less, however, about how air pollution affects influenza spread and severity, a surprising gap given the pervasiveness of air pollution around the world and the well-established policy tools available to control it. air pollution could affect influenza hospitalizations via both susceptibility and exposure (11). like smoking (10), air pollution can impair the respiratory functioning of patients, e.g., by damaging the respiratory epithelium, thereby facilitating the progression of influenza virus beyond the epithelial barrier into the lungs (12, 13, 14, 15) . existing medical research finds exposing in vitro respiratory epithelial cells to air pollution increases susceptibility and penetration of influenza (13), and experimental exposure of mice to air pollution before influenza infections increased morbid-ity and mortality (16, 17) . like humidity and temperature (5, 6, 7, 18, 19) , air pollution particles could also impact the airborne survival of viruses outside the body (18, 20, 21, 22, 23, 24) and thus increase the probability of disease transmission. we build on the existing evidence that links ambient air pollution with influenza spread and severity (13, 16, 17, 21, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) with two significant advancements toward improving causal inference (36). first, we exploit a long panel of influenza-specific hospital admissions from numerous states across the united states (u.s.) to estimate statistical models that exploit both spatial and temporal variation within counties over time, limiting threats from confounding factors. second, to better understand the causal link, we explore the role of the influenza vaccine in moderating this relationship. if the vaccine reduces infections and the probability of influenza spread, seasons in which the vaccine is more effective should weaken the link between air pollution and influenza (37). our analysis utilizes patient level data on inpatient hospitalization (39), which allows us to focus on severe cases specifically limited to influenza (for details on data, descriptives, and empirical methods see supplementary appendix s.1, s.2 and s.3). our principal outcome of interest is the number of inpatient ad-missions per county-month where the primary diagnosis is influenza according to the international classification of diseases (icd) (40). we combine this with high frequency air pollution readings of local ground monitors across the u.s., as well as data on local temperature, specific humidity, precipitation and wind speed (41). the richness of our data allows us to control for a wide variety of both regional and temporal controls. our preferred specification includes county-by-year and month-byyear fixed effects. county-by-year effects control for differences in unobserved characteristics such as demographics, socio-economic factors, and health care access and protocols that influence pollution exposure and health outcomes across counties separately for each year. the month-by-year fixed effects control for general monthly and seasonal trends within each year in both influenza and pollution (42). as our measure of pollution, we use the u.s. environmental protection agency's air quality index (aqi), which we aggregate to county-bymonth-by-year to match outcomes. the aqi is a measure of overall air quality based on the primary criteria pollutants specified in the clean air act. aggregation of pollutants means there are no real "units" for the measure. it is designed such that higher aqi values indicate worse air quality. to ensure we capture exposure to air pollution before diagnosis, we lag the aqi by one month. in all of our analyses, we focus on the influenza season (october to march). figure 1 shows the seasonality of inpatient hospitalizations in our data (figure 1a) , which matches closely with general influenza-like illnesses reported by the centers for disease control and prevention (cdc) (figure 1b ). figure 1c shows the age distribution of hospital admissions, which has important implications for vaccine effectiveness, described in more detail below. figure 2a shows a clear positive correlation between air quality and count of influenza admissions in the raw data; higher aqi correlates with more influenza admissions (43). figure 2b shows the correlation after adjusting both variables for fixed effects and weather controls. after this adjustment, a strong, positive correlation remains. table 1 shows estimates from poisson pseudo-maximum likelihood regressions given the count nature of the dependent variable. the coefficients represent the change in the expected log of inpatient admission counts, which approximates a percentage change in number of county-year-month admissions within our data (44). column (1) implies a 1-unit increase in the lagged monthly aqi results in a 0.56% increase in inpatient influenza admissions. to put this estimate in national context, a one standard deviation increase in aqi (12.79-unit increase in our data) amounts to approximately 4,064 additional inpatient hospitalizations for the 6-month influenza season in the u.s. (45). column (2) replaces our continuous measure of air quality with the count of days in a month with air quality the epa classifies as "unhealthy for sensitive groups" (aqi ≥ 100). these days are rare: in our data, the average county has around 0.4 such days per month. an additional unhealthy air quality day raises admission counts by approximately 5%. continuing with our u.s.-wide calculation, an additional unhealthy air quality day in each county generates 2,786 additional inpatient hospitalizations per influenza season. we next interact our air quality measure with a measure of influenza vaccine effectiveness. every year, the cdc reports results from small-scale studies of that season's influenza vaccine effectiveness rate by age group (see details in supplementary appendix s.1). based on the histogram in figure 1c , we use the vaccine effectiveness for the two age groups traditionally susceptible to health complications from influenza: children up to 8 and adults 65 and older. this group comprises 65% of inpatient hospitalization in our data. figure 3 shows the regression-adjusted relationship between aqi and influenza admissions separately in seasons of low vaccine effectiveness and high vaccine effectiveness for the up to 8-year-old group and 65-year-and-older group, as determined by a median sample split (46). for both age groups, the relationship between air quality and admissions rates flattens and effectively disappears in years of high vaccine effectiveness. columns (3) and (4) of table 1 show a similar story using a more continuous measure of vaccine effectiveness. a vaccine effectiveness of 53% for the up to 8year-old group or 34% for the 65-yearand-older group nullifies the link between air pollution and influenza hospitalizations (47). while our fixed effects can address many unobservable factors, there remain possible confounders in establishing a causal link between pollution exposure and influenza hospitalizations. air quality could trigger health problems in sensitive populations (e.g. asthmatics) who would then go to the hospital, where they might be observed to have influenza. for this reason, our analysis focused on patients whose primary diagnosis is influenza and ignore occurrences of influenza in secondary diagnoses. we also repeat our analysis using two alternative measures: patients where influenza is the only diagnosis and patients where any diagnosis is influenza. supplementary appendix s.4 shows that our results are robust to either of these alternatives. we perform various falsification tests by repeating our analysis using health outcomes that should not correlate with air quality and health: diabetes mellitus with complications; urinary tract infections; skull and face fractures; and osteoarthritis (48). the result of a falsification test in column (5), using the combined number of the above health outcomes, indicates a precise zero to the thousandth decimal place. we present estimates on each of these four falsification outcomes individually in supplementary appendix s.4 with similar results. supplementary appendix s.4 ex-plores heterogeneity and conducts further sensitivity analysis and robustness checks. our estimates are stable across gender and age groups. we find suggestively larger effects for blacks and hispanics, but the estimates are not statistically different from those for whites. we show robustness to (i) different weather controls, (ii) additional fixed effects, (iii) multilevel clustering of standard errors, (iv) different winsorization and interpolation of the raw aqi data, (v) including out-state patients at hospitals, (vi) focusing on states with a long time series only, (vii) using missing values instead of zeros for county-months with no hospital admissions, and (viii) using a linear ordinary least squares instead of a poisson pseudo-maximum likelihood estimator. we also show the effect of air pollution on outpatient hospitalization is larger than for inpatient hospitalizations, consistent with the notion that emergency department encounters are more frequent (but also less severe) than those requiring admission to the hospital. as a final consideration, we shift from additional influenza cases to an economic endpoint. column (6) of table 1 shows ordinary least squares estimate of the effect of aqi on hospitalization charges for influenza admissions. this suggests a one-unit increase in aqi increases hospital billing by approximately $4,929 per month in the average county during influenza season. across the u.s., a one standard deviation increase in aqi (12.79-unit increase) generates an additional $1.19 billion inpatient hospital charges per influenza season. using a rich longitudinal dataset, we provide causal evidence that air pollution increases hospitalization rates for seasonal influenza. our findings offer novel evidence important for policy making, highlighting the heightened importance of increasing vaccination rates in polluted urban centers (49). this is especially important in developing countries, which house the most polluted cities in the world and have very low baseline vaccination rates (50). they also imply pollution controls can provide an important hedge against antigenic drift or shift in the influenza virus that renders the vaccine significantly less effective in some years, helping reduce global medical spending, avoid lost productivity, and reduce loss of human life. if our results generalize to other respiratory viral infections, they will significantly understate the infectiousdisease related benefits from environmental protection (51). they may also provide important insights for the ongoing fight against the covid-19 pandemic (52). social distancing and large scale reductions in economic activity aimed at reducing viral spread have also reduced air pollution (53), which may be helping reduce the impacts of the disease. as countries relax restrictions and economic activity resumes, they may choose to reduce environmental regulations in exchange for a more rapid return to economic growth: the u.s. epa recently announced plans to suspend enforcement of environmental laws during the pandemic (54). our results suggest there could be additional disease-related social costs to consider when worsening air quality during the economic recovery. [8] a. i. barreca, j. p. shimshack, absolute humidity, temperature, and influenza mortality: 30 years of county-level evidence from the united states. american journal of epidemiology 176, s114 (2012). [11] a separate literature shows air pollution has mortality effects through and on top of concurrent influenza episodes (55, 56, 57). [12] g. diamond, d. legarda, l. k. ryan, the innate immune response of the respiratory epithelium. immunological reviews 173, 27 (2000). [ [40] for our baseline results we count patients whose primary disease icd code is influenza, but show robustness to alternative definitions. that is we exclude, for example, patients with bacteria related pneumonia or other respiratory diseases as their primary diagnosis. [41] we include weather controls to address the link between both influenza and weather (temperature and humidity can both influence influenza transmission rates) and weather and pollution (different climatic conditions can lead to different levels of air quality, all else held constant). [42] in models where we interact vaccine effectiveness, we control for county-by-influenza season effects since vaccine effectiveness varies by season. the baseline model is robust to controlling for countyby-influenza season fixed effects (see supplementary appendix s.4). [43] this relationship can be spuriously driven by external factors that affect both pollution levels and admission rates. for example, more populated counties typically have higher pollution levels and (mechanically) higher hospital admissions. seasonality can also be a factor, as particulate matter and carbon monoxide, two common lung irritants included in the aqi, peak in winter months just as influenza admissions. our set of fixed effects can address both of these issues. [44] we cluster all standard errors at the county level and provide further robustness checks in supplementary appendix s.4. [45] we multiply the 12.79-unit increase by 0.0056, by the average inpatient admissions per countymonth (3.01), the total number of us county equivalents according to the us census bureau (3142) (58) and by the 6 months within a influenza season. since reporting is voluntary, our hospital data are not exhaustive. if reporting behavior does not correlate with the likelihood of influenza infections, the 0.56% relative increase in admissions should not be affected. however, the translation into absolute admissions is likely underestimated. [46] for the up to 8-year-olds median vaccine effectiveness is 45% and for the over 65-year-olds the median is 36%. [47] vaccine effectiveness during our study period ranges from 25-57% for those up to 8 and 0-50% for those 65 and older. [48] these four diseases are a random selection of disease groups we think are unlikely to be correlated with air pollution, and also occur a sufficient number of times in the hospitalization data. see the supplementary appendix s.4 and s.1 for details on icd codes, estimation and results. [49] in a study of the spanish flu in 1918, (32) show cities with higher coal-fired power generating capacity saw higher mortality rates, potentially through exposure to higher air pollution aggravating either of these vectors. our data allows us to more narrowly investigate this link by directly assessing the impact of air pollution readings on influenza diagnosed hospitalizations across the u.s. during the modern pollution control era. [50] c. tables s.1 to s.6 1 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.20057216 doi: medrxiv preprint hospitalization data: we use hospital admission data from the healthcare cost and utilization project (hcup) and focus on the inpatient data from hospital stays (39). we exploit patient level information on diagnosed diseases per international classification of diseases (icd) codes, patient zip codes, admission months, age, gender, race as well as hospital charges. the data are available for a subset of u.s. states and years from 1991 (see table s .1). we convert monetary hospital charges to common 2018 us$ using a gdp deflator from the world bank (60) . to identify influenza hospitalizations, we count patients whose primary diagnosis is a strain of influenza. we use the clinical classifications software (ccs) from the agency for healthcare research and quality (ahrq) to classify relevant influenza icd codes. these are the following icd-9-cm codes: 4870, 4871, 4878, 488, 4880, 48801, 48802, 48809, 4881, 48811, 48812, 48819, 48881, 48882, 48889; and, for the period from october 2015 when the system was changed to icd-10-cm, the following icd-10-cm codes: j09x1, j09x2, j09x3, j09x9, j1000, j1001, j1008, j101, j102, j1081, j1082, j1083, j1089, j1100, j1108, j111, j112, j1181, j1182, j1183, j1189. we exclude patients whose primary diagnosis is not influenza, even if influenza is included among secondary diagnoses. counting primary influenza diagnoses reflects a middle ground between two extreme alternatives for which we perform robustness checks. in one robustness check, we count patients who have any (primary or secondary) influenza diagnosis. in another robustness check we only count patients for whom influenza is their only diagnosis. we exclude patients whose zip code is from a different state than the hospital in which they are treated. hospitalization data are available at the patient zip code-by-month level, which we aggregate to the county-by-month level. we assign a zero value for admissions to counties in the months with no reported influenza admission. we only do this for counties and months in states that report data in the given year. during the influenza season from october to march, 57% of county-months have no influenza related hospital admissions in the hcup data. our results are robust with and without using the zero valued county-months in our estimations. in four falsification tests, we use outcomes less likely to be affected by air quality: primary icd codes associated with (i) diabetes mellitus with complications, (ii) urinary tract infections, (iii) skull and face fractures, and (iv) osteoarthritis. we use the categories and icd codes from the clinical classifications software (ccs) from the agency for healthcare research and quality (ahrq). see section s.1.1 for details. for a further robustness check, we use outpatient data from emergency departments (61) instead of the inpatient data, with the same strategy of counting influenza patients as above. to measure air quality, we use the epa air quality index (aqi), which measures air quality derived from ground monitors (62) . the aqi captures pollution from particulate matter (pm2.5), sulfur dioxide (so2), carbon monoxide (co), nitrogen dioxide (no2) and ozone (o3). further details on aqi calculation are provided by the epa (63). we use the daily, county level, pre-aggregated data and further aggregate up to the county-by-month level. for missing county-months, we take the average value of the adjacent counties in the same month. we use the average value of the aqi within a month as well as the number of days with air at least "unhealthy for sensitive groups" according to the epa (aqi≥100). we winsorize the aqi at the top and bottom 1% for the main analysis and show robust results without winsorization. for our analysis, we take the one month lagged aqi to identify exposure to air pollution before influenza diagnosis and not afterwards. we use pre-aggregated monthly weather averages from (64, 65) , including temper-2 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.20057216 doi: medrxiv preprint ature, specific humidity, wind speed, and precipitation, and aggregate grid points up to the county-bymonth level. we use data on the timing of national influenza-like illnesses from the cdc (38) to identify the main influenza months: october through march (see figure 1b ). this coincides with the reported influenza season in various cdc publications. we restrict our main analysis to this influenza season. vaccine effectiveness: we use the estimated vaccine effectiveness, for different age groups, by influenza season, from the cdc (66) . underlying cited studies are available from 2007/2008. since vaccine effectiveness can vary across age groups during the same influenza season, we use the reported effectiveness of the two age groups most relevant for our study: children up to 8 years old and for people 65 years and older. figure 1c shows these are the main age groups observed in the hcup inpatient data with primary influenza diagnoses. we use the categories from the clinical classifications software (ccs) from the agency for healthcare research and quality (ahrq) to identify the relevant icd codes. e093392, e093393, e093399, e09341, e093411, e093412, e093413, e093419, e09349, e093491, e093492, e093493, e093499, e09351, e093511, e093512, e093513, e093519, e093521, e093522, e093523, e093529, e093531, e093532, e093533, e093539, e093541, e093542, e093543, e093549, e093551, e093552, e093553, 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e13339, e133391, e133392, e133393, e133399, e13341, e133411, e133412, e133413, e133419, e13349, e133491, e133492, e133493, e133499, e13351, e133511, e133512, e133513, e133519, e133521, e133522, e133523, e133529, e133531, e133532, e133533, e133539, e133541, e133542, e133543, e133549, e133551, e133552, e133553, e133559, e13359, e133591, e133592, e133593, e133599, e1336, e1337x1, e1337x2, e1337x3, e1337x9, e1339, e1340, e1341, e1342, e1343, e1344, e1349, e1351, e1352, e1359, e13610, e13618, e13620, e13621, e13622, e13628, e13630, e13638, e13641, e13649 , e1365, e1369, e138. 03284, 59000, 59001, 59010, 59011, 5902, 5903, 59080, 59081, 5909, 5950, 5951, 5952, 5953, 5954, 59581, 59582, 59589, 5959, 5970, 59780, 59781, 59789 , 59800, 59801, 5990; 3 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.20057216 doi: medrxiv preprint table s .1 contains states and years with available admission months and patient zip codes in the inpatient hospitalization data we use. table s.2 contains summary statistics for inpatient hospital admissions with a primary influenza diagnosis, average monthly aqi per county-month, and the number of days with aqi ≥ 100. we use the standard deviation of the aqi during the influenza season (12.79) as well as the average inpatient hospitalization numbers (3.01) for the calculation of absolute effects based on our poisson pseudo-maximum likelihood estimation. arizona 1991 arizona ,1992 arizona ,1993 arizona ,1994 arizona ,1995 arizona ,1996 arizona ,1997 arizona ,1998 arizona ,1999 arizona ,2000 arizona ,2001 arizona ,2002 arizona ,2003 arizona ,2004 arizona ,2005 arizona ,2006 arizona ,2007 arizona ,2008 arizona ,2009 arizona ,2010 arizona ,2011 arizona ,2012 arizona ,2013 arizona ,2014 arizona ,2015 1991 ,1992 ,1993 ,1994 ,1995 ,1996 ,1997 ,1998 ,1999 ,2000 ,2001 ,2002 ,2003 ,2004 ,2005 ,2006 ,2007 ,2008 ,2009 ,2011 ,2012 ,2013 ,2014 ,2015 new york 1993 ,1994 ,1995 ,1996 ,1997 ,1998 ,1999 ,2000 ,2001 ,2002 ,2003 ,2004 ,2005 ,2006 ,2007 ,2008 ,2009 ,2011 ,2012 ,2013 ,2014 ,2015 oregon 1999 ,2008 ,2009 south dakota 2009 utah 2009 vermont 2009 washington 1993 ,1994 ,1995 ,1996 ,1997 ,1998 ,1999 ,2000 ,2001 ,2002 ,2003 ,2004 ,2005 ,2006 ,2007 ,2008 ,2009 ,2011 ,2012 ,2013 wisconsin 1999 ,2009 notes: the table shows the states and years used in the main analysis. we estimate the relationship between influenza-related inpatient hospitalizations h cym and the lagged air quality index aqi cym−1 at the county c by calendar month m by year y level using a poisson model: h cym = exp(βaqi cym−1 + x cym δ + γ cy + µ ym + cym ). we include county-by-year fixed effects γ cy to control for changing factors such as population size, income, demography and influenza testing procedures across counties and time. this also captures 5 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . unobserved annual shocks at the county level that affect both air pollution and hospitalizations. calendar month-by-year fixed effects µ ym control for a flexible overall time trend. results are robust to including additional fixed effects such as state-by-calendar month or county-by-influenza season fixed effects. while county-by-year fixed effects capture the bulk of climatic differences across counties, we also control for within-year differences with a vector of weather control variables x cym . this includes temperature, specific humidity, precipitation, and wind speed in various combinations. temperature and humidity has been shown to affect both virus survival (see (5, 6, 7, 19, 67) and air pollution (18, 23, 55) . in our baseline we include three temperature (c) bins (< 0, ≥ 0 & < 15 and > 15), five bins based on the quintiles of specific humidity, and linear terms for precipitation and wind speed. we lag the aqi by one month to account for hospital admissions data at the monthly level. the goal is to capture pollution exposure prior to the influenza diagnosis, not after. in principle, air pollution could also affect patient progression after diagnosis, but we focus on the effect of pollution leading up to the diagnosis. we estimate the model with a pseudo-maximum likelihood estimator (68, 69) , which performs well with a large number of zeros and is consistent with over-or under-dispersion in the data (70) . we cluster standard errors at the county level to allow for arbitrary heteroskedasticity and serial correlation in the errors, and show robustness to two-way clustering at the added state-year level. table s .3 provides falsification tests with outcomes unlikely to be correlated with air pollution. column 1 repeats our baseline results for influenza patients. the next four columns use inpatient hospitalizations with a primary diagnosis of diabetes mellitus with complications, urinary tract infections, skull and face fractures, and osteoarthritis. coefficients and standard errors indicate a precise zero effect for these outcomes. table s .4 explores heterogeneous effects by age, gender and race. estimates across different groups are statistically indistinguishable from one another, however, the point estimates for blacks and especially hispanics are larger than for whites. table s .5 explores robustness of our main results to different controls, fixed effects, and standard error calculations. column (1) replicates the baseline results, and reports the estimates for our weather controls (reporting was suppressed in the manuscript for simplicity). temperature and humidity controls are included as dummies for separate bins. while the coefficients on temperature are not statistically significant (county-year fixed effects absorb much of the large-scale variation), the sign is as expected. temperatures below zero c as well as above 15 c lead to fewer observed hospitalizations (see also (5, 6) ). humidity decreases hospitalizations consistent with (5, 6, 7, 19) , while precipitation and wind speed have no statistically or economically significant effects. in column (2) of table s .5, we drop weather controls, and in column (3) we include alternative functional forms of the weather controls using second order polynomials in temperature and humidity with a full set of interactions. in columns (4) and (5) we include county-by-influenza season (oct -mar) fixed effects and state-by-month of the year fixed effects. columns (6) and (7) replicate (1) and (4), but cluster standard errors on the county level as well as on the state-by-year level to allow additional arbitrary spatial correlation of errors across counties within a state-year. table s.6 reports from further robustness checks. column (1) replicates the baseline results. column (2) does not winsorize the aqi data. column (3) drops county-month cells with missing aqi 6 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . measures (rather than interpolating them based on the average value of the adjacent counties). column (4) includes patients whose zip code is from a different state than the hospital in which they are treated. column (5) restricts to states with at least seven years of reported data: arizona, colorado, kentucky, massachusetts, new jersey, new york, and washington. column (6) drops county-months with no reported influenza admissions (rather than assigning a zero value for admissions). column (7) contains results from an ordinarily least square (ols) regression instead of a poisson pseudo-maximum likelihood regression. columns (8) and (9) use alternative assumptions on who to count as an influenza patient. our baseline only counts patients whose primary diagnosis is influenza. column (8) counts patients where all diagnoses are influenza, i.e., there are no other diagnosed conditions. column (9) counts all patients with any influenza diagnosis, primary or non-primary. columns (10) and (11) use the data on outpatient (instead of inpatient) hospitalizations as the outcome variable. the effect of aqi is slightly larger on outpatient hospitalizations, consistent with the notion that these are less severe but more frequent than inpatient hospitalizations. 7 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.20057216 doi: medrxiv preprint the dependent variable is the count of hospital admissions with diagnosed influenza within a county and month. we include only the influenza intensive months of october through march. the results are from a poisson pseudo-maximum likelihood regression with specified fixed effects and control variables, except the last column (9), which is an ols regression. the number of included observations can vary across different outcomes due to fixed effects and varied counts in each county-month cell. temperature controls consist of three separate bins, specific humidity controls consist of five separate bins, precipitation and wind speed are linear terms. all weather variables are based on the monthly county averages. a higher aqi means worse air quality. standard errors in parentheses are clustered at the county level. 9 all rights reserved. no reuse allowed without permission. author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.07.20057216 doi: medrxiv preprint influenza vaccines for the future estimates of global seasonal influenza-associated respiratory mortality: a modelling study epidemic influenza and vitamin d on the epidemiology of influenza influenza virus transmission is dependent on relative humidity and temperature absolute humidity modulates influenza survival, transmission, and seasonality absolute humidity and the seasonal onset of influenza in the continental united states air pollution and case fatality of sars in the people's republic of china: an ecologic study world bank emergency department databases (sedd) (healthcare cost and utilization project, agency for healthcare research and quality air quality system data mart (us environmental protection agency technical assistance document for the reporting of daily air quality (united states environmental protection agency continental-scale water and energy flux analysis and validation for the north american land data assimilation system project phase 2 (nldas-2): 1. intercomparison and application of model products nldas primary forcing data l4 monthly 0.125 x 0.125 degree v002 (goddard earth sciences data and information services center (ges disc) cdc, seasonal flu vaccine effectiveness studies (centers for disease control and prevention, national center for immunization and respiratory diseases (ncird) airborne micro-organisms: survival tests with four viruses pseudo maximum likelihood methods: applications to poisson models ppmlhdfe: fast poisson estimation with high-dimensional fixed effects the log of gravity skull and face fractures icd-9-cm codes: 80000 skull and face fractures icd-10-cm codes: s020xxa osteoarthritis icd-10-cm codes we thank luisa osang and jeffrey shaman for helpful discussions. all errors are our own. the irb for access to the hcup data through the national bureau of economic research (nber) was approved by the nber. funding: no specific grants were connected to this project. author contributions: gs, jgz, mn and ns conceptualized the study, gs analyzed the data, and gs, jgz, mn and ns wrote the manuscript. competing interests: the authors declare no competing interests. data and materials availability: the replication code and materials for both the manuscript and the supplementary materials will be made publicly available at harvard dataverse. the restricted access data can be accessed at (39). notes: the dependent variable is the count of hospital admissions with diagnosed influenza within a county-month. we include only the influenza intensive months of october through march. results are from a poisson pseudo-maximum likelihood regression with specified fixed effects and control variables. the number of included observations can vary across different outcomes due to fixed effects and varied counts in each countymonth cell. a higher aqi means worse air quality. standard errors in parentheses are one-way or two-way clustered as indicated. key: cord-000244-wrru98zg authors: pfeil, alena; mütsch, margot; hatz, christoph; szucs, thomas d title: a cross-sectional survey to evaluate knowledge, attitudes and practices (kap) regarding seasonal influenza vaccination among european travellers to resource-limited destinations date: 2010-07-07 journal: bmc public health doi: 10.1186/1471-2458-10-402 sha: doc_id: 244 cord_uid: wrru98zg background: influenza is one of the most common vaccine-preventable diseases in travellers. by performing two cross-sectional questionnaire surveys during winter 2009 and winter 2010 among european travellers to resource-limited destinations, we aimed to investigate knowledge, attitudes and practices (kap) regarding seasonal influenza vaccination. methods: questionnaires were distributed in the waiting room to the visitors of the university of zurich centre for travel' health (cth) in january and february 2009 and january 2010 prior to travel health counselling (cth09 and cth10). questions included demographic data, travel-related characteristics and kap regarding influenza vaccination. data were analysed by using spss(® )version 14.0 for windows. differences in proportions were compared using the chi-square test and the significance level was set at p ≤ 0.05. predictors for seasonal and pandemic influenza vaccination were determined by multiple logistic regression analyses. results: with a response rate of 96.6%, 906 individuals were enrolled and 868 (92.5%) provided complete data. seasonal influenza vaccination coverage was 13.7% (n = 119). only 43 (14.2%) participants were vaccinated against pandemic influenza a/h1n1, mostly having received both vaccines simultaneously, the seasonal and pandemic one. job-related purposes (44, 37%), age > 64 yrs (25, 21%) and recommendations of the family physician (27, 22.7%) were the most often reported reasons for being vaccinated. in the multiple logistic regression analyses of the pooled data increasing age (or = 1.03, 95% ci 1.01 1.04), a business trip (or = 0.39, 95% ci 0.17 0.92) and seasonal influenza vaccination in the previous winter seasons (or = 12.91, 95% ci 8.09 20.58) were independent predictors for seasonal influenza vaccination in 2009 or 2010. influenza vaccination recommended by the family doctor (327, 37.7%), travel to regions with known high risk of influenza (305, 35.1%), and influenza vaccination required for job purposes (233, 26.8%) were most frequently mentioned to consider influenza vaccination. conclusions: risk perception and vaccination coverage concerning seasonal and pandemic influenza was very poor among travellers to resource-limited destinations when compared to traditional at-risk groups. previous access to influenza vaccination substantially facilitated vaccinations in the subsequent year. information strategies about influenza should be intensified and include health professionals, e.g. family physicians, travel medicine practitioners and business enterprises. pandemic and seasonal influenza are still a challenging field of the public health system. influenza -a mild to severe respiratory infection caused by rna viruses of the family orthomyxoviridae -is one of the most common vaccine-preventable disease in travellers. worldwide, between 250'000 and 500'000 deaths are estimated to be due to seasonal influenza infection each year [1] . influenza is also responsible for tremendous economic costs both from admissions to hospital and loss of productivity [2] . influenza affects all age groups and is usually self-limited. common symptoms include acute fever, muscles pain, headache, cough and chills [3] . special risk groups, such as very young children, the elderly and those suffering from chronic lung or heart diseases are at risk for serious influenza complications, e.g. bacterial pneumonia [4, 5] . influenza reaches peak prevalence in winter in the northern hemisphere (nov-apr) -as well as in the southern hemisphere (apr-oct) and circulates yearround in the tropics [6, 7] . seasonal influenza vaccination is an effective prevention strategy and is therefore routinely recommended for special risk groups [8, 9] . of note, the seasonal influenza vaccine recommendations of the u.s. centres for disease control were recently expanded and include now about 80% of the population [10] . influenza is known to be a quite frequent infection among travellers to tropical and subtropical destinations compared to other infections, e.g. vector-borne ones. about one of hundred travellers abroad gets infected [7] . the risk of infection depends on the travel destination and the season. travellers crossing hemispheres may be confronted with different antigenic variants of the influenza virus. by returning home, the new variant may be transmitted to contact persons [11] . the first pandemic of the 21st century has highlighted the need for international influenza prevention strategies [12] . the objective of this study was to investigate the vaccination coverage as well as knowledge, attitudes and practices (kap) regarding influenza vaccination among travellers to resource-limited countries to improve or adapt current preventive strategies. two cross-sectional surveys were conducted at the university of zurich centre for travel' health during january and february 2009 and january 2010, respectively. selfadministered, anonymous questionnaires including 16 items were distributed to travellers waiting for pre-travel health advice. participation was voluntary. individuals above 17 years, understanding german or english, residing in switzerland and planning to travel to a resourcelimited destination were included. questions included demographic data (gender, age, nationality, education, profession), travel-related characteristics (destination country, duration of stay, influenza risk perception, previous travel health advice, travel purpose, travel costs) and general attitudes and practices towards influenza vaccination (vaccination coverage, reasons to be vaccinated, reasons to refuse vaccination, motivations to consider vaccination with options for multiple answers except for the vaccination coverage). in 2010, an additional question targeting the pandemic influenza a/h1n1 vaccination coverage was included. the questionnaires were checked for completeness. a written letter of exempt was received by the ethical commission of the canton of zurich. statistical analyses were conducted by using spss ® version 14.0 for windows. differences in proportions of demographics, travel-related data and attitudes and practices were compared using the chi-square test. the significance level was set at p ≤ 0.05. for the multiple logistic regression analysis the surveys were analysed as well as pooled dataset and each survey, cth-2009 and cth-2010, separately. the seasonal influenza vaccination was used as outcome and all demographic, travel-related and attitude-and practices-related factors were evaluated as independent predictors. odds ratios (or) were determined by stepwise backward elimination of variables with p > 0.150. for sensitivity analyses, each dataset of the cth studies, 2009 and 2010, was analysed separately and additionally, predictors for pandemic influenza vaccination were determined by multiple logistic regression analyses. from a total of 938 eligible individuals, 868 (92.5%) were included in the analysis ( figure 1 ). overall, 479 (55.2%) were females and 389 (44.8%) males. the great majority of participants (503, 57.9%) were between 18 and 35 years old with a median age of 32 years (range 18 -84 yrs). only 46 (5.3%) responders were above 64 years of age. in general, participants were highly educated with 480 (55.3%) being university graduates. overall, the characteristics of participants planning to travel to resource-limited destinations are presented in table 1 . of all vaccinated participants, 44 (37%) declared to be vaccinated for business reasons and 25 (21%) due to age ( travel as risk factor for an influenza infection is poorly established among international travellers when regarding the low vaccination coverage as well as the low selfperceived travel-associated risk estimates. of note, previous influenza vaccinations facilitated receiving an influenza vaccination in the following year by about 13 times. therefore, easy access to the influenza vaccine is important. high media coverage was not considered sufficient to increase the vaccination rate substantially as is indicated by the low increase of the vaccination coverage between the two surveys in 2009 and 2010 and also by the low pandemic influenza vaccination coverage of only 14.2%. therefore, multiple efforts need to complement one another including information strategies provided by family physicians and travel medicine practitioners, but also job-and age-related activities need to be considered. our sample of travellers is comparable to other studies performed at our centre for travel' health [7] with respect to the age distribution, educational level and travel duration. inherent limitations include a selection bias: frequently visited destinations such as the middle east, north africa and the caribbean are underrepresented as travellers to those destinations generally do not consider a pre-travel health consultation as indicated [11] but destinations with higher risk for faecal-orally transmitted infectious diseases, such as td or bacterial meningitis, are well represented, such as e.g. india and sub-saharan countries. therefore, our sample may represent a best practice sample. the fact, that the high proportion of university graduates indicates a health literate population may result in an even overestimated risk perception as well as influenza vaccination coverage. all data collections relied on self-reported information. hence, the results of the studies might be limited by a potential bias such as disclosure bias, although self-report of influenza vaccination status has been found to be reliable when checked against medical record documentation [13] . most seasonal influenza activity occurs during november to april on the northern hemisphere and vaccination is usually administered between october and november. therefore, travellers visiting the opposite hemisphere have to be counselled accordingly and the seasonal influ-enza vaccine also for the southern hemisphere has to be available as there is year-round influenza activity in tropical and subtropical areas. risk perception and vaccination coverage regarding seasonal and pandemic influenza was very poor among european travellers to resource-limited destinations reducing the burden of influenza-associated complications with antiviral therapy the pathology of influenza virus infections influenza: changing approaches to prevention and treatment in travelers the scientific basis for offering seasonal influenza immunisation to risk groups in europe absolute humidity and the seasonal onset of influenza in the continental united states influenza virus infection in travelers to tropical and subtropical countries awareness of vaccination status and its predictors among working people in switzerland influenza vaccination coverage rates in five european countries during season 2006/2007 and trends over six consecutive seasons centers for disease control and prevention (cdc) -cdc's advisory committee on immunization practices (acip) recommends universal annual influenza vaccination knowledge, attitudes and practices in travel-related infectious diseases: the european airport survey h1n1 influenza influenza vaccination uptake and socioeconomic determinants in 11 european countries we thank all participating travellers and we acknowledge the technical assistance of patricia blank. the authors declare that they have no competing interests.authors' contributions tds, mm and ch conceived and supervised the study. ap performed all data collection and data analysis and drafted the manuscript. mm participated in designing the study and the questionnaire and organised access to the data of the airport-study. all authors have read and approved the final manuscript. key: cord-011917-6u0t4hy8 authors: skarlupka, amanda l.; ross, ted m. title: immune imprinting in the influenza ferret model date: 2020-04-08 journal: vaccines (basel) doi: 10.3390/vaccines8020173 sha: doc_id: 11917 cord_uid: 6u0t4hy8 the initial exposure to influenza virus usually occurs during childhood. this imprinting has long-lasting effects on the immune responses to subsequent infections and vaccinations. animal models that are used to investigate influenza pathogenesis and vaccination do recapitulate the pre-immune history in the human population. the establishment of influenza pre-immune ferret models is necessary for understanding infection and transmission and for designing efficacious vaccines. nearly 500,000 annual global deaths are attributed to influenza virus infection [1] . the influenza virus consists of four different types (a, b, c, d) with types a and b causing symptoms in humans. these two types are further classified into subtypes and sub-lineages, respectively. the surface of the influenza virion consists of two major glycoproteins the hemagglutinin (ha; type a: h1-h18; type b: yamagata-, victoria-lineage) and the neuraminidase (na; type a: n1-n11), each of which are separated into antigenically distinct subtypes and together denote viral subtypes [2] . the glycoproteins undergo independent evolution leading to antigenic drift as the virus accumulates mutations over time. the ha and na gene segments reassort due to the segmented nature of the viral rna genome, leading to antigenic shifts. these shifts are unprecedented and are often met with no neutralizing antibodies in the human population, leading to heightened pandemic potential. within subtypes, there can be distinct shifts in antigenicity, such as with the seasonal a(h1n1) which circulated in humans until the emergence of the swine-origin 2009 pandemic a(h1n1). the surrogates of protection, induced either through infection or vaccination, rely on serological assays that are dependent on the presence of neutralizing antibodies to mainly the ha glycoprotein: hemagglutination inhibition, single radial hemolysis, and microneutralization [3] . initial exposure to influenza occurs during childhood; at six to nine years old, 80% of children were seropositive to a(h3n2) [4] . this initial infection primes the immune system and biases future immune responses to subsequent infections and vaccinations [5] [6] [7] [8] [9] [10] [11] . due to the cost and resources of human clinical trials, there are numerous animal models designed to study this viral pathogen [12] . the ferret model remains indispensable due to similarities in lung physiology [13, 14] , anatomical distribution of sialylated glycan receptors [15] , and glycomic profile of ferret respiratory tissues [16, 17] . these animal models are commonly used to study viral characteristics, host immune responses, and vaccine/antiviral therapies. therefore, animal models that mimic pre-existing human immunity to influenza viruses may better represent the human immune responses to infection and vaccination. the current preventative method used to combat influenza is vaccination. administered annually, the most common intramuscular split-inactivated cell-based influenza vaccine is composed of four viral strains, an a(h3n2), an a(h1n1), and two type bs, one each from the yamagata and victoria lineages. for the seasons between 2014 and 2019, the adjusted overall vaccine effectiveness (ve) in the united states ranged between 19% and 48% [28] [29] [30] [31] [32] . the ve varies between subtypes and year to year due to numerous viral (antigenic drift/shift), vaccine (egg-adaptive changes, immunogenicity) and host (age, immune-status, pre-immunity) factors. to address the viral contributions, the world health organization (who) reviews data from its global influenza surveillance network to identify circulating strains, heavily weighting ferret serological cross-reactivity data to determine antigenic drifts. vaccine manufacturers are then provided a list of recommended vaccine strains for that particular season for either the northern or southern hemisphere. methods to increase the host response to vaccination and ve include different vaccine compositions, especially in adults older than 65 years of age due to their decreased immune response to vaccination [33] . the inclusion of adjuvants, varying the amount of antigen, using live-attenuated viral vaccines and varying the route of administration are all methods used to increase ve. the gold standard for influenza vaccine research is the ferret animal model [34] . currently, there are models developed that capture infant [35] , aged [36] , naïve, and pre-immune scenarios [37] . during vaccine selection, experts consider only data generated from the naïve ferret model for determination of recommended strains for currently administered human vaccines. however, the immune response in a naïve host, compared to a pre-immune individual, differs during subsequent vaccination and/or infection [38, 39] . therefore, the use of naïve ferret sera for vaccine strain selection is potentially misrepresentative of the pre-immune human population that receives each season's influenza virus vaccines, contributing to the observed ve. a pre-immune animal model should be established through an initial viral infection, instead of through a vaccination regimen. vaccination cannot be a surrogate for viral imprinting and pre-immunity due to the inequivalences in the immune responses to an active influenza infection versus an intramuscular unadjuvanted vaccination [38] . administering a vaccine matched to the challenge virus does not produce a vaccine with 100% efficacy. healthy volunteers vaccinated with inactivated or cold-adapted live influenza vaccines were not all protected from challenge with homologous viruses; the estimated protective efficacies were 71% and 85%, respectively [40] . vaccination does not induce a robust t-cell response compared to infection, which, in ferrets, has been found to contribute to sterilizing immunity [41] . furthermore in ferrets, vaccination and viral pre-immunity differ in their protective outcomes as well [41] . significant immunological differences, such as ratios of igg and iga influenza-specific antibodies and targeted antigenic sites [26] , cannot be discerned and identified with the commonly used hemagglutinin inhibition assay (hai). a pre-immune-vaccination ferret model was used to investigate the phenomenon of low efficacy from repeat vaccination with commercial quadrivalent inactivated influenza vaccine that occurs in humans [42] . when matched to ferrets vaccinated once, repeatedly vaccinated ferrets had less protection, higher viral shedding and lower t-lymphocyte counts, whereas the serological responses, cell-mediated immunity, and histopathological changes did not differ. it was hypothesized that although the magnitude of the serological response was similar the composition differed, resulting in the difference of protection. a larger ratio of non-neutralizing to neutralizing antibodies may have been recalled in the repeat vaccination group. hence, the repeat vaccination group had lower vaccine efficacy compared to one vaccination. encouragingly, the repeat vaccination group was still better protected than no vaccine group. the relevance of using these results to explain the decreased vaccine efficacy in humans is limited due to the lack of pre-immunity establishment in ferrets. even with well-planned studies, not establishing pre-immunity creates a confounding factor when extrapolating the findings to the human population. in early ferret vaccination studies, the administration of unadjuvanted vaccines elicited no measurable antibody outcome. not all naïve ferrets seroconvert to influenza vaccination [39, [43] [44] [45] . even with the addition of an adjuvant, the immune response can be weak, especially when compared to the immune response elicited by a live homologous infection [45, 46] . the lack of an antibody response is associated with a lack of protection [47] . for instance, naïve ferrets immunized with a(h3n2)/hong kong/x31/1968 vaccine were all susceptible to homologous challenge and none produced vaccine-specific serum hai antibodies [39] . this low reactivity to the vaccine may be attributed to the outbred nature of the animal model [42] . low seroconversion ratios may also be due to the low immunogenicity of the influenza vaccine. in humans, vaccines vary in immunogenicity [48] [49] [50] , particularly in immunocompromised adults and children [51] . due to this issue, pandemic influenza vaccines can be adjuvanted to ensure an efficient immune response [52] [53] [54] [55] . with the inclusion of different adjuvants, different magnitudes of seroconversion and protection can be achieved [47] . some studies used virus-like particle (vlp) vaccines produced from insect cells using a baculoviral system which results in 100% seroconversion [56, 57] . the manufacturing process of these vlps retain insect protein that act as an adjuvant contributing to seroconversion. research groups have attempted to solve this phenomenon through multiple vaccinations, i.e., a prime-boost or prime-boost-boost regimen, or with addition of adjuvant to elicit an antibody response [58] . establishment of pre-immunity overcomes this phenomenon; pre-immune ferrets respond to vaccination at a higher proportion than immunologically naïve ferrets. with either type a homosubtypic [59] or heterosubtypic [39] pre-immunity, the vaccine-specific serum hemagglutinin inhibition titers are increased. imprinting primed the immune system towards future influenza vaccinations. this priming phenomenon is not only present in the ferret animal model, but also occurs in mice and hamsters [60] [61] [62] . one of the earliest works with pre-immunity in ferrets was conducted by webster in 1966 investigating the presence of original antigenic sin in ferrets by conducting sequential infections [63] . following this, in the 1970s, a vast amount of pre-immunity work was conducted with ferrets. these studies focused on characterizing the ferret immune system response to live and killed virus, vaccination, adjuvant and heterotypic and heterologous infections [39, 45, 46, [64] [65] [66] [67] . further, heterosubtypic immunity was shown not to wane over a period of up to eighteen months [68] . this historical collection of ferret research laid the foundation for showing that low vaccination seroconversion proportions for naïve ferrets can be overcome by the development and optimization of a pre-immune animal model. the pre-immune model never advanced after this time, potentially due to the lack of immunological reagents and tools needed to properly characterize the model and general ignorance of the magnitude imprinting and pre-immunity contributes towards vaccination and infection. after a lull in the pre-immune ferret research, the a(h1n1) 2009 swine influenza pandemic initiated the dramatic increase of the investigative effort into imprinting, pre-immunity, and heterologous protection. the early epidemiological and serological studies that inspired this interest suggested that pre-existing immunity may have altered the pandemic virus' morbidity and mortality in the human population [9, 69] . the resultant pre-immunity models were based upon the historical model: (1) establish anti-influenza virus immune memory with sub-lethal viral challenge; (2) assess for seroconversion; (3) vaccinate, if necessary; (4) challenge with a(h1n1)/california/2009. a prolonged period of rest between imprinting and vaccination or challenge allows the ferret to return to an assumed immunological baseline after the generation of an adaptive memory response and recovery from damage and local cellular activation in the lung tissue. compared to the 1970s, the drastic increase in the understanding of the immune system, the effects induced from influenza challenge and vaccination, and the ability to measure and quantify these important details allowed for a well-defined model. the worldwide 2009 pandemic inspired much research looking at the protective effects of seasonal a(h1n1) imprinting on the a(h1n1) 2009 pandemic strain. therefore, much of the published research has focused on the a(h1n1) subtype (tables s1 and s2). sterilizing immunity in ferrets, an immune state that blocks viral infection [41] , can be achieved through establishing pre-immunity [70] . whereas, with an intramuscular vaccination subsequent infection was not inhibited, although virus shedding was reduced. the gathered data from this study were restricted to viral characteristics, such as virus shedding, transmission frequency and morbidity and mortality due to the lack of ferret immunological reagents. from these data, an ideal state of protection against re-infection of influenza virus was defined along with a goal to generate a vaccine that will elicit similar protection. although not sterilizing, it was found that seasonal a(h1n1) pre-immune animals exhibited immunity and mitigated infection against the pandemic a(h1n1) virus [70] . through multiple infection and vaccination schemes, vaccination with the trivalent influenza vaccine (tiv; containing only one type b strain instead of two) was unable to lessen the resulting morbidity or contact transmission in ferrets following challenge with the pandemic a(h1n1) [38] . conversely, imprinting with a seasonal a(h1n1) virus altered the morbidity, but not the transmission characteristics of the pandemic a(h1n1) [38] . although, these viral traits were muted, there was only minimal detection of cross-reactive serum antibodies. the 2009 pandemic was characterized by distinct protective responses seen between different age groups of people. older adults were more protected than young adults and children against the a(h1n1)/california/07/2009 pandemic virus. this older population was captured in the pre-immune ferret model by imprinting with historical antigenically distinct viruses [71] . the protective responses to a pandemic challenge were then measured. the historical viruses from the 1950s and earlier elicited more protective responses than the naïve ferrets [71] . this corroborated the human data that the older population was more protected than the younger to a pandemic challenge. next, the effects of imprinting on the response to a pandemic vaccine were determined. ferrets imprinted with seasonal a(h1n1) received a pandemic a(h1n1) vaccine. this seasonal a(h1n1) priming did not diminish the antibody response to either infection or vaccination with the pandemic virus [71] . furthermore, original antigenic sin was not observed in the context of seasonal a(h1n1) to pandemic a(h1n1). additionally, priming with seasonal a(h1n1) virus provided cross-protection against the pandemic virus. however, it did not impact the transmission efficiency [72] . a human's pre-immune history can be recapitulated in ferrets by conducting repeated infections. carter et al. [73] utilized this technique by sequentially infecting two different ferret groups with seasonal a(h1n1): one with historical a(h1n1) from 1934 to 1957, and another with contemporary a(h1n1) viruses from 1999 to 2007. the abilities of the differing pre-immunities to protect against an a(h1n1)/california/07/2009 challenge were compared. both sequential groups were protected from challenge; they exhibited no weight loss, minimal recoverable virus, and no transmission compared to ferrets pre-immunized with only one of the viruses. unique to sequentially infected ferrets, the elicited antibody profile was broader and interacted with pandemic a(h1n1) ha compared to the single pre-immunity groups as measured with ha-specific elisa binding. the recall and adaptation of the antibody profile over time in response to sequential exposures is complex and still not completely understood. however, these interactions help to explain the puzzling epidemiological and serological observations surrounding the pandemic a(h1n1) outbreak. carter et al. [73] hypothesized that the older adults were exposed to more antigenically variant strains and have extensive protection compared to young adults with less exposure. in addition, the changes in the elicited antibody profile point to the possibility of achieving broad vaccine-induced protection against influenza viruses by sequential immunization with a series of antigenic variants. further analysis with these ferret samples and infection/immunization scheme confirmed the change in antibody profile observed previously. anti-ha stalk antibodies increased, even in the absence of receptor-binding site antibodies, leading to the observed cross-reactivity and reduction in clinical signs and transmission [74] . in contrast to previous reports that pre-immunity induced protection does not wane [68] , the boosts in anti-ha stalk antibodies and the cross-reactivity induced from sequential infection with antigenically distinct seasonal a(h1n1) declined over time [74] . the pandemic outbreak occurred from a transmission event of an avian-human-swine reassortant virus from a swine host into the human population [75] . whereas many groups investigated the effects of human seasonal a(h1n1) imprinting, the effects of imprinting with a classical swine virus were also determined. min et al. [76] exhibited that infection with classical swine viruses elicited cross-reactive neutralizing antibody activity and provided protection against the pandemic a(h1n1) virus. over time, a natural break point occurred between the investigation into the pandemic mystery and the examination of the immune system response within the context of imprinting and pre-immunity. within a seasonal and pandemic a(h1n1) sequential infection study, the changes in polyclonal serum antibodies responses were measured. ferrets pre-immunized with the seasonal a(h1n1)/texas/36/1991 were followed up with an a(h1n1)/california/07/2009 infection. the elicited serum antibody specificity shifted to target a different region of the ha compared to the a(h1n1)/texas/36/1991 only serum [77] . the overall antibody response moved to epitopes near the ha receptor-binding domain; sites where homology between these two strains is shared [77] . these findings resulted in research in the basic science of how imprinting and pre-immunity affects vaccination and infection and subsequent immune responses. the current pre-immunity models are moving away from investigating the differences in disease symptoms and vaccine effectiveness observed in the human population in response to the pandemic virus. resources are now focused on how pre-immunity effects vaccination responses and general vaccine efficacies to any virus, not just in terms of the pandemic a(h1n1) (tables s2 and s3) . furthermore, the pre-immune model is now being used, in replacement of a naïve model, to test novel vaccine candidates and methods currently in research and development [37] . sequential infections of antigenically distinct viruses lead to a broader antibody response than that of just one strain [59, 78] . this illustrated the importance of using a pre-immune model for antigenic characterization and vaccine testing. in fact, when a(h3n2) antigenic maps were produced using sera from naïve or pre-immune ferrets the maps poorly correlated; the classification of whether two viruses were antigenically distinct or similar varied with the model (naïve or pre-immune) [79] . the broader antibody response characterized with a(h1n1) or a(h3n2) sequential infections was extremely informative at the hetero-subtype level [78] . however, the impacts of other subtypes, such as a(h5n1) and a(h7n9) were not investigated, and neither were the nuances of how protective different antigenically drifted strains within a subtype investigated. the antibody profiles of sequentially infected ferrets with a(h3n2) viruses revealed that a(h3n2) pre-immunity affected both the quantity and quality of antibodies elicited [79] . with high hai titers against the imprinting virus, lower titers were observed toward the most recent isolate, a(h3n2)/hong kong/4801/2014. after repeated a(h3n2) infections, the antibody avidities gradually increased for a(h3n2) compared to those from a single homologous infection. this repeated a(h3n2) exposure expanded the cross-reactivity breadth against the same hai panel. with computationally optimized ha vaccines, the same increased breadth phenomenon, back-boosting, occurred. pre-immunity to a historical a(h3n2) virus helped boost the magnitude and breadth of the broadly neutralizing antibodies elicited by computationally optimized broadly reactive antigen (cobra) immunogens compared to a naïve ferret group [37] . emphasis was placed on the concept that testing vaccine candidates in naïve ferrets do not reflect the performance of the vaccines in the human population. in an a(h1n1) primed model, greater protection after vaccination was observed [18] . vaccine effectiveness varies in the human population from season to season. epidemiological data from people suggests that pre-existing immunity can result in decreased vaccine effectiveness [80] [81] [82] . the pre-immune model has been used to study how vaccines can overcome pre-existing immunity to mount a new response with m2-deficient single replicon vaccine candidates for a(h1n1) and a(h3n2) subtypes [83] . type b and a(h1n1) heterosubtypic pre-immunity followed by a(h3n2) vaccination provided protection against an antigenically distinct a(h3n2) challenge. a(h1n1) pandemic homosubtypic imprinting negatively affected the ability of a flumist-like vaccine to elicit protection towards seasonal a(h1n1) [83] . these key findings highlight the difficulty with inducing immunity to a novel ha in the presence of pre-existing heterotypic, heterosubtypic, and homosubtypic immunity. although the questions surrounding the 2009 outbreak have been sufficiently answered, there still remains the possibility for a second swine-origin pandemic. even of the same subtype and species origin, pandemic a(h1n1) pre-existing immunity was unable to induce sterilizing immunity to current circulating swine-origin a(h1n1) viruses [84] . furthermore, other subtypes-a(h3n2) and a(h1n2)-circulate in swine and transmit into the human population [85] . the swine-origin a(h3n2) subtype raises concerns due to human's documented susceptibility to a(h3n2) viruses, and transmission in ferrets being as efficient as human-origin seasonal a(h3n2) viruses [86] . differing pre-immunity may be protective against these swine-origin viruses; pre-immunity with the human a(h3n2)/perth/16/2009 cross-protected against a swine-origin variant a(h3n2), whereas other human strains did not [26] . hence, prior seasonal virus infections may be protective by limiting viral replication and reducing transmission. this may suggest that within the human population, different age groups are more susceptible to certain transmission events depending on the subtypes of influenza virus that they have previously been exposed to [4] . the ferret immune response to reinfection is strikingly different compared to the response after a primary infection [87] . when comparing a(h1n1)/mexico/4108/2009 challenge in naïve and a(h1n1)/mexico/4108/2009 pre-immune ferrets, the pre-immunity status limited viral titers. the virus was still detectable at low levels at day seven post-infection. assessment of the ferret transcriptome during this challenge provided invaluable data for unraveling the immune response to infection. in a primary challenge, innate immune system and inflammatory genes were upregulated in both the lung and lymph node tissues. comparatively, in the pre-immune ferret, the adaptive immune response genes (cxcl10, ccl5) were upregulated in the lungs, with no upregulation in the lymph nodes. the lack of lymph node gene activity suggested that influenza specific cd8+ t-cells and b-cells may have originally resided within the lungs before infection or the adaptive immune response originated from another unidentified peripheral compartment [87] . influenza virus infection also imprints on the influenza-specific t-cell memory compartment. in an a(h1n1)/a(h1n1) homologous and an a(h1n1)/a(h3n2) heterologous challenge, a(h1n1) imprinting partially protected against the a(h3n2) challenge, reducing virus shedding duration, but not the peak virus titer. the inflammatory immune response was increased, but less than that of the immunologically naive infected ferrets [88] . differences between interferon-gamma (ifn-γ) producing peripheral blood mononuclear cells (pbmcs) in naïve vs. a(h1n1) pre-immunized ferrets with a homologous a(h1n1) challenge were not discernable. however, the pre-immune ferrets had ifn-γ producing pbmcs that were stimulated by heterosubtypic viruses. the imprinting event did not lessen the quantity ifn-γ producing pbmcs compared to mock/a(h3n2) infected ferrets. however, heterosubtypic pre-immunity affected the reactivity of the ifn-γ producing lung mononuclear cells (mncs) and induced whole-blood ifn-γ producing cells quicker. a(h1n1)/a(h3n2) pre-immune ferrets had high reactivity of ifn-γ producing lung mncs to a(h1n1) virus compared to a(h1n1)/a(h1n1) ferrets that had no increase. although, the classification of these t-cells as cd8+ or cd4+ was not conducted as of yet, pre-immune ferrets can be used to model t-cell population contributions to influenza-specific memory and recognition. future studies will investigate the different cellular and humoral responses to influenza virus infection and clarify the differences between t-cell subsets. the study conducted by hay et al. [89] analyzed data derived from a previous study [70] to mathematically model the short-term antibody kinetics from either influenza infection or vaccination with and without adjuvant. their results, although limited in sample size, highlight the potential future applications and data analysis of the serological, cellular, and virological data that can be collected during a pre-immune study. the effects of a(h1n1) and a(h3n2) pre-immunity on the a(h5n1) subtype or the effect of a(h5n1) on a(h1n1) or a(h3n2) vaccination or subsequent viral challenge are not well known. although studies have been performed in pre-immune mice [90, 91] , few studies have addressed pre-immunity using the viruses of the a(h5n1) subtype [92] (table s4) . pre-pandemic vaccines for a(h5n1) influenza viruses elicited low seroconversion proportions in immunologically naïve ferrets. when primed with seasonal live attenuated influenza vaccine (laiv), a(h5n1) ha-specific igg antibody secreting cells (asc) were stimulated compared to unprimed ferrets. expansion of the a(h1n1) or a(h3n2) specific memory b-cells may cross-react with the a(h5n1) ha antigen. when imprinted with the individual vaccine strains, the igg asc levels were similar to the laiv imprinted cells, with the a(h1n1) imprinting influenza virus eliciting higher responses than a(h3n2) imprinting viruses. this effect may be a response to the ha molecule rather than the a(h1n1) na protein because an a(h1n2) reassortant virus elicited a similar response. but a synergistic or additive effect was not determined with an a(hxn1) reassortant virus to confirm the lack of protective contribution from the na. however, upon closer inspection, the protection afforded by the a(h1n1) ha was temporary and waned to levels similar to naïve ferrets three weeks after the second dose was administered. other animal models are used to study influenza vaccination and infection. the mouse model is commonly used due to the plethora of genetic tools and the availability of reagents. in addition, pre-immunity can be established in the mouse model [23, 76, 78, 91, [93] [94] [95] [96] . within mice, pre-infection compared to vaccination elicited similar innate immunity and antibody responses. sterilizing immunity was achieved in pre-immune animals that had reduced viral receptors and increased t-cell responses in the lungs [41] . although less utilized than mice or ferrets, guinea pigs produce similar results [78, 97] . the main hypothesis and question being addressed will determine strains selected for use in a pre-immune model. the priming method, whether by vaccination or infection should be evaluated based on the target population of the vaccine/study. an infant or child target demographic may warrant that priming actually occur through vaccination followed by viral challenge. this instance recapitulates if the vaccine was administered before exposure to influenza. in contrast, to target populations whose first exposure is through infection, live influenza virus would be the appropriate priming method. the initial imprinting strain should be antigenically representative for the population being modeled. therefore, the timing and order of the infections are variable. after imprinting, ferrets may be re-infected to add to the pre-immune history or be vaccinated or challenged according to study design. however, the magnitude of the contributions of a full pre-immunity history compared to just the initial imprinting virus on the immune response has not been adequately quantified. for instance, if attempting to recapitulate a person born in 1970, it is unclear if it is only necessary to pre-immunize with an a(h3n2) 1970s virus, followed by a(h1n1) influenza virus, or if all of the antigenically distinct a(h3n2) viruses are needed to establish a true pre-immune state. studies investigating the effects of heterosubtypic and heterotypic imprinting and pre-immunity can be expanded to include: (1) type b imprinting followed by an a(h1n1) or a(h3n2) vaccination or challenge, (2) a(h2n2) imprinting, (3) a(h1n1) imprinting and a(h3n2) pre-immunity and vice versa on which combination results in more protection against a(h1n1), a(h3n2), a(h2n2), or a(h5n1) challenge. limited research is available on the serological, cellular, and immunological effects of infections with varying infection doses, but there have been observed differences [98] . the immunological system should return to the baseline immunological state before attempting another repeat infection or vaccination. this period should encompass the cool-down time for both the innate and adaptive immune systems, including the induction and contraction of b-and t-cells into their memory states. studies similar to leon et al. [87] are of great importance for understanding the inner workings of the ferret immune system during imprinting and re-infection. without these studies, this cool-down component will need to continuously be stated as a study limitation. for example, pulit-penaloza et al. [84] indicated that a thirty-one day interval between primary and secondary challenges for their studies may have been too short, allowing for elevated non-specific innate responses from the primary infection to affect the secondary. without letting the immune system return to baseline, misrepresentation of cross-protection may be observed. cheng et al. [92] found that the heterologous protective titers waned to levels not significantly different than the negative naïve control ferret group providing only temporary protection. the age and gender of the animals being used may be a confounding factor especially when comparing humoral responses [99, 100] . the ages for male and female ferrets ranged from two to twelve months. ferrets were considered aged when they were greater than four years of age [36] . reporting results by gender may reveal new avenues for influenza virus research. the breeding vendor and housing conditions of the animals should also be reported. within the mouse model, the microbiome differs by vendor in the gut [101, 102] and in the lungs [103] , contributing to different responses to vaccination and influenza infection [104] [105] [106] . therefore, it is recommended that when mice or ferrets are housed in separate holding rooms, bedding or enrichment equipment is shared between the cages to merge the microbiomes together [105] . inclusion of the ferret health history is also beneficial. metadata such as whether they were castrated, spayed, de-scented, or received any previous vaccinations or treatments may shed light on immunological results. the ferret model captures special at-risk populations. ferret age was varied to encompass different age groups: young, adult and old. adult and aged ferrets, similar to humans, exhibit significant immune response differences when comparing homologous and heterologous a(h1n1) priming and challenges suggesting immune senescence in the aged ferret population [36] . therefore, the use of this model would contribute to vaccine testing and efficacy studies. currently, a vaccine specifically formulated for aged individuals already exists due to their high-risk status and substantial contributions to influenza-associated hospitalizations and deaths [33, 107, 108] ; this high dose vaccine contains 60 ug of each vaccine strain ha, compared to the standard 15 ug. the national institute of allergy and infectious diseases' (niaid) goal for the development of a universal influenza vaccine requires a protection equal to or greater than 75% against symptomatic disease lasting at least one year in all populations, including, at-risk populations [109] . the addition of pre-existing immune responses to these various at-risk population models more accurately reflects the variation of the human population and allows for appropriate testing of novel vaccine candidates. the effects of imprinting and pre-immunity on subsequent humoral and cellular responses are still under investigation. during initial pre-immune ferret infection, there are increased nasal protein secretions compared to naïve ferrets during a heterologous challenge [39] . characterization of the influenza virus infectome during different stages of the infectious process, with and without prior specific immunity to influenza, has recently been reported [87] . the differences in protection of a pre-immune animal compared to a naïve animal may be due to recall of antibodies specific to shared epitopes which do not necessary need to be neutralizing [110] . infection induces t-cell responses to t-cell epitopes within the ha and other proteins, and even neuraminidase inhibiting antibodies that are not elicited by split-inactivated vaccines [41, 111] . these responses may be either synergistic or antagonistic when paired with vaccination. for instance, vaccination may boost the non-neutralizing antibodies leading to decreased vaccine efficacy compared to a naïve animal. this back-boosting, also described as an anamnestic response, has been observed in the human population [112] [113] [114] [115] and has been recapitulated within the ferret model. this similarity makes it a useful tool for determining vaccine performance in a setting where back-boosting is present [37] . specifically, back-boosting was observed when imprinted with a(h1n1)/singapore/6/1986 followed by a(h1n1)/california/07/2009 vlp vaccination. the breadth of hai-specific antibody response was wider than just a(h1n1)/singapore/6/1986 alone and covered more viruses. the breadth increased to include viruses before and after the 1986 seasonal virus [59] . the back-boosting observed after a vlp-vaccination was dependent on the recognition of memory b-cell and t-helper cell epitopes specific to the ha of the virus. this increase in breadth was also observed after sequential infections with elisa titers to total antibody binding [78] . this back-boosting is what contributed to the difference in the naïve vs. pre-immune antigenic maps of different a(h3n2) viruses [79] . the exact mechanism of back-boosting is not completely elucidated [113] . heterosubtypic protection between the type a influenza strains suggest that cross-reactive cellular immune responses may be contributing to virus control [83] . protective t-cell responses are elicited through recognition of cross-reactive epitopes [84, 88] . cross-reactive memory b-cells can also be elicited post-imprinting [63] . characterization of the cellular and humoral responses, similar to the human-based study conducted by ryan et al. [116] , with imprinted and sequentially infected ferrets is a priority to determine if cellular immune responses differ with a(h1n1) and a(h3n2) subtype infections [116] , or during a(h3n2) and a(h5n1) influenza virus co-infections [117] , or with seasonal vs. pandemic a(h1n1) influenza virus infections [118, 119] . as the reagents for the ferret animal model continues to expand, the opportunities to investigate different correlates of infection and/or protection magnify. before this reagent development, research was limited to characterizing serum and nasal washes for neutralizing antibodies and measuring clinical signs after infection. therefore, future studies can capture the cellular immune reactions of t-cell responses with ifn-γ elisas [120] , peripheral blood leukocyte tracking [121] , and elispots [88, 122] , as well as the humoral immunity [18, 92, 123, 124] . the pre-immune ferret model is within its early stages of development; to become a mainstay within the scientific community and used reliably in the context of influenza virus research, the baseline effects of infection, re-infection, and vaccination within the ferret need to be addressed first. this involves determining the cool-down time of the ferret immune system following infection and the differences compared to a vaccination. furthermore, studies of the different immunological ferret responses such as cytokine quantity and diversity, b-and t-cell repertoire, and b-and t-cell recall responses are important to characterize. in addition, advancements in high-throughput, single cell sequencing technology allow depiction of the b-cell evolution from a single ancestral b-cell [124] . the ferret model is used to study influenza virus infection because of the ferret's natural susceptibility, shared clinical signs of illness, and possession of similarities in respiratory physiology, cell composition, and distribution of sialic acid receptors. although present, these components may not interact in the same manner as human immune system following influenza virus infection [78] . comparisons of different pre-immune influenza animal models (mice, guinea pigs, and ferrets) to human serology data emphasized that the use of different animal models should be heavily considered for pre-clinical vaccine studies due to the biases between them. basic research of the ferret physiology can be compared and validated against data from human studies. cross-validation will either bolster the ferret findings or they will provide researchers the ability to determine which immunological findings are relevant for further investigation and which are solely ferret-specific phenomena. this process focuses on the ferret model being a surrogate for the human. the decision-making process of vaccine selection and therapeutics relies on the ferret, and it is important that human-ferret shared traits are appropriately distinguished. therefore, further study, comparison, and validation are needed to fully grasp the predispositions and limitations of the model. although initially developed in the 1970s, recently, the pre-immune ferret model has rapidly progressed into a vital tool for the development of a broadly neutralizing influenza vaccine. in conclusion, care needs to be taken to begin planning studies to incorporate the effects of imprinting and pre-immunity within the animal model to apply the results to the human system. animal models used for influenza research are available and widely use. however, as more findings and reagents become available, the models need to be updated appropriately. in addition, more research on the immunological effects of imprinting in ferrets, which is then compared to the research on the effects of imprinting on humans, will contribute to the validation of using the ferret as an appropriate animal model to study influenza virus in humans. altogether, the major goal of developing a broadly neutralizing influenza vaccine by priming the immune system 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reconstruction in single-cell rna-seq data the authors would like to acknowledge z. beau reneer and ying huang for their insightful comments and critique. the authors declare no conflict of interest. key: cord-001826-av2gxfxy authors: gao, qian; wang, zhen; liu, zhenlong; li, xiaoyu; zhang, yongxin; zhang, zhizhen; cen, shan title: a cell-based high-throughput approach to identify inhibitors of influenza a virus date: 2014-07-14 journal: acta pharm sin b doi: 10.1016/j.apsb.2014.06.005 sha: doc_id: 1826 cord_uid: av2gxfxy influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus. rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors. in this study we established a 293t cell line that constitutively synthesizes a virus-based negative strand rna, which expresses gaussia luciferase upon influenza a virus infection. using this cell line, an assay was developed and optimized to search for inhibitors of influenza virus replication. biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format (z′ factor value>0.8). a pilot screening provides further evidence for validation of the assay. taken together, this work provides a simple, convenient, and reliable hts assay to identify compounds with anti-influenza activity. influenza viruses are members of the family of orthomyxoviridae and are classified into three types: influenza a, b and c viruses based on antigenic differences. influenza a virus has caused significant morbidity in the last and in this century 1 . to date, influenza-specific drugs include m2 ion channel blockers (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir), both of which were approved to prevent and treat influenza 2 . however, rapid emergence of drug-resistant mutants restricts their utilization 3, 4 , and therefore leads to an urgent need to develop novel antivirals. the use of high-throughput screening (hts) technology for antiviral discovery is a fairly recent endeavor. utilizing virusinducible reporter genes to quantitate the infectivity has been achieved for several viruses 5,6 . a reporter system mimicking the process of transcription/replication of influenza virus was constructed by lutz et al. 7 . cells were transfected with a reporter system that expressed firefly luciferase in a response proportional to the infectivity of influenza virus. this luciferase assay is more rapid and simple than the standard plaque assays. afterward de vries et al. 8 adapted this system by substituting gaussia luciferase. gaussia luciferase (hereafter referred to as gluc), discovered from the marine copepod gaussia princeps, can give high levels of light emission centered at 470 nm by catalyzing the oxidation of the substrate coelenterazine to coelenteramide 9 . this reaction requires no cofactors but o 2, and it generates over 100-fold higher bioluminescent signal intensity when compared with other frequently used luciferase reactions (e.g., renilla or firefly luciferase) 10, 11 . with the help of a secretory signal, gluc was secreted into the culture medium. lysis of the cells was not necessary, and the measurement of luciferase activity was simple and time-saving, making gluc assays a promising technique. nevertheless, transient transfection of the gluc reporter system is likely to introduce variation between the experiments. in this work, a stable cell line derived from 293t cell was established, named 293t-gluc. it synthesizes the reporter protein gluc when infected with influenza a virus. we then developed a reporter assay based on this engineered cell line. antiviral activities of three anti-influenza compounds (ribavirin, amantadine hydrochloride and nucleozin) were assessed by this assay. results demonstrated the ability of this assay to identify influenza inhibitors. several parameters (z 0 factor, cv, s/b, s/n) utilized to evaluate the quality of hts (high-throughput screening) assays also validated our approach. these findings suggest that this cell-based assay is a promising tool to identify new anti-influenza drugs. mdck and 293t cells were maintained in dulbecco's modified eagle's medium with 10% (v/v) fetal bovine serum (fbs; invitrogen). the cells were cultured at 37 1c in a humidified atmosphere of 5% co 2 . influenza a/wsn/33 (h1n1) was rescued from eight plasmids using cocultured mdck and 293t cells 12 . influenza a/ pr/8/1934 (h1n1), seasonal influenza b/beijinghaidian/1386/ 2013 (victoria) and influenza b/massachusetts/02/2012 (yamagata) were propagated in embryonated chicken eggs according to classical virological techniques. virus titers were determined on mdck cells and represented as the median tissue culture infective dose (tcid 50 ), as previously described 13 . these viruses were provided kindly by dr. yuelong shu at china cdc. coelenterazine-h was purchased from promega. nucleozin and tpck-treated trypsin were obtained from sigma. the microsource library (2000 compounds) was obtained from commercial sources and compounds from this library were dissolved in dmso at 2 mmol/l. plasmid phh-gluc (a kind gift from dr. erik de vries 8 ) was used as template for gluc reporter system (containing the rna polymerase i promoter/terminator and influenza a/wsn/33 np segment utrs) amplification. the primers used are as follows: forward primer 5 0 -tatgaattcggaaaaacgccagc aac-3 0 and reverse primer 5 0 -ataagggcccaaaatcttcttt-catccgc-3 0 . pcr products were cloned into plenti6/v5-dest vector (invitrogen) generating plenti6-gluc. 293t cells were transfected with plenti6-gluc using lipofecta-mine2000 in accordance with the manufacturer's protocol, and then were selected with 10 μg/ml blasticidin 24 h posttransfection. under antibiotic selective pressure several clonal colonies were obtained and tested for luciferase expression. one clonal cell line demonstrated high-level expression of luciferase; this cell line was named 293t-gluc and was used for subsequent experiments. 293t-gluc cells were cultured to 90% confluence, released with 0.25% trypsin-edta and resuspended in dmem with 10% fbs at 7 â 10 5 cells/ml. cells were then seeded in the wells of 96-well plate at 100 μl/well. after an overnight incubation at 37 1c and 5% co 2 , cells were infected with the indicated influenza viruses, which were contained in 10 μl serum-free dmem culture medium. post-infection medium also contained tpck-treated trypsin with the final concentration of 1 μg/ml. in specificity studies, infection was allowed to proceed for 24 h at 35 1c. for influenza a/pr/8/1934, influenza b/beijinghaidian/ 1386/2013 (victoria) and influenza b/massachusetts/02/2012 (yamagata), infections were carried out at a multiplicity of 0.1, 1 and 10, respectively, whereas a multiplicity of 0.1 and 1 was used when testing the response of influenza a/wsn/33 virus to gluc reporter assay. the dynamic signal range of the gluc reporter assay was assessed by infecting 293t-gluc cells with varying quantities of influenza a/wsn/33 virus (moi of 0.0001, 0.001, 0.01, 0.1 and 1) and determining gluc activity at various times post-infection (12, 24, 36 and 48 h post-infection). for evaluation assay of antivirals and high-throughput screening, 1 μl of each tested compound was added to cells and incubated for 2 h prior to infection, after which cells were infected with influenza a/wsn/33 virus at an moi of 0.05. after a further incubation for 24 h at 37 1c, the cell supernatant was collected and measured for gluc activity. in each 96-well plate ribavirin and dmso were used as positive and negative controls, respectively. the inhibition rate of the tested compounds was calculated with the following equation, where rlu indicates relative light unit: inhibition rate¼(rlu infected cells àrlu tested compound )/(rlu infected cells àrlu mock-infected cells ) â 100%. a stock of coelenterazine-h was prepared in ethanol to a concentration of 1.022 mmol/l and stored at -20 1c. before assay the stock was diluted into pbs by a factor of 60, which was used as working solution for luminescence. the working solution was held for 30 min at room temperature in the dark to stabilize it 14 . for luminescence analysis, 60 μl of working solution was added to 10 μl of cell-free conditioned medium and measured for 0.5 s using a 96-well microplate luminometer with automated substrate injection (berthold centro lb 960). the results were expressed in relative light units (rlus). in construction of plenti6-gluc, the open reading frame (orf) of the influenza a/wsn/33 np protein was replaced by gaussia luciferase and this rna segment was inserted in a human rna polymerase i promoter/terminator cassette in the reverse orientation and complementary sense. as a stable cell line, 293t-gluc cells that contain plenti6-gluc synthesize a viral negative strand rna constitutively, which expresses gluc under control of the untranslated regions (utrs) of the influenza a/wsn/33 np segment (fig. 1) . we assessed the response of this reporter cell line to different strains of influenza virus. as shown in fig. 2 , the infection with influenza a viruses induced much higher luciferase activity than did the two strains of influenza b virus. the observation that the 293t-gluc cell line is more responsive to influenza a virus may reflect species specificity of utr region for the corresponding rdrp, which is consistent with a previous observation 7 . among all the virus strains tested, the a/wsn/33 strain exhibited the most potent ability to induce luciferase activity when the cells were infected at identical moi. therefore, influenza a/wsn/33 virus was used for all subsequent experiments. to further optimize the assay a signal range was determined using serial 10-fold dilutions of influenza a/wsn/33 virus. as shown in fig. 3 , infection at mois of 0.1 or higher induced a gluc signal at 12 h post-infection, and signal intensity significantly increased thereafter and reached a peak at 36 h post-infection. when infected with virus at mois of 0.001 or lower, cells failed to generate a detectable gluc signal. it should be noted that viral infection at an moi of 1 induced significant cpe in 293t-gluc cells like mdck, while the cells kept a normal morphology during the 96 h time period of post-infection when the moi was less than 0.1 (data not shown). this may explain the observation that the gluc signal from the cells infected at an moi of 1 was less than that of moi of 0.1 (fig. 3) , most likely due to reduced cell viability. to ascertain assay sensitivity, three reference compounds, including ribavirin, amantadine hydrochloride and nucleozin, were examined in our assay. ribavirin is used against several dna and rna viruses including influenza viruses 15, 16 . it is converted to ribavirin monophosphate, which disturbs gtp synthesis, leading to inhibition of rna synthesis in cells 17 . amantadine hydrochloride, approved for the treatment of influenza infections, acts in the early phase of the life cycle of influenza a virus to target the m2 ion channel 18, 19 . nucleozin is a novel inhibitor with antiinfluenza activity. it impedes influenza a virus replication by triggering the aggregation of np and inhibits its nuclear accumulation 20 . we used the gluc reporter assay to evaluate these antivirals in dose-response experiments, measuring ic 50 (fig. 4) . all the compounds showed the ability to reduce the luciferase signal driven by influenza a/wsn/33 virus. ic 50 values determined from our experiments were comparable to data previously reported (table 1) , demonstrating the applicability of the gluc reporter assay for screening influenza inhibitors. furthermore, compared with the conventional plaque reduction assay (pra) 24 , the method herein is more rapid and simple for the determination of ic 50 values. at least 48 h were needed before the number of plaques could be calculated, whereas our assay can give results in 24 h after infection. moreover, this unbiased and automated method of infectivity quantitation avoids tedious plaque counting, making it an ideal approach. the above results suggest the potential to use the gluc reporter assay to screen compounds against influenza a virus. to ensure that the assay can be used in high-throughput screening, we assessed its accuracy by using several statistical parameters 25 ( table 2) . z 0 factor is a simple statistical characteristic for hts assay. it can be used to evaluate the performance of an assay. a robust assay should have a z 0 factor40.5 and the larger the value of z 0 factor, the better the data quality of the assay 26 . the z 0 factor in our experiments was 0.84 (n ¼48), which is considered to be excellent for use in hts (fig. 5) . normally, infected cells treated with a positive anti-influenza compound are used as positive control for calculating the z 0 factor. nevertheless, according to the assay validation guidance for agonist assays (http://www.ncbi.nlm. nih.gov/books/nbk83783/), the background signal is also suitable for use as a 'minimum signal'. since viral infection acts as an agonist of luminescent protein, we used mock-infected cells as positive control in the validation analysis. similarly, other work used mock-infected cells as positive control to calculate the z 0 factor 27 . in addition, as shown in fig. 4 , most of the anti-influenza compounds tested were able to achieve full inhibition in our assay. moreover, the minimum signals derived from infected cells treated with one of the positive anti-influenza compounds were close to that of mock-infected cells. cv is another parameter used for quality assessment. it reflects signal deviation within an assay and is recommended to be less than or equal to 20% (http://www.ncbi.nlm.nih.gov/books/nbk83783/). the cv of our assay was 5.79%, which meets the requirements for hts. in addition, the s/b ratio (23.24) and the s/n ratio (16.29) were comparable to those of other hts assays reported 28, 29 , proving our assay was suitable for use in a high-throughput screen. as a proof of concept, we carried out a pilot screening of the microsource library as described above and the results are plotted in fig. 6 . in each 96-well plate, ribavirin and dmso were used as a positive and negative control, respectively. majority of the 2000 screened compounds did not decrease the luminescence signal by more than 50%. seventeen showed strong luminescence reduction by 90%-100%, which translates to a hit rate of 0.85%. compared with assays exploiting the principle of cytopathic effects (cpe) protection 30 , the approach established here is rapid and simple for anti-influenza inhibitor screening. we anticipate that this approach will facilitate the discovery of compounds with anti-influenza activity and lead to drug development. influenza virus remains a health threat, and much more work needs to be done to discover new antivirals. in this work we reported a cell-based high-throughput assay to identify inhibitors for influenza virus. the assay used gaussia luciferase as a readout. several criterions for hts were evaluated, including z 0 factor and cv. results obtained validated the robustness of our assay. furthermore, we used this high-throughput assay to screen 2000 small molecules at 20 μmol/l. seventeen compounds showed 90%-100% inhibition of luminescence signal for a rate of 0.85%. utilization of this cellbased high-throughput assay will benefit identification of new antiinfluenza lead compounds in future work. influenza a viruses: new research developments antivirals targeting influenza a virus influenza virus resistance to neuraminidase inhibitors influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance three of the four nucleocapsid proteins of marburg virus, np, vp35, and l, are sufficient to mediate replication and transcription of marburg virusspecific monocistronic minigenomes a bunyamwera virus minireplicon system in mosquito cells virus-inducible reporter genes as a tool for detecting and quantifying influenza a virus replication dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway recombinant gaussia luciferase. overexpression, purification, and analytical application of a bioluminescent reporter for dna hybridization a highly sensitive protein-protein interaction assay based on gaussia luciferase gaussialuciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga chlamydomonas reinhardtii dna transfection system for generation of influenza a virus from eight plasmids a simple method of estimating fifty percent endpoints gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo broad-spectrum antiviral activity of virazole: 1-beta-dribofuranosyl-1,2,4-triazole-3-carboxamide ribavirin-current status of a broad spectrum antiviral agent the predominant mechanism by which ribavirin exerts its antiviral activity in vitro against flaviviruses and paramyxoviruses is mediated by inhibition of imp dehydrogenase antiviral activity of 1-adamantanamine (amantadine) mode of action of the antiviral activity of amantadine in tissue culture identification of influenza a nucleoprotein as an antiviral target a quantitative comet infection assay for influenza virus generation and characterization of recombinant influenza a (h1n1) viruses harboring amantadine resistance mutations design, synthesis, and in vitro biological evaluation of 1h-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza a agents targeting virus nucleoprotein elvira hsv, a yield reduction assay for rapid herpes simplex virus susceptibility testing a comparison of assay performance measures in screening assays: signal window, z 0 factor, and assay variability ratio a simple statistical parameter for use in evaluation and validation of high throughput screening assays modulation of influenza virus replication by alteration of sodium ion transport and protein kinase c activity development and validation of a high-throughput screen for inhibitors of sars cov and its application in screening of a 100,000-compound library a cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals high-throughput identification of compounds targeting influenza rna-dependent rna polymerase activity key: cord-004638-ijncfuxi authors: wang, yuheng; cheng, minna; wang, siyuan; wu, fei; yan, qinghua; yang, qinping; li, yanyun; guo, xiang; fu, chen; shi, yan; wagner, abram l.; boulton, matthew l. title: vaccination coverage with the pneumococcal and influenza vaccine among persons with chronic diseases in shanghai, china, 2017 date: 2020-03-19 journal: bmc public health doi: 10.1186/s12889-020-8388-3 sha: doc_id: 4638 cord_uid: ijncfuxi background: adults with chronic conditions such as heart disease, diabetes, or lung disease are more likely to develop complications from a number of vaccine-preventable diseases, including influenza and pneumonia. in this study, we use the data from a chronic disease management information system in shanghai to estimate vaccination coverage and characterize predictors of seasonal influenza and 23-valent pneumococcal polysaccharide vaccine (ppsv23) vaccination among people with chronic disease in shanghai. methods: the shanghai centers for disease control and prevention have information systems related to chronic disease management, hospital records, and immunizations. data from individuals with hypertension, diabetes and chronic obstructive pulmonary disease (copd) were abstracted during july 2017. the main outcome was coverage of pneumococcal and influenza vaccination. vaccination coverage was calculated across demographic groups. significance in bivariate associations was assessed through pearson’s chi-square tests, and in multivariable models through logistic regression models with a forward stepwise method to select variables. results: in the sample of 2,531,227 individuals ≥15 years, 22.8% were vaccinated for pneumonia from january 2013 to july 2017, and the vaccination coverage of influenza in the 2016/17 influenza season was 0.4%. vaccination coverage was highest in those 70–79 and lowest in those younger than 60. compared to urban areas, uptake in rural areas was higher for pneumonia vaccination (or: 2.43, 95% ci: 2.41, 2.45), but lower for influenza vaccination (or: 0.55, 95% ci: 0.51, 0.59). having a greater number of chronic diseases was associated with higher likelihood of pneumonia vaccination (3 vs 1: or: 1.68, 95% ci: 1.64, 1.71), but this relationship was not statistically significant for influenza vaccination. conclusions: we found low levels with of pneumococcal vaccination, and extremely low uptake of influenza vaccination among individuals with high risk conditions in shanghai who should be priority groups targeted for vaccination. interventions could be designed to target groups with low uptake – like younger adults, and individuals who have not yet retired. adults with chronic conditions such as heart disease, diabetes, or lung disease are more likely to develop complications from certain vaccine-preventable diseases, especially pneumonia and influenza. these complications can include long-term illness, hospitalization, and even death [1] . persons with diabetes or chronic obstructive pulmonary disease (copd) often have immune system impairment sometimes leading to greater morbidity or mortality following infection with influenza compared with healthy adults of the same age. these individuals also have an influenza-related hospitalization and excess mortality rate significantly higher than those without chronic disease [2] [3] [4] . one study showed diabetics had 3.63 times higher odds of developing serious complications from the influenza compared to non-diabetics (95% confidence interval (ci): 1.15, 11.51) [5] . in one systematic review of avian influenza, people with diabetes had 9.91 times the odds of hospitalization with influenza compared to healthy people (95% ci: 5.46, 17.99) and those with copd had 2.38 times (95% ci: 1.58, 3.57), 4.46 times (95% ci: 1.34, 14.79) and 4.02 times (95% ci: 1.69, 9.58) higher odds of hospitalization, being admitted to the icu, and requirement ventilator assistance, respectively [6] . several studies have found a benefit of administering pneumococcal polysaccharide and seasonal influenza vaccines to people with chronic illness [7] [8] [9] . simultaneous vaccination of pneumococcus and influenza in elderly copd patients could reduce pneumonia hospitalization by 63% and overall mortality by 81% [8] . influenza vaccination could substantially reduce hospitalization and mortality among diabetic patients and was well tolerated during an influenza season [10] . the combination of seasonal influenza and pneumococcal vaccine (including 23valent pneumococcal polysaccharide vaccine (ppsv23)) significantly reduced the hospitalization rate and mortality of influenza, pneumonia and other diseases such as respiratory disease, copd and congestive heart failure among the elderly compared to the uptake of influenza or pneumococcal vaccine alone [11, 12] . co-administering these vaccines could significantly reduce the rate of intensive care and prolong the survival period of elderly patients with chronic diseases [13] , and has been shown to be cost-effective [14] . the elderly and patients with chronic disease including diabetes, copd and heart disease are recommended to be priority groups for pneumococcal and influenza vaccination by the world health organization (who) [15, 16] and by the us centers for disease control and prevention (cdc) [17] . according to chinese guidelines for vaccination, adults with these chronic diseases are recommended to receive the seasonal influenza and ppsv23 vaccines [18, 19] . pneumococcal vaccines (ppsv23 and 13-valent pneumococcal conjugate vaccines) are also available to children for a fee. according to the manufacturer's instructions, children and younger adults with certain chronic conditions (cardiovascular disease, lung disease, diabetes, cirrhosis, spleen dysfunction, sickle cell disease, chronic renal failure, organ transplants, hiv, cerebrospinal fluid leakage) or who live in certain environments (individuals in long-term care facilities, staff at welfare organizations) are recommended to get the ppsv23 vaccine. these recommendations are consistent with global guidelines for prevention and treatment of chronic diseases [20, 21] . the governments of some cities in china such as beijing, shenzhen, karamay and xinxiang have published policies providing free influenza vaccination to local elderly residents, while some other cities such as chongqing and ningbo implemented subsidies for the influenza vaccine in medical insurance programs for target residents [22] . shanghai has implemented a government program providing people over 60 years old with a free pneumococcal vaccination (ppsv23) since 2013, but the influenza vaccine is not offered under the government's expanded program on immunization (epi) and is instead administered for a fee. there is a large population of chronic disease patients in shanghai [23] , but data about pneumococcal and influenza vaccination coverage among patients with chronic disease is absent. a 2010 survey from china found that influenza vaccination was actually lower in adults with highrisk health conditions (7.2%) than those without (10.8%) [24] . more information is needed about who gets vaccinated. in this study, we use the data from a chronic disease management information system in shanghai to estimate vaccination coverage and characterize predictors of influenza and pneumococcal vaccination among people with chronic disease in shanghai. we assess whether there are differences in coverage in pneumococcal vaccine and influenza vaccine across age groups, urbanicity and chronic disease diagnoses. we hypothesize that influenza vaccine has lower coverage than pneumococcal vaccine due to differentials in price, that uptake of both vaccines is lower in low age groups compared to high age groups, that uptake of both vaccines is lower in rural areas than in urban areas, and that coverage for both vaccines is higher among those with more chronic diseases. this study used a retrospective cohort design. during july 2017, the data were obtained from three distinct sources -(1) the shanghai chronic disease management information system and (2) the shanghai immunization program information system which are both housed at the shanghai cdc, and (3) the hospital record system, which is located at the shanghai health commission for hospital records. the individual's personal id was used to link the three information systems. throughout shanghai, patients ≥15 years old diagnosed with hypertension and diabetes are asked if they want to be included in a centralized databasethe chronic disease management information system. inclusion in the database means that the patients will receive more standardized management of their disease. an estimated 50% of individuals with hypertension and diabetes in shanghai are enrolled in this database. the other 50% include those who do not know they have a chronic disease, who have not gone to visit the doctor, or who are unwilling to be enrolled into the system. general practitioners follow up with patients every 3 months at community health centers and input data related to these visits into the chronic disease management information system. this database contains information on sex, birthdate, township residence, occupation and diagnostic information pertaining to hypertension and diabetes. no other individual-level information was available from the dataset. all patients from the shanghai chronic disease management information system were included in this study. data in the immunization program information system were captured and entered by vaccination providers at community health care centers. data are uploaded daily from these health centers' electronic registries into the immunization program information system. pneumococcal vaccination information from january 2013 to july 2017 and influenza vaccination information from the 2016/17 influenza season were obtained from the shanghai immunization program information system. types and dates of vaccination were extracted from the immunization program information system. the shanghai cdc and the shanghai health commission implement regular data quality checks of the immunization program information system. diagnosis of copd was obtained from the hospital record system. the international classification of diseases (icd) was used to define chronic diseases in the chronic disease management information system and the hospital record system. hypertension was defined as i10-i13, diabetes was defined as e10-e14 and copd was defined as j44. the chronic diseases in this study represent those at risk for pneumococcal disease or influenza [15, 25] . the american diabetes association recommends individuals with diabetes to have both vaccines [26] . the global initiative for chronic obstructive lung disease has similar recommendations for those with copd [27] . hypertension is not thought to be linked to either disease, but individuals with hypertension were still included because they were in the original chronic disease management information system and because many are older, and thus may be age-eligible for a free ppsv23 in shanghai. urbanicity was defined by characteristics of the township where participants resided. residency status refers to locals vs. non-locals, with locals defined as registered permanent residents of shanghai, and non-locals as migrants from other cities who have moved into shanghai for over 6 months. urban areas are those where ≤30% of locals and ≤ 35% of non-locals were engaged in agricultural work; suburban areas had ≤30% of locals but > 35% of non-locals engaged in agricultural work; and rural areas had > 30% of locals in agricultural occupations. classification of occupation was defined according to the china national standard [28] . the main outcome was receipt of pneumococcal and influenza vaccination. vaccination coverage was calculated by sex, age group, urbanicity, occupation, type and number of chronic diseases. pearson's chi-square test was used to compare the vaccination coverage among the different subgroups. we also analyzed the relationship between predictor variables (sex, age group, urbanicity, occupation, type and number of chronic diseases) and the outcomes using logistic regression models through a forward stepwise method (variable included at p-value of 0.05, excluded at p-value of 0.10, with α = 0.05). data were analyzed using spss version 20. vaccination status by township was mapped with qgis 3.6 (qgis geographic information system. open source geospatial foundation project). the shapefile map was obtained from shanghai surveying and mapping institute (https://www.shsmi.cn/info/ilist.jsp?cat_id=10098). the sample of 2,531,227 patients from the chronic disease management information system included a majority of females (53.7%), more individuals above 60 years (78.7%) than other age groups, more urban residents than other locales (59.4%), and most individuals were retired (82.6%). the majority of patients had hypertension (90.3%) with fewer diagnosed with diabetes (31.0%) and copd (10.3%); a very low proportion had been diagnosed with all three (3.2%) ( table 1) . only 22.8% patients were vaccinated for pneumococcal from january 2013 to july 2017, and vaccination coverage of influenza in 2016/17 influenza season was exceedingly low at 0.4%. vaccination coverage differed significantly across most socio-demographic characteristics. for both pneumonia and influenza vaccinations, coverage was highest in those 70-79 years (35.7 and 0.6%, respectively) compared to other age groups (p < 0.001). pneumococcal vaccination was highest in rural areas (30.3% compared to 18.7% in urban areas, p < 0.001) whereas influenza vaccination was highest in urban areas (0.4% compared to 0.2% in rural areas, p < 0.001). for both pneumococcal and influenza vaccination, coverage was highest among those with copd (30.4 and 0.9%, respectively), compared to those with hypertension (23.5 and 0.4%, respectively) or diabetes (24.1 and 0.4%, respectively) (p < 0.001, respectively). there was a dose-response relationship between number of chronic diseases and vaccination coverage; pneumococcal vaccination uptake was 36.3% among those with three conditions, compared to 27.3 and 20.9% for those with 2 or only 1 condition (p < 0.001). influenza vaccination coverage was 0.3, 0.5 and 0.9% for those with 1, 2, or 3 conditions (p < 0.001). vaccination coverage also varied geographically, with pneumococcal vaccination coverage highest in jiading and songjiang, at the periphery of shanghai, and was relatively low in the inner districts of huangpu, jing'an, hongkou, and yangpu (fig. 1) . influenza vaccination coverage was comparatively low across all districts, ranging from 0.1% in fengxian to 0.8% in xuhui. table 2 shows the multivariable logistic regression models. these models are largely in line with the unadjusted results from table 1 . individuals aged 70-79 had 1.37 times higher odds of pneumococcal vaccine uptake compared to individuals in their 60s (95% ci: 1.36, 1.38). individuals in rural areas and suburban area had higher odds of pneumococcal vaccine uptake compared to individuals in urban areas. patients with 2 and 3 chronic diseases had respectively 1.31 (95% ci: 1.30, 1.33) and 1.68 (95% ci: 1.64, 1.71) times higher odds of vaccination compared to patients with 1 chronic disease. all subjects were included in the multivariable analysis. in the adjusted model of influenza vaccination, patients aged 70-79 and above 80 had 1.73 (95% ci: 1.64, 1.81) and 1.09 (95% ci: 1.01, 1.16) times higher odds of vaccination, respectively, compared to patients aged 60-69. compared to patients in urban area, patients in suburban and rural areas had, respectively, 0.86 (95% ci: 0.82, 0.91) and 0.55 (95% ci: 0.51, 0.59) times the odds of influenza vaccination. patients with 2 chronic diseases had 1.14 times the odds of uptake influenza vaccine compared to patients with 1 kind of chronic disease (95% ci: 1.08, 1.21), but there was no significant difference in those with 1 vs 3 chronic diseases. patients with a dose of pneumococcal vaccine had 15.75 the odds of receiving the influenza vaccine compared to those with no pneumococcal vaccine (95% ci: 14.86, 16.68). influenza and pneumococcal vaccination are important for preventing illness and the elderly with chronic diseases [7] [8] [9] . in a large sample of individuals with chronic diseases residing in shanghai, china, we found low pneumococcal vaccination coverage over a 4-year study period and even lower influenza vaccine coverage. uptake of both vaccines increased in those with more chronic diseases and with older age. chronic disease patients should be targeted for attaining high vaccination coverage compared to the remaining population. there are several overriding factors for exceptionally low coverage of pneumococcal and influenza vaccination among chronic disease patients in shanghai community: (1) studies have found that individuals lack awareness of pneumococcal and influenza vaccine [29, 30] , and physicians do not often recommend vaccinations. (2) vaccination for adults is not convenient. community health care centers were responsible for implementing vaccinations in shanghai. most centers only provide 1 or 2 half days available for adult vaccination per week, while 6 half days are available for childhood vaccination. (3) some adverse news related to vaccines have made people reduce their trust in vaccination programs [31, 32] . people with chronic disease and the elderly should have priority to take these vaccines due to their risk factors, but their chronic diseases may lead them to believe they have a higher risk for adverse reactions. (4) there is a limited supply of influenza vaccine. these reasons were not assessed in the current study, but could be explored in future research. pneumococcal vaccination coverage among adults 19-64 years at increased risk for pneumococcal disease was 24.0% in 2016 in the united states although it was much higher at 66.9% among adults over 65 years old [33] . this is consistent with a study from spain showing a higher proportion of adults over 65 years had received the pneumococcal vaccine (43.8%) [34] and demonstrating that vaccination levels in both young and elderly chronic disease patients in shanghai are substantially lower than those found in the us or spain. because residents over 60 years of age in shanghai are provided with free pneumococcal vaccination, the coverage in this age groups was not surprisingly higher than younger age groups and approaching that seen in those over 65 years in hong kong in 2015 (34%) [35] which also offers free pneumococcal vaccination to the elderly [36] . in our study, less than 1% of individuals received an influenza vaccine, which is far lower than in other countries, many of which provide free vaccine through government-sponsored or private insurance programs. similar studies have shown higher influenza vaccination coverage in the united states (43.5%, among adults over 19 years, 2015/16 season) [33] , uk (56.0%, chronic disease patients, 2007/08 season) [37] , poland (11.1%, chronic disease patients, 2007/08 season) [37] , korea (45.2%, over 40 years, 2012) [38] , and hong kong (39%, over 65 years, 2015) [35] . our findings were relatively consistent with prior studies in china showing an average national vaccination coverage ranging between 1.5 and 2.2% in 2004 and 2014 [22] . the coverage among patients over 60 years was significantly higher vs younger age groups below which was almost non-existent (i.e. close to 0%). one previous study found that elderly individuals who live with other family members are more likely to get vaccinated [30] , perhaps as a result of other family members thinking the elderly, but not younger adults, need to get vaccinated or elderly individuals wanting to protect themselves against influenza as they care for their grandchildren. we found that pneumococcal vaccination coverage was higher in rural areas which distinctly contrasted with influenza vaccination coverage which was lowest in rural areas. for influenza vaccinationwhich requires payment, individuals in urban area might be more able to afford the cost of influenza vaccine while patients in rural area might not [39] . higher pneumococcal vaccination coverage in rural areas may result from individuals trusting health care workers more [40] . the study showed that patients with multiple chronic diseases would be more likely to take pneumococcal vaccination than those with only one kind of chronic disease. this association could arise for several reasons. individuals may perceive a greater personal risk of disease as they gain experience with more diseases. or individuals with more co-morbid chronic diseases may have had more opportunities to get immunized through having more healthcare encounters. the overall difference in uptake between influenza and pneumococcal vaccination could also be tied back to experiences and risk perceptions, as influenza could be seen as a nuisance disease that will quickly pass [41] . the lack of funding to influenza vaccination from the government might be another important reason. pneumococcal vaccine uptake was a strong predictor of influenza vaccine uptake, which indicates that acceptance of one vaccine probably predicts for acceptable of others. since the observation of pneumococcal vaccination was from january 2013 to july 2017 and the observation of influenza vaccination was only 2016/17 season, co-administration of both pneumococcal and influenza vaccines could reduce the incidence of various complications, hospitalization and mortality of chronic disease [8, 11, 12] . only 0.3% of total sample had taken both pneumococcal and influenza vaccine in 2016/17 season, lower than that of hospitalized persons aged over 65 years in victoria (46.6%) [42] . our study looked at vaccination coverage for influenza and pneumococcal disease including predictors for vaccination among community members in shanghai with chronic diseases. interventions or policies like government funding as a potential strategy to encourage vaccination, especially influenza vaccination among chronic disease patients, should be implemented. future studies should further examine differences in uptake of vaccines across different demographic groups. there are several strengths and limitations to this study. a strength of this study is the use of several comprehensive information systems as data sources, and the large number of individuals in the chronic disease management system. this system is opt-in for individuals with certain chronic diseases in the municipality, and an estimated 50% of individuals with chronic disease participate in it. it is possible that the individuals who participate in the chronic disease management system differ from those who do not. non-participants, for example, likely have lower health-seeking behaviors and so our estimates of vaccination coverage may overestimate trends in the entire population of those with these chronic diseases. future studies could evaluate why and how individuals participate in this database. in addition, limitations include a lack of information on key variables, like education and income. we only have data of pneumococcal vaccination coverage from 2013 onward and 1 season of influenza vaccination coverage, and inclusion of additional years would have permitted analysis of trends over time. the very low vaccination coverage, particularly for influenza vaccination, limits our ability to make recommendations beyond a general recommendation to increase coverage. we found very low levels of both pneumococcal and influenza vaccination among individuals with chronic diseases residing in shanghai. these individuals should be prioritized for vaccination with both vaccines. concomitantly, there can be greater ease of access to vaccines, and promotional materials can focus on complications of disease in those with high risk conditions. clinical evaluation of chinese guidelines for community-acquired pneumonia the influence of chronic illnesses on the incidence of invasive pneumococcal disease in adults diabetes and the severity of pandemic influenza a (h1n1) infection chronic obstructive pulmonary disease in the absence of chronic bronchitis in china clinical courses and outcomes of hospitalized adult patients with seasonal influenza in korea populations at risk for severe or complicated avian influenza h5n1: a systematic review and meta-analysis influenza vaccination of elderly persons: reduction in pneumonia and influenza hospitalizations and deaths the additive benefits of 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executive summary chinese general administration of quality supervision inspection and quarantine. classification and codes of occupations knowledge and attitude toward pneumonia and pneumococcal polysaccharide vaccine among the elderly in shanghai, china: a crosssectional questionnaire survey low coverage rate and awareness of influenza vaccine among older people in shanghai, china: a cross-sectional study china's vaccine production scare china vaccine scandal: investigations begin into faulty rabies and dtap shots vaccination coverage among adults in the united states factors associated with pneumococcal polysaccharide vaccination of the elderly in spain: a cross-sectional study. hum vaccines immunother perceptions of seasonal influenza and pneumococcal vaccines among older chinese adults vaccination coverage rates in eleven european countries during two consecutive influenza seasons influenza vaccination coverage rate according to the pulmonary function of korean adults aged 40 years and over: analysis of the fifth korean national health and nutrition examination survey rural migrant workers in urban china: living a marginalised life the free vaccination policy of influenza in beijing, china: the vaccine coverage and its associated factors cross-cultural perspectives on the common cold: data from five populations influenza and pneumococcal vaccine coverage among a random sample of hospitalised persons aged 65 years or more springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we appreciate the work of vaccination providers and primary care physicians in the city of shanghai who contributed data and who work on improving the health of populations with chronic diseases. authors' contributions yw conceived of the study, analyzed the data, and wrote the first draft. mc and cf contributed to study conception revised the manuscript for intellectual content. sw, and fw contributed to data analysis and revised the manuscript for intellectual content. qy1, qy2, yl, and xg contributed to acquiring data and revised the manuscript for intellectual content. ys, aw, and mb contributed to interpreting the data and revising the manuscript for intellectual content. all authors gave final approval for the study to be published. research was supported by the chinese association of preventive medicine (grant #20180103 to yan shi) and the shanghai health commission (grant #201840334 to yuheng wang). the funding body had no role in the design of the study and collection, analysis, or interpretation of data. the datasets analyzed for the current study are not publicly available because they contain detailed medical histories of chronic patients, but are available from the corresponding author on reasonable request. the protocol for this research was approved by the ethical review board of the shanghai municipal center for disease control and prevention (scdc). informed consent was exempted because it was limited to analysis of previously de-identified data collected for medical and public health surveillance purposes. not applicable. the authors declare that they have no competing interests. key: cord-017733-xofwk88a authors: davis, mark title: uncertainty and immunity in public communications on pandemics date: 2018-11-04 journal: pandemics, publics, and politics doi: 10.1007/978-981-13-2802-2_3 sha: doc_id: 17733 cord_uid: xofwk88a this chapter examines uncertainty in the expert advice on pandemics given to members of the general public. the chapter draws on research conducted in australia and scotland on public engagements with the 2009 influenza (swine flu) pandemic and discusses implications for communications on more recent infectious disease outbreaks, including ebola and zika. it shows how public health messages aim to achieve a workable balance of warning and reassurance and deflect problems of trust in experts and science. the chapter considers how uncertainties which prevail in pandemics reinforce the personalization of responses to pandemic risk, in ways that undermine the cooperation and collective action which are also needed to respond effectively to pandemics. uncertainty is a central challenge for public communications on matters pandemic. recent efforts to respond to outbreaks of infectious diseases, such as pandemic (swine flu) influenza (world health organization 2009), ebola (green 2014; world health organization 2014) and zika virus (world health organization 2016) have been marked by the limits of what can be known ahead of time and the challenges of responding to the particular turnings of outbreaks as they happen. the 2009 pandemic influenza-the topic of research i conducted with colleagues in australia and scotland-is a pivotal example of this problem of responding to a pandemic in real time. the 2009 pandemic put huge strain on global, national and local health systems, affecting many individuals and especially pregnant women and people with specific vulnerabilities to respiratory infections. it was a prominent, perhaps dominant, health news story of the period. but the pandemic turned out to be nothing like as severe as it was first thought to be. moreover, there was insufficient take-up of the h1n1 vaccine (bone et al. 2010; galarce et al. 2011; white et al. 2010; yi et al. 2011) and it was observed that only minorities or small majorities reported that they intended to, or did, enact recommended social isolation to avoid transmission of the virus (kiviniemi et al. 2011; mitchell et al. 2011; rubin et al. 2009 ; van et al. 2010) . like the "swine flu affair" of the 1970s in the united states (fineberg 2008) , the 2009 pandemic raised questions for the public health system of how to shape public action in light of the significant uncertainties which are particular to influenza, and without jeopardizing trust in government and the scientific knowledge on which is built public policy. central, too, was immunity, in its medical and social senses. immunity is not simply an object of biomedicine, it is also deeply entwined with collective life and the interrelations that come with, specifically, contagious diseases. it is also important to recognize that these issues are by no means settled; how individuals conduct themselves in relation to others in time of pandemic is a central and enduring concern for public health systems. in 2009 in the uk, for example, advertisements featured images of travellers on public transport and the following text: if you could see flu germs, you'd see how quickly they spread. cold and flu germs can live on some surfaces for hours. always carry tissues with you and use them to catch your cough or sneeze. bin the tissue, and to kill the germs, wash your hands with soap and water, or use a sanitiser gel. this is the best way to help slow the spread of flu. protect yourself and others (nhs swine flu information). this advice addresses responsible individuals and asks them to help limit the spread of infection. the final part of the message 'protect yourself and others' captures the idea that an easily spread influenza virus requires significant cooperation and the internalization of the idea of action on health for the collective good, as well as for oneself. this reference to altruistic action on health indicated that the social response to the 2009 pandemic exemplified biopolitics (rose 2007) . individuals are expected and encouraged to internalize the idea that they can take action on themselves to sustain and better their health and reproductive futures. this self-subjectification applies to the advice given to members of the general public on the 2009 influenza pandemic. in addition to the advice noted above, individuals were encouraged to arrange a network of "flu friends" who could be called upon in the case of illness, to stay abreast of developments in the media, and adopt expert advice (national health service 2009). publics were also advised to stay home if they suspected they were ill and to contact nhs services online or by telephone and to not attend gp surgeries of a&e, unless instructed to do so. in this view, the communications of 2009 hailed pandemic citizenship fashioned around the imperatives of action to avoid and contain the spread of infection and to make oneself available to expert advice. in what follows i explore pandemic communications under conditions of uncertainty, as exemplified by the 2009 influenza pandemic and its resonances with other recent contagions. as we will see, uncertainty has the effect of accentuating personalized responses to expert advice. it also sponsors communicative action figured around seeking the "just right" balance of warning and reassurance and related implications for trust in expert knowledge and authority to govern. the events of 2009 foregrounded many of the strengths and weaknesses of public health systems across the globe. key among these was preparedness and capacity to cope with large scale containment strategies which were used to manage the emerging pandemic. the pandemic preparedness plans in place in 2009 required that in the early phases of the pandemic, efforts should be made to sequester infected individuals and to trace their contacts so that the spreading infection could be tracked down and curbed (world health organization 2011). probably a central lesson of 2009 was that such efforts were costly and apparently ineffective. in some settings public health professionals were asked to continue this method even when they were aware that the virus was spreading quickly despite their best efforts . the 2009 pandemic therefore revealed the importance of being able to quickly assess the biological characteristics and severity of the infection so as to be able to modify the application of resources. since 2009, public health systems have attended to the development of evidence-based measures to assess seriousness and the development of local and viable responses to a global pandemic threat (australian department of the prime minister and cabinet 2011). pandemic preparedness, therefore, has demonstrated a marked shift away from uniformity and top-down governance towards local, evidence-based, approaches. for example, australia's 2009 version of its preparedness plan adopted a traditional method of top-down transmission of expert knowledge and advice to publics. government in this view was mandated to: deliver consistent and accurate public messages nationwide in the event of a pandemic. governments will make every effort to provide timely and reliable advice to the public, media, businesses and industries. (australian department of health and ageing 2008: 34) by 2014, however, the australian pandemic policy instrument referred to the need for public communications which were "two-way" and "listening" to publics (australian department of health 2014: 63). this approach to feedback on the transmission of information was said to depend on in vivo market research, the monitoring of social media, and a q&a website where publics can pose questions and air their opinions (australian department of health 2014: 63). the policy also made reference to the need for specific and tailored messages for vulnerable groups. however, during 2009 pandemic public communications faced significant challenges, not all of which are obviously addressed in the revised policies and their emphasis on feedback loops, market research and social media. surveys conducted at the time of the onset of the pandemic in 2009 show that while publics largely endorsed government action on the pandemic, they underestimated risk of infection and only minorities reported that they had adopted recommended behaviours such as social isolation and coughing and sneezing etiquette (rubin et al. 2009 ). the findings suggest that individuals interpreted public health advice with some scepticism. research shows also that espoused trust in government was associated with self-reported compliance with public health advice (lin et al. 2014; rubin et al. 2009 ). as noted, populations across the globe adopted vaccination only in small proportions, insufficient to protect the entire population. this indication of weak public engagement with the pandemic may be explained by a more general effect of risk management. it is surmised that the repetition of warnings over the last few decades-for example, hiv, bse, avian influenza, hospital superbugs, sars, h1n1, ebola and zika, to name a few-leads to weariness on the part of publics (joffe 2011) . diminishment in public engagement with risk is also thought to be an effect of risk society preoccupation with the forecasting and management of risks (giddens 1998) . public weariness can be thought of as a manufactured risk in the sense that it arises through attempts to manage risk. it is also evident that news on current risks are often framed by established patterns of meaning used to depict previous or contiguous risks (ungar 2008) . it is possible, therefore, that publics have learned to screen out global health alerts and treat media on the topic with a degree of scepticism, a perspective supported by our own davis 2017 ) and similar research (hilton and smith 2010; holland and blood 2012) . implied also is that repeated global health alerts coupled with some scepticism on the part of publics may lead them to fall back on personal knowledge and resources. the individualization of responses to pandemic risk communications was supported by our own research. individuals in our interviews and focus groups endorsed expert advice regarding coughing and sneezing etiquette and social isolation, but they did not think that these strategies would be viable in the long run . some of our respondents did adopt forms of social isolation, but they also saw in these strategies some flaws and inadequacies. they appeared, in general, to recognize the ease with which infection could occur. for these reasons, many of the people we spoke with resorted to discourse on immunity as a means of coping with a more than likely infection. almost absent was discourse on personal action as a means of protecting all, apart from among those with severe respiratory illness who were used to dealing with the threat of infection posed by others. our respondents focused on matters such as the building of immunity through consumer products, rest and exercise, and spoke of the need to cultivate and educate their personal immune system, with some reference to childhood experiences of exposure to infection. individuals seemed to accept that interaction with microbial life was inevitable and important to health and that their immune systems were shaped by their own actions. this "choice immunity" was spoken of as managing one's body and those of dependent others in ways that resonated with the well-known notion of "choice biography" which is said to characterize reflexive modernization (beck and beck-gernsheim 2002) . there are other implications of this resort to choice immunity. ed cohen has shown how immunity is a conceptual framing of subjectivity that preceded modern day microbiology (2009). with its root in the latin munis-also the root for municipal and remuneration, for example-immunity referred to the suspension of one's civic and pecuniary obligation to collective life. cohen gave examples which include, duty, gift, tax, tribute, sacrifice, and public office (2009). immunity suspends the "bond of requirement," but also, therefore, reinscribes it (p. 41). it always and necessarily marks the power of the social obligation it refuses, including in matters of health. as cohen showed, microbiology, and specifically germ theory, appropriated and reconfigured the metaphor of immunity to help narrate the emerging science of cells, microbes and pathogenesis. in particular, the idea of immunity helped to explain how the immune system destroyed cells colonized by alien microbial life and bypassed uninfected cells of the body, although autoimmunity and microchimerism complicate this understanding of biological immunity (martin 2010) . combined with germ theory, immunity operates to produce a "milieu interieur;" an imaginary of the battle with microbial invaders inside the body (cohen 2009 : 239), a metaphor which accentuates the emphasis on the individual in relation to contagious health threats. emily martin has made a similar point that media depictions of immunity have often referred to the war within the body (1994). it is therefore no surprise that individuals resort to the practical and metaphorical properties of immunity when they are asked to contend with the risk of pandemic influenza, which creates uncertainties over which they otherwise have very little apparent control. these issues are reflected in consumer products, for example, the commercial marketing of probiotic foods and supplements (burges watson et al. 2009; koteyko 2009; nerlich and koteyko 2008) , which address individual consumers in terms of "choice immunity." probiotics also raise the idea that it is important to replace bacteria that have been killed off due to antibiotic treatment and/or the idea that "good" bacteria will outcompete illness producing bacteria. the scientific underpinning and marketing of probiotics, then, depend on a division of "good" and "friendly" bacteria from disease-producing bacteria. it is against this backdrop of immunity culture that public health institutions have to shape and circulate messages on how individuals ought to conduct themselves. as with the 2009 pandemic, agencies such as the who, regional who offices, and lead national public health agencies such as the cdc and public health scotland implement communication strategies and are key sources of expert commentary in worldwide news media. a central communication challenge is how to shape messages so that they are productive of desired action on the part of members of the general public, when it cannot be known absolutely how matters will transpire. it is clear from our research with public health professionals in australia and the uk that finding a balance of motivation and reassurance was paramount (davis et al. 2011 (davis et al. , 2013 . in this context, public health experts were concerned that publics should be advised and asked to prepare for the pandemic but not in ways that promoted anxiety or promoted panic, as reflected in, for example, runs on supermarkets, pharmacies and clinics. this meant that messages also had to be reassuring but not in a way that led publics to ignore advice, or worse, to become complacent. as briggs and nichter have pointed out, pandemic messaging was carefully styled around the notion of "be alert, not alarmed" (2009). they have identified this approach as the "just right" goldilocks method, that is, the production of alert, but not panicky, reassured, but not complacent publics. for example, in a newspaper article published on 27 april 2009, in the first few days of the pandemic alert, the chief health officer of australia was quoted to have said: we should be aware but i'm not overly alarmed at this point. we don't have confirmed cases in australia but i think there will be some cases in the future. we think the population should be alert, should be aware of travellers in their midst who have the flu. but not alarmed at this point, just aware. (robotham and pearlman 2009) in this way, pandemic communications help to constitute the expert-informed, life choices of individuals. less obvious are obligations to others which also make immunity possible, such as herd immunity and the related practice of altruistic vaccination to protect vulnerable others. it is also important to recognize that explicit reference to immunity is rarely a feature of this public health advice; it is nearly always implied. the 2009 pandemic raised some other problems related to the eventual character of the pandemic as mild for most, but not all. as noted, the 2009 pandemic was quickly found to be less severe than early indications portended, though some groups faced elevated risks and the pattern of morbidity differed from that typical for seasonal influenza (presanis et al. 2011) . it therefore became necessary to manage the communications turn away from alert, but without the cessation of cautionary messaging and continued advice for those who did face higher risk of severe disease. influenza is known to return, on occasion, in a second wave which has the potential to be more severe for all or some of those affected (presanis et al. 2011) . uncertainties like these meant that it was imperative to sustain a kind of watchful, just in case, attitude, until such time as an effective vaccine became available. this particular situation of a global alert followed by revisions of preparedness and response and growing evidence of a significantly less dangerous pandemic led to new communications challenges to do with explaining to publics what was happening and how they should therefore conduct themselves. this shifting in messaging across the period of the pandemic implied "the boy who cried wolf " parable (nerlich and koteyko 2012) , which teaches in narrative form the jeopardy of trust faced by raising a false alarm, too often. one effect of false alarm is that it may amplify the importance of choice immunity, that is, recourse to the self-reliant management of the body as the means to contend with an uncertain health threat. sociological perspectives on choice biography point out that under the conditions of neo-liberal economic and political order, individuals are forced to rely on themselves and their own decision-making capacities, since there is in the end, nowhere else for them to go (beck and beck-gernsheim 2002) . they nevertheless are bound to depend on expert advice, since no one person can be expert in all the considerations that pertain to health or any other of the major life decisions (ungar 2003) . false alarm destabilizes expert authority and leaves people doubly reliant on themselves. in this view, the tendency for individuals to fall back on their immunity is a rational response to the requirement to take action and because, in the face of the uncertainties which preside in the case of influenza, the body is one apposite arena in which people are able to exercise some control. our research shows also that the communication on the pandemic had the potential to divide publics according to their vulnerability, another way in which knowledge and questions played out in the 2009 pandemic. they showed awareness of the "boy who cried wolf" dilemma but also recognized the invidious situation in which public health experts found themselves. they spoke of the needless hype of the media on the pandemic, by which they meant the extent of the reporting on the progress of the pandemic (davis and lohm forthcoming). it is important to remember, also, that some groups and individuals were affected and profoundly so, for example, women who were pregnant in 2009. public communications on the risk of pandemic influenza, therefore, had a schismatic quality in the sense that the mildness of the virus needed to be explained to publics, while some remained at risk. like the universalism of pandemic preparedness, communications were also faced with the need for nuance and provisionality. this splitting of publics according to their vulnerability , was suggestive also of the coexistence of different modes of pandemic subjectivity. the "not at risk and in general unconcerned" could look upon news media and public communications as needless and hyped, particularly as the pandemic progressed. vulnerable groups, as we have suggested , at times had trouble recognizing themselves in these messages and once they had established for themselves awareness of their immunological vulnerability, they looked upon the hype as masking what for them was a real and visceral anxiety and set of practical issues of infection control and vaccination. this schism in public engagement accentuates the sense in which people have to make up their own mind on how to act in the context of what our vulnerable interviewees suggested were confusing, mixed messages. the communications challenges of emerging, changing pandemics are considerable. messages have to, at first, inform publics without frightening them, but also reassure them without producing complacency. as the example of the 2009 pandemic indicates, as the infection progressed and evidence emerged of the health effects of the h1n1 virus, public health systems had to explain that the pandemic was mild, though this situation could change. they also had to embed in this more general message information for minorities that they remained at serious risk. this changing, complex message risked provoking accusations of false alarm and therefore mistrust, as has happened in previous outbreak situations (fineberg 2008) . as i argued, too, the mixing of a general message of a mild pandemic which might change with messages that also some particular kinds of people were at risk, placed vulnerable people in the difficult situation of having to identify themselves in these messages and take action when others were sceptical and unlikely to be acting to protect themselves and those around them. when we asked people in our research to talk about h1n1 and specifically if it could be prevented, people acknowledged that infection was unlikely to be avoided and, accordingly, they were forced to reflect on the capacity of their body to cope with infection. as indicated, this resort to personal immunity was not quite the same as the science of cellular immunity discussed by cohen and others. it more closely resembled an acceptance of the possibility of the presence of the virus in the body and fashions an arena for volitional action on the body when other forms of action seem to have less practical value, as was the case in 2009. for example, social isolation and possibly vaccination, were endorsed but by and large not extensively taken up, particularly given that the virus was in general mild and easy to catch. because the h1n1 virus was observed to be so easily transmitted, the resort to personal immunity had doubled value. it may be for this reason that publics endorsed expert advice to self-isolate and vaccinate, but did not do so, that is, they fended for themselves and the pandemic turned into a mild one, anyway, though not for everyone. appeals to the collective good and altruistic vaccination on which depend public health efforts concerning pandemics, may miss the point that individuals are led to think of their personal immunity as an arena within which they can sustain themselves in the face of deeply uncertain threats which arise in communal life. if as cohen has suggested, immunity is fused with ideas of cellular action on microbial pathogens but it is also a metaphor for freedom from obligation. it seems, then, that a key lesson from 2009 was that freedom from the dangers of infection found in personal action on immunity also implied freedom from having to act in the interest of others; the more free one is from the dangers of infection-the stronger one's immunity-the less one needs to consider the dangers which others face, particularly under conditions of uncertainty. individualized ideas of 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findings from a population-based longitudinal study in tokyo acknowledgements this chapter is based on research funded by an australian research council discovery project grant on pandemic influenza (dp110101081). i would like to acknowledge the assistance of my colleagues from the pandemic influenza project, niamh stephenson, paul flowers, emily waller, casimir macgregor and davina lohm. i am also very grateful for the time and efforts of those who participated in the interviews and focus groups for the research. key: cord-023666-r9zaf6un authors: rao, suchitra; nyquist, ann-christine; stillwell, paul c. title: influenza date: 2018-03-13 journal: kendig's disorders of the respiratory tract in children doi: 10.1016/b978-0-323-44887-1.00027-4 sha: doc_id: 23666 cord_uid: r9zaf6un influenza a and b viruses are orthomyxoviruses with three important envelope glycoproteins: hemagglutinin (ha), neuraminidase (na), and matrix proteins. influenza viruses have developed ways to evade the body's immune response using an antigenic variation known as antigenic shift (replacement of ha and na antigens with novel subtypes from noninfluenza viruses) and drift (mutations within antibody-binding sites in ha and or na). because of new influenza viruses constantly emerging from antigenic shift and drift, new influenza vaccines are required each year. human-to-human transmission of influenza occurs each winter and early spring through small-particle aerosols or droplets. the influenza virus attacks epithelial cells of the upper and lower respiratory tract, with the potential for secondary bacterial infection and acute respiratory distress syndrome (ards). the symptoms of influenza infection include fever, headache, cough, sore throat, myalgia, and nasal congestion. lower respiratory tract manifestations such as pneumonia and bronchiolitis are virtually indistinguishable from other viral infections. children with certain comorbidities, such as chronic lung disease and severe neurologic impairment, are at higher risk of influenza-related complications. the most reliable test for influenza is reverse transcription polymerase chain reaction (rt-pcr). rapid antigen tests have lower sensitivity and specificity and are not reliable during periods of low influenza activity. antiviral treatment with na inhibitors can shorten the duration of fever, symptoms, and hospitalization, especially when started within 48 hours of influenza illness onset. prevention of influenza through annual influenza vaccination is recommended for all children 6 months of age and older. the vaccines contain three or four influenza subtypes, chosen depending on the circulating strains. the two formulations approved for children are the inactivated influenza vaccine (iiv) and live-attenuated influenza vaccine (laiv). suchitra rao, mbbs, ann-christine nyquist, md, msph, and paul c. stillwell, md 27 but can be as rapid as 18 hours or as long as 5 or more days. healthy adults will shed influenza virus for 3-7 days, and young children may shed for 10 days or longer with generally higher viral titers. influenza occurs each year in winter through early spring and is associated with significant morbidity and mortality in certain high-risk populations (table 27 .1). in the united states up to 36,000 deaths per year are attributable to influenza, with people above 65 years of age accounting for more than 90% of deaths. influenza is responsible for 5%-15% of upper respiratory tract infections in children. [4] [5] [6] healthy children aged 6-23 months are at substantially increased risk of influenza-related hospitalizations, and children aged 24-59 months remain at increased risk of influenza-related clinic and emergency department visits and hospitalizations but less so than younger children. influenza virus infection begins with the attachment of viral ha to terminal sialic acids on the surface of target host cells. in the lungs, the target cells of the influenza virus are typically ciliated columnar epithelial or alveolar epithelial cells (aecs), with each ha subtype displaying a unique tropism. attachment of the influenza virus triggers receptor-mediated endocytosis by host cells and thus entry into the cell. to effectively release influenza rna into the cytosol, viral m2 forms ion channels in the viral envelope. liberated viral rna then travels to the nucleus of the infected cell, where messenger rna (mrna) and viral rna are synthesized. assembly and budding of daughter virions occurs at the cell surface, where na facilitates virion release by cleavage of sialic acid attachments to viral ha. without this cleavage, influenza virions aggregate at the cell surface and are not released, which is the mechanism by which na inhibitors are effective. ultimately, viral infection leads to apoptosis of the infected epithelial cells, which denudes the airways, resulting in acute tracheobronchitis. [7] [8] [9] [10] when type i aecs are damaged, the tight junctions of the alveolar capillary membrane allow a transudate of fluid and proteins to enter the alveolar spaces, producing acute alveolar damage with the potential of progressing to acute respiratory distress syndrome (ards). 11, 12 viral ha and m2 also inhibit the resorption of alveolar fluid by the epithelial sodium channel, further promoting alveolar edema. influenza infection and resultant cytokine induction can also activate the endothelial pole of the alveolar capillary barrier, facilitating neutrophil influx and further disruption of barrier function. 11 the host immune response to influenza infection is complex and redundant, incorporating both innate and adaptive processes (see chapter 8 for details of innate and adaptive influenza viruses are enveloped ribonucleic acid (rna) viruses and belong to the family orthomyxoviridae. there are three virus types within this family: influenza a, b, and c. influenza a and b viruses are the types that predominantly infect humans, and a viruses are responsible for pandemic outbreaks of influenza and annual epidemics. 1 the virus contains three important envelope glycoproteins: hemagglutinin (ha), neuraminidase (na), and matrix proteins (m1 and m2). ha is the viral attachment protein responsible for entry of the virus into cells; it is an important surface antigen to which virus-neutralizing antibodies are directed. na is an enzyme whose main function is to facilitate the cell-to-cell spread of virus; it is the target for the antiviral drugs zanamivir and oseltamivir. antibody against ha is very protective against infection and illness, and antibody against na can reduce illness severity. the matrix protein 2 (m2) is a structural protein linking the viral envelope with the virus core and is integral to the infectivity of the influenza virion. influenza viruses have a segmented genome, containing eight strands of rna. this property enables gene reassortment to occur among different subtypes of influenza, allowing new subtypes to form. variation in the structure of ha and na between influenza virions is the basis of the subtype h and n classification nomenclature (e.g. influenza a h1n1) ( fig. 27.1 ). influenza viruses have developed ways to evade the body's immune response using an antigenic variation known as antigenic shift and drift. antigenic shift is seen only with influenza a viruses and results from the replacement of ha (or occasionally na) with novel subtypes from other nonhuman influenza viruses. introduction of a new ha into human viruses results in a pandemic, or worldwide epidemic, with the potential to cause millions of influenza-related deaths. 2 antigenic drift results from the accumulation of mutations within the antibody-binding sites in ha, na, or both. these mutations prevent antibodies against previous strains from being effective against the current strain, enabling spread throughout a partially immune population. antigenic drift occurs in both influenza a and b viruses. 3 antigenic shift and drift must be taken into account when strains are being considered for inclusion in annual influenza vaccines. human-to-human transmission of influenza occurs through small-particle aerosols or droplets, which enter the environment from an infected individual. the virus then binds to epithelial cells of the upper and lower respiratory tract. the incubation period from exposure to illness averages 2-3 days keywords influenza respiratory virus respiratory tract illness abstract influenza a and b viruses are orthomyxoviruses with three important envelope glycoproteins: hemagglutinin (ha), neuraminidase (na), and matrix proteins. influenza viruses have developed ways to evade the body's immune response using an antigenic variation known as antigenic shift (replacement of ha and na antigens with novel subtypes from noninfluenza viruses) and drift (mutations within antibody-binding sites in ha and or na). because of new influenza viruses constantly emerging from antigenic shift and drift, new influenza vaccines are required each year. human-to-human transmission of influenza occurs each winter and early spring through small-particle aerosols or droplets. the influenza virus attacks epithelial cells of the upper and lower respiratory tract, with the potential for secondary bacterial infection and acute respiratory distress syndrome (ards). the symptoms of influenza infection include fever, headache, cough, sore throat, myalgia, and nasal congestion. lower respiratory tract manifestations such as pneumonia and bronchiolitis are virtually indistinguishable from other viral infections. children with certain comorbidities, such as chronic lung disease and severe neurologic impairment, are at higher risk of influenza-related complications. the most reliable test for influenza is reverse transcription polymerase chain reaction (rt-pcr). rapid antigen tests have lower sensitivity and specificity and are not reliable during periods of low influenza activity. antiviral treatment with na inhibitors can shorten the duration of fever, symptoms, and hospitalization, especially when started within 48 hours of influenza illness onset. prevention of influenza through annual influenza vaccination is recommended for all children 6 months of age and older. the vaccines contain three or four influenza subtypes, chosen depending on the circulating strains. the two formulations approved for children are the inactivated influenza vaccine (iiv) and live-attenuated influenza vaccine (laiv). a although all children below 5 years of age are considered at higher risk for complications from influenza, the highest risk is for those younger than 2 years, with the highest hospitalization and death rates among infants younger than 6 months. immune responses). [8] [9] [10] 13 in response to infections, activated macrophages and neutrophils work to eliminate viral particles and damaged or apoptotic epithelial cells. although this is a key element in viral elimination and the recovery of epithelial integrity, the inflammatory by-products-such as myeloperoxidase, neutrophil elastases, and increased nitric oxide synthase-may cause further injury to the airway. 8, 9 increased risk of secondary bacterial infection may be due to delayed epithelial healing after influenza infection, excessive interferon gamma production, and type i interferons. 8, 9 the majority of systemic symptoms observed in acute influenza infection are attributable to the cytokines produced and released during the host immune response. viremia is uncommon in an immunocompetent host. similarly, dysfunction in nonrespiratory organs-such as myocarditis, encephalopathy, encephalitis, and rhabdomyolysis-is not usually associated with viral infection in those tissues. 9 viral infections can also stimulate the th2 arm of the immune system by producing thymic stromal lymphopoietin (tslp), il-25, and il-33. tslp stimulates dendritic cells to induce th2 cells and predisposes the host to allergic airway inflammation and asthma. il-25 and il-33 stimulate type 2 cytokine-producing innate lymphoid cells (ilc-2) to synthesize il-5 and il-13, which are promoters of eosinophilic inflammation, excessive mucus secretion, and bronchial hyperresponsiveness. this may explain, in part, how acute influenza infection exacerbates asthma and potentiates a subsequent asthma phenotype. 14, 15 clinical features there is great overlap in the symptomatology of influenza and other respiratory pathogens. the influenza syndrome usually has a sudden onset, associated with fever, headache, cough, sore throat, myalgia, nasal congestion, weakness, and loss of appetite. 16, 17 in a retrospective study of adolescents and young adults with influenza-like illness, the best predictors of influenza infections were cough and fever, with a positive predictive value of 79%. 18 young children, however, have a less classic presentation compared with adults and tend to have higher fevers, less prominent respiratory symptoms, and more gastrointestinal symptoms such as abdominal pain, vomiting, diarrhea, and decreased appetite. 16, 19, 20 influenza infection is also an important cause of febrile seizures. 21 lower respiratory tract manifestations in young children are virtually indistinguishable from those due to other viral infections. influenza, similar to other respiratory viruses, may cause bronchiolitis, interstitial pneumonia, laryngotracheitis (croup), bronchitis, exacerbations of asthma, wheezing, and pneumonia. 22 extrapulmonary manifestations include myocarditis, hepatitis, encephalitis, myositis, renal insufficiency, guillain-barré syndrome, rhabdomyolysis, and multiorgan system failure. [23] [24] [25] [26] [27] [28] findings on examination include tachypnea, conjunctival erythema, nasal injection, edema, nasal discharge, and cervical adenopathy. rash is an uncommon manifestation of influenza, but when it occurs it is usually a generalized maculopapular rash sparing the palms and soles. other rashes associated with influenza infection have been characterized as petechial, macular, papular, reticular, or purpuric; they can be localized and pruritic or nonpruritic. [29] [30] [31] [32] [33] [34] the chest radiographic features of influenza pneumonia are indistinguishable from those of pneumonia caused by other organisms. 35, 36 the most common radiographic findings are testing for influenza is recommended if positive or negative results will influence clinical management or clinical practice for other patients. testing should be considered, regardless of immunization status, during the influenza season among children with fever and acute onset of respiratory signs and symptoms, and those with acute exacerbations of underlying chronic lung disease. infants and young children with fever and no other signs and symptoms, hospitalized children with acute respiratory symptoms who develop an acute febrile respiratory illness, and severely ill children with fever or hypothermia should also be tested. testing should occur at any time of the year for children who are epidemiologically linked to an influenza outbreak (e.g., household and close contacts of people with suspected influenza, returned travelers from countries where influenza viruses may be circulating, participants in international mass gatherings, and cruise ship passengers). 47 testing 5 days or more beyond illness onset may result in false-negative test results because of decreased viral shedding; this occurs in particular among older children. testing modalities available include influenza-specific reverse transcription polymerase chain reaction (rt-pcr), multiplex respiratory pathogen pcr, direct fluorescent antibody (dfa) tests, and rapid influenza antigen tests. rt-pcr is the most accurate testing modality for influenza; it is useful for differentiating between influenza types and subtypes and is significantly more sensitive than rapid influenza antigen detection tests (>95% vs. 10%-70%, respectively). rapid antigen tests have less sensitivity and specificity than rt-pcr tests. a meta-analysis of children demonstrated sensitivity of 64.6% for influenza a and 52.2% for influenza b. 48 these and other studies indicate that rapid antigen tests are not reliable during periods of low influenza activity. immunofluorescent antibody testing can distinguish between influenza a or b and other respiratory viruses. however, the test performance is dependent on the quality of the respiratory specimen and expertise of the laboratory. viral culture is usually unhelpful in the clinical setting because results are not available until 48-72 hours later. however, it is helpful for the confirmation of screening results, surveillance, and research. serologic testing is also not helpful in the clinical setting because acute and convalescent (obtained 10 days later) sera are required for diagnosis. testing platforms include hemagglutination-inhibition, enzyme-linked immunosorbent assays (elisa) and complement fixation assays. a fourfold rise or greater in antibody titers between acute and convalescent specimens confirms the diagnosis of influenza. the differential diagnosis of influenza infection is listed in table 27 . clinical trials and observational data show that early antiviral treatment can shorten the duration of fever, influenza bilateral, symmetric, perihilar, and peribronchial opacities, but focal opacities and asymmetric disease may also occur ( fig. 27.2) . 36 lymph node enlargement can occur, but pleural effusions are rare. the nonspecific findings make it challenging to differentiate viral from bacterial pneumonia based on the radiographic appearance alone. younger children with influenza pneumonia may have bilateral patchy opacities that probably reflect the retention of mucus. 35 progression of the pneumonia can lead to diffuse airspace disease with an ards picture and acute respiratory failure. complete resolution to a normal chest radiograph should be expected following mild disease. children with chronic lung disease, asthma, airway disease, cardiovascular disease, neuromuscular disease, and immunocompromised states are at highest risk of complicated influenza infection (see table 27 .1). 37 severe influenza infection can present with bilateral pulmonary infiltrates and hypoxemia, leading to ards and death. 11, [38] [39] [40] in particular, the 2009 h1n1 pandemic influenza virus was associated with higher rates of life-threatening lower respiratory tract illness, including ards, which was thought to be due to an increased predilection to infection of ciliated epithelial cells of the lower respiratory tract. [41] [42] [43] pneumonia and secondary bacterial infection is a common cause of hospitalization from influenza. 44 although streptococcus pneumoniae is the most common pathogen identified, staphylococcus aureus is a commonly associated copathogen. methicillin-resistant s. aureus (mrsa) coinfections with influenza are increasingly being identified and are a risk factor for mortality in previously healthy children and adolescents. 45 clinical features that support a bacterial superinfection among children with influenza include secondary fever after a period of defervescence, focal findings on pulmonary auscultation, lobar consolidation on chest imaging, and new onset of respiratory compromise occurring several days after initial symptoms. 46 neuraminidase inhibitors (nais) oseltamivir and zanamivir are the antiviral medications still recommended for the treatment and chemoprophylaxis of influenza a and influenza b virus infections owing to near universal susceptibility. they inhibit the viral na enzyme that helps progeny escape from infected cells. nais may also have efficacy against the novel influenza viruses. oseltamivir is given orally for 5 days with dose adjustments required for renal impairment and weight (table 27. 3). the most common side effects of oseltamivir are nausea and/or vomiting. transient neuropsychiatric events (self-injury or delirium) have been reported, mainly among japanese adolescents and adults. 57 zanamivir is a dry powder administered via oral inhalation. it is not fdaapproved for the treatment of children under 7 years of age. the dose is two breath-activated inhalations twice daily for 5 days. the prophylactic dose is two inhalations once daily for children 5 years of age and older. it is not recommended for children with underlying airway disease including asthma owing to lack of safety and efficacy data in these individuals. serious adverse events include bronchospasm and decline in lung function, most commonly seen in patients with underlying airway disease. (if zanamivir is used in patients with underlying airway disease, they should be instructed to have a short-acting bronchodilator available.) allergic reactions including rashes and oropharyngeal or facial edema have been reported. side effects include diarrhea, nausea, sinusitis, rhinitis, nasal congestion, bronchitis, cough, headache, dizziness, and ear/nose/throat complaints. peramivir is an intravenous nai indicated for the treatment of acute, uncomplicated influenza in patients 18 years of age and older who have been symptomatic for no more than 2 days; it is given as a single 600-mg administration. matrix protein inhibitors amantadine and rimantadine target the m2 protein and are potentially effective against only influenza a owing to the lack of a m1/m2 protein channel in influenza b viruses. these antiviral medications are not currently recommended for treatment or chemoprophylaxis since most circulating influenza a strains have developed resistance to them. [58] [59] [60] steroids have been proposed as an adjunctive therapy for influenza pneumonia; however, several studies failed to demonstrate clinical benefit in the treatment of patients symptoms, and hospitalization; it may also reduce the risk of complications from influenza (e.g., otitis media in young children, pneumonia, respiratory failure, and death). [49] [50] [51] [52] [53] [54] clinical benefit is greatest when antiviral treatment is administered early, especially within 48 hours of influenza illness onset. 49, 55, 56 treatment should not wait for laboratory confirmation of influenza but should be started as soon as possible when clinically indicated. antiviral treatment is recommended regardless of the day of illness for any patient with confirmed or suspected influenza who is hospitalized; has severe, complicated, or progressive illness; or is an outpatient who is at higher risk for influenza complications based on age or underlying medical conditions. clinical judgment-based on the patient's disease severity and progression, age, underlying medical conditions, likelihood of influenza, and time since onset of symptoms-is important in making decisions regarding antiviral treatment for high-risk outpatients. antiviral treatment may be considered for any outpatient with confirmed or suspected influenza who is otherwise healthy if treatment can be initiated within 48 hours of illness onset. the american academy of pediatrics recommended an oseltamivir treatment dose of 3.5 mg/kg orally twice daily for infants aged 9-11 months for the 2013-14 season on the basis of data indicating that a higher dose of 3.5 mg/kg was needed to achieve the protocol-defined targeted exposure for this cohort as defined in the capability acquisition and sustainment (casg) 114 study (kimberlin, 2013) . 68 it is unknown whether this higher dose will improve efficacy or prevent the development of antiviral resistance. however, there is no evidence that the 3.5-mg/kg dose is harmful or causes more adverse events to infants in this age group. provider who is familiar with the potential manifestations of egg allergy in a setting where anaphylaxis can be recognized and treated. such children should be observed for at least 30 minutes for signs of a reaction after administration of each vaccine dose. nais are 70%-90% effective in preventing influenza. yet the cdc does not recommend widespread or routine use of chemoprophylaxis owing to the possibility that resistant viruses could emerge, thus limiting the usefulness of these medications for high-risk or severely ill people. oseltamivir can be used for chemoprophylaxis of influenza among infants below 1 year of age when indicated, although, owing to limited data in this age group, children less than 3 months of age should not receive prophylaxis unless the situation is judged to be critical. chemoprophylaxis is not usually recommended if more than 48 hours have elapsed since the last exposure to an infected person. for effective prophylaxis, an antiviral medication must be taken each day for the duration of potential exposure to a person with influenza and continued for 7 days after the last known exposure. postexposure prophylaxis should be considered for family members and close contacts of infected patients if they are at high risk of complications from influenza. with or without antiviral treatment, most children have a full and uneventful recovery after an acute, uncomplicated influenza infection. unfortunately, some will have a fatal outcome. death from influenza is more frequent in children with comorbidities such as asthma or severe neurologic impairment and in those who develop ards. 66 a small proportion of children may develop asthma after experiencing an acute influenza infection, although asthma subsequent to infection seems with severe influenza infection. furthermore, studies indicate an increase in overall mortality, increased incidence of hospital-acquired pneumonia, and longer duration of mechanical ventilation and icu stay in patients treated with steroids. 61, 62 prevention routine annual influenza vaccination is recommended for all children 6 months of age and older. ideally, vaccination should occur before the onset of influenza in the community and should be administered as soon as a vaccine supply is available. vaccination should be continued as long as influenza viruses are circulating. multiple different vaccine formulations are available, and some are licensed for specific age groups or are more appropriate for particular patient populations. recommendations regarding influenza and vaccine dosing are updated by the cdc each year and are available via the centers for disease control and prevention (cdc) influenza website (http://www.cdc.gov/flu/); they are also published in the morbidity and mortality weekly report by the cdc. 63, 64 two formulations of vaccine are approved for children: inactivated influenza vaccine (iiv) and liveattenuated influenza vaccine (laiv). laiv is an intranasal vaccine and is offered solely in quadrivalent form. iiv is available in trivalent or quadrivalent formulations. the centers for disease control and prevention (cdc) has recommended that laiv should not be used until further notice due to low vaccine effectiveness against influenza a during the 2013-2014 and 2015-2016 seasons. recombinant influenza vaccine (riv) is available for adults with egg allergy. children 6 months through 8 years of age receiving vaccine for the first time require two doses of vaccine at least a month apart. influenza vaccine contraindications are provided in table 27 .4. data demonstrate that iiv and laiv 65 given in a single, age-appropriate dose is well tolerated by virtually all recipients who have egg allergy. children with anaphylaxis to eggs may receive influenza vaccine by a health care severe allergic reaction to any component of the vaccine, including egg protein, or after previous dose of any influenza vaccine. moderate to severe illness with or without fever; history of guillain-barré syndrome within 6 weeks of receipt of influenza vaccine. severe allergic reaction to any component of the vaccine. moderate to severe illness with or without fever; history of guillain-barré syndrome within 6 weeks of receipt of influenza vaccine. live-attenuated influenza vaccine (laiv 4 ) a severe allergic reaction to any component of the vaccine, including egg protein, or after previous dose of any influenza vaccine. concomitant use of aspirin or aspirin-containing medications in children and adolescents. in addition, advisory committee on immunization practices recommends laiv 4 not be used for pregnant women, immunosuppressed people, people with egg allergy, and children aged 2-4 years who have asthma or who have had a wheezing episode noted in the medical record within the past 12 months, or for whom parents report that a health care provider stated that they had wheezing or asthma within the last 12 months. moderate to severe illness with or without fever. history of guillain-barré syndrome within 6 weeks of receipt of influenza vaccine. asthma in people 5 years of age and older. medical conditions that might predispose to higher risk for complications attributable to influenza. harper sa, bradley js, englund ja, et al. seasonal influenza in adults and effect of oseltamivir on the risk of pneumonia and use of health care services in children with clinically diagnosed influenza critically ill patients with 2009 influenza a (h1n1) infection in canada rash, an uncommon but existing feature of h1n1 influenza among children. influenza other respir viruses acute fever and petechial rash associated with influenza a virus infection the spectrum of histopathologic findings in cutaneous eruptions associated with influenza a (h1n1) infection coexistence of two adamantane binding sites in the influenza a m2 ion channel leukocytoclastic vasculitis associated with influenza a virus infection radiological findings in 210 paediatric patients with viral pneumonia: a retrospective case study pulmonary infections populations at risk for severe or complicated influenza illness: systematic review and meta-analysis clinical features of the initial cases of 2009 pandemic influenza a (h1n1) virus infection in china hospitalized patients with 2009 h1n1 influenza in the united states an assessment of h1n1 influenza-associated acute respiratory distress syndrome severity after adjustment for treatment characteristics altered receptor specificity and cell tropism of d222g hemagglutinin mutants isolated from fatal cases of pandemic a (h1n1) 2009 influenza virus association of d222g substitution in haemagglutinin of 2009 pandemic influenza a (h1n1) with severe disease influenza a (h1n1) vs non-h1n1 ards: analysis of clinical course bench-to-bedside review: bacterial pneumonia with influenza-pathogenesis and clinical implications critically ill children during the 2009-2010 influenza pandemic in the united states when to consider the use of antibiotics in the treatment of 2009 h1n1 influenza-associated pneumonia seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america accuracy of rapid influenza diagnostic tests: a meta-analysis oral oseltamivir treatment of influenza in children effect of oseltamivir on the risk of pneumonia and use of health care services in children with clinically diagnosed influenza viral shedding in children with influenza virus infections treated with neuraminidase inhibitors oseltamivir treatment for influenza in hospitalized children without underlying diseases oseltamivir shortens hospital stays of critically ill children hospitalized with seasonal influenza: a retrospective cohort study orthomyxoviridae: the viruses and their replication influenza vaccine-outmaneuvering antigenic shift and drift the evolution of human influenza viruses contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study community-acquired pneumonia requiring hospitalization among u.s. children global role and burden of influenza in pediatric respiratory hospitalizations, 1982-2012: a systematic analysis virulence determinants of pandemic influenza viruses viral infection of the lung: host response and sequelae influenza a virus infection, innate immunity, and childhood novel insights into immune and inflammatory responses to respiratory viruses pathogenesis of influenzainduced acute respiratory distress syndrome case records of the massachusetts general hospital. weekly clinicopathological exercises. case 9-2004. an 18-year-old man with respiratory symptoms and shock excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity airway epithelial orchestration of innate immune function in response to virus infection. a focus on asthma clinical presentation of influenza in unselected children treated as outpatients understanding the symptoms of the common cold and influenza clinical signs and symptoms predicting influenza 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randomised controlled trial. neuraminidase inhibitor flu treatment investigator group efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group oseltamivir and delirious behavior in children with influenza incidence of adamantane resistance among influenza a (h3n2) viruses isolated worldwide from 1994 to 2005: a cause for concern adamantane resistance among influenza a viruses isolated early during the 2005-2006 influenza season in the united states surveillance of resistance to adamantanes among influenza a (h3n2) and a (h1n1) viruses isolated worldwide corticosteroids for severe influenza pneumonia: a critical appraisal effect of corticosteroid therapy on influenza-related mortality: a systematic review and meta-analysis prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) prevention and control of influenza with vaccines: recommendations of the advisory committee on immunization practices, united states, 2015-16 influenza season safety of live attenuated influenza vaccine in young people with egg allergy: multicentre prospective cohort study a review on the clinical spectrum and natural history of human influenza association between respiratory infections in early life and later asthma is independent of virus type oseltamivir pharmacokinetics, dosing, and resistance among children aged <2 years with influenza key: cord-030853-3yryw3r2 authors: vashishtha, vipin m.; kumar, puneet title: seasonal influenza vaccination and the heightened risk of coronavirus and other pandemic virus infections: fact or fiction? date: 2020-06-09 journal: indian pediatr doi: 10.1007/s13312-020-1936-1 sha: doc_id: 30853 cord_uid: 3yryw3r2 nan during this ongoing severe acute respiratory illness coronavirus 2 (sars-cov-2) pandemic, few speculative reports on significant association of influenza vaccines with an increased risk of coronavirus infection appeared both in media and academic circles. the speculation of vaccines paradoxically increasing the risk of infections possibly originated first following 2009 influenza a (h1n1pdm09) pandemic when four canadian studies suggested that receipt of seasonal influenza vaccine increased the risk of laboratory-confirmed 2009 pandemic influenza a (h1n1pdm09) virus infection [1] . this led to five additional studies, each of which substantiated these initial findings. one proposed mechanism behind this phenomenon is 'original antigenic sin' which was first used to describe how first exposure to influenza virus shapes the outcome of subsequent exposures to antigenically related strains. when an individual is volume 57 __ august 15, 2020 correspondence infected by an 'evolved' strain with a new dominant antigen, slightly different from the 'original' strain against which the person has been vaccinated, the immune system produces antibodies against the 'original' strain through preformed highaffinity memory b cells that inhibit activation of naïve b cells resulting in a weak immune response against the new 'dominant' strain. hence, the risk of infection paradoxically increased in vaccinated individuals as compared to unvaccinated individuals [2] . besides, viruses are known to interfere with the circulation of other viruses. for example, there is evidence that the circulation of rhinovirus in the community interferes and decreases the spread of seasonal and pandemic influenza viruses [3, 4] . viral interference is also well-known to interfere with "take" of oral polio vaccine. however, more recently a new phenomenon, 'vaccine-associated virus interference' has been suggested whereby a vaccine can paradoxically increase the circulation of other viruses. that is, vaccinated individuals may be at increased risk for other respiratory viruses because they do not receive the non-specific immunity associated with natural infection [5, 6] . rikin, et al. [5] found an increased incidence of acute respiratory infection in children by non-influenza respiratory viruses among 999 participants (out of which 68.8% were children) following influenza vaccination compared to unvaccinated children during the same period. in a study of 115 children [6] , a significantly increased risk of virologically confirmed non-influenza respiratory virus infections was found to be associated with receipt of inactivated influenza vaccine. coronavirus was one of the non-influenza respiratory viruses [6] . wolff, et al. [7] recently performed a large study among defence personnel to investigate respiratory virus interference during the 2017-2018 influenza season by comparing respiratory virus status with their influenza vaccination status. they concluded that overall, receipt of influenza vaccination was not associated with virus interference among the study population. however, vaccine-derived virus interference by specific respiratory viruses was significantly associated with coronavirus and human metapneumovirus [7] . however, studies that have looked into the interference of influenza vaccine with specific non-influenza viral infections are scarce. it is hypothesized that a respiratory virus infection confers immunity against the same and other respiratory viruses for a short time, perhaps a few weeks. this immune protection is associated with activation of the innate immune response to viral infection mediated by the release of type i interferons and other cytokines that have broad protective effects against a range of viruses [8] . this immunologic mechanism, known as heterosubtypic 'temporary non-specific immunity', has been proposed as the biological mechanism behind the paradoxical findings. natural influenza infection that could have provided the host with some temporary immunity against other respiratory viruses is prevented by influenza vaccination. hence, the risk of infection by non-influenza viruses (including the coronaviruses) is paradoxically increased [6] . the contentious issue of higher risk of non-influenza respiratory viruses to influenza vaccinated individuals has gained traction during the ongoing sars-cov-2 pandemic, which is also a coronavirus infection. currently, we do not have sufficient data to establish or refute the association between influenza vaccination and higher susceptibility to coronavirus infection. we need to perform systematic studies urgently to find an answer to this question with regard to sars-cov-2. this is of vital importance since it is going to have far-reaching implications. enabling delivery of essential health services during the covid 19 outbreak: guidance note sars-cov-2 disease and children under treatment for cancer. pediatr blood cancer covid -19 pandemic: the challenges for pediatric oncology government of india. advisory on the use of hydroxyl chloroquine as prophylaxis for sars-cov2 infection quarantine policy document for the health care workers (hcws) working in covid hospital/ward world health organization. health workers exposure risk assessment and management in the context of covid-19 virus: interim guidance covid-19): guidelines on rational use of personal protective equipment (setting approach for health functionaries working in non-covid areas available from:https:// www.mohfw.gov.in/pdf/additionalguidelinesonrational use ofpersonalprotectiveequipment settingapproachforhealth functionariesworkinginnon covidareas esmo guideline: cancer patient management during the covid-19 pandemic icmr strategy for covid19 testing in india real-time rt-pcr in covid-19 detection: issues affecting the results association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada original antigenic sin: how first exposure shapes lifelong antiinfluenza virus immune responses does viral interference affect spread of influenza? virus interference. did rhinoviruses activity hamper the progress of the 2009 influenza a (h1n1) pandemic in norway? med hypotheses assessment of temporally-related acute respiratory illness following influenza vaccination increased risk of non-influenza respiratory virus infections associated with receipt of inactivated influenza vaccine influenza vaccination and respiratory virus interference among department of defence personnel during the 2017-2018 influenza season respiratory virus induction of alpha-, beta-and lambda-interferons in bronchial epithelial cells and peripheral blood mononuclear cells key: cord-255807-7goz1agp authors: hak, e.; hoes, a. w.; grobbee, d. e.; lammers, j. w. j.; van essen, g. a.; van loon, a. m.; verheij, t. j. m. title: conventional influenza vaccination is not associated with complications in working-age patients with asthma or chronic obstructive pulmonary disease date: 2003-04-15 journal: am j epidemiol doi: 10.1093/aje/kwg027 sha: doc_id: 255807 cord_uid: 7goz1agp by using a nested case-control design, the authors studied the effectiveness of the influenza vaccine in reducing severe and fatal complications in 4,241 and 5,966 primary care, working-age patients aged 18–64 years who had asthma or chronic obstructive pulmonary disease during the 1998–1999 and 1999–2000 influenza epidemics in the netherlands. patients developing fatal or nonfatal exacerbations of lung disease, pneumonia, congestive heart failure, or myocardial infarction during either epidemic were considered cases. for each case, four ageand sex-matched controls were randomly sampled, and patient records were reviewed. conditional logistic regression and propensity scores were used to assess vaccine effectiveness after adjustment for confounding factors. in seasons one and two, respectively, 87% (47/54) and 85% (171/202) of the cases and 74% (155/210) and 75% (575/766) of the controls had been vaccinated. after adjustments, vaccination was not associated with reductions in complications (season one: odds ratio = 0.95, 95% confidence interval (ci): 0.26, 3.48; season two: odds ratio = 1.07, 95% ci: 0.59, 1.96; pooled odds ratio = 1.07, 95% ci: 0.63, 1.80). because influenza vaccination appeared not to be associated with a clinically relevant reduction in severe morbidity, other measures need to be explored. the risk of influenza-related morbidity and mortality during influenza epidemics is high (1) (2) (3) (4) , and nonexperimental studies have shown that vaccination against influenza prevents respiratory and cardiac complications during epidemics in elderly patients with chronic obstructive pulmonary disease (copd) (5, 6) . however, relatively little information is available regarding working-age patients with copd. some studies have shown that these patients may account for many hospital admissions for respiratory illness during epidemics, but risk estimates are largely unknown (7) (8) (9) . on the other hand, the sparse data available on acute respiratory illness in asthmatics suggest a relatively minor role for influenza (10, 11) . although the vaccine does not lead to potentially adverse effects in asthmatics (12) , the few available small-scale studies on the clinical benefits of influenza vaccination among working-age patients with copd have failed to demonstrate any effectiveness from annual vaccination (6, 13, 14) . we determined the occurrence of respiratory and cardiac morbidity during influenza periods and the clinical effectiveness of vaccination in reducing these complications in patients aged 18-64 years who had asthma or copd by using a prospective, nested case-control design. our observations covered the 1998-1999 influenza outbreak (principally type b) and the 1999-2000 epidemic (mainly type a (h3n2)) (15, 16) . since it is well known that influenza causes only part of the complications and that our outcome might therefore be nonspecific, as was the case in many previous reports (3) (4) (5) (6) , we also obtained nose and throat swabs from a sample of cases and controls to assess the relative contribution of influenza to complications. am j epidemiol 2003;157:692-700 study subjects were chosen from among primary care patients aged 18-64 years who had asthma or copd and had been targeted according to immunization guidelines for annual influenza vaccination (17, 18) . seventy-eight general practitioners in 41 computerized primary care centers across the netherlands participated in the study during the 1998-1999 influenza epidemic, and 93 general practitioners in 52 centers participated during the 1999-2000 epidemic. these general practitioners routinely integrate all patient information in text format or encoded in their computerized records by using the general practitioners information system elias (torex-hiscom, houten, the netherlands) (19) . patients eligible for inclusion in our study were selected as of october 1999 and october 2000 by means of a dedicated software module. details on the module's stepwise selection procedures have been described elsewhere (20) . briefly, patients were identified by age and the presence of copd, as indicated by international classification of primary care diagnostic codes (r91, r95, r96), anatomical therapeutic classification medical drug codes (class r03), and a tag in their computerized records indicating copd. next, the general practitioners were asked to verify whether the diagnosis of asthma or copd in the preselected patients had been made in accordance with the dutch college of general practitioners guidelines (21) . in october 1999 and october 2000, 4,241 and 5,966 eligible patients of a total of 6,011 and 8,495 study patients, respectively, preselected by using the search algorithm in the general practitioner information systems, were enrolled. since all data were supplied anonymously to the julius center for health sciences and primary care (utrecht, the netherlands), individual patient consent was not obtained. the medical ethical board of the university medical center utrecht approved the conduct of the study. subjects qualified as cases if they had a primary diagnosis of an episode of fatal or nonfatal severe exacerbation of underlying lung disease, pneumonia, congestive heart failure, or myocardial infarction during either epidemic (refer to the appendix). case criteria were verified by using a computerized questionnaire, which was integrated in the medical records of study patients and could be activated by their general practitioners during consultation. annual influenza surveillance was carried out by the national influenza center in collaboration with the sentinel practice network (15, 16) . the epidemic periods were defined as the weeks in which the incidence of influenza-like illness reported by the sentinel practices was more than four per 10,000 inhabitants per week (between week 50 of 1998 and week 12 of 1999 (season one) and between week 50 of 1999 and week 10 of 2000 (season two)). during the first and largest wave of the 1998-1999 biphasic influenza outbreak, the influenza b-harbin-type virus predominated, followed by a smaller wave of a(h3n2)sydney. clinical influenza activity during the 1999-2000 season was predominantly associated with influenza type a(h3n2)sydney. in seasons one and two, six of 60 and five of 207 cases, respectively, were deemed ineligible for the study because it was unclear whether they had asthma or copd; therefore, these patients and their controls were excluded from further consideration. in addition, 47 and 174 patients with severe exacerbation of asthma or copd, five and 26 patients with pneumonia, zero and one patient with congestive heart failure, and two and one patient who died, respectively, were considered eligible cases. no myocardial infarctions were recorded. in seasons one and two, eight and 16 cases, respectively, were hospitalized. each time that a case was identified, we randomly selected four controls from the remainder of that season's cohort, matched by age (in the same 5-year age category) and sex. of the 1,024 controls selected from the database, 50 were excluded because either no data were available for them or the baseline diagnosis was unclear or they had died or had been lost to follow-up before the relevant epidemics occurred. in the netherlands, almost all persons receive the influenza vaccine through a primary care vaccination program (17) . in both seasons, the composition of the trivalent subunit influenza vaccine complied with world health organization recommendations and matched well with circulating influenza a and b strains, as quantified by high hemagglutinin inhibition titer in ferret sera (15, 16) . a person was assumed to have been vaccinated if his or her general practitioner retrospectively confirmed receipt of influenza vaccination by reviewing the medical records. confirmed exposure/nonexposure to influenza vaccination within the 2 months before either epidemic was in high agreement with the absence/presence of the international classification of primary care r44.1 code for vaccination (kappa = 0.93). baseline demographic information, including age, sex, and health insurance coverage (private or national health service), was collected by using the software module (20) . these data are required by health insurance companies and are therefore valid and reliable. further detailed information was obtained on potential risk factors by review of medical records by the participating general practitioners, who were unaware of the role of these covariate assessments in relation to the primary aim of the study. we extracted information on the presence of concomitant high-risk disease and previous hospital admissions in the 12 months preceding the epidemic. in addition, influenza infection and influenza vaccination status in the previous season and chronic use of medications were registered, and the numbers of consultations in the preceding year were counted as an indicator of disease severity and use of medical services. some of the cases and controls in the second, 1999-2000 season (608 of 1,012) participated in an additional questionnaire study (unpublished data). kappa values, as a measure of agreement between patient and general practitioner information, were satisfactory for some important variables: 0.64 for the presence or absence of chronic comorbid disease, 0.54 for the presence or absence of respiratory medication use, and 0.54 for the presence or absence of previous influenza vaccination. six primary care centers that included 23 trained general practitioners from the utrecht academic network (6) were asked to take nose and throat swabs from their cases and from a sample of controls for virologic assessment. specimens were put into 4 ml of transport medium. swabs were vortexed for 10 seconds and were centrifuged at 2,000 × g for 15 minutes. one ml of the supernatant was used directly for rapid virus culturing and antigen testing by immunofluorescence with monoclonal antibodies against influenza virus. the other material was stored at -70°c. nested, reverse transcriptase polymerase chain reaction was carried out blindly to test for the presence of influenza a or b virus; respiratory syncytial virus; picornaviruses (rhinovirus and enterovirus); parainfluenza viruses 1, 2, and 3; and coronavirus (22) . before starting the study, we estimated that a seasonal study population of 186 cases and 744 controls would give us a statistical power of more than 80 percent to detect an odds ratio of 0.6 (i.e., reduction of 40 percent allowing for nonspecificity, as observed in other studies) (3) (4) (5) . we assumed a vaccination rate of 75 percent, a case-control ratio of 1:4, and a two-tailed α level of 0.05. we approached data analysis in two ways. first, we applied multivariate conditional logistic regression analysis for matched case-control studies to assess vaccine effectiveness independent of confounding factors. in the modeling procedure, factors that appeared to be strongly associated with both exposure to vaccination and case status were first added to the naive model that included vaccination status only. additionally, those risk factors that substantially altered the odds ratio of vaccine effectiveness further (>5 percent) were entered in the model (23) . although it has been shown that vaccine uptake is determined by patient rather than practice or physician factors (24), we extended the analysis by matching by practice, which did not materially change the results. since circulating viruses and vaccination components differed in the two seasons and only a minority of subjects were admitted to the study during both seasons, we pooled the observations and performed similar analyses on case and control person-periods (25) . moreover, we decided in advance to use statistical interaction terms to determine potential modification of vaccine effectiveness by age (18-39, 40-64 years), sex, disease (asthma or copd), and care by a pulmonologist. adjusted odds ratios, as approximations of relative risks, and their 95 percent confidence intervals were calculated. second, we applied the propensity score method, a recently introduced, powerful method of further removing "confounding by indication" (26, 27) . this technique enables assessment of the association of an intervention, that is, vaccination, with outcomes in patients who have an equal probability of receiving the vaccine. potential predictors were included in a logistic regression analysis, with vaccination as the dependent variable. the analysis was used to estimate the probability of vaccination (propensity score) for each individual patient in the full data set (256 cases, 976 controls). the fit of the model that included age and sex, health insurance, underlying disease, use of prednisolone and inhaled corticosteroids, specialist care, and cardiac and other comorbidity was appropriate (hosmer-lemeshow goodness-of-fit test: p = 0.41), and the model's discriminative ability was moderate to good, with a value of 0.71 (95 percent confidence interval (ci): 0.68, 0.75) for the area under the receiver operating curve. in a patientmatching procedure, we searched for a vaccinated person who had a propensity score closest (within a range of 0.00-0.01) to that for each unvaccinated patient. thus, in this quasi-experiment, two comparison groups that had an equal probability of vaccination were formed, and, in an analogy to the analysis of trials, cumulative incidences of complications were compared. the overall cumulative incidence of complicationsmainly respiratory-was 13 per 1,000 in the first season and 34 per 1,000 in the second season (table 1) . influenza morbidity was highest in the older age group (45-64 years), in females, and in those subjects who had copd. vaccinated subjects were older and had a higher prevalence of copd and of cardiac and other comorbidity, and they were more often insured through the national health service than were unvaccinated subjects (table 2). in addition, vaccinated subjects had higher general practitioner consultation and hospitalization rates in the 12 months preceding baseline and had more often been vaccinated against influenza in the previous season. eighty-seven percent of cases and 74 percent of controls had been vaccinated in season one compared with 85 percent of cases and 75 percent of controls in season two (table 3) . after we adjusted for the matching variables age and sex and for potential confounders, we found that the vaccine apparently was not associated with any reduction in the incidence of complications (season one: odds ratio = 0.95, 95 percent ci: 0.26, 3.48; season two: odds ratio = 1.07, 95 percent ci: 0.59, 1.96; pooled odds ratio = 1.07, 95 percent ci: 0.63, 1.80). in addition, vaccine effectiveness was not significantly modified by age, sex, or underlying pulmonary disease or by care received from a pulmonologist. in the propensity score analysis, outcome rates for the 257 vaccinated and 257 unvaccinated subjects who had been matched on their equal probability of being vaccinated were equal (relative risk = 1.03, 95 percent ci: 0.66, 1.62; refer to table 4). assessment for the presence of influenza viruses in a sample of cases and controls (refer to the materials and methods section) showed that, in seasons one and two, 10/22 cases (46 percent) and 11/20 cases (55 percent), respectively, were positive for either influenza a or b, whereas only one control had an influenza infection (table 5) . other respiratory viruses were found relatively infrequently in the cases. this study showed that, although influenza-associated respiratory morbidity is common among working-age patients who have asthma or copd, no evidence exists that the annual, conventional, inactivated trivalent subunit influenza vaccine reduces the incidence rate of these complications. since many immunization guidelines recommend influenza vaccination for patients with asthma or copd, vaccine effectiveness cannot be assessed in a placebocontrolled trial. the case-control approach enables assessment of the effects of vaccination on severe endpoints for which incidence is relatively low. an advantage of the nested case-control study includes reduction of bias due to inappropriate selection of controls. exposure rates in controls were similar in both seasons and were comparable with those in the baseline cohort. although the controls were somewhat older than the total cohort, the distribution of some important characteristics in vaccinated and unvaccinated controls was comparable with that in the baseline cohort. furthermore, potential recall bias was minimized by using computerized medical records. several potential limitations of our study need to be considered. a major issue in nonexperimental evaluation of vaccines is often that vaccinated and unvaccinated patients are not prognostically comparable. as expected, and as shown in the present and previous studies, vaccinees have more risk factors than nonvaccinees (4-6, 25, 28) . this fact may have obscured a positive effect of vaccination. however, we minimized this so-called confounding by indication in both the design and data-analysis phases of the study (23) . first, we admitted into the study cohorts only those patients who had current asthma or copd. recent studies have shown that only in a few patients registered as having asthma or copd were the diagnoses not confirmed by spirometry (29, 30) . second, since age and sex are major confounders, we matched cases and controls for these factors. in addition, matched analysis by general practice did not change our results. third, we had information on many potential confounders, and we adjusted for them by using conditional logistic regression. once we had controlled for the matching factors and just three additional risk factors (previous vaccination, specialist care, and prednisolone use in the previous year), further adjustment for eight additional risk factors did not alter the estimates of vaccine effectiveness. finally, we applied the propensity score method as an effective technique to control for confounding by indication (26, 27) . although the statistical power of the latter approach was more limited, risk factors were apparently distributed similarly in the selected vaccinated and unvaccinated subjects, and no difference was found in the incidence of outcomes. obviously, only a large, randomized controlled trial will guarantee absence of confounding, but it is very unlikely that the observed lack of vaccine effectiveness in our nonexperimental study could be explained by residual confounding in our data. most studies of the effectiveness of vaccination in the elderly have been restricted to even more severe endpoints such as death or hospitalization for influenza or pneumonia, assuming that, during influenza outbreaks, the influenza is frequently a causal component of these outcomes (31, 32) . however, from a societal point of view, the influenza-related needs for health care of patients of working-age are mainly limited to relatively less severe complications treated in primary care settings or at outpatient clinics (refer to the appendix). for example, rothbarth et al. (14) estimated that, in the netherlands, 11 excess deaths occur in this group of half a million persons during influenza epidemics. in other words, if the vaccine could prevent 50 percent of the deaths (5), more than 100,000 patients would need to be vaccinated to prevent one death. a major strength of our study is that virologic analyses of a sample of our cases and controls showed that influenza infection was frequently associated with these complications, and we found much higher prevalences than those reported in earlier influenza studies in this age group (10, 11) . furthermore, in season one, which was predominated by influenza b types, most of the positive cases had influenza a infection. this finding accords with ours and findings from others that the incidence of cases was much lower in this season compared with the influenza a season. we were not able to verify retrospectively whether case ascertainment was complete. however, occurrence rates of pneumonia, acute cardiac disease, and death in the 1999-2000 influenza a season were comparable with data from a previous study in a smaller group of similar patients followed up during the 1995-1996 influenza a epidemic (6) . although a positive relation between respiratory virus infections and exacerbations of asthma has been well established, the etiologic role of influenza viruses has long been underestimated. the underestimated role of influenza might mainly be due to the laboratory techniques used to detect these viruses; in recent years, polymerase chain reaction has become available for rapid diagnosis of influenza infection, considerably increasing diagnostic accuracy compared with conventional virologic analysis (22, 33) . this study is one of the largest so far reported, and it covered two types of influenza outbreaks. although we had limited power to detect a clinically important reduction of at least 40 percent in the first season, in the second season, and in pooled data from the two seasons combined, including 256 case person-periods and 976 control person-periods provided enough power to estimate an even smaller reduction of 35 percent. since more than 500,000 working-age patients with asthma or copd are currently indicated for influenza vaccination at a cost of 7 million euros (approximately $13 million) annually, we are convinced that, from a cost-effectiveness point of view, lower than 35 percent reductions in severe outcomes as a result of vaccination do not justify such a costly, large-scale campaign. other less severe endpoints such as productivity loss or minor exacerbations might be reduced by vaccination, although it is unlikely in light of our results. however, from a public health perspective, such possible benefits might also not justify this preventive program. our finding of a lack of benefit from influenza vaccination in respiratory patients of working-age corroborates some earlier observations. for example, paul et al. (34) observed no reduction in acute respiratory illness in a small subset of vaccinated high-risk patients less than 65 years of age during the 1985-1986 influenza epidemic. stenius-aarniala et al. (35) also found no protective effect of the vaccine in reducing asthma exacerbations in a randomized controlled trial among asthmatics, although influenza activity during the follow-up period was low. wiselka et al. (36) conducted a general practitioner-based study among more than 500 adult asthmatics and found that influenza vaccination was not associated with any substantial reduction in either asthma exacerbations or severity of symptoms. these observations seem counterintuitive in the face of the beneficial effects of conventional influenza vaccination in high-risk children and the elderly, and they do not support international recommendations to immunize working-age patients with asthma or copd against influenza (18) . although the occurrence of endpoints was twice as high in copd patients compared with asthmatics in our study, the vaccine did not reduce the incidence of endpoints in either group. it is still unclear why the vaccine is clinically not effective in both patient groups less than age 65 years. one possible explanation could be that virus-induced allergy and hyperreactivity as precipitating factors may be a much more significant pathologic mechanism in adults than in young children and the elderly (11, 37, 38) . if this explanation is true, preventive measures other than vaccination against influenza, such as self-management programs aimed at reducing the number and severity of exacerbations of asthma or copd, may have a larger impact on the influenza-related health burden in this particular group of high-risk patients than does annual influenza vaccination. the netherlands asthma foundation financially supported this study (no. 97.51). survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston impact of respiratory virus infections on persons with chronic underlying conditions reduction in mortality associated with influenza vaccine during 1989-90 epidemic influenza vaccine effectiveness in preventing hospitalization among the elderly during influenza type a and type b seasons relation between influenza vaccination and outpatient visits, hospitalization, and mortality in elderly persons with chronic lung disease is immunising all patients with chronic lung disease in the community against influenza cost-effective? evidence from a general practice based clinical prospective cohort study in utrecht, the netherlands influenza-associated morbidity and mortality in young and middle-aged women excess pneumonia-and influenza-associated hospitalization during influenza epidemics in the united states, 1970-78 acute respiratory disease hospitalizations as a measure of impact of epidemic influenza respiratory viruses and exacerbations of asthma in adults viral respiratory infection and exacerbations of asthma in adult patients the safety of inactivated influenza vaccine in adults and children with asthma vaccines for preventing influenza in people with asthma sense and nonsense of influenza vaccination in asthma and chronic obstructive pulmonary disease influenza season 1998/99; composition of vaccine for influenza season implementing dutch college of general practitioners (nhg) guidelines for influenza vaccination: an intervention study prevention and control of influenza: recommendations of the influenza vaccination and asthma or copd 699 centers for disease control and prevention the introduction of computer-based patient records in the netherlands improving influenza vaccination coverage among high-risk patients: a role for computer-supported prevention strategy? nhg standaard copd en astma bij volwassenen: diagnostiek simultaneous detection of influenza viruses a and b using real-time quantitative pcr confounding by indication in non-experimental evaluation of vaccine effectiveness: the example of prevention of influenza complications introducing a pneumococcal vaccine to an existing influenza immunization program: vaccination rates and predictors of non-compliance benefits of influenza vaccination for low-, intermediate-and high-risk senior citizens estimating causal effects from large data sets using propensity scores the effectiveness of right heart catherization in the initial care of critically ill patients use of the case-control approach in vaccine evaluation: efficacy and adverse effects spirometrie in een gezondheidscentrum. (in dutch) underdiagnosis of asthma: is the doctor or the patient to blame? the dimca project the impact of influenza epidemics on hospitalizations the efficacy and cost-effectiveness of vaccination against influenza among elderly persons living in the community rapid virological surveillance of community influenza infection in general practice acute respiratory illness among immunized and nonimmunized patients with high-risk factors during a split season of influenza a and b lack of clinical exacerbations in adults with chronic asthma after immunization with killed influenza virus influenza and asthma respiratory infection with influenza a virus interferes with the induction of tolerance to aeroallergens the incidence of respiratory tract infection in adults requiring hospitalization for asthma the participation of the general practitioners in data collection is gratefully acknowledged. the authors are indebted to f. leffers for technical assistance. they also thank dr. m. nijhuis and l. van elden for the virologic analyses. working-age patients with asthma or chronic obstructive pulmonary disease, the netherlands, 1998-1999 and 1999-2000 * copd, chronic obstructive pulmonary disease; fev 1 , forced expiratory volume in 1 second; pef, peak expiratory flow; ecg, electrocardiogram. cardiac illness death key: cord-002852-m4l2l2r1 authors: munyua, peninah m.; githinji, jane w.; waiboci, lilian w.; njagi, leonard m.; arunga, geoffrey; mwasi, lydia; murithi mbabu, r.; macharia, joseph m.; breiman, robert f.; kariuki njenga, m.; katz, mark a. title: detection of influenza a virus in live bird markets in kenya, 2009–2011 date: 2012-04-19 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2012.00365.x sha: doc_id: 2852 cord_uid: m4l2l2r1 please cite this paper as: munyua et al. (2013) detection of influenza a virus in live bird markets in kenya, 2009–2011. influenza and other respiratory viruses 7(2), 113–119. background surveillance for influenza viruses within live bird markets (lbms) has been recognized as an effective tool for detecting circulating avian influenza viruses (aivs). in sub‐saharan africa, limited data exist on aivs in animal hosts, and in kenya the presence of influenza virus in animal hosts has not been described. objectives this surveillance project aimed to detect influenza a virus in poultry traded in five lbms in kenya. methods we visited each market monthly and collected oropharyngeal and cloacal specimens from poultry and environmental specimens for virological testing for influenza a by real time rt‐pcr. on each visit, we collected information on the number and types of birds in each market, health status of the birds, and market practices. results during march 24, 2009–february 28, 2011, we collected 5221 cloacal and oropharyngeal swabs. of the 5199 (99·6%) specimens tested, influenza a virus was detected in 42 (0·8%), including 35/4166 (0·8%) specimens from chickens, 3/381 (0·8%) from turkeys, and 4/335 (1·2%) from geese. none of the 317 duck specimens were positive. influenza was more commonly detected in oropharyngeal [33 (1·3%)] than in cloacal [9 (0·4%)] specimens. none of the 485 environmental specimens were positive. virus was detected in all five markets during most (14/22) of the months. ducks and geese were kept longer at the market (median 30 days) than chickens (median 2 days). conclusions influenza a was detected in a small percentage of poultry traded in lbms in kenya. efforts should be made to promote practices that could limit the maintenance and transmission of aivs in lbms. influenza a viruses are zoonotic pathogens that infect a variety of domestic poultry such as chickens, turkeys, ducks, and geese. [1] [2] [3] from the mid-1970s, investigations have revealed reservoirs of influenza viruses present in wild bird populations and domestic poultry. 2, 4, 5 surveillance for influenza viruses within live bird markets (lbms) has been recognized as an effective tool for detecting circulating influenza subtypes in the poultry population. 6 live bird markets are ideal sites for virus mixing and transmission because of their nature of congregating birds from various farms coupled with the practices of mixing newly arrived birds with those that have been in the market for extended periods. since the 1970s, influenza viruses have been isolated from birds in lbms in multiple countries. from 7% to 30% of fecal swabs from ducks were positive for circulating h3, h4, h5, h6, h7, and h9 influenza virus subtypes in lbms in taiwan, vietnam, and hong kong in the 1980s before the onset of the h5n1, h7n2, and h9n2 poultry epidemics in southeast asia. 7, 8 influenza viruses have also been detected in various environmental specimens collected in contaminated areas in lbms including drinking water troughs, and surfaces in the delivery, holding and slaughter areas in markets. 9, 10 in a study in hong kong, influenza a (h9n2) was isolated in 1% of fecal swabs and 7% of drinking water samples collected in eight live poultry markets. 11 most (56%) of lbms in indonesia were found to have ‡6 sites contaminated with avian influenza virus (h5n1) by real-time reverse transcription polymerase chain reaction (rt rt-pcr) with slaughter and sale areas being the most heavily contaminated. 9 although avian influenza viruses (aivs) in the poultry population have not been described in kenya, qualitative risk assessment studies carried out in 2007-2008 following the 2005 threat of introduction of highly pathogenic avian influenza (hpai) h5n1 in the country suggested a significant risk of transmission of aivs if the virus were introduced into the poultry population. 12 the risk assessment identified complex marketing chains of poultry involving multiple middle men and markets coupled with unsatisfactory levels of biosecurity along the poultry chain as important factors that could contribute to the spread and transmission of influenza viruses through the poultry population and potentially to the human population. 12 in march 2009, the kenya medical research institute ⁄ us centers for disease control and prevention -kenya (ke-mri ⁄ cdc-k) in collaboration with the kenya department of veterinary services (dvs) initiated surveillance to assess the presence of avian influenza viruses in birds traded in lbms in kenya. additionally, we investigated market practices that could contribute to mixing and transmission of virus within the market. between march 24, 2009 and february 28, 2011, we conducted surveillance in five lbms in kenya: kariokor, burma, and kawangware markets, located within the capital city of nairobi; nyambari market, located 40 km north of the city along a major highway; and nakuru market, located in a major urban center in the rift valley province, about 150 km north of nairobi ( figure 1 ). we did not collect samples for the months of december because the staff were unavailable to visit the markets. we chose these markets because they are among the largest poultry markets in the country, and they trade primarily in chickens. the nyambari market trades in multiple avian species, including turkeys, geese, ducks, and doves. the kariokor market is housed in an enclosed building, while the other four are outdoor markets. in four of the five markets, birds were kept in wire mesh cages, each housing 40 birds during the day and night. in the fifth market, nyambari, birds were not kept in cages during the day but they stayed close to the feeding and watering troughs. at night, the birds at nyambari market were driven to a shelter located 500 m from the market. the five lbms receive poultry from districts across the country. the range of birds sold at the markets included chickens (indigenous chickens, spent layers and broilers), ducks, turkeys, and geese. the markets sell live poultry for restocking to farmers and for slaughter to individual homes and hotels. at kariokor and kawangware markets, poultry slaughter is carried out within the market premises. no slaughtering occurs at the other three markets. each market was visited once a month, and an oropharyngeal (op) and a cloacal (cl) swabs collected from 25 birds on every visit. birds that had stayed the longest in the market were preferentially sampled. for the market where there were multiple species, we sampled from all the poultry species. in the chicken markets where the birds were confined in cages, birds from all of the cages were sampled; on average, 3-5 birds were sampled from each cage. in addition, five environmental specimens were collected by swabbing fecal droppings on the floor of the bird cages during each monthly market visit. plastic-shafted polyester-tipped swabs were used to collect op and cl swabs from birds and to collect environmental specimens. the swabs were each placed in cryovials containing 2 ml of freshly prepared viral transport media (vtm) containing bovine serum albumin and veal infusion broth supplemented with amphotericin b and gentamycin (http://www.who.int/csr/resources/publications/surveillance/ annex8.pdf). specimens were labeled and transported at 4°c to the kemri ⁄ cdc-k laboratory and frozen at )80°c within 24 hours after collection until testing. we administered a standardized questionnaire to the poultry traders during each visit. the questionnaire included questions about the number and types of birds in each market, whether the markets had been cleaned using disinfectants, the presence of rodents and wild birds, the number of days the birds had been in the market, the source of the birds, and the health status of the birds. all specimens were tested by real-time reverse transcription polymerase chain reaction (rt rt-pcr) at the biosafety level 3 kemri ⁄ cdc-k laboratory in kisumu using the cdc protocol for influenza a virus detection. 13 briefly, total rna was isolated from 100 ll of the oropharyngeal specimens using the qiaamp rna extraction kit (qiagen inc, valencia, ca, usa) according to the manufacturer's instructions. total rna was extracted from 100 ll of each cloacal and environmental specimen using the magmax viral rna isolation kit (ambion inc, applied biosystems, foster city, ca, usa) according to the manufacturer's instructions. one step rt rt-pcr was carried out using the agpath-id rt rt-pcr kit (applied biosystems). 13 the rt rt-pcr machine was set to run at 10 minutes at 45°c for reverse transcription, 10 minutes at 95°c to activate the taq polymerase, and a typical 45 cycle pcr with denaturation at 95°c for 15 seconds and annealing ⁄ extension at 55°c for 1 minute. fluorescence was read at the annealing ⁄ extension step. the results were collected as cycle threshold (c t ) values. specimens with c t values of <40ae0 were considered positive. data were entered and stored in an ms access database and analyzed using sas version 9.1 (cary, nc, usa). descriptive statistics on number of birds sold, suppliers, and length of stay of birds were calculated. we used chisquare test for all the bivariate analysis. the average number of chickens present at the market on the day of the monthly visit was 641, 629, 381, and 218 for kawangware, burma, nakuru, and kariokor market, respectively. at the nyambari market, the average number of ducks, geese, and turkeys was 45, 52, and 60, respectively. additionally in nyambari market, doves, rabbits, and guinea fowl were occasionally present for sale and were housed in the same cages. the source of the poultry traded varied greatly for all species and markets. overall, birds were sourced by traders and middlemen from districts across five of the eight provinces in kenya. over half (55%) of the chickens traded in all the markets originated from rift valley province [bomet (33%), baringo (16%), and kericho (5ae5%) district] (figure 1 ). half (51%) of the ducks traded originated from the western province districts of bungoma, busia, kakamega, and malaba, while 16% originated from the neighboring country of uganda. forty-three percent of the geese originated from the rift valley province (nakuru district) and 22% from central province (nyandarua district). a majority (68%) of the turkeys originated from the western province [bungoma (25%), kakamega (17%), malaba (9ae4%), busia (14%), and teso (2ae6%) districts]. the five markets were open for trading for 7 days every week. rodents were reported to be present in kariokor, burma, kawangware, and nakuru markets, and disinfection was rarely carried out in any of the five markets. in all five markets, wild birds were observed mixing and feeding with the poultry. the majority of the birds sampled [2322 (88ae9%)] were supplied to the market traders by middlemen (table 1) . a small percentage (5ae0%) was bought directly from a farm by the traders, and 2ae5% of the poultry were bought from other markets (table 1) . ducks, geese, and turkeys stayed on average 15 times longer than chickens in the market ( table 2 ). over half of the ducks, geese and turkeys had been in the markets for 30 days at the time of sampling. we collected 5221 cloacal and oropharyngeal swabs. of these, 4176 (80%) were from chickens, 321 (6ae1%) from ducks, 382 (7ae3%) from turkeys, and 342 (6ae6%) from geese (table 3) . most (99ae5%) of the specimens were collected from healthy birds, but 0ae5% of samples were collected from clinically sick birds that mainly had diarrhea, difficulty in breathing, and nasal discharges. of the 5199 (99ae6%) specimens tested that could be linked to individual bird data, influenza a virus was detected in 42 (0ae8%). influenza was detected in 35 ⁄ 4166 (0ae8%) chicken op ⁄ cl specimens, 3 ⁄ 381 (0ae8%) turkey op ⁄ cl specimens, and 4 ⁄ 335 (1ae2%) geese op ⁄ cl specimens (table 3) . no virus was detected in 317 duck op ⁄ cl specimens [2ae3% upper limit at 95% confidence level (ci)] ( table 3) . the mean c t value of the 42 specimens that were positive for influenza a by rt rt-pcr was 37ae2 (standard error 0ae29); the median c t value was 38ae1 (range 33ae1-39ae6). test results for 22 (0ae4%) specimens could not be linked to individual bird data and were excluded from further analysis. all of these 22 specimens were negative for influenza a. none of 485 environmental specimens collected and tested for influenza a virus were positive (upper limit 95% ci 0ae8%) ( table 4 ). in total, we collected specimens for 22 months and influenza virus was detected in the poultry in 14 (63ae64%) of detection of influenza a virus in live bird markets ª 2012 blackwell publishing ltd these months. we did not observe any seasonal or monthly differences in influenza detection ( figure 2 ). the median monthly detection rate was 0ae4%; the highest detection rate (4ae1%) was in january 2011. influenza virus was detected in both op and cl specimens in chickens (table 3 ). in turkeys and geese, influenza a was detected in op but not in cl specimens (table 3) . in all species, virus detection was significantly higher in op [33 (1ae2%)] than in cloacal [9 (0ae4%)] specimens (p-value < 0ae01). overall, influenza virus prevalence was highest in geese 4 ⁄ 168 (2ae4%) and lowest in turkeys 3 ⁄ 191 (1ae6%). there was no significant difference in the observed prevalence of influenza among chickens, turkeys, geese, and ducks (p-value 0ae29). all the influenza positive specimens were from healthy birds. influenza a was detected in all the five markets (table 4 ). in the four markets trading primarily in chickens, the detection rate varied from 0ae5% in nakuru to 1ae2% in kariokor. the detection rate in nyambari market, which traded in mixed species (turkeys, geese and ducks), was 0ae7%. there was no significant difference in the influenza detection rate in the five markets during this period. in total, the 35 influenza a-positive specimens in chicken were distributed in 13 administrative districts. ten of 35 (28ae6%) influenza a-positive chicken specimens were from birds sourced from one district (bomet district) where 1 ⁄ 3 of the chickens sold in the four markets originated from. influenza a detection by district was variable and ranged from 0ae3% to 12ae5% for this period. however, there was no significant difference in the influenza a detection rate of the chicken, geese, or turkey specimens by district or source of the birds. we detected influenza a viruses in poultry traded in all five lbms in kenya. the influenza a viral rna was detected in geese, turkeys, and chicken. to our knowledge, this is the first time influenza a rna has been detected in poultry traded in lbms in kenya. in our study, the overall influenza a rna detection rate among the birds sampled was 1ae6%. surveillance studies on influenza viruses have recorded variable prevalence of aivs in poultry traded in lbms in different countries around the world. in a study conducted in korea in 2003, 6% of chicken specimens were positive for aivs, 14 whereas 31% and 6ae1% of duck and geese specimens, respectively, were positive for aivs in vietnamese markets in 2001. 7 in the vietnam study, aivs were not detected from chicken specimens. 7 we did not detect influenza a in any of the duck specimens. this finding was unexpected, because ducks are associated with maintenance of influenza virus in domestic birds. in fact, many studies conducted in lbms and farms reported a higher prevalence of influenza in ducks compared with other poultry species. 7,15-17 it is not clear why influenza a virus was not detected in ducks in the markets in kenya, particularly in light of the fact that the ducks stayed for longer periods of time in the market and were housed in the market together with geese and turkeys. however, all of the ducks sampled were adults of market age, and it is possible that they had already been exposed to influenza viruses early in life and therefore may have developed some immunity to the circulating viruses. screening for anti-influenza a antibodies in these species would have helped to clarify this, but this was beyond the scope of our surveillance project. in our surveillance, in all avian species, influenza a virus was more commonly detected in oropharyngeal specimens than in cloacal specimens by rt rt-pcr. in a study of ai in backyard poultry in mali in 2007, 2ae2% of tracheal swabs and 1ae3% of cloacal swabs tested positive for influenza a by rt rt-pcr. 16 experimental studies in ducks showed that viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of h5n1 influenza virus replication in ducks. 18, 19 likewise, experimentally inoculated geese and chicken shed higher virus titers in oropharyngeal swabs than in cloacal swabs. 20, 21 naturally occurring inhibitors present in cloacal and environmental swabs have been shown to limit the sensitivity of rt rt-pcr in detection of influenza a. 22 to minimize this effect in our study, we used the magmax extraction kit, which has been shown to be more effective in removing inhibitors, though the effect of inhibitors cannot be completely ruled out. 22 in the five markets, we observed several practices that could promote influenza transmission among birds. these included keeping markets open for 7 days a week, limited cleaning and disinfection of the market, mixing of new and old birds, trading multiple poultry species in the same market, and mixing with wild birds. these factors were found to be associated with transmission of low pathogenic avian influenza viruses in markets in north america. 23 environmental sampling, where specimens are collected from contaminated areas of the market, has been suggested as an effective surveillance method for influenza virus circulation. as part of our surveillance, we collected specimens from fecal droppings on the ground in the markets. however, we did not detect influenza a viruses in any of the specimens. one study in hong kong detected aivs in up to 1% of fecal swab specimens. 11 the reason for lack of detection in the environmental specimens is not clear, but we suspect that the high environmental temperatures in kenya may limit the survival of any virus shed in feces by the birds. reduced viability of several aiv subtypes has been shown to be associated with increases in temperature. [24] [25] [26] the presence of rt rt-pcr inhibitors could also have limited influenza a viral rna detection in these samples. 22 our surveillance was subject to certain limitations. we did not carry out subtyping of the influenza a specimens or virus isolation; hence, we are not able to report influenza subtypes from the birds sampled. we used rt rt-pcr for screening of the specimens for influenza a virus to determine positivity. although this method has high sensitivity and specificity for detection of type a influenza matrix gene, we may have missed some infections; in one study, virus isolation in embryonated chicken eggs was found to detect an additional 2ae3% of specimens that were negative by rt rt-pcr. 27 the authors attributed the reduced sensitivity of rt rt-pcr in part to the presence of rt rt-pcr inhibitory substances in the samples and the less volume used in rt rt-pcr assays compared with virus isolation. 27 additionally, virological studies only establish the prevalence of active infections. serology testing would have provided more information about the extent of previous exposure at the farms and markets. however, in our case, we sampled poultry in the market destined for sale, and bleeding of the birds would have been undesirable for the traders. our results show that influenza a viruses circulate regularly in lbms in kenya. continued monitoring of influenza viruses in poultry in lbms would help in detecting new introductions of aivs in the poultry population that would be of public health and socioeconomic significance to the poultry industry in the country. early detection of new potentially dangerous influenza viruses could lead to early application of control measures that could minimize the public health impact of outbreaks of hpai viruses and decrease the impact on the livelihoods along the poultry value chain. position of the us centers for diseases control and prevention. ecology, epidemiology and human health implications of avian influenza viruses: why do we need to share genetic data? evolution and ecology of influenza a viruses influenza viruses: transmission between species avian influenza in birds and mammals pathobiology of avian influenza virus infections in birds and mammals wet markets -a continuing source of severe acute respiratory syndrome and influenza? isolation and characterization of avian influenza viruses, including highly pathogenic h5n1, from poultry in live bird markets in hanoi molecular and biological characteristics of h5 and h7 avian influenza viruses in live-bird markets of the northeastern united states environmental sampling for avian influenza virus a (h5n1) in live-bird markets evaluation of routine depopulation, cleaning, and disinfection procedures in the live bird markets poultry drinking water used for avian influenza surveillance an overview of the poultry sector and status of highly pathogenic avian influenza (hpai) in kenya-background paper collaborative research on pro-poor hpai risk reduction rtpcr (rrtpcr) protocol for detection and characterization of influenza (version avian influenza viruses in korean live poultry markets and their pathogenic potential characterization of low-pathogenic h5 subtype influenza viruses from eurasia: implications for the origin of highly pathogenic h5n1 viruses avian influenza in backyard poultry of the mopti region the influenza virus gene pool in a poultry market in south central china role of domestic ducks in the propagation and biological evolution of highly pathogenic h5n1 influenza viruses in asia are ducks contributing to the endemicity of highly pathogenic h5n1 influenza virus in asia? experimental infection of swans and geese with highly pathogenic avian influenza virus (h5n1) of asian lineage virus shedding and potential for interspecies waterborne transmission of highly pathogenic h5n1 influenza virus in sparrows and chickens removal of realtime reverse transcription polymerase chain reaction (rt-pcr) inhibitors associated with cloacal swab samples and tissues for improved diagnosis of avian influenza virus by rt-pcr description of live poultry markets in the united states and factors associated with repeated presence of h5 ⁄ h7 low-pathogenicity avian influenza virus tenacity of avian influenza viruses the effect of temperature and uv light on infectivity of avian influenza virus (h5n1, thai field strain) in chicken fecal manure avian influenza virus h9n2 survival at different temperatures and phs development of real-time rt-pcr for the detection of avian influenza virus detection of influenza a virus in live bird markets ª the authors wish to express gratitude to the staff of the virology laboratory, central veterinary laboratory, kabete for their tireless participation in sampling of the birds and mr dennis odhiambo of cdc-k ieip laboratory for his excellent technical support during the surveillance, period which made this work a success. the authors thank joshua mott for his helpful advice. addendum p. munyua contributed to the overall design of the study, coordinated the field work, analyzed the data, and wrote the manuscript. l. waiboci and l. mwasi were responsible for all the rna extractions and testing and reviewed the manuscript. j. githinji, l. njagi, r. murithi and j. macharia were responsible for the field work, approval for the surveillance work, and reviewed the manuscript. g. arunga coordinated the data entry, analyzed the data, and reviewed the manuscript. r. breiman, k. njenga, m. katz contributed to the overall design of the study and reviewed the manuscript. the findings and conclusions in this report are those of the authors and do not necessarily represent the official key: cord-006252-cbelsymu authors: gross, peter a. title: current recommendations for the prevention and treatment of influenza in the older population date: 2012-11-18 journal: drugs aging doi: 10.2165/00002512-199101060-00003 sha: doc_id: 6252 cord_uid: cbelsymu influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged 65 years and older and those in long term care. currently only 30% of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine 100 mg/day should be given for 2 weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged 65 years and older and those in long term care. currently only 30% of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine 100 mgjday should be given for 2 weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. if we were more aware of the full impact of influenza on our lives, we would be more concerned about preventing it. in the united states alone more than 10 000 deaths are attributed to influenza virus in an average epidemic year (recommendations of the immunization practices advisory committee 1990). in some epidemics, the death toll has risen to more than 40 odd. the majority of deaths -80 to 90% -occur in persons 65 years of age and older . the estimated economic loss from influenza in the united states is estimated to be more than influenza is a major cause of morbidity and mortality in the elderly. influenza vaccine is recommended for people aged 65 years and older and those in long term care. currently only 30% of high risk persons are vaccinated. vaccination generally stimulates an adequate immune response, is well tolerated and is to be encouraged. prophylactic amantadine 100 mg/day should be given for 2 weeks with influenza vaccine in the aged population when they have not been previously immunised. broad application of these preventive measures would have a significant impact on reducing influenza prevalence in the elderly and other high risk groups. if we were more aware of the full impact of influenza on our lives, we would be more concerned about preventing it. in the united states alone more than 10 000 deaths are attributed to influenza virus in an average epidemic year (recommendations of the immunization practices advisory committee 1990). in some epidemics, the death toll has risen to more than 40 odd. the majority of deaths -80 to 90% -occur in persons 65 years of age and older . the estimated economic loss from influenza in the united states is estimated to be more than $usi billion each year (schoenbaum 1987) . in reality, the cost is probably 3 to 5 times greater. while much has been written on the cost-benefit ratio for influenza vaccine, in persons 65 years of age and above the benefit clearly outweighs the cost. despite the favourable impression gained from economic models, however, individual patients and their physicians do not as a rule perceive the benefit. as a result, only a small percentage of the elderly population are vaccinated each year. what is influenza? is it easy to diagnose? how effective is influenza vaccine? are the side effects a frequent concern? what efforts have been and can be made to improve vaccine acceptance? these are some of the important questions we attempt to answer in this article. i influenza virus was first isolated from humans in the 1930s (ruben 1987) . the clinical syndrome of a typical case of influenza begins with fever, cough and myalgias that last for a few days to a week . the clinical course may be complicated by laryngotracheobronchitis and pneumonia. secondary bacterial pneumonia may occur simultaneously or a few days later. hospitalisation is more likely to occur in individuals who are elderly or have pre-existing cardiac or pulmonary disease. children aged under 12 years taking aspirin during the early phases of infection are more likely to get reye's syndrome. late complications of infection such as fatigue, chronic cough, and small-airway disease may last for weeks. making a diagnosis of influenza virus infection is difficult because the acute respiratory syndromes may be confused with other viral respiratory agents such as parainfluenza viruses, adenoviruses, respiratory syncytial viruses as well as the more common rhinoviruses and coronaviruses. laboratory i educational materials on influenza are available from the centers for disease control [technical information services, center for prevention services, mailstop e06, cdc, atlanta, ga 30333, usa; phone (404) 639-18191 as well as from state and local health departments in the usa. drugs & aging 1 (6) 1991 tests can distinguish among these agents, and rapid, sensitive tests are now available to diagnose influenza virus (reichelderfer et al. 1987) . a specific diagnosis of influenza can be made in a few hours to a few days, rather than in a week or more as was the case several years ago. while influenza virus infection may be difficult to distinguish from other respiratory viruses on clinical grounds, influenza virus is unique among the respiratory viruses for the high mortality associated with it. since the sixteenth century, people have recognised the increased mortality resulting from an influenza epidemic (monto 1987) . excess mortality from influenza has been documented since 1889. on occasion, influenza outbreaks reach pandemic proportions, as occurred in 1918. influenza epidemics occur almost every winter. quantifying the mortality from influenza begins with describing the known seasonal variation in mortality from all causes, as shown in figure 1. to reduce the chance of error a dashed line equal to 1.645 standard deviations (the 'epidemic threshold') is drawn above the known season incidence line. when the mortality rate exceeds the standard deviation, it is considered significant. this so-called excess mortality is invariably associated with an outbreak of influenza. excess deaths, therefore, are considered to be due to epidemics of influenza virus. quantifying morbidity from influenza is more difficult. while excess mortality occurs in the elderly and those with certain chronic diseases, morbidity is generally more common among healthy children and young adults. the severe morbidity associated with influenza, such as hospitalisation, is most common among the elderly and chronically ill. a number of studies have documented the toll influenza exacts among the infirm and the elderly. the most recent large scale study on influenza-related mortality is by barker and mullooly (1980) . in 2 influenza a (h3n2) epidemics, they report that 11 to 13 excess deaths occurred per 100 000 persons. when they examined those aged 432 $us 1 billion each year (schoenbaum 1987) . in reality, the cost is probably 3 to 5 times greater. while much has been written on the cost-benefit ratio for influenza vaccine, in persons 65 years of age and above the benefit clearly outweighs the cost. despite the favourable impression gained from economic models, however, individual patients and their physicians do not as a rule perceive the benefit. as a result, only a small percentage of the elderly population are vaccinated each year. what is influenza? is it easy to diagnose? how effective is influenza vaccine? are the side effects a frequent concern? what efforts have been and can be made to improve vaccine acceptance? these are some of the important questions we attempt to answer in this article.! influenza virus was first isolated from humans in the 1930s (ruben 1987) . the clinical syndrome of a typical case of influenza begins with fever, cough and myalgias that last for a few days to a week . the clinical course may be complicated by laryngotracheobronchitis and pneumonia. secondary bacterial pneumonia may occur simultaneously or a few days later. hospitalisation is more likely to occur in individuals who are elderly or have pre-existing cardiac or pulmonary disease. children aged under 12 years taking aspirin during the early phases of infection are more likely to get reye's syndrome. late complications of infection such as fatigue, chronic cough, and small-airway disease may last for weeks. making a diagnosis of influenza virus infection is difficult because the acute respiratory syndromes may be confused with other viral respiratory agents such as parainfluenza viruses, adenoviruses, respiratory syncytial viruses as well as the more common rhino viruses and coronaviruses. laboratory drugs & aging 1 (6) 1991 tests can distinguish among these agents, and rapid, sensitive tests are now available to diagnose influenza virus (reichelderfer et al. 1987) . a specific diagnosis of influenza can be made in a few hours to a few days, rather than in a week or more as was the case several years ago. while influenza virus infection may be difficult to distinguish from other respiratory viruses on clinical grounds, influenza virus is unique among the respiratory viruses for the high mortality associated with it. since the sixteenth century, people have recognised the increased mortality resulting from an influenza epidemic (monto 1987) . excess mortality from influenza has been documented since 1889. on occasion, influenza outbreaks reach pandemic proportions, as occurred in 1918. influenza epidemics occur almost every winter. quantifying the mortality from influenza begins with describing the known seasonal variation in mortality from all causes, as shown in figure 1. to reduce the chance of error a dashed line equal to 1.645 standard deviations (the 'epidemic threshold') is drawn above the known season incidence line. when the mortality rate exceeds the standard deviation, it is considered significant. this so-called excess mortality is invariably associated with an outbreak of influenza. excess deaths, therefore, are considered to be due to epidemics of influenza virus. quantifying morbidity from influenza is more difficult. while excess mortality occurs in the elderly and those with certain chronic diseases, morbidity is generally more common among healthy children and young adults. the severe morbidity associated with influenza, such as hospitalisation, is most common among the elderly and chronically ill. a number of studies have documented the toll influenza exacts among the infirm and the elderly. the most recent large scale study on influenza-related mortality is by barker and mullooly (1980) . in 2 influenza a (h3n2) epidemics, they report that 11 to 13 excess deaths occurred per 100 000 persons. when they examined those aged 65 years and older, the incidence of excess deaths increased to between 68 and 104 per 100 000. these estimates should in reality be even higher because pneumonia or influenza is often omitted from the diagnoses listed on death certificates . showed that the risk ofhospitalisation for acute respiratory disease (ard) during influenza epidemics was 19.7 per 10 000 persons with high risk conditions for which influenza vaccine is indicated, compared to 9.3 per 10 000 for persons without these conditions. chronic pulmonary disorders followed by chronic cardiac conditions were the most common highrisk conditions. renal failure, diabetes and long term care residence are other risk factors. for persons over 65 years of age with a concomitant chronic pulmonary condition the hospitalisation rate for ard soared to 87.5 per 10 000. the peak of hospitalisations for ard typically followed by one week the peak of influenza virus isolations (perrotta et al. 1985) . the disease severity varies according to the influenza strain and the age ofthe patient. the h3n2 subtype of influenza a causes the most severe ilness, while the hi n 1 subtype of influenza a is associated with the mildest illness (monto et al. 1985) . type b influenza causes an illness intermediate in severity between the 2 a subtypes. a third type of influenza, type c, is rarely responsible for epidemic disease. but why is influenza a problem year after year? becoming infected with each of the 3 strains of influenza does not confer long-lasting immunity; in contrast, becoming infected naturally with, for example, measles does confer long-lasting immunity. why the difference? there is only i strain of measles and its nonsegmented genome preserves its prevention and treatment of influenza 65 years and older, the incidence of excess deaths increased to between 68 and 104 per 100 000. these estimates should in reality be even higher because pneumonia or influenza is often omitted from the diagnoses listed on death certificates . showed that the risk of hospitalisation for acute respiratory disease (ard) during influenza epidemics was 19.7 per 10000 persons with high risk conditions for which influenza vaccine is indicated, compared to 9.3 per 10 000 for persons without these conditions. chronic pulmonary disorders followed by chronic cardiac conditions were the most common highrisk conditions. renal failure, diabetes and long term care residence are other risk factors. for persons over 65 years of age with a concomitant chronic pulmonary condition the hospitalisation rate for ard soared to 87.5 per 10 000. the peak of hospitalisations for ard typically followed by one week the peak of influenza virus isolations (perrotta et al. 1985) . the disease severity varies according to the influenza strain and the age of the patient. the h3n2 subtype of influenza a causes the most severe ilness, while the hi n 1 subtype of influenza a is associated with the mildest illness (monto et al. 1985) . type b influenza causes an illness intermediate in severity between the 2 a subtypes. a third type of influenza, type c, is rarely responsible for epidemic disease. but why is influenza a problem year after year? becoming infected with each of the 3 strains of influenza does not confer long-lasting immunity; in contrast, becoming infected naturally with, for example, measles does confer long-lasting immunity. why the difference? there is only i strain of measles and its nonsegmented genome preserves its singularity. influenza virus, on the other hand, has a segmented genome. the multiple rna segments -there are 8 in all -predispose to the genetic instability of the influenza virus (table i, fig. 2 ). the nucleotide sequences in the rna segments are inherently unstable, and when 2 different strains of the same subtype infect the same host the rna segments can reassort, giving rise to progeny that are different from the 2 parents. the naming of influenza viruses is straightforward. for example, influenza a/shanghai/16/89 (h3n2) is so named because this strain is a type a influenza virus first isolated in shanghai in 1989. it was the sixteenth isolate of the subtype h3n2 detected in that year. haemagglutinin (h) and neuraminidase (n) proteins exist in types a, b, and c influenza virus. but only haemagglutinin and neuraminidase proteins of influenza a are sufficiently diverse to warrant subtype designations. for the type a influenza viruses, 3 haemagglutinins (h\, h2 and h3) and 2 neuraminidases (n \ and n2) have been described. currently, only 2 subtypes of influenza a are circulating and causing disease in humans, and they are the h3n2 and the hin\ subtypes. the genetic changes that occur have been referred to by the terms antigenic drift and antigenic shift. antigenic drift is a minor change that occurs (h3n2) is an example of this . minor changes also occur when antigens are glycosylated and camouflage the previously exposed antigenic site. antigenic shift is a major change that occurs by gene reassortment resulting in a new subtype. the change from the influenza a (h2n2) to the influenza a (h3n2) subtype that occurred in 1968 is such an example. gene reassortment probably occurs when 2 different subtypes coinfect the same host. serum antibody to the surface haemagglutinin and neuraminidase proteins is important in protection against infection and the development of disease. humoral antibody to the haemagglutinin component is the most critical, but the importance of cell-mediated immunity is less clear . the humoral immune response is highly specific for certain epitopes on the surface of the virus. the cellular response, in contrast, is less specific and more cross-reactive. the b cells which produce humoral antibody recognise antigenic determinants, or epitopes, adjacent in space but not in sequence, while t cells recognise epitopes adjacent in sequence but not necessarily in space. the 434 singularity. influenza virus, on the other hand, has a segmented genome. the multiple rna segments -there are 8 in all -predispose to the genetic instability of the influenza virus (table i, fig. 2 ). the nucleotide sequences in the rna segments are inherently unstable, and when 2 different strains of the same subtype infect the same host the rna segments can reassort, giving rise to progeny that are different from the 2 parents. the naming of influenza viruses is straightforward. for example, influenza a/shanghai/16/89 (h3n2) is so named because this strain is a type a influenza virus first isolated in shanghai in 1989. it was the sixteenth isolate of the subtype h3n2 detected in that year. haemagglutinin (h) and neuraminidase (n) proteins exist in types a, b, and c influenza virus. but only haemagglutinin and neuraminidase proteins of influenza a are sufficiently diverse to warrant subtype designations. for the type a influenza viruses, 3 haemagglutinins (hi, h2 and h3) and 2 neuraminidases (n i and n2) have been described. currently, only 2 subtypes of influenza a are circulating and causing disease in humans, and they are the h3n2 and the hin i subtypes. the genetic changes that occur have been referred to by the terms antigenic drift and antigenic shift. antigenic drift is a minor change that occurs . minor changes also occur when antigens are glycosylated and camouflage the previously exposed antigenic site. antigenic shift is a major change that occurs by gene reassortment resulting in a new subtype. the change from the influenza a (h2n2) to the influenza a (h3n2) subtype that occurred in 1968 is such an example. gene reassortment probably occurs when 2 different subtypes coinfect the same host. serum antibody to the surface haemagglutinin and neuraminidase proteins is important in protection against infection and the development of disease. humoral antibody to the haemagglutinin component is the most critical, but the importance of cell-mediated immunity is less clear . the humoral immune response is highly specific for certain epitopes on the surface of the virus. the cellular response, in contrast, is less specific and more cross-reactive. the b cells which produce humoral antibody recognise antigenic determinants, or epitopes, adjacent in space but not in sequence, while t cells recognise epitopes adjacent in sequence but not necessarily in space. the implication here is that b cell epitopes are intact external molecules such as haemagglutinins, while t cell epitopes appear to be linear sequences exposed by enzymatic breakdown of the original molecule. a killed (inactivated) vaccine against influenza virus was first developed in the 1940s. since then the manufacturing process has resulted in a more pure vaccine. the virus is grown in embryonated eggs. most of the egg protein is now eliminated and the side effects have been reduced markedly. killed vaccines are referred to as either whole virus or split virus: whole virus vaccines are inactivated by formalin; split or subunit vaccines use whole virus which is treated with a detergent to free up the haemagglutinin and neuraminidase subunits from the viral surface, then formalin is added. in children who have not previously been vaccinated, split virus vaccines cause fewer side effects such as fever and myalgias than whole virus vaccines. live attenuated vaccines have been in the development stages for over 10 years and are currently being tested; they are not commercially available. a genetically stable method of production was the initial problem but this has since been solved. however, it has been difficult to show a clear advantage of live over killed vaccines. the currently licensed influenza vaccine contains 3 strains: influenza a/shanghai/i 6/89 (h3n2); influenza a/taiwan/i/86 (hinl); and influenza b/ yamagata/i 6/88. there are 15ilg of haemagglutinin for each strain in the vaccine. it should be given intramuscularly in the deltoid in adults and in the anterolateral thigh in infants and young children. administration in the buttock is not recommended because it may result in a poorer immune response due to inadequate absorption from the subcutaneous fat. the strains to be included in the vaccine change from year to year according to world health organization (who) recommendations. 435 the primary target groups are as follows (recommendations of the immunization practices advisory committee 1990). i. persons aged 65 years or older. 2. residents of nursing homes and other chronic-care facilities housing persons of any age with chronic medical conditions. of the pulmonary or cardiovascular systems, including children with asthma. 4. adults and children who have required regular medical follow-up or hospitalisation during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, haemoglobinopathies, or immunosuppression (including immunosuppression caused by medications). i. physicians, nurses and other personnel in both hospital and outpatient-care settings who have contact with high-risk persons in all age groups, including infants. 2. employees of nursing homes and chronic-care facilities who have contact with patients or residents. 3. providers of home care to high risk persons (e.g. visiting nurses, volunteer workers). other groups merit consideration (recommendations of the immunization practices advisory committee 1990): (a) in the general population, anyone who wishes to reduce the chance of acquiring influenza infection; (b) anyone who provides essential community services and persons living or working in an institutional setting; (c) persons infected with hiv should be vaccinated, although they may not respond as well if their disease is far advanced; and (d) foreign travellers should be immunised when travelling to the trop-prevention and treatment of influenza implication here is that b cell epitopes are intact external molecules such as haemagglutinins, while t cell epitopes appear to be linear sequences exposed by enzymatic breakdown of the original molecule. a killed (inactivated) vaccine against influenza virus was first developed in the 1940s. since then the manufacturing process has resulted in a more pure vaccine. the virus is grown in embryonated eggs. most of the egg protein is now eliminated and the side effects have been reduced markedly. killed vaccines are referred to as either whole virus or split virus: whole virus vaccines are inactivated by formalin; split or subunit vaccines use whole virus which is treated with a detergent to free up the haemagglutinin and neuraminidase subunits from the viral surface, then formalin is added. in children who have not previously been vaccinated, split virus vaccines cause fewer side effects such as fever and myalgias than whole virus vaccines. live attenuated vaccines have been in the development stages for over 10 years and are currently being tested; they are not commercially available. a genetically stable method of production was the initial problem but this has since been solved. however, it has been difficult to show a clear advantage of live over killed vaccines. the currently licensed influenza vaccine contains 3 strains: influenza a/shanghai/i 6/89 (h3n2); influenza a/taiwan/i/86 (hin j); and influenza b/ yamagata/i 6/88. there are 15j.lg of haemagglutinin for each strain in the vaccine. it should be given intramuscularly in the deltoid in adults and in the anterolateral thigh in infants and young children. administration in the buttock is not recommended because it may result in a poorer immune response due to inadequate absorption from the subcutaneous fat. the strains to be included in the vaccine change from year to year according to world health organization (who) recommendations. 435 the primary target groups are as follows (recommendations of the immunization practices advisory committee 1990). i. persons aged 65 years or older. 2. residents of nursing homes and other chronic-care facilities housing persons of any age with chronic medical conditions. 3. adults and children with chronic disorders of the pulmonary or cardiovascular systems, including children with asthma. 4. adults and children who have required regular medical follow-up or hospitalisation during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, haemoglobinopathies, or immunosuppression (including immunosuppression caused by medications). i. physicians, nurses and other personnel in both hospital and outpatient-care settings who have contact with high-risk persons in all age groups, including infants. 2. employees of nursing homes and chronic-care facilities who have contact with patients or residents. 3. providers of home care to high risk persons (e.g. visiting nurses, volunteer workers). other groups merit consideration (recommendations of the immunization practices advisory committee 1990): (a) in the general population, anyone who wishes to reduce the chance of acquiring influenza infection; (b) anyone who provides essential community services and persons living or working in an institutional setting; (c) persons infected with hiv should be vaccinated, although they may not respond as well if their disease is far advanced; and (d) foreign travellers should be immunised when travelling to the trop-ics at any time of the year or from the southern to the northern hemisphere between august and march and the northern to the southern hemisphere between april and september. the vaccine is contraindicated in persons with anaphylaxis to eggs and in those with an acute febrile illness. a protocol is available for giving vaccine to allergic patients at high risk of influenza complications (recommendations of the immunization practices advisory committee 1990). other vaccines can be given along with influenza vaccine, such as pneumococcal, measlesmumps-rubella, haemophilus injluenzae type b, and oral polio vaccines. pertussis vaccine, however, should not be given within 3 days of influenza virus vaccine. for optimal effect, the vaccine should be given in november in the northern hemisphere or may in the southern hemisphere, before the winter influenza season begins. effective antibody levels develop within 2 weeks in most previously immunised persons. the levels decline a few months later, when they may be suboptimal. studies investigating vaccine efficacy are difficult to conduct and to evaluate. they are hard to conduct because randomisation to control and vaccinated groups raises ethical questions in high risk patients. evaluation is difficult because other respiratory infectious agents also cause colds, pneumonia, occasionally death, and often result in hospitalisation -the criteria used to determine efficacy. vaccine immunogenicity and protective efficacy in the elderly have had mixed reviews. immunogenicity, the vaccine's ability to stimulate protective levels of antibody, appears to be adequate in many studies. protective levels of serum antibody are considered to be equal to or greater than the reciprocal of serum dilutions at 1 : 32 or 1 : 40, depending on the dilution method. protective levels are usually achieved in 70 to 90% of vaccine recipients. reviewed 17 papers on this subject published between 1968 and 1988. a total drugs & aging 1 (6) 1991 of 30 comparisons were available in the 17 papers. in 10, the immune response was superior in young subjects; in 4, the elderly responded better, and in 16, no significant difference was noted between the young and old subjects; in this instance, the lack of differences may have been due to a type 2 error because the numbers studied were low. pointed out 3 methodological limitations. the studies included, firstly, subjects with illnesses or taking drugs, which affect the immune system; secondly, patients immunised previously with influenza vaccine; and finally, those with protective antibody levels before immunisation. inclusion of patients with any of these 3 factors would tend to underestimate the ability of elderly subjects to respond to immunisation. the association between increasing age and a poor immune response to influenza vaccine has thus not yet been convincingly made. perhaps a small subgroup of the elderly population respond poorly while most respond normally. this was originally suggested in a study by phair et al. (1978) examining the t cell response of infirm elderly to influenza antigens. more recently, in a study by , bedridden, infirm elderly patients appeared to respond less well to influenza vaccine than did healthy, ambulatory elderly patients. alternative approaches to the standard immunisation have been tried. two doses 1 month apart did not improve the immune response , neither did 2 and 3 times the standard dose . from currently available information we can conclude that the recommended single standard dose of killed influenza vaccine will stimulate an adequate immune response in most elderly persons. finally, and most importantly, in a number of recent studies influenza vaccine has been shown to reduce mortality from influenza virus infection gross et al. 1988a ). recent studies on the adverse effects of influenza vaccine show that the vaccine is typically well 436 ics at any time of the year or from the southern to the northern hemisphere between august and march and the northern to the southern hemisphere between april and september. the vaccine is contraindicated in persons with anaphylaxis to eggs and in those with an acute febrile illness. a protocol is available for giving vaccine to allergic patients at high risk of influenza complications (recommendations of the immunization practices advisory committee 1990). other vaccines can be given along with influenza vaccine, such as pneumococcal, measlesmumps-rubella, haemophilus injluenzae type b, and oral polio vaccines. pertussis vaccine, however, should not be given within 3 days of influenza virus vaccine. for optimal effect, the vaccine should be given in november in the northern hemisphere or may in the southern hemisphere, before the winter influenza season begins. effective antibody levels develop within 2 weeks in most previously immunised persons. the levels decline a few months later, when they may be suboptimal. studies investigating vaccine efficacy are difficult to conduct and to evaluate. they are hard to conduct because randomisation to control and vaccinated groups raises ethical questions in high risk patients. evaluation is difficult because other respiratory infectious agents also cause colds, pneumonia, occasionally death, and often result in hospitalisation -the criteria used to determine efficacy. vaccine immunogenicity and protective efficacy in the elderly have had mixed reviews. immunogenicity, the vaccine's ability to stimulate protective levels of antibody, appears to be adequate in many studies. protective levels of serum antibody are considered to be equal to or greater than the reciprocal of serum dilutions at 1 : 32 or 1 : 40, depending on the dilution method. protective levels are usually achieved in 70 to 90% of vaccine recipients. reviewed 17 papers on this subject published between 1968 and 1988. a total drugs & aging 1 (6) 1991 of 30 comparisons were available in the 17 papers. in 10, the immune response was superior in young subjects; in 4, the elderly responded better, and in 16, no significant difference was noted between the young and old subjects; in this instance, the lack of differences may have been due to a type 2 error because the numbers studied were low. pointed out 3 methodological limitations. the studies included, firstly, subjects with illnesses or taking drugs, which affect the immune system; secondly, patients immunised previously with influenza vaccine; and finally, those with protective antibody levels before immunisation. inclusion of patients with any of these 3 factors would tend to underestimate the ability of elderly subjects to respond to immunisation. the association between increasing age and a poor immune response to influenza vaccine has thus not yet been convincingly made. perhaps a small subgroup of the elderly population respond poorly while most respond normally. this was originally suggested in a study by phair et al. (1978) examining the t cell response of infirm elderly to influenza antigens. more recently, in a study by , bedridden, infirm elderly patients appeared to respond less well to influenza vaccine than did healthy, ambulatory elderly patients. alternative approaches to the standard immunisation have been tried. two doses 1 month apart did not improve the immune response , neither did 2 and 3 times the standard dose . from currently available information we can conclude that the recommended single standard dose of killed influenza vaccine will stimulate an adequate immune response in most elderly persons. finally, and most importantly, in a number of recent studies influenza vaccine has been shown to reduce mortality from influenza virus infection gross et al. 1988a) . recent studies on the adverse effects of influenza vaccine show that the vaccine is typically well tolerated. for example, margolis et a1. (1990a,b) reported that in elderly chronically ill patients fever and significant disability could not be attributed to the vaccine compared to a control group. a flu-like illness, however, was attributable to vaccine in 5.5%. they concluded that the overall frequency of adverse effects was low. another area of concern is the effect of vaccine on drug metabolism. the mechanism postulated is vaccine suppression of oxidative hepatic metabolism. interferon production induced by vaccination is supposed to block the oxidative cytochrome p450 system, resulting in accumulation of certain drugs. metabolism of theophylline, chlordiazepoxide, lorazepam, warfarin, phenytoin and phenobarbital (phenobarbitone) has been studied, with variable results. for theophylline, in particular, most studies seem to indicate that the effect of vaccine is minor (gomolin et a1. 1985; grabowski et a1. 1985; meredith et a1. 1985) . less than 30% of high risk persons are vaccinated annually. consequently many studies have been undertaken to determine the best strategies for immunising appropriate groups. the current strategies for implementing the influenza vaccine recommendations include immunising persons in the following settings (recommendations of the immunization practices advisory committee 1990). 1. in outpatient clinics and physicians' offices label medical records of high risk persons; use mail or telephone reminders; begin vaccinating in september (northern hemisphere) [buchner et a1. 1987] and march (southern hemisphere). 2. in facilities providing episodic or acute care (e.g. emergency rooms, walk-in clinics) healthcare providers be aware of high risk groups; provide written information in appropriate languages. 3. in nursing homes and other residential long term care facilities vaccinate all residents with the prior aggreement of attending physicians; do not require individual physician orders; obtain permission for vaccination on admission to facility. 437 4. in acute-care hospitals encourage vaccination before discharge between september and april (northern hemisphere), and march to august (southern hemisphere) of all persons aged 65 years or over, or with high risk conditions at any age . 5. in outpatient facilities providing continuing care to high risk patients (e.g. haemodialysis centres, hospital speciality care clinics, outpatient rehabilitation programmes); facilities providing services to people 65 years and older (e.g. retirement communities, recreation centres); clinics and other centres providing healthcare for travellers; healthcare workers, visiting nurses and others providing home care to high risk persons provide educational materials; encourage vaccination; emphasise to staff in intensive care units, medical/surgical units, employees in nursing homes and long term care facilities; make vaccine available for all work shifts, including night and weekend. two antiviral agents are effective in the prevention and treatment of influenza type a infections -amantadine and rimantadine. only amantadine is currently available commercially in the united states. it has no effect against influenza type b (douglas 1990). the primary use of amantadine is in preventing influenza a infection. preventive efficacy varies between 50 and over 90%. when influenza a is in the community, an unvaccinated elderly person should receive vaccine plus 2 weeks of amantadine at a dose of 100 mg/day (nicholson & wiselka 1991) . amantadine does not interfere with the immune response to vaccine, and after 2 weeks the vaccine-induced immune response should be sufficient to prevent influenza. amantadine treatment should not be stopped if the high risk person cannot be vaccinated or is immunodeficient. it should be continued for as long as influenza is epidemic -usually about 5 to 6 weeks. amantadine has also been used for the treatment of influenza a. when the drug is started within 48 hours of the onset of illness it produces prevention and treatment of influenza tolerated. for example, margolis et al. (1990a,b) reported that in elderly chronically ill patients fever and significant disability could not be attributed to the vaccine compared to a control group. a flu-like illness, however, was attributable to vaccine in 5.5%. they concluded that the overall frequency of adverse effects was low. another area of concern is the effect of vaccine on drug metabolism. the mechanism postulated is vaccine suppression of oxidative hepatic metabolism. interferon production induced by vaccination is supposed to block the oxidative cytochrome p450 system, resulting in accumulation of certain drugs. metabolism of theophylline, chlordiazepoxide, lorazepam, warfarin, phenytoin and phenobarbital (phenobarbitone) has been studied, with variable results. for theophylline, in particular, most studies seem to indicate that the effect of vaccine is minor . less than 30% of high risk persons are vaccinated annually. consequently many studies have been undertaken to determine the best strategies for immunising appropriate groups. the current strategies for implementing the influenza vaccine recommendations include immunising persons in the following settings (recommendations of the immunization practices advisory committee 1990). 1. in outpatient clinics and physicians' offices label medical records of high risk persons; use mail or telephone reminders; begin vaccinating in september (northern hemisphere) and march (southern hemisphere). 2. in facilities providing episodic or acute care (e.g. emergency rooms, walk-in clinics) healthcare providers be aware of high risk groups; provide written information in appropriate languages. 3. in nursing homes and other residential long term care facilities vaccinate all residents with the prior aggreement of attending physicians; do not require individual physician orders; obtain permission for vaccination on admission to facility. 437 4. in acute-care hospitals encourage vaccination before discharge between september and april (northern hemisphere), and march to august (southern hemisphere) of all persons aged 65 years or over, or with high risk conditions at any age . 5. in outpatient facilities providing continuing care to high risk patients (e.g. haemodialysis centres, hospital speciality care clinics, outpatient rehabilitation programmes); facilities providing services to people 65 years and older (e.g. retirement communities, recreation centres); clinics and other centres providing healthcare for travellers; healthcare workers, visiting nurses and others providing home care to high risk persons provide educational materials; encourage vaccination; emphasise to staff in intensive care units, medical/surgical units, employees in nursing homes and long term care facilities; make vaccine available for all work shifts, including night and weekend. two antiviral agents are effective in the prevention and treatment of influenza type a infections -amantadine and rimantadine. only amantadine is currently available commercially in the united states. it has no effect against influenza type b (douglas 1990). the primary use of amantadine is in preventing influenza a infection. preventive efficacy varies between 50 and over 90%. when influenza a is in the community, an unvaccinated elderly person should receive vaccine plus 2 weeks of amantadine at a dose of 100 mg/day (nicholson & wiselka 1991) . amantadine does not interfere with the immune response to vaccine, and after 2 weeks the vaccine-induced immune response should be sufficient to prevent influenza. amantadine treatment should not be stopped if the high risk person cannot be vaccinated or is immunodeficient. it should be continued for as long as influenza is epidemic -usually about 5 to 6 weeks. amantadine has also been used for the treatment of influenza a. when the drug is started within 48 hours of the onset of illness it produces a therapeutic effect. the duration of illness decreases by 50%, virus shedding decreases more rapidly, and peripheral airway resistance is reduced faster. serum antibody still develops despite the use of amantadine. amantadine treatment for the acute illness should be continued for 5 days. studies on the therapeutic efficacy of amantadine have been done in healthy adults; there are no data on its efficacy in high risk persons (recommendations of the immunization practices advisory committee 1990). resistance of influenza a virus to amantadine has been reported . the adverse effects of amantadine at a dose of 200 mg/day involve the central nervous system (nervousness, anxiety, insomnia, difficulty concentrating and lightheadedness) and the gastrointestinal tract (anorexia and nausea). they usually decrease or stop after the first week of use or when amantadine is discontinued. using a smaller dose, 100 mg/day, reduces the adverse effects, apparently without diminishing efficacy. the manufacturer's recommendations should be read carefully to adjust the dose for age, renal function, weight, and interactions with other drugs and diseases (see table ii) . rimantadine is a structural analogue of amantadine. it has 2 advantages over amantadine: it has fewer central nervous system side effects at doses of 200 mg/day, and, because most of the drug is drugs & aging 1 (6) 1991 metabolised, elimination from the body is not dependent on renal excretion. the use of influenza vaccine in the elderly is no longer an option"to be exercised only occasionally. administration of influenza vaccine and the use of amantadine when indicated should become a standard part of the care given our elderly population (nicholson 1990 ). a therapeutic effect. the duration of illness decreases by 50%, virus shedding decreases more rapidly, and peripheral airway resistance is reduced faster. serum antibody still develops despite the use of amantadine. amantadine treatment for the acute illness should be continued for 5 days. studies on the therapeutic efficacy of amantadine have been done in healthy adults; there are no data on its efficacy in high risk persons (recommendations of the immunization practices advisory committee 1990). resistance of influenza a virus to amantadine has been reported . the adverse effects of amantadine at a dose of 200 mg/day involve the central nervous system (nervousness, anxiety, insomnia, difficulty concentrating and lightheadedness) and the gastrointestinal tract (anorexia and nausea). they usually decrease or stop after the first week of use or when amantadine is discontinued. using a smaller dose, 100 mg/day, reduces the adverse effects, apparently without diminishing efficacy. the manufacturer's recommendations should be read carefully to adjust the dose for age, renal function, weight, and interactions with other drugs and diseases (see table ii) . rimantadine is a structural analogue of amantadine. it has 2 advantages over amantadine: it has fewer central nervous system side effects at doses of 200 mg/day, and, because most of the drug is drugs & aging 1 (6) 1991 metabolised, elimination from the body is not dependent on renal excretion. the use of influenza vaccine in the elderly is no longer an option to be exercised only occasionally. administration ~f influenza vaccine and the use of amantadine when indicated should become a standard part of the care given our elderly population (nicholson 1990) . experiences in the use and efficacy of inactivated influenza vaccine in nursing homes impact of epidemic type a influenza in a defined adult population underestimation of the role of pneumonia and influenza in causing excess mortality effectiveness of inactivated influenza vaccine among non-institutionalized elderly persons resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance antibody induced by influenza vaccines in the elderly: a review of the literature influenza vaccination in community elderly: a controlled trial of postcard reminders clinical manifestations and consequences of influenza antimicrobial agents: antiviral agents immunizations for health care workers and patients in hospitals survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston lack of effect of influenza vaccine on theophylline levels and warfarin anticoagulation in the elderly the effect of split virus influenza vaccination on theophylline pharmacokinetics association of in fluenza immunization with reduction in mortality in an elderly references experiences in the use and efficacy of inactivated influenza vaccine in nursing homes impact of epidemic type a influenza in a defined adult population underestimation of the role of pneumonia and influenza in causing excess mortality effectiveness of inactivated influenza vaccine among non-institutionalized elderly persons resistance of influenza a virus to amantadine and rimantadine: results of one decade of surveillance antibody induced by influenza vaccines in the elderly: a review of the literature influenza vaccination in community elderly: a controlled trial of postcard reminders clinical manifestations and consequences of influenza antimicrobial agents: antiviral agents immunizations for health care workers and patients in hospitals survey of underlying conditions of persons hospitalized with acute respiratory disease during influenza epidemics in houston lack of effect of influenza vaccine on theophylline levels and warfarin anticoagulation in the elderly the effect of split virus influenza vaccination on theophylline pharmacokinetics association of in fluenza immunization with reduction in mortality in an elderly population: a prospective study immunization of elderly people with high doses of influenza vaccine relation of chronic disease and immune response to influenza vaccine in the elderly immunization of elderly people with two doses of influenza vaccine effect of influenza vaccine on serum anticonvulsant concentrations epidemiologic implications of changes in the influenza virus genome t-cell recognition of influenza viral antigens impact of influenza epidemic on mortality in the united states from frequency of adverse reactions to influenza vaccine in the elderly frequency of adverse reactions after influenza vaccine effects of influenza virus vaccine on hepatic drug metabolism infleunza: quantifying morbidity and mortality tecumsah study of illness. xiii: influenza infection and disease influenza vaccination and the elderly amantadine for influenza a acute respiratory disease hospitalizations as'll.ijleasure of impact of epidemic influenza failure to respond to influenza vaccine in the aged: correlation with b-cell number and function prevention and control of influenza. and addendum clarification economical laboratory support system for influenza virus surveillance prevention and control of influenza: role of vaccine economic impact of influenza: the individual's perspective prevention and treatment of influenza population: a prospective study immunization of elderly people with high doses of influenza vaccine relation of chronic disease and immune response to influenza vaccine in the elderly immunization of elderly people with two doses of influenza vaccine effect of influenza vaccine on serum anticonvulsant concentrations epidemiologic implications of changes in the influenza virus genome t-cell recognition of influenza viral antigens impact of influenza epidemic on mortality in the united states from frequency of adverse reactions to influenza vaccine in the elderly frequency of adverse reactions after influenza vaccine effects of influenza virus vaccine on hepatic drug metabolism key: cord-103085-vf4qyvft authors: seitz, christian; casalino, lorenzo; konecny, robert; huber, gary; amaro, rommie e.; mccammon, j. andrew title: multiscale simulations examining glycan shield effects on drug binding to influenza neuraminidase date: 2020-11-02 journal: biorxiv doi: 10.1101/2020.08.12.248690 sha: doc_id: 103085 cord_uid: vf4qyvft influenza neuraminidase is an important drug target. glycans are present on neuraminidase, and are generally considered to inhibit antibody binding via their glycan shield. in this work we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. we created all-atom in silico systems of influenza neuraminidase with experimentally-derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. using brownian dynamics simulations, we observe a twoto eight-fold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. these glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. our work also indicates that drugs preferentially bind to the primary binding site (i.e. the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. these results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase. statement of significance the influenza glycoprotein neuraminidase is the target for three fda-approved influenza drugs in the us. however, drug resistance and low drug effectiveness merits further drug development towards neuraminidase, which is hindered by our limited understanding of glycan effects on ligand binding. generally, drug developers do not include glycans in their development pipelines. here, we show that even though glycans can reduce drug binding towards neuraminidase, we recommend future drug development work to focus on strong binders with a long lifetime. furthermore, we examine the binding competition between the primary and secondary binding sites on neuraminidase, leading us to propose a new, to the best of our knowledge, multivalent binding mechanism. it has been long appreciated that glycans on influenza membrane proteins help shield the virus from the host immune system's antibodies (1) (2) (3) (4) (5) (6) (7) . unrecognized glycosylation differences can also attenuate influenza vaccines (8) . in one study, glycans were shown to reduce epitope accessibility and drug binding to receptor proteins (9) . glycans can clearly influence antibody binding due to their presence in the antibody binding site. however, it remains to be seen whether this glycan shielding and glycoprofile variability is also a concern for influenza drugs, recognizing that these drugs are smaller than human antibodies, and the fact that glycans present themselves near, but not directly inside, the catalytic sites. currently there are three fdaapproved influenza neuraminidase (na) antivirals in the us: tamiflu (oseltamivir), relenza (zanamivir) and rapivab (peramivir), all of which have lingering questions over their efficacy, side effects, and drug resistance (10, 11) . this necessitates the need for further drug development against influenza (12) . drug developers have many hurdles to clear when designing a new influenza drug: classical admet characteristics, clinical trials and governmental regulations, among others. what is not often considered is the viral glycosylation state. the glycosylation state is the assemblage of glycans, linkages of sugars found on the surface of about half of all proteins (13) . influenza contains n-linked glycosylation sites, defined by the asn-x-ser/thr sequon, where x can be anything besides proline (14) . this leads to the so-called glycan shield, where glycans on the protein surface are capable of accessing much of the protein's surface area, and potentially shielding it from outside interactions (15) (16) (17) (18) (19) (20) (21) (22) . among their many biological functions, glycans play a crucial, but complex role in viral infection (23) . one salient example of glycan function in influenza is how they help the virus evade the immune system (1) (2) (3) (4) (5) (6) (7) . furthermore, glycans are capable of affecting receptor binding in influenza (5, 7, (24) (25) (26) (27) . traditionally, glycans have been difficult to study due to their flexibility and heterogeneity. most of the glycan characterization studies are done through mass spectrometry, which can yield highly variable glycoprofile data, such as differences in the degree of post-translational modifications, sequon occupancy, and type of glycan, for different strains of influenza (28) (29) (30) (31) (32) (33) . similarly, glycan occupancy levels are not consistent across studies, even when using the same cell line and strain of influenza (34, 35) these discrepancies may arise from differences in system setup, sample preparation, cell culturing and/or analysis method, which increases the difficulty in determining the transferability of experimental glycan results. though not well understood, the number and position of the glycosylation sites on influenza can change over time as a result of antigenic drift (36) (37) (38) (39) . this increases the glycoprofile variability, effectuating irregular but significant changes in the glycan shield over time. considering the variability and immune evasion function of the glycan shield, it remains to be seen what effect this shield has on small-molecule antiviral drug binding to viral surface proteins. previous work has shown that, depending on the viral strain and receptor mimetic used, removing viral glycans can improve binding to cell receptor mimetics (40) (41) (42) . other studies have shown that these viral glycans decrease binding of other cell receptor mimetics (27, (43) (44) (45) (46) . regardless, antiviral drugs will be much smaller than a receptor mimetic, and it is not clear whether this size difference means antiviral drugs will still be affected by the viral glycans. an earlier study by kasson and pande, using 100 ns molecular dynamics (md) simulations, showed reduced binding of α 2-3-sialyllactose trisaccharides to hemagglutinin due to glycans (43) . a recent review concluded that the viral glycosylation state should be considered when designing small molecule antivirals (47) . focusing on how small molecule antivirals are affected by the glycan shield, we combine results from distinct bd and md simulations into an integrated multiscale simulation study. we have utilized bd to estimate the rates of binding of small molecules to the primary (i.e. active/catalytic) and secondary (i.e. hemadsorption) binding sites of influenza neuraminidase in glycosylated and unglycosylated states. we see that the glycan shield is capable of moderately inhibiting drug association to the primary binding site of na on the order of two to eight times. small molecule association is faster to the primary binding site than the secondary binding site. ligand binding is independent between the primary and secondary sites -the presence of one site does not influence binding at the other site. overall, this work provides insights into the impact of glycans on small-molecule binding to na. in this study, we use brownian dynamics (bd), which has been previously used to simulate protein-small molecule association (48) (49) (50) (51) . specifically, it has also been used to simulate the association of small molecules to influenza neuraminidase (52) (53) (54) . bd makes the implicit assumption that long-range electrostatics and stochastic collisions with solvent molecules are the driving forces behind protein-ligand binding (55) . therefore, it is an efficient method to simplify binding to describe electrostatically-influenced diffusion. using bd allows for a reduction in system complexity and a focus on specific modulations of ligand association. to assess whether glycans affect small molecule binding to na, we created an in silico na model using the strain of influenza a virus, a/viet nam/ 1203/2004(h5n1) and tetrameric pdb 2hty, with uniprot id q6dpl2 (56) . building on this structure, we generated five na constructs: (i) one unglycosylated model; (ii) one glycosylated model with web server-derived glycan conformations; (iii) three glycosylated models, each with unique, biologically-relevant glycan conformations derived from all-atom md simulations that were based on (ii) as the starting structure. finally, we ran bd simulations using these models to examine binding characteristics of oseltamivir, zanamivir and sialic acid. to note, the bd input and results files are provided on github (https://github.com/cgseitz). the unglycosylated model was built using an avian h5n1 strain and was used as a basis for the other models. we picked this strain of influenza because it contains a glycosylation site at n146, a member of the 150-loop that hangs over the primary binding site, as shown in figure 1 . this close proximity provides a good test of whether glycans were capable of interfering with ligand association to influenza neuraminidase. as the bd simulations used here keep bonds rigid, it was necessary to select ligand conformations that represented a bound state and protein conformations that represented an open state, to properly approximate the initial binding contact. thus, we selected a crystallized apo head region of the strain mentioned above (pdb 2hty) (56) . the stalk region has not been crystallized for any influenza na and is unlikely to influence ligand association due to its large distance from the distal binding sites, so it was not modelled. the crystallized calcium ions were retained throughout, while the crystallized glycan fragments were removed (57) . 2hty was crystallized with a y171h mutation (pdb numbering), which was reversed for this project through pymol (58) . the histidine rotamer was chosen to be the one with the highest occurrence in proteins, according to pymol. the crystal structure contained a broken backbone between p169 and n170 which was fixed through schrodinger maestro; subsequently residues 168-171 (on each side of the fixed bond) were minimized through maestro (59) . the same procedure was done for the broken backbone between v411 and q412: the bond was created and residues 410-413 were minimized. this refitting was done for each monomer in the tetramer. the ph was set to ph 6.4, as this was done in the reference k on experiments (60, 61) . using this ph, protonation states on the neuraminidase were assigned using propka (62) . the protonation assignments were done through the pdb2pqr server (63) . partial charges on the protein were assigned according to the amber99 force field (64) . parameterizing the glycans needed special treatment as there are not glycan parameters in the amber99 force field. we used the glycam_06h-1 parameters as these would be consistent with the amber99 force field (65) . to build the first glycosylated construct (with web server-derived glycan conformations), the unglycosylated na structure was uploaded to the glyprot server, and three representative glycans were added to each na monomer, for a total of 12 glycans on the na homotetramer (66) . though there is experimental variability in glycosylation site occupancy, we decided to place a glycan in each glycosylation site to see the maximum potential effect the glycoprofile can have on ligand association. considering most of the human h5n1 transmission came directly from avian sources, the glycans used to model this structure came from an avian (hen egg) source for growing these glycans (30) . additionally, this dataset is the only one containing structures experimentally found on influenza na (30) . we chose representative glycans from this dataset, however we note that both larger and smaller glycans will exist in nature; these size differences may slightly affect the results presented here. the exact glycans were selected as shown in table 1 . table 1 . glycan structures from the glyprot web server. the "glycan structure" entries came from experimental results (30) . these structures consist of n-acetylglucosamine (glcnac), mannose (man), n-acetylhexosamine (hexnac) and hexose (hex). hexnac and hex were interpreted according to their corresponding "glyprot identifier" and the structures shown in to better diversify our system, three glycosylation sites (termed as site #1, site #2 and site #3) present on each monomer were linked to three different glycan types. importantly, the four monomers (termed as monomer a, monomer b, monomer c, and monomer d) of our homotetrameric na model were symmetrically glycosylated, meaning that sites #1, #2 and #3 were populated with the same glycan across monomers. starting with the structure containing web server-derived glycan conformations, md was then used to generate representative glycan conformations, with the assumption that md would provide realistic conformations of glycans within a microsecond's worth of sampling (67, 68) . the first step was porting the structure with web server-derived glycans into charmm-gui to prepare the structure for md (69) (70) (71) (72) (73) (74) . the disulfide bonds were taken from uniprot id q6dpl2. the system was embedded into a box described with explicit water molecules using the tip3p model (75 ). an ion model was used as described previously (76). the full system had a size of 299,732 atoms. an ionic solution of 0.15 m nacl was used, and the charmm36 all-atom additive force fields were used for the protein and the glycans (77). molecular dynamics simulations were run using gpu-accelerated amber18 with an npt ensemble (78, 79). the system was initially minimized for a total of 5000 cycles using a combination of steepest descent and conjugant gradient methods (78, 79). equilibration in an npt ensemble was performed for 125 ps, using a timestep of 2 fs and the shake algorithm to constrain all bonds involving hydrogen (80) . the equilibration temperature was set at 298 k and regulated through a langevin dynamics thermostat (81, 82) . the pressure was fixed at 1 bar through a monte carlo barostat (83) . these simulations were run using extreme science and engineering discovery environment (xsede), specifically the comet supercomputer housed at the san diego supercomputer center (84) . periodic boundary conditions were used with a non-bonded shortrange interaction cutoff of 12 å and force-based switching at 10 å. particle mesh ewald was used for the long-range electrostatic interactions (85) . for the production runs, the temperature was set at 310.15 k (61). after equilibration, this system was cloned into 50 identical replicates. each one was run in parallel for 20 ns each with a unique starting velocity, totaling 1 µs of sampling. once the md simulations finished, the trajectory of each glycan was concatenated independently of the rest of the system. each of these individual glycan trajectories were then clustered using gromacs-based gromos clustering with an rmsd cutoff of 2.5 å (86). this number was chosen so the three most populated clusters would represent at least 50% of the total glycan conformations in each of the simulations. the central structure, defined as the structure with the smallest average rmsd from all other members of the cluster, from the top cluster of each of the 12 glycans was then selected; the pyranose ring from the reducing end of the glycan was then aligned to the analogous pyranose ring of the corresponding glycan from the glyprotglycosylated structure as this should be the most stable part of the glycan (87) . the glyprot glycans were removed and the glycans from the md simulations were attached through schrodinger maestro, to create a new na system with each glycosylation site inhabited by the central structure of the most representative conformation from the md simulations. this was then repeated for the second and third most representative glycan clusters from the md simulations. the sialic acid structures used were drawn from pdb 1mwe, which crystallized the boat conformation in the active site and the chair conformation in the secondary site (88) . the chair conformation of sialic acid was crystallized with a missing carboxylate group, which was added through schrodinger maestro to model an energetically-favorable gauche conformation. zanamivir was extracted from the 3ckz crystal structure (60) . oseltamivir was extracted from the 3cl0 crystal structure (60) . a 2d comparison of these ligands can be seen in figure s1 , showing their structural similarities; we note that all mentions in this study of oseltamivir pertain to tamiflu's active metabolite oseltamivir carboxylate. these ligands were then uploaded to the prodrg server to add hydrogens (89) . charges according to the amber99 force field were added through the pdb2pqr server (63, 64) . bd simulations were run using browndye (90) . even though the md was run with the amber18 force field and the bd was run with the amber99 force field, we assume these to be sufficiently independent steps and the slight force field differences should not appreciably affect the results, especially as our k on numbers are relative, not absolute. the charges for the protein and ligands were reassigned according to the amber99 force field (64) . the temperature was set to 310.15 k, which was the temperature for the referenced k on experiments (60, 61) . the ions used are shown in table s1 . these ions were selected to mimic the ion and buffer concentration of the reference k on experiments (60, 61). the experimental assay used 5 mm cacl 2 and 32.5 mm mes buffer (60) . the ca 2+ and clconcentrations were simply calculated by finding their ionic strengths. mes buffer is prepared with na + ; the concentrations of the buffer and na + at ph 6.4 were calculated with the henderson-hasselbalch equation (91, 92) . this resulted in an overall ionic strength of 0.039 m. the calcium, chlorine and sodium van der waals radii were taken from the literature (64, 93) . the mes radius was determined by building it in schrodinger maestro and measuring it in vmd (94) . apbs was used to create the electrostatic grids needed by browndye for these simulations (95) . the grid spacings are listed in table s2 . the solvent dielectric was set to 78 while the protein dielectric was set to 4. desolvation forces were turned off. the debye length, determined from the concentration and charges of the ions in the solution, was set to 15.7 å. in browndye, the b radius is defined as the starting radius for the ligand trajectories, at a distance where the force between the protein and ligand is independent of orientation. this distance is determined from the hydrodynamic center of the receptor. because of the different glycan conformations used, the b radius differed slightly between systems. if a ligand reaches what is known as the q radius, the trajectory either ends as a non-association or is restarted from the b radius according to browndye's algorithm. the q radius is defined as 1.1 times the b radius distance. the b radius ranged between 109 å and 112 å depending on the system, and the q radius ranged from 120 å to 123 å . the exact b and q radius values for each system are shown in table s3 . bd simulations were run on all five na models generated (i.e. unglycosylated, glycosylated with web server-derived glycans, and the three systems with md-derived glycan conformations). these simulations totaled 10 million trajectories for each ligand/binding site pair, consistently giving reproducible rates within the small level of error reported and resulting in 600 million trajectories total. reproducible rates will be obtained by having a binding probability of around one in a million trajectories; we found we could roughly obtain these probabilities by using 10 million trajectories for each ligand/binding site as has been reported previously (53) . this number of trajectories produced error values comparable to those seen in the reference experimental studies, as seen in figure s2 . for systems where we saw at least one binding event, the number of binding events ranged from two to 889 (see supporting material for details). bd simulations using browndye requires the creation of reaction criteria, consisting of a list of protein-ligand atom pairs and a cutoff distance. if any three of these pairs simultaneously came closer than the cutoff distance, we assume the ligand will associate. the cutoff distance was empirically determined to be 3.228 å; this distance approximately yielded the experimental k on rates for both oseltamivir and zanamivir (60) . there are no other experimental k on rates towards the primary site of h5n1, and no referenced rates at all for the secondary site. the referenced k on experiments were done with glycans attached to na and measured to the full tetramer; this was confirmed in personal correspondence with the corresponding author (stephen martin of the mrc national institute for medical research, correspondence on july 21, 2018). considering that the reaction criteria and reaction distance were created for oseltamivir and no significant changes were made before applying them to zanamivir, we can safely assume that they are generalizable for sialic acid, an analog of both oseltamivir and zanamivir ( figure s1 ). the protein-ligand atom pairs were taken from crystal structures of ligands in the primary and secondary sites of neuraminidase for each monomer, and simulations were run for the full tetramer. the primary binding site was determined according to the crystallized binding pocket for our strain of neuraminidase (56) . this pocket is noted to have a surface area of 941.3 å 2 and a volume of 574.8 å 3 (96) . the secondary site contacts were determined from a structure of influenza a/tern/australia/g70c/75 (88) . however, all the secondary site residues are conserved between that strain and the strain used in our simulations. the combined site simulations are defined as simulations with criteria allowing for association to either the primary or secondary site; it is simply a simulation run with a concatenation of the binding criteria for these sites. in this work, we define binding site contacts to be those protein-ligand contacts seen in crystal structures. from these contacts, we created protein-ligand atom pairs in browndye to determine when a reaction has occurred in our bd trajectories. there are seven primary binding site contacts reported between oseltamivir and the 3cl0 crystal structure (60) . these binding site contacts are reported in table s4 and figure s3 . there are five primary binding site contacts reported between sialic acid and the 1mwe crystal structure; all five of these are analogous to those seen for oseltamivir (88) . the binding site contacts for sialic acid are registered in table s5 and figure s4 . there is one primary binding site contact reported between zanamivir and the 3ckz crystal structure; this one is analogous to one seen in oseltamivir (60) . the binding site contacts from oseltamivir were transferred to zanamivir retaining the one contact seen in the 3ckz crystal structure and are reported in table s6 and figure s5 . using the structural similarities of sialic acid and zanamivir to oseltamivir, analogous primary binding site atom pairs were created so that each ligand had seven primary protein-ligand atom pairs. there are five secondary binding site contacts reported between sialic acid and the 1mwe crystal structure (88) . these contacts are reported in table s7 . there are no published reports of crystal structures of oseltamivir or zanamivir in the secondary binding site, so five analogous secondary binding site protein-ligand atom pairs were created for oseltamivir (table s8) and zanamivir (table s9) to match those seen in sialic acid, so that each ligand had five secondary binding site protein-ligand atom pairs. to pare down the data from 1 µs of cumulative md sampling and pick out biologically-relevant glycan conformations, we clustered each glycan from the md simulations. the glycan trajectories were extracted and affixed on the static na crystal structure, to reveal the conformational space they can access (figure 2) . visualizing these glycan trajectories on the na structure gives a qualitative representation of how much volume and surface area the glycans are capable of accessing. keeping in mind the primary and secondary binding sites are located just beneath the glycans (figure 1) , the size and flexibility of the glycans here shows that they have the capability to "shield" the binding sites from ligand association. the three most representative clusters for each glycan were extracted from the md simulations. the central structure from each cluster was compared with the conformation generated from glyprot. these clusters show some conformational diversity, but none show a particularly similar conformation to the glyprot structure. however, the third glycan in each monomer shows a markedly decreased conformational diversity compared to the other two monomers. the clustering results from each monomer show the same trends; the results from monomer a are shown in figure 3 , while the results from monomer b (figure s6 ), monomer c (figure s7) , and monomer d (figure s8 ) are shown in the supporting material. the glycans bend away from the binding sites on their monomer towards the binding sites on the neighboring monomer. this is seen for each monomer. the primary binding sites are in purple and the secondary binding sites are in orange. the linkage between the glycans and the protein is in yellow. the na structure is in teal. the glyprot conformation is in gray, the first conformation from the md simulations is in orange, the second conformation is in blue, and the third conformation is in green. to be confident in our computed association rates, we first needed to benchmark our system against experimental results. we created empirically-derived system criteria for the association of oseltamivir to the primary binding site of glycosylated na, as described in the methods. after matching the experimental association rate with oseltamivir, the same parameters were applied to zanamivir. these are the only two experimental association rates for h5n1 na. subsequently, we investigated the association of oseltamivir and zanamivir to the primary sites of glycosylated na, obtaining association rates of 2.52 ± 0.21 /µm·s for oseltamivir and 0.47 ± 0.09 /µm·s for zanamivir. these are in agreement with the experimentally-measured rates of 2.52 ± 0.21 /µm·s and 0.95 ± 0.21 /µm·s, respectively, as visualized in figure s2 (60) . considering the experimental systems were glycosylated, we had to pick one glycan conformation to use for computing these benchmarks in our glycosylated system; for reproducibility we chose the conformation generated from the glyprot server. we note that choosing a different conformation for our computed benchmark would change the absolute association rates by a scaling factor, but the trends would remain the same. since the predicted k on for oseltamivir and zanamivir both matched up well with the experimental rates, the system proved to be transferable to ligand analogs for the primary site. we then applied the same criteria to two different conformations of sialic acid, boat and chair, to probe if the association rate was dependent on conformation. this was done in addition to analyzing how association rate was modulated by different functional groups, via comparisons of ligand analogs such as oseltamivir, zanamivir and sialic acid. with the binding criteria set up, we calculated the association rates of each of the ligands to the primary site ( figure 5a) . these results show two important findings. first, there is not a large difference in association rates between the system with glyprot glycans and the unglycosylated system. this shows that a glycan may adopt a conformation where it does not inhibit ligand binding much at all. the second finding is that the glycans from the md simulations all show a moderate level of inhibition, more than the system with glyprot glycans. this shows that biologically-relevant glycan conformations will likely exhibit a moderate level of inhibition towards ligand binding. combining the first and second finding discussed in this paragraph, glycans are capable of perturbing ligand binding to na. conf1 is the glycan structure from the most populated cluster from the md simulations. conf2 is from the second most populated cluster, and conf3 is from the third most populated cluster. the association rates using glycans structures downloaded from glyprot are shown in gray. the association rates using structures derived from the md simulations are in bright, colorful shades whereas the others are in grayscale. the association rates without using any glycans are shown in black. (a) the glycan structures from the md simulations show a moderate association rate inhibition to the primary binding site irrespective of ligand chosen. (b) little association is seen to the secondary binding site. note the different y-axis used to be able to see the small amount of binding. (c) association rates of trajectories run with either the primary site or secondary site as the trajectory end point. similar to (a), the glycans structures from the md simulations in (c) show a moderate inhibition of ligand association. the raw data for this figure is seen in table s10 (oseltamivir), table s11 (zanamivir), table s12 (sialic acid boat conformation), and table s13 (sialic acid chair conformation). there are no experimental association rates for ligands to the secondary site, so criteria were chosen based off of crystal structure data and discussed in the methods. only sialic acid has been crystallized in the secondary site of avian na, so binding site criteria for the secondary site were extracted from that structure and used to create the criteria for oseltamivir and zanamivir, as discussed in the methods (88) . a previous bd study suggested that oseltamivir can bind to the avian na secondary site (52) . a follow-up nmr study also suggested that the oseltamivir binds to the avian na secondary site (97) . however, a more recent experimental study disagreed with these findings and did not see oseltamivir binding to the avian na secondary site (98) . considering the disagreement with oseltamivir binding to the secondary site, we decided to test this and secondary site binding for zanamivir as well. the computed association rates towards the secondary site show a markedly different story than those to the primary site ( figure 5b) . none of the ligands exhibited noticeable binding towards the secondary site, with the exception of the boat conformation of sialic acid. even with this conformation, there is no consistent trend when compared to primary site binding. although the boat conformation sialic acid displays a small amount of binding, the chair conformation does not show binding. these results show that we can differentiate between these two sialic acid conformations at the bd level of theory. finally, trajectories were run where the ligand could associate to either the primary site or the secondary site ( figure 5c) . intriguingly, the results are essentiallly a concatenation of the rates seen for the primary and secondary sites individually. considering the low level of secondary site binding, the trends here are the same as seen for the primary site. as can be seen in figure s1 , there is a formal charge difference between the ligands: sialic acid contains a formal charge of -1 while oseltamivir and zanamivir are neutral. running test bd trajectories without charge treatment (results not shown), we saw analogous results to those seen in figure 5 . this meant that only the sterics of the systems affected binding, not electrostatics. clearly one or a few of the structural differences between the ligands play outsized roles in affecting the association rates. in this work we did not further probe which exact atoms in the ligands will change the association rates. biologically, the influenza replication cycle is propagated through na recognizing and cleaving sialic acid. this study compares the interplay between that molecular recognition process and na's aforementioned glycan shielding capabilities. this interplay is simplified here by approximating ligand binding as a diffusion-governed association process, modulated by protein electrostatics. previous studies have shown that viral proteins can exhibit a degree of glycosylation large enough to partially protect a variety of viruses from immune system antibodies; this is termed the viral glycan shield (18, 21, (99) (100) (101) . from static structures one can envision the shielding that glycans can provide, but a dynamic representation better depicts the steric barrier encountered by immune system antibodies and drugs (102) . in our single na protein, we see that glycans are capable of covering most of the na surface area, as shown in figure 2 . this is consistent with studies explaining how the influenza glycan shield can cloak the influenza virion from the immune system (5-7). the glycans can access a large volume, allowing for a considerable shielding potential. however, it is worthwhile to note that influenza glycoproteins are usually not as extensively glycosylated as on some other viral proteins, such as the hiv envelope protein or the sars-cov-2 s protein (15, 17, (103) (104) (105) . the exact h5n1 construct prepared here contains a glycosylation site at n146. this is part of the 150 loop that borders the primary binding site (figure 1) . the representation in figure 2 shows that the glycans present at site n146 on each monomer have the combined capability to cover both na binding sites, potentially thwarting the binding of small molecules. the results shown here display a moderate inhibitory effect due to glycans, but this effect would likely not be present in proteins whose glycans only reside far from the ligand binding sites, i.e. if the setup in figure 1 only contained the glycans at site 1 and site 3 on the bottom of the na head. when examining the effect of glycan conformation on binding inhibition, the glyprot glycans display a fairly vertical conformation. on the other hand, the glycans from the md simulations bend backwards, away from the primary binding site on their own monomer and towards the secondary binding site of the adjacent monomer, as shown in figure 4 . interestingly, this bend appears to be enough to inhibit primary site binding. it has been previously shown that specific chemical modifications on the glycans can significantly change their flexibility (106) (107) (108) . it has also been hypothesized that glycan flexibility plays a role in protein-receptor binding equilibria (87) . considering the scale of biological interactions that glycans participate in, it is likely that they would exploit their flexibility to facilitate these interactions. however, the glycan environment, and nearby steric clashes would conceivably affect this flexibility as well, introducing competing effects. revisiting the input na structure in figure 1 , we hypothesized that the glycan on top of each na monomer (the oligomannose type glycans linked to site #1) would achieve a higher degree of flexibility than the two on the bottom of each monomer (the complex and hybrid type glycans linked to sites #2 and #3, respectively). our reasoning was that these two may find steric restrictions on their flexibility, and that the placement on the glycan on the na head would be more important than the type of glycan examined. our results show this is not quite the case. the clusters in figure 3 , figure s6 , figure s7 , and figure s8 , show that, similar to the complextype glycans (a-d2), the oligomannose-type glycans (a-d1) were quite flexible even though they were situated near the hybrid-type glycans (a-d3) on the bottom of the na surface; this large degree of conformational freedom is backed up by previous work specifying that this flexibility is driven by the mannose(4)-α(1-3)-mannose(3) and the mannose(5)-α(1-6)-mannose(3) linkages (109) . these are the linkages connecting the chitobiose glycan "stalk" to the two glycan "branches". finally, the hybrid-type glycans showed noticeably less conformational flexibility than either the oligomannose-type glycans or the complex-type glycans. overall, the type of glycan and its specific linkages seemed to govern its flexibility more than potential nearby steric clashes. this agrees with previous work showing that unless there is a direct steric clash, inter-residue hydrogen bonds may have a larger effect governing glycan conformations (106, 109) . the results shown in figure 5 are consistent with diffusion controlled reactions, and show relatively high association rates. the space explored is consistent with the random walk nature of diffusion. the randomness of the ligand trajectories (from brownian motion) and the small sizes of the ligands considered here minimize the effects of the glycans on binding. the rates for each ligand are mostly of similar orders of magnitude, with or without glycosylation. however, the glycan structures from the md simulations show a moderate inhibition compared to the unglycosylated na structure and the na structure with glycan structures taken directly from the glyprot web server. the extent of this inhibition ranges from a factor of about two to eight. in general, glycans can decrease binding activity of viral proteins (3, 42, 44, 110) . due to their bulk and proximity to the primary ligand binding site, we hypothesized that, irrespective of conformation, the presence of glycans, particularly those near the binding sites, could substantially reduce ligand binding and removing these glycans would restore binding. what we found was a more nuanced picture. the na constructs with glycan conformations from the glyprot server showed similar binding rates to unglycosylated constructs. however, more realistic glycan conformations, extracted from the md simulations, showed a moderate but noticeable decrease in association rate, k on , on the order of two to eight times. one may naturally question whether glycans would have the same effect on dissociation rate, k off . one previous study testing antibody binding to cancer cells showed that antibody binding was relatively insensitive to the presence of glycans, indicating a similar dampening of k on and k off due to the presence of glycans (9) . in this study mentioned, the overall equilibrium constant k d changed by less than a factor of two irrespective of the presence or absence of glycans (9) . however, a different study done in the influenza membrane protein hemagglutinin showed that trimming the glycans from a standard length seen in hek293 cells to a single monosaccharide decreases the equilibrium constant k d by a factor of two to 48, depending on the receptor mimic used (42) . this meant that the k on and the k off were not affected in the same way by the presence of glycans (42) . glycans are present in the antibody binding sites of both of the studies mentioned above; this is in contrast to our system where glycans are situated near the catalytic sites, but not directly inside them. with this in mind, it seems likely that the slight slowing of binding small ligands by the glycans would be similarly reflected in a slight slowing of release, so that the equilibrium constants for binding these molecules are relatively insensitive to the presence of glycans. in effect, this is because the presence of glycans near the binding sites should not change the δ g in accordance with the gibbs relationship. we hypothesize that our observed decrease in association rate is due to the glycans at glycosylation site n146 (site #2) as only those glycans are capable of sterically inhibiting the binding sites (figure 2) , and we assume the glycans at sites n88 (site #1) and n234 (site #3) do not impair binding. taking the inhibition results discussed here with a different binding study using larger ligands for influenza na, there appears to be a size dependence on this inhibitory potential: smaller ligands are not as affected as larger ligands (43) . the key points here are that small molecules are not seriously impeded from binding by the glycans; future drug discovery efforts can be focused on the development of strong binders with correspondingly long lifetimes of binding. modeling studies focused on small inhibitors are likely to be helpful, even when glycans are not included. the results seen in figure 5 highlight the importance of using biologically-relevant glycan conformations relaxed on the protein structure as opposed to simply generating a glycan conformation and attaching it to the protein. though this study did use static structures as per the bd setup, we would expect similar trends if this study were repeated using a dynamic md environment since our bd trajectories already used the most highly-accessed glycan conformations gleaned from extensive md sampling. moreover, a study using mixed bd-md simulations analyzing the association of oseltamivir and zanamivir to na actually showed a less accurate k on rate than our coarser study using only bd (54) . we can rationalize that the slower binding kinetics seen in our systems with biologically-relevant glycan conformations ( figure 5 ) are due to the ligands having to maneuver around the glycans, even after running into them, and then continuing with the trajectory until reaching the binding site. this type of maneuverability can be seen in figure 6 . we generated bd trajectories that could end with the ligand binding to the primary site ( figure 5a) , the secondary site (figure 5b) , or either site ( figure 5c ) on any monomer. using this setup, we were able to differentiate binding between the primary and secondary sites, and in fact found an additive binding mode when examining both sites concurrently. by simply adding up the association rates observed for the primary site ( figure 5a ) to the analogous simulation run to the secondary site (figure 5b) , the association rate to both sites ( figure 5c ) can be roughly obtained. we do not see any evidence of a further increase in association rate using both sites, showing that the presence of a proximal binding site does not influence association rate, either for the primary site or the secondary site. our primary site binding results show two conclusions supported by literature. in figure 5a we see that oseltamivir associates faster than zanamivir, as has been seen in experimental kinetics studies (60) . moreover, we see faster binding of oseltamivir than sialic acid. this is qualitatively in agreement with an nmr study showing that oseltamivir outcompetes α (2,3)-sialyllactose in binding to the avian na active site (97) . it is not immediately clear which atoms on the ligands drive their binding differences. ligand binding to the secondary site has not been extensively studied, but it does not appear to have catalytic activity (111, 112) . focusing on the secondary site, our results show three important findings. we first see that binding to the secondary site is slower than to the primary site, if binding is seen at all (figure 5) . we do not see secondary site binding for oseltamivir and very little for zanamivir, though this may be as they are at the lower detection limit of our method. furthermore, we see that sialic acid binds faster to the secondary site than oseltamivir, which is in agreement with one study showing that α (2,3)-sialyllactose outcompetes oseltamivir for binding to the avian na secondary site (97) . a more recent study goes further and does not show any binding of oseltamivir to the avian na secondary site (98) . however, we caution that a small amount of drug binding, likely only with zanamivir, may occur to the secondary site, as seen with zanamivir bound in the secondary site in the unpublished crystal structure pdb 2cml, and also seen in figure 5b . secondly, in the small amount of secondary site binding seen (figure 5b) , glycans are actually capable of enhancing or inhibiting binding, foreshadowing the complex role glycans play in ligand binding. finally, there appears to be a small conformational dependence on association rate, but this is only seen towards the secondary site ( figure 5b) . we used two different conformations of sialic acid for these binding studies. the boat conformation was crystallized in the active site and the chair conformation was crystallized in the secondary site. in our results we see the sialic acid chair conformation actually shows fractionally higher binding to the primary site than the boat conformation ( figure 5a) . conversely, only the boat conformation shows binding to the secondary site; the chair conformation does not register binding at all (figure 5b) . however, we caution that these results may be because sialic acid was crystallized in a different strain of avian na than we used in our studies. taken together, these results show that the exact ligand conformation upon approach to the binding site may not match the crystallized binding pose, but the results we present here do not permit us to explore this note or further explain a conformational dependence on binding. comparing the association rates in figure 5 one may naturally query the competition in association rates between the primary and secondary sites. we see faster association to the primary site than the secondary site, which is not in agreement with two previous bd simulation studies (52, 53) . however, the methodology of our study differs from these two studies, and from this we can unify the difference. those bd studies showed that ligands reach a distance of 7.5 å away from the secondary site faster than to the primary site. we then show that ligands reach a distance of 3.228 å away from the primary site faster than the secondary site, though we would like to note that our paper and the sung et al. paper assigned charges for the bd trajectories according to the amber99 force field and the amaro et al. paper assigned charges according to the charmm36 force field (52, 53) . taken together, the secondary site appears to contain stronger long-range electrostatics to draw in ligands, but when the ligands approach the binding sites and sterics come into play, it appears to be more favorable for ligands to move closer to the primary site than the secondary site, assuming the rigid body approximations applied herein. considering the fact that the realistic substrates na encounters will exhibit multivalent binding, one previous study showed that the secondary site improved avian na enzymatic activity in removing sialic acid both from soluble macromolecular substrates and from cells (113) . another study confirmed that the binding in the secondary site improved catalytic activity against multivalent substrates (114) . other previous studies have suggested that the secondary site enhances the overall na catalytic activity by binding substrates and bringing them close to the catalytic primary site (111, (113) (114) (115) (116) . taking the studies above with our results, we postulate that multivalent cleavage will occur in a stepwise manner (figure 7) . the first association event of the multivalent substrate, such as sialylated cell surface receptors, will bind to the primary site, and then to the secondary site. after sialidase cleavage occurs in the primary site, the cleaved glycan branch will dissociate. then the sialylated glycan branch bound in the secondary site will be transferred to the primary site, as suggested previously (111, (113) (114) (115) . after this passage, cleavage will again occur, and the full glycan will be released, finishing the enzymatic cycle. this mechanism is in disagreement with a previously proposed mechanism, which postulates that both binding sites will not be bound simultaneously (115) . however, we feel there is a greater body of literature suggesting that binding both sites simultaneously increases catalytic activity. we note that our proposed binding mechanism may be muddied in the case of multivalent ligands with viral glycans situated near the binding sites; in this case, the glycans may sterically inhibit multivalent binding, slowing down enzymatic activity and attenuating the replication cycle. in the case of monovalent binders, such as the inhibitors oseltamivir and zanamivir, we show in figure 5 that association will happen to the primary site faster than to the secondary site. this appears to be biologically viable considering that previous studies have showed that the secondary site activity has no effect on enzymatic activity for monovalent substrates (98, (117) (118) (119) . as the primary site is the main site of enzymatic activity, it is reasonable to assume that ligands would preferentially bind to the primary site over the secondary site; reducing transfers of ligands between the binding sites would ostensibly increase catalytic activity and efficiency. taken together, abolishing the secondary site in avian na will not affect monovalent substrates such as influenza drugs as these associate faster to the primary site anyways, which our results confirm. to exposit this a different way, influenza drugs will preferentially block primary site binding over secondary site binding. a free monovalent binder will associate to the primary site over the secondary site (a). this monovalent binder will release from the primary site before a second monovalent binder will associate to the secondary site. glycans, with their sialic acid tips, are an example of a multivalent binder (b). similar to the monovalent binders, the first multivalent binding event will occur to the primary site. next, the second sialic acid tip binds to the secondary site. with both sites bound, the sialic acid in the primary site is cleaved and released. the sialic acid bound in the secondary site is then transferred to the primary site. finally, the second sialic acid is cleaved and released, and the enzymatic cycle is complete. in this work, we created na systems with varying glycan conformations, and also without the presence of glycans. these glycans are capable of covering much of the surface area of na. their conformational flexibility is dependent on their glycan type, not necessarily their spatial position. the glycosylated systems showed moderate inhibition of ligands to the primary binding site. finally, we propose a new binding mechanism for multivalent binders to na, such as cell surface receptors. these results have implications for future drug development, the overall understanding of glycans, and the na enzymatic mechanism. much sustained effort has gone into developing na inhibitors, and will continue to do so in the future. measuring the binding of a potential drug is an important step in the drug discovery process. however, most drug discovery efforts have not taken into account viral glycans. neglecting this effect can lead to a surprising drop in drug binding (9) . our work shows that glycans can have an inhibitory effect on influenza na primary site binding. there have already been a number of studies using multivalent binders as na antivirals (120) (121) (122) (123) (124) (125) . with the results shown here, we recommend future work on multivalent na drugs, to focus on developing strong binders with a long lifetime, regardless of the presence or absence of glycans. with the detection limitations of our study, we cannot conclude how glycans affect secondary site binding, although we believe binding to the secondary site will be slower than binding to the primary site ( figure 5) . however, it follows from these results that glycans could evoke a secondary site binding inhibition similar to the primary site. in summary, this work examines glycan inhibition on drug binding, compares the drug binding interplay between two binding sites, and 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biomolecular force field glycosciences.db: an annotated data collection linking glycomics and proteomics data reaching biological timescales with all-atom molecular dynamics simulations conformational analysis of furanoside-containing mono-and oligosaccharides charmm gui: a web based graphical user interface for charmm charmm: the biomolecular simulation program charmm-gui input generator for namd, gromacs, amber, openmm, and charmm/openmm simulations using the charmm36 additive force field glycan reader: automated sugar identification and simulation preparation for carbohydrates and glycoproteins glycan reader is improved to recognize most sugar types and chemical modifications in the protein data bank charmm-gui glycan modeler for modeling and simulation of carbohydrates and glycoconjugates comparison of simple potential functions for simulating liquid water numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamcis of n-alkanes langevin dynamics of peptides: the frictional dependence of isomerization rates of n acetylalanyl n ′ methylamide an analysis of the accuracy of langevin and molecular dynamics algorithms isothermal-isobaric molecular dynamics simulations with monte carlo volume sampling particle mesh ewald: an n⋅log(n) method for ewald sums in large systems gromacs: high performance molecular simulations through multi-level parallelism from laptops to supercomputers distinct glycan topology for avian and human sialopentasaccharide receptor analogues upon binding different hemagglutinins: a molecular dynamics perspective structural evidence for a second sialic acid binding site in avian influenza neuraminidases prodrg: a tool for high-throughput crystallography of protein-ligand complexes browndye: a software package for bronwian dynamics studier i affiniteten. cm forhandlinger: videnskabs-selskabet i christiana 35 über die chemische affinität ion-water interaction potentials derived from free energy 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n-glycans in solution glycan flexibility: insights into nanosecond dynamics from a microsecond molecular dynamics simulation explaining an unusual nuclear overhauser effect conformational flexibility of nglycans in solution studied by remd simulations sequence-to-structure dependence of isolated igg fc complex biantennary n-glycans: a molecular dynamics study regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety the 2nd sialic acidbinding site of influenza a virus neuraminidase is an important determinant of the hemagglutinin-neuraminidase-receptor balance influenza virus-glycan interactions functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses mutation of the second sialic acid-binding site, resulting in reduced neuraminidase activity, preceded the emergence of h7n9 influenza a virus mesoscale all-atom influenza virus simulations suggest new substrate binding mechanism substrate binding by the second sialic acid-binding site of influenza a virus n1 neuraminidase contributes to enzymatic activity n1 neuraminidase of influenza virus a/fpv/rostock/34 has haemadsorbing activity neuraminidase hemadsorption activity, conserved in avian influenza a viruses, does not influence viral replication in ducks antigenic structure and variation in an influenza virus n9 neuraminidase synthesis and anti-influenza evaluation of polyvalent sialidase inhibitors bearing 4-guanidino-neu5ac2en derivatives dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including h5n1 avian influenza attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus polymerattached zanamivir inhibits synergistically both early and late stages of influenza virus infection synthesis of multivalent difluorinated zanamivir analogs as potent antiviral inhibitors multivalent zanamivir-bovine serum albumin conjugate as a potent influenza neuraminidase inhibitor key: cord-015830-ha8oj1b3 authors: van essen, a g; berg, h f; bueving, h j; van der laan, j r; van lidth de jeude, c p; van der sande, m a b; voordouw, a c g; boomsma, j l; opstelten, w title: nhg-standaard influenzapandemie date: 2009-02-16 journal: nhg-standaarden 2009 doi: 10.1007/978-90-313-6614-9_85 sha: doc_id: 15830 cord_uid: ha8oj1b3 deze standaard is opgesteld op verzoek van het ministerie van volksgezondheid, welzijn en sport. zij geeft huisartsen richtlijnen voor een vooralsnog hypothetische situatie waarin een voor mensen nieuw influenzavirus zich wereldwijd verspreidt. omdat deze richtlijnen volledig worden bepaald door de fase waarin een influenzapandemie zich bevindt, heeft deze standaard een andere opbouw dan gebruikelijk. de paragraaf ‘achtergronden’ is relatief uitgebreid en geeft uitleg over het ontstaan en de mogelijke gevolgen van een influenzapandemie. daarna wordt het beleid uiteengezet in drie delen. eerst worden de algemene principes besproken: de virusverspreiding in de populatie indammen en de gevolgen voor de individuele patiënt beperken. de daaropvolgende onderdelen beschrijven het beleid (inclusief diagnostiek) in twee verschillende situaties: een dreigende pandemie en een manifeste pandemie. voor de uitvoerbaarheid van het geadviseerde beleid is het van groot belang dat de huisartsgeneeskundige zorg goed georganiseerd is en dat huisartsen tijdens een influenzapandemie samenwerken met andere disciplines. het nhg heeft implementatiemateriaal ontwikkeld waarin deze aspecten aan de orde komen. het is onvoorspelbaar hoe een nieuwe influenzapandemie zich zal ontwikkelen en wat de aard zal zijn van een nieuw pandemisch influenzavirus. er kunnen zich ontwikkelingen voordoen die aanpassing van het geadviseerde beleid nodig maken. deze nhgstandaard zal daarom regelmatig geactualiseerd worden op de website van het nhg (http://www.nhg.org). voor de meest recente informatie wordt verwezen naar overheidsrichtlijnen waarop deze standaard is gebaseerd.(49) deze standaard is opgesteld op verzoek van het ministerie van volksgezondheid, welzijn en sport. zij geeft huisartsen richtlijnen voor een vooralsnog hypothetische situatie waarin een voor mensen nieuw influenzavirus zich wereldwijd verspreidt. omdat deze richtlijnen volledig worden bepaald door de fase waarin een influenzapandemie zich bevindt, heeft deze standaard een andere opbouw dan gebruikelijk. de paragraaf 'achtergronden' is relatief uitgebreid en geeft uitleg over het ontstaan en de mogelijke gevolgen van een influenzapandemie. daarna wordt het beleid uiteengezet in drie delen. eerst worden de algemene principes besproken: de virusverspreiding in de populatie indammen en de gevolgen voor de individuele patiënt beperken. de daaropvolgende onderdelen beschrijven het beleid (inclusief diagnostiek) in twee verschillende situaties: een dreigende pandemie en een manifeste pandemie. voor de uitvoerbaarheid van het geadviseerde beleid is het van groot belang dat de huisartsgeneeskundige zorg goed georganiseerd is en dat huisartsen tijdens een influenzapandemie samenwerken met andere disciplines. het nhg heeft implementatiemateriaal ontwikkeld waarin deze aspecten aan de orde komen. het is onvoorspelbaar hoe een nieuwe influenzapandemie zich zal ontwikkelen en wat de aard zal zijn van een nieuw pandemisch influenzavirus. er kunnen zich ontwikkelingen voordoen die aanpassing van het geadviseerde beleid nodig maken. deze nhg-standaard zal daarom regelmatig geactualiseerd worden op de website van het nhg (http://www.nhg.org). voor de meest recente informatie wordt verwezen naar overheidsrichtlijnen waarop deze standaard is gebaseerd. 49 kernboodschappen c denk aan aviaire influenza bij een patiënt met algemene en respiratoire verschijnselen na direct contact met besmet pluimvee of vogelmest, of met een verdachte reisanamnese. c schrijf géén profylaxe met antivirale medicatie voor zonder dat er aanwijsbaar nauwe contacten zijn geweest met een patiënt met aviaire influenza (primaire profylaxe). c tijdens een dreigende influenzapandemie is virologisch onderzoek van een patiënt met mogelijke aviaire influenza altijd noodzakelijk. om verspreiding van het virus in deze fase zoveel mogelijk te beperken dient men profylactisch antivirale medicatie te geven aan contacten van patiënten met aviaire influenza (postexpositieprofylaxe). c tijdens een manifeste influenzapandemie is virologisch onderzoek niet meer nodig. iedere patiënt met klinische verschijnselen van influenza wordt dan zo snel mogelijk met antivirale medicatie behandeld. c schrijf antivirale medicatie alleen voor op medische indicatie en niet uitsluitend op verzoek van de patiënt. algemeen wordt aangenomen dat de kans op een influenzapandemie reëel is. 1 in de aanloop tot en tijdens een pandemie is de rol van huisartsen van cruciaal belang. voor hen geeft deze nhg-standaard achtergronden en algemene adviezen met betrekking tot het beleid bij een dreigende en een manifeste influenzapandemie. deze standaard is opgesteld in samenwerking met het centrum infectieziektebestrijding (cib), waartoe ook het bureau landelijke coördinatie infectieziektebestrijding (lci) behoort, en sluit zoveel mogelijk aan bij regionale draaiboeken die zijn opgesteld door de ghor-bureaus (geneeskundige hulpverlening bij ongevallen en rampen). 2 de verspreiding van vogelgriepvirussen onder treken watervogels (de zogeheten aviaire influenza) heeft geleid tot de vrees dat deze een wereldwijde influenza-epidemie (een zogeheten pandemie) onder mensen zullen veroorzaken. voordat een aviair influenzavirus een pandemie kan veroorzaken, moet aan een aantal voorwaarden voldaan worden: het virus kan mensen infecteren, vrijwel niemand heeft afweer tegen het virus, het virus is hoogpathogeen en er vindt efficiënte overdracht plaats van mens op mens. het is volledig onvoorspelbaar waar en wanneer een dergelijk virus zich zal ontwikkelen. 3 ook is onduidelijk wat de (klinische) eigenschappen van een nieuw pandemisch influenzavirus zullen zijn. er is weinig wetenschappelijke informatie beschikbaar. de adviezen in deze standaard zijn dan ook hoofdzakelijk gebaseerd op hypotheses, adviezen van experts (gezondheidsraad, cib) en consensus. om deze reden is het aangewezen om in de aanloop tot en tijdens een pandemie wetenschappelijk onderzoek te verrichten naar de aard van het pandemische influenzavirus. ook huisartsen zal gevraagd worden hieraan mee te werken. een manifeste pandemie kan leiden tot aanzienlijke morbiditeit, mortaliteit en ontwrichting van het maatschappelijke leven. naast medische hulpverlening kan daarom ook psychosociale begeleiding nodig zijn. 4 aviaire influenza: een infectieziekte die primair onder vogels voorkomt en veroorzaakt wordt door een influenza-a-virus. door uitwisseling van genen met een reeds circulerend humaan influenzavirus of door mutatie kan uit een aviair influenzavirus een nieuw humaan influenza-a-virus ontstaan. 5 influenza-achtig ziektebeeld (iaz): klinisch beeld van een acute infectie met prominente algemene (koorts, malaise, hoofdpijn en/of spierpijn) en respiratoire (hoest, keelpijn, kortademigheid en/of neusverkoudheid) symptomen. 6 influenzapandemie: een wereldwijde influenzaepidemie veroorzaakt door een (nieuw) subtype van het influenza-a-virus, waartegen in de bevolking (vrijwel) geen weerstand bestaat. primaire profylaxe: profylactische behandeling met antivirale middelen van personen die nog geen aanwijsbare nauwe contacten met een patiënt of dier met influenza hebben gehad. postexpositieprofylaxe: profylactische behandeling met antivirale middelen van personen die -waarschijnlijk -in contact zijn geweest met een virologisch bevestigd geval van influenza maar bij wie zich nog geen ziekteverschijnselen hebben geopenbaard. postexpositieprofylaxe heeft als voornaamste doel de verspreiding van het influenzavirus tegen te gaan door het reduceren van de virale replicatie en transmissie. het vermindert tevens de ziektelast van de blootgestelde personen. in deze standaard zal worden gesproken van influenza waar pandemische influenza bedoeld wordt; niet-pandemische influenza wordt aangeduid met epidemische of seizoensgebonden influenza. de who heeft een aantal fasen vóór en tijdens een influenzapandemie gedefinieerd (zie tabel 1). de actuele fase wordt vastgesteld door de who en de bijbehorende maatregelende gelden wereldwijd. 7 deze nhg-standaard beschrijft het te voeren beleid vanaf fase 3. epidemiologie bij een influenzapandemie vindt introductie en verspreiding plaats van een voor mensen nieuw influenza-a-virus, waarvan de antigene samenstelling sterk veranderd is en waartegen (nagenoeg) geen weerstand bestaat. vrijwel iedereen is dan vatbaar, met als mogelijk gevolg een explosieve verspreiding en een kans op hoge morbiditeit en mortaliteit. tijdens een seizoensgebonden influenzaepidemie treden de meeste klinische infecties op bij kinderen en verlopen de infecties het ernstigst bij ouderen. tijdens een influenzapandemie kan ook extra sterfte optreden onder personen die niet tot deze klassieke risicogroepen behoren. 12 de meeste scenario's gaan ervan uit dat circa 50% van de populatie besmet raakt en dat daarvan 50% ziek wordt. 13 tijdens een influenzapandemie kan dus verwacht worden dat in de loop van een aantal maanden ongeveer 25% van de bevolking ziek wordt, waarvan een aanzienlijk deel medische zorg zal zoeken. ter vergelijking: tijdens een seizoensgebonden influenza-epidemie consulteert de laatste jaren gemiddeld 1-2% van de bevolking de huisarts. 14 aangezien de karakteristieken van een infectie met een nieuw influenzavirus (zoals incubatietijd en klinisch beeld) en de effectiviteit van interventies nog niet bekend zijn, bestaat er onzekerheid over de omvang van de mortaliteit. het optreden van influenzapandemieën en de gevolgen daarvan zijn onvoorspelbaar. 15 de verbeteringen in hygiëne en gezondheidszorg, en de beschikbaarheid van antivirale middelen en antibiotica, wettigen de verwachting dat een nieuwe pandemie minder slachtoffers zal eisen dan de pandemieën van de vorige eeuw. anderzijds kan het toegenomen internationale (vlieg)verkeer bijdragen aan een snellere verspreiding van het virus. de pandemische influenzavirussen in de vorige eeuw deden er ongeveer zes maanden over om zich over de wereld te verspreiden. dit zou bij een volgende pandemie bekort kunnen zijn tot vier à vijf maanden. 16 vroegere pandemieën vertoonden vaak een golvend beloop, waarbij pieken en dalen in het aantal zieken en doden elkaar afwisselden. deze golven duurden zes tot acht weken en de periode tussen de golven had een wisselende duur. 17 een aviair influenzavirus zou zich kunnen gedragen als de huidige humane influenza-a-virussen, maar ook andere klinische presentaties zijn mogelijk. 18 deze onzekerheid maakt het onmogelijk de klinische symptomatologie te beschrijven van een aviair (en zich pandemisch ontwikkelend) influenzavirus. daarom volgt hieronder een beschrijving van het klinische beeld van de besmetting met een seizoensgebonden humaan influenza-a-virus. na besmetting vermenigvuldigt het virus zich in het epitheel van de (lage) luchtwegen. na een incubatietijd van één tot drie dagen (bij besmetting van mensen met het aviaire h5n1-virus is de incubatietijd gemiddeld drie tot vijf dagen) treedt een plotselinge forse temperatuurstijging op, die vaak gepaard gaat met koude rillingen. vooral bij oudere patiënten echter kan de koorts uitblijven of minder sterk zijn. binnen enkele uren ontstaat een algeheel ziekzijn met hoofdpijn, myalgieën (vooral in de ledematen) en respiratoire symptomen zoals keelpijn en hoesten. deze verschijnselen verdwijnen meestal na enkele dagen. gezonde volwassenen scheiden het virus uit gedurende ongeveer vijf dagen vanaf een dag voor de eerste symptomen, kinderen verspreiden het virus ongeveer zeven dagen en immuungecompromitteerde patiënten nog langer. de belangrijkste en meest voorkomende complicaties van een infectie met het influenza-a-virus zijn primaire virale pneumonie, myocarditis en secundaire bacteriële infectieziekten zoals otitis media, pneumonie en acute bronchitis. 19 bij een secundaire bacteriële infectie worden streptococcus pneumoniae, haemophilus influenzae en staphylococcus aureus als belangrijkste verwekkers gevonden. 20 bij preëxistente cardiale of pulmonale aandoeningen en diabetes mellitus is het risico op morbiditeit en mortaliteit ten gevolge van influenza waarschijnlijk groter. bij diabetes mellitus speelt naast ontregeling van de diabetes mellitus mogelijk ook een verminderde afweer een rol. algemene principes op populatieniveau: afwenden en vertragen van een influenzapandemie tijdens de dreiging van een influenzapandemie (who-fasen 3 t/m 5) zal men zoveel mogelijk maatregelen moeten treffen om deze af te wenden. tijdens een manifeste influenzapandemie (who-fase 6) is het vertragen van de pandemie van groot belang. dit heeft onder andere tot doel de piekbelas-ting van de gezondheidszorg en van het maatschappelijk bestel als geheel zo klein mogelijk te maken. een tweede doel is tijd winnen zodat men andere interventies kan voorbereiden, zoals het bekrachtigen van individuele preventieve hygiëne, het distribueren van antivirale middelen en het ontwikkelen en produceren van een vaccin. in principe zijn er, afgezien van agrarische en veterinaire maatregelen om het aantal geïnfecteerde vogels te verminderen, drie mogelijkheden om een dreigende influenzapandemie af te wenden of te vertragen: ingrijpen in de infectieketen, toediening van antivirale middelen en vaccinatie. isolering/quarantaine de verspreiding van een influenzapandemie wordt veroorzaakt door het snelle verloop van een infectieziektecyclus. de incubatieperiode van het huidige aviaire influenzavirus (h5n1) wordt geschat op twee tot acht (gemiddeld drie tot vijf) dagen. op basis van gegevens uit eerdere influenzapandemieën neemt men aan dat iedere influenzapatiënt één tot twee andere personen infecteert. 21 in principe leveren alle interventies die het risico van transmissie beperken een bijdrage aan het indammen van de verspreiding van een influenzapandemie. het isoleren van patiënten en in quarantaine houden van gezonde maar mogelijk besmette patiënten is echter alleen zinvol bij een dreigende introductie van een influenzapandemie vanuit verafgelegen landen (azië), of om een potentieel pandemisch influenza-a-virus (who-fasen 3 t/m 5) primair te isoleren. als een influenzapandemie in eigen land dreigt door te dringen vanuit de ons omringende landen (who-fase 6) zijn deze methoden niet langer zinvol, omdat het pandemische virus in nederland dan al te zeer verspreid zal zijn. vermijding van contacten uit een scenarioanalyse van het rivm blijkt dat sluiting van scholen tijdens de piek van een influenzapandemie kan leiden tot vermindering van het aantal geïnfecteerden. 22 de overheid zou dan ook, na advies van onder andere het rivm/cib, kunnen besluiten om tijdens een influenzapandemie scholen te sluiten en massaevenementen af te gelasten in die regio's waar influenza zich voordoet. omdat dit echter grote maatschappelijke en economische gevolgen kan hebben, wordt aanbevolen het sluiten van scholen te laten afhangen van de te verwachten ernst en omvang van de influenzapandemie. hygiëne goede hand-en hoesthygiëne dragen bij aan het tegengaan van de verspreiding van het virus. over het dragen van mond-neusmaskers bestaat minder duidelijkheid. deze lijken vooral functioneel bij werkers in de gezondheidszorg tijdens het begin van een influenzapandemie. hoewel het wetenschappelijke bewijs voor de effectiviteit van mond-neusmaskers en oogbescherming ontbreekt, wordt het gebruik onder bepaalde omstandigheden (in de prepandemische fasen) toch aangeraden. 23, 24 antivirale middelen antivirale middelen kunnen worden gebruikt zowel ter behandeling als ter preventie van influenza. daarbij dient men te kiezen voor neuraminidaseremmers en niet meer voor de oudere middelen (amantadine en rimantadine). 25 neuraminidase is een essentieel enzym op het membraan van het influenzavirus, dat het de gerepliceerde virussen mogelijk maakt om de gastheercel te verlaten. is dit enzym uitgeschakeld, dan kunnen nieuwe virussen de geïnfecteerde cellen niet verlaten en kan de infectie zich niet naar andere cellen verspreiden. de patiënt is minder en korter besmettelijk voor anderen en de verspreiding van het virus in de populatie blijft beperkt. op theoretische gronden is het waarschijnlijk dat neuraminidaseremmers ook bij een nieuw pandemisch influenzavirus effectief zullen zijn; zeker is dit echter niet. de mogelijke ontwikkeling van resistentie is een grote zorg. 26 mede om deze reden is het essentieel om terughoudend te zijn met het voorschrijven van antivirale middelen bij seizoensgebonden influenza. 27 de neuraminidaseremmers oseltamivir en zanamivir zijn geregistreerd voor zowel de behandeling als de profylaxe van influenza a. 28 primaire profylaxe primaire profylaxe met antivirale middelen (dat wil zeggen profylaxe zonder dat er aanwijsbare nauwe contacten met een patiënt met aviaire influenza zijn geweest) wordt in geen enkele fase van een pandemie geadviseerd. deze bescherming wordt immers slechts geboden zolang het middel wordt gebruikt. na het stoppen van de profylaxe is de persoon door het ontbreken van immunologische bescherming nog steeds vatbaar voor het virus. bovendien zou de beschikbare hoeveelheid antivirale middelen ontoereikend zijn voor langdurige profylaxe op grote schaal en zou het risico op de ontwikkeling van resistentie toenemen. men zou alleen tot primaire profylaxe kunnen besluiten bij bijzondere groepen patiënten, bijvoorbeeld patiënten met een onvoldoende functionerend afweersysteem ten gevolge van een beenmergtransplantatie of bewoners van een goed te isoleren afdeling van een verzorgings-of verpleeghuis waar influenza is vastgesteld, voor wie het een te groot risico zou zijn om met de behandeling te wachten tot na de eerste ziekteverschijnselen. 29 postexpositieprofylaxe postexpositieprofylaxe met antivirale middelen (dat wil zeggen profylaxe van mensen die in contact zijn geweest met een patiënt met aviaire influenza, zoals gezinsleden) is alleen geïndiceerd tijdens een dreigende pandemie (whofasen 3 t/m 5). 30 dit heeft tot doel de pandemie te vertragen of zelfs in de kiem te smoren. de beslissing om postexpositieprofylaxe in te zetten dan wel ermee te stoppen wordt door de nederlandse overheid genomen. het huidige criterium om voor postexpositieprofylaxe in aanmerking te komen is meer dan vier uur onbeschermd contact vanaf het begin van de incubatietijd. dit tijdscriterium geldt echter niet voor gezinscontacten en zorgverleners; zij komen in aanmerking voor postexpositieprofylaxe ongeacht de duur van het contact. tijdens een manifeste pandemie (who-fase 6) is postexpositieprofylaxe zinloos en wordt deze niet meer geadviseerd. behandeling alle patiënten met aviaire influenza komen in aanmerking voor behandeling met antivirale middelen, bij voorkeur te starten binnen 48 uur na het begin van de griepachtige symptomen. tijdens een dreigende influenzapandemie (whofasen 3 t/m 5) dient het klinische beeld virologisch bevestigd te zijn. tijdens een manifeste pandemie is virologische bevestiging niet noodzakelijk en wordt de patiënt behandeld op grond van de klinische symptomen. vaccinatie vaccinatie biedt de beste bescherming tegen influenza en is daarom de beste methode om een pandemie in te dammen. 31 een vaccin kan pas ontwikkeld worden wanneer het pandemische influenzavirus bekend is. vanaf dat moment (whofase 4) duurt het vier tot zes maanden voordat men met vaccinatie kan beginnen. op het gebied van vaccinaties zijn veel ontwikkelingen gaande. 32 waarschijnlijk moet aan het vaccin een hulpstof worden toegevoegd ('adjuvering') en is dubbele vaccinatie (met enkele weken interval) noodzakelijk om voldoende immunologische respons op te bouwen. het zal na de vaccinatie nog enkele weken duren voordat een gevaccineerde persoon immunologische bescherming tegen het virus heeft ontwikkeld. zodra er een pandemisch vaccin beschikbaar komt, zal de overheid de volgorde van de te vaccineren groepen vaststellen. deze volgorde zal mede bepaald worden door het dan bekende klinische beeld en door de epidemiologische karakteristieken van het pandemische influenzavirus. op theoretische gronden is het waarschijnlijk dat oseltamivir en zanamivir ook bij een nieuw pandemisch influenzavirus effectief zullen zijn, maar zeker is dat niet. deze middelen verkorten bij infectie met een regulier influenza-a-virus de duur van de symptomen met ongeveer één tot twee dagen, mits de therapie start binnen 48 uur na het optreden van de griepachtige verschijnselen. dit effect is zowel aangetoond bij gezonde volwassenen als (voor oseltamivir) bij gezonde kinderen ouder dan 1 jaar en risicopatiënten. onvoldoende is aangetoond dat deze middelen ook complicaties bij risicopatiënten voorkomen. een recent onderzoek heeft laten zien dat behandeling van influenza met oseltamivir ook bij risicogroepen kan leiden tot minder gebruik van antibiotica voor influenzagerelateerde lageluchtweginfecties. 33 men betwijfelt dat het zin heeft neuraminidaseremmers te starten als er meer dan 48 uur vertreken zijn na de eerste symptomen. 34 bij relatieve contra-indicaties voor deze middelen (zoals zwangerschap en borstvoeding) zal men de potentiële voor-en nadelen zorgvuldig tegen elkaar moeten afwegen. tijdens een manifeste pandemie (who-fase 6) wordt geadviseerd om iedereen met een griepachtig ziektebeeld te behandelen met neuraminidaseremmers, uiterlijk te starten binnen 48 uur -maar het liefst zo snel mogelijk -na het ontstaan van de eerste ziekteverschijnselen. men neemt aan dat patiënten die een neuraminidaseremmer krijgen bij de eerste symptomen van influenza -en niet profylactisch -, een immunologische bescherming tegen het virus opbouwen en daardoor bij een tweede besmetting niet (of veel minder) ziek worden. bij profylactisch gebruik van neuraminidaseremmers treedt een dergelijke immuniteit waarschijnlijk niet op. tabel 2 vat de therapeutische en profylactische toepassingen van neuraminidaseremmers bij een (dreigende) influenzapandemie samen. de distributie van antivirale middelen vindt plaats via de reguliere kanalen: de arts schrijft voor, de apotheek verstrekt. huisartsen wordt ontraden om patiënten op verzoek antivirale middelen voor te schrijven zonder medische indicatie. het in voorraad geven van deze middelen kan tot een onjuist gebruik leiden, met verminderde werkzaamheid, ontbreken van weerstandsopbouw, of vorming van resistentie tot gevolg. 27 beleid bij een dreigende pandemie (who-fasen 3 t/m 5) bij een patiënt met algemene (koorts, malaise, hoofdpijn en/of spierpijn) en respiratoire (hoest, keelpijn, kortademigheid en/of neusverkoudheid) verschijnselen na direct contact met besmet pluimvee of vogelmest, of met een verdachte reisanamnese kan sprake zijn van aviaire influenza. men moet echter bedacht zijn op eventueel afwijkende symptomen. 18 bij een vermoeden dat de patiënt aviaire influenza heeft, dient de huisarts te overleggen met de arts infectieziektebestrijding van de lokale of regionale ggd over het te voeren beleid. in een situatie waarin nog geen of slechts enkele ziektegevallen gemeld zijn, is het van groot belang om een mogelijke casus virologisch te bevestigen en verspreiding van het virus zoveel mogelijk te beperken. daarbij gelden de volgende uitgangspunten. c bij een vermoeden van aviaire influenza dient de patiënt, indien dat medisch verantwoord is, thuis te blijven. c tijdens het contact met een patiënt die mogelijk aviaire influenza heeft, moet de huisarts persoonlijke beschermingsmaatregelen in acht nemen: draag handschoenen, een mondneusmasker, een bril en een disposable schort met lange mouwen, en was na het contact met de patiënt grondig de handen en ontsmet ze met handalcohol. in who-fase 3 zijn een mondneusmasker en handschoenen afdoende. 39 c ziektegevallen dienen altijd virologisch bevestigd te worden. dit is de verantwoordelijkheid van de ggd nadat de huisarts melding heeft gemaakt van de verdenking. 40 de uitslag van virologische diagnostiek is binnen enkele uren bekend. c omdat in deze fasen de besmetting in de populatie als beperkt mag worden verondersteld, is het zinvol contactonderzoek te doen en maatregelen te nemen ten aanzien van de directe contacten van de patiënt. een patiënt met vermoeden van aviaire influenza (zie uitgangspunten) kan op verschillende wijzen onder de aandacht van de huisarts(praktijk) komen: telefonische presentatie maak een afspraak voor visite (een patiënt met aviaire influenza moet, indien dat medisch verantwoord is, thuis blijven) en bespreek dat de huisarts beschermende maatregelen moet nemen. presentatie aan de balie van de praktijk geef de patiënt een mond-neusmasker en plaats hem in een aparte ruimte. presentatie in de spreekkamer geef de patiënt een mond-neusmasker. onverwachte presentatie tijdens een visite geef de patiënt een mond-neusmasker indien dat beschikbaar is, en overleg ter plekke met de arts infectieziektebestrijding van de lokale of regionale ggd. voor alle bovengenoemde situaties gelden de volgende regels. c na het treffen van beschermende maatregelen (voor de patiënt en, indien dit niet mogelijk of afdoende is, voor zichzelf) gaat de huisarts na of de patiënt voldoet aan de criteria voor aviaire influenza. c bij blijvende verdenking op aviaire influenza neemt de huisarts contact op met de arts infectieziektebestrijding van de lokale ggd over aanvullende diagnostiek en eventuele behandeling en contactopsporing. c de huisarts geeft voorlichting over hand-en hoesthygiëne (zie kader) aan de patiënt en diens omgeving. c de huisarts geeft de patiënt de bij influenza gebruikelijke adviezen en bespreekt wanneer deze opnieuw contact moet opnemen. c na afsluiting van het contact deponeert de huisarts handschoenen, mond-neusmasker en schort in een vuilniszak die met het huisafval mee kan, en desinfecteert zijn handen met handalcohol. voorlichting zie kader. tijdens een manifeste influenzapandemie komt iedere patiënt die verschijnselen van influenza heeft in aanmerking voor behandeling met antivirale middelen. geadviseerd wordt om hiermee zo snel mogelijk te starten -uiterlijk binnen 48 uur na de eerste ziekteverschijnselen. 44 de huisarts kan kiezen tussen oseltamivir en zanamivir (zie tabel 3), beide te gebruiken gedurende vijf dagen. het is belangrijk dat antivirale middelen op de juiste indicatie worden voorgeschreven. onjuist gebruik kan leiden tot resistentievorming en tot voortijdige uitputting van de voorraad antivirale middelen (die naar men schat bij correcte indicatie-stelling toereikend zal zijn voor de nederlandse bevolking). de overheid zal in de aanloop tot een pandemie een publiekscampagne coördineren waarin voorlichting over het gebruik van antivirale middelen is opgenomen. ook bij voldoende beschikbaarheid van antivirale middelen wordt niet geadviseerd om neuraminidaseremmers profylactisch voor te schrijven, ook niet aan werkers in de gezondheidszorg. de middelen bieden immers slechts bescherming zolang men ze gebruikt en de patiënt bouwt geen immunologische bescherming op, zodat men na het stoppen van de profylaxe nog steeds vatbaar is voor het virus. in zeer bijzondere omstandigheden kan profylaxe echter worden overwogen. 47 een specifiek vaccin zal de beste bescherming bieden tegen een pandemisch influenzavirus. omdat het ontwikkelen van een specifiek tegen de pandemische virusstam gericht influenzavaccin tijd vraagt, is het vrijwel onvermijdelijk dat er in de beginfase van de pandemie een groot tekort aan vaccin zal zijn. van overheidswege wordt bepaald welke groepen dan met voorrang gevaccineerd worden. 47 daarbij kunnen (deels) andere doelgroepen gelden dan tijdens de jaarlijkse influenzavaccinatie. indien hun medische toestand het toelaat, worden patiënten zoveel mogelijk verzorgd in de thuissituatie. het doormaken van een ongecompliceerde influenza-infectie is geen reden tot opname. dit geldt ook voor patiënten in een verzorgings-of verpleeghuis. voorwaarden hiervoor zijn: c de patiënt (en/of diens gezinsleden) is geïnstrueerd om bij verslechtering van het ziektebeeld de huisarts (of huisartsenpost of in te richten zorgmeldpunt) te bellen voor advies en beoordeling; c er is voldoende zorg voor de patiënt in de thuissituatie (gezin, mantelzorg, thuiszorg). indien er voldoende opnamemogelijkheden in het ziekenhuis zijn, worden geen aanvullende opnamecriteria gehanteerd. zowel een gecompliceerd beloop van de influenza-infectie als een exacerbatie van comorbiditeit kunnen indicaties zijn voor op-name. opname vindt alleen plaats na overleg tussen de huisarts en de behandelaar in het ziekenhuis. bij een tekort aan opnameplaatsen zullen landelijk opnamecriteria worden geformuleerd. in gevallen dat deze criteria niet voldoen, is de triage in handen van een regionale triagecommissie, bestaande uit medisch deskundigen. hoewel de aard en frequentie van voorkomen van complicaties nog onbekend zijn, is al wel een aantal uitgangspunten voor het formuleren van opnamecriteria vastgesteld. 49 deze nhg-standaard geeft achtergronden en algemene adviezen met betrekking tot het beleid bij een dreigende en een feitelijke influenzapandemie. een goede organisatie van de huisartsgeneeskundige zorg en samenwerking van huisartsen met andere disciplines tijdens een influenzapandemie zijn van groot belang voor de uitvoerbaarheid van het geadviseerde beleid. daarom is in aansluiting op deze standaard een nhg-implementatieplan beschikbaar 50 waarin het beleid in meer detail en toegespitst op de huisartsenpraktijk wordt uitgewerkt. dit implementatieplan dient te worden ingebed in de lokale gezondheidszorgstructuur, waarin ten tijde van een manifeste influenzapandemie het ghor-bureau de regie voert. 2 zo moeten ook op lokaal en regionaal niveau afspraken gemaakt worden over de geneeskundige hulpverlening ten tijde van schaarste aan huisartsen of andere zorgverleners, over de geneeskundige hulpverlening buiten praktijkuren en over de eventuele instelling van categorale influenzaspreekuren of zorgmeldpunten. uitgangspunt hierbij is dat de beroepsgroep de huisartsenzorg zo lang mogelijk zelfstandig waarborgt. ghor-bureaus, huisartsenvereniging en huisartsenposten hebben inmiddels regionale samenwerkingsovereenkomsten gesloten waarin afspraken zijn vastgelegd over onder andere opleiding, training en oefening en over de instelling van zorgmeldpunten. de huisarts moet zich tijdig op de hoogte stellen van de lokaal en regionaal gemaakte afspraken. op verzoek van het ministerie van volksgezondheid, welzijn en sport begon in juni 2006 een werkgroep met het opstellen van een concept nhg-standaard influenzapandemie. in november 2006 werd de conceptstandaard met enkele kleine wijzigingen geautoriseerd door de nhg-autorisatiecommissie. de werkgroep werd begeleid door dr. w. opstelten, huisarts en wetenschappelijk medewerker van het nhg. influenzadeskundigen waarschuwen al geruime tijd voor een influenzapandemie, die in het verleden met intervallen van 11 tot 42 jaar heeft plaatsgevonden. critici vinden het absolute risico van een pandemie daarentegen overtrokken, zeker in vergelijking met 'algemeen geaccepteerde' risico's (zoals het cumulatieve risico om aan reguliere influenza te overlijden). volgens hen ontwikkelt zich dan ook een buitenproportionele angst voor een pandemie [bonneux 2006 ]. de geschiedenis leert echter dat de gevolgen van een eventuele pandemie desastreus kunnen zijn en tot een extreem grote (over)belasting van de gezondheidszorg kunnen leiden. om die reden wordt geadviseerd om plannen te maken voor het geval zich een epidemie van wereldomvang voordoet, door welk micro-organis-me dan ook veroorzaakt [kroes 2004 , bartlett 2006 ]. actuele ontwikkelingen met betrekking tot (de dreiging van) een pandemie staan vermeld op http://www.who.int. de coördinatie van de medische hulpverlening bij een pandemie berust bij een regionaal ghor-bureau (geneeskundige hulpverlening bij ongevallen en rampen). in nederland bestaan 25 ghor-bureaus. elke ghor-regio heeft een regionaal geneeskundig functionaris (rgf) die, ondersteund door het eigen ghor-bureau, verantwoordelijk is voor de afstemming van de zorgverlening tussen samenwerkende zorgorganisaties bij een pandemie. de 25 rgf'en hebben zich verenigd in een landelijke raad van rgf'en (http://www.rgf.nl). alle ghor-bureaus beschikken over drie regionale deeldraaiboeken: (1) aviaire influenza; (2) incidentele introductie nieuw humaan influenzavirus in nederland; (3) bestrijding influenzapandemie). deze deeldraaiboeken bevatten de voor de regio geldende protocollen en zijn gebaseerd op draaiboeken die door het bureau landelijke coördinatie infectieziektebestrijding [lci 2006 ] zijn opgesteld. sinds een aantal jaren richt de aandacht zich vooral op het h5n1-influenzavirus. tot op heden (november 2006) is nog geen efficiënte mens-op-mensoverdracht van dit virus beschreven. sceptici menen dat het onwaarschijnlijk is dat genetische veranderingen tot deze eigenschap zullen leiden, omdat het virus gedurende de afgelopen tien jaar niet in staat is gebleken om deze eigenschap te verwerven. het h5n1-virus zou dan primair een aviair influenzavirus blijven, dat sporadisch mensen besmet die in nauw contact met vogels leven. echter, ieder influenza-a-virus waartegen geen weerstand (meer) bestaat, kan tot een pandemie leiden. zo zou ook de herintroductie van het 'aziatische' h2n2-virus in theorie opnieuw tot een influenzapandemie kunnen leiden. de ghor-bureaus zien toe op een toereikende capaciteit van psychosociale hulpverlening bij ongevallen en rampen. de huisarts kan het ghor-bureau benaderen voor (bemiddeling bij) psychosociale hulpverlening. wij geven hier de criteria voor en de classificatie van aviaire influenza (voor h5, met als voorbeeld h5n1) zoals het ecdc die momenteel hanteert. in de who-fasen 4 en 5 vat men het virus niet meer op als een aviair virus maar als een nog niet optimaal aangepast humaan virus. gemakshalve gebruikt deze nhg-standaard toch de benaming 'aviaire influenza' ter onderscheiding van seizoensgebonden influenza [ecdc 2006 ]. het ecdc maakt onderscheid tussen een influenza-achtig ziektebeeld (iaz) en een acute respiratoire infectie (ari). voor iaz gelden de volgende drie klinische criteria: c acuut optreden van symptomen en c prominente algemene symptomen, waaronder één of meer van de volgende: koorts, malaise, hoofdpijn en spierpijn, en c een of meer van de volgende respiratoire symptomen: hoest, keelpijn, kortademigheid en neusverkoudheid. voor ari gelden de volgende drie klinische criteria: c acuut optreden van symptomen en c prominente luchtwegsymptomen, waaronder één of meer van de volgende: hoest, keelpijn, kortademigheid en neusverkoudheid, en c het oordeel van de arts dat de aandoening door een infectie wordt veroorzaakt. voor influenza moet ten minste een van de volgende laboratoriumtests positief zijn: c een influenzavirus is geïsoleerd uit een klinisch monster; c nucleïnezuur van influenzavirus is aangetoond (door middel van een pcr) in een klinisch monster; c influenza-antigeen is aangetoond in een klinisch monster met behulp van directe immunofluorescentie; c een stijging van antistoffen tegen influenza is aangetoond in gepaarde monsters. c er is een epidemiologisch verband met een bevestigd ziektegeval door mens-op-menstransmissie. classificatie van gevallen van influenza: c mogelijk ziektegeval: iedere patiënt die voldoet aan de klinische criteria voor iaz; c waarschijnlijk ziektegeval: iedere patiënt die voldoet aan de klinische criteria voor iaz en bij wie epidemiologische relatie met influenza vastgesteld is; c bevestigd ziektegeval: iedere patiënt die voldoet aan de klinische criteria voor iaz en aan de laboratoriumcriteria voor influenza. internationaal zijn richtlijnen vastgesteld waaraan alle nationale overheden zich in principe hebben geconformeerd. het is evenwel denkbaar dat een nationale overheid onder bepaalde omstandigheden van deze richtlijnen afwijkt. dit zou zich kunnen voordoen tijdens een lokale schaarste aan antivirale middelen of wanneer het pandemisch influenzavirus zich heeft verspreid in een gebied ver buiten de landsgrenzen terwijl het in het land zelf nog niet is aangetroffen. het influenza-c-virus is lastig te isoleren; daarom is er weinig onderzoek gedaan naar de klinische symptomen. het virus veroorzaakt meestal een bovensteluchtweginfectie bij kinderen jonger dan 6 jaar. vooral bij kinderen jonger dan 2 jaar is er een risico op complicaties (pneumonie) [matsuzaki 2006 ]. tot voor kort waren er vijftien h-subtypen geïdentificeerd, zoals ook de meeste reviews vermelden [nicholson 2003 ]. recent werd een zestiende h-subtype aangetoond [fouchier 2005 ]. om een infectie met influenza te voorkómen is een adequate hoeveelheid antistoffen tegen hemagglutinine (h) noodzakelijk. antistoffen tegen neuraminidase (n) en de cellulaire afweer door cytotoxische t-cellen kunnen wel de ernst en de duur van de influenza-infectie verminderen, maar haar niet voorkomen [couch 1993 , tumpey 2005 . de antistoffen worden geproduceerd door b-cellen; zij zorgen voor de zogeheten humorale immuniteit. deze is specifiek gericht tegen een bepaald subtype influenza, terwijl de cellulaire afweer kruisimmuniteit kan opwekken voor andere influenza-a-subtypes en voor influenza b [mcelhaney 1994 ]. aangezien b-cellen voor hun productie van antistoffen afhankelijk zijn van t-cellen, kan de bij het ouder worden optredende vermindering van de t-celfunctie leiden tot verminderde humorale immuniteit. momenteel wordt onderzocht in hoeverre interventies zoals jaarlijkse vaccinatie, hogere vaccinconcentraties, boostervaccinaties of vaccinadjuvering de cellulaire immuniteit kunnen verbeteren. het is nog onduidelijk of de tot nu relatief hoge incidentie van h5n1 onder kinderen en jongvolwassenen toe te schrijven is aan het feit dat ouderen in het verleden vaker zijn blootgesteld aan influenza. wel is er enig bewijs dat ouderen beschikken over een grotere (cellulaire) kruisimmuniteit voor nieuwe aviaire of pandemische influenza-a-virussen [jameson 1999 ]. het is echter onwaarschijnlijk dat de overlap in het n-subtype ervoor zou kunnen zorgen dat antistoffen tegen n1 (door voorafgaande expositie aan h1n1) ook enige kruisimmuniteit tegen h5n1 geven [ecdc 2006 ]. de pandemieën uit 1957 en 1968 zijn terug te voeren op uitwisseling van genetisch materiaal tussen een aviair en een humaan influenzavirus. het pandemische virus uit 1918 is ontstaan door geleidelijke mutatie van een aviair influenzavirus [belshe 2005 ]. in 1918-1919 trof influenza vooral jongvolwassenen, in 1957 vooral kinderen van 4 tot 14 jaar. de helft van de influenzagerelateerde sterfgevallen tijdens de pandemie van 1968-1969 en een aanmerkelijk percentage van de influenzagerelateerde sterfgevallen tijdens de pandemieën van 1957-1958 en 1918-1919 betrof personen jonger dan 65 jaar [simonsen 1998 ]. hoewel tijdens de eerste maanden van een influenzapandemie naar verwachting 50% van de bevolking met het virus besmet zal raken, wordt uiteindelijk vrijwel de gehele bevolking geïnfecteerd. naar verwachting zal een eerste besmetting met het pandemisch influenzavirus op een tijdstip dat het virus al enige maanden in de populatie gecirculeerd heeft minder ernstige klinische gevolgen hebben, omdat de pathogeniciteit van het virus geleidelijk afneemt. bovendien is de kans groot dat enkele maanden na introductie van een pandemisch influenzavirus een vaccin beschikbaar is. het percentage patiënten dat de huisarts tijdens een influenza-epidemie consulteert [de hollander 2006] is een onderschatting van het totaal aantal patiënten met influenza, omdat niet alle influenzapatiënten hun huisarts consulteren en sommige patiënten met complicaties van influenza zich buiten de huisarts om tot een ziekenhuis wenden. het is weliswaar de algemene verwachting dát er zich weer een influenzapandemie zal voordoen, maar het is niet te voorspellen wanneer dit zal zijn. de twintigste eeuw kende een aantal influenzapandemieën met intervallen van ongeveer tien tot veertig jaar: in 1918-1919 de spaanse griep (h1n1, geschatte oversterfte in nederland 600 per 100.000 inwoners), in 1957-1958 de aziatische griep (h2n2, geschatte oversterfte in nederland 50 per 100.000 inwoners) en in 1968-1969 de hongkonggriep (h3n2, geschatte oversterfte in nederland 30 per 100.000 inwoners). de oversterfte is de sterfte verminderd met de sterfte in overeenkomstige perioden in het verleden zonder aangetoonde influenza-activiteit [de jong 1999] . de omvang van de op deze manier berekende oversterfte wordt mogelijk overschat, omdat ook andere factoren (zoals de gelijktijdige circulatie van het rs-virus) kunnen bijdragen aan oversterfte. er verschijnen regelmatig onderzoeken waarbij voor verschillende wereldsteden een epidemie volgens een standaardtheorie wordt berekend. hierbij wordt de intensiteit van personenverkeer afgeleid uit de passagierslijsten van luchtvaartmaatschappijen. op basis van passagierslijsten uit het jaar 2000 verwacht men dat een influenzapandemie die begint in hong kong 120 tot 160 dagen later in west-europese steden aankomt [grais 2003 , hufnagel 2004 . een typische eigenschap van influenzapandemieën is het optreden in twee of meer golven van toenemende letaliteit [de jong 1999 , potter 1998 ]. case reports van patiënten met een h5n1-besmetting, bij wie ernstige diarree en neurologische verschijnselen domineerden in afwezigheid van respiratoire symptomen, kunnen erop wijzen dat een nieuw pandemisch influenzavirus mogelijk een ander klinisch beeld veroorzaakt dan een van de thans circulerende humane influenza-a-virussen [de jong 2005] . zeldzame complicaties zijn myo-en pericarditis en het syndroom van reye (een zeldzame maar ernstig verlopende aandoening, gekenmerkt door een initiële fase met symptomen van een gewone virale infectie zoals koorts, hoofdpijn en spierpijn, gevolgd door heftig braken, bewustzijnsverlaging en metabole ontregeling). deze complicaties kunnen optreden bij kinderen vanaf 6 maanden tot 18 jaar. het is onzeker of er een causaal verband bestaat tussen het syndroom van reye en virale infecties -met name influenza b en in mindere mate influenza a en varicella. het verband met acetylsalicylzuur is niet strikt bewezen, maar er zijn aanwijzingen dat dit middel de kans op het krijgen van het syndroom verhoogt. de mogelijkheid bestaat dat acetylsalicylzuur, op basis van een genetisch bepaald metabool defect, het syndroom uitlokt in aansluiting op een virale infectie , mcgovern 2001 . daarom wordt bij kinderen onder de 18 jaar acetylsalicylzuur afgeraden en paracetamol geadviseerd. ook myositis, encefalopathie en het syndroom van goodpasture zijn als complicaties beschreven [agyeman 2004 , studahl 2003 ]. tijdens de influenzapandemie van 1918 (en in volgende pandemieën en epidemieën) was een groot deel van de mortaliteit waarschijnlijk toe te schrijven aan bacteriële luchtweginfecties, veroorzaakt door streptococcus pneumoniae, haemophilus influenzae en streptococcus aureus [brundage 2006 ]. de waarde van het basisreproductiegetal (r 0 ) wordt voor een pandemisch influenzavirus geschat op 1,68 [longini 2004 ]. ter vergelijking: de r 0 voor sars bedraagt 3 en voor mazelen 15 [wallinga 2004 ]. alle interventies om de transmissie te blokkeren, zoals vermindering van contacten met zieken en hand-en hoesthygiëne, dragen bij tot het verlagen van de r 0 . bij een r 0 < 1 dooft de pandemie uit. de r 0 is afhankelijk van de omvang van de populatie die vatbaar is voor het virus. in de loop van een pandemie zal deze populatie, en dus de r 0 , afnemen. schoolsluiting gedurende één week tijdens de piek van de pandemie is op populatieniveau zeer effectief: het uiteindelijke aantal geïnfecteerden daalt van 50% tot 48%. schoolsluiting is het meest effectief tijdens de piek, omdat dan het grootst mogelijke aantal besmettelijken thuis komt te zitten. ter vergelijking: schoolsluiting gedurende één week in het begin van de pandemie stelt het moment van de piek enkele dagen uit en verlaagt het uiteindelijke aantal besmette personen nauwelijks [wallinga 2004 ]. het humane influenzavirus verspreidt zich aerogeen via de inhalatie van geïnfecteerde druppels, door direct contact en wellicht indirect door zelfbesmetting van de luchtwegen of de conjunctivae via stofdeeltjes die virus bevatten [salgado 2002 , bridges 2003 ]. hoe efficiënt deze transmissieroutes zijn, is on-bekend. het advies van hand-en hoesthygiëne en oogbescherming is op deze transmissieroutes gebaseerd, maar er is geen wetenschappelijke onderbouwing voor de doeltreffendheid van de geadviseerde hygiënische maatregelen [davies 1994 , who writing group 2006a . de who adviseert om het gebruik van mond-neusmaskers te laten afhangen van het risico van blootstelling aan het virus. het routinematig dragen van maskers op openbare plaatsen zou moeten worden toegestaan, maar niet aangemoedigd [who writing group 2006b] . voor informatie en advies over de preventie van infecties kan men zich wenden tot de stichting werkgroep infectie preventie (http://www.wip.nl; stwip@wip.nl). goede handhygiëne bestaat uit [lci 2006b ]: noot 27 voorschrijven van antivirale middelen zonder medische indicatie het voorschrijven van antivirale middelen aan patiënten zonder een actuele indicatie wordt ontraden. het in voorraad hebben van dit middel werkt onjuist gebruik in de hand en kan daardoor tot gevolg hebben dat de middelen minder werkzaam worden, dat de patiënt geen weerstand opbouwt of dat er oseltamivir-resistente influenzastammen ontstaan [brett 2005 ]. bovendien kan het patiënten een valse zekerheid geven, zodat klachten later gemeld en interventies later ingezet worden. de nederlandse overheid beschikt over een noodvoorraad neuraminidaseremmers die voor 10% bestaat uit zanamivir en voor 90% uit oseltamivir. de oseltamivir is voorradig als ruwe grondstof. tijdens een manifeste pandemie zal deze, in sachets, via de normale distributiekanalen aan de apotheek worden geleverd. voor gebruik moet de oseltamivir dan worden opgelost in water. deze toedieningsvorm is echter niet geregistreerd en de minister zal hieraan goedkeuring moeten verlenen [raad van rgf'en 2006]. men kan hierbij denken aan patiënten wier afweersysteem onvoldoende functioneert, bijvoorbeeld mensen die een beenmergtransplantatie hebben ondergaan en aidspatiënten met een sterk verlaagd aantal cd4+-cellen. geadviseerd wordt om de beslissing over het al dan niet geven van profylaxe over te laten aan de behandelend arts van de individuele patiënt. het verdient geen aanbeveling om alle bewoners van verpleeg-en verzorgingshuizen profylaxe te geven. in principe kan men ook hen zo snel mogelijk na het ontstaan van de eerste ziekteverschijnselen behandelen. overigens is het wel voorstelbaar dat een behandelend arts toch tot profylaxe overgaat bij zeer zwakke patiënten of in bijzondere situaties, bijvoorbeeld wanneer een gedocumenteerde influenza optreedt in een goed te isoleren afdeling van een instelling. geadviseerd wordt om zogeheten essential service workers (verplegend personeel, politie, brandweer) evenmin profylaxe te geven, maar hen bij het optreden van influenza direct te behandelen. als er voldoende voorraad neuraminidaseremmers is, wordt geadviseerd om in de periode dat het pandemische virus circuleert profylaxe te geven aan (pandemiespecifieke) risicogroepen en professionals gedurende de eerste weken nadat zij gevaccineerd zijn tegen influenza -in de fase dat hun immunologische bescherming wordt opgebouwd [gezondheidsraad 2005 ]. deze methode wordt ook wel aangeduid als ringprofylaxe [balicer 2004 ]. geadviseerd wordt om deze profylaxe zo spoedig mogelijk te starten. omdat de incubatietijd van het influenza-a-h5n1-virus bij mensen acht (meestal drie tot vijf) dagen kan bedragen, is het zinvol om tot acht dagen na mogelijke blootstelling aan een h5n1-bron met profylaxe te starten [beigel 2005 ]. postexpositieprofylaxe gedurende zeven [welliver 2001] of tien , hayden 2004 ] dagen leidt tot verminderd optreden van influenza in de behandelde gezinnen en een gereduceerde virusuitscheiding bij de personen die ondanks postexpositieprofylaxe toch ziek worden [welliver 2001 ]. uit mathematische analyses blijkt dat postexpositieprofylaxe kan leiden tot een beperking of zelfs uitdoving van een pandemie [wallinga 2004 , longini 2004 . een duur van tien dagen leidt, getuige de onderzoeksresultaten, niet tot een beter resultaat dan een duur van zeven dagen [welliver 2001 , hayden 2004 ]. het sterkste effect wordt beschreven bij postexpositieprofylaxe gedurende acht weken [longini 2004 ]. omdat dit echter een al te grote aanslag zou doen op de voorraad neuraminidaseremmers wordt een periode van zeven dagen geadviseerd. postexpositieprofylaxe met als doel de pandemie uit te doven of af te remmen is slechts zinvol bij een uitbraak van beperkte omvang [gezondheidsraad 2005] . voor het geval dat er een pandemie uitbreekt, zijn diverse scenario's ontwikkeld die uitgaan van de klinische kenmerken van het reguliere influenza-a-virus. als tijdens een pandemie 30% van de nederlandse bevolking influenza krijgt (over een periode van zes tot acht weken) en er geen preventieve maatregelen genomen worden, vinden er door de influenza 10.186 ziekenhuisopnames plaats en sterven er 4040 personen. het vaccineren van risicogroepen voor reguliere influenza (inclusief personen van 65 jaar en ouder) en personeel in de zorg (samen 3,5 miljoen personen) voorkomt 6218 ziekenhuisopnames en 2251 sterfgevallen. indien de gehele bevolking (ongeveer 16 miljoen personen) gevaccineerd zou worden, zouden daarmee ongeveer evenveel ziekenhuisopnames (6339) en sterfgevallen (2302) voorkomen worden. het geringe verschil in effect is te verklaren doordat de pandemie in dit scenario vooral in de risicogroepen tot ziekenhuisopname en/of sterfte zal leiden [van genugten 2001] . ook vaccinatie van kinderen in de schoolgaande leeftijd zou zeer effectief zijn. wanneer 80% van de kinderen jonger dan 19 jaar wordt gevaccineerd, is dit ongeveer even effectief als het vaccineren van 80% van de gehele bevolking [longini 2004 ]. vooral de schoolgaande jeugd is door de grote mobiliteit en de vele contacten een belangrijke verspreidingshaard voor influenza. het huidige onderzoek richt zich onder andere op de ontwikkeling van vaccins die een langdurige bescherming bieden tegen een breed spectrum aan influenzavirussen. deze vaccins zouden niet jaarlijks hoeven te worden toegediend en mogelijk ook (partiële) bescherming bieden tegen een pandemisch influenzavirus [hampson 2006 ]. een analyse van prospectief verzamelde gegevens van 3567 patiënten in de leeftijd van 13 tot 97 jaar laat zien dat behandeling van virologisch bewezen influenza met oseltamivir het totale antibioticagebruik met 26,7% reduceert (14,0% versus 19,1% bij placebo; p < 0,001). het gebruik van antibiotica bij luchtwegcomplicaties nam zelfs af met 55% (4,6% versus 10,3% bij placebo; p < 0,001). in de hoogrisicogroep ontwikkelde 18,5% van de patiënten in de placebogroep een luchtwegcomplicatie waarvoor antibiotische behandeling noodzakelijk was, tegen 12,2% in de oseltamivirgroep (p = 0,02) [kaiser 2003 ]. negentien rct's met neuraminidaseremmers zijn samengevat in een systematische review [jefferson 2006 ]: negen met oseltamivir (twee primaire profylaxe, vijf therapie, twee postexpositieprofylaxe) en tien met zanamivir (twee primaire profylaxe, acht therapie). in vergelijking met placebo hebben neuraminidaseremmers geen effect als primaire profylaxe van iaz (voor oseltamivir, 75 mg oraal per dag: rr 1,28; 95%-bi 0,45-3,66; voor zanamivir, 10 mg geïnhaleerd per dag: rr 1,51; 95%-bi 0,77-2,95). hogere doseringen leiden niet tot een beter effect. beide middelen zijn wel effectief in de primaire profylaxe van influenza: oseltamivir dagelijks 75 mg heeft een effectiviteit van 61% (95%-bi 15-82), oseltamivir dagelijks 150 mg heeft een effectiviteit van 73% (95%-bi 33-89) en zanamivir dagelijks 10 mg heeft een effectiviteit van 62% (95%-bi 15-83). als postexpositieprofylaxe heeft oseltamivir een effectiviteit van 59% (95%-bi 16-80) bij gezinsleden en van 68% (95%-bi 35-84) tot 89% (95%-bi 67-97) bij andere contacten van een indexpatiënt. behandeling van influenza met oseltamivir (75-150 mg) geeft snellere verlichting van symptomen dan met placebo (hr 1,20; 95%-bi 1,06-1,35; dit wil zeggen dat patiënten in oseltamivirgroep, vergeleken met patiënten uit de placebogroep, een 20% grotere kans hebben dat ze op een bepaald tijdstip verlichting van symptomen hebben). het effect van zanamivir is ongeveer even groot (hr 1,24; 95%-bi 1,13-1,36). zowel oseltamivir als zanamivir verminderen de concentratie van het influenzavirus in het neusslijmvlies (gewogen gemiddeld verschil 24 uur na starten behandeling: -0.62; 95%-bi -0,82 --0,41), een effect dat in de loop van de behandeling prominenter wordt. de excretie van virussen wordt echter niet verhinderd. in alle onderzoeken werden de middelen voorgeschreven binnen 48 uur na het optreden van de eerste symptomen. het is onbekend wat de effecten zijn bij toediening na dit tijdsinterval. betrouwbare gegevens over de effectiviteit van neuraminidaseremmers bij aviaire influenza ontbreken. van voorgaande pandemieën is niet bekend welk deel van de sterfte kon worden toegeschreven aan het optreden van secundaire bacteriële infecties. onder een (grotendeels) gezonde populatie van adolescenten en volwassenen die influenza (voornamelijk h3n2) hadden, werd bij 17% een secundaire bacteriële infectie gediagnosticeerd en met antibiotica behandeld. in de meeste gevallen betrof het acute bronchitis of sinusitis; in slechts 1-2% een pneumonie [kaiser 2000 , kaiser 2003 ]. vergelijking van de verwekkers van bacteriële infecties bij patiënten met een pneumonie tijdens de hongkongpandemie (1968) (1969) met de verwekkers in het jaar dat aan de pandemie voorafging, liet zien dat tijdens de pandemie het percentage infecties veroorzaakt door staphylococcus aureus verdubbeld was (26% versus 11%). streptococcus pneumoniae werd het vaakst (48%) gevonden [schwarzmann 1971 ]. antibiotica bij secundaire bacteriële infecties moeten werkzaam zijn tegen staphylococcus aureus en streptococcus pneumoniae. bij patiënten die buiten het ziekenhuis worden behandeld voor een community acquired pneumonia (cap), hoeft bacteriële resistentie geen overweging te zijn bij de keuze van een bepaald antibioticum. met doxycycline en amoxicilline zijn geen gerandomiseerde dubbelblinde onderzoeken verricht bij patiënten met een cap. met deze middelen is echter wel veel ervaring opgedaan, er zijn geen problemen met resistentie bekend, het bijwerkingenprofiel is bekend en de kosten zijn gering. deze middelen zijn derhalve de antibiotica van eerste keuze. in europa worden in toenemende mate infecties gerapporteerd die worden veroorzaakt door community associated methicillin-resistantstaphylococcus aureus (ca-mrsa). bij deze infecties dient ziekenhuisopname overwogen te worden. in gebieden met een hoge prevalentie van penicilline resistente streptococcus pneumoniae wordt geadviseerd bètalactamantibiotica voor te schrijven [bonten 2006 , verheij 2006 ]. dit advies is geformuleerd door de gezondheidsraad [gezondheidsraad 2003 ] en onder meer gebaseerd op de uitkomsten van een meta-analyse, uitgevoerd door het dutch cochrane centre [assendelft 2004 ]. deze meta-analyse is geba-seerd op onderzoeken met het 23-valente polysacharide pneumokokkenvaccin. inmiddels is het 7-valente geconjugeerde pneumokokkenvaccin beschikbaar, dat mogelijk een betere cellulaire immuniteit geeft. er zijn nog onvoldoende gegevens beschikbaar over de bescherming die dit vaccin biedt in deze leeftijdsgroep. er bestaan in nederland drie soorten professionele mondneusmaskers, ffp1, ffp2 en ffp3. ff staat voor filtering facepiece, p staat voor partikeldeeltje en het getal 1, 2 of 3 geeft het onderscheidend vermogen aan. alle drie de types zijn goedgekeurd en in de goedkeuring is ook de randlekkage (het lekken langs de randen van het masker) gedefinieerd. de maximaal toegestane randlekkage is bij alle typen 2%. behalve de randlekkage is er ook filterlekkage van het filtermedium (het maskermateriaal). bij de ffp1 is de maximale filterlekkage 20%, bij de ffp2 6% en bij de ffp3 is zij verwaarloosbaar klein (maximaal 1%). naast wegwerpmaskers zijn er herbruikbare maskers met verwisselbare filters. in de gezondheidszorg komen de herbruikbare maskers niet in aanmerking omdat aan het weer gebruiksklaar maken van dergelijke maskers te veel praktische bezwaren kleven [daha 2006 ]. waarschijnlijk is de besmettelijkheid van mens op mens bij de eerste gevallen van aviaire influenza (who-fase 3) zeer gering omdat het virus voor dat doel nog niet optimaal gemuteerd is. tijdens who-fase 3 kan de huisarts daarom volstaan met beperkte beschermingsmaatregelen en is het dragen van handschoenen en een ffp1-mondkapje voldoende. zodra een nieuwe influenzavariant van mens op mens overdraagbaar is (who-fasen 4 en 5) wordt altijd een ffp2-masker geadviseerd, in combinatie met een goed afsluitende bril, handschoenen en een schort met lange mouwen en manchetten [lci 2006b ]. de ggd coördineert in overleg met het lci de afname en het vervoer van de monsters; de afname van de monsters wordt of door een ggd-medewerker of door de huisarts gedaan [lci 2006b ]. bij het reinigen van de ruimte waar de patiënt verbleef, heeft de schoonmaker handschoenen aan. zichtbaar besmette oppervlakken worden huishoudelijk gereinigd en daarna met 70% alcohol gedesinfecteerd. gooi gebruikte schoonmaakdoekjes en handschoenen weg. handen worden gedesinfecteerd met handalcohol. gebruikt linnengoed reinigen op minimaal 60 graden met een totaal wasmiddel geschikt voor witte was op een volledig wasprogramma [lci 2006b ]. het lci heeft informatiemateriaal voor (potentiële) influenzapatiënten opgesteld. op http://www.rivm.nl/cib/infectieziekten/aviaire_influenza/draaiboek_aviaire_influenza.jsp zijn de volgende documenten te downloaden: -instructie preventiemaatregelen voor een patiënt; -maatregelen voor gezonde huisgenoten; -brief gericht aan een contact van een influenza verdachte patiënt; -informatie voor een contact van een van influenza verdachte patiënt. de uitslag van virologisch onderzoek met behulp van de polymerasekettingreactie (pcr) kan binnen enkele uren na monsterafname bekend zijn. influenzaassociated myositis in children pneumococcal vaccination for the elderly in the netherlands? assessment of the quality and content of available comparative studies tackling the next influenza pandemic planning for avian influenza avian influenza a (h5n1) infection in humans the origins of pandemic influenza: lessons from the 1918 virus an iatrogenic pandemic of panic antibiotics in pandemic flu the run on tamiflu: should physicians prescribe on demand? transmission of influenza: implications for control in health care settings interactions between influenza and bacterial respiratory pathogens: implications for pandemic preparedness advances in influenza virus vaccine research indicatiegebied voor het dragen van de verschillende types mondneusmakers seroepidemiological study of respiratory virus infections among dental surgeons zorg voor gezondheid: volksgezondheid toekomst verkenning 2006. houten: bohn stafleu van loghum influenzapandemieën: verleden en toekomst oseltamivir resistance during treatment of influenza a (h5n1) infection fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma report of the scientific panel on influenza in reply to eight questions concerning avian flu a prediction rule to identify low-risk patients with community-acquired pneumonia characterization of a novel influenza-avirus hemagglutinin subtype (h16) obtained from blackheaded gulls vaccinatie bij een grieppandemie. den haag: gezondheidsraad vaccinatie tegen pneumokokken bij ouderen en risicogroepen. den haag: gezondheidsraad gebruik van antivirale middelen en andere maatregelen bij een grieppandemie. den haag: gezondheidsraad reye syndrome: insights on causation and prognosis assessing the impact of airline travel on the geographic spread of pandemic influenza report of the second meeting on the development of influenza vaccines that induce broad-spectrum and long-lasting immune responses, world health organization neuraminidase inhibitor susceptibility network position statement: 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community-acquired pneumonia: pneumonie opgelopen buiten het ziekenhuis verwachtingen voor het verloop van een influenza pandemie met en zonder interventiestrategieën wiskundige modellering ten behoeve van de inspectie voor de gezondheidszorg oseltamivir (tamiflu) and its potential for use in the event of an influenza pandemic effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial oral oseltamivir treatment of influenza in children non-pharmaceutical interventions for pandemic influenza, national and community measures non-pharmaceutical interventions for pandemic influenza, international measure c handschoenen aantrekken voor ieder contact met de patiënt of zijn directe omgeving; c handen desinfecteren met handalcohol:c na het in contact komen met lichaamsvloeistoffen (snot, sputum); c na het verlaten van de kamer van de patiënten; c na het uitrekken van handschoenen; c voor en na het afzetten van een mond-neusmasker; c indien de handen zichtbaar verontreinigd zijn, vóór de desinfectie met handalcohol de handen wassen met water en zeep.goede hoesthygiëne bestaat uit:c hoesten met afgewend gezicht;c hoesten met de hand voor de mond, waarbij een papieren zakdoek wordt gebruikt; c de papieren zakdoek maar één keer gebruiken en weggooien in de afvalemmer; c na het hoesten de handen wassen met ruim water en zeep of inwrijven met handalcohol. bovenstaande geldt ook voor niezen.voor de effectiviteit van genoemde maatregelen ontbreekt echter een wetenschappelijke onderbouwing. de oudere antivirale middelen (amantadine en rimantadine) vertonen veel bijwerkingen, introduceren resistentie en blijken nauwelijks te werken tegen de h5n1-variant [gezondheidsraad 2005] . resistentie tegen oseltamivir tijdens de behandeling van h5n1-influenza is beschreven bij twee vietnamese patiënten [de jong 2005b] . bij volwassenen met reguliere influenza a (h1n1 of h3n2) is resistentie tegen oseltamivir zeldzaam; bij kinderen echter zijn resistentiepercentages tot 18% gerapporteerd [whitley 2001 , kiso 2004 , ward 2005 . dit verschil wordt verklaard door het feit dat de meeste influenza-infecties bij kinderen primair zijn en gepaard gaan met snelle virusreplicatie, omdat kinderen nog geen immuniteit hebben opgebouwd. een vergelijkbare situatie doet zich voor tijdens een pandemie, waar alle infecties met het pandemische virus als primaire infecties kunnen worden beschouwd. om deze reden is het zinvol om aanvullende antivirale middelen te ontwikkelen en onderzoek te doen naar de effectiviteit van combinaties van antivirale middelen (multi drug therapy). oseltamivir-resistente h1n1-virusstammen blijken overigens in het algemeen wel gevoelig voor zanamivir. bovendien blijken deze stammen bij dierproeven over een verminderd transmissievermogen te beschikken en minder pathogeen te zijn ]. de nederlandse overheid beschikt over een grote hoeveelheid oseltamivir als ruwe grondstof. tijdens een manifeste influenzapandemie zal deze worden geleverd in sachets. patiënten dienen zelf deze sachets te openen en de inhoud in water op te lossen. het openen en ledigen van deze sachets kan voor patiënten met een lichamelijk en/of verstandelijke handicap problemen geven, wat leidt tot verlies van poeder en dus tot een lagere dosis oseltamivir. dit laatste is in het bijzonder ongewenst omdat dit tot onderbehandeling (en dus tot resistentievorming) zou kunnen leiden. indien de huisarts vermoedt dat een patiënt de sachets niet correct kan openen, dan kan oseltamivir in capsules (tamiflu ® ) worden voorgeschreven. dit moet dan nadrukkelijk op het recept vermeld worden. omdat de hoeveelheid oseltamivir capsules beperkt is, kunnen deze alleen in uitzonderingsgevallen voorgeschreven worden.noot 45 behandeling met antivirale middelen wellicht ten overvloede dient vermeld te worden dat dit medicamenteuze beleid uitsluitend geldt in geval van besmetting met een pandemisch influenzavirus. bij infectie met een seizoensgebonden influenzavirus wordt juist een zeer terughoudend beleid met betrekking tot het voorschrijven van antivirale middelen geadviseerd [nhg-standaard influenza en influenzavaccinatie, herziening 2007 (in voorbereiding)]. definiëring van contacten [anonymus 2006b]:c intensieve contacten: gezinscontacten en contacten met medisch en verplegend personeel voor zover deze geen gebruik hebben gemaakt van persoonlijke beschermingsmaatregelen, gerekend vanaf één dag vóór het ontstaan van symptomen bij de patiënt; c gezinscontacten: gezinsleden en anderen die gezamenlijk met de patiënt een huishouden delen of deelden, én personen die gerekend vanaf één dag vóór het ontstaan van symptomen bij de patiënt in dezelfde ruimte hebben vertoefd als de patiënt; c vluchtige contacten: alle overige contacten. c hand-en hoesthygiëne; c indien vanaf één dag vóór het ontstaan van symptomen meer dan vier uur onbeschermd contact heeft plaatsgevonden: postexpositieprofylaxe met oseltamivir (voor gezinscontacten en zorgverleners geldt geen tijdscriterium); c informatie over infectiepreventiemaatregelen; c informatie over eventuele ziekteverschijnselen en hoe dan te handelen; c intensieve contacten kunnen, op voorwaarde dat ze geen symptomen hebben, hun dagelijkse activiteiten buitenshuis voortzetten.maatregelen bij vluchtige contacten:c schriftelijke informatie over ziekteverschijnselen en handelwijze (huisarts bellen, niet bezoeken) bij een infectie met influenza; c standaardbrief die overhandigd moet worden aan de huisarts bij het ontwikkelen van symptomen; c vluchtige contacten kunnen, op voorwaarde dat ze geen symptomen hebben, hun dagelijkse activiteiten buitenshuis voortzetten.noot 47 primaire profylaxe tijdens een manifeste pandemie bij voldoende beschikbaarheid van antivirale middelen kan primaire profylaxe worden overwogen in de volgende situaties [gezondheidsraad 2005 ].c bij de door een arts beoordeelde individuele patiënt met een onderliggend lijden waardoor de kans op complicaties ten gevolge van het doormaken van pandemische influenza groot wordt geacht (bijvoorbeeld aidspatiënten met een sterk verlaagd aantal cd4+-cellen, of patiënten die recent een beenmergtransplantatie hebben ondergaan). c op een goed te isoleren afdeling van een verpleeg-of verzorgingshuis met gedocumenteerde influenza binnen de afdeling. c bij gedefinieerde (pandemiespecifieke) risicogroepen en medische professionals gedurende de periode na vaccinatie met een pandemisch vaccin en in de periode dat een pandemisch virus in nederland circuleert. de profylaxe is dan bedoeld om de niet-optimale bescherming van het vaccin in het begin te ondervangen en daardoor de kans op besmetting te voorkomen. de overheid heeft een contract afgesloten met een vaccinfabrikant voor de eerste acht weken productiecapaciteit voor een pandemisch vaccin. het vroegste moment waarop we in nederland kunnen beschikken over een vaccin is vijf maanden nadat het pandemische influenzavirus geïsoleerd is. het is nog onbekend of de bescherming tegen een pandemische stam door één of twee vaccinaties wordt bereikt. bij een tekort aan vaccin komen de volgende groepen in aanmerking om met voorrang gevaccineerd te worden (in volgorde van afnemende urgentie; tussen de per klasse aangegeven groepen heersen geen voorrangsverschillen) [gezondheidsraad 2000 ]:klasse 1 c patiënten met ernstige afwijkingen en functiestoornissen aan de luchtwegen en de longen, die ondanks hun medicatie een grote kans hebben op decompensatie van de longfunctie bij een infectie met het pandemische influenzavirus. hiertoe behoren patiënten met zeer ernstige asthma bronchiale, een zeer ernstige mate van emfyseem, copd, antracosilicose, longfibrose, mucoviscidose en kyfoscoliose, alsmede patiënten die longresectie hebben ondergaan. c patiënten met een ernstige acute of chronische stoornis van de hartfunctie die ondanks hun medicatie een grote kans hebben op decompensatie van de hartfunctie bij een infectie met het pandemische influenzavirus. c patiënten met een insulineafhankelijke vorm van diabetes mellitus.klasse 2 c zwangeren die tijdens de pandemie in het derde trimester van de zwangerschap verkeren.klasse 3 c patiënten met afwijkingen en functiestoornissen van de luchtwegen en de longen bij wie -in tegenstelling tot de in klasse 1 genoemde groep -hun medicatie een redelijk stabiele compensatie van de longfunctie garandeert. c patiënten met een chronische stoornis van de hartfunctie, bij wie -in tegenstelling tot de in klasse 1 genoemde groep -hun medicatie een redelijke compensatie van de hartfunctie garandeert. deze groep omvat patiënten met stoornissen van de linker hartkamer, patiënten met aangeboren hartafwijkingen of met klepgebreken, allen voor zover hun medicatie toereikend is voor compensatie. c patiënten met chronische nierinsufficiëntie. deze groep omvat onder meer patiënten die behandeld worden met hemodialyse en chronische ambulante peritoneale dialyse (capd), en mensen die een niertransplantatie hebben ondergaan. c kinderen en adolescenten in de leeftijd van 6 maanden tot 18 jaar die langdurig salicylaten gebruiken.c patiënten die na een recente beenmerg-of orgaantransplantatie een immunosuppressieve therapie ondergaan. c patiënten met een niet-insulineafhankelijke vorm van diabetes mellitus. c verstandelijk gehandicapten in intramurale voorzieningen en verpleeghuisbewoners met een ziektebeeld dat predisponeert voor luchtweginfecties (zoals vooral het geval is bij mensen met het syndroom van down).klasse 4c personen van 65 jaar en ouder voorzover zij niet behoren tot een al genoemde groep. c personen tot 65 jaar met verminderde weerstand tegen infecties, waaronder gerekend worden degenen die met hiv geïnfecteerd zijn.de overheid heeft overigens de mogelijkheid om bij het bepalen van een vaccinstrategie af te wijken van het met voorrang vaccineren van bovengenoemde groepen. zo zou bij vaccinschaarste gekozen kunnen worden voor een doelgroep waarin de meeste transmissie van influenza te verwachten is (schoolgaande jeugd) en in eerste instantie niet voor bescherming op individueel niveau. aangezien humane influenzavirussen meestal respiratoire symptomen veroorzaken, worden de criteria voor ziekenhuisopname thans bepaald door de risicofactoren voor een gecompliceerd beloop van de influenza en die voor een pneumonie in het algemeen. omdat niet met zekerheid te voorspellen is wat de klinische eigenschappen van een nieuw pandemisch influenzavirus zullen zijn, is het mogelijk dat tijdens een toekomstige pandemie andere criteria zullen gelden.onafhankelijke risicofactoren voor een gecompliceerd beloop van influenza [fine 1997]: c leeftijd boven 65 jaar, zwangerschap, copd, ernstige stoornis van de hartfunctie, nieraandoeningen, immunosuppressie, hematologische aandoeningen, diabetes mellitus, langdurig gebruik van salicylaten bij kinderen.onafhankelijke risicofactoren voor het optreden van complicaties bij een (community acquired) pneumonie [public health agency of canada 2006]: c leeftijd, mannelijk geslacht, verblijf in een verpleeghuis, onderliggende aandoening (maligniteit, leveraandoening, decompensatio cordis, nierinsufficiëntie), afwijking bij lichamelijk onderzoek (desoriëntatie, ademhalingsfrequentie > 30/min, systolische bloeddruk < 90 mmhg, temperatuur lager dan 35°c of hoger dan 40°c, pols > 125/ min), laboratoriumafwijking (arteriële ph < 7,34, ureum > 11 mmol/l, na < 130 mmol/l, glucose > 14 mmol/l, hematocriet < 30%, partiële arteriële zuurstofdruk < 60 mmhg) en radiologische afwijking (pleurale effusie) het rivm centrum infectieziektebestrijding (cib) is verantwoordelijk voor nadere informatie aan en correspondentie naar geneeskundige hulpverleners. deze informatie is beschikbaar op: http://www.rivm.nl/cib/infectieziekten/ aviaire_influenza/draaiboek_aviaire_influenza.jsp. ook draaiboeken en protocollen over infectieziekten zijn te vinden op deze site; voor specifieke informatie kan men mailen naar: cib@rivm.nl).algemene vragen van het publiek over vogelgriep en/of influenzapandemie worden zo veel mogelijk beantwoord door de regionale ggd's en ghor-bureaus. op de website van het cib staat een uitgebreide lijst met vragen en antwoorden waarnaar men patiënten kan verwijzen (http://www.rivm.nl/ cib/infectieziekten/aviaire_influenza/ ai_nieuws.jsp).ook op de website van postbus 51 (http://www.postbus51.nl) is informatie te vinden over de vogelgriep.reisadviezen zijn te vinden op de websites van het ministerie van buitenlandse zaken (http://www.minbuza.nl) en het landelijk centrum voor reizigersadvisering (http://www.lcr.nl). key: cord-027752-xcpv9k22 authors: bresalier, michael title: uses of a pandemic: forging the identities of influenza and virus research in interwar britain date: 2011-12-15 journal: soc hist med doi: 10.1093/shm/hkr162 sha: doc_id: 27752 cord_uid: xcpv9k22 this paper counters the tendency to retrospectively viralise the 1918–19 pandemic and to gloss the important historiographical point that, in britain, such knowledge was in-the-making between 1918 and 1933. it traces the genesis of influenza's virus identity to british efforts in 1918–19 to specify the cause of the pandemic and it examines how, in the 1920s, the british medical research council used the connection between a virus and the pandemic to justify the development of virus research and to make influenza a core problem around which it was organised. it shows that the organisation of medical virus research was inextricably linked to the pandemic before the actual discovery of flu virus in 1933. recognising that the relationship between the virus and the disease itself has a history demands we rethink the pandemic's medical scientific legacy and the crucial role of virus research in shaping its history. in may 1922, walter morley fletcher, secretary of the medical research council (mrc), organized a secret meeting of pathologists at the war office to outline a new scheme of research on 'diseases probably caused by filter-passing organisms.' 1 created in 1913, the mrc had used the war to apply laboratory science to military medicine. in peace time it was seeking new challenges and the still relatively unexplored filter-passing organisms offered just such opportunities, for medical science and the health of the nation. an immediate reason for the mrc's interest was the 1918-19 influenza pandemic. in his annual report for 1921-22, fletcher stressed that investigations during the pandemic of the purported connection between a filter-passer and influenza were a key motivation for the new scheme. there could hardly be a set of problems whose solution has more potential importance for the community than this. influenza kills regularly, though its slaughter is chiefly effected during epidemics. in a few months in 1918-19 it killed more persons in india than died from the plague there during the previous twenty years …. 2 the pandemic had ignited interest in the nature of filterable viruses, however, the way forward was unclear, as fletcher observed: 'the chief problem which the investigator of [filterable viruses] meets is the difficulty of proceeding by sound experimental methods.' 3 the purported influenza agent was one of a group of pathogens that could not be seen with light microscopes or studied by the culture methods that had been so successful with bacteria. only their pathogenic effects were evident in animal models and could be traced by serological tests. the mrc's new scheme was to build on these methods to make viruses into workable laboratory objects and to develop new methods for their control. this paper traces how, in the decade after 1918, the pandemic and virus research became inextricably linked in the work supported by the mrc. fletcher and his colleagues became convinced that if questions about influenza's virus identity-and more general questions about virus diseases-were to be successfully investigated new researchers and institutions would have to be created. the 1922 plan charged the council's flagship laboratory, the national institute for medical research (nimr), with, among its primary goals, developing research on human and animal virus diseases and linking virus researchers to medical and veterinary institutions. 4 work on the influenza filter-passer was pivotal. british medical science played an important role in the battle against the pandemic, but it was also shaped by it. donald fisher has argued that the pandemic represented a 'central turning point' in efforts to modernize british medicine: 'the death of millions of persons as a result of influenza made the advancement of medical knowledge and practice urgent.' 5 according to fisher, the pandemic prompted the british state to take a new role in medical scientific administration and education, primarily through the mrc. 6 the mrc's collaboration with the rockefeller foundation to reform british medical education was an important legacy. but the pandemic reached into the very organization of medical scientific research. the mrc's virus research scheme clearly demonstrates this point. fletcher regularly invoked the idea that a filterable virus might have caused the greatest pandemic since the black death to bolster support for the scheme. more than a symbolic resource, the identities of the pandemic and virus research were forged together as part of broader mrc plans to scientifically modernise pathology. 7 experience during the war and the pandemic convinced fletcher and his colleagues that pathology needed to be founded on experimental principles, and located above all in university science departments and research institutions. 8 the nimr emerged as one of the key institutional supports for this new pathology, and virus research became closely allied to its development. my argument takes up roger cooter's recent challenge to historicise the relationship between war and epidemics. cooter claims, rightly i believe, that medical historians have taken this relationship as self-evident. 9 this is true of much work on the 1918-19 pandemic, which has retrospectively linked influenza's emergence and virulence to specific war conditions. 10 such approaches are important but fail to highlight how this relationship was understood and used by contemporary actors, particularly those involved in generating medical knowledge. my analysis critically examines ways in which the epidemic-and-war couplet was constructed through laboratory practices and their institutional organisation during and after the pandemic. in britain the mobilisation of the military medical machine against the pandemic bound the disease to the war. 11 especially important were military pathology investigations, supported by the war office and mrc, into the bacteriology of the pandemic and, in due course, into the role of a 'filterable virus'. these investigations not only connected the pandemic to a filterable virus; they also connected virus research to the wartime organisation of pathology. both were used by the mrc as a rationale for developing virus research in the 1920s. by tracing this process, my account counters the ahistorical tendency to retrospectively viralise the pandemic. ever since a virus was established as influenza's primary cause in 1933, virologists and historians have used it to explain many aspects of the pandemic. 12 such histories tend to gloss the important historiographical point that such knowledge was in-the-making between 1918 and 1933, so that there has been a tendency to ignore the historicity of the relationship between influenza, the pandemic and virus research. to properly situate the making and uses of medical knowledge of the pandemic it is important recognize that this relationship has a history that goes right back to the pandemic itself. few histories of the pandemic explore its contemporary microbiology. 13 historians now know that it struck britain in three distinctive waves. a relatively mild epidemic beginning in early spring 1918 was quickly followed by a lethal autumn epidemic after which developed a somewhat less virulent epidemic in spring 1919. an estimated 250,000 died, with most succumbing in autumn 1918. 14 while each wave presented its own clinical and epidemiological characteristics, retrospective accounts have linked them together into a single cataclysmic pandemic. at the time, however, the identities and relationship between the waves baffled medical experts. the summer epidemic shared characteristics with previous visitations, particularly that of 1889-90, with doctors describing extreme body aches, prostration, fever, sore throat, dry cough, nausea and general lassitude in most patients. yet important aspects did not fit the established picture. much confusion stemmed from uncertainties about its aetiology. between early spring and late autumn 1918, a loose bacteriological consensus built before the war fragmented, with competing pathologists and physicians backing different pathogens. resolving the aetiological problem was imperative to official pandemic strategies. the war office, army medical service, and mrc prioritised identifying the suspected influenza germ and developing preventive measures-particularly, vaccines-to control it. these strategies were initially based on the assumption that the germ was bacillus influenzae, an organism first identified in 1891 by the berlin bacteriologists, richard pfeiffer and 11 bresalier 2011. 12 burnet and clark 1942; morens and taubenberger 2009 . 13 tognotti 2003 . 14 langford 2002 , johnson and mueller 2002 shibashuro kitasato. 15 pfeiffer developed special methods for its cultivation, which, he found, required a substrate of blood-in particular, haemaglobulin. 16 he promoted the bacillus as the primary cause of influenza and his blood-agar culture technique as necessary for establishing it. identification of the germ became widely accepted as the best way to distinguish 'true influenza' from other influenza-like conditions. in britain, pfeiffer's claims were first substantiated in 1893 by e. e. klein, a founder of british bacteriology, in investigations for the medical department of the local government board (lgb). 17 pfeiffer's techniques were slowly incorporated into bacteriological practice and 'pfeiffer's bacillus' gained standing as the 'germ of influenza'. 18 both became part of the general organization of pathology for war. 19 british approaches to the pandemic were elaborated within the contexts of military medicine, and defined by military imperatives. 20 like all facets of british society, medicine had been mobilised for total war. over half the medical profession was eventually enlisted, along with many hospitals. medical science was also mobilised and the mrc played a vital role in its coordination. most important for the subsequent fight against influenza, fletcher worked closely with william boog leishman, advisor on pathology to the war office, to link pathological laboratories to base and field hospitals in france and flanders. 21 organised to collect, identify, and control pathogens, military pathology delivered therapeutic and preventive measures against a range of battlefield diseases, and its planners trusted that it could do the same with influenza. 22 signs of the spring wave were first encountered in military garrisons in france and flanders in march 1918. the epidemic perplexed medical authorities. it occurred in late spring instead of autumn. rather than the usual susceptible groups-the very young, aged, and infirm-it affected soldiers in the prime of life. 23 few typical symptoms were evident. 24 but most importantly, when pathologists ran bacteriological tests they rarely found b. influenzae. so seldom were pathologists' efforts successful that many concluded that b. influenzae was at best associated with, but not essential to, the epidemic. bacteriological evidence did not support classifying the epidemic as influenza. rather, it generated controversy. through the summer of 1918, two camps of pathologists clashed over the causal agent: the 'pfeiffer school' argued that the epidemic was influenza, and attributed failures to find b. influenzae to technical failures; the 'anti-pfeiffer school' argued that its absence indicated either that the epidemic was not influenza or that influenza was caused by another organism. 25 they proposed a range of known bacteria, including streptococci, staphylococci, and streptodipplococci. but no one could agree which played the primary role. 15 coles 1904, pp. 144-9 . 19 prüll 1998. 20 bresalier 2011. 21 prüll 1998 . 22 leishman 1923 . 23 tanner 2002 abrahams et al. 1919; french 1919. 25 ludwik rajchman, editor of the mrc's medical supplement, used the term 'pfeiffer school' those who believed that 'true influenza epidemics' were caused only by b. influenzae, 'influenza', medical supplement, 1 october 1918, p. 354. fildes and 119, used the term 'anti-pfeiffer school' to describe its critics. this situation jeopardised official prevention strategies. without an agreed agent against which to develop a specific vaccine, army medical authorities decided to produce a 'mixed' vaccine, which incorporated bacteria associated with secondary complications, including b. influenzae. 26 generally viewed as unsatisfactory, the decision added official backing to doubts about pfeiffer's bacillus. 27 these doubts also prompted leishman and his colleagues to organise research into other agents. 28 fletcher argued that, 'on the hypothesis that b. influenzae, no less than pneumococci and streptococci, are secondary [infections]', official strategy would be better served by exploring the possible role a so-called 'filter-passing virus.' 29 in november 1918, the mrc and the war office put in train a 'search for an unrecognized virus'. 30 little was known about the basic nature of filter-passing viruses. the category only emerged at the turn of the century, when it became popular among some bacteriologists as a way to explain diseases for which causes could not be ascertained by standard bacteriological methods. 31 filter-passing viruses were operationally defined by the fact that they were neither retained by standard bacteriological filters, nor susceptible to cultivation on artificial media, nor visible by available methods of light microscopy. 32 a pathogen was characterised as filterable when clinical material passed through the smallest of available filters still induced disease in a host. 33 the new category included a number of important human and animal diseases: foot-and-mouth, fowl pox, rabies, swine fever, measles and poliomyelitis. 34 the first filterable virus theory of influenza was proposed in 1914 by the german bacteriologist, wilhelm kruse, in studies of the common cold. 35 british interest grew in 1918 as a possible explanation for bacteriological failures. between october and december 1918, the mrc helped s. l. cummins, advisor in pathology to the ams, organise filterpasser studies in abbeville, flanders and etaples, france. 36 major howard graeme gibson led the abbeville team. 37 with major f. b. bowman and captain j. i. connor, gibson's team followed the work of two french bacteriologists, charles nicolle and charles lebailly, who claimed in early 1918 that they had discovered a filter-passer. as proof, nicolle and lebailly reported that they had reproduced the disease in healthy monkeys and men by serial inoculation of filtered bronchial secretions from infected cases. 38 seeking to repeat these experiments, gibson's team relied on the mrc for expertise and materials, including macaque monkeys and baboons shipped from london zoo. 39 within weeks, gibson reported that his team had isolated a filterable agent and produced an experimental disease. 40 in december 1918, they described how they had reproduced a characteristic lung haemorrhage in monkeys, similar to that seen in clinical cases, 26 'the utilisation of vaccine for the prevention and treatment of influenza', lancet, 26 october 1918 , p. 565. 27 matthews 1918 horder 1918. 28 na, fd1/530 fletcher to fildes 22 and 28 october 1918; na, fd1/533 mrc, influenza general research, 1918 , 1 november 1918 na, fd1/533 fletcher to fildes, 28 october 1918. 30 ibid. 31 van helvoort 1994. 32 rivers 1928, pp. 3-52 . 33 rivers 1928, p. 6 . 34 waterson and wilkinson 1978 . 35 kruse 1914 . 36 mrc 1919 gibson et al. 1919 . 37 cummins 1919a . 38 thomson and thomson 1933, p gibson et al. 1918, pp. 645-6. and isolated and cultured minute 'coccoid bodies' from the tissue. since the agent passed through filters and produced 'experimental influenzal' lesions, they reckoned that it was a 'filterable virus' and 'in all probability the cause of influenza' (figure 1 ). 41 tragically, gibson died from influenza, leaving the team's work unfinished, but not before pathologists at etaples, headed by major-general john rose bradford and captain james wilson, claimed to have isolated similar coccoid bodies (figures 2 and 3) . 42 although preliminary, the studies won support from the lancet and the bmj. cummins argued that the 'two series of observations, carried out independently, should confirm each other, [and] greatly strengthen the case for the new organism.' 43 f. w. andrewes, director of the department of pathology at st bart's, who had assisted on klein's 1893 investigations of pfeiffer's bacillus, pointed to gibson's experiments as providing the best evidence for the primary role of a filterable virus. 44 these endorsements did not allay criticisms. in 1919, j. a. arkwright, then working with the war office on trench fever, levelled a devastating critique of the etaples research. 45 arkwright demonstrated that the coccoid bodies were identical to those found in uninoculated tubes and that the cultures were contaminated with ordinary bacteria. in effect, the bodies were not pathogens, but either benign globoids or bacteria. this analysis forced rose bradford and wilson to reconsider, and in a stunning move, they publicly retracted their claims. 46 bradford et al. 1919, pp. 19-36. 43 gibson et al. 1919, p. 24 . 44 andrewes 1920, pp. 110-25 . 45 arkwright 1919, pp. 233-5 . 46 arkwright 1919, pp. 236-7. arkwright's criticisms coincided with renewed support for pfeiffer's bacillus. in 1920, paul fildes and james mcintosh, two leading london pathologists, published new evidence for its primary role and launched their own attack on the virus theory. 47 former colleagues at the london hospital's department of bacteriology, they shared the view that the bacillus was the agent of the pandemic, and that new techniques made it possible to demonstrate its role. mcintosh wrote: [t]he epidemic can be divided into two stages, a first in which b. influenzae was seldom demonstrated, and a second, in which this bacillus was demonstrated with great regularity. this fact is not attributable to any alteration in the epidemic itself, but to the application of new methods for the demonstration of the bacillus of influenza. 48 most important among these techniques were 'selective media', which inhibited the overgrowth of cultures by other microorganisms. 49 fildes and mcintosh argued that selective culture enabled them to establish a causal link between the bacillus and influenza. first, they could regularly identify the bacillus from large numbers of cases. second, they could isolate it from broncho-pneumonia lesions clinically associated with the disease. finally, they could use pure cultures to reproduce the disease in experimental animals. for fildes and mcintosh, the evidence generated with selective media was enough to counter the 'great revolt against the view … that b. influenzae … was the cause of the disease.' 50 building on arkwright's criticisms, they argued that filter-passing work was 'unconvincing'. 51 gibson's research was symptomatic of its shortcomings. neither the identity of the virus nor its relationship to influenza had been demonstrated. 52 moreover, inoculation experiments on humans-of the kind used by nicolle and lebailly-were easily discredited fig. 3 etaples filter-passer films. in films the organism had 'the appearance of a minute, rounded, or slightly oval, undifferentiated coccus-like body, arranged in colonies of twenty to sixty elements'. source: bradford, bashford and wilson 1919b, p. 308. 50 fildes and mcintosh 1920, p. 119 . 51 fildes and mcintosh 1920, p. 159. 174 . 52 fildes and mcintosh 1920, p. 164. because, as other observers noted, researchers either failed to isolate subjects from previous infections or simply produced a different illness. 53 the alleged filter-passer thus failed to meet any of koch's postulates. most damningly, they reckoned that researchers used the filter-passer as an alibi: 'the invisible virus concept absolves the discoverers from the necessity of producing evidence of a characteristic microbe.' 54 these criticisms cast doubt on whether influenza's cause could ever be determined. in a 1922 review, the bacteriologist robert donaldson observed that there were no good empirical grounds to support either agent. 55 while he agreed with criticisms of the filterable virus, he found little to support pfeiffer's bacillus. while frequently found in post-mortem studies, most inoculation experiments failed to produce a characteristic lesion in laboratory animals. 56 this strongly suggested that it was a secondary infection. donaldson challenged the idea that a causal inference could be drawn from its apparently high incidence during the autumn epidemic. 'we are not at liberty to claim, that because an organism is always present, it is therefore necessarily the cause.' 57 proponents of pfeiffer's bacillus had mistaken an association for a cause. some critics took the conflicting aetiological claims as indication of the failure of bacteriologically-based approaches. 58 andrew mendelshon has shown that a contingent of british physicians and epidemiologists used these conflicts to attack reductive approaches to disease causation and to promote multifactorial models to examine the role of hereditary and environmental factors in variations in the severity, incidence and susceptibility to infectious diseases. 59 but while such models found support in epidemiology, pubic health and clinical medicine, the mrc countered them. contrary to critics, official pandemic strategies were widely lauded. while war conditions may have contributed to the virulence of the epidemic, british mortality, in both the services and amongst civilians, was lower than in other countries. 60 fletcher insisted that the rapid organisation of coordinated strategies was testimony to the merits of military pathology. translating military pathology's success into peace time became an mrc goal, and both the pandemic and virus research were enrolled in this mission. the pandemic highlighted the need for a new direction in laboratory pathology, towards filterable viruses. at the same time, virus research represented a way to preserve a specific aetiology as the guiding principle of pathological research and to counter challenges to the authority of laboratory medicine. mrc interest in virus research was intimately tied to its larger goal of modernising the organisation of basic sciences in medical training, practice and research. 61 for fletcher, the pandemic demonstrated the particular need to modernise pathology. 62 this was not the old pathology of the mortuary, rather the microbiology-inspired enterprise, previously fostered in germany and france, that linked aetiologies with patho-physiology and immunology, and focused preventive and therapeutic measures on them. while the 53 maitland et al. 1921 . 54 fildes and mcintosh 1920, p. 159 . 55 donaldson 1922, pp. 139-213 . 56 maitland and cameron 1921, p. 492; maitland et al. 1921 . 57 donaldson 1922 , p. 158. 58 crookshank 1922 hamer 1927 . 59 mendelsohn 1998 . 60 johnson 2002 . 61 austoker 1989 , pp. 22-33. 62 mrc 1924 war confirmed its importance, fletcher attributed the failure to control the pandemic to a general lack of institutional support for basic pathological research. he feared that peace time would mark a return to a pre-war pathology, defined by its subordination to clinical and public health interests. 63 in his first annual report after the war, he characterised british pathology as an anathema to experimental science. 64 unlike physiology, which had generated researchers, discoveries, and university departments of international importance, pathology lacked an experimental orientation and a place in universities. in hospitals, medical schools, and public health, its practitioners played a service role, generating income but little new research and few trained researchers. 65 for fletcher, the pandemic underscored the need for an experimental approach to pathology located in dedicated laboratories, funded by the state, and coordinated by experts. 66 this vision found support in the new british journal of experimental pathology. established in 1920 by a group of mrc researchers, led by fildes, the journal emphasized the multidisciplinary nature of experimental pathology and its role as a foundation for rational medical knowledge and practice. 67 the bjep defined itself as providing an alternative to descriptive research in the morbid anatomy and epidemiology of infectious disease. it aimed to publish 'original communications describing the techniques and results of experimental research into the causation, diagnosis and cure of disease in man'. 68 no one discipline defined this approach. for its founders, experimental pathology joined together 'bacteriological, biochemical, pharmacological, physiological, serological and other subjects' in the production of new pathological knowledge. 69 multidisciplinary in character, it was bound together by the core principle of specific aetiology. fletcher built this broader vision into his campaign to create new pathology departments at oxford and cambridge. 70 but it was at the nimr that it was most completely realised. the idea of the institute was born with the creation of the mrc in 1913. only after the war was its position consolidated as the flagship for medical science. occupying mount vernon hospital in hampstead in north london, built between 1880 and 1900 and purchased by the mrc in 1914, originally it was to be modelled on the rockefeller institute for medical research in new york (rimr) (figure 4) . the mrc incorporated important aspects of the rockefeller approach-particularly the positioning of experimental sciences as foundational to medical education and to the production of medical knowledge and therapeutics-and depended on its patronage. 71 yet the mrc research system was also adapted to the british context, where clinical medicine remained dominant. as in germany, the mrc made the state an active agent of change and used its own authority and institutions to pursue its agendas. the nimr was crucial to the mrc mission. an independent government institution, with no formal affiliation with hospitals, its primary function was to foster experimental medicine, with experimental pathology one of its cornerstones. 72 experimental pathology 63 lawrence 2006 , p. 48. 64 mrc 1921 kohler 1985, pp. 55-6 . 66 alter 1987. 67 the first editorial board included fildes, mcintosh, j. a. murray and w. e. gye. 68 fildes 1920, p. i. 69 ibid. 70 weatherall 2000, p. 169-72 . 71 austoker 1989, p. 27; fisher 1978a fisher , 1978b wilkinson 2000; wilkinson and hardy 2001. 72 austoker and bryder 1989, pp. 35--38; thomson 1973, pp. 108-9. came under the remit of the department of bacteriology, headed by s. r. douglas. it shared the institute's broader organisational ideology of team work, which prioritised research that straddled disciplinary boundaries. 73 douglas and henry hallet dale, the institute's acting director, decided that experimental pathology would be primarily constructed around filterable viruses. the need for a dedicated virus scheme became readily apparent in the first years after the pandemic, when fresh investigations into the identity of the filter-passer in influenza highlighted the underdeveloped state of the field. in 1920, the mrc started funding new influenza 'virus' work by mervyn h. gordon at st barts department of pathology. an enthusiastic researcher, gordon had worked in the department since 1908. early on, he was drawn to testing the idea that filterable agents might be causes of high-profile infectious diseases for which bacterial agents could not be found. in 1912, he confirmed the claim made by the viennese pathologists karl landsteiner and erwin popper, that a filter-passer played a primary role in poliomyelitis. 74 in 1914, he announced that he had successfully isolated a filter-passer from cases of mumps. 75 when he returned to filter-passer work after the war, he and andrewes decided to focus on influenza. a member of the mrc, andrewes became convinced during the pandemic that a filterable virus held the keys to its aetiology. 76 an impetus for gordon's research was a widely reported discovery by two american pathologists, peter k. olitsky and frederick l. gates, of an alleged 'new' filter-passer from cases of influenza. 77 working at the rimr in new york, olitsky and gates first identified the agent during the pandemic. in may 1920 they reported a new technique to produce and serially transmit a 'definite and characteristic' infection in rabbits. like human influenza, the experimental infection was localized in rabbits' lungs, from where they isolated an agent. to establish its filterability, they passed solutions of ground lung material through grades of berkefeld filters, and then used the filtrate to reproduce the infection in healthy rabbits. they also employed a special method for source: wellcome library. 73 thomson 1973, p. 109 . 74 gordon 1911 gordon -1912 . 75 gordon 1914 . 76 andrewes 1920, pp. 110-15. 77 olitsky and gates 1920. cultivating the agent, developed in 1911 by their colleague, the japanese-born bacteriologist, hideyo noguchi. the so-called 'noguchi' medium was composed of fresh rabbit liver tissue, set in narrow glass tubes, sealed with wax and vaseline. 78 rockefeller researchers had used it with the common cold virus, as had british researchers at etaples in their influenza work. according to olitsky and gates, the medium enabled them to make pure cultures of the agent, to photograph it, and to study its properties. in a further fourteen papers published between 1920 and 1923, they detailed its morphological, pathogenic, and serological characteristics. they concluded that it was a minute organism, with particular affinity for the lungs, and accordingly named it, bacterium pneumosintes-'a bacterium that injures the lung' (figure 5 ). 79 the american researchers's animal model and culture system appeared to solve the two fundamental methodological problems that had hampered filter-passer studies in 1918. yet, the reception of their research among supporters of the filter-passer theory was mixed. gordon, for one, believed they had identified an agent similar to that identified by british workers in 1918, and had only 'added precision' to these original studies. 80 he tested their claims during an epidemic in late december 1921. using the noguchi method, he ran bacteriological tests on nasal and throat washings of staff at st barts hospital and the ministry of health. 81 in early january he reported to landsborough thomson, assistant secretary to the mrc, that 'something very like [olitsky and gates'] filter passer is coming up in my cultures.' the agent also looked like the one identified by british workers. 82 but gordon admitted that, 'rendering the filterable organism was difficult,' because its presence was only indicated by a rather vague 'cloudiness near the piece of kidney at the foot of the [noguchi] tube.' it became visible when gordon made films of material that had been fixed, stained, and chemically differentiated. these procedures yielded 'swarms of minute round bodies', but they were so small that they could be 'very easily missed unless especially looked for', and might be dismissed 'by an inexperienced fig. 5 olitsky and gates' bacterium pneumosintes. two stained cultures from a rabbit's lung into which were infected nasal secretions from a case of 'epidemic influenza'. on the left, the culture was magnified ×1000. on the right, it was magnified ×3000. infectivity was tested by inoculation into the lungs of rabbit. source: olitsky and gates 1921a, p. 729. 78 noguchi 1911. 79 olitsky and gates 1921b, p. 106. 80 gordon 1922a, p. 6. 81 gordon 1922a, pp. 7,10. 82 na, fd1/1297 gordon to thomson, 11 january 1922; gordon to fletcher, 3 february 1922. observer'. 83 unable to induce an experimental disease, gordon used ultraviolet photomicrographs of the culture and slide preparations to compare their morphology with those described by the british and americans ( figure 6 ). it was unclear if the agent was a 'coccoid' or a 'baccilloid', and he was unable to induce disease in experimental animals. moreover, when he consulted existing literature on filter-passers, he was struck by their similarity with bodies found in other diseases. these problems raised rather than settled questions about the filter-passer's identity. the most vexing centred on its nature and classification. gordon and his american counterparts reckoned that it was a filterable organism. its 'prodigious multiplication' convinced them that it was living and not, as earlier critics had claimed, a protein. 84 this aligned it with the dominant view of filterable viruses in medical and veterinary pathology: their ability to multiply, demonstrated by pathogenesis, was evidence of their biological nature. yet this was not the only theory available for explaining filterable viruses; an important alternative approached them as chemicals. 85 this theory had various roots, but in british pathology it was most influenced by f. w. twort's characterization of the bacteriophage as a chemically-induced lytic phenomenon, a concept he introduced in 1915. 86 h. m. woodcock, head of the department of protozoology at the lister institute, enlisted twort's concept to challenge gordon. 87 woodcock accepted the existence of a pathogenic 'virus', but not that it was living. he argued that those who viewed influenza virus as a microorganism had no direct method for distinguishing it from protein particles. gordon's microphotographs did not support this conclusion. quite the contrary, on their staining properties 83 gordon 1922a, p. 9. 84 gordon 1922b, p. 400 . 85 creager 2002, pp. 19, 32-8 . 86 twort 1915 . 87 chick et al. 1971 and physical appearance in microphotographs, they looked more like typical 'protein enzymes', the result of the breakdown of cell material by a ferment. 88 if these bodies were indeed the cause of influenza, woodcock argued, they were not exogenous organisms but by-products of an endogenous chemical process occurring within human cells. these debates spilled into a general debate about viruses and virus diseases, and the experimental methods used to elucidate them. through the early 1920s, the mrc organised several discussions on the state of virus research. the many research challenges were brought to the fore in july 1922 at a special panel on the 'bacteriology of influenza' at the annual meeting of the british medical association. concentrating on gordon's work, discussants probed his filter-passer. james mcintosh recapitulated the view that all studies had failed to establish its pathogenic identity. sympathetic researchers observed that neither adequate methods nor criteria were available for determining its nature. charles ledingham, head of bacteriology at the lister, argued that the greatest difficulty remained 'the lack of any animal, other than man, [that] was readily susceptible to the causal agent'. poor filtration methods were identified as yet another obstacle. j. h. dible, junior lecturer in pathology at manchester university, noted that filtration was 'extremely crude'; bacterial filters were 'not reliable' and the criteria for deeming an agent 'filterable' depended on an inexact calculation of the relationship between the largest hole in the filter and the smallest bit of protoplasm being filtered; much of the process was left to chance. f. w. twort stressed the need for standard methods of filtration. w. b. leishman, who chaired the meeting, concluded that, the status of the filter-passer remained a 'big unsolved problem'. 89 the bma meeting made one thing clear: too little was known about the general category of viruses to determine the identity of one. '[the] search for the primary infective agent in influenza,' agreed gordon, 'has led us into the realm of filter-passers', a realm rife with new problems. 90 supporters of the filter-passer theory viewed these problems as features of an emerging field, the boundaries of which urgently needed to be defined. preferring the bench to the office desk, he embraced the opportunity. 94 the nimr programme was moulded around two lines of work. the first employed physical and biochemical methods to create new instruments and techniques for exploring the 88 creager 2002, pp. 32-3. 89 'bacteriology of influenza ', lancet, 1922, pp. 516-18. 90 gordon 1922a , p. 11. 91 mrc 1923 , p. 11. 92 na, fd1/1297 , 17 june 1922 dale 1941 , p. 430. 94 cameron 1940 fundamental nature of viruses. its main locus was the department's division of applied optics, created in 1920 for j. e. barnard, a west-end hatter and amateur microscopist, who built the first ultra-violet microscope in britain. 95 bernard worked with william gye on the role of a filterable virus in cancer sarcomas, and explored physical problems associated with rendering viruses visible and with filtration methods for purifying and determining their size. 96 the second line of work concentrated on creating pathological and 'immunological devices' for the investigation, identification and control of virus diseases. 97 familiar to bacteriologists, these techniques included new media and experimental animals for growing viruses, and serological assays, therapeutic sera, and vaccines for typing and controlling them. both research lines were closely entwined. but it was the applied aspects of the programme that linked it to medical and public health concerns with influenza and other disease with unknown aetiologies. the public and medical profile of influenza remained high throughout the 1920s. prior to the pandemic it had been viewed as an inescapable part of modern life; but after it was viewed as a major threat. 98 no one knew if, or when, it would again become a deadly pandemic. influenza typically ranked highest amongst cases reported by general practitioners and amongst patients' complaints. 99 there was greater awareness of the disease, with 1922, 1924, 1927 and 1929 designated epidemic years. among infectious diseases, only diphtheria and scarlet fever accounted for higher annual morbidity. 100 although influenza rarely killed on its own, 'influenzal pneumonia' accounted for stunning levels of mortality killing on average nearly ten times more people than diphtheria or measles. 101 resolving influenza's aetiology remained a paramount problem. the mrc considered including it on the nimr programme. but it was deemed unsuitable for basic work because of its aetiological complexity and the lack of a viable experimental animal. rather, it would be studied indirectly through a model disease. when plans were settled in june 1922, three 'virus' diseases were chosen: measles, chicken sarcoma-a model for human cancer-and dog distemper, which served as the model for influenza. according to fletcher, distemper's apparent analogies made it 'peculiarly suitable for working out methods by which human diseases of this class might be subsequently investigated.' 102 distemper addressed a broad range of mrc interests. as fletcher highlighted in his annual report for 1921-22, its relevance as a potential model for influenza was most important: there is good reason to think that [dog distemper] offers a close parallel to human influenza. it seems probable that the infective agent is a filterable virus, and that here also the severity of the resulting disease depends largely upon secondary infections, facilitated by the primary infection. there is ground for hope that the study of dog's distemper under strict experimental conditions may throw important light 95 austoker and bryder 1989; thomson 1973, pp. 112-13 . 96 austoker 1988 , pp. 93-8. 97 mrc 1930 , p. 15. 98 honigsbaum 2010 bresalier 2010 , ch. 1. 99 digby 1999 , pp. 209, 213. 100 deutschman 1953 , p. 636. 101 ministry of health 1930 , p. 30. 102 na, fd1/1297 , 3 may 1922 upon analogous problems of human disease, and at least suggest new clues for investigation or new technical methods for the investigator. it is with the primary object of gaining knowledge of human disease that the council decided to support further study of distemper in dogs. on that ground alone they find complete justification of the expenditure of part of their funds in this direction. 103 concerned that the focus on distemper might be seen as being at odds with the nimr's mandate to work on human diseases, fletcher framed it as good for exploring influenza and general problems associated with virus infection and immunity. 104 important analogies also existed between the state of research on distemper and on influenza. veterinary pathologists were divided on whether a filterable virus or a bacillus caused distemper. its virus aetiology had been first proposed in 1905, but then widely disputed by researchers who aligned themselves behind b. bronchisepticus, identified in 1911. 105 crucially, neither side had access to dogs bred under controlled conditions, so results could always be contested. without accurate means to isolate and test either agent, there was little hope for a vaccine. 106 the prospect of resolving these problems drew together british virus researchers, veterinarians and dog owners. in late 1922, as the nimr was assembling its programme, the country magazine, the field, and the veterinary journal launched a dog distemper fund, with the aim of raising £25,000 for new research. 107 in early 1923, sir theodore cook, the field's editor and honorary secretary of the fund approached fletcher about collaborating on 'research into the causation, prevention, and treatment of distemper'. 108 while a government body, funded through the national health insurance system, the mrc actively sought out patrons for its schemes. its close collaborations with wealthy benefactors, the rockefeller foundation and dunn trust, are well known. 109 but its collaboration with the distemper fund involved a different form of patronage, one that depended on a large and varied group rather than a mighty patron. the fund relied on voluntary contributions from hunt packs, kennels, breeders, associations and middle class dog owners from across britain and the empire, and from the united states. support went well beyond monetary contributions, as many made their own dogs available as research subjects. managing these different interests required a novel research organisation. a distemper research council was created to oversee fund-raising and publicity, and an expert distemper research committee was created to coordinate scientific work at the nimr. 110 all participants shared the belief that experimental research was the best way to develop effective control technologies against distemper. the nimr's department of bacteriology and experimental pathology was made the fulcrum and laidlaw made dog distemper his main research object. 103 mrc 1923 , pp. 12-13. 104 mrc 1923 na, fd1/1275 fletcher to c.j. martin, 13 october 1922 laidlaw and dunkin 1926, pp. 222-3; laidlaw and dunkin 1928a, pp. 5-6. 106 laidlaw and dunkin 1928a, pp. 5-6. 107 worth nearly £750,000.00 in 2005. the fund eventually raised over £38,000 (£1,270,000). for details, see na, fd1/1279, 'saving the living of our dogs ', the field, 4 february 1933 , pp. i-xiii. 108 mrc 1924 , p. 85. 109 kohler 1978 lawrence 2006 . 110 na, fd1/1275 , the cure and causes of distemper, november 1924 . between 1923 and 1932 laidlaw worked with the resident veterinary pathologist, g. w. dunkin, on all aspects of the disease. the single most important obstacle was access to purpose-bred dogs. the need for controlled supplies of experimental animals was a general problem for the institute. 111 with the support of the fund, the mrc built a large-scale, animal-breeding and research facility at mill hill, an agricultural site north of hampstead. 112 completed in 1924, the 'farm laboratories' made provision for breeding and housing dogs and other large animals, a well-equipped laboratory, and an isolation compound for quarantining distempered dogs. 113 the facility put laidlaw and dunkin in a unique position to carry out distemper studies under controlled conditions and to settle the dispute over the causative agent. by 1926, they had ruled out b. bronchisepticus and had established a strong foundation for a filterable virus. 114 the telling piece of evidence came when they used bacteria-free filtrates to reproduce an experimental infection in their dogs. 115 this enabled them to determine the pathogenesis and role of the agent. its filterability partially confirmed its identity, but equally important was its resistance to cultivation. the result was in line with the emerging view that failure to grow filterable viruses in artificial culture was evidence of their unique dependence on living tissue. 116 while the nature of this dependence was not well understood, it was increasingly taken as a defining property. laidlaw and dunkin could thus legitimately claim that, 'from these three [factors] it follows that the infecting agent of dog-distemper belongs to the class of filter-passing viruses.' 117 once they settled the aetiology, they turned their attention to a vaccine. but dogs were not ideal for tackling the problem. dog experimentation was the target of vociferous antivivisection campaigns. 118 moreover, the animals were ill-suited for the work. 119 in particular, distemper varied considerably in dogs, making it difficult to diagnose. searching for a solution, laidlaw and dunkin found that the ferret was highly susceptible to distemper, and easily reproduced and identified in the animal. 120 ferrets were also easier to manage. they bred readily and quickly. unlike the dog, they were known to thrive in small spaces, which made them well suited to confinement in laboratory cages. 121 they were ideal research animals. by 1927, laidlaw and dunkin developed and tested experimental vaccines for ferrets and dogs. 122 their key innovation was a two-step immunisation process. it involved first administering a killed virus, waiting 7-14 days until the animal developed sufficient antibodies, and then injecting it with live virus. 123 following successful trials, in 1928 the distemper council arranged for its commercial production by burroughs wellcome & co in britain and two american companies in the united states. 124 the vaccine became widely available in 1931. 111 kirk 2008 . 112 mrc 1932 dunkin 1928a, p. 6. 114 laidlaw and dunkin 1926. 115 dunkin and laidlaw 1926. 116 van helvoort, 1994. 117 dunkin 1926, p. 228. 118 tansey 1994 . 119 dunkin and laidlaw 1926, p. 201. 120 na, fd1/1275 third report of the distemper research committee-'ferrets ', 7 october 1924 , thomson 1951 , pp. 471-80. 121 laidlaw 1930 laidlaw and dunkin 1927, pp. 1-16. 123 laidlaw and dunkin 1927, pp. 9-10; 1928b, p. 3; 1928a, pp. 13-15; 1928b, pp. 215-26. 124 laidlaw and dunkin 1928a, p. 11 . the american houses were lederle laboratories, in new york, and murphy laboratories in philadelphia. the distemper campaign was hailed as an enormous success. dale described it as an exemplar of 'a complete and systematic investigation of a virus disease', and its culmination of a vaccine for the nation's dog owners made it symbolise the efficacy of the nimr's approach to virus research. 125 a number of aspects ensured that it had wide-reaching significance. it established a style of virus research that linked together fundamental and applied research in ways that broke down that distinction. the innovation and commercial manufacture of a vaccine demonstrated the practical relevance of virus research. from a professional standpoint, the campaign was crucial to legitimising virus research. when laidlaw and his colleagues started their work in 1923, the challenges of rendering viruses by established bacteriological techniques of filtration, in vitro cultivation and light microscopy were well known. not only did these challenges spur technical innovations, they also gave rise to new ways of thinking, the most important of which was the concept of viruses as obligate parasites-entities dependent on living tissue for their multiplication. while this concept had been proposed as early as 1900, as researchers ran up against limitations of culture techniques in the 1920s, it grew into a basic framework. investigating viruses in experimental animals became a necessary condition of medical and veterinary virus research. work at the nimr became organised around this biological concept of viruses and the experimental approach it demanded. the distemper campaign proved that the approach was immensely productive of both expert knowledge and practical tools for tackling virus diseases. when laidlaw and dunkin handed over responsibility for their distemper vaccine to burroughs wellcome in 1931, virus research was becoming an established medical scientific field. through distemper, nimr workers fashioned their scientific identities as virus researchers and the authority of their institute. laidlaw was knighted for his distemper work in 1933. virus research was funded in universities and hospitals, and the lister had created its own programme. in 1929, the mrc devoted an entire volume of its system of bacteriology in relation to medicine to 'viruses and virus diseases', and dog distemper occupied a key place in the category. 126 the mrc had always justified distemper research in terms of its potential applications to influenza. fletcher returned to the theme in 1931, writing that, it is already clear that the usefulness of this work is not to be limited to the prevention and cure of canine distemper. in the field of medical research the work has at many points aided the development of technical methods for the study of viruses in general. 127 by the end of the campaign, the mrc had rallied much support for its convinction that virus research could conquer influenza. through the 1920s, medical and public concern about influenza's toll grew. a times editorial in 1928 observed that, at more or less regular intervals, influenza breaks out and marches across the world, claiming millions of victims and causing grievous dislocation of human enterprise. immense sums of money are spent on sickness benefits and on the care of the sick, and heavy losses are incurred by the majority of industrial undertakings; while numberless men and women lose their health permanently and become dependent on others. 128 the following year, the same paper bemoaned '[t]he sad state of unpreparedness in which the world finds itself ought to awaken determination to discover, if possible, some means of prevention.' 129 yet, hope was at hand. the editorial went on to note that an effective approach had been demonstrated with dog distemper: is it too much to ask that work on similar lines be undertaken on the cause of influenza? the work on distemper has opened a way; general studies organized by the medical research council on virus diseases have made parts, at any rate, of that way smooth. has not the time arrived to launch a campaign and to come to grips with the enemy? 130 these comments suggest that the mrc's strategy of linking influenza to distemper research had been successful in redefining the disease as a problem for virus research. fletcher's rhetoric, now echoed in the times and other general press, raised expectations that tools created to control distemper could be applied to influenza. in early 1932, sir halley stewart, an important mrc patron, offered fletcher £2,500 to launch an 'influenza campaign'. 131 linkage to the distemper campaign was both symbolic and pragmatic. the campaign's success legitimised the nimr approach to virus diseases. its primary goal had become virus identification and control through the production of serological assays, therapeutic sera and, ultimately, vaccines. new interest in applying this approach to influenza was ignited in 1931, when the american veterinary pathologist, richard e. shope announced that a combination of haemophilus bacillus (suis) and a filterable virus produced a disease in pigs-'hog flu'-that was analogous to human influenza. 132 shope's work prompted speculation that a similar type of infection might be the cause of human influenza and re-opened the possibility of finding an animal model for the disease. in late 1932, the mrc decided to concentrate its influenza efforts at the nimr, under the control of laidlaw. 133 within months, the decision paid off. in early 1933, laidlaw and two young researchers, c. h. andrewes and wilson smith, succeeded in using the ferret to isolate a filter-passer from patients in london, including smith himself, and they rapidly identified the agent they called 'w.s.' virus as the primary cause. while shope's work provided an incentive for the research, its organisation, material foundations, personnel and general reception were directly shaped by the distemper campaign. by end of the 1930s, influenza's virus identity had become part of medical and public health knowledge, and the nimr collaborated with researchers around world in developing diagnostic techniques and vaccines. the nimr's success came so quickly only because so much had been put in place in the previous decade. influenza's virus identity is now taken for granted. i have shown that the link between the virus and the disease has many histories. its genesis needs to be traced not to its 'discovery' in 1933 but to the battlefields of the first world war, to military pathology, and to debates over influenza's aetiology during and after the 1918-19 pandemic. most crucial to this process was the strategic use of the pandemic by the mrc to justify construction of virus research, which created the material conditions for laidlaw and his team to establish influenza as a virus disease. historians of virology conventionally suggest that medical and veterinary virus work of the kind pursued at the nimr was bound to a 'bacteriological paradigm', which acted as an obstacle to the development of a modern biochemical virus concept. 134 my account challenges this view. contrary to the notion that nimr virus research was defined by bacteriology, i have shown that viruses and virus diseases were construed as complex research problems requiring a multidisciplinary and collaborative approach. virus workers were committed to the principle of specific aetiology and bacteriology provided an ontological understanding of viruses and methods for tackling them. but the nimr framed virus work as a form of 'experimental pathology', and nimr reseachers, and their colleagues, identified themselves as 'experimental pathologists' rather than as bacteriologists. this distinction took on important meaning as it became evident that viruses resisted bacteriological methods. work done at the nimr, and elsewhere through the 1920s, slowly yielded a concept of viruses as obligate parasites, which set viruses apart from bacteria and forced workers to develop specific skills and knowledge, and new scientific identities. 135 my account of the nimr programme supports angela creager's observation that, rather than being bound by one disciplinary framework, interwar virus work is better understood in terms of its dependence on the interchange of bacteriological, pathological, physical and biological practices. 136 as the distemper case shows, this collaborative enterprise extended beyond the laboratory walls, involving relations with other professionals and lay constituencies. the mrc's ability to rally different groups to the cause of virus research was tied to how it mobilised the experience of the 1918-19 pandemic. my analysis challenges the standard historiographical view that the pandemic had little lasting impact on medical or social institutions. 137 the mrc's virus scheme shows that the pandemic played key roles in the creation of a research system that became an emblem of scientific modernity. rather than leading to the abandonment of laboratory-based pathology, failure to master the pandemic spurred the mrc to improve and expand it. the mrc's ability to mobilize the pandemic points to the existence of a broad consensus about the threat of influenza. 134 van helvoort 1993 . 135 laidlaw 1938 rivers 1928 . 136 creager 2002 . 137 johnson 2002 the story told here suggests we rethink how we write the history of the pandemic. rather than use virology as an explanatory resource, we need to examine its construction as a multidisciplinary field and how influenza-and other diseases-figured into this process. my analysis of the mrc's virus programme underscores the need for accounts of how different actors and institutions used the pandemic to pursue different agendas. historians have acknowledged government support for the mrc during the pandemic but not how the mrc subsequently mobilised the experience in its post-war plans. in this context, virus research emerged as a new domain for tackling infectious diseases and a vehicle for constructing a new experimental pathology. ignoring this dimension of virus research means we ignore its crucial role in shaping the history and meanings of the pandemic. a further investigation into influenzo-pneumococcal and influenzo-streptococcal septicaemia: epidemic influenzal "pneumonia" of highly fatal type and its relation to "purulent bronchitis the reluctant patron: science and the state in britain 1850-1920 the bacteriology of influenza a criticism of certain recent claims to have discovered and cultivated the filter-passing virus of trench fever and of influenza a history of the imperial cancer research fund walter morley fletcher and the origins of a basic biomedical research policy the national 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of expertise: public health policy in britain during the 1918-19 influenza epidemic an investigation of the nature of ultramicroscopic viruses a bacteriological paradigm in influenza research in the first half of the twentieth century history of virus research in the twentieth century: the problem of conceptual continuity an introduction to the history of virology gentlemen, scientists, and doctors: medicine at cambridge animal viruses, veterinary pathology, and the formulation of ideas concerning filterable viruses burgeoning visions of global public health: the rockefeller foundation, the london school of hygiene and tropical medicine, and the "hookworm connection prevention and cure: the london school of hygiene & tropical medicine: a 20th century quest for global public health many thanks to michael worboys, carsten timmerman, john pickstone and colleagues at chstm for valuable comments and suggestions. national archives, kew, surrey. key: cord-008716-38sqkh9m authors: schmidt, alexander c; couch, robert b; galasso, george j; hayden, frederick g; mills, john; murphy, brian r; chanock, robert m title: current research on respiratory viral infections: third international symposium date: 2001-06-01 journal: antiviral res doi: 10.1016/s0166-3542(01)00136-x sha: doc_id: 8716 cord_uid: 38sqkh9m nan the third international symposium on respiratory viral infections was convened by the macrae group (new york, ny) in st. lucia, windward islands, on 1 -3 december 2000. for the third time, this symposium provided a forum for virologists, vaccinologists, clinicians, pharmacologists and public health specialists to discuss recent advances in respiratory virus research in an interdisciplinary fashion (kaiser et al., 1999; munoz et al., 2000) . the spectrum of discussion ranged from basic virology and pathogenesis to vaccinology, immunology, and management strategies for respiratory viral infections. epidemiology of respiratory viral disease and possible preparations for the next influenza pandemic were also an important part of the agenda. until 1953, influenza viruses were the only known filterable human respiratory tract pathogens. in 1931, shope recovered an influenza a h1n1 virus from swine, which probably was the first human influenza virus isolated. two years later smith, andrews and laidlaw recovered the first influenza isolate from humans, and soon after, efforts to develop an inactivated influenza a vaccine began. influenza b and c were isolated in 1940 and 1947 by francis and taylor, respectively. in 1953 in the laboratory of infectious diseases (lid) at the national institutes of health, and hilleman, then working at the walter reed army medical center, recovered the first human adenoviruses and established their importance in acute febrile respiratory tract disease (rowe et al., 1953; hilleman, 1954) . in the following years, robert chanock discovered most of the remaining respiratory viruses that are considered important lower respiratory tract pathogens today. the first of them, a croup-associated myxovirus, was discovered during an outbreak of croup in cincinatti in 1954, and it was later designated human parainfluenza virus type 2 (piv2). in 1956, morris and colleagues recovered the chimpanzee coryza agent (cca) during an outbreak of a cold-like illness in a chimpanzee colony, and a year later chanock and colleagues recovered two similar isolates from an infant with bronchopneumonia and from another infant with laryngotracheobronchitis, and characterized the human virus now known as respiratory syncytial virus (rsv) (chanock et al., 1957) . the discovery in 1958 of piv1 and piv3, the single most common cause of croup and the second most common cause of serious viral pediatric lower respiratory tract disease, respectively, broadened our understanding of the etiology of acute lower respiratory tract disease (chanock et al., 1958) . the discovery of piv4 in 1960 followed in short order. in 1961, a double-blind prospective study evaluating the use of tetracycline in the treatment of cold agglutinin-positive atypical pneumonia led to the identification of the etiologic agent of this disease. the agent, originally recovered by eaton from patients with this form of pneumonia, was known to be filterable and thought to be a virus but its role as an etiologic agent was heavily disputed. a large double-blind study by chanock, kingston and mufson, in which antibodies to the eaton 'virus' were used to define a subset of patients with pneumonia, showed that tetracycline therapy decreased duration of the disease, thereby excluding a virus etiology. subsequently, the eaton agent was shown by chanock, hayflick and barile to be a mycoplasma that grew in cell-free medium; later it was named mycoplasma pneumoniae (chanock et al., 1962) . serologic analyses and studies in adult volunteers confirmed the etiologic role of this organism in cold agglutinin-positive atypical pneumonia. renewed efforts in vaccine development against respiratory viruses began in the 1960s with the observation that infants and young children, after having recovered from respiratory tract infection with adenoviruses, shed virus from their gastrointestinal tract for an extended period of time without experiencing gastrointestinal symptoms. this led to the hypothesis that one could potentially use the gastrointestinal tract to vaccinate against respiratory tract disease caused by these viruses. wild-type adenovirus type 4 and 7 administered orally in enteric-coated capsules was found to protect military recruits against respiratory tract disease caused by these viruses. gastrointestinal symptoms were not observed, and although virus was shed from the intestine, it did not infect close contacts (couch et al., 1963) . the development of vaccines against respiratory viruses suffered a major setback in 1966 when formalin-inactivated rsv vaccine not only failed to protect infants against rsv infection but instead potentiated rsv disease upon subsequent rsv infection (kim et al., 1969) . the inactivated vaccine did not induce a potent neutralizing antibody response but it stimulated an exaggerated cd4 + t cell response without stimulating cytotoxic cd8 + t cells. this unanticipated failure of a non-living vaccine reoriented the research agenda of the laboratory of infectious diseases towards the development of live-attenuated virus vaccines. a cold-passaged rsv strain (cp52) was selected in 1966 as the first candidate live-attenuated rsv vaccine strain (friedewald et al., 1968) . this candidate vaccine was safe and immunogenic in adults and older children but was insufficiently attenuated in seronegative infants (kim et al., 1971) . since then, the search for a live rsv vaccine strain has been a central focus of the lid. developing a live rsv vaccine candidate that is attenuated yet immunogenic in seronegative infants has proved to be a formidable task. since incidence and morbidity of rsv are highest in the second and third month of life, a vaccine candidate has to be safe for administration to neonates, able to stimulate an immature immune system, and able to overcome the immunosuppressive and antiviral effects of passively acquired maternal rsv antibodies. initial vaccine candidates were derived in the late 1960s and early 1970s by passage of virus at low temperature (cold passage) or by chemical mutagenesis. several different lineages of mutants, such as temperature sensitive mutants generated by 5-fluorouracil (5fu) mutagenesis, were evaluated in infants and young children but were insufficiently attenuated or genetically unstable. the cold-passaged (cp) mutant that was subsequently further attenuated by the acquisition of two missense mutations that conferred temperature sensitivity (ts) to yield cpts rsv 248/404, has provided the most promising vaccine candidate tested thus far. this candidate vaccine virus was infectious, safe and immunogenic in 1-month-old seronegative infants, conferred protection against challenge with a second dose of vaccine virus 6 weeks later, and caused only mild upper respiratory tract symptoms (wright et al., 2000) . recent development of a method for rescue of infectious rsv from cdna by collins enhanced our ability to develop rsv vaccine candidates rapidly (collins et al., 1995) . site-directed mutagenesis can now be used for the first time to construct viruses with one or more additional attenuating mutations. using recombinant cdna technology, viable rsv mutants with deletion of the ns1, ns2, sh or m2-2 gene have been constructed as vaccine candidates that bear genetically stable attenuating mutations. for these reasons, it is likely that a live-attenuated vaccine that exhibits an acceptable balance between attenuation and immunogenicity can be developed within the next several years. this vaccine virus might possess one or more gene deletion mutations together with or without the earlier characterized cold-passaged (cp) and temperaturesensitive (ts) mutations. the first piv vaccine candidates were also prepared by formalin-inactivation. similar to the experience with formalin-inactivated rsv, these vaccines did not protect against piv disease. in the early 1980s, belshe attenuated a piv3 isolate by 45 passages at 20°c (cp45) (belshe and hissom, 1982) . in clinical studies, the piv3cp45 vaccine candidate has proved to be safe, genetically stable and immunogenic in seronegative in-fants (karron et al., 1995) . this vaccine candidate is currently being tested in phase ii clinical trials. a recombinant version of piv3cp45 has been rescued from cdna, and the genetic basis of its attenuation (att), temperature-sensitivity (ts) and cold-adaption (ca) phenotypes has been determined (skiadopoulos et al., 1999) . influenza a and b virus vaccine development followed this same path of serial passage at 20°c (cold-adaptation) to generate mutants with att, ts and ca phenotypes (maassab, 1969; maassab and bryant, 1999) . in contrast to piv and rsv, live attenuated influenza a vaccine strains were virus reassortants that were generated by mating the attenuated donor virus with an epidemic wild type virus so that the reassortant virus vaccine was a chimera that contained the attenuating genes of the donor virus, while the ha and na genes were derived from the current epidemic virus (murphy et al., 1980) . this strategy, developed by john maassab, of using the cold-adapted mutant virus a/aa/6/60 as the donor of the six attenuating internal and non-structural genes for construction of reassortant vaccine strains was validated by the large series of consecutive reassortants that have proven to be attenuated and immunogenic. analysis of the genetic basis of attenuation showed that the influenza a pb1 and pb2 genes each consistently specified the ts phenotype, and pa specified the ca phenotype. however, all three of these genes of the viral polymerase complex contribute to the attenuation of the trivalent influenza a (h1n1 and h3n2) and b vaccine viruses (murphy, 1993; maassab and bryant, 1999) . the safety, protective efficacy and phenotypic stability were confirmed in large phase iii trials, and licensure is expected in the near future (belshe et al., 1998) . evidence for the prophylactic effect of serum rsv neutralizing antibodies was demonstrated in the 1980s (prince et al., 1985b) . passive transfer of homologous rsv convalescent serum to cotton rats protected them against rsv replication in the lungs following subsequent intranasal challenge with wild type virus. a serum rsv neutralizing titer of 1:300 in recipient cotton rats conferred almost complete protection. this amount of neutralizing antibody necessary for protection was later confirmed in clinical trials and today forms the basis for passive rsv prophylaxis in high-risk infants (groothuis et al., 1993) . an increased incidence and severity of rsv disease is seen in preterm infants with or without chronic lung disease (cld), in children with congenital heart disease (chd), and in immunosuppressed children and adults. respiratory disease in general is a common cause for re-hospitalization of preterm infants. cunningham and colleagues compared a cohort of preterm infants (mean gestational age at birth 289 2 weeks) to a cohort of term infants and found a 10-fold increase in readmission for respiratory disease in preterm infants without cld, and a 18-fold increase for preterm infants with cld (2.5, 25 and 45%, respectively) (cunningham et al., 1991) . cld patients in a home oxygen program were at even higher risk (53% hospitalization, 13% icu admission) (groothuis et al., 1988) . children with pulmonary disorders such as cystic fibrosis, lung malformation or recurrent aspiration pneumonitis, when admitted for rsv disease, are as likely to require icu treatment (13-50%) and mechanical ventilation (up to 33%) as children with cld (35 and 28%, respectively) (arnold et al., 1999) . earlier studies of high-risk infants admitted for rsv disease yielded similar results, with icu treatment necessary in 13-34% and mechanical ventilation necessary in 12-16% (meert et al., 1990; navas et al., 1992) . apart from preterm infants with or without cld, children with congenital heart disease (chd) are a second group of high-risk patients, particularly when they suffer from pulmonary hypertension. in a prospective study of 699 children hospitalized in five consecutive rsv seasons (1976) (1977) (1978) (1979) (1980) , 63% of 27 patients with rsv disease and chd (compared with 14% of patients without chd) required icu treatment and 37% died (macdonald et al., 1982) . in a prospective study of 214 children with chd, the incidence of rsvrelated hospitalization during one rsv season was 15% for children younger than 4 years and 24% for infants younger than 6 months of age (simoes et al., 1998) . immunocompromised children are a very diverse population and not all of them are at equal risk for severe rsv infection. children receiving corticosteroid therapy have a much lower risk for rsv-related hospitalization and death than children receiving chemotherapy for malignancies or children with primary immunodeficiencies. rsvrelated mortality was 15% for chemotherapy-recipients and 40% for children with primary immunodeficiencies compared with 0% for steroid-treated children (hall et al., 1986) . rsv grew to very high titer in children receiving chemotherapy, and more than half the patients shed rsv for 3 weeks or longer. for immunocompromised adults, the picture is not much different from that described for children. infections are often acquired nosocomially, virus shedding is prolonged, and the incidence of pneumonia and death is high. rsv is the most important viral respiratory pathogen in these patients, followed by picornaviruses, influenza and parainfluenza viruses: 60% of confirmed rsv infections in leukemia and bone marrow transplant patients resulted in pneumonia, and the fatality rate was greater than 30% . whimbey and colleagues reported rsv case fatality rates for bone marrow transplant recipients as high as 31% when therapy was administered early and adequately (ribavirin and ivig), and 100%, when therapy was initiated late or inadequately . hiv-infected children in an urban setting in south africa also have an increased burden of viral lower respiratory tract illness (lri) although respiratory viruses are less frequently isolated from nasopharyngeal aspirates of hiv-infected children than from children without hiv infection. the relative risk for severe lri caused by rsv was twice as high in hiv-infected than in uninfected children two years of age or younger (madhi et al., 2000) . little information is available regarding genetic and environmental factors in susceptibility to rsv infections. most rsv epidemiologic studies are conducted in affluent countries and temperate climates although rsv is thought to be the leading cause of severe viral acute respiratory infections (ari) in infants around the globe (weber et al., 1998) . a 3-year prospective surveillance study was conducted in the yukon-kuskokwim (yk) delta of south-western alaska to determine the rate and severity of rsv infections requiring hospitalization for infants in this yupik eskimo population (karron et al., 1999) . the annual rate of rsv hospitalization for yk delta infants less than 1 year of age was unusually high, i.e. 53-249/1000. one in 125 children born in the yk delta, compared with between one in 550 to one in 11 000 infants in affluent countries (sims et al., 1976; martin et al., 1978; glezen et al., 1981) , required ventilatory support for rsv disease. rsv infection was the single most frequent cause of hospitalization of yk delta infants. as in temperate climates, rsv epidemics in the yk delta occur annually from november through june, with peak hospitalizations for rsv disease occurring between november and february. within sub-regions of the yk delta, epidemics were as brief as 1 month, probably because there is very limited traffic between villages in the winter months. most of the infants admitted to hospital were less than 1 year of age and had no medical risk factors for rsv disease. surprisingly, 9% of the admitted infants in the yk delta and 8% of infants in a comparison group admitted to johns hopkins hospital (jhh) were less than one month old. of children with severe disease, 34% in the yk delta and 24% at jhh were less than 2 months old. disease severity in non-high risk children did not differ between children admitted to jhh or yk delta regional hospital, suggesting that differences in hospitalization practices could not account for the high rates of hospitalization for rsv in yk delta infants. in the yk delta, 19% of those admitted for rsv disease were readmitted within a single rsv season. severity of rsv disease, age at first illness and receipt of ribavirin were all associated with readmission. in infants less than 6 months of age, a low neutralizing antibody titer in cord blood samples was strongly associated with severe disease. a questionnaire-based case control study that was matched for age and sub-region in the yk delta detected three risk factors influencing rsv hospital admission. medical risk factors (prematurity, chronic lung disease, congenital heart disease) increased the risk of admission 6.25-fold. more than eight people living in one household doubled the risk of hospitalization while breastfeeding had a protective effect. smoking, food-pre chewing and economic status were not significantly associated with rsv hospitalization. this study may be useful in the continued analysis of the impact of rsv in developing countries. as in the yk delta, rsv is the leading cause of viral lower respiratory tract disease in most developing countries, but lack of access to diagnostic reagents and hospital facilities has made it difficult to quantify the impact of rsv. also, the rate of severe rsv disease in term neonates was much higher than in earlier studies, both in the yk delta population and the comparison group in baltimore. these findings should be confirmed in other populations because they have important implications for rsv vaccine development. 2.4. respiratory syncytial 6irus (rsv) and human rhino6irus (hrv) infections in children with aids and lower respiratory tract illness (lrti) acute respiratory infections (ari) cause a great burden of disease in developing countries (de arruda et al., 1991) . although a growing number of children from developing nations are hiv infected, there is little knowledge about the frequency and severity of viral ari in hiv infected children. earlier studies of viral pathogens in immunocompromised adults indicated that cmv, herpes simplex, influenza, parainfluenza, rhinovirus, adenovirus, enterovirus, and rsv cause lower respiratory infection (connolly et al., 1994) . a recent study assessed the frequency of rsv and rhinoviruses (hrv) in hospitalized children with or without aids, who presented with lower respiratory tract infection (lrti). about 73 episodes of lrti in children with aids and 73 in children without hiv infection, matched by age and sample collection month, were studied in a rural area of southern brazil. the frequency of rsv infection was highest in the fall and winter, between february and july, whereas hrv was detected throughout the year. rsv was found in 8/73 (11%) and 9/73 (12%) of lrti episodes in aids and non-hiv infected children, respectively. hrv was found in 16/73 (22%) and 12/73 (16%) of the episodes in children with aids and hiv-uninfected children, respectively. no difference was detected in frequencies of hrv and rsv infections between the two groups. hrv infections, however, tended to be more frequently associated with pneumonia in children with aids (7/16, 44%) than in the control group (1/12, 8%) (p= 0.09). other clinical presentations of lrti were observed with equal frequency. these findings did not confirm hrv as a causative agent of pneumonia in children with aids, but suggest that further studies of lrti are desirable and that interventions for hrv could be considered for immunocompromised children with lrti. the human adenoviruses (ads) are a large family of over 50 serotypes, as well as numerous variants and intermediates. they are divided into six subgroups (a-f) that exhibit different tissue tropism. the clinical manifestations of adenoviral disease are protean. the most common are respiratory syndromes in both children and adults. it is estimated that ads cause 5-15% of all respiratory disease in children, including pharyngitis, tonsillitis and pertussis-like syndrome. in children and adults, ads are also associated with lower respiratory tract infections such as bronchitis and pneumonia. subgroup d ads are associated with both epidemic and sporadic ocular infections including conjunctivitis and keratoconjunctivitis. due to their stability in the environment, these viruses are highly transmissible, particularly in nosocomial settings. in adults, ads cause largescale epidemics of acute respiratory disease (ard) in closed populations of military recruits, dormitory residents and long-term care facility occupants, which are primarily associated with ad serotypes 4 and 7a, and to a lesser extent with serotypes 2, 3, 11, 21 and 35. while replication in the gastrointestinal tract is a feature of most ad infections, only ad40 and ad41 (subgroup f) are associated with gastroenteritis in infants and young children. ad infections in immunocompromised subjects are increasing as their numbers increase, with severe consequences. clinical manifestations of ad infection in immunocompromised patients include pneumonia, hepatitis, encephalitis, and systemic and disseminated disease, with case fatality rates from 18 to 60%, depending on the nature of the immunodeficiency. nearly all ad serotypes have been associated with these infections, but the higher numbered subgroup d serotypes that are usually not associated with clinical disease in the immunocompetent host have been especially common. a number of 'new' disease associations with ad infection have recently been reported, probably due to improved highly sensitive molecular diagnostic techniques that also increase the probability of laboratory contamination. detection of viral genome in the absence of positive viral culture has been described in cases of myocarditis and pericarditis in children and adults (martin et al., 1994; bowles et al., 1999; pauschinger et al., 1999) , sudden infant death (shimizu et al., 1995) , toxic shock-like syndrome (price, 2000) and 'unexplained death' (perkins et al., 1996) . isolation of ads from patients with central nervous system manifestations of fatal acute flaccid paralysis (cardosa et al., 1999) and encephalitis with cerebral edema (chatterjee et al., 2000) has also been reported. it is, perhaps, not surprising that the clinical manifestations of ad infection are evolving because the viruses themselves are in the process of continuous evolution. the ad mutational repertoire includes homologous recombination, illegitimate recombination, and single base mutation (sbm). homologous recombination occurs in conserved regions of the genome between closely related viruses within the same subgroup, and it requires regions of homology in the two parent strands. it is the primary mechanism responsible for intermediate ads, which are mosaic viruses with shared hexon characteristics, or with the hexon characteristics of one type and fiber of another. illegitimate recombination requires only short regions of homology of one to three nucleotides (short direct repeats), and is thought to be the result of polymerase stuttering or slippage. it causes deletions, insertions and duplications of short regions of dna. in ads it occurs in noncoding regions and hypervariable regions (hvrs) of hexon capsid proteins that tolerate structural variation. the hvrs of the hexon contain the viral neutralization epitopes, so that mutations in these regions result in deletion, formation or alteration of these epitopes, leading to antigenic shift. it is the primary mechanism by which new serotypes arise, particularly among the fastest growing group, the subgroup d ads. single base mutations accumulate gradually across the viral genome but can occur at a 30-fold higher rate in the hvrs, where they cause incremental antigenic drift and the creation of variant strains. ad evolution is compounded by all three mechanisms and perhaps others that have yet to be defined. as a result, serological identification of subgroup b2 and d ads has become extremely difficult. it seems reasonable that the time has come to consider a sequence-based ad classification system, similar to that in use for papillomaviruses and enteroviruses. adenovirus has re-emerged as a leading cause of febrile respiratory disease among military recruits. large and frequent epidemics were common at trainee camps before 1971 but were eliminated with the introduction of the live enteric type 4 and 7 vaccines. in 1996, the sole vaccine manufacturer discontinued production of these live enteric coated vaccines because of contractual issues. while limited vaccine stores were still available in 1997 and 1998, vaccine stores were completely depleted by early 1999. to monitor the effect of discontinued vaccination, weekly surveillance for febrile respiratory infections (fri), defined as oral temperature \100.5°f with respiratory disease symptoms, was conducted from october 1996-june 1998 at four military training camps. during this interval, 1814 (53%) of 3413 throat cultures yielded adenovirus. during the winter of 1997-1998, adenovirus infections caused more than 90% of fri at each of the four camps. ad 4, 7, 3, and 21 accounted for 57, 25, 9, and 7% of the isolates, respectively. three training camps experienced a high prevalence of adenovirus type 4 and the fourth camp experienced a type 7 outbreak. among symptomatic trainees, those who did not receive vaccine were 28 times more likely to be infected by ad 4 or 7 than vaccinated subjects (gray et al., 2000) . surveillance was extended to eight sites in june 1998 and virus isolation was attempted for adenovirus, influenza a and b, rsv, and parainfluenza 1-3. large ad 4 epidemics were observed in six training camps throughout the us, while rsv and influenza a and b viruses were isolated less frequently. the impact of adenovirus epidemics on basic training can hardly be overestimated. recruit camps were forced to convert barracks into special infirmaries to care for the ill, and hospitals were forced to halt elective surgeries. at one camp, the number of trainees that had to repeat their basic training because of extended illness increased 20-fold. this 'recycling' has an extremely negative effect on the morale of trainees and it impacts on the military's readiness. as many as 1800 preventable adenoviral trainee medical encounters occurred during the winter months of 1999 and in 2000; two military trainees died with molecular evidence of acute adenoviral infection, one with encephalomyelitis and another with acute respiratory distress syndrome (ards). vaccination against ad 4 and 7 has proven to be extremely safe and effective, and to prevent an enormous burden of disease in military trainees (howell et al., 1998) . it is urgent that a new manufacturer for adenoviral vaccines be identified, and vaccine production must resume as soon as possible. in recent years, much progress has been made in understanding virus-induced modulations of the host immune response to viral infections. for ad, more than 20 viral gene products are known to participate in the modulation of immune responses, and many of these gene products are expressed from genes clustered in the early region 3 (e3) (wold et al., 1999; horwitz, 2001) . the overall effect of ad e3 gene products on immune responses in vivo can be appreciated from the results of three studies in mice that investigated transplant rejection and development of autoimmune disease. in the first study, the expression of the complete ad e3 cassette in pancreatic islet cells as transgenes under the control of the rat insulin promoter (rip) enabled allogeneic islet donor cells containing the h-2 bxd class i mhc to be accepted long-term by h-2 d recipient mice (efrat et al., 1995) , indicating that ad e3 gene products could potentially be used as a powerful tool in the control of transplant rejection. the second study used the lymphocytic choriomeningitis virus (lcmv) model of autoimmune diabetes mellitus, in which the lcmv proteins np or gp are expressed on the surface of islet cells, and diabetes is induced by infection with lcmv that induces cd8+ (gp) or cd8 + and cd4 + (np) t-cell mediated immune responses. in this model, the co-expression of rip-e3 with lcmv-np or gp completely prevented the onset of diabetes after lcmv infection (von herrath et al., 1997) . similar protective effects of ad e3 transgenes were seen in a third study that used the non-obese diabetic mice (nod) model of diabetes mellitus, and the underlying mechanisms are currently being investigated (efrat et al., 2001) . thus, as the understanding of the mechanism of action of the ad e3 immunoregulatory genes are being pursued in various systems, they are being utilized to control selected immune reactions that might be involved in the genesis of autoimmune diabetes. some of the better-characterized gene products of the e3 region are (in order of increasing distance from the e3 promoter) gp19, 11.6, 10.4, 14.5 and 14.7k. only the functions of gp19 and 14.7k shall be discussed here; the other ad e3 gene products have been reviewed elsewhere (horwitz, 2001) . in vitro, gp19k reduces the expression of class i major histocompatibility complex (mhc) molecules by retaining the mhc heavy chain in the endoplasmic reticulum or retrieving it back from the golgi, and also by inhibiting peptide processing (bennett et al., 1999) . in the cotton rat model of adenovirus pneumonia, gp19k deletion mutants replicate like wild-type virus but they induce a much stronger inflammatory response (ginsberg et al., 1989) , whereas in c57bl mice an increase in pulmonary pathology is not seen (sparer et al., 1996) . the ad e3 14.7k protein inhibits tnfa-induced cell death by a process that does not involve down-regulation of the tnfa-receptor. in cotton rats (ginsberg et al., 1989) and c57bl mice (sparer et al., 1996) deletions of ad e3 14.7k modify the pulmonary inflammatory response, i.e. an increase of polymorphonuclear leukocytes in cotton rats and more pronounced alveolar infiltration in mice. in order to determine how the ad 14.7k protein prevents cell death, the cell proteins that interact with this viral protein were determined. using a yeast two-hybrid system, four 14.7k-interacting proteins (fips) were identified. three of them have been characterized and have been shown to participate in quite diverse cellular pathways (li et al., 1997 (li et al., , 1998 (li et al., , 1999b . fip-1 (also known as rag-a, a ras-related small gtpase) can bind to tctel, a component of the microtubule motor protein dynein, forming 14.7k-fip-1-tctel complexes (lukashok et al., 2000) , and 14.7k has been postulated to affect microtubule dependent macromolecular transport or even modulate the transport of virus. however, because 14.7k is not a structural protein of adenoviruses and must be made de novo from early viral transcripts, it is unlikely to play a role during viral entry, even though the process is known to be microtubule dependent. the role of 14.7k during viral exit from cells has not been studied. fip-1 also binds to a second gtpase (gip-2) that localizes in the centrosome and in addition to potential effects during mitosis may be involved in transporting macromolecules between the nucleus and the cytoplasm. fip-2 binds to abnormal huntingtin, and more specifically to the expanded polyglutamine tract that appears to be associated with cell death of neurons in huntington's disease (faber et al., 1998) . whether or not ad e3-14.7k and/or fip-2 can prevent huntingtin-induced cell death is currently being investigated. over-expression of fip-3, which is also called nf-kb essential modulator (nemo) or inhibitor of kappa kinase gamma (ikkg) causes morphologic changes and eventually apoptosis in a variety of cell lines. the amount of apoptosis induced by fip-3 can be reduced by 70% when ad e3-14.7k is present. apart from 14.7k, fip-3 seems to interact with a number of key molecules in the tnf receptor and nf-kb signaling pathways such as the receptor interacting protein (rip), the inhibitor of kappa b kinase beta (ikkb) and the nf-kb inducing kinase (nik) (li et al., 1999b) . these few examples of the effects ad e3 gene products have on the pathobiology of diseases as different as autoimmune diabetes, transplant rejection and huntington's disease indicate how much remains to be learned from studying adenovirus-host interactions. the rsv (strain a2) genome is a single stranded negative-sense rna of 15 222 nucleotides that is transcribed into 11 major subgenomic mrnas. three of the eleven encoded proteins are transmembrane proteins. the g protein mediates attachment to cell surface receptors, the f protein mediates virus-cell and cellcell fusion, and the function of the sh protein is unknown. other structural proteins are the m protein, which plays a role in virion assembly, the n, p and l proteins that make up the viral polymerase, and the m2 orf1 protein that functions as a transcription anti-termination factor. the other proteins include two non-structural species, ns1 and ns2, and the m2-2 protein encoded by the second orf of the m2 mrna. using a reverse genetics system to rescue infectious rsv from cdna, five of the 11 genes of rsv can be ablated individually and in some cases in combination without rendering the virus non-viable jin et al., 2000b) . these five non-essential genes are ns1, ns2, sh, g, and m2-2. since all of these genes confer a selective advantage to rsv in vitro and/or in vivo, they can be described as virulence factors -the deletion of which will lead to attenuation of the virus. deletion of the small hydrophobic (sh) transmembrane protein yields a recombinant rsv called rsva2dsh, which replicates in vitro as well as wild type (wt) rsv and induces plaques in hep-2 cells that are larger than wt rsv plaques. there is no reduction in synthesis of rna or protein associated with the deletion of sh. in chimpanzees, however, the virus is slightly attenuated (whitehead et al., 1999a) . deletion of the ns1 or ns2 gene results in a substantial reduction in replicative efficacy in vitro, and this reduction is more pronounced in hep-2 cells than in vero cells (which lack interferon a and b genes), suggesting that these two genes act as antagonists to type 1 interferon effects. deletion of ns1 and ns2 from bovine rsv provided direct evidence that ns1 and ns2 cooperatively antagonize a/b interferon-induced antiviral responses (schlender et al. 2000) . in chimpanzees, the level of replication of both rsva2dns1 and rsva2dns2 is reduced greater than 10 000-fold in the lower respiratory tract. in the upper respiratory tract, the dns1 virus is more attenuated than the dns2 virus (teng et al., 2000) . deletion of the m2 orf2 not only identifies a markedly attenuated rsv mutant but reveals an important role for this orf in the replicative cycle of rsv (bermingham and collins, 1999) . during infection with wildtype rsv, transcription appears to shut off at approximately 12-16 h post infection while rna replication increases concurrently. in contrast, this apparent switch from transcription to rna replication was not observed for the rsvdm2-2 virus, implying that m2-2 is a regulatory protein involved in the shift. instead, transcription continued to increase while rna replication remained low compared to wild-type rsv. overall, gene expression was increased 7-18 fold. the synthesis of the g and f proteins also was increased and resulted in increased syncytium formation. replication of the rsvdm2-2 virus in vitro was attenuated, probably due to reduced rna replication (bermingham and collins, 1999; jin et al., 2000a) . in chimpanzees, comparison of the four rsv gene deletion mutants mentioned above with wt rsv and the incompletely attenuated rsv cpts-248/ 404 vaccine candidate results in a hierarchy of increasingly more attenuated viruseswt rsv b dsh b dns2 b 248/404 b dns1 b dm2-2. the final rsv gene deletion mutant, rsva2dg, was not evaluated in chimpanzees because the absence of this major protective antigen would not be desirable in a vaccine virus. rsva2dg replicates as efficiently as wt rsv in vero cells, showing that g is not essential for efficient virus replication. however, the rsvdg virus is highly attenuated in balb/c mice, indicating the importance of the rsv g protein in vivo. it is evident from the above ranking that rsv reverse genetics is able to generate mutants exhibiting gradations in their level in attenuation ). this menu of viruses with different levels of attenuation is crucial in identifying an rsv vaccine that exhibits the desired balance between attenuation and immunogenicity in seronegative infants. since clinical data indicate that rsv 248/404 is just slightly under-attenuated in the 1-month-old target population, the dns1 mutant could be exactly what is needed. in order for rsv assembly to be an efficient process, viral structural proteins must be brought together in a coordinated fashion (peeples, 1991; lenard, 1996) . compared with other paramyxoviruses rsv exhibits several unique features. the g, m2-1 and m2-2 proteins are found only in the pneumo6irus genus of the paramyxo6iridae, and the role these proteins play in rsv assembly is much less well understood than the role of the f, hn and m proteins of other paramyxo6iridae (collins et al., 1996) . comparison of multiple human, bovine and ovine rsv strains shows that the cytoplasmic domains of the f and g proteins are well conserved amongst human rsv strains and subtypes, and conserved to some degree between the three species. in analogy to other paramyxoviruses, it is likely that the cytoplasmic domain of f and g interact in the process of virion assembly with cellular proteins involved in protein trafficking and in the polarized budding process, as well as with other viral proteins. the rsv g and m proteins co-localize in the golgi apparatus, not only in rsv-infected cells but also in cells transfected with only the g and m proteins, indicating that other viral proteins are not needed for this interaction (peroulis et al., 2001) . the g-m interaction is seen with fulllength g protein but not with a secreted form of g that lacks the conserved cytoplasmic domain and transmembrane domains. systematic deletion and substitution mutagenesis of the cytoplasmic domain of g has identified a sequence-specific, six amino acid motif that directly interacts with m (peroulis et al., 2001) . during rsv infection m protein can initially be detected in the nucleus, but later in the infectious cycle it is found in inclusions within the cytoplasm (ghildyal et al., unpublished) . the n and p proteins of rsv were earlier shown to be necessary and sufficient for these inclusions to form, and the rsv m2-1 and l proteins were also shown to be present in these inclusions (garcia et al., 1993) . these same investigators were unable to identify m protein in the inclusions (garcia et al., 1993) . using confocal immunofluorescent microscopy of infected and cotransfected cells the m protein was shown to be present in these inclusions . m protein does not localize to the inclusions unless m2-1 is also present . the m and m2-1 proteins not only co-localize by confocal microscopy but also interact in a protein overlay assay (ghildyal et al., unpublished) . taken together, these data suggest that, as with other single stranded negative-sense viruses (peeples, 1991; lenard, 1996) , the rsv m protein seems to play a crucial role in rsv assembly; bringing the nucleocapsid together with the envelope proteins by binding to the cytoplasmic domains of g and f and to the nucleocapsid proteins n and p together with or via m2-1. the interaction between m, m2-1 and the nucleocapsid proteins might be more complex than outlined here, and might involve additional cellular or viral proteins. to better understand rsv assembly, the domains in the m protein that interact with g, f and m2-1 will have to be defined. while influenza viruses readily develop resistance to older antivirals such as amantadine or rimantadine, resistance to neuraminidase inhibitors occurs much less frequently. nonetheless, resistant influenza a virus mutants can be isolated from patients treated with oseltamivir (treanor et al., 2000) . one of the resistant mutants carries an arginine to lysine mutation at position 292 (r292k) of the neuraminidase protein, causing a reduction in substrate binding and enzymatic activity, as well as resistance to oseltamivir (mckimm-breschkin, 2000) . the infectivity of influenza a viruses carrying the r292k mutation was earlier found to be markedly reduced in mice and ferrets. transmission of an influenza a h3n2 clinical isolate with a r292k mutation was studied in ferrets in comparison to transmission of the parent wt h3n2 virus that was isolated from the same patient. donor ferrets (four per group) were inoculated intranasally with the r292k mutant or wt virus and housed with three naïve contact ferrets per donor ferret. the four donors inoculated with wt virus were infected and transmitted virus to each of the 12 contacts. wild type virus replicated to between 10 4 and 10 5 pfu/ml nasal aspirate. only two of four donor ferrets inoculated with mutant virus became infected, and the level of replication of mutant virus was reduced 10-100-fold compared with that of wt virus. however, both infected ferrets transmitted virus to contacts. one of them transmitted the r292k virus to one of three contacts only, with virus detected at very low titer on 1 day only. the other donor transmitted virus to all three contacts, and the transmitted virus replicated to titers greater than 10 4 pfu/ml in the contact ferrets. sequence analysis showed that the donor virus was a mixed population of mutant and wt virus and that only wt virus was recovered from contacts. these data confirm the reduced infectivity of oseltamivir resistant r292k mutants. effective transmission of the r292k mutant virus in ferrets was not observed, suggesting that the transmission of oseltamivir-resistant virus from human to human will be unlikely, even during widespread use of na inhibitors in the treatment of influenza. respiratory virus infections in immunocompromised patients are characterized by persistence of viral infection, prolonged shedding of virus, a high rate of nosocomial acquisition, and a high frequency of pneumonia and death. similarly, respiratory virus infections in the elderly are responsible for a substantial amount of morbidity and mortality. detection of respiratory virus infections in these high-risk patients is important for several reasons. it enables the initiation of specific isolation procedures, initiation of specific antiviral therapy, cessation of unnecessary therapy, tests and procedures, and it can aid in identifying and preventing potential outbreaks. respiratory virus infections can be diagnosed using serology and culture techniques, as well as newer methods such as antigen-detection by enzyme-linked immunoassay (eia) or immunofluorescence (ifa), enzymatic detection by chemical reactions, or amplification of parts of the viral genome (pcr). serology is not useful in the acute phase of most illnesses and is of limited usefulness in immunocompromised patients, the elderly or those receiving blood products. recovery of virus in cell culture is still seen as the gold standard by many but the importance of obtaining a good specimen to ensure that the culture is not falsely negative is often overlooked. a good clinical specimen is the most critical factor in ensuring a correct diagnosis regardless of the diagnostic method used, although it is perhaps most important for culture. use of nasal wash to obtain a specimen for virus isolation is well-tolerated in cooperative adults and, compared to nasal swabs or throat swabs, increases the sensitivity of cell culture for virus detection. other factors critical to laboratory success are the use of appropriate transport media, temperature of transport/incubation and time until processing (atmar and englund, 1997) . this is particularly important in the case of rsv infections in immunocompromised or elderly patients, who often have a relatively low viral titer such as 10 2 -10 3 pfu/ml, whereas children often have a titer greater than 10 6 pfu/ml of nasal wash or bronchoalveolar lavage fluid (englund et al., 1996) . for rsv, parainfluenza and influenza virus there are a number of commercially available rapid detection kits. multiple simultaneous rt-pcr for detection of rsv, influenza a and b, and piv1, 2 and 3 (hexaplex ® ) was tested in 763 pediatric samples and yielded 100% sensitivity and 98% specificity as compared with culture (fan et al., 1998) , with only 8 h processing time. in a separate study, pcr for respiratory viruses in adult patients with hematologic malignancies was found to be as sensitive as culture (van kraaj and van elden, 2000) . for influenza, several antigen detection kits ((directigen, fluoia and quick-vue) and one neuraminidase (zstatflu) assay are available to detect virus, with sensitivities ranging from 50 to 96% and specificities ranging from 52 to 99%. rapid diagnosis of influenza in pediatric patients leads to a decrease in the frequency and duration of antibiotic use and an increase in the frequency of antiviral therapy (noyola and demmler, 2000) . rapid diagnosis kits for the detection of rsv in children range in sensitivity from 61 to 87% and in specificity from 93 to 97%, with some test kits performing better than others (dominguez et al., 1993) . in immunocompromised adults, however, the sensitivity of antigen detection kits from nasal wash or throat swab sample was only 15%, while endotracheal or bronchoalveolar sampling increased sensitivity to 70 or 80%, respectively, (englund et al., 1996) . several studies in recent years have highlighted the importance of upper respiratory tract infections in the exacerbation of asthma in children. freymuth and colleagues reported human rhinovirus (hrv) (46.9%) and rsv (21.2%) as the most frequent pathogens detected in patients with exacerbation of asthma, and noted that pcr increased detection rates 5.8-and 1.6-fold in hrv and rsv infections, respectively, over that of conventional assays (freymuth et al., 1999) . in a study of wheezing children between 2 months and 16 years of age, respiratory viruses were detected in 82%, with rsv in infants (detected in 68% of subjects) and hrv in older children (71%) as the predominant pathogens. both were strongly associated with wheezing (rakes et al., 1999) . in a community-based longitudinal study, respiratory viruses were detected in 80% of episodes of acute illness with reduced peak expiratory flow, 80% of episodes of wheezing, and in 85% of episodes of upper respiratory symptoms, cough, wheezing, and a fall in peak expiratory flow (johnston et al., 1995) . in these settings, rt-pcr provides a fast and sensitive method to detect rna viruses. real time quantitative pcr assays can be similar to conventional pcr in specificity and speed. however, real-time pcr can also quantify viral load (quantity of virus in respiratory secretions) during asthma exacerbations, and it is sometimes more sensitive than conventional pcr. real time taq-man quantitative pcr allows estimation of the input viral genome copy number by including a fluorescence reporter on one end and a quenching molecule on the other. the reporter does not fluoresce until the quencher has been cleaved off by the exonuclease activity of taq polymerase, permitting an estimate of the quantity of pcr product. fluorescence is being measured continuously every seven seconds, and quantification of the target is based on the number of pcr cycles it takes to produce detectable fluorescence. a retrospective study was conducted in 117 asthmatic children 9-11 years old to compare viral loads in quiescent and exacerbation periods of asthma using the taqman technology. nasal aspirates had been collected earlier and records of peak flow measurement and clinical scoring were available. a quiescent period was defined as absence of clinical symptoms for two weeks or longer. real time quantitative pcr detected respiratory viruses (mostly rhinoviruses) in 97% of the children with an asthma exacerbation and in 42% of children in quiescence. viral load was higher in children with exacerbation of asthma than in those with quiescent asthma and higher viral loads also correlated with more severe clinical disease. although the study showed that real time quantitative pcr is more sensitive than nonnested conventional pcr and also allowed esti-mation of viral load, there is the caveat that the study had a retrospective design. modalities of immunity to acute viral respiratory infections are both specific and nonspecific, humoral and cell mediated. fever, interferon (ifn), tumor necrosis factor (tnf), natural killer cells and activated macrophages are non-specific modalities stimulated by infection that are capable of mediating antiviral effects. lung collectins such as sp-a, have also been implicated in the control of viral respiratory infections (ghildyal et al., 1999) . specific modalities are antibody in serum and secretions, lymphocyte proliferation responses with cytokine release, and cytotoxic lymphocytes (ctls). all modalities participate, to some degree, in containing an infection and promoting recovery either via inactivation of free virus or elimination of infected cells. thus, there is considerable redundancy in the mechanisms controlling the virus infection and promoting recovery. on the other hand, protection against infection is primarily conveyed by specific antibody. sufficient data is available to conclude that serum igg neutralizing antibody is the primary mediator of resistance to influenza virus infection, presumably because infection is initiated in the lower respiratory tract and igg antibody derived from serum is the dominant antibody isotype at that site (couch and kasel, 1983) . in contrast, iga antibody is the primary mediator of resistance to rhinovirus and coronavirus infection because evidence indicates these infections are initiated in the nasopharynx where iga is the dominant antibody isotype (cate et al., 1966) . since primary infections with influenza virus, rsv, or piv induce disease in both the upper and lower respiratory tract, both igg and iga antibodies are correlates of immunity to infection and disease (crowe, 1999) . although adenoviruses also cause upper and lower respiratory disease, only serum antibody has been shown to correlate with immunity. reinfection with homologous rsv, piv, rhinovirus and coronavirus can occur but is generally confined to the upper respiratory tract, presumably because igg antibody in protective quantities is more durable in serum (and lower respiratory secretions) than is iga antibody in nasopharyngeal secretions. for rsv, the risk of infection declined from 74% for naïve subjects to 56 or 33% after one or two infections, respectively; the risk of lower respiratory disease in these groups declined from 18 to 15 to 3%, respectively, (glezen et al., 1986) . for a rhinovirus, resistance to reinfection of the nasopharynx correlated with the titer of iga antibody in secretions (cate et al., 1966) . rodent models of viral respiratory disease provided much of the early data on immune modalities conferring protection (crowe, 1999) . both cd4 + and cd8 + t cells can effect clearance of influenza a virus, rsv and piv in mice in the absence of the other cell type (lightman et al., 1987) . while cd8+ cytotoxic lymphocytes (ctl) mediate immunity through lysis of infected cells and expression of antiviral cytokines, cd4+ t cells exhibit limited direct antiviral activity but play a role in activating b cells and in inducing antiviral cytokine expression (epstein et al., 1998) . there is a general consensus that ctls contribute significantly to the resolution of primary infections with influenza, rsv and piv in rodents. while the correlation between antibody response and protection in humans was established decades ago, cellular immune responses were studied much later, and understanding of the development of these responses in infants and immunosenescence of them in the elderly is still incomplete. whether or not ctls in humans convey immunity to respiratory virus infection in the lower respiratory tract and whether they promote clearance of virus in the upper respiratory tract of humans remains to be elucidated. however, for rsv disease in humans, a correlation has been observed between the presence of rsvspecific ctls in year 1 and absence of severe rsv disease in year 2 (mbawuike, in press). morever, the level of influenza-specific ctls correlated inversely with the quantity of virus in nasal secretions after challenge of humans, and the ability of ctls to function at this site was demonstrated by a reduction in influenza virus titer in nasal turbinates of mice which were adoptively immunized with purified cd8 + ctls before intranasal challenge (mcmichael et al., 1983 mbawuike, personal communication). knowledge regarding viral-bacterial interactions in the respiratory tract goes back at least to the 1918 influenza pandemic, and interactions have been described for later influenza pandemics as well. epidemics and pandemics of influenza have been followed regularly by an increase in the incidence of bacterial pneumonia (schwarzmann et al., 1971; cartwright et al., 1991) . associations between rsv and haemophilus influenzae, bordetella pertussis, neisseria meningitidis and staphylococcus aureus infections have also been described (patel et al., 1992; jiang et al., 1999) . interactions between viral and bacterial diseases are fairly complex and the underlying mechanisms are only beginning to be elucidated. it is known that up-regulation of tnfa and il-1 increases adherence and uptake of pneumococci (cundell et al., 1995) , and that several cellular receptors for bacterial adherence are up-regulated by viral infections. s. pneumoniae and h. influenzae interact with paf receptors (swords et al., 2000) , and n. meningitidis interacts with cd14 and cd18 (raza et al., 1999) . not all interactions are regulated at the level of cell surface receptors. in the mouse influenza model, for instance, severe damage and desquamation of the respiratory epithelium enables access of s. pneumonia to the basal membrane and thereby increases the risk for invasive disease (plotkowski et al., 1986) . otitis media was traditionally thought to be a purely bacterial infection, with s. pneumoniae, h. influenzae and moraxella catarrhalis as the main pathogens. more recent studies, however, indicate that the majority of acute otitis media (aom) cases are a result of mixed bacterial and viral infection. heikkinen and colleagues reported an increase of rsv, parainfluenza virus, influenza or adenovirus infection in children with otitis media (heikkinen et al., 1999) . all respiratory viral infections of the nasopharynx are thought to predispose to aom but some viruses, e.g. rsv, are frequently found in the middle ear during aom. the mechanism underlying this respiratory tract infection-aom sequence involves eustachian tube obstruction, leading to negative middle ear pressure and inspissation of bacteria into the middle ear (giebink et al., 1980) . children with rsv, adenovirus or influenza virus infections have a 30% risk of developing aom within 2 weeks of the onset of the respiratory tract infection (henderson et al., 1982) , and coinfection with bacteria and viruses also adversely influences the outcome of aom. if aom does not respond to antibiotic therapy within 48 h, it is more likely to involve a viral infection (arola et al., 1990) . the effects of viral co-infection complicate the evaluation of clinical efficacy of anti-bacterial drugs in the treatment of aom (marchant et al., 1992) . with bacterial-viral co-infection in aom, there is also delayed clearance of bacteria from the middle ear during anti-bacterial therapy compared with disease attributable to bacterial infection alone (chonmaitree et al., 1990) . it is unclear whether the decrease in efficacy of antibiotics is due to impaired host responses (poor neutrophil function) or due to poor penetration of antibiotics into the middle ear. in a recent clinical trial heptavalent pneumococcal vaccine had 90% efficacy for prevention of bacteremia and 57% efficacy for prevention of aom caused by serotypes included in the vaccine. however, the frequency of aom caused by pneumococci not included in the vaccine increased (replacement phenomenon), so that the overall effect of the vaccine was reduced (eskola et al., 2001) . in contrast to the pneumococcal vaccine, viral vaccines seem very effective in preventing aom. belshe and colleagues reported considerable efficacy for an attenuated live influenza vaccine in preventing aom, and rsv prophylaxis with rsv antibodies was also associated with a marked decrease in otitis media (belshe et al., 1998; group, 1998a ). the human adenoviruses consists of over 51 known serotypes which have been divided into six subgroups (a-f) with distinctly different organ tropism. although other factors may influence infectivity and replication of these viruses, high affinity attachment of virions to host cell receptors represents a key determinant of tissue tropism. examples of this distinctly different organ tropism are a predominance of subgroup a adenoviruses (ad) such as ad31 in pneumonia in patients with primary immunodeficiencies; the preference of subgroup b viruses such as ad11, 34 and 35 for the urinary tract in patients with kidney transplants; and the predominance of subgroup c viruses in hepatitis in liver transplant patients. receptor binding is mediated by the ad fiber protein, a homotrimeric molecule composed of an amino terminal region that anchors the fiber to the penton base capsid protein, an elongated central shaft domain (van raaij et al., 1999) , and the carboxy-terminal receptor binding knob. a high resolution structure of the ad12 fiber knob bound to its receptor, the coxsackie-adenovirus receptor (car), has recently been obtained by x-ray diffraction (bewley et al., 1999) , and amino acid residues directly involved in receptor binding were defined for several adenoviruses through mutagenesis studies (roelvink et al., 1999) . adenoviruses differ remarkably in the isoelectric point of the fiber protein knob domain, e.g. ph 8.9 for ad8 versus ph 4.5 for ad35, suggesting that these fiber proteins cannot use the same receptor. besides car, heparin sulfate proteoglycans (dechecchi et al., 2000) and sialic acid (arnberg et al., 2000) mediate adenovirus attachment. since car is only expressed on the basolateral but not the luminal surface of epithelium, car can probably not be used to target adenoviral vectors in the therapy of cystic fibrosis. the important role that charge plays in virus-cell interactions can be deduced from the differential effect that removal of sialic acid from the cell surface by neuraminidase treatment has on adenovirus attachment. while ad19p attachment is not affected by neuraminidase treatment, ad5 attachment is increased and ad37 attachment is decreased. the difference in ph optima may well be a determinant of tissue tropism. the major pathogens in adenoviral eye infections, ad8, ad19 and ad37, belonging to subgroup d, are very similar in their knob charge. chimerization of fiber proteins, whether evolved in nature or generated by mutagenesis, can cause a significant change in adenovirus pathogenesis. in 1984, a new ad7 genotype (ad7h) appeared in argentina, uruguay and chile, where it caused severe respiratory tract infections in children. analysis of the ad7h fiber protein revealed that it was a chimera containing ad7 and ad3 sequences. in 1996 this new genotype and ad7d2 also emerged in japan, where ad7 had been absent for 30 years, and caused outbreaks of respiratory disease. the fiber protein, however, is not the only factor that determines adenovirus tropism. virus uptake is thought to also be mediated by an interaction between the penton base protein with integrin avß 3 or avß 5. adenoviruses also differ in their ability to induce inflammatory responses. while ad 7 is a potent inducer of interleukin 8 (il-8), a hallmark cytokine of viral pneumonia, ad5 is not; this might explain why ad7 but not ad5 causes significant respiratory disease. in summary, it can be concluded that neither subgroup classification alone nor fiber protein knob charge alone determine adenovirus tissue tropism. key amino acids in the knob, as well as certain motifs of the penton base protein and possibly other adenovirus proteins interact in virus attachment and internalization. avian influenza viruses of the h5n1 subtype were found to be transmitted directly from poultry to humans in 1997 in hong kong. these viruses were highly pathogenic in chickens and also caused severe clinical symptoms in humans, leading to the death of six of 18 infected individuals. in order to obtain a better understanding of the pathogenesis, tropism and kinetics of replication of these viruses in primates, cynomolgus monkeys (macaca fascicularis) were infected with the highly pathogenic h5n1 a/hong kong/156/ 97 isolate that was obtained from the index case. four monkeys were inoculated with 2.5× 10 4 tcid 50 in a 5 ml inoculum that was administered intratracheally, orally (tonsils) and onto the conjunctiva. two of the monkeys were euthanized on day 4 and the remaining two on day 7 post infection. the two monkeys that were euthanized on day 7, post infection developed a respiratory distress syndrome with high respiratory rate and fever on day 5. by day 7, one of the monkeys was lethargic and severely ill, with central cyanosis. the other monkey was also ill and developed fever. the virus replicated to a titer greater than 10 6 tcid 50 per g lung tissue on day 4 but could not be isolated on day 7. macroscopic lung pathology was dominated by peribronchial consolidation and necrotic lesions. histopathologic examination of the tissues collected 4 and 7 days post infection revealed extensive pathologic changes in the respiratory tract characteristic of a viral necrotizing interstitial pneumonia. although rt-pcr for h5n1 influenza virus was positive not only in the respiratory tract but in spleen, heart and also the cerebrum and cerebellum of one monkey, virus could only be demonstrated by immunoperoxidase staining in, and isolation from, the respiratory tract. the respiratory tract seems to be the major and probably the only target for the h5n1 influenza virus. although cynomologous monkeys have been used earlier as a model for h3n2 influenza disease (rimmelzwaan et al., 1997) , this is the first time a primate model for h5n1 viruses has been described. influenza h5n1 clinical symptoms observed in this study correlate well with what was seen in human disease caused by these highly virulent viruses, and was more severe than what is seen with h3n2 viruses. therefore, cynomologous monkeys may provide a useful model for studying influenza h5n1 pathogenesis and for developing h5n1 vaccines. 4.5. sb-242235, an orally acti6e, selecti6e p38 mitogen acti6ated protein (map) kinase inhibitor impro6es pulmonary functions in a murine influenza pneumonia model influenza infections are responsible for significant morbidity, especially in high-risk groups with underlying cardiopulmonary disease and in the elderly. the pathology results from a vigorous inflammatory response in the respiratory tract and damage to respiratory epithelial cells. in cells exposed to inflammatory cytokines, p38 mitogen activated kinase (map) activation leads to the upregulation of cytokines such as il-6, il-8 and tnfa (ono and han, 2000) . a recent study examined the effect of sb-242235, a highly selective orally bioavailable inhibitor of p38 map kinase, on pulmonary function in mice infected with influenza a virus. mice were infected intranasally with influenza a/pr8/34 and treated with sb-242235 at different time points post infection. initiation of treatment on day 0, 3 or 5 post-infection resulted in a 57% (p b0.01), 32% (pb 0.01) or 10% improvement in pulmonary capacity, respectively, compared with placebotreated control animals. no effect on virus clearance, survival, or antiviral immunity was observed. the efficacy of sb-242235 in reducing pulmonary resistance and increasing blood oxygenation was similar to that of the neuraminidase inhibitor oseltamivir, the steroid dexamethasone, and the cox-2 inhibitor nimesulide. sb-242235 was superior to non-specific nsaids indomethacin, naproxen, and ibuprofen. in mice and ferrets, sb-242235 reduced airway neutrophilia, and treatment was well-tolerated without any adverse effects. these data suggest that p38 map kinase is involved in influenza-induced cell signaling and that inhibition of this enzyme might reduce the severity of pulmonary disease. fortunately, the number of viruses that cause respiratory disease severe enough to require hospitalization is limited. in children younger than five years, rsv (subgroup a and b viruses), the parainfluenza viruses (piv1, -2 and -3), influenza a and b, and adenovirus types 1, 2, 3 and 5 are the major respiratory pathogens. influenza a and b remain, due to antigenic drift and shift, important agents in all age groups with very severe disease most commonly occurring in the elderly. rsv is the single most important cause of severe respiratory disease in infancy and childhood but it also causes significant morbidity in the elderly and in immunocompromised individuals. kim, chanock, brandt and parrott were the first to quantify the contribution of these viruses to the severe respiratory tract disease leading to hospitalization of infants and young children. rsv, piv3, piv1, piv2, adenoviruses and influenza b caused 23, 12, 6, 3, 6, and 1%, respectively, of respiratory disease leading to hospitalization, while influenza a was responsible for 3% between 1957 and 1968 (h2n2 era) and 7% between 1968 and 1976 (h3n2 era) (brandt et al., 1972; parrott et al., 1973; kim et al., 1979; murphy et al., 1988) . what are the general principles underlying vaccine development for these viruses? first, the protective antigens of the virus and mediators of immunity to reinfection are largely known. it is generally accepted that neutralizing antibodies to surface glycoproteins (g and f of rsv, hn and f of piv, and ha and na of influenza) or to capsid proteins (hexon and fiber proteins) of adenoviruses are the major mediators of resistance to reinfection. second, serum and mucosal antibodies make independent contributions to immunity against reinfection. whereas serum antibody is needed to mediate immunity in the lower respiratory tract, mucosal antibody is needed to protect the upper respiratory tract (with the exception of adenovirus, where serum antibody alone can prevent uri). third, live virus vaccines are generally more immunogenic than nonliving virus vaccines in immunologically naïve subjects, but identifying a live vaccine virus that is both safe and sufficiently immunogenic can prove to be a difficult task. fourth, different age groups might need different vaccines. infants younger than four months of age make less antibody than older children due to immunosuppression by maternal antibodies and immaturity of the immune system, and only potent immunogens, e.g. live virus vaccines, will be successful in this population (murphy et al., 1986) . on the other hand, a live virus vaccine that is sufficiently attenuated for use in infants might be over-attenuated in the elderly (gonzalez et al., 2000) . fifth, viruses differ in their ability to cause disease in the presence of maternal serum antibody. while mucosally delivered rsv, piv3 and influenza are infectious in the presence of maternal antibody, piv1, piv2 and adenovirus seem to be restricted in their replication in young infants by maternal antibodies. these five principles need to be considered when determining the optimal vaccination strategy (systemic versus mucosal, vectored antigen versus live attenuated virus) and the target age for vaccination. which vaccines are available? against influenza, the vaccine currently in use in the us is mostly a non-living, subvirion vaccine made from egg-grown purified virus disrupted with detergents. for practical purposes, the only relevant protein in the vaccine is the influenza hemagglutinin. the strains to be included in the vaccine for annual vaccination are selected by the public health service based on the antigenic profile of the currently circulating strains. subivirion or subunit vaccines plus adjuvant are also being developed (minutello et al., 1999; gluck et al., 2000) . live attenuated virus vaccines based on cold-adaptation of influenza a strain a/aa/6/60-h2n2 and influenza b strain b/aa/1/66 have been evaluated in phase iii clinical trials and should soon become available as a mucosal vaccine administered by nasal spray (maassab and bryant, 1999) . to date, an rsv vaccine has not been licensed. two non-living virus vaccines against rsv are being developed. one is a purified f protein subunit vaccine and the other consists of a g protein-specific peptide conjugated to the albumenbinding site of the streptococcus g protein (cano et al., 2000) . the purified f subunit vaccine is being evaluated for use in seropositive populations such as the elderly or high-risk older children (gonzalez et al., 2000) . used by itself, the f subunit vaccine has the disadvantage of not inducing a potent mucosal antibody response. also, this antigen induces a serum antibody response that is dominated by non-neutralizing antibodies (high ratio of titer of f protein-specific binding antibody to titer of neutralizing antibody). subgroup a and b live rsv vaccine candidates are being developed . one candidate vaccine bearing multiple attenuating mutations has been evaluated in 1-2 months old infants and was found to be attenuated and immunogenic but retained mild reactogenicity for the upper respiratory tract (wright et al., 2000) . it is likely that recombinant cdna technology will be used to develop or improve live attenuated rsv vaccines in the near future (whitehead et al., 1999a) . for example, a subgroup b rsv vaccine candidate was developed by substituting the g and f glycoprotein genes of rsv b for the corresponding genes in an attenuated recombinant subgroup a virus (whitehead et al., 1999b) , thereby rapidly generating a live attenuated subgroup b rsv vaccine candidate. to date, a piv vaccine has not been licensed. subunit and vectored vaccine candidates against piv3 have been developed and tested in animal models, but there are no reports of ongoing clinical trials. two live-attenuated piv3 vaccine candidates, a bovine piv3 (bpiv3) and a cold-adapted piv3 (cp45), have been evaluated in clinical trials, and both viruses were found to be safe and immunogenic in seronegative infants (karron et al., 1995 (karron et al., , 1996 . recombinant versions of these two viruses are also being evaluated as vaccines against piv3 and as vectors for the expression of foreign viral glycoproteins such as rsv g or measles ha (karron et al., 1995; durbin et al., 2000; schmidt et al., 2000) . the recombinant piv3cp45 vaccine candidate is currently being tested in phase ii trials in seronegative infants. for adenoviruses, a live virus vaccine against serotypes 4 and 7 has been used successfully in military recruits from 1971 to 1998. the vaccine made use of site-specific attenuation, i.e. a wt virus capable of inducing respiratory disease if administered to the respiratory tract was administered orally in an enteric coated capsule. the vaccine virus replicated only in the gastrointestinal tract and did not spread to the respiratory tract. the oral vaccine was found to be com-pletely attenuated and highly efficacious in inducing serum antibodies and protecting the upper and lower respiratory tract (couch et al., 1963; howell et al., 1998) . a similar tetravalent vaccine against serotypes 1, 2, 3 and 5 should be evaluated for use in young pediatric patients since these four viruses are responsible for over 80% of the adenovirus-related respiratory tract disease in this population. in virology, reverse genetics refers to the ability to rescue infectious virus from cdna. reverse genetic systems have been developed for all major viral respiratory pathogens, i.e. influenza a virus, paramyxoviruses, adenoviruses and most recently coronaviruses. this short review will only address the impact of reverse genetics on vaccine development for selected members of paramyxoviridae. for rsv, the generation of attenuated vaccine viruses started with conventional biological methods. first, wild-type rsv was passaged extensively at low temperature, and the resulting cold-passaged (cp)rsv was demonstrated to be attenuated in chimpanzees, as well as in seropositive adults and young children (friedewald et al., 1968 ). the cprsv virus was further modified by two rounds of chemical mutagenesis and biological selection for temperature-sensitivity (crowe et al., 1994a) . a panel of resulting mutant viruses was evaluated in mice and chimpanzees and ranked according to increasing attenuation. several promising candidate vaccines then entered clinical trials. rsv cpts248/404, a cold-passaged virus with two additional attenuating point mutations, is the most promising vaccine candidate tested so far, but it still causes transient nasal stuffiness in infants (wright et al., 2000) . the development of a reverse genetics system for rsv provided a method for expediting development of further attenuated viruses. as a first step, the biologically derived mutant viruses were sequenced completely and their mutations were identified and evaluated by introduction, individually or in combination, into wt recombinant (r)rsv. the direct identification of attenuating mutations made it possible, for example, to improve the above mentioned cpts248/404 mutation by incorporating one or more additional attenuating mutations from other vaccine candidates. furthermore, in many cases, the amino acid substitutions can be engineered to involve two nucleotide substitutions relative to wt, which should confer increased genetic stability. similarly, a reverse genetics system for piv3 was established and used to analyze all 15 mutations in the biologically derived cold passaged piv3cp45 (skiadopoulos et al., 1999) . however, the capabilities of reverse genetics go far beyond the analysis of existing biological mutants. a whole new set of methods for generating attenuated paramyxoviruses has been developed as described above. gene knock-out mutations, i.e. the deletion of non-essential genes, such as the rsv sh, ns1, ns2, m2-2 or g gene, generated a number of vaccine candidates with different levels of attenuation. as another example, in some cases an attenuating point mutation was found to involve a residue conserved, for example, between the l proteins of rsv and piv3 or between the c proteins of sendai virus and piv3. transfer of the attenuating mutation between the heterologous paramyxoviruses resulted in transfer of the attenuation phenotype. host range restriction could be employed as a basis of attenuation by creating antigenic chimeric viruses that use an animal virus as a platform for the expression of human rsv or piv glycoprotein protective antigen genes schmidt et al., 2000) . in this manner, an hpiv3 vaccine candidate was generated by replacing the bpiv3 f and hn glycoprotein genes in rbpiv3 with their hpiv3 counterparts (schmidt et al., 2000) . thus, the menu of available attenuating mutations for rsv and hpiv3 include numerous attenuating point mutations (which in some cases specify temperature sensitivity and in other cases do not), gene deletions, and host range restriction elements. the attenuating mutations or elements from each menu can be combined as desired to develop appropriately attenuated vaccine candidates. reverse genetics also expedites vaccine development because attenuated platforms can be used to make vaccines against additional viruses. for ex-ample, attenuated derivatives of rsv a2 (subgroup a) can be used to generate rsv subgroup b vaccine candidates by replacing the a2 f and g glycoprotein genes with their counterparts from the b1 strain of subgroup b (whitehead et al., 1999b) . as another example, a live-attenuated vaccine candidate was developed for hpiv1 by replacing the f and hn coding regions of hpiv3 with their hpiv1 counterparts. the resulting virus thus bears the antigenic determinants of hpiv1 in a wt hpiv3 backbone, which can then be attenuated by the introduction of mutations from the hpiv3 attenuation menu. reverse genetics can potentially help us to make vaccines that are satisfactorily attenuated but more immunogenic than wt virus. protective antigen genes can be moved closer to the genomic promoter and codon usage can be optimized for translation in mammalian hosts. cytokines and/or chemokines can be co-expressed from additional genes (bukreyev et al., 1999) , and reactogenicity can potentially be reduced, for example by ablating the secreted form of rsv g, which might be a decoy for antibody and might also perturb the t helper lymphocyte response. vaccine specificity can be broadened by adding additional genes to existing vaccine viruses, e.g. the measles hemagglutinin (ha) gene can be expressed from an additional orf in rhpiv3, generating a vaccine candidate that protects against both hpiv3 and measles (durbin et al., 2000) . this use of recombinant piv3 as vaccine and as a vector has several advantages over existing vector systems. it reduces the number of viruses to be administrated to infants and enables intranasal administration and thereby mitigates neutralization and immune suppression by maternal antibodies. the eradication of measles could be facilitated by a vaccine that does not include an infectious measles virus, which might cause prolonged infection in immunocompromised hosts. in summary, vaccines can be optimized through reverse genetics in a number of ways, such as the creation of novel combinations of mutations, the fine-tuning of the attenuation phenotype, the provision of a common attenuated platforms, and the generation of multivalent vaccines. 5.3. predicti6e 6alue of animal models in rsv 6accine de6elopment rsv was initially isolated from chimpanzees that developed a common cold-like disease in 1956. since then chimpanzees and other animal models for rsv disease have been studied extensively. principally, these higher primates have been used for three different purposes: the development of live virus vaccines, the recovery of rsv antibodies, and lastly to evaluate formalininactivated and subunit vaccines, and to study enhanced disease following vaccination with formalin-inactivated vaccine. a number of animal models are used to study rsv disease. among the apes, chimpanzees have been used most extensively for several reasons. they are permissive for rsv, their core body temperature is similar to that of humans, and symptomatic scoring of uri is possible (rhinorrhea score). the permissiveness of chimpanzees makes it possible to perform quantitative virologic studies in the upper and lower respiratory tract over the time course of an acute rsv infection. also, the restriction of rsv replication in the respiratory tract of chimpanzees correlates well with attenuation in seronegative human infants. in addition, human immunologic reagents can be used in chimpanzee studies. however, only young, carefully raised chimpanzees are rsv seronegative. other primates used include old world monkeys such as african greens, rhesus, cynomologous or bonnet monkeys and new world monkeys such as marmosets, tamarins and owl monkeys. although all these monkeys are semi-permissive for rsv, their core body temperature is higher than that of humans so that the level of attenuation of temperature-sensitive rsv mutants might be overestimated. although primate studies provide essential data for vaccine development, their use can be difficult. transport and care of primates, as well as sample collection are difficult and potentially dangerous, and they require well-trained personnel. primate research centers must provide an environment similar to that of a child-care facility and, therefore, maintenance costs are very high. lambs and calves are also used to study rsv infection, mostly because ovine and bovine rsv causes significant disease in their natural host. also, these viruses are important economically. cotton rats, mice and other rodents are used as small animal models. mice have the advantage of having a body temperature similar to that of humans and immunological reagents are readily available. also, gene knockout mice, transgenic mice and various inbred strains are available to study pathogenesis. the mouse model is not without difficulties, however. mice acquire a large portion of their maternal antibodies by suckling, and, therefore, they are not an optimal model for study of immunosuppression by maternal antibodies. peak rsv titers vary 100-fold among different mouse strains, and subgroup b rsv is poorly infectious and immunogenic in mice. immune mechanisms may also vary among different mice strains. rodent models can be used to study quantitative virology, immunology and airway pathophysiology (a model of wheezing), and weight loss can be used as a surrogate marker for rsv disease. whichever animal model one chooses to study respiratory syncytial virus, it is essential to clearly define whether the ultimate goal of the study is to prepare for clinical vaccine trials or to understand basic mechanisms of disease. several cold-passaged (cp) live attenuated rsv candidate vaccines have been evaluated in clinical trials. most of these candidate vaccines were derived by further attenuating the original cprsv through chemical mutagenesis and selection of ts mutants. the first two vaccines evaluated in the cpts lineage -cpts-248/955 and cpts-530/1009, were either insufficiently attenuated or were transmitted amongst seronegative children (karron et al., 1996) . a study of a more attenuated vaccine candidate, cpts-248/404, has recently been completed (wright et al., 2000) . this virus initially was evaluated in chimpanzees where it replicated to a peak titer of only 10 1.3 pfu/ml in the upper respiratory tract. due to this restriction in the respiratory tract, it was subsequently evaluated in clinical studies. cpts-248/404 did not replicate in adults or older children. in seronegative children 6-24 months of age, however, cpts-248/404 replicated to a peak titer of 10 4 pfu/ml in the upper respiratory tract and induced an antibody response against rsv f and g glycoproteins. the virus was well tolerated in this age group, and the frequency of upper or lower respiratory tract disease, otitis media or fever was not different from that of the placebo group. based on these data cpts-248/404 was selected to be the first rsv vaccine to be administered to 1-2-month-old infants, which represent the primary target group for vaccination. in this youngest age group, 17 of 24 vaccine recipients developed a clinical syndrome characterized by nasal congestion that occurred most typically between days 8 and 12 and lasted for approximately 24 h. since young infants are obligate nose breathers, this interfered with feeding and caused fussiness and difficulty in falling asleep. most vaccine recipients shed approximately 10 3 pfu of rsv per ml nasal wash. age of the infant or level of maternal antibodies against rsv did nor affect virus shedding, indicating that virus replication in the nasopharynx was independent of these variables. in these young infants, neutralizing antibody responses and igg elisa responses to rsv f or g were rarely detected, most likely because these responses were masked by the presence of maternally derived antibody. these young infants did, however, develop serum and mucosal iga responses preferentially to the rsv g glycoprotein and detection of serum iga correlated with protection from re-infection with a second dose of vaccine. although cpts-248/404 is not an acceptable vaccine in one to two month-old infants, several lessons were learned from the study of this vaccine candidate. first, seronegative infants are a much more susceptible host to rsv than chimpanzees. this means that for further attenuated vaccine candidates, the chimpanzee model of rsv infection will not be very useful. second, only viruses that do not replicate in adults and older children are attenuated enough for vaccination of infants. and third, reverse genetics is needed to further attenuate the existing biological rsv vaccine candidates. two of these recombinant rsv vaccine candidates are currently being evaluated in clinical trials. cpts-248/404dsh was generated by deleting the sh gene from the recombinant cpts-248/404 virus, and cpts-248/404/1030dsh was engineered to contain an additional (1030) mutation in the polymerase protein. preliminary data suggest that both recombinant viruses are well tolerated in 6-24-month-old children and are not associated with lower respiratory tract illness. whereas cpts-248/404dsh replicates as well as its parent biological virus without the sh gene deletion, the cpts-248/404/1030dsh mutant appears to be much more restricted in its replication in the nasopharynx. the more restricted cpts-248/ 404dsh induced both neutralizing and elisa igg antibodies in this age group. however, since this virus replicates, as well as cpts-248/404, it is likely not to be suitable for vaccination of one to two month old infants. cpts-248/404/1030?sh is currently being evaluated in one to two month old infants. in summary, although cpts-248/404 is close to an ideal vaccine candidate, further modifications of the virus through reverse genetics, such as addition of the 1030 mutation, are likely to be needed to generate an rsv vaccine that is immunogenic yet attenuated enough to be given to young infants. antibody preparations that neutralize free virus have been used as passive immunoprophylaxis to prevent a number of viral diseases, including hepatitis a and b, varicella and respiratory syncytial virus (rsv) disease. the efficacy of antibody preparations in preventing rsv disease was established first in cotton rat and chimpanzee models, and later in extended clinical trials. therapy of rsv infections with rsv antibodies, however, have so far failed to prove efficacious (malley et al., 1998; van woensel and kimpen, 2000) . the most important mechanism of action of antibody preparations is probably neutralization of free virus. this can occur via aggregation of free virus by bivalent or multivalent antibody, via receptor blockade, via antibody-complement lysis, or via fusion inhibition. receptor blockade is thought to result from steric inhibition of receptor binding rather than binding of antibody directly to the receptor-binding site itself. even if receptor binding does occur, virus infectivity can be neutralized through fusion inhibition. most rsv neutralizing antibodies are thought to use this mechanism of action. although complete antibody is most effective in neutralizing free virus, antibody binding fragments (fabs) alone can also neutralize. the minimal unit necessary to ablate infectivity is probably a peptide corresponding to one loop of the complementarity-determining region 3 (cdr3). although neutralizing and non-neutralizing antibodies against infectious virus are often distinguished, it is not clear whether there really are antibodies that bind glycoproteins in virions without neutralizing the virion (sakurai et al., 1999) . many so-called non-neutralizing antibodies bind purified (conformationally relaxed) rsv f glycoprotein but not the (conformationally correct) f protein on infected cells. different immunoglobulin subclasses neutralize virus with varying efficacy, and mouse and human igg subclasses also bind complement with varying efficacy. the replication of sendai virus (murine piv1) and influenza a virus can be inhibited intracellularly by iga and similar effects of iga on rsv replication may occur. a last but essential determinant of neutralizing activity is defined by the amount of antibody available to neutralize virus. to prevent rsv disease in the lower respiratory tract, a serum neutralizing antibody titer of approximately 1:300 is required (groothuis et al., 1993; top et al., 2000) . upper respiratory tract infections, however, can only be prevented with serum antibody titers as high as 1:5000-1:15 000. such titers can only be achieved in experimental settings in small rodent models. there is no single monoclonal antibody directed against rsv g protein that neutralizes completely on its own but cooperative neutralization occurs when multiple mabs are used. effective treatment for rsv illness is very limited. corticosteroids, bronchodilators, ribavirin, and, more recently, rsv mabs have been used but none of these therapeutic interventions are accepted as reproducibly efficacious. it seems that neither antivirals nor anti-inflammatory agents alone improve the outcome of rsv disease. a single dose of topically administered human fabs has been shown to clear free virus from the respiratory tract of rodents, but their effect is shortlived since infected cells release newly synthesized infectious virus within a day or two (crowe et al. 1994b ). whereas peak virus titers in humans usually occur around day 4, most patients with clinical rsv disease likely present no earlier than day 7. at this point, virus load is already in decline and it may be too late for antivirals, and antiinflammatory drugs such as map kinase inhibitors may be more effective in reducing cell injury. are there immunological consequences of passive immunoprophylaxis? in chimpanzees therapy with rsv antibodies suppresses the primary antibody response to rsv. this effect is mostly antigen-specific but may also have a non-specific aspect mediated by the fc portion of the antibody. the secondary antibody response to rsv in chimpanzees is enhanced by prior antibody therapy. however, this effect was not observed in mice or in clinical studies. t cell responses do not seem to be as affected by antibody therapy as humoral responses, and t cells might fill in for absent primary antibody responses. whether this is a desirable effect or not, remains uncertain. without a safe and effective rsv vaccine available, monthly infusion of rsv-igiv (respigam ® ) was the first effective measure to prevent rsv-induced lower respiratory tract infection (lri) and hospitalization in infants. the evaluation of rsv-ivig in cotton rats correctly predicted the serum concentrations necessary to protect against rsv-induced lri (prince et al., 1985a) . protective concentrations in children were achieved by monthly infusions of 750 mg/kg of rsv-ivig. in order to increase the potency of a rsv antibody preparation, and in order to be able to replace intravenous with intramuscular administration, a humanized monoclonal anti-rsv fusion protein antibody was developed. mab1129, a mouse monoclonal antibody developed in the laboratory of infectious diseases at nih against the rsv f protein antigenic site a, one of two antigenic sites that are conserved amongst different rsv strains, was selected for humanization based on in vitro and in vivo studies, and the complementarity determining regions (cdr) were transferred from the mouse antibody to a human igg. this humanized igg should have pharmacokinetics similar to human igg and permit repeated administration at monthly intervals. the chosen mab, medi-493, had no crossreactivity with adult or neonatal tissue, broadly neutralized rsv subgroup a and subgroup b isolates at concentrations of 20 ng/ml, and proved to be 50-100 times more active on a weight basis than rsv-ivig in the cotton rat model (johnson et al., 1997) . adult volunteers tolerated doses of the humanized antibody medi-493 from 1 to 30 mg/kg well, and only some volunteers developed a low-titer, transient anti-idiotypic antibody response. medi-493 had a half-life of 17 days, as is expected for igg, and serum concentration after iv and im administration were comparable except for the initial bioavailability. medi-493 was safe and well tolerated in phase i/ii studies in high-risk children, and had no specific immunogenicity in and of itself. monthly dosage of 15 mg/kg maintained serum concentrations greater than 40 mg/ml, and a single intravenous dose of 15 mg/kg reduced rsv titers in tracheal secretions of intubated children with rsv infection (malley et al., 1998) . the impact-rsv study that led to fda approval of medi-493, also called palivizumab, was a 2:1 randomized, double-blinded, placebo-controlled phase iii multicenter study conducted at 139 sites in the us, canada and the uk. study subjects received five doses of medi-493 or placebo at intervals of 30 days and were followed for a total of 150 days. the primary endpoint of this study, overall rsv-related hospitalization, was significantly reduced (pb 0.001) from 10.6% for placebo recipients (n= 500) to 4.9% for medi-493 recipients (n=1002). for premature infants with chronic lung disease, the incidence of rsv hospitalization was reduced from 12.8 to 7.9% (p= 0.038), and for premature infants without chronic lung disease, it was reduced from 8.1 to 1.8% (pb 0.001). total rsv hospitalization days, total days with increased oxygen requirement, total days with severe lri, rate of icu admission and total days in icu were secondary endpoints that occurred less frequently in the medi-493 treated group (p b 0.05). thus, administration of 15 mg/kg medi-493 by im injection was found to be safe, well-tolerated, and to lead to a 55% reduction of rsv hospitalization in high-risk children (group, 1998b) . this conclusion was confirmed in an outcome survey conducted in nine centers in 1998 and 1999, in which 1839 high-risk children received 15 mg/kg palivizumab. although direct comparison to the impact-rsv study is not possible, rsv hospitalization rates were low and similar to those observed in the impact-rsv study: 2.1% (vs. 1.8% in the impact-rsv study) of premature infants without cld and 4.0% (vs. 7.98%) of children with cld were hospitalized in the 1998-1999 rsv season (sorrentino and powers, 2000). an alternative strategy to protect neonates and young infants against severe rsv disease is vaccination of pregnant women. transfer of high concentrations of maternal rsv-specific igg to the fetus is expected to protect the infant against severe disease (glezen et al., 1981) . the safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 (pfp-2 wyeth lederle vaccine and pediatrics, pearl river, ny) were recently evaluated for use in pregnancy. thirtyfive pregnant women were randomized at a ratio 2:1 to receive rsv pfp-2 or saline placebo at 30-34 weeks of gestation and were followed until the time of delivery. infants were followed during their first year of life and their first rsv season. rsv pfp-2 was safe and well tolerated by pregnant women, and there were no systemic reactions or serious adverse events associated with vaccine administration. all 35 infants were born healthy, and there were no differences in the frequencies and outcomes of neonatal events between the groups. during the first rsv season, there was no increase in the frequency or severity of respiratory tract illnesses in infants of vaccine recipients. there is ample material from which to draw lessons relevant to needed preparations for pandemic influenza. but, to date, the response, and specifically preparations for dealing with a serious pandemic of influenza remain more in the realm of academic reflection than meaningful action. although much time has been devoted to the development of a national response plan, we do not seem to be much better equipped to deal with a new pandemic of influenza than we were in the spring of 1957, when the h2n2 strain of influenza a virus emerged. at that time, we faced a burgeoning epidemic, whose virulence and propensity for spread were as yet unknown. a program of surveillance and field epidemiology to better define the epidemic had to be developed and, at the same time, preparations were needed for distribution and use of a new influenza vaccine, if it arrived in time. it did not. it appears today that not much progress has been made over the past 30 years, and still no real sense of urgency in dealing with the essentials of the problem can be felt. although much has been written about the 1918 epidemic, it is still perceived by most as an interesting but questionably relevant tale of death and disease during an earlier, pre-antibiotic era of medicine. many doubt that an epidemic of this severity would be possible today. but, here are a few of the facts. first, it is important to recognize that, for the us, it dwarfed all other outbreaks of the 20th century. at that time, more than 20 million died worldwide and in the united states, there were more than 500 000 registered deaths. from various studies, it is thought that, overall, perhaps 40% of the population became ill, of whom about 2% died. medical services were overwhelmed but, other than supportive care, there was little that curative medicine could offer. the deaths were so numerous that burials were greatly delayed because of the lack of morticians and grave diggers. pictures from the time provide at least a pale illumination of that catastrophic period. the effect of the disease was anything but uniform. surveys revealed morbidity rates ranging from 15% to more than 60% in different parts of the country. some remote and rural areas escaped the disease entirely but in some areas, it was remarkably lethal. western samoa, then a new zealand protectorate, registered 8500 deaths in a population of 38 000. this represented 22% of the entire population. curiously, american samoa, only 50 miles away but with a quarantine in effect, was one of the few political entities to escape the epidemic entirely. what was surprising and unique about the 1918 epidemic was that more than half of all deaths were in persons between 15 and 45 years of age -an age bracket in which death is a relatively uncommon phenomenon. pregnant women and those with cardiac problems were at highest risk but most, who died, were otherwise in good health. a substantial number in this age group died of a fulminant, rapidly progressive pneumonia marked by severe cyanosis with death occurring within a matter of 1-3 days, in brief, what seemed to be almost certainly, a primary influenza pneumonia that would have benefited little from antibiotics, had they been available. today, a better outcome might be foreseen with better ventilatory support in intensive care units; with the administration of antiviral agents; and with the administration of antibiotics for the treatment of secondary pulmonary infections. but under epidemic circumstances, the bulk of cases in a new pandemic would occur over a period of only 3-6 weeks and only a fraction of patients could be accommodated in suitable hospital settings, let alone appropriate intensive care units. what quantity of antiviral drugs is available for emergency use? where do we obtain the added quantities of the standard antibiotics that today, are produced on a just-in-time basis -when, during a period when clinical facilities are not stressed, we are regularly experiencing antibiotic shortages on a regular, rotating basis. a cogent question is whether under epidemic circumstances, our modern health care system could provide a standard of clinical care that would be better than it was in 1918. but there is still another dimension to the problem. do we appreciate that a 'pandemic', so characteristic of the emergence of a new influenza strain, means exactly that -a worldwide epidemic. many countries -developed and developing, have made no preparations and will inevitably turn to those with resources to help them in dealing with a catastrophe -a catastrophe that poses an international security threat. the rapid production and administration of large volumes of vaccine effective against the emergent new strain has been a basic building block of the strategy for dealing with pandemic influenza and, understandably so, given the limitations of curative medicine and the capacity of clinical services. the first real test of this strategy came during the 1957 epidemic. the first notice of a major outbreak occurred in mid april; specimens were received in the us on 13 may; and field testing of new lots of the vaccine began in july. not a bad record. it was foreseen that 60 million doses would be required. with good fortune in adapting the virus to grow at reasonably high titer on egg membrane and provided that sufficient fertilized eggs could be obtained, it was expected that a production target of 1 february could be met. even this was a problematical date given that the seasonal peak of epidemic influenza usually occurs between the end of december and the end of february. still, it was believed that substantial numbers would be able to be protected. however, 1957 was not a typical year, and such, one must note, was the case when the 1918 epidemic strain first appeared. widespread epidemics began occurring in mid to late september, two months before they were anticipated. more than half of all counties reported epidemics by mid-october and by the end of october, the peak incidence had past, long before any substantial quantity of vaccine was available. given these experiences, could we expect another new strain to behave differently today? over the past 30 years, extensive studies have been conducted in the search for a satisfactory live attenuated vaccine and for various approaches in production which would permit new antigenic variants to be produced rapidly and in quantity in tissue cell culture. however, today we are still producing influenza vaccine in the allantoic cavity of hens' eggs, as we were in 1957; procuring adequate supplies of fertile eggs in a timely manner remains a serious problem; and difficulties in adapting new strains to eggs remain. were we able to solve the vaccine production problem for our own country, this would not be the end to the practical quandary of dealing with the pandemic. few countries have influenza vaccine production capability and the international implications of the us having all or much of the vaccine supply are profound. following the 1976 swine influenza outbreak at fort dix, new jersey, the us had embarked on a program to rapidly produce an appropriate vaccine. vaccine production capability in europe and other countries was so marginal that a world health organization committee could only recommend that the fort dix situation be carefully monitored -a strategy of wait and hope. but as those at the who meeting commented in corridor conversations, they would not wish to face the ethical and political dilemma the united states would face were there a pandemic and the us was the only nation with a vaccine. in recent meetings with national and local hospital authorities, current capabilities of the medical system in us to deal with sudden surges in demand such as might follow release of a biological weapon were explored. such a release would result in an epidemic that would stress the system not unlike the way it would be stressed by a pandemic of severe influenza. from these meetings, it was evident that the elasticity of the nation's bed supply has been significantly reduced as drives for financial efficiency and the increasing use of out-patient procedures have sharply reduced the numbers of beds in all hospitals. meanwhile, managed care-driven market pressures and federal government reimbursement reductions have driven large numbers of hospitals into operating deficits. many of the municipal hospitals, once a primary source of care for the less prosperous and uninsured have been privatized. meanwhile, the hospitals are experiencing severe labor shortages, especially for nursing and technical personnel. few have either the resources or motivation to prepare to respond to the challenges posed by mass casualties due to any cause. reserves of antibiotics, as noted earlier, are marginal to nil; the public health infrastructure needed to deal with epidemic disease is grossly understaffed, underpaid and under trained; mechanisms for the development and implementation of community-wide plans are largely unexplored. it is not unreasonable to suggest that we are today less well-prepared to deal with an epidemic of influenza than we were 30 years ago in most parameters that one can identify. what might a new strain of influenza mean in terms of numbers of cases and deaths. estimates of past pandemics suggest that perhaps 40% of the population were affected in a first wave. with rising proportions of the population in urban areas, the greater and more rapid mixing of populations through travel, a figure of not less than 60% would seem more reasonable. thus, in a city of 3.0 million, one might expect 1.8 million cases of influenza. it is doubtful that either antibiotics or antiviral agents would be of much help given the number of cases and the dearth of reserve supplies. the number of deaths would vary greatly depending on the strain. in hong kong, a recent new strain, h5n1, resulted in death among six of 18 persons infected. it did not spread readily, however. it is estimated that the 1918 strain killed 2% of those who became ill, while the 1957 case-fatality rate was about one-tenth as large or 0.2%. thus, in a city of 3 million persons, one might anticipate between 3600 and 36 000 deaths over a period of 3-6 weeks. medical care would consist largely of supportive therapy given the numbers of those ill. public health measures, likewise, would consist of little more than reassurance given the likelihood that vaccine supplies would not be available and that stocks of antiviral drugs would be too small to be of significance. many would argue for the closing of schools, churches and other places of public gathering but experiences suggests that such actions produce little benefit. the wearing of masks was once a favored intervention but this, too, has been discredited. in brief, without vaccine, there would be little that could be done of practical benefit except to reassure the community that the epidemic would someday pass and to accept the criticism of the public and political leadership for failure to make reasonable preparations. at least four lessons can be derived from past experience. first, the threat of pandemic influenza caused by an especially virulent strain is a continuing threat that has to be taken seriously. second, adequate supplies of an effective vaccine, available in a timely manner, are absolutely critical to a preventive effort. solving this problem should command top priority for research and development funding. third, special plans, programs and funding are needed within the health care system to permit development of an adequate community-wide response to the occurrence of mass casualties whatever the cause. lastly, additional research in influenza is needed to better understand its pathogenesis and epidemiology with the expectation that better preventative measures might eventuate. pandemics are the most dramatic presentation of influenza a virus and they cause considerable excess mortality as a result of pneumonia and exacerbation of cardiopulmonary or other chronic diseases. the epidemiologic success of influenza a is in large part due to antigenic variation that takes place in the two surface glycoproteins of the virus, i.e. its hemagglutinin (ha) and neuraminidase (na) proteins. to date, 15 ha and 9 na subtypes have been defined and are used to classify influenza a viruses. antigenic variation occurs either gradually through accumulation of point mutations (antigenic drift) or more abruptly though introduction of a new ha gene into virus circulating in the human population, be it through reassortment of animal and human viruses or through a change in host-specificity of an animal virus (antigenic shift). a 'pandemic virus' can be defined as a virus with a new ha with or without a novel na gene, acquired through antigenic shift, that spreads readily from person-to-person in a population that is highly susceptible to infection. the 20th century saw three pandemics caused by new ha subtypes. influenza a h1n1 caused the 'spanish flu' in 1918, subtype h2n2 ('asian flu') was the causative agent of the 1957 pandemic and the h3n2 subtype ('hong kong flu') caused a pandemic in 1968. the excess mortality for the 1918, 1957 and 1968 pandemics in the us alone can be estimated to have been 500 000, 70 000, and 34 000 deaths, respectively. if one applies mathematical modeling to estimate the effects of a future pandemic, the us alone can expect between 89 000 and 207 000 excess deaths in the next pandemic (meltzer et al., 1999) . what are the viruses with pandemic potential? in 1997, 18 individuals in hong kong were infected with h5n1 influenza, an avian subtype earlier not known to infect humans (subbarao et al., 1998) . of the 18 patients 1-60 years of age, six died. molecular analysis established that all eight genes of the h5n1 virus were of avian origin and that reassortment with human viruses had not occurred (subbarao et al., 1998) . the reported 18 infections were most likely acquired from poultry; human-to-human spread, however, was a rare event. sequence analysis of the hong kong h5n1 virus suggests that it is a reassortant made up from two or three different avian parent viruses: ha from a goose h5n1 virus (xu et al., 1999) , na from a teal h6n1 virus (hoffmann et al., 2000) , and internal genes from a quail h9n2 (guan et al., 1999) or a teal h6n1 virus (hoffmann et al., 2000) . although this virus did not spread readily from person to person, deep concern was caused by the fact that this was the first known avian influenza a virus that caused disease in humans. until this outbreak it was thought that the receptor specificity of avian ha proteins limited their infectivity to avian species. this notion, however, needs to be revised. in march 1999, h9n2 influenza a viruses were isolated from two children with febrile upper respiratory tract illness (peiris et al., 1999) . this virus, again, was an avian virus that was able to infect humans without passage through an intermediate host (lin et al., 2000) . what are our options for vaccination against potentially pandemic viruses? in the case of the h5n1 viruses, there are four options for generating an effective influenza vaccine. a conventional inactivated vaccine can theoretically be generated by reassortment of internal genes from influenza a/pr/8/34 with h5n1 glycoprotein genes. there are, however, problems with incompatibility between certain gene segments. secondly, a cold adapted live attenuated vaccine approach could be taken. the influenza a/ann arbor/6/60 coldadapted strain has been used to generate an h5n1 vaccine in which the ha cleavage site was modified (li et al., 1999a) . this virus could also be used to produce an inactivated vaccine. thirdly, a surrogate virus that is nonpathogenic but antigenically related (takada et al., 1999) could be sought. one of the closest antigenically related viruses found to date was of the h5n3 subtype, but this virus was not highly immunogenic in humans. fourthly, purified protein could be used but again limited immunogenicity may be a problem. the development of vaccines against potential pandemic viruses poses a number of challenges. to begin with, most of the work has to be conducted in biosafety level 3+ laboratories. there is a large array of avian viruses that could potentially become pandemic viruses and those with a genotype that confers transmissibility have to be identified. h5n1, h9n2 and h6n1 viruses that were the genetic precursors of the h5n1 and h9n2 viruses that caused human infections in hong kong (guan et al., 1999; xu et al., 1999; hoffmann et al., 2000) and that continue to circulate among birds should certainly be given priority in vaccine development. an adequate strategy for vaccine development has to be selected, and safety testing must be expedited in mice, ferrets, chickens, and eventually humans. use of recently described plasmid based reverse genetics systems may lessen the technical challenges faced in generating vaccine candidates. assays to evaluate immunity have to be optimized (hemagglutination inhibition assays do not perform well with avian ha proteins, yet neutralization assays are time consuming and technically more difficult), and serologic and molecular diagnostic reagents must be made available. collaboration between veterinary and public health authorities must be maintained and new technologies such as plasmid based reverse genetics systems must be used. last but not least, alternative substrates for vaccine production and new adjuvants are urgently needed. although influenza epidemics occur every winter, we have only had three pandemics this past century -1918, 1957 and 1968 . while annual influenza epidemics generally cause an excess mortality of 20 000-40 000 people in the us, it is only the pandemics that remain in memory. of the three pandemics that occurred in the twentieth century, the 1918 pandemic was the most devastating and claimed the most victims. although there is wide spread public interest in the 1918 pandemic, the event is mostly remembered as an interesting event in the distant past. whether due to denial or not, few feel that an influenza pandemic is a current threat. most journalists see their role not only as educators and advocators but also as entertainers. the interest of the readership has to be captured, and it can only be maintained if their curiosity is satisfied. while the aids pandemic and its effect on people's lives found a wide audience through most of the early 1990s, the late 1990s were characterized by fatigue and a reduced interest in the pandemic (although it was still on the increase). as a result, editors were much less willing to include related stories. reportage on influenza pandemics suffers a similar fate: the disease itself is old, and the last pandemic occurred too long ago for most to remember. the mere warning of a coming pandemic is almost perceived as an unfulfilled promise if the event does not occur shortly after the article is published. breast cancer, in contrast, is one of a few examples, where interest in a disease process can be maintained over an extended period of time. the risk of suffering from breast cancer is felt much more acutely, it seems, and there is a sense that proactive behavior will lead to an improved outcome. whether the mass media will take on a role in changing the public's attitude toward influenza as a threat is very much in doubt as long as journalists themselves perceive the next pandemic as an event as anonymous and inevitable as an earthquake. rsv infection is initiated by g glycoprotein attachment to cell surface receptors and followed by virus-cell fusion that is mediated by the f protein. the cleavage-activated rsv f1 protein is thought to interact with the target cell membrane through its n-terminal fusion peptide, which is released from a shielded position within the f homotrimer through a major conformational change. insertion of the f1 n-terminus into the cell membrane destabilizes the cell membrane and induces lipid mixing that is followed by mixing of contents, thereby enabling the viral nucleocapsid to enter the cytoplasm. the rsv f protein was recently found to interact with a small gtpase called rhoa through a domain that lies just carboxyterminal to the f1 fusion peptide (pastey et al., 1999) . rhoa is a member of the ras superfamily that is expressed intracellularly in all cell types. it induces bundling of actin filaments into stress fibers, focal adhesion plaque formation, cell-to-cell adhesion and organization of integral membrane proteins; it has additional roles in cell morphology and motility as well as cell cycle transition from g1 to s. a series of overlapping rhoa peptides from the interaction domain were evaluated in rsv plaque reduction neutralization assays. rhoa peptide 77-95 has an ic 50 of about 1 mg/ml against an inoculum of 100 pfu. neutralizing activity is also seen against parainfluenza virus type 3 (piv3), but not against a variety of other paramyxo, orthmyxo, corona and filoviruses (pastey et al., 2000) . intranasal administration of 500 mg of peptide 77-95 prior to intranasal infection of mice with rsv reduces rsv replication more than 100-fold and also diminishes weight-loss. studies with recombinant rsv expressing green fluorescent protein indicated that rhoa peptide 77-95 inhibited rsv replication at a very early stage of infection. subsequently, it was shown that fusion of cells transfected with rsv f, g and sh was inhibited by this peptide, suggesting that it exerted an effect on rsv replication at the level of membrane fusion or entry. since rhoa is an intracellular molecule, one would have to hypothesize that the interaction of the n-terminal heptad repeat and fusion peptide with the target membrane causes enough membrane disruption to allow an interaction between rhoa and rsv f. alternatively, rsv f may not interact with rhoa during the entry process, and the rhoa-derived peptides may simply disrupt the f structure or function to render the virus noninfectious. the question of whether rhoa and rsv f interact during a natural infection is not yet resolved. rhoa can be found in the cytoplasm bound to gdp and a guanine nucleotide dissociation inhibitor, and, upon gtp exchange and isoprenylation, rhoa associates with the cell membrane. if the proposed model of rhoa-rsv f interaction at the membrane is correct, then inhibition of isoprenylation should inhibit rsv infection. indeed, inhibition of isoprenylation through hmg coa reductase inhibitors, such as lovastatin, inhibits rsv infection of hep-2 cells at an ic 50 of 3 mm and reduces peak rsv titers in lungs of mice greater than 100-fold with oral gavage of 1 mg per day (gower, t.l., graham, b.s., submitted). the antiviral effect of lovastatin is also seen with piv3 in vitro. although it has not been formally proven that rsv f interacts with rhoa in the infected cell membrane, rhoa signaling activity is triggered in rsv-infected cells (gower, t.l. et al., submitted) . while rhoa signaling is not required for rsv replication, syncytium formation is diminished when rhoa signaling activity is inhibited. in addition, rhoa signaling may play a role in other aspects of rsv pathogenesis. rhoa kinase inhibitors reduce the transcription of il-6 and il-8 mrna in rsv infected cells (cytokines abundant in the nasal secretions of rsv-infected patients), and obviate airway hyperresponsiveness, one of the cardinal symptoms of rsv disease (hashimoto, k. et al., submitted). the balb/c mouse model was used to explore whether mycophenolic acid (mpa), an inhibitor of de novo purine synthesis in t and b lymphocytes, could improve the outcome of rsv disease, which was monitored by clinical symptoms such as ruffling of fur, increased respiratory rate and weight loss. oral administration of 100 mg/kg mmf (the prodrug of mpa) daily from day 1 to 6 post-infection reduced weight loss on day 7 post-infection from 23% in untreated animals to 8% in mpa treated mice (p b 0.001). a reduction of weight loss (7.8%) was also observed when initiation of treatment was delayed until day 5 post-infection. virus titers in lungs of mmf treated mice were similar to those of untreated controls but histological changes were reduced. on day 7 post-infection, ifng levels were elevated 2.5-fold in the treatment group while il4, il5 and il10 levels were unchanged, indicating a shift toward a t helper cell type 1 response that is thought to correlate with improved rsv disease outcome. these data suggest that inflammatory responses contribute to rsv disease in mice and that an immunomodulatory approach to the treatment of human rs virus disease is worth further consideration. using a cell-based assay to identify compounds which are able to inhibit fusion of hela cells infected with rsv, more than 300 analogues of a lead compound were synthesized and one compound (termed r170591) was selected for further evaluation. the in vitro 50% inhibitory concentration (ic 50 ) of this benzimidazole derivative (mw 395) was150 pm, and thus its potency exceeds that of ribavirin almost 100 000-fold (ic 50 =10 mm). r170591 exhibits in vitro antiviral activity against human rsv (subgroup a and b) and bovine rsv but not against pneumovirus of mice or other paramyxoviruses. rsv-induced cytopathic effect was reduced by r170591 at 0.1 nm, and concentration of 10 nm reduced rsv titers 1000-fold in multi-cycle growth curves. time of addition studies indicated that both virus-cell fusion and cell-cell fusion were inhibited by this compound. selection of resistant viruses in vitro yielded two mutants with single point mutations in the f-protein: one upstream of the second heptad repeat motif and another within it (s398l and d486n). rsv titers, determined by quantitative rt-pcr, were reduced 10-fold in bronchoalveolar lavage fluid and in lung tissue of cotton rats treated once by inhalation with r170591 prior to rsv infection. rfi-641, a compound that was derived by chemical optimization of the earlier described antiviral cl-387626, is a small molecule antiviral drug that selectively inhibits rsv (wyde et al., 1998) . the molecule is water-soluble and not orally bioavailable, but it proves to be efficacious when administered intranasally or by inhalation. the in vitro ic 50 varies between 3 and 180 ng/ml for laboratory strains or clinical isolates of rsv subgroup a and b. viral specificity and the large therapeutic window of rfi-641 (\100 fold) indicate that the antiviral activity of the compound is not due to adverse effects on normal cells. addition of rfi-641 to cell culture prior to adsorption reduces rsv yield 1000-fold at 48 h post infection (wyde et al., 1998) . temperature shift experiments suggest that the rsv f protein is the target for rfi-641, and this observation is confirmed by the inhibitory effect that rfi-641 has on rsv b1 cp-52/2b5, a viable rsv mutant in which both the g and sh open reading frames are deleted. if rfi-641 is added to cell culture 5 h post infection, it inhibits syncytium formation indicating that fusion inhibition occurs both in the early and late phase of the infectious cycle. rfi-641-resistant viruses can be selected, albeit much less easily than amantadine resistant variants. resistance to rfi-641 is acquired by point mutations solely in the f protein, mostly upstream of the second heptad repeat motif, but not by mutations in the g or sh protein. when administered prophylactically by the intranasal route, rfi-641 inhibits rsv replication in vivo, with mice and cotton rats exhibiting a 10-1000-fold reduction in rsv replication on day 5 post infection. in african green monkeys, rfi-641 reduces peak rsv titers not only when administered prophylactically but also when therapy is initiated at 24 h post infection and continued for a total of 9 days. a nebulized form of rfi-641 has been shown to be active in monkeys. the preclinical profile of this drug supports its development for treatment and prophylaxis of rsv disease in pediatric, adult and geriatric populations. phase 1 clinical trials confirmed that rfi-641 is a potent antiviral, and that the safety profile of this drug is encouraging. 7.5. de6elopment of picona6irus 3c protease inhibitors ag7088 is a potent, irreversible inhibitor of human rhinovirus (hrv) 3c protease, the enzyme that is responsible for the cleavage of the viral polyprotein into its functional protein subunits (matthews et al., 1999) . ag7088 was discovered by protein structure based rational drug design, and the compound exhibited activity against a large set of different hrvs. the 50% effective concentration (ec 50 ) ranges between 3 and 81 mm. other piconaviruses such as coxsackievirus a21 or b3, enterovirus 70, and echovirus 11 are also sensitive to the compound. in a placebo controlled challenge study in which adult volunteers were infected with hrv39 and treated with ag7088 (8 mg per dose intranasally, 5 times per day), ag7088 reduced mean viral titers, as well as mucus weight, respiratory symptom score and total symptom score significantly. in this study viral titers were determined by culture and also by quantitative pcr (taqman) to exclude ex-vivo effects of ag7088 on the reduction of virus titers. a phase ii clinical trial with 868 subjects was conducted to determine the efficacy of ag7088 in naturally acquired piconavirus infections. patients selected for the study had to present within 36 h of onset of symptoms and had to suffer from at least two mild or one moderate cold symptom. a five step symptom score was used to record severity of rhinorrhea, cough, sneezing, sore throat, chills, headache, and malaise. the treatment group was stratified into two or four daily doses of 8 mg ag7088 per dose intranasally, and the mean respiratory symptom score for days 1-5 was chosen as the primary endpoint. only 29% of all enrolled patients were infected by picor-naviruses. no significant difference in respiratory or total mean symptom score for days 1-5 was detected. however, the drug was well tolerated and safe. the lack of efficacy in this particular study might have been due to a lower than expected frequency of picornavirus infections. in a retrospective analysis, stratification for start of therapy within 24 h detected a trend to fewer respiratory symptoms and fewer total symptoms. there was a trend to earlier onset of relief, but the difference between treatment and placebo group again did not reach significance. 7.6. pleconaril (p) treatment reduces the incidence of acute otitis media (om) in children with 6iral respiratory illness: a pilot study pleconaril, an orally bioavailable picornavirus 3c protease inhibitor, has in vitro antiviral activity against 93% of all rhinoviruses. to evaluate the efficacy of pleconaril in preventing acute otitis media (aom) in an outpatient population, a double-blind, placebo-controlled pilot study was conducted in children with viral respiratory illness. eighty-seven children with a median age of 3 years and a history of 2-3 episodes of aom were randomized to pleconaril treatment (5 mg/kg or 2.5 mg/kg) or placebo thrice daily for 7 days following presentation with upper respiratory symptoms. picornavirus rna was detected by rt-pcr in nasal samples in 51% of patients at baseline. the overall incidence of physician-diagnosed aom during the 14 day follow-up were 18.8% (9/48), 12.5% (3/24), and 0% (0/15) in the placebo, low dose and high dose groups, respectively. of the 87 children developing aom, 86 tested positive for picornavirus rna. pleconaril treatment reduced the frequency of nasal symptoms by 29% in the high dose (5.0 mg/kg, p= 0.006) and 9% in the low dose (2.5 mg/kg, p= 0.393) group. it reduced frequency of systemic symptoms by 40% in the 5.0 mg/kg group (p= 0.020) and 26% in the 2.5 mg/kg group (p= 0.096). pleconaril was well tolerated, and adverse events did not differ from the control group. these results encourage further expanded trials of pleconaril in children with viral respiratory infections. 7.7. oral oseltami6ir reduces influenza-related complications in all age groups influenza illness is associated with the development of secondary complications, often requiring antibiotic treatment or even hospitalization. insurance data indicate that up to 64% of influenza related hospital admissions occur in 15-64 year old individuals, often without recognized underlying disorders. influenza complications affect all age groups but the type of complication differs among them. while acute otitis media has been reported in over 20% of children with influenza, lower respiratory tract infections (lrti) such as bronchitis and less often pneumonia, are the most common complication in adults. in recent years six phase iii clinical trials were completed; two of them in pediatric populations, three in healthy adults and one in the elderly. the neuraminidase inhibitor oseltamivir (o) is already approved for the treatment of influenza in adults, and it was recently approved for treatment in children aged 1 year and older by the fda. in six phase iii trials, most of the patients were enrolled within 36 h of onset of symptoms, and oseltamivir was administered at 2 mg/kg twice daily for children and 75 mg twice daily for adults. subjects with febrile ( \37.8°c) influenza were randomized to a 5 day regimen of oseltamivir or placebo, and an important endpoint of all studies was the effect of oral oseltamivir (o) on the incidence of influenza-related complications requiring antibiotics. one thousand and twenty nine children between the age of 1 and 12 (mean age 6.4 years) were enrolled in the pediatric trials. in the adult and elderly populations, 953 patients with a mean age of 35 years and 736 elderly patients with a mean age of 73 years were enrolled. in the pediatric, adult and elderly trials 61, 64 and 65%, respectively, tested positive for influenza a or b by culture or fourfold increase in hai titers. physician-diagnosed secondary complications (bronchitis, pneumonia, lrti, sinusitis or otitis media) requiring antibiotics were assessed in patients with confirmed influenza infection. oseltamivir reduced the rate of complications compared with placebo by 40% in children (p=65/235, o =36/217; p =0.005), by 51% in adults (p =23/309, o = 11/301; p = 0.05), and by 28% in the elderly (p= 49/254, o= 31/223; p= 0.14). in the pediatric population, aom was the most common complication, followed by bronchitis, pneumonia and sinusitis. the frequency of aom was reduced from 17% in the placebo group to 10% in the oseltamivir group, a significant 40% reduction. in adults and the elderly, bronchitis and sinusitis were the most common complications of influenza. the frequency of bronchitis in the elderly was reduced from 15 to 11%. thus, oral oseltamivir reduced the incidence of secondary complications and associated antibiotic use in all age groups. when all age groups were combined, antibiotic use for any indication was also significantly reduced. twelve patients in the placebo group versus four patients in the oseltamivir group required hospitalization for probable or possible influenza-related complications, suggesting a possible effect on hospitalization. 7.8. neuraminidase inhibitors: clinical update 7.8.1. oseltami6ir oseltamivir was recently approved in the us for prophylaxis of influenza in adults and adolescents over 12 years of age after three successful phase three trials. one was conducted in healthy adults, one in a family setting with an infectious index case, and another evaluated seasonal prophylaxis in the elderly. the efficacy of oseltamivir in all three trials ranged between 70 and 90%. an application for the approval of oseltamivir for treatment of influenza in children over one year of age was recently approved by the fda. in otherwise healthy children of 1-12 years, treatment with oseltamivir reduced the median time to freedom from illness by 1.5 days (p= b 0.0001) and also reduced the relative risk of otitis media (aom) by 40%. in 6 -12 year old asthmatic children, oseltamivir treatment, started within 24 h of first symptoms, decreased the median illness duration by 1.7 days (p =0.07), and airway function was significantly improved (1 s forced expiratory volume on day 5 improved by 10.8% compared with baseline values among oseltamivir recipients and 4.7% among placebo recipients (p= 0.0147)). the incidence of development of drug resistant virus under therapy was evaluated in more than 1000 subjects. none of 542 subjects sampled on day 3 of treatment harbored resistant virus. on day 5, however, 4/451 subjects (1%) shed virus with a resistant phenotype. in children, the frequency of viral resistance was 4% (10/247). all of these subjects recovered normally despite this incidental finding. so far, resistant mutants have been less infectious than wild type virus; no evidence of transmission has been detected in an experimental infection model in ferrets. the impact study evaluated the benefit of early intervention in reducing the total time with symptoms starting from the time of first onset of fever. this reflects the total burden of illness from a patient's point of view. a total of 958 patients, 13-70 years of age were enrolled and treated within 48 h of the first onset of symptoms. treatment was initiated within 12 h in 25% of those recruited. using an accelerated time to failure model, earlier intervention was shown to strongly correlate with shorter duration of illness. the total duration of illness could be halved if treatment was started within 12 h of the onset of symptoms compared with intervention at 48 h. in phase iii trials, zanamivir reduced the duration of illness in adults over 12 years of age and in children between 5 and 12 years of age by 1.5 days (95% ci= 1-2.5 days). in high risk patients (525 subjects, 65% with asthma and 35% with chronic obstructive pulmonary disease) duration of illness amongst all influenza positive subjects was reduced by 1.5 days. duration of illness was reduced in both vaccinated (1.25 day) and nonvaccinated (2 day) subjects. during therapy, pulmonary function improved slightly between days 1 and 5. zanamivir was well tolerated, and there were no differences in adverse effects between treatment and placebo groups. with 9000 subjects studied in clinical trials, resistant mutants were not isolated by sampling on days 3 and 5. efficacy for treatment of influenza b was assessed in 300 subjects and found to be similar to that of influenza a. a prophylaxis study in a family setting showed 79% protective efficacy. finally, in a nursing home study, zanamivir exhibited a 60% in-creased efficacy in preventing influenza disease compared with rimantadine. rwj-270201, an orally available once daily neuraminidase inhibitor that is active against influenza a and b in vitro and in animals, was evaluated in phase ii trials of experimentally infected healthy adults. therapy was started approximately 24 h post infection with influenza a/texas h1n1 or influenza b/yamagata. viral load over time was the primary endpoint, and nasal wash titers were determined every 12 h on day 1-3 and daily thereafter. in the influenza a study, 90 subjects were enrolled into four treatment (100 to 400 mg per day) and one placebo arm. high dose treatment (400 mg once daily or 200 mg twice daily) reduced viral load by 68-73%, whereas 100 or 200 mg per day led to a reduction by 34%. shedding was reduced by 1-2 days in the high dose groups, and the incidence of fever declined from 25% in placebo controls to less than 5% in all treatment groups combined. to evaluate efficacy against influenza b, 56 subjects were enrolled into two treatments (800 or 400 mg per day) and one placebo group. a 61% reduction of viral load was shown for the high dose treatment (800 mg per day). no evidence for the emergence of resistant virus was detected, and oral rwj 270201 was well tolerated without excess gastrointestinal side effects. world-wide, acute respiratory infections (ari) are responsible for more than 4 million deaths per year in children under five years of age. viral respiratory infections contribute significantly to this burden of disease, not only in children but also in adults. although there is a myriad of viral respiratory pathogens, the number of virus families that cause significant burden of disease is limited. in children, the paramyxoviridae are the most important family of viruses, with respiratory syncytial virus (rsv) and the parainfluenza viruses (piv) causing most disease, followed by adeno-and influenza viruses. in adults, influenza a virus with its constantly changing antigenic composition surpasses other viral pathogens, but the impact of rsv and rhinoviruses are being increasingly appreciated. the epidemiology of respiratory virus infections very much depends on host and environmental factors. while adenovirus infections are a major concern in military training camps and in immunocompromised patients, they are not as important in the general adult population. the immunocompromised are not only at increased risk for viral respiratory disease but diagnosis and management are much more difficult in this group. newer diagnostic methods based on antigen detection or viral genome amplification make rapid diagnosis possible and thereby affect clinical management of viral respiratory disease. vaccines are still the most powerful tool in preventing viral respiratory disease but for many important pathogens there is still no vaccine available. the inactivated influenza vaccine, with its antigenic composition changing annually, is still the mainstay of influenza prevention. coldadapted live attenuated influenza virus vaccines are on the horizon but are not yet approved by the fda. adenovirus vaccines have been used successfully in the military for many years, and the recent resurgence of large outbreaks of respiratory disease after discontinuation of vaccination emphasizes the importance of maintaining a preventive strategy. live virus vaccine candidates against rsv and piv, derived biologically or by reverse genetics, are currently in clinical trials, and there is hope that safe and efficient vaccines will become available within this decade. new insight into virus-host and virus-bacteria interactions in viral respiratory illness may help us understand the complexity of these disease entities and allows a re-evaluation of management options. diseases earlier thought of as purely bacterial, such as otitis media, might be more effectively prevented by viral vaccines than by bacterial vaccines. populations at high risk for complications resulting from respiratory viral infections are now better defined and a more targeted prophylaxis is possible, be it passive prophylaxis against rsv disease with monoclonal antibody preparations or active prophylaxis with influenza-or adenovirus vaccines. in the management of influenza virus disease, much has changed for the better. neuraminidase inhibitors (ni) are now established as an effective intervention to decrease the severity and to shorten the duration of illness when therapy is initiated within 2 days of the onset of symptoms. zanamivir and oseltamivir are now licensed in the us and elsewhere for adults and adolescents twelve years of age and older, and a license for use in children over one year of age has recently been obtained for oseltamivir. both drugs are safe and well tolerated, and development of resistance occurs much less frequently than with older antivirals such as amantadine or rimantadine. rhinovirus infections, the cause of most colds, may now be amenable to treatment with the experimental 3c protease inhibitors pleconaril (given orally) and ag7088 (given intranasally), and initial studies indicate that pleconaril treatment of colds in children might reduce their risk of developing otitis media. antivirals effective against rsv and 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characterization of the pathogenic influenza a/goose/guangdong/ 1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong key: cord-008584-4eylgtbc authors: singh, david e.; marinescu, maria-cristina; carretero, jesus; delgado-sanz, concepcion; gomez-barroso, diana; larrauri, amparo title: evaluating the impact of the weather conditions on the influenza propagation date: 2020-04-05 journal: bmc infect dis doi: 10.1186/s12879-020-04977-w sha: doc_id: 8584 cord_uid: 4eylgtbc background: predicting the details of how an epidemic evolves is highly valuable as health institutions need to better plan towards limiting the infection propagation effects and optimizing their prediction and response capabilities. simulation is a costand time-effective way of predicting the evolution of the infection as the joint influence of many different factors: interaction patterns, personal characteristics, travel patterns, meteorological conditions, previous vaccination, etc. the work presented in this paper extends epigraph, our influenza epidemic simulator, by introducing a meteorological model as a modular component that interacts with the rest of epigraph’s modules to refine our previous simulation results. our goal is to estimate the effects of changes in temperature and relative humidity on the patterns of epidemic influenza based on data provided by the spanish influenza sentinel surveillance system (sisss) and the spanish meteorological agency (aemet). methods: our meteorological model is based on the regression model developed by ab and js, and it is tuned with influenza surveillance data obtained from sisss. after pre-processing this data to clean it and reconstruct missing samples, we obtain new values for the reproduction number of each urban region in spain, every 10 minutes during 2011. we simulate the propagation of the influenza by setting the date of the epidemic onset and the initial influenza-illness rates for each urban region. results: we show that the simulation results have the same propagation shape as the weekly influenza rates as recorded by sisss. we perform experiments for a realistic scenario based on actual meteorological data from 2010-2011, and for synthetic values assumed under simplified predicted climate change conditions. results show that a diminishing relative humidity of 10% produces an increment of about 1.6% in the final infection rate. the effect of temperature changes on the infection spread is also noticeable, with a decrease of 1.1% per extra degree.conclusions: using a tool like ours could help predict the shape of developing epidemics and its peaks, and would permit to quickly run scenarios to determine the evolution of the epidemic under different conditions. we make epigraph source code and epidemic data publicly available. seasonal influenza may not make headlines, but together with pneumonia, it is one of the top ten causes of death worldwide. influenza epidemics results in 3 to 5 million cases of severe illness a year, which puts a high burden on health providers and results in loss of productivity and absenteeism, such as mentioned by the world health organization in [1] . it's been long known that in temperate climates these seasonal epidemics occur mostly in winter, and typical hypotheses assigned the blame to people being in closer proximity for longer periods of time, or lowered immune systems. in general, meteorological conditions affect virus transmission due to multiple effects: virus survival rates, host contact rates and immunity, and the transmission environment (except the case of direct or short-range contact). while these factors may have an influence, the solid evidence sustains the hypothesis that the virus's best surviving conditions are low temperatures and low absolute humidity. one of the goals of the current research in this field is to understand this relationship to be able to develop a more accurate seasonal influenza model for both temperate and tropical regions. as a motivation of this work, jt et al. [2] conclude that environment factors may become more important for a future predictive model of the effects of climate change. in a previous paper [3] , some of the authors of this paper studied the interaction of the spatio-temporal distribution of influenza in spain and the meteorological conditions during five consecutive influenza seasons. the work uses real influenza and meteorological data in combination with statistical models to show that there is a relationship between the transmission of influenza and meteorological variables like absolute humidity and amount of rainfall. in this work we use the same data sources (sisss and aemet agencies) following a different approach: we study some of these relationships from a simulation perspective, considering not only the existing influenza distributions but also the ones related to the climate change. in this work we extend epigraph [4] , an influenza simulator, with a meteorological model (mm) starting from the model developed by ab and js [5] . in their paper ab and js analyze monthly weather and influenza mortality data collected between 1973 and 2002 throughout all of the 359 us urban counties. using a regression model, they conclude that there exist correlations between both absolute humidity and temperature with mortality. they report a quantitative assessment of the relation between mean daily humidity and temperature levels and mortality rates in different ranges. this is an extensive study and, as a result, we start from the assumption that their results are solid and appropriate to incorporate to epigraph in order to produce meteorological-dependent simulations based on real data. in this work we extend epigraph [4] , an influenza simulator, with a meteorological model (mm) starting from the model developed by ab and js [5] . in their paper ab and js analyze monthly weather and influenza mortality data collected between 1973 and 2002 throughout all of the 359 us urban counties. using a regression model, they conclude that there exist correlations between both absolute humidity and temperature with mortality. they report a quantitative assessment of the relation between mean daily humidity and temperature levels and mortality rates in different ranges. this is an extensive study and, as a result, we start from the assumption that their results are solid and appropriate to incorporate to epigraph in order to produce meteorological-dependent simulations based on real data. regarding other influenza simulators that consider weather conditions, ps et al. presents an agent-based simulation model [6] that evaluates the seasonal effects on the influenza propagation. although the reproductive rates are generated synthetically without considering actual meteorological data, this paper shows, in a similar way than our work, the impact of changing reproductive rates on the course of the influeza pandemic. in the article [7] , js et al. simulate influenza transmission via a sirs model modulated by climate data to obtain the basic reproduction number r 0 . both js et al. [8] and acl et al. [9, 10] study the effects of humidity on influenza transmission from the point of view of virus survival and conclude that aerosol transmission is most efficient in low humidity conditions. acl et al. [9, 10] and bx et al. [11] also conclude that aerosol transmission is more efficient at low temperatures. js et al. [8] and jm et al. [12] also deduce that virus survival increases with decreasing the humidity values. epigraph simulations use real data for modelling the population, the spatio-temporal distribution of influenza, and the meteorological conditions. this simulator consists of different components and data sources shown in fig. 1 . the previous and novel components are represented in blue and orange colors, respectively. the simulator uses input data that is obtained from different sources including: (1) the influenza data, that contains information about the initial individuals that are infected; (2) the population data, that describes the individual interactions with others; (3) the transport data, that contains information about the movement of individuals between different locations and (4) the climate data, that contains the meteorological conditions existing during the simulated time span. this data feeds the different models implemented in the simulator. we briefly describe the three models that have been previously developed and presented in [4, 13] . the epidemic model considers the propagation model of influenza extending the sir (as explained in [14] by fb et al.) to include states for latent, asymptomatic, dead and hospitalized. the infective period has different phases which may affect the dissemination characteristics of the fig. 1 overview of the data sources, processed data, and epigraph components influenza virus as ame et al. describe in [15] . each individual has a slightly different length for each infection state. we adopt most of the concrete values for the model parameters from the existing literature on flu epidemics (see [14] [15] [16] [17] ). you can find them all in [18] . the transport component models the daily commute of individual to neighboring cities (inter-city movement) and the long-distance travels for several days that represent commute of workers that need to reside at different locations or people that move at any distance for vacation purposes. the people mobility model is based on the gravity model proposed by cv et al. [19] that uses geographical information extracted from google using the google distance matrix api service. the social model is an agent model that captures individual characteristics and specifies the interaction patterns based on existing interactions extracted from social networks. these patterns determine the close contacts of each individual during the simulation, which is a crucial element to model the spread of the infection. we extract interaction patterns from virtual interactions via email or social networks (enron and facebook) and scale them to approximate a physical connection of the whole network within an urban area. these connections are timedependent to realistically capture the temporal nature of interactions, in our case modeled depending on the day of the week and time of day. the distribution of the population is in terms of four group types: school-age children and students, workers, stay-home parents, and retirees. in this paper, as main contribution, we introduce a new component of the simulator (the meteorological model), that evaluates the impact of climate parameters on influenza propagation. this component is tuned with influenza surveillance data obtained from sisss to provide realistic simulations. as far as we know, this work is the first simulator that integrate real meteorological data to predict the spatio-temporal distribution of influenza. we think that this contribution will help to better understanding the influenza propagation in real environments. in the literature we can find different influenza simulators although none of the following consider meteorological factors in the simulation. examples of them is the work of kk et al. [20] that presents an sirbased epidemic simulator that permits to parametrize both the population characteristics and the epidemic process. the goal of this work is to identify the turning point (peak of the infected population) of the infection. although the initial approaches for modelling the infection spreading across the contact network, our work consider a broader number of parameters and configuration of the network. he et al. [21] analyze, by means of simulation, the relationship between social interaction patterns at workplaces and the virus transmission patterns during influenza pandemics. the main effort is geared towards the flexible specification of the different aspects involved in a simulation, such as intervention policies, social modelling, social organization of work, etc. sim-flu [22] is different from most epidemic simulators in that it focuses on the discovery of most probable future influenza variants starting from virus sequences published by the national center for biotechnology information (ncbi). this work is complementary to the goal of most simulators, including ours, which is to understand and predict the spreading infection patterns of a known flu strand across a population. their methodology is based on observing directional changes in subtypes of influenza over time. js and ak present a framework [23] to adjust an epidemic simulation based on real-time forecasts of infections from google flu trends. the paper focuses on prediction of the timing of peak infection, but other metrics could be predicted as well. the authors of [24] simulate the spreading of influenza in an urban environment consisting of several close-by towns connected by trains. their goal is to be able to model and simulate intervention policies. epiwork [25] was a european project in fp7 whose focus was to develop a tool framework for epidemic forecast. within this project's framework, wb et al. describe gleamviz [26] , their tool for epidemic exploration which includes a simulator of transmission based on an accurate demographics of world's population over which they superpose a (stochastic) mobility model. db et al. [27] use human mobility extracted from airline flights and local commute (based on the gravity model) to predict the activity of the influenza virus based on monte carlo analysis. sm and sm [28] study the role of population heterogeneity and human mobility in the spread of pandemic influenza. in [29] , the authors reconstruct contact and time-in-contact matrices from surveys and other socio-demographic data in italy and use this matrix for simulation. epigraph uses meteorological data provided by the spanish meteorological agency (aemet) to generate environment-dependent influenza simulations. the preprocessing stage is performed to obtain clean inputs for the meteorological model. first, the weather station nearest to each simulated urban region is identified. our simulations consider 92 different urban regions with more than 100,000 inhabitants. in some cases, the station is within the city limits, while in others it is located in a nearby area (for instance at the region's airport). the data from each weather station is analyzed to reconstruct potentially missing samples. sometimes it is the case that some station data samples are missing because the station was not operational during a given time period. these represents just a small fraction of the overall samples, but they have to be properly addressed. figure 2 shows an example of how the original missing data (shown in upper figure) is reconstructed producing a complete samplingclearpage (reconstructed values are shown in the lower figure in red color). in order to add the missing samples, we have used the reconstruct data algorithm (missdata) included in the matlab's system identification toolbox. this toolbox permits the construction of mathematical models for dynamic systems, starting from measured input -output data. the resulting data is then processed to filter nonrealistic values. some weather stations produce abnormal samples corresponding to non-realistic values that are too big or too small. figure 2 shows an example of this kind of values around sample 41,000. we have corrected these cases with a matlab algorithm we implemented to detect these peaks and correct them using an interpolation of the values from the previous days. these two steps are only performed once for each new meteorological input data and the results may be used for the rest of the process. this section describes how the r0s are obtained from the meteorological conditions. in addition to the notations introduced in the introduction, for the rest of the paper we will use sh for the specific humidity and p * h2o for the equilibrium water vapor pressure. a related value to sh is absolute humidity (ah), which is the mass concentration that describes the amount of water vapour per volume of air. previous studies [30, 31] suggest that ah (and by extension sh) are one of the main factors affecting the influenza virus transmission. in epigraph we adopt the results of the regression model used by ab and js [5] . in their 2012 paper, they analyze monthly weather and influenza mortality data collected between 1973 and 2002 throughout all the 359 us urban counties. using regression, they conclude that there exists a strong correlation between absolute humidity and mortality, even when controlling for temperature, when the humidity drops below daily means of 6g/kg. temperature correlations also exist, mainly in the daily ranges between -1.1c and 15.6c. in an earlier paper ( [7] ) js et al. study the same dataset and simulate influenza transmission via a sirs model modulated by the data to obtain the basic reproduction number r 0 . they also find bestfit parameter range combinations of r 0max between 2.6 and 4, and r 0min between 1.05 and 1.3. we adopt the pair of (r 0max , r 0min ) that was found to be the best-fit parameter combinations they discover: r 0max = 3.52, r 0min = 1.12. from the definition of the specific humidity (sh) and relative humidity (rh)-see rhp and dwg [32]-we know that: (1) we also know from buck's equation that the equilibrium water vapor pressure can be calculated using the formula: where the temperature t is measured in degrees celsius. this formula works best for values of t in the range of -80c to 50c. from known values rh, p, and t, and using eqs. (1) and (2), we can calculate the specific humidity. from laboratory experiments by js et al. [7] we have: (3) where a = −180, b = log(r 0max − r 0min ) and q is the 2m above-ground specific humidity, which we approximate to sh at the given temperature. in this way, we obtain a value for p * h2o in every sample (obtained every 10 minutes) using eq. (2) . from this value in combination with the values of rh and p we obtain the value of sh using eq. (1). finally, eq. (3) computes the new r0 values for each urban region. r0s are, therefore, time-dependent values that determine, in a stochastic process, how many susceptible individuals of an infected person's connections could be potentially infected. this is the dynamic component of the infectivity of an individual with respect to the others. the other dynamic component is the stochastic transition between infective states [4] , computed with variable probabilities. our model is not different for the different types / subtypes of influenza. the values of the model parameters (basic reproduction numbers for each stage of the disease) were chosen to fall in the ranges published by ab and js, which are based on actual data for all types of influenza, over 30 years. we choose fixed r0s within the ranges, although this is a parameter that can be configured to vary. on the other hand, the evaluation was performed over data from the 2010-2011 influenza season over the whole territory of spain, for all types of influenzas that were diagnosed. we consider that both the choice of r0 (based on exhaustive data) and the evaluation against real reported cases across spain are comprehensive enough to validate our results. the spanish influenza sentinel surveillance system (sisss) comprises 17 networks of sentinel physicians (general practitioners and pediatricians) in 17 of the 19 spanish regions, as well as the network-affiliated laboratories, including the national influenza reference laboratory (national centre for microbiology, world health organization national influenza centre in madrid). more than 800 sentinel physicians participated each season covering a population under surveillance of around one million-see [33, 34] . sentinel physicians reported influenza-like illness (ili) cases-integrating virological data collected in the same population-detected in their reference populations on a weekly basis, following a definition based on the eu-ili, as described in [35] . for influenza surveillance, they systematically swab (nasal or nasopharyngeal) the first two ili patients each week and sent the swabs to the network-affiliated laboratories for influenza virus detection. the information collected by the sisss includes data on demographics, clinical and virological characteristics, seasonal vaccination status, chronic conditions, and pregnancy. data is entered weekly by each regional sentinel network in a web-based application [36] and analyzed by the national centre of epidemiology to provide timely information on the evolving influenza activity in spanish regions and at the national level. for example, during the 2011-2012 season, 651 sentinel physicians and 236 pediatricians participated to sisss and surveyed a total population of 1,142,189, which represents 2.36% of the total population of spain. we obtained the sisss data from the national center of epidemiology, institute of health carlos iii of madrid (isciii). in order to produce realistic simulations, epi-graph has to be properly configured. this configuration process consists of setting up two parameters: the date of the epidemic onset and the initial influenza-illness rates for each urban region. the first parameter is the time of onset of the epidemics, which occurs during week 50 of 2010. at this time the national average incidence values for influenza are greater than 60 cases per 100,000 inhabitants, which is the threshold determined by siss, based on data from the 2010-2011 seasonal epidemic, to be the start of the influenza season. in our simulation the exact date is the 13th of december of 2010. the second parameter values were obtained from influenza surveillance data obtained from the sisss corresponding to the influenza season 2010-11. from this data we obtained the reported weekly ili rate at national and regional level in spain. the data for the murcia and galicia communities are not available and we approximated them based on the data from the nearest community. these rates allow us to approximate the initial number of (clinically) influenza-like-infected individuals using the following formula, based on the study published online (in march 2017) in the lancet respiratory medicine by ach et al. [37] . where n report are the cases that demanded medical attention, as reported by the sisss, f pos is the fraction of positive cases, symp is the percentage of symptomatic individuals, and attend is the percentage of those with symptoms that see a doctor. for instance, for the reported n report = 90 cases per 100,000 inhabitants in week 50, and with values f pos = 33% (empirical value for 2010-2011 in spain), and symp = 23%, attend = 17% (values taken from the cited study), we calculate that the total number of infected individuals is of approximately 765 cases per 100,000 inhabitants -or 0.765% of the total population. we use this value to set up the initial conditions of the simulation (described in this section), but also to validate its results. each community has a different n report , which leads to different numbers of initially infected individuals. epigraph allows modeling at the level of each individual, and thus can simulate the effect of vaccination policies. to produce realistic results, we use different influenza vaccination coverages by age group; for those older than 65 we have used the vaccination ratios (per community) provided by the ministry of health, social services, and equality of spain. these values correspond to vaccination coverages collected by the national health system [38] . for the rest of the population (individuals younger than 65) we have used the data provided by the spanish statistical office, which is based on surveys done in each community. given that the data are available at community level, we assume that all the urban areas located in the same community have the same vaccination coverages. table 1 shows these percentages per community and age. as input of the mobility model, we use 85% workers and 15% students for short distance travel, and 50% workers, 30% students, 15% retired individuals, and 5% unemployed for long distance travel. while epigraph accounts for many of the components that influence the spreading of the virus, the behavior of these parts and the values of the parameters (such as the initial infectious individuals or the vaccination rate) are unavoidably approximate. on their website, the world health organization reports that in annual influenza epidemics, 5-15% of the population are affected with upper respiratory tract infections [1] . we have therefore introduced a scaling factor which adjusts the infection propagation rate of each individual to produce, for each urban region, a final infection rate between 5% and 15% of the total population. these values are obtained in a pre-calibration phase of epi-graph for the real climate conditions-performed only once-and are then used for all subsequent simulation experiments. note that this is the only data -which is also based on real data-that we use for the calibration process. we do not calibrate the model to an existing epidemic curve. once calibration is done, we use data from sisss, which records influenza-like-illness cases that are not confirmed by laboratory tests, for setting the initial simulation conditions of each urban area. this fact doesn't affect the validity of our results because the purpose is to compare yearly/monthly numbers under different climate conditions rather than know the accurate number of infected individuals. we have performed different tests to validate our approach and simulator. we first validated the simulator against influenza surveillance data, then we evaluated two different environmental scenarios. we believe that our simulator can be useful to predict the short-and mediumterm spread of an infection, as well as to assess the effects that changes in climate can have over influenza epidemics worldwide. the first scenario involves real climate values from aemet and allows studying the short-and medium-term propagation for influenza strands. for the second set of scenarios we generate fictitious values of rh and t by scaling the real values. our idea is to study the effects of the changing climate conditions on influenza propagation. simulations occur across the 92 largest cities in spain, which account for a population of 21,320,965 inhabitants. the time span is 7 months starting from the day identified as the onset date in our data -the 13th of december of 2010. in our experiments we have used data from 92 weather stations from the national network, distributed across the country. each weather station collects the values of temperature, atmospheric pressure, and relative humidity every 10 minutes during the entire 2011. these consists of about 157,000 data samples per station and 14.5 million data values in total. based on these values, we generate the basic reproduction numbers to obtain an r0 value per urban area at every 10 minutes. with the previously determined initial influenza-like rates per region and (year-specific) date of onset, and after calibration, each urban region data -vaccination rates, individuals' characteristics, initial infective individuals, and r0s values -are loaded from files. the validation of our simulator in terms of its capacity to predict qualitatively similar propagation results as those approximated from the influenza surveillance data recorded by sisss. the simulated values for each of the spanish regions are the aggregated values of all the urban regions belonging to it. figure 3 shows the simulated and actual estimated data. the simulated values are scaled to make the largest simulated value to be the same as the maximum real value. this allows a comparison of the evolution of the influenza propagation for each community over time. we can observe that although not perfect, the prediction shows a similar evolution with those from real scenarios. note that the simulator considers an approximation of the real conditions during the simulated period, but producing a better (unlikely perfect) fit between the two domains would need to consider all the factors of the real world that affect the flu propagation at nation-level. some of these are possibly unknown, others are not currently measured, and yet others are not possible to measure. the reason for scaling the data is that the simulated and actual estimated data reflect the population rather differently. on one hand, the simulated values correspond to the overall number of individuals infected with influenza across the considered urban areas. these take into account all the individuals within the simulated areas but only include the largest urban regions (above 100,000 inhabitants); small cities, towns, and villages are not considered. on the other hand, the influenza surveillance data are only related to a small fraction of the existing clinical cases: sisss covers a representative but small percentage of the population, in addition to the fact that there are more cases than those reported due to people not seeking medical attention. in contrast, the number of cases are collected from the complete community (including both large and small populations). it is thus not possible to compare the absolute values of the two data sources, although they should be linearly related. figure 4 shows an example of the value of these parameters for the urban region of terrasa (barcelona) over one year. we can observe strong variations of r0 that are related to the changing temperature, relative humidity, and pressure conditions. to evaluate the effect of both real and hypothetical meteorological climate changes on the spreading of influenza we evaluate temperature variations of t degrees and percentage variations of the relative humidity prh. t = 0 and prh = 1.0 correspond to the initial scenario with the original climate conditions. studies show that climate change is producing increments in the average temperature (amplified by pollution) and, in southern europe, longer periods of drought. the idea is to evaluate the impact of these changes on the influenza propagation. in this section, we consider long-term meteorological climate changes, that is, changes in the climate conditions that extend to the entire simulated period of 28 weeks. in this context, we evaluate two different scenarios, probably not as complex as future real climate changes. the first one corresponds to drought conditions, when the relative humidity values (rh) are smaller than current ones. we have considered a reduction of the relative humidity from 90% to 50% in increments of 10% (rh values half than the original ones). according to the infection model, influenza propagates easier for smaller rh values; we thus expect to observe a larger effect. figure 5 shows the overall percentage of infected individuals per community predicted by epigraph. the diminishing rh has indeed a strong impact on the number of infected individuals. on average, 12.4% of the population was infected in the base case (reduction factor equal to 1), while the average infection rate for 0.5 factor is 20.5%. we can observe that a percental reduction of rh of 10% produces an approximate increment of 1.6% in the final infection rate. the second scenario evaluates the impact of an increase of temperature on the propagation. figure 6 shows the final infection rate for an increment of the temperature between 0 degrees (current case) and 5 degrees celsius. we can observe that now there is a reduction in the infection rate when the temperature increases. now, an increment of 5 degrees reduces the average infection rate from 12.5% to 6.9%-a decrement of 1.1% per degree. both scenarios assume that the values of the parameters (rh, t) change one at a time. this is a simplification, and the idea behind this approach is to evaluate the impact of a single parameter variation on the overall influenza outcome. however, epigraph supports specifying any changing combination of climate conditions. in a more realistic scenario both parameters would change, and the climate specialists are those who should define what the concrete values are. figure 7 shows the combined effect of temperature and relative humidity change on the average nation-wide infection rate. we have plotted two planes: the first one (colored) represents the average infection rates for different increments in the temperature values and percentile reductions in the relative humidity; the second one (green) displays the infection rate of the original scenario (without climate variation) for all the coordinates and represents the baseline case. the two planes intersect in the lowerleft point, where the temperature and rh have the original values. although both parameters influence the final infection rate, relative humidity has a larger effect than temperature. figure 8 shows the effect of rh and temperature variations on the infection distribution for andalucia community. we can observe that the variation of both parameters changes the shape of the distribution, especially in terms of the peak values but also -more subtly -in terms of the propagation interval. the maximum and minimum 95% confidence intervals baseline scenario (no rh reduction nor temperature increment) ranges between 0.28 and 0.06 for urban areas in castilla la mancha and aragón, respectively. these results are produced by a simulator repeating the simulations 30 times. note that there already exist uncertainty in the input data, both with respect to the number of initially infected individuals as well as from the point of view of the epidemic model. to evaluate the effect of short-term changes in climate conditions, we modify rh and the temperature exactly like described in the previous section, only for the first week of the simulation. the rest of the simulation uses the original climate parameters. figures 9 and 10 show the final infection rate for different variations of rh and temperature. we can observe that the impact on the overall percentage of infected individuals is still important, particularly for a decrease in rh of 0.2 or more and -less evidently -for an increase in temperature of 3 degrees or more. for smaller changes in temperature the effect is less evident, but we believe that this is due to the fact that the short-term simulation of temperature increase is only one week. we achieve herd immunity in two ways: as result of vaccinating campaigns, and naturally when an individual that was infected goes to the recovery (or dead) state, in which case he becomes immune and starts acting as a propagation stopper. as a result, after certain threshold of infected vs susceptible individuals, the infection rate naturally goes fig. 7 effect of long-term changes in the relative humidity (percentil reduction) and temperature (value increment in celsius) on the influenza propagation for the average nation-wide infection rates down. this occurs at the inflection point in the propagation graph, specifically at about 16 weeks (in our data). the vaccination success rate during the 2010-2011 season was approximately 50% [39] . given the parameters shown in fig. 4 of [40] for herd immunity for influenza, we consider that the r0 considered by our model takes into consideration this type of immunity. we do not model the level at which herd immunity starts acting as a parameter, although this phenomenon occurs naturally in the simulations. the simulator is flexible enough to support different daily contact patterns for each individual. the probability of an individual getting infected during an interaction also differs (it's a stochastic process), and thus the infection can be transmitted to individuals pertaining to different groups. recently, the work in [41] suggests that rh should also be considered (together with the temperature) as a modulating factor in the influenza propagation. another study that analyses this relationship can be found in [42] . this work provides a transmission risk contour map based on the temperature and rh humidity values. note that our work addresses the problem of evaluating the influenza propagation from a different perspective. instead of analyzing the propagation mechanisms of the virus and how they are related to the environment conditions, we focus on an empirical relationship between the virus's basic reproduction number and the outdoor specific humidity. the r0 values used in this work are the combination of both outdoor and indoor virus propagations, and provide an approximation of a real scenario. note also that the fig. 8 effect of long-term parameter variation on the infection distribution shape for andalucía. in a different rh scales are evaluated (100% in red, 90% in green, 80% in blue and 70% in black); in b different temperature offsets are evaluated (0 degrees in red, +1 degrees in green, +2 degrees in blue and +3 degrees in black) effect of short-term changes in the relative humidity on the influenza propagation for the different communities considered in the simulation: in color the average infection rates for different increments in the temperature values and percentile reductions in the relative humidity; in green the infection rate of the scenario without climate variation main goal of this work is to evaluate the impact of the weather conditions on the propagation. a possible limitation is that we only model the largest 92 urban regions in spain; we could add more information related to smaller cities and towns, including rural regions. nevertheless we don't think this data would make a significant difference in the results, as the infection needs a large number of hosts to explode, and / or travel patterns between the infected areas. small town and village areas are arguably much less likely than cities to fulfill these roles. a second limitation is related to the meteorological factors affecting the infection propagation: the number and set of climate factors that the meteorological model takes into account, and the choice of the model itself. additional parameters that specialists mention as possible influencers in virus transmission are factors such as wind, precipitation, or pollution. fig. 10 effect of short-term changes in the temperature on the influenza propagation for the different communities considered in the simulation one important thing to underline is that the data that the study [5] (whose model we adopt) is based on is of real cases and spans 30 years. interactions between meteorological trends and human behavior are therefore intrinsically reflected in the data, although the rules of behavior change are not explicitly specified for the agents (i.e. individuals) involved in the simulation. the case can be made that meteorological changes were not as extreme before 2002, and that a regression model based on new data may change as well over time. while this is a definite possibility, we believe that its nature will not change in a fundamental way, such that we can still predict trends, if not absolute values. a third limitation is that we don't calibrate the model on an epidemic curve, which results in different timings of the flu peaks in some regions, such as in navarra and madrid. finally, to successfully simulate the flu epidemics requires leveraging many different types of data, most of them in large amounts, as input and calibration measurements for our tool (epigraph). for instance, we are using social network data from enron and facebook to set up the population interaction patterns, census data to extract the characteristics of the different types of individuals, google maps to initialize the transportation module, data from aemet to run simulations that are realistic from a meteorological viewpoint, and weekly ili rates obtained from the sisss to initialize and evaluate the simulator. this makes the implementation of epigraph more realistic, a strength that can lead to more accurate simulations. we have extended our simulator epigraph with a meteorological model that interacts with the rest of the system to better reflect the behavior of the influenza propagation through the entire population of spain. to produce realistic results we also take into account vaccination, with different ratios based on the individuals' ages. the simulator results are compared to real data on infection rates and across the whole country. the results for the prediction of the evolution of the influenza propagation for each community over time are similar in shape to the real data. after validating the simulator, we evaluate different scenarios that reflect changes in climate conditions, and show the predictions for variations in the relative humidity and temperature. lastly, we make epigraph's source code publicly available at [18], to be used by the scientific community. as future work, an interesting, although independent, possibility is to investigate the potential of epigraph to simulate the evolution of the virus spread for different subtypes of influenza, once the propagation model parameters (e.g. incubating period, infectious period, basic reproduction numbers, etc.) are known, or to narrow down the possible subtypes in early phases of an infection. one could also investigate the impact of new meteorological factors on the evolution of the infection. world healh organization influenza (seasonal) global influenza seasonality: reconciling patterns across temperate and tropical regions climatic factors and influenza transmission, spain leveraging social networks for understanding the evolution of epidemics absolute humidity, temperature, and influenza mortality: 30 years of county-level evidence from the united states modelling seasonality and viral mutation to predict the course of an influenza pandemic absolute humidity and the seasonal onset of influenza in the continental united states absolute humidity modules influenza survival, transmission, and seasonality influenza virus transmission is dependent on relative humidity and temperature high temperatures (30 degrees c) blocks aerosol but not contact transmission of influenza virus climatological and geographical impacts on global pandemic of influenza a(h1n1) role of absolute humidity in the inactivation of influenza viruseson stainless steel surfaces at elevanted temperatures towards efficient large scale epidemiological simulations in epigraph emergence of drug resistance: implications for antiviral control of pandemic influenza containing pandemic influenza with antiviral agents an influenza simulation model for immunization studies synchrony, waves, and spatial hierarchies in the spread of influenza coupling effects on turning points of infectious diseases epidemics in scale-free networks relevance of workplace social mixing during influenza pandemics: an experimental modelling study of workplace cultures simflu: a simulation tool for predicting the variation pattern of influenza a virus forecasting seasonal outbreaks of influenza parallel agent-based simulator for influenza pandemic the gleamviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale seasonal transmission potential and activity peaks of the new influenza a(h1n1): a monte carlo likelihood analysis based on human mobility the role of population heterogeneity and human mobility in the spread of pandemic influenza little italy: an agent-based approach to the estimation of contact patterns-fitting predicted matrices to serological data absolute humidity modulates influenza survival, transmission, and seasonality global environmental drivers of influenza perry's chemical engineers' handbook sentinel surveillance system. characterisation of swabbing for virological analysis in the spanish influenza sentinel surveillance system during four influenza seasons in the period epidemiology of the 2009 influenza pandemic in spain. the spanish influenza surveillance system amending decision 2002/253/ec laying down case definitions for reporting communicable diseases to the community network under decision no 2119/98/ec of the european parliament and of the council comparative community burden and severity of seasonal and pandemic infl uenza: results of the flu watch cohort study coberturas de vacunación en mayores de 65 años effectiveness of the 2010-11 seasonal trivalent influenza vaccine in spain: cyceva study the vaccination coverage required to establish herd immunity against influenza viruses mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence aerosol influenza transmission risk contours: a study of humid tropics versus winter temperate zone we would like to acknowledge all the sentinel general practitioners and pediatricians, epidemiologists, and virologists participating in the spanish influenza sentinel surveillance system. part of the input data used in this work have been obtained from the spanish influenza sentinel surveillance system and the meteorological information provided by spanish national meteorological agency, aemet, ministerio de agricultura, alimentación y medio ambiente. 1 authors' contributions des., mcm. and jc. designed and implemented epigraph simulator. all authors conceived and designed the experiments. des. processed the input data and run the experiments. des. and mcm. wrote the paper. cd., dgb., and al. provided insights on the validity of our assumptions, recommended additional related work, and contrasted the results with their own findings. all authors review the different manuscript drafts and approved the final version for submission. the author(s) read and approved the final manuscript. this work has been partially supported by the spanish "ministerio de economía y competitividad" under the project grant tin2016-79637-p "towards unification of hpc and big data paradigms". the work of maria-cristina marinescu has been partially supported by the h2020 european project growsmarter under project grant ref. 646456. the role of both funders was limited to financial support and did not imply participation of any kind in the study and collection, analysis, and interpretation of data, nor in the writing of the manuscript. epigraph's user manual and source code are publicly available at [18] and can be used by the scientific community. the dataset supporting the conclusions of this article is available in the [43] repository. not applicable. not applicable. the authors declare that they have no competing interests. key: cord-001289-qbct63p4 authors: lipsitch, marc; galvani, alison p. title: ethical alternatives to experiments with novel potential pandemic pathogens date: 2014-05-20 journal: plos med doi: 10.1371/journal.pmed.1001646 sha: doc_id: 1289 cord_uid: qbct63p4 please see later in the article for the editors' summary two recent publications reporting the creation of ferret-transmissible influenza a/h5n1 viruses [1, 2] are controversial examples of research that aims to produce, sequence and characterize ''potential pandemic pathogens'' (ppps) [3] , novel infectious agents with known or likely efficient transmission among humans, with significant virulence, and for which there is limited population immunity. there is a quantifiable possibility that these novel pathogens could be accidentally or deliberately released. exacerbating the immunological vulnerability of human populations to ppps is the potential for rapid global dissemination via ever-increasing human mobility. the dangers are not just hypothetical. the h1n1 influenza strain responsible for significant morbidity and mortality around the world from 1977 to 2009 is thought to have originated from a laboratory accident [4] . risk evaluations surrounding biomedical research have not kept pace with scientific innovations in methodology and application. this gap is particularly disconcerting when research involves the construction of ppps that pose risks of accidental release and global spread. we argue here that accepted principles of biomedical research ethics present a high bar to ppp experiments, requiring that risks arising from such experiments be compensated by benefits to public health not achievable by safer approaches. focusing on influenza, the object of most current ppp experimentation, we further argue that there are safer experimental approaches that are both more scientifically informative and more straightforward to translate into improved public health through enhanced surveillance, prevention, and treatment of influenza. although several pathogens may be categorized as ppps (see box 1), ''gain of function'' experiments involving influenza strains modified to be ppps are expanding [5] [6] [7] (box 2), and hence of immediate concern. in addition to the two controversial studies recently published, studies with h5n1 [8] , h7n9 [9] , and h7n1 [10] have used similar ferret passage protocols, while still others have created mammalian-transmissible strains in vitro, followed by in vivo analysis [11, 12] . related studies have genetically combined less pathogenic zoonotic avian viruses, such as h9n2, with human seasonal influenza viruses to generate strains that exhibit enhanced transmissibility, and to which humans would be immunologically susceptible [13] [14] [15] . these studies have typically been conducted in biosafety level (bsl) 3 or 3+ containment facilities. laboratory-associated infections in bsl3 facilities are conservatively estimated to occur at a rate of two per 1,000 laboratory-years [3, 16] in the united states, where protocols and enforcement are relatively stringent. globally, high-containment laboratories have variable standards and enforcement [17] . experimentation in less-regulated or unregulated laboratories, with the attendant risks of accidental or deliberate release, is facilitated by the publication of sequence and functional data on ppps, even if the original research was conducted with state-of-the-art safety and security [18] . from the conservative estimate of the rate of laboratory-associated infections of two per 1,000 laboratory-years [3, 16] , it follows that a moderate research program of ten laboratories at us bsl3 standards for a decade would run a nearly 20% risk of resulting in at least one laboratoryacquired infection, which, in turn, may initiate a chain of transmission. the probability that a laboratory-acquired influenza infection would lead to extensive spread has been estimated to be at least 10% [19] . simple branching process models suggest a probability of an outbreak arising from an accidental influenza infection in the range of 5% to 60% [20, 21] . such probabilities cannot be ignored when multiplied by the potential devastation of an influenza pandemic [22, 23] , even if the resulting strain were substantially attenuated from the observed virulence of highly pathogenic influenza a/h5n1 [24] , the subject of much of the published ppp work to date. we advocate a dispassionate review of pertinent evidence and calculations of the probabilities and magnitudes of potential risks parameterized for specific ppp research programs. thus far, experiments with novel ppps have been assessed in the context of ''dual use research of concern'' (durc), a designation for ''research that could be used for good or bad purposes'' [25] . within the broader category of durc, ppp experimentation raises ethical issues that deserve more extensive evaluation than other durc, because the scale of risk posed by ppps is much greater. while durc by definition presents a risk of malevolent use, the impact of the accidental release of many agents involved in durc-anthrax, hemorrhagic fever viruses, and, most recently, a novel clostridium botulinum toxin [26, 27] -is constrained by transmission mode or limited host susceptibility. the magnitude of accidental risk for a novel ppp is much greater. the nuremberg code, a seminal document in clinical research ethics, specifies that in research conducted on human participants, ''the degree of risk to be taken should never exceed that determined by the humanitarian importance of the problem to be solved by the experiment.'' more broadly, 74 national academies of science have stated: ''scientists have an obligation to do no harm. they should always take into consideration the reasonably foreseeable consequences of their own activities'' [28] . the ethical principles underlying both guidelines would seem to apply a fortiori to research that imposes far-reaching risk to the public [29] . given the global nature of influenza transmission, and thus implications beyond a country's borders, international agreement regarding acceptable risks is needed. ethical constructs and risk evaluations must be tailored to scientific advances in methodology and application. limited attention has been paid to the ethics of scientific experiments that pose risks beyond identified human participants [30] [31] [32] . on a practical level, however, the spirit of the nuremberg code's ''humanitarian importance'' criterion is embodied in the recent frameworks for evaluating ppp experiments from the us department of health and human services (hhs), the primary sponsor of such experiments to date. the hhs frameworks for studies anticipated to create mammalian-transmissible h5n1 [33] and h7n9 [34, 35] viruses specify that the risks and benefits should be weighed. the nuremberg code's second point states: ''the experiment should be such as to yield fruitful results for the good of society, unprocurable by other methods or means of study, and not random and unnecessary in nature.'' when projecting the benefits of experiments that put human life at risk, therefore, it is critical to compare against alternatives. what unique public health benefits do ppp experiments offer relative to the benefits of investing equivalent resources this article describes the responsible ethical scrutiny that should be applied to experimental studies creating or employing ppps. we define ppps as infectious agents with four characteristics: 1. having known or likely efficient transmission among humans 2. significantly virulent 3. unmitigated by preexisting population immunity 4. genetically distinct from pathogens currently circulating these criteria define pathogens on which experimentation would pose a risk of sparking a pandemic, placing the human population at risk of morbidity or mortality, over and above the background risk of a naturally occurring pandemic. the paradigm case is the creation of variants of influenza a/h5n1 that are readily transmissible between ferrets, a model for human transmission. such criteria would likely be applicable to experimentation with human isolates of smallpox or sars, since these pathogens are no longer known to be circulating naturally. in the future, the list may expand [57] . we do not advocate the necessity of heightened scrutiny for isolation and characterization of naturally occurring pathogens, such as wild-type h5n1 or h7n9, consistent with the hhs framework for evaluating gain-of-function studies of h5n1 viruses, which exempts characterization of naturally occurring viruses [33] . n a novel ppp research program of moderate size would pose substantial risks to human life, even optimistically assuming a low probability that a pandemic would ensue from a laboratory accident. n alternative approaches would not only be safer but would also be more effective at improving surveillance and vaccine design, the two purported benefits of gain-of-function experiments to create novel, mammalian-transmissible influenza strains. n a rigorous, quantitative, impartial risk-benefit assessment should precede further novel ppp experimentation. in the case of influenza, we anticipate that such a risk assessment will show that the risks are unjustifiable. given the risk of a global pandemic posed by such experiments, this risk assessment should be part of a broader international discussion involving multiple stakeholders and not dominated by those with an interest in performing or funding such research. in alternative research strategies? if there are unique benefits to novel ppp experiments, do they justify the risks entailed? this concept, too, is partially incorporated in the hhs frameworks, which permit funding of h5n1 [33] or h7n9 [34, 35] transmissibility gain-of-function experiments only if ''there are no feasible alternative methods to address the same scientific question in a manner that poses less risk than does the proposed approach'' [33] . the nuremberg code suggests a broader criterion: that ppp experiments should be performed if the public health benefits envisaged cannot be obtained by safer methods. we argue that alternative scientific approaches are not only less risky, but also more likely to generate results that can be readily translated into public health benefits. proponents of ppp experimentation cite two main benefits of such studies: improving our interpretation of surveillance data to detect dangerous viruses and facilitating vaccine development against future natural pandemics. both claims have been disputed. the vaccine claim has been denied by vaccine developers, who note that many, if not all, vaccines have been developed without a detailed molecular understanding of transmission [36] . advocates of ppp experimentation further argue that creating potentially pandemic strains of a particular virus, e.g., a/h5n1, could facilitate the production and stockpiling of vaccines against that variant. however, given that ppp experiments inevitably consider only a few possible genetic pathways to transmissibility, and that the precise correspondence between transmissibility in the ferret model and human transmissibility remains uncertain, we can never know whether ppp experimentation would hit upon the antigenic composition of the next pandemic strain that will emerge from nature. indeed, as described below, it is clear that there is no one-to-one mapping between a few genetic changes in a virus and its transmissibility. by contrast, universal influenza vaccines currently in preclinical and clinical trials [37] may, with further development, prove to be more worthwhile to stockpile for the purposes of pandemic preparedness than an assortment of vaccines targeting antigenic variants manufactured via ppp experimentation. current surveillance is likely inadequate to detect an emerging pandemic strain before it is too late [29, 38] , regardless of any warnings that ppp experimentation might generate about potentially worrisome mutations. between 2008 and 2013, over 1,580 highly pathogenic avian influenza (almost all h5n1) outbreaks, involving over 5 million birds, were reported to the world organisation for animal health [39] . the us national center for biotechnology information influenza virus resource [40] received about 1,400 complete or partial avian h5n1 virus sequences over this period [41] . most of these sequences were over eight months old by the time they were publicly available in the influenza virus resource. similar considerations apply to gisaid's epiflu database, the other major influenza virus sequence database [42] . given that birds [43] , like humans [44] , harbor a genetically diverse quasispecies of influenza variants, it is highly unlikely that such limited surveillance could detect a pandemic viral sequence and, furthermore, spur effective mitigation actions, before the worrisome variant was already widespread in birds. as an example of the limited public health response even when a dangerous virus has been observed, consider the global response to h7n9 avian influenza, which has proven zoonotic potential and has probably been repeatedly transmitted from human to human [45] . isolates from human cases reveal efficient binding to human sialic acid receptors and airborne transmission in ferrets [9] and guinea pigs [46] . these indicators of pandemic potential are much stronger than sequence comparisons with engineered viruses could provide, yet most live bird markets in china remain open, and human cases continue to emerge [47] . given these realities, it is difficult to envision how a surveillance signal alone would prompt swifter actions than these existing warning signs for h7n9 have. in short, the benefits for public health of the scientific findings from ppp experimentation are speculative at best. a further challenge to realizing public health benefits from ppp experimentation is that the predictability of phenotype from viral sequence is complex [38, 48, 49] , as demonstrated by a recent assessment [50] of the generality of mutations that conferred human receptor binding in engineered ferret-transmissible h5n1 strains. when these mutations were introduced into the genetic background of more recent avian isolates of h5n1, affinity to human receptors was lost [50] . instead, the phenotype of any mutation depends on interactions with its genetic background, a phenomenon known as epistasis that is observed broadly in nature and in influenza viruses specifically [51, 52] (table 1) . thus, it is unlikely that a catalog of mutations could inform reliable predictions of transmission phenotype in circulating strains. the prominent role of epistasis in influenza biology suggests that alternative approaches to studying the phenotypic impact of mutations on mammalian transmissibility would be not only less risky, but also more informative. in vivo replication and transmission of influenza in humans depend on myriad interdependent factors, including the binding affinity between hemagglutinin and human sialic acids, the ability of the virion to fuse with the endosomal membrane at the appropriate ph and temperature, as well as the stability of various viral proteins [11, 51, 53, 54] . each of these traits, in turn, is not simply determined by the presence or absence of individual amino acids at box 2. gain of function: what's in a name? the recent ferret transmission experiments with influenza a viruses have been termed ''gain of function'' experiments because they involve engineering viruses that gain transmissibility in ferrets. gain of function is a common and important approach in biological experimentation, and is not by itself cause for concern [58] . however, the elevated concern over these experiments arises from the particular function that is gained. ferret transmission is thought to be a good [59, 60] (albeit imperfect [61, 62] ) model for human-to-human transmission. consequently, strains resulting from selection for heightened ferret transmission are likely to be similarly transmissible by humans via respiratory droplets, a prerequisite for pandemic spread. in combination with demonstrated virulence for humans, this particular gain of function presents unique risks and raises special ethical issues. particular sites, but by biophysical properties arising from the interaction of many sites within and between proteins [50, 51] . consequently, the challenge of predicting transmissibility hinges on understanding the genetic determinants of each trait, coupled with the interactions of the traits from which the higher-level phenotype of transmissibility arises. an array of safer approaches ( table 2 ) to studying influenza pathogenesis and transmission focus on dissecting these interactions. some approaches start with sequence analysis and molecular dynamics modeling, which are intrinsically safe. the experimental evaluation of hypotheses raised by such studies may use viral components rather than the entire infectious virus, making these experiments simultaneously safer and more precise and mechanistic than engineering ppps. furthermore, these approaches are typically less costly than ppp experimentation, facilitating phenotypic evaluation of a greater diversity and abundance of genetic variants. ultimately, studies with intact viruses will be necessary for a full understanding of human transmissibility, a [63] [64] [65] [66] unaffected virulence or transmissibility (h1n1pdm) [67] ; decreased fitness (older h5n1) [66] na h275y oseltamivir resistance, fitness crippled (h1n1) [68, 69] oseltamivir resistance, fitness increased in absence of drug (h1n1) [68, 70] ha ls, 158,224,226 mammalian transmission (h5n1 from indonesia and viet nam) [1, 2] no switch to mammalian sialic acid binding (h5n1 from egypt) [50] polybasic ha cleavage site high avian pathogenicity (many h5 and h7 viruses) [71, 72] low avian pathogenicity (four h5 isolates) [73] ha, hemagglutinin; na, neuraminidase. doi:10.1371/journal.pmed.1001646.t001 table 2 . safer approaches to studying human adaptation of influenza a viruses, and more generally to improving vaccines and therapeutics. analysis of adaptive changes in ha of h1n1pdm [74] , lipid tail protrusion as a determinant of ha-membrane fusion [75] , and identifying determinants of inhibitorresistant na [76] biophysical basis for complex phenotypes in vitro studies of specific properties required for human adaptation, using single proteins studies of h5 or h7 receptor binding to mammalian versus human sialic acids [50, 77] ; studies of genetic determinants of optimal ph of fusion by comparing properties of natural isolates [11] higher throughput than in vivo studies; can study more sequences and define motifs required for binding, beyond individual mutations; ability to assess generality of hypothesized determinants [54] in vitro studies of genetic interactions between loci in one or several viral proteins using replication-incompetent viruses studies of epistatic interactions in nucleoprotein [51] or between nucleoprotein and polymerase [78] based on in vitro expression of markers and stability measurements of proteins higher throughput; ability to link structure to function; ability to test combinations of mutations sequence database comparisons of genetic properties of human-and avian-adapted viruses identify amino acid markers of host adaptation and quantify the extent of adaptation to a particular host [79, 80] ; search for markers of human adaptation (established from earlier studies without ppp production) in h7n9 viruses [63] very high throughput; future studies could use novel analytic methods [81] phenotype of a whole virus. elucidating the evolutionary trajectory through which existing seasonal (former pandemic) viruses became transmissible from avian precursors is safer than ppp experimentation, given that there is preexisting population immunity to seasonal strains, the products of such evolution. more generally, it should be remembered that the public health goal is to curtail influenza pandemics and seasonal transmission [55, 56] . exploring basic biology is just one scientific means to this end. other approaches, such as developing universal influenza vaccines and novel antiviral drugs and strategies to enhance host responses, as well as improving technologies for rapid vaccine manufacture, are being pursued without risks of ppp release ( table 2 ). we urge that proposals for any future experiments on ppps be evaluated according to quantitative risk-benefit analysis guided by the principles of the nuremberg code. indeed, hhs frameworks require a risk-benefit analysis to approve gain-offunction experimentation on h5n1 and h7n9 [33] [34] [35] viruses, yet no such analysis has been made public, if it has been conducted. other funding and regulatory agencies, which have not yet called for a risk-benefit analysis, should require one as well. in biomedical grant review processes, proposals compete for limited funding, and most proposals that could advance science are never supported, because of budget constraints or because funding agencies conclude that there are more promising, safer, more humane, or otherwise superior ways to achieve scientific goals. ppp experimentation poses a significant risk to public health, arguably the highest level of risk posed by any biomedical research. such experiments should be assessed on the basis of their marginal benefits, compared to those of safer approaches. in the case of influenza, given the higher throughput and lower cost of alternatives, we believe the benefits of alternative approaches will be greater than those of novel ppp experimentation, yet without the risks-thereby negating the justification for taking such risks. similarly, careful consideration should be given to analyses of novel ppp experiments beyond the study of influenza, as these are proposed. funders and regulators should evaluate the balance of risks and benefits before further novel ppp experiments are undertaken. experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets airborne transmission of influenza a/h5n1 virus between ferrets the unacceptable risks of a man-made pandemic evolution and ecology of influenza a viruses h5n1 virus: transmission studies resume for avian flu avian flu: gain-offunction experiments on h7n9 gain-of-function experiments on h7n9 effect of receptor binding domain mutations on receptor binding and transmissibility of avian influenza h5n1 viruses limited airborne transmission of h7n9 influenza a virus between ferrets airborne transmission of highly pathogenic h7n1 influenza in ferrets mutations in haemagglutinin that affect receptor binding and ph stability increase replication of a pr8 influenza virus with h5 ha in the upper respiratory tract of ferrets and may contribute to transmissibility h5n1 hybrid viruses bearing 2009/ h1n1 virus genes transmit in guinea pigs by respiratory droplet pathogenicity and transmissibility of reassortant h9 influenza viruses with genes from pandemic h1n1 virus minimal molecular constraints for respiratory droplet transmission of an avian-human h9n2 influenza a virus compatibility of h9n2 avian influenza surface genes and 2009 pandemic h1n1 internal genes for transmission in the ferret model monitoring select agent theft, loss and release reports in the united states committee on anticipating biosecurity challenges of the global expansion of high containment biological laboratories, national academy of sciences and national research council (2012) biosecurity challenges of the global expansion of highcontainment biological laboratories: summary of a workshop redaction of sensitive data in the publication of dual use research of concern containing the accidental laboratory escape of potential pandemic influenza viruses transmission dynamics and control of severe acute respiratory syndrome superspreading and the effect of individual variation on disease emergence interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early targeted layered use of nonpharmaceutical interventions. washington (district of columbia): us department of health and human services estimating age-specific cumulative incidence for the 2009 influenza pandemic: a meta-analysis of a(h1n1)pdm09 serological studies from 19 countries comment on ''seroevidence for h5n1 influenza infections in humans: meta-analysis public health and biosecurity. adaptations of avian flu virus are a cause for concern a novel strain of clostridium botulinum that produces type b and type h botulinum toxins inconvenient truths'' in the pursuit of scientific knowledge and public health interacademy panel on international issues rethinking biosafety in research on potential pandemic pathogens uncertainty in xenotransplantation: individual benefit versus collective risk probing the improbable: methodological challenges for risks with low probabilities and high stakes new directions: the ethics of synthetic biology and emerging technologies. washington (district of columbia): presidential commission for the study of bioethical issues research funding. a framework for decisions about research with hpai h5n1 viruses extra oversight for h7n9 experiments avian flu: extra oversight for h7n9 experiments gain-of-function research: unproven technique toward a universal influenza virus vaccine: prospects and challenges evolution, safety, and highly pathogenic influenza viruses world organisation for animal health (2013) wahid interface [database the influenza virus resource at the national center for biotechnology information influenza virus resource genomes/flu/database/nph-select.cgi?go = database epiflu database [database the feasibility of using high resolution genome sequencing of influenza a viruses to detect mixed infections and quasispecies viral genetic sequence variations in pandemic h1n1/2009 and seasonal h3n2 influenza viruses within an individual, a household and a community human-to-human transmission of h7n9 in a family: ''probable'' vs ''possible novel h7n9 influenza virus shows low infectious dose, high growth rate, and efficient contact transmission in the guinea pig model human infection with avian influenza virus-update accessed 28 pandemic influenza viruses-hoping for the road not taken pandemic influenza viruses: time to recognize our inability to predict the unpredictable and stop dangerous gain-of-function experiments structural determinants for naturally evolving h5n1 hemagglutinin to switch its receptor specificity stability-mediated epistasis constrains the evolution of an influenza protein prevalence of epistasis in the evolution of influenza a surface proteins development of an influenza virologic risk assessment tool receptor binding profiles of avian influenza virus hemagglutinin subtypes on human cells as a predictor of pandemic potential pandemic influenza planning biology's brave new world: the promise and perils of the synbio revolution flu season: an interview with jeffery k. taubenberger, chief of the viral pathogenesis and evolution section at the us national institute of allergy and infectious diseases the ferret as a model organism to study influenza a virus infection considerations regarding appropriate sample size for conducting ferret transmission experiments h5n1 influenza viruses: facts, not fear avian influenza: ferret h7n9 flu model questioned guiding outbreak management by the use of influenza a(h7nx) virus sequence analysis molecular determinants of adaptation of highly pathogenic avian influenza h7n7 viruses to efficient replication in the human host transmission of influenza virus in a mammalian host is increased by pb2 amino acids 627k or 627e/701n the effect of the pb2 mutation 627k on highly pathogenic h5n1 avian influenza virus is dependent on the virus lineage introduction of virulence markers in pb2 of pandemic swineorigin influenza virus does not result in enhanced virulence or transmission permissive secondary mutations enable the evolution of influenza oseltamivir resistance influenza viruses resistant to the antiviral drug oseltamivir: transmission studies in ferrets the global spread of drugresistant influenza hemagglutinin activation of pathogenic avian influenza viruses of serotype h7 requires the protease recognition motif deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza a viruses of h5 and h7 subtypes highly pathogenic avian influenza viruses with low virulence for chickens in in vivo tests analysis of adaptation mutants in the hemagglutinin of the influenza a(h1n1)pdm09 virus lipid tail protrusion in simulations predicts fusogenic activity of influenza fusion peptide mutants and conformational models quantitative predictions of binding free energy changes in drugresistant influenza neuraminidase glycan receptor binding of the influenza a virus h7n9 hemagglutinin unstable polymerase-nucleoprotein interaction is not responsible for avian influenza virus polymerase restriction in human cells identifying changes in selective constraints: host shifts in influenza charting the host adaptation of influenza viruses codon usage bias and the evolution of influenza a viruses. codon usage biases of influenza virus h7n9 influenza viruses are transmissible in ferrets by respiratory droplet comparison of the levels of infectious virus in respirable aerosols exhaled by ferrets infected with influenza viruses exhibiting diverse transmissibility phenotypes the m segment of the 2009 new pandemic h1n1 influenza virus is critical for its high transmission efficiency in the guinea pig model hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic h1n1 influenza virus in ferrets influenza virus hemagglutinin stalk-based antibodies and vaccines elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure universal vaccine against influenza virus: linking tlr signaling to anti-viral protection report of the 5th meeting on influenza vaccines that induce broad spectrum and long-lasting immune responses, world health organization the universal epitope of influenza a viral neuraminidase fundamentally contributes to enzyme activity and viral replication ifitm3 restricts the morbidity and mortality associated with influenza synthetic generation of influenza vaccine viruses for rapid response to pandemics analyzed the data: ml. wrote the first draft of the manuscript: ml. contributed to the writing of the manuscript: ml ag. icmje criteria for authorship read and met: ml ag. agree with manuscript results and conclusions: ml ag. key: cord-019057-3j2fl358 authors: afolabi, michael olusegun title: pandemic influenza: a comparative ethical approach date: 2018-08-28 journal: public health disasters: a global ethical framework doi: 10.1007/978-3-319-92765-7_3 sha: doc_id: 19057 cord_uid: 3j2fl358 community-networks such as families and schools may foster and propagate some types of public health disasters. for such disasters, a communitarian-oriented ethical lens offers useful perspectives into the underlying relational nexus that favors the spread of infection. this chapter compares two traditional bioethical lenses—the communitarian and care ethics framework—vis-à-vis their capacities to engage the moral quandaries elicited by pandemic influenza. it argues that these quandaries preclude the analytical lens of ethical prisms that are individual-oriented but warrant a people-oriented approach. adopting this dual approach offers both a contrastive and a complementary way of rethinking the underlying socioethical tensions elicited by pandemic influenza in particular and other public health disasters generally. contemporary healthcare constitutes an instinctual and institutional response to the multifaceted cycles of health, illness, and disease. 1 hence, the problems of diseases including infectious ones affect all and sundry irrespective of current "sick status". pandemic influenza is one such incident that afflicts all sectors of the society. 2 it also raises questions and issues related to utility and equity, ensuring the protection of vulnerable individuals and groups in society, the need to exercise public health powers with respect for human rights 3 as well as the just allocation of human and material resources. 4 attending to these issues, however, juggles many kinds of personal, social, political, and professional interests against one another; thus, reflecting the traditional public health dilemma of fine-tuning individual against collective good. 5 since the restrictive approach of individualism-driven moral lenses 6 is unsuitable for people-centered quandaries, it seems pertinent to employ a people-centric moral lens to engage them. in this vein, the ethical prism of communitarianism and ethics of care seem apt. by examining and contrasting the core fabric of the communitarian and care ethics frameworks vis-à-vis the attendant dilemmas of pandemic influenza; this chapter attempts to tease out a broader ethical path towards engaging the challenges of pandemic influenza. to properly set the conceptual foreground essential to articulating the ethical features of pandemic influenza, however, it is important to elaborate the associated biological, social, and global dynamics. these parameters, as macphail recently argues, are exigent in the explication and engagement of pandemic or infectious disease outbreaks. 7 there have been some speculations as to the origins of the influenza virus. it has been hypothesized that the virus originated from wild waterfowls and has only slowly evolved through multiple animal species including humans. 8 but what is known about the disease caused by the virus-influenza-is that it is a febrile illness of the upper and lower respiratory tract, characterized by a sudden onset of fever, cough, myalgia, and malaise. pneumonia is a principal serious complication 9 and local symptoms include sniffles, nasal discharge, dry cough, and sore throat. 10 pandemic influenza outbreaks describe the rapid spread of influenza infection. whereas there is some conceptual controversy about the description and definition of pandemics, 11 they generally refer to the dissemination of new infective diseases to which immunity has not been developed in a widespread manner across a significant part of the world. 12 they could break out in nations with a large geographical size (such as china, india, and the united states) or when the number of affected nations are many. the pandemic nature of influenza is historically underscored by the 1918-1919 incident that killed an estimated 20 million to 50 million people. 13 pandemic influenza is generally characterized by an alteration in the viral subtype (due to antigenic shift), higher mortality rates among younger groups, several waves of the particular pandemic, increased capacity of spread, and geographic variation in the impact of the outbreak. 14 specifically, influenza pandemics occur when an influenza virus mutates or when multiple strains combine, or re-assort to produce strains to which there is no current immunity. 15 novel outbreaks of the influenza virus occur either in large nations or across selected nations in close proximity. contemporary society experiences an increased development of new serotypes of several kinds of respiratory viruses because of the evolutionary potential afforded by the human population explosion and the great global increase in human mobility. 16 in a manner of speaking, it seems that phds such as pandemic influenza outbreaks have evolved to become recurring features of the human experience. some insights into the biological features and processes that create pandemic outbreaks support this idea. influenza viruses belong to the orthomyxoviruses family. this comprises seven genera including influenza virus a, b, c, and d. 17 although both the genus influenzavirus a and b affect humans and cause pandemics, 18 influenza a has been the principal culprit in known outbreaks to the extent that four major pandemics have resulted from it (1918-1919, 1957, 1968, and 2009) . 19 however, genetic reassortment and exchange of influenza viruses between humans and animals generate antigenic shift, which periodically introduces new viruses to the human population. this, in addition to mutation and selection, produces antigenic drift that accounts for the year-to-year variations in influenza a subtypes. 20 wild ducks, for instance, serve as the primary host for various influenza type a viruses that occasionally spread to other host species and cause outbreaks in such animals as fowl, swine, and horses. such outbreaks often lead to new human pandemics 21 due to novel viruses infecting immunologically naïve people. 22 a critical aspect of the emergence of novel virus strains is genetic variation and combination that occur at the hemagglutinin (ha) antigens (of which there are 16) and neuraminidase (na) enzymes (of which there are nine) 23 between and amongst human and animal influenza viruses. the subtypes of the ha and na surface proteins forms the basis for the classification of outbreaks. 24 for example, the 1918 through 1919 virus was h1n1, the 1957 through 1963 virus was h2n2, the 1968 through 1970 outbreak was caused by h3n2, 25 the 1996 virus was h5n1, 26 and the 2009 outbreak was caused by h1n1; 27 while the most recent virus seen in eastern china in 2013 was h7n9. 28 all of these traditional and new influenza viruses cause pandemics of differing proportions but more are projected to occur. 29 this projection is well supported by the scientific community. however, it is not known when any will occur or whether it will be caused by the h5n1 avian-derived influenza virus, newer subtypes like h7n9, or completely novel subtypes. virologists like webster and govorkova argue that given the number of cases of h5n1 influenza that have occurred in humans (more than 251) with a mortality or death rate of more than 50%, it would be prudent to develop robust plans for dealing with such pandemic influenza and its (expected) new variations. 30 such plans, however, necessarily demand attention to the associated ethical dynamics. regardless of the specific subtype of human or animal-derived influenza outbreaks, the public health challenges and the moral quandaries are essentially the same. a critical biological feature of influenza lies in its mode and pattern of transmission. this revolves around its capacity to evolve and become airborne-transmissible between and amongst human beings. 31 the influenza virus transmits from person to person primarily in droplets released by sneezing and coughing. some of the inhaled virus lands in the lower respiratory tract, the primary site of disease 21 marion russier et al., "molecular requirements for a pandemic influenza virus: an acid-stable hemagglutinin protein," proceedings of the national academy of sciences 113, no. 6 (2016) . pp. 1636 pp. -1639 anna v cauldwell et al., "viral determinants of influenza a virus host range," journal of general virology 95, no. 6 (2014). pp. 1193-1195. 23 couch. p.; shah. p. 94. 24 cauldwell et al. p. 1193 . 25 miller et al. pp. 2595 -2597 shah. p. 89. 27 rebekah h borse et al., "effects of vaccine program against pandemic influenza a (h1n1) virus, united states, 2009 -2010 ," emerging infectious diseases 19, no. 3 (2013 . pp. 439-441. 28 cauldwell et al. p. 1204. 29 macphail. p. 9. 30 robert g webster and elena a govorkova, "h5n1 influenza-continuing evolution and spread," new england journal of medicine 355, no. 21 (2006) . pp. 2174-2175. 31 russier et al. pp. 1636 -1637 being the tracheobronchial tree, and sometimes the nasopharynx. 32 largely because breathing is an essential biological need of human beings and partly because human-human associations are an inevitable part of reality, this biological feature of influenza viruses makes everyone vulnerable and susceptible to infection. specifically, crowds of people facilitate viral transmission by enabling sharp upticks in the rate of transmission. the virus also circulates for longer periods in infected persons. 33 the biological features of influenza and its mode of transmission elicit some observations. one, pandemic influenza is not a single disease for which a single and specific therapeutic intervention that will be effective all the time can be developed. in other words, while there is a general approach to engaging this public health disaster, specific interventions will usually vary by each outbreak. this gives an existential and evolutionary advantage to the influenza virus over human communities. it also engenders a disaster dynamic in the sense that every outbreak becomes "sudden" and potentially associated with large human casualties. secondly, it shows the common vulnerability to which the local and global human community are subject vis-à-vis the ease of spread of the viral infection. thirdly, the biological features of pandemic influenza demonstrate how a collective response (human material, scientific etc.) is key to engaging its social and other attendant consequences. the importance of this last remark will become clearer against the backdrop of the social and global features of pandemic influenza outbreaks, a. theme addressed in the next section of this chapter. an influenza pandemic has the potential to cause more deaths and illnesses than any other public health threat. 34 pandemic influenza a h1n1 were reported. 37 also, the h5n1 outbreak recorded a death rate of 59%, 38 and the recent h7n9 outbreak caused 251 human infections and 67 deaths. 39 in the united states, the estimated potential threat of pandemic influenza is 1.9 million deaths, 90 million sick people, and nearly 10 million hospitalizations, with almost 1.5 million requiring intensive-care units. 40 global estimates are higher. for instance, the 1918 "spanish flu" caused an estimated 20-50 million global deaths. 41 it has been projected that a recurrence of the 1918 influenza strain would probably result in the death of 51-81 million individuals. 42 these data show that substantial numbers of deaths are an inevitable consequence and feature of pandemic influenza. however, death itself often brings about certain social consequence including the death of some of the most gifted members of the society. sir william osler, one of the pioneers of scientific medicine, died of complications arising from influenza in 1919. influenza was cited by the german war general, erich von ludendorff, as a significant reason for why the initial gains of their last offensive faltered and ultimately failed during world war 1. 43 from a biological perspective, influenza exploits naïve immune systems which tend to over-respond to the influenza virus. as such, young and promising adults constitute a large part of vulnerable victims. in this regard, potential contributions to societies are nipped in the bud, young widows and widowers emerge as well as a lot of orphans. for instance, 21,000 children were orphaned due to the 1918 outbreak in new york city. 44 influenza also spread within households soon before or after the onset of symptoms in primary infected patients. 45 another associated social feature of pandemic influenza is the closure of schools with an attendant truncation of learning and educational opportunities, depending on the length of the outbreak. while some of these social features are local and exert localized effects, human beings as social animals with the aid of the increased means of locomotion transmit some of the local features into a global experience. the 1957 pandemic of influenza which occurred during a time of much less globalization spread to the united states within 4-5 months of its detection in china while the 1968 pandemic spread to the u.s. from hong kong within 2-3 months. 47 it is estimated that the burden of the next influenza pandemic will be overwhelmingly focused in the developing world. 48 however, the epidemiological notion well-known to public health experts that infectious diseases can predicate outbreaks in neighboring places and nations 49 implies that even so-called developed societies cannot be spared as long as the current interpenetration of people across the globe remains. the 2009 influenza outbreak, for instance, spread to 85 countries and caused a total of 39, 620 cases of infection. 50 in short, in a globalized world, infectious diseases travel in nodes of human, material, and animal networks. 51 data from sporadic studies suggest that influenza may be fairly prevalent in africa, albeit sub-clinically. it may, therefore, have a considerable impact on morbidity and mortality on the continent 52 should a combination of factors create a virus that is viable enough to cause a pandemic. this will have far-reaching consequences for the continent due to the material and human resource constraints, lack of preparedness plans as well as the very limited bio-therapeutic capacities that are currently available to produce vaccines. it may likewise create the dispersal of a virus novel to other continents that have experienced typical outbreaks. geographical location plays a major role in public health, 53 and disasters including health disasters are unique in that each affected region of the world has different social, economic, and health backgrounds. 54 as such, while there is a global spread, the nature of each local context and how it responds shapes pandemic influenza in some key ways. first, the nature of the "disseminating" nation influences how infection spreads elsewhere. for example, china's slow reaction to the 2003 sars outbreak as well as its limiting of access to patients and other relevant information 47 hhs, "hhs pandemic influenza plan." p. b6. 48 86, no. 9 (1996) . p. 1208 54 eric k noji, "public health issues in disasters," critical care medicine 33, no. 1 (2005) . p. s29. seemed to have deepened the global intensity of that crisis. 55 in other words, how a local public health disaster is handled shapes the local severity and how it spreads elsewhere. on the other hand, well-handled local health crises positively influence the possible impacts on contiguous nations. in this vein, radest notes that canada's rapid and coordinated response to the sars outbreak significantly limited its spread and impact in the united states. 56 the above examples echo the interconnectivity of the modern world and show how a course of action in one place, however passive, may significantly influence the course of events in another for good or bad. it supports the idea that contemporary health in the twenty-first century is now inevitably and inherently global with respect to infectious diseases. 57 at the heart of these remarks, however, is the possibility of utilizing different networks of human interconnectivity to actively foster the global good. in other words, learning about how people connect and relate at different levels (individually, communally, institutionally et cetera) and learning about the chief actors and players in such a relationship nexus may provide a powerful tool for driving global public health agenda. yet, integral to such a process is how responses to pandemic influenza are framed and implemented locally as well as their attendant limitations. this theme is addressed in the next section. the human instinct for self-preservation has, at the social plane, always resulted in some institutional responses to diseases, whether rudimentary, barely adequate, or sophisticated. in the context of phds, responses are shaped by the nature of the specific disaster, where it is taking place, and what human, material, pecuniary and technological resources are available to deal with the given emergency situation. for instance, the united states prioritizes building a system that ensures stable and economically viable vaccines to engage influenza outbreaks. 58 countries that lack the same kind of resource will clearly prioritize other approaches. however, the general approaches to pandemic influenza are therapeutic and non-therapeutic in nature. this section briefly examines them. pandemic influenza outbreaks, like most diseases, have elicited some biopharmaceutical responses geared towards mitigating its disastrous effects. due to the changing biological and social dynamics associated with the outbreak, social as well as scientific responses are always evolving to keep up. nevertheless, the therapeutic measures fashioned to combat pandemic influenza fall into two groups. these are preventive measures involving the use of anti-viral drugs as well as vaccination. in the past, drugs like rimantadine and amantadine were used as prophylaxis against influenza a. 59 but drug resistance has increasingly been observed to these m2-ion channel-blocking agents. 60 today, drugs of choice are mainly tamiflu (oseltamivir) and relenza (zanamivir). black et al. noted that early anti-viral intervention during the 2009 pandemic helped reduce the doubling time in the early stages of the outbreak. 61 the linkage between antiviral use and reduction in clinical severity and influenza infectiousness is generally supported in the extant literature. 62 hence, treatment of clinical cases with anti-viral agents constitutes the first-line of engagement for pandemic influenza and these drugs are employed to control or contain pandemic outbreaks long enough for vaccines to be made. 63 yet, drugs like oseltamivir and zanamivir, usually neuraminidase inhibitors, can only help reduce transmission if given within a day of the onset of symptoms. 64 on the contrary, delay in symptoms diagnosis, as well as intervention, favors infection dissemination. nevertheless, antiviral agents for influenza offer some protection to families and households once infection has been detected. in clinical trials, antiviral treatments have been shown to be efficacious in preventing infection, hence, slowing down transmission as well as limiting the severity of the disease. 65 but the effectiveness of neuraminidase such as oral oseltamivir and inhaled zanamivir at reducing mortality is uncertain. 66 in addition, there is some evidence of side-effects. for instance, in adults as in children, oseltamivir increases the risk of nausea and vomiting. also, treatment trials with oseltamivir or zanamivir do not settle the question of whether the complications of influenza (such as pneumonia) are reduced. 67 resistance to these anti-viral drugs has also been reported, even in people who have never been previously treated with them. 68 ultimately, the success of antiviral prophylaxis critically depends on the identification of index cases in households, pre-schools, schools, and other institutional settings. 69 this clearly highlights the importance of personal, social, and institutional cooperation in relation to dealing with the associated challenges. on the other hand, vaccination as one of the most effective and cost-saving strategies for ameliorating infectious diseases 70 offers a protective approach to limiting and/or curtailing the social and economic consequences of pandemic influenza. two types of vaccines are generally used. trivalent inactivated vaccine and live attenuated influenza virus vaccine, both of which contain the predicted antigenic variants of influenza a(h3n2), a(h1n1), and b viruses. 71 borse et al. estimated that 2009 vaccination program against influenza prevented 700,000-1,500,000 clinical cases, 4000-10,000 hospitalizations, and 200-500 deaths. they also reported that the national health effects of vaccination were greatly influenced by the timing of vaccine administration and the effectiveness of the vaccine. 72 similarly, ferguson et al. estimated that during a global outbreak, vaccination at the rate of 1% of the population per day would need to begin within 2 months of the initial outbreak. but this is not feasible under current vaccine technologies. 73 this pragmatic challenge would, however, create a biological and social climate in which infection may flourish in a logarithmic manner. the recurring antigenic variation in influenza viruses which leads to the frequent emergence of new infectious strains 74 increases the likelihood of continuous outbreaks. this and the capacity of the influenza virus to acquire amino acid changes in its viral proteins 75 implies that each outbreak will demand novel vaccines. this often delays the possible response time, again creating a window where infection can readily spread, locally and globally. for instance, it will take at least 4 months from identification of a candidate vaccine strain until production of the very first vaccine 76 during an outbreak. this biological fact makes it difficult to stockpile influenza vaccines ahead of outbreaks and, by consequence, limits the preparedness efforts geared towards confronting the public health challenges and moral quandaries. it is important to note that vaccines have some limitations. for instance, they are not entirely safe public health interventions, especially when specifics are examined. 77 this fact has increasingly come to light in relation to vaccines against pandemic influenza. besides sore arm and redness at the injection site as well as red eyes which have been reported in earlier vaccine trials, 78 there has been some association between increased incidence of narcolepsy in children and the use of the aso3-adjuvanted vaccine for pandemic h1n1 influenza in scandinavian countries. 79 in addition, anecdotal reports of fetal deaths occurring shortly after vaccination emerged in 2009 and raised public health concerns about vaccine safety. 80 another shortcoming associated with vaccination generally is vaccine failure, 81 which often creates a false sense of protection in recipients while allowing the continued spread of infection. 82 in relation to pandemic influenza specifically, vaccine failure was recently reported by manjusa et al. in people of 65 years and above as well as those who have been vaccinated against seasonal influenza. 83 this is quite troubling partly because vaccine failure vis-à-vis pandemic influenza vaccines has been little studied, and partly because there are countries like the united states where seasonal flu vaccine shots are almost the norm. another dimension to vaccine failure relates to the variation of influenza virus clades. nelson et al. recently reported that nigeria, côte d'ivoire, and cameroon exhibit more variable patterns of influenza virus seasonality, hence, there is a possibility of variants evolving locally within west africa. this, they further argue, undermines the assumption that a vaccine matched to globally dominant lineages will necessarily protect against these local lineages. 84 this notion further raises the question of whether the immune system of populations living in tropical african 76 hhs, "hhs pandemic influenza plan." p. b12. 77 environments would react similarly to a vaccine developed mainly for populations restricted to certain geographical areas of the world. 85 on this note, in the possible event that someone originally from any of these nations were present in a pandemic influenza scenario outside african shore, the likelihood of their benefiting from vaccination seems slim. hence, a significant offshoot of vaccine failure in relation to pandemic influenza (especially if newer studies show more negative results) will be the reluctance of people to receive vaccines for seasonal flu and those developed for pandemic influenza outbreaks. these have unsettling public health and moral consequences. one way of engaging the limits of influenza vaccines involve creating a vaccine type that is capable of eliciting cross-protective peptides/epitopes that would be effective against different variants. but this is very difficult. 86 besides the scientific technicalities, producing vaccines for pandemic influenza is not a cheap venture. for example, meltzer, cox, and fukuda estimated in 1999 that it would cost the united states about $166.5 billion to contain pandemic influenza. 87 whereas the economic burden of influenza in lower-and middle-income countries involves direct costs to the health service and households and indirect costs due to a loss in human productivity, 88 these countries also have limited financial capacities to pursue pandemic influenza vaccination as a public health tool. the impacts of the ensuing disease burden from such a constraint will not be locally confined, as it will ultimately seep into the trans-national and global terrains. in summary, the major and, perhaps, insurmountable constraint to vaccination as a tool for engaging pandemic influenza lies in the logistic challenge of producing a pandemic vaccine from scratch, conducting pre-clinical testing as well as generating billions of doses within a very short time for global distribution, 89 which may, however, not work across all nations. but considering the limitations associated with antiviral drugs as well as vaccines in relation to combating pandemic influenza, some form of non-therapeutic approach is necessary, at least as some adjunct to mitigate the overall impact of pandemic influenza on the local and global human community. the next section addresses this theme. 85 yazdanbakhsh and kremsner. p. e1000182. 86 the non-pharmaceutical and non-therapeutic approaches to pandemic influenza revolve around measures such as case isolation, school or workplace closure, restrictions on travel, 90 quarantine as well as contact tracing. for instance, school closure is a non-pharmaceutical intervention often suggested for mitigating influenza pandemics. the logic behind this lies in the notion that children are important vectors of transmission, more infectious, and susceptible to most influenza strains than adults. it is also tied to the idea that high a contact rate in schools fosters transmission of infection. this approach, according to cauchemez and colleagues, may bring about an estimated 40% reduction in peak attack rates. however, this reduction will be hindered if children are not adequately isolated or if the policy is not well implemented. 91 whereas school closure may only bring about a small reduction in cumulative attack rates, it can foster a substantial reduction in peak attack rates. 92 closure of schools may, however, increase anxiety and create a crisis, as was observed in france during the 1957 outbreak. 93 closure of workplaces is another non-pharmaceutical intervention for pandemic influenza. it may be warranted by the degree of the outbreak in which businesses shut down at their own discretion, and for their own safety, as was seen during the 1918-1919 outbreak. 94 however, it may also be warranted by government policy. either way, business closure incurs huge economic costs, pecuniary, and other consequences for the different people tied to and/or dependent on the affected businesses or their services and goods. different forms of quarantine measures are also used to mitigate the spread of infection during an influenza pandemic. for instance, isolation and quarantine of infected patients allow some containment of infection which consequently slows down viral transmission. 95 ultimately, quarantine contributes towards reducing the overall costs and impact of an outbreak. some medical experts see household quarantine as the most effective social distance measure, provided the level of compliance is good. 96 yet, quarantine-at least on a general note-does not always work. for example, maritime quarantine was one of the measures employed in west africa to engage the 1918 influenza outbreak as well as interning the ill. however, historians like heaton and falola note that these approaches yielded meager success in relation to quelling the spread and virulence of the pandemic. 97 measures such as cancellation of non-essential public gatherings and restrictions on long-distance travel might help to decrease influenza transmission rates as well as overall morbidity, their effectiveness has not been quantified. 98 the nature of pandemic influenza, the therapeutic and non-therapeutic approaches, and the associated limitations generate some moral concerns. the next section discusses this. ethical issues arise during outbreaks of pandemic influenza. some of these are directly tied to the nature of the virus, some in relation to human responses, some to the social responses, and others to how different human beings respond differently to the several challenges elicited by the pandemic. bioethicists have underscored the critical need to reflect on the ethical issues raised by the specter of pandemic influenza outbreaks. 99 however, what may and what may not be feasible to do will never be clear enough if these ethical quandaries are not clearly explicated. hence, this section seeks to clarify the moral quandaries elicited by pandemic influenza and show the core connecting strands that resonate amongst them. generally, contexts of uncertainty are tied to the evolving nature of knowledge. tannert et al. opine that uncertainty occurs because the more the human community gains insights into the mysteries of nature, the more they realize the limits of their knowledge about how things are. these limitations, they note, make it impossible to foresee all the associated future effects and implications of situations and decisions with certitude. 100 in relation to medicine, jean daly notes that the art of medicine seeks to abolish uncertainty. 101 regardless of the good intentions and telos of medicine, the stark reality is that this task has hardly been achieved. contexts. james marcum contends that uncertainty is largely a part of medicine because of the variability of the underlying biology. 102 uncertainty is not new in the realm of science. 103 however, in the context of public health disasters uncertainty has a strong pragmatic dimension which can influence courses of actions and decisions in multiple unfavorable ways. for example, it occurs during pandemic influenza outbreaks and generates many concerns. in this vein, borse et al. note that the public health community cannot accurately predict the arrival of a pandemic. 104 indeed, a great deal of uncertainty occurs in relation to estimating the potential impact of a pandemic such as influenza. 105 this scenario stifles preparedness efforts, especially in resource-constrained countries where there are often competing social needs to be met with limited budgets. however, the two main uncertainty issues embedded in pandemic influenza involve the nature of the virus and the types of responses available to engage outbreaks. on the one hand, the influenza virus undergoes constant variation in its antigens, creating new infectious strains. 106 the virus also acquires amino acid changes in its proteins. these scenarios increase the likelihood of pandemic outbreaks. however, the question of when, where, and of what magnitude the outbreak will be is never clear-cut. worst-case scenario analysis based on the 1918-20 pandemic provides no insight into the probability of an influenza pandemic in the next 1, 5, or 10 years 107 and how serious such an outbreak might be. this scientific uncertainty or paucity of precise knowledge ignites some social uncertainty and may prompt moral inertia in relation to the level of preparedness and the ability to mitigate the various possible ramifications of an outbreak, when it does occur. this backdrop of uncertainty creates at least three possibilities: over-preparedness, ample preparedness, and under-preparedness. assuming the level of risks remains constant, over-preparing for a pandemic will undoubtedly involve the committing and expenditure of more human and material resources to an outbreak. this will create a sense of waste (to decision and policy makers) after the incident and may affect the resources that will be committed to future outbreaks. the right amount of preparation will help curtail an outbreak while under-preparedness will barely help curtail an outbreak. however, if the level of risk increases, over-preparing may help curtail a pandemic whereas what was hitherto ample preparedness as well as what was hitherto not enough will enable the full range of the effects of a pandemic outbreak to be felt. " ibid.5, no. 5 (1999) . p. 669. 106 kuby. p. 392. 107 murray et al. pp. 221-2215. in other words, the changing nature of the virus demands a constant readjustment of the level of preparedness without a reliable frame of reference with the attendant possibility of some inevitable social harm. not surprisingly, scholars like peter doshi argue that there is a need for evidence-based ways to address hypothetical scenarios of non-zero probability such as the notion that novel influenza pathogens acquire increased virulence during successive "waves" of infection. 108 the scientific uncertainty associated with health disasters such as pandemic influenza may, however, tempt government officials to attempt some form of a cover-up, hence, raising trust issues. for instance, during the 1911 cholera outbreak in naples, italian officials paid newspapers and reporters not to report the outbreak. chinese officials tried to keep the 2003 sars outbreak a secret. saudi officials, likewise, tried to silence the virologist who discovered the coronavirus in 2012 and ultimately forced him to resign from his position. 109 incidents like these have the tendency to dissuade social cooperation during public health emergencies like influenza and have the potential to weaken the overall success of public health interventions. on the other hand, there is a lot of uncertainty surrounding the therapeutic and non-therapeutic approaches adopted vis-à-vis pandemic influenza. it is uncertain, for example, if neuraminidase antiviral drugs really cut down mortality when implemented as the first line of defense. 110 this may create some sense of hesitation in relation to using them. secondly, it is uncertain who and who will not develop some of the associated side-effects. these factors, at a pragmatic level and for less rich nations, may dis-incentivize prioritization of funds for antiviral drugs. uncertainty likewise plays out in the context of influenza vaccines. for instance, only a small amount of any vaccine can be stockpiled 111 because the scientific and public health community can hardly be sure of the efficacy of any given vaccine prior to an outbreak. this is due to possible vaccine failure which will make a new outbreak not amenable to the biological effects of hitherto effective vaccines. hence, vaccines are generally not produced until the new virus strain causing a pandemic is isolated. 112 also, there is uncertainty over who will be at highest risk of infection and complications. 113 this creates a dilemma of some sorts with the potential that a class of the people who need vaccines may not get enough, while another class of people who will benefit less from vaccination gets too much. another kind of uncertainty is linked with possible side-effects of vaccines. while some incidence of narcolepsy was reported in children after the use of aso3-adjuvanted h1n1 influenza vaccine in scandinavian countries, 114 and there have been anecdotal reports of fetal deaths 108 doshi. p. 535. 109 shah. pp. 108-111. 110 hhs, "hhs pandemic influenza plan." p. s5-6. 112 kotalik. p. 427. 113 emanuel and wertheimer. p. 854. 114 dauvilliers et al. pp. 2486 -2490 occurring shortly after the 2009 vaccination 115 ; it is not clear if these safety issues are one-off events or may recur for other pandemic vaccines. responding to influenza vaccine safety signals during a pandemic constitutes a scientific and public health policy issue since decision-makers must balance the immediate consequences of disease against uncertain risks. 116 one of the consequences of the therapeutic uncertainties associated with pandemic influenza is the validity of administering potentially ineffective antiviral drugs with side-effects or vaccines that may cause harm to people. another is the validity of withholding such drugs and vaccines because it may not be useful for some class of people, or because some people may experience certain degrees of side-effects. these issues raise concerns about human rights and whether or not they may be violated through these courses of actions, or by any other course of action associated with handling a pandemic influenza outbreak. the 1948 universal declaration of human rights and the 1966 international covenant on economic, social and cultural rights documents enunciate the rights of "everyone to the enjoyment of the highest attainable standard of physical and mental health". 117 hence, it is perhaps more than ever taken for granted that there are rights-related obligations that society, as well as healthcare providers, owe patients 118 as well as those that may potentially fall sick. since everybody is theoretically a potential victim of ill-health depending on time, placek and social or physiological circumstances, individuals can appeal to a rights-based rhetoric to garner positive action from government and healthcare professionals in relation their health. the morality of such a claim stems partly from governments' moral obligation to their citizens and partly from the fiduciary obligations that health professionals have towards fostering the health of patients (and potential patients) in a fashion that preserves their rights as human beings. many moral concerns related to human rights come to the fore in the context of pandemic influenza outbreaks. the first is related to the limited number of vaccines that can be available for each outbreak (due to reasons outlined in the preceding section) and the best sharing formula to use. whatever adopted formula in a given place or situation, some people who may benefit could be excluded. for instance, 115 pandemic influenza often generates a high number of sick people over a large geographic area who will need care at the same time. while this "need" begins at the local plane, it may evolve to be regional and/or global depending on the extent and severity of an outbreak. hence, the human and material resources of healthcare will be rapidly depleted and overwhelmed. 119 since the needs of everyone cannot be met under such a scenario, there is usually some need to ration available resources. in fact, vaccines are hardly enough during pandemics, and rationing is generally considered as the ethical option. 120 yet, the contemporary interconnection between health, the right to health and human rights 121 implies that withholding vaccines from some people who might be potential victims of a pandemic outbreak may be a human rights violation. on the other hand, administering antiviral drugs to non-vaccinated at-risk people helps reduce the severity of illness. 122 during disaster scenarios, the goal remains saving lives but a pandemic scenario in which 25-50% of the population can fall sick within a very short time 123 often demands some type of prioritization of resources. this is partly because keeping some sets of people alive, especially health workers will ultimately help society keep more people alive during a public health disaster. for instance, the traditional view is that prioritizing the vaccination of front-line healthcare workers can help reduce staff absenteeism as well as help prevent them from becoming vectors of viral infection. this is often justified by the logic that a phd situation such as pandemic influenza often makes health professionals work outside their normal scope of practice, put in extra hours, cover for ill workers, accept great risks 124 as well as incur other situational unexpected responsibilities and supererogatory duties. although adults aged 65 years or older, pregnant women, and people of any age with underlying medical conditions are at high risk of pandemic influenza and its associated complications, the notion that death is more tragic in children and young adults as opposed to elderly persons, perhaps, because younger persons have not had the chance to live and develop through all stages of life and accomplish their dreams has made some ethicists argue for the prioritization of vaccines to younger people. 125 yet, if persons are inherently born with human rights and do not have to earn rights, such an idea tends to revamp the rights to health of some class of people at the expense of others. indeed, notions such as this echo the idea that mainstream bioethical issues tend to be far-flung from the values of ordinary people and often 119 irrelevant to the decisions they experience in their encounter with healthcare. 126 in other words, an empirical approach which takes into consideration what people would want when faced with this thorny dilemma rather than an armchair speculation ought to influence the criteria for rationing vaccines. one of the non-therapeutic responses to pandemic influenza is the isolation and quarantine of infected patients. 127 whereas a visibly infected and sick person may have just a little objection to quarantine (after all, such a state mirrors the ambulatory limitations that most disease states naturally impose on people), it is often problematic for other categories of people. in this vein, isolation and quarantine raise concerns about the acceptability of confining people and preventing them from engaging in some of the social activities they otherwise would have loved. whereas restriction of movement is ethically problematic, 128 it is equally problematic to allow person a who may be infectious to roam free, thereby potentially infecting other persons who may also (without the imposition of some restriction) further spread infection. it is clear from the foregoing that pandemic influenza challenges and raises some moral concerns regarding the rights of people, 129 preempting the need to balance them against what is the optimal good of the society. but embedded in these reservations is the demand for autonomous living, broadly conceived. whereas this has been associated with western contexts, concerns about rights violations in relation to quarantine measures are not confined to the west. sambala and manderson recently commented about how ghanaians and malawians perceive public health interventions including quarantine as being intrusive. 130 but this perception seems to run contrary to the cultural norm of most african people. in relation to this strand of thought, shah notes that during epidemics, the traditional attitude of the acholi people of uganda involves working together to isolate the sick, mark homes of the sick with long elephant grass, warn outsiders not to visit affected villages, and refraining from potentially infection-transmitting practices including sexual intercourse. 131 this suggests at least two things. one, in traditional african societies there may be some fairly general consensus about the need to adopt mutual and social cooperation for the overall benefits of the society in engaging collective threats. secondly, it shows how the global village has increasingly penetrated and fragmented societies that were once non-individualized in orientation. but it seems that societies have been affected differently by the globalizing current of individualistic logic. for instance, macphail whereas europeans and americans generally view quarantine during influenza as almost worthless, asians such as hong kongers, expect it as the norm during health disasters, and demand it. 132 this probably shows how strong an influence the communal-oriented confucian idea still exerts in that country. in the context of pandemic influenza outbreaks, over-emphasizing individualism and the attendant call for autonomy (even when such does not cohere with social interests) overlooks communal values and the relational nature of social interactions. 133 it likewise ignores the complex nature of pandemic influenza and how it plays out in an equally complex web of this global age and how people more or less are susceptible to the harms of public health disasters regardless of their proximity. it has also contributed, as lachman argues, to a reduction in the fear of infectious diseases by increasing the emphasis on patients' rights, giving rise to a dangerous complacency that may do great damage to the goals of public health. 134 one of the ways to address the attendant dangers inherent in this almost pervasive trend is recognizing the vulnerabilities even to far-flung harm that is fast becoming an integral aspect of contemporary life. vulnerability-in different forms and facets-plays out in pandemic influenza, as in other public health disasters. traditionally, belonging to the human community or occupying specific facets of life constitutes sources of vulnerability. but the state of being susceptible to harm by the actions and activities of other people or by parts of nature such as viral organisms is also a potential source. in addition, the state of vulnerability may ensue from a range of social, economic, and political conditions. 135 in the context of pandemic influenza, the naturalistic, socioeconomic, epistemic, political, and biological dimensions of vulnerability arise. on the one hand, humans located in pandemic-prone cities or countries and other human beings linked to the global community by technological means of transportation (such as air travel) or non-technological ones (such as migrating birds) are generally vulnerable to influenza outbreaks. the likelihood of a novel strain of influenza outbreak occurring in a country such as china (for instance, jiangcun in guangzhou) where large numbers of people, birds, and swine mingle freely in certain markets is very high 136 ; hence, making the local population and consequently the people of such a nation more vulnerable. 132 macphail, the viral network: a pathography of the h1n1 influenza pandemic. pp. 95-95. 133 bennett and carney. p. 7. 134 peter j lachmann, "public health and bioethics," the journal of medicine and philosophy 23, no. 3 (1998) . p. 298. 135 henk ten have, "vulnerability as the antidote to neoliberalism in bioethics," revista redbioética/unesco 1, no. 9 (2014). p. 88. 136 on the other hand, the strength of health systems reflected by availability of experts, economic and technical resources will vary the extent of pandemic-related vulnerability which different societies will experience. in addition, it is widely believed within the scientific community that influenza pandemics can hardly be halted, but they can be delayed. 137 therefore, the "ignorance gap" that occurs during pandemic influenza outbreaks creates a context in which some of the preparatory strategies will inevitably fail (due to no fault of anyone), thereby leaving some people less protected. in relation to the socioeconomic dynamics, it is estimated that most influenza pandemic-associated deaths occur in poor countries or in societies with scarce health resources which are already stretched by extant health priorities and challenges. 138 farmer and campos underscore the need for bioethics to engage the growing problem posed by the gap between rich and poor nations, and how such a course of action reflects social justice. 139 politically, communist nations such as china present unique dimensions to the vulnerabilities of pandemic flu as they may control critical information traffic and access to patients, thereby deepening the crisis situation, 140 or misrepresenting it, and thereby subjecting the rest of the connected world to avoidable risks. the biological make-up of human beings both make them vulnerable to becoming infected with influenza virus as well as make them good vectors of dissemination. for instance, the virus has a surface molecule that enables it to attach firmly to cells in the mucous membranes of the respiratory tract, preventing it from being swept out by the ciliated epithelial cells. 141 but breathing is a normal aspect of human existence, and the oxygenation of the human blood and other oxygendependent biochemical processes of the human body rely on it. yet, the combination of these factors facilitates the ready transfer and exchange of the influenza viruses amongst people, especially when they are in close proximity. 142 the foregoing shows how susceptibility and vulnerability to infection during pandemic influenza reflect a combination of factors. 143 how these combine in specific localities and regions will, therefore, determine the extent of an outbreak. it is also clear that some amount of control can be exerted on minimizing some of these factors. for instance, the use of face mask (to limit infection acquisition and spread), transparency (to combat political bottlenecks), and monetary aid (to help poor nations) will exert some preventive effects on infection transmission, hence, limiting the overall burdens and severity of an outbreak. since everyone may not receive the same level of healthcare for various reasons during a public health disaster (depending on time, place, and category of persons such as adults, the aged, or children), questions about justice and what is just in the context of a pandemic outbreak arise. pandemic outbreaks exacerbate extant inequalities to the extent that certain groups of people face disproportionate risks and impacts of disease. 144 this obviously seems unfair, especially if pre-pandemic actions that would have ameliorated the situation were not done. for instance, school closure in certain districts may interrupt educational opportunities or growth of some children, and business closures will lead to financial losses. since such restrictions may not apply to every region of the nation, these measures may seem unfair to those affected, knowing that other children continue to have access to education, and other people continue to run their businesses. if this characterizes the feelings of some of the people affected by these restrictions, then it is reasonable that some form of compensation may be required to foster optimal compliance to the public health measures that are to implemented. indeed, bioethicists like michael selgelid and søren holm make explicit arguments for some form of compensation to people who suffer financial and other losses due to compliance with public health directives issued during influenza outbreaks. 145 although compensation may not be a problem in more affluent nations where other educational stimulus and business tax breaks may help alleviate any temporary pandemic-associated losses, poorer countries will find it hard to compensate people for any such losses. rationing also raises issues about justice in terms of how vaccines (if available) will be shared during an influenza pandemic. given the limited amount of supply available globally, and locally in a developed economy like the us, distributing the limited supply will require determining priority groups. 146 for people not to feel a sense of being left out during local vaccine administration, it is better to have debated and developed a preparedness plan with the consensus of the local populace. resolving vaccine distribution on a global scale will, however, involve very complex sets of factors. for instance, will countries who supply most of the technical and financial resources to develop such an influenza vaccine demand that the needs of her people be prioritized as opposed to the needs of nations that have contributed little or not at all? even if such a question were not explicitly raised, will it be fair to distribute vaccines equally if every country or affected region has not made significantly even contributions? these are unsettling questions that are bereft of simple answers. some ideas stand out when all the ethical issues generated by pandemic influenza are closely examined. four of these ideas demand attention. the first is the need to help people. secondly, the nexus of relationship that exists between people 144 henk ten have, vulnerability: challenging bioethics (routledge, 2016). pp. 70-71. 145 michael j selgelid, "promoting justice, trust, compliance, and health: the case for compensation," the american journal of bioethics 9, no. 11 (2009 146 emanuel and wertheimer. p. 854. and the influenza virus and the changing nature of what is known as well as what can be done to help people under such constraints will limit the help some people may ultimately get during an outbreak. thirdly, the threat of an outbreak presents different risks which vary by context, time, and place. lastly, regardless of the different situational dynamics that pandemic influenza presents locally, regionally, and globally; its threat will affect everyone to varying degrees. since nations theoretically care about their people, it is only reasonable that a people-centered approach offers a useful way to engage the moral quandaries elicited by pandemic influenza outbreaks. the subject matter of diseases is human populations. 147 in fact, the preoccupation of medicine remains the amelioration of the distress of people technically referred to as patients. if a people-centric approach constitutes a viable way of engaging the ethical issues embedded in pandemic influenza scenarios, one way to glean a sufficiently nuanced angle on such an approach will involve turning to ethical lenses that are, in principle, people-oriented. two principal examples of such ethical prisms are communitarianism and ethics of care. this section briefly explains each of these moral lenses, and how each may help engage the ethical issues generated by pandemic influenza. the communitarian moral lens adopts a people or community-centric perspective to moral issues. applied to public health, it offers a population-centered approach which best reflects the philosophy of public health in terms of its commitment to doing the most for the greatest number of people in a society or within a social context. bioethicists like stephen holland regard the communitarian lens as useful since it aims at realizing collective interests. this same idea offers a strong justificatory argument for adopting it in relation to public health interventions. 148 communitarianism pays attention to the social sphere, institutions, and interrelationships in relation to moral judgments that will inform public health policy and practice. its ethos provides an alternative to the dominant atomistic lens of individualism which operates via the logic of self-protection 149 and the unbridled 147 macphail, the viral network: a pathography of the h1n1 influenza pandemic. p. 196. 148 stephen holland, public health ethics (polity press, 2007) . pp. 51-55. 149 pursuance of self-interests. it holds that the social nature of life and institutional and social relationships should inform moral thinking, and by implication, the process of determining appropriate courses of actions should lie within the social space. 150 to be sure, the communitarian notion appeals to the historical traditions of communities or people who share customs, ideals, and values 151 ; and thus prioritizes common threads of thought and practices within specific communities as a strong moral basis for justifying decisions that pit different individual and social interests against one another. there is an important phenomenological aspect of communitarianism. for people raised within the traditional family structure-father, mother, children, and relatives-the family unit constitutes a micro-community which generally socializes the child into a community-oriented way of reasoning. while the strength of such an orientation is expressed in different measures by different individuals, it also provides the cognitive platform for balancing and pursuing personal interests in a feedback loop with the collective interests of other family members. yet, the ultimate measure of what level of community-oriented reasoning an individual retains in adult life will depend on their education, social experiences, whatever meanings they draw from these, and how these parameters are brought to bear in the context of specific decisions and choices. this reality partly explains the multiple versions and interpretations of communitarianism, which tends to mar its conceptual and theoretical coherence. 152 it also partly explains why community values are not generally shared by all. 153 communitarians advance three different types of claims: descriptive claims which stress the social nature of people; normative claims which celebrate the value of community and solidarity, and a meta-ethical claim which emphasizes the idea that political principles should mirror "shared understandings'. 154 two of these dynamics-the normative as well as the metaethical-are important in relation to engaging the ethical issues elicited by pandemic influenza. the significance of the meta-ethical dimension of communitarianism is its capacity to help drive and ground public health policies. this is especially so considering the reality that community and living together in today's fragmented and individualistic world is generally seen ever less as a necessity and assumes the dimensions of a choice as the default state. 155 hence, these two facets will be examined in relation to their possible 150 insights and pragmatic importance vis-à-vis engaging the quandaries associated with influenza outbreaks. healthcare focuses on helping sick people regain optimal health and healthy people maintain good health. pellegrino and thomasma remark that medicine seeks to foster social flourishing as well as the medical good of society. 156 if this is true, and if the end of the communitarian moral lens is to ensure the survival of the society by promoting the interests of people over the selfish interests of individuals, then how can this approach help engage issues of uncertainty, vulnerability, human rights and justice? this can come through appropriate educational policies and approaches carried out prior to and during influenza outbreaks. it is not known when and in whom influenza therapeutic interventions such as antiviral drugs and vaccines may cause side-effects. it is also not known when an outbreak will occur or the attendant magnitude. since public health disasters are classless in terms of who will and who may not be affected, the scenario of uncertainty affects every segment of people in the local communities and nation. hence, health workers, government officials, the rich, the poor, the educated and illiterates and other possible stratification of society are potential victims. a communitarian ethos is useful in at least two ways in relation to dealing with the uncertainties associated with pandemic influenza. generally, it can-with the right pre-disaster public education-help ensure that people understand the unavoidable scientific and knowledge-related gaps in preparedness policies and specific plans put together to engage a specific outbreak. this will help avoid or minimize blame, since scapegoating during disease outbreaks causes different shades of disruption and target important actors including health workers. 157 in fact, the better educated the public is about the challenges of stockpiling vaccines, the more cooperative they will likely be to the vaccine-supply challenges that arise during an outbreak. a communitarian ethos may also help engage the real and possible harms that may ensue due to the therapeutic uncertainties associated with pandemic influenza. these harms arise from the uncertain nature of what is knowable about a pandemic virus before it strikes as well as the biological limits of the therapeutic arsenals often produced within a very narrow time window. this is also generally tied to the reality that new health interventions including drugs and vaccines come with the possibility of some adverse events, which may be linked to the chemical/biological/physical components of the product, to genetic susceptibilities in certain individuals, or to 156 edmund d pellegrino; david c. thomasma, "the good of patients and the good of society: striking a moral balance," in public health policy and ethics, ed. michael boylan (springer, 2006) . pp. 17-25. 157 shah. p. 125. environmental triggers. 158 keeping the public aware of this fact before and during an outbreak as well as emphasizing that accepting these risks (though uncomfortable at the individual plane) will serve to ensure the society overcome a pandemic should help garner some level of support critical to ensure proper compliance. since people are born with inherent human rights and do not have to earn them, it is hard to justify trumping the rights of some for the sake of public health. this is especially so if the people whose rights may be inhibited or violated do not consent to the process. to avert this, a discursive approach involving inclusive deliberations is essential. in this vein, the communitarian lens can help foster dialogue as well as call for the need to reward people for the sacrifices they may or will bear on behalf of the community and the society. for instance, guaranteeing that some compensation will be paid for financial losses incurred through workplace closure as well as apt public education about the nature, purposes, and conditions of quarantine facilities will help convince people that such temporary rights-related inconveniences are for the benefits of the overall society. in relation to vulnerability and justice, the communitarian lens can help clarify the different kinds of social, biological, and natural vulnerabilities that face different people in different contexts. for example, it can offer a way of making the important distinction between general vulnerability that people will experience as human beings, vulnerability based on age, and occupational vulnerability seen in health professionals. based on these distinctions, it can help underscore how context-specific cooperation will help ensure the overall success of the countermeasures adopted to engage a given pandemic. critical to this, however, is the moral currency of trust. trust shapes how the public evaluates risks and benefits. it also influences the acceptance of prescribed public measures to mitigate present or perceived risks. 159 effective risk and crisis communication depend on public trust in the government during a pandemic. as such, a higher level of trust will influence a more positive level of social compliance. van der weerd and colleagues corroborated this in their empirical study of the 2009 pandemic in the netherlands. 160 in addition to trust, transparency in terms of how priorities will be made in terms of the allocation of vaccines as well as antiviral agents, and decisions pertaining to school and/or workplace closures is important. even in western climes, public health experts have sometimes pointed out the paucity of transparency in ethical reasoning and the scanty explicit ethical justification for pandemic-related policies. 161 obviously, an atmosphere of trust and transparency will be conducive to 158 discussing and addressing issues related to local justice. this is especially relevant in relation to less wealthy nations or countries with weak institutions. for instance, it will be hard to garner cooperation in hitherto abandoned communities by appealing to communitarian ethos without addressing extant disparities in the social fabric as well as the healthcare system. if human beings are located in particular communities but are willy-nilly part of a global community, 162 how well the vulnerability and justice-related issues are locally addressed will influence the extent of their regional and global dynamics. this echoes the notion that badly managed local issues associated with pandemic influenza will pose more challenges and burdens at the regional and global levels. since every nation lacks an equal capacity to deal with the local burdens of pandemic influenza, it is necessary for wealthier nations to rally around poorer ones. indeed, the transcontinental nature of health disasters including pandemic influenza and sars underscores the urgent need to strengthen how the global community deals with emerging infectious diseases, and how novel visions of global solidarity and cooperation will be key in such an endeavor. 163 this constitutes a preventive stance and falls well within the traditional agenda of public health. this approach is also a reasonable economic and health security choice as it will statistically cut down the possibility of global and transnational infection dissemination. while the communitarian ethos as argued above offers some insights into how to flexibly engage the moral dilemmas generated by influenza outbreaks, its application in non-community-oriented contexts potentially raises some difficulty at the institutional and individual planes. such possible difficulties, however, call for a global but locally nuanced moral framework. that theme, however, will be addressed in chap. 6. for now, the rest of this chapter will explore another people-centric moral lens, care ethics, in relation to resolving the quandaries of pandemic influenza. in addition to the communitarian lens, the ethics of care perspective (eoc) constitutes a people-centric method of attempting to resolve ethical issues. whereas it sometimes arrives at the same conclusions reached by traditional bioethical approaches, 164 employing it as a complimentary approach to the moral quandaries generated by pandemic influenza should yield additional nuances and insights visà-vis resolving the associated moral concerns. care ethics emphasizes varying degrees of care within relational contexts ranging from the personal sphere to the realm of moral strangers. hence, it is an other and people-centric moral lens. it has 162 henk ten have, global bioethics: an introduction (routledge, 2016). p. 113. 163 peter a singer et al., "ethics and sars: lessons from toronto," british medical journal 327, no. 7427 (2003) . pp. 1342-1343. 164 edwards, "is there a distinctive care ethics?" p. 185. been applied to diverse relational contexts including everyday lives, professional practices, social and public policies, as well as international relations. 165 for scholars like steven edwards, ethics of care uses a distinct ontological commitment to realize its outcomes as well as justify its stance. 166 it is an attempt to re-conceptualize and renegotiate the moral landscape in order to give room for a plurality of values. 167 some have argued that the removal of friendship with its altruistic emotional sequelae and the subversion of virtue ethics from the sphere of morality were some key factors that warranted the moral change which birthed the ethics of care framework. 168 while eoc is also linked with gender-based morality which undergirded campaigns for equal employment opportunities between the sexes, legal rights, reforms of family life and sexual standards, and better education 169 ; scholars like noddings have pointed out that it is broader and deeper than feminist ethics. 170 to be sure, one of its major impetus is the call for the expression of higher capabilities. 171 care ethics also encapsulates a spectrum of ideas. for kittay, care constitutes an "achievement term" such that caring occurs only when specific acts of care have been carried out. 172 in this vein, intentionality would not qualify as part of the baggage of care rhetoric. this obviously has some pragmatic appeal. most people, for instance, would only appreciate care if it helps contribute towards relieving their current distress. yet, caring may also constitute a general attitude and an orientation which may provide appropriate background conditions for shaping responses to others' needs and states of distresses. also, one may care but situational constraints may limit how a caring impulse may translate into pragmatic ends. therefore, that someone simply "lacked opportunity" to show care as apostle paul writes in his epistle to the philippians does not necessarily indicate the absence of care. 173 hence, caring cannot be reduced only to materialistic terms. one way to distinguish the general caring orientation from specific acts of care is to refer to each as "caring about" and "caring for" respectively. 174 care ethics locates morality within the ambiance of family, friends, and colleagues, and ultimately towards the public sphere. 175 it rejects the independent and atomistic notion of the self and champions an inter-dependent and inter-related view. 176 this approach grants eoc a psychological gestalt to which people brought up in caring relationships, at least in the early phases of their lives, can readily identify with. it thus partly appeals to kohlberg's theory of moral development. here, the emphasis is put on the foundational roles of trust and its place in fostering a deepened sense of reciprocity within a social context of inequality. 177 not surprisingly, some ethicists describe caring as the primary virtue which offers a general account of right versus wrong actions as well as political justice. 178 whereas the informal social contract idea underlies inter-personal and stateindividual relationships, 179 the care ethical lens may be applied to the personal sphere as well as social institutions 180 due to its multiple ways of situating relationality. 181 indeed, eoc focuses on attentiveness and sensitivity to the needs of others 182 and offers a moral compass for teasing out delicate boundaries between obligation-based ethics and responsibility-based ethics. as such, it seeks to transcend the depersonalized realm of asking "what obligations do i have to mr. x" to the humane realm of asking "how can i help mr. x" in scenarios of moral crises. 183 since caring embodies an activity, a set of activities or a labor of care from one person to the other, it presupposes that the capacity for receiving care 184 will be present in the recipient(s) of care. public health disasters including pandemic influenza with their myriad of ethical and pragmatic challenges create a spectrum of needs and contextual dependencies which some people will have to meet, directly and indirectly. it thus creates different types of carer versus cared-for relationships between and amongst victims, atrisk people, health workers, and government officials. since it is a foundational nexus like these that underlie the caring ethic, it will be insightful to examine how the ethics of care moral lens may help resolve the moral dilemmas elicited during pandemic influenza outbreaks. 175 osuji. p. 58. 176 whereas tirima recently argued that ethics of care is irrelevant to addressing the moral imperatives in disaster scenarios because it only builds off on relationships and, therefore, requires some proximity between the caring moral agent and the cared-for victim, 185 such a stance is flawed for at least three important reasons. firstly, care ethics can, through relevant public policy, 186 positively influence how victims of disasters are cared for. secondly, contexts of duty exist between some of the players and victims of disasters which form the basis of a relationship of caring. for instance, healthcare professionals incur fiduciary duties to at-risk people, victims of a public health disaster as well as the general populace that may potentially be infected and infect others. thirdly, if the care ethical prism emphasizes how individuals may offer help "in scenarios of moral crises, 187 then it should be relevant in health scenarios where different kinds of conflicting moral emergencies occur. the application of care ethics to specific disaster contexts such as influenza outbreaks, however, requires elaboration. specifically, this needs some explication with reference to issues of uncertainty, vulnerability, human rights and justice. whereas the dilemma of uncertainty that arises during pandemic influenza affects everyone, it will affect different sets of people differently. for instance, the biological uncertainties associated with an influenza outbreak are not known to the same extent by public health experts, health workers, the literate, and illiterate members of the society. caring about the potential practical consequences that may result from the attendant "ignorance" gap should, therefore, involve sharing as much useful information as possible between and amongst the different rungs of people. the relational context, in this regard, may be situated and realized through professional associations, institutional contexts, public announcements through media outlets and patienthealth professional interactions. kunin et al. recently reported on how primary care physicians helped pass on important pandemic-related information to out-patients during the 2009 pandemic in israel. this, they concluded, helped enhance the success of the national pre-pandemic preparedness plans. 188 indeed, during public health disasters, the speed at which information is needed by policymakers may be faster than is usually possible through traditional mechanisms of research dissemination. this scenario makes information sharing a norm; even possibly those provided by preliminary research findings. 189 humans instinctively show care to other humans in need. while this caring instinct has been socially modified and conditioned in some parts of the world where individualistic tendencies run rife, some communal-oriented cultures give room for a freer expression of the instinct of care. the instinct of care may, however, be counterproductive in the context of phds. for instance, during pandemic influenza, sick and dying patients remain active carriers of infection, 190 as such, will infect susceptible friends and relations who feel obligated to show care in relation to helping them. in other words, "unbridled" caring may increase the vulnerabilities elicited during pandemic influenza. yet, the care ethics moral lens may help modify and re-direct the caring impulse in a more socially useful way during a pandemic. the other-centric nature of the eoc lens implies that people should care not only about themselves but about others, perhaps, even moral strangers. how person a will care during a public health disaster will, however, differ from how b will choose to act in a manner that reflects care, depending on their levels of knowledge, resources available to them as well as their social and spatial location. in other words, how a healthcare worker will care professionally in the hospital context and supererogatorily in the non-hospital context will differ from how a lay member of the society can show care in a pandemic situation. however, appealing to the eoc may help facilitate the selflessness needed. if someone cares that their society survives an influenza outbreak, then they should be willing to play roles that will help bring about that goal. this will facilitate compliance with therapeutic measures such as vaccines and antiviral drugs as well as non-pharmaceutical measures such as contact tracing, quarantine, and workplace closure. collective adherence to these measures will help cut down the susceptibility and vulnerability of individuals, groups of people, and the society to the impact of influenza outbreaks. by enabling the willingness of people to subject themselves to the public health restrictions required to contain pandemic influenza and accept the potential risks and side-effects associated with vaccines and antiviral agents, the eoc approach may indirectly eliminate or downplay the human rights-related quandaries engendered by pandemic influenza. noddings has argued that attentiveness and responsiveness are exigent to rights, flowing from one person to the other. 191 if this is true, then the eoc may help individuals adjust the emphasis they place on articulating their rights contextually during an influenza pandemic for the sake of the collective good. finally, an appeal to the care ethical lens may help address the moral quandaries associated with local justice. although some versions of care ethics hold the posi-189 ns crowcroft, lc rosella, and bn pakes, "the ethics of sharing preliminary research findings during public health emergencies: a case study from the 2009 influenza pandemic," eurosurveillance 19, no. 24 (2014). pp. 1-3. 190 shah. p. 87. 191 noddings. p. 72. tion that it is not possible to integrate and apply justice to care, 192 such a limitation hardly applies to the context of a public health disaster such as pandemic influenza. for instance, the different conflicting priorities that arise during influenza outbreaks such as rationing of limited resources will be easier if some people are at least willing to forgo their interests for others. in non-familial carer and cared-for relationships involving at-risk government representatives and at-risk members of the society and familial relationships involving parents and children living in the same house, an appeal to a care ethical lens may help drive the moral sensitivity to the needs of others, enabling some vaccine-eligible persons (under the standard rationing criteria) to forgo their ration, preferring rather that other at-risk people (for example, ordinary people and younger family members) have them. this kind of selflessness approximates some form of humanitarian act in that person a decides to overlook their interests for others "without expecting rewards". 193 however, because human beings naturally seek their own personal interests, there may be some difficulty in achieving this other-centric goal in as many people as possible in a public health disaster situation. this implies that the care ethical lens may have some limitations in relation to sufficiently engaging the ethical dilemmas raised by pandemic influenza in particular and other types of public health disasters, in general. that theme will, however, be addressed in chap. 6. during disasters, there is the utilitarian goal of doing the most good for as many people as possible with minimal harm. 194 a people-oriented moral lens, this chapter argues, may be apt in accomplishing such an agenda. the chapter explored the strengths of the communitarian and care ethics moral lenses in relation to engaging the moral quandaries elicited during pandemic influenza outbreaks. because it is difficult to engage pandemic outbreaks with little prior preparation, 195 these moral lenses become important since they can help people develop an other-centric orientation and sensitivity to the needs of others. to systematically drive the importance of a people-centered approach to pandemic influenza, this chapter explicated the biological make-up of the influenza virus as well as the social and global features of the associated pandemic. this helped underscore the local, regional, and global seriousness of pandemic influenza as a distinct type of public health disaster. the chapter went on to show how an 192 barnes et al. p. 5. 193 195 vawter, gervais, and garrett. p. 6535. understanding of the social and biological dynamics of influenza has shaped the therapeutic and non-therapeutic approaches to engaging outbreaks. it also articulated some of the attendant limitations of pandemic influenza countermeasures including vaccines and anti-viral drugs. this chapter has also highlighted the ethical quandaries generated by influenza outbreaks. these are issues related to epistemic and social uncertainty, biological, social, geographical and political vulnerabilities, potential violations of human rights through some of the therapeutic and non-therapeutic countermeasures, as well as issues of local and global justice. against this conceptual background, the chapter pointed out how helping people is a central concern in pandemic influenza, and how the thorny ethical issues constitute difficulties encountered in accomplishing this goal. on that note, it showed how people-centered lenses such as communitarianism and ethics of care may be useful in engaging the associated practical and moral challenges. to clarify the importance of each of these approaches, the chapter elaborated each of these ethical lenses, and showed how each may help orient different players in the context of a pandemic influenza towards acquiring a sense of community and an other-centric sensitivity which will be essential to resolving the moral dilemmas as well as realizing the critical public health objective central to such a public health disaster. however, partly because there are limited grounds for deciding what the limits of practical reasoning will be ab initio, 196 and partly because of the complexities and nuances that are associated with the global dimensions of the issues at stake in pandemic influenza situations, these ethical lenses may suffer some limitations. whereas this chapter has examined none of such limits, they will be engaged in chap. 6 where the relational-ased global ethical framework will be formulated. biological features of novel avian 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influenza in africa public health ethics: an update on an emerging field biological features of novel avian influenza a (h7n9) virus key: cord-018213-w6sh9f3h authors: xue, lan; zeng, guang title: china’s institutional mechanisms for influenza a (h1n1) prevention and control date: 2018-11-24 journal: a comprehensive evaluation on emergency response in china doi: 10.1007/978-981-13-0644-0_4 sha: doc_id: 18213 cord_uid: w6sh9f3h innovation in institutional mechanisms is a fundamental issue in effectively dealing with public health emergencies. in the wake of the 2003 sars epidemic, china initially established a public health emergency management system and an emergency organization and management network, placing emphasis on “government leading, unified command, local management, responsibility on all levels, management by classifications, and inter-departmental coordination,” which strengthened the existing health emergency preparation system. and insufficient preparedness. china's fight against sars, as it were, not only posed a great challenge to the nation's socialist modernization, but at the same time offered an important opportunity for the country to improve emergency management, especially in the field of public health. in the wake of the sars epidemic, the chinese government began pushing for a national emergency management system in a systematic, planned and gradual manner, and made remarkable progress in emergency management structured on the "one plan three systems (contingency plans, institutions, mechanisms and legislation)." in regards to contingency planning, the country formed a system consisting of contingency plans at central, local, departmental, and enterprise levels as well as plans for major events, and this system played an important role in dealing with public emergencies. 1 in regards to institution building, a national emergency management system consisting of general emergency management offices as well as of special emergency management bodies were established. the emergency management office of the state council and emergency management bodies of provincial (regional and municipal) governments were set up in succession, 2 in addition to emergency management systems in specific fields such as health. in comparison with the pre-sars environment where "departments played a dominant role, and coordination was inadequate," these new institutions showed the permanent, comprehensive, and specialized nature of emergency management, 3 and laid an organizational foundation for the future. in addition, society as a whole began getting involved with the emergency management process, including: further strengthening military emergency system construction and local assistance 4 ; giving full rein to experts in 1 in march 2005, the third session of the 10th national people's congress (npc) reviewed and adopted the report on the work of the government, which stated that "… we have formulated the national contingency plan for public health emergencies, as well as 105 special-purpose and department-specific contingency plans concerning natural disasters, accidents, public health, social security, etc.; provinces (regions and cities) also have completed their own work on the development of overall contingency plans. breakthroughs have been made in building a government by the rule of law and in fully fulfilling government functions." see: report on the work of the government, delivered by wen jiabao at the third session of the 10th national people's congress on march 5-15, 2005. emergency management 5 ; and developing and implementing local emergency management plans that targeted "communities, rural areas, enterprises and schools." looking at mechanism construction, progress was also made in research on a science-based emergency management system, and an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation"-which enabled the interconnection of early warning, mass mobilization, quick response, and emergency handling-was gradually established to effectively mitigated public health emergencies. in regards to legislation, the emergency response law was took effect on november 1st, 2007. health emergency system long term. efforts would be made also to strengthen the disease control and prevention system, increase public health emergency management capabilities, boost rural health development, improve healthcare for the rural population, strengthen environmental health system, and implement national health campaigns. in february 2007, the moh outline the following overall goals for public health emergency work in the 11th five-year plan: establish and improve health emergency management legislation and the health emergency contingency planning system; build an emergency management mechanism characterized by "unified leadership, responsiveness, orderly coordination, and efficient operation" with "predominantly local management, hierarchical responsibility, and comprehensive coordination;" bolster health emergency management recruitment; improve the public health emergency monitoring and warning system; strengthen capacity for quick and effective response to health emergencies; and shape an environment of health emergency management characterized by inter-departmental coordination, collaboration, and social participation under the leadership of central and local governments. during and after the 2003 sars epidemic, china continued to establish and improve public health emergency legislation, regulations and contingency plans, and initially formed a national system for public health emergencies contingency planning (see fig. 4 the moh established a public health emergency coordination mechanism with thirty one central and national departments to deal with inter-departmental collaboration, which effectively strengthened communication and coordination between departments dealing with public health emergencies. the national government and the special administrative regions of hong kong and macau entered into a three-party emergency response collaboration agreement and decided upon implementation regulations, and established a linkage mechanism for information communication and health emergency response. additionally, the moh established a joint prevention and control mechanism with the ministry of agriculture (moa) to protect against highly pathogenic zoonotic viruses such as avian influenza and streptococcus suis; the moh established a coordination mechanism for joint prevention and control of public health emergencies at ports with the general administration of quality supervision, inspection and quarantine (aqsiq). in collaboration with the ministry of railway (mor), the ministry of transport (mot), and the aqsiq, the moh issued notices on the prevention and control of the importation of infectious diseases from abroad; and together with the moe, issued a document requiring schools to appoint part-time or full-time teachers to identify and report infectious diseases or other health emergencies at the school. this time marked the initial formation of a working inter-departmental mechanism positioned to combat health emergencies through "paying equal attention to both prevention and response, and instilling continued collaboration for any event." on april 26th, health minister chen zhu convened a meeting of the moh leading group and expert panel for influenza pandemic prevention and control, at which the attendees analyzed swine influenza situations in the united states and mexico, predicted epidemic trends, and deliberated on domestic strategies and measures to cope with a swine flu pandemic. health minister chen also held an inter-departmental meeting with the moa, the aqsiq, and other ministries to analyze epidemic trends and discuss response strategies and measures. immediately after the meeting was over, the moh reported in writing that very night to the state council on the progress of epidemic prevention and control work. on april 27th, following the emergency meeting held in geneva, the who elevated the pandemic alert level from phase 3 to phase 4, stating that the "swine flu" was widespread and was being transmitted by humans in different ways. general secretary hu jintao issued instructions to place prevention and control against this virus as the nation's top priority. on the same day, vice premier li keqiang convened the state council meeting regarding the human-swine influenza prevention working mechanism, resulting in the decision to establish a multi-departmental working mechanism for joint prevention and control of the human-swine influenza. as required by the state council meeting, the moh called together the publicity department of the communist party of china (ccppd), the ministry of foreign affairs (mfa), the ndrc, the miit, the mof, the ministry of transport (mot), the moa, the moc, the aqsiq, the china national tourism administration (cnta), the civil aviation administration of china (caac) among other departments on that very night for a meeting to deliberate on influenza a (h1n1) prevention and control; the meeting established the multi-departmental working mechanism for joint prevention and control of human-swine influenza and the notice on strengthening human-swine influenza prevention and control was drafted and published on the night of april 27th after state council review. the moh issued the notice of the moh general office on strengthening preparedness for and response to human swine influenza. on april 28th, premier wen jiabao convened a state council executive meeting which deliberated on how to strengthen national response to human-swine influenza; at the meeting they defined the overall prevention and control principles and strategies of "taking threats to public health seriously, responding actively, and coping with the epidemic in a scientific manner according to law through joint prevention and control efforts." on april 29th, the who raised its pandemic alert level from phase 4 to phase 5. on april 30th, at a press conference held at the state council information office, the moh declared the establishment of a multi-departmental working mechanism for joint prevention and control against the human-swine influenza, which would be spearheaded by the moh. under this mechanism, 33 departments and institutions (which later increased to 38) constituted 8 work groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert committee, forming a "8 + 1" pattern for joint prevention and control efforts. on the afternoon of may 1st, the joint prevention and control mechanism held its second joint conference, renaming the human-swine influenza which was occurring in mexico and the united states to "influenza a (h1n1)." the former wording of "multi-departmental work mechanism for joint prevention and control of human-swine influenza" was changed to the "joint national influenza a (h1n1) prevention and control mechanism" and roles and responsibilities were outlined for the mechanism, work groups, and the expert committee. at the same time, a meeting system for all members and liaisons was established. problems in principle which a work group encountered would be solved by the work group itself, and those which the work group struggled with would be settled through coordination under the joint prevention and control mechanism-general affairs would be solved by regular liaison meetings, and major issues decided by plenary meetings. health minister chen zhu and moh party group secretary and vice health minister zhang mao chaired the joint national influenza a (h1n1) prevention and control mechanism, and the mechanism was comprised of eight working groups-general office, ports, healthcare, support, dissemination and communication, foreign collaboration, science and technology, and animal husbandry and veterinary-and an expert advisory committee. the heads of the work groups, the leaders of the health department of the people's liberation army general logistics department (gld), the logistics department of the chinese people's armed police force (papf), and the chairman of the expert advisory committee, served as members of the joint prevention and control mechanism. the work groups, the gld health department and the papf logistics department each designated one or two departmental-level officials as liaisons for routine communication purposes. the main duties of the joint national influenza a (h1n1) prevention and control mechanism included: meet regularly to evaluate epidemic trends and determine prevention and control strategies; formulate prevention and control policies, response plans and major measures; coordinate and provide guidance on the implementation by all related departments and in various regions of prevention and control measures; and organize supervision and inspection activities concerning the implementation of prevention and control measures. see table 4 .1 for the main responsibilities of the work groups and the expert advisory committee. thirty three meetings were convened under the joint national prevention and control mechanism, in which regulations were formulated, signed, and issued for local implementation. implementation issues would be reported in real time to related state council departments for instructions. the establishment of the joint national prevention and control mechanism played a crucial role in the scientific and orderly response to influenza a (h1n1), in that (1) the mechanism raised the priority level for influenza a (h1n1) prevention and control for related departments and local governments, (2) clarified and divided responsibilities, (3) addressed investment issues, and (4) enhanced interdepartmental cooperation. a consultation system and a liaison meeting system were established under the joint national prevention and control mechanism, which were designed to ensure effective implementation of prevention and control measures. specific issues in prevention and control work would be resolved through consultation at liaisons meetings, and major issues decided by plenary meetings. each working group established a fixed meeting system where they could provide timely progress comprehensively coordinate routine affairs among departments of the joint prevention and control mechanism; organize regular meetings of the joint prevention and control mechanism and oversee the handling of top agendas; collect, sort out, and report to higher-ups about progress in prevention and control efforts; prepare progress reports on the joint prevention and control mechanism; publish information on epidemic situations and response efforts; ensure the consistency in style of writing for documents intended for outside use; and shoulder other assignments from leaders report in a timely fashion on influenza a (h1n1) situations and on progress made in major efforts under the joint prevention and control mechanism, and guide public opinion positively and correctly; arrange news releases on the joint prevention and control mechanism, and where necessary, organize press conferences; track public opinion at home and abroad, and clarify facts in a timely fashion; strengthen management and guidance on the release of (continued) 4.2 the establishment, composition and operations … reports, discuss and address problems, and push ahead with prevention and control work within their fields. the establishment and improvement of the coordination mechanism remarkably increased the efficiency of inter-departmental coordination and response efforts. this success evinces the importance of a multi-departmental coordination and collaboration mechanism based on risk communication for effective epidemic prevention and control. thirdly, horizontal collaboration between related departments was strengthened. for example, on april 29th, 2009, the moh office of health emergency sent a letter to the miit department of consumer goods industry recommending an increase in the national stockpile of supplies necessary for influenza a (h1n1) prevention and control including medical supplies and response gear. in another example, the office of health emergency, department of medical affairs, and bureau of disease control and prevention of the moh, the china cdc, and the chinese medical association, jointly formulated the technical guidance on prevention and control of human-swine influenza, and the plan for diagnosis and treatment of human-swine influenza (2009). one more example occurred on july 30th, when the foreign collaboration group for the joint prevention and control mechanism, along with the ndrc, the miit, and the sfda met to discuss donating influenza a (h1n1) prevention and control materials to the who and developing countries affected by the pandemic. during the course of the epidemic response efforts, risk evaluation, and risk management, it was necessary to build unobstructed information exchange channels to ensure the accurate transfer of data and information. under the joint prevention and control mechanism, each of the lead departments for the work groups and the expert advisory committee appointed people to collect, sort out, tabulate and analyze their work groups' epidemic information and latest progress on a daily basis, and to report in writing daily data collected by 18:00 to the general group prior to 20:00 p.m. the general group would then prioritize epidemic information and progress reports, and submit it representing the entire joint prevention and control mechanism to the general duty office of the state council. strengthened coordination and communication between the work groups and their members ensured an unobstructed flow of information as the working groups were informed of major issues as they happened. the general group was also charged with publishing information crucial for the public's knowledge on epidemic prevention and control. updates on influenza a (h1n1) prevention and control were submitted via four reporting systems: the disease monitoring information reporting management subsystem of the china information system for disease control and prevention, the public health emergency reported information management system, the china influenza information monitoring system, and administrative reporting system for health departments. reported information mainly included: ongoing epidemic situations, monitoring results from sentinel hospitals and network laboratories, progress in vaccination and results of side effects monitoring, and ongoing regional and departmental response efforts. with influenza a (h1n1) cases rising rapidly, issues with epidemic information reporting began to occur, such as overlapping reports, large discrepancies between confirmed reported cases and actual cases, and the circulation of ambiguous epidemic information. to better and more accurately reflect national epidemic situations and trends, the moh general office issued the notice on strengthening work of reporting deaths from influenza a (h1n1) on november 4th, 2009, and the notice on adjusting the work of reporting information on the influenza a (h1n1) epidemic on november 13th, 2009. by april 7th, 2010, the general group had submitted more than 700 work reports regarding influenza a (h1n1), and compiled and published over 200 response progress to influenza a (h1n1) under the joint prevention and control mechanism reports. information on epidemic trends and response efforts was released in a timely, open, and transparent manner, and by this time eight news conferences and nine press briefings had been held regarding the latest progress in influenza a (h1n1) prevention and control. to ensure that prevention and control measures were implemented effectively and efficiently, the work groups each established a supervision and inspection system by which to examine routine work on a regular basis, identify existing deficiencies and problems, and supervise and inspect response contingency plans and procedures, operational capacities, epidemic monitoring, epidemiological investigations, designated hospitals and their isolated areas, medical observation, material supplies, staff training, and information dissemination on prevention and control. in the course of influenza a(h1n1) prevention and control efforts, local governments, as instructed by the central government, examined their own conditions and established local bodies to command and coordinate response measures. the local departments worked together to implement disease prevention and control measures in priority areas and among targeted groups, and ensured continued epidemic monitoring and treatment. the following three modes mainly represent actual prevention and control measures adopted by local governments. the first work mode was similar to the national joint prevention and control mechanism. for example, shaanxi set up a leading group for influenza a (h1n1) prevention and control, whose office was located inside the provincial department of health and their local structure followed the "8 + 1" joint prevention and control mechanism model. guangdong established a joint prevention and control mechanism with the participation of thirty two departments, and nine work groups and three panels of clinical, disease prevention and control, and etiological experts functioned under the mechanism. on april 30th, fujian established an influenza a (h1n1) prevention and control work group, headed by a provincial government official; the office was located inside the building of the provincial department of health whose emergency management office was charged with performing routine work for the group. the emergency guidance on influenza a (h1n1) prevention and control in fujian (tentative), issued on may 10th, 2009, outlined response guidance as "prevention first through joint prevention and control, timely management, and level-by-level responsibility." the second work mode was the emergency operations center or leading group. after discovering their first confirmed influenza a (h1n1) case, some provinces and cities upgraded their existing disease prevention and control mechanisms and established an influenza a (h1n1) response leading group or emergency operations center. beijing was the first in the country to establish a municipal-level public health emergency operations center in may 2006, and had earlier (april 25th, 2009) launched a public health emergency response mechanism after the who declared the outbreak of swine influenza in mexico; an influenza prevention and control office (at the general office of the municipal government before it relocated to the municipal bureau of health) was established under the emergency operations center, whose members included twenty two committees, eighteen district and county governments, and the gld health department. this control office established a public health emergency response and medical rescue collaboration mechanism with the china cdc, the academy of military medical sciences, and other institutions. it established a mutual fixed epidemic communication system with local agricultural, educational, industrial, and commercial departments. a joint command response mechanism was also created with other special operations centers in beijing. on may 13th, shandong established a provincial public health emergency leading group and started level-ii response measures after discovering its first-and the country's second-confirmed imported case of influenza a (h1n1). on june 2nd, hubei established an influenza a (h1n1) emergency operations center after the province's first case was confirmed. immediately after the country's first influenza a (h1n1) case was confirmed on may 11th in sichuan, sichuan initiated level-ii public health emergency response measures and set up a provincial response leading group as per the state council's requirement of handling the virus as a category b infectious disease. the third work mode was the joint conference system. some provinces and cities established a joint conference system in response to the influenza a (h1n1) outbreak. for example, henan established an influenza a (h1n1) joint conference system on april 30th, and on the same day guangxi established a 12-department joint conference system for its own prevention and control efforts. while establishing provincial-level joint prevention and control mechanisms, health departments also set up internal expert panels. for example, fujian provincial department of health set up a provincial-level influenza a (h1n1) prevention and control expert supervision panel; guangdong provincial department of health established three expert panels for clinics, disease prevention and control, and etiology; sichuan provincial department of health established a leading group, a technical guidance expert panel, and a medical rescue panel on april 30th. the composition of local mechanisms for influenza a (h1n1) prevention and control basically followed the framework of the national joint prevention and control mechanism, with an office and several work groups collaborating under a leading group or operations center. for example, beijing's public health emergency operations center was responsible for the city's influenza prevention and control, and it was comprised of eight work groups plus an office, these groups included: immigration inspection, healthcare, epidemiological survey, material security, dissemination and communication, information, animal husbandry and veterinary, and social prevention and control supervision (referred to as "one office and eight groups"). fujian's influenza a (h1n1) prevention and control leading group consisted of thirty one departments and organizations, including the provincial department of health, a press office, and a development and reform commission. sichuan's influenza a (h1n1) prevention and control leading group (operations center) included departments from emergency management, public security, development and reform, transportation, immigration inspection and quarantine, tourism, civil aviation, foreign affairs, and publicity. the office of sichuan's influenza a (h1n1) prevention and control leading group was originally located in the provincial department of health, which was then moved to the provincial government's general office building as the epidemic worsened. its work groups consisted of emergency coordination, general support, information secretaries, epidemic prevention and control, medical rescue, supervision and inspection, press and communication, and health education. local influenza a (h1n1) prevention and control mechanisms adopted a similar communication and coordination mechanism to the joint national prevention and control mechanism, and operated in with joint offices, conferences, etc. sichuan is one example of this. the provincial operations center and the provincial leading group (headquarters) shared offices in order to strengthen the province's joint prevention and control against influenza a (h1n1), and its seven work groups were comprised of highly capable professionals from the emergency management office, third secretariat office of the general office, and the provincial health department. the health department met regularly with the departments of public security, civil aviation, immigration quarantine, economy and trade, animal husbandry, as well as pla and people's armed police troops stationed in the province. adjustments were made in real time in according with latest local epidemic situations. latest information on epidemic updates across the province were reported daily to members and related departments, and where cases were discovered, the departments of health, public security, foreign affairs, railway, transport, and others worked closely to track close contacts and ensure they were medically observed. through close collaboration between departments at various levels on joint prevention and control, sichuan ensured that cases were discovered, reported, isolated and treated at the earliest possible time, which delayed the spread of the virus and lowered epidemic intensity. government departments, enterprises, institutions, communities, and nonprofit organizations (npos) all play important roles in prevention and control of an infectious disease. with the country's response efforts entering its second phase, the 13th meeting of the national joint prevention and control mechanism, held on june 10th, 2006, proposed further improvements of existing mechanisms, in particular establishing accountability systems and mass prevention and control mechanisms with participation from urban communities, schools, enterprises and villages. these mechanisms could better implement tailored measures, disseminate self-protection knowledge for families and individuals, and improve measures that maintain the status quo and normal economic operations. when confronted with a public health emergency, under the guidance of the government, the society can effectively avoid or reduce potential damage by achieving preliminary prevention and control targets at local levels through community involvement and solidarity along with raising public awareness in self-protection. in its 1989 work report, the who mentioned two types of community participation, i.e. participation as a means, and participation as a goal, and analyzed effects of the two. in the course of china's influenza a (h1n1) prevention and control efforts, communities, the most basic social units, played an important role in knowledge dissemination and health education, the tracking and isolation of close contacts, and epidemic supervision. when uncertainties still surrounded the pathology and virulence of influenza a (h1n1) in the early days of the epidemic, local communities launched information dissemination and health education campaigns, playing a crucial role in stabilizing public opinion and raising awareness of scientific disease prevention and treatment methods. in the case of community-level outbreaks, affected communities generally adopted comprehensive response measures, which emphasized managing the sources of infection in order to contain and control the transmission of the influenza virus. measures mainly included the following: (1) sub-district offices or town governments mobilized social forces-as per laws, rules, and regulations-to provide support for isolated cases, including logistical service to personnel engaging in medical observation; (2) close contacts were medically observed centrally or at home, and healthcare workers reported daily on patients' progress; (3) patients with influenza-like symptoms were recommended to rest at home and not participate in unnecessary public gatherings or travel; (4) schools, nurseries and kindergartens, nursing homes, and construction sites were required to conduct health inspections, and enterprises with a concentrated amount of personnel or those who provided social services were required to perform morning health inspections; (5) information on epidemic trends and response measures were published in real time, and efforts were made to strengthen information disclosure within communities; and (6) health education and risk communication were carried out through multiple channels. when the virus broke out in communities, healthcare departments managed cases categorically and adopted comprehensive measures for strengthening the treatment of severely ill cases, lowering case fatality rates, and mitigating epidemic damage. with prevention and control measures in place against community-level outbreaks, response measures for priority areas mainly included the following: (1) as per related laws, rules, and regulations, local governments mobilized social forces to ensure the logistical support of measures like home-based treatment of cases with influenza-like symptoms; (2) migration was cut or restricted, recreational areas were temporarily shut down, and large-scale gatherings were canceled or postponed. enterprises and institutions within communities were permitted to grant time off for all or some of their employees; (3) schools, nurseries, and kindergartens were closed per related regulations; (4) enterprises and institutions as social services providers with large workforces implemented a health reporting system, management was enhanced where there were large flows of people, and people with influenza-like symptoms were recommended to rest and receive treatment at home; (5) when necessary, outbreak points were put under isolated control, and quarantine measures were taken in epidemic areas. at the same time, measures were taken to organize and encourage volunteers to participate in prevention and control activities, to help maintain the normal operation within communities, and provide mental health interventions to avoid adverse effects on public health. these measures, which were designed based on real community conditions, effectively guaranteed the protection of the status quo, and laid a strong foundation for local influenza a (h1n1) prevention and control efforts. in the course of influenza a (h1n1) prevention and control efforts, drug stockpiling enterprises responded actively to the government's call for material reserves and production. because influenza drugs weren't prevalent in clinical use, they were traditionally stockpiled through loans, government subsidies, business opportunities and moderate enterprise compensation. problems arose during the implementation of this mechanism such as subsidy inaccessibility and unreasonable compensation. for example, the current 10% subsidy policies regarding corporate loans and government subsidies hardly met the needs of enterprises, and the problem of unreasonable compensation to pharmaceutical enterprises still existed, partly because specific mechanisms were lacking. at the same time, more than 80% of emergency response drugs were not on the standing list of medications, and so it was necessary to build a long-term relationship between the government and enterprises to specify respective duties, and link stockpile funding with corporate social responsibility to balance compensation. reagent manufacturing was one of the government's top priorities during the entire course of its influenza a (h1n1) prevention and control efforts. some reagent manufacturers which had developed and produced reagents for biological agents such as anthrax during the 2008 olympic games already had experience in emergency response. for example, beijing kinghawk pharmaceutical co., ltd., the country's first to obtain approval for an influenza a (h1n1) testing kit, signed a strategic alliance agreement with the china cdc during the influenza a (h1n1) epidemic. the enterprise also provided its laboratories voluntarily when there was no clear policy on state funding, doing its best for society as a corporate citizen. with its technology reserve, seven production platforms and ninety approved products, kinghawk was able to perform research and development on product standardization during a critical time of the epidemic. the china cdc had access to international resources for preliminary research and development and successfully obtained information and strains from the who. this collaboration between the two parties made it possible to develop preliminary products in 72 h and thus ensured that considerable demand for clinical diagnosis was met. this played a positive role in case diagnosis during the early phases of the influenza a (h1n1) epidemic and was also quite meaningful in terms of drug use guidance. the government provided active support to research and development efforts. take kinghawk as an example. after kinghawk signed the agreement with the government on may 4th, 2009, both the beijing economic-technological development food and drug administration and the beijing food and drug administration provided recommendations. kinghawk had developed a rapid test kit by may 11th, received approval from the cfda on june 17th to launch the emergency response system, and got approved for manufacturing the reagent for 250,000 people on september 25th. kinghawk had collaborated with the china cdc in the past and had experience in reagent development and manufacturing. the development of the reagent fully demonstrated the efficiency in collaboration between the government and a commercial enterprise, and also guaranteed the timeliness of preliminary disease diagnosis. in the containment phase, hotels and other requisitioned enterprises across the country showed full support for the response measures by providing isolation zones of influenza a (h1n1) cases. many hotels suitable for isolation purposes were private firms and thus could not be requisitioned through administrative orders, which put a certain amount of pressure on local governments. however, coordination efforts by local governments did earn support and assistance from these hotels. transport enterprises shouldered the heavy task of implementing disease prevention and control for the floating population. civil aviation, railways, road and related enterprises implemented strict prevention and control measures, including disseminating knowledge about disease prevention and control, and providing necessary infrastructure support for emergency response efforts targeting the floating population. all in all, a healthy transportation environment helped lower the transmission of the disease. facing the unexpected onset of influenza a (h1n1), these transportation enterprises all established prevention and control groups. for example, the caac north china regional administration established a capital airport influenza a (h1n1) prevention and control leading group, which was based on the former capital airport public health emergency leading group; beijing capital international airport company limited, air china limited, china southern beijing company, china eastern beijing company, hainan airlines beijing company, caac air traffic management bureau all set up their own epidemic response teams to ensure the orderly implementation of prevention and control measures. telecommunications enterprises did their duties as corporate citizens and actively cooperated in influenza a (h1n1) prevention and control efforts. china mobile group beijing company limited, china telecom group beijing company limited, china unicom beijing company limited, among other telecommunications operators, suspended their normal user notification group-messaging services and mustered network resources to send messages on epidemic updates while increasing maintenance staff, strengthening network monitoring, and closely watching the impact of group messaging on their systems. nonprofit organizations, or npos, are organizations that fulfill particular social causes or missions without seeking profit for their efforts, and npos represented a crucial social force in the course of the nation's influenza a (h1n1) prevention and control. for example, the beijing red cross established a public health emergency operations center which consisted of a general information group, a rescue response group, a fundraising and aid group, a publicity group, and a public relations group. this organization actively engaged in response efforts in accordance with the beijing red cross emergency contingency plans for public emergencies. the entire nation was involved in epidemic prevention and control, including its citizens. during the response to the virus, volunteers played an important role when multiple departments suffered emergency manpower shortages. for example, medical and healthcare students in colleges and universities volunteered to work on the front lines of epidemic prevention and control. in beijing, 170 student volunteers from capital medical university assisted with response efforts at capital airport, and similar volunteering also occurred in fujian. the public actively supported the government's influenza a (h1n1) prevention and control measures, and voluntarily took part in the process via the internet and other media channels; for example volunteers called those who had just returned from abroad and informed them of potential isolation measures. at the same time, increased public health awareness was also instrumental in successfully dealing with the disease. prevention and control mechanisms at the beginning of the influenza a (h1n1) epidemic, china established a national level emergency management mechanism directly under the leadership of the state council that enabled cross-departmental joint prevention and control collaboration, which provided an effective organizational support and operation mechanism for the response efforts. though the moh had formulated the ministry of health's influenza pandemic preparedness and response plan (tentative) before the epidemic broke out, this document focused only on the duties of the moh and didn't encompass more complex coordination and collaboration with related government departments. the joint prevention and control mechanism remedied this flaw by providing a platform for coordination and collaboration between the moh and other related departments. also, because this mechanism was not like the state council's operations center, it allowed some space for strengthening the state council's leadership and collaboration once the epidemic worsened. during the prevention and control efforts, local governments adapted and innovated central policies and their implementation in light of local epidemic situations, public health trends, and demographic and economic conditions. some areas established prevention and control mechanisms with local characteristics. the main features of these mechanisms are as follows: the first was the establishment of a strong leadership system. in the process of prevention and control, local governments established their respective public health emergency leadership systems based on local epidemic situations, geographic features, and public health resources. the second was the innovation in ideas and methods. local epidemic prevention and control bodies closely monitored trends and reengineered their methods based on existing departmental systems in order to better target obstacles encountered in operations. for example, in the early days of the epidemic, the beijing government issued a notice on further specifying duties and prioritizing operations to strengthen influenza a (h1n1) prevention and control, which articulated the new public health notion of "responsibility of four sides" (government, departments, enterprises, and individuals) . this clarification brought about effective collaboration between the government and the society in public health emergency management. the beijing immigration inspection and quarantine bureau employed risk analysis methods in its prevention and control efforts and ensured electronic transfer of information on inbound passengers, which not only increased quarantine and inspection efficiency but also scientifically and efficiently improved response measures. fujian was the country's first province to implement temporary isolation measures through its local health department. henan created an epidemic prevention and control network of "three horizontal fronts"-arrangements at a government level, measures at enterprise (institution) level, and protection at a local level; and "three vertical fronts"-government supervision, inter-departmental collaboration, and public opinion guidance. these institutional innovations proved very effective in the response efforts. the third was the establishment of an inter-provincial support mechanism. on november 13th, 2009, the moh general office issued the notice on strengthening medical treatment of influenza a (h1n1) patients (no. 245, 2009) , announcing the decision to establish an inter-provincial support mechanism for medical treatment of influenza a (h1n1) patients as per the notice of the state council on strengthening the ongoing work on influenza a (h1n1) prevention and control (no. 23, 2009 ) and as needed for patients. the form of assistance was technical support, especially in regards to medical treatment of seriously and critically ill patients. throughout the entire duration of the prevention and control efforts, governments greatly heeded experts in various fields, which aided governments in creating more scientific policy adjustments and technical plans, and consequently reduced blindness and uncertainty in policy implementation. experts from cdcs, hospitals, publicity departments, and other departments took part in the decision-making process, and their input was adopted in real time. some experts even took the initiative to provide police recommendations directly to decision makers. at the same time, governments sought out expert opinions through different methods and channels, i.e., consultation at joint prevention and control meetings or direct consultation with the experts. expert recommendations ensured scientific policies and more targeted and effective policy formulation. in regards to policymaking, some local governments formulated policies and adjustments based on local conditions and epidemic trends. for example, jiangmen experimented with a home-based isolation policy, while shenzhen created corridors at ports specifically for foreigners and a separate one for students commuting between shenzhen and hong kong for school. also, in terms of policy adjustment, some local departments were able to adjust related policies in time to better suit local epidemic situations. as for issues that necessitated policy coordination, local departments also made strategic adjustments as early as possible. for example, the guangdong immigration inspection and quarantine bureau, at experts' suggestion, transferred persons who required isolation and medical observation to health departments for categorical management, which thus ensured the efficient use of epidemic prevention and control resources. over the course of influenza a (h1n1) prevention and control efforts, the government cultivated an environment of widespread social participation under the leadership of the party and government, with enterprises, communities, volunteers and other social actors playing crucial roles in the response efforts. the joint national prevention and control mechanism was essentially a command and decision-making mechanism established according to the potential amount of damage influenza a (h1n1) could inflict upon society. on the one hand, influenza a (h1n1) response required inter-departmental collaboration, and relying solely upon health departments for countermeasures wouldn't be enough; on the other hand, because the virus was not as virulent as to merit the establishment of a state council operations center (or headquarters), the state council instead instructed the moh to establish a multi-departmental joint prevention and control mechanism; and this new organization represented a relatively flexible and effective response mechanism. although local governments were already aware of the epidemic at its onset and were actively engaging with different departments in their response efforts, because there were no explicit provisions in related laws and contingency plans for the joint national prevention and control mechanism at the central level, no corresponding normative documents were available for its implementation at a local level. no unified standards on the name, content, form of establishment, and system structure for local governments' prevention and control bodies existed. although local governments adapted as they went, it was still an environment that incited disorder and confusion. on the one hand, participating departments fully endorsed the joint national prevention and control mechanism. this mechanism, they thought, possessed several advantages: firstly, the joint consultation system made it possible to directly formulate and sign policies at joint prevention and control conferences, which saved time for everyone; secondly, the joint briefing system required the work groups to send daily reports to other units and departments, thus facilitating both inter-group and inter-departmental communication; and finally, internal collaboration within groups was solid, and the briefing system allowed an unobstructed flow of information. however, the joint national prevention and control mechanism based upon consultation and communication had its limitations. on issues involving departmental interest, division of duty, and so on, this horizontal collaboration was less efficient than regulation and control by a single, high level leadership department. one contested issue dealt with the location of the local joint prevention and control office: should it be set up in the comprehensive emergency management office of the local government or in the emergency management office of a local specialized department. some provincial emergency management offices insisted that for an emergency event like the ongoing influenza a (h1n1) epidemic, a joint prevention and control office should be located in a specialized department so as to leverage the department's expertise and increase response flexibility, convenience and efficiency. in this scenario, the provincial emergency management office would be tasked with solving issues that the specialized department could not. on the other hand, some provincial health department's emergency offices argued that if the office was located in the local government, the joint prevention and control office would enjoy greater authority and more efficient collaboration. achieving a smooth and effective transition between peacetime and public emergency, and establishing mechanisms that combined crucial components from both systems, was a new challenge that arose in the influenza a (h1n1) epidemic. after the 2003 sars epidemic, local governments established permanent public health emergency response departments and corresponding working mechanisms to deal with future public health emergencies. these departments and mechanisms should have been employed upon the onset of the influenza a (h1n1) epidemic. however, most provinces established completely new leading groups only after the central government established joint national prevention and control mechanism. in one example, a provincial health department already had a permanent public health emergency operations center, but, after the central government established the influenza a (h1n1) joint national prevention and control mechanism, this province created an entirely new prevention and control leading group and a port leading group. at the same time, the health department also established new eternal mechanisms, including: the provincial cdc established an emergency response department with leaders from major sections like emergency management and vaccination planning (starting in 2005, this provincial cdc implemented a "3 in 1" meeting system with participation from emergency management, disease control, and the disease monitoring department). the main reason for this redundancy was because the central government did not provide specific conditions or qualifications for contingency planning and management for the transition period between peacetime to emergency. thus, local governments lacked a clear transition mechanism that they could utilize. it was the reason that many local governments chose to re-establish emergency management bodies when influenza a (h1n1) broke out. as public health emergency management involved multiple collaboration systems from the central government down to local governments, regions, and departments, inter-departmental collaboration in the response efforts was intrinsically complicated. the response to this epidemic revealed problems that existed both in horizontal and vertical coordination. in regards to horizontal coordination between central departments, the health, education, security, transportation and many other departments were involved in the influenza a (h1n1) response efforts, which created an environment where responsibilities could easily overlap and grey areas would occur in management. there was also a lack of coordination and standardization between central-level ministries' policy documents for influenza a (h1n1) countermeasures. for example, in regards to content standardization, the health authorities felt that using the temperature of 37.3°c as the sole standard for sending people to the hospital was unreasonable and would cause an unnecessary burden on hospitals. in regards to time standardization, on december 2nd, 2009, one province stipulated that only patients with a temperature of 38°c or higher must be sent to a hospital, and it took the country two more weeks to follow suit. in regards to inter-departmental work, port laboratories in some provinces had begun testing in the early days of epidemic, but stopped after provincial health departments decided that ports were not fit for such work. obstacles also arose in horizontal coordination and collaboration between local departments. a lack of information communication between local departments due to the unavailability of complete information in the early stages of prevention and control made it nearly impossible for effective collaboration. in regards to the division of labor and coordination between the central and local governments for disease prevention and control, some local governments held that the central government should have presented broader goals and authorized provinces and cities greater autonomy in their response measures. some felt that the central government should not have made influenza a (h1n1) prevention and control an issue of political significance but should have been objective in understanding the differences between executive leadership and scientists' opinions. while the main duty of administrative leaders should have been to organize and mobilize social resources needed to cope with the epidemic, scientists should have been the ones to handle technical issues such as epidemic analysis and response measures. at the same time, more efficient communication should have been present between central and local departments tasked with specific operations. for example, some local management departments felt that the entire process was quite political, making some documents difficult to fully implement; in the two most volatile months that lasted from april 28th to june, documents were issued frequently, and in some cases were in conflict with one another and lacked integrity and continuity. in regards to adjustment of prevention and control strategies, some regions' health departments reported the following issues: higher-level departments frequently adjusted technical guidance and strategies for prevention and control, there was a wide variety of information reporting methods and they were constantly in flux, different departments formulated their own response requirements, and differences occurred in measures and standards; all of which greatly complicated local response operations. certain communication and coordination issues also existed within the health department's internal system. the moh internal horizontal collaboration needs to be strengthened epidemiological investigations, clinical diagnoses, and laboratory testing to combine the medical treatment and disease prevention. for example, the china cdc played a crucial role as a central technical support body of influenza a (h1n1) prevention and control in epidemiological information collection, monitoring, analysis, and judgment, but at the same time it also had a lot of administrative duties, and its services and duties overlapped with those of the moh's bureau of disease control and prevention. there should be unified leadership and coordination between higher and lower-level health departments within the national epidemic prevention and control system. a certain degree of flexibility is also necessary as provinces differ in epidemic situations, medical resources, geographic features, and so on. in regards to information reporting within the health system, though the china cdc and the moh had established information systems relating to epidemic surveillance, including an epidemic direct reporting system, no information sharing mechanism was created between the china cdc and medical institutions; in particular, some county level medical institutions didn't even have sound data collection and reporting systems. this resulted in a single point of decision making and command, and their lack of network and information technology weakened the support they could've had in implementing response measures. npos such as the red cross society of china played important roles during the influenza a (h1n1) prevention and control efforts. however, by comparison with developed countries, china still lags behind in terms of public participation in public health emergencies. there still remain limitations in skill and knowledge, as no emergency volunteer systems or working mechanisms were formed, and no leveraging of npo resources really occurred. on public emergency management bodies of the chinese government key: cord-007575-5ekgabx5 authors: luby, james p. title: southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses date: 2016-01-14 journal: am j med sci doi: 10.1097/00000441-198707000-00007 sha: doc_id: 7575 cord_uid: 5ekgabx5 pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6–35% of all cases of pneumonia and are the sole pathogens in 1–17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. southwestern internal medicine conference: pneumonias in adults due to mycoplasma, chlamydiae, and viruses by james p. luby, md abstract: pneumonias in adults due to mycoplasma, chlamydiae, and viruses are a common clinical problem. these microorganisms contribute to the etiologies in 6-35% of all cases of pneumonia and are the sole pathogens in 1-17% of hospitalized cases. important trends and developments in the field include (1) the emergence of a chlamydia psittaci strain (twar) that is passaged from human to human, causes a mycoplasma-like illness, and that is relatively resistant to erythromycin, (2) the recognition of respiratory syncytial virus as a pathogen in nursing home outbreaks and in immunosuppressed adults, (3) the continuing high lethality of fully developed influenza pneumonia, (4) the efficacy of acyclovir and adenine arabinoside in limiting the complications of varicella-zoster virus infections, and (5) the increasing frequency of pneumonia caused by cytomegalovirus and the severity of this disorder in highly immunosuppressed patients. developments in the rapid diagnosis and therapy of respiratory syncytial virus infections with an aerosolized antiviral drug in children may pave the way for comparable advances in difficult pneumonias in adult patients. key population-based studies on the incidence ofpneumonia have been performed infrequently. in one study, during an 8-year interval from december 1, 1963 , through november 30, 1971 , foy and her colleagues determined the incidence of pneumonia in a prepaid medical health insurance plan comprising more than 100,000 members in seattle, washington. 1 they found that total pneumonia rates varied yearly and ranged between 7.2 and 16.8 cases/1,000 population/year. only 15% of the cases seen by the physicians caring for this group were hospitalized; 85% of the total number of cases were managed as outpatients. in adults, 35% of all cases of pneumonia were associated with cultural and/or serologic evidence of mycoplasma and/or viral infection. total rates for all cases of pneumonia increased during influenza a2 epidemic years. the highest rates were generally found in the winter quarter, followed by rates occurring during the spring quarter ( figure 1 ). the major viral and mycoplasma agents contributing to the etiology of pneumonia in this study were influenza a virus, and mycoplasma pneumoniae, followed by a smaller number of cases due to adenoviruses, influenza b virus, respiratory syncytial virus (rsv), and parainfluenza viruses. most of the parainfluenza virus infections were caused by types 2 and 3, but no attempt was made to ascertain the exact contribution of specific agents involved because of antigenic overlap in the complement fixation test. it was recognized that the majority of pneumonias associated with influenza a were related to bacterial suprainfection. rates for all pneumonia were highest in young children, followed by a peak in pneumonia due to m. pneumoniae in the 30-40-year age group. pneumonia due to influenza a virus increased in incidence above the age of 60 years. of interest is their finding that sometimes pneumonia was associated with laboratory evidence of infection with more than one respiratory nonbacterial agent. the severity of disease did not appear different between patients with a single infection and those with multiple infections, as measured by duration of illness and hospitalization rates. in individual reports, however, it has been suggested that in certain individuals, multiple infections can sometimes lead to a more severe course than would have been predicted by infection with a single agent. 2 overall rates for pneumonia as determined in this study were similar to those observed in the national health survey. in houston, texas, during the years 1975-1978, adult hospitalizations for pneumonia increased sharply during influenza a epidemics but did not change much during influenza b epidemics, a finding that was also seen in the seattle study (figure 2) . 3 although hospitalizations for pneumonia did not increase in houston during the 1976-1977 influenza b epidemic, an increased number of patients hospitalized with complications due to influenza b virus infection was seen in dallas, texas, at this time. 4 the etiology of community-acquired pneumonia in 54 adult outpatients in sweden has been determined. 5 using rises in antibody titer between acute and convalescent sera to determine etiology, these investigators found evidence of mycoplasma pneumoniae infection in 37%; streptococcus pneumoniae in 9%; hemophilus in{luenzae in 12% (6% type band 6% nontypeable); influenza a virus in 6%; chlamydia psittaci in 4%; and influenza b virus, parainfluenza 3 virus, respiratory syncytial virus, and adenovirus in 2% each. multiple infections occurred in several patients and there was no serologic evidence of infection with a particular microorganism in 41 %. the etiologic agents of community-acquired pneumonia in adult patients hospitalized for their disease can be examined (table 1) . six recent studies were selected for analysis because they had a worldwide authorship and because an attempt had been made to estimate the contribution made by both bacterial and nonbacterial agents.6-11 bacterial 46 etiologies contributed most significantly to the problem of community-acquired pneumonia in adult hospitalized patients. the most frequent microorganisms were streptococcus pneumoniae followed by staphylococcus aureus, haemophilus in{luenzae, legionella pneumophila, and other gram-negative bacteria. the role of anaerobic bacteria in the etiology of pneumonia was not studied systematically in five of the series. a nottingham, england, study accentuated the role of l. pneumophila in the etiology of pneumonia and showed the propensity of this microorganism to be associated with specific geographic sites. the major nonbacterial agents implicated in these studies included m. pneumoniae, influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, varicella, and c. psittaci. all of these latter agents could be seen as single pathogens but influenza a and b viruses, adenoviruses, rsv, parainfluenza viruses, and varicella virus also were associated with bacterial pneumonia. nonbacterial agents contributed from 6 to 35% to the etiologies of all cases. an indication of the approximate incidence of nonbacterial pneumonia without bacterial suprainfection can be ascertained in five of the series and ranged from 1 to 17% of the cases. the association of influenza a and b viruses, rubeola virus, and varicella virus with bacterial suprainfection has been well established. recently, bacterial suprainfection has been shown to occur in adults who have evidence of infection with adenoviruses and rsv. the frequency of bacterial suprainfection in association with m. pneumoniae infections is difficult to ascertain. it has been considered the concept of primary atypical pneumonia was set forth in an article by hobart a. reimann in 1938. 12 the major bacteria-causing pneumonia were known at that time with the exception of l. pneumophila. the clinical entity of psittacosis had been elucidated. influenza a virus had been grown in ferrets by laidlaw, andrews, and smith. reimann described eight cases of what he called atypical pneumonia, which he thought was due to a filterable virus. the description of cases allows a view of untreated primary atypical pneumonia. it now seems probable that m. pneumoniae was the etiologic agent in most of his cases. the illness often began in-the american journal of the medical sciences sidiously with fever, headache, and pharyngitis. with descent of the disease into the respiratory tract, the larynx became involved and hoarseness was present. finally, laryngotracheobronchitis and pneumonia occurred. a troublesome cough developed in the patient that could not be alleviated and was only slightly productive. in some patients, a pulse-temperature dissociation occurred. during the course of the disease, which often lasted several weeks, patients became dyspneic and cyanotic. two of the patients became delirious and had central nervous system dysfunction during the course of the infection. on physical examination, the patient was flushed and had evidence of pharyngitis. the physical examination of the chest usually revealed scattered rales without striking evidence of consolidation; in one patient a large pleural effusion was present. the white blood count was only modestly elevated. chest radiographs revealed mottled or diffuse areas of infiltration. attempts to isolate pathogenic bacteria and influenza a virus were unsuccessful in establishing an etiology for this syndrome. reimann considered diagnoses such as typhoid, psittacosis, and epidemic influenza, but the history in none of these cases was consistent and influenza virus could not be recovered. to summarize reimann's words, "the infection occurred in adults and began as a mild infection of the respiratory tract; this was followed by severe diffuse atypical pneumonia and in two cases by the symptoms of encephalitis. dyspnea, cyanosis, hoarseness, cough without sputum, drowsiness, and profuse sweating were the chief characteristics. the disease lasted several weeks." in 1943, finland found elevated cold agglutinin titers in cases of atypical pneumonia. eaton later isolated the agent in embryonated eggs, and chanock and colleagues were able to grow it on defined media and demonstrate it to be a mycoplasma. the entity of primary atypical pneumonia became well known and later was defined as pneumonia that did not clear with penicillin or sulfonamides, or nonbacterial pneumonia, or pneumonia with no sputum or a mucoid sputum without a predominant organism on gram's stain. we recognize today that the clinical entity of primary atypical pneumonia has multiple etiologies, particularly m. pneumoniae, but also c. psittaci, the twar strain ofc.psittaci, chlamydia trachomatis, q-fever, and viruses such as adenoviruses, rsv, influenza viruses, and para-48 influenza viruses. l. pneumophila infections often are considered in the differential diagnosis. ls early bacterial pneumonia also should be considered when the patient is first seen. in retrospect, persons with this diagnosis are often excluded from series of cases to focus specifically on the nonbacterial nature of the problem. in pertinent geographic areas, acute histoplasmosis and coccidioidomycosis may present like primary atypical pneumonia. major attempts to identify the etiologic agent on clinical grounds have been made, but the exact diagnosis usually depends on laboratory determination of the offending agent. in one study of 150 patients of all ages, 50 with viral pneumonia, 50 with mycoplasma pneumonia, and 50 with bacteremic i:meumococcal pneumonia were compared. 14 the best discriminating variables were the c-reactive protein determination, the presence or absence of predisposing disease or previous antibiotic treatment, the erythrocyte sedimentation rate, the presence of lymphocytosis, and the band neutrophile count. signs of an upper respiratory tract infection and the presence or absence of auscultatory abnormalities also aided significantly in the discrimination. determinations favoring bacteremic pneumococcal pneumonia included predisposing disease, a short duration of illness before hospitalization, alcoholism, the absence of signs of an upper respiratory tract infection, high c-reactive protein determinations and erythrocyte sedimentation rates, no prior antibiotic treatment, total leukocyte counts exceeding 15,000, relative lymphocyte counts less than 35%, relative band neutrophile counts greater than 20%, abnormal auscultatory findings, and the presence of lobar consolidation on chest radiograph. differentiation between viral and mycoplasma pneumonia could not be made easily. however, symptoms of mycoplasma pneumonia before hospitalization lasted a longer time and these patients were more likely to have received antibiotic treatment in the interval before hospitalization. patients with mycoplasma pneumonia were more likely to have lobar consolidation on chest radiograph than those with viral pneumonia, but in this study no distinction could be made between mycoplasma pneumonia and bacteremic pneumococcal pneumonia on the basis of roentgenographic findings alone. in another study comparing community-acquired pneumonias, mycoplasma pneumonia tended to occur at an earlier age than legionnaire's disease, pneumococcal pneumonia, or psittacosis.l 5 homogeneous shadowing on chest radiograph was more common in legionnaire's disease and pneumococcal pneumonia than mycoplasma pneumonia. pleural effusions were uncommon in all groups but occurred most commonly in bacteremic pneumococcal pneumonia as did multilobe disease on presentation. hilar lymphadenopathy occurred only in mycoplasma pneumonia. roentgenographic resolution was fastest in mycoplasma pneumonia, intermediate in psittacosis and nonbacteremic pneumococcal pneumonia, and slowest in legionnaire's disease and bacteremic pneumococcal pneumonia. deterioration on chest radiograph after hospital admission characterized legionnaire's disease and bacteremic pneumococcal pneumonia. because the differential diagnosis of primary atypical pneumonia at a clinical level includes pneumonia due to m. pneumoniae, chlamydial species, q fever, adenoviruses, rsv, influenza viruses, parainfluenza viruses, as well as l. pneumophila infections and early bacterial pneumonia, therapy should include an antibiotic to which the majority of these the american journal of the medical sciences luby microorganisms are susceptible. chlamydial species are more susceptible to tetracycline than erythromycin. tetracycline is effective against rickettsiae but not for l. pneumophila infections. up to 4% of pneumococcal isolates are resistant to tetracycline. a reasonable antibiotic choice is erythromycin at an equivalent dose of 30 mglkg of erythromycin base per day for 10-14 days. if legionnaire's disease is diagnosed, a higher dose of erythromycin may be necessary. if a chlamydial or rickettsial etiology is recognized, tetracycline at a dose of 2 gmiday should be given. occasionally patients with proven m. pneumoniae have been treated with erythromycin, failed to respond to therapy, but subsequently responded to a course of tetracycline therapy.ls conversely, some patients with m. pneumoniae infections have responded to erythromycin after a suboptimal response to tetracycline therapy. viruses may cause primary atypical pneumonia; however, antibiotic treatment in these instances is useless, does not prevent suprainfection, and may actually change the nature of the bacterial species suprainfecting the patient. antibiotic therapy seems reasonable in this syndrome, however, because it is usually impossible to differentiate clinically between mycoplasma pneumonia and an entity such as adenovirus pneumonia in the adult. advances in rapid laboratory diagnosis may be able in the future to influence treatment options but these techniques are still under development, are expensive, and are not widely available. mycoplasmas, the smallest free-living microorganisms, are cell-wall deficient, but have no relationship to cell-wall deficient bacteria with which they were once confused. m. pneumoniae attaches to the mucosal epithelium of the respiratory tract through a specific protein that enables the microorganism to adhere to neuraminic acid residues on respiratory epithelial cells. if mycoplasmas cannot attach, there is no damage to the host. upon adherence, mycoplasmas are able to generate hydrogen peroxide and superoxide anion, resulting in injury to epithelial cells. because infection occurs commonly in children younger than 5 years of age, although disease is rare at this time of life, mycoplasmas may induce disease primarily by immunopathologic mechanisms. l7 . lb in experimental animals not primed by prior mycoplasma exposure, inflammatory changes occur only after a long interval. with reinfection, inflammatory changes occur more briskly. the extrapulmonic manifestations of my coplasma infection have never been explained completely, but there are reports demonstrating m. pneumoniae in sites such as cerebrospinal fluid and blood. l9 alternatively, immunopathologic reactions may be the primary mechanism involved. path-ologically, the disease in man is characterized by tracheobronchial, bronchiolar and septal lymphoplasmocytic infiltrates, luminal exudates rich in polymorphonuclear leukocytes, bronchiolar and alveolar cell metaplasia, and occasionally diffuse alveolar injury.2o the bronchiole appears to be the major site of attack. the microorganisms colonize the nasopharynx and transmission of infection occurs only by close contact. especially conducive to the transmission of m. pneumoniae are situations in which persons are housed in closed quarters, such as military platoon barracks or family unit dwellings. in families, there is a high attack rate and cases continue to occur over a 3-4-month interva1. 21 . 22 the cumulative attack rate of mycoplasma infections in families may approach 90% (figure 3 ).21 mycoplasma carriage is not affected by antibiotic therapy, thereby allowing the family epidemic to continue. mycoplasma disease occurs throughout the year but is particularly frequent during fall and winter. increased numbers of cases occur with a 3-5-year periodicity. although pneumonia may occur soon after infection, the disease is usually manifested as an upper respiratory tract infection progressively descending into the lung. pharyngitis progresses into laryngitis followed by tracheobronchitis, and, finally, pneumonia. hoarseness and dysphonia may be present. middle ear involvement may occur with bullous myringitis, which usually heals without scarring. occasionally, otitis may lead to tympanic membrane perforation. sinus involvement is frequent but usually asymptomatic. the cough is often intractable and usually only slightly productive of a mucoid sputum that contains mainly polymorphonuclear leukocytes but no predominant bacterial microorganism on gram's stain. when pneumonia develops, the patient has an elevated temperature and, occasionally, a 'temperature-pulse dissociation. headache, irritation, a flushed facies, myalgias, and arthralgias are common. 2 3-26 on physical examination, the patient is febrile, appears flushed, and usually has physical evidence of pharyngitis. hemorrhagic bullous myringitis may be present in up to 5% of cases. physical findings on chest examination usually are limited to scattered rales, wheezes, and rhonchi and are often localized to the lung bases. evidence of consolidation is not striking, although m. pneumoniae infections can cause lobar pneumonia. 26 the white count is usually elevated with a shift to the left, but rarely exceeds 15,000 white blood cells/mm 3 and the neutrophile band count is usually less than 10%.14 chest radiograph reveals peribronchial infiltrates with accentuation of interstitial markings in adjacent lung segments, patchy alveolar infiltrates usually localized to the lower lobes, especially on the left, and occasionally hilar lymphadenopathy. 27 more than one lobe may be involved and a confluent lobar infiltrate may be present in some patients ( figure 4 ). less commonly, there is a diffuse interstitial infiltrate and rarely an x-ray picture indistinguishable from the adult respiratory distress syndrome. 28 . 29 without therapy, the disease course usually lasts approximately 3 weeks, but may extend up to 7 weeks. 12 extrapulmonic manifestations of mycoplasma infection often are a clue to the diagnosis and include bullous myringitis; neurologic disturbances suggesting encephalitis or aseptic meningitis and, rarely, transverse myelitis; arthritis; myopericarditis; hepatic dysfunction; splenomegaly; and skin eruptions.~u a stevens-johnson syndrome may occur. japanese workers have described typical cases of pityriasis rosea that followed mycoplasma infection. 32 the cerebrospinal fluid (csf) may be abnormal with an increased number of cells and an elevated protein concentration. hemolytic anemia may be present resulting from antibody directed against the i antigen on the red-cell membrane. 31 almost all patients recover completely after mycoplasma infection but cigarette smokers may have prolonged abnormalities in diffusion capacity.33 individual case reports have described pulmonary fibrosis, bronchiolitis obliterans, and bronchiectasis following m. pneumonia. 3~6 glomerulonephritis with continuing renal dysfunction also has been reported. 37 • 38 the diagnosis is established by culture of the microorganism or the demonstration of a fourfold rise in antibody by complement fixation or other serologic test. a single high complement fixation test antibody titer (~1: 128) may be used as presumptive evidence of infection. cold agglutinin antibody titers at low level are norispecific but very high values (~1 : 128) also can be used to support the diagnosis. treatment consists of the administration of either erythromycin or tetracycline as outlined in the therapy of primary atypical pneumonia. the patient usually responds, but it should be remembered that there are reports of inadequate resolution of the disease and the necessity to switch to the alternate drug to achieve more rapid clinical improvement. antibiotic therapy does not eliminate the carrier state. immunity is relatively short-lasting and documented episodes of repeated mycoplasma infection have been reported. a vaccine against m. pneumoniae, given present priorities, appears only a hopeful future development. mycoplasma pneumonia. an 18-year-old man was well until 8 days before admission into the hospital, at which time he developed fever, a sore throat, and a nonproductive cough. his oral temperature reached 40° c. a "pounding" headache developed. the cough persisted and became productive of a mucoid sputum. oral penicillin was prescribed but did not alleviate his symptoms. physical examination on admission into the hospital revealed a young man who was confused about time and uncertain about recent events. the oral temperature was 38.8° c and the pulse rate was 100. the pharynx was described as normal. chest examination revealed harsh breath sounds with bilateral inspiratory rales, especially on the right, anteriorly and inferiorly. there was no egophony or decreased fremitus. rhonchi were present more on the right than left. hepatosplenomegaly was present. laboratory examination revealed 12,200 white blood cells with 70% polymorphonuclear cells, 28% lymphocytes, one monocyte, and one eosinophile. the serum aspartate aminotransferase was 140 (normal <40). arterial blood gases on room air showed a ph of 7.55, pc02 of29, and p02 of 45. a lumbar puncture was performed that showed 31 white blood cells, 95% of which were mononuclear cells. the csf glucose was 69 mg/dl and the simultaneous plasma glucose 155 mg/dl. the patient was treated initially with intravenous penicillin for presumed pneumococcal pneumonia and partially treated bacterial meningitis. his condition deteriorated but finally he was placed on erythromycin therapy at the advice of a consultant. mycoplasma complement fixation test titers rose from less than 1: 8 to 1: 64. comment. encephalitis, hepatosplenomegaly, and mild hepatic dysfunction were the extrapulmonic manifestations of mycoplasma disease. typical of mycoplasma pneumonia were the long duration of illness before admission into the hospital, prior antibiotic administration, presence of a sore throat, physical examination of the chest, and characteristics of the sputum. psittacosis was first described by ritter in switzerland in 1879 as a disease of the lungs in patients in contact with sick psittacine birds. later, in 1929-1930, a pandemic of psittacosis occurred involving psittacine birds exported from south america. the clinical manifestations were described fully and the epidemiology was established, leading to control measures that have kept psittacosis or the better, more inclusive term, ornithosis, at a low level of occurrence. occasionally migrant birds can carry c. psittaci, and persons dealing with them may develop ornithosis. more importantly now, ornithosis is an occupational hazard to the farmer who manages poultry such as ducks and turkeys.17,lb,40,41 clinically, patients with ornithosis have headache, fever, pulse-temperature dissociation, pneumonia, hepatic function abnormalities, and hepatosplenomegaly. intra-alveolar inflammatory changes predominate in ornithosis with interstitial changes being secondary and less prominent. the chest radiograph reflects this and lobar consolidation may be seen. when lung involvement is minor, the disease can be diagnostically confusing, and present as a fever of undetermined origin. granuloma formation can be found in both the liver and the bone marrow and may be a diagnostic clue. ornithosis can be diagnosed by serologic tests with a chlamydial common group antigen by either complement fixation or the enzyme-linked immunoabsorbent assay (eia). treatment is with tetracycline for 10-14 days. chlamydia trachomatis can cause an afebrile pneumonia-like syndrome in young infants beginning at the age of 1-3 months, and is characterized by an afebrile state, failure to gain weight, and a staccato-like cough. on examination, there are rales, expiratory wheezing, and evidence of hyperaeration of the lungs. chest radiograph usually reveals diffuse interstitial pneumonia and hyperaerated lung fields. laboratory determinations show a modest eosinophilia and hyperglobulinemia. upon this identification, the infants can be treated with oral erythromycin syrup for 3 weeks with benefit. recently, c. trachomatis has been isolated from the lower respiratory tract of immunosuppressed patients with pneumonia, although four of the six patients reported and the only ones tested did not show a serologic response to that microorganism. 42 cases of community-acquired pneumonia in normal adults also have been reported with serologic evidence of infection with c. trachomatis. 43 fifty-two patients were studied and seven were found to have definite or suggestive serologic evidence of infection. 52 the seven ranged in age from 22 to 77. the chest radiographs of these patients have been analyzed and the infiltrates were found to be patchy and characteristically streaky with areas of plate atelectasis. there was no particular localization to a single lobe and three patients had radiographic evidence ofmultilobar involvement. 44 further studies need to be done to corroborate these reports and determine the frequency with which lung involvement occurs. in finland, an epidemic of mild pneumonia has been related to a newly described strain of c. psittaci, capable of being passaged from human to human. this epidemic occurred in adolescents and young adults and had a point prevalence of pneumonia of 15-19 cases/1,000 students at the time of x-ray survey. 45 the contribution of this particular strain of c. psittaci, designated the twar strain from tw-183 and ar-39, the first two isolates, has been examined best during a 2.5-year study at the university of washington. infected students usually presented with a mild pneumonia that simulated mycoplasma infection and was often associated with pharyngitis and laryngitis. 46 in this study, the twar strain of c. psittaci caused 12% of the pneumonias in the student population. the twar strain of c. psittaci was isolated from the students, and serial sera showed conversion to the common chlamydial group antigen by complement fixation tests. microimmunofluorescence tests revealed specific reactions to the twar strain of c. psittaci. the microorganisms isolated from the students formed typical inclusion bodies in tissue culture, were not stained by iodine, and were considered typical of c. psittaci. the clustering of cases had an epidemiology that suggested human-to-human transmission. bird-to-human transmission could not be demonstrated in any of the cases. treatment with tetracycline shortened the course, but, occasionally, patients did not respond to 1 gm of erythromycin given for 5-10 days. this new strain of c. psittaci was isolated from the patients and serologic reactions to specific antigens were demonstrated. the evidence linking c. trachomatis to lung disease has either been by isolation alone or just by serologic testing. further studies similar to the one in seattle need to be performed to link c. trachomatis to lung disease. it is clear, however, that a new strain of c. psittaci exists and can cause disease commonly. the disease due to this microorganism can be diagnosed by complement fixation or eia tests using chlamydial group antigen. specialized laboratories can isolate the organism and also perform microimmunofluorescence tests. a major new development in the evaluation of patients with primary typical pneumonia is the emergence of this c. psittaci strain that is capable of being passaged from human to human, and that may not have the desired response to erythromycin treatment. adenoviruses are ubiquitous nonenveloped dna viruses that colonize the human nasopharynx and are transmitted to other persons by close contact. types 4 and 7 are recognized for their capacity to produce epidemics in military recruit populations. because the transmission of this group of viruses is dependent upon close human contact, disease is often produced in the home or the military recruit barracks. pathogenetically, lung infection usually follows pharyngitis and a movement of the disease process down the respiratory tract. although most cases of pneumonia are not severe, cases coming to autopsy show that the tracheobronchial mucosa is denuded of the normal epithelial structures down to the basal layer. squamous metaplasia occurs along with interstitial space thickening due to the presence of chronic inflammatory cells. alveolar edema and mononuclear cell infiltrates are present. as with m. pneumoniae infections, infiltrates are often peribronchial or peribronchiolar in distribution. nuclear inclusion bodies or nuclei with a smudged appearance may be found in epithelial cells. clinically, the disease often begins with pharyngitis associated with fever and anterior cervical lymphadenopathy with or without conjunctivitis, then involves the tracheobronchial tree, and, finally, the parenchyma of the lung. pneumonia is most common in infants, young children, and military recruits. in military recruit populations, mycoplasma and adenovirus pneumonia have been found to be indistinguishable clinically except for an increased luby frequency of exudative pharyngitis with adenovirus infection. physical examination reveals pharyngitis and rhinitis and scattered rales and rhonchi. 47 evidence of consolidation is infrequent but occasionally lobar consolidation can occur, as can a pleural effusion 48 ( figure 5 ). virus rarely has been isolated from pleural fluid. 2 fatal cases of adenovirus pneumonia can occur in infants, immunosuppressed patients, and rarely in normal persons. 4 9-53 in these cases, the pneumonia is progressive with the development of diffuse bilateral alveolar infiltrates and hypoxemia requiring ventilator assistance for its correction. as the infiltrates progress, leukopenia ensues with marked lymphocytopenia. rhabdomyolysis occurs along with evidence of disseminated intravascular coagulation and renal failure. terminally, the patient becomes obtunded. bacterial suprainfection can be associated with adenovirus pneumonia. suprainfecting species of. bacteria include s. pneumoniae, group a strepto-· cocci, h. influenzae, s. aureus, and group y neisseria meningitidis. 54 in military recruits, an increased frequency of group y meningococcal suprainfection has been observed because these microorganisms commonly colonize the nasopharynx in this population. administration of antibiotics during the course of the adenovirus pneumonia does not prevent bacterial suprainfection. although most persons recover uneventfully from adenovirus pneumonia, occasional patients have residual abnormalities such as restrictive lung disease, bronchiectasis, or bronchiolitis obliterans. 55 ,56 extrapulmonic manifestations of adenovirus infection include pharyngitis, conjunctivitis, pericarditis, arthritis, skin rashes, and hepatic dysfunction. reye's syndrome has been described during the course of adenovirus pneumonia. 57 the occurrence of one or more of these manifestations during the course of pneumonia can lead the physician to order appropriate diagnostic tests to make a specific etiologic diagnosis. virus can be isolated from the nasopharynx, sputum, or endotracheal secretions. antigen can be detected by immunofluorescence tests, eia, or dna hybridization within epithelial cells derived from the respiratory tract. 58 these latter tests are specific but at the present time less sensitive than viral culture. there is no specific therapy for the infection. oral live attenuated vaccines are available against types 4 and 7 adenoviruses, and these are used now in the military to prevent epidemic disease. fatal adenovirus pneumonia. a 32-year-old man with an unremarkable past history except for hypertension was controlled on medication. two weeks before admission he developed a nonproductive, hacking cough and began to have dyspnea, which increased to the time of admission. on physical examination he appeared in moderate respiratory distress. oral temperature was 38.1° c, pulse rate was 100/min, blood pressure was 1701106, and respiratory rate was 30/min. the oropharynx was described as normal. scattered rhonchi and rales were heard diffusely through the lungs. a summation gallop was heard at the cardiac apex. laboratory examination revealed a white blood count of 5,100 with 82% polymorphonuclear cells, 11% band forms, 3% lymphocytes, and 4% monocytes. arterial blood gases on room air showed a ph of 7.44, pco. of 31, and po. of 56. ekg showed left ventricular hypertrophy. chest radiograph revealed an enlarged cardiac silhouette with patchy alveolar infiltrates ofthe entire right lung and left lower lobe. the patient was started on erythromycin 500 mg every 6 hours intravenously. he continued to spike temperatures to 40· c. cefamandole and tobramycin were added to his antibiotic therapy. two days after admission, the creatine phosphokinase value rose to 16,420 and the next day was 37,576. his creatinine rose to 4.8 mg/dl. his heart was enlarged on radiograph and the pulmonary infiltrates continued to increase. his mental status gradually deteriorated and he was transferred to the intensive care unit. the white blood count was 3,700 with 12% lymphocytes. he developed evidence of disseminated intravascular coagulation and died on the eighth hospital day of respiratory insufficiency. postmortem examination revealed changes of viral pneumonia, with some epithelial cells showing intranuclear inclusions and the appearance of "smudged" nuclei, an enlarged heart due to idiopathic myocardial disease, only minimal pathologic evidence of hypertension, and findings of disseminated intravascular coagulation. electron microscopy oflung sections revealed adenovirus. the adenovirus complement fixation test titer rose from less than 1:8 to 1:64. comment. the patient had a 2-week febrile period before admission, and pulmonary infiltrates progressed on antibiotic therapy. he developed leukopenia, lymphocytopenia, rhabdomyolysis, disseminated intravascular coagulation, and acute renal failure. his illness occurred in the setting of idiopathic myocardial disease, and it is possible that mild, chronic, left ventricular failure might have predisposed him to severe adenoviral pneumonia similar to the manner in which cardiac failure augments influenza pneumonia. pneumonia due to respiratory syncytial virus. respiratory syncytial virus is the predominant respiratory tract viral pathogen of infancy and young childhood. infection in adults usually results in no symptoms or a mild upper respiratory tract illness such as the common cold. it is now recognized that immunosuppressed patients and elderly persons can develop pneumonia because of rsv and that it can be severe and complicated by bacterial suprainfection. 5 9-61 furthermore, since immunosuppressed and elderly persons may aggregate in hospitals and nursing homes, these institutions are often sites of acquisition of infection. an epidemic of pneumonia and febrile respiratory illness took place in los angeles in february-march, 1979. 62 forty of 101 residents were affected, 22 having pneumonia. eight persons died for a case-fatality rate of 20%. other such outbreaks have been recorded. nosocomial acquisition ofrsv is very difficult to prevent. hospital personnel become colonized and may have no or mild respiratory tract symptoms. transfer of virus can occur by patient to personnel to patient transmission or directly from the personnel themselves. hands and fomites become contaminated by respiratory secretions and virus is spread to patients by direct contact with these sources. the pathology of pneumonia due to rsv is similar to that of other viral pneumonias; however, epithelial cells with intracytoplasmic inclusion bodies can be seen. the x-ray appearance of the pneumonia can be that of a diffuse interstitial process or have interstitial and patchy alveolar infiltrates in the lower lobes, or have an appearance indistinguishable from the adult respiratory distress syndrome. pneumonia due to rsv in immunosuppressed and elderly persons is a newly described phenomenon, but one that may be of increasing importance. it is also important because rsv infections can be diagnosed early by antigen detection techniques (immunofluorescence or eia) and because effective therapy has been developed recently. antigen detection tests for rsv now equal or exceed the efficacy of viral cultures for diagnosis of infection. respiratory syncytial virus infections in infancy now have been treated successfully with aerosolized ribavirin. 63 • 64 this therapy is indicated for infants and children with lower respiratory tract involvement with rsv who are exceptionally ill or who may have congenital heart disease or bronchopulmonary dysplasia. with aerosol delivery by oxygen tent, hood, or mask, concentrations of ribavirin are quite high in the upper and lower respiratory tract and exceed the minimum inhibitory concentration necessary to inhibit the growth of the virus in tissue culture. to achieve this concentration by oral administration of the drug, unacceptable toxicity would be en-countered. this toxicity would include bone marrow depression and particularly anemia related both to maturation arrest and, to a lesser extent, hemolysis. this latter event occurs because ribavirin triphosphate can accumulate in erythrocytes, having a halflife greater than 40 days, and interferes with the formation of guanosine triphosphate. aerosolized ribavirin therapy is expensive, but is presently approved by the food and drug administration for the therapy of complicated rsv infections in infants and young children. it represents the first example of an effective drug for treating a significant lower respiratory tract viral infection. it is conceivable that this technology could be applied to influenza infections. influenza and rubeola will be covered in detail but it is now recognized that other rna viruses can cause lower respiratory tract involvement in adults. these viruses include the parainfluenza viruses, respiratory enteroviruses such as coxsackie b viruses and coxsackie virus a21, rhinoviruses, and coronaviruses. 65 the magnitude of the problem, however, appears to be limited. documented instances of severe lower respiratory tract infection due to parainfluenza ii and iii viruses have occurred, however. there is presently no accepted therapy for these latter infections, although aerosolized ribavirin has been used successfully to control persisting parainfluenza virus infections of the lower respiratory tract in immunodeficient children. influenza a virus is the cause of pandemics and epidemics that occur every or every other year. all influenza a viruses possess a common group complement fixation test antigen, the nucleoprotein antigen. influenza a viruses differ in the antigenic character of the hemagglutinin and neuraminidase. the h1n1 strain of influenza a circulated in the world from 1918-1919 through 1957, when asian influenza strains (h2n2) became predominant. these strains circulated until 1968-1969, when hong kong influenza (h3n2) appeared; strains of this virus continue to be transmitted. h1n1 strains again began to circulate during 1976 and they continue to do so. influenza b strains have the same common complement fixation test antigen, and this differs from that of influenza a virus. the hemagglutinin and neuraminidase of influenza b virus are less prone to change; pandemic disease due to this virus does not occur and the interepidemic interval is longer than that of influenza a, namely, every 3-4 years. serious morbidity due to influenza a virus occurs because of host factors such as age, underlying disease, and immunosuppression; because immunity wanes with time; and because influenza a viruses are constantly changing their antigenic character. in pandemic years, when both the hemagglutinin and neuraminidase change concomitantly, the american journal of the medical sciences luby there is a tendency for more serious disease to occur than if just one of the surface proteins changes. this is well illustrated by the 1918-1919 and 1957 pandemics. influenza a viruses may, on certain occasions, be more virulent. in the 1918-1919 epidemic the pneumonia rate in persons from the ages of 25 to 40 years was approximately 10% of those who had influenza. 66 this and other facts have been cited to indicate the virulence and striking pneumotropism of the virus that led to 20 million deaths occurring throughout the world during the pandemic. influenza b viruses are more likely to cause disease in younger persons and only occasionally do epidemics occur in which there is excess mortality. pathogenetically, influenza virus attaches to cells of the respiratory epithelium and enters by a process termed "receptor-mediated endocytosis." the virion is uncoated in the endosome by fusion with the membrane of this structure, a process requiring an acidic ph. the particle then undergoes a cytoplasmic and a nuclear stage of replication. virion rna is capped and polymethylated in the nucleus so that the rna message now can be recognized by the cell and translated at the ribosome. 67 in the process of replication, the virus rapidly destroys respiratory tract epithelial structures in order to compromise natural defense mechanisms of the lung, such as mucous production and ciliary activity. in cases of severe pneumonia the epithelium of the trachea and bronchi are destroyed down to the basal layer and then metaplasia occurs, leaving the respiratory tract coated with a layer of squamous cells. there is involvement of bronchiolar structures and an intense peribronchiolar inflammatory process. in uncomplicated influenza, small airways are commonly affected, producing diffuse dysfunction in these structures, mild hypoxemia, and a compensated respiratory alkalosis. 68 . 69 in severe influenza pneumonia, there is alveolar cell destruction and disruption of the alveolar-capillary membrane resulting in hemorrhage into the alveoli along with edema, a mononuclear cell infiltrate, and the presence ofhyaline membranes. thickening of the interstitium occurs with a chronic inflammatory cell infiltrate. the process can be fulminant, occurring coincident with the onset of illness, or it can be more protracted, leading to the occurrence of progressive infiltrates over 5-7 days. when an adult respiratory distress syndrome-like picture is produced, influenza pneumonia has a high case-fatality rate, which may approximate 75%.70.71 not all influenza a pneumonia is this severe, however, and there are cases in which only an interstitial or bronchopneumonic process is apparent and the disease simulates m. pneumonia, except that in influenza the leukocyte count tends to be normal or decreased. 72.73 influenza pneumonia can coexist with bacterial suprainfection or bacterial suprainfection can occur alone. the offending bacterial pathogens may vary between pandemics; in 1889-1890, h. in{luenzae evidently was a major pathogen. in the 1918-1919 pandemic, the group a streptococcus was considered a major pathogen; more recently, s. pneumoniae has been the most common offending agent followed by s. aureus andh. in{luenzae. occasionally, other gram-negative bacteria may be involved. influenza b virus can cause a similar spectrum of pulmonary disease, but the number of patients involved is fewer. the hospitalization rate for lower respiratory tract disease nearly always increases during influenza a epidemics. this rate tends not to increase during influenza b epidemics, although total hospitalizations may be increased during this period; like influenza a virus, influenza b virus can cause a variety of disease processes outside the lung. these include myopericarditis, rhabdomyolysis, disseminated intravascular coagulation, nervous system disturbances such as encephalitis, reye's syndrome, the landry-guillan-barre : syndrome, the stevens-johnson syndrome, and others. 4 • 74 . clinically, the patient with influenza virus pneumonia has the sudden onset off ever, prostration, and myalgias followed shortly by dyspnea. blood-tinged sputum may be produced. the dyspnea progresses until hospitalization and ventilatory support are required. the illness can also assume a more protracted course leading to progressive interstitial and alveolar infiltrates over a week ( figure 6 ). some pa-tients simply have viral pneumonia with pulmonary dysfunction but do not need ventilator assistance. complicating bacterial suprainfection may coexist with viral pneumonia or more commonly presents after an afebrile interval, during which the patient appears to be recovering from the primary infection. morbidity and mortality are greatest in elderly persons, in those with chronic disease states such as chronic obstructive pulmonary disease, chronic congestive heart failure or diabetes mellitus, and in immunosuppressed patients. morbidity due to influenza a and b viruses is not limited to these groups, however. women in the third trimester of pregnancy also may have an increased rate of developing influenza pneumonia and death due to this disease process. 75 in renal transplant recipients who contract influenza a, illnesses are often prolonged, with the development of viral pneumonia, bacterial suprainfection, and myopericarditis. there may be loss of the renal allograft due · to the combination of these disease processes. 76 influenza a and b virus infections are diagnosed by serial titer rises in a suitable serologic test such as the complement fixation or the hemagglutination inhibition test. a single complement fixation test titer :::1: 128 has been shown to correlate highly with recent influenza b infections. 4 virus can be grown from the nasopharynx or endotracheal secretions by inoculation of the specimen into rhesus monkey kidney or madin-darby canine kidney tissue culture. embryonated eggs sometimes need to be used for figure 6 . influenza pneumonia. the radiograph on the lett shows combined influenza pneumonia with lett lower lobe consolidation indicating bacterial supra infection. the radiograph on the right shows diffuse alveolar infiltrates in the course of serologically documented influenza a infection. bacterial cultures from endotracheal secretions consistently showed no growth. optimal recovery of virus. virus may be able to be identified within 72 hours by using immunofluorescence. direct detection of antigen by immunofluorescence or eia can be applied to appropriate secretions, but these tests are not yet as sensitive as viral culture. 77 amantadine and rimantadine are two compounds that have both prophylactic and therapeutic efficacy against influenza a but not influenza b virus. they act by preventing uncoating of influenza a virus, perhaps by preventing the development of an acidic ph so that the envelope of the virion cannot fuse with the endosomal membrane. at the dosage given, 100 mg twice a day, amantadine has more central nervous system side effects and the dose has to be adjusted with renal failure. 78 the dose of rimantadine does not have to be adjusted with renal dysfunction because the compound is metabolized in the body. a study sponsored by the national institutes of health is underway evaluating whether rimantadine can be used effectively in the therapy of hospitalized patients with influenza a, and would include patients with influenza a virus pneumonia. ribavirin has in vitro efficacy against both influenza a and b viruses. it has multiple sites of action including interference with the formation of guanosine triphosphate and deoxyguanosine triphosphate and prevents placement of the polymethylated cap structure on the influenza a viral rna message. with the aerosolization of ribavirin, high concentrations of the drug can be produced within the respiratory tract but serum levels are low. 79 ,8o attempts most likely will be made in the future to treat influenza a virus pneumonia with aerosolized ribavirin or a combination ofribavirin and rimantadine. influenza b virus pneumonia may be able to be treated with aerosolized ribavirin. vaccines exist for both influenza a and b viruses, and standard medical care necessitates yearly immunization of elderly patients or those with underlying medical conditions. a recent emphasis of the public health service is to have medical personnel also immunized yearly, since they are. exposed to persons with influenza, may develop that illness themselves, and may then transmit the infection to sick patients within the hospital. nosocomial influenza pneumonia. a 55-year-old alcoholic man was admitted to the hospital on january 30 with alcoholic liver disease, macrocytic anemia, and symptoms of bladder neck obstruction. he was a heavy smoker and had evidence of chronic obstructive pulmonary disease. 'lwelve days after admission into the hospital, he developed fever to 39.2° c while awaiting a urologic procedure. he "felt terrible" with myalgias and developed a cough, mild diarrhea, and dyspnea. chest examination revealed diffuse rales and rhonchi. chest radiograph showed new interstitial infiltrates, more prominent on the right. the sputum was mucoid. the diagnosis of pneumonia was made and the diag-the american journal of the medical sciences luby nosis of congestive heart failure with pulmonary edema considered. however, his heart examination revealed no gallop sounds and his neck veins were not distended. he was placed on ampicillin, became afebrile after 5 days, and his dyspnea improved with low flow oxygen by face mask. influenza a complement fixation test titer on a single convalescent serum specimen was 2::1: 256. comment. influenza virus pneumonia occurred during hospitalization. the pneumonia cleared with symptomatic therapy. the public health service now recommends widespread immunization of medical personnel in an attempt to prevent nosocomial acquisition of influenza. although predictions were made that rubeola would be eradicated in this country during the early 1980s, this has not been achieved. in dallas, texas, during 1986 more than 150 cases of rubeola occurred. this marked a resurgence of cases after a relatively disease-free interval after 1971, when a large epidemic occurred in dallas, causing more than 1,000 cases, including three deaths. as a consequence ofthis and other epidemics, in 1971 texas adopted a law requiring the compulsory immunization of children against measles, mumps, rubella, poliomyelitis, diphtheria, and tetanus. present rubeola vaccines are at least 95% effective, but universal immunization of the preschool child is not practiced, particularly in lower socioeconomic class population groups. furthermore, rubeola virus has been found to violate the concept of herd immunity, a major principle on which eradication was based, because outbreaks occur in high schools and colleges in which a large percentage of the population has been immunized. an inactivated vaccine was available from 1962 through 1965. children who received the inactivated vaccine may have developed atypical measles on exposure to rubeola virus. following this, an attenuated, live strain of measles virus was used as vaccine but the high sidereaction frequency necessitated the concomitant administration of 'v-globulin. it is now recognized that the concurrent use of 'v-globulin sometimes rendered immunization ineffective. many children were immunized before the age of 12 months; for effective immunization to occur, vaccine must be given after the age of15 months. as a consequence of the lack of universal preschool immunization and difficulties related to the vaccine, there now exist two population groups who may be nonimmune with respect to rubeola, ie, preschool children, and adolescents and young adults. a few years ago rubeola epidemics were common in military recruits. following the occurrence of these outbreaks, recruits now routinely undergo serologic testing, and if antibody to rubeola virus is not detected by either hemagglutination inhibition or indirect immunofluorescence tests, live attenuated vaccine is given. this practice essentially has stopped the occurrence of these outbreaks in the military. persons who received the inactivated vaccine can develop atypical measles. the first cases of this new syndrome were misdiagnosed as rocky mountain spotted fever. they were confused with this disease because the rash began on the extremities and spread inward to involve the trunk. the rash could be maculopapular, vesicular, or petechial. in atypical measles, pulmonary involvement consists of nodular infiltrates, lobar consolidation, and the occurrence of pleural effusions. hilar lymphadenopathy may also present in these patients. s1 • s2 they have an anamnestic response in antibody production with the infection. mild eosinophilia may also be present and the virus cannot be recovered from the nasopharynx. in young adults with typical measles, approximately 5% develop clinical evidence of pneumonia. radiographic evidence of pneumonia, however, may be seen in up to 50% of the patients. the pneumonia is usually characterized by diffuse bilateral interstitial or fine reticulonodular infiltrates, particularly affecting the lower lobes. bacterial suprainfection occurs in as many as 30% of cases of recognized viral pneumonia. the types of bacteria causing infection may be determined by the circumstances in which disease occurs, such as military recruit populations. recognized pathogens include h. influenzae, s. pneumoniae, group a streptococci, group y meningococci, and s. aureus. bacterial suprainfection generally occurs between the fifth and tenth days after the rash and is heralded by clinical worsening, new or different lung infiltrates, or changes in sputum characteristics or the white blood count. antibiotic treatment of viral pneumonia does not prevent bacterial suprainfection. in immunodeficient children, measles pneumonia occurred without a rash and pathologically was called giant-cell pneumonia. it is now recognized that these children lacked cell-mediated immunity and had depressed and delayed antibody production. s3 intact cell-mediated immunity is essential for rash production. in fatal rubeola pneumonia, the entire tracheobronchial tree may be denuded of cells down to the basal layer; squamous metaplasia of the cells occurs; there is widening of the interstitial space with edema and inflammatory cells; and alveoli are filled with edema, hyaline membranes, and mononuclear cells. in addition, giant cells containing multiple nuclei are found within the tracheobronchial epithelium. extrapulmonic manifestations of measles occur and include otitis media, sinusitis, encephalitis, and the common presence of hepatic dysfunction in young adults, mostly consisting of mild elevations of the serum aspartate aminotransferase and lactic dehydrogenase. there presently is no specific therapy. some public health authorities now think that reimmunization with measles, mumps, and rubella vaccines should be 58 given before or when the child enters high school. medical personnel not sure of their rubeola immunity should have that status assessed by determination of specific antibody. varicella-zoster virus can produce pneumonia during the course of varicella or disseminated herpes zoster and can be a severe disease that can lead to mortality. varicella in childhood is not usually associated with viral pneumonia, but bacterial suprainfection can occur, necessitating appropriate antibiotic treatment. in immunosuppressed children, however, pure viral pneumonia can occur in association with varicella. in adults, there is a tendency for the virus to affect the lung relatively commonly during varicella. fifteen to twenty percent of all adults with varicella may have x-ray evidence of pneumonia but only about 5% require hospitalization. most frequently in adults, varicella pneumonia is not complicated by bacterial suprainfection; however, this can occur, particularly when patients require intubation. the virus reaches the lung both by passage down the respiratory tract and by hematogenous seeding because the rash is occurring at the same time as the pneumonia. initially, the pneumonic process appears as nodular infiltrates, 1-4 mm in diameter associated with an interstitial inflammatory infiltrate. a4 the lesions are more dense toward the hilum and are the counterpart in the lung of the pox occurring on the skin (figure 7) . peribronchial inflammatory infiltrates, hilar adenopathy, and pleural effusions may occur. the reticulonodular interstitial infiltrate can progress to widespread alveolar damage and diffuse pulmonary parenchymal infiltrates. pathologically, the pneumonia resembles influenza pneumonia except that areas of coagulative necrosis can be seen. although these necrotic areas generally clear during the course of clinical disease, they can become calcified, and the radiograph shows a picture of miliary calcifications. it has been shown that this area of coagulative necrosis can become surrounded by an inflammatory infiltrate and resemble a granuloma. a5 fibrous tissue envelopes the necrotic granulomatous process and the lesion eventually calcifies. another process occurring in varicella pneumonia is destruction of the epithelium of the trachea and bronchi. in cases in which the illness is protracted, the development of a thick, fibrinopurulent crust may occur over the lower pharynx, larynx, and upper trachea. this thick crust can cause respiratory embarrassment and can pose a problem for intubation. disseminated herpes zoster can cause the same processes in the lung. most normal adults recover from varicella pneumonia without difficulty, but there can be substantial respiratory morbidity and morfigure 7 . varicella pneumonia. the chest radiograph on the lett is from the case report described in the protocol. the close-up film on the right in another pallent shows peribronchial infiltrates and multiple 1-4-mm rounded opacities in the right lung field. tality in immunosuppressed patients or in women during the third trimester ofpregnancy. 86 clinically, the patient with varicella-zoster virus pneumonia presents with a rash followed by cough and dyspnea. the sputum is initially white and modest in amount, but can become hemorrhagic. the process can be complicated by the development of chest pain and pleural effusions, which are often blood tinged and related to the presence of pox on the pleural surface. extrapulmonic manifestations of varicella occur and consist of the characteristic skin rash, otitis media with bacterial suprainfection, myopericarditis, hepatic dysfunction, and encephalitis. reye's syndrome can complicate the course ofvaricella. there can be an associated glomerulonephritis and varicella virus can occasionally induce frank arthritis. in caring for patients with varicella-zoster virus pneumonia, it is important to realize that the external appearance of the patient or his apparent well-being may disguise underlying hypoxemia. if efforts are not made to diagnose and correct the hypoxemia, the patient may become confused, perform inappropriate activity, and become more hypoxemic. ventilator support may become necessary. deaths in varicella pneumonia occur because of respiratory insufficiency, the development of tension pneumothoraces, bacterial suprainfection, or progressive pulmonary fibrosis. two antiviral compounds, adenine arabinoside (ara-a) and acyclovir (acv), have been proven to be efficient in the treatment of significant, complicated varicella-zoster virus infections. adenine arabinoside inhibits viral dna polymerase and is given at a dosage of 10 mglkg over a 12-hour period for at least 5 days. the dosage could be increased to 15 mglkg the american journal of the medical sciences but the majority of experience with varicella-zoster virus infections is with 10 mglkg/day. the drug is sparingly soluble so that 2 ml of vehicle are required for each milligram of drug administered to the patient. at a dose of 10 mglkg, bone marrow suppression does not usually occur. if the dose is not decreased in the setting of hepatic and renal dysfunction, central nervous system disturbances, which consist of insomnia, hallucinations and tremulousness, may occur. these central nervous system manifestations usually fade with stopping the drug and rarely lead to death, but can persist for a protracted period after the drug has been discontinued. acyclovir also has been used to treat varicella-zoster virus infections. it inhibits viral dna polymerase and also acts as a chain terminator. its dosage is 500 mg/m 2 every 8 hours for at least 7 days. the only significant problem with the administration with acyclovir in this setting is the production of an obstructive nephropathy, due to salting out of the drug in the collecting tubules ofthe kidney. this is usually easily managed by administration of a fluid bolus, a diuretic, or mannitol. a comparison of the two drugs in complicated varicella-zoster virus infection has been made. 87 . 88 one group found that acyclovir was more efficacious; the other study determined that ara-a was equally as effective. because the administration of ara-a requires an increased volume of fluid and can be associated with central nervous system side effects, some authorities now consider acyclovir the drug of choice in the treatment of complicated infections due to varicella-zoster virus. oral acyclovir is absorbed poorly by the gastrointestinal tract and its efficacy in uncomplicated herpes zoster is apparent only when 800 mg are given 5 times a day for at least a 5-day period. there has been no experience with oral acv in treating varicella pneumonia. future modes of therapy may include combining ara-a with acv or administering one or other of the drugs with an interferon preparation. alpha-interferon also has been shown to be effective as therapy in complicated varicella-zoster virus infections but its use has been superceded by acv and ara-a. case report varicella pneumonia. a 39-year-old man was exposed to his two children with chicken pox. two days before admission he developed a rash, then dyspnea. on physical examination, a typical varicella rash was present. he was in severe respiratory distress. rales were present diffusely over both lung fields. the chest radiograph revealed bilateral extensive alveolar infiltrates. arterial blood gases showed a ph of 7.42, pc02 of 32, and a p02 of 24. he was intubated and begun on positive end-expiratory pressure (peep) with a fi0 2 of 70%. he was started on intravenous acyclovir 500 mg/m2 every 8 hours. he improved and was able to be extubated after 5 days. comment. severe varicella pneumonia responded to intravenous acyclovir while his oxygenation was maintained on peep with a high fi02. herpes simplex virus can cause a necrotizing bronchopneumonia in neonatal infections and can also cause pneumonia in severely immunosuppressed adult patients. the largest series of patients with herpes simplex virus pneumonia was reported from seattle in bone-marrow transplant recipients 'and consisted of 20 patients with either a focal pneumonia (12 patients) or a diffuse interstitial pneumonia (eight patients). 89 the focal pneumonia was found associated with herpetic esophagitis and tracheitis and probably resulted from contiguous spread of herpes virus to the lung parenchyma. diffuse interstitial pneumonia most probably resulted from hematogenous dissemination of virus to lung. pathologically, the process can be one of a necrotizing bronchopneumonia or of a widening of the interstitial space in the lung associated with diffuse alveolar injury. in these highly immunosuppressed patients, both bacterial and fungal suprainfection occurred and it was difficult to sort out which process was responsible for what proportion of lung damage. acyclovir has been shown to be an effective treatment of complicated herpes simplex virus infections in immunosuppressed patients. its dose in the usual patient is 250 mg/m 2 every 8 hours for at least 7 days, but if the process has been ascertained to be a rapidly progressive herpetic pneumonia, the dose could be increased to 10 mglkg every 8 hours until a therapeutic response had been obtained. with the development of potent antiviral chemotherapy, there is a need to consider and diagnose herpetic pneumonia. specific diagnosis can only be accomplished readily by lung biopsy. 60 although involvement of the lung in infectious mononucleosis due to epstein-barr virus must be considered a rare occurrence, recent studies and case reports demonstrate that it probably can happen. 90 . 91 careful attention should be given to possible coexisting mycoplasma and other viral infections, particularly since the former can be treated. radiographic abnormalities may consist of hilar adenopathy, strand-like parenchymal infiltrates, diffuse bilateral pneumonia, and a picture consistent with primary atypical pneumonia. although epstein-barr virus is susceptible to acyclovir, there are no reports treating lung involvement with this drug. cytomegalovirus (cmv) rarely causes pneumonia in normal adults as part of the cmv mononucleosis syndrome. 92 however, it is more common for cmv to induce pneumonia in normal hosts than epstein-barr virus. of 443 patients with communityacquired pneumonia, 18 had virologic,2 pathologic,2 or serologic 14 evidence of cmv infection. 93 ten of these 18 patients were not immunosuppressed. in five of the ten, cmv was the only pathogen. the remaining five patients had one or more coexisting infections; c. trachomatis in two, m. pneumoniae in one, epstein-barr virus in one, and bacteria in three, both aerobic and anaerobic. cytomegalovirus more commonly causes pneumonia in immunosuppressed patients. it occurs particularly in renal, heart, liver, and bone-marrow transplant recipients. it is now becoming an increasing problem in patients with aids. in the transplant recipient experiencing a primary cmv infection, the virus most probably reaches the lung parenchyma through the hematogenous route, and the first finding is that of a reticulonodular infiltrate and the presence of 1-4 mm opacities ( figure 8 ). pathologically, these focal areas usually consist of necrotic tissue, hemorrhage and alveolar damage with edema, a mononuclear infiltrate, and typical cytomegalic cells. the process can extend leading to diffuse interstitial and alveolar infiltrates. an attempt has been made to separate the foregoing process from that of an insidiously developing interstitial pneumonia that occurs more commonly in reactivated infections and has a better prognosis. 94 in bone marrow transplant recipients, diffuse interstitial pneumonitis due to cmv is much more common in patients receiving allogeneic transplants, and the case-fatality rate approximates 90%. 95 in some renal transplant recipients, the pneumonic process can be focal and does not have to be exceptionally severe. 96 small pleural effusions can occur occasionally. in other renal transplant recipients, however, cmv pneumonia can progress rapidly and lead to death as part of a widely disseminated infectious process. 97 in cardiac transplant recipients a variety of pulmonary opportunistic suprainfections has been well documented to occur in the course of cmv pneumonia. 98 typical microorganisms causing suprainfections include p. carinii and nocardia species. in patients with aids, there may be co-existing infection with pneumocystis. cure of the pneumocystis can be effected by drugs, leaving cmv as the major pulmonary pathogen. in patients with aids, rapid development of cmv pneumonia can occur and lead to the death of the patient. extrapulmonic manifestations of cmv in the renal transplant recipient include fever, malaise, hepatic dysfunction, splenomegaly, leukopenia, and an increase in serum creatinine. 96 with extensive cmv dissemination in heavily immunosuppressed patients, including those with aids, extrapulmonic manifestations include gastrointestinal ulceration with bleeding and perforation, hepatic dysfunction, adrenal cortical involvement, and central nervous sytem dysfunction. cytomegalovirus can be thought of as an immunosuppressive viral agent, and infection with this microorganism may lead to further immunosuppression with consequent bacterial, fungal, and parasitic suprainfection. clinically, patients with cmv pneumonia complain of dyspnea with a nonproductive cough. there can be an associated pleurisy. the process can be transient or can extend to respiratory insufficiency necessitating ventilatory support. therapy of fully developed cmv pneumonia has been shown not to be effective and this includes the use of the adenine arabinoside, acyclovir, ganciclovir (dhpg), and combinations of interferon with all the above. ganciclovir has been successful in achieving an antiviral effect in the lung, yet has not improved outcome in the american journal of the medical sciences bone marrow transplant recipients with cmv pneumonia. 99 in an occasional renal transplant recipient who has the potential of a good immune response to the virus, the cmv illness that can include localized pneumonia might be benefited by the judicious use of ganciclovir. attempts at preventing cmv pneumonia have included donor selection, avoidance of white blood cell transfusions, and prophylactic administration of alpha-interferon or 'y-globulin preparations given before and through the first 60 days after transplantation. alpha-interferon does prevent cmv viremia in the renal transplant recipient; 'y-globulin protects partially against cmv pneumonia if white blood cell transfusions have not been given. studies are in progress trying to make this latter effect more consistent, and consist of determining whether total antibody content is the necessary component or whether the effect necessitates the presence oflarge quantities of neutralizing antibody. on a priority basis, live, attenuated cmv vaccine development has been curtailed for the immediate future. a 40-year-old homosexual man presented to the hospital with fever, cough, and an erythematous rash. he had been followed in clinic with aids-related complex with lymphadenopathy, thrush, lymphopenia, anergy, diarrhea, and a positive antibody test to human immunodeficiency virus. at the time of his acute terminal illness he had a temperature of 38.5° c and had a diffuse, erythematous pruritic rash over the trunk and upper legs. the admission chest radiograph was interpreted as normal. on the second hospital day, the patient became delirious, had a worsening cough, and developed severe dyspnea. arterial blood gases on room air revealed a ph of 7.50, pco. of 30, and a po. of 44. chest radiograph now revealed bilateral diffuse reticulonodular infiltrates. he was started on sulfatrimethoprim but had a respiratory arrest and died. postmortem examination revealed cmv pneumonia without evidence ofpneumocystis. lung viral cultures rapidly grew cmv, with the cytopathic effect being present the second day. comment. explosive illness in a patient with arc revealed only cmv at autopsy and on viral culture of the lung. viral and mycoplasmal pneumonia in a prepaid medical care group during an eight-year period primary atypical pneumonia in a family due to concomitant mycoplasma pneumoniae and adenovirus type 7 infection viral pneumonia as a cause and result of hospitalization severe illness with influenza b strannegard 0, trollfors b: etiology of community-acquired pneumonia in out-patients etiologies and characteristic features of pneumonias in a municipal hospital etiology of community-acquired pneumonia in patients requiring hospitalization adult community-acquired pneumonia in central london virological investigations in adults with acute pneumonia hospital study of adult community-acquired pneumonia acute community-acquired pneumonias an acute infection of the respiratory tract with atypical pneumonia. a disease entity probably caused by a filtrable virus causes of atypical pneumonia: results of a 1-year prospective study differential diagnosis of viral, mycoplasmal and bacteraemic pneumococcal pneumonias on admission to hospital comparative radiographic features of community acquired legionnaires' disease, pneumococcal pneumonia, mycoplasma pneumonia, and psittacosis mycoplasma pneumonia: failure of erythromycin therapy lung infections caused by viruses, mycoplasma pneumoniae, and rickettsiae pneumonias due to rickettsiae, chlamydiae, viruses and mycoplasma neurologic disease associated with mycoplasma pneumoniae pneumonitis. demonstration of viable mycoplasma pneumoniae in cerebrospinal fluid and blood by radioisotopic and immunofluorescent tissue culture techniques open lung biopsy in myco-62 plasma pneumoniae pneumonia mycoplasma pneumoniae infection in families pulmonary involvement in mycoplasma pneumoniae infection in families the clinical spectrum and diagnosis of mycoplasma pneumoniae infection clinical features of mycoplasmal pneumonia in adults mycoplasmal pneumonias in the community hospital. the "unusual" manifestations become common lobar pneumonia caused by mycoplasma pneumoniae radiographic appearances of mycoplasma pneumonia mycoplasmal pneumonia and adult respiratory distress syndrome: a complication to be recognized acute respiratory failure due to atypical pneumonia the protean manifestations of mycoplasma pneumoniae infection in adults weekly clinicopathological exercises: case 39-1983 pityriasis rosea gibert and mycoplasma pneumoniae infection abnormalities in lung function following clinical recovery from mycoplasma pneumoniae pneumonia van der straeten 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virus pneumonia in three immunocompromised adults an outbreak of respiratory syncytial virus pneumonia in a nursing home for the elderly ribavirin treatment of respiratory syncytial viral infection in infants with underlying cardiopulmonary disease ribavirin aerosol treatment of bronchiolitis due to respiratory syncytial virus infection in infants coronavirus infections of man associated with diseases other than the common cold influenza: the newe acquayantance orthormyxo-and paramyxoviruses and their replication, in fields bn sanfordjp: pulmonary function in uncomplicated influenza pulmonary mechanics after uncomplicated influenza a infection studies on influenza in the pandemic of 1957-1958. ii. pulmonary complications of influenza severe influenza virus pneumonia in the pandemic of 1968-1969 mycoplasma and influenza pneumonia in a series of 214 adults the leukocyte response during viral respiratory illness in man acute myocarditis in influenza a infections. two cases of non-bacterial myocarditis, with isolation of virus from the lungs fatal influenza a pneumonia in pregnancy epidemic renal transplant rejection associated with influenza a victoria rapid diagnosis of primary influenza pneumonia pharmacokinetics of amantadine hydrochloride in subjects with normal and impaired renal function ribavirin small-particle aerosol treatment of influenza ribavirin small-particle aerosol treatment of infections caused by influenza virus trains anictoriaj7/83 (h1n1) and bltexas/1/84 measles pneumonia. bacterial suprainfection as a complicating factor measles pneumonia in young adults. an analysis of 106 cases isolation of measles virus at autopsy in cases of giant-cell pneumonia without rash report of seven cases and a review of literature persistent pulmonary granulomas after recovery from varicella pneumonia varicella pneumonia complicating pregnancy treatment of varicellazoster virus infection in severely immunocompromised patients comparative trial of acyclovir and vidarabine in disseminated varicella-zoster infections in immunocompromised patients coreyl: herpes simplex virus pneumonia. clinical, virologic, and pathologic features in 20 patients diffuse pneumonia and acute respiratory failure due to infectious mononucleosis clinical, virologic, and serologic evidence of epstein-barr virus infection in association with childhood pneumonia pneumonia associated with rising cytomegalovirus antibody titres in a healthy adult does cytomegalovirus playa role in communityacquired pneumonia? cytomegalovirus pneumonia in bone marrow transplant recipients: miliary and diffuse patterns nonbacterial nonfungal pneumonia following marrow transplantation in 100 identical twins disease due to cytomegalovirus and its long-term consequences in renal transplant recipients. correlation of allograft survival with disease due to cytomegalovirus and rubella antibody level clinical characteristics of the lethal cytomegalovirus infection following renal transplantation diagnosis of cytomegalovirus pneumonia in compromised hosts activity of 9-[2-hydroxy-1-(hydroxymethyl)-ethoxymethyl]guanine in the treatment of cytomegalovirus pneumonia key: cord-006362-7d5wzb7p authors: van riel, debby; mittrücker, hans-willi; engels, geraldine; klingel, karin; markert, udo r.; gabriel, gülsah title: influenza pathogenicity during pregnancy in women and animal models date: 2016-07-07 journal: semin immunopathol doi: 10.1007/s00281-016-0580-2 sha: doc_id: 6362 cord_uid: 7d5wzb7p pregnant women are at the highest risk to develop severe and even fatal influenza. the high vulnerability of women against influenza a virus infections during pregnancy was repeatedly highlighted during influenza pandemics including the pandemic of this century. in 2009, mortality rates were particularly high among otherwise healthy pregnant women. however, our current understanding of the molecular mechanisms involved in severe disease development during pregnancy is still very limited. in this review, we summarize the knowledge on the clinical observations in influenza a virus-infected pregnant women. in addition, knowledge obtained from few existing experimental infections in pregnant animal models is discussed. since clinical data do not provide in-depth information on the pathogenesis of severe influenza during pregnancy, adequate animal models are urgently required that mimic clinical findings. studies in pregnant animal models will allow the dissection of involved molecular disease pathways that are key to improve patient management and care. influenza a viruses (iav) belong to the major causative agents of respiratory infections posing a considerable burden for human health. the clinical course of iav infections in healthy individuals may vary from mild self-limiting disease to severe disease, which requires hospitalization with occasional lethal outcome. the annual influenza epidemic which peaks during winter in temperate climates is estimated to cause >600 million cases globally, with 3 to 5 million cases of severe illness and up to 500,000 deaths per year worldwide (www.who.int). since iav have a zoonotic origin with an unlimited reservoir in aquatic birds, they may cross species barriers and transmit to humans leading to novel virus variants with an unpredictable outcome. recently emerged avian iav belonging to the h5n1 and h7n9 subtypes are responsible for high case fatality rates in humans and pose a future pandemic threat (www.who.int). the influenza pandemic of this century was caused by an h1n1 iav in 2009-a(h1n1 )pdm09-possessing genomes of avian, swine, and human origin [1] . it is estimated that it has caused~200,000 respiratory and an additional~80,000 cardiovascular deaths predominantly in people younger than 65 years of age [2] . mortality rates upon iav infections are highest among patients with underlying medical conditions, such as those with chronic pulmonary, cardiovascular, renal, hepatic, neuromuscular, hematologic, and metabolic disorders or in patients being obese, pregnant, or having immunodeficiencies [3] [4] [5] [6] [7] . data from previous pandemics such as the 1918 h1n1 and the 1957 h2n2 pandemic as well as from seasonal influenza epidemics revealed that pregnant women have an increased risk in developing complications upon iav infection [8] [9] [10] [11] [12] [13] . the pandemic caused by a(h1n1)pdm09 highlighted again that especially pregnant women are at the highest risk to develop severe or even fatal influenza. data from the usa show that 5 % of the a(h1n1)pdm09-related deaths were among pregnant women, although they make up only 1 % of the total population [9] . this unprecedented very severe disease outcome during pregnancy led to the revision of vaccination recommendations by the world health organization (who), setting pregnant women now as the highest priority to be vaccinated again influenza regardless of gestational age. during pregnancy, women undergo various physiological changes which might contribute to disease severity upon iav infection. these physiological changes include many metabolic, hemodynamic, and immunologic changes to allow growth and development of the fetus. metabolic changes occur, e.g., in the glucose and lipid metabolism, and hemodynamic changes include an increase of plasma volume by 1000-1600 ml and alterations in the systemic coagulation system [14] . during pregnancy, there are also specific changes in both the upper and lower respiratory tract. in the upper respiratory tract, the mucosa of the nasopharynx and oropharynx changes, including hyperemia, edema, leakage of plasma into the stroma, glandular hypersecretion, and increased mucopolysaccharide content. all these physiological changes can result in nasal congestion [15] . the lower respiratory tract is altered due to the elevation of the diaphragm by up to 4 cm and a decrease in functional residual capacity. functionally, there are changes in lung function, ventilation, and gas exchange, which lead to an increase in oxygen tension required for trans-placental oxygen transfer. furthermore, changes in the cardiovascular system result in a decrease of pulmonary vascular resistance [15, 16] . all these physiological alterations might be further challenged by respiratory viral infections, such as iav and, thus, contribute to disease severity during pregnancy. immunological changes comprise the altered differentiation status of immune cells in the uterus and the formation of a tolerogenic environment at the maternal/fetal interface that prevent rejection of the semi-allogeneic fetal tissues and thereby permit pregnancy. there are also systemic changes of the immune system, mainly caused by pregnancy hormones. these changes are reflected in increased maternal disease outcome not only upon iav infections but also in patients with hiv, malaria, and toxoplasmosis [5] [6] [7] 17] . understanding the molecular basis that mediates increased mortality upon iav infection during pregnancy is key to improve patient management and care. therefore, adequate animal models are required that reflect clinical findings and allow the identification of causative disease pathways. this knowledge will allow early diagnosis and the implementation of rapid intervention strategies to improve disease outcome during pregnancy. it is estimated that vaccination compliance among pregnant women is still below the who recommendations although it has been shown that vaccination during pregnancy protects mother and child [18, 19] . however, a thorough worldwide documentation of vaccination rates is still missing [20] . thus, there is an urgent need to increase disease awareness among this most vulnerable group to improve patient management by early diagnosis as well as by increasing the currently poor vaccination compliance among pregnant women. here, we review current knowledge on the pathogenesis of iav-associated complications during pregnancy in humans and in established animal models as a basis for mechanistic studies. pathology in iav-infected pregnant women pregnant women have an increased risk of developing iavassociated complications due to infection with seasonal, pandemic, or zoonotic influenza viruses. several studies have shown that co-morbidities were present in~30 % of the cases, of which a history of asthma was seen most frequently. other co-morbidities included diabetes, cardiac diseases, hematological disorders, and low weight. since two thirds of the complications in pregnant women could not be explained by other underlying medical conditions, pregnancy itself increased the risk of developing iav-associated complications [21, 22] . interestingly, the majority of pregnant women that develop severe influenza virus-associated disease were in their second or third trimester [21, 22] . frequent symptoms in hospitalized pregnant women include fever, cough, respiratory distress, malaise, joint pain, headache, rhinorrhea, gastrointestinal symptoms, and the development of pneumonia. the latter can lead to the development of acute respiratory distress syndrome (ards) [12, 21, [23] [24] [25] [26] . ards is a rapid onset of hypoxemic respiratory failure associated with bilateral radiographic opacities without congestive heart failure. there are many causes for the development of ards during pregnancy. besides the iavmediated pneumonia, other causes include amniotic fluid embolism, pre-eclampsia, sepsis, and aspiration pneumonia [27] . treatment of a(h1n1)pdm2009-associated ards included extracorporeal life support, also known as extracorporeal membrane oxygenation (ecmo), which resulted in a survival rate of~70 % [27] . extra-respiratory tract complications are occasionally described in pregnant women. these include the development of myocarditis after infection with a(h1n1)pdm09 [28, 29] , and the development of an encephalopathy associated with a seasonal h3n2 iav infection from which viral rnawas detected in cerebrospinal fluids [30] . as in other risk groups, the most common complication of iav in pregnant women is the development of pneumonia and ards. several studies describe the histopathology in pregnant and non-pregnant fatal cases during the a(h1n1)pdm09 pandemic. these studies did not reveal differences between the two groups, although this has not been extensively studied. in pregnant women, histopathological lesions were predominantly found in the respiratory tract, characterized by multifocal desquamation of the epithelial lining of the trachea and bronchus and inflammation of the submucosal glands. within the lungs, there was evidence of diffuse alveolar damage, characterized by extensive fibrosis, hyaline membrane formation, thickening of the alveolar septa, type ii pneumocyte hyperplasia, interstitial and alveolar edema, and an influx of many inflammatory cells [31] [32] [33] [34] . virus antigen was detected in ciliated epithelial cells as well as the submucosal glands of the bronchi and bronchioles. in the alveoli, virus antigen could be detected within the alveolar epithelial cells and alveolar macrophages [31, 32, 34] . in none of these cases, there was evidence of extra-respiratory tract replication. post-mortem histopathological examination of a 24-yearold otherwise healthy pregnant women who succumbed to a(h1n1)pdm2009 after developing ards [26] revealed severe lung damage with desquamated alveolar epithelial cells, destruction of the alveolar septae and interstitial inflammatory cells as described in other cases. additional immunohistochemical staining identified inflammatory cells which mainly consisted of cd68+ macrophages and cd3+ t lymphocytes. since the patient died 28 days after hospitalization and had received antiviral therapy, viral rna could not be detected by in situ hybridization (fig. 1) . post-mortem analysis of an h5n1 highly pathogenic avian virus (hpaiv) infected 4-month pregnant women showed similar diffuse alveolar damage characterized by focal desquamation of epithelial cells in the alveoli without any evidence of type ii pneumocyte hyperplasia, influx of macrophages, neutrophils, and few lymphocytes. no lesions were described in any other parts of the respiratory tract. virus antigen was detected in tracheal epithelial cells and alveolar epithelial cells. in contrast to reports on a(h1n1)pdm2009-infected pregnant women, upon h5n1 hpaiv infection, extrarespiratory virus spread to the placenta could be detected with virus-positive cytotrophoblastic and hofbauer cells. there, lesions included scattered foci of syncytiotrophoblast necrosis, occasionally associated with dystrophic calcification. moreover, virus was detected in the fetus with virus antigen-positive kupffer cells of the liver and pneumocytes in the lungs. virus in the fetal liver was not associated with histological lesions, but the fetal lung showed edema and a few scattered interstitial neutrophils [36] . however, the pathogenesis of iav-associated complications in pregnant women is studied to a very limited extent. a b c fig. 1 post-mortem histopathological analysis of lung biopsy material obtained from a fatal pregnant case. lung biopsy material was obtained from a 24-year-old pregnant woman without any known underlying diseases who succumbed to the 2009 h1n1 influenza virus infection after spontaneous expulsion of the fetus [26] . a severe alveolitis with numerous immunohistochemically (red) stained cd68+ macrophages (a) and cd3+ t lymphocytes (b) is observed. at this late stage of infection, influenza virus rna was not detectable anymore by radioactive in situ hybridization (he staining) (c). the histochemical stainings were performed according to protocols described before [35] important host factors for the pathogenesis of influenza in mammals including humans are the distribution of specific influenza virus receptors and nuclear transport proteins, the importin-α isoforms [37, 38] which largely determine the cell tropism of influenza viruses within the respiratory tract. unfortunately, the impact of these cellular factors has not been studied during pregnancy yet. it is therefore currently unknown whether potential alterations in the distribution of receptors or importin-α isoforms might contribute to the increased risk of developing complications during pregnancy. pregnancy causes the formation of an immune-tolerant environment in the uterus that prevents rejection of fetal tissue. moreover, pregnancy has also profound effects on the peripheral immune system outside of the uterus that might interfere with the response against iav infections [5, 6] . endocrine adaptation to pregnancy, in particular the elevated levels of estradiol and progesterone, affect cytokine levels as well as their composition and function of peripheral leukocyte populations (reviewed in [6] ). numbers of peripheral neutrophils, monocytes, as well as myeloid and plasmacytoid dendritic cells (dc) are reduced [39] . upon in vitro stimulation, both nk cells and cd4 t cells produce reduced amounts of inflammatory cytokines and chemokines. interestingly, reduced cytokine production by cd4 t cells includes both t h1 (ifn-γ, tnf-α) and t h2 cytokines (il-6, il-13) [39] arguing against a shift in the t h1 -t h2 balance during pregnancy as has been postulated from mouse studies [40] . dcs from pregnant women also display higher expression of costimulatory surface molecules, such as cd40, cd80, and cd86, than those of non-pregnant women suggesting a more mature status of dcs [41] [42] [43] . it should be mentioned that results on the phenotype and function of immune cells during pregnancy differ considerably between individual studies. our understanding of immunity during pregnancy would clearly benefit from more systematic and comprehensive approaches using advanced methodology to better define and characterize immune cell subsets. despite these changes in the peripheral immune system, vaccination studies indicate that there is no general deficit in the adaptive immune response of pregnant women to influenza virus. pregnant women still generate protective antibodies against iav, which are further transferred to the fetus [44] [45] [46] [47] . moreover, pregnant women show diminished polyclonal t cell responses but enhanced virus-specific t cell responses following influenza vaccination [48] . thus, despite significant changes in their peripheral immune system, pregnant women are still able to effectively mount an adaptive immune response against iav. currently, there is only very limited information on the immune response against iav in pregnant woman with severe iav-associated complications. as stated before, complications in pregnant women are similar to those observed in other risk groups, arguing for potentially common mechanisms that mediate disease severity. there is evidence that immunopathology strongly contributes to severe courses of iav infections [49] . high virus titers in the lung, which might represent a consequence of reduced production of type i and type iii interferons in pregnant women [50] , could cause excessive production of inflammatory cytokines and chemokines and massive infiltration of granulocytes and macrophages which subsequently cause severe tissue damage. altered production of inflammatory cytokines as well as differences in the composition and response of peripheral immune cells in pregnant women could contribute to such an exacerbated response against iav [6] . however, so far, there is no direct evidence supporting such a scenario. patients with severe iav infections show lower serum levels of igg2. healthy pregnant women also have low levels of serum igg2, and levels are further reduced in pregnant women with severe iav infections [51] [52] [53] . yet again, the contribution of low serum igg2 levels to severe courses of infection in pregnant women is not clear. overall, it is currently not known why pregnant woman are more prone to develop severe courses of disease upon iav infections. iav infections can also have an effect on the pregnancy outcome or the development of the fetus. iav infections have been associated with an increase in perinatal mortality, preterm birth, cesarean section, and a low apgar score 5 min after birth [9, [54] [55] [56] . in addition, infection with iav within the first trimester of the pregnancy increases the risk by twofold on non-chromosomal congenital anomalies, such as neural tube defects, hydrocephaly, congenital heart defects, cleft lip, digestive system defects, and limb reduction defects [57] . viral antigen has been detected in the amniotic fluid (h3n2 virus; [58] ) and in fetal tissue (h5n1 virus; [36] ). however, in the majority of cases, iav could not be isolated from the placenta or fetus [31, 59, 60] . this suggests that the effect on pregnancy outcome and fetus might represent an indirect result of iav infection of the mother, such as fever and cytokine and immune response [61] . overall, there is still a lack of detailed knowledge regarding the impact of maternal iav infection on pre-or even postnatal development in the offspring. to gain more insight into the pathogenesis of iav-associated complications during pregnancy, studies have been performed in small animal models, using mice, ferrets, and pigs. these experimental settings provide insights into events early after infection and mechanisms of diseases, which are difficult to study with materials from infected patients. experimental infections in pregnant balb/c mice at a gestational age of 12-14 days have been performed with seasonal h1n1 and a(h1n1)pdm09 iav as well as with h5n1 hpaiv. intranasal infection with 10 4 or 10 5 egg infectious dose or intraperitoneal infection with 2 × 10 6 plaque forming units of pregnant mice with a(h1n1)pdm09 iav resulted in a higher mortality compared to non-pregnant mice [62] [63] [64] . virus titers in the lungs were higher and histological lesions were more severe in pregnant mice compared to non-pregnant mice at 5 days p.i. [63] . however, in another study, virus titers in bronchoalveolar lavage (bal) fluids were comparable between pregnant and non-pregnant mice at 3 days p.i. [64] . intranasal infection of pregnant mice with a seasonal h1n1 iav did not result in increased mortality, although virus titers in the lung were higher and histological lesions were more severe compared to non-pregnant mice [63] . virus transmission to extra-respiratory tract tissues, including the placenta and fetus, was not observed in any of these studies [62] [63] [64] . the importance of viral factors in the pathogenicity of iavassociated disease during pregnancy was shown in a study which compared a wild type a(h1n1)pdm09 iav to a mouse-adapted a(h1n1)pdm09 iav that contained the d222g mutation in the hemagglutinin. infection with the mouse-adapted virus resulted in increased mortality, higher virus titers in the lung, more influenza virus-infected cells in the bronchioles and alveoli when compared to pregnant mice infected with the wild type virus [62] . a study with h5n1 hpaiv showed that in pregnant mice, virus titers in the lungs were lower compared to virus titers in non-pregnant mice at 5 days p.i. however, in pregnant mice, h5n1 hpaiv spread to extra-respiratory organs and the virus could be detected in the placenta and fetus of intranasally infected mice [65] . histological lesions in the respiratory tract of a(h1n1)pdm09-inoculated pregnant mice were observed in the bronchioles and alveoli. the bronchiolar epithelium showed accumulation of mucous and detached cilia. in the alveoli, lesions were compatible with interstitial pneumonia associated with infiltration of lymphocytes and neutrophils [63] . although histological lesions were not described in detail, the location of the lesions is consistent with those observed in humans. taken together, although the number of studies is very limited, they show that virus replication, histological lesions, and mortality are increased in pregnant mice compared to non-pregnant mice. future studies should reveal more insight into virus replication and associated lesions at different time points after infection to understand the dynamics within the mammalian host in time and the duration of infection and virus shedding. infection of mice has also been used to determine the consequences of pregnancy for the immune response against infection with pandemic a(h1n1)pdm09 [62] [63] [64] . compared to infection of mice with seasonal h1n1 strains, infection with a(h1n1)pdm09 resulted in more severe interstitial pneumonia with massive cellular infiltrations, and lung pathology was further aggravated when pregnant mice were infected [63] . compared to non-pregnant mice, pregnant mice infected with a(h1n1)pdm09 iav demonstrated enhanced accumulation of neutrophils and macrophages in lung tissue and bal fluid, and increased local no production [64] . as expected, infection with a(h1n1)pdm09 was associated with increased levels of inflammatory cytokines in the lung when compared to infection with seasonal h1n1 iav [63] . in comparison to non-pregnant mice, pregnant animals had enhanced levels of inflammatory cytokines (il-1β, il-6, tnf-α) as well as neutrophil-and monocyte-recruiting chemokines in bal fluid and lung tissue, which was consistent with the fulminant accumulation of these cells in the lung [62, 64] . in contrast to the profound changes in the innate response to iav infections, the adaptive response appeared to be less affected by pregnancy. pregnant and non-pregnant mice generated similar frequencies of influenza virus-specific cd8 + t cells and these cells showed comparable activation, as indicated by cd69 expression, and accumulation in the infected lung [64] . pregnant mice also mounted strong antibody responses against a(h1n1)pdm09, although in one study, titers of antibodies were reduced compared to non-pregnant mice [62, 64] . overall, these studies indicate that disease severity in pregnant syngenically mated mice upon a(h1n1)pdm09 iav infection is mainly due to enhanced cytokine production in the lung and massive recruitment of innate immune cells which results in severe tissue damage. intranasal inoculation of a seasonal h3n2 iav in ferrets at early, mid, and late gestation resulted in virus replication within the respiratory tract comparable to that described before for non-pregnant ferrets. virus was not transmitted to the placenta, umbilical cord, amnion, and chorion of the fetus [66, 67] . of note, intracardial inoculation of h3n2 virus did result in virus transmission to the placenta and fetus, indicating that a high virus load viremia might be crucial for virus transmission to the fetus. of note, in general, seasonal influenza viruses do not cause viremia in non-pregnant women [68] . to our knowledge, there are no studies performed in pregnant ferrets with recent seasonal iav, pandemic a(h1n1)pdm09 iav, or h5n1 hpaiv. since the pathogenesis of iav is extensively studied in this mammalian species, such studies would provide useful information on the pathogenesis of iav infections during pregnancy. in addition, the pregnant ferret model could be used to study the efficacy of vaccines and antiviral therapies. intranasal or intratracheal inoculation of young pregnant gilts, 85-90 days post insemination, with swine h1n2, swine h3n2, or a(h1n1)pdm09 iav did not result in any clinical signs or gross pathological lesions 7 days post infection. in none of the inoculated animals (n = 5 per group), there was evidence for viremia or trans-placental virus transmission [69, 70] . however, another study did reveal trans-placental transmission of a swine h1n1 virus in one out of ten pigs [71] . infection with swine h1n2, swine h3n2, and a(h1n1)pdm09 induced similar titers of influenza-specific antibodies and similar proliferative response of peripheral blood cells to stimulation with influenza virus. however, serum levels of il-6, il-10, and tnf-α were enhanced and sustained for several days in pregnant pigs infected with pandemic h1n1 suggesting that infection with this virus caused a stronger innate immune response [69, 70] . there is strong evidence that immune adaptation during pregnancy contributes to influenza disease severity. it was hypothesized that there is a contradictory demand for the maternal immune system to adapt to pregnancy in order to maintain the allogenic fetus and to simultaneously mount an immune response to clear viral infection [6] . however, available animal models reveal that the severity of the infection during pregnancy also depends on viral factors since viral pathogenicity is enhanced upon pandemic rather than seasonal iav infection. the severe and even fatal disease outcome observed in pregnant women is mostly studied and reflected in mouse models so far, although these studies are restricted to one mouse strain and infection at a gestational age of 12-14 days. the pregnant ferret and pig models may also provide complementary information on viral pathogenicity observed in humans although these models have only been used in single studies so far. in general, ferrets and to a lesser extent pigs, mimic iav-mediated respiratory pathogenesis similar to that seen in humans. therefore, pathogenesis studies in pregnant pigs and ferrets-which are both outbread-should be included in future studies. in summary, future efforts in developing animal models that compare the pathogenesis of different iav strains and associated pathogenesis, including virus replication, histological lesions, and immune responses in infected dams and their impact on the offspring are urgently required. these studies should reveal iav replication dynamics and associated immune responses. in addition, these models could be used to study the pathogenesis during pregnancy of other emerging influenza viruses and evaluate vaccine or antiviral efficacy during pregnancy. thus, these studies will allow an evidence-based risk assessment to increase disease awareness and to improve patient management and care. antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza 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during abortion or preterm delivery association of foetal wastage with influenza infection during ferret pregnancy the pregnant guinea-pig as a model for studying influenza virus infection in utero: infection of foetal tissues in organ culture and in vivo pathology of human influenza revisited the influence of experimental infection of gilts with swine h1n2 influenza a virus during the second month of gestation on the course of pregnancy, reproduction parameters and clinical status pregnancy outcome and clinical status of gilts following experimental infection by h1n2, h3n2 and h1n1pdm09 influenza a viruses during the last month of gestation transplacental transmission and neonatal infection with swine influenza virus (hsw1n1) in swine this article is a contribution to the special issue on fetomaternal cross talk and its effect on pregnancy maintenance, maternal and offspring health-guest editor: petra arck acknowledgments this work was supported by research grants from the german center for infection research (dzif), the fonds nationale de la recherche luxembourg (fnr; afr 6042498), boehringer ingelheim fonds, and the german research foundation (dfg; ga1575/3; mi476/ 5). debby van riel is supported by a fellowship from the netherlands organisation for scientific research (nwo; contract number 91614115) and the erasmus mc foundation. key: cord-252443-lclxrwcm authors: lambe, teresa title: novel viral vectored vaccines for the prevention of influenza date: 2012-06-19 journal: molecular medicine doi: 10.2119/molmed.2012.00147 sha: doc_id: 252443 cord_uid: lclxrwcm influenza represents a substantial global healthcare burden, with annual epidemics resulting in 3–5 million cases of severe illness with a significant associated mortality. in addition, the risk of a virulent and lethal influenza pandemic has generated widespread and warranted concern. currently licensed influenza vaccines are limited in their ability to induce efficacious and long-lasting herd immunity. in addition, and as evidenced by the h1n1 pandemic in 2009, there can be a significant delay between the emergence of a pandemic influenza and an effective, antibody-inducing vaccine. there is, therefore, a continued need for new, efficacious vaccines conferring cross-clade protection—obviating the need for biannual reformulation of seasonal influenza vaccines. development of such a vaccine would yield enormous health benefits to society and also greatly reduce the associated global healthcare burden. there are a number of alternative influenza vaccine technologies being assessed both preclinically and clinically. in this review we discuss viral vectored vaccines, either recombinant live-attenuated or replication-deficient viruses, which are current lead candidates for inducing efficacious and long-lasting immunity toward influenza viruses. these alternate influenza vaccines offer real promise to deliver viable alternatives to currently deployed vaccines and more importantly may confer long-lasting and universal protection against influenza viral infection. influenza epidemics are associated with a nontrivial morbidity and mortality; up to one billion infections occur annually with upwards of a half a million associated deaths (1, 2) . the mortality associated with the first influenza pandemic of the 21st century, caused by the swine-origin influenza a h1n1/09 virus, was not as high as first anticipated, nor when compared with the 1918 pandemic, which claimed an estimated 15-50 million lives worldwide (3) . however, the continued risk of a lethal and transmissible influenza pandemic has generated widespread and warranted concern (4). there are three genera of influenza virus that infect humans, influenza a, b and c viruses. type a influenza viruses are the most virulent influenza viruses infecting humans and can be divided into different subtypes on the basis of the antigenic properties of the virion surface proteins, hemagglutinin (ha) and neuraminidase (na) (5, 6) . currently there are 17 ha subtypes and 9 na subtypes identified, and most of the possible combinations have been found circulating in wild birds (5) (6) (7) . however, the majority of influenza in humans is caused by only three subtypes of ha (h1, h2 and h3) and two subtypes of na (n1 and n2) (8) . indeed, recent pandemics have been caused by three subtypes, namely: h1n1 (in 1918 and 2009, the spanish and swine flu pandemics, respectively), h2n2 (in 1957, known as the asian flu) and h3n2 (in 1968, known as the hong kong flu) (8) . in contrast to influenza a viruses, b viruses mutate slowly (9) . at present, there are two antigenically distinct type b influenza lineages (victoria and yamagata), which are concurrently circulating (10, 11) , and individuals exposed to one lineage have limited cross-protective immunity because of antigenic dissimilarity between strains (11) . the burden of disease due to influenza b viral infection falls largely on children and young adults, and this influenza virus causes seasonal influenza epidemics every 2-4 years (11) . by contrast, influenza c virus, although a common cause of mild upper respiratory illness, rarely causes severe illness or local epidemics because most people acquire protective antibodies early in life (12) . southern hemisphere. routinely, formulations contain two human influenza a subtypes (h3n2 and h1n1) and one of the two influenza b lineages (yamagata or victoria) (2) . many countries have implemented influenza healthcare strategies in which the vaccination is administered to discrete population cohorts at risk of complications, the very young and the elderly. the global burden of influenza disease mainly occurs in infants aged 5 years or under; accounting for 90 million infections annually (13, 14) . children shed virus at a high rate and routinely congregate in high numbers (for example, schools, nurseries), thus facilitating easy dissemination of the virus to family members and in turn the wider public. the question of whether to mass vaccinate young children against influenza is a point of considerable debate among both the clinical immunology and public health communities (15) , and at present there is no clear consensus. supporters of vaccinating children against influenza argue that conferring protection to this segment of society will not only benefit children, but will also benefit the wider adult population (1, 15, 16) . opposition to this healthcare strategy hinges on the idea that individuals, including children, may accrue better heterosubtypic immunity through natural exposure to influenza virus as opposed to vaccination (17) (18) (19) (20) (21) (22) (23) . heterosubtypic, or cross-clade, memory can be broadly classified as protection induced to an influenza subtype or strain that confers protection against other strains and can be mediated by both humoral and cellular immunity (24) (25) (26) (27) . approximately 90% of influenza-related deaths, outside of pandemic settings, occur among people aged 65 years or older and this heightened susceptibility is increased as individuals age; persons aged 85 years or older are 16 times more likely to die of an influenza-related death compared with those aged 65 to 69 years (13, 28) . the progressive decline of the immune system is thought to be the principle cause of increased susceptibility to infection in the older adult. the age-related decline in immune responses (immunosenescence) affects both the innate and adaptive immune response (29) . although influenza vaccines have undoubtedly saved numerous lives during both pandemic and epidemic outbreaks, there remains considerable variation in how influenza vaccines are used and the efficacy achieved in targeted population cohorts. as such, influenza and its associated complications still represent a considerable global healthcare burden. for instance, in healthy adults, the efficacy of inactivated vaccines is estimated at 50-75% (30, 31) , whereas in children under the age of 2 years, efficacy associated with trivalent influenza vaccine (tiv) vaccination is comparable to placebo (32) . in the elderly, the validity of using inactivated influenza vaccines has been questioned (33), with a recent systematic review asserting that there is no conclusive evidence regarding efficacy or effectiveness of influenza vaccines for people aged 65 years or older (34) . compared with inactivated vaccines, efficacy for live vaccines in children is considerably higher (approximately 82%); however, live vaccines are also associated with low effectiveness (approximately 33%) (32) . moreover, laiv is not licensed in children under 2 years of age because it is associated with an increased risk of associated wheezing (32) , and in adults over the age of 49 years, laiv is not licensed owing to a lack of corroborated efficacy (35) . thus, there is a clear need to develop effective and efficacious influenza vaccines for both infants and the elderly. before being able to do this, however, we must understand what factors underpin the current limitations of licensed influenza vaccines. humoral immune response. currently licensed influenza vaccines induce a relatively narrow adaptive immune response with limited cross-clade immunity. cross-clade protection refers to protective immunity against one influenza isolate, which subsequently confers protective efficacy upon exposure to different isolates. the majority of humoral responses following influenza vaccination are directed toward ha, principally toward the globular head domain found in the ha1 segment. due to the constantly evolving and mutating propensity of ha, these neutralizing antibodies offer little or no protection between subtypes or even between strains, and in some cases appear to be detrimental (18) . this is a considerable disadvantage considering influenza a undergoes both antigenic drift and antigenic shift (36) . antigenic drift refers to small, gradual changes that occur through point mutations in the ha or na coding genes. antigenic shift refers to an abrupt, major alteration that generally produces a subtype change and can occur through either direct animal-to-human transmission or through a reassortment of human and animal influenza a (36). most escape mutants contain mutations in the globular head domain, and this region of the protein contains widespread immunodominant decoy domains, which can readily mutate without significantly affecting virus fitness (37) . thus, yearly vaccinations with reformulated vaccines are required because of the antigenic differences that occur between, and even within, influenza strains. natural exposure to influenza is thought to induce a broad immune response, inducing cross-clade protection through t-and b-cell responses (25, 38, 39) . indeed, antibodies directed against the stalk region of ha have previously been shown to confer broadspectrum protection toward different influenza isolates (40) . annual vaccination with tiv is thought to be a relatively poor inducer of these cross-clade immune responses (18, 23) . cellular immune response. cellular immune responses are thought to play an important protective role during influenza infection, and human challenge studies have demonstrated a negative correlation between t-cell responses to viral antigens and influenza disease (38) . t-cell responses, particularly against relatively well-conserved internal influenza antigens, can efficaciously provide crossclade protection (38, 41, 42) . although human cytotoxic t-lymphocyte (ctl) immunity declines during periods of low influenza prevalence, these cells are still detectable after 5 years, with an estimated half-life of 2-3 years (43), making these cells an ideal target for boosting through vaccination. although cellular immunity does not prevent viral infection, it is important in clearing and curtailing infection. cytotoxic cd8 + t cells play a major role and can eliminate virally infected cells (44, 45) . a direct cytolytic role for cd4 + t cells has been demonstrated (25, 46) . more importantly, cd4 + t cells can provide b-cell help, and prior cd4 + t-cell immunity can efficaciously augment haspecific antibody responses (47, 48) . unfortunately, even though inactivated influenza vaccines routinely contain internal influenza antigens, specifically nucleoprotein and matrix protein 1 (49, 50) , these vaccines do not boost cellular immunity to an efficacious level (49, 51) . prediction of the major circulating influenza subtypes on the basis of routine surveillance-required to update influenza vaccines each year-can fail. indeed, mismatch between the vaccine components and circulating virus occurs approximately every 3-4 years (52, 53) . this mismatch has a significant effect on vaccine efficacy; in healthy adults, parenteral vaccine efficacy against seasonal influenza is approximately 75% when the circulating influenza and vaccine strain match; however, efficacy decreases to 44% when mismatch occurs (30) . substantial costs are incurred because of the need for annual reformulation and given the manufacturing process, which for the recent 2009 h1n1 pandemic took over 6 months even with the implemented pandemic readiness plans (54) . each year, there is essentially a race between production, deployment and the spread of infection. one of the main challenges for inducing strong immune responses following vaccination with inactivated or subunit protein vaccines is the inherent poor immunogenicity of proteins, which can be partially overcome by the addition of adjuvants. adjuvants are a common means to augment adaptive immune responses to vaccines and can be broadly classified into vehicles or immunostimulants (55) . the former group largely enhance presentation to the immune system, whereas the latter enhance the adaptive immune response (55) (56) (57) . some adjuvants have been shown to advantageously increase and broaden the epitopes recognized by the immune repertoire (58, 59) . a number of strategies have been deployed to improve influenza vaccine efficacy for the elderly, and the inclusion of adjuvants as a means to augment humoral immunity in the elderly has demonstrated relatively encouraging results (60, 61) . another challenge is the long-term stability of recombinant proteins; the inclusion of some adjuvants can facilitate increased vaccine stability during storage or upon injection (62) . however, even with this augmented stability there are often thermostability difficulties, especially when vaccines occupy a large amount of cold-chain storage space due to relatively complex administration or dosing regimens. there are a number of platforms being explored for more efficacious influenza vaccine delivery and these can be broadly classified into: recombinant protein/ peptide, dna vaccines, viruslike particles, virosomes and viral vectors. recombinant technology has been used to generate influenza proteins in insect cells, bacteria and plants (63) (64) (65) ; and although much antigen can be pro-duced, these proteins are generally poorly immunogenic and require complex immunization regimes and/or the inclusion of adjuvants to induce efficacious humoral immunity (66) . for cellular immune responses, antigenic mapping of t-cell epitopes followed with antigen string delivery (in the form of peptides) is actively being investigated. unfortunately, peptides are generally poorly immunogenic, and repeated administration to augment adaptive immune responses can induce tolerance rather that immunogencity (67) . immunization with dna, encoding influenza proteins, has been studied for many years as an alternate vaccination strategy but clinical trials have met with limited success (68, 69) . dna vaccines do not boost preexisting immunity (the scenario for the adult human population) and are generally only weakly immunogenic in humans (70) . self-assembled viral capsid proteins, viruslike particles (vlps), have been produced from a number of viruses. a key restriction to certain vlp platforms is the small size of foreign epitopes that can be accommodated (for example, ha is a relatively big protein) (71) . for the generation of vlps appropriate protein glycosylation, correct assembly and ease of purification need to be considered. these concerns may dictate the use of mammalian cell culture systems, which is a less controllable and a more costly system (71) . although similar to vlps in many respects, virosomes, by contrast, are essentially assembled in vitro. there are a limited number of virosomal-adjuvanted-subunit influenza vaccines currently licensed; the generation of these vaccines requires good manufacturing practice (gmp)-grade influenza virus and, as such, these vaccines face similar obstacles as the production of tiv (72, 73) . vectored vaccines, such as recombinant live-attenuated or replicationdeficient viruses, are currently being developed for vaccine delivery and offer a number of advantages over other emerging technologies. viral vectored vaccines are particularly good at expressing full-length protein and generating conformationally restricted epitopes, thus driving potent humoral responses following immunization (74) . in addition, vectored vaccines can induce high levels of protective cellular immunity (75) (76) (77) . thus, viral vectors efficaciously induce both arms of the adaptive immune system. following vaccination with viral vector vaccines, virus can infect local cells and produce high levels of protein (up to 100 μg following vaccination with a modest amount of viral vector, 10 10 viral particles (vp) of adenovirus or 10 7 plaque-forming units (pfu) of modified vaccinia virus ankara) (78) . antigen has been detected at the immunization site up to 6 months after viral vectored vaccination (79, 80) , which is in contrast to the transient antigen depots following protein vaccination, even when coadministered with adjuvants (81) . the increased presence of antigen may enhance the survival of certain memory t cells and augment high-affinity antibody selection (82, 83) . viral infection results in both apoptosis and necrosis of localized tissue, facilitating the release of antigen, which is subsequently available to drive cellular and humoral immunity (84, 85) . the associated cell death following viral infection will also advantageously augment adaptive immune responses, circumventing the need for the inclusion of adjuvants. live recombinant viral vectors must remain infectious to be effective, and conventional, continuous refrigeration is required to maintain stability and viability. however, recent methodologies have allowed complete recovery of viral titer and immunogenicity after storage at up to 45°c for 6 months (86). in addition, viral vectored vaccines are relatively easy to manipulate and produce, and have well-established safety profiles. there are an increasing number of viral-based vectors currently employed as antigen delivery systems, including dna virus vectors (for example, poxviruses, herpesvirus, adenovirus and baculovirus) and rna virus vectors (for example, paramyxovirus, rhabdovirus, bunyavirus, alphavirus, coronavirus, retrovirus and flavivirus) (87) . however, the majority of human clinical trials assessing viral vectored influenza vaccines use poxviral and adenoviral technologies, and these vectors are considered lead candidates for the immunogenic delivery of antigens (77) . adenoviruses (ads) are nonenveloped dna viruses originally identified as infectious agents responsible for certain acute respiratory infections. ad infection usually manifests as mild disease, which is promptly resolved; however, infection of immunocompromised individuals can result in more severe disease (88, 89) . several properties of ad viruses are attractive for vaccine use, including relative ease of manipulation, infection of both dividing and nondividing cells, high levels of transgene expression, ability to grow to high titers in vitro and lack of integration in the host genome and stability (physical and genetic) (87, 89, 90) . ads are rendered replication defective by deletion of the e1 region genes, and although replication competent ad vectors have been demonstrated to be quantitatively (reduction of required dose) and qualitatively (induction of sitespecific immunity) more effective, the release of genetically modified ad vectors into the general public, fuelling concerns of possible carriage, has limited their systematic use (89, 90) . nonreplicating human ad (for example, adhu5) vectors have been extensively used in gene therapy applications, but unfortunately, most adults have high titers of neutralizing antibodies against a number of ad serotypes, including adhu5, which has a negative impact on their effectiveness as vaccine platforms (91) . to circumvent this limitation, alternative serotypes of adenovirus not found in human populations are being developed (92, 93) . these include ads of rare serotypes (that is, ad11, ad26, ad35, ad28 and ad49) and nonhuman ads, principally of chimpanzee origin adc (77, 87, 94) . in addition, engineered chimeric vectors in which the adhu5 capsid has been modified (thereby reducing the number of antigenic epitopes previously seen by man) are also being deployed (77, 87, 94) . a number of vaccine studies have demonstrated the feasibility of using recombinant ad vectors expressing influenza viral proteins to protect model organisms from influenza challenge. a recombinant ad vector expressing h5n1 ha induced both cellular and humoral immunity and was protective, following lethal challenge, in mice and chickens (95) . heterosubtypic immunity toward certain influenza viruses has been induced through in ovo vaccination with a nonreplicating ad vector expressing ha from h5n9 (96), and indeed long-lasting immunity, 12 months postimmunization, has been demonstrated following ad vaccination (97) . the application of ad viral vectored vaccines has progressed to human studies, and a phase i clinical trial using a nonreplicating adenovirus vectored nasally administered influenza vaccine has demonstrated that the vaccine is well tolerated and efficaciously induces humoral immunity. a follow-on trial evaluating the dose range, safety and im-munogenicity of an intranasally administered adhvn1203/04.h5 (adenovirus vectored h5 ha) in healthy adults 19-49 years of age is expected to be completed imminently (clinicaltrials.gov identifier: nct00755703). replication competent ad vectors, in general, induce stronger immune responses and a live, replication competent recombinant ad vectored vaccine, expressing the ha from h5n1 (ad4-h5-vtn), will be administered either intranasally or tonsillarly in a phase 1 randomized, dose-escalation study expected to be completed in 2014 (clinical-trials.gov identifier: nct01443936). poxviruses are among the most heavily exploited viral vectors, largely because of the overwhelming success of vaccinia virus in eradicating smallpox (87, 98) . as large dna viruses, poxviruses can easily accommodate substantial amounts (over 25 kb) of extra dna (99), thus facilitating a multivalent vaccine approach through the simultaneous expression of multiple transgenes (100, 101) . poxviruses replicate within the cytoplasm of infected cells and do not integrate into the host genome, eliminating the potential for insertional mutagenesis. they have been developed as vaccines through two different approaches; the first relies on productive replication of an attenuated strain in a permissive host. the second uses replication-defective poxviruses, which may enter cells of nonpermissive hosts and express encoded proteins, but do not produce infectious virus (98, 102) . modified vaccinia virus ankara (mva), an attenuated strain of vaccinia virus, was originally developed during the smallpox eradication campaign, because wild-type strains can produce undesirable effects in humans (103, 104) . attenuated vaccinia strains such as mva undergo very limited, or nonproductive, replication in mammalian cells (102) , although most of the virus proteins are produced, including inserted transgenes (98) . mva has an excellent safety profile in humans, having been used in many field studies (105, 106) -safety has also been demonstrated in studies with immunocompromised macaques and humans (107, 108) . one of the ongoing concerns with viral vector vaccines is their applicability for repeat vaccinations. preclinical evaluation of mva vectors has demonstrated their ability to repeatedly boost immune responses directed to recombinant antigens (109, 110) . indeed, two phase i clinical trials using repeat vaccination with mva vectored vaccines, at 6-8 months, demonstrate an ability to boost t-cell responses (111, 112) . these data suggest that vector-specific immunity does not greatly impede immune responses toward target antigens. a major advantage of mva over other vaccine preparations is that adjuvants are not required for high immunogenicity, because mva has immunostimulatory properties (84, 85) and can elicit potent humoral and antigen-specific t-cell responses (110, 113) . for the efficient activation of cd8 + t-cell responses with mva vectors, delivery of full length antigen is generally found to be superior, suggesting that cross-priming may be particularly important in mva-mediated cd8 + t-cell responses (110, 114) . there is now a body of evidence demonstrating that recombinant pox-viral vectors expressing influenza antigens are efficacious vaccines. indeed, there is a licensed pox-viral influenza vaccine, trovac aiv h5, for the prevention of influenza in chickens. this vaccine uses the fowlpox virus encoding the ha gene from a/turkey/ireland/83 and has demonstrated protective efficacy against a wide range of highly pathogenic, subtype-h5 viruses (115) . preclinical work has demonstrated the feasibility of using mva vectored influenza antigens as vaccines in mammals; mice vaccinated with mva recombinants expressing the ha of h5n1 (a/vietnam/1194/04; mva-ha-vn/04) were fully protected against infection with the homologous strain and also against infection with h5 viruses from different clades (116) (117) (118) (119) . a pentavalent vaccina vaccine with ha, na, nucleoprotein and the matrix proteins m1 and m2 was able to confer sterile cross-clade protection during stringent challenge studies (120) . studies in higher primates have assessed the feasibility of using mvaexpressing hemagglutinin as an influenza vaccine. immunization with a/vietnam/ 1194/04 (mva-ha-vn/04) induced cross-reactive antibodies and prevented virus replication in the respiratory tract of cynomolgus macaques (117) . indeed, a recent phase i clinical trial assessing an mva vector encoding nucleoprotein and matrix protein 1 (mva-np + m1) has demonstrated that this vaccine was both safe and, more importantly, immunogenic-inducing high levels of interferon (ifn)-γ production from t-cells (75, 121) . a promising avenue of influenza vaccine development is likely to come from the viral vectored vaccines currently progressing through phase i and ii clinical trials. designed to produce antiinfluenza responses, these vectored vaccines have demonstrated promising immunogenicity with good safety profiles and it is likely that these vaccines will be further used in prime-boost regimes aimed at augmenting protective immunity. these regimes involve priming the adaptive immune response toward an antigenic target through vaccination with a vector encoding the antigen of choice, followed by a boost, in which the same antigenic target is administered but in a different vector. through this, the immune response is honed toward the antigen of choice, while avoiding boosting antivector immunity. prime-boost approaches, incorporating priming with either plasmid dna vaccines or viral vector (typically ad) followed by a boost with either an ad-recombinant or mva expressing the same gene product, have been shown to enhance protective efficacy (122, 123) . indeed, the use of heterologous regimens with ad vectored vaccines boosted with mva has been shown to augment antigen-specific t-cell responses and also to efficaciously induce humoral immunity (66, (124) (125) (126) . as the supporting evidence evolves, a likely outcome is the use of viral vectored vaccines in prime-boost regimes to generate a strong and potentially heterosubtypic immune response. indeed, heterosubtypic immunity toward certain influenza viruses has been induced through in ovo vaccination with a nonreplicating ad vector expressing ha from h5n9 (96) . furthermore, vaccination with mva recombinants expressing the ha of h5n1 (a/vietnam/1194/04; mva-ha-vn/04) confers protection against infection with the homologous strain and also against h5 viruses from different clades (116) (117) (118) (119) . the induction of efficacious and long-lasting crossclade immune responses will bypass the need for annual vaccine reformulation and immunization and, most importantly, will provide protection during future influenza pandemics. despite the long-standing service of currently licensed influenza vaccines, they carry a number of real limitations that need to be addressed before population-based influenza vaccination strategies are optimized. undoubtedly, a vaccine that induced durable, sustainable and cross-clade protection against influenza would be of great medicinal and economic benefit worldwide. at the very minimum, a revision of the current vaccine regimes for at-risk populations needs to be considered to optimize health outcomes. an improved regime is likely to include different vaccines for the different ages of man: for children, a fourpronged approach including the two currently circulating influenza a subtypes and both influenza b strains has been suggested (11) . whereas for the elderly, inclusion of the two currently circulating influenza a subtypes and both influenza b strains at a higher dose or the inclusion of adjuvants to augment the lower responses should be considered. undoubtedly, the continued and concerted effort invested in developing efficacious viral vectored influenza vaccines will deliver promising and real alternatives to those currently deployed and greatly impact upon public health in the 21st century. t lambe is an oxford martin fellow and receives financial support from the medical research council, uk, and the oxford biomedical research centre. the author is grateful to s gilbert and r o'donnell for their helpful comments on the manuscript. the authors declare that they have no competing interests as defined by molecular medicine, or other interests that might be perceived to influence the results and discussion reported in this paper. the annual impact of seasonal influenza in the us: measuring disease burden and costs influenza vaccines for the future updating the accounts: global mortality of the 1918-1920 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vaccines against plasmodium falciparum msp1 key: cord-008695-y7il3hyb authors: nan title: pandemic flu: clinical management of patients with an influenza-like illness during an influenza pandemic date: 2007-01-25 journal: j infect doi: 10.1016/s0163-4453(07)60001-2 sha: doc_id: 8695 cord_uid: y7il3hyb nan • this document is intended for use in the uk in the event that the world health organisation declares that an influenza pandemic has started 1 , and the department of health in england (uk-wide lead agency on pandemic influenza, including the devolved administrations) has declared uk pandemic alert level 2 (cases of pandemic influenza identified within the uk). • these guidelines are not relevant for the management of patients affected by seasonal/interpandemic influenza, lower respiratory tract infections, community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease (copd). • once an influenza pandemic is under way, users are strongly urged to ensure that they refer to the most up-todate version of these guidelines (from web-based access points). synopsis 1. clinical management of adults referred to hospitals s1.1. severity assessment in hospital • patients with uncomplicated influenza infection would be expected to make a full recovery and do not require hospital care. • in uncomplicated infection, the illness usually resolves in seven days although cough, malaise and lassitude may persist for weeks. • patients with worsening of pre-existing co-morbid medical conditions should be managed according to best practice for that condition with reference to published disease-specific guidelines, if available, for example, the national institute of clinical excellence's copd guideline. • in hospital, patients with influenza-related pneumonia and who have a curb-65 score of 3, 4 or 5 (see box a) are at high risk of death and should be managed as having severe pneumonia. • patients with bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia should be managed as having severe pneumonia regardless of curb-65 score. • patients who have a curb-65 score of 2 are at increased risk of death. they should be considered for short stay inpatient treatment or hospital supervised outpatient treatment. this decision is a matter of clinical judgment. • patients who have a curb-65 score of 0 or 1 are at low risk of death. they can be treated as having non-severe pneumonia and may be suitable for home treatment. • patients with primary viral pneumonia or a curb-65 score of 4 or 5 should be considered for hdu/icu transfer. • general indications for hdu/icu transfer include: (1) persisting hypoxia with pao 2 < 8 kpa despite maximal oxygen administration (2) progressive hypercapnia (3) severe acidosis (ph < 7.26) (4) septic shock • patients with influenza admitted to intensive care unit should be managed by specialists with appropriate training in intensive care, respiratory medicine and/or infectious diseases. pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s3 s1.4. general investigations • the following investigations are recommended in patients referred to hospital: who this applies to full blood count all patients urea and electrolytes all patients liver function tests all patients chest x-ray all patients pulse oximetry all patients. if <92% on air, then arterial blood gases. patients with cardiac and respiratory complications or co-morbid illnesses. c-reactive protein if influenza-related pneumonia is suspected • in those patients who are subsequently followed up in a hospital outpatient clinic or by a general practitioner a repeat chest x-ray should be obtained at around six weeks if respiratory symptoms or signs persist or where there is a higher risk of underlying malignancy (especially smokers and those over 50 years of age). • further investigations including a ct thoracic scan and bronchoscopy should be considered if the chest x-ray remains abnormal at follow up. s1.5. microbiological investigations s1.5.1. early in a pandemic (uk alert levels 1, 2 and 3) • virology all patients (1) nose and throat swabs in virus transport medium. (2) if presentation is more than seven days after onset of illness, an 'acute' serum (5 10 ml clotted blood) should be collected and a 'convalescent' sample (5 10 ml clotted blood) obtained after an interval of not less than seven days. • bacteriology patients with influenza-related pneumonia (1) blood culture (preferably before antibiotic treatment is commenced) (2) pneumococcal urine antigen (20 ml urine sample) (3) legionella urine antigen (20 ml urine sample) (4) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who: (i) are able to expectorate purulent samples, and (ii) have not received prior antibiotic treatment. (5) paired serological examination for influenza/other agents. acute serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood). • virology not routinely recommended • bacteriology patients with influenza-related pneumonia in accordance to the severity of illness. (a) non-severe pneumonia (curb-65 score 0, 1 or 2) no routine testing. in patients who do not respond to empirical antibiotic therapy, sputum samples should be sent for gram stain culture and antimicrobial susceptibility tests. b severe pneumonia (curb-65 score 3, 4 or 5, or bilateral cxr changes) blood culture, preferably before antibiotic treatment is commenced pneumococcal urine antigen (20 ml urine) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who are able to expectorate purulent samples, and have not received prior antibiotic treatment. paired serological examination for influenza/other agents. 'acute' serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood). tracheal or endotracheal aspirate samples, if available, should be sent for gram stain, culture and antimicrobial susceptibility testing. s1.6. general management s1. 6 • hypoxic patients should receive appropriate oxygen therapy with monitoring of oxygen saturations and inspired oxygen concentration with the aim to maintain pao 2 8 kpa and sao 2 ges; 92%. high concentrations of oxygen can safely be given in uncomplicated pneumonia. • oxygen therapy in patients with pre-existing chronic obstructive pulmonary disease complicated by ventilatory failure should be guided by repeated arterial blood gas measurements. non-invasive ventilation may be helpful. • in patients without pre-existing copd who develop respiratory failure, niv may be of value as a bridge to invasive ventilation in specific circumstances when level 3 beds are in high demand. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted at all times. • patients should be assessed for cardiac complications and also volume depletion and their need for additional intravenous fluids. • nutritional support should be given in severe or prolonged illness. • temperature, respiratory rate, pulse, blood pressure, mental status, oxygen saturation and inspired oxygen concentration should be monitored and recorded initially at least twice daily and more frequently in those with severe illness or requiring regular oxygen therapy. an early warning score system is a convenient way to perform this. • in patients who are not progressing satisfactorily a full clinical reassessment and a repeat chest radiograph are recommended. • patients should be reviewed 24 hours prior to discharge home. those with two or more of the following unstable clinical factors should consider remaining in hospital: (1) temperature >37.8ºc (2) heart rate >100/min (3) respiratory rate >24/min (4) systolic blood pressure <90 mmhg (5) oxygen saturation <90% (6) inability to maintain oral intake (7) abnormal mental status • follow up clinical review should be considered for all patients who suffered significant complications or who had significant worsening of their underlying disease, either with their general practitioner or in a hospital clinic. • at discharge or at follow up, patients should be offered access to information about their illness, take home medication and any follow up arrangements. • it is the responsibility of the hospital team to arrange the follow up plan with the patient and the general practitioner. • individuals should only be considered for treatment with antivirals (neuraminidase inhibitors) if they have all of the following: (1) an acute influenza-like illness (2) fever (>38ºc) and (3) been symptomatic for two days or less. • treatment schedule: adults oseltamivir 75 mg every 12 hours for five days. (dose to be reduced by 50% if creatinine clearance is less than 30 ml/minute, i.e. 75 mg od). • patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, may still be eligible for antiviral treatment despite lack of documented fever. • hospitalised patients who are severely ill, particularly if also immunocompromised, may benefit from antiviral treatment started more than 48 hours from disease onset, although there is no evidence to demonstrate benefit, or lack of it, in such circumstances. s1.8. antibiotic management s1.8.1. influenza • previously well adults with acute bronchitis complicating influenza, in the absence of pneumonia, do not routinely require antibiotics. • antibiotics should be considered in those previously well adults who develop worsening symptoms (recrudescent fever or increasing dyspnoea). • patients at high risk of complications or secondary infection (appendix 2) should be considered for antibiotics in the presence of lower respiratory features. • most patients can be adequately treated with oral antibiotics. • the preferred choice includes co-amoxiclav or a tetracycline. • a macrolide such as clarithromycin (or erythromycin) or a fluoroquinolone active against streptococcus pneumoniae and staphylococcus aureus is an alternative choice in certain circumstances. • most patients can be adequately treated with oral antibiotics. • oral therapy with co-amoxiclav or a tetracycline is preferred. • when oral therapy is contra-indicated, recommended parenteral choices include intravenous co-amoxiclav, or a second or third generation cephalosporin (cefuroxime or cefotaxime). • a macrolide (erythromycin or clarithromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative regimen where required eg. for those intolerant of penicillins. currently levofloxacin and moxifloxacin are the only recommended fluoroquinolones licensed in the uk. • antibiotics should be administered within four hours of admission. • patients with severe pneumonia should be treated immediately after diagnosis with parenteral antibiotics. • an intravenous combination of a broad spectrum b-lactamase stable antibiotic such as co-amoxiclav or a second (e.g. cefuroxime) or third (e.g. cefotaxime) generation cephalosporin together with a macrolide (e.g. clarithromycin or erythromycin) is preferred. • an alternative regimen includes a fluoroquinolone with enhanced activity against pneumococci together with a broad spectrum b-lactamase stable antibiotic or a macrolide. currently levofloxacin is the only fluoroquinolone with an intravenous formulation licensed in the uk. • patients treated initially with parenteral antibiotics should be transferred to an oral regimen as soon as clinical improvement occurs and the temperature has been normal for 24 hours, providing there is no contra-indication to the oral route. • for most patients admitted to hospital with non severe and uncomplicated pneumonia, seven days of appropriate antibiotics is recommended. • for those with severe, microbiologically undefined pneumonia, ten days treatment is proposed. this should synopsis 1. clinical management of adults referred to hospitals synopsis of main recommendations pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s5 be extended to 14 to 21 days where staph aureus or gram negative enteric bacilli pneumonia is suspected or confirmed. • for those with non-severe pneumonia in hospital on combination therapy, changing to a fluoroquinolone with effective pneumococcal and staphylococcal cover is an option. • adding further antibiotics effective against mrsa is an option for those with severe pneumonia not responding to combination antibiotic therapy. • high fever (>38.5ºc) and cough or influenza-like symptoms. these children should seek advice from a community health professional. if there are no features that put them at high risk of complications they should be treated with oseltamivir, and given advice on antipyretics and fluids. children aged <1 year and those at risk of complications (appendix 2) should be seen by a gp. • high fever (>38.5ºc) and cough or influenza-like symptoms, plus at risk group. these children should be seen by a gp or in a&e. children may be considered at increased risk of complications if they have cough and fever (or influenza-like illness) and temperature >38.5ºc, plus either chronic co-morbid disease or one of following features: breathing difficulties severe earache vomiting >24 hours drowsiness these patients should be offered an antibiotic as well as oseltamivir (in those >1 year of age) and advice on antipyretics and fluids. children aged <1 year with none of the above features should be treated with antipyretics and fluids with a low threshold for antibiotics if they become more unwell. • indicators for hospital admission are: (1) signs of respiratory distress. markedly raised respiratory rate grunting intercostal recession breathlessness with chest signs (2) cyanosis (3) severe dehydration (4) altered conscious level (5) complicated or prolonged seizure (6) signs of septicaemia extreme pallor, hypotension, floppy infant • most children admitted to hospital are likely to need oxygen therapy and/or intravenous support as well as antibiotics and oseltamivir. • indications for transfer to high dependency or intensive care are: (1) failure to maintain sao 2 > 92% in fio 2 > 60% (2) the child is shocked (3) severe respiratory distress and a raised paco 2 (>6.5 kpa) (4) rising respiratory rate and pulse rate with clinical evidence of severe respiratory distress with or without a raised paco 2 (5) recurrent apnoea or slow irregular breathing (6) evidence of encephalopathy • when there are no picu beds available, children will have to be triaged on the basis of the severity of their acute and co-existing disease, and the likelihood of their achieving full recovery. • a full blood count with differential, urea, creatinine and electrolytes, liver enzymes and a blood culture should be done in all severely ill children. • a cxr should be performed in children who are hypoxic, have severe illness or who are deteriorating despite treatment. • pulse oximetry should be performed in every child being assessed for admission to hospital with pneumonia. • virology all children (1) nasopharyngeal aspirate or nose and throat swabs (2) if presentation is more than 7 days after onset of illness, an 'acute' serum (2 5 ml clotted blood) should be collected and a 'convalescent' sample (2 5 ml clotted blood) obtained after an interval of not less than 7 days. • bacteriology children with influenza-related pneumonia (1) blood culture (before antibiotic treatment is commenced) (2) sputum samples obtained from older children (3) paired serological examination for influenza/other agents. • virology not routinely recommended • bacteriology children with influenza-related pneumonia (1) blood culture (before antibiotic treatment is commenced) (2) sputum samples obtained from older children • patients whose oxygen saturation is 92% or less while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. • when children are unable to maintain oral intake, supplementary fluids should, when possible, be given by the enteral route. intravenous fluids in those with severe pneumonia should be given at 80% basal levels. • children can be safely discharged from hospital when they: (1) are clearly improving (2) are physiologically stable (3) can tolerate oral feeds (4) have a respiratory rate <40/min (<50/min in infants) (5) have an awake oxygen saturation of >92% in air. • in the setting of a pandemic, children should only be considered for treatment with antivirals if they have all of the following: (1) an acute influenza-like illness (2) fever (>38.5ºc) and (3) been symptomatic for two days or less • oseltamivir is the antiviral agent of choice. • in children who are severely ill in hospital oseltamivir may be used if the child has been symptomatic for <6 days (but there is no evidence to demonstrate benefit, or lack of it, in such circumstances). • children (a) who are at risk of complications of influenza or (b) with disease severe enough to merit hospital admission during an influenza pandemic should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae. • for children under 12 years co-amoxiclav is the drug of choice. clarithromycin or cefuroxime should be used in children allergic to penicillin. for children over 12 years doxycycline is an alternative. • oral antibiotics should be given provided oral fluids are tolerated. • children who are severely ill with pneumonia complicating influenza should have a second agent added to the regime (e.g. clarithromycin or cefuroxime) and the drugs should be given intravenously to ensure high serum and tissue antibiotic levels. to facilitate preparedness planning, this document has been written in advance of the emergence of the next influenza pandemic, at a time when the identity of the causative virus remains unknown. these guidelines are based on the best evidence available from previous pandemic and interpandemic influenza periods. the guidance may evolve as clinicopathological information on the eventual pandemic virus emerges. once an influenza pandemic is under way, users are strongly urged to refer to the most up-todate version of these guidelines (from web-based access points). seasonal influenza is a familiar infection in the uk, especially during winter. every year strains of influenza (type a or b) circulate, giving rise to clinical consultations in primary care (age-specific impact varies by season), episodes of hospital treatment (mainly in older persons and young children, but occasionally in working age adults), and deaths (mainly in the elderly). treatment in primary care and hospital may be required due to the direct effects of influenza virus infection or its possible complications, most commonly secondary bacterial pneumonia. increases in gp consultations for influenza-like illness and winter bed pressures are frequently associated with periods of known community influenza activity 6 . pandemic influenza occurs when a new influenza a virus subtype emerges which is markedly different from recently circulating subtypes and strains, and is able to: • infect humans; • spread efficiently from person to person; • cause significant clinical illness in a high proportion of those infected. because the virus is novel in humans, a high proportion of the population will have little or no immunity, producing a large pool of susceptible persons; accordingly the disease spreads widely and rapidly. influenza pandemics occur sporadically and unpredictably. in 1918, a devastating and unusual pandemic caused by influenza a/h1n1 ('spanish flu') killed between 20 and 40 million people worldwide. other pandemics that followed had a less devastating impact but were nevertheless severe. influenza a/h2n2 ('asian flu') emerged in 1957, and h3n2 ('hong kong flu') in 1968; both produced roughly 1 million excess deaths worldwide 7 . the circumstances still exist for a new influenza virus with pandemic potential to emerge and spread, and the longest interval so far recorded between pandemics is 39 years (1918 1957) . the unpredictability of the timing of the next pandemic is underlined by the occurrence of several large outbreaks of highly pathogenic avian influenza associated with epizootic transmission to humans 8 . by far the most serious has been the massive and unprecedented outbreak of highly pathogenic influenza (a/h5n1) affecting poultry in east and south east asia in late 2003, which is still continuing. this outbreak has so far been associated with a small number of human cases but a high proportion of deaths. recently, epidemiological and virological changes have been reported from northern vietnam which may indicate that the virus is beginning to adapt to humans 9 . although the emergence of an a/h5n1 strain with capacity to spread efficiently between humans is neither inevitable nor imminent, international concern has increased regarding the possibility that avian influenza a/h5n1 may evolve to produce the next pandemic. other events and developments that inform the creation of this guidance are the development and licensing of a new class of drug (neuraminidase inhibitors) active against influenza, and uk government's announcement of plans to procure 14.6 million treatment courses of oseltamivir (tamiflu ® ) 10 for use in the uk in the event of a pandemic. be involved in the management of patients with influenza. it is intended that these guidelines also be of value to health-care practitioners who do not usually manage patients with influenza but may be called upon to do so in a pandemic situation. modification of some recommendations at a local level may be necessary in specific instances. these guidelines are not relevant for the management of patients affected by seasonal influenza, sporadic acute exacerbations of chronic obstructive pulmonary disease (aecopd), lower respiratory tract infections (lrtis) or community-acquired pneumonia (cap). at the primary care level, a national operational plan including the following three broad areas is deemed important: (a) clinical management of patients with influenza (b) management of patient demand, including patients who do not have influenza (c) health service delivery plans these guidelines cover the first of these areas and will serve as the source document for the primary care operational plan. the primary care operational plan will incorporate all three areas within a single reference and is being developed by the dh in collaboration with the rcgp and the bma. even though it is impossible to predict with certainty the impact of the next pandemic, based upon the available epidemiological and modelling information, it is clear that it will generate demands for health care which may saturate or overwhelm normal nhs acute services for a period of time, perhaps several weeks or months. accordingly, it should be anticipated that the nhs (in common with all health systems around the world) will need to revert to emergency arrangements. these are laid out in further detail in operational guidance for health service planners 4 , the uk operational framework for stockpiling, distributing and using antiviral drugs in the event of pandemic influenza 5 and in the primary care operational plan. with regard to the delivery of medical care for patients with influenza this is normally achieved through: • gp treatment of community patients 'well' enough to be managed in the community • hospital care in acute medicine for persons considered too ill to be managed at home. in the event of a pandemic, the following additional care settings may have to be considered as the threshold for hospital admission rises: • treatment of patients in the community (who would normally receive care from a gp) by other health-care professionals (nurses, paramedics, pharmacists etc.) following treatment guidance laid out in this publication and using prescription-only medicines according to patient group directives (pgds). • treatment of patients in their own homes or in temporary intermediate care facilities by a gp, following treatment guidance laid out in this publication when, under normal circumstances, such patients would have been admitted for hospital care. • treatment of severely ill patients in hospital by medical and nursing teams who do not normally manage patients with influenza or community-acquired pneumonia, in areas of the hospital not normally used for providing medical care (for example, surgical teams and bed space diverted from routine elective work towards pandemic response). the recommendations offered in the current guidelines are based on a matrix of evidence centred mainly around seasonal influenza, expert opinion and group consensus. grading of these recommendations based on the strength of the evidence base was deemed inappropriate. section 2. epidemiology and health impact projections (1) the scale and severity of illness (and hence consequences) caused by pandemic influenza generally exceed those of even the most severe winter epidemics. (2) mortality in the uk is likely to exceed 50,000 deaths, possibly appreciably higher. (3) besides the elderly, excess mortality is also likely in younger adults and children. (4) modelling studies suggest that after a case occurs in hong kong, because of international travel, it will take less than one month for the virus to reach the uk. (5) once cases begin to occur in the uk it will take only two to three weeks before activity is widespread and roughly a further three weeks (six weeks after initial cases in uk) until activity peaks. (6) it is possible that there will be more than one epidemic wave (with an interval of several months) and, if a second wave occurs, it may be more severe than the first. (7) cumulative clinical and serological attack rates across all waves together may be in the order of 25% and 50% respectively. (8) increases in demand for health-care services are likely to be very substantial in both primary care and hospital settings. when an influenza pandemic occurs, a substantial proportion (possibly all) of the population is likely to be non-immune, producing a large pool of susceptible persons. in past pandemics, the scale and severity of illness (and hence consequences) have been variable but broadly of a §2. epidemiology and health impact projections introductory observations higher order than even the most severe winter epidemics. it is reasonable to expect this to be the case with the next pandemic as well. excess mortality due to influenza occurs in most winter seasons but is especially marked during epidemics. the average annual excess mortality attributable to influenza in recent years is around 12,000 deaths per annum in england and wales 11 , although there is considerable yearly variation and some years are notably much higher than the average (est. 26,000 in 1989/90 epidemic). excess mortality in england and wales associated with the three pandemics of the twentieth century has also varied widely; this was estimated at 198,000 civilians in 1918/19, and 37,500 in 1957/58. in 1968/69 and 1969/70 (both seasons considered to be associated with the influenza a/h3n2 pandemic), there were an estimated 31,000 and 47,000 deaths respectively 7 . therefore the extent of mortality associated with the next pandemic cannot be reliably predicted although it is reasonable to plan for a scenario worse than a severe winter epidemic of normal influenza. typically, there are changes in the age-distribution of cases compared with seasonal influenza. mortality, which in typical seasonal influenza is usually confined to age groups over 65 years, tends to be increased in younger age groups. the size of any increase in morbidity and mortality and the extent to which a shift in age distribution occurs depend on a variety of factors including the nature of the pandemic virus and pre-existing immunity but appears to be a consistent phenomenon 12 . therefore, clinicians can expect to see relatively larger amounts of influenza-related illness in younger adults compared with normal winter activity. at least one third of all excess deaths may be expected in persons under 65 years of age. virological and clinical surveillance of influenza have improved markedly since the last pandemic in 1968. however, the extent of international travel has also grown. modelling studies using transmission characteristics based on the 1968/69 pandemic and international air-traffic data from 2002 indicate that the approximate delay between a first case in hong kong and first introduction to uk will be less than one month 13 . in terms of the spread within the uk, it will probably take only two to three weeks from the initial introduction(s) until activity is widespread and a further three weeks (six weeks from initial uk cases) until activity peaks. the temporal and spatial spread of a pandemic strain is important, particularly in terms of the demand placed on health-care services. pandemic activity taking the form of a brief but severe peak in cases will be more difficult for all services to cope with, compared with an identical number of cases distributed over a longer time course. for example, during the a/h3n2 pandemic a long first wave occurred in the winter of 1968/9 with morbidity and mortality approximately at the same level as the previous seasonal influenza; but in the following winter of 1969/70 a short and more severe epidemic occurred with a threefold higher peak in general practice consultation rates and a four-fold higher peak in mortality attributed to influenza, bronchitis and pneumonia. the high peak in consultation rates is well illustrated in fig. 2 .1. in 1918/19, the a/h1n1 pandemic occurred in three distinct epidemic waves: early spring 1918, autumn 1918 introductory observations §2. epidemiology and health impact projections s10 provisional guidelines from bis/bts/hpa in collaboration with the department of health, version 11 (2 october 2006) and late winter 1919. the second wave was by far the largest and case-fatality rates were also higher than in the first wave. the a/h3n2 pandemic caused an epidemic wave in the winter of 1968/69 but a more severe one in 1969/70. in contrast, the second wave of the 1957/58 pandemic in the uk was very small in comparison to the first 7 . thus it should be considered a possibility that more than one wave of influenza will occur within a few months of the emergence of a pandemic virus and a subsequent wave could be worse than the first. it is impossible to predict reliably with precision the level of excess mortality that will be experienced in the next pandemic. however, table 2 .1 illustrates the broad range of excess mortality that it is reasonable to consider, based on various realistic combinations of case fatality rate and clinical attack rates derived from previous pandemics and epidemics. a case fatality rate of 0.37% corresponds to the aggregate rate observed in recent epidemic seasons (1989/90, 1991/92, 1993/94, 1995/96, 1996/97, 1997/98 and 1998/99) and the 1957 pandemic, although the overall case-fatality rate observed in the 1918 19 pandemic was in the region of 1 2%. a clinical attack rate of around 25% corresponds to the approximate clinical attack rate seen in all three previous pandemics of the twentieth century. thus, a figure of at least 50,000 excess deaths is likely. using mathematical projections, it is possible to illustrate the potential impact of the next pandemic, but these do not amount to accurate predictions. table 2 .2 summarises the number of events that might be expected by a gp with 1000 patients on his/her list and by a pct serving a population of 100,000 persons. using the same assumptions, table 2 .3 illustrates the number of events by week over an assumed 15-week (single wave) pandemic period in a typical pct population of 100,000. most major acute trusts receive patients from a catchment area spanning several pcts and the figures below require pro-rata adjustment before applying to individual hospitals. section 3. clinical features in adults (1) influenza is clinically defined as the presence of fever and new (or, in those with chronic lung disease, worsening) cough of acute onset in the context of influenza circulating in the community. this clinical definition may be modified once a pandemic occurs. (2) the spectrum of clinical disease associated with a pandemic strain cannot be forecast. (3) pneumonia, either primary viral or secondary bacterial, is the commonest complication of influenza in adults. (4) neurological complications are rare in adults. the clinical manifestations of infection by influenza viruses are diverse, ranging from asymptomatic infection to fulminant respiratory distress leading to respiratory failure and death. furthermore, the presence of an influenza-like illness (ili) comprising of a combination of fever, cough, sore throat, myalgia and headache is not specific for influenza infection. other respiratory pathogens that may present with an ili include viruses such as respiratory syncytial virus (rsv), adenovirus, rhinovirus and parainfluenza virus, as well as bacterial pathogens such as chlamydia pneumoniae, legionella sp., mycoplasma pneumoniae and streptococcus pneumoniae [14] [15] [16] . studies that have examined the value of a clinical definition of ili in the diagnosis of influenza infection have not always used the same clinical definition for an ili and have included different study populations, making comparison between studies complicated. a systematic review of the literature in this area identified the threefold combination of the presence of fever, cough and acute onset to be the most predictive clinical features. the accuracy of this clinical definition was higher in persons aged 60 years and above compared to patient groups without age restrictions [positive likelihood ratio (95% ci) 5.4 (3.8 7.7) vs 2.0 (1.8 2.1)] 17 . the probability of influenza infection also increases with increasing level of fever 18, 19 . importantly, the predictive value of clinical definitions based on an ili increases when influenza virus is known to be circulating in the community 15, 17, 20 . in cohort studies, correlation of ili with laboratory-confirmed influenza infection ranges from 25% to 45% while in clinical trials, rates of 70% have been consistently reported 15, [21] [22] [23] .. these findings relate to influenza infections during interpandemic periods. during a global influenza pandemic, when a pandemic strain is known to be circulating locally in an immunologically susceptible population, the presence of an ili would be expected to be highly predictive for influenza infection. (however, the extent to which a clinical diagnosis of ili becomes predictive during a pandemic will also be determined by the behaviour of the public. if many who would not normally present to a health professional are prompted to present, then the predictive value of a clinical diagnosis of ili will be reduced.) the following description will relate mainly to interpandemic influenza a infections. influenza b and c are not considered pandemic threats. different strains may be associated with different clinical presentations and disease severity. for instance, there is evidence to suggest that the h3n2 subtype causes more severe disease than h1n1 subtype 24 . the spectrum of clinical disease associated with a new influenza a subtype (eg. a pandemic strain) cannot be determined currently and may differ from that described for interpandemic influenza. the incubation period prior to the onset of symptoms is commonly two to four days (range 1 7 days). in adults, the illness typically presents as an abrupt onset of fever accompanied by a range of other symptoms as listed in box 3.2 [25] [26] [27] [28] [29] . fever is the paramount symptom and may reach 41ºc although more usually it ranges between 38ºc and 40ºc. the peak occurs within 24 hours of onset and lasts typically for three days (range 1 5 days) [25] [26] [27] [28] [29] . the cough is generally dry although in up to 40% of cases it may be productive. a productive cough together with chest tightness and substernal soreness is more common in patients with underlying chronic lung disease. myalgia affects mainly the back and limbs. gastrointestinal symptoms such as vomiting and diarrhoea are uncommon (<10%) in adults. abdominal pain is rare. clinical findings include a toxic appearance in the initial stages, hot and moist skin, a flushed face, injected eyes and hyperaemic mucous membranes around the nose and pharynx. tender cervical lymphadenopathy is found in a minority (~10%) of cases. wheezing or lung crackles are recognised findings (~10%) more commonly noted in patients with coexisting chronic lung disease. although the overall clinical picture of uncomplicated influenza in any specific age group is similar for different influenza a subtypes, the frequency of certain symptoms may vary. for instance, during the 'asian' pandemic of 1957 (h2n2), headache and sore throat were frequent initial symptoms 30 . in uncomplicated infection, the illness usually resolves in seven days although cough, malaise and lassitude may persist for weeks. influenza virus infection has been associated with worsening in the clinical condition of patients with a range of existing medical conditions, such as, heart failure, diabetes, coronary heart disease, asthma and chronic obstructive airways disease (copd). in addition, specific complications associated with influenza infection regardless of co-existing medical conditions are recognised (table 3 .1). based on data from interpandemic influenza, certain persons are identified as being at high risk from influenza-related complications. such patients are similar to the group currently recommended for influenza vaccination by the department of health. these include those of all ages with chronic respiratory disease including asthma, chronic heart disease, chronic renal disease, chronic liver disease, immunosuppression due to disease or treatment, or diabetes mellitus, and all those aged 65 years or older, or those in long stay residential care (see appendix 2). in the course of a pandemic, it may emerge that the patient group at high risk of complications differs from the group currently identified. in such circumstance, details of the 'high risk' patient group will be altered according to relevant clinico-epidemiological data. the incidence of pneumonia (defined as a combination of respiratory symptoms and signs supported by chest radiographic changes consistent with infection) complicating influenza infection varies widely, from 2% to 38%, and is dependent on viral and host factors [25] [26] [27] . pneumonia generally occurs more frequently and with greater severity in patients with pre-existing chronic cardiac and respiratory conditions. patients who develop pneumonia may present with symptoms and signs indistinguishable from pneumonia related to other viral and bacterial pathogens. in the context of an influenza pandemic, the presence of an ili and new or worsening dyspnoea should prompt a careful examination for the presence of complicating pneumonia. two main types of influenza-related pneumonia are recognised: primary viral pneumonia and secondary bacterial pneumonia [25] [26] [27] [28] . patients with primary viral pneumonia typically become breathless within the first 48 hours of onset of fever. an initially dry cough may become productive of blood-stained sputum. cyanosis, tachypnoea, bilateral crepitations and wheeze on chest examination and leucocytosis are usual. the commonest chest radiographic abnormality is of bilateral interstitial infiltrates predominantly in the mid-zones, although focal consolidation is also well recognised. rapid clinical deterioration with respiratory failure may ensue 31 . the mortality in hospitalised patients is high (>40%) despite maximum supportive treatment on intensive care [25] [26] [27] [28] . in the majority of fatal cases, death occurs within seven days of hospital admission. secondary bacterial pneumonia is more common (up to four times) than primary viral pneumonia. typically, symptoms and signs of pneumonia develop during the early convalescent period (four to five days from onset of initial symptoms). in others, symptoms of pneumonia blend in with the initial symptoms of influenza. chest radiography usually demonstrates a lobar pattern of consolidation. mortality rate ranges from 7% to 24% [25] [26] [27] [28] [29] 32 , although some small studies report higher mortality rates. the spectrum of pathogens implicated is similar to that observed in cap and includes streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae and groups a, c and g b-haemolytic streptococci 27, 28, [33] [34] [35] . different pathogens have predominated at different times. for instance, in the 1918 pandemic, h. influenzae, b-haemolytic streptococci and s. pneumoniae were the predominant pathogens isolated. in 1968, s. pneumoniae was the predominant pathogen (48%) followed by staph. aureus (26%) and non-typeable h. influenzae (11%) 34 . notably, staph. aureus was identified two and a half times more frequently during the 1968 pandemic compared to pneumonia occurring in the interpandemic period 34, 36 . secondary staphylococcal pneumonia is associated with a higher incidence of lung abscess formation (14% vs 2%) and carries a poorer prognosis compared to non-staphylococcal pneumonias (mortality 47% vs 16%) 25, 29, 32, 37 . during the 1957 pandemic, staph. aureus was the predominant bacterial pathogen isolated in fatal cases of influenzarelated pneumonia (up to 69% of cases in some series) 25 . bacterial and viral pneumonia can occur concurrently. in these instances, the chest radiograph may demonstrate lobar consolidation superimposed on bilateral diffuse lung infiltrates. the mortality rate in mixed viral bacterial pneumonia is high (>40%), as for primary viral pneumonia [25] [26] [27] [28] . minor abnormalities on ecg such as st segment deviation, t wave changes and rhythm disturbances have been described in uncomplicated influenza illness. they have been reported in up to 81% of patients hospitalised with influenza 25 . most do not have cardiac symptoms. myocarditis and pericarditis are occasionally encountered in severe illness 38, 39 . post mortem evidence of necrotising myocarditis has been reported in patients without clinically significant myocarditis in the antemortem period. in contrast with myalgia affecting the back and limbs which is common on initial presentation, myositis generally develops after the subsidence of the acute upper respiratory tract symptoms. the gastrocnemius and soleus muscles are typically involved with pain and tenderness to palpation. complete recovery usually occurs in three days. elevation in serum creatine phosphokinase is recognised 40, 41 . rarely, this is associated with myoglobinuria and renal failure 42, 43 . myositis is more commonly described in children than in adults. central nervous system (cns) involvement in adults is uncommon. most reports originate from japan and occur in children 44, 45 . the main clinical syndrome is an encephalitis or encephalopathy manifesting in the form of decreased consciousness and seizures about three days (range 0 7 days) following the onset of upper respiratory tract symptoms. focal neurological signs such as paresis, aphasia, choreoathetosis and cranial nerve palsies are less common. cerebrospinal fluid (csf) examination may be normal or reveal an elevation in protein or white cell count. imaging by ct or mri may be normal and if so, is indicative of a good prognosis and full recovery may be anticipated 46 . young age and abnormal ct/mri findings are associated with a poor outcome including death or recovery with severe neurological sequelae. [a fuller description is given in section 4.2. 6.] acute necrotising encephalopathy is a rare fulminant syndrome associated with multifocal brain lesions that is described mainly in japan 46 . other rare manifestations include transverse myelitis and guillain barré syndrome 47,48 . reye's syndrome, characterised by an encephalopathy, acute fatty liver, association with aspirin use and high mortality (~40%), is a special situation that is almost exclusively seen in children and adolescents 46 . nevertheless, physicians managing adults are advised to be aware of this complication. [a fuller description is given in section 4.2.6.1.] other complications rarely encountered in adults with influenza a infection include toxic shock syndrome in conjunction with secondary staph. aureus infection 49,50 and parotitis 51 . otitis media is more commonly encountered in children than adults. human infections have been caused by different avian influenza a viruses in the past, including h9n2, h7n7, h7n3 and h7n2. in recent years, outbreaks of human infections by a novel strain of avian influenza a (h5n1) have raised particular concerns globally regarding the risk of a human pandemic 52 . these concerns have been due in part to recognition that (a) avian influenza a (h5n1) can pass directly from birds to humans and (b) once in humans, avian influenza a (h5n1) causes severe disease with a high mortality. the full spectrum of human illness associated with avian influenza a (h5n1) infection is not completely known. descriptions of the clinical features of influenza a (h5n1) infection in humans are based largely on case series of hospitalised patients. subclinical infections, mild illnesses and atypical presentations of influenza a (h5n1) infections in humans have been reported, but the frequency of such infections is difficult to determine [53] [54] [55] . in hospitalised patients, an ili similar to that associated with seasonal influenza a (h1n1 or h3n2) infection is recognised. gastrointestinal symptoms are present in a relatively large proportion of both adult and paediatric cases, in contrast to the relatively low incidence of gastrointestinal symptoms in seasonal influenza. the majority of patients develop a severe primary viral pneumonia usually associated with lymphopenia, thrombocytopenia and deranged liver function tests. renal failure and multiorgan failure may develop subsequently. mortality is high. a more detailed description is given in appendix 10. should influenza a (h5n1) acquire efficient humanto-human transmission capabilities, it may result in an influenza pandemic. in such an event, the clinical features of human h5n1 disease may alter. (1) the commonest presenting features of influenza during an epidemic are fever, cough and rhinorrhoea. in infants, fever with non-specific symptoms or diarrhoea and vomiting is common; in older children pharyngitis and headache are frequent. (2) the clinical features of influenza in children during a pandemic cannot be forecast. (3) children with underlying respiratory or cardiac disease, immune compromise or who are nonambulant are more likely to be severely affected. (4) the younger the child the more likely hospital admission will be needed. the clinical features of influenza presenting in a pandemic cannot be predicted as they appear to be dependent on the strain of influenza and, in some respects, the host. a new strain of influenza a responsible for an epidemic or pandemic may result in a different spectrum of clinical features than previous strains 56, 57 . common features during previous epidemics have been described and depend on the age of the child. the studies of clinical features are hospital based and are therefore likely to reflect more severe illness. these are nevertheless informative as one of the main issues in a pandemic is which patients require hospital admission. in young children presenting to primary care in a non-pandemic influenza season there are no specific clinical features that distinguish influenza from other winter viruses 58 . neonates may present with non-specific signs of sepsis such as pallor, floppiness, (poor peripheral circulation, poor tone), lethargy, poor feeding, episodes of apnoea 59 . fever may be the only presenting feature. a north american study identified influenza as the most common reason for children aged 0 60 days being admitted to hospital during an epidemic with fever as the only clinical feature 60 . fever may be the only presenting feature in this age group too. they may also be irritable and toxic and are more likely than older children to present with gastrointestinal symptoms such as diarrhoea and vomiting. febrile convulsions, particularly repeated convulsions, are positively associated with influenza a 61 . otitis media is also a common complication in children 62 . admission rates for under two year olds are 12 times higher than for children aged 5 17 years 63 . the presentation does not differ significantly from adults. common features are sudden onset of high fever, chills (76 100%), cough, headache, sore throat, fatigue (51 75%), nasal stuffiness and conjunctivitis (26 50%) . fever tends to settle two to four days later though a dry cough and clear nasal discharge last for one to two weeks 59 . a clinical prediction model from north america for influenza in children has shown that the triad of cough, headache and pharyngitis had a sensitivity of 80% and a specificity of 78% for a positive viral culture for influenza 64 . the subjects, mean age six years, presented during an epidemic to a suburban emergency department with a febrile respiratory illness and one or more symptoms of influenza. a finnish retrospective study of children referred to hospital from 1980 to 1999 with influenza confirmed by antigen testing reported that the median age for those with influenza a was two years. the most common features were cough, fever and rhinorrhoea 62 . these were also the commonest features reported in a chinese study where the mean age of the subjects with influenza a was four years 65 . conditions these children 66 (table 4 .1) and those who are not ambulant 67 experience substantial morbidity during influenza seasons, with a disproportionate number requiring inpatient care and ventilatory support. of the 22% of (table 4 .2) as in adults, influenza can present with either primary viral pneumonia or bacterial pneumonia most commonly caused by s. pneumoniae or staph. aureus. there is much less published about pneumonia complicating influenza in children. an outbreak of severe pneumococcal pneumonia in children occurred in iowa in the winter of 1995 96. this was coincident with an epidemic of influenza (h1n1). compared with controls, patients were 12 times more likely to have rare experienced a recent influenza-like illness. they were also more likely to have family members with the illness and to have positive serology in the convalescent period. many of these patients required chest drainage 69 . another study in 2002 of 202 children with proven influenza reported that 78 who had chest radiographs had either radiographic evidence of viral pneumonia or normal radiographs. no child had lobar pneumonia reported 70 . evidence from recent outbreaks of avian influenza (h5n1) in hong kong and vietnam suggests that while some children had mild disease 71 , others appeared to have multi-organ disease including acute respiratory distress syndrome (ards) 57 . all children who developed progressive pneumonia with ards died. there were no reports of bacterial pneumonia. there is no reason to believe that, apart from ards, pneumonia complicating influenza presents differently from community-acquired pneumonia in children 72 . the general clinical indicators for severity assessment of lower respiratory tract infection are summarised in the bts guidelines 72 (appendix 8). failure to improve following 48 hours of antibiotics, or deterioration including a new, distinct spike of fever, should also be treated as severe and further complicating factors sought. the clinical course of croup caused by influenza appears to be more severe than croup caused by the more common parainfluenza virus 73 . it is more likely to be complicated by bacterial tracheitis 62 . influenza is a well recognised cause of otitis media 74 . it is the commonest bacterial superinfection of influenza and is reported in approximately 25% of patients aged <5 years 75 . influenza ranks second only to respiratory syncytial virus as a cause of bronchiolitis 76 . the clinical features are the same 77 . children with influenza may present with febrile convulsions. in a community study in the netherlands, recurrent febrile seizures were positively related to influenza a. it was recommended that children who have had a previous febrile convulsion should be immunised against influenza a 61 . these complications are described in small case series. this is defined as depressed or altered level of consciousness including lethargy and/or extreme irritability in younger children or significant change in personality or behaviour persisting beyond 24 hrs or confusion (older children). encephalopathy usually presents as seizures within several days of the onset of fever 78 . seizures at this point are usually the first symptom of involvement of the central nervous system. febrile convulsions, which are more likely to be repeated with influenza than with other causes of fever, generally occur with the onset of fever. disturbances of behaviour and neurological deficit have been reported. a rapid and severe clinical course is usual with encephalopathy and is thought to be due to brain oedema mediated by cytokines rather than by direct invasion of the brain. steroids are therefore considered. 202 children with encephalopathy were recognised in japan between 1997 and 2001. death occurred in 31%, residual neurological deficit in 26% and full recovery in 43% 79 . this is a rare childhood acute encephalopathy associated with liver dysfunction. the cause is unknown but it typically follows viral illness and there is a clear association with aspirin therapy: thus an innate susceptibility coupled with aspirin taken for relief of viral symptoms. influenza (particularly influenza b) is commonly implicated 80 . there was a dramatic fall in incidence following warnings about aspirin use in children 81 . it is possible that children on long term aspirin treatment for medical conditions may be at increased risk if they develop influenza infection. reye's syndrome is characterised by protracted vomiting and encephalopathy in afebrile patients with minimal or absent jaundice, and hepatomegaly in 50% of patients. it comprises: • acute non-inflammatory encephalopathy with an altered level of consciousness • elevation of ammonia levels 24 48 hours after the onset of mental status changes (the most frequent laboratory abnormality) • hepatic dysfunction with a liver biopsy showing fatty metamorphosis or a more than three-fold increase in alanine aminotransferase (alt), aspartate aminotransferase (ast) neurological symptoms usually occur 24 48 hours after the onset of vomiting. lethargy is usually the first neurological manifestation. diarrhoea and hyperventilation may be the first signs in children younger than two years. other investigations: head ct scanning may reveal cerebral oedema but results are usually normal. an electroencephalogram (eeg) may reveal slow wave activity in the early stages and flattened waves in advanced stages. cerebrospinal fluid may or may not have increased opening pressure with white blood cells (wbcs) fewer than 9/ml 3 (usually lymphocytes). there is no specific treatment for reye's syndrome. key aspects of management are correction of metabolic imbalance and reduction of intracranial pressure. advice should be requested from a specialist in metabolic medicine. many children have an underlying inborn error of metabolism. mortality has fallen from 50% to less than 20% as a result of earlier diagnosis and more aggressive therapy. acute necrotising encephalopathy (ane): this occurs mainly in japan where it was first described in 1995. an estimated 100 deaths per annum are related to central nervous system complications of influenza in japan 82 . this suggests either a genetic predisposition for this complication or a variation in the strains of influenza circulating in japan. ane is characterised by high fever, convulsions and coma in children aged one to five years. the onset is two to four days after the respiratory symptoms, and fewer than 10% of patients survive 83 . there are no specific markers although some patients have raised liver transaminases. in many, the csf is normal. symmetrical multi-focal brain lesions are seen and bilateral thalamic involvement is characteristic and may be demonstrated on mri 83 . this is defined as encephalopathy plus two of the following: fever of 38ºc or higher, seizures, focal neurological findings, wbc >5 cells/ml in csf, eeg findings consistent with encephalitis, abnormal neuro-imaging 84 . these must be considered when a child presents with altered level of consciousness or irritability. there is good evidence of an increased risk of meningococcal disease following influenza infection 85 . during a pandemic, the focus will be on diagnosing influenza-related illness. other neurological conditions or drug toxicity, for example, may be missed. a literature review of 316 cases of myositis 86 suggested that this was a complication mainly of schoolchildren. the calf muscles are predominantly affected. rhabdomyolysis and renal failure are rare. these are also rare complications but have been described in children with underlying medical conditions 62 . section 5. general management and investigations in primary care with widespread concern during a pandemic, a significantly increased demand for advice and consultation should be anticipated. there are likely to be significantly higher consultation rates for all types of respiratory tract infections including those which are normally managed well at home using over-the-counter remedies (e.g. febrile colds, sore throat with temperatures). consequently, demand management in both the practice and the pct will be crucial to avoid the service's capacity to triage care being overwhelmed. guidance on demand management and health service delivery is given in the primary care operational plan (see section 1.4) 87 . management decisions of patients with influenza should be based primarily on: • an assessment of illness severity • identification of whether the individual is in an 'at risk' group • current advice from doh/local public health officials based on the epidemiology of the pandemic patients who are not considered to be at high risk and who have no features suggesting severe disease or complications may not need to be seen in face to face consultations by a primary care clinician. all patients presenting in general practice with symptoms suggestive of influenza (except perhaps those in whom urgent admission is required) should be given general advice and advice on symptomatic treatment. it is important that clinicians identify and address individual concerns and expectations, provide information about the illness, and provide information about what patients can do to help themselves and when they should seek further help. some useful facts that can be provided to the patient are included in box 5.1. there is little scientific evidence for most symptomatic and self-help treatment, but experience suggests that some of the following may help, and are unlikely to cause harm: • treat fever, myalgias and headache with paracetamol or ibuprofen • rest • drink plenty of fluids • avoid smoking • consider: short course of topical decongestants, throat lozenges, saline nose drops many infants and children will have coughs and mild fevers which may be due to other infections such as respiratory syncytial virus, especially over the winter months. these children should be managed in the usual way at home by parents with antipyretics and fluids. (note: aspirin should not be used in children.) management of these children is determined by disease severity (see appendix 5). the principles of symptomatic management are similar to those for adults. box 5.1. information about influenza to provide to patients • influenza is caused by a number different types of 'influenza' viruses. • the incubation period is typically one to four days and infected adults are usually contagious from the day of illness onset to five days after. children are typically contagious for seven days, although sometimes for longer. • fever usually declines after two to three days and normally disappears by the sixth day. • cough, weakness and fatigue can persist for one to two weeks and up to six weeks. • antibiotics do not benefit most people with influenza but are sometimes needed to treat secondary infections. (important note: this information may be modified once a pandemic occurs) • fever for four to five days and not starting to get better (or getting worse) • started to feel better then developing high fever and feeling unwell again • if taking antiviral drugs (eg. oseltamivir), symptoms should start to improve within two days. lack of any improvement after two days from starting antiviral drugs is an indication to re-consult. (important note: this information may be modified once a pandemic occurs) • children under one year of age year and those at high risk of complications (see appendix 2) should be seen and assessed by a gp or at the a&e department. • children over one but under seven years of age may be seen by a nurse or a gp and those aged seven years and above may be seen by a member of the community health team (e.g. community pharmacist). • all children (and parents) should be given advice on antipyretics and fluids. • aspirin is contraindicated in children (aged under 16 years). examples of what should prompt a patient to re-consult are given in box 5.2. patients who are started on antiviral agents (see section 7 for indications for antiviral use) would be expected to begin to improve within 48 hours of starting treatment. failure to improve two days after starting an antiviral agent is an indication to re-consult. at the time of re-consultation, an alternative diagnosis should be considered as well as the occurrence of any influenzarelated complications. • any rapid deterioration following first consultation should prompt a patient to re-consult. • failure to improve two days after starting an antiviral agent is an indication to re-consult. • if the first consultation did not involve contact with a physician, re-consultation should preferably involve a physician, usually a gp. 5.5. what general investigations should be done in the community? • general investigations, including a chest x-ray, are not necessary for the majority of patients managed in the community. the aim of microbiological investigations early in a pandemic (uk alert levels 1, 2 and 3) will be to confirm that influenza a is circulating in the local community. once a pandemic is established (uk alert level 4), microbiological investigations are not recommended routinely or likely to be available readily. routine testing for bacterial pathogens is not recommended at any stage. • where possible, early in a pandemic (uk alert levels 1, 2 and 3), nose and throat swabs, or nasopharyngeal swabs (in children), in virus transport medium should be submitted to the local laboratory. • once a pandemic is established (uk alert level 4), microbiological investigations are not recommended. section 6. criteria for hospital referral 6 .1. which adults require hospital referral? adults with uncomplicated influenza infection usually do not require hospital referral. patients who might require hospital admission fall into two main groups: those with worsening of a pre-existing medical condition and those with an influenza-related complication. patients who experience a worsening or clinical deterioration of pre-existing medical problems due to influenza infection should be managed according to recommended best practice for the medical condition in question. for instance, a patient with an acute exacerbation of copd triggered by influenza infection should be managed according to current nice guidelines for copd 88 . those with a worsening of a pre-existing condition are likely to be in a group at 'high risk' of influenzarelated respiratory complications and consequently at risk §6. criteria for hospital referral part 1. clinical management in primary care pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s21 . this group should be promptly reassessed if the illness is getting worse to consider hospital referral. pneumonia is the commonest influenza-related complication requiring hospital admission. patients complaining of new or worsening dyspnoea should be carefully assessed for signs of pneumonia. if pneumonia is diagnosed, disease severity assessment is recommended and hospital referral made accordingly. there is no validated severity assessment tool developed specifically for influenza-related pneumonia. the crb-65 score (table 6 .1) is a well validated severity assessment tool developed for patients with community-acquired pneumonia (cap) 89, 90 and recommended in the british thoracic society cap guidelines 2004 for use in the community setting 72 . it is offered as an example of an assessment tool for influenza-related pneumonia. the use of any severity assessment tool does not replace clinical judgement. a patient's social circumstances should also always be taken into account. in view of the rapid and fulminant course of primary viral pneumonia, patients with pneumonia who have bilateral chest signs (crackles) should be considered for hospital referral. other influenza-related complications are uncommon. there are no specific recommendations relating to criteria for hospital admission or disease severity assessment in these cases. • patients with clinically defined uncomplicated influenza infection would be expected to make a full recovery. they require good symptomatic management, access to antiviral treatment, information about the natural history, and advice as to when to re-consult. • patients with new or worsening symptoms particularly shortness or breath or recrudescent fever not responding to treatment should be examined to assess the presence and severity of influenza-related pneumonia. • patients with worsening of pre-existing co-morbid medical conditions should be managed according to best practice for that condition with reference to published disease-specific guidelines, if available. • in patients with influenza-related pneumonia clinically, hospital referral and assessment should be considered for patients with a crb-65 score of 1 or 2 (particularly score 2) and urgent admission for those with crb-65 score of 3 or more. • patients with bilateral chest signs of pneumonia should be referred to hospital for further assessment regardless of crb-65 score. • the crb-65 score does not replace clinical judgment. • the antiviral treatment of choice is oseltamivir (tamiflu tm ). this is given as a five-day course of oral tablets; 75 mg twice daily for adults. liquid suspension is available for children from the age of one year upwards. (see table 7 .1.) from clinical trial data accrued to date and based on seasonal, interpandemic influenza, the anticipated positive effect of antivirals in a pandemic will be: (a) a reduction of illness duration by 24 hours, and therefore more rapid mobilisation of affected individuals including essential workers (b) a possible reduction in hospitalisation of infected individuals (c) a reduction of subsequent antibiotic use by infected individuals the evidence accrued to date does not suggest there will be a reduction of overall mortality, nor does it rule it out. 7.4. who should receive antiviral drugs? • ideally, antiviral treatment should be offered to every patient who is over one year of age who (a) has an acute influenza-like illness (b) has fever ( 38ºc in adults, or 38.5ºc in children) and (c) presents within 48 hours of the onset of symptoms. • exceptions: (i) patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, may still be eligible for antiviral treatment despite the lack of documented fever. (ii) immunosuppressed patients, including those on long-term corticosteroid therapy, may suffer more prolonged viraemia, and could possibly benefit from antiviral therapy commenced later than 48 hours after the onset of ili. (iii) patients who are severely ill, but who have not been hospitalised due to non-clinical reasons, may benefit from antiviral therapy commenced later than 48 hours after the onset of ili. there is no strong evidence to support antiviral use in these exceptional situations. the commonest adverse effect of oseltamivir is nausea in about 10% of patients. this can be managed with mild anti-emetic medication. other side-effects are listed in appendix 9. national distribution arrangements are laid out in the uk operational framework for stockpiling, distributing and using antiviral drugs in the event of pandemic influenza 5 and the primary care operational plan. the drug will be made available through these arrangements to pharmacies, pcts and/or gp surgeries. • pcts are encouraged to plan for the delivery of antivirals to the large numbers of previously healthy persons with an ili via community health professionals, including community pharmacists. • gps should focus their efforts on assessment and management of those persons at high risk of complications (see appendix 2) and patients developing complications. section 8. antibiotic use in primary care the use of antibiotics in adults with influenza not complicated by pneumonia is determined by (a) the presence of any co-morbid illnesses and (b) the timing of first consultation with respect to the onset of symptoms. features of an acute bronchitis, with cough, retrosternal discomfort, wheeze and sputum production are an integral part of the influenzal illness. in previously well individuals who do not have pneumonia or new focal chest signs, antibiotics are not indicated. if the patient is seen later in the course of the illness and the illness is worsening, for instance with recrudescent fever or increasing breathlessness, a worsening bacterial bronchitis §8. antibiotic use in primary care part 1. clinical management in primary care pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s23 or developing pneumonia is possible and the use of antibiotics should be considered. in selected patients, a delayed antibiotic prescription may be offered at first consultation. the antibiotic prescription should come with clear instructions that the antibiotics should be used if the illness is not starting to settle after two days or if there is worsening of symptoms. the potential advantage of this approach of delayed antibiotic prescription is to minimise rates of reconsultation 91 . there are no robust data regarding the effect of such an approach on the incidence of influenzarelated complications. those at high risk of influenza-related complications because of (a) chronic obstructive pulmonary disease (copd) and/or (b) other severe co-morbid diseases should be strongly considered for antibiotics at first consultation. if, having started antibiotics, patients do not begin to improve over the next 48 hours of antibiotic treatment (or if they get worse) they should be advised to re-contact their gp for assessment of pneumonia and its severity (see sections 3 and 6). antibiotics should cover the likely bacterial pathogens including s. pneumoniae, h. influenzae, m. catarrhalis and staph. aureus. the preferred first choice of antibiotic for nonpneumonic bronchial infections, including those patients with copd, should include an effective oral b-lactamase stable agent such as a tetracycline (e.g. doxycycline) or co-amoxiclav. a macrolide (e.g. erythromycin or clarithromycin) is an alternative for those intolerant of the preferred first choices, whilst remembering the possibility of antimicrobial resistance. clarithromycin has better activity against h. influenzae than azithromycin. further details regarding the principles of antibiotic use including antibiotic resistance patterns are given in section 14. • patients without severe pre-existing illnesses and who have uncomplicated influenza, or simple bronchitis, do not routinely require antibiotics. • patients without severe pre-existing illnesses who are seen later in the course of illness and who have developed significant worsening of symptoms (particularly recrudescent fever or increasing breathlessness) should be considered for antibiotics. • patients with copd and/or other severe pre-existing illnesses, and who are therefore at high risk of influenzarelated complications, should be strongly considered for antibiotics at first consultation. • most patients can be adequately treated with a week's course of oral antibiotics. • the preferred choice of antibiotic needs also to cover infection with staph. aureus for example either doxycycline or co-amoxiclav (see table 8 .2). • a macrolide (e.g. erythromycin or clarithromycin) is an alternative choice in certain circumstances. the principles of antibiotic selection for patients with influenza-related pneumonia who can be managed in the community are similar to those for the management of sporadic community-acquired pneumonia in general except that adequate cover for staph. aureus, in addition to cover for s. pneumoniae, should be included in any empirical regimen. for this reason a tetracycline, such as doxycycline or oral co-amoxiclav, is the preferred regimen (table 8 .2). a macrolide (e.g. erythromycin or clarithromycin) is an alternative for those intolerant of the preferred first choices. macrolide (erythromycin 500 mg qds po or clarithromycin 500 mg bd b po) a an alternative regimen is provided for those intolerant of or hypersensitive to the preferred regimen. b clarithromycin may be substituted for those with gastrointestinal intolerance to oral erythromycin and also has the benefit of twice daily dosage and better cover against h. influenzae. abbreviations: od, once daily; bd, twice; tds, 3 times; qds, 4 times. secondary bacterial infections particularly pneumonia and otitis media are common in children with influenza. s. pneumoniae, staph. aureus and h. influenzae are the most common pathogens encountered during influenza outbreaks. • children in any one of the following groups should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae: (1) those at risk of complications of influenza (see appendix 2). (2) those with one or more of the following adverse features: (a) breathing difficulties (b) severe earache (c) vomiting for more than 24 hours (d) drowsiness. part 2. clinical management of adults referred to hospital section 9. severity assessment of adults referred to hospital 9.1. what severity assessment strategy is recommended for patients referred to hospital with influenzarelated pneumonia? there is no validated severity assessment tool developed specifically for influenza-related pneumonia. the curb-65 severity assessment tool as described in the bts cap guidelines 2004 is recommended for the stratification of hospitalised patients with influenza-related pneumonia into disease severity groups 72 (table 9 .1). in addition, the presence of diffuse bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia is an adverse prognostic feature. such patients should be treated as for severe pneumonia. in all instances, clinical judgement is essential when assessing disease severity. • patients with bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia should be managed as having severe pneumonia regardless of curb-65 score. • in hospital, patients with influenza-related pneumonia who have a curb-65 score of 3 or more are at high risk of death and should be managed as having severe pneumonia. • patients who have a curb-65 score of 2 are at increased risk of death. they should be considered for short stay inpatient treatment or hospital-supervised outpatient treatment. this decision is a matter of clinical judgement. • patients who have a curb-65 score of 0 or 1 are at low risk of death. they can be treated as having non-severe pneumonia and may be suitable for home treatment. 9.2. when should transfer to a high dependency unit (hdu) or intensive care unit (icu) be considered? the indications for transfer to hdu or icu are no different in patients with influenza infection compared to other patients. most patients who might require hdu/icu care will have influenza-related pneumonia or a severe exacerbation of underlying comorbid illness, e.g. exacerbation of copd. in a pandemic situation when hdu/icu beds may not be readily available, prioritisation of patients on an individual basis matched against available resources will be expected. • patients with primary viral pneumonia or a curb-65 score of 4 or 5 should be considered for hdu/icu transfer. • general indications for hdu/icu transfer include: (1) persisting hypoxia with pao 2 < 8 kpa despite maximal oxygen administration (2) progressive hypercapnia (3) severe acidosis (ph < 7.26) (4) septic shock • patients with influenza admitted to an intensive care unit should be managed by specialists with appropriate training in intensive care, respiratory medicine and/or infectious diseases. in acute uncomplicated influenza the chest x-ray is usually normal. when primary viral pneumonia occurs as a complication, particularly in elderly adults the chest x-ray often shows multiple infiltrates or consolidation. cavitations or pleural changes suggest bacterial superinfection. in combined viral-bacterial pneumonia, the clinical features typically appear later than primary viral pneumonia and the chest x-ray often shows cavitation or pleural effusions. secondary bacterial pneumonia usually occurs after apparent improvement from the viral infection; the chest x-ray may show consolidation. • a chest x-ray should be obtained during assessment of a suspected case of influenza seen in the hospital setting (accident and emergency department or acute admissions ward). • in those patients who are subsequently followed up in a hospital outpatient clinic or by a general practitioner a repeat chest x-ray should be obtained at around six weeks if respiratory symptoms or signs persist or where there is a higher risk of underlying malignancy (especially smokers and those over 50 years of age). • further investigations including a ct thoracic scan, and bronchoscopy should be considered if the chest x-ray remains abnormal at follow up 72 . in those patients with illness severe enough to present to secondary care the following tests may be useful: full blood count: a leucocytosis with left shift may occur in those with primary viral pneumonia, mixed viralbacterial pneumonia or secondary bacterial pneumonia. (lymphopenia has been noted in human cases of severe avian h5n1 influenza.) urea and electrolytes may reveal evidence of hypo or hypernatraemia or renal impairment. liver function tests are usually normal. creatine kinase (ck) may be elevated in those with severe myalgia. c-reactive protein (crp) is unlikely to be helpful except where superimposed bacterial infection is suspected 72 . however, the diagnostic value of crp in lower respiratory tract infections remains controversial 92 . • the following blood tests should be obtained in patients admitted to hospital: (1) full blood count; (2) urea, creatinine and electrolytes; (3) liver function tests; (4) creatine kinase (if myositis is suspected). • in patients with suspected secondary bacterial infection, the c-reactive protein (crp) level may aid diagnosis. in acute uncomplicated influenza larger airway function remains normal. however, there is often an increase in bronchial reactivity which may persist for many weeks after resolution of the infection 93 . lung function tests are unnecessary in most patients. section 11. microbiological investigations for adults in hospital 11 .1. introduction the guidelines provided below are based on the assumption that when cases are first occurring in the uk as part of a global pandemic, it will be possible to perform full microbiological investigations in all new cases of influenzalike illness and influenza-related pneumonia. as case numbers rise, possibly to pandemic levels, full or indeed any microbiological investigation will become increasingly difficult. thus, data on the relative frequency of different bacterial causes of influenza-related pneumonia and their antimicrobial susceptibilities amongst investigated cases gathered earlier in the pandemic should be available to guide and refine empirical antimicrobial therapy choices for cases occurring later in the pandemic. the most likely pathogens implicated in influenzarelated pneumonia are streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae and to a lesser extent b-haemolytic streptococci (see section 3.3). in the early phases (uk alert levels 1, 2 and 3 see appendix 1) of a pandemic, microbiological diagnostic approaches should focus on confirming influenza as the primary illness, defining bacterial causes of influenza-related pneumonia, and optimizing both specific (for individual patients) and general (for populations) antimicrobial treatment recommendations. in later pandemic phases (uk alert level 4) with the much higher caseloads anticipated, microbiological investigation should be focused on patients with severe influenza-related pneumonia unresponsive to empirical antimicrobial therapy. actual and practical local level transition to less intense microbiological investigation may occur at uk alert level 3 in some regions as the number of local cases is likely to vary between regions. §11. microbiological investigations for adults in hospital part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s27 it will be necessary to perform full microbiological investigations on all hospitalised cases, including patients with severe and non-severe influenza-related pneumonia, in order to: confirm influenza as the primary infection, optimize treatment options for the patients investigated and define the most common bacterial causes of influenzarelated pneumonia and their antimicrobial susceptibility patterns. the latter data will help to inform empirical antimicrobial therapy of subsequent cases for which microbiological investigation may not be undertaken fully, or at all. in influenza, rapid virological tests, viral culture and pcr of respiratory samples will yield positive results between one and seven days after illness onset. however, if presentation is more than seven days after the onset of influenzalike illness then such sampling and testing is unhelpful. instead, serum samples for serological testing for evidence of recent influenza infection are recommended. specific detailed microbiological guidance for taking and handling specimens from individuals at risk of avian influenza prepared by prof maria zambon of health protection agency (hpa) centre for infections is available at: www.hpa.org.uk/infections/topics_az/avianinfluenza/ guidance/microbiological_guidance.htm bacteriological investigations are only recommended in patients with influenza-related pneumonia. legionella pneumophila infection is not normally associated with influenza-related pneumonia. despite this, legionella urine antigen tests should be performed on severe cap cases in the early stages of an outbreak/incident in order to confirm legionella infection is not the reason for a local increase in pneumonia admissions. these recommendations are modified from those contained in the british thoracic society community acquired pneumonia (bts cap) guidelines 2001 [thorax 2001;56(suppl iv), see sections 5.7, 5.8 and 5.9 (pp. iv23 iv28)] and the 2004 update (see pages 4 5), both available at: www.brit-thoracic.org.uk/ iqs/bts_#guidelines_pneumonia_html. sputum investigative efforts must be focused on quality samples (i.e. those from patients who are able to expectorate purulent samples, and have not received prior antibiotic treatment) and not dissipated on large numbers of poor quality samples. it is important to acknowledge that the criteria for quality samples may only be met for a minority of admissions. laboratories should offer a reliable sputum gram stain for appropriate samples, as on occasions this can give immediate indication of likely pathogens. the most likely influenzarelated pneumonia pathogens are s. pneumoniae, staph. aureus and h. influenzae, all of which may present a characteristic appearance on gram stain of purulent sputum. laboratories performing sputum gram stains should adhere to strict and locally agreed criteria for interpretation and reporting of results. a. virology all patients: • nose and throat swabs in virus transport medium should be collected from all patients and submitted to the local laboratory. the relevant laboratory should be notified of the suspected diagnosis and there should be close liaison over sample collection, handling and transport. • rapid testing by direct immunofluorescence or rapid eia test, virus culture and/or pcr should be undertaken according to local availability and/or referred to an appropriate laboratory • during uk alert level 1, when the uk is on high alert for the first cases of pandemic influenza, suspected cases are likely to be investigated by local health protection teams from the health protection agency and its partner organisations in the devolved administrations. • during uk alert levels 1 and 2, clinicians dealing with suspected cases of pandemic influenza should ensure that the local health protection team is informed and involved from the outset. • the health protection agency and its partner organisations in the devolved administrations have established a network of more than 20 laboratories across the uk which have been proficiency tested in molecular diagnosis of influenza a/h5n1. access to this service should be via local health protection teams. • if presentation is more than seven days after onset of illness, an 'acute' serum (5 10 ml clotted blood) should be collected and a 'convalescent' sample (5 10 ml clotted blood) obtained after an interval of not less than seven days. the two sera should be examined serologically for evidence of recent influenza infection. b. bacteriology patients with influenza-related pneumonia: • the following bacteriological tests should be performed: (1) blood culture (preferably before antibiotic treatment is commenced) (2) pneumococcal urine antigen (20 ml urine sample). agents. acute serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood) and the two sera stored for subsequent testing. once a pandemic is established, virological investigations are not recommended routinely and in a pandemic situation may not be readily available. the diagnosis of influenza will be based on clinical findings. if influenza-related pneumonia is present, the degree of microbiological investigation will be directed by disease severity and the presence of co-morbidities. in influenza-related pneumonia, examination of sputum should be considered for patients who do not respond to empirical antibiotic therapy. this will be particularly relevant if staph. aureus is identified as a common influenza-related pneumonia pathogen during the early phase of the pandemic as, in contrast to s. pneumoniae and h. influenzae, antimicrobial susceptibilities of this organism are less predictable and empirical choices more speculative. a. virology not routinely recommended. b. bacteriology patients with influenza-related pneumonia: (i) non-severe pneumonia (curb-65 score 0, 1 or 2) • sputum samples should be sent for gram stain culture and antimicrobial susceptibility tests in patients who do not respond to empirical antibiotic therapy. (ii) severe pneumonia (curb-65 score 3, 4 or 5) • specific investigations should include: (1) blood culture, preferably before antibiotic treatment is commenced. (2) pneumococcal urine antigen (20 ml urine). (3) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who: (i) are able to expectorate purulent samples, and (ii) have not received prior antibiotic treatment. sputum specimens should be transported rapidly to the laboratory. (4) paired serological examination for influenza/ other agents. 'acute' serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood) and the two sera stored for subsequent testing. (5) tracheal or endotracheal aspirate samples, if available, should be sent for gram stain, culture and antimicrobial susceptibility testing. section 12. general management of adults admitted to hospital 12 initial management will depend on the assessment of the reason for admission, the presence of complications, and the impact of the influenza on any pre-existing disease, or psychosocial factors. for instance, some elderly patients may require admission for social reasons. in broad terms, the most likely clinical reasons for admission will be (in order of frequency): • lower respiratory tract complications: non pneumonic bacterial exacerbation of chronic lung disease such as copd (possibly with a mixed viral infection) secondary bacterial pneumonia mixed bacterial and viral pneumonia primary viral pneumonia • cardiac complications: exacerbation of pre-existing cardiac disease with cardiac failure and/or arrhythmia primary myocarditis • other complications: exacerbation of other pre-existing disease, such as diabetes mellitus neurological complications rhabdomyolysis severe sinusitis the initial management is likely to most usually involve that of respiratory and cardiac complications, especially pneumonia and these are discussed below. management of other less common primary influenzal complications (such as rhabdomyolysis, encephalopathy) is not covered. all influenza patients admitted to hospital with abnormal cardiorespiratory symptoms and signs, including influenzarelated pneumonia, should have a chest radiograph and an electrocardiogram and should have oxygenation assessed by pulse oximetry, preferably whilst breathing air (see section 10). those with sao 2 < 92% should have arterial blood gas measurements, as should all patients with features of severe illness. knowledge of the inspired oxygen concentration is essential to the interpretation of blood gas measurements and should be clearly recorded with the blood gas result. continuous oxygen therapy is indicated for those patients with pao 2 < 8 kpa, hypotension with systolic bp <100 mmhg, metabolic acidosis with bicarbonate <18 mmol/l or respiratory distress with respiratory rate >30/min 94 . the aim of oxygen therapy should be to maintain pao 2 at >8 kpa or sao 2 > 92%. unless complicated by severe chronic obstructive pulmonary disease with ventilatory failure, high concentrations of oxygen of 35% or greater are indicated and can be safely used. high concentration oxygen therapy given to patients with pre-existing chronic obstructive pulmonary disease §12. general management of adults admitted to hospital part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s29 who may have co 2 retention can reduce hypoxic drive and increase ventilation-perfusion mismatching. in such patients initial treatment with low oxygen concentrations (24 28%) should be progressively increased on the basis of repeated arterial blood gas measurements, the aim being to keep sao 2 > 90% without causing a fall in arterial ph below 7.35, in line with the management strategy recommended in the nice copd guidelines 95 . non-invasive ventilation (niv) may be of value in patients with copd who are in acute hypercapnic respiratory failure 72,96 . the use of niv in patients with respiratory failure due to severe pneumonia but without co-existing copd has not been shown to influence mortality 72,96 . nevertheless, during an influenza pandemic when critical care level 3 beds 97 are in high demand, niv may be of value as a bridge to invasive ventilation in specific circumstances. in all instances, the risks of infection due to the dissemination of respiratory droplets related to the use of niv must be taken into account when deciding on management strategies. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted and observed at all times, including the use of personal protection equipment (ppe) (see uk infection control guidance for pandemic influenza) 3 . all patients should be assessed for volume depletion and may require iv fluids. the potential for influenza to cause cardiac decompensation, either through exacerbation of pre-existing cardiac disease or from a primary myocarditis, should be borne in mind, with any complicating heart failure and arrhythmias being managed in the usual way. physiotherapy may be of benefit in selected patients with excess bronchial secretions, particularly those with concurrent chronic obstructive pulmonary disease. in cases of severe illness requiring prolonged hospital admission, increased nutritional support whether enteral, parenteral or via naso-gastric feeding should be arranged. • hypoxic patients should receive appropriate oxygen therapy with monitoring of oxygen saturations and inspired oxygen concentration with the aim to maintain pao 2 > 8 kpa and sao 2 > 92%. high concentrations of oxygen can safely be given in uncomplicated pneumonia. • oxygen therapy in patients with pre-existing copd complicated by ventilatory failure should be guided by repeated arterial blood gas measurements. non-invasive ventilation may be helpful. • in patients without pre-existing copd who develop respiratory failure, niv may be of value as a bridge to invasive ventilation in specific circumstances when critical care level 3 beds are in high demand. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted at all times. • patients should be assessed for cardiac complications and also volume depletion and their need for additional intravenous fluids. • nutritional support should be given in severe or prolonged illness. 12.3. what monitoring should be conducted during a hospital stay? pulse, blood pressure, respiratory rate, temperature, oxygen saturation (with a recording of the inspired oxygen concentration at the same time) and mental status should be measured initially at least twice daily. this is most conveniently performed using an early warning score (ews) chart, which all ward staff should be familiar with. those with severe illness, requiring continuous oxygen or cardiovascular support, should be monitored more frequently. failure to improve clinically within 48 hours should result in a full clinical reassessment and failure to improve over 4 days is an indication to repeat the chest radiograph. • temperature, respiratory rate, pulse, blood pressure, mental status, oxygen saturation and inspired oxygen concentration should be monitored and recorded initially at least twice daily and more frequently in those with severe illness or requiring regular oxygen therapy. • an early warning score system is a convenient way to perform this. • in addition to a full clinical reassessment, a chest radiograph should be repeated in patients who are not progressing satisfactorily. there will be considerable pressure to discharge patients early during a pandemic. the type and availability of out-of-hospital facilities will dictate hospital discharge decisions. some guidance regarding simple parameters to review when considering hospital discharge can be obtained from a recent us prospective, multi-centre, observational cohort study of 680 patients admitted to hospital with cap 98 , and is offered as advice for all patients admitted with influenza-related respiratory complications. • patients should be reviewed before 24 hours of discharge home. those with two or more of the following unstable clinical factors should be considered for continued hospital management: (1) temperature > 37.8ºc, (2) heart rate >100/min, (3) respiratory rate >24/min, (4) systolic blood pressure <90 mmhg, (5) oxygen saturation <90%, (6) inability to maintain oral intake, it is usual practice to arrange 'routine' hospital clinic follow up and repeat the chest radiograph at around six weeks after discharge for acute respiratory illness such as pneumonia. however, there is no evidence on which to base a recommendation regarding the value of this practice in patients who have otherwise recovered satisfactorily. it is also not known whether there is any value in arranging clinical follow up in a hospital clinic rather than with the patient's general practitioner. during an influenza pandemic situation, it is likely that only patients who developed complications or who had significant worsening of their underlying disease will be offered clinical review at one or other venue. at discharge, patients should be offered access to information about their take-home medication, smoking and lifestyle advice as appropriate, potential future complications and action to take in the event of a relapse of symptoms. • follow-up clinical review should be considered for all patients who suffered significant complications or who had significant worsening of their underlying disease, either with their general practitioner or in a hospital clinic. • at discharge or at follow up, patients should be offered access to information about their illness, take-home medication and any follow-up arrangements. • it is the responsibility of the hospital team to arrange the follow-up plan with the patient and the general practitioner. section 13. use of antivirals in hospitalised adults 13 .1. what drugs should be used for antiviral treatment during a pandemic? oseltamivir (neuraminidase inhibitor) will be the mainstay for therapy in the pandemic. the m2 inhibitors, amantadine and rimantadine, are unsuitable for use for treatment due to the rapid emergence of resistance together with sideeffects. from clinical trial data accrued to date and based on seasonal, interpandemic influenza, the anticipated positive effect of antivirals in a pandemic will be: (a) a reduction of illness duration by 24 hours, and therefore more rapid mobilisation of affected individuals including essential workers; (b) a possible reduction in hospitalisation of infected individuals; (c) a reduction of subsequent antibiotic use by infected individuals. there is insufficient evidence accrued to date to determine the effect of antivirals, if any, on overall mortality. therefore the major utility of antivirals will be to maintain the essential workforce, and reduce hospitalisation and antibiotic treatment of complications. (neuraminidase inhibitors) during a pandemic? • individuals should only be considered for treatment with neuraminidase inhibitors if they have all of the following: (1) an acute influenza-like illness (2) fever (>38ºc) and (3) been symptomatic for two days or less • treatment schedule: adults: oseltamivir 75 mg every 12 hours for 5 days. dose to be reduced by 50% if creatinine clearance is less than 30 ml/minute. • exceptions: (i) patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, make still be eligible despite lack of documented fever. (ii) hospitalised patients who are severely ill, particularly if also immunocompromised, may benefit from antiviral treatment started more than 48 hours from disease onset. this advice reflects the lack of robust evidence to guide the use of antivirals in these exceptional circumstances and places a high value on the potential benefits of antiviral therapy. drugs available for treatment and prevention of infection by influenza are summarised in table 13 .1. there are four drugs available, the older agents amantadine and rimantadine and the neuraminidase inhibitors oseltamivir and zanamivir. older agents: the older agents, amantadine and rimantadine (rimantadine is not currently licensed in the uk), are related substances that act by blocking the ion-channel function of the influenza virus m2 protein. this protein, although a minor surface constituent of the influenza virus particles, is essential for virus replication. these agents are only active against influenza type a. amantadine is not recommended by nice for treatment and/or prophylaxis of interpandemic influenza, so in the absence of national stockpiling, supplies of amantadine can be expected to be very low. h5 viruses in south east asia are resistant to amantadine, so this agent may play no role at all depending on the nature of the pandemic strain. two neuraminidase inhibitors so far have been developed to the level of entry into the formulary: zanamivir is a modification of neu5ac2en, a dehydrated neuraminic acid derivative. oseltamivir is a similar molecule except it has a cyclohexene ring and replaces a polyglycerol moiety with lipophilic sidechains. oseltamivir can be taken by mouth, whereas zanamivir must be inhaled, using a diskhaler device. an intravenous formulation of zanamivir has been developed but its efficacy has not been established. this may be relevant for the management of ventilator cases. both drugs are active against influenza type a as well as type b viruses. older agents. both amantadine and rimantadine are effective for the treatment of type a influenza virus infection if treatment is begun within 48 hours of the onset of illness 99 . historical data show that they can shorten the illness by approximately one day but their efficacy in preventing complications, hospitalisations, or deaths has never been established. although these drugs are effective, their use in clinical influenza treatment has been limited as a result of their proclivity to induce viral resistance, and their side-effect profile. several large clinical trials have demonstrated the utility of zanamivir and oseltamivir in treatment of adults with influenza in the community ( virtually all studies on the efficacy of neuraminidase inhibitors to reduce complications have been conducted with oseltamivir, and this drug has been shown to have some effect on outcomes other than time to recovery. in a meta-analysis of adults and adolescents with a virologically proven influenza illness, oseltamivir treatment reduced overall antibiotic use for any reason by 26 105 . so far, the neuraminidase inhibitors have not been extensively investigated in patients who are at the highest risk of serious complications of influenza. such patients include the elderly and those with serious cardiopulmonary illness, such as chronic obstructive pulmonary disease. the neuraminidase inhibitors have not been associated with a reduction in mortality, but the clinical trials conducted so far have not been appropriate to measure this. it is not known for certain whether the neuraminidase inhibitors will be effective in pandemic influenza because their use has only been assessed in inter-pandemic influenza, where the virulence is moderate and there is some degree of host immunity. the antiviral activity is likely to be adequate; in vitro, all neuraminidase inhibitors have been demonstrated to have a broad spectrum of activity against multiple avian influenza viruses 106 . the older agents, rimantadine and amantadine, were studied in both the 1968 hong kong pandemic and again when h1n1 influenza appeared in a pandemic in 1977. their efficacy has been reviewed by hayden 99 . when the older agents were given for four to eight week periods as prophylaxis in a community setting, their protective efficacy against influenza illness averaged 70% compared with placebo. this compares with 80 90% efficacy observed with the same agents in studies during the interpandemic period. when amantadine or rimantadine are used to treat patients, resistant viruses emerge rapidly and approximately 30% of treated children or adults will shed resistant variants starting two to five days after the onset of treatment 104 . the resistant viruses shed from these patients retain full virulence, infectivity and transmission potential. when contacts of cases treated with amantadine or rimantadine are given post-exposure prophylaxis with these older agents, the reduction in secondary cases is minimal 107 . in contrast, the frequency of emergence of resistance during treatment with the neuraminidase inhibitors is reported to be low. however, during studies of experimentally induced influenza a/h1n1 infection in healthy adults, 4% of participants shed viruses with a histidine to tyrosine substitution at position 274 within the binding site of oseltamivir 108 . in these cases the volunteers had increased influenza viral load within the nasopharynx but there was no deterioration of symptoms. so far, there have been no proven instances of transmission of oseltamivir or zanamivir-resistant variants in field clinical trials, but the experience is relatively small currently. sequence analysis of h5n1 human isolates from north vietnam have revealed virus with a 274 y (resistant) sequence. although the isolate was not fully resistant, its ic50 for oseltamivir was shifted upwards and it is therefore less susceptible to oseltamivir than other h5n1 isolates that had been tested from the region. the patient from whom the virus was isolated was concurrently being treated with oseltamivir. both amantadine and rimantadine can cause nausea and vomiting in a small percentage of individuals receiving them (table 13 .1). unfortunately amantadine is also associated with very unpleasant central nervous system side-effects including anxiety, depression, insomnia and hallucinations. the side-effects are dose-related and do resolve with discontinuation of the drug. in the case of the neuraminidase inhibitors, both drugs appear relatively safe. zanamivir has very few side-effects, but can result in bronchospasm which might be potentially serious in patients with asthma. oseltamivir requires dose-reduction in patients with low creatinine clearance (<30 ml/min). nausea occurs in 5 15% of oseltamivir recipients but is seldom severe enough to lead to drug discontinuation (see table 13 .3). antimicrobial chemotherapy will be indicated primarily for respiratory complications due to secondary bacterial infections, principally influenza-related pneumonia. the majority of patients with exacerbations of chronic obstructive pulmonary disease (copd) and other chronic lung conditions due to secondary bacterial infections, such as bronchiectasis, will also require antimicrobial chemotherapy, as will some patients with severe sinusitis. few pneumonias and lower respiratory tract infections are defined microbiologically at initial assessment and hence most prescribing is empirical. in broad terms the antimicrobial management of these patients should follow the guidance offered in relevant national guidelines for the management of community-acquired pneumonia and copd, but modified in the light of the different range of pathogenic bacteria that may be implicated, specifically staph. aureus infection. in the minority of cases, the aetiology may be determined after hospital admission, thereby permitting modification of the initial empirical regimen. although the pathogens responsible for communityacquired pneumonia are diverse, in the case of bacterial pneumonia complicating influenza the principal pathogens which should be covered by any initial empirical antimicrobial therapy include s. pneumoniae, h. influenzae and staph. aureus. the latter is said to be more common with combined viral bacterial pneumonia, as some strains of staphylococci have synergistic effect with the virus. gram-negative enteric bacillary infection is also sometimes seen. exacerbations of copd will be largely associated with s. pneumoniae, h. influenzae, and moraxella catarrhalis. severity assessment and the association of pre-existing co-morbid disease is essential in predicting prognosis and in turn determines management, choice of antibiotic therapy and its method of administration (see section 9). during an influenza pandemic this will be principally related to concerns about the local pattern of antimicrobial resistance of staph. aureus, and assessing the possibility of methicillin-resistant s. aureus (mrsa) being present locally. clinicians should be kept closely informed of any local shift in antimicrobial resistance patterns, both at the start and during a pandemic. staphylococcus aureus is widely resistant to penicillin 109 and an increasing number are now methicillin-resistant (mrsa); when occurring in the community this generally reflects hospitalisation within the recent past or residence within a nursing home 110 . hence, b-lactamase unstable penicillins (penicillin g, aminopenicillins) and, in the case of mrsa, isoxazolyl penicillins (flucloxacillin, cloxacillin) and cephalosporins, are inappropriate for such infections. the true incidence of resistance among pathogens in the community is difficult to estimate since most laboratory samples come from selected populations. with this limitation in mind, the presence of b-lactamase production among h. influenzae varies geographically but ranges from 2% to 17% 111, 112 in various parts of the uk. m. catarrhalis has a high rate of b-lactamase production. antibiotic resistance among s. pneumoniae is of concern world wide, owing to the dominance of this organism as a cause of community-acquired pneumonia and because penicillin and macrolide resistance are frequently linked 112, 113 . however, to date it is not a common enough problem in the uk to influence initial antimicrobial management decisions. recent data provided by the hpa of antimicrobial sensitivities of respiratory pathogens isolated from blood and respiratory samples during the last three to four years (robert george, personal communication) found macrolide resistance amongst about 10 14% methicillinsensitive staphylococcus aureus (mssa) isolates and 12 19% of s. pneumoniae. macrolides, apart from clarithromycin, have poor in vivo activity against h. influenzae. by contrast, tetracycline resistance was around 5 8% for s. pneumoniae, 3% for h. influenzae and 2 8% for mssa. fluoroquinolones have activity against methicillinsensitive staphylococcus aureus (mssa), with mic 90 figures of 1.0 mg/l for ciprofloxacin, 0.5 mg/l for levofloxacin and 0.12 mg/l for moxifloxacin 114 . modern fluoroquinolones (oral moxifloxacin and oral and iv levofloxacin currently licensed in the uk) are therefore a possible choice for secondary bacterial infections following influenza where mssa is a likely pathogen. a recent pharmacokinetic and pharmacodynamic in vitro study indicated that moxifloxacin 400 mg od had advantages over ciprofloxacin 500 mg bd or levofloxacin 500 mg od in antimicrobial effects against staph. aureus 115 . the quinolones, levofloxacin or moxifloxacin, also provide cover against s. pneumoniae and h. influenzae. mrsa is an unlikely pathogen in the uk in the context of community-acquired respiratory bacterial infection following influenza, and fluoroquinolones are not sufficiently active against mrsa. there are no robust research studies available to provide evidence-based guidance on the best empirical choice of antimicrobial therapy for bacterial complications of influenza. for these reasons the recommendations for treatment have been made on the basis of assessing a matrix of laboratory, clinical, pharmacokinetic and safety data, interpreted in an informed manner and taking account of other published guidelines 116 . in those with chronic lung disease, particularly copd, bacterial exacerbation will be the commonest cause of admission. it is likely that all such patients sufficiently ill to require hospital admission with an exacerbation will require antibiotics. management of their underlying macrolide (erythromycin 500 mg qds po or clarithromycin 500 mg bd b po) or fluoroquinolone with enhanced pneumococcal activity (e.g. levofloxacin 500 mg od po or moxifloxacin 400 mg od po c ) if iv needed: co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) or levofloxacin 500 mg od iv c 3. hospital-treated, severe pneumonia co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv plus macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) fluoroquinolone with some enhanced pneumococcal activity (e.g. levofloxacin 500 mg bd iv, po c plus, either macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) or b-lactamase stable antibiotic (co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv) a an alternative regimen is provided for those intolerant of or hypersensitive to the preferred regimen. b clarithromycin may be substituted for those with gastrointestinal intolerance to oral erythromycin and also has the benefit of twice daily dosage and better cover against h. influenzae. c levofloxacin and moxifloxacin are the only currently uk-licensed fluoroquinolones with enhanced activity against s. pneumoniae, in addition to cover for staph. aureus. levofloxacin comes in an oral and a parenteral formulation and is licensed for severe pneumonia. moxifloxacin comes in an oral formulation only in the uk and is not licensed for severe pneumonia. in the future, other fluoroquinolones such as gemifloxacin and gatifloxacin are likely to extend this choice, when licensed in the uk. abbreviations: od, once daily; bd, twice; tds, 3 times; qds, 4 times: iv, intravenous; po, oral. switch from parenteral drug to the equivalent oral preparation should be made as soon as clinically appropriate, in the absence of microbiologically confirmed infection. in the case of the parenteral cephalosporins, the oral switch to co-amoxiclav 625 mg tds is recommended rather than to oral cephalosporins. condition, such as copd, should follow standard guidelines, including the use of corticosteroids if indicated. antibiotics should cover the likely bacterial pathogens, including s. pneumoniae, h. influenzae, m. catarrhalis and staph. aureus. oral therapy should be sufficient for those without adverse severity features and who are able to take oral medication. the preferred first choice of antibiotic for nonpneumonic bronchial infections should include an effective oral b-lactamase stable agent such as co-amoxiclav, or a tetracycline, such as doxycycline. a macrolide is an alternative for those intolerant of the preferred first choices, whilst remembering the possibility of antimicrobial resistance. clarithromycin has better activity against h. influenzae than azithromycin. a newer-generation fluroquinolone (e.g. levofloxacin or moxifloxacin) with enhanced activity against s. pneumoniae is an alternative choice if there is increased likelihood of resistance or local issues that dictate such a choice. • previously well adults with acute bronchitis complicating influenza, in the absence of pneumonia, do not routinely require antibiotics. • antibiotics should be considered in those previously well adults who develop worsening symptoms (recrudescent fever or increasing dyspnoea). • patients at high risk of complications or secondary infection (appendix 2) should be considered for antibiotics in the presence of lower respiratory features. • most patients can be adequately treated with oral antibiotics. • the preferred choice includes co-amoxiclav or a tetracycline. • a macrolide such as clarithromycin (or erythromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative choice in certain circumstances. patients will be suffering from primary viral pneumonia, or combined viral bacterial pneumonia, or secondary bacterial pneumonia. the features of each of these are covered in section 3. all patients with pneumonic involvement should receive antibiotics. the principles of antibiotic selection for nonsevere influenza-related pneumonia is similar to those for the management of sporadic community-acquired pneumonia in general 72 , except that adequate cover for staph. aureus should be included in any empirical regimen. it is also not felt necessary to routinely provide cover for atypical pathogens (mycoplasma pneumoniae, chlamydia sp., coxiella burnetti, legionella sp.) during a pandemic as the large majority of patients will be hospitalised as a direct result of influenza and its complications caused by bacterial infection. for these reasons oral co-amoxiclav or a tetracycline such as doxycycline is the preferred regimen (table 14 .1). when oral therapy is inappropriate, parenteral coamoxiclav or a second-or third-generation cephalosporin is offered as an alternative. based on in-vitro data, the activity of selected cephalosporins against mssa in the uk in descending rank order is cefuroxime (mic90 1 2 mg/l) > cefotaxime (mic90 2 mg/l) > ceftriaxone (mic90 16 mg/l) [robert george, personal communication]. only cefuroxime and cefotaxime are recommended as cephalosporins offering adequate mssa cover within an empirical regimen. a macrolide or one or the new fluoroquinolones are identified as alternatives in hospitalised patients, in specific circumstances. these include those intolerant of penicillins or where local microbiological surveillance suggests they are better choices. at the time of completing these guidelines, only levofloxacin and moxifloxacin are licensed and available in the uk for pneumonia. flucloxacillin is not recommended as part of an empirical regimen because its activity against a narrow spectrum of pathogens (predominantly staph. aureus) would require it to be used in combination with more than one other antibiotic. it is offered as the antibiotic of choice in confirmed methicillin-sensitive staph. aureus (mssa) infection. regardless of the regimen selected it is critical that the antibiotics be administered promptly (within four hours of admission), and in the case of the patient with severe pneumonia without delay, by the admitting doctor in the admissions ward or by the general practitioner if delays are expected in the hospital admission process. delays in administration of antibiotics are related adversely to mortality in some studies, particularly when managing elderly patients 117, 118 . following initial assessment and empirical therapy, progress should be monitored carefully. the route and choice of antibiotic treatment will require adjustment, either by stepping up and broadening the spectrum of microbiological activity in the light of clinical deterioration or as a result of positive microbiological information, or stepping down with improvement as discussed below. • most patients can be adequately treated with oral antibiotics. • oral therapy with co-amoxiclav or a tetracycline is preferred. • when oral therapy is contra-indicated, recommended parenteral choices include intravenous co-amoxiclav, or a second or third generation cephalosporin (cefuroxime or cefotaxime respectively). • a macrolide (erythromycin or clarithromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative regimen for those intolerant of penicillins. currently levofloxacin and moxifloxacin are the only recommended fluoroquinolones licensed in the uk. • antibiotics should be administered within four hours of admission. mortality is greatly increased in those with severe pneumonia (section 9). the illness may progress before microbiological information is available. preferred and alternative initial treatment regimens are summarised in table 14 .1. the recommendation of broadspectrum b-lactam regimens plus a macrolide in those with severe influenza-related pneumonia is based on the following rationale: (a) while s. pneumoniae and staph. aureus remain the predominant pathogens, gram-negative enteric bacilli, although uncommon, carry a high mortality 119 . (b) the recommended empirical regimen will offer double cover for the likely pathogens implicated in influenzarelated pneumonia and there is some evidence to indicate that combination therapy is associated with better outcomes in severe pneumonia 120 . (c) although there is no evidence of an increased incidence of infection by atypical pathogens in influenzarelated pneumonia, in severe pneumonia it is felt necessary to include cover for atypical pathogens, particularly legionella sp. as it may not be possible at the outset to distinguish between patients with sporadic severe community-acquired pneumonia in whom legionella infection is important, and influenzarelated pneumonia. parenteral administration of antibiotic is recommended in those with severe community-acquired pneumonia regardless of the patient's ability or otherwise to take oral medication. this is to ensure prompt, high blood and lung concentrations of antibiotic. a fluoroquinolone is offered as an alternative, despite limited data on their use in severe pneumonia 121 . at the time of writing, levofloxacin is the only licensed and available agent in the uk for severe pneumonia. it is marketed in parenteral and oral formulations. however, until more clinical experience is available we recommend combining it with another agent active against s. pneumoniae and staph. aureus such as a broad-spectrum b-lactam or macrolide when managing severe influenzarelated pneumonia. • patients with severe pneumonia should be treated immediately after diagnosis with parenteral antibiotics. • an intravenous combination of a broad-spectrum b-lactamase stable antibiotic such as co-amoxiclav or a second-(e.g. cefuroxime) or third-(e.g. cefotaxime) generation cephalosporin together with a macrolide (clarithromycin or erythromycin) is preferred. • an alternative regimen includes a fluoroquinolone with enhanced activity against pneumococci together with a broad-spectrum b-lactamase stable antibiotic or a macrolide. currently levofloxacin is the only such fluoroquinolone licenced in the uk. • patients who have been in hospital within the last few months have a higher chance of carrying mrsa as opposed to patients who have not been hospitalised recently. therefore due consideration should be given to the possibility of mrsa if they are known or suspected to have a staphylococcal pneumonia and/or are not responding to empirical therapy. 14.7. when should the iv route be changed to oral? there can be no rigid recommendation concerning the timing of transfer to oral therapy and further studies of this area are needed 122 . any decision must be individualised on the basis of assessing all factors, including the absence of any contraindications to oral administration, the availability of any microbiological information regarding aetiology of the infection and clear evidence that the patient is responding to initial therapy. the recommended guideline is that oral therapy be considered in a patient who has shown clear evidence of improvement and whose temperature has resolved for a period of 24 hours. • patients treated initially with parenteral antibiotics should be transferred to an oral regimen as soon as clinical improvement occurs and the temperature has been normal for 24 hours, providing there is no contraindication to the oral route. 14.8. for how long should antibiotics be given? until there are more precise methods to reliably identify microbiological and clinical end-points, the duration of therapy will remain subject to clinical judgement and custom. for these reasons the duration of therapy will vary by individual patient, disease severity and speed of resolution. • for most patients admitted to hospital with nonsevere and uncomplicated pneumonia, seven days of appropriate antibiotics is recommended. • for those with severe, microbiologically undefined pneumonia, ten days treatment is proposed. this should be extended to 14 21 days where s. aureus or gramnegative enteric bacilli pneumonia is suspected or confirmed. 14.9. failure of initial empirical therapy in those patients who fail to respond to initial empirical therapy, several possibilities need to be considered, the first of which is whether the correct diagnosis has been made. radiographic review is recommended for the community-and hospital-managed patient. this may also indicate complications of pneumonia such as pleural effusion/empyema, lung abscess or worsening pneumonic shadowing, which will be more common in the presence of staphylococcal infection. the initial empirical antibiotic regimen may need to be reassessed. however, compliance with, and adequate absorption of an oral regimen should first be considered. microbiological data should be reviewed and further specimens examined, with a view to excluding staph. aureus and gram-negative bacillary infection. in the hospital-managed, non-severely ill patient, changing to a new fluoroquinolone such as levofloxacin provides a second alternative. in the severely ill patient already receiving a b-lactam/ clarithromycin regimen, it is recommended that further staphylococcal cover is added to include cover for mrsa 123 . in addition, urgent referral to a respiratory physician should be made for clinical assessment including the possible need for bronchoscopic sampling. other rapid mrsa diagnostic techniques are in the evaluation stage. • for those with non-severe pneumonia in hospital on combination therapy, changing to a fluoroquinolone with effective pneumococcal and staphylococcal cover is an option. • adding further antibiotics effective against mrsa is an option for those with severe pneumonia not responding to combination antibiotic therapy. specific pathogen-directed antibiotic therapy 14.10 . what are the optimum antibiotic choices when specific pathogens have been identified? when a pathogen has been identified, specific therapy as summarised in table 14 .2 is proposed. in transferring patients from empirical to pathogen-targeted therapy, the regimen and route of administration will be determined by the continued need for parenteral therapy and known drug intolerance. these recommendations are again based on a synthesis of information, which includes in vitro activity of the drugs, appropriate pharmacokinetics and clinical evidence of efficacy gleaned from a variety of studies. the choice of agent may be modified following the availability of sensitivity testing or following consultation with a specialist in microbiology, infectious disease or respiratory medicine. close liaison with the local microbiology service will be essential during a pandemic. currently s. pneumoniae highly resistant to penicillin (mic 4 mg/l) is uncommon in the uk. however, it is important that the situation is monitored and in future §14. use of antibiotics in hospitalised adults part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s37 either ciprofloxacin 400 mg bd iv or piperacillin 4 g tds iv ± gentamicin or tobramycin (dose monitoring) higher doses of penicillins or alternative regimens may need to be considered. staphylooccus aureus is an uncommon cause of sporadic community-acquired pneumonia in the uk, but will assume much greater potential importance during a pandemic. most community isolates are methicillin-sensitive although the recent increase in mrsa in hospitalised patients may result in subsequent readmission with an mrsa infection, secondary to influenza. options for methicillin-sensitive and -resistant infections are based on parenteral administration in view of the serious nature of staphylococcal pneumonia. • if a specific pathogen has been identified, the antibiotic recommendations are summarised in children with high fever (>38.5ºc) and cough or influenzalike symptoms will be seen by a community health professional (a nurse or doctor if under seven years of age). if there are no features that put them at high risk of complications they should be treated with oseltamivir, and given advice on antipyretics and fluids. children under one year of age and those at risk of complications (appendix 2) should be seen by a gp. children may be considered at increased risk of complications if they have: cough and fever (or influenza-like illness) and temperature >38.5ºc and either (i) chronic co-morbid disease (see appendix 2) or (ii) one of the following features • breathing difficulties • severe earache • vomiting > 24 hours • drowsiness these patients should be offered an antibiotic as well as oseltamivir (in those over one year of age) and advice on antipyretics and fluids. children under one year of age with none of the above features should be treated with antipyretics and fluids with a low threshold for antibiotics if they become more unwell. the most severely ill children should be referred for assessment for admission. in a pandemic situation, paediatric high dependency and intensive care beds are likely to fill quickly and will be insufficient to meet demand. children will have to be triaged by the senior paediatrician on duty in consultation with tertiary specialists in respiratory medicine, paediatric intensive care or paediatric infectious diseases. triage will be on the basis of the severity of the child's (a) acute and (b) co-existing disease and the likelihood of the child achieving full recovery. where admission is not possible §16. general investigations for children in hospital part 3. clinical management of children referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s39 the tertiary specialists will provide advice and support on management to the general paediatrician. in the h5n1 cases reported from vietnam 125 all seven children had wbc < 4.0 (mean 2.44) and 6/7 had a lymphopenia <1.0 (mean 0.66). six of the seven children died. in contrast, only two of the seven children reported from hong kong died but they were both leukopenic and lymphopenic. the survivors had a mean wbc of 12.44 and lymphocyte count of 3.11 126 . four of five cases reported from thailand were lymphopenic 127 . in influenza a thrombocytopenia (<100) is found in 5 7% 71, 84 . thrombocytopenia was found in four out of seven cases of h5n1 infection in vietnamese children 125 . liver transaminases are raised in 27% of influenza a patients 124 and were raised in six out of six of those measured in the hong kong h5n1 outbreak 126 and five out of six in those measured in vietnam 125 . c-reactive protein (crp) is unhelpful in influenza with values <10 in 55% 84 ; <20 in 72% 62 and >80 in only 5% 62 . the cd4/cd8 ratio was inverted in the two children and three adults in whom it was measured in the vietnam outbreak (mean 0.7; range 0.59 1.08). two of these patients survived 125 . • a full blood count with differential, urea, creatinine and electrolytes and liver enzymes and a blood culture should be done in all severely ill children. one of the largest studies of the value of chest radiography was undertaken in children aged between two months and five years with community-acquired pneumonia managed as outpatients with time to recovery as the main outcome 128 . chest radiography did not affect the clinical outcome in these children with acute lower respiratory infection. this lack of effect was independent of clinicians' experience. there are no clinically identifiable subgroups of children within the who case definition of pneumonia who are likely to benefit from a chest radiograph. the authors concluded that routine use of chest radiography was not beneficial in ambulatory children aged over two months with acute lower respiratory tract infection (lrti). clinicians basing the diagnosis of lower respiratory infections in young infants on radiographic diagnosis should be aware that there is variation in intraobserver and interobserver agreement among radiologists on the radiographic features used for diagnosis. there is also variation in how specific radiological features are used in interpreting the radiograph. a recent study on standardization of cxr interpretation in paediatric pneumonia illustrates the importance of standardised training 129 . the cardinal finding of consolidation for the diagnosis of pneumonia appears to be highly reliable 130 and reasonably specific for bacterial pneumonia (74% of 27 patients with alveolar shadowing had bacterial proven pneumonia) 131 but overall chest radiography is too insensitive to be useful in differentiating between patients with bacterial pneumonia and those whose pneumonia is nonbacterial 132, 133 . in the context of an influenza pandemic, a cxr will not distinguish viral pneumonia from viral illness with bacterial superinfection, and all children with signs of pneumonia should be treated with antibiotics. • a cxr should be performed in children who are hypoxic, have severe illness or who are deteriorating despite treatment. oxygen saturation (sao 2 ) measurements provide a noninvasive estimate of arterial oxygenation. pulse oximetry will be a key tool in assessment and management and it is essential that it is used correctly and that users are aware of the possibility of artefactually low readings. the oximeter appears easy to use and requires no calibration. however, it requires a pulsatile signal from the patient. it is also highly subject to motion artefacts. to obtain a reliable reading: (1) the child should be still and quiet. (2) when using paediatric wrap around probes, the emitting and receiving diodes need to be carefully opposed. (3) a good pulse signal (plethysmograph) should be obtained. (4) once a signal is obtained, the saturation reading should be watched over at least 30 seconds and a value recorded once an adequate stable trace is obtained. • pulse oximetry should be performed in every child being assessed for admission to hospital with pneumonia. to be read in conjunction with the corresponding section for adults (section 11 in part 2). as with adults, the extent of virological and microbiological investigations undertaken in children should vary according to the stage of the pandemic and additionally according to the severity of an individual case. it should be noted however, that the clinical features of influenza in children are less characteristic than in adults (see section 4) and the need for special diagnostic tests is therefore greater 62, 134, 135 the utility of rapid influenza tests has been demonstrated in studies where rapid knowledge of a diagnosis of influenza (within ten minutes) has been shown to have an impact on clinicians' behaviour with respect to antibiotic use, performance of other tests and admission to hospital 136, 137 . it may be imagined that in a pandemic situation such a test could result in earlier use of antiviral therapy and a more rational approach to hospital admission and to prophylaxis of contacts. however, using a molecular reference standard, one test was shown to have low sensitivity (44%) but high specificity (97%) suggesting that its role might better be to 'rule in' influenza rather than 'ruling it out' 138 . similar conclusions have been made with other commercial rapid tests 139, 140 . as a reflection of this, rapid antigen tests were positive in only two of six patients with avian influenza a (h5n1) 125 . the need for bacteriological tests in cases of influenza with pneumonia is also logical and the range of pathogens similar to adults 36,69,141-145 except that legionella infection is extremely unlikely to occur in a previously healthy child and legionella-specific antigen testing is therefore unnecessary. the urinary pneumococcal antigen tests in children may lack both sensitivity and specificity and should be interpreted with care 146, 147 . sputum collection in children is also unreliable although in older children (e.g. over 12 years of age) it may be possible and should be handled as indicated for adults. a. virology all children: during an influenza pandemic children are likely to be admitted to hospital because of the severity of their disease and its complications or because of the impact of influenza on pre-existing disorders such as cardiac, respiratory or neurological disease. management of preexisting disorders is outside this guideline. • the most common reason for admission is likely to be: (1) lower respiratory tract disease with either a viral or bacterial or mixed pneumonia. • other reasons for admission include: (2) severe gastroenteritis (3) cardiac disease viral myocarditis (4) encephalitis children should be triaged to ward or hdu/picu after severity assessment (section 15). an influenza pandemic is likely to occur in the winter months when other winter viruses responsible for paediatric morbidity and hospital admission are circulating (such as rsv and adenovirus). particularly in the early stages of a pandemic (uk alert levels 1 3) it will be important to use rapid virological tests in an attempt to cohort influenzapositive and rsv-positive infants separately and to separate from other patients (see uk infection control guidance for pandemic influenza) 3 . hypoxic infants and children may not appear cyanosed. agitation may be an indication of hypoxia. patients whose oxygen saturation is less than 92% while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. nasal cannulae do not deliver a fio 2 more than around 40% even at flow rates of 2 l/min in infants and 4 l/min in older children. alternative methods of delivering higher concentrations of humidified oxygen such as a head box or a venturi face mask may be necessary. if sao 2 > 92% cannot be maintained with an fio 2 of 60% then additional support such as cpap, bipap or intubation and ventilation should be considered. • patients whose oxygen saturation is 92% or less while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. children who are unable to maintain their fluid intake due to breathlessness, fatigue or gastroenteritis need fluid therapy. where possible additional fluid should be by the enteral route, and where nasogastric tube feeds are used, the smallest tube should be passed down the smallest nostril to minimize effects on respiratory status. severely ill children may need intravenous fluids, and if the child is in oxygen therapy intravenous fluids should be given at 80% basal levels (to avoid complications of inappropriate adh secretion) and serum electrolytes should be monitored. the monitoring will depend on the child's condition. severely ill children will need continuous monitoring of heart rate, respiratory rate, oxygen saturation and neurological status. all children on oxygen therapy should have four-hourly monitoring including oxygen saturation. chest physiotherapy is not beneficial in previously healthy children with pneumonia. children with underlying conditions such as cystic fibrosis or neuromuscular weakness will benefit from intensive physiotherapy. children with influenza are generally pyrexial and may have some pain, including headache, chest pain, arthralgia, abdominal pain, and earache from associated otitis media. pleural pain may interfere with depth of breathing and may impair the ability to cough. antipyretics and analgesics can be used to keep the child comfortable and to help coughing. 18.9. when can children be safely discharged from hospital? in a pandemic situation there will be great pressure on hospital beds. all children should be assessed for discharge at least twice daily. children should not remain in hospital if they are receiving therapy that could be given in the community. in previously healthy children suitable discharge criteria would be: (1) child is clearly improving (2) child is physiologically stable (3) child can tolerate oral feeds (4) respiratory rate is <40/min (<50/min in infants) (5) awake oxygen saturation is >92% on air. most children will make an uneventful recovery and not require follow up. those with a prolonged illness may be followed up by their general practitioner. only children with severe disease and/or at high risk of sequelae need hospital follow up. children with lobar collapse should have a follow-up cxr. follow-up cxrs after acute uncomplicated pneumonia are of no value where the patient is asymptomatic 148, 149 . to be read in conjunction with the corresponding section for adults (section 13 in part 2) five antiviral agents are theoretically available for the therapy of influenza in children: the m2 ion channel inhibitors amantadine and rimantadine (both administered orally and for influenza a only), the neuraminidase inhibitors oseltamivir (administered orally) and zanamavir (administered through an inhaler), and ribavirin (aerosolised). the limitations of amantadine and rimantadine are detailed in section 13, particularly in the context of a pandemic where resistance may already be present 150 . both have been shown to be effective in the treatment of influenza a in children 151 . concerns exist about the development of resistance during therapy for both agents 151, 152 . a household study showed that treatment and prophylaxis with rimantadine resulted in rapid selection and transmission of drug resistant virus 153 . in a double-blind randomised, placebo controlled study, 217 children (1 12 years of age) received oseltamivir with a resultant reduction in the median duration of illness, incidence of otitis media as a complication of influenza (12% vs 21%) and the need for antibiotic prescriptions in those with influenza (68 of 217, 31% vs 97 of 235, 41%; p = 0.03) compared to placebo 103 . the most common sideeffect was vomiting (5.8%). a systematic review and meta-analyses published in 2003, which included studies up to december 2001, included only two studies of zanamivir and one study of oseltamivir 103 in which these drugs were administered for treatment of influenza a or b in children under 12 years of age 154 . the reduction in the median time to alleviation of symptoms for influenza-positive children when compared with placebo was 1.0 day (95% ci: 0.4 1.6) for zanamivir and 1.5 days (0.8 2.2) for oseltamivir. across all ages a 29% (10 44%) relative reduction in complications requiring antibiotics was observed for zanamivir, and for children specifically a 35% relative reduction was observed for oseltamivir. this was updated through to december 2002 in a cochrane review 155 . using its search criteria it identified two trials of oseltamivir (one in healthy children 103 and one in children with asthma which was later published 156 and only one with zanamivir. its conclusions were therefore the same with respect to median illness duration in healthy children. a significant reduction in complications (otitis media) was noted for oseltamivir while a trend to benefit was seen for zanamivir 155 . vomiting was significantly more common among oseltamivir recipients than placebo recipients (15% vs. 9%). the review noted that there may be a difference in efficacy according to serotype, with oseltamivir showing a significant reduction in time to resolution for influenza a (34%) but not b (8.5%) 155 . with respect to children with asthma there was a trend to reduction in time to freedom from illness for oseltamivir recipients but this did not reach statistical significance. oseltamivir appeared to result in a more rapid improvement in pulmonary function, and was well tolerated in children with asthma 155, 156 . the cochrane review concluded that oseltamivir was the preferred drug as it has shown a benefit with regard to secondary complications. it also concluded that there was no evidence of benefit in at-risk children (i.e. asthma). from the perspective of pandemic use however, it should be noted that there was no evidence of harm in this group. with regard to dosing of oseltamivir, pharmacokinetic studies have suggested that young children clear the drug faster than older children, adolescents and adults and therefore need higher doses 157, 158 . the major practical issue with regard to zanamivir is its mode of administration limiting its use to children over the age of five years (fda guidance: over seven years of age) 155 . the development of resistance to oseltamivir in children may be more common than appreciated and more common than seen in adults. in one study resistance mutations were documented in 18% of 50 children 158 . this has implications for widespread use in a pandemic situation. one particular issue with regard to paediatric use of oseltamivir is the apparent age limitation on its license (i.e. not for children under one year of age). this is particularly important because during epidemic years, of all children with influenza, it is children under six months of age who are most likely to be hospitalised 159 . the basis for this exclusion appears to be that rat data have shown high mortality in infant rats at seven days of age when given a dose of 1000 mg/kg together with high brain levels of oseltamivir, assumed to reflect the immature blood brain barrier at this age. this is reflected in product literature and an fda alert although there are no published data. as a result, there are few human data in this age group as it was felt that it would be difficult to monitor cns toxicity in this age group. however, because of a fear of encephalopathy due to influenza in young children, japanese paediatricians §19. use of antivirals in hospitalised children part 3. clinical management of children referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s43 have been using it in infants and data on 102 consecutive infants from japan revealed no encephalopathy or mortality in recipients 160 . a second japanese report where 47 children under one year were treated (4 mg/kg/day) showed similar efficacy for fever to a group of older children and no serious adverse effects 161 . there are no data on the effectiveness of oseltamivir if given more than two days from onset of illness. it is likely to be less effective and in particular to have little or no effect after five to six days of illness unless the child is immunosuppressed. giving oseltamivir to sick hospitalised patients is theoretically likely to decrease their infectivity and so may be useful but there are no data to support this. in a double blind placebo controlled study children hospitalized with influenza who had been ill for 48 hours or less and who had a temperature of 37.8ºc or more were randomised to receive either ribavirin or placebo. sixtytwo patients (35 in the placebo group, 27 in the ribavirin group) had a confirmed diagnosis of influenza. the time to reduction of temperature to 38.3ºc or less for the ribavirin group was 8.9 hours compared with 22.6 hours for the placebo group (p = 0.04). there were no other differences detected between groups 162 . there have been no further published studies in the 11 years since this report, thus ribavirin cannot be recommended at this time. • in the setting of a pandemic, children in the community should only be considered for treatment with antivirals if they have all of the following: (1) an acute influenza-like illness (2) fever (>38.5ºc) and (3) been symptomatic for two days or less. • oseltamivir is the antiviral agent of choice. • treatment schedule for children over one year: body weight 15 kg, i.e. <3 years: 30 mg every 12 h body weight >15 23 kg, i.e. 3 7 years:45 mg every 12 h body weight 24 kg, i.e. >7 years: 75 mg every 12 h • in children who are severely ill in hospital oseltamivir may be used if the child has been symptomatic for less than six days. • oseltamivir may be considered for the treatment of infants under one year of age, especially those with severe influenza. this would need to be done following appropriate discussion with the parents highlighting the concerns from the animal data and the relative paucity of human data in this age group. section 20. use of antibiotics in hospitalised children 20 .1. who should get antibiotics? secondary bacterial infections, particularly pneumonia and otitis media, are common in children with influenza. a case control study during an outbreak of severe pneumococcal pneumonia demonstrated that patients with severe pneumonia were 12 times more likely to have had an influenza-like illness and four times more likely to have positive influenza serology than controls 69 . infections with staph. aureus and h. influenzae are also more common during influenza outbreaks. a randomized controlled trial of antibiotics in 85 children aged four months to 11 years presenting with influenzalike symptoms during an influenza epidemic showed a decreased incidence of pneumonia in the antibiotictreated group (2.4% vs 16.3%, p = 0.031) 163 . there was no change in duration of fever or incidence of acute otitis media. interestingly only one out of seven of the cases of pneumonia in the placebo group was thought to be bacterial. the authors postulated that as bacterial proteases facilitate propogation and pathogenesis of influenza in a mouse model, decreasing bacterial numbers and hence protease levels in the lung may decrease viral pneumonia. another randomized trial of cephalosporins vs macrolides in 365 japanese children with influenza-like symptoms showed faster alleviation of fever (3.8±1.4 vs 4.3±1.4 days, p = 0.006) in the macrolide group and a decrease in number with cxr evidence of pneumonia (2 vs 13 cases, p = 0.002; 14/15 had interstitial changes) 164 . the authors postulate that anti-inflammatory effects of macrolides may be responsible. • children who (a) are at risk of complications of influenza or (b) have disease severe enough to merit hospital admission during an influenza pandemic should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae. the antibiotics of choice must cover the likely pathogens as above. rarely a blood culture or pleural tap will provide the pathogen. the antibiotics should then be specifically tailored, e.g. iv benzylpenicillin or oral amoxicillin for s. pneumoniae and flucloxacillin or clindamycin for staph. aureus. part 3. clinical management of children referred to hospital §20. use of antibiotics in hospitalised children s44 provisional guidelines from bis/bts/hpa in collaboration with the department of health, version 11 (2 october 2006) a recent randomized controlled trial of the equivalence of oral amoxicillin vs iv benzylpenicillin in 252 children admitted to hospital with community-acquired pneumonia showed no difference in duration of illness or complications 165 . oral antibiotics should be given provided oral fluids are tolerated. 20.5. antibiotic choice for severe or complicated pneumonia? children who are severely ill with pneumonia complicating influenza should have a second agent which provides good cover for gram positive organisms added to the regime (e.g. clarithromycin or cefuroxime) and the drugs should be given intravenously to ensure high serum and tissue antibiotic levels. section 21. acknowledgements, committee members and affiliations 21 chronic obstructive pulmonary disease (copd) including chronic bronchitis and emphysema, and such conditions as bronchiectasis, cystic fibrosis, interstitial lung fibrosis, pneumoconiosis and bronchopulmonary dysplasia (bpd). asthma requiring continuous or repeated use of inhaled or systemic steroids or with previous exacerbations requiring hospital admission. children who have previously been admitted to hospital for lower respiratory tract disease. chronic heart disease congenital heart disease, hypertension with cardiac complications, chronic heart failure and individuals requiring regular medication and/or follow-up for ischaemic heart disease. chronic renal disease nephrotic syndrome, chronic renal failure, renal transplantation. chronic liver disease cirrhosis, inflammatory bowel disease diabetes and chronic metabolic disorders diabetes mellitus requiring insulin or oral hypoglycaemic drugs. immunosuppression and malignancy due to disease or treatment: asplenia or splenic dysfunction, hiv infection at all stages, malignancy. patients undergoing chemotherapy leading to immunosuppression. individuals on or likely to be on systemic steroids for more than a month at a dose equivalent to prednisolone at 20 mg or more per day (any age) or for children under 20 kg a dose of 1 mg or more per kg per day. long-stay residential care homes residents this does not include prisons, young offender institutions, university halls of residence. others doctors retain discretion in identifying additional individual patients who they recognise as at high risk of serious complications should they develop influenza; for example patients with haemoglobinopathies, neurological diseases with muscle weakness, cerebral palsy or children on long-term aspirin who are at increased risk of reye's syndrome. a the high-risk groups described in this appendix are largely based on data from interpandemic influenza. during the course of a pandemic, the definition of 'high-risk groups' may differ. if so, details of the 'high-risk' patient group will be altered according to relevant clinico-epidemiological data. users are strongly advised to refer to the latest version of these guidelines at all times. treat as severe pneumonia antibiotics not indicated <8.5 kg -7.5 mg/kg b.d. a winter's tale: coming to terms with winter respiratory illnesses. london: health protection agency the epidemiology and clinical impact of pandemic influenza the contribution of influenza to combined acute respiratory infections, hospital admissions, and deaths in winter pandemic versus epidemic influenza mortality: a pattern of changing age distribution delaying the international spread of pandemic influenza acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings does this patient have influenza? clinical signs and symptoms predicting influenza infection predicting influenza infections during epidemics with use of a clinical case definition the predictive value of influenza symptomatology in elderly people randomized, placebocontrolled studies of inhaled zanamivir in the treatment of influenza a and b: pooled efficacy analysis efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenzavirus infections. gg167 influenza study group efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group the impact of influenza epidemics on mortality: introducing a severity index human influenza principles and practice of clinical virology clinical features of influenza age and asian influenza, 1957 rapid diagnosis of primary influenza pneumonia importance of staphylococcus aureus in pneumonia in the 1957 epidemic of influenza a should staff in long-stay hospitals for elderly patients be vaccinated against influenza? bacterial pneumonia during the hong kong influenza epidemic of influenza pneumonia the influence of bacterial superinfection on the clinical course of influenza. studies from the influenza epidemics in stockholm during pneumonia complicating asian influenza influenza a myocarditis developing in an adult liver transplant recipient despite vaccination: a case report and review of the literature rhabdomyolysis and myoglobinuric renal failure complicating influenza in a young adult acute myopathy associated with influenza a/texas/1/77 infection. isolation of virus from a muscle biopsy specimen high incidence of acute myositis with type a influenza virus infection in the elderly influenza and myoglobinuria in brothers myoglobinuria associated with influenza a infection a pregnant woman with influenza a encephalopathy in whom influenza a/hong kong virus (h3) was isolated from cerebrospinal fluid isolated lesions limited to the bilateral substantia nigra on mri associated with influenza a infection influenza virus and cns manifestations the spectrum of antecedent infections in guillain-barre syndrome: a casecontrol study myelitis associated with influenza a virus infection toxic shock syndrome complicating influenza a infection: a two-case report with one case of bacteremia and endocarditis toxic shock syndrome following influenza a infection acute parotitis associated with influenza type a: a report of twelve cases avian influenza: assessing the pandemic threat. who atypical avian influenza (h5n1) fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma avian influenza a (h5n1) infection in humans human disease from influenza a (h5n1) avian influenza virus infection of children in vietnam and thailand prospective study of the viral aetiology and clinical course among children in the community with cough and fever nosocomial influenza in children recurrence of febrile seizures in the respiratory season is associated with influenza a influenza a and b virus infections in children influenza in children clinical predictors of influenza in children clinical features of influenza a and b in children and association with myositis the burden of influenza illness in children with asthma and other chronic medical conditions the impact of influenza a on children with disabilities the impact of influenza in children severe pneumococcal pneumonia in previously healthy children: the role of preceding influenza infection impact of the rapid diagnosis of influenza on physician decision-making and patient management in the pediatric emergency department: results of a randomized, prospective, controlled trial childhood severe acute respiratory syndrome in taiwan and how to differentiate it from childhood influenza infection clinical courses of croup caused by influenza and parainfluenza viruses prevalence of various respiratory viruses in the middle ear during acute otitis media influenza virus infection in infancy and early childhood welliver sr rc. a comparison of epidemiologic and immunologic features of bronchiolitis caused by influenza virus and respiratory syncytial virus references s56 provisional guidelines from bis/bts/hpa in collaboration with the department of health encephalopathy associated with influenza a influenza surveillance system of japan and acute encephalitis and encephalopathy in the influenza season reye's syndrome and aspirin reye's syndrome in the united states from 1981 through 1997 the puzzling picture of acute necrotizing encephalopathy after influenza a and b virus infection in young children acute necrotizing encephalopathy of childhood associated with influenza type b virus infection in a 3-year-old girl clinical characteristics of children with influenza a virus infection requiring hospitalization influenza a and meningococcal disease influenza pandemic contingency planning: operational guidance on the provision of healthcare in a community setting in england effect of influenza vaccination on disease progression among hiv-infected persons validation of a predictive rule for the management of community-acquired pneumonia assessment of pneumonia severity: a european perspective information leaflet and antibiotic prescribing strategies for acute lower respiratory tract infection: a randomized controlled trial diagnostic value of c reactive protein in infections of the lower respiratory tract: systematic review airway hyperreactivity and peripheral airway dysfunction in influenza a infection american college of chest physicians/national heart, lung, and blood institute national conference on oxygen therapy acute hypercapnic respiratory failure in patients with chronic obstructive lung disease: risk factors and use of guidelines for management instability on hospital discharge and the risk of adverse outcomes in patients with pneumonia pandemic influenza: is an antiviral response realistic? randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza a and b virus infections. the mist (management of influenza in the southern hemisphere trialists) study group efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial. neuraminidase inhibitor flu treatment investigator group neuraminidase inhibitors for preventing and treating influenza in healthy adults oral oseltamivir treatment of influenza in children safety and efficacy of the neuraminidase inhibitor gg167 in experimental human influenza impact of oseltamivir treatment on influenza-related lower respiratory tract complications and hospitalizations comparison of efficacies of rwj-270201, zanamivir, and oseltamivir against h5n1, h9n2, and other avian influenza viruses study of 1-adamantanamine hydrochloride used prophylactically during the hong kong influenza epidemic in the family environment selection of influenza virus mutants in experimentally infected volunteers treated with oseltamivir a comparison of antimicrobial resistance rates in grampositive pathogens isolated in the uk from mrsa bacteraemia in patients on arrival in hospital: a cohort study in oxfordshire antimicrobial resistance in haemophilus influenzae from england and scotland in 1991 trends in the antimicrobial susceptibility of bacterial respiratory tract pathogensfindings of the alexander project antibiotic resistance among clinically important gram-positive bacteria in the uk a critical review of the fluoroquinolones: focus on respiratory infections antistaphylococcal effect related to the area under the curve/mic ratio in an in vitro dynamic model: predicted breakpoints versus clinically achievable values for seven fluoroquinolones quality of care, process, and outcomes in elderly patients with pneumonia community-acquired pneumonia in patients receiving home care prognosis and outcomes of patients with community-acquired pneumonia. a meta-analysis severe pneumonia and a second antibiotic the next generation: fluoroquinolones in the management of acute lower respiratory infection in adults comparison of 7 versus 10 days of antibiotic therapy for hospitalized patients with uncomplicated communityacquired pneumonia: a prospective, randomized, doubleblind study antibiotics in pandemic flu children with respiratory disease associated with metapneumovirus in hong kong avian influenza a (h5n1) in 10 patients in vietnam clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus cases of influenza a (h5n1) thailand randomised controlled trial of clinical outcome after chest radiograph in ambulatory acute lower-respiratory infection in children standardized interpretation of paediatric chest radiographs for the diagnosis of pneumonia in epidemiological studies reliability of the chest radiograph in the diagnosis of lower respiratory infections in young children comparison of radiological findings and microbial aetiology of childhood pneumonia accuracy of radiographic differentiation of bacterial from nonbacterial pneumonia rationalised prescribing for community acquired pneumonia: a closed loop audit comparison of clinical characteristics of influenza and respiratory syncytial virus infection in hospitalised children and adolescents influenzalike illness criteria were poorly related to laboratoryconfirmed influenza in a sentinel surveillance study effect of a rapid influenza diagnosis effect of rapid diagnosis of influenza virus type a on the emergency department management of febrile infants and toddlers near patient testing for influenza in children in primary care: comparison with laboratory test comparison of binax now and directigen for rapid detection of influenza a and b new point of care test is highly specific but less sensitive for influenza virus a and b in children and adults association of invasive pneumococcal disease with season, atmospheric conditions, air pollution, and the isolation of respiratory viruses fatal influenza a virus infection in a child vaccinated against influenza toxic shock syndrome. a newly recognized complication of influenza and influenzalike illness fulminant pneumonia caused by concomitant infection with influenza b virus and staphylococcus aureus preceding respiratory infection predisposing for primary and secondary invasive haemophilus influenzae type b disease performance of the binax now streptococcus pneumoniae urinary antigen assay for diagnosis of pneumonia in children with underlying pulmonary diseases in the absence of acute pneumococcal infection evaluation of rapid assay for detection of streptococcus pneumoniae urinary antigen among infants and young children with possible invasive pneumococcal disease the utility of chest radiography in the follow-up of pneumonia references s58 provisional guidelines from bis/bts/hpa in collaboration with the department of health value of radiological follow up of childhood pneumonia re-emergence of fatal human influenza a subtype h5n1 disease recovery of drug-resistant influenza a virus during therapeutic use of rimantadine high 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aerosolized ribavirin in young children hospitalized with influenza: a double-blind, multicenter, placebo-controlled trial efficacy of antibiotics against influenza-like illness in an influenza epidemic effect of maclorides on duration and resolution of symptoms and complication of pneumonia in children with influenza a randomised controlled equilanence trial to compare oral and intravenous treatment and the direct and indirect costs of treating children with community acquired pneumonia: pivot trial human influenza a h5n1 virus related to a highly pathogenic avian influenza virus outbreak of avian influenza a (h5n1) virus infection in hong kong in 1997 avian influenza a challenge to global health care structures in addition, antivirals may be considered in the following exceptional situations:(i) patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, may still be eligible for antiviral treatment despite the lack of documented fever. (ii) severely ill and immunosuppressed patients, including those on long-term corticosteroid therapy, may benefit from antiviral therapy commenced later than 48 hours after the onset of ili. (iii) severely ill children <1 year old. (parents must be informed that oseltamivir is not licensed for children <1 year old.) the first recorded instance of human infection by avian influenza h5n1 occurred in may 1997 in hong kong. the first patient was a 3-year old child who presented initially with symptoms of fever, sore throat and abdominal pain. he later developed reye's syndrome, ards, multi-organ failure and eventually died 166 . a total of 18 persons were subsequently infected before the outbreak ended in december 1997 126, 167 . half the patients were aged 18 years and below and only two were aged over 50 years. abdominal symptoms, such as diarrhoea, vomiting and abdominal pain, were described in ten (56%) patients. eleven (61%) had a severe illness characterised by pneumonia occurring within 14 days of symptom onset, lymphopenia, deranged liver function tests and a high mortality [six (55%) of 11 patients with pneumonia]. secondary bacterial infections were not identified as the cause of the pneumonias.the most recent human outbreak of influenza a (h5n1) infection began in december 2003. the clinical features of hospitalised patients infected by the re-emergent avian influenza a (h5n1) in 2004 were similar to those described in patients in 1997 (table a10 .1). children and young adults were the main groups affected. gastrointestinal symptoms were common. the presence of lymphopenia and deranged liver function tests was again associated with a poorer prognosis 125 .since december 2003, over 150 cases had been reported to the who 168 . the mortality rate among hospitalised patients has been generally high (>40%). death has occurred an average of ten days after the onset of illness and most patients have died of progressive respiratory failure.there has been a review of avian influenza a (h5n1) infection in humans up until september 2005 55 . updated information can be found at www.who.int/csr/disease/avian_influenza/en/. key: cord-000760-4yfohp9w authors: babapoor, sankhiros; neef, tobias; mittelholzer, christian; girshick, theodore; garmendia, antonio; shang, hongwei; khan, mazhar i.; burkhard, peter title: a novel vaccine using nanoparticle platform to present immunogenic m2e against avian influenza infection date: 2012-01-12 journal: influenza res treat doi: 10.1155/2011/126794 sha: doc_id: 760 cord_uid: 4yfohp9w using peptide nanoparticle technology, we have designed two novel vaccine constructs representing m2e in monomeric (mono-m2e) and tetrameric (tetra-m2e) forms. groups of specific pathogen free (spf) chickens were immunized intramuscularly with mono-m2e or tetra-m2e with and without an adjuvant. two weeks after the second boost, chickens were challenged with 107.2 eid50 of h5n2 low pathogenicity avian influenza (lpai) virus. m2e-specific antibody responses to each of the vaccine constructs were tested by elisa. vaccinated chickens exhibited increased m2e-specific igg responses for each of the constructs as compared to a non-vaccinated group. however, the vaccine construct tetra-m2e elicited a significantly higher antibody response when it was used with an adjuvant. on the other hand, virus neutralization assays indicated that immune protection is not by way of neutralizing antibodies. the level of protection was evaluated using quantitative real time pcr at 4, 6, and 8 days post-challenge with h5n2 lpai by measuring virus shedding from trachea and cloaca. the tetra-m2e with adjuvant offered statistically significant (p < 0.05) protection against subtype h5n2 lpai by reduction of the ai virus shedding. the results suggest that the self-assembling polypeptide nanoparticle shows promise as a potential platform for a development of a vaccine against ai. avian influenza (ai) is a devastating poultry disease with serious economic consequences to the commercial poultry industry. ai is also a significant public health concern because of recent highly pathogenic h5n1 avian influenza outbreaks causing also human deaths in asia, europe, and north africa. according to the world health organization (who) update, 2011, since 2003, 520 confirmed cases of human infection with h5n1 have been reported, of which 307 died due to disease complications. however, other avian influenza viruses including low-pathogenic avian influenza (lpai) can also be a risk to public health. for instance, the lpai subtype h9n2 infection in chickens is mild to asymptomatic and easily overlooked. however, it shares similar receptor binding epitopes with human influenza viruses and can infect humans [1] . there is a risk for lpai subtypes h5 and h7 to become high-pathogenic avian influenza (hpai) viruses in chickens due to constant virus shedding and transmission to new birds within the flock or neighboring flocks [2, 3] . vaccination is an effective way for prevention of viral diseases in poultry. however, routine vaccination against ai has not been widely practiced throughout the world mainly for surveillance reasons [1, 2] . when there is the desire for routine vaccination, constant 2 influenza research and treatment reformulation of ai vaccines is required according to the circulating field virus, which can be cumbersome in the case of an immediate outbreak. current vaccines against ai viruses can reduce mortality, clinical signs, shedding, and transmission of the virus in poultry, but they are not capable of preventing infection and virus replication [4] . the design of a universal influenza vaccine has been the major focus of researchers in the influenza vaccinology field. the external domain of matrix protein 2 (m2e) has been one of the main interests for the generation of a universal ai vaccine. the m2e is encoded by a separate open reading frame of segment 7 of the influenza virus genome, is located in the viral envelope, and projects from the surface of the virus as tetramers [5, 6] . the m2 is composed of 97 amino acids which forms 3 domains: the external domain, the transmembrane domain, and the internal domain. the external domain of m2 (m2e) is recognized by the host's immune system [7] [8] [9] . initially, vaccination of ferrets with whole m-or m2-expressing recombinant vaccinia virus showed no evidence of protection [10] . however, later vaccine constructs using plasmid and recombinant salmonella expressing m or m2 induced significant protection in terms of reduction in virus growth and mortality in mice and chickens, respectively [11] [12] [13] . a multiple antigenic peptide construct containing m2e (m2e-map) induced strong m2especific antibody titers in the serum of mice and resulted in significant protection against influenza virus challenge [13] . liang et al., 1994 [14] showed the importance of cd4 + t cells for nasal resistance and protection against the virus. it is assumed that m2e-specific memory t h cells also may have an important role in protection against the virus in the nose and trachea of mice [13] . de filette et al., 2005 [15] used the hepatitis b virus core particle (hbc) as a carrier and fused m2e (conserved region of human influenza a virus) to either the c-terminus of hbc or inserted it in the immune-dominant loop of hbc. immunization of mice with this m2e-hbc vaccine was 100% protective against lethal challenge [15] [16] [17] . antigenic epitopes of pathogens are peptides that are capable of inducing an immune response. however, their small size limits their immunogenicity. therefore, usually a larger carrier protein, such as bovine serum albumin (bsa), keyhole limpet hemocyanin (klh), or a virus-like particle (vlp), is required for optimal immunogenicity [18] . structural organization of the epitope on the carrier is critical for inducing stronger immune responses. denis et al., 2007 [19] demonstrated that a monomeric form of m2e peptide was not immunogenic and huleatt et al., 2008 [20] tried to solve that problem by adding 4 copies of the m2e peptide in their platform. here, we used peptide nanoparticles as a platform to display the m2e peptide to the host's immune system. these nanoparticles represent a novel type of repetitive antigen display system which allows presenting the m2e peptide in high density in both, either in its monomeric or its tetrameric form. this idea was first presented in raman et al., 2006 [21] ; the monomeric peptide is composed of two coiled coils connected by a short linker region. the association between the coiled coils induces self-assembly of the monomers into figure 1 : computer model (a): the pentameric-trimeric architecture of mono-m2e and the fully assembled icosahedral nanoparticle. (b) tetra-m2e, with tetrameric-trimeric architecture, and the resulting octahedral nanoparticle. in both images, green: pentameric coiled coil, turquoise: tetrameric coiled coil, and blue: trimeric coiled coil. red represents m2e in either its monomeric or tetrameric state. spherical nanoparticles with either icosahedral or octahedral symmetry ( figure 1 ) according to our computer models. the potential for these nanoparticles to serve as platforms for vaccines is apparent. as opposed to live attenuated vaccines, there is no risk of infection within the vaccinated population [21] . furthermore, the ease and speed of protein expression, purification, and self-assembly into nanoparticles reduce the cost and time of large-scale production. the concept has been successfully used for the design of malaria [22] and sars [23] vaccines prototypes. here, we present the biophysical characterization of the nanoparticles and an immunological profiling using chickens as test animals. the results suggest that the selfassembling polypeptide nanoparticle shows promise as a potential vaccine against ai. 2.1. nanoparticle synthesis. the dna coding for the nanoparticle constructs was prepared using standard molecular biology procedures. shortly, plasmids containing the peptide monomers (table 1) were constructed by cloning complementary oligonucleotides (cccgggggggcagcggc agcctgctgaccgaagtggaaaccccgacccgcaac-ggctgggaataatgaattc) encoding the avian m2e epitope with flanking residues (arggsgslltevetptrngw-e * * e) into the xmai/ecori restriction sites of the basic sapn expression construct to yield mono-m2e. to table 1 : summary of self-assembling nanoparticle peptide sequences. peptide sequence the peptide mono-m2e is composed of a pentameric coiled coil (green) and a trimeric coiled coil (blue). tetra-m2e uses the same trimer but has a tetrameric coiled coil (turquoise). in both sequences, the m2e epitope is shown in red. other amino acid residues, such as linkers and his-tags, are shown in black. m2en-gcn4 consists of m2e attached to the tetrameric gcn4 coiled coil, shown in brown. monomeric m2e, used for elisa, coating is shown in red. generate tetra-m2e, we first cloned the tetrameric oligomerization domain of tetrabrachion into the bamhi/bsshii restriction sites of ppep-t ( figure 5 ), before cloning complementary oligonucleotides (atgcatccctggttcc gcgtggaagcctgctgaccgaagtggaaaccccgac-ccgcaacggctgggaatgcaaatgcagcgatagcagc ggatcc) coding for the slightly longer avian m2e sequence (haslltevetptrngweckcsdssgs) including flanking residues into the n-terminal nsii/bamhi restriction sites. the m2e-gcn4 construct was made by replacing the nanoparticle fragment of tetra-m2e with the gcn4 sequence. the plasmids were transformed into escherichia coli bl21 (de3) cells, which were grown in luria broth with ampicillin at 37 • c. expression was induced with isopropyl β-d-thiogalactopyranoside. four hours after induction, cells were removed from 37 • c and harvested by centrifugation at 4,000 ×g for 15 min. the cell pellet was stored at −20 • c. the pellet was thawed on ice and suspended in a lysis buffer consisting of 9 m urea, 100 mm nah 2 po 4 , 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tris-2-carboxyethl phosphine (tcep). cells were lysed by sonication and the lysate was cleared by centrifuging at 30.500 ×g for 45 min. the cleared lysate was incubated with ni-nta agarose beads (qiagen, valencia, ca, usa) for at least 1 hour. the column was washed with lysis buffer and then a buffer containing 9 m urea, 500 mm nah 2 po 4 , 10 mm tris ph 8, 20 mm imidazole, and 0.2 mm tcep. protein was eluted with a ph gradient: 9 m urea, 100 mm nah 2 po 4 , 20 mm citrate, 20 mm imidazole, and 0.2 mm tcep. subsequent washes were done at ph 6.3, 5.9, and 4.3. following the ph gradient, a gradient of lysis buffer with increasing imidazole strength was used to further elute the protein. purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) as shown in figure 6 . the protein solution was filtered with a 0.1 μm polyvinylidene fluoride membrane filter (millipore billerica, ma, usa). nanoparticle self-assembly was performed by dialysis into buffer containing 8 m urea, 20 mm tris ph 7.5, 150 mm nacl, and 5% glycerol, at a protein concentration of 0.1 mg/ml. this was followed by dialysis into the same buffer containing decreasing concentrations of urea: 6 m, 4 m, 2 m, 1 m, and two changes of the same buffer without urea. following self-assembly, the nanoparticle solution was again filtered with a 0.1 μm filter. scattering. dynamic light scattering experiments were carried out on a zetasizer nano s instrument (malvern, worcestershire, uk), with a 633 nm he-ne laser. all measurements were carried out at 25 • c in a buffer containing 20 mm tris ph 7.5, 150 mm nacl, and 5% glycerol. samples were negatively stained with 1% uranyl acetate (spi supplies, westchester, pa, usa) and observed with a fei tecnai t12 s/tem at an accelerating voltage of 80 kv (fei, hillsboro, oregon). the peptide concentration of the constructs was about 0.05 mg/ml. samples were dialyzed into 20 mm sodium phosphate ph 7.5, 150 mm nacl, and 5% glycerol and concentrated or diluted to a peptide concentration of about 0.13 mg/ml for mono-m2e and about 0.05 mg/ml for tetra-m2e. circular dichroism measurements were performed at room temperature using an applied photophysics (surrey, uk) pi star 180 spectropolarimeter, taking measurements from 200 to 250 nm. the influenza virus used in the direct challenge ai study was a/turkey/ca/d0208651-c/02 h5n2 low pathogenic. influenza a/turkey/wisconsin/1/1966 h9n2 low pathogenic was used for hyperimmune serum production provided by charles river avian vaccine services (storrs, ct). viruses were grown and titered in 9-to 11-dayold embryonated specific pathogen-free (spf) chicken eggs as previously described [24] . groups. spf p2a line (b19/ b19) white leghorn chickens eggs were obtained from cornell university, ithaca, ny. the eggs were hatched in the university of connecticut poultry farm and after the hatch, the chickens were moved to the office of animal research services (oars) facilities. after 2 weeks in the brooders with free access to water and a standard starter diet, the chickens were divided into groups, bled for baseline serology, transferred to isolators equipped with high-efficiency particulate air (hepa) filters, and were provided commercial diets and water ad libitum. a previously described plaque reduction assay was modified and used to evaluate the virus neutralization activity of collected sera after vaccination [25] . briefly, serum samples from each treatment group were pooled. an equal volume of a 1 : 10 dilution of pooled serum and lpai subtype h5n2 was mixed and incubated for 30 min at 37 • c. a commercially available anti-m2 antibody (prosci-inc, poway, ca) was used in a 1 : 1000 dilution as a control for antibody activity. chicken embryo kidney cell (cekc) monolayers in 6-well plates were washed twice with prewarmed pbs and 400 μl of the above mixture was added to the cekc monolayer. the plates were incubated for 60 min at 37 • c. then, the inoculums were removed and after 2 washes with prewarmed pbs, they were overlaid with 0.8% agar (university of connecticut cell culture facility) in minimum essential medium eagle (mem). after 72 h, the plates were checked for plaque formation and for further evaluation were fixed with 99% methanol and stained with crystal violet for plaque counting. elisa. the m2e epitopes, including the nanoparticle platforms with m2e epitopes (tetra-m2e and mono-m2e) and m2e linked to gcn4, (m2en-gcn4), were used for coating of the elisa plates. briefly, individual wells of the flat-bottom 96-well immulon 1b plates (nunc/thermo fisher scientific, rochester, ny) were coated with 5 μg/ml of tetrameric m2e (m2en-gcn4) or the nanoparticle of interest. antigen adhesion was allowed to proceed at 4 • c overnight. plates were rinsed with 2% tween 20 in phosphate-buffered saline (pbs) (pbs/tween 20 ther-mofisher) and blocked with a 3% bsa in pbs solution. plates were incubated at 37 • c for 3-4 h or 4 • c overnight (preliminary studies showed that there was no difference in the result). after incubation, plates were rinsed 4 times with pbs/tween 20 and incubated for 1 h at room temperature with the previously collected sera. briefly, 2-fold serial dilutions of each serum sample were prepared in a pbs solution containing 0.2 to 0.5% bsa. hyperimmune serum from previously infected birds with the lpai subtype h9n2 or commercial anti-m2e antibody were used as positive controls; and sera from healthy, unvaccinated birds were used as a negative control. after appropriate washes, peroxidaseconjugated goat anti-chicken igy (sigma aldrich,) was prepared in a 1 : 10,000 dilution in pbs and was added to each well and plates were incubated for an additional hour at room temperature. after subsequent rinsing, the plates were developed using 3,3 ,5,5 tetramethylbenzidine (tmb) peroxidase substrate (thermo fisher scientific inc., rockford, il) followed by a room temperature incubation period of 15 to 30 min. the absorbance was read in a spectramax 250 microplate reader (molecular devices, sunnyvale, ca) at 450 nm. in order to generate a standard curve for realtime pcr, we transcribed standard rna in vitro using t7 ribomax express large-scale rna production system (promega, madison, wi). briefly, rna extraction was done by using trizol reagent (invitrogen, carlsbad, ca) according to the manufacturer. the coding region of the m gene from the lpai subtype h5n2 was amplified using previously described universal primers [26] . rt-pcr was performed using a qiagen one-step rt-pcr kit (qiagen, valencia, ca) according to the standard manufacturer's protocol. pcr products were visualized by electrophoresis through ethidium-bromide-stained (0.5 μg/ml) 1.5% (40 mm tris-acetate ph 7.8, 0.1 mm edta) agarose gels under uv light. the amplified fragment was excised from the gel and cdna was recovered from agarose gel using a qiaquick gel extraction kit (qiagen, valencia, ca) according to the manufacturer's protocol and the purified dna product was ligated with a pcr 2.1 vector (invitrogen, carlsland, ca) according to the manufacturer's protocol to generate the pcr-m5 plasmid. for further confirmation, pcr positive plasmids were sequenced in the dna biotechnology facility of the university of connecticut. six micrograms of plasmid dna was linearized using 6 units of the restriction enzyme bam hi for 4 h at 37 • c. then, linearized dna was used as a template in an in vitro transcription reaction with the t7 ribomax express large-scale rna production system (promega, madison, wi) according to the manufacturer's recommendation. after the in vitro transcription reaction at 37 • c for 1 h, the possible remaining plasmid dna was digested by dnase i and purified rna was quantified with a spectrophotometer nanodrop nd-1000 (thermo fisher scientific, wilmington, de). the copy numbers of purified rna were determined using a previously described method [27] and was used for the generation of a real time standard curve. in this study, real-time rt-pcr was performed using the previously published primers m+25: aga tga gtc ttc taa ccg agg tcg and m-124: tgc aaa aac atc ttc aag tct ctg for quantification of viral load [28] . rna extraction was done on each swab sample followed by pcr in duplicate or triplicate using 5 μl of rna per each pcr reaction. briefly, the power sybr green rna-to-ct 1-step kit (applied biosystem, foster city, ca) was used with a 20 μl reaction mixture. for each pcr run, standards were designated for the plate and viral loads were calculated using fluorescence data acquired at the end of each annealing step. the amount of unknown sample was extrapolated based on the standard curve and was reported as viral copy number. prior to vaccination and challenge study, a pilot study was performed to evaluate pathogenicity influenza research and treatment 5 upon determination of the peak of virus shedding and appropriate infectious dose in the pilot study, the vaccination and challenge trial was initiated. briefly, 42 spf chickens were divided into six groups of seven and received their first inoculation at 2 weeks of age followed by two boosters, two weeks apart, at 10 weeks and 12 weeks after hatch as described in table 2 . preceding injection, nanoparticles were concentrated using amicon centrifugal filter units with a 100 kda mwco (millipore, billerica, ma). concentration was determined by absorbance at 280 nm and nanoparticle quality was assured by dls. the nanoparticle vaccine constructs were emulsified with either freund's complete adjuvant (prime) or freund's incomplete adjuvant (boosters) and injected into the pectoral muscle of each chicken. two weeks after the second booster, the birds, except for those in the negative control group, were challenged with 10 7.2 eid 50 lpai subtype h5n2. briefly, each bird received 1 ml allantoic fluid containing 10 7.2 eid 50 lpai subtype h5n2 divided among the eyes, nasal cavity, and oropharynx, while temporarily blocking the fresh air delivery to the isolator. fresh air was resumed after 5-10 min the following challenge of the last bird in the isolator. although the clinical signs associated with lpai viruses are rare, they were observed for possible clinical symptoms daily; and the presence of the symptoms and their severity was recorded. tracheal and cloacal swabs were taken from each bird at days 2, 4, 6, and 8 after challenge and they were placed in a 3.0 ml uvt tube (becton, dickinson, nj). blood samples from each bird were collected before each booster as well as two weeks after the second booster prior to challenge. each blood sample was collected in a separating blood tube and serum was separated by placing the tubes at 37 • c for 1 h then at room temperature overnight followed by a 5 to 10 min centrifugation at 1000 rpm at 4 • c. then, the collected serum samples were stored at −20 • c until analysis. design. an obvious model for a selfassembling protein particle is a viral capsid. the capsids of spherical viruses often have icosahedral symmetry, due to their need to build a large encapsulating structure from many copies of the same, or only few, capsid proteins. an icosahedron is the most efficient way to accomplish this. by utilizing pentameric and trimeric coiled coils, we have built a self-assembling nanoparticle which uses the threefold and fivefold symmetry of an icosahedrons [21] . the pentameric coiledcoil motif of the monomer is taken from cartilage oligomeric matrix protein (comp) and the trimer is a de novo designed coiled coil. self-assembly occurs when the coiled-coil domains of different monomers associate, forming the icosahedral nanoparticle ( figure 1 ). a nanoparticle with this sort of architecture can then be used as a vaccine platform by extending the ends of the monomer with an epitope sequence. the mono-m2e species of nanoparticle follows this plan ( table 1) . as a result, it repetitively displays a monomeric form of m2e on the surface of the nanoparticles. the m2e peptide on the icosahedral nanoparticles lacks its c-terminal five residues to avoid problems with disulfide crosslinking that presumably require the native tetrameric conformation for proper formation. although the simplest icosahedral particle with t1 icosahedral symmetry is made from 60 polypeptide chains, it may also be possible that mono-m2e particles possess higher triangulation numbers, resulting in particles with an even greater molecular mass. on the other hand, the native conformation of m2 is a tetramer. hence, to elicit conformationally specific antibodies, the m2e antigen displayed by a vaccine particle should ideally be tetrameric. with that in mind, we designed the tetra-m2e peptide ( table 1) . instead of a pentameric coiled coil, this polypeptide uses the tetrameric coiledcoil motif from the protein tetrabrachion [29] . self-assembly using this peptide would result in a nanoparticle with threefold and fourfold symmetry axes or octahedral symmetry. as opposed to the larger icosahedral mono-m2e, this octahedral particle would only have 24 polypeptide chains. in addition, the epitope is now constrained to its native tetrameric conformation. the full-length m2e contains two cysteine residues. the formation of disulfide bridges between the cysteines of adjacent chains under oxidizing conditions is thought to stabilize their tetrameric conformation. the speed and ease of protein expression and purification, as well as of the self-assembly process, contribute to the overall viability of this technology as a vaccine platform. to facilitate purification, we have included polyhistidine tags at the n-terminal ends of the peptides. to enable detection of antibodies against tetrameric m2e, the peptide m2en-gcn4 was designed (table 1 ). m2e is linked to a gcn4, a coiled coil whose oligomerization state can be determined by the identity of amino acid residues in key a and d positions of the coiled coil. in this case, the tetrameric version of gcn4 was used [30] . by affixing m2e to a tetrameric protein, we can constrain it in its tetrameric conformation. the effect is similar to that experienced by the ends of the tetrameric coiled coil from tetrabrachion of the tetra-m2e nanoparticle. however, the coiled coil sequence is different. this guarantees that any antibodies bound to m2e-gcn4 are specific for the tetrameric version of m2e and not against the coiled coil or other parts of the nanoparticle. mono-m2e formed particles whose hydrodynamic diameters have a distribution which peaks at 34.5 nm, while the distribution of tetra-m2e peaks at 22.9 nm (figure 2 ). it is also noteworthy that the size distribution peak of mono-m2e is broader than that of tetra-m2e, suggesting that the former has a higher degree of polydispersity. the results were confirmed by transmission electron microscopy ( figure 3) . we can see that nanoparticles were formed and that their diameters are comparable with those measured by dynamic light scattering. it can be seen from the micrographs that neither mono-m2e nor tetra-m2e form nanoparticles with perfectly spherical morphology. this may in some way explain the polydispersity observed by dynamic light scattering. structure. the double minima found by circular dichroism confirm the alpha helical structure of the nanoparticles (figure 4 ). it appears that tetra-m2e exhibits this behavior much less than mono-m2e. this may be partly due to the larger m2e epitope sequence in the tetra-m2e peptide as compared to that used in mono-m2e. the plaque reduction assay performed by using pooled serum from chickens inoculated with tetra-m2e did not show a significant (p > 0.05) difference compared to control nonvaccinated chicken serum and commercial anti-m2e antibody. the anti-m2e immune response was monitored by determining the titer of the m2e-specific igy at three different time points (2 weeks after each inoculation). chickens after each inoculation developed high levels of antibody against the injected construct and anamnestic response clearly was seen when the plates were coated with mono-m2e and tetra-m2e nanoparticles and m2e-gcn4 (tetrameric m2e), respectively (table 1, figures 7 and 8) . for further investigation of the antibodies, the level of m2e specific antibody was measured using plates coated with tetrameric m2e-gnc4 peptide to evaluate the specific antibody against tetrameric m2e rather than the whole particle. the result of this study indicated that in chickens, after the second booster, the antibody levels are not at the same level as our previous results in mice with the same backbone but a different (malaria) epitope had been shown [23] . the dose level was also higher than what was shown to be required in mice. this could be because of the lower haplotype-specific immunogenicity of the particles in chickens, the route of administration in mice (intraperitoneal and intranasally), the body weight of the mice compared with chickens, and different immune system repertoires of mammalian and avian species. in future studies, changing the administration route can be another approach to reducing the dose of vaccine construct. we also coated the plate with inactivated purified virus to observe seroconversion of the chickens after challenge with the virus at 2 weeks after the last boost. results indicated that whole virus response was higher as expected with hyperimmune serum (figure 9 (a)), however, elisa response from chicken vaccinated with tetra-m2e and with whole virus reacted similarly on gcn-m2e coated plate (figure 9(b) ). the protective efficacy of the anti-m2e antibody responses induced by different constructs was assessed by evaluation of viral shedding post challenge. to determine the peak of shedding, viral copy number was figure 10 . we determined viral loads in tracheal and cloacal swabs samples on day 8 following challenge with 10 7.2 eid 50 lpai subtype h5n2. reduction of cloacal and oropharyngeal shedding in vaccinated birds was significant in chickens vaccinated with tetra-m2e with freund's adjuvant. virus shedding was evaluated at day 4 and day 6 after challenge; the swabs were tested for virus load ( figure 11 ). it is seen that virus shedding reduction starts at day 4 post infection with a significant decrease at day 8 post infection. currently available vaccines induce antibodies against specific field strains or closely related avian influenza strains. most of these vaccines are killed virus vaccines that induce short-lived immunity and are lacking a broad cross-reactive humoral immune response. recently, the generation of a universal influenza vaccine using conserved peptide regions among several influenza virus strains has been an area of interest in the human influenza vaccine field. m2e is a highly conserved region among influenza viruses and it has been studied as a possible universal vaccine candidate against human influenza virus infection [16, 17] . in the present study, protection efficiency of two different nanoparticle constructs harboring m2e was studied as possible vaccine candidates for low-pathogenicity avian influenza infection. biophysical analysis confirms that they are of relatively regular shape and size, but there is some degree of heterogeneity. though molecular weight measurements still remain to be carried out and we have no high resolution influenza research and treatment structural data, we assumed that nanoparticles assembled in a state close to what was expected, that is, icosahedral and octahedral nanoparticles, respectively. this will repetitively display m2e in both, either in its monomeric or its tetrameric form. there is speculation as to how the polyhistidine tag at the n-terminal end of the monomers may affect the selfassembly process, the final nanoparticle structure, or the immunogenicity of the vaccine, but attempts at producing his-tag free versions of the nanoparticle constructs either did not reliably provide pure protein or never adequately self-assembled. similarly, we attempted to include cd4 t cell epitopes to increase the immune response, but this also interfered with nanoparticle formation. since many variables can affect the host's virus shedding and the course of disease [31, 32] , prior to evaluation of vaccine constructs, the pathogenicity after challenge with the lpai subtype h5n2 virus was evaluated. a biphasic virus shedding was observed in this study. for the lpai subtype h5n2, the peaks for tracheal and cloacal shedding were at days 4 and 8 postinfection. the tetra-m2e vaccine construct provided a significant viral load reduction at the peak of viral shedding in immunized chickens. chickens immunized with tetra-m2e that harbors the tetrameric m2e with freund's adjuvant showed a clear reduction in cloacal and tracheal excretion of lpai compared to challenge control groups. the results of immunization with mono-m2e with adjuvant and tetra-m2e without adjuvant were also promising and by improving both b-and t-cell epitopes of those constructs, desirable results may be obtained. in vaccine design, repetitive bcell epitope display is considered a strategy for improving the humoral immune response [33, 34] . in addition to repetitive antigen display on the nanoparticle, we were able to present m2e in its native tetrameric conformation. the correlation between high protection and antibody response specific for tetrameric m2e elicited by tetra-m2e supports our assumption of tetrameric m2e presentation. the results of our studies show that tetrameric m2e stimulates a more specific immune response compared to the monomeric presentation and induces a significant protection against homologous virus challenge. the fact that a large portion of the antibody response is directed against the carrier and not only against the epitope(s) (figures 7 and 8) can be explained by the fact that significant portions of the core of the nanoparticles are also exposed to the immune system (compare figure 1) and hence, these portions will also induce a significant immune response. in this study, we showed that anti-m2e antibodies are not neutralizing antibodies; however they are capable of binding to the m2 proteins that are abundantly presented on the surface of the infected cells (data not shown). these can describe an efficient delayed clearance of the virus in m2e vaccinated chickens based on the previously described nk cell involvement in adcc [35] . significant improvement of virus clearance in vaccinated chickens with tetrameric m2e may be considered in a new vaccination strategy by vaccinating chickens with both a killed vaccine and a nanoparticle vaccine in order to provide robust protection, cross-reactive immunity, and clearance in case of emerging new strains of the virus. however, there remains the risk that such vaccination may cause a long-term persistence of hpai in poultry flocks, because the vaccine could not prevent the viral infection but rather suppresses the symptoms of hpai virus-infected chickens by reducing the virus shedding in chicken. thereby, especially in the case of hpai infection, the vaccination may make the infection less visible and the eradication of virus more difficult, and consequently it may provide a good opportunity for hpai virus to survive and persist in poultry flocks for a long time. for this reason, we plan to design new nanoparticle constructs that also contain fragments of hemagglutinin in addition to the m2e domain. immunization would then result in the generation of neutralizing hemagglutinin-specific antibodies in addition to the disease modulating m2e-specific antibodies. in this study, we evaluated a new approach to immunizing chickens against ai that uses a nanoparticle platform to carry an antigenic epitope. further designing and testing of new nanoparticle vaccines should demonstrate that they are effective tools for stimulation of an immune response against m2e and other b-or t-cell epitopes. therefore, application of the nanoparticle platform facilitates the development of a new generation of vaccines that harbor conserved epitopes of avian influenza viruses and would not be rendered ineffective by viral mutations such as antigenic shifts and drifts. the nanotechnology described here offers the opportunity to rapidly produce new vaccines according to the emergence of new strains of influenza virus without going through the time-consuming steps of production currently used in manufacturing commercial influenza vaccines. for future studies, the chicken's lpai infection model needs to be improved to evaluate clinical signs and higher virus shedding. this may help to better evaluate virus shedding, specifically cloacal virus shedding. also, vaccination and hpai challenge may be used to evaluate the vaccine efficiency in protection against high-pathogenicity ai viruses. avian influenza m2e: ectodomain of matrix protein 2 lpai: low pathogenicity avian influenza hpai: high pathogenicity avian influenza spf: specific pathogen free. detection of antibodies to the nonstructural protein (ns1) of avian influenza viruses allows distinction between vaccinated and infected chickens the use of vaccination to combat multiple introductions of notifiable avian influenza viruses of the h5 and h7 subtypes between 2000 and 2006 in italy avian influenza vaccination in north america: strategies and difficulties avian influenza vaccines and therapies for poultry influenza virus m2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds genetics of influenza viruses passively transferred monoclonal antibody to the m2 protein inhibits influenza a virus replication in mice a universal influenza a vaccine based on the extracellular domain of the m2 protein modified m2 proteins produce heterotypic immunity against influenza a virus mechanism of immunity to influenza: maternal and passive neonatal protection following immunization of adult ferrets with a live vaccinia-influenza virus haemagglutinin recombinant but not with recombinants containing other influenza virus proteins vaccination of chickens with recombinant salmonella expressing m2e and cd154 epitopes increases protection and decreases viral shedding after low pathogenic avian influenza challenge protective immunity against influenza a virus induced by immunization with dna plasmid containing influenza m gene protection of mice against influenza a virus challenge by vaccination with baculovirus-expressed m2 protein heterosubtypic immunity to influenza type a virus in mice: effector mechanisms and their longevity universal influenza a vaccine: optimization of m2-based constructs a "universal" human influenza a vaccine universal influenza a m2e-hbc vaccine protects against disease even in the presence of pre-existing anti-hbc antibodies innovative vaccine production technologies: the evolution and value of vaccine production technologies immunogenicity of papaya mosaic virus-like particles fused to a hepatitis c virus epitope: evidence for the critical function of multimerization potent immunogenicity and efficacy of a universal influenza vaccine candidate comprising a recombinant fusion protein linking influenza m2e to the tlr5 ligand flagellin structure-based design of peptides that self-assemble into regular polyhedral nanoparticles a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine a laboratory manual for the isolation and identification of avian pathogens a new generation of modified live-attenuated avian influenza viruses using a twostrategy combination as potential vaccine candidates universal primer set for the full-length amplification of all influenza a viruses a method for the rapid construction of crna standard curves in quantitative real-time reverse transcription polymerase chain reaction development of a real-time reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes crystal structure of a naturally occurring parallel right-handed coiled coil tetramer a switch between two-, three-, and four-stranded coiled coils in gcn4 leucine zipper mutants virulence of low pathogenicity h7n2 avian influenza viruses from the delmarva peninsula for broiler and leghorn chickens and turkeys importance of a prime-boost dna/protein vaccination to protect chickens against lowpathogenic h7 avian influenza infection the influence of antigen organization on b cell responsiveness role of repetitive antigen patterns for induction of antibodies against antibodies influenza a vaccine based on the extracellular domain of m2: weak protection mediated via antibody-dependent nk cell activity t. neef and s. babapoor contributed equally to this study. this work was supported by grants from the usda and university of connecticut research foundation. the authors wish to thank jason farnsworth and janet trombley for their veterinary technical support at the university of connecticut oac facility. key: cord-003567-h8uq5z8b authors: crank, michelle c; mascola, john r; graham, barney s title: preparing for the next influenza pandemic: the development of a universal influenza vaccine date: 2019-04-15 journal: j infect dis doi: 10.1093/infdis/jiz043 sha: doc_id: 3567 cord_uid: h8uq5z8b nan structural biology, protein engineering, and antigen delivery amenable to platform manufacturing approaches. molecularand atomic-level information about the immune-viral interface combined with new capacities for surveillance and rapid response to pandemics are shaping a new conceptual framework for vaccine development. as a result of these advances, highlevel, broad, and durable immunity against the large universe of influenza viruses may now be within reach [20] . this issue of the journal of infectious diseases was motivated by the confluence of the 1918 influenza pandemic centenary and the new opportunities afforded by technological advances and breakthroughs along with the improved understanding of influenza biology. we have gathered information, opinions, and ideas from thought leaders in immunology, virology, epidemiology, and vaccinology to address the challenge of developing a universal influenza vaccine and articulate some of the knowledge and technical gaps that remain. in 1918, there were approximately 1.8 billion persons living on earth; today, there are more than 7 billion [21] . if a pandemic similar to that of 1918 occurred today, how would the devastation compare? because of rapid international travel, the spread of a new virus would be faster than it was in 1918, when spread was largely related to the movement of troops at the end of world war i. today, population density is higher and cities are larger, providing a more favorable environment for a rapidly spreading virus. although we now have more sophisticated medical care, the availability of hospital beds and life support equipment would probably not be sufficient to manage an outbreak equivalent in magnitude to that of 1918. if there were sentinel events as in 1918 and in 2009-a small spring epidemic preceding the fall pandemic-current vaccine manufacturing approaches would not be sufficiently fast or scalable for worldwide distribution to preempt pandemic spread. therefore, development of a universal influenza vaccine that can reliably protect against drifted seasonal strains and pandemic strains without biannual reformulation is imperative. ideally, this vaccine would not need to be given every year; however, even if annual vaccination was required but antigenic components needed updating only every 5-10 years, it would still be a significant advance over the current system. s108 • jid 2019:219 (suppl 1) • crank et al there are some clear pathways to explore and knowledge gaps to fill in the immediate future using currently available technology, as described in the accompanying commentaries, outlined here: 1. by harnessing high-throughput sequencing and computational biology, more sophisticated algorithms based on sequence analysis, glycan patterns, and other features that may anticipate high transmissibility can be developed for predicting the next dominant strain [4] . the prudent study of gain-of-function mutations would allow scientists to learn more about what molecular signatures to look for. 2. improving strain selection for seasonal vaccines would increase the likelihood of an antigenic match between the vaccine and dominant circulating strains and thereby improve the utility of current vaccine technology [2] . the current vaccines could be further improved by better standardization of the neuraminidase content, adjustment of antigen doses, addition of improved adjuvants, and production in cell substrates that minimize the likelihood of viral adaptations and changes in protein sequences [2] . 3. precisely defining the b-cell repertoire and epitope-specific phenotypes involved in the response to influenza infection and vaccination would provide insight into the problem of "original antigenic sin" described by thomas francis in 1960 and the related phenomenon of immunodominance [22] . prior influenza immunity and poorly understood antigenicity patterns make it difficult to reshape and broaden the antibody response using current vaccines [7] . defining all the ways antibody can bind and neutralize influenza structurally and establishing a new nomenclature for describing antigenic sites across both influenza a groups as well as influenza b would reduce confusion and improve communication between scientists [5] . in addition, learning which features of vaccine-induced local or systemic immune responses result in sustained serum antibody responses may inform vaccine formulation and delivery approaches. 4. understanding more precisely the b-cell and antibody responses would allow the application of protein engineering for antigen design and display using molecular targets and antibody lineage end points to guide iterative design modifications [14] . 5. the role of cd4 + t cells in determining the efficacy of a b-cell response is an area of active investigation; however, more work in this area may be required to solve the problem of durability and maintenance of antibody responses [6] . 6. the direct role of cd4 + or cd8 + t-cell effector functions and whether those cells require localization in mucosal tissue or lymph nodes to effectively protect against respiratory viral pathogens are poorly understood. optimizing vaccine formulation and delivery route and modality is dependent on acquiring this type of knowledge [6] . 7. defining the importance of including specific antigenic targets, such as the head or stem domains of hemagglutinin, neuraminidase, or the m2 ectodomain in universal vaccines, and determining whether they are more effective when used in combination or alone could be accomplished through both vaccine protection and natural history studies that provide a better understanding of protective immunity [9] [10] [11] [12] . 8. understanding the mechanistic correlates of immunity generated by immunization with live attenuated vaccines may reveal the importance of secretory immunoglobulin a and intraepithelial t cells that require induction of immunity to occur at the mucosal surface [13] . 9. defining both the virological and host immune response patterns associated with transmissibility would allow better modeling of population dynamics and factors that could best interrupt transmission cycles [8] . this could be particularly important for identifying distinct vaccination strategies for different target populations, including in societal settings in which transmission dynamics and target populations vary [15] . 10. using human challenge studies and improving animal models of influenza infection and transmission may help answer some of these questions [3] . however, the utility of animal models hinges on selecting those that are most relevant to human pathogenesis and immunity. improving the characterization of and expanding the reagents for these models would not only benefit influenza vaccine development but would also provide answers to immunological questions relevant to other respiratory virus infections and emerging infectious diseases in general. recent estimates place the cost of influenza pandemics at upward of $500 billion per year [23] . in this context, an investment of at least $1 billion per year in the biomedical research effort to achieve a solution for protection against pandemic influenza seems justified. in addition, efforts to develop a universal influenza vaccine, even before reaching this goal, will likely lead to improved seasonal vaccines, with the potential to reduce morbidity rates and save tens of thousands of lives each year. solving this problem is potentially achievable with today's technology, and with an organized and sustained focus on interventions for influenza and other potential emerging infectious threats, more advanced approaches could be rapidly developed. finally, gaps remain in our understanding of influenza biology and immunity and methods to produce highly effective vaccines. to close those gaps, it will be important to align the interests of all the stakeholders preparing for the next pandemic. priorities of public health officials in lower-, middle-, and high-income countries, academic researchers, regulatory bodies, major funders, and pharmaceutical companies must be understood and collectively addressed to face the logistical and scientific challenges ahead [24] . thanks to scientific and technological breakthroughs of the past decade, vaccinology is experiencing a revolution. may we find the resolve, political will, and new business plans to take full advantage of these new opportunities and prepare ourselves before the next pandemic arrives. updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic influenza vaccines: good, but we can do better making universal influenza vaccines: lessons from the 1918 pandemic can we predict the next influenza pandemics? antibody determinants of influenza immunity the way forward: potentiating protective immunity to novel and pandemic influenza through engagement of memory cd4 t cells immunodominance and antigenic variation of influenza virus hemagglutinin: implications for design of universal vaccine immunogens dynamic perspectives on the search for a universal influenza vaccine universal influenza vaccine approaches employing full-length or head-only ha proteins universal influenza virus vaccines that target the conserved hemagglutinin stalk and conserved sites in the head domain the role of m2e in the development of universal influenza vaccines neuraminidase, the forgotten surface antigen, emerges as an influenza vaccine target for broadened protection how live attenuated vaccines can inform the development of broadly cross-protective influenza vaccines new vaccine design and delivery technologies influenza immunization in low-and middle-income countries: preparing for next-generation influenza vaccines novel vaccine technologies: essential components of an adequate response to emerging viral diseases emerging viral diseases from a vaccinology perspective: preparing for the next pandemic advances in antiviral vaccine development a universal influenza vaccine: the strategic plan for the national institute of allergy and infectious diseases historical estimates of world population on the doctrine of original antigenic sin pandemic risk: how large are the expected losses? vaccine development in the twenty-first century: changing paradigms for elusive viruses we thank our colleagues at the national key: cord-002919-6xjm7f29 authors: luo, haili; wang, shaohong; yuan, tongmei; liu, jingtao; yao, ling; pan, xianguo; long, xuemei; wu, juncheng; shen, feng title: clinical characteristics from co-infection with avian influenza a h7n9 and mycoplasma pneumoniae: a case report date: 2018-03-15 journal: j med case rep doi: 10.1186/s13256-018-1583-5 sha: doc_id: 2919 cord_uid: 6xjm7f29 background: more and more cases of human infections with avian influenza a h7n9 have been reported since it was first mentioned in 2013 in china, but concurrence of influenza a h7n9 with mycoplasma pneumoniae, however, has never been described. here, we reported the case of a woman co-infected by influenza a h7n9 and mycoplasma pneumoniae, whose treatment process was a little bit longer and a little bit complicated as well. case presentation: our patient was an 80-year-old chinese woman who presented with fever, cough, chest tightness, and shortness of breath. a computed tomography scan showed obvious infiltrations at lower parts of both lungs. arterial blood gas analysis confirmed a severe respiratory failure (type i). her sputum and throat swabs were checked for nucleic acid of influenza a and the result was positive for influenza a h7n9. she was diagnosed as having severe influenza a h7n9 and acute respiratory distress syndrome, and was admitted to an intensive care unit. she was given comprehensive treatment, including oseltamivir, methylprednisolone, immunoglobulin, gastric protection, and noninvasive mechanical ventilation. her condition improved 4 days later. however, some symptoms exacerbated again 2 days later with ground-glass changes appearing in upper area of right lung and the titer of antibody to mycoplasma pneumoniae rising from 1:80 to 1:640. she was reasonably considered to be infected with mycoplasma pneumoniae as well, and azithromycin and moxifloxacin were added to her treatment. oseltamivir was discontinued because of three consecutive negative results of nucleic acid for influenza a h7n9, but anti-mycoplasma treatment was continued. although her symptoms and abnormal changes on computed tomography scan slowly went away, she finally recovered from the mixed infection after a total of 33 days of management. conclusion: in patients with confirmed influenza a h7n9 infection whose condition worsens again, especially with new infiltration or lung ground-glass infiltration, one should suspect infection by other pathogens such as mycoplasma pneumoniae. since the case of human infection with avian influenza a h7n9 was first reported in 2013 in china [1] , many more cases of this disease have emerged in this country [2, 3] . human infection with avian influenza a h7n9 is a kind of acute respiratory disease which is caused by avian influenza a h7n9 virus, with more than 90% of the infected patients presenting with rapidly progressive pneumonia and 70% of them presenting with acute respiratory distress syndrome (ards). the mortality is as high as 30% [4] . patients with influenza a h7n9 often have some underlying diseases including chronic obstructive pulmonary disease (copd), hypertension, diabetes mellitus, and heart diseases [3] , which make patients prone to infection by some other pathogens, such as bacteria. however, concurrence of influenza a h7n9 virus and mycoplasma pneumoniae (mp) infection has never been described. mp pneumonia is an atypical community-acquired pneumonia, which is most often mild and moderate, and its prognosis is usually good [5] . here, we report a case with comorbidities of influenza a h7n9 and mp pneumonia, which made our patient's treatment process a little bit longer. our patient is an 80-year-old retired worker, whose onset started with a fever, cough, abdominal distention, weakness, and anorexia. then, chest tightness and short of breath followed. a computed tomography (ct) scan and arterial blood gas analysis confirmed the diagnosis of severe pneumonia and a respiratory failure (type i) for our patient. she was diagnosed with influenza a h7n9 by positive result of nucleic acid for influenza a h7n9 from sputum and throat swabs 10 days later and was transferred to an intensive care unit (icu) in second affiliated hospital of guizhou medical university, kaili city, guizhou province, china. during her treatment process, she was confirmed to have an infection of mp. since this is the first report of influenza a h7n9 complicated with mp, and the anti-mp treatment in such cases might be different from that in mp infection alone, it is valuable to present our case report, sharing our experiences in this patient's treatment. the main clinical characteristics of our patient and her treatment practice are reported below. an 80-year-old chinese woman began to have fever, cough, abdominal distention, weakness, and anorexia on january 5, 2017. she had been to the local poultry market with her husband to buy a live chicken and had eaten chicken meat on january 2, 2017. she is a retired teacher, and she has three children, all of whom are healthy. she is optimistic and has healthy social relations. she currently lives with her husband. she went to see a doctor at a hospital in the southeast state of guizhou province on january 11, 2017. an examination showed a fever with body temperature of 38.3°c, blood pressure of 130/78 mmhg, and pulse rate of 85 beats per minute (bpm). she was oriented and cooperative and in fine general condition. her mental status was conscious and her neurological examination was normal. she does not smoke tobacco and does not consume alcohol. her sputum and throat swabs were collected to test the nucleic acid of avian influenza a virus. then, she was given some general anti-virus drugs. fever persisted and she felt short of breath, so she went to the second affiliated hospital of guizhou medical university on january 13, 2017. an examination revealed a highest body temperature of 39.5°c, pulse rate of 83 bpm, respiratory rate of 25 to 28 breaths per minute, moist rales located at middle-lower fields of both lungs, blood oxygen saturation (spo 2 ) of 94% (oxygenation at 4 l/minute of oxygen with a nasal catheter), noninvasive blood pressure of 112/64 mmhg, white blood cell count of 3.46 × 10 9 /l (neutrophils, 69.4%; lymphocytes, 0.8 × 10 9 /l), oxygen arterial tension (pao 2 ) of 58.4 mmhg with fraction of inspired oxygen (fio 2 0.41), and carbon dioxide arterial tension (paco 2 ) of 31.8 mmhg. a ct scan showed obvious infiltrates in both lungs and some ground-glass opacity in the middle field of her right lung (fig. 1a) . in her past medical history, she had hypertension and copd. because of suspicion for contagious disease, she was admitted to the department of infectious disease of our hospital and given antibiotics, oxygen therapy, and oseltamivir. on january 14, 2017, she was definitely diagnosed as having influenza a h7n9 by the positive results of nucleic acid testing and was separated from other patients. she was transferred to the icu on the same day because of obvious dyspnea and shortness of breath. her immediate examination on icu admission showed oxygen pulse saturation of 65%, a body temperature of 38.5°c, noninvasive blood pressure of 128/75 mmhg, a maximal respiratory rate of 30 to 35 breaths per minute, and she was conscious and cooperative. her neurological examination was completely normal. arterial blood gas result revealed a ph of 7.48, paco 2 of 31 mmhg, pao 2 of 49.1 mmhg (fio 2 0.41), and bicarbonate (hco 3 − ) of 22.8 mmol/l; biochemical results consisted of total protein of 57.38 g/l, proalbumin of 69 mg/l, potassium of 3.39 mmol/l, sodium of 129.36 mmol/l, creative kinase of 162.15 u/l, creative kinasemyocardial band (mb) of 8 u/l, lactate dehydrogenase of 420 u/l, brain natriuretic peptide (bnp) of 185 ng/l, and procalcitonin (pct) of 0.056 μg/l. the titer of antibody to mp was 1:80. liver and renal functions were almost normal. urine analysis showed red blood cell of 8 cells/ low-power field (lp) and white cell of 10 cells/lp. the main diagnoses at icu admission were severe pneumonia and ards. after being delivered to the icu, our patient was given comprehensive treatments, including antibiotics, intravenously administered methylprednisolone (120 mg/day), immunoglobulin (10 g/day), and gastric protection. oseltamivir at 300 mg/day was orally administered for the first 6 days, then at 150 mg/day for the following 3 days. as she was conscious and very cooperative, she was given noninvasive mechanical ventilation (philips respironics v60, respironics california, inc, usa). her input of fluid volume was strictly limited and her hemodynamics were stable except for occasional slight fluctuations. after having been treated for 4 days, her respiratory symptoms began to alleviate, and oxygenation improved with pao 2 up to 66 mmhg (fio 2 0.4), and respiratory rate decreasing to 24 to 28 breaths per minute. however, 2 days later, she began to cough with less sputum, and tachypnea and weakness were exacerbated. the titer of mp antibodies gradually increased from 1:80 to 1:160, and then arrived at 1:640 within 9 days. a ct scan on january 26, 2017 revealed an enlarged ground-glass infiltrate in the upper area of her right lung (fig. 1b) . she was reasonably considered to be over-infected with mp, and then orally administered azithromycin (0.5 g/ day) and intravenously administered moxifloxacin (400 mg/ day) were prescribed on january 23, 2017. at the same time, oseltamivir was discontinued because of three consecutive negative results of nucleic acid for influenza a h7n9. after 5 to 6 days of anti-mp treatment, her symptoms alleviated and the titer of the antibodies decreased. a gradual reabsorption of ground-glass infiltrates was seen in upper lobe of her right lung after 9 days of treatment against mp (fig. 1c) . she was discharged on february 5, 2017 with mild cough and shortness of breath on exercises. considering that she still had a mild cough and felt somewhat short of breath, especially on exercises, and because some infiltrates in both lungs remained, she was advised to continue taking azithromycin regularly after discharge. we kept a close follow-up on her treatment. on february 7, 2017, the titer of mp antibodies decreased to 1:80. on february 13, 2017, all her symptoms had disappeared after a continuous 22 days of treatment against mp. the follow-up at 6 months after her discharge from icu revealed that she has kept very fit. her clinical course is summarized in table 1 . our patient suffered from avian influenza a h7n9 maybe because of contact history of live birds and her condition exacerbated in a short time. in her treatment process, she was confirmed to be over-infected by mp, which has not been reported before. the anti-mp process in this patient was longer than usual because of her poor underlying condition. until now, human infection with avian influenza a h7n9 has been merely confirmed to be associated with exposure to poultry [6] . this was the case for our patient, who had been to a poultry market and had eaten chicken prior to the onset of illness. surprisingly, her husband, who went to the market with her and had closer contact with the live poultry, remained healthy. this may be associated with his relatively perfect immune system and with no obvious underlying diseases. by contrast, his wife suffered from some underlying illnesses including hypertension and copd, which made her more susceptible to infection from other pathogens such as avian influenza a h7n9 virus. clinical studies have demonstrated that a proportion of patients with influenza a or b are simultaneously infected by other pathogens such as bacteria and aspergilla [3, 7, 8] because of some risk factors such as copd, use of steroids, and diabetes mellitus or even with a immunocompetent status [8] . but as far as we know, the clinical condition of co-infection caused by influenza a h7n9 and mp has not been described before. although "atypical" pneumonia was recognized in the 1930s, mp was actually isolated from the sputum of a patient with "atypical" pneumonia in 1944 [9] . early clinical observations indicated a relatively high prevalence of mp respiratory infection in younger children, especially among school-aged children from 5 to 15 years of age [10] . so far, there have been fewer reports of mp respiratory infection in older individuals. lung infection caused by mp can be diagnosed with culture of respiratory specimens and/or a fourfold rise in antibody titer to mp within 2 weeks [11] . in our patient, the mp antibody titer rose fourfold within 9 days, along with the exacerbation of some symptoms and appearance of some new ground-glass lung infiltrates. this supported our diagnosis of mp in this patient [11] . since the incubation period of mp infection is often as long as 1 to 3 weeks [5] , we speculate that mp may have already existed in our patient's respiratory system at the time when she was infected with avian influenza a h7n9. although mp infections are generally mild, patients may develop severe and fulminant disease at any age [12] . this is especially the case in patients who simultaneously suffer from another disease such as avian influenza a h7n9, as observed in our patient. the lack of a cell wall makes mp resistant to cell wall synthesis inhibitors such as β-lactam antibiotics. effective drugs against mp include macrolides, tetracyclines, and fluoroquinolones [13] . our patient was given azithromycin and moxifloxacin, and the ultimate result indicates that this therapeutic regimen was fortunately efficacious. it is generally recommended that the duration of treatment with azithromycin for communityacquired mp pneumonia is 7 to 14 days [14] . for our patient, azithromycin administration persisted for 22 days because of her poor underlying condition and her immunocompromised status. it is noteworthy that although no fluoroquinolone resistance has been described so far in mp clinical isolates, more and more macrolide-resistant mp have been reported in recent years [14, 15] . if the clinical manifestations do not resolve in 72 hours with macrolides administration, it is recommended to change antibiotics to tetracycline (doxycycline and minocycline) or fluoroquinolones (moxifloxacin and levofloxacin) [15] . in cases of confirmed influenza a h7n9, if the condition of the patient is not under initial treatment or if it even worsens with new ground-glass lung infiltrates, infection with another pathogen including mp must be suspected and sought. human infection with a novel avian-origin influenza a(h7n9) virus clinical and epidemiological analysis of the first case of human infection with avian influenza a (h7n9) virus in shenzhen, china adjuvant corticosteroid treatment in adults with influenza a (h7n9) viral pneumonia clinical findings in 111 cases of influenza a (h7n9) virus infection infection with and carriage of mycoplasma pneumoniae in children transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands the influence of corticosteroid treatment on the outcome of influenza a(h1n1pdm09)-related critical illness post-influenza aspergillosis, do not underestimate influenza b. infect drug resist studies on the etiology of primary atypical pneumonia: a filterable agent transmissible to cotton rats, hamsters, and chick embryos long-term epidemiology of infections with mycoplasma pneumoniae antibiotics for communityacquired lower respiratory tract infections secondary to mycoplasma pneumoniae in children fatal outcomes in family transmission of mycoplasma pneumoniae mycoplasma pneumoniae and its role as a human pathogen mycoplasma pneumoniae: current knowledge on macrolide resistance and treatment epidemiology of mycoplasma pneumoniae infections in japan and therapeutic strategies for macrolide-resistant m. pneumoniae we cordially appreciated the special help of professors juan du, jing yang, qi liu, zhuhong zha, and chunling zhu. there is no funding to support the case report. data sharing not applicable to this article as no datasets were generated or analyzed during the current case report. authors' contributions hl and sw took the patient in charge and were responsible for the treatments of the patient and the data collection. ty, jl, ly, xp, xl, and jw took the patient in charge. fs conceived of the article, and was responsible for the manuscript. all authors have read and approved the final version of the manuscript.ethics approval and consent to participate this is a case report and there is no need of ethics approval. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord-002972-ge7qt256 authors: torner, núria; martínez, ana; basile, luca; mosquera, mmar; antón, andrés; rius, cristina; sala, m. rosa; minguell, sofia; plasencia, elsa; carol, mónica; godoy, pere; follia, núria; barrabeig, irene; marcos, m. angeles; pumarola, tomàs; jané, mireia title: descriptive study of severe hospitalized cases of laboratory-confirmed influenza during five epidemic seasons (2010–2015) date: 2018-04-14 journal: bmc res notes doi: 10.1186/s13104-018-3349-y sha: doc_id: 2972 cord_uid: ge7qt256 objective: the plan of information on acute respiratory infections in catalonia (pidirac) included the surveillance of severe hospitalized cases of laboratory-confirmed influenza (shclci) in 2009. the objective of this study was to determine the clinical, epidemiological and virological features of shclci recorded in 12 sentinel hospitals during five influenza seasons. results: from a sample of shclci recorded during the 5 influenza epidemics seasons from 2010–2011 to 2014–2015, cases were confirmed by pcr and/or viral isolation in cell cultures from respiratory samples. a total of 1400 shclci were recorded, 33% required icu admission and 12% died. the median age of cases was 61 years (range 0–101 years); 70.5% were unvaccinated; 80.4% received antiviral treatment (in 79.6 and 24% of cases within 48 h after hospital admission and the onset of symptoms, respectively); influenza virus a [37.9% a (h1n1)pdm09, 29.3% a (h3n2)] was identified in 87.7% of cases. surveillance of shclci provides an estimate of the severity of seasonal influenza epidemics and the identification and characterization of at-risk groups in order to facilitate preventive measures such as vaccination and early antiviral treatment. electronic supplementary material: the online version of this article (10.1186/s13104-018-3349-y) contains supplementary material, which is available to authorized users. influenza is an infectious disease affecting mainly upper respiratory tract worldwide. influenza virus causes between three and five million severe cases and an estimated 250,000-350,000 deaths annually. in the european union, there are between 40,000 and 220,000 annual deaths attributable to influenza. however, mortality is only the tip of the iceberg in terms of the disease burden, since influenza also causes a decrease in functional status and increased dependency in the elderly [1] . estimating the burden of disease caused by influenza is difficult because many cases do not require medical care, or no confirmatory laboratory tests are widely performed to all influenza like illness' cases [2, 3] . in catalonia, influenza surveillance is conducted through the plan of information on acute respiratory infections in catalonia (pidirac) based on the network of sentinel physicians, who provide information on patients with influenza symptoms [4] . given the situation generated by the 2009 pandemic caused by the new influenza a (h1n1) pdm09 virus, the pidirac sentinel network included surveillance of severe hospitalized cases of laboratory-confirmed influenza (shclci) to assess severity. the pidirac sentinel surveillance network has a primary care sentinel network made up by 60 gps and pediatricians who inform on a daily basis of all ili attended and perform sampling of respiratory swabs for confirmation. this information allows to plot weekly ili incidence and 12 sentinel hospital facilities who notify on a weekly basis all influenza confirmed cases that meet the ecdc definition for severe influenza [5, 6] . this surveillance allows the clinical and epidemiological characteristics and risk factors associated with greater severity to be determined, and the emergence of influenza virus strains with clinical characteristics and behaviours outside the normal range to be detected, in order to correctly prioritize and direct preventive and control measures during the influenza season [7] . the aims of shclci surveillance are to provide an estimate of the severity of seasonal influenza epidemics to identify and characterize the risk groups that may present serious complications as a result of infection by circulating influenza viruses or their association with some underlying diseases and to identify the virological characteristics of viruses associated with these severe cases, such as genetic changes and/or antigenic changes that lead to increased virulence. the aim of this study was to describe the clinical, epidemiological and virological characteristics of shclci based on data collected in five influenza seasons in catalonia. epidemiological surveillance of severe cases of influenza in catalonia during five epidemic influenza seasons (2010-2015), beginning on week 40 of the season until week 20 of the following year, with the recording by twelve hospitals (covering 95,3% of the population) from the pidirac sentinel network of shclci reported to the epidemiological surveillance units corresponding to each hospital [5, 7] . shclci cases were cases with previous influenza like illness symptoms (sudden onset of symptoms and/ or fever; malaise; headache; muscle pain; and/or cough; sore throat; shortness of breath) who presented to a hospital facility and complyed with shclci case definition. shlcic was defined as a severe case of laboratory-confirmed influenza due to the influenza virus (a, a (h1n1)pdm09, b, c) that required hospitalization because of pneumonia, septic shock, multiorgan failure or any other severe condition, including icu admission or who developed clinical signs during hospitalization for other reasons. influenza diagnosis was confirmed by polymerase chain reaction (pcr) and/or culture of nasopharyngeal swabs. respiratory tract samples were processed within 24 h of receipt at the laboratory. a 300 μl aliquot was taken for total nucleic acids extraction and eluted in 25 μl of rnase-free elution buffer using the automatic qiasymphony system (qiagen, hilden, germany) according to the manufacturer's instructions. subsequently, two specific one-step multiplex real-time pcr was carried out using the stratagene mx3000p qpcr systems (agilent technologies, santa clara, ca, usa), were used for typing a/b influenza virus and subtyping influenza a virus [8, 9] . for each reported case, an epidemiological survey was made to collect anonymized demographic variables (age and sex); risk factors; icu admission; day of onset of symptoms, of hospital admission and discharge; vaccination history; influenza virus type and subtype; and outcome at hospital discharge. epidemiological survey was conducted by preventive medicine physician from data in medical history registry and public health epidemiologist in charge. we studied all data on shclci from five influenza seasons in pidirac sentinel network hospitals and made a comparative analysis of viral types and subtypes. the strain identified in > 50% of cases in each season was considered the predominant strain. duration of hospital stay was divided into two categories < 10 days and 10 days or more. the statistical analysis was made using the chi square test and student's t test with 95% confidence intervals (ci) for continuous variables and the anova test for categorical variables. during the 2010-2015 seasons 1400 cases of shclci were recorded, 462 (33%) required icu admission and 167 (12%) died: 778 (55.6%) were male. the median age was 61 years (range 0-101 years-mean 55.2 (sd 26.7 years). the most-affected age group was the ≥ 65 years age group with 633 cases (45.2%) ( table 1 ). the median age of the ≥ 65 years age group was 79 years (range 65-101) and the mean age was 78.7 years (sd 7.8 years): 296 (47%) were aged ≥ 80 years. of deaths, 111 (66.5%) occurred in patients aged ≥ 65 years and 55 (33.3%) in patients aged > 80 years ( table 1) . the distribution by type of influenza virus was: 87.7% (1228) influenza virus a, 531 (37.9%) of which corresponded to the a (h1n1)pdm09 subtype and 410 (29.3%) to a (h3n2), and 20.5% to influenza a that remained unsubtyped: 172 (12.3%) of cases were influenza b (additional file 1). there were significant differences in the mean age of cases according to the virus type, with a higher prevalence of virus a (h3n2) in older patients and virus a (h1n1)pdm09 in younger patients with mean age of cases 66.9 and 46.8 years (p < 0.001) and those with death as outcome 78.8 and 60.2 years, respectively (p < 0.001) ( table 1) . in 1384 (98.9%) of shclci there was a known risk factor. the most prevalent risk factors were cardiovascular disease, chronic obstructive pulmonary disease and diabetes (25.5, 23.4 and 20.5%, respectively). the most prevalent complication was pneumonia in 992 (71.7%) cases, of these 304 (30.6%) presented bacterial superinfection. for cases with known immunization for influenza, 682/967 (70.5%) of cases were not vaccinated for the current season included in the study (missing data on vaccination status: 433 (31%). the age group with highest vaccine coverage was the older than 65 age group (57%) and cases with at least one risk factor had low vaccination coverage (20.5%). vaccine proved effective in reducing intensive care unit (icu) admission [or = 0.64 (95% ci 0.47-0.88) p = 0.003] ( table 2) . of the 21 pregnant women hospitalized as shclci, all were unvaccinated, 14 (66.7%) required icu admission, 19 (90.5%) received antiviral treatment and none of them had any underlying disease or risk factor other than pregnancy. the mean hospital stay was 13.8 days (sd 17.9) with a median of 9 days (range 1-374 days). the mean stay by age group was: 0-4 years 7. a total 1125 cases (80.4%) had information on antiviral treatment, 1113 (99%) received oseltamivir and 12 (1%) zanamivir. 863 of these cases (79.6%) received treatment in the first 48 h after admission. antiviral treatment administered before 48 h on admission was associated with a shorter length of stay (los) (or 0.25: ci 0.18-0.34, p < 0,001) nosocomial cases (41) were excluded from the analysis (table 3 ). after the 2009 influenza virus a(h1n1)pdm09 pandemic, among the lessons learned was the need to expand surveillance of seasonal influenza to include severe cases in order to determine the characteristics of shclci caused by seasonal influenza viruses circulating during each season. the results obtained by the pidirac sentinel surveillance system during five post-pandemic seasons underscore the importance of prevention by vaccination in order to avoid serious complications such as ards and icu admission of the most vulnerable persons, while showing the need for increased vaccination coverages in groups such as pregnant women, in whom the proportion of icu admission is 66.7% while vaccination is zero [7, 10] . in our study no significant differences between influenza a and b virus infections among hospitalized cases was observed, except for younger age for a (h1n1) pdm09 cases; similar results also found by other studies in the united states and australia [11] [12] [13] . although the number of hospitalizations associated with influenza a virus infections was greater than the number with influenza b virus infections this fact can be explained by greater prevalence of influenza a viruses circulating in the community during the seasons included in the study. the delay in the administration of antiviral drugs at symptom onset in people with an identified risk of complications, such as the elderly or people with medical conditions that worsen the prognosis of influenza or make a longer hospital stay likely, also demonstrates the need to confirm influenza in primary healthcare and administer treatment within 48-72 h for it to be effective. influenza remains an important global public health problem in spite of scientific evidence which support immunization to protect those at high risk for complications, such as the elderly [1] . predominant influenza type/subtype circulating each season, influenza vaccination policies and coverage, influenza vaccine strain match/mismatch and vaccine effectiveness significantly influence the % of hospitalised influenza cases and cfrs in all age groups, including older age groups. however, the high percentage of hospitalizations (45.2%) and mortality (17.5%) in the ≥ 65 years age group, especially in people aged > 80 years, where mortality is higher (33.3%), reflect the consequences of increased life expectancy. early administration of antiviral treatment has proven to diminish length of stay. healthcare providers should start antiviral treatment as soon as possible, before 48 h from onset of symptoms is the recommendation, [11] unfortunately this is not feasible. yet if treated as soon as patient is admitted to the hospital facility and influenza is confirmed, shorter length of stay and prompt recovery can be attained [12, 13] . this makes it necessary to deepen our knowledge of the effect of aging and its interaction with the most prevalent chronic diseases in the elderly and the immune response in order to implement preventive measures to provide better protection of this population group [11] . it is necessary to improve some surveillance aspects, especially with regard to data collection, in order to avoid a loss of information that makes some variables impossible to assess, such as risk factors such as smoking, which was not recorded in 91% of cases as well as lack of information on the vaccination status, which was more than 30% [14, 15] . a limitation to this study is that only shclci cases were recorded during the study period. this unables global hospitalization burden estimates caused by seasonal influenza nor the estimation of seasonal differences in vaccine effectiveness to prevent severity and death. the system identifies the epidemiological and virological characteristics of severe forms of influenza that show changes in their virulence, but comparison between severe and non-severe cases is not feasible. the proportion of shclci cases admitted to icu and cfrs are potentially higher than other surveillance systems that monitor all hospitalised cases of confirmed influenza. this is particularly evident with regard to pregnant women because of the small number of cases. yet, in all shclci surveillance provides an estimate of the severity of seasonal influenza epidemics, and provides ad hoc information to identify and characterize the groups at risk of complications and take appropriate preventive measures. abbreviations ards: acute respiratory distress; icu: intensive care unit; cfr: case fatality rate; ci: confidence interval; ecdc: european center for disease control; ili: influenza like illness; los: length of stay; or: odds ratio; pcr: polymerase chain reaction; pidirac: plan of information on acute respiratory infections in catalonia; sd: standard deviation; shclci: severe hospitalized cases of laboratoryconfirmed influenza. • fast, convenient online submission ready to submit your research ? choose bmc and benefit from: vaccine effectiveness in older individuals: what has been learned from the influenzavaccine experience influenza illness and hospitalizations averted by influenza vaccination in the united states predicting clinical severity based on substitutions near epitope a of influenza a/h3n2 public health agency of catalonia. department of health. pla d'informació de les infeccions respiratòries agudes a catalunya (pidirac) estratègia de vigilància dels casos greus de grip hospitalitzats vigilancia de casos graves hospitalizados confirmados de virus de la gripe the global influenza hospital surveillance network (gihsn): a new platform to describe the epidemiology of severe influenza global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis virological surveillance of influenza and other respiratory viruses during six consecutive seasons from severe influenza in 33 us hospitals, 2013-2014: complications and risk factors for death in 507 patients early administration of oral oseltamivir increases the benefits of influenza treatment effectiveness of antiviral treatment in preventing death in hospitalized cases of severe influenza over six influenza seasons increased antiviral treatment among hospitalized children and adults with laboratory-confirmed influenza estimating the burden of seasonal influenza in spain from surveillance of mild and severe influenza disease estimated influenza illnesses, medical visits, hospitalizations, and deaths averted by vaccination in the united states seasonal influenza (flu) cdc. centers for disease control and prevention, national center for immunization and respiratory diseases (ncird) the members of the pidirac working group for the surveillance of severe nt conceived and wrote the manuscript, am and mj reviewed the manuscript and cr, ib, nf, pg, ep, sm, ms, mc, mm1, mm2, aa and tp were involved in case management. all authors read and approved the manuscript. the authors declare that they have no competing interests. the raw data supporting this study are publicly available as additional file. not applicable. ethical approval was not necessary as the study uses routinely collected, anonymous surveillance data. the study was partially funded by agaur (agència de gestió d' ajuts universitaris i de recerca) grant 1403 and ciber epidemiologia y salud pública ciberesp and by fondo de investigación sanitaria pi 11/01864 and recercaixa 2010acuo_00437i of the catalan association of public universities (acup). springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-011722-82qzf8ht authors: keitel, wendy a.; piedra, pedro a. title: influenza a(h5n1) vaccines: are we better prepared for the next pandemic? date: 2014-01-01 journal: j infect dis doi: 10.1093/infdis/jit573 sha: doc_id: 11722 cord_uid: 82qzf8ht nan the ongoing epizootic of influenza a(h5n1) infections has resulted in 641 reported human cases since 2003, 380 (59%) of which resulted in death [1] . these cases have been reported to the world health organization (who) from 15 countries in asia, africa, the pacific, europe, and the near east, and influenza a(h5n1) viruses continue circulate and evolve in poultry in asia and northeast africa. most infections have been the result of exposure to sick or dead poultry; however, limited humanto-human spread has occurred. although these viruses are capable of producing infection and illness in humans and most individuals lack of immunity, to date their pandemic potential has been limited by their inability to transmit readily from an infected person to others. international efforts are ongoing to prepare for a possible influenza a(h5n1) pandemic. four influenza pandemics have occurred since 1918, and the impact of these pandemics varied according to age group. for example, death rates in the 1918 pandemic were highest among persons aged <45 years, whereas death rates during the 1957 and 1986 pandemics were highest among persons aged >65 years. infection rates during the 2009 influenza a(h1n1) pandemic were highest among children. the epidemiology of human influenza a(h5n1) cases varies from country to country, but the median age of patients infected with influenza a(h5n1) viruses is approximately 18 years [2] . hence, a substantial burden of illness occurs in the pediatric population. should an influenza a(h5n1) pandemic occur and the epidemiological pattern of past influenza a(h5n1) infections persist, efforts must be focused on protecting this vulnerable population [3] . this is particularly important because children play an important role in the community-wide spread of influenza [4] . immunization is the cornerstone to the control of seasonal and pandemic influenza. the results of numerous clinical trials of candidate influenza a(h5n1) vaccines in adult populations have yielded several common themes. the avian h5 hemagglutinin (ha) is poorly immunogenic relative to seasonal has, 2 doses of vaccine are necessary to elicit serum antibodies in most subjects, and inclusion of an adjuvant (particularly oil-in-water emulsions) or use of whole-virus (wv) preparations can increase the frequency and magnitude of antibody responses and reduce the amount of vaccine antigen required to achieve antibody levels that have been associated with protection (ie, antigen-sparing approaches). in most cases in which mineral-containing adjuvants such as aluminum hydroxide were evaluated, little additional benefit was observed [5] . limited studies of live attenuated (laiv) influenza a (h5n1) vaccines have shown restricted replication and lower immunogenicity than that seen after immunization with seasonal laiv [6, 7] . the process of influenza a(h5n1) vaccine development is complicated by the ongoing evolution of influenza a(h5n1) viruses [8] . numerous clades now circulate among birds, and which one, if any, will acquire the ability to transmit easily from human to human is unknown. several studies have explored the effect of priming with one clade (1 or 2 doses) and boosting with another clade. these studies have shown that prior priming results in more broadly cross-reactive antibodies and higher levels of antibody than those observed after priming. the results of clinical trials of influenza a(h5n1) vaccines among children are consistent with those seen in adult populations. in this issue of the journal, van der velden et al report that a vero cell culture-derived wv influenza a(h5n1) vaccine derived from wild-type virus was well tolerated in infants and children aged 6 months to 8 years [9] . two doses containing 7.5 μg of a/vietnam(h5n1) ha elicited neutralization antibody titers associated with protection in animal models in 68.8%, 72.9%, and 85.4% in children aged 6-35 months, 3-8 years, and 9-17 years, respectively. booster immunization at 1 year with a/indonesia(h5n1) vaccine elicited the same neutralization antibody levels against both strains in ≥93%, ≥95%, and 100%, respectively. single radial hemolysis antibody assays indicated that 2 priming doses containing 7.5 μg of ha elicited responses that satisfied licensure thresholds established by european authorities (european medicines agency). high rates of seropositivity to the neuraminidase (na) also were observed following immunization. similar to studies conducted in adults with wv and adjuvanted or nonadjuvanted subvirion or purified recombinant ha influenza a(h5n1) vaccines, boosting with vaccine derived from another influenza a(h5n1) clade elicited higher and more broadly cross-reactive antibodies. immunization was reported to be well tolerated. fever was reported among 17%-19% of children in the youngest age stratum and was less frequent after the second and boosting doses. the results of this and other studies conducted among all age groups raise important questions regarding issues related to assessing the immunogenicity of both seasonal and pandemic vaccines. serum hemagglutination inhibiting (hai) antibodies have been regarded as a benchmark for assessing the immunogenicity of seasonal influenza vaccines by the food and drug administration [10] . serum hai antibody responses following immunization with the wv vaccines described in the article were low and not reported, making comparisons between results from this trial to results from other trials difficult. furthermore, neutralization antibody responses were lower than those observed following immunization of children with vaccines containing oil-in-water adjuvants (mf59 and as03). lack of standardization of hai and neutralization antibody assays is well recognized, and repeated calls for international cooperation to develop more reproducible assays have been made [11] . however, these assays will need to be clade specific, which poses a major hurdle. even if assays could be standardized, our understanding of the correlates and determinants of protection against both seasonal and pandemic influenza is far from complete. an hai titer of ≥40 has been associated with a 50% reduction in the risk of infection in adults and is used as a major immunization target. however, the level of antibody may vary with age such that hai levels of ≥110 appear necessary for children [12] . even less is known about the level of neutralization antibody required for protection. na antibody, cd4 + t-cell responses, and mucosal antibody responses all may contribute to protection against influenza a(h5n1) and/or other influenza virus infections [13] [14] [15] . identification of epitopes that stimulate broadly cross-reactive immune responses is a high priority, and vaccines prepared using these epitopes are in development [16] . another important consideration is vaccine safety. the use of an as03-adjuvanted 2009 influenza a(h1n1) vaccine was associated with an increased risk of narcolepsy, an autoimmune disease, particularly among adolescents [17] . other rare adverse events may only be observed when millions of people are exposed, as in a pandemic setting. in 1976, largescale immunization of the us population with another influenza a(h1n1) vaccine was associated with an increased risk of an autoimmune disease known as guillain-barré syndrome [18] . wv vaccines have been associated with an increased risk of fever in young children, but the outcomes have not been serious. depending on the severity of a future pandemic, risk/benefit assessments will need to be made with regard to which vaccines can be used in various age and risk groups. we will need all available manufacturing capacity and accelerated manufacturing approaches to produce and distribute vaccine to the entire population if a pandemic occurs. on the basis of the 2009 influenza a(h1n1) pandemic experience, it took about 6 months from the recognition of a pandemic virus to the start of delivery of pandemic vaccines (live and inactivated) to the population. this was truly a herculean feat by all the partners involved, although not fast enough to have an impact on the major fall wave of the pandemic. finally, several prepandemic influenza a(h5n1) vaccines have been licensed and are being stockpiled. it is time to tackle the difficult issue regarding the use of these vaccines before a pandemic occurs. the benefits include priming for robust responses to the actual pandemic strain, obviation of the need for 2 doses under difficult if not extreme circumstances, and expansion of the safety database. risks include adverse events associated with vaccination and diversion of limited resources in the absence of a pandemic. the strategic advisory groups of experts on immunization, which provides guidance on the work of the who immunization, vaccines, and biologicals department, has developed risk-based recommendations for the use of prepandemic vaccines [19] , as follows. vaccination is strongly recommended for laboratory workers who are at high risk of infection and for first responders to avian outbreaks and healthcare professionals in enzootic areas, and it may be made available to other laboratory workers and other healthcare professionals. notably, vaccination with influenza a(h5n1) vaccine is not recommended for the general population or other essential workers in enzootic or nonenzootic areas. as we learned in 2009, it is not possible to predict with any certainty what virus will cause the next pandemic. influenza a(h7n9) viruses recently caused infections in china [20] , and influenza a(h3n2) variant (swine), influenza a(h7n7), and influenza a(h9n2) viruses have caused human infections in recent years. influenza a(h2n2) was responsible for a pandemic in 1957; thus, some experts consider this subtype to be of major concern [21] . vaccines containing both seasonal and candidate pandemic vaccine antigens are being developed, but which viruses to include, in whom, and when is a matter for serious consideration. although vaccine development is a critical component of a program designed to prepare for the next pandemic, stockpiling of antivirals and antibiotics, development of plans for distribution of vaccines and antimicrobials, and ensuring the availability of adequate supplies of effective countermeasures are but a few of the critical approaches that will be necessary [22] , but are they sufficient to prepare the global community for the next pandemic? let the discussion continue! cumulative number of confirmed human cases of avian influenza a(h5n1) reported to who. www.who.int/influenza/human_ animal_interface/en_gip_20131008cumulative numberh5n1cases writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus. update on avian influenza a (h5n1) virus infection in humans prioritization of influenza pandemic vaccination to minimize years of life lost emerging infections: pandemic influenza pandemic h5n1 influenza vaccine development: an update evaluation of two live attenuated cold-adapted h5n1 influenza virus vaccines in healthy adults safety and immunogenicity of live attenuated influenza reassortant h5 vaccine ( phase i-ii clinical trials) the emergence and diversification of panzootic h5n1 influenza viruses safety and immunogenicity of a vero cell culture-derived whole-virus influenza a(h5n1) influenza vaccine in a pediatric population guidance for industry: clinical data needed to support the licensure of seasonal inactivated influenza vaccines. www.fda.gov/biologics bloodvaccines/guidancecomplianceregulatory information/guidances/vaccines/ucm074794. htm reproducibility of serologic assays for influenza virus a (h5n1) hemagglutination inhibition antibody titers as a correlate of protection for inactivated influenza vaccines in children can immunity induced by the human influenza virus n1 neuraminidase provide some protection from avian influenza h5n1 viruses? correlates of immune protection induced by live, attenuated, cold-adapted, trivalent, intranasal influenza virus vaccine preexisting influenza-specific cd4+ t cells correlate with disease protection against influenza challenge in humans correlates of protection against influenza infection in humans-on the path to a universal vaccine as03's adjuvanted ah1n1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in finland guillain-barre syndrome following vaccination in the national influenza immunization program, united states, 1976-1977 recommendations on the use of licensed human h5n1 influenza vaccines in the interpandemic period emergence of avian influenza a (h7n9) virus causing severe human illness-china vaccinate for the next h2n2 pandemic now planning for an influenza pandemic: thinking beyond the virus potential conflicts of interest. p. a. p. is on the speakers bureau for medimmune. w. a. k. certifies no potential conflicts of interest.all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-008837-74rfnt1x authors: tsang, kenneth wt; eng, philip; liam, ck; shim, young-soo; lam, wah k title: h5n1 influenza pandemic: contingency plans date: 2005-08-11 journal: lancet doi: 10.1016/s0140-6736(05)67080-8 sha: doc_id: 8837 cord_uid: 74rfnt1x nan a vaccine for h5n1 will not be available in the foreseeable months. even if pharmaceutical manufacturing begins soon after an outbreak, there would not be a sufficient supply for the countries most in need-ie, the asian nations. antiviral drugs are consequently the only specific treatment, pending availability of effective vaccines. these include m2 inhibitors (amantadine and rimantadine), which are ineffective against h5n1 in vitro, and the neuraminidase inhibitors (oseltamivir and zanamivir). 3 the neuraminidase inhibitors reduce the severity and duration of symptoms, and prevent clinical influenza as post-exposure and seasonal prophylaxis. 4 influenza contingency plans by the who and most governments generally advocate detection, isolation, staff protection, and the start of antiviral treatment for patients, and their contacts. 5 many governments, including those of hong kong, thailand, singapore, malaysia, and korea, have already stockpiled, at a very substantial expense, vast quantities of oseltamivir to prepare for an outbreak. 5 nonetheless, the efficacy of neuraminidase inhibitors, even for non-h5n1 influenza a in healthy people and taken within 48 h of disease onset, is only slight (table). [6] [7] [8] [9] [10] [11] the use of oseltamivir in five of the ten cases reported in vietnam did not show any obvious clinical efficacy, and the mortality was 80% in this cohort. 12 the two neuraminidase inhibitors, oseltamivir and zanamivir, have not been directly compared in controlled trials. their pharmacological properties are compared in the table. [6] [7] [8] [9] [10] [11] although both have similar efficacy, zanamivir has fewer adverse reactions, and a favourable resistance profile. the resistance factor would be an important consideration in a pandemic situation. the reasons for zanamivir not being chosen for stockpiling might include concern that young children and patients with intellectual or coordination impairments would not be able to inhale zanamivir properly, although there are novel ways of giving the drug to children. 13 the occurrence of bronchospasm and reduced lung function is very rare, and patients with asthma and chronic obstructive pulmonary disease (copd) seem to tolerate inhalation of zanamivir as well as the placebo. 14 the inhaled flow rate needed to give the custom-designed inhaler for zanamivir (49-110 l/min) age approved for prophylaxis 6, 7 ͼ5 years ͼ13 years age approved for treatment 6, 7 ͼ5 years ͼ1 year renal impairment 6, 7 no dose adjustment required adjustment if creatinine clearance 10-30 ml/min hepatic impairment 6, 7 no dose adjustment required safety not established reduction of influenza symptoms 8, 9 by median of 1·5 day by median of 1·3 day adverse reactions 6, 7 allergy-very rare nausea 7·0-10·7% bronchospasm and vomiting 2·1-8·0% dyspnoea-very rare diarrhoea 3·2-5·5% rash and urticaria-very rare bronchitis 0·7-3·7% headache 1·6-20·1% fatigue 0·8-7·9% frequency of drug resistance none reported 1·3 and 8·6-18·0% in adults and after treatment 10, 11 children, respectively comment is similar to that for accuhaler (30-90 l/min), and turbohaler (60-90 l/min), which are popular drypowder inhalation devices used by many asthmatic and copd patients, even during exacerbations. 15, 16 therefore governments should also consider stockpiling zanamivir as an anti-influenza agent in their pandemic plans. actual logistics for giving out antivirals to patients and close contacts need to be efficient and completed within 48 h. it seems more appropriate for communitybased health-care personnel or even pharmacists, rather than hospital-based health-care workers, to handle such procedures. governments and health agencies should also consider planning for clinical trials, for instance a combination of both neuraminidase inhibitors, with or without other potential novel drugs, such as shortinterfering rnas and interferon. 3 these trials, if initiated at the early stages of a pandemic, could provide useful information for further patient and outbreak management in later stages. the geographic location of vaccine manufacturers in developed countries would also delay poorer asian nations from obtaining the updated influenza vaccine. perhaps vaccine and neuraminidase inhibitor manufacturing activities should also begin in asia to deal with such deficiencies. the ethics of maintaining drug patents in a potential worldwide catastrophe is questionable. epidemiological modelling suggests that influenza is more infectious than severe acute respiratory syndrome, and that severe acute respiratory syndrome infection control measures might not be adequate for a pandemic of influenza. 17 there will, therefore, be an overwhelming strain on health-care workers and hospitals in an h5n1 pandemic, and staff could be rapidly demoralised and degenerate into deserters, if colleagues develop hospital-acquired h5n1 infection, especially if not given adequate intensive-care unit treatment. 18 protection of core personnel should also be planned to underpin recovery in the aftermath, when many key players in health care and governmental institutions would have perished. cumulative number of confirmed human cases of avian influenza a/(h5n1) since 28 probable person-to-person transmission of avian influenza a (h5n1) avian influenza review of the use of neuraminidase inhibitors for prophylaxis of influenza world health organization. national influenza pandemic plans issue number 5 core data sheet version 1.5 the mist (management of influenza in the southern hemisphere trialists) study group. randomised trial of efficacy and safety of inhaled zanamivir in treatment of influenza a and b virus infections efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group management of influenza virus infections with neuraminidase inhibitors: detection, incidence, and implications of drug resistance resistant influenza a viruses in children treated with oseltamivir: descriptive study avian influenza a (h5n1) in 10 patients in vietnam new application method of zanamivir with a straw efficacy and safety of inhaled zanamivir for the treatment of influenza in patients with asthma or chronic obstructive pulmonary disease: a double-blind, randomized, placebocontrolled multicentre study pharmacoscintigraphic evaluation of lung deposition of inhaled zanamivir in healthy volunteers a comparison of the performance of two modern multidose dry powder asthma inhalers are we ready for pandemic influenza? nurses' professional care obligation and their attitudes towards sars infection control measures in taiwan during and after the 2003 epidemic key: cord-001654-o2zfilcl authors: laidler, matthew r.; thomas, ann; baumbach, joan; kirley, pam daily; meek, james; aragon, deborah; morin, craig; ryan, patricia a.; schaffner, william; zansky, shelley m.; chaves, sandra s. title: statin treatment and mortality: propensity score-matched analyses of 2007–2008 and 2009–2010 laboratory-confirmed influenza hospitalizations date: 2015-03-04 journal: open forum infect dis doi: 10.1093/ofid/ofv028 sha: doc_id: 1654 cord_uid: o2zfilcl background. annual influenza epidemics are responsible for substantial morbidity and mortality. the use of immunomodulatory agents such as statins to target host inflammatory responses in influenza virus infection has been suggested as an adjunct treatment, especially during pandemics, when antiviral quantities are limited or vaccine production can be delayed. methods. we used population-based, influenza hospitalization surveillance data, propensity score-matched analysis, and cox regression to determine whether there was an association between mortality (within 30 days of a positive influenza test) and statin treatment among hospitalized cohorts from 2 influenza seasons (october 1, 2007 to april 30, 2008 and september 1, 2009 to april 31, 2010). results. hazard ratios for death within the 30-day follow-up period were 0.41 (95% confidence interval [ci], .25–.68) for a matched sample from the 2007–2008 season and 0.77 (95% ci, .43–1.36) for a matched sample from the 2009 pandemic. conclusions. the analysis suggests a protective effect against death from influenza among patients hospitalized in 2007–2008 but not during the pandemic. sensitivity analysis indicates the findings for 2007–2008 may be influenced by unmeasured confounders. this analysis does not support using statins as an adjunct treatment for preventing death among persons hospitalized for influenza. . deaths have been estimated to range between 3349 (1986) (1987) and 48 614 (2003) (2004) annually [3] . influenza-associated mortality in pandemic years can be higher still and typically shifts toward younger age groups [4] . this result was especially evident during the 2009 pandemic because cross-protective immunity to the pandemic strain was present among older adults [5] . during the recent 2009 influenza pandemic, 87% of the deaths occurred in persons <65 years of age, with children and young adults and middle-aged adults having rates of hospitalization and death 4 to 7 times and 8 to 12 times greater, respectively, than estimates from the years 1976-2001 [6] . the best available strategy to prevent and control influenza is through influenza vaccination. in the united states, all persons ≥6 months of age are recommended to receive influenza vaccine annually. however, influenza vaccine effectiveness can vary from season to season, depending on the match between the vaccine influenza strains and the circulating strains and host factors. in addition, during influenza pandemics, the development and deployment of an influenza vaccine may be delayed. influenza antiviral therapy for persons with severe influenza illness or who are at risk for complications is an important adjunct intervention to the influenza vaccination program, reducing morbidity and mortality during seasonal or pandemic influenza [7] [8] [9] [10] [11] [12] . nonetheless, there is always the possibility of widespread circulation of an influenza virus strain resistant to available antiviral agents, and, in a pandemic situation, the risk of antiviral shortages is ever present. the use of immune-modulating drugs, particularly statins, has been postulated as an additional tool for the treatment and prophylaxis of influenza, especially in countries where influenza vaccine and antiviral agents are not readily available [13, 14] . statins have wide-ranging down-regulatory effects on inflammatory and immune mechanisms [15] [16] [17] , and there is some evidence that statin treatment may beneficially alter the clinical course of some infectious diseases [18] [19] [20] [21] . to date, no randomized clinical trials have been conducted to address whether statins could reduce complications of influenza, although some observational studies have suggested protective effects [22] [23] [24] [25] . a study by vandermeer et al [23] , using data from a populationbased influenza surveillance system, found a protective effect of statin use on mortality among patients hospitalized with laboratory-confirmed influenza during the 2007-2008 influenza season. nonetheless, due to the observational nature of the study, biases could potentially explain this association, even after controlling for confounders. we sought to repeat the 2007-2008 influenza season analysis of vandermeer et al [23] and to analyze the 2009 influenza a (h1n1) pandemic data from the same surveillance platform to study the possible association between influenza-associated mortality and statin treatment. in this analysis, we take into account potential treatment indication biases through the use of propensity score-matched analysis. this study was conducted with data from the centers for disease control and prevention's (cdc) emerging infections program (eip) influenza hospitalization surveillance. the eip influenza hospitalization surveillance system collects data on persons hospitalized with laboratory-confirmed influenza from october 1 through april 30 of the following year, because influenza typically circulates in the fall to spring months in the northern hemisphere. the exception to this was the 2009 influenza pandemic, in which hospitalization data were collected from september 1, 2009 through april 30, 2010. the eip network comprises selected counties in 10 us states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, new york, oregon, and tennessee) and includes a catchment area of approximately 23 million people. cases were identified through active surveillance from reports from hospitals and review of infection control logs or hospital laboratory lists. ascertainment of cases was based on laboratory testing ordered by attending healthcare providers for clinical purposes. cases included patients (1) ≥18 years of age, (2) residing within the eip catchment area, (3) admitted to a catchment area hospital, and (4) admitted within 14 days of a positive influenza test either by viral culture, real-time reverse transcription polymerase chain reaction, immunofluorescence antibody staining (indirect or direct), rapid influenza diagnostic test, or any test of unknown type recorded in the medical chart. patients possibly infected with influenza virus during hospitalization ( positive influenza test >3 days after admission) were excluded as case subjects. demographic, epidemiologic, and clinical information were collected from chart reviews. influenza vaccination status was determined from the medical chart, primary care provider, or via phone interview (of patient or proxy). patients were considered vaccinated for influenza if a vaccine had been administered >2 weeks before hospitalization, regardless of whether the patient had seasonal vaccine, h1n1 monovalent vaccine (for the 2009 pandemic), both seasonal and monovalent vaccine, or unknown vaccine type. if antivirals were administered at any point during the course of illness, a patient was considered treated. age was categorized into 5 groups (18-34, 35-44, 45-54, 55-64, and ≥65 years). race and ethnicity were determined by chart review or by self-report during patient interviews for vaccination status information. race was categorized into 3 groups (white, black, and other), and ethnicity was categorized as hispanic or non-hispanic. race and ethnicity were analyzed separately. underlying health conditions of interest included asthma, chronic cardiovascular disease (excluding hypertension), chronic metabolic disease, renal disease, chronic lung disease, immunosuppressive disorders (including cancer diagnosis in the 12 months before hospital admission), seizure disorders, history of lymphoma or leukemia, blood disorders, neuromuscular disorders, obesity, and cognitive dysfunction. we combined all underlying chronic disease variables other than cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, and asthma into a variable for "other chronic diseases." height and weight were collected during the 2009 pandemic but not during the 2007-2008 influenza season. height and weight were used to determine body mass index (bmi), which was used to categorize patients as underweight (bmi <18.5), normal weight (bmi 18.5-24.9), overweight (bmi 25.0-29.9), obese (bmi 30.0-39.9), or morbidly obese (bmi ≥40). the exposure of interest was statin treatment, either before or during hospitalization, which was determined from hospital records. data on statin dose or frequency of administration were not collected. death within 30 days of a positive influenza test was the outcome of interest. mortality after hospital discharge was determined by linkage of hospitalization data with the social security death index (ssdi) by state. linkage was done using registry plus™ link plus (version 2.0), a probabilistic record linkage program. mortality during hospitalization was determined through chart review and data linkage with ssdi data. this study was submitted for review and approved by the institutional review boards serving the cdc and participating states. propensity scores were used to predict the probability of treatment with statins. the use of propensity scores in observational studies facilitates similar distributions of baseline characteristics between treated and untreated groups, reducing potential treatment selection bias [26] . logistic regression models were iteratively assessed to determine the balance of covariate proportions between statin treatment groups in the subsequent matched samples. the best and final model was the one that balanced covariates between treatment groups, as determined by standardized differences <0.10. the final logistic regression model for the 2007-2008 matched sample included age, sex, race, ethnicity, cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, asthma, and vaccination status as covariates. the final logistic regression model for the 2009-2010 matched sample included age; sex; race; cardiovascular disease; chronic metabolic disease; chronic lung disease; renal disease; weight category; long-term care residence; vaccination status; and interaction terms for age and race, age and sex, and age and cardiovascular disease. after calculation of propensity scores, a greedy matching algorithm was used to identify 1 untreated patient for each treated patient in the respective matched samples [27] . the χ 2 test was used to assess differences in characteristics between the statin treatment groups in the unmatched cohort. categorical variables were transformed to indicator variables to facilitate assessment of balance of covariates between statin treatment groups after matching. standardized differences [26] were used to evaluate measured baseline covariate distributions between statin treatment groups in the matched cohorts. mcnemar's test for matched pairs was used to assess the difference in proportion of deaths between treated and untreated groups. we used cox proportional hazards models with robust standard errors, stratified on matched pairs, to determine the effect of statin treatment on mortality within 30 days of a positive influenza test. we used the method described by rosenbaum [28] for survival outcomes for determining the sensitivity of point estimates to hidden bias. we conducted statistical analysis using sas software (version 9.3; sas institute, cary, nc), and openepi (version 2.3.1) was used for post hoc sample size calculation [29] . table 1 shows demographic and clinical characteristics of treatment groups before matching, for both the 2007-2008 influenza season (statin treatment group n = 1013, nontreatment group n = 2030) and the 2009 pandemic (statin treatment group n = 980, nontreatment group n = 3458) cohorts. in the 2007-2008 cohort, the statin treatment group was older, with a greater proportion of males, a greater proportion of whites, and a higher prevalence of chronic medical conditions (with the exception of asthma and "other chronic diseases") compared with the nontreatment group. in the 2009 pandemic cohort, the statin treatment group compared with the nontreatment group was older, with a greater proportion of males and whites. the statin treatment group had a higher prevalence of cardiovascular disease, chronic metabolic disease, chronic lung disease, and renal disease, but not of other chronic conditions. the statin treatment group also had a greater proportion of persons considered obese and morbidly obese. influenza vaccination was more prevalent among those in the statin treatment group, for both cohorts. there was no significant difference between the proportion of those treated with antivirals for either cohort. after matching on propensity score (table 2) , both the 2007-2008 and 2009 pandemic matched samples were balanced on treatment groups. for all covariates, the standardized differences after matching were <0.10 for both cohorts. there were 670 pairs in the 2007-2008 sample. for 21 pairs, the treated case subject died within 30 days, but the untreated case subject did not. there were 51 pairs in which the untreated case subject died within 30 days but the treated subject did not. for 1 pair, both the treated and untreated case subjects died, and for 597 pairs, neither case subject died. the results of mcnemar's test indicate that the 30-day mortality rates between the treated (3.28%) and untreated (7.76%) groups were significantly different (p < .001). there were 439 pairs in the 2009-2010 cohort sample. for 17 pairs, the treated case subject died within 30 days but the untreated case subject did not. there were 23 pairs in which the untreated case subject died within 30 days but the treated subject did not. for 4 pairs, both the treated and untreated case subjects died, and for 395 pairs, neither case subject died. the results of mcnemar's test indicate that the 30-day mortality rates between the treated (4.78%) and untreated (6.15%) groups were not significantly different (p = .43). cox proportional hazards models with robust standard errors were fit to the matched samples, stratified on matched pairs. the sole predictor variable for the models was statin treatment. we tested post hoc whether logistic regression on the unmatched 2009 pandemic cohort would have resulted in a significant point estimate for an effect of statins on mortality. we used a logistic model with backward deletion (and all first-order covariates). because statin treatment was not a covariate selected through stepwise selection, we forced statin treatment into the final model. in a final model that included age, sex, and renal disease as covariates, the adjusted odds ratio (or) for statins was not significant (or = 0.74; 95% ci, .52-1.07). a sensitivity parameter and corresponding bounds were calculated for the observed point estimate from the 2007-2008 matched sample. the results (gamma = 1.47; maximum p value = .049) indicate that the point estimate is sensitive to hidden bias. an unmeasured confounder that could explain a 47% difference in the odds of statin treatment between groups could explain the observed association between statin use and mortality. sensitivity analysis for the point estimate for 2009-2010 data was not calculated due to the lack of an observed significant effect. in this analysis, using data from a population-based laboratoryconfirmed influenza hospitalization surveillance platform, we evaluated the effects of statin use over 2 influenza seasons. we found that statins had no statistically significant effect on mortality during the 2009 pandemic season, but there was a significant statin effect on reducing mortality during the 2007-2008 b other chronic diseases is a combination of any underlying chronic illnesses mentioned in patient medical records other than cardiovascular disease, chronic metabolic disease, chronic lung disease, renal disease, asthma, and obesity. c any antivirals administered during the course of illness. influenza season. the differential impact of statin treatment on mortality between seasons could be due to differences in circulating strains, because the predominant influenza a virus subtype in 2007-2008 season was h3n2, whereas influenza a(h1n1)pdm09 virus predominated during the pandemic period. age group-specific attack rates among different influenza subtypes can vary, and differences in immune histories by age could have an impact on the different results between the 2007-2008 season and the pandemic. statins have been found to modulate anti-inflammatory effects [30] ; whether the difference in apparent efficacy of statins between the 2 seasons could be explained by variation in age-specific immune histories or variation in the degree of cytokine dysregulation caused by the different influenza virus subtypes is an area for additional investigation. however, on further examination, the latter association was measurably sensitive to bias and therefore could reflect omissions of covariate measurement rather than an actual relationship between statin treatment and death after influenzaassociated hospitalization. data from the 2007-2008 season was previously analyzed using a multivariable logistic regression model [23] . results from that analysis showed that after controlling for demographic characteristics, underlying medical conditions, vaccination, and antiviral treatment, the use of statins reduced the odds of death (adjusted or, 0.59; 95% ci, .38-.92). however, a limitation of observational studies is the lack of random treatment assignment, which can result in sizeable differences in the distribution of covariates between treatment groups. we sought to balance the covariates between the statin-treated and untreated groups by using propensity score analysis. nonetheless, we also found a protective effect of statins on death among laboratoryconfirmed influenza patients hospitalized during the 2007-2008 season. questions persist about the efficacy of statin medications in reducing severe complications of influenza because we showed that our findings could be driven by unmeasured biases. four other studies that looked at statin use and influenza outcomes also found equivocal results. two of them examined the impact of statins in reducing severe disease in adults hospitalized with laboratory-confirmed influenza during the 2009 h1n1 pandemic [22, 31] . neither found a significant association between use of statins and severe disease (classified as either intensive care unit admission or death), although both were potentially hampered by small sample size. two studies evaluating statin use during multiple influenza seasons in the decade before the 2009 pandemic found a protective effect on influenza mortality, although 1 of the studies found only a modest (10% reduction in deaths from pneumonia) effect [24, 25] . these latter 2 studies relied entirely on administrative claims data; patients identified with influenza were not laboratory-confirmed, raising concern for misclassification of outcome, and may have been additionally biased by misclassification of exposure, because both studies abstracted data on previous history of statin use but not actual use at the time of the influenza hospitalization. information about whether there is benefit to initiating statin use in patients without other indications for statins at the time of influenza diagnosis would certainly be of clinical and public health interest. in this context, randomized controlled trials (rct) that address initiation of statin treatment in patients with influenza would offer clear advantages. recent rct data have been pessimistic regarding the role of statins in modulating inflammatory responses in infectious disease processes. one rct observed no clinical effect of statins on 28-day mortality among patients with ventilator-associated pneumonia [32] ; another showed that statins did not improve clinical outcomes among patients with acute respiratory disease syndrome associated with sepsis [33] . in contrast, a recent in vitro study showed that statin treatment can protect host cells against influenzainduced inflammation by reducing the production of tumor necrosis factor-α, interleukin-8, and interferon-γ, and therefore inhibit influenza a virus replication [34] . further studies to evaluate the effect of immunomodulatory agents in reducing influenza-related complications may still be warranted, but they may be better suited for settings where these drugs are not used widely. this study has several limitations. we could not determine whether a patient's statin treatment continued throughout the 30-day follow-up period because we only had medical data for the period of time the patient was hospitalized. the length of exposure to statins before hospitalization was not measured nor was the dose or frequency of statin use before or during hospitalization, which could have a possible effect on the outcome. in addition, identification of death after hospital discharge via data linkage between the ssdi and eip data could result in an underdetection of deaths; the probabilistic linkage algorithm used is highly accurate, but it is not a match-merge of data by, for example, a unique identification number. however, there is no reason to expect that either mortality status would be misclassified or that deaths would be undetected as a result of treatment status or any particular covariate. emerging infections program influenza surveillance sites do not collect detailed data on socioeconomic status or baseline functional status, and data on bmi were not collected during the 2007-2008 influenza season. certainly, an unmeasured covariate such as insurance coverage could hypothetically increase the likelihood of statin treatment prehospitalization while simultaneously reducing the likelihood of death after hospitalization. healthy user bias may certainly apply to the current study as well as most studies cited above [22] [23] [24] [25] 35] . patients being treated with statin medication have been found to have better access to preventive services such as screening services and vaccinations, and therefore they may be healthier at baseline than patients not taking statins [36] . moreover, those with limited access to care would be more likely to be hospitalized later in the course of illness, and they would have reduced opportunities to respond to medical interventions. during an influenza pandemic, we may have very few tools to prevent and treat influenza virus infection and therefore reduce mortality, because vaccine development can occur very late in the course of the pandemic and antivirals may be in limited quantity and of unknown effectiveness to a novel strain. our study results do not find a definite protective effect of statins on influenzaassociated death. promotion of the use of statins as part of public health pandemic preparedness or for an individual patient's benefit is not warranted based on current available data. influenza illness and hospitalizations averted by influenza vaccination in the united states influenza-associated hospitalizations in the united states estimates of deaths associated with seasonal influenza -united states influenza and the winter increase in mortality in the united states, 1959-1999 cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study oseltamivir and risk of lower respiratory tract complications in patients with flu symptoms: a meta-analysis of eleven randomized clinical trials outcomes of adults hospitalised with severe influenza antiviral therapy and outcomes of influenza requiring hospitalization in ontario treatment with neuraminidase inhibitors for critically ill patients with influenza a (h1n1)pdm09 impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza a(h1n1) pandemic: a systematic review and meta-analysis in hospitalized patients patients hospitalized with laboratory-confirmed influenza during the 2010-2011 influenza season: exploring disease severity by virus type and subtype pandemic influenza: a potential role for statins in treatment and prophylaxis how will physicians respond to the next influenza pandemic? beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide statins in the intensive care unit do statins have a role in preventing or treating sepsis? the effect of statin therapy on infection-related mortality in patients with atherosclerotic diseases prior statin therapy is associated with a decreased rate of severe sepsis statins and sepsis in patients with cardiovascular disease: a population-based cohort analysis statins for the prevention and treatment of infections: a systematic review and meta-analysis pre-admission statin use and inhospital severity of 2009 pandemic influenza a(h1n1) disease association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study influenza and copd mortality protection as pleiotropic, dose-dependent effects of statins influenza morbidity and mortality in elderly patients receiving statins: a cohort study an introduction to propensity score methods for reducing the effects of confounding in observational studies performing a 1:n case-control match on propensity score observational studies openepi: open source epidemiologic statistics for public health statins as a newly recognized type of immunomodulator effect of immunomodulatory therapies in patients with pandemic influenza a (h1n1) 2009 complicated by pneumonia effect of statin therapy on mortality in patients with ventilator-associated pneumonia: a randomized clinical trial rosuvastatin for sepsis-associated acute respiratory distress syndrome protective effect of fluvastatin on influenza virus infection association between statins and mortality adherence to lipidlowering therapy and the use of preventive health services: an investigation of the healthy user effect we thank the following personnel: erin parker, mph potential conflicts of interest. w. s. reports personal fees from merck and personal fees from sanofi-pasteur during the conduct of the study; j. m. reports grants from the cdc during the conduct of the study; p. r. reports grants from the cdc during the conduct of the study; and s. m. z. reports grants from the cdc during the conduct of the study. all authors have submitted the icmje form for disclosure of potential conflicts of interest. conflicts that the editors consider relevant to the content of the manuscript have been disclosed. key: cord-000131-ugbwvy6j authors: jones, james holland; salathé, marcel title: early assessment of anxiety and behavioral response to novel swine-origin influenza a(h1n1) date: 2009-12-03 journal: plos one doi: 10.1371/journal.pone.0008032 sha: doc_id: 131 cord_uid: ugbwvy6j background: since late april, 2009, a novel influenza virus a (h1n1), generally referred to as the “swine flu,” has spread around the globe and infected hundreds of thousands of people. during the first few days after the initial outbreak in mexico, extensive media coverage together with a high degree of uncertainty about the transmissibility and mortality rate associated with the virus caused widespread concern in the population. the spread of an infectious disease can be strongly influenced by behavioral changes (e.g., social distancing) during the early phase of an epidemic, but data on risk perception and behavioral response to a novel virus is usually collected with a substantial delay or after an epidemic has run its course. methodology/principal findings: here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the influenza a(h1n1) outbreak during the first few days of widespread media coverage (april 28 may 5, 2009). we find that after an initially high level of concern, levels of anxiety waned along with the perception of the virus as an immediate threat. overall, our data provide evidence that emotional status mediates behavioral response. intriguingly, principal component analysis revealed strong clustering of anxiety about swine flu, bird flu and terrorism. all three of these threats receive a great deal of media attention and their fundamental uncertainty is likely to generate an inordinate amount of fear vis-a-vis their actual threat. conclusions/significance: our results suggest that respondents' behavior varies in predictable ways. of particular interest, we find that affective variables, such as self-reported anxiety over the epidemic, mediate the likelihood that respondents will engage in protective behavior. understanding how protective behavior such as social distancing varies and the specific factors that mediate it may help with the design of epidemic control strategies. an ongoing outbreak of novel influenza a(h1n1), colloquially referred to as ''swine flu,'' has caused over 200,000 confirmed cases (as of 28 august 2009 [1] ). because of under-reporting, the actual number of people infected is substantially larger, particularly considering that many countries have ceased testing for a(h1n1) [1] . as human-to-human transmission became widespread in at least one region of the world, who rapidly declared the outbreak an imminent pandemic [2] and with widespread human infection, who declared a phase 6 pandemic on 11 june 2009, where it remains at the time of submission [3] . the virus appears to have a higher reproduction number and somewhat higher case fatality ratio than recent seasonal influenza viruses [4, 5] , and has certainly caused great concern in the population, fueled by both extensive media coverage and an initially high level of uncertainty about mortality rates and transmissibility of the virus. mathematical and computational models are useful for predicting the fate of an epidemic, and while such models have become increasingly complex and realistic in recent times, a key ingredient is often ignored: human behavioral responses to the threat and/or presence of a disease [6] . how people assess risk of infection and how such risk assessment drives behavioral change is of great interest as individual social distancing can greatly affect the spread of an epidemic [7, 8, 9] . while the effect of behavioral change in response to perceived health threats on the spread of infectious diseases has been investigated theoretically for some time, particularly in the context of sexually transmitted diseases [8] , recent work has started addressing the problem in a broader context that is also applicable to the spread of influenza [6, 7] . this work has a strong, though as yet under-explored relationship to work on risk perception and health threats [10, 11, 12] . data on risk perception and behavioral response in the general population have rarely been collected right from the outset of an epidemic. instead, they are usually gathered with a substantial delay in the case of influenza a(h1n1) [13] , after the epidemic has run its course, as in the case of sars [9] , or before sustained human to human transmission is established, as in the case of highly pathogenic avian influenza a(h5n1) [14] . however, the feasibility of halting or mitigating the spread of an infectious disease is highest during the very early phases of an outbreak, and thus data on behavioral response during this time would provide valuable information for public health policy and research. here, we report the results from an online survey that gathered data (n = 6,249) about risk perception of the outbreak during the first few days of widespread media coverage (april 29 -may 5, 2009) of the emergence of novel swine-origin influenza a(h1n1). the research presented here was approved by the stanford university non-medical human subjects institutional review board on 28 april 2009 (protocol #16782). we fielded in internet-based survey starting on 29 april 2009 using opinio survey software [15] . the url for the survey is (https://opinio.stanford.edu/opinio/s?s = 1403). the sampling universe for this study was adults 18 and older with access to a networked computer. the initial seed for the sample was generated using social networking software, and a request sent to a standing subject pool comprised of stanford alumni and social science students at a nearby community college maintained by the institute for research in the social sciences at stanford university. the survey was picked up by a variety of internet media sources including several science general media blogs. directly following publicity in these blogs, we received the most responses. table 1 summarizes the sample. the survey was designed to get a rapid assessment of respondents' affective state, sources of information on the emerging pandemic, and the behaviors undertaken for protection while minimizing respondent burden. as such, it included only 17 questions. questions probing subjective assessment of risk perception, level of anxiety, and ability to avoid flu infection were asked on a 9 point ordinal scale with anchors at the extrema (''very high'', ''very low'') and the center (''intermediate''). subjective emotional status (i.e., respondents' affective state regarding the epidemic) was anchored by the terms ''very calm'' through ''intermediate'' to ''very anxious.'' comparative questions of subjective risk percep-tion for eight health threats were asked using a five-point ordinal scale with anchors at all points: ''very low,'' ''low,'' ''intermediate,'' ''high,'' and ''very high.'' questions regarding media (both respondents' frequency of getting information from a particular source and their judgment of each source's accuracy) were asked on a five point ordinal scale with anchors at all points (''very often/ accurate'' to ''never/almost certainly inaccurate''). respondents' knowledge of swine flu was assessed with a series of six true/false questions. respondents gave free-text responses to questions about their current age, the number of people currently living in their household (including themselves) and their zip code if they currently live in the united states. respondents who reported not currently living in the united states were asked to report their current country of residence in a free-text field. a screen-shot of the full survey instrument is included in the supplementary material. for our analysis of participants' response to the threat of swine flu, we use a variable we call ''survey day.'' the survey went online in the evening of 28 april, pacific time, so we combined responses from 28 and 29 april into a single day. this combined day of 28-29 april represents survey day 1. subjects were asked to state the number of contacts in the past 24 hours. contacts were defined by close physical contact as operationalized by a face to face conversation of more than two words in the presence of another individual or physical exposure involving skin contact such as a handshake, hug, or contact during sporting activities. respondents were provided five ordered categories: less than 5, 5-10, 11-20, 21-50, 51-100, more than 100. handcock and jones [16] discuss the phenomenon of heaping and related problems for statistical inference in answering epidemiological questions regarding contact number. structuring responses within broad ordinal categories avoids many of the pitfalls of contact-heaping encountered in epidemiological investigations. to measure the response in epidemiologically relevant behavior to information on the potential pandemic, we asked a series of questions about protective actions taken by the respondents. in the survey, we asked: ''given the current status of the epidemic, which of the following precautionary actions will you take?'' avoid people who cough/sneeze avoid large gatherings of people wash hands more often avoid people who are in contact with infected people avoid public transportation avoid school/work avoid travel to infected areas use disinfectant wear a mask not all of these behaviors are necessarily effective or recommended protective measures (e.g., wearing a mask), but our aim was to gauge people's attempt at self protection so even non-efficacious behavioral change is interesting in that it indicates willingness to act on the part of the respondent. we constructed an index of protective behavior by summing the answers to the questions regarding actions taken to avoid influenza infection. the index ranged from 0-9, with larger values indicating more protective measures taken. using a binomial glm with canonical logit-link, we modeled the protection index as a function of covariates. our primary interest was the possible mediating effect of affective variables on action taken to protect against swine flu infection. to evaluate the hypothesis that respondents' affective state (subjective anxiety, fatalism about infection) predicts protective measures, we include in the model demographic (age, gender), epidemiological (household size, number of contacts, survey day), and media (source of information on the outbreak) conditioning variables. for the media variables, we constructed dummies with a value of 1 corresponding to answers of ''very often'' or ''often'' and a value of 0 for all other responses to the question of ''how often do you use the following sources to get information about swine flu?'' with such a large number of conditioning variables, in addition to the affective variables of greatest interest, there is a distinct danger of overfitting the glm. to address this problem, we used likelihoodbased model selection [17] to search the model space set up by our conditioning variables [18] . of the nine protective behaviors, increased hand-washing is both the simplest and probably most effective at curbing transmission. as such, it is strongly advocated in infection control educational material [19] . in addition to our tests for predictors of the protection index, we therefore also tested the effect of measured covariates on the odds of increased hand washing using a binomial glm again with canonical logit-link. a concern regarding the relationship of people's self-reported anxiety and their protective behavior is that some people might generally be more anxious regarding health. we probed general anxiety by asking about respondents' anxiety with regard to a number of infectious, chronic, and violent threats to health. we asked a series of questions probing respondents' perceived subjective risk on a 5-point scale for a variety of health threats, including other infectious diseases (a(h5n1) ''bird flu'', seasonal flu, hiv/aids), chronic diseases (heart disease, diabetes, cancer), and violence (unintentional accidents, terrorism). we calculated the correlation matrix for answers to these threat questions and used principal components analysis (pca) to explore potential structure in the responses to different categories of threat [20] . we begin by presenting descriptive results of the survey and follow with our primary analytical questions from the survey, namely, testing the hypothesis that respondents' affective state mediates their protective action. we gathered 6,249 responses from 28 april to 5 may 2009. table 1 presents descriptive statistics of the sample. figure 1 presents the distributions of respondents' contacts within the 24 hours prior to taking the survey. figure 2 presents the means of the subjective threats. swine flu had a mean second only to injury, and the highest among the infectious sources of threat. the mean of perceived threat from swine flu fell above the bonferroni-corrected 95% confidence interval for all other threats but unintentional injury. figure 3 presents the frequency distribution of perceived personal risk. there is a notable bimodality to this plot. this apparent bimodality is not simply attributable to sampling error since the difference between the responses = 4 vs. those = 5 vs. those = 6 is in excess of 300. further analysis using finite mixture models [21] provides strong statistical support for the reality of the bimodal pattern (results not shown). while the majority of respondents felt that their personal risk was low, there is a second mode rating their risk as intermediate ( = 5) . this same bimodal pattern can be seen in the frequency distribution of personal empowerment (i.e., ability to avoid infection) shown in figure 4 . while most respondents indicate that they are confident they can avoid infection, a substantial second mode appears at the intermediate value. figure 5 shows the frequency distribution of protective behaviors. we can see that nearly 80% of respondents report washing hands more frequently, while very few avoid work or school or wear protective masks. figure 6 shows the means for respondents' information sources. not surprisingly, the most common source of information reported was the internet. again, mean values are plotted with their 95% bonferroni-corrected confidence intervals. with the exception of social-networking tools (e.g., facebook, twitter), all other media sources are statistically indistinguishable from each other, with the social-networking tools being used significantly less. the results of the model for the protection index show a number of robust trends (table 2). in particular, we find that age increases and male gender decreases the protection index. receiving a large amount of information from the internet, television, and health officials all increase the protection index while receiving large amounts of information from print media, friends, or social networking media has no effect. the number of household members has no discernible effect, though the number of contacts outside the home does. for the ordered factor ''contacts,'' the first category (,5 contacts in the past 24 hours) is the reference category. interestingly, relative to respondents with the fewest number of contacts, all other contact categories have reduced protection indices, indicating that people with fewer contacts take more protective actions. not surprisingly, residence in mexico has a large positive effect, while residence in canada or europe decreased the index. the day that the survey was taken (29 april = 1) had a negative effect on the index, indicating that respondents took less protective action as the epidemic proceeded. respondents' reported subjective anxiety has a substantial impact on the index with high anxiety increasing protection, supporting our hypothesis that affective state mediates protective behavior. increased hand-washing showed similar trends to the model for the protection index (table 3) . male gender decreases while age and survey day increase the odds of increasing hand-washing. receiving a large amount of information from the internet, radio, television, and health officials increase, while living in europe or australia/new zealand decrease the odds. as with the overall protection index, perception of risk and subjective anxiety significantly increase the odds of increased hand-washing modestly. a key epidemiological question is how people's affective status and protective behaviors undertaken change as the epidemic proceeds. to develop a measure for this, we cross-tabulated individual values of the protection index and affective status by survey day. pearson's chi-square test for independence of both tables was strongly significant (affective: x 2 = 135.6, df = 48, p,0.001; protection: x 2 = 113.1, df = 54, p,0.001), indicating substantial departure of cells from the expected values. to visualize the pattern of departure from the expected values, we calculated an expected tables taken as the cross-product of the marginals of the observed table normalized by the grand sum. we combined rows of these tables to simplify the presentation, plotting the difference between observed and expected tables for a high, medium and low emotional status/protection index respectively. for example, a value of 251 on the calm affective status on day one means that there were 51 fewer responses in the calm categories than would be expected by the overall marginal distribution of responses across all days. in figures 7 and 8, we plot the change in respondent's protective behavior and emotional status over the first week of the survey. the lines represent the differences between observed and expected frequencies of responses for the 9-point scale simplified to three levels each. we see that by day three of the survey (may 1st), the relative number of people reporting a calm emotional state was very high, while the number of people reporting high values of the protective index declined dramatically. we interpret these results to indicate that people's response to a potential pandemic is quite sensitive to media reports. in general, individuals' survey responses to perceived risk for the eight health threats were only moderately correlated, with pairwise correlations typically well under 0.5. pca did not reveal that a substantially reduced number of dimensions explained these correlated data -six principal components were required to explain 85% of the variance. nonetheless, some intriguing pc loadings revealed themselves. in particular, the second pc, which explained 15.6% of the variance in the data, showed strong positive correlations with swine flu (r = 0.516), bird flu (r = 0.530), and terrorism (r = 0.467). all three of these threats receive a great deal of media attention and their fundamental uncertainty are likely to generate an inordinate amount of fear vis-a-vis their actual threat [22] . our results indicate that respondents' behavior varies systematically with covariates from demographic, epidemiological, media, and affective domains. people's anxiety about swine flu and the preventative actions they took to avoid infection declined as the perceived gravity of the novel outbreak waned. overall, subjective risk perception was low and people's belief in their ability to avoid infection was high. both of these distributions nonetheless showed a marked bimodality, with a large proportion of respondents indicating a higher subjective risk and more protective actions taken than the majority (figures 3-4) . the results of our statistical modeling suggest that respondents' deployment of protective behavior is mediated by their subjective anxiety level, controlling for demographic, epidemiological, and geographic variables. there is good and bad news in this result. the literature on risk perception and public health shows that there is generally a very weak correlation between people's anxiety over a particular risk and the probability of death or disability arising from that risk [11, 12] . overall, it is unclear whether anxiety over perceived risk will lead to efficacious protective behaviors [10] . this said, by far the most common protective behavior reported in our survey was increased hand-washing, which has been shown to be effective at removing influenza a(h1n1) virus from subjects' hands [23] and is promoted by cdc and other health organizations as an effective infection control intervention [19] . one curious result from the model for the protection index is that people with the fewest contacts have marginally higher protection indices. there are two potential explanations for this finding: (1) individuals with small social support networks (and consequently, few contacts outside the home) are more anxious, making it more likely that they will take greater protective actions or (2) people concerned about infection and taking relatively many protective actions also reduce the number of contacts they have and therefore had a small number of contacts in the past 24 hours. the first explanation is consistent with work in social epidemiology on the role of social networks in mediating infection risk [24, 25] because of the nature of the sample, we are unable to evaluate the direction of causality that leads to this result. nonetheless, it remains an intriguing hypothesis. many questions about this novel swine-origin influenza a(h1n1) virus remain. of particular concern is the possibility that the virus could mutate during the flu season in the southern hemisphere and selection could drive it to become more virulent as it returns to the northern hemisphere in autumn. worryingly, such a pattern of multiple waves with an increased proportion of the total influenza-associated mortality burden has been reported for all three past influenza pandemics [26, 27] . finding a means to simultaneously communicate to the public the structural uncertainty inherent in projecting pandemics and the seriousness of a pandemic after the media frenzy about its emergence has died down remains a major challenge to public health. pharmaceutical interventions such as vaccines and antiviral drugs may form a strong line of defense, but the efficacy of such measures remains unclear and depends on the particular biology of a given pathogen. this is exacerbated by people's reluctance to be vaccinated [28] . with more than 300 infectious diseases emerging within less than a century [29] , the threat of pandemic influenza is only the latest out of many public health threats posed by infectious diseases in a globalized world. unlike pharmaceutical interventions, non-pharmaceutical interventions like social distancing may be effective in halting or at least mitigating an epidemic independent of the specific biology of a pathogen, and thus provide a reliable set of strategies to combat infectious diseases that warrant further attention [30] . our results that people do not rely on social networking tools to the extent that they rely on other media may have implications for cdc strategies for spreading public health information via social media [19] . in particular, public health messages spread via social media will need to backed up by information spread via more traditional channels, which respondents list as being common sources of trusted information on the outbreak. our study is subject to a number of weaknesses. the advantage of our internet-based sampling strategy is the ability to quickly deploy a survey and thereby track responses in near real-time. the clear disadvantage of this strategy is a sacrifice of population representativeness. despite its general availability on the internet, our sample shows a pronounced bias for highlyeducated respondents living in the western united states. these biases clearly limit the generality of our results, but the large number of respondents filing out the survey as information on the potential pandemic changed nonetheless provides a uniquely valuable data source. within one week (the cutoff point for the current analysis), we had a sample of 6,249 responses. in contrast, the telephone-based study of rubin et al. [13] employed a random-digit-dial sampling design, allowing a more representative sample of the general uk population, but their sample was only 997 respondents and the survey was undertaken after media attention had abated, beginning 8 may 2009. nonetheless, the results reported in this paper are largely congruent with our own results and we see the studies as strongly complementary. our respondents began filling out the survey on the day that who upgraded the pandemic threat category of the h1n1 outbreak from 4 to 5 and spans the week in which the number of who-confirmed cases increased tenfold. while our sampling design is subject to many of the usual criticisms of internet-based surveys and is not necessarily representative of the general population, the unparalleled immediacy, longitudinal nature, and the large number of respondents it contains make our data set unique and scientifically important for the study of the spread of information and distribution of risk perception and 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influenza vaccination at different who alert levels: two questionnaire surveys global trends in emerging infectious diseases interim pre-pandemic planning guidance: community strategy for pandemic influenza mitigation in the united states-early, targeted, layered use of nonpharmaceutical interventions. atlanta: department of health and human services, cdc we thank chris thomsen, karen cook, and especially vijoy abraham from the stanford institute for research in the social sciences for help getting the survey online so quickly. dan salkeld provided helpful comments. we are also very grateful to two anonymous reviewers for substantially improving the original manuscript. conceived and designed the experiments: jhj ms. analyzed the data: jhj ms. wrote the paper: jhj ms. key: cord-001154-7k59ogn0 authors: memoli, matthew j.; athota, rani; reed, susan; czajkowski, lindsay; bristol, tyler; proudfoot, kathleen; hagey, rachel; voell, jocelyn; fiorentino, charles; ademposi, angela; shoham, shmuel; taubenberger, jeffery k. title: the natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts date: 2013-11-01 journal: clinical infectious diseases doi: 10.1093/cid/cit725 sha: doc_id: 1154 cord_uid: 7k59ogn0 introduction. medical advances have led to an increase in the world's population of immunosuppressed individuals. the most severely immunocompromised patients are those who have been diagnosed with a hematologic malignancy, solid organ tumor, or who have other conditions that require immunosuppressive therapies and/or solid organ or stem cell transplants. materials and methods. medically attended patients with a positive clinical diagnosis of influenza were recruited prospectively and clinically evaluated. nasal washes and serum were collected. evaluation of viral shedding, nasal and serum cytokines, clinical illness, and clinical outcomes were performed to compare severely immunocompromised individuals to nonimmunocompromised individuals with influenza infection. results. immunocompromised patients with influenza had more severe disease/complications, longer viral shedding, and more antiviral resistance while demonstrating less clinical symptoms and signs on clinical assessment. conclusions. immunocompromised patients are at risk for more severe or complicated influenza induced disease, which may be difficult to prevent with existing vaccines and antiviral treatments. specific issues to consider when managing a severely immunocompromised host include the development of asymptomatic shedding, multi-drug resistance during prolonged antiviral therapy, and the potential high risk of pulmonary involvement. clinical trials registration, clinicaltrials.gov identifier nct00533182. during the past half-century, medical advances have led to an increase in the world's population of immunosuppressed individuals. this includes those receiving immunosuppressive therapies, those with acquired immunosuppressive diseases such as the 34 million worldwide with human immunodeficiency virus (hiv) and aids [1] , and individuals living longer with any of the over 150 known primary immunodeficiencies [2] . the most severely immunocompromised are those who have been diagnosed with a hematologic malignancy, solid organ tumor, or who receive other immunosuppressive therapies such as chemotherapy and/or solid organ or stem cell transplants. over 100 000 individuals annually receive solid organ transplants (sot) worldwide [3] and more than 50 000 with hematologic malignancies and other diseases are treated with hematopoietic stem cell transplants (hsct) annually [4] , leaving most of these individuals immunosuppressed for long periods. even larger numbers of individuals are receiving immunosuppressive chemotherapies making immunocompromised individuals a larger part of the population than during any of the influenza pandemics of the twentieth century. epidemiologic studies have shown that severe immunosuppression is a major comorbidity that places individuals at the highest risk of severe morbidity and mortality due to influenza infection. patients with aids have increased duration of disease due to influenza and higher incidence of pneumonia leading to increased mortality [5] [6] [7] . a study of hospitalized patients with leukemia and influenza reported 80% with pneumonia and 33% mortality [8] . more recently during the 2009 pandemic, studies reported similarly high levels of lower respiratory tract disease and need for hospitalization in those with hematologic malignancies and solid tumors undergoing chemotherapy [9] . similarly, in a large retrospective study of hsct recipients, 1.3% of all patients developed influenza infection and 29% developed pneumonia [10] . a more recent study of hsct patients demonstrated that up to 75% developed pneumonia after influenza infection with mortality as high as 43% [11] . in sot, nosocomial outbreaks [12] and severe complications of influenza such as myocarditis [13] and severe pneumonitis [14] have been reported, even in those previously vaccinated. multicenter studies of a(h1n1)pdm09 infection in sot recipients have reported that 30%-40% of individuals infected developed pneumonia, 16%-17.5% required intensive care, with mortality as high as 7% [15] . influenza following sot leads to higher morbidity and mortality rates [16] , with increased rates of influenza pneumonia following lung transplantation, and a >20% mortality rate observed in sot recipients infected with influenza [17, 18] . in addition to increased morbidity and mortality following infection, severely immunocompromised patients have been reported to show prolonged influenza shedding [19] [20] [21] [22] [23] [24] [25] . this has been associated with intrahost viral evolution, including antigenic drift within a single immunosuppressed host [24] , the development of antiviral resistance after therapy [19, 21-23, 25, 26] , and simultaneous coinfection with 2 influenza subtypes [27] . rigorous vaccination programs and improved pharmacotherapy have decreased the impact of influenza on the general population; however, influenza still remains a serious threat to severely immunocompromised individuals. the 2009 pandemic was a reminder that it is still unclear how well current vaccination strategies and current pharmacotherapy can perform in preventing and mitigating illness in immunocompromised individuals. the primary goal of this study was to compare the natural history of influenza infection in those who were severely immunocompromised to individuals who were not immunocompromised. careful examination of symptoms and signs of infection, virological measurements, immunological studies, and clinical parameters were performed to investigate the natural pathogenesis of influenza in this group of severely immunocompromised hosts. subjects were recruited at the national institutes of health (nih) clinical center in bethesda, md, and washington hospital center in washington, dc, under an irb approved protocol "influenza in nonimmunocompromised and immunocompromised hosts" (clinicaltrials.gov identifier nct00533182). all participants or legal guardian signed informed consent. medically attended patients with a diagnosis of influenza were recruited. history and physical was performed, initial diagnostic specimen collected, and further nasal washes and blood samples collected every 2 days during the first 7 days postdiagnosis. nasal washes were performed using the method described by neclerio [28] . follow-up examinations and sample collection at both 2 weeks and 4 weeks were performed. subjects who shed virus for an extended period of time were followed throughout the course of their infection, and sampling was performed until the infection resolved. clinical data were compiled and statistical analyses performed using graphpad prism software. initial diagnosis was made by the clinical laboratories at the facility where samples were collected. further samples were tested using a matrix one-step taqman real-time reverse transcription polymerase chain reaction (rt-pcr) assay as described elsewhere [29] and/or the luminex multiplex respiratory viral panel kit (luminex corp. austin, tx). viral isolation used standard mdck culture methods. type and subtype were determined by rt-pcr and sequencing of the hemagglutinin (ha) and neuraminidase (na) genes and comparison to known influenza sequences in genbank. total nucleic acid was extracted from samples using the bio-mérieux nuclisens easymag system. viral complementary dna (cdna) was reverse transcribed using the influenza specific uni-12 primer, and pcr was performed using custom primers to amplify the viral gene segments (table 1) . sanger sequencing was performed on all pcr products. serum and nasal wash cytokines were measured using the bio-plex pro human cytokine 17-plex assay on a bio-rad bio-plex 200 (bio-rad hercules, ca). the following cytokines were measured: interleukin 1β, interleukin 2, interleukin 4, interleukin 5, interleukin 6 (il-6), interleukin 7, interleukin 8 (il-8), interleukin 10, interleukin 12, interleukin 13, interleukin 15, interleukin 17, granulocyte colony-stimulating factor (g-csf), granulocyte macrophage colony-stimulating factor (gm-csf), interferon γ, monocyte chemoattractant protein 1 (mcp-1), macrophage inflammatory protein 1β (mip-1β), and tumor necrosis factor α (tnf-α). controls used were collected from healthy volunteers. results were compiled and statistical analysis performed using graphpad prism software. eighty-six influenza patients were enrolled over a 3-year period between 2008 and 2011. of these, 32 (37.2%) were classified as severely immunocompromised, primarily due to a malignancy or condition being treated with either a recent hsct or immunosuppressive therapy. the remaining 54 patients (62.8%) were classified as nonimmunocompromised and included individuals both with and without underlying comorbidities. the demographics of these individuals are summarized in table 2 . of note, 46.5% of the individuals in the study had been vaccinated within 1 year of becoming infected with influenza. a majority of the nonimmunocompromised individuals were overweight or obese (81.4%). although most patients in the study were nonsmokers, 22.2% of nonimmunocompromised participants were current smokers, whereas there were no current smokers in the immunocompromised group. hematologic conditions were the most common underlying diagnosis (78.1%) and 59.4% of patients had undergone hsct within 1 year of influenza infection in the immunocompromised group. most had some form of graft-vs-host disease (gvhd; table 3 ). those who did not undergo hsct (21.9%) were undergoing immunosuppressive therapy (table 3 ). in the nonimmunocompromised group 17 patients (31.5%) had chronic comorbidities including hypertension, diabetes mellitus, coronary artery disease, asthma or chronic obstructive pulmonary disease, or well-controlled hiv infection. a full symptom evaluation at the time of diagnosis was performed in 80 of 86 patients ( table 4 ). the most common symptoms noted were dry cough (90%), fever (83.8%), and headache (82.5%). chills, coryza, productive cough, and sweats were also common. a range of other symptoms were observed including shortness of breath and chest pain. of interest was the prevalence of multiple gastrointestinal and neurologic symptoms. the majority of symptoms were more prevalent in the nonimmunocompromised group including those that were statistically higher: dry cough, chills, sweats, myalgia, shortness of breath, chemosis, and neurologic symptoms. gastrointestinal symptoms were slightly more common in the immunocompromised group with decreased appetite observed as the only symptom statistically more prevalent (table 4) . fifty-seven of the patients had complete physical exams documented on diagnosis, and an overall trend of physical exam abnormalities was observed more in the nonimmunocompromised group, with pulmonary abnormalities in particular being more statistically prevalent (table 5 ). pulmonary abnormalities on exam were the most common finding (44% overall). cardiac abnormalities were noted in 14% of patients, mostly consisting of tachycardia. other abnormalities were minimal, and many individuals had no significant findings. all of the immunocompromised patients required hospitalization upon diagnosis. six patients (18%) in the immmunocompromised group required intensive care and mechanical ventilation. in contrast, only 16 patients (29.6%; p = .0001) of the nonimmunocompromised group required hospitalization, and none required intensive care treatment (p = .0019). there was 1 death observed in the immunocompromised group (3%). the immunocompromised patients also exhibited a significantly longer length of illness (p = .0466) with a mean shedding of 19.0 days, whereas nonimmunocompromised patients were observed to shed for a mean of 6.4 days (figure 1 ). consistent with these differences were the radiologic examinations observed. chest computed tomography (ct) or chest radiograph (cxr) was performed at the time of diagnosis on 58 of the 86 patients recruited. a majority of immunocompromised patients (87.5%) had radiologic studies performed, whereas less (55.5%) were performed on nonimmunocompromised patients. of these 58 radiologic examinations, 23 (39.7%) showed new abnormalities at the time of diagnosis. the immunocompromised patients were significantly more likely to have new imaging abnormalities with 16 of 28 (57.1%) compared to 7 of 30 (23.3%) in the nonimmunocompromised group (p = .015). viral diagnoses of the 86 patients enrolled are summarized in table 6 . of the 80 influenza a virus strains identified, 6% were seasonal h1n1, 19.7% h3n2, and 66.3% a(h1n1)pdm09 subtypes. of the 80 patients identified as having influenza a infection, 9 (11.3%) of these patients either were infected with a neuraminidase inhibitor resistant virus or developed resistance during the course of infection and treatment. when adamantane resistance is considered, 100% of the influenza a isolates collected in this study were resistant to at least one class of antiviral drug, and 3 patients (4%) had viral infections developing resistance to both available classes of antivirals ( table 6 ). the a (h1n1)pdm09 virus was the most prevalent subtype of influenza a virus isolated (66.3%), and as expected, all of these viruses were resistant to the adamantanes as were the h3n2 viruses. all 6 of the seasonal h1n1 isolates identified contained the h275y nai resistance mutation conferring resistance to oseltamivir and possibly peramivir [22] . this mutation was noted at the time of initial identification of virus and remained stable in all consecutive isolates collected from those patients. three immunocompromised patients were infected with influenza viruses that developed neuraminidase inhibitor resistance during treatment. one patient with an h3n2 infection developed a novel deletion mutation in the na gene. the other 2 cases were a(h1n1)pdm09 infections that developed h275y mutations. in all of these cases the mutations arose after treatment with oseltamivir and in one case peramivir during a prolonged clinical course [21] [22] [23] . cytokine response during acute infection was measured both from serum and nasal washes. of the cytokines measured il-6, il-8, tnf-α, g-csf, gm-csf, mcp-1, and mip-1b were found to be elevated in serum and/or nasal wash compared to controls during infection (figures 2 and 3) . overall, no significant differences were noted in these responses between the immunocompromised and non-immunocompromised groups. influenza infection has a major effect on the population in all regions of the world, differing from season to season and emerging unpredictably during a pandemic. the patient population identified in this study reflects the population of the washington, dc, metropolitan region with the majority of subjects identifying themselves as either white or black. given the nature of a prospective study of immunocompromised patients, it was not possible to have extremely well matched comparator groups, but they were reasonably well matched ( table 2) . most participants were between the ages of 18 and 65 with a mean age of 25.55. this mean age of approximately 25 is consistent with the fact that the 2009 a(h1n1)pdm09 virus was the most prevalent virus identified in this study, and that much of this study was performed during the 2009 pandemic and postpandemic period when a slightly younger population was more affected by influenza than during a typical influenza season [30] a number of significant differences were observed between the severely immunocompromised and nonimmunocompromised groups. the immunocompromised patients experienced more hospitalizations, longer lengths of influenza virus shedding, and more severe disease and complications requiring intensive care and mechanical ventilation. these differences highlight that not only are these patients an important at-risk group for enhanced morbidity and mortality but also should be considered a group that could contribute to viral spread in the general population. the immunocompromised patients in this study shed virus for a mean of approximately 19 days, likely making them contagious for a significant length of time. many of these patients were asymptomatic for a significant portion of the time in which they were shedding. importantly, in a number of these cases the viruses they were shedding had developed drug resistance mutations while maintaining their transmissibility [21, 23] , making it possible that these individuals could also be a source for community spread of drug resistant viral strains. although many of the immunocompromised patients were asymptomatic during some portion of the time they were infected, most exhibited some typical influenza symptoms during the first few days of infection. this was consistent with the cytokine measurements observed, as the acute innate immune response is likely principally responsible for many of the acute symptoms observed. cytokine measurements in nasal wash specimens demonstrated similar cytokine responses in both the immunocompromised and nonimmunocompromised groups. these cytokine elevations were consistent with what has been reported previously during influenza infection [31] . in serum, similar elevations were noted in both groups as well, although to a somewhat lesser degree when compared to noninfected controls. despite the similarity in acute innate response between the 2 groups, important differences were noted in overall symptoms between the immunocompromised and nonimmunocompromised groups. fewer overall symptoms were noted in the immunocompromised group, and all influenza symptoms were found more commonly in the nonimmunocompromised group except for gastrointestinal symptoms, that were likely related to underlying illnesses or therapeutics as many of these immunocompromised individuals suffered from gvhd of the gut or were receiving a toxic chemotherapeutic agent. the overall trend of the more inflammatory and systemic symptoms occurred more often in the nonimmunocompromised group suggesting that although the innate immune response is similar, the clinical manifestations may not be, and the possibility of a blunted illness should be taken into account when evaluating immunocompromised patients with an influenza-like illness. a significantly higher number of radiographic abnormalities were noted in the immunocompromised group, pointing to their increased risk of pulmonary involvement and complications. these radiologic changes were often in the absence of pulmonary abnormalities on physical exam, which were more prevalent in the nonimmunocompromised group. this is important to consider clinically as the presence or absence of symptoms and signs may not be adequate indicators of disease, especially in immunocompromised individuals who are likely at higher risk of pneumonia, secondary bacterial infections, and other pulmonary complications. vaccination remains the cornerstone of prevention for influenza infection and its complications. in this study, approximately 47% of all individuals recruited had been vaccinated within the last year prior to infection yet still became ill with influenza. a larger number of the nonimmunocompromised (59%) were vaccinated compared to only 25% of the immunocompromised. this fact makes this observation even more concerning as a significant number of immunocompetent individuals became infected despite a vaccine that closely matched the infecting virus. the observations of vaccination made in this study are somewhat consistent with recent observations by the cdc that during recent years the effectiveness of the influenza vaccine, especially in certain groups of individuals has been moderate to poor [32] . clearly influenza vaccines have variable efficacy that is hard to predict, especially when factoring in emerging viruses and special populations. this highlights that further work needs to be undertaken to find novel vaccine/prevention strategies to reduce the disease burden in the general healthy population and especially in special populations such as the severely immunocompromised, where vaccination is even further hampered by the inability of the host to respond. this may require reconsidering prevention of disease as our goal and shifting our focus to reducing severe disease and complications such as the prolonged shedding and severe illness observed in the immunocompromised patients in this study. when vaccines do not prevent infection or are not given, the last step in preventing morbidity and mortality in influenza is therapeutic treatment, of which antivirals are the current gold standard. the prevalence of antiviral drug resistance found in this and other studies is a warning that should not be ignored. consistent with recent observations, all influenza viruses identified in this study were resistant to at least one class of antiviral. in this group of patients, 4% of viruses were resistant to both major classes of antivirals including some isolates of h3n2 and a(h1n1)pdm09. the majority of these viruses were identified in immunocompromised patients after they had received prolonged courses (beyond 5 days) of antiviral therapy. these data in conjunction with observations that these viruses are transmissible and not attenuated in animal models [21, 23] suggest that the development of multi-drug resistance is a significant concern and that immunocompromised patients may be at risk of acting as hosts for the development and community spread of resistance. further research, the development of novel therapies, and reevaluation of how immunocompromised individuals are treated for influenza should be undertaken. study of the natural history of influenza is extremely difficult because in almost all cases it is impossible to identify exactly when a patient was infected. although a relatively small cohort was observed in this study, it is one of the largest cohorts of severely immunocompromised individuals with influenza infection studied prospectively to date. the comparison of these individuals with nonimmunocompromised individuals during influenza infection demonstrated that the immunocompromised patients are at risk of more severe or complicated disease, which may be difficult to prevent with current vaccines and treat with current antivirals. specific issues to consider when managing severely immunocompromised hosts include the development of asymptomatic shedding that could increase the risk of transmission, the development of multi-drug resistance during prolonged antiviral therapy, and the potential high risk of pulmonary involvement of their disease leading to secondary infections or complications. a better understanding of the basic human pathogenesis of influenza will be necessary if we are to address how to prevent and treat influenza in a more individualized way, especially for those who are immunocompromised. it is clear that current antivirals are not adequate in all circumstances, current vaccine strategies must be improved, and careful evaluation of each individual case may be necessary to tailor treatment and prevention for not only severely immunocompromised individuals but likely those in other important at-risk populations. global hiv/aids response: epidemic update and health sector progress towards universal access incidence and temporal trends of primary immunodeficiency: a population-based cohort study transplantation immunology: solid organ and bone marrow hematopoietic stem cell transplantation: a global perspective excess mortality due to pneumonia or influenza during influenza seasons among persons with acquired immunodeficiency syndrome influenza in human immunodeficiency virus-infected patients 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maryna eichelberger, division of viral products, center for biologics evaluation and research, food and drug administration for her contribution to the preparation of this article.financial support. this work was supported by the intramural research program of national institute of allergy and infectious diseases. dr memoli was the lead scientist for this work leading all aspects including the design, clinical protocol implementation, laboratory studies, and data analysis/manuscript preparation.potential conflicts of interest. key: cord-005314-p7hzoz5d authors: wong, li ping; sam, i-ching title: knowledge and attitudes in regard to pandemic influenza a(h1n1) in a multiethnic community of malaysia date: 2010-09-11 journal: int j behav med doi: 10.1007/s12529-010-9114-9 sha: doc_id: 5314 cord_uid: p7hzoz5d background: assessment of general public’s knowledge and attitudes toward the development and prevention of new disease outbreaks is imperative because they have profound effects on health behaviors and may contribute to the control of the epidemic. purpose: to investigate the level of knowledge and attitudes towards the influenza a(h1n1) outbreak across various ethnic groups and socio-demographic backgrounds in malaysia. method: a cross-sectional, population-based, computer-assisted telephone interview exploring knowledge and attitudes regarding influenza a(h1n1) was conducted in malaysia. between july 11 and september 12, 2009, a total of 1,050 respondents were interviewed (response rate 69.3%). results: the mean total knowledge score for the overall sample was 7.30 (sd ± 1.961) out of a possible score of 13 (chinese had the highest scores, followed by indians, then malays). some erroneous beliefs about the modes of transmission were identified. the majority of the participants (73.8%) perceived the a(h1n1) infection as often deadly. despite the overestimation of the severity of a(h1n1) infection, high confidence in preventing infection and low perceived susceptibility of infection were reported. influenza a(h1n1)-related stigma was prevalent and exhibited differences across ethnic groups. conclusions: findings suggest that provision of education and clear information are essential to correct the misconceptions, and increase perceived susceptibility to infection so that the general public will take precautions against a(h1n1) infection. in april 2009, a new strain of human influenza a(h1n1) virus appeared in mexico, and has spread rapidly around the world, causing the first pandemic for 40 years. malaysia confirmed its first case of influenza a(h1n1) on may 15, 2009 . in response to increasing local transmission, malaysia moved from containment to mitigation stage on july 9, 2009 [1] . since then, the a(h1n1) pandemic has grown exponentially. on july 23, 2009 , approximately 2 weeks after the commencement of this study, the first death from influenza a(h1n1) was reported. on september 13, 2009 , the date on which our data collection finished, the total fatality count was 74 [2] . public knowledge and attitudes of past outbreaks, such as sars and avian influenza, have been studied in many western [3] [4] [5] and asian countries [6] [7] [8] [9] [10] . it is imperative to assess the general public's knowledge and attitudes of the development and prevention of new disease outbreaks [9, 11] . during the sars epidemic, public perceptions affected health behaviors such as handwashing and wearing face masks, which in turn contributed to the control of the epidemic [11, 12] . in both the sars and h5n1 avian influenza outbreaks, public fear and level of distress was associated with perceptions and knowledge [8, 13] . currently, there are few studies on knowledge and perceptions of the new influenza a(h1n1) epidemic. a recent study in hong kong showed inadequate knowledge among the general public and misconceptions in the modes of transmission of a(h1n1) [14] . another study conducted in the uk in the early stage of the pandemic showed that risk perception and perceived seriousness of the influenza a(h1n1) were associated with the practice of recommended health behaviors [15] . an internet-based survey, conducted during who pandemic stage 5, revealed that emotional concerns about infection were significant predictors of behavioral responses to the h1n1 pandemic [16] . in the immediacy of the new emerging a(h1n1) influenza virus, we conducted a baseline survey to examine knowledge, attitudes, psychosocial consequences, and impact of the pandemic in malaysia. of note, the survey was carried out over a period of escalating reported deaths, generating much public and media concern. malaysia is a fast-developing country in southeast asia with a population of 27.7 million, which encompasses a majority malay (50.8%) and other ethnic groups, mainly chinese (23.0%), non-malay bumiputra (11.0%), and indian (6.9%) [17] . each ethnic group has its own culture and religions. this multiethnic, multi-religious society allow for a diverse exploration of cultural variation in expression of attitudes and perceptions towards the new a(h1n1) pandemic. the aim of this study was to investigate the level of knowledge and attitudes towards the influenza a(h1n1) outbreak across various ethnic groups and socio-demographic backgrounds. a cross-sectional, population-based, computer-assisted telephone interview (cati) survey was carried out between july 11 and september 12, 2009. the study population comprised malaysian adults randomly sampled from the latest electronic version (2008/2009) of the residential telephone directory of selangor state and the federal territory of kuala lumpur. kuala lumpur, the capital city and federal territory of malaysia, has a population of 1,629,400, and covers an area 243 km 2 . the state of selangor surrounds the capital city of kuala lumpur, has a population of 5,017,100 and an area of 8,154 km 2 . one person aged 18 years and older from each household was randomly selected and invited to participate. interviews were conducted between 5:30 p.m. and 10:00 p.m. on weekdays and from 12:00 p.m. to 7.00 p.m. on weekends or public holidays to avoid over-representation of unemployed participants. unanswered numbers were called at least two more times on separate days before being regarded as uncontactable. the sample was stratified by the three main ethnic groups in the malaysian population, malays, chinese, and indians. the chinese and indians were slightly over-sampled to assure that analyses of disparities could be conducted with sufficient statistical power for each of the ethnic groups. on the knowledge assessment section of the questionnaire, each respondent was first asked to list the symptoms of influenza a(h1n1). a score of 1 was given for each correctly identified symptom. the subsequent knowledge questions (six items) were posed in which the answers were either yes, no, or don't know. a score of 1 was assigned to a correct answer and a value of 0 to an incorrect answer or don't know responses. questions on perception were divided into two parts. the first part explored perceived susceptibility towards influenza a(h1n1) (three items), in which participants indicated their level of susceptibility on a four-point likert-type scale (very high, high, low, very low). the second part examined a(h1n1)-related stigma (six items), with answers of either yes or no. demographic questions (nine items) were asked after completion of the survey questions. the survey questions were adapted and modified from previous published literature [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] . the questionnaires were in three languages: bahasa malaysia (the national language), english, and chinese. a team of trained multiethnic interviewers performed the interviews; each of the ethnic group interviewers was assigned to interview respondents of a similar ethnic group. in the pilot study, interviews were conducted with 20 various ethnicity respondents from the telephone directory. the test respondents commented that the questions were easily understood, and the average completion time was 15 min. informed consent was obtained verbally. respondents were assured that their responses would be confidential and reminded that their participation in the interview was voluntary. the study was approved by the medical ethics committee, university malaya medical center, kuala lumpur, malaysia. all statistical analyses were performed using spss version 17.0 for windows software (spss, inc., chicago, il, usa). non-response and irrelevant (non-applicable) responses (for example, questions regarding pork and pork products to muslim respondents) were treated as missing values and, therefore, excluded from the analyses. t tests and one-way analysis of variance (anova) were used for comparisons of means; the chi-squared test was used to test the significance of differences in percentages. post hoc comparisons using the tukey's hsd test were conducted to evaluate pairwise differences among the means. multiple linear regression analysis using the enter method was carried out to explore relationships between knowledge scores and socio-demographic variables that had a p value less than 0.10 in bivariate comparisons. multivariate logistic regression analyses were used to determine factors associated with perceived susceptibility to a(h1n1), and perceived confidence in a(h1n1) prevention. a backward stepwise logistic regression selection method was carried out to select significant independent variables and make adjustments for covariates among variables. wald chisquared statistics were used to test the significance of individual coefficients in the model. goodness of fit was assessed with the hosmer-lemeshow test. statistical significance was set to p<0.05. figure 1 provides the flowchart of the cati process. a total of 6,230 call attempts were made, resulting in 1,050 (16.9%) responding households. the low response rate was due to the large number of uncontactable households. however, of the total successful calls (1,750), 60.0% (n= 1,050) completed the interview. by ethnic group, the proportions that completed interview upon successful calls were 64.8% (436/673), 52.3% (352/673), and 64.8% (262/ 404) for malay, chinese, and indian samples, respectively. table 1 shows socio-demographic characteristics of the 1,050 respondents in the survey. the majority of respondents in this sample were malays (41.5%), followed by chinese (33.5%) and indians (25.0%). the majority was able to name three (33.2%) or four (25.6%) influenza-like illness symptoms, and only a total of 23 respondents (2.2%) were unable to name even one of the symptoms. a fifth of respondents (20.4%) were able to name only one to two symptoms. as shown in table 2 , fever, running nose, and coughing were the three most commonly identified symptoms of influenza a(h1n1). half of the respondents (50.4%) could not identify the symptom of sore throat. the six-item close-ended knowledge questions had an inter-item correlation range of−0.043 to 0.203 with cronbach's alpha of 0.233. a considerable number of participants (n=329, 31.3%) erroneously believed that the a(h1n1) influenza can be transmissible to people through eating improperly cooked pork and pork products, the majority of whom were of malay ethnicity 47.8%, followed by indians (31.5%), and the lowest were the chinese (13.7%) [χ 2 (5, 1030)=199.60, p<0.001]. with regard to the severity of influenza a(h1n1), a considerable number of participants had the misconception that a(h1n1) infection is often deadly (73.8%). more specifically, people who were female, malay, employed, had a high income, graduated from university, and living in urban areas were more likely to perceive that a(h1n1) infection is often deadly (p<0.01). the mean total knowledge score for the overall sample was 7.30 (sd±1.961) out of a possible score of 13. statistically significant differences in the mean total knowledge score were observed among ethnic groups, gender, education attainment, employment status, average household income, and locality ( perceived susceptibility and confidence in prevention table 4 shows that 64.5% and 67.2% of the respondents, respectively, rated perceived susceptibility and perceived family members' susceptibility to a(h1n1) infection as "low" or "very low". the majority (73.0%) were also "very confident" or "quite confident" in preventing themselves from getting a(h1n1). figure 2 shows the temporal trends for all responses to "very" and "quite" confident in prevention and "very high" and "high" perceived susceptibility to infection. the first death was reported 2 weeks after the commencement of this study, and increased steadily and reached a peak in week 6, before a rapid decline. the levels of perception of "very high" and "high" susceptibility to infection did not vary significantly over the study period. in contrast, rates of those who were "very" and "quite" confident in prevention of h1n1 infection decreased significantly as the number of reported deaths increased. as indicated in with regard to perceived confidence of preventing a (h1n1) infection, females were more confident than male respondents. highly significant differences between the three ethnic groups was found; malay respondents reported the highest levels of perceived confidence in prevention of a(h1n1), followed by the indians and chinese. older respondents reported higher confidence of prevention. multiple logistic regression suggested that respondents were more likely to have high perceived confidence in prevention of a(h1n1) if they were female (or=1.61, vs. (table 4) . the internal consistency of the six-item stigma related to a (h1n1) questions, evaluated by cronbach's alpha was 0.632, inter-item correlation ranged from 0.110 to 0.389. the mean number of stigma was 1.94 (mode 1, median 2) out of a maximum of 6. as indicated in table 5 , three most commonly reported stigmas were: "afraid to be in contact with people with flu symptoms" (60.8%), "afraid to be in contact with people who have just returned from overseas" (54.1%), and "afraid of visiting hospitals" (36.2%). a minority indicated fear of eating pork and pork products (7.2%). some erroneously related the outbreak with avian influenza and expressed fear of eating birds (chicken) and bird products (7.3%). there were significant differences in the responses to stigma items among the three ethnic groups studied. on the whole, the stigmatizing responses were more common among malay ethnicity except for "afraid of visiting hospitals". among those that believed the a(h1n1) infection to be often deadly, more than half (53.2%) were not "afraid to be in contact with people with flu symptoms", two thirds (67.0%) were not "afraid to be in contact with people who have just returned from overseas" the majority of respondents were able to recognize the three common influenza-like illness namely fever, runny nose, and cough. however, less than half were able to name the symptom of sore throat. a considerable minority of respondents that was unable to name even one symptom was of concern. although many knew of the mode of transmission of influenza a(h1n1), transmission via eating improperly handled and cooked pork and pork products was misperceived by a sizable minority of respondents. this was likely due to the frequent references of a(h1n1) virus as "swine flu" in the early stages of the pandemic. compared to a recent hong kong study [14] , more respondents in malaysia thought that a(h1n1) could be transmitted by eating pork products (31.3%, vs. 6.9%), more did not know that the a(h1n1) virus is transmittable via droplets (8.7%, vs. 2.0%), while fewer did not know that the a(h1n1) virus is transmittable via contact with contaminated objects (16.8% vs. 21.1%). results of the multiple linear regression analysis using total knowledge score as the dependent variable indicated that provision of information and education should focus on the male gender, malay ethnic group, people of lower education and income, and those living in rural areas. participants who were female, malay, employed, of high socio-economic status, and living in urban areas appeared to be more likely to overestimate the severity of a(h1n1) as they believed the a(h1n1) infection as often deadly. an overestimation of the severity of a(h1n1) should be prevented as it may cause unnecessary panic and distress to the community and may lead to interference of a wide range of daily activities. thus, these specific groups of people may require targeted education [8, 10] . despite the overestimation of the severity of a(h1n1), a high proportion of the participants in this study reported low perceived susceptibility to a(h1n1) infection, which did not change significantly over the study period, despite the increasing number of deaths. because perceived susceptibility is expected to begin the process of adoption of preventive behaviors, health authorities and media should stress to the public to continuously adopt precautionary measures to prevent infection even if the number of reported deaths decreases. many participants in this study perceived high confidence in preventing themselves and family members from getting the disease. this is of particular concern because an individual may not engage in preventive behavior because of lack of perceived susceptibility to the disease [18] . of interest, this confidence declined and remained lower even after the number of reported deaths peaked. it has been shown that perceived vulnerability for sars was significantly associated with perceived threat [19] . additionally, perceptions of risk and severity were found to influence public's behavioral responses to pandemic influenza [9, 20] more research is needed to investigate why the malaysian public overestimates the severity of this new disease and yet under-perceives its risk of infection, to provide insight into how the health authorities or media should disseminate information to counteract these misconceptions in the future. the findings of our multivariate logistic regression analyses indicated that education to enhance perceived susceptibility to a(h1n1) infection should focus on people of older age and people living in suburban areas. influenza a(h1n1) related stigma was prevalent and exhibited differences across ethnic groups. finding of significant ethnic disparities in a(h1n1)-related stigma suggest further in-depth qualitative exploration on cultural values and beliefs related to each ethnic group. in this study, we also found that perceived severity of a(h1n1) infection was not associated with stigma. despite widespread information about the control measures in response to the emergence of pandemic influenza a(h1n1), in particular, near one third of the study participants (including a majority of those that believed a(h1n1) infection is often deadly) did not fear being in contact with people with influenza symptoms, and approximately 50% were not afraid to be in contact with people who had just returned from overseas. although health authorities have repeatedly emphasized that the a(h1n1) virus is not transmitted by food, a substantial minority avoided eating pork-related products for fear of infection. our recently published data on temporal changes in psychobehavioral responses during the h1n1 influenza pandemic [21] showed a noticeable increase in fear of visiting hospitals and fear of eating out in concordance with the escalating number of reported deaths. similar trends were observed during the sars outbreak, whereby anxiety levels closely mirrored the daily number of new cases and were strongly associated with the intensity of the outbreak [22] . moderate fear has been shown as most effective at motivating health-protective behaviors. according to janis' fear-as-acquired drive model, low levels of fear lead to inaction, moderate levels produce self-protective behavior, whereas high levels result in counterproductive defensive responses [23] . therefore, health authorities should disseminate accurate information and a realistic view of associated risks to induce a moderate level of fear in order to encourage people to take precautions [21] . there are several limitations of this study. the cronbach's alpha values of <0.70 in this study indicate low consistency, so the scores should be interpreted cautiously. telephone surveys do not include households without fixed-line telephones, which are more likely to be from socio-economically disadvantaged groups [24] . furthermore, it may not be possible to generalize our findings to the whole country, as the study sampled from one state (out of 13) and one federal territory (out of three). as a result, the study population had a monthly household income which was higher than the national average of rm3,686 [25] (us$1=rm3.49, as of oct 5, 2009), and slightly different proportions of ethnic groups [17] . despite these methodological caveats, the study has the advantage of a high response rate of 60.0%. additionally, the issues highlighted by this study are of considerable importance for the understanding of the public's view on the new influenza a(h1n1) outbreak and have significant implications for information communication efforts. the study can serve as a baseline for future national-level surveys. the study also adds to the relatively scarce knowledge of the new influenza a(h1n1) in a malaysian context. in summary, some misconceptions and erroneous beliefs about the modes of transmission need to be addressed. high confidence in preventing infection and low perceived susceptibility of infection were reported despite an overestimation of severity of the a(h1n1) infection. findings suggest that provision of education and clear information are essential to correct the misconceptions. the perceived susceptibility of infection needs to be increased so that public will take precaution against a(h1n1) infection and increase health protective behaviors. pandemic influenza a (h1n1) 2009 in malaysia-the next phase ministry of health malaysia press release a survey of knowledge, attitudes and practices towards avian influenza in an adult population of italy avian influenza in the uk: knowledge, risk perception and risk reduction strategies the public's response to severe acute respiratory syndrome in toronto and the united states the singaporean response to the sars outbreak: knowledge sufficiency versus public trust attitudes, and practices regarding avian influenza (h5n1) sarsrelated perceptions in hong kong perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population anticipated and current preventive behaviors in response to an anticipated human-tohuman h5n1 epidemic in the hong kong chinese general population monitoring community responses to the sars epidemic in hong kong: from day 10 to day 62 sars transmission, risk factors, and prevention in hong kong differences in public emotions, interest, sense of knowledge and compliance between the affected area and the nationwide general population during the first phase of a bird flu outbreak in israel widespread public misconception in the early phase of the a(h1n1) influenza epidemic public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey initial psychological responses to swine flu department of statistics the health belief model and personal health behavior perceived threat, risk perception, and efficacy beliefs related to sars and other (emerging) infectious diseases: results of an international survey risk perception related to sars and avian influenza: theoretical foundations of current behavioral research temporal changes in psychobehavioral responses during the 2009 h1n1 influenza pandemic longitudinal assessment of community psychobehavioral responses during and after the 2003 outbreak of severe acute respiratory syndrome in hong kong effects of fear arousal on attitude change: recent developments in theory and experimental research emerging advantages and drawbacks of telephone surveying in public health research in ireland and the economic planning unit, prime minister's department acknowledgement the study is funded by university of malaya (research grant rg186/10htm). the authors declare no conflict of interest. key: cord-020466-hdcke0d4 authors: hammel, jean m.; chiang, william k. title: commentary date: 2004-11-19 journal: ann emerg med doi: 10.1016/j.annemergmed.2004.10.005 sha: doc_id: 20466 cord_uid: hdcke0d4 nan influenza is one of the costliest infectious diseases worldwide. not only do yearly endemic outbreaks lead to significant local morbidity and mortality, but less frequent pandemicsd4 in the 20th century alonedcause death tolls in the millions. the ''spanish flu'' of 1918 to 1919 resulted in 20 to 40 million deaths worldwide. 1 in the late 1990s, after several decades of fairly stable human influenza viruses, highly pathogenic avian influenza infected humans in southeast asia for the first time. in the setting of the severe acute respiratory syndrome (sars) epidemic and emerging human infections with these highly pathogenic avian influenza strains, the prospect of a pandemic looms large for public health authorities and should prompt preparative measures from emergency care providers. the ability of influenza to cause repeated outbreaks lies in its viral structure and ease of genetic reassortment. influenza viruses belong to the orthomyxoviridae family and contain singlestranded rna. these enveloped viruses can be classified into a, b, and c types on the basis of their internal nucleoprotein and matrix protein identity. only influenza a and b are associated with major outbreaks. 2 influenza a is more diverse, associated with more outbreaks, and includes all avian influenza strains. influenza a contains 8 distinct genes. two surface glycoproteins, hemagglutinin and neuraminidase, account for its infectivity as well as trigger the host immune response. hemagglutinin binds sialic acid receptors on the host cell membrane to gain access to the cell; this is the main target of antibodies and vaccines. neuraminidase facilitates release of progeny virions from host cells after replication and is the target of antiviral therapies. these proteins are also used to identify different strains; to date, 15 hemagglutinin (h1-15) and 9 neuraminidases (n1-9) have been identified. only 3 hemagglutinin (h1-3) and 2 neuraminidase (n1, n2) subtypes of influenza a persist in humans. certain hemagglutinin and neuraminidases subtypes are associated with pigs (h1, h3, n1, n2) and horses (h7, n7, n8). all hemagglutinin and neuraminidase subtypes are established in aquatic birds. 2, 3 the epidemiology of influenza is closely linked with the genetics of these 2 proteins. local epidemics, like the severe ''flu season'' of 2003 to 2004 in the united states, are caused by antigenic drift. this means that random point mutations in the genetic sequence escape repair, leading to gradual ''drift'' of the gene pool. the genes coding for hemagglutinin and neuraminidase are inherently unstable and constantly undergoing genetic drift. people are more susceptible to these slightly altered strains but also retain some degree of crossimmunity from prior influenza exposures. thus, resultant epidemics are limited in scope and severity. antigenic shift is potentially far more dangerous. shift occurs when 1 of the 8 genes is exchanged in its entirety for another during replication. this requires a host cell, called a ''mixing vessel,'' to be coinfected with 2 influenza strains. pigs are an ideal mixing vessel because they are readily infected with both human and avian forms. 4, 5 alternatively, a person infected with a conventional human influenza virus could be coinfected with a highly pathogenic avian influenza strain. global influenza pandemics result from such antigenic shift because there is no pre-existing immunity to the new strain. aquatic birds serve as the primary reservoir for influenza a, carrying the viruses largely without adverse effects. 4,6 domestic poultry, however, are highly susceptible to avian influenza. depending on the virulence of the particular strain, poultry may manifest mild upper respiratory infections or rapidly fatal illness. 4 formerly known as ''fowl plague,'' highly pathogenic avian influenza is now identified as strains h5 and h7, which cause severe disease in terrestrial birds. 6, 7 since the hong kong pandemic of 1968, the human strains of influenza a had remained fairly stable. 5 however, in the late 1990s, new avian strains began to infect humans in relatively small numbers. eighteen people were infected with influenza a (h5n1) in hong kong in 1997; 6 died. 4, 8 in 1999, h9n2 was isolated for the first time from 2 children in hong kong, who both recovered after mild respiratory illnesses. 4, 8 most concerning, however, is this year's re-emergence of influenza a (h5n1)dslightly different genetically than the 1997 strain. [9] [10] [11] affecting poultry across southeast asia, this is the most widespread outbreak of avian influenza in history. more than 100 million poultry have died or been killed as a result. 12 as of september 28, 2004, in thailand and vietnam, 42 cases were reported in healthy young adults and children, with a 71% mortality rate. 13 the h5n1 strain of influenza transmitted to humans this year has 2 of the 3 classic features of a potential pandemic: high mortality and an immunologically naive population. 14 lacking, up to this point, is efficient human-to-human transmission. 5 of note, some evidence shows limited human-to-human spread during the 1997 hong kong outbreak. a case-control study of health care workers in 3 hospitals caring for infected patients showed that more exposed health care workers were seropositive for h5n1 antibodies than nonexposed workers (3.7% versus 0.7%). 15 none exhibited signs of illness. the clinical presentation of highly pathogenic influenza a (h5n1) includes fever, cough, and shortness of breath. sputum production, pleuritic chest pain, and diarrhea were variably present. 4, 10, 11 almost all patients had contact with poultry; the mean incubation period was 3 days. 4,10 on examination, tachypnea, respiratory distress, and rales were found in the first 10 patients in vietnam; 10 a similar but less dramatic presentation was found in the patients in thailand. 11 in all, the chest radiographs were markedly abnormal: patchy or interstitial infiltrates, lobar collapse, focal consolidation, or air bronchograms. 10, 11 laboratory testing revealed leukopenia (2,100/mm 3 ) with pronounced lymphopenia (700/mm 3 ), with an inversion of the cd4:cd8 ratio. 10, 11 some patients also exhibited thrombocytopenia and elevated transaminases. the natural history of influenza a (h5n1) is one of progressive respiratory distress and multiple organ system involvement. the h5n1 strain, more so than others, directly infects macrophages and causes excess cytokine release. 2, 4 this creates a clinical picture akin to systemic inflammatory response syndrome. at the same time, h5n1 is resistant to the antiviral action of these excess cytokines. 16 also unique to influenza a strain h5n1 is its propensity to affect other organ systems, renal, hematologic, and gastrointestinal in particular. 6 in the acute setting, definitive diagnostic testing is not yet available. rapid nasopharyngeal antigen assays can quickly identify the presence of influenza a but cannot differentiate between strains. reverse transcriptase polymerase chain reaction is the diagnostic test of choice for viral typing but is not helpful acutely, as results require 10 to 14 days. this test may be available through the local health department or by calling the centers for disease control and prevention (404-639-3747). two basic classes of antiviral agents are available for the management of influenza a, m2 membrane protein inhibitors, amantadine and rimantadine, and neuraminidase inhibitors, zanamivir and oseltamivir. these agents reduce the severity and duration of symptoms when initiated within 2 days of symptom onset. 2 these antiviral medications have not been well studied for avian influenza but may play a role in preventing concomitant human and avian influenza infection. the current h5n1 avian strain is resistant to m2 protein inhibitors. 4, 9, 11 neuraminidase inhibitors, steroids, and broad-spectrum antibiotics were variably used in the recent h5n1 outbreaks. given the relatively small number of patients and the completely uncontrolled manner in which the medications were used, however, no conclusions can be drawn regarding their efficacy. influenza vaccination appears to be the most logical method of controlling the spread and prevent potential epidemics, but current vaccines provide no protection against avian h5 or h7 strains. because h5 and h7 strains are highly lethal to chicken embryos, current vaccine production methods cannot produce avian vaccine efficiently. newer vaccine production techniques, including the use of reverse genetics technology that can tailor rapidly to specific influenza strains, appear to be promising. 17 full respiratory isolation precautions, similar to those for sars, are recommended in all cases of suspected human infection with highly pathogenic avian influenza viruses. these include hand washing, gloves and gown, n95 masks, eye protection, and a negative pressure isolation room, if available. those discharged home should be isolated in the home setting, as is recommended for sars. these precautions should be continued for 14 days after onset of symptoms until an alternative diagnosis is established or test results indicate the patient is not infected with influenza a. the diagnosis of influenza a h5n1 should be considered and isolation precautions instituted for any patient with respiratory symptoms and either contact with poultry or recent travel to southeast asia. because a definitive diagnosis of highly pathogenic avian influenza cannot be made in the emergency department and these patients will appear ill, treatment should be initiated early and broadly. antibiotics for severe pneumonia would be appropriate, as well as antiviral agents. nasopharyngeal swabs for influenza a should be sent and blood drawn for reverse transcriptase-polymerase chain reaction typing (contact the local health department). lastly, for both testing and surveillance purposes, the health department should be contacted for all suspected cases. it is important for those involved in disaster planning to incorporate large-scale infectious outbreaks into their preparations. bioterrorism, sars, and pandemic influenza represent threats that could quickly overwhelm our health care system. preparations should include infection control plans for rapid influx of infectious patients into health care facilities, personal protective equipment for health care workers, and access to stockpiles of antimicrobials, vaccines, and supportive care equipment. the emergency medicine continuous certification (emcc) program started january 1, 2004. all diplomates who want to maintain their certification with abem beyond their current certification expiration date must participate fully in the emcc program. effective 2004, the licensure requirement for all diplomates changed. diplomates must now continuously maintain a current, active, valid, unrestricted, and unqualified license in at least one jurisdiction in the united states, its territories, or canada, and in each jurisdiction in which they practice. inactive medical licenses voluntarily held by physicians are in compliance with the policy on medical licensure. cases of influenza a (h5n1)dthailand prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 the 1918 ''spanish'' influenza pandemic and characterization of the virus that caused it pandemic threat posed by avian influenza a viruses avian influenza: a new pandemic threat? human influenza a h5n1 virus related to a highly pathogenic avian influenza virus clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus avian influenza and human health human infection with influenza h9n2 outbreaks of avian influenza a (h5n1) in asia and interim recommendations for evaluation and reporting of suspected casesdunited states avian influenza a (h5n1) in 10 patients in vietnam cases of influenza a (h5n1)dthailand cdc health update: update on avian influenza a (h5n1) confirmed human cases of avian influenza a (h5n1) pandemic risks from bird flu risk of influenza a (h5n1) infection among poultry workers, hong kong, 1997-1998 lethal h5n1 influenza viruses escape host anti-viral cytokine responses influenza vaccines: present and future key: cord-000262-4owsb0bg authors: leung, gabriel m.; nicoll, angus title: reflections on pandemic (h1n1) 2009 and the international response date: 2010-10-05 journal: plos med doi: 10.1371/journal.pmed.1000346 sha: doc_id: 262 cord_uid: 4owsb0bg gabriel leung and angus nicoll provide their reflections on the international response to the 2009 h1n1 influenza pandemic, including what went well and what changes need to be made in anticipation of future flu pandemics. there is general consensus that the only predictable characteristic of influenza viruses and pandemics is their unpredictability [1] . given such uncertainty, reasonable application of the precautionary principle should prevail in the responses. indeed many of the initial responses to the 2009 pandemic went well. once isolated, the pandemic virus strain was shared immediately, specific diagnostic assays were produced and distributed worldwide, antivirals were available in many countries, vaccine development started promptly, and clinical trials demonstrating vaccine safety and immunogenicity were conducted rapidly. there were many inherently favourable features of the pandemic itself, not all of which were immediately apparent (table 1) . this was not 1918 spanish flu. the impact has been mostly confined to the health sector. but that impact has been significant and heterogeneous, with pressure experienced by primary and hospital care (especially intensive care and paediatric services). distilling descriptions of the impact of a complex public health threat like a pandemic into a single term like ''mild,'' ''moderate,'' or ''severe'' can potentially be misleading [2] . certainly the experience of hospital clinicians indicated that this pandemic, sometimes described as ''mild to moderate,'' was not limited to only mild or moderate illness. many patients were severely ill and died, and undoubtedly, high-quality clinical management of patients with severe complications in intensive care units saved many lives of the critically ill, who often required prolonged hospitalisation [3] . the epidemiology of this pandemic is different than for seasonal influenza epidemics, but not unlike previous pandemics. young people have been disproportionately affected in terms of hospitalisation and deaths compared to seasonal influenza in which complications and mortality are predominantly borne by the elderly [4] . similarly, the risk to pregnant women has been higher than for seasonal influenza [5, 6] , which was also noted in previous pandemics. the attributable premature mortality may remain unclear for some time. recent american analyses have estimated many more deaths than those officially reported with laboratory confirmation of infection and that years of life lost were equivalent to the 1968 pandemic. the lower bound of such estimates is equivalent to the annual burden caused by a typical h3n2 seasonal epidemic in temperate climates [7, 8] . the years-of-life-lost metric captures the impact of a different agespecific mortality pattern which death counts cannot. deaths involving the young and healthy incur many more potential years of life lost compared to those of older adults and of chronically ill individuals. there are also a number of ''firsts'' for the 2009 pandemic after an interpandemic period of more than four decades (box 1). these brought both opportunities and challenges. under the auspices of the world health organization (who), the process of a global review by public health specialists from around the world has recently begun. they were nominated by national authorities and are led by an elected chair who assessed the handling of the 1976 swine influenza event among us military personnel at fort dix [9] . here we offer some initial reflections on the first 12 months of the present pandemic. considerable effort in recent years had been dedicated to preparing for surveillance during a pandemic and to incorporating modelling in planning in some countries. the pandemic virus was detected and isolated reasonably early, although too late for any attempt at containment. it remains unclear precisely when or where it first emerged, but the earliest human infections were detected in north america and the best estimates of the timing of emergence are variously mid-february from field epidemiology in southeast mexico or mid-january from a molecular clock model [10] . situational awareness during the early phase allowed quick assessment by countries, notably those affected first (mexico, us, canada, and southern hemisphere temperate countries). the integration of clinical, laboratory, and epidemiologic data proved essential and gave important insights into disease severity, transmission dynamics, and anticipated impact of interventions. focused local or national studies with analyses shared through who or regional bodies proved more valuable than relying on collection of primary data for analysis in some regions [2] . although there were modelling efforts underway, only a few governments incorporated such data for policy decisions. data from seroepidemiological studies have been limited, primarily due to the lack of routine influenza serosurveys, and the essay section contains opinion pieces on topics of broad interest to a general medical audience. technical challenges with the assays, interpretation, and validation of results. available serological data on prevalence or seroincidence of humoral immunity yielded age-specific attack rates that indicated a substantial proportion of asymptomatic infections and mild illnesses, similar to or greater than past pandemics and seasonal outbreaks. this was confirmed by a recent hong kong study showing the proportion of asymptomatic infection, secondary attack rates, viral shedding, and course of illness among household members were largely similar between infections with seasonal and pandemic influenza virus strains circulating during 2009 [11] . the few published serosurveys revealed heterogeneities in infection rates among different groups and between different places [12] [13] [14] . in particular there appears to be serological evidence of substantial preexisting humoral immunity among older adults, ranging from 23% (1:32 titre by haemagglutination inhibition in those 65 years or over) [14] to 34% (1:80 titre by microneutralisation assay in those 60 years or over) [15] in different studies. further data on population susceptibility by age or the availability of a rapid and accurate serological test could allow health services to further target vaccine efforts for subsequent waves, as has been done in a few countries [14] . early on, some airports installed thermal screening and others asked travellers to declare fever or respiratory symptoms at disembarkation. the utility of these interventions has been repeatedly challenged [16] , although if executed well could delay the start of community transmission by a few weeks [15, 17] (table 2) . similarly, during the early stages of global or local spread, quarantine, isolation, school closures, and other social distancing measures were variously implemented in some populations (e.g., mexico [18] , western japan [19] , uk), although most have not yet been formally evaluated and published [20] . two exceptions are in hong kong and the uk. in the former, it was estimated that transmission fell by 25% when schools closed [21] . in settings like hong kong, with the infrastructure and resources to implement such measures and n decisions regarding pandemic response during the exigencies of a public health emergency must be judged according to the best evidence available at the time. n revising pandemic plans-to be more flexible and more detailed-should wait for who leadership if national plans are not to diverge. surveillance beyond influenza should be stepped up, and contingencies drawn up for the emergence or re-emergence of other novel and known pathogens. [22] but some countries attempted containment in phases 5 and 6. some countries even instituted a ''containment phase'' using case-finding and various measures such as isolation and antiviral treatment of ill suspected and confirmed cases, and quarantine of exposed persons with or without antiviral chemoprophylaxis, while others never attempted or quickly moved from resource-intensive containment to mitigation [22] . a preliminary evaluation of intensive containment undertaken in parts of the uk during its spring/summer wave of 2009 demonstrates how resource-and labour-intensive community containment could have been and also how even with a lot of resources the measures had to be abandoned [23] . it is now recognised that the phrase ''containment'' was unfortunate and potentially misleading since at best the actions were only mitigating impact [24] . this pandemic virus transmitted efficiently among children and at least one study has shown that school closures were associated with reduced population transmission when implemented early [21] . closures appear to have stopped school outbreaks in western japan and might have also mitigated impact initially on the local communities [25] . however, decisions on this intervention were contextually specific, dependent on feasibility and their potential downsides [26] . in europe and the us the judgement was generally that proactive school closures would not be justified as a community mitigation intervention in the context of a perceived mild-to-moderate pandemic among the general population, and reserve plans for widespread closure have not been activated in most jurisdictions. however, local decisions were made to close schools in some areas as a response to prevent transmission and high attack rates among schoolchildren or simply where there was too much illness and absenteeism to sustain teaching [21, 27] . personal protective interventions such as face masks, hand hygiene, and early isolation may have been beneficial in reducing transmission at the individual level in the home [27, 28] , although household secondary attack rates during the pandemic were similar to those with seasonal influenza [13, 29] . their population level impact remains to be assessed. there was much debate over whether to use conventional masks or respirators in health care settings. one well-conducted canadian trial on seasonal influenza virus transmission published during the pandemic suggested no additional advantage from n95 respirators [30] . oseltamivir and zanamivir (and later peramivir in some countries) played a role in the mitigation effort, sometimes drawing on national stockpiles. except for japan, widespread use of antivirals had not been the norm previously. it became standard to recommend neuraminidase inhibitors for treatment of inpatients and high-risk outpatients, and in restricted circumstances for chemoprophylaxis. innovative delivery schemes were sometimes developed. those who fell sick in england could have a telephone assessment (taking pressure off primary care) and then if appropriate receive empiric oseltamivir treatment from a local pharmacist. in norway oseltamivir was made available ''over the counter.'' however in many european settings, reluctance remained among primary care providers to prescribe a drug they were unused to. another controversy was whether to offer oseltamivir to all those with symptoms or target those at higher risk for complications. the observational data so far suggest that early treatment with neuraminidase inhibitors have worked to reduce severe disease and have not been linked to significant adverse risks [31, 32] . late clinical presentation and delayed initiation of antiviral treatment have been implicated with more severe complications worldwide, indicating gaps in identifying and treating patients before disease severity increases. while sporadic cases of oseltamivir resistance have been reported in association with a specific mutation (h275y in neuraminidase), such oseltamivir-resistant viruses have rarely transmitted [3] . indeed, the pandemic virus has remained genetically and antigenically stable so far. the core pharmaceutical preventive intervention was vaccines and this has box 1. a series of ''firsts'' about pandemic (h1n1) 2009 n the first pandemic to emerge in the twenty-first century. it has been more widespread and remains ongoing, compared to sars. n the first pandemic to occur after major global investments in pandemic preparedness had been initiated. n the first pandemic for which effective vaccines and antivirals were widely available in many countries, thus requiring public health authorities to earn and retain the confidence of health care providers through whom such are usually distributed. n the first influenza pandemic to coincide with the ongoing hiv/aids pandemic and for which preliminary data do not suggest a substantial, disproportionate impact on hiv-infected patients. n the first pandemic that took place within the context of a set of international health regulations and global governance, which had not been widely tested until the present. n the first pandemic with early diagnostic tests that led to rapid diagnosis but also an early obsession in the media and of policymakers with having reports of the numbers of those infected. n the first pandemic with antivirals available in many countries that led to a hopeful expectation that the pandemic might be containable, leading to the preparation for and implementation of a ''containment phase'' in some places. n the first pandemic in which intensive care was available in many countries to treat critically ill patients, fostering an expectation that everyone could be treated and cured. n the first pandemic with a ''blogosphere'' and other rapid social media messaging tools that challenged conventional public health communication. been a particular focus for critics citing the uneven and suboptimal uptake across countries. development of a pandemic vaccine was a scientific success, but limited availability until after the autumn/winter wave had nearly peaked in the northern hemisphere contributed to lower coverage than anticipated [33] . vaccination coverage depended on many factors, including availability, preordering, licensing and bureaucratic hurdles, logistics, convenience, and, most crucially, public and professional perceptions. this pandemic presented a particular risk communication challenge, since while infection usually results in mild illness, occasionally it is lethal, even in the young and previously healthy despite optimal treatment [34] [35] [36] . in the absence of any excess risk of serious side effects compared to annual seasonal vaccines [37] (despite the intensive effort to look for such) the benefits of immunisation far outweighed any potential down-sides at the individual level, particularly for those at higher risk for complications. notwithstanding such evidence, the cost of pandemic vaccines was considerable and a loss of public confidence has sometimes been triggered by unsubstantiated media reports of serious side effects with a ''new vaccine'' that utilised the same manufacturing technology as for years of seasonal vaccines. uptake among health care providers as role models has been mixed, as has their expression of the need for vaccination at all. this sometimes cast doubt in the minds of the public. conversely, pandemic deaths in young healthy people abruptly changed public perception (such as in canada, romania, and finland); supply and organisational issues then became crucial. another more fundamental criticism challenges whether vaccines should have been procured at all given an eventual surplus in the developed north. the unexpected finding that a single dose was immunogenic among all persons except for younger children, which reduced the required number of doses by half from the projected number needed in most countries, but this was not known in advance of countries placing vaccine orders. had there been ''overpreparation''? the prior worry had been the reverse -would there be sufficient production capacity to meet needs [38] ? even in retrospect, and with the observed burden of the pandemic, a vaccine was clearly justified for countries where annual vaccines for seasonal influenza are routinely recommended. field and pharmacovigilance data so far have shown that these vaccines were immunogenic, effective, and very safe [39] . however, the frailty was timing and availability. generally supplies came in later and in smaller amounts than forecasted, in part due to lower yield in growth of the vaccine virus strain than expected. reduce and delay community spread somewhat at the earliest stage to allow better preparation for mitigation response [15] completely prevent entry of infected individuals due to suboptimal sensitivity and asymptomatic (including infected and within incubation period) or subclinical presentation [16] many countries did not attempt these measures because of logistics, stage of pandemic [22] or other cost-benefit considerations [16] china hong kong sar japan personal protective measures (e.g., face masks, hand hygiene, cough etiquette, early self-isolation when ill) reduce risk of infection to self and close contacts (if self is ill and infected) [27, 28] have not been evaluated whether they can provide significant populationlevel protection virtually all countries implemented these measures to varying degrees in health care settings according to the risk of the situation. almost all encouraged hand hygiene, cough etiquette, and early self-isolation most countries recommended adoption of hand hygiene, cough etiquette, and early self-isolation when ill, but use of face masks in the community was uncommon except in east asia. antivirals for treatment and chemoprophylaxis [21, 22] mitigation: reduce illness severity and complications if administered early; reduce transmission from those receiving treatment; sometimes also used as chemoprophylaxis in high-risk circumstances provide significant population-level protection or allow containment attempts at source containment were not possible, as the pandemic was effectively already in who phase 5 when what became the pandemic virus was first identified [22] . initial observational studies suggest antivirals were successful when early treatment was administered canada germany hong kong sar japan uk us (these populations attempted the intervention initially but effort was not sustained towards the later stages of the pandemic) vaccines mitigation (a) at individual level by conferring immunity to infection in those at higher risk of severe disease or (b) at a population level by immunizing population groups especially those who are transmitting most (i.e., children) in most countries vaccine was not available early enough and/or arrived in insufficiently large amounts to achieve mitigation at a population level. greater population benefit may occur in the next season most of the orders arrived after the peak of the autumn/winter wave in the geographic north (whose countries had received most vaccines). therefore, judgement on their impact in averting serious morbidity and deaths may come only after the second winter. perhaps then, differential use by countries will allow for comparisons where there is good surveillance for severe disease and deaths. there have been claims of extraneous influence on the independent and objective judgment of expert advice that in turn influenced decision-making [40] . these claims have been robustly countered as they relate to who's advisory and decision-making process [41] . as harvey fineberg, chair of who's external review, pointed out, when assessing any allegations of impropriety or bias, or the perception of such, it would be important to distinguish between financial or other conflicts with potential pecuniary gains versus predispositions arising from an individual's background and experience. rather than aiming for a complete purge of any and all experts who had worked with vaccine manufacturers and received sponsorship, as these are often the very same group who possess the most relevant and useful expertise precisely because they have been closely involved in the research and development process, the focus should be on making the declaration of such interest wholly transparent and comprehensive according to a set of robustly established procedures that can withstand the strictest scrutiny. it is reassuring that who has honoured its commitment to making public the names and declarations of interest of the pandemic emergency committee when the pandemic was declared over on 10 august 2010. additionally, receiving advice should be differentiated from making decisions. the people entrusted with undertaking the latter task should then judge the validity of the advice rendered by experts, having taken into account their interest declarations. the decision makers should also be prepared to justify their actions. it is important to learn from our experience through the first year and beyond as we move into the new seasonal influenza [42, 43] . it is theoretically possible, although unlikely, that the second winter of this pandemic will be worse than the first, as happened for the 1968 pandemic when transmissibility increased [44] . equally, if the pandemic virus outcompeted the a(h3n2) virus strains responsible for more intense seasonal epidemics, there may even be a diminution of disease burden in older people. as of this writing, seasonal influenza a (h3n2) and b virus strains continue to cocirculate. antigenic drift in the 2009 h1n1 virus is expected to occur in the future, especially under the pressure of so many people now being immune through infection or immunisation, although the timing is unpredictable. the pandemic virus is included in the trivalent seasonal influenza vaccine composition for both hemispheres. clear communication of public health messages will remain a particular challenge and not confusing what could happen (and should be prepared for) with what is most likely to happen. in assessing the pandemic response, decisions made during the exigencies of a public health emergency must be judged according to the best evidence available at the time. hindsight always gives perfect vision and using post-hoc information to evaluate prior decisions at best confuses and often produces unfair conclusions. preparedness plans will have to be revised in due time, after the many lessons learned have been gathered. this should be done quickly in case the worst is not yet over [45] . however, rewriting plans should best wait for who leadership if national plans are not to diverge. a strong argument exists for making future plans more flexible and having extra descriptions including the many aspects of severity when a pandemic is emerging that then determine the consequential public health actions [2] . broadening surveillance for a range of influenza a viruses among a wide range of animals (e.g., swine), not just in avian species, as well as strengthening the monitoring of seasonal influenza virus infections in humans will facilitate identification of novel influenza a viruses of pandemic potential, and earlier detection of the emergence of a pandemic virus. more broadly we should look beyond influenza and draw up contingencies for the emergence or re-emergence of other novel and known pathogens [45] . one challenge faced initially in this pandemic was for timely collection and sharing of clinical data to inform optimal management of critically ill patients worldwide. establishing clinical research infrastructure prior to a pandemic and a central institutional review board will facilitate data collection and analyses [46] , whether for the next influenza pandemic, sars outbreak, or next novel respiratory pathogen of global importance. clinical management of severe influenza disease should not be limited to the current antiviral regimen, and include the development of other therapeutics (e.g., novel antivirals and immunotherapy). ongoing improvements in the routine and timely monitoring of hospital admissions and deaths attributable to influenza, as well as representative serological surveys at regular intervals can provide epidemiological data with which to reduce uncertainty around the true burden of influenza and thus inform policy choices [47] . assessment of the humoral and cellular immune response over time in a subset of vaccinated individuals could reveal how vaccine-induced immunity differs from natural infection, and whether cross-reactive responses to other influenza virus strains are modulated by the two types of immunological response [48] . the latter could become important as the pandemic strain has already been cocirculating with other interpandemic influenza a virus strains in some parts of the world. greater access to antivirals and influenza vaccines worldwide is an ongoing challenge. although who secured pledges of 200 million vaccine doses and monies for operations, and more than 80 less-resourced countries have signed agreements with who for supply of vaccines, this gap remains. it is an indefensible fact that these vaccines started to flow to the poorer countries well after they began going to the countries with advance purchase arrangements. delivering timely pandemic influenza vaccination in countries without existing seasonal vaccine programmes is proving difficult. the longterm solution has to be improved surveillance, expanded monitoring of disease burden, and better prevention and control of influenza, including the development of seasonal vaccine use and production in all regions of the world [49] . increased coverage of available bacterial vaccines (hib, pneumococcal) will help prevent secondary invasive bacterial coinfections with either seasonal or pandemic influenza. finally accusations of ''overreaction'' can be countered by the observation that investment in fire services or insurance is usually judged against their ability to respond to conflagrations. if the first test is a lesser fire, that experience should be used for improvements rather than as a reason to scrap the fire engines and cancel the insurance [40] . influenza pandemics of the 20th century experience and lessons from surveillance and studies of the 2009 pandemic in europe writing committee of the who consultation on clinical aspects of pandemic influenza (2010) clinical aspects of pandemic 2009 influenza a (h1n1) virus infection severe respiratory disease concurrent with the circulation of h1n1 influenza pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states critical illness due to 2009 a/h1n1 influenza in pregnant and postpartum women: population based cohort study estimates of the prevalence of pandemic (h1n1) 2009, united states preliminary estimates of mortality and years of life lost associated with the 2009 a/h1n1 pandemic in the us and comparison with past influenza seasons the swine flu affair: decision-making on a slippery disease pandemic potential of a strain of influenza a (h1n1): early findings comparative epidemiology of pandemic and seasonal influenza a in households incidence of 2009 pandemic influenza a h1n1 infection in england: a cross-sectional serological study 2009 influenza a(h1n1) seroconversion rates and risk factors among distinct adult cohorts in singapore cross-reactive antibody responses to the 2009 pandemic h1n1 influenza virus entry screening to delay local transmission of 2009 pandemic influenza a (h1n1) entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation a clinical virological and epidemiological analysis what mexico taught the world about pandemic influenza preparedness and community mitigation strategies epidemiological characteristics and low case fatality rate of pandemic (h1n1) 2009 in japan europe's initial experience with pandemic (h1n1) 2009 -mitigation and delaying policies and practices school closure and mitigation of pandemic (h1n1) 2009, hong kong who interim protocol: rapid operations to contain the initial emergence of pandemic influenza the role of the health protection agency in the 'containment' phase during the first wave of pandemic influenza in england influenza pandemic: an independent review of the uk response to the 2009 influenza pandemic influenza (h1n1) 2009 outbreak and school closure closure of schools during an influenza pandemic facemasks and hand hygiene to prevent influenza transmission in households: a cluster randomized trial household transmission of 2009 influenza a (h1n1) virus after a school-based outbreak household transmission of 2009 pandemic influenza a (h1n1) virus in the united states surgical mask vs n95 respirator for preventing influenza among health care workers: a randomized trial antiviral treatment for patients hospitalized with 2009 pandemic influenza a (h1n1) the truth about tamiflu? neuraminidase inhibitors in pandemic a/h1n1 flu interim results: state-specific influenza a (h1n1) 2009 monovalent vaccination coverage -united states hospitalized patients with 2009 h1n1 influenza in the united states pediatric hospitalizations associated with 2009 pandemic influenza a (h1n1) in argentina mortality from pandemic a/h1n1 2009 influenza in england: public health surveillance study preliminary results: surveillance for guillain-barre syndrome after receipt of influenza a (h1n1) 2009 monovalent vaccine -united states preparing for the next pandemic pandemic influenza a(h1n1) 2009 breakthrough infections and estimates of vaccine effectiveness in germany conflicts of interest. who and the pandemic flu ''conspiracies the international response to the influenza pandemic: who responds to the critics. pandemic (h1n1) 2009 briefing note 21 how well are we managing the influenza a/h1n1 pandemic in the uk? a new decade, a new seasonal influenza: the council of the european union recommendation on seasonal influenza vaccination estimates of the transmissibility of the 1968 (hong kong) influenza pandemic: evidence of increased transmissibility between successive waves emerging infectious diseases: a 10-year perspective from the national institute of allergy and infectious diseases early observational research and registries during the 2009-2010 influenza a pandemic studies needed to address public health challenges of the 2009 h1n1 influenza pandemic: insights from modeling association between the 2008-09 seasonal influenza vaccine and pandemic h1n1 illness during spring-summer 2009: four observational studies from canada global pandemic influenza action plan to increase vaccine supply we thank timothy m. uyeki of the us centers for disease control and prevention for substantial discussion and input. icmje criteria for authorship read and met: gml an. agree with the manuscript's results and conclusions: gml an. wrote the first draft of the paper: gml. contributed to the writing of the paper: gml an. contributed to the review from the experience of the pandemic in european countries: an. key: cord-001219-517gka4h authors: timpka, toomas; spreco, armin; gursky, elin; eriksson, olle; dahlström, örjan; strömgren, magnus; ekberg, joakim; pilemalm, sofie; karlsson, david; hinkula, jorma; holm, einar title: intentions to perform non-pharmaceutical protective behaviors during influenza outbreaks in sweden: a cross-sectional study following a mass vaccination campaign date: 2014-03-07 journal: plos one doi: 10.1371/journal.pone.0091060 sha: doc_id: 1219 cord_uid: 517gka4h failure to incorporate the beliefs and attitudes of the public into theoretical models of preparedness has been identified as a weakness in strategies to mitigate infectious disease outbreaks. we administered a cross-sectional telephone survey to a representative sample (n = 443) of the swedish adult population to examine whether self-reported intentions to improve personal hygiene and increase social distancing during influenza outbreaks could be explained by trust in official information, self-reported health (sf-8), sociodemographic factors, and determinants postulated in protection motivation theory, namely threat appraisal and coping appraisal. the interviewees were asked to make their appraisals for two scenarios: a) an influenza with low case fatality and mild lifestyle impact; b) severe influenza with high case fatality and serious disturbances of societal functions. every second respondent (50.0%) reported high trust in official information about influenza. the proportion that reported intentions to take deliberate actions to improve personal hygiene during outbreaks ranged between 45–85%, while less than 25% said that they intended to increase social distancing. multiple logistic regression models with coping appraisal as the explanatory factor most frequently contributing to the explanation of the variance in intentions showed strong discriminatory performance for staying home while not ill (mild outbreaks: area under the curve [auc] 0.85 (95% confidence interval 0.82;0.89), severe outbreaks auc 0.82 (95% ci 0.77;0.85)) and acceptable performance with regard to avoiding public transportation (auc 0.78 (0.74;0.82), auc 0.77 (0.72;0.82)), using handwash products (auc 0.70 (0.65;0.75), auc 0.76 (0.71;0.80)), and frequently washing hands (auc 0.71 (0.66;0.76), auc 0.75 (0.71;0.80)). we conclude that coping appraisal was the explanatory factor most frequently included in statistical models explaining self-reported intentions to carry out non-pharmaceutical health actions in the swedish outlined context, and that variations in threat appraisal played a smaller role in these models despite scientific uncertainties surrounding a recent mass vaccination campaign. although encouraging the public to undertake specific protective behaviors has proved useful in containing outbreaks of infectious disease [1] , more research has been called for examining the social, demographic, and cultural factors that influence these efforts [2] . this is particularly important to understanding people's hesitations to heed official advice, particularly in the absence of clear scientific evidence regarding the disease outbreak [3] . the as03-adjuvanted pandemrixh was the most commonly used vaccine in response to the influenza a(h1n1)pdm09 outbreak in europe [4] ; finland and sweden recommended this vaccine to their entire populations. in august 2010 reports of a possible association between exposure to the vaccine and occurrence of narcolepsy in children and adolescents emerged in both the latter countries, which led to a review of the vaccine by the european medicines agency (ema). subsequently, increased narcolepsy diagnoses associated with the start of the campaign have been confirmed [5] . in sweden, scientific uncertainty regarding the safety of this mass vaccination was both publicly discussed [6] and questioned by researchers [7] . beliefs that the interventions suggested are effective and safe [8] , that the illness has severe consequences [9] , and that there is a high likelihood of exposure [10] have been associated with compliance with behavioral recommendations. it has also been pointed out that behavioral research in epidemics should not only identify determinants of individual and population behavioral responses, but also clarify the mechanisms underpinning these [11] . protection motivation theory (pmt) [12, 13] posits that an intention to perform protective activities is determined by perceptions of threat and the ability to cope. in addition to intentions and preceptions, a recent review concluded that protective behavior needs to be investigated with regard to sociodemiograpic characteristics in order to identify the ''contagious'' effect and contextual nature of perceptions and mediating mechanisms [14] . for instance, coping appraisals are made in interaction with environmental resources, which vary in availability across population subgroups. protective behavior associated with influenza outbreaks has also been investigated with regard to general estimates of health status [15] , but few studies have used validated measures of self-rated health as a means for the sub categorization. at present, several such measures are available for use in population-based research [16] . to provide a snapshot of intended self-protective behaviors during a period when scientific uncertainty pervaded public discussions addressing infectious disease control, we carried out a cross-sectional telephone survey of a demographically representative sample of the swedish population. the specific aim was to examine to what extent self-reported intentions to improve personal hygiene and increase social distancing during influenza outbreaks can be explained by perceptions of threat and the ability to cope as outlined in pmt, self-reported assessments of health, trust in official information, and sociodemiographic factors. the study used a cross-sectional design to analyze associations between intended protective behaviors during influenza outbreaks and items in a theoretical model of explanatory factors [14, 17] . a random sample of 1,011 persons ranging between 20-90 years of age was drawn from the swedish national population register. a combined telephone and questionnaire survey was carried out during the first quarter of 2012. the study was conducted according to the world medical association's declaration of helsinki from 1964 regarding ethical principles for medical research involving human subjects, revised in 2008. potential study sample participants were informed about the study by letter via postal mail and invited to participate in a telephone survey on protective behaviors during influenza outbreaks. those agreeing to participate returned their consent in writing. all collected data were managed confidentially and analyzed anonymously. the study design was approved by the institutional (ethics) review board at umeå university (dnr 2011-314-31ö ). a hypothetical explanatory model was constructed to inform the analysis of the main research question; i.e. to what extent selfreported intentions to perform protective behaviors during influenza outbreaks can be explained by perceptions of threat and the ability to cope as outlined in the pmt, self-assessments of health status, trust in official information, and sociodemiographic factors. in this model, protective behaviors during outbreaks are restricted to two categories: increased personal hygiene (use of disinfectants and other handwash products; frequent washing of hands when having touched common objects, such as door knobs) and social distancing (staying home from work or school; avoiding use of public transportation). the intentions to carry out a protective behavior are assessed by asking whether the respondent would try to perform the behavior during a mild and severe influenza outbreak, respectively. both outbreak scenarios described personal risk of infection as high (i.e., 1 in 3 people infected). the mild influenza description details moderate health consequences (less than 1 in 1000 infected people dying) and a minor lifestyle impact (services mainly operating normally). the severe scenario describes serious health consequences (1 in 50-100 infected people dying) and services no longer being able to operate normally. the first set of explanatory factors concerned perceptions of threat and the ability to cope. based on the notion of subjective expected utility [18] , which postulates that people's choices are a product of assessments of probability and utility of options, healthrelated methodologies such as the pmt and the health belief model [19] have included formally quantified models of subjective health risk perceptions, i.e., as the likelihood of contracting a disease multiplied by disease severity. together with different types of cost-benefit valuations and self-efficacy expectations, these perceptions of risk are presumed to determine health-protective behaviors. in the present study, the collection and analysis of data on protection motivation in relation to influenza outbreaks are structured according to the pmt. this theory suggests that threat appraisal will generate an intention to act, while coping appraisal determines the type of action. threat appraisal is in this study characterized in its three dimensions [11, [20] [21] : -perceived relative risk of catching influenza; measured by one item assessing personal likelihood of infection, if no preventative action was taken, -anxiety about catching mild and severe influenza; measured by one item for each influenza type, and -perceived severity of the consequences of catching mild and severe influenza; measured by one item for each influenza type. coping appraisal is also represented in its three dimensions: -response efficacy; assessed by one item asking about protecting oneself from influenza by employing enhanced personal hygiene and one item asking about social distancing, -self-efficacy; measured by two items asking whether the respondent felt it is possible to carry out protective behaviors by social distancing and increased personal hygiene, respectively, and whether they were confident they could carry out these actions if they so desired [22] , and -response costs; defined as the estimated efforts needed to overcome perceived barriers on carrying out protective actions. for social distancing, this dimension was assessed by asking for 'work concerns', i.e. guilt and anxiety about not completing work. response costs for increased personal hygiene were assessed through items asking for concerns associated with acquiring adequate soaps and disinfectants (handwash products) and learning the correct techniques to use them. self-reported health assessments have in epidemiological studies been found to be valid indicators of health status as measured by prediction of future physician contacts and all-cause mortality [23] . in this study, self-reported health is measured by the sf-8 tm 24-hour recall questionnaire in order to examine associations with intentions to carry out protective behaviors. this general selfreported health instrument contains eight health-related questions that, in turn, can be summarized in two overall measures of physical and mental health: physical component summary (pcs) and mental component summary (mcs), respectively [24] . it is derived from the sf-36 for the purposes of yielding comparable scores for the 8 health dimensions and 2 summary measures of the sf-36 with minimal respondent burden. trust in government information during influenza outbreaks has in previous studies been found to be associated with greater self-efficacy and personal hygiene [25] . trust in official information was therefore included in the explanatory model, asking for agreement with a single statement about trust in government information during outbreaks. the sociodemiographic factors included in the model were marriage status, number of children living at home, formal education, employment status, and ethnicity. prior to the telephone call, the subjects were asked to complete a paper-based survey, querying for sociodemiographic data and data elements from the sf-8 tm . the remaining data were collected in the telephone interview. to catalyze their considerations about the research topics, each subject was presented with brief scenarios of mild and severe influenza outbreaks. interview data were derived from open statements, and the respondents were asked to score their agreement along a seven-point scale from 1 (strongly disagree) to 7 (strongly agree). the collection of data on perceptions associated with precautionary behaviors was structured in accordance with the pmt (text s1). to assess trust in official information in this study, the single statement ''for information during influenza outbreaks i do rely on government sources'' was used. we conducted a drop-out analysis based on the demographic variables available for the entire sample, i.e. gender, age and place of residence. all collected data were first subjected to descriptive statistics, i.e. mean, median and standard deviation for continuous data and frequency and proportions (%) for categorical data. the primary end points for the ensuing analyses were intentions to increase social distancing (staying home while not ill; avoid public transportation), and enhance personal hygiene (use of handwash; frequent washing of hands after touching common objects) during mild and severe influenza outbreaks, respectively. the theoretical model of potential explanatory factors was used as the basis for the analysis. for each endpoint, logistic regression analyses were applied using the items in the model as explanatory variables. these included trust in official information; variables corresponding to pmt items (the threat appraisal items of perceived personal risk, emotional response (worry), perceived severity; and the coping appraisal items of general response efficacy, self-efficacy, and response costs); variables representing the sf-8 summary items (pcs and mcs); and sociodemographic characteristics (age, gender, educational level, living with partner, living with child, and employment). when used as response variables, ordinal variables were dichotomized (agree/do not agree). to contrast expected perceptions against other perceptions, the variables were converted with the agreement scores in the expected extreme as one category. for threat appraisal, agreement scores in the low extreme were contrasted against other opinions, except for the estimates of the severity of the consequences of getting infected where the scores in the high extreme were contrasted against the other opinions. regarding coping appraisal, the personal hygiene scores in the high extreme were contrasted against other opinions for response efficacy and self-efficacy and in the low extreme for response costs. for social distancing, agreement scores in the low extreme were contrasted against other opinions for response efficacy and in the high extreme for self-efficacy and response costs. the area under the roc curve (auc) was used as model performance indicator and nagelkerke r 2 to estimate the determination level for each model. the limits for interpreting the auc (or c-statistic) were set to 0.90, 0.80, and 0.70, denoting very strong (outstanding), strong (excellent), and acceptable discriminatory performance, respectively [26] . all tests were twosided and p,0.05 was regarded as statistically significant. all calculations were done using spss version 18 or higher. two-hundred and fifty-four persons in the total population sample (n = 1,011) could not be reached by a telephone call. of the 757 persons reached, 443 provided a complete response, leading to a 59% response rate to the telephone survey and a 44% participation rate with regard to the total sample. the age category 65-90 years was slightly over-represented (54% response rate) among the study participants when compared to the total population sample (p = 0.039). however, the effect size of this difference in participation was small (cramer's v = 0.08). thus, while elderly individuals were overrepresented in our data, the impact of this deviation from the reference population was of a small magnitude. in terms of place of residence, those living in small labor market regions (with a total population of less than 100,000 inhabitants) exhibited the highest participation rates: 51%, compared to 41% in large regions (with a population greater than 1 000,000 inhabitants). the basic sociodemiographic characteristics of the final study participants are displayed in table 1 . the general level of health in the study population as measured by sf-8 scores was above the reference values for all items except for physical functioning and vitality (lower scores) and general health (equal scores) ( table 2 ). there was no statistically significant difference between men and women regarding the mean scores of any sf-8 item or summary component. every second respondent (50.0%) reported high or very high trust (scores 6-7) in information about influenza provided by official sources (mean score 5.3; median 5.5; standard deviation (sd) 1.7)). neither age, education, employment nor any component of self-rated health was associated with trust in official information about influenza. however, the level of trust was associated with gender, with men reporting lower trust levels than women (p = 0.018; odds ratio (or) 0.60 (95% confidence interval (ci) 0.40;0.91)). regarding social distancing measures, 9% of the respondents scored strong (strong or very strong) agreement with the stated intention to stay home when not ill during mild influenza outbreaks, and 11% of the respondents scored strong agreement with this intent during severe outbreaks. more than twice as many respondents (23%) scored strong agreement with avoiding use of public transportation during a mild outbreak, while 29% of the respondents scored strong agreement with this intention during a severe outbreak. regarding measures related to personal hygiene, 77% of the respondents scored strong agreement with the stated intention to use handwash products during mild outbreaks, while 85% of the respondents scored strong agreement with this intention during severe outbreaks. regarding the intention to frequently engage in handwashing, 46% reported strong agreement in association to mild influenza outbreaks and 60% in association to severe outbreaks. a model describing the intention to stay home without being ill during a mild influenza outbreak included eight significant variables and displayed a strong discriminative performance (auc 0.85 (95% ci 0.82;0.89)) ( table 3 ). this self-reported intention was strongly associated with coping appraisal; low perceived response costs associated with staying home and selfefficacy with regard to social distancing; and, interestingly, to a disbelief in the general efficacy of social distancing as an infectious disease control measure. planning to stay home was also strongly associated with male gender and, with a weaker association, to being unemployed and living with a partner. the intention was also associated with threat appraisal, although with a weaker strength; with worry about getting infected and high perceived severity of the influenza threat. in comparison, the intention to stay home without being ill during a severe outbreak was represented by a model including only four significant variables, but that also displayed a strong discriminative performance (auc 0.82 ((95% ci 0.77;0.85)). as for the mild outbreak scenario, this intention was strongly associated with coping appraisal; to response costs and perceived self-efficacy with regard to social distancing. however, staying home during a severe outbreak was also strongly associated with threat appraisal related to concerns about getting infected. regarding sociodemographic factors, this intention was only associated with not having employment. the intention to avoid using public transportation during a mild influenza outbreak was represented by a model including six significant variables and an acceptable discriminative performance (auc 0.78 (95% ci 0.74;0.82)) ( table 4 ). this self-reported intention was, also, strongly associated with coping appraisal; to perceived response costs associated with avoiding public transportation and to self-efficacy with regard to social distancing. the intention was also strongly associated with threat appraisal in terms of worry about getting infected. in addition, avoiding use of public transportation was associated with a lower level of formal education, living with a partner, and high trust in official information. in contrast, the intention to avoid public transportation during a severe influenza outbreak was described by a model including four significant variables and an acceptable discriminative performance (auc 0.77 (95% ci 0.72;0.82)). as for the mild outbreak scenario, avoiding public transportation during severe outbreaks was strongly associated with coping appraisal; to response costs; and, with weaker strength, to perceived self-efficacy with regard to personal social distancing. with regard to threat appraisal, avoiding public transportation during a severe outbreak was associated with worry about getting infected and a high perceived severity of the influenza threat. planning to use handwash products during a mild influenza outbreak was described by a model including three significant variables and an acceptable discriminative performance (auc 0.70 (95% ci 0.65;0.75)) ( table 5 ). planning to use handwash was strongly associated with female gender. this intention was, for mild outbreaks, also explained by self-efficacy with regard to personal hygiene and trust in official information. for the severe outbreak scenario, planning to use handwash products was represented by a model including four significant variables and an acceptable discriminative performance (auc 0.76 (95% ci 0.71;0.80)). this intention was, too, strongly associated with female gender. in addition, it was strongly associated with coping appraisal; to a belief in the general efficacy of increased personal hygiene; and low response costs associated with acquiring of suitable products. contrary to any of the other intended behaviors studied, the intention to use handwash products during severe outbreaks was associated with low self-rated physical health. an intention to frequently engage in handwashing after having touched common objects during a mild influenza outbreak was represented by a model including four significant variables and an acceptable discriminative performance (auc 0.71 (95% ci 0.66;0.76)) ( table 6 ). the intention was strongly associated with coping appraisal in terms of self-efficacy with regard to personal hygiene. it was also associated with female gender, higher age, and lower education. in comparison, planning to frequently wash hands during a severe outbreak was represented by a model including three significant variables and an acceptable discriminative performance (auc 0.75 (95% ci 0.71;0.80)). similar to the mild influenza scenario, it was strongly associated with coping appraisal in terms of a high self-efficacy with regard to personal hygiene. the intention was also associated with female gender and being born in the country. despite the fact that the safety of the mass vaccination during the a(h1n1)pdm09 outbreak had been questioned by national mass media in a campaign-like manner, two years after the outbreak every second respondent in a representative sample of the swedish adult population reported high trust in official information about influenza. while the proportion of persons reporting intentions to improve personal hygiene during influenza outbreaks ranged between 45-85%, the proportion reporting intentions to increase social distancing did not exceed 25%. this pattern can generally be explained by the notion that the initial behavioral changes during an influenza outbreak are more likely to resemble familiar reactions and well-known routines [27] , such as increasing personal hygiene, rather than changes that require deductive planning, such as increasing social distancing. the explanatory models developed in this study showed statistical associations ranging from strong (staying home without being ill) to acceptable (avoiding public transportation and increasing personal hygiene). among the explanatory factors considered, coping appraisal was the factor most frequently showing associations (as displayed by odds ratios) with the reported intentions. in a validation analysis (data not shown), we fitted each model fully (including all terms in the five explanatory factors categories trust of information, threat appraisal, coping appraisal, sociodemographic factors, and self-rated health) and calculated the proportion of correctly classified cases for these full models for all eight scenarios. then we left the terms from one of the five categories out separately, and calculated the proportion of correctly classified cases for each of these subset models. we found that the proportion of correctly classified cases without coping appraisal was lower than the corresponding proportion for all full models and lower or equal to the corresponding proportion for 28 of the 32 models excluding one of the other four categories. we interpret these observations combined as indicative evidence that of the explanatory factors considered, coping appraisal was the factor strongest associated with the reported intentions. analogous to our results, a recent british web-based survey of university employees found that coping appraisal was the principal predictor of variability in protective intentions during pandemics [21] , and response costs have been reported as the largest predictor for emergency nurses not reporting to work during an influenza pandemic [28] . a contributing influence to the lesser relative importance of threat appraisal suggested by our results may be a scandinavian tendency to perceive risks lower than in other countries [29] [30] [31] . one of the explanations for this tendency is that the media in scandinavia appear to report more about risks abroad with less attention to risk inside the country [29] . in contrast to our results, self-efficacy during the a(h1n1)pdm09 outbreak in hong kong was found to be only weakly associated with social distancing [25] . however, hong kong residents are limited in their ability to avoid crowds, and the relatively mild impact of the outbreak could have led to the notion that people saw no reason to jeopardize their economic well-being and curtail other social activities. a socio-geographic theory of protective behaviors during infectious disease outbreaks suggested that efficacy beliefs of chinese living in the uk and the netherlands were comparable to those of native uk and dutch residents during the sars outbreak in 2003 [32] , indicating that country of residence is more important than ethnicity or country and culture of origin in determination of protective behaviors. however, with coordinated regional disease control efforts and increasing influence from social media, this may change. gender was the sociodemiographic characteristic that showed the strongest association with the observed variation in reported intentions. as also found in a norwegian study from the same time table 4 . simple and multiple logistic regression models of explanatory factors for the intention to avoid using public transport displayed by influenza outbreak scenario. period [33] , the swedish women in this study were more disposed to enhance their protective behaviors related to personal hygiene than were men. one explanation of this finding could be an interaction with concerns about the consequences of getting infected. a recent study from the u.s. reported that women were more worried than men about getting seriously ill or even dying during a severe influenza outbreak [34] . however, no gender differences with regard to threat appraisal were reported from the norwegian study [33] . originally, we did not include interaction terms in our statistical analyses. a secondary analysis (data not shown) did not reveal any statistically significant interaction between gender and any threat or coping appraisal item such that omitting the interaction from the model would disturb the estimation of the main effects. therefore, an alternative explanation of our findings is that the female respondents were more disposed to enhance their protective behaviors related to personal hygiene than the male respondents because swedish women purchase and use hygiene products more often than men [35] , and, in consequence, were more confident about the practical handling of handwash and liquid soap. conversely, men were more inclined to stay home without being ill during influenza outbreaks. this could be explained by the fact that fewer of the employed swedish men (12%) than women (46%) were at the time of the study working in caring or educational occupations that require physical presence at the workplace, such as nursing, child care, and teaching [36] . in other words, a larger proportion of men could consider the possibility of staying home while continuing to work during an ongoing influenza outbreak, which was not an option for many women. these findings indicate that more research is needed to understand gender-related differences in protective behavior during influenza outbreaks. the main strengths of this study are its foundation on a current theoretical model [14] and a relatively large representative sample of the swedish population. however, the study has also important limitations that must be taken into consideration when interpreting the results. the demographic characteristics available may not be the most important factors biasing the results. for instance, it is possible that individuals with low trust in official information about influenza were under-represented, and anxious individuals worrying about disease risks were over-represented, among the participants. moreover, interpreting cross-sectional data on protective behaviors is difficult because they confound the motivation and accuracy-associated aspects regarding the causaltemporal relationship between perception and behavior [37] . the motivational hypothesis assumes that high perceived risk leads people's intention to adopt protective behaviors, while the accuracy hypothesis suggests that people who act in a more risky way should also feel more at risk. as an example, individuals having physical contact with many people through their occupation may have been aware of that daily routines are associated with a higher risk for getting infected. accordingly, a negative correlation may indicate accurate relative risk perceptions, i.e. that people are aware of their risk status [20, 37] . further longitudinal studies of protective behaviors during influenza outbreaks are thereby warranted [38] . another limitation is that we assessed self-reported intentions rather than objectively measured behavior. nevertheless, intentions are a well-validated proxy for behavior predicting a moderate amount (30-42%) of the variance in actual behavior across a wide range of contexts [39, 40] . moreover, proponents of dual-process health behavior models have suggested that analytic central and emotional-heuristic processes work in concert to select decisions [14] , and under certain circumstances emotions may even be the dominant force [41] . while the pmt used in this study does include an emotional component, it still represents a cognitive appraisal model in assuming that cognitive risk assessment determines experience of fear. such a model is naturally applicable for the study of behaviors aimed at fending off long-term disease, where fear is likely to be less imminent and therefore secondary to more rational reflections about gains and losses related to protective behavior. however, in an acute threat situation, like a severe influenza outbreak, emotional aspects might gain more immediate importance. this would even be more likely during periods of scientific uncertainty, when fewer facts are available. it is in this context interesting to note that coping appraisal in this study was found to be the motivation factor that contributed most to the discriminatory performance despite the fact that threataffect was included in the general model, although indirectly through cognitive assessment. however, what role affect-or emotion-based judgments play in interaction with threat and coping appraisals is still an issue in need of clarification. finally, it should be noted that there were relatively small differences between the reported intended behaviors associated to the mild and severe scenarios, respectively. one explanation of this observation can be the fact that the a(h1n1)pdm09 outbreak was relatively mild in sweden, and that the respondents, wrongfully, related the severe scenario to their recent personal experience rather than the scenario description. however, the lack table 6 . simple and multiple logistic regression models of explanatory factors for the intention to wash hands after touching common objects displayed by influenza outbreak scenario. of difference can also be seen as a sign of its own, i.e. that the swedish population may not be fully aware of the seriousness of a full influenza pandemic. failure to monitor the beliefs and attitudes of the public has recently been identified as a weakness in preparedness strategies against infectious disease outbreaks [42] . we examined how items in a general explanatory model of intended health behavior were associated with personal hygiene and social distancing practices following a questioned mass vaccination campaign against influenza in the swedish population. we observed a relatively high trust in official recommendations and a higher proportion of intentions to improve personal hygiene than those used to increase social distancing. among the explanatory factors considered, coping appraisal was the factor most frequently included in models explaining self-reported intentions. variations in threat appraisal played a smaller role in these models despite the uncertainties surrounding the mass vaccination during the a(h1n1)pdm09 outbreak. the results also show that not just from a third world perspective [43] it is necessary to consider that not all population sub groups have the same predispositions to enact specific behaviors to protect their health. for instance, they suggest that further studies are needed of gender differences in protective behaviors during influenza outbreaks. we conclude that developing interventions that support the general population's efforts to perform self-protective behaviors during influenza outbreaks and longitudinal studies of such interventions across several influenza seasons are warranted also in european countries. text s1 interview guideline for collection of data on perceptions associated with precautionary behaviors. 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netherlands, compared to the general population in these countries influenza-like illness in norway: clinical course, attitudes towards vaccination and preventive measures during the 2009 pandemic deriving behavior model parameters from survey data: self-protective behavior adoption during the 2009-2010 influenza a(h1n1) pandemic energy consumption by gender in some european countries statistics sweden (2012) women and men in sweden. facts and figures 2012. stockholm: statistics sweden use of correlational data to examine effects of risk perceptions on precautionary who takes precautionary action in the face of the new h1n1 influenza? prediction of who collects a free hand sanitizer using a health behavior model how well do the theory of reasoned action and theory of planned behaviour predict intentions and attendance at screening programmes? a metaanalysis does changing behavioral intentions engender behavior change? a meta-analysis of the experimental evidence responses to information about psychosocial consequences of genetic testing for breast cancer susceptibility: influences of cancer worry and risk perceptions developing pandemic preparedness in europe in the 21st century: experience, evolution and next steps delivering interventions to reduce the global burden of stillbirths: improving service supply and community demand item specific for intention to wash hands after touching common objects. 1 formal education past high school/secondary school. doi:10.1371/journal.pone.0091060.t006 key: cord-257489-ruf4rzxm authors: kee, sae yoon; lee, jin soo; cheong, hee jin; chun, byung chul; song, joon young; choi, won suk; jo, yu mi; seo, yoo bin; kim, woo joo title: influenza vaccine coverage rates and perceptions on vaccination in south korea date: 2007-06-28 journal: j infect doi: 10.1016/j.jinf.2007.04.354 sha: doc_id: 257489 cord_uid: ruf4rzxm objective: this survey was performed to assess the level of influenza vaccine coverage, to understand the driving forces and barriers to vaccination and determine vaccination interventions for the following year in korean population. methods: a national sample of 1720 community dwelling adults of age 18 and older were surveyed by individual visits during april 2005. demographics, state of influenza vaccination, reasons for vaccination or non-vaccination and perceptions on vaccinations were asked by questionnaire. results: influenza vaccination coverage in general population and high risk group was 34.3% and 61.3%, respectively. predictors for vaccination were ≥65 of age, performance of regular exercise, vaccination in the previous season, experience of influenza-like illness, belief that vaccine can prevent common cold and opinion that vaccine must be taken annually. the most common reason for vaccination for both whole population and high risk groups was to prevent both influenza and common cold, while the most common reason for non-vaccination was the thought that he/she was healthy enough not to be in need for vaccination. having more information on influenza and vaccination as well as doctor's recommendation for vaccination appeared to be the most important modus operandi to encourage influenza vaccination among non-vaccinees. conclusions: doctor's recommendation was the most important factor in encouraging people to be vaccinated against influenza. doctors should be geared up with precise information and actively encourage high risk population in order to increase vaccination coverage. summary objective: this survey was performed to assess the level of influenza vaccine coverage, to understand the driving forces and barriers to vaccination and determine vaccination interventions for the following year in korean population. methods: a national sample of 1720 community dwelling adults of age 18 and older were surveyed by individual visits during april 2005. demographics, state of influenza vaccination, reasons for vaccination or non-vaccination and perceptions on vaccinations were asked by questionnaire. results: influenza vaccination coverage in general population and high risk group was 34.3% and 61.3%, respectively. predictors for vaccination were !65 of age, performance of regular exercise, vaccination in the previous season, experience of influenza-like illness, belief that vaccine can prevent common cold and opinion that vaccine must be taken annually. the most common reason for vaccination for both whole population and high risk groups was to prevent both influenza and common cold, while the most common reason for non-vaccination was the thought that he/she was healthy enough not to be in need for vaccination. having more information on influenza and vaccination as well as doctor's recommendation for vaccination appeared to be the most important modus operandi to encourage influenza vaccination among non-vaccinees. influenza causes significant morbidity in both healthy population and patients with high risk conditions. healthy adults may suffer from high fever, headache and myalgia, whereas clinical manifestations are more serious in high risk patients such as elderly or patients with comorbid conditions and may even cause death due to respiratory complications. 1e3 the clinical course of influenza differs by age, immune status, characteristics of circulating influenza strains, comorbidities and pregnancy status. 4 changes at antigenic sites of influenza virus render a new strain that can avoid the immunity induced by previous strains, thus causing influenza epidemics. 5 the most effective way of preventing influenza is to immunize with vaccines made after prediction of antigenic variation. in one study, inactivated vaccine showed efficacy of 86% reduction in influenza-like illness in healthy adults when vaccine strain was well matched with predominant circulating strain. 6 although antibody production rate is lower in people over the age of 65, various studies proved influenza vaccine to be effective in reducing influenza related diseases and complications, hospitalizations and mortality in this group. 7e11 the priority group who are recommended for annual vaccination includes persons aged !65 years, persons with chronic illness such as chronic cardiopulmonary disease, diabetes, chronic liver disease and malignancy, residents of long term care facilities, health-care personnel and pregnant women. 12 the center for disease control and prevention is expanding the priority group for vaccination in recognition of the significance of influenza and importance of vaccination. the priority group for influenza vaccination have been also expanded in korea; pregnant women and persons aged 50e64 years were newly added in 2003 and children of age 6e23 months were added in 2004. 13 people working in organizations dealing with sars (severe acute respiratory syndrome) have been newly added in response to the movement of cdc. influenza vaccine production and import are increasing in korea; while vaccines for 8e10 million people, which can cover about 19% of total population, were supplied in the season 2002e2003, vaccine for 15 million people were distributed in the season 2003e2004. in the season 2004e 2005, vaccines for 17 million people were supplied, and according to the sales statistics, it is estimated that 33% of total population have been vaccinated. 13 these percentages are comparable to other countries: fedson 14 reported in 2000 that influenza vaccine distribution per 1000 population was 183 doses in korea, and this number is relatively high compared to northern america (265 doses), western europe (170 doses), southeast asia (0.04 dose) and worldwide (37 doses). 15 vaccine distribution rate grew even higher to 359 doses per 1000 population in 2004. 13 korea shows relatively high influenza vaccine distribution rate, however, exact vaccination coverage among total population or priority group have not yet been studied. the korea centers for disease control and prevention set a goal to increase vaccination rate in the priority group to reach at least 60%. 13 nevertheless, vaccination coverage rate has been calculated according to the sales record, and nationwide vaccination rate by self-report of the whole population or priority group has never been studied. precise identification of vaccination rate in the whole population as well as high risk groups is urgently needed in order to accomplish objectives of influenza vaccination policy. therefore, the aim of this study was to investigate the level of influenza vaccination coverage in adults and high risk groups, identify factors related to vaccination and opinions about influenza and influenza vaccine, and discover the way to increase vaccination coverage. this is a population based cross-sectional descriptive study. the target study population included non-institutionalized persons aged !18 years living in south korea. the survey was conducted by gallup korea â , a professional research company, and face-to-face interviews were performed by 80 trained professional interviewers from 19 to 29 april 2005. in order to represent the total population, multistratified random sampling according to the principle of proportionate probability sampling was adopted to select the subjects. south korea is divided into eight provinces and seven cities and each province or city is further subdivided and stratified into 4e5 units. the number of households to be interviewed in each administrative district was calculated and decided proportionately according to the location and sizes of the district, age and gender. the statistics of 2005 from the national statistical office was used for the calculation. 16 if the selected household could not be surveyed, an alternative household was chosen in the same manner. before the interview, the interviewer explained the purpose of the study to all the subjects and verbal informed consent was obtained by respondents who agreed to participate. the questionnaire contained 22 questions. data on demographics such as age, gender, level of education, and level of income were obtained. questions about drinking, smoking and exercise habits and comorbid conditions were asked. the interview continued with asking whether or not the respondent was vaccinated in the season 2004e2005 and 2003e2004. if the respondent was vaccinated in the season 2004e2005, further questions on the reason of vaccination was asked. thirteen reasons were presented, and respondents were to choose as many as they wish. for non-vaccinated respondent, the reasons of non-vaccination were asked in a form of multiple choice questions with 15 reasons. six yes-or-no questions on opinions about influenza vaccine were presented to all respondents. further 11 yes-or-no questions about opinions on influenza and influenza vaccination were presented to high risk group. all respondents were asked whether they intended to have vaccinated in the following season. regular exercise was defined as performing exercise more than once a week, smoker as currently smoking, and regular alcohol consumer as drinking alcohol more than twice a week. high risk group was defined as either age ! 65 years or having comorbid conditions. comorbid conditions included cardiovascular diseases such as congestive heart failure and myocardial infarction, diabetes, lung diseases including asthma and chronic obstructive pulmonary disease, chronic liver diseases including chronic hepatitis and liver cirrhosis, and malignancy. univariate analysis of factors associated with vaccination/non-vaccination was performed using c 2 test and fischer's exact test. to describe statistical significance, the 95% confidence interval (ci) was computed. in logistic regression, gender, presence of comorbid conditions, age (!65), level of education, size of dwelling town, monthly income, smoking habit, drinking habit, exercise habit, vaccination in previous season, history of influenza-like illness and six opinions about influenza were included. all statistical analyses were performed using the spss 10.0ko for windows (spss inc.) of the total responses from 1730 subjects, 10 insincere responses were excluded and, therefore, data of 1720 (99.4%) subjects were analyzed. mean age was 43.1 ae 14.59 years and 848 subjects (49.3%) were male. one hundred and seventy-four subjects (10.1%) were !65 years and 224 subjects (13.0%) had one or more comorbid conditions; 328 subjects (19.1%) were classified as high risk group. the coverage rates for influenza vaccination were 34.3%, 61.3%, 79.7%, and 54.9% among total adult population, high risk group, persons aged !65 years and persons with comorbid conditions, respectively (table 1) . influenza vaccination coverage was higher in females, increasing with age (18.3% in age 18e29 years versus 84.8% in age ! 70 years), and in persons with any comorbid condition. as for the socio-demographic variables, the likelihood of receiving the vaccine increased when the education level was lower, the size of town was smaller, and the income level was lower. persons on regular exercise, non-smokers and not so regular alcohol consumers showed higher vaccination rates compared to subjects not doing regular exercise, current smokers and regular alcohol consumers, respectively. persons who had been vaccinated in the preceding season (2003e2004) and those with a history of influenza-like illness also showed higher rates (table 1) . reasons for vaccination among vaccinees are described in table 2 . the most common reason for vaccination in total population was 'to prevent not only flu but also common cold' (79.5%), followed by 'influenza being a serious disease' (29.0%), 'recommendations from friends or family members' (22.2%) and 'received information from mass media' (11.5%). reasons such as 'having seen people get sick/ die from flu' (11.2%), 'not in good health' (10.7%), 'doctor's advice' (7.8%), 'have chronic disease' (5.4%) were among less common reasons. the most common reasons for vaccination were not different in high risk group, however, 'have interest in vaccination because of bad health status' showed higher rank (18.4%) than the total population. reasons for refusal among non-vaccinee are described in table 3 . in total population, 'perception of good health' was the most common cause of non-vaccination (70.5%) followed by 'not enough time' (26.0%), 'troublesomeness of vaccination' (18.4%), 'distrust in the effectiveness of vaccine' (11.3%), and 'missed vaccination time' (10.5%). the rank of reasons for non-vaccination was not different in high risk group but less people (58.3%) chose 'in good health' as the reason. factors influencing future vaccination are summarized in table 4 . 'more information on importance of vaccination' (27.7%) was the most common factor to increase the drive for vaccination, followed by 'recommendation from doctors or nurses' (27.4%). in the high risk group, this rank was reversed, and doctors or nurses' recommendation was the most influential factor for future vaccination (38.1%). other options included 'if vaccine is cheaper' (18.8% in total population and 21.3% in high risk group), 'if more information is provided about influenza' (18.8% and 15.0%, respectively), 'if i have enough time' (18.0% and 18.9%, respectively), 'if i can get vaccinated at workplace' (8.1% and 10.2%, respectively) and 'if there is a way other than shots' (2.9% and 5.5%, respectively). of the total population 17.3% and of the high risk group 16.5% were negative about getting vaccinated and answered 'i would not take it in any situation'. opinion about influenza vaccine is described in table 5 . more than 60% of both vaccinees and non-vaccinees agreed that 'vaccine can prevent influenza' and 'vaccine is safe'. more vaccinees compared to non-vaccinees agreed that 'vaccine can prevent common cold' and 'vaccine should be taken annually'. however, more non-vaccinees thought vaccine was expensive. less than 20% of vaccinees and non-vaccinees thought that 'you never get influenza once you are vaccinated'. in all opinions, the difference in the percentages of vaccinees and non-vaccinees were statistically significant. further questions were presented to persons of the high risk group. most of both vaccinees and nonvaccinees agreed that complications of influenza might be serious (93.5% and 90.6%, respectively) and that they had chance to hear about influenza and influenza vaccination from mass media (77.1% and 70.9%, respectively). however, more vaccinees of the high risk group compared to non-vaccinees agreed on the following opinion; influenza might be dangerous to high risk group, influenza might aggravate underlying diseases, vaccination might reduce chances of hospitalizations, and vaccination might reduce expenses for extra medication. furthermore, more than 60% of vaccinees agreed that they were at high risk of catching influenza, and at bad health, and that acquaintances advised them to get vaccinated, however, less than 50% of non-vaccinees agreed on the same opinion. in comparison, nearly 50% of non-vaccinees thought themselves to be in good health whereas only 12.4% of vaccinees thought the same way. also, although more vaccinees than nonvaccinees were advised to get vaccinated, it was less than 50% in both groups (table 6) . results of multivariate analysis to determine factors associated with vaccination are summarized in intention for vaccination in the next season was as follows: 43.3% of total subjects and 68.9% of the high risk group were willing to get vaccination. self-reported influenza vaccination coverage of 34.3% in this study corresponded well to the percentage estimated from the number of vaccine doses sold (33%). 13 moreover, the coverage in high risk group met the target set by korean cdc (>60%). these coverage rates in korea in the season 2004e2005 is relatively high, compared to the coverage in the united states (8.8% in whole population and 42% in high risk groups) and europe (19e24% in priority group 17 ). nevertheless, the rates are not satisfactory enough, because who set the goal of attaining vaccination coverage of the elderly population to at least 50% by 2006 and 75% by 2010 18 and more efforts are needed to increase the coverage rates. in univariate analysis, people of older age or persons having comorbid condition were more likely to get vaccinated, which is in concordance with studies from other countries. 19e21 since these two groups are the main target for vaccination, it implies that vaccination program in south korea is quite successful. people with healthy lifestyle habits such as regular exercise, non-smoking and no regular alcohol consumption also had higher vaccination rate. people with healthy lifestyle may have more interest in general health, seek for preventive health care and therefore are more willing to get vaccinated. vaccination coverage in females was significantly higher in univariate analysis, and similar result was shown in another study. 17 the fact that more females (56% versus 44% males) were !65 years who had higher vaccination rate might be the explanation in south korea. interestingly, vaccination coverage was higher among people of lower education level, and lower income and living in smaller towns. this may be partially explained by the fact that both persons !65 years and persons with chronic illnesses are more likely to be undereducated and have lower income, as is shown by south korean statistics, 22 and similar results were also presented by jimenez et al. 23 the government policy to administer influenza vaccine free of charge to low income group at public health centers may be another explanation: survey showed that people vaccinated at public heath centers were older, and had lower level of education and were living in a smaller town (data not shown). to prevent common cold as well as flu was the most common reason for vaccination. this is concordant with the high percentage of agreement (62.2%) that vaccine can prevent common cold. also, the perception that 'influenza vaccine can prevent common cold' was a predictor for vaccination. this idea might have been responsible for the increase of vaccination rate, however, wrong attitude due to wrong knowledge must be corrected. self-perception of bad health, interest in vaccination and chronic illness were common reasons for vaccination in high risk group, showing their interest in health. 'confidence in health' was the most common reason for non-vaccination (60%) in both all adults groups and high risk groups, followed by 'being too busy', 'because it is troublesome' and 'miss vaccination time'. among nonvaccinees with non-vaccination reason of 'miss vaccination time', 49% were willing to get vaccination in the following season. 'not believing in the effectiveness of vaccination' accounted for about 11% of the responses. these results led us to suggest some intervention to increase vaccination uptake: more efforts should be paid to convince people in the priority group who are at high risk, and to provide information on influenza and effectiveness of vaccination to increase vaccination motive. also, improvement of accessibility to vaccines such as providing vaccination at workplace may contribute to an increase of vaccination uptake. less than 1% of non-vaccinees reported 'side effects of vaccination' or 'fear of getting influenza by vaccination' as the reason for non-vaccination. the above result is different from other studies 21, 24 and implies that people have correct knowledge on side effects of vaccines. health-care workers' recommendation for vaccination was the most important factor to influence future vaccination habit in high risk group, in agreement with other studies. 20,21,23,25e27 booth et al. 28 reported that 71e82% of general physicians recommend vaccination to priority group, and song et al. 29 showed that reminding persons of age ! 65 to get flu shots by telephone calls or postcards significantly increased vaccination rate. in this present study, recommendations to the high risk groups by doctors and public health centers were 10% and 3%, respectively, inferring that recommendation rate from doctors in clinical practice is very low. perenboom and davidse 30 reported that active recommendation to persons with chronic illness increased the rate of vaccination from 42% to 75.5%. therefore, the role of health-care workers, especially doctors, appears to be very important in increasing vaccination rate, and therefore, they should give active recommendations. in the high risk group most of the persons were aware of the fact that influenza is a serious disease and it may be more dangerous or produce more complications in persons with chronic illness. furthermore, more than half of them believed that influenza vaccination might reduce hospital admission and extra medical expenses, showing that they have correct perception on influenza and influenza vaccine. however, while more than 60% of vaccinees in the high risk group agreed that they were not in good health and at high risk of catching influenza, and they are interested in vaccination because of bad health, only 28.3% and 42.5% of non-vaccinees, respectively, agreed on that. also, 48.3% of vaccinees were advised to get vaccinations while only 28.3% of non-vaccinees did receive the advice. this shows apparent difference in the perception of one's health between vaccinees and non-vaccinees and, therefore, efforts should be made to inform people about the priority group of vaccination in order to increase coverage rate. forty-three and three-tenth of total subjects and 68.9% of the high risk groups were willing to get vaccination in the coming season, and the percentage in high risk groups exceeds the rate in the season 2004e2005 as well as the target of korea cdc (61.3% and 60%, respectively). 13 persons who had been vaccinated previously were more willing to have vaccination in the following season (table 7) , and this correlates with other studies 24,31,32 that previous vaccination is the most significant predictive factor for future vaccination. moreover, belief that 'vaccine must be taken annually' was a predictor for vaccination. efforts to increase vaccination rate in priority group for at least one season may have influence over vaccination for several years. this may be particularly useful in the situation of vaccine shortage, when it is recommended by authorities that supply of vaccines should take precedence to priority group. 33 the strength of this study lies in the fact that survey was conducted on individual interview basis and meanings of questionnaire were explained thoroughly even to the elderly, and thus receiving precise answers. there are some limitations in the study. first, high risk group consisted of only persons !65 or persons with comorbid condition, and therefore, the whole priority group were not included in the analysis. secondly, the survey was conducted in april, when it was past the influenza season and therefore recall bias might have occurred. thirdly, the presence of comorbid condition and vaccination uptake were totally relied on self-reports of the subjects and therefore actual presence of illness or vaccination uptake might have been over-or under-estimated. in summary, the significance of influenza and importance of vaccination were well perceived, especially, among the high risk groups and 43.3% in total population and 68.9% of the high risk group showed intention to have vaccination, which is very encouraging. since giving correct information and health-care personnel's recommendation to vaccination would greatly influence vaccination rate, doctors should be geared up with precise information and actively recommend them to get influenza vaccinations. impact of influenza on mortality in relation to age and underlying disease influenza-attributable mortality among the elderly in switzerland population-based study on incidence, risk factors, clinical complications and drug utilisation associated with influenza in the united kingdom 1918 influenza pandemic caused by highly conserved viruses with two receptor-binding variants effectiveness and cost-benefit of influenza vaccination of healthy working adults: a randomized controlled trial benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens effects of a large-scale intervention with influenza and 23-valent pneumococcal vaccines in adults aged 65 years or older: a prospective study influenza vaccination in community-dwelling elderly: impact on mortality and influenzaassociated morbidity influenza vaccination and reduction in hospitalizations for cardiac disease and stroke among the elderly clinical effectiveness of influenza vaccination in persons younger than 65 years with high-risk medical conditions: the prisma study prevention and control of influenza, recommendations of the advisory committee on immunization practices pandemic influenza and the global vaccine supply the macroepidemiology of influenza vaccination (miv) study group. the macroepidemiology of influenza vaccination in 56 countries korea statistics information system influenza vaccination coverage rates in five european countries-a population-based cross-sectional analysis of two consecutive influenza seasons world health organization. prevention and control of influenza pandemics and annual epidemics (agenda item 14 prevalence and predictors of influenza vaccination among frail, community-living elderly patients: an international observational study influenza vaccination coverage rates in germany factors associated with influenza vaccination among elderly spanish women influenza coverages in spain and vaccination-related factors in subgroup aged 50e64 predictors of influenza vaccine acceptance among healthy adults crosssectional study on influenza vaccination factors affecting influenza vaccination among attendees at a senior center factors associated with influenza and pneumococcal vaccination behavior among high-risk adults implementation of influenza immunisation policy in general practice: 1997 to 1998 effectiveness of telephone and postcard reminders for the influenza vaccination: a study in the elderly who have visited a family practice center in a tertiary care hospital increasing the coverage of vaccination against influenza by general practitioners patient acceptance of influenza vaccination influenza vaccination. knowledge, attitudes, and behavior among high-risk outpatients update: influenza vaccine supply and recommendations for prioritization during the 2005e06 influenza season. mmwr morb mortal wkly rep:850 key: cord-015646-tt2p9uue authors: xue, lan; zeng, guang title: global strategies and response measures to the influenza a (h1n1) pandemic date: 2018-11-24 journal: a comprehensive evaluation on emergency response in china doi: 10.1007/978-981-13-0644-0_2 sha: doc_id: 15646 cord_uid: tt2p9uue as an infectious respiratory disease, influenza is prone to cause pandemics for its fast mutation, easy dissemination, susceptibility to humans, and its elusive nature in terms of treatment. three influenza pandemics occurred in the 20th century which caused huge losses worldwide. analysis by the who, after the global peak in the winter of 2009, there were no signs of any further widespread dissemination of the virus, thus proving the end of the influenza pandemic. nevertheless, the organization warned that entering the post-pandemic period didn't mean the influenza a virus would disappear completely, as epidemic outbreaks were still likely to occur in some regions. additionally possibilities of virus variation were evident and so countries were advised to be on alert during this time. in response to the threat of a global influenza pandemic, the who as per the international health regulations 2005 (ihr 2005) , put a large amount of work into global prevention and control efforts, and also adjusted prevention and control strategy priorities to fall in line with this global influenza outbreak. countries worldwide have been proactive in their responses to the who's strategies and recommendations. in order to tackle possible influenza pandemics and minimize losses, in 1999 the who published its official guidance, the influenza pandemic plan: the role of the who and guidelines for national and regional planning, which was then later revised in 2005 and 2009, respectively. 2 in the 2005 revised who global influenza preparedness plan, an influenza pandemic was divided into six different phases: phases 1-2 are interpandemic, i.e., no new influenza viruses have been detected in humans but an influenza virus subtype is circulating among animals and could potentially pose a threat to humans; phases 3-5 consist of the pandemic alert phases where a new influenza virus has been detected in humans but its spread among humans remains limited; phase 6 is the warning phase, declaring that the new influenza virus has spread widely across human populations. in its 2009 revision of the pandemic influenza preparedness and response, the who retained the use of a six-phase approach, but made some changes to the criteria. phases 1-3 are characterized by the transmission of an influenza virus among animals and few humans, and correlate with preparedness, including capacity building and response planning activities. phase 4 is characterized by sustained human-to-human transmission of an influenza virus, while in phases 5-6 the virus becomes widespread and prevalent among humans. phases 4-6 clearly signal the need for response, prevention, and control measures. during the post-peak period, pandemic activity drops, but there are still possibilities of recurrent outbreaks, before levels finally return to those seen in seasonal influenza 3 periods. these plans from the who were made mainly based on the threat levels from the highly pathogenic avian influenza (h5n1), which are much different from the threats posed by influenza a (h1n1) in 2009, and which are not likely to be the same as future influenza threat levels. these documents have nevertheless played a crucial role in pandemic response efforts and have provided some basic guidance that can be utilized in the outbreak of any infectious disease. the pandemic influenza preparedness and response also summarized the lessons learned from coping with sars and the highly pathogenic avian influenza, which will be a great asset in responding to future outbreaks of infectious diseases. on may 2nd, 2009, the who published its first ever list of countries and laboratories with the capacity to perform pcr (polymerase chain reaction) testing used to diagnose the influenza a (h1n1) virus in humans, which was updated and re-published on may 4th, 2009. the who's criteria for diagnostic capabilities are: "scoring 100% in the last two or more who external quality assurance programme panels (eqap) received by the laboratory; or scoring 100% in the last panel and having a history of consistent results for earlier panels." on the list published were 98 institutions in 73 countries which were able to perform pcr to diagnose the influenza a (h1n1) virus in humans. in response to the outbreak and spread of influenza a, in the initial stages of the pandemic, the who began working on various alert and preparedness plans. on april 25th, 2009, the who held an emergency meeting, swiftly determining the severity of the pandemic situation and announced that it constituted a public health emergency of international concern. 4 on the evening of april 27th, 2009, the who raised the influenza pandemic alert level from phase 3 to phase 4, 5 and again to phase 5 on the evening of april 29th. 6 level was raised to phase 6, the highest level the who has declared in the past 41 years-signalling the onset of a global influenza pandemic. on august 10th, 2010, based on its global assessment, the who removed the phase 6 alert level and announced that the world was moving into the post-pandemic period. 7 while adjusting pandemic alert levels, the who proposed that countries stay flexible in tailoring their specific response measures to their local epidemic situations, and warned that influenza a (h1n1), as highly infectious as it is, would continue to do harm in the infected countries and could potentially spread to more countries. as the virus continued to spread in the southern hemisphere, which was at that time entering winter, the risk of its combination and mutation with other local epidemic influenza viruses increased, and so the international community was still required to closely monitor the situation. in the early days of the pandemic, the who's influenza pandemic assessment team published its assessment results on may 11th, 2009, in which a comparison was made with the 1957 and 1918 pandemics. 8 the assessment came to the following conclusions: this was a new subtype of the influenza a virus; the influenza a (h1n1) virus was likely to become more contagious than seasonable influenza viruses; differences in clinical symptoms were related to the patient's overall health situation; young people were more susceptible to the virus; the mortality rate was expected to be far lower than the 1918 pandemic; and there were still many uncertainties surrounding the pandemic. after the pandemic tapered off, on april 12th, 2010 the international health regulations review committee held its first meeting in geneva to assess the global response and the functioning of the ihr in relation to the pandemic, as well as to summarize related experiences and lessons learned. the assessment work is still under way and completion is expected in may 2011. 9 in addition to its preparation and alert efforts, the who also strengthened pandemic monitoring and introduced a series of strategies and measures relating to pandemic response, treatment, vaccine development, inoculation, and distribution. director-general's opening statement at virtual press conference. h1n1 in post-pandemic period. beginning on april 24th, 2009, when it first published information on the outbreak of human swine influenza in the u.s. and mexico, the who continually released pandemic and epidemiological information to the globe with the intention of facilitating international communication and sharing. from april 24th through july 6th, 2009, during the early days of the pandemic, every day or every other day, the who published new laboratory-confirmed cases and deaths in affected countries, and at the same time it closely tracked the global transmission of influenza a (h1n1). as the pandemic developed, who experts considered that as far as pandemic risk monitoring and response strategies were concerned, continued laboratory virus testing to all patients was no longer necessary, as it could overburden laboratories and thus influence their capacity in caring for critically ill patients and other unusual circumstances. 10 on july 16th, 2009, the who announced that countries affected by the epidemic were no longer required to report new confirmed cases, and recommended that attention be placed on monitoring influenza viruses and unusual epidemic events. but countries where influenza a was not present still needed to report cases as they were discovered. after april 2010, although the increasing rates of the fatality were on the decline and the pandemic activity remained relatively low, the who continued the monitoring of the pandemic and remained in close contact with public health experts in countries across the globe in order to determine whether the virus activity had returned to levels and patterns normally seen for seasonal influenza. global pandemic activity had remained low over the past few months, and there was little evidence of higher pandemic influenza activity than what was normally caused by the seasonal influenza. the transmission of the influenza a virus still persisted in the southern hemisphere, but it was still impossible to determine if countries there had transitioned to levels and patterns expected for seasonal influenza. therefore, the who continued conducting epidemiological monitoring of the global pandemic situation and reported on relevant information. 11 10 zhang (2009 after the outbreak of influenza a (h1n1), the who consulted related pharmaceutical manufacturers about developing vaccines, encouraging worldwide support of influenza a (h1n1) vaccination production. the organization also collaborated with drug authorities in related countries ensuring that newly developed influenza a vaccines met as many safety standards as possible. meanwhile, the organization helped china in efficiently obtaining live strains of the influenza a (h1n1) virus, which accelerated the country's research and development of relevant vaccines and drugs. while ensuring an adequate amount of seasonal influenza vaccines were available, the who also initiated research and development for influenza a (h1n1) vaccinations in the early stages of the pandemic. 18 given that global limited production capacity for antiviral drugs and influenza vaccines could never meet the healthcare needs of 6.8 billion people, the who recommended governments to have clear and targeted prevention and control measures to avoid waste of resources. 19 on july 2nd, 2009, a meeting of the world's health ministers was held in mexico to assess the influenza pandemic and discuss countermeasures and inoculation distribution. at the meeting, who director-general margaret chan called for international collaboration and solidarity, while stressing that special attention must be paid to high-risk groups like pregnant women and patients with chronic diseases. the who also called on vaccine manufacturers to provide them a certain amount of free vaccines so as to help developing countries better cope with their epidemics. 20 in response to the ongoing global pandemic, the who stressed the importance for countries to carry out inoculations and to set forth three goals for their vaccination strategies, i.e. ensuring the normal operation of national healthcare systems, lowering morbidity and mortality, and minimizing possibilities of community-level outbreaks. to ensure continued normal operations of healthcare systems, the who recommended medical workers first be vaccinated, then pregnant women, patients aged six months and older with such chronic illnesses like asthma and obesity, healthy people aged 15-49, healthy children, healthy people aged 50-64, and people aged 65 and older-in that exact order. 21 the who also urged pharmaceutical manufacturers to produce vaccines at full capacity, to ensure fair distribution among developed and developing countries. countries such as china, italy, france, the united states, germany, the united kingdom, norway, sweden, finland, australia, and japan took steps to vaccinate domestic residents, based on their own epidemic situations, healthcare resources, and ability to acquire vaccines. some of the countries placed orders for more vaccines in order to cope with potential outbreaks. 22, 23 response strategies varied widely across countries (see a detailed description in the next section) because each was faced with outbreaks and developments with different characteristics, in addition to political, economic, and cultural dissimilarities, especially in their public health systems which varied in both management and operation. while developed countries already had fairly effective response measures in place thanks to their advanced economic and social development as well as robust healthcare systems, some developing countries with poor economic foundations and weak public healthcare had a much harder time dealing with public emergencies. therefore, they had an even harder time in dealing with influenza a (h1n1). after the pandemic broke out, countries showed varied responses to the who's recommended response strategies and measures; in particular developing countries that had greater reliance on these strategies and measures as well as technical assistance from the who, were much more proactive. there is no doubt that the who played a crucial role in helping countries worldwide-especially developing ones-in coping with the pandemic, whether it is pandemic monitoring, clinical diagnosis and treatment of the virus, or vaccine development and distribution. however, because this pandemic originated in north american countries, taking into account the political, economic and cultural differences between countries as well as their different response capabilities, the who was also faced with new challenges like how to provide tailored guidance to developed and developing countries. the purpose of this guidance was to increase the effectiveness of related strategies and measures, mitigate and contain the spread of the pandemic, and minimize the negative effects of the virus on society and populations. such targeted guidance was not particularly prevalent in their guidance regarding response strategies and measures as the requirements placed on developed countries were quite low, resulting in an overall devaluation of said proposed strategies and measures. therefore, when confronting similar public health emergencies in the future, the who should present more pertinent strategies and tailored measures which could play greater roles in pandemic preparation and response. the outbreak in late april 2009 of influenza a (h1n1) in several north american countries quickly attracted attention in related countries. responding promptly to the crisis, government agencies and related departments in multiple countries immediately initiated public health emergency mechanisms and put into action a wide range of prevention and control strategies and measures. considering the serious economic, social, and public health consequences that could happen due to the outbreak, coping with the pandemic would demand participation, coordinated preparation, and enhanced collaboration from governments and different departments. some countries specifically established unified leadership bodies and related mechanisms to deal with the pandemic, while others did so through existing government bodies or departments. for example, countries like the united kingdom, india, japan, and mexico set up a special coordination and management mechanism, and established an emergency decision-making, command and coordination body which was directed by the heads of government with the guidance and participation of relevant agencies. the british government specifically established a ministerial committee consisting of related government departments to strengthen inter-departmental communication and coordination and ensure the formulation and execution of preparation and response policies. the indian ministry of health and family welfare established the inter-ministerial task force and joint monitoring group for ai/pandemic to direct and coordinate the national response to the pandemic. 24 france's public health emergency mechanism was run by the "inter-ministerial risk group" with 24 dr. shashi khare. pandemic influenza a h1n1: preparedness & response in india. cdc new delhi. http://209.61.208.233/linkfiles/rce_day02_h1n1_india-dr_shashi_khare.pps. the responsibility of decision making, situational tracking, and publicity, and the minister of the interior acted as the lead and was responsible for approving and initiating such decisions. japan established the new influenza response headquarters directed by the prime minister, and transformed the risk management center's information liaison office under the prime minister's official residence into the official residence's liaison office for directing and coordinating national pandemic response efforts. mexico, whom in the past responded to public health emergencies mainly through direct government interventions and temporary emergency groups, established the national committee for health security (cnhs) for analyzing, monitoring, and assessing the security issues of national health policies and for proposing relevant policies. the united states, australia and some other countries didn't specifically establish a governing body in response to the pandemic. after its incorporation in 2003 into the united states department of homeland security (dhs), the federal emergency management agency's (fema's) responsibilities were expanded from natural disaster response to counter terrorism and pandemic diseases. the fema director, appointed by the president, reports directly to the secretary of homeland security and may, in response to a crisis, be summoned by the president to attend ministerial-level meetings and take part in the decision-making process. after the influenza pandemic outbreak in 2009, the united states launched its standard emergency response procedures, which included close collaboration and coordination among the federal, state and local governments along with the private sector. the u.s. congress was charged mainly with funding public health efforts at the federal, state, and local levels, 25 while it was the responsibility of the federal government to update response plans, strengthen the development and revision of community-based plans, and enhance response capabilities. the dhs oversaw the distribution of antiviral medications and the dissemination of pandemic information to the public. 26 the u.s. department of health and human services (hhs), the executive body of pandemic preparation and response, was in charge of deploying, directing, and overseeing various response efforts, and they also completed the following: issued guidance on the influenza pandemic, 27 provided technical, financial, and medical support to states, and based on pandemic analysis announced a national state of emergency. as the national public health institute under the hhs, the center for disease control and prevention (cdc) played a crucial role in virus monitoring, prevention, and control. similarly, australia established a mechanism in which an inter-agency committee under the leadership of the prime minister and the cabinet was in charge of determining the federal government's preparation and 25 weissman (2009 2.3 national and regional response strategies and measures response strategies as well as pandemic countermeasures, 28 with state governments making and implementing relevant policies under the guidance of the federal government. whether or not a governing body was established for management of the pandemic, countries worldwide attached great importance to collaboration among government institutes and departments. for example, interim pandemic assessment reports by u.s. departments all mentioned that the timely response to, and rapid progress made in coping with the influenza pandemic, were due in large part to the clear divisions of labor and close collaboration among federal government institutes, departments, and state, and local governments. 29, 30 the indian government also stressed that pandemic responsibilities did not fall solely on the health department, and that it was necessary for multiple departments to collaborate with one another; the following departments of india were involved in pandemic preparation and response: the ministry of finance which provided cash, budgets, risk management, and insurance; the ministry of commerce and industry which provided medical equipment; the ministry of road transport and highways which was charged with handling relevant transportation and communication issues; the ministry of defense and related military departments which was charged with public services, laws and regulations, security, and human rights; the ministry of information and broadcasting which guaranteed the transparency of strategic communication, the dissemination of information, etc.; the ministry of environment and forests and the ministry of health and family welfare which ensured biosafety, sanitation, wildlife conservation, etc. to effectively curb the transmission of the pandemic and its negative effects on society, many countries formulated a national strategy or plan against possible influenza outbreaks from 2003-2005, outlining the duties and division of labor among government departments as well as their preparation and response strategies. their policies on influenza a (h1n1) were generally built on these strategies. in 2005, pursuant to the pandemic preparedness guidance published by the who, the united states developed the hhs pandemic influenza plan and the national strategy for pandemic influenza, according to which preparation and response strategies and measures would be chosen based upon phases that measured the pandemic's development. included in the documents are detailed provisions about the duties along with preparation and response strategies of related government departments and mechanisms, i.e.: inter-departmental collaboration, 28 council of australian governments/working group on australian influenza pandemic prevention and preparedness. national action plan for human influenza pandemic. 2010. 29 sebellus (2009a) . 30 sebellus (2009b). public risk communication, vaccine production and distribution, and the stockpiling of antiviral medications. in accordance with the who pandemic preparedness guidance, the united kingdom published their influenza pandemic contingency plan in 2005, and their national framework for responding to an influenza pandemic in 2007, which stipulated that strategies and measures for both preparation and response would be selected based upon pandemic phases. in 2005, australia formulated the australian heath management plan for pandemic influenza and later revised it in 2008, and it remains as the country's national-level health plan for an influenza pandemic. 31 india formulated the influenza pandemic preparedness and response plan in 2005, which was used as a foundation for prevention and control policies against influenza a (h1n1). in 2006, the mexican government issued the national preparedness and response plan for pandemic influenza, 32 on which the country's prevention and control policies against influenza a were built. on may 11th, 2009, the japanese government swiftly issued the action plan for measures against influenza a (h1n1) to curb its domestic transmission. this plan contained response measures formulated according to four phases of distinct pandemic phases, i.e. occurrence overseas, early occurrence at home, infection expansion-spread-recovery, and stabilization. for countries across the globe, central governments primarily provided the funds for prevention and control efforts against influenza a (h1n1), and these funds were made available to related departments in the different pandemic phases. during the initial period and at the peak of the pandemic, these funds were mainly used for stockpiling antiviral drugs; purchasing relevant equipment, facilities, protective supplies and other materials; establishing points of distribution for antiviral drugs; providing patients with free antiviral drugs; and carrying out pandemic monitoring. during post-peak periods, funds were mainly utilized to purchase unified influenza a vaccines from manufacturers, which were then distributed to the public with no charge. 33 some developed countries also specifically established foreign assistance funds that provided developing countries both monetary and material assistance in combatting the pandemic. the united states congress invested heavily in pandemic prevention and control. in 2006, the congress provided an appropriation of more than seven billion u.s. dollars (usd) for implementing the pandemic preparedness strategy. on april 28th, 2009, the u.s. president received another appropriation of 1.5 billion usd from congress which was specifically designated for combatting the swine flu. in july of that year, congress provided 1.85 billion usd to be used as funds for emergency resource deployment and an additional 5.8 billion usd for emergency preparation and response against the influenza pandemic. 34 in september, the congress went on to make 1444 million usd available to states and hospitals for carrying out vaccination programs. 35 meanwhile, the united states agency for international development (usaid) provided mexico with five million usd in emergency aid funds, 900,000 sets of personal protective equipment for virus monitoring personnel, and tamiflu for 400,000 courses of treatment. additionally, the hhs provided 147 countries with 769 laboratory diagnostic kits, and donated to the pan american health organization (paho) medications for 420,000 courses of treatment in aid of latin american and caribbean countries. in australia, funds for prevention and control against influenza a (h1n1) originated mainly from the federal government, which was used specifically for monitoring pandemic development, stockpiling and distributing antiviral drugs, training medical personnel, providing free vaccinations for citizens, and assisting developing countries with prevention and control efforts. the federal government spent 43 million usd on antiviral drugs, 1.4 million usd on the purchasing of automatic detection equipment for the national influenza center and other public health laboratories, 4 million usd on training general practitioners across the country, and 3 million usd on a donation to the who which was used in aiding developing countries, especially those neighboring australia, with pandemic monitoring, detection, preparation and response. in the united kingdom, funds for responding to influenza a (h1n1) came mainly from the british government; by january 20th, 2010, the department of health had dispensed to the nation 1.26 million doses of pandemrix, an influenza vaccine developed by glaxosmithkline, and 370,000 doses of a baxter-developed vaccines. 36 the indian government established a one billion rupee disaster response fund in accordance with the disaster management act, which was administered by the ministry of home affairs, and this disaster fund accepted donations from individuals and organizations. in addition, a national disaster fund was specifically established to finance disaster relief and recovery efforts. state governments also 34 william corr (2009 established disaster response funds and relief funds in accordance with the law at the state and regional levels. 37 mexico invested a total of 350 million usd in influenza a (h1n1) preparation and response, including the purchasing of drugs and vaccines, and the adoption of other prevention and control efforts. 38 declaring a state of emergency helped the hhs prepare for and respond to the influenza pandemic, and prompted the food and drug administration (fda) to issue emergency use authorizations (euas) for the use of antiviral drugs and therapeutic tools-i.e. they approved the use of relenza and tamiflu as stockpiled antiviral drugs for prevention and control of the virus, rt-pcr for virus detection, and n95 masks, which protected pandemic-affected communities. on april 29th, 2009, in light of the who's pandemic alert phases and its national pandemic situation, singapore raised their alert level in its five-level disease warning system from green to yellow, and again to orange the next day. to prevent the influenza virus from spreading into and circulating within their territories, many countries adopted strict inspection and quarantine measures in the early days of the pandemic. baggage and raw meat products from epidemic affected areas-were strictly quarantined; many airlines required their service staff to observe and question passengers suspected of illness, and when necessary, have them examined. american border officials between the united states and mexico also were required to examine the physical condition of travelers crossing the border and be prepared to take necessary measures. additionally, citizens were asked to stop all unnecessary travel into epidemic areas. australia also implemented strict border control, requiring all flights from the americas to report the health status of passengers on board before landing; any individual with influenza-like symptoms had to be assessed by australian quarantine authorities in order to determine if further treatment was required; eight major airports across the country were equipped with body temperature measuring instruments, and every incoming passenger was required to complete a health declaration card. india adopted pandemic monitoring measures at airports, sea ports, and inland ports across the country; all incoming passengers to the twenty two international airports were screened, especially those from epidemic areas or with influenza symptoms, who were then quarantined and treated for at least three days. medical personnel were trained in advance, and were required to wear masks, gloves, and protective clothing at work. influenza a (h1n1) inspection standards and operational rules were formulated and implemented national widely at that time. japan's ministry of health, labor and welfare required all flights from mexico, united states, and canada arriving at the narita, kansai and chå«bu centrair international airports be inspected while aboard the plane. local airports not included on the list of airports for quarantine measures, for example in niigata, akita and hiroshima, also decided to follow suit and expanded the scope of quarantine to include flights from south korea, hong kong, and some other countries and regions. japanese border inspection and quarantine authorities screened people from mexico, the united states and had cargo strictly quarantined, especially baggage and raw meat products from epidemic areas. while applying strict control measures against the importation of the virus, in the early days of the pandemic countries also began strengthening preparation capacity building. for example, in the united states, during the initial stage of the outbreak, the hhs dispensed medication from the strategic national stockpile enough to treat three million people, the department of defense (dod) separately readied enough medication for seven million soldiers, and the cdc allocated antiviral drugs, protective equipment, and testing kits. at the same time, the hhs provided training for medical personnel with the goal of enhancing their abilities in treating and handling the pandemic. the german government required each state to stockpile enough antiviral drugs to use for 20% of their populations. south korea increased budget spending so that by the end of october the country's had enough drugs stored for 10% of its population. in an effort to mitigate the spread of the virus, the indian government designated specific hospitals to treat influenza a (h1n1) cases. to increase public awareness of the pandemic, countries developed large scale health education and communication projects. the u.s. cdc provided health recommendations to society, communities, clinical workers and other professionals, and launched an online live-broadcast health education program, "know what to do about the flu," to help strengthen the public's abilities in protecting themselves against the virus. the united kingdom updated pandemic situations and work priorities on a regular basis via an official government website, and provided technical support relating to virus prevention and treatment. india published a "public notice' through national media channels with the aim of disseminating knowledge and increasing public awareness of influenza a (h1n1) prevention and control. the government also set up a toll-free service hotline to answer questions about the influenza pandemic. in japan, an information, education and communication campaign was launched targeting high-risk groups of people arriving at and departing from the country's international airports, and the ministry of health, labour and welfare opened an information window to answer questions from the public. as the pandemic developed and more cases emerged, it was found that the majority of cases were coming from local communities instead of from abroad. at this point in time, the continued use of containment strategies had been ineffective, and medical personnel were having to dedicate more time and energy to the increasing number of patients. according to the national response framework, the hhs in the united states needed to stockpile enough antiviral drugs for one-fourth of the country's population during the pandemic, and to prepare at least six million treatment courses during the pandemic's initial phase. in the spring of 2009, the hhs allocated eleven million treatment courses that could be used for rapid response against the pandemic. the cdc and the fda also worked together to address potential options for treatment of severely hospitalized patients. 41 in october, the hhs shipped an additional 300,000 bottles of the antiviral oseltamivir in oral suspension formula to 41 anna schuchat (2009a). 2.3 national and regional response strategies and measures states in order to mitigate a predicted national shortage. the fda worked closely with the cdc, the office of the assistant secretary for preparedness and response (aspr), manufacturers, and others to increase production and availability of personal protective equipment such as gloves, masks, and respirators. at the same time, the 2009 influenza (h1n1) consumer protection team, established by the fda, put in place an aggressive strategy to combat fraudulent influenza products. 42 the british secretary of state for health declared on july 2nd, 2009, that the united kingdom's response efforts were transitioning from a "containment phase" to a "treatment phase." in order to cure patients more efficiently, the british government created a national stockpile by the purchasing of more antiviral drugs, and drug distribution centers were also established across the country, with the national health service (nhs) playing a leading role in treatment provisions. to relieve pressure on medical institutions, on july 23rd, 2009, the british government launched the national pandemic flu service (npfs). 43 the npfs was a self-help healthcare system which, through a dedicated website and call centers, provided people worried about flu-like symptoms with professional assessment services, including the suggestions on whether they should receive treatment or contact a general practitioner, etc. a person, if assessed as indeed having influenza a (h1n1) symptoms, would be given an authorization number by the system, which he or she could use to pick up antiviral drugs from one of local distributions centers. the launch of this system effectively mitigated the pressure on primary healthcare institutions and allowed general practitioners to dedicate their attention to critically ill patients. in order to quickly detect and treat critically ill patients, and also to ensure an adequate number of hospital beds as the number of cases increased, the japanese government readjusted its guidelines on pandemic response efforts, and discarded the practice of classifying regions according to rate of transmission in that area. according to the revised guidelines, regular hospitals received patients infected with influenza a (h1n1); all mildly ill patients were instructed to medicate and rest at home, rather than being hospitalized. for patients with asthma or other illnesses whom had contracted influenza a (h1n1) and whose conditions were likely to worsen, a pcr (polymerase chain reaction) test or other influenza a virus test was performed, and effective antiviral drugs were administered as early as possible. when necessary, decisions would be made to get them hospitalized. japan gradually used the confirmed cases reported from a certain number of hospitals as estimations and predictions for that area's infected population. australia used antiviral drugs from their national medical stockpile to treat moderately and critically ill patients, especially those with severe breathing difficulties or those whose conditions were rapidly worsening. all medical personnel, who contracted influenza a (h1n1) and developed moderate symptoms of 42 jesse goodman (2009 infection, or were more prone to develop serious symptoms, were eligible for antiviral treatment. patients with fairly mild symptoms were encouraged to self-medicate. india issued clinical management guidelines, where the indian committee on infectious diseases published guidance on the screening and clinical treatment of laboratory-diagnosed cases of influenza a (h1n1); the ministry of health and family welfare issued guidelines on family isolation, clinical examinations, and hospitalization by categories of influenza a (h1n1) cases-where categories a/b patients were asked to be isolated and reduce contact with their family and others and category c patients required immediate hospitalization. all suspected cases were tested at the national institute of communicable diseases (nicd) in new delhi, or at the national institute of virology in pune, and then examined further at relevant laboratories. india currently has forty four laboratories dedicated to the early management of controlling confirmed cases. 44 given the dynamic nature of the pandemic, involving each and every citizen in its mitigation became a very important part of global response efforts. to contain the pandemic, mexico mobilized a large force of police officers and soldiers to execute the following: distribute masks among citizens for free, shut down public places, cancel or delay large-scale events, halt teaching activities in all schools-including universities, primary and secondary schools, and kindergartens -in mexico city and in the state of mexico. on april 29th, 2009, the mexican government declared a suspension of all nonessential public affairs and economic activities from may 1st through may 5th. moreover, the mexican government also adopted a wide range of measures to strengthen pandemic information communication and sharing, i.e.: reporting pandemic developments via media channels, setting up 800 hotlines, launching influenza prevention websites, giving out leaflets on pandemic information that called for personal hygiene and increased public awareness of the virus. 45 in the united states, the hss launched a one-stop influenza information website (www.flu.gov), which gathered information from regular media briefings conducted by the hhs and other federal agencies, 46 and provided the public with scientific and effective information services. in collaboration with federal, state, and local partners, the hhs also developed a wide range of community-based intervention guidelines which were being evaluated simultaneously. the cdc and the dhs provided specific recommendations targeted to a wide variety of groups, including the general public, people with certain underlying health conditions, infants, children, parents, 44 john and moorthy (2010) 2.3 national and regional response strategies and measures pregnant women, seniors, health care workers, workers in relevant industries, laboratory workers, and homeless people. with these recommendations, people were equipped to take appropriate action in reducing the transmission of the virus, especially in early autumn before vaccines were widely disseminated. the cdc also provided, and updated on a regular basis, scientific guidance on influenza prevention and control to schools, daycares, universities, large and small businesses, and federal agencies. these comprehensive guidelines provided not only advice on how individuals and institutions could protect themselves against the virus and mitigate its spread, but also recommendations for healthcare providers about the appropriate use of anti-viral drugs, especially in treating patients who were at the highest risk of suffering complications from the influenza. 47, 48 in japan, after the alert level transitioned from an "overseas pandemic" phase to the heightened "early onset of a domestic pandemic" phase, the local governments of osaka and hyå�go prefectures required the following for areas where infections had occurred: gatherings and collective recreational activities be suspended, entertainment venues be temporarily closed, social service workers be required to wear masks, teaching activities of varying levels at more than one thousand educational institutions be suspended for one week, citizens avoid trips and gatherings, and business activity be reduced for the time being. in australia, patients with mild symptoms were allowed to stay at home as a means of isolation. with the rapid spread of the pandemic, the united states didn't take stock in counting cases, but instead focused on the evolution process of the virus. 49 the united states' advanced and unique monitoring system for bacteria and viruses uses dynamic and standardized methods to collect data related to virus occurrence, virus developments, and basic medical trends, and employs national demographic data to compute virus incidence and describe its epidemiological characteristics. this system brings together and facilitates cooperation within the cdc, state health authorities, academic partners, hospitals and infection control centers. moreover, it contains special research platforms, i.e., socio-economic evaluations of disease risk factors, effects of the disease and vaccinations, data on resources for vaccine research and development, and data on approved vaccines. in australia, laboratory testing focused on critically ill patients, high-risk groups with severe diseases, and personnel in relevant institutions. monitoring was also conducted to see if any resistance or mutations of the virus had occurred. understanding that vaccinations were the best means for combatting the virus, countries focused a large amount of resources on vaccination development and inoculation methods. in its influenza pandemic preparedness and response plan, the hhs in the united states set two objectives for vaccine preparation 50 : to stockpile twenty million vaccinations for key personnel, and to increase manufacturing capacity to cover the population in the united states, in other words, produce 300 million doses within six months of the pandemic outbreak. immediately following the outbreak, the national institute of allergy and infectious diseases (niaid) subordinate to the u.s. national institutes of health (nih) began its research on the virus and vaccination development. in july 2009, the niaid initiated a series of clinical trials on the effectiveness of newly developed vaccines. in september, the fda approved manufacturing for four vaccination types, which were then made available for distribution among the states. the federal government then identified priority groups for vaccination and formulated an inoculation policy. 51 starting on october 5th, a national influenza a (h1n1) voluntary inoculation program begun targeting high-priority groups including pregnant women; people between the ages of 6 months through 24 years of age; people aged 65 years or older with chronic health disorders like asthma, diabetes and heart disease; and healthcare and emergency services personnel. 52 during the two months that followed, vaccine manufacturers provided 10-20 million vaccination doses each week, an amount which reached roughly 250 million by the end of 2009. 53,54 according to statistics, the federal government ordered a total of 229 million doses of the vaccine with the plans of vaccinating 158 million people, and in the end 90 million people were actually inoculated. on october 21st, 2009, the united kingdom launched its national influenza a (h1n1) inoculation program. the first phase of the plan provided the vaccine to the high risk population of fourteen million people, including critically ill patients, pregnant women, and healthcare personnel working in hospitals. soon afterwards, general practitioners across the country began encouraging people with health disorders or immunity problems, and pregnant women to get vaccinated. on december 8th, 2009, the british government went on to include children ages six months to five years old in the vaccination program. in august 2009, australia approved a national vaccination program and began providing free vaccinations to healthcare workers, pregnant women, and individuals with chronic health disorders who were susceptible to the virus. on september 30th, the australian government announced that all adults and children aged ten years and older could also receive free vaccinations. in may 2009, the mexican government announced an appropriation of 6.2 million usd for the establishment of a dedicated committee composed of 12 authoritative medical experts, and this committee's mission was to mobilize and coordinate research efforts for carrying out etiological, epidemiological, diagnostic reagent and vaccine research relating to influenza a (h1n1). it was also responsible for providing policy recommendations on pandemic prevention and control and medical treatment options to the government. 55 in july 2009 japan began distributing permits authorizing the utilization of influenza a (h1n1) vaccines, and they also launched a national vaccination program. the first groups to receive it included healthcare personnel, police officers, as well as high-risk groups like pregnant women, patients with chronic diseases, and seniors. as influenza a (h1n1) cases gradually declined, some countries readjusted their pandemic response levels as well as their measures for virus prevention, control, and treatment. countries set about making summaries and conducting evaluations while continuing their pandemic monitoring and information sharing. in 2010, most regions across the globe saw a decline in influenza a (h1n1) activity, and though in some regions the virus still sustained its intensity (level), the overall virus transmission dropped. additionally, it was discovered in most cases that the influenza a virus only caused mild infections, and that its virulence had not increased since it was first reported in april 2009. effective vaccinations had been in circulation since november 2009. it was for these reasons that the singaporean ministry of health decided on february 12th, 2010, to downgrade its alert level from yellow to green. beginning in february 2010, the united kingdom deactivated the national pandemic flu service (npfs), an act done in line with ensuring the operational response was appropriate to the threat level posed by the virus and also because general practitioners and primary care trusts could now manage the clinical 55 mexico sets up special committee for influenza a (h1n1) research. xinhuanet.com, may 12, 2009 . http://news.xinhuanet.com/world/2009 caseload by themselves. 56 anyone concerned about flu-like symptoms were advised to contact their doctor for assessment, who could then issue an antiviral authorization voucher if needed. the npfs would be reactivated should the pandemic virus regained its virulence. starting on april 1st, 2010, free antiviral medication from the national stockpile was no longer available to patients with influenza a (h1n1). normal treatments and prescription charges were reinstated for those suffering from influenza. 57 in june 2010, the united states declared the end of the public health emergency. as confirmed cases declined and the spread of the virus continued to slow, the u.s. federal, state, and local health authorities began to readjust their response strategies. in addition to continued efforts in strengthening public health education and inter-agency collaboration, other measures included bolstering the vaccination campaign, 58 strengthening virus monitoring, and continuing focus on virus mutations. as the pandemic developed in the united states, especially after the wide distribution of vaccinations to the public, some u.s. agencies and institutions evaluated the results of a range of their prevention and control measures. the purpose of these evaluations were to identify problems that existed in the national pandemic response measures, and correct them to better the response in the future. (upmc's) center for biosecurity held a conference to summarize important lessons learned from pandemic responses and raised policy suggestions in mitigating future infectious disease emergencies. on may 4-5th, 2010, the cdc, the national association of county and city health officials (naccho), and other stakeholders met to review the federal, state and local policies that had an impact on local health departments' pandemic detection, response, and recovery efforts. while modernized health care systems, antiviral drugs and vaccines represented the advantages of global response efforts this time around, factors like globalization and urbanization allowed the fastest transmission of any pandemic ever witnessed. after outbreaks occurred in multiple countries, governments worldwide immediately adopted a wide variety of proactive containment measures. while there were many successful responses, shortcomings were also exposed which incited doubt and controversy surrounding the pandemic. in regards to prevention and control measures, governments in most of the affected countries did not look lightly upon the pandemic, and they played leading roles in policy making, resource collection and allocation, as well as organization and coordination. firstly, governments identified and allocated prevention and control organizations and accountability mechanisms at the national level. as mentioned before, some countries such as the united kingdom and india specifically established bodies for comprehensive coordination in response to the influenza pandemic, while others like the united states-where established emergency response agencies were already in existence-launched their emergency response efforts upon the outbreak of the pandemic. the u.s. government then oversaw an organized response from varying agencies. secondly, countries developed national-level pandemic strategies or response plans as general outlines for prevention and control efforts. thirdly, funds for response efforts in most cases originated from the central government, where the capital was then allocated to appropriate departments based upon their responsibilities. lastly, central governments were in charge of across-the-board organization and coordination in all aspects of the response efforts, especially in the provision of services, drug supplies, and vaccinations, while at the same time playing a crucial role in communication and coordination with other social service organizations, businesses, and the general public. in the course of global responses to the sudden outbreak of the influenza pandemic, the who made good use of its expertise and networking strengths. with a global approach, the organization disseminated information, pushed coordination, and strengthened guidelines. it played an important role in coordinating and guiding countries' efforts to raise awareness, develop technical guidance, release pandemic information, develop vaccines, etc. most countries possessed an influenza prevention and control system comprised of a variety of collaborative relationships, i.e.: partnerships between central, provincial (state), and local governments, the private sector, and individuals, as well as international partnerships established through bilateral or multilateral collaboration. each party within this system had its function and standard operating procedures, with the division of labor already institutionalized; and in implementing specific prevention and control measures, these parties were expected to fulfill their expectations and duties as stakeholders. each stakeholder understood their role to play during the preparation, prevention, and control of the pandemic, and no major changes occurred in that respect during the pandemic. at the same time, capacity building and positioning was constantly being improved according to the different functions of each party. in addition to inter-departmental coordination and collaboration, countries like the united states also called upon the public for participation and global collaboration, which expanded collaboration as it brought in community and societal involvement. during different phases of the pandemic, countries emphasized the integration of comprehensive measures and key response issues, and efforts were adjusted according to the development of the pandemic. in the early phases, prevention and control strategies were "strict," as they focused largely on containment with inspection and quarantine measures. cases diagnosed early were treated in a timely manner to better the odds of developing a successful vaccination. at the spreading period of the pandemic, the focus shifted to clinical treatment of patients, alongside strengthening virus monitoring. during the post-peak period, while some countries quickly revised alert levels which reduced social impact, others had no readjustment mechanisms for policy changes in place which resulted in inefficient prevention and control. during this time, most countries recognized the importance of international collaboration. firstly, faced with the grim situation of a pandemic gripping the globe, affected countries followed the who's pandemic strategies and recommendations. combining the domestic situation with who's proactive policies and recommendations, most countries adopted relevant response measures. however, there were many countries that didn't adopt all of the who's policies and recommendation, nor did they follow all of the policy readjustments. instead in light of their domestic situation, governments formulated their own response strategies and measures. secondly, relatively close collaboration between countries did occur. due to the many uncertainties surrounding the occurrence and development of the influenza a (h1n1) pandemic, the level of "appropriateness" of response strategies -i.e. were they considered "lax" or "strict," "ineffective" or "overreacting"became a major controversial point surrounding the pandemic prevention and control policy. on the one hand, based on their own pandemic situations, their preparation evaluation, and cost-benefit analyses, developed countries such as the united states, canada, the united kingdom, and france, adopted policies that focused more on treatment than on control. the united states, for example, in the early days of the pandemic considered influenza a (h1n1) no bigger a threat than the seasonal influenza, so the government failed to take strict response measures, like quarantine and medical observation, which resulted in a spike in domestic infections. on october 23rd, 2009 the united states declared a national health emergency, sparking questions about the government's response efforts. while some critics questioned whether there indeed existed such an emergency, others argued that a state of emergency should have been declared from the very beginning. an article published in the new york times in early january 2010, gave full recognition to the country's response strategy, insisting that apart from luck, the federal government's appropriate, rapid, and conservative response successfully contained the virus and minimized potential harmful effects it could've had on the economy. on the other hand, some countries began with strict measures and relaxed them later on, causing difficulties in latent response efforts. for example, countries like mexico declared a state of high alert immediately upon the outbreak, leading to a certain extent, a public panic. but after the who elevated the pandemic alert phase, the mexican government rushed to lower its domestic alert level in order to ease public anxiety. thus the public became careless, causing the increased transmission rate. moreover, media in japan, france and other countries exaggerated pandemic situations that embellished "the widespread transmission" of the virus in home countries through imported cases. people became panic-stricken and it became increasingly difficult to implement proper response measures. japan and other countries failed in resource management as they placed too much emphasis on border control and quarantine, and not enough on domestic control and detection, thus making it difficult to contain the spread of the pandemic. these actions also led to widespread criticism of government response efforts. though the who's role in the global pandemic response efforts was widely recognized, the organization also suffered criticism as there were varied opinions about the timeliness of alert level changes and their investments in personnel and equipment. reuters reported on april 12th, 2010, that the who admitted to having problems in their response efforts, including its failure to communicate the uncertainty of the new virus before it swept the globe. some critics held that from the perspective of pandemic development, the influenza a (h1n1) pandemic was not as dreadful as it was initially anticipated, and it was the who that created a global panic in its response-which caused an excess in vaccination stockpiling among some countries. some even suspected that the ihr emergency committee might have had an "affair" with some drug manufacturers and was suspected of helping them seek profit by deliberately exaggerating pandemic situations so that the who would raise its pandemic alert to the highest level. 61 in response, on april 12th, 2010, the who commissioned a panel of external experts to conduct an overall evaluation of the global response to the influenza pandemic in the hope of providing lessons for the future, and simultaneously to assess the global implementation of the ihr 2005. the who's policy evaluation comprised three main parts, i.e. capacity and preparedness, pandemic alert and risk assessment, and response. on june 10th, 2010, the who officially responded to and clarified such issues as to the influenza a (h1n1) virus met the criteria for a pandemic, the severity of the pandemic, and related conflicts of interest. 62 61 central people's government of the people's republic of china. who experts warn global h1n1 pandemic still not over yet. http://www.gov.cn/jrzg/2010-04/15/content_1581776.htm. 62 who. the international response to the influenza pandemic: who responds to the critics. http:// www.who.int/csr/disease/swineflu/notes/briefing_20100610/en/index.html. there are no international standards for vaccine allocation in mitigating the global burden of disease. while the united states began vaccinating its citizens in early october 2009 after the fda approved on september 15th the marketing of influenza a (h1n1) vaccines produced by csl, medimmune, novartis vaccines and diagnostics, and sanofi pasteur, mexico, which had been suffering a severer pandemic situation, was unable to launch a vaccination program until january 2010. building a powerful global vaccine production infrastructure for influenza pandemics where countries and regions in need could acquire adequate vaccines at affordable prices became one of the hot international topics at this time. the who stated that although antiviral drugs used at that time to combat influenza enjoyed complete patent protection, the organization proposed that these drugs be acquirable in the cases of public health crises. the use of antiviral drugs was hit heavily upon in the who's guidance documents, but, given cost issues, the use of such drugs and vaccines had little operability in most middle and low-income countries. moreover, some international media held that the outbreak in the united states brought to the forefront the many flaws in their healthcare system, most notably the use of old-fashioned vaccine technology and excessive reliance on vaccine manufacturers abroad. a highly controversial event also occurred during vaccination distribution: the new york city department of health and mental hygiene decided to give the small amount of vaccine available in the early phases of the pandemic to big corporations on wall street such as goldman sachs and citibank, an act which experts believe only exacerbated public relation issues. vaccine production and distribution became a controversial focal point during prevention and control of the pandemic as it involved multi-faceted issues such as vaccine patents, mass psychology, and social justice. h1n1 preparedness: an overview of vaccine production and distribution u.s. global health response to a novel 2009-h1n1 hhs' effort to provide science-based pandemic influenza guidance for the u.s. workforce lessons from previous influenza pandemics and from the mexican response to the current influenza pandemic h1n1 preparedness: an overview of vaccine production and distribution 2009 pandemic influenza in india who director-general margaret chan says international community cannot afford to take influenza a (h1n1) pandemic lightly preparing for the 2009-2010 influenza season 2009 h1n1 influenza: monitoring the nation's response protecting the protectors: an assessment of front-line federal workers in response to the 2009-h1n1 influenza outbreak global surveillance during an influenza pandemic. version1, updated draft assessing the severity of an influenza pandemic 2009-h1n1 influenza: hhs preparedness and response efforts characteristics of the india's public health emergency management system: from a perspective of influenza a (n1h1) preparedness and response. global science, technology and economy outlook influenza a pandemic moves into a new phase, who changes way of epidemic reporting key: cord-018811-zhwr3h07 authors: oxford, john; gilbert, anthony; lambkin-williams, robert title: influenza vaccines have a short but illustrious history of dedicated science enabling the rapid global production of a/swine (h1n1) vaccine in the current pandemic date: 2010-06-18 journal: influenza vaccines for the future doi: 10.1007/978-3-0346-0279-2_6 sha: doc_id: 18811 cord_uid: zhwr3h07 vaccines for the swine flu pandemic of 2009 have been produced in an exquisitely short time frame. this speed of production comes because of 50 years of hard work by virologists worldwide in pharma groups, research laboratories, and government licensing units. the present chapter presents the background framework of influenza vaccine production and its evolution over 50 years. isolation of the causative virus of influenza in 1933, followed by the discovery of embryonated hen eggs as a substrate, quickly led to the formulation of vaccines. virus-containing allantoic fluid was inactivated with formalin. the phenomenon of antigenic drift of the virus ha was soon recognized and as who began to coordinate the world influenza surveillance, it became easier for manufacturers to select an up-to-date virus. influenza vaccines remain unique in that the virus strain composition is reviewed yearly, but modern attempts are being made to free manufacturers from this yolk by investigating internal virus proteins including m2e and np as “universal” vaccines covering all virus subtypes. recent technical innovations have been the use of vero and mdck cells as the virus cell substrate, the testing of two new adjuvants, and the exploration of new presentations to the nose or epidermal layers as dna or antigen mixtures. the international investment into public health measures for a global human outbreak of avian h5n1 influenza together with a focus of swine influenza h1n1 is leading to enhanced production of conventional vaccine and to a new research searchlight on t-cell epitope vaccines, viral live-attenuated carriers of influenza proteins, and even more innovative substrates to cultivate virus, including plant cells. when the influenza a virus first emerged from a presumed avian reservoir at the end of the ice age 10,000 or so years ago, there was a distinct difficulty in finding new human victims. for example, at that time, only a few hundred settlers were in the london region near the royal london hospital, now a community of four million people. at that time a traveler would have to walk a 100 miles to find another small settlement, perhaps at stonehenge near salisbury. nowadays we have a truly global community of six billion people, linked so that two million people are moving each day by plane, while perhaps ten million are journeying in their homelands. influenza, like all viruses, is opportunistic. in 1918, it had the unprecedented opportunity to spread at the end of the first global war. ten million soldiers began the move homewards, and every steamship was packed as they fanned out from france to england, europe, the usa, canada, australia, india, and se asia [1] [2] [3] . how perfect for a virus spread by aerosol droplets, close contact, and contamination of towels, cups, and every day utensils. a virgin population, which had never before encountered the avian virus (h1n1), was on the stage of this theater of infection. perhaps, a billion people were infected in the next 18 months, and 50-60 million died, making this by far the biggest outbreak of infectious diseases ever recorded, with an impact many times greater than the so-called bubonic plague outbreaks in medieval europe. however, more than two billion people survived. the overall mortality was less than 1%, although in a few semi-closed societies of hunter-gatherers in the arctic, the mortality from the disease and subsequent starvation as young hunters died and husky dogs attacked and ate the survivors exceeded 90% [4] [5] [6] [7] . it is well to remember that when h1n1 emerged in 1916/1917 and became pandemic in 1918 everyone except for the over 70s were fully susceptible. this is different from today where most people on planet earth have immune memory to the h1n1 family of viruses and by definition to a/swine flu. this explains why the current h1n1 vaccine is immunogenic. while most people in the world were infected, we are forced to view the innate protective power of our immune system with awe [8, 9] . we are equipped with 100,000 genes, seven million years of evolution, and 80,000 years of specialization since our emergence from africa. in contrast, influenza is a miniscule eight-gene vehicle. a recent study [10] of the reproductive number (r0) of the 1918 virus suggests that, unexpectedly, it may have been quite low, not exceeding three persons infected with a single case. the current pandemic a/swine h1n1 virus is not so different. this would place pandemic influenza not far above the lowly group of viruses such as small pox and sars and not reaching the heights that measles has attained. however, this unexpected theoretical analysis, if it is not flawed, gives us more practical opportunities to break a chain of infection of a pandemic with antivirals, hygiene, and vaccines [11] [12] [13] . we are experimenting with these approaches at the present moment. the new world of the twenty-first century, although harboring in some countries a few old-fashioned attitudes, akin to "influenza and pneumonia is the old person's friend" nevertheless has the capability for the first time to defend itself against mother nature and her threat of influenza. for the first time in history, intense surveillance by the world health organization (who), early identification of a new pandemic influenza virus by molecular diagnostics, application of vaccination and antiviral chemoprophylaxis, and possible quarantine and masks could actually prevent a pandemic arising. for the expressed intention of who and the world community of infectious disease researchers is to deflect the first wave of the first pandemic of the twenty-first century. in this endeavor, our huge resources of natural innate immunity, assisted by new vaccines, are already helping us. the formulation of the vaccines and their stockpiling alongside antineuraminidase (ni) antivirals has needed significant investment of time and money, and this started with a three billion euro investment from the usa and eu. we are presently gathering the fruits of this investment with the outbreak of a/swine (h1n1) virus. baroness findlay of glandaff put the epidemiology of influenza h5n1 situation succinctly in the house of lords report of pandemic influenza [14] "we believe the risk of a pandemic of human-to-human transmissible virus is to be taken very seriously. we believe that it may not happen in the very short time. to explain why we came to this stance; we believe that the problem, if it does emerge is more likely to emerge in asia. asia is where fire fighting must be done today." the baroness had just heard the background science that china alone holds 700 million domestic ducks, a possible trojan horse of virus persistence, which approximates to 70% of the world's domestic duck population. expert evidence from fao had summarized that china, indonesia, and vietnam represented the core of the problem, but only 160 million dollars were available at that point in 2005/2006 to help, and biosecurity is not imposed strictly, while veterinary services are haphazard. the current pandemic virus emerged from pigs but a continuing threat is another reassortant event with h5n1 most likely in a coinfected child in egypt or se asia where h5n1 viruses are endemic and where swine h1n1 viruses are spreading. we are not the first generation of virologists to recognize the influenza pandemic threat, but we are the first to have the knowledge of the avian and pig reservoir and the tools to deal with the problem in a scientific manner. the world capacity for influenza vaccine today of one billion doses did not arrive by accident: it came to us from the hard work and dedication of four generations of dedicated scientists and doctors. the intention here is to give just tribute to these pioneers and their new discoveries. using the vaccine methods developed over six decades, we can for the first time confront influenza as it emerges, surround it, and actually prevent a pandemic. we no longer need to be passive observers at a theater of infection. churchill coined the phrase "give us the tools and we will finish the job." well, we now have them and we will. such is the essence and spirit of this chapter. the serendipitous discovery of infection of ferrets, which produce clinical signs, and the cross-infection of a student from a ferret was the first technology foundation stone [9] . ferrets are used today as a key model to investigate new vaccines. the two most important technologies, which form the granite-like foundation of influenza vaccine research, are the hemagglutination inhibition test (fig. 1 ) and the cultivation of virus in embryonated hen's eggs (fig. 2) , first reported in 1941 and 1946, respectively [15, 16] . if one adds two other vital scientific observations that of hobson et al. [17] who correlated a hi titer of 40 with protective efficacy in volunteers in 1972 and then the discovery of a single radial diffusion for standardization of the hemagglutinin (ha) content of vaccines by schild in 1973, it is quite apparent that the technologies are all now well tried and tested [18] . the elucidation of the structure of the fragmented influenza genome [19] has quickly led to techniques, genetic reassortment, and correlation of functions with certain genes (fig. 3) . from a practical viewpoint, some old much passaged viruses such as a/pr/8/34 (h1n1) grew to extraordinary infectious titers in the egg allantoic cavity, exceeding a new wild-type virus by 100fold or more. why not create a reassortant in the laboratory with six replicative genes of a/pr/8/34 to give high replication while having the two new ha and neuraminidase (na) genes of the new epidemic virus? this technique proved to be a masterstroke and in the last quarter of a century three laboratories, csl in fig. 1 the classic hemagglutination inhibition test. the test depends upon interaction of eight ha units of virus that would normally agglutinate 0.5% turkey red blood cells. preincubation of this standard virus with dilutions of serum antibody abrogates the agglutinating property of the virus (vertical rows 5 and 9). no antibody is detectable in rows 1-4, [6] [7] [8] melbourne, nibsc in london, and ed kilbourne's laboratory in new york, have rushed each year to produce the new candidate vaccine viruses prefixed ivr-, nib-, and x-, respectively. the almost made-to-order technique of gene reassortment fig. 2 inoculation of embryonated hen's eggs to grow influenza virus for vaccine. virus is inoculated through the shell of a 10-day-old embryonated hen's egg and more rarely in the research laboratory into the amniotic cavity (top). after 2 days of incubation at 37 c, the clear fluids are removed and titrated for ha by hemagglutination fig. 3 the influenza genome is in eight fragments. the genome could be labeled with 32p extracted and separated on polyacrylamide gels with influenza was also central to producing host range mutants with attenuation genes for live vaccines. some of the starter and seed viruses for the current production of a/california/4/09 h1n1 (swine) vaccine used this biological technology, while others used reverse genetics to make a gm starter virus. undoubtedly the simultaneous discovery of the reverse genetics [20, 21] by the three laboratories in new york, wisconsin, and oxford was a masterstroke in technical advance, which has enabled mutations to be placed, at will, into the genomes of the negative-strand viruses. the conjunction of older and newer techniques with the licensing of the mammalian cell lines from monkey kidney (vero) [22] , dog kidney (mdck) [23] , or human tissue (per-6) has led directly to the newly emerging influenza vaccines of the twenty-first century. we are using all these techniques of the last 50 years to produce the a/swine h1n1 vaccines of 2009 for the current pandemic. the first experiments on the attempted immunization of animals were made in the usa by francis magill [24] and in england by andrewes and smith in 1937 [25] . the model is still vital today and the first experimental assessment of a/california/ 04/09 h1n1 vaccine was made in this model. mouse lung suspensions or filtrates were used after inactivation with formaldehyde, and it was found relatively easy to protect mice against intranasal infection with influenza. immunization experiments in man were accelerated when allantonic fluid preparations of virus formed the starting material soon after the technique of allantoic inoculation of fertile hen's eggs was discovered [16] . the first field trial demonstrating short-term protection by inactivated vaccine took place in the usa during a sharp epidemic of influenza in 1943 (commission influenza 1944) [26] . progress with the development of purer, more potent vaccines has proceeded steadily since those early days, and technical advances with ultracentrifugation and chromatography, by methods producing richer cultures and chemical inactivation avoiding too great a modification of the surface ha and na antigens have all helped. to avoid the relatively high rate of local and general systemic reactions caused by the older egg-grown inactivated whole-virus vaccines, chemical treatment to disrupt the particle and to separate the wanted antigens (ha and na) from other constituents of the virus has led to a variety of different split or subunit vaccines . ether extraction [27, 28] , deoxycholate treatment [29] , and treatment with other detergents have been introduced. some methods have provided subunit vaccines causing fewer clinical side reactions than the older whole-virus particle vaccines, but drawbacks have appeared, including that of reduced antigenicity. adjuvants of oily emulsions promised potent vaccines with excellent antibody responses, and a few reactions were first encountered. however, a rare abscess at the site of inoculation caused much distress and this early approach had to be abandoned. in spite of attempts to develop safer materials, none have yet the whole virus is disrupted with detergent, which dissolves the lipid membrane releasing ha, na, and internal np, seen as "lamb tails" been developed commercially until very recently when mf59 and a50 have been formulated. thus, after 60 years of work, the hope of an ideal inactivated vaccine free from the induction of clinical reactions and yet potent immunogenically has just been fulfilled with pandemic h5n1 vaccines and swine h1n1 vaccines. in 1946, a major antigenic deviation of influenza a virus occurred with the appearance of a/cam/46 (h1n1) virus in australia. in the usa and europe, outbreaks of influenza occurred early in 1947, which were due to the same virus; some communities previously receiving vaccine containing pr8 and weiss viruses (h0n1 in the old classification and now reclassified h1n1) were attacked. this time the vaccine did not protect against the new virus typified by the prototype a/fm/1/47 (h1n1) [30, 31] , and this led to realization of the enormous importance of the updated antigenic make-up of inactivated vaccine. yet other difficulties have become appreciated, one of which is the inappropriate antibody response occurring sometimes after inoculation, when the vaccine induces cross-reacting antibody to heterologous viruses or the first virus in the subtype which the vaccine first experienced, rather than that appropriate to the specific antigen, ha, of the vaccine virus. this response is probably allied to the phenomenon of "original antigenic sin." sometimes this aberrant response can be useful as with a/swine h1n1 vaccine. it is likely that the over 65s will produce recall antibody to h1n1 viruses which infected them in the 1940s and that this virus is somewhat related to the current a/swine virus. the starting materials for almost all types of inactivated vaccine are allantoic fluids from fertile hen's eggs previously inoculated with a seed culture, the yield of which is enhanced using a recombinant virus, one parent of which is a high-yielding laboratory strain (a/pr8/34) and the other acts as the donor of the requisite surface ha and na antigens from a wild-type virus [32] . the a/pr/8/34 virus donates six genes and the wild-type virus two genes: the ensuing reassortant high growth viruses are called 6/2 reassortants. purification from unwanted egg material is accomplished by ultracentrifugation on a zonal ultracentrifuge [33] . whole-virus particles thus separated are inactivated by formalin or b-propiolactone, the ha content being as high as possible commensurate with the necessity to avoid febrile reactions after inoculation. children were sensitive to the older egg-grown wholevirus vaccines; as many as 30% under 2 years developed fever after 0.25 ml of vaccine and up to 8% of 6-year-old children were similarly affected after 0.5 ml [34] . the precise constituent producing the fever was not clearly identified, but the viral proteins were believed to be concerned [35, 36] . more modern whole-cell virus vaccines produced in cell culture are more purified and produce fewer side reactions. separation of the ha and na by means of detergents such as tween 80 or triton n101 produced split-virus or subunit vaccine, and general experience suggested that these materials are less pyrogenic, but less immunogenic, than whole-virus vaccine [37] . this was particularly well demonstrated by studies during the swine influenza campaign in the usa in 1976, when many observers reported results, which ultimately led to the recommended use in children of two doses of split-type rather than whole-virus vaccines. such recommendations continue at the present time. in adults, too, the older egg-grown whole-virus vaccines gave a higher proportion of febrile reactions than split virus [38] . however, this situation is changing as whole-virus vaccines produced in vero cells for example come to the fore. former methods for assays of the potency of inactivated vaccine depended on measuring the ha activities of the vaccines with erythrocyte suspensions using the salk pattern technique of miller and stanley [15] . in retrospect, this technique was not hugely accurate especially for subunit and split viruses. in a major technological breakthrough, schild et al. [18] proposed a method of assay based on single radial immunodiffusion (srd) (fig. 7) . the ha antigen content of vaccines was estimated using srd tests in agarose gels containing specific hi antibodies. the srd method was modified and refined by wood et al. [39] . it may be gradually replaced now by hpmc technologies. the srd technique was valid for both whole-virus and split-virus vaccines and was quickly adopted for international use and is still the gold standard. in this test, vaccine virus preparations and reference antigen calibrated in terms of micrograms of ha are disrupted with detergent, and dilutions of the treated antigens are introduced into wells in srd immunoplates. the size of the precipitation ring obtained for the vaccine is compared with that obtained with a reference antigen of calibrated ha content titrated on the same plate. the vaccine potency is measured in terms of micrograms of ha per vaccine dose. inactivated influenza vaccines frequently contain two or more virus strains and the ha content of each component (15 mg) is assayed independently. it has been known for many years that the serological response to inactivated vaccine depends on the previous experience of the recipient to infection by viruses of the same subtype of influenza a virus as that present in the vaccine. although a single subcutaneous injection of (h1n1) vaccine gave as good a response as two doses prior to 1957, the advent of the new pandemic a/asian (h2n2) virus produced a different effect. thus, holland et al. [40] demonstrated that two doses at an interval of two or more weeks produced a better response to one dose and in this regard the vaccine-induced immune response was much inferior to that noted single radial diffusion (srd) test to standardize ha. vaccine antigen is pipetted into 3-mm wells in an agar plate containing specific anti-ha, -na, and -np antibodies. after a few hours incubation, a zone of precipitation is quantified and the area is proportional to the quantity of ha in the vaccine before the change in virus subtype. such an experience was again noted during the first year of circulation of a/hong kong (h3n2) virus and also when the a/new jersey/76 (hsw1n1) vaccine was used in children and young adults. also, in the circumstances of 1977-1978, when most persons under 25 years of age had no previous antibody to the recirculating h1n1 virus, a two-dose regimen for children and young adults produced a more satisfactory response than a single injection [41] . to reiterate, in 1977 an "old" h1n1 virus from the 1950s was accidentally released from a laboratory and established itself as an epidemic virus. it was called a "pseudo pandemic." everyone over 24 years had previous immunity. the contrast between the effects of a single dose of vaccine in persons infected with h1n1 viruses at least 20 years earlier was very striking. these data have immediate relevance today in terms of h5n1 vaccine and of course with the a/swine vaccine. the world is full of immune virgins as regards h5n1 but not in the case of a/swine h1n1. most persons have immune memory to the h1n1 family, and therefore it comes as no surprise that vaccines induce high levels of hi antibody. several factors are of importance in the determination of the quantity and the precise composition of the antibody response to the surface antigens of the virus present in inactivated vaccine. first and foremost, the quantities of hi and ni antibodies induced by vaccine are broadly related to the quantity of antigen present in a single dose. second, the precise composition of the antibodies formed in response to influenza a virus is important. thus, reinforcement of previously acquired antibodies by the orientation of the b-lymphocyte response to the first infection by the particular subtype of virus experienced in childhood or later may take precedence over the strain-specific antibody response to the vaccine virus. third, the precise response is influenced by the route by which the vaccine is presented to the body's immune system. first then, several earlier studies reported a graded relationship between the quantity of antigen inoculated and the antibody response that results. this was so in the study of mostow et al. [42] , who gave increasing doses of vaccine in a single injection containing 300-4,600 chick cell agglutination (cca) units containing a/japan/57 (h2n2) virus groups of volunteers. the serum hi response was tested with four different h2n2 viruses isolated 1962-1967 and also the homologous virus. with more than a tenfold increase in ha from the least to the highest dose, the geometric mean titer (gmt) of antibody increased only fivefold. similar results were obtained by potter et al. [43] , who inoculated student volunteers with vaccines ranging in dosage from 5 to 400 iu and containing a/port chalmers/73 (h3n2) virus. the vaccine was a surface-antigen detergent-treated material [44] adsorbed to aluminum hydroxide gel. gmt hi serum titers increased against homologous virus from 8-to 174-fold with the increase in dose of vaccine ha. three other h3n2 strains and a/singapore/57 (h2n2) virus were also tested, and all three h3n2 viruses showed graded hi antibody responses proportional in magnitude to increase in antigen dose, as did the homologous virus. the pandemic working group of the mrc committee on influenza vaccine [45] gave graded doses of whole-virus vaccine containing the a/new jersery/76 (hsw1n1) strain to groups of volunteers in 1976. those less than 44 years of age, who did not possess significant serum hi antibody to the virus before immunization, showed a postvaccination antibody titer ranging from 64 to 148 gmt with a nearly eightfold increase in dose from 8 to 61 mg of ha. above this age, in those 45-64 with preexisting hsw1 antibody, there was an increase in antibody titer from 7 to 36 times (gmt) with a change in ha concentration from 4 to 61 mg. thus, the effect of increasing the potency of this vaccine on the antibody response was much greater in those sera, which indicated that they had been exposed to the antigen, presumably by infection with a related virus, than in those with no such exposure. both whole and detergent-split-virus vaccines showed a relatively poor hi response in volunteers less than 25 years of age whose initial serum had no significant amount of prevaccination or postinfection hi antibody. in this group of subjects, two doses of vaccine gave a better antibody response than did one, but the resultant postvaccination gmt was half that obtained with a single dose of the vaccineover 25 years of age. this historical data is very relevant to us today as we analyze the hi data from the current batches of a/swine h1n1 vaccine where in the over 5 years a single dose of vaccine is sufficient because of wide preexposure to members of the h1n1 family of viruses. the younger groups had no prior immune memory to the h1n1 family of virus, their experience being more orientated to h2n2 and h3n2 families. these examples underline the practical importance of a considerable degree of antigenic drift within a subtype comprising hi antibody response. also, the recall of antibodies induced by previous infection illustrates the general rule that an up-todate monovalent vaccine reinforces antibodies against former members of the subtype, while also inducing specific antibodies to the vaccine virus. this was clearly shown by direct comparison of monovalent and polyvalent vaccines such as the mrc committee on influenza vaccine's trials [46] [47] [48] [49] . the quantitative dose response already described for hi is also found with ni antibody but is less consistent. thus, potter et al. [50] noted that there was a two-to sixfold increase in ni antibody as vaccine potency was increased from 5 to 400 iu of ha. yet the trial of a/new jersey/76 (hsw1n1) vaccine conducted by the pandemic working group of the mrc influenza vaccine committee [45] found only a slight increase in ni antibody after an increased dose from 100 to 200 iu using 100 iu of ha in the vaccine. nicholson et al. [41] gave a whole-virus vaccine of the a/ussr/77 (h1n1) virus, which ranged in potency up to sixfold, and found, in those under 25, a threefold increase in ni antibody. however, in those over 25 years of age, an increase in dose of vaccine had a less constant effect on ni antibody formation. one possible reason for the variation in the effect of different vaccines on the ni antibody is the lack of consistency in the na content [51] ; however, another possibility may be that immunological priming to the ha in the vaccine can in some way suppress the immunogenicity of the na antigen, which may be physically associated with the ha. the second important variable in the immune response to inactivated vaccine arises from the relative amounts of cross-reactive and strain-specific antibodies that are generated. the differentiation of these require special techniques such as srd and the adsorption studies. webster et al. [52] compared, in adults, the response to an a/port chalmers/73 (h3n2) subunit vaccine to homologous and heterologous h3n2 viruses. most of the antibody was cross-reactive with a/hong kong/68 virus but when higher doses of the vaccines were used, strain-specific a/port chalmers/ 73 antibody was produced in addition to that against heterologous virus. oxford et al. [53, 54] compared whole-and split-virus vaccines containing a/victoria/75 or a/scotland/74 viruses and using single radial hemolysis and adsorption techniques showed that in an immunized adult, cross-reactive antibody was induced much more frequently than specific antibody against homologous virus. they showed the same phenomenon in adults during infection with a/port chalmers/73 virus, who frequently also developed antibody rises to a/hong kong antigens from 1968. oxford et al. [54] used similar techniques to analyze sera from children aged 3-6 years immunized with a surface-antigen vaccine containing a/victoria/75 (h3n2) antigens. most children produced a strain-specific serum antibody to the vaccine antigens, whereas adults similarly vaccinated tended to produce antibody cross-reacting with all variants of the h3n2 subtype tested. postepidemic sera from those of various ages recently infected by a/texas/77-like strain showed crossreactive antibody in adults but in contrast mostly strain-specific responses in children. strain-specific antibody is considered to be more protective. the influence of the route of immunization with inactivated vaccine has been studied in the past by many observers. the chief alternative to the subcutaneous-intramuscular route is intradermal injection using a reduced amount of vaccine. the advantages of this route are economy and the avoidance of febrile reaction. the principal disadvantage is the fact that the antibody response is less consistent. it was shown by appleby et al. [55] that the gmt after intradermal vaccine was less than half that obtained with subcutaneous vaccine, and this seemed logical in that only one-tenth of the vaccine dose was given intradermally. mccarroll and kilbourne [56] found little difference in the antibody responses to intradermal and subcutaneous vaccines in equivalent doses. tauraso et al. [57] reinvestigated the question using a two-dose regime before the arrival of the a/hong kong/68 (h3n2) epidemic. in the equivalent amount of 0.1 ml of vaccine, antibodies formed in higher titer after intradermal than subcutaneous vaccine. however, the titers after 0.5 ml of vaccine subcutaneously were little different from intradermal injection of 0.1 ml. it is considered advisable, however, in practice to limit intradermal vaccination when the vaccine is in short supply or when, in children or the aged, reactions after subcutaneous vaccine might pose problems. the nasal route of inoculation either by instillation of drops or by spray was first studied in detail by waldman et al. [58] . compared with the subcutaneous vaccine in a dose of 0.5 ml, antibodies capable of neutralizing the virus a/taiwan/64 (h3n2) increased to a greater extent in sputum and nasal secretions after repeated nasal inoculation with a total volume of 3.6 ml vaccine. in contrast, the intranasal vaccine produced a much lower rise in serum antibody, the gmt being only onesixth that after subcutaneous vaccine. waldman et al. [59] , using an aerosol spray, found that a better serum antibody response occurred with a small-sized particle spray than a larger one, but the nasal antibody response was better after the latter or with nasal drops. absorption studies showed that a majority of the secretory antibody (iga) response in nasal secretion was cross-reactive with heterologous viruses (a/hong kong/68 h3n2). phillips et al. [60] compared subcutaneous or intradermal vaccine in nurses with vaccine dropped intranasally. the subcutaneous route produced the best serum antibody rises, and intradermal vaccine was superior to the intranasal route in terms of antibody response. the nasal antibody titers after immunization by either subcutaneous or respiratory routes paralleled those in serum. the fact that nasal antibodies increase after subcutaneous vaccine [61, 62] is important because the lack of a good response in serum antibody in those given the same vaccine intranasally is a limitation hardly offset by local nasal secretory changes. challenge of immunized groups of persons by live-attenuated virus also supports the view that nasal antibodies play a supplementary role to serum hi antibody [63] . [51] . after a second dose of the same vaccine, fewer volunteers experienced reactions than seen after the first dose. later studies of the endotoxin content of various pools of inactivated type a or b vaccines using the limulus lysate test gave no hint of a parallel between the occurrence of general reactions and the endotoxin content [64] . neurological illness is a recognized sequel to immunization with a variety of vaccines but had not previously been observed with any frequency after influenza virus vaccines. wells [65] noted the rare instance of guillain-barré syndrome (gbs), which appeared in excess among the persons vaccinated with a/swine vaccine compared with the numbers in unvaccinated individuals. of 1,098 persons with gbs reported from october 1, 1976, to the january 31, 1977, 532 had received vaccine before the onset of neurological symptoms. the overall risk of gbs was calculated as ten cases per million vaccinated. the rate of occurrence during the 10-week swine vaccine period was five to six times greater than in unvaccinated persons. however, the excess in number was greater in the second and third weeks after inoculation than either the first or subsequent weeks. as reported by langmuir [66] , gbs was not associated with a particular variety of vaccine or age group. however, that numbers were slightly greater in those aged 25-44 than in middle aged or elderly persons, which appears to rule out the possibility that the syndrome was, in some way, related to the absence of antibodies to the swine virus before immunization, for most of those aged over 45 would have been exposed to antigens of this virus many years before. after the swine influenza campaign was terminated, surveillance was continued, and during the period 1978-1979, when 12.5 million doses of ordinary inactivated vaccine were estimated to have been used, the related risk of gbs was 1.4 times the incidence in unvaccinated persons. this risk was regarded as not significant [67] . no clue to the cause of the marginally increased risk of gbs in immunized persons in 1976 has yet been obtained but could be virus strain related. no untoward effects have been noted in the billions of vaccines used since 1979 to the present day. at the time when a/hong kong/68 (h3n2) virus was spreading in asia, plans were made by the mrc committee on influenza vaccine to protect children in residential schools and other groups in a controlled manner. inactivated polyvalent vaccine containing two h2n2 viruses (a/england/64 and a/england/66) and a b strain were compared with an h3n2 a/hong kong whole or deoxycholate-treated virus vaccine in initial serological trials. antibody formation even in those without detectable serum hi antibody gave gmts over 100 in those receiving a/hong kong vaccine intramuscularly. however, controlled trials in two boarding schools showed no convincing evidence of protection. in uncontrolled trials in other schools either the polyvalent or the a/hong kong vaccine were given or no vaccine at all. there were 12 schools where epidemics of influenza occurred in january and february 1969 but no evidence of protection was found in those receiving a/hk vaccines. the only clue obtained concerning the vaccine failure was first that only one dose of vaccine had been given, and this is known to be inadequate to give a satisfactory antibody response in previously seronegative persons, and second, there was an interval between vaccine administration and infection of 2-4 months. these two factors may have combined to explain the absence of protection because of the inadequacy of the antibody response at the time of challenge. it would be fair to add that others [68, 69] did obtain protection from a/hong kong/68 whole-virus vaccine during the first outbreak of influenza due to this virus in the usa. the use of modern adjuvanted h5n1 vaccine in two doses is anticipated to give protective effects. the current a/swine vaccines produce protective hi antibody (>40) in most persons over 5 years of age following a single dose. as emphasized above, this reassuring situation is because most persons have prior immunity to the h1n1 family of viruses which circulated between 1918 and 1957 and then again from 1977 to the present day. the use of living but attenuated virus as an immunizing agent developed slowly from the initial studies of mawson and swan [70] in australia and the ussr. the major difficulty of the lack of a laboratory test to indicate that cultured virus had lost its pathogenicity, while retaining infectivity for man, meant that deliberate intranasal inoculation of volunteers furnished the only way to select a suitable strain for infection without causing clinical reaction. in spite of the widespread adoption of live vaccines selected by this method and given as an intranasal spray in the ussr, little interest was exhibited in most other countries. from 1956 onwards, trials took place in volunteers in england and wales to provide evidence of safety and immunogenicity of cultured viruses and the drawback of a reduced infectivity of well-attenuated viruses handicapped progress. the necessity to observe a match between the antigens of epidemic viruses and those present in the vaccine was a further drawback until the technique of reassortment of characters between two strains, one of which was of proven attenuation, was utilized to yield seed viruses with the desirable clinical and antigenic properties. other disadvantages of live viruses appeared during the intensive researches of the 1980s particularly in the usa and in england [71, 72] . it cannot yet be claimed that the ideal live-attenuated virus vaccine has been formulated, but reverse genetics and increased knowledge of virulence genes have now lead to a resurgence of interest. in the 1980s, genetic studies were intensively pursued in attempts first to define the particular gene or combination of genes, donated by the attenuated virus that confers the property of attenuation upon the reassortant strain. it was found that the biological properties of excreted virus may be altered compared with those of the original virus in the vaccine and the manner of this alteration was also studied genetically. such work is essential in achieving the goal of an effective and safe vaccine virus for human use. experimental inoculations were carried out initially in small-scale tests in volunteers under semi-isolation to permit close observation (see below). multiple cultivation and passage of viruses either in animal hosts, such as ferrets and mice, or in developing chick embryos or tissue cultures had been practiced even before the use of temperature-sensitive (ts) or cold-adapted (ca) mutants was suggested. early workers in britain used the pr8/34 virus as a host range mutant, which, although noninfective for man, has retained animal pathogenicity even after many passages in eggs. as a donor parent with good powers of multiplication in the laboratory, pr8 was mated with various strains of wild-type influenza a viruses to obtain recombinants with up-to-date surface ha and na antigens. this method was preferable to simple laboratory cultivation because some viruses failed to alter in pathogenicity after as many as 30 serial passes in cultures [73] , although other virus strains appeared to become attenuated with only a few passages in eggs. pr8 virus was chosen also by workers in belgium who prepared reassortants from a number of viruses, some of which were licensed for human use [74] . to select recombinants with as high proportion of rna components as possible derived from the host range mutant pr8, florent et al. [75] used rna-rna hybridization to identify gene origins. later the gene constellation of four of the candidate vaccine viruses was determined, and florent [76] found that some clones of beare and hall's [77] recombinants of pr8 and a/englannd/69 (h3n2) containing five genes from pr8 were satisfactorily attenuated. however, one clone though containing six pr8 genes was nevertheless clinically virulent to volunteers. a further genetic study of pr8 host range recombinants using viruses tested clinically by beare and reed [78] was made by oxford et al. [79] . it was again found that recombinants from pr8 and a/england/69 viruses could contain only the surface ha and na genes from wild-type virus and yet retain virulence for man. additional attempts to stabilize the attenuation of candidate viruses were made both by beare at the medical research council's laboratories at salisbury and the rit workers by rendering the virus resistant to an inhibitor present in normal horse serum. this property was present in the rit series of recombinants. it seems strange that stabilization has not been pursued since nor has cultivation of host range mutant viruses, such as pr8, at abnormally low temperatures, such as 25 c. this method was found by sabin [80] to be preferable to normal temperatures when attenuating polio viruses, and it was exploited by both workers in the usa and ussr. marker tests, which can be equated with attenuation of virulence for man, were sought with relatively variable results. one such test used weanling rats that were inoculated intranasally first with virus and later with cultures of haemophilus influenzae. virulent virus induces bacteremia and meningitis, and using this method jennings et al. [81] successfully separated a number of reassortant viruses and obtained some correlation with clinical virulence. yet the host range mutant parent pr8/34 and rit 4050, which are both attenuated in man, were classed as virulent by the rat. a new approach at that time used an avian (duck) virus, which was found to have only low pathogenicity for squirrel monkeys inoculated intranasally and was proposed as a donor of attenuation. a reassortant with a virulent human a/udorn/ 72 (h3n2) virus behaved as did the avian parent in the squirrel monkey, although immunizing the latter against the virulent parent. clinical trials have suggested that this virus is attenuated for man and is immunogenic but has not been investigated since [82] . most work on the development of viruses with restricted multiplication at temperatures above the normal range for cultivation has been affected by chanock, murphy, and associates at the national institutes of health, bethesda [83] . the technique used chemically produced mutation in virus rna by cultivation in the presence of the mutagenic agent 5-fluorouracil. after cultivation and plaquing at 33 c, 37 c, and 38 c, mutant viruses with the requisite temperature sensitivity were obtained. intranasal inoculation of hamsters confirmed temperature restriction, in that much lower titers of virus were found in the hotter lungs than in the cooler upper respiratory tract. spread from inoculated volunteers to adults in contact was not observed, and no evidence of a change in virulence was found in viruses recovered from adult recipients of vaccine [84] . however, in seronegative children, the a/hong kong/ 68-ts-l [e] virus produced mild febrile reactions and a virus that had lost its properties was recovered from some who were infected. a second series of ts-1a2 was then developed by combining two defective ts viruses, each of which belonged to a different complementation group in respect of the genetic defect. the progeny exhibited greater temperature restriction than the ts-1[e] line of viruses. it was termed a/udorn/72 ts-1a2, and it was recombined with three further viruses; wild-type a/victoria/3/75, a/alaska/77 (h3n2), and a/hong kong/77 (h1n1). these ts-1a2 viruses were highly immunogenic and exhibited temperature restriction of multiplication in cell cultures and reduced replication in the hamster lung. the a/victoria/3/75-ts-1a2 recombinant retained its ts properties after inoculation into doubly seronegative children. unfortunately, when the a/alaska/77-ts-1a2 virus was similarly tested in a single child after tests in adults had shown genetic stability, the nasal secretions of the vaccine yielded a ts-positive virus that produced plaques at 39 c even though the child had shown no symptoms or fever. the recombinant 1a2 virus with a/hongkong/77 (h1n1) parent exhibited a capacity to infect 70% of doubly seronegative adults and was attenuated compared with the wild-type parent. nevertheless, it appeared possible that a virus such as the a/alaska-ts-1a2 might, if transferred to contacts from an inoculated child, result in clinical illness, and clinical studies with this particular virus were not pursued. beginning with a strain of h2n2 virus recovered in ann arbor, michigan, in 1960 by cultivation of throat washings in tissue cultures at 36 c, maassab [85, 86] evolved a virus, a/ann arbor/6/60 (h2n2), which has acted as a donor of attenuation to other viruses by genetic reassortment. earlier passages were made in chick kidney tissue cultures followed by intranasal passages in mice and then a gradual adaptation to lower temperatures, in tissue cultures and in developing hens' eggs inoculated allantoically, led to a virus with good powers of multiplication at 25 c. the ca variant was found to retain the infectivity of the original strain for both the mouse and the ferret, although it produced no deaths in mice and no fever or turbinate lesions in ferrets, whereas the original virus was pathogenic for both species. the virus proved to be temperature sensitive with a shut-off temperature of 37 c [87] . recombinants with wild-type viruses of both h2n2 and h3n2 subtypes were prepared, studied in the laboratory and in volunteers, and analyzed genetically. the original a/ann arbor/6/60 (h2n2) virus was not, however, tested in fully susceptible persons presumably because of the difficulty in that period of finding seronegative adults. a few persons with low titers of serum neutralizing antibodies (1:4 to 1:6) were inoculated and as judged by antibody responses, became infected without undergoing clinical illnesses. more rigorous clinical studies have been pursued with recombinants, in particular, those with h3n2 antigens, and details of the results have been brought together and earlier data summarized by kendal [72] . the donor ca parent has been more recently reassorted with h5n1 genes. it is clear that infectivity and immunogenicity were fully retained for seronegative adults of whom 111 received h3n2 recombinants. among those receiving three of four recombinants, clinical reactions were minimal or negligible but with the fourth, derived from the a/scotland/74 parent, in 4 of 12 volunteers receiving 10 8.5 and in 1 receiving 10 7.5 tcid 50 , there were clinical illnesses. viruses reisolated from the vaccines retained ts properties and so did those given recombinants of a/victoria/75 (h3n2) and a/alaska/77 (h3n2). however, some loss of ca restriction was found in virus re-isolated from volunteers given the a/scotland/74 recombinant. cold-adapted recombinants with a/ussr/77 (h1n1)-like virus have also been studied in adult volunteers and found to be less immunogenic as judged by hi antibody responses. a better response was obtained by wright et al. [88] in children in nashville given 10 6.5 tcid 50 of strain cr 35 (h1n1) and none of 11 children developed adverse clinical reactions even though eight became infected. all reisolated viruses retained the ts phenotype. the failure to elicit serum antibody response in adults given this same virus recombinant is puzzling. using the elisa enzyme-linked assay, murphy et al. [89] found that by this more sensitive method antibody rises could be demonstrated and the results tallied better with the ability to re-isolate viruses from the inoculated volunteers than did the serum hi responses. the leningrad group of workers led by smorodinstev [90] was the first to obtain a virus indirectly attenuated by cultivation at 25 c. the group used strains selected by inoculating volunteers with several viruses derived from cultures repeatedly incubated at 25-26 c to speed up attenuation. approximately 5-7 months were required for the preparation and production of new strains even using genetic recombination to incorporate new surface ha and na antigens. although alexieva et al. [91] found that cold cultivation was not successful in producing reliably attenuated viruses for use in children, the technique was adopted for general use. genetic studies of the leningrad viruses are described briefly by kendal et al. [72] , and these parent ca viruses are currently the center of new interest for attenuated h5n1 vaccines. usually, preliminary studies were made in the ussr in 18-21-year-old seronegative adults who receive virus twice at intervals of 10-14 days administrated by nasal spray. viruses were attenuated by passage for varying periods at 25 c and both donor viruses and recombinants proved temperature sensitive. in 1961-1964, when h2n2 viruses were circulating, 5,165 children aged from 1 to 6 received the ca a/leningrad/57 (h2n2) virus. some febrile reactions occurred but only in less than 1% of the children. further studies of recombinants with h3n2 or h1n1 antigens and the same leningrad h2n2 parent after 47 serial passages under cold conditions of cultivation (25 c) were conducted in children, half of whom had no detectable serum antibody to the vaccine strain. no reactions occurred and over 90% of the children responded with antibody production. it is clear from the earlier papers by alexieva et al. [91, 92] that intranasal administration of children aged 7-15 were too reactogenic and that this is the reason why the peroral route has been chosen for routine administration in the ussr. a japanese virus recovered in 1957, a/okuda/57(h2n2), was found to be attenuated for children and served as a donor of attenuation both in japan and in england. zhilova et al., japanese workers, [92] developed a recombinant virus (ko-1) from ultraviolet-irradiated a/okuda/57 and wild-type a/kumamoto/22/76 (h3n2). serial passaging in eggs in the presence of normal horse serum was followed by plaque purification and later clinical tests in a few children. the m (membrane) gene was found to have been donated by the okuda parent. from reassortants with other human viruses, a candidate wrl 105 virus was selected and underwent clinical trials without harmful clinical effects [93] but has been little investigated since that time. cultivation of influenza viruses in mammalian cells rather than eggs initially encouraged two manufactures to invest in cell culture fermenters for vaccine production [22, 23] . many more groups are now using these technologies to produce the current a/swine h1n1 vaccine. capacity can be increased to cope with a surge in demand for a pandemic virus vaccine. moreover, the final vaccine has the theoretical advantage of the absence of egg proteins. the cell culture vaccine virus is also easier to purify. where clinical isolates of influenza viruses are cultivated in mammalian cells and eggs in parallel, different antigenic variants may be selected [94] . the biological variants have amino acid substitutions in the receptor binding site in proximity to an antigenic site on the ha, and an amino acid change in this region can alter antigenicity. of the two virus subpopulations that can be selected, the virus which is grown on mdck (or vero) cells rather than in eggs appears more closely related to the wild-type clinical virus. there is some indication that cell-grown virus vaccines offer greater protection in animal models than the corresponding egg-grown vaccine. these are all powerful arguments in favor of the new generation of influenza vaccines being cultivated currently in vero [22] or mdck [23] or per 6 cells. alongside 50 years of experience producing an immunogenic and safe vaccine, the world capacity for influenza monovalent vaccine manufacture has expanded to the present two billion doses. the preparation work and investment for h5n1 are showing rewards with the current pandemic of a/swine h1n1. most manufacture is still located within the eu, but production is increasing in the usa, korea, japan, china, and most recently, india. the international collaboration in face of the outbreak of a/california/4/09 (h1n1) in mexico around christmas 2008 to the present, the exchange of clinical data and viruses enabled vaccine manufacture to start production by may/june 2009. by october 2009, the production of hundreds of millions of doses of a monovalent vaccine containing 15 mg of ha and immunogenic after a single ion dose in the over 5-year olds is a quite remarkable achievement. many countries have started to immunize at-risk groups, namely younger people <65 years of age with diabetes, obesity, chronic heart, or lung problems, and the immunosuppressed including pregnant women. it is forecast that up to 40-50% of some countries could volunteer for the vaccine. it is especially important that medical and nursing staff take the vaccine to protect both themselves and their patients. however, such large vaccination campaigns open schisms in modern societies,which on the one hand become very concerned about young persons dying but on the other hand have prejudices about vaccines in general. in the first winter wave the over 65s, unusually for a pandemic virus, are protected by prior experience of the h1n1 family and hence overall mortality is likely to be less than a seasonal year but the mortality is likely to be in younger persons, thus exposing our achilles heel. additionally nearly half the deaths to date have been in young persons without comorbidities. finally, within a year the virus is likely to mutate to allow it to infect the over 65 group, so, paradoxically, mortality in the second pandemic year could easily exceed the first. twenty-first century vaccines induce protection across the different virus subtypes? there are 16 known subtypes of the ha of influenza a virus. only three subtypes have caused pandemics in humans, h1, h2, and h3, while h5, h7, and h9 predominantly circulating in birds have crossed the species barrier into humans and caused human outbreaks. we do not know whether these latter three subtypes could mutate into human-to-human transmitters and thereby acquire pandemic potential. at present, h5n1 is causing considerable concern in se asia. an important question therefore is whether a vaccine could be engineered to give socalled heterotypic or cross-subtype immunity to protect against all these potentially pandemic viruses. it is well known that the internal proteins of influenza a virus such as m1, m2 and np are shared by all influenza a viruses. these internally situated proteins are certainly immunogenic (particular np) but could the immunity induced, either t cell or antibody, be broadly reacting? to back up the central core of this approach, it has been known for 40 years that mice infected with an influenza a (h1n1) virus would later resist a lethal challenge from an influenza a (h3n2) virus. given the lack of genetic and antigenic relatedness between the h1 and h3 proteins, or indeed the corresponding n1 and n2 proteins, this strong cross-immunity was attributed to an internal protein such as np or m. however, it has been difficult to construct a solid database and there has been a lingering doubt about this so-called cross-protective immunity. most virologists deduced, virtually by elimination, that a cross-reactive portion of the ha (ha2) could have provided the cross protection. furthermore, this cross protection is particularly seen in the mouse model, leading some to conclude that the mouse recognized cross protection epitopes that perhaps humans did not. fundamental studies to correlate the genetics and immunology of np and m established the cytotoxic t-cell response to portions of these proteins. however, the work clearly showed that m2 could be a cross-reactive immunogen, although a relatively weak one [95] . the m2 protein is an integral membrane protein of influenza a viruses that is expressed at the plasma membrane of virus-infected cells and is also present in small amounts on virions. the important extracellular domain, potentially targeted by antibodies and t cells, is conserved by virtually all influenza a viruses. even the 1918 pandemic virus differs only in one amino acid. the first indication that the m2 was immunologically active was the observation that an anti-m2 monoclonal antibody reduced the spread of virus cell culture. not unexpectedly, the antibody reacted with the extracellular domain of m2. even more excitingly, the antibody reduced the replication of virus in mouse lungs. immunization studies with m2 constructs, however, have given more mixed results. immunization of mice with dna plasmid of m1 and m2 gene gave protection mainly via t-helper cell activity. an alternative approach utilized a hepatitis b core and m2 fusion protein. the cross protection resided in antibodies, although m2specific antibodies did not neutralize the virus in vitro. presumably, protection was mediated by an indirect mechanism such as complement-mediated cytotoxicity or antibody-dependant cytotoxicity. however, the protection induced in the mouse model was considerably less than that induced by a conventional sub unit ha/na vaccine. it could be argued that weak heterotypic immunity may be present already in the community and that this is helping to prevent the emergence of chicken influenza a (h5n1) in se asia [96] . certainly with evidence of tens of millions of domestic birds infected since late 2003 in 13 countries in se asia, with only a handful of human infections and only human-to-human transmission in family groups, there is a possibility that the unique cocirculation since 1977 of two influenza a viruses (h1n1 and h3n2) may have enhanced heterotypic immunity in most communities, which in turn abrogates the emergence of chicken influenza a (h5n1) into humans. it would be foolhardy, though, to take this argument to a fuller conclusion and relax preparations for a new pandemic influenza a virus. at present, with the unprecedented research investment into influenza vaccines, there are new discoveries of adjuvants and vaccine formulations to be tested as well as fundamentals of virus transmission, infectiousness, and pathogenicity. the ultimate test is in influenza-infected volunteers. this specialized work was initiated over 60 years ago. during the great pandemic of 1918, when the precise nature of the causative microbe of the spanish influenza had not been established, a group of american scientists asked for young volunteers from the army and navy. the quest was to probe the nature of the microbe that was already causing devastation in their own country and where, by 1919, 500,000 young people were to die. however, this was not the first study into the precise nature of the microbe. the infection had first been documented a year earlier as a herald wave in the great city-sized military base and encampment of etaples [6, 12] . here the british army constructed the largest establishment [97] in its history, where 100,000 newly recruited soldiers each day intermingled with thousands of wounded soldiers, pigs and, in the nearby villages and markets, with ducks, domestic chickens, and geese. these are now recognized as the necessary biological features of an epicenter for the creation of a pandemic virus. we surmise, in retrospect, that an avian virus from a silently infected goose or duck could have crossed species either to a pig or to a soldier already infected with a human strain of influenza. this is the mixing bowl hypothesis. indeed, common epidemic influenza was known to be circulating in the winter of 1916-1917 in etaples. another factor in etaples could have been the hundreds of tons of gases of 25 varieties contaminating the landscape of the nearby somme battlefield, as well as many of the wounded soldiers brought by the night trains into the 12 hospitals on site and causing respiratory distress. a group of pathologists there and at abbeville, led by g. gibson, raised the question of the nature of the microbe. could it be a gram-negative bacterium such as h. influenzae, already described by pfeiffer as the cause of the previous influenza pandemic of 1889? or could it be a virus? viruses were rather unknown entities at that time but had been identified by their filter-passing nature. hence, gibson's experiment was quite simply to take sputum from a soldier victim and filter it through a berkefield candle filter, which would hold back any known bacterium but allow the passage of the much smaller ultrafilterable virus. but what then? gibson had not even considered that a human volunteer would receive the filtrate. in fact, he gave it to a series of macaques and, inadvertently, to himself. he died and the macaques became ill. his premature discovery of new virus influenza has lain undiscovered and hitherto unquoted in the archives of the first world war [98] . meanwhile, in the usa, a more vigorous decision had been taken, and army and navy volunteers were infected intranasally with filtered material from spanish influenza victims. some volunteers were placed 0.5 m from dying servicemen, who coughed in their faces. the incredible result of this heroic endeavor is that not a single volunteer became ill, whereas all around the usa their companions were dying. it is more than possible that the volunteers had already been subclinically infected in the early summer outbreak of 1918, which was less virulent than the autumn virus and would be expected to give cross-immunity. unit in salisbury (uk) as soon as the second world war was over, the medical research council in the uk established the common cold unit in salisbury at the harvard hospital. the hospital was a donation from the usa to cope with expected bomb casualties from london. in the event, this fully equipped multibuilding facility was used as an acute surgical hospital for servicemen. with christopher andrewes as its first chief scientist, the unit recruited volunteers to unravel the virological mysteries of respiratory disease. for the next 40 years, a small team of virologists and clinicians infected volunteers and discovered the first human coronavirus, the common cold virus, and were the first to describe the clinical effects of interferons. essentially similar units were set up in the usa and ussr. the considerable difficulties encountered in mounting field trials led to experiments in which immunized volunteers were subjected to deliberate inoculation with live virus in the form of either attenuated strain or modified wild-type strain. this protocol was suggested by henle et al. [99] , who immunized a group of children with inactivated influenza a (h1n1) virus vaccine and then inoculated them with egg-cultured virus of the same subtype but recently isolated, by inhalation of an aerosol. high rates of infection (75%) were produced in 28 unimmunized children of whom 10 became ill. those receiving vaccine either escaped subsequent infection or developed serological changes; only 1 child of the 42 vaccinated children thus challenged became ill. although this study illustrated the outstanding success of the immunized protocol, there are probably few observers today who would be prepared to submit their children to a similar risk of deliberately induced illness. ideally young adults 18-45 are used for quarantine experiments. such a risk is, of course, experienced during epidemics and bell et al. [100] undertook a similar experiment in adult volunteers some of whom were immunized with a single dose of inactivated a/japan/305/57 (h2n2) virus vaccine soon after the a/asian epidemic began. the volunteers were isolated before being given intranasally pooled nasopharyngeal washings from patients with influenza and this caused clinical illness in 87% of volunteers previously given a placebo. as 50% of the vaccinated volunteers developed fever after challenge in this experiment, the single injection of inactivated vaccine proved relatively ineffective, presumably because of its inadequate immunogenicity. the information obtained by deliberate challenge of immunized volunteers has been explored in the past using modified attenuated virus strains. beare et al. [73] did this in their comparison of inactivated or live influenza b vaccines in which a challenge from the live virus b strain was used to assess the comparative efficacy of the two vaccines. reinoculation with live virus was resisted better by those receiving the same material a month previously than by those injected with inactivated vaccine. couch [101] has reported a number of trials in volunteers after inactivated vaccine using a low dose of an essentially unmodified h3n2 virus that had received one or two passages in human embryonic kidney culture. it was first established by greenberg et al. [102] that previous infection by homotypic h3n2 virus gave protection against deliberate exposure for up to 4 years after the original infection. comparison of inactivated vaccine a/hongkong/68 (h3n2) given intranasally or subcutaneously showed that following challenge with live virus only those who had developed a serum antibody response after vaccine by either route resisted infection. in a further trial of an anti-na inactivated vaccine made from an heq1n2 virus, it was shown that a reduced frequency of illness and a reduced titer of virus in nasal wash specimens resulted following live h3n2 virus challenge compared with the findings in control subjects. the number of those who contracted infection was also reduced somewhat by the inactivated na vaccine, thus supporting the suggestion of schulman et al. [103] that na antibody, although incapable of neutralizing viral infectivity, could limit the extent of viral replication. beutner et al. [104] also immunized children with an na-specific vaccine and noted that antibody to na had a role protecting against illness rather than against infection. slepushkin et al. [105] and monto and kendal [106] came to similar conclusions with regard to na vaccine and the clinical evidence of protection from illness. a series of experiments on volunteers, designed to obtain evidence of protection from vaccines containing viruses that were homotypic or heterologous to the challenge virus, is important in relation to the determination of the best composition of inactivated vaccine. potter et al. [43] gave one of four inactivated monovalent h3n2 virus vaccines to groups of students, measured their pre-and postimmunization antibodies by hi and ni tests, and later challenged all the groups with a live intranasal h3n2 virus (wrl 105). this virus was antigenically nearest to the a/port chalmers/73 virus and vaccine from this latter strain and also that containing a/scotland/74 virus gave better protection against infection than earlier h3n2 virus vaccines; the result thus correlated with the induced hi antibody titers. larson et al. [107] also challenged the immunity produced by inactivated vaccine made from a/port chalmers/73 (h3n2) virus with that from a strain developed by the pasteur institute [108] . this virus (30c) with an antigen closely similar to a/england/72 (h3n2) was selected in the laboratory by a method analogous to natural selection by antigenic drift, and thus represents the first human attempt to anticipate antigen variation in nature. challenge of those immunized with one or the other vaccines showed that protection by the heterologous 30c virus was about one-quarter as effective as that produced by the homologous a/port chalmers/73 virus. experiences related by couch also confirm [101] that antibody effective against the homologous ha of the challenging virus is more protective than that formed by heterologous antigen. protection was also compared after inactivated vaccine by intranasal or subcutaneous routes, which showed that the important mediator of immunity was the serum igg content of anti-ha rather than the respiratory secretion content of specific iga. we have established a new quarantine unit, based in london (http://www.retroscreen. com), but very much centered upon the experience and ethos of the common cold unit of the past [109] . in a series of experiments over the past 2 years, we have infected over 250 young volunteers with influenza a (h3n2), influenza b, and influenza (h1n1) virus and more recently respiratory syncytial virus, and we now have fully characterized virus pools [110] . in the usa, a quarantine unit had already been established in virginia and also at baylor and pioneered work into the new na inhibitors of influenza using an influenza a virus isolated in 1991 [111] . so far our own unit has focused on evaluating new influenza vaccines [112] . we use groups of 20 young volunteers and quarantine them in a student hostel or hotel or phase i clinical unit along with clinicians and scientists (fig. 8) . the mrc common cold unit was rooted strongly in the postwar era with deck chairs, free run rabbits, country walks, afternoon cream teas, and two-course english meals. our new unit reflects a more diverse community, so chicken tikka is as common on the menu as roast lamb and baked potatoes, but the wish of many of the volunteers is the same: to contribute to knowledge. influenza a virus has a proven record as a "bioterrorist" virus but driven not in churchill's words by the "evil forces of perverted science" but by the vast unfathomable laws of nature and emergence, reemergence, and resurgence of natural disease. we are experiencing the attacks on pregnant women and younger persons at the present moment with a/swine h1n1 [113] [114] [115] . information from the human genome project, whereby a significant proportion of the 30,000 active genes are already known to be involved in innate and acquired immunity, provides reassurance that the immune system will continue to provide some protection against new fig. 8 a volunteer room at the common cold and influenza unit, harvard hospital, salisbury, in the 1980s. volunteers would stay for 2 weeks in this country-placed unit to be infected and carefully studied for clinical symptoms viruses. this is excellently illustrated with a/swine where most of the population has immune memory to this h1n1 family of viruses. gauguin in his last great painting "who are we, where have we come from, where are we going?" asks crucial questions about the future of humankind. but it was the medieval painter breugel who asked the major question, yet to be answered in the twenty-first century. his medieval painting "the triumph of death" shows a horseman on a white charger scything at random and gathering souls during an outbreak of pasteurella pestis in medieval times. the question haunting the painting is "why do some persons survive while others die." even in 1918 in most communities 99% of persons infected with the virus survived. but why did some die and exactly how were they killed by such a minute and fragile form of life that we know as the orthomyxovirus influenza? was the immune reaction and ensuing cytokine storm overwhelming or was virus replication in the endothelial cells of the air sacs more important? an extraordinary clear message is emerging, which tells us to build our public health infrastructure and continue and expand our epidemiological vigilance and surveillance against all these infectious viruses and bacteria. the virus cannot be permanently dislodged from its avian and swine reservoir. for pandemic influenza, every country needs a detailed and practical plan and a supply of antiviral drugs and new vaccines at hand for an emergence of h5n1. this virus will be a lot more difficult to deal with than a/swine h1n1. we would then be "at the end of the beginning" as regards protection of all citizens. influenza was the twentieth century's weapon of mass destruction. nature is the greatest bioterrorist of our world and emerging viruses could do for 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to vaccination with live influenza a2 hong kong vaccine effect of neuraminidase antibody on hong kong influenza immunity to challenge in volunteers vaccinated with an inactivated current or earlier strain of influenza a (h3n2) sélection par pression immunologique de mutants dominants du virus de la grippe a (hong kong) cold wars: the fight against the common cold fluinsure tm , an inactivated trivalent influenza vaccine for intranasal administration, is protective in human challenge with a/panama/2007/99 (h3n2) virus. in: kawaoka y (ed) options for the control of influenza volunteer challenge studies dna vaccination protects against an influenza challenge in a phase 1b double blind randomised placebo controlled clinical trial the emerging influenza pandemic: estimating the case fatality ratio assessing the severity of the novel a/h1n1 pandemic cdc. novel h1n1 influenza vaccine acknowledgments we are pleased to receive grant income from the eu to develop new influenza vaccines. key: cord-002438-b8t4a57r authors: cheng, wei; yu, zhao; liu, shelan; zhang, xueying; wang, xiaoxiao; cai, jian; ling, feng; chen, enfu title: comparison of influenza epidemiological and virological characteristics between outpatients and inpatients in zhejiang province, china, march 2011–june 2015 date: 2017-02-22 journal: int j environ res public health doi: 10.3390/ijerph14020217 sha: doc_id: 2438 cord_uid: b8t4a57r given the rapid rate of global spread and consequently healthcare costs related to influenza, surveillance plays an important role in monitoring the emerging pandemics in china. however, the characteristics of influenza in southeast of china haven’t been fully studied. our study use the surveillance data collected from 16 sentinel hospitals across zhejiang province during march 2011 through june 2015, including the demographic information and respiratory specimens from influenza-like illness (ili) patients and severe acute respiratory illness (sari) patients. as analysis results, most sari and ili patients were in the age group of 0–4 years old (62.38% of ili and 71.54% of sari). the respiratory specimens have statistically significantly higher positive rate for influenza among ili patients than that among sari patients (p < 0.001). the comparison between ili patients and sari patients shows no statistically significantly difference in detecting influenza virus type and influenza a virus subtype. the sari and ili patients were found to be positively correlated for overall positive rate (r = 0.63, p < 0.001), the weekly percentage of a(h1n1)pdm09 (r = 0.51, p < 0.001), influenza b virus (r = 0.17, p = 0.013), and a/h3n2 (r = 0.43, p < 0.001) among all the positive numbers. our study demonstrated that the activities of influenza virus, including its subtypes, had a similar temporal pattern between ili and sari cases. influenza virus is estimated to cause 3 to 5 million cases of severe illness and 250,000 to 500,000 deaths each year, while 5%-10% of adults and 20%-30% of children are infected with the influenza virus worldwide [1] . in lower and middle-income countries, influenza could result in large economic burden encompassing direct costs to the health service and households, and indirect costs of productivity losses [2, 3] . vaccination is a cost-effective way to reduce the public health and economic impacts caused by influenza. because of the antigenic shift and drift of the virus, the influenza vaccine composition needs regular updates. currently, the selection of strains for the annual influenza vaccine are primarily 2 of 12 based on the predominant strain of influenza virus detected in influenza-like illness (ili) [4] . however, ili only represents a proportion of acute respiratory infections with mild clinical manifestations. severe acute respiratory illness (sari) surveillance is another type of surveillance for severe influenza-associated disease. it is recommended to be monitored with established surveillance systems with ili according to the guidelines of the world health organization (who) [5] . in china, patients meeting with the definition of sari are required to have inpatient observation and necessary treatment. due to the emerging high healthcare cost and severe consequences of sari, effective vaccination is needed to reduce the incidence of sari caused by influenza [6] [7] [8] . therefore, whether influenza epidemiological and virological characteristics among sari patients were consistent to those of ili patients is critical to the effectiveness of current vaccination plan. zhejiang province located in the southeastern china, featuring by its blooming economy and high density of population. it has over than 55 million permanent residents, which are distributed in 11 metropolitan areas. influenza surveillance in zhejiang province launched at the same time with national surveillance in the year of 2001, and expanded to 16 ili and four sari sentinel hospitals by the year of 2011. both ili and sari are monitored in the surveillance system. although studies describing influenza surveillance with both ili and sari are well documented in the northern and southern hemispheres [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] , few studies have fully compared the influenza epidemiological and virological characteristics between ili and sari cases. in this study, we used four-year continuous surveillance data to compare the epidemic and virological characteristics of influenza virus between ili cases and sari cases in zhejiang province. the influenza surveillance network in zhejiang province is a part of the national influenza surveillance system [20] . the ili surveillance in zhejiang province was initially launched in 2001. as to 2009, the ili surveillance has been expanded into 16 sentinel hospitals that cover all of the 11 metropolitan areas in zhejiang province. the types of sentinel hospitals include general hospital, specialized clinic as well as maternal and children hospital. the sari surveillance started in 2009. as to march 2011, the surveillance has expended to four sentinel hospitals including three general hospitals and one children's hospital. all four sari sentinel hospitals were selected from the existing ili surveillance network ( figure 1 ). ili is defined as any person with sudden onset of fever >38 • c and cough or sore throat in the absence of other diagnosis [21] . the definition of sari is varied by ages. a patient >5 years old is defined as having sari if, upon or during admission, presenting an acute onset of elevated temperature (axillary temperature ≥38 • c) and cough or sore throat, as well as tachypnea (respiratory rate ≥25/min) or dyspnea (difficulty breathing). a patient ≤5 years old is defined as having sari if, upon or during admission, presenting with acute onset of cough or dyspnea, and at least one of the following six signs or symptoms: (a) tachypnea(respiratory rate >60/min for ages <2 months, respiratory rate >50/min for ages 2 to <12 months, and respiratory rate >40/min for ages 1 to ≤5 years); (b) inability to drink or breastfeed; (c) vomiting; (d) convulsions; (e) lethargy or unconsciousness; (f) chest in-drawing or stridor in a calm child [4] . ili is defined as any person with sudden onset of fever >38 °c and cough or sore throat in the absence of other diagnosis [21] . the definition of sari is varied by ages. a patient >5 years old is defined as having sari if, upon or during admission, presenting an acute onset of elevated temperature (axillary temperature ≥38 °c ) and cough or sore throat, as well as tachypnea (respiratory rate ≥25/min) or dyspnea (difficulty breathing). a patient ≤5 years old is defined as having sari if, upon or during admission, presenting with acute onset of cough or dyspnea, and at least one of the following six signs or symptoms: (a) tachypnea(respiratory rate >60/min for ages <2 months, respiratory rate >50/min for ages 2 to <12 months, and respiratory rate >40/min for ages 1 to ≤5 years); (b) inability to drink or breastfeed; (c) vomiting; (d) convulsions; (e) lethargy or unconsciousness; (f) chest in-drawing or stridor in a calm child [4] . each week, physicians from departments of pediatrics and respiratory, as well as emergency rooms in all of the 16 ili sentinel hospitals were required to report the number of total visits to outpatient, as well as the number of outpatients who presented with non-specific symptoms that meet a case definition of ili. as for four sari sentinel hospitals, the number of sari and total admissions from pediatrics ward, the respiratory medicine ward and the intensive care unit were also reported. all of the reported numbers in the above-mentioned departments were required to record by age classified as 0-, 5-, 15-, 25-, and 60-years old. nasopharyngeal or throat swabs were required to collect from all of sari cases and 5-15 ili cases of each sentinel hospital on every week. the collection of swabs was conducted by trained nurses from who had not received antiviral each week, physicians from departments of pediatrics and respiratory, as well as emergency rooms in all of the 16 ili sentinel hospitals were required to report the number of total visits to outpatient, as well as the number of outpatients who presented with non-specific symptoms that meet a case definition of ili. as for four sari sentinel hospitals, the number of sari and total admissions from pediatrics ward, the respiratory medicine ward and the intensive care unit were also reported. all of the reported numbers in the above-mentioned departments were required to record by age classified as 0-, 5-, 15-, 25-, and 60-years old. nasopharyngeal or throat swabs were required to collect from all of sari cases and 5-15 ili cases of each sentinel hospital on every week. the collection of swabs was conducted by trained nurses from who had not received antiviral drugs. in addition, a standardized case report form containing demographic and sample information was also required to complete among whose biological samples had been collected. due to the outbreak of avian influenza a(h7n9) in april 2013, ili surveillance was strengthened by increasing the weekly number of the biological samples collected in each hospital from 5-15 to 20. the biological samples were collected and saved in cryovial tubes, stored at 4 • c at the sentinel site, and then sent to regional center for disease control and prevention (cdc) within 48 h after the sample collected. the regional cdc laboratory tested influenza using real-time reverse transcription polymerase chain reaction (rrt-pcr) assay following the standard protocols. specimens tested as positive for influenza a were further tested for subtypes (i.e., a(h1n1), a(h3n2), a(h1n1)pdm09, and a(h7n9)) using specific rrt-pcr. once the laboratory complete, regional cdc submitted the laboratory results to the online surveillance system. data obtained from the surveillance system were reported weekly by the staff of the sentinel hospitals and laboratories. the mean and standard deviation or median and interquartile range (iqr) were calculated for continuous variables, and percentages were calculated for categorical variables. chi-squared test and fisher's exact test were used to assess the differences of age, sex, season, influenza virus type/subtype, and influenza positive rate between all the sampled ili and sari cases. the weekly number of positive influenza by subtype and the percentage of specimens tested positively were plotted to describe seasonality and circulation of influenza types/subtypes among sari and ili cases. spring, summer, autumn, and winter were defined from week 11 to 21, 22 to 38, 39 to 48, and 49 to 10 of the next year, respectively [22] . cochran-armitage trend test was used to analyze the trend change of influenza virus positivity with the increase of age. spearman correlation was applied to analyze the linear relationship of the influenza virus positive rate, weekly percentage of influenza virus subtypes accounted for all the positive numbers between sari and ili patients. ili percentage was calculated as the percentage of total outpatient visits that were due to ili and sari percentage was calculated as the percentage of total admissions that were due to sari. to compare ili percentage and sari percentage of which can better reflect the activity of influenza virus among outpatients and inpatients, spearman correlation analysis was used to assess the linear relationship between weekly ili influenza-positive rate and ili percentage, as well as weekly sari percentage and sari influenza-positive rate. a two-sided p-values were considered as statistically significant if it was found less than 0.05. all the statistics were conducted using sas version 9.2 (sas institute, cary, nc, usa). verbal consent was obtained from all patients in prior to survey and specimen collection. for children aged under 15 years old, verbal consent was obtained from at least one parent or legal guardian. the influenza surveillance were a national-wide, governmental public health activity. therefore, institutional review board approval was not required in china. in this study, the personal identifiers (e.g., names, address, occupations and so on) were not disclosed in order to maintain patient confidentiality, all the patient information was analyzed anonymously. during the study period, 52,293 patients completed both the standardized case report and laboratory sample test, of which 46,868 (89.63%) were ili patients and 5425 (10.37%) sari patients. the median age of the tested patients was 15 years (iqr: 3-34), and the median age of ili patients was significantly older than it of sari patients (p < 0.001). in addition, the group of sari patients had higher proportion of children at 0-4 years old (71.54% for sari versus 27.82% for ili), male during the study period, 52,293 patients completed both the standardized case report and laboratory sample test, of which 46,868 (89.63%) were ili patients and 5425 (10.37%) sari patients. the median age of the tested patients was 15 years (iqr: 3-34), and the median age of ili patients was significantly older than it of sari patients (p < 0.001). in addition, the group of sari patients had higher proportion of children at 0-4 years old (71.54% for sari versus 27.82% for ili), male (61.25% for sari versus 50.67% for ili), and patients enrolled in the winter (34.14% for sari versus 28.32% for ili) than those of ili patients (table 1) . the most identified influenza virus were influenza a virus (61.97% among ili, and 63.05% among sari), followed by influenza b virus (37.92% among ili, and 36.95% among sari), and mixed type virus (0.11% among ili; 0.00% among sari), with no statistical significance between the two groups (p = 0.774). for influenza a virus in the ili group, a(h3n2) was the most identified subtype (75.00%), followed by a(h1n1)pdm09 (24.87%), a(h7n9) (0.10%), and a(untype) (0.04%). those proportions were correspondent to that in the sari group-a(h3n2) (73.49%), a(h1n1)pdm09 (25.58%), a(h7n9) (0.93%), and a(untype) (0.00%) with p-value 0.067 (table 1) . influenza viruses were found in the specimen of 8601 of 52,293 (16.44%) all patients, with 8260 of 46,868 (17.62%) ili patients and 341 of 5425 (6.29%) sari patients. table 2 shows the specific positive rate of influenza virus by age groups, genders, and seasons. for ili patients, the highest (24.74%) rate is in the 40-59 years age-group, followed by >60 years age-group (23.01%). meanwhile, the positive rate of influenza viruses of sari patients was highest in the >60 years age-group (11.07%), followed by 5-14 years age-group (10.06%). among both ili and sari cases, influenza virus was found in all age groups, and cochran-armitage trend test showed that the influenza virus positive rates tend to be higher at older ages (figure 3) . the positive rate was highest in the winter, and lowest in the autumn. overall, the influenza virus positive rate among ili cases was significantly higher than that among sari cases across different groups of age, sex and season ( table 2 ). mixed type virus (0.11% among ili; 0.00% among sari), with no statistical significance between the two groups (p = 0.774). for influenza a virus in the ili group, a(h3n2) was the most identified subtype (75.00%), followed by a(h1n1)pdm09 (24.87%), a(h7n9) (0.10%), and a(untype) (0.04%). those proportions were correspondent to that in the sari group-a(h3n2) (73.49%), a(h1n1)pdm09 (25.58%), a(h7n9) (0.93%), and a(untype) (0.00%) with p-value 0.067 (table 1) . influenza viruses were found in the specimen of 8601 of 52,293 (16.44%) all patients, with 8260 of 46,868 (17.62%) ili patients and 341 of 5425 (6.29%) sari patients. table 2 shows the specific positive rate of influenza virus by age groups, genders, and seasons. for ili patients, the highest (24.74%) rate is in the 40-59 years age-group, followed by >60 years age-group (23.01%). meanwhile, the positive rate of influenza viruses of sari patients was highest in the >60 years age-group (11.07%), followed by 5-14 years age-group (10.06%). among both ili and sari cases, influenza virus was found in all age groups, and cochran-armitage trend test showed that the influenza virus positive rates tend to be higher at older ages (figure 3) . the positive rate was highest in the winter, and lowest in the autumn. overall, the influenza virus positive rate among ili cases was significantly higher than that among sari cases across different groups of age, sex and season ( table 2 ). due to the outbreak of avian h7n9 virus in april 2013, ili surveillance was strengthened by increasing the number of samples for testing from 5-15 to 20. figure 4 shows that although the weekly number of the samples tested has increased since the week 14 of year 2013, the weekly number of all ili patients remained at similar level from year 2011 to 2014 (figure 4 ). due to the outbreak of avian h7n9 virus in april 2013, ili surveillance was strengthened by increasing the number of samples for testing from 5-15 to 20. figure 4 shows that although the weekly number of the samples tested has increased since the week 14 of year 2013, the weekly number of all ili patients remained at similar level from year 2011 to 2014 (figure 4) . figure 5a ). consistently, this influenza activity was also observed among sari patients ( figure 5b ). seven a(h7n9) viruses (five in the ili group and two in the sari group) were detected during the early year of 2014 ( figure 5 ). we found that the weekly percentage of influenza virus types/subtypes among all the identified influenza cases were significantly correlated between sari and ili patients, with a(h1n1)pdm09 (r = 0.51, p < 0.001), influenza b virus (r = 0.17, p = 0.013), and a(h3n2) (r = 0.43, p < 0.001) ( table 3) . table 3 . correlation analysis of weekly influenza virus type/subtype constitution among total positive numbers between influenza-like illness (ili) and severe acute respiratory illness (sari). figure 5a ). consistently, this influenza activity was also observed among sari patients ( figure 5b ). seven a(h7n9) viruses (five in the ili group and two in the sari group) were detected during the early year of 2014 ( figure 5 ). we found that the weekly percentage of influenza virus types/subtypes among all the identified influenza cases were significantly correlated between sari and ili patients, with a(h1n1)pdm09 (r = 0.51, p < 0.001), influenza b virus (r = 0.17, p = 0.013), and a(h3n2) (r = 0.43, p < 0.001) (table 3 ). 3.6. correlation analysis between weekly ili influenza-positive rate and sari influenza-positive rate, ili percentage and ili influenza-positive rate, sari percentage and sari influenza-positive rate table 4 shows the results of spearman correlation analysis among influenza virus positive rate and percentage of people with ili or sari. the positive rate for influenza virus of ili cases and those of sari cases was statistically significantly correlated (r = 0.63, p < 0.001). the percentage of ili cases and ili influenza-positive rate was statistically significantly correlated (r = 0.53, p < 0.001), which was higher than it of sari (r = 0.19), whose coefficient was statistical significant though (p < 0.001). to our knowledge, this is the first study to compare the epidemic characteristics of influenza between outpatients and hospitalized inpatients in zhejiang province. children less than 5 years of age were found to be the largest group of both ili and sari patients, which was consistent with those reported by other studies [10, 16, 17] . besides, we found a good agreement between sari and ili patients for the weekly proportion of samples tested positively for influenza virus and the distribution of the influenza virus types/subtypes among all the identified patients. this demonstrated that the seasonal pattern and predominant circulation types of influenza were similar between ili and sari patients. finally, we found that the correlation between the weekly influenza positive rates and percentages of patients meeting the definition of ili and sari is higher among ili patients than it among sari patients, which indicated that compared to ili, influenza may less important in causing sari. although the largest age groups were same (0-4 years old) among ili patients and sari patients, this age group had higher proportion within sari patients than it within ili patients. this difference might be caused by the different behaviors when a child or an adult is found to be sick. compared to adults, children are more likely to be taken to hospital, especially for sari cases. therefore, children have sari were more likely to be involved in the surveillance. similar results have been obtained in mongolia [10] , philippines [11] , jordan [23] . our findings further demonstrated that young children are vulnerable for both mild and severe respiratory infection, and the low influenza detection rate among 0-4 years age-group in both sari and ili patients foreshadow the need of expand the respiratory illness surveillance to more types of pathogens [12, 24] . being consistent with other studies, influenza virus was detected in all age groups among both ili and sari cases [10, 14] . and overall, the proportion of samples tested positively for influenza viruses in different age groups presented to be higher with the increase of age. therefore, all persons aged 6 months and older are recommended for vaccination and elders should be considered with priority [25] . to understand the temporal characteristics of influenza epidemics is essential for planning influenza vaccination programmes because vaccine effectiveness wanes over time, a boost of vaccine is essential to prevent the spread of diseases [26] . the simple and preferred measure to assess and compare seasonality patterns was the proportion of influenza positives [27] . the highly correlated influenza virus positive rate between sari and ili patients demonstrated the similar temporal pattern of influenza activity in the two groups. similar findings were recently reported in a description of influenza surveillance in egypt, which showed that the seasonality of influenza among ili cases and sari cases was consisted in november-february [19] . moreover, the comparison between ili patients and sari patients shows no statistically significantly difference in detecting influenza virus type and influenza a virus subtype, which was similar to the findings from nigeria [16] . finally, the correlation of weekly percentage of influenza virus type/subtypes accounted for all the positive numbers between sari and ili patients indicated that the predominant influenza types/subtypes among ili and sari cases was corresponded. these findings are essential for planning influenza vaccination programmes for those severe cases given recommendations for strain inclusion within the vaccine are based on the ili surveillance system. our results were consistent to the study conducted by peng et al. in the year of 2015 [4] . but in that study, the detailed analyses such as correlation analysis between weekly ili influenza-positive rate and sari influenza-positive rate, ili percentage and ili influenza-positive rate, sari percentage and sari influenza-positive rate were not performed. therefore, we believe the present study can provide more comprehensive information on the comparison of influenza epidemiological and virological characteristics between outpatients and inpatients. in line with the findings of other studies [11, 13, 14] , we also found higher influenza detection rate among ili patients compared to that among sari patients. moreover, we also found the correlation coefficient of ili percentage and ili influenza-positive rate was higher than that of sari percentage and sari influenza-positive rate. these phenomena may due to the higher specificity of the ili diagnosis compared to the sari diagnosis [28] . some studies have demonstrated that influenza played an important role in the viral aetiologies of ili cases [29] [30] [31] , while other respiratory virus such as respiratory syncytial virus, rhinovirus, human bocavirus were essential for the cause of sari, especially in children [32, 33] . this indicated that it is more necessary to conduct pathogen spectrum test for sari cases so as to accurately understand the cause of those severe cases. of note, we detected seven cases of a(h7n9) viruses (five in the ili group and two in the sari group) during early year of 2014, when this virus was outbreak in zhejiang province [34] . the detection rate of a(h7n9) virus in the sari patients was higher than that in the ili patients because this strain frequently cause severe syndromes. however, our study found that the surveillance network has low sensitivity on the capture of patients infected with a(h7n9) virus. although one of the critical functions of influenza surveillance is to detect novel strains of influenza, the rapid detection of emerging novel influenza strains or outbreaks of respiratory disease calls for other surveillance with standardized methodology [5] . this study has several limitations. first, although sari surveillance was required to catch all patients in accordance with the definition, sometimes the cases may not be fully recorded because of physicians' oversights and absenteeism. in the ili surveillance, to get all ili patients surveyed was impossible due to the limited amount of resources and personnel. therefore, the subject chosen for ili sampled may prone to sicker patients or younger patients. second, compared to ili surveillance, sari surveillance may be less representative due to its sparse surveillance sites. in the future, we should consider to expand and to enhance the coverage of the surveillance network, with priority to choose hospitals from ili surveillance. third, we did not test for pathogens other than influenza, which made us unable to exclude other viral, bacterial, and fungal pathogens that could be the causes of ili and sari. this study demonstrated circulating types/subtypes of influenza strains and seasonality pattern of ili cases were similar to that of sari cases in zhejiang providence. this reassured the effectiveness of influenza vaccine as strain selection based upon ili surveillance. our study results suggest that compared to ili patients, it is more necessary to conduct pathogen spectrum detection among sari patients. in the future, the expanded and enhanced ili and sari surveillance in the province may contribute to identify the novel virus, detect pandemics at early stage, and then improve the understanding of the etiology of ili and sari. a systematic review of the social and economic burden of influenza in low-and middle-income countries the economic burden of influenza-associated outpatient visits and hospitalizations in china: 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respiratory infections in china identification of viral and bacterial pathogens from hospitalized children with severe acute respiratory illness in lusaka, zambia epidemiology of human infections with avian influenza a(h7n9) virus in the two waves before and after october 2013 in zhejiang province acknowledgments: this work was financially supported by grants from zhejiang province (major science and technology programme, grant number 2014c03039), (medical research programme, grant number 2015rcb011, 2016rca008). we would like to thank physicians and staff in the 16 influenza surveillance hospitals across zhejiang province for their dedication in completing the countless numbers of data forms that made this work possible. we also want to thank physicians and staff of hangzhou, ningbo, huzhou, shaoxing, jiaxing, quzhou, taizhou, zhoushan, wenzhou, jinhua, lishui and yiwu centers for disease control and prevention for their invaluable assistance with samples collection and detection. the authors declare no conflict of interest. key: cord-009137-wj5vhvxx authors: fananapazir, l.; edmond, elizabeth; eccleston, maureen; anderton, j.l. title: raised urinary fibrin-degradation products, complement, and igg during an influenza-like illness date: 1977-04-30 journal: lancet doi: 10.1016/s0140-6736(77)92227-9 sha: doc_id: 9137 cord_uid: wj5vhvxx urine from eight normal controls in whom an influenza-like illness developed contained high concentrations of fibrin-degradation products (f.d.p.), igg, and c(3). the study was carried out when influenza a was prevalent in the community. however, a wide range of serological investigations revealed no evidence for influenza a or other viruses. the infection may have been caused by other viruses which produce upper-respiratory-tract infections and which are not readily diagnosed by serology. urinary fibrin-degradation products are a well-known marker of glomerulonephritic activity and viral antigens may have induced an immune-complex glomerulonephritis in the 8 controls in whom an influenza-like disease developed. a larger normal population should be investigated during a virus epidemic. of male and female blood-donors. their serum-bilirubin data were skewed but women with a bilirubin z 7 mg/dl (12.0 p.1110j/l) and men with a bilirubin > 1.4 mg/dl (24.0 p.11101/l) were claimed to constitute a second, minor population who, owens and evans suggested, had gilbert's syndrome; if so about 6% of the population would have this disorder. they found two distinct linear sections when their data were plotted on normal-probability as distinct from log-probability paper. our data plotted on normal-probability paper yielded a smooth logarithmic curve with no evidence of bimodality. this difference could be due to sampling error in earlier study ; the upper end of owens and evans' distribution contains very few data points. in fact their data can be accommodated by a single log-normal distribution, thus weakening their case for bimodality. the men with a bilirubin >25 jjonol/1 who were recalled had the usual criteria of gilbert's syndrome-i.e., their serum-bilirubin rose when they fasted,11 13 the bilirubin was mainly unconjugated, and gross haemolysis was excluded. the minor haemolysis demonstrated in some patients with gilbert's syndrome is not enough to account for the hyperbilirubinaemia.14 it is not surprising that the repeat bilirubins were slightly different from the first ones. in patients with a raised bilirubin the concentration does vary and the patients had not fasted before the second examination. is gilbert's syndrome a disease or merely an extreme expression of normality? a low uridine-diphosphate glucuronyl transferase,'s altered bilirubin kinetics,4 slightly reduced red-blood-cell survival,14 or even a genetic predisposition3 do not mean that it represents a disease state. one would expect to find differences in factors which control bilirubin concentration in a group of people with a high bilirubin if they are compared with those with a lower bilirubin. were it feasible to measure them in a large normal population these factors could also be expected to have a skew distribution. the frequency of symptoms in patients with gilbert's disease probably reflects the way they were diagnosed-i.e., by blood tests done to investigate vague lassitude or discomfort. our recalled patients had no such symptoms. we suggest that "constitutional hyperbilirubinaemia" be used in preference to "gilbert's disease" so that the patient can be reassured that the condition produces no symptoms and so that unnecessary investigations will not be ordered during any future non-associated illness in which the bilirubin may be found to be further increased. certainly, treatment with phenobarbitone'6 to lower the bilirubin and diminish vague symptoms seems misplaced. failure to recognise that the distribution of bilirubin concentration is skewed and the subsequent misapplication of gaussian statistical techniques can yield a misleadingly low upper limit of normal. an upper limit of 14 &micro;mol/l (or 0-8 mg/dl) seems too low. the 98th percentiles in our population were 19 plmol/1 (1-1 1 mg/dl) for females and 25 mol/1 (1-5 mg/dl) for males, and these values more realistically define the upper limit of "normality". even on these strict criteria 2% of our screened men had abnormal bilirubin concentrations; almost all of them will have had no hepatocellular disease, but they would, in the past, have been diagnosed as having gilbert's disease. requests for reprints should be addressed to d. r., medical centre, 210 pentonville road, london n1 9ta. hospital, and regional virus laboratory, city hospital, edinburgh summary urine from eight normal controls in whom an influenza-like illness developed contained high concentrations of fibrin-degradation products (f.d.p.), igg, and c3. the study was carried out when influenza a was prevalent in the community. however, a wide range of serological investigations revealed no evidence for influenza a or other viruses. the infection may have been caused by other viruses which produce upper-respiratory-tract infections and which are not readily diagnosed by serology. urinary fibrin-degradation products are a well-known marker of glomerulonephritic activity and viral antigens may have induced an immune-complex glomerulonephritis in the 8 controls in whom an influenza-like disease developed. a larger normal population should be investigated during a virus epidemic. introduction the immunological basis for glomerulonephritis is well established and immunofluorescence studies have shown immunoglobulins in the glomeruli of patients with glomerulonephritis. immunoglobulins are produced in response to an antigen, but the nature of the antigen is often obscure and its demonstration difficult. however, numerous bacterial antigens (especially streptococci) may cause glomerular damage through immunological mechanisms. chronic viral infection of animals with lymphocytic choriomeningitis, the agent of aleutian dis-ease of mink 2 coxsackie b4, adenovirus/ and other viruses6 can result in immune-complex nephritis. in man the role of viruses in the aetiology of glomerulonephritis has been studied chiefly in cases or renal disease developing after viral illnesses caused by echovirus 9,7 coxsackie b,8 and varicella infections.9 raised titres of anti-epstein-barr have been demonstrated in patients with systemic lupus erythematosus glomerulonephritis, and parainfluenza type-3 antibody titres were raised in patients with immune-complex glomerulonephritis.10 intravascular coagulation with renal involvement has been reported in patients with influenza-a virus infection" 12 and in a case of goodpasture's syndrome after influenza-a2 virus infection. 13 urine fibrin-degradation products are an accurate marker of the activity of the glomerulonephritic process. 14 in the present study high concentrations of urine fibrin-degradation products and immunoglobulins suggested that an immunological process consistent with a subclinical attack of glomerulonephritis had occurred during an epidemic of an influenza-like disease. sixteen normal healthy adult volunteers agreed to take part in an investigation to define the normal concentration of urine fibrin-degradation products (f.d.p.) for our laboratory. 20 ml of urine was collected daily in the early morning for a month, dialysed against tap-water, concentrated in polyethylene glycol at 4&deg;c overnight, and stored at -18&deg;c until assay. the tanned red-cell haemagglutination-inhibition im-munoassayl5 was used with the following modifications. the immunoassay was performed in the microtitre system (cooke engineering co.) and all reagents, fibrinogen standards, and sensitised cells were from the same batch. three concentrations of human fibrinogens (kabi pharmaceuticals ltd., lots no. 5 and 175) were included in each assay. the concentration of "clottable" protein in these standards was estimated by the method of ratnoff and menzie. 16 the mean sensitivity of the assay calculated with these values was 0. 35 mg/1. the concentrationofantisera was 1/40 000 (burroughs wellcomelot k 24482). the antigen/antibody incubation period was 4 h at 20&deg;c. the incubation period after the addition of the sensitised cells was 18 h (overnight) at 20&deg;c. human group-0-negative, glutaraldehyde-fixed, fibrinogen (10 g/ml) (kabi pharmaceuticals ltd., lot no. 58175) sensitised cells were used." the plates were read by two observers who agreed to within half a well. igg, igm, and c3 were measured by the mancini technique. 18 because of the limited amount of urine available for analysis, urine was examined for protein content and blood by 'labstix' only. during the investigation clinical evidence of an influenzalike syndrome with acute upper-respiratory-tract symptoms (cough, sore throat, runny nose), general malaise, and arthralgia developed in eight controls. lymphadenopathy developed in one, and three were absent from work for 2 or 3 days. during the study there had been an epidemic of influenza in the community caused by parainfluenza a and b viruses. blood was taken for virus studies during the attack and one month later, and serum was investigated for antibodies to a wide range of viruses. complement-fixing antibody titres to the following antigens were determined: influenza a, b; parainfluenza type 1; adenovirus; chlamydia group b; coxiella burneti ; mycoplasma pneumonice, and respiratory syncytial virus. antibodies to coxsackie bl-6 viruses were measured by a metabolic inhibition test. serum was tested for heterophil paul bunnell antibody and streptolysin 0 antibody. during the influenza-like illness blood was also taken to measure serum-p.d.p., blood-urea, serum-creatinine, platelets, reticulocytecount, haptoglobulins, prothrombin index, and thrombin-time and a blood-film was studied for fragmented cells or burr cells to detect any evidence of intravascular coagulation. urinary f.d.p., igg, and c3 concentrations are shown in two of the controls who had an influenza-like illness ( fig. 1 ). the mean urine f.d.p. concentration during the illness in eight controls was 2.37 mg/1 (s.n.&plusmn;1263) and this was significantly higher than the mean concentration in those controls who did not have the illness proteinuria was found in only two controls (30 and 300 mg/dl), but the method used to measure urinary protein was poor, there being insufficient urine to carry out a detailed protein estimation by the biuret method. blood not associated with menstruation was detected by microscopy in two consecutive urine samples from one woman during the illness. blood urea and creatinine were not raised in any of the controls at any time. serum antibody titres to the antigens tested were not raised in any of the eight controls. throat and nasal swabs were not taken. the paul bunnell test for heterophil antibody was negative in all eight cases. routine bacteriological cultures were negative and the anti-streptolysin 0 titre was not raised in any of the eight controls. the results of all haematological investigations for disseminated intravascular coagulation were within normal limits. urine f.d.p., complement, and igg were raised in eight of sixteen adult controls studied. in all eight, the urinary changes were associated with an influenza-like illness. there were no changes in the urine of the other eight controls who remained well throughout the study. the possibility that a virus was responsible for the influenza-like illness was investigated. the infection occurred at the beginning of april, 1976, when influenza-a infection was prevalent in the area. 111 however, in the eight controls affected there was no serological evidence for influenza-a infection or for infection by a wide range of other viruses. however, the infection may have been caused by viruses&mdash;e.g. rhinovirus, echovirus, coronavirus, all known to cause upper respiratory infection-which are not readily diagnosed by serology. although there was no laboratory confirmation of viral infection, the clinical illness with upper-respiratory-tract symptoms, general malaise, fever, and arthralgia was compatible with such an infection. the excretion of f.d.p., complement, and igg in the urine is associated with glomerulonephritis.2o immune complexes consisting of viral antigen and antibody may have been responsible for a glomerular nephritic process in the eight controls in whom urinary fibrin-degradation products, igg, and complement were raised. in man, as in laboratory animals, immune-complex disease due to deposition of viral antigen/antibody complexes in glomeruli may be of importance, since coxsackie-b antigen8 and australia antigen 21 22 have been observed in glomeruli of patients with nephritis. others have used explant cultures in an attempt to isolate viruses directly from renal tissue obtained either at necropsy or by diagnostic biopsy.z3 z4 cytomegalovirus, adenovirus, measles, varicella, and coxsackie virus bl 1 have been isolated from infant kidneys obtained at necropsy, but not from kidneys of older patients.23 particles with the characteristics of coronaviruses have been seen in the kidneys of patients with endemic (balkan) nephropathy.25 c-type r.n.a. viruses have been implicated in the aetiology of systemic lupus erythematosus,26 and kidneys from patients with lupus nephropathy have been found to contain antigens related to c-type virus from human cells (hel-12 virus).'" urine fibrin-degradation products, complement, immunoglobulin, and protein excretion should be measured prospectively and detailed virus studies should be carried out during an epidemic of influenza or other viral infection in a larger normal population. requests for reprints should be addressed to j. l. a., department of medicine, western general hospital, edinburgh eh4 2xu. diseases of liver and biliary system communicable diseases scotland: weekly reports 76/14 and 15. c. d. s. unit, ruchill hospital nowos-&lstrok;awski, a. ibid. 1974, ii key: cord-026982-1igz6i8u authors: li, yanbo; ye, xiaofang; zhou, ji; zhai, feng; chen, jie title: the association between the seasonality of pediatric pandemic influenza virus outbreak and ambient meteorological factors in shanghai date: 2020-06-17 journal: environ health doi: 10.1186/s12940-020-00625-7 sha: doc_id: 26982 cord_uid: 1igz6i8u background and objectives: the number of pediatric patients diagnosed with influenza types a and b is increasing annually, especially in temperate regions such as shanghai (china). the onset of pandemic influenza viruses might be attributed to various ambient meteorological factors including temperature, relative humidity (rh), and pm(1) concentrations, etc. the study aims to explore the correlation between the seasonality of pandemic influenza and these factors. methods: we recruited pediatric patients aged from 0 to 18 years who were diagnosed with influenza a or b from july 1st, 2017 to june 30th, 2019 in shanghai children’s medical centre (scmc). ambient meteorological data were collected from the shanghai meteorological service (sms) over the same period. the correlation of influenza outbreak and meteorological factors were analyzed through preliminary pearson’s r correlation test and subsequent time-series poisson regression analysis using the distributed lag non-linear model (dlnm). results: pearson’s r test showed a statistically significant correlation between the weekly number of influenza a outpatients and ambient meteorological factors including weekly mean, maximum, minimum temperature and barometric pressure (p < 0.001), and pm(1) (p < 0.01). while the weekly number of influenza b outpatients was statistically significantly correlated with weekly mean, maximum and minimum temperature (p < 0.001), barometric pressure and pm(1) (p < 0.01), and minimum rh (p < 0.05). mean temperature and pm(1) were demonstrated to be the statistically significant variables in the dlnm with influenza a and b outpatients through time-series poisson regression analysis. a u-shaped curve relationship was noted between the mean temperature and influenza a cases (below 15 °c and above 20 °c), and the risks increased for influenza b with mean temperature below 10 °c. pm(1) posed a risk after a concentration of 23 ppm for both influenza a and b. high pm(1), low and the high temperature had significant effects upon the number of influenza a cases, whereas low temperature and high pm(1) had significant effects upon the number of influenza b cases. conclusion: this study indicated that mean temperature and pm(1) were the primary factors that were continually associated with the seasonality of pediatric pandemic influenza a and b and the recurrence in the transmission and spread of influenza viruses. seasonal pandemic influenza, attributable to both types a and b, is particularly prevalent in temperate regions [29] . the onset of influenza viruses is usually characterized by an explicit and predictable annual winter epidemic as a sharp contrast with a less distinct pattern of sporadic outbreaks throughout the tropical regions [8] . three to five million severe influenza-related illness is reported annually, leading to 250,000-500,000 deaths each year, affecting 20-30% of children and 5-10% of adults worldwide [16, 29, 34] . young children are one of the most vulnerable groups associated with the highest morbidity and mortality as they are more prone to infections from lacking both prior exposure and immunity to the virus [13] . previous studies have revealed the correlation between influenza outbreak and ambient meteorological risk factors such as temperature and relative humidity (rh). a time-series study comparing the incidence of influenza to three climatic parameters including the mean temperature, rh, and rainfall, in five different cities found an association between the rh and the incidence of influenza a, and a key association between the mean temperature and the incidence of influenza, in most cities [31] . this corresponded with another study, which showed similar results on the dependence of the dynamics of influenza transmission (or pathology) on temperature and rh under controlled experimental conditions [19] . another study indicated that specific humidity plays a significant role in the seasonal transmission of influenza viruses, with two types of environmental conditions, "cold-dry" and "humid-rainy," being identified as associated with seasonal influenza [30] . however, the relationships between the incidence of influenza and more ambient meteorological factors such as accumulative precipitation, barometric pressure, wind speed and pm 1 concentrations remained to be elucidated. these factors, together with temperature and rh, are the most common and standard parameters for measuring atmosphere and climate by the meteorological instrumentation. barometric pressure is strongly associated with temperature as a low temperature usually indicates high-pressure zone and vice versa. seasonal variation of wind speed and aerosol concentrations like pm 1 may influence the influenza seasonality. specifically, wind dispersion (of the atmospheric aerosols) and the transport by ambient fine particulate matter may significantly affect transmission and concentrations of the airborne influenza virus, and hence increases the exposure risk of the population to the virus [7, 11] . shanghai features a subtropical monsoon system with higher wind speeds in early spring and autumn, a low subtropical pressure system in summer and a high-pressure system in winter. in this highly polluted city, a high level of atmospheric particulate matter pollution is much more commonly observed in late autumn and winter than summer. it is therefore worthy to investigate how the ambient climate conditions correlate with the influenza seasonality in shanghai, thereby providing more insights into the interactions between influenza and future climate change. this study focused on establishing the relationship between the seasonality of pediatric pandemic influenza (a and b) and ambient meteorological factors. by understanding the etiopathogenesis of influenza in relation to ambient meteorological factors, appropriate public health interventions could be implemented to control, mitigate or prevent the outbreak of influenza epidemics among young children. in this study, we recruited pediatric patients aged from 0 to 18 years who were diagnosed with influenza a or b for over 2 years from july 1st, 2017 to june 30th, 2019 in shanghai children's medical centre (scmc). ambient meteorological data including weekly mean temperature, maximum and minimum temperature, rh, minimum rh, atmospheric pressure, wind speed, accumulative precipitation and pm 1 concentrations were collected from shanghai meteorological service (sms). the outpatient and ambient meteorological data from mid-2017 to mid-2019 were sorted and categorized according to weeks, thereby allowing for preliminary simple correlation analysis (pearson's r) and the further time-series poisson regression analysis of non-linear and delayed exposure-response relationship using the distributed lag non-linear model (dlnm). all the ambient meteorological factors as independent variables (ivs) of the pearson's correlation were taken into the evaluation of r and their associated p values for further non-linear and delayed exposure-response analysis. ivs that were statistically significant were incorporated into the dlnm with a significance level of 0.05 (α = 0.05). factors with probability lower than the critical level (p < 0.05) were accepted and taken into account of the model. two separate dlnms were developed for separate analyses of the relationship with weekly influenza a and b outpatients, and the potential effects from the only time-varying confounding variable which is seasonality (expressed as year and month in the formula) were minimized by controlling this effect moderator in our models. we chose the degree of freedom (df) of 4 for the ambient meteorological factors and a maximum lag of 7 days associated with these factors for the best model fitting based on the akaike information criterion (aic), and 7 lag days were also determined since the study was based on a weekly analysis. the reference level was defined at the median values of weekly mean temperature and pm 1 concentrations to calculate the relative risks (rrs) and associated 95% confidence intervals (cis). the extreme effects of high values and low values were determined at 95th and 5th percentiles respectively with reference to the median values. specifically, the rrs from high temperature (hot effects) for the dlnm of influenza a and high pm 1 concentrations (high pm 1 effects) for the dlnms of both influenza types were calculated by comparing the 95th percentile to the median values. the rrs and cis from low temperature (cold effects) for the dlnms of influenza a and b were calculated by comparing the 5th to the median values. to estimate the cumulative effects (or rrs) of extreme values, a maximum lag of 7 days was also selected for all extreme effects including high and low temperatures, and high pm 1 concentrations. all results with p < 0.05 were considered statistically significant. all the above data collection, collation and presentation, were carried out by microsoft excel 365, the morbidity of influenza in children by months as shown in fig. 1a , there was a significant increase in the monthly number of type a outpatients from mid-2017 to mid-2019, particularly in winter (december-february), where a larger peak was observed in 2018-19 than 2017-18. as shown in fig. 1b , a slight decline in the monthly number of type b outpatients, was observed from mid-2017 to mid-2019. a winter peak or high-morbidity period was observed from 2018 january to february. there was a delayed effect (or response lag) to the 'supposed' winter outbreak of influenza b viruses in 2018-19. specifically, a relatively smaller peak was noted in spring (march to may) in 2019, compared with the larger winter peak from 2018 january to february. compared to type a (fig. 1a) , the overall number of type b outpatients was significantly lower. the number of male and female pediatric type a outpatients from july 1st, 2017 to june 30th, 2019, were 12, 196 and 9740, respectively, with a ratio of 1.25: 1. male children from 0 to 6 years accounted for 43.6% of the total outpatient populations. the highest number of outpatients was seen in the age group of 0 to 3 years, followed by 4 to 6 years, implying that preschoolers and early school-aged children were more susceptible to the infections caused by influenza a viruses than schoolaged children (table. 1 ). the number of male and female pediatric type b outpatients from july 1st, 2017 to june 30th, 2019, were 4102 and 3277 respectively, with a ratio of 1.25: 1. male children from 0 to 6 years accounted for 37.9% of the total outpatient populations. the highest number of outpatients was noted in the age group of 4 to 6 years, followed by 0 to 3 years, implying that preschoolers and early school-aged children were more susceptible to the infections caused by influenza b viruses than schoolaged children (table. 2). pearson's r correlation test was carried out between the number of influenza a and b outpatients and ambient meteorological factors. as shown in table. 3, weekly mean temperature, along with maximum and minimum temperature, exhibited a significant negative correlation (r) with the weekly number of influenza a outpatients (p < 0.001). weekly mean barometric pressure and pm 1 concentrations, on the contrary, showed significant positive correlations (p < 0.001 and p < 0.01 respectively) with influenza a outpatients. however, there was no significant correlation between influenza a outpatients and weekly mean rh, minimum rh, wind speed and accumulative precipitation (p > 0.05). as shown in table. 4, weekly mean temperature, along with maximum and minimum temperature, exhibited a significant negative correlation (r) with the weekly number of influenza b outpatients (p < 0.001). both barometric pressure and pm 1 concentrations also showed significant positive correlations (p < 0.01), while the weekly mean minimum rh showed moderately significant negative correlation (p < 0.05). however, there was no significant correlation between influenza b outpatients and weekly mean rh, wind speed and accumulative precipitation (p > 0.05). the degree to which the weekly number of influenza b outpatients and ambient meteorological factors were correlated (table. 4 ) was lower and less discernible than the case of influenza a as shown in table. 3. additionally, barometric pressure was excluded from the probability range of p < 0.001 (table. 4 ), which differed from the case of influenza a (table. 3). only statistically significant variables that were common to both types of influenza (aka tables 3 and 4 ) were included in fig. 2 (black lines represent smoothed conditional means, grey regions represent 95% cis, and solid dots represent weekly values) and dlnms for subsequent non-linear and delayed regression analysis. additionally, strong collinearity was also observed in the correlation test among all three temperature-related factors, specifically 0.99 between mean and maximum or minimum temperature, followed by 0.97 between the maximum and minimum temperature. this was also true for barometric pressure as perfect negative collinearity of − 0.92 was noted with mean temperature. hence, only weekly mean temperature and pm 1 were included in subsequent models for analysis. since the weekly number of influenza a and b outpatients followed a poisson distribution, and the ambient meteorological factors including mean temperature and pm 1 had lag effects, dlnms were used in analyzing the effects of these factors on the incidence of seasonal influenza. the effects of mean temperature and pm 1 on pediatric influenza seasonality for an effective visual interpretation, the rrs and 95% cis of weekly influenza outpatient numbers were plotted against the covariates of mean temperature and pm 1 for both influenza types, relative to the median values of these meteorological factors and over the corresponding lag days (fig. 3a-b) . for the case of influenza a, the relationship between mean temperature and influenza outpatient numbers was non-linear and could be interpreted as a u-shaped curve (fig. 3a) . the rrs increased with temperatures below 15°c and above 20°c, with a continuously higher rate of change either below or above these thresholds (fig. 3a) . a positive relationship was observed between pm 1 and influenza a cases as there were no known rrs or levels of danger until 23 ppm which was the breakpoint for the onset of influenza incidence risks, in which the rrs increased at a higher rate with higher pm 1 concentrations (fig. 3b) . for influenza b, the rrs increased below 10°c (no risks above), with a more significant increase below 5°c (fig. 3c) . similar to influenza a, a positive relationship was also observed between pm 1 and influenza b cases as the rrs also increased above 23 ppm of pm 1 , and there were no risks below the breakpoint (fig. 3d) . to identify the cumulative extreme effects on influenza a cases, the estimated effects of mean temperature comparing the 5th and 95th percentile to the median value and pm 1 comparing 95th percentile to the median value were plotted against lag days in fig. 4a -c. to identify the cumulative extreme effects on influenza b cases, the estimated effects of mean temperature comparing only 5th percentile to the median value, and pm 1 comparing 95th percentile to the median value were plotted against lay days in fig. 4d -e. as shown in fig. 4a -c, all cold, hot and high pm 1 were significant and thus considered as risk factors associated with the seasonal occurrence of pediatric influenza a. cold and high pm 1 effects were also significant and risk factors associated with the seasonality of pediatric influenza b as shown in fig. 4d -e. since all data were analyzed weekly, the maximum lag days selected for the calculation of cumulative extreme effects (or cumulative rrs) of meteorological factors were all 7 days. we also calculated the cumulative extreme effects of meteorological factors including mean temperature and pm 1 on pediatric influenza a and b cases (tables 5 and 6 ). in terms of influenza a, for the total population of all age children, the rrs of cold and hot effects at 7th lag day were (tables 5 and 6 ). additionally, the cumulative extreme effects of both mean temperature and pm 1 were higher for the case of influenza b than a (tables 5 and 6 ). however, the factor of mean temperature showed no cumulative extreme hot effects in table. 6 since there were no risks above 10°c (fig. 3c) . overall, all cumulative extreme effects were statistically significant with p ≤ 0.05. the pediatric influenza outpatient trend from july 1st, 2017 to june 30th, 2019 by time series, age and sex ( fig. 1a-b ; tables 1 and 2) conformed to the general trend of pediatric influenza morbidity. winter (december/january to march) is the prevailing season for the outbreak of influenza epidemics, whereas april to november is the low-morbidity season of influenza viruses [12, 26] . there was a response lag to the outbreak of influenza b in 2019 winter as delayed to march-may due to differences in antigenic changes and transmission rates between influenza types a and b [1, 33] . influenza a viruses are more likely to have an antigenic mutation and more efficient transmission than b viruses [1, 33] , leading to higher pediatric morbidity and mortality [24, 27, 33] . the sex ratio of the number of male to female pediatric outpatients aged 0-18 was 1.25: 1 for both influenza types. preschoolers (0-3 years) and early school-aged children (4-6 years) were more susceptible to the infections caused by influenza viruses than older (school-aged) children, which is consistent with findings from other studies [6, 7, 15, 17, 18, 21, 35, 37] . preschoolers (including infants) are particularly susceptible to influenza virus infections during the epidemic season due to lack of immunity and incapability of their lungs to fight off virus infections, which causes widespread hospitalizations in children diagnosed with influenza a or b [7, 15, 21, 35] . additionally, school-aged children play a critical role in the transmission of influenza viruses by attending school or daycare, which would lead to greater exposure to adverse ambient meteorological factors and hence the more efficient spread of epidemics [6, 14, 15, 17, 18, 35] . the results from pearson's r test revealed an evident negative correlation between the weekly number of pediatric influenza outpatients and corresponding weekly mean temperature, along with maximum and minimum temperature for both influenza types, which has been widely reported [3, 12, 15, 33, 35, 36] . hence, ambient temperature (mean, maximum and minimum) was closely related to the seasonality of pediatric pandemic influenza a and b [15] . barometric pressure and pm 1 concentrations exhibited a weak positive correlation with the weekly number of pediatric influenza a and b outpatients (with the addition of weak negative correlation from between the weekly number of influenza b outpatients and corresponding weekly mean minimum rh). the barometric pressure was highly negatively correlated with mean, maximum and minimum temperature, as shown by their respective pearson's r correlation coefficients-(0.92, − 0.92 and − 0.91), which plays a vital role in the spread and transmission of influenza viruses [15] . other studies also reported the positive correlation between the number of pediatric influenza outpatients and pollutant levels including pm 2.5 , pm 10 and o 3 concentrations as the pollutants could affect the spread of viruses [7, 35] , but they did not mention the relationship between pm 1 concentrations and pediatric influenza incidence, suggesting a potential research deficit on the aspect. moreover, influenza seasonality was highly associated with minimum rh [15, 25, 30, 33] as proved by a negative correlation with the number of influenza b outpatients which supports the findings from one study conducted in beijing [3] . however, the negative correlation contradicts with findings from other studies as minimum rh was regarded positively correlated with influenza infections [15] due to loss of infectivity under low minimum rh [10, 11, 15, 23] . overall, the correlation coefficients between the number of pediatric influenza b outpatients and ambient meteorological factors (table. 4 ) were lower and less distinct (or discernible) than the case of influenza a (table. 3) due to difference in virologic basis including the accumulation of surface antigenic changes and transmission rates [1, 33] . the results and findings from the dlnms also confirmed that the influenza seasonality was attributed to the effects of ambient meteorological factors including mean temperature and pm 1. specifically, we found both lower (below 5°c) and higher temperature (above 25-30°c) (a u-shaped curve relationship) were strongly associated with a higher incidence of pediatric influenza a, which corresponds to two previous studies [5, 33] . one study conducted in jiangsu province, china, which is also a temperate region, indicated influenza a peaked at − 4°c and 28°c respectively [5] . another study conducted in two subtropical regions and one temperate region including shanghai, hong kong, and british columbia respectively found a similar consistent relationship with influenza incidence peak at low and high temperature in all three regions [33] . a negative relationship between mean temperature and pediatric influenza b incidence was observed as the rr increased significantly below 5°c, which is consistent with other previous studies [5, 9, 20, 22, 28] . one study in jiangsu explicitly linked the mean temperature to the occurrence of influenza b by indicating a peak in pediatric influenza b incidence at 5°c [5] . other studies from guangzhou (a subtropical region in se china) and seoul (a temperate region in korea) reported similar findings that there was a significant increase in influenza incidence below 20°c and 0-5°c respectively [9, 22] , along with a study using a guinea pig model which also explored a link between an increase in influenza transmission rate and temperature below 5°c [20] . however, no relationship was found in the high-temperature range in these studies [9, 20, 22, 28] , except the one in jiangsu [5] . apart from mean temperature, we also found a significant increase in pm 1 concentrations higher than 23 ppm, which is particularly prevalent in winter. due to limited literature on the non-linear and delayed exposure-response relationship between pm 1 concentrations and pediatric influenza incidence, we have not yet found supports of this finding from other studies. regarding the cumulative extreme effects at 0-7 lag days, we found the cumulative rrs were significantly higher in the relationship overall, these findings provided a useful insight into the seasonal influenza in relation to the current atmospheric setting which is highly uncertain given what the future climate change can hold. there have been several prior research studies investigating the relationship between climate change and the frequency of seasonal influenza onset [2, 4, 32] , but only one research indicated a higher likelihood of an early onset of severe seasonal influenza pandemics (commonly known as the flu seasons) for both influenza a and b following warmer than average winters due to larger fraction of susceptible populations being left in the face of a potential outbreak in the next colder season than during normalcy [32] . this specific finding corroborated with one of ours that pediatric influenza patients were more susceptible during winter when the temperature is low, suggested a winter epidemic among children. in the realm of climate change and global warming, it could be anticipated that more severe flu seasons would occur given the increasing frequency of 'warmer' winters. other studies were still uncertain on the exact and more detailed effects of climate change on the seasonality influenza pandemics [2, 4] . hence, it is certain that the current findings from this study will change in future 'climate change' scenario but uncertain regarding the process of changes that would occur given the rising frequency of anthropogenic climate change and associated global warming due to the enhanced greenhouse effect. our study suggested that mean temperature and pm 1 were continually associated with the seasonality of pediatric pandemic influenza a and b and recurring in the transmission and spread of influenza viruses. largesample and multi-centre research are required to obtain a more comprehensive understanding of the interactions between these meteorological variables and the seasonal morbidity of pediatric pandemic influenza. the biology of influenza viruses change of influenza pandemics because of climate change: complex network simulations weather conditions and spatio-temporal spreading risk of the beijing 2009 influenza a (h1n1) epidemic el niño effects on influenza mortality risks in the state of california the effect of ambient temperature on the activity of influenza and influenza like illness in jiangsu province low influenza vaccination rates among child care workers in the united states: assessing knowledge, attitudes, and behaviors impact of ambient fine particulate matter (pm 2.5) exposure on the risk of influenza-like-illness: a time-series analysis in beijing 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seasonality: lifting the fog influenza seasonality: underlying causes and modeling theories influenza virus transmission is dependent on relative humidity and temperature high temperature (30 c) blocks aerosol but not contact transmission of influenza virus the effect of influenza on hospitalizations, outpatient visits, and courses of antibiotics in children effects of temperature, humidity, and diurnal temperature range on influenza incidence in a temperate region. influenza other respir viruses inactivation of respiratory syncytial virus in aerosol influenza and the winter increase in mortality in the united states, 1959-1999 absolute humidity modulates influenza survival, transmission, and seasonality analysis of atmospheric environment change during flu rage and flu occurrence forecast the impact of influenza epidemics on hospitalizations a four year seasonal survey of the relationship between outdoor climate and epidemiology of viral respiratory tract infections in a temperate climate a comprehensive review of the epidemiology and disease burden of influenza b in 9 european countries. hum vaccines immunother environmental predictors of seasonal influenza epidemics across temperate and tropical climates comparison of the incidence of influenza in relation to climate factors during 2000-2007 in five countries climate change and influenza: the likelihood of early and severe influenza seasons following warmer than average winters different responses of influenza epidemic to weather factors among shanghai, hong kong, and british columbia influenza (seasonal) air pollution, temperature and pediatric influenza in the spatio-temporal response of influenza a (h1n1) to meteorological factors in beijing epidemiological analysis of influenza in nanjing children's hospital from 2013 to 2017 publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to acknowledge the shanghai children's medical centre for providing the patient data and the shanghai meteorological service for providing the meteorological data. authors' contributions yl organized, analyzed, and interpreted the meteorological and patient data and was a major contributor in writing the manuscript. xy and jz collected meteorological data from shanghai meteorological service. xy and fz have substantively revised the work. jc collected patient data from shanghai children's medical centre. all authors read and approved the final manuscript. this study was supported by school of medicine, shanghai jiaotong university (grant no. yg2017qn32), and shanghai science and technology committee (to j.c.: grant no. 16411960700). the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. key: cord-252974-pwx27kdi authors: fornek, jamie l.; korth, marcus j.; katze, michael g. title: use of functional genomics to understand influenza–host interactions date: 2007-08-31 journal: adv virus res doi: 10.1016/s0065-3527(07)70003-9 sha: doc_id: 252974 cord_uid: pwx27kdi infection with influenza typically results in mild‐to‐moderate illness in healthy individuals; however, it is responsible for 30,000–40,000 deaths each year in the united states. in extreme cases, such as the influenza pandemic of 1918, tens of millions of people have died from the infection. to prepare for future influenza outbreaks, it is necessary to understand how the virus interacts with the host and to determine what makes certain strains of influenza highly pathogenic. functional genomics provides a unique approach to this effort by allowing researchers to examine the effect of influenza infection on global host mrna levels. researchers are making increasing use of this approach to study virus–host interactions using a variety of model systems. for example, data obtained using microarray technology, in combination with mouse and macaque infection models, is providing exciting new insights into the pathogenicity of the 1918 virus. these studies suggest that the lethality associated with this virus is in part due to an aberrant and unchecked immune response. progress is also being made toward using functional genomics in the diagnosis and prognosis of acute lung infections and in the development of more effective influenza vaccines and antivirals. increasing use of this approach to study virus-host interactions using a variety of model systems. for example, data obtained using microarray technology, in combination with mouse and macaque infection models, is providing exciting new insights into the pathogenicity of the 1918 virus. these studies suggest that the lethality associated with this virus is in part due to an aberrant and unchecked immune response. progress is also being made toward using functional genomics in the diagnosis and prognosis of acute lung infections and in the development of more effective influenza vaccines and antivirals. for centuries, influenza virus has plagued humankind. while influenza infection typically causes mild-to-moderate illness in healthy individuals, it still results in 30,000-40,000 deaths per year in the united states. those most susceptible to influenza infection are infants, the elderly, and those individuals that are immunocompromised due to hiv/aids infection or organ/tissue transplant (cdc, 2006) . in extreme cases, such as the 1918 pandemic, it is estimated that 50 million people died as a result of influenza infection (taubenberger and morens, 2006) . what was unique about this pandemic is that the most susceptible to this disease were young, otherwise healthy, individuals. since 1918, multiple influenza pandemics have occurred, although none nearly as deadly. another influenza pandemic is inevitable and much effort is being placed on disease surveillance and monitoring of transmission across species (pandemic flu, 2007; subbarao and joseph, 2007) . of particular concern is the h5n1 family of avian influenza viruses (horimoto and kawaoka, 2005) . while the transmission rate of h5n1 viruses from birds to humans is extremely low, the case mortality rate in humans is greater than 50% (who, 2007) . fortunately, human-tohuman transmission is extremely rare (who, 2005) . it is difficult to predict for how long this will be the case and there is increasing concern that h5n1 viruses will recombine with human viruses. this could result in an h5n1 virus with the capacity for human-to-human transmission and perhaps generate a catastrophic pandemic (subbarao and joseph, 2007) . understanding the ways in which influenza interacts with the host is an important component of preparing for the next pandemic. it is necessary to understand these interactions in order to improve existing vaccines, to develop new and more efficacious vaccines that will provide protection against multiple strains and subtypes, and to develop new antiviral therapeutics (subbarao and joseph, 2007) . because of its ability to provide a global view, functional genomics is one of the most useful approaches for studying virus-host interactions. our laboratory is using functional genomics to study a variety of viruses, including hcv, siv/hiv, ebola virus, hsv, sars coronavirus, west nile virus, and influenza virus (baas et al., 2006a,b; baskin et al., 2004; fredericksen et al., 2004; geiss et al., 2000 geiss et al., , 2001 geiss et al., , 2002 geiss et al., , 2003 kash et al., 2004 kash et al., , 2006a kobasa et al., 2007; lederer et al., 2006; pasieka et al., 2006; smith et al., 2003a smith et al., ,b, 2006 thomas et al., 2006; walters et al., 2006 walters et al., , 2006a . this chapter will focus on how microarray technology is being utilized to uncover the mysteries of influenza pathogenesis. we will explore increasingly complex models for studying influenza-host interactions using functional genomics, including cell culture systems, murine models of infection, and nonhuman primates (fig. 1) . finally, we will discuss the promise of using genomics to define molecular signatures of the disease that could lead to the evolution of the microarray as a diagnostic tool. a. cell culture models initial functional genomic endeavors in our laboratory utilized established cell lines to understand the ways in which influenza virus disrupts cellular processes. we first performed a series of experiments to determine replication-dependent and -independent events during influenza infection. hela cells were mock infected or infected with either active or heat-inactivated a/wsn/33 (h1n1). using the dual-labeling technique, cdna arrays were hybridized with rna from mock versus active or heat-inactivated virus or with rna from heat-inactivated versus active virus, allowing us to determine which genes were regulated by actively replicating virus (geiss et al., 2001) . we found that while there are distinct subsets of genes whose regulation is replication dependent or independent, more gene expression changes were observed in the presence of replicating virus. further analysis revealed that these genes could be classified in five major categories: protein synthesis, cytokine and growth factor signaling, transcription factors and dna-binding proteins, processing and export of mrna, and the ubiquitin pathway. in contrast, genes whose regulation was replication independent were grouped representative of the following categories: metallothioneins, cell cycle related, transcriptional regulators, part of the ubiquitin pathway, or cellular kinases (geiss et al., 2001) . although specific aspects of influenza replication-independent and -dependent events could have been assessed using conventional laboratory techniques, our gene expression studies allowed us to examine numerous gene expression changes at the same time. from this data, it was possible to identify specific processes that are related to influenza replicationindependent and -dependent events and speculate on how these events work together in influenza pathogenesis. further studies using a cell culture system were aimed at discerning viral determinants of virulence. of particular interest was the viral ns 1 protein. ns 1 appears to play a role in subverting the host response to the virus. it has been suggested that ns 1 attenuates the interferon response to the virus through its double-stranded (ds) rna-binding domain . it has also been suggested that the dsrna-binding domain of ns 1 functions to inhibit the 2 0 5 0 -oas/rnasel antiviral response (min and krug, 2006) . this may indicate that ns 1 from different influenza viruses plays distinct roles in subverting the host response to the virus. to better understand the effect of ns 1 on virus-host interactions, we infected an established human lung epithelial cell line, a549, with a/pr/ 8/34 (h1n1), a/pr/8/34 in which ns 1 was deleted, or with a/pr/8/34 in which ns 1 contained a deletion in the c-terminus (geiss et al., 2002) . the latter two viruses were reconstructed using plasmid-based reverse genetics (fodor et al., 1999) . these studies allowed us to examine the global host response to influenza infection in the absence of ns 1 or in response to infection with a virus exhibiting attenuated ns 1 function. numerous genes were preferentially upregulated in response to infection with the mutant viruses compared to the parental strain. many of these genes were related to the antiviral and interferon responses. these data suggest a role for the ns 1 of a/pr/8/34 as an antagonist of the interferon response to the virus (geiss et al., 2002) . antagonism of this crucial defense response to influenza most likely contributes to the lethality of this virus in mice. therefore, these initial studies from our laboratory were crucial in understanding the importance of the interferon response in the host response to influenza. this study was also the first to use functional genomics to examine the role of specific genes from the 1918 strain. in addition to the viruses mentioned above, a549 cells were infected with a/wsn/33 or a recombinant in which the ns 1 of a/wsn/33 was replaced with the ns 1 from the 1918 virus. we noted that there was greater suppression of interferonstimulated genes in cells infected with the 1918 ns 1 recombinant virus than in cells infected with the parental strain. the host response to a/wsn/33 virus containing the 1918 ns 1 was also compared with the response to wild-type a/pr/8/34 and with the a/pr/8/34 ns 1 mutant viruses. from these analyses, we determined that the expression of numerous interferon-stimulated genes was anti-correlated between these viruses and a/wsn/33 containing the 1918 ns 1 . for example, nmi and stat1 expression were upregulated in cells infected with the a/pr/8/34 viruses but downregulated in cells infected with a/wsn/33 containing the ns 1 from the 1918 virus. these studies suggest that the ns 1 from the 1918 virus is more adept at suppressing key interferon responses. it will be interesting to use functional genomics to compare the effect of the ns 1 from the 1918 virus on host-virus interactions to that of the ns 1 s from modern day low pathogenicity human influenza viruses and both low and high pathogenicity avian h5n1 viruses. such comparisons will lend a global view into how different influenza ns 1 s affect the host response and lead to important observations as to the role of ns 1 in influenza pathogenicity (geiss et al., 2002) . interestingly, mice infected with a virus containing the ns 1 of 1918 and the other seven genes from a/wsn/33 did not succumb to the infection. in contrast, all mice infected with a/wsn/33 died by 10 days postinfection (basler et al., 2001) . these data, in conjunction with the array studies described above, suggest that the ns 1 of the 1918 virus is an important virulence factor, but it is not solely responsible for the high lethality of the 1918 virus. therefore, it was imperative to study the effect of other 1918 genes on mortality and examine the critical interplay of all of the 1918 genes. such studies will be discussed in the following section covering murine models of influenza infection. using cell culture systems in the application of functional genomics is crucial to the understanding of how influenza infection affects antiviral responses on the cellular level. however, these systems are limited in that the data obtained from them can only lead to inferences as to what is occurring in the host as a whole. for this reason, it is necessary to study influenza infection in the context of the whole organism. the use of functional genomics in conjunction with various mammalian models of infection, and in humans, will be discussed in the next sections. in order to study the effects of influenza in the context of the whole organism, many scientists have utilized mouse models of infection. although mice are not a natural host for influenza virus, their accessibility and the vast repertoire of genetically altered species makes them a useful tool in many areas of research, including functional genomics. since laboratory strains of mice are inbred, this reduces host variation, making it easier to clarify how influenza is affecting the host. of particular interest to our laboratory is how the host response induced by highly pathogenic influenza infection differs from that induced by viruses with lower pathogenicity. of all the influenza viruses that have surfaced in the last century, very few have caused as much intrigue as the 1918 pandemic strain. among the most perplexing questions surrounding the influenza pandemic of 1918 is what made this virus so deadly. environmental, biological, or demographic factors could have contributed to its virulence; however, the most pertinent factors may be related to how this virus interacts with the host innate immune response. as mentioned in the previous section, we used functional genomics to study the effect of the 1918 ns 1 on global gene expression using a cell culture system. while this study provided an important first step in understanding this deadly virus, it only hints at what might be occurring in the whole host. with the sequencing and reconstruction of the 1918 virus using reverse genetics (tumpey et al., 2005a) , our laboratory and others have been able to study the effects of various genes from this virus and the fully reconstructed virus on the host (basler et al., 2001; reid et al., 1999 reid et al., , 2000 reid et al., , 2002 reid et al., , 2004 taubenberger, 1998; taubenberger et al., 1997) . in initial studies, we infected mice with the lethal, a/wsn/33 stain or with a recombinant of this virus containing the ha and na from the 1918 virus. a recombinant a/wsn/33 virus containing the ha and na of a contemporary human strain (a/new caledonia/99) was also included in these studies. both ha and na are major virulence factors and ha is the major viral factor against which host antibodies are produced (lamb and krug, 1996) and evidence suggests that the ha of the 1918 virus is necessary for transmission (tumpey et al., 2007) . gene expression profiling was then performed on lungs isolated from these mice. increased gene expression in the lungs of mice infected with either a/wsn/33 or the recombinant virus containing the ha and na from the 1918 virus was observed at 24 h postinfection. in contrast, relatively few gene expression changes were observed in the lungs of mice infected with the a/wsn/33 recombinant strain containing the ha and na from a/new caledonia/33. by 72 h postinfection, gene expression changes were similar between the two infection groups, indicating that the ha and na of the 1918 virus were sufficient to accelerate the host response to the virus (kash et al., 2004) . a subset of genes was preferentially upregulated in mice infected with the a/wsn/33 recombinant virus containing the 1918 ha and na. among this group were genes that are indicative of t cell activation, macrophage activation, and cell death (kash et al., 2004) . in support of these findings, tumpey et al. demonstrated that mice infected with a/texas/36/91 containing the ha and na from the 1918 virus or with a/wsn/33 containing these genes developed severe lung pathology, including varying degrees of necrotizing bronchitis, alveolitis, and pulmonary edema. strikingly, there was also an increase in neutrophils and alveolar macrophages in the lungs of these animals. to analyze the importance of these immune cells in the context of a/texas/36/91 recombinant virus, animals in which neutrophils and/or alveolar macrophages had been depleted were infected with a sublethal dose of the virus. infected neutrophil-depleted mice had a 60% survival rate. in contrast, all animals in which alveolar macrophages or both alveolar macrophages and neutrophils were depleted died by 9 days postinfection with the recombinant virus (tumpey et al., 2005b) . taken together, these data emphasize the importance of certain immune mediators in combating infection with a recombinant virus containing the ha and na from the 1918 virus. however, as discussed below, these findings also suggest that an inappropriate activation of the host response to the virus may contribute to its pathogenicity. we have also used functional genomics to analyze the host response of mice infected with the fully reconstructed 1918 virus. these studies, led by john kash, revealed that genes related to various immune cells, notably nk cells, neutrophils, macrophages, and t helper 1 (th1) cells, were upregulated in mice infected with the fully reconstructed 1918 virus as early as 1 day postinfection. these genes were persistently activated in the lungs of r1918-infected mice throughout the course of the experiment (5 days) (kash et al., 2006b) . key mediators of the immune response to influenza virus are cytokines and chemokines that are responsible for the activation of and recruitment of immune cells into the infected tissue (julkunen et al., 2001) . in agreement with early and persistent activation of immune cells in r1918-infected mice, this same gene expression pattern was observed for genes related to pro-inflammatory cytokines and chemokines such as tnf, il6, and ccl5 (kash et al., 2006b) . it is crucial that a delicate balance of immune responses is maintained during infection in order to limit excessive damage to the host. if these responses go unchecked, or are insufficient, it can result in dire consequences for the host (la gruta et al., 2007) . our data suggest that a hyperactive and persistent host response is associated with the 1918 virus and that this is a key contributor to the high mortality associated with this virus. another important aspect of our study was an examination of what effect the full constellation of genes from the 1918 virus had on gene expression and virus-induced morbidity and mortality. in order to accomplish this, mice were infected with the fully reconstructed 1918 virus (r1918), with a/texas/91/36 containing the ha and na from 1918 (2:6 1918), or with a/texas/91/36 containing the ha, na, m, np, and ns 1 genes from the 1918 virus (5:3 1918) (fig. 2) . compared with the response of mice infected with the r1918 virus, which exhibited early and persistent upregulation of genes related to nk cells, neutrophils, macrophages, and t helper 1 (th1) cells, mice infected with either the 5:3 1918 or 2:6 1918 virus exhibited a delay in the upregulation of these genes. however, expression levels of genes related to these immune cells was similar in all three 1918 recombinant viruses by day 5 postinfection. interestingly, animals infected with either the 5:3 1918 virus or the 2:6 1918 virus exhibited lung pathology intermediate to mice infected with a/texas/91/36 and those infected with r1918 at day 3 postinfection. the differences in gene expression not only correlated with lung pathology, but also with viral titers and morbidity, demonstrating the usefulness of gene expression profiling in understanding molecular mechanisms of disease and disease outcome (kash et al., 2006b) . studies in our laboratory are now focusing on combining genomics with the use of knockout or transgenic mice to further understand the complex host-virus interactions that occur in response to infection with the 1918 virus. we are also interested in using genomics to examine the effects of h5n1 infection on mice and to determine if there are molecular signatures of disease that are present as a consequence of infection with highly pathogenic strains of influenza. in addition, we are taking advantage of the vast repertoire of transgenic and knockout mice available to gain further insight into key regulators of the innate and/or adaptive immune response to influenza infection in general. for example, in collaboration with michael gale, we are working to understand the role of the pattern recognition receptor, retinoic acid inducible gene i (rig-i), during influenza infection. rig-i plays an important role in the interferon response to many viruses, including influenza fredericksen and gale, 2006; kato et al., 2005 kato et al., , 2006 liu et al., 2007; sumpter et al., 2005; yoneyama et al., 2005 yoneyama et al., , 2004 . we recently examined the gene expression profiles in rig-i deficient mouse embryonic fibroblasts (mefs) infected with a/pr/8/34. global gene expression profiles revealed significant differences in gene expression between wildtype and rig-i deficient mefs. we are in the process of furthering analyzing data, but preliminary analyses have revealed an important role for rig-i in the host response to the virus (loo et al., submitted). even though mouse models have provided critical insights into the pathogenesis of influenza, the information gained from these studies is limited since mice are not natural hosts for the virus. for this reason, data obtained from mouse studies can be difficult to translate to human infection. numerous studies have utilized nonhuman primate models to study influenza pathogenesis (berendt, 1974; grizzard et al., 1978; liu et al., 1997; rimmelzwaan et al., 2001; van riel et al., 2006) . unlike mouse models of infection, influenza infection in nonhuman primate models mimics human infection. for example, nonhuman primates can be infected with human influenza strains without prior adaptation and the virus is transmissible between animals. in addition, nonhuman primates and humans are close evolutionary relatives. as a consequence, nonhuman primates are increasingly being utilized to examine influenza pathogenesis, and with the sequencing of the rhesus macaque genome (rhesus macaque genome sequencing and analysis consortium, 2007), genomic and proteomic resources for working with these animals are becoming progressively more available (magness et al., 2005; spindel et al., 2005; wallace et al., 2007) . although there are numerous advantages to using nonhuman primates in influenza research, certain considerations must be taken before working with them. nonhuman primates exhibit host variation similar to that in humans, a factor that needs to be taken into account when analyzing genomics data. additionally, the numbers of nonhuman primates available for research are limited. therefore, most nonhuman primate studies are restricted in their sample size. lastly, considerable ethical concerns must be taken into account when using nonhuman primates in research studies. our laboratory, along with others, has diligently worked to address these concerns and yet still obtain the insights into influenza-host interactions that only studies in nonhuman primates can provide. in a seminal study led by carole baskin, pigtailed macaques (macaca nemestrina) were infected with the reconstructed h1n1 human influenza strain, a/texas/36/91. physical symptoms, such as throat inflammation, loss of appetite, and weight loss, correlated with the upregulation of interferon-stimulated genes at days 4 and 7 postinfection. gene expression profiling also revealed the upregulation of genes related to neutrophil and monocyte/macrophage function. accordingly, an influx of neutrophils and macrophages into the lungs of infected monkeys was observed. although not the first to use nonhuman primates as a model of influenza infection, this study was unique for two reasons. it was the first to use pigtailed macaques and it was the first in which functional genomics was used to examine influenza infection in nonhuman primates (baskin et al., 2004) . to expand upon the above study, we have also employed functional genomics to assess the effect of influenza infection on the early innate immune response in the lungs of pigtailed macaques, how genes related to this response were regulated over time, and whether gene expression signatures of infection could also be detected in the blood. finally, we examined the correlation between genomic and proteomic data collected for both lung and pbmc samples. significantly, this study was also the first to use macaque-specific oligonucleotide arrays, which were developed in our laboratory (wallace et al., 2007) . as in the previous study, animals were infected with a/texas/36/91. subsequent analysis focused on gene expression changes present at day 2 postinfection in order to determine molecular correlates of early influenza infection. in lesions where viral mrna was present, there was increased expression of interferon-stimulated genes and antiviral-related genes. notably, the majority of these genes were significantly upregulated, suggesting a robust host response against the virus. differential expression of cytokine, chemokine, and immune-related genes was also present in samples isolated at 7 days postinfection (baas et al., 2006a) . these data indicate a robust and sustained host response in the lungs of influenza-infected pigtail macaques. we also compared the signatures of infection in the lung with those found in whole blood. this analysis identified numerous genes whose expression was upregulated in the lung and in the blood throughout the time course of the infection. there was an upregulation in interferonstimulated genes and antiviral-related genes such as irf7, ifit2, oas1, and oas3. our findings suggest that there are common signatures of influenza infection between the lung and whole blood, indicating that gene expression profiling of blood may eventually prove useful for diagnostic or prognostic applications. this subject is further discussed in the following section. we also worked with richard smith's group at pacific northwest national laboratory to perform the first ever global proteomic analyses on macaque lung samples. side-by-side comparison of genomic and proteomic data from infected macaque lung samples revealed that there were many correlations between the two sets of data. of particular interest, were the correlations observed for interferon-stimulated genes and antiviral-related genes. members of these families, such as ifit1, ifit2, stat1, and mx1, were identified by both genomics and proteomics. in further support for the use of whole blood as a surrogate marker of influenza pathogenesis in the lungs, gene expression data for the above markers and others correlated with the lung genomics and proteomics data (baas et al., 2006a) . we would like to further these studies by determining if similar proteomic results are observed in whole blood. from these analyses, we also identified an increase in the abundance of certain proteins in influenza-infected lung that would not have been predicted by our genomics data. this observation points to the need for the integration of genomics and proteomics data to gain a more complete understanding of influenza pathogenesis. furthermore, integration of genomic and proteomic data will enhance our understanding of the differences between mrna levels and protein abundance. we have also recently used functional genomics and a macaque infection model to study the pathogenesis of the 1918 virus. for these studies, cynomolgus macaques (macaca fasicularis) were infected with the human h1n1 virus, a/kawasaki/173/01, or with the reconstructed 1918 virus (kobasa et al., 2007) . microarray analysis on bronchi from infected animals revealed a robust activation of numerous pro-inflammatory chemokine and cytokine genes in both a/kawasaki/173/01 and 1918-infected animals at day 3 postinfection. additionally, there was an increased activation of genes related to the interferon-a response in response to infection with either of these viruses at this time-point. strikingly, many of the genes related to these responses exhibited a more robust upregulation in the a/kawasaki/173/01-infected animals at day 3 postinfection, but returned to baseline levels or were downregulated later in infection compared with r1918-infected animals. in contrast, animals infected with the r1918 virus exhibited an increased and robust upregulation of expression of interferon-stimulated genes and chemokines and cytokines through the study endpoint. in support of our genomics observations, ccl2, ccl5, il-8, and il-6 levels were increased in the serum of r1918-infected macaques compared with the levels present in a/kawasaki/173/01-infected animals at days 3 and 6 postinfection. viral titers were substantially greater in both the upper and lower respiratory tracts of macaques infected with r1918 at all three time-points postinfection. r1918-infected animals also exhibited severe lung pathology at 8 days postinfection (kobasa et al., 2007) . taken together, these data agree with data obtained using mouse models, suggesting that the pathogenesis induced by infection with the 1918 virus is associated with, and potentially caused by, an aberrant and unchecked immune response to the virus. as a consequence, this response turns from one that is beneficial to the host to one that is extremely detrimental. the studies described above demonstrate how nonhuman primate models can be used in combination with functional genomics to understand influenza-host interactions. our studies, in conjunction with those of others, firmly demonstrate that nonhuman primate models of influenza provide crucial information into disease progression and pathogenesis. currently, we are focused on using functional genomics to assess the effectiveness of influenza vaccination in nonhuman primates (baskin et al., submitted) . these studies illustrate a novel use for functional genomics in influenza vaccine development. genomic analyses during vaccine trials may reveal gene expression markers of protective immunity or gene expression changes that are indicative of a predisposition to a particular response to immunization and subsequent challenge. as mentioned previously, a major promise of genomics is the capacity to use this technology in the more precise and efficient diagnosis of disease. of major interest, is the use of functional genomics to ascertain molecular signatures of infection that permit the distinction among diseases. discussed below is how this technology is being tested for influenza diagnosis. to identify the gene expression signatures induced by various pathogens, chaussabel et al. examined peripheral blood mononuclear cells (pbmcs) obtained from pediatric patients presenting with various illnesses (2005) . specifically, they examined diseases with distinct immunological components such as systemic lupus erythematosus (sle), influenza a, staphylococcus aureus, escherichia coli, and streptococcus pneumoniae. they also examined adult patients who received liver transplants with immunosuppressive therapy or patients who received bone marrow transplants and experienced graft versus host disease. these samples were compared with pbmcs from healthy volunteers. the authors were able to identify unique gene expression patterns for patients presenting with influenza and sle. they then determined expression profiles common to all of the diseases using genes that were either up-or downregulated in patients infected with influenza or sle. analyses also demonstrated that the genes whose expression was regulated in a similar manner in both influenza and sle patients fell into distinct categories such as defense response, interferon induction, and heavy metal binding. furthermore, the authors were able to determine how many genes related to these processes were expressed in individual patients (chaussabel et al., 2005) . in an extension of the above studies, the authors examined the gene expression profiles of pbmcs from young patients presenting with acute infections including influenza a, s. aureus, s. pneumoniae, and e. coli (ramilo et al., 2007) . analysis of these samples was performed in a methodical manner using statistical comparison, sample classification, validation of classifier genes using a test set, and validation of microarray platforms and chips. the authors were able to identify subsets of genes that distinguished patients with influenza (viral infection) from those that presented with either e. coli or s. pneumoniae (gram-negative and grampositive bacterial infections, respectively). the same was found for patients infected with influenza compared to those infected with s. aureus (gram-positive bacterial infection). distinct expression patterns were also present in pbmcs from patients infected with e. coli or s. aureus. using sets of classifier genes obtained from the above analyses, the authors examined the gene expression profiles of pbmcs isolated from patients presenting with lower respiratory infections the same as those listed above or from healthy volunteers. from these analyses, the authors were able to classify the samples from these new patients into the correct disease categories. in addition, the authors tested a separate set of samples using a different array platform. these studies also demonstrated that patients presenting with these illnesses could be accurately classified into distinct groups based on gene expression profiles (ramilo et al., 2007) . through these painstaking efforts, the authors convincingly used functional genomics to discriminate between patients with a variety of acute infections, including influenza. while these studies provide evidence that genomics can be used to define molecular signatures of disease associated with certain pathogens, they also have significant limitations. for example, these studies were performed on samples that had been taken from patients that had already been diagnosed with a particular illness and genomic analyses only had to distinguish between a relatively few possibilities. however, in order to be effective in a clinical setting, gene expression profiling will need to provide a high degree of accuracy and overcome numerous confounding factors such as age, race, gender, immune status, and co-infection with more than one pathogen. all of these issues must be addressed before functional genomics can function in disease diagnosis. however, once these challenges have been met, genomic diagnosis may decrease the amount of elapsed time between sample collection and disease diagnosis thereby allowing doctors to treat patients more quickly. this is particularly important for patients presenting with acute infections. additionally, the use of microarrays in this manner may eliminate the need for patients to undergo certain painful and potentially dangerous diagnostic procedures, such as tissue biopsies. functional genomics has clearly provided critical information regarding virus-host interactions and has made significant contributions to influenza research. as we have described, functional genomics has been utilized to study influenza infection in a variety of model systems including cell culture, mice, and macaques. researchers are also utilizing functional genomics to study influenza infections in chickens, but these endeavors are still in their infancy (degen et al., 2006) . it will also be desirable to use functional genomics to examine influenza infection in ferrets. unlike mice, ferrets can be productively infected with human influenza viruses and ferret-to-ferret transmission occurs. due to these characteristics, ferrets provide a useful and unique model for influenza infection studies. unfortunately, genomic studies using ferrets are currently limited due to the lack of ferret nucleotide sequence information. we therefore strongly recommend that the ferret genome be sequenced and that ferret-specific microarrays be developed. from the functional genomics experiments published so far, we have been able to gain invaluable insight into influenza pathogenesis. perhaps the most critical use of this technology has been in the study of the virus responsible for the deadly 1918 influenza pandemic. in regards to highly pathogenic influenza, future experiments should also focus on the effect of avian h5n1 infection on global gene expression, using multiple model systems such as those that are being used to study the 1918 virus. functional genomics has provided us with numerous insights into influenza-host interactions. in particular, we have utilized this technology to discern how low and high pathogenicity viruses affect host responses. however, there are many challenges facing our laboratory and others that use functional genomics. of utmost concern, is the integration of the vast amounts of genomics data that is available and has yet to be generated. among the major obstacles are microarray platform differences, species differences, cell type differences, and annotation differences (wallace et al., 2006) . data from microarray studies also needs to be integrated with conventional biological approaches and with data that will be obtained from the burgeoning field of proteomics. as demonstrated throughout this manuscript, our laboratory has worked tirelessly to achieve these goals. we firmly believe that functional genomics will be crucial to the development of novel therapies necessary for the prevention of influenza infection and spread. integrated molecular signature of disease: analysis of influenza virus-infected macaques through functional genomics and proteomics sars-cov virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffinembedded tissues integration of clinical data, pathology, and cdna microarrays in influenza virus-infected pigtailed macaques (macaca nemestrina) sequence of the 1918 pandemic influenza virus nonstructural gene (ns) segment and characterization of recombinant viruses bearing the 1918 ns genes simian model for the evaluation of immunity to influenza cdc flu shot analysis of significance patterns identifies ubiquitous and disease-specific gene-expression signatures in patient peripheral blood leukocytes molecular immunophenotyping of lungs and spleens in naive and vaccinated chickens early after pulmonary avian influenza a (h9n2) virus infection rescue of influenza a virus from recombinant dna control of antiviral defenses through hepatitis c virus disruption of retinoic acid-inducible gene-i signaling west nile virus evades activation of interferon regulatory factor 3 through rig-i-dependent and -independent pathways without antagonizing host defense signaling the host response to west nile virus infection limits viral spread through the activation of the interferon regulatory factor 3 pathway influenza a virus lacking the ns 1 gene replicates in interferondeficient systems large-scale monitoring of host cell gene expression during hiv-1 infection using cdna microarrays global impact of influenza virus on cellular pathways is mediated by both replication-dependent and -independent events cellular transcriptional profiling in influenza a virus-infected lung epithelial cells: the role of the nonstructural ns 1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza gene expression profiling of the cellular transcriptional network regulated by alpha/beta interferon and its partial attenuation by the hepatitis c virus nonstructural 5a protein experimental production of respiratory tract disease in cebus monkeys after intratracheal or intranasal infection with influenza a/victoria/3/75 or influenza a/new jersey/76 virus influenza: lessons from past pamdemics, warnings from current incidents molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression global host immune response: pathogenesis and transcriptional profiling of type a influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus global suppression of the host antiviral response by ebola-and marburgviruses: increased antagonism of the type i interferon response is associated with enhanced virulence genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus cell type-specific involvement of rig-i in antiviral response differential roles of mda5 and rig-i helicases in the recognition of rna viruses aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus a question of selfpreservation: immunopathology in influenza virus infection orthomyxoviridae: the viruses and their replication distinct cellular responses differentiating alcohol-and hepatitis c virusinduced liver cirrhosis immunization of non-human primates with dna vaccines retinoic acid-inducible gene i mediates early antiviral response and toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells analysis of the macaca mulatta transcriptome and the sequence divergence between macaca and human the primary function of rna binding by the influenza a virus ns1 protein in infected cells: inhibiting the 2 0 -5 0 oligo (a) synthetase/rnase l pathway functional genomic analysis of herpes simplex virus type 1 counteraction of the host innate response gene expression patterns in blood leukocytes discriminate patients with acute infections origin and evolution of the 1918 ''spanish'' influenza virus hemagglutinin gene characterization of the 1918 ''spanish'' influenza virus neuraminidase gene characterization of the 1918 ''spanish'' influenza virus matrix gene segment novel origin of the 1918 pandemic influenza virus nucleoprotein gene the rhesus macaque genome sequence informs biomedical and evolutionary analyses pathogenesis of influenza a (h5n1) virus infection in a primate model identification of novel tumor markers in hepatitis c virus-associated hepatocellular carcinoma hepatitis c virus and liver disease: global transcriptional profiling and identification of potential markers gene expression patterns that correlate with hepatitis c and early progression to fibrosis in liver transplant recipients leveraging human genomic information to identify nonhuman primate sequences for expression array development scientific barriers to developing vaccines against avian influenza viruses regulating intracellular antiviral aefense and permissiveness to hepatitis c virus rna replication through a cellular rna helicase, rig-i influenza virus hemagglutinin cleavage into ha1, ha2: no laughing matter influenza: the mother of all pandemics initial genetic characterization of the 1918 ''spanish'' influenza virus functional gene analysis of individual response to challenge of sivmac239 in m. mulatta pbmc culture characterization of the reconstructed 1918 spanish influenza pandemic virus pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission virology in the 21st century: finding function with functional genomics high-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations host-specific response to hcv infection in the chimeric scid-beige/alb-upa mouse model: role of the innate antiviral immune response application of functional genomics to the chimeric mouse model of hcv infection: optimization of microarray protocols and genomics analysis identification of a specific gene expression pattern associated with hcv-induced pathogenesis in hcv-and hcv/hiv-infected individuals avian flu facts www the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity we thank tracey baas, carole baskin, gary geiss, john kash, and our many other colleagues past and present that contributed to the studies discussed in this review. we apologize for not being able to list them all by name. we also thank sean proll for figure preparation. funding for genomic and proteomic studies in our laboratory is provided by public health service grants r01ai022646, r01hl080621, r21ai017892, r21ai063436, r24rr016354, p01ai052106, p01ai058113, p30da015625, and p51rr000166 from the national institutes of health. key: cord-256432-53l24le2 authors: yang, honglin; pang, xiaoping; zheng, bo; wang, linxian; wang, yadong; du, shuai; lu, xinyi title: a strategy study on risk communication of pandemic influenza: a mental model study of college students in beijing date: 2020-09-04 journal: risk manag healthc policy doi: 10.2147/rmhp.s251733 sha: doc_id: 256432 cord_uid: 53l24le2 purpose: to understand the characteristics of risk perception of influenza pandemic in college students with prominent frequency and the differences between these risk perceptions and professionals. then, offering a proposal for the government to improve the efficiency of risk communication and health education. methods: according to the mental model theory, researchers first draw a framework of key risk factors, and then they ask these students about the understanding of the framework with questionnaire and then making concept statistics and content analysis on the respondents’ answers. results: researchers find some students’ misunderstanding of pandemic including excessive optimism to the consequences of a pandemic, a lack of detailed understanding of mitigation measures, and negative attitudes towards health education and vaccination. most students showed incomplete and incorrect views about concepts related to the development and exposure factors, impact and mitigation measures. once threatened, it may lead to the failure of decision-making. the majority of students we interviewed had positive attitudes towards personal emergency preparedness for a pandemic influenza and specialized health education in the future. conclusion: researchers suggest that the government should make a specific pandemic guidance plan by referring to the risk cognitive characteristics of college students shown in the research results, and update the methods of health education to college students. influenza, which is a highly variable infectious disease that can quickly evolve into a pandemic, can pose a significant threat to people's health. 1 the corresponding emergency response measures require the active cooperation of the public to work effectively. because of its wide range of impact and potential mortality, effective risk communication will help the public understand information related to influenza. 2 compared to risk communication in other fields, when public health events occur, the government often turns to experts to ask them what the public should know. so, it is a challenge that how to effectively transform scientific knowledge into useful structures and non-professional backgrounds. 3 our researchers use influence diagrams from mental model interview to analyze the critical risk factors of flu, which can improve student`s ability of decision-making to maintain their physical health. [4] [5] [6] [7] [8] morgan et als monograph on mental model theory argues that everyone relies on their mental models to understand information. it grows into a unique and intrinsic pattern as individuals grow, similar to a workflow chart. splitting the outside world into multiple components to help us understand may not be perfect; however, it affects our way of thinking and behavior choice. 3, 9, 10 a person's mental model is influenced by various factors, including personal experience, acquired learning, and living environment, and these factors are changeable and also important in affecting our health-related behaviors. 3, [11] [12] [13] [14] [15] therefore, targeted education can help an individual correct misunderstanding in their mental models and then improve their risk management. in china, there is no application of mental model theory in the field of health education and no special pandemic preparedness guideline for the general public. however, in western countries, particularly the united states, many scholars have conducted substantial research in this area. lazrus et al have studied the public mountain flood communication framework in boulder county, colorado state. 16 casman et al 17 used the influence map to establish a dynamic risk model for waterborne cryptosporidiosis, which defines "key awareness variables" in risk communication and assigns scores for evaluation. our researchers hope to use the mental model theory to analyze the most critical risk factors of influenza pandemic from a broader perspective and find out college students` risk perception of these factors. the understanding and cognitive characteristics help improve the communication work of the government, which is the aim of this article. this study refers to the impact map formed by morss et al in the flood risk communication of boulder county 18 and draws the risk factor framework of the influenza pandemic. the entire frame is an analysis of disaster events from a macro perspective, including "causes," "development," "response," "event impact" and "risk information dissemination." then, through literature research and expert consultation, the researchers summarized the concept of the communication framework and initially formed its content suitable for the influenza epidemic. the content of the whole frame consists of the causes of influenza epidemics, the impact of pandemics, emergency preparedness and strategies of different groups, risk information, and emergency response decisions, as shown in figure 1 . the researchers subsequently searched for the corresponding supporting documents according to the content of the framework and conducted expert seminars. combined with the materials of the literature and expert opinions, the authors initially wrote identical concept items under each part of the frame. finally, we used the delphi method to invite 18 experts from the related fields to judge the structure, importance and scientific nature of these items. 19 the purpose of mental model interviews is to determine which concepts or beliefs are "out there" with sufficient frequency such that in smaller samples, these concepts or beliefs become reasonable. there is no standard method for determining sample size in relevant theories and research practice. 3 according to professor morgan's monograph and related research examples, the sample size for a mental model interview should be 20~30, at which point new information has reached saturation. 3 based on these research facts, combined with the research design of lazrus and morss, 16, 18 we recruited the first 30 respondents from 5 randomly selected non-medical college by telephone and posters. to avoid confounding bias, these students are also from non-medical majors (including russian, finance, urban planning and marketing) and have not studied medical related professional courses. after all the investigations have been completed, we discussed the results and deleted two poor interview results, and then drew a line chart of information saturation according to the number of concepts mentioned by the respondents in figure 2 . we found that after the 22nd interviewee, information saturation began to show a downward trend, and subsequent respondents did not propose new concepts. we believe that the information provided by these 28 respondents can meet the sample size required for the analysis of this study, because the purpose of mental model study is not to use statistical methods to analyze the distribution of some risk cognition in the population, but to find out which concepts or beliefs, are "out there" with some reasonable frequency, 3 so as to help government departments identify what should be focused on when developing guidance programs and health education materials for this population. the interview began with an open question, such as "please tell us about the pandemic." our investigators guided the respondents to elaborate on their main concepts, then details of the outbreak, as well as the mitigation measures that should be employed. if the interviewer had experienced emergencies, then they were encouraged to talk about the decision or idea at that time. the interview results were subsequently transcribed, encoded and classified using the coding software atlas.ti. we also conducted a quantitative analysis of the results of the compilation, then created a statistical chart, observed the degree of attention of the respondents, and compared these results with the risk perception of experts to determine the interviewee's understanding of the related concepts and other features. the questions used in this interview refer to a questionnaire in the study of skarlatidou et al. 20 the interview covers the content in figure 1 . two researchers simultaneously coded the results of the interviews. the classification consistency index (holsti reliability) of the coder was subsequently calculated, 21 which fluctuated between 0.624 and 0.965, and the average reliability statistic was 0.749. according to the study of boyatzis and burrus, the coding reliability of trained different coders ranges from 0.74 to 0.80; 22 therefore, the reliability of the coder was within the normal range and displayed adequate consistency. information saturation trend provided by 28 respondents. for each of the respondents' answers to the number of concepts noted, the researchers first mapped the scatter plots. then, to better show the increase and decrease in the information provided by the respondents, polylines were used to connect the points. the content of the concept is derived from the framework of figure 3 and is described by the responses of all 28 respondents. here is the result of two rounds of delphi expert consultation. the value of the authority coefficient is 0.885 (>0.70), which indicates that the study has a good expert score. 19, 23, 24 as shown in table 1 , in the first round of expert consultation, the coordination coefficient of each item was 0.291 (p<0.001), and in the second round of expert consultation, the coordination coefficient was 0.324 (p<0.001), which was better than the first round and indicates that the opinions of the experts are consistent from the perspective of significance test. 25 finally, we created a communication framework for an influenza pandemic, as shown in figure 3 . it serves as the basis for our investigation of the problem content of college students and can also be regarded as a kind of "standardized communication content". the respondent may have a higher probability of taking the correct protective measures if he has a good understanding of the entire framework. communication framework of pandemic influenza. the frame is composed of six main conceptual dimensions; the central concept is the bold label, and the 2ndlevel concept in the box is the part. more complicated concepts in the framework are omitted; refer to the coding manual in the appendix. the whole frame contains 79 concepts, and the arrowhead represents the influence relationship of each part. the analogy part is listed separately to describe the events associated with the respondents. note: table 1 shows the statistical coefficient calculation results of the two delphi studies, and the p values of the two coefficients all meet the requirements. the researchers counted the percentage of respondents that mentioned a concept item. also, this study used a stacked bar chart to show the number of concepts mentioned by the 28 respondents ( figure 3 ). as shown in the graph, we distinguish the concept of different attributes in terms of dimensions (risk factors). the richness of the color can visually distinguish the depth of the mental model of each interviewee [the number of concepts mentioned by an interviewee], and we can determine which dimensions of the expert`s risk perception the public is highly aware of and in which areas the public lacks awareness. furthermore, the length of the bar graph reflects the number of concepts mentioned in the dimension: a taller bar graph reflects more relevant concept items indicated by the respondents and a deeper degree of understanding of the related content. for example, respondents 12, 16 and 21 knew more about the emergency response decisions during the pandemic, whereas interviewee #24 was less aware in this regard. figure 4 shows the differences in thinking about the risk of and coping with the influenza pandemic among different groups. even with a higher education level, each college student interviewee displayed a significant difference in the depth and detail of their mental model. some of the respondents' mental models appear particularly "scarce" (such as respondents #2 and 25). nearly all respondents discussed less information than the risk perception of experts. only one interviewee (interviewee #9) cited concepts that reflected almost all the parts of the communication framework in figure 2 . the other students did not suggest many more new concepts in the interview. their conceptual descriptions reflect the concern for specific content and common cognitive deficiencies and misunderstandings. the following sections discuss these best features of the interview answers. the interactions between multiple factors may affect the formation and development of pandemic influenza. several factors mentioned by our respondents are shown in table 2 ; 39% of the respondents believed that influenza virus variation was an essential cause of the pandemic. they used statements such as "new virus," "virus mutation," and "an unknown virus." additionally, 32% of the respondents referred to disease surveillance, which included "poor supervision of the source of infection" and "unchecked work", and they were more inclined to use terms to express their views (for example, "gene mutation", "isolation treatment", "infrared surveillance", and "take the body temperature"). forty-six percent of the respondents cited characteristics related to the international spread of the pandemic. interviewee #6 indicated "foreign virus carriers from foreign places into beijing." however, some respondents believed that climate factors could lead to flu cases because they confused pandemic influenza with seasonal flu, such as interviewee #7, who answered "when the seasons change, people may catch a cold easily. if they do not pay attention, a pandemic will happen if they don`t do that." many respondents (46%) also cited the impact of population density, including densely populated places and more floating cities with higher risk areas for influenza. other factors were less frequently cited by less than 17% of interviewee, including virus resistance, viral power, avian influenza immunity, and a human lack of immunity to new viruses. compared to the experts, the mental models of many of the students interviewed contained only part of the communication framework. although some key factors were cited by most of the respondents, other essential factors were rarely cited or were misunderstood by the respondents. for example, interviewee #16 believed that the flu was a "foodborne disease" and "caused by drugs." for individuals infected with influenza, no respondents discussed the impact of vulnerable groups on the development of the pandemic, and there was no further detailed description of the virus variation. a full understanding of these information can help people to evaluate the risk level in the environment, including which situations may have a higher risk of infectious diseases. another neglected concept is the lethality of the virus. no respondents mentioned this concept or discuss the content related to us. in fact, the lethal rate is also an important indicator of a new infectious virus. 26 from the perspective of scientific disease control, the lethal rate affects whether the virus has the characteristics of limited regional transmission (for example, ebola virus, its lethal rate is 50-90%, making the virus only intermittently epidemic in individual countries and regions, with certain limitations in time and space.) 27 from the perspective of promoting public participation in disease response, highrisk events can promote individual polar to make protective decisions. 28 knowing the virulence of the virus can avoid the negative attitude to personal disease prevention caused by fluke psychology. as shown in table 3 , approximately 29% of the respondents discussed the fatality of the flu, while only 14% of the respondents described the severe symptoms that could occur after the infection, such as interviewee #5: " . . . if there goes a pandemic, it would be more than a common cold. runny nose and sneezing or, maybe, pneumonia?" none of the respondents cited complications related to influenza infection. even if a real pandemic is only composed of common symptoms of fever and fatigue, complications such as pneumonia, myocarditis, and bronchitis are the real causes of death in some vulnerable patients. 29, 30 therefore, although most of the respondents understood that the flu could have serious health threats, they did not understand how people die as a result of the flu. these misunderstandings may be related to some respondents' personal and onesided understanding of the pandemic and the lack of targeted health education. for example, interviewee #10 stated "that is, people usually do not pay attention to clothes, then they catch a cold. it is quite a normal situation every year." most respondents also discussed the social and economic impacts of the pandemic, and 46% of the respondents referred to negative effects on schools, shops, public transport, and other infrastructure during the pandemic, such as interviewee #14: "schools may shut down . . . the shops outside may be closed because of this disease, and the economy may be seriously affected because everyone will hide at home." most of the types of infrastructure, of which transportation was the most frequently cited, were generally quoted as examples of people during the sars or bird flu period, such as interviewee #11 who stated, "everyone is not going out at the time of the outbreak . . . wearing a mask if you have to go outside." thirty-two percent of the respondents were worried about overburdened hospital patients during the pandemic. some of the respondents (28%) also imagined disastrous consequences, including the impact of the pandemic on the community. according to interviewee #16, for a long time . . . our life may be threatened, many people steal food and drugs and will be locked inside their house . . . not just the direct impact, it will bring other serious problems. although the respondents mentioned the relevant concepts in the communication framework, they fail to understand the severe damage that pandemic influenza could cause to individual health; moreover, they are not fully aware of panic actions during the outbreak. the most common panic behavior is to escape from the epidemic area. to avoid disaster is people's instinctive behavior, especially in the outbreak of infectious diseases. 31 in fact, during the outbreak of the novel coronavirus (covid-19) in china, people in some areas fled the outbreak area. and it happened to be the chinese new year's holiday. lots of college students returned home to celebrate festival, which strongly increased the risk of virus transmission. although these situations have not caused irreparable serious consequences, they have also brought great interference to the case investigation and disease monitoring in all provinces of the country. surprisingly, there are 30% of the respondents believed that a negative impact of a flu pandemic would be minimal or more positive, and nearly all of them stated that it "feels like the pandemic is far away from me." according to interviewee #23, it "is a kind of epidemic disease, but speaking of cold and flu, what is generally not a major disease, easier to treat the feeling, plus the pandemic, it is only a larger scope of infection, right?" the content reflects that some students do not pay substantial attention to public health and their health. more people choose to passively wait and accept the strategies and measures employed by the school or the state government; they lack the initiative to understand the relevant information and take preventive actions. the coping strategies in table 4 are essential to pandemic emergency work and a necessary part of the communication framework in figure 2 . twenty-nine percent of the respondents cited the importance of personal hygiene habits, such as wearing masks and isolating patients; however, there are not many people who provided detail regarding these aspects. a few respondents described these strategies on the government, organization, and individual levels. most of them referred to "masks" and "be far away from the cough" in the relevant description and noted details of whether to use a special mask or separate the patient from the family. for example, interviewee #6 stated: "if it is a more serious situation, we will wear a mask, and then the hospital will be more nervous about the flu . . . " another 18% of the respondents believed that there was no need to isolate the suspected patients, such as interviewee #19: "you cannot go to the hospital first because most of the cases are not true flu, to the hospital may be isolated, so look first." for the government's decision-making, 57% of the respondents cited health education and counseling. most of them were willing to accept the necessary emergency response; over 1/4 of the respondents referred to influenza surveillance, public disinfection, and hospital treatment. these answers demonstrate these students still make mistakes and lack of understanding of the most effective protection decisions, although they have better educational backgrounds and a high degree of potential coordination. moreover, although vaccination is the most effective way to prevent the flu, only two of the respondents said they were willing to receive the flu vaccine, and the other respondents said they would not vaccinate themselves if it were not compulsory. "there is no need for voluntary vaccination" (respondents 3 and 17), "some vaccines may have side effects . . . it will hurt me" (interviewee 26). notably, interviewee #9, who originated from hong kong, was able to describe all the individual and government contingency strategies and discussed his own experience of avian influenza in hong kong in addition to elaborating on the entire process of emergency work. this fully embodies the maturity and perfection of the hong kong government in the risk communication of emergencies and the higher risk awareness. thus, the related communication and publicity strategies are worth referencing. the risk of pandemic influenza can be reduced by timely warning, access to correct information, and attitudes towards communication and interest in the face of threats. as shown in table 5 , 50% of the respondents had a specific information identification ability; 43% of the respondents chose to obtain their information on pandemic risk from the official channels. all respondents were willing to take several methods to search for risk information, including using the internet. however, although knowing first-hand influenza warning and decision support information originates from the cdc, very few of the respondents (10%) were able to clarify what types of communicators can provide help and detailed descriptions on this topic, including the specific types of early warning information that is available, where the information is, and how it is transmitted. individuals have only mastered the general concept, such as interviewee #11: " . . . go to the official website or wechat (to) find how to prevent." for health education and publicity, most of the respondents indicated that they would not take the initiative to participate in similar activities. the reasons were "traditional lectures are boring," "the publicity manual was not attractive". moreover, as interviewee #25 indicated, "i think all of them are theoretical knowledge which can be seen on the internet. if they can tell us something that you need to deal with when an event comes, it would be better." regarding suggestions for future risk communication. most of the respondents were satisfied with the current government's work and had a positive attitude towards the emergency plan of the official guide form; they were more focused on "the details of the emergency work" (cited by 25% of the respondents) and "hope to get official plan" (cited by 21% of the respondents). for example, interviewee 20 indicated that " . . . the way must be change, not as before, because the flu is not like a common cold, people will not pay much attention to it. communication, whether it is a family or school, it is best to have some specific suggestions, such as how to wash hands and disinfection, everyone can refer to themselves to do it." in general, there was a clear difference in the breadth and depth of the overall understanding of the pandemic-related information and communication framework among each student interviewed. as expected, in the context of the communication framework, most of the students' mental models were not as rich as those of the experts. they were more concerned with the critical information necessary to make individual decisions in the interpretation of risk information, for example, interviewee #10 says: "now, i want to know what type of impact will it cause, and what type of protection measures can protect me?" most respondents only referred to the critical concepts in the communication framework, without a detailed description or in an inaccurate or unclear manner; therefore, these gaps may reduce the ability of people to manage their behavior and their compliance with expert opinions. compared to the communication framework in figure 2 , the respondents used personal experience and analogies to produce more related concepts to establish the information base they needed to make decisions. table 5 the discussion of related items complete negation of self-media 5 18% dialectical view of self-media 14 50% willing to participate in publicity 8 29% refusing to participate in publicity 20 71% access to information from the authority 12 43% access to information from other mass media 14 50% obtaining information from other trusted sources 13 46% differences between pandemic and seasonal influenza 4 14% the influence of rumors 7 25% note: table shows the concept of risk information mentioned by respondents and their suggestions on current government risk communication. the infection of flu often brings many complications, in the heart and lung systems, to those who have low immunity, such as infants and young children, and these are also the significant causes of virus' potential lethality. 30, 32 the interview results show that some students do not pay sufficient attention to the impact of pandemic influenza and remain optimistic, particularly the lethality of virus, serious complications, and identification of vulnerable populations. our respondents trust in the country's sound epidemic prevention system. however, because we still have a lot of unknown information to explore about the virus, the outbreak of a new virus often brings challenges to the health system of a region. for example, virus identification, targeted program formulation, and information release all need time. for the existence of these time lags between case generation and interventions, if we want to carry out successful disease control actions, it is more important for the public to actively carry out personal protection rather than passively wait for the intervention of government departments. moreover, the desalination of the history of the epidemic, and the lack of targeted health education may also be reasons for the over-optimism of the pandemic. consequently, those who have inaccurate risk perception will estimate themselves as "the strongest young people" or "a person who having enough understanding about the flu." once a new virus outbreaks, these people may also bring misleading information to other individuals in their social circle, which will affect others' emergency decisions. in particular, for those who have experienced influenza pandemic without being negatively affected, luck may cause them to have a more positive response to future pandemics. 33, 34 furthermore, although the h1n1, h5n1, and other influenza outbreaks have been derived from new viruses following mutation, the repetition of the old virus and the prevention of the flu season risk becoming a pandemic. 31 being able to distinguish the key differences between the pandemic and common flu can effectively improve the level of personal risk cognition. among the respondents, we found that some students remained confusion: they believed that a pandemic is the mass spread of seasonal influenza or a pandemic is an almost impossible "super calamity". moreover, a pandemic is often unpredictable and generally involves international outbreak. therefore, it is important for the public to understand that the pandemic is not far away from us. we need to pay attention to our own prevention during the flu season, and at the same time, we need to be alert to unusual cold symptoms, especially when we go abroad. otherwise, patients may mistakenly think that they are suffering from common influenza, choose to place or take medicine, thus delaying the diagnosis and treatment time, infecting others and causing serious consequences. finally, concerning vaccination, our respondents have negative views regarding this issue. only 2 of the 28 respondents cited the importance of the vaccine and had a history of active vaccination, and the reasons mainly focused on the conventional "i feel good and don`t need vaccination" and "doubts about the safety of vaccines." therefore, our risk communication at present seems inadequate in promoting the necessity of vaccination. the public is not aware of the importance of the vaccine for influenza prevention or the misperceptions caused by its one-sided understanding of the pandemic, as discussed in "the countermeasures of the pandemic". in an investigation of the willingness of the elderly to be vaccinated, shaoliang geng 35 found that the primary sources of influenza and related knowledge in elderly adults were family, relatives, friends, and television, and the most trusted means of knowledge were doctors. there are cracks in clinical and public health knowledge, and patients lack knowledge about the importance of vaccination. the correction of this misunderstanding is vital for college students and because it can promote the dissemination of inoculation knowledge of young students in the family, thus improving the injection of the recommended groups (old people and young children). as discussed in the acquisition of risk information and public suggestion, in the absence of relevant knowledge and information, the respondents applied personal experiences and analogies to compose the foundation of their mental model and help themselves understand the risk of the pandemic. understanding differences in causality between risk factors can also lead to substantial differences in risk perception and coping between individuals. 33 many students only know a few general concepts and have not formed a complete emergency preparedness mode of thinking in a communication framework, knowing what one can do during the pandemic but not much about what to do and what is truly meaningful. for example, although nearly all respondents cited wearing masks and bringing in patients in time for medical treatment, the most basic measures can be limited in the presence of a real pandemic, which is only a result of a personal experience analogy (compared to a cold or related disease). what ` s more, for those in the outbreak area, especially those with suspected symptoms, it is the right and effective decision to stay at home and seek the help of local medical institutions to protect personal health than to conceal facts and escaping from outbreak area in panic. but none of our respondents know that. also, most respondents have only basic concepts (the government and the health department) regarding the types of communicators who provide the relevant risk information. these overly broad understandings may limit their ability to rapidly identify critical information or influence their knowledge of specific report under the threat of severe flu, mainly when their typical sources of information or communication channels are not available, or the necessary information is not provided. if the government is unable to offer exact messages or be out of protection from the spread of information. public trust in official authority may be reduced. students always prefer health education with new styles and systematic content. the appeal of traditional lectures and guideline books full of academic words is far less attractive, and it is hoped that the government will "reduce the over the generality of the description" and "release relevant data to increase persuasion" in future communication work. foltz's research confirms that it is necessary to use various mechanisms in the risk communication of emergencies. individuals with nonprofessional backgrounds tend to think in more specific terms, their vocabulary is less expansive, and subtle expressions cannot be well understood. bright colors and charts easily attract them. complex text information transmission will make people feel tired and irritable. 2 if possible, two student respondents also suggested organizing practical exercises, which they think is more helpful to deepen the impression and understand self-protection measures used to cope with the pandemic. information consistency is the decisive factor in understanding and perceiving personal risk. in terms of communication effectiveness, multiple sources of consistent messages are typically more effective than messages from a single source or with different contents. 36 the earlier the warning people receive and the higher the threat of information is, the higher the possibility that people take active preventive measures. therefore, the government department should incorporate the outbreak situational information and the proposed measures into influenza warnings, while maintaining the consistency of multiple communication messages. first, the results of this research reflect some misunderstanding in the respondents with a more prominent frequency: 1) influenza virus mutation and seasonal influenza have the potential to evolve into a pandemic, and the prevention of common influenza cannot be ignored. 2) the impact of an influenza pandemic is often unprecedented, and influenza virus infection can be lethal; in addition to severe cold symptoms, it also results in severe complications in patients. 3) influenza vaccination plays an active role in pandemic prevention and should be actively vaccinated, particularly children with low immunity and elderly adults, a vulnerable group. 4) for suspected patients in the family, the first choice is a social isolate, and it is very dangerous for family members to remain in close contact with their protection work. it is imperative for individuals to have common knowledge regarding influenza, the correct personal response and the degree of risk in our area for making the right decisions. therefore, we suggest that the government should put the above content as the focus of communication when communicating the risks related to the pandemic, or formulating the corresponding health education materials, so as to improve the compliance of the audience. on the other hand, the content of government risk communication should not be limited to medical advice. the public health department should develop a response plan for individuals and organizations. in terms of organization, a pandemic does not directly damage related facilities in contrast to many other catastrophic events. however, the regular work of employees within the organization will be affected. the absence of ill employees in central positions will have a severe impact on the regular operation of the organization. therefore, we need to develop a "continuous work plan" for these particular circumstances. the government should release relevant risk information on an influenza pandemic in the form of a preparation plan, or, use the network for distance health education or guiding emergency response work through local radio or television stations. finally, we should update the channels and methods of risk communication and health education. the government should strengthen the application of new media to adapt to young people's information acquisition preferences. in the form of communication, it can be gradually changed from traditional lectures to novel approaches, such as public welfare videos, songs, and scene construction experiences. moreover, scene effects can play an essential role in 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an integrated risk model of a drinking-water -borne cryptosporidiosis outbreak flash flood risks and warning decisions: a mental models' study of forecasters, public officials, and media broadcasters in application of delphi method in screening self-rated health evaluation index system what do lay people want to know about the disposal of nuclear waste? a mental model approach to the design and development of an online risk communication validity in the qualitative research interview the competent manager: a model for effective performance delphi method and its application in medical research and decision making research on the structure of public risk communication ability of influenza pandemic in health sector coordination coefficient w test and its spss implementation influenza century: review and enlightenment of influenza pandemic in the 20th century discrete logistic dynamic model and its parameter identification for the ebola epidemic modern epidemiology methods and applications beijing: beijing medical university peking union medical college joint publishing house research on monitoring and evaluation index system of national essential medicine system in primary health care institutions. hubei: hua zhong university of science and technology analysis of the clinical characteristics of influenza a (h1n1) how does the general public evaluate risk information? the impact of associations with other risks prevalence and characteristics of children at increased risk for complications from influenza analysis of the information demand characteristics of public health emergencies of infectious diseases investigation on knowledge and willingness of influenza vaccination among the elderly over 60 years old in xuchang city social and hydrological responses to extreme precipitations: an interdisciplinary strategy for post-flood investigation the authors would like to acknowledge linxian wang for helping compiling interview questionnaires, making suggestions on interview skills and finding supporting documents. we also express the sincere gratitude to students involved in the interviews of this research. this research did not involve any experiments or investigation which need ethical approval, and did not receive any specific funding too. the authors report no conflicts of interest for this work. risk management and healthcare policy is an international, peerreviewed, open access journal focusing on all aspects of public health, policy, and preventative measures to promote good health and improve morbidity and mortality in the population. the journal welcomes submitted papers covering original research, basic science, clinical & epidemiological studies, reviews and evaluations, guidelines, expert opinion and commentary, case reports and extended reports. the manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. visit http://www.dovepress.com/testimonials.php to read real quotes from published authors. key: cord-007681-vhghhvnu authors: schwartz, benjamin; orenstein, walter a. title: prioritization of pandemic influenza vaccine: rationale and strategy for decision making date: 2009-06-15 journal: vaccines for pandemic influenza doi: 10.1007/978-3-540-92165-3_24 sha: doc_id: 7681 cord_uid: vhghhvnu few catastrophes can compare with the global impact of a severe influenza pandemic. the 1918–1919 pandemic was associated with more than 500,000 deaths in the usa and an estimated 20–40 million deaths worldwide, though some place the global total much higher. in an era when infectious disease mortality had been steadily decreasing, the 1918–1919 pandemic caused a large spike in overall population mortality, temporarily reversing decades of progress. the us department of health and human services, extrapolating from the 1918–1919 pandemic to the current us population size and demographics, has estimated that a comparable pandemic today would result in almost two million deaths. vaccination is an important component of a pandemic response. public health measures such as reduction of close contacts with others, improved hygiene, and respiratory protection with facemasks or respirators can reduce the risk of exposure and illness (germann et al. 2006; ferguson et al. 2006), but would not reduce susceptibility among the population. prophylaxis with antiviral medications also may prevent illness but depends on the availability of large antiviral drug stockpiles and also does not provide long-term immunity. by contrast, immunization with a well-matched pandemic vaccine would provide active immunity and represent the most durable pandemic response. however, given current timelines for the development of a pandemic influenza vaccine and its production capacity, vaccine is likely not to be available in sufficient quantities to protect the entire population before pandemic outbreaks occur, and thus potentially limited stocks may need to be prioritized. this chapter reviews information on influenza vaccine production capacity, describes approaches used in the usa to set priorities for vaccination in the setting of limited supply, and presents a proposed strategy for prioritization. few catastrophes can compare with the global impact of a severe influenza pandemic. the 1918-1919 pandemic was associated with more than 500,000 deaths in the usa and an estimated 20-40 million deaths worldwide, though some place the global total much higher. in an era when infectious disease mortality had been steadily decreasing, the 1918-1919 pandemic caused a large spike in overall population mortality, temporarily reversing decades of progress. the us department of health and human services, extrapolating from the 1918-1919 pandemic to the current us population size and demographics, has estimated that a comparable pandemic today would result in almost two million deaths. vaccination is an important component of a pandemic response. public health measures such as reduction of close contacts with others, improved hygiene, and respiratory protection with facemasks or respirators can reduce the risk of exposure and illness (germann et al. 2006; ferguson et al. 2006 ), but would not reduce susceptibility among the population. prophylaxis with antiviral medications also may prevent illness but depends on the availability of large antiviral drug stockpiles and also does not provide long-term immunity. by contrast, immunization with a well-matched pandemic vaccine would provide active immunity and represent the most durable pandemic response. however, given current timelines for the development of a pandemic influenza vaccine and its production capacity, vaccine is likely not to be available in sufficient quantities to protect the entire population before pandemic outbreaks occur, and thus potentially limited stocks may need to be prioritized. this chapter reviews information on influenza vaccine production capacity, describes approaches used in the usa to set priorities for vaccination in the setting of limited supply, and presents a proposed strategy for prioritization. an influenza pandemic occurs with the introduction and spread of a new influenza a virus subtype among people. although some cross-protection against antigenically different influenza viruses within a subtype occurs following prior infection or vaccination, the entire population is likely to be susceptible to an influenza a virus subtype that has not circulated (or has not circulated recently) among people. consequently, in an influenza pandemic, rates of illness are higher, severity is greater, and the distribution of mortality is more widespread compared with seasonal influenza (simonsen et al. 1998) . given the susceptibility of the entire population, the goal of the united states' pandemic vaccination program is to offer vaccination to everyone living in the usa. there are several potential approaches to implementing pandemic influenza vaccination when vaccine supplies are inadequate to rapidly vaccinate the entire population: vaccine could be administered on a "first come, first served" basis or could be targeted first to individuals and groups based on specified criteria. criteria for targeting in other mass vaccination campaigns have included geographic area (e.g., group a meningococcus in the african meningitis belt), exposure or proximity to a case (e.g., smallpox), age (e.g., polio), risk of infection (e.g., h. influenzae type b), risk of complications from infection (e.g., seasonal influenza), risk for transmitting infection (e.g., rubella), or (most often) a combination of these factors. targeting has been justified as providing earliest protection to those who are most vulnerable to infection, most at risk of severe or fatal disease, or whose protection may prevent or reduce further transmission (heymann and aylward 2006) . when vaccine supply, the capacity to administer it, or funding is limited, so that the optimal strategyrapid universal vaccination-is impossible to implement, targeting mass vaccination becomes more important to achieve the best possible outcomes. in the 2005 national strategy for pandemic influenza, the president defined a goal of establishing domestic manufacturing capacity that produces sufficient vaccine to vaccinate the entire us population within six months of the emergence of a virus with pandemic potential (the white house 2005). to achieve this, over $1 billion has been allocated (1) to expand domestic egg-based influenza vaccine production, (2) to support advanced development of new vaccine production technologies, such as growth of influenza virus in cultured cells or development of recombinant vaccines, and (3) to support the advanced development of "antigen-sparing" approaches, such as new adjuvants, that can stimulate a more robust immune response, allowing manufacturers to reduce the amount of antigen in each dose and formulating the antigen produced into more vaccine doses. until the promise of these approaches is realized, however, pandemic influenza vaccine supply is likely to be far less than pandemic response needs. for the 2007-2008 influenza season, most of the influenza vaccine administered in the usa was produced in other countries, and these sources of supply may not be available during a pandemic. moreover, the amount of antigen needed to achieve a protective immune response could be substantially greater for a pandemic virus compared with seasonal influenza viruses. a clinical trial of an unadjuvanted candidate h5n1 vaccine showed that two doses containing 90 mg of hemagglutinin antigen were needed to achieve an immune response that may correlate with protection in more than half of healthy adult recipients (treanor et al. 2006) . this per dose concentration is sixfold higher than the quantities of hemagglutinin antigen included for each strain in the seasonal trivalent inactivated vaccines (tiv), and twofold higher than the total hemagglutinin in a standard dose of tiv. since two doses of the h5n1 vaccine were needed to achieve adequate immunogenicity, the quantity of antigen needed to immunize an adult would be 12-fold higher than the amount of antigen to vaccinate against a seasonal strain. initial trials with other candidate h5n1 vaccines that contain alum, novel lipid-based adjuvants, or that use the inactivated whole virus documented immunogenicity with two doses of 30 mg, 3.8, and 10mg, respectively (bresson et al. 2006; leroux-roels et al. 2007; lin et al. 2006 ). while additional studies are needed, these results suggest a potentially wide range of antigen quantities needed in different vaccine formulations, which will directly impact how quickly the population can be effectively vaccinated in the event of a pandemic. vaccine supply, therefore, would depend on the production capacity for different vaccine formulations at the time a pandemic occurs. under some scenarios, vaccine supply would be very limited, whereas under others, assuming success in evaluating and licensing new formulations and producing them in the usa, supply may be robust. the time required to develop, license, and manufacture pandemic influenza vaccine is also an important variable. using current technologies, at least 20 weeks would be required from the time the pandemic virus was identified until the first vaccine doses become available. depending on a combination of factors, including where the pandemic begins, how quickly it is detected, the effectiveness of containment measures and the season, the first us pandemic wave may occur before any pandemic vaccine becomes available or after sufficient lead time such that vaccination is already widespread (ferguson et al. 2005; longini et al. 2005 ). in the 1957 pandemic, the first us cases occurred in june but no community outbreak occurred until august and the first pandemic wave did not peak until the end of october; by this time almost half of the approximately 60 million vaccine doses eventually produced had been delivered. by contrast, in 1968, the pandemic was not recognized until later in the year, and at the time initial us outbreaks began few persons had been vaccinated (schwartz and wortley 2006) . because influenza vaccine production capacity, vaccine formulation, and the time from pandemic recognition to onset of us outbreaks all are uncertain for the next pandemic, we are unable to predict how many people will be vaccinated before pandemic disease is widespread. thus, prioritizing who is vaccinated earlier and who later will best target available supply to achieve national pandemic response goals. us pandemic response goals include slowing the spread of pandemic disease and reducing the health, societal, and economic impacts of the pandemic (the white house 2005). the approach to using a limited supply of pandemic vaccine may differ depending on which goals are considered most important. results of mathematical models suggest that vaccinating school-aged children can best reduce transmission of influenza, slowing disease spread and reducing overall community attack rates (germann et al. 2006) . while studies of vaccination for seasonal influenza support a strategy of vaccinating children to protect others in the community through herd immunity (monto et al. 1969; piedra et al. 2005; reichert et al. 2001) , uncertainty in the amount of vaccine that will be available or its timeliness make reliance on trying to induce indirect protection a risky strategy. hospitalizations and deaths from pandemic illness can be reduced by directly vaccinating those at highest risk for these severe outcomes. based on age-specific mortality rates in the 1957 and 1968 pandemics, vaccinating persons ³65 years old would have prevented substantially more deaths compared with vaccinating other age groups, despite the lower vaccine efficacy among the elderly (in 1918 this would not have been the case because of the high mortality rate among young adults). another approach to reduce the health impacts of a pandemic would be to vaccinate healthcare workers so that they can continue to provide care to others. in an unmitigated pandemic, the demand for healthcare services will be overwhelming at a time when healthcare workers may be out of work due to illness, the need to care for sick family members, or because they are afraid of becoming infected at the workplace. a survey of county health department workers in maryland found that 46% of respondents indicated they would not report to work in a pandemic. in a multivariable analysis, confidence in one's personal safety was significantly associated with a willingness to work (balicer et al. 2006) . whether response to a survey is predictive of actual behavior is unclear; anecdotally, virtually all healthcare workers in toronto reported to work during the sars outbreak, despite the fear associated with a new disease and the spread that occurred within hospitals. whether vaccinating healthcare providers to maintain effective care or vaccinating those at highest risk of illness would better reduce the health impacts of a pandemic is unknown. the potential societal and economic impacts of a pandemic are associated with pandemic severity, although even in a severe pandemic these impacts cannot accurately be predicted. historical experience does not provide a guide, as a severe pandemic has not occurred for almost a century. a report by the us department of homeland security's national infrastructure advisory committee (niac) analyzed the components of 14 critical infrastructure sectors that would be essential to society in a pandemic and the workforce needed to maintain those products and services (national infrastructure advisory council 2007). the report identifies significant interdependency between sectors, expresses concern about the maintenance of supply chains, many of which stretch overseas, and emphasizes the importance and challenges of implementing a targeted vaccination program. of the approximately 85 million workers in these sectors, 16.9 million were defined by niac as essential in a pandemic. about nine million of these workers are in the healthcare and emergency services (emergency medical services, law enforcement, and fire protection) sectors. in other sectors, the proportion of the workforce defined as critical ranges from almost 50% in the nuclear sector to less than 5% of the food and agriculture sector. because the availability of pandemic vaccine before disease outbreaks is not assured, business planning includes other measures such as "social distancing," improved hygiene, use of facemasks or respirators, and possibly antiviral drug prophylaxis to protect workers in essential operations. because of the uncertainties about the severity and epidemiology of the next pandemic, vaccine supply, and the best approach to using vaccine to reduce health, societal and economic impacts, there is no scientific method to define the optimal use of pandemic influenza vaccine. in 2005, a working group from two us advisory committees, the advisory committee on immunization practices (acip) and the national vaccine advisory committee (nvac) met to develop a pandemic vaccine prioritization strategy. the working group considered the epidemiology and impacts of pandemics, the groups at highest risk for complications and death from influenza, vaccine efficacy, critical societal functions, and ethical issues. the prioritization strategy proposed by the committees included vaccinating groups defined in tiers and subtiers, depending on vaccine supply. groups that were prioritized for earliest vaccination included healthcare workers, manufacturers of pandemic vaccine and antiviral drugs, and persons at high risk of severe illness and death. personnel in critical infrastructure sectors other than healthcare were prioritized after these groups, which include over 100 million persons. this strategy was published in the department of health and human services' pandemic plan to provide guidance to state planners and stimulate further discussions (us department of health and human services 2005). shortly after publication of the plan, a federal working group was created to reassess and potentially revise pandemic vaccine prioritization guidance. factors contributing to the decision to reassess the recommendations included a shift in national pandemic planning assumptions to a more severe pandemic scenario extrapolated from the 1918 pandemic (table 1 ); recognition that the hhs guidance did not include groups that could be considered for prioritization such as border protection personnel or the military; a broader understanding of the risk to essential services stimulated by the niac report; and a series of public engagement meetings convened by the cdc, where participants identified protecting essential community services as the most important goal for pandemic vaccination rather than protecting those who are at highest risk (public engagement pilot project on pandemic influenza 2005). the federal working group process included consideration of the scientific issues reviewed in the earlier prioritization process, assessment of mathematical modeling results, and discussion with public health officials, critical infrastructure providers and homeland and national security experts. recognizing that science alone cannot define the best approach to pandemic vaccine prioritization, key elements of the process were consideration of ethical issues, input from the public and stakeholders, and a formal decision analysis. ethical input into the working group process was achieved through the participation of public and private sector ethicists and an analysis conducted by the ethics subcommittee of cdc's advisory committee to the director (ethics subcommittee of the advisory committee to the director, cdc 2007). a strategy of targeting pandemic influenza vaccination to reduce health, societal and economic impacts was considered ethically appropriate. although a strict utilitarian principle could not be applied because of uncertainty about what strategy would provide the most benefit, targeting protection of society in a broad sense was given higher priority than protecting individuals at high risk of complications from influenza. fairness and equity are important principles where everyone is recognized to have equal value, and all table 1 national pandemic planning assumptions. note that planning for some responses such as nonpharmaceutical community mitigation strategies is done across a range of pandemic severities, as defined by the pandemic severity index (cdc, community mitigation guidance) clinical illness attack rate of 30% (rates highest among school-aged children, about 40%, and declining with age); us national estimate: 90,000,000 cases° care seeking by about half of those who are clinically ill° hospitalization of 11% of clinical cases; us national estimate: 9,900,000° case fatality rate of 2.1%; us national estimate: 1,900,000 • risk groups for severe illness and death will depend on the pandemic virus and are likely to include infants, pregnant women, persons with chronic and immunosuppressive medical conditions, and the elderly • outbreaks will last 6-8 weeks in affected communities; effective use of nonpharmaceutical community mitigation strategies (e.g., social distancing) will prolong community outbreaks but reduce their overall magnitude • multiple waves of illness will occur, with each wave lasting 2-3 months persons within a targeted group should have similar access to vaccination. reciprocity, which posits that protection should be afforded to those who assume increased risk in an occupation that benefits society, also was considered important, and a reasonable corollary to healthcare providers' "duty of care" where one is committed to provide care even in settings that increase personal risk. procedural ethical principles of inclusiveness and transparency were met through a process of engaging with the public and stakeholders in meetings, and through a request for comments posted in the federal register and on the government's pandemic influenza website. the goal of the public and stakeholder meetings was to identify the objectives of a pandemic vaccination program that participants felt were most important to pursue. public meetings were held in two demographically different communities with participants recruited by community groups. stakeholder representatives from government, healthcare, business, and community organizations participated in a third meeting. each meeting included initial presentations to educate participants on influenza and influenza vaccine, pandemics, and the rationale for vaccine prioritization. participants discussed potential objectives of pandemic vaccination in small groups and then met in a plenary session where the objectives were discussed further. finally, participants rated the importance of each of ten proposed objectives using a seven-point likert scale ranging from "extremely important" (a score of 7) to "not important" (a score of 1). despite the differences between groups in terms of geographic location, demographic characteristics, and occupational background, the values expressed at each meeting were similar (table 2) . table 2 importance of pandemic vaccination program objectives based on scores assigned by participants at public engagement meetings in las cruces, new mexico, and nassau county, new york, and a stakeholders meeting in washington, dc. scores were assigned from a seven-point likert scale ranging from 7 = extremely important to 1 = not at all important key outcomes of this process included the importance of achieving multiple objectives with the pandemic vaccination program, the value given to protecting critical services and exposed workers, and the preference for vaccinating children before those who are most likely to become sick or to die-older adults and those who have underlying medical conditions. results from the public and stakeholder engagement process provide insight into the values and preferences of the population but do not translate directly into a prioritization strategy for pandemic vaccine. we therefore conducted a formal decision analysis to assess the priority of different population groups. we identified 53 potential target groups for pandemic vaccination defined by their occupation or by their age and health status. the degree to which each group met each of the ten vaccination program objectives was then assessed and scored: how well each group met objectives related to occupational role or exposure was scored by representatives on the federal working group; for objectives where clinical trial or epidemiological data can be used to assess how well a group met an objective, scoring was done by influenza experts from cdc and academic medical centers. the score assigned to each group for each objective was then weighted by the average rating of the objective's importance from the public engagement and stakeholders meetings ( table 2) . a total score was calculated for each group as the sum of the objective scores multiplied by their weights for the ten vaccination program objectives, as described by s x = o 1 w 1 + o 2 w 2 + ... + o 10 w 10 , where s x is the total score for group x; o 1-10 are the scores the group received for each of the ten objectives; and w 1-10 are the weights for each of the objectives. 1 as an example, medical care practitioners received high scores from the working group for objectives of fighting the pandemic and providing care, providing an essential community service, being vulnerable due to their jobs, and being at risk of spreading infection to those who are unprotected (their patient population). because most healthcare workers are healthy adults who would respond well to vaccination, they also received high scores for the objective of being most likely to be protected by the vaccine. medical care practitioners score lower for providing essential economic services, protecting homeland and national security, and being most likely to get sick or die (as some may have underlying medical conditions or be 65 years old or older). this group would receive no points for keeping the pandemic out of the usa or being children. based on this analysis, groups scoring highest for vaccination were front-line public health workers involved in the pandemic response (for example, providing vaccinations), medical care practitioners, emergency medical service personnel, law enforcement personnel, and emergency relief workers. occupational groups invariably scored higher than general population groups defined by their age and health status because more of the ten program objectives were relevant (i.e., they would receive some score for objectives related to one's occupational role and exposure risk as well as one's age-and health-related risk of influenza, ability to be protected by vaccination, and potential role in disease spread). by contrast, general population groups received no score for the occupationally-related objectives. to control for this difference, we stratified potential vaccination target groups into four categories: those that provide healthcare and community support services; those that provide critical infrastructure services; those that protect homeland and national security; and the general population. within these categories, target groups were clustered based on their scores, with breakpoints between clusters defined by difference between scores. groups scoring highest among each of these categories are shown in table 3 . the us pandemic vaccine prioritization guidance incorporates both the tier structure from the guidance included in the 2005 hhs pandemic plan and the target group categorization used in the decision analysis. reflecting the similar value placed by the public on protecting persons who provide pandemic healthcare, who maintain essential community services or are at high occupational risk, and protecting children, each of the highest vaccination tiers for a severe pandemic includes groups from each category (table 4) . generally, the specific groups included in each tier track closely with the results of the decision analysis. some groups, such as deployed military forces and those who provide support for their mission, are placed in a higher tier in recognition that they may be affected in a pandemic earlier than persons in the usa due to their geographical locations, their increased risk because of crowded living conditions, and the impact of illness on their ability to function effectively. in some critical infrastructure sectors, target groups are prioritized in a lower tier because their expected occupational burden would likely decrease in a pandemic (e.g., passenger transportation), they can largely be protected by changes in work practices such as teleworking, and/or the workforce or work is "fungible;" essential support and sustainment pers. 650,000 intelligence services 150,000 border protection personnel 100,000 national guard personnel 500,000 other domestic national security pers. 50,000 other active duty and essential suppt. that is, the impact of absenteeism or reduced function can be mitigated by the redundancy within the sector (e.g., trucking, food processing). workers in infrastructure sectors are targeted for early pandemic vaccination to maintain the essential services they provide in recognition of the interdependencies between sectors. healthcare, for example, relies on the sectors that provide electricity, clean water, communications, information technology, transportation, pharmaceuticals, food, and chemicals. in a less severe pandemic, however, historical experience suggests that these services are unlikely to be substantially affected. in both the 1957 and 1968 pandemics, essential services were maintained without targeting pandemic vaccination. therefore, the us strategy differs for severe, moderate, and less severe pandemics, with some of the occupational groups not targeted in moderate and less severe pandemics, and those workers being vaccinated with their age and health status group in the general population category. pandemic severity is classified using the pandemic severity index, which defines five categories based on the case fatality rate of pandemic illness (cdc 2007) . a category 1 pandemic, defined by a case fatality rate of <0.1%, would result in a mortality only slightly greater than a severe seasonal influenza epidemic, and the proposed us vaccine prioritization guidance for less severe pandemics (categories 1 and 2) is formulated to be more similar to recommendations for annual influenza vaccination. pandemic vaccine prioritization strategies developed in other industrialized countries are generally based on similar ethical principles and target similar groups to those in the us plan. while healthcare providers and those critical to a pandemic response are the groups targeted first in many plans, workers in other infrastructure sectors may not be targeted. this may reflect national planning assumptions for a less severe pandemic, lower predicted rates of worker absenteeism, and a belief that infrastructures can be protected by planning to protect workers using nonpharmaceutical interventions and antiviral medications to treat or prevent illness. some countries, such as canada or australia, which have substantial domestic influenza vaccine manufacturing capacity and small populations, may choose not to prioritize vaccination because of the ability to vaccinate everyone over several months. to our knowledge, only the us strategy explicitly presents different vaccine targeting based on pandemic severity, although every country is likely to reassess and potentially modify their national plan based on the epidemiology of the pandemic. prioritizing pandemic vaccination addresses only a single component of planning an effective pandemic influenza vaccination program. plans are also needed on how the vaccine supply will be allocated among the states or other jurisdictions, how it will be distributed, and how the program will be implemented. key implementation issues include the method of identifying persons who are in target groups, validation at the vaccination site, vaccine administration and tracking, and monitoring for the occurrence of adverse events. a major problem could be having to turn away persons who are panicked about the severity of a pandemic yet do not meet the criteria for vaccination at that time under the prioritization strategy. currently, no comparable program exists and each step will need to be planned and tested in preparedness exercises. effective communications also will be important. while substantial public involvement in the development of the vaccine prioritization strategy increases the chance that the approach will be acceptable to the public, communications goals will be to assure the public that the entire population will have the opportunity to be vaccinated, to communicate the rationale for prioritization and the prioritization strategy, and to inform people when it is their turn to be vaccinated. rationing of healthcare is not an issue that most americans have had to face in the past. outside of military settings, healthcare services generally have not been limited by availability as much as by economic or geographic factors. prioritizing pandemic influenza vaccine introduces a new paradigm. the approach taken by us planners considering science, ethics, and public values and preferences creates a model for how such rationing can take place. nevertheless, the optimal solution is to pursue preparedness activities that will obviate the need to prioritize. ongoing programs to increase influenza vaccine production capacity, to stretch vaccine supply through the use of new adjuvants, and to develop influenza vaccines targeted at antigens that are conserved across the different influenza a subtypes may all lead to a time when pandemic influenza vaccine prioritization will be unnecessary. local public health workers' perceptions toward responding to an influenza pandemic safety and immunogenicity of an inactivated split-virion influenza a/vietnam/1194/2004 (h5n1) vaccine: phase 1 randomised trial community strategy for pandemic influenza mitigation ethics subcommittee of the advisory committee to the director, cdc (2007) ethical guidelines in pandemic influenza strategies for containing an emerging influenza pandemic in southeast asia strategies for mitigating an influenza pandemic mitigation strategies for pandemic influenza in the united states mass vaccination: when and why antigen sparing and cross-reactive immunity with an adjuvanted rh5n1 prototype pandemic influenza vaccine: a randomised controlled trial safety and immunogenicity of an inactivated adjuvanted whole-virion influenza a (h5n1) vaccine: a phase 1 randomised controlled trial containing pandemic influenza at the source effect of vaccination of a school-aged population upon the course of an a2/hong kong influenza epidemic the prioritization of critical infrastructure in a pandemic influenza outbreak in the united states: final report and recommendations by the council herd immunity in adults against influenza-related illnesses with use of the trivalent-live attenuated influenza vaccine (caiv-t) in children public engagement pilot project on pandemic influenza (2005) citizen voices on pandemic flu choices the japanese experience with vaccinating schoolchildren against influenza mass vaccination for annual and pandemic influenza pandemic versus epidemic influenza mortality: a pattern of changing age distribution national strategy for pandemic influenza safety and immunogenicity of an inactivated subvirion influenza a. (h5n1) vaccine pandemic influenza plan: appendix d key: cord-007733-zh8e76w7 authors: dimenna, lauren j.; ertl, hildegund c. j. title: pandemic influenza vaccines date: 2009-06-15 journal: vaccines for pandemic influenza doi: 10.1007/978-3-540-92165-3_15 sha: doc_id: 7733 cord_uid: zh8e76w7 since their compositions remain uncertain, universal pandemic vaccines are yet to be created. they would aim to protect globally against pandemic influenza viruses that have not yet evolved. thus they differ from seasonal vaccines to influenza virus, which are updated annually in spring to incorporate the latest circulating viruses, and are then produced and delivered before the peak influenza season starts in late fall and winter. the efficacy of seasonal vaccines is linked to their ability to induce virus-neutralizing antibodies, which provide subtype-specific protection against influenza a viruses. if pandemic vaccines were designed to resemble current vaccines in terms of composition and mode of action, they would have to be developed, tested, and mass-produced after the onset of a pandemic, once the causative virus had been identified. the logistic problems of generating a pandemic vaccine from scratch, conducting preclinical testing, and producing billions of doses within a few months for global distribution are enormous and may well be insurmountable. alternatively, the scientific community could step up efforts to generate a universal vaccine against influenza a viruses that provides broadly cross-reactive protection through the induction of antibodies or t cells to conserved regions of the virus. against influenza a viruses. if pandemic vaccines were designed to resemble current vaccines in terms of composition and mode of action, they would have to be developed, tested, and mass-produced after the onset of a pandemic, once the causative virus had been identified. the logistic problems of generating a pandemic vaccine from scratch, conducting preclinical testing, and producing billions of doses within a few months for global distribution are enormous and may well be insurmountable. alternatively, the scientific community could step up efforts to generate a universal vaccine against influenza a viruses that provides broadly cross-reactive protection through the induction of antibodies or t cells to conserved regions of the virus. influenza viruses belong to the family of orthomyxoviridae, which includes negative single-stranded rna viruses with segmented genomes. among the three genera of influenza viruses (a, b, and c), influenza a and c viruses infect humans as well as other species, while influenza b virus mainly infects humans. the most common and serious infections of humans are caused by influenza a virus. influenza a viruses are further divided into subtypes based on their hemagglutinin (ha) and neuraminidase (na) genes, which encode the two viral surface proteins. influenza a virus typically infects epithelial cells that line the respiratory tract, but may also replicate in other tissues in different hosts, including conjunctiva, intestine, brain, liver, kidney, and gut. in general, influenza a virus infections are self-limiting in healthy human adults, and mainly cause life-threatening disease in the very young and in the elderly. notwithstanding, this depends on the circulating type. aquatic birds serve as the main reservoir of influenza a viruses and carry all of the known subtypes (h1-16, n1-9) without necessarily developing disease upon infection. the virus can adapt to other species such as poultry, pigs, horses, or humans. in humans, thus far the h1, h2, or h3, and n1 or n2 influenza viruses have established transmittable infections. influenza viruses mutate rapidly, and these mutations affect mainly (but not exclusively) the genes encoding the surface proteins. point mutations that cause gradual changes are referred to as antigenic drift, and allow the virus to evade protective neutralizing antibody responses induced by previous infections. most annual epidemics are caused by antigenic drift variants. rearrangements of the ha-or na-encoding gene segments between viral types circulating in humans and those endemic in animals result in more dramatic changes, also called antigenic shifts, and the pandemics of 1957 with h2n2 and 1968 with h3n2 were caused by such new types of influenza virus. according to the world health organization (who), a pandemic is the emergence of a serious new disease caused by an agent that spreads easily among humans. who recommends three measures to lessen the impact of the next influenza virus pandemic: (1) increased surveillance to allow for the earliest possible warning that a human pandemic has started; (2) early intervention to stall global spread and prevent further adaptations; and (3) development of an effective pandemic vaccine. available vaccines against influenza virus are seasonal vaccines that are updated annually to incorporate the latest circulating viruses. seasonal vaccines are composed of three different influenza viruses, which are typically two subtypes of influenza a virus and one strain of influenza b virus. the vaccine composition is generally agreed upon in spring to allow for manufacturing and distribution before onset of the influenza season in late fall to winter. seasonal vaccines are currently derived from egg-grown viruses that are either inactivated and then given systemically or attenuated by cold adaptation and given directly to the airways. pandemic vaccines, the focus of this chapter, are at this stage virtual vaccines of an unknown composition. they aim to protect against a newly evolved pandemic influenza virus. a pandemic vaccine may thus have to be manufactured at the onset of a pandemic, or alternatively one would need to devise a vaccine that induces broadly cross-reactive protection, unlike the current vaccines. influenza a viruses are enveloped spherical viruses which contain eight segments of single negative-stranded rna. segments 1, 2, and 3 encode the transcriptase complex composed of basic polymerases (pb)2 (segment 1) and pb1 (segment 2) and acid polymerase (pa, segment 3). segment 4 encodes the hemagglutinin (ha), which has receptor-binding activity, promotes cell fusion, and is the major target for neutralizing antibodies. segment 5 encodes the nucleoprotein (np) which complexes the viral rna to form the nucleocapsid. np is a major target for cross-reactive cd8 + t cells in mice and humans (falk et al. 1991) . segment 6 encodes the viral neuraminidase (na), a cell surface protein with enzymatic activity, which also provides a target for neutralizing antibodies. drugs such as zanamivir and oseltamivir, which block the enzymatic cleavage of sialic acid residues by na, are available and can be used to treat or prevent infections (garman and laver 2004) . segment 7 encodes matrix (m) protein 1 and 2. m2 has ion channel activity, which is blocked by the antiviral drug amantadine (ison and hayden 2001) . m protein is also a target for cross-reactive cd8 + t cells in humans, while the m2 ectodomain is a target for nonneutralizing but nevertheless protective antibodies . segment 8 encodes nonstructural proteins (ns) 1 and 2. the twentieth century experienced three major influenza virus pandemics (table 1) and several small abortive pandemics, as well as pandemic threats and numerous outbreaks in animals also called epizootics or panzootics. the first pandemic of the twentieth century started in 1918 in the usa and then spread to africa and europe, first to france and then spain, and subsequently to every part of the globe. this pandemic was caused by an h1n1 virus and is paradoxically and unfairly referred to as the spanish flu. the pandemic that started in march of 1918 and lasted until june of 1920 killed half a million americans and somewhere between 50 and 100 million humans worldwide (johnson and mueller 2002) . this virus infected nearly 50% of the population and killed 2.5% of all of those that became infected. it is estimated that 25 million people died during the first 25 weeks of the pandemic. death rates were high in humans between the ages of 20-40, an age group which generally recovers easily from influenza a virus infection. during the initial stages of the pandemic, the early symptoms of infection, which included hemorrhages and lung edema followed by death within 24-48 h, were commonly misdiagnosed. the severity of the symptoms is assumed to have been caused by an excessive release of cytokines in response to the virus (kash et al. 2004) , which was most severe in healthy adults with sturdy immune systems. the 1918 h1n1 virus was recently isolated from victims preserved in permafrost, and upon sequencing the virus was rederived through genetic engineering . this allowed for an extensive characterization of the virus using modern tools of science. the 1918 h1n1 virus has several distinct features that may explain its unique virulence. most types of influenza a virus require trypsin-like enzymes for cleavage of the viral ha, which in turn restricts their cellular tropism. the na of the h1n1 virus of 1918 can directly or indirectly cleave ha, thus rendering this virus independent of trypsin-like enzymes (steinhauer 1999) . increased virulence was further enabled by ns proteins, which allow the virus to disable the interferon (ifn) pathway (seo et al. 2004 ), a crucial component of both innate and adaptive immunity. human-to-human transmission, a prerequisite for a human pandemic, appears to have involved a switch in preferential binding of the ha protein from a-2,3 sialic acid found in the avian enteric tract to a -2,6 sialic acid present in the human respiratory tract ). this altered receptor binding activity can be achieved experimentally through a single amino acid exchange at position 190 of the ha of the 1918 h1n1 subtype. additional changes in the viral pb and pb2 proteins, which contain four amino acids that are conserved in human viruses and that differ from those prevalent in avians, are likely to have affected transmission between humans (russell and webster 2005) . the 1957 pandemic, also referred to as the asian flu, originated from a recombination between a circulating human virus and a virus endemic in ducks. the virus was first isolated early in 1956 in guizhou, china, and by february of 1957 had spread to singapore, and to the usa by june of that year. this virus, an h2n2 virus, caused an estimated 1-4 million deaths worldwide (dunn 1958) . death rates were highest in the elderly. the 1968 pandemic, also called the hong kong flu, was caused by an antigenic shift of an h2n2 virus to an h3n2 virus. this pandemic was comparatively mild, causing an estimated 500,000 human deaths (cockburn et al. 1969; kilbourne 2006) . again mortality was high in those above 65 years of age. in 1946 an h1n1 virus that was first seen in japan and korea spread to military bases in the usa (lessler et al. 2007 ). further spread was not observed. in 1977, an h1n1 virus spread rapidly from china and caused epidemic disease in children and young adults (<23 years) worldwide. older humans were not affected, presumably due to protection from previous exposure to h1n1 viruses. in the winter of 1976, a novel swine influenza virus subtype was detected in military recruits at fort dix, new jersey. a total of 13 soldiers became symptomatically infected and one died. there was only limited spread to humans living outside the military base. fearing a major pandemic, a vaccine was rapidly generated and administered to 40 million humans. a few months after mass vaccination had started, reports of guillain-barré syndrome in vaccine recipients started to accumulate, and by early 1977 (when vaccination was stopped) more than 500 cases of gbs had been reported, 25 of which were fatal (langmuir et al. 1984 ). a highly pathogenic form of avian h5n1 virus was first detected in asian poultry in 1997 (centers for disease control and prevention 1997) . during this year, a total of 18 human cases were reported from hong kong, of which six were fatal. the virus rapidly caused pneumonia and multiple organ failure in infected individuals, which were mainly young adults. culling of infected flocks of poultry initially appeared to have stopped further spread, but then in 2003 additional human cases with a similar h5n1 virus were recorded in vietnam (tran et al. 2004 (steel et al. 2008; cristalli and capua 2007) . other countries rejected the idea of bird vaccination due to fears that this may mask infections and allow for further mutations that may promote human transmissibility. similar to the 1918 h1n1 virus, pathogenic h5n1 virus activates ha through a trypsin-independent mechanism (hulse et al. 2004 ). pathogenic h5n1 virus has a multibasic cleavage site that can be digested by furin and furin-like proteases, which are more ubiquitously present in human tissues than the trypsin-like enzymes that cleave ha of current human influenza viruses. the ns1 protein of pathogenic subtypes of h5n1 virus renders the virus resistant to the activity of ifns and tumor necrosis factor (tnf)-a (seo et al. 2002) . the h5n1 virus has changed since its first isolation in 1997. such changes include resistance to the antiviral drug amantadine due to a m2 mutation first reported in 2004 from thailand (cheung et al. 2006) . the virus has become more lethal for humans and mice, and has gained robustness against destruction in the environment. the virus has increased its host range and has been shown to cause disease in felines such as tigers (keawcharoen et al. 2004) , which are otherwise resistant to influenza a virus infections. in 1999, an h9n2 virus, which also originated from poultry, caused illnesses in two children in hong kong. both children survived and there was no serological evidence that the virus spread to their contacts. a number of epizootics and panzootics have been caused by a wide variety of influenza viruses. in poultry, numerous outbreaks with highly pathogenic influenza viruses have been reported from all over the globe within the last 50 years. these outbreaks were caused by a variety of subtypes, such as h5n1, h7n2, h1n7, h7n3, h13n6, h5n9, h11n6, h3n8, h9n2, h5n2, h4n8, h10n7, h2n2, h8n4, h14n5, h6n5, h12n5, and others. h5n1 virus, which is currently endemic in asia, africa, and europe, has within the last eight years caused the deaths of millions of birds, many of which were culled to prevent further spread and to protect humans. influenza virus outbreaks have been observed in other species. for example, from 1979 to 1980, several hundred harbor seals died along the coast of new england due to infection with a h7n7 virus (geraci et al. 1982) . as of 1997, h3n2 circulates in pigs (gramer et al. 2007) . horses have been infected with h7n7 and h3n8 viruses (amonsin et al. 2007; oxburgh and hagström 1999) . the latter can also infect and kill canines. h5n1 has caused the deaths of felines, including tigers and domestic cats (cristalli and capua 2007; steel et al. 2008) . several of these viruses have infected humans without achieving the capacity for human-to-human transmission. in 2003, 89 people were infected with h7n7 influenza virus from poultry in the netherlands (koopmans et al. 2004) . in 2002-2003, two residents of us mid-atlantic states showed serologic evidence of infection with h7n2 . in 2004, two poultry farm workers in british colombia became infected with h7n3 virus (tweed et al. 2004) . in 2004, egypt reported human infections with h10n7. any subtype of the influenza virus thus has the potential to infect humans and to evolve into a pandemic virus, which has to be taken into account when designing pandemic vaccines. more than 90% of deaths during seasonal influenza virus outbreaks occur in the elderly (³65 years of age). immunosenescence during aging leads to impaired immune responses, which increases the susceptibility of the aged to infectious agents. the elderly are affected by primary immunological changes, which are part of the natural aging process, and secondary immunological changes caused by underlying diseases and unhealthy life styles (malaguarnera et al. 2001) . primary changes of the immune system in healthy elderly involve mainly t cells, though changes in natural killer (nk) cells and nk t cell function with age have been noted (ginaldi et al. 1999c; solana and mariani 2000) . t cells show clonal senescence, their potential for expansion is decreased, and their ability to produce certain cytokines and to respond to cytokines decreases. the proportion of t cells with a memory cell phenotype increases while numbers of naïve t cells decrease. stimulation with new antigens appears to result in shortened immunological memory (ginaldi et al. 1999b ). the t cell repertoire loses diversity (effros et al. 2003) due to chronic antigenic stimulation, leading to continued clonal expansion of some t cells, which undermines the homeostatic balance of the immune system. primary b cell responses in the elderly are commonly low and short-lived, resulting in antibodies with low affinity (ginaldi et al. 1999a ). formation of germinal centers is decreased, antigen transport is impaired, and follicular dendritic cells show atrophy and their capacity to form antigen depots is reduced (zheng et al. 1997; aydar et al. 2004 ). autoantibodies are more common and the b cell repertoire becomes more restricted. many of these changes reflect secondary effects due to an age-related decline of helper functions from cd4 + t cells, which show reduced expression of critical costimulatory receptors that are essential for activation of b cells, germinal center formation and rearrangement, and hypermutation of immunoglobulin genes. underlying chronic diseases dramatically increase the risk of serious complications of an influenza virus infection. patients with one or two chronic diseases have 40or 150-fold (respectively) greater risk for developing pneumonia upon influenza virus infection (janssens and krause 2004; stott et al. 2001) . underlying chronic heart, lung, or liver diseases increase the risk of serious influenza virus infection in all age groups, not just the elderly. vaccines perform poorly in the elderly, commonly resulting in inadequate and short-lived titers of protective antibody responses (biro 1978; saurwein-teissl et al. 2002) . current influenza virus vaccines provide 70-90% protection against a closely related virus in those <65 years of age, but only 30-40% protection in humans above the age of 65. young children, pregnant women, and immunosuppressed individuals also have an increased risk for influenza a virus-associated morbidity. another risk factor is superinfection of the airways with bacterial pathogens, which can enhance virulence of the influenza virus through bacterial proteases (callan et al. 1997 ). on the other hand, influenza virus can increase bacterial infection by destroying respiratory epithelium and increasing bacterial receptor (mccullers 2006) . other risk factors include living in institutionalized settings such as prisons or nursing homes, or working in healthcare, where the risk of exposure and the risk of further spread are increased. vaccines aim to induce memory immune responses that, upon encountering the virus, are rapidly reactivated or recruited to either completely prevent an infection by causing so-called sterilizing immunity, or to rapidly control viral spread. it is thus important to understand which type of immune response provides reliable protection in order to specifically design immunogens that elicit this type of a response. influenza virus pandemics unfortunately have an element of surprise on their side by their very nature, and it may be unrealistic to expect that at the onset of a pandemic, which can potentially spread around the globe within less than six months, sufficient doses of a reliable vaccine or efficacious antiviral drugs will be available to protect the entire human population. other preventions, such as activation of protective innate immune responses in those at immediate risk for infection, may add to the repertoire we can call upon to combat the next influenza virus pandemic. innate immunity can provide resistance to influenza virus infection, as has been demonstrated in animals treated with immunomodulators such as baculovirus, lentidan, double-stranded rna, or modified heat-labile toxin of escherichia coli prior to infection (abe et al. 2003; irinoda et al. 1992; saravolac et al. 2001; williams et al. 2004) . clinical trials in children who were vaccinated with an attenuated influenza a virus vaccine after the onset of an influenza a virus outbreak also suggested that protection was at least in part mediated by an innate immune response to the vaccine (piedra et al. 2007 ). influenza a virus infection leads to the rapid increase of proinflammatory cytokines in nasal and pulmonary secretions (jao et al. 1970; gentile et al. 1998) . the virus causes the activation and maturation of dendritic cells and stimulates plasmacytoid dendritic cells to secrete large amounts of type i ifns (lópez et al. 2004; cella et al. 2000) . influenza virus activates macrophages to secrete il-1, 6 and 12 and tnf-a (mak et al. 1982; pirhonen et al. 1999 ). il-12 in turn induces ifn-g production by nk cells. the early cytokine response to influenza virus can be pronounced and can result in significant pathology (van reeth et al. 2002) . nevertheless, early cytokines such as interferons also provide resistance to influenza a viruses (beilharz et al. 2007; fattal-german and bizzini 1992) . ns1 of h5n1 renders the virus resistant to the antiviral activity of ifns and tnf-a (sekellick et al. 2000) . reassortant influenza a viruses carrying the ns1 of h5n1 induce increased levels of cytokines in mice and decreased levels of il-10 (lipatov et al. 2005a ). both macrophages and nk cells can kill infected cells and are crucial to early infection control (zychlinsky et al. 1990; tsuru et al. 1987) , as are natural igm and the early components of the classical pathway of complement, which together can neutralize influenza virus (jayasekera et al. 2007 ). inhalation infection with influenza a virus triggers a mucosal immune response in the upper respiratory tract that is initiated within nasal-associated lymphoid tissue (nalt) in mice and within waldeyer's ring (tonsils) in primates. in the lower respiratory tract, responses are induced in bronchus-associated lymphoid tissues. responses can also be detected in distant lymphoid tissues such as spleen or blood. infection causes a local secretory iga response as well as igm and igg antibodies directed mainly against the viral ha. antibody-secreting cells can be detected in mice in the respiratory mucosa and in lung tissue within five days after infection. dimeric iga (diga) antibodies which are transcytosed across epithelial cells upon binding to their receptors can bind to de novo synthesized viral antigens and block viral assembly, thus contributing to viral clearance (tamura and kurata 2004) . influenza virus-specific cd8 + and cd4 + t cells are induced upon intranasal application of influenza a virus (roti et al. 2008; swain et al. 2004 ). viral clearance following a primary infection is mediated in part by cd8 + t cells and in part by antibodies, which in turn require the activity of cd4 + t helper cells for their induction. lack of cd4 + t cells does not affect induction of a primary cd8 + t cell response to influenza a virus (yap and ada 1978; mozdzanowska et al. 2005) , although absence of cd4 + t cells in general reduces the magnitude of the memory cd8 + t cell pool and the cd8 + t cell recall response. neither ifn-g nor ifn-a/b appear to be essential for viral clearance (price et al. 2000) , although loss of both ifn pathways has been reported to exacerbate disease. perforin is essential for viral clearance, and mice lacking perforin show delayed viral clearance and increased mortality to influenza a virus infection (topham et al. 1997) . increased mortality was also observed in il-1 receptor knockout mice (szretter et al. 2007 ); these mice developed normal cd8 + t cell responses and viral titers were only modestly above those of normal mice. il-1 receptor knockout mice showed a defect in recruitment of inflammatory cells to the site of infection, most notably neutrophils and cd4 + t cells. a secondary infection with influenza a virus can be prevented by local siga and can be blunted by rapid activation of memory b cells. neutralizing iga antibodies are thought to primarily prevent infection of the upper respiratory tract, while serum igg plays a role in protecting against viral pneumonia (tamura and kurata 2004) . protective neutralizing antibody responses induced by infection or vaccination are subtype specific and do not provide protection against heterotypic challenge. their ability to provide resistance to an antigenic drift subtype depends on the degree of antigenic variation between the viruses (kaye et al. 1969) . it must pointed out, however, that although the role of neutralizing antibodies in providing resistance to influenza virus is not debated, it remains far from clear-cut. some mouse studies showed that adaptive transfer of neutralizing secretory iga protected the animals, while transfer of neutralizing antibodies of the igg isotype was inefficient (renegar and small 1991) . other mouse studies showed that protection by h5-specific igg1 monoclonal antibodies can be achieved against h5n1 infections (hanson et al. 2006 ). yet others reported protection by igg antibodies that bound ha but failed to neutralize the virus (mclain and dimmock 1989). one monoclonal neutralizing antibody was described that cross-reacted between h1 and h2 and consequently protected animals upon passive transfer against infection with either virus (okuno et al. 1994 ). in other virus infections, such as those with rabies virus, where neutralizing antibodies are known to play a dominant role in protection against infection and disease, protective titers of neutralizing antibodies have been defined. for rabies virus, a titer of or above 0.5 international units protects against challenge; this knowledge has greatly facilitated vaccination efforts. in contrast, it is still not known what titer of influenza a virus-neutralizing antibodies reliably provides protection against disease. in general, it is assumed that titers above 1:40 are protective, although numerous clinical trials have demonstrated that humans with lower titers were protected while others with higher titers developed symptomatic infections. protection against heterotypic challenge (i.e., challenge with a different subtype of influenza virus than that used for immunization) can be mediated by a number of mechanisms. as already mentioned above, some neutralizing antibodies can cross-neutralize several subtypes of influenza a virus. nonneutralizing antibodies to the ectodomain of matrix protein (m2e) can protect against heterotypic challenge in animal models (mozdzanowska et al. 2003) . the 23 amino acid (aa) long m2e is conserved in its nine n-terminal amino acids and shows relative minor variability in the remaining sequences. this is likely to reflect a lack of selective pressure, as natural infections or traditional vaccines induce only low antibody responses to m2e ). the currently circulating avian h5n1 and h7n2 subtypes show sequence variability with previous human isolates that affect m2e antibodybinding sites. for example, they show changes in amino acids at positions 10-16 of m2e (h5n1: pirnewg to ptrngwg, or ptrnewe) (liu et al. 2005) . cd8 + t cells induced by repeated infections appear to contribute little to natural resistance to influenza virus infection in humans. this may be linked to suboptimal stimulation of this t cell subset upon natural infection, as human volunteers with exceptionally high levels of circulating influenza a virus-specific cd8 + t cells showed reduced viral shedding upon an experimental infection compared to those with low levels of pre-existing influenza a virus-specific cd8 + t cells (epstein 2006; murasko et al. 2002) . in mice, a number of studies showed that cd8 + t cells protect, while other showed that they fail to protect. early studies from the group of g. ada showed that adoptive transfer of influenza virus immune cells provided protection against challenge with a heterotypic subtype of the virus (yap and ada 1978) . these studies were confirmed by r. dutton and colleagues, who studied the efficacy of passively transferred, in vitro activated cd8 + t cells isolated from mice transgenic (tg) for a t cell receptor (tcr) to the influenza a virus ha (cerwenka et al. 1999) . transfer of naïve tcr-tg cd8 + t cells failed to provide resistance to challenge. protection against a lethal infection could be provided by the transfer of rested memory-like or effector tcr-tg cd8 + t cells, although the latter effected more rapid viral clearance, which may indicate that the rested cd8 + t cells needed to expand before they assumed effector functions. protection was only mediated by cd8 + t cells that were able to home to the infected respiratory tissues. poxvirus vectors expressing the influenza a virus np, which induce a cd8 + t cell response (andrew et al. 1986 ), were shown to induce some protection against heterotypic challenge (endo et al. 1991; altstein et al. 2006) . further studies showed that although vaccinia virus vectors expressing the influenza virus np induced only limited protection in mice, adoptive transfer of t cells isolated from np-immune mice and expanded in vitro were highly effective (mbawuike et al. 2007 ). yet another group reported that a vaccinia virus vector which expressed a sequence of np that induced a sturdy cd8 + t cell response in mice, including in their lungs, completely failed to induce protective immunity as assessed by peak viral loads, morbidity or mortality (lawson et al. 1994 ). heterotypic t cell-mediated protection was also reported after immunization of mice with an adjuvanted influenza virus vaccine (sambhara et al. 1998) or with dna vaccines expressing internal proteins of influenza virus (saha et al. 2006; fu et al. 1997) . another group reported that protection upon intranasal immunization with an adjuvanted nucleoprotein vaccine was mediated by t helper cells of the th1 type rather than by cd8 + t cells (tamura et al. 1996 ). yet another group reported protection with an adenovirus vector expressing nucleoprotein (roy et al. 2007 ). in our hands, subunit vaccines expressing the nucleoprotein induced strong cd8 + t cell responses that could readily be detected in spleen, blood, or even lungs of vaccinated mice. nevertheless, vaccinated mice were not reliably protected against disease or death following challenge with influenza a virus (unpublished). overall t cell protection studies largely agree that adoptive transfer of in vitro expanded cd8 + t cells provides protection against influenza virus. results on the protective nature of in situ activated influenza virus-specific cd8 + t cells range from solid protection to complete absence of protection, even under circumstances where high numbers of influenza virus-specific cd8 + t cells were present in the airways at the time of challenge. the lack of consistency of protection through cd8 + t cells may reflect genetic differences in the mouse strain used for the experiments, differences in the dose or type of challenge virus, differences in the interval between vaccination and challenge, and/or differences in the functionality of cd8 + t cells induced by various approaches. the take-home message for developing an influenza vaccine that is useful for preventing or ameliorating a pandemic therefore remains ambiguous. neutralizing antibodies protect against ha provided there is sufficient homology between the vaccine and the infecting virus. antibodies against m2e protect against a wider array of subtypes, as m2e is more conserved; nevertheless, m2e shows some variability, and protection through m2e-specific antibodies is not as robust as protection provided by neutralizing antibodies. the rules that govern cd8 + t cell-mediated protection against influenza virus remain ill-defined. influenza virus was first isolated in 1933 (smith et al. 1933) , and effective vaccines were developed and tested by 1943-1944 and became available by 1945 (francis et al. 1945a,b) . vaccines were thus not available during the spanish flu pandemic, but rapidly became available during the 1957 asian flu pandemic (gundlefinger et al. 1958) , when they were mainly used in military personnel. although the 1968 hong kong flu subtype was identified rapidly, vaccine production was delayed, and a vaccine was not available during the outbreak. until recently, all available influenza vaccines were trivalent inactivated (killed) virus vaccines. initially whole-virus vaccines were used, which were then replaced by 2001 by the less reactogenic split-virus vaccines. in june of 2003, a live attenuated, cold-adapted, temperature-sensitive, trivalent influenza virus vaccine was licensed in the united states for use in humans between 2 and 49 years of age. multiple clinical trials have been performed in adults (demicheli et al. 2004 ), children , and the elderly (jefferson et al. 2005) to assess the efficacy of influenza vaccines. studies on live vaccines are still limited, but to date they suggest that such vaccines may be more effective than inactivated vaccines in some cohorts (treanor et al. 1999) . one manuscript published an analysis of trials involving a total of 59,566 adults (demicheli et al. 2004 ) which showed that the live attenuated vaccines reduced the number of cases of serologically confirmed influenza by 48% while the inactivated vaccines had a vaccine efficacy of 70%. the yearly recommended vaccines had low effectiveness against clinical influenza cases or time off work, the later a nonspecific outcome that included illness caused by influenza as well as other agents. the authors concluded that universal immunization of healthy adults is not supported by their results. fifty-one studies involving 263,987 children were included in an analysis of influenza virus vaccine efficacy in children ). the attenuated vaccines showed an efficacy of 79% in children older than two years, while inactivated vaccines had a lower efficacy of 59%. in children under two, the efficacy of inactivated vaccine was similar to placebo. in another study, results from 19 randomized clinical studies covering a total of 247,517 children were analyzed and reported to show an overall vaccination efficacy of 36% against clinical disease, 67% against laboratoryconfirmed cases, and 51% against acute otitis media. between-study variability was related to the children's age and study quality. for example, when studies from the ussr were excluded from the analysis, the overall efficacy of the vaccine in preventing clinical cases increased from 36% to 61% (manzoli et al. 2007) . indirect evidence for the effectiveness of annual influenza virus vaccination of children can be gained from japan, where as of 1957 school children were vaccinated annually. vaccination became mandatory in the 1970s and was discontinued in 1994. during the time of mandatory vaccination, mortality among the elderly declined markedly, presumably due to reduced exposure to their infected grandchildren. sixty-four studies were analyzed to determine the efficacy of influenza vaccination in the elderly (jefferson et al. 2005; rivetti et al. 2006 ). in homes for elderly individuals, the effectiveness of vaccines against disease caused by influenza virus could not be demonstrated. when the vaccines were closely matched to the circulating virus subtype, they prevented pneumonia, hospital admission, and deaths. in elderly individuals living in the community, vaccines were not significantly effective against clinical influenza or pneumonia that were not laboratory confirmed. the authors concluded that vaccination was useful in long-term care facilities but not necessarily in community settings. another large analysis of community-living elderly came to the opposite conclusion. this analysis showed that vaccination was associated with a 27% reduction in the risk of hospitalization for pneumonia and a 48% reduction in the risk of death (nichol et al. 2007 ). other smaller studies showed that immunization of frail elderly did not reduce the rate of hospital admissions due to acute respiratory illnesses (jordan et al. 2006) , and that vaccination failed to reduce the overall mortality of the elderly (rizzo et al. 2007 ). in summary, although annual influenza virus vaccination is highly recommended, especially for high-risk populations, results of clinical trials designed to prove their efficacy remain controversial and thus far do not fully support the notion that vaccination affords reliable protection against influenza virus infection and its sequelae. who has summarized a number of global pandemic phases that have been adopted in federal and regional response plans and serve to define the type of responses required. details on these phases and suggested courses of action can be obtained online (see also as of early 2008, the usa is currently in an interpandemic period, while parts of asia, africa, and eurasia have entered phase iii(/iv) of a pandemic alert period; pathogenic avian h5n1 virus has repeatedly infected humans without causing proven human-to-human transmission yet. small clusters of human infections that may reflect human-to-human transmission have been observed. in anticipation of an influenza virus pandemic that would kill up to an estimated 1.9 million americans and require the hospitalization of an estimated 10 million americans, in november of 2005 the department of health and human services issued a pandemic influenza plan, 3 and state governments developed blueprints for local pandemic response plans. funding was provided to increase infrastructure, enhance vaccine production capability, and to augment basic knowledge on influenza virus pathogenesis and host responses. how much experience do we have with pandemic influenza virus vaccines? as mentioned above, vaccines for the spanish and hong kong flu pandemic were not available at that time, and the vaccine that was available during the asian flu pandemic was mainly used in military personnel (dull et al. 1960) . in summary, our experience with the global use of a vaccine for pandemic influenza virus is nonexistent. one could envision four scenarios for the role of a vaccine in the next influenza virus pandemic: (a) an ideal outcome in which the world could be vaccinated with a universal vaccine that would never allow another pandemic to strike, (b) an optimistic outlook in which sufficient doses of a vaccine are produced in advance in order to rapidly immunize those at the epicenter of the pandemic and those at high risk, before additional vaccine for global immunization could be produced and distributed, (c) a pragmatic attitude that prepares as effectively as possible for the next pandemic without necessarily expecting that a vaccine will be on hand at the start of the pandemic, and (d) a worst-case scenario, in which the next pandemic influenza virus will outsmart us. in an ideal scenario, scientists would develop a universal vaccine for influenza virus, industry would rapidly get involved in conducting large-scale trials needed for licensure, and then, with the aid of governments and philanthropic agencies, initiate a worldwide vaccination program before the next pandemic subtype of influenza virus evolves. ideally, the vaccine would be adjuvanted to induce robust, long-lasting immunity not only in healthy adults but also in high-risk populations such as the elderly, infants, or those suffering from chronic diseases. it is hoped such a vaccine would prevent the development of any future influenza virus pandemics (table 3) . in experimental animals, some vaccines affect protection against heterotypic challenge with influenza virus, such as vaccines based on m2e (mozdzanowska et al. 2003; liu et al. 2004; slepushkin et al. 1995; fan et al. 2004; frace et al. 1999; neirynck et al. 1999; de filette et al. 2006; eurekalert 2007) . protection through m2e-expressing vaccines is mediated by humoral immunity and can be achieved by passive transfer of monoclonal m2e-specific antibodies prior to virus challenge (mozdzanowska et al. 2003) . one vaccine developed by w. gerhard and colleagues was based on an m2e peptide linked to universal t helper cell epitopes. others developed m2e vaccines based on papilloma virus-like particles (ginaldi et al. 1999a) , or fusion proteins linking m2e to hepatitis b virus core protein (de filette et al. 2006 ). all of these subunit vaccines elicited antibodies to m2e in animals that protected against subsequent challenge with different types of influenza a virus, and the m2e-hepatitis b virus core fusion protein vaccine has now entered a phase i trial (eurekalert 2007) . the immunogenicity of an m2e vaccine could be increased by adjuvants such as toll-like receptor 5 ligands (huleatt et al. 2008 ). in one study, passively immunized animals were challenged with influenza a viruses that were identical or that differed in their m2e sequence; animals were protected against viruses that expressed the same m2e sequence but not against subtypes with m2e variants, (fan et al. 2004) . several m2e sequences corresponding to the h1n1, h5n1, and h9n2 influenza subtypes were formulated using a liposome-based vaccine technology and evaluated as potential immunogens for the development of a "universal" influenza vaccine. mice immunized with the polyvalent liposomal m2e survived challenges with different subtypes of influenza virus, and antiserum from immunized mice provided passive protection to naïve mice (ernst et al. 2006) . one study on a dna vaccine expressing m2e fused to the nucleoprotein of influenza a virus reported increased mortality in vaccinated pigs, indicating that a poorly immunogenic vaccine (and dna vaccines are commonly poorly immunogenic, especially in larger species) may exacerbate influenza virus-associated pathology (heinen et al. 2002) . in most studies, vaccines expressed one sequence of m2e. notwithstanding, although m2e is far less variable than ha, it is not completely conserved, and mutants such as those present in recent h5n1 variants have been observed, suggesting that a universal m2e-based vaccine for influenza a virus should incorporate several common variants of m2e, including those that are present (aydar et al. 2004 ). m2e vaccines induce some protective immunity, although this protection wanes against high challenge doses of virulent virus. m2e vaccines thus need to be optimized further, either through the use of novel adjuvants, or by their incorporation into more immunogenic vaccine carriers. once this is achieved, m2e vaccines may well become part of an ideal universal vaccine for influenza virus, alleviating the need for a pandemic vaccine. under some circumstances, cd8 + t cells directed against conserved sequences of influenza a virus provide protection against heterotypic challenge; however, under other circumstances, they fail to protect. influenza virus antigens such as np and m proteins (which carry conserved epitopes of influenza a virus) and vaccine carriers that induce robust cd8 + t cell responses to such epitopes are readily available-the missing link remains a solid knowledge of what distinguishes a protective cd8 + t cell from one that is ineffective or, even worse, exacerbates disease. once this knowledge is gained, a universal influenza vaccine based on antigens that aim to induce t cell responses could be developed and deployed, either alone or in combination with an m2e-expressing vaccine. currently there is no universal vaccine for influenza virus in the industrial pipeline, and who estimates that it will take at least another 5-10 years before such vaccines become available. 4 the highly pathogenic h5n1 virus that is endemic in wild birds in asia, africa, and europe, and has spread to poultry and from there to humans, is currently viewed as a major candidate to evolve into the next pandemic subtype, through mutations that allow for efficient human-to-human transmission. several entities have started to develop vaccines based on current subtypes of avian influenza virus under (a) the assumption that h5n1 would evolve into a pandemic virus, and (b) the optimistic conjecture that the pandemic virus would have sufficient homology with currently circulating viruses to allow for cross-protective immunity ( table 3) . the asian highly pathogenic avian h5n1 virus has divided into two antigenic clades. clade 1 includes human and bird isolates from vietnam, thailand, and cambodia and bird isolates from laos and malaysia. clade 2 viruses include bird isolates from china, indonesia, japan, south korea, the middle east, europe and africa, and were primarily responsible for human h5n1 infections during 2005-2006. clade 2 is further subdivided into six subclades with a distinct geographic distribution. over time, the pool of h5n1 viruses that could potentially evolve into a pandemic form is diversifying rapidly, making it very difficult to decide on a specific virus as the basis for a vaccine. initial vaccines were developed for protection against the h5n1 subtype that was isolated from humans in hong kong in february 2003, but this virus has changed substantially, so these vaccines are now most likely no longer useful (suguitan et al. 2006) . in april 2004, who made a h5n1 prototype seed virus available to manufacturers. in august 2006, who changed the prototype and now offers three new prototype viruses. future changes of the reference virus to accommodate additional mutations are expected. developing vaccines to h5n1 based on traditional approaches was a challenging task. the highly virulent h5n1 viruses rapidly kill embryonated chicken eggs, which are used to propagate the influenza a viruses for the annual vaccines. a number of manufacturers thus started to develop cell culture systems based for example on vero or mdck cells to propagate h5n1 influenza virus. cell-grown influenza virus vaccines were tested in humans and showed immunogenicity and safety profiles that were comparable to those of egg-grown vaccines (halperin et al. 2002) . others used reverse genetics to develop reassortant viruses in which gene segments encoding ha and na were derived from highly pathogenic h5n1 virus, and all other genes were derived from the h1n1 virus a/pr/8/34, which was isolated in puerto rico in 1934 and is commonly used in animal studies (lipatov et al. 2005b; subbarao et al. 2003) . the ha gene was further modified to replace the stretch of six basic amino acids at the cleavage site that can be digested by furin (shi et al. 2007) , and the resulting virus is avirulent in chickens and can be grown readily in eggs. most vaccines for highly pathogenic h5n1 tested to date were based on inactivated or attenuated virus used with or without adjuvant subbarao et al. 2003; lipatov et al. 2005b; stephenson et al. 2005) . these vaccines achieved protection in mice, ferrets, or birds against pathogenic subtypes of influenza a virus expressing the same or a closely related ha through the induction of neutralizing antibodies. in a human clinical trial with an inactivated h5n1 influenza virus vaccine attenuated through reverse genetics and changes of the ha cleavage site to allow propagation in eggs, protective titers of neutralizing abs could be induced in volunteers after two doses of the vaccine (treanor et al. 2006) . unfortunately, the dose that was needed to induce immune responses was six times that used for current influenza a virus vaccines. in a subsequent larger trial, the vaccine was adjuvanted with aluminum hydroxide, which did not improve the vaccine's immunogenicity (bresson et al. 2006) . others reported the opposite results (leroux-roels et al. 2007 ). additional clinical trials were conducted with inactivated whole-virus vaccine, which caused seroconversion in ~80% of vaccines that received the highest vaccine dose (10 mg), again indicating that the ha of h5n1 viruses is not a potent inducer of neutralizing antibody responses (lin et al. 2006) . the immunogenicity of h5n1 vaccine could be increased by adding mf59 adjuvant (nicholson et al. 2001) . clinical trials have also been initiated with an attenuated h5n1 vaccine. 5 a number of groups have developed subunit vaccines for h5n1 virus. a dna vaccine encoding h5 provided partial protection against challenge with h5n1 virus (bright et al. 2003) , while dna vaccines encoding np or m were comparatively ineffective (epstein et al. 2002) . dna vaccine priming followed by a booster immunization with a replication-defective vector of adenovirus of the human serotype 5 (adhu5), both expressing np, augmented specific t cell responses and provided superior protection against challenge (epstein et al. 2005) . two groups explored adhu5 vectors expressing h5; they were shown to induce b and t cell responses against ha which protected against challenge with a pathogenic h5n1 virus hoelscher et al. 2006 ). nevertheless, it should be pointed out that seroprevalence rates of neutralizing antibodies to adhu5 are high in humans, especially those living in asia or africa, and that such antibodies strongly dampen antibody responses to the transgene product expressed by an adhu5 vector. fowl pox vectors (qiao et al. 2006) and alpha virus replicons (schultz-cherry et al. 2000) expressing h5 were also shown to induce protective immunity against h5n1 influenza viruses. in 2003, a h7n7 virus caused an outbreak in poultry in the netherlands during which 88 humans became infected and mainly developed conjunctivitis, while one died of complications due to pneumonia. the virus isolated from the fatal case showed a mutation in the polymerase gene that was similar to that of highly pathogenic h5n1 (munster et al. 2007) . a reassortant vaccine expressing h7 and n7 on the a/pr8 background was developed, and an inactivated adjuvanted form of this vaccine induced neutralizing antibodies and protection in mice after two doses (de wit et al. 2005) . a low-pathogenic subtype of h7n2 has been circulating in poultry in the northeastern usa since 1994, while highly pathogenic avian influenza has sporadically appeared, such as in an outbreak in chile (h7n3) in 2002, an outbreak in the united states (h5n2) in 2004, and an outbreak in canada (h7n3) in 2004 (senne 2007) . these viruses readily become pathogenic through some mutations (lee et al. 2006 ) and thus pose a pandemic threat. a reassortant vaccine has been generated against h7n2 and was shown to induce protective immunity in mice and ferrets . thus far, only one h5n1 influenza vaccine has been licensed by the united states food and drug administration (fda), while a number of other candidate vaccines against h5n1 avian influenza are in clinical trials and should be licensed in the near future. whether or not these vaccines will be protective against the next pandemic virus is unknown. initiating widespread vaccination before the actual pandemic starts would thus raise ethical questions-any vaccine, even one that is well tolerated, carries risks for the recipients. without any clear indication that h5n1 is turning into a pandemic virus, the risk of vaccination would surpass the benefit to the vaccinated individual. this was demonstrated during the swine flu vaccine debacle of 1976, when vaccination against a virus that never spread caused a serious, crippling disease in hundreds of recipients. in addition to causing harm to these unfortunate individuals, this incident continues to provide ammunition to the vocal community of vaccine opponents that seem to have forgotten the haunting images of humans disfigured by poxviruses or wards full of polio virus-infected children on iron lungs. in contrast, a universal vaccine could be given before a pandemic, as such a vaccine would prevent seasonal influenza, thus providing a tangible benefit to its recipients. scio me nihil scire (i know that i don't know) is a famous saying attributed to the greek philosopher socrates by plato. if we take a socratic view of the form and shape of the next pandemic influenza virus, mass production of a vaccine that induces protection through subtype-specific antibodies before a pandemic virus has actually evolved makes no sense. making sure that an infrastructure is in place to rapidly and efficiently respond to a pandemic is, on the other hand, a prudent approach, and global agencies such as who in concert with governments are preparing for the next pandemic. constant monitoring of evolving subtypes, new human infections and potential human-to-human spread in order to detect a pandemic at the earliest possible time is a vital task, and this been established. the sharing of virus isolates to identify potential vaccine candidates is important and requires international collaboration. indonesia, which has the highest incidence of h5n1related human deaths, initially refused to provide h5n1 samples to who in order to focus attention on their concern that while developing countries provide new viral isolates, any resulting vaccines produced by commercial companies would likely be used primarily in developed countries. by march 2007, indonesia, which was the only country that took this stance, reversed its policy. to date, global vaccine production capacity is insufficient, as seasonal influenza vaccines are only used by a small portion of the global population. a number of vaccine manufacturers have started to increase their production capacity, and it is expected that the current capacity will double by 2009. the long-term goal is to increase production capacity to three billion doses per year. it is also expected that manufacturing will commence in less developed countries. idiosyncrasies of the actual pandemic vaccine, such as the required dose and the potential need for repeat injections, will determine whether this capacity will suffice. this is being addressed by attempts to increase the immunogenicity of influenza virus vaccines through novel adjuvants. poorly growing vaccine subtypes could also offset the speed of production, and this is being tackled by developing cell-culture-based systems and through the use of reverse genetics to achieve rapid attenuation of influenza viruses. recent studies indicated that antibodies to currently circulating viruses show some cross-reactivity with h5n1. 6 these studies must be confirmed. if indeed annual vaccinations with certain types of vaccine offer some degree of heterotypic protection a broadening of seasonal influenza vaccine coverage would certainly be warranted, especially in countries that have advanced to a "pandemic alert period." progression through all of the steps of vaccine development, from preclinical trials in rodents through to clinical trials in humans and then licensing, generally takes 5-10 years. the fda, which regulates vaccine licensure in the usa, has formulated guidelines for industry for the accelerated licensure of pandemic influenza vaccines based on the induction of neutralizing antibodies to hemagglutinin in order to ensure that regulatory aspects do not hinder the rapid deployment of a pandemic vaccine. vaccines that do not contain viral hemagglutinin are not covered by these guidelines. nevertheless, even if the production of a vaccine starts on the day that a pandemic virus has been identified, it will still take 4-6 months until the very first dose of vaccine is available. other control measures are therefore needed to limit damage until the vaccines become available for everyone. in the usa, the federal government and state governments have formulated pandemic preparedness plans to be followed in the event of a pandemic. these plans not only list the responsibilities of government entities and individuals, but also address the use of limited pharmacological agents and other types of control measures. similar to vaccines, the availability of antiviral drugs is expected to be limited. antiviral drugs may slow the pandemic if used in a timely manner at the epicenter of the pandemic. they may also be extraordinarily useful for protecting persons that provide essential healthcare and for maintaining vital infrastructure. assuming a delay of at least 6-12 months before sufficient doses of vaccine are available for global mass vaccination, vaccines will have to be rationed at the beginning of the pandemic. governments will issue lists of high-priority personnel that are to be vaccinated first. although these lists vary from state to state in the usa, they typically include hospital and health department staff, emergency medical service personnel and household members, law enforcement personnel, fire fighters, medical laboratory workers, emergency management personnel, long-term care facility staff, utility workers (gas, electric, water, waste management, etc.), communications personnel, fuel and food suppliers, public transportation and air travel personnel, corrections workers, morticians/coroners/medical examiners, pharmacists, red cross field workers, us postal service staff, persons involved with vaccine production and delivery, etc. it has been suggested that once more vaccine becomes available, healthy working adults should be vaccinated before high-risk populations such as children or the elderly. it is still to be decided who will ultimately purchase the pandemic vaccinefederal or state governments, who could clearly facilitate orderly distribution, or the private sector. if the private sector carries the cost, insurance companies will have to take a stance on cost coverage, and plans will have to be developed for the uninsured. once a vaccine becomes available, other problems will arise. some of these issues are being addressed by governments, and the examples below apply to the usa. a pandemic vaccine would not be expected to undergo the vigorous safety testing typical of other vaccines. manufacturers that develop pandemic vaccines can request indemnification from the secretary of health and human services for "an activity that involves unusually hazardous risks and for which insurance is not available or sufficient to cover those risks." a vaccine will have to be distributed rapidly and in an orderly manner. the us government ruled that it may mobilize the phs commissioned corps to distribute vaccines to federal agencies with direct patient care responsibilities, or to states, tribes, and other localities through the national disaster medical system and through agreements between the federal government, states, and localities. liability protection must be put in place. the us government has ruled that federal employee administrators are covered by the federal government and could make claims through the federal tort claims act. state employees may be covered for malpractice or tort claims coverage under state law. federal contractor and private sector employees distributing the vaccine would be expected to carry malpractice insurance or they could be covered by the volunteer protection act, state good samaritan act, or state emergency compact provisions. if a person is injured following administration of a pandemic vaccine or antiviral medication in connection with his/her employment, compensation may be available under a state's worker's compensation program. for federal employees, compensation may be available under the federal employees' compensation act. assuming that vaccines will not be available at the onset of the next pandemic, other nonpharmaceutical measures are being discussed to (1) limit international spread, (2) reduce spread within national and local populations, and (3) reduce an individual person's risk for infection. influenza viruses are typically shed 24-48 h before the onset of disease, and virus titers peak within the first three days after onset of symptoms and then decline by day 7-8. it is possible but not yet proven that the virus spreads by shedding a small amount of virions before the first symptoms occur, which could markedly reduce the effectiveness of most quarantine measures. infection occurs predominantly via droplets formed by coughing or sneezing individuals. infection by aerosolized virus is less common. the virus can also be transmitted via infected hands or surfaces. the wearing of masks and the employment of appropriate sanitizing measures to clean hands and infected surfaces are thus useful actions for protecting an individual (jefferson et al. 2008) . it is thought that temporary protection of populations may in part be achieved by implementing quarantine measures. in the pandemic of 1918, some island countries enacted maritime quarantines (markel et al. 2007 ). australia and madagascar were able to delay the start of the pandemic by several months, while samoa and new caledonia remained completely free of the pandemic. quarantine measures were attempted on land, but they were unsuccessful. quarantine was tried again in 1957, but it largely failed. quarantine was very successful in stopping the sars epidemic of 2003 (hsieh et al. 2007 ). the sars virus has a longer incubation time than influenza virus, and peak virus titers are not reached until several days after the onset of symptoms. isolating cases thus proved an effective way to prevent the further spread of sars, but it is unlikely to work against influenza virus. who recommends exit screening for international travelers that leave countries that are affected by an influenza virus pandemic. it is unclear if and to what extent this may delay the spread of the virus. however, any delay would be useful in allowing extra time for vaccine production. the us government has issued an executive order adding potentially pandemic influenza viruses to the list of quarantinable diseases, which empowers the centers for disease control and prevention (cdc) to detain, medically examine, or conditionally release individuals that are reasonably believed to be carrying a communicable disease. the intent of this order is to enable the united states to respond efficiently and effectively in the case of an outbreak by pandemic influenza viruses. this order gives legal authority to the department of health and human services to isolate a passenger arriving on board an international vessel that show evidence of infection with a novel influenza virus, even if that passenger refuses to cooperate. the federal government (such as the cdc) generally defers to the state and local health authorities in the use of their own quarantine powers. state-implemented interventions would likely include the isolation and treatment of infected individuals, voluntary home quarantine for members of the households of infected individuals, the closure of schools and universities, and the encouragement of social distancing through the cancellation of large public gatherings. who estimates that in the worst case scenario more than 70 million people could die as a consequence of the next influenza virus pandemic. other estimates are higher: 180-360 million deaths. to put this number into perspective, 360 million is approximately the total population of south america, or half of the population of europe. death tolls will primarily depend on the virulence of the next pandemic-current clades of highly pathogenic h5n1 virus kill >60% of those infected. it would also depend on the ease with which the virus is transmitted between humans, and last but not least on the effectiveness of control measures. late detection of a pandemic, which could evolve in a war-ravaged country that lacks surveillance, would shorten the time interval available for the development of a vaccine. vaccine production could be further delayed by problems with the seed virus, such as toxicity toward eggs or cell substrates, poor immunogenicity of the vaccine, or unacceptable reactogenicity. the modern world, with its high degree of social and economic interdependency, has not yet experienced a major pandemic that disrupts crucial aspects of local and global infrastructure. lack of available workers and restricted movement could threaten essential services, and the failure of any one system could trigger others to fail too, causing cascading breakdowns. highly pathogenic h5n1 influenza viruses have been circulating for more than ten years and many have come to believe that they are unlikely to jump species. although this may well be the case, the consequences of relaxing our efforts to prepare for the next pandemic could be horrific. in 1969, when smallpox virus was on the brink of extinction and polio was disappearing from developed countries, the us surgeon general, william stewart, told congress that it was time to "close the books on infectious diseases." unfortunately, congress listened and shifted federal funding from microbiology/virology to cancer and cardiovascular diseases. ever-increasing liability costs made the industry more and more reluctant to stay involved in vaccine production, and the number of companies that produce vaccines has now become so limited that annual vaccine shortages are common (markel et al. 2007 ). infectious agents have continued to take a major toll on human lives. since 1969 more than 30 new microbes have emerged, such as hiv-1, which has claimed over 22 million lives thus far. even old microbes such as influenza virus continue to take a toll on human lives; for example, in the usa alone, seasonal influenza causes over 200,000 hospitalizations and over 30,000 death each year. the world will experience a new influenza virus pandemic. no-one can predict when it will happen, where it will start, what virus will cause it, and what the global and local impacts will be. the only aspect of the next pandemic we can predict with certainty is that it will happen. complacency, not only in the usa but worldwide, has weakened the infrastructure for efficiently combating newly emerging infectious agents, and this infrastructure needs to be rebuilt. communications with the public must be improved globally in a manner that informs accurately without alarming unduly. it is clear that this has not yet been achieved, as exemplified by a recent h5n1 outbreak in west bengal in india, 7 where children were reported to have unprotected contact with birds that died due to infection with h5n1. our continued lack of knowledge about the very basic question of the immunobiology of influenza viruses and the efficacy of vaccines in different cohorts is mind-boggling. we still do not fully understand correlates of protection against influenza virus, and debates on the role of cd8 + t cells in providing protection are continuing. this knowledge needs to be generated, especially in order to enable the development of a universal influenza virus vaccine, the "holy grail" that could prevent future influenza virus pandemics. baculovirus induces an innate immune response and confers protection from lethal influenza virus infection in mice immunization with influenza a np-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses genetic analysis of influenza a virus (h5n1) derived from domestic cat and dog in thailand cell-mediated immune responses to influenza virus antigens expressed by vaccinia virus 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pandemic virus a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission human illness from avian influenza h7n3, british columbia in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding innate imprinting by the modified heat-labile toxin of escherichia coli (ltk63) provides generic protection against lung infectious disease the recovery of mice from influenza virus infection: adoptive transfer of immunity with immune t lymphocytes fine specificity and sequence of antibodies directed against the ectodomain of matrix protein 2 of influenza a virus immunosenescence and germinal center reaction a homogeneous population of lymphokineactivated killer (lak) cells is incapable of killing virus-, bacteria-, or parasite-infected macrophages key: cord-003523-byxuruk1 authors: fritsch, annemarie; schweiger, brunhilde; biere, barbara title: influenza c virus in pre-school children with respiratory infections: retrospective analysis of data from the national influenza surveillance system in germany, 2012 to 2014 date: 2019-03-07 journal: euro surveill doi: 10.2807/1560-7917.es.2019.24.10.1800174 sha: doc_id: 3523 cord_uid: byxuruk1 introduction: recent data on influenza c virus indicate a possible higher clinical impact in specified patient populations than previously thought. aim: we aimed to investigate influenza c virus circulation in germany. methods: a total of 1,588 samples from 0 to 4 year-old children presenting as outpatients with influenza-like illness (ili) or acute respiratory infection were analysed retrospectively. the samples represented a subset of all samples from the german national surveillance system for influenza in this age group in 2012–14. the presence of influenza c virus was investigated by real-time pcr. for positive samples, information on symptoms as well as other respiratory virus co-infections was considered. retrieved influenza c viral sequences were phylogenetically characterised. results: influenza c viral rna was detected in 20 (1.3% of) samples, including 16 during the 2012/13 season. the majority (18/20) of influenza c-positive patients had ili according to the european union definition, one patient had pneumonia. viruses belonged to the c/sao paulo and c/kanagawa lineages. most (11/20) samples were co-infected with other respiratory viruses. conclusion: our data are the first on influenza c virus circulation in germany and notably from a european national surveillance system. the low detection frequency and the identified virus variants confirm earlier observations outside a surveillance system. more virus detections during the 2012/13 season indicate a variable circulation intensity in the different years studied. influenza c virus can be considered for ili patients. future studies addressing its clinical impact, especially in patients with severe disease are needed. influenza viruses are a major threat to human health and are therefore in the focus of national and international health authorities. among these, influenza virus types a and b are the main considered, as they cause annual epidemics with high morbidity and considerable mortality [1] . in contrast, influenza c virus has been regarded as a pathogen of minor relevance, causing mild or clinically unapparent disease [2, 3] . nevertheless, in recent years, detections of influenza c in hospitalised young children with (severe) lower respiratory tract disease were reported [4] [5] [6] [7] [8] [9] . thus, the clinical and epidemiological significance of this virus species might have been underestimated and needs to be reassessed. in europe, the burden of influenza c virus infection in children and adults is largely unknown, as no systematic surveillance data are available. the few studies published mainly focus on clinical data, mostly from hospitalised children [4, 9, 10] . in germany, no surveillance data and no sequence information on circulating influenza c viruses have ever been reported. therefore, we decided to search for influenza c in our outpatient sample collection assembled for the purpose of influenza virus surveillance in germany. as young children are described to have the highest infection rates [6, 7, 11] , we confined our study to the 0-4 year-old age group. we furthermore sequenced the haemagglutinin esterase (he) gene from influenza c-positive samples to phylogenetically characterise the detected viruses. all samples were collected from practitioners participating in the national influenza surveillance, who are distributed over the complete german territory and represent a statistically valid proportion of the german population [12] . these practitioners continuously collect nasal or throat swabs from non-hospitalised patients presenting with symptoms of influenza-like illness (ili) according to the european union (eu) definition or an acute respiratory infection (ari). an ili case is defined by a sudden disease onset with at least one of four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of three respiratory symptoms (cough, sore throat, shortness of breath) [13] , while ari is an acute respiratory disease with at least one of the four following symptoms: fever, cough, rhinorrhoea or sore throat. the samples are sent to the german national influenza centre, accompanied by a completed questionnaire on patient characteristics, sampling date, disease symptoms, influenza vaccination status and therapeutic intervention i.e. antiviral treatment. they are routinely analysed for influenza virus types a and b, human respiratory syncytial virus (rsv) as well as -since april 2013 -human adenovirus (adv), metapneumovirus (hmpv) and rhinovirus (hrv). all samples are stored at -80 °c afterwards. for this study, a subset of 1,588 samples (66.9%) was selected from a total number of 2,377 samples taken from children ≤ 4 years of age in the years 2012-14 as described in the supplementary file (supplement s1). briefly, at least every second sample in a chronological order was retrospectively analysed for influenza c virus. to extend the basis for the co-infection data, all influenza c-positive samples were additionally tested for human parainfluenza viruses types 1-4 and coronaviruses oc43, nl63, hku1 and 229e. positive samples taken before april 2013 were furthermore retrospectively examined for adv, hmpv and hrv. the conduct of a sentinel surveillance is covered by german legislation ( §13, §14, protection against infection act). the german national surveillance of influenza and other respiratory viruses was furthermore approved by an ethical committee of the charitè berlin (application number ea2/126/11). additionally, for all samples a written consent was given for their inclusion in research studies. all analyses were done with pseudonymised data. after their arrival in the laboratory, 3ml of cell culture medium (minimum essential medium (mem) with for sequence analyses, cdna was synthesised with the accuscript hi-fi reverse transcriptase (agilent, santa clara, us) and a primer that binds to the conserved 3' end of the rna gene segments (uni11, see table 1 ). assay validation was performed with synthetic double stranded dna strings (gblocks; idt, skokie, us) in singleplex as well as duplex format (including the internal control fcv) on 96-well as well as 384-well plates. for the determination of the linear detection range and the correlation (r 2 ) of quantification cycle (c q ) values, a 10-fold dilution series (10 6 -10 1 copies per reaction) was examined in duplicates. pcr efficiency was calculated by inserting the slope value of the standard curve into the formula e = 10 (-1/slope)-1 . the limit of detection (lod) was established as 95% detection probability, calculated by probit analyses of the results of a 10-fold examination of low copy numbers (50-0.1 genome equivalents per reaction) applying the ibm spss statistics 20 software. for intraassay precision, gblocks were examined sixfold in a single run, while for interassay precision the intraassay data were extended by two additional runs with double reactions. all reproducibility runs were performed on consecutive days in independent experiments, and precision was described as standard deviation of the observed c q values. conventional pcr for sequence determination of the he gene was carried out in a total reaction volume of 50µl. the reaction contained 1x extaq buffer, 1.25mmol/l dntp (thermo fisher scientific, waltham, us) with dutp (ge healthcare, chicago, us), 1.25u extaq polymerase (takara, kusatsu, japan), 500nm primers (metabion, planegg, germany) as listed in table 1 , and 5µl of the prediluted cdna. alternatively, the superscript monthly distribution in 0-4 year-old children of (a) the number of samples tested for influenza c, as well as testing coverage among samples received by table 1 ) were used for amplicon sequencing in cases where the nested pcr primers did not yield a sequence spanning the complete amplicon. all he sequences were processed and assembled in the geneious software before their deposition at the global initiative on sharing all influenza data (gisaid; www. gisaid.org) database (epi1183982-epi1183998). the applied amino-acid numbering includes the signal peptide. all he sequence analyses were performed with geneious version 10.0.5. multiple sequence alignments were compiled on the basis of the mafft algorithm. the n-terminal sequences including the signal peptide sequence (mffslllmlgltea [16] ) as well as the c-terminal region with incomplete sequence information (last 13 nt including the stop codon) were excluded. the alignment for phylogenetic analyses thus covered the nt 43 to 1,955 of the complete coding sequence and was calculated including reference sequences downloaded from the gisaid database (see supplement s2). maximum likelihood trees were constructed applying the hky85 model and the spr tree topology search. branching reliability was estimated by performing 1,000 bootstrap replicates. trees were manually edited in corel draw x6. a qpcr assay for the detection of influenza c viruses was established as singleplex as well as duplex qpcr including our routine internal control fcv. the assay proved to be a robust and sensitive tool and furthermore did not show any cross-reactivity to a variety of viral respiratory pathogens and to human genomic dna (validation results summarised in table 2 ). the duplex qpcr approach was applied to retrospectively examine 1,588 throat or nasal swabs, of which 1,570 samples gave valid qpcr results, i.e. yielded either an influenza c or a fcv signal (or both) in duplex qpcr runs. twenty samples (1.3%) were found positive for influenza c virus rna, with c q values ranging from 19 to 39. the positive samples predominantly were taken between october 2012 and april 2013, reaching an average positivity rate of 2.6% (16/604) in these months (0.7-7.1% per month, figure 1 ). outside of this particular winter season, viruses were identified only sporadically with detection rates of 1.2% (3/249, january-april 2012) or 0.2% (1/414, october 2013 -april 2014 . no virus detection was achieved from may to september of any year studied. also, no particular age distribution could be observed ( figure 2 ). all influenza c-positive samples were additionally examined by qpcr to identify other respiratory viruses. more than half of the influenza c-positive patients (11/20; 55%) proved to be co-infected with diverse other respiratory pathogens (table 3) , with influenza c c q values covering the complete range of 19 to 39. all 20 patients with influenza c virus infection reported fever and cough. fifteen patients reported a maximum temperature between 38.5 °c and 40.2 °c, while for the remaining five patients the maximum temperature was not provided. additionally, a sudden disease onset (18/20) , rhinitis (18/20) , sore throat (8/20) and muscular pain and/or headache (6/20) were predominant symptoms. clinical signs of pneumonia were reported for one patient with an influenza c c q value of 25, but also low amounts of influenza a(h3n2) were detected in this sample. in patients with a sole influenza c virus infection, the sudden disease onset (7/9), the maximum fever (38.9 °c -40.2 °c in 6 patients), rhinitis (8/9), sore throat (4/9) and muscular pain and/or headache (2/9) were reported in similar proportions. the sequencing of the he gene was achieved for 17 samples, of which three yielded only partial sequences. two of the incomplete sequences covered a consecutive stretch of 1,071 and 1,218 nt, respectively, while the three incomplete sequences were only characterised based on the nt homologies to other sequences. the two fragments of sample 13-04691 (775nt, 557nt) are 100% identical to our sample sequence 13-04332, which belongs to the c/victoria/2/2012 subgroup of the c/sao paulo clade. similarly, sample sequence 13-05486 (1,037nt) is 100% identical to the sequence of c/sao paulo samples 13-04022 and 13-05206, which group into the c/miyagi/6/2014 subgroup of the c/aichi/1/99 subclade. sample sequence 13-00631 has a similarity of > 99.2% to the same sao paulo lineage cluster, while the similarity to c/victoria/2/2012 (98.1%) and the prototype strain sequences for the other five he lineages is lower (≤ 95.4%). although influenza c virus was discovered 70 years ago, there is only little knowledge on the biology and epidemiology of this virus type. some studies indicated a low clinical impact with only mild symptoms [2, 3, 17] , and in spite of a high seroprevalence in the population, virus detections were rare [6, 11, 18] . these findings led to the conclusion that influenza c infection is common, but clinically inapparent or too mild to require a visit to a doctor [2] . additionally, the low detection rate may be in part due to the fact that in earlier times virus diagnostics were mainly based on virus culture, which is difficult for influenza c [6, 19] and necessitates conditions that differ from influenza a and b virus cultivation [20] . as a consequence, influenza c virus diagnostics were restricted to specialised laboratories and correspondingly rare [4] . with the introduction of molecular methods, influenza c has been increasingly included into studies on respiratory pathogens and clinical diagnostics. thereby, the low detection rates in the general population were confirmed, but a higher clinical impact for paediatric patients was indicated, as influenza c was described to also cause lower respiratory tract disease [6] [7] [8] [9] [10] . in a 6-month prospective study (december 2009 -may 2010 including japanese children with communityacquired pneumonia, bronchiolitis or bronchitis, influenza c infection was identified even with a prevalence approximating those of influenza a or hmpv [5] . further studies, mostly in children, described the symptoms of influenza c infection to be indistinguishable from influenza a and b infections [4, 6] , although the maximum body temperature may be lower and the fever shorter compared with influenza a [6, 21, 22] . in finnish military recruits, influenza c virus caused common cold-like symptoms, but occasionally resulted in pneumonia and bronchitis [3] . for europe, only little information on influenza c circulation has been published. in adults, a seroprevalence of ca 70% and more was found in france [23] , finland [3] , and united kingdom [17] . applying pcr on samples from all age groups, a virus detection rate of ≤ 1% was reported for normandy/france [10] , scotland [24] and spain [21] , but higher detection rates of 3.5 -4.2% were found in two adult studies from finland [3, 25] . outside europe, a similar seroprevalence as well as comparable detection rates have been described for australia, canada, japan, nigeria, the philippines, peru and the us [6] [7] [8] 11, 15, 18, 19, 22, [26] [27] [28] . in view of the lack of knowledge on influenza c virus circulation in germany, we decided to generate the first systematic data on the basis of our national influenza virus surveillance. we chose to examine the age group of 0-4 years, as young children have been shown to have the highest infection rates [6, 7, 11, 19] . we analysed a representative subset of the 2,377 samples received in this age group between 2012 and 2014 (52.1 -100% of all samples in the corresponding month). first, we validated a previously published qpcr [15] and duplexed it with our routine internal control, fcv. in an extensive validation effort according to international standards [29] , we found the singleplex as well as the duplex format to perform with high sensitivity, specificity and precision. we therefore applied it to our sample compilation and identified influenza c rna in 20 of 1,570 samples with valid qpcr results (1.3%). the vast majority of virus detections (16 of 20) was found in samples that were collected between october 2012 and april 2013, signalling a more pronounced virus circulation during these months with positivity rates of up to 7.1% (5/70) in november 2012. as these samples were collected in 10 of 16 german federal states, virus circulation was not confined to a region, but widespread, maybe even nationwide. the virus prevalence however was markedly lower during the other winter seasons observed in this study, and no virus could be detected during the summer months. this absence of summer circulation is in congruence with reports from japan, france, finland and spain [3, 4, 6, 10, 25] , but is in contradiction to a report from catalonia in spain, in which the majority of positive samples were taken in august and september of the observed time span [21] . an upsurge of influenza c virus circulation in the spring of 2013 was also observed in the philippines [8] , but did not occur in japan, from where virus circulation in even numbered years was reported, including the years 2012 and 2014 [11, 30] . however, a biennial pattern of virus circulation with increased or time-shifted profile has also been described for other respiratory viruses in germany [31, 32] and therefore is conceivable, but remains open in our study due to the short study period, which presents a limitation. in total, the proportion of influenza c-positive patients was small, but within the expected range. it needs to be emphasised though, that the obtained overall positivity rate is largely based on only few months during the winter season 2012/13 with substantial virus circulation in our study population. because of our limited access to clinical data, only few conclusions can be drawn with regard to the clinical relevance of influenza c virus. the vast majority of patients (18/20) carrying the virus fulfilled the eu ili definition. although, due to our study design, there may be a bias to ili cases during periods of influenza a and b virus circulation, our findings are in concordance with other studies, in which ili was described for the majority or all of influenza c infected patients [21, 27] . bronchitis or bronchiolitis was not reported for any patient, but one child (infected also with an influenza a virus) presented with symptoms of pneumonia. however, the proportion of pneumonia in our influenza c-positive samples does not differ considerably from that of our complete sample collection of this age group spanning the years 1999 to 2017 (data not shown). in our ambulant setting, we thus do not see an indication for an accumulation of lower respiratory tract disease in influenza c infected patients, but an influenza-like clinical presentation is common. interestingly, a substantial share of influenza c-positive samples showed co-infection with other pathogens, as reported also in other studies [3, 4, 15, 27] . in these cases, the cause for ili symptoms cannot clearly be attributed to influenza c. yet, we used qpcr assays with comparable performance characteristics (lod and efficiency), so that a comparison of the obtained c q values can be semiquantitatively interpreted for the different pathogens within one sample. in our study, the majority of co-infected samples (6/11; 54 .5%) exhibited the highest viral load for influenza c, including the pneumonia case for whom it was ca 10,000-fold higher than that of influenza a at the time point and the sample site examined. in four samples, the influenza c c q was close to the detection limit (≥ 35) and thus influenza c was presumably of minor relevance. repetitive sampling from the same patients and continuous parallel assessment of the patients clinical presentation could clarify the role of the single pathogens in the disease course, but is not included in our routine influenza surveillance system. therefore, we cannot judge on the temporal dynamics of virus replication and the clinical impact of each virus detected. due to the slow evolution and thus high antigenic homology of influenza c virus [11] , we decided to characterise the german sequences only on the basis of the he gene sequences. the he glycoprotein has a variety of functions in the viral replication cycle and greatly determines the antigenicity of the virus [33] . based on antigenic and phylogenetic characteristics of this protein, distinct virus lineages have been described that were named after their prototype strains c/taylor/1233/47, c/kanagawa/1/76, c/mississippi/80, c/aichi/1/81, c/ yamagata/26/81 and c/sao paulo/378/82 [30, 34] . all influenza c lineage clusters are comprised of isolates from a multitude of continents, indicating a global circulation of virus lineages [34] . however, four lineages seemingly disappeared (c/taylor, c/aichi, c/ mississippi, c/yamagata), and only the c/kanagawa and c/sao paulo lineage have been detected within the last decade [8, 9, 19, 21, 27, 30, 34] . from our positive samples, a total of 17 partial and near full-length he sequences could be generated. we almost exclusively detected c/sao paulo lineage viruses, and only two c/kanagawa viruses were identified. our c/ sao paulo sequences add to both lineage subclades described by matsuzaki et al. and represented by c/ aichi/1/99 and c/victoria/2/2012 [30] . a total of 11 sequences (9 complete, 2 incomplete) group into the c/aichi/1/99 subclade and were sampled between march 2012 and april 2013, while three additional sequences (2 complete, 1 incomplete) group into the c/ victoria/2/2012 subclade and were sampled between november 2012 and march 2013. thus, viruses of both subclades co-circulated during the 2012/13 winter season. in contrast, the two c/kanagawa lineage viruses were sampled in march 2012 and april 2014, thus outside of the period with increased infection rates. they form a distinct cluster within the c/kanagawa clade, most closely related to c/miyagi/9/96. both c/ kanagawa viruses are almost identical to each other showing a nt homology of 99.1%, although they were sampled with a 2-year distance. their closest neighbour, c/miyagi/9/96 even has a homology of 99.5% and 99.3% on the nt level. this further supports the described genetic stability of this virus type compared with influenza a and b viruses [30] , possibly also reflecting their antigenic properties. to summarise, our study is the first report on influenza c circulation in the context of a nationwide outpatient influenza surveillance system in europe. we found influenza c in a proportion of samples that was in accordance with previous reports. an increased and widespread virus circulation was observed during the winter and spring months of 2012/13, with viruses predominantly belonging to c/aichi/1/99 subclade of c/ sao paulo lineage viruses. infected patients showed symptoms of ili including upper as well as lower respiratory tract infection, although its association to the observed clinical symptoms remain uncertain in the majority of cases due to the identified co-infections. 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diagnostics confirm that influenza c is an uncommon cause of medically attended respiratory illness in adults minimum information necessary for quantitative real-time pcr experiments genetic lineage and reassortment of influenza c viruses circulating between 1947 and 2014 human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany genetic variability of group a human respiratory syncytial virus strains circulating in germany from 1998 to hemagglutinin-esterase-fusion (hef) protein of influenza c virus analyses of evolutionary characteristics of the hemagglutinin-esterase gene of influenza c virus during a period of 68 years reveals evolutionary patterns different from influenza a and b viruses we thank susi hafemann, nathalie tollard and uwe kozian for excellent technical assistance. we also acknowledge all laboratories that contributed influenza c virus sequences to the gisaid database and thereby enabled our analyses. none declared. af: screening and sequencing of patient samples, manuscript preparation; bs: design of study, manuscript preparation; bb: design of study, data analyses, manuscript preparation. this is an open-access article distributed under the terms of the creative commons attribution (cc by 4.0) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord-000390-qav5okgk authors: omer, saad b.; goodman, david; steinhoff, mark c.; rochat, roger; klugman, keith p.; stoll, barbara j.; ramakrishnan, usha title: maternal influenza immunization and reduced likelihood of prematurity and small for gestational age births: a retrospective cohort study date: 2011-05-31 journal: plos med doi: 10.1371/journal.pmed.1000441 sha: doc_id: 390 cord_uid: qav5okgk background: infections during pregnancy have the potential to adversely impact birth outcomes. we evaluated the association between receipt of inactivated influenza vaccine during pregnancy and prematurity and small for gestational age (sga) births. methods and findings: we conducted a cohort analysis of surveillance data from the georgia (united states) pregnancy risk assessment monitoring system. among 4,326 live births between 1 june 2004 and 30 september 2006, maternal influenza vaccine information was available for 4,168 (96.3%). the primary intervention evaluated in this study was receipt of influenza vaccine during any trimester of pregnancy. the main outcome measures were prematurity (gestational age at birth <37 wk) and sga (birth weight <10th percentile for gestational age). infants who were born during the putative influenza season (1 october–31 may) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature compared to infants of unvaccinated mothers born in the same period (adjusted odds ratio [or] = 0.60; 95% ci, 0.38–0.94). the magnitude of association between maternal influenza vaccine receipt and reduced likelihood of prematurity increased during the period of at least local influenza activity (adjusted or = 0.44; 95% ci, 0.26–0.73) and was greatest during the widespread influenza activity period (adjusted or = 0.28; 95% ci, 0.11–0.74). compared with newborns of unvaccinated women, newborns of vaccinated mothers had 69% lower odds of being sga (adjusted or = 0.31; 95% ci, 0.13–0.75) during the period of widespread influenza activity. the adjusted and unadjusted ors were not significant for the pre-influenza activity period. conclusions: this study demonstrates an association between immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. however, no associations were found for the pre-influenza activity period. moreover, during the period of widespread influenza activity there was an association between maternal receipt of influenza vaccine and reduced likelihood of sga birth. please see later in the article for the editors' summary infections during pregnancy have the potential to adversely impact birth outcomes and fetal growth and development. respiratory infections-particularly those resulting in pneumonia-have been associated with low birth weight and increased risk of preterm birth [1, 2] . influenza virus is an important respiratory pathogen that causes substantial burden of diseaseincluding morbidity and mortality among pregnant women, with greater risk of influenza-related morbidity among pregnant women than among non-pregnant and postpartum women [3] . vaccination against influenza is the most effective tool to prevent morbidity and mortality due to influenza. influenza vaccination during pregnancy provides protection for the infant as well as the mother. a randomized controlled clinical trial in bangladesh demonstrated that vaccination of pregnant women with the inactivated influenza vaccine had 63% effectiveness in reducing laboratory-confirmed influenza in their infants [4] . since there is evidence of adverse fetal/newborn outcomes after infection during pregnancy [5, 6] , including influenza infection [2] , it is plausible that influenza vaccination in pregnancy could mitigate adverse birth outcomes such as prematurity and small for gestational age (sga) births. the potential impact of maternal influenza immunization on birth outcomes could have important public health implications for developed as well as developing countries and may be of particular interest during influenza pandemics. the objective of this study was to evaluate whether there is an association between receipt of inactivated influenza vaccine during pregnancy and birth outcomes using a multiyear, populationbased, state-wide dataset from the state of georgia (in the united states). we conducted a retrospective cohort analysis of a large surveillance dataset. the primary exposure variable was receipt of inactivated influenza vaccine during any trimester of pregnancy by mothers of infants born between 1 we analyzed pregnancy-and birth-related data from the georgia pregnancy risk assessment monitoring system (prams) and influenza activity information compiled by georgia for the council of state and territorial epidemiologists (cste) reports. prams is a multistate surveillance system managed by the us centers for disease control and prevention and state health departments, including the georgia department of community health [7, 8] . the prams sample is drawn monthly from the state's birth file and includes resident women who have experienced a live birth. georgia prams oversamples women based on race (black) and birth weight (,2,500 g). we adjusted for the oversampling by using analysis weights described elsewhere in the methods section. prams data are collected primarily by mailed questionnaires, with telephone follow-up among nonresponders. the georgia prams dataset contains information on maternal influenza vaccination (during any trimester); maternal attitudes, behaviors, and experiences before, during, and shortly after pregnancy; and newborn birth certificate data, including birth date and birth weight. prematurity was defined as clinical estimate of gestational age (at birth) as less than 37 wk. newborns with birth weight below the10 th percentile for their gestational age were considered to be sga. we used gender-specific reference values for the us published by oken at al. [9] to assign sga (yes/no) categories. in order to model the impact of the intensity of influenza activity in georgia on the association between maternal influenza vaccination and birth outcomes, we used a modified version of the cste report categories of influenza activity. cste reports assess the spread of influenza within each state for each week based on lab-confirmed and syndromic data [10] . the influenza activity is considered local if there are influenza outbreaks or an increase in cases of influenza-like illness in a single region of the state along with recent identification of laboratory-confirmed influenza from that region. in the case of influenza outbreaks or increases in influenza-like illness with recent laboratory-confirmed influenza in at least two but fewer than half the regions of the state, the influenza activity level is considered to be regional. if the influenza outbreaks or increased numbers of influenza-like illness cases (plus laboratory-confirmed cases) are reported in at least half of the regions of the state, the influenza activity is considered to be widespread ( figure 1 ). we defined the pre-influenza period as the period between the start of the putative influenza season (october 1) and the beginning of local influenza activity (per cste reports). the pre-influenza period is characterized by the availability of the vaccine and the absence of influenza activity ( figure 1 ). the definition for the pre-influenza period was similar to the one used by other authors [11, 12] . stratified analysis was performed for the overall study period, the putative influenza season (1 october-31 may), the preinfluenza period (during the putative influenza season), the period of at least local activity, the period of regional activity, and the period of widespread activity ( figure 1 ). in georgia prams, live births to black women and those that are low birth weight are oversampled in order to provide enough statistical power for stratified analyses for groups of interest relevant to prams objectives. for combined analyses, in accordance with standard practice, analysis weights developed by the centers for disease control and prevention [13] were used to adjust for the sample design (e.g., to account for oversampling of the two risk groups) and differential response rate across groups. we used logistic regression to evaluate the association of maternal influenza vaccine and (a) prematurity and (b) sga. linear regression was used to evaluate the statistical significance of differences between infants born to vaccinated and unvaccinated women in terms of mean gestational age at first antenatal visit and mean birth weight. confounding due to differences between the vaccinated and unvaccinated individuals is a recognized issue in observational studies of influenza vaccination-particularly studies evaluating vaccine effectiveness for reducing all-cause mortality in the elderly [12] . one approach to account for confounding is to choose a period when the vaccine was available but the influenza virus was not circulating as the ''control'' pre-influenza period [11, 12] . in the pre-influenza period, there should be no vaccine effect, and observed effects during this period are assumed to be due to confounding. therefore, as our primary strategy for confounder adjustment, we identified a group of covariates that would move the odds ratios (ors) of association between maternal influenza immunization and birth outcomes during the pre-influenza period to 1.0 (i.e., no effect), hence arriving at a set of covariates that could effectively control for confounding due to the differences between the vaccinated and the unvaccinated women in analyses of all influenza activity periods. we selected covariates for the separate multivariate models for each of the birth outcomes (i.e., prematurity and sga) using a modified version of the approach described by jackson et al. [11] and nelson et al. [12] . briefly, variables for each birth outcome were evaluated for potential confounding by selecting the covariates that, in bivariate models, modified the association between maternal influenza vaccine and the birth outcome and moved the or towards 1. from this initial group of variables, we arrived at a parsimonious model by sequentially dropping each covariate and observing a change in or of the association between maternal immunization and the birth outcome. we excluded the variable whose removal moved the or the most towards 1. if dropping a covariate resulted in moving ors away from 1 and a change in magnitude of less than 1%, we removed the covariate that caused the least change and then repeated the process. if the change was more than 1%, we considered the current set of covariates as the smallest group of variables required to account for confounding. in order to address the possibility that the confounders in the preinfluenza period were different from the confounding factors in the influenza activity period, we also developed secondary multivariate models using a traditional analytic approach for confounder adjustment. in these secondary models, the covariate list (for both the pre-influenza-period-based and traditional adjustment) was based on evidence in the literature regarding associations with birth outcomes and availability of data in the georgia prams dataset. the covariate list included the following: gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12 th grade, mother's marital status, and maternal weight before pregnancy. the ors for association between the different covariates and receipt of influenza vaccine were computed using logistic regression. we also computed the population prevented fraction of prematurity for the various periods of influenza activity using the formula:p p c (1{or) p p c (1{or)zor wherep p c is the proportion of cases vaccinated and or is the odds ratio approximating relative risk (we verified this assumption for our data). the formula, based on the approach described by kleinbaum et al. [14] , is suitable for computing the population prevented fraction when adjusted measures of association are used. the population prevented fraction for a vaccine estimates the reduction in an outcome given the efficacy/effectiveness of the vaccine and the specific vaccine coverage. we used stata version 10 (stata corporation) for statistical analysis. identified associations were evaluated for statistical significance at a = 0.05 using two-tailed tests, and taylor series linearization methods [15] were used to estimate variance. the study was reviewed and approved by the emory university institutional review board and the georgia department of human resources institutional review board. a total of 4,326 women (and their newborns) were included in georgia prams during the 28-mo study period. influenza vaccine information was available for 4,168 (96.3%) women in prams (study population); of these, 578 women (14.9% [weighted]) had received the influenza vaccine during pregnancy. the vaccine coverage was 19.2% (weighted) among mothers of infants born during the putative influenza season. out of the 122 wk of the study, at least local influenza activity was detected during 27 wk (22.1%)-including widespread activity in 8 wk ( figure 1 ). there were 1,547 premature newborns (10.6% [weighted]) and 1,186 newborns with sga (11.2% [weighted]) in our study population. the odds of having received influenza vaccine during pregnancy were lower (a) for black women than for all other ethnic groups (or = 0.78; 95% ci, 0.62-0.98), (b) for women with diabetes (or = 0.30, 95% ci, 0.10-0.95), and (c) for mothers who used multivitamins in pregnancy (or = 0.64; 95% ci, 0.50-0.83) ( table 1) . insured women were more likely to have received the influenza vaccine (or = 1.41; 95% ci, 1.09-1.81). likelihood of having received an influenza vaccine during pregnancy was not associated with any of the other binary covariates (table 1) . similarly, gestational age at first prenatal visit was similar for vaccinated women and unvaccinated women (mean: 5.2 wk versus 5.3 wk; p = 0.23), and maternal weight before pregnancy was similar for vaccine recipients and non-recipients (mean: 68.3 kg versus 68.1 kg; p = 0.88). based on the approach of identifying covariates that produce adjusted ors of 1 during the pre-influenza period, the group of covariates in the prematurity multivariate models included gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. the covariates in the primary multivariate models for sga included maternal age less than 19 y, maternal medical risk factors, labor/delivery complications, hypertension (treated or untreated), birth defects, and history of alcohol use during pregnancy. in unadjusted models, and in models with covariates based on lack of effects in the pre-influenza season, infants born during the putative influenza season (1 october-31 may) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature than infants of unvaccinated mothers born in the same period (adjusted or = 0.60; 95% ci, 0.38-0.94). the magnitude of effect of maternal influenza vaccine on prematurity increased during the period when there was at least local influenza activity in any part of the state (adjusted or = 0.44; 95% ci, 0.26-0.73) and was the highest for those born during the period of widespread influenza activity (adjusted or = 0.28; 95% ci, 0.11-0.74) ( table 2 ). the adjusted and unadjusted ors were not significant for the association between receipt of maternal influenza vaccine and prematurity for the pre-influenza activity period or for the analysis without consideration of influenza activity ( table 2) . compared with newborns of unvaccinated women, those born to vaccinated mothers had lower odds of sga (adjusted or = 0.31; 95% ci, 0.13-0.75) during the period of widespread influenza activity (table 3) . although the magnitude of the ors of the association between maternal influenza vaccine and sga generally increased with the increase in the intensity of influenza activity, these associations were not statistically significant (other than for the period of widespread activity) ( table 3 ). the associations observed in the secondary multivariate models using the traditional approach were qualitatively similar to the associations in the primary multivariate models (tables 2 and 3) . newborns of vaccinated women were, on average, 96.7 g heavier than newborns of unvaccinated women (3,348 g versus 3,251 g; p = 0.002). during the putative influenza season, the difference between the two groups increased to 113 g (3,360 g for the vaccinated group versus 3,247 g for the unvaccinated group; p = 0.004). there were no significant differences in birth weights outside the putative influenza season (3,317 g [vaccinated] versus 3,255 g [unvaccinated]; p = 0.233). there was no statistical interaction by specific influenza season (i.e., 2004-2005 and 2005-2006) for all analyses of prematurity, sga, and birth weight (for all interaction terms: p.0.05-detailed data available on request). moreover, the association between maternal influenza vaccine and birth outcomes was qualitatively similar for the two influenza seasons. for example, during the period of widespread influenza activity, the adjusted ors for prematurity were 0.17 (95% ci, 0.03-0.86) for the 2004-2005 season and 0.32 (95% ci, 0.10-1.14) for the 2005-2006 season. the fraction of prematurity prevented in the population during the study period (population prevented fraction of prematurity) was 0% for the pre-influenza activity period and 7.9% for the putative influenza season. the population prevented fraction increased during the periods of influenza activity (at least local activity, 15.7%; at least regional activity, 17.5%; widespread activity, 17.5%). this study demonstrates an association between immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. for births during the 8 wk of widespread influenza activity, the odds of prematurity were approximately 70% lower among the newborns of the vaccinated mothers compared to mothers who did not receive the influenza vaccine. during the period of widespread influenza activity there was also an association between maternal receipt of influenza vaccine and reduced likelihood of sga. the magnitude of association between influenza vaccine and prematurity (as measured by the values of ors) increased with the increase in the intensity of influenza activity and was higher for the 2004[16] . although the sga-related ors were not statistically significant for influenza activity periods except for the period of widespread activity, the overall ''gradient'' of effect in the point estimates of the ors was qualitatively similar to that of prematurity. the increase in the impact of maternal influenza vaccines on birth outcomes by influenza activity, both in terms of ors and population prevented fractions, supports the validity of our findings. the possibility of confounding due to differences between vaccinated and unvaccinated individuals included in observational studies of influenza immunization is well recognized [12] . the most significant type of confounding in influenza studies is due to a higher likelihood of individuals with high functional capacity (i.e., healthier the primary adjusted models were based on the approach of identifying covariates that produce adjusted ors of 1 during the pre-influenza period and included the following covariates: gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. b in the secondary adjusted models, the covariates included gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12th grade, mother's marital status, and maternal weight before pregnancy. the ors for association between the different covariates and receipt of influenza vaccine were computed using logistic regression. c ratio of the odds of prematurity in newborns of mothers who received influenza vaccine during pregnancy compared to mothers who did not receive the vaccine by intensity of influenza activity, e.g., in the analysis of all seasons/periods, the (unadjusted) odds of prematurity were 25% lower among the infants of mothers who received the influenza vaccine during pregnancy than among infants whose mothers who did not receive the vaccine. doi:10.1371/journal.pmed.1000441.t002 table 3 . ors of being sga by maternal influenza vaccine status (ors,1 imply a protective association of the vaccine). the primary adjusted models were based on the approach of identifying covariates that produce adjusted ors of 1 during the pre-influenza period and included the following covariates: gestational age for first antenatal visit, maternal diabetes (gestational and/or non-gestational), multivitamin use in pregnancy, history of alcohol use during pregnancy, education less than 12th grade, and mother married. b in the secondary adjusted models, the covariates included gestational age at first antenatal visit, maternal age less than 19 y, maternal age more than 35 y, multiple births, maternal medical risk factors, labor/delivery complications, birth defects, maternal diabetes (gestational and/or non-gestational), hypertension (treated or untreated), mother insured, multivitamin use in pregnancy, history of smoking during pregnancy, history of alcohol use during pregnancy, black race, education less than 12th grade, mother's marital status, and maternal weight before pregnancy. the ors for association between the different covariates and receipt of influenza vaccine were computed using logistic regression. c ratio of the odds of being sga among newborns of mothers who received influenza vaccine during pregnancy compared to those born to mothers who did not receive the vaccine by intensity of influenza activity, e.g., in the analysis of all seasons/periods, the (unadjusted) odds of being sga were 84% lower among the newborns of mothers who received influenza vaccine during pregnancy than among infants whose mothers who did not receive the vaccine. doi:10.1371/journal.pmed.1000441.t003 individuals) to get vaccinated-a phenomenon often known as the ''healthy user effect.'' however, most observational studies where significant confounding has been documented were conducted in the elderly and included a relatively nonspecific end point of all-cause mortality. it is reasonable to assume that, compared to older individuals, women of reproductive age may be less likely to have significant functional limitation even in the presence of co-morbidities. therefore, influenza vaccine studies in this age group may be less likely to suffer from bias due to the healthy user effect. moreover, we found no statistically significant difference between the vaccinated women and the unvaccinated women in terms of gestational age at which they sought antenatal care. on the other hand, the possibility of other confounders cannot be discounted in studies involving pregnant women. in order to address confounding, we used the pre-influenza period (i.e., the season where vaccine was available but there was minimal circulation of influenza virus) as the ''control'' period. the use of the pre-influenza period for selecting confounders from a broad set of covariates is an approach suggested by nelson et al. [12] and jackson et al. [11] that takes advantage of the seasonality of influenza circulation. the associations observed in our study were robust to adjustment for confounders identified using this approach (and the more traditional approach)-supporting the validity of our findings. influenza virus, particularly seasonal influenza virus, rarely crosses the placenta [3, 17, 18] . however, the effect of infection on prematurity is thought be at least partially mediated through inflammatory pathways [5, 6] . increase in pro-inflammatory cytokines (e.g., il-1, il-6 and tnf-a) and reduction in antiinflammatory cytokines (e.g., il-10) have been linked to preterm labor [6, 19, 20] . il-1 stimulates the amnion and the decidua to produce prostaglandins and can stimulate myometrial contractions [20] . prostaglandins are known to play an important role in the initiation and progression of labor [21] . moreover, in animal models, administration of il-1 results in preterm birth [20] . similarly, tnf-a induces the amnion, the decidua, and the myometrium to produce prostaglandins, and administration of tnf-a to pregnant animals can induce preterm labor [19, 22] . recent studies have shown that influenza virus infection induces gene expression of pro-inflammatory cytokines including il-1b, il-6, tnf-a, interferon (ifn)-b, ifn-a, and granulocyte macrophage colony-stimulating factor (gm-csf) [19] . in addition to biological plausibility, there is epidemiological evidence of an association between maternal infection and preterm birth [5] . the association is strongest for intrauterine viral infections and systemic and intrauterine bacterial infections [5, 6] . viral infection outside the reproductive tract, including influenza infection, may also play a role in the etiology of prematurity. for example, in an analysis of 1957-1958 data, newborns of women who had serological evidence of ''asian'' (pandemic) influenza during pregnancy were 50% more likely to be premature compared to newborns of uninfected women [2] . moreover, a recent literature review found that sars infection in the second or third trimester of pregnancy may be associated with spontaneous preterm delivery and early cesarean sections for deteriorating medical condition, although only 16 such cases were identified in the literature [23] . moreover, in studies in china and hungary, birth defects were associated with history of influenza [24, 25] . however, a few observational studies have failed to demonstrate an association between influenza infection and birth outcomes [26, 27] . the lack of observed effect in some studies could be due to a true lack of association, small or difficult to measure effect size, challenges related to the study population (e.g., administrative datasets), or non-differential misclassification due to challenges in retrospectively identifying influenza infection. although less than ideal, modeling receipt of influenza vaccine as the exposure/ independent variable reduces the likelihood and the intensity of non-differential misclassification bias. preterm births represent a significant burden to health care and society [5] . like several developed countries, there has been an increase in the rate of preterm births in the us, which rose from 9.5% in 1981 to 12.8% in 2006 [28, 29] . although the etiology of prematurity is complex [5] and not completely understood, our results suggest that at least a fraction of preterm births may be preventable through maternal influenza vaccination. the association between maternal influenza vaccination and sga was only statistically significant (and the highest in magnitude) for the period of widespread influenza activity. possible reasons for the effect being limited to the period of highest influenza activity include the following: (a) in a developed country setting, the effect of maternal influenza infection on fetal growth is milder than the effect on prematurity; (b) sga represents fetal compromise resulting from infection that is insufficient to trigger early parturition, but may result in the delayed observation of growth restriction (i.e., the observation in the widespread activity period may be the cumulative effect of previous periods). moreover, in the vaccinated group, the birth weight distribution in the pre-influenza period was different from the distribution in the period of widespread activity (see text s1). however, the difference in mean birth weights (in the vaccinated group) between these two periods was not statistically significant (p = 0.74). since the ostensible increase in birth weight in the widespread activity period compared to the pre-influenza period in the vaccinated group cannot easily be explained by vaccine action, this difference-although non-significant-may suggest confounding vis-à-vis the birth weight outcome. this study has a few limitations and strengths. although we assessed and adjusted for many covariates, like any observational study, there is a possibility of residual confounding and selection bias. moreover, data on influenza infection during pregnancy were not included in the prams dataset. although the primary explanation of the effects of influenza immunization in pregnancy on birth outcomes is through prevention of infection, having influenza infection data would have provided additional support for our findings. another issue is that the information regarding maternal influenza immunization was based on recall and could be susceptible to information bias. however, the vaccination rates in our study are similar to the rates computed by other authors for georgia, and to the united states national level coverage estimated by the national health interview survey [30, 31] . the prams dataset does not contain information regarding the precise trimester of vaccination. therefore, the effect of vaccination in a specific trimester could not be evaluated. moreover, it is possible that mothers of premature infants had less time to receive influenza vaccine than mothers of term infants (i.e., reverse causality). on the other hand, since this was a population-based study with a sampling strategy aimed at producing representative estimates, the temporal distribution of influenza vaccination in pregnancy would be similar to that of the general population, hence adding to the generalizability of our findings. the results of this study, nevertheless, need to be replicated in other populations as it is plausible that the impact of vaccines on birth outcomes would vary with the underlying influenza epidemiology and demographic characteristics. text s1 impact of maternal influenza immunization on likelihood of prematurity and sga births. editors' summary background. maternal infections during pregnancy can have harmful effects on both mother and baby. for example, influenza is associated with increased morbidity and mortality among pregnant women compared to women who are not pregnant or who acquire influenza infection after delivery. and some respiratory infections, especially those that can cause maternal pneumonia such as influenza virus, are known to be associated with the baby being small-below the 10th percentile-for gestational age and with an increased risk of preterm birth-birth before 37 weeks of gestation. previous studies have shown that inactivated influenza vaccination during pregnancy provides protection against influenza virus for both mother and baby. as there has been an increase in the rate of preterm birth the united states from 9.5% in 1981 to 12.8% in 2006, the impact of maternal influenza immunization on birth outcomes has important public health implications and is of particular interest during influenza pandemics. why was this study done? given that maternal vaccination can protect babies from influenza virus, it is plausible that influenza vaccination in pregnancy could mitigate adverse birth outcomes such as prematurity and the baby being small for gestational age. the researchers of this study set out to evaluate this hypothesis by investigating whether there was an association between women receiving inactivated influenza vaccine during pregnancy and positive birth outcomes for their babies in the population of the state of georgia, in the united states. what did the researchers do and find? the researchers conducted a retrospective cohort analysis of a large surveillance dataset (the georgia pregnancy risk assessment monitoring system) to analyze the relationship between receipt of inactivated influenza vaccine during any trimester of pregnancy by mothers of infants born between june 1, 2004, and september 30, 2006 , and their baby being premature or small for gestational age. the study period encompassed the 2004-2005 and 2005-2006 influenza seasons-the two most recent seasons for which the data were available. the researchers did a stratified analysis for the overall study period, and various periods during it, and also weighted their analysis to adjust for possible oversampling. they used logistic regression to evaluate the association of maternal influenza vaccine and (a) prematurity and (b) small for gestational age, and also used linear regression to evaluate the statistical significance of differences between vaccinated and unvaccinated women for mean gestational age at first antenatal visit and mean birth weight. during the study period, 4,168 mother-baby pairs were included in the analysis. local influenza activity was detected during 27 weeks (22.1%), and 578 women (14.9% [weighted]) had received the influenza vaccine during pregnancy, giving a vaccination coverage of 19.2% (weighted) among mothers of infants born during the assumed influenza season. in the study sample, 1,547 babies (10.6% [weighted]) were born premature, and 1,186 babies (11.2% [weighted]) were small for gestational age. infants who were born during the assumed influenza season (october-may) and whose mothers were vaccinated against influenza during pregnancy were less likely to be premature than infants of unvaccinated mothers born in the same period, with an adjusted odds ratio of 0.60. the effect of maternal influenza vaccine on reducing prematurity was the highest for infants born during the period of widespread influenza activity, with 72% lower odds of prematurity in infants of vaccinated mothers than infants of unvaccinated mothers. compared with newborns of unvaccinated women, babies of vaccinated mothers also had 69% lower odds of being small for gestational age during the period of widespread influenza activity, but the adjusted and unadjusted odd ratios were not significant for the pre-influenza activity period. what do these findings mean? these results show that there was an association between maternal immunization with the inactivated influenza vaccine during pregnancy and reduced likelihood of prematurity during local, regional, and widespread influenza activity periods. in addition, during the period of widespread influenza 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during pregnancy and risk of congenital abnormalities in offspring maternal morbidity and perinatal outcomes among pregnant women with respiratory hospitalizations during influenza season impact of maternal influenza vaccination during pregnancy on the incidence of acute respiratory illness visits among infants annual summary of vital statistics annual summary of vital statistics prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) receipt of influenza vaccine during pregnancy among women with live births-georgia and rhode island we would like to thank ms. deanna west-tankoo for her administrative help and for her help with retrieving the relevant articles. key: cord-000759-36dhfptw authors: uribe-sánchez, andrés; savachkin, alex title: predictive and reactive distribution of vaccines and antivirals during cross-regional pandemic outbreaks date: 2011-06-05 journal: influenza res treat doi: 10.1155/2011/579597 sha: doc_id: 759 cord_uid: 36dhfptw as recently pointed out by the institute of medicine, the existing pandemic mitigation models lack the dynamic decision support capability. we develop a large-scale simulation-driven optimization model for generating dynamic predictive distribution of vaccines and antivirals over a network of regional pandemic outbreaks. the model incorporates measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. the performance of the strategy is compared to that of the reactive myopic policy, using a sample outbreak in fla, usa, with an affected population of over four millions. the comparison is implemented at different levels of vaccine and antiviral availability and administration capacity. sensitivity analysis is performed to assess the impact of variability of some critical factors on policy performance. the model is intended to support public health policy making for effective distribution of limited mitigation resources. as of july 2010, who has reported 501 confirmed human cases of avian influenza a/(h5n1) which resulted in 287 deaths worldwide [1] . at the same time, the statistics for the h1n1 2009 outbreak has so far included 214 countries with a total reported number of infections and deaths of 419,289 and 18,239, respectively [2] . today, an ominous expectation exists that the next pandemic will be triggered by a highly pathogenic virus, to which there is little or no pre-existing immunity in humans [3] . the nation's ability to mitigate a pandemic influenza depends on the available emergency response resources and infrastructure, and, at present, challenges abound. predicting the exact virus subtype remains a difficult task, and even when identified, reaching an adequate vaccine supply can currently take up to nine months [4, 5] . even if the existing vaccines prove to be potent, their availability will be limited by high production and inventory costs [6, 7] and also will be constrained by the supply of antiviral drugs, healthcare providers, hospital beds, medical supplies, and logistics. hence, pandemic mitigation will have to be done amidst limited availability of resources and supporting infrastructure. this challenge has been acknowledged by who [7] and echoed by the hhs and cdc [8, 9] . the existing models on pandemic influenza (pi) containment and mitigation aims to address various complex aspects of the pandemic evolution process including: (i) the mechanism of disease progression, from the initial contact and infection transmission to the asymptomatic phase, manifestation of symptoms, and the final health outcome [10] [11] [12] , (ii) the population dynamics, including individual susceptibility [13, 14] and transmissibility [10, [15] [16] [17] , and behavioral factors affecting infection generation and effectiveness of interventions [18] [19] [20] , (iii) the impact of pharmaceutical and nonpharmaceutical measures, including vaccination [21] [22] [23] , antiviral therapy [24] [25] [26] , social distancing [27] [28] [29] [30] [31] and travel restrictions, and the use of low-cost measures, such as face masks and hand washing [26, [32] [33] [34] . recently, the modeling efforts have focused on combining pharmaceutical and nonpharmaceutical interventions in search for synergistic strategies, aimed at better resource utilization. most of such approaches attempt implementing a form of social distancing followed by application of pharmaceutical measures. for significant contributions in this area see [33, [35] [36] [37] [38] [39] [40] [41] . one of the most notable among these efforts is a 2006-07 initiative by midas [42] , which cross-examined independent simulation models of pi spread in rural areas of asia [43, 44] , usa and uk [45, 46] , and the city of chicago [47] , respectively. midas crossvalidated the models by simulating the city of chicago, with 8.6m inhabitants and implementing a targeted layered containment [48, 49] . the research findings of midas and some other groups [12, 33] were used in a recent "modeling community containment for pandemic influenza" report by iom, to formulate a set of recommendations for pi mitigation [50] . these findings were also used in a pandemic preparedness guidance developed by cdc [51] . at the same time, the iom report [50] points out several limitations of the midas models, observing that "because of the significant constraints placed on the models . . . the scope of models should be expanded." the iom recommends "to adapt or develop decision-aid models that can . . . provide real-time feedback . . . and include the costs and benefits of intervention strategies." our literature review yields a similar observation that most existing approaches focus on assessment of a priori defined strategies, and virtually none of the models are capable of "learning," that is, adapting to changes in the pandemic progress, or even predicting them, to generate dynamic strategies. such a strategy has the advantage of being developed dynamically, as the pandemic spreads, by selecting a mix of available mitigation options at each decision epoch, based on both the present state of the pandemic and its predicted evolution. in an attempt to address the iom recommendations, we present a simulation optimization model for developing predictive resource distribution over a network of regional outbreaks. the underlying simulation model mimics the disease and population dynamics of each of the affected regions (sections 2.1 and 2.2). as the pandemic spreads from region to region, the optimization model distributes mitigation resources, including stockpiles of vaccines and antiviral and administration capacities (section 2.3). the model seeks to minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks, using measures of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. the methodology is calibrated and implemented on a sample outbreak in fla, usa with over 4m inhabitants (section 3). the strategy is compared to the reactive myopic policy, which allocates resources from one actual outbreak region to the next, each time trying to cover the entire regional population at risk, regardless of the resource availability. the comparison is done at different levels of vaccine and antiviral availability and administration capacity. we also present a sensitivity analysis for assessing the impact of variability of some critical factors, including: (i) antiviral efficacy, (ii) social distancing conformance, and (iii) cdc response delay. the objective of our methodology is to generate a progressive allocation of the total resource availability over a network of regional outbreaks. the methodology incorporates (i) a cross-regional simulation model, (ii) a set of single-region simulation models, and (iii) an embedded optimization model. we consider a network of regions with each of which classified as either unaffected, ongoing outbreak, or contained outbreak ( figure 1) . the cross-regional simulation model connects the regions by air and land travel. the single-region simulation models mimic the population and disease dynamics of each ongoing region, impacted by intervention measures. the pandemic can spread from ongoing to unaffected regions by infectious travelers who pass through regional border control. at every new regional outbreak epoch, the optimization model allocates available resources to the new outbreak region (actual distribution) and unaffected regions (virtual distribution). daily statistics is collected for each ongoing region, including the number of infected, deceased, and quarantined cases, for different age groups. as a regional outbreak is contained, its societal and economic costs are calculated. in sections 2.1-2.3, we present the details of the simulation and optimization models. a testbed illustration and a comparison of our strategy to the myopic policy is given in section 3. a schematic of the cross-regional simulation model is shown in figure 2 . the model is initialized by creating population entities and mixing groups, for each region. a pandemic is started by an infectious case injected into a randomly chosen region. the details of the resulting regional contact dynamics and infection transmission are given in section 2.2. as the infected cases start seeking medical help, a new regional outbreak is detected. a resource distribution is then determined and returned to the single-region model. the outbreak can begin cross-regional simulation the single-region model subsumes the following components (see figure 3 ): (i) population dynamics (mixing groups and schedules), (ii) contact and infection process, (iii) disease natural history, and (iv) mitigation strategies, including social distancing, vaccination, and antiviral application. the model collects detailed statistics, including number of infected, recovered, deceased, and quarantined cases, for different age groups. for a contained outbreak, its societal and economic costs are calculated. the societal cost includes the cost of lost lifetime productivity of the deceased; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined [52] . each region is modeled as a set of population centers formed by mixing groups or places where individuals come into contact with each other during the course of their social interaction. examples of mixing groups include households, offices, schools, universities, shopping centers, entertainment centers, and so forth, [53] . each individual is assigned a set of attributes such as age, gender, parenthood, workplace, infection susceptibility, and probability of travel, among others. each person is also assigned δt time-discrete (e.g., δt = 1 hour) weekday and weekend schedules, which depend on: (i) person's age, parenthood, and employment status, (ii) disease status, (iii) travel status, and (iv) person's compliance to social distancing decrees [54] . as their schedules advance, the individuals circulate throughout the mixing groups and come into contact with each other (see section 2.2.2). it is assumed that at any point of time, an individual belongs to one of the following compartments (see figure 4 ): susceptible, contacted (by an infectious individual), infected (asymptomatic or symptomatic), and recovered/deceased. in what follows, we present the infection transmission and disease natural history model, which delineates the transitions between the above compartments. process. infection transmission occurs during contact events between susceptible and infectious cases, which take place in the mixing groups. at the beginning of every δt period (e.g., one hour), for each mixing group g, the simulation tracks the total number of infectious cases, n g , present in the group. it is assumed that each infectious case generates r g per δt unit of time new contacts [46] , chosen randomly (uniformly) from the pool of susceptibles present in the group. we also assume the following: (i) during δt period, a susceptible may come into contact with at most one infectious case and (ii) each contact exposure lasts δt units of time. once a susceptible has started her contact exposure at time t, she will develop infection at time t + δt with a certain probability that is calculated as shown below. let l i (t) be a nonnegative continuous random variable that represents the duration of contact exposure, starting at time t, required for susceptible i to become infected. we assume that l i (t) is distributed exponentially with mean 1/λ i (t), where λ i (t) represents the instantaneous force of infection applied to susceptible i at time t [55] [56] [57] . the probability that susceptible i, whose contact exposure has started at time t, will develop infection at time t + δt is then given as a schematic of the disease natural history is shown in figure 5 . during the incubation phase, the infected case stays asymptomatic. at the end of the latency phase, she enters the infectious phase [44, 46, 48] . she becomes symptomatic at the end of the incubation period. at the end of the infectious phase, she enters the period leading to a health outcome, which culminates in her recovery or death. mortality for influenza-like diseases is a complex process affected by many factors and variables, most of which have limited accurate data support available from past pandemics. furthermore, the time of death can sometimes be weeks following the disease episode (which is often attributable to pneumonia-related complications [58] ). because of the uncertainty underlying the mortality process, we adopted an age-based form of the mortality probability of infected i, as follows: where μ i is the age-dependent base mortality probability of infected i, ρ i is her status of antiviral therapy (0 or 1), and τ is the antiviral efficacy measured as the relative decrease in the base probability [44] . we assume that a recovered case develops full immunity but continues circulating in the region. mitigation is initiated upon detection of a critical number of confirmed infected cases [59] , which triggers resource distribution and deployment. the model incorporates a certain delay for deploying field responders. pharmaceutical intervention (phi) includes vaccination and antiviral application. vaccination is targeted at individuals at risk [60] to reduce their infection susceptibility. the vaccine takes a certain period to become effective [61] . vaccination is constrained by the allocated stockpile and administration capacity, measured in terms of the immunizer-hours. we assume that as some symptomatic cases seek medical help [62, 63] , those at risk of them will receive an antiviral. the process is constrained by the allocated stockpile and administration capacity, measured in terms of the number of certified providers. both vaccination and antiviral application are affected by a number of sociobehavioral factors, including conformance of the target population, degree of risk perception, and compliance of healthcare personnel [64] [65] [66] . the conformance level of the population at risk can be affected, among other factors, by the demographics and income level [67] [68] [69] [70] [71] as well as by the quality of public information available [54] . the degree of risk perception can be influenced by the negative experience developed during previous pharmaceutical campaigns [72, 73] , as well as by public fear and rumors [74, 75] . nonpharmaceutical intervention (npi) includes social distancing and travel restrictions. we adopted a cdc guidance [51] , which establishes five categories of pandemic severity and recommends quarantine and closure options according to the category. the categories are determined based on the value of the case fatality ratio (cfr), the proportion of fatalities in the total infected population. for cfr values lower than 0.1% (category 1), voluntary at-home isolation of infected cases is implemented. for cfr values influenza research and treatment 5 between 0.1% and 1.0% (categories 2 and 3), in addition to at-home isolation, the following measures are recommended: (i) voluntary quarantine of household members of infected cases and (ii) child and adult social distancing. for cfr values exceeding 1.0% (categories 4 and 5), all the above measures are implemented. as the effectiveness of social distancing is affected by some of the behavioral factors listed above [54] , we assume a certain social distancing conformance level. travel restrictions considered in the model included regional air and land border control for infected travelers. figure 2 , the optimization model is invoked at the beginning of every nth new regional outbreak epoch (n = 1, 2, . . .), starting from the initial outbreak region (n = 1). the objective of the model is to allocate some of the available mitigation resources to the new outbreak region (actual distribution) while reserving the rest of the quantities for potential outbreak regions (virtual distribution). by doing so, the model seeks to progressively minimize the impact of ongoing outbreaks and the expected impact of potential outbreaks, spreading from the ongoing locations. mitigation resources can include stockpiles of vaccines and antivirals, administration capacity, hospital beds, medical supplies, and social distancing enforcement resources, among others. the predictive mechanism of the optimization model is based on a set of regression equations obtained using single-region simulation models. in what follows, we present the construction of the optimization model and explain the solution algorithm for the overall simulation-based optimization methodology. we introduce the following general terminology and notation: the optimization criterion (objective function) of the model incorporates measures of expected societal and economic costs of the pandemic: the societal cost includes the cost of lost lifetime productivity of the deceased; the economic cost includes the cost of medical expenses of the recovered and deceased and the cost of lost productivity of the quarantined. to compute these costs, the following impact measures of morbidity, mortality, and quarantine are used, for each region k: to estimate these measures, we use the following regression models obtained using a single-region simulation of each region k: where δ i ·· denotes the regression coefficient associated with resource i and δ im ·· is the regression coefficient for the interaction between resources i and m. similar models are used for y hk , d hk , and v hk . the above relationships between the impact measures and the resource distributions ought to be determined a priori of implementing a cross-regional scenario (see section 3). here, we consider each region k as the initial outbreak region. we assume, however, that as the pandemic evolves, the disease infectivity will naturally subside. hence, the regression equations need to be re-estimated at every new outbreak epoch, for each region k ∈ c n , using the singleregion simulation models, where each simulation must be initialized to the current outbreak status in region k in the cross-regional simulation. as an alternative to using a computationally burdensome approach of re-estimating the regression equations, a modeler may choose to use a certain decay factor α n [76] to adjust the estimates of the regional impact measures at every nth outbreak epoch, in the following way: in addition, we use the following regression model to estimate the probability of pandemic spread from affected region l to unaffected region k, as a function of resources allocated to region l, which, in turn, impact the number of outgoing infectious travelers from the region: where γ i ·· denotes the regression coefficient associated with resource i, γ im ·· is the regression coefficient associated with interaction between resources i and m, and γ 0 ·· represents the intercept. consequently, the total outbreak probability for unaffected region k can be found as p k = l∈b n p lk . as in the case of the impact measures, the estimates of the regional outbreak probabilities need to be progressively re-estimated or adjusted using a scheme similar to (4), as follows: 6 finally, we calculate the total cost of an outbreak in region k at the nth decision epoch as follows: where m h is total medical cost of an infected case in age group h over his/her disease period, w h is total cost of lost wages of an infected case in age group h over his/her disease period, w h is cost of lost lifetime wages of a deceased case in age group h, and w h is daily cost of lost wages of a non-infected case in age group h who complies with quarantine. the model. the optimization model has the following form. minimize tc n j q 1 j , q 2 j , . . . , q r j + s∈c n tc n s q 1s , q 2s , . . . , q rs · p n s subject to the first term of the objective function represents the total cost of the new outbreak j, estimated at the nth outbreak epoch, based on the actual resource distribution {q 1 j , q 2 j , . . . , q r j } (see (7)). the second term represents the total expected cost of outbreaks in currently unaffected regions, based on the virtual distributions {q 1s , q 2s , . . . , q rs } (7) and the regional outbreak probabilities p n s (6) . the set of constraints assures that for each resource i, the total quantity allocated (current and virtual, both nonnegative) does not exceed the total resource availability at the nth decision epoch. note that both the objective function and the availability constraints are nonlinear in the decision variables. (1) estimate regression equations for each region using the single-region simulation model. (2) begin the cross-regional simulation model. (4) select randomly the initial outbreak region j. set n = 1. (c) re-estimate regression equations for each region k ∈ b n ∪ c n using the single-region simulations, where each simulation is initialized to the current outbreak status in the region (alternatively, use (4) and (6)). (d) solve the resource distribution model for region j. (e) update the total resource availabilities. (10) calculate the total cost for each contained region and update the overall pandemic cost. to illustrate the use of our methodology, we present a sample h5n1 outbreak scenario including four counties in fla, usa: hillsborough, miami dade, duval, and leon, with populations of 1.0, 2.2, 0.8, and 0.25 million people, respectively. a basic unit of time for population and disease dynamics models was taken to be δt = 1 hour. regional simulations were run for a period (up to 180 days) until the daily infection rate approached near zero (see section 3.3). below, we present the details on selecting model parameter values. most of the testbed data can be found in the supplement [77] . models. demographic and social dynamics data for each region [77] were extracted from the u.s. census [78] and the national household travel survey [79] . daily (hourly) schedules [77] were adopted from [53] . each infected person was assigned a daily travel probability of 0.24% [79] , of which 7% was by air and 93% by land. the probabilities of travel among the four regions were calculated using traffic volume data [80] [81] [82] [83] , see table 1 . infection detection probabilities for border control for symptomatic cases were assumed to be 95% and 90%, for air and land, respectively [84] . the instantaneous force of infection applied to contact i at time t ((1), [57] ) was modeled as influenza research and treatment 7 where α i is the age-dependent base instantaneous infection probability of contact i, θ i (t) is her status of vaccination at time t (0 or 1), and δ is the vaccine efficacy, measured as the reduction in the base instantaneous infection probability (achieved after 10 days [61] ). the values of age-dependent base instantaneous infection probabilities were adopted from [46] (see table 2 ). the disease natural history included a latent period of 29 hours (1.21 days), an incubation period of 46 hours (1.92 days), an infectiousness period from 29 to 127 hours (1.21 to 5.29 days), and a period leading to health outcome from 127 to 240 hours (5.29 to 10 days) [85] . base mortality probabilities (μ i in (2)) were found using the statistics recommended by the working group on pandemic preparedness and influenza response [52] . this data shows the percentage of mortality for age-based high-risk cases (hrc) ( table 3 , columns 1-3). mortality probabilities (column 4) were estimated under the assumption that highrisk cases are expected to account for 85% of the total number of fatalities, for each age group [52] . single-region simulation models were calibrated using two common measures of pandemic severity [35, 45, 46] : the basic reproduction number (r 0 ) and the infection attack rate (iar). r 0 is defined as the average number of secondary infections produced by a typical infected case in a totally susceptible population. iar is defined as the ratio of the total number of infections over the pandemic period to the size of the initial susceptible population. to determine r 0 , all infected cases inside the simulation were classified by generation of infection, as in [33, 43] . the value of r 0 was calculated as the average reproduction number of a typical generation in the early stage of the pandemic, with no interventions implemented (the baseline scenario) [33] . historically, r 0 values for pi ranged between 1.4 and 3.9 [37, 43] . to attain similar values, we calibrated the hourly contact rates of mixing groups [77] (original rates were adopted from [46] ). for the four regions, the average baseline value of r 0 was 2.54, which represented a high transmissibility scenario. the values of regional baseline iar averaged 0.538. mitigation resources included stockpiles of vaccines and antiviral and administration capacities (section 3.4). a 24-hour delay was assumed for deployment of resources and filed responders [59] . phi. the vaccination risk group included healthcare providers [66] , and individuals younger than 5 years (excluding younger than 12 months old) and older than 65 years [60] . the risk group for antiviral included symptomatic individuals below 15 years and above 55 years [60, 86] . the efficacy levels for the vaccine (δ in (9)) and antiviral (τ in (2)) were assumed to be 40% [44, 87] and 70%, respectively. we did not consider the use of antiviral for a mass prophylactic reduction of infection susceptibility due to the limited antiviral availability [9] and the risk of emergence of antiviral resistant transmissible virus strains [26] . we assumed a 90% target population conformance for both vaccination and antiviral treatment [64] . the immunity development period for the vaccine was taken as 10 days [61] . a version of the cdc guidance for quarantine and isolation for category 5 was implemented (section 2.2.4, [51] ). once the reported cfr value had reached 1.0%, the following policy was declared and remained in effect for 14 days [51] : (i) individuals below a certain age ξ (22 years) stayed at home during the entire policy duration, (ii) of the remaining population, a certain proportion φ [88] stayed at home and was allowed a one-hour leave, every three days, to buy essential supplies, and (iii) the remaining (1 − φ) noncompliant proportion followed a regular schedule. all testbed scenarios considered the quarantine conformance level φ equal to 80% [54] . an outbreak was considered contained, if the daily infection rate did not exceed five cases, for seven consecutive days. once contained, a region was simulated for an additional 10 days for accurate estimation of the pandemic statistics. a 2 5 statistical design of experiment [89] was used to estimate the regression coefficient values of the significant decision factors and their interactions (see section 2.3; the values of adjusted r 2 ranged from 96.36% to 99.97%). the simulation code was developed using c++. the running time for a cross-regional simulation replicate involving over four million inhabitants was between 17 and 26 minutes (depending on the initial outbreak region, with a total of 150 replicates) on a pentium 3.40 ghz with 4.0 gb of ram. the performance of the dpo and myopic policies is compared at different levels of resource availability. table 4 summarizes the total vaccine and antiviral requirements for each region, based on the composition of 8 influenza research and treatment average daily cost of lost productivity of a non-infected quarantined case (20-99) $432.54 theregional risk groups (see section 3.3). table 5 shows the per capita costs of lost productivity and medical expenses, which were adopted from [52] and adjusted for inflation for the year of 2010 [90] . comparison of the two strategies is done at the levels of 20%, 50%, and 80% of the total resource requirement shown in table 4 . figures 6(a) and 6(b) show the policy comparison in the form of the 95% confidence intervals (ci) for the average number of infected and deceased, respectively. figure 7 also shows the policy comparison using the 95% ci for the average total pandemic cost, calculated using the pandemic statistics, and the per capita costs from table 5 . for illustrative purposes, we also show the average number of regional outbreaks, for each policy, at different levels of resource availability, in the testbed scenario involving four regions, with the hillsborough as the initial outbreak region ( table 6) . it can be observed that the values of all impact measures exhibit a downward trend, for both dpo and myopic policies, as the total resource availability increases from 20% to 80%. an increased total resource availability not only helps alleviating the pandemic impact inside the ongoing regions but also reduces the probability of spread to the unaffected regions. for both policies, as the total resource availability approaches the total resource requirement (starting from approximately 60%), the impact numbers show a converging behavior, whereby the marginal utility of additional resource availability diminishes. this behavior can be explained by noting that the total resource requirements were determined assuming the worst case scenario when all (four) regions would be affected and ought to provided with enough resources to cover their respective regional populations at risk. it can also be seen that on average, the dpo policy outperforms the myopic approach at all levels, which can attest to a more efficient resource utilization achieved by the dpo policy (see also table 6 ). the difference in the policy performance is particularly noticeable at the lower levels of resource availability, and it gradually diminishes, as the resource availability increases and becomes closer to be sufficient to cover the entire populations at risk in all regions. it can also be noted that the variability in the performance of the dpo strategy is generally smaller than that of the myopic policy. in general, for both strategies, the performance variability decreases with higher availability of resources. in this section, we assess the marginal impact of variability of some of the critical factors. the impact was measured separately by the change in the total pandemic cost and the number of deaths (averaged over multiple replicates), resulting from a unit change in a decision factor value, one factor at a time. factors under consideration included: (i) antiviral efficacy, (ii) social distancing conformance, and (iii) cdc response delay. we have used all four regions, separately, as initial outbreak regions for each type of sensitivity analysis. the results (patterns) were rather similar. due to limited space, we have opted to show the results for only one initial region, chosen arbitrarily, for each of the three types of sensitivity studies. while duval county was selected as the initial outbreak region to show the sensitivity results on antiviral efficacy, hillsborough and miami dade were used as the initial regions to show the results on, respectively, social distancing conformance and cdc response delay. figure 8 depicts the sensitivity of the average total cost and average total deaths to antiviral efficacy values between 0% and 80%. as expected, for both policies, the curves for the average number of deaths exhibit a decreasing trend which is almost linear for the values of τ between 0% and 40%. as the value of τ approaches 70%, the curves start exhibit a converging behavior. the curves for the average total pandemic cost exhibit a similar pattern for both policies. it can be noted that the performance of both policies is somewhat identical for low antiviral efficacy (between 0% and 30%). however, the performance of the dpo policy improves consistently as τ increases which can be attributed to a more discretionary allocation of the antiviral stockpile by the dpo policy. reduction of the contact intensity through quarantine and social distancing has proven to be one of the most effective containment measures, especially in the early stages of the pandemic [27, 30, 31, 41] . figure 9 shows the sensitivity of the average total cost and average total deaths to the social distancing conformance ranging between 60% and 80%. we observed that for both impact measures, the dpo policy demonstrated a better performance with the difference ranging from $3b to $26b in the total cost and from 1,400 to 20,000 in the number of fatalities. the biggest difference in performance was achieved at the lower-to-medium levels of conformance (between 65% and 72%). as the conformance level approached 80%, the dominating impact of social distancing masked the effect of better utilization of vaccines and antivirals achieved by the dpo strategy. the cdc response delay corresponds to the interval of time from the moment an outbreak is detected to a complete deployment of mitigation resources. depending on the disease infectivity, cdc response delay may represent one of the most critical factors in the mitigation process. figure 10 shows how the performance of both policies was significantly impacted by this factor. the dpo policy showed a uniformly better performance with the difference ranging between $3b to $4b in the average total cost, and between 800 to 1,800 in the average number of mortalities, over the range (24figure 10 : sensitivity analysis for cdc response delay. as recently pointed by the iom, the existing models for pi mitigation fall short of providing dynamic decision support which would incorporate "the costs and benefits of intervention" [50] . in this paper, we present a large-scale simulation optimization model which is attempted at filling this gap. the model supports dynamic predictive resource distribution over a network of regions exposed to the pandemic. the model aims to balance both the ongoing and potential outbreak impact, which is measured in terms of morbidity, mortality, and social distancing, translated into the cost of lost productivity and medical expenses. the model was calibrated using historic pandemic data and compared to the myopic strategy, using a sample outbreak in fla, usa, with over 4 million inhabitants. summary of the main results. in the testbed scenario, for both strategies, the marginal utility of additional resource availability was found to be diminishing, as the total resource availability approached the total requirement. in the testbed scenario, the dpo strategy on average outperformed the myopic policy. as opposed to the dpo strategy, the myopic policy is reactive, rather than predictive, as it allocates resources regardless of the remaining availability and the overall cross-regional pandemic status. in contrast, the dpo model distributes resources trying to balance the impact of actual outbreaks and the expected impact of potential outbreaks. it does so by exploiting regionspecific effectiveness of mitigation resources and dynamic reassessment of pandemic spread probabilities, using a set of regression submodels. hence, we believe that in scenarios involving regions with a more heterogeneous demographics, the dpo policy will likely to perform even better and with less variability than the myopic strategy. we also note that the difference in the model performance was particularly noticeable at lower levels of resource availability, which is in accordance with a higher marginal utility of additional availability at that levels. we thus believe that the dpo model can be particularly useful in scenarios with very limited resources. contributions of the paper. the simulation optimization methodology presented in this paper is one of the first attempts to offer dynamic predictive decision support for pandemic mitigation, which incorporates measures of societal and economic costs. our comparison study of the dpo versus myopic cross-regional resource distribution is also novel. additionally, our simulation model represents one of the first of its kind in considering a broader range of social behavioral aspects, including vaccination and antiviral treatment conformance. the simulation features a flexible design which can be particularized to a broader range of phi and npi and even more granular mixing groups. we also developed a decision-aid simulator which is made available to the general public through our web site at http://imse.eng.usf.edu/pandemics.aspx. the tool is intended to assist public health decision makers in implementing what-if analysis for assessment of mitigation options and development of policy guidelines. examples of such guidelines include vaccine and antiviral risk groups, social distancing policies (e.g., thresholds for declaration/lifting and closure options), and travel restrictions. limitations of the model. lack of reliable data prevented us from considering geo-spatial aspects of mixing group formation. we also did not consider the impact of public education and the use of personal protective measures (e.g., face masks) on transmission, again due to a lack of effectiveness data [91] . we did not study the marginal effectiveness of individual resources due to a considerable uncertainty about the transmissibility of an emerging pandemic virus and efficacy of vaccine and antiviral. for the same reason, the vaccine and antiviral risk groups considered in the testbed can be adjusted, as different prioritization schemes have been suggested. the form of social distancing implemented in the testbed can also be modified as a variety of schemes can be found in the literature, including those based on geographical and social targeting. effectiveness of these approaches is substantially influenced by the compliance factor, for which limited accurate data support exists. it will thus be vital to gather the most detailed data on the epidemiology of a new virus and the population dynamics early in the evolution of a pandemic, and expeditiously analyze the data to adjust the interventions accordingly. cumulative number of confirmed human cases of avian influenza a(h5n1) 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international airport: daily traffic volume data tallahassee regional airport: daily traffic volume data how thermal-imaging cameras spot flu fevers writing committee of the world health organization (who) antivirals for pandemic influenza: guidance on developing a distribution and dispensing program safety and immunogenicity of an inactivated subvirion influenza a (h5n1) vaccine attitudes toward the use of quarantine in a public health emergency in four countries design and analysis of experiments inflation calculator nonpharmaceutical interventions for pandemic influenza, national and community measures the authors would like to acknowledge with thanks the many helpful suggestions made by professor yiliang zhu, department of epidemiology and biostatistics at the university of south florida, tampa, fla, usa. key: cord-004060-nxw5k9y1 authors: zhang, yewu; wang, xiaofeng; li, yanfei; ma, jiaqi title: spatiotemporal analysis of influenza in china, 2005–2018 date: 2019-12-23 journal: sci rep doi: 10.1038/s41598-019-56104-8 sha: doc_id: 4060 cord_uid: nxw5k9y1 influenza is a major cause of morbidity and mortality worldwide, as well as in china. knowledge of the spatial and temporal characteristics of influenza is important in evaluating and developing disease control programs. this study aims to describe an accurate spatiotemporal pattern of influenza at the prefecture level and explore the risk factors associated with influenza incidence risk in mainland china from 2005 to 2018. the incidence data of influenza were obtained from the chinese notifiable infectious disease reporting system (cnidrs). the besag york mollié (bym) model was extended to include temporal and space-time interaction terms. the parameters for this extended bayesian spatiotemporal model were estimated through integrated nested laplace approximations (inla) using the package r-inla in r. a total of 702,226 influenza cases were reported in mainland china in cnidrs from 2005–2018. the yearly reported incidence rate of influenza increased 15.6 times over the study period, from 3.51 in 2005 to 55.09 in 2008 per 100,000 populations. the temporal term in the spatiotemporal model showed that much of the increase occurred during the last 3 years of the study period. the risk factor analysis showed that the decreased number of influenza vaccines for sale, the new update of the influenza surveillance protocol, the increase in the rate of influenza a (h1n1)pdm09 among all processed specimens from influenza-like illness (ili) patients, and the increase in the latitude and longitude of geographic location were associated with an increase in the influenza incidence risk. after the adjusting for fixed covariate effects and time random effects, the map of the spatial structured term shows that high-risk areas clustered in the central part of china and the lowest-risk areas in the east and west. large space-time variations in influenza have been found since 2009. in conclusion, an increasing trend of influenza was observed from 2005 to 2018. the insufficient flu vaccine supplements, the newly emerging influenza a (h1n1)pdm09 and expansion of influenza surveillance efforts might be the major causes of the dramatic changes in outbreak and spatio-temporal epidemic patterns. clusters of prefectures with high relative risks of influenza were identified in the central part of china. future research with more risk factors at both national and local levels is necessary to explain the changing spatiotemporal patterns of influenza in china. influenza is associated with notable mortality and morbidity worldwide, as well as in china 1-3 . the behaviours of major epidemics and pandemics of influenza were complicated due to dramatic genetic changes, subtype circulation, wave patterning and virus replacement 4 . influenza vaccination is the most effective means to prevent infection, severe disease and mortality 5 . the world health assembly recommends vaccinating 75% of key risk groups against influenza 6 . although seasonal influenza vaccination was introduced in 1998, influenza vaccination is not yet included on the national immunization program (nip) in china 7 . the average national vaccination coverage was reported to be just 1.5-2.2% between 2004 and 2014 7, 8 . the overall number of flu vaccines approved for sale by china's national institute for food and drug control (nifdc) has decreased in recent years 9, 10 . the low coverage rate and reduction in flu vaccine supplementation have raised much concern about the increased risk of influenza incidence in china. although new emerging influenza virus types and subtypes, such as avian influenza a h5n1 [11] [12] [13] [14] , influenza a (h1n1)pdm09 [15] [16] [17] , and influenza a h7n9 18, 19 , have been reported continuously in china, the disease burden of influenza has been dominated by a(h3n2), a(h1n1)pdm2009 influenza viruses, pre-pandemic a(h1n1) or influenza b in recent years, which account for the majority of cases 20 . the influenza a(h1n1)pdm2009 virus was first introduced to mainland china on may 9, 2009 21 , and has been one of the dominant viruses in the seasonal influenza epidemics since then 20 . the effect of newly emerging influenza a(h1n1)pdm2009 viruses on the geographic patterns and temporal trends of influenza across the whole country is still unknown. covariates associated with the reported incidence cases of influenza. the table 2 . the crude odds ratios (ors) and adjusted ors in both the univariate poisson models and multivariate adjusted poisson model are statistically significant. after adjusting for other covariates, a spatially unstructured random effect term (v i ), a spatially structured conditional autoregression term (υ i ), a first-order random walk-correlated time variable (γ 1j ), and an interaction term for time and place (δ ij ) in the multivariate adjusted spatiotemporal model, the flu vaccines (per million doses), flu surveillance protocols, rate of influenza a (h1n1)pdm09, latitude and longitude still remain statistically significant. holding all other covariates to zero and adjusting for spatiotemporal variation, every one million increase in the number of influenza vaccines for sale approved by the china food and drug administration was associated with a 12.7% decrease in the influenza incidence risk (95% ci = 0.825-0.923). similarly, the new update of the influenza surveillance protocol in 2017 was related to a 65.6% increase in the influenza incidence risk (95% ci = 1.097-2.496) compared to the protocol used in 2005 to 2008. for every 10% increase in the rate of influenza a (h1n1)pdm09 among all processed specimens from ili patients, there was a 19.5% increase in the influenza incidence risk (95% ci = 1.005-1.413). every one degree increase in the latitude and longitude was associated with a 1.5% (95% ci = 0.980~0.991) and 0.2% (95% ci = 0.997~0.999) increase in the influenza incidence risk, respectively. the spatial and temporal effects in spatiotemporal models with covariates. the spatial effects. the map of the spatially structured conditional autoregression term demonstrated areas of spatial patterning and similarity among prefectures. the spatially structured relative risk and posterior probabilities of spatially structured relative risk greater than 1.0 are presented in figs. 3 and 4, respectively. table 1 . deviance information criterion (dic) for five spatiotemporal models. abbreviations: d, posterior mean of the deviance; pd, the number of effective parameters; dic, the deviance information criterion, as a measure of the trade-off between model fit and complexity. note: model terms used in four models include an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); uncorrelated time (γ j ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ 1j ). θ ij represents the relative risk of area i at time j. * model 1, convolution + uncorrelated time (time iid), e.g., θ α ν table 2 . risk analysis of covariates associated with reported cases of influenza. abbreviations: or, odds ratio; ci, confidence interval. * univariate poisson analysis models. ** multivariate adjusted poisson analysis model, which included all variables in the univariate analysis models. † multivariate adjusted spatiotemporal models, which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). ‡ total number of flu vaccines approved for sale by china's national institute for food and drug control (nifdc), which were rescaled to one million doses as one unit. data were collected from nifdc. # the convolutional spatial risk term, which includes both the spatially structured conditional autoregression term (υ i ) and the spatially unstructured random effect term (ν i ) at the prefecture level, identified areas at increased risk of influenza throughout the 14-year study period (fig. 5) . posterior probabilities for an area's spatial risk estimate exceeding 1.0 are presented in fig. 6 . the proportion of the total spatial heterogeneity explained by the spatially structured conditional autoregression term was 73.51%. after adjusting for fixed covariate effects and time random effects, both the map of the spatial structured term and the convolutional spatial term show that high-risk areas clustered in the central part of china and the lowest-risk areas in the east, northwest and southwest. the higher-risk prefectures were mostly distributed in guangdong, guangxi, guizhou, hunan, jiangxi, zhejiang, hubei, anhui, henan, hebei, beijing, tianjin, gansu, ningxia, and inner mongolia. the lower-risk areas in the east included some prefectures in the shandong peninsula and the prefectures of heilongjiang, liaoning, and jilin provinces in the northeast. the northwest areas are composed of prefectures in tibet, qinghai and xinjiang, while the southwest areas include chongqing and prefectures in sichuan and yunnan provinces. the temporal trend. the relative risks of the 14-year study period, holding the covariates and spatial risk constant, were calculated by exponentiating the marginal first-order random walk-correlated time term (γ 1j ) in the spatiotemporal models of influenza risk with and without covariates. for the spatiotemporal model without . ** adjusted by convolutional spatial term, space-time interaction term, and covariates, e.g., . figure 3 . map of the spatially structured relative risk ( υ e i ), spatiotemporal model of influenza incidence risk with covariates, china prefectures, 2005-2018. note: the linear terms in the model of spatiotemporal model of influenza incidence risk with covariates were , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ covariates, an overall increasing trend was found in the temporal trend term in the 14-year study period. the risk of influenza remained low between 2005 and 2008. a steep increase was observed in 2009. it dropped slightly back to a low level and remained stable in 2010 and 2011. a rapid increase was obvious in the last 3 years (table 3) (fig. 7) . for the temporal trend term in the spatiotemporal model with covariates, the relative risks in the years from 2005 to 2016 were not significantly different from that in the spatiotemporal model with covariates. the relative risks in the model with covariates in 2017 and 2018 were significantly lower than those in the model without covariates. the lower boundary of the 95% confidence intervals in the model with covariates showed some levelling off in recent years. the differences between the spatiotemporal model with and without covariates indicated that the recent increases in influenza incidence risks could be partially explained by the fixed covariate effects. space-time interactions. the probability exceedances for the yearly space-time interactions are presented for the study period (fig. 8) . these identify areas with residual spatial risk greater than 1.0 compared to the prefecture-wide risk after the fixed effects, unstructured, spatially structured, and time random effects are held constant. changing patterns and large variations among the yearly specific spatial distributions are shown in fig. 8 . it is interesting that most of the higher-risk areas were western areas of china before 2009, and most of the higher-risk areas are eastern or northern areas of china after 2009. , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). (2019) 9:19650 | https://doi.org/10.1038/s41598-019-56104-8 www.nature.com/scientificreports www.nature.com/scientificreports/ based on the incidence data of influenza gained from the chinese notifiable infectious disease reporting system, we used the bayesian spatiotemporal model in this study to assess the space-time patterns of the influenza epidemic at the prefecture level in mainland china from 2005 to 2018 and explored several factors that may be associated with the changing spatial and temporal patterns in the influenza incidence risk. several potential factors may be associated with the rapid increasing trend of influenza in china. first, insufficient flu vaccine supplements and a low uptake rate might be associated with an increase in influenza incidence. the results of the final spatiotemporal model showed that every million increase in the number of influenza vaccines approved for sale by the china food and drug administration was associated with a 12.7% decrease in the influenza incidence risk (95% ci = 0.825-0.923). the rapidly increased crude rates of influenza from 2016 to 2018 coincided with a large reduction in the numbers of vaccines approved for sale at the same time. the reductions in the numbers of vaccine supplements were mostly due to the outcomes of vaccine scandals related to improper vaccine storage and production in 2016 and 2018, respectively 9,10,37 . previous studies reported that uptake figures of the influenza vaccine averaged 1.9% nationally and 4.3% among urban elderly aged 60 years and above in 9 cities of china during the 2008-2009 and 2011-2012 influenza seasons, respectively 7, 8, 20 . it is expected that the uptake may be even lower, as people lost their faith in the safety of domestically produced vaccines after the vaccine scandals in china 38 . our results are consistent with the study in italy, which reported an association between vaccination coverage decline and influenza incidence among italian elderly 39 . , which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ second, currently circulating influenza strains in humans include influenza a (h1n1)pdm09, influenza a (h3n2) and influenza b viruses, (b/victoria and b/yamagata) 5, 40, 41 . influenza a (h1n1)pdm09 has been reported to be the predominant subtype in recent years according to ili surveillance and is more likely to be the major cause of regional and widespread outbreaks 40 . our study showed that for every 10% increase in the rate of influenza a (h1n1)pdm09 among all processed specimens from ili patients, there was a 19.5% increase in the influenza incidence risk (95% ci = 1.005-1.413). shu et al. reported that the predominant subtype of seasonal influenza a (h1n1) and b/yamagata could circulate from the south to the north of china from 2006 to 2009 34 . our study also found that every one degree increase in latitude and longitude was associated with a 1.5% (95% ci = 0.980~0.991) and 0.2% (95% ci = 0.997~0.999) increase in the influenza incidence risk, respectively. this result was consistent with the role of climatic factors in influenza transmission dynamics 20, 42 . third, the greater effort in influenza surveillance and the use of new technologies may account for the rise in influenza incidence. in recent years, especially after the 2009 pandemic season, influenza surveillance has been expanded worldwide, as recommended by the world health organization (who) [43] [44] [45] 33, 34, 41 . as cnidrs includes all sentinel hospitals, sentinel hospitals are likely to report more cases of influenza to cnidrs. in addition, more hospitals have used electronic health information systems, which may improve both the quantity and quality of data collection and exchange from hospitals to cnidrs [46] [47] [48] [49] . fourth, the reporting on influenza a (h1n1)pdm09, avian influenza a (h7n9), highly pathogenic avian influenza (hpai) h5n1 and avian h6 influenza has increased in recent years 12 , which included all variables in the univariate analysis models; which included all variables in the univariate analysis models; an intercept (α); a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). www.nature.com/scientificreports www.nature.com/scientificreports/ media and public health campaigns against the new emerging virus have caused both the government and the public to be more concerned about influenza. the improved public perception of influenza may change people's health-seeking behaviours, especially in the epidemic seasons 52, 53 . furthermore, enlarged coverage of health care insurance in both urban and rural areas in recent years in china may also induce people to use more health services 54, 55 . a rapid increase in the numbers of airlines and high-speed railway transports in china has been reported in recent years 56 . these factors would make it easy to transmit the influenza virus at a larger scale and in a shorter time across the country 56-58 . the spatial pattern. the bym model includes both a spatial conditional autoregression component and a heterogeneous random effect component. this structure allows us to know how much of the residual disease risk is due to spatially structured variation and how much is unstructured overdispersion 22 . the spatially structured conditional autoregression term demonstrated areas of spatial patterning and similarity among prefectures. the results of spatially structured variation show a distinguished spatial pattern of risk of influenza across prefectures in china. the highest-risk areas clustered in the middle part of china, while the lowest-risk areas were distributed in the east, northwest and southwest. different patterns of influenza between the north and south in china were well reported 3, 16, 20, 34, 41, 59 . in china, the line following the qinling mountain range in the west and the huaihe river in the east is often used to split the mainland into the north and the south 34 . in this study, we observed clustering in both the north and the south in the middle part of china. the unique structured spatial patterns may be attributed to the shared risk factors among the neighbouring areas. this may be associated with similarities in the climatic zone, the predominant subtype of the virus at the time of epidemics, socioeconomic background or lifestyles. the last important factor should not be ignored. some studies reported that clustering of diseases may be a consequence of spatial heterogeneity in surveillance efforts 60,61 . the space-time interaction. the space-time interaction is a random effect term, which is interpreted as the residual effect after the unstructured, spatially structured and time effects are modelled and represent sporadic short-term outbreaks or clusters. the changes and circulations of virus subtypes may determine the characteristics of the space-time interaction terms. the year 2009 was the critical point according to the results of the spatiotemporal analysis. there are four types of ili activities: sporadic, local outbreak, regional outbreak and widespread outbreak in flunet (www. who.int/flunet), global influenza surveillance and response system (gisrs) 62, 63 . since the first case of influenza a (h1n1)pdm09 was reported on may 9, 2009, in mainland china, the type a (h1n1)pdm09 virus has been detected in all ili activities according to the data from flunet. the yearly ili activities may be partially associated with the changes and similarities in the patterns of the space-time interactions from 2005 to 2018. from the flunet data mentioned above, we found that sporadic ili activities were dominant in 2005, 2006, 2007 and 2008. correspondently, we found more areas with high relative risk in these 4 years in the space-time term. this implies that the more sporadic the activities are, the larger the variations in the spatiotemporal distribution of the risk of influenza. in contrast, the large outbreaks account for most ili activities in the years 2009, 2010, 2017 and 2018. few prefectures were observed to have a relative risk greater than 2 or 3 during that period. large outbreaks, especially large regional and widespread outbreaks, may reduce the differences in the incidence risk of influenza among the areas and times on a large scale. strengths. this work adds to the existing research on influenza epidemiology in the following ways. first, the study initially presents the spatiotemporal distributions with higher-resolution spatial data than has been reported in china for the last 14 years, which allows more opportunity for focused investigations and interventions. next, we used the exceedance probabilities instead of the observed risk estimates to identify those areas for which the increased risk was highly unlikely to be due to chance. then, this study also provided a baseline model that can be extended to include social, economic, ecological, and environmental factors, as well as intervention measures to explore their associations with influenza. finally, the methods in this study offer practical tools for spatial analysis of other notifiable infectious diseases in cnidrs. there are some limitations to this study. cnidrs is a passive surveillance system, and accessibility to health facilities and patient visit behaviour may affect the number of cases reported. we collected both clinically diagnosed and laboratory-confirmed cases in cnidrs, so misdiagnosis and misreporting are unavoidable because it is difficult to distinguish influenza from other respiratory viruses without laboratory testing, especially in the non-epidemic seasons. this paper outlined the application of the bayesian spatiotemporal model to assess the relative disease risk of influenza at the prefecture level in mainland china. we observed an increased incidence trend of influenza from 2005 to 2018 that was fairly steady in the first 4 years and increased rapidly in the last 3 years. clusters of prefectures with high relative risk values concerning influenza incidence were identified in the central part of china. the identification of high-risk areas is especially a priority in china because the limited resources available for disease control need to be focused on the places most in need. we hypothesize that the insufficient flu vaccine supplements, low vaccine uptake, the newly emerging influenza a (h1n1)pdm09 and expansion of influenza surveillance efforts might be the major causes of the dramatic changes in outbreak and spatiotemporal epidemic patterns. future research with more risk factors at the national and local levels is necessary to explain the changing spatiotemporal patterns of influenza in china. model specifications for spatiotemporal analysis. the besag york mollié (bym) convolution model was used as a baseline model 22 . using the notation of banerjee et al. 65 , the bym model is as follows: • n is the number of areas. the y i counts of influenza cases in area i are independently identically poisson distributed. θ i is the risk for area i. e i is the number of expected cases of influenza in area i, which acts as an offset. • α quantifies the average incidence risk of influenza in all the prefectures. • ν i is a spatially unstructured random effects component that is i.i.d normally distributed with mean zero. • υ i is a spatially structured component using an intrinsic conditional autoregressive structure (icar). the random effect for each area ζ i is thus the sum of a spatially structured component υ i and an unstructured component ν i . it is termed a convolution prior 22, 66 . the bym model was extended to include a linear term for space-time interaction and a nonparametric spatiotemporal time trend. possible random effects specifications for the temporal term include a linear time trend (β j ), a random time effect (γ j ), a first-order random walk (γ 1j ), a second-order autoregression (γ 2j ), etc. 25 . four types of interactions are proposed in knorr-held (2000) 28 , see knorr-held (2000) 28 for a detailed description. in this study, we assume no spatial and temporal structure on the interaction, and therefore, δ ij ∼ normal(0; τ δ ). four candidate models were tested and compared: in model 4, the space-time interaction is a random effect term and is interpreted as the residual effect after the unstructured, spatially structured and time effects are modelled and represent sporadic short-term outbreaks or clusters. model selection was based on deviance information criteria (dic), which take into consideration the posterior mean deviance, a bayesian measure of model fit, and the complexity of the model. a smaller dic indicates a better fit of the model 67 . the final linear model consisted of an intercept (α); a vector of national-level explanatory variables ∑ β = ( x ) k 1 n k k for the yearly total number of lot release of influenza vaccines by the china food and drug administration, the positive rate of influenza a (h1n1)pdm09 among the number of ili specimens processed, the percentage of influenza a (h1n1)pdm09 among all the positive influenza specimens, and protocol changes; a spatially unstructured random effect term (ν i ); a spatially structured conditional autoregression term (υ i ); a first-order random walk-correlated time variable (γ 1j ); and an interaction term for time and place (δ ij ). the prefecture-specific structured and unstructured spatial risks of influenza compared to the whole spatial risk of all prefectures are obtained by applying an exponential transformation to the components of ν i and υ i , respectively. the relative risk of space-time interaction is computed by the exponentiation of the term δ ij . the exceedance probabilities of spatial risk and risk of space-time interaction were also calculated. the exceedance probability represents the posterior probabilities for an area's spatial risk estimate exceeding some pre-set value and has been proposed as a bayesian approach to hotspot identification 68, 69 . all spatial models were computed using integrated nested laplace approximations (inla), which have been developed as a computationally efficient alternative to mcmc 70 . all spatial analyses were conducted within microsoft r open version 3.5 using the r-inla package (version 18.07.12). ethics approval. the authors assert that all of the procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and the helsinki declaration of 1975 as revised in 2008. this article does not contain any studies of human or animal subjects performed by any of the authors. since this analysis was based on anonymous aggregated statistical data, patient informed consent and ethical committee approval were not required in china. disclaimer. the views expressed are those of the authors and do not necessarily represent the official policy of the chinese center for disease control and prevention. the burden of influenza: a complex problem the substantial hospitalization burden of influenza in central china: surveillance for severe, acute respiratory infection, and influenza viruses estimates of global seasonal influenza-associated respiratory mortality: a modelling study pandemic influenza: certain uncertainties temporal patterns of influenza a and b in tropical and temperate countries: what are the lessons for influenza vaccination? plos one 11, e0152310 seasonal influenza vaccine supply and target vaccinated 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models: development and evaluation with a focus on cluster detection approximate bayesian inference for latent gaussian models by using integrated nested laplace approximations this study was supported by grants from the key joint project for data center of the national natural science j.q. ma. conceived, designed, and supervised the study. y.w. zhang., x.f. wang. and y.f. li. collected and cleaned the data. y.w. zhang. analysed the data and wrote the drafts of the manuscript. j.q. ma. and y.w. zhang. interpreted the findings. all authors read and approved the final manuscript. the authors declare no competing interests. correspondence and requests for materials should be addressed to j.m. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-010959-sigw7yxk authors: lampejo, temi title: influenza and antiviral resistance: an overview date: 2020-02-13 journal: eur j clin microbiol infect dis doi: 10.1007/s10096-020-03840-9 sha: doc_id: 10959 cord_uid: sigw7yxk influenza affects approximately 1 billion individuals each year resulting in between 290,000 and 650,000 deaths. young children and immunocompromised individuals are at a particularly high risk of severe illness attributable to influenza and these are also the groups of individuals in which reduced susceptibility to neuraminidase inhibitors is most frequently seen. high levels of resistance emerged with previous adamantane therapy for influenza a and despite no longer being used to treat influenza and therefore lack of selection pressure, high levels of adamantane resistance continue to persist in currently circulating influenza a strains. resistance to neuraminidase inhibitors has remained at low levels to date and the majority of resistance is seen in influenza a h1n1 pdm09 infected immunocompromised individuals receiving oseltamivir but is also seen less frequently with influenza a h3n2 and b. rarely, resistance is also seen in the immunocompetent. there is evidence to suggest that these resistant strains (particularly h1n1 pdm09) are able to maintain their replicative fitness and transmissibility, although there is no clear evidence that being infected with a resistant strain is associated with a worse clinical outcome. should neuraminidase inhibitor resistance become more problematic in the future, there are a small number of alternative novel agents within the anti-influenza armoury with different mechanisms of action to neuraminidase inhibitors and therefore potentially effective against neuraminidase inhibitor resistant strains. limited data from use of novel agents such as baloxavir marboxil and favipiravir, does however show that resistance variants can also emerge in the presence of these drugs. the world health organization estimates that annually there are approximately 1 billion human influenza cases of which 3 to 5 million are considered severe (especially in children, the elderly and in the immunocompromised) and result in 290,000 to 650,000 deaths [1] . influenza can be transmitted through the following routes: 1. respiratory droplets (> 5 μm) generated e.g. by coughing and sneezing. these do not remain suspended in the air and settle to the ground within 1-2 m 2. contact transmission either through direct transfer of infectious particles from an infected to an uninfected individual or indirectly via contaminated surfaces or objects (i.e. fomites) and influenza can survive for hours on nonporous surfaces 3. possibly by airborne transmission via small aerosols (< 5 μm) generated from breathing/talking (and can remain suspended in the air for minutes to hours) [2] ; however, there is limited data to suggest that infectious particles can be transmitted over long distances (and special air handling and ventilation systems are not considered necessary to prevent spread) influenza belongs to the orthomyxovirus family and there are four influenza types a to d of which only influenza a, b and c can infect humans (influenza c is rare and usually causes a mild upper respiratory tract illness) [3] . influenza a and b contain 8 pieces of segmented single-stranded rna which encode various proteins including haemagglutinin (which facilitates attachment to the host cell) and neuraminidase (which facilitates release of new virus particles from the host cell). influenza a has the broadest host range of the influenza viruses and significant interspecies transmission occurs [4] . eighteen haemagglutinin (h) and 11 neuraminidase (n) subtypes have been described in influenza a (of which 16 h and 9 n subtypes have also been detected within avian species) [5] . influenza b is far less genetically diverse than influenza a and has no distinct antigenic subtypes (mutates 2 to 3 times slower than influenza a and apart from humans, only seals and ferrets have demonstrated susceptibility) [6] [7] [8] . influenza achieves antigenic diversity via two main mechanisms: 1. antigenic drift where mutations readily occur in ha and na resulting in new antigenic variants (thus avoiding preexisting host immunity); the error prone nature of the viral polymerase is a significant factor in this 2. antigenic shift due to reassortment of gene segments between two distinct influenza viruses within the same host giving rise to a novel strain the 1918 influenza a h1n1 pandemic is thought to have arisen from reassortment between human and avian strains (based on sequencing of fixed, frozen lung tissue from victims) and similarly, the most recent 'swine flu' influenza a h1n1 pandemic was thought to arise from a series of reassortment events between human influenza a h3n2, swine influenza a h1n1 and avian influenza a h1n2 [9, 10] . lack of influenza b infection in several other species may explain why antigenic shift is not seen with influenza b [11] . this potential for vast genetic variability within influenza viruses and their highly error-prone rna dependent rna polymerase does raise concerns regarding the possible emergence of treatment resistant strains and generates further questions regarding their viral fitness and transmissibility as well as which strategies to employ in rapidly identifying and effectively treating these resistance variants. this article discusses these issues including novel agents and experimental strategies that have been used in an attempt to treat as well as prevent the emergence of resistant influenza viruses in humans. the mechanism of action of the adamantanes is by blocking the m2 ion channel of influenza a thus preventing viral uncoating and the subsequent release of influenza a viral rna into the host cell. they have activity against influenza a but not influenza b (due to their lack of the m2 protein, influenza b has an alternative ion channel called bm2) [12] . amantadine was approved for clinical use in 1966 and subsequently rimantadine in 1993. both drugs were initially very effective in treating and preventing influenza a infection with efficacy rates of up to 90%. the resistance of influenza a to amantadine was first recognised during the 1980 influenza a epidemic [12] . however, resistance to both drugs in seasonal influenza a subtypes was rare (1-2% frequency) until after 2000 when there was a dramatic rise in rates of resistance. by 2013, approximately 45% of all influenza a subtypes in circulation globally were resistant to the adamantanes (> 69% of h1 subtypes and 43% of h3 subtypes) [11] . resistance (as a result of the s31n mutation in the m2 protein) to the adamantanes occurs rapidly within 3-5 days of use and occurs in 30-50% of both immunocompetent and immunocompromised patients [13, 14] . due to such high levels of resistance, the adamantanes are no longer recommended for treatment of influenza a [15] . neuraminidase inhibitors are currently first-line treatment for both influenza a and b in the united kingdom (uk) and most (if not all) parts of the world. they competitively inhibit neuraminidase on the surface of influenza a and b. they act by preventing cleavage of sialic acid residues on budding newly formed virus particles thus preventing release of new virus particles from infected host (ciliated epithelial) cells. resistance occurs much less readily in comparison with the adamantanes [16] . the neuraminidase inhibitors, if given within 36 h of onset, have been shown to reduce the duration of illness by 30% (with an approximately 40% reduction in illness severity) and, if given within 24 h of symptom onset, even greater reductions in the duration of illness attributable to influenza (approximately 44% reduction) have been observed [17, 18] . a decrease in the incidence of secondary complications, such as otitis media, sinusitis and pneumonia, with the use of neuraminidase inhibitors was also demonstrated [17] . additionally, when used as prophylaxis (before or shortly after exposure), neuraminidase inhibitors can reduce the incidence of infection by approximately 70-90% [19] . in the uk, oseltamivir (oral agent; typically first line for influenza a and b treatment) and zanamivir (inhaled and also available as an aqueous solution which can be administered intravenously or via nebuliser) are licensed for the treatment of influenza a and b and also for prophylaxis. oseltamivir is licensed in the uk for use in all ages including neonates, whereas zanamivir is not licensed for children under the age of 5 years. peramivir (a single-dose intravenous infusion) was licensed in the uk in 2018 but has not been marketed/launched (it is used in the usa, japan and south korea). laninamivir (an inhaled neuraminidase inhibitor) is licensed for use in japan. based on the data from animal models which demonstrated that oseltamivir-resistant viruses were unfit and poorly transmissible, resistance to neuraminidase inhibitors was not envisaged to become an important clinical issue [20] . prior to 2007, oseltamivir resistance was rarely seen in clinical practice (and low resistance rates of 1-5% were reported in clinical trials) [21] . human cases of oseltamivir-resistant influenza a h1n1 began emerging during the 2007-2008 influenza season. many of the cases reported were individuals who had not taken oseltamivir demonstrating that resistant virus could be efficiently transmitted between humans [22] . during the 2008-2009 influenza season, in some parts of the world, such as the usa, canada, the uk and australia, very high rates (> 90%) of oseltamivir-resistant seasonal influenza a h1n1 strains were seen [23, 24] . in 2009, the pandemic (pdm09) influenza a h1n1 strain emerged worldwide but in the majority of cases (< 1.5% resistance) remained oseltamivir susceptible initially. oseltamivir resistant h1n1 pdm09 cases did emerge but this was mostly immunocompromised patients that had received oseltamivir. subsequently, however, in the 2010-2011 influenza season in the uk (and other parts of the world), increasing numbers of oseltamivir resistant cases were identified with no previous oseltamivir exposure [25] . resistance to oseltamivir was still seen with much greater frequency in immunocompromised individuals receiving oseltamivir (with very little cross-resistance seen with zanamivir). the neuraminidase mutation responsible for the oseltamivir resistance that emerged in the seasonal influenza a h1n1 (a/brisbane/59/2007-like) strain in 2007 and then in the 2009 h1n1 pdm09 and also subsequent h1n1 strains is the h275y mutation; a histidine to tyrosine substitution at amino acid 275 of the influenza a n1 neuraminidase [26, 27] . normally when oseltamivir binds to the neuraminidase on the influenza virion, the neuraminidase active site changes shape to accommodate oseltamivir. a neuraminidase mutation, such as h275y, prevents this conformational change in the active site and therefore, oseltamivir is unable to bind. zanamivir, however, does not require this structural change in the neuraminidase active site in order to bind [28] . the h275y mutation reduces the susceptibility (ic 50 ; the half maximal inhibitory concentration) of influenza a h1n1 to oseltamivir by approximately 400-fold but not zanamivir [13, 29] . peramivir binds to sialic acid residues in a similar manner to oseltamivir and is also affected by the h275y mutation [30] . this mutation has been shown to persist even after cessation of treatment, and strains harbouring this mutation are capable of causing outbreaks and significant morbidity and mortality in a similar fashion to their wild-type counterparts [14, 27] . children and severely immunocompromised patients are at higher risk of developing resistance most likely due to higher viral loads and prolonged viral shedding [31] . due to certain difference in the neuraminidase enzyme structure, neuraminidase resistance is less likely to occur in influenza a h3n2 and influenza b compared with a h1n1 pdm09 without causing significant loss of neuraminidase enzymatic function and reduced viral fitness [32, 33] . in vitro and in vivo studies have demonstrated that influenza a h3n2 and influenza b neuraminidase inhibitor resistant strains have a lower replicative capacity and less ability to transmit. in many of the case reports of influenza a h3n2 and b resistant strains, resistance only occurs after prolonged treatment (> 10 days but over 1 month in many) and these resistant variants often disappear once treatment is ceased [34, 35] . public health england (phe) publishes the most frequently observed influenza a and b mutations and their neuraminidase inhibitor resistance profiles in their 'surveillance and laboratory testing of influenza neuraminidase inhibitor resistance' reports [36] . table 1 summarises the centers for disease control and prevention (cdc) and phe resistance data for the usa and uk, respectively. the influenza resistance information study (iris) was a multicentre global observational study of neuraminidase inhibitor resistance and clinical outcomes in immunocompetent patients conducted from 2008 to 2013 [37] . this study included patients over 1 year of age presenting within 24 hours of an influenza-like illness and/or had a positive rapid influenza test. nose throat swabs were collected on days 1, 3, 6 and 10 for influenza typing/subtyping, sequencing and neuraminidase inhibitor phenotypic susceptibility testing. there were 3230 influenza a and b reverse-transcription polymerase chain reaction (rt-pcr) positive patients in the study. except for 30 patients with pre-treatment (i.e. transmitted) resistant influenza a h1n1 strains, no resistance was detected in day 1 samples. emergence of oseltamivir resistance after day 1 was detected in 43/1207 (3.56%) of oseltamivir-treated influenza a positive patients; a higher frequency was seen in 1-5 year old (11.8%) compared with those over 5 years (1.4%). all resistant h1n1 viruses had the h275y mutation and all resistant h3n2 viruses had the r292k mutation (conferring reduced susceptibility to both oseltamivir and zanamivir). virus clearance was a median of 8.1 days for treated patients with oseltamivir-resistant virus vs 9.9 days for untreated patients vs 10.9 days for treated patients with oseltamivir-resistant virus. time to alleviation of symptoms was 1 day shorter in treated patients as compared with untreated patients. interestingly, the oseltamivir-resistant treated group exhibited the shortest duration of symptoms (symptoms resolved by day 6 or earlier). dual resistance to oseltamivir and zanamivir is rare. a literature review by abed et al. identified 14 published cases of human influenza a and b infections with mutations conferring reduced susceptibility to both oseltamivir and zanamivir [38] . seven had influenza a h1n1 pdm09, 4 had influenza a h3n2, 1 had avian influenza a h7n9 and 2 had influenza b. the age range was 8 months to 88 years and 12 out of the 14 patients were immunocompromised. the other two patients had underlying chronic lung disease. thirteen out of 14 patients had received neuraminidase inhibitor therapy before emergence resistance mutations (5 oseltamivir alone, 2 oseltamivir then zanamivir, 2 zanamivir alone, 3 zanamivir and oseltamivir simultaneously, 1 oseltamivir then peramivir). there was a mean of 12.76 days (range 0-72 days) treatment before detection of the first mutation. mortality was high at 71% (10/14 patients). of the 4 survivors, there was an immunocompetent asthmatic child with h1n1 pdm09 who received no treatment, 1 immunocompromised adult with h1n1 pdm09 who received oseltamivir for 14 days then inhaled zanamivir for 40 days, 1 immunocompromised adult with h3n2 who had 5 days of oseltamivir and 1 immunocompromised child with h3n2 who received oseltamivir for 3 months and then inhaled zanamivir for 72 days. in a recently published uk series of three cases of oseltamivir-resistant influenza a h1n1 pdm09 that occurred in england in immunocompetent patients in the 2018-2019 influenza season, two of the patients (a 7-week old previously well boy and a 39-year old asthmatic woman) made a good recovery with 5 days of oseltamivir despite whole genome sequencing revealing h275y mutations in 44% and 100% of the virus population, respectively [39] . the third case was a 15-month old girl with a developmental condition (najer syndrome) admitted with a 1-day history of respiratory illness who showed minimal clinical improvement with 15 days of oseltamivir after which she was switched to intravenous zanamivir (as well as receiving antibiotics) and died soon after. phe sequencing data later revealed h275y mutations in 35% of the virus population in a day 5 nasopharyngeal aspirate specimen and this rapidly rose to 80% of the virus population harbouring h275y mutations 1 day later in a day 6 nasopharyngeal aspirate sample. the treatment of influenza as recommended by phe is oseltamivir as first line for immunocompetent children and adults [40] . for immunosuppressed patients, neuraminidase inhibitor choice is based on the dominant circulating strain in that particular season; oseltamivir is recommended when the dominant circulating strain is of lower risk for oseltamivir resistance (i.e. influenza a h3n2 or b) and zanamivir is recommended when the dominant strain has a higher risk of oseltamivir resistance (e.g. h1n1 pdm09). currently, available phe date for the 2019-2020 influenza season indicates that influenza a h3n2 is the dominant circulating strain [41] . in many laboratories, typing/subtyping results are becoming available early in the treatment course therefore guiding selection of treatment. neuraminidase inhibitor susceptibility testing should be considered particularly in young children and immunocompromised patients being treated with a neuraminidase inhibitor for influenza (especially h1n1 pdm09), who are not responding to treatment and/or have persistently high viral loads (low cycle threshold values using rt-pcr) and/or exposed to a suspected or confirmed resistant case. given that resistance variants can emerge within only 1-2 days of treatment (as well as can be transmitted), resistance testing can be performed at any time prior to, during or after treatment. in england, phe offers both genotypic and phenotypic influenza a and b susceptibility testing which is summarised in table 2 [36] . relatively newer techniques also exist such as pyrosequencing, a high-throughput sequencing method that is able to type/subtype, screen for mutations and delineate the relative proportions of the various influenza variants [42, 43] . another molecular technique, digital pcr (using a droplet-based system) has been shown to be highly accurate and precise in the identification and quantification of influenza sequence variants with the ability to detect rare single nucleotide polymorphisms present at levels as low as 0.001% of the virus population [44] [45] [46] . from a direct clinical perspective, identification of variants at such low proportions may not be relevant but these techniques may provide further insights into the viral dynamics in the emergence of resistant influenza viruses. baloxavir marboxil is a single dose oral agent for the treatment of influenza a and b (no data for it is used of prophylaxis). it was licensed in japan and usa in 2018 but is not currently licensed in the uk. it suppresses influenza replication by inhibition of cap-dependent endonuclease (an enzyme required for initiation of influenza mrna synthesis) and therefore, its mechanism of action is different to that of the neuraminidase inhibitors [47] . there is limited data on resistance but a recent us/japan randomised controlled study of healthy adults/adolescents with influenza a and b treated with baloxavir marboxil found that 9.7% (36/370) developed a specific mutation (pa/i38x) 3-9 days after treatment and that the emergence of these pa/i38x variants was associated with higher viral loads, prolonged detection of virus and a longer duration of symptoms compared with baloxavir marboxil treated individuals who did not develop the pa/i38x mutation [48] . favipiravir is an oral (and intravenous) antiviral which inhibits rna-dependent rna polymerases [49, 50] . it has been approved for the treatment of influenza a and b in japan with very strict regulation for clinical use and is intended to be reserved for pandemics causes by novel/re-emerging influenza strains resistant to other antivirals. a recent study has demonstrated that a specific k229r mutation in the pb1 subunit of the influenza virus polymerase results in reduced susceptibility to favipiravir in vitro and in cell culture. viral fitness, which was demonstrated to be impaired by this mutation, can be restored by a compensatory second (p653l) mutation [51] . the effects or these mutations in a clinical setting are yet to be determined. human clinical trials of dual therapy with oseltamivir plus zanamivir for h1n1 have been investigated in various settings (including in ecmo patients) with varying outcomes and no clear benefit in terms of clinical outcome or prevention of drug resistance [52] [53] [54] . studies in mice also failed to show a benefit [55] . double dose oseltamivir did not reduce the risk of emergence of oseltamivir resistance in patients with influenza a h1n1 pdm09 [56] . this study was a small (n = 52), randomised trial of patients treated in community. one patient in the single dose group and one in the double dose group developed oseltamivir resistance. there was no mention of any immunocompromised patients in the study. a triple-combination of amantadine, oseltamivir and ribavirin (tcad regimen) was shown in vitro and in the mouse model to have synergistic activity against sensitive and resistant influenza viruses (with greater synergy than any double antiviral regimen) [57] . there was a phase i pilot study of tcad in 2013 to assess pharmacokinetics and safety in 6 immunocompromised patients with influenza a (h1n1 & h3n2). five out of 6 tolerated and completed the 10-day course of treatment (1 patient had worsening respiratory failure and tcad was stopped) [58] . a clinical response and a corresponding viral load reduction in the 5 patients that completed treatment. no drug-drug interactions were seen and no haematological toxicity was seen with ribavirin. no new resistance mutations emerged on treatment. a study of tcad vs oseltamivir in critically ill mechanically-ventilated patients with pandemic h1n1 showed that tcad was well tolerated but did not improve outcomes compared with oseltamivir alone [59] . an in vitro study in 2016 of a zanamivir-oseltamivir hybrid inhibitor (ms-257) showed effectiveness against neuraminidase inhibitor-resistant influenza strains but there have been no further published studies to date [60]. oseltamivir resistance is rare and zanamivir resistance is extremely rare. the presence of resistant virus does not necessarily mean a more severe infection and/or worse outcome, particularly in immunocompetent adults. in some instances, neuraminidase inhibitor resistant virus may actually be less fit (especially with h3n2 and influenza b). there is an increased risk of resistance in the immunocompromised (but resistance does occur in the immunocompetent) and young children (< 5 years); this is primarily with influenza ah1n1 pdm09 but can occur less commonly with influenza a h3n2 and b. neuraminidase inhibitor susceptibility testing should be considered primarily in h1n1 infected immunocompromised patients and young children failing to respond to treatment, and clinicians should consider zanamivir in the intravenous form for patients that are critically ill/developing severe complications. there should potentially be a higher threshold for neuraminidase inhibitor susceptibility testing in influenza h3n2 or b infected patients. this should generally be reserved for those that have had extended treatment (at least 10 days) and not responded/deteriorated with persistent low ct values and no other identifiable cause. finally, there is no clearly proven benefit from 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neuraminidase inhibitor-resistant influenza a(h3n2) viruses detection of rare drug resistance mutations by digital pcr in a human influenza a virus model system and clinical samples quantitative and sensitive detection of rare mutations using droplet-based microfluidics characterization of oseltamivir-resistant influenza virus populations in immunosuppressed patients using digital-droplet pcr: comparison with qpcr and next generation sequencing analysis baloxavir: first global approval treatment-emergent influenza variant viruses with reduced baloxavir susceptibility: impact on clinical and virologic outcomes in uncomplicated influenza mechanism of action of t-705 against influenza virus favipiravir (t-705), a novel viral rna polymerase inhibitor the mechanism of resistance to favipiravir in influenza failure of combination oral oseltamivir and inhaled zanamivir antiviral treatment in ventilator-and ecmo-treated critically ill patients with pandemic influenza a (h1n1)v efficacy of oseltamivir-zanamivir combination compared to each monotherapy for seasonal influenza: a randomized placebo-controlled trial oseltamivir-zanamivir combination therapy suppresses drug-resistant h1n1 influenza a viruses in the hollow fiber infection model (hfim) system oseltamivirzanamivir combination therapy is not superior to zanamivir monotherapy in mice infected with influenza a(h3n2) and a(h1n1)pdm09 viruses a randomized study of standard versus double dose oseltamivir for treating influenza in the community triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro combination therapy with amantadine, oseltamivir and ribavirin for influenza a infection: safety and pharmacokinetics triple-combination antiviral drug for pandemic h1n1 influenza key: cord-003232-nquw7qga authors: kuchipudi, suresh v.; nissly, ruth h. title: novel flu viruses in bats and cattle: “pushing the envelope” of influenza infection date: 2018-08-06 journal: vet sci doi: 10.3390/vetsci5030071 sha: doc_id: 3232 cord_uid: nquw7qga influenza viruses are among the major infectious disease threats of animal and human health. this review examines the recent discovery of novel influenza viruses in bats and cattle, the evolving complexity of influenza virus host range including the ability to cross species barriers and geographic boundaries, and implications to animal and human health. influenza viruses are known to constantly evolve and cross species barriers. the genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. the purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. in addition, we discussed the growing complexity of influenza virus-host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses. influenza is among the major infectious disease problems affecting animal and human health globally. several human influenza pandemics have been recorded since 1590 ad [1] , with the most significant of those being the "spanish flu" of 1918, often referred to as the "mother of all pandemics" [2] . spanish flu pandemic is believed to have affected approximately 25-30 percent of the world's population and caused more than 50-60 million human deaths globally [3] . influenza infections in humans occur either as epidemic (seasonal or interpandemic) influenza caused by influenza a and b viruses, or as sporadic pandemic influenza caused by influenza a viruses [4] . study of influenza pandemics has been of great interest to epidemiologists. influenza epidemics and pandemics have been repeatedly occurring for centuries, but to date the ability to predict a pandemic has not been achieved [5] . influenza viruses belong to the family orthomyxoviridae (from the greek orthos, meaning "standard, correct", and myxa, meaning "mucus"). members of orthomyxoviridae are characterized by virions that are either spherical or pleomorphic measuring 80-120 nm in diameter. genomes are comprised of single-stranded negative-sense rna that is arranged in either eight, seven or six segments depending on the genus. classification of the historically described a, b and c influenza viruses based on antigenic differences in nucleocapsid (np) and matrix (m1) proteins. influenza a viruses (iavs) are further classified into subtypes based on the antigenic properties of the external glycoproteins namely iavs are known to infect humans and a wide variety of animals including pigs, horses, minks, seals, whales and birds [19] . avian influenza viruses (aivs) continue to spread, evolve and cause significant economic losses to poultry industries globally. unlike low pathogenic avian influenza viruses (lpaivs) that cause mild clinical signs in domestic poultry, infection of gallinaceous poultry with highly pathogenic avian influenza viruses (hpaivs) results in severe fatal disease often causing up to 100% mortality within two-three days [20] . aivs have also been implicated in the generation of influenza viruses that have caused all human influenza pandemics. in particular, human infection with certain contemporary eurasian lineage hpai h5n1 viruses frequently caused severe disease with a case fatality rate of up to 50% [21] . ability to infect a wide range hosts is a key contributing factor to the complex and seemingly expanding genetic diversity of iavs. it is now well established that iavs infect domestic pets such as dogs and cats, adding to the list of host species that could potentially expose humans to influenza viruses. an h3n8 equine influenza virus in 1999 and an avian virus-like h3n2 strain around 2005 or 2006 were transmitted to dogs, and these canine influenza viruses have been circulating in the u.s. dog population ever since [22] . after the first report of pandemic h1n1 influenza a virus (ph1n1) infection of cats in italy in 2011 [23] , more than 500 cats became infected with influenza subtype h7n2 in animal shelters in new york, ny, usa during 2016-2017 [24] . while transmission of these animal viruses to humans has not yet been documented, the close contact of many humans with pet dogs and cats presents an increased risk of opportunity for such a host jump. transmission of iavs between animals and humans is bidirectional such that humans are infected by animal influenza viruses and human influenza viruses have been shown to transmit to animals. in particular, studies established that domestic pets are susceptible to infection by human influenza viruses. for example, dogs experimentally inoculated with a human seasonal h3n2 or pandemic (pdm) h1n1 (2009) showed nasal shedding and seroconversion. however, dogs inoculated with influenza b virus did not exhibit virus shedding or seroconversion highlighting that interspecies transmissibility is a key feature of iavs and not ibvs [25] . aquatic birds such as ducks, gulls, and shorebirds have historically been identified as the primary reservoir of all the known iav subtypes [26] . recently, northwest atlantic gray seals were suggested as an endemically infected wild reservoir population for diverse influenza viruses [27] . arguably, influenza virus host range could be much broader than currently known, with additional reservoirs that are yet to be revealed. prior to 2011, 16 antigenically different ha (h1-h16) and nine different na (n1-n9) types had been described, all of which were found in the aquatic bird reservoir. however, the number of ha and na types have now expanded after the identification of two novel influenza-like viruses in fruit bats. the first was in a frugivorous yellow-shouldered bat (sturnira lilium) in guatemala [28] . soon after, a second virus was found in the flat-faced fruit bat (artibeus planirostris) in peru [29] . the newly identified bat flu viruses are genetically distinct from all previously known iavs and hence are designated as novel subtypes namely h17n10 and h18n11 [29] . serological surveys showed wide spread prevalence of h17/h18-specific antibodies among bat populations in central and south america [29] . finding novel bat-influenza viruses is not surprising as bats represent 24% of all known mammalian species [30] and have been known to be natural reservoirs of many deadly zoonotic rna viruses including ebola virus [31] [32] [33] . it is believed that bats have the capacity to harbor more influenza virus genetic diversity than all the other mammalian and avian species combined [29] . notably, little yellow-shouldered bats in central america have been proposed as a potential sylvatic mammalian reservoir of influenza [28] . discovery of novel bat iavs raises numerous questions including the host range evolution of iavs and the role of bats in evolution of iavs. the potential for the newly discovered bat iavs to reassort with previously known iav types is being explored and providing novel information about the iav life cycle. bat flu viruses have been reconstructed by reverse genetics using synthetic dna which has allowed for more functional understanding of these viruses [34] . multiple experimental studies have shown that reassortment of bat iavs and conventional iavs does not occur under experimental conditions [35] [36] [37] . this is partly explained by functional differences between bat iavs and conventional iavs. iav nonstructural protein ns1 is a multifunctional protein that plays a crucial role in evading host immune responses and serves as a key virulence factor [38, 39] . unlike the ns1 of conventional iavs, bat ns1 fails to bind to the host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme phosphoinositide 3-kinase (pi3k) [40] . in addition, the surface glycoprotein ha and na genes of bat iavs only show low nucleotide sequence identity with those of the conventional iavs [28, 29] . notably, it has been found that the bat iav hemagglutinin (ha) and neuraminidase (na) proteins lack the receptor binding activity that are characteristic of the conventional iav [28, 41] . ha of bat iavs does not bind to the classical avian (saα2,3-gal) or human (saα2,6-gal) iav receptors highlighting the possibility of unique entry mechanisms that are yet to be identified [42] . consequently, it was found that bat iavs initiate infection at the basolateral membrane of cells unlike conventional iavs which preferential initiate infection on the apical surface of cells [34] . these findings highlight that there are major evolutionary constraints to bat iavs to effectively reassert with conventional iavs and/or infect other species including humans. while the zoonotic potential of bat iavs is not yet fully established and continues to be a focus of ongoing research, bats have been found to be susceptible to infection by conventional iavs. a recent study found serological evidence of iav h9 subtype infection in 30% of frugivorous bats tested in africa [43] . a key source of iav genetic diversity could come from the replication of iavs in a non-native host species that initiate evolution of new virus variants [44] . the receptors for previously known iavs are sialic acids (sa) on host cells [45] . consequently, the expression of these receptors on host cells is a key determinant of the ability of iavs to infect a host [46] . aivs preferentially bind to sa receptors that are linked to galactose by an α 2,3 linkage (saα2,3-gal), while human and classical swine influenza viruses show preference to α2,6 linked sas (saα2,6-gal). it is widely believed that hosts that co-express both saα2,3-gal and saα2,6-gal receptors could support reassortment of iavs and hence play a major role in the evolution of iavs [45, 47] . we have demonstrated abundant co-expression of both saα2,3-gal and saα2,6-gal receptors that are compatible with avian and human iav binding in respiratory and digestive tracts of little brown bats, the most widespread bat species in north america [48] . the potential for bats to support infection by multiple conventional iav types and thus serve as a source of novel genetic variants remains to be fully explored. although influenza viruses infect humans and a wide range of animals and birds, cattle were never considered to be susceptible to influenza virus infection. however, a novel influenza virus has recently been identified in several animals including swine, cattle, sheep, and goats. the virus was first isolated as an influenza c-like virus from pigs with respiratory illness in oklahoma, usa, in 2011 [49, 50] . the virus was subsequently classified as influenza d virus (idv), and the virus has now been reported from many countries including united states, france [51] , italy [52] , china [53] , japan [54] ireland [55] and countries in north and west africa [56] . the international committee on taxonomy of viruses (ictv), which is responsible for developing, refining, and maintaining a universal virus taxonomy, has recently released revised classification of orthomyxoviridae [57] . in earlier classification, orthomyxoviruses belonged to any one of the five genera: influenzavirus a, influenzavirus b, influenzavirus c, thogotovirus and isavirus [58] . however, the most recent classification of orthomyxoviridae includes eight genera and nine species (table 1) . genus alphainfluenzavirus comprises the species influenza a virus, betainfluenzavirus comprises the species influenza b virus, gammainfluenzavirus comprises the species influenza c virus, and delatinfluenzavirus comprises the species idv [58] . like other influenza viruses, idvs possess a negative-sense single-stranded rna genome. however unlike iavs that contain 8 segments, idvs comprise seven genomic segments that are predicted to encode nine proteins, including glycoprotein hemagglutinin-esterase fusion (he), polymerases pb2, pb1, and p3, nucleoprotein, matrix protein (m1 and cm2), and nonstructural proteins (ns1 and nep) [59] . idvs use 9-o-acetylated sialic acid as their cellular receptor on host cells such as icvs [60] . it is well established based on several epidemiological studies that cattle are the primary reservoirs of idvs [50, 59] . in addition, idvs have also been isolated from a range of animals including pigs, sheep, goat, horses and camelids. while the precise role of idvs in clinical disease in animals is not yet fully investigated, their role in causing respiratory infections in cattle has been implied. two recent studies carried out metagenomic characterizations of the virome associated with bovine respiratory disease in feedlot cattle and found correlation of idv presence with bovine respiratory disease (brd) clinical signs, raising exciting new prospects for understanding and combatting this complicated disease [61, 62] . brd complex is one of the major diseases affecting the cattle industry in the usa and around the world. productivity losses due to brd are estimated to be $23.60 per calf with an annual economic impact of more than one billion dollars to the u.s. cattle industry [61] . brd is associated by multiple pathogens and accounts for approximately 70-80% of the morbidity in the usa [63] and 84.5-99.9% of the morbidity in mexican feedlot cattle. brd results in the use of widespread therapeutics and antibiotics in feedlots, which increasingly raises public health concerns of promoting antibiotic resistance [64, 65] . pathophysiology of brd involves complex interactions between host, pathogen, environment and management factors. in feedlot cattle, brd is initiated by viral infection followed by stress due to travel which is typically followed by a secondary infection by resident bacteria [66] . viral infection can cause increased susceptibility to secondary bacterial infections by either immunosuppression or by damaging the epithelium of upper airways and injuring lung parenchyma which facilitates the migration of bacterial pathogens and colonization of the lower respiratory tract. depending on several factors, the clinical outcome of brd can be variable; however, higher morbidity and mortality are observed in the event of mixed viral and bacterial infections [67] . many viral pathogens have been implicated in brd, which include bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus and bovine parainfluenza 3. experimental studies in calves with idv showed damage that results in the induction of inflammation in the trachea. idv could be a significant player in brd and could facilitate coinfections with other bovine pathogens [59] . in addition to the animal health implications of idvs, a recent study found idv antibodies in 34 out of 35 persons that had contact with cattle and only 2 out of 11 that did not have any exposure to cattle [68] . the results of this study raised the possibility that idv could be relevant from a public health stand point and that it could pose a zoonotic risk to cattle-exposed workers. with much still to be characterized about the new idv species, exploring its impact on human an animal health through epidemiological studies will be vital to understanding spread of this virus. emerging and novel zoonotic infections often result from pathogens jumping from their original host into novel host species [69] . the host range evolution of mammalian viruses typically involves more closely related hosts [70, 71] . in particular, rna viruses with broad host range are more likely to jump between distantly related species [70, 72, 73] . a key determinant in the host range evolution of a virus is the mechanism of viral entry used by the viruses. analysis of 64 human viruses revealed that the viruses that use receptors that are highly conserved in their amino acid sequence across species have the broadest host range [74] . the ability of viruses to bind to an alternative receptor is sometimes key in species jumping. aivs need to change to preferentially bind to saα-2,6-gal receptor to efficiently transmit between humans. in experimental studies, it was shown that the shift from saα-2,3-gal to saα-2,6-gal binding requires four mutations for a ha of hpai h5n1 viruses [75] . however, several newly emerged h5n1 aivs in egypt have been found to have acquired the human receptor saα-2,6-gal binding ability during their emergence in birds [76] . influenza a, b, c and d viruses have varying susceptible host range ( figure 1) . notably, the newly discovered idvs have the widest host range after iavs. further, humans and pigs are susceptible to infection by all four types of influenza viruses. with the extensive host range that continues to grow and the zoonotic potential, influenza viruses remain a major challenge to epidemiologists. owing to their tremendous potential to affect animal and human health, there is a need to carry out in-depth and comprehensive studies to unravel the ecological complexity of influenza virus host range evolution. in particular, we feel that researchers should focus on answering the key questions, "what is the role of bats in the ecology and evolution of iavs?", "are idvs involved in the epidemic influenza infections in people?", and "are birds susceptible to infection by idvs?" vet. sci. 2018, 5, x for peer review 6 of 10 need to carry out in-depth and comprehensive studies to unravel the ecological complexity of influenza virus host range evolution. in particular, we feel that researchers should focus on answering the key questions, "what is the role of bats in the ecology and evolution of iavs?", "are idvs involved in the epidemic influenza infections in people?", and "are birds susceptible to infection by idvs?" a history of influenza influenza: the mother of all pandemics the origin and virulence of the 1918 "spanish" influenza virus reviewing the history of 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evolution and genetics of virus host shifts phylogeny and geography predict pathogen community similarity in wild primates and humans a tale of two phylogenies: comparative analyses of ecological interactions emerging pathogens: the epidemiology and evolution of species jumps host range and emerging and reemerging pathogens human viruses: discovery and emergence experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets acquisition of human-type receptor binding specificity by new h5n1 influenza virus sublineages during their emergence in birds in egypt acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest.acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest. key: cord-004280-c470nlie authors: coleman, kristen k.; sigler, william v. title: airborne influenza a virus exposure in an elementary school date: 2020-02-05 journal: sci rep doi: 10.1038/s41598-020-58588-1 sha: doc_id: 4280 cord_uid: c470nlie influenza contributes significantly to childhood morbidity and mortality. given the magnitude of the school-aged child population, a sizeable proportion of influenza virus transmission events are expected to occur within school settings. however, influenza virus activity in schools is not well-understood, likely due to our limited ability to accurately monitor for respiratory viruses without disrupting the school environment. in this study, we evaluated the use of a bioaerosol sampling method to noninvasively detect and quantify airborne influenza a virus (iav) densities in a public elementary school. air samples were collected from multiple locations in the school, two days per week, throughout an eight-week sampling period during influenza season. real-time rt-pcr targeting the iav m gene revealed detectable iav on five occasions in densities ranging from 2.0 × 10(−1) to 1.9 × 10(4). no significant differences in iav densities were related to student presence/absence. the majority of iav-associated particles were ≤4 μm in diameter, and theoretical calculations indicate infectious thresholds after minutes of exposure. our study represents the first identification and quantification of airborne influenza virus in an elementary school, and the results suggest that airborne iav has the potential to circulate in schools during influenza season, in large enough doses known to cause infection. a total of 128 air samples were collected over an eight-week period (february-march), followed by qrt-pcr targeting the influenza a virus (iav) m gene, which revealed detectable iav in 5% (5/96) of the air samples collected indoors, in densities of 2.0 × 10 −1 , 1.9 × 10 3 , 3.8 × 10 3 , 1.5 × 10 4 and 1.9 × 10 4 m gene copies m −3 air (table 1) . iav was not detected in any of the outdoor reference samples. an r 2 value of 0.97 for each standard curve was achieved for the qrt-pcr assays and the detection limit for iav m gene was 1 rna copy per reaction volume (25 μl) when the cut-off for positive result was set at 40 cycles. because each influenza virus particle packages one copy of the m gene 34 , our results can also directly reflect the number of virus particles m −3 air. however, our results are reported in copy numbers m −3 air to remain consistent with other quantitative studies measuring airborne iav densities. significantly different (p = 0.049) airborne iav densities were detected between all three indoor locations (i.e., gymnasium, classroom, and corridor) and all positive samples were collected during the last two weeks of 66 , and a 20-30% relative humidity level; descriptive of an average elementary school student in the usa weighing ~23-32 kg with an assumed tidal volume (v t ) of 7 ml per kg of body mass. † irrespective of iav target gene; based on the assumption that one tcid 50 is equivalent to ~300 pcr-detectable iav rna copies [47] [48] [49] [50] . the study (month of march, table 1 ). sample location was strongly correlated with iav density (r 2 = 0.95), with the highest density of iav detected in the corridor. two (6%) of the air samples collected from the gymnasium were positive for iav, as well as two (6%) from the corridor, and one (3%) from the classroom. no significant difference in iav densities was observed between samples collected during and after school (p = 0.14). indoor relative humidity (rh) and temperatures remained relatively consistent (19-29% rh and 20-23 °c) throughout the study. no significant difference in iav densities was observed between samples collected at different rh levels (p = 0.74) or temperatures (p = 0.10). significantly different (p = 0.049) airborne iav densities were detected among particle size fractions. particle size was strongly correlated with iav density (r 2 = 0.95), with 91% of virus-laden particles detected in respirable size fractions (≤4 μm in diameter). the largest virus-laden particles (>4 μm, 9% of total particles) were detected only in the gymnasium, while the smallest (<1 μm; 5% of total particles) were detected only in the classroom. no significant difference in virus-laden particle size was observed among samples collected at different rh levels (p = 0.82) or temperatures (p = 0.44), nor during and after school hours (p = 0.44). theoretical exposure threshold calculations accounting for body mass, tidal volume, and breathing rate predict that exposure to an equivalent of 1.1 × 10 4 -2.8 × 10 5 iav rna copies m −3 air for one minute is sufficient to induce infection in a student ( table 2 ). based on the airborne iav densities detected in the school, we estimated that students in the classroom (day 80), gymnasium (day 80), and corridor (day 84 and 87) were at risk of infection following 6, 3 and 1 minute(s) of breathing, respectively (table 1 ). aerosolization is an important mechanism for spread of iav 25 . since children are significantly burdened by influenza and play a key role in transmission 5, 29, 35, 36 , we established a protocol through which iav densities could be monitored in a school setting. viral densities were compared to theoretical iav exposure thresholds, above which students are expected to become infected. student illness and absenteeism caused by respiratory diseases is thought to occur, in part, because of prolonged time spent in school buildings, 50% of which experience impaired indoor air quality 31 . therefore, understanding how the student environment influences disease transmission can not only help to better address student health, but also improve sanitation/cleaning practices and inform predictive efforts. to our knowledge, our work represents the first identification and quantification of airborne iav in an elementary school, showing that schools can not only harbour airborne iav densities similar to those found in clinical settings 37, 38 , but also in infectious doses. furthermore, the detection and quantification of airborne iav in the school airshed warrants future investigations to determine the relationship between iav densities and student illness. three locations in an elementary school were sampled for airborne iav, including the main corridor, gymnasium, and a classroom. because each location featured different iav densities and particle sizes, the exposures of students to iav differed according to sample location. for example, students were likely at an elevated risk of iav infection by breathing in the classroom on day 80, especially since the particles were <1 μm in diameter. infection could have also arisen from breathing for 3 minutes in the gymnasium on day 80, but is less likely, as particles were >4 μm in diameter. while no specific activity can explain elevated iav densities in the classroom, two defined activities in the gymnasium throughout the school day are thought to promote elevated iav densities. first, the gymnasium is used by approximately 25 students during each class period for physical education instruction. physical activity/exercise can result in aerosolized iav through increased respiratory rates, and increased respiratory distress due to bronchoconstriction 39 , which has been demonstrated to affect up to 16% of children 40 . second, the gymnasium hosts the student lunch period, which lasts for 50 minutes, during which two cohorts (approximately 235 students each) eat for 25 minutes. the entire student body is not only represented in the gymnasium during the lunch period, but is also moving, en masse, as each cohort enters and exits the space, theoretically creating turbulence to maintain suspension of iav (if present) throughout the period, and likely into the post-lunch hours. airborne iav densities were highest in the school corridor, which was expected, and can be partially explained by two factors. first, student lockers are situated along the walls of the corridor which encourages air turbulence as students pass through the corridor and open and shut locker doors. second, the corridor represents the only available passage between classrooms and therefore each student passes through the corridor multiple times per day, which dually increases the probability that an infectious student will shed virus in the area, and that other students will be exposed. in a college student office setting, zhang and li (2018) 41 demonstrated that the frequency of close contact (within 1 m) is 9.64 contacts per hour per student, which contributed to 45% of reported iav infections. additionally, previous studies have suggested that influenza illness and death rates could be decreased by as much as 50% by reducing the contact rates of infected persons 42 . given the high airborne iav densities detected in the school corridor, along with elevated student contact rates, it is plausible to conclude that the school corridor is a "hotspot" for influenza virus transmission. studies focusing on clinical environments have demonstrated that a considerable proportion (48-53%) of total airborne iav-laden particles are respirable 37, 38 . in the current study, the majority (91%) of airborne particles associated with iav were respirable (≤4 μm in diameter), representing the most infectious fraction based on size. particles >4 μm in diameter are deposited predominantly in the nasal cavity or trachea, and are subject to mucociliary clearance before initiating infection 43 . in contrast, particles ≤4 μm in diameter can be deposited deep into the lungs, and are more likely to result in lower respiratory tract infections, which disproportionately impact children during influenza pandemics 24 . therefore, identifying the particle size distribution of airborne iav is critical for understanding the potential transmission and infectious impact of the virus. although we successfully detected airborne iav in the school with appropriate sensitivity to accurately quantify iav densities, environmental factors created sample processing challenges and difficulty interpreting the data. airborne pathogen densities in nonclinical environments can be several orders of magnitude lower than (2020) 10:1859 | https://doi.org/10.1038/s41598-020-58588-1 www.nature.com/scientificreports www.nature.com/scientificreports/ those detected in clinical environments 37, 38, 44 . furthermore, desiccation stress is known to limit the stability of airborne iav 45 . therefore, in the school, lower iav densities and high variability were anticipated, and sampling parameters were chosen to maximize detection, including short sampling durations to preserve the integrity of captured iav rna. we chose a flow rate of 3.5 l/min for four hours at a time, which was previously demonstrated to efficiently capture and preserve airborne iav rna for rt-pcr detection 37, 38 . next, iav packages a single copy of our rna target, the m gene 34 , and therefore a 1:1 relationship between the number of gene copies detected and number of virions is a valid assumption. however, brown et al. (2015) indicated that iav densities can be overestimated when quantifying gene copies 46 , and studies have assessed the relationship between rna copy number and the number of viable viruses, suggesting that one tcid 50 is equivalent to ~300 pcr-detectable iav rna copies [47] [48] [49] [50] . while iav viability was not directly assessed in the current study, our quantitative results do provide an estimate of the potential for iav transmission in the school environment, consistent with the prevailing public health proposals for conservative estimates of disease transmission risk 51 . lower densities of airborne iav were expected in the absence of students from the building. however, no significant difference in airborne iav density, as a function of student presence or absence, was observed. this result was unexpected, as children are thought to be key vehicles of iav transmission and are viewed here as the major factor contributing iav to the school airshed. we did not collect individual student health data during the study, and therefore could not definitively link the prevalence of influenza among the student population with virus detections. however, the detection of iav during the absence of all students from the building was nonetheless an important observation, indicating persistence of the virus in the airshed. airborne iav can remain viable for up to 36 hours 52,53 and is likely facilitated by two factors. first, it has been demonstrated that temperature and rh influence the viability and transmission of influenza viruses [54] [55] [56] [57] [58] . however, research has recently demonstrated that rh does not influence influenza virus viability 59 , but rather the rate of aerosol deposition, which influences the concentration of virus particles in the air. in the current study, the environmental conditions during the school day (19-29% rh and 20-23 °c) were optimal for iav persistence. second, children shed iav for a longer duration than adults shed the virus 60 , encouraging prolonged iav aerosolization in a school setting where children predominate. overall, our findings demonstrate that on the short term, iav is not fully cleared from the school environment upon removal of students, and support the assertion that schools should be considered an iav transmission hotspot, even in the absence of students. although airborne influenza virus has been detected in select indoor settings 37, 38, 44, [61] [62] [63] [64] [65] , the ability to consistently detect the virus in the airshed remains limited in environments featuring low iav densities. we now have the first molecular evidence of airborne iav in an elementary school, during a portion of the influenza season when students were exposed for appropriate durations to densities of influenza-laden particles that could facilitate infection. furthermore, given the magnitude of the school-aged population, our data provide justification for considering schools as influenza hotspots, warranting further study to determine the relationship between airborne iav densities and student health to improve influenza management in the greater community. bioaerosol sampling. air samplings were performed four times per week during an eight-week sampling period (february-march) in an elementary school (toledo, oh area) that enrolls approximately 470 students, in grade levels k-6. airborne iav was sampled in the school gymnasium, a classroom, main corridor, and an outdoor reference, using two-stage bioaerosol cyclone samplers provided by the national institute for occupational safety and health (niosh) and chosen for their portability, durability, minimal preparation time, and efficiency that equals that of commercial samplers 14 . each sampler was placed 1.2 m from the ground, simulating the average elementary school student's breathing level, and connected to an skc airchek xr5000 pump (skc, eighty four, pennsylvania) with 6.35-mm tygon tubing, operating at a flow rate of 3.5 l of air min −1 , collecting a total of 840 l of air for each sample. the pump flow rate and sampling duration was based on previous studies that demonstrated efficient capture of airborne influenza virus rna for rt-pcr detection 14, 37, 38 . each sampler collects particles >4 μm in diameter into a 15 ml centrifuge tube, particles 1-4 μm in diameter into a 1.5 ml centrifuge tube, while particles <1 μm in diameter are collected onto a 37-mm diameter, polytetrafluoroethylene filter with 2-μm pores. the influence of student presence/absence on airborne influenza virus detection was determined by collecting samples early in the school day (8:00 am-12:00 pm, students present), and in the afternoon/evening (3:00-7:00 pm, students absent). after sampling, collection tubes and filter cassettes were transported to the laboratory on ice and stored at −80 °c, if not immediately processed. samplers were washed with isopropanol and air dried between sample collections. temperature and rh were continuously recorded inside the school building near each sampler using hobo dataloggers (onset; bourne, ma, usa). sample rna was extracted and purified using the magmax viral rna isolation kit (ambion) following the manufacturer's instructions with slight modifications, including the addition of lysis/binding solution directly to (i) sampler tubes, and (ii) 50-ml falcon tubes containing the ptfe filters. xeno rna control (ambion), a synthetic rna transcript, was added to the sample lysis solution to act as an internal, positive control target for assessing the efficiency of rna recovery. purified rna was eluted in 30 μl of elution buffer. all analysis materials were purchased rnase-and pyrogen-free, if possible, and otherwise depyrogenated by autoclaving at 250 °c for 30 minutes. one-step, real-time, rt-pcr targeting the influenza a virus m gene was performed in an applied biosystems step-one real-time pcr system with commercial taqman aiv-matrix reagents (ambion) in a total reaction volume of 25 μl. reverse transcription was performed at 65 °c for 5 min, 50 °c for 2 min, and 95 °c for 10 min, followed by 65 cycles of qpcr analysis at 95 °c for 15 s, and annealing/elongation at 60 °c for 1 min. three negative control reactions (no template) were included in each qrt-pcr assay. to quantify the viral load present in scientific reports | (2020) 10:1859 | https://doi.org/10.1038/s41598-020-58588-1 www.nature.com/scientificreports www.nature.com/scientificreports/ each sample, the ct value from each reaction was compared to those of a standard curve derived from a dilution series of known quantities of iav m gene copies. iav exposure threshold calculations. based on observed densities of airborne iav, we estimated the breathing time, above which a student could theoretically become infected. to calculate the time, we used a known range of 0.6-3.0 tcid 50 (tcid 50 is the number of iav particles that induce infection in 50% of inoculated tissue culture cells) 66 , which has previously been used to estimate the risk of airborne iav infection after exposures consistent with a one-hour clinical visit, an eight-hour workday, and after 24 hours indoors 44 . since approximately 300 rna copies is equivalent to one tcid 50 47-50 , the resulting threshold iav density capable of initiating an infection is theoretically equivalent to 50-900 rna copies. assuming the average elementary school student weighs 23-32 kg, has a respiratory rate of 20 breaths min −1 , and a tidal volume (air volume displaced in a single breath) of 7 ml per kg of body mass, we calculated an inhalation volume range of 3200-4480 ml air min −1 for a typical student, which was then compared with our estimated iav densities to determine the number of minutes of breathing necessary to cause an infection. statistical analysis. data were imported into stata version 15.0 (statacorp, college station, tx, usa) and a two-sample t-test or one-way analysis of variance was performed to test for significant differences in iav densities and particle sizes between indoor sample locations (gym, classroom, corridor) and environmental conditions (temperature, rh, and student presence). regression analyses were then performed to test for correlations between variables demonstrated to be statistically significant. datasets generated during this study are available from the corresponding author upon reasonable request. outbreak of 2009 pandemic influenza a (h1n1) at a new york city school epidemiological and clinical characteristics of the outbreak of 2009 pandemic influenza a (h1n1) at a middle school in luoyang effectiveness of non-pharmaceutical interventions in controlling an influenza a outbreak in a school pandemic (h1n1) 2009 virus outbreak in a school in london influenza and school-based influenza-like illness surveillance: a pilot initiative in maryland interrupting the transmission of respiratory tract infections: theory and practice using data on social contacts to estimate 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viruses indoors and dependence on humidity indirect health effects of relative humidity in indoor environments the effect of environmental parameters on the survival of airborne infectious agents influenza virus infectivity is retained in aerosols and droplets independent of relative humidity amantadine therapy of epidemic influenza a2-hong kong bioaerosol sampling in clinical settings: a promising, noninvasive approach for detecting respiratory viruses molecular surveillance of respiratory viruses with bioaerosol sampling in an airport. tropical diseases detection of influenza and other respiratory viruses in air sampled from a university campus: a longitudinal study monitoring for airborne respiratory viruses in a general pediatric ward in singapore bioaerosol sampling for respiratory viruses in singapore's mass rapid transit human influenza resulting from aerosol inhalation this study was approved by the university of toledo irb (ref no: 200094). we thank the ottawa hills elementary school principal, kori kawczynski, and superintendent, dr. kevin miller, for graciously allowing us access to their school; dr. bill lindsley (national institute for occupational safety and health, niosh) for loaning the aerosol samplers used in this project and guiding us in their use; drs. april ames, daryl dwyer, sheryl milz, and daryl moorhead for their helpful insight and support throughout the study; and former ms student, marcus keller, and undergraduate student jessica dilworth for volunteering their time in the laboratory and out in the field. the authors declare no competing interests. correspondence and requests for materials should be addressed to k.k.c.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-016475-7ldxvbpz authors: pleschka, stephan; ludwig, stephan; wolff, thorsten; planz, oliver title: anti-viral approaches against influenza viruses date: 2006 journal: new concepts of antiviral therapy doi: 10.1007/978-0-387-31047-3_5 sha: doc_id: 16475 cord_uid: 7ldxvbpz nan influenza is a highly contagious, acute respiratory disease with global significance that affects all age groups and can occur repeatedly in any individual. the etiological agent of the disease, influenza virus is responsible e. bogner and a. holzenburg (eds.) , new concepts of antiviral therapy, 115-167. nordufer 20, d-13353 berlin, germany; friedrich-loeffler-institute (fli) , germany; ä for an average between three and five mill. cases of severe influenza leading to about 250,000-500,000 mortalities annually in the industrialized world according to who estimations. compared to otherwise healthy persons, death rates in patients of risk groups (s. 2.4) are 50-100 fold higher in patients with cardiovascular or pulmonary disease as compared to healthy individuals. annual health cost, costs, e.g. due to work absenteeism (also related to parental care of infected children) or costs related to death, increased disabilities etc. can be higher than 40 mil. € in european countries. furthermore. for a pandemic outbreak the centers for disease control (cdc) estimates that in the usa that 85% of all death will be caused by 15% of the population which are at high-risk. this will result in a financial burden of up to 166.5 billion us$ not including the commercial impact. the death rate would be up to 207,000 accompanied by up to 734,000 hospitalizations, 18-42 million outpatient visits and 20-47 million additional illnesses wilschut and mcelhaney, 2005) . this clearly would overrun the capacity of current supply and management of vaccines available. since waterfowl represents the natural reservoir for the virus (lamb and krug, 2001; webster, 1999; wilschut and mcelhaney, 2005; wright and webster, 2001 ) and many other animal species can be infected, the eradication of the virus is impossible and a constant reemergence of the disease will continue to occur. epidemics appear almost annually and are due to an antigenic change of the viral surface glycoproteins (fig. 1) . furthermore, highly pathogenic strains of influenza-a-virus have emerged unpredictably but repeatedly in recent history as pandemics like the "spanish-flu" that caused the death of 20-40 millions people worldwide (taubenberger et al., 2000; webster, 1999) . since these pandemic virus strains usually possess different antigenic characteristics, current vaccines will be ineffective once such a virus emerges. regarding the vast possibilities for such a strain to "travel" around the world (hufnagel et al., 2004) it becomes evident that effective countermeasures are required for the fight against these foes. in recent outbreaks of avian viruses that infected humans (1997, 1999, 2003/4/5) hatta and kawaoka, 2002; li et al., 2004 ) from a total of 108 confirmed cases 54 people died (07/2005) (world health organization, 2005) . fortunately, until now these particular viruses have not acquire the ability to spread in the human population. however, any novel virus strain emerging in the future may have such a capability (webby and webster, 2003) . here, we give an overview of current and new anti-influenza strategies, such as immunization methods and drugs against the virus. since every virus depends on its host cell, cellular functions essential for viral replication may also be suitable targets for anti-viral therapy. in this respect intra-cellular signaling cascades activated by the virus, in particular mapk pathways, have recently come into focus ludwig et al., 1999) . influenza viruses belong to the order of the orthomyxoviridae. they possess a segmented, single stranded rna-genome with negative orientation. they are divided into three types, a, b and c based on genetic and antigenic differences. among the three types influenza-a-viruses are clinically the most important pathogens since they have been responsible for severe epidemics in humans and domestic animals in the past. thus the focus of this chapter will be on type-a influenza viruses. a detailed description of the viral proteins and the replication cycle of influenza-a-viruses can be found elsewhere (lamb and krug, 2001; wright and webster, 2001) . therefore we will only give a brief overview on these topics without referring to individual references. the influenza-a-virus particle is composed of a lipid envelope derived from the host cell and of 9-10 structural virus proteins ( figure 1 and table 1 ). the components of the rna-dependent rna-polymerase complex (rdrp), pb2, pb1 and pa are associated with the ribonucleoprotein complex (rnp) and are encoded by the vrna segments 1-3. the pb1 segment of many, but not all, influenza-a-virus strains also contains a +1-reading frame encoding the recently discovered pb1-f2 protein (chen et al., 2001) . the viral surface glycoproteins hemagglutinin (ha) and neuraminidase (na) are expressed from vrna segments 4 and 6, respectively. the nucleoprotein (np) is encoded by segment 5 and associates with the vrna segments. it is the major component of the rnps. the two smallest vrna segments each code for two proteins. the matrix protein (m1) is colinear translated from the mrna of segment 7 and forms an inner layer within the virion. a spliced version of the mrna gives rise to a third viral transmembrane component, the m2 protein, which functions as a ph-dependent ion channel. employing a similar coding strategy segment 8 harbors the sequence information for the nonstructural ns1 protein and the nuclear export protein ns2/nep. ns2/nep is a minor component of the virion and is found associated with the m1 protein. figure 1 . the influenza-a-virus particle schematic representation of the spherical influenza-a-virus particle that has a diameter of about 100nm. the eight viral rna segments were separated by urea-polyacrylamide gel electrophoresis and visualized by silver staining (left) . the corresponding gene products and their presumed location in the virus particle are indicated (right). ns1 is not a structural part of the mature virion. for details see text. table 1 summarizes details of the genome segments, the encoded viral proteins and their according function. the viral replication cycle is initiated by binding of the ha to sialic-acid (neuraminic acid) containing cellular receptors and subsequent endocytosis of the virus (figure 2 ) (for references: (lamb and krug, 2001; wright and webster, 2001) ). the active ha molecule consists of two subunits (ha 1 / ha 2 ) derived from the uncleaved precursor ha 0 , which becomes proteolytically processed after release of the virion by extra-cellular proteases. this cleavage is absolutely essential for ha-function and cell infection. virus disassembly occurs in the acidic environment of late endosomal vesicles and involves two crucial events. first, the conformation of the ha is changed to a low-ph form, which results in exposure of a fusion active protein sequence within the ha 2 . this fusion peptide is thought to contact the endosomal membrane and to initiate fusion with the viral envelope. second, the low ph in the endosomes activates the viral m2 ion channel protein resulting in a flow of protons into the interior of the virion. acidification facilitates dissociation of the rnps from the m1 protein. the rnps are subsequently released into the cytoplasm and rapidly imported into the nucleus through the nuclear pore complexes. the viral genomic segments are replicated and transcribed by the viral rdrp associated with the rnps in the nucleus of the infected cell. the vrna is directly transcribed to mrna and, in addition, serves as a template for a complementary copy (crna), which itself is the template for new vrna. in the late phase of infection newly synthesized viral rnps are exported to the cytoplasm. ns1 protein functions as a regulatory factor in the virus infected cell. the na, the m2 and the precursor ha (ha 0 ) proteins follow the exocytotic transport pathway from the rer via the golgi complex and the trans golgi network. the mature ha and na glycoproteins and the nonglycosylated m2 are finally integrated into the plasma membrane as trimers (ha) or tetramers (na, m2), respectively. m1 assembles in patches at the cell membrane. it is thought to associate with the glycoproteins (ha and na) and to recruit the rnps to the plasma membrane in the late phase of the replication cycle. finally the viral rnps become enveloped by a cellular bilipid layer carrying the ha, na and m2 proteins resulting in budding of new virus particles from the apical cell surface. replicative viral proteins enter the nucleus to amplify the viral genome. in the late stage of the infection cycle newly synthesized rnps are exported from the nucleus and are assembled into progeny virions that bud from the cell surface. the polymerase complex of influenza viruses does not possess a proof reading activity, thus numerous mutations accumulate in the viral genome during ongoing replication (lamb and krug, 2001) leading to changes in all proteins. this includes conformational alteration of ha-and na-epitopes against which neutralizing antibodies are generated. influenza-a-viruses are categorized by antigenic differences of the ha-and na-proteins. the high mutation rate combined with the high replication rate results in a multitude of new variants produced in each replication cycle, thus allowing the virus to rapidly adapt to changes in the environment. this results in an escape of the existing immunity and in resistance to drugs acting directly against viral functions. gradual changes of the antigenic properties that make existing vaccines less or non effective are described as antigenic drift and demand for new compositions of the yearly vaccines. due to the nature of their segmented genome influenza virus can independently recombine segments upon the infection of a cell with two different viruses. this is described as genetic reassortment. today 16 hasubtypes (h1-h16) and 9 na-subtypes (n1-n9) are known, which can mix and lead to new antigenic properties. (lamb and krug, 2001; webster et al., 1992; wright and webster, 2001) . not all combination will ultimately be advantageous, but can lead to the generation of a virus that combines the ability to replicate in humans with novel antigenic properties (antigenic shift). this has happened at least three times in the last century resulting in the pandemics of 1918 ("spanish flu"), 1957 ("asian flu") and 1968 ("hong kong flu") that caused up to 40 million death. therefore, the question is not "if" but "when" will such a pandemic occur again (horimoto and kawaoka, 2001; webby and webster, 2003; webster, 1997b) . a vaccine against such "new" viruses can not be generated in advance and as vaccine production would need significantly more time than it takes for a pandemic virus to spread around the world (hufnagel et al., 2004) , alternative weapons in the fight against these enemies are urgently needed. besides pandemic variants that can occur when human and avian influenza virus reassort in porcine hosts (regarded as "mixing vessels") (webster, 1997a; webster et al., 1997) , avian influenza virus strains have directly infected humans, as happened in hong kong in 1997 (claas et al., 1998; de jong et al., 1997; subbarao et al., 1998 subbarao et al., ) and recently (2004 subbarao et al., /2005 koopmans et al., 2004) during vast outbreaks of avian influenza. these viruses show an extremely high virulence in humans with case fatality rates up to 70%. the virus that normally causes a respiratory disease (for references: (wilschut and mcelhaney, 2005) ) is transmitted by aerosol droplets and contaminated hands and can already be shed before onset of symptoms . therefore, high population density and dry air leading to reduced protection of respiratory epithelium by the mucus are conditions that promote transmission of the virus. the infection with influenza viruses is normally limited to the respiratory tract. here proteases released by clara cells in the epithelium are present that activate the ha to allow further infections (s. 2.2) (for review (ludwig et al., 1999) ). innate immunity as well as the adaptive immune system will normally restrict virus propagation. therefore population groups, that have a less protective immune system, such as young children up to two years and older persons over 65 as well as immunocompromised or chronically diseased persons are especially of risk. the replication of the virus leads to the lysis of the epithelial cells and enhanced mucus production causing running nose and cough. furthermore, inflammation and oedema at the replication site are due to cytokines released. this can lead to fever and related symptoms. bacterial super-infections of the harmed tissue can further complicate the situation. normally onset of systemic (fever, myaglia, headaches, severe malaise) and respiratory (coughing, sore throat, rhinitis) symptoms occur after about two days incubation period and can last for about seven to ten days. coughing and overall weakness can persist for up to two weeks. if the virus spreads from the bronchiolar tract to the aveolars, viral pneumonia and interstitial pneumonitis with mononuclear and haemorrhage infiltration and finally lysis of the inter-aveolar space is possible (wilschut and mcelhaney, 2005) . this scenario is a likely picture in case of infection with a pandemic influenza strain, where the individual has not had a prior exposure to the virus and the innate immunity reaction can lead to a strong immunpathogenesis. high virus replication will induce secretion of large quantities of cytokines by the infected epithelia and will stimulate inflammatory processes. together with the destruction of the epithelia this results in an influx of fluid into the aveolars leading to hypoxia and acute respiratory distress syndrome, that may cause the death within a short period of time (1-2 days after onset). this scenario might also be caused by additional viral factors enhancing pathogenicity. such factors that are yet not well defined probably have contributed to the devastating outcome of the "spanish flu" (wilschut and mcelhaney, 2005) . accurate and rapid diagnosis of the disease is essential for an effective treatment, especially with anti-viral substances, as virus replication and therefore illness progresses rapidly. samples can be tested serologically, by cell culture or rt-pcr for strain typing and should be done within four days after onset of symptoms (wilschut and mcelhaney, 2005) . there are two main methods of influenza prophylaxis: the use of antiviral drugs and vaccines. several drugs are available for influenza prophylaxis functioning either as m2-ion channel inhibitors (amantadine and rimantadine) or as inhibitors of the na (zanamivir and oseltamivir). despite these anti-viral drugs, which are a useful adjunct to influenza vaccines, vaccination itself remains the cornerstone of prophylaxis. vaccination induces a good degree of protection and is in general well tolerated by the recipient. nevertheless, while resistant virus variants can emerge after antiviral drug treatment the disadvantage of vaccination is that immunization needs to be refreshed almost every year, since the vaccine must reformulated to take account of the changing virus. in the immune response to influenza infection both the humoral and cell mediated immunity are involved. from the side of the humoral immune system, both the mucosal and the systemic immunity contribute to resistance to influenza infection. the cellular immune response is involved in recovery from influenza virus infection by eliminating virus-infected cells and by providing help for antibody production woodland et al., 2001) . consequently, the humoral immune response is the primary target of vaccination. after influenza virus infection antibodies directed against all major viral proteins can be detected in humans and the level of serum antibodies correlate with resistance to disease (couch, 2003; couch and kasel, 1983; coulter et al., 2003; nichol et al., 1998; potter and oxford, 1979) . only antibodies specific for the surface glycoproteins ha and na are associated with resistance to infection. in contrast, antibodies to the conserved internal antigens m and np are not protective (de jong et al., 2003; tamura and kurata, 2004) . the mucosal tissues of the respiratory system are the main portal entry of influenza virus and consequently the mucosal immune system functions as the first line of defense against infection apart from innate immunity (see paragraph 4). antibodies secreted locally in the upper respiratory tract are a major factor in resistance to natural infection. secretory immunoglobulin a (siga) and to some extent igm are the major neutralizing antibodies directed against the entering virus. furthermore, these antibodies can function intra-cellular to inhibit influenza replication. iga and igm are involved in protection of the upper respiratory titre ≥40) can be detected in approximately 80% of subjects after natural influenza virus infection and correlates with protection against the flu. plasma cells producing all three major ig classes are present in the peripheral blood in normal subjects (cox and subbarao, 1999; laforce et al., 1994) . tract while serum igg acts in protection of the lower respiratory tract ). an anti-ha antibody response (haemagglutination-inhibition (hi) the immune response induced by infection protects against reinfection with the same virus or an antigenically similar viral strain. cell mediated immunity plays a role in recovery from influenza virus infection and may also prevent flu-associated complications, but it does not seem to contribute significantly in preventing infection. influenza specific cellular t cells have been detected in the blood and the lower respiratory tract secretions of infected subjects . influenza virusspecific cytotoxic t-lymphocytes (ctl) regognize both external and internal proteins of virus on infected cells. in humans a major component of this response is directed toward the np-and m1-protein. even though influenza virus specific ctl's are not able to protect against the infection, these cells are important for the clearance of the virus. futhermore, cytolysis of influenza virus-infected cells can be mediated by influenza virus-specific antibodies and complement mcmichael et al., 1983; mcmichael et al., 1986; townsend et al., 1989) . cd4+ t cells function as helper cells for antibody production. moreover, it is suggested, that cd4 cells might act as direct effectors in protection against influenza virusinfection (brown et al., 2004) . inactivated vaccines (iv) are availeble for about 60 years. because of the antigenic drift observed in influenza ha-and na-glycoproteins these vaccines need to be matched with the randomly mutating molecular structure of the new occurring "drift" strain. besides these vaccines there are various new approaches for influenza vaccines in promising developmental stages. these new stratagies include vaccines with immunomodulators, virosomes and dna-vaccines. ivs vaccines are administered world wide each year with millions of doses. these vaccines have good safety and tolerance profiles, with very low number of adverse reactions reported. these reactions are tenderness and redness that arise locally at the injection site and are more frequent in healthy (<50%) than in elderly recipients (25%) . ivs are produced by propagation of the virus in embryonated chicken eggs. the currently used bacterial endotoxin-free trivalent ivs (tiv) are formulated with 15µg ha each from a current influenza virus a/h1n1, a/h3n2, and a b-virus strain. the seed strain is prepared by co-infecting the allantoic sac of the chicken embryo with a laboratory-adapted high-growth phenotype of h1n1 (a/pr/8/34) and the epidemic strain. this results in viral replication and genetic re-assortment leading to high growth reassortants. thereafter the new hybrid viruses are screened for the absence of genes encoding pr/8 or pr/8-like surface glycoproteins. the selected seed strain containing ha-and na-components of the epidemic strain is mass propagated in chicken eggs to obtain sufficient quantities of vaccine virus. the allantoic fluid is harvested, and the virus is concentrated and highly purified by zonal centrifugation. as a next step the virus is inactivated. depending on the nature of inactivation the vaccine is used as whole inactivated vaccine after treatment with formalydehyde or β-propiolactone or as split vaccine (chemically disrupted by ethyl either or sds). furthermore, the vaccine is used as subunit vaccines (purified surface glycoproteins). even though influenza vaccines have excellent tolerant profiles, since propagation in chicken eggs may lead to contamination of the vaccine with trace amounts of residual egg proteins, they should not be administered to persons who have anaphylactic hypersensitivity to eggs. whole inactivated influenza vaccine is more immunogenic than split vaccine or subunit vaccine, but is also associated with more frequent side reactions. consequently, split or subunit are given to children younger than age 9 and two half doses are recommended given at least 1 month apart for naïve persons to develop protective immunity (bridges et al., 2003) . protection after vaccination against influenza virus infection is dependent on the antigenic match between the vaccine strains and circulating the influenza virus strain. moreover, protection is also dependent the age and the previous exposition to influenza of the vaccine recipient. if ivs have a good antigenic match they are 60-90% effective in the prevention of morbidity and mortality among healthy adults (beyer et al., 2002) . in elderly people the effect of protection is reduced to 50-70% because of decreased immune function. since the immune system is naïve in young children, they also show a reduction in protection against influenza vaccination (nichol et al., 1998) . immunosuppressed individuals, elderly people and subjects with underlying chronic diseases are at increased risk for influenza and related complications. for these people conventional influenza vaccines provide only limited protection. in order to enhance the immune reaction after influenza vaccination, several adjuvants (latin verb: adjuvare -to help) that function as immunopotentiators have been evaluated. the liposomal influenza vaccine (influsome-vac) consists of liposomes containing the viral surface proteins ha-and na-derived from various influenza strains and il-2 or granulocyte-macrophage colonystimulating factor (gm-csf), as an adjuvant (babai et al., 2001) . in clinical trails with either young adults or elderly vaccination of influsome-vac appeared to be both safe and more immunogenic than the currently used vaccine (ben-yehuda et al., 2003a; ben-yehuda et al., 2003b) . furthermore adjuvant emulsions combined with subunit influenza antigens are in use, such as the "oil in water"-emulsion containing squalene, mf59, (fluad). this commercially available product was tested in clinical trials in comparison with non-adjuvanted conventional vaccines. again in elderly individuals the addition of the mf59-adjuvant to subunit influenza vaccines enhances significantly the immune response without causing clinically important changes in the safety profile of the influenza vaccine (podda, 2001) . other adjuvants that increase immunoreactivity after influenza vaccination are immune stimulating complexes (iscoms). they are 30-40 nm cage-like structures, which consist of glycoside molecules of the adjuvant quil a, cholesterol and phospholipids in which the antigen can be integrated. . in animal models, even in the presence of pre-existing antibodies they function as a potent adjuvant system by inducing cellular and humoral immune responses. (coulter et al., 2003; rimmelzwaan et al., 2001; windon et al., 2001) . as mentioned, gm-csf has a potential role as a vaccine adjuvant. it may enhance the response to vaccination in immunosuppressed individuals. gm-csf stimulates maturation of hematopoietic progenitor cells, induces class ii major histocompatibility complex antigen expression on the surface of macrophages, and enhances dendritic cell migration and maturation (jones et al., 1994) . nevertheless, in various clinical trails with immunosuppressed individuals and cancer patents it was shown, that it is unlikely that gm-csf improves the immune response (ramanathan et al., 2002) . for production of influenza vaccines in large-scale cell culture systems several continuous cell lines have been tested for the production of influenza vaccines (kistner et al., 1998; pau et al., 2001; seo et al., 2001; youil et al., 2004) . production of influenza vaccine in mammalian cell lines has some advantages but also has disadvantages compared to production in chicken egg. (tree et al., 2001; youil et al., 2004) . process controllability, scalability and supply of substrates are much easier in cell culture systems. furthermore, cell culture production reduces the risk of microbial contamination. in contrast, the greatest disadvantage of cell culture based influenza vaccine is the relative low viral yield. on the other hand and a major disadvantage of production in chicken eggs is their supply and possible bacterial contaminations. additionally the lethality of h5n1 influenza virus to chicken embryos (s. 3.1.3). at the present (2005) two cell line derived vaccines have been licensed in europe . estimated time for production of such vaccines is about 6 months. the power of this time gaining approach to generate a great variety of specific influenza-vaccines under controlled safety conditions is achieved by the direct use of field strains (kistner et al., 1998) as well as seed strains specifically designed by reverse genetics systems and the large scale cell culture system. nevertheless, the application of these techniques largely depends on meeting the needs of high viral yield, appropriate permissiveness, and ability to support replication of all influenza virus strains to immunopotentiating reconstituted influenza virosomes (irivs) possess several characteristics defining them as vaccine adjuvants. they are a liposomal carrier system. these reconstituted virus-like particles (vlp; diameter 150nm) contain a lipid bilayer of phosphatidylcholine and phosphatidylethanolamine. ha and na are intercalated into the lipid bilayer and give the irivs their fusogenic activity, but lack the viral genetic material. irivs are able to deliver proteins, rna/dna and peptides to immunocompetent cells. in addition, virosomes, as vaccine delivery systems, have been shown to be safe and not to engender any antibodies against the phospholipid components. therefore, their use in vaccination of children and elderly people is recommended. the system is already registered for human use and allows a specific targeting of antigens by a cellular or a humoral immune response. a virosome vaccine, inflexal-v, is used in switzerland and italy. (gluck et al., 2004; langley and faughnan, 2004; zurbriggen, 2003) . dna-vaccines are non-infectious and non-replicative plasmid constructs that encode either only the proteins of interest or the protein of interest in combination with immunomodulatory proteins. this kind of vaccination by direct intra-muscular injection of dna was first demonstrated in 1990 in a mouse model system et al. (wolff et al., 1990) . directed intra-muscular dna-vaccination is not very common. the creation of recombinant influenza vaccines based on dna-plasmids is more appropriate. with this technique rapid and flexible construction of dna-plasmid vectors can be achieved, which can address the problems of antigenic drift induced by the circulating influenza virus strain (ljungberg et al., 2000) . these above described techniques have a potential for the development of live and inactivated vaccines. the efficacy of the plasmid based dnavaccines expressing the immunogenic influenza virus genes alone or in combination with dna encoding various cytokines has also been demonstrated in several animal models (for detail see: bardiya and bae, 2005) . during dna-vaccination, the foreign genes are endogenously high titers in short time (reviewed in: bardiya and bae, 2005) . expressed in the host, the proteins subsequently processed, and recognized by the immune system of the host. dna-vaccines elicit a broad-based humoral and cellular immunity against influenza virus proteins (justewicz et al., 1995) . in addition, alterations in the vector, dose of the dna, inclusion of cpg-odn motifs, fusion with influenza virus-specific helper t cell or ctl-epitopes, and appropriate vaccine delivery mechanisms will further improve the efficacy of these vaccines (bowersock and martin, 1999; joseph et al., 2002) . an alternative to ivs are attenuated "live" vaccines such as cold-adapted vaccines (cav: caiv-t, flumist ® ) and ns1-defective strains used as intranasal influenza vaccine, that may lead to long-lasting, broader immune response (humoral and cellular) that resembles more closely the natural immunity derived from viral infection. for example, ctls, which are important for the clearance of the virus are activated during an productive infection . additional cytokines produced by the infected cells during the innate immunity response enhance and support reaction of the humoral system. compared to ivs, that are strain-and subtype-specific the cavs (that also have to be adapted to circulating strains) can provide a broader immunity against circulating viruses (belshe et al., 2000; king et al., 1998; nichol, 2001; stepanova et al., 2002; treanor et al., 1999; wareing and tannock, 2001) . cavs that already have been used successfully in russia and are now licensed in the usa kendal, 1997) can be administered intra-nasally for example as aerosols (abramson, 1999) . this results in a limited viral replication in the upper and lower respiratory tract and circumvents the need for syringes. it also supports protective mucosal immunity, which is an important property of nasally applied live influenza virus vaccines. for the generation of a cav a donor and a wild type strain are reassorted in such a way, that the ha-and na-segments are wild type (wt) derived and the remaining six segments originate from the donor strain. for this purpose two master strains are currently used as donors in the usa. one to generate a-type and one b-type influenza cavs (mendelman et al., 2001; murphy and coelingh, 2002) . these strains are cold adapted (25°c) (kendal, 1997; maassab and bryant, 1999) and therefore temperature sensitive (ts) and attenuated, meaning that these viruses will not propagate efficiently at body temperatures. to prevent easy reversion of the genetic markers, that encode the ts-defect and allowing the virus to regain full virulence, all six donor-derived segments carry mutations. for the production of such cav strains embryonated eggs are infected with both viruses (wild type and donor strain) under the selection of antibodies directed against the ha and na of the donor strain. the attenuated donor strain by itself is unable to cause significant illness in humans, but is able to donate the ha-and na-proteins of the contemporary epidemic strain to produce live attenuated vaccine by the traditional egg-based process (belshe, 2004; clements and murphy, 1986; jin et al., 2003) . the live attenuated vaccines were shown to be safe and effective in the general population kendal, 1997; langley and faughnan, 2004) . cav are trivalent like the ivs and are composed according to the who recommendations (mendelman et al., 2001) . new possibilities of reverse genetic techniques will certainly improve production of vaccine strains in time and quality (s. 3.1.3). even though one should consider the possibility of reassortment with another human strain in the vaccinated person, which could produce an aggressive virus, cavs have been successfully used in russia without reports of severe side effects and seem to be safe. they show a comparable effectiveness to trivalent iv's (tivs) and both vaccines can also be used in combination belshe et al., 2004; boyce and poland, 2000; edwards et al., 1994; glezen, 2004; jackson et al., 1999; mendelman et al., 2001; swierkosz et al., 1994; treanor and betts, 1998; treanor et al., 1992) . in addition to the traditional live attenuated vaccines, production by reverse genetics (s. 3.1.3) of replication-incompetent influenza virus-like particles (vlps) by deletion of either the entire ns gene (encoding both the ns1 and ns2 protein) or only the ns2 gene has also been reported. these vlps were entirely produced from cdnas (watanabe et al., 2002b) . although, these technologies are in the very early stages of development and so far only tested in animal models, the vlp incapable of replication and spread to other cells due to deletion of a major protion of the ns1 or m2, are expected to be good novel influenza vaccine candidates (galarza et al., 2005; watanabe et al., 2002a) . a variation of the theme is presented by influenza virus strains (generated by reverse genetics (s. 3.1.3)) that express a modified ns1 (palese and garcia-sastre, 2002; palese et al., 1999; talon et al., 2000) . this non-structural protein is the major viral interferon (ifn)antagonist (s. tab. 1). even though ns1 is a multifunctional viral protein that supports viral replication it seems to be an accessory protein as a virus without the ns1-gene can replicate in ifn-deficient systems (garcia-sastre et al., 1998a; garcia-sastre et al., 1998b; ludwig et al., 1999) . ifnα/β are two important cytokines expressed in primary infected epithelia cells, that induce innate immunity. ifnα/β-induction severely reduces viral replication even in the presence of ns1. therefore recombinant viruses expressing altered ns1 with reduced capacity to suppress cellular ifn-induction could raise protective immunity and might represent interesting attenuated live vaccine candidates (talon et al., 2000) . such viruses have been generated by reverse genetic techniques (s. 3.1.3) and have been successfully tested in experimental settings . after initial experiments that implied the in vivo reconstitution of rnps from plasmid-expressed rdrp, np and vrna it became possible to generate recombinant influenza virus de novo totally from plasmid dna (fodor et al., 1999; , allowing complete genetic manipulation. this manipulation can either concern the combination/mixture of the genomic rna-segments and/or the genesequences themselves. the technique involves the transfection of four plasmids expressing the viral rdrp and the np together with eight plasmids (for all eight genomic rnas) that generate a vrna-like transcript. this again results in the in vivo reconstitution of active rnp-complexes, which will replicated and transcribe the vrnas. thereby all viral rnas and proteins are generated and the viral replication cycle is established resulting 1998; palese et al., 1996 ) . reverse genetics technique can be used to produce influenza vaccines based on recombinant virus (for detail see: bardiya and bae, 2005) . these methods do not require selection procedures and eliminate the need for multiple passages in eggs, thereby reducing the time required for vaccine production. it is known that interference among the vaccine viruses of type-a and -b can occur that affect the efficacy of the live attenuated vaccines by restricting their replication. to overcome that problem a chimeric virus (a/b) possessing chimeric (a/b) ha, and full-length b-type na in the background of a type-a vaccine virus was created (horimoto et al., 2004) . this study provided a novel method for creating live attenuated vaccine from a single donor strain. for different reasons the technique of reverse genetics has become highly relevant for anti-viral vaccine approaches. (i) for the production of regular ivs against wild type strains, that either grow poorly or are too pathogenic in eggs (s. later) one can generate strains carrying the ha and na needed in the background of an egg adapted virus. this is normally done by reassortment of the wild type with the egg-adapted strain and can not be well controlled. this problem can be circumvented by plasmid based reverse genetics that allow the controlled design of the reassortant. (ii) as mentioned the cav are composed of ha-and na-genes from the wild type in the production of infectious influenza viruses (for review: garcia-sastre, strain and a mixture of the other six segments from wild type and donor virus. by choice of the according plasmids one can compose a cav-strain that carries all six segments from the donor strain each with an adaptive mutation. this way it is less likely that a revertant virus will arise by mutation in one of the donor strain segments (s. 3.1.2) (maassab and bryant, 1999; schickli et al., 2001) . (iii) it is possible to specifically design viruses with altered ns1-genes that could be used as highly attenuated life vaccines (s. 3.1.2), additionally modification of other viral genes (murphy et al., 1997; parkin et al., 1997) or of the replications efficiency of the genesegment (muster et al., 1991) can be applied to further attanuate the virus. (iv) viruses could be produced that lack an essential gene (e.g. nep) (watanabe et al., 2002b) . the missing gene-product can be transcomplemented from an expression-plasmid in the transfected cell during virus generation. the recombinant viruses would be still infectious and lead to expression and presentation of viral proteins, but could not themselves establish a productive propagation as they are lacking the according gene. currently used ivs are prepared from egg-grown viruses (wilschut and mcelhaney, 2005) . this method is not without limitations but has proven to be efficient. as mentioned earlyer (3.1.1), one particular problem that could arise would be production of a vaccine strain against an highly pathogenic avain influenza virus like the types that have recently infected humans. besides bio-safety questions they pose further problems. the ha of these viruses is activated within the infected cell by ubiquitous proteases allowing the virus to spread through out the organism. due to the special hacharacteristics these viruses themselves are highly pathogenic birds and eggs as well as a vaccine strain that would carry the according ha. therefore efficient virus production in embryonated eggs will be problematic (lipatov et al., 2004) . by plasmid based reverse genetic techniques recombinant viruses can be produced that have lost the pathogenic character of the ha and can replicate well in eggs li et al., 1999; liu et al., 2003; subbarao et al., 2003) . this could additionally be combined with virus production in cell culture systems (s. p. 12) romanova et al., 2004) thereby overcoming the limitation posed by the number of embryonated eggs available at a given time (stephenson et al., 2004) . it should also be mentioned that not only type-a influenza viruses but also type-b influenza viruses can be generated and manipulated by reverse genetic systems and can therefore also be engineered to fit the circulating wild type strains (dauber et al., 2004; hatta et al., 2004; jackson et al., 2004; maassab and bryant, 1999) . inhibitors of viral functions (treatment and anti-viral chemoprophylaxis of influenza) anti-viral treatment is generally considered a supporting measure to prevent and control outbreaks of epidemic influenza in addition to immunoprophylaxis. however, chemotherapy is the only option to combat the disease when there is no type-specific vaccine available as for instance upon the emergence of a pandemic shift variant. two classes of substances are currently licensed in many countries for the treatment and/or prophylaxis of influenza, which includes the adamantane compounds amantadine and rimantadine, and the na-inhibitors oseltamivir and zanamivir. other small inhibitory compounds that target the viral polymerase complex are also introduced in this section, although none of them has been converted into a pharmaceutical product so far. amantadine (1-amino adamantane hydrochloride) and its derivative rimantadine (α-methyl-1-adamantane methylamine hydrochloride) have potent anti-viral activity against most influenza-a-viruses, because they block the viral m2 ion channel protein during the early stage of viral uncoating (pinto et al., 1995) . specifically, the adamantane compounds inhibit the acidification of the virion inside the endosome, which prevents the intra-cellular release of the viral rnps. the 50% inhibitory concentration (ic 50 ) of most natural influenza-a-virus strains against adamantane compounds is in the range of 0.2 to 0.4 µg/ml as determined by plaque reduction assay (appleyard et al., 1977; hayden et al., 1980; scholtissek and faulkner, 1979) . amantadine and rimantadine have proven effectiveness in the treatment of uncomplicated influenza-a-virus infection. they can reduce the duration of fever and system symptoms by approximately one day when given within two days after onset of disease signs (demicheli et al., 2000; tominack and hayden, 1987) . furthermore, both substances also have prophylactic effectiveness in reducing influenza-associated morbidity and clinical symptoms. a survey of studies undertaken with healthy adults demonstrated average effectiveness of 61% for amantadine and 72% for rimantadine in preventing laboratory confirmed influenza (demicheli et al., 2000) . during long-term prophylaxis amantadine was found to cause mild reversible adverse effects in a small proportion of the recipients, which involved central nervous system (cns) and minor gastrointestinal complaints. no increase in side effects was observed during treatment with rimantadine compared to placebo (n.n., 1985) . an early recognized limitation for widespread clinical use of m2-blockers is the rapid emergence of drug-resistant viruses in tissue culture, in animal models and in patients (appleyard et al., 1977; hayden et al., 1989; oxford et al., 1970) . one study found that a total of 27% of children with laboratory-confirmed influenza shed resistant viruses after seven days of treatment with rimantadine (hall et al., 1987) . unfortunately, such selected drug-resistant viruses are virulent, as they can transmit to family members and cause disease even when the contact persons were treated prophylactically with rimantadine (hayden et al., 1989) . viruses that become insensitive to amantadine show complete cross-resistance to rimantadine and vice versa. thus, the clinical usage of adamantane amine compounds has been limited by the reported adverse effects, the induction of viral drug resistance and the inactivity towards influenza-b-viruses. nevertheless, these drugs are still recommended as a cost-effective choice particularly in influenza chemoprophylaxis (harper et al., 2004) . it is noteworthy, that amantadine resistance has also been detected in the highlypathogenic h5n1 viruses currently circulating in south east asia (puthavathana et al., 2005) . thus, adamantane compounds are not an option to treat such infections. amantadine and rimantadine are approved for treatment of adults and children older than 12 years at two daily 100 mg doses. the substances should carefully be used in individuals above 64 years in age and patients with impaired renal functions and halving of the daily doses is recommended. only amantadine is licensed for treatment of children between 1 and 9 years and should be dosed with 5 mg/kg per day. in order to avoid emergence and transmission of drug-resistent viruses, treatment should be kept to a minimal time of 3 to 5 days until disease symptoms disappear. chemoprophylaxis can be considered for protection among high-risk groups including children and adults with chronic pulmonary or cardiac disease, immunocompromised persons with a reduced response to vaccines or in the case of a poor match between an epidemic virus strain and the current vaccine. since the adamantane compounds do not interfere with the development of neutralizing antibodies (tominack and hayden, 1987) , they can also be used for the protection of persons at high risk to bridge the time gap between vaccination and the establishment of an efficient immune status. for adults and children older than 9 years two 100 mg doses of amantadine or rimantadine per day are recommended. children between 1 and 9 years should receive a maximum of 150 mg per day in two divided doses. two anti-viral drugs that inhibit both influenza-a and b-viruses, zanamivir (relenza™, glaxosmithcline) and oseltamivir (tamiflu™, roche pharmaceuticals) have recently been approved for general use in the usa, australia, europe and japan. the current knowledge suggests that nainhibitors (ni) will have a better clinical utility than the m2-blockers, because these substances are broadly effective against type-a and -b influenza viruses including highly virulent avian virus strains. further, they appear to have a very low frequency of adverse effects and are less prone to induce drug resistance. zanamivir and oseltamivir function as slow binding, substrate competitive inhibitors that strongly reduce viral na-activity by interacting with five sub-sites close to the enzymatic pocket of the na. the ic 50 values of these inhibitors were found to be in the range of 0.8 -8.8 nm depending on the virus types and subtypes (mckimm-breschkin et al., 2003) . targeting of the viral na does not require the delivery of an inhibitor into the cell interior, because the enzyme is a surface glycoprotein. influenza viruses attach to the host cell through binding of the viral ha to sialic acid moieties that are conjugated to cellular glycoproteins. by the time of progeny virus budding these receptor determinants need to be removed to allow efficient release from the host cell. this is accomplished by the viral na (acylneuraminyl hydrolase, ec 3.2.1.18) that hydrolyzes glycosidic linkages adjacent to n-acetyl-neuraminic acid (neu5ac, sialic acid). thus, blockade of na-activity by antibodies, temperature-sensitive mutation or inhibitory substances results in the aggregation of budding virions at the cell membrane and, hence, reduction of virus release (compans et al., 1969; palese and compans, 1976; palese et al., 1974) . in infected animals or humans, na probably also enhances penetration of the virion through the viscous mucus on respiratory epithelia, which contains sialic acids (matrosovich et al., 2004) . thus, inhibition of viral na-activity was the rationale behind several efforts to identify substances that would reduce influenza virus spread and replication. the development of the current nis was based on early characterizations of the sialic acid transition state analogue 2-deoxy-2,3 dehydro nacetylneuraminc acid (neu 5ac2en) (meindl et al., 1974) and the determination of the three-dimensional structure of the na by x-ray crystallography varghese et al., 1983; varghese et al., 1992) . neu 5ac2en had been shown to inhibit viral na-acitvity but was not protective in a mouse model of influenza (palese and schulman, 1977) . based upon computer-assisted drug design, von itzstein et al. demonstrated that the introduction of positively charged amino-or guanidino moieties at position 4 of neu 5ac2en increased na inhibition by two to four orders of magnitude (von itzstein et al., 1993) . importantly, the inhibition of naactivity by 4 guanidino-neu5ac2en that is now also termed zanamivir translated into efficient reduction of viral replication of type-a and binfluenza viruses in the nanomolar range in vitro and dose-dependent decrease of viral titers in infected animals (von itzstein et al., 1993; woods et al., 1993) . zanamivir has low oral bioavailability, but shows high antiviral activity in humans or animals when administered topically by inhalation of dry-powder aerosol (cass et al., 1999) . the second currently approved na inhibitor compound oseltamivir (3r,4r,5s-4acetamido-5-amino-3-(1-ethylpropoxyl)-1-cyclohexene-1carboxylic acid¸ also termed gs4071/ro64-0802) has similarly potent activities against type a and b influenza viruses . oseltamivir emerged from an independent na structure-based study and is based on a cyclohexen ring structure in which the polar glycerol side chain of the sialic acid analogues is replaced by a lipophilic 3-pentyloxy moiety (kim et al., 1997) . importantly, oseltamivir has high oral anti-viral activity when administered as its methylester pro-drug, gs4071/oseltamivir phosphate, that is converted to the active drug by hepatic enzymes (hayden et al., 1999b; li et al., 1998; mendel et al., 1998) . zanamivir and oseltamivir have potent anti-viral effectiveness against community-acquired influenza and are in general safe to use in healthy adults (abramson, 1999; boivin et al., 2000; hayden et al., 1997; makela et al., 2000; monto et al., 1999; n.n., 1985) . in clinical trials the nis significantly shortened disease duration and reduced symptoms and viral loads when treatment was initiated within 26 hours post infection (hayden et al., 1996; hayden et al., 1999b) . even, when inhalation of zanamivir was begun within 30 hours after onset of symptoms the time to alleviation of major disease signs (cough, myalgias, fever, headache) was shortened by one to two days and patients were able to resume normal activities earlier (hayden et al., 1997; monto et al., 1999) . initiation of therapy later than 30 hours after disease onset still reduced viral loads but was less beneficial for symptom recovery. two 75 mg daily doses of oseltamivir for five days were shown to reduce shedding of virus and the severity and duration of influenza symptoms by one to two days when therapy was begun within 36 hours after onset of disease signs (nicholson et al., 2000; treanor et al., 2000) . some side effects that included diarrhea, nausea and nasal symptoms were observed during clinical testings of zanamivir but were similar in placebo groups (glaxowellcome, 2001) . the ni substances are also highly effective to prevent spread of the disease. a post-exposure protection study with zanamivir demonstrated 79% efficacy in preventing transmission of influenza to family members, when the index case was treated with zanamivir . oseltamivir had a comparably high efficacy in preventing laboratory-confirmed influenza by 74% and influenza with fever by 82% (hayden et al., 1999a) . within households, one 75 mg dose oseltamivir per day was 89% protective against clinical influenza even when the index cases were not treated (welliver et al., 2001) . thus, to prevent the spread of the flu within household contacts the nis appear to be preferable compared to the m2-blockers that can induce the emergence and transmission of virulent drug-resistant viruses. during the development of nis for clinical use it was recognized that viruses with a reduced drug sensitivity could be selected in tissue culture (summarized in (mckimm-breschkin, 2000; tisdale, 2000) ). resistance can be characterized by various methods including ic 50 -determination of the viral na, by plaque reduction assays (number and size) and yield reduction assay in tissue culture (matrosovich et al., 2003; tisdale, 2000) . under laboratory conditions several passages are usually required to select such variants, which is different to amantadine-resistant viruses that can emerge in a single cycle experiment. drug-resistant viruses were also isolated from diseased persons treated with nis (gubareva et al., 2001; gubareva et al., 1998; kiso et al., 2004; zambon and hayden, 2001) . however, the available data on the pathogenicity of these mutant viruses in animal models suggest that they have reduced replication capability in vivo and may therefore be clinically less relevant in humans. resistance to nis was found to be complex, because it can be associated with mutations in the na, the ha or synergistically in both genes. namutations that confer reduced drug sensitivity were identified at amino acid residues 119, 152, 274, 292 and 294 (based on n2-na numbering) (gubareva, 2004; zambon and hayden, 2001) . these amino acids are part of or cluster around the conserved catalytic pocket and their mutation can decrease the enzymatic activity to below 5% and some also destabilize the enzyme (varghese et al., 1998) . the various ni-molecules slightly differ in their interactions with the enzyme. thus, a given na-mutant enzyme may show a range of sensitivity against different inhibitors (gubareva et al., 2001) . interestingly, some viruses with a reduced sensitivity to nis were found to carry mutations in the ha, which affected the receptor binding site in the globular head region, the stalk region and the ha 2 -subunit (mckimm-breschkin, 2000) . apparently, the ha-mutations reduce drug sensitivity by decreasing the affinity for cellular sialic acid receptor molecules and thereby easing the release of budding viruses from the plasma membrane. these findings corroborate the concept that efficient viral replication requires a carefully balanced interplay between the strength of ha/receptor binding and the activity of the na that removes these receptor determinants . the use of zanamivir (relenza™) and oseltamivir (tamiflu™) is recommended for the treatment of uncomplicated influenza caused by type-a and b-viruses (harper et al., 2004) . therapy with either drug should be initiated within 48 hours after the onset of disease signs and should be continued for five days (glaxowellcome, 2001; roche, 2001) . it is important to consider that bacterial superinfections may occur that would not be affected by these anti-virals. neither substance has been shown to prevent serious complications of influenza like pneumonia. zanamivir is approved for treatment of influenza in persons aged 7 years and older. the recommended dosage is two inhalations of 5 mg doses twice a day using the inhalation device provided by the manufacturer. zanamivir is not recommended for persons with underlying respiratory conditions like asthma or chronic obstructive pulmonary disease, because of the risk of precipitating bronchospasm in such patients (glaxowellcome, 2001) . oseltamivir can be used for treatment of patients of 1 year or older. depending on the age, the recommended doses for children above 12 years and adults are two 75 mg capsules a day. two daily doses of 15 -30 mg is recommended for children under 15 kg, 2 x 45 mg for children between 15-23 kg and 2 x 60 mg for persons weighing >23-40 kg. currently, tamiflu™ but not relenza™ is licensed for chemoprophylaxis in children older than 12 years and in adults. for persons with creatinine clearance of 10-30 ml/min, halving of the usual dosage for therapy or prophylaxis is recommended. two approaches are possible, a seasonal prophylaxis that provides a 92% reduction of confirmed influenza infection in a vaccinated population of frail elderly persons (mcclellan and perry, 2001) , and a short-term prophylaxis for controlling institutional outbreaks by breaking the virus circulation. several further compounds that inhibit the influenza virus na were identified in independent efforts and have been evaluated as anti-influenza agents. thus, the cyclopentane derivatives bcx-1812 (rwj-270201), bcx-1827 , bcx-1898 and bcx-1923 as well as the pyrrolidine-based a315675 (from abbott laboratories) showed strong potent anti-viral activies at least in vitro (kati et al., 2002; smee et al., 2001) . thus, although development of bcx-1812 has been halted after showing a lack of activity in a phase iii clinical trial (chand et al., 2005) , additional nis may emerge as anti-influenza drugs in the future. two unique properties of the trimeric rna-dependent rna-polymerase of influenza viruses, which are not shared by cellular enzymes, provide attractive opportunities for anti-viral interference with possibly little disturbances of the host cell. first, the polymerase exhibits an endonuclease activity that cleaves the first 10 -13 nucleotides including the 5'-cap structure from nascent host rna-polymerase ii cap transcripts and use them to prime viral mrnas (lamb and krug, 2001) . second, the viral polymerase replicates the negative-sense viral rna-segments via unprimed synthesis of a complementary positive-strand rna-intermediate. for both of these activities, inhibitory small molecule compounds have been identified, some of which were also shown to reduce viral propagation in tissue culture and/or in infected mice. however, further clinical development has not been reported for any of those substances so far. the viral endonuclease activity is associated with the pb1-subunit and depends on binding of the polymerase to the terminal ends of the vrnatemplate and the cap structures of nascent mrna-transcripts (li et al., 2001) . the endonuclease most likely utilizes a two metal ion mechanism for cleavage of the cellular nucleic acid (klumpp, 2004a) . it has been shown that derivatives of the fungal metabolite flutimide as well as a class of 4substituted 2,4-dioxobutanoic acids specifically inhibited the cap-dependent endonuclease, presumably by interaction with the active catalytic site of the enzyme (hastings et al., 1996; parkes et al., 2003; tomassini et al., 1994; tomassini, 1996) . the most potent compounds of these two classes had ic 50 values in the range of 0.2 -6 µm when tested in virus yield assays in tissue culture experiments. further, intranasal instillation of the l-735,882 compound was reported to inhibit viral titers in nasal washes of mice infected with influenza virus a/pr/8/34 virus, but the effects on disease progression were not studied (hastings et al., 1996) . another screening effort has identified t-705 (6-fluoro-3-hydroxy-2pyrazinecarboxamide) to have potent and selective anti-influenza activity. t-705 showed ic 50 values of less than 0.5 µg/ml in virus yield assays in mdck cells against all three influenza virus types (a, b, c) with no signs of cytotoxicity (furuta et al., 2002) . importantly, t-705 was also orally active in a mouse model and shown to significantly reduce viral lung titers and enhance survival rates from 20% to 100% after infection with influenza virus a/pr/8/34 virus at a dose of 200 mg/kg per day (furuta et al., 2002; takahashi et al., 2003) . although the basis for its anti-viral activity was unclear at that time, t-705 was found to inhibit replication of an oseltamivirresistant mutant virus in vitro suggesting that this inhibitor targets a different viral function (takahashi et al., 2003) . indeed, recent analyses showed that the compound is metabolized inside the cell into t-705-ribofuranosyl-5'triphosphate (t-705-rtp), which is a potent and selective inhibitor of apgprimed viral rna-polymerase activity (furuta et al., 2005) . these findings show that t-705 may have the potential to become a novel oral antiinfluenza drug that targets a viral function not blocked by one of the currently licensed nis or m2-blockers. influenza viruses only have a limited coding capacity. thus, these viruses employ functions of their host-cell for efficient replication. these dependencies create opportunities to design novel anti-viral strategies by targeting specific host cell functions. cell fate decisions in response to extra-cellular agents, including pathogenic invaders are commonly mediated by intra-cellular signaling cascades that transduce signals into stimulus specific actions, e.g. changes in gene expression patterns, alterations in the metabolic state of the cell or induction of programmed cell death (apoptosis). thus, these signaling molecules are at the bottleneck of the control of cellular responses. in this section we will review the recent advances in the analysis of influenza virus induced signaling pathways and first attempts to use signaling mediators as targets for anti-viral approaches. mitogen activated protein kinase (mapk)-cascades have gained much attention as being critical transducers to convert a variety of extra-cellular signals into a multitude of responses (english et al., 1999; hazzalin and mahadevan, 2002; widmann et al., 1999) thereby, these pathways regulate numerous cellular decision processes, such as proliferation and differentiation, but also cell activation and immune responses (dong et al., 2002) . four different members of the mapk-family that are organized in separate cascades have been identified so far: erk (extra-cellular signal regulated kinase), jnk (jun-n-terminal kinase), p38 and erk5/bmk-1 (big map kinase) (garrington and johnson, 1999; widmann et al., 1999) . these mapks are activated by a dual phosphorylation event on threonine and tyrosine mediated by mapk-kinases (mapkk also termed meks or mkks). the mapk "erk" is activated by the dual-specific mapkk mek1 and -2 that are controlled by the upstream serine threonine mapkkkinase raf. raf, mek and erk form the prototype module of a mapk-pathway and are also known as the classical mitogenic cascade. the mapk p38 and jnk are activated by mkk3/6 and mkk4/7, respectively, and are predominantly activated by pro-inflammatory cytokines and certain environmental stress conditions. the mek5/erk5 module is both activated by mitogens and certain stress inducers. there is evidence that all these different mapk-cascades are activated upon infection with rna-viruses, including influenza viruses. thus, these signaling cascades may serve different functions in viral replication and host cell response. another important signaling pathway, which is commonly activated upon virus infection is the iκb-kinase (ikk)/nfκb-signaling module (hiscott et al., 2001) . the nfκb/iκb family of transcription factors promote the expression of well over 150 different genes, such as cytokine or chemokine genes, or genes encoding for adhesion molecules or anti-and pro-apoptotic protein (pahl, 1999) . the canonical mechanism of nfκb activation includes activation of iκb-kinase (ikk) that phosphorylates the inhibitor of nfκb (iκb) and targets the protein for subsequent degradation (delhase and karin, 1999; karin, 1999b) . this leads to the release and migration of the transcriptionally active nfκb factors to the nucleus (ghosh, 1999; karin and ben-neriah, 2000) . the ikk-complex consists of at least three isozymes of ikk: (i) ikk1/ikkα, (ii) ikk2/ikkβ and (iii) nemo/ikkγ. the most important isozyme for nfκb-activation via the degradation of iκb is ikk2 (karin, 1999a) . nemo acts as a scaffolding protein for the large ikk complex (courtois et al., 2001 ) that contains still other kinases such as mekk1 (mapkk-kinase 1) (lee et al., 1998) , nik (nfκb inducing kinase) (nemoto et al., 1998; woronicz et al., 1997) and the dsrnaactivated protein-kinase (pkr) (gil et al., 2000; zamanian-daryoush et al., 2000) . both nfκb and the jnk mapk-pathway regulate one of the most important anti-viral gene expression events, the transcriptional induction of interferon beta (ifnβ) . ifnβ is one of the first antiviral cytokines to be expressed upon virus infection, initiating an autoamplification loop to cause an efficient and strong type-i ifn response. the ifnβ enhanceosome, which mediates the inducible expression of ifnβ, carries binding sites for transcription factors of three families, namely the ap-1 family members and jnk targets c-jun and atf-2, the nfκb factors p50 and p65, and the interferon-regulatory factors (irfs) (hiscott et al., 1999; thanos and maniatis, 1995) . in the initial phase of a virus infection this promoter element specifically binds the constitutively expressed and specifically activated irf3-dimer (taniguchi and takaoka, 2002) . ap-1and nfκb-transcription factors are activated by a variety of stimuli. however, a strong irf3-activation is selectively induced upon infection with several rna-viruses, in particular by the dsrna, which accumulates during replication yoneyama et al., 1998) . thus, irf3 is the major determinant of a strong virus-and dsrna-induced ifnβ-response. interestingly all four so far defined mapk-family members are activated upon an influenza virus infection (kujime et al., 2000; ludwig et al., 2001; has helped to get a clearer picture of the importance of the erk-signaling pathway for influenza virus replication. the activation of the map-kinase erk upon productive influenza virus infection (kujime et al., 2000) appears to serve a mechanism that is beneficial for the virus . strikingly, blockade of the pathway by specific inhibitors of the upstream kinase mek and dominantnegative mutants of erk or the mek-activator raf resulted in a strongly impaired growth of both, influenza a-and b-type viruses . conversely, virus titers are enhanced in cells expressing active mutants of raf or mek olschlager et al., 2004) . this has not only been demonstrated in cell culture but also in vivo in infected mice expressing a constitutively active form of the raf-kinase in the alveolar epithelial cells of the lung (olschlager et al., 2004) . while in the wt-situation influenza viruses primarily infect bronchiolar epithelial cells, there is efficient replication in the alveolar layer most exclusively in the cells carrying the transgene. as a consequence this results in an earlier death of the transgenic animals (olschlager et al., 2004) . this indicates that activation of the raf/mek/erk pathway is required for efficient virus growth. noticeably, inhibition of the pathway did not significantly affect viral rna-or protein-synthesis . the pathway rather appears to control the active nuclear export of the viral rnp-complexes that are readily retained in the nucleus upon blockade of the signaling pathway. most likely this is due to an impaired activity of the viral nuclear export protein nep . this indicates that active rnp-export is an induced rather than a constitutive event, a hypothesis supported by a late activation of erk in the viral life cycle. so far the detailed mechanism of how erk regulates export of the rnps is unsolved. there are two likely scenarios: either it does occur directly via phosphorylation of a viral protein involved in rnp-transport or by control of a cellular export factor. although in the initial studies no alteration of the overall phosphorlyation status of the np, m and nep proteins was observed there are now first indications that certain phosphorylation sites of the np indeed are affected by mek-inhibition (s.p., unpublished data). it remains to be shown, whether this is of functional relevance for the rnp-export process. it is striking that mek-inhibitors are not toxic for the cell, while more general blockers of the active transport machinery, such as leptomycin-b exert a high toxicity even in quite low concentrations. this may indicate that mekinhibitors are no general export blockers but only block a distinct nuclear export pathway. indeed there are first evidences that the classical mitogenic cascade specifically regulates nuclear export of certain cellular rna-protein complexes. in lps-treated mouse macrophages mek-inhibition results in a specific retention of the tnf-mrna in the nucleus (dumitru et al., 2000) . this is also observed in cells deficient for tpl-2, an activator of mek and erk. in these cells the failure to activate mek and erk by lps again correlated with tnf mrna retention while other cytokines are normally expressed (dumitru et al., 2000) . thus the erk-pathway may regulate a specific cellular export process but leaves other export mechanisms unaffected. it is likely that such a specific export pathway is employed by influenza-a and b-viruses. the finding of an anti-viral action of mek-inhibitors prompted further research showing that replication of other viruses, such as borna disease virus , visna virus (barber et al., 2002) or coxsackie b3 virus (luo et al., 2002) is also impaired upon mek-inhibition. requirement of raf/mek/erk-activation for efficient influenza virus replication may suggest that this pathway may be a cellular target for antiviral approaches. besides the anti-viral action against both, a-and b-type viruses , mek-inhibitors meet two further criteria which are a prerequisite for a potential clinical use. although targeting an important signaling pathway in the cell the inhibitors showed a surprisingly little toxicity (a) in cell culture planz et al., 2001; pleschka et al., 2001) (b) in an in vivo mouse model (sebolt-leopold et al., 1999) and (c) in clinical trials for the use as anti-cancer agent (cohen, 2002) . in the light of these findings it was hypothesized that the mitogenic pathway may only be of major importance during early development of an organism and may be dispensable in adult tissues (cohen, 2002) . another very important feature of mek-inhibitors is that they showed no tendency to induce formation of resistant virus variants . although targeting of a cellular factor may still raise the concern about side effects of a drug, it appears likely that local administration of an agent such as a mek-inhibitor to the primary site of influenza virus infection, the lung, is well tolerated. here the drug primarily affects differentiated lung epithelial cells for which a proliferative signaling cascade like the raf/mek/erk-cascade may be dispensable. following this approach it was recently demonstrated that the mek inhibitor u0126 is effective in reducing virus titers in the lung of infected mice after local administration (o.p., s.p. and s.l., unpublished). activation of the classical mitogenic raf/mek/erk-cascade is initiated by yet other phosphorylation events. the kinase raf is known to be regulated by phosphorylation of different upstream kinases including members of the protein kinase c (pkc)-family (cai et al., 1997; kolch et al., 1993) . the pkc-superfamily consists of at least 12 different pkc-isoforms that carry out diverse regulatory roles in cellular processes by linking into several downstream signaling pathways (toker, 1998) . beside a regulation of the raf/mek/erk-cascade and other downstream pathways, pkcs may have additional functions during viral replication. a role of pkcs in the process of entry of several enveloped viruses has been proposed based on the action of protein kinase inhibitors h7 and staurosporine (constantinescu et al., 1991) as well as by the calcium-channel blocker verapamil (nugent and shanley, 1984) . influenza virus infection or treatment of cells with purified viral ha results in rapid activation of pkcs upon binding to host-cell surface receptors (arora and gasse, 1998; kunzelmann et al., 2000; rott et al., 1995) . in a recent study it was shown that the pan pkc-inhibitor bisindolylmaleimide-i prevented influenza virus entry and subsequent infection in a dose dependent and reversible manner (root et al., 2000) . using a dominant-negative mutant approach this function was assigned to the pkcßii-isoform. overexpression of a phosphorylation-deficient mutant of pkcßii revealed that the kinase is a regulator of late endosomal sorting. accordingly, expression of the pkcßii-mutant resulted in a block of virus entry at the level of late endosomes (sieczkarski et al., 2003; sieczkarski and whittaker, 2002) . thus, a specific inhibition of pkcßii may be a suitable approach to blunt virus replication at a very early time point in the replication cycle. activation of the transcription factor nfκb is a hallmark of most infections by viral pathogens (hiscott et al., 2001 (hiscott et al., ) including influenza et al., 1999 . influenza viral nfκb-induction involves activation of iκbkinase (ikk) and is also achieved with isolated influenza virus components. this includes dsrna (chu et al., 1999) or over-expression of the viral ha, np or m1 proteins (flory et al., 2000) . since gene expression of many pro-inflammatory or anti-viral cytokines, such as ifnβ or tnfα, is controlled by nfκb the concept emerged that ikk and nfκb are essential components in the innate immune response to virus infections (chu et al., 1999) . accordingly, influenza virus-induced ifnβpromoter activity is impaired in cells expressing transdominant negative mutants of ikk2 or iκbα (wang et al., 2000; wurzer et al., 2004) . nevertheless, ikk and nfκb might not only have anti-viral functions as two recent studies demonstrate that influenza viruses replicate much better in cells where nfκb is pre-activated (nimmerjahn et al., 2004; wurzer et al., 2004) . conversely, influenza virus titers from different host cells in which nfκb-signaling was impaired by means of specific inhibitors or dominantnegative mutants, a dramatic reduction could be observed (nimmerjahn et al., 2004; wurzer et al., 2004) . thus, in the context of an influenza virus infection a function of nfκb to support virus replication appears to be dominant over the function as a transcription factor in the anti-viral response. on a molecular basis this was shown to be due to the nfκbdependent expression of pro-apoptotic factors, such as tnf-related apoptosis inducing ligand (trail) or fasl . inhibition of virus induced expression of these factors results in strongly impaired viral growth. this links the pro-viral action of nfκb to the induction of apoptosis, a process that will be discussed in the next section. finally, viral need for nfκb-activity suggests that this pathway may be suitable as a target for anti-viral intervention. to this end we have shown recently that several pharmacological inhibitors of nfκb act anti-viral in vivo, without toxic side effects or the tendency to induce resistant virus variants (i. mazur, w. wurzer, c. erhardt, t. silberzahn, t.w., o.p., s.p. and s.l, unpublished). another cellular signaling response commonly observed upon virus infections, including influenza virus is the induction of the apoptotic cascade. apoptosis is a morphological and biochemical defined form of cell death (kerr et al., 1972) and has been demonstrated to play a role in a variety of diseases, including virus infections (razvi and welsh, 1995) . apoptosis is mainly regarded to be a host cell defense against virus viruses (reviewed in: julkunen et al., 2000; ludwig et al., 2003; infections since many viruses express anti-apoptotic proteins to prevent this cellular response. the central component of the apoptotic machinery is a proteolytic system consisting of a family of cysteinyl proteases, termed two groups of caspases can be distinguished: upstream initiator caspases such as caspase-8 or caspase-9, which cleave and activate other caspases and downstream effector caspases, including caspase-3, -6 and -7, that cleave a variety of cellular substrates, thereby disassembling cellular structures or inactivating enzymes (thornberry and lazebnik, 1998) . caspase-3 is the most intensively studied effector caspase. work on caspase-3 deficient mcf-7 breast carcinoma cells has revealed a caspase-3 driven feedback loop, that is crucial to mediate the apoptotic process (janicke et al., 1998; slee et al., 1999) . thus, caspase-3 is a central player in apoptosis regulation and the level of pro-caspase-3 in the cell determines the impact of a given apoptotic stimulus. although it is now well established that influenza virus infection induces caspses and subsequent apoptosis, the consequence for virus replication or host cell defense is still under a heavy debate (reviewed in (lowy, 2003; ludwig et al., 1999; schultz-cherry et al., 1998) . with the identification of pb1-f2, a new influenza virus protein expressed from a +1 reading frame of the pb1 polymerase gene segment, a pro-apoptotic influenza virus protein has been discovered (chen et al., it is long known that influenza virus infection with a-and b-type viruses results in the induction of apoptosis both in permissive and un-permissive cultured cells as well as in vivo (fesq et al., 1994; hinshaw et al., 1994; ito et al., 2002; mori et al., 1995; takizawa et al., 1993) . interestingly, viral activation of mapks or upstream kinases has been linked to the onset of apoptosis. in a mouse model for a neurovirulent influenza infection, jnkactivity correlated with apoptosis induction in the infected brain (mori et al., 2003) . in embryonic fibroblasts deficient for the mapkk-kinase ask-1 the virus-induced jnk-activation was blunted concomitant with an inhibition of caspase-3 activation and virus-induced apoptosis (maruoka et al., 2003) . as an extrinsic mechanism of viral apoptosis induction it has been noted quite early on that the fas receptor/fasl-apoptosis inducing system (fujimoto et al., 1998; takizawa et al., 1995; takizawa et al., 1993; wada et al., 1995) is expressed in a pkr-dependent manner in infected cells (takizawa et al., 1996) . this most likely contributes to virus-induced cell death via a receptor mediated fadd/caspase-8-dependent pathway (balachandran et al., 2000) . another mode of viral apoptosis induction might occur via activation of tgf-β that is converted from its latent form by the viral na (schultz-cherry and . within the influenza virus infected cell the apoptotic program is mediated by activation of caspases (lin et al., 2002; takizawa et al., 1999; zhirnov et al., 1999) with a most crucial role of caspase-3 (wurzer et al., 2003) . although it is now well established that influenza virus infection induces although it is now well established that influenza virus infection induces caspases (for review see: cohen, 1997; thornberry and lazebnik, 1998) . 2001). pb1-f2 induces apoptosis via the mitochondrial pathway if added to cells and infection with recombinant viruses lacking the protein results in reduced apoptotic rates of lymphocytes (chen et al., 2001) . however, most of the avian virus strains are lacking the pb1-f2 reading frame and pb1-f2deficient viruses do not affect apoptosis in a variety of other host cells (chen et al., 2001) . these results have let to the assumption that apoptosis induction by pb1-f2 may be required for the specific depletion of lymphocytes during an influenza virus infection, a process which is observed in infected animals (tumpey et al., 2000; van campen et al., 1989a; van campen et al., 1989b) . a recent study adds a new aspect to the open discussion by the surprising observation that influenza virus propagation was strongly impaired in the presence of caspase inhibitors (wurzer et al., 2003) . this dependency on caspase activity was most obvious in cells where caspase-3 was partially knocked-down by sirna (wurzer et al., 2003) . consistent with these findings, poor replication efficiencies of influenza-a-viruses in cells deficient for caspase-3 could be boosted 30-fold by ectopic expression of the protein. mechanistically, the block in virus propagation appeared to be due to the retention of viral rnp-complexes in the nucleus preventing formation of progeny virus particles (wurzer et al., 2003) . interestingly the findings are consistent with a much earlier report showing that upon infection of cells over expressing the anti-apoptotic protein bcl-2 the viral rnp-complexes were retained in the nucleus (hinshaw et al., 1994) resulting in repressed virus titers (olsen et al., 1996) . furthermore the recently identified proapoptotic pb1-f2 (chen et al., 2001) is only expressed in later phases of replication consistent with a later step in the virus life cycle that requires caspase activity (wurzer et al., 2003) . the observation of a caspase requirement for rnp-nuclear export was quite puzzling since this export process was shown before to be mediated by the active cellular export machinery involving the viral nuclear export protein (ns2/nep) (neumann et al., 2000; o'neill et al., 1998) and the anti-apoptotic raf/mek/erkcascade . caspase activation does not support, but rather inhibit the active nuclear export machinery by cleavage of transport proteins. this suggests an alternate strategy by which caspases may regulate rnp-export, e.g. by directly or indirectly increase the diffusion limit of nuclear pores (faleiro and lazebnik, 2000) to allow passive diffusion of larger proteins. such a scenario is supported by the finding that isolated nps or rnp-complexes, which are nuclear if ectopically expressed, can partially translocate to the cytoplasm upon stimulation with an apoptosis inducer in a caspase-3-dependent manner (wurzer et al., 2003) . these findings can be merged into a model in which the rnps are transported via an active export mechanism in intermediate steps of the virus life cycle. once caspase activity increases in the cells, proteins of the transport machinery get destroyed, however, widening of nuclear pores may allow the viral rnps to use a second mode of exit from the nucleus (faleiro and lazebnik, 2000) . that would be a likely mechanism to further enhance rnp-migration to the cytoplasm in late phase of the viral life cycle and thereby support virus replication. such a complementary use of both "active" (raf/mek/erkdependent) and "passive" (caspase-dependent) transport mechanisms is supported by the observation, that at concentrations of mek-and caspaseinhibitors, which only poorly block influenza virus replication alone, efficiently impaired virus propagation if used in combination (wurzer et al., 2003) . thus, while both pathways do not interfere with each other (wurzer et al., 2003) they appear to synergize to mediate rnp-export via different routes. therefore one may conclude that influenza virus has acquired the capability to take advantage of supposedly anti-viral host cell responses to support viral propagation. this includes early induction of caspase activity but not necessarily execution of the full apoptotic process that most likely is an anti-viral response. this dual role of "early" versus "late" apoptotic events during virus replication may exclude the use of caspase-inhibitors as anti-flu agents, although in cell culture these inhibitors may have a beneficial outcome for the host cell. besides mediators of signaling and apoptosis a variety of other cellular enzymes are required for efficient virus growth. there are also some initial attempts to use these components as target for an anti-viral intervention. another important requirement for a cellular enzyme is the proteolytic cleavage of the ha by proteases. the infectivity and pathogenicity of influenza virus is based on the proteolytic cleavage of the precursor ha 0 into ha 1 and ha 2 chains by an arginine-specific, trypsin-like host protease. the viral glycoproteins are glycosylated in the endoplasmic reticulum (er) and the er-α-glycosidase-i is responsible for the removal of terminal α-1,2 glucose residues from precursor oligosaccharides in the er. a variety of viruses such as hiv, hsv and dengue-virus have been shown to be highly sensitive to inhibitors of these enzymes (mehta et al., 2001; mehta et al., 2002) . one of these inhibitors, castanopermine, has been demonstrated to inhibit replication of influenza virus a/hongkong/11/88 in mdck cells with an ic 50 value of <6 µm (klumpp, 2004b) . the inhibitor also acted antiviral in vivo in a mouse model and reduced lung titers of a/pr8/34 infected mice by tenfold when administered intranasal. the compound has reached phase ii clinical trials for the treatment of hiv and has been licensed for a potential treatment of hepatitis-c-virus infections (reviewed in (klumpp, 2004b) ). several exogenous protease inhibitors were investigated with respect to their anti-influenza activity: camostat, a serine protease inhibitor; was shown to exhibit strong anti-influenza effects in vitro and in vivo in mice and in chicken embryos. the compound also showed strong anti-influenza effects in amantadine-resistant type-a and -b virus infection in vitro (lee et al., 1996) . other protease inhibitors nafamostat mesilate, camostat mesilate, gabexate mesilate and aprotinin also inhibited virus replication in vitro protease inhibitors gordox, contrycal and epsilonaminocapronic acid were tested in both animal and clinical experiments. inhalation of aminocapronic acid-containing aerosols exerted the most effective therapeutic effect, reducing the duration of viral antigen in the nasopharyngeal epithelium 1 1/2 to 2 fold (zhirnov et al., 1984) . recombinant human mucus protease inhibitor (mpi) was investigated for its anti-viral activity in rat lungs in vitro. the c-, but not the n-terminal domain of mpi was shown to inhibit the proteolytic activity of tryptase clara and of virus activation at nm concentrations (beppu et al., 1997; kido et al., 1999) . however, the current understanding is that protease inhibitors -mainly used in hiv therapy -may produce serious toxic side effects. recent investigations showed that protease inhibitors can cause diabetes, hepatic and renal failures and mutagenic (potentially carcinogenic) effects. a further disadvantage of protease inhibitors is the rapid development of viral resistance, and a variable strain sensitivity to these anti-viral agents still under evaluation as potential anti-influenza therapeutics (kido et al., 2004; savarino, 2005) . regarding the continues threat caused by seasonal flu-epidemics and the immanent danger of re-occurring pandemic outbreaks that both impose a great burden on human and animal health, and considering the fact, that influenza viruses can not be eradicated, the possibilities to fight this disease have been greatly improved by novel molecular biological techniques in recent years. vaccination is still by far the best prophylactic measure, but new drugs, which attack the virus directly, will further support to combat these foes. nevertheless the viral tactic to escape direct intervention by resistance is a major drawback of current therapeutic interventions. this (discussed in luscher-mattli, 2000) . nevertheless protease inhibitors are zhirnov, 1994) (reviewed in luscher-mattli, 2000) . the : : problem might be overcome by innovative methods that target cellular functions essential for efficient virus replication 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influenzavirus infections. gg167 influenza study group safety and efficacy of the neuraminidase inhibitor gg167 in experimental human influenza use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment mapk-regulated transcription: a continuously variable gene switch? apoptosis: a mechanism of cell killing by influenza a and b viruses hostile takeovers: viral appropriation of the nf-kappab pathway triggering the interferon response: the role of irf-3 transcription factor influenza a viruses possessing type b hemagglutinin and neuraminidase: potential as vaccine components pandemic threat posed by avian influenza a viruses forecast and control of epidemics in a globalized world virulent influenza a viruses induce apoptosis in chickens reduced incorporation of the influenza b virus bm2 protein in virus particles decreases infectivity safety of a trivalent live attenuated intranasal influenza vaccine, flumist, administered in addition to parenteral trivalent inactivated influenza vaccine to seniors with chronic medical conditions caspase-3 is required for dna fragmentation and morphological changes associated with apoptosis multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (flumist) derived from cold-adapted a/ann arbor/6/60 potential role of granulocyte-macrophage colonystimulating factor as vaccine adjuvant liposomal immunostimulatory dna sequence (iss-odn): an efficient parenteral and mucosal adjuvant for influenza and hepatitis b vaccines inflammatory responses in influenza a virus infection antibodyforming cell response to virus challenge in mice immunized with dna encoding the influenza virus hemagglutinin the beginning of the end: ikappab kinase (ikk) and nf-kappab activation how nf-kappab is activated: the role of the ikappab kinase (ikk) complex phosphorylation meets ubiquitination: the control of nf-[kappa]b activity in vitro characterization of a-315675, a highly potent inhibitor of a and b strain influenza virus neuraminidases and influenza virus replication novel generations of influenza vaccines cold-adapted live attenuated influenza vaccines developed in russia: can they contribute to meeting the needs for influenza control in other countries? apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics the human mucus protease inhibitor and its mutants are novel defensive compounds against infection with influenza a and sendai viruses secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza a virus infection in the airway and chemical agents up-regulating their levels may have therapeutic potential influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors safety and immunogenicity of low and high doses of trivalent live cold-adapted influenza vaccine administered intranasally as drops or spray to healthy children resistant influenza a viruses in children treated with oseltamivir: descriptive study development of a mammalian cell (vero) derived candidate influenza virus vaccine recent advances in the discovery and development of anti-influenza drugs recent advances in the discovery and development of anti-influenza drugs protein kinase c alpha activates raf-1 by direct phosphorylation transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands p38 mitogen-activated protein kinase and c-jun-nh2-terminal kinase regulate rantes production by influenza virus-infected human bronchial epithelial cells influenza virus inhibits amiloride-sensitive na+ channels in respiratory epithelia influenza: virology, epidemiology, disease, and prevention orthomyxoviridae: the viruses and their replication prevention of influenza in the general population mekk1 activates both ikappab kinase alpha and ikappab kinase beta evaluation of anti-influenza effects of camostat in mice infected with non-adapted human influenza viruses genesis of a highly pathogenic and potentially pandemic h5n1 influenza virus in eastern asia the active sites of the influenza cap-dependent endonuclease are on different polymerase subunits recombinant influenza a virus vaccines for the pathogenic human a hong kong 97 (h5n1) viruses identification of gs 4104 as an orally bioavailable prodrug of the influenza virus neuraminidase inhibitor gs 4071 caspase activation in equine influenza virus induced apoptotic cell death virus-dependent phosphorylation of the irf-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation influenza: emergence and control preparation of a standardized, efficacious agricultural h5n3 vaccine by reverse genetics effective construction of dna vaccines against variable influenza genes by homologous recombination influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms influenza virus-induced ap-1-dependent gene expression requires activation of the jnk signaling pathway influenza-virus-induced signaling cascades: targets for antiviral therapy? a fatal relationship-influenza virus interactions with the host cell mek inhibition impairs influenza b virus propagation without emergence of resistant variants coxsackievirus b3 replication is reduced by inhibition of the extracellular signal-regulated kinase (erk) signaling pathway influenza chemotherapy: a review of the present state of art and of new drugs in development the development of live attenuated cold-adapted influenza virus vaccine for humans clinical efficacy and safety of the orally inhaled neuraminidase inhibitor zanamivir in the treatment of influenza: a randomized, double-blind, placebo-controlled european study structure and function of the interferon-beta enhanceosome ask1 regulates influenza virus infection-induced apoptotic cell death overexpression of the alpha-2,6-sialyltransferase in mdck cells increases influenza virus sensitivity to neuraminidase inhibitors neuraminidase is important for the initiation of influenza virus infection in human airway epithelium oseltamivir: a review of its use in influenza neuraminidase sequence analysis and susceptibilities of influenza virus clinical isolates to zanamivir and oseltamivir resistance of influenza viruses to neuraminidase inhibitorsa review cytotoxic t-cell immunity to influenza recognition of influenza a virus nucleoprotein by human cytotoxic t lymphocytes inhibition of hepatitis b virus dna replication by imino sugars without the inhibition of the dna polymerase: therapeutic implications imino sugars that are less toxic but more potent as antivirals inhibition of neuraminidase activity by derivatives of 2-deoxy-2,3-dehydro-n-acetylneuraminic acid oral administration of a prodrug of the influenza virus neuraminidase inhibitor gs 4071 protects mice and ferrets against influenza infection safety, efficacy and effectiveness of the influenza virus vaccine, trivalent, types a and b, live, cold-adapted (caiv-t) in healthy children and healthy adults efficacy and safety of the neuraminidase inhibitor zanamivir in the treatment of influenza a and b virus infections differential activation of the c-jun nterminal kinase/stress-activated protein kinase and p38 mitogen-activated protein kinase signal transduction pathways in the mouse brain upon infection with neurovirulent influenza a virus in vivo induction of apoptosis by influenza virus principles underlying the development and use of live attenuated cold-adapted influenza a and b virus vaccines an influenza a live attenuated reassortant virus possessing three temperature-sensitive mutations in the pb2 polymerase gene rapidly loses temperature sensitivity following replication in hamsters an influenza a virus containing influenza b virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice current status of amantadine and rimantadine as anti-influenza-a agents: memorandum from a who meeting coordinate regulation of ikappab kinases by mitogen-activated protein kinase kinase kinase 1 and nf-kappab-inducing kinase influenza a virus ns2 protein mediates vrnp nuclear export through nes-independent interaction with hcrm1 genetic engineering of influenza and other negativestrand rna viruses containing segmented genomes generation of influenza a viruses entirely from cloned cdnas live attenuated influenza virus vaccines: new options for the prevention of influenza benefits of influenza vaccination for low-, intermediate-, and high-risk senior citizens efficacy and safety of oseltamivir in treatment of acute influenza: a randomised controlled trial active nf-kappab signalling is a prerequisite for influenza virus infection verapamil inhibits influenza a virus replication the influenza virus nep (ns2 protein) mediates the nuclear export of viral ribonucleoproteins lung-specific expression of active raf kinase results in increased mortality of influenza a virus-infected mice bcl-2 alters influenza virus yield, spread, and hemagglutinin glycosylation induction of virus-specific immunity by iscoms aprotinin aerosol treatment of influenza and paramyxovirus bronchopneumonia of mice in vivo selection of an influenza a2 strain resistant to amantadine generation of high-yielding influenza a viruses in african green monkey kidney (vero) cells by reverse genetics activators and target genes of rel/nf-kappab transcription factors inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action influenza vaccines: present and future learning from our foes: a novel vaccine concept for influenza virus inhibitors of viral neuraminidase as potential antiviral drugs characterization of temperature sensitive influenza virus mutants defective in neuraminidase negativestrand rna viruses: genetic engineering and applications use of a pharmacophore model to discover a new class of influenza endonuclease inhibitors genetically engineered live attenuated influenza a virus vaccine candidates the human cell line per.c6 provides a new manufacturing system for the production of influenza vaccines 271. the wild-type m2 channel was found to be regulated by ph. the wild-type m2 ion channel activity is proposed to have a pivotal role in the biology of influenza virus infection mek-specific inhibitor u0126 blocks spread of borna disease virus in cultured cells a plasmid-based reverse genetics system for influenza a virus influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade the adjuvanted influenza vaccines with novel adjuvants: experience with the mf59-adjuvanted vaccine determinants of immunity to influenza infection in man molecular characterization of the complete genome of human influenza h5n1 virus isolates from thailand randomized trial of influenza vaccine with granulocyte-macrophage colony-stimulating factor or placebo in cancer patients apoptosis in viral infections a single dose of an iscom influenza vaccine induces long-lasting protective immunity against homologous challenge infection but fails to protect cynomolgus macaques against distant drift variants of influenza a (h3n2) viruses tamiflu™ patient information live cold-adapted influenza a vaccine produced in vero cell line entry of influenza viruses into cells is inhibited by a highly specific protein kinase c inhibitor b cell superstimulatory influenza virus (h2-subtype) induces b cell proliferation by a pkcactivating, ca(2+)-independent mechanism expanding the frontiers of existing antiviral drugs: possible effects of hiv-1 protease inhibitors against sars and avian influenza plasmid-only rescue of influenza a virus vaccine candidates amantadine-resistant and -sensitive influenza a strains and recombinants influenza virus neuraminidase activates latent transforming growth factor beta induction of apoptosis by influenza virus blockade of the map kinase pathway suppresses growth of colon tumors in vivo characterization of a porcine lung epithelial cell line suitable for influenza virus studies role of protein kinase c betaii in influenza virus entry via late endosomes dissecting virus entry via endocytosis serial killers: ordering caspase activation events in apoptosis cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities the humoral response to live and inactivated influenza vaccines administered alone and in combination to young adults and elderly confronting the avian influenza threat: vaccine development for a potential pandemic evaluation of a genetically modified reassortant h5n1 influenza a virus vaccine candidate generated by plasmid-based reverse genetics characterization of an avian influenza a (h5n1) virus isolated from a child with a fatal respiratory illness multidose, live attenuated, cold-recombinant, trivalent influenza vaccine in infants and young children vitro and in vivo activities of t-705 and oseltamivir against influenza virus activation of the apoptotic fas antigen-encoding gene upon influenza virus infection involving spontaneously produced beta-interferon induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells possible involvement of double-stranded rna-activated protein kinase in cell death by influenza virus infection recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death influenza a and b viruses expressing altered ns1 proteins: a vaccine approach defense mechanisms against influenza virus infection in the respiratory tract mucosa the interferon-alpha/beta system in antiviral responses: a multimodal machinery of gene regulation by the irf family of transcription factors the 1918 influenza virus: a killer comes into view virus induction of human ifn beta gene expression requires the assembly of an enhanceosome caspases: enemies within monitoring of viral susceptibility: new challenges with the development of influenza na inhibitors signaling through protein kinase c inhibition of cap (m7gpppxm)-dependent endonuclease of influenza virus by 4-substituted 2,4-dioxobutanoic acid compounds expression, purification, and characterization of orthomyxovirus: influenza transcriptase rimantadine hydrochloride and amantadine hydrochloride use in influenza a virus infections recognition of influenza virus proteins by cytotoxic t lymphocytes evaluation of live, cold-adapted influenza a and b virus vaccines in elderly and high-risk subjects efficacy and safety of the oral neuraminidase inhibitor oseltamivir in treating acute influenza: a randomized controlled trial. us oral neuraminidase study group evaluation of trivalent, live, cold-adapted (caiv-t) and inactivated (tiv) influenza vaccines in prevention of virus infection and illness following challenge of adults with wild-type influenza a (h1n1), a (h3n2), and b viruses protective efficacy of combined live intranasal and inactivated influenza a virus vaccines in the elderly comparison of large-scale mammalian cell culture systems with egg culture for the production of influenza virus a vaccine strains depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h5n1) virus isolated from humans destruction of lymphocytes by a virulent avian influenza a virus virulent avian influenza a viruses: their effect on avian lymphocytes and macrophages in vivo and in vitro structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 a resolution the structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase rational design of potent sialidase-based inhibitors of influenza virus replication transcription stimulation of the fas-encoding gene by nuclear factor for interleukin-6 expression upon influenza virus infection interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics influenza a virus ns1 protein prevents activation of nf-kappab and induction of alpha/beta interferon live attenuated vaccines against influenza; an historical review influenza a virus with defective m2 ion channel activity as a live vaccine immunogenicity and protective efficacy of replication-incompetent influenza viruslike particles are we ready for pandemic influenza? influenza virus: transmission between species and relevance to emergence of the next human pandemic predictions for future human influenza pandemics spanish influenza: the secrets remain elusive evolution and ecology of influenza a viruses interspecies transmission of influenza viruses influenza: interspecies transmission and emergence of new pandemics effectiveness of oseltamivir in preventing influenza in household contacts: a randomized controlled trial mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human local immune responses to influenza antigen are synergistically enhanced by the adjuvant iscomatrix direct gene transfer into mouse muscle in vivo cellular immunity and memory to respiratory virus infections 4-guanidino-2,4-dideoxy-2,3-dehydro-n-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza a and b viruses in vitro communicable disease surveillance & response (csr) ikappab kinase-beta: nf-kappab activation and complex formation with ikappab kinase-alpha and nik fields virology nf-kappab-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand (trail) and fas/fasl is crucial for efficient influenza virus propagation caspase 3 activation is essential for efficient influenza virus propagation direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 comparative study of influenza virus replication in vero and mdck cell lines nf-kappab activation by double-stranded-rna-activated protein kinase (pkr) is mediated through nf-kappab-inducing kinase and ikappab kinase position statement: global neuraminidase inhibitor susceptibility network caspase-dependent nterminal cleavage of influenza virus nucleocapsid protein in infected cells immunostimulating reconstituted influenza virosomes we would like to thank all the colleagues who have helped in many invaluable ways in the production of this chapter, in particular, dr. c. erhardt, dr. w. wurzer, h. marjuki, b. daubner, v. oehlschlaeger, j. lampe and k. oesterle. this work is dedicated to prof. dr. c. scholtissek's 75 th birthday. key: cord-030279-pv770doe authors: novossiolova, tatyana title: twenty-first century governance challenges in the life sciences date: 2016-11-29 journal: governance of biotechnology in post-soviet russia doi: 10.1007/978-3-319-51004-0_4 sha: doc_id: 30279 cord_uid: pv770doe the chapter explores the rapid advancement of biotechnology over the past few decades, outlining an array of factors that drive innovation and, at the same time, raise concerns about the extent to which the scope and pace of novel life science developments can be adequately governed. from ‘dual-use life science research of concern’ through the rise of amateur biology to the advent of personalised medicine, the chapter exposes the limitations of the existing governance mechanisms in accommodating the multifaceted ethical, social, security, and legal concerns arising from cutting-edge scientific and technological developments. so far as to suggest that the 'life sciences knowledge, materials and technologies are advancing worldwide with moore's law-like speed.' 3 and whilst some commentators have questioned the extent to which the ongoing progress of biotechnology has translated into practical applications and novel products, 4 there is some consensus that the biotechnology landscape has been fundamentally transformed over the recent decades with the possibilities now unlocked holding revolutionary potential. indeed, rapid advances in the field have produced a knowledge base and set of tools and techniques that enable biological processes to be understood, manipulated and controlled to an extent never possible before 5 ; they have found various applications in numerous spheres of life, generating enormous benefits and offering bright prospects for human betterment; and they have come to be regarded as a key driver of economic development with potential to close the gap between resource-rich and resource-poor countries. 6 the progress of biotechnology has been largely driven by three sets of forces, namely social, political and economic. 7 the social dynamics at work in this context are understood as the efforts to improve public health and overall wellbeing of individuals both in the global north and global south, boost agricultural yields and encourage environment-friendly practices to mitigate the adverse effects of climate change. several factors account for the significant value attached to the life sciences in the context of intense globalisation and continuous change. surging population numbers and extended life expectancy are augmenting the demand for developing effective and affordable medications, novel approaches for the treatment of chronic diseases and additional cost-effective sources of energy and food production. at the same time, rising global trade and travel, coupled with increased urbanisation, and an uneven distribution of wealth are creating optimal conditions for disease outbreaks, pandemics and environmental degradation. 8 against this backdrop, biotechnology appears full of promise and critical to tackling social and natural concerns; enhancing disease prevention, preparedness and surveillance; promoting development; and alleviating human suffering. 9 economic dynamics include national expenditure on research and development, purchasing power, trends in consumerism and market pressures and fluctuations. besides public funding for r&d which remains a key factor in the growth and flourishing of bioindustry in developed and emerging economies alike, private investment from venture capital firms, start-up companies and transnational corporations (tncs) have also played an indispensable role in capturing new markets and further facilitating the extension of bioeconomy on a global scale. dupont's significant footprint in india is indicative in this regard, not least because of the depth and diversity of the activity that the company has undertaken via its offshore r&d centres ranging from crop science to biofuels. 10 likewise, merck has outlined a 1.5 billion dollar commitment to expand r&d in china, as part of which it intends to establish an asia headquarters for innovative drug discovery in beijing. 11 political dynamics are triggered by states' increasing commitment to support the progress of biotechnology as a way of maximising their power and boosting their status in the international arena. 12 in the aftermath of 9/11 and the 'anthrax letters' attack of october 2001, substantial effort has been given to harnessing life science research for the purposes of national security. biodefence and bioterrorism preparedness are thus considered high-priority areas for national investment by government agencies and the military alike. an illustrative example of this two-tiered approach is the funding policy in the usa, where biodefence research is financed by the nih, department of homeland security (dhs) and defense advanced research projects agency (darpa), to name a few. under the synergistic influence of these three sets of forcessocial, economic and politicalbiotechnology has been transformed into a truly global fast-evolving enterprise encompassing a multitude of stakeholders, delivering considerable benefits and holding out still greater promise, with profound and far-reaching implications for virtually every aspect of human well-being and social life. the pharmaceutical industry is a case in point, for its steady expansion would hardly be possible were it not for the vast array of techniques and methods enabled by the progress of the life sciences. worth roughly 400 billion dollars, the global pharmaceutical market dominates the life sciences industry and arguably determines the trajectory of life sciencesrelated technological development and global spread. 13 gene cloning, dna sequencing and recombinant construction of cell lines, to name a few, are all deemed indispensable for the development of novel medicines and therapeutics. it suffices to mention that more than half of the top selling commercially available drugs in the usa would not exist without those methods. 14 agriculture, too, has been heavily influenced by the ongoing biotechnology revolution, as evidenced in the rapid growth and dispersion of commercialised transgenic crops (biotech crops) and the efforts to use gmos (both animals and plants) for the production of vaccine antigens and other biologically active proteins ('biopharming'). 15 indeed, the increase in the area of farmland planted with transgenic crops rose dramatically from 1.7 hectares in 1996 to about 60 million hectares in 2002 16 and is still growing. in addition, technological convergence 17 between biotechnology, nanotechnology, information technologies and cognitive science has unlocked a broad scope of opportunities for maximising public (and private) welfare, offering substantial benefits in wideranging areas such as medicine, pharmacy, crime investigation and national security by ensuring precision and reliability, while at the same time, reducing the amount of time previously required for the performance of certain tasks. four key features of biotechnology make it so appealing to the majority of stakeholders involved. first, biotechnology innovation is characterised by duality, whereby research yields results that simultaneously lead to advances in basic knowledge and stimulate product development. 18 second, the output that the life sciences generate in the form of new medicines, improved nutrition products, enhanced yields and novel materials, is 'strongly positive'. 19 the increasing utility of tools and strategies for human enhancement, whether in professional sport, for cosmetic and aesthetic purposes, or on the battlefield, vividly reflects the firm conviction that the transformative capacity of biotechnology, even at the most fundamental level, is something to be welcomed and vigorously embraced. what is more, biotechnology possesses proven economic viability, as illustrated in the burgeoning industries and new markets it has spurred. against this backdrop, the high rate of biotechnology expansion is anything but surprising, since every increment in biological capability pays back the researcher and the researcher's sponsors in short order. payback comes in the esteem of peers, in promotions, and in increases in the academic or corporate salaries of the researchers whose work generates knowledge and new therapies. payback comes in the form of profits for the manufacturers of kits to perform the manipulations, royalties for the writers of the methods manuals profits for the drug industry. payback comes for the public in the form of new drugs and therapies. 20 fourth, besides being cost-effective, many of the benefits that biotechnology offers are easy to obtain and disseminate. in other words, many of the various prospects for public (and private) betterment are not situated at some distant moment in the future but can be realised immediately, as a result of which pressing problems can be alleviated, if not fully resolved, and substantial revenue can be generated in the short term. last but not least, while there are some risks and concerns associated with the advancement of biotechnology, few of those are deemed urgent or significant enough to impact on the pace of innovation. as the actual manifestation of such risks is often contingent upon the interplay of a variety of factors, this renders the likelihood of a major crisis unfolding as a result of the progress of biotechnology low. moreover, there is a genuine belief that any challenges that may arise from the proliferation of novel technologies can either be foreseen or dealt with on a case-by-case basis. given the enormous potential of biotechnology for addressing societal, economic and environmental challenges, it is unsurprising that most states have readily endorsed scientific and technological innovation and embarked on largescale generously-funded r&d programmes in the life sciences. given the powerful multifaceted impetus for biotechnology advancement, it is possible to identify at least five key trends in the governance of biotechnology that are common for highly industrialised and developing countries alike. those include: high-level coordination, facilitation and funding; synergies within and between both the public and private sector; emphasis on strategic and competitive interests at the expense of precaution; regulations that seek to promote rather than restrict scientific and technological progress; and overreliance on technical solutions. at international level, the on-going expansion of biotechnology has been hailed not only as an inherently positive development but also as an essential prerequisite for enhancing human welfare and addressing various socio-economic, environmental and health concerns. in its 2013 world health report, the who called for: increased international and national investment and support in [life science] research aimed specifically at improving coverage of health services within and between countries. 21 the who has also strived to promote research on specific diseases, such as hiv/aids, cancer, pandemic influenza, tuberculosis and malaria, with the goal to improve methods for prevention and diagnostics and facilitate the development of effective therapeutics and vaccines. 22 in a similar fashion, the un food and agriculture organisation (fao) has highlighted the positive impact that biotechnology could have on the development of agriculture: . . . biotechnology could be a major tool in the fight against hunger and poverty, especially in developing countries. because it may deliver solutions where conventional breeding approaches have failed, it could greatly assist the development of crop varieties able to thrive in the difficult environments where many of the world's poor live and farm. 23 it is not difficult to see how those assertions have been translated into national policies and practical steps across the globe. the us nih that provide the bulk financial support for medical and health-oriented r&d in the us spent over 30.9 billion dollars during the fiscal year 2012, about a third of which was allocated for funding biotechnology and bioengineering projects. 24 within its sixth framework programme for research and technological development spanning the period 2002-2006 the european union (eu) distributed more than 2.5 billion euro for projects under the theme 'life sciences, genomics and biotechnology for health'. 25 developing countries, too, are increasingly investing in 'red' biotechnology as part of their efforts to address public health concerns. according to a recent who report, support for biotechnology and particularly, for cancer research, in cuba has soared over the past 20 years, amounting to over one billion dollars. 26 as a result, the cuban biotechnology industry is burgeoning, holding around 1200 international patents and exporting vaccines and pharmaceuticals to more than 50 countries. the prospect of climate change coupled with rising population numbers has compelled governments in the global north and south alike to explore 'green' biotechnology as a means of ensuring food security. the usa remains by far the largest commercial producer of gm crops. several eu member states (france, germany, spain, poland, romania, czech republic, portugal and slovakia), canada and australia further feature in the list of industrialised nations that have embarked on growing gm plant breeds. more and more emerging economies are striving to expand their agrobiotechnology sector, most notably brazil, india, argentina, south africa, mexico, burkina faso, myanmar and chile. 27 in 2008, the chinese government launched a major r&d initiative worth 4 billion dollars to develop new plant varieties by 2020 that will enhance yields, have improved nutritional value and be resistant to pests. 28 public-private partnerships underpinned by access to early-stage risk capital and strong linkages between business, universities and entrepreneurial support networks constitute an important vehicle for promoting innovation and fostering technology transfer and product development. for instance, the chinese government has launched a major initiative mobilising 2.5 billion dollars in venture capital to support start-ups in the immense zhangjiang science park outside shanghai 29 ; russia's rusnano has entered a 760 million dollar partnership with the us venture capital firm domain associates to fund 'emerging life science technology companies and establish manufacturing facilities in russia for production of advanced therapeutic products'; and cleveland's university hospital has allocated 250 million dollars for setting up a 'non-profit entity to fund and advise physician-scientists on transitional research and a related for-profit accelerator that will develop selected compounds to proof of concept.' 30 the kauffman foundation in the usa, a wealthy philanthropic establishment dedicated exclusively to the goal of entrepreneurship has been particularly zealous in its quest for promoting university-based entrepreneurial activities nationwide. its kauffman campuses initiative launched in early 2003 enjoyed so much popularity among universities that following the initial round of grants totalling 25 million dollars, the foundation announced its resolve to leverage a 100 million dollar investment for the creation of new interdisciplinary education programmes. 31 university-industry partnerships, while not a novel phenomenon in the area of biotechnology, have considerably intensified over the past several decades, thus facilitating the widespread commercialisation of life science research. indeed, 90 per cent of the companies in the us surveyed by blumenthal et al. in 1996 had relationships with an academic institution in that year and in more than half of those cases industry provided financial support for research in such institutions. 32 according to another study, the total industry investment in academic life science research in the usa tripled between 1985 and 1998 reaching almost 2 billion dollars and has been growing ever since. 33 against this backdrop, some commentators have put forward the 'triple helix' model, which serves both as a conceptual tool and a policy blueprint. in the former case, it is used to elucidate the academic-industry-government relationships that underpin the institutional arrangements and changing practices in the processes of production, transfer and application of knowledge in post-industrial societies; in the latter, it is promoted as a framework for economic development through state investment and knowledge sharing between academia and industry. 34 others, however, have remained sceptical of the close integration of universities and the private sector voicing concerns about the possible deleterious effects arising therefrom: as in other activities, when big money flows fast, temptations and opportunities arise for risky behaviour and stealthy or even brazen wrongdoing in pursuit of personal or institutional advantage. the new world of academiccommercial dealings is characterised by some grey areas and evolving rules for permissible and impermissible conduct. the people who manage and conduct research in scientific organisations are not immune to the weaknesses and foibles so plentiful elsewhere, despite the accolades for probity that science bestows upon itself. 35 with more and more universities joining the biotechnology 'gold rush' and corporate values and goals steadily penetrating the professional academic cultures, scholarship turns into a result-oriented activity subject to the priorities and interests of business partners and industrial sponsors. strategy and careful planning deemed essential to the pursuit of for-profit knowledge can have a restraining effect on the spontaneous vigour characteristic of academic research, limiting the range of problems that could be studied to those defined by the market. 36 at the same time, scientists often find themselves under tremendous pressure striving to satisfy the demands of their industrial clients without utterly neglecting their academic duties ranging from mentorship through filing grant applications to publishing. the extensive workload coupled with the bright prospects for securing long-term research funding and achieving some individual gain and prominence provide a favourable environment in which instances of dubious, sometimes fraudulent, behaviour, conflicts of interest and lack of transparency, unless too severe, are unlikely to encounter widespread opprobrium and may even go unnoticed. 37 in the race for patents and venture capital, the business mentality dulls scientific rigour and the ethics threshold appears not too difficult to cross. interests at the expense of precaution given the tremendous benefits that biotechnology is expected to generate in virtually any sphere of human activity, it is not difficult to understand why its progress is predominantly viewed through an explicitly positive lens by policy-makers. since the opportunities for achieving public betterment and enhancing state prestige and international standing are too tempting and too abundant, there is a powerful urge to dedicate both will and resources to promoting the large-scale expansion of the life sciences. for one thing, the prospect of conquering disease and maximising human wellbeing provides solid justification for a deliberate and sustained investment in fostering scientific and technological prowess. lack of commitment and reluctance to support r&d in the life sciences then becomes an unfavourable option in the political calculations of states regardless of their level of economic development and international status. within the context of political calculus pervaded by realist fears, competition and power, the perceived risks of inaction with regard to scientific and technological development justify vast expenditure, lower regulatory barriers to innovation and product development. political choices concerning biotechnology support are therefore frequently made at the expense of calls for caution and potential social, environmental and ethical concerns. the regulation of genetic engineering is a case in point. as discussed in the previous chapter, from the outset, the attempts of governments to impose strict controls on research involving rdna faced a severe backlash from academic scientists and business executives alike. by the 1980s, the various legislative initiatives put forward in the usa were abandoned in favour of the regime established by the nih guidelines, which virtually exempted the biotechnology industry from formal regulation. while the leading us-based companies pledged to 'voluntarily comply' with the guidelines, behind the scenes they craftily continued to push for a system that would insulate them from governmental and public scrutiny. 38 indeed, during the 1990s when the states parties to the btwc strived to strengthen the treaty by negotiating a binding verification mechanism corporate interests proved too big and too important to be ignored. both the pharmaceutical research and manufacturers of america (phrma) which represented the country's major research-based pharmaceutical and biotechnology companies, and the biotechnology industry organisation (bio) which at that time represented some 1400 biotechnology firms, became vocal opponents of any measures designed to promote international arms control which seemed to hinder in any way the protection of proprietary information and intellectual property. 39 in the period between 1994 and 2001 the associations invested considerable effort, time and ingenuity in lobbying the us government and influencing the diplomatic talks in geneva to secure an outcome that was in line with the demands of their constituencies. of course, it would be naive to ascribe the us resolve to reject in 2001 both the text of the protocol and its utility in general for providing adequate verification and enhancing confidence among states parties solely to the activity of the biotechnology industry; nevertheless, it would be equally naive to suppose that corporate interests played no significant role in the process. 40 besides economic priorities, national security and military calculations can also provide a compelling rationale for downplaying the potential risks associated with biotechnology expansion. following the 'anthrax letters' attack in october 2001, the us government embarked on a massive financial investment to boost its bioterrorism preparedness and enable the prevention, early detection, monitoring and emergency response to biological threats. as outlined in biodefense for the 21st century, a presidential directive that set out a comprehensive framework for national biodefence policy, between 2001 and 2005 the federal government provided roughly 6 billion dollars 'to state and local health systems to bolster their ability to respond to bioterrorism and major public health crises'. 41 along with the highly controversial vaccination programme that the government envisaged, 42 another important development designed to enhance america's biodefence preparedness and capability was the drastic increase both in the number of high-containment labs (bsl-3 and bsl-4) and the number of researchers with access to some of the most dangerous pathogens known to mankind, including the causative agents of ebola, plague and q fever. some commentators have questioned the logic behind this policy highlighting the heightened risk of accidental or deliberate release of pathogens. 43 far from being ill-founded or hypothetical, such fears stemmed from a range of high-profile cases that occurred after 2001 across the us in which the lack of proper training and professional negligence resulted in scientists being exposed to or infected with deadly microbes. 44 real-life horror stories about vials of plague being transported in the hand-luggage of researchers on passenger aircraft without the required authorisation, and deadly cultures gone missing from what appeared to be secure laboratories further fuelled the criticism toward the us biodefence policy raising difficult questions about its appropriateness and actual goals even before the 'anthrax letter' investigation revealed that the attack was 'insider's business'. 45 life science research, just as any other sphere of professional activity, is subject to a range of institutional, national and international regulations. along with the more general rules such as those related to occupational health and safety, fair pay and job competition, conflict of interests, labour rights, and professional liability, there are also specific ones addressing particular aspects of the research process including project clearing (e.g. review by local biosafety committees), safe laboratory practice and transport of pathogens (e.g. 2005 international health regulations), exchange of viral strains (e.g. pandemic influenza preparedness framework, 2011), handling of dangerous pathogens (e.g. us select agent programme) and ethical treatment of human subjects and samples obtained therefrom (e.g. the 2004 human tissue act in the uk). 46 while hardly exhaustive, this list suffices to convey the idea that the regulatory regime governing the practice of life science research is dense and comprehensive. with more than 30 international organisations overseeing biotechnology from various perspectives, 47 there is a prima facie reason to assume that the regime in its current form is sufficiently flexible to accommodate novel advances and hold any potential risks, which they may pose, at bay. yet in reality over the past decade the opposite trend has prevailed, that is, the existing governance mechanisms have struggled to respond adequately to the proliferation of new scientific developments with multiple adaptive uses and the multiplicity of cutting-edge developments posing profound ethical quandaries. how to account for this discrepancy? part of the problem stems from the fact that since at least the late 1970s the regulation of biotechnology has been streamlined so as to become compatible with and not a restriction on continued technological change and economic growth. 48 as such, it rests upon the barely questioned assumption that the progress of biotechnology is inherently good and needs to be harnessed and vigorously promoted. needless to say, any measures that seem to slow down or restrain its advancement are deemed undesirable and even detrimental to socio-economic development. hence, when developing regulations, policy-makers have generally pursued a twofold objective: first, to promote the safe practice of life science research by reducing any risks arising therefrom both to scientists and the general public; and second, to ensure that any issues that may hinder the expansion of biotechnology are not subject to restrictive legislation. a vivid manifestation of this approach is the way in which the ongoing debate on 'dual use research of concern'benignly-intended research that seeks to maximise human welfare by responding to health, societal and environmental ills but could also facilitate to the development of more sophisticated and potent biological weapons and enable bioterrorism 49has been handled. for more than a decade, researchers, journal editors, security experts and policy-makers have strived to devise oversight mechanisms and governance initiatives that could adequately tackle the challenge of dual use without stifling innovation. unfortunately, to date their efforts have met with little success, as a result of which virtually each experiment of dual use concern is dealt with separately on a case-by-case basis. this is not to say that there are no similarities across the studies of this kind. on the contrary, a few of the most notable examples follow a similar paradigm, including the creation of a vaccine-resistant strain of the mousepox virus, the artificial synthesis of the polio virus, the recreation of the 1918 spanish influenza virus and, most recently, the production of a mammalian-transmissible h5n1 avian influenza virus (see fig. 4 .1). 50 all four of them were performed in strict compliance with the rules and procedures in place for laboratory biosafety, biosecurity and biorisk management and under appropriate physical containment conditions; all had passed thorough review by the respective local biosafety and bioethics committees; and all of them were deemed essential in terms of public health benefits. above all, the ethical and security concerns that the studies have raised go far beyond the laboratory door, posing fundamental questions about how life science research is reviewed, conducted and communicated. yet none of the high-profile experiments of concern has proved critical enough to provoke a radical change in the way dual-use research is governed. 51 three points merit consideration in this regard. the first pertains to the manner in which the dominant discourse on dual use is framed, that is, in purely ethical terms as a dilemma. while bioethics undoubtedly has a role to play in the discussions on dual use, the language in early 2001, the journal of virology published a report of the creation of a highly virulent strain of the ectromelia virus, the causative agent of mousepox. the work described in the report was carried out by a group of australian scientists based in canberra. its original goal was the development of an infectious immunecontraceptive that could be used against wild mice for the purpose of pest control. to achieve this, the group drew upon previously published work. during the course of the experiment, the researchers unexpectedly discovered that the newly engineered strain of the mousepox virus, which they created, killed 60% more mice than the parent virus, including mice that had been vaccinated or that had natural immunity. when the research was published, concerns were raised that it could potentially be misapplied for hostile purposes, or even that the same technique could be utilised for creating a more virulent strain of the variola virus, which causes smallpox in humans. in 2002, a team of scientists led by dr eckard wimmer from the university of new york at stony brook announced that they had successfully created a polio virus 'from scratch'. to carry out the research, the scientists 'followed a recipe they downloaded from the internet and used gene sequences from a mail-order supplier'. a once the virus created, it was tested on mice, as a result of which the infected animals became paralysed and died. the study spurred a wide-ranging debate, not least because it drew attention to the possibility of using synthetic biology for constructing de novo viruses for the purposes of bioterrorism. in 2005, it was announced that cdc scientists, together with colleagues from several research institutions across the usa, had successfully recreated the influenza virus, that was responsible for the 1918 pandemic, which killed between 20 and 50 million people worldwide. using dna from a tissue of a flu victim buried in the permafrost in alaska, the researchers managed to reconstruct the influenza virus and thus study its pathogenesis and properties that contributed to its virulence. despite the scientific justification that was put forward, critics have argued that the study is 'a recipe for disaster', not least because the availability of the virus' full-genome sequence and detailed method for its reconstruction on the internet may facilitate its synthesis by a of 'dual-use dilemmas' is too abstract to offer appropriate analytical tools for dealing with the issues at play. as discussed above, the questions that dual-use research poses such as data sharing, research funding and project planning are far from hypothetical but they feature explicitly in everyday professional practice. however, the 'dilemma framework' automatically strips them of the complex socio-technical arenas in which they have actually presented themselves by laying an emphasis on what action should ideally be taken, rather than what is practically feasible given the circumstances. 52 moreover, such issues are typically structural in nature for they constitute fundamental elements of the life sciences professional culture, and as such, could hardly be adequately addressed solely at the level of individual researchers. yet framing social, legal and security concerns in terms of moral dilemmas allows for structural issues to be omitted from the discussion, rendering life scientists the chief, if not the only, moral agents expected to reach what is deemed to be the 'right' answer. 53 assigning abstract duties then comes to be regarded as an appropriate 'solution', even if those are virtually impossible to fulfil given the complexities of the working environment within which researchers operate. the second point is related to the reductionist view that dominates the discourse of what counts as a risk in life science research. perhaps one of the most significant legacies of the asilomar conference on rdna (see chapter 3) is the emphasis on laboratory risk that could be effectively managed by dint of physical containment and rules and procedures for safe laboratory practice. 54 it suffices to mention that the bulk of guidelines and formal regulations published by the who focus exclusively on promoting and refining measures that aim to maximise laboratory biosafety and prevent the accidental release of pathogens. hence, it is hardly surprising that the concept of dual use and the idea of risks beyond the laboratory door implicit in it seem alien to the majority of practising researchers. striking as it may appear, even though dual use research has been debated for more than a decade now, the level of awareness among life scientists of the broader social, security and legal implications of their work remains low. 55 the third point deals with the way in which risks in life science research are assessed and mitigated. given the narrow definition of risk encompassing technical particulars, physical containment and biosafety, risk assessment is considered an appropriate and reliable tool for ensuring research safety. the heavy reliance upon risk assessing tools is underpinned by two underlying assumptions. one is that it is possible to foresee and calculate most, if not all, things that could potentially go wrong both during the development phase of the project and after its completion. the other is that it is possible then to use the produced data as a basis for devising measures and strategies for eradicating, or at least, mitigating the risks likely to occur. attractive as it may seem, this 'new alchemy where body counting replaces social and cultural values' presupposes a clear distinction between the risk assessment 'experts' and the general public, whereby the former are granted a licence to make decisions about the risks that the latter cannot do without. 56 likewise, costbenefit analysis on the basis of which research proposals are screened for potentials risks and security concerns has attracted some serious criticism. in the view of some commentators, besides being sometimes deeply inaccurate, the cost-benefit analysis is 'ethically wrong' since 'applying narrow quantitative criteria to human health and human life' is unacceptable. 57 but there are other problems, too. as pointed out by dickson, the cost-benefit analysis distorts political decision-making by omitting any factors that cannot be quantified, thus obscuring questions of equity, justice, power, and social welfare behind a technocratic haze of numbers. 58 as a result, complex and politically charged decisions are reduced to a form that fits neatly into the technocratic ways of making regulatory decisions, whereby calculations and approximations made by the few substitute for the judgements of many. 59 the wide-ranging controversy that unraveled in late 2011 when two teams of scientists working independently in the netherlands and the usa managed to produce an air-borne strain of the h5n1 avian influenza virus, a highly pathogenic and lethal microbe with over 60 per cent mortality rate in humans arguably constituted the pinnacle of the deliberation on dual use research. both studies set alarm bells ringing for the security community who almost immediately jumped in the debate voicing concerns over the possibility of biological proliferation and bioterrorism. some commentators even argued that the experiments ran counter to the spirit if not to the letter of the 1975 btwc. 60 against this backdrop, the resultant controversy was deemed at least initially to offer a timely opportunity to evaluate the existing governance mechanisms, determine their gaps and weaknesses, and broaden the scope of deliberation inviting participation of a wide range of stakeholders. unfortunately, the outcome of the debate proved far more moderate, signalling preference for preserving the status quo without disrupting the established systems for governance and oversight. despite the extensive mass media coverage of the controversy, only few public consultations were held and none of those was designed as a platform for making policy proposals or developing action plans. moreover, the denselypacked agenda prepared duly in advance left very limited scope for posing 'tricky' questions which the participating 'experts' might have struggled to answer. needless to say, all consequential decisions were made behind closed doors away from public scrutiny and on some occasions the people with the greatest vested interest in the publication of the studies were also the ones with the greatest say in the process. 61 there were no significant changes in terms of governance initiatives, either. far from being ground-breaking developments, the us government policy for oversight of life sciences dual use research of concern 62 and the decision of the dutch government to invoke export control legislation before allowing the publication of the study conducted within its jurisdiction were little more than desperate moves that aimed to obscure the inadequacy and shortcomings of the measures already in place. 63 overall, the manner in which the h5n1 debate was handled could be treated as a missed opportunity, whereby those in charge of the decision-making process did little to address or even acknowledge the broader issues underpinning dual-use research of concern but simply 'kicked the can down the road to the next manuscript' waiting for the next controversy to erupt. 64 technology seems to play a significant role in the governance of life science research. high-containment laboratories, well-equipped biosafety cabinets, sophisticated waste management systems, enhanced personal protective equipment and secure containers for the safe storage and transportation of biohazard materials are just a few of the tools and systems in place that allow the safe handling of dangerous pathogens and toxins and, at the same time, protect both laboratory personnel and the general public from exposure to deadly microbes. that said, the effectiveness of technical solutions should not be overstated if only for the fact that 'problems' of governance are barely technical matters per se but rather constitute complex issues of human relatedness. nevertheless, the attractiveness of technological fixes as offering reliable risk mitigation and reassurance in the safety of biotechnology is ever growing. it suffices to mention that the h5n1 controversy discussed above was in part resolved after the lead researchers in the netherlands and the usa respectively agreed to add a detailed section on the technical specificities and laboratory biosafety and biosecurity measures taken during the experiments. 65 the strategy has proven effective in diverting attention from the rather inconvenient questions regarding the utility and significant potential for hostile misuse of the so called 'gain-of-function' (gof) research and concentrating it on more mundane issues dealing with in-house precautions and safety procedures. once the latter were deemed adequately resolved, the former were effectively forgotten. still, the value of technical means in ensuring reliable risk management should not be taken for granted. for one thing, laboratory biosafety precautions, however sophisticated, are far from perfect and accidents do occur. such is the case with the pirbright site in the uk which was at the centre of a major outbreak of foot-and-mouth disease in 2007, as a result of which over 2100 animals were slaughtered. 66 in 2012 the bioterrorism bsl-3 laboratory at the us cdc in atlanta suffered repeated problems with airflow systems designed to help prevent the release of infectious agents. 67 the faulty system could perhaps be regarded as an exception had it not been for the authoritative investigation report of the us government accountability office (gao) released in march 2013. according to the report, the cost of building and maintaining high-containment laboratories, coupled with the absence of national standards for their design, construction, operation, and maintenance 'exposes the nation to risk'. 68 far more critical is the situation in the developing world and emerging economies where lax regulations and technical failures have significantly heightened the risk of accidental release of pathogens, as demonstrated by the numerous 'escapes' of the severe acute respiratory syndrome (sars). 69 but even if technology functions impeccably, this hardly reduces the likelihood for a human error or inappropriate behaviour. unlocked doors in high-containment facilities hosting deadly pathogens, eating and drinking in laboratories and poor waste disposal practices are just a small part of the otherwise long list of mundane mishaps that may result in severe consequences. it is worth mentioning that the us cdc came under the spotlight after internal e-mail correspondence revealed that doors in the bsl-3 block where experiments involving the causative agenets of anthrax, sars and influenza were performed were left unlocked on numerous occasions, thus increasing the risk of unauthorised access or theft. 70 given the chance of technical flaw and the potential for human error, some life scientists have begun to question the reliability of existing laboratory precautions and demand thorough review and evaluation. in a recent letter to the european commission the foundation for vaccine research has asked for 'a rigorous, comprehensive risk-benefit assessment' of gof research that 'could help determine whether the unique risks posed by these sorts of experiments are balanced by unique public health benefits which could not be achieved by alternative, safe scientific approaches'. 71 engines that drive biotechnology momentum by and large, the ongoing progress of biotechnology is largely viewed and assessed through an explicitly positive lens which allows focusing almost exclusively on the benefits likely to be accrued notwithstanding the risks, actual and potential. the resultant distorted image is problematic, not least because it precludes any comprehensive discussion on the potential side effects and negative implications of novel life science advances. above all, it sustains the barely questioned assumption that the existing governance mechanisms are adequate and sufficient to cope with the stresses and strains of the rapidly evolving biotechnology landscape. yet given the complex and multifaceted dynamics shaping the life science enterprise, the rapid pace of innovation, and the limits to predicting the synergistic and cumulative effects of the proliferation of new technologies, the uncritical acceptance of such assumptions is at best naïve and at worst dangerous. arguably the advancement of the life sciences has greatly benefited from the fascinating breakthroughs made in other areas of study, such as chemistry, engineering, computing, informatics, robotics, mathematics and physics. some commentators even talk about a third revolution in biotechnology underpinned by scientific and technological convergence: convergence does not simply involve a transfer of tools sets from one science to another; fundamentally different conceptual approaches from physical science and engineering are imported into biological research, while life science's understanding of complex evolutionary systems is reciprocally influencing physical science and engineering. convergence is the result of true intellectual cross-pollination. 72 the resultant 'new biology' has opened up a range of marvellous possibilities enabling the manipulation of living matter at the full range of scales, as well as the application of biological systems principles for the development of novel materials, processes and devices. 73 as such, it has been largely hailed as possessing the 'capacity to tackle a broad range of scientific and societal problems.' 74 this is not an exaggeration. as noted by a recent report of the us nas, the precipitous decline in the cost of genome sequencing would not have been possible without a combination of engineering of equipment, robotics for automation, and chemistry and biochemistry to make the sequencing accurate. 75 likewise, it is the combination of expertise from fields as diverse as evolutionary biology, computer science, mathematics, and statistics that has allowed both the analysis of raw genomic data and the subsequent use of these data to other fields. 76 at the same time, advances in nanoscience and nanotechnology have considerably enhanced drug delivery making it more accurate by targeting specific parts of the body. 77 yet the transformative potential of scientific and technological convergence comes at a price, not least because parallel to the benefits it offers, there are risks the effects of which could be truly devastating. 78 take drug delivery, for instance. thanks to the technological breakthroughs over the past decade, doctors have gained unprecedented access to the human body which, in turn, has facilitated the treatment of previously incurable disease and conditions (e.g. some forms of cancer). nanoparticles and aerosols are now utilised for delivering a precise dose of therapeutics to tissues and cells via novel pathways circumventing body's natural defences and evading immune response. it is not difficult to imagine how such knowledge could be misapplied for malicious ends, including incapacitating and killing. research on bioregulators is a case in point. bioregulators are natural chemicals in the human body that play a vital role in the maintenance of the homeostasis but when administered in large quantities or in healthy individuals could be toxic and lead to serious disorders, even death. given their properties, bioregulators constitute the perfect bioweapon: efficient and virtually impossible to detect. and if in the past, security analysts discounted the risk of their weaponisation due to the instability of the compounds when released in the atmosphere, the emergence of novel drug delivery techniques has significantly altered the security calculus. 79 this is just but one example of the challenges that the increasing convergence between biology and chemistry poses to the integrity of the international biological and chemical non-proliferation regimes. 80 even though some effort has been made over the recent years to address those and other areas of concern and strengthen the international prohibition against biological and chemical warfare, in practical terms little has been achieved, as a result of which the risk of the hostile exploitation of novel scientific developments remains far from hypothetical. along with the risk of misuse of new knowledge, there is the risk posed by the lack of sufficient scientific knowledge. cross-disciplinary convergence opens a multitude of opportunities for manipulation and modification of living matter but, at the same time, it precludes almost any sensible assessment of the potential interactions likely to occur in the process. nano-based medicine is but one area that has attracted criticism in this regard. since some elements behave differently at nano-scale, it becomes extremely difficult to assess their level of toxicity or other negative side effects that they may exert. such is the case with long carbon nanotubes, which having been initially praised for their potential to improve implant development 81 were later blamed for exhibiting asbestos-like behaviour that could lead to cancer. 82 another area of converging science with far-reaching implications is synthetic biology, a cross-disciplinary field that draws upon strategies and techniques from molecular biology, chemistry, engineering, genomics, and nanotechnology and thus enables the design and modification of biological systems at a fundamental level. empowered by the tools of synthetic biology, in 2002 scientists managed to assemble a polio virus 'from scratch' in the absence of a natural template. and in 2010 craig venter and his team announced the construction of the first self-replicating synthetic cell which, in their view, was 'a proof of the principle that genomes can be designed in the computer, chemically made in the laboratory and transplanted into a recipient cell to produce a new self-replicating cell controlled only by the synthetic genome.' 83 the controversial work has attracted criticism on several grounds, including the potential negative effects of the accidental or deliberate release of the novel organism in the environment and the arrogance of scientists to 'play god'. 84 more broadly, both the polio and synthetic cell studies have exposed the obstacles to the regulation of synthetic biology. 85 while some commentators dismiss the risk of bioterrorism, underscoring the key role of tacit skills and knowledge and the difficulties that the lack thereof poses to the replication of the experiments, 86 other issues still merit attention. consider the question of access to commercially available genomic sequences. even though the oversight system for screening base pair orders has improved since the 2006 guardian report that exposed the lax regulations under which virtually anyone could order gene sequences, 87 gaps still remain leaving scope for abuse by those with malign intent. for example, schmidt and giersch have outlined at least three areas of emerging challenges that the existing governance regimes would struggle to accommodate, including 'split orders', 'outsourcing', and the potential for non-natural biological systems. 88 the human genome project completed in 2003 lasted over ten years and cost close to 3 billion dollars; by contrast, about a decade later, wholegenome sequencing can be performed within hours at a price of roughly 1000 dollars or less. 89 while still in its infancy, personalised medicine and individual genetic testing are steadily gaining popularity. indeed, 'up to 100 000 people in england are expected to have their entire genetic makeup mapped in the first stage of an ambitious public health programme' launched by the national health service in 2012 that aims to 'revolutionise the treatment and prevention of cancer and other disease.' 90 according to its proponents, genomic testing offers numerous advantages vis-à-vis traditional evidence-based medicine, including the possibility of early diagnostics of disease, of individually-tailored treatment and, perhaps most importantly, of disease prevention, as illustrated in the resolve of the hollywood actress angelina jolie to undergo double mastectomy after discovering she has an inherited genetic mutation that puts her at high risk of breast and ovarian cancer. 91 but this is just the beginning. in 2012 scientists managed to sequence a foetus's entire genome using a blood sample from the mother and a saliva specimen from the father, a development that could potentially allow for a range of genetic disease conditions to be detected prenatally. 92 and laboratory experiments have already demonstrated the efficacy of genetic therapy to cure mitochondrial disease by creating an embryo with genetic material from both parents and a third person acting as a donor. 93 while truly breathtaking, the advances outlined above raise a host of thorny issues of ethical, social, and legal concern that merit public scrutiny and extensive deliberation before decisions regarding their widespread application are made. at a very basic level, there is the question of whether and to what extent we as individuals are capable of assimilating the information that our own genetic makeup may reveal. are we sufficiently resilient to cope with the emotional distress, anxiety, shame, stigma and guilt that the awareness of severe medical conditions that we or our closed ones are suffering or likely to develop? far from hypothetical, this question has prompted the establishment of a novel profession, that of the genetics counsellor whose task is to help patients overcome any negative effects, stress, or psychological trauma that the disclosure of their genomic map may create. 94 this is just a partial solution though, for the crux of the matter lies in finding a way to deal effectively with risk and probabilities and we as humans are yet to demonstrate a capacity for understanding or relating them to our own lives. 95 individual emotional turmoil, however significant, constitutes only the tip of the iceberg. according to daniel kevles, the torrent of new genetic information has already begun to fundamentally reconfigure social practices and inter-personal relations: it has been rightly emphasised that employers and medical or life insurers may seek to learn the genetic profiles of, respectively, prospective employees or clients. employers might wish to identify workers likely to contract disorders that allegedly affect job performance while both employers and insurers might wish to identify people likely to fall victim to diseases that result in costly medical or disability payouts. whatever the purpose, such genetic identification would brand people with what an american union official has called a life-long 'genetic scarlet letter' or what some europeans term a 'genetic passport'. 96 linking genetic makeup with human identity would ultimately set the scene for the proliferation of technologies aimed at human enhancement: after all, if a gene therapy could allow one to stand a chance in a job competition, boosting one's capabilities would potentially make them a more desirable candidate. other issues of more immediate concern are also likely to arise. one is privacy. gene-sequencing companies usually hold the genetic data of their clients in digital format on online platforms, which automatically creates a risk that personal information may be leaked, hacked or stolen. 97 further, there is the question of ownership. consider, for instance, the controversial issue of human gene patenting, whereby patented genes are treated as research tools and, as such, are controlled by the patent holder who may restrict and charge for their use. 98 thus created, the system often operates to the detriment of patients by hindering research practice, elevating diagnostics prices and denying access to second and independent medical opinion. 99 gene identification alone has a potential 'dark side' too, for it could enable the development of weapons targeted at groupspecific gene markers (e.g. ethnicity). 100 pre-natal genetic testing is yet another significant bone of contention, not least because it evokes notions of state-mandated eugenic programmes and assaults on human rights and dignity. while a nazi-like campaign for a superior race seems improbable in the twentieth-first century, this is not to say that other forms of eugenics may not be encouraged. indeed, some commentators have highlighted the rise of 'homemade eugenics', 101 whereby individual families can make decisions on the attributes of their progeny: the lure of biologically improving the human race, having tantalised brilliant scientists in the past, could equally seduce them in the future, even though the expression of the imperatives may differ in language and sophistication. objective, socially unprejudiced knowledge is not ipso facto inconsistent with eugenic goals of some type. such knowledge may, indeed, assist in seeking them, especially in the consumer-oriented, commercially driven enterprise of contemporary biomedicine. 102 it is plausible to assume that when presented with the opportunity of having their future child tested for genetic disorders, many parents would barely hesitate to accept. such a resolve could have far-reaching implications though. for instance, some genetic therapies entail the use of donor dna different from that of the parents, whereby any genetic modifications in the embryo will pass down to future generations. 103 despite the government support for the 'three-parent babies' in the uk, local religious organisations have protested vociferously against the legalisation of the technique. 104 at the same time, there are certain genetic disorders that can be diagnosed at an early stage but, as of yet, cannot be cured, which inevitably poses the tough choice between raising an unhealthy child and abortion. to be sure, such questions constitute more than individual parents' dilemmas, for they touch upon established social and cultural values, something evident in the profound differences across national reproductive policies. more broadly, there are concerns that reproductive genomics may remain a prerogative of those affluent enough to afford it, thus further exacerbating the divide between the global rich and the global poor. 105 the growth of life science capacity over the past few decades across the globe has been truly astonishing, leading to the emergence of a vibrant research community that brings together researchers from various parts of the world. indeed, a 2011 nas report highlights the extension of both north-south and south-south partnerships, which has played a key role in synergising strengths and maximising competitiveness by improving the quality and effectiveness of research and facilitating data sharing. 106 at the same time, increasing collaboration in the realm of biotechnology industry has offered companies situated in emerging economies access to the global market, thus contributing to economic development and growth. 107 recent advances in technology and laboratory and experimental equipment have further impacted on the practice of life science research in profound ways. improvements in dna sequencing technology have significantly shortened the time required for the preparation of nucleic base-lines, thus relieving scientists of the burden of completing the task themselves and allowing them to focus on their actual project instead. studies and experiments once performed by senior researchers with extensive experience are now carried out by masters students. aided by specially designed genetic engineering toolkits, children as young as the age of ten start exploring the realm of biology in an interactive and engaging manner. needless to say, their notion of science and the world in general would differ significantly from that of their parents whose primary sources of knowledge used to be textbooks and encyclopaedias. indeed, the increasing commercialisation of synthetic biology offers anyone curious enough to fiddle with biological systems the chance of doing so in the comfort of their own home. 108 such modern gene hackers often lack formal background in biology and come from various walks of life. driven by an insatiable appetite for knowledge and the vision of a ground-breaking discovery that could be turned into a multimillion dollar profit, they take up the rather unusual hobby of biohacking which entails the redesign of existing and the creation of novel biological systems. for just few hundred dollars bio enthusiasts set up laboratories easily obtaining all essential requisites and equipment from online sales. and if to some biohacking equates to little more than an unusual hobby, others highlight its potential to generate substantial revenue and fuel economic development. 109 contrary to popular expectation, biohackers are not just eccentric individuals who work in solitude away from public attention. rather, they are members of a wide global movement dedicated to the ideal of do-it-yourself biology (diy), which has branches in 45 locations on four continents. 110 the movement has been partially institutionalised through the establishment of the biobricks 111 and international genetically engineered machine (igem) foundations, which seek to promote the open and ethical conduct of biological engineering and stimulate innovation and creativity. to this end, igem holds an annual competition open to high school students, university undergraduates and entrepreneurs from all over the world. with more than 200 participating teams, the competition constitutes the premiere forum at which biohackers can showcase their skills through project presentation. exciting as it may seem, the ongoing diffusion of life science expertise poses an array of governance conundrums. at the level of professional practice, the proliferation of research facilities around the world has exposed the urgent need for laboratory biosafety and biosecurity training, especially in developing states where a tradition of handling dangerous pathogens is lacking. the issue is further complicated, for such countries often lack the required legal and institutional infrastructure to ensure that professional practice is in compliance with relevant international regulations. foreign aid has gone some way in helping overcome those deficiencies but it has given rise to new problems, too. for instance, it is far from unlikely for a donor state to provide material support for the construction of a state-of-theart laboratory eventually leaving its maintenance to the local government, which can hardly afford the subsequent costs. a similar trend is observed in the area of capacity building and human resource development. most projects that aim to promote biorisk management and a biological security culture tend to be severely constrained in terms of time and funding and overly ambitious in terms of agenda and expected outcomes. lack of adequate mechanisms for quality assessment hinders progress evaluation and sometimes leads to duplication of effort and resources. the emergence of the diy biologists in the life science arena has further added to the challenge of ensuring that novel scientific and technological developments are utilised in a safe and ethical manner. even at the level of everyday practice, difficulties still persist. for instance, many amateur scientists have complained of the lack of manuals and guidelines regarding the safe operation and maintenance of home laboratories. issues such as waste disposal, safe handling and storage of biological material and prevention of contamination pervade the work of biohackers who unlike professional researchers conduct experiments in a much more volatile environment. 112 potential security concerns are also present. with more and more individuals gaining access to biological engineering technologies, ensuring appropriate oversight of what goes on in garage laboratories becomes increasingly difficult. the experience of the us fbi is a case in point. back in 2004 the fbi arrested steven kurtz, a professor at the university of buffalo under the suspicion of plotting a bioterrorist attack. 113 the subsequent investigation revealed that all laboratory and dna extraction equipment found in kurtz's house was legitimately obtained and used in his artwork. in an attempt to avoid mistakes of this kind, the fbi has drastically changed its approach to dealing with the diy movement launching a series of outreach activities that seek to raise awareness of the potential security implications of biohacking. 114 while undoubtedly necessary, such initiatives may well be seen as too little, too late in light of the wide spread of materials, tools and devices that could facilitate the malign misuse of the life sciences. indeed, it is worth noting that as early as the late 1990s the us defence threat reduction agency (dtra) managed to build a research facility that simulated the manufacture of weaponised anthrax using only commercially available materials and equipment. 115 the role of states: both a poacher and gamekeeper structural factors have an important bearing on the development and growth of biotechnology. economic considerations, power interests and realist fears generate potent dynamics that shape and influence and sometimes direct the life science trajectory. within this context, states assume a dual role. on the one hand, they are expected to act as gamekeepers and regulate, monitor and control the process of life science research and the dissemination of novel technologies. on the other hand, though, they also have powerful incentives to act as 'poachers', not least because of the fascinating opportunities for enhancing their prosperity, prestige and security that scientific and technological development open up. 116 the following passage effectively outlines states' dual function: government has an important role in setting long-term priorities and in making sure a national environment exists in which beneficial innovations will be developed. there must be a free and rational debate about the ethical and social aspects of potential uses of technology, and government must provide an arena for these debates that is most conducive to results that benefit humans. at the same time, government must ensure economic conditions that facilitate the rapid invention and deployment of beneficial technologies, thereby encouraging entrepreneurs and venture capitalists to promote innovation. 117 given that the agent (i.e. state governments) in charge of initiating ethical debates on the progress of biotechnology is also the one expected to provide the conditions that would allow this progress to generate outcomes likely to contribute to economic growth and political superiority, it is hardly surprising that any issues likely to slow down or otherwise hinder the enormous momentum of the life sciences are omitted from public discussion. this duality further informs how risks are perceived, framed and addressed. for instance, even though most of the developing countries lack capacity to manage dual use research of concern, they do not see this as an immediate priority and prefer to invest effort and resources in improving their laboratory biosafety and laboratory biosecurity infrastructure and capacity. 118 in the view of their governments, the dangers of naturally occurring and circulating diseases constitute a far greater worry than the potential for misuse of cutting-edge research. by contrast, some developed countries, most notably the usa, have embarked on building their biological defence systems highlighting the grave threat posed by the potential use of bioweapons by non-state actors. their activities have encountered severe opprobrium as some analysts see them as a contravention of the norms embedded in the btwc. 119 the evolution of the chemical and biological non-proliferation regime epitomises the attempts of states to avert the hostile exploitation of the life sciences whilst promoting their use for 'peaceful, prophylactic and protective purposes'. the entry into force of the btwc and the chemical weapons convention (cwc) in 1975 and 1997, respectively, is indicative both of states' renunciation of chemical, biological, and toxin weapons and of their commitment to the goals of arms control and disarmament. that said, the imperfections and shortcomings of these treaties signify the influence of realist fears and political calculations that pervade international negotiations. in the case of the btwc, two points merit attention. the first pertains to the lack of verification mechanism when the treaty was first agreed back in the early 1970s. subsequent revelations of secret state-led offensive biological programmes in the former soviet union, south africa and iraq up until the early 1990s have significantly undermined the convention. second, the failure to negotiate a binding protocol in 2001 has further dimmed the prospects for strengthening the regime and thus ensuring universal compliance with its prescriptions. less acute but just as worrying is the situation regarding the cwc. even though the convention is exemplary in many respects, not least because of its verification system, almost universal membership and implementing body -organisation for the prohibition of chemical weapons (opcw)it still faces serious challenges that need to be considered. for instance, while the treaty bans the development, production, acquisition, and retention of chemical weapons, the definition of 'purposes not prohibited under th[e] convention' entails 'law enforcement including domestic riot control purposes' (article ii.9d). some commentators have argued that given the lack of a universally agreed definition what kind of activities count as 'law enforcement', this text opens a major loophole in the convention. 120 several states parties of the convention have voiced concerns in this regard. australia has noted that: the weaponisation of [central nervous system] acting chemicals for law enforcement purposes is of concern to australia due to the health and safety risks and the possibility of their deliberate misuse, both of which have the potential to undermine the global norm against the use of toxic chemicals for purposes prohibited by the convention. [ . . . ] australia's position is that it is not possible for a state party to disseminate anaesthetics, sedatives or analgesics by aerial dispersion in an effective and safe manner for law enforcement purposes. 121 critics highlight the possibility for the deployment of novel chemical weapons for the purposes of countering terrorism, something evident in the 2002 moscow theatre siege (dubrovka) when the russian security forces used a fentanyl-derivative agent, as a result of which about a sixth of the hostages and all of the terrorists involved died. 122 in 2011 the european court of human rights ruled with regard to the dubrovka operation that: there had been no violation of article 2 (right to life) of the european convention on human rights concerning the decision to resolve the hostage crisis by force and use of gas. 123 the court, nonetheless, noted that: even if the gas had not been a 'lethal force' but rather a 'non-lethal incapacitating weapon', it had been dangerous and even potentially fatal for a weakened person [ . . . ]. 124 the court further confirmed some of the earlier criticisms that were levelled against the government, particularly in terms of preparedness and provision of medical assistance. 125 according to the ruling, russia had to pay damages to all the 64 applicantsrepresentatives of siege victims. to date, russian officials have withheld information concerning the exact formula of the gas, which was used during the dubrovka operation, on security grounds. 126 given the lack of an internationally agreed definition of what constitutes 'terrorism' on the one hand, and the rise of irregular/asymmetric warfare and sporadic conflicts, on the other, some commentators have warned against the possibility of a 'grey area' which may enable states to utilise non-traditional methods of war to gain advantage. 127 deliberative systems encompass a vast array of practices, processes and mechanisms, both formal and informal, whereby a polity considers the 'acceptability, appropriateness and control of novel developments in or impacting on, shared social and physical arenas'. 128 by design, they reflect and are informed by the values, beliefs and standards shared among the group, or in other words, by the prevalent culture. as such, deliberative systems vary across societies with their intensity, inclusiveness and structure depending on the established political and social norms. yet their chief purpose and function remain virtually the same, namely to help societies adapt to the changing circumstance of their milieu in a way that ensures stability, sustainability and safety. public deliberation requires time; and wide-ranging life science advances, current and planned, offer profound challenges to shared ideas and ideals about the foundations of human relatedness and of social coherence, justice, human dignity and many other norms, both formal and informal. 129 yet given the ruminative nature of deliberative processes, on the one hand, and the fast speed at which biotechnology innovation is evolving on the other, the danger of the former being steadily outpaced and overburdened by the latter is far from hypothetical. consider the following passage sketching the scale of social changes likely to arise from the increasing convergence between nanotechnology, biotechnology, cognitive neuroscience and information technology: in the foreseeable future, we will be inundated with new inventions, new discoveries, new start-ups, and new entrepreneurs. these will create new goods and new services. [ . . . ] as expectations change, the process of politics and government will change. people's lives will be more complex and inevitably overwhelming. keeping up with the changes that affect them and their loved ones exhausts most people. they focus most of their time and energy on tasks of everyday life. in the future, when they achieve success in their daily tasks, people will turn to the goods and services, the new job and investment opportunities, and the new ideas inherent in the entrepreneurial creativity of the age of transitions. no individual and no country will fully understand all of the changes as they occur or be able to adapt to them flawlessly during this time. 130 this vision of a 'brave new world' merits attention on two important grounds. first, it implies that the changes likely to occur in the not too distant future as a result of the rapid progress of science and technology are imminent and unavoidable in the sense that their advent hardly depends on or even requires extensive public deliberation. second, given that our capacity for adaptation to and grasp of those changes will be considerably impaired, the age of transitions leaves little space for public deliberation. to add to this gloomy picture, there is already some evidence that the progress in the life sciences is overwhelming the existing deliberative mechanisms. for instance, kelle et al. argue that the rapidity of biotechnology advancement coupled with the immensity and complexity of the knowledge accumulated therefrom complicates efforts to deal with potential risks, something evident in the regulatory gap that the convergence of chemistry and biology has created in the area of arms control. 131 this is problematic, for the reduced resilience of deliberative systems provides favourable conditions in which scientific and technological innovation can continue unabated. a vicious circle is thus created in which the inability of deliberative systems to cope with the strain exerted by biotechnology advancement fuels the latter turning it into a self-propelling force. the proliferation of contentious 'gain-of-function' research is a case in point. even though the h5n1 controversy discussed in the preceding sections exposed the limitations of existing governance mechanisms for addressing the potential security, ethical, and legal implications arising from such studies, it hardly precluded scientists from conducting similar experiments. indeed, less than four months after the moratorium on research involving contagious h5n1 virus was lifted, a team of chinese researchers announced the creation of a hybrid of the h5n1 strain and the h1n1 virus that caused the 2009 flu pandemic. 132 and it was not long until the newly-emerged h7n9 influenza virus became airborne, as well. 133 if anything, those examples indicate that in light of the rapid pace of life science progress, addressing governance concerns on a caseby-case basis is not only self-defeating but given the number and variety of conundrums, it is likely to become unsustainable in the long run. given the significant potential of biotechnology to bring about multifaceted changes in different spheres of life and generate considerable benefits in the form of new products, enhancement of public and private capital and alleviation of social ills, there is a powerful urge to allow the ongoing expansion of the life sciences to proceed largely unfettered. risks are carefully calculated and, where possible, downplayed as hypothetical at the expense of comprehensive deliberation. and even when proposals for risk mitigation measures are entertained, preference is usually given to those unlikely to hinder the progress of life sciences. by and large, there is a genuine belief that the existing governance mechanisms in the area of biotechnology can accommodate and cope with the wide-ranging pressures exerted by scientific innovation and the rapid diffusion of technologies with multiple uses, by offering 'solutions' and handling concerns on a case-by-case basis. in particular, the technology of safety is still 'celebrated as an unadulterated improvement for society as a whole'. 134 yet there are reasons for scepticism toward the adequacy and effectiveness of the governance approaches currently in place. much of the discussion in the preceding sections has focused on the ways in which the increasing pace, growth and global diffusion of biotechnology advances are beginning to expose the limits of the existing measures for control and risk management by challenging accepted values and beliefs and redefining established norms of practice. as the multifaceted dynamics driving the biotechnology momentum continue to intensify and multiply, it becomes more and more difficult to comprehend, let alone foresee, the various impacts that the large-scale deployment and proliferation of novel scientific and technological advances have both on our social systems and the environment. given the tight coupling between human-made and natural systems and their complex, often unanticipated interactions with catastrophic potential, the existing narrow definitions of risk are rendered inadequate. 135 at the same time, the advent of new technologies with multiple adaptive applications opens up an array of possibilities for hostile exploitation thus compelling governments to make tough decisions in an attempt to reconcile the benefits of biotechnology with the potential security concerns arising therefrom. while the advancement of biotechnology promises tremendous public health benefits, it also holds a considerable catastrophic potential, as the case of 'gain-of-function' experiments illustrate. as scientific capabilities and work involving dangerous pathogens proliferate globally, so do risks and the prospects of failures, whether technical or arising from human error. indeed, assessing the rapidly evolving life science landscape some security commentators argue that 'current genetic engineering technology and the practices of the community that sustains it have definitively displaced the potential threat of biological warfare beyond the risks posed by naturally occurring epidemics' 136 . laboratories, however well equipped, do not exist in isolation but are an integral part of a larger ecological system. as such, they constitute a 'buffer zone' between the activities carried out inside and the wider environment. and despite being technically advanced and designed to ensure safety, this 'buffer zone', just as other safety systems is far from infallible. for one thing, mechanical controls leave room for human error and personal judgement, both of which are factors that could be highly consequential but which could hardly be modelled or predicted with exact certainty. 137 the speed at which the transformation of the life sciences is taking place is yet another factor that adds to the complexity of life science governance. stability is a fundamental condition for the development and preservation of human and natural systems alike. in social systems, culture is the primary source of stability, for it determines what values, beliefs, practices and modes of behaviour are deemed acceptable and, as such, lays the foundations of order. all forms of governance therefore are cultural artefacts and manifestations of culture. culture also provides the tacit standards whereby change is assessed and treated as acceptable or unacceptable. hence, any state of affairs in which the rate of change precludes regulation disrupts the ordinary functioning of the system and jeopardises its preservation: the breakdown of human regulation does not extinguish regulation of a simpler sort. [ . . . ] the system formed by men and the rest of the natural world will continue to regulate itself after a fashion, even if human regulation wholly fails at all levels above the primary group. but the resulting 'order' would exclude all those levels of human order which man-made stability makes possible. 138 to be sure, a world characterised by a runaway biotechnology would be far different from the one we know. the main challenge to averting this prospect lies in ensuring that the systems of governance are in sync with the progress of life sciences. history has shown that even highly developed, long-standing systems of governance can fail for reasons as diverse as disasters; loss of authority/legitimacy of governing bodies; and pervasive corruption. one further source of failure includes the inability of a society to adapt to its changing milieu: men are adaptable; they can learn to live even in harsh and hostile environmentsso long as the environment remains constant enough to give them time to learn. [ . . . ] if they form the habit of adapting by constantly changing that to which they are trying to adapt, they build uncertainty into the very structure of their lives. they institutionalise cluelessness. 139 the process of adaptation is closely connected to cultural patterns and any serious disruptions in the latter could have detrimental effects and impair it severely. the extent to which change is taking place within the framework of the prevalent culture defines the borderline between system evolution and system disintegration. the governance mechanisms currently in place, both formal and informal, are all a function of historical, cultural, and sociopolitical contingencies. as such, their capacity for adaptation largely depends on our ability to comprehend and assimilate the complex changes that the progress of biotechnology brings about. they can only evolve as fast as our shared standards, values, routines and perceptions allow them to. and that is why governance can hardly be reduced to a technocratic exercise; on the contrary, to be effective, it requires extensive deliberation and full appreciation of the far-reaching implications of novel life science advances. trends and drivers of change in the biomedical healthcare sector in europe: mapping report (dublin: european foundation for the improvement of living and working conditions chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template the test-tube synthesis of a chemical called poliovirus: the simple synthesis of a virus has far-reaching societal implications craig venter creates synthetic life form', the guardian moore's law pertains to the rapid rate of technological development and advances in the semiconductor industry, specifically the doubling of the number of transistors on integrated circuits that occurs approximately every 18 months. although advances in the life sciences occur at more random intervals and are driven by new conceptual breakthroughs in understanding of biological processes, it is a useful metaphor for the exponential growth of knowledge related to biology. see committee on the advances in technology and the prevention of their application to next generation bioterrorism and biological warfare threats, an international perspective on advancing technologies and strategies for managing dual-use risks the myth of the biotech revolution: an assessment of technological, clinical and organisational change globalization, biosecurity and the future of the life sciences innovation in global industries: u. s. firms competing in a new world biotechnology and the un's millennium development goals top ten biotechnologies for improving health in developing countries rising to the challenge: u.s. innovation policy for global economy innovating in the new austerity, burrill & co's 26th annual report on the life sciences industry globalization and the future of the life sciences, op cit an international perspective on advancing strategies for managing dual-use risks the valley of the shadow of death', dspace@mit globalization and the future of the life sciences the biotechnology promise: capacity-building for participation of developing countries in the bioeconomy converging technologies for enhancing human performance: science and business perspectives converging technologies -shaping the future of european societies taking care of the symbolic order: how converging technologies challenge our concepts enhancement technologies and the modern self the ethics of nbic convergence bringing converging technologies closer to civil society: the role of precautionary principle', innovation: the innovation in global industries, op cit in the valley of the shadow of death see the 'infectious diseases' section of the who website for an overview of the nih budget for 2015, see david malakoff and jeffrey mervis, 'first look: us spending deal a mixed bag for science within nih's flat 2015 budget, a few favourites', scienceinsider for information on the eu sixth framework programme and the activity area of life sciences cuba -battling cancer with biotechnology global status of commercialised biotech/gm crops plant genetic engineering: china hesitates on the brink', gmo safety rising to the challenge, op cit government academic, and venture firms come together in march to fund translational and early-stage development', fiercebiotech science for sale: the perils, rewards and delusions of campus capitalism relationships between academic institutions and industry in the life sciences -an industry survey the expanding role of university patenting in the life sciences: assessing the importance of experience and connectivity university-industry relationships and the design of biotechnology research entrepreneurial science in the academy: a case of the transformation of norms the triple helix of university-industry-government relations the triple helix: university-industry-government innovation in action the dynamics of innovation: from national systems and "mode 2" to a triple helix of university-industry-government relations commercialisation of the university and the problem choice by academic biological scientists see also dina biscotti et al. 'the "independent investigator": how academic researchers construct their professional identity in university-industry agricultural biotechnology research collaborations toward more secrecy in science: comments on some structural changes in science -and on their implications for an ethics of science varieties of secrets and secret varieties: the case of biotechnology for a detailed analysis on the us decision to reject the draft btwc protocol, see malcolm dando the white house the pitfalls of bioterrorism preparedness: the anthrax and smallpox experiences bioterrorism and smallpox planning: information and voluntary vaccination taking biodefence too far new labs, more terror a plague of researchers mixing bugs and bombs lab loses trio of plague mice', nature news anthrax letters' attack and the controversy of the us biodefence programme see jeanne guillemin army suspends germ research at maryland lab back to bioweapons? international health regulations (who, 2005), specifically section 'laboratory pandemic influenza preparedness framework for the sharing of influenza viruses and access to vaccines and other benefits (who for further discussion on the implementation of biotechnology regulations, see bo sundqvist et al. 'harmonisation of european laboratory response networks by implementing cwa 15793: use of gap analysis and an "insider" exercise as tools on the development of health and safety regulations on the use of nanotechnology, see eileen kuempel et al. 'risk assessment and risk management of nanoparticles in the workplace: translating research into practice there is a debate on whether the 'american model' of science-policy making underpinned by neoliberal ideology is fully embraced in europe. see, for example, gabriele abels, 'the long and winding road from asilomar to brussels: science, policy and the public in biotechnology regulation further, a policy paper issued by a business taskforce appointed by the uk government issued a policy paper in late 2013 demanding the liberalization of the existing eu legislation which, in their view, 'places restrictions on products and technologies without adequate evidence of risk'. see department for business, innovation & skills and the prime minister's office, cut the eu red tape: report form the business task force proposed framework for the oversight of dual use life science research: strategies for minimising the potential misuse of research information biosecurity reconsidered: calibrating biological threats and responses some commentators have expressed scepticism toward the claim that scientific and technological advancement poses serious threats underscoring the importance of other factors, such as socio-economic and socio-technic contexts. see, for example, kathleen vogel, 'intelligent assessment: putting emerging biotechnology threats in context others, however, argue that advances in modern biology and medicine have implications for the evolution of biological weapon programmes. see malcolm dando, 'the impact of the development of modern biology and medicine on the evolution of offensive biological warfare programmes in the twentieth century expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox the efficacy of cidofovir treatment of mice infected with ectromelia (mousepox) virus encoding interleukin-4 disaster in the making creation of killer poxvirus could have been predicted chemical synthesis of poliovirus cdna: generation of infectious virus in the absence of natural template a tale of two studies: ethics, bioterrorism and the censorship of science first synthetic virus created', bbc news university of bradford, available at www.brad.ac.uk/ acad/sbtwc (accessed 8/03/2013). see also german ethics council, biosecurity -freedom and responsbility of research on the framing of risk in biotechnology, see geert van calster, 'risk regulation, eu law and emerging technologies: smother or smooth? ethics of risk analysis and regulatory review: from bio-to nanotechnology european biotechnology regulation: framing the risk assessment of a herbicide tolerant crop framing the uncertainty of risk: models of governance for genetically modified crops denmark's regulation of agri-biotechnology: co-existence bypassing risk issues a distorted regulatory landscape: genetically modified wheat and the influence of non-safety issues in canada on the shortcomings of the existing models for public deliberation on the risks of biotechnology, see les levidow, 'european public participation as risk governance: enhancing democratic accountability for agrobiotech policy for critique of cost-benefit analysis, see also brian rappert the creation of contagious h5n1 avian influenza virus: implications for the education of life scientists all meetings of the us national science advisory board for biosecurity (nsabb) convened to discuss the manuscripts were restricted to selected individuals and full proceedings were never published. moreover, the consequential consultation meeting organised by the world health organisation (who) in february 2012 which rejected the nsabb recommendation for a redacted publication of the manuscripts featured the lead scientists who conducted the experiments and representatives of the us national institutes of health flu experts -and one ethicist -debate controversial h5n1 papers', scienceinsider fight over dutch h5n1 paper enters endgame', scienceinsider fight over dutch h5n1 paper enters endgame', scienceinsider dutch appeals court dodges decision on hotly debated h5n1 papers', scienceinsider bias accusation rattles us biosecurity board experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets airborne transmission of influenza a/h5n1 virus between ferrets: materials/methods, supporting text, tables, figures, and/or refences safety incidents" at animal lab', bbc news notes 155 airflow problems plague cdc bioterror lab high-containment laboratories: assessment of the nation's need is missing the scientist lab infection blamed for singapore sars case between 2014 and 2015, several high-containment facilities in the us have experienced serious biosafety lapses. accidents involving dangerous pathogens such as the causative agents of anthrax and bird flu were reported biosafety in the balance' anthrax? that's not the real worry cdc closes anthrax and flu labs after accidents about the same time, there were also reports about smallpox vials being retrived after having been left unaccounted for over 50 years. see ap, 'forgotten vials of smallpox found in storage room gain-of-function' influenza research and proposal to organise a scientific briefing for the european commission and conduct comprehensive risk-benefit assessment the third revolution: the convergence of the life sciences see committee on biomocular materials and processes, national research council, inspired by biology: from molecules to materials to machines a new biology for the 21st century nanotechnology: what it can do for drug delivery facing the truth about nanotechnology in drug delivery carbon nanotubes -bullets in the fight against cancer on the governance challenges brought about by the convergence between biology and other fields of science, see francis fukuyama and caroline wagner, information and biological revolutions: global governance challenges -summary of a study group report of the first workshop the body's own bioweapons innovation, dual use, and security: managing the risks of emerging biological and chemical technologies life sciences and related fields: trends relevant to the biological weapons convention building better implants some nanotubes could cause cancer and breeding: panacea or pandora's box?', news release on the security implications of synthetic biology, see international council for the life sciences synthetic biology and biosecurity awareness in europe on the social and ethical aspects of synthetic biology, see presidential commission for the study of bioethical issues, new directions: the ethics of synthetic biology and emerging technologies synthetic biology: social and ethical challenges framing biosecurity: an alternative to the biotech revolution model vogel's view are contested in jonathan tucker 'could terrorists exploit synthetic biology lax laws, virus dna and potential for terror on the issue of commercial order screenings, see stephen maurer et al. making commercial biology safer: what the gene synthesis industry has learned about screening customers and orders on the governance of synthetic biology, see catherine lyall dna synthesis and biological security industry self-governance: a new way to manage dangerous technologies unpacking synthetic biology: identification of oversight policy problems and options synthetic biology, security and governance'¸biosocieties dna synthesis and security science enters $1,000 genome era', bbc news happened when i had my genome sequenced', the observer for information about commercial companies offering full-genome sequencing genome-based therapeutics: targeted drug discovery and development: workshop summary dna of 100,000 people to be mapped for nhs', the guardian systems cancer medicine: towards realisation of predictive, preventive, personalised and participatory (p4) medicine' grateful and moved" by reaction to her mastectomy decision', the guardian dna blueprint for fetus built using tests of parents cure for illness raises ethical fear', the guardian what happened when i had my genome sequenced another point that cadwalladr raises is the danger of a negative placebo effect whereby doubts about certain genetic disorder may lead to psychosomatic symptoms from eugenics to patents: genetics, law, and human rights unhidden traits: genomic data privacy debates heat up from eugenics to patents see also harriet washington, deadly monopolies: the shocking corporate takeover of life itself -and the consequences for your health and our medical future taking life: private rights in public nature benefits and threats of developments in biotechnology and genetic engineering' in sipri yearbook, armaments ethnic-affiliation estimation by use of population-specific dna markers racial differences in the response to drugs -pointers to genetic differences the achilles' helix from eugenics to patents three-person" embryos to combat genetic disease', the guardian innovative genetic treatment to prevent mitochondrial disease, press release three-parent embryos for mitochondrial disease? twelve reasons for caution february 2015, the uk passed legislation allowing the use of the technique. see james gallagher, 'uk approves three-person babies', bbc news building baby from the genes up one commentator distinguishes between 'big science' which was 'todown, hierarchical, vertical' and 'networked science' characterised by 'open systems, open software, open participation'. see diane rhoten biopunk: solving biotech's biggest problems in kitchens and garages life hackers biology is technology: the promise, peril, and new business of engineering life diffusion of synthetic biology: a challenge to biosafety do i understand what i can create: biosafety issues in synthetic biology charge dropped against artist in a terror case strategies to educate amateur biologists and scientists in non-life science disciplines about dual use research in the life sciences governing amateur biology: extending responsible research and innovation in synthetic biology to new actors, research report for the wellcome trust project 'building sustainable capacity in dual use bioethics secret project manufactured mock anthrax', the washington times biological weapons and america's secret war global governance and the twenty-first century technology converging technologies for improving human performance: nanotechnology, biotechnology, information technology and cognitive science report of the who informal consultation on dual use research of concern biological threat assessment: is the cure worse than the disease? dangerous ambiguities: regulation of riot control agents and incapacitants under the chemical weapons convention weaponisation of central nervous system acting chemicals for law enforcement purposes, xix session of the conference of the states parties moscow theatre siege: questions remain unanswered', bbc news press release: use of gas against terrorists during the moscow theatre siege was justified, but the rescue operation afterwards was poorly planned and implemented spasenie zalozhnikov ili unichtozhenie terroristov?', novaya gazeta the 2002 dubrovka and 2004 beslan hostage crises: a critique of russian counter-terrorism sekretov bol'she net', rossiskaya gazeta see also national research council, avoiding surprise in an era of global technology advances the challenge to deliberative systems of technological systems convergence converging technologies for improving human performance, op cit preventing a biochemical arms race, op cit bringing the bwc conventions closer together', the cbw conventions bulletin h5n1 hybrid viruses bearing 2009/h1n1 virus genes transmit in guinea pigs by respiratory droplet infectivity, transmission, and pathology of human-isolated h7n9 influenza virus in ferrets and pigs limited airborne transmission of h7n9 influenza a virus between ferrets shifting the blame: literature, law, and the theory of accidents in nineteenth-century america normal accidents, op cit building a bio world shifting the blame, op cit freedom in a rocking boat: changing values in an unstable society some commentators have critiqued the work of diamond on the grounds of simplicity. for a summary of some of the criticisms levelled at his work, see eric powell key: cord-002137-j5sfiyz8 authors: ward, kirsten; seale, holly; zwar, nicholas; leask, julie; macintyre, c. raina title: annual influenza vaccination: coverage and attitudes of primary care staff in australia date: 2010-10-12 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2010.00158.x sha: doc_id: 2137 cord_uid: j5sfiyz8 please cite this paper as: ward et al. (2011) annual influenza vaccination: coverage and attitudes of primary care staff in australia. influenza and other respiratory viruses 5(2), 135–141. background annual influenza vaccination is recommended for all australian health care workers (hcws) including those working in primary health care. there is limited published data on coverage, workplace provision, attitudes and personal barriers to influenza vaccination amongst primary health care staff. the aim of this study was to contribute to the limited literature base in this important area by investigating these issues in the primary health care setting in new south wales (nsw), australia. methods a postal survey was sent to general practitioners (gps) and practice nurses (pns) from inner city, semi‐urban and rural areas of nsw, australia. there were 139 responses in total (response rate 36%) from 79 gps (response rate 30%) and 60 pns (response rate 46%). results reported influenza vaccination coverage in both 2007 and 2008 was greater than 70%, with gps reporting higher coverage than pns in both years. the main barriers identified were lack of awareness of vaccination recommendations for general practice staff and concern about adverse effects from the vaccine. conclusions rates of influenza vaccination coverage reported in this study were higher than in previous studies of hospital and institutional hcws, though it is possible that the study design may have contributed to these higher results. nevertheless, these findings highlight that more needs to be done to understand barriers to vaccination in this group, to inform the development of appropriate strategies to increase vaccination coverage in primary health care staff, with a special focus on pns. influenza is a serious respiratory virus which costs the australian healthcare system $115 million annually. 1 primary health care workers (hcws) like general practitioners (gps) and practice nurses (pns) have found to be at higher risk for influenza than the general population. 2 this may be because of: (i) exposure to influenza infection in both the general community and the workplace; and (ii) close proximity to visitors and patients. 1, 3 vaccines remain the cornerstone of influenza prevention in many countries worldwide 4 and are considered to be 50-80% effective in healthy persons aged 16-65 years. 5 annual influenza vaccination of australian hcws is recommended by the national health & medical research council (nhmrc), 3 the australian committee on safety and quality in healthcare 6 and various jurisdictional health departments, including new south wales (nsw). 7 the royal australian college of general practitioners 8 recommend that general practice staff members are offered immunisation appropriate to their duties. whilst there have been numerous australian studies on influenza vaccine uptake amongst hospital and institutional hcws 6, [9] [10] [11] [12] [13] and some studies on attitudes of primary care clinicians to influenza vaccination for their patients 14, 15 , there has been limited published studies to date on influenza vaccination coverage, barriers and enablers amongst primary health care staff in australia. influenza vaccination coverage amongst gps in australia was 50% in 1998. 16 in neighbouring new zealand, coverage amongst gps was 68% and 64% in pns in 2001. 17 other countries have reported lower coverage estimates, with only 35% of canadian family physicians from québec 18 and 36% of gps from the netherlands 19 vaccinated in 1996 and 2008, respectively. a study across all primary health care professions in israel reported an average of 30% coverage across physicians, nurses, pharmacists and administration staff. 20 factors associated with influenza vaccination status have been examined in primary health care clinicians in other countries. [17] [18] [19] [20] [21] amongst this group of hcw's, significant predictors for vaccine acceptance include the following: agreement that hcw's have professional responsibility to be vaccinated, on-site access to free vaccine, workplace recommendation for staff influenza vaccination, desire for self-protection and belief that the benefits of vaccination outweigh the risk of vaccine side effects. 17, 21 furthermore, previous influenza vaccination has been significantly associated with current vaccine acceptance in both hospital hcws and primary care physicians. 20 factors significantly associated with lack of vaccine acceptance include the following: no medical indication for vaccination, belief that regular medical exposure will protect against the disease, low risk of contracting influenza, fear of vaccine side effects and lack of time or priority. 17, 19, 20 some of these factors are similar to those cited by hospital hcws whilst others differ. in a review of attitudes and predictors to influenza vaccination of hospital hcw's, lack of convenient access to vaccine and poor knowledge about influenza infection were prominent reasons for lack of vaccination with the desire for self-protection and belief in the vaccine's effectiveness the most prominent reasons for vaccine acceptance in this group. 22 to the best of our knowledge, there have been limited studies which specifically examine: influenza vaccination coverage, workplace provision of vaccination, knowledge, attitudes and personal barriers to influenza vaccination amongst gps and pns in australia. the aim of this study therefore was to contribute to the limited literature base in this important area by investigating this in the primary health care setting in nsw, australia. a paper-based survey was developed based on pilot work undertaken by the authors and commonly identified barriers to vaccination from the literature. 6, 23, 24 it elicited demographic data about the respondents, their influenza vaccination status from 2007 to 2009 and identified barriers to being immunised. it also posed questions intended to determine the respondent's attitude towards vaccination, based on seven statements about efficacy, safety, adverse events and recommended target groups for influenza vaccine and was part of a wider survey that incorporated questions on pandemic influenza. the survey was piloted with four gps and pns from outside the study area. feedback from this process contributed to enhanced content, altered survey structure and modified wording. our sample was drawn from divisions of general practice (dgp) in nsw. dgps are government-funded organisations that provide support to a defined geographical catchment of general practices in australia. they are classified by population and locality into five categories based on rural, remote metropolitan areas (rrma). 25, 26 nsw has the highest number of dgps, with 32. purposive sampling by the authors was used to select four dgps in nsw to represent a diverse sample from metropolitan, semiurban and rural areas. the final sample size for each participating division was weighted according to how many gps and pns were practicing in the area. the study was undertaken from the 1st february to 1st april 2009, prior to the pandemic (h1n1) influenza 2009 which was identified in late april 2009. 27 authors were blinded to participant selection, as they were randomly selected from de-identified dgp databases of gps and pns. surveys were posted by dgps and were accompanied by personalised explanatory letter and a reply-paid envelope. non-responders were sent a second letter and survey by the dgps within 4 weeks. quantitative data was entered into microsoft access and was analysed using microsoft excel. responses to the geographical location question were categorised into inner city, semi-urban and rural areas. questions about influenza vaccine-related barriers, attitudes and beliefs were categorised into either agree, disagree or uncertain. the categories were compared with demographic characteristics and selfreported vaccination status of respondents using categorical data analysis. ethical approval was granted by the university of new south wales ethics committee. of the 390 staff that was sent a survey, 139 completed and returned it, giving an overall response rate of 36%. fifteen surveys were returned uncompleted as the staff member was no longer at the practice. response rates were higher amongst pns (46%) than gps (30%). the demographic and occupational characteristics of the respondents are summarised in table 1 . there was some variation in geographical location of respondents, with 58ae3% (81 ⁄ 139) located in semi-urban areas, 36% (50 ⁄ 139) in the inner city and the remaining 5ae7% (8 ⁄ 139) in rural locations. of the respondents, 45% (62 ⁄ 139) were working in practices with £4 gps. our sample parallels the findings of the 2007-2008 bettering the evaluation and care of health (beach) survey in terms of the spread in gp age, years worked in general practice and geographical location. the beach program is a continuous national study of general practice activity in australia. it provides a reliable, ongoing, representative description of general practice activity nationwide. 28 just over 70% of respondents were vaccinated against influenza in 2007 (70ae5%, 98 ⁄ 139, 95% ci: 62ae9-78ae1) and in 2008 (72ae7%, 101 ⁄ 139, 95% ci: 65ae3-80ae1). differences in vaccination coverage between gps and pns for both 2007 (p = 0ae74) & 2008 (p = 0ae22) (see table 1 participants indicated that free influenza vaccine was most commonly provided at the practice for gps (79ae1%, 110 ⁄ 139, 95% ci: 72ae3-85ae9), administration staff (74ae8%, 104 ⁄ 139, 95% ci: 67ae6-82ae0) and pns (72ae7%, 101 ⁄ 139, 95% ci:65ae3-80ae1). of the gps working at a practice which provided free influenza vaccine for gps, 84ae5% (49 ⁄ 58) were vaccinated in 2008 in contrast to 58ae3% (7 ⁄ 12) coverage in gps from practices that did not provide the vaccine free of charge for them. for pns, 69ae8% (37 ⁄ 53) vaccinated in 2008 worked at a practice where the vaccine was provided free and 66% (2 ⁄ 3) were vaccinated despite the vaccine not being provided free for pns at their practice. respondents' knowledge, attitudes and perceptions of influenza vaccination are summarised in table 2 . over 90% of the participants believe that the influenza vaccine is attitudes towards vaccination barriers amongst the respondents are presented in table 3 . while there was a low level of agreement with all of the statements provided (<10%), the most commonly identified barriers were lack of awareness of any recommendation for general practice staff to receive influenza vaccination (10ae1%, 14 ⁄ 139, 95% ci: 5ae1-15ae1) and unacceptable nature of vaccination side effects (7ae2%, 10 ⁄ 139, 95% ci: 2ae9-11ae5). having to pay for the vaccine was identified as a barrier to getting vaccinated by 25ae2% (35 ⁄ 139) of respondents. five of the twelve gps, who worked in a practice that did not provide free vaccine, felt that paying for a vaccine was a barrier. our study of primary health care staff found much higher influenza vaccination coverage than hospital hcws in australia. overall reported coverage of gps and pns was markedly higher than those for australian institutional and hospital hcws which have been found to range from 18% to 58%. 6, [9] [10] [11] [12] 23 self-reported vaccination coverage for gps in our study (for 2007) was higher than the percentage reported for australian hospital-based doctors from the northern territory (72ae7% versus 28%) 12 and western australia (72ae7% versus 51ae9%). 9 pns in our study had higher coverage when compared to nurses in residential aged care facilities (racf) (68ae3% versus 62%) 11 , and hospital-based nurses (68ae3% versus 49ae6%) yet had lower coverage than pns from the australian capital territory (act) (68ae3% versus 79%). 29 the australian national influenza & pneumococcal survey provides the earliest available influenza vaccination coverage estimates for gps in australia. 16 results of this survey found coverage for nsw gps was the lowest of any jurisdiction, with 29% vaccinated in 1998, and just over 20% for the preceding 3 years. comparing these rates to those observed in our study, influenza vaccination coverage amongst gps in nsw appears to have risen substantially from 1998 to 2008. more recently, a national survey from the australian general practice network (agpn) 23 assessed influenza vaccination coverage in gps and pns in the same years as our study (2007 ⁄ 2008) with similar response rates (34% versus 36%). comparing vaccine uptake between the studies for both gps and pns in nsw only, the agpn study reported slightly lower coverage (5%) across both years in both groups. 23 as data was collected at a practice level in the agpn study, individual vaccination status may have been incorrectly reported and may be underestimating the actual coverage rate. however, our results may overestimate actual coverage because of a number of reasons including the low response rate and use of self-reported vaccination status. studies into gp influenza vaccination coverage have been performed in other countries. [17] [18] [19] [20] [21] 30 cowen et al. 21 in the united states (us) had a similar response rate to our study (30% versus 38%), but reported a much higher vaccination coverage rate for us family physicians (84%). semaille et al. 30 and brunton et al. 17 reported influenza vaccination coverage as 67% amongst french gps and 68% amongst new zealand gps, respectively. in contrast, studies from the netherlands 19 and israel 20 found lower coverage rates for gps with 36% and 40%, respectively. even though the majority of our respondents worked in a practice that provided free vaccine for their staff, many felt that paying for the vaccine was a barrier to getting vaccinated. the wording of the question may have impacted on the result, as respondents could have taken it to mean for gps in general and not for them personally. however, it is interesting to note, of the gps who said their practice does not provide free vaccine, 40% stated that paying for the vaccine was a barrier to receiving it. further qualitative research would assist in addressing these gaps in understanding. the gps and pns in our study largely disagreed with the common vaccination myths presented in the survey (see table 3 ). in contrast, the most frequently reported reason amongst dutch gps for not being vaccinated was having no medical indication for influenza vaccination 19 and in israel; physicians were much less frequently influenced by the fear that vaccination would cause influenza when compared to other practice staff. 20 there was almost no consistency of agreement with both these misconceptions in our sample or that of litt et al., 16 who found that the main reasons indicated by australian gps for being vaccinated against influenza were concern about getting influenza or its complications and to prevent having time off work because of influenza. live viruses in the influenza vaccine was the most common myth supported by respondents (21ae6%, 30 ⁄ 139) yet a decade ago, less than 4% of australian gps gave this as a reason for not getting influenza vaccine. 16 the reasons behind this shift in belief is unknown; however, technology could play a part, with increased access to a variety of information sources. in the light of this, gp education should continue to focus on dispelling this myth through use of evidence-based information. other barriers to influenza vaccination identified in our study were lack of awareness of any recommendations for general practice staff to receive the influenza vaccine (10%) and unacceptable side effects of vaccination (7%). these are similar to those commonly cited by hcws in other countries. [19] [20] [21] for hospital doctors, being too busy has been identified as a major barrier to getting vaccinated against influenza. 6, 12, 24 in contrast, only a small number of participants, all of whom were gps, identified lack of time as a barrier as did approximately 25% of gp respondents the australian influenza and pneumococcal vaccination survey (2003) in the elderly. 16 a previous investigation of general practice staff across 53 dgps in australia found a significant association between workplace influenza vaccination policy and staff vaccination. 23 we found that interrelationship between staff beliefs and practice policies may be an important determinant of hcw immunization behaviours in these practices. office policies demanding immunisation and operating within an effective hierarchy can lead staff members to re-evaluate their beliefs about influenza immunization in the light of their own experience. 31 continued efforts at the practice level to make formal commitments to staff health by developing policies for influenza vaccination of staff may assist in increasing coverage in this group. provision of the influenza vaccine to patients along with consistent, direct, exposure to influenza like illness by this population 32 may further impact on their decision to be vaccinated. pandemic (h1n1) influenza 2009 is also likely to increase awareness of influenza vaccination and instigate new policies and practices surrounding vaccination of primary health care staff as has been seen in other countries during heightened awareness of an impending influenza pandemic threat. 33 there are a number of limitations to this study including sample size, generalisability and use of self-report for vaccination status. although the sample size was small, compared to other general practice-based surveys, it would be considered reasonable in the light of the challenges with surveying this population. 34, 35 there is potential for selection bias in this study towards those who are particularly concerned about influenza and ⁄ or vaccination or those who accept vaccination. furthermore, using self-reported vaccination status in adults has been shown to overestimate coverage. 36, 37 there may also be limitations with generalisability because our study was conducted only in one state of australia. this study did not collect any data on nonrespondents or outcomes for those who intended to be vaccinated in 2009. in addition, the barriers in our survey may not have covered all possible options, thus may have influenced participants response. qualitative research is needed to further explore these findings. despite these limitations, influenza vaccination coverage was found to be relatively high amongst the gps and pns in our study; however, there is still room for improvement. understanding barriers to vaccination is the first step to developing effective strategies to overcome them. for institution-based hcws, there is now an extensive literature base around knowledge, attitudes and practices towards influenza vaccination, whereas, there are only a few studies exploring these topics in the primary health care setting in australia. to enhance development and targeting of strategies to increase coverage, a more complete and current understanding of coverage in this group is needed to build on the estimates presented here. we believe this study to be a basis for future investigations and interventions to increase influenza vaccination rates in primary health care staff in australia. influenza-related disease: the cost to the australian healthcare system the role of influenza vaccine in health care workers in the era of severe acute respiratory syndrome national health and medical research council (nhmrc). the australian immunisation handbook, 9th edn. canberra: australian government world health organization. influenza vaccines vaccines for preventing influenza in healthy adults discussion paper: influenza vaccination amongst health care workers policy directive: occupational assessment, screening & vaccination against specified infectious diseases royal australian college of general practitioners. infection control standards for office based practices co-ordinated approach to healthcare worker influenza vaccination in an area health service influenza vaccine coverage among health care workers in victorian public hospitals influenza vaccination of staff in aged care facilities in the act: how can we improve the uptake of influenza vaccine? 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(cat. no.gep22). canberra: australian government influenza vaccination in act nurse immunisers evaluation of the vaccine coverage of the general practitioners in the french community organizational culture influences health care workers' influenza immunization behaviour annual report of the national influenza surveillance scheme high coverage of influenza vaccination among health care workers can be achieved during heightened awareness of impending threat response rates of victorian general practitioners to a mailed survey on miscarriage: randomised trial of a prize and two forms of introduction to the research the effect of cash and other financial inducements on the response rate of general practitioners in a national postal study sensitivity and specificity of patient self-report of influenza and pneumococcal polysaccharide vaccinations among elderly outpatients in diverse patient care strata validity of self-reported influenza and pneumococcal vaccination status among a cohort of hospitalized elderly inpatients influenza vaccine coverage and attitudes in australian primary care staff ª thanks to the following for their support in this study; central sydney general practice network, albury wodonga regional gp network shire gps (sutherland division of general practice) and went west ltd. j leask is an investigator on a grant which is part funded by sanofi pasteur. c. r. macintyre receives funding from influenza vaccine manufacturers gsk and csl biotherapies for investigator-driven research. k ward has received funding from wyeth to attend an immunisation conference. all other authors of this manuscript have no conflicts of interest to declare. national centre for immunisation research and surveillance is supported by the australian government department of health and ageing, the nsw department of health and the children's hospital at westmead. key: cord-002407-25cawzi0 authors: nogales, aitor; martínez-sobrido, luis title: reverse genetics approaches for the development of influenza vaccines date: 2016-12-22 journal: int j mol sci doi: 10.3390/ijms18010020 sha: doc_id: 2407 cord_uid: 25cawzi0 influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. however, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. the segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. with the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary dna copies of the viral genome by transfection of susceptible cells. these reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. in addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. in this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. influenza a (iav) and b (ibv) viruses belong to the orthomyxoviridae family of enveloped viruses [1] . iav is able to infect several species and mostly exists in the wild aquatic fowl reservoir [2] [3] [4] . on the other hand, ibv is mainly restricted and adapted to humans, although sporadic infections of seals have been documented [5, 6] . iav and ibv genomes contain eight negative sense, single-stranded viral (v)rna segments [1] (figure 1 ). iav and ibv vrnas contain a central coding region that is flanked at both terminal ends by non-coding regions (ncrs), which serve as promoters to initiate genome replication and gene transcription by the viral polymerase complex [1, 7] . influenza vrnas in the virion are found as viral ribonucleoprotein (vrnp) complexes encapsidated by the viral nucleoprotein (np) and a single copy of the viral polymerase complex. influenza virus-encoded rna-dependent rna polymerase (rdrp) [8] is a trimeric complex consisting of the polymerase basic 1 (pb1) and 2 (pb2) and acidic (pa) proteins and, together with the viral np, are the minimal components involved in viral replication and transcription [9] . iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and noncoding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from iav and ibv share many features, but they differ in their host range, virion structure, genomic organization and glycan binding specificities [1, 10] . despite having similar genomes encoding homologous proteins, iav and ibv are distinguished by the different lengths of proteins and non-coding regions (ncrs) that serve as promoters for genome replication and gene transcription [5, 11, 12] (figure 1 ). likewise, they can also be distinguished by the presence of accessory proteins encoded from overlapping open reading frames (orfs) and by the antigenic differences of internal proteins [13] (figure 1a ,b). for instance, iav and ibv both encode ion channel proteins from the gene m segment 7, m2 and bm2, respectively. the m2 and bm2 proteins of iav or ibv are encoded together with the matrix protein 1 (m1) and both are incorporated into virions and expressed on the surface of virus-infected cells [1] . however, the m2 protein of iav is translated from a spliced mrna [14] , while the ibv bm2 protein is translated using a different strategy, where the initiation codon of bm2 protein overlaps the termination codon of m1 protein (uaaug, a stop-start pentanucleotide) [15] . in addition, ibv expresses the nb ion channel, which is absent in type a influenza virus [1] ( figure 1b) . however, both influenza viruses encode two surface glycoproteins, hemagglutinin (ha) and neuraminidase (na) ( figure 1a ,b). iav and ibv ha proteins are involved in binding to cellular receptors and responsible for the fusion of the viral and endosomal membranes [16] . infection with iav or ibv induces a protective immunity mediated, at least partially, by antibodies directed against the viral ha, which is the main immunogenic target in both natural infections and vaccine approaches. influenza na glycoprotein is responsible for the cleavage of sialic acid moieties from sialyloligosaccharides and facilitates the release of newly produced virions from infected cells [17, 18] . iavs are classified on the basis of the antigenic properties of ha and na into 18 ha (h1-h18) and 11 na (n1-n11) subtypes [1, 19, 20] . however, only iav h1n1 and h3n2 subtypes are currently circulating in humans. on the other hand, two major lineages of ibv are circulating in humans, the victoria-like and yamagata-like subtypes that are divergent from the ancestral ibv (b/lee/1940) and have been co-circulating in humans since the 1980s [5, 21, 22] . these two subtypes are the predominant circulating virus strains about once every three years [23] [24] [25] . infection with influenza viruses begins when the viral ha protein binds to its cellular receptor, a sialylated glycoprotein containing α-2,3 or α-2,6 linkages [16] . upon the binding to the receptor, the uptake of the virus by receptor-mediated endocytosis is initiated, and the cell membrane engulfs the virus particles in an endosome. after endocytosis and upon acidification of the endosome, influenza viral ha undergoes a conformational change responsible for the fusion of the viral and the endosome membrane [16] . then, the m2 (iav) or bm2 (ibv) ion channel proteins promote the release of the vrnp complexes from the virion core into the cytoplasm of infected cells [14, 26] . the vrnps are translocated from the cytoplasm to the nucleus of infected cells to initiate viral genome replication and gene transcription [27] . the nuclear export (nep) and matrix 1 (m1) proteins are responsible for the nuclear export of newly-synthesized vrnps into the cytoplasm of infected cells. notably, a single copy of each of the eight vrnas is packaged into new virions [28, 29] . selective package of influenza vrnas into nascent virions is mediated by rna-rna interactions of vrna packaging signals present at the terminal ends of each of the vrna segments [30] . finally, the receptor-destroying enzymatic activity of na is responsible for the release of newly-synthesized viral particles from the surface of infected cells [17] . among the first battles between the host and the virus, cellular type i interferon (ifn-i) plays an important role in controlling viral infection [31] . therefore, viruses have developed multiple strategies to hijack the host cellular immune response. influenza vrna segment 8, or the nonstructural (ns) gene, encodes two distinct proteins through a direct or alternative splicing mechanism [1] . influenza virus segment 8 produces the nonstructural protein 1 (ns1) as a primary transcript, whereas nep is produced by alternative splicing of the ns mrna [32] . ns1 has multiple functions during the replication cycle of influenza virus, but most notably inhibits induction of ifn-i response and innate immune activation [31] . influenza viruses pose a threat to human health and are responsible for global epidemics every year [33] [34] [35] [36] [37] . in fact, influenza virus is one of the most significant causes of morbidity and mortality yearly, leading to a significant economic impact [38] . despite the implementation of effective and comprehensive vaccination programs, the world health organization (who) estimates that seasonal influenza virus infections results in about one billion infections, 3-5 million cases of severe disease and between 300,000 and 500,000 deaths around the world annually [39] . moreover, just in the united states (u.s.), influenza viral infections result in an average of 87 billion dollars of cost due to prophylactic, therapeutic and hospitalization costs, as well as missed school or work days [38, [40] [41] [42] . in addition to seasonal influenza, iav can cause sporadic pandemics of great consequences when novel viruses are introduced into humans [43] . the mechanisms responsible for the emergence of seasonal and pandemic iavs are antigenic drift and antigenic shift, respectively [12, [44] [45] [46] . in the case of seasonal influenza, mutations in the viral genome result in the selection of antigenic variants with changes in viral tropism, increased levels of viral fitness or in the ability to escape neutralizing antibody (nab) responses induced upon previous natural infections or vaccinations [44, 45] . moreover, antigenic drift can lead to variant viruses resistant to antivirals [47] . on the contrary, antigenic shift results from co-infection of a host cell or organism with two or more iavs where vrnas in viral progenies are reassorted [12, 46] . by obtaining viral segment constellations that confer influenza virus optimal replication, transmissibility and immunologic escape, reassortant iavs can lead to a pandemic in immunologically-naive populations. iavs and ibvs can reassort intratypically between subtypes or lineages, but intertypic reassortment, or genetic swapping of segments between iav and ibv, has not been reported [21, 48, 49] . the absence of intertypic reassortment is mediated by the lack of compatible packaging signals between iav and ibv [12] . in the last century, three iav pandemics have occurred: the h1n1 spanish flu of 1918, the h2n2 asian flu of 1957 and the h3n2 hong kong flu of 1968 [44, 50] . of these three, the 1918 h1n1 spanish flu was particularly fatal and responsible for approximately 50 million deaths around the world [51] . although not classified as true pandemics, three epidemics of influenza h1n1 viruses in 1947, 1976 and 1977 were feared to have pandemic potential. in 2009, a swine-origin h1n1 iav was responsible for the first influenza pandemic of the 21st century and infected, in less than one year, more than 600,000 individuals around the world [52, 53] . although influenza virus infections are primarily spread by person-to-person transmission via aerosolized droplets, infection with new avian-or swine-origin iav can take place and may have great risk for pandemic potential if the virus acquires the ability to be transmitted between humans [54, 55] . ibvs usually contribute less to seasonal epidemics than iavs of the h3n2 subtype, but they contribute more than type a h1n1 influenza strains and are the predominant circulating virus strains once every three years [23] [24] [25] . moreover, during the last decade, ibv has been the cause of several acute respiratory illness outbreaks [56] [57] [58] [59] [60] . to date, no influenza pandemics have been linked to ibvs. public health concerns posed by influenza virus infections are aggravated by the ability of influenza viruses to efficiently transmit and the limited therapeutic options to treat viral infections [37] . thus, vaccination remains our best medical intervention to protect humans against seasonal influenza virus. however, the efficiency of current influenza vaccines is suboptimal [61] . the segmented genome of influenza viruses provides an evolutionary advantage of reassortment, or the exchange of viral genome segments between different viral strains within the same type. in addition to this exchange of genome material (or antigenic shift), influenza viruses can introduce mutations in their genomes (or antigenic drift), leading to viral mutants with resistance against current antivirals or nabs [62] [63] [64] . because of the antigenic drift, influenza vaccines need to be reformulated yearly to ensure that the ha and na present in the vaccine match those present in circulating seasonal viruses. to date, three types of influenza virus vaccines are approved by the food and drug administration (fda) for human use: recombinant viral ha, inactivated virus and live-attenuated viruses [65] [66] [67] [68] [69] . regardless of the type of vaccine, seasonal influenza vaccines contain antigens from the three circulating influenza virus strains: iav subtypes h1n1 and h3n2; and ibv (victoria-like or yamagata-like lineage) [67, 70] . recently, to improve the efficacy of seasonal influenza vaccines, a quadrivalent influenza vaccine formulation that includes both ibv lineages components (victoria-like and yamagata-like lineage) has been approved by the fda [14] . the most common influenza vaccine is the inactivated influenza vaccine (iiv). iiv, which is administered intramuscularly, has been shown to elicit protective humoral immunity by producing nabs that target epitopes on ha [61, 71] . in contrast to iiv, the live-attenuated influenza vaccine (laiv) and its administration mimic the natural route of virus infection, which consequently having risks and benefits [72] . an advantage is that laiv elicits both more rapid and efficient innate and adaptive immune responses [65] and can provide more efficient cross-reactive t-cell-mediated protection against heterologous influenza viruses [72, 73] . although both iiv and laiv have been shown to be efficient for the treatment of influenza viral infections, there is an unmet need to increase the effectiveness of seasonal influenza vaccines. likewise, there is an urgent need to develop effective vaccines for the treatment of potential pandemic influenza viruses. although vaccination is the main method to prevent influenza infections in humans, antivirals offer an additional countermeasure against new rapidly-spreading and/or potentially pandemic influenza viruses [74] [75] [76] . therapeutic choices to control influenza infection are currently limited to two classes of fda-approved antivirals targeting either the viral m2 ion channel (amantadine, rimantadine) [77] [78] [79] or the sialidase active site of na (oseltamivir, zanamivir) [80] . the first inhibits the initial steps of the virus life cycle, while the second inhibits virus release. however, na inhibitors are the only type of antivirals approved for the prophylaxis and treatment of ibv infections [81] , although data from clinical trials have shown that oseltamivir is less effective against ibvs than against iavs [82] . importantly, mutations in the viral genome can lead to influenza antivirals being ineffective, like in the case of m2 blockers, which are no longer recommended against circulating seasonal influenza viral strains [83] . therefore, the emergence of drug-resistant influenza variants is an increasing concern for controlling influenza infections [76, 84] , and there is a significant need for the identification of novel compounds with antivirals properties. influenza viruses evade human pre-existing immunity by accumulating mutations (antigenic drift), allowing reinfection of individuals previously exposed to natural infections or vaccinated. thus, vaccine companies have to reformulate the composition of influenza vaccines yearly to ensure a good match between the viruses present in the vaccine and those seasonally circulating in humans [33, 34, 85] . the who global influenza surveillance network (gisn) [35] , which includes more than 120 national influenza centers in over 90 countries [86] , tracks the evolution and epidemiology of influenza viruses and uses the collected data for the vaccine strain selection process. these studies also help to understand virus evolution and epidemiology in different geographic areas [19] . (4) the national institute of infectious disease in tokyo, japan. basically, the degree of immunity induced by one influenza strain that is effective against another strain is mainly dependent on the antigenic difference between both viral strains [87, 88] . the influenza viral glycoprotein ha, the primary target of the protective neutralizing immune responses [88, 89] , is the focus of influenza virus surveillance and the primary component targeted by currently licensed influenza vaccines [72, 89] . influenza vaccine production is challenging because of the wait time that is needed to identify the predominant circulating virus and the production time that is needed to manufacture the vaccine. the national influenza centers perform virus isolation on certain samples obtained from patients to identify circulating viruses and to determine if the viruses grow efficiently in culture. normally, influenza viruses are isolated using madin darby canine kidney (mdck) cells instead of chicken embryonated eggs because higher isolation rates, especially for h3n2 iav strains, have been reported using mdck cells [90] [91] [92] . although most influenza viruses are isolated from mammalian cells, viruses must be generated in embryonated eggs for vaccine manufacturing due to regulations [93] . nowadays, the vaccine strains must be selected almost 7-9 months ahead of the influenza season in which they will be used. the recommendation for the strains included in the vaccine composition for the northern hemisphere is made in february to allow time for the~300 million doses of vaccine to be produced in time for vaccinating people in october/november. this allows for influenza season preparation, which typically peaks sometime between december and march [86] . on the other hand, for the southern hemisphere, recommendations are provided in september, and vaccination takes place in march/april of the following year [86] . seasonal influenza vaccines must protect against h1n1, h3n2 and b viral strains currently circulating in humans globally [34, 86] . the main goals of influenza vaccines are the protection against infection and disease caused by influenza infections and to restrict virus transmission within the population [67, 94] . nab responses, commonly assessed by measuring hemagglutination inhibition (hai) titers, are currently used as a serological marker of the efficient immunological response to the vaccine. the effectiveness of influenza vaccines is variable and usually higher in children and in healthy adults under the age of 65. in individuals above 65 years of age, lower effectiveness has been observed [61, 71, 95] . recommendations for influenza vaccination differ between countries, but since influenza vaccines do not induce long-lasting antibody protection, annual influenza vaccinations are recommended. nowadays, the most used influenza vaccines can be divided into iiv and laiv [65, 66, 72, 73, 96 ]. killed virus vaccines or iiv are generally administered intramuscularly and can be classified as whole virus vaccines or split vaccines [66, 71, 97, 98] . whole virus vaccines were the first to be developed. the influenza virus is grown in embryonated chicken eggs, subsequently purified, concentrated and chemically inactivated with formaldehyde [98] (figure 2 ). whole virus vaccines are safe and well tolerated, with an efficacy of 60%-90% in children and adults. on the other hand, the split-virus vaccine exposes all viral proteins and subviral elements upon dissociation of the virions by a nonionic detergent treatment step [97, 99] . most influenza vaccines in the u.s. and europe are egg-produced, formaldehyde-inactivated, then chemically disrupted with nonionic detergents after purification. unlike virus-based vaccines, subunit influenza vaccines consist of purified viral ha or ha/na proteins without the other viral components [68] . these subunit vaccines can be produced in eggs if the viral proteins are prepared from viruses where ha/na have been purified by removal of other viral component. in addition, the subunit vaccines can be generated using novel manufacturing technologies [98, 100] , which allows for the production of large quantities of the viral ha using baculovirus expression systems and recombinant dna technologies [100] . the iav strains that are used in vaccine manufacture are high-growth 6 + 2 reassortants containing the ha and na gene segments from the target strains in the backbone of influenza a/puerto rico/8/1934 h1n1 (pr8) or other high growth virus. influenza pr8 replicates at high titers in eggs and cells and also has a favorable safety profile in humans [98, 101] (figure 2a ). to generate the reassortant viruses, eggs are co-infected with pr8 and seasonal strains (figure 2a ). selection of appropriated seed vaccine viruses is made by amplification in the presence of pr8 ha and na nabs [98] (figure 2a) . then, selected viruses are cloned and sequenced for confirmation. the ibv vaccines are typically wild-type (wt) viruses. however, ibv reassortants are used if wt ibv growth properties are not optimal for efficient growth and vaccine production [98] . the remaining class of vaccines consists of live-attenuated influenza viruses. attenuated human laivs were developed in the 1960s by serial passage of the virus in eggs using suboptimal conditions of temperature. the resulting attenuated viruses displayed a temperature-sensitive (ts) cold-adapted (ca) attenuated (att) phenotype that grew at 25 • c, but not at temperatures found in the lower respiratory tract (>35 • c) [102] [103] [104] [105] . because this ts, ca, att phenotype restricts virus replication to the upper respiratory track, these viruses could induce local protective immunological responses [65, 95, 96, 98] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cellmediated immune response and provide a cross-reactive and longer lasting immune response [106] . laiv have been available in the u.s. since 2003 and are administrated intranasally. the advantages of a live virus vaccine, as compared to the inactivated virus vaccine, is that it is applied to the nasal mucosa where the vaccine can induce local immunity (including nabs), generate a cell-mediated immune response and provide a cross-reactive and longer lasting immune response [106] . the current laivs consist of the internal viral segments (pb2, pb1, pa, np, m and ns) of an attenuated master donor virus (mdv) and the ha and na viral segments from the selected seasonal virus strain ( figure 2b ). the mdvs used are a/ann arbor/6/60 (h2n2) and b/ann arbor/1/66 for iav and ibv, respectively [103, 104, [107] [108] [109] [110] [111] [112] [113] . similar to mdv iav, mdv ibv was originally derived by serial passage of the parental wt virus and isolated at successively reduced temperatures in primary chicken kidney (pck) cells [114] . the resulting ibv mdv grows efficiently at 25 • c (ca), but its growth is restricted at 37 • c (ts). the genetic changes in the mdv strains have been recently characterized. the mdv iav includes five mutations in two of the viral polymerases (pb2 n265s; and pb1 k391e, d581g and a661t) and np (d34g) [103, 104, 115] . the mdv ibv has been reported to contain two mutant amino acids in np (a114 and h410) and one in pa (m431) that are responsible for the ts, ca signature [108] . two additional residues in m1 (q159 and v183) provide the mdv ibv an attenuated (att) phenotype [108] . although laivs have been approved for clinical use, to date, their mechanism of attenuation has not been completely understood. however, the tolerability of laivs in specific populations is an important concern because of the inherent risk of immunizing with live viruses. thus, laivs are not recommended for immunocompromised patients or asthmatics [116] and are not approved for use in children under two years of age [72, 117] . moreover, laivs harboring different ha and na viral segments can be unequally safe or immunogenic from year to year as the viral ha and na are different. similar to u.s. laivs, in russia, two ts, ca mdv iavs have been obtained by using a similar temperature adaptation approach. the two russian mdvs were originated from the same parental a/leningrad/134/57 h2n2 (len/57) influenza strain [118, 119] . similarly to the u.s. mdv, the russian mdvs have been selected by growing len/57 in embryonated chicken eggs at lower (25 • c) temperatures. the mdv len/17 was obtained after 17 passages of len/57 at 25 • c and has been used in the preparation of the russian laiv for use in adults. the genetic changes in the mdvs len/17 have been identified, and these include four mutations in three viral proteins (pb2 v478l; pb1 k265n and v591i; nep m100i) [120] . the second donor strain (len/47) was obtained after a total of 47 passages and has been used for vaccinating children less than 16 years of age, although the genetic changes responsible for the further ts, ca phenotype have not been well characterized [121] . in all cases, like for iiv, the mdvs contain six internal genes to generate vaccine strains in combination with the seasonal recommended ha and na genes from the circulating strains ( figure 2b ). laivs are generated either by classical reassortment in eggs (as previously described for the iiv) ( figure 2b ) or by reverse genetics, as indicated below [98] . most of the influenza virus vaccines have been traditionally produced in eggs, but with the progress made in mammalian cell culture technologies, influenza vaccine manufacturers have invested in these novel cell culture systems for the mass production of influenza vaccines without the need of eggs. the majority of the currently licensed influenza vaccines that are made by biotechnology companies use fertilized chicken egg-based production technology, but this process has multiple drawbacks. this form of manufacturing depends on the access to embryonated eggs, relies on the ability of influenza viruses to efficiently grow in eggs and is a resource-and time-intensive process [91, [122] [123] [124] . moreover, the risk of egg contamination by avian pathogens or microbes represents a risk for the production of influenza vaccines [125, 126] . importantly, in the case of an iav pandemic, the egg supplies can be compromised. for iiv, one dose for adults contains approximately 45 µg of ha (15 µg of viral ha for each of the three antigenic h1n1, h3n2 and ibv components), meaning one egg = 7-10 dose of vaccine. laivs share similar egg-based production process steps. the laiv is recovered from infected eggs and then purified and concentrated [90] . importantly, new vaccine production approaches that do not depend on the propagation of influenza viruses in eggs (e.g., cell cultures) represent an excellent option to increase influenza vaccine production. mammalian cell cultures have been used in the biopharmaceutical industry for the production of therapeutic proteins and/or vaccines [127, 128] . in 2012, the fda approved a cell-based production process for influenza vaccines, but the manufacturing process begins with egg-grown vaccine viruses per fda regulations. influenza vaccine production using fda-approved mdck or vero (african green monkey kidney) cells may eventually and completely replace the use of eggs for the production of influenza vaccines in the future. influenza vaccine production in mammalian cell lines offers several advantages over egg-based production: it allows faster and greater production capacity, improved availability of substrate for virus growth [122, 127] and eliminates reliance on the supply of embryonated chicken eggs [129, 130] . in addition, cell cultures can be cryopreserved and scaled up in bioreactors at any time. adjuvants have been shown to enhance the immune response elicited by an antigen and could be used to improve the immunogenicity of iiv [131, 132] . the use of adjuvants could also reduce vaccine dose, stretching antigen and vaccine supplies. currently, fda-licensed adjuvants for influenza vaccine usage include aluminum salt (alum) and the squalene oil-in-water emulsion systems mf59 (wadman 2005 (novartis)) [133] and as03 (glaxosmithkline) [134] . however, most of the current iivs do not contain any type of adjuvant, but many are under investigation. genetics techniques to generate recombinant viruses were first developed for dna viruses and based on the transfection of cells with plasmids encoding the viral genome or by heterologous recombination between plasmids bearing viral sequences with the virus genome and the helper virus [135] . initial genetics approaches for dna viruses were followed by manipulations of positive-sense rna viral genomes [136, 137] . transfection of plasmid dna, or rna transcribed directly in vitro from plasmids, containing the genome of poliovirus into susceptible cells led to the generation of recombinant infectious poliovirus [138] . however, the genomes of negative-sense rna viruses, including influenza, were less suitable to molecular biology manipulations in comparison with dna or positive-sense rna viruses since their genomes are complementary to mrna in their orientation and, therefore, not infectious by themselves [1, 7] . they require the presence of vrna(s) and the viral rdrps to initiate the replication cycle of the virus [33, 44] . the advent of reverse genetics and molecular engineering has transformed the influenza field, allowing multiple questions to be answered using genetically-engineered recombinant influenza viruses [139] . such studies include mechanisms of viral genome replication and gene transcription, pathogenicity and virulence, virus-host interactions or host range and transmissibility [12, 13, 139, 140] . moreover, these technologies have been implemented to develop influenza vaccines [141] and to generate recombinant influenza viruses expressing foreign proteins as vaccine vectors [142] [143] [144] or harboring reporter genes to easily track viral infections [142, [145] [146] [147] . plasmid-based reverse genetics for influenza virus allows for the simultaneous expression of the viral components involved in viral genome replication and gene transcription (pb2, pb1, pa and np) and the eight negative-stranded vrnas in transfected susceptible cells, which together generate de novo, recombinant iavs or ibvs (figure 3 ) [48] . the goal to generate vrna in vivo from cloned complementary (c)dnas was achieved when the rna polymerase i (pol i) system for influenza vrna synthesis was established [148, 149] . pol i is a nuclear enzyme that transcribes ribosomal (r)rna, which like influenza vrna does not contain a cap structure on the 5 or poly (a) structures on the 3 ends [150, 151] . importantly, pol i initiates and terminates transcription at defined promoter and terminator sequences allowing the generation of vrnas without additional nucleotides at their 5 or 3 ends, which is required for efficient generation of recombinant viruses using reverse genetics. nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . nevertheless, the pol i promoter is species specific [152, 153] and was originally established for influenza rescue in human cells [135, 140, 149, 154] . currently, the pol i promoters of different species have been identified, allowing the generation of recombinant influenza viruses using reverse genetics techniques in avian, canine, equine or murine cells lines [155] [156] [157] [158] . influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that influenza viruses require the presence of eight vrna segments for efficient virus fitness and successful production of virion progeny. the initial description of influenza reverse genetics, originally established in 1999 for iav [154, 159] , required the use of 12 plasmids to generate recombinant influenza viruses: four polymerase ii (pol ii) protein expression plasmids, encoding the viral rdrp complex (pb2, pb1 and pa) and np for vrnp reconstitution; and eight pol i-driven plasmids for expression of the eight vrna segments [154, 159] . however, it was later described that only eight ambisense and/or bidirectional plasmids were needed for complete reconstitution of influenza viruses ( figure 3a ) [150, 151] . the eight plasmid-based rescue system is now the most common method for the generation of recombinant influenza viruses. because fewer plasmids are required, the eight-plasmid approach is more successful than the initial twelve-plasmid reverse genetic technique. the core of the eight-plasmid rescue system is that each plasmid contains an "ambisense cassette" that includes rna pol i and/or ii sequences, which drives the transcription of vrnas (pol i) and protein (pol ii) expression from the same viral cdnas ( figure 3b ). using a similar technology in 2002, this reverse genetics technique allowed the recovery of ibv entirely from ambisense plasmids [13, [160] [161] [162] . now, iav and ibv reverse genetics techniques are well established and commonly used in multiple research laboratories for different research purposes. influenza reverse genetics techniques have had an important effect on expanding our knowledge of the molecular biology and pathogenesis of influenza viruses, allowing researchers to answer important questions in the biology of iav and ibv that were not possible using conventional virological or biochemical procedures [12, 144, 147] . scientists can now mutate specific nucleotides in the influenza viral genome to elucidate the nature of regulatory sequences or the contribution of specific amino acids to the function of influenza viral proteins. for instance, reverse genetics technologies have enabled the identification and characterization of the cis-acting elements required for virus cell entry, uncoating, genome replication and gene transcription, encapsidation, packaging and viral release [13, 135, [163] [164] [165] . moreover, by engineering viral vectors suitable for the expression of foreign proteins in infected cells, multiple recombinant influenza viruses harboring reporter fluorescent and/or luminescent genes have been generated and used to identify antivirals or nabs in vitro and/or in vivo [142, 146, 147] . finally, reverse genetics have allowed the creation of single-cycle infectious iavs (sciiavs) that are restricted to one cycle of replication in parental cell lines. however, in complementing cell lines, sciiavs can replicate efficiently and to levels comparable to wt forms of influenza viruses [144] . highly virulent iav have the potential to pose a greater human threat than many other biosafety level (bsl)-3 and bsl-4 pathogens because of their efficient transmission and limited therapeutic options [47] . traditional immunological approaches (e.g., hai or microneutralization assays) to identify the presence of iav nabs rely on the manipulation of live forms of viruses and need the use of special bsl conditions. thus, novel approaches that allow the detection of viral nabs without the use of live forms of iavs and highly contained bsl laboratories would facilitate these serological assays. in this regard and because of their safety profile, sciiavs represent an excellent alternative to identify new antivirals and/or nabs [144] . moreover, several sciiavs have been shown to represent an excellent option for their implementation as safe, immunogenic and protective vaccines and/or vaccine vectors [69, 144] . influenza plasmid-based reverse genetics represent a better alternative to circumvent the process of generating reassortant virus by co-infection of chicken embryonated eggs for the generation of influenza vaccines (figure 2 ) [135] . moreover, the generation of recombinant influenza viruses using plasmid-based reverse genetics approaches is simple, well established and currently in use in several laboratories around the world [139, 166] (figure 4) . briefly, the eight-ambisense plasmids are co-transfected into susceptible fda-approved cells, and viable virus can be recovered from the tissue culture supernatants [139, 166] , then amplified in either embryonic eggs or fda-approved for vaccine production cells (figure 4) . for many years, iivs have been produced by reassortment in eggs ( figure 2) [90, 98, 122] . however, strains with the desired genotype (six internal genes of a high-growth virus and the ha and na glycoproteins of the seasonal influenza virus) could be produced easily and more quickly by implementing reverse genetics approaches. this process would overcome the need of chicken embryonated eggs to generate the desired reassortant virus and, therefore, minimizing the time associated with the selection process of the reassortant iiv ( figure 4a) . moreover, the improvements in surveillance, as well as the de novo gene synthesis for the production of the ha and na viral segments from the selected strains could reduce considerably the time of vaccine production. although influenza reverse genetics could be useful for the production of vaccine seed strains, to date, it has not been possible to predict which gene segment(s) constellations are required for efficient growth of different vaccine viruses. moreover, the time to produce vaccine viruses using reverse genetics versus the time needed to generate them using the classical reassortment approach could be a major consideration for the cost effectiveness in vaccine production and manufacturing. thus, the traditional viral reassortment has remained preferred because it allows the generation of a number of diverse gene(s) combinations in order to select recombinant viruses with better fitness [98] . during the last few decades, considerable improvements have been accomplished in the development of influenza vaccines. however, novel approaches to increase the effectiveness of seasonal influenza vaccines are needed. reverse genetics technologies have proven a valuable tool to develop reassortant strains for the production of laiv candidates ( figure 4b ). currently, seed viruses containing six gene segments from the mdv a/ann arbor/6/60 (h2n2) and the ha and na from the selected seasonal virus can be quickly generated using reverse genetics systems [98, 131] ( figure 4b ). the ha and na from selected seasonal influenza viruses can be amplified by rt-pcr or quickly chemically synthetized and then cloned in ambisense plasmids used for virus rescue. this technology could speed up the development of new laivs, bypassing the need to isolate the exact virus reassortment in eggs ( figure 2 ). the best way to combat influenza virus infection is to prevent it. thus, an urgent need exists to develop novel and more effective influenza vaccines. plasmid-based reverse genetic technologies have allowed the engineering of recombinant influenza viruses that contain single or multiple mutations in the viral genome, which can be potentially implemented as novel or improved vaccine approaches. in fact, several novel vaccine candidates have been developed with promising results in animal models of experimentation. here, we review and discuss some of them. because of ns1's ability to hijack the host innate immune ifn-i response, a variety of potential vaccine strategies have been developed, which are based on the use of modified ns1 proteins as a means for virus attenuation [167] [168] [169] [170] [171] [172] . equine [170] , swine [169, 173, 174] , avian [167, 175, 176] , canine [177] and human [178, 179] iavs with partial truncations in or deletions of the viral ns1 protein are all attenuated in vitro and in vivo [167, 169, 170, 179] . importantly, these recombinant iavs can induce a protective immune response upon a single intranasal vaccination in mice [177, [179] [180] [181] , horses [170] , pigs [169, 173, 174] , birds [167, 175, 176] and macaques [178] ; therefore, they represent excellent laiv candidates to prevent iav infections. in addition, a similar approach has been employed to develop attenuated ibvs with similar results [180] . mice inoculated with the ns1-truncated or -deleted mutants elicited an antibody response and showed protection against wt virus challenge [180] . thus, these ns1-truncated influenza a and b viruses represent excellent candidates as safe, immunogenic and protective laivs for multiple virus strains in different animal models. the genetic code in animals encodes for 20 different amino acids (aa) using 61 codons. this degeneracy of the genetic code allows amino acids, except tryptophan (w) and methionine (m), to be encoded by more than one synonymous codon [46] . viruses, including influenza, rely on the host cell translation machinery to synthesize their viral proteins for the formation of infectious virus progeny. as an evolutionary consequence, viruses have modified their codon usage according to the host they infect [182] . experimentally, protein synthesis can be downregulated by synthetically deoptimizing the codon usage of a gene [141, 182] . the generation of recombinant viruses containing genes with deoptimized codons is now feasible [141, 182] , and their level of attenuation depends on the viral gene targeted and the number of codon changes made during the codon deoptimization process [182] . for influenza viruses, many regions in the viral genome cannot be altered because of their important role in viral replication and transcription (e.g., ncrs), packaging (e.g., packaging signals), the presence of multiple overlapping orfs (e.g., segments 7 and 8), etc. [48] . to date, recombinant iavs that have been attenuated using a codon-pair [183, 184] or a codon bias [182] deoptimization approach to decrease expression levels of the viral pb1, ha and np [183] , na and ha [184] or ns1 and nep [182] have been generated. importantly, influenza viruses generated by codon deoptimization showed similar viral replication kinetics to wt virus in mdck cells, which is important for their effective use for vaccine production. however, to date, the ability of these recombinant iavs containing codon-pair or codon bias deoptimized viral segments to replicate in eggs has not yet been evaluated. importantly, all of the codon deoptimized iavs were attenuated in mice and able to provide, upon a single immunization dose, protection against a lethal challenge with a wt form of the virus, showing that laivs were safe, immunogenic and protective. however, mice do not accurately reflect virus pathogenesis and immunological responses seen in humans [81] and do not have the same codon usage bias as humans. therefore, studies aimed to demonstrate the safety, immunogenicity and protection efficacy of codon deoptimized recombinant iavs in other well-established animal models of influenza (e.g., guinea pigs, ferrets or nonhuman primates) could lead to their implementation as laivs in future vaccinations [141] . other promising approaches for the development of laivs relate to the use of sciiavs [144] . sciiavs based on their safety profile, ability to elicit protective humoral and cellular responses and protective effectiveness represent a feasible alternative to current influenza vaccines for the treatment of influenza viral infections [69, 144] . however, to date, no single-cycle infectious ibvs (sciibv) have been reported. when delivered intranasally, sciiav has been shown to be safe in a mouse model of influenza infection, without signs of illness or mortality [69, [185] [186] [187] [188] [189] . moreover, and similar to the current laivs, intranasal immunization with a single dose of sciiavs elicited localized mucosal immune responses and recruitment of influenza-specific cd8 t-cells into the lungs of vaccinated animals [145, 185, 186, 190] , the latest being the main contributor of immunity against challenge with heterologous influenza viruses [69, 190] . importantly, sciiavs protected mice against lethal influenza virus challenges [69, 144] . moreover, similar safety, immunogenicity and protection efficacy of sciiavs were observed in ferrets [69] . vaccination of pigs with sciiav has also been shown to be immunogenic and protective against challenge with swine influenza viruses [191] . moreover, sciiavs expressing foreign genes represent an excellent option for their implementation as bivalent vaccines. for instance, sciiavs expressing the surface protein a (pspa) of streptococcus pneumoniae, the hemagglutinin-neuraminidase (hn) protein of hpiv-3 or the fusion (f) protein of respiratory syncytial virus (rsv) [192] [193] [194] , were able to induced abs against the foreign polypeptides and reduced the viral load of the heterologous pathogen while retaining their ability to protect against challenge with iav. it is worth indicating that, for vaccine purposes, it is important to consider what influenza viral gene is replaced, since it was shown that sciiav replication is required for protection [69, 185, 189] . although substitution of the polymerase (pb2, pb1 and pa) segments may allow insertion of larger foreign genes, removing the viral polymerase from sciiavs limits their ability to express more polymerase during the single-cycle round of infection, decreasing total viral antigens and, thus, limiting their protective efficacy. thus, sciiavs where the viral ha or na has been removed were able to confer, upon a single immunization, protection against a lethal challenge with influenza. [190] . on the contrary, a single dose of a sciiav where the viral pb2 has been removed was only as efficacious as an inactivated virus [189] . it is worth noting that sciiavs combine the advantages (better immunogenic properties of the laiv and the safety profile of the iiv) and circumvent the disadvantages (safety of the laiv and poor immunogenicity of the iiv) of current influenza vaccine approaches [144] . while sciiavs have been shown to be safe, immunogenic and protective against lethal challenges with wild-type forms of iavs in animal studies, no human trials have yet been performed. more recently, the rearrangement of the influenza virus genome has been shown to have great potential for the development of improved laivs against influenza virus, as well as vaccine vectors against other pathogens [195, 196] . avian influenza virus subtypes h5n1 and h9n2 have pandemic potential [197, 198] . however, h5n1 iiv induces limited adaptive immune responses, and in the case of laiv, there are safety concerns about the possibility of reassortment between the viral segments in the laiv and circulating h5n1 strains. to overcome these concerns, a bivalent laiv against influenza a/vietnam/120320/04 h5n1 and a/guinea fowl/hong kong/wf10/1999 h9n2 was generated using viral genome rearrangement [195] . to that end, the nep was removed from the ns viral segment of the h9n2 virus and substituted by the ha of the h5n1 virus. h9n2 ns1 and h5n1 ha were separated by the foot-and-mouth disease virus (fmdv) 2a autocleavage site to allow co-linear expression of both viral proteins. then, nep was cloned down-strain of the h9n2 pb1 segment separated by another fmdv 2a autocleavage site. the rearranged h9n2 virus expressing the h5n1 ha was able to provide complete protection against challenge with a/vietnam/1203/2004 h5n1 and also against a potential pandemic h9:ph1n1 iav reassortant virus in both mice and ferrets [195] . segments 7 (m) and 8 (ns) of iavs use an alternative splicing mechanism to express two different viral proteins from the same viral segment. recently, we have generated recombinant influenza a/puerto rico/8/34 h1n1 viruses containing modified m and/or ns segments, in which the overlapping orfs of the m1 and m2 viral proteins (m segment) and/or the ns1 and nep proteins (ns segment) were separated with the porcine teschovirus 1 (ptv-1) 2a autocleavage site [196] . recombinant viruses with a rearranged m segment were affected or impaired in replication in vitro at nonpermissive temperatures (37 and 39 • c, respectively), whereas high viral titers were obtained at permissive low temperatures (33 • c) [196] . notably, viruses containing the m split segment were highly attenuated in vivo, but able to confer, upon a single immunization dose, complete protection against a lethal homologous challenge with wild-type pr8 [196] . importantly, viruses with a reorganized m segment were able to confer better protection than a temperature-sensitive, laiv pr8 virus [103, 104, 115, 196] . these studies demonstrate that the rearrangement of the influenza viral genome can be used for the generation of safe, immunogenic and protective laivs. current influenza vaccines induce immunity to the influenza virus strain-specific ha antigen and are not very effective against new pandemic viruses, given that ha is highly susceptible to frequent changes by antigenic drift and shift [12, [44] [45] [46] [74] [75] [76] . to overcome these drawbacks, different approaches aimed to develop a universal influenza vaccine able to induce cross-protective broadly neutralizing immunity against conserved viral antigens, such as the ectodomain of m2 (m2e) [199] , the ha stalk domain [200] or na [201] , have been explored. the m2e antigen is a linear peptide that is very well conserved across iav strains. although the mechanism of m2e-specific immunity is unclear, protective anti-m2 antibodies have been observed in multiple animal models, including mice, ferrets and primates [36] . however, these conserved antigenic targets need to be presented in a carrier system or conjugated to adjuvant molecules. promising results have been obtained with virus-like particles (vlps), which are morphologically similar to the virus and present surface proteins in a highly immunogenic form. because vlps do not contain the viral genome, they are considered safer than viral vaccines, yet still induce strong humoral and cellular immune responses [202] . vlps are most commonly made by expression of ha, na and m1 [203, 204] , although ha and na alone may be sufficient for vlp production [205] . reverse genetics approaches to generate recombinant viruses, including influenza, have been described for representative family members of negative-sense, single-stranded rna viruses. these plasmid-based reverse genetics methods have provided scientists with a unique opportunity to study different aspects of the biology and pathogenesis of these viruses, both in vitro and in vivo, as well as to generate attenuated forms to be used as vaccines [135] . in this review, we discussed the use of reverse genetics for the generation of influenza vaccines with a special focus on laivs. in general, laivs are highly immunogenic, and immunization usually induces faster and substantially higher levels of both systemic and local mucosal antibody and t-cell responses, providing better protection than their inactivated counterparts. prevention of influenza virus infection requires seasonal vaccinations, and identification of the correct virus subtype to include in the vaccine leaves little time for vaccine development, scale up and distribution. although influenza vaccines work well most of the years, if a new viral variant emerges after the strain to be included in the vaccine has been selected, the efficacy of the vaccine would be sub-optimal for this new variant strain. this usually results in decreased efficacy of the vaccine. reverse genetics approaches have also allowed the development of live-attenuated viruses that could be implemented, in the near future, as laivs. these new advances in reverse genetics approaches are reducing the potential time of recovery and production from months to weeks and represent an excellent alternative for the rapid development and implementation of laivs for the treatment of both seasonal and potentially pandemic influenza strains. acknowledgments: influenza virus research in the luis martínez-sobrido and aitor nogales laboratory was partially funded by the national institute of allergy and infectious diseases (niaid) centers of excellence for influenza research and surveillance (ceirs hhsn266200700008c). the authors declare no conflict of interest. the viruses and their replication transmission of influenza virus in a mammalian host is increased by pb2 amino acids 627k or 627e/701n the origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza a viruses evolution of 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authors: elderfield, ruth; barclay, wendy title: influenza pandemics date: 2011-09-22 journal: hot topics in infection and immunity in children viii doi: 10.1007/978-1-4614-0204-6_8 sha: doc_id: 7784 cord_uid: fq2urilg the recent h1n1 pandemic that emerged in 2009 has illustrated how swiftly a new influenza virus can circulate the globe. here we explain the origins of the 2009 pandemic virus, and other twentieth century pandemics. we also consider the impact of the 2009 pandemic in the human population and the use of vaccines and antiviral drugs. thankfully this outbreak was much less severe than that associated with spanish flu in 1918. we describe the viral factors that affect virulence of influenza and speculate on the future course of this virus in humans and animals. influenza is a seasonal respiratory illness associated with more serious consequence and even death in the very young, old and immunocompromised. annual epidemics are predictable and affect a relatively small percentage of the global population at any one time. pandemics differ from epidemics in that they are a global phenomenon, affecting large numbers of people in multiple countries simultaneously. pandemics tend to arise swiftly often out of the normal season, and affect a wider age group and spectrum of individuals than seasonal influenza. the first recorded influenza pandemic was in 1510 [1] . since then human populations have been subjected to at least 15 pandemics, most notably in 1918 when estimates of the human deaths that resulted vary between 40 and 100 million [1, 2] . the influenza virus, the etiologic agent, is a member of the family orthomyxoviridae. there are three categories of influenza known as types a, b and c. only type a causes pandemics and thus is the most widely studied. influenza b viruses cause typical seasonal infections restricted to humans. influenza c viruses also infect humans and have been isolated from clusters of children, but are often not recognised and may be dismissed as an untyped influenza like illness, due to the lack of diagnostic tests [3] . the virus particle is enveloped, whereby the genome is protected by a lipid bilayer derived from the host cell membrane. the appropriated membrane is studded with viral glycoproteins: the attachment spike protein haemagglutinin (ha) which binds to sialic acid (sa) receptors on the cell surface; the integral m2 protein, an ion channel involved in the uncoating of the virus inside the infected cell, and the neuraminidase protein (na) which cleaves the cell membrane sas that would otherwise tether the budding nascent virus particle to the infected cell. lining the inside of the virion membrane is the abundant matrix protein (m1), surrounding the eight genomic negative-sense rna segments which are intertwined with nucleoprotein (np) and each associated with one set of the three viral polymerase subunit proteins (pb1, pb2 and pa) (fig. 1 ). the viral ribonucleoproteins (vrnps) are the replicative units that are transported into the nucleus of the host cell after virus entry. there, the virus co-opts a number of host factors to assist the polymerase in transcribing viral mrna, and replicating new genomes via crna intermediates. finally the newly synthesized viral proteins and genomes are transported to the host cell's external membrane where the progeny virions assemble and bud. the 1918 pandemic dealt a devastating impact on a global population. several features of the era, in addition to the extraordinary virulence of the virus itself that will be discussed below, contributed to the impact of the 1918 pandemic. at that time, although influenza was known as an infectious disease in terms of symptoms, the virus itself had not yet been identified. indeed for a good proportion of the pandemic, hemophilius influenzae (known as bacillus influenzae at the time) was suggested as the causative agent. the influenza virus was eventually isolated from pigs by richard shope in 1931 [4, 5] , then from humans by andrewes, laidlaw and wilson smith in 1933 [6, 7] . at the start of the influenza pandemic in 1918, the world was at war. the situation in america is absorbingly described in john m. barry's "the great influenza" (penguin, 2004) complied from military records, personal papers, oral history and newspapers of the period. there is some epidemiological and historical evidence that the outbreak may have begun in army camps either in the usa or in europe where large numbers of young susceptible hosts were living in very crowded conditions. there is also evidence of at least two waves of disease, and indications that the second wave was more virulent than the first. for example, by the second wave historical accounts by the medical and scientific staff at the time describe symptoms such as: cyanosis which started as mahogany spots over the cheek bones, and could expand until the patient turned black, caused by the lack of oxygen transfer in the lungs, leading to blue unoxygenated blood; extreme chills and fever, severe joint pains, vomiting and abdominal pains; earaches, headaches often localised around the eyes; and disturbing blood loss from nose, stomach, intestine and eyes. at post mortem the lungs were often filled with the debris of destroyed cells and blood, which today would be diagnosed as acute respiratory distress syndrome (ards). in philadelphia, where the virus had been introduced from the local port, health workers and scientists were requesting widespread restrictions on gatherings and provision of information to the media. unheeding, the governor and the senior health official sanctioned a large city wide liberty loan parade in order to gather funds for the war effort. within 72 h of the parade every bed in the city's 31 hospitals was filled. the daily death rates for the city rose at an alarming rate, 3 days after the parade 117 died in one day, on day 11 more than 400 people died. the second and third waves of the 1918 pandemic resulted in a cumulative case fatality rate (expressed as a ratio of the number of people infected to the number of people who died) of > 2.5%. later pandemics of the twentieth and twenty-first century only reached case fatality rates of less than 0.1% [8] . remarkably, although it has not been possible to isolate infectious virus directly from stored samples of that era, we do today have access to the causative agent of this pandemic following the elegant application of modern science. in 2005, jeffrey taubenberger and colleagues used the polymerase chain reaction to amplify small fragments of viral rna isolated from formalin-treated post-mortem pathological slides and also from frozen lung tissue obtained from a person who died in alaska and was buried in the permafrost, from this material the nucleotide sequence of the 1918 virus was deduced [9, 10] . taubenberger joined forces with terence tumpey and colleagues at the cdc. they used the 1918 virus sequence information to generate plasmids containing the viral cdna which when transfected into suitable mammalian cells, allowed the recovery of infectious 1918 virus [9, [11] [12] [13] [14] [15] [16] . the reconstituted virus was more virulent in animal models than any other influenza virus strains studied previously [11, [16] [17] [18] [19] [20] [21] [22] [23] . thus although there is strong evidence that secondary bacterial infection contributed significantly to deaths from 1918 virus in humans [24, 25] , the virus itself, in the absence of bacteria, is remarkably pathogenic to animals. studies have been carried out to map the genetic determinants of this virus in the hope that this will help us to predict the virulence of future influenza strains as they emerge. the polymerase genes and the virus ha gene have been implicated in the extreme virulence of this virus, but work continues to understand the mechanisms by which this particular influenza strain is so deadly [26] [27] [28] [29] . phylogenetic studies suggest that the genome of the 1918 virus is most similar to viruses found in birds. however there are a number of key amino acid changes that indicate that, although it originated in an avian host, the virus underwent adaptation in order to replicate and transmit within human and swine hosts [8] . interestingly the 1918 virus exhibits low pathogenicity in experimentally infected swine [20] . the 1918 pandemic virus has been called the 'mother of all pandemic viruses' [8] as all of the twentieth century pandemics are derived from virus lineages descended from the 1918 virus. after 1918, viruses derived from that outbreak continued to circulate in humans causing annual epidemics of moderate or mild severity [30] . however, because of their segmented genomes, influenza viruses are particularly prone to a special form of recombination known as reassortment that occurs if one host is coinfected by two different viruses. such mixing events allow the introduction of genetic material from viruses that usually circulate in birds with the human adapted viruses, and new viruses thereby created may be able to cause a novel outbreak. we know that the two major pandemics in the second half of the twentieth century were formed in this way: the 'asian' pandemic of 1957 was caused by the emergence of an h2n2 sub-type virus that retained the m, np, pb2, pa and the nonetheless, perhaps because antibiotics were available by that time or perhaps the h2 virus itself had a milder phenotype, the death toll of this pandemic was much lower at only ~two million people. the 1968 h3n2 'hong kong' pandemic that followed just 11 years later resulted in between one and two million deaths. this virus was a recombinant between the circulating human h2n2 virus and an avian strain with h3 ha. the reassortant virus still retained five segments originally derived from the 1918 strain, but acquired the ha and pb1 from the avian virus. each of the h2 and h3 pandemic events were so universal that the virus displaced the previously circulating strains [31, 32] . in 1977, the h1n1 strain re-emerged as a circulating human strain (not to be confused with the 1976 swine h1n1 outbreak at fort dix). the colourfully named 'red' or 'russian' flu appeared initially in china in may of 1977 with isolates found in russia soon after [33] . this strain produced a relatively mild disease mainly in young children. however, subsequent genetic analysis indicated a 27 year gap in the evolutionary history of this virus. in fact it was genetically similar to virus isolated in 1950. the eventual conclusion drawn was that this virus had been deep frozen in a laboratory and its release was accidental. the h3n2 and h1n1 subtypes have continued to co-circulate and to cause human seasonal influenza outbreaks into the twenty-first century (fig. 2 ) [32] [33] [34] [35] [36] . the influenza a virus naturally circulates in aquatic birds, where it replicates in the gut [37] [38] [39] . all of the 16 ha and 9 na subtypes have been isolated from either or both of the anseriformes (an order which includes ducks and geese) and from the charadriiformes (the order to which shore birds and gulls belong). no other species has been infected by all the influenza types. different subtypes predominate in the different orders of birds; h3 and h6 for example, are found mainly in anseriformes, whereas in charadriiformes h4, h9, h11 and h13 are the predominant ha subtype. the virus is excreted in high titres into the water bodies that are home to domestic and migratory birds [40, 41] . the virus can then be picked up by migratory birds and spread along the migratory routes. however, the relative geographic isolation of some flocks has been proposed as one cause of genetic divergence within the ha and na subtypes [42] . whilst the disease caused by influenza is mainly asymptomatic in aquatic birds, some isolates are capable of developing from low pathogenic avian influenza (lpai) into a highly pathogenic influenza (hpai) capable of killing domestic poultry. the two subtypes that are prone to change pathogenicity are h5 and h7, as seen in eurasia with the hpai h5n1 viruses and the netherlands with the h7n7 virus. economically, the now widely-distributed h5n1 virus has been responsible for the death of over a billion head of poultry either directly through the disease or indirectly through preventative culling measures [39] other subtypes h9, h1, h3, h4 and h14, whilst still appearing mild in aquatic birds, can be fatal in domesticated flocks [39] . only viruses of the h1, h2 or h3 subtypes are known to have circulated among humans or pigs. the drastic recombination events that result in novel pandemic viruses described above are called antigenic shift. after the introduction of the new subtype and its wide circulation in humans, the increasing prevalence of specific immunity among human hosts exerts selection pressure that drives evolutionary change in the ha protein via the accumulation of point mutations that block the antibody recognition through conformational changes or glycosylation events on the antigenic epitopes. this process is called antigenic drift. as the virus continues to circulate in humans in the interpandemic periods, mutations accumulate that confer antigenic drift as well as other adaptive mutations that alter the nature of the virus and may be associated with loss of virulence. this, along with increased wide vaccination campaigns for the elderly and immuno-compromised populations, could explain the gradual decrease in influenza-like illness since the hong kong pandemic of 1968, particularly in recent years ( fig. 3 ) [43, 44] . the ability of influenza a viruses to recombine so readily has worried virologists and public health planners alike, because of the risk that a seasonal strain of influenza with human adapted components might recombine with one of the highly pathogenic avian influenza (hpai) viruses such as the notorious h5 or h7 subtypes. these two subtypes are lethal in poultry because of an extended tropism conferred by mutation in the ha gene that allows them to infect and propagate in many organs and tissues, rather than being restricted to areas where the appropriate host cell proteases exist. consequently infection with these viruses carries a high mortality in humans of more than 60%. h5n1 virus has been responsible for over 500 cases of human infection and 300 deaths (as of 31st august 2010 according to the world health organisation avian influenza surveillance system) but thankfully has not yet reassorted with a human-adapted influenza virus, nor given rise to a pandemic outbreak [45] . because the research community was so focused on surveillance of and strategies to control h5 and h7 infections, the outbreak of the 2009 'swine' influenza was a surprise. we had largely overlooked the idea that the next pandemic would originate in pigs even though an outbreak in fort dix, new jersey in 1976 associated with the death of a soldier from infection with an h1n1 swine flu had led to mass vaccination campaigns at that time. the fort dix incident did not give rise to a pandemic, the virus remained contained within the military and transmission of the virus had fizzled out by the time the now-infamous vaccination campaign began [47, 48] . most text books propose that swine are the mixing vessel in which influenza viruses of avian and human origin reassort. it has been evident that pigs can be infected with influenza since the early days of virus isolation. indeed, shope initially isolated influenza from a pig [4, 5] and kida et al. showed that pigs could be infected by many different subtypes of avian influenza [49] . the 2009 pandemic virus illustrates just how good a mixing pot the swine host can be. the origins of its eight gene segments come from at least four different sources and three different hosts. the pb2 and pa segments appear to have originated in an avian reservoir and transmitted to the swine host around 1998. the pb1 segment derives from a human virus but was transferred to swine in 1998. the ha, np and ns segments once again can trace their lineage back to the 1918 pandemic influenza, when the virus infected pigs and subsequently circulated through the years to become a classical swine virus. the na and m segments are from circulating swine viruses, of the eurasian lineage, believed to have transferred from birds in 1979 ( fig. 4) [50, 51] . why do swine make such good mixing vessels? ito et al. showed in 1998 that the pig respiratory tract displayed sa receptors that are bound by viruses isolated from birds as well as those used by human-adapted viruses, implying that the pig was capable of being infected by an avian and a human-adapted virus at the same time [52] . in addition, the co-expression of the avian-like and human-like receptors in swine potentially allows for the selection of avian viruses with small mutations that adapt them to bind to and replicate in mammalian cells, a process known as 'receptor switching.' for avian influenza viruses to adapt to and transmit between humans, it is now apparent that in addition to the reassortment events that occur during antigenic shift, their ha proteins must also undergo modifications that alter their fine receptor binding specificity. the influenza ha protein binds to sa residues on the host cell surface as a prelude to cell entry. in the avian gut, these are predominantly a-2,3 linked receptors but in the human upper respiratory tract a-2,6 linked receptors predominate. avian influenza viruses would therefore preferentially bind a-2,3 linked receptors and human-adapted influenza viruses have changed key residues at the receptor binding site allowing greater affinity for a-2,6 linked receptors (fig. 5 ) [42] . in h3 ha proteins, receptor switching occurs if there is a change from glutamine (q) at position 226 to leucine (l) (h3 numbering) and is enhanced by glycine (g) at 228 to serine (s) [53, 54] . for other subtypes the changes required for human adaptation are not exactly the same. some h2 viruses still bind to a-2,6 human-like receptors even when the q226 is present, a trait shared by avian h6 and h9 proteins. the h1 subtype tends to show changes at residues 225 (aspartate [d] to g) and 190 (d to glutamic acid [e]) rather than 226 and 228 but they achieve the same end [9, 10, [53] [54] [55] . fortunately, in the case of h5 ha, none of the changes found in other subtypes have completely mediated a receptor binding switch, suggesting that the barrier to human adaptation may be particularly high for this subtype [56, 57] . one important difficulty in understanding these adaptive events is that the nature of the influenza virus receptor is not completely clear, but it is certainly more complex than a single sugar moiety [58] . the nature of carbohydrate to which influenza virus might attach has been recently studied using glycan arrays. glycan arrays present hundreds of different carbohydrates. different viruses or expressed ha proteins are then given the opportunity to bind to a favourite residue [59] [60] [61] [62] [63] . this type of experimental procedure was used recently to elucidate the receptor binding preferences of the novel pandemic h1n1 2009 virus. interestingly, this virus along with two other swine viruses tested, was able to bind both a-2,6 and a-2,3 sa, whereas seasonal h1n1 influenza virus had a strong preference to bind carbohydrates with a-2,6 linkages and showed no binding to those with a-2,3 [63] there multiple recombination events of classical swine influenza (black) resulted in a triple reassortant (trig), when recombined with eurasian swine influenza (grey) generated the ph1n1 virus [51] 6 the emergence of swine origin influenza (ph1n1) the swine origin 2009 pandemic influenza virus appears to have emerged from san luis potosi, mexico in late february 2009 [31] , though it has been suggested that the virus was circulating at low levels in humans for some months prior to this. indeed the most common ancestor may have emerged between august 2008 and january 2009 [31, 51, 64] . the pandemic threat of the new virus was realised as the first wave peaked in mexico in late april 2009. the virus quickly spread across the globe. who moved to pandemic phase 4 after confirming human to human transmission on the 27th april, and just two days later, phase 5 was declared as the outbreak was found in two or more countries within one who region. finally passage of the virus into a second who region triggered escalation to phase 6 on the 11th june. the h1n1 virus responsible for the 2009 pandemic is not the same h1n1 virus that had been circulating in humans in recent years causing seasonal h1n1 outbreaks. both viruses have ha proteins originating from the 1918 pandemic virus. however the ha of the seasonal h1 had been under antigenic and other selective pressure as it circulated in the human population over a total of seven decades. on the other hand, since pigs are short lived, they exert little antigenic pressure to drive evolution of ha because the likelihood that a pig will be re-infected by the same influenza virus during its brief life time is very low. the rather genetically static 1918-derived pig virus became known as 'classical' swine influenza virus and it was the predominant influenza virus of swine on the north american continent throughout the twentieth century. at the time this classical swine h1 ha recombined into what was to become the 2009 pandemic virus, it still retained 90% amino acid sequence identity to its 1918 progenitor. however, the human seasonal virus had changed so dramatically that it shared only 79% amino acids with the 1918 ha protein, and this did not allow for any antigenic cross protection for humans who had been infected with seasonal h1 in recent years against the novel 2009 pandemic strain. in the uk, the first reported case was on the 26th april 2009, brought back by those returning from holidays in mexico. by may 29th there were 215 uk cases, of whom 52 were returning travellers, 39 were direct contacts of those travellers and 108 were people who had links to the secondary cases. already at this early stage there were eight sporadic cases which were not linked to travel. on 21st june, the influenza-like-illness (ili) incidence baseline was crossed and the first wave in the uk was clearly underway. this wave peaked in july and then fell well below baseline by mid-august, after schools closed for the summer holidays. nevertheless 17% of the deaths in the uk occurred during this wave. the second wave was far shallower, but endured for a longer period from september 2009 until february 2010, and was responsible for the remaining 83% of fatal cases [65, 66] . the height of the peak of ili cases in the second wave was much lower than had been predicted. several factors contribute to the explanation for this: a proportion of the population (>30%) most vulnerable to seasonal influenza infection, the elderly, were already immune. in blood samples of those over 80 years old, collected in 2008 before the 2009 pandemic virus emerged, it was possible to detect antibodies that cross react with 2009 pandemic virus' ha in >30% of the samples (haemagglutination titre 1/32). in those aged 65-79 years, the seropositive frequency drops to ~20%, whereas in the 4-14 year old age bracket, the proportion with significant hai was just ~4% [67] . these neutralising antibodies in sera collected from the elderly exist because many people in older age groups were infected in early life by closer derivatives of the 1918 h1n1 virus. the second factor was the surprisingly mild nature of the 2009 pandemic virus in most people. serology conducted retrospectively detected neutralising antibodies in a far larger percentage of the population than could be expected from the reports of ili. one in three of the children in london and birmingham were seropositive for the virus by september 2009 [67] suggesting that many people who had the infection did not report it. this could be because the symptoms were sufficiently mild that they did not feel they needed the flu service that was on offer at the time, or they did not even realise they had the infection. in this respect, the population was fortunate; this virus spread effectively but did not produce overtly pathogenic effects in most people. the case fatality rate was 0.02%, far lower than the 2.5% of the 1918 pandemic or the 0.1% of the 1957 and 1968 pandemics. despite that, there were over 1,500 hospitalizations due to influenza like illnesses in the uk and 474 deaths [68] . globally mortality far exceeds 18,000 deaths [69]. in the event of a pandemic, such as in 2009, difficult decisions need to be made by health authorities to prioritise limited supplies of drugs and vaccines. pharmaceutical companies may generate millions of doses of vaccine (300 million for the 2009 pandemic) [70] to protect the population, but this is still only enough to immunize a small proportion of the globe, so provision of vaccine to those most vulnerable to this virus needed to be prioritized. usually vaccine is given primarily to those most susceptible to poor outcome from infection by seasonal influenza, namely the elderly. in the uk, from 2001 to 2009, 69% of those who died from seasonal influenza were >65 years old. however, with the 2009 pandemic virus only 15% of deaths were in the > 65 demographic. true to form, those elderly individuals who did succumb to the 2009 pandemic influenza suffered a severe illness, with a case fatality rate of 0.9%, indicating the virus was able to cause serious morbidity in those who were immunologically susceptible [65] . the first infection with an influenza virus in children can often be quite severe. johnson et al. showed that the high incidence of unexpected paediatric fatalities from the fujian h3n2 seasonal drift variant in 2003 was linked with a higher than usual infection rate for seasonal influenza in the young in that year, possibly explained by a more drastic antigenic drift than in immediately previous years [71] . similarly when a novel pandemic virus circulates widely, the incidence rate in the very young is particularly high and their clinical course in the face of lack of any relevant immunological experience is often severe. indeed during the 2009 pandemic, the highest mortality rates were observed in those under 1 year of age [72] . in addition since school age children are major transmitters of influenza, there is good logic in targeting them in pandemic and seasonal immunization campaigns because overall community incidence may be curtailed in this way [73] [74] [75] [76] . the 2009 pandemic virus was certainly able to infect and transmit well within the paediatric cohort. in the uk one in three children and in hong kong half of the children had been infected after the first wave [77, 78] , supporting the global observations that school aged children and young adults were most likely to contract influenza [79] . generally seasonal influenza causes two paediatric deaths per million people, while ph1n1, by mid-2010, had been responsible for 5-6 deaths per million in the netherlands and the uk respectively and 11 per million in argentina in the paediatric cohort [72, [80] [81] [82] . in their 2010 study of paediatric mortality from pandemic influenza in the uk, sachedina and donaldson identified 70 deaths in the 0-18 year old age group directly attributable to infection with the virus. as in similar studies across the world, they described common symptoms including fever, cough and shortness of breath [72, 79, [83] [84] [85] [86] . the uk study observed that a combination of neurological, gastrointestinal and respiratory disease was present in more than half the deaths, an observation again echoed in other countries. indeed pre-existing neurological disorders have frequently been listed as a co-morbidity [65, 66, [83] [84] [85] [87] [88] [89] . half of those of school age who died in the uk attended schools for those with special needs [72] . as with adults, bacterial coinfections were often observed; in the us 43% of paediatric deaths were associated with secondary bacterial infections and in the uk 20% were associated with laboratory confirmed cases [72, 73] . pregnant women appear to be especially at risk from complications of influenza of either a seasonal or pandemic nature, especially during the second and third trimesters [90] . this increased susceptibility has been ascribed to mechanical changes within the body, which act to increase the pressure on the cardiovascular system, including an increased heart rate, stroke volume and oxygen consumption set against a decreased lung capacity [91, 92] . this may also account for increased risk in obesity. in addition, hormonal changes in pregnancy cause what might be broadly termed a swing away from cell mediated immunity and a bias towards the humoral system that may affect the ability to clear the virus [92, 93] . on the other hand humoral immunity is also not complete in pregnancy and depletion in the levels of igg2 were observed in pregnant women who died in the 2009 ph1n1 pandemic [94] . there is evidence from previous pandemics that the mortality rate was high amongst pregnant women: in the 1918 pandemic between 27% and 47% of those pregnant who contracted influenza died. in 1957 the percentage of deaths was lower, but still considerable at 20% [92] . there is also evidence of complications; of those pregnant women who developed pneumonia but survived during the 1918 pandemic, more than 50% did not carry the foetus to full term [92] . in the 2009 pandemic it was very evident that other co-morbidities increased susceptibility to severe influenza infection. these included obesity, asthma and chronic obstructive pulmonary disease (copd), diabetes, immunosuppression, heart conditions and neurological complications. whilst only a third of those who were admitted to intensive care had co-morbidities, over three quarters of those who died did [73] . in the uk, the flucin database collected data from all those admitted to hospital with influenza during the pandemic first wave. co-morbidities were described for around 50% cases, leaving a significant number of young healthy adults that suffered severe disease and even death despite no obvious prior predisposition for bad outcome [95] . in the us, a study of the first 100 fatal cases caused by ph1n1 found that 25% of them had bacterial coinfections, with staphylococcus aureus and streptococcus pneumoniae being the most common pathogens [96] . other fatal case studies have put the incidence of bacterial coinfections between 28% and 36% and also included streptococcus pyogenes in the list of common bacterial coinfections [89, 97, 98] . in argentina, coinfection with streptococci increased the likelihood of severe outcome with an odds ratio of 17 [99] . however in the uk, high incidence of bacterial coinfection was less evident [95] . the effect of bacterial superinfection on the outcome of infection with pandemic h1n1 2009 is likely to have been affected by differences in the bacterial strains circulating in communities around the world at the time and this may, in part, account for the widely different case fatality rates seen in different areas. there is a well-established system in place for the generation of seasonal influenza vaccines; the dominant circulating strains are carefully monitored and predictions are made annually about which viruses are likely to predominate in the forth-coming 'flu season. the chosen seasonal viruses are recombined with the internal segments of the high growth a/puerto rico/08/34 (pr8) vaccine backbone strain, to create viruses with ha and na antigens from seasonal strains that can be readily amplified in eggs. similarly for the 2009 pandemic vaccine, a reassortant virus bearing the h1 ha and n1 na genes of the pandemic strain a/california/07/2009 on a high growth body was used to generate high yield virus in eggs. however, vaccine production problems became apparent early during production phase when manufacturers realized that the growth of the cal/07/09 reassortant was only 30-50% of that seen for the seasonal strains. eventually a higher growth variant was obtained. in all about 30 versions of h1n1/2009 pandemic vaccine were generated in multiple countries by different manufacturers, with either wild type or reassortant viruses grown traditionally in eggs or in cell based systems and vaccines produced as spilt (just the ha and na genes) preparations, whole inactivated virion preparations or, in one case, a live attenuated virus. using a strategy based on development of h5n1 pandemic vaccines, the most widely used pandemic vaccine in the uk was an inactivated vaccine generated by gsk that was administered combined with as03 adjuvant (composed of a-tocopherol, squalene and polysorbate 80 emulsion) a chemical mix added to enhance and prolong the immunogenic response and reduce the amount of ha protein required per dose to achieve immunity (antigen sparing). the immunogenicity of the pandemic vaccine was in fact much higher than expected based on experience of clinical trials with the h5n1 equivalent. in the end, a single dose of adjuvanted vaccine was sufficient to achieve seroconversion in adults. although it was expected that two doses would be needed in children, who usually require a prime boost regimen for effective levels of antibody to be achieved, a single dose was eventually used as it turned out to be adequately immunogenic and significantly less reactogenic after the first dose than the second [100] . this vaccine was recorded as having a 72% effectiveness despite the relatively small doses of 3.75 mg of ha protein (unadjuvanted vaccines typically contain 15-30 mg). other adjuvants were also trialled globally including alum (aluminium hydroxide) in china and russia and another oil-in-water adjuvant broadly similar to aso3 called mf59 in korea and italy. clinical trials were run using the proprietary sanofi-pasteur af03 adjuvant in the usa, europe and asia [70] . live attenuated vaccine (laiv) for pandemic 2009 was widely administered in the us. other vaccine strategies that were not yet licensed have been researched using the 2009 h1n1 virus as antigen. these include: the use of virosomes (lipid vesicles) that have the ha and na proteins scattered through the bilayer, live recombinant adenovirus vaccines that express the ha protein, virus like particles in which the ha, na and m are expressed in insect cells which are infected with a recombinant baculovirus, these are purified and self assembled into immunogenic particles, finally plants infected with a transformed agrobacterum vector that generate ha proteins [70] . although the outcome of infection in most people infected with pandemic 2009 virus was mild, in animal models this virus causes more severe disease than recent seasonal h1n1 viruses. itoh et al., compared seasonal h1n1 (a/kawasaki/utk-4/09) and pandemic h1n1 (a/california/04/2009) viruses in a number of animal models. interestingly, infected mini-pigs remained relatively asymptomatic. in contrast, ph1n1 virus caused severe lung pathology in mice, ferrets and macaques including lung lesions and damage caused by the infiltration of inflammatory mediators to a greater extent than was observed with infection with the seasonal viruses [17, 101, 102] . van de brand et al., infected ferrets intratracheally with very high doses of seasonal h1n1, ph1n1 or hpai h5n1 virus and found that infection with ph1n1 caused pathology intermediate between seasonal influenza and h5n1 and could lead to severe pneumonia and death in this model [17] . infection of alveolar pneumocytes, not observed with the seasonal virus [17, 102] may correlate with the more profound binding of the ph1n1 ha to a-2,3 linked sa [63] which tends to be located deeper into the lung [103] . the difference between the animal models and the epidemiology in humans suggests that a basal level of existing immunity in the human population has protected against the moderately severe disease this virus can cause in immunologically naive experimental animals. there are two antiviral drug classes currently available to treat influenza. adamantanes (amantadine and rimantadine) are directed against the ion channel m2 protein and prevent the uncoating of the virus genome early in infection. the second class of drugs, the neuraminidase inhibitors (oseltamivir -tamiflu â® and zanamivir-relenza â® ) were rationally designed to block the active site of the neuraminidase of the influenza virus. na acts to cleave the sa receptors on the surface of the host cell, allowing the release of newly formed virions which can then infect uninfected cells. unfortunately, influenza viruses readily developed resistance to the adamantanes through point mutations at residues 26, 27, 30, 31 or 34 in the m2 protein, with no compromise in viral fitness [104, 105] . 90% of the seasonal h3n2 isolated in the us and asia contain a resistant phenotype and the s31n mutation was already present in the 2009 ph1n1 at the time it crossed into humans [106, 107] . the nai drug class has therefore become a favourite for stockpiling anti-influenza therapies. the first of these drugs to reach the clinic was zanamivir (relenza) but use of this drug is hindered by the necessity to inhale it because it is not orally bioavailable. the second nai oseltamivir (tamiflu â® ) benefits from a convenient oral formulation, good bioavailability and is suitable for use in paediatric and adult populations. data have not yet emerged fully for its effects in those > 65 years of age or the immunocompromised [108] . according to the cochrane review of efficacy in 2005, if zanamivir or oseltamivir are used to treat an infection 48 h after onset of symptoms, there is a 'modest' reduction in influenza symptoms within 0.78 days in adults and 1 day in children for zanamivir and 0.86 days in adults and 0.87 days in children for oseltamivir [108, 109] . as prophylactics, both drugs fare well, in control groups taking the medications during seasonal influenza, there was a reduction in incidence of 69% and 74% for zanamivir and oseltamivir respectively. for post-exposure prophylaxis there were 81% and 90% relative reductions in infection after zanamivir and oseltamivir administration respectively [108, 109] . resistance to oseltamivir can emerge, but early experiments indicated that mutations such as h275y (n1 numbering, h274y in n2), would confer a fitness cost to the virus by reducing na affinity for the sa substrate [110, 111] . thus, it was assumed that the resistance mutation would not persist in the community. however, by 2008, ~99% of seasonal h1n1 viruses had acquired the h275y mutation that was associated with the resistant phenotype without fitness cost [108, 112] . the lack of fitness cost can be ascribed to compensating mutations in the na protein [113, 114] . the obvious fear was that the ph1n1 virus would develop oseltamivir resistance given the widespread use of the drug in the early waves of the pandemic. ph1n1 viruses with the h275y mutation have been isolated, however these were predominantly in those undergoing prolonged treatment regimens (often in immunocompromised patients) or through the use of low dose prophylaxis [115, 116] . at the time of writing, there had not been widespread transmission spread of the resistant strain. indeed the fitness cost to this strain of virus is still under debate, with different labs publishing conflicting results [117] [118] [119] . there is historical evidence that implies that during the 1918 pandemic, the second and third waves were more severe than the first. it has been suggested that the virus acquired mutations as it circulated in its new human host and these 'hotted up' its virulence. thus it is important to identify any mutations that may similarly increase virulence of the 2009 ph1n1 strain. in particular it was possible that critically ill or deceased patients had been infected with a virus variant that had more pathogenic potential than the viruses that predominated in the community. the sequence of viruses from such cases has been analysed in a number of studies [120] [121] [122] [123] [124] [125] . one of the interesting mutations observed is a d225g (h3 numbering, d222g in h1) mutation in the ha protein. 18% of critical cases in norway and 12.5% of the critically ill in hong kong had this mutation [123] . in scotland this mutation was only found in patients who were critically ill (4.1%) [120] . however it is not clear that this mutation alone is responsible for poor outcome: the d225g mutation was found in the virus from a nasopharyngeal swab and tracheal aspirate from a 25 year old man admitted to intensive care with pneumonia and ards. however the same virus transmitted to a contact case, but did not lead to severe illness despite the latter individual having two hallmark co-morbidities, namely obesity and diabetes [121] . although d225g was detected predominately in viruses from critical cases in greece, it was also isolated in two mild cases of the disease [122] . it has been suggested that some of the reported isolates with this change are the results of egg adaptation during the culture period [126] . in addition, the prevalence of this mutation in the critically ill has been ascribed to factors such as sampling bias, the critically ill being more likely to be genotyped than the mild cases. the presence of this mutation enhances binding of h1 ha to a2,3 linked sa receptors [124] . the proposed mechanism by which such a mutation may enhance virulence is that the ability to bind more efficiently to a2,3sa receptors extends the lung tropism of the virus to bind ciliated cells that may then be unable to clear virus efficiently via the mucociliary escalator [124] . additionally, increased binding to type ii pneumocytes and macrophages in the alveoli and to submucosal glands in the trachea and bronchi may enhance lung damage [127] . however using reverse genetics to engineer this point mutation into an otherwise isogenic background, it was shown that the d225g change was not associated with an increase in virulence in the ferret or guinea pig models and remains easily transmitted between guinea pigs [127] . the mutation did result in a lower infectious dose for infection of mice who predominately express the a2,3, linked form of sa receptor [128] . the pb1 segment has a second reading frame (+1) which encodes a small protein (87-90 amino acids), pb1-f2 [129] . this protein has been assigned two functions, induction of apoptosis through its mitochondrial targeting c terminal domain and a role in lung inflammation [129] [130] [131] [132] [133] . a proposed third function, relating to polymerase function and reflected by the retention of the pb1 protein in the nucleus appears to be strain specific [134, 135] . viruses with intact pb1-f2 genes cause increased pathology in the mouse model [131] and also predispose the host to secondary bacterial infections and subsequent pneumonia. mice infected with a pr8 virus containing the full length pb1-f2 suffered greater weight loss and increased mortality when subjected to a secondary bacterial infection than mice infected with a pr8 with a truncated form of pb1-f2 [133] . however in natural isolates, particularly those from swine, the pb1-f2 gene is not always full length. zell et al. analysed the influenza a sequences available in genbank in 2007 [136] . they found that 96% avian strains possessed the full length pb1-f2, but in contrast only 75% of swine viruses and 81% of human viruses had the full length gene. classical swine influenza strains have truncated forms of pb1-f2 with premature stop codons after 11, 25 and 34 residues [136] . truncation of the pb1-f2 gene to just 57 amino acids also occurred in seasonal h1n1 viruses in the 1950s. loss of the c terminus of pb1-f2 removes the mitochondrial targeting sequence of the protein, abrogating its interaction with host proteins ant3 and vdac1 and reducing its ability to trigger apoptosis in immune cells [130, 137] . in addition this region of the protein appears to harbour pro-inflammatory properties. indeed, peptides generated to contain amino acid sequence from the c-terminal region of pb1-f2 generated an inflammatory response when administered to mouse lung. two days post exposure mice lost up to 15% body weight. interestingly the same peptide derived from recent h3n2 seasonal virus contains 5 amino acid differences from early h3n2 homologues that appear to abrogate the pro-inflammatory function. inflammation triggered by pb1-f2 peptides from highly virulent strains such as 1918 influenza may predispose to secondary bacterial pneumonia [138] . indeed, some highly virulent viruses such as h5n1 and 1918 h1n1 viruses possess a point mutation in this region of pb1-f2, n66s, that is partly responsible for their enhanced morbidity and mortality in mice [139] . the 2009 ph1n1 influenza virus has a pb1-f2 gene truncated to just 11 amino acids in length which is inactive. using reverse genetics to engineer viruses in which full length protein was restored, hai et al. noted no increase in virulence in mice or ferrets [140] . even the introduction of the notorious 1918 like n66s point mutation did not affect the outcome of infection in these models. thus acquisition of virulence by restoration of this gene to the pandemic virus seems unlikely [140] . the tropism of influenza virus is not only determined by its receptor use. sa is a widely distributed cell surface sugar but influenza in humans is largely restricted to the respiratory tract. the reliance on host cell proteases to cleave and thus activate the fusogenic properties of the ha protein determines the organs in which the virus can undergo productive infection. in humans the abundance of clara tryptase in respiratory secretions allows the virus efficient replication in the lung [150] . in highly pathogenic avian influenza viruses such as h5 and h7 strains, the insertion of a polybasic motif allows the ha to be cleaved by ubiquitous proteases such as furin, facilitating systemic infection [151] . despite the high mortality rates of those afflicted with the 1918 virus, it does not contain the polybasic cleavage site found in the highly pathogenic h5n1 viruses. the acquisition of this virulence motif in h5 and h7 subtypes of ha occurs during amplification in poultry [152, 153] . this motif has not been seen in any ph1n1 isolates in 2009 or 2010, and ph1n1 viruses remain dependent on the addition of trypsin to growth media for their propagation in cell culture. the influenza virus counteracts the otherwise suppressive effect of the interferon response using a nonstructural protein ns1, reviewed in detail by hale et al., [141] . ns1 works in at least two ways to prevent induction of interferon. firstly in the cytoplasm ns1 binds dsrna and other rnas that are the likely triggers of innate immunity as well as forming a complex with the host cell pattern recognition receptor rig-i and its controlling protein trim25. secondly in the nucleus some ns1 proteins can bind to the host cell factor cpsf30 and in doing so they suppress the processing of newly synthesized mrnas and prevent their export to the cytoplasm. this latter function is strain specific. it has been suggested that viruses that have enhanced ability to perform both these functions may induce a more severe disease because they can evade the innate immune response more efficiently. indeed introduction of cpsf30 binding ability to the lab adapted pr8 vaccine strain that usually lack this function enhanced its virulence in mice [142] . the ph1n1 virus lacks cpsf binding capacity. however hale et al. have shown that reintroduction of this phenotype did not affect virulence of ph1n1 in ferrets or mice [143] . despite lacking cpsf30 binding capacity, the ph1n1 virus induces very low levels of interferon in infected cells [144] . the 1968 and 1957 pandemic viruses both displaced the previously circulating subtypes. in contrast the re-emergence of h1n1 in humans in 1977 was not associated with subtype displacement likely because a large cohort of the older population was not susceptible to the virus and therefore remained viable hosts for the contemporary h3n2 viruses. similarly due to residual immunity in the elderly, ph1n1 2009 has not displaced the h3n2 subtype. initially it was believed that the seasonal h1n1 subtype may have gone extinct after ph1n1 emerged, as there was a period of many months where this virus was not isolated, however recently seasonal h1n1 isolates have been detected in texas [145] . the trivalent vaccine administered in 2010 contains ph1n1, h3n2 and influenza b virus antigens but no seasonal h1n1 component. influenza is a seasonal disease. infections peak once a year in the cold, dry season in the northern or southern hemispheres, although in the tropics the seasons are less clearly separated and it may be that virus continually circulates [146] . the royal college of general practitioners (rcgp) scheme in the uk has monitored the incidence of ili since the emergence of the h3n2 subtype in the 1968 pandemic (fig. 3) , the re-emergence of related h3n2 and seasonal h1n1 strains has been observed year on year and is due to the capacity of the virus to accumulate small point mutations in ha and na antigens, the process called antigenic drift. these mutations occur at antigenic sites and allow the circulating virus to evade immune suppression by throwing off antibody binding through confirmation changes or glycosylation events. accumulated drift mutations may ultimately change the phenotype of the virus. the virus may alter its affinity or specificity for the receptors on the host cell surface in its efforts to avoid the immune response [147] . indeed it is clear that, as it has evolved over four decades in humans, the h3n2 virus has changed its receptor binding affinities with phenotypic consequence [43, 148] . as herd immunity increases against the newly emerged ph1n1 virus, it is not in doubt that antigenic drift will occur. however, the resulting phenotypic changes are unknown and currently unpredictable. moreover since the virus has re-infected swine, a species in which frequent reassortments occur [149] , the evolution of this 2009 h1n1 virus and the consequence of reassortment events in animals for human disease remain to be observed in the coming years. the 1918 influenza pandemic: lessons for 2009 and the future updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic. bulletin of the history of medicine study of 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and vdac1 influenza a virus pb1-f2 protein contributes to viral pathogenesis in mice the influenza a virus pb1-f2 protein targets the inner mitochondrial membrane via a predicted basic amphipathic helix that disrupts mitochondrial function expression of the 1918 influenza a virus pb1-f2 enhances the pathogenesis of viral and secondary bacterial pneumonia the effects of influenza a virus pb1-f2 protein on polymerase activity are strain specific and do not impact pathogenesis the proapoptotic influenza a virus protein pb1-f2 regulates viral polymerase activity by interaction with the pb1 protein prevalence of pb1-f2 of influenza a viruses. the journal of general virology the proapoptotic influenza a virus protein pb1-f2 forms a nonselective ion channel improving therapeutic strategies for secondary bacterial pneumonia following influenza. future microbiology a single mutation in the pb1-f2 of h5n1 (hk/97) and 1918 influenza a viruses contributes to increased virulence pb1-f2 expression by the 2009 pandemic h1n1 influenza virus has minimal impact on virulence in animal models the multifunctional ns1 protein of influenza a viruses. the journal of general virology glycine 184 in the non-structural protein ns1 determines virulence of influenza a virus strain pr8 without affecting the host interferon response inefficient control of host gene expression by the 2009 pandemic h1n1 influenza a virus ns1 protein pandemic h1n1 2009 influenza a virus induces weak cytokine responses in human macrophages and dendritic cells and is highly sensitive to the antiviral actions of interferons texas state health services the global circulation of seasonal influenza a (h3n2) viruses hemagglutinin receptor binding avidity drives influenza a virus antigenic drift changes in in vitro susceptibility of influenza a h3n2 viruses to a neuraminidase inhibitor drug during evolution in the human host reassortment of pandemic h1n1/2009 influenza a virus in swine isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial clara cells. a possible activator of the viral fusion glycoprotein influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease molecular analyses of the hemagglutinin genes of h5 influenza viruses: origin of a virulent turkey strain the overall evolution of the h7 influenza virus haemagglutinins is different from the evolution of the proteolytic cleavage site key: cord-003466-599x0euj authors: nickol, michaela e.; kindrachuk, jason title: a year of terror and a century of reflection: perspectives on the great influenza pandemic of 1918–1919 date: 2019-02-06 journal: bmc infect dis doi: 10.1186/s12879-019-3750-8 sha: doc_id: 3466 cord_uid: 599x0euj background: in the spring of 1918, the “war to end all wars”, which would ultimately claim more than 37 million lives, had entered into its final year and would change the global political and economic landscape forever. at the same time, a new global threat was emerging and would become one of the most devastating global health crises in recorded history. main text: the 1918 h1n1 pandemic virus spread across europe, north america, and asia over a 12-month period resulting in an estimated 500 million infections and 50–100 million deaths worldwide, of which ~ 50% of these occurred within the fall of 1918 (emerg infect dis 12:15-22, 2006, bull hist med 76:105-115, 2002). however, the molecular factors that contributed to the emergence of, and subsequent public health catastrophe associated with, the 1918 pandemic virus remained largely unknown until 2005, when the characterization of the reconstructed pandemic virus was announced heralding a new era of advanced molecular investigations (science 310:77-80, 2005). in the century following the emergence of the 1918 pandemic virus we have landed on the moon, developed the electronic computer (and a global internet), and have eradicated smallpox. in contrast, we have a largely remedial knowledge and understanding of one of the greatest scourges in recorded history. conclusion: here, we reflect on the 1918 influenza pandemic, including its emergence and subsequent rapid global spread. in addition, we discuss the pathophysiology associated with the 1918 virus and its predilection for the young and healthy, the rise of influenza therapeutic research following the pandemic, and, finally, our level of preparedness for future pandemics. influenza viruses have posed a continual threat to global public health since at least as early as the middle ages, resulting in an estimated 3-5 million cases of severe illness and 291,243-645,832 deaths annually worldwide, according to a recent estimate [1] . regional influenza epidemics occur on an annual basis, resulting in millions of illnesses and hospitalizations despite intensive vaccination and awareness programs [2, 3] . moreover, influenza pandemics arise sporadically due to the introduction of an antigenically-distinct influenza a virus within a population, which can result in devastating effects on global public health and healthcare networks. the emergence of influenza subtype h1n1 in 1918, which ultimately resulted in an estimated 50-100 million deaths worldwide, would forever change the course of human history and will be discussed in detail in the following sections [4] [5] [6] . the aims of this short review are to discuss: i) the emergence and spread of the 1918 virus; ii) the unique severity of disease in young, healthy individuals; and iii) the subsequent influence of the pandemic on influenza virus therapeutic and future preparedness. it is postulated that 10% of the worldwide population is infected by an influenza virus each year, resulting in a total economic burden of $87.1 billion usd [7, 8] . as a testament to the significant toll posed by influenza on public health and healthcare systems, the us centers for disease control and prevention (cdc) estimated that from 2010 to 2015, influenza infections resulted in 9.23-35.6 million illnesses and 139,000-707,000 hospitalizations annually in the us alone [9] . it has been suggested that children are likely the primary transmitters of influenza [10] . lethal influenza infections are primarily associated with high risk populations, including infants (< 1 year), the elderly (> 65 years), and individuals with pre-existing comorbidities, including chronic respiratory abnormalities, cardiac disease, immunodeficiency, and pregnancy [11, 12] . mortality in children and young adults is generally low [3] . symptoms manifest as a sudden high fever, headache, pharyngitis, cough, myalgia, nausea, vomiting, and fatigue, which generally resolve within 7 days in healthy adults [11, 13] . severe and/or lethal disease is typically associated with viral pneumonia or secondary bacterial infections in the lower respiratory tract [3] . to be considered a pandemic, an influenza virus must: i) spread globally from a distinct location with high rates of infectivity resulting in increased mortality; and ii) the hemagglutinin (ha) cannot be related to influenza strains circulating prior to the outbreak nor have resulted from mutation [14, 15] . it should also be appreciated that prior to the first isolation of a human influenza virus in 1933, the cause of influenza outbreaks and pandemics could only be inferred based on physiological symptoms of disease, along with the speed and breadth at which illness was spread [15] . as early as 412 bc, hippocrates, the father of modern medicine, described the first known account of an influenza-like illness in his sixth "book of epidemics" [16, 17] . here, he recounted an annual recurring upper respiratory tract infection characterized by pharyngitis, coryza, and myalgia which peaked around the winter solstice [18] . this seasonal epidemic occurred in perinthus, a northern port town located in what is now turkey, and is referred to as the "cough of perinthus" [16] . it has been suggested that potential pandemics may have occurred in 1510 and 1557; however, it is unanimously agreed that the first documented influenza pandemic occurred in 1580, resulting in high morbidity [15, 19] . the virus originated in asia, before spreading to africa, and then simultaneously spreading from both continents to europe. it reportedly spread across the entire european continent within 6 months, before eventually reaching the americas [19, 20] . two pandemics were recorded in the eighteenth century. the first began in russia in 1729, eventually moving across the entirety of europe within 6 months and, ultimately, across the known world over the following 3 years [20] [21] [22] [23] . the second pandemic began in china in 1781, before spreading to russia and, subsequently, across all of europe. interestingly, this second pandemic had a high proclivity for young adults [24] . two major pandemics also occurred throughout the nineteenth century. the first began in 1830 in china, with subsequent spread to southeast asia, russia, europe, and north america and had a low overall mortality rate [15, 19, 20, 23] . a second pandemic emerged in russia in 1889 and spread rapidly to europe and north america, circumnavigating the globe in just 4 months [25, 26] . the virus, suggested to be of subtype h3n8, reappeared at least 3 more times in successive years resulting in an estimated 1 million global fatalities [20, 23, 26, 27] . four influenza pandemics have occurred over the past century ( fig. 1) . the 1918-1919 spanish flu pandemic, subtype h1n1, resulted in an estimated 50-100 million deaths worldwide and will be discussed in detail in the following sections. the 1957-1958 asian flu pandemic, subtype h2n2, originated in china in february 1957 and spread throughout asia and then globally by the summer. case fatality rates were approximately 0.67% with 1-2 million deaths worldwide [20, [28] [29] [30] [31] . just a decade later, the 1968-1970 hong kong flu pandemic, subtype h3n2, emerged in china in july 1968 and spread throughout europe, north america, and australia by early 1969 [25] . although mortality rates were low, the pandemic would ultimately claim between 500,000 and 2 million lives [25] . in april 2009, the 2009-2010 swine flu pandemic, subtype h1n1, began with nearly simultaneous outbreaks in mexico and the us, before spreading globally over the next 6 weeks. while the 2009-2010 pandemic had a low associated case fatality rate, resulting in 284,000 deaths worldwide, it had devastating effects on global economies and healthcare networks [25, 32] . conventionally, influenza pandemics result in the extinction of previously circulating virus strains; however, this view was complicated by events in 1977. although h1n1 was replaced by h2n2 as the circulating strain following the 1957-1958 asian flu pandemic, a descendant of the 1918 virus "re-emerged" suspiciously in 1977, likely as a result of a man-made event, and established itself as a co-circulating strain, along with the reassortant h3n2 virus (following the 1968-1970 hong kong flu pandemic) [4, 33] . the suspicious "re-emergence" of a descendant of the 1918 virus in 1977 has been postulated to have been the result of a man-made event. this hypothesis has gained traction, as both the ha and na of the re-emerged virus show incredible similarity to a 1950 reference virus, and it is unlikely that this strain was maintained in an animal reservoir for almost two decades without having undergone detectable mutation [33] . in 2009, a triple reassortment (made up of avian, swine, and human influenza genes) pandemic h1n1 jumped from pigs to humans, resulting in the co-circulation of three influenza strains [34] . the first wave of the 1918 pandemic one hundred years following its emergence, the origin of the 1918 pandemic influenza virus remains shrouded in mystery. the 1918 pandemic began early in the final year of the first world war. whereas prior pandemics had spread largely along trade routes, the global context of the war enabled greater viral spread facilitated by the mass mobilisation of military personnel and civilians [25, 35] . this was further augmented by the poor health and sanitation conditions found within trenches along the frontlines of the war, facilitating disease transmission [36] . public knowledge regarding the severity of the pandemic was hindered, as many news agencies were barred from writing about the global health threat, instead reporting solely on morale boosting subjects [37] . however, as spain was a neutral party in the war, newspapers were able to report on the devastating effects that the 1918 pandemic virus was exhibiting in spain. thus, it was generally perceived that this devastating illness originated in spain, resulting in the pandemic being incorrectly labeled as "the spanish flu" [37] . a century following its emergence, there remains a relative paucity of knowledge regarding the ancestry and regional origin of the 1918 virus. sequence analysis suggests that the virus was derived from an avian-like influenza virus and that all eight gene segments likely evolved in parallel [34, 38] . analyses of influenza virus genome sequences also suggest that the initial entry of the 1918 precursor virus into human circulation began in 1915 and did not appear to have jumped directly from an avian source [4, 38, 39] . however, improved understanding regarding the emergence of the 1918 virus, as well as factors (biological, social, environmental) that contributed to viral transmission and pathogenesis, have been vital to the development of current epidemic and pandemic influenza outbreak response efforts. descendants of the 1918 pandemic influenza virus strain have been the cause of almost every seasonal influenza a infection worldwide over the past century [4] . additionally, each of the pandemics occurring in 1957, 1968, and 2009 were caused by descendants of the 1918 pandemic (1918, 1957, 1968 and 2009 ). circulation of h1n1 was reinitiated in 1977 and has therefore been added to this timeline. grey arrows designate the circulating or co-circulating strains during the interpandemic periods influenza virus strain, earning the 1918 viral strain the nickname "the mother of all pandemics" [4] . investigations concerning the origins of the first wave of the pandemic, beginning in march 1918, have primarily focused on the us and china, though recently it has been suggested that the origin may have been an outbreak of a respiratory disease misidentified as pneumonic plague in china [15, 36, 40] . humphries suggests that the dissemination of labourers from china to assist allied war efforts during this outbreak resulted in the inadvertent spread of the virus to europe [36] . from 1916 to 1918, the route of travel to europe for the labourers included checkpoints in singapore, durban, cape town, north africa, and canada. additional reports of the first wave of the virus in the spring of 1918 suggest that the pandemic originated with chinese workers at camp funston, kansas, where the workers began suffering from 2 to 3 day fevers, gastrointestinal symptoms, and general weakness [37, 41] . within 3 weeks 1100 soldiers had been hospitalized, and thousands more had received out-patient treatment [41] . the illness was able to spread to other military camps within the us, before traversing the atlantic ocean via soldiers supporting allied operations in europe. the us army reported that from march-may 1918, 11.8% of us soldiers were hospitalized due to this unidentified respiratory illness [41] . while illness rates were high during this initial wave, mortality rates were largely similar to seasonal outbreaks of influenza. spain reported that the mortality rates for pneumonia and influenza was only 0.065% [37] . although there was some acceptance that this new illness was indeed influenza, this was not generally accepted [37] . radusin reported that although the physiological symptoms were similar to influenza, the illness was too mild and short-lasting with minimal complications for it to be influenza [37] . infections began to subside in many regions by the early summer [41] . the generally accepted lines of spread of the first and second waves of the 1918 virus are provided in fig. 2 . in mid-august of 1918, reports suggesting a second wave of this severe illness began to surface [35] . in some regions, primarily northern europe, the period between the end of the first wave and the beginning of the second wave was incredibly short, making the two waves almost indistinguishable [4, 42] . this second wave, occurring from september-november 1918, was responsible for the majority of illnesses and fatalities associated with the pandemic. although the origins of the first wave [80] continue to be debated, the origin of the second wave is generally agreed to be the harbour town of plymouth in southern england, which allowed the pandemic influenza virus strain to easily spread to the rest of the world [25] . ships from plymouth were dispatched to freetown, sierra leone in august 1918, which allowed the virus to spread across the african continent [25] . new zealand soldiers, who stopped in freetown on their way to and from the war front in europe, facilitated transfer of the pandemic virus to new zealand [25] . from plymouth, the virus also spread to boston, from which it was able to disseminate across the rest of north america resulting in > 1 million fatalities over the ensuing four months [5, 25] . this second wave spread globally throughout the fall of 1918 with illness seen first amongst military personnel and, subsequently, within the general population [25, 35] . the second wave of the 1918 pandemic differed from the first in that much higher morbidity and mortality rates were reported, with the majority of all fatalities associated with the pandemic occurring during this wave [4] . ultimately, the pandemic would result in an estimated 500 million infections worldwide (~1/3 of the world's population at the time) and a case fatality rate > 2.5%, more than 25 times higher than any other pandemic [4, 37] . as a testament to the severity of this second wave, during the fall of 1918, the first 4-5 pages of spanish newspapers were filled with obituaries of those who had succumbed to the pandemic virus [35] . further, reports from philadelphia, pennsylvania stated that across 31 hospitals in the city, every hospital bed was occupied by patients with influenza [35] . the pandemic was especially problematic in highly isolated communities where many individuals had limited contact with prior influenza strains, thus lacking any pre-existing immunity. for example, some inuit settlements reported case mortality rates as high as 70%, while certain communities in africa were completely decimated [35] . interestingly, individuals who had been infected throughout the first wave seemed to be protected against this secondary wave, and recent analyses have suggested that these individuals had up to 94% protection throughout the fall wave [4, 41] . a third and final wave of the pandemic appeared in most of the world in the early months of 1919 [4, 5, 35] . this final wave generally overlapped the first wave in terms of regional distribution; however, it seemed to spare areas where the second wave had been especially severe. overall, morbidity rates were lower throughout this final influenza wave; however, mortality rates are believed to have been just as severe as the second wave [4, 35] . three successive annual winter post-pandemic recurrences occurred following the third wave of the pandemic with continually decreasing mortality rates, in particular within those 20-40 years of age [43] . classically, fatal influenza infections are primarily associated with the very young (< 5 years) and the elderly (> 65 years) resulting in a characteristic "u"-shaped mortality curve (fig. 3) . interestingly, however, the 1918-1919 h1n1 influenza pandemic mortality curve exhibits a "w"-shape due to excess mortality in young adults 20-40 years of age due to influenza-related illness. it has been postulated that the increased disease severity in young adults was likely associated with immune status due to the lack of pre-existing immunity in this population [44] . further, more than 99% of fatal infections occurred in those < 65 years of age and nearly 50% of all influenza-related deaths during the 1918 pandemic were in those aged 20-40 years [4] . influenza and pneumonia fatality rates in those aged 15-34 years were more than 20 times higher than in previous years and absolute risk of influenza-related death was higher in those < 65 years of age than those > 65 years old [4] . it is still not fully understood why this occurred, but it is possible that an antigenically similar influenza strain circulated prior to 1889, providing a level of protection against the novel h1n1 pandemic strain to those born prior to 1889 [4] . additionally, archaeserological and epidemiological evidence have shown that an h3 subtype influenza virus may have been responsible for the 1889 influenza pandemic, which circulated until the emergence of the 1918 pandemic virus, leaving those individuals who had not been exposed to an h1 subtype virus highly susceptible to the pandemic virus [34] . it has also been suggested that the generation of an excessive inflammatory response ("cytokine storm") in healthy, young adults infected with the 1918 virus may have contributed to the excess mortality seen within this age group [34] . recent in vivo studies with the 1918 virus have shown a marked upregulation of inflammatory cytokines, along with the suppression of important antiviral immune responses [34, 45] . in addition, other influenza strains, such as fatal h5n1 infections in humans, have also been associated with the deleterious consequences of an excessive inflammatory response [46] . ultimately, the case fatality rate was so severe in young adults during the 1918-1919 pandemic that the average life expectancy rate in the us dropped by~12 years [47] . physiological symptoms of the 1918 pandemic virus generally lasted for 7 days and were described as feeling cold, shivering, high fever, weakness, nausea, loss of appetite, pharyngitis, cough, and bloodshot eyes [35] . in some patients, a short "rebound" to normal health would occur that was followed by an aggressive recrudescence of disease and, ultimately, death [35] . similar to the 1889 pandemic, the majority of fatal infections resulted from respiratory complications. however, it has also been demonstrated that excess influenza fatalities during the 1918-1919 pandemic were associated with an acute aggressive bronchopneumonia (including epithelial and vascular necrosis, hemorrhage, edema, and bacterial-associated variant pathology within the lungs) and a severe acute respiratory distress-like syndrome associated with severe facial cyanosis [43] . autopsies performed on preserved lung tissues in the modern era have revealed acute pulmonary hemorrhage and secondary bacterial infections associated with pulmonary lesions in nearly all the fatal cases examined [41, 43, 47] . streptococcus pneumoniae was present in many cases; however, staphylococcus aureus, haemophilus influenzae, and streptococcus pyogenes also appeared to complicate fatal cases [48, 49] . neutrophilic pulmonary infiltration was seen in cases of pneumococcal pneumonia, while cases of staphylococcal pneumonia were marked by multiple microabscesses infiltrated by neutrophils [48] . however, alveolar cell damage was seen in each case along with pulmonary repair and remodelling [48] . tissues from each of the fatal cases examined had similar pathologic presentation, independent of which pandemic wave they were associated with. despite the difference in mortality rates, each wave showed similar cellular tropism, infecting both type i and type ii pneumocytes, as well as the bronchiolar respiratory epithelium [48] . a multitude of scientific and technological advances have occurred over the past century, allowing for a greater understanding of the dynamic relationship between the host and influenza viruses during infection. these advances, along with access to autopsy samples and the reconstitution of the 1918 pandemic virus, have facilitated a greater understanding of how the pandemic virus differs from other seasonal and pandemic influenza virus strains. moreover, technological advancements following the 1918-1919 influenza pandemic virus have facilitated the development of preventative measures, including vaccines and antivirals, to limit widespread illness due to influenza infections. the determination of the genomic sequence of the 1918 pandemic virus, and the subsequent reconstruction of the virus, has provided us with the opportunity to decipher the viral-and host-specific properties that contributed to the severity of the 1918-1919 pandemic. it has been demonstrated that in contrast to other influenza viruses, the 1918 pandemic virus is highly virulent and pathogenic in multiple animal species without prior adaptation [45, 50] . while obvious knowledge gaps remain, in particular with respect to the origin of the virus and the molecular mechanisms (host and/or viral) underlying differential pathogenesis as compared to other influenza viruses, there have been considerable advances in our understanding of the 1918 pandemic virus. [81, 82] . means with standard deviations are presented for the prepandemic mortality curve. adapted from taubenberger and morens [4] since the isolation of the first human influenza virus in 1933, researchers have worked to develop an effective influenza vaccine [16] . current influenza vaccines are reformulated seasonally and provide protection against circulating influenza a and b viruses [13] . the world health organization conducts worldwide surveillance studies throughout the year on currently circulating influenza strains, and thus recommends which strains should be included in each influenza vaccine [13] . while the seasonal influenza vaccine is approximately 60% effective, this protection is dependent on the characteristics of the individual being vaccinated, including age and overall health, as well as the match between the strains included in the vaccine formulation and currently circulating strains [13] . individuals who have been vaccinated are generally protected from illness and provide a measure of protection for those who are not able to be vaccinated due to their age or other health issues through herd immunity [13] . there has also been increasing interest in the development of "universal" influenza vaccines designed to provide protection against a wide range of antigenically-distinct influenza viruses, including those currently in circulation and those that may emerge in the future [51] . these will not be discussed in detail as recent reviews have provided excellent discussions of this topic [51] [52] [53] [54] [55] [56] [57] . two major classes of antivirals have emerged for therapeutic treatment of severe influenza virus infections. adamantane antivirals target the matrix-2 (m2) surface protein, while neuraminidase (na) inhibitors target the na viral surface protein. adamantane compounds were the first licensed influenza antivirals and block the m2 ion channel protein from properly functioning, thus effectively blocking membrane fusion [58, 59] . unfortunately, adamantane antivirals are only able to target influenza a viruses limiting their application for influenza b virus infections [58] . further, more than 90% of influenza a viruses are resistant to this class of drugs due to the high mutation rate of the virus [58, 60] . thus, the use of na inhibitors is recommended [60] . na inhibitors block the na surface protein and prevent the release of progeny virus and infection of additional cells [60] . while resistance to na inhibitors has been observed in some influenza virus strains, they are still highly effective in the majority of patients [60] . studies have shown that both adamantane antivirals and na inhibitors provide protection against the 1918 virus [50] . although outside the auspice of this commentary, it should be mentioned that advances in mechanical ventilation modalities, including non-invasive positive pressure ventilation, from the 1950s onwards, have provided an additional support mechanism for treatment of severely ill patients [34] . the routine clinical use of antibiotics in the early twentieth century also heralded a new era for combating influenza viruses. as a testament to this, excess influenza mortality declined significantly from 1942 to 1951 onwards [61] [62] [63] . however, the widespread general administration of antibiotics has resulted in an escalating public health crisis due to multi-drug resistance. this has impacted the treatment of severe influenza infections, as methicillin-resistant s. aureus (mrsa) is the most frequently isolated bacteria from patients with severe influenza-bacterial co-infections in the us [64, 65] and complicated up to 55% of fatalities during the 2009 pandemic [66] [67] [68] [69] . influenza preparedness and lessons for the future although it has now been a century since the start of the spanish flu pandemic, lessons from this global health catastrophe continue to inform modern-day pandemic preparedness. investigations of the pandemic, including those with the reconstructed virus, have allowed researchers, as well as the global public, to understand the mechanisms that underlie pandemic emergence and escalation to public health crisis. it also allows researchers to predict the potential public health risks which may be caused by new pandemic viruses. for example, sequencing of the 1918 pandemic virus revealed similarities in the h1 protein of the 2009 pandemic virus, allowing researchers to predict that a lack of protection, and thus a high mortality rate may be seen in healthy, young adults throughout the 2009 h1n1 pandemic [45] . thus, when vaccines were limited during the early stages of the 2009 pandemic, young adults were prioritized over the elderly, who demonstrated some degree of protection to this influenza strain, resulting in a lower mortality rate in young, healthy adults [45] . the average age for laboratory-confirmed fatalities during the 2009 pandemic was 37 years in the us, supporting this vaccine prioritization initiative [70] . additionally, the awareness of the complications caused by secondary bacterial co-infections from the 1918 pandemic ensured that the medical community was aware of this threat throughout the 2009 pandemic, likely resulting in a reduced mortality rate due to severe influenza infections with complications [45] . however, the 2009 pandemic, albeit milder than previous pandemics in terms of overall mortality, resulted in significant strains on global healthcare networks and economies [25] . in canada, direct healthcare costs (including hospitalizations, outpatient visits, and therapeutics) related to the 2009 pandemic have been estimated at $2 billion cad, with $250 million cad related directly to hospital care [71] . a computational modeling study by smith and colleagues suggested that direct costs related to illness would be between 0.5-4.3% of gdp in the uk for pandemics ranging from low to extreme [72] . further, the 2002-2004 severe acute respiratory syndrome outbreak resulted in~$1 billion total gdp loss in toronto alone [73] . this highlights the importance of pandemic preparedness beyond a healthcare-centric approach to one that also includes downstream economic effects. the 1918-1919 pandemic resulted in incredible improvements to public health as well as scientific advances. however, our current understanding of influenza viruses, and their ability to cause illness in humans is still in its infancy in many aspects, and further underlines our inherent need for continued influenza research. the identification of key molecular determinants involved in the pathophysiology of severe influenza infections will also assist drug discovery and development strategies, including insights on appropriate timing for administration of antivirals and/or antibiotics. the development of efficacious broader-spectrum or "universal" influenza vaccines is also of incredible importance. the emergence of novel highly pathogenic avian influenza (hpai) viruses, including h5 and h7 subtypes, are of particular concern due to their pandemic potential. circulating hpai viruses are of potential concern to global public health [74] . asian lineage avian influenza a (h5n1), which circulates in fowl, is rarely found in humans but has resulted in life-threatening cases when able to establish stable lineages [74] and h7n9 has resulted in sporadic human infections in china resulting in > 1500 infections with an estimated 39% case fatality rate since 2013 [75] . because hpai viruses can arise from previously known low-pathogenicity viruses with only minor mutations, it is important to be vigilant concerning these potential pandemic viruses [76, 77] . in spite of the public health advancements in the 100 years following the 1918-1919 pandemic, including widespread access in the developed world to an efficacious influenza vaccine, influenza viruses remain a global public health threat. this pas year, there were > 55,000 reported influenza infections, 5155 influenza-associated hospitalizations, and 303 deaths across canada [78] . further, during the 2016-2017 influenza season, vaccination rates in those 18-64 years of age was only 37 and 69% in those ≥65, both below the national vaccination target of 80% [79] . these data suggest that our continued vigilance against influenza must not only include a "research"-driven focus but also include public outreach and awareness campaigns that increase the general understanding of the healthcare burden associated with influenza infections. estimates of global seasonal influenza-associated respiratory mortality: a modelling study influenza: the mother of all pandemics updating the accounts: global mortality of the 1918-1920 "spanish" influenza pandemic characterization of the reconstructed 1918 spanish influenza pandemic virus influenza: molecular virology the annual impact of seasonal influenza in the us: measuring disease burden and costs prevention cfdca: estimated influenza 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like emergence of a highly pathogenic avian influenza virus from a low-pathogenic progenitor public health agency of canada. 2016/17 seasonal influenza vaccine coverage in canada textbook of influenza vital statistics rates in the united states: 1940-1960. washington: office ugp vital statistics rates in the united states: 1900-1940. washington: us government printing office not applicable. availability of data and materials not applicable. authors' contributions men and jk conceived of the ideas presented herein and made substantial contributions to the drafting and revising of the manuscript. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors have declared that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-006172-ndmf5ekp authors: akins, paul taylor; belko, john; uyeki, timothy m.; axelrod, yekaterina; lee, kenneth k.; silverthorn, james title: h1n1 encephalitis with malignant edema and review of neurologic complications from influenza date: 2010-09-02 journal: neurocrit care doi: 10.1007/s12028-010-9436-0 sha: doc_id: 6172 cord_uid: ndmf5ekp background: influenza virus infection of the respiratory tract is associated with a range of neurologic complications. the emergence of 2009 pandemic influenza a (h1n1) virus has been linked to neurological complications, including encephalopathy and encephalitis. methods: case report and literature review. results: we reviewed case management of a 20-year old hispanic male who developed febrile upper respiratory tract signs and symptoms followed by a confusional state. he had rapid neurologic decline and his clinical course was complicated by refractory seizures and malignant brain edema. he was managed with oseltamavir and peramavir, corticosteroids, intravenous gamma globulin treatment, anticonvulsants, intracranial pressure management with external ventricular drain placement, hyperosmolar therapy, sedation, and mechanical ventilation. reverse transcriptase polymerase chain reaction analysis of nasal secretions confirmed 2009 h1n1 virus infection; cerebrospinal fluid (csf) was negative for 2009 h1n1 viral rna. follow-up imaging demonstrated improvement in brain edema but restricted diffusion in the basal ganglia. we provide a review of the clinical spectrum of neurologic complications of seasonal influenza and 2009 h1n1, and current approaches towards managing these complications. conclusions: 2009 h1n1-associated acute encephalitis and encephalopathy appear to be variable in severity, including a subset of patients with a malignant clinical course complicated by high morbidity and mortality. since the h1n1 influenza virus has not been detected in the csf or brain tissue in patients with this diagnosis, the emerging view is that the host immune response plays a key role in pathogenesis. the current pandemic of 2009 influenza a (h1n1) (2009 h1n1) virus has presented challenges for clinicians the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. worldwide. neurologic complications of seasonal influenza are likely under-recognized by neurologists and the frequency of acute or post-infectious neurologic complications with 2009 h1n1 virus infection is unknown. it is worth noting the historical relationship between h1n1 and neurology. following the 1918-1919 h1n1 pandemic, an increase was observed in encephalitis lethargica cases [1] . what have neurologists learned about complications of 2009 h1n1 virus infections worldwide? we present a case report of 2009 h1n1-associated encephalopathy and review neurologic complications associated with seasonal influenza and 2009 h1n1 virus infection. the kaiser permanente inpatient neurosurgery service maintains ongoing institutional review board approval for a prospective database registry for clinical research purposes. we identified a case of acute encephalopathy associated with 2009 h1n1 virus infection of the upper respiratory tract referred from an outside kaiser community hospital for management. we conducted a detailed review of the electronic medical records. we also conducted a literature review using pubmed. mesh search terms included influenza, encephalitis, encephalopathy, h1n1, acute necrotizing encephalopathy, and meningitis. a previously healthy 20-year old male college student had 5 days of non-productive cough, rhinorrhea, myalgias, and fever but no headaches or neck stiffness. on the 6th illness day, he presented to the emergency department of a community hospital with lethargy and confusion. he was electively intubated for airway protection. his chest x-ray (cxr) was normal. routine admission laboratory tests including hepatic transaminases were within normal range. a non-contrast head computed tomography (ct) did not reveal any abnormalities (fig. 1, top row) , and he underwent lumbar puncture. cerebrospinal fluid (csf) analysis showed 53 wbc/ll with 91% lymphocytes, 6 rbc/ll, protein 113 mg/dl, and glucose 59 mg/dl. he was diagnosed with meningoencephalitis and started on vancomycin, ceftriaxone, acyclovir, and oseltamivir (150 mg twice daily per nasogastric tube). on the morning of the third-day of hospitalization, he experienced tonic-clonic seizures and remained comatose with extensor posturing afterwards. repeat head ct (fig. 1 , bottom row) demonstrated diffuse brain edema and effaced basal cisterns. he received fosphenytoin, mannitol, and propofol. the treating physicians contacted the neuro-intensive care unit at kaiser sacramento for additional assistance. he was emergently transferred to the kaiser permanente sacramento neuro-intensive care facility (nicu). on arrival, his initial examination demonstrated a glasgow coma scale of 3 (e1v1m1). his repeat cxr did not demonstrate on the bottom row, the small arrow points to effacement of basal cisterns (left) and subcortical brain edema (larger arrows, bottom row, left and right). this subcortical edema is confirmed on mr imaging (fig. 2) any infiltrates or signs of acute respiratory distress syndrome (ards). an external ventricular drain was placed by the neurosurgeon at the bedside. he reported that the csf pressure noted at the time of initial catheter placement was elevated. the first recorded intracranial pressure (icp) was 10 mm hg, and this reading was taken after the expected loss of csf during the procedure. on the second day of nicu hospitalization, his glasgow coma scale (gcs) score was 4 (e1v1m2) and average icp was 7 mm hg. throughout the remainder of the hospitalization, the recorded icp remained below 20 mm hg. initial icp was maintained with external ventricular drainage at 0 cm relative to the external auditory canal and a midazolam infusion (5 mg/h). electroencephalogram (eeg) monitoring demonstrated diffuse, severe slowing in the delta range and no electrographic seizures. on hospital day 3, mri of the brain was obtained (see fig. 2 ). he received 20 days of dual neuraminidase inhibitor treatment (oseltamivir 150 mg twice daily per nasogastric tube, peramavir 600 mg iv daily); intravenous gamma globulin (1 gm/ kg 9 2 days); dexamethasone (10 mg iv load, 6 mg iv every 6 h with taper over 4 weeks); icp monitoring and management; ventilator support; and anticonvulsants (fosphenytoin, levetiracetam). his weekly glasgow scale scores showed delayed improvement (3, e1v1m1, admission): 5 (e2v1m2, week 1), 5 (e2v1m2, week 2), 5 (e2v1m2, week 3), 9 (e3v2m4, week 4). the midazolam infusion was discontinued on hospital day 4, after clinical observation and eeg confirmation that he was not having electrographic seizures. thereafter he received intermittent doses of lorazepam as needed for sedation while on the ventilator. over 3 weeks, neuroimaging demonstrated improvement in his brain edema with restoration of his basal cisterns, and the external ventricular drain was successfully weaned and removed. more rapid weaning of his external ventricular drain was not attempted due to severe neurologic impairments with gcs less than eight and radiographic appearance of diffuse brain edema and effaced basal cisterns. his nicu course was complicated by ventilatorassociated klebsiella pneumoniae and spontaneous pneumomediastinum on day 6 of intensive care. chest ct demonstrated subcutaneous emphysema, mediastinal emphysema, bilateral lower lobe atelectasis, and no pulmonary interstitial emphysema, or pneumothorax. he did not develop adult respiratory distress syndrome or suffer periods of hypoxemia. rt-pcr of an admission nasopharyngeal swab was positive for 2009 h1n1 virus at the california department of public health virology laboratory. rt-pcr analysis of csf samples was negative for influenza a and b viruses, herpes virus type 1, 2, and 6, varicella, enterovirus, and epstein barr virus. nasopharyngeal samples were negative for enterovirus and mycoplasma pcr. bacterial and viral cultures of csf were negative. test results from clinical specimens (blood, endotracheal aspirate, serum, and csf) sent to the california encephalitis project did not reveal an alternative cause. follow-up mri brain imaging (fig. 2b, d) was repeated at 1 month. after 6 weeks, he transitioned to acute rehabilitation, and 1 month later returned home. because he had improved upper extremity use without recovery in his legs, the physiatry staff performed spine mr imaging and no specific cause was identified. at the time of this case report, the patient has returned home with his family. he is talking and interacting with his family normally. he has not returned to college. his gastromy tube has been removed. he has generalized rigidity without tremor or dyskinesia. he is ambulatory but requires a walker due to reduced endurance and leg weakness. fig. 2 magnetic resonance imaging was done at the time of patient transfer a, c to the neuro-intensive care center and at 1 month of treatment b, d with influenza-specific antiviral therapy, corticosteroids, and intravenous gamma globulin therapy. a coronal flair image shows diffuse brain edema with sulcal effacement and symmetric hyperintensities selectively affecting the white matter and sparing cortex and subcortical nuclei such as basal ganglia and thalami. b coronal flair image at 1 month shows resolution of sulcal effacement, marked reduction in white matter hyperintensity, and relative brain atrophy (20 year old patient). c diffusion-weighted imaging on admission showed some increased signal in the periventricular zones that were also bright on t2 and flair sequences consistent with t2 shine-through. d diffusionweighted imaging at 1 month revealed hyperintensity in the caudate and putamen with corresponding decreased signal in adc map and lack of hyperintensities on t2 and flair sequences (see fig 1b) we present a case of a patient with acute encephalitis associated with febrile upper respiratory tract illness due to 2009 h1n1 complicated by seizures and malignant cerebral edema. few adult cases of 2009 h1n1 influenzaassociated acute encephalitis or encephalopathy have been reported to date. descriptions of 2009 h1n1-associated neurologic complications are limited to case reports and small case series, and have been more commonly reported among young children. given the current influenza pandemic, we provide an overview of neurologic complications associated with seasonal influenza and h1n1 (fig. 3 ) and review clinical management and rationale. influenza virus infections can cause human respiratory disease and have been associated with a variety of central nervous system disorders [2] . influenza virus has been rarely detected in csf of patients that developed acute encephalitis/encephalopathy [3] [4] [5] . the systemic inflammatory response syndrome (sirs) to influenza virus infection of the upper respiratory tract is hypothesized to play a prominent role in the more severe stages leading to cytokine dysregulation (''cytokine storm'') in influenzaassociated encephalopathy or encephalitis (iae) patients [6] . elevated cytokines in serum and csf have been reported in patients with seasonal influenza-associated encephalopathy [4, [7] [8] [9] [10] . elevated csf to plasma ratios suggest activation of cytokine production within the cns may have occurred along with the respiratory tract and systemic cytokines [7, 11, 12] . microglia and astrocytes are capable of producing cytokines in the cns [13, 14] . it is known that influenza virus infects and replicates at the nasopharyngeal epithelium leading to extensive damage during infection. below the mucosa, the free nerve endings of the olfactory nerves may also become infected. as seen with herpes simplex viruses, some postulate that influenza virus could penetrate and replicate at the olfactory mucosa and the free nerve endings with resultant axonal transport of virions to the olfactory bulbs, to the olfactory tract, and finally to the brain [15] . there is some literature to support this mechanism when one looks at h5n1, or avian influenza, where mice inoculated intranasally with h5n1 developed cns lesions in the pons, medulla oblongata, and cerebellar nuclei. astrocytes and glial cells were positive for viral antigen but viral replication ceased before 7 days [16, 17] . further study is needed to elucidate the pathogenesis of cns disease complicating influenza a infection. neurologic symptoms associated with influenza can arise at different intervals after the initial influenza illness (fig. 3 , table 1 ). when assessing patients clinically, it is important to determine if the patient has active or recent symptoms (within days) of influenza or if the neurologic symptoms have appeared in a subacute manner. we will first discuss neurologic complications in the setting of recent influenza virus infection and then proceed to complications that present in a delayed manner the development of a confusional state in the setting of influenza illness symptoms and fever raises the possibility of influenza-associated encephalitis or encephalopathy. the degree of encephalopathy varies from a confusional state to obtundation. it is important to recognize that a small portion of cases can rapidly deteriorate to coma and subsequent brain death due to diffuse, malignant cerebral edema. focal and generalized seizures often occur and can be present with either mild or severe cases. the presence of fever and altered mental state should prompt clinicians to pursue csf analysis unless neuroimaging or laboratory studies reveal a contraindication. influenza illness may include upper respiratory symptoms, pneumonia, or diarrhea (more commonly in young children with seasonal influenza). a thorough medical assessment to exclude other causes such as sepsis, metabolic or toxic disorders, structural cns diseases, and other cns infections is warranted. we define encephalitis by the presence of inflammation in the csf or demonstration of viral infection in brain biopsy or autopsy specimens. we define encephalopathy when csf is acellular and brain biopsy or autopsy specimens have failed to demonstrate viral infection. in some cases, this distinction is arbitrary and the case has borderline csf pleocytosis or csf analysis was not performed due to malignant brain edema. a consistent observation is that patients with seasonal influenza-associated encephalopathy rarely ever have evidence of influenza viral rna in csf based on rt-pcr analysis of csf. furthermore, there is no evidence of seasonal influenza virus infection of brain specimens. in one case series, only one out of 18 patients with acute seasonal influenza-associated encephalitis had influenza viral rna detected [5] . terminology for post-infectious encephalitis can be confusing. for example, the international pediatric multiple chronic condition * sometimes classified as adem [18] sclerosis study group [18] listed ten terms that have been used to describe acute disseminated encephalomyelitis (adem). some terms focus on the triggering event, such as post-infectious encephalomyelitis; others on pathologic or pathophysiologic features such as acute demyelinating encephalomyelitis or hyperergic encephalomyelitis. these authors also classify acute hemorrhagic leukoencephalitis, acute necrotizing hemorrhagic leucoencephalitis (also known as acute necrotizing encephalitis, (ane)), and acute hemorrhagic encephalomyelitis as hyperacute forms of adem. these diagnostic terms are of great historical interest. they generally preceded modern neuroimaging and relied more on the clinical and pathologic details. the study group also lumps a diversity of neuroimaging findings under the diagnosis of adem including: ring-enhancing lesions; diffuse and multi-focal regions of t2 hyperintensity with and without associated hemorrhage; multi-focal lesions with associated mass effect (tumefactive lesions); and images with symmetric, bithalamic edema. while we prefer one term (adem) rather than ten terms to describe post-infectious encephalitis, we are concerned that the pathophysiology and outcome of a process leading to the formation of ring-enhancing lesions (demyelinating, for example, acute demyelinating encephalomyelitis) must be radically different than that causing bithalamic edema (necrotizing, for example, ane). in reality, iae presents along a spectrum ranging from milder cases with normal neuroimaging to more malignant cases with abnormal neuroimaging and less favorable outcomes. for the sake of discussion and literature review, we present a simplified classification scheme based on clinical and imaging findings. the iae benign variant can present with fever, confusional state, and seizures but neuroimaging with ct brain or mri brain does not demonstrate any acute abnormalities. csf analysis is within normal limits or has borderline findings. rt-pcr testing for 2009 h1n1 influenza viral rna is positive in upper respiratory secretions but negative when csf is tested [19] [20] [21] . these patients typically recover within 1 week, and most cases have received oseltamavir and anticonvulsants. the initial reports of pediatric cases of 2009 h1n1 encephalopathy in the us were not severe [19] . similarly, other reported adult cases of 2009 h1n1 iae without ards have not been severe with complete recovery [20, 21] . a more recent pediatric case series of 2009 h1n1 iae reported that 2/4 patients had imaging abnormalities and neurologic sequelae [22] , so the treating physicians need to be aware that full recovery is not a certainty. the iae with splenial sign presents with acute febrile respiratory illness and additional neurologic symptoms with a characteristic mri abnormality. we found case reports associated with seasonal influenza but not with h1n1. it has been reported in children, but rarely in adults [23] [24] [25] [26] [27] [28] . encephalopathy is always present and can be severe. seizures are often present. mri imaging demonstrates increased t2 and flair signal and restricted diffusion in the splenium of the corpus callosum. this finding is reversible. the mri finding is not specific and has been reported with other infections, high-altitude brain edema, and certain metabolic states such as hypernatremia [29] . csf analysis is unremarkable. these patients have been treated with oseltamavir and anticonvulsants, and typically recover within 1 month. the iae with posterior reversible leucoencephalopathy syndrome (pres) presents as moderate to severe febrile encephalopathy. this subtype has been reported with seasonal influenza but not specifically with h1n1. the mri imaging appears radiographically identical to pres caused by more typical causes such as pregnancy or malignant hypertension [30, 31] . vascular caliber changes have been observed in these cases; this is non-specific and can be related to infectious vasculitis or pres. given the diverse causes of pres including malignant hypertension, pregnancy, metabolic disorders, and certain medications such as chemotherapeutics and immunosuppressants; it is often difficult to distinguish the pathophysiology of iae in this clinical setting. therapy is focused upon antiviral treatment, corticosteroid administration, and supportive care. iae with malignant brain edema is one of the most challenging subtypes to diagnose and treat. both seasonal influenza and h1n1 can be complicated by severe forms of acute encephalopathy and malignant brain edema [32] [33] [34] [35] . survival in some cases has been achieved with aggressive neuro-intensive case management with other therapies, including administration of antivirals, corticosteroids, immunoglobulin (2 gm/kg in adult patients), hyperosmolar therapy, plasmapheresis, and hypothermia in some cases. one of the goals of treatment is to reduce viral expression with early antiviral treatment and thereby to reduce stimulation of the host inflammatory response. our case presentation illustrates the rapid time course for this complication (see fig. 1 ) and neurocritical care treatment approaches. because of diffuse brain edema, a broad treatment approach using hyperosmolar therapy, intubation, fever control, and sedation were important. to the best of our knowledge, this is the only case description of iae in which an external ventricular drain was utilized, probably because it is difficult to place a catheter into the small, compressed ventricles of patients with diffuse brain edema associated with influenza. another adult case of h1n1 encephalitis has been reported with radiographic findings similar to ours. fugate et al. [35] described an adult with h1n1 influenza-associated acute hemorrhagic leukoencephalitis. like our patient, their case also showed confluent areas of increased t2 signal in the periventricular white matter and centrum semiovale. because of the additional finding of microhemorrhages demonstrated on gradient echo mri sequences, they diagnosed acute hemorrhagic leukoencephalitis or hurst disease. their patient also had restricted diffusion in the basal ganglia (see fig. 2 ). because their patient had severe adult respiratory distress syndrome (ards) with oxygen saturation readings in the range of 70-80%, the authors attributed the basal ganglia findings to hypoxic brain injury. our patient did not have advanced pulmonary disease, hypoxia, or hypotension. care should be taken to distinguish iae with malignant edema from reyes' syndrome in which patients may present with lethargy, confusion, seizures, or coma accompanied by brain edema. reyes' syndrome most commonly occurs in children but has been reported in adults following influenza and aspirin ingestion [36] . it can be distinguished based on the accompanying hepatic abnormalities, hyperammonemia, and hypoglycemia. caution should be taken with any neurosurgical procedures in reyes' syndrome due to increased risk of perioperative bleeding. one of the most devastating complications of seasonal and pandemic influenza is ane [37] [38] [39] . patients develop rapid neurologic deterioration to coma. seizures are often present. initial brain ct may show decreased density in the thalami, and mri of brain demonstrates the characteristic bilateral thalami lesions. this finding may be initially mistaken for ischemic strokes (top-of-basilar syndrome) or venous infarction secondary to thrombosed internal cerebral veins, vein of galen, or straight sinus. it is interesting that there have been case reports for recurrent ane and also familial ane. this suggests that there may be a genetic susceptibility and a gene associated with familial seasonal influenza ane cases has been reported (nuclear pore gene, ranbp2; [40] ). this condition is often fatal or accompanied by permanent neurologic sequelae in surviving cases. it is intriguing that the neuroanatomical changes found in the thalami, midbrain, and cerebellum on neuroimaging correlated with the clinical symptoms reported for encephalitis lethargica, specifically ''sleeping sickness'', ophthalmoparesis, quadriparesis, and delayed parkinsonism (see below). it is conceivable that survivors with less fulminant involvement could manifest a clinical syndrome with symptoms and signs that localize to brainstem structures. a pediatric case of 2009 h1n1-associated ane with bilateral thalamic imaging findings without associated malignant brain edema has been published [41] , but detailed clinical follow-up was not reported. during the subacute period, additional classic neurologic syndromes associated with influenza have been described. post-influenzal cerebellitis is quite uncommon and has been reported rarely in adults [42] [43] [44] . this syndrome was diagnosed in a 31-year old woman who developed ataxia, dysarthria, and truncal titubation 1 month after influenza b virus infection, with neurologic symptoms that resolved gradually after an additional month. ct and mri brain imaging were unrevealing. csf studies detected evidence of the persistence of the np gene of influenza b virus in the csf from samples taken 7 and 9 weeks after the onset of initial influenza illness. a 25-year old woman gradually developed gait and speech problems after influenza a illness that was treated with oseltamavir. csf showed pleocytosis. the cerebellar cortex had increased t2 signal which resolved over an 80 day period. she received pulse intravenous corticosteroid therapy. her symptoms resolved [42] . plasmapheresis [45] and ivig [46] have also been used for this condition. some cases of cerebellitis following viral and mycoplasma illness have developed fulminant cerebellar swelling with secondary brainstem compression, obstructive hydrocephalus, with fatal outcome [47] . interventions with posterior fossa decompression and external ventricular drain placement may lead to a favorable outcome in a child with this severe condition. antibodies to the glutamate receptor have been reported in patients with post-infectious influenza viral cerebellitis [44] . guillain-barre syndrome (gbs) is a subacute, immunemediated disease predominantly affecting the peripheral nervous system. the diagnosis and treatment are wellknown to most neurologists and this condition has been extensively described and reviewed. gbs has been rarely reported in association with seasonal influenza virus infection [48] , but it should be noted that influenza testing is rarely pursued in gbs cases and may be unrevealing. treatment for influenza-related gbs is identical to treatment for other gbs due to other associated causes. monitoring for respiratory compromise due to neuromuscular weakness with timely respiratory support if needed is critical. plasmapheresis or gammaglobulin treatments are also helpful. the precise pathophysiology is uncertain, but molecular mimicry of the infectious agent is presumed to stimulate autoimmune responses. this has been demonstrated to occur in campylobacter jejuni-associated gbs [49] . influenza-associated myositis has been reported with seasonal influenza [50] and h1n1 variant [51] . myalgias are a common symptom of influenza, but some patients develop frank weakness and have elevated serum levels of creatine phosphokinase (cpk). it is more common in children but has been seen in all age groups. the calf muscles are most suspectible, and patients may walk with a stiff gait or toe walk. onset is usually within the first week of infection and spontaneous improvement typically occurs within 2 weeks in most cases. rarely, severe cases can result in myoglobinuria-associated renal failure and compartment syndromes requiring fasciotomies. influenza can also selectively attack specific muscle groups such as the heart (myocarditis). muscle biopsy shows necrosis, regenerating fibers, and occasionally inflammation. post-viral parkinsonism has been reported after an assortment of infections including influenza virus [52] . an outbreak of these cases was temporally noted following the great influenza (h1n1) pandemic of 1918-1919 [53] . patients with this condition respond poorly to medical therapy, and it has an unfavorable prognosis. encephalitis lethargica is also known as von economo encephalitis and sleeping sickness [53] . a wave of such cases was reported following the 1918-1919 influenza a (h1n1) virus pandemic. the cardinal features of this condition are altered consciousness with prolonged somnolence and ophthalmoplegia. after intervals of months to years, survivors are at risk of developing parkinsonism. pathological findings include nerve cell destruction primarily in the midbrain, subthalamus, and hypothalamus [53, 54] . using modern laboratory techniques, formalin-preserved autopsy brain specimens of encephalitis lethargica cases analysed for influenza viral rna were negative [54] . scientists have proposed a ''hit-and-run'' model of early viral-mediated injury with late sequelae [54] . the neurologist oliver sacks [55] drew attention to this mysterious disorder and the discovery of l-dopa, in his book, awakenings later converted to a feature-length movie. the delayed appearance of restricted diffusion in the basal ganglia in our patient and others [35] is concerning for this condition (fig. 2) . we do not know if this indicates that our patient with 2009 h1n1 is at risk of developing postviral parkinsonism, but long-term clinical follow-up will be important. a delayed diffusion neuroimaging abnormality was also reported in the dentate nucleus of a patient with seasonal influenza encephalopathy/splenial sign [42] . we present a case of acute encephalitis associated with 2009 pandemic influenza a (h1n1) virus infection, complicated by malignant brain edema. the emerging hypothesis about acute neurologic complications of seasonal influenza is that the immune response triggered by influenza virus infection of the respiratory tract plays a prominent role in the pathogenesis of neurological manifestations. this hypothesis regarding the development of acute encephalopathy and brain edema is analogous to current theories about the role of the immune system and cytokines in the development of ards with 2009 h1n1 virus infection. we have also provided an overview of the spectrum of acute and post-infectious neurologic complications reported in association with seasonal and pandemic influenza virus infection of the upper respiratory tract. neurologists should be aware of the potential for a wide range of neurologic complications in association with the current 2009 h1n1 pandemic and seasonal influenza. 1918 influenza, encephalitis lethargica, 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consecutive cerebellitis produced by glutamate receptor delta2 autoantibody plasmapheresis improves outcome in postinfectious cerebellitis induced by epstein-barr virus brain spect imaging and treatment with ivig in acute post-infectious cerebellar ataxia: case report acute near-fatal parainfectious cerebellar swelling with favourable outcome guillain barre syndrome and influenza virus infection carbohydrate mimicry between human ganglioside gm1 and campylobacter jejuni lipooligosaccharide causes guillain-barre syndrome benign acute childhood myositis: laboratory and clinical features melting muscles: novel h1n1 influenza a associated rhabdomyolysis viral parkinsonism lack of detection of influenza genes in archived formalin-fixed, paraffin waxembedded brain samples of encephalitis lethargica patients from 1916 to 1920 new york: random house, inc key: cord-001634-mi5gcfcw authors: davis, mark d m; stephenson, niamh; lohm, davina; waller, emily; flowers, paul title: beyond resistance: social factors in the general public response to pandemic influenza date: 2015-04-29 journal: bmc public health doi: 10.1186/s12889-015-1756-8 sha: doc_id: 1634 cord_uid: mi5gcfcw background: influencing the general public response to pandemics is a public health priority. there is a prevailing view, however, that the general public is resistant to communications on pandemic influenza and that behavioural responses to the 2009/10 h1n1 pandemic were not sufficient. using qualitative methods, this paper investigates how members of the general public respond to pandemic influenza and the hygiene, social isolation and other measures proposed by public health. going beyond the commonly deployed notion that the general public is resistant to public health communications, this paper examines how health individualism, gender and real world constraints enable and limit individual action. methods: in-depth interviews (n = 57) and focus groups (ten focus groups; 59 individuals) were conducted with community samples in melbourne, sydney and glasgow. participants were selected according to maximum variation sampling using purposive criteria, including: 1) pregnancy in 2009/2010; 2) chronic illness; 3) aged 70 years and over; 4) no disclosed health problems. verbatim transcripts were subjected to inductive, thematic analysis. results: respondents did not express resistance to public health communications, but gave insight into how they interpreted and implemented guidance. an individualistic approach to pandemic risk predominated. the uptake of hygiene, social isolation and vaccine strategies was constrained by seeing oneself ‘at risk’ but not ‘a risk’ to others. gender norms shape how members of the general public enact hygiene and social isolation. other challenges pertained to over-reliance on perceived remoteness from risk, expectation of recovery from infection and practical constraints on the uptake of vaccination. conclusions: overall, respondents were engaged with public health advice regarding pandemic influenza, indicating that the idea of public resistance has limited explanatory power. public communications are endorsed, but challenges persist. individualistic approaches to pandemic risk inhibit acting for the benefit of others and may deepen divisions in the community according to health status. public communications on pandemics are mediated by gender norms that may overburden women and limit the action of men. social research on the public response to pandemics needs to focus on the social structures and real world settings and relationships that shape the action of individuals. conclusions: overall, respondents were engaged with public health advice regarding pandemic influenza, indicating that the idea of public resistance has limited explanatory power. public communications are endorsed, but challenges persist. individualistic approaches to pandemic risk inhibit acting for the benefit of others and may deepen divisions in the community according to health status. public communications on pandemics are mediated by gender norms that may overburden women and limit the action of men. social research on the public response to pandemics needs to focus on the social structures and real world settings and relationships that shape the action of individuals. the re-emergence of infectious diseases is a leading public health problem. pandemics and epidemics [1] including avian influenza, sars, ebola, and pandemic influenzaand the rise of anti-microbial organisms [2] now threaten the health of populations around the globe. it has been argued that the re-emergence of these diseases marks the end of the golden age of medicine and the dawning of a period where health and security will be undermined by resurgent infectious diseases [3] . pandemic influenza stands out in this situation because: it spreads quickly around the globe affecting many millions of people; it is associated with, potentially, high mortality, and; the world experienced a highly publicised, though ultimately mild for most, pandemic influenza in 2009/10. it is believed that another, more serious influenza pandemic is inevitable, though no-one, as yet, can predict when it will occur. for these reasons, explaining infectious diseases threats to the general public and encouraging them to adapt their health behaviours is high on the public health agenda. in relation to pandemic influenza, public communications feature in preparedness and response planning which requires that members of the general public adopt measures during a public health emergency, including: hygiene (e.g., covering the mouth and nose when sneezing or coughing, washing hands, keeping surfaces clean, avoiding sharing personal items) and the avoidance of close contact with others [4] . understanding how populations respond is also crucial for the science that supports response planning. for example, mathematical models, which underpin pandemic response planning, factor in biological, psychological and sociological assumptions of how populations respond to infectious diseases [5, 6] . effective communications with the general public and understanding how they respond, therefore, have a pivotal role to play in the management of pandemic influenza, in particular, and in the area of emerging infectious diseases, in general. however, knowledge of how to best communicate on pandemics with the general public and how they take up these messages is an emerging field with some inconsistencies [7] . evaluations of the public health response to the 2009/10 pandemic influenza claim that public communications were largely successful in preparing and reassuring publics during the emergency [8, 9] . these findings need to be read against the fact that the pandemic was a short-lived and ultimately mild public health emergency for most people. there is a view, also, that members of the general public are resistant to pandemic risk messages. some commentary has suggested that the general population is increasingly resistant to public policy on global threats, including climate change and emerging infectious diseases [10] . surveyswhich dominate the social scientific view on public responsesconducted during the 2009 pandemic indicate that populations in the uk and australia were complacent with regard to h1n1 and reported insufficient behavioural responses [11] [12] [13] [14] [15] . broad brush, risk communication research has identified that material circumstances and symbolic framing of risk [7] , inequalities in education and access to media [16] , (mis)trust in media and governmental advice [17, 18] , all shape how members of the general public respond to communications on pandemics. close-focus, qualitative research offers the view that while the general public endorses governmental advice, in the circumstances of the 2009/10 pandemic they were also unlikely to act in the ways advised by governments [19, 20] . there are additional explanations for the apparent resistance on the part of the general public. for example, because they are bombarded with so many messages, including those pertaining to pandemics, members of the general public may by subject to 'health threat fatigue' [21] . this is not the same as resistance. it is, instead, a dulling of alertness seated in screening out of overwhelming and competing risk messages. members of the general public appear to digest and critically reflect on risk communications messages [22] , and tailor risk reduction strategies to their personal circumstances [23] . it is also argued that the general public is only too aware of the 'boy who cried wolf ' syndrome [24] , where too frequent assertion of danger leads publics to dismiss public health warnings. in addition, audience reception of communications on health is framed by the historic rise of individualism in society [25] and health systems [26] . individualism implies that members of the general public take on the view that responsibility for their health is a matter of personal volition and effort. this view is often utilised in health communications that call on people to take care of themselves, but it is a perspective that can obscure factors that are not within the control of the individual. it is also an approach to risk that has a moral loading and therefore a negative effect for those who are unablethrough choice or otherwiseto avoid health harms. exactly how individualism plays out in relation to pandemic influenza warrants further inquiry. because it is so vital that public health authorities communicate with members of the general public as effectively as possible and as there are competing explanations and routes of inquiry available in the literature, it is necessary to re-examine the apparent resistance to communications and advice on the part of the general public. a central challenge is to get beyond prevailing assumptions and build up a theory of public engagement informed by the life worlds of the general public [7] . understanding why populations fail to sufficiently enact precautions must involve taking account of how lives are lived and the meanings ascribed to the threat of infectious diseases. indeed, what might look like lack of precaution may turn out to be reasonable given the material and symbolic circumstances of affected individuals and populations. a related challenge, then, is re-examining how public health characterises the general public in research on pandemics and in the more general area of emerging infectious diseases. taking these steps is vital to ensure that the public health response and its communications with the general public are as resonant, meaningful and effective as possible. this paper, therefore, uses inductive, qualitative research methods to develop new knowledge on how members of the general population respond to pandemic influenza, set against the backdrop of the assumed resistance on the part of the general public and related critiques, including, health risk fatigue, the risk communication dilemma and individualism. the analysis poses the question: how do members of the general public respond to the threat of pandemic influenza and to the hygiene, social isolation and other measures proposed by public health? by addressing this question in the manner indicated, the paper offers an alternative framing of pandemic influenza perceptions and behaviours in an effort to contribute to the better health of individuals and populations facing risk of infectious diseases. the following analysis was generated in international research (australian research council discovery project dp1101081) focusing on the responses of members of the general public to the events of 2009 alongside interviews with researchers, clinicians and policy-makers [27, 28] and analyses of the public policy texts on pandemic influenza control [29] . this research has examined general public data in light of sociological and psychological perspectives on responses to pandemic influenza [23, [30] [31] [32] [33] . the present paper synthesises and builds on the research undertaken on the general public, in particular, and introduces new data analysis to address the public health challenge of effective communication and engagement with members of the general public. interview and focus group participants were recruited through community sampling in melbourne, sydney and glasgow. generating data in australia and scotland addressed the international dimension of pandemic influenza and the events of 2009. australia was closely observed by other nations as early stages of the global pandemic in 2009 coincided with the southern hemisphere influenza season. the pandemic quickly affected melbourne, which reported a high and early peak of known infections [34, 35] . the city, for a time was known as the 'flu capital of the world.' the first confirmed cases in the uk were in scotland among passengers on a flight from mexico to glasgow [36] . the uk and australia reported 457 [8] and 191 [37] deaths, respectively, associated with the 2009 h1n1 pandemic. our analysis of interview and focus group texts reveals more convergence than difference between melbourne, sydney and glasgow, perhaps because the pandemic was managed in those cities by public health professionals who were members of a global pandemic response network. the research aimed to identify how members of the general public respond to pandemic influenza so that public health communications can be designed to engage with how its audiences respond to risk messages and how they enact hygiene, social isolation and related measures. four purposive criteria were used to select respondents in each city: women who were pregnant during 2009 (or with a new baby); older members of the community (71 years of age and older); people with compromised immune systems and or respiratory illness such as asthma; and people who self-identified as being 'healthy' (e.g., no disclosed health problems) and who did not belong to one of the former categories. in addition, selection of participants was conducted to ensure: a balance of male and female participants and a range of ages from 18 years upwards. drawing on interviews and focus groups ensured depth and breadth. interviews explored in-depth discussion of personal experiences of living through the h1n1 pandemic, seasonal influenza and related concerns. focus groups examined social norms concerning precautionary behaviours regarding pandemic influenza. between april 2011 and may 2012, 116 people participated in the research (see table 1 ) in 57 interviews and ten focus groups (with 59 participants). interviews included people from the purposive criteria (pregnant = 14; 71+ = 3; hiv/respiratory illness = 17; healthy = 23); a gender mix (women = 34; men = 23), and; an age range of 18 to 71+ years. focus groups included people from the 71+ group (10); hiv/respiratory illness (37) and the healthy group (22); a gender mix (women = 36; men = 23), and; an age range of 18 to 71+ years. this pattern of participation reflects the challenges of recruiting women who were pregnant in 2009, the very elderly and men. seven respondents reported having been diagnosed with h1n1; none through a laboratory-confirmed test (a reflection of our community sampling). a further eleven interviewees reported that a relative, friend or other social contact had been diagnosed, clinically. it needs to be acknowledged, however, that, as influenza is not ordinarily diagnosed with a laboratory confirmed test [38] , public health professionals and members of the general public identify and manage the infection on the basis of symptoms. indeed, respondents noted difficulty determining whether they had had influenza participants were asked to speak about their experiences with influenza and the public health response to the 2009 pandemic. topics for discussion included: health background (including pre-existing medical conditions, other infectious diseases, influenza vaccination); influenza experiences (including knowledge of pandemic influenza, sources of knowledge, experiences with the 2009 pandemic and seasonal influenza, prevention of infection, caring for self and/or someone else with infection); public communications (including broadcast and electronic media, public health advice, advice from gps, workplace and schools). verbatim transcripts of interviews and focus groups were analysed using an inductive, theory-building method. all transcripts were open coded to generate themes for analysis. interpretive memoranda were generated which explained each theme and how it connected with existing perspectives on the general public response to pandemic influenza. the research team reviewed these themes and memoranda to ensure that the themes were understood and that they could withstand refutation. this discussion also provided the basis for an agreed coding scheme that was used to re-code all data. key themes were identified for subsequent, in-depth written analysis in the form of technical reports and draft manuscripts. our approach to coding, memo writing and in-depth analysis sustains a dialogue between theory (pre-existing categories derived from social science theory and the relevant literature) and data (inductively-derived themes). this approach avoids the traps of overly dataor theory-driven analysis and ensures that the research has relevance to the field. this paper, therefore, is based on in-depth, nuanced analysis of interview and focus group texts that offers new perspectives and propositions, which provide the basis for interrogating prevailing assumptions regarding the general public response to pandemic influenza. this approach is consistent with social inquiry of the highest standard [39] . the assumed complacency and resistance on the part of members of the general public was not in evidence in the narratives provided by our research participants. other factors, centred around health individualism and contextual factors such as gender and biomedical situation do appear to influence how people respond to the threat of pandemic influenza. in what follows, we focus on themes that establish and complicate the role of health individualism and its effects in the responses of members of the general public to pandemic influenza. the interviews and focus groups revealed a tension to do with self and other in relation to the threat of pandemic influenza. as we have discussed elsewhere, respondents endorsed the pandemic control measures advocated by public health authorities [23] . they agreed that hygiene control measures (coughing and sneezing etiquette) and social distancing were valuable. this endorsement held in australia and scotland. characteristically, however, respondents did not believe that pandemic influenza could be prevented in the long run. they believed that the influenza virus was easy to catch and that hygiene measures and social isolation were difficult given that social interaction was needed to sustain work, schooling, the family and daily life. for this reason, respondents focused on strengthening their immunity through, for example, taking vitamins and eating healthy food: i think if you're healthy, keep up your vitamins and eat the right foods, drink healthily, eat healthily and live healthily. exercise. you've got to do all those things. (heather, 71+, melbourne) this immunity boosting was seen as a prudent defence against the seemingly inevitable moment of exposure and a means of coping with infection when and if it occurred. importantly, this focus on one's body and immunity in the face of seemingly inevitable infection accentuated health individualism, encouraging members of the general public to focus on their body's abilities to resist and cope with infection. there was evidence that immune boosting has the status of a social norm as those who were seen to succumb to infection were sometimes judged as failing to adequately care for themselves, even though it was admitted that the virus was easy to catch. to some extent individualism is an asset for public health interventions that seek behaviour change at the individual level. however, an individualistic approach to pandemic risk may obscure factors that the individual cannot control and, as indicated by the judgement of those who acquired infection, health individualism may be moralising. health individualism was not the only factor influencing how members of the general public perceived risk for pandemic influenza and took action. respondents who had responsibilities for others (e.g., pregnant women, people in couples or caring for people with health problems, families with children) or who saw themselves as vulnerable to influenza (e.g., respiratory illness, immune disorders) focused on social units such as the couple, family and colleagues at work: well given that the flu broke out at xxxx street primary school and my son was three and he was at xxxx street childcare, i pulled him out. so when my husband picked him up that day i was at work. i said, 'take him home. give him a bath. wash his clothes.' yeah. i stopped sending him and i was one week off my maternity leave so i stopped work a week early … i didn't go to the supermarket, didn't really mix. (gill, pregnant, melbourne, 31 -40 years) it appears, then, that both health individualism and relationships with important others influence what people do. in this regard, social proximity appears to be important, that is, those others who are close to oneself in terms of social and emotional ties and living situation are factored into health precautions. this social proximity also showed up in the ways in which respondents saw geographical distance and low population density as protective. those respondents living further away from the populous 'epicentres' of infectioncentral melbourne, for examplebelieved that they were less likely to encounter someone with the virus. ' we're familiar with chest infections' one important way in which this tension between responsibility to oneself and to others came to light in interviews and focus groups related to differences between the responses of those with pre-existing conditions and those who identified as 'healthy.' those who faced increased risk of serious disease focused on their relationships with othersincluding strangers they might encounter in public spaceslargely in an effort to protect themselves. those with no vulnerabilities showed themselves to be archetypally focused on their individual health. for example, people with severe respiratory illness reported that engagement with the risks of influenza was a 'well trodden path' for them: as lung patients, we're, we're familiar with chest infections and, as joy says, we could, we could have a flu and not know it. and the gp checks us over. and the only way that i know that they'll know whether it's a chest infection or flu, or pneumonia, is for an x-ray. (arthur, lung disease, melbourne, 71+ years) people with pre-existing lung conditions, then, were commonly hyper-vigilant during the 2009 pandemic and their accounts were peppered with examples of how social interaction was imbued with risk for them and also some resentment that the healthy majority seemed to not understand the significant threat that influenza infection might pose to their health [33] . people with immune disorders in our sampleprimarily hivunderstood they needed to be vigilant but saw influenza as a lower priority than their hiv infection and its effective management. older respondents (71+) conveyed judicious vigilance tempered with an unwillingness to be seen to overreact. important in these accounts was awareness of the vectors of transmission and that one's health was to some extent dependent on those with whom one interacted. in contrast, the healthy majority of our respondents saw pandemic influenza as a personal, though distant, health threat. they saw themselves 'at risk' and possibly as 'a risk' to close family, but not as 'a risk' to unknown others (e.g. a person sitting beside them on public transport). this focus on the 'at risk' self to the exclusion of the self as 'a risk' to others underlines how health individualism manifests in the responses of the 'healthy' majority of the general public. this focus of the healthy on their own health risks (at the expense of others) surfaced in narrative on expectations of recovery from influenza: like you sort of just, you think, maybe you just think influenza as a common cold sort of thing. and it's like, 'it'll pass. i might go to the doctor's and get some, something to help me get through it, ' or something. but yeah, i don't know … it's just like, 'just ignore it and push through.' (chris, healthy, melbourne, 18 -30 years) this interview participant shows how a healthy individual engages with pandemic influenza as a commonplace and personal risk, in contrast to those with pre-existing conditions who have to take pandemic, and even seasonal, influenza seriously. this expectation that one can 'push through' reinforces the previous theme noted with regard to the focus on the capacity of one's body to deal with infection. it is also an orientation to influenza risk that sets the scene for individuals to determine that infection is a risk worth taking since recovery is likely. also, recovery expectations synergise with the belief that infection is difficult to avoid in the long run. this means that people may assume that, while non-pharmaceutical strategies of pandemic control are sensible, their limited utility is set against the likelihood of recovery. this nexus of risk calculation helps explain why segments of populations appear to be complacent in surveys, as noted above. they may in fact be making multi-layered risk assessments of the likelihood of infection, their health status and expectations of recovery. another important provision on health individualism was the gendered meanings of one's response to infection. particularly in domestic settings, the management of respiratory illness was largely feminised. women provided elaborate accounts of managing the respiratory infections of family members while men did not. importantly, the pejorative term 'man flu' was used to denote the over-inflation of mild symptoms to gain sympathy and respite from normal activities, with connotations of questionable masculinity: it's always a little difficult to tell when you're moving from, sort of, a cold through the man flu to proper influenza. (vincent, healthy, sydney, 41 -50 years) these findings imply that responses to pandemic influenza in real world settings areas with other health problemsassociated with gender roles which shape behaviour, for example, women may be expected to perform infection control and symptom management, while men are expected to not show their symptoms and 'soldier on' or face accusations of 'man flu.' the uniform implementation of social distancing and other protective measures may therefore be compromised. accentuating the role of gender in response to messages concerning pandemic influenza, pregnant women found themselves thrust into a position of particular risk during the 2009/10 pandemic, at a time when they were already taking responsibility for the well-being of their unborn child. in particular, the prospect of vaccination elicited varied, often emotion-laden, responses: well, (sigh) when you're pregnant everything is about the baby … you just want to try and make your baby as healthy as possible and you want to try and keep your baby safe. (rebecca, pregnant in 2009, glasgow, 31 -40 years) the imperatives of good motherhood and responsibility for their unborn children placed these women into the emotionally-charged position of having to make decisions regarding virus protection in circumstances of intense uncertainty [32] . some distress was apparent among the pregnant women respondents, but also great resilience and active use of public policy information to protect themselves and their babies. as rebecca's account, above, indicates, health individualism in tension with responsibilities to others, gender and one's life situation played out in engagements with vaccination. though recollection was variable, 64 respondents in the present research (55%) reported that they had had an influenza vaccination at some point in their lifetime and there was no evidence of 'in principle' resistance to vaccination. this is a notable finding given that participants were sought in community settingswhere those with anti-vaccine views are thought to be locatedand in light of commentary that members of the general public are resistant to the science and technology used to manage global threats. indeed, endorsement of public health measures and attempted compliance characterised the respondents' accounts, with the provisos on the practical value of non-pharmaceutical strategies of infection control and management, as already discussed. but, taking on vaccination was not always straightforward: i saw in the press releases about the vaccine and i remember ringing the clinic and they said,'well if we were to give it to you, you'd have to come to the hospital and that's gonna put you at risk of getting exposed to it so we'd rather you not come in for the, for the vaccine.' and i was thinking,'well that's a bit of a catch importantly, though, vaccination, like non-pharmaceutical infection control, was mostly discussed as a personal strategy of health protection. apart from those with pre-existing vulnerabilities, vaccination was not readily understood as a method for protecting others and therefore society. this individualistic focus on one's own health implies that efforts to promote 'herd immunity' may not accord with perceptions and behaviours of the healthy majority. the findings question the prevailing view that the general public resists risk communication with regard to pandemic influenza. nor do the related ideas of complacency and fatigue seem relevant. more salient was multi-layered risk management informed by health individualism and to some extent tempered by interpersonal responsibilities, one's personal circumstances, gender, expectations of recovery, and prior experiences with influenza. as others using qualitative methods have also suggested [19] , respondents did not reject what was done by governments in 2009. they show interest in pandemic influenza, though their mode of engagement with it varied. they indicated that they wished to be informed but reserved the right to interpret and apply advice according to their own situation. public health guidance on hygiene and social isolation was endorsed, though its utility was largely found to have practical, long-term limitations given that social interaction was fundamental to daily life and the transmission of the virus. resistance and the related notions of complacency and fatigue, then, appear to have limited value for explaining how members of the general public respond to pandemics. part of the reason for this inapt attribution of research results to public resistance concerns research approach. forced choice surveys produce measures of hypothesised variables thought to influence behaviour. in-depth interviews and focus groups yield a different picture, where general public perceptions of the dangers of pandemics are placed in the context of what appears to be endorsement of the efforts of public health, tempered with awareness of the practical difficulties of managing influenza on a local basis. personal experience narratives reveal members of the general public to be engaged and willing to apply guidance in real world settings, though also aware of limits on what might be possible in time of pandemic. going beyond the idea of resistance, our analysis offers an alternative framing of how members of the general public respond to pandemic influenza. health individualism complicated by life circumstances (family life, health status) and the gendering of the meanings and practices surrounding the experience of influenza and how to deal with it in real world settings, appear to be important. risk communications are likely to benefit by addressing these influences on risk management behaviours. in particular, emphasising individual responsibility in risk communication may amplify divisions between people with different biomedical vulnerabilities and encourage those who consider themselves healthy to think of themselves as 'at risk' but not 'a risk' to others. this is a major hurdle for public health, particularly when hygiene, social isolation and vaccination are likely to become more important methods for controlling the spread of re-emerging infectious diseases. the pejorative, gendered meanings of influenza, of which 'man flu' stands as exemplary, point towards the deeply inscribed gendering of responses to infectious diseases. the role of gender in social aspects of health care is no surprise, but fully-fledged gender analysis is yet to be acknowledged in the public health address to the general population with regard to pandemics. in particular, messages to enact hygiene and social isolation are likely to accentuate already feminised health care in the domestic sphere. further, it is not simply that women are burdened with the labour of influenza care and men not. if men do find themselves unwell they risk accusations of 'man flu' and may therefore avoid making themselves available for health care interventions, a dynamic which keeps men out of the gp clinic in general [40] . as recent reviews have indicated [7, 16] , the influences of social factors on responses to pandemics need to be foregrounded in the social research agenda for better public health. our research indicates that health individualism and gender need to be part of this new research agenda. our findings also point to several further, specific, challenges for risk communication: ideas of proximity to risk; expectations of recovery, and; vaccination. proximity appears to be a blind spot in risk communications. public health messages of emergency are filtered by perceptions of proximity to threat, consistent with psychological theory [41] and cultural constructs where the source of contagion is placed at a distance from self [42] . we found that these ideas of proximity did surface in the narratives of members of the general public. yet, we know that, for example, within six weeks of the infection being detected in australia, people in remote communities in australia were found to be infected [43] . risk communication needs to attend to these ideas of distance from risk and the related underestimation of the speed with which the influenza virus can travel in a hyper-connected world. expectations of recovery from influenza also appear to dominate narratives. as others have argued [44] , healthy respondents recognised influenza infection as severerequiring bed and restbut thought that they would eventually recover. this finding implies that members of the general public may interpret infection as a risk worth taking, that is, that they can cope with infection if prevention fails them, due to their own choices or otherwise. members of the general public appear to be actively engaged with manifold risks that they juggle and prioritise in real world settings. our findings also suggest that taking up vaccination is not a simple matter, even among those who endorse the use of the biotechnology. survey findings have found that approximately 42% of australians are concerned about general vaccine safety [14] and that australian [45] and worldwide [46] rates of h1n1 vaccination have been found to be insufficient, prompting concerns that the 'anti-vaccine lobby' and other detractors are influencing use of this biotechnology. as noted, a slight majority of our respondents reported that they had been vaccinated in their lifetime and none spoke of vaccination as dangerous, though, of course, some may have held these views and not revealed them or opted out of our community-based recruitment strategies. our research, however, points to more immediate and practical considerations that shape how and when people vaccinate, including considerations of relative risk and whether or not a new vaccine should be used in pregnancy. attending to these more immediate concerns may be beneficial for public health, though we acknowledge that public perception of vaccine technologies is also an important public health agenda. the analysis presented is retrospective as the interviews and focus groups were conducted after the end of the pandemic on 10 august 2010 [47] , and therefore when it was known that the mortality rate had at first been overestimated [48] . importantly, too, the respondents were volunteers selected according to purposive criteria, implying that the sample is not representative and that generalisations to populations are not strictly tenable. what the analysis offers, however, is the opportunity to drill down into how people make sense of pandemic influenza, therefore providing the basis for building theory on how members of the general public, think, feel and act in the contemporary era of efforts to manage global health threats. the perspectives identified here help situate what we know in social context and alert public policy to some dilemmas and alternative explanations of why members of the general public do what they do. for public health to shape the actions people take prior to and during a pandemic, we need to understand and engage with the perspectives of those acting. viewed from the outside, the behaviour of the general public has been cast as resistant. however, viewed from the perspective of ordinary people involved in anticipating and responding to infection, it is clear that public health has engaged its publics. this engagement is frequently informed by individualistic ways of assessing and responding to risk, social norms (e.g. gender roles), knowledge of the clinical uncertainties of influenza infection, and reasoned thinking about the limits of preventing influenza transmission. the current challenge for pandemic influenza preparedness and response is not so much to address public disinterest, but to acknowledge and engage with members of the general publics' experiences of influenza and how they make sense of, and act on, pandemics in real world settings. factors in the emergence of infectious diseases antibiotic resistance: long-term solutions require action now world 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the 2009-2010 influenza pandemic: effects on pandemic and seasonal vaccine uptake and lessons learned for seasonal vaccination campaigns world health organization. h1n1 in post-pandemic period: director general's opening statement a virtual press conference influenza a(h1n1): lessons learned and preparedness submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www this research was funded by an australia research council discovery project grant (dp11010181) with additional funding from glasgow caledonian university. we are grateful to casimir macgregor for assisting with interviews and to everyone who agreed to participate in interviews and focus groups. the authors declare that they have no competing interests.authors' contributions md helped conceive of this research, drafted this manuscript, managed the data collection and analysis in melbourne and integration with all data, and is a grantholder. ns helped conceive of this research, contributed sociology of public health perspectives to the manuscript and edited it, managed data collection and analysis in sydney and integration with all data, and is a grantholder. dl collected and analysed data used in this paper, conducted a literature review used in this paper, and contributed to the draft manuscript. ew collected and analysed data used in this paper and contributed to the draft manuscript. pf helped conceive of this research, contributed health psychology perspectives to the manuscript and edited it, managed data collection and analysis in glasgow, and is a grantholder. all authors read and approved the final manuscript. key: cord-002408-bbtslrrt authors: almogy, gal; stone, lewi; bernevig, b. andrei; wolf, dana g.; dorozko, marina; moses, allon e.; nir-paz, ran title: analysis of influenza and rsv dynamics in the community using a ‘local transmission zone’ approach date: 2017-02-09 journal: sci rep doi: 10.1038/srep42012 sha: doc_id: 2408 cord_uid: bbtslrrt understanding the dynamics of pathogen spread within urban areas is critical for the effective prevention and containment of communicable diseases. at these relatively small geographic scales, short-distance interactions and tightly knit sub-networks dominate the dynamics of pathogen transmission; yet, the effective boundaries of these micro-scale groups are generally not known and often ignored. using clinical test results from hospital admitted patients we analyze the spatio-temporal distribution of influenza like illness (ili) in the city of jerusalem over a period of three winter seasons. we demonstrate that this urban area is not a single, perfectly mixed ecology, but is in fact comprised of a set of more basic, relatively independent pathogen transmission units, which we term here local transmission zones, ltzs. by identifying these ltzs, and using the dynamic pathogen-content information contained within them, we are able to differentiate between disease-causes at the individual patient level often with near-perfect predictive accuracy. ltzs are defined by the property that the transmission rate of an infectious disease within an ltz is significantly greater than the average transmission rate of the disease between the different ltzs. this means that the population within an ltz is in relative terms highly connected, and we suppose sufficiently connected to justify the assumption of 'random mixing, ' so that an invading pathogen could come into contact reasonably rapidly with most of members of the ltz. however, an invading pathogen may have difficulty in spreading beyond the confines of the relatively isolated ltz. extreme examples of ltzs have been documented in the literature and include confined military bases or cruise ships [21] [22] [23] [24] or other small isolated communities (e.g. religious or ethnic) through which diseases rapidly propagate, reaching most members of the population. to investigate the possibility of ltzs in a large regional area, we analyze clinical data from a healthcare medical center in the city of jerusalem, containing the clinical test results for influenza virus and respiratory syncytial virus (rsv) from patients presenting with ili symptoms. using a 'k-means clustering' algorithm, putative ltz groups are identified solely based on the physical distance between home locations of the patients. the methodology is akin to the procedure of 'community detection' applied in the study of complex networks and designed to locate highly connected clusters of nodes 25, 26 . our analysis finds that while influenza and rsv incidences tend to overlap and show more or less equal number of cases over the whole region, individual ltzs show a far more homogeneous disease content at most given times, with some being dominated by rsv while others by influenza. we use these findings to arrive at a prediction algorithm that, applied to patients presenting at the hospital with ili, is capable of differentiating between cases of influenza and rsv, often with near-perfect accuracy. defining local transmission zones. the transmission dynamics of respiratory pathogens in a population are constrained by the physical distance between infected and susceptible individuals. an ltz for a given pathogen represents a group of individuals within the general population, such that the transmission probability of the pathogen within the group is greater than transmission probability between that group and any of the other groups. thus we suppose the population of a region can be subdivided into a set of k groups or ltzs, such that for any two ltzs i and j: here p(ltz i , ltz j ) is the average probability that an individual from ltz i infects an individual from ltz j . in our case we are given the geographic coordinates of a group of individuals, and we assume that the probability of transmission between two individuals is proportional to the euclidean distance between them. to divide the population into a set of k distinct ltzs we make use of an optimization technique known as k-means clustering that calculates the k different geographic zones while attempting to ensure that eq. 1 holds in an optimal fashion 25 . for any preassigned k we are able to divide the set of all patients home-locations into a set of k ltzs using the aforementioned clustering method, after determining all pair-wise euclidean distance between address locations of the full patient set (see also methods). thus the resulting ltzs represent k groups of patients, partitioned purely on the basis of the physical proximity between these patients' home locations 27, 28 . note that when examined over the entire period , the spatial distributions of influenza and rsv are very similar and we have found that the ltzs obtained using only the influenza or rsv data for clustering purposes are very similar to those obtained when using the entire data (not shown). the clustering method used was effective at identifying geographically distinct areas as clusters, e.g. neighborhoods outside the jerusalem municipal boundary (fig. 1 , highlighted area). here we chose k = 36 ltz groups as a representative example because it guaranteed that the smallest ltz consisted of at least 100 individuals. interestingly, the clustering algorithm was also able to make meaningful distinctions within the municipal boundaries, e.g. between the 'bet-safafa' and 'gilo' neighbourhoods (indicated on map as circles). hadassah-hebrew university medical centers between 2009 and 2012 is shown (fig. 2a) . the unusual dynamics in 2009-10 may be the result of the unusual ili dynamics during this season, caused by the then emerging h1n1 influenza pandemic strain (h1n1pdm, ref. 12). in the 2009-10 season, we note the occurrence of first an influenza epidemic followed by an rsv epidemic, with very small overlap between the two epidemics. as such there is a notable time-delay between the peaks of the epidemic curves of influenza and that of rsv. in contradistinction, over the 2010-11 season, the influenza and rsv epidemics peak at almost the same time and overlap almost totally. ideally we expect to find that within a single well defined working ltz, the two disease signals show relatively small overlap. the underlying concept is that any pathogen arriving at a susceptible ltz is able to spread rapidly through the entire local population. this domination within an ltz is to be expected since disease transmission within an ltz is stronger than transmission between them. such a situation would be particularly favoured if only a limited number of infected individuals invade a susceptible but heterogeneous region. the pathogen dominating an ltz is likely to be the first successfully invading pathogen. in this extreme case, there would be zero overlap of the diseases in any ltz, because each ltz has only a single pathogen. here, our working assumption is that transmission between ltzs has relatively minor impact at these time-scales. hence, even if at the whole region scale (i.e. k = 1) disease signal overlap is high, as say in 2010-11, in the individual ltzs within the region the disease signal overlap should be expected to be far smaller, ideally close to zero. this motivates us to develop a quantitative index for measuring disease signal overlap (so). to do so, we let the number of influenza and rsv cases at time t be represented by i(t) and r(t) respectively. let ρ i,r (τ) be the lagged cross-correlation between i(t) and r(t − τ), that is, the cross-correlation between the influenza and rsv time series when there is a time-delay τ between the signals. the signal overlap so i,r between the two time-series is then defined as (see also methods): , , here ρ i,r (0) is precisely the usual pearson correlation between i(t) and r(t) and this is divided by the maximum such correlation possible when the time-series are delayed for a time τ, ranging from minus to plus 13 weeks. the index is first used to examine the disease signal overlap when the whole region is considered a single ltz (i.e., k = 1). the overlap for influenza and rsv ranges from a minimal value of so = 0. disease ratios. disease signal overlap is a useful index for studying and comparing the intersection of two diseases over a season. for shorter term dynamics we make use of the disease ratio index (dr) of the weekly incidence of the two pathogens. for a particular week, the disease ratio for influenza and rsv is defined here as (see also methods): where [i] and [r] are the number of new cases of influenza and rsv respectively, for that week. the disease ratio quantifies the degree to which one pathogen is dominant over the other during a period of one week. when only influenza is detected over a week, the disease ratio is dr = + 1, when only rsv is detected then dr = − 1, and hence the absolute value of the dr is maximal when only one pathogen is present (|dr| = 1). if the pathogen incidences are equal over the week then dr = 0. we show the disease ratio for the whole region (k = 1) colour-coded and plotted as a function of time in consecutive weeks (fig. 3a) . in the first half of the 2009-10 season there are only cases of influenza and dr = + 1 (red), whereas for the second half of the season there are only rsv cases (dr = − 1, blue). in contrast, during most of the 2010-11 season dr = 0 (green), as expected given the almost complete overlap of the influenza and rsv disease signals. in the 2011-12 season, despite the high degree of overlap in the entire season, there is initially a period of rsv dominance, reflected in a negative dr (blue). this dominance erodes into a disordered, mixed pattern where neither pathogen achieves strong dominance for any contiguous period. it is informative to repeat the above qualitative examination of the whole region but in terms of its individual ltz groups. plotting the disease ratio for k = 36 ltzs as a representative example (fig. 3b) , reveals substantial variation in the dr of different ltzs. in the first season (2009-10), nearly all ltzs reflect the pattern observed for the k = 1 analysis. namely, for the first part of the year dr = + 1 (influenza dominance) while for the second half of the year dr = − 1 (rsv dominance). note however, that the individual ltzs show a large degree of variability in terms of when the appearance of the first pathogen was detected in a patient, and when the dominance pattern changed from influenza to rsv. the 2011-12 season shows a different phenomenon. most importantly, even when there are roughly equal number of influenza and rsv cases in the whole region, the dr in individual ltzs will often show a more binary pattern, with long periods where some ltzs contain only influenza (red), while others only rsv (blue). the inset in fig. 3b shows in detail a period of over 2 months (2011-12 season) where the single area dr for k = 1 is near zero while 3 of the 4 highlighted ltzs contain (almost) only rsv, and the remaining ltz contains only influenza consistently over the entire season. we make these visual observations concrete by calculating the average per-season absolute disease ratio (|dr|) and making quantitative comparisons between analyses based on the whole area (k = 1) and analyses based on an ltz approach (k > 1). we are particularly interested in knowing when the ltz is dominated entirely with influenza or dominated entirely with rsv. for either case the absolute disease ratio attains the value |dr| = 1. dividing the whole region into ltz groups (k > 1) led to a significant increase (p < 0.01, n = 30, using 2-sided student t-test) in the absolute value of the disease ratios compared to the whole region (k = 1) results for all k values (fig. 3c) . for the 2009-10 season the ratio increased from |dr| = 0.7 in the whole region to |dr| = 0.9 using k > 12 ltzs. for the 2010-11 season, there was an increase from |dr| = 0.5 to |dr| = 0.8 for k > 24, and from |dr| = 0.6 to dr > 0.9 for k > 12 in the 2011-12 season. shortly, we use the concept behind this index to assess the likelihood of someone residing in that ltz to be infected by influenza or infected with rsv. frequency-based differentiation of influenza and rsv. the above results show that compared to the regional level (k = 1), there is a reduced coexistence of influenza and rsv when examined at the ltz level (k > 1), leading to lower signal overlap (fig. 2b) , and increased disease ratios within ltzs (fig. 3) . together, these results support our hypothesis that patients from the same ltz will have a strong propensity to carry the same pathogen. this suggests that the pathogen incidence within an ltz may be better predicted by considering the data from that ltz, rather than the more abundant, yet less specific data collected at the whole region level (k = 1). we now make use of the ltz concept to implement a test-algorithm designed to predict whether a patient has influenza or rsv given that a person arrives at hospital with ili symptoms, based solely on previous data kept in the hospital database. the test depends on determining the specific ltz that the patient resides in and recent information about the pathogens present in that ltz. if the ltz hypothesis is correct, ltz-based predictions should significantly outperform predictions based on the whole region data, where ltzs are ignored. if on the other hand, the ltz hypothesis is incorrect, then an attempt to predict a patient's disease by using ltz-specific pathogen content would be no better than predictions where the whole region is considered as a single unit. the test proceeds on a day-by-day basis beginning from the earliest time-point in the data (january 2009) and advances in chronological order (up to may 2012). predictions are made on each day t for all patients that arrive in the hospital with ili symptoms on that day. predictions for a newly presenting patient arriving on day t are made as follows: (i) the ltz associated with the patient is determined. it is to be expected that the per season accuracy of the predictions will improve with smaller signal overlap and increased disease ratio. the per season predictive accuracy for the three seasons as a function of the number of ltzs k are presented (fig. 4a) . during the first season, where signal overlap was minimal, the predictive accuracy was greater than a = 80% in all tests, ranging from a minimum of approximately a = 83% for the whole region, up to a = 96% for k = 48 ltzs. this high accuracy, even for the whole region, reflects the low signal overlap between rsv and the pandemic influenza strain that emerged in 2009 12 . during the 2011-12 season, the whole region (k = 1) predictions were accurate only in about a = 60% of cases, whereas the ltz-based predictions (k > 1) reached accuracy of over a = 90%. this is consistent with what might be expected given the great reduction in signal overlap during that season for larger k values (fig. 2b) . during the 2010-11 season, where the signal overlap was almost unity, predictions based on the whole region were similar to predictions made at random, with an accuracy level of a = 50%. the ltz-based approach (k > 1) preformed significantly better but only achieved an accuracy of a = 60% correct predictions (k = 48). the increase in predictive accuracy when using the ltz-based approach was significant (p < 0.01) for all values of k in 2010-11 and 2011-12, but only for k = 24, 36 and 48 during the 2009-10 season (fig. 4a, significant changes marked with asterisk) . these results are consistent with the differences in the signal overlap between the whole region and ltzs groups, which we describe above (fig. 2b) . in summary, the increased predictive accuracy using ltz-data compared to the whole region data is sufficient to prove the ltz concept, and that home-location can provide a good basis for identifying ltzs. increasing the value of k did not always improve the accuracy of the predictions; e.g. k = 48 performed better than k = 84 in 2009-10 and in 2010-11, and k = 24 provided better accuracy than k = 36 in 2011-12. however, higher |dr| (and lower so) values were associated with improved predictive accuracy over a given season (compare examination of the predictive performance of individual ltzs made clear that a small proportion of ltzs were far less accurate than others; by removing 10% of the ltzs that have the worst performance, the remaining ltzs, representing approx. 90% of patients, provide predictions that are nearly 100% accurate (fig. 4c) for the 2009-10 and 2011-12 seasons (k = 36, 48, 84). while the majority (approx. 60%) of ltzs included in the top 90% (fig. 4c) were common to all 3 seasons, we did not find any obvious characteristics that distinguish these ltzs from those included in the 10% worst performers. poor predictive accuracy in a specific ltz might be the result of limitations in the data itself, or difficulties in achieving perfect clustering by the k-means clustering method, i.e. it is quite possible that some ltzs were poorly identified or in fact 'misidentified' due to errors introduced by the k-means clustering 25 . this study is the first of its kind in using a combination of clinical and geographical data to demonstrate that from a 'pathogen's perspective' even a geographic unit as small as one urban area is not a single, perfectly mixed ecology, but is in fact comprised of more basic transmission components, i.e. ltzs, which should be seen as tightly connected micro-community structures that allow for rapid pathogen transmission. specifically, we proposed the hypothesis that in terms of the spread of ili, the city of jerusalem and its surrounding areas are not a single disease transmission unit but may be more appropriately described by a set of multiple smaller-scale component ltzs. these ltzs, due to spatial fragmentation and isolation effects, will tend to have extended time-periods in which only a single disease dominates, often completely. to test this hypothesis we examined the geographic distribution of influenza and rsv cases in jerusalem over 3 seasons, from 2009-10 to 2011-12. analysis of the putative ltz groups showed unusually small signal overlap and large absolute disease ratio (figs 2 and 3 ) as compared to the whole region values (i.e. k = 1). these results show that the spatial distribution of influenza and rsv incidences may be better differentiated in location-based groups of patients than it is in the entire area as a whole. this confirms our hypothesis that the jerusalem region is comprised of multiple ltzs, and is not by itself a single transmission unit. we also measured the performance of a simple algorithm for predicting whether incoming hospital patients with ili symptoms were infected with rsv or influenza. the prediction scheme made use of a frequency based approach and at the regional (k = 1) level, the predictions simply matched the relative prevalence of the diseases. dividing the whole region into ltzs led to a substantial increase in predictive accuracy in all seasons (fig. 4a) and moreover, after taking into account that a proportion (10%) of the ltzs used may have been misidentified, when removed the predictions for two of the seasons attained routine prediction accuracy often of 100% in differentiating between cases identified as associated with influenza and those that were associated with rsv (fig. 4c ). an important part of the work preformed by hospitals and health organization consists of the diagnosis and surveillance of communicable disease, which may be carried out at the molecular and/or the symptomatic levels. the results presented here hint at the potential epidemiological importance in examining disease data at the ltz scale,and the possibility for generating 'maps' of the real-time distribution of pathogens at resolution levels sufficient to guide improved public health policies. furthermore, the improved predictive accuracy achieved by focusing the analysis at the patient-specific ltzs (fig. 4) indicates a potential utility in data-driven diagnostics, where more sophisticated algorithms taking into account ltz information as well as other relevant population parameters, such as the patients' age, medical background etc. is expected to further increase the predictive accuracy. we hope to explore this further in future research. scientific reports | 7:42012 | doi: 10.1038/srep42012 our choice of ltzs was based entirely on patients' approximate home-locations, without any reference to urban motifs, e.g. socioeconomic divisions, public transportation etc. 28, 29 and clearly provides only a partial picture of the complexity of urban transmission ecology. the social and spatial structures differ among cities, and jerusalem may not be representative of any 'generic' city; e.g. it might be that in some areas the population connectivity pattern is far more homogenous, and hence the ability to leverage ltz-based identification of a pathogen might be limited, or provide better results at other values of k. the "optimal value" of k will be the true number of ltzs in a given area, and for a given choice of pathogens, and as such is expected to vary. unfortunately, there is no simple way of determining this optimal k in advance of the analysis, which is a key problem with many clustering algorithms and "community detection" network algorithms in particular 26 . it would be of great interest to test the methodology presented here in other geographical locations, on a more comprehensive list of pathogens, and include a wider dataset of human social activities 20, 27, 28, 30 , which together could provide a finer, and more dynamic definition of area-and pathogen-specific real life ltzs. settings. the study was performed using data obtained from the computerized database of hadassah-hebrew university medical center, jerusalem, israel, a tertiary care medical institution serving a population of approximately 1 million people from the entire jerusalem metropolitan. the demographics and microbiological data included in this study were collected from the institutional database between january 2009 and may 2012. the dataset contained 16,000 positive and negative clinical test results for two causing agents: influenza virus (influenza) and rsv. the area considered in the analysis is a square shape of approximately 700 sq. km. with vertices at: (32, 35); (31.6, 35); (31.6, 35.4); (32, 35.4). the whole jerusalem urban area population is well covered by this sample since the hospital provides care to over 60% of the referrals from the population in all parts of the city homogeneously. diagnosis of both influenza and rsv at the hadassah-hebrew university was done on routine basis for patients arriving either to the emergency room or being admitted. the diagnosis was based on direct immunofluorescence assay using commercial monoclonal antibodies (chemicon, temecula, california) until 7th march 2010, and by in house rt-pcr assay after that date 31 . the original data contained patient addresses in free text format collected from patients during admission (e.g. 'house number x, street y, town z'), which were converted into standard coordinate system using a process of reverse geocoding. in order to maintain patient anonymity the last 3 digits of the obtained coordinates were omitted, resulting in a location resolution of 110 on 110 meters. the study was approved by the hadassah institutional ethics committee. clustering. patients were partitioned into k groups using the k-means clustering method 25 , applied to the (approximate) home locations of the entire patient set. the minimal number of individuals in any ltz was reduced as k increased, however at the highest k value tested (k = 84) there was still a minimum number of 37 individuals in any ltz (with minimum numbers of 776, 196, 121, and 55 individuals for k = 12, 24, 36, and 48, respectively). thus our schemes provides reasonably sufficient data for statistical estimates even for k-values as large as k = 84. the k values tested were arbitrarily set to multiples of twelve. results for the values k = 60 and k = 72, or of other intermediate k-values we tested (e.g., k = 13 or k = 31) provided qualitatively similar results and are not presented in this study for clarity. note the number of meaningful clusters that may be found is limited by data type/availability. as k is increased, more data is required to accurately extract ltzs. this is because a minimum number of data-points is needed in an ltz before it can be identified accurately by the algorithm. adding a temporal element to the clustering process would be critical for detecting "pathogen hotspots" (using satscan, www.satscan.org, kulldorff, harvard medical school, boston, ma), typically characterized by a significantly elevated incidence of one disease/pathogen. there is no question that detection of disease hotspots is an important activity in modern disease surveillance where it may be used as a real-time warning alert of any atypical activity. our aims however are quite different, since here our goal is to determine if a city-area (i.e. jerusalem) is essentially a single, well-mixed ecology in terms of pathogen transmission, or if there are additional spatial partitions within this limited geographic scope. to address the question in the most direct way, we make use of the robust k-means clustering algorithm, based only on proximity between home locations addresses. disease-signal overlap. we define the general form of a disease signal (ds) for some causing agent v as a function: v ds v (a, t) is the number of positive cases of infection v in a monitored area a over a time period t. for our analysis below, v is a boolean variable, where 1 denotes the influenza virus and 2 denotes rsv. from the data available to us, this function can be immediately constructed for any chosen area a and time period t. the time period t is usually defined to be a week at a time, hence ds v (a, t) represents the total number of positive cases of disease v in area a during week t. we choose ∈ + t z . our data runs from january 2009 to may 2012, and hence ∈ … t (1 173). generically, any area analyzed may contain different disease signals at (or over) the same (period of) time, e.g. 10 positive clinical test results for influenza and 10 for rsv. in this case the signals from the v = 1, 2 are termed 'overlapping' . we calculate the per season overlap so v1,v2 of two disease signals v1 and v2 using the following methodology: global epidemiology of influenza: past and present. annual review of medicine 51 incidence of medically attended influenza during pandemic and post-pandemic seasons through the influenza incidence surveillance project emergency department syndromic surveillance providing early warning of seasonal respiratory activity in england demonstrating the use of high-volume electronic medical claims data to monitor local and regional influenza activity in the us ten-year performance of influenzanet: ili time series, risks, vaccine effects, and care-seeking behaviour infectious disease epidemiology infectious disease dynamics seasonality of viral infections: mechanisms and unknowns seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center syndromic surveillance for influenza in the emergency department-a systematic review clinical signs and symptoms predicting influenza infection the 2009 a (h1n1) influenza virus pandemic: a review spatial transmission of 2009 pandemic influenza in the us unifying viral genetics and human transportation data to predict the global transmission dynamics of human influenza h3n2 modeling human mobility responses to the large-scale spreading of infectious diseases transmission of sars and mers coronaviruses and influenza virus in healthcare settings: the possible role of dry surface contamination influenza a virus transmission via respiratory aerosols or droplets as it relates to pandemic potential community detection in graphs a high-resolution human contact network for infectious disease transmission understanding individual human mobility patterns emergence of new norovirus variants on spring cruise ships and prediction of winter epidemics norwalk-like virus infection in military forces: epidemic potential, sporadic disease, and the future direction of prevention and control efforts outbreak of acute gastroenteritis associated with norwalk-like viruses among british military personnel-afghanistan hepatitis e outbreak on cruise ship data clustering: 50 years beyond k-means dynamics and control of diseases in networks with community structure find me if you can: improving geographical prediction with social and spatial proximity inferring social ties from geographic coincidences structure of urban movements: polycentric activity and entangled hierarchical flows interplay between telecommunications and face-to-face interactions: a study using mobile phone data association of human metapneumovirus with radiologically diagnosed community-acquired alveolar pneumonia in young children we would like to thank mor amitai, gideon almogy, nadav harel, amit gruber, amos cahan and nicolas regnault for their critical reading and helpful suggestions, and the packard foundation for funding. the support of the australian research commission grant dp150102472 is gratefully acknowledged. here ds a ( ) v1 is the average over the season of ds v1 (a, t), and where the maximization is done over a time-shift ∆ ∈ … 0 13 t weeks, spanning all events over each entire season. this is also called the ratio of the cross correlation coefficients of the unnormalized vectors ds v1 (a, t) and ds v2 (a, t):, which is a quantity depending on the area in question (see eq. 2, main text). the maximization over the time-shift δ t is done in absolute value, i.e. we are maximizing the absolute value of the time-shifted cross correlation. in the context of the current paper v1 and v2 represent influenza and rsv respectively. the resulting degree of signal overlap per a given period varies between − 1 and 1, where 1 indicates ds v1 and ds v2 completely overlap (same frequency) and zero time shift, and a − 1 score indicates anticorrelation and zero time shift. the overlap is calculated per period, in the results presented here, per 'winter season' , defined here as august year x to may year x + 1, that is three seasons in total: 2009-10, 2010-11, and 2011-12. notice that our definition of the ratio in eq. [5] is different from the usual cross correlation: a score of 1 implies two possibilities: 1. that the data is perfectly correlated at zero time shift or 2. that the data is perfectly correlated and periodic with some period δ t.by dividing the whole region into k = …k, we can compute the signal in eq. [5] for each ltz. this provides a more local way to analyze the correlations between diseases. the total signal is then the weighted sum of the signal of the individual ltzs with weight factors w i equal to the percentage population size n i of the i' th ltz:we define the resulting overall signal overlap as a weighted sum over all ltzs that comprise the area analyzed. given some (number n of) ltzs … ∈ … i i k the signal overlap in each ltz, and consequently the overall ratio in the whole region are bound between negative one -anticorrelation) and one (complete overlap).disease signal ratios. we define the relative disease ratio dr of the incidence of two viruses using the following formula:the relative signal ratios are bound between a value of one (all cases are influenza) and negative one (all cases are rsv) and are specific per time-period, here calculated per one week time periods; when the incidence of influenza is equal to that of rsv the ratio is equal to zero.the per-season absolute |dr| values (presented in fig. 3c) were calculated as the average of the absolute values of the per-week |dr| values; when the per-week incidences of influenza and rsv are identical, the average value is minimal, i.e. zero, and when influenza and rsv do not coincide on any week the average |dr| is 1. when calculating the per week relative disease ratio over multiple ltzs, the overall ratio is the weighted average of the ratios in the ltzs. key: cord-003571-upogtny6 authors: viboud, cécile; lessler, justin title: the 1918 influenza pandemic: looking back, looking forward date: 2018-10-20 journal: am j epidemiol doi: 10.1093/aje/kwy207 sha: doc_id: 3571 cord_uid: upogtny6 in commemoration of the centennial of the 1918 influenza pandemic, the american journal of epidemiology has convened a collection of 12 articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these 12 articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the 1918 pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. initially submitted august 28, 2018 ; accepted for publication september 6, 2018 . in commemoration of the centennial of the 1918 influenza pandemic, the american journal of epidemiology has convened a collection of 12 articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge again. in the present commentary, we place these 12 articles in the context of a growing body of work on the archeo-epidemiology of past pandemics, the socioeconomic and geographic drivers of influenza mortality and natality impact, and renewed interest in immune imprinting mechanisms and the development of novel influenza vaccines. we also highlight persisting mysteries in the origins and severity of the 1918 pandemic and the need to preserve rapidly decaying information that may provide treasure troves for future generations. age patterns; history of epidemiology; influenza; mortality; pandemic; prior immunity one hundred years after the fact, the 1918 influenza pandemic remains one of the most important epidemics of the modern medical era; it was significant for its impact on both human health and the development of epidemiology and other medical sciences. still, as we mark its centennial, it is sobering to realize how little we understand about the origins and lethality of this unusual outbreak despite decades of intense multidisciplinary research. although it would be 80 years before it was possible to fully characterize the virus responsible for the 1918 pandemic (1), contemporaneous medical authorities put commendable effort into reporting detailed epidemiologic data on the progression of the pandemic that ranged from individual-level clinical records to aggregated city-level vital statistics (2, 3) . in addition to quantitative epidemiologic data, there exist many anecdotal reports from clinicians, particularly those who served military populations, that have been mined to provide modern audiences a comprehensive account of the pandemic (4). yet, many important questions remain about the evolutionary origins of the pandemic virus; the contribution of world war i and troop displacements to pandemic emergence and progression; the unique age profile of pandemic deaths, with its signature of high mortality rate among healthy young adults; the consequences of such a large mortality event on natality; and the heterogeneity of the pandemic experience around the world. such mysteries have captured the attention of the lay public and scientific community alike. in commemoration of the centennial of the 1918 pandemic, the american journal of epidemiology has convened a collection of 12 articles that further illuminate the epidemiology of that pandemic and consider whether we would be more prepared if an equally deadly influenza virus were to emerge today. five of the 12 articles touch on the origins of the 1918 pandemic virus, addressing the role of swine as mixing vessels in this and other pandemic events (5), the age-specific mortality patterns of the pandemic (6) (7) (8) , and prior population immunity (9) . others include reports on geographic and social heterogeneities in the pandemic experience in which the authors describe the spatial diffusion of the pandemic in india and portugal (10, 11) , the socioeconomic predictors of high mortality risk in sweden and globally (12, 13) , and the consequences of the pandemic on us natality rates (14, 15) . finally, 2 commentaries address preparedness for future influenza pandemics (16, 17) . the influenza virus is remarkable for its ability to infect a variety of animal species, from bats to birds to mammals. although successful cross-species transmission events may be rare, they play a key role in the genesis of new pandemic strains. nelson and worobey (5) discussed different lines of evidence informing the origins of the 1918 virus, including the genetic make-up of the 1918 virus and other pandemic strains, the characteristics of influenza receptors across different influenza hosts, and the frequency of cross-species transmission events. they concluded that the pandemic virus must have emerged in mammals just before 1918, most likely from the avian reservoir, with onward transmission from humans to swine. more broadly, a re-analysis of virologic data from the 1957 and 1968 pandemics, together with a modern understanding of the swinehuman interface, suggested a twist on the long-standing concept of swine as a "mixing vessel" for influenza virus. the authors proposed that swine should be viewed as a repository of historic human viruses rather than a conduit for reassortment of genetic material between avian and human viruses. van wijhe et al. (6) returned to the question of the origins of the 1918 virus by exploring the epidemiologic imprint of the 1918 virus on danish mortality records, echoing recent work on immune imprinting (18) (19) (20) . they identified several age breakpoints in pandemic mortality that were suggestive of the cycling of different influenza strains between the mid-19th century and the 1918 pandemic. most notably, they argued for co-circulation of 2 subtypes of influenza virus (carrying type i and ii hemagglutinin surface antigens) between 1873 and 1908. as a result, persons born between 1873 and 1908 (aged 10-45 years during the 1918 pandemic) may have been primed by either hemagglutinin type, potentially explaining the intriguing age profile of pandemic mortality in adults. cilek et al. (7) used a similar epidemiologic approach to explore the 1918 pandemic mortality patterns in madrid, spain. madrid is particularly interesting because a lethal pandemic wave was reported in the city in june 1918, the earliest such event recorded. similar to other regions of the world, madrid experienced a signature pandemic pattern of higher mortality rates among young adults. however, seniors in madrid suffered equally high rates of excess influenza mortality. this is unlike the experience of the rest of europe and north america, where seniors were reportedly spared, presumably because of antigenic recycling (i.e., exposure to a related strain in childhood that conferred partial protection) (20, 21) . this is an intriguing finding, and it will be important for future work to reconcile the well-accepted idea that a 1918-like virus may have circulated in europe and north america in the second half of the 19th century, with the notion that madrid would have escaped this virus. to understand the unique epidemiology of the 1918 virus, it can be useful to document the experience of remote populations, in which prior immunity to influenza would be expected to be low because of less frequent circulation of the virus. rice (8) built on a rich literature in this area to document mortality patterns in new zealand between 1890 and 1918. he found that the 1890s were a decade associated with high rate of influenza mortality in new zealand, despite the low global connectivity of this island in the era before air travel. he also noted that influenza mortality in 1918 was highest among young adults, with a more pronounced intensity in males than in females. these patterns are broadly consistent with the those among young adults in europe and the americas, pointing to the near universality of increased influenza mortality risk in this age group in 1918. chuah et al. (9) used seroepidemiology and structural equation modeling to answer the inverse question: how did early-life exposure to the 1918 pandemic virus impact how people responded to the 2009 pandemic, which was caused by an antigenically similar virus? they found evidence for immunologic priming from the 1918 virus in the oldest people they studied (individuals 80 years of age or older) that impacted both baseline titers and vaccine response in 2009. this work adds to a growing body of evidence that early-life exposures can have profound effects on immune response and mortality patterns decades after they occur (18) . information about global connectivity in the 19th century is tenuous, and influenza records before 1890 are scarce. epidemiologic reconstructions of "modern" pandemics of the type presented here (6-9) provide indirect information on the exposures of populations that are now long gone, generating valuable hypotheses about influenza circulation patterns and disease dynamics well into the 19th century. such reconstructions offer precious insights into what influenza may have looked like 200 years ago in a very different world and how long-term changes in human demography and mobility may affect disease dynamics (22) . active research topics in the field of archeo-epidemiology include the search for predictors of influenza mortality, such as socioeconomic indicators or geography, and the drivers of influenza spatial diffusion. in 2 articles in the present issue, the authors concentrated on the spatial diffusion of influenza, focusing on british india and portugal, 2 countries that have been poorly studied in the context of the 1918 pandemic (10, 11) . both studies revealed a highly heterogeneous spread of the pandemic and geographic variation in pandemic mortality impact, albeit at different spatial scales. although portugal as a whole was severely hit by the pandemic compared with other european countries, some provinces nearly fully escaped (10). analysis of district-level mortality records in india revealed a northeastward wave of infection from september to november 1918 that was associated with climate and population density (11) . diffusion was driven by long-distance jumps via the railroad network, superimposed on local diffusion between neighboring provinces. further, the authors found moderate heterogeneity in the mortality experiences of different indian provinces. spreeuwenberg et al. (12) also made use of recently unearthed data from india to revisit the global mortality impact of the 1918 pandemic. india is a particularly important country for global burden estimation because it was the one most severely hit by the 1918 pandemic, with annual pandemic excess mortality rates that were 40-fold higher than those in denmark for instance (23) . in the new study, the authors placed the burden of the pandemic at a much lower number than did previous work (12) , in part by using more detailed data to better adjust for high background mortality unrelated to flu. the results of the portuguese study by nunes et al. (10) echoed these conclusions-that careful analyses of more detailed data tend to decrease estimates of pandemic burden. the risk factors responsible for increased mortality and morbidity from influenza remain elusive, whether at the population level (e.g., effect of population density or weather on transmission) or the individual level (socioeconomic status, comorbid conditions, etc.). this is still an active area of contemporary influenza research, with direct applications to design targeted intervention strategies. in the present issue, bengtsson et al. (13) explored the role of social class on pandemic mortality by linking individual death records with historical census data on occupation in a powerful study that captured the entire swedish population. the authors found that low-skilled or unskilled adults had higher death rates than did more skilled workers during the pandemic period relative to prepandemic years, whereas farmers (especially men) fared particularly well. social differences tended to be smaller in women, and there was no clear gradient between social class and mortality. the authors hypothesized that these social differences were linked to differential crowding in the workplace (hence an effect on transmission) rather than differences in income or nutrition. this is a topical issue because the effects of socioeconomic status and baseline health on influenza mortality are still debated today (24) . researchers have long thought that the 1918 pandemic could have affected birth rates (25) because of the large impact of this event on young adult mortality, the increased risk of severe flu outcomes during pregnancy, and a possible association between influenza infection and miscarriage. two papers in this issue address the topic of natality (14, 15) . key questions here include the trimester of pregnancy during which the risk of death is highest for the mother and/or the unborn child and the impact of influenza on (increased) stillbirths and (decreased) live births. the duration of the pandemic effect on natality is also important because it informs the biological mechanism at play. if influenza impacts the probability of conception or fetal deaths, one would expect a temporary natality drop in the aftermath of the pandemic, followed by a rebound in births a few months later. in contrast, a high mortality rate among young women of childbearing age due to influenza infection would result in a long-lasting natality trough. dahal et al. (14) explored these questions using individual birth and death certificates from arizona, where there was a drop in natality 9-11 months after pandemic mortality peaked. this was a temporary depletion, consistent with a detrimental effect of influenza early in pregnancy. in a larger study of populationlevel vital statistics in us states, chandra et al. (15) found a 10% drop 9-10 months after peak influenza mortality, which they ascribe to a drop in conception during the period of intense pandemic activity. they also found a natality drop in the 3 months after peak mortality, which they linked to excess preterm births and stillbirths due to influenza infections in the last trimester. interestingly, these patterns were also found in the aftermath of the 1920 influenza pandemic wave, albeit with a less pronounced effect. one reason we still look back at the 1918 pandemic 100 years later is because doing so will make us better able to prepare for the future. the last 2 articles of this collection are focused on preparedness for future pandemic threats (16, 17) , building on the lessons learned in 1918 and later pandemics, and on new tools to protect populations, including the very active (but still elusive) topic of universal influenza vaccines. jester et al. painted an optimistic picture of progress made in influenza surveillance domestically and internationally, antiviral treatments, and robustness in the infrastructure for vaccine production (16) . epstein reviewed the progress of the development of a broadly cross-protective flu vaccine, focused on conserved parts of the influenza virus, such as the matrix protein, nucleoprotein, the hemagglutinin stem, and various cocktail combinations (17) . these vaccines offer promising broad protective effects against new influenza antigenic variants and could potentially be used in pandemic situations. however, some of the candidate vaccine formulations permit limited viral replication and may foster the emergence of escape mutants fit enough to cause disease. these features could have adverse epidemiologic consequences, and these risks need to be projected and monitored carefully. the 1918 pandemic is traditionally considered a worst-case scenario for pandemic preparedness, but there were many other pandemics before 1918 about which we know very little regarding mortality impact, circulating strains, or prior immunity. in fact, the 1889 pandemic has only recently drawn attention among epidemiologists (26, 27) . most european and north american countries began formal collection of vital statistics in the mid-to-late 19th century, so that any pandemic predating 1850 can only be explored using church or cemetery records (or indirectly through reconstruction of modern pandemics). digitization of historic records is time consuming, data lack standardization, and information is generally limited to small populations. even with regard to the 1918 pandemic, crucial questions may never be answered, including which specific virus (or even subtype) circulated before 1918 and further back into the 19th century and what the population immunity profile was before the pandemic. the search for archival influenza specimens predating 1918 has remained elusive, and to our knowledge no archived sera exist from this period. in the absence of further virologic evidence, our understanding of the origins of the pandemic is limited to a handful of influenza virus sequences collected during may to november 1918 and to the epidemiologic signature of the 1918 virus in different populations. as nelson and worobey noted (5), more work can be done in this area, particularly to explore the uracil content of post-1918 viruses in different hosts, reconstruct their evolutionary trajectories, and better characterize host receptors and barriers to cross-species jumps. further, as rice (8) and dahal et al. (14) noted, a systematic analysis of the age mortality profiles of the 1918 pandemic in a sample of remote and wellconnected locations would be most useful, together with modeling of plausible biological hypotheses and immune histories most consistent with the data. databases, which are often crowdsourced or maintained by state health departments (14) . further analyses of such data could shed light on the mortality profile of the pandemic in understudied locations and would also allow identification of family linkage and host genetic risk factors, which could be tested among descendants. many other library archives exist, although paper-based records rapidly decay and need to be digitized as quickly as possible. the detailed, sometimes freeform, notes typically kept by the scientists at the time mean that careful examination of these archives can sometimes yield surprising fruit. one such resource is the work of wade hampton frost, who was the first chair of the department of epidemiology at johns hopkins university and a critical figure in the fight against the 1918 pandemic. modern reanalysis of dr. frost's detailed work (3) has already yielded abundant insights, and we included digital copies of his papers on the 1918 pandemic from the chesney archive in the web appendix (available at https://academic. oup.com/aje). the 1918 pandemic is remarkable for the large amount of extremely detailed epidemiologic data collected by public health officials (2, 3), in part because it was an era that valued epidemiology, at a time when analytical approaches and knowledge of infectious agents were limited. these exquisitely detailed records have been particularly useful in the attempt to understand the pandemic retrospectively. as a thought experiment, we can imagine ourselves in 2118: we may ask how scientists would look back at the large amount of data we archive on a daily basis in 2018. on the one hand, much if not all of the modern data are digital, meaning that they do not run the risk of being destroyed by fire or floods and they can be more accessible to a wide audience, spurred by the open access movement. however, digital data can also be corrupted (intentionally or unintentionally) and disappear. much from the floppy-disk era has already been permanently lost, and it is unclear if modern cloud-based archives would survive a major disruption (whether technological or civil). further, even today, there is a systematic dearth of epidemiologic and molecular data from low-and middle-income settings (including data on the 2009 pandemic (24)). we are just beginning to scratch the surface of the intricate relationship between the influenza virus and the complex immune history of a host who has had repeated influenza exposures (9, (18) (19) (20) 28) . it is unclear whether we will be able to fully understand these interactions in the foreseeable future; in the meantime, population birth cohorts carrying important influenza immune histories disappear. we echo earlier calls for a time-stamped global repository of human sera and pathogen specimens, ideally together with epidemiologic information (biobanks) for current and future use (29) . we also applaud the push by the us national institute of allergy and infectious diseases to fund international influenza birth cohort studies and help untangle the complex mechanisms of influenza immunity (30) . if again confronted with a deadly flu pandemic, we would be in a better place than we were in 1918 because of the availability of drugs, vaccines, and antibiotics and the general improvements in health and nutrition. there are high hopes for the development of universal vaccines, but we need to keep in mind that influenza is a rapidly evolving virus that has a large and diverse animal reservoir and presumably many tricks in store. we can only anticipate another hundred years of very active, and always surprising, influenza research. structure of the uncleaved human h1 hemagglutinin from the extinct 1918 influenza virus preliminary statistics of the influenza epidemic influenza transmission in households during the 1918 pandemic the great influenza: the story of the deadliest pandemic in history origins of the 1918 pandemic: revisiting the swine "mixing vessel" hypothesis loose ends in the epidemiology of the 1918 pandemic: explaining the extreme mortality risk in young adults age-specific excess mortality patterns during the 1918-1920 influenza pandemic in madrid, spain influenza in new zealand before 1918: a preliminary report investigating the legacy of 1918 pandemic on age-related seroepidemiology and immune responses to subsequent influenza a(h1n1) viruses through a structural equation model the 1918-1919 influenza pandemic in portugal: a regional analysis of death impact spatiotemporal patterns and diffusion of the 1918 influenza pandemic in british india reassessing the global mortality burden of the 1918 influenza pandemic social class and excess mortality in sweden during the 1918 influenza pandemic natality decline and spatial variation in excess death rates during the 1918-1920 influenza pandemic in arizona, united states short-term birth sequelae of the 1918-1920 influenza pandemic in the united states: state-level analysis readiness for responding to a severe pandemic 100 years after 1918 universal influenza vaccines: progress in achieving broad cross-protection in vivo potent protection against h5n1 and h7n9 influenza via childhood hemagglutinin imprinting re-examining the evidence regarding circulation of a human h1 influenza virus immediately prior to the 1918 spanish flu age-and sex-specific mortality associated with the 1918-1919 influenza pandemic in kentucky epidemiological evidence of an early wave of the 1918 influenza pandemic in new york city influenza epidemics in iceland over 9 decades: changes in timing and synchrony with the united states and europe estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918-20 pandemic: a quantitative analysis global mortality estimates for the 2009 influenza pandemic from the glamor project: a modeling study natality decline and miscarriages associated with the 1918 influenza pandemic: the scandinavian and united states experiences transmissibility and geographic spread of the 1889 influenza pandemic age-specific excess mortality patterns and transmissibility during the 1889-1890 influenza pandemic in madrid, spain evidence for antigenic seniority in influenza a (h3n2) antibody responses in southern china use of serological surveys to generate key insights into the changing global landscape of infectious disease the ghost of influenza past and the hunt for a universal vaccine this article does not necessarily represent the views of the national institutes of health or the us government.conflict of interest: none declared. key: cord-003122-a3f4l6iu authors: dou, dan; revol, rebecca; östbye, henrik; wang, hao; daniels, robert title: influenza a virus cell entry, replication, virion assembly and movement date: 2018-07-20 journal: front immunol doi: 10.3389/fimmu.2018.01581 sha: doc_id: 3122 cord_uid: a3f4l6iu influenza viruses replicate within the nucleus of the host cell. this uncommon rna virus trait provides influenza with the advantage of access to the nuclear machinery during replication. however, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. the segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral rna (vrna) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. to accomplish this goal, influenza a viruses (iavs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vrna gene segments in and out of the cell. the aim of this review is to present the current mechanistic understanding for how iavs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the iav infection process. influenza viruses belong to the orthomyxoviridae family and are classified as either type a, b, c, or the recently identified type d (1, 2) . influenza a viruses (iavs) and type b viruses (ibvs) contain 8, negative-sense, single-stranded viral rna (vrna) gene segments ( figure 1a ) (3, 4) , which encode transcripts for 10 essential viral proteins, as well as several strain-dependent accessory proteins ( figure 1b) . in comparison, influenza type c and d viruses only possess seven vrna gene segments, as the hemagglutinin-esterase fusion protein vrna replaces the hemagglutinin (ha or h) and the neuraminidase (na or n) vrnas (1, 2) . iavs will be the main focus of this review since they are the primary agents responsible for influenza pandemics, and a major contributor to the annual influenza epidemics in the human population (5) . the natural reservoir for iavs is wild aquatic birds, but they commonly infect other species, including humans, and have even been isolated from penguins in antarctica (12) (13) (14) (15) . the ability to adapt to multiple species is a major reason why iavs are more diverse than ibvs, which are essentially exclusive to humans. despite the host-range differences, many similarities do exist between these two viruses. both possess a host-derived lipid membrane, referred to as an envelope, which is decorated on the surface with the viral membrane proteins ha, na, and to a lesser extent the matrix 2 (m2) protein ( figure 1c ) (16) (17) (18) . the envelope is supported underneath by the matrix 1 (m1) protein, and inside, the eight vrnas are found as individual viral ribonucleoprotein (vrnp) complexes ( figure 1c , bottom). each vrnp is comprised of a vrna that is wrapped around numerous red circles represent the 5′ m 7 pppg cap, black lines denote the 10-13 nucleotide, host-derived primers that are obtained by the cap-snatching mechanism of the viral polymerase. a(n) corresponds to the 3′ poly-a tail produced by reiterative stuttering of the viral polymerase. the smaller mrnas (empty boxes) represent transcripts that encode nonessential accessory proteins found in many strains, whereas those that are less prevalent (pb2-s1, m42, and ns3) are not illustrated (6) (7) (8) (9) (10) (11) . (c) diagram of an influenza a or b virus. the viral membrane proteins ha, na, and m2 are shown, along with the eight viral ribonucleoproteins (vrnps), and the matrix protein m1 that supports the viral envelope. to highlight the vrnp components, the illustration beneath the virus is not to scale. a single vrna gene segment is shown wrapped around multiple nucleoprotein (np) copies with the conserved promoter regions in the 5′ and 3′ utrs forming a helical hairpin, which is bound by a single heterotrimeric viral rna-dependent rna polymerase (pb1, pb2, and pa). (d) top view of an influenza virus cross-section showing the vrnp "1 + 7" configuration. vrnps are depicted with black circles as it is not known if the positioning of a particular vrnp is conserved or interchangeable. 2 frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 copies of the viral nucleoprotein (np) and bound by a single copy of the heterotrimeric viral polymerase, consisting of pb1, pb2, and pa (19) (20) (21) . the polymerase binds the vrnas at a helical hairpin that results from the base pairing between the conserved semi-complimentary 5′ and 3′ ends (21) (22) (23) . morphologically, iavs can either form spheres with a diameter of ~100 nm or filaments that can reach up to 20 µm in length [reviewed in ref. (24) ]. however, upon passaging in eggs, or mdck cells, the filamentous form is generally lost (25, 26) . several studies have attributed the morphology change to m1, presumably through its function in supporting the envelope (27) (28) (29) . regardless of the virion shape, ha is the most abundant viral envelope protein, followed by na, and m2 (30) . recent work has shown that the viral envelope also contains host membrane proteins (30, 31) . these proteins are likely recruited based on the lipid composition at the plasma membrane budding site, which can differ between cell types (32, 33) . through possible interactions with each other and m1, the eight vrnps typicallly form a 1 + 7 configuration inside the virus ( figure 1d ) (34, 35) . the 1 + 7 configuration may have a mechanistic function, as it is also conserved in type c and d viruses that only possess 7 vrnas (36) . further supporting the mechanistic concept, it was recently shown that iavs can package cellular ribosomal rna (as a vrnp) when one of the vrnas is made unavailable (37) , possibly explaining how type c and d viruses acquire their "eighth" vrna. the classification of iavs into subtypes is based on the genetic and antigenic properties of the surface antigens ha and na, which mediate viral entry and release, respectively (17, 18) . to date, 16 ha (h1-16) and 9 na subtypes (n1-9) have been found in iavs isolated from aquatic birds (13) . two additional subtypes for ha (h17 and h18) and na (n10 and n11) have recently been identified in bats (38, 39) , but in contrast to the ha and na subtypes from the more traditional avian iavs, these do not appear to recognize sialic acid (sa) (40) (41) (42) . despite the numerous possible subtype combinations, only three have consistently persisted in the human population, causing the following pandemics in the process: 1918 and 2009 (h1n1), 1957 (h2n2), and 1968 (h3n2) (43) . currently, only the h1n1 and h3n2 subtypes, as well as the two antigenically distinct ibv lineages (victoria and yamagata), are endemic in the human population (44) , which is why many iav vaccines include two representative iav and ibv strains (5) . a significant challenge in battling iavs is the constant evolution of the surface antigens (ha and na) in response to pressure from the host immune system, which is referred to as antigenic drift and antigenic shift. antigenic drift is most evident in circulating seasonal iavs, where substitutions by the polymerase that cause mutations in the surface antigen epitopes have continuously been selected to enable reinfection of the same host (45) . antigenic shift is responsible for the development of the iav pandemics, and it relies on the less the terminal sialic acid residues are displayed with an α-2,3 linkage, as well as an α-2,6 linkage, to illustrate the "linear" and "bent" presentations. (b) illustration of iav cell entry. (i) iavs initiate cell entry by using the ha receptor-binding domain (located in the ha1 region) to associate with sialylated glycoconjugates on a host "receptor." binding to the "receptor" triggers endocytosis. (ii) the virus then traffics to the endosome where the lower ph facilitates a conformational change in ha, exposing the fusion peptide (located in the ha2 region) for insertion into the endosomal membrane. (iii) the ha pre-hairpin conformation begins to collapse, forming a six-helix bundle that promotes hemifusion of the viral envelop with the endosomal membrane. at some point, the m2 channel opens to release the viral ribonucleoproteins (vrnps) from m1 by acidifying the viral interior. (iv) ha further collapses into a trimer of hairpins to promote the formation of the fusion pore, which (v) releases the vrnps into the cytosol. (vi) the exposed nuclear localization signals (nls) on the vrnps are recognized by the adaptor protein importin-α, leading to the recruitment of importin-β that (vii) facilitates the transport through the nuclear pore complex (npc) and into the nucleus. frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 frequent process of reassortment, which involves the exchange of vrnas between two iavs during co-infection of a cell (43, 46, 47) . while reassortment can happen between two related iavs, antigenic shift occurs when the reassortment process yields a new iav subtype. iavs are also under constant negative selection due to the functional requirements of the viral proteins, and the constraints of the limited genome. several roles have been reported for most of the iav proteins. these include the function of ha in receptor binding, as well as membrane fusion, and viral release by the sialidase activity of na. to perform these functions, the proteins need to correctly fold, oligomerize, and as for the genome itself, they have to be properly trafficked and packaged into new virions. thus mutations that benefit one property may hinder another. the goal of this review is to highlight these functional requirements by providing a summary of the mechanisms iavs have evolved to facilitate cell entry, replication, virion assembly and movement, with particular attention to how iavs coordinate the infection process. iavs initiate the infection process by using the ha molecules on the viral envelope. upon reaching a potential host cell, the ha receptor-binding site attaches the virus to surface glycoconjugates that contain terminal sa residues (figure 2a ) (18, 48, 49) . iavs then scan the cell surface for the proper sialylated "receptor" by using the sialidase function of na to remove local sas and liberate nonproductive ha associations (50) . currently, the "receptor's" identity remains unknown, but it is generally thought that has from avian iavs have higher specificity for receptors with α-2,3-linked sas that have a "linear" presentation (51, 52) , whereas has from human iavs prefer an α-2,6 linkage, which results in a more "bent" presentation ( figure 2a ) (53, 54) . while these preferences correlate with sa linkages in the respective hosts (55) , several studies have shown that matching ha receptor binding preferences with the sa linkages in a particular host is not essential for infection, but is more critical for transmission (56) (57) (58) (59) . this implies that the iav "receptor" either displays significant cell tropism in the airways or that iavs can potentially use more than one receptor. despite the unknown identity of the receptor, it is clear that ha-mediated binding to the receptor triggers endocytosis of the virion ( figure 2b , step i). the endocytosis can either occur in a clathrin-dependent manner, involving dynamin and the adaptor protein epsin-1 (60-62), or by macropinocytosis (61, 63, 64) . once inside the cell, the virus is trafficked to the endosome, where the low ph activates the m2 ion channel (61, 65, 66) , and causes a large conformational change in ha that exposes the fusion peptide ( figure 2b , step ii) (67) (68) (69) . opening of the m2 ion channel acidifies the inside of the viral particle, releasing the packaged vrnps from m1 ( figure 2b , step iii), which enables the transfer of the vrnps to the host cytoplasm following ha-mediated fusion (70, 71 fusion of the viral-endosomal membranes by ha occurs through multiple steps [reviewed in refs. (72, 73) , and requires cleavage of ha by host cell proteases into two subunits, ha1 and ha2 (55, 74, 75) ]. the cleavage (see ha proteolytic activation at the golgi or plasma membrane) is required to enable the exposure of the fusion peptide on the n-terminus of the ha2 upon the ph change in the endosome (69). once exposed, the fusion peptide inserts into the endosomal membrane, while the c-terminal transmembrane domain (tmd) anchors ha2 in the viral membrane, creating a pre-hairpin conformation (see figure 2b , step ii "box"). the ha2 trimers then fold back on themselves creating a hairpin that begins to position the two membranes in close proximity to each other (see figure 2b , step iii "box"). the hairpin bundles then further collapse into a six-helix bundle, and in doing so, the two membranes come closer together enabling the formation of the lipid stalk, and the subsequent fusion of the two membranes ( figure 2b , step iv). to date, not all of these stages have been observed with ha and some have been inferred based on observations of related fusogens from other viruses. in contrast to the early steps in iav entry, vrnp trafficking to the nucleus following the fusion event is highly dependent on the host cell machinery and transport pathways [reviewed in ref. (76) ]. supported by numerous studies, the current model is that the newly released cytoplasmic vrnps use the importin-αimportin-β nuclear import pathway to gain entry to the host cell nucleoplasm ( figure 2b , steps vi and vii) (77) (78) (79) (80) (81) (82) (83) . to initially engage this pathway, it is thought that the vrnps use the surface exposed nuclear localization sequences from the numerous np molecules to recruit the adapter protein importin-α (80) (81) (82) . upon binding to the vrnp, importin-α is recognized by the importin-β transport receptor, which directs the vrnp to the nuclear pore complex, where it is transported into the nucleoplasm. recent improvements in imaging and rna labeling techniques have made it possible to monitor the entire entry process in single cells (61, 62, (83) (84) (85) . the cumulative results from these studies show that iavs can deliver their vrnps from the cell surface to polyadenylation "reiterative stuttering" the nucleus in approximately 1 h, with entry and fusion occurring rather quickly (~10 min), and nuclear import requiring the bulk of the time (85) . a striking observation from these studies is the efficiency with which the eight vrnas reach the nucleus, indicating how effectively vrnps recruit the host nuclear import factors. supporting this observation, it was shown that np adaptation to the importin-α isoforms of a particular species is crucial for productive iav infections (86) . while the bulk of the vrnp trafficking work has been carried out using various immortalized cell lines, the potential species related differences, and the essential role of vrnp trafficking in reassortment, emphasize the need for further methodology development to examine the details of iav entry in primary cells and tissue explants. inside the nucleus, the heterotrimeric viral rna-dependent rna polymerase carries out the transcription and replication of the vrnas [reviewed in refs. (87, 88) ]. the replication of the influenza genome involves two steps: transcription of complimentary rna (crna), followed by transcription of new vrna copies using the crnas as templates. the crnas are produced by an unprimed process that relies on the correct complementation of free rntps (generally gtp and atp) with the 3′ end of the vrna template ( figure 3a ) (89, 90) . the nucleotide complementation locks the vrna template into the polymerase active site within the pb1 subunit and results in the formation of an a-g dinucleotide from which the crna is elongated (91) . upon exiting the polymerase, the crna associates with newly synthesized np molecules and a single copy of the viral polymerase to assemble into a crnp (90) . currently, it is thought that the newly produced viral polymerases, which are incorporated into the crnps, generate multiple vrna copies in a manner similar to crna transcription ( figure 3b) . however, there is one distinction related to the difference in the positioning of the longer 3′ end of the positivesense crna. due to the increased length, the crna is positioned in the polymerase such that the rntp annealing and dinucleotide formation is likely to occur at the nucleotides located 4 and 5 bases from the crna 3′ end (figure 3b , pathway i) (90, (92) (93) (94) . the dinucleotide primer then has to dissociate and reanneal to the nucleotides at the 3′ end prior to elongation ( figure 3b) . alternatively, the crna 3′ end could reposition within the polymerase due to rntp bin ding, resulting in the generation of full-length vrna transcripts directly ( figure 3b , pathway ii). the transient nature of the rntp annealing and dinucleotide formation makes it technically challenging to exclude either possibility. the remaining task of assembling a vrnp is analogous to crnp formation. viral mrna transcription from the vrna templates is primed, making it significantly more efficient than crna and vrna transcription (95) . the viral polymerase obtains the primers through a mechanism termed cap snatching (96) , which is aided by the association with the cellular rna polymerase ii c-terminal domain (figure 4 ) (97) (98) (99) . for cap snatching, the viral polymerase uses the pb2 subunit to bind to 5′ caps of nascent host transcripts (100) and the pa subunit endonuclease domain to cleave 10-13 nucleotides downstream of the 5′ cap (101) (102) (103) . the pb2 cap-binding domain then rotates to position the newly acquired capped primer into the pb1 catalytic center where it is extended using the vrna as a template (95) . finally, each transcript is polyadenylated through a reiterative stuttering′ process, which occurs when the polymerase encounters the short poly-u sequence at the vrna 5′ end (figure 4 "box") (104, 105) . this process likely involves multiple cycles of dissociation, repositioning, and reannealing of the mrna to this template region of the vrna to achieve polyadenylation. during the course of infection, mrna synthesis occurs before crna and vrna transcription, and mrna transcription is much more abundant because the use of primers significantly increases the initiation efficiency (106) . the initial mrnas are transcribed by the vrnp-associated polymerases and exported from the nucleus for translation by cytoplasmic ribosomes (93) . however, the m and ns transcripts also possess donor and acceptor splice sites that match well with those in human transcripts (107) . these sites recruit the cell spliceosome, which produces the spliced transcripts that encode for the m2 and ns2 proteins, respectively (108) (109) (110) (111) (112) . the ns transcript has been reported to maintain a similar ratio of non-spliced and spliced transcripts throughout infection (113) , whereas the ratio of the spliced m transcripts (encoding m2) have been shown to increase during infection (114) . these observations imply that ns1 and ns2 are always equally expressed, while m2 expression is more biased toward the later stages of infection. however, it is likely that the splicing efficiency of the ns and m transcripts differs between iav strains (115, 116) . iav protein synthesis is entirely dependent on the translation machinery of the host cell. following nuclear export [reviewed in ref. (117) ], the translation of the viral mrnas is divided between cytosolic ribosomes (for pb1, pb2, pa, np, ns1, ns2, and m1) and endoplasmic reticulum (er)-associated ribosomes for the membrane proteins ha, na, and m2 (figure 5 , steps i and ii). nuclear localization sequences on the newly synthesized np proteins and polymerase subunits (pb1, pb2, and pa) target these proteins into the nucleus by recruiting the importin-α-importin-β pathway that is utilized for vrnp nuclear import (figure 5 , step iii). the np and pb2 proteins are imported individually, whereas the pb1 and pa proteins are imported as a heterodimer (81, 118) . in the nucleus, these newly synthesized proteins assist in viral mrna transcription and vrna replication. np monomers bind to 12 nucleotide stretches with a partial g bias in vrnas, and presumably crnas, to assemble vrnps and crnps through a process that may be regulated by the np phosphorylation (figure 5 , steps v and vii) (119) (120) (121) . the heterotrimeric polymerase assembles and binds to the newly formed crnps to transcribe vrnas ( figure 5 , step vi) that upon formation into vrnps can generate additional viral mrna (figure 5, step viii) , or crna transcripts (figure 5 , step ix) (90, 93) . the viral rna-binding protein ns1 is synthesized early and also imported into the nucleus, where it can act as an inhibitor of interferon signaling [reviewed in ref. (122) ]. in addition, ns1 may contribute to viral mrna export from the nucleus by linking the viral transcripts to the cellular nuclear export components tap/nxf1, p15, rae1, e1b-ap5, and the nucleoporin nup98 (123) . ns2 (alternatively known as the nuclear export protein) and m1 are imported into the nucleus as well. multiple studies have implicated these two proteins in the nuclear export of vrnps (70, 71, (124) (125) (126) (127) . while the mechanism remains unclear, current data support a model where m1 acts as an adaptor protein linking ns2 to vrnps (figure 5 , step x) (128, 129) . through established interactions with crm1, ns2 is then able to target the vrnp complex to the crm1 nuclear export pathway for transport to the cytoplasm (127), where m1 potentially prevents the re-import of vrnps by blocking access to the np nuclear localization sequences (figure 5 , step xi) (71) . within the cytoplasm the vrnps are trafficked toward the plasma membrane for viral assembly by rab11. rab11 facilitates the interaction by associating with the viral polymerase pb2 subunit (130) , potentially providing a quality control mechanism that ensures new virions incorporate vrnps carrying a polymerase. earlier studies proposed that vrnps specifically associate with rab11 on recycling endosomes, which use microtubules for transport toward the cell surface ( figure 5 , step xiia) (130) (131) (132) ). an alternative model has recently been proposed where infection causes tubulation of the er membrane network and the vrnps bind to rab11 molecules that have localized to this network for trafficking toward the plasma membrane ( figure 5 , step xiib) (133) . currently, it is not known how vrnps are transferred to the plasma membrane in either model, or how iavs incorporate all eight of the different vrnps in a "1 + 7" configuration. while several studies have indicated that specific vrnp associations likely contribute to the packaging of the eight vrnps (35, 134, 135) , the underlying mechanisms remain to be established. the iav membrane proteins, which are ultimately destined for the viral envelope, are synthesized by ribosomes associated with the er membrane. similar to cellular secretory proteins, ribosome-nascent chain complexes containing na, ha, or m2 are co-translationally directed to the er by interactions of their hydrophobic targeting sequences with the signal recognition particle (srp) (figure 6 , step ii) (136) (137) (138) (139) . the cleavable signal sequence on ha facilitates the interaction with srp, whereas na and m2 use their respective tmd as an er targeting sequence. once bound, srp targets the ribosome-nascent chain complexes to the srp receptor in the er membrane (figure 6, step iii) , which transfers the ribosome to a sec61 protein-conducting channel known as the translocon (140-142) . linked to the dependence on srp, mutations that alter the targeting sequence hydrophobicity of cellular secretory proteins have been shown to decrease their er targeting and subsequent synthesis (143, 144) . although this aspect has not been examined for the iav membrane proteins, there is evidence that the hydrophobicity of their er-targeting sequences change (138, 148) , which suggests iavs potentially use this mechanism to titrate na and ha expression. the translocon enables passage of the elongating na, ha, and m2 polypeptides into the er lumen and facilitates the membrane partitioning of their respective tmd segments through a lateral gate (145, 146) . to activate the membrane integration, the tmd segments have to be of the appropriate length and hydrophobicity (146, 147) . in human h1n1 and h3n2 viruses, these criteria are conserved in the tmds of ha and m2, but not in the tmd of na, as it has become progressively less hydrophobic in the h1n1 viruses (148) . the uncharacteristic hydrophobicity loss was shown to be possible because of the na tmd being positioned at the n-terminus (138) . the positioning (~435 amino acids from the c-terminus), combined with the slow rate of ribosomal translation (~5 amino acids per second), likely provides these nontypical tmds with significant time to properly orientate and facilitate membrane insertion during the co-translational translocation process. during translocation, the n-terminus of ha and m2 is directly translocated into the er lumen, whereas na inverts, positioning the c-terminus in the er lumen (137, 138) . in addition, ha and na receive multiple n-linked glycans. the glycans are transferred by the oligosaccharyltransferase to asn-x-ser/thr sequences, and vary in number as well as positioning based on the strain, or subtype (149) . one function of the glycans is to increase the folding efficiency of na and ha by recruiting the lectin chaperones (calnexin and calreticulin) and the associated oxidoreductase erp57, which aids in disulfide bond formation (136, (150) (151) (152) . this is especially crucial for the ha and na proteins that possess a significant number of intramolecular disulfide bonds (e.g., six in has, eight in n1, and nine in n2) (153) (154) (155) . by contrast, m2 possesses two intermolecular disulfide bonds in its tetrameric conformation (156) . depending on the subtype, na tetramers also possess 2 or more intermolecular disulfide bonds. oligomerization of ha involves the trimerization of independently folded monomers, whereas na tetramerization has been proposed to result from the pairing of two co-translationally formed dimers, which assemble through a process involving the n-terminal tmd of na (150, 157) . in line with this model, it has been shown that the tmd is essential for proper na folding, and that the decreasing hydrophobicity in the n1 tmds functions to frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 support the folding and oligomerization of the enzymatic head domain (158, 159) . iavs easily achieve the protein concentrationdependent requirement for oligomerization due to the abundance of ha and na that is synthesized during an infection. however, these high synthesis levels at the er can also be deleterious by activating the er-stress response. indeed, several studies have shown that iav replication does activate the er-stress induced unfolded protein response (160, 161) , but this response is also mitigated by the inhibition of the eif2α-kinase and stress granule formation through the functions of other viral proteins (162) . despite everything that is known about the synthesis and assembly of the iav membrane proteins, several aspects have yet to be addressed. these include obtaining atomic structures of fulllength ha and na in a membrane, something that should become easier to address with the advances in cryo-electron microscopy structure determination. identifying if the na protein removes sa residues directly from substrates within the golgi, as this could decrease the effectivity of nonmembrane permeable na inhibitors. it is also unclear how iavs regulate the timing and expression levels of the viral proteins as viral mrna transcription shows little temporal variation (163, 164) . while it is likely that m2 is regulated in part by splicing (112, 114) , this does not apply to ha and na. recent work has linked na and ha regulation to the nucleotide composition of the 5′coding regions for their er-targeting sequences, which dramatically differ from the profile of corresponding regions in human secretory protein mrnas (165, 166) . an obvious candidate for post-transcriptional regulation is the viral rna-binding protein ns1. indeed, many studies have shown that ns1 can increase translation of particular mrnas, possibly by enhancing the translation initiation rate through the recruitment of eif-4g to the 5′region of viral mrnas (165, (167) (168) (169) (170) (171) . however, a clear mechanistic picture for influenza protein regulation is lacking. ha traffics from the er as a fusion incompetent precursor termed ha0. to gain its fusion function, ha must be cleaved into the subunits ha1 and ha2 (74, 172, 173) . the cleavage occurs in either a monobasic, or a multibasic, cleavage site (55) . multibasic sites are commonly found in highly pathogenic avian iavs and are cleaved by furin, a calcium-dependent serine endoprotease that is located within the trans-golgi network (174) . furin is also ubiquitously expressed (175) , which is one of the major reasons why avian iavs with a multibasic cleavage site are generally more pathogenic. by contrast, human (and low pathogenic avian) iavs encode for has with a monobasic cleavage site, which have been shown to be processed by different proteases in human respiratory epithelial cells. these include the transmembrane protease serine s-1 member 2 (tmprss2), human airway trypsin-like protease (hat), and possibly tmprss4 (176, 177) . hat localizes at the plasma membrane where it can either cleave newly synthesized ha or the ha found in cell-associated virions (178, 179) . similar to furin, tmprss2 resides in the trans-golgi network, where it cleaves ha en route to the plasma membrane. the m2 ion channel is thought to prevent the premature activation of ha following cleavage by equilibrating the slightly acidic ph of the golgi (180, 181) . distinct from furin, tmprss2 expression has been found to be more restricted to the upper and lower respiratory tract, whereas hat was mainly shown to be expressed in the upper respiratory tract (182) . these cell tropisms suggest that lower respiratory infections are likely mediated by tmprss2, and could be one of the primary reasons human iavs are confined to the epithelial layer of the respiratory tract. compared with the bulk lipid profile of the plasma membrane, iav envelopes are enriched in cholesterol and sphingolipids (32) , indicating that they bud from distinct apical plasma membrane regions often referred to as "rafts" (183) . however, infectious iavs must possess mechanisms to target the eight vrnps, m1, ha, na, and m2 to these sites in the membrane (184, 185) . ha is believed to localize to these distinct regions based on fatty acid modifications of the c-terminal cysteine that occur in the golgi (186) (187) (188) (189) , whereas na enrichment has previously been attributed to a property in the c-terminus of the tmd (190) . in contrast, m2 has been shown to accumulate at the boundaries of these budding domains (191) , and the cytosolic protein m1 has been proposed to localize to the budding region by associating with the short cytoplasmic tails of ha and na (192) . however, it is equally plausible that na and ha create membrane domains with a unique lipid profile that have a high affinity for m1. finally, the vrnps, delivered to the cell periphery by rab11, are thought to localize to the budding site by binding to m1 (193, 194) . in addition to orchestrating the assembly of the correct viral components at the apical budding site, iavs also have to remodel the membrane to induce bud formation, and ultimately scission of the viral envelope from the plasma membrane. to promote bud formation, the virus must first induce significant curvature in the membrane and then constrict the two opposing membranes of the viral envelope to help to facilitate membrane scission. curvature can be induced by (i) protein or "molecular" crowding on one leaflet of a bilayer, (ii) association of curved or "bending" proteins with the bilayer, (iii) biased accumulation of cone shaped lipids in one leaflet of the bilayer, or (iv) the cytoskeleton (195) . based on cumulative data regarding budding, iavs appear to induce membrane curvature through a combination of these mechanisms. indicative of using molecular crowding and bending proteins, several studies have demonstrated that ha and na expression is sufficient to induce budding, and that the efficiency and shape uniformity benefit from the presence of m1 (196) (197) (198) (199) . these results indicate that the abundance of ha and na on one side of the membrane can contribute to curvature. it also is intriguing to speculate that the asymmetric (154) shape of na plays a role in this process as it is often seen clustering in the viral membrane (16, 199) . by contrast, m1 appears to be analogous to a membrane-bending protein as it recruited to the cytosolic side of the membrane budding site, oligomerizes upon reaching the membrane, and these oligomers have been modeled to form curved structures (200) (201) (202) . based on these properties, it is plausible that m1 significantly influences the membrane curvature at the budding site, potentially explaining its role in discerning whether iavs form spheres or filaments (27, 203) . the ion channel m2 localizes to the budding site boundary and has also been shown to contribute to iav scission by functioning as a membrane-bending protein (191, 204) . the membrane-bending property of m2 is localized in an amphiphilic α-helix that can incorporate the amino acid side chains from its hydrophobic face into a leaflet of the bilayer. with this domain positioned in the cytosol, the intercalation results in negative membrane curvature, which has been proposed to facilitate viral bud neck formation and scission, presumably by decreasing the distance between the two opposing membranes of the viral envelope (204) . while much of the framework concerning iav budding has been established, it has been difficult to identify the details of the budding process, in part due to the mobility and heterogeneity of the plasma membrane. the lack of strong phenotypes from domains proposed to contribute to budding could also imply that iavs have built redundancy into the budding process (205) (206) (207) . the possibility of redundancy is certainly plausible, as iavs contain the necessary components to allow for a combination of lipid recruitment, molecular crowding, and a membrane-bending protein. iav cell release and movement once the newly assembled iavs bud, their release is highly dependent on the sialidase activity of na. na is a homotetramer, and each subunit is comprised of a short n-terminal cytoplasmic tail (six amino acids), followed by a tmd, a length variable stalk, and a globular enzymatic head domain (208) . the globular head domain forms a 6-bladed propeller structure, where each blade is comprised of four antiparallel β-sheets that are stabilized by disulfide bonds (155, 209, 210) . the catalytic tyr residue is found in a highly conserved active site that forms a deep pocket in the center of each monomer (211) . all of the residues necessary for catalysis exist within each monomer (212) , which has made it difficult to reconcile why na evolved to function as a tetramer (208, 213, 214) . structures of the enzymatic head domain indicate that na tetramers bind up to five calcium ions and calcium has been shown to contribute to na activity (155, 208, 215) . however, it remains unclear why influenza na has evolved to position a calcium ion at the tetrameric interface. na facilitates viral release by catalyzing the hydrolysis of the glycosidic linkage that attaches sa to underlying sugar molecules (216) (217) (218) . by removing local sa residues, na prevents ha binding at the cell surface, which facilitates the release of the virus during budding (figure 6a and step vi) (219, 220). na has also been shown to promote the separation of iavs by removing sa residues from the n-linked glycans located on the ha and na molecules in the viral envelope ( figure 6b and step vii) (221) . in contrast to ha, nas from human iavs show a general preference for α2,3-linked sa with variable abilities to cleave α2,6-linked sa residues (208, 222, 223) . however, a thorough analysis of na sa preference is lacking. more recent studies have found that some strains possess nas that are inefficient enzymes, but still capable of sa binding, raising the question of whether a poor na enzyme could contribute to, or replace, the ha receptor-binding function (224, 225) . the movement of iavs from cell to cell in the respiratory epithelium is significantly different from that in immortalized cell lines grown in liquid culture due to the presence of different cell types and a mucus layer. the mucus layer provides a protective barrier for the epithelium and is rich in heavily glycosylated mucins that can interact with iavs and limit cell binding (226, 227) . studies measuring viral movement through mucus and respiratory epithelial cells have shown that na-mediated cleavage of sas from mucins enhances iav movement through the mucus layer and infectivity ( figure 6c and step viii) (226, 228, 229) . recent work showed that this function may also apply to transmission, as iavs that possess low na activity, and are inhibited by mucus, are deficient in aerosol and contact transmission (230) . iavs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves. the functional requirements of each iav protein, such as enzyme catalysis, substrate binding, oligomerization, and domains that perform essential interactions with host proteins all combine to create substantial negative selection pressure that often manifests in the form of sequence conservation. negative pressure can also come from functions within the vrna sequences. these include promoters and "packaging signals, " but are also likely to involve aspects such as the formation of structural elements, or possibly mediating vrnp interactions that generate the 1 + 7 assembly in viral particles. in addition, the exposure of iavs to the immune response and constantly changing environments such as host, temperature, ph, cell type, and antivirals result in positive selection pressure. experimentally, addressing each type of selection has its caveats, but clearly a holistic picture of both iav and host functions are required to begin predictions of evolutionary constraints on the virus. most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. however, all the viral transcribed rnas are subject to replication errors by the viral polymerase, which are estimated at 1 per 2,000-10,000 nucleotides (231) (232) (233) . consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection. as many iav proteins are homo-oligomers this can potentially generate heterogeneity within individual protein complexes that could have functional advantages. by applying single particle and single cell analysis, these types of aspects are beginning to be investigated (234) . another interesting approach is deep mutational scanning, which has been used to examine the site-specific amino acid tolerance of iav proteins in general, and in the context of different selection pressure (235) (236) (237) (238) . currently, the best characterized protein in iavs is ha, which has two primary functions, (i) to initiate binding to the host cell and (ii) to deliver the vrnps to the host cell cytosol by fusing the viral and endosomal membranes. these functions are efficiently divided between the two domains of ha (ha1 and ha2), created by proteolysis. the receptor-binding site responsible for entry is located in the considerably larger ha1 subunit that is known to be immunodominant, explaining the high sequence variability in this region (239) . by contrast, the smaller ha2 subunit, containing the fusion peptide that is necessary to deliver the viral genome to the host cell, shows considerably higher sequence conservation. this organization is logical from the viral perspective as the large ha1 subunit likely blocks antibody recognition of ha2. the viral downside is the need to escape antibodies that inhibit the receptor-binding pocket without losing specificity and the binding function. based on this knowledge, several exciting new strategies are being developed to elicit the production of antibodies that target the more conserved region of ha (240) (241) (242) . the hope is that these strategies will generate broadly neutralizing antibodies that recognize multiple ha subtypes from iavs and the distinct lineages in ibvs, providing longer lasting immunity and alleviating the threat of potential pandemics. a similar approach using na would likely provide additional benefits. however, our knowledge of na lags behind ha. currently, it is still not known why na has evolved to function as a tetramer, which is relevant because this property presumably restricts the potential antigenic drift (mutations) it can accommodate and still function. iav replication and spreading frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 a relatively overlooked feature in the replication process is the contributions of host rna-binding proteins (rbps). human cells are predicted to encode over 1,500 rbps, 700 of which are predicted to interact with mrnas (243) . as a rna virus, it is highly likely that iavs have evolved to utilize this enormous network of rbps, which is supported by observations that some rbps inhibit iav replication, whereas others contribute (244) (245) (246) . it should also be considered that changes in rbps have been associated with various cancers, which could possibly influence the susceptibility to influenza infections (247, 248) . with the growing interest in rna biology, this aspect of iav infections is likely to receive considerable attention in the future. in terms of iav antivirals, the recent progress in determining the structures and mechanisms of the viral polymerase should significantly aid in the current development of drugs aimed at inhibiting different aspects of iav transcription (249) . through continued progress in defining the fundamental mechanisms that are necessary for iav infections, replication and intercellular movement, it should become possible to minimize the annual burden caused by iavs. rd wrote the review with input from dd, rr, hö, and hw. 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the influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity the cytoplasmic tail of the neuraminidase protein of influenza a virus does not play an important role in the packaging of this protein into viral envelopes mutations in the membrane-proximal region of the influenza a virus m2 protein cytoplasmic tail have modest effects on virus replication influenza neuraminidase influenza virus neuraminidase: structure, antibodies, and inhibitors the 2.2 a resolution crystal structure of influenza b neuraminidase and its complex with sialic acid mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity structure of the catalytic and antigenic sites in influenza virus neuraminidase conversion of a class ii integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms a 2 (n2) neuraminidase of the x-7 influenza virus 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variants of human influenza a(h3n2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment? influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors influenza a penetrates host mucus by cleaving sialic acids with neuraminidase mucoproteins in relation to virus action a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus neuraminidase is important for the initiation of influenza virus infection in human airway epithelium pandemic swine h1n1 influenza viruses with almost undetectable neuraminidase activity are not transmitted via aerosols in ferrets and are inhibited by human mucus but not swine mucus comparative mutational analyses of influenza a viruses rates of spontaneous mutation among rna viruses rapid evolution of rna viruses extreme heterogeneity of influenza virus infection in single cells deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by mxa the inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin how single mutations affect viral escape from broad and narrow antibodies to h1 influenza hemagglutinin immunogen design for hiv-1 and influenza is it possible to develop a "universal" influenza virus vaccine? toward a universal influenza virus vaccine: potential target antigens and critical aspects for vaccine development hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection structure and function analysis of an antibody recognizing all influenza a subtypes a census of human rna-binding proteins cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing cellular ddx21 rna helicase inhibits influenza a virus replication but is counteracted by the viral ns1 protein multiple nuclear-replicating viruses require the stress-induced protein zc3h11a for efficient growth dissecting the expression landscape of rna-binding proteins in human cancers rna-binding proteins in cancer: old players and new actors inhibitors of influenza a virus polymerase the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-018089-m94q75xn authors: mubareka, samira; palese, peter title: influenza virus: the biology of a changing virus date: 2010-06-18 journal: influenza vaccines for the future doi: 10.1007/978-3-0346-0279-2_1 sha: doc_id: 18089 cord_uid: m94q75xn influenza viruses are members of the family orthomyxoviridae and include influenza virus types a, b, and c. this introduction provides an overview of influenza virus classification, structure, and life cycle. we also include a brief review of the clinical manifestations of influenza and the molecular determinants for virulence. the genetic diversity of influenza a viruses and their capability to successfully infect an array of hosts, including avian and mammalian species, are highlighted in a discussion about host range and evolution. the importance of viral receptor-binding hemagglutinins and host sialic acid distribution in species-restricted binding of viruses is underscored. finally, recent advances in our understanding of the seasonality and transmission of influenza viruses are described, and their importance for the control of the spread of these viruses is discussed. h1n1 influenza virus arose from an unpredicted source; precursors of the pandemic influenza a (h1n1) 2009 virus have been circulating among pigs for over a decade [1, 2] . additional reassortment events have led to the current pandemic influenza a (h1n1) 2009 virus. features observed in past pandemics, including atypical seasonality and shifting of the burden of disease to younger populations, are evident during the influenza pandemic of 2009. our understanding of the biology of influenza virus and its effect on the host has advanced considerably in recent decades. recent events in influenza virus research have contributed to this progress [3] . these include the development of plasmidbased reverse genetics systems [4, 5] , the generation of the 1918 pandemic h1n1 influenza virus [6] , improved access to biosafety level 3 facilities, the establishment of international influenza virus sequence databases, and bioinformatics [7, 8] . advances have also led to the production of fda-approved antivirals for influenza, and a heightened understanding of host-virus interactions resulted in the exploration of novel therapies including immunodulatory approaches [9] . new vaccine technologies such as the use of live-attenuated vaccines [10] [11] [12] [13] and the development of novel vaccine production methods, including cell culture-based approaches, are the benefits of scientific progress. continued acceleration of influenza virus research has direct implications for the development of improved vaccines, infection control, and clinical management during pandemic and interpandemic periods. influenza viruses are members of the family orthomyxoviridae and include influenza virus types a, b, and c. influenza viruses possess seven (influenza c) or eight (influenza a and b) genome segments composed of negative sense single-stranded rna. these types differ in various aspects, the most important of which include antigenicity, host range, pathogenicity, transmission, and seasonality. standard nomenclature for human influenza viruses includes type, geographic location of isolation, isolate number, and year of isolation. for example, an influenza a virus isolated in panama in 1999 would be referred to as a/panama/2002/1999. subtypes of influenza a viruses are described by hemagglutinin (ha) and neuraminidase (na) designations. to date, 16 ha and 9 na subtypes have been described. influenza a viruses are mostly responsible for seasonal epidemics, global pandemics, and the burden of disease attributable to influenza. clinical disease includes systemic and respiratory manifestations, and rarely may be complicated by central nervous system involvement, toxic shock, or multiorgan system failure [14, 15] . circulating strains of influenza a viruses are targets for annual vaccination to mitigate morbidity and mortality imparted by these viruses. in addition to infecting humans, influenza a viruses circulate in other mammals, including swine and horses. waterfowl harbor several lineages of influenza a viruses and serve as a reservoir. transmission among wild and domestic fowl and mammalian species is an important characteristic of influenza a, enabling viral reassortment and emergence of novel subtypes in susceptible human populations. in contrast, influenza b virus has a restricted host range, circulating only in humans, although the virus has been isolated in seals [16] . influenza b virus demonstrates seasonality and is responsible for human disease, although the clinical manifestations are generally less severe compared with influenza a virus-associated illness. nonetheless, rare cases of encephalitis and septic shock have been described in children [17, 18] . at present, the two major lineages are represented by influenza b/victoria/2/1987 and b/yamagata/16/1988 viruses [19] . re-emergence of the victoria lineage after a decade of absence was associated with an outbreak during the 2001-2002 influenza season, affecting healthy but immunologically naive children [20] . influenza b virus is included in inactivated and live-attenuated annual influenza vaccines. unlike influenza a and b, influenza c virus lacks neuraminidase and codes for a single-surface hemagglutinin-esterase-fusion (hef) glycoprotein. this virus does not demonstrate marked seasonality and is not included in the annual influenza vaccine, although it has been responsible for occasional outbreaks, predominantly in children [21] . illness in humans is generally mild and consists of an upper respiratory tract infection. influenza c has also been isolated in swine, raising the possibility that this species may serve as a reservoir [22] . influenza viruses are enveloped, deriving the lipid bilayer from the host cell membrane during the process of budding. viral particles are pleomorphic in nature and may be spherical or filamentous, ranging in size from 100 to over 300 nm [3] . spikes consisting of ha and na project from the surface of virions at a ratio of roughly 4:1 in influenza a viruses ( fig. 1 ) [3] . the viral envelope is also associated with the matrix (m2) protein which forms a tetrameric ion channel. the polymerase proteins pb1, pb2, and pa, the nucleoprotein (np), and the virion rna comprise the ribonucleoprotein (rnp) complex. this complex is present in the core of virions, which also includes the nuclear export and nonstructural protein (nep/ns1). influenza virus genes, gene products, and primary functions are summarized in table 1 . in humans, influenza viruses are transmitted by the respiratory route. host cellular receptors consist of oligosaccharides residing on the surface of respiratory epithelial cells. specificity of binding is imparted by the linkage of the penultimate galactose (gal) to n-acetylsialic acid (sa). a2,6 linkage (saa2,6gal) is distributed in the human respiratory tract and is associated with binding to human influenza virus ha. in contrast, avian hosts including waterfowl and domestic poultry harbor sialic acid with a2,3 linkage (saa2,3gal) which is distributed in the gastrointestinal tract, reflecting the fecal-oral mode of transmission of avian influenza strains in these species [23] . specificity of viral ha binding is imparted by the receptor-binding pocket on the surface of the ha molecule (fig. 2) . the ha is a rod-shaped trimer anchored in the virion's envelope and contains three primary ligand-binding sites on a globular head [24, 25] . specificity of binding has been linked to certain amino acid residues in the ha receptor-binding domain. in h3 subtypes, amino acid 226 is one such residue, where the presence of leucine allows binding of saa2,6gal, whereas the presence of glutamine at this position permits binding of saa2,3gal. amino acid changes in the ha of other subtypes, such as h1 viruses (including the h1n1 virus responsible for the 1918 pandemic), have been associated with adaptations in receptor-binding specificity, translating into a switch in host specificity with disastrous consequences [26, 27] . specifically, changes at amino acid position 225 impart the ability of a/new york/1/18 to bind both avian and human host influenza virus receptors [26] . strains of the 2009 pandemic h1n1 influenza viruses retain amino acids (aspartic acids) at positions 190 and 225 of the ha consistent with human sialic acid receptorbinding specificity, although conflicting data exist regarding binding specificity for these viruses. one approach utilizing carbohydrate microarrays suggests that dual (human and avian) sialic acid receptor binding occurs [28] ; data obtained using a different approach, namely biotinylated a2,3and a2,6-sialylated glycans, suggest currently circulating pandemic viruses preferentially bind human sialic acid receptors with a2-6 linkage [29] . the importance of these amino acid residues to respiratory droplet transmission has recently been described using the ferret transmission model. h1n1 viruses containing aspartic acids at residues 190 and 225 were capable of aerosol transmission. this contrasted with h1n1 viruses with glutamic acid and glycine at residues 190 and 225, respectively (consistent with avian sialic acid receptor-binding specificity), which did not transmit through the air [30] . furthermore, other changes in the ha (and na) of an avian h9n2 after adaptation in the ferret conferred a more efficient respiratory transmission phenotype [31] . several possible pathways for the entry of influenza viruses into host cells have been postulated and recently reviewed [32] . endocytosis is a multistep process consisting of surface receptor-mediated binding, internalization, and intracellular trafficking. clathrin-mediated and clathrin-independent internalization via caveolae and caveolae-independent endocytosis have been demonstrated [33, 34] . an initial acidification step in early endosomes is followed by trafficking to low-ph late endosomes, a process mediated by members of the rab host protein family. fusion of influenza virus to the endosome is triggered by low ph conditions and mediated by the fusion peptide of ha2 after cleavage of ha, creating a pore in the endosome through fusion of viral and endosomal membranes ( fig. 3 ) [3] . subsequent steps in the uncoating process involve the influenza virus tetrameric m2 protein, which is involved in the release of rnp into the host cell cytoplasm through ion channel activity [35, 36] . viral rna (vrna) synthesis occurs in the nucleus, and viral rnps must therefore be imported. this process is primarily mediated by viral np, which coats viral rna and possesses nuclear localization signals (nlss), including an unconventional nls which binds host karyopherin-a and is essential for energy-dependent rnp nuclear import [37, 38]. viral rna serves as a template for the production of messenger rna (mrna) and subsequent transcription, as well as for the generation of complementary rna (crna), which is positive sense and functions as a template for the generation of more vrna (viral replication). rna segments are coated by np through nonspecific interactions between the arginine-rich positively charged np and the negatively charged rna phosphate backbone [3] . the viral polymerase complex consists of tightly associated pb1, pb2, and pa and associates with np-coated rna without disrupting this interaction [39] . pb1 is an endonuclease involved in both replication and transcription and binds the promoter region of rna segments [40] . it functions as an rna-dependent rna polymerase and catalyzes rna chain elongation. interaction with pa is required for this function and viral replication [41]. pb2 binds both np and pb1 via separate binding sites [42] . initiation of transcription is reliant on pb2, which binds the cap on host pre-mrna, and this cap serves as a primer for transcription [43, 44] . in addition, interactions between pb2 and host proteins may be species specific and potentially plays a role in restricting host range synthesis of mrna begins with a host cell 5 0 -capped primer, generated by host cell rna polymerase ii and obtained from host pre-mrna [44] . transcription is thus initiated and synthesis on the template occurs in a 3 0 to 5 0 direction. a polyadenylation signal consisting of 5-7 uridines at the 5 0 end of vrna prematurely terminates transcription after inducing stuttering of the viral polymerase [48] [49] [50] . the generation of np and ns1 tends to occur earlier after infection compared with the generation of surface glycoprotein and m1 mrnas [3] . mechanisms for the regulation of gene expression remain evasive, although np has been implicated in the control of gene expression [51] . viral replication requires the synthesis of vrna, which is primer independent and occurs through a crna intermediate. nascent crna is therefore not capped or polyadenylated upon termination. the notion that crna synthesis is initiated after a switch from mrna synthesis has been challenged [52] . rnp complexes subsequently associate with m1 at its c-terminal domain, and aggregation of this complex leads to inhibition of transcription [53] . m1 also interacts with nep at its c-terminal domain [38, 54] . nep, in turn, associates with host nuclear export receptor crm1 via the nep n-terminal domain [54], thus orchestrating the export of viral rnp from the nucleus. posttranslational modification of the ha consists of glycosylation in the golgi apparatus [55] . along with viral rnp, protein components of the virion are coordinately trafficked to the apical surface of the host cell for assembly into progeny virus. two models for the packaging of viral rna segments exist and include the random incorporation [56, 57] and the selective incorporation models [58, 59] . the latter implies that each rna segment possesses a packaging signal, resulting in virions with exactly eight segments. putative packaging signals in coding regions of polymerase genes, spike glycoprotein genes, and the ns gene have been identified [58, [60] [61] [62] [63] . viral assembly is coordinated by the m1 protein, which associates with the cytoplasmic tails of the viral glycoproteins [19, 64, 65] , as well as rnp and nep, as described above. lipid rafts navigate viral membrane glycoproteins to the apical surface of the host cell [66, 67] . in addition, there is evidence that targeting of np and polymerase proteins to the apical surface also involves lipid rafts [68] . genomic packaging and viral assembly occurs at the apical membrane and is associated with accumulation of m1 and the formation of lipid rafts. the m1 protein has also been implicated in viral morphology [69, 70] . because the ha binds cell surface sialic acid receptors, virions must be released. the na functions as a sialidase and cleaves sialic acids from the host cell and viral glycoproteins to minimize viral aggregation at the cell surface [71] . balance between the ha and na is thus required for optimal receptor binding and destruction [64, 72] . in addition to its receptor-destroying activity, na is a viral spike glycoprotein and important surface antigen [73] . among the influenza virus types, influenza a demonstrates the most genetic diversity and is capable of successfully infecting an array of hosts, including avian and mammalian species. influenza a viruses exhibit an evolutionary pattern, which is complex and consists of antigenic drift and shift. drift occurs on an annual basis and has been attributed to low fidelity of the rna polymerase and subsequent selection from immune pressure exerted by the host [74] . this results in antigenic diversity of the hemagglutinin and neuraminidase glycoproteins and is one of the major challenges to vaccine production, requiring annual changes to vaccine components. the ha1 domain contains several epitopes and is the most dynamic as a consequence, demonstrating clusters of antigenic variance over time [75] . antigenic shift results after a viral reassortment event where exchange of one or more of the viral segments with that of another strain may result in a novel serotype, potentially diversifying the host range of the virus. it is in this setting that pandemic strains have emerged in immunologically naïve populations in the past, including the h2n2 (with new ha, na, and pb1 segment) subtype in 1957 and the h3n2 influenza virus (with new ha and pb1 segments) which caused a pandemic in 1968 (fig. 4) . since 1997, several avian influenza viruses, including h5n1, h7n2, h7n3, h7n7, h9n2, and h10n7 subtypes, have infected humans [76], though limited evidence for person to person spread exists [77, 78] . lack of transmission among humans remains a barrier to pandemic spread of these viruses. the h5n1 subtype isolated from avian species has undergone genetic reassortment, and several genotypes exist. genotypes z and v are largely responsible for outbreaks of highly pathogenic influenza viruses (hpai) in domestic birds in southeast asia beginning in 2003 [77] . h5n1 viruses may also be divided into clades based on the genomic analysis of the ha genes, and clade 2 is further divided into subclades; up to ten clades have been identified in avian species, four of which have infected humans [79, 80] . less than 1% divergence from avian isolates has been reported in viruses isolated from humans in asia [7] . the pandemic influenza a (h1n1) 2009 virus has been described as a "triple reassortant" of swine, human, and avian influenza viruses; the h1 gene from this virus has been circulating among swine for decades, with limited drift compared with genes of h1 viruses that have been circulating in humans, and is thus antigenically different from seasonal human h1n1 viruses. the pandemic influenza a (h1n1) 2009 virus is composed of six segments from the triple reassortant, including a human pb1 segment, classical swine-origin ha, np, and ns, and avian-origin pb2 and pa segments that have been circulating in swine since approximately 1998. the na and m segments originate from a eurasian lineage of swine influenza viruses [1, 2, 81] (fig. 5) . in order to tackle the challenge of understanding the evolution of influenza virus, large-scale collaborative efforts such as the influenza genome sequencing project have been undertaken. the presence of several cocirculating clades in the human population has been described, accounting for reassortment. this can result in limited vaccine effectiveness, as seen with a/fujian/411/2002-like virus during the 2003-2004 season [8] . genetic evolution appears to be a relatively gradual process; however, antigenic changes in the ha1 domain tend to cluster [75] . ongoing changes of the h3 hemagglutinin in the human population result from selective pressure exerted by the host immune system. in contrast, the h3 lineage in birds has remained relatively stable [82] . the rate of change of the h3 subtype is greater when compared with h1 viruses and influenza b, with estimated nucleotide changes per site per year of 0.0037 for h3, 0.0018 for h1, and 0.0013 for influenza b [83] . as greater numbers of influenza virus genome sequences become available and we gain insight into antigenic patterns of change, this knowledge may be applied to annual vaccine development. prediction of future influenza sequences could lead to more timely development of effective vaccines [84] though modeling methods have yet to be validated. influenza a virus is a zoonotic pathogen capable of infecting birds (waterfowl and chickens), swine, horses, felines, and other species. host range restriction of different types of influenza viruses is observed. species-restricted binding of viruses is mediated by different types of receptor-binding hemaglutinins [85] [86] [87] [88] [89] . the distribution of different types of sa linkages has recently been elucidated in humans though the type of cell infected (ciliated vs. nonciliated) is under debate [90, 91] . sa with a2,6gal linkage predominates on epithelial cells of the upper airway, including nasal mucosa, sinuses, bronchi, and bronchioles [92] . in human tracheobronchial epithelial (htbe) cells, oligosaccharides with sa with a2,6gal linkage predominate on nonciliated epithelial cells [91] although these oligosaccharides have been described on ciliated and goblet cells in the human airway [93] . lower airways contain sa with mostly a2,3gal linkage, in addition to sa with a2,6gal linkage [92, 94] . host restriction is not absolute, and human infections with avian influenza viruses (including h5n1, h9n2, and h7n7 viruses) have been extensively described [95] [96] [97] [98] [99] [100] . h5n1 binds type ii pneumocytes and macrophages of the lower respiratory tract in humans [92, 94, 101] . h5n1 infection of ciliated cells in htbe cell culture with limited cell-to-cell spread [90] and of human nasopharyngeal, adenoid, and tonsillar ex vivo cell cultures has been shown [102] . binding of h5n1 viruses to saccharides terminating in a2,6gal sa linkage has been achieved by mutating ha amino acid residues at positions 182 and 192, suggesting potential for adaptation to the human host [103] . differences in influenza virus receptors among avian species have been described and are reflected in differential binding of different types of avian influenza viruses. although chicken and duck influenza viruses preferentially bind a2,3gal-linked sa, viruses from chickens had greater affinity for sa where the third sugar moiety was a b(1-4)glcnac-containing synthetic sialylglycopolymer. duck viruses preferred b(1-3)galnac sugar moieties in the third position [104] . distribution of influenza virus receptors reflects the sites of replication. in chickens and waterfowl, sa with a2,3gal linkage is found in the upper respiratory tract and intestines. some species demonstrate the ability to support replication of both human and avian influenza viruses. the respiratory tract and intestines of quail contain both a2,3galand a2,6gal-linked terminal sialic acids [105] . in swine, oligosaccharides with both types of linkages may be found and suggest this species serves as a mixing vessel where human, avian, and swine influenza viruses can reassort [106, 107] . uncomplicated influenza in humans is an upper respiratory tract infection characterized by cough, headache, malaise, and fever (influenza-like illness). these symptoms are nonspecific and are not predictive of influenza virus infection, particularly in individuals <60 years old [108] . pulmonary and extrapulmonary complications may arise. the latter consist of central nervous system involvement (encephalitis, acute necrotizing encephalopathy, reye's syndrome, and myelitis) [14] , myositis/rhabdomyositis [109] , myocarditis [109, 110] , increased cardiovascular events [111] , disseminated intravascular coagulation [109] , and toxic and septic shock (bacterial and nonbacterial) [15, 18, 109] . pulmonary complications include primary viral pneumonia, secondary bacterial pneumonia (see below), and exacerbation of chronic lung disease [109, 112] . acute lung injury (ali)/acute respiratory distress syndrome (ards), multiorgan failure, profound lymphopenia, and hemophagocytosis have been associated with h5n1 infection and carry high mortality rates [15, 95, [113] [114] [115] . bacterial pneumonia following influenza virus infection is a well-recognized complication of influenza since the pandemic of 1918 [116] . more recently, pediatric deaths have been attributed to copathogenesis between influenza virus and staphylococcus aureus, accounting for 34% of pediatric deaths reported to the cdc during the 2006-2007 influenza season [117] . in one case series, 43% of coinfected cases involved methicillin-resistant s. aureus, thus contributing to management challenges for these patients. coinfection was also associated with a worse prognosis compared with influenza virus or s. aureus infection alone [118] . to date, secondary bacterial lower respiratory tract infection has not been a dominant feature in adults during the current 2009 pandemic but has been described in children [119] . severe pandemic 2009 influenza has been predominantly associated with viral pneumonitis and subsequent ali, particularly in pregnant women in their third trimester [120] and indigeous people including aborigines in australia [121] , maoris and pacific islanders in new zealand [122] , and first nations people in canada [123] . few human histopathological studies of uncomplicated influenza exist. pathological findings from postmortem examination of 47 fatal pediatric influenza a cases included major airway congestion (90%), inflammation (73%), and necrosis (50%) [112] . lower airway pathology included hyaline membranes (67%), interstitial cellular infiltrates (67%), and diffuse alveolar damage (dad). secondary pneumonia, intraalveolar hemorrhage, and viral pneumonitis were noted in a quarter of cases [112] . fulminant dad with acute alveolar hemorrhage and necrosis followed by paucicellular fibrosis and hyaline membrane formation is observed in h5n1infected human lungs [124] . extrapulmonary pathology includes reactive hemophagocytosis in the hilar lymph nodes, bone marrow, liver, and spleen [125] ; white matter demyelination [124] and cerebral necrosis [101] ; and acute tubular necrosis of the kidneys [113] . despite the presence of diarrhea and h5n1 virus replication in the gastrointestinal tract of humans, no pathological lesions have been described in the bowel [101, 114] . immune dysregulation has been implicated in the pathogenesis of ards and reactive hemophagocytosis. elevated levels of neutrophil, monocyte, and macrophage chemoattractants (il-8, ip-10, mig, and mcp-1) and proinflammatory cytokines (il-10, il-6, and ifn-g) are observed in h5n1-infected humans [95] . in addition, increased levels of il-2 (in a human case) [113] and rantes (in primary human alveolar and bronchial epithelial cells) [126] have also been reported. contribution of proinflammatory mediators to lung pathology has also been demonstrated using toll-like receptor 3 knockout mice infected with mouse-adapted wsn influenza a virus. these mice demonstrated enhanced survival despite higher virus replication and lower levels of rantes, il-6, and il-12p40/p70 compared with wild-type mice [127] . likewise, host response has been implicated in the copathogenesis of bacterial pneumonia post-influenza virus infection. specifically, sensitization by type i interferons [128] , induction of il-10 [129] , and upregulation of interferon-a [130] have been linked to secondary bacterial pneumonia after influenza virus infection. viral determinants for copathogenesis have also been elucidated and include pb1-f2 and viral neuraminidase [131, 132] . the ha, pa, pb1, pb2, pb1-f2, na, and ns1 gene products have been implicated in virulence. virulence determinants have been explored using the reverse genetic system for influenza viruses and mammalian (ferret and mouse) models for influenza virus pathogenicity. the polymerase gene complex, consisting of pa, pb1, and pb2 genes, is involved in replication and transcriptional activity. a single-gene reassortant containing the pb2 from a/hong kong/483/97 (h5n1, which is fatal in mice) in the background of a/hong kong/486/97 (h5n1, causing mild respiratory infection in mice) demonstrated a lethal phenotype in this animal model [133] . in addition, reassortants containing polymerase complex genes from a/chicken/vietnam/c58/ 04 (h5n1), a nonlethal virus, in the background of a/vietnam/1203/04 (h5n1) influenza virus isolated from a fatal human case were attenuated in an animal model [134] . when a single point mutation k627e in the pb2 gene was generated in a/ vietnam/1203/04 [134] and in a/hong kong/483/97 [133] , virulence was reduced in mice, although in other studies this substitution did not reduce virulence substantially [135] . the molecular mechanism(s) responsible for virulence have yet to be completely elucidated. enhanced replication of viruses retaining a lysine at position 627 in pb2 at the lower temperatures of the upper respiratory tract (33 c) [136] may be responsible for robust transmission in mammals [137] . this theory is supported by recent work demonstrating that replacement of the lysine at position 627 with glutamic acid (avian consensus sequence) abrogates aerosol transmission of a 1918 influenza a virus [30] . currently circulating strains of pandemic h1n1 influenza virus have a glutamine in pb2 at position 627. this may account for reduced efficiency of aerosol transmission of this virus in ferrets, compared with a seasonal h1n1 virus [29] . pb1-f2 is the gene product arising from a second reading frame of the pb1 gene and has been implicated in immune cell apoptosis through the vdac1 and ant3 mitochondrial pathways [138] . knockout of pb1-f2 did not alter viral replication, but enhanced clearance of the virus and reduced lethality in mice was demonstrated, suggesting that pb1-f2 may play a role in viral pathogenesis [139] . enhanced pathogenicity was observed in mice infected with recombinant influenza virus containing the pb1-f2 gene from a highly pathogenic h5n1 virus isolated from a fatal human case in hong kong in 1997 [139] . currently circulating strains of the pandemic influenza a h1n1 2009 virus do not express pb1-f2. evasion of the host immune response is a key virulence determinant, permitting viruses to establish sustainable infection. the innate immune system is the first line of host defense, and the influenza virus possesses the ability to interfere with this response. type i interferons (ifn-a/b) are central to establishing an antiviral state in host cells. interferon antagonism has been primarily attributed to the ns1 protein of influenza virus, which plays a multifunctional role in preventing the activation of ifn transcription factors (for review, see [140, 141] ). the effect of avian influenza virus ns1 on ifn production has also been explored. a/goose/guangdong/1/96 virus with an ns1 that differs by one amino acid from a/goose/guangdong/2/96 at position 149 is lethal in chickens and antagonizes ifna/b [142] . in addition, the c-terminus of the ns1 protein contains a pdz ligand domain, capable of binding pdz protein interaction domains of host proteins, thus potentially disrupting host cellular pathways. viruses causing pathogenic infection in humans between 1997 and 2003 contained avian motifs at the ns1 pdz ligand-binding site. these and the motif found in the 1918 influenza virus ns1 had stronger binding affinities to pdz domains of human cellular proteins compared with low pathogenicity influenza viruses [143] . neurovirulence has been associated with glycosylation of the na glycoprotein [144] . the ha glycoprotein has also been associated with virulence. although cleavability of the ha gene has been primarily implicated in pathogenicity in chickens, lethality has also been demonstrated in mice. basic amino acids at the ha cleavage site are determinants for ha cleavage and ha2 fusion activity [145] . enhanced cleavage of the ha by ubiquitous host proteases is made possible by the presence of a polybasic cleavage site, contributing to the virulence of highly pathogenic avian influenza viruses [146, 147] . replacement of the polybasic cleavage site in a high pathogenicity h5n1 virus from hong kong (hk483) with an amino acid sequence typical of low pathogenicity viruses resulted in attenuation [133] . pandemic influenza a h1n1 2009 virus strains do not appear to have the polybasic cleavage site. virulence determinants for the pandemic 2009 h1n1 virus are currently investigation. data obtained from mammalian models early in the course of the spread of this virus indicate that compared with a seasonal h1n1 influenza virus, strains of the pandemic virus replicate more efficiently in the lower respiratory tract, and are stronger inducers of proinflammatory mediators, and induce bronchopneumonia [148] . influenza a and b viruses exhibit marked seasonality, and this pattern dictates the annual vaccination schedule. several theories with respect to the mechanism(s) responsible for this seasonal pattern have been proposed (for review, see [149] ). year-round human influenza virus activity in equatorial regions may be a reservoir for annual outbreaks in the northern and southern hemispheres. as research progresses in this area, factors determining seasonality may be exploited for the control of the spread of influenza virus [150] . transmission of influenza virus among humans is poorly understood and the mode(s) of spread are currently under debate [151, 152] . it is widely accepted that influenza virus is transmitted by the respiratory route in humans, though the contribution of small particle aerosols relative to large respiratory droplets is unknown. in addition, the role of fomites is questionable. until recently, ferrets have served as the only animal model for the study of influenza virus transmission. a novel mammalian model using the guinea pig has recently been developed to overcome the limitations of the ferret model. guinea pigs are highly susceptible to infection with an unadapted human h3n3 (a/panama/2002/1999, or pan99) influenza virus, with a 50% infectious dose of 5 pfu, and this virus grows to high titers in the upper respiratory tract and to moderate titers in the lungs. transmission of pan99 by direct contact and aerosol in this system is 100% (fig. 6) [153] ; however, transmission efficiency may vary among influenza virus subtypes [154] . environmental factors such as temperature and relative humidity also appear to play a substantial role [155, 156] . control of influenza virus spread during interpandemic and pandemic periods through vaccination [157] and physical means will be paramount to abrogating person-to-person transmission and is crucial where viruses are resistant to currently available antivirals. effective and timely vaccine development depends on in-depth understanding of influenza virus biology. although recent advances have been made, ongoing research will be required to fulfill this goal. identification and characterization of days post-infection nasal wash titre (log 10 pfu/ml) air flow exposed inoculated exposed inoculated exposed inoculated fig. 6 close range transmission of human influenza a among guinea pigs. inoculated animals placed in proximity to uninoculated animals (without direct contact) spread pan99 to all exposed animals. adapted from [109] the molecular signatures required for transmission will be of utmost importance to preventing further influenza virus pandemics. globalization of h1n1 infection in humans requires parallel efforts on behalf of virologists in conjunction with epidemiologists and other members of the public health community to translate the growing body of knowledge into means by which influenza spread can be controlled. antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans triple-reassortant swine influenza a (h1) in humans in the united states orthomyxoviridae: the viruses and their replication rescue of influenza a virus from recombinant dna generation of influenza a viruses entirely from cloned cdnas characterization of the reconstructed 1918 spanish influenza pandemic 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indonesian clusters of h5n1 virus infection in 2005 avian influenza a (h5n1) infection in eastern turkey in 2006 human infection with an avian h9n2 influenza a virus in hong kong in 2003 transmission of h7n7 avian influenza a virus to human beings during a large outbreak in commercial poultry farms in the netherlands avian influenza a virus (h7n7) associated with human conjunctivitis and a fatal case of acute respiratory distress syndrome influenza a h5n1 replication sites in humans tropism of avian influenza a (h5n1) in the upper and lower respiratory tract haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to human-type receptors differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 h5n1 chicken and human influenza viruses from hong kong quail carry sialic acid receptors compatible with binding of avian and human influenza viruses evidence for interspecies transmission and reassortment of 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relationship: a lesson in detente the multifunctional ns1 protein of influenza a viruses the ns1 gene contributes to the virulence of h5n1 avian influenza viruses large-scale sequence analysis of avian influenza isolates glycosylation of neuraminidase determines the neurovirulence of influenza a/wsn/33 virus sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza a virus survey of the hemagglutinin (ha) cleavage site sequence of h5 and h7 avian influenza viruses: amino acid sequence at the ha cleavage site as a marker of pathogenicity potential in vitro and in vivo characterization of new swine-origin h1n1 influenza viruses influenza seasonality: underlying causes and modeling theories seasonal dynamics of recurrent epidemics review of aerosol transmission of influenza a virus transmission of influenza a in human beings the guinea pig as a transmission model for human influenza viruses transmission of influenza virus via aerosols and fomites in the guinea pig model influenza virus transmission is dependent on relative humidity and temperature high temperature (30 c) blocks aerosol but not contact transmission of influenza virus blocking interhost transmission of influenza virus by vaccination in the guinea pig model acknowledgments the work completed in this laboratory was partially supported by the w.m. keck foundation, national institutes of health grants p01 ai158113, the northeast biodefense center u54 ai057158, the center for investigating viral immunity and antagonism (civia) u19 ai62623. s.m. is grateful for the ruth l. kirschstein physician scientist research training in pathogenesis of viral diseases award (5t32a1007623-07) and support from sunnybrook health sciences center, toronto, on, canada. key: cord-020756-d9f5fd7x authors: de jong, menno douwe title: avian influenza viruses and pandemic influenza date: 2007 journal: new and evolving infections of the 21st century doi: 10.1007/978-0-387-32830-0_9 sha: doc_id: 20756 cord_uid: d9f5fd7x nan global outbreaks with high death tolls. these pandemic strains can certainly be regarded as (re)emerging pathogens. the "athen's plague" described by hippocrates' contemporary thucydides is believed by some to constitute the first account of such a devastating influenza epidemic. since the 16th century, many large-scale outbreaks of influenza-like illnesses have been described in europe. in 1580, one of such outbreaks spread from europe into africa and asia, possibly making it the first recorded influenza pandemic. the most devastating influenza pandemic in modern recorded history, known as the "spanish flu," occurred in 1918-1919, killing up to 100 million people worldwide. other less destructive pandemics during the past century occurred in 1957 and 1968. avian influenza a viruses are key to the emergence of human influenza pandemics. the virus strains implicated in the 20th century's influenza pandemics originated directly from avian influenza viruses, either through genetic reassortment between human and avian influenza strains (1957, 1968) or possibly through adaptation of purely avian strains to humans (1918) . it was long thought that the host range of avian influenza viruses precluded direct transmission to humans and that the emergence of pandemic strains required genetic reassortment between avian and human strains. however, occurrences of direct bird-to-human transmission of avian influenza viruses have increasingly been reported in recent years, culminating in the ongoing outbreaks of influenza a (h5n1) among poultry and wild birds in several asian, european and african countries with continuing instances of human infections. these unprecedented developments have resulted in increasing global concerns about the (re)emergence of pandemic influenza a strains and the role of avian influenza viruses in this. influenza viruses are pleomorphic, enveloped rna viruses belonging to the family orthomyxoviridae. protruding from the lipid envelope are two distinct glycoproteins: the hemagglutinin (ha) and neuraminidase (na) . ha attaches to cell-surface sialic acid receptors, thereby facilitating entry of the virus into host cells. because it is the most important antigenic determinant to which neutralizing antibodies are directed, ha represents a crucial component of current vaccines. na is the second major antigenic determinant for neutralizing antibodies. by catalyzing the cleavage of glycosidic linkages to sialic acid on host cell and virion surfaces, this glycoprotein prevents aggregation of virions thus facilitating the release of progeny virus from infected cells. inhibition of this important function represents the most effective antiviral treatment strategy to date. a third membrane protein, the m2 protein, is present in small quantities in influenza a viruses. by functioning as an ion channel, this protein regulates the internal ph of the virus, which is essential for uncoating of the virus during the early stages of viral replication. this function is blocked by the antiviral drugs amantadine and rimantadine. the genome of influenza viruses is segmented, consisting of eight single-stranded, negative-sense rna molecules that encode 10 proteins. the rna segments are contained within the viral envelope in association with the nucleoprotein (np) and three subunits of viral polymerase (pa, pb1, and pb2), which together form the ribonucleoprotein (rnp) complex responsible for rna replication and transcription. additional proteins contained within the virion include m2 and the viral nuclear export protein (nep), which function in assembly and budding and in export of rnp from the nucleus, respectively. the only nonstructural protein of influenza a viruses is ns1, which has multiple functions in viral replication and is also thought to counteract interferon activity of the host thereby evading the immune response. based on antigenic differences in np and m proteins, influenza viruses are classified as types a, b, and c. influenza b and c viruses are not divided into subtypes. all avian influenza viruses are classified as type a. further subtyping of influenza a viruses is based on antigenic differences between the two surface glycoproteins ha and na. to date, 16 ha subtypes (h1-h16) and 9 na subtypes (n1-n9) of influenza a viruses have been identified (fouchier et al., 2005) . the standard nomenclature for influenza viruses include the influenza type, the host of origin (excluding humans), the place of isolation, the strain number, the year of isolation, and finally the influenza a subtype in parentheses (e.g., a/duck/vietnam/11/04 [h5n1]). the natural reservoir of influenza a viruses are aquatic birds, in which the viruses appear to have achieved an optimal level of host adaptation and do not cause disease . from this principal reservoir, viruses are occasionally transmitted to other animals, including mammals and domestic poultry, causing transitory infections and outbreaks. through adaptation by mutation or genetic reassortment, some of these viruses may establish species-specific permanent lineages of influenza a viruses and cause epidemics or epizootics in the new host. in the human population, the establishment of these lineages in the 20th century was preceded by influenza pandemics. transmission of viruses and transitory infections may also occur among the new hosts (e.g., between humans and pigs or chickens and humans). although all ha and na subtypes are found in aquatic birds, the number of subtypes that have crossed the species barrier and established stable lineages in mammals is limited. only three ha and two na subtypes (i.e., h1n1, hin2, h2n2, and h3n2) have circulated in humans since 1918. in horses, only two influenza a subtypes (h7n7 and h3n8) are found, while, despite susceptibility to all avian subtypes in experimental settings, the only subtypes recovered from pigs in nature are h1, h3, n1, and n2. the molecular, biological, or ecological factors determining the apparent subtype-specific ability of viruses to cross species barriers and spread among a range of hosts remain largely unresolved. although interspecies transmission does occur at times, there certainly are host-range restrictions. for example, avian influenza viruses usually do not replicate efficiently in humans and vice versa (hinshaw et al., 1983; beare and webster 1991) . relatively little is known about the viral and host factors governing the host range of influenza viruses and the mechanisms by which species barriers are crossed. however, in view of their role in entry of the virus, the viral ha glycoproteins and their sialic acid receptors on host cells clearly are important determinants of host-range restrictions. human influenza strains preferentially bind to sialic acid residues linked to galactose by the α2,6 linkage, and avian and equine influenza strains recognize sialic acid linked to galactose by α2,3 linkage rogers and d'souza 1989; connor et al., 1994; gambaryan et al., 1997; matrosovich et al., 1997 matrosovich et al., , 2004 . correspondingly, human respiratory epithelial cells predominantly contain α2,6 sialic acid-galactose linkages, whereas the host cells in birds and horses mainly contain α2,3 linkages (couceiro et al., 1993; ito et al., 1998; matrosovich et al., 2004) . interestingly, in contrast with the human respiratory tract, epithelial cells in the human eye predominantly contain α2,3-linked sialic acid receptors, which may explain why conjunctivitis is a common symptom of human infections with avian influenza viruses (paulsen et al., 1998; terraciano et al., 1999; diebold et al., 2003) . it has been hypothesized that, by serving as the main port of entry and site of initial replication, the eye may play a role in the adaptation of avian influenza viruses to humans (olofsson et al., 2005) . the presence of α2, 3-linked sialic acid receptors has also recently been demonstrated in the lower respiratory tract of humans, i.e. on bronchiolar and alveolar cells, which may explain the propensity of avian h5n1 viruses to cause pneumonia and not upper respiratory illnesses, in humans (shinya et al., 2006; van riel et al., 2006) . respiratory epithelial cells in the pig contain both α2,3 and α2,6 linkages, which explains why this animal is susceptible to both human and avian influenza viruses . because of this trait, the pig is widely regarded as a potential source of new pandemic strains, because it could serve as a nonselective host in which mixed infection of avian and human strains efficiently occurs, potentially resulting in new reassortant viruses, or in which purely avian strains can adapt to human receptor recognition (figure 9 .1). the receptor specificity of ha for either of the two sialic acid-galactose linkages is determined by the structure of the receptorbinding site of ha. although several residues have been implicated, the amino acids at positions 226 and 228 particularly seem to determine ha receptor specificity, that is, glu-226 and gly-228 are predicted to have affinity for avian and equine receptors, whereas leu-226 and ser-228 confer specificity for human receptors (wilson et al., 1981; naeve et al., 1984; weis et al., 1988; suzuki et al., 1989) . albeit less important, substrate specificity of na for either α2,3or α2,6-linked sialic acid also contributes to the efficiency of viral replication in different hosts (hinshaw et al., 1983) . this is illustrated by the fact that during its evolution in humans, the na of h2n2 viruses, which were of avian origin and therefore highly specific for hydrolization of α2,3-linked sialic acids, acquired high affinity for the human α2,6-linked sialic acids (baum and paulson, 1991) in addition to the surface glycoproteins, laboratory experiments with reassortant viruses suggest that the genes encoding internal proteins, such as m, np, pb1 and pb2, may also play a role in determining the host range (almond 1977; scholtissek et al., 1978a; snyder et al., 1987; subbarao et al., 1993) . however, because most of these experiments evaluated reassortant viruses with different constellations of gene segments, it remains difficult to interpret whether the proteins themselves contribute to host-range restrictions or whether certain combinations of gene segments from different origins are incompatible. manipulation of the genome using reverse genetics approaches will undoubtedly provide more definitive insight in the role of other host range determinants. antigenic variation of influenza a viruses can occur gradually by accumulation of point mutations (antigenic drift) or drastically by genetic reassortment (antigenic shift). antigenic drift, driven by immunological pressure on ha and na, allows the virus to evade the immune response and is the reason that influenza viruses manage to cause yearly epidemics. it is also because of antigenic drift that periodic replacements of human vaccine strains are needed. in contrast with human and other non-avian influenza strains, antigenic drift in avian viruses is very limited despite similar mutation rates (austin and webster, 1986; kida et al., 1987; liu et al., 2004) . most likely, this reflects optimal adaptation of these viruses to the host resulting in limited immunological pressure and consequent evolutionary stasis of these viruses in their natural reservoir. drastic changes in antigenicity can occur through the acquisition of completely new surface proteins by genetic reassortment . the segmented nature of the influenza virus genome facilitates the exchange of genes between two viruses (e.g., human and avian strains) that coinfect a host cell. although such exchange can result in 256 possible combinations of the eight different genomic segments of the virus, antigenic shift only arises when the reassortment at least includes the ha gene. provided that the reassortant virus is efficiently transmissible from infected to noninfected hosts, such an antigenically novel virus strain has pandemic potential when introduced in a population that completely lacks immunity against the new surface protein (figure 9 .1a). the pig is regarded as the ideal host for reassortment in view of its equal susceptibility for human and avian influenza strains . however, the increasing reports of bird-to-human transmissions of avian viruses indicate that coinfections, and consequently reassortments, could also take place in humans. beside genetic reassortment, antigenic shift is also caused by direct transmission of non-human influenza viruses to humans, as occurred or is still occurring on a relatively large scale in hong kong in 1997 (h5n1), in the netherlands in 2003 (h7n7), and in asia, the middle east, europe and africa since 2004 (h5n1) (yuen et al., 1998; fouchier et al., 2004; hien et al., 2004a) . as is true for reassortant viruses, these viruses are of pandemic potential when acquiring the ability for efficient transmission between humans through adaptation in either humans or an intermediate host (figure 9 .1b). finally, antigenic shift can occur when a previously circulating human influenza virus reemerges after an extended period of time. this happened in 1977 when h1n1 virus, which circulated in the 1950s, reappeared in the human population ("russian flu"), possibly after escaping a laboratory (nakajima et al., 1978; scholtissek et al., 1978b) . the reemergence of this virus gave rise to a relatively mild pandemic affecting mainly young persons who were still immunologically naive to this subtype. the same could have happened in 2005, when h2n2 virus, which had disappeared from the human population after the emergence of h3n2 viruses in 1968, was inadvertently sent to more than 3000 laboratories worldwide as part of an external quality assurance scheme (enserink, 2005) . avian influenza viruses can infect a wide range of domestic and wild birds, including (but not restricted to) chickens, ducks, turkeys, geese, quail, pheasants, seabirds, shore birds, and migratory birds. in these natural hosts, influenza viruses replicate in the gastrointestinal tract and are secreted in large amounts into the feces (webster et al., 1978) . transmission between birds occurs directly or indirectly through fecally contaminated aerosols, water, feed, and other materials. the spectrum of disease in birds ranges from asymptomatic infection, to mild respiratory illness, to severe and rapidly fatal systemic disease. most avian influenza viruses isolated from birds are avirulent (i.e., result in asymptomatic infection or only mild disease). avian influenza viruses capable of causing outbreaks of severe disease (fowl plague) in chickens or turkeys are classified as highly pathogenic and are currently restricted to h5 and h7 subtypes. typically, these highly pathogenic strains do not cause disease in ducks or geese. infection of poultry by highly pathogenic avian influenza viruses is characterized by disseminated infection and clinically manifested by decreased egg production, respiratory signs, excessive lacrimation, edema of the head, diarrhea, neurological symptoms, and death. the knowledge concerning the viral factors that determine the pathogenicity of influenza viruses is limited and is primarily derived from studies of highly pathogenic avian influenza viruses. a broad tissue tropism and the ability to replicate systemically are the hallmarks of these viruses. the most important and well-studied molecular correlate of these properties resides in the cleavability of the ha precursor glycoprotein (webster and rott, 1987; garten and klenk, 1999; steinhauer, 1999) . in the viral life cycle, post-translational cleavage of the precursor ha molecule into two subunits (ha1 and ha2) by host proteases is essential for infection to proceed. this cleavage generates a fusogenic domain at the amino terminus of ha2 that mediates fusion between the viral envelope and the endosomal membrane. has of avirulent avian influenza strains are cleaved only in a limited number of cell types, resulting in localized respiratory or gastrointestinal infections and mild illness. in contrast, has of highly pathogenic h5 and h7 strains can be cleaved in several different host cells, resulting in a broad cell tropism and the ability of causing systemic infection (klenk and garten, 1994; senne et al., 1996) . this apparent promiscuity of ha for a broad range of cellular proteases is determined by the structure of the ha cleavage site: has with high cleavability (i.e., from highly pathogenic strains) have multiple basic amino acid residues immediately upstream of the cleavage site, whereas has from avirulent subtypes usually have only a single arginine residue at this site (bosch et al., 1981; walker and kawaoka, 1993; senne et al., 1996; chen et al., 1998) . evidence for the correlation between a multibasic cleavage site, susceptibility for proteases and virulence has been provided by experiments in which viruses were generated with altered cleavage sites in otherwise unchanged genetic backgrounds (ohuchi et al., 1991; horimoto and kawaoka, 1994) . the reason why multibasic cleavage sites seem restricted to the has of h5 and h7 subtypes is unclear but may suggest that the number of basic residues is limited by structural features of ha. analyses of nucleotide sequences of h5 and h7 ha genes has shown the occurrence of direct repeats of purine-rich sequences (aagaaa) at the cleavage site in many cases . such repeats may arise because of pausing of the transcriptase-complex at a region of secondary structure, resulting in slippage of the complex and insertion of a short repeat sequence. additionally, recombination events between two genes of the same virus (e.g., from m or np to ha) may result in the insertion at the cleavage site of short sequences that code for multibasic amino acid residues (orlich et al., 1994; suarez et al., 2004) . in addition to the presence of multiple basic amino acids, susceptibility to ubiquitous proteases is also determined by the loss of a glycosylation site in the vicinity of the cleavage site (deshpande et al., 1987; webster, 1988, 1989) . although ha clearly is an important determinant of viral pathogenicity, animal studies indicate that virulence in mammals is a polygenic trait involving a constellation of other genes that can vary with the specific virus strain and host . however, besides ha, two genes have specifically been implicated in viral pathogenicity in mammals; that is, pb2 and ns. by reverse genetics experiments, it has been shown that a lysine residue at position 627 (lys627) of pb2 seems essential for high virulence and systemic replication in mice of highly pathogenic influenza h5n1 viruses responsible for the outbreak among poultry and humans in hong kong in 1997 (h5n1/97) (hatta et al., 2001) . the presence of lys627 in pb2 of h5n1/97 viruses appears to determine the viral replicative efficiency in mouse cells (and not avian cells) but does not increase the tissue tropism of the virus in mice . lys627 has also been found in pb2s of some, but not all highly pathogenic h7n7 and h5n1 viruses isolated from humans during the outbreaks of these viruses among poultry and humans in 2003 (the netherlands) and 2004 (viet nam, thailand), respectively li et al., 2004) . interestingly, a lysine residue at position 627 of pb2 is also present in all human influenza subtypes (h1, h2, h3) (webster, 2001) . furthermore, single-gene reassortant viruses carrying a pb2 gene of avian origin and all other genes from a human virus showed efficient replication in avian but not mammalian cells (subbarao et al., 1993) . this host cell restriction could be traced to a glutamic acid residue at position 627 of the avian pb2 instead of a lysine residue at the same position in the human virus (subbarao et al., 1993) . these observations suggest that an amino acid change to lys627 in pb2 may help avian viruses to adapt to efficient replication in mice and possibly other mammals, thereby increasing the virulence in these hosts. reverse genetics experiments with highly pathogenic avian influenza h5n1 and h7n7 viruses have indicated that other members of the viral polymerase complex beside pb2, i.e. pa and pbi, likely also play a role in adaptation of avian viruses to the mammalian host, suggesting that host factors are important for viral polymerase activity (gabriel et al., 2005; salomon et al., 2006) . in addition to pb2, the ns gene seems to play a role in the pathogenesis of avian and human influenza virus infections. this gene encodes two proteins: the nuclear export protein (nep) and the only nonstructural protein of influenza viruses, ns1. the ns1 gene or its product contribute to viral pathogenesis by allowing the virus to evade the interferon response of the host (garcia-sastre, 2001 krug et al., 2003) . this evasion may occur through multiple mechanisms, including interference with the activation of cell-signaling pathways and protein kinases involved in interferon induction or interference with the maturation of cellular pre-mrna at the post-transcriptional level. the ns gene has also been implicated in determining the high pathogenicity of influenza h5n1/97 viruses in mammals. experiments in pigs using recombinant viruses showed that the presence of the ns gene of h5n1/97 viruses greatly increased the pathogenicity of an h1n1 virus, possibly by escaping the antiviral effects of interferons and tumor necrosis factor alpha (tnf-α) seo et al., , 2004 . this enhanced virulence in pigs required the presence of glutamate instead of aspartate at position 92 (glu-92) of the h5n1 ns gene, but this amino acid change has not been found in all highly pathogenic h5n1 viruses isolated from humans or animals . beside the apparent cytokine resistance of h5n1/97 viruses, in vitro studies in human macrophages and respiratory cells showed that these viruses also seem to induce the transcription of proinflammatory cytokines, in particular tnf-α and interferon-β, and that the ns gene contributes to this induction (cheung et al., 2002; chan et al., 2005) . similar results were obtained in mice, in which infection with a recombinant h1n1 virus containing the h5n1/97 ns gene caused a cytokine imbalance in mouse lungs, characterized by increased concentrations of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines (lipatov et al., 2005a) . cytokine dysregulation by h5n1/97 viruses is also suggested by observations in human infections. pathological examination of patients who died of influenza h5n1 infection during the 1997 outbreak in hong kong showed reactive hemophagocytic syndrome, which is believed to be a cytokine-driven condition, as the most prominent feature . in addition, exceptionally high levels of certain chemokines were observed in the serum of human cases with avian influenza h5n1 . together, these observations may suggest that a combination of increased resistance against, and high induction of cytokines by the virus synergistically lead to a profound cytokine dysregulation that may play a role in explaining the severity of illness in mammals, including humans. although the ns gene seems to play a crucial role in this, it is likely that other particular gene constellations involving different internal genes also contribute. the virulence of highly pathogenic avian influenza viruses is clearly influenced by the specific host. two variants of the h5n1/97 virus, one of which was isolated from a human patient with mild repiratory illness and the other from a fatal human case, displayed similar differential pathogenicity in mice (zitzow et al., 2002) . however, in ferrets, both variants caused indistinguishable severe systemic disease (zitzow et al., 2002) . conversely, experimental infection with h5n1/97 viruses exhibiting high virulence in mice caused localized respiratory illness without systemic spread in primates and only viral replication in the respiratory tract without clinical illness in pigs rimmelzwaan et al., 2001) . the host factors determining the clinical outcome in animals are unclear. the clinical outcome of human influenza is influenced by factors such as the patient's age, the level of preexisting immunity, immunosuppression, comorbidities, pregnancy, and smoking habits, indicating that host-related factors certainly contribute to pathogenesis in humans. most of the above factors may be explained by differences in local, innate, or specific immunity at different stages of life or under specific circumstances, but other factors likely also play a role. for example, the observation that influenza-related encephalopathy seems well-recognized in japan but less so in other countries may suggest that there are differences in proneness for certain disease manifestations among populations, possibly related to genetic differences (morishima et al., 2002; sugaya, 2002) . although evidence is lacking at present, it is not unlikely that host factors also play a role in the the susceptibility and pathogenesis of human infections with avian influenza viruses. introduction of an influenza a virus with a novel ha gene in a population that lacks immunity to this ha has the potential to cause a pandemic when the virus posesses the ability to spread efficiently among humans (figure 9 .1). during the 20th century, this has happened three times, in 1918, 1957, and 1968 , killing millions of people worldwide. in all three pandemics, the viruses originated from avian influenza viruses. the virus strains responsible for the influenza pandemics of 1957 and 1968 both first emerged in southeastern asia, and both arose through reassortment of genes between avian viruses and the prevailing human influenza strain (scholtissek et al., 1978c) . the "asian influenza" pandemic of 1957 was caused by an h2n2 virus that had acquired three genes (h2, n2, and pb1) from avian viruses infecting wild ducks, in a backbone of the circulating h1n1 human influenza strain. as the asian flu strain emerged and established a permanent lineage, the h1n1 strains soon disappeared from the human population for unclear reasons. similarly, the h3n2 virus causing the "hong kong influenza" pandemic of 1968 (a) (b) figure 9 .1. mechanisms for generation of a pandemic influenza a strain. pandemic influenza a strains could result from genetic reassortment involving the hemagglutinin gene between avian and human strains in coinfected pigs or humans, followed by adaptation to human receptors in either host and human-to-human transmission (a); or through adaptation to humans of a purely avian influenza strain, either in humans or in an intermediate host such as the pig (b). mechanism a was implicated in the "asian" (1957) and "hong kong" (1968) influenza pandemics. the h1n1 virus that caused the "spanish flu" influenza pandemic of 1918 likely resulted from mechanism b. consisted of two genes from a duck virus (h3 and pb1) in a background of the human h2n2 strain circulating at that time. the latter virus disappeared with the emergence of the h3n2 virus and since then has not been detected in humans. sequence analysis of the hypothetical precursor strain that immediately preceded the pandemic h3n2 virus suggested that fewer than six amino acids in ha had changed during the avian-to-human transition . interestingly, a number of these changes may reflect adaptation to the new host because they modified the area surrounding the receptor-binding pocket of ha, including a glu to leu change at position 226, which is particluarly implicated in determining specificity for human receptors (see section 9.2.4). the fact that beside one or two novel surface glycoproteins, both pandemic strains also posessed a pb1 gene of avian origin is intriguing and may suggest a role of this gene in interspecies transmission . although millions of people died during the 1957 and 1968 pandemics, the viruses involved did not appear particularly virulent, suggesting that lack of immunity was the main reason for the excess mortality. this was different during the "spanish flu" pandemic of 1918, in which lack of immunity in the human population was combined with an apparent extremely high virulence of the virus, resulting in the demise of up to 100 million people worldwide. because the 1918 pandemic occurred before viruses were identified as the causative agents, no intact virus has been available for analysis. this and the similar lack of available human and animal influenza strains circulating before 1918 has made it difficult to determine the exact origin of the pandemic h1n1 virus and the reason for its extreme virulence. however, valuable insight has been provided by the recovery of fragments of viral rna isolated from archived autopsy specimens and tissue from alaskan flu victims buried in the permafrost (taubenberger et al., 1997) . this enabled sequence analysis of all eight genes of the virus (reid et al., 2004; taubenberger et al., 2005) . phylogenetic analyses of these genes suggest that the 1918 h1n1 virus may not have arisen by the same mechanism as the 1957 and 1968 pandemic viruses (i.e., by reassortment of avian and human influenza viruses) but perhaps by direct transmission from an avian source after adaptation in humans or another permissive mammalian host, such as the pig (reid et al., 2004; taubenberger et al., 2005) . this is supported by the observation that human h1n1 strains, including the 1918 pandemic strain, have retained the amino acid residues at positions 226 and 228 of ha predictive for binding to avian receptors (see section 9.2.4) (taubenberger et al., 1997) . recent crystallographic studies showed that structural changes in the h1 ha allowed the virus to recognize human receptors despite the presence of these avian-like residues, which may explain why the virus could nevertheless efficiently infect and spread among humans (gamblin et al., 2004; stevens et al., 2004) . the possibility that the 1918 strain had retained the structure and biological properties of its avian ancestors while acquiring the ability to recognize and efficiently infect human cells may explain the high virulence of this virus. mathematical modeling studies have suggested that the transmissability of the 1918 virus was not remarkably different than regular human influenza strains, indicating that extremely efficient spread did not account for the high morbidity and mortality (mills et al., 2004) . although part of the high mortality of the 1918 pandemic could be explained by the lack of antibiotics to treat secondary bacterial pneumonia and poor living conditions, the extremely rapid and severe clinical course implies high pathogenicity of the virus as the major cause. the molecular basis for this high virulence remains unclear. the 1918 h1 ha lacks the multibasic cleavage site characteristic of highly pathogenic avian influenza viruses (see section 9.3.2) (taubenberger et al., 1997; reid et al., 1999) . there are conflicting observations concerning the role of the ns gene in the 1918 pandemic strain. in mice, the presence of the complete ns or only the ns1 segment seemed to confer decreased, rather than enhanced pathogenicity of reassortant h1n1 viruses (basler et al., 2001) . in contrast, in vitro experiments in human lung cells suggested more efficient inhibition of interferon-regulated genes by h1n1 virus in the presence of the 1918 ns gene (geiss et al., 2002) . the most convincing evidence implicates ha as an important determinant of the high virulence. the presence of ha of the 1918 virus conferred high pathogenicity in mice to human strains that were otherwise non-pathogenic in this host . furthermore, these infections were associated with severe hemorrhagic pneumonia and the induction of high levels of macrophage-derived cytokines and chemokines, strikingly reminiscent of clinical observations in humans during the spanish flu pandemic, as well as of recent in vitro and in vivo observations of infections with highly pathogenic avian influenza h5n1 viruses (oxford 2000; cheung et al., 2002; peiris et al., 2004 ). before the year 2003, a few isolated cases of human infections with highly pathogenic h7n7 viruses were reported. these infections concerned laboratory accidents involving exposure to viral cultures or infected animals, and in one case presumed exposure to infected waterfowl in the absence of an overt outbreak (delay et al., 1967; campbell et al., 1970; taylor and turner, 1977; webster et al., 1981; kurtz et al., 1996) . all reported cases, except one, were clinically characterized by selflimiting conjunctivitis. influenza h7n7 virus was isolated from blood of one patient wih hepatitis, but the clinical relevance of this finding was unclear as was the source of this infection (delay et al., 1967; campbell et al., 1970) . in 2003, a large-scale outbreak of h7n7 viruses decimated the poultry industry in the netherlands and was associated with several human infections koopmans et al., 2004) . after diagnosing the first case of human infection with h7n7 virus, active case finding among exposed persons and their close contacts identified a total of 89 laboratory-confirmed infections in humans . this amounted to approximately 2% of the estimated number of people potentially exposed to the virus. highest infection rates were observed in veterinarians and chicken cullers. of the 89 h7n7 cases, 83 persons presented with conjunctivitis, of whom 5 also complained of influenza-like symptoms koopmans et al. 2004 ). it cannot be excluded that the remaining six patients also had conjunctivitis. although two individuals reported an influenza-like illness only, one had suffered a previous eye injury that precluded evaluation of conjunctivitis, while the other had chronic blepharitis. four individuals did not fit any case definition, either because of missing data or because they complained of red eyes only and therefore did not meet the criteria for a diagnosis of conjunctivitis. the prominence of conjunctivitis as the presenting syndrome in these and other reported human cases of influenza h7n7 is striking and may be explained by the presence of α2,3-linked sialic acid receptors in the eye, which are preferentially recognized by ha of avian influenza viruses, including h7n7 subtypes (see section 9.2.4) (olofsson et al., 2005) . the observation that, in contrast with human influenza strains, viral loads in conjunctival specimens of the dutch h7n7-infected individuals seemed higher than in respiratory specimens, supports the notion that h7n7 virus may replicate efficiently in cells in or near the eye and not in the respiratory tract . six of the seven cases of influenza-like illnesses were mild. one patient, a previously healthy 57-year-old veterinarian, complained of fever and headache 2 days after visiting an infected farm . he subsequently developed pneumonia complicated by acute respiratory distress syndrome and multiorgan failure, of which he died 13 days after the onset of illness. autopsy revealed pathologic changes in the lungs similar to those found in influenza h5n1-infected humans and no significant abnormalities in other organs. direct human-to-human transmission of h7n7 virus during the dutch outbreak is suggested by the fact that three individuals with confirmed infections had not been in direct contact with infected poultry but were family members of poultry workers with h7n7 conjunctivitis . the h7n7 virus causing the outbreak in the netherlands most likely evolved from a low pathogenic virus from wild ducks after the introduction of this virus into the poultry population . in agreement with its classification as a highly pathogenic avian influenza virus, the ha contained multiple basic amino acids at the cleavage site. sequence comparison of virus isolates from chickens and humans, including those implicated in human-to-human transmission, revealed virtually no differences, indicating no significant accumulation of mutations on bird-to-human or human-to-human transmission. the only exception was the virus isolated from the fatal case, which showed a total of 14 amino acid substitutions not seen in the other isolates. most of these substitutions involved the ha, na, and pb2 genes, which have all been implicated as determinants of host range and pathogenicity. intriguingly, the mutations in pb2 included a glutamine to lysine change at positions 627, associated with high virulence of h5n1 viruses in mice (see section 9.3.2) . the h7n7 outbreak in poultry was effectively contained by the culling of approximately 30 million chickens, which amounts to about 28% of the total chicken population in the netherlands . after the first human infections were identified, individuals exposed to potentially infected chickens were vaccinated with the available human vaccine to prevent possible dual infection with human and avian strains and the resulting risk of reassortment. as the outbreak progressed, the recommendation for vaccination was extended to all poultry farmers in a 3-km radius of infected farms and to persons suspected of h7n7 infection. in addition, a prophylactic regimen of the neuraminidase-inhibitor oseltamivir was started for all people handling potentially infected poultry to prevent bird-to-human transmission and human-to-human transmission of avian viruses. prophylactic treatment was to be continued for 2 days after the last exposure. these control measures may serve as a model for the control of emerging influenza viruses because they, at least theoretically, minimize the possibility that the virus spreads among the human population. in early 2004, an outbreak of highly pathogenic h7n3 viruses occurred in poultry farms in british columbia, canada . the causative virus probably had evolved by homologous recombination between the ha and m genes in a low pathogenic h7n3 virus . this recombination event resulted in the introduction of a multibasic sequence at the cleavage site of ha. surveillance among potentially exposed people identified two laboratory-confirmed cases of h7n3 infection. in these cases, conjunctivitis and mild influenza-like symptoms (coryza, headache) developed 1-3 days after exposure . both were treated with oseltamivir and recovered fully. no secondary cases were identified. the outbreak among poultry was contained by extensive culling. control measures in potentially exposed people were similar to those during the dutch h7n7 outbreak. in 1999, human infections with h9n2 viruses were reported in two unrelated children from hong kong, aged 1 and 4 years (peiris et al., 1999) . both children had a mild influenza-like syndrome, associated with mild lymphopenia in one, and slightly raised transaminase levels in the other child. neither child developed pneumonia and both recovered uneventfully within 3-7 days. there was a history of probable contact with live chickens in one of the patients, but otherwise the source of transmission was unclear. no serological evidence of h9n2 infection was found in the children's family members or health care workers. three serum samples from 150 volunteer blood donors in hong kong showed the presence of neutralizing antibodies against h9n2 virus, suggesting that additional infections had occurred in hong kong (peiris et al., 1999) . around the same time as the infections in hong kong, five additional, similarly mild cases of human h9n2 in humans were reported from mainland china . the human infections in hong kong and mainland china were caused by non-highly pathogenic h9n2 viruses of two disinct lineages. the hong kong virus was related to a quail h9n2 virus (a/quail/hk/ g1/97 [h9n2]) and possessed internal genes similar to the h5n1 virus that caused the outbreak in poultry and humans in 1997 (guan et al., 1999 lin et al., 2000) . this may suggest that the quail h9n2 virus has been the donor of all internal genes to the h5n1 outbreak strain (guan et al., 1999) . the strains isolated from humans in mainland china were related to a different lineage of h9n2 viruses found in ducks and chickens (a/duck/hk/y280/97 [h9n2] and a/chicken/hk/g9/97 [h9n2]) (guo et al., 2000) . most h9n2 strains isolated since 1999 seem to be related antigenically to the latter virus but posess a variety of internal gene constellations, including those of h5n1/97-like origin (choi et al., 2004; lipatov et al., 2004) . interestingly and perhaps concerning, h9n2 viruses isolated from poultry have acquired a preference for binding to the human-like α2,6 sialic acid-galactose linkages, which may suggest that certain species of poultry could act as an intermediate host in the zoonotic transmission of influenza viruses from their natural reservoir in acquatic birds to mammals, including humans (matrosovich et al., 2001) . indeed, h9n2 viruses have also been isolated from pigs in southeastern china, indicating widening of the host range (peiris et al., 2001) . in addition, contemporary human h3n2 strains are cocirculating in southeastern chinese pigs, providing ideal circumstances for potential genetic reassortment leading to the emergence of viruses with pandemic potential (peiris et al., 2001 ). in recent years it has become clear that, in contrast with the usually mild illnesses caused by h7 and h9n2 viruses, human infections with highly pathogenic influenza h5n1 viruses are associated with severe, often fatal disease. in may 1997, after outbreaks of influenza h5n1 among poultry on three farms in the new territories of hong kong, an influenza h5n1 virus was isolated from a 3-year-old boy in hong kong, who died of severe pneumonia complicated by acute respiratory distress syndrome and reye syndrome (subbarao et al., 1998) . in november and december of the same year, concomittant with outbreaks of influenza h5n1 among chickens in poultry markets and on farms in hong kong, 17 additional cases of human h5n1 infections were identified, five of which were fatal (yuen et al., 1998; chan 2002) . the outbreak was contained after the slaughtering of all 1.5 million chickens in hong kong. in response to the outbreak, influenza surveillance in poultry was intensified permitting early recognition of other outbreaks of avian influenza in 2001 and 2002. no further human h5n1 infections were reported until february 2003, when two laboratory-confirmed cases and one probable case were identified in one family from hong kong . the daughter died of an undiagnosed respiratory infection while visiting fujian province in mainland china. upon their return to hong kong, the father and son developed severe respiratory illnesses of which the father died. h5n1 virus was isolated from both patients. in december 2003, an outbreak of highly pathogenic h5n1 virus was identified among poultry in the republic of korea (lee et al., 2005) . subsequently, outbreaks by antigenically related viruses were reported among poultry in thailand, viet nam, japan, china, cambodia, laos, malaysia, and indonesia. the reason for this apparent simultaneous occurrence of h5n1 outbreaks in many asian countries remains unclear. however, h5n1 viruses have also been found in dead migratory birds, which may suggest a role of wild birds in the spread of h5n1 viruses in the region .since 2005, migratory birds indeed have also been responsible of spreading the virus to regions outside asia, including several countries in the middle east, europe and africa. human infections during the southeast asian outbreaks were first reported in early 2004 from viet nam and thailand, (hien et al., 2004a; chotpitayasunondh et al., 2005) . since then, concurrent with the spread of the virus by migrating birds and consequent poultry outbreaks elsewhere, human h5n1 infections have been reported in several other countries in asia (china, cambodia, indonesia), eurasia and europe (azerbaijan, iraq, turkey), and africa (egypt, djibouti). at the time of this writing (may 2006) more than 200 human infections have been reported worldwide of which more than half were fatal (who, 2005) . it cannot be excluded and may even be likely that additional cases have gone unnoticed in affected countries due to a lack of clinical awaress, active surveillance, or diagnostic facilities (hien et al., 2004b) . although many countries initially affected by poultry outbreaks in 2004 have been declared free of the virus, h5n1 virus seems to have reached endemic levels in poultry and aquatic birds in several asian countries, despite attempts to contain the outbreak by extensive culling of poultry. this is also suggested by the establishment of multiple geographically distinct sublineages of h5n1 influenza viruses in asia . continuing occurrences of bird-to-human transmissions increase the opportunity of the virus to adapt to humans and acquire the ability to spread between humans. in addition, continuing cocirculation of avian and human viruses in countries, where humans live in close proximity with poultry and pigs, increases the risk of reassortment between both in coinfected humans or other mammalian hosts, such as the pig. the isolation of h5n1 viruses from pigs in china and indonesia is concerning in this respect . for all these reasons, the current developments in asia and other regions in the world seem to justify the global concern that, similar to 1957 and 1968, a new pandemic influenza strain may emerge in the near future. at presentation, most cases of human h5n1 infections were characterized by a severe influenza syndrome, clinically indistinguishable from severe human influenza, with symptoms of fever, cough, and shortness of breath, and radiological evidence of pneumonia (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . abnormalities on chest radiographs at presentation included extensive, usually bilateral infiltration, lobar collapse, focal consolidation, and air bronchograms (figure 9 .2). radiological evidence of pulmonary damage could still be observed in surviving patients several months after the illness (t.t. hien, personal communication). beside respiratory symptoms, a large proportion of patients also complained of gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain, which are common in children with human influenza, but not in adults. in some cases, diarrhea was the only presenting symptom, preceding other clinical manifestations . unlike human infections with h7 or h9 viruses, conjunctivitis was not prominent in h5n1infected patients. the clinical course of the illness in severe cases was characterized by rapid development of severe bilateral pneumonia necessitating ventilatory support within days after onset. complications included acute respiratory distress syndrome, renal failure, and multiorgan failure. evidence that the clinical spectrum of human h5n1 infections is not restricted to pulmonary symptoms was provided by a reported case of possible central nervous system involvement in a vietnamese boy who presented with diarrhea, followed by coma and death. influenza h5n1 virus was isolated from throat, rectal, blood, and cerebrospinal fluid specimens, suggesting widely disseminated viral replication . his sister had died of a similar illness 2 weeks earlier, but no diagnostic specimens were obtained. although highly virulent h5n1 viruses have shown neurotropism in mammals such as mice and cats (lipatov et al., 2003; tanaka et al., 2003; keawcharoen et al., 2004) , these cases may be similarly rare as central nervous system manifestations associated with human influenza (morishima et al., 2002; sugaya, 2002) . genetic predisposition of the host to such manifestations may play a role. striking routine laboratory results in h5n1-infected patients, especially in severe cases, were an early onset of lymphopenia, with a pronounced inversion of the cd4+/cd8+ ratio, thrombocytopenia, and increased levels of serum transaminases (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . high levels of cytokines and chemokines have been observed in several h5n1-infected patients, suggesting a role of immune-mediated pathology in the pathogenesis of h5n1 infections (see section 9.3.2) peiris et al., 2004) . this was supported by pathological examination in two patients who died during the outbreak in hong kong, which showed reactive hemophagocytosis as the most prominent feature . other findings included diffuse alveolar damage with interstitial fibrosis, hepatic central lobular necrosis, acute renal tubular necrosis, and lymphoid depletion. although the gastrointestinal, hepatic, renal, and hematologic manifestations could suggest wider tissue tropism, there was no evidence of viral replication in organs outside the respiratory tract . although many laboratory-confirmed h5n1 infections were associated with severe, often fatal disease, milder cases have also been reported, especially during the outbreak in hong kong (yuen et al., 1998; chan 2002 ). an increasing number of milder cases also seemed to occur in viet nam, as the outbreak progressed in 2005 (who, 2005) . although increased clinical awareness and surveillance may account for such observations, progressive adaptation of the virus to humans is the dreaded alternative explanation. detailed monitoring of virus evolution during outbreaks is obviously important and may help to distinguish between both possibilities. the occurrence of mildly symptomatic and asymptomatic infections have also been suggested during the outbreak in hong kong by seroepidemiological studies in household members of h5n1infected patients and health care workers. in these studies, 8 of 217 exposed and 2 of 309 nonexposed health care workers were seropositive for h5n1-specific antibodies (bridges et al., 2002) . seroconversion was documented in two exposed nurses, one of whom reported a respiratory illness 2 days after exposure to an h5n1-infected patient. more importantly than showing the occurrence of asymptomatic infections, these data indicated that nosocomial person-to-person transmission had occurred, albeit limited to a few cases. an additional case of possible human-tohuman transmission during the hong kong outbreak was suggested by h5n1-seropositivity in a household contact of a patient, who had no history of poultry exposure . seroepidemiological studies in health care workers involved in the care of h5n1-infected patients in thailand and viet nam in 2004 have not shown evidence of person-toperson transmission, despite the absence of adequate infection control measures in the vietnamese cohort at the time of study liem and lim, 2005; schultsz et al., 2005) . during the outbreak in thailand in 2004, extensive epidemiological investigations have suggested person-to-person transmission from a child, who died of presumed h5n1 infection, to her mother who had no history of exposure to poultry and had provided prolonged unprotected nursing care to her daughter . an aunt of the child may have been infected by the same route because her last exposure to poultry before infection had been 17 days, considerably longer than the estimated incubation period of 2-10 days. there have been several similar family clusters of h5n1 cases in other affected countries, which have all ignited concerns about the possibility of human-to-human transmission, but most of which could be explained by common exposure to poultry. although there has been no evidence of efficient transmission of influenza h5n1 virus between humans to date, caution and detailed investigations obviously remain warranted in case of any cluster of infections, especially in view of the relatively rapid evolution h5n1 viruses have exhibited in recent years. 9.4.5.3. the evolution of h5n1 viruses, 1997 in 1996, an h5n1 virus was isolated from geese during an outbreak in guangdong province in china (influenza a/goose/guangdong/1/96 [a/g/gd/96]) (xu et al., 1999) . this virus proved to be the donor of the ha gene of the reassortant h5n1 viruses causing the outbreak among poultry and humans in hong kong in 1997. the internal genes of the hong kong h5n1 viruses were closely related to those of an h9n2 virus isolated from quail (see section 9.4 .3) (guan et al., 1999) . the origin of the na gene remains unclear but was notable for a 19-amino-acid deletion in the stalk region (subbarao et al., 1998) . such deletions may be associated with adaptation of influenza viruses to land-based poultry (matrosovich et al., 1999) . the ha gene contained multibasic sequences at the cleavage site, in accordance with its classification as a highly pathogenic strain (claas et al., 1998; matrosovich et al., 1999) . the role of other genes potentially involved in its pathogenicity is reviewed in section 9.3.2. after the eradication of the 1997 hong kong strain, the goose precursor viruses continued to circulate in geese in southeastern china (cauthen et al., 2000; webster et al., 2002) . through reassortment between this virus and other avian viruses, multiple antigenically similar genotypes, which were highly pathogenic in chickens but not in ducks, emerged and again were eradicated in hong kong in 2001 and 2002 . then, in late 2002, h5n1 strains isolated from wild migratory birds and resident waterfowl in two hong kong parks showed marked antigenic drift and exhibited high pathogenicity in ducks sturm-ramirez et al., 2004) . the latter property is rarely found in nature and had not been observed in strains isolated during previous years. an antigenically and molecularly similar virus caused the two confirmed human infections in early 2003 in a family from hong kong (see section 9.4.5.1) peiris et al., 2004) . h5n1 influenza viruses isolated from healthy ducks in southern china between 1999 and 2002 were all antigenically similar to the precursor influenza a/g/gd/96 virus . it is thought that these ducks played a central role in the generation of the virus responsible for the outbreaks in southeast asia since 2003. detailed genetic analyses of h5n1 strains isolated during the period 2000-2004 from poultry and humans in china, hong kong, indonesia, thailand, and viet nam demonstrated that a series of genetic reassortment events, all traceable to the a/g/gd/96-precursor virus, ultimately gave rise to a dominant h5n1 genotype (genotype z) in chickens and ducks . this genotype is implicated in the human cases in hong kong in 2003 and the outbreaks among poultry and humans since 2004. the evolution of h5n1 viruses in recent years has been associated with increasing virulence and an expanding host range, which beside terrestrial poultry and wild birds also includes mammals. although all h5n1 viruses isolated from ducks in china between 1999 and 2002 were highly pathogenic in chickens, an increasing level of pathogenicity was observed in mice with the progression of time: virus isolated in 1999 and 2000 were less pathogenic than those isolated in 2001 and 2002 . it has been suggested that the increasing ability to replicate in mammals has resulted from transmission between ducks and pigs the expanding host range is also illustrated by successful experimental infection of domestic cats and natural infections of cats, tigers and leopards with recent h5n1 strains (keawcharoen et al., 2004; kuiken et al., 2004; songserm et al., 2006) . in summary, continued evolution of h5n1 viruses since 1997, involving multiple genetic reassortment events between a/g/gd/96-like viruses and other avian viruses and perhaps transmission between birds and pigs or other mammalian hosts, has resulted in a highly virulent genotype with an expanded host range that is causing widespread outbreaks among wild birds, poultry and humans affecting several regions in the world. although transmission between birds and humans at present still seems inefficient, as does transmission between humans, this may change when the virus is allowed to continue its evolution through adaptation and reassortment. in clinical trials, human influenza has been clinically diagnosed correctly in approximately two-thirds of adults with influenza-like symptoms, despite the lack of pathognomomic features (monto et al., 2000) . although virus isolation remains the gold standard of diagnosis and indispensable for virus characterization, rapid laboratory confirmation of suspected human influenza in routine diagnostic laboratories is usually performed by immunochromatographic or immunofluorescent detection of influenza virus antigens or by reverse transcriptase (rt)-pcr detection of viral nucleic acids in respiratory specimens. in addition, serological evidence of human influenza a virus infection can be obtained by commercially available elisa kits that detect antibodies to conserved viral antigens, such as the nucleoprotein. in the absence of cocirculating avian influenza strains in the human population, further subtyping of influenza viruses or detection of subtype-specific antibodies are usually not done by routine diagnostic laboratories but are restricted to reference laboratories involved in epidemiological analyses and planning of vaccine strains. however, in case of an outbreak of avian influenza, efforts to further subtype the virus (e.g., by subtype-specific rt-pcr methods) should be made by routine laboratories because immediate knowledge about the infecting influenza subtype is essential for infection control and timely epidemiological investigations. dependence on reference laboratories, which in the case of many southeast asian countries affected by avian influenza outbreaks are situated abroad, potentially results in unacceptable delays and hampers timely recognition of outbreaks and institution of adequate control measures (hien et al., 2004b) . however, the reality is that diagnostic facilities in many affected countries are scarce and often not sufficiently equipped for virological diagnostics, let alone subtyping of influenza viruses. global efforts to improve diagnostic capacity in resource-poor countries may prove an important step toward the prevention and control of pandemic influenza (hien et al., 2004b ). similar to human influenza viruses, avian viruses can be isolated in embryonated eggs or in cell culture, using permissive cells such as madin darby canine kidney (mdck) cells or rhesus monkey kidney (llc-mk2) cells. unlike human strains and avirulent avian strains and in accordance with their promiscuity for cellular proteases, highly pathogenic avian viruses do not require the addition of exogenous trypsine for efficient replication in cell culture. for safety purposes, the isolation of highly pathogenic avian influenza virus requires biosafety level 3 laboratory facilities or higher. cytopathic effects in cell culture are nonspecific. initial identification of influenza a virus can be performed by immunofluorescence staining with monoclonal antibodies against the nucleoprotein. further ha and na subtyping is performed by subtype-specific rt-pcrs of culture supernatant or hemagglutination inhibition and neuraminidase inhibition assays using a panel of reference antisera against various subtypes. in human infections, avian influenza viruses have mostly been isolated from conjunctival swabs and respiratory specimens such as throat or nasal secretions or washings (yuen et al., 1998; fouchier et al., 2004; hien et al., 2004a) . in one case of h5n1 infection, virus was also isolated from serum, cerebrospinal fluid, and a rectal swab . detection of influenza a viral antigens in clinical specimens by direct immunofluorescence or by rapid immunochromatographic assays is widely used for diagnosis of human influenza because of their ability for rapid diagnosis. however, in patients with avian influenza, the usefulness of these assays seems limited due to low sensitivity, possibly because of lower viral loads than during human influenza (yuen et al., 1998; peiris et al., 2004) . in addition, some rapid antigen detection kits do not distnguish between influenza types a and b, and none of the currently available immunofluoresence and immunochromatographic assays distnguishes between influenza a subtypes. however, developments of h5n1-specific rapid antigen detection tests are ongoing . rt-pcr methods allow for sensitive and specific detection of viral nucleic acids and have shown to increase the diagnostic sensitivity for many viral pathogens when compared with culture or antigen detection methods. during the h5n1 outbreaks in hong kong and southeast asia, rt-pcr methods for specific detection of h5n1 viral nucleic acids proved valuable and seem to be the diagnostic methods of choice in case of an outbreak of avian influenza (yuen et al., 1998; hien et al., 2004a; chotpitayasunondh et al., 2005) . especially when using real-time pcr technology, a reliable subtype-specific diagnostic result can be generated within a few hours after specimen collection. a disadvantage of rt-pcr methods is its proneness for contamination and the consequent risk of false-positive results, which should be minimized by proper precautions, including physical separation of laboratories for pcr preparation and amplification. in addition, the inclusion of an internal control in rt-pcr assays is highly desirable to monitor for false-negative results due to inefficient nucleic acid extraction, cdna synthesis, or amplification. during outbreaks of avian influenza, the detection of subtypespecific antibodies is particularly important for epidemiological investigations. hemagglutination inhibition (hi) assays are the gold standard for detection of antibodies against human influenza viruses. however, their usefulness for detection of antibodies against avian viruses in mammalian species, including humans, seems limited (hinshaw et al., 1981; beare and webster, 1991; kida et al., 1994) . several studies have shown a failure to detect hi antibodies against avian viruses in mammals, even in cases where infection was confirmed by virus isolation. possible reasons for this failure include poor immunogenicity of some avian viruses and lack of sensitivity to detect low-titered or less avid antibodies induced by avian viruses (hinshaw et al., 1981; lu et al., 1982; kida et al., 1994; rowe et al., 1999) . it has been demonstrated that hi testing with subunit ha, but not with intact virus, could detect antibodies against an avian h2n2 virus (lu et al., 1982) . however, neutralizing antibodies against this virus could readily be detected with intact virus. a direct comparison of hi testing with a microneutralization assay in h5n1-infected persons from the 1997 hong kong outbreak indeed showed the latter to be more sensitive . although an indirect elisa assay using recombinant ha from h5n1/97 showed at least equal sensitivity as the microneutralization assay, the specificity in adult sera was inferior, most likely due to the presence of cross-reactive epitopes common to all has . based on these observations, neutralization assays are the methods of choice for detection of antibodies against avian viruses in humans. using these assays, it has been shown that the kinetics of the antibody response against h5n1 virus in patients infected during the hong kong outbreak are similar to the primary response to human influenza viruses . neutralizing antibodies were generally detected 14 or more days after the onset of symptoms, and titers equal to or higher than 1:640 were observed 20 or more days after onset. using neutralization assays, antibodies against h9n2 could be detected in a small number of blood donors from hong kong (peiris et al., 1999) . however, in two laboratory-confirmed h7n3-infected patients with conjunctivitis, no neutralizing antibodies could be detected in sera obtained more than 20 days after onset of the illness . similarly, no hi antibodies could be detected in an h7n7-infected patient with conjunctivitis . although the reason for this apparent failure to mount an antibody response remains unclear, it has been suggested that this could be secondary to the highly localized nature of the infection in these cases . currently, two classes of drugs are available with antiviral activity against influenza viruses: inhibitors of the ion channel activity of the m2 membrane protein, amantadine and rimantadine; and inhibitors of the neuraminidase, oseltamivir and zanamivir. the therapeutic efficacy of amantadine in human influenza is unclear due to a paucity of reliable clinical studies, but reductions of fever or illness by 1 day have been observed in adults and children (nicholson et al., 2003) . major disadvantages of amantadine include neurotoxicity and a rapid development of drug resistance during treatment. resistance is conferred by single nucleotide changes resulting in amino acid substitutions at positions 26, 27, 30, 31, or 34 of the m2 protein. rates of resistance against amantadine in human influenza viruses has increased from less than 0.5% in 1994-1995 to more than 12% in 2003-2004 . particularly high resistance frequencies of up to 61% were observed in viruses isolated in asia (bright et al., 2005) . rimantadine causes less neurological side effects but is not available in most parts of the world. although several h5n1-infected patients have been treated with amantadine during the 1997 h5n1 outbreak in hong kong, the numbers were too small to draw any meaningful conclusions concerning its activity against this virus (yuen et al., 1998) . in vitro sensitivity testing of virus isolated from the first patient during this outbreak showed normal susceptibility to amantadine (subbarao et al., 1998) . strikingly, the sublineage of genotype z h5n1 viruses prevalent in thailand, viet nam, cambodia and malaysia in 2004 invariably showed an amantadine-resistance conferring amino acid substitution at position 31 of the m2 protein, while this mutation was mostly not present in sublineages of h5n1 viruses isolated in other geographic regions puthavathana et al., 2005; cheung et al., 2006) . both oseltamivir and zanamivir have proven efficacy in the treatment of human influenza when started early during the course of illness and are particularly effective as seasonal or postexposure prohylaxis (nicholson et al., 2003) . zanamivir has poor oral availability and is therefore administered by inhalation, which has limited its use in the elderly and may induce bronchospasm. oseltamivir can be given orally. the development of drug resistance during treatment has been reported for both drugs and is associated with mutations in the active site of neuraminidase or in the hemagglutinin. the latter mutations decrease the affinity of ha for the cellular receptor, thereby obviating the need for neuraminidase to escape the cells. data on the efficacy of neuraminidase inhibitors in avian influenza virus infections are scarce. the h5n1 strains implicated in the 1997 hong kong outbreak were susceptible in vitro to oseltamivir and zanamivir (leneva et al., 2000; govorkova et al., 2001) . oral oseltamivir and topical zanamivir also showed therapeutic and protective activities against hong kong h5n1 isolates in murine animal models (gubareva et al., 1998; leneva et al., 2001) . recent murine studies suggest that, perhaps due to higher virulence, higher doses of oseltamivir and longer durations of treatment are necessary to achieve antiviral effects in mice against h5n1 strains causing the southeast asian outbreak since 2004, when compared with the 1997 hong kong h5n1 strain (yen et al., 2005) . in vitro sensitivity testing of h7n7 isolates during the 2003 outbreak of this virus in the netherlands showed normal susceptibility to zanamivir and oseltamivir . h7n7 infection was detected in 1 of 90 persons who reportedly received prophylactic treatment with oseltamivir during that outbreak, compared with 5 of 52 persons who had not taken oseltamivir prophylaxis . oseltamivir treatment has been given to several patients infected with avian influenza viruses, including h7n7, h7n3, and h5n1 subtypes, but no conclusions can be made concerning its efficacy. however, the timing of antiviral treatment may not have been optimal in many human cases of avian influenza so far. beneficial effects of antiviral treatment in human influenza are optimal when started within 48 h after onset of the illness. during the h5n1 outbreak in viet nam in 2004, h5n1infected patients were admitted 5 days or later after onset of symptoms (hien et al., 2004a) . earlier recognition of avian influenza in humans may improve the efficacy of antiviral treatment. nevertheless, favourable virological responses associated with a beneficial clinical outcome have been reported in h5n1-injected patients despite late initiation of treatment . the emergence of drug-resistant h5n1 variants during prophylaxis or treatment with oseltamivir has also been reported, and may be associated with clinical failure of treatment le et al., 2005) . treatment strategies which minimize the risk of resistance development, such as antiviral combination treatment, deserve attention. in addition, parenteral formulations of antiviral drugs may be desirable to guarantee systemic drug levels in h5n1 patients with severe disease. a novel intravenously administered neuraminidase inhibitor, peramivir, is currently in clinical development. although several h5n1-infected patients have received steroids in addition to oseltamivir, the potential benefits of this need formal evaluation in clinical studies (hien et al., 2004a) . considering the observed cytokine dysregulation in h5n1-infected animals and humans, a beneficial effect of immunomodulating agents could be hypothesized and perhaps requires further study. finally, neutralizing monoclonal antibodies have been shown effective in treating established influenza a virus infection in mice with severe combined immunodeficiency (palladino et al., 1995) . although mice are not men, this strategy deserves attention in the treatment of a severe illness such as influenza h5n1. birds infected with avian influenza excrete large amounts of virus in feces and other secretions, which contaminate the direct environment, such as dust, soil, water, cages, tools, and other fomites. avian influenza virus may remain infectious in soil, water, or contaminated equipment for weeks to months, depending on the temperature and humidity (i.e., longer in colder climates). illness in birds caused by highly pathogenic avian influenza viruses results in systemic replication and the presence of infectious virus in their eggs and many tissues and organs. transmission of avian influenza viruses between birds occurs directly or indirectly through contact with fecally contaminated aerosols, water, feed, and other materials. bird-to-human transmission likely occurs via the same route (i.e., direct contact with birds or contaminated fomites). most, but not all human infections with avian influenza viruses involved handling of affected poultry or direct exposure to live poultry in the week before onset of the illness (mounts et al., 1999; hien et al., 2004a; koopmans et al., 2004) . case-control studies during the 1997 h5n1 outbreak in hong kong identified visiting a stall or market selling live poultry during the week before the illness as a risk factor, whereas eating or preparing poultry products were not risk factors (mounts et al., 1999) . in cases in which no apparent direct exposure to poultry could be identified, contact with contaminated environment, such as water, has been suggested . of note, it has been shown that ducks infected by the currently circulating h5n1 strain in southeast asia remain healthy but excrete large amounts of virus for prolonged periods of time (hulse-post et al., 2005) . because water in ponds and canals in which large flocks of ducks reside is widely used for bathing and drinking in rural areas of many southeast asian countries, it may not be unlikely that such water represents a source of transmission when contaminated by infected ducks. in fact, contact with contaminated water is regarded as the most important mode of transmission between aquatic birds. a limited number of possible human-to-human transmissions have been reported, which involved prolonged, close, and unprotected contact with infected patients koopmans et al., 2004; ungchusak et al., 2005) . similar to human influenza, droplet and contact transmission are probably the most effective means of transmission of avian influenza virus between humans, should the the virus acquire the ability for efficient spread, but airborne transmission remains a possibility. the occurrence of diarrhea in h5n1-infected patients, which may contain infectious virus, represents a potential nonrespiratory route of transmission that needs to be considered in infection control practices hien et al., 2004a; . data concerning excretion patterns and periods of potential infectivity are lacking for human infections with avian influenza viruses. based on exposure histories, the incubation time for human h5n1-infections has been estimated at 2-10 days, but it is not known whether excretion of virus occurs during this time (yuen et al., 1998; hien et al., 2004a) . based on the current (lack of) knowledge, infection control measures during contact with potentially infected birds or environment or with patients with suspected or confirmed infection should prevent contact, droplet, and airborne transmission. these measures include mask (preferably high-efficiency masks, with surgical masks as a second alternative), gown, face shield or goggles, and gloves. the efficacy of neuraminidase inhibitors as seasonal or postexposure prohylaxis against human influenza is high (nicholson et al., 2003) . offering prophylactic treatment to potentially exposed people in the setting of a poultry outbreak of avian influenza, as has been done during h7outbreaks in the netherlands and canada tweed et al., 2004) , is rational but hardly feasible during the ongoing outbreak in asia and africa for logistical and financial reasons. postexposure prophylaxis to unprotected health care workers and close contacts of infected patients needs serious consideration. the potential use of specific monoclonal antibodies for prophylaxis warrants further investigation. eliminating the source of infection (i.e., infected birds) remains the most effective infection control measure. culling of all infected poultry has proved succesful during avian influenza outbreaks in hong kong, the netherlands, and canada (chan, 2002; koopmans et al., 2004; tweed et al., 2004) . however, considering the geographic extensiveness of the outbreak, the different farming practices in affected regions, and the occurence of infection in migratory birds, it is doubtful whether culling of poultry will be able to contain the outbreaks in the various regions. the bulk of human influenza vaccines are produced from inactivated viruses grown in embryonated eggs. vaccine production against highly pathogenic avian influenza viruses is complicated because of the requirement for high biosafety containment facilities and the difficulty, in some cases, to obtain high virus yields in embryonated eggs because of the virus' pathogenicity (stephenson et al., 2004; wood and robertson, 2004) . several other approaches have been used in an attempt to overcome these obstacles, including the use of reverse genetics techniques, generation of recombinant hemagglutinin, dna vaccination, and the use of related apathogenic h5 viruses with and without different adjuvants (nicholson et al., 2003; stephenson et al., 2004; webby et al., 2004; wood and robertson, 2004) . experimental h5n1 vaccines in which important virulence determinants were altered using plasmid-based reverse genetics have shown protective efficacy to homologous and heterologous h5 strains in animal models and may prove an attractive approach takada et al., 1999; lipatov et al., 2005b) . studies in humans using an h5n3 vaccine developed from a 1997 apathogenic avian virus showed high rates of seroconversions to the vaccine strain and heterologous h5n1 strains after 3 doses, but only when the vaccine was given with the adjuvant mf59 . in animal models, baculovirus-derived recombinant h5 vaccines were immunogenic and protective, but results in humans were disappointing even when using high doses (crawford et al., 1999; treanor et al., 2001) . in a study using a subvirion influenza h5n1 vaccine, neutralizing antibody responses were observed in approximately half of the subjects receiving the highest dose of the vaccine (two intramuscular injections of 90 micrograms) (treanor et al., 2006) . h5 dna vaccines protected mice from infection by homologous, but not by heterologous h5n1 viruses (kodihalli et al., 1999; epstein et al., 2002) . the increasing frequency of outbreaks with highly pathogenic avian influenza viruses among poultry and wild birds, and direct transmission of these viruses to humans, has ignited grave concerns about an imminent influenza pandemic. indeed, two of three prerequisites for a human pandemic have been met in the h5n1 outbreaks since 1997: the emergence of an antigenically novel strain to which the population has no immunity, and the transmission of this strain to humans in whom it can cause severe disease. to date, there fortunately is no evidence of efficient spread of h5n1 virus between humans, but continued circulation of this strain, which now has reached levels of endemicity among poultry in several asian countries, increases the opportunity to adapt to humans through mutation or genetic reassortment in humans or intermediate mammalian hosts. as suggested by the "spanish flu" pandemic of 1918, extremely high transmissibility is no prerequisite for a severe pandemic killing tens of millions of people, and as shown by the severe acute respiratory syndrome (sars) virus epidemic in 2003, viruses can rapidly spread across the globe in the current age of intense global travel. as a consequence of all this, pandemic preparedness has become an increasingly important issue, and pandemic plans are being developed by an increasing number of countries worldwide. control measures based on case identification (e.g., contact tracing and quarantine) were essential for the control of sars. however, during an influenza epidemic, such measures may not be as effective because of short incubation periods and the potential infectivity before the onset of case-defining symptoms. much of the preparedness therefore will rely on clinical management and vaccination. mathematical modelling studies have suggested the possibility of containing an influenza pandemic at the source by antiviral prophylaxis and other preventive measures (ferguson et al., 205; longini et al., 2005) . many developed countries are now stockpiling antiviral drugs for initial management of illness or prophylaxis during the first months of a pandemic, when vaccines are in development and not yet available. in case of an influenza pandemic, there will be limitations on the timeliness and availability of vaccines (stohr and esveld, 2004) . it has been estimated that it could take at least 6 months for the first vaccine doses to be produced after identifying a pandemic strain. currently, global production capacity for influenza vaccines is insufficient for worldwide coverage in case of a pandemic, especially because vaccination for the novel influenza strain likely requires two doses, and interruption of annual production of the human influenza vaccine is undesirable (schwartz and gellin, 2005) . in response to the pandemic threat by the h5n1 outbreak in southeast asia, plans have been made to stockpile candidate h5n1 vaccines based on the currently circulating strain. however, there is no certainty whether the next pandemic will indeed be caused by h5n1 virus, and if so, whether antigenic drift will not have rendered the stockpiled vaccine less effective by the time pandemic spread occurs. in the latter event, such vaccines may still mitigate the illness, which is beneficial for vaccinated people but may also carry a risk of prolonged excretion and increased spread of the virus. similar worries exist when poultry would be vaccinated with a suboptimal vaccines. in case of an influenza pandemic, all possibilities for rapid production of vaccines, as well as potential methods to reduce doses without affecting immunogenicity should be considered. this would require the use of alternative, currently not officially approved methods for vaccine production, such as reverse genetics techniques and cell culture-based vaccine production, and the use of alternative adjuvants that may enable dose reduction (webby and webster, 2003; stephenson et al., 2004; wood and robertson, 2004; schwartz and gellin, 2005) . in addition, vaccine doses may be spared by alternative administration routes. it has been shown that intradermal, instead of intramuscular vaccination for human influenza may require less antigen by recruiting efficient antigen-presenting cells present in the dermis (belshe et al., 2004; kenney et al., 2004) . notwithstanding the importance of current efforts to prepare for a possible h5n1 pandemic, more structural and longer term global efforts are needed to allow for early recognition of emerging novel influenza viruses in the future. in 2002, a who global agenda for influenza surveillance and control has been adopted, of which the main objectives are to strengthen surveillance, improve knowledge of the disease burden, increase vaccine use, 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receptor, sialic acid cumulative number of confirmed human cases of avian influenza a/(h5n1) reported to who structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution from lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza genetic characterization of the pathogenic influenza a/goose/guangdong/1/96 (h5n1) virus: similarity of its hemagglutinin gene to those of h5n1 viruses from the 1997 outbreaks in hong kong latex agglutination test for monitoring antibodies to avian influenza virus subtype h5n1 virulence may determine the necessary duration and dosage of oseltamivir treatment for highly pathogenic a/vietnam/1203/04 (h5n1) influenza virus in mice clinical features and rapid viral diagnosis of human disease associated with avian influenza a h5n1 virus pathogenesis of avian influenza a (h5n1) viruses in ferrets key: cord-003099-a0acr28o authors: koch, r. m.; diavatopoulos, d. a.; ferwerda, g.; pickkers, p.; de jonge, m. i.; kox, m. title: the endotoxin-induced pulmonary inflammatory response is enhanced during the acute phase of influenza infection date: 2018-07-05 journal: intensive care med exp doi: 10.1186/s40635-018-0182-5 sha: doc_id: 3099 cord_uid: a0acr28o background: influenza infections are often complicated by secondary infections, which are associated with high morbidity and mortality, suggesting that influenza profoundly influences the immune response towards a subsequent pathogenic challenge. however, data on the immunological interplay between influenza and secondary infections are equivocal, with some studies reporting influenza-induced augmentation of the immune response, whereas others demonstrate that influenza suppresses the immune response towards a subsequent challenge. these contrasting results may be due to the use of various types of live bacteria as secondary challenges, which impedes clear interpretation of causal relations, and to differences in timing of the secondary challenge relative to influenza infection. herein, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with bacterial lipopolysaccharide (lps) in either the acute or the recovery phase of infection. methods: male c57bl/6j mice were intranasally inoculated with 5 × 10(3) pfu influenza virus (ph1n1, strain a/netherlands/602/2009) or mock treated. after 4 (acute phase) or 10 (recovery phase) days, 5 mg/kg lps or saline was administered intravenously, and mice were sacrificed 90 min later. cytokine levels in plasma and lung tissue, and lung myeloperoxidase (mpo) content were determined. results: lps administration 4 days after influenza infection resulted in a synergistic increase in tnf-α, il-1β, and il-6 concentrations in lung tissue, but not in plasma. this effect was also observed 10 days after influenza infection, albeit to a lesser extent. lps-induced plasma levels of the anti-inflammatory cytokine il-10 were enhanced 4 days after influenza infection, whereas a trend towards increased pulmonary il-10 concentrations was found. lps-induced increases in pulmonary mpo content tended to be enhanced as well, but only at 4 days post-infection. conclusions: an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increase our insight on influenza-bacterial interplay. combing data of the present study with previous findings, it appears that this enhanced response is not beneficial in terms of protection against secondary infections, but rather damaging by increasing immunopathology. patients with influenza infection often suffer from severe secondary bacterial infections, which are associated with high morbidity and mortality rates [1, 2] . a striking example of this relationship was provided by bacteriological and histopathological analysis of infected lung tissue obtained from people who died of influenza during the 1918-1919 "spanish flu" pandemic, in whom bacterial pneumonia was found to be the predominant cause of death [1] . these data suggest that an influenza infection profoundly influences the immune response upon a secondary bacterial infection. several studies have evaluated immunological interactions between influenza and bacterial infections, including infections with gram-negative bacteria [3] . in vitro studies in which influenza-infected alveolar macrophages were subsequently stimulated with bacterial lipopolysaccharide (lps), a bacterial compound that induces a profound innate immune response, revealed increased levels of pro-inflammatory cytokines tumor necrosis factor (tnf) α, interleukin (il)-1β, and il-6 [4] [5] [6] [7] [8] , indicative of a priming effect on these cells by influenza. data from in vivo animal studies are ambiguous. similar to the in vitro data, some report enhanced responses. for instance, influenza infection in mice was shown to enhance the inflammatory response and neuropathogenicity resulting from lps administration on days 3 and 4 after influenza inoculation [9] . likewise, murine influenza infection resulted in increased levels of pro-inflammatory cytokines in both plasma and lungs, and enhanced pulmonary neutrophil influx upon pneumococcal infection 7 days later [10] . similar results were observed in mice 14 days after influenza infection [11] . however, two otherwise largely comparable studies demonstrated reduced pulmonary pro-inflammatory cytokine concentrations upon streptococcus pneumoniae and staphylococcus aureus infections in mice infected with influenza 7 days before, indicative of influenza-induced immunosuppression [12, 13] . these equivocal results may be due to differences in the severity or kinetics of the influenza infection or the use of different bacteria as secondary challenges, thereby targeting various complex multi-receptor signaling pathways. also, the use of live bacteria could have contributed to these ambiguous results. for instance, if influenza would induce immunosuppression and thereby facilitate outgrowth of bacteria upon a secondary live infectious challenge, the increased bacterial burden can eventually result in fulminant inflammation, which would wrongfully suggest influenza-induced augmentation of the immune response. in the present study, we investigated whether influenza infection results in an enhanced or suppressed innate immune response upon a secondary challenge with lps. furthermore, we assessed the kinetics of these influenza-induced effects by performing the lps challenges in either the acute or the recovery phase of influenza infection. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive 86/609, and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec 2013-029). forty-eight male c57bl/6j mice (charles river, sutzfield, germany) aged 10-12 weeks and weighing 23-29 g were used. mice were housed in individually ventilated cages, with five mice per cage at the central animal facility of the radboud university. at day 0, six groups of eight mice (total n = 48) were anesthetized by isoflurane and intranasally inoculated with a sublethal dose of influenza virus (ph1n1, strain a/ netherlands/602/2009, 5 × 10 3 pfu) or mock treated (nacl 0.9%) in a volume of 50 μl. following infection, all mice were monitored and weighed daily. the temperature was recorded with an infrared thermometer on the skin, and physical condition was scored using a scoring and weight sheet (weight, body temperature, ruffled coat, hunched back, reduced mobility, and moribund). at either day 4 (acute phase) or day 10 (recovery phase), mice were placed in a temperature-controlled chamber to receive lps (e coli, serotype 0111:b4, 5 mg/kg) or nacl 0.9% by intravenous injection in the tail vein. ninety minutes after lps or nacl administration, mice were deeply anesthetized with isoflurane and exsanguinated through orbital extraction, followed by cervical dislocation after which organs were collected. ethylenediaminetetraacetic acid (edta)-anticoagulated blood was centrifuged at 13000×g for 2 min at room temperature after which plasma was stored at − 80°c until analysis. subsequently, perfusion of the lungs was performed by intracardiac injection with phosphate-buffered saline (pbs), after which lung lobes were harvested and snap frozen in liquid nitrogen and stored at − 80°c until homogenization. lung tissue was placed in 1 ml lysis buffer containing pbs, 0.5% triton x-100, and a protease inhibitor cocktail (complete edta-free tablets, roche, woerden, the netherlands, 1 tablet per 50 ml lysis buffer). subsequently, lung lobes were homogenized at 50 hz, using a polytron homogenizer, and subjected to two rapid freeze-thaw cycles using liquid nitrogen. finally, homogenates were centrifuged (10 min, 14,000×g, 4°c), and the supernatant was stored at − 80°c until cytokine analysis. concentrations of tnf-α, il-1β, il-6, and il-10 in plasma and lung homogenates were measured using a luminex assay (milliplex, millipore, billerica, ma) according to the manufacturer's instructions. the lower detection limit of the assay was 32 pg/ml for all cytokines. plasma il-1β levels were below the detection limit in the majority of animals. lung homogenate cytokine concentrations were normalized to total protein content determined by bicinchoninic acid assay (bca protein assay; thermo fisher scientific). myeloperoxidase (mpo) content was measured in lung homogenates using an enzyme-linked immunosorbent assay (hycult biotech, uden, the netherlands) according to the manufacturer's instructions. concentrations were normalized to total protein content as described above. all data were normally distributed according to the shapiro-wilk test. the grubbs test (extreme studentized deviate method) was used to exclude significant outliers from analysis (maximum of one exclusion per dataset). to determine the number of animals required per group, we performed a power calculation based on a minimal detectable difference of 50% in lps-induced plasma tnf-α levels between influenza-infected and non-influenza-infected mice. mean ± sd (533 ± 163 pg/ml) tnf-α plasma levels were obtained from previous work from our group, in which male c57bl/6j mice were also injected intravenously with 5 mg/kg lps and sacrificed 90 min later [14] . using a two-sided α of 0.05 and a power of 80% (β of 0.2) in an unpaired t test design, six animals per group were required. to account for potential loss of animals due to influenza infection, eight animals per group were used. the effect size was based on previous work [9] , in which influenza infection modulated the plasma cytokine response to lps administration by at least 50%. comparisons were analyzed using unpaired student's t tests and repeated measures one-way analysis of variance (anova) as indicated in the figure legends. statistical analyses were performed in graphpad prism 5.03 for windows (graphpad software, san diego, ca). two-tailed p values < 0.05 were considered statistically significant. all influenza-inoculated mice showed clinical signs of infection, including weight loss, lethargy, and pyrexia. four influenza-infected mice were prematurely taken out of the experiment because of signs of severe infection. body weight decreased in all influenza-infected mice in the acute phase of infection, whereas it remained stable in mock-inoculated mice (fig. 1) . from day 7 onwards, body weight started to increase, marking the recovery phase of influenza infection (fig. 1) . influenza infection by itself did not result in increased plasma levels of any of the cytokines measured at both 4 and 10 days post-infection (fig. 2) . expectedly, lps fig. 1 body weight of influenza-or mock-inoculated mice. data are presented as mean with sem. dagger indicates the two time points at which mice in the respective groups were sacrificed administration led to profoundly increased plasma concentrations of tnf-α, il-6, and il-10. although tnf-α and il-6 plasma levels appeared to be somewhat higher in mice challenged with lps 4 days after influenza infection compared with mock-inoculated mice, this did not reach statistical significance (p = 0.12 and p = 0.86, respectively). plasma concentrations of the anti-inflammatory cytokine il-10 were however significantly enhanced in mice challenged with lps 4 days after influenza infection. no differences in any of the plasma cytokine levels were measured between influenza-infected and mock-inoculated mice at 10 days. in lung homogenates, influenza by itself caused mildly elevated levels of tnf-α, il-1β, il-6, and il-10 at 4 days post-infection and to a lesser extent at 10 days after infection (fig. 3) . similar to what was found in plasma, lps challenge also led to increased concentrations of all measured cytokines in lung tissue. a synergistic increase of all pro-inflammatory cytokines in the lungs was found in influenza-infected mice challenged with lps 4 days later and, to a lesser extent, in mice challenged with lps 10 days post-influenza infection. for il-10, the potentiating effect was additive rather fig. 2 plasma levels of tnf-α, il-6, and il-10 in mice that received influenza/mock followed by lps/nacl 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001 (calculated by unpaired student's t tests) fig. 3 levels of tnf-α, il-1β, il-6, and il-10 in lung homogenates of mice that received influenza/mock followed by lps/nacl 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001, # p = 0.05-0.10 (calculated by unpaired student's t tests) than synergistic, only observed at 4 days post-influenza infection, and reached a trend towards statistical significance. in accordance with pulmonary cytokine levels, influenza infection by itself led to increased mpo content in the lungs 4 days after infection and tended to result in increased mpo content 10 days post-infection (fig. 4) . again, lps administration also resulted in increased mpo levels in lung tissue, and there was a trend towards enhanced mpo content in influenza-infected mice challenged with lps 4 days after infection. in the present study, we demonstrate that a systemic lps challenge in the acute phase of influenza infection (4 days post-infection) results in an enhanced pulmonary, but not systemic pro-inflammatory cytokine response. this effect was synergistic rather than additive, indicating that influenza infection actually modulates the immune response to a subsequent challenge with lps. furthermore, this effect remained present, although less pronounced, in the recovery phase of influenza infection (10 days post-infection). the lps-induced increase in mpo content in lung homogenates, reflecting pulmonary neutrophil influx or sequestration, tended to be enhanced in the acute phase of influenza infection as well. these results suggest that influenza infection, especially in the acute phase, may cause a more pronounced pulmonary pro-inflammatory immune response upon a secondary bacterial infection. our results are in accordance with in vitro data reporting a cellular priming effect of influenza observed upon secondary stimulation with lps [4] [5] [6] [7] [8] , as well as with other murine in vivo studies that report increased inflammation and pulmonary neutrophil influx or sequestration upon a secondary bacterial infection or lps challenge in the acute phase of influenza infection [9, 10] . for example, a preceding influenza infection in mice gravely enhanced lung injury induced by a secondary infection with streptococcus pneumoniae 7 days later, resulting in a severe necrotic pneumonia accompanied by increased mortality [10] . also, fig. 4 mpo content in lung homogenates of mice that received mock/influenza followed by nacl/lps 4 or 10 days later. data are presented as scatter-dot plots with horizontal lines indicating the mean value. *p < 0.05, **p < 0.01, ***p < 0.001, # p = 0.05-0.10 (calculated by unpaired student's t tests) the increased mpo content observed in our study is an important hallmark of acute respiratory distress syndrome (ards) [15, 16] , a severe complication of influenza infection caused by excessive pulmonary inflammation. these and our study reveal that the enhancing effect on the pro-inflammatory innate immune response is most evident in the lungs, probably because the influenza-induced damage and consequent inflammatory effects are most pronounced at this site. in this context, our data are in line with the recommendation to use corticosteroids in patients with severe influenza infections in the intensive care unit to counteract the pulmonary hyperinflammatory response causing ards. several underlying mechanisms may contribute to the observed effects. at the cellular level, studies have shown that influenza and certain bacterial pathogens, such as haemophilus influenzae and streptococcus pneumoniae, utilize similar immunological pathways and that the overlap in the inflammatory mediators produced thereby creates augmentation of the immune response during sequential infection, in turn causing immunopathology [17, 18] . furthermore, it has been hypothesized that influenza stimulates tnf-α gene transcription activators or may interfere with labile transcription repressor proteins and stabilizes tnf-α mrna by delaying its degradation [8] . alternatively, the increased lung mpo levels observed do not necessarily reflect pmn infiltration into the lungs, but may (also) result from pmns trapped in the vasculature, as circulating activated neutrophils become rigid and can be trapped within the small capillaries of the lung [19] . as such, increased entrapment of leukocytes in the pulmonary vasculature during influenza infection could also contribute to the enhanced inflammatory cytokine levels upon lps challenge. we can only speculate on this, because no histological data are available, which represents a limitation of this work. it may be argued that the enhanced pro-inflammatory immune response induced by influenza serves as a means to efficiently eliminate the primary pathogen and to enhance host defense towards a secondary infection. for instance, pro-inflammatory cytokines are induced in influenza-infected cells to limit viral replication and to initiate downstream immune responses [20] . however, this is not supported by previous work, where an increased bacterial burden was observed irrespective of an enhanced or suppressed response [11] [12] [13] . several explanations for this observation may be put forward. first, next to potentiating pro-inflammatory cytokine responses, the present study and work by others [11] have shown that influenza infection also potentiates production of the key anti-inflammatory cytokine il-10, which was demonstrated to be crucial in facilitating bacterial outgrowth upon secondary challenge with streptococcus pneumoniae [11] . second, influenza may on the one hand prime for production of innate cytokines produced by myeloid cells, but impair t cell-derived cytokines that are instrumental for the adaptive immune response. this was elegantly demonstrated by kudva et al., who showed that, in line with our results, infection with staphylococcus aureus 6 days after influenza resulted in increased pulmonary levels of innate cytokines such as il-6 and mcp-1, and increased neutrophil influx to the lungs, but decreased concentrations of t-cell-derived il-17 and il-22, which were demonstrated to play a pivotal role in fending off the staphylococcal infection [21] . whereas the enhancing effects of influenza on pro-inflammatory innate immune parameters were less pronounced at 10 days post-infection, a suppressed response was neither evident. this could be partly biased by the exclusion of two mice in both recovery groups due to severe influenza infection. however, it might also be argued that 10 days post-infection is too soon for these effects to manifest. for example, profound desensitization towards lps and flagellin, another toll-like receptor (tlr) ligand, was observed in alveolar macrophages obtained from mice up to 6 weeks after influenza infection [22] . furthermore, the direction of the response upon a secondary challenge is probably highly dependent on the pathogen or stimulus used, each using distinct intracellular signaling pathways. with regard to this, it is well-known that influenza virus particularly predisposes to aspergillus fumigatus, which is present in 25% of all influenza patients [23, 24] , causing infections such as invasive pulmonary aspergillosis that are associated with very high mortality rates. as different mechanisms may be important in host defense towards various pathogens, the specific response towards aspergillus fumigatus could be suppressed by a preceding influenza infection. the use of corticosteroids may be another important factor in the observed vulnerability towards particular secondary infections, as steroid use was shown to be independently associated with the presence of aspergillus fumigatus in sputum of cystic fibrosis patients [25] and with a substantially increased risk of community-acquired staphylococcus aureus bacteremia [26] . furthermore, a meta-analysis revealed that the use of corticosteroids was significantly associated with nosocomial infections [27] . to the best of our knowledge, these putative detrimental effects of corticosteroid treatment during influenza on secondary infections have yet to be studied systematically in animal models. in any case, it remains to be determined whether the overall effects of corticosteroid treatment are beneficial or not, as they may lead to increased susceptibility in a subset of influenza virus-infected patients but may also provide health benefits in another subset of influenza virus-infected patients. an lps challenge in the acute phase of influenza infection results in an enhanced pulmonary pro-inflammatory innate immune response. these data increases our insight concerning viral-bacterial interplay. combined with previous findings, it appears that this enhanced pro-inflammatory response does not lead to protection against secondary infections but rather causes immunopathology leading to damage, and thereby to organ failure. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness secondary bacterial infections associated with influenza pandemics influenza-induced type i interferon enhances susceptibility to gram-negative and gram-positive bacterial pneumonia in mice infection of macrophages by influenza a virus: characteristics of tumour necrosis 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inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung spleen-derived ifn-gamma induces generation of pd-l1(+)-suppressive neutrophils during endotoxemia cxcl10-cxcr3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection insights into the interaction between influenza virus and pneumococcus patterns in bacterial-and viralinduced immunosuppression and secondary infections in the icu the neutrophil in vascular inflammation new fronts emerge in the influenza cytokine storm influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice sustained desensitization to bacterial toll-like receptor ligands after resolution of respiratory influenza infection invasive pulmonary aspergillosis is a frequent complication of critically ill h1n1 patients: a retrospective study influenzaassociated aspergillosis in critically ill patients inhaled corticosteroids and aspergillus fumigatus isolation in cystic fibrosis use of glucocorticoids and risk of community-acquired staphylococcus aureus bacteremia. a population-based case-control study mayo corticosteroids for the treatment of human infection with influenza virus: a systematic review and meta-analysis. clinical microbiology and infection: the official publication of the european society of clinical microbiology and infectious diseases the authors thank fred van opzeeland, elles simonetti, francine van der poll, ilona van de brink, and jelle gerretsen for their help with the mouse experiments and laboratory analyses. this work was supported by an efro (dutch: "europees fonds voor regionale ontwikkeling," english: "european regional development fund") grant (2011-013287). efro had no role in the design of the study; in the collection, analysis, and interpretation of data; and in writing the manuscript. data sharing is not applicable to this article as no reusable datasets were generated or analyzed during the current study.authors' contributions rk designed and conducted the study, analyzed and interpreted the data, and drafted the manuscript. dd and gf aided in the study design and conduct, interpreted the data, and critically revised the manuscript. pp and mdj supervised the study, interpreted the data, and critically revised the manuscript. mk designed and supervised the study, interpreted the data, and critically revised the manuscript. all authors read and approved the final manuscript. all procedures described were in accordance with the requirements of the dutch experiments on animals act, the ec directive 86/609, and approved by the animal ethics committee of the radboud university nijmegen medical center (ru-dec 2013-029). not applicable. the authors declare that they have no competing interests. key: cord-002939-6a3ga6v9 authors: ribeiro, ana freitas; pellini, alessandra cristina guedes; kitagawa, beatriz yuko; marques, daniel; madalosso, geraldine; fred, joao; albernaz, ricardo kerti mangabeira; carvalhanas, telma regina marques pinto; zanetta, dirce maria trevisan title: severe influenza a(h1n1)pdm09 in pregnant women and neonatal outcomes, state of sao paulo, brazil, 2009 date: 2018-03-26 journal: plos one doi: 10.1371/journal.pone.0194392 sha: doc_id: 2939 cord_uid: 6a3ga6v9 to investigate the factors associated with death and describe the gestational outcomes in pregnant women with influenza a(h1n1)pdm09, we conducted a case-control study (deaths and recovered) in hospitalized pregnant women with laboratory-confirmed influenza a(h1n1)pdm09 with severe acute respiratory illness (sari) in the state of são paulo from june 9 to december 1, 2009. all cases were evaluated, and four controls that were matched by the epidemiological week of hospitalization of the case were randomly selected for each case. cases and controls were selected from the national disease notification system-sinan influenza-web. the hospital records from 126 hospitals were evaluated, and home interviews were conducted using standardized forms. a total of 48 cases and 185 controls were investigated. having had a previous health visit to a healthcare provider for an influenza episode before hospital admission was a risk factor for death (adjusted or (or(adj)) of 7.93, 95% ci 2.19–28.69). although not significant in the multiple analysis (or(adj) of 2.13, 95% ci 0.91–5.00), the 3(rd) trimester deserves attention, with an or = 2.22, 95% ci 1.13–4.37 in the univariate analysis. antiviral treatment was a protective factor when administered within 48 hours of symptom onset (or(adj) = 0.16, 95% ci 0.05–0.50) and from 48 to 72 hours (or(adj) = 0.09, 95% ci 0.01–0.87). there was a higher proportion of fetal deaths and preterm births among cases (p = 0.001) and live births with low weight (p = 0.019), compared to control subjects who gave birth during hospitalization. after discharge, control subjects had a favorable neonatal outcome. early antiviral treatment during the presence of a flu-like illness is an important factor in reducing mortality from influenza in pregnant women and unfavorable neonatal outcomes. it is important to monitor pregnant women, particularly in the 3(rd) trimester of gestation, with influenza illness for diagnosis and early treatment. introduction pregnancy constitutes an important risk for the development of influenza-related complications and hospitalization. the 1918 and 1957 influenza pandemics showed increased mortality in pregnant women [1, 2] . the reasons for the increased risk during pregnancy probably derive from a combination of immunological and physiological factors. an increased susceptibility to certain intracellular pathogens has been described [3] . the identification of a new viral subtype, influenza a(h1n1)pdm09, in mexico and the united states in april 2009 and its worldwide dissemination led the who to announce the beginning of a pandemic in june 2009 [4] . a study developed during the first month of the outbreak in united states estimated that the rate of admission for pandemic h1n1 influenza in pregnant women was higher than in the general population (0.32 per 100,000 pregnant women, 95% ci 0.13-0.52 vs 0.076 per 100,000 population at risk, 95% ci 0.07-0.09) [5] . since 2012, the world health organization (who) has defined this group as the highest priority for vaccination [6] . a study conducted in california among hospitalized pregnant women and women of reproductive age with influenza a(h1n1)pdm09 showed that pregnant women in the 2 nd and 3 rd trimesters who delayed treatment(! 48 hours) were more likely to undergo admission to the icu or death [7] . most studies evaluate risk factors for increased severity in pregnant women [8, 9, 10, 11] and few studies have analyzed the risk factors for death in pregnant women from influenza a(h1n1)pdm09. a systematic review study showed a higher risk of hospitalization in pregnant women with influenza a(h1n1)pdm09, with relative risks ranging from 4.3 to 7.2. however, only one study showed a higher risk of death in pregnant women with influenza a (h1n1)pdm09 (rr 10.2) and seven studies presented no significant risks (0.3 to 1.3) [12] . meta-analysis also showed no higher risk of death in pregnant women with pandemic influenza, or 0.99 (95% ci 0.67 to 1.46) [8] . a higher risk of fetal abnormalities in pregnant women with both seasonal influenza virus infection and a(h1n1)pdm09 infection was reported. in addition, women with a diagnosis of a(h1n1)pdm09 infection had a higher risk of placental problems, antepartum haemorrhage, and antepartum complications [13] . during the pandemic, there were a significant number of deaths in pregnant women from influenza a(h1n1)pdm09 in são paulo, which justifies this study. the objective of this study was to analyze factors associated with death in pregnant women with influenza a(h1n1) pdm09 and severe acute respiratory illness (sari) and describe the gestational and neonatal outcomes. the state of são paulo has a population of more than 41 million inhabitants, with 598,473 live births in 2009, according to data from the information department of the unified public health system (datasus) of the ministry of health. a case-control study was conducted that evaluated pregnant women living in são paulo with confirmed infection of influenza a(h1n1)pdm09 and hospitalized with sari, defined as: fever and cough and dyspnea or pneumonia or respiratory failure or tachypnea or radiological alterations consistent with pneumonia or oxygen therapy or mechanical ventilation. the definition of sari has been adapted from the one stated by the who to increase sensitivity in the detection of cases and controls [14] . in 2009, the ministry of health of brazil established the compulsory notification of any hospitalized case of influenza associated with sari and the inclusion of an epidemiological investigation into the influenza-web database of the national disease notification system (sinan). all hospitalized pregnant women who were notified with influenza associated with sari and eventually died during the epidemic period from july 9 th to december 1 st 2009 in são paulo were included in the analysis. for each death (case), four controls were randomly selected from those who recovered. the cases and controls were identified from the sinan, using the following variables for selection: rt-pcr (positive for influenza a/h1n1pdm09), final classification (confirmed), evolution (recovered or death), hospitalization (yes), date of hospitalization, hospital and residence in the são paulo state. the controls were matched by epidemiological week of admission date of the case to adjust for possible variations in access to treatment and clinical protocols. all pregnant women had a laboratory confirmation of influenza a(h1n1)pdm09 from a sample of respiratory secretions using the rt-pcr method, performed at the adolfo lutz institute, the public health laboratory [15] . for data collection, trained health professionals used two standardized forms: one to collect hospital record information and the second was used for home interviews. for the cases, the interviews were conducted with close family members, and for the controls, the interviews were conducted with the patients themselves. the hospital form included the following variables: pathological history, health care, symptoms on admission, admission to the intensive care unit, antiviral treatment (the ministry of health released the antiviral oseltamivir), use of antibiotics, complications, laboratory tests, radiological examinations, evolution, gestational and neonatal outcomes. the home form included the following variables: sociodemographics, history of a previous health visit to a healthcare provider for the influenza episode that resulted in hospitalization (after the onset of symptoms and before hospital admission date), vaccination history and gestational and neonatal outcome. education level was classified as low (no schooling or incomplete primary), medium (complete primary or incomplete high school) or high (complete high school or university). occupations were grouped using the occupational risk pyramid for the pandemic in the occupational safety and health act of the national institute for occupational safety and health-cdc, which classified the occupation risk for influenza infection as low (professional managers and other university and technical professionals without close contact with the population), medium (professionals in the areas of education, trade, service and administration with close contact with the population), or high and very high risk (doctors, nurses, other health professionals and support staff in the health services) [16] . a pre-test with 10 patients with influenza a(h1n1)pdm09 was performed to identify and correct any errors. the questionnaires are presented in s1 data collection form and the complete data used in this study are shown in s1 database. the study was started during the epidemic to support the actions of epidemiological surveillance, and the use of the data was approved by the ethics committee of the school of public health of usp (protocol 2283, of.coep/312/11). data collection complied with the recommendations of the national health council for research in human beings, including the signing of a consent form. clinical and demographic variables are presented as medians and interquartile ranges or percentages, mann-whitney u or chi-square tests were used for comparisons, as appropriate. odds ratios and 95% confidence intervals (95% ci) were calculated to evaluate factors associated with death. for the multiple logistic regression, variables were selected with a p of <0.20 in the univariate analysis and those considered important for the adjustment. the initial model included: health plan; previous visit to a healthcare provider; the presence of at least one of the high-risk medical conditions for developing influenza-related complications (adapted from the centers for disease control and prevention): asthma, neurological and developmental disorders, heart disease, kidney disease, liver disease, hemoglobinopathies, endocrine disorders, immunosuppressive diseases and obesity, with the absence of these conditions as reference; the use of an antiviral [no use (as reference), 48 hours from the first symptoms, > 48 and 72 hours, > 72 hours], pregnancy trimester [1 st and 2 nd trimesters (as reference) and 3 rd trimester]. the data were controlled for education level and age. the wald and hosmer-lemeshow tests were used to evaluate the significance of the variables and test the fit of the model, respectively. analyses were performed using spss (ibm, armonk, ny, usa), version 17.0. p values of <0.05 were considered significant. in são paulo, in 2009, 51 pregnant women with confirmed influenza a(h1n1)pdm09 infection notified in sinan eventually died. a total of 204 controls were randomly selected among the 525 pregnant women hospitalized with confirmed influenza a(h1n1)pdm09 infection notified in sinan who recovered. the investigation was conducted in 126 hospitals where all cases and controls were hospitalized. with regard to the 51 cases and 204 controls initially identified in sinan for the study, 22 files were missing or did not meet the case or control requirements, resulting in 48 cases and 185 controls reviewed. two pregnant women who died were identified in another study [13] , and included in the present. the home interviews were performed for 42 cases (87.5%) and 165 controls (89.2%), as shown in fig 1 . table 1 presents the socio-demographic characteristics of the pregnant women. there were no significant differences in socio-demographic distribution between cases and controls, and median age, family income, education level, smoking history, occupational risk for influenza infection, previous pregnancy and existence of private health insurance were not associated with death. table 2 shows the distribution of cases and controls according to clinical variables and visits to a healthcare provider. a total of 92.7% of cases sought medical care for the influenza episode prior to hospitalization. this proportion was higher than that of the controls. when evaluating the preconditions for admission, the presence of other risk conditions for developing complications related to influenza did not differ in cases and controls. asthma and obesity were the most common conditions, among cases (8.3% each) and controls (8.9% vs. 3.2%, respectively). the use of an antiviral during hospitalization was an important protective factor against death, with proportions of treatment at 77.1% and 91.4% in cases and controls, respectively. the proportions of women who received antiviral within the first 48 hours of symptoms were 27% and 63.4% in cases and controls, respectively. the protective effect was also observed for treatment starting within 48 to 72 hours (5.4% and 13% in cases and controls, respectively). the initiation of treatment with an antiviral more than 72 hours after the onset of symptoms did not present significant protection. the median number of days between the date of the first symptoms and hospitalization was four for the cases and two for the controls (p = 0.003). treatment with antibiotics was used in 100% of cases and 64.9% of controls. the average number of antibiotics used was 3.9 for cases and 1.1 for controls. among the cases, there was a higher proportion of patients admitted to the intensive care unit (95.8% vs. 16.2%), and a higher proportion of cases underwent mechanical ventilation compared to controls (100% vs. 13%). the cases exhibited higher rates of complications than the controls (100% vs. 10.3%), predominantly: respiratory distress syndrome (72.9% vs. 4.9%), shock (75.0% vs. 0.5%), sepsis (64.6% vs. 3.2%), infections (35.4% vs. 3.8%) and renal alterations (35.4% vs. 2.7%). there were three episodes of pre-eclampsia, which evolved to death (cases) and one in the control group. none of them fulfilled the definition of hellp syndrome, named for three features of the disease (hemolysis, elevated liver enzyme levels, and low platelet levels). all cases that evolve to death had influenza a(h1n1)pdm09 as the main cause in the death certificate. co-infection with other infectious agents occurred in 20.4% of cases and 1.6% of controls, with the following pathogens found: acinetobacter baumannii (n = 4), klebsiella pneumoniae (n = 3), staphylococcus aureus (n = 3), pseudomonas aeruginosa (n = 2), enterococcus spp. (n = 2), candida spp (n = 2), candida albicans (n = 1), klebsiella spp. (n = 1) and streptococcus pneumoniae (n = 1). the results of chest radiology were assessed in 93.8% of cases and in 80% of controls. among the cases, 91.6% presented with alterations, with a consolidation pattern evident in 50% of cases. among the controls, 59.5% presented alterations, with a consolidation pattern evident in 14.4% of cases. table 3 shows the findings from the laboratory examinations at the time of hospital admission. the cases presented lower median platelet, hemoglobin and hematocrit counts and severe influenza a(h1n1) in pregnant women higher median levels of creatine phosphokinase-cpk, lactate dehydrogenase-ldh, glutamic oxaloacetic transaminase-got, urea and creatinine, with statistical significance. table 4 presents the variables in the final multiple logistic regression model. having had a previous health visit to a healthcare provider for the influenza episode before hospitalization was a risk factor for death, or 7.93 (95% ci 2.19-28.69). antiviral treatment was a protective severe influenza a(h1n1) in pregnant women factor for death when administered within the first 48 hours after the onset of symptoms, or 0.16 (95% ci 0.05-0.50), and when administered 48 to 72 hours after the onset of symptoms, or 0.09 (95% ci 0.01-0.87). the third trimester of gestation, which was a significant risk factor in univariate analysis, lost significance in the multiple analysis, or 2.13 (95% ci 0.91-5.00), when antiviral treatment was included in the model. the proportion of women who did not receive any antiviral treatment was similar in the three trimesters of gestation (11.1, 11.1 and 12% respectively) and the proportion of those who received the treatment after 72 hours was higher in the 3 rd trimester (14.8%, 25.9% and 32%, respectively for the 1 st , 2 nd and 3 rd trimester of gestation. as shown in table 5 , among the cases, 45.8% of pregnant women had live births, with one twin birth, and 54.1% of the women experienced fetal deaths, of which fetal deaths later (! 23 weeks) represented 65.4% of the total number of deaths. among the controls, 13.5% delivered during hospitalization, with one twin birth. regarding the neonatal outcome in this group, there were 7.7% fetal deaths and 92.3% live births. considering the live births that occurred during hospitalization, in 100% of cases and in 75.0% of controls a cesarean delivery was performed. the distribution of gestational outcomes shows a concentration of miscarriages and premature births among cases compared to controls who delivered during hospitalization: 82.6% and 45.8%, respectively (p = 0.001). among the 144 controls who were discharged before delivery and who completed a home interview, 100% had live births, 62.5% by cesarean delivery and 86.8% at term. regarding the live births, among the cases, there was predominance of gestational age at birth between the 32 nd and 36 th weeks of pregnancy (65.2%), and among the controls who delivered during hospitalization, 54.2% occurred at 37 weeks and over (p = 0.003). among the controls that gave birth after hospital discharge, 86.8% of births were full term (!37 th week). analyzing the weight of live births, there was a higher proportion of low birthweight (<2,500 g) among cases (73.9%) than among controls who gave birth during hospitalization (37.5%), p = 0.011. considering control women who delivered after discharge, low birthweight occurred in only 6.3% of the births. during hospitalization, 8.7% (2 in 23; 28 and 38 days after birth) of the live births of cases and 4.2% (1 in 24; 12 days after birth) of live births of controls evolved to death after giving birth. none of the live births of control women who delivered after discharge evolved to death. the median gestational age, birth weight and apgar score in the 1 st minute were significantly lower among cases than among controls who delivered during hospitalization. among the cases, 73.7% of the newborns were admitted to the intensive care unit (icu), contrasting to only 35.0% of newborns from controls who delivered during the hospitalization. when the weight of the newborns was compared with the gestational age, 27.3% of newborns from cases were classified as small for the gestational age, and 12.5% and 7.5% of those from controls who gave birth during and after hospitalization, respectively, as shown in table 6 . severe influenza a(h1n1) in pregnant women the case control design, including all reported deaths of hospitalized patients who presented with laboratory confirmed influenza a(h1n1)pdm09 with sari in the state of são paulo, and the random selection of four controls for each case, with the collection of hospital data and home interviews, allowed for the expansion of the analysis of risk factors for death. it was possible to evaluate the gestational and neonatal outcomes, including those of pregnant women who delivered after hospital discharge. the main results suggest that an early search for care, the training of physicians for the proper treatment of pregnant women, and early antiviral administration can be protective factors against death. another noteworthy result was the presence of unfavorable neonatal outcomes, with a significant proportion of stillbirths and miscarriages, low birth weights and lower apgar scores among pregnant women who died. after hospital discharge, the patients had a favorable neonatal outcome. pre-eclampsia was present in six percent of women who evolved to death and in less than one percent of those who survived. despite the fact that all women who died had the influenza a (h1n1)pdm09 infection as the main cause of death in their death certificate, we cannot rule out the possibility that pre-eclampsia may have contributed to their unfavourable outcome. age distribution and race were not associated with a risk of death in pregnant women, similar to results in other studies [9, 10, 11] . the third trimester of gestation lost its significance as risk factor for death in the multiple analyses, when antivirus treatment was included in the model. a higher proportion of women in the final period of gestation received the treatment after 72 hours of symptoms than those who were at earlier stages of gestation. a meta-analysis study showed that the third trimester of gestation was a risk factor for death (or 1.22, 95% ci 1.01-1.48) for pandemic influenza, when compared with those in the first or second trimester [12] . although the present study did not confirm the association with the third trimester of gestation, its result indicates that it is important to monitor pregnant women with influenza illness, with special attention during this trimester, for diagnosis and early treatment. the pregnant women who had a previous health visit to a healthcare provider for the influenza episode before hospitalization had a higher risk of death. this increased risk could be an indication of difficulties in accessing hospitalization or lack of perception of the severity of the case by doctors or lack of recognition that, even in cases that are not serious, considering that pregnant women are in a high risk group for severity of the disease, early antiviral therapy should have been introduced. the median time between the first symptoms and hospitalization was twice as high among pregnant women who died. similar results were found when patients in general with influenza during the pandemic were evaluated in são paulo [17] and in mexico [18] . these findings also reinforce the need for pregnant women to have access to health services, particularly hospitalization in serious cases. the training of physicians concerning the proper care for pregnant women and the need to start early treatment are as important as the early search for care. the use of an antiviral medication was a protective factor death when administered within 72 hours of symptom onset. several studies have shown an increased risk of death or worsening disease in pregnant women who started treatment late [10, 11, 19, 20, 21, 22, 23] and in patients in general [17, 18, 24, 25, 26, 27, 28, 29] . regarding the history of previous diseases, no differences in the presence of risk conditions for developing influenza-related complications were found between pregnant women who eventually died and those who recovered. however, surveillance showed a higher proportion of risk conditions among pregnant women who eventually died compared to those who survived [10] . a study in china showed that obesity (bmi ! 30) was a factor associated with mortality in patients with severe disease [9] . this result is in line with the current study. although it was not possible to calculate bmi, a higher rate of obesity among the cases than among the controls was reported. there were a higher proportion of cases with co-infections than controls, consistent with other reported results [17] . the patients who eventually died presented significant alterations in laboratory values when compared to controls. the alterations observed in cpk, ldh, platelets and creatinine were similar to those reported for patients in general [27, 29, 30, 31, 32] . in pregnant women, there was also a decrease in the number of red blood cells. in relation to neonatal outcomes, there were a greater proportion of fetal deaths in patients who died than in controls that delivered either during hospitalization or after discharge. among the live births in the cases, there was a greater proportion of low birth weight, gestational age less than 32 weeks, admitted to icu, lower scores on the apgar scale, when compared to the live births of controls that delivered during or after hospitalization. similar results were reported in studies that evaluated pregnant women infected with influenza a(h1n1) pdm09 compared to women of childbearing age without infection [33] , pregnant women with influenza a(h1n1)pdm09 with severe disease [9, 20] or women who gave birth to live newborns during hospitalization [10, 34] . control women who delivered after discharge from the hospital had favorable outcomes in their offspring, with a median apgar of 9 both in the 1 st and 5 th minutes of life. considering all births in the state of são paulo during 2009, according to the national system of live births-sinasc, 58% were delivered by cesarean, 9% were preterm birth (<37 weeks) and 9% had low birthweight ( 2500 g). in the control women of the study who gave birth after hospitalization, these proportions were also high (62.5%, 13.2% and 7.7%, respectively). this study has limitations. underreporting of sari by health professionals as well as gaps in the sinan may have occurred. the notification of influenza through a new viral subtype associated with sari was initiated during the pandemic; consequently, the sensitivity may have varied with time. the quality of information from medical records can differ between hospitals. the use of standardized hospital and home questionnaires minimized these difficulties during data collection. although all hospital reports were reviewed, we were not able to establish the roles of other factors, such as secondary infections or obstetric factors, which could have contributed to death in the cases. the results presented in this study indicate that early treatment can prevent unfavorable outcomes in pregnant women and in their offspring and reinforce the need for the proper training of doctors for the clinical management of pregnant women and early administration of antiviral treatment. these findings also support interventions in situations of future pandemics and seasonal influenza with the goals of preventive measures and the organization of health services for the appropriate clinical management of pregnant women. influenza occurring in pregnant women maternal mortality in the epidemic of asian influenza emerging infectious and pregnancy pandemic (h1n1) 2009-world now at the start of 2009 influenza pandemic h1n1 2009 influenza virus infection during pregnancy in the usa world health organization vaccines against influenza who position paper california pandemic (h1n1) working group. severe 2009 h1n1 influenza in pregnant and postpartum women in california pandemic influenza in pregnant: a systematic review of the literature clinical features and risk factors for severe and critical pregnant women with 2009 pandemic h1n1 influenza infection in china maternal and infant outcomes among severally ill pregnant and postpartum women with 2009 pandemic influenza a(h1n1)-united states pandemic 2009 influenza a (h1n1) in 71 critically ill pregnant women in california populations at risk for severe or complicated influenza illness: systematic review and meta-analysis a large, population-based study of 2009 pandemic influenza a virus subtype h1n1 infection diagnosis during pregnancy and outcomes for mothers and neonates pan american health organization-world health organization. operational guidelines for intensified national sari surveillance implantação e otimização da pcr em tempo real para o diagnóstico de influenza a(h1n1) no instituto adolfo lutz e perspectiva para guidance on preparing workplaces for an influenza pandemic risk factors for death from influenza a(h1n1)pdm09, state of são paulo, brazil impact of antiviral treatment and hospital admission delay on risk of death associated with 2009 a/h1n1 pandemic influenza in mexico influenza a(h1n1) virus illness among pregnant women in the united states severity of 2009 pandemic influenza a (h1n1) virus infection in pregnant women french experience of 2009 a: h1n1v influenza in pregnant women evaluation of pregnancy as a risk factor in the outcomes of influenza a (h1n1)/ 2009 in women of childbearing age influenza-like illness in hospitalized pregnant and postpartum women during the 2009-2010 h1n1 pandemic hospitalized patients with 2009 pandemic influenza a (h1n1) virus infection in the united states severe hospitalised 2009 pandemic influenza a(h1n1) cases in france hospitalized patients with 2009 h1n1 influenza in the united states critically ill patients with 2009 influenza a(h1n1) in mexico factors associated with death or hospitalization due to pandemic 2009 influenza a(h1n1) infection in california fatalities associated with the 2009 h1n1 influenza a virus pneumonia and respiratory failure from swine-origin influenza a (h1n1) in mexico critically ill patients with 2009 influenza a(h1n1) infection in canada factores asociados a ingreso en unidad de cuidados intensivos en pacientes hospitalizados por influenza pandémica a/h1n1 maternal and neonatal outcomes among pregnant women with 2009 pandemic influenza a(h1n1) illness in florida, 2009-2010: a population-based cohort study the anzic influenza investigators and australasian maternity outcomes surveillance system. critical illness due to 2009 a/h1n1 influenza in pregnant and postpartum women population based cohort study we would like to thank the sao paulo state secretary of health, the santa casa de são paulo medical school for the collaboration in the preparation of the project. this study would not possible without the collaborations of the researchers and field coordinators, the epidemiological surveillance center, the disease control coordination, the adolfo lutz institute, municipal departments of health for data collection. we would also like to thank all those who contributed to this study, especially luiz roberto barradas barata (in memoriam). key: cord-003302-vxk7uqlc authors: fedson, david s title: influenza, evolution, and the next pandemic date: 2018-10-03 journal: evol med public health doi: 10.1093/emph/eoy027 sha: doc_id: 3302 cord_uid: vxk7uqlc mortality rates in influenza appear to have been shaped by evolution. during the 1918 pandemic, mortality rates were lower in children compared with adults. this mortality difference occurs in a wide variety of infectious diseases. it has been replicated in mice and might be due to greater tolerance of infection, not greater resistance. importantly, combination treatment with inexpensive and widely available generic drugs (e.g. statins and angiotensin receptor blockers) might change the damaging host response in adults to a more tolerant response in children. these drugs might work by modifying endothelial dysfunction, mitochondrial biogenesis and immunometabolism. treating the host response might be the only practical way to reduce global mortality during the next influenza pandemic. it might also help reduce mortality due to seasonal influenza and other forms of acute critical illness. to realize these benefits, we need laboratory and clinical studies of host response treatment before and after puberty. an evolutionary perspective in public health has been important in explaining associations between different human phenotypes and chronic diseases [1] . the same perspective might help us understand many forms of acute critical illness. it might also suggest better ways to manage critically ill patients. two recent studies of influenza virus infection and endotoxemia in mice have shown that survival is better before puberty than after puberty. these studies help explain the lower mortality in children compared with adults seen in the 1918 influenza pandemic and in many other types of acute illness. this difference is probably the heritage of human evolution. understanding the scientific basis for this difference suggests an alternative way to respond to the next pandemic. instead of relying on vaccination and antiviral treatment, we might be able to treat patients with inexpensive generic drugs that modify the host response to acute critical illness. unlike pandemic vaccines and antiviral treatments, these drugs will be available in any country with a basic healthcare system. if laboratory and clinical research convincingly demonstrates this approach works, it would benefit people everywhere. this idea has been discussed several times in the past decade [2] [3] [4] [5] [6] [7] [8] [9] . the 1918 influenza pandemic is remembered because it killed as many as 50-100 million people worldwide: an estimated 2.5% or more of the global population [9] . remarkably, the mortality rate was much higher in younger adults than it was in children, giving rise to the familiar w-shaped pandemic mortality curve ( fig. 1 ) [6, 10] . a similar mortality pattern has not been described for subsequent pandemics (1957, 1968 and 2009 ). some investigators have attributed high pandemic mortality in young adults to secondary bacterial pneumonia [11] . this explanation is incomplete and unsatisfactory for several reasons. children were infected with the 1918 virus more frequently than adults ( fig. 1 ) and they were almost certainly colonized with the same bacteria that were associated with bacterial pneumonia in adults, yet their pneumonia mortality rates were much lower [6, 12] . more important, lower mortality among children compared with adults was not unique to the 1918 pandemic. children have lower mortality than adults due to infections caused by many bacteria (e.g. group a streptococcus, s. pneumoniae, s. aureus and m. tuberculosis) and viruses (e.g. mumps, varicella, poliomyelitis, epstein barr virus, hepatitis e, yellow fever, sars and smallpox). similar mortality differences have been seen in several other conditions (e.g. disseminated c. albicans infection, acute lung injury accompanying severe malaria, sickle cell chest syndrome, multi-organ failure following severe trauma, severe burn injury and febrile neutropenia) [6, 10] . in short, lower mortality rates among children were not unique to the 1918 pandemic. most influenza scientists have sought to explain the 1918 mortality difference by studying the virus. numerous reports have shown that the virulence of individual influenza viruses differs markedly in laboratory models of infection. for example, influenza a (h3n2) viruses are generally more virulent than h1n1 viruses, and the 1918 virus (h1n1) was more virulent than the ordinary seasonal h1n1 influenza viruses seen today. antigenic changes in human influenza viruses are frequently reported over the course of a single influenza season [13] , and in 1918 the mortality impact of the second pandemic wave in the fall was much greater than it was during the first wave the preceding spring [10] . several investigators have sought to explain the greater mortality among adults in 1918 by studying influenza disease in human populations. during the 1918 influenza pandemic, children 5-14 years of age who were living in remote, isolated communities were often the only ones to survive [14] . there is general agreement that pandemic mortality peaked in young adults, but it was much lower in adults 45 years of age (fig. 1) . their lower mortality is thought due to the protective effect of residual immunity following exposure to h1n1-like viruses that circulated before an h3n2-like virus (russian influenza) appeared in the early 1890s [10] . influenza virologists agree that antigenic priming following infection with these earlier h1n1viruses (known as 'original antigenic sin') provided some measure of long-lasting protection against the highly virulent h1n1 virus that emerged in 1918 [15] . accordingly, individuals born after the early 1890s were exposed only to h3n2-like viruses and would have missed antigenic priming with pre-1890 h1n1-like viruses. as young adults in 1918, they were susceptible to infection with the new pandemic h1n1 virus. influenza a viruses are categorized into two groups. group 1 includes h1, h2 and h5 subtypes, whereas group 2 includes h3 and h7 subtypes ( figure 1a in ref. [16] ). this phylogenetic understanding has informed recent age-specific analyses (based on individual birth year) of mortality patterns before, during and after the 1918 pandemic [17] [18] [19] [20] [21] . worobey et al. have used a hostspecific molecular clock approach to demonstrate that high mortality in young adults may have been due to childhood exposure to a doubly heterosubtypic putative h3n8 virus that circulated from 1889 to 1900 [19] . they think that young children (but not infants) were protected by childhood exposure to a newly emerged (post-1900) h1variant or n1 antigens. miller et al. interpret the historical data more cautiously [18, 20] . they agree with worobey et al that early life antigenic imprinting might have led to a dysregulated t-cell response that increased the risk of death following infection in 1918 with a new and antigenically dissimilar influenza virus. however, they question whether a new h1 virus emerged in the early 1900s to replace the h3 virus that first appeared in 1890. worobey et al have also analyzed cases and deaths due to h5n1 and h7n9 influenza. they have shown that ha imprinting was "the dominant explanatory factor for observed incidence and mortality patterns for both h5n1 and h7n9" [21] . for example, individuals born during the period when h1n1 viruses circulated were protected against h5n1 infection (both are group 1 viruses) but were at increased risk of h7n9 infection (a group 2 virus) [21] . however, individuals born during the 1968-2015 period (the h3n2 era) were protected against h7n9 infection (both are group 2 viruses), whereas those born before 1957 (the h1n1 era) were not. the importance of antigenic imprinting was also shown for the 2009 h1n1 pandemic. individuals born before 1957 were exposed to seasonal h1n1 viruses before the emergence of the new h2n2 pandemic virus in 1957. as older individuals, they experienced a lower incidence of ph1n1 disease in 2009 than those born after 1957 [22] . however, miller et al. have argued that early life h2n2 virus infection may have actually increased the risk of death during the heterotypic 2009 h1n1 pandemic [23] . the importance of antigenic imprinting for human influenza is undeniable. 'first flu' may indeed be 'forever' [24] , but whether antigenic imprinting is helpful or harmful for every individual is still an open question. a similar uncertainty has arisen about influenza vaccination. the doctrine of 'original antigenic sin' was developed following observations that influenza vaccination led to the anamnestic recall of antibodies to earlier influenza viruses [15] . recent epidemiologic studies, however, suggest that repeated influenza vaccinations may lead to reduced vaccination effectiveness [15, 23] . moreover, repeated infection of ferrets with h3n2 viruses affects both the quantity and quality of their antibody responses [25] . thus, there appear to be two sides to the host response following repeated influenza virus infections and vaccination. moreover, these observations leave open the larger question of whether antigenic interaction among influenza viruses is the only determinant of how an individual will respond to influenza virus infection. writing about the 1918 pandemic, ahmed et al. observed that "children were not protected from infection, but, for reasons that are as mysterious today as they were in 1918, they were able to cope with the disease much better than their adult counterparts" [10] . they added, ". . . this change in disease susceptibility occurs around the time of puberty, and it is possible that sex-associated hormones are involved in this transition" [10] . in trying to understand the 'mystery' of greater mortality among young adults during the 1918 pandemic, scientists have studied influenza viruses and the human response to previous infection. in essence, they have asked 'why did young adults die?' they could also have asked 'why did children live?' [6, 9, 12] . influenza scientists have never created an experimental model of the mortality experience seen in the 1918. recently, however, suber and kobzik experimentally replicated the different susceptibility of children and adults to influenza-related mortality [26] . they infected groups of c57bl/6 mice with influenza a(h1n1) (pr8) virus. male and female mice were infected on either postnatal day 25 (p25, prepubertal) or postnatal day 28 (p28, pubertal). (in c57bl/6 mice, puberty usually starts on postnatal day p27 or p28.) mortality was much greater in pubertal (p28) than in prepubertal (p25) mice ( fig. 2a and b) . deaths began to occur 9 days following infection. by this time, pulmonary virus titers had fallen to levels much lower than they were on days 3 and 6, and they were similar in prepubertal and pubertal mice ( fig. 2c) . high-dose, exogenous estrogen treatment is known to protect adult female mice infected with influenza virus [27] . to determine the role of sex hormones in the prepubertal/pubertal mortality differences between p25 and p28 mice were not significant by the kruskal-wallis with dunn's multiple comparisons test. from ref. [26] with permission difference, suber and kobzik castrated male and female mice on day p21 and infected them on day p28 [26] . over the next 3 weeks, mortality was reduced in castrated but not in sham-operated mice. they then blocked the onset of puberty in p28-infected female mice by pretreating them (starting on day p21) with leuprolide, a gonadotropin-releasing hormone (gnrh) analog. leuprolide desensitizes gnrh receptors and decreases the secretion of gonadotropins, and this blocks the normal pubertal increase in estrogen. they also pretreated male and female p28infected mice with acyline, a gnrh antagonist. both leuprolide and acyline pretreatment improved survival [26] . in addition, when p21-ovariectomized mice were infected on day p28, estrogen treatment given 0-2 or 6-8 days following infection abrogated the protective effect of ovariectomy. protection was also reversed in castrated males by treatment with both estrogen and testosterone (normally, testosterone is converted to estrogen by aromatase). transcriptome profiling over the course of infection showed marked enrichment of estrogen, b-estradiol and estrogen receptor 1 [26] . for this reason, estrogen receptor blockade was carried out using fulvestrant. pretreating p28-infected male and female mice reduced subsequent mortality, and when fulvestrant was given to females 3 days following infection, survival was greatly improved. in addition, in older postpubertal male and female mice infected on day p42, survival also improved when fulvestrant treatment was started 3 days following infection [26] . transcriptome analysis also showed increased expression of il-1b in the lungs and blood leukocytes of pubertal (p28) mice 9 days after infection [26] . when pubertal mice were infected on day p28 and then treated with anti-il-1b blocking antibody 5 and 9 days later, survival was greatly improved. early treatment on the day of infection, however, was not beneficial, suggesting that il-1b activity was expressed differently at different stages of disease [26] . as discussed above mortality rates in many infectious diseases are lower in children than they are in adults [6, 10] , for this reason, joachim and kobzik studied mice with endotoxin (lps)-induced sepsis before and after puberty [28] . the conditions for these experiments differed slightly from those in the influenza experiments [26] ; all mice were female and postpubertal mice were given lps on postnatal days 33-35, not earlier. prepubertal mice injected intraperitoneally with lps on day p24-26 had significantly better survival than postpubertal mice 20 h after injection (fig. 3a) , although endotoxin levels in the blood were similar in both groups (fig. 3b) [28] . as in the influenza experiments, survival in prepubertal mice improved when the onset of puberty was delayed by pretreatment with estrogen (3 days before and on the day of lps injection). similarly, in postpubertal mice injected with lps, blocking the onset of puberty by pretreatment with leuprolide (administered daily from prepubertal day p24 to postpubertal day p35) substantially improved survival. in addition, fulvestrant was administered to pre-and postpubertal mice to determine whether the increase in lps-induced mortality was specifically due to the onset of puberty or the lack of estrogen activity, but the results were indeterminate. finally, in lps-treated postpubertal mice, adoptive transfer of peritoneal cells (macrophages and b and t cells) harvested from lps-naive pre-and postpubertal mice had different effects on survival: mice that received cells from prepubertal mice had significantly lower mortality than those treated with postpubertal cells (fig. 3c) [28] . investigators have presented many arguments for and against the use of murine models to explain aspects of acute critical illness in humans [29, 30] . for comparative studies of puberty, mice present special problems; humans live much longer than mice and human puberty extends over several years, not a day or two as in mice. nonetheless, murine studies before and after puberty can be of considerable value. for example, estrogens have an mice were injected intraperitoneally with lps (salmonella enterica) and followed for 5 days. mortality in mice injected with prepubertal peritoneal cells was significantly lower than it was in control mice or those that received postpubertal peritoneal cells. from ref. [28] with permission important role in puberty and they can also modify the response to acute critical illness. kobzik et al. have shown that unlike other studies, estrogen treatment reversed protection against influenza mortality in prepubertal and castrated mice [26] . these apparently contradictory findings might reflect the known pro-inflammatory activities of low-dose estrogens and their anti-inflammatory effects when higher doses are used [26, 27] . suber and kobzik have shown that the different influenza mortality rates in pre-and postpubertal mice have nothing to do with previous exposure to influenza viruses and/or control of virus replication. this observation is critically important. although antigenic imprinting clearly influences human outcomes in both pandemic and seasonal influenza [15, 18-20, 22, 23] , differences in antigenic imprinting might not be the only explanation for better childhood survival during the 1918 pandemic. in attempting to understand the 'mystery' of better survival among children compared with adults during the 1918 pandemic, investigators have focused exclusively on infection with influenza viruses and ignored the better survival of children with other infectious diseases and noninfectious critical illnesses [6, 10] . the better survival of prepubertal mice following endotoxin treatment shown by kobzik et al. suggests that this is a general phenomenon. long-lasting, age-specific antigenic imprinting seen with influenza is not known to occur with most other forms of acute critical illness [6, 10] . instead, changes in the host response to critical illness associated with increased mortality appear to begin with the onset of puberty. it follows that factors associated with prepuberty might somehow contribute to the better survival of children compared with adults. infection with influenza viruses initially targets respiratory epithelial cells [31, 32] . in response, myeloid and lymphoid cells mount a brisk pro-inflammatory response, often called a 'cytokine storm'. patients who develop severe illness are unable to control what becomes a systemically dysregulated immune response. after several days (usually a week or more), they develop evidence of immunosuppression [6, 32] . death occurs in those who are unable to resolve their illness and restore homeostasis. the pathogenesis of acute lung injury, including severe influenza, involves (among other things) mitochondrial dysfunction [33] , oxidative stress [34, 35] , endothelial dysfunction [36, 37] and molecular mechanisms (e.g. specific lipid mediators) that initiate the resolution of pulmonary and systemic injury and the restoration of homeostasis [38, 39] . the evolutionarily conserved process of autophagy is central to the host response [40] ; it contributes to both influenza virus replication [41] and the evolution of influenza-related lung injury [42, 43] . the host response to infection may involve mechanisms that enhance resistance (which reduces pathogen burden) or tolerance (which reduces the impact of infection) [44, 45] . both resistance and tolerance are driven by a multiplicity of metabolic changes in immune and other host cells [46] [47] [48] [49] , some of which include estrogen signaling [50] . some of these immunometabolic changes have been documented in mice with experimental influenza virus infection [51] . the molecular mechanisms that account for the difference in the mortality rates of children and adults with different forms of acute critical illness (seen in humans and now replicated in mice) are largely unknown. working together, the endocrine, nervous and immune systems integrate and regulate the availability of energy. evolutionary biologists have developed the theory of life history, which emphasizes trade-offs in how energy is allocated to storage, activity, maintenance, and the anabolic activities of growth and reproduction [52] . according to life history theory, the transition to puberty is accompanied by an overall switch in the allocation of energy from growth to reproduction. nonetheless, although this theory has given us a better understanding of changes in energy metabolism that occur over extended periods of time, it has yet to explain the sudden and intense increase in energy expenditure that accompanies the host response to acute critical illness or whether tradeoffs in energy allocation in critical illness are different before and after puberty. in outbreaks of seasonal and pandemic influenza, only a small number of individuals who are infected develop severe or fatal illness. this was true during the 1918 pandemic; although approximately one-third of the human population was infected, only a small proportion died. some of this protection is due to cd8 + t-cell immunity, especially immunity directed against the evolutionarily conserved np antigen [53] . this immunity reflects previous exposure of populations to influenza virus antigens, but importantly it encompasses both group 1 and group 2 viruses, unlike antigenic imprinting discussed above. t-cell immunity does not prevent the occurrence of infection, but it modifies the course of illness, reducing virus shedding and in some instances limiting or preventing the occurrence of symptoms [53] . inborn genetic variants can also account for life-threatening infections [54] , but the susceptibility of most individuals probably depends more on variations in host defense mechanisms that are expressed only after infection has occurred [53, 55] . the importance of these post-infection variations was demonstrated in 25 ha-seronegative healthy young adults who were experimentally infected with influenza h3n2 virus [56] . (a similar study was undertaken with h1n1 challenge infection [57] .) following infection, peripheral blood cytokine responses were determined every six hours for the next five days. in nine subjects who developed symptomatic illness, there were early increases in cytokines associated with fever, leucocyte recruitment and markers of innate antiviral immunity, and some of these increases appeared as early as two days before the onset of symptoms [56] . these cytokine findings were also demonstrated in a parallel genomic analysis. in contrast, the 17 subjects who remained asymptomatic showed early and persistent down regulation of the same inflammatory markers. symptomatic subjects developed cytokine profiles similar to those that have been seen in patients who develop severe illness, while those who remained asymptomatic showed host responses indicating rapid control of infection. these findings suggested that the "inflammatory pathway an individual will follow is probably determined at (a) very early, even presymptomatic time" [56] . which pathway is followed is probably determined (at least in part) by epigenetic factors [58] [59] [60] . in a study of populations in sweden and japan during the early and late years of the 20 th century, evolutionary biologists showed that the increase in the annual probability of death due to all causes was greatest during the second decade of life, not in later years (fig. 4 ) [61] . other studies have shown that stress differentially allocates energy resources between reproduction and immune function [62] , and estrogens contribute to the energy trade-offs that help maintain homeostasis [52, 63] . at least some of the mechanisms responsible for maintaining homeostasis during puberty are epigenetically regulated [64] . numerous laboratory and clinical studies have described biological pathways that are associated with the susceptibility of neonates and the elderly to acute critical illnesses, but very few studies have compared host responses before and after puberty [65, 66] . one such study documented the responses of weanling and adult ferrets following infection with the 2009 h1n1 pandemic virus [65] . compared with adult ferrets, weanlings developed much milder clinical illnesses and had less evidence of pulmonary damage, yet rapid virus clearance from the respiratory tract was seen in both groups. like adults, the immune responses of weanlings to infection were robust, but they were different. pro-inflammatory cytokine responses in the two groups were similar, but regulatory response genes for il-10 and tgf-1 were more highly expressed in weanlings [65] . because influenza in ferrets closely mimics the disease in humans, this study suggests that the milder response to influenza in children compared with adults is due to a more strongly expressed regulatory response. very few studies have directly compared the cell signaling responses of children and adults to acute critical illness. nonetheless, in 2004, surgeons reported two studies that directly compared the inflammatory responses of peritoneal macrophages harvested from the sterile abdomens of children and adults [67, 68] . pro-and anti-inflammatory responses were elicited by exposing the macrophages ex vivo to endotoxin and il-1. unlike adults, responses in children were dominated by an il-10 anti-inflammatory pattern. recent research has shown that dna methylation stably reduces the expression of il-10 in th1 cells [69] . short-term reversal of this epigenetic mechanism can bring about an increase in il-10 gene expression. a decade ago, surgeons who were involved in liver transplantation in children and adults sought to better understand inflammatory responses in their patients by studying hepatic ischemicreperfusion injury in mice of different ages [70] . they found evidence of less inflammation but greater autophagy in the livers of younger (4-5-week old) mice compared with older (10-12-week old) mice, and the response of younger mice was associated with greater nuclear retention of pparg activity. following pretreatment (for three days) of older mice with the pparg agonist rosiglitazone, their highly inflammatory response was changed to the less inflammatory response seen in younger mice, and this change was associated with the autophagy pattern seen in younger mice. it is important to recognize that in this study younger mice were not clearly shown to be pre-pubertal and treatment was given before, not after the ischemic episode. nonetheless, pre-treatment with rosiglitazone was able to "roll back" the damaging response of "adults" to the more benign response of "children". pparg agonists have important effects on energy metabolism, and there is considerable "crosstalk" among these agents (glitazones) and other drugs that also have immunometabolic activities (e.g., statins [71] , ace inhibitors and angiotensin receptor blockers [72] , metformin [73] ) [5] [6] [7] [8] [9] . these findings suggest that many if not all of these drugs could be used to change the host response of adults to that seen in children. the drugs would probably work better if given in combination rather than by themselves [8] . for a severe infection like pandemic influenza, treatment with these drugs could improve an adult's tolerance of infection [44] [45] [46] [47] and might improve survival. the drugs might also have similar effects in children who develop life-threatening illness. ever since the emergence of highly virulent avian h5n1 influenza in 1997, virologists have warned of the possibility of a new and devastating influenza pandemic. in 2006, epidemiologists provided an estimate of what global mortality might be if the next pandemic is like the one in 1918 [74] . this estimate (51 to 81 million deaths) seems low because in 1918 pandemic mortality is thought to have been 50-100 million and today the global population is four times larger. a recent study from the institute for disease modeling estimates that during the first six months of a 1918-like pandemic, almost 33 million people could die [75] . moreover, if the next pandemic is caused by an h5n1-like virus, which has a high case fatality rate, its impact on global mortality could be much worse [3] [4] [5] [6] . influenza virologists are now concerned about the possibility of an h7n9 pandemic [9, 76] . nonetheless, even in the absence of pandemics, yearly outbreaks of seasonal influenza cause appreciable mortality worldwide [77] . most of these influenza-related deaths (pandemic and seasonal) occur in developing countries [74, 77] . current strategies for national and global pandemic preparedness focus on influenza vaccination for populations and antiviral treatment for individuals [9] . influenza virologists hope to develop universal influenza vaccines that will provide long-lasting protection, making it unnecessary to vaccinate against seasonal influenza each year [78] . vaccination with a universal vaccine might conceivably provide protection against infection with a future pandemic virus. recent virologic studies, however, raise important questions about whether this strategy will work [79, 80] . moreover, much of the world will lack the human infrastructure to guarantee administration of a universal vaccine [9] . this means that for the foreseeable future, health officials responsible for pandemic preparedness will have to count on using conventional pandemic vaccines. unfortunately, none of these vaccines will be available during the first six pandemic months [9] . moreover, when they eventually become available, it is unlikely they will be equitably distributed to low-and middle-income countries that don't produce their own influenza vaccines [81] . antiviral treatment of individual pandemic patients could also be problematic. supplies of one of the drugs (oseltamivir) are limited and the drug is not widely used. there is also concern about the development of antiviral resistance. moreover, a recent report on 1220 patients hospitalized in china with laboratoryconfirmed h7n9 influenza showed that although 70% of all patients were treated with oseltamivir, case fatality rates were still 40% [82] . an alternative strategy for reducing pandemic mortality would be to develop effective treatments that target the host response of patients who develop severe illness [2] [3] [4] [5] [6] [7] [8] [9] . these drugs might have some effect on the development of symptomatic illness [83] , but their potential impact on pandemic mortality would be far more important. agendas for laboratory and clinical research to evaluate their potential have been published in several articles ( table 6 in reference 8, table 1 in reference 9, and more generally in references 2 and 4-6). although influenza scientists often regard host response treatment as an adjunct to antivirals [84] , some of this research must be limited to generic drugs that target the host response because most of the world's people won't have access to antivirals but will have access to generics. these studies must go beyond documenting cytokine responses following infection and examine immunometabolic and epigenetic factors that affect (among other things) cellular immunity, endothelial function and energy metabolism [8, 9] . all of these studies should include comparisons before and after puberty. treating the host response holds promise for not only reducing pandemic mortality but also for reducing the appreciable mortality associated with seasonal influenza and other forms of acute critical illness (e.g., ebola virus disease [8] ). many of the candidate drugs are produced as generics in developing countries, and supply chains for their worldwide delivery are already in place [5] [6] [7] [8] [9] . physicians are familiar with these drugs because they use them every day. if this treatment strategy were shown to work, it could be used in any country with a basic healthcare system. for a pandemic, treatment could start in all countries on the first pandemic day. influenza virologists have expanded our understanding of the molecular biology and epidemiology of influenza viruses. laboratory and clinical investigators have deepened our understanding of the host response to critical illness. evolutionary biologists have suggested that evolution provides insights that could help public health. all of these developments should shape the way we respond to the next influenza pandemic. charles darwin wrote ". . . observation must be for or against some view if it is to be of any service" [85] . his view -the hypothesis that evolution is guided by natural selection -was supported by extensive observations made over several decades, and its explanatory power (i.e., 'service') has withstood challenge for almost 160 years. the idea (hypothesis) of treating the host response to pandemic influenza was introduced in 2004 [2] . its potential explanatory power has also been supported by experimental and clinical observations, although treating the host response has received little attention from scientists and health officials [4] [5] [6] [7] [8] [9] . yet in a practical sense this approach to treatment could be of great service (in darwin's word) if it could reduce global mortality during the next pandemic. kobzik and colleagues have shown age-specific differences in influenza mortality in mice before and after puberty that are not affected by previous infection (antigenic imprinting) with influenza viruses [26] . considered with evidence from endotoxemic mice [28] and other studies [4] [5] [6] [7] [8] [9] , their findings suggest that the mortality impact of pandemic and seasonal influenza and other forms of acute critical illness might be reduced by treating the host response. by reducing the damage caused by influenza in adults to the more tolerant response seen in children, treatment could in effect "roll back" evolution. physicians will inevitably be called upon to manage seriously ill patients during the next pandemic, but there is a real risk they will relive the experiences of physicians 100 years ago [9, 86] . this is sure to occur if influenza scientists and public health officials continue to reject the possibility that host response treatment could reduce pandemic mortality [7] [8] [9] 87 ]. if we are to take seriously the challenge of preparing for the next pandemic, it is self-evident that a "top down" approach based on vaccination and antiviral treatment, driven by the decisions of elite scientists, health officials and corporate executives, will not meet the world's needs [2] [3] [4] [5] [6] [7] [8] [9] . instead, an effective response must include a "bottom up" approach to individual patient treatment by ordinary doctors working in ordinary healthcare systems who use ordinary, widely available and inexpensive generic drugs that modify the host response. the studies reviewed here suggest that the biological basis for treating the host response reflects our evolutionary heritage. this idea might not be revolutionary [88] , but its practical implications for health, equity and security during the next pandemic could be immense. consequently, investigators must undertake laboratory and clinical research to convincingly show whether it will be effective. if it is effective and is put into practice, it would represent a striking application of the idea of evolutionary public health [1] . conflict of interest: none declared. evolutionary public health: introducing the concept pandemic influenza: a potential role for statins in treatment and prophylaxis commentary: from scarcity to abundance: pandemic vaccines and other agents for "have not" countries meeting the challenge of influenza pandemic preparedness in developing countries confronting the next influenza pandemic with antiinflammatory and immunomodulatory agents: why they are needed and how they might work treating influenza with statins and other 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laboratory-confirmed case series statins, influenza vaccination and influenza the role of adjuvant immunomodulatory agents for treatment of severe influenza darwin and the scientific method influenza in 1918: recollections of the epidemic in philadelphia herding, social influences and behavioural bias in scientific research: simple awareness of the hidden pressures and beliefs that influence our thinking can help to preserve objectivity revolutionary science key: cord-331714-2qj2rrgd authors: lvov, dimitry konstantinovich; shchelkanov, mikhail yurievich; alkhovsky, sergey vladimirovich; deryabin, petr grigorievich title: single-stranded rna viruses date: 2015-05-29 journal: zoonotic viruses in northern eurasia doi: 10.1016/b978-0-12-801742-5.00008-8 sha: doc_id: 331714 cord_uid: 2qj2rrgd in this chapter, we describe 73 zoonotic viruses that were isolated in northern eurasia and that belong to the different families of viruses with a single-stranded rna (ssrna) genome. the family includes viruses with a segmented negative-sense ssrna genome (families bunyaviridae and orthomyxoviridae) and viruses with a positive-sense ssrna genome (families togaviridae and flaviviridae). among them are viruses associated with sporadic cases or outbreaks of human disease, such as hemorrhagic fever with renal syndrome (viruses of the genus hantavirus), crimean–congo hemorrhagic fever (cchfv, nairovirus), california encephalitis (inkv, tahv, and khatv; orthobunyavirus), sandfly fever (sfcv and sfnv, phlebovirus), tick-borne encephalitis (tbev, flavivirus), omsk hemorrhagic fever (ohfv, flavivirus), west nile fever (wnv, flavivirus), sindbis fever (sinv, alphavirus) chikungunya fever (chikv, alphavirus) and others. other viruses described in the chapter can cause epizootics in wild or domestic animals: geta virus (getv, alphavirus), influenza a virus (influenzavirus a), bhanja virus (bhav, phlebovirus) and more. the chapter also discusses both ecological peculiarities that promote the circulation of these viruses in natural foci and factors influencing the occurrence of epidemic and epizootic outbreaks single-stranded rna viruses the bunyaviridae family was named after the prototypical bunyamwera virus (bunv) isolated in 1943 from mosquitoes (aedes spp.) in bunyamwera, uganda. 1 currently, the bunyaviridae family includes four genera of animal viruses (orthobunyavirus, phlebovirus, nairovirus, and hantavirus) and one genus (tospovirus) of plant viruses. 2 bunyavirus virions are spherical in shape (size, about 80à120 nm) and have an outer lipid bilayer with the viral envelope glycoproteins gn and gc exposed on the surface. the genome consists of three segments of single-stranded, negative-sense rna with a total length from 11,000 to 19,000 nt. depending on the size, the segments are designated l (large), m (medium), and s (small). the viral proteins are synthesized on the mrna that is produced during replication and that is complementary to the genomic rna. the length of segments varies for different genera, but in general, they have a common structure. the l-segment, whose length is from 6,400 nt (phlebovirus) to 12, 200 nt (nairovirus) , has a single open reading frame (orf) encoding rna-dependent rna polymerase (rdrp). the m-segment of all of the genera also has a single orf, which encodes a polyprotein precursor of envelope glycoproteins gn and gc. the length of the m-segment ranges from 3,288 nt for some of the phleboviruses to 4,900à5,366 nt for the nairoviruses. the mature glycoproteins gn and gc of the bunyaviruses are derived during complex endoproteolytic events leading to cleavage of the polyprotein precursor by cellular proteases. the s-segment of the bunyaviruses encodes a nucleocapsid protein. additional nonstructural (nss) protein is encoded by the s-segment of viruses of the phlebovirus, tospovirus, and orthobunyavirus genera. 2, 3 the bunyaviruses are widely distributed in the world and are one of the most numerous known zoonotic viruses. most of the zoonotic bunyaviruses are transmitted to animal or humans by bloodsucking arthropod vectors, usually mosquitoes or ticks. viruses of the hantavirus genus are the exception, being transmitted mainly by aerosol formed from virus-laden urine, feces, or saliva of infected rodents or insectivores that are their natural hosts. 4à6 the genus hantavirus consists of those bunyaviruses of vertebrates which do not have the ability to replicate in an arthropod's cell and which are transmitted by respiratory route through the formation of aerosols from urine or feces containing the virus. 1 the morphology of the virion and the genome structure of the hantaviruses are common to all bunyaviruses. the size of the negative-sense ssrna genome of the prototypical hantaan virus (htnv) is 6,533 nt for the l-segment, 3,616 nt for the m-segment, and 1,696 nt for the s-segment (figure 8 .1). 1 in nature, hantaviruses persist asymptomatically in rodents and insectivores, with each type of hantavirus associated predominantly with one host species. the phylogenetic relationships of hantaviruses enable virologists to divide them into three lineages, which correspond in general to their main hosts. in the s-segment of some hantaviruses carried by arvicolinae and sigmodontinae rodents, there is an additional orf-encoded nonstructural protein nss. but nss is absent in the hantaviruses of the murinae rodents. 2à4 history. hemorrhagic fever with renal syndrome (hfrs) was originally described as a separate nosological category (called "endemic (epidemic) hemorrhagic nephroso-nephritis" at that time) by anatoly smorodintsev (figure 2 .11) during 1935à1940 in the far east. later, japanese scientists described hfrs in northeastern china as "songo fever" and swedish scientists as "epidemic nephropathy"; a similar disease was described in 1960 in china. 1 the abbreviation "hfrs" was suggested by mikhail chumakov (figure 2 korea. 2 hantaviruses. the hantaviruses are members of the hantavirus genus of the bunyaviridae family. the first serotype, -htnv, included strains isolated from mouselike rodents (muridae) in south korea, china, and the southern part of the russian far east (primorsky krai). 2à4 the second serotype, puumala virus (puuv), was isolated from hamsterlike rodents (cricetidae), mainly the bank vole (myodes glareolus) in finland and then in other european countries and the western part of russia, as well we from maximowicz's vole (microtus maximoviczii) in the far east). 5à8 the third serotype, seoul virus (seov), was isolated from brown rats (rattus norvegicus), black rats (rattus rattus), and laboratory albino rats (rattus norvegicus f. domestica) in south korea and elsewhere, including the united states. 3, 4 the fourth serotype, dobravaàbelgrade virus (dobv), was isolated from the striped field mouse (apodemus agrarius) in slovenia 9 and yugoslavia. 10 the fifth serotype, sin nombre virus (snv), literally "nameless virus" in spanish, was isolated from the meadow vole (microtus pennsylvanicus). 8 in addition to the 5 main serotypes, 15 other serotypes are known today, including 6 in eurasia: amur virus (amrv), isolated from asiatic forest mice (apodemus peninsulae) in the far east of russia 11 and in china 12 ; tula virus (tulv), from common voles (microtus arvalis) in central russia 13, 14 ; khabarovsk virus (khav), from from reed voles (microtus fortis) and siberian brown lemmings (lemmus sibiricus) in the far east 15 ; thottapalayam virus (tpmv), from asian musk shrews (suncus murinus) in india 16 ; thailand virus (thaiv), from bandicoots (bandicota indica) in thailand 17 ; and a newfound hantavirus, from chinese mole shrews (anourosorex squamipes) in vietnam. 18 virion and genome. the size of the negative-sense ssrna genome of the prototypical htnv is 6,533 nt for the l-segment, 3 ,616 nt for the m-segment, and 1,696 nt for the s-segment (figures 8.1 and 8.2 ). epizootiology. rodents (order rodentia) are the main natural reservoir of hantaviruses. nevertheless, strains have been isolated from birds in the far east 19 and from bats in china. 20 infection in rodents is asymptomatic, but the virus is expelled with saliva, urine, and excrement, most intensively during the first month after inoculation. (during this period, virus antigen can be detected in the lungs.) 4 the evolution of hantaviruses is closely related to that of its rodent host (figure 8 .2). 4, 6, 21 at least 34 species of rodents (rodentia), 2 species of lagomorphs (order lagomorpha), 7 species of insectivores (order insectivora), 1 species of predators (order carnivora), and 1 species of artiodactyls (order artiodactyla) are known to take part in hantavirus circulation on the territory of northern eurasia. 8, 21, 22 the main species of rodents, which are the hosts of hantaviruses in russia, are presented in table 8 .1. the infection rate of mouselike rodents and insectivores lies within the limits 3.3 6 0.5%. 23 hantavirus antigens have been detected in birds as well: the oriental turtle dove (streptopelia orientalis), coal tit (parus ater), marsh tit (parus palustris), daurian redstart (phoenicurus auroreus), nuthatch (sitta europaea), black-faced bunting (emberiza spodocephala elegans), eurasian jay (garrulus glandarius), hazel grouse (tetrastes bonasia), pheasant (phasianus colchicus), ural owl (strix uralensis), green-backed heron (butorides striatus), and grey heron (ardea cinerea). 19 hantavirus (magboi virus, or mgbv) was isolated in 2012 from the hairy slit-faced bat (nycteris hispida) in africa (sierra leone), 24 but the role of bats in the circulation of hemorrhagic fever with renal syndrome virus (hfrsv) is yet to be investigated in detail. in western siberia, the main natural reservoir of hfrsv is rodents of the hamsterlike (cricetidae) family-in particular, bank voles (myodes glareolus), with a susceptibility up to 70%; red-backed voles (myodes rutilus), susceptibility 9%; and, in the north, siberian brown lemmings (lemmus sibiricus), 14%. the infection rate of other rodents and insectivores is about 0.4à3.0%. 8, 22 in eastern siberia, the maximum susceptibility is demonstrated in grey red-backed voles (myodes rufocanus), 70%; house mice (mus musculus), 15%; water voles (arvicola terrestris), 8%; and tundra voles (microtus oeconomus), 8%. 8 in the far east, hfrsv was revealed to circulate among field mice (apodemus agrarius) with a susceptibility of about 35%; asiatic forest mice (a. peninsulae), susceptibility 30%; reed voles (microtus fortis), 4à18%; grey redbacked voles (myodes rufocanus), 12%; and other rodents (rodentia), 0.7à4.3%. 21, 22, 25 epidemiology. hfrsv infection starts by aerogenic penetration of the virus during the inhalation of waste products (saliva, urine, excrement) of latently infected animals. an alimentary pathway (with contaminated food and water) of the infection is also possible. 4, 8, 22, 26, 27 hfrs is distributed over eurasia (russia, belarus, ukraine, moldova, the baltic countries, the czech republic, slovakia, bulgaria, romania, serbia, slovenia, england, france, germany, belgium, hungary, denmark, fennoscandia, kazakhstan, georgia, azerbaijan, china, north and south korea, japan), as well as american and african countries. 7, 28, 29 during 2000à2009, in 58 of 83 regions in russia, 74,890 cases of hfrs were registered (table 8 .2). 8 annual morbidity of hfrs in russia is in the range from 2,700 to 11,400 cases (1.3à7.8%) and is decreasing. about 95% of cases take place in european forest landscapes. puuv associated with the bank vole (myodes glareolus) provokes about 90% of hfrs cases in russia (especially in bashkortostan, udmurtia, mari el, tatarstan, the chuvash republic, orenburg, ulyanovsk, and the penza region). 8, 30 morbidity in the urban population is higher (65%) than in the rural one. the peak of the disease occurs during julyàoctober in forests and in gardens and kitchens closely situated to the forests. 4 ,31à33 dobv associated mainly with field mice (apodemus agrarius) and small forest mice (a. uralensis) is of leading epidemiological significance in the central and southwestern sectors of the european part of russia (the voronezh, lipetsk, orel, and belgorod regions), as well as in georgia. 8, 31, 34, 35 puuv and tulv are associated with the common vole (microtus arvalis) and the bank vole (myodes glareolus) and are also distributed over this territory. 4, 8, 36 a similar situation is observed in other regions of the central federal district: in the moscow, yaroslavl, ryazan, tver, kaluga, vladimir, ivanov, kostroma, smolensk regions. hfrs morbidity in the moscow region is associated with puuv, 31 the infection rate of which is 12à57% among bank voles (myodes glareolus), 10à20% in the common vole (microtus arvalis), 11% in major's pine vole (microtus majori), and in 4à6% other rodent species. 1 in krasnodar krai, the black sea field mouse (apodemus ponticus) and major's pine vole (microtus majori) play the main role in human morbidity. 31, 37 human morbidity in the european part of russia is registered beginning at a relatively low level in marchàapril, decreasing to yet a lower level in mayàaugust, increasing in septemberànovember, and then increasing again during decemberàjanuary. 1 the hyperendemic territory is the southwestern ural region (especially the bashkortostan republic and the chelyabinsk and orenburg regions), the volga-vyatka economic region (especially the udmurt republic), the chuvash republic, and the tatarstan, mari el, samara, penza, saratov, and ulyanovsk regions. 4, 8 the main human morbidity occurs among those 20à40 years old (chiefly men). in russia, hfrs represents a significant part of all naturalfoci zoonotic diseases. the immune layer to hfrsv in the european part of russia is a mean 4.7%; in the bashkortostan republic, it reaches up to 40% (mean, 17%). 4 the immune layer among the populations of western and eastern siberia is about 2% for the entire region, 0.2% in krasnoyarsk krai, 1.1% in the irkutsk region, 3.1% in the omsk region, and 12.6% in the tyumen region. 1, 4 the far east provides about 2% of all hfrs cases in russia. 23 the highest morbidity was revealed in khabarovsk krai, primorsky krai, and the amur region. 1 in khabarovsk krai and primorsky krai, las in china and japan, -htnv is associated with grey red-backed voles (myodes rufocanus). 2, 3, 21, 37 the morbidity of seov (the third serotype) associated with the synanthropic brown rat (rattus norvegicus) and black rat (r. rattus) was examed in both the far east and the european part of russia. the researchers found that seov provoked hfrs more often among the urban population, whereas htnv did so more often among the rural population, of primorsky krai. 21 morbidity in the far east has a small uptick in mayàjuly and reaches its main peak in novemberàdecember. the immune stratum in the far east is about 1% (ranging from 0.3% in the amur region to 1.5% in primorsky krai). 1, 21 pathogenesis. capillary damage is the basis of hfrs pathogenesis. in the first part of the disease, toxicoallergic phenomena predominate, caused by viral infection of the walls of vegetative centers, venules, and arterioles. lesions on the sympathetic nodes of the neck are followed by hyperemia of the face and neck. irritation of the vagus nerve leads to bradycardia and a fall in arterial pressure. damage to the vascular permeability is accompanied by hemorrhages in mucous membranes and the skin. the cause of death is cardiovascular insufficiency, massive hemorrhages into the vital organs, plasmorrhea into the tissues, collapse, shock, swelled lungs, spontaneous rupture of the kidneys, a hypertrophied brain, and paralysis of the vegetative centers. 4, 22 clinical features. the incubation period is 4à30 days. hfrs starts with fever, headache, muscular pain, dizziness, nausea, vomiting, hyperemia of the face and neck, bradycardia, and a fall in arterial pressure. abnormalities of the central nervous system (cns) in the form of block, excitement, hallucinations, meningeal signs, and visual impairments often occur. hemorrhagic syndrome becomes apparent as plasmorrhea into the tissues, together with microthrombosis; exanthema; petechial skin rash; nasal, pulmonary, and uterine bleeding: vomiting blood, hematuria, and visceral bleeding. in some cases, pasternatsky syndrome, pain in the kidneys, oliguria, and albuminuria become morphologically apparent as interstitial and tubular nephritis. the duration of fever is 3à9 days. two-wave temperature dynamics is possible. 22, 38 analyses of 5,282 cases of hfrs etiologically linked with puuv in sweden during 1997à2007 found 0.4% mortality in the first three months of the disease. 39, 40 defense immunity remains for at least 30 years. 8, 22 diagnostics. laboratory diagnostics are based on the fluorescent antibody method (fam), enzyme-linked immunosorbent assay (elisa), and reverse transcription polymerase chain reaction (rt-pcr) testing. the virus can be isolated with the use of vero e6 (green monkey kidney cell line), 2bs (diploid human embryo lung cell line), a-549 (human lung carcinoma cell line), or rlc (rat lung tissue primary cell culture). 8, 22 control and prophylaxis. treatment of hfrs can be symptomatic, pathogenetic, or etiotropic (or any combination thereof). during the fever period, early hospitalization, disintoxical therapy, and strengthening of the walls of vessels are necessary. during the oliguria period, transfusion with desalinated human albumin, hemodes, a 5% glucose solution, and an isotonic nacl solution (under the control of the emitted volume of urine) are given. in case of shock, antishock therapy is applied, and hemodialysis is prescribed for kidney insufficiency. 4, 22 vaccination is the most effective approach to the prophylaxis of hfrs. the efficacy of vaccination was demonstrated in china and in north and south korea. nevertheless, it must be mentioned that vaccines in these countries are produced from htnv and seov stains and do not defend against puuv infection, which is the main etiological agent of hfrs in the european part of russia (where 98% of all russian morbidity occurs) 8 . for a long time, anti-hfrs vaccine was difficult to produce because there were no sensitive cell lines to accumulate hantavirus. however, the recent adaptation of puuv and dobv to the certified vero e6 cell line affords an opportunity to produce candidate vaccines against hfrs. experimental series of "combi-hfrs-vac" vaccine have passed compliance tests for medical immunoglobulin preparations for use in humans. 8, 41, 42 the genus nairovirus includes the ticktransmitted bunyaviruses, whose genome is the largest in the family bunyaviridae. the size of l-segments of the dugbe virus (dugv), a prototypical species of the nairoviruses, is 12,255 nt. the m-and s-segments are 4,888 and 1,716 nt, respectively (figure 8 . 3) . as with other bunyaviruses, the l-segment of the nairoviruses encodes an rdrp, the m-segment encodes a polyprotein precursor of the envelope glycoproteins gn and gc, and the s-segment encodes the nucleocapsid (n) protein. 1, 2 the genus nairovirus was established on the basis of antigenic relationships among viruses of the six antigenic groups of arthropod-borne viruses: the crimeanàcongo hemorrhagic fever (cchf), nairobi sheep disease (nsd), qalyub (qyb), sakhalin (sak), dera ghazi khan (dgk), and hughes (hugv) groups. 3à6 subsequently, a seventh, thiafora (tfa), group was assigned to the genus. 7, 8 currently, about 35 viruses are assigned to the genus nairovirus, now united in the aforementioned seven groups. 1 sequence analysis of previously unclassified bunyaviruses revealed that the nairoviruses actually number much more than 35 . three additional groups of nairoviruses-issyk-kul (isk), artashat (artsv), and tamdy (tam)-were established on the basis of phylogenetic analysis (table 8 .3). cchfv belongs to the nairovirus genus of the bunyaviridae family and is the etiological agent of crimeanàcongo hemorrhagic fever (cchf). history. cchf was first mentioned as "hunibini" and "hongirifta" by tajik physician abu-ibrahim djurdjani in the twelfth century. the viral nature of cchf was originally established in 1945 during an expedition to crimea headed by mikhail chumakov at the time of an outbreak. 1à3 the modern history of cchfv investigation starts in june 1944 with an epidemic of the disease in the northwestern steppe part of the crimean peninsula. more than 200 severe cases of the disease broke out, all exhibiting hemorrhagic syndrome, known in that time as "severe infectious capillary toxicosis." mikhail chumakov headed an expedition to the region, and much research revealed that the disease is transmitted by hyalomma plumbeum (marginatum) ticks of the ixodidae family. the disease 1 in 1963, the historical hodzha strain was isolated from a patient with hemorrhagic fever in uzbekistan, as was a set of strains from h. marginatum larvae and nymphs in the astrakhan region, near the caspian sea. 2, 3 in 1967, the similarity between the etiological agent of crimean hemorrhagic fever and that of congo virus, isolated from a patient in 1966 in zaire (congo), was demonstrated, so the virus was renamed cchfv. 4, 5 genome and taxonomy. like the genomes of all nairoviruses, that of cchfv consists of three segments of negative ssrna: a signed small (s) (1,672 nt) segment, a medium (m) (5,366 nt), and a large (l) (12,108 nt) segment. each segment has a single orf that encodes the nucleocapsid protein (n, 482 aa, s-segment), a polyprotein precursor of envelope glycoproteins gn and gc (1,684 aa, m-segment), and rdrp (3,945 aa, l-segment). genetic diversity among cchfv strains may reach 31% nt and 27% aa differences for m-segment sequences, a reflection of pressure on the immune system and adaptation to various ecologic zones with different prevalences of hyalomma tick species. the s-and l-segments are more conservative: the level of divergence of s-segment sequences is 20% nt and 8% aa, and that for l-segment sequences is 22% nt and 10% aa. phylogenetic analysis based on sequence data comparisons of s-segments shows that cchfv isolates from different regions can be clustered into seven phylogeographic groups: west african isolates (group i), as well as isolates from central africa (uganda and the democratic republic of the congo) (group ii); south africa and west africa (group iii); the middle east and asia (group iv) (the asian strain can be divided to two distinct subgroups: asia 1 (iva) and asia 2 (ivb)); europe and turkey (group v); and greece (group vi), a separate group detached from the rest of europe (figure 8.4) . 6à8 in general, the genotypic structure defined on basis of the s-segment analysis is correlated strictly with geography. cases of isolation of strains not typical for a given territory were attributed to possible transmission of the virus by infected ticks carried by migratory birds. the tree topology based on the l-segment comparison is, on the whole, similar to that generated on the basis of the s-segment. exceptions are the viruses from senegal, which represent a separate lineage in the s-segment analysis, and those clustered within group iii in the l-segment analysis. similarly, the division of group iv into group iva (asia 1) and ivb (asia 2) is not clear (figures 8.5 and 8.6) . in russia, most of the strains of cchfv that were isolated were isolated in the country's southern regions (astrakhan, volgograd, and stavropol districts). phylogenetic analysis showed that all of them are closely related to european and turkish strains (group v). 9à12 epizootiology. up to today, cchfv has been found to circulate in 46 countries in europe, africa, and asia. 4,13à15 cchfv was isolated from at least 27 species of mainly ixodidae ticks, but their roles in maintaining virus circulation are different (tables 8.4 and 8.5). the main significance for cchfv reservation and transmission belongs to ticks of the hyalomma genus: h. marginatum in the south of he european part of russia, h. anatolicum and h. detritum in the middle east and asia, and h. asiaticumin kazakhstan. according to our data, the viral load among imagoes of h. marginatum in the astrakhan region in 2001à2005 was 1.33%; among nymphs, the load was 0.2%. the presence of transphase and transovarial transmission of cchfv provides a reservation for viruses during the interepidemic period. three hostsfor larvae (ground birds, mainly corvidae; table 8.4 isolation of cchfv from ixodidae ticks mouselike rodents; and hares), nymphs (also ground birds, mouselike rodents, and hares), and imagoes (large mammals-mainly cattle, sheep, and camels)-provide a variety of ecological links of cchfv to vertebrates. 1,16à19 in nigeria, cchfv was isolated from midges (culicoides sp.) 4 the distribution of h. marginatum is limited by the isotherm of effective temperatures such that sum (σ t $ 10 c ) 5 3,000 c, or 120 days with mean temperature $20 c per year. 20 so, the northern boundary of the distribution of cchfv in the south of the european part of russia lies in the dry steppe subzone. 1 in russia and south africa, cchfv is often isolated from hares. 1, 21 cchfv was isolated from hedgehogs (atelerix spiculus) in nigeria. hares and mouselike rodents play the main role in cchfv circulation. 1, 21, 22 viremia in birds is not sufficient for vector transmission (although specific antibodies appear); nevertheless, ground birds are an important element of cchfv transmission because they are the hosts for the preimaginal phases of h. marginatum development. 16, 18, 23 during field investigations of chatkalsky ridge in kirgizia, nymphs and larvae of h. marginatum dominated among field-collected materials from birds. the highest number of ticks was found on rollers (coracias garrulus), crested larks (galerida cristata), tree sparrows (passer montanus), and blackbilled magpies (pica pica). in the astrakhan region, rooks (corvus frugilegus) are the main hosts for h. marginatum preimaginal phases. 16 during migrations, birds can take part in dispersing preimago ticks that carry the virus. for example, in spain in 2010, cchfv of african origin (probably introduced by migrating birds) was isolated from h. lusitanicum. 24 european birds overwintering in africa were also found to harbor ticks that carried the virus. 25 cchfv infection rates found as the result of an investigation of 40,711 domestic animal sera are presented in table 8 .6. 20 domestic animals are one of the main reservoirs of cchfv among vertebrates. viremia (2.6à3.7 (log 10 ld 50 )/20 mcl) sufficient for the infection of ticks was detected 5à8 days after experimental inoculation of sheep. viremia after up to 10 days post inoculation was detected in small gophers (citellus pygmaeus), long-eared hedgehogs (hemiechinus auritus), and wood mice (apodemus sylvaticus). experimental infection was revealed only in nymphs, and that is why hares and corvidae birds-the main hosts for nymphs-play the chief role in cchfv circulation. astrakhan 1 5 11 13 9 4 37 16 20 5 6 7 10 7 1 152 volgograd 0 18 9 3 3 2 6 16 30 7 2 3 0 0 6 105 dagestan 0 6 10 7 3 1 3 3 2 2 1 3 2 0 2 rostov 27 0 5 7 9 9 16 55 53 83 27 16 48 41 38 434 stavropol 10 48 21 54 30 41 38 41 63 80 66 28 26 24 32 602 krasnodar 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 total 38 85 59 97 77 76 139 200 234 193 119 67 97 75 79 1,635 of ixodidae (at first, h. marginatum) ticks in this region as the result of climatic changes. during 1999à2010, 13,838 cases of cchf 44 were recorded in russia, including 520 in stavropol krai, 45 307 in the rostov region, 46 276 in the kalmyk republic, 47 134 in the astrakhan region, 48 99 in the volgograd region, 49 41 in the dagestan republic, 5 in the ingush republic, and 1 in the karachaevoàcherkesskaya republic 50 (table 8 .7). in 2013, 80 cases of cchf were recorded on the territory of the southern federal district and the north caucasian federal district (table 8 .8, figure 8 .8). the absence of any recorded cases of cchf in krasnodar krai could be explained by a lack of attention to cchf diagnostics. a decrease in the proportion of severe clinical forms with hemorrhagic syndrome occurred after 2006. the drop could have been due to the introduction of high-grade express diagnostics methods into clinical practice and an intensification in seeking out and diagnosing those suspected of having cchf. at the same time, the disease extended its incidence into the new territories of the volgograd region, with nosocomial cchf cases recorded there once again. 52 pathogenesis. pathogenesis is defined by lesions of the vascular and nervous systems. 17, 51, 53 clinical features. the incubation period after transmissive cchfv inoculation (as the result of a tick bite) is 2à7 days, whereas that after contact inoculation is 3à4 days. the difference is due to a much higher quantity of virus entering the system during contacts inoculation. 17, 50, 53 cchf starts rapidly, with the temperature increasing to 39à40 c and the appearance of fever, skin hyperemia in the top half of the trunk, headache, lumbar pain, abdominal and epigastric pains, generalized arthralgia, conjunctivitis, pharingitis, and diarrhea. about 50% of cases have two obvious waves of increasing temperature, with the temperature decreasing in 6à7 days after the end of the incubation period. petechial rash appears in the majority of all cchf patients in 3à4 days after the incubation period and is a marker of the second increasing-temperature wave. hemorrhagic diathesis with nasal bleeding (in two-thirds of cases), bloody vomiting, blood in the sputum, and hematuria, all starting 3à5 days after the end of incubation period, are characteristic in 85% of cases. the duration of the hemorrhagic period is 8à9 days. meningitis symptoms and signs of psychosis (depression, sleepiness, lassitude, photophobia) could develop as well. lethality is 16à20% for transmissive inoculation and up to 50% for contact inoculation. nevertheless, lethality is decreasing as the result of the introduction of modern testing systems and treatment with ribavirin. the convalescent period is about a month. 17, 50, 51, 53 e.v. leshchinskaya has suggested the following clinical classification of cchf: (i) severe form with hemorrhagic syndrome (1.a. without band bleeding; 1.b. with band bleeding); (ii) without hemorrhagic syndrome (2.a. medium-severe form; 2.b. light form). 50, 53 diagnostics. diagnosis is based on the detection of both specific antibodies via elisa (igm after 8 days post disease progression and igg) and virus rna via rt-pcr testing (earlier than 8 days post disease progression). 43, 54 both tests must be conducted for a definitive diagnosis of cchf to be made. during the first week of infection with cchf, positive results via rt-pcr are obtained in 93% of cases; during the second week, the percentage is 40%. during the second week of the disease, positive results in igm via elisa are obtained in 93% of cases; during the third week, the percentage of positive reults in igg via elisa is 80%. 55à58 control and prophylaxis. ribavirin is the most effective drug prescribed today. 53 ,59à61 ribavirin is used for 5 days after symptoms first appear: 2,000 mg (10 capsules) or 30 mg/kg for the first time, then 600 mg 3 2 times a day if the weight of the patient is more than 75 kg or 500 mg 3 2 times a day if the weight of the patient is is less than 75 kg). the duration of treatment is 4à10 days. ribavirin must not be used by pregnant women, except when the disease is considerd life threatening. vaccine development is currently just in the experimental stages, 62à64 so prophylaxis involves early detection of sick humans and the prevention of further contact infections. nonspecific prophylaxis includes the eradication of ixodidae ticks on livestock and acaricide treatment of locations inhabited by domestic animals. in pastures with large numbers of ixodidae ticks, animals have to be led into box stalls and the humans leading them there must use special suits. history. artashat virus (artsv, strain leiv-2236ar) was originally isolated from ornithodoros alactagalis ticks (family argasidae) collected in the burrows of a small five-toed jerboa (allactaga elater) near arevashat village (40 02 absence of antigenic relationships with any known viruses, it was referred to as an "unclassified bunyavirus." 1à3 taxonomy. three strains of artsv were sequenced. 4 a full-length genome comparison revealed that artsv has 42à60% nt similarity to other nairoviruses. phylogenetic analysis revealed that the virus is a new species in the nairovirus genus and forms a distinct genetic lineage on the nairovirus tree, which was constructed for all three segments of the genome (figures 8.10à8 . 12) . the phylogeny of the nairoviruses is based mainly on analyses of the partial sequence of the conservative catalytic core domain of rdrp. 5, 6 the similarity of this domain of artsv to other nairoviruses is 42à65% nt and 58à70% aa. the phylogenetic tree constructed by the maximum-likelihood method on the basis of the amino acid alignment of the rdrp catalytic core domain of nairoviruses confirms the topology of artsv on a newly formed genetic lineage (figures 8.10à8.12 ). the nairoviruses on the tree can be divided into two main phylogenetic groups. the first group includes the nairoviruses, which are transmitted predominantly by ixodids: the crimeanàcongo hemorrhagic fever group (hyalomma and haemaphysalis, as well as dermacentor, rhipicephalus, and ixodes), the dugbe group (mainly amblyomma, but also hyalomma, rhipicephalus, and haemaphysalis), the sakhalin group (ixodes), and the tamdy group (hyalomma). the first group also includes erve virus (ervev), whose vectors are unknown. 7, 8 the second phylogenetic lineage includes the nairoviruses from the hughes, issyk-kul, dera ghazi khan, and qalyub groups, whose vectors are argasids: argas and ornithodoros. the tree topology of artsv shows that the virus is in the lineage of the nairoviruses transmitted predominantly by ixodidae ticks, although all isolations of artsv were obtained from the argasidae ticks o. alactagalis and o. verrucosus (table 8.9 ). it can be assumed that the adaptation of artsv to argasids is the result of the the narrow ecologic niche occupied by those ticks, which are ticks of the subgenera theriodoros and pavlovskyella. note that, although ervev, a european nairovirus, is phylogenetically close to the nairovirus transmitted by ixodids, the association of ervev with ixodes spp. ticks has not been established in endemic areas (southern europe). 8 ervev has been isolated from shrews (crocidura russula). 9 arthropod vectors. the adaptation of viruses to argasidae ticks facilitates the possibility of survival of viral populations in winter at low temperatures and in dry periods. the ability of argasids to fast (up to 9 years and more), the long life cycle of these ticks (up to 20à25 years), and their polyphagia and ecological plasticity determine the stability of the natural foci of arboviruses transmitted by argasids. these foci are confined mainly to the arid regions of the southern part of the temperate and subtropical zones. 1, 2, 10 the northern border of the range of argasids coincides with isolines denoting a frost-free period of 150à180 days per year and an average daily temperature above 20 c for no less than 90à100 days per year. 11 tick species from the subgenera theriodoros (ornithodoros alactagalis, o. nereensis) and pavlovskyella (o. papillipes, o. verrucosus, o. cholodkovskiy, o. tartakovskiy) are associated mainly with burrows of rodents. 11 this ecological peculiarity narrows the possibility of the spread of viruses that are adapted to ticks from the theriodoros and pavlovskyella subgenera. 2 it also applies to artsv associated with burrowà shelter biomes and found only in transcaucasia. history. caspiy virus (casv, prototypical strain leiv-63az) was originally isolated from the blood of a sick herring gull (larus argentatus) caught on gil island in the baku archipelago, off the western coast of azerbaijan in the caspian sea (40 17 0 n, 49 55 0 e; figure 8 .13) in 1970. 1à4 on the basis of electron microscopy, casv was classified as a member of the bunyaviridae family, but antigenic relationships with known bunyaviruses have not been found. thus, casv was categorized into the unclassified bunyaviruses. 5,6à8 at the same time, and in the same place, three strains of casv were isolated from ornithodoros capensis (family argasidae) ticks taxonomy. the genome of the prototypical strain leiv-63az of casv was sequenced, and it has been shown that casv is a member of the hugv group of the nairovirus genus. 11 the s-segment of casv is about 1,594 nt in length and has a single orf that encodes the nucleocapsid protein (n, 497 aa). the second start codon, in position 7, is located in the n-protein orf of casv. the identity of the amino acid sequence of the n-protein of casv with those of other nairoviruses is only 28%, on average. the cleavage site for caspase-3 (d285evd288) that has been found in the n-protein of cchfv is absent in casv. cleavage of n by caspase-3 is required for effective replication of cchfv. 12 note that caspase cleavage sites in the nucleocapsid protein are also necessary for replication of human influenza a viruses. 13 the m-segment of casv, like that of the other nairoviruses, has a single orf-encoded polyprotein precursor of the envelope glycoproteins gn and gc. the length of the gn/gc precursor of casv is 1,376 aa. according to the results of an analysis of polyprotein in the program signalp server 4.1, the first 32 aa constitute the signal peptide that is cleaved on the ssa/sy site. the cleavage site between pre-gn and pre-gc is in position 699 (vsg/ik). these data are confirmed by the location of transmembrane domains in mature proteins gn and gc that was defined with the use of the program tmhmm server 2.0. six potential sites of n-glycosylation are predicted in the mature gn protein of casv, only one in the gc protein. in general, the level of identity of polyprotein in casv is 25à27% aa with that of other members of the nairovirus genus (table 8. 3). the l-segment of casv has an orf (4,001 aa) that encodes the viral enzyme rdrp, which is the most conservative viral protein. the similarity of the rdrp of casv to that of other nairoviruses for which complete genome sequences were available is 38.8à43.0% aa. phylogenetic analysis based on the predicted full-length amino acid sequences revealed that casv is equidistant from other nairoviruses, and forms a distinct branch, on the trees (figures 8.10à8.12). for many nairoviruses, only short sequences of the catalytic core domain of rdrp are available in genbank. this domain of rdrp is very conservative and relevant to phylogenetic analysis. 1, 14, 15 the highest level of similarity (80% aa) that the rdrp core domain of casv has is with the same sequences in viruses of hug. on the dendrogram, constructed on the basis of a comparison of rdrp core domains, casv is located on the branch of the hug group (figures 8.10à8.12 ). note that viruses of this group (as well as casv) have been isolated from ornithodoros (carios) ticks that are associated with seabirds on the coasts and islands of the world's oceans. 2, 16 thus, the phylogenetic relationship of casv with hug group viruses reflects the ecological features of those coasts and islands. arthropod vectors. ornithodoros capensis ticks inhabit the coasts and islands of the atlantic, indian, and pacific oceans from the southern part of the temperate zone to the equator, as well as some large inland ponds. 3 3, 4, 17 o. capensis ticks feed on many bird species, mainly those of the order charadriiformes: gulls (family laridae) and terns (sturnidae), but also cormorants (phalacrocoracidae) and pelicans (pelecanidae). 4, 17 these argasid ticks have a life cycle made up of six to eight stages: egg, larva, three to five stages of nymphs, and imago. according to laboratory study, the cycle is from 43 to 83 days and so can be completed during a single breeding season. these ecological peculiarities provide stability to the natural foci of the viruses, which are adapted to the o. capensis tick viruses and their transcontinental transfer by migrating birds. 5 vertebrate host. in 1970, during the collection of field material on islands in the baku archipelago, an epizootic among herring gulls was observed. the first strain of casv was isolated from sick birds. migrations in search of food, including migration between the western and eastern coasts of the caspian sea, result in a sharing of the argasids and viruses ranging over the area. history. the prototypical strain leiv-858uz of the chim virus (chimv) was isolated from ornithodoros tartakovskyi ticks collected in july 1971 in the burrows of great gerbils (rhombomys opimus) in the vicinity of the town of chim in the kashkadarinsky region of uzbekistan) (38 47 0 n, 66 18 0 e; figure 8 .14). 1à3 isolation of chimv was carried out during monitoring of these arboviruses' foci on the territory of central asia and kazakhstan. chimv was investigated through serological testing with viruses from different families and with unclassified viruses isolated earlier in the ussr. because no antigenic relationships of chimv were (and still have not been) found, chimv was assigned to the category of unclassified viruses. 3, 4 later, four strains of chimv were isolated from the ticks o. tartakovskyi, o. papillipes, and rhipicephalus turanicus (rhipicephalinae) respectively collected in the burrows of great gerbils in the kashkadarya, bukhara, and syrdarya districts of uzbekistan in 1972à1976. 5, 6 three strains of chimv also were isolated from hyalomma asiaticum (hyalomminae) ticks and from the livers of great gerbils, which were collected in the floodplains of the or river and karatal river (dzheskazgan district, kazakhstan) in april 1979 (figure 8 .14). 7, 8 taxonomy. the genome of the prototypical strain leiv-858uz of chimv was sequenced, and, on the basis of sequence analysis, the virus was classified as a novel member of the nairovirus genus. 9 phylogenetic analysis based on a partial sequence of a catalytic center of rdrp placed chimv on the genetic branch of the qybv group. 9, 10 the amino acid sequence of this domain of chimv has an 87% identity with qybv, geran virus (gerv), and bandia virus (bdav), the other members of the qybv group. 11à14 all these data are consistent with the fact that viruses of the qybv group, as well as chimv, have an environmental connection to ticks of the ornithodoros genus and to the burrows of rodents. qybv has repeatedly been isolated from o. erraticus ticks, collected in burrows of the african grass rat (arvicanthis niloticus) in the nile valley and the nile delta in egypt. 13 to date, only short sequences of the rdrp of qybv are available in genbank, but recently we gave a genetic characterization of gerv, isolated in transcaucasia and, apparently, closely related to qybv. 11 the full-length amino acid comparison of chimv with gerv showed that their nucleocapsid proteins n (s-segment) have only a 55.6% identity. the similarity of complete amino acid sequences of rdrp (l-segment) is 74.8%. the similarity of the polyprotein precursor of gn/gc is 55.6%. the proteins of chimv have 30.3à42.4% aa (n-protein), 27.5à45.1% aa (gn/gc precursor), and 48.1à62.3% aa (rdrp) identities with their counterpart proteins in other nairoviruses. among these nairoviruses, chimv has the highest level of similarity with iskv, which is associated with bats in central asia (figures 8.10à8.12) . 15 arthropod vectors. most isolations of chimv were obtained from ornithodoros tartakovskyi ticks. these ticks are common in the irano-turanian and mountain provinces of asia (kazakhstan, the central asian republics, northeastern iran, and china (xinjiang)). the western border of the area in question is the eastern shore of the caspian sea (53à54 e), the eastern border is in xinjiang (87 e) , and the northern border is 44à47 n. the typical biotopes that o. tartakovskyi ticks inhabit are the foothills of dry steppes with loess soils. the ticks also inhabit meadow steppes and deserts (floodplain terraces and canals). o. tartakovskyi ticks prefer burrows of small diameter (inhabited by rodents, including jerboas, ground squirrels, small predators, and hedgehogs, as well as by turtles and birds). synanthropic biotopes are rarely inhabited. 16 vertebrate hosts. the great gerbil (family muridae, subfamily gerbillinae, genus rhombomys) is distributed from the shores of the caspian sea on the plains of central asia and southern kazakhstan, to the deserts of central asia, iran, and afghanistan, and on eastward to northern china and inner mongolia. great gerbils are typical inhabitants of sandy deserts and form a colony with complex multistory burrows that have a large number of entranceways and egresses (up to 200à500). these burrows are a specific biotope that exists for many decades, and they maintain natural foci (in particular, of plague) in arid areas. 6, 8 animal infection. the significance of chimv in the pathology of humans is unknown. antibodies to chimv have been found in camels (9.5%) in the kashkadarya region in uzbekistan. 5 this finding shows the ability of chimv to infect camels, as does qybv, but additional studies are necessary to clarify the pathogenicity of chimv in humans and cattle. 17 history. grnv (strain leiv-10899az) was isolated from ornithodoros verrucosus (family argasidae, subfamily ornithodorinae) ticks collected in a burrow of red-tailed gerbils (meriones (cricetidae) erythrurus) near geran station, goranboy district, azerbaijan; figure 8 .15). serological methods have failed to identify grnv, but the virus has been sequenced and classified into the nairovirus genus (family bunyaviridae). 1 taxonomy. the genome of grnv was sequenced by a next-generation sequencing approach. 1 full-length genome analysis revealed that the genetic similarity of grnv to other known nairoviruses is, on average, 30à40% aa for the nucleocapsid protein (n, s-segment), 27à33% aa for the polyprotein precursor of the proteins gn and gc (m-segment), and 48.0à74.8% aa for rdrp (l-segment). the highest level of similarity all three proteins of grnv have is to that of chimv (54.2à74.8% aa identity) and that of iskv (42.4à62.3% aa identity). 2,3 further analysis based on a comparison of partial sequences of the conservative core domain of rdrp of the nairoviruses showed that grnv and chimv were most closely related to qybv, which is the prototypical virus of the group of the same name. 4 the nucleotide sequence of the rdrp core domain of grnv has 74.3% nt and 97.1% aa identities with the counterpart sequence of qybv. the data obtained allow grnv to be classified as a virus of the qybv group (figures 8.10à8.12). the phylogenetic relationship between grnv and qybv corresponds to their similar ecological characteristics. qybv was first isolated in 1952 by r. taylor and h. dressler from argasid ornithodoros erraticus ticks collected in a rodent burrow in the nile river delta near qalyub village, egypt (30 n, 32 e). 5à7 complementbinding antibodies to qybv were found in humans (1.5%), camels, donkeys, pigs, buffalos, dogs, and rodents. 1, 7 the antigenic group of qalyub, a group that includes qybv and antigenic-related bdav, is one of the prototypical groups of the nairovirus genus. 5, 8 previously, qybv had been repeatedly isolated from o. erraticum collected in the burrows of rodents (arvicanthis) in africa. the second member of the qybv group, bdav, was isolated from o. sonari (a member of the o. erraticus group) collected in the burrows of rodents (mainly mastomys) in senegal. 9, 10 the isolation of gerv, which is closely related to qybv, is the first confirmation of the circulation of qybv group viruses in transcaucasia. arthropod vectors. the area of distribution of o. verrucosus ticks covers the southern part of moldova as well as ukraine and the caucasus region, and is limited by 47 30 0 n latitude. the area includes the southern part of russia (the krasnodar and stavropol regions), the northern and eastern foothills of dagestan, the foothills and lowland hills of georgia, the valleys of the hrazdan river in armenia, the foothills of the lesser caucasus mountains in azerbaijan, and the gobustan plateau and the absheron peninsula, also in azerbaijan. o. verrucosus ticks inhabit shelter biotopesin particular, the burrows of red-tailed gerbils (meriones (cricetidae) erythrurus), animals that are common in central asia, southern kazakhstan, and eastern transcaucasia. redtailed gerbils tends to inhabit desert and semidesert landscapes. their burrows are deep and may have 5à10 entranceways and egresses. history. iskv (prototypic strain, leiv-315k) was originally isolated from a pool of internal organs (liver, spleen, brain) of nyctalus noctula bats, and their ticks (argas (carios) vespertilionis) were collected near issyk-kul lake in kyrgyzstan in 1970 (figure 8 .16). 1, 2 subsequently, iskv was isolated from other bat species of the vespertionidae family (vespertilio serotinus, vespertilio pipistrellus, myotis blythii, rhinolophus ferrumequinum), and from birds, in different regions of kyrgyzstan and tajikistan. 3à11 two strains were isolated from anopheles hyrcanus mosquitoes and culicoides schultzei biting midges, respectively (figure 8.16, table 8 .10). 3, 12, 13 complement-fixation testing showed that iskv is closely related or identical to the keterah virus, which was isolated from scotophilus temminckii bats and a. pusillus ticks in malaysia in 1960. 14, 15 a strain that has a close, one-sided antigenic relationship to iskv, leiv-218taj (named garm virus), was isolated from a common redstart (phoenicurus phoenicurus) caught in the village of garm, tajikistan, morphological studies by electron microscopy characterized iskv as a member of the bunyaviridae family, and because no antigenic relation to any known viruses was found, it was assigned to the unclassified bunyaviruses. 16 taxonomy. the genome of the prototypical strain of iskv, leiv-315k, was sequenced, and, on the basis of sequence analysis, the virus was classified into the nairovirus genus. 17 like the genomes of other nairoviruses, that of iskv consists of three segments of rna (in negative polarity), each of which has a single orf-encoded nucleocapsid protein (n, 485 aa, s-segment), a polyprotein precursor of the envelope glycoproteins gn and gc (1,631 aa, m-segment), and a rdrp (3,950 aa, l-segment). a pairwise comparison of the full-length nucleotide and deduced amino acid sequences of the iskv orfs with those of other nairoviruses revealed 48.2à51.1% nt (39.0à42.1% aa), 37.3à39.7% nt (23.2à26.5% aa), and 43.1à47.0% nt (31.9à34.5% aa) identity for rdrp, the precursor of gn and gc, and the n protein, respectively (table 8.10) . phylogenetic analysis carried out for the fulllength amino acid sequences by the maximumlikelihood nearest-neighbor method showed that iskv occupies a new and distinct branch on the phylogenetic trees relevant to all three nairovirus proteins (rdrp, gn/gc, and n) (figures 8.10à8.12) . for the many known nairoviruses (i.e., qybv, dgkv, and hugv, as well as for a new nairovirus that was found in european bats by a metagenomics approach), there are only partial sequences of the conservative catalytic core domain of rdrp. 16, 18, 19 the level of identity for this domain of iskv with other nairoviruses ranged from 59.6à66.1% for the nucleotide sequence and 64.8à75.2% for the amino acid sequence (table 8 .10). the iskv rdrp core domain has the highest level of identity with qybv (66.6% nt and 74.5% aa). the phylogenetic tree constructed on the basis of the amino acid alignment of the rdrp core domain of nairoviruses confirms the topology of iskv on a new genetic branch of the nairoviruses (figures 8.10à8.12) . arthropod vectors. most isolates of iskv were obtained from argas vespertilionis ticks, and we can assume that these ticks are the main natural reservoir of the virus. the range of ticks of the a. vespertilionis group covers territory in central asia, africa, oceania, and australia ( figure 8.17) . vertebrate hosts. the natural vertebrate hosts of iskv are apparently bats-specifically, the genera nyctalus, vespertilio, rhinolophus, and myotis (family vespertilionidae). these bats are common in the temperate and subtropical zones of europe, asia, and north africa, and widespread iskv transmission and the appearance of an emergency are possible in all of their territories. human pathology. the first case of issyk-kul fever was registered in tajikistan in august 1975 when a staff member became ill after catching bats during surveillance for arbovirus. iskv was isolated from his blood on the second 21 the disease occurs with fever (39à40 c), headache (94%), dizziness (50%), hyperemia of the throat (48%), cough (25%), and nausea (31%). the outcome is generally favorable, and no deaths have been registered. 18 most of the cases were associated with the presence of bats in the attic of the residence. the primary route of human infection was apparently by argasid ticks, but respiratory or alimentary routes (via the feces and urine of bats) could not be excluded. furthermore, a laboratory experiment showed that iskv can be transmitted by aedes caspius mosquitoes. 22 the percentage of the population immune to iskv in the southern part of tajikistan is 7.8%. in kyrgyzstan, antibodies to iskv have been found in 0.7à3.2% of the human population. the highest percentage (9%) with antibodies to iskv was found in the southeastern part of turkmenistan. 12 history. uzun-agach virus (uzav), strain leiv-kaz155, was isolated from the liver of a myotis blythii oxygnathus (order chiroptera, family vespertilionidae) bat caught in the vicinity of the village of uzun-agach, alma-ata district, kazakhstan, during the virological sounding of territory in central asia and kazakhstan in 1977 (figure 8.18 ). 1à3 on the basis of virion morphology, uzav was classified into the bunyaviridae family. no serological study of uzav was ever conducted, but the place of uzav isolation, uzun-agach, is close to where iskv was originally isolated, namely, near issyk-kul lake, and the source of both viruses is the same: bats. 4, 5 taxonomy. the full-length genome of uzav was sequenced, and, on the basis of phylogenetic analysis, the virus was classified into the nairovirus genus. 6 the genome of uzav, like those of other nairoviruses, consists of three segments of ssrna with negative polarity. the l-segment encodes rdrp (3,988 aa), the m-segment encodes a polyprotein precursor of the envelope glycoprotein gn and gc (1,621 aa), and the s-segment encodes the nucleocapsid protein n (485 aa). a pairwise comparison of the sequence of the uzav genome with those of other nairoviruses showed that the virus is related most closely to iskv. full-length sequences of the l-and m-segments of uzav have, respectively, 69.3% nt and 64.1% nt identities with those of iskv. amino acid sequences of rdrp (s-segment) of uzav and iskv have 76.2% aa similarity. the similarity of the amino acid sequences of the precursor of gn and gc for uzav and iskv is 66.7% aa. a comparison of the s-segments of uzav and iskv revealed that they are almost identical (99.6%). thus, we can conclude that uzav is a reassortant virus that got an s-segment from iskv. phylogenetic analysis based on l-and m-segments placed uzav in the lineage of iskv (figures 8.10à8 .12). 6, 7 vertebrate hosts. the vertebrate host of uzav is apparently bats, but because only a single isolation was obtained, this assertion is speculative. the finding that uzav is a reassortant virus closely related to iskv suggests that uzav occupies the same ecological niche as iskv and therefore is associated with bats and their argasid ticks. myotis blythii oxygnathus, the bat from which uzav was isolated, is common in the southern parts of the russian plain and in western siberia, caucasia, kazakhstan, southern europe, northern africa, middle and central asia, iran, and iraq. bats are important natural reservoir of emerging viruses. 8à11 iskv and uzav are the first nairoviruses that appear to be associated with bats. sakhalin virus (sakv) has been isolated from ixodes (ceratixodes) uriae (family ixodidae, subfamily ixodinae) ticks, which are obligate parasites of auks (family alcidae). the prototypical strain of sakv (leiv-71c) was isolated in 1969 from i. uriae ticks collected in a colony of the common murre (uria aalge) on tyuleniy island near the southeastern coast of sakhalin island in the sea of okhotsk (48 29 0 n, 144 38 0 e; figure 8 .19). 1à4 subsequently, 52 strains of sakv were isolated from i. uriae ticks on tyuleniy island and iona island in the sea of okhotsk, the commander islands in the barents sea, and the southeastern coast of the chukotka peninsula in the bering strait (table 8 .11). 4à7 on the basis of virion morphology, sakv has been classified into the bunyaviridae family. sakv was the first of the eponymous viruses, which together have formed a basis for the nairovirus genus. 8 paramushir virus (pmrv), prototypical strain, leiv-2268, a virus of the sakv group, was originally isolated from ixodes signatus ticks collected in 1972 in a colony of cormorants (phalacrocorax pelagicus) on paramushir island (in the kuril islands) (50 23 0 n, 155 41 0 e; figure 8 .19). 9,10 later (in 1972à1987), 18 strains of pmrv were isolated from i. uriae ticks, collected in the nests of auks (family alcidae) on tyuleniy island in the sea of okhotsk and on the commander islands in the bering sea (table 8 .11). 11à14 at least five nairoviruses are included in the sakv group. 3,10,15à17 avalon virus (avav), which was isolated from engorged imagoes and nymphs of i. uriae collected in l. argentatus nests on great island, newfoundland, , in 1972, is apparently identical to pmrv. 15, 18 several strains of avav were isolated in 1979 in cap sizun, brittany, france. 19 clo mor virus (cmv) was isolated in 1973 from nymphal i. uriae ticks collected in a uria aalge colony of clo mor, cape wrath, scotland. 20 cmv was found to be closely related to sakv in a complementfixation test. two strains of cmv were isolated from i. uriae collected in seabird colonies on lundy island (england) and the shiant isles (scotland) ( table 8.12) . 18, 20 rukutama virus (rukv) (strain leiv-6269s), which previously had been included in the sakv group, is now classified into the uukuniemi virus (uukv) group in the phlebovirus genus. 9, 21 taxonomy. complete genomes of sakv (strain leiv-71c) and pmrv (leiv-1149k) were sequenced. 9 also, partial sequences of rdrp of tillamook virus (tillv, identical to sakv), isolated from i. uriae ticks on the pacific coast (oregon) of the united states, are available (table 8 .12). 18 a full-length genome comparison showed that sakh and pmrv respectively share 75.6% nt and 88.0% aa identities in rdrp (l-segment), 59.7% nt and 57.9% aa in the precursor of gn and gc (m-segment), and 62.3% nt and 62.2% aa in the nucleocapsid protein (s-segment). sakv n-protein ranges from 30% (casv, hugv) to 43% (cchfv) similarity to other nairoviruses. the similarity of rdrp and the precursor of gn and gc proteins of sakv to other nairoviruses ranges from 42.8% (casv, hugv) to 50.8% (cchfv), respectively, and from 25.9% (ervev, tfav) to 28.9% (nsdv, dugv), respectively. 9 arthropod vectors. it has been shown that the infection rate of infected ixodes uriae imagoes is 2 times higher than of the species' nymphs and 10 times higher than that of the larval stage. transovarial transfer of sakv has been found to be 10%. the infection rates of male and female ticks are approximately the same. the hypostome of male i. uriae ticks is vestigial; therefore, they cannot be infected by breeding on infected birds. the infection rate of i. uriae imagoes is at least 20 times higher than that of i. signatus imagoes. 4à6,22,23 some other species of ixodes ticks are parasites of seabirds and may be an additional reservoir of sakv. i. auritulus and i. zealandicus ticks are distributed from alaska to cape horn in south and north america. 24 laboratory experiments have demonstrated that aedes aegypti and culex pipiens molestus mosquitoes can be infected by sakv as they suck blood. the virus was found in mosquitoes on 9, 14, and 19 days after infection in titers 1.0, 1.5, and 2.0 log 10 (ld 50 )/10 μl, respectively. however, it was shown that infected mosquitoes could not transmit the virus to mice through a bite. 6, 22 vertebrate hosts. ixodes uriae ticks and their host, the common murre (uria aalge), are a natural reservoir of sakv. pelagic cormorants (phalacrocorax pelagicus) and their obligate parasites (i. signatus) likely have only an additional influence. antibodies to sakv have been found in the common murre (u. aalge), pelagic cormorants (p. pelagicus), fulmars (fulmarus glacialis), tufted puffins (lunda cirrhata), and black-legged kittiwakes (rissa tridactyla) in the far east. 4à6,22 a serological examination of birds via an indirect complement-fixation test revealed that the northern boundary of the range of sakv is the commander islands, where antibodies have been found in 2.2% of birds. the southernmost place where antibodies have been detected (1.1% birds) is kunashir island in the kuril islands. antibodies were found most often (in 4.1à17.8% of birds) in the central part of the basin of the sea of okhotsk (on sakhalin island, tyuleniy island, and iona island). antibodies were also found in the red-necked phalarope (phalaropus lobatus), sanderling (calidris alba), the long-toed stint (c. subminuta) (up to 8.4% of the population), fulmars (f. glacialis) (4.9%), leach's petrels (oceanodroma leucorhoa), tufted puffins (l. cirrhata) (4.6%), the common murre (u. aalge) (3.8%), japanese 5, 6, 22 neutralizing antibodies to avav, a virus closely related to pmrv, have been found in 27.6% of puffins (fratercula arctica), petrels (calonectris leucomelas), and herring gulls (larus argentatus) in canada. 24, 25 findings of antibodies to sakv in seabirds carrying out their annual seasonal migration to the southern hemisphere suggest the possibility of transcontinental transfer of the virus to the southern hemisphere. the closely related taggert virus (tagv) was isolated from ixodes uriae ticks in penguin colonies on macquarie island, a phenomenon that may indicate a transfer of viruses by birds and their ticks between the northern and southern hemispheres. human infection. three human cases of cervical adenopathy associated with avav were described in france. 25 serological examination of farmers in cap sizun, brittany, france, found only 1% of the population positive. 18 history. tamv (prototypal strain, leiv-1308uz) was originally isolated from hyalomma asiaticum asiaticum (family ixodidae, subfamily hyalomminae) ticks collected from sheep in the arid landscape near the town of tamdybulak subsequently 52 strains of tamv were isolated in uzbekistan, 4à7 turkmenistan, 8à11 kyrgyzstan, 12,13 kazakhstan, 11, 14, 15 armenia, 6, 16 and azerbaijan 8,17à19 in 1971à1983 (table 8.13) . most of the strains were obtained from h. asiaticum ticks, but several were isolated from birds, mammalians (including bats), and sick humans. on the basis of virion morphology, tamv has been classified into the bunyaviridae family. serological studies by complement-fixation and neutralization tests revealed no antigenic relationships of tamv with any known viruses. 2 taxonomy. three strains of tamv isolated in uzbekistan (leiv-1308uz), armenia (leiv-6158ar), and azerbaijan (leiv-10226az) were completely sequenced. 20 phylogenetic analysis of the full-length sequences showed that tamv is a novel member of the nairovirus genus, forming a distinct phylogenetic lineage (figures 8.10à8.12 ). the similarity of the amino acid sequence of tamv rdrp (l-segment) with those of other nairoviruses is 40% aa, on average. the similarity of the rdrp of tamv with that of the nairoviruses associated predominantly with ixodid ticks (cchfv, hazara virus (hazv), and dugv) is higher (40% aa) than that with viruses associated with argasid ticks (iskv and casv) (38% aa). the similarity of the tamv polyprotein precursor of cn and gc with that of other nairoviruses is less than 25% aa. the similarity of the amino acid sequence of the nucleocapsid protein (s-segment) of tamv is 33% aa with ixodid nairoviruses and 28% aa with argasid nairoviruses. phylogenetic analysis of the catalytic core domain of the rdrp of the nairoviruses confirms that tamv forms a novel group in the nairovirus genus (figures 8.10à8.12 ). 20 genetic diversity among the three sequenced strains of tamv is low. the prototypic strain leiv-1308uz, isolated in central asia, has 99% nt identity in the l-segment with leiv-10226az from transcaucasia. the l-segment of the strain leiv-6158ar has 94.2% nt and 96.3% aa identity with the l-segment of leiv-1308uz. the similarity of the m-segment of leiv-1308uz with those of leiv-10226az and leiv-6158ar is 93% nt and 89% aa, respectively. the similarity of the s-segment among the three strains is 93à95% nt. 20 arthropod vectors. h. asiaticum ticks are apparently a main reservoir of tamv. more than half (57%) of tamv isolations were obtained from h. asiaticum asiaticum ticks, 6% from h. asiaticum, 8% from h. anatolicum, 6% from h. marginatum, 6% from rhipicephalus turanicus, and 2% from haemaphysalis concinna. the infection rates of male and female ticks in endemic territory were 1:210 and 1:200, respectively. the infection rate of h. asiaticum nymphs was 20 times lower. 7, 10, 14, 16 furthermore, tamv was isolated from larvae of h. asiaticum, which were hatched from eggs in the laboratory, indicating transovarial transmission of the virus. h. asiaticum asiaticum ticks are the most xerophilous subspecies of the hyalomma genus (ixodinae subfamily), 21 a characteristic that allows tamv to be distributed over the karakum desert in turkmenistan, the moinkum desert in kazakhstan, and the central part of the kyzyl kum desert in kazakhstan and uzbekistan. 7 . animal hosts. the larvae of h. asiaticum feed on ruminants, hoofed animals, small predators, hedgehogs, birds, and reptilians. one of the major hosts of h. asiaticum preimagoes is the great gerbil (rhombomys opimus). wild animals, as well as sheep and camels, are the hosts for h. asiaticum imagoes and may be involved in the circulation of tamv (table 8 .13). human pathology. sporadic cases of the disease associated with tamv was registered in kyrgyzstan in october 1973, when tamv was isolated from the blood of a patient with fever (39 c), headache, arthralgia, and weakness. 16 h. asiaticum asiaticum ticks rarely attack humans, and no outbreaks of tamv fever have been registered; however, human infection by h. asiaticum ticks is still possible concinna (ixodidae, haemaphysalinae) during 1971à1975. 1,2 according to preliminary information, burv is not able to agglutinate erythrocytes of birds and mammals and has no antigenic relationships with 59 arboviruses from different groups of the togaviridae, taxonomy. the genome of burv was sequenced, and the virus was classified into the nairovirus genus, family bunyaviridae. the genome consists of three segments: an l-segment (orf, 11,919 nt; encodes rdrp); an m-segment (orf, 4,035 nt; encodes a polyprotein precursor of the envelope proteins gn and gc); and an s-segment (orf, 1,482 nt; encodes the nucleocapsid protein n). 3, 4 a comparison of rdrp sequences of burv with those of other nairoviruses demonstrated that the virus is distantly related to tamv (59% aa similarity). the similarity of the rdrp catalytic core domain of burv to that of tamv is 82% aa, compared with about 60% aa for viruses in other phylogenetic groups. the level of similarity for the nucleotides sequences of this part of the rdrp of burv is 68% nt with those of tamv and 45à50% nt with those of other viruses (figure 8 .10). 3 the m-segment of burv has a long orf and encodes a polyprotein precursor of the envelope glycoproteins gn and gc. 4 the size of the polyprotein precursor is 1,344 aa. the mature gn and gc proteins of nairoviruses are formed by complex processes involving cellular peptidases. by the netnglyc 1.0 server, 11 potential glycosylation sites were predicted, with only 5 within mature gn or gc proteins. 5, 6 the level of similarity of the amino acid precursor of gn and gc in burv is 45% with that of tamv and no more than 27% with viruses of other phylogenetic groups. phylogenetic analyses based on a comparison of the full-length polyprotein precursor demonstrated the position of burv on the tamv branch and was consistent with the rdrp data ( figure 8 .11). 3 the s-segment of nairoviruses encodes a nucleocapsid protein (n). 4, 7 the size of the burv nucleocapsid protein is 493 aa, corresponding to the average size of the n protein of other nairoviruses (480à500 aa). the level of similarity of the amino acid sequence of burv n protein with that of tamv is 44%, and that with the amino acid sequences of other nairoviruses is30à32%. phylogenetic analyses of burv n protein are represented in figure 8 .12. the phylogenetic position of burv is on the tamv branch, despite the virus's having the lowest level of similarity of the n protein compared with that of other virus proteins. arthropod vectors. as mentioned earlier, six strains of burv were isolated from the ticks haemaphysalis punctata (five strains) and haem. concinna (one strain) in 1971à1975. the rate of infected ticks was 2.2à2.6%. burv is associated with haem. punctata and haem. concinna ticks in pasture biocenoses. the virus is phylogenetically close to tamv, which is also associated with ixodes ticks in pasture and desert biocenoses. 8 the orthobunyavirus genome consist of three segments of single-stranded negative-sense rna designated as large (l), medium (m), and small (s) (figure 8 .22). 1 the l-segment of the prototypical bunv (6,875 nt in length) encodes the viral rdrp. 2 the m-segment (4,458 nt) encodes two surface glycoproteins (gn and gc) and a nonstructural protein (nsm). 3, 4 the s-segment (961 nt) encodes the nucleocapsid protein (n) and a nonstructural protein (nss). the nss protein is considered a pathogenic factor for vertebrates, because it may act as an antagonist of interferon, which is involved in blocking the host's innate immune responses. 5à7 6, 7 to olyka virus, isolated in 1973 from an. maculipennis mosquitoes collected in western ukraine; 8à11 and to chittoor virus, isolated in 1957 from an. barbirostris mosquitoes collected in brahmanpally, chittoor district, andhra pradesh state, india. 12 the african ngari virus (nriv) is reassortant between batv and bunv. 12, 13 in russia, batv was repeatedly isolated in different regions (figure 8 .23). anadyr virus (anadv), strain leiv-13395, was isolated by s.d. lvov from a pool of aedes mosquitoes collected in september 1986 in a swamp tundra landscape near the village of ukraine, and russia are members of the european group. two strains of batv-leiv-ast04-2-315 and leiv-ast04-2-336-isolated in russia were completely sequenced and placed into the cluster of the european strains. 14 within this group, they are phylogenetically close to strain 42, isolated in the volgograd region in 2003 from anopheles messeae (maculipennis) mosquitoes, for which the partial nucleotide sequences of the l-and m-segments are known. between the strains leiv-ast04-2-315 and leiv-ast04-2-336, there is very high level of nucleotide and amino acid identity of three segments of the genome: 99.6/99.0% (l-segment/rdrp), 99.9/100.0% (m-segment/ polyprotein predecessor), and 99.7/100.0% (s-segment/nucleocapsid). the levels of nucleotide identity of strain 42 with these strains on partial sequences of l-and m-segments are 98.6/98.8% and 100/100%, respectively; that is, for the m-segment, all available nucleotide polymorphisms are synonymous. the lowest observed genetic differences and the temporal and geographical proximities of the various strains of these viruses suggest a common origin as different isolates of the same strain of batv circulating in the southern part of russia. phylogenetic analysis of anadv (strain leiv-13395) revealed its similarity to batv. the l-segment of anadv is from 76.5% to 79.7% identical with those of the different batv strains (figure 8 .26, table 8 .15). the identity of the l-segment of anadv with the l-segments of other viruses of the bunyamwera group is 73.5% (bunv), 74.1% (cvv), and 73.9% (tenv). the amino acid and is about 82.5% with tenv and cvv. the amino acid similarity of the nucleocapside protein is 98.7% with that of batv from uganda. phylogenetic analysis of the nucleotide sequences of the s-, m-, and l-segments conducted with the use of a maximum-likelihood algorithm placed anadv (leiv-13395) on a distinct branch of the dendrogram that considers it a new representative of the bunyamwera group. arthropod vectors. batv has been reported in sudan, africa. 15 the distribution of batv in southeastern asia includes malaysia, india, sri lanka, thailand, cambodia, and japan, 5,16 while in europe batv is distributed over austria, germany, yugoslavia, moldova, ukraine, belarus, and other countries. 2,17à19 in central europe, batv was isolated from anopheles claviger, an. maculipennis (an. messeae), coquillettidia richiardii, aedes (ochlerotatus) punctor, and ae. communis. 6, 7, 20 a wide distribution of batv in different landscape belts of the european part of russia, as well as in siberia and the far east, was demonstrated: in the temperate belt the main source of batv isolation was the zoophilic anopheles genus, whereas in high latitudes (tundra, northern taiga) it was the aedes genus. 18,21à24 in the european part of russia, batv has been isolated in the northern (komi republic), middle (vologda region), and southern (leningrad, yaroslavl, and vladimir regions; 2, 6, 17, 18, 20 in the southern hyperendemic regions of russia, the main vector of batv is an. messeae. according to our data, the infection rate of an. messeae in the middle belt of the volga delta (astrakhan region) reaches 0.188% (approximately 1 infected mosquito out of 500). because this species of mosquito attacks mainly domestic animals, it serves as a biological barrier, reducing risk of infection to humas. in the northern areas (the subarctic, the northern taiga), batv circulation is due mainly to aedes mosquitoes: ae. communis complex and ae. punctor. under experimental conditions, batv was isolated from hibernating females of an. messeae. hibernation is one of the mechanisms by which batv survives during the winter. 20, 25 vertebrate hosts. in anthropogenic biocenoses of the southern regions of russia, domestic animals are the main vertebrate reservoir, because they (especially cattle) are the main hosts for an. messeae. batv-neutralizing antibodies were found in india among rodents (mus cervicolor (55.2%), rattus exulans (36.4%), rattus rattus (19.5%), bandicota indica (15.5%)) and bats (cynopterus sphinx) (2.6%). 2, 5 this indicator is significantly higher in india among domestic animals: goats (41.8%), camels (100%), cows (60.9%), and buffalos (23.3%). in finland, anti-batv antibodies occasionally were found among cows (0.9%), but not among reindeers. 19 the chittoor strain is associated with mild illness, but is pathogenic to sheep and goats. 12 batv was isolated from birds: crows (corvus corone), coots (fulica atra), and grey partridges (perdix perdix). 9 persistent avian infection was established experimentally with reactivation of viremia by cortisone six months after the acute infection period. 10 an investigation of 5,000 sera of domestic animals in russia during 1982à1992 revealed anti-batv antibodies among these animals significantly more often than among people (table 8 .16). the largest immune layer was found in populations of horses (up to 80%), cattle (35à60%), sheep (up to 80%), and camels in forestàsteppe, semidesert, and desert landscape belts. in contrast to the situation in finland, antibodies were found in reindeer sera in a tundra landscape belt of the chukotka peninsula. no examinations of vertebrates in natural biocenoses were conducted. epidemiology. epidemic outbreaks and sporadic cases caused by batv, as well as outbreaks of hemorrhagic infection caused by ngari virus, have been reported. 13, 15, 18, 26, 27 to date, no cases of laboratory infection are known. according to a serological examination of 10,000 people in the endemic regions of russia, about 3à10% withstand batv infection in an asymptomatic form. the highest infection rate was established in forestàsteppe and steppe belts. (however, as a rule, the rate is higher for domestic animals than humans.) some northern areas in russia became hyperendemic for no apparent reason. 18, 21, 22 pathogenesis. no pathogenetic mechanism during batv infection in humans has yet been described in detail. there are experimental data, however, on batv infection in primates: 28 green monkeys (chlorocebus sabaeus) were found to be carriers of the virus 50 days after inoculation (the observation period); the virus was pantropic, destroying small vessels and producing vasculitis and perivascular focal lymphohistiocytic infiltrates. clinical features. the disease etiologically linked with batv proceeds mainly as influenzalike disease complicated by meningitis, malaise, myalgia, and anorexia. 13, 15, 18, 26, 27 at the same time, ngari virus (reassortant between batv and bunv) infection in east africa appears as outbreaks of hemorrhagic fever. 13 diseases associated withtheclosely related ilev in africa and madagascar also proceed with hemorrhagic phenomena and with lethal outcomes. 29, 30 diagnostics. a highly specific test based on rt-pcr has been developed, as have elisa tests for the detection of specific anti-batv igm and igg. 24 genome and taxonomy. the genome of the ce group of viruses consists of three segments of ssrna with negative polarity. the l-segment of lacv, a prototypical virus of the group, is 6,980 nt in length, the m-and s-segments 4,527 and 984 nt, respectively. as in other bunyaviruses, the l-segment encodes rdrp, the m-segment a polyprotein precursor of the envelope glycoproteins gn and gc, and the s-segment nucleocapsid protein (n). two nonstructural proteins are found in infected cells: nss, which encodes by adding an orf in the s-segment; and nsm, which forms during the maturation of the gn and gc proteins from the precursor. 11 25, 26 but viruses of the ce serocomplex were isolated from ae. albopictus (a known vector for at least 22 arboviruses), which was imported from southeastern asia and spread into 30 states of the united states. 27, 28 transovarial transmission was established in ae. vexans 29 and cs. annulata. 16 overwintering of tahv was documented in cx. modestus and cs. annulata females. 16 mosquito species have been defined and classified only partially in connection with the huge volume of this laborious work. the majority of strains were isolated from pools of mosquitoes belonging to different species. of 250 strains that were isolated (1 strain was isolated from a wild population of the common house mouse, mus musculus), only 112 were isolated from strictly defined species (table 8 .18). the other 138 strains were isolated from aedes mosquitoes of unidentified species: 34% of strains were from ae. communis, 18% from the mixed pools, in which ae. communis prevailed. strains were isolated from other species significantly less often. only one strain was isolated from anopheles maculipennis (an. messeae) and culiseta alaskaensis. 30 the dynamics of the seasonal infection rate of mosquitoes was investigated for two years on the model of the northern part of the russian plain and the eastern part of fennoscandia. in tundra, the epizootic period begins with the second decade of july and proceeds to the beginning of august, when the activity of mosquitoes comes to an end. in forest tundra, the epizootic period begins with the first decade of july and proceeds for 1.5 months; in the northern taiga, this period lasts at least 2 months (julyàaugust); in the middle and southern taiga, the first strains began to be isolated in the second decade of june. the mosquito infection rate increases significantly in the third decade of july and reaches a maximum in the middle to end of august, when the total number of mosquitoes decreases. 30, 31 the data collected testify to an almost universal distribution of ce serocomplex viruses in all landscape belts, except the arctic, in all six physicogeographical lands examined in the north of russia, 32 located on a territory of more than 10 million km 2 . the infection rate of mosquitoes increases (р , 0.01) in moving from the subarctic (tundra) (0.0090 6 0.0018%) to the landscape belt of the middle taiga (0.0196 6 0.0020%). this indicator in tundra and in the forest tundra is close to that in the southern taiga of the russian plain (0.0122%), in north america (0.01%), and in the forest steppes of the russian plain (0.0100à0.0017%). in the steppe belt of the russian plain, the infection rate of mosquitoes appeared to be the smallest (0.001%). in the leaf forests of the russian plain (0.0148%) and of the former czechoslovakia (0.0210%), the infection rate of mosquitoes is comparable to that for landscape belts of the northern and middle taiga. to date, at least s63 ce serocomplex virus strains were isolated from mosquitoes in the central and southern parts of the russian plain. among them, 4 strains were isolated from the blood and spinal fluid of patients, and 3 strains from the internal parts of rodents (2 from the bank vole, myodes glareolus; and 1 from the wood mouse, apodemus sylvaticus). the infection rate of mosquitoes depends on the landscape belt and the particular season in which field material was collected. the rate decreases, as a rule, from the north to the south. data indicating an absence of viruses in semideserts can be explained by an insufficient quantity of mosquitoes collected, but in wet subtropical zones in azerbaijan ce serocomplex viruses were isolated from anopheles hyrcanus. 33 in the southern taiga belt and mixed forests, the infection rate of mosquitoes was defined to be from the third week of may to the second week of august and two peaks were noted: at the end of june (the emergence of the first generation of aedes mosquitoes) and at the end of july to the beginning of august (the emergence of the second generation of aedes mosquitoes). in the majority of the southern belts, the infection rate was registered from the second week of june until the end of august with a small peak in the first week of august caused by the emergence of the second generation of aedes mosquitoes and by the peak of activity of culex, coquillettidia, and anopheles mosquitoes. 17 in steppe and forestàsteppe belts, ce serocomplex viruses were isolated from mosquitoes collected in the rostov and orenburg regions, as well as in the foothills of the caucasus mountains (krasnodar krai). most of the strains were obtained from aedes mosquitoes, which play the leading role in virus circulation. in these regions, anopheles mosquitoes join the virus population maintenance (three strains were isolated), being ecologically connected with agricultural animals and, because of that connection, playing an important role as an indicator species in anthropogenic biocenoses. in the center and south of the russian plain, there is a mix of populations of inkv, tahv, khtv. 31, 32 vertebrate hosts. the principal vertebrate hosts of tahv in europe are lagomorpha (hares (lepus europaeus), rabbits (oryctolagus cuniculus), hedgehogs (erinaceus roumanicus), and rodents (rodentia)). experimental viremia has been established in lagomorphs, hedgehogs, ground squirrels (citellus citellus), muskrats (ondatra zibethicus), squirrels (sciurus vulgaris), martens (martes foina), polecats (putorius eversmanni), foxes (vulpes vulpes), badgers (meles meles), bats (vespertilio murinus), piglets, and puppies. 14, 15, 34, 35 in total, 251 strains of ce serocomplex viruses were isolated within all landscape belts of all physicogeographical lands (figure 8 .30, table 8 . 19 ). according to our data, the susceptibility of mosquitoes increased from the tundra to the northern and middle taiga; however, the highest indicators were noted to be in the forestàsteppe and the steppe of western siberia (in altai krai). identification of these strains revealed at least three viruses of the ce complex: 2 strains of tahv, 44 of inkv, and 183 strains of khtv. 30 in all landscape belts east of the yenisei river (central and northeast siberia and the physicogeographical lands bordering the north pacific ocean), only khtv strains have been isolated. west of the yenisei river, inkv strains predominated in the tundra and the forestàtundra of western siberia, whereas khtv prevailed in other landscapes located to the south. in the eastern part of fennoscandia and in the north of the russian plain, inkv and khtv strains were isolated in about equal proportions. 30 the pattern of distribution of tahv, inkv, and khtv over northern eurasia suggests that the emergence of the ancestor of ce serocomplex viruses probably is connected to oligocene chineseàmanchurian fauna of the deciduous forests of eastern siberia evolving into okhotsk fauna during the upper tertiary period. the okhotsk fauna, in its turn, extended in early glacial times to the north, the west, and partially to the east in tundra through ancient beringia and on into north america. the ancestral virus could then penetrate into north america together with this fauna and gradually extend in the southern direction, in the process laying the foundation for the appearance of some other viruses of the ce serocomplex now circulating mainly in north america. mercurator, nigripes, excrucians 1 0.9 maculipennis b 1 0.9 total 112 100 a one strain was isolated from the genus culiseta. b one strain was isolated from the genus anopheles. the introduction of the virus population to the western hemisphere probably occurred through two pathways around the central siberian plateau: (i) through the tundra lying to the north of the plateau and (ii) through southern taiga and forestàsteppe territories. these pathways can explain the modern predominance of khtv in the forestàsteppe belt of siberia and in a taiga belt west of the yenisei river. in moving to other ecological systems further to the west, khtv could have been transformed partially to inkv and tahv. the inkv population penetrated into the western part of the eurasian subarctic through the taiga belt and occupied that part of eurasia, whereas tahv proceeded into the deciduous forests of europe, where it now prevails. 36 epidemiology. cev is endemic in the united states in california, new mexico, texas, the southwestern part of virginia, tennessee, and kentucky. 26, 37 sporadic morbidity with cns lesions occurs in those states, but the main morbidity is linked to lacv, which is endemic in 20 states, predominantly the u.s. census bureauàdefined east north central states (ohio, wisconsin, minnesota, iowa, and indiana), where morbidity reaches 0.1à0.4%. 26 cases of lacv-associated encephalitis are within the distribution of the main vector-aedes triseriatus-eastward from the rocky mountains. 38 during the last few decades, natural foci in west virginia, north carolina, and tennessee, with sporadic cases occurring in louisiana, alabama, georgia, and florida, joined with previously known ones in wisconsin, illinois, minnesota, indiana, and ohio. thus, having traversed the distance from southeastern asia to north america, ae. triseriatus is now part of the north american virus circulation. 39 the clinical picture varies from an acute fever syndrome (in some cases with pharyngitis and other symptoms of acute respiratory disease) to encephalitis. lethality is about 0.05%. from 40 to 100 cases occur annually. generally, the virus attacks children age 10 and under (60%), a phenomenon that may be explained by the existence of a layer of immunity in up to 40% of adults. 40 jcv (in the united states and canada) and sshv (in the northern part of the united states and in canada) are associated with sporadic cases of fever and encephalitis. 26 domestic dogs are susceptible to lacv, which provokes encephalitis. 21, 34, 41, 42 the role of deer in virus circulation has been established as well. horizontal and vertical transmission of viruses provides an active circulation of the virus, a high rate of infection in mosquitoes, and stability of natural foci under the relatively rough conditions of the central and northern parts of the temperate climatic belt. 43 all three viruses (inkv, khtv, and tahv) of the ce serocomplex distributed in eurasia have significance in human pathology. 43, 44 these viruses were found in czechoslovakia in 1959, 4, 45 austria in 1966, 13 finland in 1969, 6, 46 romania in 1974, 12 norway in 1978, 24 the former ussr(in transcaucasia) in 1972, 47 and elsewhere in the european and asian parts of russia. 9,30,32,33,36,44,48à50 in europe, human disease associated with tahv presents as an influenzalike illness mainly in children with sudden-onset fever, headache, malaise, conjunctivitis, pharingitis, myalgia, nausea, gastrointestinal symptoms, anorexia, and (seldom) meningitis and other signs of cns lesions. 16,42,51à56 the circulation of ce serocomplex viruses was established in china, 57 where they provoke human diseases with encephalitis 58 as well as acute respiratory disease, pneumonia, and acute arthritis. 59 in north america (the united states and canada), lacv is the most important of these viruses, 60 but sshv also is associated with human disease. 61 between 1963 and 1981 in the united states, 1,348 cases of ce were reported. 60, 62 so, ce serocomplex viruses have circumpolar distribution. in russia, these viruses are found from subarctic to desert climes ( figure 8 .30, table 8 .18). 32, 44 according to our summary data for 8,732 sera, the number of people with specific antibodies to ce serocomplex viruses in the tundra and forestàtundra belts (27.8%) is significantly lower than the number in the north and middle taiga belts (48% and 47%, respectively). these data correlate with the infection rate of mosquitoes in those landscape belts. 31, 49 results obtained from serological investigation of the human population correlate with those obtained from virological investigation of the mosquitoes (figure 8 .31). the maximum immune layer of the healthy population is registered in the southern taiga. in the landscape and geographical zones located south of that landscape, a gradual decrease in this indicator takes place. specific antibodies to inkv are seen everywhere that this virus circulates. in forest-àsteppes, specific antibodies to tahv and inkv are marked out with an identical frequency. in semideserts, anti-tahv antibodies are found twice as often as anti-inkv ones. the small number of strains isolated in these natural zones precludes establishing a relationship between the circulation of viruses and an immune layer of the population. active circulation of ce serocomplex viruses on the territory of russia results in regular registration of the diseases caused by these viruses. more than 7% of all seasonal fevers are etiologically linked to such viruses, and in some natural zones (the southern taiga and the mixed forests), this indicator increases to 10à12%. in mixed forests, the main etiological role most often belongs to inkv (50.4%), and in semideserts (astrakhan region) to tahv (76.5%). the diseases caused by ce serocomplex viruses in the center and south of the russian plain start appearing during the middle of may and reach a maximum in almost equal titers of specific antibodies to more than one virus were revealed in 65 patients (35.5%) in a neutralization test. 31, 43, 49 diseases were registered from may to september: in may, 22 cases (12.02%); in june, 35 (19.13%); in july, 67 (36.61%); in august, 54 (29.51%); and in september, 5 (2.73%). the seasonal dynamics in all landscape zones were identical: the maximum number of diseases is noted in julyàaugust. diseases were registered everywhere in the form of sporadic cases and small outbreaks, but more often in the taiga and the deciduous forests of the european part of russia and western siberia. most patients were 15à40 years old, with those up to 30 years making up 52.5% of all people infected. 9 pathogenesis. a systematic destruction of small vessels, together with the development of vasculitis and perivascular focal lymphohistiocytic infiltrates, underlies the pathogenesis of the diseases caused by ce serocomplex viruses. lesions in the lungs, brain, liver, and kidneys are the most frequent complications. 31, 49 clinical features. the incubation period lasts from 7 to 14 days, but in some cases is only 3 days. three main forms of disease linked with ce serocomplex viruses have been proposed: (i) influenzalike; (ii) with primary compromise of the bronchiopulmonary system; (iii) neuroinfectious, which proceeds with a syndrome of serous meningitis and encephalomeningitis. analysis of the clinical picture of cases examined showed that 79.8% of cases proceeded without signs of cns lesion, 20.2% with a syndrome of acute neuroinfection, and 8.9% with radiologically uncovered signs of changes in the bronchiàlung system. a comparison of clinical forms and etiologic agents showed that inkv and tahv often cause disease without cns lesions (65.6% and 92.5%, respectively) and that inkv plays the leading role in acute neuroinfection (34.4%). the etiological role of khtv was established in 14 cases without cns symptoms of lesions. 63 eighty-three patients had an influenzalike form of the disease etiologically linked to ce serocomplex viruses. the incubation period was 7à14 days. the disease began abruptly, with a high temperature that reached a maximum of 39à40 c in 98.9% of patients on the first day. the duration of the fever was 4.48 6 0.30 days. one of the main symptoms was an intensive headache (3.62 6 0.26 days in duration) that developed in the first few hours and was often accompanied by dizziness, nausea (31.3%), and vomiting (21.7%). 43,63à66 a survey of patients revealed infection of the sclera (59.0 6 3.4%), hyperemia of the face and the neck (10.8 6 3.4%), and, in some cases (3.6%), a spotty and papular rash on the skin of the trunk and the extremities. violations of the upper respiratory airways were characterized by hyperemia of the mucous membranes of the fauces (95.2 6 2.3%)and congestion of the nose and a dry, short cough (13.2 6 3.7%). with regard to the lungs, 26.5 6 4.8% of patients exhibited rigid breathing a dry, rattling cough during auscultation, and a strengthening of the bronchovascular picture on roentgenograms. among cns symptoms, the most common were a decrease in appetite, a stomachache without accurate localization and with liquid stool, and a small increase in the size of the liver with a short-term increase in aminotransferase activity in the blood. inflammatory changes in the bronchiàlung system (bronchitis and pneumonia) occurred as well. in all cases in which it appeared, pneumonia had a focal character with full the etiological role of different ce serocomplex viruses has been established in 8% of 463 cases with acute diseases of the nervous system (serous meningitis, encephalomeningitis, arachnoiditis, acute encephalomyelitis, and seronegative tick-borne encephalitis (tbe)): inkv (56.7 6 8.1%), tahv (8.1 6 4.5%), and unidentified (35.1 6 7.8%). the age of patients with cns lesions was from 3 to 61 years, with the majority (51.5%) from age 21 to 30. serous meningitis was observed in 29 patients who arrived at the hospital a mean 3.3 days after symptoms appeared. the disease began abruptly. the majority (58.6%) of patients complained of a high temperature that reached a maximum the first day, the duration of the fever was 4.54 6 0.05 days, with a critical (37.9%) or steplike (62.1%) decrease. headache was noted in 100% of patients and was accompanied by dizziness in 31%. vomiting developed on the first (53.6%) or the second (46.4%) day and continued in 67.7% of patients. meningeal signs appeared in 96.5% of patients but were weak and dissociated in most cases, with only 37.9% of patients exhibiting rigidity of the occipital muscles. the duration of the meningeal signs was 3.50 6 0.4 days. the cells of the spinal fluid (investigated on the 4.57th 6 0.54 day of the disease) was lymphocytic, mostly reaching three digits and up to 500 cells (55.6%); the protein concentration was reduced (0.15 6 0.02 g/l) in 41.4% of cases but was within the normal range (0.31 6 0.01 g/l) in other cases. in 34.5% of patients exhibiting acute neuroinfection symptoms of bronchitis and focal pneumonia, their condition was confirmed radiologically. encephalomeningitis caused by inkv was characterized by an abrupt beginning and fast development of focal symptomatology (ataxy, horizontal nystagmus, and discoordination) against a background of common infectious and meningeal syndromes, including inflammatory changes to the spinal fluid. 43,63à66 the variability of the clinical picture of the diseases caused by ce serocomplex viruses and its similarity-especially at early stagesto that of other infections suggest the necessity of carrying out differential clinical diagnostics with a number of diseases. the influenzalike form needs to be differentiated, first of all, from influenza, especially in the presence of symptoms of neurotoxicity, as well as from other acute respiratory diseases (parainfluenza, adenoviral and respiratoryàsyncytial diseases), pneumonia (including a mycoplasma and chlamydia etiology), and enteroviral diseases. the main epidemiological features and clinical symptoms that lend themselves to carrying out differential clinical diagnostics for the influenzalike diseases described here are presented in table 8 .20. note that considerable difficulties arise in implementing differential clinical diagnostics of the diseases that proceed with acute neuroinfection syndrome (serous meningitis, encephalomeningitis), especially when those diseases occur in the same season (tables 8.20 and 8.21). 31, 44, 66 the main criteria in differential clinical diagnostics of the disease etiologically linked with ce serocomplex viruses are as follows (see tables 8.20 and 8.21): acute onset; high short-term fever (4à8 days, on average) reaching a maximum on the first day and decreasing critically at the end of the feverish period; and intensive headache, nausea, vomiting, and weakness. also observed are insignificant catarrhal phenomena (nose congestion, rare dry cough) or their complete absence. a radiograph of the chest reveals signs of bronchitis and focal pneumonia with poor clinical symptomatology. an examination of the liver shows that its size, as well as its aminotransferase activity, has increased. changes in urine, such as albuminuria and, in some cases, cylindruria, are frequently reported. finally, symptoms relating to the vegetative nervous system (hyperemia of the face and the neck, subconjunctival hemorrhage, bradycardia, and persistent tachycardia) can be observed, as can both cns lesions in the form of serous meningitis and encephalomeningitis in combination with compromise of the bronciopulmonary system, liver, and kidneys. diagnostics. specific diagnostics of the diseases etiologically linked with ce serocomplex viruses could be based on virological testing (using sensitive biological models of newborn mice or cell lines to isolate the strains) or on serological testing. in the presence of the sera taken from patients during the acute period of the disease (the first 5à7 days) and in 2à3 weeks, the best method of retrospective inspection is a neutralization test. a hemagglutination inhibition test is considerably less sensitive. both complement-binding reactions and diffuse precipitation in agar have no diagnostic value today. for serological reactions, it is necessary to utilize hktv, tahv, and inkv antigens simultaneously. (in reference labs, sshv antigen should be used as well.) a quadruple (or greater) increase in the titers of specific antibodies or the detection of specific antibodies in the second serological test in their absence in the first test are diagnostic criteria. elisa for igg indication and monoclonal antibody capture elisa (mac-elisa) for igm indication provide good diagnostic opportunities. control and prophylaxis. supervision of morbidity and of the activity of natural foci linked with ce serocomplex viruses offers the following instructions: (i) monitor the patient clinically and the disease epidemiologically. (ii) provide well-timed diagnostics and seroepidemiological investigations. (iii) track the number and specific structure of mosquito vectors and possible vertebrate hosts. history. khurdun virus (khurv), strain leiv-ast01-5 (deposition certificate n 992, 04.11.2004, in the russian state collection of viruses), was isolated from a pool of internal parts of the coot (fulica atra; order gruiformes, family rallidae), collected august 3, 2001 , in natural biomes in the western part of the volga river delta, in khurdun tract, ikryaninsky district, astrakhan region. 1 later, nine more strains of khurv were isolated from f. atra and the cormorant phalacrocorax pygmaeus; order pelecaniformes: family phalacrocoracidae) in 2001à2004 (figure 8 .32). at least six viruses associated with birds have been shown to circulate in the volga river estuary. 2,3 khurv has not been identified by any serological method, 1 including sera against viruses of the flaviviridae, togaviridae, bunyaviridae, and orthomyxoviridae families. 4 taxonomy. the genome of khurv was sequenced, and phylogenetic analysis revealed that it is a new representative of the orthobunyavirus genus (figures 8.33à8.35). 5 the genome consists of three segments of ssrna with negative polarity-an l-segment (6,604 nt), an m-segment (3,161 nt), and an s-segment (950 nt)-and has only 25à32% identity with those of other orthobunyaviruses. the terminal 3 0 -and 5 0 -sequences of khurv genome segments, determined by rapid amplification of cdna ends, are canonical for the orthobunyavirus (3 0 -ucaucacaug and cgtgtgatga-5 0 ). 6 the l-segment of khurv has a single orf (6,526 nt) that encodes rdrp (2,174 aa). the similarity of khurv rdrp with those of the orthobunyaviruses is 32%, on average. the similarity of the conservative polymerase domain iii (a, в, c, d, and e motifs) 7 in rdrp reaches 62% (in bunv). the м-segment of khurv is shorter than those of the orthobunyaviruses (3,161 nt vs. 4,451 nt for bunv). the м-segment of khurv has a single orf (2,997 nt), which encodes a polyprotein precursor (998 aa) of the envelope glycoproteins gn and gc. apparently, the m-segment of khurv does not contain a nonstructural protein nsm, which is common in most of the orthobunyaviruses. 8, 9 the putative cleavage site between gn and gc of khurv was found in position 319/320 aa (asa/en). this site corresponds to the cleavage site between nsm/gc of the orthobunyaviruses and the conservative amino acid a/е (vaa/ee in bunv). the size of the gn protein of khurv is the same as that of the other orthobunyaviruses, 320 aa. the similarity of khurv gn is 23à29% aa, on average, to that of the other orthobunyaviruses (28.5% aa to bunv). the size of the gc protein of khurv, 679 aa, is shorter than that of the other orthobunyaviruses (cf. 950 aa for the gc protein of bunv). the c-part (approx. 500 aa) of the gc protein, which includes the conservative domain g1 (pfam03557), has about 30% aa similarity to the c-part in the other orthobunyaviruses, whereas the n-part (approximately 170 aa) has no similarity to that of any proteins in the genbank database. the s-segment of khurv is 950 nt in length and encodes a nucleocapsid protein (227 aa). the similarity of the n protein to that of the orthobunyaviruses is 22à26 aa%. most orthobunyaviruses have an additional orf that encodes arthropod vectors. there are no known arthropod vectors of khurv; the virus has been isolated only from birds. more than 20,000 aedes, culex, and anopheles mosquitoes were examined during the survival period for arboviruses in this region, and no khurv isolations were obtained. the family ceratopogonidae of biting midges is a potential vector of khurv, but these insects have not been surveyed. vertebrate hosts. all isolations of khurv were obtained from birds. nine strains of the virus were isolated from coots (fulica atra). (one hundred seventeen birds were examined and were found to have an infection rate of 8.5%.) one strain was isolated from the pygmy cormorant (phalacrocorax pygmaeus). (two hundred eighty-nine cormorants, mostly ph. carbo, were examined and were found to have an infection rate of 0.3%.) the phlebovirus genus comprises about 70 viruses that are divided into two main groups based on their ecological, antigenic, and genomic properties: mosquito-borne viruses and tick-borne viruses. 1, 2 the genome of the phleboviruses consists of three segments of ssrna with negative polarity: l (about 6,500 nt), m (about 3,300à4,200 nt), and s (about 1,800 nt) (figure 8 .36). in general, the structure of the genome is the same for mosquito-borne and tick-borne phleboviruses, but the m-segment is shorter in tick-borne viruses and it does not encode the nonstructural protein nsm. 3 phylogenetically, the phleboviruses can be divided into two branches in accordance with their ecological features. the tick-borne phleboviruses comprise viruses of the uukuniemi group, the bhanja group, and the two novel related viruses severe fever with thrombocytopenia syndrome virus (sftsv) and heartland virus (hrtv), which form separate clusters and are unassigned to any group (figures 8.37à8.39). the uukv serogroup currently comprises 15 viruses, but the status of some of them may be revised with the accumulation of more genomic and serological data. history. bhanja virus (bhav) was originally isolated from haemaphysalis intermedia ticks that were collected from a paralyzed goat in the town of bhanjanagar in the ganjam district in the state of odisha, india, in 1954 and was assigned to the unclassified bunyaviruses. 1 in europe, the first isolation of bhav was obtained from adult haem. punctata ticks collected in italy (1967) and then in croatia and bulgaria. 2,3,4 palma virus (palv), a virus closely related to bhav, was isolated from haem. punctata ticks in portugal. 5 two viruses-kismayo virus (kisv) and forécariah virus (forv)-antigenically related to bhav were isolated in africa. 6, 7 these viruses have been merged into the bhanja group on the basis of their serological cross-reactions. 8, 9 in transcaucasia, bhav (strain leiv-1818az) was isolated from ixodidae ticks rhipicephalus bursa collected from cows in ismailli district, azerbaijan, in 1972 ( figure 8 .40). closely related to bhav, razv (strain leiv-2741arm) was isolated from ixodid ticks dermacentor marginatus collected from sheep near the village of solak in the razdan district of armenia ( figure 8 .40). 10, 11 serological methods (detection of antibodies in animals and humans) have shown that bhav circulates in many mediterranean countries, the middle east, asia, and africa. 12, 13 taxonomy. viruses of the bhav group are not antigenically related to any of the other bunyaviruses, but they were assigned to the phlebovirus genus on the basis of a genetic analysis of their full-length genome sequences. 14, 15, 16 weak antigenic relationships were found between bhav and sftsv, a novel phlebovirus isolated in china. 16, 17, 18 sftsv, in its turn, is antigenically related to viruses of the uukuniemi group. 19 the genomes of certain viruses of the the m-segment of bhav (3,307 nt) encodes a polyprotein precursor (1,069 aa) of the envelope glycoproteins gn and gc. like the m-segments of other tick-borne phleboviruses, that of bhav has no nsm proteins that are common to mosquitoes-borne phleboviruses. the predicted cleavage site between gn and gc proteins has been found by signal ip software (http://www.cbs.dtu.dk/services) to be in position 559/560 of the polyprotein precursor (motif mhmalc/cdesrl). a dipeptide cd in the cleavage site is also typical for sftsv and hrtv, which were associated with human disease in china and the united states, respectively. 17, 18, 22 other phleboviruses, including uukv and rvfv, contain a dipeptide cs in this position. the s-segment (1,871 nt) of bhav has two orfs (n and nss proteins) disposed in opposite orientations (an ambisense expression strategy) and separated by an intergenic spacer (139 nt) . the similarity of the nucleocapsid vertebrate hosts. the ungulates, including domestic cows, sheep, and goats, are apparently involved in the circulation of bhav. 24 usually, bhav infection in adult animals is asymptomatic, but it is pathogenic to young ones (lamb, calf, suckling mouse), causing fever and meningoencephalitis. 13 ,25à27 experimental infection of rhesus monkeys by bhav induced encephalitis. 28 several strains of bhav were isolated from the four-toed hedgehog (atelerix albiventris) and the striped ground squirrel (xerus erythropus) in africa. antibodies have been detected in dogs, roe deer (capreolus capreolus), and wild boars (sus scrofa). 12 human pathology. bhav infection in human is mainly asymptomatic, but several cases of fever and meningoencephalitis caused by bhav have been described. 29à31 history. gissar virus (gsrv) was isolated from argas reflexus ticks collected in a dovecote in the town of of gissar in tajikistan (38 40 0 n, taxonomy. the genome of gsrv (strain leiv-5995taj) has been sequenced. 4 phylogenetic analysis shows that gsrv is a member of the phlebovirus genus of the uukuniemi group (figures 8.37à8.39 ). gsrv is closely related to grand arbaud virus (gav), which was isolated from a pool of argas reflexus ticks collected in a dovecote near gageron in arles in the rhô ne river delta in the camargue region of france in 1966. 5 gav is classified as virus belonging to the uukuniemi group. 6 the identity of the nucleotide and amino acid sequences of gsrv and gav is 76% nt for the s-segment (94% aa for the nucleocapsid protein), 73% nt for the m-segment (82% aa for the polyprotein precursor of gn/gc), and 76% nt for the l-segment (87.5% aa for rdrp). arthropod vectors. regardless of their geographical distribution, gsrv and gav occupy a narrow ecological niche associated with ticks (argas reflexus) and birds (most likely, pigeons (columbidae)). in laboratory experiments, gsrv reproduced in a. reflexus ticks in 30 days with titers up to 2.0 log 10 (ld 50 )/20 mcl. 7 the distribution of argas reflexus ticks is limited between 51 n on the north and 31 n on the south. the a. reflexus metamorphosis cycle is about three years. the ticks inhabit pigeons' habitats, which are also used by other birds, such as swallows and swifts. a. reflexus larvae were found in europe on a rock swallow (ptyonoprogne rupestris), in egypt on a little owl (athene noctua), in israel on a rock dove (columba livia) and a fan-tailed raven (corvus rhipidurus), and in crimea on the western jackdaw (corvus monedula). the mass reproduction of mites in a dovecote has a negative impact on pigeons' bereeding behavior. worse, at night the ticks can go down to the living space and bite people if the dovecote is built into a house. 8 vertebrate hosts. the main vertebrates involved in the circulation of gsrv are apparently birds, particularly the columbidae. in laboratory experiments, gsrv was isolated from the blood of small doves (streptopelia senegalensis) 5, 9, 22, and 30 days after infection. the virus titer in the blood was 1.5à2.5 log 10 (ld 50 )/20 mcl, on average. serological examination of birds in tajikistan found antibodies to gsrv 2% of doves (columba livia). 7 history. khasan virus (khav) was isolated from haemaphysalis longicornis ticks collected from spotted deer (cervus nippon) in 1971 in the forest in khasan district in the south of primorsky krai, russia (figure 8.43 ). 1 morphologic studies showed that khav belongs to the bunyaviridae family. the virion of khav has structural elements (filaments up to 10 nm) that are typical for uukv, but no antigenic relationships between khav and uukv (as well as zaliv terpeniya virus, ztv) have been found. 1, 2 in a complement-fixation test, khav did not react with serum used in the identification of certain bunyaviruses, so it was categorized in with the unclassified bunyaviruses. 3 taxonomy. the genome of khav (strain leiv-prm776) was sequenced, and the virus was classified into the phlebovirus genus of the bunyaviridae family. 4 the genome of khav consists of three segments of ssrna whose size and orf structure correspond to the size and orf structure of the other tick-borne phleboviruses. a full-length pairwise comparison of l-segments revealed a 53.1% nt identity between khav and uukv and 45.3% between khav and rvfv. the predicted amino acid sequence of rdrp of khav has 48.6% and 35.3% aa identities with uukv and rvfv, respectively. as in other tick-borne phleboviruses, the m-segment of khav does not contain any nsm protein. the similarity between the m-segments of khav and uukv is 45.6% nt, and that between the polyprotein precursors of khav and uukv is 35.9% aa. the s-segment of khav has 35% nt (25.5% aa for the n-protein) identity, on average, with that of the uukuniemi group viruses and 35% nt (27.8% aa), on average, with the mosquitoborne phleboviruses. on phylogenetic trees constructed on the basis of the alignment of full-length genome segments, khav forms a distinct branch external to the uukuniemi group viruses (figures 8.37à8.39 ). at least 14 viruses with unsettled taxonomy are included in the uukuniemi group. 5 some of them can be considered variants of the species uukv, manawa virus (mwav), precarious point virus (ppv), and gav. two tick-borne phleboviruses, sftsv and hrtv, are more closely related to the bhanja group than the uukuniemi group. 6, 7 arthropod vectors. only a single isolation of khav was ever obtained, and the ecology of the virus has not been studied. haemaphysalis longicornis ticks, from which khav was isolated, are distributed in the far east of russia, the northeastern part of china, the northern islands of japan, korea, fiji, new zealand, and australia. 8 haem. longicornis ticks also are the main vector of sftsv (oterwise called huaiyangshan virus, hysv), which caused a large outbreak of febrile illness with a high mortality rate (30%) in 2009 in china. 9 vertebrate hosts. the principal vertebrate host of khav is unknown. khav was isolated from ticks collected on deer. 1 haemaphysalis longicornis ticks are repeatedly found on cows, goats, horses, sheep, badgers, and dogs. 8 history. the sandfly fever virus group includes naples and sicilian subtypes. 1 epidemics of the comparatively mild acute febrile disease of short duration brought on by these viruses in countries bordering the mediterranean have been known since the napoleonic wars. 2 the same disease was common among newly arrived austrian soldiers on the dalmatian coast each summer. 3 experiments conducted by an austrian military commission proved that the disease was caused by a filterable agent in the blood of patients and that the sandfly phlebotomus papatasi can serve as a vector to transmit the disease. 4 during world war ii, epidemics occurred among troops in the mediterranean and two antigenically distinct strains were isolated from the blood of patients in 1943 in sicily and naples. these strains have been designated the sandfly fever sicilian virus (sfsv) and sandfly fever naples virus (sfnv), with prototype virus tosv. 5,6 dr. a. sabin gave a clinical description of the disease and demonstrated that immunity developed to one type of virus does not protect from infection caused by the other type. later, several viruses related to sfnv (anhanga (anhv), bujaru (bujv), candiru (cduv), chagres (chgv), icoaraci (icov), itaporanga (itpv), and punta toro (ptv)) were isolated from humans and rodents in south america. 2, 3, 7 to date, viruses related to tosv have been found in all regions of the world, including the palearctic, neotropical, ethiopian, and oriental zoogeographical regions. 2 the prototype strain of tosv was isolated from phlebotomus papatasi sandflies in 1971 in monte argentario in central italy. 8 two viruses antigenically related to tosv-karimabad virus (karv) and salehabad virus (salv)-were isolated from phlebotomus flies collected in 1959 near karimabad village and salehabad village, respectively, in iran. 9, 10 several related viruses were isolated in the mediterranean: sandfly fever cyprus virus (sfcv; 11 adria virus (adrv, salehabad-like), isolated in saloniki (alternatively, thessaloniki), greece; 12 and massilia virus, isolated near marseilles, france. 13 epidemic outbreaks of sandfly fever whose agents could not be typified occurred in some central asian countries and in crimea during and after world war ii and in turkmenistan after the devastating earthquake of 1948. antibodies to sfsv, sfnv, and karv were found in the blood of humans in tajikistan, azerbaijan, and moldova. 14 antibodies were also found in wild animals in turkmenistan: the great gerbil (rhombomys opimus), the long-clawed ground squirrel (spermophilopsis leptodactylus), and the hedgehog (erinaceus auritus). three strains of sfnv and two strains of sfsv were isolated in 1986à1987 from the blood of patients in afghanistan. 14, 15 taxonomy. the genome of tosv consists of three segments of negative-polarity ssrna: l-segment (6,404 nt in length), m-segment (4,214 nt) and s-segment (1,869 nt). phylogenetic analysis revealed that viruses of the sfnv complex are divided into five genetic clades that differ in their geographical distribution: (i) from africa (saint floris virus and gordil virus (gorv)); (ii) from the western mediterranean (punique virus (punv), granada virus (grv), and massilia virus); (iii) tosv; (iv) viruses from italy, cyprus, egypt, and india; (v) strains from serbia and tehran virus. 16 distribution. sfnv and sfsv are distributed over those areas of the southern parts of europe and asia, and over those areas of africa, which are within the range of the vector. 15 ,17à22 tosv is distributed over italy; spain; portugal; the south of france; slovenia; greece, including the ionian islands: cyprus; sicily; and turkey. 13,17,23à32 both the naples and sicilian strains were isolated from the blood of patients with febrile illness in the vicinity of aurangabad, maharashtra state, in northern india. sandfly virus fever also circulates in western india, as well as in pakistan. 33 the cocirculation of two tosv genotypes was uncovered in the southeast of france. 13, 15, 33 a case of disease associated with tosv befell a tourist returning from elba to switzerland in 2009, and another struck an american tourist returning from sicily the same year. 27 tosv from france is genetically different from that in spain. 3, 13, 33, 34 periodic outbreaks of sandfly fever occurred in the first half of the twentieth century in some central asian republics, transcaucasia, moldova, and ukraine. arthropod vectors. the primary vector of sfnv and sfsv is phlebotomus papatasi; for tosv, the primary vectors are ph. perniciosus and ph. perfiliewi. the viruses can be transmitted by the transovarial route and therefore may not require amplification in wild vertebrate hosts. 35 the infection rate of sandflies can reach 1:220. 36 the active period of phlebotomus in the southern part of europe lasts from may to september. sandflies are peridomestic; the immature stages feed on organic matter in soil and do not require water, but are sensitive to desiccation and therefore are often found in association with humid rodent burrows. vertebrate hosts. the main vertebrates involved in the circulation of sfnv are rodents, particularly the great gerbil (rhombomys opimus) and the long-clawed ground squirrel (spermophilopsis leptodactylus), as well as a hedgehog (erinaceus auritus). the great gerbil is distributed over areas ranging from near the caspian sea to the arid plains and deserts of central asia. the northern border of the animal's distribution is from the 213 8.1 family bunyaviridae mouth of the ural river on northward to the aral karakum and betpak-dala deserts, to the southern coast of lake balkhash, and thence to northern china and inner mongolia. the habitats of rh. opimus are sandy and clayey deserts. tosv was isolated from the brain of the bat pipistrellus kuhlii. 8 animal and human pathology. sandfly virus fever does not cause disease in domestic or wild animals. the hosts of phlebotomus sandflies are usually rodents, which may develop antibodies. over 100 human experimental volunteers were infected at the time of world war ii. 36, 37 the incubation period is between 2 and 6 days, and the onset of fever and headache in those patients was sudden. nausea, anorexia, vomiting, photophobia, pain in the eyes, and backache were common and were followed by a period of convalescence with weakness, sometimes diarrhea, and usually leucopenia. viremia was present 24 h before and 24 h after the onset of fever. 37 tosv was established as the cause of one-third of previously undiagnosed human aseptic meningitis and encephalitis cases examined in central italy. sfcv was associated with a large outbreak in the ionian islands of greece. 28 adrv is associated with serious illness with tonic muscle spasms, convulsions, difficulty urinating, and temporary loss of sight. human disease frequently goes unrecognized by local health-care workers. studies of antibodies in people indicate that the most infections occur in children. when large numbers of unimmunized adults are introduced into an endemic area, the incidence of disease can be high. human exposure to sandflies can be reduced by repellents, air-conditioning, and screens on windows. because sandflies have a flight range of not more than 200 m, human habitats can be constructed at a distance from potential domestic sandflies' breeding places, such as chicken houses and quarters for other farm animals. 19 history. uukv was originally isolated from ixodes ricinus ticks collected in 1959 from cows in southeastern finland. 1,2 antigenically similar isolates (strains leiv-540az and leiv-810az) have been obtained from blackbirds (turdus merula) and i. ricinus ticks collected in the foothills of the talysh mountains in the southeast of azerbaijan in 1968 and 1969, respectively. 3à5 uukv is distributed in the mid-and southern boreal zones of fennoscandia and adjacent areas of the russian plain. twelve strains of uukv were isolated from i. ricinus ticks (the infection rate was 0.5%), and one strain from aedes communis mosquitoes, in landscapes in the mideastern region of fennoscandia. 6, 7 three strains were isolated from i. persulcatus ticks collected in belozersky district, vologda region, russia, in 1979. 8, 9 uukv was also isolated from the mosquitoes ae. flavescens and ae. punctor in the west of ukraine, as well as at the border between poland and belarus. 10, 11 twenty-eight strains of uukv were isolated from i. ricinus ticks collected in lithuania and estonia in 1970à1971. 6,7,12à14 uukv was isolated as well from birds and i. ricinus ticks in western ukraine and belarus. 11, 15, 16 in central europe, uukv was found in the czech republic, slovakia, and poland. 17à20 the prototypical strain leiv-21c of ztv was isolated from ixodes uriae ticks collected in 1969 in a colony of common murres (uria aalge) in tyuleniy island in zaliv terpeniya bay in the sea of okhotsk). 21, 22 in accordance with the results of electron microscopy, ztv was assigned to the bunyaviridae family. complement-fixation testing revealed that ztv is most closely related to uukv, but the two viruses are easily distinguishable in a neutralization test. 21, 22 more than 60 strains of ztv were isolated from i. uriae ticks collected in colonies of seabirds on the shelf and islands of the sea of okhotsk, the bering sea, and the barents sea (table 8 .23, figure 8 .44). 9, 21, 23, 24 two strains of ztv were isolated from i. signatus ticks collected on ariy kamen island in the commander islands, but their infection rate was less than 1:10,000 (,0.01%). 9 a similar virus was found in norway. 25 one strain of ztv (leiv-279az) was isolated from the mosquito culex modestus collected in 1969 in a colony of herons (genus ardea) in the district of kyzylagach in the southeastern part of azerbaijan (figure 8.44 ). 3 natural foci of ztv and uukv associated with bloodsucking mosquitoes (subfamily culicinae) are found in continental areas in the european part of russia, particularly murmansk region. 7 taxonomy. the viruses of the phlebovirus genus can be divided into two main ecological groups: those transmitted by bloodsucking mosquitoes (subfamily culicinae) and midges (subfamily phlebotominae), together called mosquito borne, and those transmitted by ticks (tick borne). uukv is a prototypical virus of the uukuniemi antigenic group, which includes at least 15 related tick-borne phleboviruses (figures 8.37à8.39 ). 26 the genome of uukv consists of three segments of ssrna: an l-segment 6,423 nt long, an m-segment 3,229 nt long, and an s-segment 1,720 nt long. the m-segment of uukv, and indeed, that of all tick-borne phleboviruses, is shorter than the m-segment of mosquito-borne phleboviruses, owing to the absence of the nonstructural protein nsm, which is common in the mosquitoborne phleboviruses. originally, ztv was described as a virus closely related to uukv. a full-length sequence comparison showed that the similarity of ztv to uukv is 77.3% nt identity of the l-segment (90.9% aa of rdrp) and 70.9% nt identity of the m-segment (81.5% aa). arthropod vectors. most isolations of uukv and ztv were obtained from ixodes ricinus and i. uriae ticks, respectively. the infection rates of nymphs and larvae of i. uriae are 5 and 13 times lower, respectively, than that of the imago. these rates indicate a high frequency (8à10%) of transovarial transmission of ztv. 7, 9 probably, ztv has a more pronounced ability to replicate in mosquitoes that are active in the subarctic climate zone (tundra landscapes) in july through the first half of august at temperatures sufficient for the accumulation of virus in the salivary glands. 7 islands. in the murmansk region, which lies to the north of the european part of russia, antibodies were found in 6% of common murres (u. aalge), 4% of black-legged kittiwakes (rissa tridactyla), and 1% of voles (microtus oeconomus). 7, 9 apparently, ruminants could be infected by mosquitoes or by eating fallen birds. on the north coast of the kola peninsula, antibodies were found in 6% of thick-billed murres (u. lomvia), in 7% of blacklegged kittiwakes, and in 1% of voles. 7, 9 in central and eastern europe, a number of vertebrate hosts are involved in the circulation of uukv: forest rodents (myodes glareolus, apodemus flavicollis) and terrestrial passerine birds-the blackbird (turdus merula), pale trush (t. pallidus), ring ouzel (t. torquatus), european robin (erithacus rubecula), hedge sparrow (prunella modularis), wheatear (oenanthe oenanthe), european starling (sturnus vulgaris), carrion crow (corvus corone), magpie (pica pica), brambling (fringilla montifringilla), hawfinch (coccothraustes coccothraustes), yellow bunting (emberiza sulphurata), turtle dove (streptopelia turtur), and ringnecked pheasant (phasianus colchicus). 20,28à32 viremia and long-term persistence of the virus were demonstrated in experimentally infected birds of many species. specific antibodies were detected in cows and reptiles. human pathology. an association was revealed between uukv and different forms of disease, including neuropathy. 33, 34 a serological survey of 1,004 people in lithuania concluded that antibodies existed in 1.8à20.9% of the population. human antibodies to uukv were detected in less than 5% of the human population in central europe 33à35 and 13à14% in belarus. 16 the people living in the tundra landscape had antibodies to ztv in 3.3% of cases, while in the forest no such antibodies were detected (via a neutralization reaction). (table 8 .24). in previous studies, rukv was mistakenly included in the sakhalin serogroup in the nairovirus genus. 1 taxonomy. the genome of komv (strain leiv-13856) and rukv (strain leiv-6269) were completely sequenced, and the two viruses were classified into the phlebovirus genus. 2,3 a full-length comparison showed that the genetic similarity between komv and rukv is 93.0à95.5% nt. among other tick-borne phleboviruses, komv and rukv are most closely related to mwav, which was isolated from argas abdussalami ticks in 1964 in pakistan. 4 the similarities of the genomes of komv and rukv to that of mwav are 67.1% nt for the l-segment (73.0% aa for rdrp), 59.6% nt of the m-segment (58% aa for the polyprotein precursor), and 66.8% nt for the s-segment (58.4% aa for the n-protein). in phylogenetic trees, komv and rukv were placed into the uukuniemi group (figures 8.37à8 .39). 5 the ecology and area of distribution of komv and rukv are the same as those of ztv, which is closely related to uukv. several strains of ztv isolated on the commander islands were sequenced, and no reassortants of ztv with komv were found. 6, 7 arthropod vectors. all isolations of komv and rukv were obtained from ixodes uriae ticks, the obligate parasite of alcidae birds. the commander islands are located on the border of the temperate and subarctic climatic zones, and many different viruses belonging to the bunyaviridae (ztv, sakv, pmrv), flaviviridae (tyuleniy virus, tyuv), and reoviridae (okhv) families have been isolated from i. uriae ticks collected from birds living in colonies there. 8à11 note that the komv infection rate of the i. uriae ticks in the commander islands is 10 times less than the ztv (1:900) and tyuv (family flaviviridae, genus flavivirus) infection rates of the same ticks. vertebrate hosts. the main vertebrate host of komv and rukv is apparently alcidae birds, especially the common murre (uria aalge), but their involvement in the circulation of komv and rukv has not been studied sufficiently. human pathology. uukv group viruses, in general, do not play a role in human infectious pathology, although serological studies have detected antibodies to various viruses of this group in people. the flaviviridae family (from the latin flavus, "yellow," as well as from yellow fever virus (yfv)) includes three genera: flavivirus, pestivirus, and hepacivirus. 1 the flaviviridae are small (40à60 nm) enveloped viruses. the genome is represented by ssrna the flavivirus genus includes more than 70 viruses classified into 15 antigenic groups. 1, 3 the flavivirus virion is spherical (50 nm) and consists of a nucleocapsid (30 nm) and a lipid bilayer envelope covering it. the lipid envelope contains two transmembrane glycoproteins: m (matrix protein, 8 kd) and e (envelope protein, 50 kd). the genome of the flaviviruses is a single molecule of rna about 11,000 nt in length and capped on the 5 0 terminus. the genomic rna encodes a long orf of a polyprotein precursor flanked by 5 0 and 3 0 untranslated regions. mature viral proteins are produced during a complex process of proteolytic cleavage of the polyprotein precursor by cellular and viral proteases. structural proteins (core, m, and e) occupy one-third of the rna (the n part of the polyprotein) on the 5 0 part of the genome, followed by nonstructural proteins (ns1-ns5b) (figure 8 .46). 2, 4 most of the flaviviruses are arboviruses; that is, they can be transmitted to vertebrate hosts by bloodsucking arthropod vectors (figure 8.47 ). approximately 50% of known flaviviruses are transmitted by mosquitoes, about 30% by ticks. the arthropod vectors of some flaviviruses are unknown. there is also a group of flaviviruses that infect only insects and not vertebrates. some flaviviruses (e.g., west nile virus, wnv) have ecological plasticity and can be transmitted either by mosquitoes or by ticks. flaviviruses are distributed over all continents, with mosquito-borne viruses found mainly in regions with an equatorial and tropical climate and tick-borne viruses found mostly in regions with a temperate climate zone. many flaviviruses are associated with birds, which can transfer them during the birds' seasonal migration. flaviviruses belongs to natural foci zoonoses. certain flaviviruses, such as yfv, dengue virus (denv), and west nile virus (wnv), pose a serious threat to humans. 5à7 history. the first hint that omsk hemorrhagic fever (ohf) was etiologically linked figure 8 .48) an area with a wide network of lakes. about 200 cases with two lethal outcomes ("atypical tularemia" and "atypical leptospirosis") were investigated (without the expedition produced prodigious results: the prototype strain ohfv/kubrin was isolated from the blood of one patient; the mechanism of transmission of the virus by the ixodidae tick dermacentor reticulatus was established; the epidemiological and clinical features of ohf, as well as its pathogenesis and pathomorphology, were described; and inactivated vaccine from mouse brain was developed and prepared for epidemiological trials. 5 later, the role of another species of ixodidae ticks (d. marginatus) as an ohfv vector was revealed. 8, 9 taxonomy. ohfv belongs to the phylogenetic branch of the mammalian tick-borne virus group (figure 8 .47). the ohfv genome has a length of 10,787 nt, and its organization is common to the flaviviruses. two genotypes of ohfv are known today: prototypical strains for the first one are ohfv/kubrin and ohfv/bogolubovska, which have an extremely small genetic distance between them; the prototypical strain for the second genotype is ohfv/uve. 10à12 only six nucleotide substitutions, which encode four amino acids, have been found in the entire genome. three of four amino acid changes were located in the envelope glycoprotein e. 11 phylogenetic analysis based on a comparison of partial sequences of the e gene available in genbank showed that ohfv isolates can be divided to three genetic lineages (figure 8 .49). the genetic diversity among strains of different lineage is up to 11.8%. arthropod vectors. the natural foci of ohfv are found in the forestàsteppe landscape zone of western siberia, an area with numerous bogs and a wide network of lakes within the omsk, novosibirsk, kurgan, and tyumen regions (figure 8 .48). the natural foci border the area of distribution of tbev, and the two virus's natural foci are intermingled. 13à15 the principal ixodidae tick vectors for ohfv are dermacentor pictus (in the northern forestàsteppe subzone) and d. marginatus (in the southern forestàsteppe subzone). 3, 8, 9 the infection rate of d. pictus in epidemic years reaches 8%, in interepidemic years 0.1à0.9%. the main host for preimago phases of d. pictus is the narrow-headed vole (microtus gregalis). this species of rodent is host to 70à90% of d. pictus nymphs and larvaein the northern forestàsteppe subzone. in 1959à1962, when the number of microtus gregalis voles fell significantly, there was a concomitant decrease in the number of d. pictus ticks in the center of an epidemic zone that was accompanied by a sharp decrease in the infection rate of ticks and an attenuation of the meadow natural foci of ohfv. in some of those years, however, a high number of ixodes apronophorus, all phases of which feed on the water vole (arvicola terrestris), become involved in the virus's circulation on a par with d. pictus ticks. ar. terrestris makes fodder migrations in juneàaugust from damp locales (where their infection takes place) to coastal meadows (where peak activity of the larvae and nymphs of d. pictus is observed during those months). small animals living in those meadows become infected as they feed on the d. pictus larvae and nymphs. in damp locales, i. apronophorus could infect muskrats. also, d. marginatus, whose optimum zone lies in a steppe landscape belt, plays some (though largely insignificant) role in the lake areas of the southern forestàsteppe subzone. 16 during epizootic and epidemic activity of ohf natural foci, gamasidae ticks, as well as aquatic organisms belonging to the hydracarinae, take part in ohfv circulation. their involvement is confirmed by the identity of isolated strains with those isolated from muskrats and sick humans. experiments with experimentally and spontaneously ohfvinfected gamasidae ticks testify to the ability of longitudinal (more than six months) virus preservation. 17 vertebrate hosts. the principal vertebrate host of ohfv, which is able to directly infect humans, is the muskrat (ondatra zibethicus). this species was introduced into western siberia from canada in 1928. their population density reached a modern-day high in the 1940s. close interactions among o. zibethicus and local populations of arvicola terrestris emerged. ar. terrestris has periods of rapid population growth followed by epizootics of tularemia, leptospirosis, and ohfv. muskrats suffered these epizootics together with other local species of rodents: microtus oeconomus, m. gregalis, myodes rutilus, apodemus agrarius, and ar. terrestris. 13 the ofv infection rate among muskrats is about 15% in both the autumnàwinter and the springàsummer periods. 16 latent infection was established in all rodents except the muskrat. 18 ohfv was detected in birds and in mosquitoes, but the role of these two animals in virus circulation is not clear. 18à21 epidemiology. ohfv is transmitted both by ixodidae tick bites and as the result of direct contact with infected muskrats, their flesh, and fresh fells. 1, 5 ohf morbidity during 1945à1949 reached 1.5à5.0%. then there was a gradual decrease down to single cases. most ohf cases (96.8%) were detected in the lake forestàsteppe, in the south of the forestàsteppe landscape zone, which occupies 14.5% of the territory where 15.3% of country people in the omsk region live. the northern forestàsteppe landscape zone is the youngest landscape of western siberia, having evolved in place of the former southern taiga landscape zone. 22, 23 in the south of western siberia, the following territorial zones can be marked out: (i) the 223 8.2 family flaviviridae preferred territory of tick-borne encephalitis virus (tbev) (the southern taiga); (ii) intermediate territory (the boundary of the southern taiga with the northern forestàsteppe); (iii) the preferred territory of ohfv (the northern and southern forestà steppe); and (iv) the territory of sporadic cases of ohf (part of the southern forestàsteppe and steppe). 13, 23 in the first zone, more than 90% of all cases of tbe in western siberia are registered and only single ohf cases are found; in the second zone, 1% each of cases of tbe and ohf; in the third zone, 4% of tbe and 96% of ohf; and in the fourth zone, 4% of tbe and single cases of ohf. 13 the seasonal incidence of ohf distinctly correlates with the activity of the principal ixodidae tick vectors. cases (a few) of ohf acquired by direct contact with muskrats occur mainly during the season in which the animals are hunted, in octoberàjanuary. in the springà summer season, ohf cases occur chiefly in rural areas. the age of patients ranges from 5 to 70 years, but cases occur mainly among middle-aged persons (40à50 years old). in the autumnàwinter period, ohf occurs mainly among muskrats trappers (60%), adult members of their families (28%), and children (12%). it appears that all patients infected directly from muskrats develop symptomatic illness. seroprevalence ranges from 0 to 32% in populations of endemic regions. 3, 7, 23 in the last decade of the twentieth century, an increase in ohf natural foci activity took place in the tyumen (1987), omsk (1988, 1999à2007), novosibirsk (1989à2002; regular epidemic activity took place on the territory of only four administrative districts), and kurgan (1992) regions. in the absolute majority of laboratoryconfirmed cases, the nontransmissive pathway (direct contact with muskrats) of the infection dominated. 17 pathogenesis is determined first of all by the destruction of capillaries, the vegetative nervous system, and the adrenal glands. 16, 24 clinical features. the incubation period of ohfv is 2à4 days long. the disease begins abruptly, with fever, head and muscular pain, hyperemia, and injection in the sclera. the body temperature increases up to 39à40 c and stays that way for 3à4 days, then decreases a little and critically falls on the 7th to 10th day after symptoms appear. from the first days of the illness, there are diapedetic bleedings, especially in the nose. recovery is usually complete, without any residual phenomena; lethal outcomes are possible, but are rare. 16,24à26 control and prophylaxis. ohfv survives up to 20 days in lake water. water can be contaminated by urine and feces of the infected muskrats or some other rodents. the water pathway in human infection has been discussed in the literature. 13, 14 prevention of the infection depends on the use of protective respirators and rubber gloves in processing muskrat pelts and on personal protective measures against tick bites. tbe vaccine offers a high degree of protection against ohf. 10, 23 cases of laboratory-acquired ohf have been reported in unvaccinated persons, and tbe vaccine is recommended for laboratory personnel working with either virus. 23 interferon and its inductors have shown a high efficiency in preventing ohf in experiments using animal models. 27 the genome of powv is a about 10,835 nt in length. the virus comprises two genetic lineages, formed by powv (lineage i) and the closely related deer tick virus (dtv, lineage ii) (figure 8 .51). 8 phylogenetic analysis based on partial sequences of the e gene showed that the population of powv in russia has a low genetic diversity. 9 the strains of powv isolated in russia have a high genetic similarity to the strains of lineage i isolated in north america. a full-length genome comparison revealed that far eastern isolates (leiv-3070prm, spassk-9, and nadezdinsk-1991) have a 99.5% identity with strain powv/lb from canada (figure 8.51) . arthropod vectors. powv was isolated from ixodidae ticks collected in the russian far east and in the u.s. states of california, colorado, connecticut, massachusetts, south dakota, and west virginia. serological investigations of wild mammals indicate that powv also circulates in the canadian provinces of alberta, british columbia, and nova scotia. 3,10à12 in north american natural foci, powv was isolated from ixodes cookei, i. spinipalpus, i. marxi, and dermacentor andersoni ticks. 3, 10, 11 in the far east, known vectors of powv are haemaphysalis longicornis, haem. concinna, haem. japonica, d. silvarum, and i. persulcatus ticks. 5, 9, 13, 14 transphase and transovarial transmission of powv in ixodidae ticks has been established. vertebrate hosts. in north america, powv was isolated from wild mammals: the woodchuck (mormota monax, the main reservoir), american red squirrel (tamiasciurus hudsonicus), deer mouse (peromiscus maniculatus), red fox (vulpes fulva), eastern gray squirrel (sciurus carolinensis), north american porcupine (erethizon dorsatum), striped skunk (mephitis mephitis), raccoon (procyon lotor), long-tailed weasel (mustela frenata), and gray fox (urocyon cinereoargenteus). 2, 4, 15 infection of wild vertebrates most often is inapparent. 2, 10 in the south of the russian far east (in primorsky krai), powv was isolated from aquatic birds: the common teal (anas crecca) and the mallard (anas platyrhynchos). 9, 13, 14, 16 epidemiology. human infections of powv were reported in canada (ontario and quebec), the united states (new york and pennsylvania), 2 and russia (primorsky krai). 14, 17, 18 nevertheless, human infection rarely develops. clinical features. the clinical picture of developing meningitis and encephalomeningitis includes high temperature, dryness in the gullet, drowsiness, headache, disorientation, convulsions, vomiting, difficulty breathing, coma, and paralysis, with 11% lethality in the severe phase of the disease. autopsy has revealed widespread perivascular and focal parenchymatous infiltration. in 50% of recoveries, consequent damage to the cns develops, which could lead to death in 1à3 years. 2, 18 control and prophylaxis. the vaccine against tbev is not effective against powv. 2 history. in 1931à1934, the russian military medical doctoràneuropathologist a.g. panov, together with his colleagues a.n. shapoval and d.a. krasnov, described a neuroinfection with a high level of mortality in the far east. this neuroinfection later was called "springà summer encephalitis." 1,2 during field expeditions in 1937à1940, the historical strain tbev/ sofjin was isolated from the brain of a patient with encephalitis who died in khabarovsk krai (figure 8.52) . in that period, the main vector of tbev-ixodes persulcatus tickswas established, epidemiological peculiarities of tbe were studied, and the first anti-tbev vaccine was developed on the basis of intracerebrally infected mouse brain and was successfully used in medical practice. 2 complex expeditions were undertaken by a number of prominent virologists (l.a. zilber (figure 2.9 strain tbev/leiv-1380kaz (the former aav) was isolated from ixodes persulcatus in the low-mountain part of southeastern kazakhstan (alma-ata region) in 1977. 11 preliminary investigation revealed a one-sided antigenic relation between aav and powv. 12 aav was associated with human cases of meningitis. specific antibodies to aav were found among ground squirrels (citellus fulvus), agricultural animals, and humans. later, the aav genome was sequenced (genbank id: kj 744033). 13 a full-length genome comparison showed that aav has the highest similarity (94.6% nt and 98.3% aa identities) to the tbev/ chita-653, tbev/irkutsk-12, tbev/aino, and tbev/vasilchenko strains belonging to the siberian genotype (figure 8.53) . recent genetic studies of tbev revealed two additional genotypes of this virus on the territory of eastern siberia (irkutsk region): for the first one, only a single strain is known today; for the latter, there are five strains in mongolia. 14 thus, tbev has a high level of genetic diversity in northern eurasia. tbev-sib genotype dominates in europe, western siberia, and eastern siberia, tbev-fe in the far east. 15, 16 the tbev-fe genotype, which was widely distributed in siberia and northeastern europe, is now being displaced by tbev-sib. tbev-fe strains are often pathogenic to laboratory mice, whereas tbev-sib frequently provokes severe and lethal disease. 15 local populations of all genotypes of tbev could be stable for a long time. 16 distribution. tbev is distributed within the areas of distribution of its main vectors: ixodes persulcatus and i. ricinus ticks (figure 8 .54-see details in the detailed work of e.i. korenberg 17 norway; 29à31 in the rest of europe, the czech republic, 8, 32 slovakia, 6, 33, 34 bulgaria, 35 hungary, 36, 37 poland, 38, 39 croatia, 40 latvia, 41 lithuania, 42 estonia, 43, 44 denmark, 31 germany, 45à48 austria, 49 slovenia, 50 france, 51 italy, 52,53 and spain 54 (table 8 .25); and in asia, the russian far east and siberia, 1,16,55 japan (hokkaido), 55 north and south korea, 56, 57 china, 58 mongolia, 59 kazakhstan, 13 and kyrgyzstan. 60 arthropod vectors. natural tbev infection has been observed in 16 species of ixodidae ticks. the principal arthropod vectors for tbev in russia are the ixodidae ticks ixodes persulcatus (in the far east, siberia, and the north of the european part of the country) and i. ricinus (in the south of the european part) (figure 8.54 ). 69 the northern boundary of i. persulcatus and i. ricinus lies within the limits of an effective temperature sum isoline of about 1,000à1,300 c (the middle taiga landscape belt). the most suitable climatic conditions for these ticks are within the south taiga. imago tick activity begins in the third d decade of april and reaches a maximum in the second and third decades of may or in the first and second decades of june, with activity beginning to decrease in the third decade of june. this time frame correlates with morbidity dynamics having an 8-to 10-day lag (figure 8 .55). 70 the ecological links of tbev during its circulation in natural foci are extremely diverse as the result of wide distribution of this virus (figures 8.52 and 8.54 ). ixodidae ticks, mainly i. persulcatus, are the natural reservoir of tbev and the core of natural foci. 12, 62, 71, 72 from the very beginning of the tick's larval stage, a suctional, tarlike liquid appears around the hypostome and becomes rosin. 62, 73 the quantity of virus in this rosin plug is comparable to that in the tick's body (10 3 à10 4 pfu/mcl). 74 the place of suction on the body of the host is significant for the development of infection; for example, suction in the axillary hollow results in the highest lethality (16.1%, 1.5 times more in comparison to suction in the neck and in the head. 73 ticks become infected as they suck blood from a vertebrate host with a level of viremia that is equal to or higher than the threshold required for infection. ticks can also become infected from an uninfected vertebrate host as they suck blood together with infected ticks. 73, 75 transovarial and transphase transmission of tbev has been described in the literature; nevertheless, the effectiveness of vertical transmission of tbev is low. (about 1% of progeny turn out to be infected). 52, 76 the sexual pathway of tbev transmission from male to female is quite effective (about 50%). 77à79 the aggressiveness and activity of tbev-infected ixodidae ticks increases with the tbev titer in their bodies. 62, 75 infected ticks have been found on the clothing of figure 8.55 trends in the incidence of tbe in russia, by month (as a percentage of the amount of disease for the year, according to long-term data). humans at a fequency 5à20 times higher than uninfected ticks have been found. 48, 62, 75 tbev has been isolated from the mosquitoes anopheles hyrcanus in kyrgyzstan 80 and aedes sp. in western siberia. 81 the strain tbev/malyshevo was isolated from aedes vexans nipponii mosquitoes collected in 1978 on the coast of petropavlovskoe lake in khabarovsk krai in the russian far east (48 40ʹn, 135 41ʹe ). 82à84 a preliminary investigation 82 concluded that this strain belonged to negishi (negv) virus, 85 and later the possibility was discussed that the strain belonged to a separate, malyshevo virus. then, phylogenetic analysis using a next-generation sequencing approach revealed that malyshevo is a strain of tbev and is closely related to tbev strains isolated in the far east: tbev/1230, tbev/ spassk-72, tbev/primorye-89. 13 tbev has been isolated many times fromticks and fleas of the superfamily gamasoidea living in nests of rodents and birds (table 8 .27), even during the winter period. 2,47,86à89 vertebrate hosts. hosts for the preimago stage of ixodidae ticks-asian chipmunks (tamias sibiricus), shrews (members of the soricidae family), bank voles (myodes glareolus), field voles (microtus agrestis), mountain hares (lepus timidus), and 74 species of birds (table 8 .28)-have great significance in tbev circulation. 10, 12, 62, 64, 71, 72, 90, 91 persistent tbev infection in bank voles and field voles has been found during the winter period. 26 infection among vertebrates occurs mainly by tick bites. in rare instances, alimentary transmission of tbev through milk containing viruses is possible. 34, 92 epidemiology. there are two basic modes of human infection by tbev: (i) as the result of being bitten by infected ixodidae ticks (the main mode); and (ii) as the result of consuming infected raw goat, sheep, and cow meat, milk, or dairy products (mainly in natural foci linked to ixodes ricinus). 23, 32, 93 the latter pathway of tbev distribution often involves whole families. as much as 70% of cases in belarus have been alimentary. 70 tbev can persist in milk at 60 c for more than 10 min, and some of the viruses can remain viable even after pasteurization at 62 c for 20 min. nor is tbev inactivated after 24 h at 4 c and ph 2.8. many laboratory infection cases (usually by aerosol) have been described. several hundred cases are recorded in europe (table 8 .25) and in russia (table 8.26 ) each year, with considerable interannual variation. 17,70,94à96 the highest level of tbe morbidity is registered in the baltic states (latvia, 6.2à10.8 per 100,000 population); lithuania, 6.5à13.5; and estonia, 10.4à13.5) and in slovenia (10.2à18.6) and the czech republic (5.0à10.0). in neighboring austria, where the vaccination rate is higher, the index is lower (0.6à1.2). 97 seasonal tbe morbidity in russia is connected with seasonal activity of the ixodidae tick vectors (figure 8.55) . the risk of infection depends upon the frequency of tick bites, which is different for populations living in the different landscape belts. results of an investigation of almost 200,000 people demonstrate that the highest risk is for the population living in the southern taiga belt, where about 20% of adults were found to have tick bites during one epidemic season (table 8. in rural localities of the southern taiga belt, about half of schoolchildren and about 80% of adults have antibodies to tbev. for comparison, only 14à20% of adult citizens of kemerovo, a city of about half a million in western siberia, and 2à3% of citizens of moscow have antibodies specific to tbev (table 8.29) . 98 a mathematical model for evaluating the infection rate and the probability of developing the disease as a function of the density of the tick population, its infection rate and biting activity, and the level of the immune human layer was developed by d.k. lvov and coauthors. 98à102 the same approach, which is also suitable for other arboviral infections, was used for landscape-epidemiological zoning of tbev natural foci in altai krai in the southern part of western siberia: more than 10,000 residents living in the different landscape belts on a territory about 250,000 km 2 were tested by serological methods (figure 8.56) . the tests produced a good fit between calculated and registered morbidity data (table 8.30) . pathogenesis. tbe can be realized in several pathogenetic variants. an inapparent clinical form is characterized by short-term localization of tbev in lymph nodes and immune cells, as well as by extranervous reproduction without viremia. infection is terminated by the development of stable immunity. about 95% of cases of infection are inapparent. 102 clinical fever is expressed as a common infectious process, but both the central and the peripheral nervous system are involved in the pathology. 103 neuroinfection is characterized by lesion of the envelope and substance of the spinal cord and cns. clinical features. the incubation period ranges from 1 to 30 days, but usually is 7à12 days. the onset of illness in typical cases is abrupt and with a headache. the temperature clinical symptoms of tbe, as well as the severity of the disease, are at least partially determined by biological properties of the virus. 104 there are two main clinical forms of tbe: the far eastern variety, associated with far eastern and siberian strains of the virus, and the european variety (also known as western biphasic meningoencephalitis or biphasic milk fever), associated chiefly with european strains. human disease of the first type is usually clinically more severe in the acute phase, but is associated with a lower rate of chronic cns sequelae. the first phase starts with sudden fever, flulike symptoms (pronounced headache, weakness, nausea, myalgia, arthralgia), and conjunctivitis. the second phase appears after 4à7 days of apparent recovery, but then the cns is affected (meningoencephalitis appears), accompanied with fever, retrobulbar pain, photophobia, stiff neck, sleeping disorders, excessive sweating, drowsiness, tremors, nystagmus, meningeal signs, ataxia, pareses of the extremities, dizziness, confusion, psychic instability, excitability, anxiety, disorientation, and/or memory loss. tbev produces diffuse degenerative changes in neurons, perivascular lymphocytic infiltration, and damage to purkinje cells in the cns. mortality ranges from 1% (tbev-eur), to 8% (tbev-sib), to 20à40% (tbev-fe). convalescence is prolonged, and neurological and psychotic sequelae often include paresis and atrophic paralysis of the neck and shoulders. 27, 45, 104 a chronic form of the disease occasionally combines with a progressive course (called kozhevnikov's epilepsy), in which progressive neuritis of the shoulder plexus, multiple sclerosis, and progressive muscle atrophy often develop. 105, 106 the chronic form is registered in 1à2% of all tbe cases and is said to be the result of virusàimmunity interactions. 19 many authors have noted a decreasing number of severe tbe cases. 103 diagnostics. laboratory diagnosis of tbe involves both serological (elisa, hemagglutination inhibition test (hit), neutralization testing) and virological methods (virus isolation using a biological model of intracerebrally inoculated newborn mice, 5à6 g mice, cell culture), as well as highly sensitive rt-pcr and real-time rt-pcr. control and prophylaxis. specific and nonspecific prophylaxis tools are highly efficient if they are utilized correctly. personal safety includes protection against ticks. vaccination against tbev has a long history of success. mass vaccination of populations in the endemic territory is necessary. a full course of vaccination provides 98% safety. 102 all vaccines produced in russia are effective in the entire area of distribution of tbev, independently of the strain used to prepare the vaccine. vaccination has reduced tbe morbidity down to single cases in austria, the czech republic, and slovakia. 107 single cases of tbev among vaccinated persons need to be investigated because possible causes are personal peculiarities of the immune system and errors in the control of vaccine production. 108 the presence of brain tissue in vaccines produced on the basis of intracerebrally inoculated newborn mice was a source of danger for a long time: demyelinating encephalitis could develop. this danger was eliminated after vaccines were developed which used tbev strains that reproduced in cell cultures. in the 1960s, cell culture vaccines against tbev were developed by e.n. levkovich history. japanese encephalitis virus (jev) was originally isolated by h. hayashi in 1933 from a patient who died with encephalitis and then, again, in 1935 from a patient who died with a fever in tokyo. 1, 2 before that, however, japanese encephalitis (je) epidemics was documented in japan in 1903 and onward as "ioshiwara cold." in the south of the russian far east, strains of jev were known since the end of the 1930s (figure 8.57 ). je played a role in the historical events of world war ii. american military personnel massed on okinawa and preparing to invade japan were demoralized by an outbreak of encephalitis among the indigenous people. a fictionalized account of the risk from je for american soldiers during world war ii underscores the military risk. 3 taxonomy. phylogenetic studies indicated that jev isolates be divided into five genotypes, the distributions of which overlapped (figure 8.58 ). genotypes i, ii, and iii are most prevalent and are spread throughout asia (japan, china, india, korea, malaysia, and vietnam), the far east of russia, and northern australia. genotypes iv and v are rarer and were isolated in indonesia and india, respectively. genotypes i and iii are found mostly in temperate zones, whereas genotypes ii and iv predominate in tropical zones. 4à6 genetic diversity between strains of the different genotypes ranges from 9.1% to 16.6%. arthropod vectors. jev circulation in the equatorial and subequatorial climatic zones is year-round and is seasonal in the tropical, subtropical, and temperate belts, with a peak at the end of summer and the beginning of fall. jev is brought from the equatorial and tropical climatic belts to the subtropical and temperate belt during the spring migration of birds. about 30 species of mosquitoes are able to transmit jev; nevertheless, only some of them are effective vectors. the main vector in japan, the philippines, the korean peninsula, china, the indochinese peninsula (except malaysia), indonesia, sri lanka, india, and nepal is epidemics usually develop after plentiful precipitation and a long rise in environmental temperatures until they are no less than 25 c (but within the range 25à32 c). 7 for a long time, the main vector for jev in the south of primorsky krai in russia was culex tritaeniorhynchus. in the 1940s, as a result of both improvements in agriculture and meteorological changes, this species of mosquitoes consisted about 80% of all field collections. in subsequent years, however, their numbers abruptly declined, and by the 1960s the species represented only 0.15à0.75% of all mosquitoes collected. cx. pipiens is an accessory vector, and aedes togoi transmits jev in seashore areas. jev was also isolated in 1989 from ae. vexans. 8, 9 vertebrate hosts. aquatic and semiaquatic birds (especially herons) have the main significance in the natural cycle of jev circulation. regular transfer of jev in migratory birds from endemic territories with year-round circulation of the virus to regions of the southern part of the temperate climatic belt (in particular, the southern part of primorsky krai, to the south from lake khanka 11 ) is likely. 10 jev transfer over hundreds of kilometers by infected mosquitoes is possible as well, especially in areas with a monsoonal climate (e.g., in australia through the torres strait 12à14 ). birds transfer jev from natural to synantropic biocenoses, where, thanks to culex tritaeniorhynchus mosquitoes willingly attacking wild birds, pigs, persons, synantropic birds, and domestic animals (chiefly pigs), these all join into jev circulation. 7 infection in pigs could be inapparent, or it could be clinically expressed with encephalitis and a lethal outcome. the level of viremia in infected pigs is enough to infect mosquitoes. such epizootics among pigs are, in effect, amplifiers for jev, serving as prerequisites for the development of epidemics, first of all among people living in the countryside, but then among city dwellers as well. antibodies to jev specifically were revealed among wild boars (83%), raccoons (59%), 14 and dogs (17%). 7 in the south of china, jev was isolated from both leschenault's rousette (rousettus leschnaulti), a species of fruit bat, and the little tube-nosed bat (murina aurata), 15 and anti-jev antibodies were identified in the blood of those animals. 16 jev preservation in bats could be one of the mechanisms of the year-round circulation of the virus in its natural foci, with activation in the spring and subsequent replication and spreading in the summer and autumn. in natural foci, birds are the principal vertebrate hosts contributing to transmission of the virus; in synantropic foci, pigs are the most important vertebrate hosts. 10,11 jev has been isolated from the grey-headed bunting (emberiza fucata), great cormorant (phalacrocorax carbo), japanese thrush (turdus cardis), azure-winged magpie (cyanopica cyana), japanese wagtail (motacilla grandis), barn swallow (hirundo rustica), and night heron (nicticorax nicticorax). natural foci are situated in meadows. of late, culex tritaeniorhynchus has become more abundant in connection with intensive rice cultivation, portending the possibility of increased jev circulation and epidemics. 17, 18 epidemiology. all the territory of japan, except for northern part of hokkaido, 7 is endemic, but most diseases are registered near islands in a closed sea, as well as in tokyo and adjacent prefectures. 3 before 1966, outbreaks of je emerged in japan practically every year, with 1,200à2,700 patients seen. later, morbidity began to decrease to tens of cases per year. in the 1970a and 1980s, morbidity fell to the level of single cases per year. the main cause of the decrease was a significant drop in the population of the main jev vector-culex tritaeniorhynchus mosquitoes-as the result of a reduction in the acreage of rice fields as well as water pollution in places of mosquito habitation. in addition, the program of mass vaccination carried out annually among children of school age and a change in the structure of pork farms lessening the availability of pigs played a significant role in the falloff in the mosquito population. je is a serious problem in 20 countries of southeast asia and oceania. 19 during the last few years, more than 50,000 cases per year were registered, with about 20% lethality. 19 morbidity increases annually in bangladesh, indonesia, laos, myanmar, north korea, and pakistan. 19, 20 in addition, , the occurrence of an epidemic in southeastern asian countries is becoming more and more likely because those countries are now seeking to increase their production of rice. the greatest risk of je is said to be in china, nepal, sri lanka, thailand, 21 laos, and vietnam. je is of the highest importance among all kinds of endemic encephalitis, potentially threatening nearly 50% of the population of our planet. 3 the disease especially affects military contingents, as it did the american army during the concentration of armies in okinawa 3 and the soviet army during the battle of lake khasan (also called the changkufeng incident) in the south of primorsky krai. precursors of jev circulated in indonesia and then evolved into six genotypes. 22 genotype iii is widespread in a moderate climatic belt and often provokes epidemic outbreaks in eastern and southeastern asia. genotype i originated in indonesia, circulated in thailand and cambodia in the 1970s and in south korea and japan in the 1990s, and has now completely replaced genotype iii. 23 genotype i got into japan in two ways: from southeastern asia and from mainland china. 24, 25 two island territoriesthe philippines and taiwan, in both of which genotype iii circulates-were free of genotype i-and the philippines remains free-but the genotype appeared in taiwan in 2008. 26 the evolution of jev led to the emergence of two new subclusters in 2009à2010; the two together have replaced genotype iii. until recently, the qinghai-tibet plateau, in china, was free of jev, but in august 2009 the virus was isolated from culex tritaeniorhynchus mosquitoes there. 27 during an epidemic in septemberànovember 2009, genotype i circulated in japan. 28 in nepal, on the northern border of india, je has been known since 1978, after which outbreaks were observed annually. 9 jev circulates in the north of australia as well. 12, 21 je claimed morbidity in the south of the russian far east (in primorsky krai) in 1938 during an expedition headed by p.g. sergiev and i.i. rogosin. epidemics of jev broke out in the region in 1938, 1939, and 1943. more than 800 cases were recognized between 1938 and 1943, with 68% reported in the extreme south of primorsky krai. the northern extent of this area is limited by the southern part of the ussuri lowland (about 42à43 n, 130à133 e). enzootic jev circulation without human morbidity has been documented, with the seroprevalence of residents estimated at about 10à20%. 11,18,29,30 je cases occur mainly in augustàseptember (but also when heavy rains are combined with high temperatures from april to september: $21 c in april, $23 c in june, $25 c in august, and $21 c in september). clinical features. the clinical picture of je varies from asymptomatic and easy feverish forms to an encephalitis syndrome. the ratio of clinical to asymptomatic forms is from 1:300 to 1:1,000, although the ratio in india in the 1970s and 1980s was from 1:20 to 1:30. 31à33 the start of the disease is sudden, with fever (80%), headache, vomiting (24%), and symptoms of cns destruction (most often, hemiplegia and articulation lesions)-in 12% of cases, and at the height of the illness in 65% of cases. about one-third of patients with cns lesions recover completely. 34 lethal outcomes are preceded by unconsciousness and then coma (20à44% of the total number of patients). death comes in two-thirds of cases during the first week, in one-fourth during the second week, and in the rest of the cases in one month, from the onset of symptoms. after the disease, residual phenomena in the form of paralysis and mental issues are quite often observed. 28, 32 control and prophylaxis. inactivated vaccines are used to immunize people, 19,29,33,35à37 live vaccines to immunize pigs and horses. 31 vaccination and protection of pigs from mosquito attack and protection of humans from mosquitoes (through the use of repellents, mosquito nets, bed curtains, etc.) are recommended during epidemics among people. mass vaccination has been carried out successfully in japan, south korea, china, and india. 19,28,33,35à37 live vaccine manufactured on the basis of the chinese strain sa 14à22 is is given in china, south korea, and other countries in government programs aimed at expanding immunization of children. 19 24 the complete genomes of tyuv and kamv (genbank id: kf815939 and kf815940, respectively) were presented in a 1973 article in the journal of medical entomology, 25 and it was established that kamv was a new virus within the tyuv group of the flavivirus genus. virion and genome. tyuv is a prototypical virus of the tyuleniy antigenic complex. the viruses of that complex belong to the ecological group of seabird tick-borne flaviviruses, which forms a distinct branch on the phylogenetic tree. 26 four species are known in the tyuleniy antigenic complex: tyuv (in russia and the united states), meav (in europe), srev (in oceania) and kamv (in russia). the genetic similarity between the seabird tick-borne flaviviruses and the mammalian tick-borne flaviviruses is about 42% nt. a full-length genome comparison showed that the similarity among the four viruses in the tyuleniy antigenic complex is 70% nt and 85% aa, on average. tyuv leiv-61c, isolated in the russian far east, has 86% nt and 97% aa identities with tyuv isolated on the pacific coast of the united states. kama virus (strain leiv-tat20776) has 70% nt identity with the other viruses of the tyuleniy antigenic complex (meav, srev, tyuv). the similarity of the polyprotein precursor of kamv is 74% aa with each of tyuv and srev, 78% aa with meav. 25 arthropod vectors. tyuv is distributed over the basins of the sea of okhotsk and the bering and barents seas. the infection rate of ixodes uriae in the pacific part of the virus's distribution is 4.5 times greater than in the atlantic part (table 8 .31). 16 ,18à23 outside of northern eurasia, tyuv is distributed over the west coasts of the united states (chiefly in oregon) and canada. 27, 28 the infection rate of nymphs and larvae of i. uriae is one-twentieth to one-half the infection rate of the imago. the infection rates of i. uriae females and males (the males have only a rudimentary hypostome and do not feed) are practically the same. 21 these data testify to the transphase and transovarial transmission of tyuv. (the efficiency of this type of transmission is about 5%.) attempts to isolate tyuv from i. signatus ticks were unsuccessful. the presence of antibodies to tyuv among local cows and indigenous people of the commander islands 19,21 indicates the possible role of sanguivorous mosquitoes (e.g., aedes communis, ae. punctor, and ae. excrucians) in infection. mosquitoes could also take part in virus circulation: their infection rate from the end of july to the beginning of august reaches 0.3% in nesting colonies of seabirds and 0.1% on the seacoast. experimental infection of tyuv on the model of aedes aegypti demonstrated the presence of the virus 4à31 days after inoculation, with 1.5à2.0 lg ld 50 /10 mcl on days 4à17; 3.0à3.5 lg ld 50 /10 mcl on days 23à27; and 1.5 lg ld 50 /10 mcl on day 31. the transmission of tyuv during the feeding of infected mosquitoes on mice was established 7à19 days after infection of the mosquitoes. in culex pipiens molestus, tyuv was detected 5à21 days (the period of observation) after infection, with 1.0à2.0 lg ld 50 /10 mcl. 20 vertebrate hosts. migratory seabirds play a role in the exchange of tyuv group flaviviruses between the northern and southern hemispheres. 18, 29 investigation with the help of indirect complement-binding reactions of sera samples from 2,500 birds collected in the far east revealed that the maximum tyuv infection rate takes place in brü nnich's guillemots (uria lomvia), common murres (u. aalge), and tufted puffins (fratercula cirrhata). lower rates were seen in pelagic cormorants (phalacrocorax pelagicus), redfaced cormorants (ph. urile), glaucous-winged gulls (larus glaucescens), kittiwakes (rissa tridactyla), northern fulmars (fulmarus glacialis), and sandpipers (scolopacidae). 17, 18, 20, 21, 23, 30 the presence of specific anti-tyuv antibodies among sandpipers-red-necked phalaropes 27, 28, 31 considering the annual migrations of these birds, tyuv can be found within the i. uriae area of distribution in nesting colonies of puffins. about 90% of adult and 10% of juvenile northern fur seals (callorhinus ursinus) on the commander islands have specific anti-tyuv antibodies, implying that these animals are involved in the circulation of that virus. a tyuv strain was isolated from the arctic ground squirrel (citellus (urocitellus) parryii) on the southeastern coast of the chukotka peninsula (63 n, 180 e). this event is one more argument for virus splash into the continent, with rodents included in virus circulation. in the tundra of the kola peninsula seacoast, antibodies specific to tyuv were detected among cattle (28.1%) as well as red-necked phalaropes (phalaropus lobatus), snow buntings (plectrophenax nivalis), ruffs (philomachus pugnax), and rodents: tundra voles (microtus oeconomus). 21 thus, in the atlantic part of its distribution, tyuv also tends to penetrate into the continent. experimental infection of kittiwakes (rissa tridactyla), herring gulls (larus argentatus), and brü nnich's guillemots (uria lomvia) was followed by the development of clinical features with cns lesions and lethal outcomes. 32 epidemiology. the indigenous population in the far eastern part of tyuv distribution has specific anti-tyuv antibodies: 8.4% in tundra on the coast of the chukotka peninsular, 4.2% in forestàtundra on the coasts of the sea of okhotsk and the bering sea, 7.4% -in taiga on sakhalin island, and 9.1% in tundra on the coast of the kola peninsula. 21 the development of fever in humans visiting nesting colonies of seabirds on the coast of the barents sea has been described in the literature. 33 ecological peculiarities of tyuv and kamv distribution. penetration of tyuv from the northern to the southern hemisphere is carried out by about 20 species of birds, mostly turnstones (arenaria interpres), that nest in the north of asia and overwinter in australia and new zealand. wedge-tailed shearwaters (puffinus pacificus) nest in the southern hemisphere and carry out an annual migration along the coasts of the pacific ocean up to northern eurasia and north america. 23, 34 close genetic relations found between tyuv and kamv have not been explained yet because information is lacking about ecological links between alcidae birds in the north and bank swallows in the central part of the russian plain. nontheless, the closeness demonstrates an ancient link between the flaviviruses and ixodidae ticks-obligatory parasites of colonial and burrow-shelter birds not only on the ocean coast, but also on the continental part of the distribution of those viruses. 19, 20, 23, 35, 36 meav and srev, which are genetically close to tyuv, 25,26 could be intermediate evolutionary branches between tick-borne viruses of seabirds and later mammalian viruses transmitted by ticks. 13, 15 the main vector of tyuv in subarctic regions-ixodes uriae, adapted to seabirds-is replaced by the ornithodoros capensis complex or argas spp. in the subtropics and tropics. 18, 27 the northern boundary of the argas genus distribution is limited by a july isotherm of 15à20 c and of the ornithodoros genus by 20à25 c in europe and 25à30 c in asia. 37 the vector of kamv-the i. lividus tick-has transpaleoarctic distribution, from the british isles in the west to japan in the east and from 62 n down to 43 s. this species of tick has an extrazonal distribution in the diggings of bank swallows (riparia riparia) made in the soft ground of steeps along the banks of rivers and lakes in taiga, leaf forest, forestàsteppe and 247 8.2 family flaviviridae steppe climatic belts. i. lividus ticks are typical parasites of-burrow-shelter birds and relate strictly to the life cycle of the host: after the appearance of birds in the nesting areas in may, larvae begin to feed. in june, nymphs feed on the nestlings; female imagoes also feed on the nestlings, but male imagoes do not. 38 given the presence of kamv-a virus closely related to tyuv-in the central part of the russian plain, it is worthwhile, and even necessary, to carry out a wider search for tyuv analogues on the continental part of northern eurasia. history. dengue fever (denf), etiologically linked to dengue virus (denv) (family flaviviridae, genus flavivirus), has been known in asia, africa, and america since the end of the eighteenth century. 1,2 wide epidemics of denf appeared in southeastern asia after world war ii. 3 according to who data, denf morbidity, including imported cases, has been detected in more than 100 countries of asia, africa, and europe. more than 2.5 billion people on earth are under the threat of denf. about 50 million people fall victim to denf annually. 4 american armies sustained heavy losses as the result of denf during world war ii, 3 as well as during 1960à1990 in vietnam, the philippines, somalia, and haiti. 5 simultaneous outbreaks of denf and chikungunya fever often occur. 6 the virus etiology of denf and its transmission by mosquitoes was established by p.m. ashburn and c.f. craig in experiments using volunteers at the beginning of the twentieth century. 7 denv-1 was isolated in 1944 from the blood of patients with fever on the hawaiian islands, 8 denv-2 in 1944 from the blood of patients with fever on new guinea, 8 denv-3 in 1956 from the blood of patients with fever in the philippines, 9 and denv-4 in 1956 from the blood of patient with fever during epidemics in manila. 9 taxonomy. four different serotypes of denv form a distinct phylogenetic lineage on the mosquito-borne flavivirus lineage (figure 8.47 ). genetic variation among different strains suggested that denv be divided into distinct genetic clusters considered as genotypes. the genetic diversity of denv is best exemplified in denv-2, the different strains of which are divided into four genotypes: asian 1, asian 2, american/asian and so-called cosmopolitan. 10 denv-3 strains are divided into five genotypes (iàv), 11 and denv-4 strains form three genotypes. 12 in general, a particular genotype is linked to specific geographical regions and that genotype may be used in describing imported cases of denv infection. arthropod vectors. denf belongs to natural-foci diseases. its vectors are anthropophilic species of mosquitoes: aedes aegypti and ae. albopictus in synantropic natural foci. humans are the only vertebrate hosts in synantropic natural foci, whereas wild mammals are involved in virus circulation in sylvatic natural foci. vectors in equatorial africa are ae. furcifer, ae. vittatus, ae. tailori, and ae. luteocephalus. vertebrate hosts. in southastern asia, the vertebrate hosts of denv are macaques (genus macaca) and surilis (genus presbytis) living in the rain forests of equatorial climatic belts; the main vector is aedes niveus; a circulation of denv-{1, 2, 4} has been identified. natural foci of denv were also found in the eastern part of equatorial africa, in senegal and nigeria. the vertebrate hosts are patas monkeys (erythrocebus patas); wild strains are considered possible precursors of epidemic ones. among humans, wild strains provoke slight clinical forms of dengue fever. 13à15 epidemiology. denf has an epidemic character involving tens of thousands of people in southeastern asia, oceania, the caribbean basin, central and south america, and africa. the transmission pathway is a mosquito bite, mainly by members of the aedes genus. these mosquitoes are able to transmit denv in 8à10 days after feeding on a sick person. about 60à70% of the human population falls victim to denf during epidemics. 15 denv continues to circulate actively and to provoke wide epidemics. for example, all four types of denv exist in sri lanka, with new clades replacing old ones, accompanied by a severe clinical picture. 16, 17 in the 1980s, a new wave of denf epidemics began to develop in sri lanka, india, pakistan, and central and south america. 18, 19 these epidemics were linked mainly to the relatively new denv-3, but to denv-1 and denv-2 as well. 20 in some cases-for instance, in myanmar 21 and china 1 -all four types of denv circulated simultaneously. clinical features. the incubation period is 2à7 days. the start of the disease is quick, with fever and with frontal and retroorbital headache. lymphadenopathia, rash in macule and papule forms (not always), leukopenia, skin hyperesthesia, changes in taste, loss of appetite, and muscle and joint pains gradually develop. then, after 1à2 days of normal body temperature, the second wave of fever develops, accompanied by a measleslike rash. the palms and soles are rash free. severe cns complications have been described to arise in endemic regions (e.g., brazil). 3 the hemorrhagic clinical form of denf, with shock and a high level of lethality (especially among children), was originally seen in the philippines in 1953. later, this clinical form was registered in india, malaysia, singapore, indonesia, vietnam, cambodia, and sri lanka, as well as on islands in the pacific. according to who data, more than 1.3 million patients had hemorrhagic denf from 1956 to 1992, with 14,000 lethal outcomes. starting from 1975, hemorrhagic denf has become the main cause of hospitalization and deaths among children in the countries of southeastern asia. 1 the hemorrhagic form of denf usually develops after a secondary infection by a type of denv different from the primary one. the primary type of denv is not neutralized, but fragments antigen binding (fab)associated enhancement of the infection occurs. for example, in french polynesia in 2000, two years after epidemics of denv-2, an outbreak etiologically linked to denv-1 emerged and hemorrhagic denf was detected among children 6à10 months and 4à11 years old. 16 five symptoms are characteristic of the hemorrhagic clinical form of dengue: high temperature, rash, hemorrhagia, hepatomegalia, and insults to the circulatory system. thrombocytopenia with blood condensation also occurs. 4 hemorrhagic denf can be without shock or can precede it. shock develops in 3à7 days of the disease, wheninsults to the circulatory system appear: the skin becomes cold, sticky, and cyanochroic; the pulse rate increases; and drowsiness appears. in the absence of antishock actions, patients die within 12à24 h. the severity of the disease depends on a number of factors: the infection titer in the blood, the type of denv, its biological properties, and more. 22à24 imported cases of dengue. there is a high risk of denv infection for visitors to endemic regions, with consequent penetration of the virus into nonendemic regions. 1, 25 denf has occurred in spain in the past (e.g., in cádiz in 1778). several tens of human cases are introduced into the country each year from equatorial and subequatorial regions. denv-1 and denv-2 caused a huge outbreak in greece in augustàseptember of both 1927 and 1928: in those periods, about 650,000 of 700,000 inhabitants of athens and piraeus contracted denf, including hemorrhagic forms and about 1,000 lethal outcomes. 26 penetrations of denv also took place in the netherlands in 2006à2007 27 and in japan, 28 france, 29 northern italy, 30 and germany in 2010. 31 during 2002à2011 in russia, among patients with fever from the risk group that visited tropicalàequatorial countries, 48 cases of denf were identified with the help of serological investigation (22 cases arrived from indonesia; 11 from thailand; 3 each from vietnam and india; 2 each from venezuela and the dominican republic; and 1 each from sri lanka, malaysia, singapore, sierra leone, and costa rica). 32à34 in 2013 in russia, 30 cases of denf were identified in moscow, 8 in st. petersburg, and 8 imported strains of denv were isolated. the risk of denf for europe has appeared again with the introduction of aedes albopictus and ae. aegypti mosquitoes in the countries of the mediterranean and black sea basins. 35 stable populations of both these species were found on the southeastern coast of the black sea (in krasnodar krai, russia, as well as in abkhazia). 36à38 control and prophylaxis. the main approach to prophylaxis is to struggle against mosquito vectors. during the 1950s and 1960s, a program against ae. aegypti mosquitoes that was unprecedented in terms of scale and expense was conducted in america, but it was stopped in 1970; as a result, in 1995 the number of ae. aegypti mosquitoes was estimated to be same as that before the program began. 39 the struggle against mosquito vectors in singapore turned out to be more successful, but still did not prevent denf morbidity. 40 investigations into four-component vaccines are far from completion today. 22, 41 express methods of denf diagnostics are used in airports. 42 who issues a reference guide for the diagnosis, treatment, prophylaxis, and control of denf. 43 (table 8 .32, figure 8 .60). further serological investigations with the help of hit revealed that sokv belongs to the flaviviridae family, and with the help of complement-fixation testing (but not neutralization testing), to the entebbe bat serogroup. 1à3 a prototypical strain of this serogroup was isolated from a kenyan big-eared free-tailed bat (tadarida lobata) collected near entebbe, uganda, in july 1957. 5 taxonomy. the genome of sokv was sequenced, and genome analysis showed that the virus is related most closely (71% nt and 79% aa identities) to entebbe bat virus (entv). sokv has about 50% nt and 55% aa identities with other flaviviruses, except viruses of the rio bravo (rbv) and modoc (modv) groups (,50% similarity). 6 no arthropod vector of entv and sokv has been established; however, phylogenetic analysis based on a full-length genome comparison placed sokv and entv together on a distinct branch of mosquito-borne flaviviruses related to yfv and sepik virus (sepv) (figure 8.47) . arthropod vectors. according to serological data, domestic animals do not take part significantly in sokv circulation, although antibodies to sokv were detected among cows and sheep. isolation of sokv from birds that were known not to have made contact with obligatory parasites of bats, as well as the presence of positive sera from humans and domestic animals, suggest the participation of mosquitoes in sokv circulation. transmission of the virus by bats could be carried out by argas vespertilionis and ixodes vespertilionis. 7à10 vertebrate hosts. more than 20 flaviviruses were isolated from bats (order chiroptera); about half are unique to these mammals. 11 24 ; and yokose virus (yokv). 25 the insectivorous bats vespertilio pipistrellus, from which sokv was isolated, belong to the evening bats family (vespertilionidae), which is active during the evening and at night. their daylight shelters are situated mostly in house garrets. v. pipistrellus is distributed over europe, the mediterranean, the caucasus region, and central asia. a part of the population overwinters in africa, where infection by local viruses (e.g., bbv, dbv, entv) could occur. experimental infection of sparrows (passer montanus) resulted in sokv being detected in internal parts of infected birds on the 8th and 25th days after inoculation. 26 epidemiology. there are no laboratoryconfirmed human cases of sokv infection. nevertheless, the proximity of sokv hosts (bats) to human habitats, as well as the presence of encephalitis and hemorrhagic fever agents among the flaviviruses, suggest that sokv may be dangerous to humans. complement-binding specific anti-sokv antibodies were detected among humans in kyrgyzstan and turkmenistan (6.2% and 4.0%, respectively), testifying to recent infection events. 1à4,7,9,10,16,27à31 history. wnv (family flaviviridae, genus flavivirus), theetiological agent of west nile fever (wnf), was first isolated during research on yfv in 1937 from the blood of a native of uganda who was suffering a mild fever. 1 the strain isolated, b956, belongs to genetic lineage ii. (see "taxonomy" next.) strain eg101, isolated from the sera of a child without clinical signs in egypt, 2 is the prototype for african genetic lineage i, widely used for investigations. wnv belongs to the jev group, has the broadest antigenic properties, and, on theoretical grounds, appears to be the most ancient member of the flavivirus genus. 3 lowpassaged wnv strains are known by many investigators to be common causes of laboratory infection, apparent or inapparent. 4 taxonomy. phylogenetic analysis revealed that different geographic isolates of wnv could be grouped into two major genetic lineages (figure 8.61 ). lineage i includes strains from africa, southern and eastern europe, india, and the middle east. lineage ii includes isolates from west, central, and east africa, as well as madagascar. lineage 1 can be subdivided into three clades: clade 1a consists of strains from europe, africa, the united states, and israel. the topotypic isolates of wnv in australia-kunjin virus (kunv)belong to clade 1b, and clade 1c is formed by isolates from india. 5 subsequently, two genetically divergent rabensburg strains-97à103 (isolated in the czech republic) and leiv-krnd88-190 (isolated in russia)-were proposed to form novel lineages iii and iv, respectively. 6à8 a fifth lineage was formed by strains from india. 9 phylogenetic analyses based on complete genomic sequences revealed that the various lineages differed from each other by 20à25%. a putative novel sixth lineage has been detected in spain in 2006, but only a partial sequence of the ns5 gene of this isolate is available in genbank. 10 world distribution. the distribution of wnv in northern eurasia, and indeed, in the whole world, covers vast territories within the equatorial, tropical, and temperate (the southern part) climatic belts in africa, europe, asia, australia, and north america (the last starting from 1999). in africa, it is very difficult to find a country or landscape in which wnv has not been detected by either a virological or serological approach. the isolation of this virus from a wide array of species of birds, mosquitoes, ixodidae and argasidae ticks, and domestic animals as well as humans testifies to the ecological plasticity of the virus and therefore to its ability to adapt to different ecological conditions. two genetic lineages circulate in africa: the first, which dominates, and the second. sporadic morbidity and epidemic outbreaks permanently take place in a number of african countries, especially the republic of south africa, where a wide outbreak with at least 3,000 human cases occurred in 1974 after an active period of rain. according to a report from the pasteur institute, during the last 10à15 years alone, epidemic outbreaks were registered in algeria (in 1994, with more than 50 cases and 8 deaths, and in 1997, with 173 cases), in tunisia (during 1997à2003, with 173 cases), morocco (in 1996 and 2002; the epidemic reached both humans and horses), in senegal (in 1993), and in kenya (in 1998). 11 new centers of infection continue to be arise in africa-for example, in 2009 in morocco, where morbidity among people and horses was observed and 3.5% of birds had specific anti-wnv antibodies, 12 and in 2010 in the republic of south africa, where there were a number of lethal outcomes. 13 the wide distribution of wnv in africa and its circulation among populations of the majority of the continent's species of local and migrating birds indicates that the virus is able to penetrate to southern europe and western siberia through the birds' migration pathways. most of the birds nesting in or migrating through the volga delta overwinter in africa. 14 thus, africa is the main source of penetration of wnv genotypes i and ii into southern europe and western siberia. in asia, a peculiar third genotype of wnv appears to be circulating in the indian subcontinent. 11 a prototypical strain of wnv genotype 3 was isolated from xculex vishnui mosquitoes in southeastern india, and human morbidity was identified in india, pakistan, and israel. taking into account the fact that most of the birds from western siberia and many from eastern siberia overwinter in india and other countries of southern asia, there is a high probability that wnv genotype 3 has penetrated into siberia. also in asia, both epidemics and sporadic cases etiologically linked with the first genotype of wnv have arisen regularly in israel since at least1958. one such outbreak was observed in 1999à2000. 15 surveillance in south korea does not indicate any wnv circulation in that country. 16 in australia and oceania, the kunjin variant of the first genotype of wnv appears to be circulating. 17à19 kunv could be introduced into northern eurasia (in eastern siberia and the far east) by migrating birds overwintering in southeastern asia and australia. 11, 14 in 2011, an outbreak among horses in new south wales, australia, was identified. 20 in central europe, for a long time only two strains of wnv were known: one isolated in from aedes cantans in 1972 in western slovakia and the other isolated from ae. vexans, ae. cinereus, and culex pipiens in 1997 in the czech republic, near the austrian border. anti-wnv antibodies were identified in the czech republic among 1.4à9.7% of birds, including crows, daws, turtle doves, common kestrels, ducks, coots, and thrushes. later, two strains of the so-called rabensburg genotype of wnv were isolated from cx. pipiens in 1997 and 1999 in the czech republic. 21à23 the strain belonging to the second lineage of wnv was isolated from a goshawk in hungary. 7 in 1996 in tuscany, italy, usutu virus (usuv), which is closely related to wnv, was isolated during an epizootic episode among birds, especially thrushes (turdus merula), and then, again, in 2001 in austria. later, this virus was found in hungary, switzerland, and germany. 24 practically all of the southern european countries are endemic for wnv. 25, 26 especially tragic events unfolded in romania, where there was an epidemic in julyàoctober 1996 with a peak at the end of august to the beginning of september in the southeastern part of the country, downstream of the danube river. six administrative units and bucharest were affected, among other jurisdictions. human morbidity reached 12.4%, and 835 patients with cns insult were hospitalized. the number of patients with fever was at least 10 times more, and the number of infected individuals 100à300 times more. the outbreak, which dragged on until 2000, 27 testifies to the development of a city epidemic form of wnf. the virus belonged to the first genotype of wnv and probably was brought to romania by birds from africa. wnv distribution in europe indicates an especially high risk of a wvf outbreak in deltas of the large rivers-the rhô ne in france and the danube in romania-through which the main migratory paths of birds overwintering in africa lie. 14 in the recent past, wnv has been active in europe in italy, 28, 29 greece, 30, 31 spain, 10 poland, 26 the czech republic, 3, 22 and france. 22 infected mosquitoes were imported into great britain from the united states by airplane travel. 32 as for north america, before 1999 that continent was free of wnv. penetration of wnv into america most likely happened by infected mosquitoes in the holds of ships from ports in the mediterranean sea or black sea. 11 fifty-six cases of human wnf were revealed in new york city and its surroundings at the end of julyàseptember 1999, with a peak in the second half of august. seven cases (12.5%) had a lethal outcome. the virus was found in culex sp. and aedes vexans mosquitoes caught in septemberàoctober in new york city and in the states of new jersey and connecticut. positive results were obtained by rt-pcr during an investigation of brain tissues of dead birds: crows, seagulls, storks, herons, ducks, cuckoos, pigeons, jays, robins, hawks, and eagles. the genomes of the strains that were isolated were found to belong to the first genotype and were close to the strains isolated in 1996 in romania and in 1998 in israel. 33 in 1999, wnv was registered in the united states, probably translocated there by migrating birds or by infected mosquitoes inhabiting the holds of visiting ships. wnv was found in by 2003à2004, practically all the territory of the united states, southern canada, and latin america became endemic with high morbidity and mortality. 34 the greatest morbidity in the united states was found in the states of northdakota, south dakota, and nebraska. 27, 35 the number of diseased individuals reached 4,000à9,000 cases in separate years. during 1999à2006 in the united states, more than 16,000 wnf cases were identified, with more than 600 (4%) succumbing to the disease. the economic damage was estimated in billions of dollars. 36, 37 today, wnv continues to circulate in the united states. 38, 39 morbidity grew in the states of louisiana and mississippi after hurricane katrina. 40 in montana, the infection rate of people living in close proximity to a colony of pelicans (pelecanus erythrorhynchos) is five times higher than in other regions of the state. 41 in a sea park in texas, grampuses (orcinus orca) contracted encephalitis and died, 42 and previous episodes of polyencephalomyelitis were revealed among seals (phoca vitulina). also in texas, three new genetic clades of wnv were found, testifying to rapid evolution of the virus on the american continent. 43 in 2012, an epidemic arose again, accompanied by a large number of lethal outcomes. in texas, a state of emergency was declared. northern eurasia. in northern eurasia, on the basis of the results of multiple investigations, the distribution of wnv includes moldova, ukraine, belarus, armenia, azerbaijan, georgia, kazakhstan, tajikistan, kyrgyzstan, uzbekistan, turkmenistan, the south of the european part of russia (the desert, semidesert, steppe, and forestàsteppe landscape belts), and western siberia. 11, 35, 44 the first data on wnv isolation were obtained from hyalomma marginatum ticks collected in the astrakhan region in 1963. data were also obtained in azerbaijan from a blackbird (turdus merula) and a european nuthatch (sitta europaea) and, later, from a herring gull (larus argentatus) and argasidae ticks (ornithodoros coniceps) parasitizing it. 14 wnf morbidity is now a permanent feature in the astrakhan region, kazakhstan, central asian countries (republics of the former ussr), ukraine, and azerbaijan. virological, entomological, zoologicoornithological, and epidemiological investigations of wnv in the astrakhan region and the kalmyk republic were conducted especially actively. 8,39,45à61 virus activity in the volga river delta was found at least as far as 50 years ago. 11, 35, 60 but interactions between wnv, on the one hand, and animal and vector populations, on the other, were not investigated in detail as well as genetic characteristics of the virus; indeed, the latter began to be studied well only during the first decade of twenty-first century, when suckling mice and vero-e6 cell culture were used to isolate the virus and serological investigations were employed to detect viral rna (neutralization testing, elisa, hit) and to sequence genes (rt-pcr). wnv endemic territories in southern russia were known from the moment the virus was isolated in the astrakhan region in 1964. (the number of cases confirmed by elisa in the southof the european part of russia is presented in table 8 .33.) sporadic cases with a moderate clinical picture and minor outbreaks were observed in the area practically annually, as well as in other southern regions of the former soviet union. the immune structure to wnv among humans in the ussr was also known, with the most immunity occurring in the south of russia, mainly the astrakhan region (figure 8 .62, table 8 .34). all this familiarity with wnf is why an outbreak in 1999 in volgograd was not exactly unexpected, 62 even though it originally was identified by regional experts as an enterovirus infection. still, laboratory-confirmed wnf cases reached more than 500 that year, and according to our estimations, the number of infected patients exceeded 200,000 (table 8.35) . mortality (about 10%) was also unusually high. large deltas of european rivers such as the rhô ne, danube, and volga rivers are known to be transit hubs for migrating birds and places of introduction of viruses linked with birds. 14 the main natural focus in russia is the volga delta. the volga delta and contiguous territories around the northern caspian basin have been endemic for wnf for many years (tables 8.33à8 . 35) , and other arboviruses have been ecologically linked with aquatic and semiaquatic birds frequenting the region. ninety percent of these species of birds overwinter on the african continent. up to 100,000 birds pass over the region daily during their seasonal migrations via the volga delta main line of the eastern europe migratory route. (see figure 3.2 .) the problem is that the volga delta is the place from which viruses are introduced into anthropogenic biocenoses in close vicinity to human habitation. one consequence of this scenario was epidemic outbreaks in the astrakhan and volgograd regions in 1999à2001. the volga delta consists of three basic belts, each with its own unique ecosystem features (figure 8.63) . the lower volga delta borders the caspian sea and is characterized by extensive exposed spaces with water. the water depth usually does not exceed 1.0à1.5 m, a situation that is highly conducive to the mass propagation of mosquitoes and one that also provides nesting opportunities for aquatic and semiaquatic birds. near where it empties into the caspian sea, the volga bed turns significantly to the west, so the western part of the delta, including both the reed bed of the northwestern caspian coast (up to lagan in the kalmyk republic) and some flooded islands, is more extensive than the central and eastern parts. the extreme eastern part of the delta lies in kazakhstan. a number of hunters and fishermen could be infected in the lower delta of the volga. the middle volga delta is more distant from the sea, has powerful currents, and consists of shallow lake ecosystems with reeds and shrubs. water ecosystems adjoin semidesert ones. within the limits of this zone, wild biocenoses combine with anthropogenic areas around a number of settlements, whose inhabitants keep cattle, sheep, and camels. wnf is widely registered among the native population. the upper volga delta adjoins the volgaà akhtuba lowlands and semideserts. large cities, including astrakhan, are located within the limits of this zone. some species of wild birds that are common in the middle delta also occur in this zone, coming into close contact with domestic animals and synanthropic birds. analysis of retrospective data collected before 1999 revealed that the main locus of native-population morbidity by wnf is in the volga delta (table 8 .35). viruses could be introduced into the northern part of the volgaàakhtuba lowland up to volgograd and maybe even higher. thus, in the future it will be necessary to control the introduction of the virus into the volgaàakhtuba lowland from astrakhan to volgograd. arid landscapes occupying contiguous terrian to the west of the volgaàakhtuba system and the volga delta are situated within the boundaries of the caspian seaàturanian basin physicogeographical area (figure 8.63) . every year at the end of july, a group of specialists from the d.i. ivanovsky institute of virology in moscow has traveled to the astrakhan region and the kalmyk republic to organize and conduct a joint scientific expedition with local centers of sanitaryàepidemiological inspection for ecologo-virological monitoring of the northwestern caspian region (figures 8.64à8.66) . the main goal of the expedition is to contain the ecological and epidemiological situation after suppression of wnv circulation in the previous epidemiological season as the result of a combination of natural factors. the plan for the collection of field material took into account the results of previous expeditions, when key milestones and marker species of mosquitoes and wild and domestic animals were identified. in particular, the researchers planned to investigate the role of the ixodidae tick hyalomma marginatum (figure 8 .67) in wnv and other arbovirus circulation in anthropogenic and wild biotopes. both federal and local heads of various services, as well as virologists, epidemiologists, veterinarians, hunters, and frontier guards, were supplied with materials containing evaluations of ecologo-virological monitoring of their respective territories in the previous epidemiological season. practical recommendations were given for prophylaxis of wnf, cchf, and other arboviral diseases. field materials-bloodsucking mosquitoes, ixodidae ticks, internals (blood, serum, liver, spleen, and brain) of wild birds and mammals, and sera from donors and domestic animalswere collected on the territory of the astrakhan region and the kalmyk republic from the end of july to the beginning of august 2000à2004 within the boundaries of the volga delta, the volgaàakhtuba valley, and adjacent arid landscapes. field materials were collected in the biotopes of the west volga coast and the east akhtuba coast, including internal wateràmeadows of the upper and lower volgaàakhtuba zones, hydromorphic and adjacent meadowàsteppe biotopes of the upper and meddle belts of the volga, the volga avandelta, the territory of the sarpa lakes, and the east side of ergeny (see figures 8.64à8.66 ). during 2000à2004, the expedition collected 504,731 bloodsucking mosquitoes (of the order diptera and family culicidae: genera culex, aedes, coquillettidia, and anopheles); 11,266 ixodidae ticks (of the taxon acari and family ixodidae: genera hyalomma, rhipicephalus, and dermacentor), mainly h. marginatum; internal parts of 2,794 birds and 67 hares (lepus europaeus); sera from 4,500 human donors (2,500 in the astrakhan region and 2,000 in the kalmyk republic); and sera from 5,300 domestic animals (2,900 in the astrakhan region and 2,400 in the kalmyk republic) (figure 8.68 ). the field materials that were collected were stored and transported to the d.i. ivanovsky institute of virology in liquid nitrogen in dewars, in accordance with all requirements for the handling and transport of infectious samples. internal parts of 2,794 wild birds were investigated by virological methods (table 8 .36). twelve wnv strains (tables 8.36 and 8.37) were isolated. according to the bioprobe method used, the total wnv infection rate among wild birds is about 0.4%, with the highest level (0.7%) reached in the middle and rt-pcr testing for any indication of wnv rna was carried out on 108 samples of internal parts collected from wild mammals on the territory of the northwestern caspian region. positive results are presented in tables 8.42 detected in anopheles messeae (0.028%), a common visitor to houses with domestic animals in anthropogenic biocenoses, as well as in an. hyrcanus (0.026%) in rushes in natural biocenoses. as is illustrated in figure 8 .73, the highest intensity of wnv circulation takes place among sanguivorous mosquito populations in anthropogenic biocenoses on the territory of the volga delta (figure 8.74 ). rt-pcr testing was carried out for the detection of wnv rna in 11,266 samples of hyalomma marginatum ticks (taxon acari, wnf cases began to be registered starting in june 2001, with the maximum reached in august (figure 8.76) . durint the first three 27 .0% of patients in the latter group had intracranial hypertension syndrome. there were two cases of severe disease: a 71-year-old patient with seromeningitis and an 8-year-old child with neurotoxic syndrome during the acute period. all of the cases had a favorable result: no lethal cases were registered. sera from 2,884 farm animals collected in the astrakhan region during 2001à2004 were tested by hit and neutralization testing in order to detect specific anti-wnv antibodies. in addition, hit-positive sera underwent neutralization testing. anti-wnv antibodies were found by hit in all species investigated: horses (mean positive result for the entire observation period, 9.8%; coincidence with neutralization testing, 94.1%), cattle (6.4%; 72.0%), camels (5.2%; 41.7%), pigs (3.1%; cattle are the main host of anopheles messeae, and cowsheds offer favorable conditions for the mosquitoes to reproduce. cattle-specific antigens could often be found in the intestines of culex pipiens females (but not an. messeae females), which inhabitat damp basements. town utilities adjoin with farm utilities in all settlements of the astrakhan region, so cattle are the hosts both for an. messeae and for cx. pipiens. both species of mosquitoes are active vectors of wnv in anthropogenic biocenoses. horses were the only species of farm animals with clinically expressed wnf. in contrast to cattle, whose pastures are situated close to human settlements, horses browse far from settlements, often grazing in natural biocenoses. a significant portion of horse livestock in the astrakhan region are of the kushum breed, bred for meat and racing, and browse freely all year. pedigree horses (don, akhaltekinsky and arabian race horses) are kept in bloodstock farms in a stall, or they browse locally. draft horses are kept in settlements. horse-specific antigens have been found in the intestines of replete females of all mosquitoes species (except for culiseta annulata, which are relatively fewer). the total (2001à2004) distribution of hitpositive horses increases from the upper volgaàakhtuba to the lower, with the highest number found in the middle belt of the volga delta (where the epicenter of the natural foci is located). pigs are the animals closest to human settlements, so pig-specific antigens are often found in the intestines of replete females of the anthropogenic mosquito species anopheles messeae and culex pipiens. pigs are kept in individual yards or on pig farms. the latter are situated far from human settlements. as they are in cattle housing, an. messeae are the main mosquito species on the pig farm; nevertheless, all mosquitoes collected here by probe were negative for wnv. in 2003, we collected sera on the pig farms, and all probes were hit negative. in 2004, we collected sera both on pig farms and in individual yards. sheep are the most numerous species of farm animal in the astrakhan region. sheep pastures are in the dry steppe, where conditions are favorable for the ixodidae tick hyalomma marginatum. only a couple of species of mosquito could live in the saltish, dry steppe il'mens: aedes caspius and cx. modestus. the latter is an active vector for wnv. a stable and low level of infection rate among sheep (about 2%) reflects the low level of intensity of wnv circulation in arid landscapes of the astrakhan region. kalmyk racing camels inhabit more arid landscapes than sheep inhabit; consequently, one might expect a lower level of seropositive camels. however, hit often demonstrates a high percentage of positive results: 33.3% in 1989 and 13.9% in 2001. so, we instead collected sera from camels during 2002à2004 in semiwild pastures, and the percentage of seropositive results decreased. the coincidence between the results of hit testing and neutralization testing is presented in table 8 .46. horses are the best marker of wnv circulation, because they have the largest percentage of hit-positive results and the greatest coincidence between hit and nt results. kushum race horses are the most significant marker. monitoring the infection rates among farm animals will be continued, taking into account the relationships and phenomena described. it has been found that wnv can remain viable during interepidemiological periods in overwintering imagoes of sanguivorous mosquitoes (e.g., anopheles messeae, culex pipiens and culiseta annulata) as well as overwintering imagoes of the ixodidae tick hyalomma marginatum. the scheme of wnv circulation on the territory of the northwestern caspian region is presented in figure 8 .77. after the 1999à2006 outbreak of wnf in four administrative units in southern russia, a significant outbreak with more than 500 cases arose in the summer and autumn of 2010. the disease spread up to 500 km to the north and northeast from an earlier known endemic area and now includes an additional two administrative units (tables 8.47 the orthomyxoviridae includes six genera of enveloped viruses with a segmented, negative-polarity ssrna genome. the genome of the orthomyxoviruses consists of six (thogotovirus and quaranjavirus), seven (influenza c virus), or eight (influenza a virus, influenza b virus and isavirus) segments. 1,2 all orthomyxoviruses encode three enzymes formed of viral rdrp: pb1 (figure 8.79) , pb2, and pa. these proteins are about 30% similar among viruses of different genera. common structural proteins are np, associated with genomic rna; matrix protein; and two envelope proteins: hemagglutinin, or ha (possesses hemagglutinating activity) and neuraminidase, or na (also called sialidase) in the influenza viruses. viruses of the thogotovirus and quaranjavirus genera are transmitted by arthropod vectors, predominantly ixodidae and argasidae ticks, respectively. viruses of the influenza a virus, influenza b virus and influenza c virus genera are important human pathogens transmitted by a respiratory route. 1 genus isavirus has only one species: infectious salmon anemia virus, which strikes fish in the salmonidae family. genus influenza a virus has just one named species: influenza a virus, represented by numerous antigenic and genetic subtypes. the genome of influenza a virus consists of 8 segments of ssrna that encode 11 or more proteins. 1à5 influenza a viruses are divided into distinct subtypes based on the antigenic and genetic properties of their ha and na proteins. sixteen subtypes of ha (ha1à16) and 9 subtypes of na (na1à9) have been found worldwide in aquatic birds. two additional subtypes of ha (ha17 and ha18) and na (na10 and na11) are seen in new world bats. 4,6,7 h17 and ha18 form a clade distinctly history. influenza as a human disease was originally described in 412 b.c. by hippocrates (figure 8 .82) in his book epidemics, but the "father of medicine" did not consider influenza to be an infectious disease. instead, the famous english physician thomas sydenham (figure 8.83 ) was the first who suggested the infectious nature of the disease. 1, 2 the term "influenza" has been around since the first half of eighteenth century and derives from the italian "influenza di freddo" ("influence of the cold") or from spanish "influencia de las estrellas" ("influence of the stars"), the latter reflecting the contemporaneous belief in astrological reasons for the emergence of disease. 3 up to the nineteenth century, the archaic terms "catarrhus epidemicus," "cephalgia contagiosa," "febris catarrhalis" and "febris comatose" had wide currency. 4 the english word "grippe" (related to the russian "грипп") is related to the german "greifen" ("to catch hold") and derived from the french "gripper" ("to catch hold," "paralyze"); the word gained currency at the beginning of nineteenth century. (cf., e.g., the passage from volume 1, chapter 1 of tolstoy's famous novel war and peace: "she was, as she said, suffering from la grippe; grippe being then a new word in st. petersburg, used only by the elite."). 5 before the nineteenth century, influenza a epidemics were described only qualitatively. subtypes of the etiological agent were retrospectively revealed for the 1889à1892 epidemic (h2n2), the 1897à1900 epidemic (h3n8), and the 1918à1919 pandemic (h1n1, the so-called spanish flu) 5à9 -retrospectively only because influenza a virus wasn't found until 1930 by richard shope (figure 8 .84) on the model of swine (sus scrofa) flu. 10, 11 human flu was found two years later 12,13 by a group of english scientists: wilson smith (figure 8 .85), christopher andrewes (figure 8 .86) and patrick laidlaw (figure 8.87) . during the pandemic of 1918à1919, it was suggested that the etiological agent of influenza a was the socalled afanasievàpfeiffer bacillus," 14à16 named after the russian bacteriologist mikhail afanasiev (figure 8 .88) and the german bacteriologist richard pfeiffer (figure 8 .89)-the modern haemophilus influenzae bacillus. 17, 18 three influenza a pandemics were described after the discovery of the etiological agent: the avian flu has been known under the name "lombardian disease" since the beginning of the nineteenth century. 31à34 in 1878, the italian veterinarian edoardo perroncito (figure 8 .90) described a highly contagious disease (previously named "exsudative typhus of chickens") among chickens, with 100% lethality in the vicinity of turin. 35 the terms "classic fowl plague" and "bird pest" came into wide use in 1901, when a large epizootic outbreak in tyrol province, italy, did away with the population of farm birds there. 33 the term "braunschweig disease" was used to identify an analogous disease among guinea fowls in europe. in 1901, the italian scientists eugenio centanni and ezio savonuzzi demonstrated that the etiological agent of classic fowl plague is a filtrated substance. 34 nevertheless, classic fowl plague wasn't identified as influenza a virus until 1955, by werner shäfer (figure 8 .91) on the example of the historical strain a/chicken/brescia/1/1902 (h7n7). 36, 37 w.b. becker was the first who identified influenza a virus among wild birds when he subtypes of influenza a virus in northern eurasia. at present, we know that numerous avian influenza viruses are abundant in the bird populations of northern eurasia. however, until the end of the 1960s, these data were absent. at that time in the former ussr, avian influenza a virus was being isolated only from poultry. one of the first avian viruses isolated in the ussr-a/duck/ukraine/1/1963-was destined to play an important role in the development of the theory of influenza virus evolution. 43 in 1960à1964, a group of researchers in the ukrainian soviet republic isolated several influenza virus strains from ducklings affected with sinusitis. the first three strains were isolated in 1960 in crimea and in the kharkov astrakhan region 95 24 49 31 11 25 73 16 1 1 5 22 18 67 70 508 volgograd region 380 32 15 15 3 12 63 2 5 411 57 institute of virology in moscow. as early as 1964, the duck strains ya-60, b-60, z-61, and c-61 were analyzed with respect to their antigenic specificity by hit and were found to be antigenically distinct from the human h1 and h2 viruses. 47 after the appearance of the h3 pandemic virus in 1968, some of the ukrainian duck strains were shown to be antigenically 48, 49 moreover, hit testing also showed that the b-60 and bv1 strains of the virus reacted with human sera, including those collected in 1881à1886 and in 1905à1908. on the basis of this phenomenon, the authors suggested that an avian virus similar to the strains b-60 and bv1 was the precursor of the human pandemic strain and that this antigenic variant had appeared in humans several times in the past. 48 formerly known as ya-60, strain a/ duck/ukraine/1/1960 was shown 50 to belong to the h11n2 subtype, whereas a/duck/ ukraine/2/1960 was identified as h3n6 and a/duck/ukraine/1/1963 as h3n8. the highly pathogenic h5n2 and h7n2 strains were isolated from chickens in the moscow region. 51, 52 several virus strains producing enteritis in chickens were isolated in 1972 and in 1974 in chicken farms and identified as h6n2 strains, 51,53,54 an unusual antigenic formula for a pathogenic virus affecting poultry. six h3n2 isolates were obtained in a chicken farm in kamchatka from chickens affected with rhinitis, conjunctivitis, and laryngotracheitis. 51, 55 in 1977, isolates identified as h3n1 viruses were isolated from sick chickens and ducks in the russian federation 25 and uzbekistan in the former ussr. 26 in 1984, h8n4 strains were isolated in the western part of the ukrainian soviet republic from the lungs of ducklings affected with pneumonia. the isolation was the only one of an h8 influenza virus in the ussr (lvov dk, unpublished data). in 1970, a large-scale series of virus isolations from wild birds, combined with some serological studies, was initiated as a part of the coordinated program of the national committee on the studies of viruses ecologically linked to birds together with the virus ecology center of the d.i. ivanovsky institute of virology. by the end of the 1970s, the pattern of circulation of avian viruses in the territory of the ussr was identified. 3, 11, 26, 30 in the ensuing years, the pattern of the influenza a virus subtypes (including h15 and h16) circulating in northern eurasia was amplified (figure 8.92 ). blood sera collected in the spring and autumn of 1970 near lake khanka and peter the great bay (both in primorsky krai) from 262 birds-mainly mallards (anas platyrhynchos), common teals (an. crecca), baikal teals (an. formosa), garganeys (an. querquedula), falcated ducks (an. falcata), pintails (an. acuta), grey herons (ardea cinerea), coots (fulica atra), black guillemots (cepphus grylle) and blacktailed gulls (larus crassirostris)-were hittested against h1, h4, h5, h6, h10, and h11 avian influenza viruses. no antibodies were found in the sera of grey herons and coots, nor were any found against h11 in any species. antibodies against all the other subtypes tested were encountered occasionally in the sera of gulls, black guillemots, and ducks. in some species, such as teals, falcated ducks, and black guillemots, antibodies against several subtypes were detected. 27 in 1972, sera were collected from gulls, cormorants, murres, and tufted puffins in the commander islands. antibodies against h2, h3, h5, and h7 viruses were detected. 28 in 1970à1972, sera from gulls, cormorants, and murres were collected in the kamchatka, sakhalin, and magadan regions and antibodies to h1, h2, h3, h5, h6, and h7 viruses were detected. 30 antibodies against h1, h3, h4, h5, and h7 were identified in sera taken from arctic terns (sterna paradisaea), black-throated loons (gavia arctica), mallards (anas platyrhynchos), common teals (anas crecca), tufted ducks (aythya fuligula), greylag geese (anser anser), skuas (stercorarius sp.), and a blue whistling thrush (myophonus caeruleus) collected in the white sea basin in the estuary of the pechora river in the arkhangelsk region of russia in 1969à1972. 56 the serologic studies suggested a wide range of avian influenza viruses circulating in wild birds in northern eurasia. this suggestion was confirmed and extended by the isolation of virus strains from other wild birds. many avian species proved to be hosts of h1 viruses. a virus belonging to the h1n3 subtype was isolated in 1977 from a tern in the southern part of the caspian sea basin. 57 in 1978, an h1n4 strain was isolated from a common teal (anas crecca) in the russian republic of buryatia in eastern siberia. 41 several h1n1 viruses were isolated in kazakhstan in 1979 from waterfowl, including the common teal (an. crecca), garganey (an. querquedula), shoveler (spatula clypeata), and coot (fulica atra), 58 as well as in 1980 from tree sparrows (passer montanus) and hooded crows (corvus cornix). 41 in 1979, an h1n1 virus was isolated from a hawfinch (c. coccothraustes) in mongolia. 59 in the same year, an h1n2 strain was isolated from a black-headed gull (larus ridibundus) on an island in the northern part of the caspian sea. 41 the avian viruses belonging to the h2 subtype seem not to be abundant in russia. in fact, for a long time the only virological evidence of the presence of this subtype in russia was the isolation of an h2n3 virus in 1976 from a pintail (anas acuta) in primorsky krai. 60 however, serological data suggested that h2 viruses circulated in wild birds not only in primorsky krai, but also in other regions of the far east, including the commander islands as well as the kamchatka, sakhalin, and magadan regions. 54, 61 avian influenza a viruses belonging to the h3 subtype are widespread in northern eurasia. an h3n2 virus was isolated from a common murre (uria aalge) in 1974 on sakhalin island, 62 and another h3n2 strain was isolated in 1976 from a pintail (anas acuta) in primorsky krai. 63 two h3n2 strains were isolated in 1974 in the ukrainian soviet republic from unusual hosts for avian viruses: the white wagtail (motacilla alba) and the european turtle dove (streptopelia turtur). 64 h3n2 strains were also isolated from grey crows (corvus cornix) in 1972 in the volga basin and from a shelducks (tadorna ferruginea) in 1979 in kazakhstan. 65 an h3n2 virus was isolated from a tree sparrow (p. montanus) in 1983 in the ukrainian soviet republic. 66 in 1972à1973, h3n3 and h3n8 viruses were isolated from ducks and herons in khabarovsk krai. one of the viruses closely resembled a strain isolated a year later in central asia. this resemblance demonstrated that h3n3 viruses circulated in regions fairly distant from one another. 67 in 1972à1973, h3n8 viruses were isolated in khabarovsk krai from wild ducks (anas sp.), tufted puffins (fratercula cirrhata), and horned puffins (f. corniculata) 65 and in the arkhangelsk region in the pechora river estuary (white sea basin) from arctic terns (sterna paradisaea) and black-throated loons (gavia arctica). 68 in 1978, h3n8 strains were isolated in the republic of buryatia from a mallard (an. platyrhynchos) and a pintail (an. acuta), 65 as well as in khabarovsk krai from the common murre (u. aalge) 67 and from black-headed gulls (larus ridibundus). 69 avian viruses of the h4 subtype were isolated in 1970à1980 mostly in a narrow belt stretching from the lower volga, through kazakhstan, and on to the south of eastern siberia. several h4n6 strains were isolated in 1976 from slender-billed gulls (chroicocephalus genei) in the volga delta 70 and from great black-headed gulls (ichthyaetus ichthyaetus) on the islands in the northern part of caspian sea. 41 in 1977, h4n8 virus was isolated from the black tern (chlidonias niger) in central kazakhstan. 71 in the republic of buryatia, h4n6 strains were isolated in 1978 from the common goldeneye (bucephala clangula). 41 isolations of h5 influenza viruses from wild birds were scarce. in 1976, several h5n3 strains were isolated from terns (common terns and little terns) and a slender-billed gull in the volga river delta. 70 a detailed description of the penetration of the h5n1 strain of of highly pathogenic avian influenza (hpai) a into northern eurasia and its further dissemination is presented shortly. the strains belonging to the h6 subtype seem not to be abundant, but their geographic distribution is wide. an h6n2 strain was isolated in 1972 from the arctic tern (sterna paradisaea) 68 in the arkhangelsk region (white sea basin). one h6n4 strain was isolated in 1978 from the pintail (anas acuta) in primorsky krai, 41 and an h6n8 strain was isolated from the common tern (s. hirundo) in 1977 in the caspian sea basin. 57 in 2010, two h6n2 strains were isolated on kunashir island (the southernmost of the kuril islands) and four were isolated on sakhalin island. an h7n3 strain was isolated in 1972 from a sandpiper (a member of the scolopacidae family) in the arkhangelsk region of russia. 68 one strain of h8n4 was isolated in 2001 in the republic of buryatia, and one strain in 2003 in mongolia. an h9n2 strain was isolated from a mallard (anas platyrhynchos) 72 in primorsky krai in 1982 and in khabarovsk krai in 2013. over 40 h10n5 strains were isolated from a wide array of bird species near alakol lake in east central kazakhstan in 1979. the strains were isolated from several species of ducks (anas sp.), from shorebirds (members of the order charadriiformes), to passerine birds (members of the order passeriformes), to coots (fulica atra), plovers (members of the family charadriidae, subfamily charadriinae), and chukars (alectoris chukar). 41 this situation is a rare case of an isolation of closely related viruses from an extremely wide array of avian species. the viruses identified as h11n8 strains were isolated in 1972 from the arctic tern (sterna paradisaea) and the red-throated diver (gavia stellata) in the estuary of the pechora river in the northern part of european russia. 54 several h11n6 strains were isolated from the common teal (anas crecca), the european widgeon (an. penelope), and the european golden plover (pluvialis apricaria) in 1979 in eastern siberia. 41 in 1987, h12n2 strains were isolated from mallards, a pintail, and european widgeons south of issyk-kul lake in kyrgyzstan. 72 two strains of h12n2 were isolated from wild ducks (subfamily anatinae) in kyrgyzstan. the results of virus isolation and serological studies in the territory of the ussr in 1970à1980 suggested a wide circulation of avian influenza viruses in wild birds and enabled researchers to construct a map of avian influenza viruses encountered in different regions of northern eurasia. the general pattern of distribution of influenza virus subtypes in wild birds was fairly evident by the end of the decade. virus isolation was continued in the ensuing years, and it brought 290 8. single-stranded rna viruses several major results. isolations were performed mostly in the central and southern parts of european russia, in western and eastern siberia, and in the russian far east. 72 overall, 1,005 strains were isolated from wild birds in russia in 1980à2013 (table 8 .49). about 250 samples were taken yearly from 50 to 100 birds in each geographic region. the mean percentage of successful isolations ranged from 3.5% to 5.7%. over 50% of the isolates were h13 viruses (h13n2, h13n3, h13n6, and h13n8) isolated mostly from gulls and shorebirds in the northern part of the caspian sea. the viruses of the h3 subtype (over 25% of the total number of isolates) were isolated in several regions. many strains isolated in 1979à1985 from great black-headed gulls (ichthyaetus ichthyaetus), herring gulls (larus argentatus) and caspian terns (hydroprogne caspia) on the island of maly zhemchuzhny in the northern part of the caspian sea were not identified at the time of isolation with respect to the subtype of their ha. as it turned out, the strains belonged to the subtype h13, was first described in 1982, 73 and in 1989 the mysterious caspian isolates were identified 74 as h13n2, h13n3, and h13n6. to characterize the h13 subtype molecularly and antigenically, the complete nucleotide sequence of the ha of the strain a/great black-headed gull/astrakhan/ 277/84 was used for comparison with the has of two american strains isolated from a gull and a pilot whale. 75 virus isolation studies in the northern caspian basin were continued in the 1990s and 2000s. materials were collected from wild birds in the area of the northern coast of the caspian sea (including maly zhemchuzhny island) from the delta of the terek river in the north caucasus region to the emba river in western kazakhstan. most of the strains that were isolated belonged to the h13 subtype, including h13n2, h13n3, h13n6, and h13n8 isolates; besides these strains, only single isolates belonging to the h4n3, h4n6, h6n2, and h9n2 subtypes were isolated. 76, 77 in 1990, a new, previously unrecognized, subtype of influenza virus h14 ha was described 78 on the basis of the characterization of two strains isolated in 1982 from mallards (anas platyrhynchos) in the ural river delta. the h14n5 and h14n6 subtypes were isolated from mallards and gulls in astrakhan. 76 a partial sequencing revealed that ns gene of the h14 strains isolated from the gulls was closely related to the ns gene of h9 and h13 strains isolated previously from gulls and terns in the caspian sea basin and to the h9n4 strain isolated in the russian far east. the ns gene of an h14n5 strain isolated from a mallard was much more distantly related to the ns gene of the viruses isolated from gulls. 76 the results suggest that reassortment events play a significant role in the evolution of h14 viruses, with the ns gene being an important determinant of the range of the host. a large-scale isolation of avian influenza viruses from fecal samples was performed in 1995à1998 in eastern siberia and the far east by a group that included both russian and japanese researchers. 79 scientific contacts between russian and japanese researchers of avian influenza a virus were ongoing during the eighth russianàjapanese consultations at a conference titled "protection of migratory wild birds in the asiaàpacific region" held at the russian ministry of natural resources in moscow april 01à05, 2011. at the conference, the d.i. ivanovsky institute of virology took the initiative to renew the international meetings on medical ornithology at the level of experts of asiaàpacific countries that had been taking place regularly during the 1970 and 1980s. as a result, the first international meeting for medical ornithology in the asiaàpacific region was held in tokyo, japan, on june 23, 2011. the meeting was devoted to the topic of hpai h5n1 distribution in asia. a second meeting was conducted in moscow at the d.i. ivanovsky institute of virology march 15à16, 2012 (figure 8.93) . 80 in the summer of 2000 in a valley in the sayan mountains in southeastern siberia, the strains h3n8, h7n1, h7n8, h13n1, and h13n6 were isolated. 81 the h3n8 and h7n8 strains were isolated from ruddy shelducks (tadorna ferruginea) and common redshanks (tringa totanus), the h7n1 strains from common pochards (aythya ferina), and the h13n1 strains from northern shovelers (anas clypeata) and great crested grebes (podiceps cristatus). the h13n6 strains were isolated from all of the aforementioned species, as well as from teals, ducks, and terns. in 2000à2002, the subtypes h3n8, h4n2, h4n6, h4n8, h4n9, h5n2, h5n3, h9n2, and h13n6 were isolated in the same region; 1,750 samples were taken from 48 bird species. 72 a strain isolated from the muskrat (ondatra zibethicus) 81 in 2000 in the republic of buryatia was identified as an h4n6 virus closely resembling the h4n6 strains isolated from ducks in the same year and the same region. 72 the has of the h4 strains (including the muskrat strain) isolated in buryatia formed a separate group of the eurasianàaustralian branch in the phylogenetic tree of h4 ha (figure 8.94 ). they had a c-terminal proline residue in their ha1 subunit, in contrast to the serine residue of most eurasian strains. the ha genes of the h5n2 isolates turned out 82 to have cleavage peptides lrnvpqretr/gl identical to the ones of the low-pathogenic strains isolated from ducks in hong kong and malaysia. in contrast, the has of h3 and h4 strains isolated from teals in 2002 and from mallards in 2003 near lake chany in novosibirsk region western siberia, were related to the has of the european h3 and h4 strains. 83, 84 interestingly, the has of the h3 strains were closely related to the ha of a/duck/ukraine/1/1963 (h3n8). 83 however, unlike the has of h3 and h4, the has of h2 strains isolated in the same area in 2003 from mallards resembled the has of h2 strains isolated in 2001 in japan from mallards (anas platyrhynchos). 84 in 2003, influenza a virus strains belonging to a rare subtype h8n6 were isolated in mongolia from the great cormorant (phalacrocorax carbo), white wagtail (motacilla alba), and magpie (pica pica). 85 penetration of hpai h5n1 into northern eurasia: reasons and consequences. during longitudinal wide-scale monitoring of influenza a viruses among wild bird populations in northern eurasia, several h5n2 and h5n3 strains were isolated in 1976 and 1981 in the caspian sea basin. 70, 74 more recently, in 1991à2001, strains belonging to the same subtypes were isolated in siberia, and their features proved to be relevant to h5 virus circulation. onn the one hand, the has of the strains isolated from teals in 2001 in primorsky krai, as well as the has of strains isolated from a mallard in lake chany in western siberia in 2003, were shown to be closely related to has of h5 strains isolated in 1997 in italy from poultry. 79, 82 on the other hand, the ha of the h5n3 strain isolated from a wild duck as early as 1991 in altai krai in southwest siberia was closely related to the ha of a/duck/malaysia/f119-3/ 1997 (figure 8 .95). the ha of the altai (1991) and lake chany (2003) viruses had a monobasic ha1àha2 cleavage site, and, accordingly, it had a low-pathogenic avian influenza (lpai) phenotype. 72, 79, 82, 86 besides the amino acid sequence of the ha, the sequences of other genes of the h5 viruses isolated in russia proved to be relevant. the np genes of the h5n2 and h5n3 strains isolated in primorsky krai in 2001 formed a separate cluster in the phylogenetic tree, together with the np genes of the h4n6 strains isolated from common shelducks (tadorna ferruginea) and common pochards (aythya ferina) in the republic of buryatia in 2000, the h2n3 strain isolated from the northern pintail (anas acuta) in primorsky krai in 1976, and the 43, 72 however, they were very distantly related to the np genes of h3n8, h6n1, and h5n1 strains isolated from poultry and humans in southeast asia in 1996à2001 and to the np genes of h4n8 viruses isolated from wild ducks in the caspian sea basin in the european russia in 2002. by contrast, unlike the np genes, ns genes of the strains from primorsky krai were closely related to the ns genes of the h5n1 and h4n8 viruses isolated in southeastern asia in 1997à2001, as well as to the ns genes of an h4n8 virus isolated in the caspian sea basin in 2002 (figure 8.97) . 43, 72 an abundance of influenza a subtypes in the avian populations of northern eurasia provides excellent conditions for gene exchange. the extent of the exchange is demonstrated by the relatedness of different genes of the russian isolates to the genes of european strains, on the one hand, and south asia isolates, on the other. 72, 76, 83, 84 the exchange is to a certain extent restricted by host specificity, but this restriction is not rigid, and the virus genes frequently traverse interspecies barriers. avian migration routes crossing russian territory are an important factor in the gene flow. the extensive intra-and interspecies contacts in the natural habitats of wild birds in russia stimulate rapid virus evolution and the appearance of new variants through reassortment events and, presumably, through the postreassortment adjustment of genes, thereby restoring the functional intergenic match. 87, 88 another factor may be the occurrence of avian influenza viruses in lake water, first registered in 1979 in eastern siberia. 41 this phenomenon might provide a means for the temporal as well as territorial transfer of genes, as suggested by the recent detection of influenza 89 thus, the sequencing data suggest that there exists an extensive exchange of genes of the avian influenza viruses circulating in europe, siberia, and southeast asia along the avian migration routes connecting europe, through the russian territory, with southeastern asia, the cradle of potentially pandemic reassortant viruses. after the highly pathogenic h5n1 viruses began disseminating from southeastern 82 our second prediction was that overwintering migrating birds could transmit the hpai virus into northern eurasia during their spring migration. we discussed two possible routes by which the birds might introduce the virus: the dzungarian (indianàasian) migration route and the asianàpacific route. preparing for these two possibilities, we increased our surveillance in the southern part of western siberia (through the russian foundation for basic research project 03-а04-49158) and in primorski krai (through the international scienceàtechnical center project 2800) in the spring of 2004. in april of 2005, a wide epizootic outbreak emerged at kukunor lake (also called qinghai lake) in qinghai province, china, and from this location the virus could spread through the dzungarian gate, which links the northwestern mountain ranges of tibet with the western siberian lowland. our second prediction was confirmed as well, when hpai h5n1 first appeared in northern eurasia, in western siberia (novosibirsk region, russia) in the summer of 2005 (figure 8.98 ). although the official start of the epizootic among poultry was dated july 10, 2005 (table 8 .50), that one occurred among wild birds about 2 weeks before was retrospectively established. 5 the outbreak spread quickly and caused over 90% lethality among poultry. the virus isolations in the area were performed independently by two groups of researchers. a number of strains were isolated in zdvinsky district, novosibirsk region, by a group of researchers from the d.i. ivanovsky institute of virology in moscow. the materials for isolation (cloacal and tracheal swabs, pools of internal organs, and blood) were taken from dead, sick, and healthy birds at the farm where the epizootic occurred and from wild birds in the vicinity. 90, 91 three strains were isolated from dead chickens (gallus gallus domesticus), two strains from sick or dead ducks (anas platyrhynchos domesticus), and one strain from a healthy great crested grebe (podiceps cristatus). all of the strains were deposited into the russian state collection of viruses functioning under the auspices of the d.i. ivanovsky institute of virology ( 93 several features of the primary structure of virus proteins, such as lys627 residue in pb2 and glu92 residue in ns1, characteristic of highly virulent variants of h5n1 viruses, correlated with the high pathogenicity of the novosibirsk isolates. a deletion in the na gene in amino acid positions 49à60 indicated that the strains belonged to the genotype z, which dominated in 2004 in southeastern asia. 94 the other group of strains was isolated by a team of researchers from the state research center of virology and biotechnology vector (also known as the vector institute) in koltsovo, novosibirsk region. two strains were isolated from chickens and one strain from a turkey in the village of suzdalka, dovolnoe district, in july 2005. the viruses were isolated from homogenates guangdong province, china. 95 the viruses were highly pathogenic to chickens in a laboratory test. 96 our third prediction was that the virus would move with the migrating birds to their overwintering locations. as it turned out, coincident with this prediction, epizootic outbreaks occurred along the main migration routse in the urals, the russian plain, europe, africa, central asia, and india figure 8 .99), 102 indicating the distribution of the virus through the eastern european flyway of birds (figure 8 .100), connecting western siberia, the russian plain, eastern europe, the middle east, and africa. 54 our fourth prediction was that the virus would return in birds migrating from their overwintering places to northern eurasia in the spring of 2006, with a widening of the epizootic. dramatic events occurred june 10à28, 2006, at uvs-nuur lake, which is situated on the boundary between the great lakes depression of mongolia and the tyva republic of russia (figure 8.98 ). an estimated 3,000-plus birds died in the russian part of this lake, which is only about 1% of the total area of the lake. the species most affected was the great crested grebe (podiceps cristatus); as also affected were coots (fulica atra) and cormorants (phalacrocorax carbo). terns and gulls were involved in the epizootic to a significantly less extent. the absence of poultry farms in the vicinity of uvs-nuur lake precluded outbreaks among poultry. the tyva strains appeared to be the beginning of a new genetic lineage in the qinghaiàsiberian genotype 2.2. the lineage was designated as a tyvaàsiberian subgroup 104 (figure 8 .99) that was isolated not only in siberia, but also in europe. it is believed (table 8 .51) from dead and sick poultry, and all the isolates were identified as hpai h5n1 (table 8 .52) with a high level of sequence similarity to the qinghaiàsuberian genotype 2.2 (figure 8.99) . this outcome implied a common source of infection for all the local outbreaks ( figure 8.101) , and subsequent epidemiologic investigation demonstrated a link to live-bird markets in moscow, where the affected farmers had purchased poultry several days before. a complete genome analysis of the prototype a/ chicken/moscow/2/2007 revealed 105 group of strains is shown with the use of braces: designations common to all strains in the given group are shown outside the braces; the variable part of the designations is cited inside the braces; the asterisk "*" means "any designation." only mutations that are found in all the strains of the given group are listed in the table. b bold font indicates substitutions with respect to hpai/h5n1/2.2 consensus; the frame -substitutions unique to northern eurasian strains (tables 1à2)-that is, they did not occur among northern eurasian strains previously; the frame with grey background -substitutions unique to all hpai/h5n1/2.2 genotypes (strains isolated in both northern eurasia and other places); {kc-substitution that takes place in the strains of the given epizootic outbreak only; {£c-substitution that takes place in the strains of both the given and later or previous epizootic outbreaks. valley ecosystem in the north or south caucasus in the winter of 2007 and was introduced into the live-bird market through contaminated poultry cages or contaminated grain. in september 2007 , an outbreak was detected in the northeastern part of the basin of the sea of azov on a chicken farm called "lebyazhje-chepiginskaya" in the krasnodar region of russia (figure 8.98) . the virus isolates-a/ chicken/krasnodar/300/2007 from poultry and a/cygnus cygnus/krasnodar/329/2007 from a sick whooper swan (cygnus cygnus) found in a "liman" (shallow gulf) near the farm-were closely related to each other (they had two synonymous nucleotide substitutions in pb1, two synonymous in pb2, one nonsynonymous in m1, two nonsynonymous in na, and one nonsynonymous in ns1) and belonged to the iranànorth caucasian subgroup of qinghaiàsiberian genotype 2.2 (figure 8 .99). the isolated strains contained 10 unique amino acid substitutions with respect to a qinghaiàsiberian consensus in pb2, pa, ha, na, and ns1, suggesting that regional variants were continuing to emerge. 106 in december 2007, a poultry farm called "gulyai-borisovskaya" in the rostov region became infected (figure 8.98) . unfortunately, the infection was not reported in time, and infected poultry manure was spread on adjacent fields, where wild terrestrial birds could be infected. 107 this exposure is thought to have contributed to the infection of a number of species. including rooks (corvus frugilegus), jackdaws (corvus monedula), rock doves (columba livia), common starlings (sturnus vulgaris), tree sparrows (passer montanus), house sparrows (passer domesticus), and more. surveillance of these species by rt-pcr detected h5 virus in 60% of pigeons and crows, in around 20% of starlings, and in 10% of tree sparrows, all without clinical features. these results were confirmed by viruses isolated from wild birds and poultry (table 8 .51). birds whose infection was confirmed by rt-pcr and virus isolation seemed reluctant to move and had ruffled feathers. on necropsy, the birds were observed to have had conjunctivitis; hemorrhages on the lower extremities and in muscle, adipose, intestine, mesentery, and brain tissue; and changes in the structure of the pancreas and liver. wide involvement of wild terrestrial birds in virus circulation, presumably from the exposure to infected chicken manure, distinguished this outbreak from others. genome analysis ( the qinghaiàsiberian clade includes viruses that have infected and caused severe disease and mortality in humans, but currently they do not appear to be transmitted efficiently in humans. upon analyzing representative viruses in our collection for their potential to replicate in mammals, we found that isolated strains replicated effectively in mammalian cell culture lines bhk-21, lech, vero e6, mdck, and spev. 5, 108, 111 pb2 has consensus k627 that promotes virulence in mammalian cells. 93 on the basis of the amino acid sequence of ha receptor-binding sites of qinghaiàsiberian isolates containing e202, q238, and g240, its affinity of qinghai siberian isolates for α2 0 -0 à3 0sialic acids was predicted. however, a double mutation q238 l and g-240 s or just a single mutation e-202 d could switch ha receptor-binding affinity from avian to human receptors. 113 all the qinghaiàsiberian isolates are sensitive to amantadine, rimantadine, and oseltamivir, as has been confirmed by both direct biological experiments in vitro 114 54 the first overwintering area could be the source for the iranànorth caucasian subgroup, the second for the tyvaàsiberian subgroup. returning to their nesting areas in northern eurasia in the spring of 2006, wild birds afforded a mixed virus population the opportunity to spread (figure 8.100) . 5, 24, 28, 42, 43, 108 a decrease in the potential of isolated strains to reproduce in vitro (figure 8 .102) is more evident in poultry (tcid 50 5 11.847à0.272 3 t) than in to wild birds (tcid 50 although hpai h5n1 has penetrated into northern eurasia through the dzungarian flyway of wild birds, this fact did not exclude the possibility of the virus transferring through other flyways -(e.g., through the far eastàpacific flyway). 54 indeed, in april with wild waterfowl. one initial theory of the introduction of the virus to poultry was from the birds' exposure to hunted ducks, but the direct interaction of wild birds with poultry seems more likely. the isolates (see table 8 .51) from dead chickens and the common teal (anas crecca) collected in the vicinity of epizootic farms were identical and indicated a direct role of migrating birds in the introduction of the virus. the teal, which appeared to be the most likely source of infection of poultry, had no obvious behavior changes but did have hemorrhagic lesions in the intestines on necropsy. it is interesting to underline the fact that common teals were the source of isolation of h5 (figure 8.99) . 117, 118 fortunately, both clades (2.2 and 2.3.2.1) of hpai h5n1 that had penetrated into northern eurasia had low epidemic potential because their receptor specificity did not switch from α2 0 à3 0 -to α2 0 à6 0 -sialoside affinity, a fact that was revealed by the primary structure of the ha receptor-binding region and direct testing in sialoside-based experiments in vitro. 5, 80 thus, we discuss the epizootic event provoked by hpai h5n1 in northern eurasia during 2005à2010 as a model of an emer-gingàreemerging situation in need of permanent ecologo-virological monitoring. influenza a viruses among mammals. the circulation of influenza a viruses among swine (order artiodactyla: family suidae, genus sus) was originally established in 1930 by richard shope (figure 8 .84): his investigations not only established the viral etiology of swine flu and isolated the first historical strain a/swine/iowa/15/1930 (h1n1), but also serologically demonstrated the close relation between human infection agents and those of swine. 11 shope's findings gave rise to a number of isolations of swine respiratory disease agents. many of these agents later turned out not to be influenza a virus; for example, "kö be porcine influenza virus," isolated in germany; 119 "infectious pneumonia of pigs;" 120,121 "beveridgeàbetts virus" 122 (more often, these pathogens belonged to chlamydia sp.); and "hemagglutinating virus of japan," 123,124 which initially was named "influenza d virus" and was later identified as sendai virus (sev) (family paramyxoviridae, genus respirovirus). 125 nevertheless, a number of strains isolated at the end of 1940s in korea (strain oti), 126 and in the 1950s and 1960s in lithuania (prototype a/swine/kaunas/353/ 1959), 127 estonia, 128 poland, 129 and russia 130 were identified as influenza a (h1n1) virus. also, in the middle of twentieth century, influenza a strains closely related to a/ swine/iowa/15/1930 (h1n1) were isolated in czechoslovakia 131,132 and hungary. 133 finally, after the beginning of the "asian flu" pandemic in 1957, swine influenza a (h2n2) virus strains were isolated initially in china 134 and later in czechoslovakia 135 the principal peculiarity of pigs is the presence of both α2 0 à6 0 -sialosides (typical of human cells) and α2 0 à3 0 -sialosides (typical of avian cells) on the surface of respiratory tract cells. this feature permits both human (or adapted swine) and bird influenza a virus strains to circulate simultaneously, giving rise to conditions favorable to the reassortment and emergence of virus variants with suddenly appearing new properties. 42,136à143 avian influenza a virus strains have been demonstrated to initiate productive infection in swine under experimental conditions. 31,144à147 the great number of reassortment forms of influenza a viruses isolated from swine constitute evidence of the extremely high reassortment potential of the swine viral population. thus, a/swine/england/191973/1992, isolated from nasal swabs of sick pigs in great britain in 1992, belongs to the unique h1n7 subtype, which was formed by the reassortment of a/ussr/90/1977 (h1n1) (the source of pb2, pb1, pa, ha, np, and ns segments) and a/equine/prague/1/1956 (h7n7) (the source of na and m segments). 148 151 the most evident illustration of the reassortment potential of swine populations is the emergence of the pandemic "swine flu" h1n1 pdm09 in 2009 as the result of the reassortment of two swine genotypes of the h1n1 subtype: the "american swine genotype" (the source of pb2, pb1, pa, ha, np, and ns segments) and the "european swine genotype" (the source of na and m segments) (figure 8.104 ). 24à29 using different receptor-mimicking sialosides (table 8 .55), we investigated the evolution of receptor specificity in influenza a (h1n1) pdm09 virus during pandemic and postpandemic epidemiological seasons. different types of sialoside specificity spectra are presented in figure 8 .105. to compare α2 0 à3 0 -and α2 0 à6 0 -sialoside specificities, we introduced the special parameter w 3/6 , which is the ratio of the optical density for flat α2 0 à3 0 -sialosides (3 0 sl and 3 0 sln) to the optical density for flat α2 0 à6 0 -sialosides (6 0 sl and 6 0 sln): if w 3/6 is ,1 (w 3/ 6 , 1.00), then α2 0 à6 0 -specificity dominates. in contrast, if w 3/6 . 1.00, then α2 0 à3 0 -specificity dominates. (strains with w 3/6 % 1.00 have approximately equal α2 0 à3 0 -and α2 0 à6 0specificities.) 152 the sialoside specificity of the first pandemic strains isolated in our study, a/california/04/2009 (h1n1) pdm09, demonstrates dual affinity to both α2 0 à3 0 -and α2 0 -6 0 -sialosides (figure 8.106) . therefore, such strains might be able to effect swineàhuman and humanàhuman transmission, and their pathogenicity is higher than that of seasonal influenza viruses (w 3/6 % 1 pigs could be the source of influenza a virus not only in humans, but also in synantropic animals. s. agapov published an article on the pathogenic properties of influenza a virus specimens isolated from brown rats (rattus norvegicus) in pigsties. 161 experimental infection of swine influenza a virus strains in rodentsmice (subfamily murinae) and hamsters (subfamily cricetinae)-has been described in a number of publications. 3,133,146,161à163 rodents have become a widely used laboratory model for influenza a virus. productive infection in laboratory mice (order rodentia: family muridae, genus mus) was revealed in a pioneer publication 13 of w. smith (figure 8 .85), c. andrewes (figure 8 .86) and p. laidlaw (figure 8.87 ). adapted to mice, influenza a virus strains are widely used to investigate infectious process, pathology, and the efficiency of antivirals. 161,164à168 in 2000, the strain influenza a/muskrat/ buryatia/1944/2000 (h4n6) was isolated from muskrat (ondatra zibethicus) hunted in the selenga river delta, near where it empties into lake baikal. despite mountain relief along the lake coast, the delta represents a sandbank wedge overgrown with low reeds where the conditions are conducive to a mass nesting of ducks and a high density of population of muskrats. as a result, there is a high level of interaction between the populations of aquatic birds and muskrats. in particular, a/muskrat/buryatia/ 1944/2000 (h4n6) has the highest homology with a/pochard/buryatia/1903/2000 (h4n6). the strain from muskrat turned out to be virulent to mice without any preliminary adaptation, like the majority of h4 strains from siberian ducks. it was suggested that virulence was promoted by an r220g mutation in ha. 72, 81 the russian state collection of viruses contains the influenza a/sciurus vulgaris/ 6su-6 0 sln 6-su-6 0 -sialyllactose: 6-su-neu5acα2-6galβ1-4glcβ primorje/1004/1979 strain with an undetermined subtype isolated from a red squirrel (sciurus vulgaris). 5 weasels (order carnivora: family mustelidae) are another sensitive group of hosts for influenza a viruses. the sensitivity of the domestic ferret (mustela putorius furo), an albino form of the forest polecat (mustela putorius), to the virus was explored even in the earliest scientific publications devoted to influenza a virus. 13, 14 today, ferrets are the best animal model of influenza a virus infection. in particular, sera of infected ferrets (as well as infected rats) are widely utilized for influenza a virus subtype identification. in 1985, japanese scientists demonstrated that the epidemic strain a/kumamoto/ 22/1977 (h3n2) was able to provoke disease in the european mink (mustela lutreola), 169 and perhaps it was this virus that caused a respiratory disease epizootic on japanese fur farms during 1977à1978. in 1984à1985, during an epizootic among minks in sweden, six strains of influenza a (h10n4) virus (prototype a/ mink/sweden/e12665/1984) were isolated and turned out to have an avian origin. 170 in 2007, an influenza a/stone marten/germany/r747/06 (h5n1) strain was isolated from the internals of a stone marten (martes foina) that was found dead in a place where there was mass mortality of birds in germany. 171, 172 the circulation of influenza a virus among cats (order carnivora: family felidae) was originally established in 1942 by the japanese virologists j. nakamura and t. iwasa: strain a/cat/fusan/1/1942 (known as "chiba virus") 173 turned out to be an avian strain of the h7n7 subtype. 168 in 1970, c.k. paniker and c.m. nair described the successful experimental infection of adult cats and eight-monthold kittens by a/hong kong/1/1968 (h3n2), of the "hong kong flu" pandemic strain. 174 a number of h5n1 strains from felidae members-tigers (panthera tigris), 175à177 leopards (p. pardus), 176 and domestic cats (felis catus) 178à180 -were described after 2005. the first experiment involving the infection of dogs (order carnivora: family 178 this strain had an avian origin, but provoked lethal pneumonia in dogs. 186 it is noteworthy that influenza a virus can be isolated from nasal swabs of dogs during inapparent infection, 187 so this virus might be more widely distributed among dogs than is usually considered. influenza a virus is often the cause of pericarditis in dogs. 188 the circulation of influenza a viruses among horses (order perissodactyla: family equidae, genus equus) was originally explored in 1956 by a group of czechoslovakian scientists headed by bella tumova (figure 8 .107). in that year, a widespread epizootic emerged among horses (equus ferus caballus) and the historical strain a/equine/prague/1/1956 was isolated. 189 a subtype of this strain was given an initial designation h eq1 n eq1 and later was identified as h7n7 (but, for a long time, veterinarians designated this subtype as equine influenza type 1). 146 later, influenza a (h7n7) strains were isolated in other european countries 190 and the united states. 191 during the "asiatic flu" pandemic of 1958à1961, a number of strains of influenza a (h2n2) were isolated from sick horses in the moscow region of the former ussr 192 hungary. 133, 163 it was shown that these strains were significantly different from a/equine/ prague/1/1956 (h7n7), belonged to the h2n2 subtype, and had a human origin. equine influenza a type 2 was originally found in 1963 in miami, florida, in the united states, when the prototypical strain a/equine/ miami/1963 was isolated and designated as subtype h eq2 n eq2 . 193 later, this subtype was identified as h3n8 and was multiply isolated 194à196 in both north and south america. in the former ussr, influenza a (h3n8) virus strains were isolated from horses in the ukrainian soviet republic during a widespread epizootic in 1970 in the vicinity of kiev. 31 the russian state collection of viruses contains the influenza a/equine/mongolia/3/ 1975 (h5n3) strain, which originates from birds and over came the interspecies barrier to penetrate into the equine population. the circulation of influenza a virus among camels (suborder tylopoda: family camelidae, genus camelus) was originally established by d. k. lvov 59 (figure 2 .36) in 1980. in december 1979, an epizootic of "contagious cough" among bactrian camels (camelus bactrianus) emerged in mongolia. thirteen strains were isolated from nasal swabs; 59 145, 198 tajikistan, 199 and the ukrainian soviet republic in the former ussr. 31 the circulation of influenza a viruses among cattle has been confirmed by multiple serological data. 31,200à204 the first isolation of influenza a strain from sick sheep (ovis aries) (order artiodactyla: family bovidae, subfamily caprinae) was carried out in 1959 by a group of hungarian scientists under the direction of g. takatsy during an epizootic among farm animals. 133, 163 the strain a/sheep/hungary/b111/59 (h2n2) isolated by takatsy was later utilized by j.l. mcqueen and f.m. davenport for experimental infection in lambs, but they observed no clinical symptoms. 205 the circulation of influenza a viruses among deer (order artiodactyla: family cervidae) was originally established by t.v. pysina and d.k. lvov when they isolated the a/rangifer tarandus/chukotka/1254/77 (h6n2) strain from slowed reindeer (rangifer tarandus) in the chukotka peninsula. 206 the russian state collection of viruses in the d.i. ivanovsky institute of virology contains the strains a/ deer/primorje/1201/78 (h1n1), isolated from red deer (cervus elaphus) in primorsky krai, and a/rangifer tarandus/yamal/865/90 (h13n1), isolated from reindeer (r. tarandus) on the coast of the barents sea. specific antibodies towards influenza a (h1n1) and a (h3n2) were detected in the sera of red deer (c. elaphus) and elks (alces alces) in the north of germany. 207, 208 s.q. li established the presence of about a 10% immune layer toward influenza a (h1n1) and a (h3n2) among cervidae in the northeastern provinces of china. 209 the strain influenza a/whale/pacific ocean/19/1976 (h1n3) (or, alternatively, a/ whale/po/19/1976) from a whale belonging to the balaenopteridae family (order cetacea, suborder mysticeti) and bagged in the south pacific ocean was isolated by a group of soviet virologists under the direction of d.k. lvov 210 (figure 2 .36) in 1976. this strain turned out to be reassortant between human and avian virus variants. 211 two strains of influenza a virus were isolated by a group of american virologists under the direction of r. webster 212 (figure 2 .20) from slowed long-finned pilot whales (globicephala melaena) near portland, maine, in the united states in 1984: a/whale/maine/1/84 (h13n9) (from periapical lymph nodes in the lungs) and a/whale/maine/2b/84 (h13n2) (from the lungs). further molecular genetic investigation, carried out by a russianàamerican group of scientists, revealed that influenza a variants in gulls (family laridae) were the source of these strains. 75 a number of influenza a virus strains were isolated on the coast of north america: h4n5, 213 h4n6, 214 and h7n7. 215, 216 thus, one could expect to find influenza a viruses among seals in northern eurasia as well. pathogenesis. epithelial cells of mucous membranes are the main targets of influenza a viruses. degeneration, necrosis, and further apoptosis, followed by tearing away of the epithelial cell layer take place as a result of the infection. nevertheless, the main element of influenza a virusàinduced pathogenesis is lesions on the system of vessels; the lesions emerge as the result of the toxic effect of the virus, an effect that includes the multiple formation of active oxygen forms. the latter provoke the generation of hydroperoxides, which interact with lipids and phospholipids of the cell wall to oxidize their peroxide, thereby hindering transport across the cell membrane. 217à219 a subsequent increase in the permeability of vessels, the fragility of their walls, and a violation of the body's microcirculation result in hemorrhagic manifestations, from nasal bleeding to hemorrhagic hypostasis of the lungs and hemorrhages in the substance of the brain. 219, 220 frustration of the circulation, in turn, defeats the nervous system. the pathomorphological picture is characterized by the existence of lymphomonocytic infiltrates around small and average-size veins, hyperplasia of glial elements, and a focal demyelinization that testifies to the toxic and allergic nature of the pathological process in the cns during influenza. 219, 221, 222 the most significant factors involved in cell tropism of the influenza a virus are the receptor assembly on the surface of the potential target cell and the ability of cell proteases to cleave ha into two subunits (ha1and ha2) followed by fusion peptide rescue. 223à227 for example, for avian influenza a virus variants, there is an obvious threshold in the virulence level: so-called lpai and hpai. hpai strains strike vascular endothelial and perivascular parenchymal cells as well as the cardiovascular system, quickly reproduce high titers in practically all internal organs, and cause systemic disease leading to death of a bird 1à7 days after infection. lpai strains, to the contrary, reproduce in low titers, have a narrow tropism toward mucous in the digestive and respiratory tracts (figure 8 .108), and cause enteritis or rhinitis with low mortality. (however, bird diseases connected with lpai also cause significant damage to agriculture and can break the interspecies barrier, resulting in diseases that are dangerous to people). wild aquatic and semiaquatic birds, which are natural reservoirs of influenza a viruses, can have inapparent disease during either lpai or hpai infection. 5,24,27,28,39,41à43,53,226,228à230 the ability of ha to be cleaved by proteases depends on the amino acid composition of the proteolytic cleavage site: lpai strains contain only one or two positively charged basic amino acids (k or r), whereas hpai strains have an enriched amount of basic amino acids. 5,24,27,28,39,41,228à230 nevertheless, pandemic strains with extremely high virulence in humans have only single basic amino acids within the limits of the proteolytic cleavage site (table 8 .58). still, it is noteworthy that lpai could provoke human disease as well. except for the amino acid composition of the proteolytic cleavage site of ha, the efficiency of the cleavage process depends on glycosylation of ha in the vicinity of this site. 231, 232 amino acid substitutions that switch virus tropisms from avian to mammalian cells in different influenza a virus proteins have been described: e627k, 112 144, 146, 219, 221 the classic diagnostic approach is to isolate the virus with the use of sensitive biological models (ferrets, developing chicken embryoa, and cell lines). influenza a virus infection could be retrospectively detected by hit 239 or neutralization testing, but the most effective diagnostic methods are rt-pcr and biological microchips. control and prophylaxis. vaccination, together with the forced slaughter of livestock. is the most effective and accessible approach to influenza a prophylaxis among domestic animals. each country chooses its own strategy for combining these methods. for example, in russia only livestock in small and individual farms is to be vaccinated whereas birds in poultry farms are not vaccinated, but are killed if either hpai or lpai is detected. 32 the genome of the quaranjaviruses consists of six segments of negative ssrna. segments 1à3 encode the proteins of a replicative polymerase complex (polymerase basic protein 2, or pb2; polymerase acidic protein, or pa; and polymerase basic protein 1, or pb1, respectively). the pb1 protein (polymerase 1 basic protein, rdrp) is one of the most conservative proteins of all viruses with a segmented rna genome. the amino acid sequence similarity of the pb1 protein among the viruses of different genera in the orthomyxoviridae family is 25à30%, on average, but the similarity of the functional domains of rdrp (pre-a, a, b, c, d, and e motifs) is 40à50% (figure 8.110) . the envelope glycoprotein gp (ha, segment 5) of the quaranjaviruses has a very low similarity to the homologous protein (ha, segment 4) of influenza viruses. however, it has some similarities tgo the surface glycoprotein of the baculoviruses. 1 the amino acid sequences of thogotovirus genus members have about 20% identity with qrfv and tlkv. two other segments of the genome (segments 4 and 6) of the quaranjaviruses encode two proteins whose function is unknown. these proteins are probably structural proteins, which act as nucleocapsid (n) and matrix protein (m), respectively, but currently their function is not well known. other viruses of the quaranjavirus genus have been found in south africa, nigeria, egypt, iran, afghanistan, and oceania. the quaranjaviruses are associated with argasidae ticks (argas arboreus, a. vulgaris, ornithodoros capensis), which are obligate parasites of birds. 3 tlkv has been classified into the quaranfil group of the orthomyxoviridae family on the basis of its antigenic reactions. 4à14 taxonomy. like the other members of the quaranjavirus genus, tlkv has a genome that consists of six ssrna segments. 13 the pb1 protein amino acid sequence of tlkv has 86% and 84% identities with qrfv and jav, respectively (table 8 .61). the similarity of the pb2 and pa proteins of tlkv to those of orf virus (orfv) is 70%, on average. the envelope glycoprotein (gp, segment 5) of the quaranjaviruses has very low similarity to the homologous protein (ha) of influenza viruses. however, it has some similarities to the surface glycoprotein of the baculoviruses. 4 the similarity of the gp of tlkv to that of qrfv is 72% nt and 80% aa (table 8 .62). segment 5 of tlkv has one orf and encodes a protein with unknown function (524 aa). its similarity to the same protein of qrfv is 85% aa. segment 6 encodes a protein 266 aa long, which has no homology with any of the virus's proteins that are deposited in the database genbank. the similarity of this protein in tlkv and the same protein in qrfv is 60%. figures 8.110 and 8.112 show the results of phylogenetic analysis based on a comparison of pb1 and the envelope protein (gp and ha, respectively). on the phylogenetic trees, tlkv is grouped with qrfv and jav within the quaranjavirus genus. 13 arthropod vectors. natural foci of tlkv associated with argas vulgaris ticks in kyrgyzstan are located below the northern border of the area of distribution of argasidae ticks (43 % on). this boundary coincides with the line of a frost-free period of 150à180 days a year and an average daily temperature above 20 for no less than 90à100 days per year. the ability of these ticks to withstand prolonged starvation (up to 9 years), as well as their long life cycle (25à30 years), polyphagia, and ability to transfer viruses transovarially, provides stability of the virus's natural foci. 1à3,15à18 animal hosts. tlkv was isolated from argasidae ticks collected in the nesting burrows of birds. complement-fixation testing of the birds from these colonies revealed that qrfv have been found in 2.6% of the human population. 11 the genus thogotovirus currently includes four viruses: thogoto virus (thov), dhori virus (dhov), araguari virus (argv), and jos virus (josv). 1, 2 the viruses of thogotovirus are arboviruses, transmitted mainly by ixodidae ticks; therefore, the genus had previously been called orthoacarivirus, to emphasize these viruses' association with ixodids (taxon acari: order parasitiformes, family ixodidae). thov was originally isolated from the ticks rhipicephalus (boophilus) decoloratus and rh. evertsii collected from cattle in thogoto forest, nairobi, kenya, in 1960. subsequently, it was isolated from human, cows, camels, and ticks in many countries in africa. 3, 4 the genome of the thogotoviruses consists of six segments of negative-polarity ssrna that encode seven proteins. (segment 6 encodes two forms of matrix protein.) 1, 2 the most conservative proteins of the replicative complex (pb1, pb2, pa) of thogotoviruses have 25à30% identity with those of the influenza a virus genus. history. dhori virus (dhov) was originally isolated from hyalomma dromedarii ticks collected from camels in india. 1 dhov has also been isolated in egypt, portugal, russia, and transcaucasia. 2à7 in russia, several strains of dhov were isolated from h. plumbeum ticks, anopheles hyrcanus mosquitoes, and lepus europaeus hares, all in the volga river estuary. 5, 7 one strain of dhov was isolated from the cormorant phalacrocorax carbo in maly zhemchuzhnyi island in the caspian sea (45 00 0 n, 48 18 0 e; figures 8.113 and 8.114 ). 4 the prototypical strain of batken virus (bknv), leiv-k306, was isolated from hyalomma marginatum ticks collected from sheep near the town of batken in kyrgyzstan in april 1970. 8 other strains of bknv were isolated from a mixed pool of aedes caspius and culex hortensis mosquitoes in kyrgyzstan 9 and from ornithodoros lahorensis and dermacentor marginatus ticks in transcaucasia. 10 antigenic studies showed that bknv is closely related to dhov, but differs from it. 8 taxonomy. the similarity of the structural homologous proteins of the thogotoviruses (thov, dhov, argv, and josv) ranges from 25% (m-protein, segment 6) to 45% (np, segment 5). the envelope protein ha (segment 4) has 35à45% identity, on average. the similarity of the nonstructural proteins (pb1, pb2, and pa) ranges from 60% to 74%. bknv has a high similarity to dhov. the proteins are 96% (pb2, pa, np, m) and 98% (pb1) identical. the similarity of the envelope protein ha of bknv to that of dhov is 90%, a percentage that explains the antigenic differences between these two closely related viruses. because the homology of the other structural and nonstructural proteins of bknv and dhov is 96à98%, it can be concluded that bknv is a variant of dhov, typical to central asia and transcaucasia. a phylogenetic analysis based on a comparison of the pb1 and ha proteins is presented in figures 8.110 and 8.112 . arthropod vectors. apparently, the main arthropod vector of dhov and bknv is hyalomma sp. ticks-in particular, h. marginatum. dhov has also been isolated from h. dromedarii, dermacentor marginatus, and ornithodoros lahorensis ticks. rare isolations of dhov and bknv from mosquitoes (anopheles hyrcanus, aedes caspius, and culex hortensis) suggest that they play some role in the circulation of these viruses. 9 vertebrate hosts. antibodies to dhov were found in 100% of camels, 19% of horses, and 2% of goats in the indian state of gujarat, where the virus was first isolated. antibodies to bknv were found in 1.0% of sheep and 1.3% of cattle in kyrgyzstan. 9 two strains of dhov were isolated from a hare (lepus europaeus) and a cormorant (phalacrocorax carbo) in natural foci of the virus. 4, 5 the bird, from which dhov was isolated on maly zhemchuzhnyi island, was ill with respiratory failure, inability to fly, and loss of coordination 4 (figure 8.114c) . human disease. several cases of disease caused by dhov have been registered. 11 the disease occurred with fever, encephalitis (40%), headache, and weakness. antibodies to dhov were found in 4à9% of the population in the volga river delta (in the south of russia) and in 0.8% in the south of portugal. 12 antibodies to bknv were found in the sera of 0.3% of the human population of kyrgyzstan. five cases of laboratory infection were identified. 11 the togaviridae family consists of two genera (alphavirus and rubivirus) of enveloped rna viruses. the virion of the togaviruses (70 nm) contains a core particle (40 nm) formed by a capsid protein and comprising a single-stranded, positive-sense genomic rna 11,400à11,800 nt long. the lipid bilayer contains the heterodimers of two surface glycoproteins e1 and e2, which form an icosahedral surface of the virion. the genomic rna has a cap structure at the 5 0 -and poly-a tail at the 3 0 -end, as well as two orfs encoding nonstructural and structural proteins. the nonstructural proteins are encoded by the 5 0 -orf (which occupies two-thirds of the genome), whereas the structural proteins are encoded by the subgenomic 3 0 -orf. 1 most viruses of the alphavirus genus are arboviruses and can replicate in either a vertebrate host and or an invertebrate vector. 2, 3 the rubivirus genus consists of one species-rubella virus-that is transmitted by aerosol and is the causative agent of disease known as rubella. 4,5 the genome of the alphaviruses is a singlestranded rna with positive polarity about 11,500 nt in length. the viral rna has a cap at the 5 0 -end and a poly-a tail at the 3 0 -end. a large part of the genome of the alphaviruses (about two-thirds, beginning from one-third into the genome and extending to the 5 0 -end) encodes nonstructural proteins that form the viral replicative complex nsp1, nsp2, nsp3, and nsp4). structural proteins (core, e3, e2, 6k, and e1) are translated from subgenomic rna (26s rna), which is formed in the process of replicating the virus and corresponds to the other one-third of the genome (figure 8.115 ). 1 the alphaviruses can infect a wide range of vertebrates. most of the alphaviruses are arboviruses and are associated with mosquitoes (genera culex, culiseta, aedes, coquillettidia, and haemogogus) and birds, the latter of which can transfer viruses during migration. 2à4 other vertebrate hosts of the alphaviruses are ruminants, reptiles, amphibians, and fish. 5, 6 the alphaviruses are divided into 10 antigenic complexes. among the alphaviruses are dangerous pathogens of humans or animals, such as eastern equine encephalitis virus (eeev), western equine encephalitis virus (weev), sindbis virus (sinv), chikungunya virus (chikv), and others. 7 history. chikv (family togaviridae, genus alphavirus, semliki forest group) is the etiological agent of a fever that is mortally dangerous to humans. this disease is accompanied by joint and muscle pains (right up to complete immobilization of the patient) and a two-wave course of the fever, together with a macu-laràpapular rash emergency (usually during the second wave). 1 the etymology of the name "chikungunya" is {chee-kungunyalac, which, in the makonde local language, means "doubled up," owing to the severe joint pains. chikv was originally isolated by r.w. ross from the serum of a patient with fever during the decoding of an epidemic outbreak in tanzania in februaryàmarch 1956. 2à4 the close relation of chikv to mayaro virus (mayv), from the semliki forest group, was demonstrated in 1957 by serological methods. 5, 6 distribution. chikv was also isolated in cambodia in southeastern asia in 1963, 7 in hindustan in 1964, 8, 9 and in the eastern part of new guinea in 2012. 10 the basic area over which chikv is distributed (table 8. taxonomy. chikv belongs to the togaviridae family, alphavirus genus, semliki forest group. on the basis of comparative analysis of the e1 gene, chikv was classified into three genotypes: a (asian), cesa (centre, east, and south african), and wa (west african) 1,12à14 (table 8. vertebrate hosts. rodents, bats, and monkeys are the natural reservoir of chikv. 1,11à14,46 there is substantial evidence, that, in africa, wild primates play an important role in the natural transmission cycle, but it is not clear whether infection in primates is incidental to or necessary for the maintenance of the virus. in uganda, chikv was frequently isolated from aedes africanus mosquitoes, which preferto feed on monkeys in the forest canopy. 47 specific anti-chikv antibodies were found among chimpanzees (pan troglodytes) in equatorial and savanna forests in the democratic republic of the congo (kinshasa) 48 and in savannas in southern africa. antibodies were found over a wide area in vervet monkeys (cercopithecus aethiops) and baboons (pipio ursinus), and in both species the virus could circulate in the blood for two to three days at high concentrations without evidence of illness. 49 so, wild animals could play an important role as amplifying hosts. 49 chikv was isolated in dakar , senegal, from bats, which developed viremia after experimental infection. but in india, inoculation of the virus into two species of fruiteating bats was followed by low virulence. 50, 51 antibodies were found among donkeys, bats, and wild rodents in africa 52 and among domestic animals in asia. 49, 50 inoculation of african strains into cattle, sheep, goats, and horses failed to produce viremia. apart from chickens, adult fowl and several species of wild birds did not develop viremia after experimental infection. but experimental infection of vervet monkeys and baboons led to high viremia (up to 8 log 10 pfu/ml) during six days, which is sufficient for the infection of mosquitoes. 53 arthropod vectors. chikv is transmitted by bloodsucking mosquitoes. the main vectors for this virus during epidemics are aedes aegypti and ae. albopictus in urban regions and mosquitoes from the aedes, culex, and coquillettidia genera in rural landscapes. 1,11à14,46 chikv has been multiply isolated from ae. africanus, ae. luteocephalus, ae. furciferàtaylori, cx. fatigans, and coq. fuscopenatta, all of which could preserve the virus and realize virus circulation in natural foci. 1, 54, 55 epidemiology. a high level of viremia in humans (up to 8 log 10 pfu/ml) makes it possible for mosquitoes to transmit chikv from human to human 1 -a plausible reason that large epidemic outbreaks have been known in big cities of southern and southeastern asia since the 1960s. 11,13,56à58 beginning in the middle of the 1980s, epidemiological processes linked to chikv have intensified (table 8 .63), although this fact could be explained by improvements in laboratory diagnostics: previously, chikungunya fever was often confused with dengue. in any event, chikvprovoked lethality has increased, in some cases up to 4.5%). 1, 59 increases in the frequency of imported chikungunya fever cases seen at the beginning of the twenty-first century (table 8 .63) are most dangerous, especially when the possibility of chikv penetration into local mosquito populations is taken into account. since 2006, imported cases of chikungunya fever have become more frequent in europe (italy, 15, 38, 60, 61 spain, 39 france, 35, 44, 62 belgium, 35 switzerland, 35 germany, 35 the czech republic, 35 norway 35 ); the americas (canada, 13 the united states, 35, 63 brazil 44 ); eastern asia (hong kong, 36 south korea, 40 japan 37,45 ); and australia. 33 outbreaks in brazilian cities emerged with infections from aedes aegypti, whereas in rural regions aedes albopictus was the vector, introduced from southeastern asia, 44 including japan. 64 imported cases of chikungunya fever in russia. a 59-year-old patient arrived in russia september 22, 2013 , and suddenly fell ill, with a body temperature of 38.7 c. antipyretic drugs were not effective. early in the morning on september 24, 2013 , the patient was delivered to a moscow infection hospital with a diagnosis of "fever with unknown etiology." the fever had mid-level severity, and the patient complained of shivering, headache, and asthenia. hyperemia of the conjunctivae, papularàhemorrhagic rash on the abdomen, and cruses were found. a medical radiolograph (figure 8 .116) of the lungs of the patient revealed decreased clarity at the back of the lung field and diffuse reticular pneumosclerosis in the right lower lobe pyramid, as well as local changes with expressed peribronchial and perivascular alterations. a round shadow was detected near (i.e., peribronchially to) the intermediate bronchus. the roots were intensified. the heart was enlarged at the left. thus, the medical radiography portrait was consistent with rightside pneumonia with lymphadenopathy. several peculiarities of the case were the bareness of clinical symptoms (pneumonia was diagnosed only via medical radiography), a rapid progression of changes in the lungs, and the absence of inflammation markers in the peripheral blood. three days later, positive dynamics were detected: the basal parts of the right lung were restored to their previous level of clarity, although the shadow indicating a hypertrophic lymph node and right root broadening remained. bioprobes (blood swabs and nasopharyngeal swabs) were delivered to the d.i. ivanovsky institute of virology. the absence of influenza a and b viruses was established by rt-pcr. the strain chikv/leiv-moscow/1/2013 was isolated with the use of intracerebrally inoculated newborn mice and was identified with the help of a completegenome (genbank id: kf872195) nextgeneration sequence approach. phylogenetic analysis (figure 8 .117, table 8 .64) revealed that the chikv/leiv-moscow/1/2013 strain belonged to an asian genotype. this strain was deposited into the russian state collection of viruses (deposition certificate n 1239 with a priority of november 11, 2013). 65 serological methods revealed eight cases of imported chikungunya fever that had previously been described in russia: 66 from indonesia, singapore, india, the island of réunion, and the maldives islands. the chikv/leiv-moscow/ 1/2013 strain was found to belong to the a-genotype, whereas most of the cases imported into europe belong to the cesa genotype, reflecting the "bridge" role of russia between europe and asia. the modern-day intensification of both international links and transport flows among countries increases the probability of imported cases of infection emerging. the penetration of aedes aegypti and aedes albopictus to the russian black sea coast 1,67,68 suggests the emergence of seasonal outbreaks in the dynamically developing greater sochi region as well. history. getah virus (getv) was originally isolated in western malaysia from culex gelidus and cx. tritaeniorhynchus mosquitoes. 1à3 this virus is widespread in southeastern asia and in australia. 3à5 the first isolation of getv in northern eurasia was carried out by m.p. the genome of getv is 11,598 nt long. the strains of getv, circulating in different geographical regions of northeastern and southeastern asia, have a high level of similarity. 8à11 a pairwise comparison of complete genome sequences revealed that isolates from malaysia, south korea, china, mongolia, japan, and russia have 96à98% nt identities, suggesting that the rate of getv evolution is low. phylogenetic analysis of the e2 gene ( figure 8.118) is not conducive to dividing the getv strains into distinct clusters. analyses of numerous strains isolated in japan showed that genetic differences were determined by the time of isolation more than the place of isolation. 8 an analysis of 21 strains of getv isolated in different regions of russia revealed their high degree of similarity, but still, they could be divided into three groups on the basis of minimal differences. the first group comprises strains from tundra and for-estàtundra in the magadan region and the sakhaàyakutia republic in the north of asia. the second group encompasses strains from leaf-bearing forests of khabarovsk krai. the third group consists of isolates from forestàsteppe and steppe landscape belts of khabarovsk krai, the republic of buryatia, and mongolia. 10, 12 distribution. according to our data, 6,10,12à22 getv is distributed over eastern siberia and north pacific physicogeographical lands (figure 8.119) . the most intensive virus circulation was revealed in the steppe landscape belt of mongolia, as well as in the mixed forests of khabarovsk krai and in the northern taiga of the magadan region and the sakhaàyakutia republic. getv circulation intensity is significantly lower in tundra and forestàtundra landscapes, a phenomenon that could be explained by the temperature there. getv is the only member of the alphavirus genus whose distribution extends to the rough climatic conditions of the high latitudes of northern eurasia. 18, 19 getv has penetrated to the north of asia from the overwintering places of birds, which regularly migrate by the east asian flyway 17, 23 (figure 3.2) . the distribution of the virus in the north coincides with that of aedes mosquitoes, which are the effective vector of getv. getv and closely related viruses are known outside of northern eurasia in japan, various countries in southeastern asia, and australia. 1à3,5,24à29 human infection. the pathogenicity of getv to humans has not yet been described. nevertheless, the antigenically close rrv has been associated with large epidemic outbreaks of polyarthritis in australia and sarawak. 2, 4 vertebrate animal infection. symptomatic and subclinical infections of animals were reported in 1998 in japan, where there was a large outbreak involving 722 racehorses. 30, 31 among the clinical features seen were fever, rash on various parts of the body, and edema on the hind legs. virus isolates were more similar to the prototypical malaysian strain than to the japanese sagiyama strain. getv has been implicated in illness and abortion or stillbirths in pigs. 32,33 disease among horses was described in india. 34 infection in cattle is usually subclinical. 3 arthropod vectors. getv has been isolated from culex gelidus, cx. tritaeniorhynchus (malaysia, cambodia, china), cx. bitaeniorhynchus, anopheles amictus (australia), cx. vishnui (philippines); the sagiyama subtype of getv was isolated from cx. tritaeniorhynchus and aedes vexans, as well as from pigs with fever, in japan. 27, 35 although their natural transmission cycle is not known in details, mosquitoes acquire getv mainly while feeding on domestic mammals and fowl. there may also be a jungle cycle involving wild vertebrates. 5 the bebaru subtype was isolated from culex lophoceratomyia and aedes spp. mosquitoes collected in mangrove swamp forests of western malaysia. 32 the main vectors in russia (i.e., in northern eurasia) are aedes nigripes, ae. communis, ae. impiger, ae. punctor, and ae. excrucians. 18 4, 13 kfv was first noted in the summer of 1981 in the central and southwestern parts of fennoscandia, including russia, finland, sweden, and southern norway (figure 8 .120). 14 the prototypical strain leiv-65a of kyzv was first isolated from culex modestus mosquitoes collected in a colony of ardeidae birds (herons) in kyzylagach reservation, located on the coast of kyzylagach bay in the caspian sea (39 10 0 n, 48 58 0 e; figure 8 .120). 15 taxonomy. on the basis of a comparison of a partial sequence of the e2 gene, isolates of sinv can be divided into five genotypes (figure 8 .121). 9 genotype i includes viruses from europe and africa, genotype ii isolates from australia and oceania, and genotype iii viruses from india and the philippines. together with the chinese strain sinv xj-160, kyzv was assigned to genotype iv. genotype v consists of only the strain m78 from new zealand. the strains of genotype i form two subclusters, one of which comprises sinvs from northern europe and sub-saharan africa and the second of which consists of strains from the mediterranean region (southern europe, northern africa, and the middle east). 9 the genetic distance between the viruses of the different genotypes of sinv (e.g., between the european and australian isolates) is not more than 23% nt (table 8 .65). at the same time, sinvs isolated in the same geographic region are characterized by a high degree of similarity (figure 8.122) . thus, sinv strains isolated in russia, germany, sweden(ockv), and finland have about 99% similarity (table 8 .65). 3, 5, 6, 11 babanki virus, which is from cameroon, has 98% similarity to the european strains of sinv. despite the high degree of similarity among the different genotypes of sinv, known cases of human disease are caused only by strains of the europeanàafrican subcluster of genotype i (karelian fever, a disease of ockelbo, a disease of babanki). kyzv has a high similarity (99%) to the chinese isolate sinv xj-160, isolated from anopheles sp. mosquitoes in the xinjiang uighur autonomous region in the northwest of china. 16 the divergence of kyzv and xj-160 from the european isolates of sinv is 18% nt and 7% aa of the entire genome sequence (table 8 .65). the geographic isolation of kyzv and xj-160 and their genetic divergence from the european and australian isolates suggest that kyzv is a variant of sinv that is typical to central asia. distribution. sinv has been isolated in many regions of southern europe, the middle east, africa, southeastern asia, the philippines, and australia. 2, 17, 18 the african continent is almost all endemic for sinv: strains are known from egypt, the republic of south africa, uganda, the central african republic, sudan, nigeria, and zimbabwe. as for asia, there are strains from turkey, india, malaysia, and the philippines. in australia, sinv strains were multiply isolated in the north of the continent. in europe, sinv has been isolated in sicily (italy) and slovenia. on the territory of the former ussr, sinv strains were multiply isolated in belarus, ukraine, azerbaijan, tajikistan, and western siberia (in the areas around the central region of the ob river valley). 17à19 . vertebrate hosts. the main vertebrate hosts of sinv are different species of birds, predominantly of the orders passeriformes, pelecaniformes, ciconiiformes, and anseriformes. sinv infection in birds can chronic, allowing them to transfer the virus during their seasonal migration. 17à20 migratory birds play an important role in the wide distribution of this virus. sinv has been known to persist for as much as two months after experimental infection. sinv strains have been multiply isolated from aquatic and semiaquatic birds in the delta of the nile river in egypt, from the white wagtail (motacilla alba) and the common hill myna (gracula religiosa) in india, and from the reed warbler (acrocephalus scirpuceus) in the western part of slovakia. in zimbabwe, sinv has been isolated from insectivorous bats of the rhinolophidae and hipposideridae families. 2 occasionally, sinv has been isolated from rodents and amphibians. on the territory of the former soviet union, sinv was originally isolated from a yellow herons (ardeola ralloides) caught out of a bird colony in the southeastern part of azerbaijan in 1968. serological methods have revealed sinv circulation in the astrakhan region among aquatic and semiaquatic birds, especially those of the orders pelecaniformes (18%), ciconiiformes (15%), and anseriformes (11%). neutralizing antibodies to sinv were found in coots (fulica atra) (16.7%) from natural foci of the middle belt of the volga river delta. in the kuban river delta in krasnodar krai, specific anti-sinv antibodies were found among eight species of aquatic and semiaquatic birds, most frequently mallards (anas platyrhynchos) and purple herons (ardea purpurea). in belarus, anti-sinv antibodies were detected in 4% of birds in the summer and in 0.4% in the fall. 21 antibodies to sinv have been detected among farm animals (table 8. cattle (17.5%) and horses (15.0%) in the middle belt of the volga river delta. arthropod vectors. sinv is closely associated with ornithophilic mosquitoes. in egypt, this virus was isolated from culex univittatus, cx. antennatus, and anopheles pharoensis; in uganda, from coquillettidia spp.; in sarawak, (malaysia), from cx. bitaeniorhynchus; in australia, from cx. annulirostris, aedes normanensis, and ae. vigilax; in india, from coq. fuscopennata; in sudan, from cx. quinquefasciatus; and in europe, from cx. pipiens, cx. torrentium, culiseta morsitans, coq. richiardii, ochlerotatus communis, oc. excrucians, ae. cinereus, and an. hyrcanus. 22, 23 according to our data, in the volga river delta sinv is transferred by culex pipiens in anthropogenic biocenoses and by anopheles hyrcanus and coquillettidia richiardii in natural ones. in the natural foci of the middle belt of the volga delta, 1 strain can be isolated from approximately 3,800 an. hyrcanus or 3,300 coq. richiardii mosquitoes; in the low belt of the delta the ratio is 1 in about in a power less, and in anthropogenic biocenoses it is 1 strain per 1,500 cx. pipiens mosquitoes. sinv strains from gamasidae ticks (ornithonyssus bacoti) in india and from ixodidae ticks (hyalomma marginatum) in sicily (italy) are known. 2 productive experimental infections were described in the argasidae ticks ornithodoros savignyi and argas persicus (although infected ticks did not transmit the virus during feeding). 23 most likely, ticks do not play an important role in sinv circulation or as a reservoir for this virus. human pathology. sinv causes acute fever in humans but has a favorable outcome. antibodies to sinv are widely detected in human sera (table 8 .66), although in eastern siberia and the far east cross-reactions with getv (another member of the semliki forest serogroup) can take place. the start of the 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in uzbekistan and turkmenia genetic characterization of caspiy virus (casv) (bunyaviridae, nairovirus), isolated from seagull larus argentatus (laridae vigors, 1825) and ticks ornithodoros capensis neumann in eastern and western cost of caspian sea structure of crimean-congo hemorrhagic fever virus nucleoprotein: superhelical homo-oligomers and the role of caspase-3 cleavage influenza virus pathogenicity is determined by caspase cleavage motifs located in the viral proteins ovarian tumor domain-containing viral proteases evade ubiquitin-and isg15-dependent innate immune responses the high genetic variation of viruses of the genus nairovirus reflects the diversity of their predominant tick hosts immunofluorescence studies on the antigenic interrelationships of the hughes virus group (genus nairovirus) and identification of a new strain ticks (ixodoidea) on birds migrating from africa to preliminary data about isolation of three novel arboviruses in caucasus and central asia chim virus, 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of alphaviruses and flaviviruses-experimental infections key: cord-003898-y6zpvw84 authors: tan, kai sen; andiappan, anand kumar; lee, bernett; yan, yan; liu, jing; tang, see aik; lum, josephine; he, ting ting; ong, yew kwang; thong, mark; lim, hui fang; choi, hyung won; rotzschke, olaf; chow, vincent t; wang, de yun title: rna sequencing of h3n2 influenza virus-infected human nasal epithelial cells from multiple subjects reveals molecular pathways associated with tissue injury and complications date: 2019-08-27 journal: cells doi: 10.3390/cells8090986 sha: doc_id: 3898 cord_uid: y6zpvw84 the human nasal epithelium is the primary site of exposure to influenza virus, the initiator of host responses to influenza and the resultant pathologies. influenza virus may cause serious respiratory infection resulting in major complications, as well as severe impairment of the airways. here, we elucidated the global transcriptomic changes during h3n2 infection of human nasal epithelial cells from multiple individuals. using rna sequencing, we characterized the differentially-expressed genes and pathways associated with changes occurring at the nasal epithelium following infection. we used in vitro differentiated human nasal epithelial cell culture model derived from seven different donors who had no concurrent history of viral infections. statistical analysis highlighted strong transcriptomic signatures significantly associated with 24 and 48 h after infection, but not at the earlier 8-h time point. in particular, we found that the influenza infection induced in the nasal epithelium early and altered responses in interferon gamma signaling, b-cell signaling, apoptosis, necrosis, smooth muscle proliferation, and metabolic alterations. these molecular events initiated at the infected nasal epithelium may potentially adversely impact the airway, and thus the genes we identified could serve as potential diagnostic biomarkers or therapeutic targets for influenza infection and associated disease management. the global burden of inter-pandemic influenza is high. it is estimated to affect 1 billion people annually, with 3-5 million severe cases requiring hospitalization or intensive care treatment, resulting in approximately 0.5 million deaths [1] . worryingly, drug-resistant influenza strains are emerging at a rapid rate that will severely hamper the ability of our healthcare systems to contain influenza outbreaks [2] . therefore, alternative strategies are needed against severe influenza infections during both seasonal and pandemic influenza outbreaks. the normal human airway epithelium is a pseudo-stratified layer of ciliated and non-ciliated columnar cells, goblet cells, club cells, and basal cells [3] . the airway epithelium protects against airway infection via efficient mucociliary clearance (mcc), the production of inflammatory mediators and chemokines against viruses, and the recruitment of immune cells [4] . when the influenza virus breaches the defense of the human airway epithelium, it causes a myriad of innate responses by the infected host in response to viral invasion [5, 6] . among these changes are critical factors that can determine disease severity, and which may lead to the development of diagnostic, prognostic prediction markers, or anti-influenza therapies [7] [8] [9] [10] . however, few studies have hitherto been performed in relevant models [11] and human models of influenza are not feasible due to potential severity of the infection. therefore, the mechanistic study of viral-induced airway changes using relevant models can lead to better understanding of the development of severe complications. additionally, we need greater clarity on the different immune responses in view of the rising prevalence of chronic diseases such as diabetes mellitus and asthma. patients with these disorders are especially susceptible to severe influenza complications compared to healthy subjects [12] . thus, the establishment of a baseline response against influenza infection of "healthy" tissue is beneficial to facilitate future comparative studies to better manage influenza in patients with co-morbidities. although the study of host responses in influenza infection is not new, current in vitro cell lines cannot accurately represent human airway infection due to the lack of key mucociliary features [13] . hence, we have previously developed an air-liquid interface (ali) human airway epithelial cell culture that is able to sustain influenza infection [5, 6] . we have also further compared the transcriptomic responses of our infected human nasal epithelial cells (hnecs) with 15 other in vitro and in vivo influenza infection transcriptomic studies [6] . the comparison revealed that at their peak responses against influenza, the differential transcriptome signature in hnecs was highly similar to the signatures from other influenza infection models [6] . interestingly, compared to the homogenous cell lines tested, our heterogenous hnec model exhibited a more comparable response to the clinical influenza studies, indicating that most responses were initiated at the nasal epithelium [6] . therefore, in this study, we aim to further utilize the hnec model as a physiologically relevant in vitro model to clarify the nasal epithelial responses against influenza h3n2 infection, which would then facilitate the identification of the key host factors that are significant for future studies. to establish host factors that are significantly altered in the nasal epithelium as a reference of early innate responses against influenza, the dynamic expression of the genes needs to be clearly elucidated. while there are many studies that utilize microarray analysis to identify the host responses against influenza, the limitation of the microarray is its inability to determine the full extent of gene changes due to its hybridization-based protocol [14] . the aim of this study was to utilize rna sequencing (rnaseq) technology to not only reveal the hnec responses (from multiple individuals) against influenza infection, but also to identify those genes with high magnitude changes to serve as potential reference markers of the innate responses of influenza infection. given that rnaseq functions by reading virtually all the rnas present in the samples tested, we can also discern the magnitude of each rna change and mark them as the canonical responses. in addition, as rnaseq is not constrained by probe usage as in microarrays, they are therefore more reliable in detecting novel interactions during influenza infections of hnecs. hence, rnaseq analysis will further augment the transcriptomic data established previously by microarray analysis. the augmented baseline can then be applied to future clinical studies and practice against influenza infection, especially for comparison against patients with other underlying co-morbidities that may be affected by more severe disease. approval to conduct this study was obtained from the national healthcare group domain-specific board of singapore (dsrb ref: d/11/228) and the institutional review board of the national university of singapore (irb ref: . written consent was obtained from donors prior to the collection of the tissue biopsies. at the time of collection, all subjects were free of symptoms of urti. the medical backgrounds of the subjects are summarized intable s1. the hnespcs were isolated and enriched from the tissue biopsies according to a previously standardized protocol [5, 15] , which normalized the hnespcs to a baseline state that differentiates into hnecs resembling healthy tissues if they pass the quality control checks for their differentiation [5] . following enrichment, the hnespcs were expanded further and subjected to ali culture in transwells for in vitro differentiation according to previous protocol as well [5, 15] . briefly, primary cells were subjected to isolation for selection of hnespcs, which were enriched and expanded with dulbecco's modified eagle medium: nutrient mixture f-12 (dmem/f12) (gibco-invitrogen, carlsbad, ca, usa) containing 10 ng/ml of human epithelial growth factor (egf, gibco-invitrogen, carlsbad, ca, usa), 5 µg/ml of insulin (sigma, st. louis, mo, usa), 0.1 nm of cholera toxin (sigma, st. louis, mo, usa), 0.5 µg/ml of hydrocortisone (sigma, st. louis, mo, usa), 2 nm/ml of 3,3 ,5-triiodo-l-thyronine (t3) (sigma, st. louis, mo, usa), 10 µl/ml of an n-2 supplement (gibco-invitrogen) and 100 iu/ml of antibiotic-antimycotic (gibco-invitrogen, carlsbad, ca, usa). the expanded hnespcs were then transferred onto 12-well 0.4 µm transwell inserts (corning, corning, ny, usa). once confluent, growth medium was discarded and 700 µl of pneumacult™-ali medium with inducer supplements (stemcell technologies inc., vancouver, canada) was added to the basal chamber to establish ali conditions. the cells were cultured in ali culture for 4 weeks, with media change every 2-3 days. after 3-4 weeks of differentiation, hnecs from a total of seven donors were then subjected to influenza h3n2 virus infection. the influenza a strain used in this study is of the h3n2 subtype (a/aichi/2/1968) (atcc, manassas, va, usa). the virus was propagated using embryonated egg culture and used for all the infection in the hnecs. prior to infection, fully differentiated hnecs were washed with 1× dpbs and infected with the h3n2 influenza virus at a multiplicity of infection (moi) of 0.1 and incubated for 1 h at 35 • c. after the 1 h incubation, the viral inoculum was removed and the hnecs were incubated back in 35 • c. the control hnecs were harvested for apical wash and rna prior to the infection at 0 h post-infection (hpi). the infected hnecs were then harvested for the apical wash and rna following 8, 24, and 48 hpi incubation at 35 • c. at each infection time point, 150 µl of 1x dpbs was added and incubated in the apical chamber for 10 min at 35 • c to recover progeny viruses as the apical wash. the plaque assay for viral quantification was performed using overnight mdck cultures (atcc, manassas, va, usa) at 85-95% confluence in 24-well plates. the mdck cells were incubated with 100 µl of serial dilutions (from 10 −1 to 10 −6 ) of virus from apical washes at 35 • c for 1 h, where plates were rocked every 15 min to ensure equal viral distribution. after incubation, the inocula were removed and replaced with 1 ml of avicel (fmc biopolymer, philadelphia, pa, usa) overlay, and incubated at 35 • c for 65-72 h. after incubation, avicel overlay were removed, and cells were fixed with 4% formaldehyde in 1× pbs for 1 h. formaldehyde was then removed, and cells were washed with 1× pbs prior to staining with 1% crystal violet for 15 min before washing the stain away. the plaque-forming units (pfu) were calculated as follows: number of plaques × dilution factor = number of pfu per 100 µl. at each time point after the collection of apical wash, the hnecs were lysed using rna lysis buffer. total rna was then extracted from the lysate using mirvana mirna isolation kit (life technologies, grand island, ny, usa). the extracted total rna was first subjected to nanodrop analysis to first ensure the rna quality, before being submitted for rnaseq analysis. then, 500 ng from the remaining rna was subjected to cdna synthesis using maxima first-strand cdna synthesis kit (thermoscientific, pittsburgh pa, usa). after this, qpcr analysis was performed to evaluate the transcriptional levels of host response genes selected based on previous microarray analysis using pre-designed primers (sigma aldrich). each qpcr reaction was performed in duplicate using gotaq-qpcr master mix kit (promega, san luis obispo, ca, usa), and relative gene expression was calculated using the comparative method of 2-∆∆ct normalized to the housekeeping gene pgk1. relative gene expression levels were presented as median values and interquartile ranges, while statistical significance was determined using the wilcoxon signed-rank test. all human rnas were analyzed on the agilent bioanalyzer (agilent, santa clara, ca, usa) or the perkin elmer labchip gx system (perkin elmer, waltham, ma, usa) for quality assessment with rna integrity number (rin) or rna quality score range from 6.8-9.7 and median of 9.0. cdna libraries were prepared using 2 ng of total rna and 1 µl of a 1:50,000 dilution of ercc rna spike in controls (ambion ® thermo fisher scientific, waltham, ma, usa) using smartseq v2 protocol [16] , except for the following modifications: fastq files were mapped to the human genome build hg38 using star. gene counts were computed using featurecounts (part of the subread package) using annotations from gencode version 26. differential gene expression analysis was performed using edger in a paired fashion under r version 3.3.3. multiple testing correction was done using the method of benjamini and hochberg and p-values (false discovery rate; fdr) less than 0.05 was deemed to be significant. geneset enrichment analysis using data from gene ontology (go) was performed using the bioconductor package topgo, while the analysis using reactome pathway was performed using the vioconductor package reactomepa. both analyses were run in r version 3.3.3 using multiple testing-corrected significant differentially-expressed genes. tnf-α, tnf-β, vegf, eotaxin/ccl11, and pdgf-aa. samples and standards were incubated with fluorescent-coded magnetic beads which had been pre-coated with the respective capture antibodies. after an overnight incubation at 4 • c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for 1 h, and streptavidin-pe was then added and incubated for another 30 min. plates were washed twice again, then beads were re-suspended with sheath fluid before acquiring on the flexmap ® 3d (luminex) using xponent ® 4.0 (luminex) acquisition software. data analysis was done on bio-plex manager™ 6.1.1 (bio-rad). standard curves were generated with a 5-pl (5-parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than 0.05 were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points (figure 1 ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [6] . the respective capture antibodies. after an overnight incubation at 4 °c, plates were washed twice. biotinylated detection antibodies were incubated with the complex for 1 h, and streptavidin-pe was then added and incubated for another 30 min. plates were washed twice again, then beads were resuspended with sheath fluid before acquiring on the flexmap ® 3d (luminex) using xponent ® 4.0 (luminex) acquisition software. data analysis was done on bio-plex manager™ 6.1.1 (bio-rad). standard curves were generated with a 5-pl (5-parameter logistic) algorithm, reporting values for both mfi and concentration data. results were then expressed as mean fold change compared with uninfected control, and p-values (fdr) of less than 0.05 were considered significant. prior to analysis, the responses of all seven hnecs donors following influenza infection were plotted on a principal component analysis (pca) plot. the pca plot indicated a degree of variability in the responses between donors and time points ( figure 1 ). nonetheless, the responses were clustered tightly enough following infection to signify their consistency of infection for further transcriptomic analysis-similar to those observed in our previous study [6] . significant gene expression changes (fdr < 0.05) of infected hnecs were detected as early as 8 hpi, and further increased at 24 and 48 hpi (table 1; figure 2a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the 10x fold change genes significant gene expression changes (fdr < 0.05) of infected hnecs were detected as early as 8 hpi, and further increased at 24 and 48 hpi (table 1; figure 2a ). also, the number of genes decreased in a linear fashion as the fold change in expression increased, as seen in the 10x fold change genes indicated in figure 2b , where about 10% of the significantly altered genes remained. at 8 hpi, there were 31 upregulated genes and 13 downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at 8 hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of 704 and 1080 genes, and downregulation of 217 and 758 genes at 24 and 48 hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl10 and cxcl11 was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo2, klk12, and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at 24 and 48 hpi. tables s2-s4 list the complete set of significant gene expression changes, arranged according to their fold change (log 2 fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the 10 genes tested by qpcr at 48 hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of <0.05 (il4i1, ifnl1(il29), cxcl10, tnfsf10, ifi6, ccl24, and cyp26a1), one gene had a p-value of <0.1 (ctgf), while only two genes were not statistically significant (tgfa and ano5) ( figure s1 ). indicated in figure 2b , where about 10% of the significantly altered genes remained. at 8 hpi, there were 31 upregulated genes and 13 downregulated genes. the major upregulated genes were the antiviral sensors and early response genes such as ifns, ifits, and ifis. interestingly enough, interferon lambda (ifnλ) gene ifnls was the earliest response interferon of infected hnecs, as opposed to interferons alpha or beta, at 8 hpi. at later time points, the number of gene expression changes increased substantially, with upregulation of 704 and 1080 genes, and downregulation of 217 and 758 genes at 24 and 48 hpi, respectively. there was augmented expression of antiviral effectors and inflammatory genes at both time points. ifnl remained the interferon gene with highest expression at both time points, while a marked elevation of cytokines such as cxcl10 and cxcl11 was also observed. considering downregulated genes, proliferative and transcriptomic functions appeared to be suppressed, with diminished expression of genes such as fmo2, klk12, and fosb. genes associated with metabolism, cell cycle, and dna repair were further suppressed following infection at 24 and 48 hpi. tables s2-4 list the complete set of significant gene expression changes, arranged according to their fold change (log2fc). in addition, we have also verified that the genes showing major expression changes by rnaseq generally concurred with rt-qpcr analyses. of the 10 genes tested by qpcr at 48 hpi, all of them showed the same directional changes in expression as observed by rnaseq. hence, seven of these genes showed a p-value of <0.05 (il4i1, ifnl1(il29), cxcl10, tnfsf10, ifi6, ccl24, and cyp26a1), one gene had a p-value of <0.1 (ctgf), while only two genes were not statistically significant (tgfa and ano5) ( figure s1 ). we then further compared the transcriptomic alterations in the hnecs over time, following influenza h3n2 infection. the number of gene expression changes mirrored the viral titer changes, which peaked at 48 hpi, and were consistent between donors ( figure 3a ). approximately two thirds of genes at 8 and 24 hpi overlapped with other time points, while about one third of genes at 48 hpi overlapped ( figure 3b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at 48 hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure 3c , which portrays the heatmaps of the top 10 genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that 48 hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further compared the transcriptomic alterations in the hnecs over time, following influenza h3n2 infection. the number of gene expression changes mirrored the viral titer changes, which peaked at 48 hpi, and were consistent between donors ( figure 3a ). approximately two thirds of genes at 8 and 24 hpi overlapped with other time points, while about one third of genes at 48 hpi overlapped ( figure 3b ). the overlapping genes displayed similar directional consistency at the significant time points. in addition, congruent with the consistent viral titer with most gene expression changes at 48 hpi, we also noted the most consistent alterations in expression of genes across donors. this is highlighted in figure 3c , which portrays the heatmaps of the top 10 genes with the smallest p-value, together with their direction and magnitude of change. based on these analyses, we proposed that 48 hpi represents the optimal time point for the subsequent pathway analysis to ascertain influenza-specific pathway changes. we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points 8, 24, and 48 hpi, there were 3, 41, and 30 significant (adjusted p-value < 0.05) go biological processes (table s5 ) and 3, 93, and 74 significant (adjusted pvalue < 0.05) reactome pathways (table s6) , respectively. at the early time of 8 hpi, interferon we then further subjected the significant gene changes to gene set enrichment using both go and reactome databases. at time points 8, 24, and 48 hpi, there were 3, 41, and 30 significant (adjusted p-value < 0.05) go biological processes (table s5 ) and 3, 93, and 74 significant (adjusted p-value < 0.05) reactome pathways (table s6) , respectively. at the early time of 8 hpi, interferon-mediated antiviral responses were elevated as expected. at 48 hpi, the pathways appeared to be more stabilized and consistent for both go and reactome analyses, despite displaying more gene expression changes at this time point. responses to influenza virus skewing towards type i immunity were predominant in the go analysis. the expected interferon-mediated functions by the epithelium validated the authenticity of our model, where we found enriched type i interferon (go) and rig-i (reactome) pathways with upregulation of nearly all significant gene members (data not shown). besides the interferon and antiviral pathways, we identified several functions of interest initiated by the nasal epithelium that may contribute to the pathology and pathogenesis of influenza. at 48 hpi, go pathway enrichment analysis revealed that the nasal epithelium was actively involved in initial ifnγ signaling (go:0060333), despite not directly producing ifnγ. we also observed enriched function in apoptosis and necroptosis (go:0008637 and go:0060544), immune evasion (go:0045824), and other pathways that may lead to complication events such as smooth muscle proliferation (go:0048661) and response to fatty acid (go:0070542) ( table 2 ). for the reactome pathway analysis, we selected pathways that were enriched with more than 30 significant genes present in the enriched pathway, and these were generally in agreement with the go analysis (table 3 ). in addition to ifnγ signaling (877300) and apoptosis (109581), it also revealed changes in epithelial-initiated b cell receptor signaling (983705 and 1168372) and amino acid metabolism (71291) following influenza infection. it is noteworthy that these pathways were initiated at the epithelial level without the participation of immune cells, thus highlighting the relevant genes of interest for future studies. given that rnaseq analysis facilitates more accurate expression changes following infection compared to hybridization technology such as microarray, we conducted further analysis on the levels of gene expression changes to enable more stringent and accurate transcriptomic analyses for future studies. by comparing these results to a previous study that identified 11 influenza-specific signatures, we verified that these 11 genes were all expressed in infected nasal epithelium later at 24 hpi, but not at 8 hpi. furthermore, at both 24 and 48 hpi, all but one of the 11 gene signatures exhibited elevated expression of >2.5-fold change (>1.33 log 2 fc) compared to uninfected control hnecs (table 4 ). when we applied the higher fold change cutoff, the number of significant genes decreased by approximately 50% (figure 4a) , which was also congruent with the linear association observed earlier. therefore, future studies on early transcriptional alterations could consider adopting the 2.5-fold change in expression as a more stringent threshold, which may be more feasible, especially for large transcriptomic studies that yield large numbers of data points. in addition, when compared to the previous microarray study on a similar hnec model [6] , both rnaseq and microarray shared a high degree of overlap, with about one third and half of total genes from rnaseq and microarray overlapping, respectively ( figure 4b ). the overlap was generally observed in genes with highly altered expression, such as cxcl10, cxcl11, and rsad2, which were changed to a similar magnitude in both rnaseq and microarray (table s7 ). when we compared the 11 influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at 48 hpi, the magnitude of the gene change was generally higher than that of the microarray (table 5 ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only (193 genes versus 22 genes with elevated expression greater than 2.5-fold). genes such as heatr9, pdcd1, il4i1, art3, and kcnh7 were altered to a higher magnitude than the 2.5-fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. from rnaseq and microarray overlapping, respectively ( figure 4b ). the overlap was generally observed in genes with highly altered expression, such as cxcl10, cxcl11, and rsad2, which were changed to a similar magnitude in both rnaseq and microarray (table s7 ). when we compared the 11 influenza signature genes, rnaseq revealed a more consistent increase in magnitude, i.e., at 48 hpi, the magnitude of the gene change was generally higher than that of the microarray (table 5 ). in addition, rnaseq was also able to detect novel genes with expression changes of high magnitude that were generally higher than those found by microarray only (193 genes versus 22 genes with elevated expression greater than 2.5-fold). genes such as heatr9, pdcd1, il4i1, art3, and kcnh7 were altered to a higher magnitude than the 2.5-fold threshold. hence, rnaseq-based transcriptomic analysis may augment transcriptomic findings to identify novel gene responses against influenza in the future. after deriving the transcriptomes by rnaseq, we then further investigated whether the changes in expression of genes resulted in alterations in secretory cytokines and chemokines early in the infection of hnecs. initially, we detected significant reductions in multiple cytokines at 8 hpi, with the exception of il-15 which was increased ( figure s2 ). this may reflect the initial immune suppression during influenza infection. however, at 24 and 48 hpi, less significant changes were observed, i.e., only increase in tnf-a and decrease in mdc and pdgf-aa were noted at 24 hpi. this was followed by increase in ip-10 (cxcl10) and tgf-a and decrease in pdgf-aa seen at 48 hpi. this analysis highlights changes in ip-10, tgf-a, and pdgf-aa to be significant early responses in secretory cytokines/chemokines following influenza infection. our study has identified epithelium-initiated host responses which are found to be involved in both innate and adaptive responses. the finding is significant as we can now focus on the primary point of contact of influenza-the nasal epithelium in the study of early host responses for identifying host factors that can be utilized for diagnostic and therapeutic purposes [7] . in addition, our study also showed that it is important for reference databases to use relevant human models like the hnecs model, which contains the mucociliary component of the airways, in order to provide closely representative host responses. while there exists a high number of microarray studies that showed the host responses using similar hnec models, there are only a small number of equivalent rnaseq studies. compared to microarrays, rnaseq analysis can provide a more comprehensive picture of the transcriptomic landscape, and is not limited by the hybrid library variant and concentrations [14] . hence, in order to derive accurate magnitude of gene expression changes, we performed an rnaseq analysis of h3n2 infection using the hnecs model. h3n2 influenza virus was selected, given that it is a major circulating subtype over long periods of time. in addition, relatively lower efficacy of vaccines against this subtype prompted us to study its interactions with the primary host target to elucidate the immune responses and association with adaptive immunity [17, 18] . this model has been previously evaluated to be a highly clinically-relevant model that can facilitate controlled infection of nasal cells from multiple individuals. in addition, we have also previously shown-by microarray analysis-that the nasal epithelium is responsible for the initiation of host responses following influenza infection [6] . this renders the hnecs to serve as a valuable tool to analyze transcriptomics from different individuals infected under the same conditions to ensure consistent and relevant responses in humans. once the magnitude of gene expression changes was considered, several interesting findings emerged. firstly, the infected hnecs were observed with strong activation of antiviral genes and early inflammatory genes leading to type i immune responses. a large number of gene expression changes were of magnitude of over 100-fold difference (log 2 7 to log 2 9 fold change). most of the genes with high-magnitude expression changes were verified by qpcr, with statistical significance congruent with the rnaseq analysis. secondly, despite the absence of immune cells, the infected hnecs were able to generate strong type i responses that may likely aid the recruitment of cytotoxic cells to clear the infected cells. thirdly, in early responses of the hnecs, ifnλ genes, which represent type iii interferons, were more strongly induced than the more frequently observed type i interferons (ifnα and ifnβ), while type ii interferons were not produced by hnecs, in agreement with previous studies [5, 19] . the induction of type iii interferons may reflect an important event within the hnecs where ifnλ, the initial responders against the infection, may be more beneficial in the antiviral response [20, 21] . moreover, we also observed notable suppression of expression of certain genes following influenza infection, including suppression of proliferation and dna repair genes, which may contribute to the pathology and pathogenesis of influenza [22] . finally, rnaseq also unraveled expression changes of certain newly-discovered genes in response to influenza infection of the upper airway cells. genes such as heatr9 [23] , il4i1 [24] , tnfsf13b (baff) [25] , and pdcd1 (pd-1) [26] are recently implicated in influenza pathogenesis and mucosal defense, thereby signifying the role of the nasal epithelium against influenza infection. furthermore, rnaseq identified altered expression of art3 and kcnh7 genes that were not previously detected in influenza transcriptomes. these findings hence further reiterate the value of rnaseq in enhancing data on influenza transcriptomes for reference in future studies. via pathway enrichment analysis, we have identified known antiviral pathways to validate the hnecs responses against influenza. in addition, we have also documented the potential pathways initiated by the nasal epithelium that may contribute to influenza pathogenesis as represented by the gene expression changes listed in tables 2 and 3 . by analyses using literature-inferred go and reactome databases, we have demonstrated that the nasal epithelium can play a role in the main antiviral signaling, i.e., ifnγ responses despite not being a direct producer of ifnγ. the pathway enrichment indicated that hnecs may serve as important regulators of type ii interferons. even though the effects of ifnγ are vital to the robust clearance of influenza viruses [27] , there are reports of unregulated ifnγ being a contributor to inflammatory damage [28, 29] . therefore, the over-production of ifnγ response factors such as icam1 and cd44 may contribute to inflammatory damage of the epithelium. hence, production of factors such as stat1 [28] by the hnecs is also crucial in ensuring appropriate regulation of ifnγ-mediated expression of influenza response genes to modulate inflammation and to minimize damage. the primary contact of influenza virus with the nasal epithelium may subsequently lead to damage to the airway epithelium as well. this is apparent with the clear enrichment of the pathways of apoptosis, mitochondrial apoptotic processes, and necroptosis that contribute to cell death and mechanical barrier loss during infection [5, 30] . genes such as ifi6, bak1, caps8, tnfsf10, and fas suggest active apoptotic cell death that not only destroys cells in the epithelial barrier, but may also serve to propagate the virus and to perpetuate the damage [31] [32] [33] . furthermore, during virus infection, aberrant regulation of apoptosis may also lead to further injury to the epithelium and surrounding tissues [34] . on the other hand, necroptosis pathways have also been observed to be enriched in influenza-infected hnecs. compared to apoptosis, the study of necroptosis in influenza infection is relatively new with contradicting findings [34] . ripk3/necroptosis studies appear to generate contradictory results as to whether necroptosis protects against or is detrimental during influenza infection [35, 36] . hence, its increased expression during infection of hnecs warrants further investigation on its role in influenza-induced damage. in addition, we also noted enrichment of b-cell signaling pathways by the infected hnecs which may be vital for b-cell responses during the adaptive immune response [37] . we noted that most genes enriched in the b-cell pathways were related to antigen recognition such as proteasome subunits (psme2, psmb9, psma6, etc.) and b-cell receptor-associated genes such as dapp1 and card11 [38] . however, changes in expression of certain growth factors (including ereg and fgfs) following influenza infection may lead to complications involving airway remodeling and recruitment [39, 40] . further, the effects of the growth factors were further confirmed by the enrichment of pathways related to the proliferation of smooth muscle cells also induced by the infection. changes to airway smooth muscle cells are usually implicated in airway remodeling [41] [42] [43] , and may also contribute to post-influenza complications. hence, the genes found in this study may be crucial for elucidating the nasal-initiated responses that may contribute to the pathology and pathogenesis of influenza infection of the airways. finally, another interesting pathway that may contribute to epithelial damage is the negative regulation of innate immune responses. these genes may serve as proviral factors and aid in immune evasion. for example, adar is a proviral factor that works in synergy with influenza ns1 to enhance viral replication [44] . trafd1 is a negative regulator of toll-like receptor signaling which is upregulated in influenza-infected hnecs [45] . dhx58 is a negative regulator of rig-i/mda5 signaling pathway [46] . ceacam1 is involved in regulation of liver inflammation [47] and its expression appears to exert antiviral effects on influenza virus [48] . nmi binds to influenza virus ns1 and inhibits irf7-mediated interferon signaling [49, 50] . therefore, aberrant expression of genes in this signaling pathway may directly contribute to immune evasion of influenza, culminating in viral propagation and increased epithelial damage. we summarized the identified pathways (listed in tables 2 and 3) that alluded to immune evasion (negative regulation of innate immune responses), antigen processing (metabolism of amino acids and derivatives), and immunomodulation (interferon gamma signaling, b cell receptor signaling, and response to fatty acid) that may contribute to severity of influenza. there was evidence of direct pathway enrichment of potential influenza evasion strategies and/or immunomodulation with accompanying transcriptomic changes. the genes in the pathway may be analyzed for their immunomodulatory activity and whether their expression is beneficial to the virus (immune evasion) or the host (preventing cytokine storm). in addition, the infected hnecs also revealed modified responses associated with fatty acid, with many lipid signaling molecules such as leukotrienes that mediate antiviral responses and subsequent inflammation of the airway [51, 52] . such modified responses may also determine the afforded in the airway and the severity of airway inflammation and damage. in addition, the modification may also affect the lower airway responses to inflammatory mediators; hence, the changes in these pathways may also suggest a potential mechanistic link to the pathogenesis of viral-induced exacerbation of chronic inflammatory diseases. lastly, we also noted enrichment of pathways related to amino acid metabolism, which is important in antigen processing and proteasomal degradation of foreign protein. the changes in these genes at the hnecs, the target site of influenza infection, may determine the effectiveness of antiviral responses mounted and may therefore influenza disease severity. in addition, we also compared our rnaseq analysis against previously reported influenza-specific signatures in order to improve future transcriptomic analysis [53] . in vitro transcriptomic analysis yields a large number of differentially-expressed genes that would require additional criteria to identify functionally significant genes. by means of this comparison, we discovered that almost all influenza-specific signatures exhibited differences in expression of above 2.5-fold. hence, we propose applying fold change of >2.5 as a threshold for future in vitro transcriptomic systems analyses, in order to increase the stringency in detecting functionally significant gene changes. finally, we observed that, unlike the transcriptome, there were notably fewer cytokines that were readily secreted during the acute phase of infection. expression of cytokines was reduced at 8 hpi, except for il-15, which interestingly is implicated in influenza-induced acute lung injury [54] . even fewer cytokines showed altered expression at later time points. among them, only tgf-a, ip-10 (cxcl10), and pdgf-aa were significantly altered at 24 and 48 hpi. these may be significant markers that can be detected in the secretion of influenza-infected mucosal surface that may influence the severity of influenza. ip-10 is a well-established ifnγ response gene, and serves as a useful marker for response against influenza [5, 6] . tgf-a represents an important factor involved in the secretion of il-8 in response to influenza, and may determine the early appropriate innate responses to prevent severe disease [55] . on the other hand, it is also involved in pulmonary fibrosis as a ligand of epidermal growth factor receptor (egfr) and may contribute to complications in the lower airway [56] . pdgf-aa was found to be elevated in the cerebrospinal fluid of influenza-associated encephalopathy [57] , but was consistently reduced in hnec secretory fluid, thus warranting further investigation into its role in the infected nasal mucosa. the establishment of a reference transcriptome based on early responses of the human nasal epithelium model serves a key role in research on critical host factors involved in influenza. as the primary host contact with the virus, not only are immune responses against influenza important, but also the alterations in non-immune functions such as metabolism, cell content, and cell cycle, which may contribute to disease severity. in terms of translational potential, the model system identified gene expression changes of significant magnitude and pathways that impact responses against influenza and its severity. these genes may represent novel targets for future diagnostic and therapeutic development. under controlled conditions, the hnecs clinically establish the baseline for "normal" innate immune responses of the nasal epithelium against influenza viral infection. such a baseline can be particularly crucial when studying the changes in innate immune responses against influenza, especially in patients with underlying chronic diseases who may have aberrant airway responses against influenza. their antiviral responses may differ from "normal" subjects, and this study thus provides the basis for comparing the differential responses that culminate in more severe infections in patients with co-morbidities such as diabetes and chronic airway inflammatory diseases. such comparative clinical studies can potentially enhance the management of influenza viral infection in patients with chronic diseases. in conclusion, rnaseq technology allowed us to accurately quantify the magnitude of gene expression changes, as well as the relevant enriched pathways during h3n2 influenza virus infection of hnecs, which can serve as a baseline for future clinical studies. the establishment of this baseline under controlled condition elucidated the antiviral innate response by the infected nasal epithelium, and highlighted the molecular factors and abnormalities in the upper airway that may contribute to influenza severity. furthermore, this study also culminated in the identification of novel gene signatures and host factors that may be harnessed for future research to develop influenza diagnostic markers and therapeutic targets. supplementary materials: the following are available online at http://www.mdpi.com/2073-4409/8/9/986/s1, figure s1 . real-time quantitative pcr validation of genes selected from rnaseq analyses. pcr data are expressed as log2 fold change using median and interquartile range. statistical significance was determined using wilcoxon signed-rank test. * p < 0.05, # p < 0.1; figure s2 . luminex assay of secreted cytokines/chemokines in the apical supernatant. luminex data are expressed as mean fold change from uninfected control. statistical significance was determined using fdr. * p < 0.05; table s1 . information of seven donors of hnecs; table s6 . significant enriched pathways based on reactome pathway database analysis; table s7 . list of significant genes with altered expression at 48 hpi of influenza h3n2 infection of hnecs analyzed by rnaseq and microarray [6] . oseltamivir resistance-disabling our influenza defenses role of il-13ralpha2 in modulating il-13-induced muc5ac and ciliary changes in healthy and crswnp mucosa epithelial damage and response human nasal epithelial cells derived from multiple individuals exhibit differential responses to h3n2 influenza virus infection in vitro comparative transcriptomic and metagenomic analyses of influenza virus-infected nasal epithelial cells from multiple individuals reveal specific nasal-initiated signatures systems-biology approaches to discover anti-viral effectors of the human innate immune response uncovering the global host cell requirements for influenza virus replication via rnai screening. microbes infect cellular networks involved in the influenza virus life cycle cd151, a novel host factor of nuclear export signaling in influenza virus infection propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures distinction between rhinovirus-induced acute asthma and asthma-augmented influenza infection a novel three-dimensional cell culture method enhances antiviral drug screening in primary human cells comparing bioinformatic gene expression profiling methods: microarray and rna-seq the use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro full-length rna-seq from single cells using smart-seq2 predicting clinical severity based on substitutions near epitope a of influenza a/h3n2 effectiveness of seasonal influenza vaccinations against laboratory-confirmed influenza-associated infections among singapore military personnel in 2010-2013. influenza other respir in vitro model of fully differentiated human nasal epithelial cells infected with rhinovirus reveals epithelium-initiated immune responses ifnlambda is a potent anti-influenza therapeutic without the inflammatory side effects of ifnalpha treatment interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness influenza infection induces host dna damage and dynamic dna damage responses during tissue regeneration heatr9 is upregulated during influenza virus infection in lung alveolar epithelial cells the il4i1 enzyme: a new player in the immunosuppressive tumor microenvironment. cells cigarette smoke inhibits baff expression and mucosal immunoglobulin a responses in the lung during influenza virus infection highly pathological influenza a virus infection is associated with augmented expression of pd-1 by functionally compromised virus-specific cd8+ t cells new fronts emerge in the influenza cytokine storm inflammatory impact of ifn-gamma in cd8+ t cell-mediated lung injury is mediated by both stat1-dependent and -independent pathways production of interferon-gamma by influenza hemagglutinin-specific cd8 effector t cells influences the development of pulmonary immunopathology h3n2 influenza virus infection enhances oncostatin m expression in human nasal epithelium nf-kappab-dependent induction of tumor necrosis factor fas/fasl is crucial for efficient influenza virus propagation nucleoprotein of influenza a virus negatively impacts antiapoptotic protein api5 to enhance e2f1-dependent apoptosis and virus replication influenza a virus enhances its propagation through the modulation of annexin-a1 dependent endosomal trafficking and apoptosis programmed cell death in the pathogenesis of influenza dai senses influenza a virus genomic rna and activates ripk3-dependent cell death zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways the multifaceted b cell response to influenza virus influenza virus-induced type i interferon leads to polyclonal b-cell activation but does not break down b-cell tolerance neutrophils induce smooth muscle hyperplasia via neutrophil elastase-induced fgf-2 in a mouse model of asthma with mixed inflammation respiratory syncytial virus infection provokes airway remodelling in allergen-exposed mice in absence of prior allergen sensitization cd151, a laminin receptor showing increased expression in asthma, contributes to airway hyperresponsiveness through calcium signaling regulation of human airway smooth muscle cell migration and relevance to asthma airway smooth muscle in asthma: phenotype plasticity and function. pulm pharm the interactomes of influenza virus ns1 and ns2 proteins identify new host factors and provide insights for adar1 playing a supportive role in virus replication yoshimura, a. fln29, a novel interferon-and lps-inducible gene acting as a negative regulator of toll-like receptor signaling rna-and virus-independent inhibition of antiviral signaling by rna helicase lgp2 ceacam1 in liver injury, metabolic and immune regulation ceacam1-mediated inhibition of virus production subcellular proteomic analysis of human host cells infected with h3n2 swine influenza virus negative regulation of nmi on virus-triggered type i ifn production by targeting irf7 mast cells and influenza a virus: association with allergic responses and beyond leukotriene b4 enhances nod2-dependent innate response against influenza virus infection multi-cohort analysis identifies conserved transcriptional signatures across multiple respiratory viruses interleukin-15 is critical in the pathogenesis of influenza a virus-induced acute lung injury influenza induces il-8 and gm-csf secretion by human alveolar epithelial cells through hgf/c-met and tgf-alpha/egfr signaling overactive epidermal growth factor receptor signaling leads to increased fibrosis after severe acute respiratory syndrome coronavirus infection vascular endothelial growth factor (vegf) and platelet-derived growth factor (pdgf) levels in the cerebrospinal fluid of children with influenza-associated encephalopathy this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the surgeons and staff in the department of otolaryngology, national university hospital, singapore. we thank h.h. ong and t.t. he for the subject selection and recording. we thank m.c. phoon and s.h. lau for technical assistance in viral experiments. the authors would like to acknowledge the staff of the immunomonitoring platform at sign. the authors declare no conflicts of interest. key: cord-003598-m2fsrwvw authors: elbahesh, husni; gerlach, thomas; saletti, giulietta; rimmelzwaan, guus f. title: response modifiers: tweaking the immune response against influenza a virus date: 2019-04-12 journal: front immunol doi: 10.3389/fimmu.2019.00809 sha: doc_id: 3598 cord_uid: m2fsrwvw despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza a virus (iav) infections remains limited and is challenged by the virus itself. while vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. in addition, antigenic variation of iav complicates the production of efficacious vaccines. similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. like many viruses, iav is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. while host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as iav antivirals. future strategies to combat iav infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (brms). in principle, brms are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. brms are already being used in the clinic to treat malignancies and autoimmune diseases. repurposing such agents would allow for accelerated use against severe and potentially fatal iav infections. in this review, we will address the potential therapeutic use of different brm classes to modulate the immune response induced after iav infections. influenza viruses (ivs) are responsible for significant morbidity and mortality in the human population with ∼500,000 annual deaths worldwide. ivs can cause severe acute respiratory disease especially in high-risk populations like children, the elderly and the immunocompromised. while both influenza a and b viruses (iav and ibv, respectively) cause annual epidemics, the majority of severe human infections are caused by iav. ivs have segmented negative-sense single-stranded rna genomes. the lack of proof-reading activity of the viral rna-dependent rna polymerase (rdrp) and successive replication can lead to the accumulation of nucleotide mutations which drive antigenic drift. in addition, the segmented nature of their genome allows genetic reassortment between iv's to take place, which can produce novel strains that have acquired alternative antigenically distinct hemagglutinin, also known as antigenic shift. both antigenic drift and antigenic shift contribute to the iv's ability to evade pre-existing host immunity induced by previous infections. early recognition and responses to iv infection are largely mediated by innate immune sensors expressed by its primary target, the alveolar epithelial cells (1, 2). recognition of ivs is mediated by pattern recognition receptors (prrs) that include toll like receptors (tlrs), retinoinc acid inducible gene-i (rig-i), and nucleotide oligomerization domain (nod)-like receptor family pyrin domain containing 3 (nlrp3); all of which can recognize viral rnas during various stages of the infection cycle (3-5). activation of these sensors triggers signaling cascades that lead to the production of interferons as well as pro-inflammatory cytokines and chemokines ultimately resulting in an antiviral state within the surrounding cells/tissue (6). accordingly, ivs have multiple mechanisms to evade these responses mediated by the viral nonstructural 1 protein (ns1), polymerase basic 1 protein (pb1), polymerase basic 2 protein (pb2), polymerase acidic (pa) and nucleoprotein (np) [ in otherwise healthy individuals, iav infections are mild and the ensuing pro-and anti-inflammatory responses are balanced. in contrast, a "cytokine storm" is typically associated with severe infections including those caused by highly pathogenic iv strains. during a cytokine storm, chemokine and cytokine responses are dysregulated in both intensity and kinetics resulting in excessive damage to the host due to infiltration of inflammatory immune cells. acute lung injury (ali) caused by this inflammatory response is typically characterized by significant damage or destruction of the respiratory epithelium leading to acute respiratory distress syndrome (ards) (7, 8). clinical treatment options for severe influenza virus infections remain limited and relying heavily on the administration of antiviral neuraminidase inhibitors (nais) and supportive critical care (9). however, nais have not been effective in patients with severe h7n9 or h5n1 infections and there is evidence that fatal outcomes are associated with development of antiviral resistance in patients (10-12). while virus-targeted therapies remain the standard approach, iv's mutability and adaptation to current antivirals has highlighted the need for new therapeutic options that target host factors that regulate iv infections and resulting immune responses. in either approach, the focus is to prevent or limit damage to the lung epithelium due to exaggerated or dysregulated immune cell responses. biological response modifiers (brms) can alter the immune response thereby offering an additional therapeutic approach to treating severe infections. in this review, we highlight several studies that have shown the viability of brms as potential treatment options. for clarity, brms are categorized based on the type of biological agent (table 1) . therapeutic antibodies iav infections and some vaccines elicit broadly-neutralizing antibodies (abs) that target the viral ha-stem. however, their abundance and immune-subdominance is overshadowed by abs targeting the ha-head domain. the effectiveness of these hastem abs against a broad range of iav subtypes, makes them an attractive target not only for vaccine development but also as antivirals. indeed, several ha-stem specific human monoclonal abs are now being evaluated in clinical trials [reviewed in davidson (34) ]. mhaa4549a, medi8852, and vis410 are human monoclonal abs that have been shown to control viral replication and improve symptoms of human patients in phase 2 clinical trials (13-15). while virus-specific abs aim to reduce antigenic load, abs to host targets aim at limiting the secondary wave of cytokines and reduce prolonged damaging cellular infiltration during severe infections. host-target directed antibodies have been utilized to target key regulators of this inflammatory wave and could potentially be used to dampen these overt responses. angiopoietin-like 4 (angptl4) is a soluble angiogenicregulating protein. following proteolytic cleavage, the cterminal portion (cangptl4) is involved in integrin-dependent wound repair and can regulate vascular permeability (35, 36) . angptl4 was significantly elevated in lung biopsies from iav-induced pneumonia patients (16). in mouse studies, neutralizing anti-angptl4 abs reduced pulmonary tissue leakiness significantly accelerating lung recovery and improved lung tissue integrity (16). neutrophil infiltration into the alveolar space occurs within 1 day following iav infections (37) . neutrophil extracellular traps (nets) released during iav-induced pneumonia into the alveolar space caused alveolar damage (38) . the complement protein c5a was shown to induce nets release and administration of anti-c5a abs (ifx-1) reduced h7n9-induced ali due to reduced infiltration of lung macrophages and neutrophils as well as reduction of viral load in african green monkeys (17, 39). tumor necrosis factor alpha (tnfα) is a key cytokine for controlling severe iav infections. it regulates two main antiviral functions: the induction of (i) the nfkb pathway, which ultimately controls expression of several inflammatory cytokines and (ii) apoptosis through multiple signaling cascades (40, 41) . tnf upregulation during iav infections correlates with infection severity, especially following highly pathogenic iav-infections (42) (43) (44) . mice treated with anti-tnf abs showed reduced disease burden; however, the authors of that study reported no effect on viral replication (20). tnf-related apoptosis inducing ligand (trail) can trigger apoptosis in iav-infected cells. iav-infected human epithelial cells are sensitized to trail-mediated apoptosis while peripheral blood mononuclear cells upregulate trail expression. moreover, administration of monoclonal abs against trail increases survival rate following iav infections in mouse studies (18, 19). antimicrobial peptides (amps) are host proteins that have direct antibacterial and antiviral activities and can modulate immune responses to infections. while the literature is largely focused on the antibacterial aspects of amps, several studies have highlighted the antiviral potential of amps against several viruses including ivs [reviewed in hsieh and hartshorn (45) anti-angptl4 -reduced pulmonary tissue leakiness, significantly accelerated lung recovery and improved lung tissue integrity in mice. -mouse-adapted laboratory iav (h1n1) c5a ifx-1 antibody -reduced viral load and virus-induced ali due to reduced infiltration of lung macrophages and neutrophils in iav-infected african green monkeys. -highly-pathogenic avian iav (h7n9) trail anti-trail -increased survival rate following iav infections in mouse studies. -improved symptoms and increased survival of iav infected mice. ed survival after h1n1 or h5n1 mouse infections. -1957 pandemic iav (h2n2); mouse-adapted laboratory iav (h1n1); 2009 pandemic iav (h1n1) (31) (32) (33) and albericio and kruger (46)]. ll-37 is a human cathelicidin derived amp that is found predominantly in neutrophils and its expression can also be induced in epithelial cells and macrophages (47) . aerosol administration of either human ll-37 or its mouse counterpart mcramp led to reduced morbidity and mortality to similar levels as the neuraminidase inhibitor zanamivir that is used for the treatment of human influenza patients (21). both cellular and viral fadd-like il-1β-converting enzymeinhibitory protein (cflip and vflip, respectively) protect cells from death receptor mediated apoptosis. kα2 is a vflip-derived peptide that consists of 10 amino acids from the α2 helix of the kaposi's sarcoma herpes virus (kshv) death effector domain 1 protein. a synthetic version of this peptide, tat-kα2, was generated by fusing kα2 to a portion of the hiv tat protein (22, 48) . in mouse challenge studies, intranasal administration of tat-kα2 at the time of infection with highly pathogenic avian h5n1 virus resulted in protection of the treated mice. no replicating virus was detected in the lungs at either 3 or 5 days after infection suggesting complete protection from infection (22). it should be noted that this effect is largely due to direct destabilization of the virions by the tat-kα2 peptide and it is likely that infection in treated mice was not established; the efficacy of this amp has not been determined during an established infection and warrants further investigation. host kinases regulate not only iav entry and replication but also initiate antiviral signaling cascades that regulate expression of pro-inflammatory chemokines and cytokines during infections and present viable targets for intervention (24, (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) . iav infection has been shown to upregulate c-jun n-terminal kinases 1 and 2 (jnk1/jnk2). these kinases directly regulate the induction of pro-inflammatory responses. iav-induced jnk1/jnk2 activation mediates production of chemokines and cytokines including tnf-α, interferon β (ifn-β), and interleukin 6 (il-6) (24) . in vivo inhibition of jnk1/jnk2 resulted in reduced levels of pro-inflammatory cytokines and reduced viral titers (23, 24). the mitogen activated protein kinase (mapk), p38, regulates viral entry and replication (55, 59) . furthermore, p38 regulates ifn stimulated gene (isg) gene expression and ultimately cytokine production via stat1 phosphorylation (25) . using either of two specific p38 inhibitors (sb 202190 or sb 203580), mice were protected from lethal h5n1 infection exhibiting reduced mortality and pro-inflammatory responses (25) . activation of another mapk, mek, is required for efficient iav replication and its inhibition results in viral ribonucleoprotein (vrnp) retention and reduced titers of progeny virus (26, 60, 61) . importantly, treatment of mice with the clinically approved mek inhibitor (ci-1040) showed reduced lung viral load and mortality of mice following infection with a lethal dose of pandemic h1n1 iav; interestingly, this inhibitor significantly out-performed the clinically recommended oseltamivir in these studies (26) . another central regulator of immune responses at the epithelium as well as immune cells is the nf-κb signaling pathway. accordingly, iav has evolved several mechanisms to modulate this pathway to counteract antiviral responses including directly targeting the ikb kinase (ikk) (62, 63) . sc75741 is a potent nfkb inhibitor that functions by reducing the ability of the p65 subunit of the nfkb complex to bind dna; thereby limiting its transcription-regulating functions (64, 65) . in vivo administration of sc75741 at 4 days after lethal infection with either h5n1 or h7n7 avian viruses resulted in significant protection with most mice surviving and showing little to no clinical symptoms; similar results were obtained by prophylactic administration (27) . g-protein coupled receptor kinase 2 (grk2) is best known for its phosphorylation of gpcrs in cardiac tissue resulting in recruitment of β-arrestin to facilitate rapid receptor internalization and lysozomal degradation (66) . recent phosphoproteomic studies identified grk2 as a potentially proviral host protein for iav that plays a major role in virion uncoating (28) . although in vivo inhibition of grk2 using paroxetine led to a significant reduction in upper respiratory tract viral load and to a modest reduction in lower respiratory tract titers at 4 days post infection, this inhibition was not protective from lethal infections (28) . however, it is possible that the route of administration (intraperitoneal vs. intranasal) and dosing regimen influenced the results. sphingosin kinases (sphk) are lipid kinases that mediate conversion of sphingosine to bioactive lipid sphingosine 1phosphate (s1p) (67), a known modulator of central apoptotic pathways (68) . iav infections leads to increased expression and activation of sphk1 and sphk2 (29) and in vitro inhibition of sphk1 was shown to decrease iav rna synthesis via suppression of nfkb activation (69) . treatment of mice with specific inhibitors to either sphk1 or sphk2 or a pan-sphk inhibitor led to prolonged survival of mice following lethal iav infection (29) . peroxisome proliferator-activated receptors (pparα, pparβ, and pparγ) regulate metabolic homeostasis and are important mediators of the inflammatory response. several ppar agonists have been investigated for efficacy during iav infections with varying results. gemfibrozil (pparα agonist) not only improved symptoms when administered 4 days after infections with an h2n2 virus, but also increased survival of iav infected mice (31) . prophylactic treatment of h1n1-infected mice with pioglitazone (pparγ agonist) resulted in increased survival (32) . combined activation of pparγ and its downstream target ampk improved survival of mice infected with pandemic iav strains (33) . protease activated receptor (pars) link protease activity to inflammatory cellular responses (70) . par1 expression is upregulated in the mouse airways following iav infections (71) . intranasal administration of a par1 antagonist (sch79797) at the time of infection with various iav strains including highly pathogenic avian h5n1 and pandemic h1n1 viruses led to increased survival and a decrease in inflammatory responses. moreover, this effect was also observed when sch79797 was administered 48-72 h after infection (30) . the use of statins, angiotensin ii receptor blockers (arbs) and angiotensin converting enzyme inhibitors (acei) has been proposed to regulate the iav-induced cytokine storm in severe infections (72, 73) . retrospective studies conducted separately in mexico, netherlands, uk and usa reported an association of reduced iav-related pneumonia and lower case fatality due to lower respiratory tract iav infections with statin treatment (74) (75) (76) (77) . however, this association was contested in two additional studies that found no benefit of statin treatment on iavinduced disease burden (78, 79) . this uncertainty regarding the iav therapeutic potential of these widely used compounds warrants further investigations at the basic science level and in clinical trials. the continuous accumulation of adaptive mutations and the introduction of novel viruses in the human population continue to pose a threat to public health, especially to individuals at high risk to influenza. the emergence of strains resistant to existing classes of antiviral drugs and reduced vaccine effectiveness highlights the need for the development of alternative intervention strategies. therefore, therapeutic approaches that can diminish the potential for drug-resistance while being effective against multiple iav subtypes/strains are highly desirable. targeting host cell factors meets these criteria and is more likely to avoid overtly robust immune responses thereby reducing disease severity and improve patient outcome (figure 1) . a large effort has been made in recent years to identify host proteins to serve as intervention targets against iv infections. several genetic and proteomic screens have identified several promising hits with potential roles in the iv replication cycle (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) . in addition to these genome-wide screens, viral and host protein interactions can be mapped into networks that can also be used to identify host factors critical for iv replication (91, 92) . interestingly, meta-analysis of some these studies shows limited overlap in the genes/proteins identified as required host factors (87, (93) (94) (95) . this is likely due to study-specific variations in iv types/strains and cell-lines used, inclusion/exclusion criteria, limited hit-validations and methods used to "knock-down/out" these genes. local microenvironment within a given tissue can dictate the quality and intensity of an immune response. inhibition or activation of critical signaling pathways expressed in both respiratory tract epithelial and immune cells by brms can have opposite and unintended consequences. as discussed above, trail regulates immune cell-mediated apoptosis of infected cells and several studies have shown that blocking trail signaling by genomic deletion or depletion by monoclonal antibody administration can improve infection outcome in iavinfected mice. indeed inhibition of trail signaling in alveolar macrophages and other monocytes limits their ability to induce apoptosis in alveolar cells, prevents lung tissue damage and promotes survival (19, 96, 97) . however, cd8+ t cells from trail−/− mice are less able to protect mice from severe infections, consistent with impaired trail-mediated effector functions of cd8+ t cells (18). similarly, opposing beneficial and detrimental outcomes have also been observed in studies using bcl-2 inhibitors to treat iav infections (98, 99) . brm delivery should be guided by immune system "compartmentalization" to ensure they elicit balanced immune responses. ideally, mucosal delivery deposits brms that reduce viral titers at the site of iav replication; however, systemic delivery of certain brms might be required to dampen dysregulated responses. this not only depends on the brms used but also on the timing of their administration. moreover, the duration of treatment with brms must be considered because sustained inhibition of certain inflammatory responses can result in an immune status that increases susceptibility to secondary opportunistic infections. repurposing of clinically approved drugs could potentially be used as brms for the treatment of severe iav infectious and should be explored (86, 89, 90) . considering that susceptibility to severe iav infections is influenced by host genetics and hostspecific immune responses, selection of therapeutic brms should be carried out using in vivo model systems that are representative of the immune status spectrum and underlying conditions of high-risk influenza patients (young, immunocompromised, nonnaive, obese, pregnant, or aged). using these model systems will increase the likelihood of identifying brms with clinically relevant antiviral and immunomodulatory potentials. he, tg, gs, and gr conceptualized and composed the manuscript. gr and he oversaw all aspects of the manuscript preparation. this work was funded by the alexander von humboldt foundation in the framework of the alexander von humboldt professorship endowed by the german federal ministry of education and research. we apologize to any investigators whose relevant work was not included due to space limitations. regulatory roles of c-jun in h5n1 influenza virus replication and host inflammation inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h5n1 infection the mek-inhibitor ci-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to 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protein kinase is a determinant of respiratory virus entry and tropism antiviral activity of the mekinhibitor u0126 against pandemic h1n1v and highly pathogenic avian influenza virus in vitro and in vivo combination of mek inhibitors and oseltamivir leads to synergistic antiviral effects after influenza a virus infection in vitro jnk2 and ikkbeta are required for activating the innate response to viral infection influenza a virus-encoded ns1 virulence factor protein inhibits innate immune response by targeting ikk a novel class of potent nf-kappab signaling inhibitors the nf-kappab inhibitor sc75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance grk2 in the heart: a gpcr kinase and beyond regulation of sphingosine kinase and sphingolipid signaling sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases sphingosine kinase 1 serves as a pro-viral factor by regulating viral rna synthesis and nuclear export of 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for influenza virus replication the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the use of random homozygous gene perturbation to identify novel host-oriented targets for influenza genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication antiviral effects of inhibiting host gene expression influenza virus-host interactomes as a basis for antiviral drug development genomewide crispr/cas9 screen identifies host factors essential for influenza virus replication repurposing host-based therapeutics to control coronavirus and influenza virus repurposing of drugs as novel influenza inhibitors from clinical gene expression infection signatures comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction network-guided discovery of influenza virus replication host factors cellular networks involved in the influenza virus life cycle genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery meta-and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding the magnitude of the t cell response to a clinically significant dose of influenza virus is regulated by trail pathogenic potential of interferon alphabeta in acute influenza infection anticancer compound abt-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins key: cord-005081-kxrzv16n authors: kiselev, o. i. title: progress in the development of pandemic influenza vaccines and their production technologies date: 2010-11-12 journal: appl doi: 10.1134/s0003683810090024 sha: doc_id: 5081 cord_uid: kxrzv16n this article analyzes the current situation in the field of construction and production of pandemic influenza vaccines. the main task of protecting the population against influenza pandemics requires state-of-the-art approaches to the construction of influenza vaccines to be based on reassortment and genetic engineering techniques, including the analysis of primary structures of influenza viral genes, synthesis and cloning of the main viral genes, reverse genetics techniques, and banks of plasmids bearing basic viral genes. reassortant technologies are now giving way to new approaches for objective reasons. the state-of-the-art technologies provide safety not only at the laboratories where vaccine viruses are constructed but also make the production process wholly safe. we are using the following approaches to the development of industrial production: use of nanoparticles and nanoemulsions as functional adjuvants, construction of totally-safe strains for live attenuated influenza vaccines with deletions of molecular determinants of pathogenicity, application of protein and chemical chaperones to provide self-assembly of haemagglutinin molecules of the h1n1v-2009 virus, and impregnation of whole-virion preparations with nanoparticles to enhance antigenicity. the level of protection against the avian flu epi zootic and the influenza pandemic caused by the swine lineage h1n1v virus can be characterized as insufficient for russia's population. the experience in preventing an outbreak of sars in the territory of russia has evidenced that organized contraepidemic measures produce a temporary, albeit appreciable, effect. the who recognizes the functional priority of epidemiological services, including quarantine mea sures, in localizing epicenters of infections and pre venting their cross border spread to other countries and continents, that is, the very principles, on which the activities of public health services in our country have always been based. however, the most successful part of contraepidemic measures in preventing the spread of infections with respiratory transmission mechanism is vaccinoprophylaxis [1, 2] . supply of vaccines critically depends on the level of technologies used in the preparation of vaccine strains and mass production of vaccines. the technologies now in use in russia and abroad are those developed more than 15 years ago. however, this sphere has made significant progress over the last 5 years. the prepared ness activities related to the influenza pandemic and its global outbreak in 2009 substantially increased the demand for vaccines. according to who's estimate, the annual global demand accounts for 2 to 3 billion doses. russia's influenza vaccine production potential is higher than in other countries and sufficient for con ducting annual vaccinations in more than 50% of the population. at the same time, the problems associated with the supply and quality of influenza vaccines remain the most important for russia and the rest of the world and strongly depend on the promptness with which new technologies are adopted. vaccination is an important part in the complex of contraactions organized by public health services against influenza pandemics. like the soviet union used to be, the russian federation remains a leader in conducting mass prophylactic immunizations, in par ticular, antiinfluenza vaccinations. this must be marked as one of the most important achievements of the past. the country's complex of vaccine producers has demonstrated increased growth rates in recent years against the difficult situation in the pharmaceu tical industry. despite certain technological lagging, our country remains a leading producer of practically a whole spectrum of important vaccines in the world's sector of this industry. in fact, from the late 1960s up to the late 1980s, all contemporary influenza vaccines were pioneered in the soviet union. then basic tech nologies used to spread globally [1] [2] [3] [4] [5] [6] [7] . the initial impulse for scaling up the industrial supply came with the hong kong influenza pandemic of 1968 [5] [6] [7] [8] . the avian flu epidemic also brought a serious challenge to the world community, but that was already the period when a necessity to employ novel biotechnological achievements in influenza vaccine production was realized [1] . medical services have acquired much experience in the application of influenza vaccines. in the russian federation, the population has already been vacci nated using seasonal influenza trivalent and monova lent vaccines against the pandemic h1n1v 2009 virus. in russia and globally, two basic types of vaccines are used for prophylaxis [8] [9] [10] [11] [12] [13] : inactivated influenza vaccines and live attenuated influenza vaccines. the main types of vaccine preparations with their properties and compositions are given in fig. 1 (on the colored inset). we give also novel types of vaccines; their properties and technology are described below; generation iv represents the contribution made by nanotechnologies playing a principal role in revolu tionized vaccine production development. inactivated influenza vaccines contain influenza virus strains preliminarily subjected to uv and chem ical inactivation or fragmentation with subsequent iso lation of antigen containing components. the effi cacy of inactivated vaccines in preventing the disease onset accounts for 70-80% for healthy recipients under 65 years of age and 30-40% for patients over 65. on the contrary, the efficacy of inactivated vaccines in preventing influenza related lethal outcomes accounts table 2 ). for 80% even among such a risk group as patients over 65 years of age [7, 12] . by their production technologies inactivated vac cines can be subdivided into three groups: whole vir ion vaccines (generation i), split vaccines (generation ii) and subunit vaccines (generation iii) (see fig. 1 ). whole virion vaccines consist of inactivated (chemical or ultraviolet inactivation) whole virions of influenza viruses and contain, apart from the required antigens for immunization, a considerable amount of ballast in the form of viral nucleocapsid. their advan tage is that the surface antigens in the composition of inactivated virions are in conformation close to the natural one. split vaccines are products of detergent fragmenta tion of virions' membranes with removal of all compo nents of nucleoid and contain, along with surface anti gens, internal antigens, such as protein m1. at the same time, they are real fragments of highly purified membranes of viral particles (see fig. 1 , phase 2). subunit vaccines contain only surface protective antigens (haemagglutinin and neuraminidase, pre dominantly, the former) and are preparations with the highest degree of purification from influenza virus components. to the group of subunit vaccines, for example, belongs grippol, a polymer, which cannot be characterized as an ideal preparation (see below). the main requirements for the vaccines and their pro duction technologies were first specified under the who's special document [8, 13] . live attenuated influenza vaccines, proposed at first by a.a. smorodintsev in 1938, have successfully been applied in russia for decades. in the united states, a live intranasal vaccine was approved by the fda in june 2003 and permitted its use in age groups ranging from 5 to 49 years. repetitive passages on chicken embryos in decreased temperature conditions allowed researchers to obtain a weakened virus that does not replicate at high temperatures, characteristic of human lungs, but can propagate in the nasopharynx at a temperature of 34°c, causing a topical infection leading to the development of a secretory and general ized immune response [1, 14] . since the laiv vacci nation resembles a naturally occurring transient infection, it has a sufficient efficacy and is character ized, according to different sources, by 94% serocon version in children, while, in combination with an inactivated vaccine, it elicits a positive immune response in 68% of the elderly [1, 6] . the overwhelming majority of seasonal and pan demic viruses are products of reassortment 1 [1, 14] . first of all, this applies to the current h1n1v 2009 virus, the agent of the influenza pandemic. therefore, application of laiv for preventing the emergence of reassortants with novel properties is restricted on a rise 1 reassortment is an exchange of viral genome fragments between two or more viruses. of morbidity and the more so in the outbreak of an influenza pandemic. the data on the age related lethality from the pan demic h1n1v 2009 influenza has shown that the morbidity and mortality from influenza related com plications in the period from march to december 2009 were significantly lower among patients born after 1957. a relatively high level of antibodies to the swine lineage h1n1viruses was revealed in patients over 50 to 55 years of age, which defines a sufficiently high level of protection against the current pandemic h1n1v 2009 influenza [12, 15, 16] . these data evi dence in favor of conducting a total vaccination in rel atively young groups of the population and children in the coming period prior to the second wave of the pan demic expected in 2010-2011. table 1 gives a group of 12 influenza vaccine prep arations by domestic and foreign producers, which are now manufactured or registered in russia. construction studies on developing influenza vaccines and their production technologies have been extended in the most recent 5 years thanks to the deployed program on influenza pandemic preparedness. the who has adopted a special technology transfer program on developing influenza vaccine production in third world countries. europe and the united states agreed to allot grants and worked out programs for developing novel technologies and vaccine preparations with improved qualities: a new generation of laiv, that is, delns1 vaccines with a limited replicative potential, laiv with deletions of pathogenicity factors in genes, latest variations of subunit vaccines enhanced by adju vants, and capsid nanovaccines and nanovaccines based on inactivated viruses and virus like particles [14] . what are basic prospects opening before the vac cines constructed using state of the art genetic engi neering techniques? using the reverse genetics technique the genome of influenza type a viruses consists of 8 segments encoding 11 genes (table 2) . vaccines against especially pathogenic strains of influenza viruses are particularly difficult to develop, since the conventional approaches, based on the reas sortment of "wild" isolates (epidemiologically impor tant viruses) and donors of attenuation, practically do not work for obtaining this type of candidate vaccine strains. usually, in reassortment, the genes that encode surface antigens in the donor are replaced by ha and neuraminidase of the epidemic's virus. a can didate vaccine strain is unable to cause a disease but in immunization it supports the development of immu the open reading frame in the pb1 pb1 f2 gene, which encodes the amphiphilic transmembrane protein with signaling peptide of mitochondrial local ization. (3) mutations in the pb2 gene (polymerase com plex). (4) mutations in the gene encoding the nonstruc tural ns1protein-an antagonist of the interferon system of the first type. (5) mutations in the m2 protein gene that controls key stages of a reproductive cycle through the so called proton pump and drug resistance to rimantadine. as was mentioned, to construct a new vaccine, the surface antigens, primarily ha, should be replaced. replacing ha in the donor of attenuation by the ha of the avian virus a (h5n1) will lead to a transference of the pathogenicity determinant. a candidate vaccine strain should contain at least one sign of pathogenicity. to obtain a safe vaccine, this determinant should be removed. however, this is impossible to realize at the rna level. the viral genome should be converted to the dna form. this technique was realized for the first time by d. baltimore, a nobel prize laureate, for the polio virus rna more than 20 years ago; relative to the rna containing influenza viruses, this approach was termed "reverse genetics" (in nature this process is realized by reverse transcriptase) and patented [14, [17] [18] [19] [20] [21] . several research teams throughout the world construct viruses using the removal technique at the level of the dna sequences encoding pathogenicity sites (the ha cleavage site in the case of avian influ enza viruses). six basic plasmid dnas encode internal genes of a known donor of attenuation. constructing plasmids is a specific process, since, in each plasmids, bidirectional transcription is provided for the virus specific mrna and the complimentary virion rna with a negative polarity-a necessary factor for the synthesis of virus specific proteins and accumulating genomic fragments when obtaining infectious viral progeny. in 1973 researchers from the national institute for biological standards and control (nibsc), london, common cold research unit (mrc, england, salis bury), as well as from the rjc research and production therapeutic corporation, rixensart, belgium, offered the a/pr/8/34 strain as the master donor for con structing influenza vaccines. eight reassortants of the influenza h1n1 and h3n2 virus serosubtypes were prepared on the basis of this donor and then thor oughly tested in sensitive subjects from different age groups at laboratories in the united states, west europe, and australia. the a/pr/8/34 strain was offered as a donor of attenuation, because the reassor tants tested lacked reactogenicity and showed very high activity indicators in adults and children (3-4 fold excess over the generally accepted minimum pro tection level for humoral antibodies). the reverse genetics technique was successfully applied in con structing the candidate vaccine strains on the basis of the avian flu h5n1virus isolates having the most important pathogenicity determinant in the ha gene as a cluster of residues from lysine and arginine at the site of proteolysis with furine proteases. after the site was removed from the cloned gene its pathogenicity significantly decreased, being in conformity with the requirements for candidate vaccine strains. the a/pr/8/34 virus is still used as a successful donor of high reproductive activity. this very strain served as the donor for constructing important viruses for inactivated, predominantly whole virion vaccines at the research institute of influenza (st. petersburg). the vaccine passed through preliminary trials for effi cacy, genetic stability, and antigenic homogeneity; the preparation is being prepared now for clinical trials. development of inactivated vaccines or vaccines with modified ns1 and pb2 genes, replication defi cient in the human body, is recommended for obvious reasons. such vaccines are obtained by reverse genet ics techniques mastered at the international laboratory of the research institute of influenza, rams (fig. 2) . a further attenuation of the virus through genetic modifications is possible by obtaining strains with lengthy deletions in the ns1 gene encoding the antag onist of the first type interferons and the signaling domain pdz localized at c end of this viral protein [7, 13] . the level of this technology is so sophisticated that using the 8 plasmid technique complete copies of highly pathogenic viral isolates can be constructed. in particular, within the european project fluvac, we obtained a complete copy of the kurgan h5n1 2005 virus isolate. the representative scheme (see fig. 2 ) for the vac cine ni brg 14 virus assembly shows the removal of the proteolysis site rerrrkkr and insertion of a new "attenuated" site retr. the na gene in this construction remained intact. the vaccine strain on this base has specially been prepared for the use in the territory of the russian federation. manipulations with the ns1 gene were put into the basis of a new technology for obtaining influenza viruses with limited replication potentialreplicatively deficient strains [20, 21] -aimed to replace laiv as more safe and efficacious. the safety of these vaccines is assessed primarily by their high sensitivity to the endogenous interferon. an influenza table 2 ; n1-neuraminidase 1; see explanations in the text. virus is a strong inductor of interferon and, therefore, viruses deprived of the interferon antagonist-full value protein ns1-are quickly eliminated from the body under the action of interferon i and ii types after a short cycle of reproduction. this fact enables us to use the influenza virus as a safe vector. in our work the influenza virus was used as a vector for obtaining the strain expressing the esat 6 protein of the tb caus ing bacteria [22] [23] [24] . the intranasally administered modified virus manifested in animals the properties of an antitubercular vaccine [23] . the arsenal of approaches to the technological development and solutions in constructing and manu facturing influenza vaccines has become considerably more substantial over recent years. the annual prepa ration of conventional vaccines (live attenuated, whole virion, and split and subunit vaccines) includes, in the first place, the phase of obtaining vac cine strains, which usually takes up to 6 months. many technological techniques and innovations are oriented to the prompt preparation of antigen producers and the possibility to modify them in correspondence with the data on antigenic drifts of important seasonal or pandemic viruses. thus, the vaccines now in use can be supplemented by the following list: dna vaccines, recombinant subunit vaccines, vector based vaccines (their obtaining is connected with the use of one of the most attractive technologies for virus like particles), and a universal vaccine based on highly conservative antigens (m2, m1, and np proteins). in fact, the term recombinant vaccines can define any type of vaccine preparations obtained through genetic engineering. at the same time, considering the advance of these approaches to constructing influenza vaccines, it is more appropriate to specify recombinant vaccines by two types: recombinant influenza viruses used as vectors or deficient viruses for obtaining non pathogenic vaccine strains and constructs bearing individual viral genes for obtaining different types of vaccines. we considered above the first approach and now we consider the second approach. in practice, the process of constructing individual recombinant vaccines comprises the following actions: (1) cloning and expression of individual viral genes in different vectors to obtain viral antigens in different expression systems as highly purified vaccine prepara tions. (2) cloning and expression of individual viral anti gens to obtain virus like particles, to achieve each of the above results, one is to make a well grounded selection. the following antigen proteins are localized on a viral particle's surface: haemagglutinin (ha), neuraminidase (na), and the m2 protein. it is estab lished that five antigenic sites are localized in the "head" of the apical part of the ha molecule and that the antibodies' virus neutralizing activity is associated with the immune response to these sites. antibodies to ha block the interaction of the virus with cellular receptors, that is, infecting the cells. antibodies to na block the secretion of viruses by the infected cells (i.e., propagation of infection). antibodies to the m2 pro tein also considerably block the dissociation of infec tious viral particles from the infected cells' mem branes. we see further that, of the three main surface proteins, only ha is of a principal significance, the consideration of which the production technology for subunit vaccines predominantly containing ha is based. immunity to na is believed to be accessory. the m2 protein, considering its supreme conserva tiveness, became the model object for constructing a universal antiinfluenza a vaccine. thus, recombinant vaccines with individual com ponents of influenza type a viruses are mainly ori ented to the key and highly variable viral compo nent-the ha molecule. among the internal antigens of a viral particle, the np, m1, and ns1 proteins are considered as material for obtaining vaccines. another, no less important problem consists in selecting a substrate for growing antigenic substances. scientists have been discussing for many years the option of replacing chicken embryos with cell cul tures. as previously, conventional technologies are oriented to growing viral material on chicken embryos. it applies to laiv and iiv of the current generations. this technological process consumes millions of chicken embryos (in fact, one dose of a sea sonal influenza vaccine is equivalent to one embryo). the scale of the problem is realized by everyone, if to consider that in the event of a pandemic only in russia the demand for vaccines accounts for at least 100 mil lion doses. this scale of mass production relying on the conventional substrate cannot be promptly deployed (by the coming of a season and more so by the outbreak of a pandemic). the use of cell cultures in producing antigenic components of vaccines allows specialists to decide the above task rapidly and in sizable volumes. vaccine production based on the vero and mdck cell cultures have already been licensed in the united states and european countries. moreover, the use of this approach is in full correspondence with the complete technological chain for obtaining recombinant viruses using the 8 plasmid technique and, in fact, it allows producers to rapidly deploy mass production from the laboratory scale capacity. in the last 7 or 8 years, can didate vaccine strains for a majority of seasonal viruses have already been obtained using the so called reverse genetics technique (see the above description). this approach corresponds also with the dna vaccine technology. although dna vaccines have been under development for more than 15 years (their develop ment continues) none of such vaccines are so far per mitted for use. a marked breakthrough in the construction of recombinant vaccines is related to the use of insect cells and the obtaining of virus like particles (vlps) based on baculovirus expression vectors. this tech nique deserves our consideration in detail because of the high technological and quality indicators demon strated by vaccine preparations. best solutions on the composition of vaccines, their technologies, high immunogenicity, and safety are actually united under the project. in accordance with the classification pre sented, thanks to the absence of ballast components and preservation of the spatial structure of surface antigens, vlps excel recombinant vaccines, dna vectors based vaccines, and whole virion vaccines grown on a cell substrate. a positive advantage is, evi dently, that the sizes of vlps and virions also corre spond. we will consider, one by one, projects and achieve ments in the field of new approaches to constructing influenza vaccines and the use of different systems for their production. we will discuss the projects that are now believed to be the most important for realizing a technological breakthrough in the mass production of safe influenza vaccines. use of vectors for constructing and producing virus like particles as influenza vaccines. adenoviruses have been used as a tool in model studies on gene therapy for many years. in recent years, specialists engaged in constructing recombinant vaccines have become interested in the advantages promised by adenovirus based vectors. a particularly attractive feature of ade noviruses is their high productivity and efficiency in gene transfer and expression. vectors for vaccines are constructed with deletions by early genes e1 and e2. in some research works, adenovirus vectors are used for the expression of individual viral antigens and, in particular, ha of influenza viruses. a serious disadvantage for these vectors is the presence in the human population of widespread immunity, which leads to a rapid limitation in the expression of target antigens. during epidemics, 15-25% of the human population suffers from adenoviral infections. hexon is the strongest antigen among adenoviral proteins. antibodies to this protein are recorded in more than 75% of healthy subjects. therefore, when planning the use of these vectors for some works one should consider, first of all, their population receptiv ity. it equally refers to gene therapy and recombinant vaccines intended for a wide application against mass infections, such as influenza. one of the projects of the russian corporation of nanotechnologies is devoted to the use of adenoviruses as vectors for constructing influenza vaccines. the vectors based on alpha viruses are a relatively new tool for constructing recombinant vaccines. the venezuelan equine encephalomyelitis virus, specifi cally pathogenic for horses, is especially attractive to virologists as an object of research. the author of this survey studied the basic problems of replication and expression of the rna containing virus genome in the early 1970s. the expression of this virus genome is supported by two operons: early and late genes. for eign genes are inserted in the reading frames of the late genes, thus providing a high level of their expression, comparable with that of the surface major proteins of alpha viruses. alphavax inc. is the world's leader in using alpha viruses as vectors for constructing vac cines. at the same time, we are underlining the safety of vectors based on alpha viruses: they are nonpatho genic for humans, and the human population has no immunity to these viruses, since they have never circu lated among people. constructing recombinant vector influenza vaccines based on alpha viruses is one of the independent and promising strategies in this field. the project offered by novavax inc. and protein science inc. is one of the most prospective for constructing a new type influenza vaccines and organizing their production. these companies use var ious vectors for the expression of ha and other influ enza virus proteins in insect cells. the latter grow at a temperature of approximately 22°c; the temperature regime for their growth is controlled by special equip ment. at the same time, culturing insect cells is a sim pler and less expensive process, compared to the growth of animal or human cells, and, at the same time, not associated with a risk of malignancy devel opment. human safe vectors based on baculoviruses are used to express antigens and other foreign proteins in insect cells [25] . baculoviruses express polyhedrin protein in considerable amounts under the control of a strong promoter. thus, a polyhedrin promoter is used for the expression of foreign antigens (fig. 3 , on the colored insert). with the above constructs, a high level of expression is achieved in insect cells for ha of different subtypes. at present, two approaches are applied for mass production of influenza vaccine components. one system (protein science) synthesizes ha, the main influenza virus component, for a vaccine. the other system (novavax) synthesizes simultaneously three proteins: ha, na, and m1. synthesis of individual ha allows using this antigen, purified, as the main component of a vaccine identical in its properties to a subunit vaccine. at the same time, it remains unclear if the protein folding with the formation of ha "rosettes" is used to prepare the vaccine, which is a very important process for increasing immunogenicity and ensuring the quality of the immune response, comparable to the native ha in the composition of viral particles. the simultaneous expression of viral proteins ha, na, and m1 is accompanied by their self assembly in insect cells. this process results in the formation of vlps having high immunogenicity, allowing the antigenic burden to be reduced, com pared to the individual ha. the system of obtaining vlps using the sf9 insect cell line with a baculoviral vector expressing three main proteins ha, na, and m1 (see fig. 3 ) was tested in the pandemic h5n1 and h9n2 viruses and the sea sonal influenza h3n2 virus. in all cases, the vlps had high immunogenicity in conformity with the require ments for vaccine preparations. proteins. for many reasons, including economic ones, the strategy of using systems producing vaccine com ponents in plants is very attractive. attempts are now being taken to obtain plant producers for influenza virus antigens [7, 14] . russia is conducting such stud ies within a few projects. there are attempts jointly it is natural that edible vaccines are important not only for controlling zoonozic pools of viruses and bac teria, but we believe that they are principally signifi cant for early vaccination in babyhood when the immune system of the intestines in neonatals is still in the process of formation. therefore, large scale works on obtaining vaccine preparations based on rice culti vars are worthy of attention [26] . a progress in these studies may principally change our children's vaccina tion calendar and make vaccination in infancy com pletely safe. genetic properties of the influenza a viruses are characterized by extreme antigenic variability. the strains should be constantly monitored and studied as to their genetic structure and antigenic properties. a special commission of who considers, on an annual basis, data on the properties of isolates from all over the world and makes decisions on the strains in the composition of a next vaccine. at the same time, an outbreak of a pandemic brings a sharp change in the agent's antigenic structure or antigenic shift. in 2009, an antigenic shift occurred within a subtype of h1n1, and a new swine lineage virus emerged with a princi pally different structure of the surface ha and na antigens. the global population in its majority proved unprotected against this virus; its relatively high pathogenicity explains the increased mortality from the infection caused by this agent (table 3) . on the other hand, for the majority of other rna containing viruses, researchers have obtained vaccine strains not requiring seasonal replacement. in particu lar, it refers to the agents of measles and poliomyelitis. the natural question has been raised for years: is it possible to develop some universal anti influenza a vaccine based on the agent's conservative proteins? if we take surface antigens, only three can partici pate in the selection (see above). the m2 protein's principal difference from the other two is its high con servatism admitting only insignificant replacements in the gene structure. the surface of virions exposes approximately 20 copies of the m2 protein tetramer that performs the functions of ion channel-proton pump. the functional activity of the proton pump is important at the onset of infection for decapsidation of the virus in endosomes and, subsequently, under final phases for gemmation and release of viral particles. thus, the m2 protein controls the process of infecting cells and propagation of infection. it has recently been established that the sign of contagiousness, or the virus spreading speed in human to human transmission, is associated with the m2 protein [27, 28] . according to other data, the m2 protein is signifi cantly responsible for the development of oxidative stress accompanying influenza infections. therefore, induction of an immune response to this surface pro tein is believed to be very important for the protection against influenza infections. at present, the construction, preclinical trials and, in some cases, clinical trials have been completed on the following preparations: (1) recombinant preparation of the apical (extramembraneous) m2e domain in the capsid car rier of the hepatitis b protein particles. (2) recombinant preparation, including in tan dem repetitive copies of 24 amino acid residues of the m2 protein in the composition of e. coli flagellin. (3) recombinant fused protein m2e conjugated with a residue of cysteine at the 3 position and con taining palmitine acid as an adjuvant. in fig. 4a (on the color insert), the m2 protein is represented by a physical map, where the transmem braneous domain is underlined by alpha spiral (given is the most spread sequence with indicating amanta dine and rimantadine resistant mutations; the aman tadine molecule is designated in yellow in the region of adamant binding site); given on the map's left is the extramembraneous domain at n end (positions 1-25), the blue arrow points to variations of this domain's sequences in different subtypes of influenza a viruses (see fig. 4c ). on the top right is a tetramer model of the transmembraneous domain in a lipid membrane (see fig. 4b ). considering the first importance of projects on cre ating a universal vaccine, we give below the m2 protein sequences for important pandemic viruses character ized by a high level of conservativeness, a very valuable quality for constructing a universal vaccine (fig. 5) . the high conservativeness of the main the m2 pro tein domains used in constructing recombinant vac cines allows us to rely on reaching a sufficient efficacy of the vaccines against any possible pandemic strain, including avian flu and swine lineage h1n1 influ enza. this type of vaccine has been created in russia on the base of the center bioengineering, russian academy of sciences [14] . vaccines production of vaccines, as well as other biophar maceutical processes, includes two main phases: prep aration and main technological process. the preparation phase consists in obtaining a sow ing material-a virus in cell cultures-in a limited quantity, its subsequent cultivation inside voluminous reactors, and isolation of the virus from cells and main cellular components. as a result, a raw suspension of a viable virus is obtained, which is subjected to further purification. the main technological process includes the follow ing key phases of purification of the virus: concentra tion, chromatography, ultradiafiltration, isolation of separate components (for split or subunit vaccines), mixing with adjuvant (if the latter is included in the vaccine) and preparation of the final product for pack aging. below we are discussing the current level and prob lems in the influenza vaccine production realization. the key phase of the preparatory technological process is fermentation. the development of wave bioreactors used for fermentation has become a turn ing point in the field of cell cultivation and has signif icantly increased cell culture yields in growing viruses. a majority of companies trading in modern techno logical equipment recommends this type of reactors. the joint project of the research institute of influenza of the rams with the ge company (sweden) includes a series of important solutions for the key part of the production cycle. the system of cell selection, concentration, and filtration with separation of the fractions containing the end product is successfully realized using the uniflux system developed within the above project (fig. 6 ). uniflux systems can be used for concentrating virus suspensions, vaccine preparations, protein aggregates, monoclonal antibodies, and pure recom binant proteins. concentration of virus suspensions is a necessary phase for preparing the material for chro matographic purification. the use of standard monolith columns for the chromatography of viral particles is the most impor tant technological solution (fig. 7) . under a majority of technological processes, viral particles are subjected to purification through sucrose gradient ultracentrifu gation. the chromatographically purified vaccine was obtained in columns with macroporous glass. how ever, this vaccine contained hydrodynamically and mechanically destroyed virions. exposing the viral nucleotide on the surface of the destroyed viral parti cles leads to the induction of immunity against the native viral ribonucleoproteid (rnp). this phenome non is not so unsafe, since a cross reaction of antibod ies with rnp of the body's cells cannot be excluded, while this kind of phenomena may lead to autoim mune processes that may trigger systemic diseases. application of state of the art techniques for chromatography of viral particles permits one to obtain highly purified samples of both viruses and subunit preparations or vlps. chromatographic puri fication helps solve another, more important problem. electron microscopy analysis of the current vaccines as to their structure reveals that antigens in the major ity of vaccine preparations are in a denatured or heavily disorganized state (fig. 8) . the research institute of influenza of the rams is now developing, within some international projects, production technologies for a new generation of vac cine preparations: (1) live replicatively deficient vaccines del ns1 with additional genetic defects within a wide spectrum of molecular determinants of pathogenicity for opti mizing their safety and preserving their high immuno genicity; (2) protein nanovaccines based on the controlled folding technology; 100 nm 50 nm (d) (3) antigen and virion complexes with metalized nanoparticles (fig. 9 ). first of all, we should underline that the principal strategy in improving influenza vaccines has ever been their increased safety and immunogenicity. this explains the transition from whole virion to split vac cines and from the latter to subunit vaccines. a high level of antigen purification generated the problem of decreased immunogenicity of vaccines and a search for immunoadjuvants. approximately 15 years ago polyoxidonium was selected to perform this function. the polyoxidonium based vaccines were termed poly mer subunit vaccines. however, such an approach has grown old now. a number of new adjuvants based on the known mechanism of stimulating the immune response through toll receptors have been tested in recent years [12, 29] . discussions are still ongoing concerning the safety of laiv. the current strategy is oriented to a search for inactivated analogues of many live vaccines. firstly, this is connected with a probable reversibility of attenuated strains of viruses towards wild types, or reassortment of strains, having a fragmentary genome, with wild viruses. using a live or even attenuated virus for mass vaccination, we, certainly, create a temporary circulatory background, considerably increasing a probability for the reassortment of viral genomes with viruses actively circulating in a current period of time in human population. despite the fact that the phe nomenon of reassortment between laiv and wild viruses has not been confirmed and viruses with signs of pathogenicity have not been isolated in the majority of complicated vaccination cases, the application of laiv is limited in the period when seasonal influenza is on the rise. mass immunization with laiv in the event of a pandemic should be conducted with maxi mum care. at the same time, improvement of laiv safety is possible through genetic techniques-in particular, we have achieved this in our studies within the euro pean fluvac project. the team led by professor a.yu. egorov has developed so called delns1 vac cines. vaccine strains carrying the nsi gene deletion can quickly be eliminated, since they are highly sensi tive to the endogenous interferon (the latter is the antagonist of type i and type ii interferons). there fore, this type of live vaccine was classified as replica tively deficient. a probability of recombination or reassortment is ultimately low in this case. another approach to a genetically controlled reduction in the laiv reactogenicity consists in the removal of the reading frame in the pb1 gene. the pb1 f2 reading frame encodes a short protein of 87 amino acids-the factor of macrophage apoptosis causing the death of cells during translocation in mito the analysis of development strategies in vaccine production technology has shown that genetic engi neering technologies are moving ever more to the fore front of this development. on the other hand, there is an understanding, initially absent in immunologists, that the tertiary structure of the antigen is equally important for adequate immunogenicity and the func tioning of enzymes. significantly improved instru mental provision of technological processes enables creating typical multipurpose vaccine productions, including vaccine preparations based on nanotechnol ogies. the current discussions about the scale of mortality due to the h1n1sw1 pandemic are held with not quite correct argumentation. even without very strict crite ria, it is an emergency when a healthy young or mid dle aged person dies from influenza. vaccination and timely administration of efficacious antiinfluenza means, within a complex antiinfluenza therapy, offer good chances to avoid, in an overwhelming majority of cases, severe complications and, certainly, a lethal outcome. according to the latest statistics, the h1n1sw1 related mortality rate is reliably higher than the share of lethal outcomes in the 1968-1969 pan demic, while lower than during the 1957 asiatic influ enza. of course, medical science has made a signifi cant progress since 1957, continuously introducing from 1968 antiviral therapeutic means into clinical practice. based on the above, we have to draw the fol lowing not very comfortable conclusion: high influ enza related mortality rates are, to a considerable degree, explained by our poor performance in orga nizing medical aid to patients. russia is also no exclu sion in this aspect. at the same time, availability of developed facilities for producing influenza vaccines allows russia, in contrast to other countries, to promptly take decisions on a national vaccination pro gram of scale. returning to the problem of genetic properties of pandemic viruses and the discussion about the molec ular determinants of pathogenicity, we have to repeat that the 2009 influenza pandemic was caused by the mixed h1n1v 2009 virus with a genome that included genes of swine, avian, and human viruses. based on the specific structure of its genome and pathogenicity level in animals (experimental models) and humans, we can characterize this etiological agent as a moder ately pathogenic virus. in particular, ha of the h1n1v 2009 virus does not contain the proteolysis sites characterizing the highly pathogenic h5n1 viruses. the pb1 f2 reading frame in the genome's second fragment (encoding the pb1 protein and the supplementary pb1 f2 protein resembling bacterial toxins by structure) is three times interrupted by stop codons, which leads to the absence of protein synthe sis in the infected cells. the absence of pb1 f2, as the most important factor of pathogenicity, in the influ enza a viruses is an additional argument in support of specifying this virus as moderately pathogenic. in addition, this very genetic sign is a principal difference of the current pandemic h1n1v 2009 virus from the 1918 h1n1 virus that caused the hispanic influenza pandemic. moreover, the pandemic h1n1v 2009 virus is defi cient in the ns1 protein, the antagonist of i and ii types interferons. in the ns1 protein from north america and the russian federation's isolates, researchers have found c terminal deletion of an important regulatory site playing a key role in the acti vation of the signaling cell systems controlling the antiviral response of the body's cells (the deletion of so called pdz binding domain) in influenza infections. this deletion is probably related to the increased sen sitivity of the pandemic h1n1v 2009 influenza to interferons and, in particular, to gamma interferon. summarizing the above analysis of the functional domains of the h1n1v influenza virus protein, we have to state that the current pandemic influenza viruses are characterized by the following structural defects: the ha proteolysis site-psiqsr/glf gai-is a substrate for the membrane type serin pro tease tmp/sst, the presence of the three stop codons in the pb1 f2 protein, and the presence of cooh terminal deletion of the pdz binding domain of ns1. the above genetic defects of pandemic viruses should additionally be characterized by the following supplementary properties: (1) resistance to the preparation tamiflu, which is often manifested by seasonal h1n1 viruses and which is characteristic of a pandemic virus. (2) cooperative specific properties of the ha and na proteolysis and their dependence on membrane bound cellular proteases [12] , a systemic induction of a "cytokine storm". (3) ns1 and ns2 conditioned opposition of the interferon system and immunosuppression. (4) presence of the "weakened" consensus sequence of the ebola like suppressive domain [12] . however, it should be underlined that the above defects may in part or fully be repaired in the process of active circulation of the h1n1sw viruses, carrying a risk of enhanced pathogenicity. therefore, in con structing vaccines for the forthcoming pandemic sea sons we shall have to foresee the constructive specific features associated with a probable emergence of viruses with modified properties. as the most marked achievements in domestic sci ence, we should note the obtaining of a full functional copy of the kurgan isolates for the avian influenza h5n1 virus using the recursive pcr and reverse genetics techniques (8 plasmid system variation [12]) and the technology created for producing a universal vaccine based on the m2 protein [14] . gripp ptits: proiskhozhdenie infektsionnykh biokatastrof (avian influenza: the origin of infectious biocatastrophes) gripp. na poroge pandemii (influenza: on the brink of pandemia) etiologiya, immunologiya i klin ika aziatskogo grippa mezhdunarod naya konferentsiya (bull. who. hong kong flu. int. conf.), 1969 pandemiya-2009. novyi virus h1n1 (pandemia-2009. new virus h1n1) note for guidance on harmonization of requirements for influenza vaccines. 1997 (comp/bwp/214/96). euro pean agency for the evaluation of medicinal products the lan cet who guid ance on development of influenza vaccine reference viruses by reverse genetics a review of production technologies for influenza virus vaccines and their suit ability for deployment in developing countries for influ enza pandemic preparedness. world health organiza tion initiative for vaccine research proc. natl. acad. sci. usa key: cord-003870-hr99dwi7 authors: clohisey, sara; baillie, john kenneth title: host susceptibility to severe influenza a virus infection date: 2019-09-05 journal: crit care doi: 10.1186/s13054-019-2566-7 sha: doc_id: 3870 cord_uid: hr99dwi7 most people exposed to a new flu virus do not notice any symptoms. a small minority develops critical illness. some of this extremely broad variation in susceptibility is explained by the size of the initial inoculum or the influenza exposure history of the individual; some is explained by generic host factors, such as frailty, that decrease resilience following any systemic insult. some demographic factors (pregnancy, obesity, and advanced age) appear to confer a more specific susceptibility to severe illness following infection with influenza viruses. as with other infectious diseases, a substantial component of susceptibility is determined by host genetics. several genetic susceptibility variants have now been reported with varying levels of evidence. susceptible hosts may have impaired intracellular controls of viral replication (e.g. ifitm3, tmprs22 variants), defective interferon responses (e.g. gldc, irf7/9 variants), or defects in cell-mediated immunity with increased baseline levels of systemic inflammation (obesity, pregnancy, advanced age). these mechanisms may explain the prolonged viral replication reported in critically ill patients with influenza: patients with life-threatening disease are, by definition, abnormal hosts. understanding these molecular mechanisms of susceptibility may in the future enable the design of host-directed therapies to promote resilience. the normal response to infection with influenza a virus (iav) is to remain asymptomatic. during the 2009/2010 pandemic, serosurveillance studies revealed that a majority of volunteers who tested positive for antibodies to the new h1n1pdm09 virus did not report any symptoms [1] . the majority of people newly exposed to one of the most dangerous viruses to circulate in human populations in recent history, which in the same population created an overwhelming burden of critical illness [2] , did not notice any symptoms. wide variation in susceptibility is a general feature of human and animal populations exposed to any pathogen [3] . explaining the mechanisms of susceptibility may enable effective targeting of vaccine therapies, may reveal new therapeutic approaches [4, 5] , and, in theory, may contribute to future clinical risk prediction models. as with any infectious disease in a given host, the site of infection, the scale of the initial exposure, and the virulence, degree of pathogenicity, of the pathogen determine the nature of the disease in iav infection. although the alimentary tract is a common site of infection in other species (for example the natural hosts, water fowl [6] ), initial infection in humans is through the respiratory tract. the number of viable iav virions transmitted has a direct effect on the probability of symptoms, both in animal models [7] and human challenge studies [8] . this may explain a proportion of the variation in individual responses to the virus. the virulence of the virus itself varies greatly. perhaps fortunately, there is a general trend for the most virulent iav strains to be less transmissible; that is, those that cause the most severe disease are less likely to be passed on to others. while highly transmissible iav strains, such as h1n1pdm09, replicate well in the upper respiratory tract, viruses associated with higher rates of severe disease, such as h5n1 and h7n9 avian iav, exhibit tropism for the lower respiratory tract [9, 10] . within a given strain, not all iav viruses are the same. in fact, it is statistically unlikely that any two iav virus particles will have exactly the same genome sequence. small changes, such as a single amino acid change in the hemagglutinin protein, can significantly alter the tropism of the virus for example, increasing the likelihood of spread to the lower respiratory tract and establishing a more severe infection [11] . iav viruses change rapidly by two mechanisms: shift and drift. shift is the exchange of viral segments between strains, occasionally resulting in a new iav subtype to which a large proportion of the population does not have existing immunity. this shuffling of the viral genes contributes to the sudden and dramatic change in virulence that may occur from season to season, and to zoonoses, as iav jumps from its natural avian host to mammalian pig and human hosts. drift refers the accumulation of small mutations in the viral genome that occur on a continuum. because of the short genome (around 13,500 bases of rna are carried by a functional virion particle) and a very high error rate when this genome is replicated [12, 13] , viral quasispecies arise, leading to a heterogenous swarm of virions [14] . this variation enables iav to evolve extremely rapidly where a selective pressure exists. for example, it is likely that iav can evolve de novo resistance to antivirals during treatment of a single patient [15] [16] [17] . studies of viral whole genome sequence during outbreaks have failed to identify consistent viral factors associated with severe disease [18] . it is therefore likely that viral factors do not explain the vast spectrum of variation observed in the disease. variation attributable to the host previous exposure to iav due to the remarkable memory of the adaptive and innate immune systems, previous exposure to iav has a strong effect on future susceptibility. adaptive immune memory is highly strain-specific and provides targeted antibodymediated defence against iav [19] . the first iav strain to which a child is exposed has a profound effect on subsequent immunity-a concept known as original antigenic sin [20] . the host immune system is extensively programmed by this first iav exposure, such that the susceptibility of whole populations of adults can be predicted using the patterns of circulating iav in each patient's year of birth [21] . this has been proposed as one reason why the burden of mortality for the 2009/2010 outbreak was shifted towards patients younger than 65 years of age [22] -patients over 65 years old are more likely to have been exposed at a young age to an iav strain similar to the h1n1pdm09 strain, and were hence protected. interestingly, the lifelong immunity provided by this first iav exposure has broad protective effects against different iav strains [21] . cell-mediated immunity may play an important role in this protection. an iav challenge study in healthy volunteers found that preexisting cd4(+) t cell responses to iav nucleoprotein and matrix proteins were present prior to infection [23] . the magnitude of this cd4(+) t cell response when challenged correlated with reduced symptoms and reduced virus shedding. regardless of prior exposure, the most reliably quantified risk factors for life-threatening seasonal and pandemic iav are advanced age (> 65 years), obesity, immunosuppression, cardiovascular disease, and neuromuscular disease [24] . a number of well-recognised host factorsbest summarised by the broadly understood but poorly defined term, "physiological reserve"-increase the chance of organ failure and death following any severe injury or infection. these factors are extensively discussed elsewhere in the critical care literature; here, we focus on host factors that are thought to confer some element of specific susceptibility to iav (fig. 1) . studies dating back to the 1918-1919 pandemic have suggested that pregnancy, particularly in the third trimester, increases the risk of death from iav [25] . additionally, pregnant women have a higher rate of hospitalisation with seasonal iav [26] . however, in the largest systematic review of clinical risk factors for iav, pregnancy was not fig. 1 conceptual visualisation of variation in specificity of host susceptibility factors. factors predicted to confer more specific susceptibility to influenza are placed higher in the diagram independently associated with severe disease from either seasonal or pandemic iav [24] . the immunological changes that occur in pregnancy are theoretically compatible with increased severity of iav: in particular, an increase in innate immune activation and a decrease in the number and activity of cells associated with cytotoxic immunity-in which infected cells are killed to limit the dissemination of the virus [27] . these changes may lead to an increased propensity to develop ards [28] and a decreased ability to eliminate iav-infected cells, which is a core component of anti-iav immunity. some indices of severity used in epidemiological studies are themselves directly affected by pregnancy. the cardiovascular adaptations to pregnancy, combined with an increased metabolic rate, a decrease in functional residual capacity, and increased basal ventilation to perfusion mismatch, are expected to worsen hypoxaemic respiratory failure following any insult. in parallel, admission to hospital or critical care may be in part biased by elevated concern for a pregnant patient, and by the perception of high risk of severe iav [29] . obesity was identified as a risk factor for iav infection over a decade ago and confirmed during the swine flu pandemic [30, 31] when it was associated with an increased risk of death [32] . although comorbidities associated with obesity-specifically diabetes mellitus and cardiovascular disease-compromise pulmonary host defence and increase the chance of death following any severe systemic injury [33] , an independent association between obesity and severe iav is robust and replicated [24] . in parallel with the immune changes associated with pregnancy, obese patients are more likely to have impaired cell-mediated immunity and excessive chronic activation of the innate immune system [34] . this is reflected in a study which demonstrated that among vaccinated adults, those who are obese are more likely to suffer severe consequences of iav [35] . furthermore, it has been shown that obese adults have an impaired antibody response to iav vaccination [36] , and impaired cd4(+) and cd8(+) t cell responses iav in vitro [37] . obese patients have a prolonged period of viral replication and shedding, even in the absence of clinical disease [33] . extremes of age are well-recognised risk factors for severe disease. children under the age of 5 years, and particularly those under 2 years, have consistently been found to be at high risk for severe disease and serious complications following iav infection [38] [39] [40] . functional immaturity of the immune system, together with a failure to recognise iav-related antigens, may largely explain this effect. in industrialised countries, the group at highest risk of death from seasonal iav is those over 65 years of age [22, 41, 42] . senescence affects antiviral immunity in complex ways; it is difficult in clinical epidemiological studies to distinguish the effect of these immune changes from the effects of frailty and antigenic exposure. baseline markers of systemic inflammation are elevated [43] and circulating t cell counts are reduced. naive t cells, a key component of cell-mediated adaptive immunity, are lost from the circulation due to the process of thymic involution, which begins very early in life [44] . in mouse models of iav infection, aged mice exhibit slower antiviral and adaptive immune responses, and more severe disease [45] . expansion of clonal t cell populations, driven by cytomegalovirus (cmv), occurs in older adults and may impair t cell responses to new pathogens [46] . in contrast, in the young, a multi-omic systems analysis demonstrated that cmv infection is associated with an enhanced t cell mediated response to iav vaccination [47] . integrating systems studies of host response to iav infection with markers for genetic susceptibility (see below) may in the future reveal new biological pathways and patterns of disease [48] . as with pregnancy and obesity, ageing is associated with both an increase in the basal activation of the innate immune system (sometimes referred to as "inflammaging") and a decrease in cell-mediated immunity. this combination of mechanisms may explain the particularly strong effects on susceptibility. susceptibility to death from any infection is strongly inherited by children from their parents [49] . in iav, numerous genetic studies in humans and animal models have revealed specific genes associated with susceptibility, which are extensively reviewed elsewhere [50] [51] [52] . in addition to the specific genetic variants discussed below, there is direct evidence, from a study of death records in utah, that susceptibility to iav is heritable at a population level [53] . much of what is known about human genes associated with iav susceptibility has been discovered from lossof-function mutations in the immune system, which lead to loss of the gene product or a substantial reduction in gene function. these often lead to severe defects that are likely to present in childhood. such variants can reveal key components of the immune response to a specific infection. in considering the biological lessons from such discoveries, it is important to consider that, in most people, these components of the immune system function perfectly well and may not be suitable targets for therapy. secondly, there is little that can be inferred from the absence of any particular gene, or immune process, from the list of loss-of-function defects associated with susceptibility to iav. the conditions that must be met for such a gene to be discovered are not restricted to disease susceptibility. many variants that confer susceptibility to iav have broader pleiotropic effects that may be terminal in utero or in early life, or may lead to susceptibility to other infections or autoimmune conditions that obscure the clinical picture. alternatively, some variants may lead to strain-specific susceptibility and will only be detected following exposure to the right virus. the full range of genetic defects associated with susceptibility to iav in animal models is reviewed elsewhere [54, 55] . so far, three known human genes, all transcription factors active primarily in myeloid cells, have been found to have loss-of-function variants that increase susceptibility to iav. since transcription factors function as master regulators of large numbers of genes, functional deficiencies are expected to have broad, nonspecific effects. in 2015, ciancanelli et al. identified a patient with a mutation in the transcription factor interferon regulatory factor 7 (irf7) that led to severe infection and ards when she was 2.5 years old [56] . irf7 is a transcription factor and a key regulator of the type i interferon response. this was the first published example of a singlegene inborn error of immunity that was specific to iav. both parents were heterozygous for different loss-offunction alleles, but each had sufficient functional irf7 activity allowing them to avoid severe iav. the patient inherited these two different loss-of-function alleles (compound heterozygosity) leading to complete loss of functional irf7. leukocytes and plasmacytoid dendritic cells from this patient produced very little interferon type i (α/β) and iii (γ) in vitro indicating that expression and production of these interferons in these cell types is specifically irf7-dependent in iav infection in humans. whole exome sequencing of 20 children identified a variant in the gene encoding interferon regulatory factor 9 (irf9) in a 2-year-old child who had previously suffered from bronchitis and biliary perforation [57] . the child inherited a mutation on both alleles from consanguineous parents leading to a single change in the dna sequence (single nucleotide polymorphism, snp) in the irf9 gene. this snp occurs at an essential site leading to aberrant processing of the gene transcript and thus the expression of a truncated, functionally defective, protein product. in this case, irf9 was only partially defective. activation of interferon-stimulated gene 3 (isg 3) was impaired in response to iav infection or interferon α stimulation, but other irf9-dependent pathways remained intact. the consequence of this appears to be a global reduction in type i interferon responses, a key mechanism of early mucosal resistance to infection, in all cell types. unrestricted viral replication was observed in cells from the patient and was also shown for parainfluenza virus and respiratory syncytial virus. gata2 is a zinc finger transcription factor, part of the gata family, so named because they bind a g-a-t-a pattern (also called a motif) in dna sequence. transcription factor binding at sites bearing this motif alters the probability that a given gene will be transcribed, and ultimately controls the amount of the encoded protein that is made. gata2 deficiency results in primary immune cell deficiency and affects a wide range of cell types. decreased circulating counts of b lymphocytes, nk cells, monocytes and plasmacytoid dendritic cells have been observed, along with reduced t cell thymic output. in 2018, sologuren et al. published a case study of a father and son who contracted and subsequently died from severe iav [58] . both patients were heterozygous for a novel mutation in gata2 that led to a dysfunctional protein. despite the known effects of gata2 deficiency on primary immune development, the first, older patient had suffered few health problems prior to his 30th year, after which frequent respiratory illnesses and a single incidence of viral pneumonia is reported prior to his severe illness. the second patient had been hospitalised with pneumonia at 16 without recurrence until hospitalisation with severe iav at 31. the authors attribute protection from viral and bacterial infection observed in the lifetime of these patients to long-living memory t and b cells. genetic variants with less drastic effects on susceptibility can be detected by comparing flu-susceptible populations with control populations (table 1) . these studies generally look for candidate genes or take a genomewide approach. candidate gene association studies have a long but troubled history in human genetics. genes are selected because of some underlying hypothesis; single variants within these genes are then chosen because they are believed to have an effect on the expression or function of the gene. genotype frequencies (that is, the proportion of a population who have a given variant) at these genomic positions are then compared between a case and control group. this has the advantage of economy, since only one or two variants need to be genotyped for each participant, and has the superficial appearance of statistical efficiency, since fewer comparisons are made. the fundamental limitation is that, in a human genome composed of 3 × 10 9 bases, of which 4 − 5 × 10 6 are different between any random pair of people [59] , the probability of choosing the right base is very low. in the event that a given variant meets a nominal level of significance, the evidence for an association is easily misinterpreted. looking backwards from a single small p value, it is common to focus on the fact that probability of seeing such an association by chance alone is very low. what is easy to forget is that the probability of such an association existing is also very low. an understanding of this methodology is important for the interpretation of such studies. many of the positive studies reflect more the biases of well-informed investigators in the choice of target genes. the additional value of an unreplicated genetic association on this background is often small. lgals1 galectin-1 cell-cell interactions rs4820294 [82] rs2899292 [82] rs4820294 [82] st3gal1 (*) st3 beta-galactoside alpha-2, [85, 88] gene: gene symbol. gene name: gene name and alternate name. function: summary function of gene product. snp: snps associated with host susceptibility to influenza a associated with gene. (*) represents genes not addressed in the text nonetheless, candidate gene approaches in various forms detected numerous real and informative associations with disease before the advent of genome-wide genotyping technology [60] . we focus here on larger studies, those that have been replicated, and studies with particular relevance to the pathogenesis of severe iav. genome-wide approaches seek to eliminate the aforementioned bias. in the most widely used design, the genome-wide association study (gwas), hundreds of thousands of common variants are genotyped in each patient. this is expensive and requires correction for multiple comparisons. a widely used convention is to correct for 1 × 10 6 independent comparisons in each study, requiring a p value <5 × 10 −8 for significance. large numbers of patients are needed to detect associations at this level above the background noise of variation in human populations. however, genome-wide approaches use no preconceptions about the pathogenesis of disease. hence, such methods have the potential to teach us something that we did not already know. because of the stringent threshold for statistical significance, and the burden of multiple testing, statistical power to detect small effects is usually lacking unless many tens of thousands of patients are included. for this reason, the expected outcome is false-negatives. hence, we would caution against drawing any conclusion from the absence of significant associations within a given gene. genome-wide in vitro knockdown screens can also be used to limit bias and enable genome-wide discovery. in this approach, although a candidate gene is often selected from cell culture results and tested for genetic associations in patients, there is an important difference from single-gene candidate studies: the pool of genes from which the candidate is chosen comprises the entire protein-coding part of the genome. a role for interferon-induced transmembrane protein 3 (ifitm3) in iav replication was discovered in an in vitro genome-wide knockdown screen in cultured cells [61] . the protein product of this gene restricts iav entry by blocking the fusion of host and viral membranes [62] and acts as a restriction factor in viral infections, along with family members ifitm1 and ifitm2 [61] . ifitm proteins were also shown to inhibit the early replication of other virus types, for example the west nile virus [63] . based on this genome-wide knockdown screen, a candidate gene sequencing approach was conducted to test for an association with severe illness. the 2009/2010 pandemic provided a colossal natural experiment-a large proportion of the population were exposed to a new pathogen, but only a small minority developed lifethreatening illness requiring critical care. focusing on these previously healthy adults with life-threatening iav (in the genisis and mosaic studies) may have increased the effect size seen [64] . genotypes at every variant within the ifitm3 gene were compared with population controls, identifying a single variant (rs12252-c) associated with severe iav. this variant is rare in the european cohorts in which it was discovered, but is frequent in the han chinese cohorts hospitalised with severe h1n1pdm09 infection [65] . the association has been replicated in independent studies in different populations [66] . a second snp associated has been shown in populationlevel studies to regulate ifitm3 expression. rs34481144-a encourages the transcription factor ctcf to bind to the regulatory region of ifitm3 and repress gene expression in response to iav infection [67] . this snp can also disrupt the methylation pattern (a key modification of dna that usually silences genes) in the regulatory region leading to cell type-specific effects. ifitm3 expression in memory cd8(+) t cells in response to viral infection has been found to protect and encourage survival of these cells allowing for the establishment of adaptive immunity. loss of methylation at this site prevents ctcf from binding to the dna and inducing expression of ifitm3 in response to the pathogen, thus reducing cell survival. this is estimated to lead to a 2.6-fold increased risk of a severe outcome upon iav virus infection. ifitm3 has also been recently shown to have a protective effect on the heart during severe iav infection. myocarditis has been associated with iav infection since the 1918 pandemic [68] , and iav has been shown to lead to a sixfold increased risk of myocardial infarction in the 7 days post infection [69] . so far, ifitm3 is the only gene for which snps have been identified and independently confirmed in vivo and in vitro to restrict iav replication [70] . however, this gene is not specific to iav replication and the full extent of the antiviral actions remains to be discovered. unbound complement is rapidly inactivated in plasma. where this process is defective, uncontrolled complement activation can damage host cells. cd55 prevents the formation and accelerates the decay of c3 and c5 convertases. these proteases are part of the complement system and have roles in opsonisation and the release of inflammatory molecules. cd55 polymorphisms were associated with severe h1n1pdm09 infection (defined as requiring supplementary oxygen, admission to intensive care or death) [71] . this study found carriers of the rs2564978-t/t polymorphism had significantly lower levels of surface cd55 on their circulating monocytes compared to the more common c allele. further work identified a deletion in a nearby regulatory region as the element responsible for the specific effect on both protein and mrna levels of cd55 in monocytes. a more recent study of han chinese individuals that looked at several genes confirmed an association between cd55 rs2564978 t/t and death from severe iav infection [72] . the cumulative effects of multiple snps (ifitm3, cd55, and the immune cell receptors tlr3 and tlr4) on iav susceptibility have been examined in a targeted study [72] . this independently confirmed the association of the cd55 rs2564978 polymorphism with severity, and the ifitm3 rs12252-c and tlr3 rs5743313-cc genotypes were both over represented in fatal cases. in a small-scale pilot study, genome-wide genotypes of 42 patients with severe iav were compared to 42 controls with mild iav. the rs2070788-g allele of tmprs22 was significantly overrepresented in severe compared with mild cases of h1n1pdm09, with a > 2fold higher risk of severe infection. there was higher tmprs22 expression in human lung tissues with the high-risk gg genotype [73] . this was replicated in a targeted study of 162 severe and 247 mild iav patients. this genetic association in humans is highly biologically plausible: tmprs22 has been shown to play a role in haemagluttinin cleavage, an important step in iav replication. additionally, mice lacking this gene are strongly protected from iav infection [74] [75] [76] . this genome-wide array also identified a snp in pulmonary-surfactant-associated protein b (sp-b), rs1130866, as a potential association. this snp was genotyped in a targeted study of 111 severe and 185 mild iav patients to replicate the finding [77] . again, this is a plausible association with severe disease: sp-b forms a key part of pulmonary surfactant and is essential for lung function. a subset of the same protein family, sp-a and sp-d, have been shown to initiate and enhance immune cell ingestion and killing (opsonisation) of pathogens and play a role in the progression of iav in mice [78] . a polymorphism associated with sp-b, rs1130866 [77] , has also been associated with copd in several cohorts [79] . susceptibility to severe h1n1 infection was analysed in a recent genome-wide study (integrated with data on genetic variants associated with altered gene expression) which implicated an intronic snp of gldc, rs1755609-g [80] . the gldc gene encodes glycine decarboxylase, also known as the p protein of the glycine cleavage system, a pathway in glycine metabolism [81] . the association was replicated by targeted genotyping in a larger cohort of 174 patients suffering severe iav infection and 258 mildly infected controls. the risk variant corresponds to higher gldc expression in lymphoblastoid cell lines and human lung tissues. consistent with this effect, inhibition of gldc in cultured bronchial epithelium using sirna or a specific inhibitor, aminooxyacetic acid (aoaa), leads to an increased type i ifn response and a restriction of viral replication in vitro. this effect on viral restriction was seen with both h1n1 and h7n9, and the allele genotype was replicated in susceptibility cohorts for both viruses. the protective effect of aoaa against h1n1 was shown in mice to be comparable with that of zanamivir. susceptibility to severe h7n9 was examined in a gwas performed with 102 patients and 106 controls who worked with poultry. this study identified rs13057866, associated with galectin-1 (lgals1), as a potential susceptibility factor. lgals1 is a lectin that may have a role in modulating cell-cell and cell-matrix interactions. the study further demonstrated that genetic variants of lgals1, including rs4820294 and rs13057866, lead to higher expression of lgals1 protein in human cells, possibly leading to a protective effect. carriers of the rs4820294/rs2899292 gg haplotype were found to have higher lgals1 protein in lymphoblastoid cells and expression levels of lgals1 in human lung correlated with the rs4820294 snp [82] . the role of host factors in susceptibility suggests a clinically important conclusion: there is something unusual about the small minority of patients who develop critical illness following iav. therefore, extrapolating from human challenge and primary care studies of viral clearance is very likely to lead to error. viral clearance among critically ill patients is slow and incomplete [83] . hence, the critically ill population should be regarded-by definition-as highly abnormal hosts. susceptible hosts may have impaired intracellular controls of viral replication (e.g. ifitm3, tmprs22 variants), defective interferon responses (e.g. gldc, irf7/9 variants), or defects in cell-mediated immunity with increased baseline levels of systemic inflammation (obesity, pregnancy, advanced age). in the context of any of these susceptibility mechanisms, failure to clear the virus is an expected consequence, indicating that a full course of effective antiviral therapy is likely to benefit this population. in the future, understanding the biological mechanisms of susceptibility to severe iav may yield therapeutic targets to modify the biology of the susceptible hosts in critical care and render them resilient. comparative community burden and severity of seasonal and pandemic influenza: results of the flu watch cohort study the swine flu triage (swift) study: development and ongoing refinement of a triage tool to provide regular information to 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otherwise healthy children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests. key: cord-002136-mkl89qkt authors: nunes, sandro f.; murcia, pablo r.; tiley, laurence s.; brown, ian h.; tucker, alexander w.; maskell, duncan j.; wood, james lionel n. title: an ex vivo swine tracheal organ culture for the study of influenza infection date: 2009-12-09 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2009.00119.x sha: doc_id: 2136 cord_uid: mkl89qkt background the threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. ex vivo organ culture (evoc) systems have been successfully used in the study of both human and animal respiratory pathogens. objectives we aimed to develop an air interface evoc using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. methods tracheal explants were maintained in the air interface evoc system for 7 days. histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. explants were infected with a swine h1n1 influenza virus. influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time rt‐pcr. results histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. challenge with swine h1n1 influenza virus showed that the evoc tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. conclusion the air interface evoc system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. influenza a virus is the most significant worldwide respiratory pathogen, infecting a range of species that includes humans, pigs, horses, sea mammals and birds. 1 influenza viruses are divided into different subtypes based on the antigenic reactivity of their two surface glycoproteins, the haemagglutinin and the neuraminidase. so far, 16 different haemagglutinin (h1-h16) and nine neuraminidase (n1-n9) subtypes have been identified among all influenza a viruses. 2 waterfowl, shorebirds and gulls are considered the natural hosts of all influenza subtypes, from which mammalian influenza viruses are directly or indirectly derived. 1, 3 influenza infection in humans and pigs is primarily restricted to the upper and lower respiratory tract with viral replication occurring in the epithelial cells present on the surface of the respiratory mucosa. 4 the acute phase of infection is characterized by a multifocal destruction of the epithelium with cellular desquamation to the luminal space, which may often lead to patches along the mucosa with few or no epithelial cells over the basement membrane. it is also associated with the presence of oedema, vascular congestion, hyperaemia and infiltration of inflammatory cells into the submucosa underlying the epithelial layer. 4, 5 the presence of both n-acetylneuraminic acid-a2,3-galactose (neu-aca2,3gal) and neuaca2,6gal receptors in the pig respiratory tract makes this species susceptible to most human and avian influenza viruses. 6, 7 for this reason, pigs have been proposed as a 'mixing-vessel' for reassortment between human and avian viruses. 3, [6] [7] [8] the significance of cross-species transmission of swine influenza viruses to humans is underscored by the recent human influenza outbreak with an h1n1 of possible swine origin. 9, 10 in addition to being important natural hosts, pigs constitute a valuable animal model for the study of influenza infection, 5, 11 as virus may be transmitted experimentally to in-contact animals and they can exhibit clinical signs, histopathology and cytokine expression profiles comparable to influenza infection in other species. [3] [4] [5] 11, 12 in vivo studies have provided valuable information on clinical course, viral pathogenesis, innate and acquired immune responses, vaccine efficacy and antiviral treatment. 4, 5, [13] [14] [15] however, such studies are greatly affected by animal welfare considerations and logistical constraints. to overcome such constrains, in vitro systems that involve either established or primary cell lines have been widely used, 16, 17 but such systems usually lack the natural physiological context present in the respiratory mucosa, such as cell polarization, coordinated ciliary activity, mucus production and apical contact with air. ex vivo organ cultures (evoc) of tracheal explants with an air interface system have been successfully developed and used in the study of both human and animal respiratory pathogens. [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] such systems reproduce, to a great extent, the physiological conditions and the cellular complexity of the respiratory mucosa of the host in a highly controlled setting. the three-dimensional structure and cell diversity of the respiratory mucosa is preserved together with important physiological features like normal expression levels of cell receptors, effective mucus production and ciliary activity. 18, 23, 27 moreover, evoc systems allow a quantitative analysis of the effects of pharmacological agents (i.e. antiviral drugs) and ⁄ or the host innate response on infection dynamics. 23, 27, 31 in addition, tissue collected from a single animal can be used in numerous replicas, which reduces the experimental variability and the number of animals used, following the principles of (reduction, replacement and refinement (3rs) in animal experimentation. 32 in this study, we developed a swine tracheal evoc system for the study of influenza virus infection. swine tracheal explants infected with a contemporary avian-like swine h1n1 virus supported productive infection and exhibited histopathological changes consistent with in vivo influenza infection. overall, this system constitutes a useful physiologically relevant model for the study of influenza infection in a scalable, well-defined and controlled experimental setting. tissue collection and preparation weaned pigs (6-8 weeks old), sourced from a specific pathogen free (spf) closed herd serologically negative against influenza virus, were killed by intravenous administration of sodium pentobarbitone (0ae8 mg ⁄ kg i.v. to effect). whole tracheas were aseptically collected and transported in pre-warmed culture medium consisting of a 1:1 mixture of dulbecco's modified eagle's medium (dmem) and roswell park memorial institute (rpmi) 1640 medium supplemented with penicillin (100 u ⁄ ml; sigma), streptomycin (50 lg ⁄ ml; sigma, dorset, uk), l-glutamine (10 mm; sigma) and amphotericin (2ae5 lg ⁄ ml; sigma). once in the laboratory, tracheas were kept at 37°c in a 5% co 2 -95% air mixture in a humidified incubator. four to six washes were performed, over a period of 3 hours, by immersing the tissue in fresh, warm dmem ⁄ rpmi medium. the presence of bacterial flora was evaluated by inoculation of the final washing medium in brain heart infusion broth (oxoid, hampshire, uk) with further incubation at 37°c for 72 hours. after the washing steps, the surrounding connective tissue present exterior to the tracheal cartilage was removed and tracheas were opened and cut lengthways into four strips. each segment, incorporating the respiratory mucosa and underlying cartilage, was then cut into approximately 1ae0 · 0ae5 cm explants and placed on a square section of sterile filter paper, which was in turn placed onto agarose plugs in 6-well plates with the medium bathing the filter paper and the basal portion of the explants, as described previously. 29 explants were maintained at 37°c in a 5% co 2 -95% air mixture in a humidified incubator for up to 7 days. tissue samples were fixed in 10% (v ⁄ v) buffered formalin and paraffin-embedded. five micrometre sections from across each explant were subject to haematoxylin-eosin, periodic acid-schiff and van gieson staining. haematoxylin-eosin stained slides were used to measure the thickness of the epithelial layer using imagej software (http://rsbweb.nih.gov/ij/). five randomly selected fields across each section were measured and statistical significance was assessed by analysis of variance using prism 5 (graphpad software, la jolla, ca, usa). five microlitres of an emulsion of 1 lm diameter polystyrene microsphere beads (polybead microspheres, polysciences inc., northampton, uk) were pipetted onto the epithelial surface of the explants, and bead clearance was evaluated at 10, 20 and 30 minutes post-inoculation. the assay was considered positive if the microbeads had been completely cleared to one edge of the tissue. virus a stock of avian-like swine h1n1 influenza virus (a ⁄ swine ⁄ england ⁄ 453 ⁄ 06) was grown in embryonated chicken eggs, aliquoted and stored at )80°c. the virus was isolated at the veterinary laboratories agency (vla) from pigs suffering a respiratory disease outbreak in england in 2006. viral titres were determined by haemagglutination assays using adult chicken erythrocytes and plaque assays on madin-darby canine kidney (mdck) cells. 33 organ culture inoculation viral inoculations were performed in triplicate in three independent experiments, with doses ranging from 2ae5 · 10 1 to 2ae5 · 10 6 plaque forming units (pfu). five microlitres of viral suspension was applied onto the epithelial surface of the explants. culture medium was used for mock-infected controls. inoculated explants were sampled every 24 hours for histology, bead clearance assays and viral quantification. re-hydrated sections were treated with 3% h 2 o 2 in methanol for 15 minutes at 22 ± 3°c for endogenous peroxidase blocking, washed twice in tap water and incubated with pronase xxiv (menapath, wokingham, uk) for 10 minutes at 22 ± 3°c for antigen retrieval. after two washes with distilled water, sections were blocked with 1ae5% donkey serum in tris base saline buffer (tbs) for 20 minutes and incubated overnight at 4°c with a chicken anti-swine influenza polyclonal antibody (1:4000) kindly provided by vla-weybridge. slides were washed twice for 5 minutes with 0ae05% tween 20 in tbs (tbs ⁄ t) and incubated for 1 hour at 22 ± 3°c with a donkey anti-chicken igy biotin-labelled antiserum (1:200 in blocking buffer; jackson immunoresearch europe ltd, newmarket, uk). sections were washed twice in tbs ⁄ t, incubated for 30 minutes with vectastain elite abc reagent (vectastain elite abc kit, vector laboratories peterborough, uk) prepared following the manufacturer's instructions, then washed twice with tbs ⁄ t (5 minutes each). colour development was carried out by incubation with sigma-fastô solution (sigma) for 10 minutes. slides were counterstained with mayer's haematoxylin, dehydrated and mounted with dpx mounting medium. inoculated explants were immersed in 1ae5 ml of sterile phosphate-buffered saline (ph 7ae4) and shaken in a mixer mill mm300 (retsch, leeds, uk) at 20 hz for 10 minutes at 4°c, followed by centrifugation at 16 200 g for 10 minutes. the collected supernatant was used in plaque assays (performed in triplicate) in mdck cells. results were expressed as arithmetic mean pfu ⁄ ml. quantification of viral copy number by real-time rt-pcr rna extraction and reverse transcription. inoculated explants were disrupted in 1 ml of trizol ò reagent (invi-trogen) using a mixer mill ò mm300 (retsch, leeds, uk). rna extractions were performed according to the manufacturer's instructions, with slight modifications: after the phase separation step, the aqueous phase containing the rna fraction was transferred to a new tube and purified with rneasy mini kit (qiagen, crawley, uk). cdna synthesis from 50 ng of total rna was performed with superscriptô iii reverse transcriptase (invitrogen, paisley, uk), according to the manufacturer's instructions, using the specific primer for the influenza matrix segment described in hoffmann et al. 34 real-time rt-pcr. viral copy numbers were estimated using a real-time quantitative rt-pcr (qpcr) assay 35 run in a corbett rotor gene 3000 (corbett research, mortlake, australia). cycle parameters were: 15 minutes at 95°c, followed by 45 cycles of 15 seconds at 94°c and 60 seconds at 60°c, with fluorescence acquisition in the last step. standard curves were generated using 10-fold dilutions of a plasmid containing the target sequence of the influenza matrix segment, ranging from 1 · 10 2 to 1 · 10 8 copies per microlitre. for each run, all samples, as well as the no-template controls, plasmid standards, and positive and negative controls were run in triplicate and expressed as the arithmetic mean number of vrna copies per microlitre of cdna. tracheal explants maintain normal histological architecture for up to 7 days, with progressive loss of ciliary activity uninfected tracheal explants preserved their normal histology without major alterations for up to 7 days after removal from the animal ( figure 1a -e), with signs of sporadic cell degeneration in the transitional layers between the epithelium and the submucosa. the overall structure of both the pseudostratified columnar epithelium, and the underlying lamina propria were maintained. a progressive increase in the amount of mucus on the epithelial apical surface was observed from day 3 onwards (figure 1d,e) . periodic acid-schiff staining showed that explants maintained overall carbohydrate levels (figure 2a-d) . van gieson staining showed that the elastic fibres present in the submucosa had a wave-like form after day 4 indicating a small level of fibre relaxation ( figure 2e-h) . the average thickness of the epithelium did not change significantly (p = 0ae4) for the duration of the organ culture ( figure 3a) . a bead clearance assay was used to evaluate co-ordinated ciliary activity. tracheal explants were tested immediately after collection and every 24 hours thereafter. beads were cleared in less than 30 minutes in all pieces examined. however, a progressive increase in the time for complete tracheal explants were infected with avian-like swine h1n1 influenza virus (a ⁄ swine ⁄ england ⁄ 453 ⁄ 06). a high dose (2ae5 · 10 6 pfu) was initially used to attempt to ensure viral infection of the majority of susceptible cells. mock-infected pieces displayed normal histological appearance as described above ( figure 4a ,c). infected explants ( figure 4b ,d) exhibited histopathological changes compatible with in vivo influenza infection, 4,36 including loss of cilia at the apical surface, shrinking and vacuolization of epithelial cells with signs of destruction and desquamation and the presence of cellular debris in the mucus of the epithelial surface. furthermore, a significant difference in the thickness of the epithelial layer between mock and virus-infected was observed (p < 0ae0001) with a marked reduction at day 1 post-infection (pi) (figure 4e ). ciliary-mediated bead clearance was reduced 24 hours pi and completely absent at day 5 ( figure 4f ). viral antigen was detected in infected explants by immunohistochemistry. the distribution of positive cells was relatively widespread throughout the whole section and was limited to the epithelial layer ( figure 5a ). positive staining was predominantly seen in the nucleus, but with some also in the cytoplasm ( figure 5b ). viral antigen was also detected between the cilia and in aggregates of mucosal material present at the surface of the epithelium. in subsequent infections with a lower dose of virus (2ae5 · 10 2 pfu) the pattern of cells with positive staining remained evenly distributed across the tissue section (figure 5c,d) . to determine if the swine tracheal explants supported productive viral replication, explants were infected with 2ae5 · 10 2 pfu of swine influenza virus and maintained in organ culture for 5 days. infectious extracellular virus extracted from the epithelial surface was quantified in plaque assays in mdck cells. a marked increase in the amount of infectious virus was observed 24 hours pi, when the number of infectious units increased by approximately 10-fold when compared with virus recovered immediately after infection. viral titres consistently increased by 3 log 10 on day 2 and remained stable up to day 4 pi (at c 6ae4 log 10 ) followed by a small decline by day 5 pi (figure 5e) , showing that tracheal explants support productive viral infection. a qpcr assay to measure intracellular and extracellular virus was used as a complementary method to assess infection kinetics. to this end, explants were inoculated with 2ae5 · 10 1 , 2ae5 · 10 2 or 2ae5 · 10 4 pfu, and maintained in organ culture for 5 days. viral quantification showed that infection kinetics was dose dependent (figure 5f-h) . in explants inoculated with 2ae5 · 10 4 pfu, the peak viral load was observed at day 1 pi. this was reduced at day 3 pi but then there followed a resurgence in copy number by day 5 pi ( figure 5f ). inoculation with 2ae5 · 10 2 pfu showed a different pattern of infection in which the peak shifted to day 3 pi, with a higher viral copy number than the one observed when a higher dose was used ( figure 5g ). viral rna was detected at day 2 pi in explants inoculated with the lowest dose (2ae5 · 10 1 pfu) and a marked increase in viral copy number was observed at days 4 and 5 pi (figure 5h) . we have established an ex vivo air interface tracheal organ culture for the study of influenza viruses which could also be used to study other respiratory pathogens. evoc systems have been developed previously for the study of respiratory pathogens using human, swine, bovine, canine and horse respiratory tissue. 18, 20, 23, 25, 27, 29, 30 the experimental value of the tracheal explants described here is illustrated by the preservation of good levels of both histological and functional features for seven consecutive days, and by showing productive infection with an avian-like swine h1n1 virus. a vast body of knowledge in influenza biology has been generated using diverse experimental systems, each possessing advantages and disadvantages. in vivo studies have provided valuable data on influenza pathogenesis, although detailed information about quantitative infection dynamics and the host innate immune response is still scarce. 1, [3] [4] [5] 15, 37, 38 for respiratory pathogens in particular, samples are usually collected by nasal swabs or bronchoalveolar lavage, which may not fully reflect the tissue-specific bacterial and viral infection dynamics occurring throughout the respiratory tract, making it difficult to dissect regional tissue-specific effects from generalized systemic responses. cell culture systems, using epithelial cells from the respiratory mucosa, may lack key physiological features such as cell polarization, reliable expression levels of cellular surface receptors or the intricate relationships between the different cell types present in the respiratory tract. 39 primary cell cultures are difficult to establish, possess an inadequate distribution of the different cell types and become altered after a limited number of passages. established continuous cell lines are by definition aberrant and usually belong to a cell type different to the natural target cells and, in some cases, are derived from a species different from the natural host. ex vivo organ cultures provide an alternative solution. nasal mucosa 27 and tracheal explants 39 immersed in cell culture medium have been used previously for the study of influenza infection. however, using this technique cells are subjected to submersion-related stress and to a continuous source of infecting pathogen as the inoculum remains in the cell culture medium. 22 the tracheal organ culture described here exhibits several advantages compared with submerged organ cultures, cell cultures or in vivo studies. first, in an air interface system the explants are exposed basolaterally to culture medium and the apical cell surface is exposed to the air. the threedimensional structure of the tissue as well as the interactions among cells is preserved. in particular, the cells of the epithelial lining of the trachea maintain the same morphological appearance, differentiation and polarity as described in the living host. in contrast to submerged systems, once explants are inoculated, re-infection from the culture medium or other components of the system is rare. the absence of a blood supply constitutes a weakness of the air interface evoc system as recruitment of cells of the immune system to the infection site is abrogated. however, such a potential pitfall might be turned to advantage and used to dissect the effects of innate and intrinsic immunity. 40 in this study, the overall histology of the explants was preserved for 7 days with no significant differences in the thickness of the uninfected epithelium. this is consistent with previous reports of similar systems in which explants of respiratory tissue from different species have been maintained for variable periods of time, ranging from 60 hours to 5 days. 23, 25, 27 mucus production provides an important nonspecific defence against viral infections. 41, 42 during the initial washing steps in setting up the explants, a majority of the mucus is removed from the epithelial surface. however, mucus is produced de novo by goblet cells throughout the duration of the organ culture, and thus the physiological conditions present in the living host are maintained in this regard. we also evaluated the function of the mucociliary escalator system, and thus also of co-ordinated ciliary function of the tracheal explants, using a simple bead clearance assay. for beads to be cleared from the surface of the organ culture piece, individual cilia must beat in a coordinated manner across the epithelial surface with mucus acting as a vehicle to transport unwanted particles away from where the alveoli would have been in a wave-like motion towards where the larynx would have been. 43 by using the bead clearance assay, we were able to distinguish between small patches of ciliary movement and truly coordinated ciliary activity, an important measure of the robustness of the system. the increase in the clearance time observed at later time points in uninfected explants may be associated with changes in ph, or energy limitations of the tissue. although the supplemented culture medium contains most of the essential components required for survival of the explants, it may not constitute a complete substitute for the nutrients available from the living host. infection of swine tracheal explants with a ⁄ swine ⁄ england ⁄ 435 ⁄ 06 (h1n1) resulted in productive infection with histopathological and immunohistochemical patterns consistent with those observed in influenza infections of both humans and pigs. 3, 4, 13, 36 as expected, infection kinetics was dose dependent: when using a high dose of virus we observed a mild infection that peaked bimodally at day 1 and day 5 pi. with an intermediate dose, a single peak occurred at day 3 pi, whereas the lowest infection dose resulted in detectable virus at day 2 pi, followed by a subsequent rise in copy number at later time points. such influenza infection in a pig tracheal evoc system ª 2009 blackwell publishing ltd, influenza and other respiratory viruses, 4, 7-15 distinct patterns might be related to differential levels of innate immune response, as well as to the initial number of susceptible target cells. a large inoculum would be expected to infect most of the susceptible cells and viral replication may also trigger a massive innate immune response, resulting in a small number of susceptible cells available for a second round of viral replication. once the antiviral signalling molecules are depleted, the remaining infectious particles can infect the susceptible cells, and this would be consistent with the second rise in virus titres observed at day 5 ( figure 5f ). although a second round of infection could not be ruled out, the peaks are too separated in time for this to be likely. a smaller dose of virus might trigger a milder innate immune response, restricted to neighbouring cells, allowing the virus to infect a larger number of cells over multiple rounds of infection. accordingly, the kinetics of infection with the lowest viral dose displayed a marked shift to the right because more replication cycles were required to infect all or most of the susceptible cells. further experiments that incorporate the quantification of cytokine expression will help to test this hypothesis. in our study, an influenza virus of swine origin was used in the validation of the pig trachea evoc system. future studies, including viruses of different subtypes isolated from diverse hosts, should be performed to evaluate the susceptibility of pig trachea to influenza infection and to study the mechanisms underlying any possible host-specific specificity. in summary, the evoc system constitutes a useful biological tool with a wide range of possible applications for the study of influenza infection, including the evaluation of mutants obtained by reverse genetics, the study of natural variability and reassortment events, the effects of antiviral drugs in natural target cells, investigation of host susceptibility and range of influenza viruses and the development of mathematical models of infection dynamics. when possible, evoc systems should be considered as an alternative to in vivo experiments, applying the principles of 3rs in animal experimentation. evolution and ecology of influenza a viruses characterization of a novel 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reverse transcriptase pcr assay for type a influenza virus and the avian h5 and h7 hemagglutinin subtypes localization of swine influenza virus in naturally infected pigs influenza type a in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission realities and enigmas of human viral influenza: pathogenesis, epidemiology and control cultures of equine respiratory epithelial cells and organ explants as tools for the study of equine influenza virus infection intrinsic immunity: a front-line defense against viral attack defense mechanisms against influenza virus infection in the respiratory tract mucosa effective mucus clearance is essential for respiratory health efficient mucociliary transport relies on efficient regulation of ciliary beating we thank yvonne spencer, sara marsh, alejandro nunez (vla-weybridge) and carina ferrari for their assistance with the immunohistochemistry protocol and also t. j.mckinley for his assistance in the statistical analysis of data. this work was supported by a grant from defra and hefce under the veterinary training and research initiative to the cambridge infectious diseases consortium (cidc) through a fellowship awarded to s. f. nunes. j.l.n.w. is funded by the alborada trust and p.r.m. is funded by the wellcome trust. the swine influenza virus was obtained through a programme of influenza surveillance in gb pigs funded by defra (project ed1204). key: cord-002337-8v907g24 authors: lipsitch, marc; barclay, wendy; raman, rahul; russell, charles j; belser, jessica a; cobey, sarah; kasson, peter m; lloyd-smith, james o; maurer-stroh, sebastian; riley, steven; beauchemin, catherine aa; bedford, trevor; friedrich, thomas c; handel, andreas; herfst, sander; murcia, pablo r; roche, benjamin; wilke, claus o; russell, colin a title: viral factors in influenza pandemic risk assessment date: 2016-11-11 journal: nan doi: 10.7554/elife.18491 sha: doc_id: 2337 cord_uid: 8v907g24 the threat of an influenza a virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. to date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. the goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. we describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin ph of activation, and polymerase complex efficiency) that contribute to pandemic risk. doi: http://dx.doi.org/10.7554/elife.18491.001 aquatic birds are the main reservoir of influenza a viruses in nature (krauss and webster, 2010) . influenza viruses from aquatic birds sporadically enter terrestrial bird and mammalian host populations and achieve sustained circulation in these new hosts (vandegrift et al., 2010) , sometimes after reassortment with influenza viruses already circulating in the new host (webster et al., 1992) . adaptation of viruses from aquatic birds to mammals involves a change in tissue tropism from intestinal to respiratory epithelia (hinshaw et al., 1979; hénaux and samuel, 2015) . multiple influenza a subtypes-defined by the patterns of antibody recognition of two surface proteins, hemagglutinin (ha) and neuraminidase (na)-circulate in avian species and swine at any given time. among these, a number are known to cause sporadic zoonotic infections in humans (peiris, 2009 ). more than one thousand human infections with avian influenza viruses were detected in the last decade, for example h5n1 and h7n9 (qin et al., 2015) as well as swine influenza viruses, e.g. an h3n2 variant that spilled over into humans attending agricultural shows in the early 2010s, h3n2v (jhung et al., 2013) . in addition, zoonotic infections with other viruses from poultry or wild birds have occurred, including for example h7n7 (fouchier et al., 2004) , h10n8 (wohlbold et al., 2015) , h6n1 (wei et al., 2013) , h9n2 (butt et al., 2005) , and h5n6 ; for more examples and a fuller discussion see (short et al., 2015) . the severity of zoonotic influenza a infections ranges from clinically inapparent (gomaa et al., 2015; to et al., 2016) to fatal (de jong et al., 2006; gao et al., 2013) . although secondary transmission can occur following some of these spillover events (kucharski et al., 2014) , only a very small proportion of them-four in the last hundred years, which seems to be close to the historical average (patterson, 1986 )-led to sustained person-to-person transmission with global spread (box 1). there are 18 known ha types and 11 known na types (tong et al., 2013) , which could theoretically be found in any combination. so far, sustained spread in humans has been limited to the h1n1, h2n2, and h3n2 subtypes , though it is possible that other subtypes circulated prior to 1918, the year of the first pandemic from which viruses are available for study (worobey et al., 2014) . multiple virus-host interactions are necessary for replication and onward transmission; the differences in the genetic requirements to accomplish each of these interactions in humans versus other animals provide a barrier to sustained transmission following spillover . experiments in ferrets have been used to measure viral transmissibility via respiratory droplets (in this review we use this term to refer to any transmission through the air between ferrets that are not in direct or indirect physical contact). droplet transmission in ferrets is a useful, albeit imperfect, correlate of the potential of influenza strains to transmit efficiently in human populations (buhnerkempe et al., 2015) . for this reason, some have argued that there is a box 1. steps in pandemic emergence. for an avian-adapted strain of influenza a to become a pandemic strain, several events are required: 1. the avian-adapted strain must become sufficiently widespread in wild or domestic birds, swine or other reservoir species to expose at least one human to infection. 2. one or more humans must acquire infection from the reservoir species. 3. the infection must replicate sufficiently in a zoonotic case to produce infectious virus in respiratory or other secretions. 4. the infection must be transmitted to additional humans, avoiding an "early" termination of the transmission chain due to chance. such early termination is a significant risk given the relatively low infectiousness of influenza and the moderate degree of overdispersion in the number of secondary cases infected by each case, both of which contribute to the probability that a transmission chain will terminate by chance (lloyd-smith et al., 2005; lipsitch et al., 2003) . it must also avoid extinction due to deliberate control efforts put in place by public health authorities (ferguson et al., 2006; merler et al., 2013) . 5. finally, the infection must spread beyond the local area to infect members of distant populations, a process accelerated by modern global travel (cooper et al., 2006) . this step and the one before are enhanced if the level of population susceptibility is high, as occurs when the surface proteins of the new strain are dissimilar to those on any currently or recently circulating human influenza a strains. we know from serologic studies and human infections that several different influenza a viruses have achieved steps 1 and 2 at any given time (gomaa et al., 2015; to et al., 2016) . steps 3 and/or 4 appear to be the rare, rate-limiting steps; that is, the conditional probability of achieving step 3 and 4 given the previous steps is low, so that sustained human-to-human transmission of a novel strain occurs a few times per century while zoonotic infections must occur thousands or more times per year. no case is known in which an influenza a strain has reached stage 4 but failed to reach stage 5, although it may have happened undetected. the appearance -by mutation or reassortment -and selection of genetic changes that encode human-adaptive viral traits may be seen as a process that can accelerate or increase the probability of one or more of these steps (though there is no guarantee that a given change that enhances one of these steps will enhance all of them. this is why the detection of phenotypes associated with human adaptation in avian or zoonotic isolates of novel influenza a viruses is thought to correlate with increased risk of pandemic emergence. as we describe throughout the paper, the process of human adaptation need not be complete to initiate a pandemic, so it may continue to occur at various stages throughout this progression. doi: 10.7554/elife.18491.002 general phenotype of 'transmissibility by respiratory droplets in mammals' such that experiments to select for such transmission in droplets in ferrets are important models of the process of adaptation to human transmission (imai et al., 2012; herfst et al., 2012) . this view is not universally shared . starting from an zoonotic highly pathogenic avian influenza isolate from a human case of infection (or a reassortant of the ha from a different zoonotic h5n1 highly pathogenic avian influenza isolate, with the other segments from the 2009 pandemic h1n1 strain), it was shown that certain specific traits that had been previously associated with mammalian host adaptation were required to achieve respiratory droplet transmission. these ferret-transmission phenotypes in turn were associated with certain genetic changes relative to the original avian viruses (imai et al., 2012; herfst et al., 2012; linster et al., 2014) . these specific changes occur both in ha and in polymerase-complex proteins. the rationale for these experiments was that, because the ferret model recapitulates many features of human infection, changes identified in adaptation to ferret transmission would also be important for adaptation to sustained transmission in humans schultz-cherry et al., 2014) , though this can never be known with certainty . notably, viruses isolated from humans who were infected by contact with birds show some of these changes (russell et al., 2012; bi et al., 2015) , particularly the change at amino acid 627 of the pb2 gene (jonges et al., 2014; fonville et al., 2013) , which often affects polymerase complex efficiency (see below). this indicates that even the first generation of human infection from nonhuman hosts can initiate a process of host adaptation. it also indicates that not all the human-adaptive changes must be in place in the avian reservoir to initiate this process. some human infections, including some zoonotic cases (de jong et al., 2006; chen et al., , 2006 sha et al., 2016) and some cases early in a pandemic (rogers and d'souza, 1989; connor et al., 1994; glaser et al., 2005; pappas et al., 2010) , involve viruses that are not yet fully human-adapted (see below and tables 2-4). the interpretation of some of these isolates is complicated by uncertainty about whether they were passaged in hen's eggs at some point in their history. certain types of countermeasures against an influenza pandemic are effective only against one lineage of viruses -for example, creating stockpiles or seed stocks of vaccines against a particular subtype, or culling poultry infected with that subtype. it is not currently feasible to invest in such countermeasures against all viruses circulating in avian or other reservoirs, or even against all those causing known zoonotic cases. therefore, there would be value in an accurate system to assess the relative pandemic risks posed by each virus and prioritize them for the development of such strain-specific countermeasures, while directing fewer resources to strains of lower concern (kaplan et al., 2016) . this consideration has motivated calls for comprehensive analysis of all available data to assess the threat to public health posed by these strains. one response is the cdc's influenza risk assessment tool (irat) (trock et al., 2015) , which incorporates elements including properties of the virus, field and epidemiological findings, and attributes of the human population to provide a framework to differentiate among novel influenza viruses thought to possess pandemic potential. such risk assessments can help focus pandemic prevention and response efforts on the viruses thought to pose the highest risk of pandemic spread , in the most worrisome cases providing a rationale for costly measures such as poultry culling or vaccine seed stock development, or even stockpiling of large quantities of vaccine. a guiding question of this article is to examine the degree to which it is justified to rely on measurements and predictions of viral genetic and phenotypic traits in prioritizing responses to particular viral subtypes and within-subtype lineages. there are several hurdles to evaluating the accuracy of such predictions . factors limiting our ability to identify high-risk viruses and predict the risk they pose include: . limited surveillance of nonhuman influenza viruses, such that high-risk viruses may not be detected and hence cannot be assessed (butler, 2012) . limitations include both the number, geographic and species diversity of hosts sampled, and the difficulty in sampling all genetic variants present in a given infection (poon et al., 2016; varble et al., 2014) ; . failure to fully characterize some viruses that are detected (hoye et al., 2010) ; . imperfect public health systems lacking capacity to detect zoonotic infections presenting in patients (sanicas et al., 2014; simonsen et al., 2013) ; . epistasis and other complexities that prevent straightforward prediction of viral traits from genotype kryazhimskiy et al., 2011; gong and bloom, 2014; bloom et al., 2010; wu et al., 2016; raman et al., 2014; tharakaraman et al., 2013; gong et al., 2013) ; . technological limitations in molecular modeling and phenotypic assays that limit confidence in predicting and measuring viral traits (russell, 2014) ; . uncertainties about the taxonomic level at which risk predictions should be made (box 2); . practical, ethical and cost limitations of animal transmission experiments, as well as some exceptions to the correlation between human transmissibility and droplet transmissibility in nonhuman animal models (buhnerkempe et al., 2015) ; . lack of data on immediate animal precursors of viruses that caused previous pandemics; . multiple scales at which viral strains compete and hence experience selection (i.e. replication within hosts, transmission between hosts). evolutionary theory for such multi-scale selection is incomplete. viral fitness components are rarely measured at both scales for the same strain and are imperfectly correlated across scales (gog et al., 2015; park et al., 2013; beauchemin and handel, 2011) ; . the role of stochastic events in the ecology and evolution of influenza viruses during and after host-switching to humans (gog et al., 2015; lloyd-smith et al., 2015) , including the potential for transmission bottlenecks to either promote or inhibit emergence of human-adapted viruses (varble et al., 2014; moncla et al., 2016; wilker et al., 2013; zaraket et al., 2015) . these difficulties are exacerbated by the fact that influenza pandemics are rare events, and that risk assessments are not yet made with enough quantitative precision to formally evaluate their practical application. even perfect information about the viral determinants of pandemic risk might only be enough to distinguish between strains with a low risk of causing a pandemic (say, 0.1% per year) and those with an extremely low risk (say, less than 0.01% per year), with unpredictable ecological or evolutionary contingencies determining which of these low-probability events will actually occur. one such contingency is that an avian influenza virus could acquire one or more of the determinants of pandemic potential by reassorting with a human seasonal influenza virus. with only one pandemic every few decades, the data set for testing the prediction of such rare events is inadequate, a problem that challenges predictions in many fields beyond infectious diseases (king and zeng, 2001; hansson, 2006) . evolutionary events in which a strain increases human-to-human transmissibility, but not enough to spark a pandemic, are extremely hard to observe, but if we could do so it would increase our ability to characterize the process of adaptation (kucharski et al., 2015) . despite these challenges, there has been tremendous interest and investment in making and using such predictions, and a number of new ideas to improve predictions are in various stages of development (box 3). building on the findings of a previous workshop , we considered in detail the present state of knowledge concerning three phenotypic traits: ha receptor binding specificity, and ha ph of activation, and polymerase complex activity, (figure 1 ). these were chosen from a longer list of candidate traits (table 1 ) because they span the viral life cycle ( figure 1 ) and their role in host adaptation has been extensively studied. all three are believed to be required for an influenza virus to cause a pandemic; consistent with this assumption, all three traits have been present to some degree in the earliest viruses isolated in pandemics since the 20th century, though some have been enhanced by subsequent evolution during seasonal transmission in humans. moreover, for each of these three traits, viruses isolated from avian hosts typically do not show the mammalian-adapted phenotype, reflecting divergent selection pressures in the two classes of hosts (tables 2, 3, and 4). all three traits changed in the adaptation of zoonotic h5 influenza viruses to droplet transmission in ferrets (imai et al., 2012; linster et al., 2014) . we emphasize that each of these traits is quantitative, and that human-adaptation is not a threshold criterion but a continuum; in this review when we speak of human adaptation we mean a tendency to be better adapted to humans, rather than an absolute yes-or-no property. this review starts with a summary of our knowledge about the role of each of the three functional traits in conferring pandemic potential on a virus strain. following these case studies, we draw some generalizations about the prospects of predicting pandemic risk from virus genotype or from assays of particular viral traits. for each trait we present a table showing the degree to which the sequence changes or phenotypic properties associated with avian or human adaptation are present in isolates from birds and humans, respectively. if the avian traits were always found in avian isolates and human traits always in human isolates, only the shaded cells on the main diagonal would be filled. in such a case, however, it is hard to see how viruses would ever make the jump from birds to humans, since so many traits would have to change simultaneously, and indeed the off-diagonal cells are not empty. finding avian-adapted traits in viruses isolated from humans most often occurs in zoonotic cases, showing that not all human-adapted traits are required for the first human infection. in some cases there are also box 2. granularity of pandemic risk prediction -for what taxonomic level does it make sense? determining the appropriate taxonomic level for influenza virus risk assessment is a challenging endeavor. influenza virus subtype is a convenient classification but there can be substantial variation in estimable risk within subtype. for example, h5n1 viruses can be roughly segregated into high pathogenicity and low pathogenicity phenotypes with the high pathogenicity variants generating substantially greater concern for both human and animal populations. even within the high pathogenicity h5n1 variants, risk to animal populations and potential for adaptation to humans is likely to vary by phylogenetic lineages or clades of viruses. much of the difficulty for predicting the threat posed by subtypes or coarse grained concepts of virus variants stems from two factors: first, a lack of understanding of how genetic context affects the ability of a virus to adapt for efficient spread in humans; and second, the critical, and geographically variable, role of ecology in determining likelihood of cross species transmission. phylogenetic clade is a practical unit for risk prediction. however, in species where reassortment is frequent, phylogenetic clade must be considered on a gene by gene basis. the definition of phylogenetic clades can be challenging and arbitrary, but recent efforts to develop a unified nomenclature for highly pathogenic h5n1 viruses may offer a transferrable framework for the classification of other viruses (smith and donis, 2015 ; who/oie/fao h5n1 evolution working group, 2008) . clades of viruses circulating in poultry, swine or other domestic animals with extensive human interactions should be prioritized for surveillance and further study. foundational efforts are required to assess the diversity of viruses present in these animal populations, particularly for low pathogenic avian influenza viruses. further study will then be required to assess the abundance and prevalence of different virus subtypes and clades, along with geographic spread and overlap with ecological risk factors (hill et al., 2015; gilbert et al., 2014) , e.g. live animal markets, cohabitation of humans and animals, and biosecurity in animal processing facilities. antigenic characterization of animal influenza viruses should form part of a comprehensive risk assessment, particularly of viruses from swine and possibly dogs. swine influenza virus diversity is driven in large part by introductions of viruses from humans to swine (nelson et al., 2015 lewis et al., 2016) . the substantial antigenic diversity of viruses circulating in swine and antigenic differences with viruses circulating in humans poses an ever increasing risk for re-introduction into humans. much of the antigenic variation in swine has a strong relationship to phylogenetic clade (lewis et al., 2016) . similarly, the high contact rates between humans and dogs, combined with increased circulation of h3n2 canine influenza viruses, may present increasing opportunities for reassortment (na et al., 2015) and for zoonotic infections (flanagan et al., 2012) . doi: 10.7554/elife.18491.003 viruses isolated from humans after a pandemic starts that retain some degree of avian-like traits, and we discuss these in more detail in the text -these represent the greatest challenge to use of genotypic or phenotypic information for pandemic prediction because they run the risk of false negatives. the other off-diagonal cell, which represents avian isolates with some human-like traits, simply shows that some circulation of viruses in birds is possible without the classical 'avian' phenotypes. how this happens is a phenomenon worthy of further study. we conclude with some recommendations for future research and for the practice of pandemic risk assessment. trait 1: hemagglutinin receptor binding specificity attachment of an influenza virus to a host cell requires binding of the viral ha to a sialylated glycan receptor (sialic acid) on the surface of the host cell. cells of the avian gut and a minority of cells in deep lung in mammals predominantly express receptors terminated with an a2,3linked sialic acid: hereafter, a2,3 glycans or avian receptors gambaryan et al., 1997; van riel et al., 2006; shinya et al., 2006) . by contrast, in humans and other mammals, upper respiratory epithelial cells express mainly glycan receptors terminated by a2,6-linked sialic acid: a2,6 glycans or human receptors shinya et al., 2006; chandrasekaran et al., 2008) . the human upper respiratory epithelium is the primary target site for infection of human-adapted viruses, and infection at this site is thought to be a prerequisite for efficient human-to-human transmission via respiratory droplets. thus, it appears that human adaptation of an ha is associated with a switch in its binding preference from avian to human receptors. receptor binding is not either-or; human-adapted influenza virus ha may show some binding to avian receptors, and vice versa. receptor binding preference is defined as the ratio of affinity (or avidity) of an ha molecule for an a2,6 glycan relative to that for an a2,3 glycan, with higher values associated with greater human adaptation. the evolution of receptor binding specificity is driven by the host environment, with selection for specificity during the infection process within a host and during the process of transmission. the error-prone replication of influenza genomes can facilitate rapid emergence of viruses with amino acid substitutions that alter the receptor binding characteristics of the ha (lakdawala et al., 2015) . increased transmissibility may result from mammalian receptor adaptation, either because the virus shedding form the infected donor host is increased, or because the ability of virus to infect the recipient host at a low dose is enhanced, or for both of these reasons. recent experimental evidence in ferrets implicates the soft palate as an important site of selection for a2,6 specificity (lakdawala et al., 2015) . preference for binding human or avian glycan receptors is determined by the structure of the viral ha. except for a few conserved amino acids in the sialic acid receptor binding pocket, the influenza ha has considerable structural plasticity to evolve variation at the rim of the pocket to engage different sialic acid linkages. importantly, antigenic regions of the ha are located nearby regions that determine receptor-binding preference, meaning that selection for antigenic escape may be constrained by the need to maintain receptor preference (koel et al., 2013) . more speculatively, selection for changes in receptor preference might also alter recognition of the ha by host antibodies. conformation of hemagglutinin as a determinant of receptor binding preference. although the co-crystal structures of ha and sialylated glycans have not been solved for all pairs, there is evidence that avian-or human-adapted ha bind to different conformations of the avian and human receptors: the cis conformation of human receptors and the trans conformation of avian receptors ha et al., 2001 ha et al., , 2003 gamblin et al., 2004; liu et al., 2009; xu et al., 2010; yang et al., 2010; lin et al., 2012; yang et al., 2012; zhang et al., 2013) . this finding has led to the concept of "hallmark" residues within the receptor-binding site of avianand human-adapted has. avian-adapted has typically carry glu at position 190, gln at position 226, and gly and position 228 (h3 numbering), and the gln226->leu, gly228->ser substitutions have been associated with a switch to human receptor preference in has of h2, h3 (matrosovich et al., 2000) , and h5 (chutinimitkul et al., 2010) viruses. in h1 ha, glu190fiasp and gly225fiasp have been considered as hallmark amino acid changes to switch receptor specificity leading to greater human box 3. novel approaches to identifying genomic predictors of traits and transmission phenotypes. the advent of inexpensive, large-scale sequencing, combined with improved computing power and novel algorithms to interpret nucleotide and protein sequences, have generated new approaches to characterizing the genotype-trait and genotype-transmission phenotype maps in influenza viruses. some are well-established, while others are under active development. they include: protein structural analysis to identify properties of individual amino acid residues and pairs of residues. a number of approaches have been devised to make use of databases of genome sequences and inferred protein sequences of influenza virus isolates, alone or in combination with metadata on the source (species), date of isolation and passage history of the isolates. characterizing the predictors -at the level of individual amino acid residues within a proteinof variability or conservation can assist in identifying the major selection pressures on that protein. evolutionary analysis of the predictors of high rates of nonsynonymous substitutions within ha showed solvent accessibility and proximity to the sialic acid receptor binding site are the strongest predictors of high nonsynonymous evolutionary rates (meyer and wilke, 2015) . comparisons of residue-specific evolutionary rates in avian and human lineages can help to assess which sites are specifically involved in human adaptation and which may be evolving in avian reservoirs with potential consequences for human adaptation (meyer et al., 2013) . innovative use of metadata associated with sequences deposited in databases will be required to ensure that computational inferences from these databases are reliable. for example, methods that aim to identify sites under positive selection in the ha protein frequently find regions or sites that seem to contradict experimental evidence (meyer and wilke, 2015; tusche et al., 2012; kratsch et al., 2016) . several of these apparent contradictions can be resolved by accounting for viral passaging. for example, passaging in regular mdck cells produces a strong signal of positive adaptation underneath the sialic-acid binding site; this signal is entirely absent in unpassaged virus or virus passaged in siat1 mdck cells (mcwhite et al., 2016) . at the same time, passage bias mutations are assumed to increase fitness of the strain in the respective species and are often necessary to grow in culture at all. therefore, sites associated with isolates passaged in mammalian cultures vs. those passaged in embryonated hen's eggs have the potential to further identify sites associated with mammalian or human adaptation. metadata can also help to point to individual amino acids associated with human adaptation. for example, one proposed computational approach is to find potentially zoonotic human-isolated sequences when the majority of their database hits from preceding years were of animal origin. this serves, on one hand, as systematic survey to derive lists of times and places of likely zoonotic events and, on the other hand, provides close sequence pairs of zoonotic human and their putative animal precursors. in those pairs, common sites that repeatedly changed from the animal to the human zoonotic isolates could be reasoned as being involved in human adaptation. combining these sites with those from passage changes, provides strong evidence for the involvement of a particular site in host adaptation. network analysis of the level of sequence covariation of pairs of residues among protein sequences in the database has led to the identification of groups of mutually covarying sites, which have been used to define features of the ha protein that play a role in determining glycan receptor usage . complementary to such covariation analysis is the analysis of predicted molecular interactions. using x-ray co-crystal structures or modeled structural complexes of ha-glycan receptors, molecular features have been defined as distinct networks of inter-residue interactions involving key residues that make contacts with the different glycan receptor topologies. these features go beyond hallmark residue analyses and more accurately predict how amino acid variations in the receptor binding site impact the inter-residue interactions and glycan receptor binding specificity (raman et al., 2014) . similarly, network analysis of amino acid residues predicted to have significant interactions has shown that antigenic sites on the ha interact with residues controlling glycan receptor binding specificity, and that changes in these antigenic residues can then lead to changes in receptor-binding affinity (soundararajan et al., 2011) . it seems likely that as these different lines of evidence -structural location, biophysical interaction, sequence covariation, sequence evolutionary rates, association with zoonotic or in vitro adaptation, etc.-begin to be understood at the resolution of individual amino acids within an influenza protein, such overlapping approaches will yield clearer understanding of the genetic and structural bases of host adaptation to human infection and transmission. a significant step toward such integration is the recent release of the flusurver online tool which automatizes influenza sequence and structure analysis and highlights mutations that could alter the discussed traits based on extensive literature-derived genotype to phenotype lists, structural visualization of the mutation positions and their geographic and temporal frequency of occurrence and cooccurrence for epidemiological relevance (http://flusurver.bii.a-star.edu.sg and directly from within gisaid http://www.gisaid.org). in particular, the tool has been successful in picking up mutations affecting host receptor binding as well as ph dependency (cotter et al., 2014; maurer-stroh et al., 2010) . however, also in this approach, annotations of the effects of mutations are based on inference from similarity to mutations studied in specific sequence contexts, which in most cases will not be identical to the investigated input sequences. association studies. understanding the genetic basis of adaptive phenotypic change is a central goal in biology, and influenza poses special challenges and advantages relative to other organisms. association studies have begun mapping the genomes of arabidopsis thaliana to over 107 quantitative traits and the genomes of humans to over 100,000 (bergelson and roux, 2010; leslie et al., 2014) . these studies often investigate genetic variation at the scales of single nucleotide polymorphisms, alleles, and loci. motif-based approaches have already proven useful in influenza (e.g., the insertion of multiple basic amino acids indicates highly pathogenic h5 and h7), and such simple, robust correlations simplify the prediction of phenotypic traits. recent investigations of influenza (thyagarajan and bloom, 2014; ashenberg et al., 2013; pinilla et al., 2012) have shown that many mutations have roughly consistent impacts across diverse backgrounds. a complication of all association studies is confounding from genetic linkage and diverse environmental selective pressures. although influenza's genes might be tightly linked over short time scales, the virus evolves quickly, and many traits can be assumed to be under stabilizing selection. thus, association studies that appear statistically impractical now may be feasible with a few more years of expanded surveillance. as reviewed here, however, influenza often breaks simple genetic rules, perhaps due to epistasis (e.g., [bloom et al., 2010] ). high-dimensional genotype-phenotype relationships obscure simple correlations from association studies. a relevant lesson comes from the cancer genome atlas (tcga), which amassed sequences from thousands of diverse tumors to investigate the mutations leading to different cancer types. although metastatic cancers are typically conceptualized as possessing six main phenotypic traits (hanahan and weinberg, 2011) , tcga revealed that the genetic commonality among tumors of any given type is shockingly low (kandoth et al., 2013; ledford, 2015) . human genomes are much larger and more complex than influenza's, however, and so it is possible that an influenza atlas might reveal more patterns, which could inspire hypothesis-driven experiments (weinberg, 2010) . binding of upper-respiratory-tract glycans by the influenza virus hemagglutinin is one of the best-understood ingredients in making a virus capable of efficient human transmission. yet the viral sequence determinants of this trait have been mapped only for a limited number of variants. a systematic screening strategy to scan the genetic "landscape" for sequences with a preference for human glycan receptors might include four components: (1) selection of viral genetic background, (2) large-scale mutagenesis, (3) screening and selection, and (4) confirmatory assays. because both mutations near and far from the sialic-acid-binding site on hemagglutinin have been shown to alter glycan specificity, this should be based on a minimally biased approach to mutagenesis: screening combinations of all possible substitutions at all hemagglutinin residues that are not absolutely conserved across known subtypes. critical considerations include choice of viral genetic background (both subtype and strain identity), extent of combinatorial screening (if conserved sites are omitted, every mutant containing changes at up adaptation (glaser et al., 2005; tumpey et al., 2007) . the determinants of specificity are reviewed in much more detail in (paulson and de vries, 2013) . additional structural features involved in receptor binding preference. the cis and trans definition of glycan conformation does not fully describe ha binding to a range of structurally diverse glycans displayed on human respiratory cells and tissues (chandrasekaran et al., 2008) . this limitation motivated studies that revisited the definition of glycan conformation, extending the conformational analysis beyond the terminal sialic acid linkage to describe overall topology and dynamics of the glycan receptor upon binding to the receptor-binding site of avian and human-adapted has (chandrasekaran et al., 2008; xu et al., 2009) . ha sequence determinants of preference for the "cone"-like topology of avian receptors, versus the "umbrella"-like topology of human receptors, are still being defined (raman et al., 2014). c. experimental assays to measure hemagglutinin receptor binding specificity experimental evidence on differential binding of avian and human viruses to sialic acid receptors in avian and human conformations, respectively, was first obtained by hemagglutination assays with erythrocytes whose surfaces had been chemically modified to display glycans terminating with either homogeneous a2,3 or homogeneous a2,6-linked sialic acids (paulson and rogers, 1987) . subsequent analysis of the repertoire of glycan structures in erythrocytes of various animal species informed the use of cells from different species as probes of ha receptor binding preference in hemagglutination assays (ito et al., 1997) . greater precision and reproducibility has been achieved with the use of purified sialylated glycans to create solid-phase binding assays with fluorogenic or enzymatic detection (gambaryan et al., 2006; gambaryan and matrosovich, 1992) . with these assays, it is possible to characterize the relative direct binding of whole virions or recombinant trimeric ha oligomers to glycans attached to a solid phase or the competition of such glycans with binding to a generic glycoprotein attached to the solid phase (gambaryan and matrosovich, 1992) . in recent work, biosensor interferometry and thermophoresis have been used to measure glycanbinding avidities and affinities in a more precise manner and to relate the two (xiong et al., 2013a) . the development of glycan microarrays represented a turning point in the analysis of influenza virus receptor binding specificity, because it allowed simultaneous evaluation of virion or recombinant ha binding to a large repertoire of sialoglycans blixt et al., 2004; childs et al., 2009) . several measures of preference for an ha molecule or whole virus have been defined, including the ratio of the number of a2,6 to a2,3 glycans bound (stevens et al., 2006a (stevens et al., , 2006b or the corresponding ratio of binding affinity or avidity (imai et al., 2012; xiong et al., 2013a) . a limitation to predictive power is that glycans tested on current arrays may not match those present in the human respiratory tract (walther et al., 2013) . these arrays may also not present glycans in the same fashion as respiratory epithelial cells, so strategies such as measuring the binding of labelled viruses to human respiratory tissues (chutinimitkul et al., 2010) or explant cultures ) may be promising alternatives, although challenges remain in standardization and quantification of such assays. structural studies of wild-type and mutant ha in complex with representative sialoglycans provide the ultimate level of detail by characterizing interactions at the atomic level. x-ray to 4 simultaneous sites could be screened with substantial effort), and design of highly parallel screening, selection, and confirmatory assays. the mutagenesis and screening involved would be extremely large in scope: (before eliminating conserved residues, all 4-site mutnats~[550 residues x 20 amino acids] 4 = 1.4 x 10 16 variants for each subtype tested). however, some computational pre-screening to narrow the set of residues tested combined with contemporary mutagenesis and screening technologies such as deep scanning codon mutagenesis (thyagarajan and bloom, 2014; bloom, 2015; fowler and fields, 2014 ) make such an endeavor feasible. doi: 10.7554/elife.18491.004 crystallography advances in recent years have accelerated structural determination, and similar progress in recombinant protein purification techniques combined with robotic crystal screening have reduced the amount of protein and labor required. in summary, genetic and protein sequence analysis, glycan arrays, and x-ray crystallography studies provide complementary data towards understanding the sialoglycan interactions of emerging viruses, with tradeoffs of equipment and reagent costs and throughput against level of precision and detail provided. at present, estimating the contribution of receptor specificity to the pandemic risk posed by a novel virus relies primarily on the similarity between the receptor binding characteristics of the emerging virus and that of the most closely related ha with known transmissibility among humans or a surrogate animal model. as noted above, hallmark residues have substantial predictive power. these distinct sets of hallmark residues in the h1, h2 and h3 subtype (paulson and de vries, 2013) correlate with human-adaptation in known sequences collected from birds or humans (connor et al., 1994; paulson and de vries, 2013) ; they induce changes in receptor-binding specificity when introduced experimentally (chen et al., 2012; leigh et al., 1995) ; and experimental selection for receptor binding in vitro (chen et al., 2012) or in ferrets (imai et al., 2012) cause these changes to appear. however, hallmark residue predictions of receptor-binding specificity are imperfect, as evidenced by a failure to switch in vitro receptor-binding preference from avian to human when changes observed in h5n1 strains after selection in ferret gain-of-function experiments were introduced to other h5n1 viruses (tharakaraman et al., 2013) . the involvement of other features in human adaptation, such as the topology of the bound ha-receptor complex, further complicate the genetic prediction of human adaptation, as the residues involved in these features are less well characterized (shriver et al., 2009) . in principle, phenotypic assays that directly measure the receptor-binding preference of ha -if performed under realistic conditions that capture the interaction of the ha trimer with the receptor (gambaryan et al., 1997; takemoto et al., 1996; collins and paulson, 2004 )-may better capture the trait of interest than genetic predictions of this preference. however, even here, a simple equivalence between binding preference for a2,6-linked glycans and pandemic risk could be misleading. several viruses circulating in humans during the early phase of previous pandemics were found to show either a preference for avian receptors (rogers and d'souza, 1989; connor et al., 1994) or a mixed preference for both human and avian receptors (rogers and d'souza, 1989; glaser et al., 2005; childs et al., 2009 ). in the case of early 2009 pandemic viruses, findings are mixed (childs et al., 2009; chen et al., 2011) . some of the findings of dual or avian specificity may reflect artifacts introduced when human isolates were passaged in eggs before receptor specificity was assayed; alternatively, they may genuinely reflect a transitional stage in the evolution of ha genes in human populations after transmission from other species (connor et al., 1994; glaser et al., 2005; stevens et al., 2010) , ( table 2) . consistent with this latter possibility, an h5n1 virus isolated from a human zoonotic case in vietnam displayed strong avian receptor preference . this preference changed in the course of experiments to adapt it to respiratory droplet transmission in ferrets (imai et al., 2012) . taken together, these findings confirm that there is a strong correlation between measured receptor preference and the host from which a virus is isolated. however, they raise questions about the predictive value of human receptor binding preference. indeed, the examples of mixed receptor preference in human isolates from the early phase of the h1 or h2 pandemics suggest that the ability to evolve human receptor specificity over a chain of human infections, which may be present in many avian-receptor-adapted viruses, may be sufficient for pandemic emergence. in summary, detection of a human receptor preference in a spillover virus may be an indication of increased risk, but exclusive human receptor preference is probably not necessary for an influenza a virus to initiate a pandemic. with several possible exceptions noted above, most viruses isolated to date fall within the shaded cells in table 2 , which indicates concordance between the source of the isolate and the virus trait. thus, prioritizing pandemic countermeasures against virus lineages with inferred or measured human receptor preference will likely lead to better targeting of such figure 1 . key phenotypic traits for the adaptation of avian influenza viruses to replicate efficiently in humans. (a) a switch in receptor binding preference from avian-like (a2,3-linked sialic acid) to human-like (a2,6-linked sialic acid) receptors. the human form on the left shows the typical distribution of human adapted influenza viruses determined by their receptor binding preference for a2,6, linked sa that is predominantly expressed in the upper respiratory tract but also in the lungs. the human form on the right shows that infection with avian influenza viruses is concentrated in the lungs where their preferred a2,3 linked sa receptor is expressed. (b) lower ha ph of activation and increased polymerase complex efficiency. free-floating viruses that enter the human respiratory tract (upper part of figure) encounter mucus and a mildly acidic extracellular environment that act as innate barriers to virus infection. if na is able to desialylate decoy receptors on mucus and ha has a sufficiently low ph of activation, then the virus particle may reach the apical surface of the respiratory epithelium intact. there through a multiplicity of interactions between ha and cell-surface sialic acid, the virus enters the target cell. after the virus is internalized, it passes through the endosomal pathway where the ph is progressively decreased. the low ph of the endosomal environment triggers an irreversible conformational change in ha that fuses the viral and endosomal membranes and ultimately results in the release of virus genetic material in the form of the viral ribonucleoprotein complex (vrnp) into the cell cytoplasm. the eight vrnps are subsequently imported into the cell nucleus by interactions between the vrnps and cellular nuclear import machinery. inside the nucleus the virus polymerase complex replicates the virus genome in conjunction with co-opted cell proteins. doi: 10.7554/elife.18491.005 table 1 . influenza virus adaptations that appear to be required for human-to-human transmission. preference for a2,6-linked mammalian sialic acid receptors over a2,3-linked avian ones ha ph of activation ha avoids extracellular inactivation and undergoes conformational changes leading to membrane fusion at appropriate ph for human cells (5.0-5.4 or perhaps 5.5) (russell, 2014) polymerase complex efficiency efficient replication in human cells (cauldwell et al., 2014; naffakh et al., 2008) virus morphology filamentous morphology associated with several adaptations to mammals (seladi-schulman et al., 2014; seladi-schulman et al., 2013; campbell et al., 2014; beale et al., 2014) length of na stalk longer stalk of na required to penetrate human mucus and deaggregate virions (blumenkrantz et al., 2013) antagonism of interferon production species-specific binding of the ns1 protein to host factors (rajsbaum et al., 2012) ha-na "balance" substrate selectivity and catalytic rate of na are calibrated to "balance" avidity of ha for the cell-surface glycan receptor (zanin et al., 2015; baum and paulson, 1991; yen et al., 2011; handel et al., 2014) doi: 10.7554/elife.18491.006 glaser et al., 2005) ; most human h2 and h3 seasonal isolates (connor et al., 1994; matrosovich et al., 2000) *these anomalous results are speculated by the authors to be possibly, or even probably the result of laboratory adaptation to egg passage and may not reflect the properties of the primary isolate. a possible counter to this interpretation is that it is seen only in the earliest isolates from human pandemic viruses, while nearly all isolates from after the pandemic year, which should also have been passaged in eggs, show human-adapted phenotypes. avian h1-h4, h11 isolates (galloway et al., 2013; russier et al., 2016; dubois et al., 2011; reed et al., 2010) avian h5, h8, h9,h10,h14,h15 isolates (galloway et al., 2013) found in human isolates h5n1 (imai et al., 2012; linster et al., 2014) and h7n9 (schrauwen et al., 2016) human zoonotic isolates with ph 5.6. one human h1n1 (2008) * the role of amino acids 590 and 591 in adaptation was not recognized until after the 2009 strain had already emerged (mehle and doudna, 2009) ; it has the residues associated with avian adaptation at sites 627 and 701 that were known at that time (herfst et al., 2010) . ** complete sequence information not given in the paper *** the rarity of these raises questions about possible sequencing errors. doi: 10.7554/elife.18491.009 greater acid stability (lower ph of activation) is associated with greater human adaptation. the ha is synthesized and folded such that the fusion peptide is buried and inactive until specific activation signals are provided. the structural changes that expose the fusion peptide and lead to fusion have been described in detail (skehel and wiley, 2000) . if the virion is exposed to sufficiently low ph outside of a host or host cell, the ha protein undergoes irreversible structural changes too early and is unable to mediate virus entry; such virions become inactivated. thus the term acid stability is more broadly used to define the threshold for acidification that triggers membrane fusion (in the endosome) or inactivation (if triggered outside of the cell for an ha that is not sufficiently stable). during endocytosis, an influenza virion is exposed to sequentially lower ph values in early endosomes (ph 6.0-6.5), late endosomes (ph 5.0-5.5), and lysosomes (ph 4.6-5.0) (mellman et al., 1986) . if the ha is too stable, and fusion is not triggered in the acidic endosome of the host cell, further traffic into lysosomes results in virus inactivation by lysosomal proteases (skehel and wiley, 2000) . based on surveillance studies, human-transmissible influenza isolates appear to have ha proteins that are more acid stable (have a lower activation ph) than avian influenza viruses (russell, 2014). the ha activation ph values for h1n1, h2n2, and h3n2 seasonal viruses during box 4. ferret model: validity and limitations in pandemic risk assessment. the use of small mammalian models in influenza virus pathogenesis and transmission has proven invaluable for the study of these complex, polygenic traits. the ferret model is particularly valuable, as ferrets are highly susceptible to most influenza a viruses without the need for prior host adaptation. however, even this gold-standard model is not a true substitute for humans. below, we summarize the benefits, drawbacks, and alternatives to the ferret model for the study of influenza. validity. influenza is a respiratory pathogen in humans, and employing mammalian models that possess comparable lung physiology permits a greater extrapolation of results from the laboratory. importantly, the linkage types and distribution of sialic acids throughout the ferret respiratory tract are generally comparable to humans (jayaraman et al., 2012; jia et al., 2014) : like humans, ferrets express the sialic acid n-acetylneuraminic acid ( neu5ac), but not the sialic acid n-glycolylneuraminic acid ( neu5gc), on respiratory epithelia. as a result, ferrets are uniquely suited for the study of influenza viruses compared with other small mammalian models which express neu5gc (ng et al., 2014) . furthermore, human and avian influenza viruses exhibit comparable binding to upper and lower respiratory tract tissues in ferrets and humans (van riel et al., 2006; shinya et al., 2006) . secondly, ferrets infected with influenza viruses demonstrate numerous clinical signs and symptoms of infection associated with human disease. ferrets infected with human influenza viruses often exhibit transient weight loss, transient fever, and sneezing, whereas infection with selected hpai viruses in this species can lead to pronounced weight loss, sustained fever, lethargy, dyspnea, and neurological complications (belser et al., 2009 ). thus, ferrets represent a preclinical model to assess the ability of novel vaccine and antiviral treatments to mitigate influenza virus. as ferrets are a suitable model for the coincident study of pathogenesis and transmission, this model allows for a greater understanding of virus-host interactions and the interplay between both of these parameters. finally, the ferret model can yield valuable insights about the potential human-to-human transmissibility of influenza viruses -the critical determinant of pandemic risk. a recent meta-analysis showed that estimates of transmissibility derived from ferret respiratory droplet transmission studies could explain 66% of measured variation in human transmissibility, for influenza subtypes that have been detected in humans (buhnerkempe et al., 2015) . furthermore, there is a strong statistical relationship between the attack rates measured in particular ferret experiments and the probability that the influenza strain in question is capable of sustained transmission among humans: if two-thirds or more of contact ferrets become infected via respiratory droplets, then the strain is likely to have pandemic potential (see figure) . however, extrapolation of this relationship to novel strains is inherently risky, and variable outcomes observed for h7n9 influenza transmission in ferrets highlight the potential for false alarms. further analysis of ferret transmission experiments, ideally in concert with molecular and virological research, could raise their sensitivity and specificity for identifying pandemic threats. limitations. there is no 'perfect' small mammalian model for influenza. a longstanding challenge of the ferret model has been limited availability of ferret-specific commercial reagents compared with other models, though recent sequencing of the ferret genome should improve this situation . ethical considerations, and the size and cost of ferrets, necessitate generally small sample sizes in ferret experiments, limiting statistical power (belser et al., 2013) . like other vertebrate models, the ferret is not appropriate for high-throughput screens, so research in the ferret model is most potent when complemented with in vitro and computational approaches. finally, ferrets are not well suited to model the multiple influenza exposures over several years that may be experienced by humans and may mold their immune responses in ways that affect the infection risk with subsequent viruses (andrews et al., 2015) . studies of first influenza infection in ferrets may thus overestimate infection and/or transmission risk relative to that in populations with a history of prior infection with related viruses (belser et al., 2016) . the 20th century range from ph 5.0 to 5.4 (galloway et al., 2013) . in 2009, emerging pandemic h1n1 viruses had ha activation ph values of approximately 5.5, but numerous subsequent isolates have acquired mutations that lower the activation ph to the range of the 20th century human influenza viruses (cotter et al., 2014; maurer-stroh et al., 2010; russier et al., 2016) . broad surveys of avian and swine influenza isolates have shown that ha activation ph can vary substantially with a range from ph 4.6-6.0 (galloway et al., 2013; scholtissek, 1985) . among avian viruses, low-pathogenic duck viruses appear to range in acid stability from ph 5.3-6.0 and highly pathogenic avian viruses range from 5.6-6.0 (galloway et al., 2013) . consistent with observed patterns in natural isolates, some experimental evidence indicates that within the range of natural variation, lower activation ph is adaptive for mammalian replication while higher activation ph is adaptive for replication in avian hosts. for isolates of h5n1 alternatives. the ferret is but one of several well-characterized mammalian models for influenza virus. mice are widely used in the field as they offer a greater availability of commercially available species-specific reagents, permit studies with greater statistical power due to larger sample sizes, and offer the advantage of transgenic animals. however, not all human influenza viruses replicate well in mice without prior adaptation due to a predominance of avian-like receptors in the murine respiratory tract; also mice do not display clinical signs and symptoms of influenza that mimic humans, and are not a reliable model for virus transmission studies. the guinea pig is another model, and offers several comparable advantages to ferrets, including generally similar lung physiology to humans and potential for transmission studies. experiments in guinea pigs are often less expensive than in ferrets, because of lower husbandry costs and reduced drug costs when dosing is based on body weight (lowen and palese, 2007) . however, guinea pigs do not exhibit clinical signs and symptoms of infection similar to humans, and do not exhibit severe disease following infection with hpai or pandemic influenza viruses, limiting their utility for viral pathogenesis studies. box 4-figure 1. ferret respiratory droplet transmission experiments predict the potential for sustained human-to-human transmission of influenza viruses. the solid line shows the weighted logistic regression relationship predicting the probability that a given strain is supercritical (i.e. capable of sustained spread among humans), and the dashed lines show the 95% confidence interval for the prediction. filled circles show the measured secondary attack rates (sar) in ferrets for influenza subtypes that are known to be subcritical (blue) or supercritical (red) in humans. the filled pink area shows the range of sar for which the virus is significantly likely to be supercritical. reprinted from (buhnerkempe et al., 2015) . box 5. role of seroepidemiology in pandemic risk assessment. pandemic threat assessment can also be enhanced by immunological surveys of human populations in geographical area where strains of concern are known to be circulating (van kerkhove et al., 2011) . serological surveys can help to estimate the frequency of spillover infections from non-human to human hosts and also to assess the degree of cross reactivity arising from endemic human strains that share recent genomic ancestors with non-human strains of concern (figure for box 4). attempts have been made to use serological surveys to estimate the rate of spillover infections to humans for recent strains of concern van kerkhove et al., 2012) . sometimes blood samples are obtained from the general population (chao et al., 2011) and other times only high risk individuals are tested. inherent measurement error and cross-reactivity between human and non-human strains make the measurement of low rates of incidence problematic (van kerkhove et al., 2012) . confidence that serologic responses truly reflect zoonotic transmission, rather than cross-reactivity with antibodies generated in response to human influenza infection, may be enhanced by comparison of high-risk persons to those without known exposure to zoonotic sources (huang et al., 2015; gomaa et al., 2015) . although there is evidence of exposure of poultry workers to h5n1 influenza viruses in china, rates are much lower than for other endemic non-human influenza viruses (kim et al., 2011) , such as h9n2 (blair et al., 2013) . more recent studies of exposure of high risk workers to the h7n9 lineage suggest even higher rates of exposure to this new strain than has been observed in similar studies of h5n1 or h9n2 . even when rates of spillover can be estimated accurately, the use of such information in pandemic threat assessment is not obvious. clearly, the first detected presence of human infections for a given strain is of concern because the degree of transmissibility among humans is unknown. should the emergent strain fail to achieve sustained transmission, it is not immediately clear how best to use further information on the frequency of human spillover infections. for example, should we interpret high sustained levels of human spillover as evidence of increased risk because of the number of human infections, or as evidence of decreasing risk because of the number of times the strain has failed to achieve sustained transmission? cross reactivity between non-human and human influenza strains has implications beyond the measurement of spillover infections. often levels of cross reactivity in humans may indicate some degree of reduced population susceptibility (worobey et al., 2014) . all else equal, such evidence of lower population susceptibility should reduce our level of concern about a pandemic threat from a particular virus, because even if it gains efficient human-to-human transmissibility, its effective reproductive number and the proportion of the population at risk will be less than for a virus to which there is no cross-reaction in the population. for example, older individuals are thought to have been far less susceptible to pandemic h1n1 than were younger individuals, because they had previously been exposed to similar strains early in life (yu et al., 2008) . the low average age of infection with a swine variant form of h3n2 (h3n2v) in north america (jhung et al., 2013) is likely driven by reduced susceptibility in adults because of early exposure to similar strains. such immunological overlaps are likely to be a general feature of influenza emergence because human strains frequently emerge into swine populations (nelson et al., 2015) . data on reduced human susceptibility due to cross-reactivity must be synthesized with other data used for threat assessment. in some cases, the aging of the part of the population with prior exposure to a closely related strain could be the most important known factor increasing the risk of an emergence event. mechanistic models could be used to estimate the degree of increased risk of emergence due to the aging of partially immune cohorts. highly pathogenic avian influenza virus, an increase in ha activation ph within the range of 5.2-6.0 has been associated with increased replication and pathogenicity in chickens (dubois et al., 2011) . conversely, a mutation that decreased the ha activation ph of a/ chicken/vietnam/c58/2004 (h5n1) from 5.9 to 5.4 has been shown to attenuate virus growth and prevent transmission in mallard ducks (reed et al., 2009 ) but increase virus growth in the upper respiratory tracts of mice and ferrets (zaraket et al., 2013a (zaraket et al., , 2013b . therefore, for h5n1 viruses, a higher ha activation ph (5.6-6.0) has been associated with a component of fitness in birds, and a lower ha activation ph (ph 5.0-5.4) has been linked to greater replication in the mammalian upper respiratory tract. two h5n1 viruses were adapted to transmit by the airborne route between ferrets (imai et al., 2012; herfst et al., 2012) . after a switch in receptor-binding specificity from avian to human receptors (as described above) and deletion of a glycosylation site, in both studies a final mutation that decreased the ha activation ph was shown to be necessary for airborne transmissibility in ferrets. however, these and other studies have shown that this acid stability change is not sufficient in the absence of human receptor-binding specificity (zaraket et al., 2013a; shelton et al., 2013) . recently, an ha protein whose activation ph was 5.5 or lower was shown to be required for the pandemic potential of 2009 ph1n1 influenza virus . nearly 100 mutations have been described to alter the ha activation ph values of various influenza a virus subtypes (russell, 2014; mair et al., 2014) . these acid stabilizing/destabilizing residues are located throughout the ha1 and ha2 subunits and tend to be positioned in regions of the molecule that undergo large-scale changes in structure during ph-activated protein refolding (russell, 2014; bullough et al., 1994; wilson et al., 1981) . mutations that modify the activation ph do not appear to alter the prefusion ha protein box 5-figure 1. transmission genomics of non-human transmission (top), spillover transmission (middle) and sustained human transmission (bottom). haemagglutinin and neuraminidase gene segments have been color-coded to show an example shared infection history in humans who are current spillover hosts for h7n9 and h9n2. these shared evolutionary histories make it challenging to interpret serological studies of human spillover infections. humans infected by h2n2 or h3n2 will likely have cross-reactive antibodies to h9n2, because of the similarity between the neuraminidase in those viruses. because incidence of spill-over infection is likely to be low, even low-levels of cross-reactivity can make the interpretation of serological studies of the general population challenging. doi: 10.7554/elife.18491.014 doi: 10.7554/elife.18491.013 backbone in x-ray crystal structures (dubois et al., 2011; weis et al., 1990; de vries et al., 2014) . therefore, an experimental determination or modeling of intermediate structures may be required in order to reliably predict ha ph of activation. further complicating genetic prediction of ha activation ph values are observations that the na and m proteins can also modulate ha acid stability in some cases (huang et al., 1980; su et al., 2009; reed et al., 2010; o'donnell et al., 2014) . a variety of experimental techniques have been developed to measure the activation ph of the ha protein, quantified as the highest ph at which the ha protein is activated to undergo the irreversible structural changes that mediate membrane fusion (hamilton et al., 2012) , or alternatively the highest ph at which, in the absence of a membrane with which to fuse, the ha protein is inactivated (inactivation ph). classical membrane fusion assays have measured the property in bulk (hoekstra et al., 1984) . the ph of inactivation can be measured using aliquots of virions that are exposed to buffers of progressively lower ph and, after restoration to neutral ph, assayed for retention or loss of infectivity (scholtissek, 1985) . in many classical fusion assays, fluorescent probes are used to label virions, ha-expressing cells, and/or target liposomes or cells. in these in vitro assays, habound target cells are typically exposed to buffers of various ph values and then lipid and/or contents mixing are measured by fluorescence (loyter et al., 1988; hoekstra and klappe, 1993) . alternatively, cell monolayers expressing cleaved ha proteins can be pulsed by low-ph buffers and then incubated to readout hamediated cell-to-cell fusion either microscopically by syncytia formation or by reporter gene expression. if ha conformation-specific monoclonal antibodies are available for the subtype being studied, ha-expressing cells can be pulsed with low ph and then analyzed for conformational changes by flow cytometry (reed et al., 2009 ). if such antibodies are lacking, ha-expressing cells can be assayed for trypsin susceptibility after low-ph exposure, with prefusion ha being resistant and postfusion ha susceptible to trypsin degradation (steinhauer et al., 1996) . recently, methods have been developed to study ha activation and membrane fusion by individual virions, including single virion fusion using total internal reflection fluorescence microscopy (hamilton et al., 2012) . although the biological trigger for ha's conformational change is a drop in ph, ha refolding can also be triggered by other destabilizing agents such as heat and urea (scholtissek, 1985; ruigrok et al., 1986; carr et al., 1997) . stability at a lower ph is associated with stability at higher temperatures and higher urea concentrations, permitting the use of these agents instead of, or in addition to, ph in assays of stability. thermal stability has been determined by measuring the threshold temperature at which denatured ha protein loses its ability to bind erythrocytes and cause hemagglutination (linster et al., 2014) . many questions remain regarding whether ha activation ph plays a similar role in all influenza subtypes isolated from a wide variety of avian species. for early isolates of the h1n1pdm lineage in 2009, the ha protein has an activation ph of 5.5, which appears intermediate between the canonical human (lower) and avian (higher) ranges. subsequent h1n1pdm isolates have ha activation ph values ranging from 5.2-5.4, suggesting ph 5.5 may be the upper limit for human pandemic potential and a lower value may be preferred. indeed, a destabilizing ha mutation in the background of h1n1pdm results in a lossof-function of airborne transmissibility in ferrets and has been reported to be followed by regain-of-function by mutations that lower the ha activation ph to 5.3, a value representative of human-adapted h1n1pdm viruses . for the moment, it appears that while ha ph of activation that is shown experimentally to be suitable for human infection is highly typical of isolates from human pandemic and seasonal influenza ( table 3 , bottom right) (galloway et al., 2013) , it is possible for humans to have symptomatic infection with (though not extensively transmit) viruses with activation ph closer to the range associated with terrestrial birds ( table 3 , bottom left). conversely ( table 3 , top right), there are avian h9, h10, h14, and h15 isolates that display activation ph typical of human viruses (galloway et al., 2013) . the existence of these human-like avian viruses is perhaps unsurprising, as they may lack other essential adaptations for human transmission. as in the case of receptor binding, reliance on this trait to prioritize pandemic prevention measures should consider this property in conjunction with other properties associated with pandemic potential and will likely enrich the coverage of truly high-risk strains on average. systematic assessment of the predictive value of ha activation ph will require broad empirical testing, since nearly 100 residues throughout the ha molecule have been implicated in regulating ha ph of activation. predicting activation ph from sequence will therefore require more extensive data. to address this issue, sequencing studies combined with phenotypic assays could be performed on a large range of ha variants to determine the effects of ph-altering mutations in different ha subtypes. high-resolution determination of ha structural intermediates may assist in developing molecular modeling approaches to calculate ha stability from sequence. in the interim, there is a pressing need to develop high-throughput assays for ha ph of activation, along with other properties believed important to interspecies adaptation, in the thousands of surveillance samples obtained annually. the heterotrimer of influenza polymerase subunits (pa, pb1, pb2 gene products, together forming the rna-dependent rna polymerase) and the nucleoprotein (np gene product) is required to transcribe and replicate the viral genome (huang et al., 1990) . the polymerase genes of viruses isolated from avian hosts show a number of genetic differences from their counterparts in viruses isolated from humans (chen et al., 2006) , and avian virus polymerase typically performs inefficiently in replicating the viral genome in human cells (cauldwell et al., 2014; naffakh et al., 2008) . adaptation to efficient human-to-human transmission requires efficient activity of this complex of proteins, which we refer to as the polymerase complex, in human cells (cauldwell et al., 2014; naffakh et al., 2008) . some mutations in pb2 are consistently associated with efficient function of the polymerase complex in mammalian cells ( figure 2 ). as long ago as 1977, it was shown that an avian influenza virus could achieve efficient replication in mammalian cells by acquiring mutations solely in the pb2 subunit of the viral polymerase (spooner and barry, 1977) . the most famous of these mutations was later described as pb2 residue 627 (subbarao et al., 1993) , which is a glutamic acid (glu) in avian influenza viruses but a lysine (lys) in human-adapted viruses, including those that emerged in the pandemics of 1918, 1957 and 1968 , and their seasonal descendants. an important exception is the virus that sparked the pandemic of 2009. in this virus, the pb2 segment had been introduced from an avian precursor into swine viruses in the 1990s, and mammalian adaptation had been achieved by a different set of pb2 mutations including changes at residues at 271, 590 and 591 (mehle and doudna, 2009) . now that the 3-dimensional structure of the viral polymerase has been elucidated, we can see that residue 627, 271, 590 and 591 lie on the same external surface. mammalian-adapting mutations increase the positive charge of this domain, suggesting that they either adapt the virus for interaction with an enhancing host factor or enhance its ability to repel a restriction factor (mehle and doudna, 2009) . recently a host factor, anp32a, that differs between mammals and flighted birds was shown to be a cofactor of the influenza polymerase, and the species specific difference could explain the inefficient function of avian virus polymerase and the stringent selection for the 627glu->lys adaptive mutation in mammals (long et al., 2016) . another residue implicated in mammalian adaptation of the polymerase is residue 701 of pb2, which lies close to but is distinct from the 627 cluster. it has been suggested that this mutation and others in this domain at residues 702 and 714 affect the interaction between pb2 and importin-alpha isoforms either in a way that enhances nuclear import of newly synthesized pb2 or that affects polymerase function once inside the nucleus, the site of viral rna replication (resa-infante et al., 2008; gabriel et al., 2005; sediri et al., 2015) . other mutations have been described that adapt pb2 for the mammalian nucleus (for example the triplet threonines at positions 147, 339 and 588) but whether they affect interaction with anp32a, importins or as yet unidentified host factors is not yet elucidated. the adaptive value of these mutations is shown by experimental or observational data in which a mammalian host is infected with a virus whose pb2 is not adapted for efficient mammalian replication, but such a mutation becomes common in the virus population over the course of infection. such evolution has been observed in a fatal human case of influenza a/h7n7 (jonges et al., 2014) and in mouse experiments following serial lung passage using an isolate from this outbreak . lys at position 627 has also been associated with greater severity in zoonotic h7n9 (sha et al., 2016) and h5n1 (de jong et al., 2006) cases however, reverse genetics experiments show that certain strains of avian influenza may be less able to accept these mutations than others (long et al., 2013) . polymerase complex efficiency in human cells can be measured by an in situ assay in which the influenza polymerase is reconstituted from cloned cdnas in plasmids and then coexpressed with "minigenome," a viral-like rna encoding a reporter, such as luciferase. by measuring the rate of reporter accumulation in the transfected human cell line, specific combinations of rna sequences for the polymerase-complex viral genes can thereby be screened directly for their efficiency in producing the mrna encoding the reporter gene product, providing a measure of human adaptation of the polymerase complex (moncorgé et al., 2010) . the original form of the in situ reconstituted polymerase assay requires expression of just the minimal set of four viral proteins to replicate the minigenome rna: pb1, pb2, pa and np. however, recent work showed an important additional role for another protein, the nuclear export protein (nep), which is translated from a spliced mrna derived from rna segment 8 (that also encodes the major interferon antagonist ns2) (robb et al., 2009) . in human h5n1 isolates that do not contain pb2 host-adapting mutations, the inefficient activity of these avian polymerases in human cells could also be compensated for by certain mutations in nep (mänz et al., 2012) . it appears that nep is an important regulator of the balance between transcription and replication (chua et al., 2013paterson and , and can thus enhance fitness in viruses containing otherwise inefficient polymerases. the mechanism of this is as follows: the polymerase-enhancing domain of nep is masked when nep is folded in one conformation. however, mutations that increase the ability of nep to rescue avian polymerase function allow more ready unfolding of the protein, unmasking the "activating" domain at the lower temperature of the mammalian respiratory tract. interestingly, nep overexpression in cells in which human-adapted polymerase is reconstituted is inhibitory because excess complementary rna accumulates at the expense of messenger rna and further viral rna replication (robb and fodor, 2012) . thus although a short-term adaptation of avian virus polymerase to mammalian cells can be achieved in this way, it may be that further compensatory changes rebalance nep function in the face of polymerase adaptation during continued circulation in humans, although direct evidence for this selection is lacking. indeed, although the rescue of low polymerase activity by nep may explain the human infections by h5n1 viruses that lack other polymerase adaptations, it is not clear that such rescue is sufficient to create a level of transmissibility consistent with pandemic spread. nonetheless, this finding shows that the minimal polymerase assay is not always sufficient to predict viruses that have functionally adapted polymerase activity to human cells and that a role for other viral proteins including at least nep should also be considered in assessment of polymerase function. alternatively, polymerase activity could be measured in the context of viral infections (although this will require proper containment). this could be achieved by measuring intracellular levels of viral transcripts using transcriptomics or qrt-pcr. such experiments would provide important information if they are performed using appropriate cell lines (or respiratory explants) at the temperature of the human airway (33˚c). it has been suggested that plaque size at 33˚c can be used as a surrogate measure of polymerase function but plaque size is a mutligenic trait. the predictive value of such assays for transmissibility is limited. ultimately, it would be valuable to develop a simple screen to assess the ability of a viral polymerase to support replication and transmission in humans. this phenotype is influenced by at least 4 different viral genes and involves interactions with several different human host factors. if all the relevant host factors were enumerated, one could imagine quickly converting sequence information into an assay that tested for interactions that should support activity. along these lines the recent description of a host factor, anp32a that differs between flighted birds and mammals and explains the poor activity of avian polymerase in mammalian cells is a step forward (long et al., 2016) . the inefficient polymerase of avian influenza viruses in mammalian cells is one of the hostrange barriers that likely diminishes pandemic risk. unlike the requirement for adaptive mutation in the novel ha, this polymerase barrier can be rather readily overcome by reassortment in which an avian virus with novel antigenicity can acquire one or more polymerase genes from mammalian-adapted viruses. in addition, adaptation of avian virus polymerase by accumulation of adaptive mutations in either the polymerase genes or possibly in other viral genes such as the ns segment encoding nep can enhance avian virus polymerase function sufficiently to support a host range jump. many h5n1 viruses that circulate today in the avian reservoir already have mutations in pb2 at 627 (long et al., 2013) or 701 (de jong et al., 2006) , likely resulting from the reintroduction of mammalian-adapted strains back into the wild bird reservoir. these have been associated in human infections with more severe cases (de jong et al., 2006) . the fact that these strains have not achieved sustained human-tohuman transmission demonstrates that while polymerase adaptations to humans are likely necessary, they are not sufficient for a strain to spark a pandemic. moreover, the absence of the signature pb2 627k mutation in the 2009 h1n1 pandemic strain demonstrates the limitations of relying on any single mutation for risk prediction (herfst et al., 2010) ; viruses with the avian-like residue have also been isolated from zoonotic human cases of h5n1, h7n9, and h9n2 infections ( table 4 , bottom left). on the other hand, the concept that adaptation of the polymerase is necessary for sustained human transmission is validated by findings that the 2009 pandemic strain had adapted to replication in human cells by changes other than e627k within the polymerase (mehle and doudna, 2009) . identification of biophysical mechanisms common to mammalian-adaptive mutations may in the future provide the basis for new biological or biophysical assays of polymerase function to inform risk predictions. in summary, no single polymerase mutation appears to be predictive of pandemic risk for all viruses, but the concept that the polymerase must adapt to human cells before it can cause extensive human-to-human transmission appears consistent with the four pandemic jumps that have occurred in modern times. there has been tremendous progress in understanding the traits involved in the adaptation of avian influenza viruses for efficient human-tohuman transmission and the genetic and structural basis of each of these traits. while the ability to use virus sequence data to inform risk assessment of pandemic potential is improving, it remains essential to consider these data alongside other experimental and epidemiological data. for example, in 2013 there was a substantial increase in the number of human infections with a/h5n1 viruses in cambodia. the increase in infections was cause for substantial concern by itself. enhancing the level of concern was the finding that some of the viruses collected from infected humans contained previously identified genetic mutations suggestive of human adaptation . these findings led to extensive epidemiological and experimental investigations and then to the decision to produce a candidate vaccine virus from a virus representative of the 2013 cambodian outbreaks. while predictions of virus phenotypes from sequence data can be informative, they are not infallible, for several reasons, notably the large number of sites involved in determining such traits (raman et al., 2014; russell, 2014; cauldwell et al., 2014; mehle and doudna, 2009; mänz et al., 2012; herfst et al., 2010) , the important role of epistasis (dependence of a mutation's effect on the genetic background in which it appears) in determining these traits kryazhimskiy et al., 2011; gong and bloom, 2014; bloom et al., 2010; wu et al., 2016; raman et al., 2014; tharakaraman et al., 2013; gong et al., 2013) , and the consequent imperfections in our ability to map single sequence polymorphisms to a trait value. for example, the hallmark ha amino acid residues 190, 226 and 228 are important to human adaptation, but "human-adapting" mutations at these residues do not always change receptor-binding specificity; it depends on the genetic background. similarly, amino acid residues 627 and 701 of the pb2 protein are often involved in human adaptation, but both carried the "avian-adapted" residue in the 2009 h1n1 pandemic strain. when these changes were introduced individually or together in the laboratory, the resulting polymerase showed greater activity in a minigenome assay, but replication was unchanged or attenuated in vitro, in mice, and in ferrets (herfst et al., 2010; jagger et al., 2010) . after the pandemic strain was identified and its anomalous residues at these sites noted, other sites within pb2 were identified and found to be responsible for human adaptation (mehle and doudna, 2009; yamada et al., 2010) . based on the evidence to date, it seems clear that all three of the traits considered in this review, and possibly others in table 1 , must be simultaneously present at least to some degree for a strain to cause a pandemic. yet with only a few instances of pandemic strains emerging per century, it should not be surprising that a new pandemic strain would violate an apparent rule of human adaptation that applied perfectly to previous pandemic strains, as in the case of the pb2 residues associated with human adaptation in 2009, or as in the case of activation ph of ha in early 2009 isolates , which had a value outside the range previously seen in human influenza viruses. it is unclear whether the list of sites and phenotypic traits associated with human adaptation is nearly complete or will continue to grow as we experience additional pandemics. at least for the traits of receptor binding (childs et al., 2009) (table 2) and acid stability (table 3) , full human adaptation may not be required to initiate a pandemic in a virus that is otherwise well-adapted for humans. thus, whether or not the list of traits required for pandemic is now complete, our understanding of where the threshold lies for being sufficiently humanadapted continues to change. there are three complementary approaches to address these limitations: improving genetic prediction of biological traits, improving assays of these traits, and improving animal models of human transmission; all approaches are currently progressing in parallel simon et al., 2011) . the first approach aims to further refine our understanding of sequence-totrait relationships by continued studies of diverse, naturally or artificially produced mutations and their effects on the traits of interest. such research could use all of the approaches described above and higher-throughput assays that could be developed with improved technology, for example as described in box 3. this will include generating mutations not found in known strains in nature to probe for those that could be involved in human adaptation in the future thyagarajan and bloom, 2014) . the goal would be to identify classes of functionally equivalent substitutions, sufficient individually or in defined combinations to confer a trait of interest when introduced into a defined, avian-adapted genetic background. use of in vitro approaches with noninfectious viruses or viral components, or infectious viruses containing surface proteins to which there was already population-wide immunity would reduce the possible biosafety and biosecurity risks of such studies (lipsitch and inglesby, 2014) . the second approach is to develop and improve the throughput and accuracy of biochemical and cell-biological assays of these traits, so that virus isolates can be characterized phenotypically in a routine manner, reducing reliance on sequence-based predictions. it seems feasible to develop high-throughput versions of many of the assays described in this review for each of the three traits discussed, which could then be routinely run on surveillance isolates to contribute to risk prediction. for none of these three traits is there a single gold standard assay, and different assays may provide different estimates of risk. the third approach is to improve animal models to more precisely study phenotypes that are important for human adaptation, and to clarify whether the notion of 'mammalian adaptation' is in fact a valid category. ferrets are the closest known model for human transmission (see box 4). respiratory-droplet transmission studies in ferrets, and potentially in other animal models, have shown a remarkably strong correlation with human transmissibility of influenza a strains (buhnerkempe et al., 2015) . while these assays are not perfectly predictive, they may be the most reliable way at present to assess the transmission potential of a virus in human populations. here a partial counterexample to their overall strong predictive value is h7n9 avian influenza isolates from human zoonotic cases. these viruses transmit in ferrets, albeit less efficiently than human seasonal strains, yet humanto-human transmission has been extremely rare in the hundreds of human zoonotic cases caused by h7n9 (buhnerkempe et al., 2015) . a challenge is the expense and practical challenge of using large enough numbers of ferrets (buhnerkempe et al., 2015; belser et al., 2013; nishiura et al., 2013) to assess transmissibility; this will remain a technique of limited throughput for the foreseeable future. nonetheless, the value of ferret testing for risk assessment can be enhanced in at least two ways: first, by standardizing the conditions for ferret transmission experiments, so these can be more confidently compared between laboratories; and second, by continuing to combine ferret studies with studies of viral traits and sequence/structural studies to further identify correlates of transmissibility in ferrets. while the biological properties of a virus certainly play a large role in determining the pandemic risk posed by a strain, it is possible that even a virus perfectly adapted for human-tohuman transmission might fail to transmit extensively, due to ecological factors, chance, or both. initiation of a pandemic requires not only a well-adapted virus but ecological opportunity to spill over into humans (perhaps multiple times if the first introduction is not "successful" [mills et al., 2006] ), as well as a human population that is immunologically susceptible and sufficiently connected to establish ongoing transmission (box 5). additional complexity arises from the fact that selection pressures for within-host proliferation and competition may diverge from those needed for efficient transmission (park et al., 2013) . genetic bottlenecks at the time of transmission (poon et al., 2016; varble et al., 2014; zaraket et al., 2015) may further enhance the role of chance, as a highly adaptive variant arising in a host may not get transmitted in a particular event. however, selective bottlenecks, which have been observed in experimental transmission of h5n1 and avianlike h1n1 viruses in ferrets, could lower the barrier to emergence of human-adapted viruses (moncla et al., 2016; wilker et al., 2013) . both ecological and host factors are considered in the cdc's irat (trock et al., 2015) . evolutionary factors also play a role in pandemic risk evaluations. even with excellent surveillance, we may never isolate exactly the virus that is destined to cause a pandemic from an animal reservoir or a zoonotic human case; rather, we may isolate its evolutionary precursor. understanding the potential of a strain to produce pandemic-capable progeny is yet a further scientific challenge. perhaps the most startling finding of the gain-of-transmissibility experiments with h5n1 avian influenza viruses was that so few mutations were required to convert a strain circulating in birds to mammalian transmission. this concern was reinforced by a finding that many of these mutations, including combinations of some of them, were already present in strains isolated during surveillance (russell et al., 2012) . the interpretation of the latter finding, however, is complicated by the problem of epistasis: the effect of these mutations in the genetic background of field strains may or may not be the same as in the strain studied in the laboratory. it seems clear that a pandemic risk assessment informed by genetic sequence data is better than one uninformed by such sequence data, but the thought experiment of considering the 2009 swine-origin virus, had it been seen prior to initiating the pandemic, shows that such efforts may fail to identify the risk posed by strains that subsequently cause a pandemic. according to the knowledge at the time, early human isolates of 2009 (and presumably their swine precursors) would have had an ha intermediate between human and avian adaptation in terms of receptor binding. they had an activation ph outside the range previously seen in human viruses and more typical of avian viruses. moreover, these viruses lacked the amino acid residues then thought to confer human adaptation for the polymerase complex. we must imagine that had this strain been detected in swine surveillance prior to the pandemic emergence, genetic as well as phenotypic considerations would have marked it as low risk, creating a false-negative risk assessment. given that this virus did in fact create a pandemic, it is evident that failure of a nonhuman influenza virus to fully meet the three criteria discussed in this review does not disqualify it from posing a significant risk of a pandemic. whether false positive predictions of high pandemic risk are also possible is more difficult to determine, because even a strain that is truly high risk may fail to cause a pandemic for any number of reasons; thus it is challenging to prove that an assessment of high risk for a particular strain was erroneous. from a decisionmaking perspective, a false positive is perhaps less worrisome than a false negative, as a false positive may motivate expenditure on prevention measures directed at a strain that would not have caused a pandemic, while a false negative may lead to a failure to respond to a strain that would. as we seek to improve our understanding of genetic and phenotypic bases of efficient human transmission of influenza viruses, there are multiple possible approaches. one approach that has received considerable attention recently is to perform gain-of-transmissibility studies in highly pathogenic avian viruses; this has been controversial because of concerns about the unusual biosafety and biosecurity risks entailed in such studies (for contrasting perspectives on these risks, see the exchange in 2014-5 between lipsitch and inglesby, and fouchier inglesby, 2014, 2015; fouchier, 2015] ). there are alternative approaches to ferret gain-of-transmission experiments in highly pathogenic avian influenza viruses, though disagreement remains about the level of evidence such alternatives provide. one alternative is to perform similar experiments starting from avian viruses that are not highly pathogenic in mammals, and/or are related to currently circulating human seasonal viruses, so that immunity would already be present in the population. such experiments can provide the same degree of causal rigor as gain-of-function in highly pathogenic avian viruses with novel surface antigens and can elucidate general principles of mammalian adaptation, but they cannot confirm that the same changes would be observed in other strains that are not used in the experiment. a recent report shows a related way forward: the recreation of the steps of mammalian adaptation using viruses whose ha and na are already circulating in humans (lakdawala et al., 2015) . such loss+gain-of-transmissibility experiments reconstruct the properties of naturally occurring seasonal human strains, from laboratory-generated, avian-adapted (or at least human-deadapted) precursors. reconstructing such seasonal strains should pose a risk similar to that of working with the seasonal strains themselves, less than that of a novel subtype. a 2011 report employed a similar strategy, demonstrating the importance of ha activation ph in mouse adaptation by selection experiments on a live attenuated h5n1 vaccine strain lacking the ns1 gene (krenn et al., 2011) . more recently, a 2009 h1n1 pandemic virus was modified to express a mutation that increased ph of ha activation, then selected in ferrets for droplet transmission, and it was found that a second site mutation partially restored the lower ph of activation of the selected virus . one limitation of de-adaptation strategies is that the acquisition of transmissibility is perhaps most likely to evolve by reversion of the de-adaptation changes. as with all gain-of-function and loss-offunction studies, epistatic effects of other loci in the genetic background of the viruses used in such studies set limits to the generalizability of such experiments. another kind of alternative is simple characterization of ferret transmissibility of naturally occurring highly pathogenic strains without selection for airborne transmission. this approach can provide correlative evidence for the importance of genetic differences but cannot prove the mechanistic role of any particular change. even if strain-based assessment methods were much better, surveillance would be a key rate-limiting step for pandemic risk assessment to direct countermeasure development. if a virus about to cause a pandemic is not found in surveillance it cannot be assessed. the fact that we have yet to identify a pandemic strain in nonhuman hosts or in human spillover cases before the pandemic starts indicates there is much work to be done. although surveillance has expanded since the 2009 pandemic, it has not been designed to optimize the chances of detecting a pandemic strain before it becomes pandemic; indeed, how to do so is not clear at present. some possible considerations would be to maximize the diversity of isolates collected, to preferentially sample strains that are known to cause human infections, and to feed back information from risk assessments to inform choice of sampling. rapid sequencing and phenotypic characterization of strains and dissemination of this information, along with interpretations of the risk profiles implied, is also important to maximizing the value of surveillance. further thought should be given to the possibility of using high-throughput sequencing as a screen for which viruses should be subjected to phenotypic testing, which for the moment is typically more costly, slower, and lower-throughput than sequencing. more deliberate approaches to the design of surveillance systems would also depend on answering the question addressed in box 2: how different must a virus be from previously characterized viruses to merit separate evaluation of its pandemic risk? the uncertainties noted above about the phenotypic characteristics of viruses isolated from previous pandemics (which may have been present in the primary isolate or may have arisen during egg passage in the laboratory) underline the need for careful attention to passage histories of surveillance isolates to avoid altering their genotype and phenotype post-isolation (bush et al., 2000) . expanding and rationalizing surveillance in this way would require overcoming political, logistical and financial constraints that vary between countries and regions. even with all of the foregoing suggestions in place, it may be improbable that we can reliably identify the 'needle in the haystack' that is the next pandemic influenza strain. ultimately, the goal is not risk assessment for its own sake, but preparedness and early response to pandemic threats. in other areas where security is at stake, it has been argued that making and improving predictions should be accompanied by a systematic effort to design responses that will not fail even if the predictions are wrong (danzig, 2011) . in the influenza context, the value of some countermeasures is strongly reliant on our ability to identify truly high-risk prepandemic threats: notably, preparation of seed vaccine stocks for candidate pandemic strains, stockpiling of subtype-specific vaccines, and culling of poultry infected with such strains. other types of countermeasures, ranging from strengthening local public health departments to stockpiling antivirals or ventilators to developing faster processes for vaccine manufacture to universal vaccines that should be effective against any influenza a strain, should provide benefits whether or not we have advance notice of the strain causing the next pandemic. a comprehensive assessment of priorities to prevent or mitigate the next influenza pandemic should consider the balance between improving our risk assessment capacity and developing responses robust to the possibility that we will once again be caught by surprise. we thank jesse bloom, ruben donis, judith fonville, scott hensley, and benjamin tenoever for contributing valuably to the workshop on which this paper is based and ruben donis for significant assistance in improving the manuscript. we thank james hay for the figure for box 5. this work and the workshop from which it originated were supported in part by the research and policy for infectious disease dynamics (rapidd) program of the science and technology directorate, u.s department of homeland security, and the fogarty international center, nih. the findings and conclusions in this report are those of the authors and do not necessarily reflect the views of the centers for disease control and prevention, the department of health and human services or its components, or the nih or its institutes. this paper is dedicated to the memory of ellis mckenzie, cofounder of rapidd and senior scientist at the fogarty international center of nih, who was a valued friend and colleague and who enthusiastically supported the workshop that produced his paper. immune history profoundly affects broadly protective b cell responses to influenza mutational effects on stability are largely conserved during protein evolution the n2 neuraminidase of human influenza virus has acquired a substrate specificity complementary to the hemagglutinin receptor specificity a lc3-interacting motif in the influenza a virus m2 protein is required to subvert autophagy and maintain virion stability a review of mathematical 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influenza a(h5n6) virus. infection molecular basis of the receptor binding specificity switch of the hemagglutinins from both the 1918 and 2009 pandemic influenza a viruses by a d225g substitution competing interests: ml: reports the following financial disclosures for topics unrelated to this manuscript: consulting income from pfizer and affinivax (both donated to charity) and research funding from pfizer and path vaccine solutions. these entities had no role in the preparation of this work or in the decision to submit the work for publication. reviewing editor for elife. car: during the prepartion of this manuscript, caab received funding through a research contract with astrazeneca inc., but for distinct, unrelated research. astrazeneca inc. had no role in the preparation of this work or in the decision to submit the work for publication. the other authors declare that no competing interests exist. key: cord-345848-s84lxe6l authors: everitt, aaron r.; clare, simon; pertel, thomas; john, sinu p.; wash, rachael s.; smith, sarah e.; chin, christopher r.; feeley, eric m.; sims, jennifer s.; adams, david j.; wise, helen m.; kane, leanne; goulding, david a.; digard, paul; anttila, verneri; baillie, j. kenneth; walsh, tim s.; hume, david a.; palotie, aarno; xue, yali; colonna, vincenza; tyler-smith, chris; dunning, jake; gordon, stephen b.; smyth, rosalind l.; openshaw, peter; dougan, gordon; brass, abraham l.; kellam, paul title: ifitm3 restricts the morbidity and mortality associated with influenza date: 2012-03-25 journal: nature doi: 10.1038/nature10921 sha: doc_id: 345848 cord_uid: s84lxe6l the 2009 h1n1 influenza pandemic showed the speed with which a novel respiratory virus can spread and the ability of a generally mild infection to induce severe morbidity and mortality in a subset of the population. recent in vitro studies show that the interferon-inducible transmembrane (ifitm) protein family members potently restrict the replication of multiple pathogenic viruses. both the magnitude and breadth of the ifitm proteins' in vitro effects suggest that they are critical for intrinsic resistance to such viruses, including influenza viruses. using a knockout mouse model, we now test this hypothesis directly and find that ifitm3 is essential for defending the host against influenza a virus in vivo. mice lacking ifitm3 display fulminant viral pneumonia when challenged with a normally low-pathogenicity influenza virus, mirroring the destruction inflicted by the highly pathogenic 1918 'spanish' influenza. similar increased viral replication is seen in vitro, with protection rescued by the re-introduction of ifitm3. to test the role of ifitm3 in human influenza virus infection, we assessed the ifitm3 alleles of individuals hospitalized with seasonal or pandemic influenza h1n1/09 viruses. we find that a statistically significant number of hospitalized subjects show enrichment for a minor ifitm3 allele (snp rs12252-c) that alters a splice acceptor site, and functional assays show the minor cc genotype ifitm3 has reduced influenza virus restriction in vitro. together these data reveal that the action of a single intrinsic immune effector, ifitm3, profoundly alters the course of influenza virus infection in mouse and humans. protection rescued by the re-introduction of ifitm3. to test the role of ifitm3 in human influenza virus infection, we assessed the ifitm3 alleles of individuals hospitalised with seasonal or pandemic influenza h1n1/09 viruses. we find that a statistically significant number of hospitalised subjects show enrichment for a minor ifitm3 allele (snp rs12252-c) that alters a splice acceptor site, and functional assays show the minor cc genotype ifitm3 has reduced influenza virus restriction in vitro. together these data reveal that the action of a single intrinsic immune effector, ifitm3, profoundly alters the course of influenza virus infection in mouse and man. ifitm3 was identified in a functional genomic screen as mediating resistance to influenza a virus, dengue virus and west nile virus infection in vitro 1 . however, the role of the ifitm proteins in anti-viral immunity in vivo is unknown. therefore, we infected mice that are homozygous for a disruptive insertion in exon 1 of the ifitm3 gene that abolishes its expression 8 (ifitm3 −/− ), with a low-pathogenicity (lp) murine-adapted h3n2 influenza a virus (a/x-31). lp strains of influenza do not normally cause extensive viral replication throughout the lungs, or cause the cytokine dysregulation and death typically seen after infection with highly-pathogenic (hp) viral strains 9 , at the doses used (fig. 1a) . however, lp-infected ifitm3 −/− mice became moribund, losing >25% of their original body weight and exhibiting severe signs of clinical illness (rapid breathing, piloerection) 6 days after infection. in comparison, wild-type (wt) litter mates shed <20% of their original body weight, before fully recovering (fig. 1a, b) . there was little difference in virus replication in the lungs during the first 48 hours of infection. however, virus persisted and was not cleared as quickly in ifitm3 −/− mice, whose lungs contained 10-fold higher levels of replicating virus than the wt mice at 6 days post-infection (fig. 1c) . no viral rna was detected in the heart, brain or spleen of infected wt or ifitm3 −/− mice over the course of infection, revealing that systemic viremia was not occurring. full genome sequencing of virus removed from the lungs of wt and ifitm3 −/− mice showed no genetic variation. we demonstrated that ifitm3 protein expression after influenza infection was absent in ifitm3 −/− mice but increased substantially in wt controls (fig. 1b, supp. fig. s1 ). infection of wt and ifitm3 −/− mice with a human isolate of pandemic influenza a h1n1 (ph1n1/09) resulted in the same severe pathogenicity phenotype in the ifitm3 −/− mice (fig. 1a, b) . mouse embryonic fibroblast (mef) lines generated from multiple matched littermates demonstrated that ifitm3 −/− cells are infected more readily in vitro, and lack much of the protective effects of interferon (ifn). importantly, the stable restoration of ifitm3 conferred wt levels of restriction against either x-31, or the more pathogenic puerto rico/8/34 (pr/8) influenza strain (fig. 1d, supp. fig. s2 ). further to ifitm3's role in restriction of hp h5n1 avian influenza 7 , we also show that it limits infection by recent human influenza a isolates and influenza b virus (supp. fig. s3 ). therefore, enhanced pathogenesis to diverse influenza viruses is attributable to loss of ifitm3 expression and consequential changes in immune defence of the lungs. examination of lung pathology showed fulminant viral pneumonia with substantial damage and severe inflammation in the infected ifitm3 −/− mice. lung pathology was characterised by extensive oedema and red blood cell extravasation, as well as pneumonia, hemorrhagic pleural effusion and multiple, large lesions on all lung lobes (fig. 2a, b, supp. fig. s4 ). we note this pathology is similar to that produced by infection of mice and primates with 1918 h1n1 virus [9] [10] [11] . given the higher viral load in ifitm3 −/− mice and increased replication of influenza a virus in ifitm3 deleted cells in vitro (fig. 1d) , we examined both viral nucleic acid and protein distribution in the lung. influenza virus infection penetrated deeper into the lung tissue in ifitm3 −/− compared to wt mice whose infection was primarily restricted to the bronchioles, with minimal alveolar infection. influenza virus was detected throughout the entire lung in ifitm3 −/− sections, spreading extensively in both bronchioles and alveoli ( fig. 2c) . histopathology showed marked infiltration of cells and debris into the europe pmc funders author manuscripts bronchoalveolar space of ifitm3 −/− mice (fig. 2b, supp. fig. s4b ). the extent and mechanism of cell damage was investigated by tunel assay, showing widespread cellular apoptosis occurring 6 days post-infection in ifitm3 −/− mice, whereas apoptosis in wt lungs was very limited (supp. fig. s4c ). together, the ifitm3 −/− mouse pathology is consistent with infection by hp strains of influenza a virus, where widespread apoptosis occurs by day 6 post-infection, whilst lungs from lp infections were similar to those of wt mice, displaying minimal damage 9, 12, 13 . analysis of cell populations resident in the lung tissue on day 6 post-infection showed that ifitm3 −/− mice displayed significantly reduced proportions of cd4+ (p=0.004) and cd8+ tcells (p=0.02) and natural killer (nk) cells (p=0.0001) but an elevated proportion of neutrophils (p=0.007) (fig. 3a) . despite the extensive cellular infiltration, (supp. fig. s4b , s5a) the absolute numbers of cd4+ t-lymphocytes in the lungs of the ifitm3 −/− mice were also lower and neutrophils increased compared to wt mice (supp. fig. s6 ). the peripheral blood of infected ifitm3−/− mice showed leukopenia (supp. fig. s5c ). blood differential cell counts indicated marked depletion of lymphocytes on day 2 post-infection in the ifitm3 −/− mice (p=0.04) (fig. 3b ) reflecting changes observed previously in hp (but not lp) influenza infections in both humans and animal models 9, 12, 14, 15 .heightened cytokine and chemokine levels are also hallmarks of severe influenza infection; having been observed in both human and animal models 9, 16 . we observed exaggerated pro-inflammatory responses in the lungs of ifitm3 −/− mice, with higher levels of tnfα, il-6, g-csf and mcp-1 showing the most marked changes (fig. 3c , supp. fig. s7 ). this is indicative of the extent of viral spread within the lungs, as tnfα and il-6 are released from cells upon infection 17 . consistent with the immunopathology data above, these changes are comparable in level to those seen with non-h5n1 hp influenza infections 9 . neutrophil chemotaxis, together with elevated proinflammatory cytokine secretion, has previously been reported as one of the primary causes of acute lung injury 18 . to further investigate the extensive pathogenesis observed with lp influenza a virus infection in the absence of ifitm3, we infected both wt and ifitm3 −/− mice with a pr/8 influenza strain deficient for the multi-functional ns1 gene (delns1) 19, 20 . ns1 is the primary influenza virus interferon antagonist, with multiple inhibitory effects on host immune pathways 20, 21 . we found that delns1 virus was attenuated in both wt and ifitm3 −/− mice, whilst the isogenic pr/8 strain expressing ns1 exhibited typical high pathogenicity in all mice tested, lower doses of pr/8 influenza (whilst lethal in both genotypes of mice) caused accelerated weight loss in ifitm3 −/− compared to wt mice (supp. fig. s8 ). as delns1 influenza a virus retains its pathogenicity in ifn-deficient mice 19 , this suggests that ifitm3 −/− mice can mount an adequate ifn-mediated anti-viral response without extensive morbidity. therefore, unchecked lung viral replication and an enhanced inflammatory response accounts for the profoundly deleterious effects of viral infection in ifitm3 −/− mice. the human ifitm3 gene has two exons and is predicted to encode two splice variants that differ by the presence or absence of the first n-terminal 21 amino acids (fig. 4a) . currently, 13 non-synonymous, 13 synonymous, one in-frame stop and one splice site acceptor-altering single nucleotide polymorphisms (snps) have been reported in the translated ifitm3 sequence (supp . table s1 ). using tests sensitive to recent positive selection, we can find evidence for positive selection on the ifitm3 locus in human populations acting over the last tens of thousands of years in africa (fig. 4b, c) . we therefore sequenced 1.8kb of the ifitm3 locus encompassing the exons, intron and untranslated regions from 53 individuals who required admission to hospital as a result of pandemic h1n1/09 or seasonal influenza virus infection in 2009-2010. of these, 86.8% of patients carried majority alleles for all 28 snps in the coding sequence of the gene, but 13.2% possessed known variants. in particular, we discovered over-representation in cases of the synonymous snp rs12252, wherein the majority t allele is substituted for a minority c allele, which alters the first splice acceptor site and may be associated with the ifitm3 splice variant (enst00000526811), which encodes an ifitm3 protein lacking the first 21 amino acids due to the use of an alternative start codon. the allele frequencies for snp rs12252 vary in different human populations (supp. table s2 ). the ancestral (c) allele, reported in chimpanzees, is rare in sub-saharan african and european populations (derived allele frequency (daf) 0.093 and 0.026-0.036 respectively), but more frequent in other populations (supp . table s2 ). snp rs12252 is notable for its high level of differentiation between europeans and east asians, although the fixation index (f st , a measure of population differentiation) does not reach statistical significance. the genotypes associated with rs12252 in caucasians hospitalised following influenza infection differ significantly from ethnically matched europeans in 1000 genomes sequence data and from genotypes imputed against the june 2011 release of the 1000 genomes phased haplotypes from the uk, netherlands and germany (wtccc1: p=0.00006, netherlands: p=0.00001, germany: p=0.00007; fisher's exact test). patients' genotypes also depart from hardy weinberg equilibrium (p=0.003), showing an excess of c alleles in this population (fig. 4d) . principal components analysis of over 100k autosomal snps showed no evidence of hidden population structure differences between wtccc controls and a subset of the hospitalised individuals from this study (supp. fig s9a, b) . to test the functional significance of the ifitm3 rs12252 polymorphism in vitro, we confirmed the genotypes of hapmap lymphoblastoid cell lines (lcls) homozygous for either the majority (tt) or minority (cc) variant ifitm3 alleles (supp. fig. s9c ). we next challenged the lcls with influenza a virus and found that the minority (cc) variant was more susceptible to infection, and this vulnerability correlated with lower levels of ifitm3 protein expression as compared to the majority (tt) variant cells (supp. fig. s10 ). although we did not detect the ifitm3 splice variant protein (enst00000526811) in the cc lcls, we nonetheless investigated the possible significance of its presence by stably expressing the n-terminally truncated (nδ21) and wt proteins to equivalent levels in human a549 lung carcinoma cell lines before infection with influenza a virus (a/wsn/1933 (wsn/33)). we found that cells expressing the nδ21 protein failed to restrict viral replication when compared to wt ifitm3 ( we show here that ifitm3 expression acts as an essential barrier to influenza a virus infection in vivo and in vitro. the fulminant viral pneumonia that occurs in the absence of ifitm3 arises because of uncontrolled virus replication in the lungs, resulting in profound morbidity. in effect, the host's loss of a single immune effector, ifitm3, transforms a mild infection into one with remarkable severity. similarly, the enrichment of the rs12252 callele in those hospitalised with influenza infections, together with the decreased ifitm3 levels and the increased infection of the cc-allele cells in vitro, suggests that ifitm3 also plays a pivotal role in defence against human influenza virus infections. this innate resistance factor is all the more important during encounters with a novel pandemic virus, when the host's acquired immune defences are less effective. indeed, ifitm3-compromised individuals, and in turn populations with a higher percentage of such individuals, may be more vulnerable to the initial establishment and spread of a virus against which they lack adaptive immunity. in light of its ability to curtail the replication of a broad range of pathogenic viruses in vitro, these in vivo results suggest that ifitm3 may also shape the clinical course of additional viral infections in favour of the host, and may have done so over human evolutionary history. background-matched wt (>95% c57bl/6) and ifitm3 −/− mice 8 19 , made as described 23 . their weight was recorded daily and they were monitored for signs of illness. mice exceeding 25% total weight loss were killed in accordance with uk home office guidelines. littermate controls were used in all experiments. lungs from five mice per genotype were collected on days 1, 2, 3, 4 and 6 post-infection, weighed and homogenised in 5% weight / volume (w/v) of leibovitz's l-15 medium (invitrogen) containing antibiotic-antimycotic (invitrogen). samples were quantified for viral load by plaque assay in 10-fold serial dilutions on madin-darby canine kidney (mdck) cell monolayers overlaid with 1% avicell medium 24 . lungs were subjected to two freeze-thaw cycles before titration. virus was also quantified by rt-qpcr, wherein rna was first extracted from lung, heart, brain and spleen using the rneasy mini plus kit (qiagen). purified rna was normalised by mass and quantified with sybr green (qiagen) using the manufacturer's instructions and 0.5μm primers for influenza matrix 1 protein (m1) fw: 5′-tgagtcttctaaccgaggtc-3′, rv: 5′ggtcttgtctttagccattcc-3′ (sigma-aldrich) and mouse β-actin (actb) fw: 5′ctaaggccaaccgtgaaaag-3′, rv: 5′-accagaggcatacagggaca-3′. qpcr was performed on a steponeplus machine (applied biosystems) and analysed with stepone software v2.1 (applied biosystems). lungs were homogenised in 5% w/v of tissue protein extraction reagent (thermo scientific) containing "complete protease inhibitor" (roche). total protein was quantified by bca assay (thermo scientific) and was normalised before loading into wells. proteins were visualised with the following indicated primary antibodies: mouse ifitm2 rabbit polyclonal was purchased from santa cruz biotechnology (cat# sc-66828); anti-fragilis (ifitm3) rabbit polyclonal antibody was from abcam (cat # ab15592). the ifitm3 and nδ21 western blot using the a549 stable cell lines were probed with the anti-ifitm1 antibody from prosci (cat# 5807), which recognises a conserved portion of the ifitm1, 2 and 3 proteins which is still present even in the absence of the first twenty one n-terminal amino acids. the lcl blots (including the a549 cell line lysate controls) were probed with either an antibody which is specific for the n-terminus of ifitm3 (rabbit anti-ifitm3 (nterm aa 8-38) (abgent, #ap1153a)), or with anti-ifitm1 antibody from prosci (cat# 5807), as well as rabbit anti-mx1 (proteintech, #13750-1-ap) and mouse anti-gapdh (clone gapdh-71.1) (sigma, #g8795). for the lcl immunoblots all antibodies were diluted in dpbs (sigma) containing 0.1% tween 20 (sigma) and 5% non-far dried milk (carnation) and incubated overnight at 4°c. all primary antibodies were consequently bound to the corresponding species-appropriate hrp-conjugated secondary antibodies (dako). actin antibody was purchased from either abcam or sigma, mouse monoclonal, cat# a5316. 5-μm sections of paraffin-embedded tissue were stained with hematoxylin and eosin (sigma-aldrich) and were examined and scored twice, once by a pathologist under blinded conditions. the tunel assay for apoptosis was conducted using the tacs xl dab in situ apoptosis detection kit (r&d systems). lung tissue was embedded in glycol methacrylate (gma) to visualise the spread of viral protein, as described previously 25 . briefly, 2-μm sections were blocked with 0.1% sodium azide and 30% hydrogen peroxide followed by a second block of rpmi 1640 (invitrogen) containing 10% fetal calf serum (sigma-aldrich) and 1% bovine serum albumen (invitrogen). viral antigen was stained using m149 polyclonal antibody to influenza a, b (takara) and visualised with a secondary goat anti-rabbit antibody conjugated to ap (dako). sections were counterstained with hematoxylin (sigma-aldrich). murine ifitm1 and ifitm3 protein expression in lung sections from either uninfected mice, or those two days postinfection with a/x-31, were immunostained with either anti-ifitm1 antibody (abcam, cat# ab106265) or anti-fragilis (anti-ifitm3) rabbit polyclonal antisera (abcam, cat# ab15592). sections were also stained for dna with hoechst 33342 (sigma). viral rna was visualised in 5-μm paraffin-embedded sections using the quantigene viewrna kit (affymetrix). briefly, sections were rehydrated and incubated with proteinase k. they were subsequently incubated with a viewrna probe set designed against the negative stranded vrna encoding the np gene of a/x-31 (affymetrix). the signal was amplified before incubation with labelled probes and visualised. single cell suspensions were generated by passing lungs twice through a 100μm filter before lysing red blood cells with rbc lysis buffer (ebioscience) and assessing for cell viability via trypan blue exclusion. cells were characterised by flow cytometry as follows: tlymphocytes cd4 + or cd8 + , t-lymphocytes (activated) cd4 + cd69 + or cd8 + cd69 + , neutrophils cd11b hi cd11c − ly6g + , dendritic cells cd11c + cd11b lo ly6g lo mhc class ii high, macrophages cd11b + cd11c + f4/80 hi , natural killer cells nkp46 + cd4 − cd8 − . all antibodies (supp . table s3) were from bd bioscience, except cd69 and f4/80, which were from abd serotec. samples were run on a facsaria ii (bd bioscience) and visualised using flowjo 7.2.4. data were analysed statistically and graphed using prism 5.0 (graphpad software). mice (n=3 per genotype per day) were bled on days 0, 1, 2, 3, 4 and 6 by tail vein puncture. leukocyte counts were determined by haemocytometer, whilst blood cell differential counts were calculated by counting from duplicate blood smears stained with wright-giemsa stain (sigma-aldrich). at least 100 leukocytes were counted per smear. all blood analyses were conducted in a blinded fashion. data were analysed statistically and graphed using prism 5.0 (graphpad software). lungs were collected and homogenised days 0, 1, 2, 3, 4 and 6 post-infection from four mice of each genotype. g-csf, gm-csf, ifnγ, il-10, il-1α, il-1β, il-2, il-4, il-5, il-6, il-9, ip-10, kc-like, mcp-1, mip-1α,. rantes and tnfα were analysed using a mouse antibody bead kit (millipore) according to the manufacturer's instructions on a luminex flexmap3d. results were analysed and quality control checked using masterplex qt 2010 and masterplex readerfit 2010 (miraibio). data were analysed statistically and graphed using prism 5.0 (graphpad software). adult ifitm3 −/− mice 8 were intercrossed and fibroblasts (mefs) were derived from embryos at day 13.5 of gestation, as described previously 1 . mefs were genotyped by pcr (thermo-start taq dna polymerase, abgene; epsom, uk) on embryo tail genomic dna using primers and the cycle profile described previously 8 we recruited patients with confirmed seasonal influenza a or b virus or pandemic influenza a ph1n1/09 infection who required hospitalisation in england and scotland between november 2009 and february 2011. patients with significant risk factors for severe disease, and patients whose daily activity was limited by co-morbid illness were excluded. 53 patients, 29 male and 24 female, average age 37 (range 2-62) were selected. 46 (88%) had no concurrent comorbidities. the remaining 6 had the following comorbid conditions: hypertension (3 patients), alcohol dependency and cerebrovascular disease (1 patient), bipolar disorder (1 patient) and kyphoscoliosis (1 patient). four patients were pregnant. where assessed, 36 patients had normal body mass (69%), one had a bmi <18.5 and 10 had a bmi between 25 and 39.9 and one a bmi>40. seasonal influenza a h3n2, influenza b and pandemic influenza a ph1n1/09 were confirmed locally by viral pcr or serological tests according to regional protocols. consent was obtained directly from competent patients, and from relatives/friends/welfare attorneys of incapacitated patients. anonymised 9ml edta blood samples were transported at ambient temperature. dna was extracted using a nucleon kit (genprobe) with the bacc3 protocol. dna samples were re-suspended in 1 ml te buffer ph 7.5 (10mm tris-cl ph 7.5, 1mm edta ph 8.0). human ifitm3 sequences were amplified from dna obtained from peripheral blood by nested pcr (genbank accession numbers jq610570 -621). the first round utilised primers fw: 5′-tgagggttatgggagacggggt-3′and rv: 5′-tgctcacggcaggaggcc-3′, followed by an additional round using primers fw: 5′-gctttgggggaacggttgtg-3′and rv: 5′-tgctcacggcaggaggcccga-3′. the 1.8kb ifitm3 band was gel extracted and purified using the qiaquick gel extraction kit (qiagen). ifitm3 was sanger sequenced on an applied biosystems 3730×l dna analyzer (gatc biotech) using a combination of eight sequencing primers (supp . table s4 ). single nucleotide polymorphisms were identified by assembly to the human ifitm3 encoding reference sequence (acc. no.: nc_000011.9) using lasergene (dnastar). homozygotes were called based on high, single base peaks with high phred quality scores, whilst heterozygotes were identified based on low, overlapping peaks of two bases with lower phred quality scores relative to surrounding base calls (supp. fig. s9 ). we identified snp rs12252 in our sequencing and compared the allele and genotype frequencies to allele and genotype frequencies from 1000 genomes sequencing data from different populations (supp . table s3 ). in addition, we used the most recent release of phased 1000 genomes data 27 to impute the region surrounding snp rs12252 to determine allele frequencies in the publicly available genotype dataset of wtccc1 controls (n=2,938) and four previously published datasets genotyped from the netherlands (n=8,892) and germany (n=6,253) 22 . in the imputation, samples genotyped with illumina 550k, 610k and 670k platforms were imputed against the june 2011 release of 1000 genotypes phased haplotypes using the impute software 28 we assessed for population stratification by principal component analysis. genotype data from the wtccc1 1958 birth cohort dataset were obtained from the european genotype archive with permission, reformatted and merged with genotype data from the genisis study to match 113,819 snps present in both cohorts. suspected strand mismatches were removed by identifying snps with more than 2 genotypes and using the ld method as implemented in plink (v1.07) 29 , resulting in 105,362 matched snps. quality control was applied in genabel version 1.6-9 to genotype data for these snps for the genisis cases and 1499 individuals from wtccc. thresholds for quality control (deviation from hardy-weinberg equilibrium (p<0.05), maf<0.0005, call rate<98% in all samples) were applied iteratively to identify all markers and subjects passing all quality control criteria, followed by principal component analysis using genabel. we tested for positive selection using both a haplotype-based test (|xp-ehh-max|) and allele frequency spectrum-based test statistics, namely tajima's d, fay and wu's h and nielsen et al.'s clr on 10 kb windows across the entire genome as described previously 27, 30 . the three statistics were combined and the combined p value was plotted corresponding to the 10 kb windows. author contributions a.r.e., g.d., a.l.b. and p.k. designed the study; a.r.e., p.o., g.d., a.l.b. and p.k. the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 interferon-induced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms distinct patterns of ifitm-mediated restriction of filoviruses, sars coronavirus, and influenza a virus a diverse range of gene products are effectors of the type i interferon antiviral response ifitm3 inhibits influenza a virus infection by preventing cytosolic entry normal germ line establishment in mice carrying a deletion of the ifitm/fragilis gene family cluster pathogenesis of pandemic influenza a (h1n1) and triple-reassortant swine influenza a (h1) viruses in mice characterization of the reconstructed 1918 spanish influenza pandemic virus aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h5n1) virus isolated from humans enhanced virulence of influenza a viruses with the haemagglutinin of the 1918 pandemic virus pathogenesis of emerging avian influenza viruses in mammals and the host innate immune response h5n1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice the pathogenesis of influenza virus infections: the contributions of virus and host factors author manuscript; available in pmc inflammatory responses in influenza a virus infection involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury influenza a virus lacking the ns1 gene replicates in interferon-deficient systems the multifunctional ns1 protein of influenza a viruses the ns1 protein of the 1918 pandemic influenza virus blocks host interferon and lipid metabolism pathways genome-wide association study of migraine implicates a common susceptibility variant on 8q22.1 efficient generation and growth of influenza virus a/pr/8/34 from eight cdna fragments mutational analysis of cis-acting rna signals in segment 7 of influenza a virus virological analysis of fatal influenza cases in the united kingdom during the early wave of influenza in winter 2010/11 a map of human genome variation from population-scale sequencing a flexible and accurate genotype imputation method for the next generation of genome-wide association studies plink: a toolset for whole-genome association and population-based linkage analysis gallego romero i, 1000 genomes project consortium we would like to thank c. brandt for maintaining mouse colony health and wellbeing and t. hussell for provision of a/x-31 virus. we also thank d. gurdasani for statistical analysis of genotype frequencies. we also thank m. hu and i. gallego romero for calculating genome-wide selection statistics. this work was supported by the wellcome trust. the refer to web version on pubmed central for supplementary material.footnotes † correspondence and requests for material should be addressed to p.k (pk5@sanger.ac.uk) and a.l.b (abrass@partners.org). key: cord-003701-i70ztypg authors: chow, eric j.; doyle, joshua d.; uyeki, timothy m. title: influenza virus-related critical illness: prevention, diagnosis, treatment date: 2019-06-12 journal: crit care doi: 10.1186/s13054-019-2491-9 sha: doc_id: 3701 cord_uid: i70ztypg annual seasonal influenza epidemics of variable severity result in significant morbidity and mortality in the united states (u.s.) and worldwide. in temperate climate countries, including the u.s., influenza activity peaks during the winter months. annual influenza vaccination is recommended for all persons in the u.s. aged 6 months and older, and among those at increased risk for influenza-related complications in other parts of the world (e.g. young children, elderly). observational studies have reported effectiveness of influenza vaccination to reduce the risks of severe disease requiring hospitalization, intensive care unit admission, and death. a diagnosis of influenza should be considered in critically ill patients admitted with complications such as exacerbation of underlying chronic comorbidities, community-acquired pneumonia, and respiratory failure during influenza season. molecular tests are recommended for influenza testing of respiratory specimens in hospitalized patients. antigen detection assays are not recommended in critically ill patients because of lower sensitivity; negative results of these tests should not be used to make clinical decisions, and respiratory specimens should be tested for influenza by molecular assays. because critically ill patients with lower respiratory tract disease may have cleared influenza virus in the upper respiratory tract, but have prolonged influenza viral replication in the lower respiratory tract, an endotracheal aspirate (preferentially) or bronchoalveolar lavage fluid specimen (if collected for other diagnostic purposes) should be tested by molecular assay for detection of influenza viruses. observational studies have reported that antiviral treatment of critically ill adult influenza patients with a neuraminidase inhibitor is associated with survival benefit. since earlier initiation of antiviral treatment is associated with the greatest clinical benefit, standard-dose oseltamivir (75 mg twice daily in adults) for enteric administration is recommended as soon as possible as it is well absorbed in critically ill patients. based upon observational data that suggest harms, adjunctive corticosteroid treatment is currently not recommended for children or adults hospitalized with influenza, including critically ill patients, unless clinically indicated for another reason, such as treatment of asthma or copd exacerbation, or septic shock. a number of pharmaceutical agents are in development for treatment of severe influenza. annual seasonal influenza epidemics of variable severity result in significant morbidity and mortality in the united states (u.s.) and worldwide [1] [2] [3] . in temperate climate countries, including the u.s., influenza activity peaks during the winter months whereas in tropical regions influenza activity may be more variable [4] [5] [6] . most persons with symptomatic influenza virus infection have self-limited uncomplicated upper respiratory tract illness. one study estimated that during 2010-2016, approximately 8.3% of the u.s. population experienced symptomatic influenza each year [7] . however, complications may result in severe illness, including fatal outcomes. during 2010-2018, an estimated 4.3-23 million medical visits, 140,000-960,000 hospitalizations, and 12, 000-79,000 deaths were associated with influenza each year in the u.s. [8] . another study estimated that 18, 000-96,000 influenza-related intensive care unit (icu) admissions occur annually in the u.s. [9] . there are an estimated 291,000-646,000 respiratory deaths attributed to seasonal influenza each year worldwide [2] . here, we review strategies for prevention, diagnosis, and treatment of influenza virus infections in the icu (table 1) . influenza vaccination is the primary method for preventing influenza and reducing the risk of severe outcomes. in the u.s., the advisory committee on immunization practices (acip) recommends annual influenza vaccination for all persons aged 6 months and older and prioritizes those at higher risk for influenza complications [10] . high-risk groups include adults aged > 65 years [11, 12] , children aged < 5 years (particularly those aged < 2 years) [13, 14] , pregnant women (up to 2 weeks postpartum) [15] [16] [17] [18] , persons with certain chronic medical conditions, native americans/alaska natives, 1 and residents of nursing homes and other long-term care facilities (table 2) . studies have specifically highlighted that those with chronic pulmonary, cardiovascular, renal, hepatic, neurologic, hematologic or metabolic disorders, immunocompromised persons, children and adolescents receiving aspirin-or salicylate-containing medications and who might be at risk for experiencing reye syndrome with influenza virus infection, and those who are extremely obese (bmi > 40) are at increased risk for influenza-related complications [10, [19] [20] [21] [22] [23] . many studies evaluated risk factors for severe influenza during the 2009 h1n1 influenza pandemic. adult icu patients with influenza a(h1n1)pdm09 virus infection were primarily non-elderly, were obese [24] [25] [26] [27] [28] , and had higher odds of death, invasive mechanical ventilation, acute respiratory distress syndrome (ards), septic shock, and multi-lobar pneumonia when compared with seasonal influenza patients [24, 29] . in children, independent risk factors for influenza a(h1n1)pdm09-related mortality included chronic neurologic condition or immune compromise, acute myocarditis or encephalitis, and early presumed mrsa co-infection of the lung [30] . female gender was also identified as a risk factor; however, there was no gender difference in overall mortality. bacterial coinfection was identified in approximately one third of fatal influenza a(h1n1)pdm09 cases in the largest autopsy case series [31] . bacterial co-infections in the interpandemic period are also common in critically ill influenza patients [32] . one study identified past or current tobacco use as a risk factor associated with icu admission [33] . a recent multicenter cohort study reported that mortality was higher in immunosuppressed patients with influenza a(h1n1)pdm09 than in immunocompetent patients [34] . severity of influenza seasons varies from yearto-year based on the predominant influenza viruses, and between seasonal and pandemic influenza [35, 36] . one study reported that patients with influenza a(h1n1) pdm09 had higher odds of severe disease than patients with either influenza a(h3n2) or influenza b virus infections [37] . however, influenza b virus infection has been shown to increase the odds of in-hospital mortality in children compared with influenza a virus infection [38] . influenza vaccination is recommended each fall for all persons aged > 6 months in the u.s. and should continue • there are an estimated 291,000-646,000 seasonal influenza-associated respiratory deaths every year worldwide. • annual influenza vaccination is the primary method of preventing influenza and influenza-related complications, especially in high-risk persons. • influenza molecular diagnostic testing is recommended for all patients requiring hospitalization with suspected influenza. • influenza antiviral treatment should be started as soon as possible in hospitalized patients with suspected influenza, including critically ill patients, and should not be delayed while awaiting results of influenza diagnostic tests. • enterically administered oseltamivir is recommended for influenza patients except for those with contraindications (e.g., gastric stasis, ileus, malabsorption). • repeat virologic testing in lower respiratory tract specimens may be required to determine therapeutic endpoints in ventilated patients with influenza • corticosteroids are not recommended for the routine treatment of influenza except when indicated for treatment of underlying medical conditions (e.g., copd or asthma exacerbation) or septic shock. [39] , and reducing in-hospital mortality and icu admissions for those aged 18-49 years and > 65 years compared to unvaccinated individuals [40] . one study reported that duration of icu hospitalization was reduced a half-day in patients aged 50-64 years who had received influenza vaccination compared with unvaccinated patients [41] . a study across all age groups in spain reported influenza ve of 58% in reducing the risk of severe influenza requiring hospitalization [42] . a southern hemisphere study reported influenza ve of 82% in reducing influenza-associated icu admissions among adults [43] while a study in spain showed an adjusted influenza ve of 23% in preventing icu admission and death [44] . despite the benefits of influenza vaccination, there continues to be low vaccine coverage among adults admitted to the icu who often have a high prevalence of high-risk comorbidities [45, 46] . in children, low influenza vaccination coverage has also been reported among those admitted to pediatric icus, even among those with underlying high-risk conditions [47] . full influenza vaccination was shown to result in a 74% reduction in pediatric icu admissions compared to unvaccinated or partially vaccinated influenza patients [47] . furthermore, one study showed that influenza ve was 65% in reducing the risk of mortality in children aged 6 months to 17 years in the u.s. [48] . these data further emphasize the benefits of influenza vaccination in reducing severe influenza complications, especially in high-risk persons. persons with uncomplicated influenza typically experience acute onset of respiratory symptoms (cough, rhinorrhea, congestion), myalgias, and headache with or without fever. during influenza season, clinicians should also consider influenza when there is only fever present or in patients who are afebrile and have respiratory symptoms [49] . complications of influenza vary by age, underlying comorbidities or high-risk conditions such as pregnancy, and immune function; elderly and immunocompromised persons may not always manifest fever. critically ill patients may be admitted with respiratory or multi-organ failure, exacerbation of an underlying condition such as chronic lung disease [50, 51] , heart failure [52] , or other extrapulmonary complications including stroke, encephalopathy, or encephalitis [30, 49, 53] . influenza testing is recommended for all patients requiring hospitalization with suspected influenza, including those admitted to the icu during influenza season with acute respiratory illness and community-acquired pneumonia, without a clear alternative diagnosis. furthermore, all individuals requiring critical care outside of influenza season should be tested for influenza if there is a possible epidemiological link to an individual with recent influenza, such as travel to areas with influenza activity or exposure to an institutional influenza outbreak. special consideration should be given to elderly and immunocompromised patients, as influenza virus infection may not present with typical acute respiratory illness signs and symptoms (e.g., absence of fever). the infectious diseases society of america (idsa) 2018 influenza clinical practice guidelines also recommend influenza testing for patients at high risk of complications such as exacerbation of chronic cardiopulmonary disease [49] . diagnosis of influenza should be made as soon as possible in critically ill patients, and initiation of antiviral treatment should not be delayed while awaiting results of diagnostic tests. studies have reported an increase in mortality of icu patients with influenza a(h1n1)pdm09 virus infection when diagnosis was delayed [54] , and shorter hospital length of stay when antiviral treatment was initiated within 6 h of admission [55] . several kinds of influenza diagnostic tests are available in clinical settings with variable sensitivities and specificities, including antigen detection assays, and molecular assays (nucleic acid detection) using respiratory tract specimens (table 3) . within each of these testing categories, there is a wide range of available tests with varying diagnostic accuracy, and understanding the limitations of each diagnostic tool will allow the clinician to properly interpret their results. most studies of influenza diagnostic accuracy have been conducted on specimens from patients with uncomplicated influenza, and few have assessed the performance of influenza tests in critically ill patients. the idsa guidelines recommend molecular influenza assays for testing respiratory specimens from all hospitalized patients with suspected influenza because of their high sensitivity, specificity, and time to results (15 min to several hours) [49] . the use of rapid influenza molecular diagnostic testing can result in better outcomes for patients and reduce the amount of resources required to care for patients in the emergency room [57] . serology and viral culture are not recommended for clinical decision making, because timely results will not be available to inform clinical management. serology requires collection of appropriately paired acute and convalescent sera performed at specialized public health reference laboratories, and results based upon a single serum specimen are not interpretable [49] . although viral culture can confirm the presence of infectious virus with very high sensitivity and specificity, it must be performed at public health laboratories and requires 3-10 days to yield results. a recent meta-analysis reported that influenza antigen detection tests that produce rapid results had very high specificities (> 98%), but sensitivities were highly variable compared with rt-pcr [58] . rapid influenza diagnostic tests (ridts) without an analyzer device had only moderate sensitivity (53-54%), ridts that utilize an analyzer device (digital immunoassays) had moderately high sensitivity (77-80%), and rapid influenza molecular assays (nucleic acid detection) had high sensitivity (92-95%) [58] . low sensitivity of ridts for detecting influenza virus in icu patients has been reported [59] . recently, a systematic analysis of rapid influenza molecular tests from 29 studies reported pooled sensitivity and specificity of 87.9% and 97.4%, respectively [60] . therefore, antigen detection assays, such as rapid influenza diagnostic tests and immunofluorescence assays, are not recommended for hospitalized patients with suspected influenza because of their lower sensitivities, unless molecular assays are not available [49] . negative results for influenza based on tests with low sensitivity (e.g., ridts, immunofluorescence assays) should not be used to make clinical decisions. instead, negative test results should be followed up with reverse transcription polymerase chain reaction (rt-pcr) or other influenza molecular assays to confirm results, and antiviral treatment should continue until results are available. preferred respiratory specimens for influenza testing in hospitalized patients without lower respiratory tract disease include nasopharyngeal, mid-turbinate nasal, or combined nasal-throat swabs. collection of lower respiratory tract specimens should be considered in hospitalized patients with suspected influenza if upper respiratory tract specimens are negative and a positive test would result in a change of clinical management [61] , because viral replication in the lower respiratory tract may be ongoing and prolonged after virus is no longer detectable in the upper respiratory tract [24, 25] . influenza a(h1n1)pdm09 virus in particular has been shown to have affinity for infecting the lower respiratory tract [24, 31] . in hospitalized patients receiving invasive mechanical ventilation in whom influenza is suspected, but not yet diagnosed, influenza testing should be performed on endotracheal aspirate specimens instead of those collected from the upper respiratory tract [61] . molecular testing, including rt-pcr for influenza viruses can also be performed on bronchoalveolar lavage (bal) fluid if collected for the testing of other pathogens. blood, plasma, serum, cerebrospinal fluid, urine, and stool samples have very low diagnostic yield and are not recommended for influenza testing [49] . diagnostic test results on specimens collected from non-respiratory sites should not be used for clinical decision making even for patients with extra-pulmonary complications of influenza. novel influenza a viruses are typically of animal origin, differ antigenically and genetically from currently circulating seasonal influenza a viruses (including h1n1pdm09 and h3n2 subtypes) and have infected at least one person. novel influenza a viruses can cause a wide clinical spectrum of illness, ranging from asymptomatic infection, uncomplicated illness, to fulminant pneumonia, ards, and multi-organ failure [62] and human infection with a novel influenza a virus is of public health concern. in the u.s., human infection with a novel influenza a virus is nationally reportable to the centers for disease control and prevention; globally, treatment of severe influenza presents multiple challenges. the mainstay of therapy for patients with influenza is initiation of antiviral medication as soon as possible after illness onset [49] . currently available fdaapproved antiviral medications include neuraminidase inhibitors (nais) (e.g., oral oseltamivir, inhaled zanamivir, and intravenous peramivir); cap-dependent endonuclease inhibitor (baloxavir marboxil); and adamantanes (e.g., amantadine and rimantadine) ( table 4 ). nais and baloxavir have activity against both influenza a and b viruses. adamantanes only have activity against influenza a viruses and are not recommended for treatment of influenza due to widespread resistance among currently circulating strains of seasonal influenza a viruses. notably, fda-approved antiviral medications for treatment of influenza are approved for early treatment of uncomplicated influenza in outpatients based upon randomized placebo-controlled clinical trials conducted among previously healthy outpatients. meta-analyses of randomized placebo-controlled clinical trials of early oseltamivir treatment of influenza in pediatric and adult outpatients have reported clinical benefit in reducing duration of illness and risk for some complications associated with influenza [65, 66] . no completed randomized, placebo-controlled trials of antiviral treatment have been conducted in hospitalized influenza patients to establish the efficacy of oseltamivir or other nais. a number of observational studies have reported clinical benefit of neuraminidase inhibitors in hospitalized patients, including reduction in duration of hospitalization and risk of death, including in icu patients [67] [68] [69] [70] [71] [72] [73] [74] . additionally, a systematic review of published reviews/meta-analyses reported survival benefit of nai treatment in hospitalized patients [75] , although another meta-analysis of observational studies did not [69] . in particular, a large pooled individual patient-level meta-analysis of observational studies from 38 countries identified a 38% reduction in risk of mortality in critically ill adults and those aged ≥ 16 years old when comparing early nai treatment (< 48 h) with later treatment (> 48 h), and a 69% reduction in mortality risk between influenza patients receiving early nai treatment and those who did not receive nais [72] . the mortality risk reduction of nai treatment at any time versus no treatment was 28% for critically ill patients aged ≥ 16 years old; while a similar reduction in mortality was identified in critically ill children aged < 16 years, the result was not statistically significant [72] and was likely underpowered because death is less common in hospitalized children with influenza than in adults. although studies have shown the greatest clinical benefit when antivirals are started within 2 days of illness onset, some observational studies have shown clinical benefit of neuraminidase inhibitors when started up to 5 days following symptom onset [15, 55, 76, 77] . the large metaanalysis mentioned above also identified a significantly reduced mortality risk reduction (35%) in critically ill patients aged ≥ 16 years old who received nai treatment > 48 h after symptom onset compared with those who did not [72] . a cohort study of early versus late oseltamivir treatment reported a significant reduction in mortality and median duration of icu hospitalization in severely ill patients with influenza a(h3n2), but not a(h1n1pdm09) or b virus infection in greece [78] . one french study reported delays in initiation of oseltamivir treatment prescribed to hospitalized influenza patients and suggested empiric administration of oseltamivir treatment in the emergency department for patients being admitted with lower respiratory tract disease during influenza season [79] . overall, based upon available observational data to date in hospitalized patients with influenza, including icu patients, initiation of neuraminidase inhibitor antiviral treatment is recommended as soon as possible for hospitalized patients with suspected or confirmed influenza. data on optimal dosing and duration of therapy with neuraminidase inhibitors are limited in critically ill influenza patients. enterically administered oseltamivir is the preferred treatment for most hospitalized patients, given the lack of data for intravenous peramivir in this population. the use of inhaled zanamivir is not recommended in in critically ill patients due to the lack of data in hospitalized patients and the risk of bronchospasm in patients with underlying lung disease. studies indicate that oseltamivir administered orally or via oro/naso-gastric tube is well absorbed in critically ill patients and reaches plasma levels comparable to those in ambulatory patients [80] . similarly, several observational studies indicate that enteric oseltamivir reaches comparable plasma concentrations to non-critically ill patients in those receiving extracorporeal membrane oxygenation (ecmo) and renal replacement therapy [80] [81] [82] [83] [84] [85] [86] [87] , although dosing should be reduced in patients with significant renal impairment. there is scant evidence that increased nai dosing (e.g., twice daily dosing) in critically ill patients provides additional clinical benefit than standard dosing [80, [88] [89] [90] [91] [92] . of note, studies also suggest that increased oseltamivir dosing does not provide additional clinical benefit in obese adults, including extreme obesity (bmi > 40) [93, 94] . duration of therapy can be difficult to define, as prolonged influenza viral replication and shedding from the both upper and lower respiratory tract can occur in critically ill patients [95, 96] . for this reason, it may be beneficial to continue antiviral therapy beyond 5 days, and repeat virologic testing may be beneficial in determining appropriate therapeutic endpoints [97] . continuing antiviral treatment in critically ill patients until virus is not detectable in the lower respiratory tract may also help reduce the pro-inflammatory dysregulated cytokine response triggered by influenza virus infection and reduce nosocomial influenza virus transmission to healthcare personnel in the icu. consultation with a specialist with training in infectious diseases for the potential emergence of antiviral resistant virus infection should be considered for icu patients with evidence of persistent influenza viral replication after nai treatment, particularly in severely immunocompromised patients [49, 98] . for patients who cannot tolerate or absorb enteric oseltamivir due to gastric stasis, malabsorption, or other gastrointestinal processes, intravenous peramivir may be an alternative [99, 100] ; however, studies have not identified an advantage for intravenous peramivir in comparison with enteric oseltamivir [101] . notably, a randomized trial conducted in three influenza seasons found similar clinical outcomes between iv peramivir and enteric oseltamivir in hospitalized adult influenza patients [102] ; a separate trial did not identify significant additional clinical benefit of peramivir in combination with standard-of-care therapy (which often included an nai) [103] . a more recent, multicenter randomized controlled trial also found similar clinical benefit between enteric oseltamivir and intravenous peramivir in hospitalized influenza patients [104] . in 2018, a novel antiviral agent, baloxavir marboxil, was fda-approved for early treatment of uncomplicated influenza in outpatients aged ≥ 12 years old. baloxavir acts via inhibition of the influenza virus cap-dependent endonuclease, a different mechanism than the neuraminidase inhibitors, and can treat nai-resistant influenza virus infections. randomized controlled trials of single-dose oral baloxavir showed similar clinical benefit to 5 days of twice-daily oral oseltamivir [105] . however, because these studies were limited to patients with uncomplicated influenza, the role of baloxavir monotherapy or in combination with an nai for treatment of hospitalized influenza patients is unclear. specifically, optimal dosing, duration of therapy, and appropriate endpoints have yet to be determined for baloxavir treatment of hospitalized influenza patients. in the outpatient rct, patients treated with single-dose baloxavir showed significant reduction in influenza viral levels in the upper respiratory tract at 24 h compared with those receiving placebo or oral oseltamivir [105] . however, it is unknown whether this reduction in influenza viral shedding correlates with reduced transmissibility. a potential concern for the use of baloxavir in critically ill patients is the rapid development of resistance observed during the outpatient clinical trials [106] . a trial to assess the efficacy and safety of baloxavir in combination with oseltamivir versus oseltamivir monotherapy in hospitalized influenza patients is currently enrolling participants [107] . there are no completed randomized clinical trials of adjunctive corticosteroid treatment in influenza patients. a trial of corticosteroid therapy was planned during the 2009 h1n1 pandemic, but was halted due to limited number of enrolees [108] . one observational study in china during the 2009 h1n1 pandemic reported that administration of parenteral glucocorticoids within 72 h of illness onset tripled the risk of developing critical illness or death from influenza a(h1n1)pdm09 virus infection [109] . a re-analysis of prospectively collected data on 1846 influenza patients admitted with primary influenza pneumonia to 148 icus in spain during 2009-2014 using propensity scoring matching reported that corticosteroid use was significantly associated with icu mortality [110] . meta-analyses of observational studies have concluded that that corticosteroid treatment of hospitalized influenza patients does not result in better outcomes and may be associated with adverse outcomes including increased mortality [111] [112] [113] . similarly, a retrospective observational study conducted on critically ill children during the 2009 h1n1 pandemic found that high-dose (equivalent to 2 mg/kg per day of methylprednisolone) corticosteroid treatment was associated with mortality in the icu, although a causative relationship was not determined [30] . a selection of individual observational studies in critically ill children and adults have also reported potential association between corticosteroid treatment and adverse influenza outcomes [30, 114, 115] . a recent cochrane review of available observational studies suggested increased mortality when adjunctive corticosteroid therapy is used for influenza patients; however, the available evidence was of low quality and the authors suggest interpreting these results with caution [116] . multiple studies have reported that corticosteroid treatment is associated with prolonged influenza viral shedding in hospitalized patients [117] [118] [119] , including in sporadic human infections with avian influenza a(h7n9) virus in china [120] , and increased rates of secondary bacterial and fungal co-infections [121, 122] , which may lead to adverse clinical outcomes. however, there is some evidence to suggest that the increased risk attributed to corticosteroid treatment is a result of bias in observational studies. a large, retrospective study of critically ill adults in canada found an increased risk of mortality in patients receiving corticosteroids; however, after adjusting for time-dependent differences between groups, no significant differences in mortality were observed with corticosteroid treatment [123] . moreover, potential differences between low-dose and medium-/ high-dose corticosteroid treatment are not well understood. one observational study of hospitalized patients with viral pneumonia due to avian influenza a(h7n9) virus infection in china reported that high-dose, but not low or moderate-dose corticosteroids, was associated with increased 30-day and 60-day mortality [124] . currently, on the basis of available observational data to date, adjunctive corticosteroid treatment is not recommended for children or adults hospitalized with influenza, including critically ill patients, unless clinically indicated for another reason, such as treatment of asthma or copd exacerbation or septic shock [49] . further studies are required to understand the clinical benefit or harms associated with corticosteroid treatment of critically ill influenza patients. although neuraminidase inhibitors (oseltamivir) are currently recommended for antiviral treatment of influenza in hospitalized patients based on observational studies, including in critically ill patients, there are a number of novel strategies and products for treating influenza in various stages of development. one approach under investigation is triple-combination antiviral drug (tcad) therapy, which combines amantadine, ribavirin, and oseltamivir for treatment of influenza in critically ill and high-risk patients. unfortunately, studies to date have not shown a benefit of tcad over oseltamivir monotherapy [125] [126] [127] . several novel antiviral compounds are in various stages of investigation, including small-molecule polymerase inhibitors such as pimodivir [128] and favipiravir [129] . a number of monoclonal and polyclonal antibodies, targeted against a variety of influenza viral proteins, are also in development [130] [131] [132] [133] . similarly, convalescent plasma has shown potential benefit in the treatment of severe influenza, and further trials are underway [134] [135] [136] . another area of intense interest is the modification of the host antiviral response to influenza virus infection. there are ongoing preclinical and clinical studies of a variety of other immunomodulatory agents for treatment of influenza, including celecoxib [137] , statins, etanercept, pioglitazone, azithromycin [138] , and interferons [139] . influenza vaccination can reduce the risk of complications from influenza, including reducing illness severity and the risks of hospitalization, icu admission, and death. the elderly, young children, pregnant women, and those with underlying medical conditions are most at risk for severe complications of influenza. a diagnosis of influenza should be considered in critically ill patients admitted with complications such as exacerbation of underlying chronic comorbidities, community-acquired pneumonia, and respiratory failure during influenza season. influenza molecular assays are recommended for testing upper respiratory tract specimens in patients without signs of lower respiratory tract disease. however, because critically ill patients with lower respiratory tract disease may have cleared influenza virus in the upper respiratory tract, but have prolonged influenza viral replication in the lower respiratory tract, an endotracheal aspirate (preferentially) or bronchoalveolar lavage fluid specimen (if collected for other diagnostic purposes) should be tested by molecular assay. antiviral treatment with standard-dose oseltamivir delivered orally or enterally by oro or naso-gastric tube is recommended as soon as possible for patients with suspected influenza without waiting for testing results. corticosteroids should not be routinely administered for treatment of influenza and should only be given for other indications (e.g., exacerbation of asthma or chronic obstructive pulmonary disease, or septic shock), because of the risk for prolongation of influenza viral shedding and ventilator-associated pneumonia in critically ill influenza patients with respiratory failure. future directions for treatment of influenza in critically ill patients include novel antiviral compounds, combination antiviral treatment with drugs with different mechanisms of action, immunomodulatory agents, and strategies for multimodality, combination antiviral, and host-directed immunomodulatory therapies. endnotes 1 these risk factors are included in the u.s. cdc's advisory committee on immunization practices recommendations for influenza vaccination. this may also apply to indigenous people from other countries, 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disease control and prevention. key: cord-004348-4jdn4kw6 authors: chen, juine-ruey; liu, yo-min; tseng, yung-chieh; ma, che title: better influenza vaccines: an industry perspective date: 2020-02-14 journal: j biomed sci doi: 10.1186/s12929-020-0626-6 sha: doc_id: 4348 cord_uid: 4jdn4kw6 vaccination is the most effective measure at preventing influenza virus infections. however, current seasonal influenza vaccines are only protective against closely matched circulating strains. even with extensive monitoring and annual reformulation our efforts remain one step behind the rapidly evolving virus, often resulting in mismatches and low vaccine effectiveness. fortunately, many next-generation influenza vaccines are currently in development, utilizing an array of innovative techniques to shorten production time and increase the breadth of protection. this review summarizes the production methods of current vaccines, recent advances that have been made in influenza vaccine research, and highlights potential challenges that are yet to be overcome. special emphasis is put on the potential role of glycoengineering in influenza vaccine development, and the advantages of removing the glycan shield on influenza surface antigens to increase vaccine immunogenicity. the potential for future development of these novel influenza vaccine candidates is discussed from an industry perspective. seasonal influenza outbreaks cause 3 to 5 million cases of severe illness and 290,000 to 650,000 respiratory deaths each year [1, 2] . the orthomyxoviridae are a family of enveloped viruses with a genome consisting of 6~8 segments of negative-sense single-stranded rna, including four genera of influenza virus: a, b, c and d [3] . influenza a and b are the main cause of annual flu outbreaks in humans, with influenza a further classified into subtypes based on their surface glycoproteins hemagglutinin (ha) and neuraminidase (na). 18 ha subtypes (h1~h18) and 11 na subtypes (n1~n11) are currently known, most notable today are the h1n1 and h3n2 subtypes that cocirculate in the human population. since the 1970s influenza b has diverged into two lineages based on antigenicity, the yamagata and victoria lineages, with little or no serum cross-reactivity [4] . in contrast to the severity and epidemic potential of influenza a and b, influenza c infections induce only mild flu symptoms in children, while influenza d is not known to infect humans [5] . recurrent influenza epidemics with pre-existing immunity occurs because the influenza virus employs two mechanisms to escape recognition: antigenic drift and antigenic shift. antigenic drift is the gradual accumulation of point mutations on the influenza virus' surface glycoproteins ha and na, driven by high error rates (estimated at 1.5 × 10 − 5 per nucleotide per replication [6] ) of the virus' rna-dependent rna polymerase (rdrp). mutations that allow the virus to evade the host immune system are positively selected for and become fixed, resulting in the rise of new strains that are antigenically different from what the host was vaccinated against. the second escape mechanism, antigenic shift, is the reassortment of gene segments across different strains infecting the same host, resulting in a wholesale change in antigenicity [7, 8] . antigenic shift have historically been associated with influenza pandemics, the most recent example being the 2009 swine-origin h1n1 that included segments from classical swine h1n1, eurasian swine h1n1, and a triple reassortant from 1998 [9] . the rise of new strains through antigenic drift and shift is followed by cross-immunity mediated competition between antigenically similar strains, which results in a progressive replacement of existing strains with new variants [10, 11] . unfortunately, current seasonal influenza vaccines are strain-specific and have a very narrow range of coverage, meaning extensive surveillance, accurate predictions and annual vaccination are needed as circulating strains evolve continuously over time. this task is coordinated by the world health organization (who) global influenza surveillance and response system (gisrs), which gathers year-round data from hundreds of national influenza centers around the world and issue vaccine formulation recommendations for each upcoming flu season [12] . when vaccine strains are well-matched with circulating strains, vaccination provides healthy adults younger than 65 years with 70-90% protection [13] , and reduced hospitalizations in the elderly and those with chronic illnesses by 30-70% [14] [15] [16] . however, in years when there is a mismatch between the vaccine and circulating strains, the vaccine effectiveness (ve) tends to be much lower [17] . here we discuss various challenges the current seasonal flu vaccine is facing, and how a universal influenza vaccine approach through carbohydrate design to elicit broadly neutralizing antibodies (bnabs) targeting the influenza ha glycoprotein can potentially play a role in the future of influenza prevention. despite the first influenza vaccine being commercially available as early as 1945, influenza outbreaks continue to be a major public health concern today. it is imperative for health authorities, researchers and the pharmaceutical industry to work together on improving the efficacy of influenza vaccines. traditional trivalent influenza vaccines include two inactivated influenza a strains (h1n1 and h3n2) and one influenza b strain, but this has recently been overtaken by quadrivalent influenza vaccine comprised of h1n1, h3n2 and both influenza b lineages that offers a more complete coverage [18] . commercially available vaccine options include egg-or cell-based inactivated influenza vaccine (iiv), a live attenuated influenza vaccine (laiv), and a recombinant ha vaccine produced in insect cells [16] . the production of egg-based influenza vaccines has remained virtually unchanged since the advent of split (subvirion) vaccines in the 1970s, and still commands 88% of the global market share in 2018 [19] . the main advantages of the egg-based platform include an excellent production capacity that is capable of producing an estimated 1.5 billion doses annually, and a low production cost that allows global access to the vaccine [20] . the strain-specific nature of current vaccines necessitates the annual selection of candidate vaccine viruses (cvvs), including screening the antigenicity of isolates, preparing reassortant viruses, and adaptation of the virus to eggs (fig. 1) . for egg-based manufacture, the entire process from strain selection to vaccine availability typically takes 6~8 months with tight time constraints, and any unexpected circumstance such as a delayed who strain recommendation [21] or unexpected low virus yield [22] , can snowball into significant production delays and directly affect vaccine supply. this lengthy interval also gives circulating influenza viruses time to mutate, as it did during the 2014-2015 flu season when late-emerging h3n2 variants rendered the recommended vaccine strain ineffective [8] . a second drawback of using an egg-based platform stems from the adaptation process of culturing a human virus in avian tissue, where adaptive mutations may accumulate and potentially change the strain's antigenicity [23] [24] [25] . ha, apart from being the primary target for neutralizing antibodies, is the main facilitator of influenza virus entry by binding to sialic acids on the surface of the host cells. human influenza ha preferentially bind to α-2,6 linked sialic acids commonly found on epithelial cells in the human upper respiratory tract [26, 27] . however, in egg-based production vaccine strains are inoculated into the allantoic cavity of embryonated chicken eggs which only contain α-2,3 linkages [28] . with successive passages, this becomes a selective pressure that can cause the acquisition or a total shift in receptor specificity, with its accompanying mutations and antigenic changes on ha's receptor binding site. a recent example of this occurred during the 2016-2017 flu season, when egg-adapted vaccine strains were found to lack a glycosylation site (t160, h3 numbering) on h3n2 ha antigenic site b, one of the five major antigenic sites that induce neutralizing antibodies [24] . a third concern is the egg-based platform relies on a steady supply of embryonated eggs. this egg supply can be overwhelmed by sudden increases in demand, such as during a pandemic. laiv is generated by combining the ha and na of currently circulating strains with the internal proteins of an attenuated cold-adapted strain. this results in a reassortant vaccine virus that can be administered intranasally and has some limited replicative ability in the human upper respiratory tract. as the entire influenza replication cycle is utilized at the site of infection, laiv has also been reported to elicit cell-mediated immunity [29] and local mucosal immunity [30] besides the induction of a robust antibody response. clinically, laiv has shown variable but overall comparable efficacy to iiv in adults and better efficacy in children. recently however, the necessity of effective replication in human respiratory tissue has emerged as an area of concern. the us advisory committee on immunization practices (acip) recommended against laiv between 2016 to 2018 due to low efficacy of the h1n1 component [31] , although this phenomenon was not noted in europe and canada [32] . the reason for this lack of efficacy is still unclear, but possibilities include viral interference of tetravalent vaccine strains resulting in reduced virus shedding for the weakest strain, strong cross-reactive antibodies from previous seasons preventing virus replication, and inherent lower replication in host tissue by the h1n1 pandemic strain [33] , among others. acip has since resumed recommendation for laiv in 2018 following a change in the h1n1 vaccine component [34] . secondly, as currently-available laiv is also produced in embryonated chicken eggs, it is plagued by many of the same concerns as egg-based iiv. in 2019 astrazeneca's laiv product flumist experienced manufacturing issues due to low yields in two strains, resulting in a reduction in shipments worldwide [35] . in order to overcome limitations of the egg-based manufacturing process, production systems using mammalian or insect cell cultures have emerged [36, 37] . the manufacturing process for cell-based iiv is similar to egg-based iivs, but has several advantages over the latter (fig. 1) . viral production in a cell culture bioreactor is more flexible, more scalable and unaffected by egg shortages. additionally, recent comparisons have shown that cell-based vaccines provided a moderately higher ve for elderly individuals (≧65 years old) than egg-based vaccines, possibly due to less egg-adapted mutations [38] . for recombinant ha production in insect cells, the baculovirus expression system is utilized to manufacture recombinant ha, which is then purified and formulated into ha trimer "rosettes" [39] . this not only has the same benefits of speed, flexibility and scalability as cellbased iiv, but also eliminates the reliance on influenza virus replication for vaccine production and the timeconsuming process of strain selection. flublok, a recombinant ha vaccine developed by sanofi pasteur, was found to be 30% more efficacious than traditional iiv for people ≧50 years old [40] . high yielding vaccine strains for egg-or cell-based production are generated by either classic or reverse genetic reassortment. these adapted viruses go into mass production, either in embryonated chicken eggs or mdck cells with a production timeline of approximately six to eight months. in recombinant ha (rha) vaccines, the ha sequence is cloned into baculovirus and expressed by insect cells, significantly shortening production time however, the comparatively high cost of these alternatives to egg-based influenza vaccines have prevented them from taking a bigger share of the influenza vaccine market. according to the us centers for disease control (cdc) adult influenza vaccine contract pricing for 2019-2020, the cost of the cell-based vaccine flucelvax is approximately 40% higher than an inactivated egg-based vaccine produced by the same manufacturer. the recombinant ha vaccine flublok can be more than twice as expensive as egg-based vaccines [41] . additionally, while cell-based and recombinant vaccines have the benefit of speed and flexibility that is critical for pandemic preparedness, it does not translate to a competitive advantage on the seasonal vaccine market [42] . so far slow progress has been made to transition away from egg-based production, and more support from governments around the world is needed. various next-generation influenza vaccines under development aims to broaden or lengthen the human immune response with novel antigens and adjuvants, gradually expanding the strain-specific nature of current vaccines to include all strains within a subtype (eg all h1 strains), multiple subtypes (eg h1/h5/h9), or incorporating all subtypes within a group (influenza a group 1 or group 2), with the ultimate goal of creating a truly "universal" pan-influenza vaccine that can elicit lifelong immunity against all influenza a and b viruses [43] . from a public health perspective influenza continues to be the only human disease that requires annual vaccination. it is estimated that replacing just 10% of seasonal vaccines with a universal vaccine would avert 6300 influenza-related deaths and save 1.1 billion us dollars in direct healthcare costs per year in the united states alone [44] . in 2017, the national institute of allergy and infectious diseases (niaid) in the us laid out a detailed strategic plan for the development of a universal influenza vaccine, highlighting knowledge gaps and research areas in pursuit of this common goal [43] . in their outline, they established four criteria for a universal influenza vaccine as: 75% effectiveness against symptomatic influenza infection, protection against both group i and group ii influenza viruses, durable protection that last at least 1 year, and be suitable for all age groups. it is with these criteria in mind that we discuss various vaccine candidates being developed (table 1) . historically, a crucial strategy of influenza virus' escape from pre-existing immunity is the addition of nglycosylation sites on the immunodominant ha head domain [75] . these bulky but poorly-immunogenic n-glycans allow the virus to hide antigenically-conserved domains from host immune system recognition [76] , a mechanism known as "glycan shielding". when h1n1 first emerged in 1918, it carried only one conserved glycosylation site at position 104 (h1 numbering) on the ha head. but as the virus continued circulating in the human population up to the 1950s, it sequentially acquired glycans at positions 144, 172 and 177, all at or adjacent to the major antigenic site sa on the ha head. this was followed by a 20-year hiatus as h1n1 was supplanted by h2n2, before re-emerging in 1977 carrying the same three acquired and one conserved glycosylation sites as before. the following decades saw n144 replaced by n142, the disappearance of n172, and the acquisition of n71 before the glycan shield was ultimately reset due to the emergence of 2009 pandemic h1n1, carrying only the original conserved glycosylation site on 104 [77] . conversely, h3n2 circulated in 1968 carrying two glycans on its ha head, n81 and n165 (h3 numbering). although the glycosylation site at position 81 was subsequently lost, positions 63, 122, 126, 133, 144, and 246 were accrued and retained [78] . overall, the continued circulation of an influenza subtype in the human population corresponds to a steady increase in n-glycans on its ha head domain. evidence that these acquired n-glycans provide a shielding effect comes from not only the observation that they tend to appear on or near major antigenic sites, but also studies have shown the acquisition of sites 177 and 142 on h1n1 slow genetic drift in the shielded areas [79] , and mutational deletion of 177, 142 and 71 on a pre-pandemic h1n1 strain elicited a protective immune response against the 2009 pandemic h1n1 strain [77] . similarly, in h3n2 positive selection disappeared when an antigenic site becomes shielded by n-glycans [78] , and the introduction of five recent glycosylation sites at positions 63, 122, 126, 133 and 246 allowed a 1968 h3n2 strain to evade polyclonal human serum raised against it [80] . these observations indicate that exposing the comparatively conserved, glycan-shielded regions of viral hemagglutinin could be a potential strategy to increase the breadth of influenza vaccine protection [52, 81, 82] . however, previous attempts have shown complete deglycosylation of all carbohydrate moieties on influenza ha by either prokaryotic production [52] , tunicamycin treatment [83] or pngase f digestion [53] does not appear to be a viable strategy. conserved n-glycosylation sites on the ha stem are essential for intracellular transport, correct glycoprotein folding and ha trimerization [84] , and a completely unglycosylated ha would have a high chance of altered antigenicity. therefore, our group focuses on harnessing glycoengineering techniques to alter n-glycan composition on the ha, creating recombinant has that retain only a single n-acetylglucosamine (glcnac) attached to asparagine per n-glycosylation site (monoglycosylated ha, or ha mg ). to accomplish this, n-acetylglucosaminyltransferase i deficient (gnti − ) human embryonic kidney cells that are unable to synthesize complex type n-glycans were used to produce secreted, transmembrane domain truncated has that have only high mannose residues on their nglycosylation sites. these high mannose has were then further trimmed with the high mannose-cleaving enzyme endoglycosidase h leaving a single glcnac residue, dramatically decreasing the size and shielding effect of these n-glycans while still maintaining the native ha structure in its trimeric state. antibodies raised against ha mg inoculation demonstrated better binding affinity, neutralization and crossreactivity than the unprocessed ha (fully glycosylated ha, or ha fg ) [52, 53] . ha mg also induced the maturation of dendritic cells, more splenic granzyme bsecreting cd8 + t cells, and elicited a more diverse haspecific b-cell repertoire than that of ha fg when used as a vaccine (fig. 2) . in terms of cross-protection, inoculation with an h1n1 pre-pandemic bris/07 ha mg not only provided better protection in mice against laboratory strains wsn and pr8, but also offered 70% protection against a pandemic strain [52, 53] . while a recombinant ha mg vaccine would have all the advantages of a cell culture production system including speed, flexibility and safety, egg-based production remains the mainstay of influenza vaccine manufacture today. devising a simple method to apply the monoglycosylation concept to egg-based vaccines with minimal modification will allow this procedure to be integrated into established production methods. extensive testing found that kifunensine, an α-mannosidase i inhibitor, can be injected into embryonated eggs to convert influenza virus membrane glycoproteins to a uniformly high mannose composition. after harvesting these virions their high mannose n-glycans were then trimmed with endoglycosidase h to create intact monoglycosylated virus particles, and all participating reagents are removed in subsequent purification steps [54] . like the recombinant ha mg before, monoglycosylated split inactivated influenza vaccines produced by kifunensine and endoglycosidase h treatment were shown to have higher neutralization and cross-neutralization activity, higher hemagglutination inhibition (hai), more ha stem selectivity, and higher antibody dependent cellular cytotoxicity (adcc) (fig. 2) . a monoglycosylated pandemic h1n1 split virus vaccine offered cross-protection against strains as diverse as the pre-pandemic nc/99 and the laboratory strain wsn [54] . aside from having simplified glycans, this procedure produces antigens that are virtually identical to the current influenza vaccine, and would presumably offer a similar safety profile. an adjuvanted recombinant ha trivalent nanoparticle influenza vaccine (tniv) has been developed by novavax using the baculovirus expression system to produce recombinant has, which were then purified and mixed with polysorbate 80 to form protein-detergent nanoparticles of 2~7 ha trimers [45] . the administration of this tniv with a saponin adjuvant (matrix-m) in ferrets induced higher levels of neutralizing antibodies against a panel of a (h3n2) strains than a commercial inactivated vaccine (trivalent fluzone). a phase i/ii clinical trial showed similar results in patients, where tniv induced significantly greater hai responses compared to trivalent fluzone against not only previous strains, but a forward-drifted a/singapore variant [46] . another candidate is a chimeric ha (cha) vaccine born from a collaboration between icahn school of medicine at mount sinai and gsk/nih. this strategy originates from the observation that our immune system tends to focus on the immunodominant but highly variable ha head domain, while the subdominant conserved stem region has a better ability to elicit bnabs. by sequential immunization with a cha protein consisting of a stem from circulating strains coupled to an irrelevant ha head from exotic influenzas, the strategy is devised to re-direct our immune system to better stimulate stemspecific responses [48] . in a preclinical study, ferrets sequentially immunized with heterologous influenza strains including live attenuated influenza vaccine (laiv) bearing an h8 head domain and an h1 stem domain (ch8/1) and a split-inactivated vaccine bearing an h5 head domain and an h1 stem domain (ch5/1), conferred superior protection against challenge with pandemic h1n1 virus following different prime-boost combinations and immunization regimens [49] . this approach is currently in collaboration with gsk in a phase i study, and clinical data will be obtained by the end of 2019. multimeric-001 (m-001) is a vaccine currently being developed by biondvax pharmaceuticals consisting of nine conserved b and t cell epitopes from ha, nucleoprotein (np) and matrix 1 (m1) protein arranged in triplicate and put onto a single recombinant protein [57] . phase i/ii clinical studies have shown the m-001 vaccine induced both cellular and humoral immunity to influenza a and b strains as a standalone vaccine [58] , and also enhanced seroconversion when used as a primer for elderly patients before inoculation with inactivated trivalent vaccines [85] . flu-v is another epitope-based vaccine developed by seek (peptcell) based on in silico multiple alignment of influenza sequences and prediction of possible t-cell epitopes. six consensus sequences from influenza np, m1 and matrix 2 (m2) proteins were identified and synthesized into a candidate vaccine. flu-v has been shown to induce a specific cd8 + response against these conserved epitopes and confer protection against heterotypic infection in mice [59] , and a phase ib challenge trial also showed that the blood cells from immunized subjects exhibited cross reactive immunity against different influenza viruses [62, 63] . codavax is an laiv being developed by codagenix that takes advantage of inherent human codon pair bias to reconstruct the influenza viral genome with synonymous but sub-optimal codons. this results in viral proteins that have the same amino acid sequence and antigenicity as wild type strains but attenuated due to excessive use of rare codons [64, 65] . in animal models, the vaccine is shown to be effective at lower doses than conventional laiv [66] . codavax has scheduled a phase i/ii trial in the first quarter of 2017. m2sr is an m2 deficient single-replication laiv being developed by flugen. in this strategy the m2 sequence in the viral genome (critical for viral uncoating and assembly) is largely deleted, but viruses are produced in m2-expressing cells to generate infective virions. therefore, after inoculation into a host the attenuated virus is unable to propagate infective progeny, limiting the infection to a single round of replication [67] . in a ferret model m2sr was found to be less susceptible to the negative effects of pre-existing immunity on drifted strains [68] . initial results from a placebo-controlled phase ii trial indicate that the vaccine was effective against a mismatched h3n2 challenge. inovio has made efforts to apply their syncon® synthetic dna vaccine platform to influenza. by sequence alignment and cluster grouping of ha they have generated four "micro-consensus" sequences within an influenza subtype, which were then cloned onto expression vectors and delivered to the vaccine recipient via in vivo electroporation [72] . in mouse and ferret models these micro-consensus sequences against h1n1, h3n2 and h7n9 were found to elicit protective immunity against lethal challenges. acam-flu-a is an influenza m2 ectodomain vaccine developed by acambis (now sanofi pasteur). due to overlapping nucleotides with m1, the m2 ectodomain is highly conserved in influenza a viruses, but poorly immunogenic [74] . acam-flu-a utilizes the hepatitis b core (hbc) as a carrier to fuse three tandem repeats of the m2 ectodomain onto each hbc subunit, creating an immunogenic virus-like particle (vlp). initial results showed intramuscular injection of the vaccine was able to generate anti-m2 ectodomain seroconversion in 90% of healthy volunteers [73] . however, after immunization m2-specific antibody titers steadily declined over a 1year period [86] , so combination with the other antigens or adjuvants might be necessary. the need for accurate surrogate markers of ve for clinical study and licensing approval precisely characterizing influenza immunity and correlates of immune protection is one of the three major areas for improvement outlined in niaid's strategic plan for a universal influenza vaccine [87] . serological assays such as hemagglutination inhibition (hai) and single radial hemolysis (srh) have long been held by regulatory agencies as a correlate of protection for inactivated influenza vaccine licensure. european medicines agency's (ema) committee for human medicinal products (chmp) criteria indicates that for seasonal influenza vaccine approval one of three conditions must be met: seroprotection (hi titer of ≧1:40 or srh of 25 mm 2 ) rate of over 70%, seroconversion (4-fold increase in titer) rate more than 40%, or a geometric mean increase (pre-vs post-vaccination) of 2.5 times in healthy adults, and 60, 30%, 2.0x respectively for elders [88] . the us fda center for biologics evaluation and research fig. 2 the production and immune response of monoglycosylated influenza vaccine. the production of monoglycosylated split virus vaccine adds two key steps to the traditional egg-based platform. kifunensine, a mannosidase i inhibitor, is added during egg inoculation to arrest viral glycoprotein processing, resulting in a uniformly high mannose composition. endoglycosidase h is added after harvest to trim high mannose residues down to a single glcnac. the resultant monoglycosylated split vaccine provides a more diverse immune response and more effective cross-strain protection than conventional egg-based vaccines. ha fg , non-modified egg-based vaccine with complex type n-glycans attached to ha; ha hm , ha with only high mannose type n-glycans; ha mg , ha with a single glcnac at its n-glycosylation sites. models of ha fg , ha hm and ha mg are created with protein data bank id code 3lzg and 6fyt by adding glycan with glyprot (http://www.glycosciences.de/modeling/glyprot/ php/main.php), coot (https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/) and pdb of lipid bilayer from lipid bilayer membranes for rasmol (https://www.umass.edu/microbio/rasmol/bilayers.htm). the images were displayed with program pymol (www.pymol.org) (cber) follows a similar criterion for accelerated approval [89] . however, hai and srh assays may not always be applicable when it comes to laiv or novel nextgeneration vaccines currently under development. hai measures the antibody-mediated inhibition of erythrocyte agglutination caused by ha binding to sialic acids on the erythrocyte surface. as such, the assay only detects antibodies directed at the ha head domain where its receptor binding site is located. universal vaccine strategies based on eliciting immune response against conserved epitopes on the ha stem domain, m2, m1 or np would not be detected by the hai assay. srh detects the concentration of influenza-targeting antibodies by measuring a ring of hemolysis caused by the antibody-virus-erythrocyte complex activating the complement system [90] . while this method measures all serum antibodies against influenza surface antigens, it still does not recognize local mucosal immunity or cellmediated immunity, such as immunization strategies that target m1 or np [91] . this has led to the recognition that non-hai or srh assays need to be taken into account for regulatory approval of next-generation influenza vaccines [87, 88] , though challenges in standardization of assays and reproducibility between laboratories still need to be overcome. finally, human challenge trials are gaining acceptance by regulatory agencies for universal vaccine development which may lack traditional serological correlates for protection [87, [92] [93] [94] . there is increasing recognition that utilizing all aspects of our immune system are needed to control influenza outbreaks. elderly people often have more serious complications from influenza infections and a less robust immune response to vaccination [95] . currently, high dose or adjuvanted iivs are recommended for people 65 years and older, while laiv is only approved for healthy adults up to the age of 49. on the other end of the spectrum, maternally-derived antibodies generated from inoculation during pregnancy are expected to provide protection for infants < 6 months, so vaccination that elicit a predominantly cell-mediated immune response are unlikely to be of use. novel strategies for a universal flu vaccine will have to take into account differences in immune response from specific populations that are at higher risk for influenza complications. with traditional seasonal flu vaccine human immunity wanes in 6-8 months of time, enough to last through the influenza season [96, 97] . but if a universal vaccine were to break the cycle of yearly vaccinations, long-term protection will be needed. having durable protection for at least 1 year and preferably through multiple seasons is one of the four criteria set by the niaid for a universal influenza vaccine [87] , but how to achieve that goal is still unknown. immunization schedules, formulations, dosages, and adjuvants will all likely have to be considered. the evolution of influenza vaccine development has shown a trend of cell-based vaccines gradually taking the place of traditional egg-based manufacture. with the plethora of next-generation vaccines currently under development, who expects a universal influenza a vaccine to be in advanced clinical trials as early as 2027 [98] . although many candidates have shown promising results in preclinical studies, demonstrating clinical safety and efficacy in a human population remains the most significant hurdle towards regulatory approval. our group has pioneered the strategy of exposing previously-shielded conserved epitopes on the ha through enzymatic trimming of n-glycans. this technique has been shown to elicit cross-neutralizing antibodies against antigenically diverse strains of influenza viruses within a subtype [52, 53] , and thus hypothetically a trivalent or tetravalent monoglycosylated vaccine containing the three influenza subtypes (h1, h3, and influenza b) circulating in the human population would be, for all intents and purposes, a universal flu vaccine. we believe this monoglycosylated split virus vaccine strategy has three unique qualities that give it a significant advantage in the new drug developmental process: the monoglycosylated split vaccine provides multiple conserved epitopes for immune recognition due to the rapid mutation rate of the influenza virus, using only a single conserved epitope as the antigenic target for universal vaccine runs the risk of generating escape mutants [99, 100] . in our previous studies we have only demonstrated the concept that monoglycosylated split virus vaccine induces more stem-specific antibodies directed against conserved epitopes on the ha stem [54] . however, in theory by trimming off oligosaccharides on every n-glycosylation site on the ha, multiple conserved epitopes would be exposed, inducing a multi-faceted immune response that imposes a higher evolutionary barrier for escape mutant generation. another influenza glycoprotein that could potentially benefit from the monoglycosylation process is na. the preparation of monoglycosylated split virus vaccine would remove glycans from not only ha but also na, hypothetically inducing more anti-na antibodies that interfere with virus budding, disease progression and severity of symptoms [101] . the monoglycosylated split vaccine induces a similar immune response to current iivs, meeting established surrogates of ve although a more diversified criteria encompassing cmi, neutralization assays and na antibodies is encouraged, traditional serological assays remain the gold standard for regulatory approval. by incorporating our monoglycosylation technology onto the existing inactivated split vaccine platform, we could invoke a similar humoral response as conventional iivs. serological surrogates of vaccine efficacy such as hai or srh can be measured and noninferiority comparisons with conventional vaccines can be made, opening up a well-trodden path towards licensure. the monoglycosylated split vaccine is suitable for all age groups whether novel vaccine strategies that are effective on healthy adults are equally suitable for all age groups remains a concern. due to having the same constituents as an iiv, the monoglycosylated split vaccine can be expected to offer a similar safety profile as the conventional flu vaccine. as such, it is possible that formulations suitable for different age groups, such as reduced dosage for children and high dose/adjuvanted vaccines for the elderly, can also be applied to our monoglycosylated split vaccine. furthermore, the robust humoral immunity induced by iiv assures sufficient protection for infants < 6 months by maternal vaccination. 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next generation influenza vaccines two escape mechanisms of influenza a virus to a broadly neutralizing stalk-binding antibody how single mutations affect viral escape from broad and narrow antibodies to h1 influenza hemagglutinin contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions jrc, yml, y-ct, and cm wrote the manuscript. jrc and yct designed and illustrated table and figures. all authors read and approved the final manuscript. funding was provided by academia sinica genomics research center summit project as-summit-108.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. key: cord-001521-l36f1gp7 authors: nan title: oral and poster manuscripts date: 2011-04-08 journal: influenza other respir viruses doi: 10.1111/j.1750-2659.2011.00209.x sha: doc_id: 1521 cord_uid: l36f1gp7 nan pandemic influenza h1n1 (h1n1pdm) virus of swine-origin causes mild disease, but occasionally is associated with acute respiratory distress syndrome and death. 1, 2 it is important to understand the pathogenesis of this new disease. previously we showed a comparable virus tropism and host innate immune responses between h1n1pdm and seasonal h1n1 influenza virus in the human respiratory tract, 3 however h1n1pdm virus differed from seasonal h1n1 influenza virus in its ability to replicate in human conjunctiva, suggesting subtle differences in receptor-binding profile and highlighting the potential role of the conjunctiva as an additional route of infection. we now compare the tropism and host responses elicited by pandemic h1n1 with that of related swine influenza viruses and a pandemic-swine reassortant virus in ex vivo and in vitro cultures of the human respiratory tract and conjunctiva. we have used recombinant virus to investigate the role of the hemagglutinin (ha) and neuraminidase (na) of h1n1pdm virus in its conjunctival tropism. these findings are relevant for understanding transmission and therapy. fragments of human conjunctiva, bronchi, and lung tissues were cut into 2-3 mm fragments within 2 h of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. viruses investigated included h1n1pdm (a ⁄ hk ⁄ 415742 ⁄ 09), swine h1n2 virus (a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04), which shares a common derivation for seven genes with h1n1pdm, a natural swine reassortant h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10), which has acquired the na gene from h1n1pdm and other swine influenza h1n1 viruses. reverse genetics derived recombinant viruses with ha and na gene segments of seasonal h1n1 and pandemic h1n1 swapped were also studied. lung fragments were cultured at 37°c in culture plates; conjunctival and bronchial biopsies were cultured in air-liquid interface at 33 and 37°c respectively. tissue fragments were infected for 1 h and incubated for 1, 24, and 48 h post infection. infectious viral yield was assessed by titration in mdck cells. the infected tissues were fixed with formalin and analyzed by immunohistochemistry for influenza antigen. cytokines profiles induced by influenza virus infected respiratory epithelial cells in vitro were measured by quantitative rt-pcr and elisa. we found comparable replication in seasonal and pandemic h1n1 viruses in human respiratory tract, while the swine influenza a ⁄ swine ⁄ hk ⁄ 4361 ⁄ 99 (h1n1) virus and a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2) virus failed to infect and replicate in human lung ex vivo culture, but it replicated productively in human bronchus ex vivo. interestingly, the swine reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus (with the na from h1n1pdm) infected and productivity replicated in lung ex vivo and in vitro. pandemic h1n1pdm virus, but not seasonal h1n1 virus, was able to infect ex vivo cultures of human conjunctiva, suggesting subtle differences in receptor binding profile in h1n1pdm, seasonal viruses, and the swine related h1n2 viruses. using reverse genetics derived recombinant viruses, we were able to demonstrate that the ha and na segments of h1n1pdm, but not the polymerase genes, were required for the conjunctival tropism of h1n1pdm ( figure 1 ). in contrast with highly pathogenic influenza h5n1 virus, which induced high cytokine and chemokine decretion, the related swine viruses, a ⁄ swine ⁄ hk ⁄ 915 ⁄ 04 (h1n2), as well as the swine pandemic reassortant virus, a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10 (h1n1) we studied were similar to h1n1pdm and seasonal influenza viruses in their intrinsic capacity for cytokine dysregulation. collectively, our results suggest that pandemic h1n1pdm virus differs in modest but subtle ways from seasonal h1n1 virus in its intrinsic virulence for humans, findings that are in accord with the epidemiology of the pandemic to date. the ha and na gene segments are key to the conjunctival tropism manifested by the h1n1pdm virus. the pandemic reassortant influenza h1n1 (a ⁄ swine ⁄ hk ⁄ 201 ⁄ 10) virus isolated from swine with the na from h1n1pdm shares with h1n1pdm the capacity for productive replication in lung ex vivo and in vitro. these findings are relevant for understanding transmission and therapy. isolation of influenza viruses from specimens is traditionally performed in two classical systems: embryonated chicken eggs and mdck cell culture. nevertheless, several publications are dedicated to the theme of alternative cell culture systems, which may be used for influenza virus isolation and cultivation. [1] [2] [3] this is in part because mdck cells are of animal origin, which means that they cannot be used as a proper model for estimating interactions between a human virus and a human cell culture as a host. a variety of human monolayer and suspension cell cultures have been tested on their capability to support influenza virus replication. among them, some support influenza a virus growth as well as mdck cells do, 4 others support replication of a virus, but do not enable the formation of mature viral particles, 5 whereas others show only a weak level of replication or are not permissive at all. 2 caco-2 cells, for example, represent a good substitute for mdck cells, because it has been shown that the rate of viral isolation in caco-2 cells is as effective as in mdck, and sometimes is even better. 6 the success of viral replication is determined not only by the cell culture type, but also by the virus itself. despite the accepted view that it is the type of receptor that defines the interaction between the virus and the host cell, there is evidence that it is not the only factor that predetermines the fate of the cell. 7 the fate of the infected cell can also differ. a series of articles show that apoptosis is the most probable mechanism of cell killing by influenza viruses. 8, 9 influenza a viruses of different subtypes induce apoptosis to a different extent (e.g. h3 viruses provoke more strong apoptotic response than h1 viruses do 10 ). nevertheless, it has been demonstrated that caco-2 cells do not follow the apoptotic pathway and die through necrosis. 11 the sjpl cell line also dies through necrotic pathway and not apoptosis. 12 the aim of our work was to compare growth characteristics of different flu viruses (e.g. avian, swine, and human) in various human and animal cell cultures and to evaluate their influence on cell culture growth. the parameters measured in the study were as follows: cytopathic changes of cell cultures following virus infection, hemagglutinin production, np synthesis, the dose-dependent effect of infection on cell proliferation, and the ability of viruses to induce apoptosis. influenza viruses used included: highly pathogenic avian h5n1 a ⁄ kurgan ⁄ 5 ⁄ 05, low pathogenic avian h5n1 a ⁄ gull ⁄ kostanai ⁄ 7 ⁄ 07, swine h1n1 a ⁄ swine ⁄ 1976 ⁄ 31, human h1n1v a ⁄ california ⁄ 07 ⁄ 09, human h1n1v a ⁄ saint-petersburg ⁄ 5 ⁄ 09, human h1n1 a ⁄ brisbane ⁄ 59 ⁄ 07, and human h3n2 a ⁄ brisbane ⁄ 10 ⁄ 07. the viruses were propagated in 10-days embryonated chicken eggs, the allantoic fluid was collected, the aliquots were made and stored at )70°c for further use. to evaluate tcid50 for each virus on all cell cultures, 96-well plates were used. the cells were seeded 0ae2 ml per well (concentration of 1-1ae5 · 10 cells ⁄ ml). the confluent 24-h old monolayer was used for viral inoculation. the cells were washed twice with serum-free medium, then 0ae05 ml of tenfold viral dilutions from viral aliquots were added and left for 45 min for contact at 37°c. the cells were then washed to remove the non-attached particles, and the wells were filled with tpck-trypsin (2 lg ⁄ ml)-containing medium without bovine fetal serum. the plates were observed daily for cytopathic effect, and the results were evaluated at 72 h after infection for cytopathic effect and by reaction of hemagglutination with suspension of chicken erythrocytes (0ae75%). infection of suspension cell cultures was done in centrifuge tubes. cells (concentration 3-5 · 10 5 ) were inoculated with viral dilutions (moi = 1-10). after 45 min of contact, cells were washed, resuspended in rpmi with trypsin and fetal serum, and seeded in 24-well plates (1 ml in each well). the results were fixed after 72 h, calculating the number of cells grown and estimating the rate of apoptosis by hoechst-33258 staining. 13 cells were grown in 24-well plates with seeding concentration 1 · 10 cells ⁄ ml. one millilitre of cell suspension was placed in each well, inoculated with viral dilution (moi = 1-1000) and left for 72 h. after, the cells were detached from plastic with versene and calculated in fuks-rosental camera to evaluate the number of cells. the monoclonal antibodies obtained in research institute of influenza towards viral nucleoprotein np were used following the standard protocol described in. 13 for all viruses tested, mdck turned out to be more permissive than sp cell culture. avian viruses, independently of their pathogenicity, replicated efficiently on both animal cultures tested. human h1n1 and h3n2 viruses demonstrated weaker replication in sp cells. the most significant differences were seen for swine influenza and pandemic h1n1v viruses which replicated in mdck cells at the rates comparable with other viruses, but showed poorer growth in sp cell line (see table 1 ). human cell lines displayed clear differences in their susceptibility to viruses of various origins. avian influenza viruses replicated in all cell lines except girardi heart, and the most intense replication rate was observed for ecv-304, l-41, and rd lines. a-549 and a-172 were poorly infected, as well as all suspension cell lines tested. seasonal human h1n1, as well as h3n2 viruses, replicated in all cell cultures tested, but the rate of infectivity was rather low in practically all cultures tested with the exception of rd and t-98g cell lines. strikingly, swine influenza virus and human pandemic h1n1v viruses didn't replicate well in any of human lines tested. a weak replication rate was observed in ecv-304, rd, and t-98g, but in general, human cell lines were the titers produced by swine and pandemic influenza viruses are shaded in grey. *low-pathogenic avian influenza virus; **highly-pathogenic avian influenza virus poorly susceptible to pandemic h1n1v. swine influenza virus differed because it infected weakly a-172 and girardi heart cell cultures, which was not the case for h1n1v viruses. our study has shown that all influenza viruses were able to induce apoptosis in the cell cultures tested. the degradation of chromatin found in the nucleus with hoechst-33258 staining was seen before the first symptoms of cytopathic effect (cpe) in monolayer of cells. in cell cultures where the cpe was not visible, high doses of virus still induced apoptotic response. the process of apoptosis is rather well studied in mdck cells and some other cell types, so we've focused on three human monolayer cell cultures that are relatively poorly studied: a-549, ecv-304, and flech. these cell cultures are less susceptible to viral infection, and besides, it was interesting to find out whether the viruses that do not cause any cpe do infect these cultures. a-549 turned out to be most sensitive to apoptotic response, while flech turned out to demonstrate weak reaction. time needed for apoptosis induction by different flu viruses also varied. the earliest apoptosis was noted for h5n1 and h3n2 viruses and h1n1 viruses induced apoptosis at about 20 h postinfection. it is well-known that apoptosis can be induced only by a reproducing virus, and that uv-kills viruses that are not capable of it. we tested whether swine and pandemic h1n1v viruses (that do not show cpe in these cultures) do replicate in them and induce apoptosis with the help of monoclonal antibodies against viral np. the obtained data show that they indeed do replicate in these cell cultures, as we observed np fluorescence, and that they also induce apoptosis (see table 2 ). we've shown earlier 13 thus, we've tested the ability of swine and pandemic h1n1v viruses in this aspect. it was shown that these viruses were comparable with the effect seen for seasonal h1n1 virus. moreover, swine influenza virus induced stronger apoptotic response in hemablastoid cell lines in comparison with pandemic h1n1v viruses, which also have a swine origin. we also checked the ability of flu viruses to influence monolayer cell cultures growth. the data clearly indicated that only ecv-304 endothelial line and t-98g glioblastoma line displayed cell proliferation in response to low moi. apoptosis wasn't registered in these stimulated cultures, apparently because the moi was very low. all the other monolayer cultures didn't respond to low moi by stimulation of their proliferation. interaction between an influenza virus particle and a host cell can follow several scenarios. cpe seen in infected cells is accompanied with high rates of viral particles production and leads to cell death. the death itself may be through apoptotic or necrotic pathways. 4, 8 also, infection process in low doses can stimulate cell proliferation -the effect seen for hemablastoid lines, histiocytes, peripheral blood cell lines, 14, 15 and in glioblastoma and endothelial cell lines as it was described here. considering the origin of ecv line, 16 these cells bear all the antigenic, biochemical, and physiological traits of umbilical cord and are actively used in pharmacological tests as well as glioblastoma cells; they also are of special interest for oncogenesis studies. table 2 . replication, apoptosis induction, and np synthesis of influenza viruses in a-549, ecv-304 and flech cell cultures. the numbers represent the log 10 tcid 50 ⁄ 0ae2 ml calculated by reed-muench method as described in. 17 the ()) symbol means that no cpe could be observed in any dilution and no hemagglutination could be registered. the (+) symbol means that apoptosis was observed with hoechst-33258 staining though the productive replication and production of progeny viruses in human cell lines was generally low, it is evident that viral infection does occur in these cells, even for swine and h1n1v viruses. it can be demonstrated by the presence of np de novo synthesis and by stimulation of virus-induced apoptosis. in fact, we observe a contradiction: avian influenza viruses actively reproduce in human cell lines, but we do not see their vast spreading in human population, while h1n1v viruses that hardly replicate in all human cultures tested have caused the latest pandemic. influenza viruses continue to cause problems globally in humans and their livestock, particularly poultry and pigs, as a consequence of antigenic drift and shift, resulting frequently and unpredictably in novel mutant and reassortant strains, some of which acquire the ability to cross species barriers and become pathogenic in their new hosts. long-term surveillance of influenza in migratory waterfowl in north america and europe have established the importance of anseriformes (waterfowl) and charadriiformes (gull and shorebird) in the perpetuation of all known subtypes of influenza a viruses. the available evidence suggests that each of the 16 hemagglutinin (ha) and nine neuraminidase (na) subtype combinations exist in harmony with their natural hosts, cause no overt disease, and are shed predominantly in the feces. 1, 2 in this study we determined the subtypes and prevalence of low-pathogenic influenza a viruses present on the territory of kazakhstan in 2004-2006 and further analysed the ha and na genes of these isolates in order to obtain a more detailed knowledge about the genetic variation of influenza a virus in their natural hosts. (institute for biological safety problems, gvardeiskiy, zhambyl oblast, kazakhstan)). samples that were identified as influenza a virus positive by matrix rrt-pcr were thawed, mixed with an equal volume of phosphate buffered saline containing antibiotics (penicillin 2000 u ⁄ ml, streptomycin 2 mg ⁄ ml, and gentamicin 50 lg ⁄ ml), incubated for 20 minutes at room temperature, and centrifuged at 1500 g for 15 minutes. the supernatant (0ae2 ml ⁄ egg) was inoculated into the allantoic cavity of four 9-day old embryonated hens' eggs as described in european union council directive 92 ⁄ 40 ⁄ eec. 3 embryonic death within the first 24 hour of incubation was considered as non-specific, and these eggs were discarded. after incubation at 37°c for 3 days the allantoic fluid was harvested and tested by haemagglutination (ha) assay as describe in european union council directive 92 ⁄ 40 ⁄ eec. in the cases where no influenza a virus was detected on the initial virus isolation attempt, the allantoic fluid was passaged twice in embryonated hens eggs. the number of virus passages in embryonated eggs was limited to the maximum two to limit laboratory manipulation. a sample was considered negative when the second passage ha test was negative. the subtypes of the virus isolates were determined by conventional haemagglutination inhibition (hi) test and neuraminidase inhibition (ni) test, as describe in european union council directive 92 ⁄ 40 ⁄ eec. 3 rna extraction and pcr with specific primers rna was extracted from infective allantoic fluid using rneasy mini kit (qiagene, gmbh, germany) according to the manufacturer's instructions. the rna was converted to full-length cdna using reverse transcriptase. the rt mix comprised 2ae5 ll of dmpc water, 5 ll of 5· first strand buffer (invitrogen), 0ae5 ll of 10 mm dntp mix (amersham biosciences), 2 ll of 50 mm uni12 primer, 32 u of rnaguard (amersham biosciences), 200 u of mmlv reverse transcriptase (invitrogen) and 5 ll rna solution in total volume of 25 ll. the reactions were incubated at 42°c for 60 minutes followed by inactivation of the enzyme at 95°c for 5 min. pcr amplification with ha and na gene specific primers was performed to amplify the product containing the full length ns gene. twenty-five microliter pcr-mix contained 1· platinum taq buffer (invitrogen), 200 lm dntp, 2ae5 mm mgcl 2 , 240 nm each of fw primer and rw primer, 1 u platinum taq dna polymerase (invitrogen) and 3 ll cdna. reactions were placed in a thermal cycler at 95°c for 2 min, then cycled 35 times between 95°c 20 seconds, annealing at 58°c for 60 seconds, and elongation at 72°c for 90 seconds and were finally kept at 8°c until later use. sequences of the purified pcr products were determined using gene specific primers and bigdye terminator version 3ae1 chemistry (applied biosystems, foster city, ca), according to the manufacturer's instructions. reactions were run on a abi310 tm dna analyzer (applied biosystems). sequencing was performed at least twice in each direction. after sequencing, assembly of sequences, removal of low quality sequence data, nucleotide sequence translation into protein sequence, additional multiple sequence alignments, and processing were performed with the bioedit software version 7ae0ae4ae1 with an engine based on the custal w algorithm. the phylogenetic analysis, based on complete gene nucleotide sequences were conducted using molecular evolutionary genetics analysis (mega, version 4ae0) software using neighbor joining tree inference analysis with the tamura-nei c-model, with 1000 bootstrap replications to assign confidence levels to branches. [4] [5] [6] [7] ha and na sequences obtained from genbank the ha and na gene was analyzed both with selected number of influenza isolates and in comparison with virus genes obtained from genbank were used in phylogenetic studies [22] . the nucleotide sequence data obtained in this study has been submitted to the genbank database and is available under accession numbers fj434373, fj436942ae1, fj434369, fj434370, gu982281-gu982284 for ha and fj434374, fj436943ae1, fj434371, fj434372, gu982285-gu982288 for na. avian influenza prevalence in our study h4, h5, and h13 influenza a virus subtypes were found to circulate at the same time, in the same geographic region in the kazakhstan. this finding most likely indicates the existence of a large reservoir of different influenza a viruses in kazakhstan. we analyzed the ha and na gene sequences of the eight influenza a viruses isolated in kazakhstan together with selected number of isolates, reported between year 1941 to 2008, and previously published in the genbank. 8 phylogenetic analysis of the h4 ha gene showed that all viruses separated into the american and eurasian lineages ( figure 1 ). an evolutionary tree suggests that north american isolates have diverged extensively from those circulating in other parts of the world. geographic barriers which determine flyway outlay may prevent the gene pools from extensive mixing. the lack of correlation between date of isolation and evolutionary distance suggests that different h4 ha genes co circulate in a fashion similar to avian h3 ha genes and influenza c genes, implying the absence of selective pressure by antibody that would give a significant advantage to antigenic variants. analysis of phylogenetic relationships among the ha5 ha genes reported in this study clearly shows that viruses belong to the western pacific flyway, one of the major migratory flyways in this region that have subsequently spread throughout eurasia. 9 these findings provide further evidence of the dynamic influenza virus gene pool in this region. along the western pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern china has 'mixed' longitudinally with viruses isolated from japan, mongolia, and siberia. however, it appears that there has also been 'mixing' latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the eurasian continent. this helps to explain the latitudinal spread of the qinghai-like (clade 2ae2) h5n1 virus in the last 2 years, while h5n1 outbreaks in korea and japan may represent the longitudinally transmitting pathway. 10 ha of subtype h13 so far has been found exclusively in shorebirds, such as gulls, and in a pilot whale (potentially a spillover from shorebirds), but not in other avian species that are natural hosts of influenza a virus, such as ducks and geese; therefore the study of the evolution of these viruses is very interesting. phylogenetic analysis h13 ha gene revealed three significantly different evolutionary lines: an american line, a european line, and a line comprising the isolates from america and eurasia. 11 further we analyzed na genes of influenza viruses (figure 2) . the na gene is important both because of its functional role in promoting the dissemination of the virus during infection, and because, like ha, it is a principal target of the immune system. it was shown that phylogeny of na genes of influenza have the same properties as hemagglutinin. na genes of kazakhstanian viruses belong to eurasian lineage of virus evolution. obtained data are important for surveillance and diagnostics because some of the lpai viruses examined in this study can infect and be shed by chickens and turkeys and may have epidemiology potential during further recombination with other influenza viruses. influenza virus is divided into different subtypes based on hemagglutinin (ha) and neuraminidase (na) on the virus surface. within each subtype, ha continues to mutate and produce immunologically distinct strains, as antigenic drift. the continuous mutation of influenza virus (iv) is important for annual epidemics and occasional pandemics of disease in humans. antigenic drift requires vaccines to be updated to correspond with the dominant epidemic strains. in humans, ivs show both antigenic drift frequently. in contrast, ivs from birds are in evolutionary stasis, 1 and they show little amino acid changes. 2, 3 the reason is that ivs in bird intestine are not subjected to strong immune selection. hemagglutinin (ha) gene of influenza a virus encodes the major surface antigen, which is the target for the protective neutralizing antibody response that is generated by infection or vaccination. in humans, influenza a viruses show antigenic drift with amino acid changes in the globular head of the ha so as to evade herd immunity of the population. on the contrary, avian influenza a viruses show evolutionary stasis in wild birds. h6 aivs have occurred frequently in chicken farms in the world. 4 although vaccination is not permitted, h6n1 aivs have circulated in taiwan for a time. 5 the seroprevalence in chicken flocks reaches about 50% in the field. h6n1 aivs invades internal organs, such as kidney and lung. 6 thus, viruses in chicken flocks are pressured into antibody selection. here, we report that h6n1 aivs in the field have showed evolutional changes instead of evolutional stasis. in response to requests from poultry farmers for diagnostic investigations of illness in poultry flocks, the authors did necropsy at the pen-site. after careful examination, tracheae were taken and kept in cold for virus isolation in the laboratory. for avian influenza virus isolation, trachea was homogenized 1:10 in tpb with antibiotics. the homogenate was frozen and thawed three times and then centrifuged at 1157 g for 15 minutes. the supernatant was passed through a 0ae45 lm filter. the homogenate was examined for the presence of virus by inoculation into five 9-to 11-day-old specific-pathogen-free (spf) chicken eggs for two passages. thirteen h6n1 aivs were isolated in this laboratory during 2000 and 2008 from different parts of taiwan. besides the viruses isolated in this laboratory, the ha sequences of 27 chicken h6n1 aivs were from the genbank. the accession numbers of hemagglutinin of aiv reference strains included in this study were as the following: g2 ⁄ 87, dq376619; g23 ⁄ 87, dq376620; 0824 ⁄ 97, dq376621; na3 ⁄ 98, dq376622; 165 ⁄ 99, dq376626; 0705 ⁄ 99, dq376624; ns2 ⁄ 99, dq376623; sp1 ⁄ 00, dq376628; 0329 ⁄ 01, dq376633; 1205 ⁄ 01, dq376630; 1212 ⁄ 01, dq376631; 1215 ⁄ 01, dq376632; 0208 ⁄ 02, dq376641; 0408 ⁄ 02, dq376642; pf1 ⁄ 02, dq376635; pf2 ⁄ 02, dq376636; pf3 ⁄ 02, dq376637; a37 ⁄ 02, dq376639; 0320 ⁄ 02, dq376638; 0107 ⁄ 02, dq376640; 0222 ⁄ 02, dq376634; 0706 ⁄ 03, dq376644; 1203 ⁄ 03, dq376645; ch1006 ⁄ 04, dq376649; 0114 ⁄ 04, dq376647; a342 ⁄ 05, dq376653 and 0204 ⁄ 05, dq376652. the viruses isolated were propagated in the allantoic cavities of 10-day-old embryonated spf eggs for 72 hour. the virus rna was extracted using qiaamp viral rna miniprep kit (qiagen) . six-week-old balb ⁄ c mice were injected emulsion intraperitoneally with 100 lg of purified and concentrated a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) virion with complete freund's adjuvant. every two weeks, the mice were boosted supplementary five times with 50 lg of virion in incomplete freund's adjuvant. when the mice were boosted, blood was collected from tail vein and tested by the western blot assay to check the antibody titers. the mice were then injected intraperitoneally with 50 lg of virion at week 8. five days after the last injection, the splenocytes in the mice were fused with myeloma cells (sp2 ⁄ 0-ag14). one week before fusion, the myeloma cell line was expended in dmem medium (hyclone laboratories, logan, ut) with 10% fetal bovine serum at 37°c to ensure they were in the exponential growth phase. the spleen cells from immunized mice were washed, harvested, and mixed with the previously prepared myeloma cells and fused by gradually adding 50% polyethylene glycol-1500. the resulting pellet was plated into 96 well tissue culture plates. only the fused cells grew in medium with hypoxanthine-aminopterin-thymidine (hat). with fresh medium replacement over 2 weeks, the hybridomas were ready for screening. hightiter monoclonal antibody (mab) preparations were obtained from the ascetic fluid of mice injected with the selected hybridoma clones. the antibody from mouse ascetic fluids was purified by precipitation with ammonium sulfate, then aliquoted and frozen at )70°c, avoiding repeated freezing and thawing. eventually, six mabs were obtained and named ch11-d10, eb2-b3, eb2-e5, eb2-f9, ff9-f5, and ff9-f7, respectively. the hi test was performed following a standard method. all the viruses were diluted twofold and reacted with 1% chicken erythrocytes in the v-bottomed microtiter plate by the hemagglutination test. after agglutination, four hemagglutinating units of a ⁄ chicken ⁄ taiwan ⁄ 2838v ⁄ 00 (h6n1) and ascetic fluids from the immunized mice of the six mabs were prepared for hi test. hi titers of 2 4 or more were regarded as positive. the cases submitted for diagnosis from chicken farms had respiratory signs, increase in mortality, or drop in egg production (e.g. egg production dropped from 10% to 40%). the extent of drop in egg production depended on the chicken ages. for example, the age of case 3511 was 27 weeks, a stage of increasing egg production. however, after h6n1 aiv infection, the egg production decreased 1% instead of increasing and then stayed at 65% for a week. the infected chickens showed signs of decreasing activity, anorexia from 150 g per bird to 90 g per bird, and respiratory signs. case 2829 showed infection in the second floor first and then transmitted to third and fourth floor, indicating that the virus transmitted by air or human movement. however, most cases showed air borne transmission from one flock to another in spite of enforcing restrictions of persons entering the poultry pens and changing clothes and booths. in most cases, males' mortality was higher than that of female pen mates. by comparing the sequences of ha of those h6n1 viruses, we found that amino acid changes in ha1 were higher than those in ha2, showing that antigenic changes on the globular head of ha molecule rather than randomly on the whole ha protein, indicating that h6n1 viruses in taiwan had been selected in the presence of antibody pressure. the aa residues and changes that showed yearly trends were the followings: a-5s, i19s, v30i, n35s, e39k, l45m, e54d, q66k, a94v, or t, s127n, s128r, k133n, y138d, n139t, s140i, g141d, l149v, i152v, g155e, t188n, g226s, a285v, k304e, d386n, i533m, and m535i. however, their significance on antigenic variation was previously unknown. by hemagglutinination inhibition (hi) assays, except mab ch11-d10, all other monoclonal antibodies elicited from 2838v ⁄ 00 showed different hi titers with the different h6n1 viruses (table 1 ). however, those 5 mabs showed negative hi to 2829 and 2831, the early h6n1 strains. this indicated that the epitopes recognized by those mabs were undergoing antigenic drift. introduction aquatic birds are recognized as the natural reservoirs of the influenza a virus as all known subtypes (h1-h16, n1-n9) have been found in them. phylogenetic analyses of influenza viruses found in other animals revealed that all were directly or indirectly derived from viruses resident in aquatic birds. 1 however, the prevalence, movement, and evolutionary dynamics of influenza viruses in these avian hosts have not been well defined. southern china was hypothesized to be an 'epicenter' for the generation of human pandemic influenza viruses as all major influenza pandemic viruses in the 20th century emerged from this region. 2 the ecological background that facilitates the occurrence of these pandemic influenza strains has not been fully explored. in the past two decades, four lineages, belonging to h5n1, h9n2, and h6n1 viruses, have become established and long-term endemic in different types of poultry in this region. [3] [4] [5] some of these viruses were disseminated to many countries in eurasia and africa and have continued to cause sporadic human infection, posing a persistent pandemic threat to the world. 6 in the mean time, the endemic influenza lineages have undergone extensive genetic reassortment events giving rise to many variants, dramatically increasing the genetic diversity of the influenza virus in this region. questions remain as to how and where these viruses emerged, and what were the sources of the gene segments incorporated within the novel reassortant variants of the h5n1, h9n2, and h6n1 virus lineages. to address these questions, surveillance of influenza in migratory and domestic (sentinel) ducks has been conducted since 2002 at poyang lake, the biggest fresh-water lake and the major migratory bird aggregation site in southern china. the aim of this study is to identify the prevalence, seasonality, and movement of virus between migratory and domestic ducks. migratory ducks were captured during over-wintering, from november to march. cloacal swabs and blood samples were collected from each individual bird. all birds were released after sampling. to observe the interaction between migratory ducks and domestic birds, we also sampled domestic ducks from two duck farms (designated as sentinel ducks) surrounded by rice fields and inaccessible to other types of poultry, but accessible to migratory birds. that is, the sentinel ducks share the same water body with migratory ducks and have the chance to spread viruses to each other. for sentinel ducks, sampling was conducted fortnightly, all year-round, on the two farms from august 2002 onwards. cloacal swabs and fresh fecal droppings were taken. about 200 birds were randomly sampled fortnightly from these farmed ducks. all swabs were soaked in vials containing 1ae5 ml transport medium with antibiotics and kept on ice-packs during sampling and immediately stored in )80°c freezers for further use. blood samples from migratory ducks were treated according to methods previously described. 7 serological survey and virus subtyping in migratory and sentinel ducks used hemagglutination inhibition (hi) and neuraminidase inhibition (ni) tests as previously described. 3 for isolates that were not identified by reference antisera, subtypes were determined by rt-pcr using subtype specific ha and na diagnostic primers. prevalence and seasonal patterns of influenza virus in migratory and sentinel ducks during 2002 during -2007 a total of 11 508 cloacal swabs from migratory ducks and 36 475 cloacal or fecal swabs from sentinel ducks were collected at poyang lake. from these specimens, 90 influenza isolates were obtained from migratory ducks and 1681 from sentinel ducks; isolation rates of 0ae78% and 4ae61%, respectively (table 1) . it was noted in sentinel ducks that virus occurrence formed a seasonal peak from november to february, which completely overlapped the over-wintering months of migratory ducks. this suggests that virus movement or transmission between migratory and sentinel ducks occurred during this period at poyang lake. thirty positive samples (hi titer ‡20) were identified from 715 blood samples collected during november and december in 2004. among these, 15 samples were positive to h6, 8 were positive to h9, 5 were positive to h1, and 2 were positive to h4. one serum sample was positive to both h4 and h6, which suggested co-infection of influenza virus in migratory ducks might occur in natural conditions. poyang lake, which is located in the northeastern part of jiangxi province, is the largest freshwater lake in china and is part of the eastern asia-australia migration route. 9 every year, hundreds of thousands of migratory ducks congregate at poyang lake during the migration season. 9 recent farming practice involves raising domestic waterfowl in dense populations in the poyang lake region. farmraised domestic waterfowl are allowed to feed in and share the same water body with migratory birds, thereby facilitating direct interactions between domestic waterfowl and freeranging migratory birds. this makes poyang lake an ideal site to observe the dynamics of influenza virus interactions between migratory and sentinel ducks in southern china. in our longitudinal surveillance during [2002] [2003] [2004] [2005] [2006] [2007] , the overall virus isolation rate from migratory ducks was less than 1%, which suggests a low prevalence of viral infection during the birds' southern migration. similar results have been observed in taiwan, which is also an important stopover site for migratory birds along the eastern asia-australia migration route during 10 years of surveillance. 10 the overlap in seasonal patterns of virus infection between migratory and sentinel ducks found in our study suggests that virus movement or transmission between migratory and sentinel ducks occurred during the period of time migratory birds were at poyang lake. the ha subtypes harbored in migratory and sentinel ducks were similar in our study. for migratory ducks, h4, h6, h10 were the predominant subtypes, while h3, h4, and h10 were the major subtypes in sentinel ducks. hpai h5n1 was only detected from migratory ducks in early 2005 on two sampling occasions. from phylogenetic analyses the h5n1 viruses isolated from migratory ducks were closely related to the viruses endemic in domestic poultry in southern china. 8 therefore, it appears that h5n1 viruses endemic in domestic poultry could be transmitted to migratory ducks via close contact in southern china. only lp h5 viruses were detected from sentinel ducks at poyang lake during this period. whether h5n1 virus infection was absent from sentinel ducks at poyang lake needs further investigation. serological surveys provided further evidence for the prevalence of aiv in migratory ducks at poyang lake. the serological results in 2004 did not match well with the epidemiological results during [2002] [2003] [2004] [2005] [2006] [2007] , which suggests that influenza virus infection in migratory birds could be influenced by multiple factors, such as host immune status, population size, spatial and temporal variations, and migration routes. southern china has the biggest domestic duck population in the world. our study demonstrates that dynamic interactions between migratory ducks and sentinel ducks occurred frequently throughout the surveillance period. thus, sentinel ducks could be treated as intermediate hosts between the ''real gene pool'' from migratory ducks and domestic poultry in the whole influenza virus ecosystem. a sentinel duck sampling system may be a feasible method to represent the viruses in the natural gene pool and a baseline for virus or gene interactions between migratory and domestic ducks. 11 further investigations and surveillance are required to better understand the role of the domestic duck population in facilitating virus interactions and the generation of genetic diversity. two distinct lineages of h9n2 influenza viruses represented by a ⁄ chicken ⁄ beijing ⁄ 1 ⁄ 94 (ck ⁄ bei-like) and a ⁄ quail ⁄ hong kong ⁄ g1 ⁄ 97 (g1-like) have become established and endemic in poultry in southern china. 1 these established h9n2 lineages continue evolving to generate many different reassortant variants (or genotypes) 1,2 and are causing sporadic cases of human infection. 3, 4 studies of h9n2 viruses isolated from pigs in hong kong and shandong province have also raised the possibility of reassortment with human-like viruses from pigs. 5, 6 in addition, h9n2 viruses isolated beyond the late 1990s had preferential binding with a-2,6-neuacgal human-like receptors. 7 these observations suggest that the h9n2 influenza viruses still have pandemic potential. unlike highly pathogenic h5n1 influenza viruses that have been rarely detected in the live-poultry markets in hong kong since 2002, h9n2 viruses are still frequently isolated in our surveillance program. therefore, we try to understand the continuing evolution of h9n2 viruses through genetic characterization and phylogenetic analyses of the viruses isolated in hong kong live-poultry markets from 2005 to 2009. a total of 47 421 terrestrial poultry were sampled at different live-poultry markets in the hong kong sar between january 2005 and december 2009. of those samples, 29 691 were from chickens and the others were from minor poultry species including chukar, pheasant, guinea fowl, silky chicken, and pigeon. fecal droppings, cloacal and tracheal swabs, drinking water, and environmental samples from cages were collected into transport medium. viruses were isolated in 9-to 11-day old embryonated eggs as described previously. 8 virus isolates from positive sampling occasions were selected for sequence analysis. rna extraction, cdna synthesis, and pcr were carried out as described previously. 8 dna sequencing was performed using bigdye terminator v3ae1 cycle sequencing kit on an abi 3730 dna analyzer (applied biosystems) following manufacturer's instructions. all sequences were assembled and edited with lasergene 7ae0 (dnastar, madison, wi) software. sequence alignment and residue analysis were performed with the bioedit sequence alignment editor, version 7ae0. 9 all eight gene segments of sequenced viruses were characterized and analyzed phylogenetically together with virus sequence data available in public databases. maximum-likelihood trees were constructed using garli 0ae96. 10 estimates of the phylogenies were calculated by performing 1000 neighbor-joining bootstrap replicates using paup*4ae0. 11 systematic surveillance of live-poultry in hong kong from 2005 to 2009 resulted in 1088 h9n2 isolates from 47 421 samples (overall isolation rate, 2ae3%) ( table 1 ). there were 966 strains isolated from 42 253 chicken samples (isolation rate, 2ae3%). of these viruses, four were isolated from 1556 tracheal swabs (isolation rate, 0ae3%), while 541 isolates were isolated from 29 030 cloacal or fecal swabs (isolation rate, 1ae9%). an additional 421 isolates were collected from 6926 drinking water samples (isolation rate, 6ae1%). there were 122 strains of h9n2 viruses isolated from 5168 minor poultry samples (isolation rate, 2ae4%) ( table 1) . of these viruses, only one was isolated from 1209 tracheal swabs (isolation rate, 0ae1%), whereas 72 strains of viruses were isolated from 3610 cloacal or fecal swabs (isolation rate, 2ae0%). the isolation rate in drinking water in minor poultry was again higher when compared with other sampling methods with 49 strains isolated from 297 drinking water samples (isolation rate, 16%). taken together, these findings suggest that the h9n2 viruses mainly replicated in the intestinal tract of chickens and minor poultry species. also, the high isolation rate in drinking water samples could be a sensitive indicator for monitoring the prevalence of h9n2 viruses in the field. to better understand the evolutionary pathway of h9n2 viruses in southern china, 50 representative viruses, isolated from hong kong live-poultry markets from 2005 to 2009, were sequenced and genetically characterized. phylogenetic analysis of the h9 ha gene revealed that ck ⁄ bei-like viruses were predominant and one chicken isolate had a g1-like ha gene ( figure 1 ). this is the first time the g1like h9 ha gene has been detected in chickens from livepoultry markets in hong kong. the ck ⁄ bei-like lineage is further divided into two subgroups as previously described. 1 subgroup 1 is represented by qa ⁄ st ⁄ 243 ⁄ 00 and subgroup 2 is represented by dk ⁄ hk ⁄ y280 ⁄ 97. all h9n2 viruses in this study belonged to subgroup 2 of the ck ⁄ bei-like lineage except for the virus with the g1-like ha gene. phylogenetic analysis of the na gene also showed a similar evolutionary pattern to the ha gene with all viruses clustered within the ck ⁄ bei-like lineage. these results revealed that ck ⁄ bei-like viruses are predominant in both chickens and minor poultry. all of the pb1, pa, np, ns and m genes clustered with those of h9n2 lineage viruses previously prevailing in terrestrial poultry in southern china. phylogenetic analysis of the pb2 gene revealed three different lineages; g1-like (n = 1), ck ⁄ sh ⁄ f ⁄ 98-like (n = 15), and unknown avian (n = 34). the sh ⁄ f ⁄ 98-like lineage (or f ⁄ 98-like) was previously reported in eastern china 12 and was used previously for vaccine production in an intensive vaccination program. 13 this pb2 gene lineage was also distinguishable from the ck ⁄ bei-like lineage and its presence in the viral genome may be due to reassortment between the vaccine strain and field isolates, followed by selective establishment in terrestrial poultry. gene constellation analyses of the 50 viruses revealed six genotypes. thirty-four of the viruses analyzed belonged to two genotypes, b14 and b15, which were also the prevailing reassortants found in other provinces in southern china since 2005. 2 the remaining sixteen viruses belonged to four novel genotypes that have not been identified before in this region. characterization of h9n2 influenza viruses isolated from live poultry in hong kong markets from a 5 year surveillance program revealed that ck ⁄ bei-like viruses were predominant in southern china and were continuing to evolve. two recognized and four novel genotypes were identified in this study. one characterized virus, ck ⁄ hk ⁄ nt499 ⁄ 07, had a g1like ha gene (the first time this has been detected in hong kong poultry markets) that showed a close relationship with two human h9n2 strains isolated in 2009. g1-like viruses were usually detected and caused outbreaks in chickens of middle eastern and european countries, [14] [15] [16] and minor poultry, mainly quail, in southern china. 1 whether the g1-like virus was transmitted from china to middle eastern and european countries, as the highly pathogenic h5n1 virus did in the last five years, or vice versa, is still unknown. since the ck ⁄ hk ⁄ nt499 ⁄ 07 strain clustered with other g1-like strains isolated previously in minor poultry in southern china, the g1-like viruses in chicken may be due to interspecies transmission from minor poultry species. genetic studies demonstrated that reassortants with genotypes b14 and b15 persistently occurred in either chickens or other minor poultry species from 2005 to 2009. other genotypes that were prevalent in southern china might be being gradually replaced and four novel genotypes were identified in this study. these novel genotypes were generated through reassortment of viruses with different lineages. a newly emerged f ⁄ 98-like lineage originating from eastern china is responsible for generation of some of the novel genotypes found in this study. 12 the ck ⁄ bei-like lineage is gradually being replaced by f ⁄ 98-like lineages which are becoming dominant in northern and eastern china. 12, 17 animal experiments have also demonstrated that f ⁄ 98-like viruses are more effective in replication and transmission in chickens compared with ck ⁄ bei-like viruses. 18 since the f ⁄ 98-like lineage of the pb2 gene has been introduced into southern china, this newly emerged lineage may have a higher tendency to replace the rnp genes in the circulating ck ⁄ bei-like viruses and subsequently become the endemic virus in terrestrial poultry. in vietnam, the modelling of the pandemic h1n1 progression estimates that 460 000 (260 000-740 000) pigs might be exposed to the virus on the basis of 410 000 cases among swine owners (220 000-670 000). 1 a poor level of biosecurity, high animal densities, and a mix of species could increase the risk of influenza virus flow, persistence, and emergence on swine and poultry farms. this study was set up in the red river delta, where a third of the national pig husbandry is produced. 2 the aims are to give preliminary information of the epidemiological state of swine influenza and in order to further assess the risk of infection of swiv, through cross-species transmissions from poultry to pigs. this paper will present the preliminary results on swiv and the risk factors of pig seropositivity in vietnam. a cross-sectional study was conducted in two provinces of the red river delta in april 2009. pig farms were randomly selected from nine communes representative of at risk area of avian h5n1. in each farm, pig and poultry were sampled and collected to virological and serological analyses. interviews were conducted in all farms by trained interviewees. questionnaires included closed and open questions on ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 60-78 livestock husbandry ⁄ management and household characteristics, such as herd size and structure, health history and vaccination, pig housing, watering and feeding system, reproduction, purchasing of animals, biosecurity measures, pig contact with poultry, and environmental factors. the virological detection assay was performed on pools of nasal swab specimens from pigs. we investigated whether real-time rt-pcr assay could detect gene m on pools of nasal swab specimens before attempting virus isolation from individual nasal swab specimens. the poultry and pig sera were tested against influenza type a with an enzyme-like immunosorbant assay (elisa) competition test idvetª. this commercial kit is designed to specifically detect antibodies directed against the np protein antigen of influenza type a viruses. the positive serum samples were examined in hemagglutination inhibition (hi) to determine antibody titers and subtypes. the hi test was tailored for h1, h3, and h9 subtypes in pigs and h6 and h9 subtypes in poultry. seroneutralization tests by pseudo particles were used to test the presence of antibodies directed against h5 subtype. we analysed the data for relationships between influenza a serological status (the outcome variable) and possible risk factors using r version 2ae11ae1 (r development core team). the statistical unit was the individual. initially, the quantitative variables were encoded into categorical variables according to the quartiles or median. descriptive statistics (e.g., means or medians, proportions, standard deviations) were calculated for all herd-level and commune level predictors to assist in the subsequent modeling process. we also performed the independence test among all variables to determine if variables were dependant. then, univariate analysis of potential risk factors for the pigs being positive for swiv and estimation of odds ratios were performed using generalised linear mixed models with binary outcome and logit link function for each herd-level and commune-level variable to determine which variables were individually associated with influenza a seropositivity at a significance level of p < 0ae30. herd and commune of residence were included as a random effect to account for the correlation of observations at the herd level. the third stage of the analyses included the four herdlevel variables found to be significantly (p < 0ae30) associated with influenza a seropositivity. an automatic process using all possible associations between the selected variables was computed into a mixed logistic regression models, with random effects. when two variables were collinear, as determined before, only one variable was likely to enter the multivariable model, and therefore, the selection of which collinear variable to enter the model was guided by biological plausibility and statistical significance. all of the 146 pools of nasal swabs were rt-pcr negative. the maximal possible prevalence considering perfect diagnostic tests would be of 2ae03% at a confidence level of 95%, in an infinite population within these regions (win-episcope 2ae0). six hundred-and-nine pig sera were tested in 76 nonvaccinating farms. the herd seroprevalence of swine influenza in the commune previously infected by the avian h5n1 in the red river delta raised by 17ae1% [8ae7; 25ae6] in april 2009. but among 13 seropositive farms, only four had at least two seropositive pigs. the within-herd seroprevalence is very low, and no seropositivity was detected in the majority of farms. estimates had large confidence intervals due to small sample sizes. the individual seroprevalence raised 3ae62% [1ae98; 5ae27]. the subtyping of seropositive sera is still in process. descriptive statistical analyses on five major risk factors of swiv: farm size, breeding vs. fattening, purchasing, percentage of family income, and poultry production, were conducted. based on this analysis, three types of farming systems were identified and included in mixed models ( table 1) . percentage of family income by pig production and poultry production were not differentiating factors for this typology. whereas types 1 and 2 seem to be specialized in fattening, the type 3 produces and might sell piglets on the farm site. the exploration of the different variance components indicated that the random effect variances were mainly associated with the herd, while the commune did not seem to have any effect. therefore we included in all models only the herd as a random effect. the random effect term for herd was modelled, assuming a normal distribution with a table 1 . typology of farming system type 1: large fattening farms largest scale production, with more than 40 pigs per year specialized in fattening, and purchase more than 20 pigs per year type 2: small fattening farms small scale of production, with less than 20 pigs per year specialized in fattening, and purchase less than 20 pigs per year type 3: medium breeding-fattening farms medium scale of production, with less than 40 pigs per year breeding and fattening piglets, with rare purchase common variance [$n(0,r2herd)]. 3 the univariate analyses were conducted on 22 variables and typology variables, with herd as random effect. some coefficient or confidence intervals were inconsistent because of small effectives, especially for the percentage of self-product culture or the pig freegrazing because of the lack of positive results in the dataset. the only one significant (p value < 0ae1) parameter was the percentage of pig sales in the familial annual income. surprisingly, common risk factors of swine influenza infection, such as farm size, animal movements, and sanitary parameters got low odds ratio individually (without being significant); the typology provides the hypothesis of complex interactions effects that increase the risk of infection. as shown in table 2 , the farming system type 3 got a higher seroprevalence of 6ae47% [3ae00-11ae94] and a higher risk indicator, with or = 5ae26 (p-value = 0ae18) in comparison with type 1. this finding was not significant. in the multivariate mixed model, the percentage of familial income provided by pig production was the only one significant variable, with or = 0ae22 [0ae04-1ae25]. the focus on diseased animals in the winter-time is usually required in order to increase the likelihood to isolate the virus, although the isolation rate on healthy or clinical samples never exceed 6%. 4 the season and the lack of disease reports might explain the difficulties to detect influenza viruses. additionally, the pooling method tends to decrease the isolation rate because of a dilution effect, potential presence of pcr assay inhibitors, or uneven distribution of virus in the sample. 5 our seroprevalence results must be confirmed and the subtypes identified, especially because we found only one positive animal in a few farms that could be attributed to false positive results of the elisa test (performances are not known). these preliminary results are in favor of a virus circulation at low level in the spring, but must be completed by further surveys in the winter and before the new year (têt celebration) when pig production, trade, and movement increase at their maximum. no clear prior information on the expected prevalence of swine influenza in vietnam, tests sensitivity, and speci-ficity could be obtained from literature or reliable sources. bayesian methods will be carried out in the future in order to compute prevalence and ⁄ or to estimate the probabilities of freedom. the risk factors analysis was limited by the lack of positive results. further studies are necessary to identify the at-risk season and type of farming systems at risk of swine influenza infection. however, this investigation of risk factors leads to the hypothesis that medium size breeding-fattening farms had a higher risk than large or small size fattening farms. further investigation are needed to precise this typology. the risk of swiv infection increases with a combination of three major factors. poultry production does not seem to play any role on swine infection. the generalized linear mixed model afforded to take into account all the non investigated parameters at the herd level. although we investigated the most common risk factors of swine influenza infection covering different kind of fields, the herd random effect might explain risk variations. mixed models have become a frequently used tool in epidemiology. due to software limitations, random effects are often assumed to be normally distributed. since random effects are not observed, the accuracy of this assumption is difficult to check. 6 further studies, such as case-control or cohort studies could help to identify more precisely risk factors of swine influenza seropositivity, as these study designs are more adapted than cross-sectional studies. the concept that swine are a mixing-vessel for the reassortment of influenza viruses and for the emergence of pandemic influenza viruses has been re-enforced by the emergence of the recent pandemic. 1 the pandemic h1n1 virus of 2009 (h1n1pdm) is believed to have emerged through the reassortment of north american triple reassortant and eurasian avian-like swine influenza viruses. 2 since the immediate precursor of this pandemic virus has not yet been identified, it is not possible to be definite whether the reassortment leading to the pandemic occurred in swine, but swine influenza viruses are the nearest known ancestors of each gene segment of h1n1pdm. 1, 2 the mechanisms of pandemic emergence are not clear. it is believed that the pandemics of 1957 and 1968 arose through reassortment of the pre-existing human seasonal influenza virus with avian influenza viruses, and swine have been proposed to be a possible intermediate host where such reassortment between human and avian viruses may take place. 4 the 2009 pandemic was the first to arise for over 40 years and the first to occur after the understanding that pandemics arise from animal influenza viruses. 3 systematic studies of influenza virus ecology and evolution in swine are, therefore, important in order to understand the dynamics of pandemic emergence. furthermore, since swine are the likely host within which h1n1pdm virus originated, it was predicted that this virus would readily infect swine and may reassort with endemic swine influenza viruses. these predictions have now been confirmed with reports of h1n1pdm being detected in pigs in many countries and reassortment with endemic swine influenza virus being confirmed. 5 while h1n1pdm has been genetically and antigenically stable in humans, reassortment between h1n1pdm, which is well adapted to transmission in humans, and other avian or swine viruses may lead to the origin of novel viruses posing a threat to public health. in addition to endemic swine virus lineages, avian influenza viruses such as h9n2 and highly pathogenic avian influenza (hpai) h5n1 have also been occasionally identified in pigs in parts of asia. 6, 7 it has been shown that h1n1pdm readily reassorts with h5n1 to generate viable progeny in vitro. 8 it is therefore essential to monitor the ecology, evolution, and biological characteristics of swine influenza viruses so that their continued evolution and zoonotic and pandemic potential can be monitored. there is however, a paucity of surveillance data on swine influenza viruses worldwide. this is in part related to the negative commercial consequences that may arise from detection of influenza in a swine herd leading to a major economic loss to the producer. here we outline a surveillance system that has been in place in hong kong for the last decade, based on sampling animals arriving at an abattoir in hong kong. we demonstrate the feasibility of such surveillance in an abattoir setting and compare methods used for detection influenza viruses in swine. virus isolation was carried out by inoculation into mdck cells and by allantoic inoculation in embryonated eggs as previously described. 6 virus isolates were subtyped by haemagglutination inhibition tests using specific antisera and genetically characterized by sequencing and phylogenetic analysis of the haemagglutin gene. 2, 6 virus detection by rt-pcr a subset of 1154 recent specimens was tested in parallel by real time pcr using the biorobot universal system (qiagen) that enables fully-automated viral nucleic acid extraction and downstream reaction setup in a 96-well plate format. total viral nucleic acids were extracted in a 96-well plate format with the qiaamp virus biorobot mdx kit (qiagen) on the biorobot universal system (qiagen) according to the manufacturer's instructions. briefly, 140 ll of sample was lysed in 280 ll buffer al, supplemented with 5ae6 lg carrier rna in a s block (qiagen), which placed the samples into a 96 well plate format. after protease digestion, samples were transferred to silica based membrane in 96 well plate format for binding. following two washing steps, rna was eluted in 80 ll of elution buffer (buffer ave) into a 96 well elution microplate cl (qiagen) . for the synthesis of cdna, 10 ll of purified rna was used in a 20 ll reaction containing 4 ll of 5· buffer, 0ae75 nm of each deoxynucleotide triphosphate (dntp), 10 mm dithiothreitol, 3 lg random primer, 40 u of rnaseout recombinant ribonuclease inhibitor, and 200 u of superscript iii reverse transcriptase (all from invitrogen). reactions were performed in the geneamp 9700 thermocycler (applied biosystems) with the following parameters: 60 minutes at 50°c, 15 minutes at 72°c, and soak at 4°c. subsequent to the reactions, 20 ll of cdna was diluted 1 ⁄ 10 by adding 180 ll of ae buffer (qiagen) . real-time pcr was performed using the power sybrò green pcr master mix (applied biosystems) according to the manufacturer's instructions. briefly, 5 ll of 1⁄ 10 diluted cdna was amplified in a 25 ll reaction containing 12ae5 ll of 2· power sybr green pcr master mix, 800 nm of forward primer m52c (5¢-ctt cta acc gag gtc gaa acg-3¢) and 800 nm of reverse primer m253r (5¢-agg gca ttt tgg aca aag ⁄ t cgt cta-3¢). the primers have been designed to amplify the sequences in the conserved region of influenza a virus matrix gene, thereby detecting viruses from different species including swine influenza viruses. 8 real-time pcr was performed in the abi 7500 fast system (applied biosystems) with the following cycling conditions: 10 minutes at 95°c once, 30 seconds at 95°c, and 1 minutes at 50°c for 40 cycles, followed by melting curve analysis with 15 seconds at 95°c, 1 minutes at 50°c, and 15 seconds at 95°c. in each assay, serially diluted plasmids containing the full length m gene cloned from a ⁄ vietnam ⁄ 1204 ⁄ 2004 (h5n1) were included as standards to perform absolute quantification. a manual baseline was set from cycles 3-15 and a manual cycle threshold (ct) was set at 0ae2. samples that were positive or unequivocal results from the real-time pcr were confirmed by performing gel electrophoresis on the pcr products. positive visual identification was made in the presence of the target pcr product at 244 bp in length. a total of 16 566 tracheal and 11 100 nasal swabs were processed during the years january 2002-april 2010 and yielded 393 influenza virus isolates, an overall virus isolation rate of 1ae4%. of these, 352 were subtype h1 (classical swine, eurasian avian-like swine, and triple-reassortant), 25 were human-like h3 viruses, and 16 were eurasian avianlike swine h3n2 viruses. culture in mdck cells yielded 95% of h1 subtype viruses, 100% of the human seasonal-like h3n2 viruses, and 68ae8% of the avian-like eurasian swine h3n2 viruses. culture in embryonated eggs yielded 17ae9% of the h1 subtype viruses, 0% of the human seasonal-like h3n2 viruses, and 62ae5% of eurasian avian-like swine h3n2 viruses ( figure 1 ). tracheal and nasal swabs each gave comparable overall virus isolation rates (1ae4%). however, isolation rates for human-like h3n2 viruses were 4ae8 fold higher in nasal swabs (0ae17% versus 0ae036% respectively; p = 0ae002) ( figure 1) . a parallel evaluation of rt-pcr and culture was carried out in 1154 specimens. rt-pcr detected 10 ⁄ 12 (83%) of the culture positive specimens. rt-pcr was also positive in 8 ⁄ 1154 (0ae7%) culture negative specimens, but all these specimens had very low virus load in the rt pcr tests. virus could not be cultured from these culture negative specimens even by attempts at virus re-isolation from the frozen specimen. surveillance in an abattoir setting provides an acceptable yield of influenza viruses and is a feasible method of swine influenza surveillance. sampling in a large abattoir setting allows surveillance to be carried out anonymously with no negative consequences to the supplier. the supply-chain of pigs to the hong kong abattoir involves pigs being trucked in over long distances and may provide opportunity for virus amplification during transport. thus, virus isolation rates may be lower in more vertically integrated and homogenous production and slaughter systems where less mixing of pigs occurs. our results indicate that mdck cell culture is essential for optimizing virus isolation during swine influenza surveillance. allantoic inoculation of embryonated eggs by itself is sub-optimal for isolation of swine influenza viruses. it is however possible that inoculation of embryonated eggs by the amniotic route may lead to better isolation rates than allantoic inoculation. rt-pcr detection is an alternative method for virus detection. but the additional specimens detected by rt-pcr did not yield culturable virus, even following attempts at re-isolation and sequential passage. the rt-pcr positive ⁄ virus isolation negative specimens had very low virus load, and this may be the explanation for the inability to isolate such viruses. in addition, rt-pcr did not detect all viruses isolated by culture. tracheal and nasal swabs gave comparable isolation rates with the exception of human-like h3n2 viruses which were more frequently isolated from nasal swabs. this may suggest that, in contrast to endemic swine influenza virus lineages, these human-like h3n2 viruses are less adapted to replication in the lower respiratory tract. in summary, collection of nasal or tracheal swabs in an abattoir setting together with virus isolation in mdck cells provides a feasible approach to surveillance of swine influenza viruses. kong, 2003 kong, -2010 introduction wild waterfowl are the natural reservoir of influenza a viruses (aiv), and they play an important role in the genesis of pandemic influenza. it is suggested that the 1918 pandemic virus was purely derived from avian virus, which adapted to humans and caused efficient human-to-human transmission, 1 while the pandemics of 1957 and 1968 had acquired the viral haemagglutinin, pb1 polymerase, and in 1957, the neuraminidase gene segments from the avian gene pool. 2 the major regional outbreaks of highly pathogenic avian influenza (hpai) h5n1 in asia, europe, and africa highlight the potential role played by migratory waterfowl in disseminating highly pathogenic influenza viruses. 3 therefore defining the influenza virus gene-pool in wild birds is of vital importance. surveillance was carried out 2-3 times weekly from 2003 to 2010 during the winter months of october to april in the hong kong mai po nature reserve and lok ma chau, hong kong. the hong kong mai po nature reserve and lok ma chau are along the east asia-australian flyway where a peak of more than 30 000 ducks and grebes congregate every winter. 4 fecal droppings were collected and transported in vials containing 1ae0 ml of vtm, which was prepared from m199 (9ae5 g ⁄ l), penicillin g (2 · 10 6 u ⁄ l), polymyxin b (10 · 10 6 u ⁄ l), gentamicin (250 mg ⁄ l), nystatin (0ae5 · 10 6 u ⁄ l), ofloxacin hcl (100 mg ⁄ l), and sulfamethoxazole (1 g ⁄ l). an aliquot of 100 ll from each swab sample was inoculated into the allantoic cavity of a 9-to 11-day-old chicken embryonated egg, and incubated for 3 days at 37°c. positive ha isolates were subtyped using standard antisera 5, 6 and rt-pcr was performed with the used of one-step rt-pcr assay (invitrogen) described earlier, 7 followed by sequencing on abi prism 3730xl dna analyzer. the determination of species of origin was performed by dna barcoding of the mitochondrial cyto-chrome oxidase i gene from dna extracted from the fecal droppings. 8 during the 7-year surveillance period, a total of 193 influenza viruses were isolated from 39 354 samples collected, an overall isolation rate of 0ae49%. a total of 192 isolates were obtained from 39 152 specimens collected during the winter period coinciding with the southern migration of waterfowl along the east asian flyway and one isolate obtained from 202 samples collected in spring during the period when northern migration of waterfowl took place along the east asian-australasian flyway. the isolation in hong kong was slightly lower than a similar study conducted in south korea in which the isolation rate of migratory birds was 0ae8% in 2003-2008. 9 this suggested a slightly lower prevalence of influenza virus present in hong kong as the birds migrated southwards. the viruses isolated in hong kong, representing hemagglutinin (ha) subtypes of h1-h12 and neuramidinase (na) subtypes of n1-n9, were all from wild waterfowl ( table 1) . out of the twelve ha subtypes isolated, h10 and h6 were the two subtypes that were isolated frequently every year for h10 and in six out of seven years for h6, respectively. h10 and h6 viruses accounted for 16ae7% and 22ae4% of all virus isolated, respectively. on the other hand, h3, h9, and h12 were the least prevalence (0ae5%) and were only isolated once in 7 years. of the na subtypes, n1 and n2 were isolated most often (24ae4% and 19ae2% of all isolates, respectively) and n5 was the least (0ae5%). november was the month that had the highest prevalence of influenza virus (0ae83% of samples being positive) compared to only 0ae3% in march. the subtype's variation was the most diverse in december during our 7 years of surveillance. this suggested that more of these wild migratory birds may be carrying influenza virus when they arrive in hong kong. however the continued isolation of viruses suggests continued circulation of these viruses in the vicinity of mai po. the study of dna barcoding for the mitochondrial cytochrome oxidase i gene retrieved from fecal droppings revealed that the isolates originated mainly (89ae2%) from birds of the order anseriform, family anatidae including eurasian wigeon, northern shoveler, northern pintail, common teal, and garganey. non-anseriformes which were found to have shed aiv viruses were cormorant, grey heron, and stint. none of the water samples collected from the ponds where these birds congregate were found to be positive for the virus. phylogenetic analyses of the ha gene of the lpai h5 viruses isolated in this study clustered with that of the other lpai h5 viruses isolated from hokkaido, mongolia, and siberia and were not closely related to the hpai h5n1. satellite tracking of 47 eurasian wigeons and northern pintails in dec 2008 and 2009 revealed their flyway from hong kong to as far north as eastern russia, eastern mongolia, and northern china. no hpai h5n1 viruses were isolated in this study from apparently healthy birds. however, as part of the surveillance of dead wild birds carried out by the department of agricultural, fisheries and conservation of the government of hong kong during this same period, over 50 dead wild birds were tested positive for hpai h5n1 and has been reported elsewhere. 10 our influenza surveillance in hong kong has revealed a diversity of influenza virus subtypes the migratory waterfowl infected within the region. the result of the phylogenetic analysis correlated with the findings from satellite tracking that viruses isolated in hong kong were closely related to those isolated in areas along the migratory route. no healthy bird was isolated with hpai h5n,1 although dead wild birds have been regularly found to have hpai h5n1 virus, suggesting that infected birds might not live for a long period. introduction a novel swine-origin h1n1 influenza virus emerged in mexico in april 2009 and rapidly spread worldwide, causing the first influenza pandemic of the 21st century. 1 most confirmed human cases of h1n1 ⁄ 2009 influenza have been uncomplicated and mild, but the increasing number of cases and affected persons worldwide warrant optimal prevention and treatment measures. today, almost all of the pandemic h1n1 ⁄ 2009 viruses tested are resistant to m2blockers. 2 therefore, only the neuraminidase (na) inhibitors are currently recommended for treatment of this pandemic influenza. for the control of influenza infection, the clinical use of oseltamivir has increased substantially during the 2009 pandemic. to date, the majority of tested clinical isolates have remained susceptible to na inhibitors, oseltamivir and zanamivir, 2 but oseltamivir-resistant variants with h275y na mutation (n1 numbering) have been isolated from individuals taking prophylaxis, from immunocompromised patients, and from a few community clusters. 3, 4 in view of the high prevalence of oseltamivirresistant seasonal h1n1 influenza viruses in 2007-2008, the isolation of resistant h1n1 ⁄ 2009 viruses without known oseltamivir exposure raised great concern about the transmissibility and fitness of these resistant viruses. here we studied the transmissibility of a closely matched pair of pandemic h1n1 ⁄ 2009 clinical isolates, one oseltamivir-sensitive and one resistant, in both direct contact and respiratory droplets routes among ferrets. viral fitness was evaluated by co-infecting a ferret with both the oseltamivir-sensitive and -resistant viruses. the viruses were also characterized by full genome sequencing, susceptibility to na inhibitors, and growth in mdck and mdck-siat1 cells. oseltamivir-resistant influenza a ⁄ denmark ⁄ 528 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 528 ⁄ 09) was isolated from the throat swab of a patient who had influenza-like symptoms and received post-exposure oseltamivir prophylaxis (75 mg once daily). 5 wild-type influenza a ⁄ denmark ⁄ 524 ⁄ 09 (h1n1) virus (a ⁄ dm ⁄ 524 ⁄ 09) was isolated from a patient in the same cluster of infection as the a ⁄ dm ⁄ 528 ⁄ 09 virus. to assess growth kinetics of viruses, confluent mdck or mdck siat1 cell monolayers were infected with viruses at a multiplicity of infection (moi) of approximately 2ae0 pfu ⁄ cell (single-step) or 0ae001 pfu ⁄ cell (multi-step). supernatants were collected every 2 h or 12 h p.i. for 6 time points. a modified fluorometric assay using the fluorogenic substrate 2¢-(4-methylumbelliferyl)a-d-n-acetylneuraminic acid (munana) was used to determine viral na activity. 6 the drug concentration required to inhibit 50% of the na enzymatic activity (ic 50 ) was determined by plotting the percent inhibition of na activity as a function of compound concentration calculated in the graphpad prism 4 (la jolla, ca) software from the inhibitor-response curve. na enzyme kinetics were determined by measuring na activity every 60 seconds for 60 minutes under the same conditions as above, when all viruses were standardized to an equivalent dose of 10 6ae0 pfu ⁄ ml. the k m and v max were calculated by fitting the data to the appropriate michaelis-menten equations using nonlinear regression in the graphpad prism 4 software. young adult ferrets (4-5 months of age) were obtained from the ferret breeding program at st. jude children's research hospital. all ferrets were seronegative for influenza a h1n1 and h3n2 viruses and for influenza b viruses. for transmission studies, the donor ferrets were lightly anesthetized with isoflurane and inoculated intranasally with 10 6 tcid 50 virus in 1ae0 ml sterile pbs . after the donor ferrets were confirmed to shed virus on day 2 p.i., each donor was then housed in the same cage with two naïve direct-contact ferrets. two additional recipient ferrets were placed in an adjacent cage isolated from the donor's cage by a two layers of wire mesh (approximately 5 cm apart) that prevented physical contact but allowed the passage of respiratory droplets. ferret weight and temperature were recorded daily for 21 days. nasal washes were collected from donors and recipients on day 1, 2, 4, 6, 8, 10, 12, and 14 p.i. by flushing both nostrils with 1ae0 ml pbs, and tcid 50 titers were determined in mdck cells. serum samples were collected 3 weeks after virus inoculation, and were tested for seroconvention by hi assay. full genome sequencing revealed that the pair of h1n1 ⁄ 2009 viruses differed only at na amino acid position 275, where the pandemic a ⁄ dm ⁄ 528 ⁄ 09 virus had an h275y amino acid mutation caused by a single t-to-c nucleotide substitution at codon 275. the wild-type a ⁄ dm ⁄ 524 ⁄ 09 was susceptible to oseltamivir carboxylate (mean ic 50 : 5ae0 nm), but the a ⁄ dm ⁄ 528 ⁄ 09 carrying the h275y na mutation had ic 50 values approximately 200-300 times of the wild-type viruses (mean ic 50 : 972 nm). the ic 50 of zanamivir was comparable for both viruses and were uniformly low (mean ic 50 £ 1ae3 nm). the h275y na mutation confers resistance to oseltamivir carboxylate but did not alter susceptibility to zanamivir. to understand the impact of the h275y mutation on the na enzymatic properties, na enzyme kinetics was determined. the na of the oseltamivir-resistant virus had a slightly higher k m (mean = 55 lm) and lower v max (mean = 101 u ⁄ sec) than na of the sensitive virus (k m , mean = 80 lm; vmax, mean = 86 u ⁄ sec). the results suggested that the h275y na mutation reduced na affinity for substrate and na catalytic activity, although the function of na was not severely impaired. to further evaluate the impact of the h275y na mutation on virus growth in vitro, single-and multi-cycle growth studies of both viruses were performed in mdck and mdck-siat1 cells. in the both single-and multiple-cycle growth curves, the two viruses reached comparable levels eventually, but the initial growth of the resistant virus was significantly delayed by at least 1-2 logs in comparison to that of wild-type virus (p < 0ae05). the donor ferrets inoculated with wild-type a ⁄ dm ⁄ 524 ⁄ 09 or oseltamivir-resistant virus shed virus productively until day 6 or day 8 p.i., with a peak virus titer comparable to that of a ⁄ dm ⁄ 524 ⁄ 09 virus (table 1 ). in a ⁄ dm ⁄ 524 ⁄ 09 virus group, two of 2 direct-contact ferrets the weight loss in ferrets is the maximum percentage loss compared with the initial weight. virus shedding is indicated as number of virus-shedding animals ⁄ total number; mean peak virus titer (log 10 tcid 50 ⁄ ml) in nasal wash samples is indicated in parentheses. serum hemagglutination inhibition (hi) titer to homologous virus in ferret serum was determined on day 21 p.i. duan et al. and 1 of 2 respiratory droplet-contact ferrets were infected through virus transmission, as indicated by the virus titers and inflammatory cell counts in their nasal washes and also by sero-conversion. under identical conditions, in a ⁄ dm ⁄ 528 ⁄ 09 group, only 2 of 2 direct-contact ferrets were infected through virus transmission, but neither respiratory droplet-contact ferrets was infected, as confirmed by the absence of sero-conversion (table 1) . virus shedding in the direct-contact ferrets was lower and peaked after a longer interval in this group than in the oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 group (table 1) , but the resistant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. these results showed that an oseltamivir-resistant h275y mutant of pandemic h1n1 virus, a ⁄ dm ⁄ 528 ⁄ 09 virus could be only transmitted efficiently by direct contact. to compare the relative fitness, growth capability, and transmissibility of the sensitive and resistant h1n1 ⁄ 2009 viruses within host, a donor ferret was co-inoculated with a 1:1 ratio of the sensitive and resistant viruses, and another two naive ferrets were housed with the donor to test direct contact. during co-infection, the pattern of virus shedding and the clinical signs were similar to those in ferrets inoculated with either a ⁄ dm ⁄ 524 ⁄ 09 or a ⁄ dm ⁄ 528 ⁄ 09 virus (table 1 ). in the inoculated donor ferret, the virus population in the nasal washes remained mixed but wild-type viruses outgrew the resistant virus progressively ( figure 1 ). two of 2 direct-contact ferrets were infected through virus transmission, but only wild-type virus was detected in both direct-contact ferrets ( figure 1 ). in summary, oseltamivir-sensitive a ⁄ dm ⁄ 524 ⁄ 09 virus possessed better growth capability in the upper respiratory tract than did resistant a ⁄ dm ⁄ 528 ⁄ 09 virus, and thus had an advantage in directcontact transmission. our study determined the comparative transmissibility of two naturally circulating oseltamivir-sensitive and -resistant pandemic h1n1 ⁄ 2009 viruses; we demonstrated inefficient respiratory-droplet transmission of an oseltamivir-resistant h275y mutant of pandemic h1n1 virus among ferrets, although it retained efficient direct-contact transmission. we suggest that the lower fitness of resistant virus within the host along with its reduced na function and delayed growth in vitro may in part explain its less efficient transmission. notably, the h275y mutant of h1n1 ⁄ 2009 used in this study was the first oseltamivir-resistant h1n1 ⁄ 2009 isolate from a patient on oseltamivir prophylaxis to be characterized for transmissibility. our observation in the animal model is consistent with the epidemiological data collected from humans, which showed no evidence of predominant or continued circulation of oseltamivir-resistant viruses. 4 as this study was undertaken, additional h275y mutants of h1n1 ⁄ 2009 viruses have emerged in the absence of oseltamivir use. 7, 8 the emergence of these viruses should raise concerns as to whether resistant h1n1 ⁄ 2009 viruses will acquire greater fitness and spread worldwide as the naturally resistant h1n1 viruses did during the 2007-2008 season. two independent studies have evaluated the pathogenecity and transmission of other oseltamivir-resistant pandemic h1n1 ⁄ 2009 clinical isolates in the animal models. 9, 10 one of the studies, 9 which also used an oseltamivir-resistant virus isolated from a patient under oseltamivir prophylaxis, observed similar results as ours: although the respiratory-droplet route of transmission was not investigated, it was shown that the resistant isolate was transmitted though direct-contact route and was as virulent as wild-type virus in ferrets. 9 in another study, 10 two oseltamivir-resistant isolates were transmitted through the respiratory-droplet route in ferrets, and the dynamics of transmission were different between the two isolates. apparently, these two oseltamivir-resistant isolates were still unequal in their transmissibility and were disparate from the resistant isolate in our study. the isolation history of the two resistant isolates was unclear in this study, and this would be an important factor to understand the fitness of drug-resistant viruses. further studies with more clinical isolates of diverse isolation background are warranted to identify how these novel h275y mutants of pandemic h1n1 ⁄ 2009 virus have changed to retain their full transmissibility. taken together, all these related studies underline the necessity of continuous monitoring of drug resistance and characterization of potential evolving viral proteins. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of pigs have been considered as hypothetical ''mixing vessels'' facilitating the genesis of pandemic influenza viruses. 1, 2 the pandemic h1n1 ⁄ 2009 virus (ph1n1 ⁄ 09) contained a very unique genetic combination and was thought to be of swine origin, as each of its eight gene segments had been found to be circulating in pig populations for more than a decade. 3 however, such a gene constellation had not been found previously in pig herds all around the world. only after its initial emergence in humans has this virus been repeatedly detected in pigs, and found to further reassort with other swine influenza virus. [3] [4] [5] a primary question remaining to be answered is whether the ph1n1 ⁄ 09-like and their genetically related viruses could become established in pig populations, thereby posing novel threats to public health. despite the fact that ph1n1 ⁄ 09 first appeared in mexico and the united states, and six of its eight gene segments were derived from the established north american triple reassortant swine influenza virus (trig), its neuraminidase (na) and matrix protein (m) genes belonged to the eurasian avian-like swine lineage (ea), which had never been detected in north america previously. 3, 6 likewise, the trig-like viruses were never reported in europe. 6 in contrast, both lineages of virus were frequently detected in asia, and reassortants between them have also been documented in recent years. 3, 5 this has given rise to a complicated ecological situation, i.e. the simultaneous prevalence of multiple genotypes of h1n1 and h1n2 viruses in pigs. 3, 5 among them, two representative reassortants showed the most similar genotypic characterization to the ph1n1 ⁄ 09 virus, the sw ⁄ hk ⁄ 915 ⁄ 2004 (h1n2) and sw ⁄ hk ⁄ 201 ⁄ 2010 (h1n1), which respectively harbor seven and six gene segments closely related to the pandemic strains. 3 , 5 to understand their in vivo characteristics and zoonotic potential, these two viruses, together with a human prototype strain and a swine ph1n1 ⁄ 09-like isolate, were chosen for a study of their pathogenicity and transmissibility in domestic pigs, ferrets, and mice. the prototype ph1n1 ⁄ 09 virus, a ⁄ california ⁄ 04 ⁄ 2009 (ca04), was provided by the world health organization collaborating centers for reference and research on influenza (atlanta, ga, usa). three ph1n1 ⁄ 09-related swine influenza viruses were isolated through our surveillance program in south china as previously described. 3, 5 the a ⁄ swine ⁄ guangdong ⁄ 106 ⁄ 2009 (h1n1, gd106) virus was a ph1n1 ⁄ 09-like swine isolate. a ⁄ swine ⁄ hong kong ⁄ 915 ⁄ 2004 (h1n2, hk915), the closest pandemic ancestor known to date, possesses an m gene derived from the ea lineage, with the other gene segments from trig viruses. 3 a ⁄ swine ⁄ hong kong ⁄ 201 ⁄ 2010 (h1n1, hk201), a recent pandemic reassortant progeny, had a ph1n1 ⁄ 09like na gene (also belonging to the ea lineage), an ea-like hemagglutinin (ha) gene, and six trig-like internal genes. 5 all viruses were propagated in madin-darby canine kidney (mdck) cells for three passages, and their titers were determined by plaque assays. all experiments with live viruses were conducted in biosafety level 3 (bsl-3) containment laboratories. pigs (4-6 week old, n = 5-6) and ferrets (5 month old, male, n = 3) were intranasally infected with 10 6 pfu of each virus, and mice (8) (9) week old, female balb ⁄ c, n = 10) with a dose of 10 4 pfu. naïve uninfected pigs (n = 2) were co-housed in the same cage with the inoculated ones from each group. body weights and clinical signs were recorded daily. virus replication was determined by titration of the virus in nasal and rectal swabs (pigs), nasal washes (ferrets), as well as from lungs and other organs (pigs and mice). seroconversion was tested by hemagglutination inhibition (hi) assays. histopathological and immunohistochemical analysis were performed as previously described. 7 statistical analysis was performed by mean analysis with pasw statistics 18 (spss inc., chicago, il, usa). the probability of a significant difference was computed using anova (analysis of variance). results were considered significant at p < 0ae05. the pathogenicity of the four viruses tested differed significantly in inoculated mice. animals infected with 10 4 pfu of hk915 experienced the most severe body weight loss (25ae1 ± 4ae7%) but started to recover after 8 days post-infection (dpi). hk201 caused similar peak body weight loss (16ae9 ± 4ae6% on 8 dpi) in mice as did ca04 (17ae3 ± 2ae4%, on 7 dpi), but the onset of clinical signs and weight loss (on 4 dpi) was 1 day later than those caused by the other three viruses. the gd106-infected group suffered the least body weight loss (6ae9 ± 1ae9%, 5 dpi) and was the earliest to recover. although all four viruses were detected in the lungs with comparable virus titers on 3 dpi (p > 0ae05), mice inoculated with gd106 consistently showed the lowest lung index (lung weight ⁄ body weight, %) on 3, 6, and 14 dpi (p < 0ae01), suggesting the slightest injury and consolidation of the lungs. in concordance with the body weight change, the lung index from the hk915 group was higher than that from any other groups on 6 and 14 dpi, indicating the marked virulence of hk915 in mice. notably, virus titer of hk201 in the nasal turbinate was lower than the other groups both on 3 and 6 dpi (p < 0ae01), but virus replication in the lower respiratory tract was either higher (in the trachea) or similar (in the lungs). observations of the body weight changes caused by infection of ph1n1 ⁄ 09 or its genetically related swine viruses in ferrets have come to a similar conclusion as that for the mouse experiment. after nasal inoculation with 10 6 pfu of each virus, all groups of ferrets experienced transient body weight loss for 2-3 days, except for those infected with gd106, which showed no significant weight loss (p > 0ae05). although ferrets from the ca04-infected group reached their peak weight loss (6ae2 ± 0ae8%, 2 dpi) one day earlier than those from the hk201 and hk915 groups, they began to regain body weight quickly thereafter. hk201-infected ferrets also recovered rapidly and their body weights reached the same level as those of the gd106-infected group at 6 dpi. comparatively, ferrets inoculated with hk915 had the most retarded body weight recovery, which did not get back to the baseline level until 11 dpi. hk201 was only detectable in the nasal wash on 2 dpi, whereas the duration of virus shedding for gd106, hk915, and ca04 was 4-6 days. by combining the data obtained from the virus titration in the mouse turbinate and ferret nasal washes, a possible conclusion can be made that hk201 may have lower transmissibility than the other three viruses. after inoculation or exposure by direct contact (physical contact) with the ph1n1 ⁄ 09 virus and its close relatives, most pigs experience no or mild symptoms, such as slight loss of appetite and inactivity. body weight loss was only recorded in pigs inoculated with hk915 during the second week post-inoculation, but not in their contact pigs or in the other groups. diarrhea was observed intermittently in each of the inoculated or contact groups throughout the experiment, and viruses could be recovered in the rectal swabs, saliva, drinking water, and environmental swabs (inner cage walls accessible to the pigs) at various time points. however, virus titers in the positive rectal swabs were just slightly above the detection limit, while those from the environment sometimes could be higher. whether these viruses can replicate in the digestive tract or were just carried-over by contaminated foods and water requires further investigation. although virus could be detected in the nasal swabs of all infected or contact animals, the lowest peak titer was from pigs inoculated or in contact with hk201 (0ae5-1ae5 log tcid50 ⁄ ml lower than the other groups), suggesting unfavorable replication in the nasal cavity for this virus. postmortem examination on 4 and 7 dpi revealed that pigs infected with hk915 had the most extensive gross lesions in the lungs, and histochemical staining of viral nucleoprotein (np) in lung tissues on 4 dpi also suggested the best replication for hk915 in the lower respiratory tract. on 11 days post-contact (dpc), all pigs exposed to the inoculated animals developed sero-conversions (hi = 80-160) except for one from the gd106 contact group. however, on 17 dpc, its hi titer reached 40, indicating slower seroconversion. this study revealed that both the 2009 pandemic h1n1 and its genetically related swine viruses could readily infect mice, ferrets, and pigs causing mild to moderate clinical symptoms. they could also transmit efficiently between pigs. when compared with the pandemic stains and its reassortant progeny (hk201), the hk915 (h1n2) virus containing the ea-like m gene in the genetic context of the trig virus showed consistently higher virulence in all three mammalian models tested, but it is still unknown what might happen if such a virus further reassorts to obtain the pandemic-like or ea-like na gene. however, our findings suggest that pigs could likely maintain the prevalence of different genotypes of pandemic-related influenza viruses, and highlight the zoonotic potential of multiple strains of swine influenza virus. pandemic influenza viruses emerge from the animal reservoirs. 1 among the three pandemics that occurred in the last century, we learned that the 1957 h2n2 and the 1968 h3n2 pandemic viruses emerged by reassortment between circulating human virus and avian-origin influenza virus(es). 1 studies on the emergence of the catastrophic 1918 spanish h1n1 virus suggest that the virus may have obtained all of its eight gene segments from the avian reservoir, 2,3 or alternatively is a reassortant between mammalian and a previously circulating human influenza virus. 4 over 40 years since the last pandemic, the first pandemic in the 21st century arose in 2009 and was caused by a swine-origin influenza virus containing a unique gene combination, with gene segments derived from the circulating north america ''triple reassortant'' (pb2, pb1, pa, ha, np, and ns) and the ''eurasian'' (na and m) swine influenza viruses. 5, 6 analysis of the pandemic h1n1 viruses failed to identify known molecular markers predictive of adaptation to humans. 6 the ''triple reassortant'' swine influenza viruses emerged in late 1990s in north america is a reassortant between classical swine (descendent of the 1918 virus after adaptation in swine population), avian, and human influenza viruses. 7 the eurasian influenza virus was originally an avian influenza virus that was introduced into the european swine population in the late 1970s. 8, 9 while incidents of zoonotic infection with triple reassortant or eurasian influenza in humans have been reported, 10, 11 sustained human-to-human transmission has never been established. these results suggest that the unique gene combination seen with the pandemic h1n1 viruses may confer its transmissibility among humans. we have carried out systematic prospective surveillance of swine influenza in southern china over that last 12 years through samples routinely collected at an abattoir in hong kong. during this time, the surveillance results suggest co-circulation of classical swine h1n1, triple reassortant h1n2, eurasian swine h1n1, and a range of reassortants between these three virus lineages. 4, 12 ferrets have been reported as a suitable model for the study of influenza transmission as they are naturally susceptible to influenza infection, exhibit similar clinical signs (including sneezing), and possess receptor distribution in the airway similar to that of humans. [13] [14] [15] to identify molecular determinants that enable sustained human-to-human transmission, we compared the pandemic virus with genetically related swine influenza viruses obtained from this surveillance program for their ability to transmit from ferret to ferret by direct contact or aerosol transmission. viruses human h3n2 influenza virus [a ⁄ wuhan ⁄ 359 ⁄ 95 (wuhan95)] and pandemic h1n1 influenza viruses [a ⁄ california ⁄ 4 ⁄ 09 (ca04)] were included for the study. swine influenza viruses that are genetically related with the pandemic h1n1 virus were selected from our surveillance system, including classical swine-like influenza virus a ⁄ sw ⁄ hk ⁄ 4167 ⁄ 99 (h1n1) (swhk4167), triple reassortant-like a ⁄ sw ⁄ arkansas ⁄ 2976 ⁄ 02 (h1n2) (swar2976), and one reassortant between triple reassortant and eurasia swine influenza viruses [a ⁄ sw ⁄ hk ⁄ 915 ⁄ 04 (h1n2) (swhk915)]. swhk915 contains seven gene segments (pb2,pb1,pa,ha,np,m,ns) closely related to the pandemic h1n1 viruses. transmissibility was tested in 4-to 6-month-old male ferrets obtained from triple f farm (sayre, pa); all ferrets were tested to have hi titer £40 against human seasonal influenza h1n1 (a ⁄ tennessee ⁄ 560 ⁄ 2009), h3n2 (a ⁄ brisbane ⁄ 10 ⁄ 2007), and influenza b (b ⁄ florida ⁄ 4 ⁄ 2006) prior the experiments. in each virus group, three ferrets were inoculated with 10 5 tcid 50 of the virus. at 1 day postinoculation (dpi), we introduced one naïve direct contact ferret to share the cage with inoculated ferret, and one naïve aerosol contact ferret into the adjacent compartment of the cage separated by a double-layered perforated divider. nasal washes were collected every other day and tested for influenza virus antigen and to determine viral titers (tcid 50 ). weight changes, temperature, and clinical signs were monitored daily. transmission is defined by detection of virus from nasal washes and ⁄ or by seroconversion (>4fold rise in the post-sera collected after 16-18 days post contact). experiments were performed in the p2+ laboratory at st. jude children's research hospital. all studies were conducted under applicable laws and guidelines and after approval from the st. jude children's research hospital animal care and use committee. at 10 5 tcid 50 inoculation dose, all viruses replicated efficiently in the ferret upper respiratory tract with peak titers detected from inoculated ferrets at 2 dpi. lower peak titers were detected from swhk4167 and swhk915 inoculated ferrets, however, the differences were not statistically significant (table 1) . tissues collected from inoculated ferrets at 3 dpi showed that pandemic h1n1 and swine influenza viruses replicated both in the upper and lower respiratory tract of the ferrets, while the replication of human seasonal influenza wuhan95 was restricted in the upper respiratory tract. direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. human seasonal influenza (wuhan95) and pandemic h1n1 viruses (ca04) transmitted most efficiently via direct contact route as the virus can be detected on 2 dpi from direct contact ferrets, and the peak titers were detected on 4 dpi from direct contacts. moderate direct contact transmission efficiency was detected from swar2976 and swhk915 viruses as the virus can be detected from direct contact ferrets at 4 dpi, with peak titers detected at 4 dpi or 6 dpi. classical swine-like swhk4167 showed least efficient contact transmission as virus could be detected from all direct contacts only at 6 dpi, and the peak titer detected on 8 dpi. aerosol transmission was detected in groups of human seasonal influenza virus wuhan95 (2 ⁄ 3), pandemic h1n1 influenza virus ca04 (3 ⁄ 3), as well as swine precursor virus swhk915 (1 ⁄ 3). transmission of wuhan95 and ca04 to aerosol contacts was detected at 4 dpi or 6 dpi, while transmission of swhk915 was detected later at 8 dpi, suggesting that the swhk915 virus possessed aerosol transmission potential, but may require further adaptation to acquire efficient aerosol transmissibility. in addition to viral detection from nasal washes, we also detected viruses from the rectal swabs of ferrets inoculated or infected with pandemic h1n1 viruses (ca04) or classical swine-like virus (swhk4167), which share the common origin for the ha, np, and ns gene segments. while many of the swine influenza viruses studied were able to transmit via the direct contact route, swhk915, which shares a common genetic derivation for seven genes with h1n1pdm, possessed capacity for aerosol transmission, albeit of moderate efficiency. swhk915 differed from swine triple reassortant viruses in the origins of its m gene. it is possible that the m gene derived from eurasian avianlike swine viruses also contributes to the transmissibility of h1n1pdm influenza viruses. outbreaks of highly pathogenic avian influenza (hpai) of the h5n1 subtype are of extreme concern to global health organisations as human infection can result in severe acute respiratory distress syndrome, multi-organ failure, and coma. hpai viruses of either h5 or h7 subtypes contain a characteristic multi-basic cleavage site in the hemagglutinin glycoprotein 1 as well as other virulence factors 2 that expand the viral tropism beyond the respiratory tract of poultry. there is also emerging evidence of viral rna or antigen in multiple organs and the cns of humans infected with h5n1 that is consistent with systemic infection 3, 4 and raises the question of the role of the cleavage site in dissemination of the virus in this species. the majority of human cases with h5n1 have involved contact with sick or contaminated poultry and exposure to respiratory secretions of birds that can be inhaled and ingested. particular risk factors for h5n1 infection include bathing with sick birds, improper hand washing after handling sick birds, or slaughtering poultry. 5 viral inoculum may also be consumed directly during a variety of religious and cultural practices, such as drinking contaminated duck blood and kissing of merit release birds. h5n1 infection is lethal in 60% of human cases, and the pathogenetic mechanisms leading to this level of mortality are unclear. to date 505 cases have been reported to the who, although many more people have potentially been exposed to h5n1 through contact with infected bird populations. 6 some studies have suggested that genetic factors may predispose an individual to severe h5n1 disease, 7 but little is known about the influence of route of virus exposure on morbidity and mortality. in ferrets, an animal model frequently used to study influenza because of its similar disease profile to humans, 8 swayne et al. 9 observed that exposure to a virulent h5n1 strain a ⁄ vietnam ⁄ 1203 ⁄ 2004 by intra-gastric gavage did not lead to disease and did not generate an antibody response, whereas ferrets that experienced a more natural exposure by being fed contaminated meat developed severe signs of infection. in this study we further assessed the disease profile of h5n1 following a natural oral exposure in the ferret model. to achieve this inoculation condition, conscious ferrets voluntarily consumed a liquid inoculum of h5n1 hpai strain a ⁄ vietnam ⁄ 1203 ⁄ 2004. as a comparison anesthetised ferrets were exposed by intranasal administration of inoculum and the ensuing disease profiles of the different routes of infection were compared. eight ferrets per group were inoculated with 10 6 egg infectious dose 50 of a ⁄ vietnam ⁄ 1203 ⁄ 2004 in a volume of 500 ll that was given to the nares of anaesthetized ferrets to establish a total respiratory tract (trt) infection or voluntarily consumed by conscious ferrets to establish an oral infection. ferrets were culled at a predetermined humane endpoint that was defined as either a > 10% weight loss and ⁄ or evidence of neurological signs, discussed in 10 ; animals that did not reach the humane endpoint were euthanased on day 14 after challenge. nasal washes and oral swabs collected during the course of infection and organ homogenates were assessed for the presence of replicating virus by growth in embryonated-chicken eggs; viral loads were determined by titration on vero cells and expressed as tcid 50 . tissue samples were fixed with formalin and embedded in paraffin for sectioning. viral lesions were identified by hematoxylin and eosin staining of the sections and the presence of viral antigen in the sections was determined by staining with antibody to influenza a nucleoprotein. pre-and post-exposure antibody responses were assessed by hemagglutination-inhibition assays using irradiated a ⁄ vietnam ⁄ 1203 ⁄ 2004 virus. the majority (75%) of ferrets infected by the trt route rapidly became inactive, developed severe disease, and were euthanased at the humane endpoint following infection ( figure 1 ). ferrets infected orally had an improved chance of survival, as only 25% of animals developed severe disease (figure 1 ), and the surviving ferrets were more active than ferrets infected by the trt throughout the stage of acute infection (data not shown). the improved survival rate and wellbeing of ferrets infected orally was not a result of poor infection rates by this route, as 5 of 6 surviving ferrets developed h5 specific antibodies by day 14 post-infection, and they did not have pre-existing antibodies to h5n1 (data not shown). the two ferrets that developed severe disease after oral infection had similar disease profiles to ferrets infected by the trt route; they both progressed to a > 10% weight loss and exhibited neurological signs (data not shown). viral loads in organs of these two ferrets confirmed dissemination to extra-pulmonary sites (table 1) : replicating virus was detected at high titres in the spleen, pancreas, liver, and brain. similar findings were recorded in ferrets with trt infections in this study (not shown) and elsewhere. 10 viral load in nasal washes and oral swabs taken at days 3, 5, and 7 post-infection by the oral route did not correlate with the development of severe disease, and virus was isolated only sporadically and at low titre from the nasopharynx of these animals (data not shown). interestingly, the two ferrets with severe disease after being infected orally had no detectable viral antigen or lesions in the olfactory epithelium and bulb (table 1) , whereas 5 of 6 ferrets culled after infection by the trt route had lesions and viral antigen in both the olfactory epithelium and bulb (data not shown). trt oral 50 100 figure 1 . percentage of ferrets that survived infection after oral or trt infection. ferrets were exposed to a ⁄ vietnam ⁄ 1203 ⁄ 2004 by the total respiratory tract (trt) route (circles) or the oral route (triangles). the percentages of ferrets that survived infection are indicated at each day following challenge. ferrets exposed orally were more likely to survive h5n1 infection than ferrets exposed to the same dose of virus by the trt. the improved survival rates that were observed after an oral infection could be a consequence of low-level viral replication in the upper respiratory tract in combination with delivery of a substantial portion of the inoculum directly to the stomach where it may have been inactivated by the harsh environment of the gastro-intestinal tract. 11 most ferrets infected orally developed an h5-specific antibody response which differs from the studies of swayne et al. 6 in which ferrets gavaged with a liquid inoculum neither developed signs of disease nor an antibody response. however swayne et al. administered virus to anaesthetized ferrets by gastric gavage that would have bypassed the oropharynx. in our study virus was administered to the oral cavity directly and would have had access to the oropharynx. low level of replication at this site may have been sufficient to trigger an antibody response. the two ferrets that developed severe disease following oral infection had a similar profile of viral dissemination as ferrets infected by the trt route. differences were seen in the olfactory epithelium and bulb as lesions, and viral antigen did not occur in these sites following oral infection, although cerebral involvement was identified. one route of dissemination of h5n1 into the cns may be by transport within nerves through the olfactory bulb into the cerebrum. 12 due to the absence of lesions and antigen in these sites following oral infection the spread of virus into the brain in these two animals may be occurring through involvement of other cranial nerves or the hematagenous routes. nasal turbinates ) 3ae00 ) ) ) ) pharyngeal lymph node interactions of oseltamivir-sensitive and -resistant highly pathogenic h5n1 influenza viruses in a ferret model <2ae5 b ) + + + + olfactory epithelium nd a nd ) ) ) ) olfactory bulb nd nd ) ) ) ) trachea <2ae5 ) ) nd ) nd lung ) <2ae5 + + ) + spleen 3ae75 ) + + ) + small intestine ) ) ) ) ) + pancreas ) 3ae50 + ) + + the pandemic potential of highly pathogenic h5n1 influenza viruses remains a serious public health concern. while the neuraminidase (na) inhibitors are currently our first treatment option, the possibility of the emergence of virulent and transmissible drug-resistant h5n1 variants has important implications. clinically derived drug-resistant viruses have carried mutations that are na subtype-specific and differ with the na inhibitor used. 1 the most commonly observed mutations are h274y and n294s in the influenza a n1 na subtype (n2 numbering here and throughout the text); e119a ⁄ g ⁄ d ⁄ v and r292k in the n2 na subtype; and r152k and d198n in influenza b viruses. 2 h5n1 influenza viruses isolated from untreated patients are susceptible to the na inhibitors oseltamivir and zanamivir, 3 although oseltamivir-resistant variants with the h274y na mutation have been reported in five patients after 4,5 or before 6 drug treatment; and the isolation of two oseltamivir-resistant h5n1 viruses with n294s na mutation from an egyptian girl and her uncle after oseltamivir treatment were described. 6 the impact of drug resistance would depend on the fitness (i.e., infectivity in vitro, virulence, and transmissibility in vivo) of the drug-resistant virus. if the resistance mutation only modestly reduces the virus' biological fitness and does not impair its replication efficiency and transmissibility, the effectiveness of antiviral treatment can be significantly impaired. the recombinant wild-type h5n1 influenza a ⁄ vietnam ⁄ 1203 ⁄ 04 (vn-wt), a ⁄ turkey ⁄ 15 ⁄ 06 (tk-wt) viruses, and oseltamivir-resistant viruses with h274y na mutation (vn-h274y and tk-h274y) were generated by using the 8-plasmid reverse genetics system. susceptibility to na inhibitors was tested by using a fluorescence-based na enzyme inhibition assay with munana substrate at a final concentration of 100 lm. viral fitness was studied in vivo in a ferret model: groups of three ferrets were lightly anesthetized with isoflurane and inoculated intranasally with vn-wt, vn-h274y, or mixtures of the two at a different ratios at a dose of 10 2 pfu in 0ae5 ml pbs; they were inoculated with tk-wt, tk-h274y, or mixtures of the two at a different ratios at a dose of 10 6 pfu in 0ae5 ml pbs. respiratory signs (labored breezing, sneezing, wheezing, and nasal discharge), neurologic signs (hind-limb paresis, ataxia, torticollis, and tremor), relative inactivity index, weight, and body temperature were recorded daily. virus replication in the upper respiratory tract (urt) was determined on days 2, 4, and 6 p.i. the competitive fitness (i.e., co-inoculation of ferrets with different ratios of oseltamivir-resistant and -sensitive h5n1 viruses) was evaluated by the proportion of clones in day-6 nasal washes that contained the h274y na mutation. na mutations were analyzed by sequence analysis of individual clones ($20 clones ⁄ sample) created by ligation of purified pcr products extracted from nasal wash samples into a topo vector. introduction of the h274y na mutation conferred high resistance to oseltamivir carboxylate in vitro; the mean ic 50 of the vn-h274y and tk-h274y viruses was 3375 and 1208 times, respectively, that of the corresponding wildtype viruses. the oseltamivir ic 50 of the tk-wt virus was $16 times that of the vn-wt virus. all four recombinant h5n1 viruses were susceptible to zanamivir. introduction of the h274y na mutation reduced $90% and 60% of the na activity of vn-h274y and tk-h274y viruses, respectively, as compared to the wild-type virus activity (p < 0ae01; two-tailed t-test). all ferrets inoculated with either vn-wt or vn-h274y virus exhibited acute disease signs (high fever, marked weight loss, anorexia, extreme lethargy), rapid progression, and death by day 6-7 p.i., and no differences in clinical signs and replication in the urt of ferrets were observed between wild-type and oseltamivir-resistant viruses ( table 1) . both of the tk viruses caused milder illness than did the vn viruses, despite a much higher dose (10 6 pfu ⁄ ferret), and the tk-h274y virus caused less weight loss and fever than the tk-wt virus (table 1) . however, competitive fitness experiments revealed a disparity in the growth capacity of vn-h274y and tk-h274y viruses as compared to their wild-type counterparts: clonal analysis established the uncompromised fitness of vn-h274y virus and the impaired fitness of tk-h274y virus (table 2) . although, the trend towards an increase ⁄decrease in the frequency of the h274y na mutation relative to the wild-type was statistically significant (p > 0ae05) for two studied groups only. mutations within the na catalytic (r292k) and framework (e119a ⁄ k, i222l, h274l, n294s) sites or near the na active enzyme site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged spontaneously (without drug pressure) in both pairs of viruses (results not shown). the na substitutions i254v and e276a could exert compensatory effect on the fitness of vn-h274y and tk-h274y viruses. the lethality and continuing circulation of h5n1 influenza viruses warrants an urgent search for an optimal therapy. our results showed that the h274y na mutation affects the fitness of two h5n1 influenza viruses differently: the oseltamivir-resistant a ⁄ vietnam ⁄ 1203 ⁄ 04-like virus outgrew its wild-type counterpart, while the oseltamivir-resistant a ⁄ turkey ⁄ 15 ⁄ 06-like virus showed less fitness than its wild-type counterpart. we used a novel approach to compare the fitness of oseltamivir-sensitive and -resistant influenza viruses that included analysis of virus-virus interactions within the host (competitive fitness) during co-infection with these viruses. although mixed populations were present in the urt of ferrets on day 6 p.i., the fitness of vn-h274y virus was uncompromised as compared to that of its drug-sensitive counterpart, while that of tk-h274y virus was impaired. a minor population of na inhibitor-resistant variants may gain a replication advantage under suboptimal therapy in two ways: (i) preexisting variants less sensitive to the drug are selected from the quasispecies population, leading to an increase of the number of resistant clones, and (ii) outgrowing variants may acquire additional compensatory mutations that enhance their fitness. it is possible that use of antiviral drugs (particularly at suboptimal concentration) against mixtures of oseltamivir-resistant and sensitive viruses will promote the spread of drug-resistant variants * ferrets in all groups inoculated with a ⁄ vietnam ⁄ 1203 ⁄ 04 virus died by day 6-7 p.i. and were observed once daily for 7 days. ** results obtained from one ferret. *** by inhibiting drug-sensitive variants that are competing with them for the dominance in the infected host. the influence of multiple genes on the fitness of viruses carrying h274 na mutation cannot be excluded. in our study we focused on additional na mutations, and sequence analysis of individual na clones 7 was done to identify potential host-dependent and compensatory na mutations. we found that the na mutations e119a and n294s, which confer cross-resistance to oseltamivir and zanamivir, 1,2 can emerge spontaneously in clade 2.2 h5n1 influenza virus in ferrets. further, we observed that mutations at na catalytic (r292k) and framework (i222l and n294s) sites and in close proximity to the na enzyme active site (v116i, i117t ⁄ v, q136h, k150n, a250t) emerged without drug pressure in both pairs of h5n1 viruses. compensatory mutations in na or other genes may mitigate any fitness cost imposed by resistance mutations. our study identified six potential compensatory na changes (d103v, f132s, i254v, e276a, h296l, and f466s) that may affect the fitness of viruses with the h274y na mutation. we suggest that na mutations at residues i254v and e276a are of importance. interestingly, we observed differences in predominance of i254v and e276a na mutations in different genetic backgrounds: i254v mutation was identified in a ⁄ vietnam ⁄ 1203 ⁄ 04 (h5n1)-like and e276a in a ⁄ turkey ⁄ 15 ⁄ 06 (h5n1)-like genetic background. moreover, i254v na mutation was identified only when ferrets were inoculated with the mixtures of vn-wt and vn-h274y viruses, but not in ferrets inoculated with vn-h274y virus. none of the potential compensatory na mutations was identified in the original inoculum used to infect ferrets. the h274y na mutation causes a large shift in the position of the side chain of the neighboring e276 residue, 8 which must form a salt bridge with r224 to accommodate the large hydrophobic pentyl ether group of oseltamivir. residue i254 is located near the na active site, and although it does not alter polarity, it results in a shorter side-chain and, thus, may indirectly affect the residues in the na active site. we suggest that antigenic and genetic diversity, virulence, the degree of na functional loss, and differences in host immune response and genetic background can contribute to the observed differences in the fitness of h5n1 influenza viruses. therefore, the risk of emergence of drugresistant influenza viruses with uncompromised fitness should be monitored closely and considered in pandemic planning. this study was supported by contract hhsn266200700005c from the national institute of allergy and infectious diseases, national institutes of health, and by the american lebanese syrian associated charities (alsac). the data presented in the manuscript have been published at: govorkova ea, ilyushina na, marathe bm, mcclaren laninamivir (r-125489) is a strong na inhibitor against various influenza viruses, including oseltamivir-resistant viruses. [1] [2] [3] [4] [5] [6] we discovered a single intranasal administration of laninamivir octanoate (cs-8958), a prodrug of laninamivir, showed a superior anti-virus efficacy in mouse and ferret infection models compared to repeated administra-tion of oseltamivir and zanamivir. [2] [3] [4] 7 this suggested that cs-8958 works as a novel long-acting na inhibitor of influenza virus in vivo. a single inhalation of cs-8958 proved noninferiority in adult patients 8 and significantly superior in child patients, 9 compared to an approved dosage regimen of oseltamivir for treatment. cs-8958 has been commercially available as an inhaled drug, inavir ò , for the treatment of influenza in japan since october 2010. the long-acting characteristics of cs-8958 are explained by several reasons. first, cs-8958 was quickly hydrolyzed to an active metabolite, laninamivir, after an intranasal administration to mice, and was retained for a long time as laninamivir in target organs, such as lung and trachea. 10 however, with an intranasal administration of laninamivir, it disappeared quickly and did not demonstrate its longlasting characteristics. 10 another reason is a strong binding of laninamivir to nas of seasonal influenza viruses compared to other three na inhibitors, oseltamivir carboxylate, zanamivir, and peramivir. 3 in the following, the tight-binding ability of laninamivir to pandemic (h1n1)2009 na, as well as to the seasonal influenza virus nas, was demonstrated. in addition, we present a hypothesis of the mechanism of the long-lasting property of cs-8958 in mouse based on a localization of an enzyme that hydrolyzes cs-8958 to laninamivir. the influenza viruses, pandemic(h1n1)2009 (inf139), a ⁄ new caledonia ⁄ 20 ⁄ 99 (h1n1), a ⁄ panama ⁄ 2007 ⁄ 99 (h3n2), and b ⁄ mie ⁄ 1 ⁄ 93 were treated with excess na inhibitors, such as oseltamivir carboxylate, zanamivir, peramivir, and laninamivir, and then unbound na inhibitors were removed from the mixtures with a bio-spin column bio-gel p-6 (bio-rad laboratories, hercules, ca, usa). the na substrate, 4-methylumbelliferyl-n-acetyl-a-d-neuraminic acid (nacalai tesque, japan) was added to the virus-na inhibitor complex, and the na activities were followed for 6 hours at room temperature by measuring the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. the enzyme which hydrolyzes cs-8958 to laninamivir was partially purified from rat lungs using ion exchange column chromatography, and almost all bands separated by an sdspolyacrylamide gel electrophoresis were identified by mass spectrometry. the gene expression profiles of the enzyme were investigated by the bioexpress database (genelogic inc., gaithersburg, md, usa). the enzyme gene cloned from mouse lung mrna was transiently expressed in cos cells. antiserum to the esterase was prepared by immunizing rabbits, and immunostaining was done using histomouse-tm-max kit (invitorgen corp., carlsbad, ca, usa) according to the manufacturer's manual. binding stability of na inhibitors to the four viruses are shown in figure 1 the enzyme that hydrolyzes cs-8958 to laninamivir in rat lungs was identified as carboxyesterase. this esterase was shown to be expressed in epithelial cells of rat lung by in situ hybridization. 11 the mouse homolog of the rat esterase was carboxylesterase 3 (ces3). the mrna of the mouse ces3 was shown to be highly expressed in lung and liver by the gene expression profile, and ces3 was also found to contain signal sequences for retention in endoplasmic reticulum (er) and golgi at the c-terminus. the cloned ces3 gene and the ces3 gene lacking the signal sequence were exogenously expressed in the cos cells. the cs-8958-hydrolyzing activity associated with the cos cells expressing ces3 was recovered from the culture sup of the cos cells expressing ces3 lacking the retention signal sequence. localization of ces3 was immunohistologically confirmed inside the airway epithelium cells of mice, which are the target cells for influenza virus infection. the long acting property of intranasal administration of cs-8958 in mice can be explained both by the long retention of laninamivir in the respiratory tract and by the stable binding of laninamivir to influenza virus na. again, stable binding of laninamivir to na of pandemic (h1n1)2009 virus was also observed similar to that of seasonal h1n1 virus. the following are speculated as the mechanisms for the long-lasting characteristics of cs-8958 in mice. we explain the mechanism by clarifying a cs-8958 hydrolyzing enzyme and its localization inside cells. the hypothesis of the mechanism is presented in figure 2 . briefly, hydrophilic laninamivir may not enter easily inside cells, whereas hydrophobic cs-8958 may enter inside cells. ces3 with er ⁄ golgi retention signal hydrolyzes octanoate of cs-8958 figure 1 . difference of binding stabilities of various na inhibitors to influenza virus neuraminidases. the na substrate was added to the influenza virus-na inhibitor complex (oseltamivir carboxylate, n; zanamivir, h; peramivir, s; laninamivir, •; distilled water, ¤), and the na reaction was followed for 360 minutes. the background (only the na substrate [d] ) is also shown. a part of data from. 3 to generate the hydrophilic drug, laninamivir, and then it is trapped inside er ⁄ golgi because of its high hydrophilicity. the glycoprotein, na, which matures in er ⁄ golgi, meets laninamivir there and efficiently makes a stable complex with it. there are some questions that remain. how does cs-8958 move from the cell membrane to er ⁄ golgi? is laninamivir indeed trapped inside er ⁄ golgi, and does it make a complex with na in mice? we are now making an attempt to clarify these concerns. in our study, we have explored the antiviral potential of two newly synthesized compounds to provide protection against the novel pandemic influenza virus h1n1 (2009) strain. the compounds were reconstituted in dimethylsulphoxide (dmso), and so the initial studies began with cytotoxicity determination of solvent on uninfected and untreated madin-darby canine kidney (mdck) cells. on obtaining an upper limit for dmso, the compounds were tested for estimation of their maximum non-toxic dose to the mdck cells. thereafter, the effective dose of the compounds was evaluated and validated by a number of assays and gene expression profiling at both nucleic acid and protein level. we found that these newly synthesized compounds possess potent inhibitory activity towards the novel pandemic influenza h1n1 (2009) virus. these findings are being evaluated in vivo for a better understanding of their inhibitory capabilities and also their effect on the host metabolism. this will be required in the course of development of new drugs for use in the prophylaxis and treatment against the influenza virus. the mdck cell line (from nccs, pune) was maintained in 1· dmem media (sigma, st. louis, mo, usa) supplemented with 10% fetal calf serum and antibiotics viz. 100 unit ⁄ ml penicillin and 100 lg ⁄ ml streptomycin at 37°c ⁄ 5% co 2 . 5 the synthesized compounds used in this study were kindly provided by the department of chemistry, university of delhi, delhi, india. the pandemic influenza h1n1 (2009) virus was isolated and propagated in the allantoic cavities of embryonated chicken eggs during the pandemic period. the virus stocks were prepared and stored at )80°c. plaque assay was performed as previously described by hui et al., 2003. 6 briefly, 0ae5 · 10 6 mdck cells ⁄ ml were seeded in six-well plates and maintained in dmem for 24 hours at 37°c ⁄ 5%co 2 . the monolayer of the cells was inoculated with serially diluted virus samples for 45 minutes at 37°c ⁄ 5%co 2 . subsequently, a mixture of agar overlay was added, and the plates were incubated at 37°c for 5 days or until formation of plaques. the plaques were visualized after removal of the agar plug and staining with 0ae1% crystal violet or neutral red solution. the virus titre was expressed as plaque forming unit (pfu) per milliliter. the in vitro cytotoxicity analysis was performed to determine the 50% cytotoxic concentration (cc 50 ) of the compounds on mdck cells. the compounds were dissolved in dimethylsulfoxide (dmso), and so a prior cytotoxicity analysis was performed to determine the toxic concentration of dmso on the cells. various concentrations of compounds were mixed with dmem containing 1% fcs before addition to the preformed monolayer of mdck cells in 96-well plates. a series of suitable controls for in vitro cc 50 determination was included in every plate, and the plates were incubated in the optimum environment for mdck cell culture. the cc 50 of test compounds was analyzed by estimation of percentage cell viability of the compound-and mocktreated mdck cells by performing a colorimetric assay using tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl tetrazolium bromide (mtt) at end-point of 48 hours post-incubation. the assay was performed as described by mosman 1983. 7 briefly, mtt stock at a concentration of 0ae5 mg ⁄ ml was prepared in 1· pbs. the media was aspirated from the wells and 100 ll of mtt dye from the stock was added to each well. following incubation at 37°c ⁄ 5% co 2 for 2-4 hours, the dye was very carefully removed from the wells, and the cells were incubated with 100 ll of stop solution (dmso) per well at 37°c ⁄ 5% co 2 for 1 hour. the absorbance of the supernatants from each well was measured at 540 nm, and the percentage cell viability was calculated. madin-darby canine kidney cells were maintained overnight in a 12-well tissue culture plate at 37°c ⁄ 5% co 2 . the cells were inoculated with various virus dilutions at 37°c ⁄ 5% co 2 for 45 minutes and observed for cytopathic effect (cpe). the media from the experimental wells were aspirated after 48-72 hours of infection and were subjected to plaque assay. the percentage cell viability was determined by performing mtt assay. the results of both these tests were used to assess tcid 50 of the virus. the pre-formed monolayer of mdck cells was inoculated with the 10-fold dilution corresponding to tcid 50 of the virus for 1 hour at 37°c ⁄ co 2 . the experimental setup included control wells for the cells, virus, and compound. meanwhile, the concentrated stocks of the synthesized compounds were diluted with dmem (with1% fcs) to various concentrations within their respective cc 50 ranges. one hour post-infection, the cells were incubated with these diluted solutions. the cells were observed at various time intervals post-inoculation for cpe, and 200 ll media was collected from each experimental well for performing hemagglutination test. after 48 h, the media was collected for plaque assay and the cells were subjected to mtt cell viability assay. preformed monolayers of mdck cells were infected with virus and treated with the respective inhibitory concentration of the compounds. forty-eight to 72 hours post-incubation, total cellular rna was isolated using ribozol (amresco, solon, oh, usa) and treated with 50 lg ⁄ ml of dnase (promega, madison, usa). the concentration and quality of the rna from each well were determined by measuring their absorbance at 260 and 280 nm. one microgram of the cdna synthesized from each rna sample was used for sybr green-based real-time pcr detection of the ha gene of pandemic influenza h1n1 (2009) virus. as a control, human glyceraldehyde-3-phosphate dehydrogenase (hgapdh) was also amplified using gene specific primers. 8, 9 immunoblotting immunoblotting was performed to further validate the antiviral potential of the compounds. the experimental protocol was the same as for real time rt-pcr analysis. the cells were harvested 48 hours post-treatment with the compounds to prepare whole cell lysates in mammalian cell lysis buffer [0ae1 m nacl, 0ae01 m tris cl (ph 7ae6), 0ae001 m edta (ph 8ae0), 1 m m protease inhibitor cocktail, 100 lg ⁄ ml pmsf]. the protein concentration was determined by bca protein assay. the cell lysates were fractionated on 12% polyacrylamide for western blotting. the blot was developed using sheep monoclonal antibody (santa cruz biotechnology, ca, usa) against ha protein of influenza virus and horseradish peroxide conjugated rabbit-anti sheep igg (1:1000 dilutions) as secondary antibody. 10 the median cytotoxic concentration for compound meuh came out to be 150 lm, and that for flh was 160 lm. compounds showing potent antiviral effect on the pandemic influenza h1n1 (2009) virus propagation in madindarby canine kidney cells ( figure 1 ). the viral titres remained constant in cells treated with the compounds, while they increased in the untreated virus infected cells. ed 50 for the compounds meuh and flh were 150 and 160 lm, respectively. fifty-two percent (meuh) and 45% (flh) inhibition against the pandemic influenza h1n1 (2009) virus was achieved using ed 50 of the test compounds. both the compounds were able to reduce the rna levels of the ha gene by approximately 40-45%, whereas approximately 50% inhibition was seen when both the compounds were used in combination. similar results were obtained by the immunoblotting analysis ( figure 2 ). antiviral therapy has shown to be a promising tool in the management of various respiratory diseases, including those caused by influenza viruses. we have already shown inhibition of influenza virus replication in our earlier studies using catalytic nucleic acids, 11 which can be used as an approach in the development of new therapeutic strategy. these therapies are very useful as the influenza virus vaccines need annual renewals due to frequent genetic drifts in the viral surface proteins. in pandemic situations the existing vaccines do not provide complete protection against the novel virus as the population generally remains naïve for the newly mutated surface antigens. the antiviral drugs play an important role in the control of novel viral strains for which there are no vaccines available. however, the key obstruction in the extensive use of antiviral drugs is their cost and relative therapeutic efficacy provided. two classes of drugs were being used for treatment and control of the influenza virus infection in humans, the m2 ionchannel blockers 12, 13 (amantadine and rimantadine), which prevent viral uncoating, and the neuraminidase inhibitors 14, 15 (zanamivir and oseltmivir), which prevent the release of influenza virions from the cytoplasmic membrane. but widespread resistance to these antiviral drugs 16, 17 has limited their use. thus, novel drugs are required for the effective therapy against the emerging strains of influenza virus. the novel chemical compounds used in our study were tested for their antiviral efficacy against the pandemic influenza h1n1 (2009) virus. a reduction in the cpe in compound treated virus infected mdck cells indicated presence of antiviral activity in chemical compounds. the persistence of constant viral titers in the compound treated cells provided evidence for the interference posed by the compounds in the replication of influenza virus. inhibition in the ha gene expression further validated our hypothesis for the antiviral effect of compounds. the efficacy of these compounds in animal models is currently being validated in our laboratory. further, molecular studies are required to ameliorate the awareness regarding the mode of action of these chemical compounds against the viruses. 4 and is now licensed in japan, 5 while another, laninamivir, is being developed as an inhaled prodrug. 6 resistance to nais among circulating influenza viruses was previously low (<1% worldwide). [7] [8] [9] however, the 2007-2008 influenza season was marked by a worldwide emergence of oseltamivir-resistant seasonal influenza a (h1n1) viruses with the h275y (h274y in n2 numbering) in the na. [9] [10] [11] [12] [13] [14] the prevalence of oseltamivir resistance was even higher in the subsequent 2008-2009 influenza season with many countries reporting up to 100% oseltamivir resistance, 15 seasonal and pandemic influenza viruses collected globally between october 1, 2008 and september 30, 2009 were submitted to the who collaborating center for surveillance, epidemiology and control of influenza at the centers for disease control and prevention (cdc) in atlanta, ga, usa, and propagated in madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa). reference viruses representative of oseltamivir-sensitive and -resistant seasonal and pandemic viruses were also propagated in mdck cells. susceptibilities of virus isolates to the nais oseltamivir carboxylate (hoffman-la roche, basel, switzerland) and zanamivir (glaxosmithkline, uxbridge, uk) were assessed in the chemiluminescent ni assay using the na-star tm kit (applied biosystems, foster city, ca, usa) as previously described. 9 additionally, subsets of virus isolates were tested for susceptibility to peramivir (biocryst pharmaceuticals, birmingham, al, usa). fifty percent inhibitory concentration (ic 50 ) values were calculated using jaspr curve fitting software, an in-house program developed at cdc. curve fitting in jaspr was done using the equation: v = vmax · (1 ) ([i] ⁄ (ki + [i]))), where vmax is the maximum rate of metabolism, [i] is the inhibitor concentration, v is the response being inhibited, and ki is the ic 50 for the inhibition curve. box-and-whisker plot analyses 7 of log-transformed ic 50 s were performed for each virus type ⁄ subtype and nai using sas 9.2 software (sas institute, cary, nc, usa) to identify viruses with extreme ic 50 values (outliers). outliers were characterized based on a statistical cutoff of ic 50 greater than three interquartile ranges from the 75th percentile. outliers were subjected to genetic analysis by pyrosequencing 17 and ⁄ or conventional sequencing 18 to detect known or novel markers of nai resistance. those harboring previously characterized mutations in the na associated with nai resistance were considered drug-resistant; their descriptive statistics were determined separately from naisusceptible viruses. descriptive statistics to compute the mean, median, and standard deviation (sd), and a one-way analysis of variance were performed on original scale ic 50 data, using sas 9.2 software (sas institute) for each nai and virus among seasonal influenza a (h1n1) viruses tested for oseltamivir susceptibility (n = 1533), 1431 (93ae3%) were outliers for the drug (table 1 ) and harbored the oseltamivir-resistance conferring h275y mutation in the na. by contrast, only a small proportion (0ae7%) of tested h1n1pdm viruses (n = 2259) were resistant to oseltamivir. all influenza a (h3n2) viruses (n = 834) were sensitive to oseltamivir except for one outlier, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, whose ic 50 of 2ae50 nm was beyond the statistical cut-value off and >10-fold the mean ic 50 for the drug (0ae24 nm). all influenza b viruses (n = 914) were sensitive to oseltamivir with exception of an outlier b ⁄ texas ⁄ 38 ⁄ 2008, with d197e (d198e in n2 numbering) mutation in the na, whose ic 50 was beyond the cut-off, but only fourfold greater than the mean ic 50 for the drug. all virus types ⁄ subtypes tested for zanamivir were sensitive to the drug (table 1) , except for some outliers among seasonal influenza a (h1n1) and a (h3n2) outliers. the seasonal influenza a (h1n1) outliers included a ⁄ thailand ⁄ 1035 ⁄ 2008 (h1n1) and a ⁄ hawaii ⁄ 20 ⁄ 2008 (h1n1), both with combined h275y and d151d ⁄ g mutations in their na. the presence of concurrent mutations at na residues h275 and d151 in seasonal influenza a (h1n1) virus isolates substantially enhances resistance to oseltamivir and peramivir and ⁄ or zanamivir, however, the changes at d151 are typically cell-derived and not present in clinical specimens. 19 influenza a (h3n2) outliers for zanamivir included a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, as well as a ⁄ maryland ⁄ 02 ⁄ 2009 and a ⁄ vladivostok ⁄ 53 ⁄ 2009 with d151g and mixed d151d ⁄ g mutations, respectively. some mild outliers for zanamivir among a (h3n2) viruses with ic 50 beyond the statistical cutoff but <10-fold mean ic 50 for the drug were also identified; their genetic analysis revealed presence of wildtype and mutant sequences at residue 151 namely, d151d ⁄ g, d151d ⁄ n, or d151d ⁄ a. mutations at residue d151 of the na are associated with reduced susceptibility to zanamivir in a (h3n2) viruses, 9 but were reported to be cell-culture derived in recent h3n2 viruses. 20 all virus isolates tested for peramivir (n = 1058) were sensitive to the drug, except for h275y variants among seasonal influenza a (h1n1) and h1n1pdm viruses, which exhibited reduced susceptibility to the drug. in addition, one influenza a (h3n2) isolate, a ⁄ ontario ⁄ rv0442 ⁄ 2009 with d151v mutation in the na, showed reduced susceptibility to peramivir. the ic 50 values determined in functional ni assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. 21 nevertheless, combining elevated ic 50 values with the presence of established molecular markers of resistance in the na of virus isolates and their matching clinical specimens provides a reliable and reasonably comprehensive approach of identifying nai-resistant isolates for surveillance purposes. in this study, outliers with elevated ic 50 values for oseltamivir among seasonal influenza a (h1n1) and h1n1pdm viruses were confirmed to be oseltamivir-resistant based on the presence of the h275y mutation in the na. outliers for oseltamivir and ⁄ or zanamivir among influenza a (h3n2) viruses in this study were shown to harbor mutations at d151, which were earlier associated with reduced susceptibility to zanamivir, 9 and were cell-culture derived. 20 the effects of d151 mutations on nai susceptibility appear to be strain-specific; however, there are no conclusive supporting data and further investigations are required. outliers among the influenza a viruses in this study exhibited changes in the na, derived naturally or through cell-culture, which altered their susceptibility to nais. however, mild outliers for oseltamivir and ⁄ or zanamivir among influenza a viruses with slightly elevated ic 50 s, but without apparent changes in the na are sometimes identified. in such instances it is imperative to exclude the potential presence of influenza b among such outliers, using conclusive genetic tests such as real time pcr, since influenza b viruses exhibit higher ic 50 values for oseltamivir and zanamivir than influenza a viruses. 21 viruses exhibiting such mixes are typically excluded from statistical analyses of ic 50 s for respective drugs and virus type ⁄ subtype. establishment of a clinically relevant ic 50 cutoff value which could be used to differentiate statistical outliers from truly resistant viruses is imperative. global surveillance for nai susceptibility of influenza viruses circulating globally should be sustained to reflect the impact of seasonal and pandemic of influenza, given the limited pharmaceutical options available for control of influenza infections. nasopharyngeal swab specimens from patients with acute respiratory infection were collected at 164 influenza sentinel surveillance units (outpatient and hospital-based) all over mongolia. specimens were transported to the virology laboratory, nccd, ulaanbaatar, and rt-rt pcr positive samples were grown in a mdck cell culture according to the protocol developed by cdc. 5 6 and influenza virus gene segment 7 (m genes) sequencing (7 strains-genbank accession numbers: cy053364, cy053365, cy054547, cy054549, cy055171, cy065990, and cy065998) and influenza virus gene segment 6 (na gene) sequencing (2 strains genbank accession numbers: cy073447 and cy073448) by the standard methods with applied biosystems 3130xl genetic analyzer using primers supplied by who collaboration centers. a chemiluminescent na inhibition assay was performed with veritas microplate luminometer using the commercially available kit, na-star (applied biosystems, foster city, ca, usa), according to the manufacturers protocol. the na inhibitor susceptibility of influenza virus isolates was expressed at the concentration of na inhibitor needed to reduce na enzyme activity by 50% (ic 50 ). oseltamivir carboxylate, was provided by f. hoffman-la roche ltd (basel, switzerland). na inhibition assay data were analyzed using robosage software comparing test data with the data produced by the reference na inhibitor sensitive and resistance strains, which were provided by the who influenza collaboration center, melbourne, australia. all viruses tested were sensitive to oseltamivir with two exceptions: a seasonal influenza virus a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) with 137ae1 nm ic 50 value and a pandemic influenza virus a ⁄ dundgovi ⁄ 381 ⁄ 2010(h1n1) with 65ae6 nm ic 50 value ( figure 1 ). there was oseltamivir resistance detected in 2ae7% (37 ⁄ 1) of seasonal a (h1n1) and in 0ae4% (262 ⁄ 1) of a (h1n1) pdm viruses. the oseltamivirresistant viruses were collected from untreated patients. in total, 18 influenza b viruses were analyzed by na inhibition assay and all were sensitive to oseltamivir. the na of both oseltamivir-resistant strains contained h275y mutation based on the sequencing analysis. the difference in the na amino-acid sequences between the mongolian oseltamivir-resistant viruses and the respective oseltamivir-sensitive reference viruses is shown in table all 37 a(h1n1) viruses analyzed for m2 channel inhibitor resistance by pyrosequencing contained the s31n mutation and, thus, were resistant to this class of anti-influenza drugs. the segment 7 sequencing revealed that 6 seasonal a(h1n1) viruses possess the common s31n mutation. of note, a single strain a ⁄ zavkhan ⁄ 8299 ⁄ 2009(h1n1) contained an unusual s31d change in the m2 protein. our study shows that the same prevalence [2ae7% (1 ⁄ 37)] of seasonal a(h1n1) viruses with h275y mutation in 2008 ⁄ 2009 season in mongolia with the published data for 2007 ⁄ 2008 season from japan. 7, 8 however the prevalence of oseltamivir resistance in japan has dramatically increased in 2008 ⁄ 2009 season to 100% (77 ⁄ 77). the observed double mutations: h275y and d354g in a ⁄ ulaanbaatar ⁄ 1735 ⁄ 2009(h1n1) strain, which have been also found in japan in 2008 ⁄ 2009 season. 7 the patient from whom the oseltamivir resistant seasonal influenza h1n1 virus has been isolated was a 1-year-old boy, living in ulaanbaatar, the capital city, without history of using oseltamivir. the patient from whom the oseltamivir resistant a(h1n1)pdm virus was isolated was a 59 year-old man, residing in the dundgovi, the southern province, also without history of antiviral treatment. according to the who data, isolation of the pandemic viruses carrying h275y change from untreated patients has been uncommon. circulation of amantadine-resistant seasonal a (h1n1) viruses has been increasing in mongolia since 2006 ⁄ 2007 influenza season. 4 all pandemic influenza a(h1n1) strains (16) tested were resistant to m2 channel inhibitors due to the presence of the s31n mutation in the m2 protein. among seasonal a(h1n1) viruses, one contained a s31d change whereas the others had s31n, the well established marker of resistance to both amantadine and rimantadine. this is the first report of detecting the s31d change in the seasonal a(h1n1) viruses. according to the cdc data (unpublished), the s31d change conferred the drug resistance in the a(h3n2) viruses according to the virus yield reduction assay. it is essential to continue the antiviral resistance surveillance of influenza virus strains circulating in mongolia to ensure the efficiency of a proper clinical management of influenza patients. (conferred by the s31n mutation). of note, the genotype 2 and genotype 3 dual resistant viruses from asia appear to be genetically similar to those previously reported dual resistant viruses from hong kong, sar. 4, 5 the genotype 4 virus was the only dual resistant virus with a nearly complete 2c genome. oseltamivir-resistance for this virus appears to be the result of a reassortment as demonstrated by the presence of the oseltamivir-resistant clade 2b na gene. although the detection of dual resistant seasonal influenza a (h1n1) viruses is still rare, there has been an increased prevalence of dual resistance viruses during the last three seasons: 0.06% (1 of 1753 tested in 2007-2008), 1.5% (21 of 1426 in 2008-2009) , and 28% (7 of 25 in 2009-2010) (v 2 p < 0.001). while the continued circulation or co-circulation of seasonal a (h1n1) viruses is uncertain, the emergence of dual resistant influenza viruses in five countries does present a public health concern, especially since dual resistant viruses would limit the options for antiviral treatment to a single licensed antiviral drug: zanamivir. moreover, the markers of resistance seen in seasonal a (h1n1) viruses also confer resistance in the more widely circulating 2009 pandemic a (h1n1) virus. and, since the acquisition of mutations in influenza a viruses typically occur through drug selection, spontaneous mutation, or genetic reassortment with another drug resistant influenza a viruses, the detection of influenza a (h1n1) viruses that are resistant to both adamantanes and oseltamivir warrants close monitoring, even if only detected at low frequency. new antiviral agents and strategies for antiviral therapy are likely to be necessary in the future. 1 heightening concern that drug resistance will likewise become prominent in pandemic viral strains and highlighting the need for antiviral drug resistance surveillance. the h275y mutation in h1n1 neuraminidase is the most common mutation conferring resistance. however, due to the high mutation rates of viruses, new mutations can be expected that will also render viral neuraminidase less sensitive to antiviral drugs. pcr methods can be used to detect previously identified mutations; however, functional neuraminidase enzyme activity inhibition testing is necessary for detecting drug resistance that results from novel mutations. the two neuraminidase enzyme inhibition assays using either the fluorescent munana or chemiluminescent na-star ò substrate are robust tools for ni susceptibility testing. the munan-a-based assay is broadly used by many groups, including many regional health organizations for ni susceptibility testing, yet no standardized protocol or dedicated kit has been in place for this assay, making comparison of data generated between different laboratories difficult. borrowing from multiple neuraminidase inhibitor susceptibility network (nisn)-published munana-based neuraminidase assay protocols, 2 we have developed a kit-based fluorescent neuraminidase assay that offers both standardization and off-the-shelf quality-controlled reagents for ni susceptibility testing and other neuraminidase assay applications. the na-fluor tm influenza neuraminidase assay reagents and protocols were optimized in comparison to published nisn protocols according to the criteria of assay performance, ease-of-use, consideration of historically used assay conditions, reagent storage stability, and environmental impact. our optimized assay conditions consists of 100 lm munana, 33ae3 mm mes, 4 mm cacl 2 , and ph 6ae5 in a 100 ll assay volume, and performing the assay for 60 minutes at 37°c following a 30 minutes preincubation of drug with the virus. these conditions are consistent with the majority of published influenza ni screening data in publication. 2 the standard na-fluor tm assay workflow for screening viral isolates for sensitivity to nis includes first titering the viral sample by neuraminidase activity to determine optimal virus concentration to be used in subsequent ic 50 determination assays. the na-fluor tm assay is an ideal tool for titering virus based on neuraminidase activity in the viral coat. titering of viral samples prior to running the ic 50 determination assays insured that assays would be performed within the fluorescence detection dynamic range of both the assay and the fluorometric instrument being used. viral titers giving rfus in the range of 2000-5000 were used for subsequent assays. comparison to traditional munana assays a primary goal of developing a standardized munana assay was to provide a standardized protocol and set of reagents that would allow for comparison of ni surveillance data between laboratories and over time. in addition, the assay should provide data comparable to historical data sets based on traditional munana-based protocols. to insure that our newly developed na-fluor tm assay met these criteria we performed side-by-side comparisons of the na-fluor tm assay to munana-based nisn protocols, as well as our na-xtd tm and na-star ò chemiluminescent neuraminidase assays to compare assay sensitivity and dynamic range and for ni ic50 determination with multiple viral isolates. for all assay comparisons, assays were performed according to respective published protocols. for direct comparison of results, an equivalent amount of virus (and concomitant neuraminidase activity) was used for each assay. the na-fluor tm assay provides low-end sensitivity (by signal to noise ratio) and dynamic range similar to nisnpublished, munana-based protocols (data not shown). these assays all show a low-end detection of approximately 0ae002 u ⁄ well and dynamic range of 2-3 orders of magnitude when performed simultaneously side-by-side using serial dilutions of bacterial (clostridium perfringes) neuraminidase. these assays show approximately onefold less dynamic range and approximately fivefold less low-end sensitivity than chemiluminescent assays under these conditions. given the large amount of archived ni inhibition data for viral isolates over the past decade, it is very important for a standardized assay to generate data similar to established protocols so that data can be compared in relative terms. when run side-by-side, na-fluor tm assay provided oseltamivir carboxylate and zanamivir ic 50 values similar to nisn-published, munana-based protocols. ic 50 values vary somewhat for munana assays versus chemiluminescent assays depending on the viral isolate, as previously described. 3 the na-fluor tm assay also exhibited similar sensitivity for detecting ni sensitive virus compared to nisn-published fluorescent assays as shown in figure 1 . the large shift in ic 50 values between oseltamivir-sensitive and resistant virus using the na-fluor tm assay enables detection of mutant virus in mixed viral samples ( figure 2 ). this capability is critical for identifying resistant virus in clinical isolates presenting mixed populations of resistant and sensitive virus during ni susceptibility surveillance. several characteristics of the na-fluor tm assay make it an ideal assay for processing large numbers of viral isolates for ni sensitivity surveillance or for using the assay for high throughput screening for lead discovery of new antiviral reagents. the na-fluor tm assay signal was found to remain stable for up to 4 hours after stop solution addition when stored at room temperature and for several days when stored at 4°c (data not shown). ic 50 values did not change over these times, indicating that the assay is compatible with processing many samples in a short time frame. the na-fluor tm assay was also found to be highly reproducible giving a z' of 0ae78 or above indicating that the assay can be used confidently to identify nis in high throughput screening mode. 4 the assay can tolerate up to 5% dmso, a common compound delivery reagent used in high throughput screens (data not shown). we have developed a standardized na-fluor tm assay suggested protocol that gives data similar to established mun-ana protocols. however, we have also found that several protocol adaptations can be made that generate comparable data while allowing the user more flexibility in assay mode, use of additional reagents, and to meet user-specified assay time requirements. the na-fluor tm assay can be run in either the standard 60 minutes ⁄ 37°c endpoint mode described above or as real-time kinetic assay 5 with repeated reads taken over time without the addition of stop solution, which both serves to terminate neuraminidase activity and to enhance the fluorescence of the product. for typical ni-sensitive viral strains, the rate of munana substrate turnover at 37°c is linear for at least 4 hours (data not shown). as would be expected, rates of substrate turnover decrease in the presence of nis reflected in a decreased slope exhibited by real-time kinetic reads. real-time acquired rfus are typically 5-6 fold lower than rfus acquired after addition of stop solution at the same time point. ic 50 values obtained using slope analysis for real-time assays are similar to values obtained by endpoint analysis. whether run in real-time or end-point mode, the linear rate of substrate turnover allows the user to run the assay for shorter or longer assay times than the standard protocol without compromise to assay performance. the na-fluor tm assay is also compatible with standard methods used in many laboratories to inactivate virus. 6 we have shown that ni ic 50 values for multiple viral strains remain unchanged when the assay is performed in the presence of 0ae1% np-40 or 1% triton x-100 (data not shown). similar results are also obtained by adjusting the na-fluor tm stop solution to 40% ethanol prior to addition for assay termination. the assay is unaffected by phenol red concentrations present in cell culture media. we have developed a standardized munana-based fluorescent neuraminidase assay, the na-fluor tm influenza neuraminidase assay kit, which has been optimized for ni susceptibility screening. the assay provides data that can be compared to data generated using traditional munanabased protocols. the assay is economical, highly reproducible, easy to use, and environmentally friendly. the assay is flexible and amendable to user-specific adaptations including assay mode, assay timing, and reagent compatibility. trademarks ⁄ licensing ª 2010 life technologies corporation. all rights reserved. relenza is a registered trademark of glaxo to test the prophylactic potency of h5-vhhb, mice were treated intranasally with pbs, 100 lg of h5-vhhb, or negative control rsv-vhhb at 4, 24, or 48 hours before infection with one ld 50 of nibrg-14ma virus. body weight loss was monitored daily, and on day 4 mice were sacrificed to determine the viral load in the lungs. all mice that received h5-vhhb retained their original body weight, whereas those receiving pbs or rsv-vhhb gradually lost weight (data not shown). intranasal administration of h5-vhhb at 4 or 24 hours before challenge resulted in undetectable lung virus titers. when animals were treated with h5-vhhb 48 hours before challenge, virus titers were 50fold lower compared to pbs and rsv-vhhb treated mice, and three out of seven animals still had undetectable virus titers ( figure 1 ). we next determined if h5-vhhb nanobody ò could be also be used therapeutically. we administered 60 lg of this nanobody ò intranasally to mice up to 72 hours after chal-lenge with 1 ld 50 of nibrg-14ma virus. four days after challenge, animals that received h5-vhhb 4, 24, or 48 hours after challenge had significantly higher body weight (data not shown) and lower lung virus loads than control mice. although mice treated with h5-vhhb nanobody ò 72 hours after challenge were not clinically protected compared to control mice, they had significantly lower lung virus titers (figure 1 ). to identify the ha amino acid residues that are potentially involved in h5-vhh binding, escape viruses were selected by growth and plaque purification of nibrg-14ma virus in the presence of h5-vhhm or h5-vhhb nanobodies ò . the ha sequences of six independently isolated h5-vhhm escape viruses revealed substitution of a lysine by a glutamic acid residue at position 189 in ha1 (h5 numbering). in addition, two h5-vhhm escape mutants carried an n154d and four carried an n154s substitution. the three-dimensional structure of nibrg-14 ha shows that n154d ⁄ s and k189e are close to each other as part of the corresponding antigenic site b in h3 ha. 4, 5 interestingly, the n154d ⁄ s mutations remove an n-glycosylation site, which is surmised to have evolved in h5n1 ha as a strategy to mask an antigenic site. 6 escape viruses selected in the presence of h5-vhhb carried k189n (n = 4) or k189e (n = 2) substitutions. these results indicate that residues in antigenic site b, at the top of ha and very close to the receptor binding domain (rbd), are essential for neutralization of the virus by h5-vhhm ⁄ b nanobodies ò (figure 2 ). the virus titer was measured in lung homogenates prepared on day 4 after challenge. the x axis refers to the time points in hours relative to the challenge (time = 0 hours) when ha-specific nanobodies (h5-vhhb), control nanobodies (rsv-vhhb) or pbs was administered to the mice. # below detection limit, n not determined [n = 4-7 mice per condition: p values < 0ae05 (*)]. here we demonstrated that prophylactic and therapeutic treatment with llama-derived immunoglobulin single variable domain fragments is effective to control infection with h5n1 influenza virus in a mouse model. we demonstrate that pulmonary delivery is a highly effective route of administration to treat or prevent influenza virus infection. in addition, we demonstrate that a homobivalent h5-vhhb has powerful h5n1-neutralizing activity in vivo. it is important to note that we used a mouse-adapted derivative of the non-highly pathogenic nibrg-14 virus in our challenge model. nevertheless, this virus induces severe morbidity and lethality in mice. compared to conventional neutralizing monoclonal antibodies, vhhs offer the advantage that they are easy to produce in escherichia coli, typically with high yield. in addition, their small size (15 kda for a monovalent vhh) and high folding capacity allow the generation of oligovalent vhh derivatives. in vitro escape selection revealed that a k189e substitution in ha1 abolished the neutralizing effect of h5-vhhm ⁄ b. a lys or arg residue at this position is conserved in all human h5n1 virus isolates. of note, all selected escape mutants contained a glutamic acid or serine residue at position 189, which suggests that the conserved positively charged amino acid is important for neutralization by h5-vhh nanobodies ò . interestingly, escape mutants selected with h5-vhhm also carried an n154d ⁄ s co-mutation that removes an n-glycosylation site in this antigenic site of ha. the predicted n-glycosylation site at n154 in a ⁄ hong kong ⁄ 156 ⁄ 97 ha was shown to be glycosylated and may have evolved to mask an antigenic site near the rbd. 7, 8 the selected amino acid changes are located near the receptor binding site of ha. therefore, it is possible that enhanced receptor binding properties of these escape viruses contribute to or are responsible for the loss of neutralizing activity of h5-vhh nanobodies ò . 9, 10 we conclude that influenza virus neutralizing nanobodies ò have considerable potential for the treatment of h5n1 virus infections. although we focused on vhhs that presumably recognizes an epitope near the rbd, it is possible to select vhh molecules that bind to other epitopes in ha, including more conserved domains. more, a novel na (i117m) substitution was discovered in a series of specimens from a patient. for the amantadine resistance, 1113 samples were tested, and all of them were confirmed to be resistant. we collected respiratory specimens from patients who had been clinically refractory to antiviral treatment since october 2009 upon ethical approval from the relevant institutions. to investigate the resistant pattern, sequence analysis to the na and matrix (m2) genes were conducted by reverse transcription (rt)-pcr and sequencing reaction. the obtained sequences were analyzed by the influenza sequences and epitopes database, which was developed in korea. eleven patients were found to be having oseltamivir-resistant pandemic (h1n1) 2009 viruses with the h275y substitution in the viral na genes (tables 1 and 2 ). some cases were associated with oseltamivir treatment on the basis of h275y change from the oseltamivir-sensitive genotypes to oseltamivir-resistant genotypes in consecutive samples from the same patient. furthermore, a novel na (i117m) substitution that may be associated with oseltamivir resistance was detected in specimens from one patient (patient g) who had myelodysplasia and received oseltamivir and peramivir (tables 1 and 2 ). in addition, we obtained viruses from clinical specimens (patients a and c) and evaluated antiviral susceptibility by measuring the dose of oseltamivir and zanamivir required for 50% inhibition (ic 50 ) of na activity. these viruses (from patients a and c) were resistant only to oseltamivir (ic 50 713ae2 and 359ae4 nmol ⁄ l, respectively). susceptibility to zanamivir was not altered whether na contained y275 or h275 (ic 50 0ae13 and 0ae78 nmol ⁄ l, respectively). one isolate of pandemic (h1n1) 2009 virus with an oseltamivir-sensitive genotype (h275 in its na) was susceptible to oseltamivir (ic 50 1ae18 nmol ⁄ l) and zanamivir (ic 50 0ae42 nmol ⁄ l). patients with oseltamivir-resistant pandemic (h1n1) 2009 were treated during hospitalization with oseltamivir alone or with a combination of other antiviral drugs ( we found 11 patients of oseltamivir resistance with h275y mutation in the na gene of pandemic (h1n1) 2009 virus through the surveillance of patient refractory to antiviral treatment. in addition, novel amino acid change (i to m) at position 117 in the na gene, which might influence oseltamivir susceptibility, was detected in sequential specimens of a patient. these data showed that generation of oseltamivir resistance could be associated with oseltamivir treatment. therefore, it needs to strengthen the antiviral monitoring by supplementation of the clinical data including antiviral treatment. during the pandemic, oral oseltamivir was the primary antiviral medication used for treatment of hospitalized patients with ph1n1 infection. many physicians worried that clinical deterioration or failure to respond to treatment with oseltamivir was due to either oseltamivir resistance or oseltamivir failure. in the united states, two investigational intravenous (iv) nais were available during 2009-2010: peramivir through emergency use authorization and zanamivir by investigational new drug application. peramivir would be an option for patients with oseltamivir failure, but would not be appropriate for patients infected with h275y oseltamivir resistant mutants. iv zanamivir was available in limited supply, but would be appropriate for severely ill patients infected with an oseltamivir-resistant ph1n1 virus. during the pandemic, clinicians had few options for antiviral resistance testing in the united states. to respond to this need, the us centers for disease control and prevention (cdc) offered antiviral resistance testing for patients suspected to have clinical failure due to oseltamivir resistance. we describe the methods that cdc used to prioritize patients for testing during the pandemic and to detect markers for oseltamivir resistance, as well as the results from this testing. to facilitate decisions on which patients to test, we developed testing algorithms that were shared with state labora-tories, epidemiologists, and the emergency operation center at cdc. we prioritized patients who might benefit the most from antiviral testing given the inherent delay in providing antiviral results, e.g. patients who might have prolonged ph1n1 shedding. patients that were critically ill [intensive care unit (icu) admission] or patients with severe immunocompromising conditions with clinical evidence for oseltamivir treatment failure (persistent detection of virus and clinical unresponsiveness to the drug) were prioritized. in addition, we tested specimens from patients that failed oseltamivir chemoprophylaxis. standard forms with information regarding specimen and minimal clinical information were collected on all patients. all protocols were validated and approved by clinical laboratory improvement amendments, e.g. quality standards to ensure accuracy, reliability, and timeliness of patient test results. information collected on patients was deemed public health response, not research, at cdc. clinical specimens, confirmed as 2009 pandemic influenza a (h1n1), were tested for the h275y mutation in the na using pyrosequencing. 5 results were returned to sender within 24-48 hours of specimen receipt. from october 2009 until july 2010, a total of 116 specimens from 73 patients were submitted for testing. viruses from 26 (36%) of 73 patients had h275y mutation in the na in at least one submitted specimen. clinical information was available for 66 patients (table 1) . most patients had received oseltamivir for treatment prior to obtaining the specimen sent for antiviral testing. four patients received oseltamivir for chemoprophylaxis, all were immunosuppressed, and all had the h275y mutant; duration of chemoprophylaxis until ph1n1 infection was detected varied (4-39 days). among the patients with an h275y mutant who were treated with oseltamivir, the median time on oseltamivir prior to collection of specimen with h275y mutation was 18 days (range 3-36 days). three patients were part of a hospital cluster of oseltamivir-resistant virus infections and were infected with h275y mutants prior to oseltamivir treatment. 6 patients with immunocompromising conditions accounted for almost half of all patient specimens tested, but they accounted for the majority of oseltamivir-resistant ph1n1 virus infections (table 1) ; among 32 individuals with severe immunocompromising conditions and clinical failure while on oseltamivir therapy, 23 (72%) had the h275y mutant detected. among the immunosuppressed patients with an oseltamivir-resistant virus, 16 (70%) had hematologic malignancies reported. in contrast, among the subset of icu patients without immunocompromising conditions and clinical failure while on oseltamivir therapy, we found little resistance: 2 (5ae9%) of 34 icu patients had oseltamivir resistance detected. during the 2009 pandemic, we were able to provide timely and useful information to clinicians regarding suspected cases of oseltamivir resistance. our testing algorithm limited the number of specimens to specimens from the highest risk patients that would benefit the most from antiviral treatment. such an approach allowed us to offer this service without compromising our public health duties. in addition, the information we collected on patients from this service complimented our data on the national surveillance for antiviral resistance. we also performed national antiviral resistance surveillance from april 2009 to july 2010. overall, 67 resistant ph1n1 viruses were identified from april 2009 to july 2010 in the united states among 6812 tested samples, including specimens described above, surveillance specimens, and resistant viruses reported in the literature. 7 further studies to understand risk factors for oseltamivir-resistant ph1n1 infection in patients with severe immunocompromising conditions are needed. while efforts to provide antiviral testing technology and materials to state laboratories are ongoing, clinicians still have limited options for such testing. rapid and inexpensive assays that could be performed by clinical laboratories, especially those caring for immunosuppressed patients, would be useful to inform patient care. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit provides the next-generation na-xtd tm 1,2-dioxetane chemiluminescent neuraminidase (na) substrate, together with all necessary assay reagents and microplates, to quantitate sensitivity of influenza virus isolates to neuraminidase inhibitors. like the na-star ò influenza neuraminidase inhibitor resistance detection kit, the na-xtd tm influenza neuraminidase assay provides highly sensitive detection of influenza neuraminidase activity. in addition, the na-xtd tm assay provides extended-glow light emission that eliminates the need for reagent injection and enables signal measurement either immediately or up to several hours after assay completion. the na-xtd tm assay is also used to quantitate influenza na activity directly in cellbased virus cultures to monitor viral growth or inhibition. global monitoring of influenza strains for resistance to neuraminidase inhibitors (nis) is essential for understanding their efficacy for seasonal, pandemic, or avian influenza, and studying the epidemiology of viral strains and resistance mutations. functional neuraminidase inhibition assays enable detection of any resistance mutation, making them extremely important for global monitoring of virus sensitivity to nis. the first-generation chemiluminescent na-star ò influenza neuraminidase inhibitor resistance detection kit has been widely used for virus ni sensitivity assays, 1-8 including identification of a ⁄ h1n1 pandemic virus resistant to oseltamivir. 9, 10 in addition, this assay has been used for identification of new ni compounds, 11 ni characterization, 12 studies of virus transmission, 13 drug delivery, 14 na quantitation of virus-like particles, 15 and cell-based virus quantitation. 16 neuraminidase assays performed with chemiluminescent 1,2-dioxetane substrates, including na-star ò and na-xtd tm substrates, typically provide 5-to-50-fold higher sensitivity by signal-to-noise ratio than assays performed with the fluorescent munana substrate. in addition, chemiluminescent assays provide linear results over 3-4 order of magnitude of neuraminidase concentration compared to 2-3 orders of magnitude with the fluorescent assay. 2 the high assay sensitivity achieved with chemiluminescent assays enables use of lower concentrations of viral stocks, and the wide assay range minimizes the need to pre-titer virus stocks prior to ic 50 determination. chemiluminescent reactions result in conversion of chemical energy to light energy, as light emission. the na-xtd tm substrate is a 1,2-dioxetane structure bearing a sialic acid cleavable group. to perform the na-xtd assay, virus dilutions (from cell culture supernatant) are pre-incubated in the presence of neuraminidase inhibitor. then na-xtd substrate is added and incubated for 30 minutes for substrate cleavage to proceed. finally, light emission is triggered upon addition of na-xtd accelerator, which provides a ph shift and a proprietary polymeric enhancer, both required for efficient light emission. chemiluminescent assays are performed in solid white microplates, and light emission is measured in a luminometer. the na-xtd tm substrate has a single structural difference from the na-star ò substrate that provides a much longer-lasting chemiluminescent signal, with a signal half-life of approximately 2 hours (not shown), compared to $10 minutes with the na-star assay, eliminating the need for luminometer instruments equipped with reagent injectors and enabling more convenient batch-mode processing of assay plates. the na-xtd tm assay kit also provides a new accelerator solution, containing a next-generation polymer enhancer, and a triton ò x-100-containing sample prep buffer providing enhanced na activity. read-time flexibility is demonstrated by determination of oseltamivir ic 50 values using data collected over 3 hours after addition of na-xtd tm accelerator. although signal intensity slowly decreases over time, the ic 50 curves and values are identical at each time point, shown using influenza b ⁄ lee ⁄ 40 ( figure 1) . triton x-100 detergent at 1% has been shown to inactivate flu virus while increasing neuraminidase activity. 17 the addition of na sample prep buffer (containing 10% triton x-100) to virus stocks (at 1 ⁄ 10 volume, achieving a final concentration of 1%) provides increase in na activity up to fourfold, but is not consistently observed, and seems to be most effective with more concentrated virus stocks. ic 50 values are unaffected by the addition of triton x-100 to the virus stock prior to virus dilution (not shown), so the assay is compatible with known virus inactivation reagents. assay sensitivity and ic 50 values determined with the na-xtd assay have been compared to those obtained with both the chemiluminescent na-star assay and the fluorescent na-fluor assay (not shown). the chemiluminescent assays provide 2-to 50-fold higher sensitivity by signal-to-noise ratio, depending on the virus strain, wider assay dynamic range, and better low-end detection limit than the fluorescent assay. the wide assay range with the chemiluminescent assays enables determination of ic 50 values over a range of virus concentrations, eliminating the need to titer virus prior to performing ic 50 determination assays. ic 50 values obtained with the na-xtd assay are nearly identical to those obtained with the na-star assay, with both oseltamivir and zanamivir neuraminidase inhibitors, and tend to be slightly lower than ic 50 values obtained with the fluorescent assay. viral na quantitation provides a convenient read-out to measure viral growth or inhibition, including inhibition in the presence of inhibitory compounds or antibodies, described as accelerated viral inhibition with na as readout assay (avina). 16 bation in the presence of varying concentrations of oseltamivir carboxylate. samples of culture media were assayed 24 hours later. quantitation of na activity with the na-xtd tm assay demonstrates inhibition of viral growth by oseltamivir carboxylate in cell culture ( figure 2 ). different volumes of culture media were assayed with the na-xtd assay, either in the culture plate or in a separate assay plate (not shown). performing the assay using the entire well contents (100 ll) reduces assay sensitivity due to the high concentration of phenol red. assaying a smaller volume of culture medium (either in culture plate or a separate assay plate) provides higher sensitivity, and enables temporal monitoring or use of remaining culture medium for other assays. the applied biosystems ò na-xtd tm influenza neuraminidase assay kit is a next-generation chemiluminescent neuraminidase assay providing high assay sensitivity and ''glow'' light emission kinetics for improved ease-ofuse. the applied biosystems ò na-fluor tm influenza neuraminidase assay kit, based on the fluorescent mun-ana substrate, has also been developed to complement the na-xtd tm and na-star ò chemiluminescence assays, for users lacking luminometer instrumentation or choosing to use fluorescence assay detection. together these kits offer: • standardized reagents and protocols • choice of detection technology • simple instrumentation requirements • high sensitivity for use with low virus concentrations • compatibility with batch-mode processing and largescale assay throughput • broad specificity of influenza detection • flexibility in assay format • additional na assay applications -cell-based viral assays, screening for new nis, detection of na from other organisms functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for ni resistance mutations, including known and new mutations. together, these assays provide highly sensitive, convenient and versatile assay systems with standardized assay reagents, and simple assay protocols for influenza researchers. 15 over 200 000 hospitalizations and 36 000 deaths in the us annually are attributable to seasonal influenza, primarily in chronically ill persons and the elderly. 1-3 following the emergence of pandemic 2009 h1n1 influenza, severe illnesses have also been observed in children and young healthy adults. 4 the occurrences of staphylococcal and pneumococcal pneumonia complicating influenza pandemics are well described. [5] [6] [7] although temporal associations of bacterial pneumonia and influenza circulation have been reported, there is little precise data on rates of bacterial complications of seasonal or pandemic influenza. the study of bacterial lung infection has been hampered by insensitive tests for invasive disease and the difficulty of interpreting routinely obtained sputum culture results. 8, 9 procalcitonin (proct), the prohormone of calcitonin, can discriminate viral and bacterial infections. 10 this 116-aminoacid precursor protein normally produced by neuroendocrine cells of the lungs and thyroid gland was first shown to be elevated in bacterial infections in patients with pulmonary injury and pneumonitis. 11 stimuli of proct include tnf-a, endotoxin, and other bacterial products. 12 several studies indicate that bacterial infections commonly induce hyperprocalcitonemia, but that viral infections, including 2009 h1n1, are associated with only minimal increases. 10, 12, 13 of note, proct induction is attenuated by viral-induced interferon-c. 14 a meta-analysis of studies comparing proct and crp as markers for bacterial infection found that proct was more sensitive and specific than crp for differentiating bacterial from other causes of inflammation. 15, 16 therefore, we measured proct levels in patients with seasonal and pandemic influenza and compared results with conventional methods for bacterial diagnosis. adults ‡21 years of age admitted to rochester general hospital (rgh) from november 1st to june 30th for two winter seasons (2008-2010) with an admitting diagnosis compatible with acute respiratory tract infection were recruited for the study. patients were screened within 24 hours of admission, and those with prior antibiotic use, immunosupression, or pregnancy were excluded. subjects or their legal guardian provided written informed consent. the study was approved by the university of rochester and rgh research subjects review board. at enrollment demographic, clinical and laboratory information was collected. influenza testing included nosethroat swabs (nts) for rapid antigen, viral culture, and reverse transcription-polymerase chain reaction (rt-pcr) and serology. testing for bacterial pathogens included blood cultures, sputum for culture and gram stain, nts for mycoplasma pneumoniae and chlamydophila pneumoniae pcr, s. pneumoniae antigen testing, and pneumococcal serology. if patients were unable to expectorate, sputum was induced with normal saline and bronchodilators. specimens were considered adequate by the standard criteria of >25 neutrophils (pmns) and <10 epithelial cells per high power field. serum was collected at admission and hospital day 2 for proct measurements. influenza infection was defined a positive result for any of the following tests: . cloned proteins were coated on eia plates at 2 ug ⁄ ml in bicarbonate buffer. after overnight incubation, plates were washed and two-fold dilutions of serum were incubated overnight at room temperature. plates were washed and incubated with alkaline phosphatase conjugate for 3 hours, followed by substrate. a greater than or equal to fourfold rise in titer was considered evidence of infection with s. pneumoniae. urinary antigen for s. pneumoniae samples were assayed for antigen using the binax now kit. (binax inc, scarborough, me, usa). the proct was measured using time resolved amplified cryptate emission technology (kryptor pct; brahms, henningsdorf, germany). functional sensitivity is 0ae06 ng ⁄ ml (normal levels are 0ae033 ± 0ae003 ng ⁄ ml). 18 mycoplasma and chlamydia pcr real-time pcr targeting the p1 adhesion gene for m. pneumoniae and the ompa gene for c. pneumoniae was used to detect atypical bacteria. 19 results fifty-one of 529 (9ae6%) illnesses evaluated tested positive for influenza virus. of these, 20 were due to ''seasonal influenza'' (16 influenza a ⁄ h3n2 and 4 influenza b), and 31 were identified as ''pandemic influenza'' (2009 h1n1). demographics of both groups were similar: mean ages 55 ± 18 and 50 ± 11 years, respectively, and equivalent sex and racial characteristics. other than a higher incidence of underlying lung disease in the seasonal group (75% versus 45%, p = 0ae05), pre-existing medical conditions including obesity were similar. symptoms, physical findings, and discharge diagnoses did not differ, and chest radiographs (cxr) showed infiltrates in 20% and 13% of seasonal and pandemic subjects, respectively. two pandemic and one seasonal influenza patient developed respiratory failure, and none died. overall, bacterial infections were diagnosed in 8 (16%) subjects (4-seasonal and 4-pandemic), and none were bacteremic. bacterial infections included: 5-s. pneumoniae, 1-m. pneumoniae, 1-s. aureus, and 1-h. influenzae. all seasonal patients were diagnosed with asthma or bronchitis, whereas three pandemic patients had pneumonia. mean serum proct (ng ⁄ ml) levels in seasonal versus pandemic patients on admission and day 2 were: 0ae14 ± 0ae17 versus 1ae05 ± 2ae86 and 0ae17 ± 0ae17 versus 1ae39 ± 4ae37, respectively, and were not significantly different (table 1) . several patients in the pandemic group had high proct levels, and there was a trend toward more pandemic patients having admission proct values ‡0ae5 ng ⁄ ml than seasonal subjects [1 (5%) versus 6 (19%), p = 0ae22] ( figure 1a , b). of the four patients with proc-t > 1ae5 ng ⁄ ml, two had dense infiltrates on cxr, one had a peripheral wbc of 17 200 ⁄ ml with a threefold increase in s. pneumoniae antibody, and one developed respiratory failure associated with copd exacerbation. reliable sputum samples (within 6 hours of antibiotics) were collected in only 22 (43%) subjects. of these, proct was ‡0ae5 ng ⁄ ml in two with influenza alone and three associated with bacterial infection, and <0ae5 ng ⁄ ml in 12 with influenza alone and five associated with bacterial infection. in the 22 with reliable sputa and accepting the conventional bacterial diagnosis, sensitivity of a proc-t ‡ 0ae5 ng ⁄ ml for bacterial infection was 38%, specificity 86%, positive predictive value 60%, and negative predictive value 71%. notably, one patient considered to have influenza alone (proct -0ae87 ng ⁄ ml) had group a streptococcus and s. aureus in a contaminated sputum and bilateral infiltrates on cxr. three of five patients with bacterial infections and proct < 0ae25 ng ⁄ ml had a clinical diagnosis of bronchitis. mean proct values were significantly higher in patients with infiltrates versus those with atelectasis or no acute disease on cxr (2ae4 ± 4ae8 ng ⁄ ml versus 0ae36 ± 1ae2 ng ⁄ ml, p = 0ae02). combining patients with proct values ‡0ae5 ng ⁄ ml with those having positive bacterial tests, rates of bacterial infection associated with seasonal and pandemic influenza were 20% and 26%, respectively. notably, antibiotics were administered to 88% of subjects despite 73% having no acute disease on cxr. in our study, bacterial infections were diagnosed in approximately 25% of adults hospitalized with influenza with no significant difference in rates noted between seasonal and pandemic influenza infected subjects. previous reports of bacterial infection rates of 4-15% with seasonal influenza are difficult to compare with recent studies of pandemic influenza, because the latter tended to focus on more severely ill patients. [20] [21] [22] bacterial pneumonia has been suspected or diagnosed in 13-24% of patients in intensive care associated with 2009 h1n1 infection and up to 38% of patients who died. 23, 24 despite aggressive pursuit of specimens for bacterial testing, diagnoses could be confirmed in only 8 (15ae6%) of patients using conventional methodology. given the difficulty in establishing a diagnosis of bacterial infection, elevated proct values may be helpful to identify patients at high risk for invasive disease. in a study of 25 patients with severe 2009 h1n1 or bacterial infection necessitating intensive care, a threshold proct level of 0ae8 ng ⁄ ml, demonstrated 100% sensitivity and 62% specificity for bacterial infection. 25 among 38 patients with 2009 h1n1 associated pneumonia, many of whom had respiratory failure, a threshold proct value of 0ae5 ng ⁄ ml provided a sensitivity of 100% and specificity of 53% for bacterial infection. 26 access to samples from lower airways in ventilated patients in these studies may have improved recovery of bacteria and account for the different results we observed. it should be noted that none of our patients were bacteremic, which is a very strong stimulus for proct release. proct levels have been used successfully to guide therapy in community acquired pneumonia, and our data showing high proct levels in patients with infiltrates on cxr suggests proct may be most useful for excluding invasive disease. 27, 28 elevated proct levels were not observed in patients with purulent sputum and clear cxr. it is notable that a proct level of <0ae25 ng ⁄ ml did not exclude patients with bacterial bronchitis since proct has been used to guide antibiotic therapy in copd exacerbations. 29 while it could be argued that healthy patients with bacterial bronchitis do not require antibiotic treatment, physician behavior in our study indicates antibiotics are frequently prescribed. combining patients with proct values ‡0ae5 ng ⁄ ml and those with a positive bacterial test, approximately 25% in patients in our study had bacterial complications associated with influenza infection. efforts should be made to curtail antibiotic use in hemodynamically stable patients with clear cxrs. given physician discomfort regarding discontinuing antibiotics, proct measurements in combination with routine bacterial cultures should be useful tools to guide therapy. influenza, mrsa, cytokines: diagnosis, treatment, prevention -a possible strategy for outpatient care we started the antiviral treatment of influenza in humans using neuraminidase inhibitors on january 29, 1999 in a successful attempt to cure a 96-year-old patient. since then, we have used the inhalant antiviral drug zanamivir, and later (october 1, 2002) changed to the use of oseltamivir with systemic bioavailability for treating patients with influenza. after 10 years of experience with antiviral treatment of outpatients, we highlight the importance of early diagnosis and early treatment. 1 the necessity of an earliest possible diagnosis was confirmed in the pandemic of 2009. large hospitals reported that patients with an h1n1 ⁄ 09 infection had to be treated with extracorporeal membrane oxygenation. we are convinced this is due to delayed recognition of infection in most cases. valuable time is lost when the patient with a sudden onset has to be brought to a hospital for emergency treatment. the point at which the patient goes to the doctor is decisive, and this problem of timing and the delivery of early treatment is not specific to germany. in our medical office, we assessed patients with suspected influenza (to date 262 seasonal infections, and in 2009, 25 h1n1 ⁄ 09) through clinical diagnosis, 2 and then proven by point of care rapid test (quickvue; quidel, san diego, ca, usa) followed by pcr. all of the patients undergo concomitant lab tests: leukopenia, serum iron level, and the humoral inflammation status [sum of the c-reactive protein (crp) and fibrinogen levels]. because of the constant threat of a bacterial superinfection, a bacterial swab and antibiogram is carried out on every patient. in all cases positive for influenza, oseltamivir was given immediately. nowadays it is important that a double infection with influenza and mrsa must be recognized immediately and treatment started at once with antivirals and, when appropriate, with a suitable antibiotic. we pay particular attention to an extremely low iron level (signum mali ominis). in addition we monitor oxygen saturation and the course of the humoral inflammation status every 1-2 hours for every of our outpatients. among our patients with seasonal influenza, we saw 132 within 12 hours, 103 within 36 hours, and 27 within 48 hours after disease onset. for pandemic influenza, it was 12 patients within 12 hours, 11 within 24 hours, and two within 36 hours. for all patients, we measured crp < 3ae5 mg and fibrinogen < 350 mg ⁄ dl (12 hours), crp < 10 mg and fibrinogen < 450 mg ⁄ dl (36 hours), and crp > 10 mg and fibrinogen < 500 mg ⁄ dl (48 hours, only seasonal cases). antibiotics were necessary in 130 cases, heparin and oxygen administration in 27 cases. one hundred forty-eight patients had a superinfection following influenza. the most common strains were haemophilus parainfluenzae and staphylococcus aureus. the subsequent use of a suitable antibiotic was only necessary in 50% of the patients. in all cases diagnosed, treatment (including heparin and oxygen administration) and monitoring were conducted in our medical office. none of our patients (seasonal and pandemic) had to be admitted to hospital. the early decision of whether or not antiviral and antibacterial treatment is taking effect is the only way the threat of a cytokine storm can be averted. not only does the primary care physician have to be aware of the pathophysiology involved, but also the necessary diagnostic and therapeutic options have to be made available to him. the result will lead to a saving of both lives and healthcare costs. this applies both in epidemic as well as in pandemic times. today we know that influenza leaves behind a defenceless immune system, and that the proteases of s. aureus contribute to influenza associated pneumonia. mark von itzstein, who discovered neuraminidase inhibitors, emphasized the synergistic cooperation of viruses and bacteria (personal communication, 2009). mrsa and influenza viruses are posing problems worldwide. the case of a 17-year-old boy with h1n1 ⁄ 09 infection demonstrates how fatal developments can be prevented. due to his constantly recurring colds, we had already detected the mrsa colonization years earlier and had always worked on boosting his general health and resistance. both the patient and his family were included in dealing with the problem. the patient was, and is, always vaccinated early with a virosomal vaccine (baxter). during the oktoberfest in munich in september 2009, when h1n1 infections were increasingly occurring, we learned that our patient had come down with an extremely acute feverish illness. with the help of the rapid test, we diagnosed an h1n1 ⁄ 09 virus infection and started treatment with oseltamivir immediately. the humoral inflammation status, which had increased very rapidly to more than 4ae5 mg ⁄ dl within hours, was treated with the effective cotrimoxazol from the antibiogram. at the same time, the patient was heparinized. the following day the patient had no fever and was symptom-free. it was only through our early knowledge of what could develop pathophysiologically that we were in a position to make the right decision at the right time. every doctor treating outpatients can follow this procedure if he is familiar with the pathophysiology of the disease and has the available tests on hand: virus rapid test, additional laboratory parameters (leukopenia, iron), and the humoral inflammation status. the decisive factor, however, is the constant clinical alertness towards the course of every acute feverish cold with acute onset. the patient has to remain in the care of the attending physician, and the chosen treatment has to be administered and monitored. this means constant spo2 measurements and checking the humoral inflammation status every 2 hours. if a clinical worsening occurs during monitoring, the treatment regime has to be changed immediately, which means the administration of an appropriate antibiotic. this outpatient care on the part of the doctor has to be available 7 days a week so that no time will be lost. reports from the netherlands and denmark show that, with the help of this preventive strategy under the motto 'search and destroy,' the dangerous, fatal course of infections reported in germany with at least four deaths a day, can be avoided. however, the doctor has to be adequately remunerated for the elaborate amount of time this intensive outpatient care requires. with our strategy, we have moved from divergence to convergence in the care of our patients. we reported on our 10 years of clinical experience with this approach at the antivirals congress in peking. our main message was early diagnosis and early treatment. we were able to demonstrate this in 287 outpatients with seasonal influenza and 25 h1n1 ⁄ 09 outpatients. our creed is: as much outpatient care as possible and as little hospitalization as possible. virological and autopsy findings in suspected and confirmed fatal cases of 2009 h1n1 pandemic influenza in the czech republic -preliminary results influenza viruses cause substantial morbidity and mortality. pandemic influenza may have a serious impact on certain (mainly younger) age groups in comparison with seasonal flu. influenza is one of few viral infections capable of causing a pneumonia that is difficult to cure and ⁄ or leads to sudden death. the aim of this study was to analyze and compare virological and autopsy findings in patients who died with suspected or confirmed 2009 h1n1 pandemic influenza virus infection. there were 2477 virologically confirmed cases of pandemic influenza and 102 deaths in the czech republic during pandemic wave. more than 400 influenza strains belonging to the new pandemic variant were isolated in the national influenza reference laboratory. postmortem biological samples were collected from any patient who died with suspected influenza infection to test for respiratory viruses. the samples were screened for 2009 h1n1 pandemic influenza virus by real-time pcr (rt pcr), and when rt pcr positive, by virus isolation assay. no immunohistochemical staining for influenza antigen was done on the rna pcr positive cases. other important respiratory viruses such as respiratory syncytial virus, parainfluenza viruses, and adenoviruses were detected by virus isolation assay in a suitable cell culture. epidemiological analysis of postmortem histopathologic findings in the airway tissue was carried out in 57 of 61 fatal cases. virological findings were subsequently correlated with histological changes and available demographic and clinical data. statistical analysis was performed by t-test using spss software. sixty-one deaths (34 males, 27 females) were analyzed. the rna of the 2009 h1n1 pandemic influenza virus was detected by pcr in 38 cases, while 23 cases remained negative. five respiratory syncytial viruses and two adenoviruses were detected in the influenza negative group. the mean age of 38 confirmed 2009 h1n1 pandemic influenza victims was 46ae0 years, age range 7-74 years and median 47ae5 years. the mean age of 23 influenza negative victims was 55ae1 years, age range 1-89 years and median 57ae0 years. the 95% ci for the difference in the age between the two groups is )1ae7; 19ae9. the test is statistically significant at the 10% level. the obtained significance (p = 0ae07) can be explained by the relatively small size of the study group. the most common postmortem histopathologic finding in the lung tissue of the 2009 h1n1 pandemic influenza virus-positive victims was diffuse alveolar damage (often bilateral) and ⁄ or hyaline membrane formation, possibly with signs of respiratory distress syndrome (in 18, i.e., 51ae4%, of 35 autopsied patients). in the 2009 h1n1 pandemic influenza virus negatives, the most common finding was pneumonia or bronchopneumonia with the detection of various bacterial species (in 12, i.e., 54ae5% of 22 autopsied patients). the cause might be either primary bacterial infection or superinfection following primary infection with influenza virus that remained undetected. the 2009 h1n1 pandemic influenza victims were younger than the patients who died with suspected but undetected 2009 h1n1 pandemic influenza. the majority of deaths were primarily linked to rapidly developing respiratory failure. this result supports the previous reports of severe respiratory outcomes in younger age groups that are typically linked to the spread of a pandemic strain of influenza. 1 due to limited amount of pandemic vaccine, especially at the beginning of pandemic, it is advisable to assess experiences with antiviral treatment, mainly dosing, and way of antiviral administration. primers specific for each of the eight genes of pandemic h1n1 ⁄ 2009 were adopted from assays as described previously to discriminate against seasonal human h1n1 and h3n2 viral segments (table 1) . 9 the primers were allowed to cross-react specifically with the sister clade viral segments of pandemic h1n1 ⁄ 2009. 2 the method we employed in this study was a 2-step singleplex sybr green-based real-time rt-pcr. this approach helped lower the running cost of the assays and facilitated downstream molecular analyses (e.g., sequencing) by using screened cdna samples. viral rna was extracted from viral cultures or clinical samples as described 3, 10 and was converted to cdna in a universal rt-pcr. each 10 ll rt reaction containing 5ae5 ll of purified rna, 2 ll of 5· firststrand buffer (invitrogen), 100 u of superscript ii reverse transcriptase (invitrogen), 0ae1 lg of uni12 (5¢-ag-caaaagcagg-3¢), 11 0ae5 mm of deoxynucleoside triphosphates and 10 mm of dithiothreitol was incubated at 42°c for 50 minutes, followed by 72°c for 15 minutes for heat inactivation. for each segment-specific real-time pcr, the 20 ll reaction contained 1 ll of a 10-fold diluted cdna samples, 10 ll of fast sybr green master mix (applied biosystems), and 0ae5 lm of the corresponding primer pair. the thermocycling conditions of all eight segment-specific pcrs were optimized as 95°c for 20 seconds, followed by 30 cycles of 95°c for 3 seconds and 62°c for 30 seconds, and all eight assays were performed simultaneously in a 7500 sequence detection system (applied biosystems). at the end of the amplification step, pcr products went through a melting curve analysis to determine the specificity of the assay (60-95°c; temperature increment: 0ae1°c ⁄ seconds). cdna of a ⁄ california ⁄ 04 ⁄ 2009 virus was used as a positive control. robust and specific amplification was achieved in all eight segment-specific real-time rt-pcr reactions. 9 pcr product for each segment of pandemic h1n1 ⁄ 2009 yielded unique melting curve pattern with distinctive melting temperature (tm), which was not observed in negative and water controls ( figure 1 ). reactions with tm value within 2 sds of the mean tm were determined as positive. we evaluated the assays with a number of serologically confirmed human clinical samples. all pandemic h1n1 ⁄ 2009 samples (n = 67) were positive in all eight assays, while all seasonal samples (h1n1 = 38; h3n2 = 66) were negative in all assays, as expected ( figure 1 and data not shown). these results showed that no reassortant of pandemic h1n1 and seasonal viruses was present in the tested human isolates. we applied these assays to our on-going influenza virus surveillance program in swine. nasal and tracheal swab samples were collected at an abattoir in hong kong and cultured in madin darby canine kidney cells or embryonated eggs as described. 12 positive viral cultures in hemagglutination assays were tested with the established segmentspecific real-time rt-pcr assays. among 59 swine viral isolates collected from 2009 to september 2010, 10 of them were recognized as pandemic h1n1 ⁄ 2009 in all eight segments. they were confirmed to be of pandemic h1n1 ⁄ 2009 origin by subsequent full genome sequencing analyses, showing that there were interspecies transmissions of the virus from humans to pigs. 13, 14 the remaining 49 viruses had one to seven gene segments positive in the segment-specific real-time rt-pcrs. thirty of them were selected as representative samples for full genome sequencing analyses based on the genotyping data generated in our assays. they were swine h1n1 or h1n2 viruses with their gene segments derived from tr or eurasian avian-like swine lineages. it should be highlighted that all of their positive gene segments in our assays belonged to the sister groups of pandemic h1n1 ⁄ 2009. their melting curve patterns were very similar to those derived from segments of pandemic h1n1 ⁄ 2009, except for ha of tr lineage. 9 our results successfully demonstrated the use of these segment-specific real-time rt-pcrs to recognize gene segments of contemporary tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses. 2 the ha-specific assay was able to discriminate pandemic h1n1 ⁄ 2009 from other contemporary swine viruses in the same lineage. nevertheless, to confirm the identity and to examine all the genetic variations in the viruses of interest, full genome sequencing analyses were necessary. in this study, the biggest obstacles in primer design were sequence similarity and diversity of influenza viruses. we attempted to use degenerated primers, but they were highly non-specific. the finalized non-degenerated primers crossreacted with genes from pandemic h1n1 ⁄ 2009 and its sister clade tr (pb2, pb1, pa, ha, np, and ns) and ea (na and m) swine viruses with some minor sequence mismatches. three avian (h5n1, h7n7, and h9n2) and 1 classical swine (h1n1) were also tested with our assays. all of these animal viruses were negative, except for ns gene of the classical swine virus. our segment-specific real-time rt-pcr assays might be used in high throughput genotyping. they detected pandemic h1n1 ⁄ 2009 viruses and acted as a preliminary screen-ing tool to select virus reassortants of interesting genotypes for further sequencing analyses. in fact, we identified a novel reassortant in january 2010 during the course of this study. this sw ⁄ hk ⁄ 201 ⁄ 2010 has a previously unidentified viral gene combination as shown in figure 1 . it was confirmed to be a reassortant between pandemic h1n1 ⁄ 2009 and other swine viruses in full genome sequencing characterization. it has a pandemic h1n1-like n1 gene, an ea-like h1, and the other six internal genes derived from tr swine viruses. 13, 14 the eight established real-time rt-pcrs can rapidly reveal the gene-origins of influenza viruses. we are currently using these assays in influenza surveillance in humans and other animals. it is believed that similar strategy might be applied to detect and genotype other influenza viruses and possible reassortants in the future. pandemic influenza a ⁄ h1n1 ⁄ 2009 infects millions of people around the world. a significant fraction of the world's population may also already have been exposed to the virus and, although asymptomatic, may be at least partially immune to the disease. a precise assessment of the number of people exposed to the influenza a ⁄ h1n1 ⁄ 2009 virus is epidemiologically relevant. however, assays typically used to estimate antibody titers against a particular influenza strain, namely hi and neutralization, require use of the actual virus. this seriously limits broad implementation, particularly in regions where high biosafety facilities are unavailable. we developed an elisa method for the evaluation of presence of specific 2009 h1n1 influenza virus-antibodies in serum samples. mouse anti-histidine tagged antibodies (100 ll; 5 lg ⁄ ml; abd serotec ò , uk) in pbs (ph 7ae2) were dispensed into standard 96-well plates and incubated for 12-16 hour at room temperature. excess antibody was removed by at least two successive alternate washings with pbs-tween 0ae05% and pbs. commercial blocking solution (300 ll, superblock ò t20; pierce ò , usa) was added and incubated for at least 2 hour at room temperature. after successive washing steps with pbs-tween 0ae05%, non-glycosylated histidine-tagged recombinant protein (100 ll; 10 lg ⁄ ml) was added to each well. this protein consisted of the receptor-binding domain of the hemagglutinin of the influenza a ⁄ h1n1 virus. 1,2 after 1 hour incubation, wells were washed for at least two alternating 5 minutes cycles with pbs-tween and pbs. a 1:50 dilution of the serum or plasma sample to be assayed (100 ll) was added to each well and incubated at room temperature for 1 hour. after repeated alternating 5 minutes pbs-tween 0ae05% and pbs washes, anti-human igg antibody solution (100 ll ⁄ well; 1:30 000 dilution in pbs-tween 0ae05%) marked with horse radish peroxidase (pierce ò , usa) was added and incubated for 1 hour at room temperature. after repeated alternate washes with pbs-tween 0ae05% and pbs), substrate solution (100 ll; 1-step ultra tmb-elisa; pierce ò ) was added to each well. after incubation for 15 minutes at room temperature in darkness, the enzymatic reaction was stopped by addition of 1 m h 2 so 4 (50 ll ⁄ well). yellow color produced by the enzymatic reaction was evaluated by absorbance at 450 nm in a biotek ò microplate reader (usa). blank assays using albumin in place of human sera established the elisa background signal, which was subtracted from sample absorbance signals: abs serum sample ¼ abs serum sample before correction à abs albumin sample : absorbance values were normalized based on the average signal of 103 non-exposed subjects (uninfected subjects), and expressed as normalized absorbance (abs norm ): where abs serum ample is the sample absorbance signal, abs albumin sample is the albumin control absorbance signal, abs non exposed subjects is the average absorbance signal of non-exposed subject samples. for ferret serum samples, the same basic protocol was followed, with minor modifications. an anti-igg anti-ferret polyclonal antibody preparation was used at a dilution of 1:30 000 in pbs-tween 0ae05%. 2 a recombinant receptor-binding domain of the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus, expressed in escherichia coli strains, 2 was used as the elisa antigen. this 27 kda protein, designated here as ha 63-286 -rbd, contained amino acids 63-286 of the influenza a ⁄ mexico ⁄ indre4114 ⁄ 2009(h1n1) hemagglutinin. a sequence coding for a series of six histidines at the n-terminus of the protein was included in the genetic construct to allow purification using immobilized metal affinity chromatography (imac) and attachment to assay surfaces treated with anti-histidine antibodies (or alternatively co +2 or ni +2 ). a panel of four samples (kindly provided by st. jude from ferrets exposed to different influenza strains, namely h3n2, h1n1 swine, and h5n1, was also tested by the elisa method using 1:50 dilutions. protein ha 63-286 -rbd specifically and selectively recognizes antibodies from serum samples from convalescent h1n1 ⁄ 2009 influenza subjects. dubois et al. 4 demonstrated that this protein, produced in e. coli, folds properly into a 3-d structure practically indistinguishable from the analogous region in the ha of the influenza a ⁄ h1n1 ⁄ 2009 virus. ha 63-286 -rbd preserves three of the conformational immunogenic epitopes (sa, sb, and cb) described for influenza a ⁄ h1n1 hemagglutinins. 5 the recombinant protein was used as the antigen, attached through histidine tags to microplate surfaces treated with anti-histidine antibodies to discriminate between serum samples from subjects exposed and non-exposed to influenza a ⁄ h1n1 ⁄ 2009. samples collected before the pandemic onset, and therefore presumed to exhibit low specific antibody titers against influenza a ⁄ h1n1 ⁄ 2009, were analyzed by elisa using the antigen ha 63-286 -rbd. the histogram of normalized absorbance values from this sample set displayed a normal behavior with a standard deviation of 0ae57 units. only 12ae5, 9ae7, and 3ae88% of these samples exhibited normalized absorbance values higher than 1ae5, 1ae75, and 2ae0, respectively. no sample from non-exposed individuals presented an absorbance value higher than 2ae5. variability among samples from non-exposed subjects was much lower than in samples with high specific serum antibody titers from convalescent h1n1 ⁄ 2009 patients. exposure to the h1n1 ⁄ 2009 influenza virus with this elisa method can be predicted by absorbance values normalized to those of abs norm ¼ ðabs serum ample à abs albumin sample þ=ðabs non exposed subjects à abs albumin sample þ ð1þ serum from uninfected subjects. consequently, for reliable results, inclusion of samples from non-exposed subjects on every assay microplate is necessary. figure 1 shows the analysis of 20 human serum samples, including 17 samples from convalescent patients with positive diagnosis by rt-pcr. three positive (dark gray bars) and two negative controls (light gray bars) were included in the same microplate. all serum samples corresponding to convalescent subjects exhibited absorbance values 1ae75-4ae5 times higher than negative samples ( figure 1 ). normalized absorbance values above 1ae5 suggested exposure to the virus, although, a more conservative threshold value of 1ae75 units is proposed for discrimination between exposed and non-exposed subjects. the elisa method described here yields adequate reproducibility and a high signal ⁄ noise ratio within determinations in the same microplate and among different microplates. using a normalized absorbance value of 1ae75, the method was able to discriminate samples from convalescent patients, preferably after the third week of infection, and at least up to the twentyfourth week of exposure. assay sensibility was further validated against results from hi assays. a previously reported study showed that all members in a pool of fourteen samples diagnosed as positive by hi exhibited normalized absorbance values higher than 1ae5, and 85% of them exhibited normalized absorbance values higher than 2ae0. 1 in general, high hi titers (>320) were correlated with normalized absorbance values higher than 4ae0. figure 2a shows results using the ha-rbd elisa method and the hi assay on a pool of seventeen known positive serum samples corresponding to convalescent h1n1 ⁄ 2009 patients. all samples determined as positive by hi (10 samples) were also positive by elisa. while sensitivity of the hi assay was 10 ⁄ 17 = 58ae88%, the elisa method recognized all samples correctly as positive (100% sensitivity) when a threshold of 1ae5 or 1ae75 was used. figure 2b shows that sera from ferrets infected with other influenza strains (h3n2, h1n1 swine, and h5n1) showed no cross-reactivity when analyzed by elisa. in summary, the ha-rbd elisa method presented here consistently distinguished influenza a ⁄ h1n1 ⁄ 2009 infected and non-infected individuals, particularly after the third week of infection ⁄ exposure. since no actual viral particles are required, this assay can be readily implemented in any basic laboratory. in addition, should sufficient vaccine be unavailable, this elisa could determine the level of specific antibodies against the virus and presumably the extent of partial protection in a subject. therefore, the elisa protocol might allow better administration of vaccination programs during pandemic or seasonal influenza outbreaks. in april 2009, a novel h1n1 influenza virus emerged in north america and caused the first influenza pandemic of the 21st century. [1] [2] [3] [4] the 2009 pandemic h1n1 (pdmh1n1) has a unique gene constellation that was not previously identified in any species or elsewhere. it is genetically related to the triple reassortant swine h1n1 influenza viruses currently circulating in north america, with the exception of the neuraminidase (na) and matrix (m) genes, which are derived from a eurasian swine influenza virus. swine h1n1 influenza viruses were first isolated in 1930 and continued to circulate in north america with very little antigenic changes (classical swine h1n1) until 1998. since 1999, however, the antigenic make up of swine h1 viruses has shown increased diversity due to multiple reassortment events and the introduction of h1n1 genes from human influenza viruses. currently, four swine h1 clusters (a, b, c, d) are found endemic in the north american swine population. 5, 6 these swine h1 viruses show substantial antigenic drift compared to the classical swine h1 viruses. cluster d swine h1 is derived from current human h1 viruses, and there is a substantial antigenic divergence between classical swine h1 and human seasonal h1 viruses. epidemiological evidence shows a two-way transmission of influenza viruses between swine and humans, and such events lead to the emergence of the pdmh1n1 virus. 5, 7, 8 phylogenetic analysis have suggested that possible ancestors of the eight genes of pdmh1n1 were circulating in the swine population for at least 10 years prior to the emergence of the pdmh1n1 virus in humans, although the pdmh1n1 virus itself was not isolated from pigs until after the pandemic. interestingly, pdmh1n1 infections have been reported not only in humans and pigs, but also in other animal species such as turkeys, cats, ferrets, cheetahs, and dogs. [9] [10] [11] after the first report of pdmh1n1 infection in swine in canada, other countries, including argentina, australia, singapore, northern ireland, finland, iceland, england, united states, japan, and china reported outbreaks of pdmh1n1 in swine as well. 9, [12] [13] [14] the ample geographic range of pdmh1n1 outbreaks in swine, its apparent broad host range, and the possibility of two-way transmission between swine and humans poses a tremendous challenge for controlling the virus. therefore, to differentiate pdmh1n1 from other h1 strains, particularly in swine and human populations, is an important issue to ascertain the magnitude of the disease caused by the pdmh1n1. in this study, we developed an elisa assay to discriminate pdmh1n1 strains from other swine and human h1 viruses. madin-darby canine kidney (mdck) cells (atcc, manassas, va, usa) were maintained in modified eagle's medium (mem) containing 5% fbs. a ⁄ california ⁄ 04 ⁄ 09 ⁄ h1n1 virus (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc), atlanta, georgia. other viruses are listed in table 1 . viruses were propagated in mdck cells and stored at )70°c until use. viruses were titrated by the reed and muench method to determine the median tissue culture infectious dose (tcid 50 ). 15 three monoclonal antibodies (3b2, 5h7, and 12f3) against ha of 2009 pandemic h1n1 were prepared in our laboratory following previously described methods (shao and perez et al., unpublished). purification and labeling of mabs mab 3b2, 5h7 and 12f3 were purified on a protein g-sepharose affinity column (upstate biotechnology, lake placid, ny, usa). biotinylation of the detection antibody in the elisa was performed using sulfo-nhs-lc-biotin (sulfosuccinimidyl-6-(biotinamido)hexanoate; pierce, rockford, il, usa) according to the manufacturer's instructions. purified 5h7 and 12f3 were selected as the capture antibody, and biotin-conjugated 3b2 was selected as the detection antibody, and hrp-conjugated streptavidin (abcom, cambridge, ma, usa) was developed using the tmb substrate system (kpl, gaithersburg, md, usa). in brief, the mixture of the purified 5h7 and 12f3 (2ae0 and 2ae2 lg ⁄ ml respectively, in carbonate ⁄ bicarbonate buffer, ph 9ae6) was coated to 96-well plates (test well, t) for 12 h at 4°c. at the same time, a control antibody was coated to 96-well plates (control well, c). after blocking the plates with 5% (w ⁄ v) non-fat milk in pbs for 1 hour at 37°c, the samples were diluted in extract buffer (1%tween-20, 0ae5%bsa in pbs) and added to the wells (100 ll ⁄ well, each sample was table 1 . specificity assay of the sandwich elisa result (t ⁄ c) added to four wells-two for t wells and two for c wellsand the mixture was incubated at 37°c for 1 hour. after four washes, 100 ll biotin-conjugated 3b2 (0ae25 lg ⁄ ml) in dilution buffer (0ae5% bsa in pbs) was added to the wells and the mixture was incubated for 1 h at 37°c. following three washes, 100 ll diluted hrp-conjugated streptavidin (62ae5 ng ⁄ ml) in dilution buffer was added to the plates. after incubation for 1 h at 37°c, the plates were washed five times, and the binding developed using the tmb substrate system for 30 minutes. the ratio of the average od 650 value of the t wells to that of the c wells (t ⁄ c) of individual samples was calculated. t ⁄ c values >1ae5 were considered positive in the sandwich elisa. we developed three monoclonal antibodies, 3b2, 5h7, and 12f3, against a prototypical pdmh1n1 strain, a ⁄ california ⁄ 04 ⁄ 2009 (h1n1) (ca ⁄ 04). these monoclonals were used to develop a rapid sandwich elisa for specific diagnosis of pdmh1n1 strains. purified 5h7 and 12f3 were used as capture antibodies, whereas the biotin-conjugated 3b2 was used as detection antibody. the sandwich elisa showed strong reaction with different pdmh1n1 strains as described in in order to evaluate if the sandwich elisa could distinguish the pdmh1n1 from other swine h1 clusters (a, b, c, d), 14 swine influenza strains spanning these clusters were tested. these viruses were first diluted 1:10 in extract buffer, and then added to the coated plates. as shown in table 1 , the t ⁄ c ratios of these viruses were <1ae5, and therefore showed negative elisa result. likewise, testing of human seasonal virus strains a ⁄ brisbane ⁄ 59 ⁄ 2007 (h1n1), a ⁄ malaya ⁄ 302 ⁄ 1954(h1n1), a ⁄ wsn ⁄ 1933 (h1n1), and a ⁄ brisbane ⁄ 10 ⁄ 2007 (h3n2) also showed negative elisa results. furthermore, the sandwich elisa showed no cross reaction with avian influenza viruses, including strains of the h2, h3, h5, h6, h7, h8, h9, h10, h11, h12, and h13 subtypes. more recently, the mutation d222g in the ha of some pdmh1n1 strains has been associated with exacerbated disease and altered receptor binding. [16] [17] [18] [19] [20] to evaluate if such mutant could be detected in our sandwich elisa, we tested a mutant of a ⁄ netherland ⁄ 602 ⁄ 2009 (h1n1) carrying the d222g mutation (engineered by reverse genetics). as described in table 1 , our elisa could still capture the d222g mutant virus and showed a positive reaction, which highlights the specificity of our assay for pdmh1n1 strains, even those with mutations. to evaluate the sensitivity of the elisa, we used the serially diluted pdmh1n1 viruses to determine the limit of detection (lod). as shown in table 2 , in our elisa the highest positive dilutions of nl ⁄ 602 and ca ⁄ 04 were 1:320 and 1:160, respectively. the lod of the sandwich elisa by tcid 50 was 3ae2 · 10 3 and 1ae5 · 10 4 tcid 50 ⁄ ml, for nl ⁄ 602 and ca ⁄ 04, respectively. it is important to note that the t ⁄ c ratio from nl ⁄ 602 and ca ⁄ 04 viruses showed clearly a dose dependent effect, while the t ⁄ c ratio of a ⁄ swine ⁄ iowa ⁄ 30 (h1n1) did not show the same dependence and was always <1ae5, corroborating the high specificity of the sandwich elisa for pdmh1n1 strains. although we did not compare our elisa with other current commercial rapid influenza detection kits, the lod of our elisa assay is similar to other commercial kits that detect human seasonal influenza virus. 21 comparison of the sandwich elisa with the ''gold standard'' -virus isolation in order to further evaluate the feasibility of the application of the elisa to clinical samples, 70 nasal wash samples 1ae58 · 10^6 )(0ae8) )(0ae8) )(0ae9) )(1ae0) )(0ae9) )(0ae8) )(1ae1) )(0ae9) -from ferrets, 56 of those previously infected with ca ⁄ 04 and shown positive by virus isolation, were tested. the samples were diluted 1:1 in extract buffer and then tested using the sandwich elisa. result showed 51 out of 56 positive samples by virus isolation were positive also by the sandwich elisa (sensitivity 90ae1%). the 14 samples tested that were negative by virus isolation were also negative in the elisa, indicating 100% specificity for our assay. these results show not only that our elisa has high compatibility with the virus culture method, but also indicates this application can be used for clinical samples. although real time rt-pcr targeting the ha gene has been used for specific diagnosis of pdmh1n1 with high sensitivity, [22] [23] [24] [25] [26] [27] it is a method that requires manipulation of the sample to extract viral rna, and it is prone to crosscontamination during the pcr steps. in this study, we described a convenient sandwich elisa based on three mabs developed against the pdmh1n1 strain. the elisa not only shows high specificity for pdmh1n1 strain, but also shows great sensitivity. the elisa could distinguish pdmh1n1 strains from human seasonal h1 and h3 viruses and, more importantly, from other swine h1 viruses. we must note that current rapid diagnostic tests cannot be used to differentiate pdmh1n1 from swine or human h1 viruses. 28 it is also worth noting that the sensitivity of commercial rapid antigen-based diagnostic tests for detecting pdmh1n1 is lower than that for human seasonal influenza viruses. 28, 29 a study by kok et al. 32 showed that sensitivity of the current rapid antigenic tests for pdmh1n1 is only 53ae4%, whereas that for seasonal influenza a is 74ae2%. chen et al. 33 developed a dot-elisa and increased the sensitivity for influenza rapid antigen detection. however, the dot-elisa developed by chen cannot distinguish among subtypes. the lod of our elisa is between 3ae2 · 10 3 to 1ae5 · 10 4 tcid50 ⁄ ml, comparable to the lod of rapid diagnostic tests for human seasonal influenza viruses. 21 compared to the ''gold standard''-virus isolation-our sandwich elisa showed 90ae1% sensitivity using ferret nasal washes. our results highlight the potential application of our sandwich elisa for the specific diagnosis of pdmh1n1 viruses. 17 the timely and reliable laboratory evidences are vital factors for field epidemiologists trying to control outbreaks of infectious diseases and for the practicing clinicians to properly manage disease cases. therefore, analysis of new detection methods in comparison to the routine ''classical'' methods is essential to select new methods to be introduced into health service practices, especially in developing countries. in this study we have compared rt-rt-pcr detection of influenza viruses and direct fluorescent-antibody assay using r-mix hybrid cells (a549&mv1lu) with the ''classical'' cell culture methods in developing country settings. in this study, we analyzed 503 nasopharyngeal swabs col the detection of influenza h1, h3, b, and pandemic influenza (h1)pdm virus-specific nucleic acids was performed by rt-rt-pcr in abi7500 fast real time pcr system using primers recommended by cdc, usa, and super-scriptô iii one-step rt-pcr and platinum ò taq dna polymerase kits (invitrogen). the cycling protocol was: 30 minutes at 50°c, 2 minutes at 95°c, and 45 cycles of 15 seconds at 95°c, 30 seconds at 55°c. 1 rapid detection of influenza infected cells has been performed by dfa using the infected hybrid cells of r-mix within 48 hours after inoculation, according to the manufacturers instruction (diagnostic hybrids, inc., usa). 2 the isolation of influenza viruses was performed on mdck cell culture by the protocol recommended by cdc, usa. 3 we detected 1891(25ae2%) influenza virus-specific nucleic acid fragments from all tested samples by rt-rt-pcr. among the positive samples, there were 25ae6% a(h1n1), 0ae4% a(h3n2), 5ae6% influenza b, and 19ae5% a(h1n1)pdm with different distributions by time series in different age-groups. inoculation of the cell lines by rt-rt-pcr positive samples selected randomly has detected influenza virus in 52ae3% (521 ⁄ 995) on mdck cell culture and 51% (77 ⁄ 151) on r-mix hybrid cell culture with varying distribution for different strains. in other words, mdck cell culture technique was better for isolation for pandemic influenza viruses and dfa using r-mix hybrid cell culture technique for detection of seasonal influenza viruses (table 1) . average times needed for the final results for different methods were: 4 hours for rt-rt-pcr, 48 hours for dfa on r-mix and 10 days for mdck cell culture with two passages at least. the peak of the seasonal influenza a virus detection occurred in the 7-8th weeks of 2009, however the pandemic influenza detection peak was observed in the 43-44th weeks of 2009 ( figure 1 ). the outbreaks by seasonal influenza viruses was observed mostly among the children of 0-15 years of age, and pandemic influenza virus outbreak was observed mostly in the adults of 16-64 years of age. the results of this study indicate that rt-rt-pcr is the most suitable method for decision makers in epidemiological and clinical settings by sensitivity and timeliness. the final results show that r-mix dfa requires 12 times longer, and by mdck cell culturing, 60 times longer periods, than by rtrt-pcr. mdck cell culture technique has a higher isolation of pandemic influenza viruses, and r-mix dfa has a greater detection rate of seasonal influenza viruses by our results. according to our study, with rtrt-pcr, the isolation of positive samples by tissue culture of influenza a viruses was 24% and influenza b viruses was 66ae5%, which is lower than in similar spanish study. 4 however our study illustrates similar results with a canadian study 5 where the sensitivity of dfa method and tissue culture technique was shown to be lower than rtrt-pcr sensitivity. as recorded by a study of american researchers, 6 r-mix hybrid and conventional cell culture techniques have had similar sensitivity, which does not match the results of our study. however, the results of our study match with the results of italian and american scientists 7, 8 where the r-mix hybrid method for seasonal influenza viruses is higher than mdck cell culture technique. background: viral kinetics is increasingly used to study influenza infectiousness. the choice of the study design, i.e. when and how many times nasal samples are to be collected in individuals depending on the sample size, is crucial to efficiently estimate the viral kinetics (vk) parameters. material and methods: we performed a model based optimal design analysis in order to determine the minimal number of nasal samples needed to be collected per subject and when to collect them in order to correctly estimate the vk parameters. the model used was a non linear mixed effect model developed with data collected from 44 patients sampled nine times in 9 days (initial design -504 samples collected), and we used d-optimization for design identification. we also computed the minimal number of participants necessary. results: considering that 25% of the influenza-like illness cases are not due to volunteer challenge studies have been used since the 60's to provide data on virus shedding from the respiratory tract during influenza infection. 1 recently, vk was studied in naturally acquired influenza infection. 2, 3 these data are invaluable to describe the natural history of influenza-infection and to compute natural history parameters such as the latent period, generation time, or the duration of infectiousness. [1] [2] [3] [4] however, among the 56 studies used in a meta-analysis about viral shedding kinetics, 1 the designs varied greatly from one to another. these differences led to variable amount of available information concerning the vk. the lack of adequate sampling leads to imprecise estimates. on the other hand, intensive sampling or over-sampling, while associated with highly informative data, may lead to unnecessary discomfort for the patient and cost to the investigator. optimal design is increasingly used to conceive studies 5 and provides cost-efficient designs. here we propose an optimised design to model vk in the case of influenza infection. we defined the number of participants, the number of samples to collect and their allocations. this design allows, at a minimum cost and discomfort, accurate vk curves and allows the natural history parameters to be well described. model a vk population model was proposed for influenza infection. this model describes with eight parameters the relations between free virus, uninfected target epithelial cells, infected epithelial cells, and early immune response. this model was built on a dataset of 44 volunteers from which nasal samples were collected once a day over 9 days. we call this dataset the ''original dataset''. three parameters, the induction of the early immune response, the virus production rate, and the virus clearance, did not show inter-individual variability and were precisely estimated (relative standard error below 10%). we considered them as fixed in this research work. five parameters were hence considered here: b the infection rate, d the infected cell mortality rate, w the effect of early immune response on virus production rate and v init the initial value of virus titre. in order to correctly estimate these parameters it is crucial to determine a design to collect informative data. optimal designs maximise the amount of information provided by the study. it involves the determination of the number and allocation of sample times per subject as well as the number of participants. 6 d-optimization is based on the maximization of the determinant of the fisher information matrix and thus minimizes the variance of the parameters. 7 we used the fedorov-wynn algorithm implemented in pfim 3.0 to maximize this determinant, 8 which implies to pre-define a set of possible sample times. with the hypothesis that the inoculation occurred at 8:00 am, we chose three possible hours (8:00, 14:00, and 20:00) for each day with respect of the sleep-time. 8 to validate the design, we simulated 100 datasets of 44 volunteers with the optimised design obtained. we then estimated the population parameters using monolix 3.1 for each of the datasets. we compared the estimated parameters obtained with the simulated datasets to the parameters used to build the optimal design. we computed the relative bias as: with n: number of successful estimations among the 100 simulated datasets. h i : parameter value obtained with the ith dataset. h: parameter value obtained with the original dataset. we also compared the observed rse from these simulations with the rse predicted by pfim and the rse obtained with the original dataset. the rse is proportional to ffiffiffi n p , where n is the population size. we can hence deduce the smallest number of participants necessary to obtain rse below 50%. where rse predicted is the highest predicted rse (here rse for w) with 44 participants and n predicted = 44 and rse min is equal to 0ae5. considering that 25% of the influenza-like illness cases are not due to influenza virus, the total number of participants should be multiplied by 1ae25. we found that the best design was when all the participants are sampled five times: three times during the second day post-inoculation at 8:00, 14:00, and 20:00 hours and twice on the third day post-inoculation at 8:00 and 20:00 ( figure 1 ). the comparison of the relative bias and rse predicted by pfim and those obtained after simulation and re-estimation of the parameters are shown in figure 2 . v init and d in a lesser extent present bias. fixed effect parameters are precisely estimated and accordingly to pfim except for v init . we found that 16 participants shedding virus or 20 participants with ili symptoms are necessary if 25% of them are not infected with influenza virus. we propose an optimised design to accurately study the vk of influenza virus with the minimal number of samples. this design is well balanced between the amount of necessary information and the precision of estimation. we found that 100 samples are necessary to precisely fit the vk curves, which is five times less than the number of samples collected in the original study. 4??? the samples should be collected during the second and third days after inoculation. yet we showed in a previous work that the incubation period lasted 1ae9 days. 4??? hence, the optimised sample times correspond to the two-first days of symptoms and this design could be applied to naturally acquired infections studies in which the inoculation time is unknown. an advantage of this design is its practicality and convenience. all samples are collected during the daytime and after the onset of symptoms. it can thus be used for studies with naturally acquired infections. the design was validated with several criteria concerning the accuracy of the estimation with the optimised design. the parameters estimates were generally satisfactory. the parameter describing the effect of the early immune response on the virus production rate was, however, less precisely estimated (predicted rse = 62%), and the initial value of the viral titre was very different of the one obtained with the original dataset (bias v init on figure 2 ). this is probably due to the fact that it was measured at day 2 post inoculation, and that the inter-individual variability is much higher than at day 0. furthermore, d (the infected cell mortality rate) seems also to be biased. this may be due to the fact that three parameters were fixed. the model used was developed from experimentally inoculated healthy volunteers with low serum haemagglutinin antibody titre and with virus inoculation time at 8:00 am. the applicability of the design to naturally acquired infection would depend on the pathogenicity of the virus as well as pre-existing immunity and the relevance of challenge method to natural influenza acquisition. 1 our design could be directly used to accurately study vk during influenza infections and would reduce the discomfort of patients and the cost of the experimentation. usefulness of a self-blown nasal discharge specimen for use with immunochromatography based influenza rapid antigen test introduction influenza rapid antigen tests (irat) have become very popular and are widely used for confirming suspected clinical diagnosis of influenza in japan. 1 most of the currently used irat that are based on immunochromatography (ic), nasopharyngeal swab, nasopharyngeal aspiration, and throat swab have been approved as specimens for japanese national health insurance purposes. but the specimen collection by these methods gives patients considerable discomfort, and sometimes appropriate specimens cannot be obtained due to patient resistance, especially by children. in the present studies, self-blown nasal discharge was used as the specimen for an irat, and the results were compared with the results of viral isolation and an identical kit primed with nasopharyngeal swab specimens for seasonal influenza viruses and pandemic (h1n1)2009 virus. patients who visited any of the 10 clinics that belong to the influenza study group of the japan physicians association in the 2006-2007 and the 2009-2010 influenza seasons with influenza-like illnesses exhibiting findings were registered after providing informed consent. a square plastic sheet of 20 · 20 cm was handed to the patient. nasal discharge was collected by blowing the nose into the plastic sheet as a specimen for irat, i.e. self-blown specimen. two nasopharyngeal swab specimens were also obtained at the same time for irat and virus isolation. self-blown specimens were obtained successfully by 152 (82ae2%) of 185 consecutive outpatients in the 2006-2007 season, as seen in table 1 the sensitivity and specificity of various influenza rapid antigen tests have been reported in various settings. [1] [2] [3] [4] direct comparison of the results is difficult because of differences in patient or influenza virus, characteristics such as age, study designs, and other features. in this study of the 2006-2007 influenza season, the sensitivity, specificity, and accuracy of the ic kit primed with nasopharyngeal swab specimens were 87ae0%, 94ae0%, and 90ae8%, respectively. these results were quite comparable to our results of the 2005-2006 season, 3 in which the overall results of other ic kits were 82ae9%, 81ae0%, and 82ae3%, respectively, indicating that the ic kit used is quite reliable. the sensitivity, specificity, and accuracy of an ic kit will vary by the method of specimen collection. in general, virus titer is considered to be highest with nasopharyngeal aspiration, lower with nasopharyngeal swabs, and lowest with throat swabs. practically, nasopharyngeal swab is the most popular. the sensitivity, specificity, and accuracy of the ic kit with self-blown discharge specimens compared well with those of an identical ic kit primed with nasopharyngeal swab specimens. for self-blown specimens, sensitivity and specificity were 87ae0% and 94ae6% for influenza a, 85ae7% and 99ae1% for influenza b, 88% and 88ae9% for pandemic (h1n1) 2009. self-blown specimens display sensitivity, specificity, and accuracy comparable to that of conventional nasopharyngeal swab specimens. there was no significant difference in sensitivity, specificity, or accuracy between self-blown specimens and nasopharyngeal swab for influenza a, influenza b, and pandemic (h1n1) 2009. these results suggest that selfblown specimens are as useful as nasal cavity swab specimens for the diagnosis of influenza in the clinical settings. nasal discharge, obviously, cannot be collected from infants incapable of blowing their own nose or patients who do not develop a nasal discharge. in this study, self-blown specimens were obtained from 82ae2% of the patients. the rate of successful collection was over 70% in the age groups of 0-6 and 7-15 years. these rates would seem to be sufficient for clinical use. the procedure of self-blown specimen collection using a plastic sheet is easy and causes no pain or discomfort. it seems to be more acceptable and safe than the other methods, especially for children. furthermore, this procedure reduces the risk of influenza transmission from patients to the medical staff members involved in sample collection. self-blown sample collection may be superior to other sample collection methods in these respects. we previously reported an inverse correlation between the amount of virus in a specimen and the time to a positive reaction. 4 in this study, there was no significant difference in the mean time to a positive between self-blown self-blown specimens enough to be examined were obtained 152 from 185 consecutive outpatients, and specimens showed a tendency to be obtained large amount from children rather than the aged. there were no statistically significant differences between the ic kit results primed with self-blown discharge and nasopharyngeal swab specimens for influenza a, influenza b and pandemic (h1n1) 2009. and nasal swab specimens, suggesting that the self-blown specimens contained sufficient viral antigen for the ic kits. the influence of the presence or absence of nasal congestion on the results of the kit was assessed. the sensitivity of selfblown specimens from patients with nasal congestion was significantly lower than that from patients without nasal congestion. it is possible that insufficient capability to blow the nose due to nasal congestion might tend to lead to false negatives. the observation that the time to positive is longer for patients with nasal congestion than for patients without nasal congestion is concordant. application of self-blown specimen collection only to appropriate patients would increase the sensitivity, which would be important in a clinical setting. we tested only two commercial antigen detection kit, the quick vue rapid sp influ kit and quicknaviô-flu (denka-seiken co., ltd). the resulting sensitivity, specificity, and accuracy of the ic kit primed with self-blown specimens were considered adequate for clinical use. to confirm the usefulness of self-blown nasal discharge specimens, further investigation is necessary using other kits and in different settings. the usefulness of a self-blown nasal discharge specimen for an influenza rapid antigen test based on immunochroma-tography was evaluated in the 2006-2007 and 2009-2010 influenza season. results suggest that self-blown nasal discharge specimens are useful as specimens for influenza rapid antigen tests based on immunochromatography for not only seasonal influenza viruses, but also pandemic (h1n1) 2009 virus. the specimen collection by the patients themselves will reduce the burden of other collection methods and the risk of infection to the medical staff. in april 2009, a mixed-origin h1n1 influenza virus was recognized as a new causative agent of influenza-like illnesses (ili) in humans. since its emergence, the virus has spread rapidly throughout the world and caused a pandemic. most commercial rapid antigen tests (rat) can detect influenza a or b viruses, but cannot specifically distinguish pandemic (h1n1) 2009 virus with seasonal influenza. recent studies have indicated that the poor performance of the rat approach and nonspecific detec-tion of the pandemic (h1n1) 2009 virus was the main obstacle to their widespread use in private clinics. 1,2 with the need for a new rapid kit with reasonable sensitivity and specificity for pandemic (h1n1) 2009 virus, we developed a new rat kit in collaboration with company, standard diagnostics, inc., (yongin-si, gyonggi, korea). monoclonal antibody (mab) against haemagglutinin (ha) of the pandemic (h1n1) 2009 virus was developed using korean isolate and applied to the new kit with the mab to seasonal influenza virus. we examined the detection limit of the kit using the serial dilution of korean pandemic virus isolate (a ⁄ korea ⁄ 01 ⁄ 2009). during december 2009, 432 clinical specimens from patients with ili were collected at 11 sentinel clinics of six provinces in korea. the specimens were tested by the new rat, and the results were compared with those of real-time reverse transcription polymerase chain reaction (rrt-pcr) by us cdc and virus isolation in mdck cell culture to determine the sensitivity and specificity for the diagnosis of pandemic (h1n1) 2009. the detection limit of the new kit against ha of a ⁄ korea ⁄ 01 ⁄ 2009 virus was confirmed to be 10 4 pfu ⁄ ml. by contrast, the detection limit against the np protein was 10 3 pfu. however, when the kit was applied to clinical specimens, no difference between the two targets was found. using rrt-pcr and viral culture as the references, the performance of the ridt is shown in table 1 . among 432 specimens, 178 were tested positive by rrt-pcr and 186 were tested positive by viral culture. among the 178 rrt-pcr confirmed cases, 122 were positive, and among the 186 viral culture confirmed cases, 120 were positive with the new rat. using rrt-pcr as the reference standard, the overall sensitivity of rat was 68ae5% (95% confidence interval (ci): 61ae7-75ae3%) and specificity was 98ae4% (ci: 96ae9-99ae9%). with viral culture as the reference, the rat sensitivity and specificity was 64ae5% (ci: 57ae6-71ae4%) and 97ae6% (ci: 95ae7-99ae5%), respectively. when analyzed by the regions tested, the sensitivity ranged between 57ae1% and 95ae5% for rrt-pcr and between 53ae3% and 87ae0% for viral culture as a reference. among 340 patients who had a record of their symptom onset and sample collection date, 86 (25ae3%) visited the clinic on the day of symptom onset, and 158 (46ae5%) visited 1 day later. when the rat performance was evaluated by day of onset, the sensitivity was lower at three or more days after the onset of symptoms; however, the sensitivity was highest at 2 days after onset and reasonable on the day of onset or at 1 day after ( table 2 ). we found that this new rat had reasonable sensitivity and high specificity compared with rrt-pcr and viral culture for detecting the pandemic (h1n1) 2009 virus. in one recent study, the sensitivity and specificity of the new rat kit was 77% and 100%, respectively, and the ha protein for pandemic (h1n1) 2009 was detected more sensitively than the np protein for influenza a virus. 3 the sensitivity and specificity of our new rat were lower than those of that study. we found that the test performance varied depending on the clinics in which the tests were performed, and this might be attributable to the persons who collected the specimens. although the clinicians were trained well for *ci, confidence interval. **ppv, positive predictive value. ***npv, negative predictive value. collecting specimens, there might be some differences in performance. the new rat kit could detect pandemic (h1n1) 2009 virus specifically. although the sensitivity was lower than those of rrt-pcr and virus culture, and negative rat results should be confirmed with more sensitive methods, this kit could be useful in sentinel clinics if used with caution. determination of infectious virus titres is central to many experiments designed to study the biology of influenza virus. assays based on the measurements of viral components, whether viral protein or nucleic acid, does not differentiate infectious virus from non-infectious or defective viral particles, which may have no infectivity or biological *three hundred and forty samples with a known date of onset and sample collection were analyzed. ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 132-158 activity. therefore the ''gold standard'' of virus measurement requires bioassays that examine the ability of viral particles to replicate and further infect other cells. titration on madin-darby canine kidney (mdck) cells in a 96 well plate format is commonly used to measure influenza virus titre. this method is labour intensive, subjective in their read out of cytopathic effect, and takes several days to obtain a result. microneutralization tests that quantitate neutralizing antibody titres and assays of drugs for antiviral activity also require 96 well based assays of residual virus infectivity. therefore, technologies that improve on the titration of infectious virus will be of great benefit. this study utilized the xcelligence system (roche applied science), which adopts microelectronic biosensor technology to monitor dynamic, real-time label free and non-invasive analysis of cellular events. 1 the system measures electronic impedance using an array of microelectrodes located at the bottom of each culture well (e-plate 96). adherent cells are attached to the sensor surface of electrode arrays, and changes in impedance can be detected and recorded. the xcelligence system can monitor cell events induced by viral infection, such as changes in cell number, adhesion, viability, morphology, and motility. measured electrode impedance is expressed as dimensionless cell index and is graphically represented using software to show the phenotypic changes of a cell population over time. the aim of this study is to demonstrate that using this platform to measure real-time cell index has potential to circumvent many of the limitations of the currently established procedures of end point titration of virus infectivity and for microneutralization assays. madin-darby canine kidney cells were propagated in growth medium consisting of minimum eagle's medium (invitrogen) supplemented with 10% fetal bovine serum (invitrogen), 0ae6 mg ⁄ l penicillin (invitrogen), and 60 mg ⁄ l streptomycin (invitrogen), with incubation at 37°c in a 5% co 2 humidified atmosphere. influenza a ⁄ hong kong ⁄ 54 ⁄ 1998 (h1n1), a seasonal influenza virus from a patient who suffered from a mild febrile illness, was propagated in mdck cells maintained in virus medium consisting of minimum eagle's medium (invitrogen) supplemented with 0ae6 mg ⁄ l penicillin (invitrogen), 60 mg ⁄ l streptomycin (invitrogen), and 2 mg ⁄ l np-tosyl-l-phenylalaninechloromethyl ketone-treated trypsin (sigma, st louis, mo, usa), with incubation at 37°c in a 5% co 2 humidified atmosphere. virus stocks were aliquoted and stored at 70°c until use, and the 50% tissue culture infectious dose (tcid 50 ) of the virus stock was determined by titration in mdck cells according to standard procedures, 2 and the tcid 50 of the stock virus was calculated by the method of reed and muench. 3 to perform a microneutralization assay, mdck cells seeded at a density of 1000 cells ⁄ well in an e-plate 96 was removed from the xcelligence system after approximately 48 hour; growth medium was then removed, cells washed, and replaced with 100 ll virus-medium. a human serum, which is known to contain high titre antibody against the h1n1 virus was heat inactivated for 30 min at 56°c, and twofold serial dilutions were performed in virus medium. the diluted serum was mixed with an equal volume of virus medium containing influenza virus at 400 tcid 50 ⁄ 100 ll. after incubation for 2 h at 37°c in a 5% co 2 humidified atmosphere, 50 ll of virus-antibody mixture was added to the mdck cells to give each well an equivalent virus dose of 100 tcid 50 . a back titration of the virus challenge dose was performed, and a cell control (free of virus) was performed in quadruplicates. after incubation at room temperature for 30 minutes, the e-plate 96 was then placed back onto the xcelligence system in the incubator and maintain at 37°c with 5% co 2 , and the cell index values were measured every 15 minutes for at least a further 72 hour. the same procedures were performed with cells seeded in conventional 96 well cell culture plates for parallel comparison with the currently used standard method. in this case, cells were examined for cytopathic effect under an inverted microscope after 3 days of infection and the lowest virus dilution, which protected the cells from viral induced cytopathic effect taken as the neutralizing end point. after 48 hour of seeding mdck cells at 1000 cells ⁄ well, standard microneutralization assay for influenza virus was performed. integral to this assay, a serial titration of the input virus at 0ae5 log 10 increments was carried out. wells infected with the undiluted virus (100 tcid 50 ⁄ well), the cell index commenced dropping at a steeper gradient than the no-virus cell control after approximately 3 hour of infection ( figure 1 ). this drop in cell index continues at a consistent slope until it flattened out when approaching zero cell index. this steep decrease in cell index with constant gradient was also observed for virus dilutions up to and including 2 log 10 (100-folds), and the profile shifted with increased time in proportion to the dilution made to the virus. virus dilutions beyond 2 log 10 have cell index profiles similar to the no virus input control, and this corresponds to the absence of cytopathic effect as determined by microscopic observation at 72 hour after infection. hence, there was a correlation between the amount of virus used for infection, the onset of the influenza virus-mediated cytopathic effect, and the steep decline in cell index. a human serum with known microneutralization antibody titre to h1n1 virus was used in this study to investigate the real time cell index changes that occur during the assay ( figure 2 ). using influenza virus treated with serum dilutions up to and including a dilution of 1:160, the cell index profile remained essentially the same as the no virus cell control, which correlates with the lack of cytopathic effects under microscopic observation at 72 hour of infection. at a serum dilution of 1:320, the steep decrease in cell index, which is characteristic of cellular cytopathic effect induced by the virus, became evident at around 50 hour post infection, and this was reduced to 40 hour when serum dilution of 1:940 was used. in contrast, for the virus -no antibody control, the onset time for this steep decrease in cell index occurs at approximately 7 hour. for both serum dilutions of 1:320 and 1:940, full cytopathic effect was observed microscopically at 72 hour of infection. from microscopic observation of cytopathic effect, according to the current standard procedures, the neutralizing titre of the human serum used in this study is at 1:160 as it is the last dilution of the serum that prevented cytopathic effect from being detected. an essential part of the microneutralization assay is to confirm the titre of the input virus (normally 100 tcid 50 ⁄well) by performing a titration assay with decreasing serial dilutions of the virus. under normal procedures, cells are examined microscopically after 72 hour of infection for sign of cytopathic effects. in the case of mdck cells, the cytopathic effect is cell death, which is indicative of the presence of live influenza virus infecting and replicating in the cells. therefore, the titre of the virus is taken as the last dilution in which cytopathic effect is present. parallel realtime cell index measurements demonstrated that for wells with cytopathic effects, the profile exhibits a steep gradient linear decrease in cell index after infection with the virus, which can be termed the ''cpe plunge.'' the time in which the cpe plunge became evident appears to be inversely proportional to the amount of virus, therefore the opportunity exists to utilize this aspect to calculate or compare quantitatively different virus concentrations. for unequivocal assignment of cytopathic effect, it normally requires 2-3 days after infecting the cells, with 3 days after infection being the standard time to read virus titration and microneutralization assays. using the real-time cell index monitoring, it is found that apparent cytopathic effect can only be observed microscopically when the cell index has dropped to near zero. as the time of onset of the ''cpe plunge'' becomes evident many hours prior to observable cytopathic effect, it is possible that the time to results can be drastically reduced after some formulation of the method. we compared the current standard method in perfoming a microneutralization assay with one utilizing the real-time cell index measurement to investigate whether this approach is able to offer better performance over the existing one. the current standard neutralization assay is the microscopic observation of antibody mediated protection from virus cytopathic effect in mdck cells. this study showed that this may also be achieved by examining the profile generated from the real-time measurements of the cell index. using real-time cell index monitoring, it is possible to detect inhibitory activity at higher dilutions of the anti-serum than can be detected by the standard microscopic observation of cytopathic effect. therefore, the realtime cell index monitoring could potentially be developed to be a more sensitive method for measuring anti-viral activity. as drug resistant strains of influenza a viruses 4 including the 2009 pandemic h1n1 are being reported, the real-time cell based monitoring system may also have the potential to be developed for use as a diagnostic platform for drug resistance assays. this study suggests that real-time cell index monitoring has the potential to substantially reduce human resources in reading results, as well as reducing time-to-result of these assays from 3 days to two. the saving could be substantial for work involving bio-hazard level 3 ⁄ 4 pathogens such as h5n1 viruses as personnel working with these organisms are require to be highly trained and experienced. in addition, the reduction in transferring plates to and from the microscope in reading cytopathic effect will substantially reduce the possibility of accidents from occurring. furthermore, the system provides objective digital data to an otherwise subjective assay method, which can improve standardization, data exchange, and hence collaboration between different laboratories. with more detailed validation and development, real-time cell index monitoring could transform the way we study and diagnose infection with pathogens such as influenza viruses. the emergence of a novel h1n1 influenza a virus of swine origin, the pandemic a(h1n1) 2009, with transmissibility from human to human in april 2009 posed pandemic con-cern and required modifications to laboratory testing protocols. a new protocol for universal detection of influenza a and b viruses and simultaneous subtyping of influenza a (h1n1) 2009 virus, composed of two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy), was evaluated and compared to the reference protocol recommended by who. fast set infa ⁄ infb was able to detect influenza a and b viruses circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. the who assay was found to have a slightly lower end-point detection limit (10 )5 dilution) in comparison to the new protocol (10 )6 ). specificity of the assays was 100% as assessed on a panel of stored clinical samples including adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, s. pneumoniae, n. meningitidis, h. influenza, and human influenza viruses. the new assay panel allows the detection, typing, and subtyping of influenza viruses as requested for diagnostic and surveillance purposes. the high sensitivity of the protocol is coupled with capacity to detect viruses presenting significant heterogeneity by fast set infa ⁄ infb and with high discriminatory ability by fast set h1n1v. a rapid and sensitive assay for the detection of influenza virus in clinical samples from subjects with ili or low respiratory tract infections is a fundamental tool for epidemiological and virological surveillance, management of hospitalized patients, and control of virus nosocomial transmission. the emergence, in april 2009, of a novel h1n1 influenza a virus of swine origin, the pandemic (a(h1n1) 2009), with transmissibility from human to human poses pandemic concern and required modifications to the laboratory testing protocols. 1 molecular diagnosis of influenza is generally achieved through a twophase process: a screening phase for the detection of virus, and the subsequent strain characterization performed by either sub-type-specific rt-pcr or entire ⁄ partial genome sequencing. 2 during a pandemic, simultaneous implementation of both the detection of influenza a and b influenza viruses and identification of the new subtype is useful for clinical and epidemiological reasons. here, we describe a new protocol including two-one-step rt-pcrs, fast set infa ⁄ infb and fast set h1n1v (relab, italy) that allows universal detection of all influenza a viruses and, simultaneously, all subtypes that are influenza a(h1n1) 2009. specificity and clinical sensitivity of the two-one-step rt-pcrs (fast set infa ⁄ infb and fast set h1n1v; relab, italy) were evaluated by testing 306 selected specimens, including: • fifty samples collected from nasopharyngeal swabs representative of influenza viruses, belonging to differ-ent subtypes and lineages, and other respiratory viruses and bacteria circulating in italy between 1995 and 2008. • six purified a(h5n1), a(h7n7), and a(h9n2) strains, kindly supplied by alan hay, who influenza centre, london, uk. • two hundred-fifty influenza positive samples selected according to type, subtype, clade and viral concentration from >2500 specimens received by the liguria influenza reference laboratory between january 1st and december 31st, 2009. since 1995, nasopharyngeal swabs sampled from patients suspected of having contracted the influenza virus have been collected in viral transport medium, and upon arrival into the laboratory, the samples were divided in ‡3 aliquots. those not immediately processed were stored frozen at )80°c. stored samples were used for this evaluation, and all specimens were re-extracted for the study. samples collected between 1995 and 2008 included specimens positive for: no seasonal a(h1n1) have been detected since january 1st, 2009. furthermore, 1 weak positive sample using fast set infa ⁄ infb, but negative at block pcr and typing ⁄ subtyping assays was tested. the analytical sensitivity of the test under investigation was determined testing ten-fold serial dilutions of seasonal influenza a(h1n1), seasonal influenza a(h3n2), new pandemic influenza a(h1n1) 2009, and b cell culture-grown viruses. the intra-assay reproducibility was measured by testing the same a(h1n1) 2009 positive sample 15 times in the same experiment, while the inter-assay reproducibility was confirmed by testing the same samples in 3 independent experiments. to evaluate the performance of the protocol, all samples were tested using a block pcr confirmation test (seeplex ò rv12 ace detection), 3 and all specimens collected between january 1st and december 31st, 2009 and dilutions were also assayed using the recommended who ⁄ cdc protocol of real-time rtpcr for influenza a(h1n1). typing and sub typing were performed using the who protocol and ⁄ or sequencing. 4 viral rna was extracted from swabs using the qiaamp viral rna mini kit (qiagen) according to the manufacturer's protocol. fast set infa ⁄ infb and fast set h1n1v are two multiplex one-step real time pcr assays developed and evaluated by the liguria regional reference centre for diagnosis and surveillance of influenza in collaboration with relab diagnostics. both assays contain primers and a dual-labelled hydrolysis probe that targets two regions of the matrix gene (table 1) . amplification conditions were as follows: reverse-transcription 50°c for 15 minutes, denaturation 94°c for 2 minutes, then 40 cycles of 95°c for 15 seconds, 58°c for 30 seconds. the entire amplification process extended for 110 minutes. an internal control real-time assay was also incorporated in order to detect pcr inhibition, failed extraction ⁄ pcr and technical error. the cdc realtime rtpcr (rrtpcr) protocol for detection and characterization of swine influenza includes a panel of oligonucleotide primers and dual-labelled hydrolysis (taqman ò ) probes to be used in real-time rtpcr assays for the in vitro qualitative detection and characterization of swine influenza viruses in respiratory specimens and viral cultures. this protocol recommends three primer-and-probe sets: infa, amplifying a conserved region of the matrix gene from all influenza a viruses; sw infa, designed to specifically detect the nucleoprotein (np) gene segment from all swine influenza viruses and sw h1, designed to specifically detect the hemagglutinin gene segment from a(h1n1) 2009. 5 the seeplex ò rv12 ace detection for auto-capil-lary electrophoresis is a multiplex block rt-pcr that applies dpoô (dual priming oligonucleotide) technology and is designed to detect 12 major respiratory viruses, 11 respiratory rna (influenza a and b virus, parainfluenza virus type 1, 2 and 3, respiratory syncytial virus a and b, rhinovirus a ⁄ b, coronavirus oc43 and 229e ⁄ nl63) viruses and 1 dna (adenovirus) virus, from patients' samples including nasopharyngeal aspirates, nasopharyngeal swabs and bronchoalveolar lavage. 3 conventional viral culture was performed inoculating 0ae1 ml of each specimen into mdck-siat1 seeded into 24-well plates for influenza isolation. virus detection was performed by the hemagglutination test using 0ae75% guinea pig red blood cells (rbc). specificity and clinical sensitivity results of the new protocol are reported in table 2 . fast set infa ⁄ infb was able to detect influenza a and b virus circulating between 1995 and 2008 belonging to different subtypes and lineages, and no cross-reactions were observed by either fast set infa ⁄ infb or fast set h1n1v. among specimens collected between january 1st and december 31st, 2009, all 60 fast set infa ⁄ infb and fast set h1n1v high titre positive samples resulted positive using the who ⁄ cdc assay and showing reactivity using infa and sw infa primer-andprobe sets. among 90 low titre a(h1n1) 2009 positive samples at fast set infa ⁄ infb, 28 (31ae1%) were not detected by the who ⁄ cdc assay, but were positive using seeplex ò rv12. the who ⁄ cdc sw h1 primer-and-probe set works in 96ae7% (58 ⁄ 60) and 1ae1% (1 ⁄ 90) of high and low titre a(h1n1) 2009 positive samples, respectively. all a(h3n2) strains collected during 2009 and initially detected by fast set infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12 and who ⁄ cdc assay showing reactivity using the infa primer-and-probe set. all infa ⁄ infb were confirmed after rna re-extraction by seeplex ò rv12. one influenza a case identified by the who ⁄ cdc kit (infa primer-and-probe set, ct values: 37ae5, sw infa primerand-probe set: negative) and new protocol (a primer-andprobe set, ct values: 34ae1, a(h1n1) 2009 primer-and-probe set, ct values: 34ae6) was not detected by either seeplex ò rv12 or by who subtyping protocol and ⁄ or sequencing, suggesting a very low viral load or unspecific results by real time assays. the analysis of serial dilutions of cell culturegrown a(h1n1) 2009 showed that the detection limit of fast set infa ⁄ infb, fast set h1n1v, and seeplex ò rv12 was identical (10 )6 ) and 1log 10 lower than that using the who ⁄ cdc protocol (10 )5 ). a similar analysis with respect to a(h1n1) and a(h3n2) strains indicated that fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 (10 )5 and 10 )6 , respectively). in comparison with the new protocol, the who ⁄ cdc assays, considering infa primer-and-probe set, was found to have a slightly lower end-point detection, detecting the 10 )5 a(h1n1) and a(h3n2) dilution. also in detecting influenza b virus, fast set infa ⁄ infb sensitivity (10 )6 and 10 )7 , respectively) was 1log 10 lower than that showed by seeplex ò rv12 and the who ⁄ cdc protocol. data on intra-assay and inter-assay precision, measured as cv% of ct showed that the dispersion indices observed had values of less than 3%. since 2009 samples were detected using the new protocol that resulted negative using the who ⁄ cdc assays. the unfortunately low quantity of low titre a(h3n2) samples collected during 2009 did not allow us to highlight differences between assays fast set infa ⁄ infb, and fast set h1n1v positivity was always confirmed by seeplex ò rv12, which demonstrated high sensitivity, showing a detection limit comparable or lower when compared with those observed using the who ⁄ cdc assays. the high analytical sensitivity of seeplex ò rv12 is reported by kim 3 who observed a detection limit of 10 copies per reaction for each type ⁄ subtype of influenza viruses. the high sensitivity of the new protocol is coupled with its capacity to detect viruses presenting a significant heterogeneity by fast set infa ⁄ infb and high discriminatory ability by fast set h1n1v. fast set infa ⁄ infb was able to identify representative influenza viruses of circulating strains during the last decade belonging to different subtypes, lineages, and clusters, and fast set h1n1v primerand-probe set reacted selectively with a(h1n1) 2009 target. a recent report demonstrated that the sw infa assay is not specific to a(h1n1) 2009 and is able to detect both human and avian (h5n1) influenza a viruses and so there is the potential for misidentification. 15 high titre (ct 8ae5 and 15ae6 at fast set infa ⁄ infb) a(h5n1) viruses did not react with fast set h1n1v primer-and-probe set (data not shown). available human a(h5n1) sequences are similar within the h1n1v primer-and-probe regions, but having 4-5 mismatches in the forward primer and, more notably, two of the mismatches occurred within 9 nucleotides of the 3 end, an important determinant for primer specificity. in conclusion, this protocol can be a powerful tool in the diagnostic laboratory setting for specific simultaneous analysis of several samples in minimal time, showing enhanced sensitivity in detecting influenza viruses, and high discriminatory ability in identifying the new pandemic a(h1n1) 2009. a university-corporate partnership to enhance vaccination rates among the elderly: an example of a corporate public health care delivery public health campaigns usually rely on governmental infrastructure and finance for vaccine implementation programs. however, there are many financial and physical barriers which preclude widespread and effective vaccine administration, especially among the elderly. on an international scale, both government agencies and citizen groups have a vested interest in searching for more resourceful methods of attaining significant immunization levels (>75% of the population). in fact, it seems to have become both a grassroots civic and governmental goal, especially among developing countries. we implemented the unique strategy of enlisting the assistance of a privately-owned food market chain to address the public health issue of mass vaccination for the elderly. in this context, publix pharmacy and the university of south florida (usf) recently developed both a handbook and a training program to facilitate the administration of vaccinations. between 2008 and 2009, the publix-usf partnership resulted in administration of over thirty thousand influenza a (h3n2) vaccinations, 76% of which were given to adults over 55 years of age. consequently, vaccine administration costs were decreased by using corporate resources and bypassing overly strained municipal resources. this unique university-corporate partnership successfully delivered h3n2 vaccine to a vulnerable cross-section of society at a lower cost and with minimal side effects and morbidity. it may be safely projected that university-corporate partnerships could result in an effective method for rendering a vital service to an aging and especially vulnerable segment of the population. government policy and funding are the foundation of immunization programs on an international scale. for example, in the united states, governmental programs account for over 50% of the monetary outlay used for immunization. 1 until 2006, the global alliance for vaccines and immunizations (gavi) acted as a catalyst for implementing vaccine and immunization programs in each targeted country. 2 under the auspices of gavi-collaborations between governments, charitable organizations, and multinational health agencies (such as uncief and the who)-many countries have increased their spending for vaccination programs. however, development of financially sustainable immunization programs geared toward reaching the majority of the population are still at a nascent level of evolution. 3 the development of more innovative and costeffective approaches has become imperative in order to reach a greater number of vaccination candidates. administering the influenza vaccine only to the subpopulation of over 65 year olds would save an estimated 220 000 quality-adjusted life years in a cohort of approximately half the world's population. 4 widespread public vaccination programs are made more complex by the continuing development of newer vaccines, concomitant specialized administration costs, and the logistical challenge of conveying recipients to vaccination points of service. 1, 5 in spite of the increasing complexity of mass vaccination, cost-benefit analyses clearly favor annual influenza vaccination in the elderly population on an international scale. 4, 6 recently, in 2008, influenza vaccine administration was reported to reach between 32% and 82% of the elderly population, which denotes varying degrees of success within each particular country. 7, 8 however, there was also a report of a uniform plateau effect at around 75% of the population, beyond which additional vaccination coverage was difficult to achieve. 9 physical limitations to vaccination seem to be more insurmountable for the elderly. unfortunately, this is the population segment which could experience the most significant vaccination-associated mortality reduction. 10 we employed the unique strategy of involving the resources of publix supermarkets, a corporate food market chain, to address the public health issue of widespread vaccination for the elderly. we took advantage of recent 2005 changes in the florida statutes, which expanded the scope of pharmacists' practice to include administration of vaccines. subsequently, publix pharmacy and the university of south florida (usf) developed a handbook and training program to facilitate and enhance vaccine administration by publix pharmacists. by using proprietary pharmacists and more practical supply storage, we were able to decrease the costs of vaccine administration. the consumer was charged $10 for administration costs plus the cost of the injection itself, regardless of insurance or eligibility for governmental subsidy. although patients were initially self-selected, they were ultimately excluded if they had demonstrated prior adverse effects to influenza vaccinations or to any of the components of such vaccinations. between 2008 and 2009, the publix-usf partnership vaccinated 30 063 people against influenza a (h3n2), of which 29 404 were florida residents. the age range was 1-105 years old with a median age of 65 years old. seventysix percent of the participants were over 50 years old (see figure 1 ). within the population surveyed, the reported side effects of the vaccine in this study were not serious, but included: vertigo, cold sweats, chills, vomiting, syncope, rash, nausea, stomach pain, elevated blood pressure, injection site reaction, inflamed bursa, and bilateral thigh discomfort. participants from all socioeconomic classes were vaccinated. an income-by-zip code analysis revealed 44% of those vaccinated resided in zip code areas where the average household income was <$50 000 per year. of those remaining, 25% had an average income of $50 000-$70 000 per year, and 31% had an income of >$70 000 per year. each person vaccinated was charged ten dollars for administration costs. this represents a decrease in the administration costs ranging from one dollar to ten dollars saved per vaccine. 11, 12 conclusion this unique university-corporate partnership successfully delivered h3n2 vaccine to a high-risk population with decreased vaccine administration costs. the influenza vaccine is well-tolerated, with minimal side effects when patients who have a history of adverse reactions are excluded. we can postulate that university-corporate partnerships may indeed be effective at reaching the aging population which is a challenge in most communities. this delivery model may prove to be another tool for improving the efficiency of mass immunization by facilitating accessibility, which results in wider coverage. this model also enhances delivery of healthcare by decreasing costs of immunization regardless of whether the payer is a government, insurance company, or self-pay consumer. the gavi initiative stressed three goals for accomplishing sustainability and independence in immunization programs. 3 the goals were to: (i) mobilize additional resources from governmental and non-governmental sources; (ii) improve program efficiency to minimize additional administration resources needed; and (iii) increase the reliability of funding. empowering privately owned corporations within the community, such as food markets or pharmacies, to administer vaccines mobilizes additional resources to readily achieve the first goal of gavi. mobilizing resources of non-healthcare, corporate vaccination locations enhances accessibility due to travel convenience. in our study, participants came from all socioeconomic classes, suggesting that ease of access is independently hindering mass vaccination, and that people of all incomes are more likely engaged when access issues are eliminated. the second and third goals were also accomplished by recruiting a corporation's resources for vaccine administration (refrigeration, storage, and employees). this minimizes the money spent from vaccine program funds to support the infrastructure of immunizations, thus improving financial efficiency and sustainability. financial efficiency implies that money is spent to safely reach as large a portion of the population as possible. by using corporate storage facilities instead of paying for independent facilities, money can be spent elsewhere. more vaccines can be purchased and more money can be spent on media communications to encourage vaccination. sustainability requires the ability to fund annual vaccination programs which reach 75% of the population or greater. key to the control of pandemic influenza are surveillance systems that raise alarms rapidly and sensitively. in addition, they must minimise false alarms during a normal influenza season. we develop a method that uses historical syndromic influenza data from the existing surveillance system 'servis' 1 monitoring seasonal ili activities in scotland. we develop an algorithm based on wcr of reported ili cases to generate an alarm for pandemic influenza. wcr is defined as the ratio of the number of reported cases in a week to the number of cases reported in the previous week. from the seasonal influenza data from 13 scottish health boards, we estimate the joint probability distribution ( figure 1) we compare our method, based on our simulation study, to the mov-avg cusum and ili rate threshold methods and find it to be more sensitive and rapid. the wcr method detects pandemics in larger fraction of total runs within the same early weeks of pandemic starting than does any of the other two methods ( figure 2 ). as shown in the table, for 1% pandemic case reporting rate and detection specificity of 95%, our method is 100% sensitive and has mdt of 4 weeks, while the mov-avg cusum and ili rate threshold methods are, respectively, 97% and 100% sensitive with mdt of 5 weeks. at 99% specificity, our method remains 100% sensitive with mdt of 5 weeks. although the threshold method maintains its sensitivity of 100% with mdt of 5 weeks, sensitivity of mov-avg cusum declines to 92% with increased mdt of 6 weeks. for a two-fold decrease in the case reporting rate (0ae5%) and 99% specificity, the wcr and threshold methods, respectively, have mdt of 5 and 6 weeks with both having sensitivity close to 100%, while the mov-avg cusum method can only manage sensitivity of 77% with mdt of 6 weeks. the first cases of the 2009 pandemic were reported in scotland in the 29th week of the season. the wcr algorithm as well as the mov-avg cusum method detects the pandemic 12 weeks later in week 41. the ili threshold method detects it 1 week later in week 42. both the wcr and mov-avg cusum methods therefore outperform the ili threshold method by 1 week in the retrospective detection of the 2009 pandemic in scotland. while computationally and statistically very simple to implement, the wcr method is capable of raising alarms rapidly and sensitively for influenza pandemics against a background of seasonal influenza. although the algorithm has been developed using the servis data, it has the capacity to be used at large scale and for different disease systems where buying some early extra time is critical. more generally, we suggest that a combination of different statistical methods should be employed in generating alarms for infectious disease outbreaks. different detection methods would provide cross-checks on one another, boosting confidence in the outputs of the surveillance system as a whole. real-time evidence being created worldwide will greatly contribute to the full understanding of influenza pandemics. here we report the real-time epidemiology and virology findings of the influenza a(h1n1)2009 pandemics in mongolia. the epidemiological and virological data collected through isss of nic, nccd, mongolia (real-time information on registered ili cases and virological laboratory results are available from the weekly updates in the nic, mongolia website: http://www.flu.mn/eng/index.php?option=com_ content&task=category&sectionid=5&id=36&itemid=51) were used for analysis in relation to the previous seasonal influenza activities in the country. influenza viruses were detected in naso-pharyngeal samples from ili patients by rt-rt-pcr with applied biosystems fast real time pcr system 7500, using primers and instructions supplied by cdc, usa. 1 influenza viruses were isolated by inoculation of rt-rt-pcr-positive samples of mdck cell culture according to the standard protocol. 2 ten representative strains of a(h1n1)pdm viruses were selected for sequencing of different gene segments, namely: a ⁄ ula, and a ⁄ dundgovi ⁄ 381 ⁄ 2010. sequencing of influenza virus gene segments was performed in applied biosystems 3130xl genetic analyzer using primers and instructions supplied by cdc, usa, 3 and bioinformatic analysis was performed with abi ⁄ seqscape v.2.5 and mega4 programs. the pandemic alert in mongolia was announced by the government on april 28, 2009, just after the who announcement of the pandemic alert phase, and planned containment measures were intensified. despite intensive surveillance, no a(h1n1)pdm virus was detected in mongolia until the beginning of october 2009. around 40 suspected cases, mostly arriving from the a(h1n1)pdm epidemic countries, tested zero by rt-rt-pcr for a(h1n1)pdm virus. the first a(h1n1)pdm case detected by the routine surveillance system in ulaanbaatar city, the capital of mongolia, was confirmed by rt-rt-pcr on october 12, 2009 (41st week of 2009). the reported ili cases escalated rapidly, reached the peak in the 43-44th week of 2009, and gradually decreased thereafter ( figure 1 ). week of 2010. however, the registered ili cases increased again from the 5th week of 2010, and peaked at the 8-9th weeks of 2010. the viruses isolated during this 2nd peak were influenza b strains ( figure 1 and table 1 ). for the genetic characterization of the mongolian pandemic isolates, 3 gene segments i (pb2), 2 gene segments ii (pb1), 3 gene segments iii (pa), 9 gene segments iv (ha), 3 gene segments v (np), 4 gene segments vi (na), 7 gene segments vii (m), and 3 gene segments viii (ns) of the representative a(h1n1)pdm mongolian strains were sequenced, and all sequences have been deposited in the genbank (accession numbers: cy050844, cy0533648, cy050846, cy050845, cy065987, cy065989, cy065990, cy 065988, cy065991, cy065985, cy065984, cy065986, cy052366 , cy053365, cy054546, cy054547, cy055169, cy 055170, cy055171, cy056363, cy055308, cy057191, cy057083, cy065995, cy065997, cy065998, cy065996, cy 065999, cy065993, cy065992, cy065994, cy054548, cy054549, cy073448). all 8 genes of mongolian strains were possessing 99ae4-99ae9% similarity with the genbank deposited gene sequences of the original pandemic strain a ⁄ california ⁄ 072009(h1n1). the who declared the pandemic alert phase (phase iv) on april 27, 2009, 4 and was prompted to announce the pandemic phase (phase v) two days later. 5 after 43 days, the who declared the beginning of the pandemic peak period (phase vi) on june 11, 2009. 6 however, in mongolia, the pandemic alert period continued for 168 days. mongolia was free of the pandemic virus during the whole first wave of the pandemics in the northern hemisphere. with the confirmation of the 1st influenza a(h1n1)pdm case on october 12, 2009 in ulaanbaatar, mongolia entered into the pandemic phase (phase v), and after just 2 weeks, the registered ili cases peaked, confirming mongolia shifted into the pandemic peak period (phase vi), which i i i v i i v i v v v i i i i iii iv v vi vii viii worldwide by who in mongolia coincided with the 2nd wave of pandemics in many countries of the northern hemisphere (see, picture 1 and table 1 ). despite the relatively milder clinical manifestations, the disease burden for the health service was enormous, while the morbidity per 10 000 population at the peak period was 5-6 times higher above the upper tolerant limit, and 3-4 times higher above the seasonal influenza outbreaks. in contrast to the seasonal influenza outbreaks where over 80% of the registered ili cases have been in the age group under 15, 2 it has been observed that over 50% of the registered cases in this pandemic peak period were in the age group of 16-60. on january 18, 2010, we regarded the pandemic had entered into the post-peak period (phase vii) when the registered ili cases became lower than the upper tolerant limit, during which time mongolia experienced an influenza b outbreak. on may 24, 2010 we determined that mongolia entered the post-pandemic period (phase viii) as the influenza virus isolations were almost stopped, and after no pandemic virus detected for 3 months. who announced pandemic vii and viii phases much later. 7, 8 this first ever real-time laboratory confirmed influenza pandemics in mongolia and confirmed some variations of pandemic spread in different parts of the world. the comparison of deduced amino-acid sequence changes have shown that the mongolian strains belong to the clade 7, according to the classification of a(h1n1)pdm influenza strains suggested by m. nelson, 9 which has circulated worldwide since july 2009. this is also evidence that the 1st wave of the pandemics did not hit mongolia. the who public health research agenda for influenza 1 is aimed to support the development of evidence needed to strengthen public health guidance and actions essential for limiting the impact of influenza on individuals and populations. each stream-specific group reviewed and discussed the proposed organization, content, rationale, and global health importance of their designated research stream. specific research recommendations were made for topics within each stream: background: a syndromic surveillance system using nonclassical data sources for detection and monitoring evolution of flu and flu-like illness (ili) in djibouti is reported here as part of the preliminary report of djibouti who-copanflu international study (wcis)**. methodology: clinical reports, over-the-counter drug sales, lab diagnosis report, and health communication trends were obtained for an integrated statistical analysis. results: transition to winter is concomitant with upsurge of ili cases and ili drug sales. in addition, more rural folks manage ili infections on self medicament than through clinical consultancy. inefficient and vague data collections were observed. a successful implementation of wcis will create a platform upon which challenges faced in djibouti health department in routine surveillance will be addressed to achieve a near-real time surveillance of flu pandemic. conclusion: innovations, prompt reporting, and instituting open source syndromic surveillance system software's in resource limited environment like djibouti will enhance early detection and evolution monitoring of pandemic flu. the spanish flu in 1918 ⁄ 1919 infected and killed millions of people, and threatened to wipe humanity off the face of planet. however, the recent scenarios of influenza h1n1 (2009) pandemics' worldwide occurrence fell short of most scientific prediction on its magnitude and intensity. this dampened their confidence; they cannot state precisely as to when, how, where, and which of the spanish flu-like pandemic will occur in the future. in support of scientific community and governments, the who hasn't gone to slumber, but is reminding its member states to up their post pandemic surveillance and monitoring of influenza virus in circulation for advance preparedness in case of an outbreak. 1 despite all uncertainty around the pandemic flu h1n1 2009, there remains a common knowledge and understanding that this flu has shown a great potential to evolve and cause huge morbidity and mortality. although its future magnitude may be unpredictable, its recurring events have severe consequences on human health and the economic well being of everyone. 1 and therefore, advance planning and preparedness is critical in protecting any population in the future, especially those located in resource limited environment without universal health cover and generous disaster emergency funds. 2 . two collapsed sets of a weekly and monthly mean data (of four years period) were clustered in five categories of ili cases, drug sales, lab results, vaccine consumption, and health promotion. this was followed by a descriptive statistics analysis of cumulative weekly and monthly data to establish presence or absence of trend. time series analysis was not done due to data limitation. copanflu program: as at the time of going to press, the cohort study is at the household recruitment and inclusion phase and the study covers the djibouti city. it is in our intention to use the cohort study findings to validate or improve the niph ministry of health djibouti ili surveillance effort for better preparedness. clinical service: 83% of all health facilities are in djibouti city. of the 39ae9% (326 445) of the population that seeks medical care on influenza and influenza like illness each year, 58ae2% (187 586) and 11ae9% (38 389) of them are attended to at the city's public and private clinics, respectively ( figure 1 ). the rest are attended from the regional health centers. the majority of ili incidence sharply rise with the onset of the winter season (october to april), affecting mostly the middle age group (1-24 years). pharmaco-surveillance: 2% (99 350) of total prescriptions were antipyretic and antiflu drugs, 91ae4% (90 765) of which were consumed by peripheral regions, the non djilab diagnostics: the annual ili lab diagnosis was negligible 0ae0072% (250), which can be attributed to less equipped virology laboratories to warrant routine service utility. documented cases were from previous bouts of avian influenza that had a human incidence from 2006 and 2009. 5 with support of egypt-based naval army medical research unit three (namru 3), clinicians were motivated to sample all ili patients and submit to collaborating international reference influenza lab in cairo, egypt. vaccination: influenza vaccinations were undocumented, but at least 0ae4% (3122) of population sought the service (for yellow fever and meningitis) as mandatory travel advisory or as childhood immunization need. at the time of going to the press, there were at least 80 000 vaccine doses of h1n1 (2009) virus donations yet to be administered. health promotion and hygiene: print and audiovisual risk communication remained favorite means of reaching out to urban dwellers (70ae6%). while to the rural and nomadic population, person to person communications was the preferable means. to increasing public awareness that will encourage reporting of ili cases and entrench risk aversion health behavior that limits flu spread, who-copanflu international study djibouti has incorporated basic training on ili infection and personal hygiene by interviewers during household inclusion. improving national epidemic surveillance capacity and response under new international health regulation 6 is important for any nation, including djibouti. our finding indicates the winter season predisposes one to ili infections; they therefore opt for medical services or self medication depending on their capability and ⁄ or understanding. in djibouti, almost no city dwellers favors self medication over clinical consultation, suggesting the presence of inhibitory factors like distance from the health centers and the cost of accessing consultancy. common in the absence of universal primary health care setting, it therefore calls for active innovativeness in outbreak detection, disease reporting, and preventive medicine on the part of health authority so as to achieve good population health. 7 in respond to these, niph has turned resource limitation to a motivation instead and is working towards institutionalizing a near-real-time syndromic surveillance system as a core functional unit. it capitalizes on three major aspects within its reach: prompt accurate data generation for analysis, ehesp wcic-study input, and information technology use. prompt accurate data generation for analysis: data used in our analysis suffered from un-timeliness (weekly instead of daily basis), incompleteness (vague over-counter drug sales records), entry errors (incidence case reports), and poor collection format (most of data collection forms). use of satellite handset phones for regional health centers and mobile phones for city sentinel clinics will reduce unnecessary data delivery delays. in addition, creating awareness to data entry personnel on the importance of careful and completeness of entries is important, as is the need to reformat data collection forms to capture exact aspects of surveillance needs for relevant executable analysis. 8 besides alerting for immediate impending epidemics, these data can also be adopted for projective predictive modeling of annual epidemics, including that for influenza. 9 ehesp wcic-study input: djibouti wcic-study is complementary to the existing syndromic surveillance system, but with emphasis on flu and flu-like illness. various innovations as suggested above are used in seeking to overcome the prevailing challenges. while every attempt is made to realize its (wcic-study) objective and for global comparison, lessons learned from successful implementation will form a platform for future refined syndromic surveillance protocol as equally reported elsewhere in asian countries. 7, 10 information technology: national institute of public health djibouti has an informatics department with sufficient working pcs and personnel to execute efficient data collection and management for epidemiological analysis. however, licensing cost of near-real time syndromic surveillance software is prohibitive, but the open access software with capacity to generate custom graphs, maps, plots, and temporal-spatial analysis output for specific syndromes should make implementation a lot easier. such output for conditions like flu (or gastroenteritis) will be essential to cause prompt response of the local public health office and international partners in saving lives and suffering of djibouti people. 11 pandemic flu surveillance and preparedness requires multifaceted, interdisciplinary, and international approach whose efficiency and efficacy can only be refined over time. building on the health care system's swot for preparedness, the ehesp wcic-study promises to refine surveillance system operation and knowledge on individual's risk determinants to swine flu (h1n1) 2009 virus infection at the household level in djibouti. these efforts are ultimately creating available control options at the time of need (pandemic occurrence), and at the same time exploring investment in quality data profiling and information technology, which will include syndrome surveillance software systems like essence, ewors, or other open sourced ones. the antibody efficacy -which compares the illness frequency between those with and those without a protective level of pre-epidemic hi antibodies ( ‡1:40) -has been proposed 1 ; however, this index has rarely been used due to practical difficulties in confirming the strain-spe-cific disease corresponding to each of the vaccine-induced antibodies. we followed 114 elderly individuals residing in a nursing home, whose serum specimens were obtained before and after undergoing trivalent influenza vaccination, in 2002 ⁄ 2003 influenza season (medium-scale mixed [a ⁄ h3n2 and b] epidemic in study area, and a ⁄ h3n2 was circulating at the nursing home). 2 the serum antibody titre to each strain of influenza virus was measured by the hi method, using the same antigens as those in the vaccine. all participants' body temperatures, respiratory symptoms, other general symptoms, hospitalization, discharge, and death were recorded daily from 1 november 2002 to 30 april 2003 in a prospective manner. when the participants suffered any influenzalike symptoms, such as sudden fever ‡37ae8°c, throat swabs were collected and tested using a rapid diagnosis kit for influenza, which utilizes an immunochromatographic method. the adjusted odds ratios (or adj ) for febrile illness and kit diagnosed influenza were evaluated using multiple logistic regression models adjusting for possible confounders (i.e., age, sex, coexisting conditions, and vaccine strains). after vaccination, the proportion of subjects achieving an hi antibody titre ‡1:40 (seroprotection level) were 61ae4% (51ae8-70ae4%) for a ⁄ h1n1, 79ae8% (71ae3-86ae8%) for a ⁄ h3n2, and 26ae3% (18ae5-35ae4%) for b. during the follow-up period, the a ⁄ h3n2 strain was isolated therein, and 44 subjects experienced sudden-onset fever ( ‡37ae8°c), and eight subjects were positive for rapid diagnosis kit. patients with a seroprotection level of the hi antibody titre ( ‡1:40) had lower incidences of febrile illness (or adj , 0ae35; 95% ci, 0ae09-1ae28) and rapid kit diagnosed influenza (or adj , 0ae35; 95% ci, 0ae03-4ae64) than those with a lower titre. thus antibody efficacy (1 ) or adj ) against fever related to a ⁄ h3n2 and kit diagnosed influenza were both estimated to be 65%. although statistical significance was not detected due to limited sample size, these results lend support for the usefulness of antibody efficacy. some data presented within this manuscript was also published in hara et al. asia via a regional network from which epidemics in the temperate regions were seeded. 1 the virus isolates obtained from nasopharyngeal swab specimens from outpatients were typed and subtyped by the hemagglutination (ha) inhibition assay. 2 the emergence of a ⁄ fujian ⁄ 411 ⁄ 2002 coincided with higher levels of influenza-like illness in korea than what is typically seen at the peak of a normal season. most of the intermediates and fujian-like strains were isolated from asian countries, and the mutational events associated with the fujian strains took place in asia. closely dated phylogeny from december 26, 2001 to august 11, 2002 showed that the antigenic evolution of the h3n2 fujian strains had periods of rapid antigenic changes, equivalent to 10 amino acid changes per year ( figure 1 ). the fujian-like influenza strains were disseminated with rapid sequence variation across the antigenic sites of the ha1 domain. the antigenic evolution of the fujian strains was initiated by exceptionally rapid antigenic change that occurred in asia, which was then followed by relatively modest changes. some of the data presented in this manuscript was previously published in kang et al. we compared reactivity to the novel virus strain using haemagglutination inhibition (hi) assays performed on discarded plasma specimens left over from routine testing. samples were taken from healthy adult blood donors (>16 years) before and after the ph1n1 influenza epidemic that occurred during the southern hemisphere winter of 2009, and again prior to onset of the 2010 southern hemisphere influenza season. reactivity to the novel h1n1 2009 strain of influenza was relatively uncommon among the healthy adult population during the first australian winter wave, rising from a baseline of 12% to 22%. a further increase in the seropositive proportion from 22% to 43% was observed over the summer months, most likely attributable to immunisation. this level of immunity appears to have been sufficient to constrain the 2010 winter epidemic. together with a final serum collection, planned for late 2010, these data will aid evaluation of the extent and severity of disease in this 'second wave' of ph1n1. assessment of the extent of disease due to novel influenza a(h1n1) virus (ph1n1) during the 2009 winter outbreaks in australia was made difficult by the generally mild nature of disease. the epidemic was experienced in a staggered fashion around the country, 1 reflecting the considerable geographical distances between state and territory capital cities ( figure 1 ). differences in the intensity of case-finding during the evolving pandemic response and between jurisdictions hindered comparisons of disease burden in distinct geographical regions. 2 rates of reported hospitalisations and deaths appeared fairly similar across states 1 but, without a consistent exposure denominator, assessment of relative severity was difficult. we conducted a national serosurvey of antibody to ph1n1 using residual plasma from healthy blood donors collected before and after the 2009 epidemic to estimate ph1n1 exposure. 3 here we report the findings of that first collection, together with new data on seroprevalence of ph1n1 antibody in specimens gathered in march-april 2010. these latter samples were collected prior to onset of seasonal influenza activity to assess the impact of a national ph1n1 vaccine program conducted in spring ⁄ summer 2009 ⁄ 10 on the proportion of individuals with antibody titres deemed protective. 4 findings informed estimates of population susceptibility to ph1n1 prior to the 2010 influenza season and provided a baseline for a subsequent serosurvey that will be collected at the end of 2010 to assess the extent of exposure during the 'second wave.' tralian red cross blood service (the blood service) for dengue fever surveillance studies. these samples were used to provide a baseline estimate of prevalence of cross-reactive antibody to ph1n1 in the australian population. discarded plasma specimens, taken for virologic testing from healthy adult blood service donors, were prospectively collected at two additional timepoints for measurement of antibody to ph1n1. collection periods were as follows: approximately 120 plasma samples were randomly selected from donors in each of brisbane, hobart, melbourne, newcastle, perth, sydney, and townsville on each occasion. up to 20 specimens were identified in each of the following age strata: 16-24, 25-34, 35-44, 45-54, 55-64, and >65 years. at the last collection timepoint, there was deliberate over-sampling of the oldest and youngest age strata in which approximately 40 specimens were collected (i.e., up to 160 specimens per site). in accordance with the provisions of the national health and medical research council's national statement on ethical conduct in human research, individual consent was not required for use of these specimens, given the granting of institutional approval by the blood service human research ethics committee. reactivity of plasma against ph1n1 was measured in haemagglutination inhibition (hi) assays using turkey red blood cells (rbc). 5 egg-grown a ⁄ california ⁄ 7 ⁄ 2009 virus was purified by sucrose gradient, concentrated and inactivated with b-propiolactone, to create an influenza zonal pool preparation (a gift from csl limited). plasma samples were pretreated with receptor destroying enzyme ii (denka seiken co. ltd), 1:5 (volume ⁄ volume) and tested as previously described. 6 following 1 hour incubation, 25 ll 1% (volume ⁄ volume) of rbc was added to each well. hi was read after 30 minutes. any samples that bound to the rbc in the absence of virus were adsorbed with rbc for 1 hour and reassayed. samples in which background activity could not be eliminated by these means were excluded from the analysis. titres were expressed as the reciprocal of the highest dilution of plasma where haemagglutination was prevented. a panel of control sera and plasma samples was included in all assays. it comprised paired ferret sera pre-and postinfection with the pandemic virus or seasonal influenza a(h1n1), a(h3n2), or influenza b viruses and paired human plasma and sera collected from donors before april 2009 or after known infection with the pandemic virus or after immunisation with the australian monovalent pandemic 2009 vaccine. all assays were performed by the who collaborating centre for reference and research on influenza. for each of the three study timepoints and within each age group, the proportion of seropositive individuals (hi titres ‡40) was calculated, with exact (clopper-pearson) confidence intervals. the contribution of individual variables (age, gender) and location to seropositive status was assessed in separate multivariate logistic regression models developed to assess the post-pandemic and pre-influenza season 2010 collections. all statistical analyses were conducted in stata 10. locations of specimen collection are shown in figure 1 , together with the number of samples tested from each centre. samples with high background hi titres or discrepancies between assays were excluded at each timepoint as follows: 5 at baseline, 27 from the post-pandemic collection, and 0 in early 2010. pared with baseline was 10% overall, rising from 12% to 22% (table 1 ). the only jurisdictions in which seropositive proportions were higher in october ⁄ november than in the baseline collection were hobart [31% (95% ci 22ae3, 39ae7)], perth [24% (16ae3, 31ae7)], and sydney [24% (16ae3, 31ae7)]. in the multivariate regression model, the only jurisdiction in which exposure appeared somewhat higher than the reference population of brisbane was hobart [or 1ae83 (95% ci 0ae99, 3ae4), p = 0ae06]. a marked age effect on antibody status was observed at this timepoint, with an increase in the proportion of seropositive individuals in relation to the baseline collection only noted for those aged between 16 and 34 years (table 1) . according to the multivariate model, the youngest and oldest cohorts had similar titres, with all other groups showing significantly lower seropositive proportions than the reference population of 16-24 years [e.g. 45-54 years or 0ae36 (95% ci 0ae21, 0ae64, p < 0ae0001)]. an overall increase in the seropositive proportion from 22% to 43% was observed between october 2009 and april 2010, distributed throughout all jurisdictions ( (17, 25) ]. antibody titres prior to the 2010 influenza season rose in all age groups, but remained significantly lower among [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] year olds than in the youngest age cohort (table 1) . adjusted ors for the seropositive proportion in the multivariate model in these age groups were: 35-44 years [or 0ae49 (95% ci 0ae31, 0ae77)]; 45-54 years [or 0ae48 (0ae31, 0ae75)]. the relatively low titres observed in these groups reflected small incremental increases in the seropositive proportion across each of the time points studied, suggestive of both low rates of infection and vaccination. the rise in immunity observed across the population was most likely attributable to immunisation in the majority, given the absence of observed outbreaks and very few notified cases of ph1n1 during the period between the two plasma collections. this study suggests that, while adult exposure to ph1n1 during the 2009 southern hemisphere winter was uncommon at around 10%, vaccine uptake in the australian population over the period november 2009-may 2010 was in the order of 20%. this latter estimate is in keeping with recently published figures for adult ph1n1 vaccine coverage from a national immunisation survey conducted by the australian institute of health and welfare. 4 in that survey, vaccine coverage was significantly higher in tasmania than in other states, but mostly in those over 65 years of age, possibly in a subgroup whose health status may have differed from that of the donor population. 4 no allowance has been made in this analysis for likely waning of natural or vaccine induced immunity, possibly resulting in lower estimates of natural and ⁄ or vaccine exposure than may have occurred over the period. regardless of such intervening processes, the seropositive proportion among australian adults at the start of the 2010 winter season appeared likely to be sufficient to constrain transmission of infection in the age groups tested. this assertion has been borne out in practice, with only modest levels of influenza reported during the late and protracted 2010 season. 7 a final serum collection is planned for the end of the 2010 influenza season in australia from which to assess the level of exposure in relation to the baseline observed here. the need for epidemiologic studies such as this has been highlighted by groups such as the european centre for disease control to aid evaluation of the extent and severity of the 'second wave,' 8 known to be variable from historical reports of past pandemics in disparate populations. 9 in 2009-2010, the first wave of the swine-origin novel h1n1 flu (h1n1) pandemic swept across the world, including japan. to examine the epidemiological nature of this novel infectious disease among school children within and among small regional communities, we have carried out a complete survey on the incidence of h1n1 among school children using absentee reports provided by school health teachers in two small administrative districts (population: about 140 000 in total) in japan. we then examined the epidemiological diversity on the inci-dence of h1n1 within and among small regional communities. we investigated seventeen elementary and ten junior high schools in moroyama-town and sakado-city located in the central part of saitama prefecture. populations are: all ages, 37 015 and 100 634; elementary schools, 1873 and 5389; junior high schools, 1131 and 2568, respectively. the number of school children in each school ranges from 137 to 876. the surveillance system was built on an apache-and mysql-based web server using html, php, and java-script. 1 school health teachers enter information on children absenteeism due to school infectious diseases via web browsers at each school infirmary on a daily basis. in addition to the trend graphs shown on the web browser, detailed analyses were reported to the schools and local educational boards weekly. the basic reproduction number (r 0 ) of h1n1 was estimated according to becker. 2 agentbased modeling and simulations were also performed using a multi-paradigm simulator anylogic version 6.5 (xj technologies, st. petersburg, russia). by the end of march 2010, cumulative incidence (ci) of h1n1 among school children in moroyama and sakado reached 30% and 34%, respectively. the overall r 0 among school children in this area was 1ae43. vaccination rate of children in this area during the surveillance period was reported to be very low (<10%). there was no considerable difference between the epidemic curves in this neighboring town and city. on the other hand, in the individual schools, the cis as of the end of march 2010 scattered from 16% to 51% ( figure 1 ) even though the schools are closely located. to examine the cause of this diversity, we built an agent-based community model 3 consisted of the same numbers of agents as those of children in the actual schools and people in moroyama and sakado to simulate the infection. the ratio of probability of infection in schools and the remaining places were assumed to be 1:1 or 10:1. using a heuristic optimization scheme, we estimated the parameters for the simulations to give the overall ci of 33% (the ci as of the end of march 2010). we then performed simulations repeatedly. the cis obtained with the repetitive simulations with the assumption of higher probability of infection in schools scattered from 23% to 44%, indicating that the cis of the small population communities may vary considerably, even though all the agents were assumed to have the same susceptibility to infection at the beginning, and the other conditions were the same. the policies for surveillance ⁄ analyses ⁄ prevention of communicable diseases in local communities have generally been decided on governmental-and ⁄ or each local administrative district-basis (populations: several hundred thousands to several millions) in japan. we found the considerable variations in the cis of h1n1 for children among much smaller areas, i.e., the school districts (populations: all ages, several thousands; school children, several hundreds). we thus conclude that the granularity of surveillance ⁄ analyses ⁄ prevention should be finer than in the past to achieve the most effective policies against influenza and similar communicable diseases in the local communities. the cause of this diversity can be explained in part by the stochastic nature of infection transmission processes in the small populations shown by the agent-based simulations. we have already conducted a complete questionnaire survey for the school children and their parents to clarify the relevance of the other issues including differences in environmental factors, preventive policies (e.g., vaccination, school closures), etc., in each school. the detailed analyses will be reported elsewhere. a www-based surveillance system for transmission of infectious diseases among school children within and among small regional communities. j epidemiol 2010; 20(s1):s151. this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. the first cases of the 2009 a ⁄ h1n1v pandemic influenza were reported in mexico and the united states in april 2009. given the context of this new influenza virus and considering the likelihood of its pandemic spread, the cohorts for pandemic influenza (copanflu) international consortium was created in order to study individual and collective determinants of pandemic a ⁄ h1n1v influenza across different countries by setting up prospective cohorts of households, followed during 2 years. this study relies on the first available data from the copanflu-france project, which is part of the copanflu international consortium. we studied factors associated with elevated haemagglutination antibody titers against a/h1n1v at entry in the copanflu-france cohort. we focused in this primary analysis on the association between the titers and influenza vaccination (seasonal or pandemic) across age groups. the copanflu-france cohort was set up in fall 2009. inclusions began on december 4, 2009 and ended on july 23, 2010. households were sampled using a random telephonic design (mitofsky-waksberg method) 1 in a stratified geographical sampling scheme, aimed at including a sample of subjects representative of french general population. all household members were eligible to the cohort, without any age limit. the inclusion of a household required the participation of all members: the refusal of one or more member(s) prevented the inclusion of other members. the protocol was approved by a research ethics committee and written informed consent was obtained for all subjects. this study requires several visits to the households by nurses who collect written data with questionnaires and biological samples. during the inclusion visits, nurses collected from all subjects detailed data regarding medical history, including vaccination and preventive measures against influenza. blood samples were collected at entry and centralized. a standard hai technique was adapted to the detection and quantification of antibodies to the 2009 a ⁄ h1n1v virus. 2 the titration endpoint was the highest dilution that exhibited complete inhibition of haemagglutination in two independent readings. the lowest read dilution was 1 ⁄ 40. geometric mean titers were calculated for hai assays with the use of generalized estimating equations for interval-censored data, 3, 4 taking into account a within-household correlation. multivariate models were derived from this method to identify factors associated with elevated gmts. we defined the ''gmt ratio'' (gmtr) as the multiplicative factor applied to the gmt in presence of an explanatory variable. for qualitative explanatory variables, a gmtr of n means a predicted n-fold higher gmt for subjects exposed to the considered factor compared to others. for continuous explanatory variable, the same interpretation applies to a unit difference. the following variables were included in the multivariate models: age, history of pandemic or seasonal influenza vaccination, and history of ili. age was categorized in three groups: 0-15 years (reference group), 15-50 years and over 50 years. the definition of ili was that used by the cdc 5 : fever ‡37ae8°c and cough and ⁄ or sore throat without another known cause. history of ili was defined as an ili reported by the subject between september 12, 2009 (beginning of the influenza epidemic in france) and the date of inclusion. this preliminary analysis included 1304 subjects belonging to 544 households. results reported hereafter do not account for missing data. participating households were sized 1-7 subjects, mean size = 2ae4. in comparison, the mean size of french households is 2ae3 according to the latest national census. 6 the median age of subjects at entry was 40ae6 years [iqr: 16ae5; 58ae6] versus 34ae4 [15ae2; 52ae5] for french population. 7 the proportion of subjects reporting a history of ili since the beginning of the epidemic varied from 2ae9% for subjects over 50 years to 9ae1% for subjects below 15 years (table 1) . vaccination with the pandemic strain was the highest in subjects below 15 (16%) whereas vaccination with the seasonal strain was the highest in subjects over 50 (44ae9%). detailed data regarding vaccination is given in table 1 . this study confirms previous findings that age, pandemic influenza vaccination, and history of ili are associated with elevated post-seasonal gmt. [8] [9] [10] [11] [12] [13] among non-vaccinated subjects, elevated gmt in the elderly may be the result of exposure to similar viruses in early life, whereas children and young adults with elevated gmt are likely to have been infected by the 2009 a ⁄ h1n1v virus. [13] [14] [15] [16] interestingly, a significant drop in the hai titer is observed during the months following vaccination with the pandemic strain. this study also shows that seasonal influenza vaccination may have contributed to an increase of the hai titer, especially in the elderly. the reason for this association is not obvious: although we cannot discard the hypothesis of a higher incidence of a ⁄ h1n1v infections in seasonal vaccine recipients, as described by several other studies, 17-19 the main explanation may be a cross-reaction between pandemic and seasonal strains. 16, 20, 21 further analyses in this cohort are needed to confirm and explain these first results. the follow-up of subjects involved in the copanflu-france cohort will provide data to study the risk factors for infection by the influenza virus. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown virulence. infants under 5 years old had higher risks of hospitalization, complications, and rate of death for sari. materials and methods: a cross-sectional study was executed from may to december in 2009. the sources were: mandatory reporting form of the province surveillance system, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports. inclusion criteria: children under 5 years old with diagnostic of ili or sari and confirmed cases with epidemiological nexus or laboratory confirmation (rrt-pcr, ifi). the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. results: the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356) being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years (95% ci 1426-1722) ( table 1) . infants had less risk of getting sick in relation to the rest of the population (rr 0ae40 [95% ci 0ae33-0ae47]) (p < 0ae05). the chance of sari in infants was 2ae89 (95% ci 2ae46-3ae39) compared to the rest of the population. the lethality rate was higher in infants under 1 year old (14 ⁄ 100 000 people [4 ⁄ 28 364]). discussion: the evidence suggests that the infants under 5 years old had lower risk of getting sick than the rest of the population, but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. in april 2009, the cdc alerted about the appearance of a new strain of ia h1n1 with unknown dissemination and virulence. in june, the world health organization declared the pandemic. 1, 2 the ili often presents an unspecific clinical picture in infants under 5 years old, from mild symptoms to sari, especially in the newborn babies. infants under 5 years old have higher risks of hospitalization, complications, and rate of death for sari. 3, 4 on may 7th, argentina declared the first imported case of ia h1n1, and by the end of the month, it announced the viral circulation in the country. the epidemiological surveillance system of the province arranged that all the patients with influenza diagnosis made by a doctor must be reported. from april 24th to november 14th, 20 212 suspected cases of ili in the province of tucumán were reported. the ili rate was 1353 ⁄ 100 000 people, and ia h1n1 comprised 40 ⁄ 100 000 people. the lethal rate of sari ia h1n1 was 306ae7 ⁄ 100 000 people (62 ⁄ 20 212). 5 the objective of this research was to determine the epidemiological characteristics of the pandemic ia h1n1 in infants under 5 years old in the province of tucumán between may and december in 2009. the province of tucumán is placed in the center of the northwest of the republic of argentina. it has a population of 1 493 488 inhabitants of which 138 821 are infants under 5 years old. the crude birth rate for 2008 was 19ae9&. the infant mortality rate was 13ae8&. respiratory pathologies in infants under 5 years old were the third cause of death in the province (12%). the public health system of the province is composed by three sectors: public, private, and welfare. with 91 health facilities as a total, the average of available beds is 3& per inhabitants and 6& per neonates. a cross-sectional study was executed from may to december in 2009 in the province of tucumán, argentina. the following sources were used: mandatory reporting form of the surveillance system of the province filled by a doctor, databases of the hospital management information system, clinical pictures reviews, and telephone daily medical reports (patients with sari). inclusion criteria: • suspected case of ili: sudden appearance of fever higher than 38°c, cough, or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nausea or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea. ) were used for the analysis. the odds rations, risk ratio and 95% confidence interval were calculated to compare ambulatory with hospitalized patients, confirmed and dismissed, <5 years old and the rest of the population. it was considered significant a rate of p < 0ae05. the age specific mortality rates were calculated with an estimated population for the province according to the national statistics and census institution. the epidemiological surveillance system of the province received 21 012 ili reports, 11ae77% (2474 ⁄ 21 012) were infants under 5 years old. twenty seven percent were dismissed (676 ⁄ 2474), and 34% (623 ⁄ 1798) of suspected cases were confirmed. the first ia h1n1 case was a child of 2 years from the province of buenos aires, in 22th epidemiological week, and the last suspected case was reported in october 26, 2009 ( figure 1 ). the ili rate in infants under 5 years old was 1295ae19 ⁄ 100 000 people (95% ci 1236-1356), being higher in infants of 4 years old (1569 ⁄ 100 000 people of 4 years, [95%ci 1426-1722]). the higher ili rates in confirmed the pandemic of ia h1n1 (2009) was detected for the first time in the province of tucumán. the evidence suggests that infants under 5 years old had lower risk of getting sick than the rest of the population (protective factor), but had higher risk of sari if they had some past illness. the highest lethality rate was presented in infants under 1 year old. towns with the highest demographic density had superior proportion of cases. non-medical interventions had an important role in the epidemic containment for not having a specific vaccination available. as this age group had high risks of hospitalization, it would be advisable to prioritize their vaccination. outbreak of h1n1 influenza -2009: behavior of influenza h1n1 in school children in the province of tucumá n, argentina criteria: patients treated with antiviral medication for prophylaxis, respiratory pathologies which did not justify specific medication, and incomplete forms. results: from all notifications, 6342 were cases of ili in the group aged 5-17 years old; 53% were males. the incidence rate in this group was 17ae4 per thousands of inhabitants. the 13% of laboratory samples were influenza a h1n1, 34% were confirmed as unspecific influenza, and 49% were dismissed. the school aged children group had a high risks of getting sick (r.r. 1ae78 [95% c.i. 1ae55-2ae04]), especially males. it appeared that school aged children had a protective factor for presenting sari (or 0ae66 [95% c.i. 0ae66-0ae89], p < 0ae05). the lethality rate in this group was 9ae32 ⁄ 10 thousands. headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. it was reported a decrease in the ew 28 coinciding with winter holidays (ew 27). the epidemic curve was different in males compared to females during the winter holidays. discussion: school aged children got sick more than the rest of the population, although they presented less proportions of sari. however, comorbidities were decisive in order to present sari or death. the epidemic curve was different in males compared to females. through its analysis, the beneficial effect of school closure was observed, as long as children meet the recommendation to stay home. in april 2009, different countries reported cases of influenza a h1n1; mexico reported a high mortality rate associates with this disease. 1 the world health organization (who) declared the phase 6 influenza pandemic alert on june 11. 2 several reports from different countries describe the behavior of the pandemic in school aged children. this group plays an important role in the transmission of influenza. in germany, during the summer peak, pandemic hardly spread within this group. this might be explained by the timing of the summer school holidays, which started between ew 27 and 31. since mid october, after the autumn holidays, the school-aged children began to be more affected, and the proportion increased from 16% in the initiation period to 43ae8% in the acceleration period. 3 in australia, 55% of h1n1 cases were school aged children (5-17 years), with a median age of 16 years (29% of cases were aged 13-17 years and, and 26% between 5-12 years). 4 in canada, the infection rate was highest in this group. 5 in chile, the incidence rate was 4500 ⁄ 100 000 inhabitants, although in general they had mild desease. 6 school closure can operate as a proactive measure, aimed at reducing transmission in the school and spread into the wider community, or reactive, when the high levels of absenteeism among students and staff make it impractical to continue classes. the main health benefit of proactive school closure comes from slowing down the spread of an outbreak within a given area and, thus, flattening the peak of infections. this benefit becomes especially important when the number of people requiring medical care threatens to saturate health care capacity. it has its greatest benefits when schools are closed very early in an outbreak, before 1% of the population falls ill. school closure can reduce the demand for health care by an estimated 30-50% at the peak of the pandemic under ideal conditions, but too late in the course of a community-wide outbreak, the resulting reduction in transmission is likely to be very limited. policies for school closure need to include measures that limit contact among students when they are not in school. 7 tucumán is placed in northwest argentina and has a total area of 22 524 km 2 . the population (2001 census, projection 2009) was 1 493 488 inhabitants; of wich 421 638 were 5-19 years old. the health system of the province is composed of 3 sectors: public, private, and welfare. it has a total of 91 health facilities with internement available and an average of 3 & inhabitants. influenza-like illness (ili) has seasonal and endemic behavior in this province, as evidenced by past records from the national health surveillance system and influenza sentinel surveillance unit of the province. an increase of ili was reported in 2009, with a peak in the ew 28. the objectives were: general objective to describe the behavior of the influenza a h1n1 2009 epidemic in school aged children from the province of tucumán, argentina. specific objectives • to explore the response to preventive measures by school aged population. • to assess the effect of the suspension of classes in this group. • to estimate the magnitude and severity of the disease. • to observe the effect of co-morbidities in this group. a cross-sectional study was executed from may to december 2010. data were gathered through mandatory reporting forms, wich were collected from all public and private health centers. inclusion criteria: patients with compatible symptoms with influenza a; school aged children 5-17 years old. exclusion criteria: patients treated with antiviral medication as prophylaxis, respiratory pathologies which did not justify specific antiviral medication, and incomplete forms. • suspected case of ili: cases considered by clinical criteria (fever higher than 38°c, cough or sore throat. it may or may not be accompanied by asthenia, myalgia or prostration, nauseas or vomiting, rhinorrhea, conjunctivitis, adenopathy, or diarrhea). • confirmed case: person with positive laboratory results for influenza a h1n1 or unspecificed influenza a (by laboratory results through rrt-pcr or immunofluorescence techniques). • dismissed case: by negative or different laboratory results, or different clinical evolution. • comorbidities: chronic illnesses like arterial hypertension, diabetes, asthma, recurrent obstructive bronchial syndrome (robs), smoking, chronic obstructive pulmonary disease (copd), immunosuppression, hiv ⁄ aids, cancer, nephropathy, obesity; pregnancy was also considered. data were analyzed using epi2000 software (epi infoô cdc, atlanta, eeuu). rates were calculated and rr was estimated with their respective confidence interval (ci). population data were taken from 2001 national census projections. an estimation based on the same census was used for the group between 5 and 17 years old. to observe the effects of other co-variables, the or and their ci were calculated. logistic regression was used to evaluate the influence of the comorbidities. x 2 was used to compare proportions. respiratory samples (nasopharyngeal and faryngeal swabs) were obtained. they were analyzed at influenza sentinel surveillance unit of tucumán, and ⁄ or sent to national reference laboratory dr. c. malbrán (rt-pcr). from all notifications (20 212), 6342 were cases of ili in the group aged between 5 and 17 years old, 53% (3340 ⁄ 6342) of which were males. the incidence rate was 17ae4, and it differed according to the sexes: 18ae0 males and 16ae7 females per thousands of inhabitants (p < 0ae05). of all laboratory samples (370) 13% were confirmed as influenza h1n1, 34% were confirmed as unspecificied influenza, and 49% were dismissed. the remaining percentage corresponded to the isolation of other viruses (parainfluenza, respiratory syncytial virus, and adenovirus). the school aged group had higher risk of getting sick, in relation to the rest of the population (rr 1ae78 [95% ci 1ae55-2ae04]), especially males (rr 1ae97) compared with females (rr 1ae66). the highest attack rate was observed in the capital of tucumán (42 ⁄ 1000 inhabitants). according to the rest of the population, it looked like being school aged children meant a protective factor for presenting sari (severe acute respiratory infection) (or 0ae66 [95% ci 0ae66-0ae89], p < 0ae05). the lethality rate was 9ae32 ⁄ 10 thousand. the risk of dying was low compared to other ages. persons with comorbidities had significantly higher risk of presenting sari (or 1ae8 [95% ci 1ae35-2ae58], p < 0ae05) and of dying (or 8ae1 [95% ci 19ae6-3ae34], p < 0ae05). respiratory comorbidities were the most frequent: asthma 2ae5% (162 ⁄ 6342) and 2% rors (122 ⁄ 6342). the symptoms headaches, myalgia, coryza, and sore throat were very common and significantly different (p < 0ae05) than the rest of the population. if we compared the group aged 5-17 years with 18-39 years old, the epidemic curve of the first group showed a decrease in the ew 28, coinciding with winter holidays (ew 27) (figure 1 ). there was a slight increase in the tendency when classes began, but it showed a clear declination afterwards. the analysis of rates in school aged children by ew showed a reduction of 22ae2% in males and 26ae6% in females (p < 0ae05) at ew 28. however, after the first week of winter holidays, the curve in males had a significant increased to 65ae6% compared to ew 27, reaching the highest weekly rate of the epidemic (18 ⁄ 10 000 inhabitants). the reopening of classes coincided with a significant decrease of the rate (34ae9%), from 18 to 11ae7 ⁄ 10 000 inhabitants in ew 31 (p < 0ae05). in females, the school closure coincided with a plateau-shaped curve, and the reopening with a significant decrease of 43ae4% of the rate, from 12ae1 to 6ae9 in ew 32 ( figure 2 ). the school children got sick a lot more than the rest of the population, although they presented less proportions of sari. however, comorbidities were determined in order to present sari or death. symptoms like headache, myalgia, coryza, and sore throat were considered more conducting for the definition of cases in this population in tucumán. the epidemic curve was different in males compared to females during the winter holidays. the beneficial effect of school closure was observed as long as persons met the recommendations. the difference between males compared to females during winter holidays could mean that women would have carried out social distance recommendations much better, for example, remained at home. the significant reduction after the opening of classes is a factor to be considered as an effective intervention in the declining stage of the curve. here, we report pdmh1n1 infection attack rate (iar) during the first wave of the pandemic. we used our iar estimates to infer the severity of the pandemic strain, including the age-specific proportion of infections that led to laboratory confirmation, hospitalization, intensive care unit (icu) admission, and death. [1] [2] [3] [4] part of these results are now available in ref. 5 subjects of a community study, 5-14 years old between 1 november 2008 and 31 october 2009, we conducted a cohort study of pediatric seasonal influenza vaccination and household transmission of influenza. one hundred fifty-one children aged 5-14 were recruited and provided baseline sera in november and december 2008. between september and december 2009 a further 766 children aged 5-14 were recruited and provided baseline sera for the second phase of the study. for this serologic survey, we tested the 151 sera collected before the first wave and the 766 sera collected after the first pandemic wave. written informed consent was obtained from all participants. parental consent was obtained for participants aged 15 or younger, and children between the ages of 8 and 15 gave written assent. all study protocols were approved by the institutional review board of the university of hong kong ⁄ hospital authority hong kong west cluster. age-stratified data on virologically confirmed outpatient consultations, hospitalizations, icu admissions, and deaths associated with pdmh1n1 from 29 april 2009 to 15 november 2009 were provided by the hong kong hospital authority (the e-flu database). 6 since may 2009, patients admitted with acute respiratory illnesses routinely underwent laboratory testing for pdmh1n1 virus by molecular methods. sera were tested for antibody responses to a ⁄ california ⁄ 4 ⁄ 2009 by viral microneutralization (mn). 7 most individuals infected with influenza develop antibody titers ‡1:40 by viral microneutralization after recovery. 8 we defined the pdmh1n1 seroprevalence rate as the proportion of individuals who had antibody titers ‡1:40. while mn antibody titers of ‡40 are not by themselves conclusive evidence for pdmh1n1 infection, we have assumed that the increase in cross-sectional seroprevalence between the pre-and post-first wave time periods are evidence of recent pdmhn1 infection. the iar was defined as the proportion of individuals infected by pdmh1n1 during the first wave. the case-confirmation rate (ccr), case-hospitalization rate (chr), case-icu-admission rate (cir), and case-fatality rate (cfr) were defined as the proportion of pdmh1n1 infections that led to laboratory-confirmation, hospitalization, icu admission, and death. due to containment efforts until june 29, 2009 all laboratory-confirmed cases were required to be hospitalized for isolation regardless of disease severity. as such, only surveillance data from june 30 onwards were used to estimate severity measures. we estimated the iar as the difference between the prefirst-wave and post-first-wave seroprevalence rate. we used the estimated iar as the denominator for calculating the ccr, chr, cir, and cfr. we used an age-structured sir model with 5 age classes (0-12, 13-19, 20-29, 30-59, and ‡60) to describe the transmission dynamics of pdmh1n1 in hong kong between 10 june and 15 november 2009. we assumed that the mean generation time was 2ae3 days. using the age-structured transmission model, we estimated the following transmission parameters from the serial cross-sectional serologic and hospitalization data: (i) r o , the basic reproductive number; (ii) p 1 and p 2 , the reduction in within-age-group transmission for 0-12 and 13-19 years old during summer vacation (compared to school days during september-december 2009); (iii) d r , the average time for neutralization antibodies titer to reach ‡1:40 after recovering from infection; (iv) h a , the age-specific relative susceptibility with 20-29 years old adults as the reference group. we assumed non-informative priors for all parameters and used monte carlo markov chain methods to obtain posterior distributions of the parameters. sources of specimens: [1] pediatric cohort study (2-29 april 2009 virological surveillance data suggested that the first wave of pdmh1n1 in hong kong occurred from august to october 2009. most of the laboratory-confirmed infections in this first wave occurred in individuals aged below 25 years old accounting for >72% of the lab-confirmed cases and hospitalizations, 32% of icu admissions, and 6% of deaths. taking into account a delay of 2-3 weeks for antibody titers to appear during convalescence, 8 we found that these virological surveillance data were consistent with our serial cross-sectional seroprevalence data, which indicated a sharp rise in seroprevalence among the 5-25 years old from september to november and a plateau thereafter (data not shown). among individuals aged 5-14 years, the seroprevalence rates were similar across time between pediatric outpatient subjects and pediatric cohort study subjects (data not shown). similarly, for older age groups, the seroprevalence rates were largely similar between blood donor subjects and hospital outpatient subjects (except for the 20-29 years old in november-december). this provided some evidence that despite biases in our convenience sampling scheme, the resulting serologic data provided a reasonably representative description of seroprevalence in the community. the estimated pre-and post-first-wave seroprevalence rates and the corresponding iar estimates are shown in table 1 . the severity estimates (ccr, chr, cir, and cfr) are shown in table 2 . in summary, we estimated the iar was 43ae4% among 5-14 years old, 15ae8% among 15-19 years old, 11ae8% among 20-29 years old, 4ae3% among 30-39 years old, 4ae6% among 40-49 years old, and 4ae0% among 50-59 years old. overall, we estimated a population-weighted iar of 10ae7% (9-12%) among individuals aged 5-59 years through the first wave in hong kong. ccr were around 2ae8-5ae4% among the 5-59 years old. chr were around 0ae47-0ae87% among the 5-59 years old. cir increased from 7ae9 (5ae2-12ae6) per 100 000 infections in 5-14 years old to 75 (32ae7-281) per 100 000 infections in 50-59 years old. cfr followed a similar trend with 0ae4 (0ae1-2ae3) death per 100 000 infections in 5-14 years old to 26ae5 (10ae4-109) deaths per 100 000 infections in 50-59 years old. compared to children aged 5-14, adults aged 50-59 were 9ae5 and 66 times more likely to be admitted to icu and die if infected. the best-fit age-structured transmission model gave the following parameter estimates: 1. the basic reproductive number was 1ae38 (95%ci, 1ae36-1ae41). 2. it took an average of 14 (9-21) days for recovered individuals to develop neutralization antibody titer ‡1:40. table 2 . estimated age-specific proportions of individuals with pdmh1n1 infections that were laboratory-confirmed, were hospitalized, were admitted to icu, and died. case-icu and case-fatality rates are expressed as number of episodes per 100 000 infections 3. compared to 20-29 years old, 0-12 years old children and 13-19 teenagers were 3ae7 (3ae2-4ae5) and 1ae6 (1ae3-2) times more susceptible to pdmh1n1 infection, respectively. 4. compared to 20-29 years old, 30-59 years old older adults and 60-79 years old elderly were only 0ae42 (0ae3-0ae6) and 0ae33 (0ae18-1ae5) times as susceptible as the 20-29 years old, respectively. 5. compared to the school period during september-december 2009, summer vacation reduced within-agegroup transmission by 61% (53-72%) among 0-12 years old, but only 12% (3-18%) among 13-19 years old. using computer simulations, we estimated that if preexisting seroprevalence is zero, real-time serologic monitoring with about 1000 specimens per week would allow accurate estimates of iar and severity as soon as the true iar has reached 2% (data not shown). we estimated that during the first wave in hong kong, 43ae4% of school-age children and 10ae7% of individuals aged 5-59 were infected by pdmh1n1. a serologic survey in england found similar iars in london and the west midlands. 8 both studies highlight the importance of including serologic surveys in pandemic surveillance. the geographically compact and well-mixed population in the urban environment of hong kong permits some degree of confidence in the validity of our iar and severity estimates. the completeness of the pdmh1n1 surveillance system, welldefined population denominator, and our large-scale serologic survey provide accurate numerators and denominators for the severity measures. we based severity estimates for pdmh1n1 on the iar as the denominator. in most previous studies of pdmh1n1 severity, the denominator was clinical illness attack rate, which depends on the probability of symptoms as well as medical care seeking behavior of the population. 2, 9 our estimated cirs and cfrs are broadly consistent with presanis et al.'s 2 'approach 2' severity estimates, but around 7-9 times lower than their 'approach 1' estimates. our estimates of chr are 2-10 times higher than their approach 2 estimates of symptomatic chr. however, the hospitalization-death ratio was 4253 ⁄ 27 = 164 as of november 15 in hong kong, but 996 ⁄ 53 = 19 as of june 14 in new york, 2 suggesting that the clinical threshold for admission in terms of disease severity at presentation may have been lower in hong kong. our study has a number of limitations. first, we have used antibody titers of ‡1:40 by viral microneutralization as an indicator of recent infection, correcting for pre-existing seroprevalence levels, but this may lead to underestima-tion of the iar if some infections led to antibody titers <1:40, or if some individuals with baseline titers ‡1:40 were infected. second, our estimates of the iar would be biased upwards if infection with other circulating influenza viruses led to cross-reactive antibody responses resulting in antibody titers ‡1:40. however between august and october 2009, 83% of influenza a viruses detected in hong kong were pdmh1n1, and only 3% of isolated viruses were seasonal h1n1 viruses. 10 third, a minority of severe illnesses associated with pdmh1n1 infection might not be identified by molecular detection methods, for example if admission occurred after viral shedding from the primary infection has ceased, in which case we may have underestimated the disease burden of pdmh1n1. finally, our analyses are primarily based on seroprevalence among blood donors to the hong kong red cross, who may not be representative of the whole population. we do not have detailed data on donors to compare their risk of infection with the general population, but we did observe very similar seroprevalence rates across the three groups of subjects in our study, i.e., blood donors, hospital outpatients and participants in a community cohort (data not shown). in conclusion, around 10ae7% of the population aged 5-59 and half of all school-age children in hong kong were infected during the first wave of pandemic h1n1. compared to school-children aged 5-14, older adults aged 50-59, though less likely to acquire infection, had 9ae5 and 66 times higher risk of icu-admission and death if infected. thus, although the iar of pdmh1n1 is similar to that of a seasonal epidemic, the apparently low morbidity and mortality of 2009 pandemic influenza (h1n1) appears to be due to low infection rates in older adults who had a much greater risk of severe illness if infected. the reasons why older adults appear relatively resistant to pdmh1n1 infection even though they appear to lack neutralizing antibody remains unclear. if antigenic drift or other adaptation of the pdmh1n1 virus allows these older age groups to be infected more efficiently, the morbidity and mortality of subsequent waves of the pandemic could yet become substantial. and the national institute of allergy and infectious diseases, national institutes of health (contract no. hhsn266200700005c; adb no. n01-ai-70005). the funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish. bjc reports receiving research funding from medimmune inc., a manufacturer of influenza vaccines. the authors report no other conflicts of interest. some data presented in this manuscript were previously published in wu et al. 5 it is well known that a primary goal of vaccination is to generate immunological memory against the targeted antigen to prevent disease in a vaccinated person. this ensures an accelerated immune response in the event of future contact with the pathogenic agent, such as a virus. therefore, it is very important to develop criteria for the assessment of vaccine immunogenicity by measuring both t and b memory cell levels from the vaccinated host. in contrast to inactivated influenza vaccines, live attenuated influenza vaccines (laivs) have been shown to provide primarily cellular and local immune responses. 1-3 to date, however, the hemagglutination-inhibition (hai) test (i.e. detection of serum antibodies) remains the method widely accepted for evaluation of an influenza vaccine's immunogenicity. improved understanding of the role of cellular and mucosal immunity and their contribution to protecting against severe illness caused by influenza infection has emphasized the need to reconsider methodologies used to evaluate the immunogenic impact of various influenza vaccines. such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses. 4 the aim of this study was to assess the ability of new russian pandemic laivs a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) ('ultragrivak,' registered 25ae03ae2009) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) ('influvir,' registered 13ae10ae2009) to induce memory t-cells in naïve human subjects and to compare results to levels of hai antibodies from each subject. a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was generated by 7:1 genetic reassortment of low-pathogenic avian influenza virus a ⁄ duck ⁄ potsdam ⁄ 1406-86(h5n2) and master donor strain a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2). 5, 6 the vaccine strain contains ha gene from avian virus, as well as na and internal genes from the master donor virus. a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was generated by classical (6:2) reassortment of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) with the master donor virus. 7 the vaccine strain contains ha and na genes from a 'wild-type' h1n1 strain and internal genes from the master donor virus. participants were aged 18 to 20 years and were without contra-indication of laiv vaccination. immunogenicity of a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv was assessed in ten vaccinated persons and ten volunteers inoculated with a placebo (sterile physiological saline solution). immunogenicity of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv was estimated in 16 vaccinated volunteers and nine volunteers inoculated with placebo. viruses or placebo were administered intranasally twice with an interval period of 21 days at a dosage of 0ae25 ml per nostril for each vaccination. physical examination, venous blood and nasal swab samples were collected at four time points during the study: (i) before vaccination (day 0); (ii) 21 days after first vaccination (day 21); (iii) 21 days after the second vaccination (day 42); and (iv) 6 weeks after the second vaccination (day 63). serum hai antibodies were measured by standard hai assay 8 using 1% human red blood cells. test antigens for the assay were a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) to match the appropriate vaccine antigen. local iga antibodies in nasal swabs were evaluated by elisa 9 using whole purified a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) or a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) viruses at 16 hau per 0ae05 ml for absorption to elisa plates. endpoint elisa titers were expressed as the highest dilution of sera that gave an optical density (od) greater than twice the mean od of six negative controls in the same assay. percentages of virus-specific cd3 + cd8 + ifn-c + and cd3 + cd4 + ifn-c + peripheral blood memory cells were determined using a flow cytometry iccs assay performed by the published method. 3 pbmcs were prepared with standard histopaque-1077 gradient centrifugation from heparinized whole blood. wilcoxon matched pair test, mann-whitney u test and the students t-test were used for statistical data analysis. prior to the first vaccination (day 0), gmts of hai antibodies to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laivs were 1 ⁄ 5ae4 and 1 ⁄ 5ae7, respectively. in addition, gmts of siga against these specific antigens from nasal swabs were 1 ⁄ 5ae7 and 1 ⁄ 9ae1, respectively. no hai antibody titers greater than 1:40 were observed prior to vaccination. background levels of virusspecific t-cells varied significantly within groups. mean levels of virus-specific cd8 + ifnc + cells were 0ae151% to a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) and 0ae173% to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). for cd4 + ifnc + cells, initial levels were 0ae240% and 0ae336%, respectively. thus, background levels of virus-specific antibodies were low, but prior vaccination or virus exposure in some volunteers produced some pre-existing levels of t cells, thus they were not absolutely immunologically naïve in this sense. preexistence of h5n1-crossreactive antibodies and t-cells has been observed previously. [10] [11] [12] effect of vaccination antibody immune responses both influenza a (h5n2) and influenza a (h1n1) laivs stimulated production of serum hai antibodies and local iga antibodies in nasal swabs. following the first vaccination with influenza a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92 (h5n2) laiv, 20% percent of volunteers exhibited seroconversion of hai antibodies; after the second vaccination, 30% of volunteers exhibited seroconversion. after the first vaccination, a 10% conversion rate of siga was observed; after the second vaccination, 60% showed conversions in levels of siga. the first vaccination with a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) laiv showed 6ae3% of hai antibodies seroconversions vaccination, and 50% seroconversion after second vaccination. for local siga, those results were 18ae8% and 31ae3% following the first and second inoculation, respectively. figure 1 summarizes cellular immune responses observed in the vaccinated versus the placebo group. after the influenza a (h5n2) laiv inoculation, significant differences in both cd4 and cd8 ifnc-producing t-cells were observed at day 42 after the second vaccination (d63). these data indicate that healthy young people who never received such avian influenza vaccines and were not exposed to h5n1 wild-type viruses were able to respond to the live attenuated h5n2 influenza vaccine. after the first influenza a (h1n1) laiv vaccination, reliable increases were observed in cd8 + cells only. after the second vaccination, increases in both cd4 + and cd8 + fold changes were significantly higher in vaccinated volunteers compared to the placebo group. it is noteworthy that cellular immune responses (cd4 + and cd8 + cells) were more marked in the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1). considering the long-term circulation of h1-subtype viruses among humans in contrast to the novelty of h5viruses, such a result would be expected. similar data were also observed following vaccination with the h5n2 laiv. after first vaccination, the percent of people with notable increases in virus-specific cd4 + and cd8 + t-cells was 20% and 10% to h5n2 and 38% and 75% to h1n1, respectively. after the second vaccination, these results were 40% and 30% to h5n2 and 69% and 69% to h1n1, respectively. importantly, a significant number of vaccinated volunteers without remarkable increases ( ‡4-fold) in hai antibodies had notable increases in cd4 + and ⁄ or cd8 + memory cells. the percent of people with notable increases in virus-specific t cells after the second vaccination among hai()) volunteers was 40% and 75% to h5n2 and h1n1, respectively. these results indicate that laivs were able to induce broadly responsive, key antiviral immune responses that would not have been detected by the hai assay alone. thus, it can be deduced that hai data alone fails to reveal important broad and specific immune responses to laiv. consequently, the hai test alone is not suitable for assessment of laiv immunogenicity. furthermore, vaccination with h5n2 laiv was able to induce cross-reactive memory t-cells to a seasonal vaccine strain, a ⁄ 17 ⁄ solomon islands ⁄ 06 ⁄ 9 (h1n1) ( table 1) . reliable increases to a (h1n1) were observed in up to 20% of volunteers. there was an inverse dependence between levels of memory t cells before and after vaccination. authors are thankful to path for the financial support of these studies. we are also thankful to jessica d'amico and dr. rick bright for their editorial review. options for the control of influenza vii background: increased susceptibility of older populations to secondary bacterial pneumonia-like infections following influenza infection has been well documented. 1 recent evidence in mouse models suggests that this increased risk from secondary bacterial infection occurs through a desensitization of the innate immune response. 2 this recent finding, however, does not account for potential differences in immune responsiveness due to age. materials and methods: to address this parameter, we used three age groups (aged, adult, and young mice) to evaluate the role of age in influenza-mediated vulnerability to secondary bacterial challenge with pseudomonas aeruginosa. all mice were evaluated for multiple parameters including: (i) survival; (ii) lung bacterial load; (iii) total lung protein content; (iv) immune cell infiltration; (v) cytokine ⁄ chemokine expression; and (vi) toll-like receptor (tlr) rna expression profiles. results: prior challenge with influenza contributed to aberrant cytokine ⁄ chemokine profiles and increased lung cellular infiltrate in response to secondary bacterial infection across all age groups, supporting a critical role for influenza infection in the alteration of immune responses to other pathogens. also similar to human influenza, these changes were exacerbated by age in mice as demonstrated by increased bacterial load, mortality, and total lung protein content (an indicator of lung damage) after p. aeruginosa challenge. conclusions: these data support a potential role for virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and the increased susceptibility of influenza virus infected mice to secondary bacterial infection. the understanding of the complex interaction of host and pathogen -and the role of age -in human influenza is critical in the development of novel therapeutics and improved vaccine approaches for influenza. our results support further examination of influenza-mediated alterations in innate immune responses in aged and non-aged animals to allow elucidation of the molecular mechanisms of influenza pathogenesis in humans. there is considerable evidence in the clinical literature to support the role of influenza infections with an enhanced risk for secondary bacterial pneumonias. [3] [4] [5] given the increased pneumonia-related morbidity and mortality in both the young and elderly populations, there is rationale for gaining a deeper understanding as to the systemic changes in the pulmonary microenvironment. although there are some recent reports that account for some of the molecular mechanisms at work in this disease process, 2 there is a paucity of experimental evidence that considers the potential effects of age. developmental changes in the immune system that occur in the aged environment have been well documented with regard to senescence of the adaptive immunity, global changes in myeloid cell function, and the establishment of a general pro-inflammatory state. 6, 7 the aim of this work was to provide evidence for the contribution of the aged immune environment to the pathology of influenza mediated secondary bacterial infections. animals used in this study were housed under conditions approved by tulane university's institutional animal use and care committee. female balb ⁄ c mice used in these studies were divided into three age groups: aged (18 months old), adult (6 months old), and young (2 months old). each age group was subdivided into two groups: influenza infected and naïve (control). mice were infected by the intranasal route with 4 · 10 5 pfu of mouse-adapted influenza a ⁄ pr ⁄ 8 ⁄ 34. clinical disease was measured by body weight changes over a 6 week period post influenza challenge, and recovery was determined as return to pre-infection weight. all mice were subsequently challenged intransally with1 · 10 7 cfu pseudomonas aeruginosa strain pao1. twenty-four hours post-pseudomonas challenge, bal with sterile pbs was performed on all mice in all groups. total rna from the cellular fraction was pooled from three experimental animals from each group. tlr mrna was detected by qrt-pcr, where expression levels were determined as relative to b-actin mrna levels. cdna was synthesized from total cellular rna from bal samples using iscript cdna synthesis kit (biorad). pcr reactions were composed of 0ae1 lg cdna forward and reverse primers according to optimized conditions and 12ae5 ll of 2 · syber green icycler supermix (biorad), in a total vol-ume of 25 ll and were run using a biorad icycler utilizing melting point determination. primers and concentrations used in this study included: mus_tlr2f: tgctttcct-gctggagattt-600 nm, mus_tlr2r: tgtaacgcaac agcttcagg-900 nm, mus_tlr3f: atatgcgcttcaa tccgttc-300 nm, mus_tlr3r: caggagcatactggt gctga-600 nm, mus_tlr4f: ggcagcaggtggaattg tat-600 nm, mus_tlr4r: aggccccagagttttgttc t-900 nm, mus_tlr5f: ctggggacccagtatgctaa-600 nm, mus_tlr5r: acagccgaagttccaagaga-900 nm, mus_tlr7f: ggagctctgtccttgagtgg-900 nm, mus_tlr7r: caaggcatgtcctaggtggt-600 nm, mus_ b-actinf: agccatgtacgtagccatcc-600 nm, mus_b-actinr: ctctcagctgtggtggtgaa-900 nm. as a measure of protein leakage into the alveolar space, total protein content in each bal was measured by bca assay of each supernatant fraction according to manufacturer's instructions (pierce). cytokine and chemokines levels were measured by multiplexed bead array (bioplex, biorad). immune cell characterization of bal was estimated by flow cytometry. lymphocyte populations were gated by forward versus side scatter and characterized as b cells (f4 ⁄ 80 ) , cd19 + ) or t cells (cd11b ) , cd4 + ). the myeloid population that is composed of macrophages, neutrophils, dendritic cells, and natural killer cells was enumerated by gating all but those found in the lymphocyte gate using forward versus side scatter plots. flow cytometry data was analyzed using flojo software (treestar). statistical analysis, where appropriate, was performed using a two-way analysis of variance (age versus influenza infection status) supported by bonferonni's correction for multiple comparisons. 8 a recent finding by didierlaurent, et al., 2 described an influenza mediated desensitization of tlr function as a primary contributor to an increase in bacterial burden when challenged after resolution of the primary influenza infection. this finding, however, was obtained using animals that were 6-8 weeks of age, where our study included two cohorts of older mice (6 months and 18 months). using whole protein content of the bal as an estimate of protein leakage into the lumen of the lung, we found elevated protein content in aged mice as compared to young and adult mice. in aged mice, a slightly lower total lung protein when comparing influenza infected to protein in the bal from influenza naïve mice challenged with p. aeruginosa (table 1) . supporting previously published studies showing a generalized pro-inflammatory cytokine environment in the aged immune system, we provide evidence for significantly (p = 0ae0465) and an increase in ifnc (p = 0ae0323) was detected. the decrease in gm-csf correlates well with a previous report that gm-csf is less prevalent in influenza resolved animals (table 1 ). 2 we also report a noticeable change in the immune cell populations with respect to b-cells, cd4 + t-cells, and the myeloid cell populations. there is a trend of increased prevalence in cd4 t-cells in the post-influenza environment across all ages. b-cell numbers also trend toward increase in influenza treated animals in young and adult animals; however, there is a noticeable decrease in the bcells in aged animals. across all age groups, there is a general decrease in frequency of cells that would normally make up the myeloid cellular fraction of the bal (macrophages, neutrophils, dendritic cells, and natural killer cells) ( table 1 ). our study also shows, as cited by others, that toll-like receptor (tlr) gene expression in the post-influenza environment is decreased in cells found in the bal 2 after both influenza and pseudomonas infection. our data support the previous finding of a reduced expression of tlr mrna in influenza-cleared mice when we measured tlr 2, 3, 5, and 7. only tlr4 showed differences with respect to age with young mice showing little or no detectable change in tlr4 mrna expression. our results show an increase in the expression across all tlrs examined in the aged mice group (table 1) irrespective of influenza infection status. these data support earlier studies performed with adult mice that showed reduced tlr mrna expression in the post-influenza environment. this study also expands the current understanding of the potential role of age in influenza mediated bacterial infection-induced mortality. the impact of these alterations in the immune microenvironment across age groups and infection status is highlighted by the ability of bacterially challenged animals to clear infection. assessment of bacterial load in the lungs of p. aeruginosa challenged mice indicated a difference in young and adult mice if previously infected with influenza virus. in aged mice, both influenza challenged and influenza-naïve mice had higher bacterial loads and less variability when comparing within the age group, supporting the risk of age alone in susceptibility to bacterial pneumonia (table 1, figure 1 ). taken together, these data support the potential role for both virus-mediated and age-mediated alteration of innate immune effectors in the pathogenesis of influenza and increased the susceptibility to secondary bacterial infection that results from influenza infection in mice. these findings highlight distinct differences in the immune environment between age groups and thus reveal necessity for further examination as to the mechanisms of immunity across age with respect to current infection status. garnering a clearer understanding as to the complex interaction of host and pathogen with respect to age in influenza infections is central to the development of increased efficacy in vaccine and therapeutic strategies. prospective estimation of the effective reproduction background pandemic influenza a (h1n1) virus (ph1n1) emerged in early 2009 and rapidly spread to every continent. an urgent priority for international and national public health authorities was to estimate the transmissibility of the pandemic strain for situational awareness and to permit calibration of mitigation strategies. the basic reproductive number, r 0 , is defined as the average number of secondary cases that 1 index case generates in a completely susceptible population, and is a common measure of transmissibility. however, it is difficult to estimate r 0 without an understanding of the degree of any pre-existing immunity in the population. the effective reproductive number, r, is defined as the average number of secondary cases that 1 index case generates, and can be estimated over time (i.e. r t ). wallinga and teunis 1 described a method to estimate r t based on illness onset dates of the cases while assuming that all secondary cases would have been detected, and cauchemez et al. 2 extended the method to permit prospective estimation by adjusting for secondary cases that have not yet experienced illness onset at the time of analysis. we describe how the method can further be extended to account for reporting delays, allowing true real-time estimation of r t during an epidemic, and we illustrate the methodology on notifications of ph1n1 and associated hospitalizations in hong kong. we obtained data on all laboratory-confirmed ph1n1 infections ('cases') reported between may 1 and november 15, 2009 to the hospital authority and center for health protection in hong kong collated in the eflu database. a subset of the cases was hospitalised. the database also included information on age, sex, illness onset date, laboratory confirmation date, and contact history (for the early cases). laboratory-confirmed ph1n1 infection was a notifiable condition throughout our study period. we extended existing methods for estimating r t over time to allow for reporting delays between illness onset and notification, and between illness onset, notification, and hospitalisation for those cases that were hospitalised, where the reporting delay distribution were estimated empirically from the data. 3 we further extended the methodology to allow for imported cases (infected outside hong kong) contributing to the estimation of r t as infectors but not infectees. we used multiple imputation to allow for missing data on some symptom onset dates to make best use of all available data. 4 we used a serial interval with mean (standard deviation) of 3ae2 (1ae3) days, 5 and in sensitivity analyses, we used serial intervals with mean 2ae6 days 6 and 3ae6 days. 7 statistical analyses were performed in r version 2.9.2 (r development core team, vienna, austria). in late april 2009 following the who global alert, hong kong initiated containment protocols to attempt to delay local transmission of ph1n1 for as long as possible. these measures included screening at ports, airports, and border crossings, and enhanced surveillance for people with influenza-like illness, particularly for those who had recently returned from abroad. laboratory testing capacity was substantial due to heavy investment in local infrastructure following previous experiences with avian influenza a ⁄ h5n1 in 1997 and severe acute respiratory syndrome in 2003. laboratory-confirmed ph1n1 cases were isolated until recovery, and their close contacts were placed under quarantine for 7 days. imported cases were identified sporadically through may and early june 2009. the first case of ph1n1 not traceable to importation (i.e. a local case) was identified on june 11 and triggered a change to mitigation phase measures. some containment measures, including isolation of cases, were continued until the end of june to allow a soft transition between containment and mitigation phases. as an immediate measure to try to reduce community transmission of ph1n1, all childcare centres, kindergartens, and primary schools were proactively closed for 14 days (subsequently extended for another 7-14 days to summer vacation in early july). 8 any secondary schools in which one or more confirmed ph1n1 case was identified were reactively closed for 7 days. on june 13 the government opened eight designated flu clinics across the territory to provide free medical consultation for outpatients with influenza-like illness and free laboratory testing for ph1n1. these clinics resumed regular chronic disease services in mid-august, and laboratory testing and antiviral treatment was restricted to high risk groups in september. the various interventions are highlighted in figure 1 (a), superimposed on the epidemic curve of laboratory-confirmed ph1n1 cases and ph1n1-associated hospitalizations. around 15% of the cases were hospitalised, and this proportion increased somewhat towards the end of the epidemic. 3 figure 1(b) shows the estimates of r t based on laboratory-confirmed ph1n1 cases. the estimated r t peaked at 1ae5 on june 12, and fell below 1 between 20 june and 3 july (which was within the school closure period). r t fluctuated between 0ae8 and 1ae3 through the school summer vacations in july and august, it subsequently increased to around 1ae2-1ae3 after schools reopened in september until the epidemic peaked in late september, and then fluctuated below 1 as the epidemic declined. the trends in r t based on h1n1-associated hospitalizations were similar, although with wider confidence intervals due to the smaller number of events ( figure 1c ). the extension of the methods to allow for reporting delays avoided substantial bias in realtime estimates of r during the epidemic for the most recent 7 days, and closely tracked the final estimates of r t . 3 our results suggest that ph1n1 may have had slightly lower transmissibility in hong kong than elsewhere. for example, estimates of r t were around 1ae5-2ae0 in new zealand 9 and australia. 10 lower transmissibility in hong kong has been associated with school closures in june and july followed by summer vacations from july through august. 8 furthermore, in hong kong the influenza virus usually does not circulate after august, 11 and therefore seasonality could also be a cause for the lower r t . on the other hand, the interventions applied during the mitigation phase, such as the widespread use of antiviral treatment in hong kong and the pre-existing immunity in the ageing population in hong kong, may also be associated with lower transmissibility. there are some limitations to our work. first, we only used aggregated data, and we did not consider the heterogeneity among the cases in terms of sex and age or other factors. therefore our estimates can only provide a snapshot of the overall trend, but limited information for any specific subset of population. secondly, we did not consider the possibility that cases might be infected in hong kong and exported to other countries, which could lead to slight underestimation of the transmissibility. one has to be careful in translating the estimated r t to the effectiveness of any specific interventions, as interventions may not be the only factor influencing the transmissibility; for example, a depletion of the susceptible population during an epidemic can also be a factor for the decline in r t . 12 in conclusion, real-time monitoring of the effective reproduction number is feasible and can provide useful information to public health authorities for situational awareness and planning. in affected regions, laboratory capacity was typically focused on more severe cases, and changes in laboratory testing and notification rates meant that that case counts may not necessarily reflect the underlying epidemic. a useful alternative to case-based surveillance is surveillance of the subset of severe infections, for example hospital admissions, or icu admissions, 13 and our results show that it was feasible to monitor ph1n1-associated admissions in real-time to estimate transmissibility. influenza antigenic cartography projects influenza antigens into a two or three dimensional map based on immunological datasets, such as hemagglutination inhibition and microneutralization assays. a robust antigenic cartography can facilitate influenza vaccine strain selection since the antigenic map can simplify data interpretation through intuitive antigenic map. however, antigenic cartography construction is not trivial due to the challenging features embedded in the immunological data, such as data incom-pleteness, high noises, and low reactors. to overcome these challenges, we developed a computational method, temporal matrix completion-multidimensional scaling (mc-mds), by adapting the low rank mc concept from the movie recommendation system in netflix and the mds method from geographic cartography construction. the application on h3n2 and 2009 pandemic h1n1 influenza a viruses demonstrates that temporal mc-mds is effective and efficient in constructing influenza antigenic cartography. the web sever is available at http://sysbio.cvm. msstate.edu/antigenmap. as a segmented, negative stranded rna virus, influenza virus is notorious for rapid mutations and reassortments. the mutations on the surface glycoproteins (ha and na) of influenza viruses are called antigenic drifts, and these antigenic drift events allow the virus to evade the accumulating immunity from previous infection or vaccination and lead to seasonal influenza epidemics. a reassortment event with a novel influenza antigen may result in antigenic shift and cause influenza pandemic. for instance, the 2009 h1n1 pandemic virus is a reassortant with a swine origin ha antigen. vaccination is the primary option for reducing the effect of influenza, and identification of the right vaccine strains is the key to development of an effective vaccination program. the antigenicity of an optimal vaccine strain should match that of the epidemic strain. in influenza surveillance program, the influenza antigenic variants are generally identified by the immunological tests, such as hemagglutination inhibition (hi) assay, microneutralization (mn) assay, or elisa. these immunological assays measure the antigenic diversity between influenza viruses by comparing the reaction titers among the test antigens and reference antisera. however, data interpretation of the data from these assays is not trivial due to the embedded challenges such as data incompleteness, high noises, and low reactors. by mimicking geographic cartography, influenza antigenic cartography projects influenza antigens into a two or three dimensional map using immunological datasets. 1 antigenic cartography can simplify the data interpretation, and thus, facilitate influenza antigenic variant identification. recently, we developed a novel computational method, temporal matrix completion-multidimensional scaling (mc-mds), in antigenic cartography construction. 2 in this paper, we described the details of temporal mc-mds, especially the original concepts introduced in this method, and how they can achieve the robustness in antigenic cartography construction. our method included two integrative steps: it first reconstructs the hi matrices using low rank mc method, and then generates antigenic cartography using mds with a temporal regularization. the mc concept was adapted from the movie recommendation system in netflix and the cartography concept from geographic cartography. in 2006, netflix, an online dvd and blu-ray disc rentalby-mail and video streaming company, held a 3-year netflix prize contest (http://www.netflixprize.com/) on computational methods for improving its recommendation system. 3 in its recommendation system, netflix collected the rating data from the individuals. based on his or her renting history and the ratings in the systems (e.g., from evaluators and other renters), netflix recommendation system suggests certain movies to a renter. apparently, no individuals would be feasible to provide ratings for all of the movies, as it will take hundreds of years for a single person to rate over 50 000 movies available from netflix. thus, the resulting rating data is an incomplete matrix, and it can be as sparse as less as 1%. 4 the challenge in netflix recommendation system is a classic mc problem. [4] [5] [6] [7] [8] as the inspiration of netflix prize contest, many efficient low rank mc algorithms were developed, for instance, opt-space, 7 svt, 5 cf, 9 bellkor, 10 pf, 11 and fwls. 12 eventually, the team bellkor's pragmatic chaos won this contest. their methods combines nonlinear probe blending and linear quiz blending to come up with a predictor bigchaos. 13 matrix completion estimates the unobserved values based on the observed values. the users can refill the missing data without repeating the experiments. furthermore, mc will help reduce the noises in the data, for instance, those biases by different individuals performing experiments. in influenza antigenic characterization, hi assay is a commonly used assay for antigenic analysis, since hi assay is relatively economic and easy to perform. however, hi is labor intensive, and it is almost impossible for any individual lab to complete the hi assays for all pairs of antigens and antisera during influenza surveillance. in addition, both testing antigens and the reference antisera are dynamic. for instance, in seasonal influenza surveillance, generally only contemporary antisera are used in experiments. thus, we will have to integrate multiple hi tables in order to evaluate the overall antigenic changes for influenza vaccine strain selection. the resulting hi tables will be incomplete, and the observed entries in the integrated hi data can be as less as 3%. the completion of this matrix can be formulated as a typical mc. briefly, given the combination of hi matrix with m antigens and n antisera, the hi matrix can be represented as m m·n = (m ij ) m·n , where m ij denotes the hi values from the reaction between testing antigen i and antiserum j. the low rank mc assumes that both antigen and antiserum can be embedded into a low rank space. to be specific, the low rank mc method is to seek matrix u m·r , v n·r and a diagonal matrix r r·r , where m = u m·r r r·r (v n·r ) t . in order to achieve this goal, the optimization formulation has been employed, which can be represent as following, where e denotes the observed entries in hi matrix and g(x) is a regularization function. the eqn (1) is the standard format of a low rank mc formulation. the geographic cartography is a common technique to display the cities and their geographic distances in a map. this cartography can be generated using mds based on a geographic distance matrix. figure 1(a) shows the antigenic cartography generated using a distance matrix with seven cities, and figure 1 (b) is a map for comparison. as an analog of geographic cartography, the influenza antigenic cartography maps the influenza antigens into a two or three dimensional map based on the distance matrix generated using immunological data. this incomplete matrix can be filled through mc algorithm discussed in section mc and netflix. low reactors, non-random date incompleteness, and temporal model generally, three types of data are present in a combined hi matrix: high reactor, low reactor, and missing values. among these three data types, high reactors are the most reliable data points. the low reactors are those values present in the hi matrix as ''equal to or less than a threshold h'', where h can be 5, 10, 20, or 40. low reactors have similar values in the affinity dataset but could be from different binding settings. these low reactors are present due to the detection limits of biotechnology, and they are not reliable. both these missing values and low reactors make it very difficult to analyze and interpret antigenic correlations amongst tested antigens and reference antigens. to our best knowledge, none of the existing mc method can handle the threshold values. in addition, the non-random incompleteness of influenza immunological datasets generates an additional challenge in traditional mc methods, which are based on the assumption that the observed values are randomly distributed among the matrix. in a typical combined antigenic hi data, most of the off-diagonal entries are missing values or low reactor values. 1 in order to overcome the above issues, we incorporated a regularization function into the eqn (1), where this indicator function is only valid for those entries with low reactor values. an alternating gradient decent method is applied to solve the optimization problem in eqn (2) . in addition, a temporal mds method is proposed to project the antigens into a 2 or 3 dimensional map. x where d ij is the average distance between virus i and virus j, t i is the isolation year of virus i, d ij is the distance between virus i and virus j in cartography, d ac i is the distance between virus a and center of group i, and d c i c j is the distance between the centers of group i and group j. all the parameters are tuned by cross validation. we named this method as temporal mc-mds. by applying temporal mc-mds method in an h3n2 dataset, 2 low reactors. figure 2 (a) is a three-dimensional influenza antigenic map based on this data by using mc-mds method. the reported 11 clusters (hk68, en72, vi75, tx77, bk79, si87, be89, be92, wu95, sy97, and fu02) were displayed in the core of a spiral s-shape, and bk79 and be92 are located at the turning point of this s-shape. however, the antigenic distances between some viruses are incorrect. for example, the distance between hk68 and fu02 in the projection is 7ae1223 units, which is close to the distance between hk68 and bk79 (6ae5113 units). the main reason leading to those inaccurate distances is the unique distribution of hi datasets described in section 2.3. in comparison, with the temporal model, not only the viruses in 11 clusters have been clearly separated, but also the antigenic distances between each cluster are proportional to their isolation time interval. in this updated cartography ( figure 2b ), the antigenic distance between hk68 and fu02 is 15ae0633 units, where the distance between hk68 and fu02 is 6ae3984 units. this result suggested that the temporal information is critical for antigenic cartography construction for immunological datasets spanning a long time period. the hi data from seasonal influenza surveillance belong to this category. for seasonal influenza virus ⁄ pandemic influenza viruses within a short time span, the temporal model is probably not necessary, as there is lack of long-term immunological pressure present in the population. figure 2 (c) is an antigenic cartography generated using a hi dataset with 2009 h1n1 influenza viruses spanning from april of 2009 to june of 2009. this map demonstrates that there is lack of antigenic drifts during the first wave of this pandemic influenza as all of these viruses are mixed altogether. our limited studies on h5 and h7 avian influenza viruses suggested the temporal model is not needed for avian influenza viruses. however, extensive studies are required to investigate whether there is any special data structure present in this type of data. in this study, we described in details the concepts and applications of new computational method, temporal mc-mds for influenza antigenic cartography construction. we formulate the influenza cartography as two integrative steps: low rank mc problem from the concept of netflix movie recommendation system and mds from geographic cartography construction. in order to handle two additional challenges, including low reactor and non random distribution of antigenic data, a temporal model is incorporated into mc-mds as temporal mc-mds. our applications demonstrated that temporal mc-mds is effective in constructing influenza antigenic cartography. the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus without temporal model, and the antigenic clusters were defined in ref. [2] ; (b) the three dimensional antigenic cartography for a ⁄ h3n2 seasonal influenza virus with temporal model; (c) the two dimensional antigenic cartography for 2009 a ⁄ h1n1 pandemic influenza without temporal model, and these viruses were labeled in shape by the corresponding month for them to be detected. one grid is corresponding to a twofold change in hemagglutination inhibition experiment. the mechanisms driving the three waves of infection and mortality in the uk in 1918-1919 are uncertain. although the circulation of three distinct viruses could have generated three waves of infection, 1 the virological evidence required to prove or disprove this hypothesis is lacking. social distancing, an alternate mechanism for generating fluctuations in the effective susceptible pool and therefore explaining multiple waves of infection, 2, 3 was not generally imposed in the uk as it was in the us and australia. we are therefore motivated to explore the possible role of continual population-level changes in the average protective response against the circulating virus in generating a multi-wave pandemic, within a biologically motivated deterministic model for influenza transmission. the nature and duration of protection against further infection following recovery from influenza is uncertain and depends on the mode and tempo of viral evolution, as well as the response of the cellular and humoral arms of the adaptive immune system. 4 for a given seasonal ⁄ pandemic strain, memory b-cells may generate a specific antibody response in a portion of the adult ⁄ elderly population, depending on the exposure to related antigenic sub-types. 5 however neutralising antibodies are unlikely to be a widespread immunological response to a novel (pandemic) strain. memory t-cells which recognise conserved internal viral proteins may be a more common mechanism for protection; the generation of very high levels of cytotoxic cd8 + t-cells potentially facilitates rapid viral clearance, 6, 7 and lower levels of cd8 + t-cells perhaps provide partial protection. 8 in this work we explore key drivers of multi-wave pandemics within phenomenological models that incorporate different immune response mechanisms building on existing models 9,10 incorporating the role of evolving population-level protection in multi-wave pandemics. we use weekly reports of influenza mortality rates 11 for five administrative units in the uk (blackburn, leicester, newcastle, manchester and wigan) where records from block censuses instigated by local medical officers to record the cumulative incidence of reported symptoms in each wave in a sample of 1000 or more households are also available. 10 the symptom reporting data allows us to estimate the case fatality rate and thus use the mortality time series to constrain our transmission model. furthermore, the incidence of individuals reporting symptoms in multiple waves provides information about the acquisition and loss of immunity. we extract the death rate and symptomatic (re)infection rates predicted by our model prevalence for a given set of parameters and estimate a likelihood-based on a comparison to all the death and cumulative reported incidence data assuming a negative binomial error distribution. we utilise monte carlo markov chain (mcmc) methods with parallel tempering algorithms to maximise this likelihood and obtain parameter estimates. parallel tempering -which concurrently searches for maximal likelihood parameter solutions on a set of scaled likelihood surfaces -allows for relatively rapid exploration of the parameter space. we use bayesian information criteria (combined with qualitative assessment of biological plausibility) to aid model selection. we have implemented a deterministic compartmental transmission model, which allows for a variety of phenomenological modes of protection against the pandemic virus. to facilitate this, we stratify the population into two groups; the 'experienced' population (stratum 1) who have had been exposed to an influenza virus and the 'naive' population (stratum 2) who have not. in each stratum, i hosts may be classified as either susceptible s i , exposed e1 i and e2 i , having (recovered from) a symptomatic i i (r i ), or asymptomatic a i (ra i ) infection. note that the states tq i , tq2 i , e2 i , t i , and t2 i are included so that the hosts move between the key epidemiological states with a peaked (rather than exponential) distribution of waiting times. hosts in the experienced stratum may exhibit reduced susceptibility, infectiousness, and symptomatic proportion compared to naive hosts, parameterised by e i , e s , and e a , respectively; however note that depending on the model parameters, there may be fully susceptible hosts within the experienced stratum. in addition, we assume homogeneous population mixing and a constant basic reproduction number r 0 with the force of infection: modulated by a sinusoidal seasonal term with amplitude b 1 with phase chosen to maximise transmission in the winter season. here n is the total population size, and x e is the initial fraction in the experienced strata. the proportion of symptomatic cases a and the case fatality rate l are permitted to vary from wave to wave (and given indices 1, 2 or 3 accordingly). the transmission dynamics is described by the following set of coupled ordinary differential equations. where s in,1 = p utq2 i and s in,2 = 0 in order to divert recovered infectious hosts from the naive stratum into the experienced stratum. the probabilities of gaining permanent protection are q 1 = q and q 2 = 0. the latent exposed period is fixed to be c = 1 ⁄ 1ae3 days, and the rate of recovery is parameterised by m = 1 ⁄ t inf , where t inf is the infectious period. hosts with prior sterilising protection begin in q 1 and move into s 1 at rate u q = 3 ⁄ t wq . recovered hosts (r i ) migrate back to s 1 at a rate u = 3 ⁄ t w . the state p 1 contains hosts with permanent protection. the modes of protection captured in this model are: i. permanent prior protection (beginning in state p 1 ), ii. waning prior protection (beginning in state q 1 ), iii. permanent acquired protection with probability q (moving into state p 1 ), iv. waning acquired protection with probability 1 ) q, and, v. partial prior protection (beginning in state s 1 ) resulting in reduced infectiousness (e i ), susceptibility (e s ), and symptomatic proportion (e a ). in the context of this model, 'permanent' protection refers to protection which lasts for the duration of the epidemic. here we explore the results of parameter fitting to two models which differ in the nature of the assumed pre-existing protection in the community at the beginning of the pandemic. protection hypothesis 1 assumes that the prior protection is sterilising but temporary, whilst protection hypothesis 2 assumes that the prior protection is partial but permanent and may act on susceptibility, infectiousness, and ⁄ or asymptomatic proportion. each model allows waning acquired protection and for a proportion q of the experienced population to gain permanent protection following infection. fitted parameters common to each model are t inf , b 1 , q, t w , a, l and the proportion beginning in p x i . prior protection hypothesis 1: sterilising, waning prior protection we fix x e = 1 and fit for q 1 (t = 0) ⁄ n and t wq so that protective modes i, ii, iii, and iv are enabled ( figure 1 ). it is important to note that due to the slow convergence of the mcmc chains, we cannot guarantee that our parameter estimates correspond to the global minimum. furthermore, parameter estimates can only be meaningfully interpreted for good fits to the data. due to the prediction of a fourth (unobserved) wave for the model fit to blackburn, we do not report these parameter estimates here. the fits to the leicester data are generated with the parameter set r 0 = 5ae7, a 1 = 0ae25, a 2 = 0ae65, a 3 = 0ae65, t w = 0ae28years, t wq = 0ae45 years, we fix q 1 (t = 0) ⁄ n = 0 and fit for x e , e a , e i , and e s so that protective modes i, iii, iv, and v are enabled (figure 2) . the parameters corresponding to the fit in figure 2 for leicester are r 0 = 7ae4, a 1 = 0ae09, a 2 = 0ae50, a 3 = 0ae61, t w = 0ae22 years, p 1 (t = 0) ⁄ n = 0ae01, s 2 (t = 0) ⁄ n = 0ae48, b 1 = 0ae021, t inf = 0ae94 days, q = 0ae561, e a = 0ae9966, e i = 0ae946, and e s = 0ae594. our model with protection hypothesis 1 -which, similarly to the model discussed in ref. [9] , assumes that a sub-population has waning sterilising prior protection -is able to generate multiple waves of infection via the continual replenishment of s 1 from an initially large proportion (over 40%) of hosts with prior protection in q combined with the waning of acquired immunity in around 23% of cases on a time-scale of 3 months. disease severity as measured by symptomatic proportion increases from 25% in the first wave to above 60% for the second and third waves. over a quarter of the population are initially permanently immune, and a large r 0 value of 5ae7 drives transmission in the remaining population. protection hypothesis 2 -which assumes that prior protection offers partial susceptibility and ⁄ or reduced infectiousness or symptomatic disease -performs slightly more poorly; the fit to the leicester data has an inferior likelihood (although the mortality data only likelihood is a little larger), despite the higher dimensionality of the model. nevertheless, the model fit still mirrors many characteristics of the data, particularly for leicester. we note that for this model, a 1 is very near the lower limit, corresponding to ubiquitous exposure in the first wave. in this scenario, refuelling of the susceptible pool to generate secondary and tertiary waves is still possible due to a shorter waning time of acquired protection (well within 3 months) and a lower probability of gaining permanent protection following infection, when compared with the parameter estimate for hypothesis 1. the parameter estimates suggest that approximately 50% of the population initially experiences reduced disease severity (e a $ 0ae66), but similar susceptibility and infectiousness. a larger value for r 0 $ 7ae4 is required to drive transmission despite low numbers beginning in p 1 , due to the large number of hosts who acquire temporary or permanent immunity early on in the pandemic. it is clear that, at least mathematically and perhaps biologically, there are multiple possibilities for the structure of population-level protection which are compatible with the generation of multiple pandemic waves. however, whilst the models considered here are able to explain the observed mortality and reinfection data for some patterns of infection and mortality (e.g. leicester), they are not consistently able to reproduce a pandemic which dies out after three waves across the connected populations we are studying (e.g. for blackburn). it is challenging to construct a deterministic model for the spread of disease within multiple locations in the uk in 1918, which assumes homogeneous mixing without modulation of the transmission rate by social distancing. an improved model working with these assumptions likely requires a richer structure for the host protection response than the structures we have explored thus far. we are currently seeking improved fits to the data by implementing a number of biologically defensible exten-sions to our model, including incremental immunity whereby t w increases by a factor v after each exposure to the pandemic flu, and incremental loss of prior protection whereby a increases as hosts lose their sterilising prior protection. it is important to note that the mechanism(s) generating differences in the pandemic experience recorded in geographically connected locations is an open question; true differences in demography, varying degrees of reactive social distancing, inhomogeneities in the circulation (or circulation history, i.e. prior immunity) of viral strains, stochastic variations, and ⁄ or unique socio-cultural ⁄ behavioural conditions may all contribute to this effect. the 2009 h1n1 experience in australia and elsewhere highlighted the difficulties faced by public health authorities in diagnosing infections and delivering antiviral agents (e.g. oseltamivir) as treatment for cases and prophylaxis for contacts in a timely manner. consequently, forecasts from mathematical models of the possible benefits of widespread antiviral interventions were largely unmet. we summarise results from a recently developed model that includes realworld constraints, such as finite diagnostic and antiviral distribution capacities. we find that use of antiviral agents might be capable of containing or substantially mitigating an epidemic in only a small proportion of epidemic scenarios given australia's existing public health capacities. we then introduce a statistical model that, based on just three characteristics of a hypothetical outbreak [(i) the basic reproduction number, (ii) the reduction in infectiousness of cases governments and public health agencies worldwide, spurred by outbreaks of sars and h5n1, have developed preparedness strategies to mitigate the impact of emerging infectious diseases, including pandemic influenza. pandemic response plans are presently being revised in light of the 2009 h1n1 experience. [1] [2] [3] many developed countries amassed large stockpiles of neuraminidase inhibitors (nais) with the expectation that they could be used to not only treat the most severely ill, but curb transmission in the community. without relevant field experience indicating how nais should be distributed, mathematical and computational modelling has been used to inform optimal deployment policy in a pandemic scenario. 4-10 models of population transmission were used to infer likely effects on epidemic dynamics, using data from human and animal studies of experimental infection and nai efficacy trials. in the australian (and wider) context, models indicated the potential for substantial benefit at the population level if nais were distributed in a liberal manner, targeting close contacts of indentified cases. 11 furthermore, results indicated that use of limited nai resources in this way may improve the impact of case treatment due to the effects on epidemic dynamics. 11 however, these models did not take into account logistic and other real-world constraints, such as finite diagnostic and antiviral distribution capacities, which were identified as limiting factors during the australian 2009 h1n1 pandemic response. [12] [13] [14] in particular, if using positive pcr diagnosis as a 'decision to treat' test, delays to confirmation of diagnosis, particularly once total laboratory capacity was exceeded, prevented timely delivery of nais to both cases and contacts of cases. 12 in previous work, 15 we have extended our existing models to examine how diagnostic strategies [e.g. using pcr confirmation versus syndromic influenza-like illness (ili) presentation as a decision to treat], diagnostic-capacity, and nai distribution capacity each impact on the ability to deliver an effective intervention. the model uses case severity (the proportion of infections deemed severe) to determine the overall presentation proportion, and so the ability to identify individuals eligible for nai treatment and contact prophylaxis. figure 1 (a) shows a key result from the model. for each curve shown, we simulated thousands of epidemics, sam-pling across plausible ranges of parameters describing virus, population, and intervention characteristics using a latin hypercube sampling (lhs) approach. without intervention, the proportion of the population infected either symptomatically or subclinically by the end of the epidemic is around 50%. if a syndromic strategy (ili presentation) is used to determine provision of nais as treatment and prophylaxis, excessive distribution of drug to individuals who are not infected with influenza occurs early in the epidemic. early stockpile expiry accounts for a marginal impact of the antiviral intervention on the final outbreak size, in the order of a few percent. the second strategy modelled (pcr ⁄ syndromic) is one where pcr confirmation of diagnosis is required early in the epidemic to make treatment decisions until such time as laboratory capacity is exceeded. from this point, individuals are treated on the basis of symptoms alone -during an epidemic phase in which a substantial proportion of ili presentations will be attributable to influenza. under this strategy, the intervention is able to control the outbreak in approximately 10% of the simulated epidemics given the 'base case' constraints on diagnosis and delivery assumed in the model. the results highlight that a successful antiviral intervention requires a highly sensitive diagnostic strategy in the initial stages of the epidemic and comprehensive distribution of post-exposure prophylaxis. a pcr ⁄ syndromic strategy for decision to treat and provide contacts with prophylaxis is thus optimal. the surface in figure 1(b) shows the percentage of simulation runs for the pcr ⁄ syndromic strategy that have a final population attack rate of <10% (a substantial reduction from the no intervention case of approximately 50%) as a function of pcr capacity and nai daily distribution capacity. as indicated by the arrow, the estimated australian pcr laboratory capacity appears to be sufficient, while significant benefits for the public health outcome may be achieved if logistical delivery constraints for nai distribution can be ameliorated. however, the probability that such an interventioneven with substantial increases in pcr and nai distribution capacity -would successfully mitigate an epidemic is low (12-25%), and consequently it is difficult to universally recommend an antiviral intervention. 15 in this study, we introduce a statistical model that predicts whether or not an nai distribution strategy based on a pcr ⁄ syndromic antiviral distribution policy will be successful in mitigating an epidemic. we thereby provide proof-of-principle for the design of a decision support tool that may be used by public health policy makers during an epidemic when faced with formulation of context specific nai distribution policy. synthetic data of hypothetical outbreaks and interventions were generated using the lhs simulations developed in ref. [15] . we selected a random sample of 100 outbreaks from a total of 2000 simulated epidemics (5% of model simulations). using these data, we identified independent model parameters that were most highly rank-correlated with the final attack rate. these parameters were included in a logistic regression model to assess their ability to predict whether an influenza epidemic would be successfully mitigated by an antiviral intervention (ar < 10%). model predictions were then validated against the full simulated dataset. full details of the simulation model, its structure, parameterisation and parameter distributions are available in ref. [15] . use of the lhs simulation approach, and the method of model analysis and evaluation was similar to that previously described. 16 matlab 2010a (mathworks, natick, ma, usa) was used for the analysis and statistical model fitting. table 1 shows results from our logistic regression model. key parameters sufficient to predict whether or not an outbreak may be controlled by the deployment of av agents are: 1. r 0 , the basic reproductive number of the outbreak (assigned values between 1ae35 and 1ae45 for this example). as the value of r 0 increases, the epidemic progresses more rapidly and is more difficult to control, explaining the negative correlation coefficient. 2. e t , the relative infectiousness of treated individuals (assigned values between 0ae8 and 1ae0). higher values for this parameter indicate only modest drug effects on transmission, explaining the negative correlation coefficient. 3. g, the proportion of infections that are severe (assigned values between 0ae001 and 0ae1), and which in turn determines the presenting proportion (derived values between 0ae11 and 0ae56). as the presenting proportion increases, the ability to identify and treat cases and deliver prophylaxis to contacts also rises, increasing the impact of the antiviral intervention. the roc curve (1-specificity versus sensitivity, not shown) for the logistic regression model specified in table 1 has an area under the curve of 0ae937, demonstrating that the model predicts the success of an antiviral intervention extremely well. for example, with a sensitivity of 95% we still have a specificity of approximately 80%. evaluation of the 2009 pandemic response has emphasised the need for early informed decision-making to implement proportionate disease control measures. our model identifies a low probability of successful epidemic mitigation using targeted antivirals alone (figure 1 and ref. 15 ), in distinction to results from models that fail to account for the diagnosis and delivery constraints inherent in any public health response. the decision support tool (table 1) highlights key epidemic characteristics that are predictive of a high likelihood of effective mitigation. the reproduction number was one of the earliest parameters estimated from early outbreak data during the 2009 h1n1 outbreak. 17, 18 our findings reinforce the importance of characterising epidemic severity as early and as accurately as possible, in order to inform a proportionate pandemic response. critically, a typically mild pandemic (low g), such as that experienced in 2009, is predictably difficult to contain using a targeted antiviral strategy due to the low proportion of infectious cases that present to health authorities. the relative infectiousness of treated individuals, e t , is strongly negatively correlated with successful mitigation, perhaps a surprising result given the model's underlying assumption (based on available epidemiological and human clinical trials data) that e t lies in the range [0ae8, 1]. that is, nais provided as treatment have a maximum impact of just a 20% reduction in infectiousness. however, our previous results 4 show a strong synergistic effect of treatment when overlayed on a contact prophylaxis strategy, explaining the observation here that e t is critical in determining likely success of an intervention. despite the limited impact of treatment at the individual-level, the model outcomes are highly sensitive to the value of the relative infectiousness of treated cases. it follows that determination of e t is important for predicting the population-level outcome of a control effort. a 'small' reduction (of the order approximately 10%) may be extremely valuable in terms of success of a public health control strategy, and so should not be discounted. using a mathematical model which takes into account some of the key logistic constraints that are inherent to healthcare responses, we have derived a logistic regression model for estimating the probability that an antiviral intervention based on liberal distribution of nais as treatment and prophylaxis could successfully mitigate an influenza epidemic. the model demonstrates an excellent degree of accuracy when applied to synthetic data. the choice of parameters for the regression model was restricted to those that were both highly correlated with the success of the intervention and hopefully feasible to measure during the early stages of an emerging epidemic. the model could therefore be a useful near real-time decision support tool for public health policy in the face of an influenza epidemic, although further validation on a range of synthetic data (and real-world data where available) is required. influenza to seasonal flu status to avoid overstretching the demands on healthcare services. a great deal of information has emerged as the result of the pandemic response exercises conducted by affected countries. however, uncertainties remain regarding the effectiveness of intervention measures, as well as the feasibility and the timing of their implementation. mathematical and computational models [2] [3] [4] have been used to project the outcomes of influenza outbreaks under various scenarios and epidemiological hypotheses. motivated by the events of 2009 and public health measures adopted by the taiwan cdc, we use a stochastic, individual-based simulation model 5 to study the spatio-temporal transmission characteristics of the h1n1 virus, so as to quantitatively assess the effects of early intervention strategies. our stochastic disease simulation model 5 builds upon a highly connected network of individuals interacting with each other via social contact groups. to represent the daily interactions of approximately 23 million people living in taiwan, we constructed a computer-generated mock population based on national demographic and employment statistics (to derive daily commute patterns) from the 2000 taiwan census (http://www.stat.gov.tw/). each individual is created with a set of attributes, including age, sex, residence, family structure, and social standing (employment status, etc.). based on their attributes and the time of day, each individual is assigned to miscellaneous contact groups, where the potential of interactions between any two individuals resulting in flu virus transmission occurs. such epidemiological properties are defined by empirically parameterized attributes such as basic reproduction number r 0 , transmission probability, contact probability and associated probability distributions outlining the disease's natural history. additionally, intervention measures are implemented as scheduled events that could alter control parameters during the course of a simulation run. the targeted basic reproduction number (r 0 ) in all our simulations is 1ae6, following the suggested range by who of 1ae2-1ae7. 6 as the latent ⁄ incubation and infectious periods for h1n1 have not yet been reliably ascertained, we adopt the natural history of the 1957 and 1968 pandemic influenza viruses. 2, 7 here, the latent period ranged from 1 to 3 days, with a median value of 1ae9 days. the infectious periods begin 1 day prior to symptom onset and can continue for 3-6 days, with a median value of 4ae1 days. twothirds of the infected individuals will develop clinical symptoms, and the asymptomatic cases will have half the infectious strength. the efficacy of antiviral drugs (oseltamivir) and vaccines are based on these studies. 8, 9 for the source region of the infected cases, we use the north american continent (canada, mexico and united states) with an estimated total population of 450 527 697 and an average 16 hours of flight time to taiwan. the average daily passenger number is 2489 based on the 2009 annual statistical report on tourism, tourism bureau, taiwan (http://admin.taiwan.net.tw/english/statistics/year.asp? relno=61). each simulation lasts 365 days and starts with a baseline simulation of r 0 % 1ae6 h1n1pdm outbreak at the source region. the outbreak was adjusted to approximate clinical attack rate (car) in the united states, april 2009-march 13, 2010. 10 we estimate the daily number of imported cases according to average daily passenger numbers and their probability of holding a disease status. we then apply airport exit ⁄ entry screening per corresponding success rates, by subtracting the number of identified symptomatic cases. we also consider latently infected passengers with inflight disease progression, by fitting a gamma distribution to the cumulative distribution of time to onset data with 16 hours average flight-time, as presented by pitman et al. 11 the daily imported cases are seeded according to the traveling patterns of foreign tourists and residents returning home. from the disease's natural history, we derive that roughly 50% of the infected travelers present no symptoms; the percentage increases if most symptomatic individuals elect not to travel in their condition, or are stopped by airport screening. we use the official epidemic data provided by the taiwan cdc to calibrate the simulation model and perform regression analysis on scenario parameters. this data is a close estimation of the weekly new clinical cases of h1n1pdm patients. it consists of weekly opd (outpatient department) icd-9 code 487 (influenza) tallies collected by the bureau of national health insurance, taiwanadjusted to exclude seasonal flu patients and to account for uninsured patients. we formulate our scenario settings according to 2009 events in taiwan, and establish settings to approximate the actual events. with domestic events and intervention schedules fixed in time, the start date determines the simulation outcomes and the data range for selected indicators, such as the mean car, the epidemic peak, and several significant dates for the incoming index case events. we plot the 2009 taiwan weekly h1n1 opd487 cases alongside the weekly new clinical cases from our simulation results in figure 1 . our simulations not only capture the epidemic trend, but also pick out the most likely date, may 20, for identifying the first symptomatic case at airport screening based on practical assumptions. we further analyze the effectiveness of various mitigation measures with february 6, 2009 as the empirical start date for h1n1pdm in north america. the simulation result confirms that by the time we identified the first symptomatic case at the border screening, infected cases had already made their way to the public. by our calculation, roughly four such cases had passed in each of our scenario settings, with the first case happening as early as 3 weeks before detection. figure 1 also highlights the importance of the timing for the implementation of mitigation measures; for example, a 6-day-delay of the identical intervention plan results in nearly an additional 1% of the population being infected. therefore, the rule of thumb for healthcare officials is to implement intervention measures as early as possible. in our study, we have ignored the possibility of inflight transmission and any false positive results by airport screening procedures. to assess the effectiveness of each mitigation strategy of interest and their combinations, we take the calibrated simulation model and perform 100 simulation realizations for groups of scenarios containing only those intended mitigation measures, and analyze the averaged results. for example, in the airport exit screening policy only scenario, the first imported symptomatic case can be delayed up to 2 months, and the epidemic peak can be delayed up to 13 days. as the data suggests, the exit screening policy alone has very little impact on car. combining various screening success rates for both exit and entry screening allows us to quantitatively assess their beneficial ramifications on the epidemic. for example, there is very little additional benefit between 100% and 80% suc-cess rates for entry screening policies when exit screening policies are adequate, as the enhanced border screening only delayed the epidemic peak by 1 day, and reduced car by <0ae01%. base on this result, the government should not attempt to exhaust all its resources in securing the border during a pandemic event, because the return of such a policy will be disappointing. instead, a response plan with a shifting focus on health resource allocation and the capacity of adjusting intervention strategies in line with the developing epidemic will be most effective. based on the same principle, we perform experiments with assorted scenarios, including relaxing entry screening policies after identifying the first imported symptomatic case, mass vaccination based on the actual vaccination schedule of h1n1pdm in taiwan, and altering the start dates of the vaccination schedule. our results show that with a reasonable reduction in the airport entry screening success rate, we conserve valuable healthcare resources, but loose a few days for the strategic planning and preparation of subsequent response measures. in other simulation scenarios, a national vaccination campaign has very little impact on the outcome, due to the late start of the vaccination schedule. we then explore the effect of a national vaccination campaign with various starting dates. the simulation results are illustrated in figure 2 , where the benefit of an early start date for mass vaccination is clearly demonstrated. considering a scenario with an 80% airport exit screening success rate, 80% airport entry screening success rate and 80% symptomatic case tracing success rate, the combined intervention strategy results in: a 2% reduction in car if the vaccination campaign starts in mid-november; 11% reduction if the campaign starts in mid-october; 26% reduction if the campaign starts in mid-september; and 37% reduction if the campaign starts in mid-august. in retrospect, the taiwanese government's response to h1n1pdm proved to be effective. first and foremost, it initiated enhanced border monitoring and on-board quarantine inspection as soon as the threat of a flu pandemic became clear. at the same time, the domestic preparations towards h1n1pdm were escalated, such as antiviral drug stockpiling and distribution, and vaccine acquisition. as the h1n1 cases increased worldwide, various revised plans were adopted and implemented; such as the shift from labor-extensive on-board quarantine inspection to the notifiable infectious disease reporting system and realtime outbreak and disease surveillance system in order to effectively track down symptomatic and exposed passengers, apply prophylaxis treatment and mandatory in-home quarantine. as a result, all h1n1pdm related statistics are well below the international average. in modern society, countries rely heavily on the global economy for their own prosperity. shutting down the border for any length of time is not only costly, but could have disastrous economic effects that linger long after the event is over. moreover, with nearly 50% of the infected passengers presenting no symptoms whatsoever, they are not detectable by any port authority's screening procedures, and the importation of the novel flu virus is therefore inevitable. many studies conclude that entry screening is unlikely to be effective in preventing or delaying the importation of influenza, and has negligible impact on the course of subsequent epidemic. however, these studies are based on the assumption that effective exit screening is in place. our study shows that as the exit screening success rate decreases, the sensitivity of the entry screening policy becomes more pronounced. with the same methodology, we can also study the effects of varying the length of flight time, or the disease's incubation time. lastly, the benefit of entry screening is even more crucial for a small island country such as taiwan, since all incoming traffic must go through the port authority where entry screening can be enforced. in england and wales, three waves of the pandemic struck in summer, autumn, and winter seasons of 1918-1919. although the proportion of people reporting symptoms was often greater in the first wave, 1-3 a puzzling feature was the much higher mortality in the second wave, in which 0.27% of the population died, compared with 0.03% in the out-of-season first wave and 0.10% in the third wave. 4 an obvious hypothesis to explain the changes in mortality from wave to wave would be that the 1918 virus mutated to higher virulence after the (lower mortality) first wave. although pandemic virus reconstituted from the high mortality waves has proven to have high virulence in animals, 5 it has not been possible to recover virus from the first wave in 1918 for comparative purposes. indeed it is questionable whether virulence mutation(s) occurring between wave 1 and wave 2 could have spread to so many different populations in the time-frames observed. furthermore, in all three pandemic waves, there was the same agedistribution of mortality, with more deaths occurring amongst younger adults than older adults. [1] [2] [3] this 'pandemic signature', arguably due to immune protection of older adults who were exposed to a similar virus in the years before 1890, 6, 7 suggests that the 1918-1919 viruses were at least immunologically similar in all three waves. a second hypothesis would be that the higher case fatality in the later waves was due to higher rates of complicating bacterial pneumonia, 8 to increased transmission of influenza virus in the cooler months of the year, or to other seasonal effects. 9 we have considered a third (immunological) hypothesis to explain the greatly increased mortality in waves 2 and 3. the underlying idea is that the mortality rate in the first wave was lower than in later waves because most persons were protected by prior immunity in the first wave, and that the mortality was higher in later waves because of waning of that short-lived immunity. this hypothesis builds on our earlier modelling papers suggesting that even before the first wave in 1918, military, 10 school, and urban 11 populations in england and wales apparently had (short-lived) immune protection, presumably induced by recent prior exposure to seasonal influenza. [10] [11] [12] we suggest that this short-lived strain-transcending protection was in addition to the longer-lasting immunity, presumably induced by exposures to a similar virus circulating prior to 1890, that arguably reduced pandemic mortality for older adults in 1918-9. 6,7 cumulative mortality rates attributed to pandemic influenza were available for each of the three waves in 1918-1919 for 330 populations in england and wales. 13 we have built immunological models to potentially explain the variation in mortality rates across waves and populations. to show proof of principle, we have fitted these models to mortality data from a randomly selected sub-set of twenty populations. our key assumption was that the risk of a fatal infection would be limited to persons with inadequate immunity who were being exposed to the pandemic virus for the first time. persons who were exposed and who survived an earlier wave were assumed to be protected against death in a later wave. model a and assumptions (see figure 1) before the first wave, we assumed that people could be fully susceptible (s 0 ), or partially protected (q 0 ), or fully protected (p) by prior immunity which was not necessarily specific for the new virus. we assumed that exposure to the new pandemic virus would be fatal (m) in a proportion h of fully susceptible persons who were actually exposed (e) in the relevant wave. for those surviving that first exposure, it was assumed that they would be permanently protected against death in later waves by an immune assumed that viral exposure and multiplication would induce an immune response specific for the pandemic virus that would protect them against death in that wave and in subsequent waves. in contrast, for persons with strong prior immune protection, p, the virus would not be able to multiply to induce pandemic-specific immune protection. between waves, it is assumed that due to the waning of non-specific prior immunity, persons in the p state can move to the q state, and persons in the q state can move to an s state before the next wave. the proportion (e) of susceptible persons exposed to productive infection in each population was estimated by applying the following version of the final size equation 11 to the proportion susceptible (s & q) in each wave, for each population: note: in both figures 1 and 2 , we have omitted the flows out of the q and e states that removed persons from the risk of death. parameters: s 0 = proportion fully susceptible to infection and death before wave 1; q 0 = proportion susceptible to immunising infection, but not to death from exposure in wave 1; p 0 = proportion temporarily protected against both immunising infection and death from exposure in wave 1; n 0 = proportion even more protected against both immunising infection and death from exposure in wave 1 (model b only); r 0 = basic reproduction number (the average number of secondary cases for each primary case) in a fully susceptible population; f = proportion moving from q to s between waves; g = proportion moving from p to q between waves; d = proportion moving from n to p between waves (model b); h = proportion of e that actually move to m and die. model a could provide a very good fit for the summer, autumn, and winter waves of the 1918-1919 pandemic (results not shown). however, because of the replenishment of the pool of susceptible persons over time, model a also predicted a fourth wave of influenza in the spring season of 1919. as no such wave was seen, and as we could not find parameters values for model a that did not predict a fourth wave, we must regard model a as inadequate. model b was similar to model a, but with an additional stage of prior immunity (n), which could wane to p. model b allowed us to not only fit the three observed waves, but also to fit the imputed data (zero cases) corresponding to the absent fourth wave. following earlier work, 10,11 we used a bayesian approach with markov chain monte carlo (mcmc) procedures to estimate model parameters, and we used hyper-parameters to allow for parameter variation between populations. 11 the initial conditions were specified by the parameters: p 0 , q 0 , s 0 and n 0 . from these and the other parameters, it was possible to simulate the behaviour of model a over three waves, and of model b over four waves, and to estimate the expected numbers dying in each wave in each population. we calculated the log likelihood of the observed numbers of deaths given the parameter estimates, and we used mcmc simulation to generate the posterior distributions of parameters. although we obtained an excellent fit between observed and expected numbers of deaths in each of the three waves for the 20 populations for model a, we could not find parameter values for model a that would fit the three observed waves without giving rise to a fourth wave in the spring of 1919. accordingly, in the modified model b, we allowed for an additional stage of prior immunity (figure 2) , and we fitted the model to the same data, plus imputed data corresponding to 'the absent fourth wave'. we obtained a very good fit to the three observed waves and the absent fourth wave in each population. the 95% credibility intervals for parameter estimates, derived from the posterior distributions of the hyper-parameters were: h = 0.05-0.08, s 0 = 0.015-0.032, q 0 = 0.40-0.47; n 0 = 0.2 (fixed); p 0 = 1 ) s 0 ) q 0 ) n 0 ; f = 0.33-0.44; g = 0.87-0.94; d = 0.94-0.97 and r 0 = 2.6-3.6. this analysis had allowed all parameters to vary from population to population under the constraints of the hyper-parameters. however, several of the biologically determined parameters might be expected to be more constant from population to population, whereas those dependent on mixing history and other social characteristics which vary more widely from population to population. to test this possibility, we fixed the mean values for the more biological parameters (f = 0.385; d = 0.955; g = 0.905) and estimated the 95% credibility intervals for the others as: h = 0.05-0.10; s 0 = 0.012-0.032, q 0 = 0.36-0.44; and as before n 0 = 0.2 (fixed); in a subsequent paper we will be able to provide more details of the method, the robustness of the assumptions, and the results from fitting to many more populations. this short report suggests that the observed patterns of mortality in england and wales over the three waves of the 1918-1919 influenza pandemic 4,13 can be explained by an immunological model. in particular, the lower mortality in wave one can be explained by the assumption of protective immunity antedating the first wave, arguably induced by prior exposure to seasonal influenza. 10, 11 the much greater mortality in wave two can be explained by the waning, between wave one and wave two, of that short-lived and less-specific immune protection. the somewhat lesser mortality in wave three and the 'absent fourth wave' can be explained in terms of the progressive acquisition of immunity specific to the pandemic virus. the credibility estimates for parameters are of potential interest. for example, r 0 estimates of 3.1-4.5 across different populations are consistent with our earlier findings. 10, 11 if all persons had been susceptible, such r 0 values imply that the virus would have infected most people in all populations. however, even in the first wave, the proportion susceptible, s 0 + q 0 , was <50% in all populations, so that a considerable number of persons escaped productive infection in that wave; as their immunity waned, they became susceptible to infection in the later waves. it is likely that the variation in r 0 between populations is due to different rates of population mixing. estimates for h indicate that between 5% and 10% of infections in the most susceptible persons were fatal; the higher values of h could reflect higher rates of secondary bacterial infection in the most socially disadvantaged and overcrowded populations. 4 although we have shown the plausibility of an immunological explanation for wave to wave changes in pandemic mortality, we cannot assume that our particular model is even approximately correct. nor can we exclude the possibility that the higher mortality in the later pandemic waves in 1918-1919 was because of genetic change in the virus in later waves, or because of changing rates of secondary bacterial infection 8 or seasonal effects. 9 nevertheless, there is growing evidence that the population spread of pandemic influenza, whether in 1918-1919 10, 11 or in 2009, 12, 14 can be constrained by significant prior immunity, even for viruses that are ostensibly novel. previous reports, reviewed in ref. [10, 11] , support the idea of strain-transcending immune protection, which can wane over periods of a few months. this form of protection, probably induced by recent exposure to seasonal influenza, may not be mediated by hi or neutralizing antibody. 11 in contrast, strain-specific immunity, most often mediated by hi or neutralizing antibodies can be so long-lasting that after several decades it will still provide significant protection against any closely-related virus that re-appears in the population. 14 it has not escaped our notice that although attack-rates in the h1n1 2009 pandemic were low in many countries, with generally mild symptoms, the virus did cause lifethreatening illness in a small proportion of younger affected persons. 15 it seems likely that those who were most severely affected in 2009 were doubly unlucky: they had missed out on seasonal influenza infection or vaccination in the preceding season(s), and they were born too late to have been protected by the closely-related viruses that are thought to have circulated before 1950. 14 during the early phases of the 2009 influenza pandemic in italy, real-time modeling analysis were conducted in order to estimate the impact of the pandemic. in order to evaluate the results obtained by the model we compared simulated epidemics to the estimated number of influenza-like illness (ili) collected by the italian sentinel surveillance system (influnet), showing a good agreement with the timing of the observed epidemic. by assuming in the model mitigation measures implemented in italy, the peak was expected on week 44 (95% ci: 44, 45). results were consistent with the influnet data showing that the peak in italy was reached in week 46. these predictions have proved to be a valuable support for public health policy makers for planning interventions for mitigating the spread of the pandemic. mathematical models have recently become a useful tool to analyse disease dynamics of pandemic influenza virus can-didates. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] as of april 2009, after the pandemic threat emerged worldwide, 12 it was crucial for policy makers to have early predictions on the possible spread of the 2009 pandemic influenza virus in order to support, with quantitative insight into epidemic, policy decisions. thus, after the first pandemic alert was announced by the world health organization (who) in late april 2009, a national crisis management committee headed by the minister of health was established in italy in order to provide weekly advice to the italian ministry of health. real-time analyses using an individual based model were undertaken. the transmission model was previously used for evaluating the effectiveness of the control measures adopted in the national pandemic preparedness plan 3 and for assessing the age-prioritized distribution of antiviral doses during an influenza pandemic. 2 to parameterize the transmission model, we used data derived from the national surveillance system until 17 june 2009 and estimates of key epidemiological parameters as available at that time. in order to provide a preliminary assessment of the model predictions performed during the early stages of the epidemic, we compare model predictions with surveillance data of influenza-like illness (ili) available since august 2009. after the first pandemic alert was announced by the who in late april 2009, a national active surveillance system for the 2009 pandemic influenza was set up from 28 april to july 2009. 13 however, over the period from april to october 2009, surveillance systems, laboratory testing, and diagnostic strategies have varied considerably in italy. since end of july 2009, following who recommendations, 14 the focus of surveillance activities has changed in reporting requirements, as active case-finding became unsustainable and unnecessary. for this reason, the ministry of health (ministry of health, available in italian at the website: http://www.normativasanitaria.it) requested regional health authorities to report the weekly aggregated ili cases according to a new case definition (sudden onset of acute respiratory symptoms and fever >38°c plus at least one of the following systemic symptoms: headache, malaise, chills, sweats, fatigue; plus at least one of the following respiratory symptoms: cough, sore throat, nasal obstruction). by october 2009, following the increasing number of cases, the sentinel influenza surveillance system (influ-net available at: http://www.flu.iss.it) became the official surveillance system for ili cases in italy (ministry of health, available in italian at the website: http://www. normativasanitaria.it). since 1999, influnet is routinely based on a nation-wide, voluntary sentinel network of sentinel community based physicians in the 21 regions and autonomous provinces of the country. incidence rates are, therefore, not based on consultations, but on the served population of each reporting physician each week. influ-net usually consists of an average of 830 (range 648-902) general practitioners (including physicians and pediatricians) per year, covering about 1ae5-2% of the general population, (representative for age, geographic distribution, and urbanization level) reporting ili cases (according with a specific case definition). italian influnet surveillance system is part of the european influenza surveillance scheme (eiss). a stochastic, spatially explicit, individual-based simulation model 3 was used. individuals are explicitly represented and can transmit the infection to household members, to school ⁄ work colleagues, and in the general population (where the force of infection is assumed to depend explicitly on the geographic distance). the national transmission model was coupled with a global homogeneous mixing susceptible-exposed-infectious-removed (seir) model accounting for the worldwide epidemic, which is used for determining the number of cases imported over time. regarding the epidemiological assumptions (e.g., length and shape of the infectivity period, which lead to an effective generation time of 3ae2 days), this study is consistent with refs [3, 4, 11, 15] , but for the proportion of symptomatic individuals, which is assumed to be 66ae7%. 16 the basic reproductive number of the national transmission model was set to 1ae4, according to the early estimates as obtained during the initial phase of the epidemic in mexico in a community setting. 17, 18 we initialized our simulations through the global homogeneous mixing model in such a way that 158 imported cases were generated until 7 june 2009. this gives a reliable way for fixing the time in the simulations and thus determining the timing of school closure and vaccination in the simulations. the model accounts for school closure for both summer and christmas holidays: we assumed that in these periods contacts among students decrease, while contacts in the general community increase, as in ref. [19] . we also considered scenarios accounting for partial immunity in the population. 20 in order to investigate the effects of recommendations of the ministry of health (confirmed cases coming from affected areas were isolated for 7-10 days, either in hospital or at home) established in the early phase of the pandemic (april-july 2009), we assumed that a fraction of the imported symptomatic cases were isolated on the first day after the symptoms onset. this recommendation was in place until 27 july 2009. we also assumed, according to the italian school calendar, that schools were closed from 10 june 2009 to 10 september 2009 for the summer holidays, and from 22 december 2009 to 6 january 2010 for christmas holidays. the effects of prolonged school closure were also investigated. when considering vaccination, we assumed 6 weeks for the logistical distribution of doses of pandemic vaccine. since at the time of simulation specific recommendations regarding the administration of a single dose of pandemic vaccine from ema were not available yet, we considered the administration of 2 vaccine doses 1 month apart). the pandemic vaccine was considered effective after the administration of the second dose with a vaccine efficacy of 70%. we assumed the vaccine to be administered by priority, vaccinating first the target population accounting for essential services workers (including health care workers and blood donors), pregnant women at the second or third trimester, and at risk patients (with chronic underlying conditions) younger than 65 years old. the vaccination coverage was assumed 90%. regarding antiviral treatment and prophylaxis, recommendations of the ministry of health in the initial phase of the epidemic were to administer antivirals to all confirmed cases and to their close contacts. we assumed that the surveillance system would be able to detect 90% of symptomatic cases. after 8 july 2009, recommendations changed and antiviral treatment was considered only for cases with severe complications and in case of local clusters. since it was difficult to establish the proportion of treated cases, we considered different scenarios: antiviral treatment from 0% to 30% of the symptomatic cases. consistently with ref. [3] , both treatment and prophylaxis were assumed to start 1 day after the clinical onset of symptoms in the index case. treatment was assumed to reduce infectiousness by 70%, whereas antiviral prophylaxis was assumed to reduce susceptibility to infection by 30%, infectiousness by 70%, and the occurrence of symptomatic disease by 60%. as of 26 july 2009, approximately 1238 confirmed cases have been reported to the italian surveillance system for pandemic influenza. during july, the sudden increase of ili confirmed cases suggests for sustained autochthonous transmission in italy. by analyzing the number of ili cases reported to the surveillance during the weeks from 39 to 44, we found that the exponential growth rate was 0ae832 ⁄ week and thus we estimated the national reproductive number to be r 0 = 1ae38. this estimate of the basic reproductive number supports the choice of the value adopted in the model simulations (r 0 = 1ae4). in the absence of intervention measures, the predicted cumulative attack rate was 30ae6% (95% ci: 30ae6, 30ae7), and the peak was expected on week 42 (95% ci: 41, 42) with a peak day incidence of 0ae35% (95% ci: 0ae329%, 0ae37%). by assuming case isolation, antiviral treatment, and prophylaxis to 90% of symptomatic cases until 8 july 2009, the peak was expected on week 43 (95% ci: 43, 44). when considering 33ae3% of natural immunity in the population aged more than 59 years, the peak was expected 1 week later than in the previous scenario, i.e., on week 44 (95% ci: 44, 45). to validate the model, we compared model predictions (which are based only on the available information on the early phases of the epidemic) with ili data (figure 1 ). based on model predictions, we estimated the underreporting factor of influnet ranging from 3ae3 to 3ae7, considering different scenarios. by aligning the simulations with the ili data adjusted by the underreporting factor, we can observe that almost all the points in the increasing phase of the epidemic lie within the 95% ci of the model results (both considering or not natural immunity). the decay phase of the simulated epidemics shows a small delay with respect to the ili data. when introducing single and combined mitigation measures, such as case isolation, antiviral treatment, prophylaxis, and vaccination in the model, results showed that even a low proportion of symptomatic cases treated with antiviral drugs could have led to a relevant reduction in the epidemic size (table 1) . we simulated the planned italian vaccination strategy (begun on 15 october 2009), obtaining a limited but not negligible reduction in the attack rate with respect to the scenarios accounting only for antiviral treatment. moreover, the effect of vaccination would be higher if coupled with antiviral treatment; vaccination would have no effect on delaying the peak incidence. model predictions produced in italy during the early phase of the 2009 pandemic influenza are in excellent agreement with italian surveillance data on the beginning of the epidemic (when case isolation, antiviral treatment of index cases, and antiviral prophylaxis to close contacts were implemented by the italian regional public health authorities) and are basically consistent with the influnet data during the course of the epidemic. the model has been useful for predicting the timing of the epidemic, while it has overestimated the impact of the 2009 influenza pandemic for adult and elderly individuals. however, the disalignment is probably due to the model parameterization. based on literature values, 4,5 we assumed a similar fraction of cases in the different social contexts considered in the model (namely 1 ⁄ 3 in households, 1 ⁄ 3 in schools ⁄ workplaces, and 1 ⁄ 3 in the general community), since analysis on the relative transmissibility of the virus was not carried out for any country yet. we were also able to estimate an underreporting factor for the influnet data in the range 3ae3-3ae7. if we focus our attention on the reporting factor computed by considering the total number of cases (instead of symptomatic cases), the resulting value lies in the range 18-20ae2%, which is in excellent agreement with the range estimated in ref. [21] on previous a ⁄ h1n1 influenza seasons, namely 16ae2%-21ae6%. moreover, based on our results showing that vaccinating 40% of the italian population was more than adequate to mitigate the pandemic, the ministry of health decided to stockpile a limited number of vaccines. we have also shown that starting the vaccination program in october (or later) could have had only a limited effect on reducing the impact of the epidemic, although it may have been useful to prevent a possible second wave and to protect essential workers and at-risk patients. finally, our results have shown that antiviral treatment would have been the most efficient strategy to reduce the impact of the influenza pandemic, even with a limited antiviral stockpile. a population-wide passive immunotherapy program in this paper, we assume that convalescent plasma (cp) is efficacious in treating severe cases of pandemic influenza. under this premise, we test the hypothesis that a population-wide passive immunotherapy program that collects plasma from a small percentage of convalescent individuals can harvest sufficient cp to treat a substantial percentage of severe cases during the first wave of the pandemic. the proposed program involves recruiting adults (individuals age 20-55 years) to donate blood if they have experienced influenza-like symptoms more than 2 weeks ago (to account for the time needed for neutralizing antibodies to build up). the blood samples would be screened for infectious diseases (including hiv, hbv, hcv, htlv, and syphilis, etc., as in routine blood donation screening) and neutralizing antibodies against the pandemic virus. donors whose blood samples are free of known infectious agents and contain a sufficiently high titer of neutralizing antibodies would then be invited to donate plasma by plasmapheresis or routine whole blood donation. qualified donors with higher titers may be given higher priority for plasma donation. in this paper, we use the demographic and logistical parameters of hong kong as a case study. see figure 1 for a schematic of the proposed passive immunotherapy program. we examine the following questions regarding the logistical feasibility and potential benefits of the proposed passive immunotherapy program: (i) what percentage of convalescent individuals (donor percentage) is needed in order for the program to significantly reduce pandemic mortality? (ii) how many severe cases can be offered passive immunotherapy? (iii) what are the ratelimiting factors in the supply of passive immunotherapy? (iv) what are the epidemiologic and logistical factors that determine the demand-supply balance of passive immunotherapy? a more detailed presentation of our results is now available in ref. [ 5 ] . transmission and natural history model for pandemic influenza we use an age-structured disease transmission model to simulate the spread of pandemic influenza. the natural history model is similar to that used by basta et al. 6, 7 the most important parameter in characterizing the growth of an epidemic is the basic reproductive number r 0 , which is defined as the average number of secondary cases generated by a typically infectious individual in a completely susceptible population. we consider values of r 0 between 1ae2 and 2, which is consistent with recent estimates. 6, [8] [9] [10] logistical model for the passive immunotherapy program we assume that q d (%) of 20 to 55 year-old individuals who have recovered from symptomatic infections of pandemic influenza donate their blood for screening t r = 14 days after cessation of symptoms. follow-ups of convalescent individuals infected with h1n1pdm in an ongoing clinical trial of passive immunotherapy suggested that neutralizing antibodies level reaches maximal level around 14-21 days after recovery and stays at that level for months after. 11 we assume that q s (%) of these donors are qualified for plasma donation of which q r (%) are recurrent donors who return to donate plasma every t w = 14 days. screening involves both detection of infectious agents and neutralizing antibodies against the pandemic virus. the latter is the rate-limiting step because neutralization tests of pandemic viruses can only be done in a bsl3 setting. we assume that five bsl3-trained technicians are available to test the blood specimens, each running 150 viral neutralization tests in 3 days. therefore, the capacity and turnaround time of blood screening are u s = 750 and t s = 3 days, respectively. hong kong currently has nine plasmapheresis machines which allow a maximal throughput of 162 plasma donations per day (assuming 12-hour daily operation with each donation taking 40 minutes). therefore, the capacity and turnaround time of plasmapheresis are u p = 9 and t p = 1 ⁄ 18 days, respectively. collected cp are ready for use in transfusion after final quality check, which takes t q = 2 days. we assume that r t plasma donations are required to treat one severe case on average. the expert panel of the abovementioned study of passive immunotherapy for h1n1pdm in hong kong suggested that r t < 10. we assume that p h (%) of symptomatic cases will be severe cases for whom passive immunotherapy is suitable. although p h will be smaller than the case-hospitalization rate (passive immunotherapy may not be suitable for some hospitalized cases), we assume that the two have similar ranges and consider p h ranging from 0ae1% to 1%. because each severe case requires r t plasma donations on average, demand for cp is simply r t p h times the number of symptomatic cases. therefore, r t p h can be regarded as a single parameter, which we refer to as the lumped demand parameter. we define the outcome as the percentage of severe cases that can be offered passive immunotherapy by the proposed program during the first wave of the local epidemic. we refer to this outcome as treatment coverage and denote it by q. we consider the base case scenarios assuming q r = 20% and q s = 80%. in general, the treatment coverage q increases sharply as the basic reproductive number r 0 and the lumped demand parameter r t p h decrease (figure 2a ). in particular, when r 0 is large and r t p h is small, q is very sensitive to r t p h , but insensitive to r 0 . similarly, when r 0 and r t p h are small, q is very sensitive to both. with a donor percentage of q d = 15%, the proposed program can supply passive immunotherapy to more than 82% of severe cases (q > 82%) if r 0 < 1ae4 and r t p h < 1ae5%, but <35% if r 0 > 1ae8 and r t p h > 1ae5%. in general, the treatment coverage q increases sharply as the donor percentage q d rises from 0%, but with rapidly decreasing marginal increase ( figure 2b ). when r 0 < 1ae4 and r t p h < 1ae5%, q > 67% even if q d is as low as 5%, which is comparable to the current average blood donation rate of 38ae1 donations per 1000 population in developed countries. 12 when q d is >15%, q becomes largely insensitive to further increase in q d in most scenarios. the treatment coverage q for q d = 15% is more than 81% that for q d = 50% across all values of r 0 and r t p h considered in the base case. therefore, increasing the donor percentage q d beyond 15% has a relatively small impact on cp supply. this is because increasing q d can boost supply only when plasmapheresis is not yet the supply bottleneck. for the same reason, once the donor percentage q d has reached 15%, the treatment coverage q is insensitive to further increase in q d even when the plasmapheresis and screening capacity are doubled ( figure 2b , lower panel). we conduct an extensive multivariate sensitivity analysis to test the robustness of our base case observations against uncertainties in parameter values. we generate 15 000 epidemic scenarios by randomly selecting parameter values from their plausible ranges using latin-hypercube sampling. although there are numerous model parameters, the treatment coverage q is mainly determined by three lumped parameters: (i) r t p h , which indicates the magnitude of demand; (ii) q s q d , which indicates the magnitude of supply; (iii) the initial growth rate of the epidemic r (results not shown). while the dependence of q on r t p h and q s q d is readily comprehensible, it is not obvious a priori that q depends on the natural history and transmission dynamics of the disease via only the initial epidemic growth rate. when the plasmapheresis and screening capacity are very large, the supply-demand dynamics is further simplified: the treatment coverage q depends on lumped demand parameter r t p h and the lumped supply parameter q s q d only via their ratio. finally, q becomes insensitive to q s q d when the latter increases beyond 15-20%, which is consistent with our base case observations. our results suggest that with plasmapheresis capacity similar to that in hong kong, the proposed passive immunotherapy program can supply cp transfusion to treat 67-82% of severe cases in a moderate pandemic (basic reproductive number r 0 < 1ae4, lumped demand parameter r t p h < 1ae5%) when the donor percentage is 5-15%. increasing the donor percentage beyond 15% has little additional benefit because cp supply is constrained by the capacity of plasmapheresis during most stages of the epidemic. increasing plasmapheresis capacity could significantly boost cp supply, especially when there is a substantial pool of recurrent donors to alleviate the dependence of cp supply on donor percentage. in an ongoing clinical trial of passive immunotherapy for h1n1pdm virus infection in hong kong, 20% of convalescent individuals agreed to donate their plasma for the study. therefore, the donor percentage required by the proposed passive immunotherapy program (5-15%) is likely to be feasible. in view of the logistical feasibility of such program, we recommend that further clinical studies are conducted to evaluate the safety and efficacy of passive immunotherapy as a treatment for severe cases of pandemic influenza virus infection. our study is based on the premise that cp will be efficacious in reducing morbidity and mortality associated with pandemic influenza. in theory, the polyclonal nature of neutralizing antibodies in cp would lower the probability of an escape mutant emerging in treated patients. further, besides providing neutralizing antibodies against the pandemic virus, cp also might carry antibodies to other bacterial pathogens, which might decrease the severity of coexisting bacterial infections. 4 as such, cp not only might reduce the case fatality rate but might also increase the recovery rate and shorten duration of hospitalization of severe cases. the proposed passive immunotherapy program can thus significantly reduce the burden on the healthcare system, especially the intensive care unit, which will likely be stressed, if not overloaded, at the peak of an influenza pandemic wave, hence benefiting the general public and not only those receiving passive immunotherapy. although the hypothesized efficacy of cp has yet to be proven in clinical trials, our modeling results show that a public health system similar to that in hong kong has the capacity to support a population-wide passive immunotherapy program that can supply cp treatment to a substantial percentage of the severe cases in a moderately severe pandemic. we estimate that compared to other developed countries, hong kong has a relatively low plasmapheresis capacity. our conclusions regarding donor percentage needed and rate-limiting factors remain valid for plasmapheresis capacity ranging from 50% to 400% of what we have assumed in the base case (results not shown). our conclusions are robust against uncertainties in the natural history and transmission dynamics of pandemic influenza. our sensitivity analysis shows that the outcome depends on these epidemiological characteristics only via the initial growth rate of the epidemic. as such, our results are applicable not only to pandemic influenza, but also to other emerging infectious diseases for which the time-scales of disease transmission and antibody response are similar to that for influenza virus. the three determinants of treatment coverage (the initial epidemic growth rate, the lumped demand parameter r t p h , and the lumped supply parameter q d q s ) are all readily measurable in real-time during an epidemic. therefore, our methods and results can be used as a general reference for estimating the treatment coverage of the proposed passive immunotherapy program for a given plasmapheresis capacity. background highly pathogenic h5n1 virus continues to pose a serious threat to human health and appears to have the capacity to cause severe disease in previously healthy young children and adults. at present, antiviral therapy by oseltamivir remains the mainstay for managing h5n1 patients. while early treatment improves survival, approximately 50% of patients treated within 4 days of illness still succumb to the disease. in addition to the role of viral replication, there is good evidence that the host proinflammatory responses contributes to h5n1 pathogenesis. this suggests that both antiviral and immune-modulatory drugs may have a role in therapy. we previously demonstrated that cyclooxygenase 2 (cox-2) plays a regulatory role in h5n1 hyperinduced pro-inflammatory responses, and its inhibitor has potent effects at modulating this host response. now we demonstrate that, in addition to its immune-modulatory effect, a selective cox-2 inhibitor, ns-398 has a direct antiviral effect against h5n1 infection. materials and methods human primary monocytederived macrophages or alveolar epithelial cells (a549) were pre-treated with ns-398 or drug-vehicle for 1 hour before h5n1 virus infection. h5n1 viruses at multipicity of infection (moi) of 2 was used to infect the cells. following virus adsorption for 30 mins, the virus inoculum was removed, and the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls for 3, 6, 24, 48, and 72 hours post-infection. cells were harvested for rna isolation at 6 hours post-infection to study viral matrix (m) gene expression. supernatants were collected for 50% tissue culture infection dose (tcid 50 ) assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. results ns-398 was found to suppress virus gene transcription and infectious virus yield in h5n1-infected human cells. conclusion we demonstrate that a selective cox-2 inhibitor, ns-398, shows an inhibitory effect on h5n1 viral replication in addition to its immune-modulatory effect that could counter the detrimental effects of excessive proinflammatory cytokine production. the findings suggest that selective cox-2 inhibitors may be a therapeutic target for treating h5n1 disease in combination with appropriate antiviral therapy. the emergence and spread of the highly pathogenic avain influenza viruses (h5n1) in poultry and wild birds with repeated zoonotic transmission to humans has raised pandemic concern. at the time of writing, 507 human cases have been reported with 302 fatalities, an overall case fatality rate of around 60% (cumulative number of confirmed human cases of avian influenza a ⁄ (h5n1) reported to world health organization updated to 18 october 2010). our previous data demonstrated that cox-2 was markedly up-regulated in h5n1-infected primary human macrophages, and that it played a regulatory role in the h5n1hyperinduced host pro-inflammatory responses. 1 such cytokine dysregulation is proposed to be a major contributor to the pathogenesis of h5n1 disease in humans. 2 with the use of selective cox-2 inhibitors, we found that the h5n1-hyperinduced cytokine response was significantly suppressed by the drug in a dose-dependent manner. 1 selective cox-2 inhibitor is a form of a non-steroidal anti-inflammatory drug that selectively targets cox-2, and it is an inducible enzyme responsible for inflammatory process and immune response. here, we report a novel finding of a direct antiviral effect of a selective cox-2 inhibitor, ns-398, against h5n1 infection in human primary macrophages and alveolar epithelial cells. taken together with our previous findings that suggest an immuno-modulatory effect that can modulate virus driven cytokine dysregula-tion, these findings highlight a role for cox-2 and its downstream signaling as potential novel targets for adjunctive therapy of severe viral pneumonia, such as that caused by h5n1. such therapy may be combined with conventional antiviral drugs. the h5n1 virus used was a ⁄ vietnam ⁄ 3212 ⁄ 04 (3212 ⁄ 04) (h5n1), a virus from a patient with h5n1 disease in vietnam during 2004. the viruses were grown and titrated in madin-darby canine kidney cells cells as described elsewhere. 3 virus infectivity was expressed as tcid 50 . all experiments were performed in a biosafety level 3 facility. monocyte-derived macrophages: peripheral-blood leucocytes were separated from buffy coats of healthy blood donors (provided by the hong kong red cross blood transfusion service) by centrifugation on a ficoll-paque density gradient (pharmacia biotech) and purified by adherence as reported previously. 3 the research protocol was approved by the ethics committee of the university of hong kong. macrophages were seeded onto tissue culture plates in rpmi 1640 medium supplemented with 5% heat-inactivated autologous plasma. the cells were allowed to differentiate for 14 days in vitro before use in the infectious experiments. alveolar epithelial cells: a549 cells were obtained from atcc and maintained in culture using dulbecco's modified eagle medium supplemented with 10% fetal calf serum, 0.6 mg ⁄ l penicillin, and 60 mg ⁄ l streptomycin. differentiated macrophages or a549 cells were pre-treated with a selective cox-2 inhibitor, ns-398 (cayman), at concentrations as indicated or drug-vehicle for 1 hour before infection. cells were infected with h5n1 viruses at moi of 2. following virus adsorption for 30 min, the virus inoculum was removed, the cells were washed and incubated in corresponding medium with ns-398 or drug-vehicle as controls throughout the experiments. cells were harvested for rna isolation at 6 hours post-infection to study viral m gene expression. supernatants were collected for tcid 50 assay to determine the virus titers at 3, 24, 48, and 72 hours after h5n1 infection. total rna was isolated using the rneasy mini kit (qiagen) according to the manufacturer's instructions. the cdna was synthesized from mrna with poly(dt) primers and superscript iii reverse transcriptase (invitrogen). transcript expression was monitored by real-time pcr using power sybr ò green pcr master mix kit (applied biosystems) with specific primers. the fluorescence signals were measured using the 7500 real-time pcr system (applied biosystems). the specificity of the sybr ò green pcr signal was confirmed by melting curve analysis. the threshold cycle (ct) was defined as the fractional cycle number at which the fluorescence reached 10 times the standard deviation of the base-line (from cycle 2 to 10). the ratio change in target gene relative to the b-actin control gene was determined by the 2 )ddct method as described elsewhere. 4 ns-398 reduced the viral m gene expression in h5n1infected human macrophages in a dose-dependent manner ( figure 1) . similarly, production of infectious virus yield in h5n1 infected macrophages was found to be suppressed in the presence of ns-398 at 100 lm compared to vehicletreated cells (figure 2a) . a comparable effect of ns-398 was observed in h5n1-infected human alveolar epithelial cells ( figure 2b ). we have previously demonstrated that cox-2 expression was dramatically upregulated following h5n1 infection in human macrophages in vitro and in epithelial cells of lung tissue samples obtained from autopsy of patients who died of h5n1 disease. 1 this suggests that cox-2 may be an important host factor involved in h5n1 pathogenesis and also provide a possible explanation on why h5n1 virus replication is susceptible to a selective cox-2 inhibitor. cox-2 was previously reported to play an important role in the pathogenesis of other influenza a viruses. 5 an in vivo study has highlighted the importance of cox-2 in h3n2infected mice. findings showed that infection induced less severe illness and reduced mortality in cox-2 knock-out mice than in wild-type mice. on the other hand, cox-1 knock-out mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the bal fluid, whereas the inflammatory and cytokine responses were dampened in cox-2 knock-out mice. these data suggests that cox-1 and cox-2 may lead to opposite totally contrasting effects on influenza h3n2 infected mice. cox-1 deficiency is detrimental, whereas cox-2 deficiency is beneficial to the host during influenza viral infection. therefore in the present study, instead of blocking cox enzymes in general as reported by others, 5 we have chosen ns-398 that selectively block cox-2 but preserve cox-1 activity and showed that this drug significantly reduced h5n1 virus replication in a dose-dependent manner. taken together with our previous report suggesting its immuno-modulatory effects, 1 we believe that selective cox-2 inhibitors and cox-2 signaling pathways deserve investigation as a promising approach for targeting therapy in h5n1 diseases. however, a few reports have suggested the importance of cox-2 in the late stage of inflammation for the resulution of inflammation, [6] [7] [8] and this raises concern whether inhibition of cox-2 may be harmful in treating diseases related to dysregulation of host inflammatory response such as acute lung injury, 9 which is a leading cause of death in h5n1 patients. we previously looked at the autopsy samples of lung tissues from h5n1 patients and found that cox-2 expression was markedly up-regulated compared with that from persons who died of non-respiratory causes. 1 moreover, data also demonstrated that pro-inflammatory cytokines, such as tnf-a, was markedly elevated in the h5n1 infected lung autopsies. 10 taken together, with the histo-pathological findings, which showed predominant features of exudative inflammatory phase in autopsy lung samples from h5n1 patients, 11, 12 we may therefore speculate that people who had fatal h5n1 infection died during acute inflammation phase, and before the resolution could occur, especially for the cases with a short disease duration (<10-12 days). 13 in conclusion, the roles of cox-2 in both pro-inflammation and pro-resolution phases deserves detailed investi-gation. the timing of selective cox-2 inhibitor therapy in h5n1 infected patients may be extremely critical. therefore a time-dependent study using selective cox-2 inhibitors on h5n1-infected animal models will be particularly important in order to address the effectiveness of this drug in treating h5n1 disease. avian antibodies to combat potential h5n1 pandemic and seasonal influenza highly pathogenic avian influenza a virus (hpaiv) strain a ⁄ h5n1 with unprecedented spread through much of asia and parts of europe in poultry remains a serious threat to human health. passive immunization (transfer of protective immunoglobulins) offers an alternative and ⁄ or additional strategy to prevent and cure influenza. here, we report that virus-specific immunoglobulin y (igy) isolated from eggs of immunized hens provide protection in mice against lethal h5n1 virus infection by neutralization of the viruses in the lungs upon intranasal administration. importantly, chicken eggs obtained from randomly selected supermarkets and farms in vietnam, where mass poultry vaccination against a ⁄ h5n1 is mandatory, contain high levels of igy specific for a ⁄ h5n1 virus. when administered before or after the infection, igy prevented and significantly reduced replication and spread of hpaiv h5n1 and related h5n2 strains. thus, the consumable eggs readily available in markets of countries that impose poultry vaccination against a ⁄ h5n1 could offer an enormous source of valuable biological material that provides protection against a ⁄ h5n1 virus with pandemic potential. the approach could be used to control seasonal influenza. since 2004, hpaiv of the h5n1 subtype has resulted in more than 430 cases of laboratory-confirmed human infection in 15 countries with a death rate of more than 50% (http://www.who.int/csr/disease/avian_influenza/). h5n1 influenza virus remains a global threat because of its continued transmission among domestic poultry and wild birds. passive immunization (the transfer of antigen-specific antibodies (abs) to a previously non-immune recipient host) offers an alternative and ⁄ or additional countermeasure against influenza. 1 development of human monoclonal antibodies (mabs) against h5n1 influenza haemagglutinin (ha) using epstein-barr virus (ebv) immortalization of b cells isolated from patients infected with h5n1, 2 phage display, 3 humanized mabs, 4 and human recombinant abs 5 has been attempted. chickens produce a unique immunoglobulin molecule called igy that is functionally equivalent to mammalian igg. 6 igy is found in the sera of chickens and is passed from hens to the embryo via the egg yolk. 7 egg igy has been used to prevent bacterial and viral infections (see review 8 ) of the gastrointestinal tract and recently for protection against pseudomonas aeruginosa infection of the respiratory tract of patients with cystic fibrosis (cf). 9 the epidemic of hpaiv h5n1 virus has resulted in serious economic losses to the poultry industry, mostly in southeast asia. therefore, many countries including china, indonesia, thailand, and vietnam have introduced mass vaccination of poultry with h5n1 virus vaccines that controls the h5n1 epidemic to some extent. 10 chickens immunized with recombinant h5 and ⁄ or inactivated h5n1 reassortant vaccines produced a high level of virus-specific serum antibodies (abs) and were protected from h5n1 virus challenge. 11 theoretically, these abs could be found in egg yolk and separated for use in humans to prevent and cure h5n1 hpaiv infection and disease, respectively. here, we examined the possibility that igy isolated from consumable eggs available in supermarkets in vietnam, where mandatory h5n1 vaccination has been implemented, provide prophylaxis and therapy of hpaiv h5n1 infection in mice. six-to 8-week-old female balb ⁄ canncrl (h-2d) mice (charles river and jackson laboratory) and hy-line . igy abs were extracted from egg yolks as previously described. 12 the 50% egg infectious dose (eid 50 ) was determined by serial titration of virus stock in eggs, and eid 50 ⁄ ml values were calculated according to the method of reed and muench. 13 human virus stocks were grown in mdck cells as described previously ,14 with viral titers determined by standard plaque assay. the 50% tissue culture infectious dose (tcid 50 ) of virus was determined by titration in mdck cells. 15 the standard elisa was performed for detection of anti-igy in the sera of igy-immunized mice. fifty percent lethal dose (ld 50 ) titers were determined by inoculating groups of eight mice i.n. with serial 10-fold dilutions of virus as previously described. 16 for infection, ketamine-anesthetized mice were inoculated intranasally with a lethal dose with 250 pfu (5 · ld 50 ) of a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) virus as previously described, 17 10 · ld 50 of vn ⁄ 1203 (h5n1) or 5 · ld 50 a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2) resuspended in 50 ll pbs per animal. ketamine-anesthetized mice were treated intranasally with 50 ll of igy before or after infection. mice were observed for weight loss and mortality. subsets of animals were scarified for virus titre. we found comparable hai titers in the sera and egg yolks obtained from a farm in vietnam that was participating in a national mass vaccination program. furthermore we found 90% of eggs purchased in randomly selected supermarkets in hanoi, vietnam containing h5-specific igy. the hai and vn titers of pooled egg yolk igy are comparable with those of sera obtained from hens selected randomly from the farm that underwent supervised h5n1 vaccination. in contrast, igy separated from eggs purchased in korean markets where poultry are not vaccinated against avian influenza h5n1 has no detectable h5-specific hai or vn activity. we first treated naïve mice intranasally with h5n1-specific igy before infection with hpaiv h5n1 strain, a ⁄ vietnam ⁄ 1203 ⁄ 2004, isolated from a fatal case. such treated mice displayed mild weight loss and recovered completely by the end of the first week after inoculation ( figure 1a ). when animals were treated once with h5n1specific igy after h5n1 inoculation they exhibited minimal weight loss during the first week after inoculation, and virus titers in the lungs were substantial reduced at day 3 after infection; however, 50% of treated mice succumbed to infection during the second week after inoculation ( figure 1b) . it is possible that not all the hpaiv a ⁄ h5n1 viruses were neutralized upon the single treatment with igy, and escaping viruses can spread systemically to organs outside of the lungs. these viruses may reappear in lung tissue later when specific igy is absent. indeed, vn ⁄ 1203 virus injected intravenously or into the brain can spread to the lungs. 18 to circumvent the virus escape, we administered multiple treatments with h5n1specific igy after the infection. as a result, all infected mice recovered completely by the second week post-infection ( figure 1c) , and virus titers in the lungs were substantially reduced to the level that seen in protected mice that received single prior-infection treatment ( figure 1d) . similarly, the protective efficacy of h5n1-specific igy was observed in mice infected with lethal dose of mouseadapted avian influenza virus strain a ⁄ aquatic bird ⁄ korea ⁄ w81 ⁄ 2005 (h5n2). this virus shares 94.4% nucleotide sequence homology with ha (h5) but has different na (n2) from the one used for mass immunization in vietnam (reassortant avian h5n1 influenza virus a ⁄ goose ⁄ gd ⁄ 96-derived, strain re-1). the results indicate that h5n1-specific igy isolated from eggs purchased in markets have preventive and therapeutic effects against infection with hpaiv h5n1 and the related strain h5n2. the findings suggest that while a single treatment with igy prior to lethal infection was sufficient to protect the animals from the infection, multiple treatment is required for complete therapeutic effect after infection with hpaiv such as vn ⁄ 1203 strain. we further examined the protective efficacy of igy isolated from eggs laid by hens immunized in the laboratory with heat-inactivated human influenza a ⁄ h1n1 virus, a ⁄ pr ⁄ 8 ⁄ 34. we found substantial levels of hai and vn abs in the sera and yolks derived from immunized hens. when naïve mice were administered intranasally with such anti-pr ⁄ 8 igy at 6-8 hours before or after infection with lethal dose of pr ⁄ 8 virus, they were protected from the infection or lethal disease, respectively. the virus titers in the lungs of a ⁄ pr8 specific igy-treated mice at day 3 after infection were also significantly lower than those seen in untreated mice or mice receiving normal igy. intranasal administration is the most effective route as compared to oral or peritoneal or intravenous administration for protection against lethal challenge, and the presence of virus-specific igy in bronchoalveolar lavage (bal) is required for the protection. the results provide a proof-of-concept that intranasal administration of virus-specific igy prevents influenza virus infection and cures the disease. the concept could be applied to control influenza outbreaks including seasonal and pandemic influenza. the protection was correlated with hai and vn activities of the igy and reduced virus titers in the lungs after treatments, suggesting that the protection is mediated by vn. we asked if administration of igy in the respiratory tract induces anti-igy ab response in mice. if this is the case, the next question is whether pre-existing anti-igy abs block igy-mediated protection. indeed, significant levels of anti-igy were observed in animals that received single or multiple administration of igy. when igy-immune mice were treated with virus-specific igy before or after lethal challenge, the results were identical to those obtained from treated naive mice, indicating that pre-existing anti-igy abs do not interfere with the protection mediated by virus-specific igy. consistently, incubation with anti-igy serum did not interfere with hai and vn activity of the virus-specific igy, indicating that anti-igy abs do not block virus binding by virus-specific igy (figure 2 ). the finding suggests that the igy treatment could be applied to persons who have developed anti-igy during the individuals' life, and such treatment strategy could be repeated if multiple treatment is required and ⁄ or necessary later on to protect infections with other pathogens. the approach using specific igy for prevention and therapy of hpaiv h5n1 infection offers a practical alternative to immunotherapy using convalescent plasma 19 and an additional therapeutic option to antiviral drugs since widespread drug resistance has been recently reported among influenza virus strains. igy is relatively stable. we found no change in protective activity after at least 12 months storage at 4°c, and lyophilization does not affect the activity, making production of igy practical. the use of igy immunotherapy has many advantages, since igy does not activate the human complement system or human fc-receptors, which all are well-known cell activators and mediators of inflammation. 20 we chose the water dilution method for preparation of igy. the method is simple, efficient and does not require any toxic compounds or any additives. such igy preparations by this method have been used in other human study. 9, 21 eggs are normal dietary components, so there is minimal risk of toxic side effects, except for those with egg allergy. thus, our study demonstrated that influenza virus-specific igy can be used in passive immunization that provides great help for immunocompromised patients and elderly who have weaken immune response to influenza vaccines. importantly, the consumable eggs readily available in the markets of countries that impose mandatory h5n1 vaccination offer an enormous source of valuable, affordable, and safe biological material for prevention and protection against potential h5n1 pandemic influenza. parts of the information and data presented in this manuscript were previously published in http://www.plosone.org/ article/info:doi%2f10.1371%2fjournal.pone.0010152. the polyphenol rich plant extract cystus052 is highly introduction the 2009 ⁄ 2010 h1n1influenza a virus pandemic clearly demonstrates that influenza is still a major risk for the public health. although the pandemic swine origin influenza a virus (soiv) caused only mild symptoms, the control of the outbreak still remains difficult. even as vaccine is available against this virus, the possibility of reassortment between the pandemic and a seasonal or avian a ⁄ h5n1 influenza virus strain is indeed a frightening, but a likely event. this reassortant strain might be able to transmit easily between humans causing fatal infections, and the current soiv vaccine might no longer be sufficient to protect against the reassorted virus. in such a case, we can only rely on effective antiviral drugs. today, neuraminidaseinhibitors, such as oseltamivir, represent the most common clinically approved medication against influenza a viruses. unfortunately, the frequency of reports describing the appearance of drug-resistant seasonal h1n1 and also h5n1 influenza a viruses dramatically increased in the recent past. [1] [2] [3] [4] drug resistance to the known antivirals highlights the urgent need for alternative antiviral compounds with novel defense mechanisms. recently, we have reported that a polyphenol rich plant extract, cystus052, which showed antiviral activity against influenza a viruses in cell culture and in mice. 5, 6 moreover, the antiviral activity of cy-stus052 against seasonal influenza virus and common colds was also demonstrated in humans. 7 however, the efficiency of cystus052 against soiv and a ⁄ h5n1 isolates was unknown so far. therefore, we investigated cy-stus052 effectiveness against the pandemic strain and seven natural influenza a ⁄ h5n1 isolates detected in several avian species during 2006 ⁄ 2007 avian influenza outbreak. additionally, the potency of the most common neuraminidase inhibitor oseltamivir was also investigated against these isolates. here, we show that cystus052 treatment was effective in in vitro studies against soiv and a ⁄ h5n1 influenza virus. viruses avian h5n1 isolates were originally obtained from the bavarian health and food safety authority, oberschleissheim, germany. the soiv a ⁄ hamburg ⁄ 4 ⁄ 2009 was obtained from the robert-koch-institut, berlin, germany. all h5n1 viruses were further propagated in embryonated chicken eggs or mdck ii (h1n1v) cells at the friedrich-loeffler-institut, tübingen, germany. for the cytopathological effect (cpe) inhibition screening, in accordance with sidwell, 8 mdck ii cells were infected with different viruses at moi of 0ae005. virus-infected cells were then treated with antiviral compounds cystus052 from 0ae1 to 1000 lg ⁄ ml or oseltamivir from 0ae01 nm to 1 mm. after incubation for 48 hours at 37°c and 5% co 2 , cells were fixed, and viable cells were stained with crystal violet. after extraction of crystal violet from viable cells with 100% methanol, the extinction was measured with an elisa reader. immediately before infection, mdck ii cells (8 · 10 4 cells ⁄ well) were washed with pbs and subsequently incubated with virus diluted in pbs ⁄ ba (0ae2% ba) 1 mm mgcl 2 , 0ae9 mm cacl 2 , penicillin and streptomycin to a multiplicity of infection (moi) of 0ae001 for 30 minutes at 37°c. cystus052 was added in a concentration of 50 lg ⁄ ml directly to the virus-stock and on the cell monolayer simultaneously with the infection. after 30 minutes incubation period, the inoculums were aspirated and cells were incubated with either mem or mem containing 1 lm oseltamivir. at indicated time points, supernatants were collected. infectious particles (plaque titers) in the supernatants were assessed by a plaque assay under avicel as described previously. 9 in order to investigate the antiviral potential of cy-stus052, ec 50 values based on the inhibition of the cpe on mdck ii cells were determined for cystus052 and in addition for oseltamivir. the ec 50 values for cystus052 ranged from 1ae53 to 18ae88 lg ⁄ ml. cystus052 demonstrated the highest sensitivity against the soiv, sn1 and mb1 isolates with ec 50 values below 5 lg ⁄ ml. compared to these virus strains, cystus052 showed a slightly increased ec 50 value for gsb1 (18ae88 lg ⁄ ml). in contrast the ec 50 values for bb1 and bb2 were notably elevated (65ae68 and 76ae22 lg ⁄ ml). thus, the weakest antiviral effect of cystus052 was observed against these two isolates. the ec 50 values evaluated for oseltamivir ranged from 0ae07 to 512ae76 lm ( table 1 ), indicating that bb2 (512ae76) and gsb1 (356ae92 lm) can be considered resistant against oseltamivir. to confirm these results we investigated the ability of cystus052 to block virus replication as published before. 1 as a control, virus infected cells were treated with oseltamivir as described earlier. 6 in the absence of the drugs all influenza strains showed similar growth properties (figure 1, black squares) . first progeny viruses were detectable between 8 and 20 hours post infection (figure 1, black squares) . treatment with cystus052 resulted in reduction of virus titers of all influenza virus strains (fig. 1a-h, open triangles) . surprisingly, oseltamivir failed to inhibit the replication of two h5n1 influenza virus strains (gsb1 and bb2), supporting the data of ec 50 values ( figure 1d+h , grey rhombes). we assessed the antiviral activity of cystus052 against the newly emerged soiv and seven avian h5n1 influenza viruses. cystus052 showed efficient antiviral activity against the pandemic h1n1v strain and was effective to a wide range of h5n1 viruses. furthermore, cystus052 demonstrated a broader and more efficient antiviral potential than oseltamivir. cystus052 treatment leads to a stronger reduction of progeny virus titers, and more importantly, cystus052 was effective against all tested viruses, while oseltamivir was unresponsive against two of seven a ⁄ h5n1 viruses. even though the pandemic strain in general is still sensitive to oseltamivir treatment, there are increasing numbers of reports of emerging resistant variants. the treatment with cystus052 does not result in the emergence of viral drug resistance since the mode of action is an unspecific physical binding of the virus particle that is also beneficial to reduce opportunistic bacterial infections. 5,7,10 cystus052 is an extract from a special variety of the plant cistus incanus, and it is very rich in polymeric polyphenols. 11 it is well known that polyphenols exhibit protein-binding capacity. 12 however, cystus052 exhibited no neuraminidase inhibiting activity. therefore, ingredients of cystus052 may act in a rather unspecific physical manner by interfering with the viral hemagglutinin at the surface of the virus particle as demonstrated before. 5 while this prevents binding of the virion to cellular receptors, it does not block accessibility and action of the viral neuraminidase. since, infections with influenza a viruses are still a major health burden and the options for control and treatment of the disease are limited, plant extracts such as cystus052 should be considered as a new candidate drug for a save prophylactic and therapeutic use against influenza viruses. attenuation of respiratory immune responses by antiviral neuraminidase inhibitor treatment and boost of mucosal immunoglobulin a response by co-administration of immuno-modulator clarithromycin in paediatric influenza the antiviral neuraminidase inhibitor osv and zanamivir are widely used treatment options for influenza infection and are being stockpiled in many countries. although mucosal immunity is the frontline of defense against pathogens, the effects of neuraminidase inhibitor treatment on airway mucosal immunity have not been reported. the suppression of viral rna replication and viral antigenic production by these drugs may result in a limited immune response against influenza virus. 1 macrolides, such as cam and azithromycin, have anti-inflammatory and immunomodulatory properties that are separate from their antibacterial effects. [2] [3] [4] this study examined the impact of osv treatment on immune responses in the airway mucosa and plasma in mice infected with iav and pediatric influenza patients. we also assessed the immuno-modulatory effects of cam in influenza patients who were treated with or without osv. female 3ae5-week-old weanling balb ⁄ c mice were nasally inoculated with 25 pfu of iav ⁄ pr8 ⁄ 34 h1n1 at day 0. immediately after infection, mice were given 50 lg of osv orally or vehicle at 12-hours intervals for 11 days. the levels of virus-specific siga in nws and bronchoalveolar fluids (balf) and igg in plasma were measured by elisa as reported previously. 5 a retrospective clinical study was conducted. for the study, 40 children with acute influenza were recruited and grouped according to the treatment received: 5 days treatment with osv (n = 14), cam (n = 8), osv + cam (n = 12), and untreated (n = 6). since parents in japan are well aware of the adverse effects of osv especially the neuropsychiatric complications, 6 the decision on whether to administer osv or not and to prescribe cam was made by the parents and the attending paediatricians, based on their anti-viral and immuno-modulatory activity. 3, 6 comparisons were made of the levels of siga against iav ⁄ h3n2 and iav ⁄ h1n1, total siga, in nws and disease symptoms before and after treatment. anti-ha siga and total siga in nws of patients were determined from the standard regression curves with human iga of known concentration in a human iga quantitation kit (bethy laboratories). because an affinity purified human anti-ha-specific siga standard of each influenza a subtype is not available, the relative value of anti-haspecific siga amount was expressed as unit (u). one unit was defined as the amount of one lg of human iga detected in the assay system as reported previously. 6 the concentrations of siga in individual nws were normalized by the levels of total siga (lg ⁄ ml). oseltamivir suppresses viral rna replication and viral antigenic protein production. to investigate the influence of daily treatment with osv on ha-specific mucosal and systemic immune responses, we analyzed ha-specific siga levels in nws and balf as well as igg levels in plasma at days 8 and 12 post-infection in mice treated orally with osv or methylcellulose (mc) as vehicle. the osv treated mice showed lower antibody responses in nws and balf than control mice treated with mc solution (table 1) . significantly reduced ha-specific siga responses were particularly noted in the osv group at day 12, the period of maximal mucosal siga induction. the airway secretions and plasma from mice at day 0 did not contain detectable levels of ha-specific antibodies. these findings were supported by other data whereby mice treated with osv displayed significantly lower numbers of ha-specific iga antibody-forming cells (afcs) in the nasal lamina propria, mediastinal lymph nodes, and lungs compared with mc-treated mice. these results clearly indicate that oral administration of osv downregulates ha-specific siga responses in mucosa. on the other hand, there were no significant differences in the elevated levels of ha-specific plasma iga and igg antibodies or the increased numbers of ha-specific iga and igg afcs in the spleen between osv-and mc-treated mice. taken together, these results implicated the oral administration of osv in a suppressed induction of haspecific siga responses in respiratory lymphoid tissues, although systemic ha-specific antibody responses were not significantly affected by osv. since cam up-regulates il-12, a mucosal adjuvant cytokine in the airways, and promotes the induction of siga and igg in the airway fluids of mice infected with iav, 4, 7 we assessed the impact of treatment with osv and ⁄ or cam on the levels of anti-influenza siga in nws and clinical status of influenza patients. the concentration ratio of table 1 . anti-ha-specific siga to total siga in nws was expressed as titer: anti-ha-specific siga (u ⁄ mg) ⁄ total siga (lg ⁄ mg) · 100. figure 1 shows changes in the anti-ha(h3n2) siga ratio (titer) and fold of increase in siga titer in each patient during the 5-days' treatment for the four different treatment groups. it is noteworthy that, upon admission to the hospital, the siga titers were <3 in 93% of patients. during the 5 days of treatment, rapid increases in the titers were observed in almost all patients in cam, osv + cam, and no treatment groups. in contrast, in the osv group, the anti-ha-specific siga titers remained unchanged or decreased in the majority of patients. the finding of significant low induction of anti-viral siga in the osv group was supported by the results of animal experiments. however, the addition of cam to osv augmented siga production and restored mucosal siga levels; 75% of patients treated with osv + cam showed >5-fold increase in the titers during treatment. these observations suggest that cam stimulated the local mucosal immunoresponse in the nasopharyngeal region of patients treated with osv. the prevalence of disease manifestations was also analyzed. 6 among the symptoms listed, a significant decrease in the prevalence of cough was recorded between the no treatment group and the osv + cam group and between the osv group and the osv + cam group (**p < 0ae01), despite the limited number of patients in each group. the duration of the febrile period was significantly shorter in the osv and osv + cam groups than the no treatment group. however, no significant difference was observed between the osv group and osv + cam group. it has been reported that osv does not affect the cellular immune responses, such as cytotoxic t lymphocytes and natural killer cells. 8 however, the effects of osv on mucosal immunity have not been studied so far. the present study showed that osv treatment of mice infected with iav induced insufficient protective mucosal siga responses in the respiratory tract, although treated mice showed the similar levels of systemic igg and iga antibody responses in plasma to those in mice treated with vehicle (table 1) . 1 the observed effect of osv on mucosal immunity was probably due to a suppression of viral replication and viral antigen production in the mucosal layer. these observations in mice are further supported by our clinical reports of siga in nws and balf of osv treated influenza patients. 6 the 14 membered-and 15 membered-ring macrolides have been found to possess a wide range of anti-inflammatory and immuno-modulatory properties, 2,3 and to be effective in the treatment of respiratory syncytia and iav infection. 9,10 the efficacy of low doses administered on the long term against pathogens that are insensitive to macrolides indicates a mode of action that is separate from their antibacterial activity. 2, 3, 9, 11 in the present study, we evaluated the immunomodulatory effects of cam on mucosal immune responses in pediatric influenza. a decrease in the proportion of total siga that was anti-ha-specific siga during treatment was observed in 21.4% of patients in the osv group (those represented by the dotted lines and closed diamonds in figure 1 ), whereas an increase in the proportion was observed in most patients of the other groups (except for one patient of the untreated group). despite the low or unchanged induction of anti-ha-specific siga in the majority of osv-treated patients, the additional use of cam with osv boosted the mucosal immune response and restored local mucosal siga levels. we are currently engaged in detailed immunological studies of the effects of cam and osv on the levels of mediators controlling iga class switching in nws of influenza patients and airway secretion of mice infected with iav. further studies should clarify the boost mechanisms of cam and the suppression mechanisms of osv in iga class switching. our findings suggest the risk of re-infection in patients showing a low mucosal response following osv treatment and cam effectively boosts the siga production for protection of re-infection. to date there is an urgent need to develop new antivirals against influenza. most of the molecules reported target influenza proteins that acquire rapid mutations of resistance. the development of new molecules that have a broad antiviral activity and are not subjected to influenza mutation is of particular interest. our laboratory and others recently showed that proteases can participate to the innate immune response in the airways through the activation of a family of receptors called par. in particular, through the release of interferon, par2 agonists curbed viral replication significantly in infected cells. in this study, since erk activation is crucial for virus replication, we investigated whether par2 could inhibit virus replication through inhibition of the erk pathway. results showed that while influenza a infection alone or par2 stimulation alone induced erk activation, par2 stimulation does not inhibit erk activation in influenza infected cells. thus, par2 agonists may be a potential new drug against influenza viruses that could be used in combination with other anti flu therapy such as the inhibition of the erk pathway. respiratory tract-resident proteases are key players during influenza virus type a infection. 1, 2 in addition to their direct activating effect on surface viral proteins, lung mucosal proteases can regulate cellular processes by their ability to signal through protease-activated receptors (pars). 3 after cleavage of the receptor by proteases, the new aminoterminal sequence of par binds and activates the receptor internally. these receptors are highly expressed at epithelial surfaces, in particular in the lung, where human influenza virus replicate in vivo. pars are thus directly exposed to proteases present in the airways. among the four different pars, par2 acts as an antiviral through an interferondependent pathway. 4, 5 thus, agonists of par2 are potential new drugs against a broad range of influenza viruses, which is in accordance with the broad antiviral action of interferon. however, the signalling pathway induced by par2 agonists in influenza a infected cells has still to be investigated. in this manuscript, we showed that influenza infection or activation of par2 induced erk activation, a crucial step for efficient virus replication. 6, 7 however, par2 agonists do not impaired erk activation in influenza a virus infected cells. since the pathway of par2 protection is likely to be erk-independent, the use of anti erk molecules in combination with par2 agonists maybe of potential interest in future anti-influenza therapy. influenza viruses a ⁄ wsn ⁄ 33 (h1n1) (a kind gift from nadia naffakh) was used in the present study. mdck (madin-darby canine kidney) and the human alveolar type ii a549 cell were obtained from atcc and grown as previously described. 8 for western blot analysis, the following antibodies were used: monoclonal antibody for phospho-erk1 ⁄ 2 (t202 ⁄ y204) and for erk1 ⁄ 2 antibodies from cell signaling technology (beverly, ma), horseradish peroxydase (hrp)-coupled rabbit polyclonal antibodies against mouse or rabbit igg from paris (compiègne, france). a549 cells were infected with iav at an moi of 1 in emem medium, as previously described. 9,10 at various time points post infection, cells were collected and proteins were analysed as previously described. 6,11 par2 stimulation was performed at 37°c in emem medium as previously described. 4 after infection and ⁄ or stimulation, cells were lysed in ice-cold lysis buffer. lysates were centrifuged at 12 000 g for 20 min, and total proteins of the supernatants were analyzed by western blot analysis as previously described. 12, 13 results since activation of the erk pathway is essential for efficient influenza replication, 7 we first investigated the kinetics of erk activation after influenza infection in human a549 alveolar epithelial cells. for this purpose, a549 cells were infected with influenza viruses at a moi of 1 at different time point post-infection, and activation of erk1 ⁄ 2 pathway was assessed by western blot analysis using an anti-erk antibody. results showed that erk was phosphorylated after influenza infection in a time course depen-dent manner when compared to uninfected cells. in contrast, erk phosphorylation was not observed with heatinactivated viruses, suggesting that productive infection is needed for erk activation ( figure 1a ). antibodies against erk1 ⁄ 2 were used as controls. since erk is activated after influenza infection, we then tested whether activation of par2 in uninfected cells also leads to activation of this pathway. for this purpose, a549 cells were stimulated with the selective human (h) or mouse (m) par2 agonist or a control peptide for the indicated time ( figure 1b ). when exposed to the par2 agonists and compared to controltreated cells, erk phosphorylation increased over the time course of stimulation. thus, influenza infection or stimulation of par2 without infection in a549 cells induced activation of the erk pathway at different time point post-infection. since influenza infection and par2 stimulation induced erk activation, we then investigated whether par2 could inhibit erk activation in influenza infected a549 cells. results in figure 2 showed that in influenza infected cells, par2 activation for ten minutes does not inhibit erk activation after influenza infection. thus, erk activation is not inhibited by par2 activation in influenza stimulated cells. in this manuscript, we studied the activation of the erk pathway after par2 stimulation and or influenza infection. particularly interesting is the fact that either influenza infection or par2 stimulation alone induce erk phosphorylation in a549 epithelial cells, while erk activation is not inhibited in a549 infected cells compared to uninfected ones after par2 stimulation. proteases are key factor in the pathogenicity of influenza viruses. in addition to the cleavage of ha, necessary for iav replication, extracellular proteases also play a role in the modulation of the immune system against influenza viruses through the activation of pars. particularly par2, activated by extracellular trypsin-like proteases, could inhibit virus replication through the release of interferon, 4,5 thus, strengthening the immune system via agonist peptides and providing new therapeutic potential against a broad range of influenza strains. in addition, targeting the host instead of the virus could provide a way to escape from virus resistance. 14 thus, a better understanding of how virus escapes from immune surveillance may provide new therapeutic strategies to block iav. in addition, combinations of drugs that block virus replication via different pathways are of interest. the non classical molecules hla-g maybe an interesting new target as we recently showed that it is upregulated after influenza infection, 13 and it is a well known immunotolerant molecule. 15 indeed, it inhibits the innate immune response 16 as well as the adaptive immune response. 17, 18 also, as previously suggested, the erk signal transduction cascade is also of potential interest since it is crucial for virus replication and particularly influenza replication. 6, 7 as shown here, it is unlikely that par2 protection occurs through an erkdependent pathway. thus strengthening the immune response with par2 agonists and blocking nuclear retention of the viral ribonucleoprotein complexes with inhibitors of the mek ⁄ erk pathway may be alternative combinatory approaches for influenza therapy. in addition, since those potential drugs target the host instead of the virus, this could help in the design of new antivirals molecules more resilient to iav mutations and thus to virus resistance. the initial waves of the first influenza pandemic of the 21st century have passed. in june 2009, vaccine companies estimated they could produce in 6 months almost 2.5 billion doses of pandemic vaccine. 1 instead, they actually produced only 534 million doses, of which 64% were non adjuvanted preparations. had these doses been produced with adjuvants (i.e., 3.75 lg instead of 15 lg ha per dose), an additional 1 billion doses could have been made available. yet there was public opposition to adjuvants in many countries, especially by regulatory officials in the united states. misperceptions about the safety of both adjuvanted and nonadjuvanted vaccines were widespread. added to this, shortfalls in vaccine production, delays in vaccine delivery, and the ''mildness'' of the pandemic itself meant that only a few countries achieved reasonable levels of vaccine coverage. millions of doses went unused and had to be destroyed. supplies of antiviral agents were even more limited. thus, despite the best efforts of influenza scientists, health officials, and companies, more than 90% of the world's people did not have timely access to affordable supplies of vaccines and antiviral agents. instead, they had to rely on 19th century public health ''technologies.'' given current understanding of biology in the early 21st century, they should have had -and probably could have had -something better. this report reviews evidence for an alternative approach to serious and pandemic influenza that could be used in all countries with basic health care systems. instead of confronting the influenza virus with vaccines and antiviral agents, it suggests that we might be able to modify the host response to influenza virus infection by using anti-inflammatory and immunomodulatory agents. this idea was introduced several years ago 2 and has been reviewed in several publications. [3] [4] [5] [6] [7] [8] the central importance of the host response in the 1918 pandemic, young adults had high mortality rates. ever since, influenza virologists have sought to answer the question ''why did young adults die?'' by defining the molecular characteristics of the 1918 virus that were responsible for its virulence. 8 in doing so, they have overlooked a crucial piece of clinical evidence from the 1918 pandemic: compared with young adults, children were infected more frequently with the same virus, yet they seldom died. consequently, the more important question is ''why did children live?'' this can only be explained by recognizing that children must have had a different host response to the 1918 influenza virus than adults. physicians have long recognized that for several other medical conditions, both infectious (e.g., pneumococcal bacteremia) and non-infectious (e.g., multiple trauma), children have a more benign clinical course than adults. 6, 8 a corollary of this observation is that secondary bacterial pneumonia, although commonly found in young adults in 1918, could not have been the primary cause of death. children must have had the same or higher rates of nasopharyngeal colonization with the same bacteria that were associated with pneumonia deaths in adults, yet children seldom died of secondary bacterial pneumonia. 6 if young adults died with secondary bacterial pneumonia, underlying host factors must have made them more susceptible. few people who die of influenza do so during the first few days of illness when pro-inflammatory cytokine levels are high. instead, like patients with sepsis, they usually die in the second week, when anti-inflammatory cytokines and immunosuppression dominate. 6, 8, 9 influenza deaths occur more frequently in older persons with cardiopulmonary conditions, diabetes, and renal disease, but as seen in the 2009 h1n1 pandemic, they also occur in younger adults with obesity, asthma, and in women who are pregnant. regardless of age, people with all of these conditions share one characteristic in common: they have chronic low-grade inflammation. in effect, their ''innate immune rheostats'' have been set at different, and perhaps more precarious, levels that make them more vulnerable to influenza-related complications. 10 laboratory studies of influenza virus infection confirm the importance of the host response. in several studies in mice in which the host response has been modified (e.g., cytokine knockout), survival has been improved without increasing virus replication in the lung. 5 in fact, severe disease can be induced without any influenza virus replication. for example, fatal acute lung injury has been induced in mice by inactivated (not live) h5n1 virus. 11 in this model, antiviral agents would be useless; only the host response could be responsible for disease. these observations raise the following question: could the host response be modified so patients with severe seasonal and pandemic influenza might have a better chance of surviving? influenza is associated with acute coronary syndromes, and influenza vaccination and statins reduce their occurrence. these associations led to the suggestion in 2004 that statins might be used to treat pandemic influenza. 2 other agents that might also be effective include ppara and pparc agonists (fibrates and glitazones, respectively) and ampk agonists (e.g., metformin). 5, 8 these agents have been studied in laboratory models of inflammation, sepsis, acute lung injury, ischemia ⁄ reperfusion injury, energy metabolism, mitochondrial function, and programmed cell death. the results of these studies cannot be reviewed in detail here, but the major findings for cell signaling are summarized in the table 1 . unfortunately, the results of experimental studies are not always clear cut. for example, in one study of influenza virus infected mice, il-10 was necessary for containing infection, 12 but in another study il-10 appeared to be harmful. 13 nonetheless, overall understand-ing of cell signaling pathways in influenza virus infections and the actions of statins, glitazones, fibrates, and ampk agonists strongly suggest that these agents could benefit patients with severe influenza. laboratory studies in mice infected with pr8 (h1n1) h2n2 and pandemic h1n1 viruses show that resveratrol, fibrates, glitazones, and ampk agonists reduce mortality by 30-50%, often when treatment is started 2-4 days following infection. 14-17 (resveratrol is a polyphenol found in red wine. it shares with these other agents many of the same cell signaling effects.) in h5n1-infected mice, treatment with celecoxib and mesalazine, together with zanamivir, showed better protection than zanamivir alone. 18 remarkably, these immunomodulatory agents have not increased virus replication. even more remarkable, in another model of a highly inflammatory and frequently fatal conditionhepatic ischemia ⁄ reperfusion injury -glitazone treatment ''rolled back'' the host response of ''young adult'' mice (8-10 weeks old) to that of ''children'' (3-4 weeks old). 19 this unique study suggests that immunomodulatory treatment might roll back the damaging and sometimes fatal host response of young adults with influenza to the more benign and rarely fatal response of children. several, but not all, observational studies have shown that outpatient statins decrease hospital admissions and mortality due to community-acquired pneumonia. 8 for influenza itself, preliminary evidence presented in october 2009 suggests that immunomodulatory treatment of influtable 1 . cell signaling targets that might be affected by immunomodulatory treatment of severe seasonal and pandemic influenza* down regulate pro-inflammatory cytokines (e.g., nf-kappab, tnfa, il-1, il-6) up regulate anti-inflammatory cytokines (il-10, tgfb) up regulate pro-resolution factors (lipoxin a4, resolvin e1) up regulate ho-1 and decrease tlr signaling by pamps and damps up regulate enos, downregulate inos, restore inos ⁄ enos balance and stabilize cardiovascular function decrease formation of reactive oxygen species and decrease oxidative stress improve mitochondrial function and restore mitochondrial biogenesis decrease tissue factor and its associated pro-thrombotic state stabilize the actin cytoskeleton in endothelial cells and intracellular adherins junctions, and thereby increase pulmonary barrier integrity and decrease vascular leak differentially modify caspase activation and apoptosis in epithelial and endothelial cells, macrophages, neutrophils and lymphocytes in the lung and other organs increase the bcl-2 ⁄ bax ratio in influenza virus-infected cells and prevent the apoptosis necessary for virus replication. *see references 2,5,7,8 for details. nf-kappab, nuclear factor kappab; tnfa, tumor necrosis factor alpha; tgfb, transforming growth factor beta; ho-1, heme oxygenase -1; tlr, toll-like receptor; pamp, pathogen-associated molecular pattern; damp, damage associated molecular pattern; enos, endothelial nitric oxide synthase; inos, inducible nitric oxide synthase. enza patients with severe illness could be beneficial. in a study of almost 4000 patients hospitalized with laboratoryconfirmed seasonal influenza, inpatient statin treatment reduced hospital mortality by 66%. 20 in these patients, the cell signaling effects of statin treatment, summarized in the table 1 , probably acted to reduce pulmonary infiltrates, maintain oxygenation, stabilize myocardial contractility and the peripheral circulation, reverse immunosuppression, restore mitochondrial biogenesis, and prevent multi-organ failure. achieving these clinical effects led to a decrease in mortality. because of the molecular cross-talk between statins, fibrates, glitazones, and ampk agonists, 5,8 similar clinical benefits might be expected from other members of this ''family'' of immunomodulatory agents. simvastatin, pioglitazone, and metformin are produced as inexpensive generics in developing countries. they are used throughout the world in the daily treatment of millions of patients with cardiovascular diseases and diabetes. global supplies are huge. because most people with influenza recover without specific treatment (this was true in 1918), not all patients would require immunomodulatory agents. instead, only those at risk of ards, multi-organ failure, and death would need to be treated. importantly, the cost of treatment for an individual patient would be less than $1.00 (d.s. fedson, unpublished observations). moreover, unlike vaccines they could be used on the first pandemic day. thus far, influenza scientists and the institutions that support their work (e.g., nih and cdc, national health agencies in many countries, the bill and melinda gates foundation, the welcome trust, and the world health organization) have shown little interest in immunomodulatory treatment. nonetheless, when more than 90% of the world's people have no access to influenza vaccines and antiviral agents, their physicians must have access to an effective ''option,'' especially one that might be lifesaving. research on immunomodulatory agents for influenza must involve investigators in many fields outside influenza science -those with expertise in the molecular and cell biology of inflammation, immunity, sepsis, cardiopulmonary diseases, endocrinology and metabolism, ischemia ⁄ reperfusion injury, mitochondrial function, and cell death. laboratory studies needed to identify promising treatment agents would probably cost $5-15 million (d.s. the results of these studies would inform clinical trials that critical care physicians are already eager to undertake. 21, 22 this work will be especially important for people in developing countries where critical care capacity is extremely limited and not likely to improve. 23 like critical care physicians, influenza scientists too must recognize that they cannot afford not to undertake research to determine whether generic immunomodulatory agents might be useful in managing severe seasonal and pandemic influenza. the nf-kappab-inhibitor sc75741 efficiently blocks h5n1 influenza virus propagation in vitro and in vivo without the tendency to induce resistant virus variants introduction influenza is still one of the major plagues worldwide. the appearance of highly pathogenic avian influenza (hpai) h5n1 viruses in humans and the emergence of resistant h5n1 variants against neuraminidase inhibitors highlight the need for new and amply available antiviral drugs. we and others have demonstrated that influenza virus misuses the cellular ikk ⁄ nf-kappab signalling pathway for efficient replication, suggesting that this module may be a suitable target for antiviral intervention. 1 here, we show that the novel nf-kappab inhibitor sc75741 efficiently blocks replication of influenza a viruses, including avian and human a ⁄ h5n1 isolates in vitro in concentrations that do not affect cell viability or metabolism. in a mouse infection model with hpai a ⁄ h5n1 and a ⁄ h7n7 viruses, we were able to demonstrate reduced clinical symptoms and survival of sc75741 treated mice. moreover, influenza virus was reduced in the lung of drug-treated animals. besides this direct antiviral effect, the drug also suppresses h5n1-induced overproduction of cytokines and chemokines in the lung, suggesting that it might prevent hypercytokinemia we hypothesise to be associated with pathogenesis after infections with highly pathogenic influenza viruses, such as the a ⁄ h5n1 strains. thus, a sc75741-based drug may serve as a broadly active nontoxic anti-influenza agent. to assess the number of infectious particles (plaque titers) in organs a plaque assay using avicel ò was performed in 96-well plates as described by mastrosovich and colleagues. 2 virus-infected cells were immunostained by incubating for 1 hour with a monoclonal antibody specific for the influenza a virus nucleoprotein (serotec) followed by 30 minutes incubation with peroxidase-labeled anti-mouse antibody (dianova) and 10 minutes incubation with true blueô peroxidase substrate (kpl). stained plates were scanned on a flat bed scanner and the data were acquired using microsoft ò paint software. the virus titer is given as the logarithm to the basis 10 of the mean value. the detection limit for this test was <1ae7 log 10 pfu ⁄ ml. organs of infected and control mice were homogenized and incubated over night in 1 ml trizol ò reagent (invitrogen) at 4°c. total rna isolation was performed as specified by the manufacturer (invitrogen). rna was solubilised in 50 ll rnase free water and diluted to a working concentration of 50 ng rna ⁄ ll. reverse transcription real-time pcr was performed using quantifastô sybr ò green rt-pcr kit and quantitect primer assays (qiagen) . all samples were normalized to gapdh and fold expression analyzed relative to uninfected controls. 3 ct values were obtained with the smartcycler ò (cepheid). to answer the question whether the nf-kappab inhibitor sc75741 shows antiviral properties against influenza virus, h5n1 infected mdck cells were treated with different concentrations of the inhibitor (figure 1 ). already treatment with 1 nm of sc75741 led to a reduction of viral cpe of more than 70%. almost 100% protection of cells was achieved when cells were treated with 50 lm sc75741. the results indicated that sc75741 has antiviral properties at concentrations ranging from 1 to 5 nm. we next tested whether sc75741 would also be effective in the mouse model of influenza virus infection. when h7n7 mice were treated i.v. once daily for 5 days with 5 mg ⁄ kg sc75741, survival rate of the animals increased significantly (p < 0ae05). the same results were found when h7n7 influenza virus infected mice were treated i.p. with 15 mg ⁄ kg sc75741 (data not shown). moreover, sc75741 treatment was not only effective when the inhibitor was given prior to h5n1 influenza virus infection, but also in a therapeutic setup when sc75741 was applied to the animals 4 days after infection (data not shown). since influenza virus infected mice showed increased survival after lethal infection, we next questioned whether the amount of influenza virus was reduced in the lung. therefore, we performed quantitative real-time (qrt) pcr to detect viral mrna. mice were treated with either sc75741 or the solvent, and 48 hour later the lungs were prepared to perform qrt-pcr. as shown in figure 2a the amount of viral mrna was reduced by 90% in sc75741 treated mice compared to solvent treated controls, indicating that sc75741 leads to a reduced expression of h5n1 specific mrna in the lung of infected mice. since infection of mice with h5n1 leads to hypercytekinemia, 4 we also investigated the expression of cytokines in sc75741 treated mice. as shown in figure 2b the amount of il-6 specific mrna was drastically reduced in sc75741 treated mice compared to solvent treated controls. moreover, also the expression of ip-10 was altered in sc75741 treated h5n1 influenza virus infected mice. here, roughly 90% reduction of specific mrna was detectable ( figure 2c ). thus, sc75741 leads to a reduced transcription of il-6 and ip-10 in h5n1 infected mice. there is an urgent need for new concepts to develop antiviral drugs against influenza virus. targeting cellular factors is a promising but challenging approach, and the concerns about side effects are obvious. however, it should be considered that drugs targeting viral factors, such as amantadine or oseltamivir, also exhibit a wide range of side effects in patients. thus, drug safety has to be rigorously tested in clinical trials regardless whether a drug targets a cellular or a viral factor. moreover, resistance against human h1n1 influenza viruses and highly pathogenic avian h5n1 virus strains to oseltamivir and amantadine have been reported. 5 in that respect, the strategy to target cellular factors 6,7 might be one way to ensure that new drugs against influenza virus will be useful and effective for a long time without causing the development of resistant virus variants. we were able to demonstrate that the nfkappab inhibitor sc75741 is able to reduce influenza virus activity in cell culture. moreover, the compound was also effective against highly pathogenic avian influenza viruses of the h5n1 and h7n7 subtypes in the mouse model. next to the reduction of virus sc75741 was also able to reduce h5n1-induced overproduction of cytokines and chemokines in the lung in the lung of mice after infection with h5n1. most importantly, the drug did not show any tendency to induce resistant virus variants (data not shown). thus, a sc75741based drug may serve as a broadly active non-toxic antiinfluenza agent. [1] [2] [3] [4] [5] in hong kong, the first confirmed case was a tourist from mexico reported on may 1, 2009. the local government made its first attempt to contain the spread of h1n1 in the local community by closing the metropark hotel where that tourist was staying, and quarantining 350 guests and staff for 7 days. following identification of the first local case around 6 weeks later on june 11, 2009, the government closed all kindergartens and primary schools from june 12 until early july. fever clinics were also opened, the alarm levels in hospitals were raised to the highest, and a public education campaign was implemented. previous studies of the community responses to severe acute respiratory syndrome (sars) and human-to-human h5n1 avian flu identified the importance of understanding the background perceptions of risk and psychological impact on the community. [6] [7] [8] [9] [10] in this study we investigated the psychological and behavioral responses of the general local community throughout the first wave of ph1n1, and we also examined the factors associated with greater use of preventive measures. 11 a total of 13 surveys were conducted between april and november 2009, covering the entire first wave of the ph1n1 pandemic. computer generated random-household telephone numbers from all land-based local telephone numbers covering over 98% of hong kong households were used to recruit a total of 12 965 local adults. one cantonese-speaking adult (age ‡18) was invited for interview in each selected household on the basis of a kish grid. the survey instrument was based on previous experience in sars and avian influenza projects. information, including knowledge on modes of transmission, psychological responses to pandemic influenza, preventive behaviors, attitudes towards the new vaccines and socio-demographics, was collected. informed consent was obtained prior to the interview. ethics approval was obtained from the institutional review board of the university of hong kong. descriptive statistics were weighted by sex and age based on the reference population data provided by the hong kong government census and statistics department. 12 multivariable logistic regression analyses were used to examine the association between the use of preventive measures and knowledge, perceptions and behaviors, sociodemographic characteristics, and psychological responses to pandemic influenza. multiple imputation was used to cope with a small proportion of missing data and make the best use of all available data. 13 statistical analyses were conducted in r version 2.9.1 (r development core team, vienna, austria). twelve thousand and nine hundred and sixty-five local adults were recruited throughout the study period, with a total of 127 715 telephone calls being made; the response rate among eligible participants was 69.9%. 11 hong kong entered the containment phase after the world health organization (who) announced a global alert, and policies including border screening, tracing, and quarantine of doi:10.1111/j.1750-2659.2011.00219.x www.influenzajournal.com suspected cases were implemented. hong kong transitioned to the mitigation phase on june 10, 2009 when the first local case was reported. the chronology of these and other events plus the epidemic curve of laboratory-confirmed ph1n1 cases are shown in figure 1(a) . the anxiety scores and risk perception of the respondents are shown in figure 1(b,c) . anxiety, measured by the state trait anxiety inventory, remained steady throughout the study period. in response to the announcement made by who and the unknown nature of the new virus, a higher proportion of the respondents expressed worry (more, much more, or extremely more worried than normal) if developed ili and perceived ph1n1 severity (same, more, or much more serious than sars) initially in early may 2009. fewer respondents reported worry if they developed ili as the pandemic proceeded, with a slight perturbation around the first deaths in july 2009 and a steady decline to 40.0%, while perceived severity of ph1n1 declined more dramatically after an early high. perceived risks of infection of respondents (absolute susceptibility) and risk relative to others (relative susceptibility) were also investigated and found to remain relatively stable throughout the first wave, with no indication of an increase during the period of peak ph1n1 activity in september (figure 1c) . as the first wave of ph1n1 progressed, knowledge on modes of transmission did not improve. on the contrary, later in the epidemic increasing proportions of respondents reported oral-fecal and cold weather as modes of transmission of ph1n1. around 35-40% of the respondents did not recognize direct and indirect contact or touching infected persons and contaminated objects as transmission routes for ph1n1 throughout the first wave ( figure 1d ). higher proportions of respondents avoided crowded places and rescheduled travel plans in the second half of june 2009 when local kindergartens and primary schools were closed and the first ph1n1-associated deaths were announced. social distancing measures such as avoiding crowded places and rescheduling travel plans remained stable with slightly decreasing trends thereafter. the use of hygiene measures and other social distancing strategies was relatively stable with slightly decreasing trends during the study period ( figure 2 ). female sex and older age were generally associated with greater reported use of hand hygiene measures, home disinfection, avoidance of crowded places, and rescheduling of travel plans. 11 female sex was also positively correlated with use of face masks and cough etiquette. we found a negative correlation between anxiety and use of all hand hygiene measures and cough etiquette, but a positive correlation between anxiety and use of home disinfection and (c) proportion of the respondents reporting higher worry if developed flu-like symptoms (more, much more, or extremely worried), higher perceived seriousness of h1n1 compared to sars (much more or more severe), higher probability to contract h1n1 over the next 1 month (certain, much more, or more likely), higher probability to contract h1n1 over the next 1 month compared to others outside family (certain, much more, or more likely). (d) proportion of the respondents identifying 5 possible modes of transmission as the actual modes of transmission of h1n1. social distancing measures. 11 other significant factors contributing to greater use of preventive measures were worry and knowledge. 11 greater worry was associated with higher probability of home disinfection, social distancing measures, and use of face masks. knowledge that h1n1 could be spread by indirect contact was associated all the investigated preventive measures, and knowledge that h1n1 could be spread by droplets was associated with cough etiquette, but not face masks. there were no consistent trends between all the investigated preventive measures and absolute and relative susceptibility. 11 community transmission emerged in hong kong in mid-june 2009, and prior to emergence of community transmission, perceived risk and perceived severity were high. as ph1n1 spread in hong kong, risk perception declined, even at the same time as incidence was increasing. anxiety was low throughout, at around 1.8 on the 4-point scale, compared to a maximum of 2.5 during sars on the same scale. 9 anxiety has been showed to be positively correlated to personal hygiene measures and social distancing in previous studies; 9,14 however, we found a negative correlation between anxiety and use of all hand hygiene measures, cough etiquette, and face masks, and a positive correlation between anxiety and home disinfection. 11 the differences in findings may be due to the fact that our anxiety measure was not specific to h1n1, and the score could be affected by other factors including economics. unlike hygiene measures, higher anxiety level, greater worry, and higher risk of perception were all associated with more social distancing. 6, 7, 9, 14 social distancing is the most direct strategy in avoiding infection from other people, and it is commonly observed in an outbreak that the general public avoids crowded places, travelling to other countries, and social gatherings, 9, 14 but the economic impact could be substantial. 15 as community incidence of h1n1 peaked, we did not observe any increase in use of preventive measures (figure 2) . we found that face mask use peaked at the early stage of the pandemic, while hand hygiene remained fairly constant, and the knowledge on the modes of transmission of ph1n1 did not improve over time. the lack of substantial change in preventive measures or knowledge about the modes of ph1n1 transmission in the general population suggests that community mitigation measures played little role in mitigating the impact of ph1n1 in hong kong. on the other hand, knowledge that ph1n1 could be spread by indirect contact was associated with all of the preventive measures studied. consistent with reports during the sars period, 9, 16 this study also showed that females and those of older age were more likely than others to use hygiene measures, avoid crowded places, and reschedule travel plans. this study has some limitations. first, this was a crosssectional study that was carried out at different time points, rather than a longitudinal study following the same individuals over time, and so the inferences on changes in behavior may need to be interpreted more cautiously. second, we recruited samples from all land-based local telephone numbers that cover 98% of hong kong households, but the response rate was not high enough to guarantee a representative sample, and this could be a source of selection bias. third, the responses were self-reported, and this may lead to social desirability bias in estimating knowledge, attitudes, and preventive behaviors. fourth, since the hong kong population has previously gone through unique experiences from sars in 2003 and avian flu in 1997, our results may not be comparable to other countries or settings. in conclusion, this study revealed that the ph1n1 pandemic failed to generate an increase use of preventive measures in the local community. there was no association between anxiety level and the events of the pandemic. with a relatively low mortality and morbidity rates compared to sars, ph1n1 was not a matter of concern in the hong kong community. the lack of substantial change in the use of preventive measures and improvement in knowledge on the modes of transmission of ph1n1 suggested that public health campaigns during the pandemic may not have had substantial effects on the general public. london is a major tourist destination, the seat of government and finance in the uk, and in 2012 will host much of the olympic and paralympic games. along with the rest of the global community, in 2009 and early 2010 london faced the challenges of responding to the first pandemic of the 21st century. at the time, nhs in london was composed of 72 organisations, including the london ambulance service, acute hospitals, mental health and primary care trusts, and the strategic health authority. while london's nhs is well practiced at responding to large, big bang incidents, the influenza a ⁄ h1n1v pandemic was a rising tide event that lasted many months. significant preparatory work had been undertaken prior to april 2009, which meant that the nhs in london was ready to respond. nhs london (the strategic health authority for london) led the response in partnership with local managers in all nhs organisations. the first uk cases of influenza a ⁄ h1n1v were reported in scotland on 27 april, with the first in london on 30 april. cases continued to increase, and the first wave peaked in london in july. cases reduced over the school summer holidays, but increased again when children returned to school at the start of september, and a second, smaller wave occurred. it is essential that the nhs learns from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic to ensure it is prepared for future challenges. nhs london provided a standardised debriefing pack to all nhs organisations in the region to identify, capture, and learn lessons. each debrief event involved health and inter-agency partners to ensure all viewpoints were considered and brought together in a single local report. all local reports were compiled in an over-arching document, which brings together common themes to inform ongoing preparedness in the region. 1 the debrief process identified a number of common themes, such as the need for clear and appropriate communication, the importance of working with partners, and the benefits of strong and early leadership. however, differences between and within organisations were also highlighted; for example, some wanted more freedom for local decision making, whereas others would have preferred more stringently applied central direction. the following paragraphs considers individual areas assessed in the debrief process. command and control was in the main effective, with clear direction delivered from the national centre through nhs london to local nhs organisations. effective leadership is essential; the identification of senior local individuals to lead the response with teams of people to support them was critical. appropriate use of technology to communicate messages and coordinate command and control processes greatly aided the response. this included the development of the nhs london noon brief, a daily digest and associated web portal, and regular teleconferencing. key points are: • operational management at all levels must be considered in pandemic planning. • appointing an executive lead in each organisation was invaluable in the response. • pandemic flu planning for london must continue to be regionally led. communication is an essential component of the response to any incident. it must be clear, timely, and accurate. in the main, communication was excellent and met these criteria. one of the most challenging aspects was when messages from partner organisations differed, which occasionally led to confusion, unnecessary work, or frustration. the use of technology greatly aided communication across the region and supported the response; this included secure web sites, bluetooth, and text messaging etc. key points are: • regular internal communications and staff briefings are critical in the response to emergencies. • regular teleconferencing should be incorporated into future plans. • organisations should consider proactive and innovative methods for communicating during emergencies. robust partnership working was an essential component of pandemic preparedness work; however in the event, the a ⁄ h1n1v pandemic had little impact on sectors in london other than health. resilient communication networks between organisations, a common understanding, and the ability to make decisions were essential to the response at local level. ipcs proved an excellent mechanism to maintain local working relationships and resolve problems. clarity on the seniority of those attending these meetings and whether multi-site organisations such as mental health trusts should attend every ipc should be considered on a local and regional basis. key points are: • pandemic planning must remain part of inter-agency working. • social care resilience and planning must be embedded and integrated in health planning. 'vulnerable groups' is a universal term that covers a large and fluid group of individuals with different needs. ensuring access to healthcare during the pandemic for those who became vulnerable due to the situation, or those identified as such prior to the event, was the role of the pct in partnership with the local authorities. work continues to ensure that communication with vulnerable people is appropriate and timely in all incidents, and that organisations work together to achieve this. key points are: • planning to support the breadth of vulnerable people must continue. • pandemic preparedness for the prison sector should be further developed. • red ⁄ amber ⁄ green ratings for assessing vulnerabilities of mental health service users in an emergency should be further developed across the region. correct and appropriate usage of ppe is an essential component of reducing influenza spread, particularly in healthcare settings. london's nhs had been working towards developing local stockpiles of ppe when the pandemic commenced; however, there was little in place. the unanticipated national stockpile, while providing ppe to all organisations, was accompanied with some challenges in that it was often unfamiliar stock. key points are: • work around local stockpiling of non-standard consumables should continue. • regular training and fit testing of respirators should be embedded in all organisations. antiviral treatment was a core component of the response to influenza a ⁄ h1n1v, and was provided free of charge from a national stockpile. npfs reduced pressure on frontline nhs services once it was activated; however, there were concerns that patients could 'cheat' the system and obtain the drugs prior their clinical need. information about storage requirements of countermeasures must be clearly explained when they are delivered to frontline services, and the potential for recall into national stockpiles should be planned for. key points are: • regular exercising of local mass countermeasures centres and antiviral collection points (acps) should continue. • the use of community pharmacies as acps should be further considered in the capital. pandemic influenza vaccine uptake by healthcare workers was better than usual seasonal influenza uptake in the majority of nhs organisations, but could have been even better. this was largely due to the second pandemic wave not being as significant as expected, lack of clarity around when the vaccine would be delivered, and limited amounts being available initially. • gp-led and mass vaccination models for pandemic vaccination should be considered in local plans. • local lessons from the pandemic vaccination campaign should be applied to seasonal flu vaccination. the ability to maintain or increase capacity in response to a surge in demand, no matter what the cause, must be planned for. any of a number of situations could result in reduced staff or more patients, such as industrial action, transport disruption, disease outbreak, major incident, or poor weather. the work undertaken during planning for and responding to the pandemic will stand organisations in good stead for future disruptions. the importance of robust business continuity planning locally cannot be overlooked, as this is a key component of maintaining and increasing capacity. key points are: • local gp 'buddy schemes' should be encouraged for response to extreme pressure events. • organisations should regularly run staff skills audits so as to be aware of their overall capability for managing emergencies. • less emphasis should be placed on the use of retired staff when planning service continuity. reporting is a necessary but onerous task, and is often one of the most time-demanding parts of any incident response. it is also the aspect least likely to be tested through exercising. nhs london worked with organisations to endeavour to reduce reporting pressures, but much of this was dictated by central government. it is essential that future reporting requirements are proportional, informative, and realistic. while recognising it is not possible to predict the detail of information that may be requested, some broad assumptions can be made. key points are: • organisations should consider how they would collect and collate data from disparate parts of their organisation, rather than focussing on the detail of what that might be. • national and regional planning should consider the need for information and how this is balanced with the demand this places on organisations. • the introduction of the concept of a daily dashboard to identify areas of pressure should be incorporated into pandemic flu planning. the winter and pandemic influenza resilience assurance process undertaken in autumn 2009 was a useful process to inform planning for the first winter when the pandemic virus would be circulating in the uk. this consisted of a regional inter-agency exercise and a comprehensive review of the winter and pandemic plans of all nhs organisations in london. • regular assurance of pandemic flu preparedness should be maintained. • future resilience assurance processes should be undertaken in a timely and measured manner. • local organisations should continue to undertake regular pandemic flu exercises. the recovery period is as important as the response, but often receives minimal attention and has the potential to suffer as staff return to their normal jobs. one of the aspects that was not anticipated during the pandemic was the amount of stock (ppe, antivirals, and vaccine consumables) that would be recalled into national stockpiles. this proved particularly challenging for pcts who had to coordinate the process across their local areas. key points are: • the recovery period of an emergency must be given the same status and importance as the response. • future pandemic flu planning must include the recovery of national stockpiles of equipment and medicines. it is essential the lessons from the 2009 ⁄ 10 influenza a ⁄ h1n1v pandemic are learnt and embedded into business-as-usual and emergency response processes in preparation for the next pandemic and other incidents. even though the a ⁄ h1n1v pandemic was generally milder than previous pandemics, it still presented challenges to the nhs in london. the biggest challenge that remains is to ensure that the public and nhs staff are aware that a more virulent virus could cause significantly more illness, death, and disruption, and that we must maintain our preparedness should this happen. the influenza a ⁄ h1n1v pandemic has been a major stimulus to business continuity planning and emergency preparedness across health in london, and many of the experiences during the pandemic proved invaluable in the unusually severe weather in early 2010. it is important that this impetus and focus is maintained. changes to the nhs landscape in london will be considered in ongoing pandemic and emergency preparedness to ensure we remain as well prepared as possible for future events, particularly as london approaches the 2012 olympic and paralympic games. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. the way national health systems operate during inter-pandemic and the pandemic alert periods and the methods they use to address potential threats posed by zoonotic viruses with pandemic potential, as well as sea-sonal influenza epidemics, can clearly indicate whether the countries have enough capacities to respond adequately to unexpected influenza outbreaks. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during a pandemic scenario, the group prioritized topics and questions relating to rapid action and response. five to 10 key public health needs associated with a pandemic scenario have been identified for each of the research agenda streams: five priority public health topics were identified for a pandemic scenario as follows: • examination of host range and transmission dynamics of animal influenza viruses to guide surveillance, control strategies, and risk communication. • enhanced surveillance in animals and humans to monitor virus evolution: o early detection of novel reassortants or changes in genotype and ⁄ or phenotype related to virulence. o development of epidemiological and laboratory diagnostic tools and capacity building to optimize case finding. o develop a framework for surveillance in animals that address ethical, legal, and social barriers to intra-pandemic surveillance and reporting. • deconstruct the origins of the pandemic virus to identify factors that permitted efficient human transmission. • develop strategies to limit economic, social, and cultural disincentives of animal-based interventions to reduce intra-and inter-species transmission. • operational research to optimize risk communication in the early phases of the pandemic linked to animal husbandry and food safety. stream 2: limiting the spread of pandemic, zoonotic and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: transmissibility of influenza across the progression of infection and spectrum of disease: • relative contributions of the different modes of transmission for influenza. five priority public health topics were identified for a pandemic scenario as follows: • identification of groups at higher risk of infection and severe disease outcome through enhanced surveillance. • understanding disease severity and identification of predictors of severe outcomes. • investigation of vaccine effectiveness, especially in high risk groups in diverse geographic areas. • establishment ⁄ enhancement of pharmacovigilance, particularly for adverse events among at-risk groups. • optimization of strategies for rapid and targeted vaccine deployment. • rapid assessment to optimize acceptance of pandemic vaccine. six priority public health topics were identified for a pandemic scenario as follows: • collaboration and coordinated sharing of data, protocols, regulatory, and other implementation strategies and databases from different countries on all aspects of patient management and outcome to accelerate improvements in patient care. • development of best practices in patient management in different settings, including checklists and algorithms for clinical care and treatment, prognostic parameters, and tests to predict potential for the development of severe disease. • rapid, reliable, simple, low-cost point-of-care diagnostic tools for influenza. • best use of current antiviral drugs and optimal formulations in different target populations, such as parenteral and other routes of administration for severe infections. • use of combination therapies, including use of adjunctive therapies (e.g., use of convalescent serum and immunomodulators). • role of ongoing viral replication, host responses, and the effect of co-infections in the pathogenesis of severe disease. modern tools for early detection and monitoring of disease the group on surveillance tools concluded that the agreed topics of interest were equally applicable during a pandemic or inter-pandemic period: • studies to appraise and adapt modern technologies for early detection of influenza outbreaks in surveillance at the human-animal interface. • develop, integrate, and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems. • study efficient mechanisms on sharing data, clinical specimens, and viruses with consideration for local, ethical, legal, and research perspectives. • examine the timeliness and quality of data required for early detection from local to national and global levels for the respective stakeholders. five priority public health topics were identified for a pandemic scenario as follows: • identify environmental determinants of seasonal variation in influenza transmissibility in tropical and temperate regions. • estimate the transmission risk associated with types of contacts by comparing measured contact patterns with outbreak data. • incorporation of validated models of behavioral responses to risk and control measures in virus transmission. • development and implementation of novel technology for real-time sero-surveillance during a pandemic. • develop experimental and theoretical framework to assess host adaptation to study host receptor, antigenicity, and virulence. modern tools for strategic communication three priority public health topics were identified for a pandemic scenario as follows: • evaluate tools to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups to guide future communication efforts; develop tools and methods to more rapidly and accurately assess and monitor knowledge, attitudes, beliefs, and practices in different population groups, and thereby, guide future communication efforts. for communicating in different cultural settings, which engage and empower individuals and communities to practice and promote appropriate risk reduction measures. implementation of the identified research priorities is expected to underpin public health decision making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs and economic loss, and mitigate potential social disruption. complemented by an analogous research roadmap for a pandemic influenza scenario, the research recommendations for an interpandemic period represent a framework to provide evidence to guide public health policies on influenza control. one of the major lessons learnt from all global pandemic events is that better preparedness of national health systems to deal with influenza viruses could make a significant difference. these public health decisions to ensure the maximum of efficiency require a robust scientific knowledge base. the who public health research agenda for influenza 1 developed by the global influenza programme (gip) in cooperation with international influenza experts identified specific research topics and their importance in meeting public health needs for inter-pandemic periods according to its five key research streams: • stream 1. reducing the risk of emergence of pandemic influenza. • stream 2. limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza. • stream 3. minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza. • stream 4. optimizing the treatment of patients. • stream 5. promoting the development and application of modern public health tools. stream-specific breakout discussion groups during the global consultation meeting included representatives of researchers and public health professionals. funding organizations were invited to observe the process with no direct participation in the deliberations. the methods used to design the research roadmap for an influenza inter-pandemic scenario are closely related to the process of development of the final document of who public health research agenda for influenza. during an inter-pandemic phase, a more comprehensive approach was applied to establish research topics and prioritizing a range of questions that will build a solid foundation to guide research activities to support public health decision making. five to ten key public health needs associated with an inter-pandemic scenario have been identified for each of the research agenda streams: stream 2: limiting the spread of pandemic, zoonotic, and seasonal epidemic influenza ten priority research topics were identified for both pandemic and inter-pandemic scenario as follows: 1. transmissibility of influenza across the progression of infection and spectrum of disease 2. relative contributions of the different modes of transmission for influenza 3. biological, behavioral, and social host factors that influence the risk of transmission and infection 4. patterns, drivers, and mechanisms affecting the seasonality of transmission 5. viral and population factors that influence transmission and spread of different influenza types, subtypes, and strains 6. strategies to reduce the transmission of influenza in community, household, and health care settings, especially in less-resourced areas 7. impact and cost effectiveness of social measures, such as school closures, and the role of surveillance in assessing timing of these interventions 8. impact, effectiveness, and cost effectiveness of individual measures, such as isolation and quarantine 9. role of vaccination in limiting the spread of influenza and strategies for its use 10. impact of antiviral treatment and prophylaxis in reducing transmission of influenza stream 3: minimizing the impact of pandemic, zoonotic, and seasonal epidemic influenza 1. identify higher risk groups and severe disease through surveillance; disease severity and identification of predictors of severe outcomes 2. evaluate vaccination preventable disease burden and the potential impact of immunization programs through vaccine demonstration projects 3. enhancement of the properties of existing vaccines, including duration and breadth of protection, safety, immunogenicity, and dosesparing 4. development of new vaccines and vaccine platforms, especially suitable for under-resourced country settings 5. study the effectiveness of vaccine strategies to reduce disease burden in children and other high risk groups in a wide range of settings 6. improved uptake and acceptability of vaccines for both seasonal and pandemic influenza seven priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: inter-pandemic seasonal influenza scenario 1. research on the burden of severe disease with a focus on regionalspecific factors, such as the burden of tb and hiv and optimization of pandemic and management 2. development of new antiviral strategies and validation of surrogate endpoints which may aid in advancing understanding of disease progression 3. further clinical evaluation of current antiviral drugs, particularly in populations at risk 4. integration of seasonal influenza with pandemic preparedness; strengthen surveillance, health care systems, capacity, and preparedness planning 5. improving diagnostics (e.g., multiplex assays for viruses and bacteria), including antiviral resistance testing at point-of-care 6. dissemination of best practices, situation analysis, preparation for next epidemic (e.g., establish protocols for rotating stockpiles of antiviral drugs) 7. increased attention to basic science research such as studying immunomodulatory drugs five priority public health topics were identified for an inter-pandemic zoonotic influenza scenario as follows: inter-pandemic zoonotic influenza 1. antiviral susceptibility of circulating zoonotic viruses (e.g., h5, h9, h7 influenza viruses) 2. reassortment between zoonotic and human influenza viruses and the potential for inter sub-type spread of antiviral resistance and virulence modern tools for early detection and monitoring of disease the group focusing on surveillance tools concluded that the agreed topics of interest were equally applicable during both pandemic and inter-pandemic period: 1. identify modern technologies for early detection of influenza outbreaks as well as their application in surveillance at the human-animal interface 2. develop and evaluate innovative approaches for influenza surveillance and monitoring with other existing disease monitoring systems 3. studies to address challenges on data, clinical specimens, and viruses sharing with consideration for local, ethical, legal, and research perspectives 4. examine the timeliness and quality of data required for early detection from local to regional, national, and global levels role of modeling in public health decision making five priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. integration of genetic and epidemiological data to understand spatiotemporal spread to forecasts evolution for vaccine strain selection and to anticipate likely burden of disease 2. quantifying the relative contributions of different modes of transmission of human influenza and developing mechanistic modeling of transmission processes 3. research using data-capture technologies to characterize human contact and mobility patterns at local, regional, and global scales, and their correlation with transmission risk 4. integration of genetic, antigenic, and epidemiological analyses to optimize surveillance for newly emerging pathogens at the animal ⁄ human interface 5. identifying and quantifying human and environmental ecological, behavioral, and demographic determinants of the risk of cross-species transmission and pandemic emergence modern tools for strategic communication four priority public health topics were identified for an inter-pandemic seasonal influenza scenario as follows: 1. review of evidence and experience related to health crisis communication from fields to organize knowledge and support evidencebased practice in strategic communication 2. identify and develop tools to rapidly and accurately monitor knowledge, attitudes, and practices in different population groups and guide future communication efforts 3. identify and develop communication tools and approaches for cultural settings and communities to practice and promote appropriate risk reduction measures 4. understand the potential ethical, social, economic, and political communication in crisis and develop strategies to work within constraints while maximizing opportunities complemented by an analogous research roadmap for a pandemic influenza scenario, the research topic recommendations for an inter-pandemic period represent an important outcome of joint international efforts by who, academicians, and public health experts. implementation of the identified research priorities is expected to underpin public health decision-making at all levels with proven knowledge that will help to save large numbers of lives, reduce health costs, and economic loss and mitigate potential social disruption over a medium-tolong term period. the impacts of school resumption on the incidence of pandemic (h1n1) 2009 in school students introduction school closure is one non-pharmaceutical intervention that is often suggested in pandemic preparedness plans, and it was widely implemented in pandemic (h1n1) 2009 to reduce transmission amongst school students. however, from past epidemiological studies, the effect of school closure in reducing respiratory disease transmission was inconclusive. 1 given this public health intervention causes major disruption to the education system and potentially raises childcare issues to working parents, evaluating its effect in the recent pandemic is necessary to improve future pandemic planning. in hong kong, since school closure was implemented early in the pandemic and closure was effectively continued with the commencement of summer holiday, the lack of incidence data in the absence of school closure makes it difficult to analyse its effect directly. this has prompted us to analyse the situation indirectly from the angle of school resumption after summer holiday. in hong kong, public health surveillance on pandemic (h1n1) 2009 was effective from 25th april-30th september 2009: healthcare professionals were advised to report suspected cases of infection to centre for health protection, department of health, hksar, for further laboratorial confirmation. demographics of reported cases were subsequently recorded into a computerised system (the ''e-flu'' database). following institutional approval, a dataset of all confirmed cases diagnosed from may to september 2009 was obtained, which included the age, gender, confirmation date, and notification date of each report. all cases were classified into four defined socio-economic classes by age: pre-schoolers (0-5), school students (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) , adults (20-60), and retirees ( ‡61). assuming cases had contracted infection on the earlier date between confirmation and notification, daily incidence in each age class was counted for epidemic curve construction. upon observing an unusual rise in the epidemic curve of school students when school season resumed in september, interrupted time series analysis (also known as intervention analysis) 2 was applied to obtain the statistical significance of this observation. the analysis was applied to the incidence in school students from 12th july to 26th september 2009, which covered the period from the start of summer holiday to the end of the 4th week of new school season. incidence in school students before summer holiday was deliberately dropped since not all schools were closed when the school closure policy was effective: all primary schools were closed proactively, whereas secondary schools were individually closed on a reactive basis if students were identified to have contracted the infection. school activity was formulated as a step function, which takes value from 1st september 2009 onwards (st = 0: t < 1st september, st = 1 otherwise). a range of times series models were fitted by the maximum likelihood method and aic (akakine information criterion) was used to select the one with best fit. all computations were performed in sas version 9.2. a total of 3905 (14ae8%) pre-schoolers, 13758 (52ae1%) school students, 8383 (31ae8%) adults, and 338 (1ae3%) retirees were diagnosed with the infection in the surveillance period. the epidemic curves of preschoolers, school students, and adults showed a steady rise from 11th june onwards when local transmission of pandemic influenza was identified. an upsurge in the epidemic curve of school students can be observed in early september, coinciding with the commencement of the new school year (figure 1) . interrupted time series analysis on the epidemic curve of school students returned an arima(0,1,0) model with equations: where st, yt, yt denote school activity, predicted and actual incidence in school students on day t, respectively. standard error and significance for model constants were: 60ae27 (se = 33ae5, p = 0ae08), 2ae73 (se = 3ae84, p = 0ae48). in short, the model can be interpreted as: the number of infected school students rose by 2ae73 per day on average during the entire study period, with a sharp increase by 60ae27 coming into effect when the new school year began. time series analysis showed, at the marginally significance level, that daily incidence in school students had a major increase when school season resumed. on the assumption that the increase was not caused by any change in health seeking behaviour, this result suggests that school resumption had facilitated transmission amongst school students. on the basis that school activity significantly increases incidence of pandemic influenza in school students, this study suggests closures of schools in the early phase of pandemic (h1n1) 2009 and subsequently in the summer holiday probably had a major effect in mitigating transmission amongst school students. youngsters were postulated to be major vector for transmission in pandemic (h1n1) 2009. if this were true, it would be reasonable to expect the epidemic curves of the other age classes to show a similar upsurge when one is observed in school students. the absence of such observation in the epidemic curve of hong kong suggests school students were mostly disseminating the virus amongst themselves, but not to the other age groups. in november 2009, gip convened the first global consultation on a public health research agenda for influenza to identify key research topics in each of the five main streams of public health research. during this meeting, the scientific working group (swg) of the sub-stream in ''modern tools for risk communication'' identified the requirements in research during influenza pandemics and inter-pandemic periods to provide clear, credible, and appropriate messages which meet the needs of diverse communities. the swg suggested that who hold a follow-up workshop to assess the use of modern tools related to strategic and risk communication and to further promote research in these areas. communication'' in may 2010. one of the main objectives of the meeting was to generate a roadmap of public health research priorities related to strategic and risk communication. the research roadmap was developed by the group of invited experts on the basis of an analysis of available evidence and experience on public health and health crisis communication from relevant disciplines across global regions, as well as critical assessment of existing communication methods related to influenza control in different cultural, social, and ethnic settings. the workshop consisted of a series of presentations by experts in relation to experiences and lessons learned about communication during the sars, h5n1 epidemic, and h1n1 pandemic. there were also a series of group discussions on identifying research needs for pandemic and interpandemic periods in order to strengthen the research agenda. the expert group identified important public health needs in relation to communication during pandemics as well as in the inter-pandemic times. the main topics of discussion centered on communicating issues of influenza virus transmission, the use of influenza vaccines safety and efficacy, and use of antivirals as well as definition of the severity of the pandemic and the phase changes. in this context a number of research areas were identified, which can be broadly classified into four areas: understanding of communication principles and mechanisms is associated with an array of research topics covering different subject areas. one of the key questions here relates to the link between communication and ''behaviour change'' models and their application and appropriateness for different settings. the expert group defined the term ''behaviour change'' in this context as the modification of behaviour towards better health practices that are supported by clinical and scientific evidence for personal protection against infectious diseases and other adverse health risks. research topics related to these models require understanding and differentiating information and ''behaviour change'' needs of different audience segments, such as stakeholder mapping, target audience analysis, research into behaviour motivation, social norms, and the cultural, religious, social, legal, and political barriers and enablers of particular behaviors that are beneficial in influenza control. this research area also includes the analysis of media consumption among different audiences, role models, including ways to analyse how rumours and misinformation are spread, and ways to provide evidence-based information correctly. other important areas of investigation embrace methods to communicate uncertainty, learning how to build trust while communicating about a pandemic, and understanding what needs to be done before, during, and after a pandemic in order to create the best environment for influenza pandemic communication. critical key audiences identified for more intensive analysis were health workers, religious, public health, and societal (political and community) leaders. • investigation of the role of different communication channels and communication formats for different target audiences in a pandemic, particularly for groups that are ''hard-to-reach.'' • determining effects of perceptions related to pandemic influenza (severity, susceptibility, response efficacy, self efficacy, perceived social norms) on protective behaviours in different groups. • understanding audience in terms of their knowledge, preventive activities, and reasons why engaged ⁄ not engaged. • developing mechanisms to synergies between risk communication and behavior oriented approaches in the pandemic and inter-pandemic phases. • determining social, economic, cultural, and religious factors which support behaviours to limit spread and minimize impact in different settings. • identification of the key predictors ⁄ factors that influence people's behavior among different groups and populations vis-à -vis pandemic flu behaviors. • identification of elements that contribute to trust among populations and in different settings (country, public, professional, community), particularly where trust was previously compromised. • understanding psychology of different groups regarding their response to uncertainty, and finding the best way to communicate uncertainty. the research questions in this section relate to the planning, development, and evaluation of tools that can be quickly accessed and used in a pandemic situation. these may include communication materials and channels; the setting up of key stakeholder and champion communication networks; research protocols that are ready for rapid assessment during a pandemic or new communication tools. the use and understanding of terminology and language by both lay and professional groups and communities in planning for and ⁄ or reacting to a pandemic are important areas of research. acute examples, such as the naming of the viruses or the use of the word ''pandemic,'' illustrate this need well. the research focus of this area is to look at lessons learned from the a(h1n1) 2009 pandemic and to document and evaluate case studies, both looking at best practices, challenges, and barriers that were experienced. different communication strategies need to be evaluated and models to be built not only in terms of reach, but also in terms of impact on thinking, emotional response, and behavioural modification. a key question was how to prepare communication for a pandemic and how can the pandemic communication contribute to longer term ''behavioural change.'' mathematical modelling on gauging outcomes of such ''behaviour change'' would provide strategic approaches in risk communication. this section aims to answer the question whether the modeling, mapping, and scenario planning are actually useful in the pandemic situation. the expert group agreed that the research on the above issues should use a variety of methods and engage a number of disciplines. this would include literature reviews, case studies, trials, ethnographic studies, modelling, surveys, network analysis, as well as any other useful methodology. in an inter-pandemic situation for actual behaviour under pandemic conditions. • study the synergies and develop priority research topics on strategic ⁄ risk communication for influenza under inter-pandemic situations that includes zoonotic and seasonal infections. the who public health research agenda for influenza initiated and facilitated a multi-disciplinary discussion for communication during pandemic and inter-pandemic situations. it focused on both theoretical and practical issues to improve practice and ensure the health of the public for influenza. critical areas for research were identified to build evidence in this field. it was recognized that there are extensive bodies of knowledge in a number of disciplines, 2,3 such as health promotion, behavioural psychology, social sciences, social and behaviour change communication, social marketing, and communication for development relating to these questions, and that these should be explored. outcomes of these research activities are expected to widen the evidence base which will support developing communication strategies for influenza by countries, institutions, and individuals and will, consequently, help to improve public health world-wide. abstract background: cytokine dysregulation contributes to the unusual severity of h5n1 (reviewed in 1 ). previously, we demonstrated that interferon regulatory factor 3 (irf3) and p38 map kinase (p38) signaling pathways separately contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 here we investigate the role of innate sensing receptors in the induction of these cytokines and chemokines in response to h5n1 and seasonal h1n1 infection. materials and methods: human macrophages derived from peripheral blood monocytes were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses. the role of innate sensing receptors in cytokine and chemokine induction by h5n1 virus was investigated using transient knock-down of these receptors with sirnas. the expression of innate sensing receptors in infected cells, and as a result of paracrine activation (by virus free supernatants of infected cells) of adjacent uninfected cells were also monitored by real-time pcr and ⁄ or western blotting. the involvement of janus kinase (jak) signaling pathways in these autocrine ⁄ paracrine cascades was investigated using a jak inhibitor. results: we previously showed that tnf-alpha, ifn-beta, and ifn-lambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with other cytokines and chemokines being induced as part of a secondary autocrine and paracrine cascade. here we demonstrated that retinoicacid-inducible gene i (rig-i) rather than toll-like receptor 3 (tlr3) plays the predominant role in h5n1-induced cytokines and chemokines in human macrophages via the regulation of irf3 and nf-kb nuclear translocation. in addition to the effects on virus infected cells, paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the upregulation of sensing receptors. conclusions: h5n1 directly induced tnf-alpha and ifnbeta mainly via rig-i signaling, and the subsequent activa-tion and nuclear translocation of irf3 and nf-kb in human macrophages. in addition to the effects on cytokine signaling, the innate immune sensing regulators themselves were also up-regulated by h5n1 infection, much more so than by seasonal influenza infection, via jak signaling. the up-regulation of innate sensing receptors was not limited to the infected cells, but was also found in adjacent uninfected cells through paracrine feedback mechanisms. this may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. a more precise understanding of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. human h5n1 infection is associated with a mortality rate of more than 60%. the basis for the unusual severity of h5n1 disease has not been fully explained. cytokine dysregulation has been suggested to contribute to the disease severity of h5n1 (reviewed in 1 ). however, signaling pathways involved in the cytokine induction by h5n1 virus are not fully understood. previously, we demonstrated that irf3 and p38 map kinase (p38) are separate signaling pathways which contribute to the induction of pro-inflammatory cytokines and chemokines in h5n1-infected cells. 2 rig-i and melanoma differentiation-associated gene 5 (mda5) are important cytosolic sensors of nucleic acid of pathogens, while tlr3 and tlr8 also recognize nucleic acid species of pathogens, but they are localized at the endosomal membrane. 3 rig-i was found to be responsible for the recognition of influenza a virus infection, 4 and the transfection of vrnps induces ifn-beta expression. 5 while many studies have shown the role of rig-i in the induction of ifn-beta by influenza virus infection, the majority of these studies used either immortalized cell lines or mouse embryonic fibroblasts. there is a lack of data on the role of these innate sensing receptors in highly pathogenic avian influenza h5n1 infection in primary human cells in vitro, which are more physiologically relevant. furthermore, there is little data on the autocrine and paracrine up-regulation of these innate immune sensors following virus infection. human macrophages were obtained from peripheral blood monocytes by adhesion and differentiation in vitro for 14 days in rpmi medium supplemented with 10% autologous plasma. the cells were infected with h5n1 (483 ⁄ 97) or seasonal h1n1 (54 ⁄ 98) viruses at a moi of 2ae0. a549 cells were obtained from atcc and cultured in mem medium supplemented with 10% fcs and 1% penicillin and streptomycin. the role of innate sensing receptors in cytokine induction by h5n1 and h1n1 viruses was investigated using transient knock down of these receptors with sirnas in human macrophages as previously described 2 using specific sirnas purchased from qiagen. immunofluorescence staining assay of irf3 and nf-jb was employed to detect the nuclear translocation of these transcription factors after h5n1 infection. rabbit polyclonal antibodies against human irf3 and and nf-kb were obtained from santa cruz biotechnology. goat anti-rabbit igg antibody conjugated with alexa fluor 488 was a product of molecular probes. for investigation of paracrine effects on rig-i and tlr3 expression, culture supernatants collected from mock, 54 ⁄ 98 or 483 ⁄ 97 infected human macrophages were used to treat uninfected cells. the supernatants were first passed through a filter with 100-kda cut-off. virus particles as well as molecules with a molecular weight higher than 100 kda were retained and removed, while the filtrate was collected for treatment of uninfected cells. the expression of innate sensing receptors in infected cells and in adjacent uninfected cells following paracrine activation by virus free supernatants of infected cells was monitored by real-time pcr. the involvement of jak signaling pathways in these paracrine cascades was investigated using a jak inhibitor (calbiochem). we previously showed that tnf-alpha, ifn-beta, and ifnlambda 1 are the key mediators directly induced by the h5n1 virus in primary human macrophages with others being induced as part of a secondary autocrine and paracrine cascade. 2 in this study, we demonstrate that knockdown of rig-i or tlr3 led to the reduction of ifn-beta and tnf-alpha in human macrophages by both 54 ⁄ 98 (h1n1) and 483 ⁄ 97 (h5n1) infection. as shown in figure 1a , 483 ⁄ 97 virus induced higher level of ifn-beta mrna expression than 54 ⁄ 98 infection. cells transfected with rig-i or tlr3 sirna significantly reduced the expression of ifn-beta after 483 ⁄ 97 infection, by 63% and 29%, respectively. rig-i silencing also significantly reduced the ifn-beta expression in 54 ⁄ 98 infected cells by 52%. in contrast, silencing of mda5 or tlr8 did not suppress the induction of ifn-beta by either 54 ⁄ 98 or 483 ⁄ 97 infection; in fact, there was a slight (15%) increase of ifn-beta in cells transfected with mda5 sirna. based on these results we conclude that while both rig-i and tlr3 contribute to h5n1-induced interferon-beta induction in human macrophages, rig-i plays the dominant role. in order to investigate the relationship between these innate sensing receptors and the activation of transcription factors irf3 and nf-jb, we next measured the nuclear translocation of irf3 and nf-jb in cells with rig-i or tlr3 silencing after h5n1 infection. immunofluorescence staining assay on irf3 and nf-jb was performed and the number of cells with nuclear translocation was quantitated. the percentages of cells with nuclear translocation were plotted in figure 1b . we demonstrated that rig-i knockdown led to a significant reduction of irf3 nuclear translocation after 483 ⁄ 97 infection, whereas the nuclear translocation of nf-jb after 483 ⁄ 97 infection was significantly suppressed by rig-i or tlr3 silencing. these results suggest that the involvement of rig-i and tlr3 in the cytokine induction by 483 ⁄ 97 was via the regulation of irf3 and nf-jb nuclear translocation. since rig-i and tlr3 are important in influenza a virus-induced cytokine expression, we next explored the expression of these innate receptors in neighboring uninfected human macrophages by treating the uninfected macrophages with the filtered culture supernatants collected from mock, 54 ⁄ 98, or 483 ⁄ 97 infected macrophages. as shown in figure 2a , 483 ⁄ 97 supernatant differentially induced the mrna expression of rig-i, mda5, and tlr3 compared to 54 ⁄ 98 supernatant treated human macrophages. the induction of rig-i was higher than the induction of mda5 and tlr3. in the presence of 1 lm of jak inhibitor, the up-regulation of all three innate sensing receptors was significantly reduced showing their induction was dependent on jak activity. human lung epithelial a549 cells were also treated with the supernatants collected from macrophages infected with mock, 54 ⁄ 98, or 483 ⁄ 97 virus. differential induction of rig-i, mda5 and tlr3 by 483 ⁄ 97 supernatant compared to 54 ⁄ 98 supernatant treated cells was observed (figure 2b) . 483 ⁄ 97 supernatant dramatically induced all three innate sensing receptors, while 54 ⁄ 98 supernatant only marginally induced rig-i and mda5, but not tlr3. as in human macrophages, treatment with 1 lm of jak inhibitor caused a significant suppression of 483 ⁄ 97 supernatantinduced rig-i, mda5, and tlr3 expression in a549 cells. these results, taken together with the direct effects on virus infected cells, suggest that paracrine interactions between macrophages and alveolar epithelial cells contributed to cytokine cascades via modulation of jak signaling and by the up-regulation of innate sensing receptors. h5n1 directly induced ifn-beta ( figure 1 ) and tnf-alpha (data not shown) mainly via rig-i signaling and the consequent activation and nuclear translocation of irf3 and nf-kb in human macrophages. these results were consistent with a previous study using beas-2b cells showing the essential role of rig-i in ifn-beta reporter activity by h3n2 influenza virus infection. 4 while tlr3 also played a role in induction of ifn-beta and the activation of irf3 and nf-kb, it plays a less important role compared to rig-i. the reduction of irf3 and nf-kb activation was also confirmed with the study by le goffic 4 showing differential regulation of irf3 and nf-kb by rig-i and nf-kb can also be regulated by tlr3. in addition to the direct role of rig-i and tlr3 in sensing and signaling the presence of influenza virus, the innate immune sensing regulators were themselves also highly upregulated in both infected (data not shown) and adjacent uninfected cells by influenza virus infection. compared with seasonal h1n1 virus, the h5n1 viruses had a much more dramatic effect on inducing innate sensing receptors via jak signaling pathways activated by autocrine and paracrine mediators. the up-regulation of rig-i, mda5, and tlr3 was markedly induced by virus free culture supernatants from h5n1-infected macrophages, while supernatant from 54 ⁄ 98-infected cells induced the expression of these receptors only to a lesser degree. the soluble mediators in the virus infected cell supernatant caused paracrine upregulation of rig-i, mda5, and tlr3 in uninfected macrophages as well as human lung epithelial cells. these effects may lead to broadened and amplified cytokine signals within the microenvironment of the infected lung. taken together these results provide, at least, part of the explanation on the hyper-induction of cytokines in h5n1 infection. a more precise identification of the signaling pathways triggered by h5n1 virus leading to cytokine induction may provide novel options for the design of therapeutic strategies for severe human h5n1 influenza and also for treating other causes of acute respiratory disease syndrome. we generated mutants of y55 (h9n2) and a ⁄ duck ⁄ hokkaido ⁄ vac generation and characterization of mutant viruses rgy55sub (h9n2), rgvac1sub (h5n1), and rgvac1ins (h5n1), which have a serial basic amino acid residues at their ha cleavage sites were generated by site-directedmutagenesis and reverse genetics. rgy55sub (h9n2) and rgvac1ins (h5n1) required trypsin to replicate in mdck cells, and showed similar levels of growth to their parental viruses (table 1) . chickens intravenously inoculated with rgy55sub (h9n2) or rgvac1ins (h5n1) did not show any signs of disease. rgvac1sub (h5n1) replicated in mdck cells without exogenous trypsin, and one of the eight chickens inoculated with the virus showed slight depression at 1 day post-infection. the h9 and h5 mutant viruses were serially passaged in the air sacs of chicks to assess their ability to acquire pathogenicity. plaque formation in mdck cells and pathogenicity in 3-day-old chicks and 4-week-old chickens are shown in table 1 . rgy55sub (h9n2) replicated in mdck cells in the absence of trypsin and killed all of the chicks after six consecutive passages. two of the eight-four-weekold chickens inoculated intravenously with rgy55sub-p8 (h9n2) died within 5 days. eventually, over 75% of the chickens intravenously infected with rgy55sub-p10 (h9n2) died by 2 days post inoculation, and its pathogenicity was comparable to that of hpaivs. 10 rgvac1sub-p1 (h5n1) was pathogenic to both chicks and 4-week-old chickens, and mortality increased after one more passage. rgvac1ins-p1 (h5n1) replicated in mdck cells in the absence of trypsin, killed all of the chicks, and caused 75% mortality among 4-week-old chickens. the lethal effect of rgvac1ins-p1 (h5n1) on chickens increased with one additional passage in the air sacs of chicks, as in the case of rgvac1sub (h5n1). to examine whether the pathogenicity of each virus via the natural route of infection correlated with that by intravenous infection or not, three 4-week-old chickens were challenged intranasally with the viruses at an eid 50 of 10 6ae5 and observed for clinical signs until day 14 post-infection (data not shown). all chickens inoculated with rgy55sub-p10 (h9n2) or its parental viruses survived without showing any clinical signs, and serum antibody responses were detected in the hi test. on the other hand, rgvac1sub-p2 (h5n1) and rgvac1ins-p2 (h5n1) were pathogenic as in the intravenous experiment, killing two of three chickens by day 11 post-inoculation. one of three chickens were not infected with rgvac1sub-p2 (h5n1) or rgvac1ins-p2 (h5n1) via intranasal route (data not shown), indicating these p2 viruses had not been completely adapted to the host. to investigate the possibility of these p2 viruses to acquire further pathogenicity for chicken, rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were obtained from the brain homogenates of the chickens that died on 11 days post intranasal inoculation with the p2 viruses. although mortality rate of chickens inoculated with the p3 viruses was equal to that with p2 viruses, enhancement of pathogenicity was observed in intranasal inoculation study; all of the chickens inoculated with rgvac1sub-p3 (h5n1) were infected, and time to death was shortened to 4-6 days post inoculation in chickens with rgvac1ins-p3 (h5n1) (data not shown). to investigate whether tissue tropism of the viruses was involved in their pathogenicity, we determined viral titers in the tissue and blood samples from 4-week-old chickens intranasally inoculated with each virus on 3 days post infection ( table 2) . rgy55 (h9n2) and rgvac1 (h5n1) were scarcely recovered from the samples, and the mutant strains before passage showed broader tissue tropism than the parental viruses. none of the chickens inoculated with rgy55sub-p10 (h9n2) showed any signs of disease, and viruses were recovered from each of the samples except the brain and the blood. one chicken inoculated with rgvac1sub-p2 (h5n1) showed clinical signs such as depression, and viruses were recovered from virtually all of its organs and blood samples. two of three chickens inoculated with rgvac1ins-p2 (h5n1) showed disease signs, and one died 2 days post inoculation. the viruses were recovered from almost all samples of the two chickens showing signs of disease. p3 viruses were efficiently replicated in systemic organs of the chickens as compared with p2 viruses. throughout the study, the viruses were recovered from the brains of all of the chickens showing clinical signs. here, we demonstrated that the h9 influenza virus acquired intravenous pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the ha and passaged in chicks. rgy55sub-p10 (h9n2) killed 75% of chickens infected intravenously, and its pathogenicity was comparable to that of hpaivs (table 1) . however, chickens intranasally inoculated with rgy55sub-p10 (h9n2) did not show any clinical signs of disease (data not shown). these results are consistent with a previous study in chickens that found some h10 influenza viruses did not show intranasal pathogenicity although their intravenous pathogenicity index was over 1ae2, classified as hpaiv according to the definition by european union. 11 ohuchi et al. 12 reported that the insertion of additional basic amino acids into the h3 ha cleavage site resulted in intracellular proteolytic cleavage. other groups reported that h3 and h6 has tolerated amino acid mutations into their cleavage sites, and the viruses with the mutated has replicated in mdck and ⁄ or qt6 cells in the absence of trypsin. 13, 14 the results in the present study is in agreement with these, namely, cleavage-based activation by a ubiquitous protease is not restricted to the h5 and h7 has. the intranasal pathogenicity of the h9 and h5 mutants were different (data not shown), although these viruses similarly replicated in mdck cells in the absence of trypsin and killed chickens by intravenous inoculation ( table 1) . the viruses were recovered from the brain and the blood of some chickens infected with rgvac1 mutants (h5n1), and morbidity was closely associated with viral titers in the brain (table 2) . on the other hand, no viruses were recovered from the brain of chickens infected with rgy55 mutants (h9n2), explaining why rgy55sub-p10 (h9n2) did not show intranasal pathogenicity. all the viruses passaged in the air sacs of chicks killed chicken embryos by 48 hours post allantoic inoculation (data not shown). rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) were more pathogenic to chicken embryos than rgy55sub-p10 (h9n2); the allantoic fluid obtained from the embryonated eggs inoculated with the h5 viruses passaged in air sacs was turbid. it has been reported that infection of a highly pathogenic h7 virus were strictly confined to endotherial cells in chicken embryos or chickens. 15, 16 therefore, it is suggested that endotheliotropism differed between the h9 and h5 viruses passaged in air sacs and affected their intranasal pathogenicity. taken together, it is assumed that rgvac1sub-p3 (h5n1) and rgvac1ins-p3 (h5n1) showed marked intranasal pathogenicity with high levels of viremia caused by replication in vascular endothelial cells, leading to invasion of the brain. in the intravenous experiment, rgy55sub-p10 (h9n2) easily reached systemic organs, including the brain hematogenously, replicated through the cleavage of ha by a ubiquitous protease, and then exerted its pathogenicity. further study including a pathological analysis is currently underway to test this hypothesis. for all hpai viruses of subtypes h5 and h7 known to date, the cleavage of ha occurs at the c-terminal r residue in the consensus multibasic motifs, such as r-x-k ⁄ r-r with r at position p4 and k-k ⁄ r-k ⁄ t-r with k at p4, and leads to a systemic infection. early studies demonstrated that the ubiquitously expressed furin and pcs are activating proteases of hpai viruses. 1 furin and pcs cleave the consensus multi-basic motif r-x-k ⁄ r ⁄ x-r with r at position p4. 2 however, replacement of p4 r by k and a nonbasic amino acid significantly suppresses the processing activities of furin and pcs. 2 most of the type ii transmembrane serine protease identified so far recognize a single r at position p1, but the newly isolated mspl and its transcript variant tmprss13 preferentially recognize paired basic residue, particularly r and k at position p4, at the cleavage site. [3] [4] [5] thus, mspl and tmprss13 can activate various bioactive polypeptides with multibasic residue motifs, including fusogenic viral envelope glycoproteins. the present study was designed to characterize the proteolytic processing of the hpai virus ha by mspl and tmprss13 in comparison with furin. hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) 6 was isolated from embryonated eggs inoculated with tracheal homogenates from dead crows. then, the mutant ha sequence was constructed by changing r residue to k residue (n'-rkkr-c' to n'-kkkr-c') at the ha cleavage site by sitedirected mutagenic pcr as described. 7 we used human cell line ecv304, which expresses mspl and tmprss13 at levels below detection, and established the cells stably expressing mspl and tmprss13, such as ecv304-mspl and ecv304-tmprss13. 7 to determine the cleavage specificities of mspl ⁄ tmprss13 and furin, peptides (20 lg each) were incubated with 0ae25 mu mspl ⁄ tmprss13 for 1 hour and furin for 8 hours at 37°c, respectively. after incubation, the samples were separated by reverse-phasehigh-performance liquid chromatography (rp-hplc) with the use of a c 18 column. the elution samples were then identified by amino acid sequence analysis and by maldi-tof-ms. we analyzed the cleavability of 14-residue synthetic peptides derived from ha cleavage sites of hpai strains, such as a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 (h5n2) 8 and a ⁄ fpv ⁄ rostock ⁄ 34 (h7n1), 9 and low pathogenic strain a ⁄ aich ⁄ 2 ⁄ 58 (h3n2). after incubation with human mspl or human furin, the digested samples were separated by rp-hplc, and peptide fragments were characterized by mass-spectrometry and protein sequencing. in contrast to the low cleavage efficiencies of the h3 ha peptide with a single r at the cleavage site ( figure 1a) , both the h5 ha peptide with the k-k-k-r motif ( figure 1b ) and the h7 ha peptide with the r-k-k-r motif ( figure 1c) were fully processed at the correct positions by mspl within 1 hour. in the case of h7 ha peptide with multiple basic residues, mspl cleaved two carboxyl-terminal sides of r in the cleavage site sequence of n'-k-k-rfl-k-k-rfl-g-c', while furin cleaved only at a single site of r with r at position p4, n'-k-k-r-k-k-rfl-g-c' in the presence of 1 mm cacl 2 . these cleavage site specificities of furin were consistent with that reported for the h5 ha peptide of hpai virus a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) with r-k-k-r motif. 1 however, the h5 ha peptide with k at position p4 ( figure 1b) was hardly cleaved by furin under the same experimental conditions. tmprss13 showed similar results (data not shown). these findings suggest that mspl and tmprss13 cover diverse cleavage specificities, including non-susceptible specificity to furin. full length recombinant ha of hpai virus with kkkr cleavage motif was converted to mature ha subunits with membrane-fused giant cell formation in mspl or tmprss13 transfectant cells. 7 in addition, this conversion was suppressed by bowman-birk trypsin inhibitor, a membrane non-permeable highmolecular mass inhibitor against mspl ⁄ tmprss13. to test for the generation of infective virus, the conditioned media of 1-day culture of ecv304-wt and ecv304-mspl cells infected with wt and mutant hpai h5n1 viruses were inoculated into newly prepared cells and cultured for 24 hours. although spreading of wt virus infection with ha cleavage motif of r-k-k-r was detected from the conditioned medium of both ecv304-wt and ecv304-mspl cells, that of mutant virus with ha cleavage motif of k-k-k-r was only detected from the condition medium of ecv304-mspl cells. these results strongly suggest that the expression of mspl, but not furin, potentiates multicycles of hpai virus with k-k-k-r ha cleavage motif. seasonal human influenza a virus has have consensus monobasic cleavage site sequence, n'-q ⁄ e-x-rfl-g-c', and all hpai virus has have two types of cleavage site sequences with multiple basic amino acids, n'-r-k ⁄ r-k ⁄ r ⁄ x-rfl-g-c' with r at position p4 in a large number of hpai viruses and n'-k-k ⁄ r-k ⁄ t-rfl-g-c' with k at position p4 in a small number of hpai viruses. figure 1 shows furin efficiently cleaved synthetic hpai a ⁄ hong kong ⁄ 156 ⁄ 97 (h5n1) ha cleavage site peptide with the r-k-k-r motif, but hardly cleaved the hpai virus a ⁄ chick ⁄ penn ⁄ 1370 ⁄ 83 ha cleavage site peptide with the k-k-k-r motif. furthermore, cleavage of the full-length ha of hpai virus with r-k-k-r motif was detected, but cleavage of hpai virus ha with k-k-k-r motif was hardly detected in ecv304-wt cells containing furin ( figure 2 ). these substrate specificities of furin suggest that proteases other than furin and pc5 ⁄ 6 play a role in the processing of has of hpai virus with k-k ⁄ r-k ⁄ t-r cleavage motif. mspl and tmprss13 show unique cleavage site specificities of the double basic residues at the cleavage site, and r or k at position p4 greatly enhanced the efficiency, which none of the other ttsps have shown similar substrate specificities so far. furthermore, infectious and multicycle viral replication along with ha processing was also noted in genetically modified mutant recombinant live hpai virus a ⁄ crow ⁄ kyoto ⁄ 53 ⁄ 2004 (h5n1) with k-k-k-r cleavage motif in ecv304-mspl cells (figure 2) . these results were supported by the data of two cleaved peptides by mspl in figure 1c . these findings suggest that mspl has diverse cleavage specificities and may cleave ha at least two sites, although multiplicity of the mutant hpai virus was observed under the conditions. these results also suggest that mspl and tmprss13 in the membrane might potently activate the ha membrane fusion activity of hpai viruses and promote their spread. highly pathogenic avian influenza viruses replicate in various organs in birds, and the ha processing proteases might be widely distributed in these organs. indeed, tmprss13 and mspl are ubiquitously expressed in almost all human organs tested and are highly expressed in lungs, leukocytes, pancreas, spleen, and placenta. 4, 5 in addition, mspl and tmprss13 are strictly localized in the plasma membranes, suggesting that proteolytic activation of hpai virus ha occurs not only through the trans-golgi network by furin and pc5 ⁄ 6, but also on the cell surface by mspl and tmprss13. the pb1-f2 protein, which is translated from the +1 reading frame of the pb1 gene segment, has been linked to the pathogenesis of both primary viral and secondary bacterial infections in a mouse model. 1-3 a mitochondrial targeting sequence is located in the c-terminal portion of the pb1-f2 open reading frame, and expression of full length pb1-f2 has been associated with mitochondrial targeting and apoptosis in a monocyte dependent manner. 1, 4 it has been theorized that enhanced virulence could result from mitochondrial disruption with subsequent cell death mediated by pb1-f2. 4, 5 a suggested second function of the pb1-f2 protein is that it enhances immunopathology by triggering the inflammatory response. 3, 6 in earlier studies from our group, the pro-inflammatory phenotype was markedly upregulated when the pb1-f2 from the 1918 pandemic strain was expressed, arguing that this protein may be an important virulence factor for highly pathogenic pandemic viruses. 3, 6 in this report we analyze the pb1-f2 protein's contribution to pathogenesis in a mouse model, examining both inflammation and cell death. pb1-f2 proteins from a variety of epidemiologically important iav strains including all pandemic strains from the 20th century, a highly pathogenic avian influenza virus of the h5n1 subtype, and representative seasonal strains were utilized to determine the relevance to pandemic disease. we demonstrate that macrophage mediated immunopathology, but not apoptosis, are relevant functions of pb1-f2 proteins from past or potential pandemic influenza viruses. using the predicted amino acid sequences of the pb1-f2 proteins from pr8, a ⁄ brevig mission ⁄ 1 ⁄ 1918, 1 ⁄ singapore ⁄ 1 ⁄ 1957, a ⁄ hong kong ⁄ 1 ⁄ 1968, a ⁄ wuhan ⁄ 359 ⁄ 1995, and a ⁄ vietnam ⁄ 1203 ⁄ 2004, peptides from the c-terminal end were synthesized as described. 3 an additional n-terminal peptide was synthesized from the pr8 sequence as a positive control (mgqeqdtpwilstghistqk) as described. 3 a panel of viruses were reverse engineered as described 7, 8 and included laboratory strain pr8, a virus unable to express pb1-f2 (dpb1-f2 ⁄ pr8), or expressing the pb1-f2 of the 1918 pandemic strain (1918 pb1-f2 ⁄ pr8) or the truncated 1956 h1n1 strain (beij pb1-f2 ⁄ pr8). 3, 7 in addition, 7:1 reassortants encoding pb1 gene segments from a *current address: department of immunology and microbiology, university of melbourne, melbourne, vic., australia. 2004 highly pathogenic avian influenza of the h5n1 subtype (h5n1 pb1 ⁄ pr8), or from a 1995 human h3n2 strain (h3n2 pb1 ⁄ pr8) were utilized along with their isogenic deletion mutants for pb1-f2 (h5n1 dpb1-f2 ⁄ pr8 and h3n2 dpb1-f2 ⁄ pr8). cell lines and cell death assays raw264.7 cells were grown under conditions as described. 9 cells were infected with one multiplicity of infection (moi) of virus for 2-12 hours, or exposed to 50 lm (final concentration) of peptides derived from the c-terminal portion of pb1-f2 for 1 hour. cells from the supernatant and monolayers were harvested, washed, and stained with annexin (apc) and propidium iodide (pi) (becton dickinson, san jose, ca, usa), then analysed for cell death as described. 3 six-to eight week old female balb ⁄ cj mice (jackson laboratory, bar harbor, me, usa) were maintained in a biosafety level 2 facility in the animal resource center and procedures approved by the animal care and use committee at sjcrh. infectious agents and peptides were diluted in sterile pbs and administered intranasally to anesthetized mice (n = 6-10) in a volume of 100 ll (50 ll per nare) and monitored for overt signs of illness and weight loss daily. following euthanasia by co 2 inhalation, the trachea was exposed and cannulated with a 21 gauge plastic catheter (bd insyte; becton dickinson, sandy, ut, usa). bronchoalveolar lavage fluid (balf) was collected, red blood cell depleted, and cellular content analyzed via flow cytometry as described. 3 one way analysis of variance (anova) was used for multiple comparisons of cell death and cellularity of balf. a p-value of <0ae05 was considered significant for these comparisons. graphpad prism version 5.00 for windows (graphpad software, san diego, ca, usa) was utilized for all statistical analyses. to assess the contribution of pb1-f2 to inflammation, we utilized a panel of previously described reverse engineered viruses in the mouse infection model. 3, 7 the effect of pb1-f2 expression was observed clearly in the inflammatory infiltrate in response to infection in the lungs. deleting pb1-f2 from pr8 or expression of the c-terminally truncated beij pb1-f2 had a significantly reduced influx of macrophages ( figure 1a) . expression of the 1918 pb1-f2 caused similar inflammatory effects as the pr8 virus. disruption of pb1-f2 expression the virus containing the h5n1 pb1 gene segment in a pr8 background also significantly decreased the inflammatory response compared to the virus maintaining the ability to express full length pb1-f2 ( figure 1a) . however, no differences were seen that could be attributed to the 1995 h3n2 derived pb1-f2. the lungs of mice infected with the panel of pb1-f2 variant viruses were examined at 72 hours. pathologic changes typical of pr8 viral infection were observed in all lungs. these typical findings included perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris (figure 2 ). in the lungs of mice infected with pr8 or 1918 pb1-f2 ⁄ pr8, however, significantly more perivascular cuffing was noted, with a prominent increase in numbers of macrophages (figure 2a, c) . the overall number of inflammatory cells throughout the lungs, including both airways and alveoli, was quantitatively greater in these mice than in mice infected with dpb1-f2 ⁄ pr8 or beij pb1-f2 ⁄ pr8 ( figure 2b, d) . as the function and influence of pb1-f2 protein on normal viral function is not currently understood, and given the abrogation of enhanced inflammation induced by the truncated pb1-f2 beij ⁄ pr8 virus, we sought to elucidate whether the c-terminal domain of pb1-f2 could alone induce this inflammatory response. mice were exposed to a panel of peptides and were euthanized 24 hours later for collection of balf. significant influxes of macrophages into the balf were seen following exposure to c-terminal pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2), and 1968 (h3n2), and the 2004 h5n1 virus compared to controls ( figure 1b) . similar effects were not seen with the peptide derived from a more recent h3n2 strain, a ⁄ wuhan ⁄ 359 ⁄ 1995. when peptide exposed mice were followed for morbidity for 7 days, peptides proven to induce a heightened inflammatory response correlated strongly with overt clinical signs of illness (data not shown). thus, the ability to cause lung inflammation appears to be a property of pb1-f2 proteins of viruses containing pb1 gene segments reassorted directly from the avian reservoir. the pb1-f2 protein may contribute to virulence by rendering the host cellular immune response ineffective through inducing apoptosis. 5 we sought to determine whether this was an epidemiologically important function for combating the host immune response to infection by testing the ability of pb1-f2 proteins from several different iav strains to cause cell death. we therefore infected raw264.7 cells with the panel of recombinant viruses at an moi of 1 for 2-12 hours. as has been demonstrated previously, 1,4,5 pr8 virus induces significant cell death compared to uninfected controls ( figure 1c ). when raw264.7 cells were infected with pr8 virus, necrotic death peaked 8 hours after infection. viruses lacking the c-terminal portion of pb1-f2, including the dpb1-f2 ⁄ pr8 and the beij pb1-f2 ⁄ pr8 were unable to cause cell death ( figure 1c ). in addition, expression of the 1918 pb1-f2 also did not cause significant increases in cell death over controls. expression of pb1-f2 or deletion of pb1-f2 in either an h3n2 or h5n1 pb1 gene segment background similarly did not alter the cell death phenotype. to examine additional strains for which we did not have isogenic virus pairs, we next exposed the balbcj mouse derived macrophage cell line raw264.7 to the panel of pb1-f2 peptides derived from pr8, the pandemic strains from 1918 (h1n1), 1957 (h2n2) and 1968 (h3n2), and the 2004 h5n1 for 1 hours. cell death in raw264.7 cells was caused only by the peptides derived from the laboratory strain pr8 and the peptide derived from the 1918 pandemic strain ( figure 1d ). viability was not affected by exposure of raw264.7 to peptides derived from other virus strains. we conclude from these data that the mechanism by which pb1-f2 contributes to the pathogenicity of pandemic influenza is unlikely to be through its reported ability to cause cell death. these data presented here demonstrate that the lung inflammatory response is enhanced by the influenza a virus pb1-f2 protein in a mouse model. this inflammatory response was characterized by increased cellular infiltration of macrophages into the interstitial and alveolar spaces of the lungs, as well as enhanced perivascular inflammation, airway necrosis, hemorrhage, and deposition of cellular debris. this augmentation was shown to be induced by pb1-f2 proteins only from those strains contributing to the formation of all pandemic strains of the 20th century and from the currently circulating, highly virulent h5n1 strains that constitute an imminent pandemic threat. the iav h1n1 strains circulating in humans since around 1950 code for a truncated pb1-f2. these viruses may lack the cterminal residues responsible for the inflammatory effects demonstrated in this publication. additionally, recently circulating h3n2 strains, in contrast to their pandemic forbear from 1968, have lost the capacity to cause pb1-f2 mediated inflammation through mutation of the c-terminus of this protein. in 2009 a novel h1n1 iav emerged from an animal reservoir and caused a human pandemic. disease burden from this strain has been considered mild 10 in contrast to the three pandemics of the 20th century. 11 the reasons for this disparity in pathogenesis are unclear. an examination of the origins of the three 20th century pandemics shows that only 2 the hemagglutinin (ha) and pb1 gene segments were reassorted directly from the avian reservoir in every case, suggesting gene products of one or both of these may be important. 12 the ha surface glycoprotein provided the antigenic novelty required for the each virus to achieve pandemic status. however, the significance of inclusion of a novel pb1 gene segment in each of the 20th century pandemics is not yet understood. we show here that the pb1-f2 of these pandemic strains contributes to virulence through induction of inflammatory responses. thus pb1-f2 may serve as a marker of the pathogenicity of pandemic strains. since the 2009 h1n1 strain codes a truncated pb1-f2 of only 11 predicted amino acids, the lack of pb1-f2 mediated inflammation may account in part for its relatively lower virulence. 13, 14 of the panel of pb1-f2 proteins studied, only that from the laboratory strain pr8 was capable of rendering responding host-immune cells ineffective by induction of cell death. we therefore hypothesize that molecular signatures specific to induction of apoptosis may have been lost through genetic mutation of the pb1-f2 gene throughout the evolution of the iavs. our findings suggest that this apoptotic function is unlikely to be important for the virulence of any of the known pandemics. rather, the inflammatory phenotype appears to be the dominant contribution of pb1-f2 to pandemic disease. influenza virus-cytokine-protease cycles are principal mechanisms of multi-organ failure in severe influenza and therapeutic approaches introduction influenza a virus is the most common infectious pathogen in humans, causing significant morbidity and mortality, particularly in infants and the elderly. mof with severe edema is observed in the advanced stage of influenza pneumonia. 1 however, the relationships amongst factors that induce vascular hyper-permeability in severe influenza remain unclear. it is reported that significant increases in levels of pro-inflammatory cytokine levels, such as tnf-a, il-6, and il-1b, affect host survival both positively and negatively. 2 the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and mof. 3 in addition, iav infection up-regulates several cellular proteases including ectopic trypsin 4 and mmp-9. 5 up-regulated ectopic trypsin mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin (ha), 6 which is crucial for viral entry and replication 7 and the subsequent tissue damage in various organs. 8 the aim of the this study was to define the pathogenic impact of cytokine storm in iav infection and the molecular mechanisms by which pro-inflammatory cytokines and proteases cause vascular dysfunction in animal model. weanling female mice aged 3 weeks (c57bl ⁄ 6crslc) were infected with iav ⁄ wsn ⁄ 33 (250 pfu) with and without treatment of pdtc (2.5 mg ⁄ kg), nac (10 mg ⁄ kg), and ndga (10 mg ⁄ kg). these inhibitors were administrated once daily for 4 days after infection. the levels of cytokines in tissue homogenates were measured by elisa kits. the effect of inhibitors on viral replications was determined by real-time pcr. gelatin zymography and western blotting were conducted as reported previously. 9 host cellular responses in the airway after iav infection figure 1 shows schematic view of typical biological responses in the airway of mice after iav infection. an initial response before viral proliferation is significant increases in pro-inflammatory cytokine levels. immediately after cytokine inductions, there is a marked up-regulation of ectopic trypsin along with an increase in virus titer in the airway, lung, and brain. 4 ectopic trypsin mediates the post-translational proteolytic cleavage of iav ha, which is crucial for viral entry and replication and the subsequent tissue damage in various organs. we also found that iav infection markedly induces mmp-9 and matrix degradation. 5 just after the peak of viral proliferation, the innate and adaptive immune responses of protective immunity are induced for defense and recovery, or oppositely on rare occasions, mof with vascular hyper-permeability is started into the advanced stage of influenza. the levels of tnf-a and il-6 in the lungs were increased persistently for 6 days after iav wsn infection, and that of il-1b peaked at days 4-6 post-infection (figure 2a ). since these cytokine responses are associated with activation of nf-jb and ap-1, we treated mice once daily for 4 days with anti-oxidant inhibitors: pdtc and nac against nf-jb activation, and ndga against ap-1 activation. pdtc and ndga significantly suppressed the up-regulation of tnf-a and il-1b (p < 0.001), and nac suppressed tnf-a (p < 0.001), and il-6 (p < 0.01) at day 4 post-infection. gelatin zymography showed up-regulation of ectopic trypsin and mmp-9 in mice lung, brain, and heart during infection for 4 days ( figure 2b ). trypsin and mmp-9 induction was inhibited by treatment with pdtc, nac, and ndga, probably via blockade of nf-jb and ap-1 binding in the promoter region of the genes. viral rna replication in various organs at day 4 post-infection was suppressed by more than one order of magnitude by pdtc, nac, and ndga ( figure 2c ). suppression of viral multiplication and induction of cellular factors by pdtc, nac, and ndga, significantly improved the survival of mice at day 14 post-infection, the late stage of infection ( figure 2d ). to elucidate the mechanisms underlying brain vascular dysfunction of influenza-associated encephalopathy, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 (zo-1) and transmembrane occludin, and the matrix protein laminin, were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 post-infection, which were partly rescued by pdtc, nac, or ndga (figure 2e ). 9 no other tight-junction protein, claudin-5 or matrix fibronectin and type iv collagen, were affected. the present study reports several new observations: (i) proinflammatory cytokines, tnf-a, il-1b, and il-6, when up-regulated by iav infection, induce trypsin and mmp-9 expression in various organs in mice; (ii) inhibitors of nf-jb and ap-1 effectively suppress the up-regulation of proinflammatory cytokines, trypsin, and mmp-9 and improve survival rates of infected mice. based on these results, we propose the 'influenza virus-cytokine-protease cycle' hypothesis as one of the mechanisms of vascular dysfunction in mof with cytokine storm in severe influenza and influenza-associated encephalopathy. 9 the significance of pro-inflammatory hyper-cytokinemia, or 'cytokine storm,' in the pathogenesis of iav infection remains unclear. on the positive effects, cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, on the negative effects of excess cytokines, the hyper inflammatory process evoked by viral infection may become harmful through intracellular activation of nf-jb, ap-1, and the janus kinase-signal transducers and activators of transcription signaling pathways. 3, [10] [11] [12] the in vivo experiments presented here showed that nf-jb and ap-1 inhibitors markedly suppress the expression of cytokines, trypsin, mmp-9, and viral replication, resulting in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production ⁄ activation of vasodilatory mediators such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs. 3 the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyper-permeability following the 'cytokine storm' remain unclear. tnfa up-regulation alters the cellular redox state, reduces the expression of four complex i subunits by increasing mitochondrial o 2 ) production and depleting atp synthesis, decreases oxygen consumption thereby resulting in mitochondrial damage, 3, 13 and increases [ca 2+ ] i 14 atp depletion dissociates zo-1 from the actin cytoskeleton and thereby increases junctional permeability. 15 endothelial dysfunction induced by 'influenza virus-cytokine-protease cycle' in the early stage of severe influenza may further affect various circulating factors, coagulation factors and complement systems, and vascular interacting cells, such as neutrophils, macrophages and lymphocytes. mof is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway. 16 in addition to these data, up-regulated trypsin and pro-inflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of iav infection. further studies are required on the role of the 'influenza virus-cytokine-protease cycle' in the pathogenesis of mof, particularly in the late stage of viral infection. though influenza a virus replication kinetics and host responses have been previously studied in umbilical vein endothelial cell or transformed endothelial cell lines, the tropism of influenza a virus including h5n1 and pandemic h1n1pdm for primary human lung microvascular endothelial cell has not been well defined. 1 in this study we employed primary human lung microvascular endothelial cells, which are more physiologically relevant for understanding pathogenesis of influenza in the lung as to obtain a better understanding of the links of endothelial cell infection to systematic virus dissemination and multiple organ involvement in severe human influenza. supernatants of cells infected at moi of two were collected for cytokine protein assays, and total rna was extracted for gene expression analysis using qpcr. we found that seasonal influenza h1n1 and h3n2 viruses initiated viral gene transcription and viral protein expression, but did not produce infectious progeny, while the highly pathogenic avian influenza h5n1 and the pandemic influenza h1n1pdm virus could replicate even with the absence of exogenous protease (figure 1) . furthermore, when compared to seasonal h1n1 and h3n2, the h5n1 virus was a more potent inducer of cytokine and chemokine including ifn-b, mcp-1, rantes, ip-10 (figure 2) , and il-6, in virus infected endothelial cells, whereas h1n1pdm induced intermediate levels of cytokine and chemokine. avian influenza h5n1 and pandemic h1n1pdm virus (but not the seasonal h1n1 and h3n2 virus) can productively replicate in human lung microvascular endothelial cells. this is likely to be of relevant to pathogenesis and provides a possible explanation for the extra-pulmonary infection seen in animal infection models. this extra-pulmonary spread may support the previous speculation and anecdotal evidence that h5n1 and h1n1pdm virus can infect the gastrointestinal tract through the virus dissemination from the infected respiratory tract as the first target cells for influenza infection. [2] [3] [4] in addition, the release of proinflammatory cytokine and chemokine induced by influenza h5n1 and h1n1pdm virus infection in lung microvascular endothelial cells may be important contributors to the pathogenesis of severe human influenza disease leading to endothelial cell dysfunction that contributes to severe pulmonary disease symptoms. during its replication, influenza virus utilizes the host cellular machinery for many aspects of its life cycle. characterization of such virus-host protein-protein interactions is a must to identify determinants of pathogenesis. the m2 ion channel protein plays a crucial role during the entry and late stages of the viral life cycle where its c-terminal domain, well conserved among influenza a viruses, is accessible to cellular machinery after fusion with endosomal membrane and during its trafficking along the secretory pathway prior to assembly and budding. 1 the aim of the study is to identify cellular interactants of m2 that play important regulatory roles during influenza infection. to identify cellular partners of m2 we performed a genome-wide yeast-two-hybrid (y2h) screening approach 2 using the cytosolic domain of m2 as bait and a human placenta random primed cdna library as prey and tested more than 60 million interactions. from the y2h screening, an interesting interaction with the human annexin a6 (anxa6) protein, 3 a member of annexin family proteins that binds to phospholipds in a ca 2+ -dependent manner, was identified. co-immunopre-cipitation of myc-tagged anxa6 and viral m2 proteins coexpressed in hek293t cells after transfection and infection confirmed the direct interaction between anxa6 and m2. we further investigated whether this interaction had any functional significance with regards to influenza life cycle. using a rna interference strategy to silence the anxa6 gene in human lung epithelial a549 cells, we observed increased progeny virus titers either in a single or multiple viral growth kinetics study, suggesting a negative regulatory role for anax6 during viral infection (figure 1 ). a novel interaction between m2 and anxa6 was identified. more functional studies are in progress to define precisely the potential negative regulatory role of this interaction during viral infection. a systematic dissection of the viral life cycle will be performed to identify the step(s) affected by the anxa6 cellular factor using specific assays such as real-time quantitative rt-pcr in a single or multiple viral growth kinetics study, cell transduction with ha-and m2-pseudotyped lentiviral particles, virion attachment and internalization assay, immunofluorescence staining of np protein as a marker of viral ribonucleoproteins localization, viral polymerase activity measurement, and viral budding observation by electron microscopy. rna extraction was achieved by qiagen biorobot ez1 prior to respiratory multiplex pcr analysis. what remained of the extracted material of each specimen was stored by refrigeration at 4°c. electronic patient records were searched for parameters, such as c-reactive protein (crp), white cell count (wcc), length of admission in days, and patient co-morbidities. patients were divided into three groups according to clinical severity: mild, moderate, and severe. the 'mild' group comprised of those admitted for three days or fewer, or not admitted at all. the 'moderate' group comprised those who required admission to hospital for more than 3 days as a result of swine flu, but who did not require admission to an intensive care unit (itu). the 'severe' group comprised those who had required itu admission. invitrogen '2· reaction mix': 0ae4 mm of each dntp + 6 mm magnesium sulphate. primer ⁄ probe mix recipe applied biosystems 7500 fast real-time pcr system, 'respiratory multiplex' program. well content 25 ll; thermocycler initial stage 50ae0°c for 15 minutes, then 95°c for 2 minutes. subsequent cycles of 95ae0°c for 15 seconds followed by 60°c for 33 seconds for 45 cycles. sequence detection software version 1.4 (applied biosystems). of 126 clinical isolates analyzed, all samples produced amplification of pdh material; 100 produced amplification of both swine flu and pdh material. human male dna (lot no. 36048611039 at 10 ng ⁄ l, applied biosystems) at concentration calculated at 16 666ae6 cells ⁄ ll was diluted from 10 )1 to 10 )4 , yielding mean average ct values of respectively 30ae10, 33ae60, 37ae78, and 37ae22. plotting log of cell number versus ct gave a y = mx + c line from which ct could be interpolated into cell numbers. for swine flu quantification, a sample of swine flu ct 19ae28 was diluted through 10 )1 to 10 )10 . it must be noted that due to variability in resultant swine flu ct values, repetitions at these dilutions were done using an rna carrier (1350 lg ⁄ l, qiagen; cat no. 1017794) in place of rnasefree water. the 10 )4 concentration was positive in nine out of 15 assays; this fraction was used in the calculation described by simmonds 2 to obtain a copy number of targets per reaction by the equation copy value = )ln(f), where f is decimal fraction of failure rate. here, f = 6 ⁄ 15 = 0ae4; )ln0ae4 = 0ae916 copies. a control curve was generated with ct values of 26ae73, 30ae02, 33ae67, and 37ae23 giving copy values of 916, 91ae6, 9ae16, and 0ae916, respectively. using excel (microsoft office, 2010), these control series were adapted into formulae to convert swine flu and pdh ct values into copy numbers of these elements per reaction. simple division derived a value for swine flu copy per pdh copy, but this was chosen to be expressed as swine flu copy number per 100 human cells. this will be referred to as the 'c' value. forty-two patients had known clinical details; average age was 29ae74, female to male ratio 63:37, and average admission length of 7 days. of the mild group (n = 26), nine cases were not admitted to hospital. of the remainder, the mean average admission length was 2ae5 days. mean average c value for all samples was 1ae49 · 10 6 , with a standard deviation of 1ae49 · 10 7 ; geometric mean was 4ae28, and median average was 6ae45. log(mean average c value) is shown for each severity group and for identified risk factors in the 'mild' severity group (figures 2a, b respectively) . in each case variation was too great to yield statistical significance. figure 1 shows the range of c values observed in the 'moderate' severity group. > -4 · 0 ; < -4 · 5 > -3 · 5 ; < -4 · 0 > -3 · 0 ; < -3 · 5 > -2 · 5 ; < -3 · 0 > -2 · 0 ; < -2 · 5 > -1 · 5 ; < -2 · 0 > -1 · 0 ; < -1 · 5 > -0 · 5 ; < -1 · 0 > -0 · 0 ; < -0 · 5 > 0 · 0 ; < 0 · 5 > 0 · 5 ; < 1 · 0 > 1 · 0 ; < 1 · 5 > 1 · 5 ; < 2 · 0 > 2 · 0 ; < 2 · 5 > 2 · 5 ; < 3 · 0 in a study by duchamp et al., 3 no significant correlation was observed between viral ct value and presence or absence of cardiaorespiratory disease, myalgia, digestive symptoms, or upper or lower respiratory tract infection (although a trend was observed towards patients presenting with signs of upper respiratory tract infection). to our knowledge, no other study has used a dual pcr for analysis of respiratory virus concentrations, and no study has attempted to correlate biochemical markers with respiratory virus concentration. the data exhibited a spectrum of c values, from values <5 · 10 )5 to over 1 · 10 8 . the three severity group standard deviations all overlapped with each other, preventing statistical significance. analysis of co-morbidities showed a high mean average c value when asthma was present (6ae05 · 10 7 ), but again this was associated with an excessive standard deviation. whereas the median average c value in the presence of asthma was higher than the overall average c value (9ae09 versus 5ae85), it was significantly lower than the median c value when no co-morbidity was documented (15ae55). there are multiple caveats that may be the cause of such variety of c values obtained. the duration between initial rna extraction and study pcr had a range of 21 to 285 days, with mean average delay of 211 days. the degradation of viral rna is an important contributor to assay variance and failure; rna degradation in clinical samples has been studied. [4] [5] [6] degradation of human dna in clinical samples may have occurred. several studies have chartered degradation of stored human dna. 2, 7 with regards to sampling, the clinical collection of throat swabs is naturally variable according to the method of the collector. a small number of bronchoalveolar lavage samples were analyzed, yet did not amplify, presumably due to rna degradation. the upper respiratory tract may be only a physical stepping stone for the virus, and take no further role in pathogenesis of severe disease (although undoubtedly is crucial for transmission). interestingly, a ferret study of pathogenesis observed that swine flu yields from the upper respiratory tract were greater than those given by ordinary seasonal h1n1, with consequently increased shedding. 8 the review by mansfield 9 cites significant findings regarding influenza pathogenesis, including the predilection of h5n1 strains for type ii pneumocyte cells and alveolar macrophages. it also highlights the limitation of knowledge through dearth of human autopsy studies; an exception is the recognition of haematophagocytic syndrome in severe cases. it is known that specific immunoglobulin is effective against establishment of infection in the upper respiratory tract, whereas specific cytotoxic t lymphocytes (ctls) are necessary for clearance of the virus from the lower respiratory tract. 10 it is also suggestive that a gap of two whole days transpires between initial infection and instigation of a specific immune response. 11 it is plausible that in the healthy individual, virus progression is confounded by efficient natural mucosal immunity, in part through good secretory immunoglobulin levels. airway inflammation associated with asthma exacerbation is known to increase both risk of respiratory viral infection and poorer outcome. it is unproven but likely that the local inflammatory processes give rise to increased virion burdens in the upper airways; however, the same effect is conceivable for epithelial cell turnover. there will likely be variance within each clinical category due to patient circumstances and clinicians' judgment of required admission. unfortunately, the duration of symptoms prior to swab collection was often omitted in the clinical notes. finally, stratification of patient group by receipt of antiviral treatment was not studied. no correlations were observed with c values and crp, wcc or admission length. trends were observed towards higher c values in 'mild' cases, but without statistical significance. the relative small study size, coupled with the intrinsic variability of the parameters studied, warrants larger, better controlled, prospective studies to elucidate clinical use of the c value for influenza illness prediction and management. in mid-april 2009 a novel variant of a(h1n1) influenza virus began to spread rapidly throughout the world, causing the first pandemic of the 21st century. the majority of the cases associated with this new virus show to be mild, but severe and fatal cases have been reported. molecular markers associated with severity have already been identified, as is the case of the mutation d222g. 1 resistant viruses to antiviral drugs have also been identified, highlighting the importance of rapid determination of the antiviral drug profile. global a(h1n1) 2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. the objective of this ongoing research study was, primarily, to thoroughly characterize the genetic profile and evolution of the emergent influenza a(h1n1) 2009 virus circulating in portugal and its phenotypic expression on antiviral drugs susceptibility. the cases considered in this study were obtained from the community and from two collaborating hospitals in lisbon -a reference hospital for adults (hospital de curry cabral) and a reference hospital for children (hospital dona estefânia). the cdc real-time pcr protocol, recommended by world health organization (who), was the method used to confirmed all influenza a(h1n1) 2009 cases. from a total of 577 a(h1n1) 2009 positive cases diagnosed and confirmed, 163 were selected for this study, taking in consideration that they should cover the period of epidemic activity in portugal and include cases from persons belonging to risk groups and cases associated with more severe clinical features. ninety-six a(h1n1) 2009 strains were isolated in mdck-siat1 cells, from combined naso-oropharyngeal swabs. for the evaluation of the genetic profile of a(h1n1) 2009 virus circulating in portugal, 37 of the 96 isolates were characterized by genetic analysis of the ha, na, and mp genes. the remaining five gene segments (pb1, pb2, pa, ns, and np) were also sequenced for six of this 37 isolates. briefly, sequencing was performed according to the protocol developed by cdc and recommended by who, 2 using bigdye terminator v.1.1 technology. nucleotide sequences were determined in a dna automatic sequencer abi prism 3130xl genetic analyzer. for each genomic segment, genetic analysis was performed with lasergene v.4.05 software (dnastar inc, usa) using an average of 4-6 overlapping readings, including sense and antisense, for precise nucleotide and amino acid sequence determination. genetic mutation and phylogenetic analysis were performed by neighbor-joining method, using mega4.0 software, against published sequences from the vaccine strain (a ⁄ california ⁄ 7 ⁄ 2009) and from selected a(h1n1) 2009 strains available on gisaid epiflu database. all mutations were identified with reference to the vaccine strain genome sequence. antiviral drug susceptibility profile of a(h1n1) 2009 influenza virus circulating in portugal was evaluated both phenotypically and genotypically for nais and genotypically for amantadine. phenotypic evaluation to nais, oseltamivir and zanamivir, was performed for all 96 isolates by ic 50 determination through munana fluorescence assays. 3 genotypic evaluation was performed by searching for mutations associated with resistance to nais in all 37 na gene sequences. amantadine susceptibility profile was performed for all 96 isolates by searching on m2 sequence for the 5 molecular markers associated with resistance to this antiviral drug (l26f ⁄ i; v27a ⁄ d; a30t; s31n; g34e). genetic characterisation of the ha1 subunit of ha reveals point mutations in different strains. all 37 analysed strains present p83s and i321v mutations, which distinguish them from the vaccine strain ( figure 1a ). thirty-three of the 37 sequenced strains group in the s203t branch. this mutation is referred in the literature as being associated with the putative antigenic site ca. 4 most of these strains (19) further subgroup in the d222e branch, this mutation being associated with one loop of the receptor-binding site. 1 from the early to the late epidemic period, an increased circulation of virus carrying the mutation s203t was observed. this is in agreement with the association between this mutation and an enhanced viral fitness that is described in the literature. 5 additional mutations were also observed in a small number of virus, of which we highlight: regarding the genetic characterisation of na, the majority of strains analysed (34 of 37) presents the mutations n248d and v106i ( figure 1b) . as mutation s203t in ha gene, these two na mutations are described in the literature as associated with enhanced viral fitness. 5 the few strains not carrying these mutations have circulated in the beginning of the epidemic period. fifteen of the 37 analysed strains further subgroup in y155h branch. additionally, mutation i223v was identified in two strains. for the remaining gene segments available for the six analysed strains, the observations include: (i) no previously described virulence markers in pb2, pb1-f2, and ns1 were detected; (ii) pb1-f2 protein is present in the truncated form of 11 amino acids; (iii) the presence of mutations i123v and l122q in ns1 and v100i in np; (iv) the described association of mutation i123v in ns1 and v100i in np genes with viral fitness. phenotypic evaluation of nais susceptibility revealed the existence of three minor and two major outliers to oseltamivir ( figure 2 ). the two minor outliers exhibited a reduction of approximately twofold in the susceptibility to this antiviral drug, comparing to the baseline level, while the reduction exhibited by the two major outliers was of approximately three-and fourfold. regarding zanamivir, two minor outliers were identified with a reduction of approximately twofold in the susceptibility, compared to the baseline level. these two minor outliers (a ⁄ portugal ⁄ 17 ⁄ 2009 and a ⁄ portugal ⁄ 82 ⁄ 2009) correspond to the two major outliers identified for oseltamivir. genetic analysis revealed the presence of the mutation i223v in the na sequence of these two strains. the contribution of this mutation for the profile of reduced susceptibility identified for both nais is not known, but a mutation in the same na position (i223r) has been referred to as being associated with a reduction in nais susceptibility. 6 full genome sequence analysis of these strains shows that both strains also present the v480i mutation in pb2 gene. however, no association of this mutation with antiviral drug susceptibility is referred in the literature. concerning genetic evaluation of susceptibility to amantadine, all 96 analysed strains present a serine in position 31, which is a molecular marker of resistance to m2 inhibitors. these preliminary results allow us to discuss several points. however, the additional data that is being obtained through this ongoing study will be essential for a more complete analysis. for example, more information is needed to determine if the mutations found alter the biology and the fitness of the virus or if there are associated with an increased prevalence of the virus. the majority of the mutations identified in ha1 subunit have been detected in a(h1n1)2009 strains distributed throughout the epidemic curve, not evidencing a specific evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1)2009 virus. 7 the occurrence of mutations in the position 222 of the ha1 subunit of a(h1n1)2009 virus have been described. however, more studies are needed to clarify the outcome of these mutations, as for example in patients with severe complications. it could also be relevant to investigate the presence of single and mixed variants in viruses and in clinical specimens and the possibility of these mutations affecting the binding specificity. regarding the susceptibility of a(h1n1)2009 pandemic viruses to antiviral drugs, all analysed strains were found to be resistant to amantadine. this resistant profile was not unexpected since the mp gene from this new variant had originated in the eurasian swine lineage, which is characterised by being resistant to this antiviral drug. 8 the majority of the a(h1n1)2009 strains analysed revealed to be susceptible to both nais, with only five strains exhibiting a profile of reduced susceptibility, three to oseltamivir and two to both nais. for these last two, the presence of the i223v mutation in the na sequence could explain the reduction observed, but a more complete analysis is needed to confirm this. the french national pandemic plan includes an early containment phase followed by a limitation phase. the efficacy of such a plan depends on pre-existing surveillance and laboratory networks. the grog community surveillance network and the hospital lab networks organized by the two french nics carried out the virological monitorthe efficacy of such plan depends on pre-existing influenza surveillance and laboratory networks. in france, the community surveillance is carried through the grog surveillance network. in addition, surveillance is also carried out in hospitals by the renal network. this renal network is divided in two sub-networks: the so-called h5-labs network, activated during the containment phase and the extended renal lab network activated in the limitation phase. the h5-labs have bsl-3 facilities that can be used for diagnosis purposes. as part of the national influenza surveillance system led by the french institute for public health surveillance (invs), the grog community surveillance network and the lab networks linked to the two french nics carried out the virological monitoring of the a(h1n1)2009 pandemic from the early containment phase up until the end of the pandemic phase. during the containment phase, all suspected cases were hospitalized and declared to invs. each patient was tested on the same day by specific virological diagnosis. hospital admission was not mandatory during the limitation phase, (i) the clustered cases were monitored to study transmission chains, and (ii) the circulation of the virus in the community was monitored through grog swabs collected by practitioners. the nics organized the influenza surveillance to fulfill several objectives according to the epidemiological situation. first, rt-pcr tools (influenza a m gene rt-pcr and a(h1n1)2009 specific h1 and n1 genes rt-pcrs) were developped and distributed to the lab networks on the 14th of may 2009. 1 from the early phase, the nics and the h5-lab network analyzed all the samples collected from hospitalized and community patients. during the early phase of the limitation phase, an increasing number of labs were performing the specific assays. when the pandemic wave started, all hospital labs could do the testing. results were centralised by nic and reported on a weekly basis. in addition, nics carried out the monitoring of antiviral resistance emergence (na pyrosequencing, specific h275y rt-pcr, and phenotypic assays), and real-time surveillance of genetic changes involved in virus adaptation (pb2) virulence factors or antigenic variations (ha). this sequencing was carried out by the pf8 sequencing platform of the institut pasteur. the first imported a(h1n1)2009 influenza cases were observed from the 28th of april 2009. a limited number of cases have been reported in may. local transmission could be detected end of may. clusters were observed in schools in june and in summer camps during summer. as opposed to the epidemiology of the a(h1n1)2009 virus in other european countries, no summer wave was observed in france. only a limited number of sporadic cases were reported up until october. early september, a significant number of cases presenting with influenza-like illness was reported (figure 1 ). the virological investigation of these cases showed high prevalence of rhinovirus infection. this circulation of rhinovirus was a counfounding factor of the pandemic. the pandemic wave lasted 11 weeks between mid-october and the end of december (week 43 to week 53, figure 1 ). the pandemic wave started week 42-43 in the ile-de-france area, and only week 44-45 in the rest of france. the peak was recorded week 48 ( figure 1 ). the impact of the pandemic was mainly observed in the 5-15 years group of age. overall, 1334 severe cases have been admitted to the hospital, and 308 deaths have been recorded by the end of the pandemic wave. the major impact was observed in the 15-65 years group of age (66% of deaths recorded). amongst the severe cases and the deceased cases, 20% and 16% of cases had no risk factor, respectively. these specimens, 24 279 were positives for h1n1, representing 99ae7% of total influenza virus detections. only nine brisbane-like h1n1, 62 brisbane-like h3n2, and eight b viruses have been detected in the same period of time. the weekly positive rate ranged from 0% to 48%. phylogenetic and antigenic analyses of the viruses collected during the pandemic wave did not show any emerging genetic or antigenic variants (figure 2a,b) . eight patients, all among cases presenting with severe illness, were infected by a virus harbouring the d222g mutation in the ha. 2 amongst the virus tested for antiviral susceptibility or screened for the h275y mutation by or specific rt-pcr, only 11 oseltamivir-resistant viruses related to the na h275y mutation have been detected. one of these cases also had an i223r mutation associated to a reduced sensitivity to zanamivir. all but one resistant virus were detected in treated immunocompromised patients. overall, eight patients presented a virus with the d222g mutation in the ha. all these patients had a severe infection; one of these had also a h275y mutation in the na asociated to oseltamivir resistance. the pandemic started by the end of april 2009. although the first cases recorded were as early as the 28th of april, the epidemic wave associated with a widespread spread of the virus was only recorded in october. the french population did not have to face a summer wave, as observed in north america and in numerous european countries. 3, 4 it is difficult to speculate the reasons for the lack of summer wave; the specimens collected were negative for influenza. moreover, during september, it was anticipated that school openings would be the trigger for the beginning of the pandemic wave. as a matter of fact, a significant increase of influenza-like syndromes were observed at that time, but the virological investigation carried out by the laboratories showed thta is was related to a very large epidemic of rhinovirus. 5 the epidemic circulation of other respiratory viruses can be counfounding factors for the surveillance of the influenza epidemic clinical when the survellance is only based on collection of clinical information. the starting of the pandemic wave was heterogeneous in france. the ilede-france region (paris and its suburbean area), where the population is dense, experienced an early start as compared to the rest of france. however, once the pandemic started in the rest of the county, the epidemic curves were quite similar. the peak was reached at identical times, although it may have been delayed in some remote places in france. overall, we estimate that 10% of the french population consulted for an ili presentation. the impact was mainly observed in the 5-15 years groupe of. however, this age groupe represented only a limited number of severe cases and deaths. on the other hand, the 15-65 years groupe of age, where the prevalence was not high, was the age group where the majority of severe cases and deaths was recorded (74% and 66%, respectively). 6 this data is consistent with the observational data reported by numerous other countries. 7 according to the profile of hospitalized cases, a(h1n1)2009 was more aggressive than seasonal viruses. the number of admission to the hospital was ten-fold that observed during a normal influenza epidemic. even if the mortality was limited (312 cases), the age distribution of the deceased patients was different as compared to seasonal influenza (75% mortality in <65 years of age). the lack of recordeable excess mortality has been interpreted to be the consequence of a very mild pandemic, milder than some seasonal epidemics. however, the median age of the fatal cases was much younger than those observed during the seasonal flu, leading to a mis-interpretation of the real impact of the pandemic. when the impact is measurered in loss of years of life, the impact of this pandemic is larger than seen with seasonal influenza, and is quite comparable to these of the two last pandemics. 8 the pandemic preparadness of numerous countries, the develoment of new intensive care techniques and equipment, and the large use of antivirals have reduced the overall impact of this pandemic. these are new factors that should be taken into account when evaluating the real impact of the 2009 h1n1 virus. 8 the virological monitoring of the pandemic was achieved by the community-based and hospital-based seasonal influenza networks, reminding the importance of maintining such networks. the diagnosis of influenza in most of the patients was carried out by molecular techniques. it has been clearly stated from the beginning of the pandemic that near-patient tests were lacking of susceptibility and could not be used for patient management. the distribution of a set of validated and comprehensive techniques by the two nic was very helpfull for the monitoring of the pandemic and the patients. however, this diagnostic procedure change should not preclude maintaining virus isolation that is necessary for whole genome analysis, monitoring of antigenic changes, and phenotypic testing for antiviral testing. some of the mutants that have been recorded, including viruses with antiviral resistance phenotype or genotype, could be analysed from grown virus strains. it is striking that despite a large antiviral usage, only a limited number of isolates had mutations associated to resistance. however, the frequent isolation of such resistant virus was observed in immunocompromised patients that presented severe infections and long virus shedding. 9 the impact of the pandemic is still under evaluation. sero-epidemiological analysis will be performed to asses for the real attack rate of the 2009 pandemic virus. as in other countries, it has been recorded that asymptomatic infections could be observed frequently. 10 it is quite unlikely that the impact of the pandemic was reduced by the vaccination campaign, although this vaccination started on the 12th of november, just when the pandemic started in france. it is estimated that 6 millions received the vaccination. pandemic strains of the influenza virus sporadically emerge, deviating from the regular endemic strains of seasonal influenza. in april 2009, a novel pandemic influenza virus a ⁄ h1n1 emerged, swiftly spreading across the world. immediately, domestic and international public health agencies were forced to develop containment and mitiga-tion strategies in response to the pandemic. however, the dynamics and transmission patterns of this novel virus are yet to be fully understood. simultaneously, seasonal strains of influenza (a ⁄ h1n1, a ⁄ h3n2, and b) continued to circulate in many nations. both pandemic and seasonal variants of influenza are responsible for significant morbidity and mortality. 1 to characterize the dynamics of this disease and the variation within strains, a more detailed understanding of the patterns in viral shedding during natural infection is required. the majority of data on the patterns of viral shedding during influenza infection are a result of volunteer challenge studies. 2 in these studies, volunteers are commonly screened for pre-existing immunity against the challenge strain and are of a certain demographic and age. information on the patterns of viral shedding in natural influenza infections, pandemic or seasonal, is limited but should provide greater generalizability. we describe the trends of viral shedding and clinical illness in community acquired cases of pandemic and seasonal strains of influenza. in 2008, a community-based study was conducted to analyse the effectiveness of non-pharmaceutical interventions to prevent the spread of influenza in households. 3 in 2009, a similar community-based study was initiated to collect comparative data from individuals infected with seasonal and pandemic influenza. 4 both studies were conducted with very similar protocols, involving 617 households in total. the specimens and symptom data required for this study all arise from secondary infections ascertained in these two community-based studies. the recruitment process in both studies was essentially identical. index cases were first recruited from their healthcare provider if they presented with influenza-like illness (ili). this individual would be included in the follow-up if he ⁄ she tested positive for influenza virus infection by rapid antigen test (quickvue) and was the first person in his ⁄ her household that showed signs of ili in the previous 2 weeks. follow-up consisted of three home visits that spanned approximately 7-10 days. at each home visit, nasal and throat swab (nts) specimens were collected from all household members, regardless of the presence or absence of symptoms. symptoms were recorded in daily symptom diaries provided for every household member, and digital thermometers were provided to record daily tympanic temperature. the symptoms recorded were fever ‡37ae8°c, headache, myalgia, cough, sore throat, runny nose, and phlegm. influenza virus infection and subtype was identified by reverse transcription polymerase chain reaction (rt-pcr) on the nts specimens. viral shedding was quantified from the same specimens by rt-pcr to determine viral loads, as well as by quantitative viral dilutions to determine median tissue culture infectious dose (tcid 50 ). the details concerning laboratory methods have been described in a previous study. 5 all analyses in this study focus exclusively on secondary cases; these are household contacts of recruited index cases who acquire influenza virus infection following the initial home visit. index cases generally presented with a certain threshold of illness severity requiring medical attention, whereas infections among household contacts can vary from asymptomatic to severe representing naturally acquired influenza infections. these secondary cases must be negative for influenza for their first nts specimen, and subsequently tested positive. we analysed mean viral loads measured by rt-pcr and quantitative culture by plotting by day since acute respiratory illness (ari) onset according to strain of influenza (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, seasonal a ⁄ h3n2, and seasonal b). ari is the reference time point, because the day of infection is unknown and is defined as the presence of ‡2 of the symptoms mentioned above. average symptom scores were also plotted according to ari onset and grouped into upper respiratory symptoms (sore throat and runny nose), lower respiratory symptoms (cough and phlegm), and systemic signs and symptoms (fever ‡ 37ae8°c, headache, and myalgia). mean daily tympanic temperatures were also plotted since date of ari onset and according to strain of influenza virus. all analyses were conducted using r software (version 2.10.1; r development core team). 6 a total of 617 households and 2499 individuals were followed-up in the two studies. of 1887 household con-tacts tested by rt-pcr, 153 were found to be influenza positive. among these influenza infections, 13 (8ae5%) were asymptomatic (rt-pcr positive plus 0 symptoms recorded), 20 were subclinical (rt-pcr positive plus 1 symptom recorded), and 88 presented with an onset of ari during the follow-up period. from the cases with ari onset, seven pandemic a ⁄ h1n1, 40 seasonal a ⁄ h1n1, 22 seasonal a ⁄ h3n2, and 19 seasonal b influenza virus infections were identified. the age distribution among secondary cases was observed to be largely comparable across the four strains of interest (table 1 ). there were a lower proportion of males who acquired pandemic a ⁄ h1n1 compared to the seasonal strains of the virus. cough was the most commonly reported symptoms during follow-up in cases of pandemic a ⁄ h1n1 and seasonal b, whereas runny nose was most common in seasonal a ⁄ h1n1 and a ⁄ h3n2 cases. cumulatively, fever ( ‡37ae8°c) was reported in approximately half (51%) of the secondary cases. patterns of viral shedding were analysed in a subset of 88 influenza positive individuals who recorded an onset of ari in their symptoms diaries (figure 1 ). household contacts that were asymptomatic, subclinical, or did not have an ari onset were excluded from the analysis. viral shedding in all three influenza a strains were recorded to occur on the day of ari onset or 1 day post-ari onset. following the peak, measured levels of viral shedding declined steadily to undetectable levels over 5-6 days. the trend of viral shedding in influenza b infected individuals rose 2 days before ari onset, fluctuated for around 6 days before eventually resolving. the patterns of viral shedding over time measured by quantitative viral culture were generally similar to the patterns measured by rt-pcr. the patterns of symptoms and signs were comparable in the four strains of influenza included in this study, peaking on the day or 1 day post-ari onset, and gradually declining over a period of 5-7 days. in all strains, systemic symptoms and signs were observed to resolved faster than upper and lower respiratory symptoms. the trend of tympanic temperature in each influenza strain was comparable to the respective symptom pattern. patterns of viral shedding observed in influenza a strain infections (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were broadly similar. the pattern differed from the observed pattern of viral shedding in seasonal influenza b infections. the majority of viral shedding in influenza a strains occurred at and near ari onset, whereas there were variable amounts of viral shedding preand post-ari onset for those with influenza b. the biological reason for this difference is yet to be clarified. these differences are consistently observed regardless of laboratory method used to quantify the viral loads. it was observed that viral shedding measured by tcid 50 resolved more quickly than when measured by rt-pcr, suggesting that rt-pcr is more sensitive, but it could be detecting inactivated fragments of rna instead of active virus. the trends observed for the seasonal strains of influenza in this study were similar to those reported in literature. 2 the patterns of symptoms and signs as well as tympanic temperature in the four different strains of interest in this study were found to be comparable. these patterns closely resemble the patterns of viral shedding observed in the influenza a virus strains, but not in the influenza b virus strain. the trends of viral shedding, symptom scores, and tympanic temperature for pandemic a ⁄ h1n1 were similar to trends observed for seasonal a ⁄ h1n1 and seasonal a ⁄ h3n2 infections, suggesting that the dynamics of these viruses are largely the same. the clinical course of infection with pandemic a ⁄ h1n1 influenza virus appeared to be similar to the seasonal b influenza virus, but the patterns of viral shedding over time diverges. in general, our results suggest that the dynamics of the pandemic a ⁄ h1n1 virus were similar to the seasonal a ⁄ h1n1 and a ⁄ h3n2 viruses, and clinically similar to the seasonal b virus. this study faced sample size limitations; very few cases of pandemic a ⁄ h1n1 were detected and the secondary attack rate in general was low, though a total of 617 households were followed up. this lack of power led to the inability to analyse the differences between adult and children and other characteristics that could be correlated with amount of viral shedding. there are also biases that must be factored in during recruitment. the eligibility criteria of only healthy households could select for households with higher innate immunity. on the other hand, recruitment at health care providers can be biased towards index cases that had more severe illness that required medical attention. the strength of the study is the broad generalizability of the results due to the strict classification of secondary cases. the infections reported in this study were all community-based and should represent true natural infections. pandemic potency of the influenza virus is largely determined by its transmissibility. the first objective of this study was to model the transmission of influenza h1n1 and h5n1 viruses. at present, vaccination with laiv has been used as a widespread, effective public health measure for influenza prophylaxis. some unsubstantiated concerns have been raised about a potential possibility of reassortment of circulating influenza viruses with laiv viruses following vaccination with laiv. thus, another objective of this study was to assess the probability of pig-to-pig transmission of cold-adapted viruses and their potential reassortment with wt influenza strains. female albino guinea pigs weighing 300-350 g were inoculated intranasally with 10 5 eid 50 of virus without anaesthesia. transmission studies were then performed 24 hours after inoculation. inoculated animals were housed at 25% relative humidity and 22°c in the same cage with noninfected guinea pigs or in cages placed 3 m away from non-infected pigs. virus replication was determined by virus isolation in hen eggs and by pcr. sera were collected at 0 and 28 days post inoculation. seroconversions were assessed by routine hai test. genome composition of reassortants was monitored by rflp analysis. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated, and virus growth properties were observed following virus titration in hen eggs. when infected pigs were co-caged with non-infected (naïve) individuals, vn1203, indo ⁄ 5, a ⁄ california ⁄ 7 ⁄ 2009, and nibrg-23 were isolated in 0%, 25% 83ae3%, and 100% of contact animals, respectively. serological confirmation of virus transmission was higher than virological data (25%, 100%, 100%, and 100%, respectively). in addition, it was shown that when pigs inoculated with a ⁄ california ⁄ 7 ⁄ 2009 were co-caged with animals inoculated with nibrg-23, they got infected with both viruses ( table 1) . the ability of direct transmission of cold-adapted viruses was also investigated. data show that the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 laiv candidate was detected in the upper respiratory tract of 87ae5% vaccinated pigs. the mdv was identified in 100% of infected animals. however, neither group of contact pigs, co-housed with the vaccinate pigs, had evidence of infection with cold-adapted viruses. in addition, none of the contact pigs had any evidence of seroconversion to the coldadapted viruses as determined by hai assay. it was also most interesting to note that pig-to-pig transmission of the highly transmittable nibrg-23 reassortant virus was not seen when pigs, vaccinated with mdv, were co-caged with animals infected with nibrg-23 virus (table 1) . this strongly implies a form of interference or protection from transmissibility that was provided by the cold-adapted virus. the results show that nibrg-23 and indo ⁄ 5 viruses were able to spread between cages over the 3 m distance (100% and 50% naïve animals were successfully infected, respectively). a ⁄ california ⁄ 7 ⁄ 2009 influenza and vn1203 viruses did not transmit between infected and non-infected guinea pigs housed in separated cages (table 1) . pigs with confirmed a ⁄ california ⁄ 7 ⁄ 2009 virus replication were also infected with nibrg-23 virus if h1n1-and h5n1-infected animals were separated by a space. thus, influenza virus transmission from h5n1-to h1n1-infected pigs has been shown, but the reverse pattern did not occur. transmission of nibrg-23 or a ⁄ california ⁄ 7 ⁄ 2009 viruses was not observed when contact pigs were first vaccinated with the mdv and housed at a 3 m distance ( table 2) . it was also shown that efficiency of transmission of nibrg-23 was much higher than of other studied h5n1 viruses; it can be transmitted between naïve guinea pigs separated from infected animals at a distance of 4-5 m (data not shown). five reassortants were isolated from animals which were infected with a ⁄ california ⁄ 7 ⁄ 2009 virus and co-caged with pigs inoculated with nibrg-23. two reassortants possessed different combinations of pr8, nibrg-23, and a ⁄ california ⁄ 7 ⁄ 2009 genes and demonstrated the non-ca ⁄ non-ts phenotype typical of wt viruses. unexpectedly, two other reassortants inherited ha gene from nibrg-23, na gene from a ⁄ california ⁄ 7 ⁄ 2009, and other genes from pr8 became ca and ts. 7:1 non-ts reassortant inherited pa gene from pr8 and seven other genes from a ⁄ california ⁄ 7 ⁄ 2009, gained ca properties. in spite of aforesaid experimental data, we cannot exclude the theoretical possibility of simultaneous infection of human host with cold-adapted and wt influenza viruses. to better understand possible consequences of such a reassortment event, we co-infected guinea pigs with a mixture of mdv and nibrg-23 viruses. nasal washes were collected and cloned by limited dilutions in hen eggs in the presence or absence of immune serum to the mdv. cloning of nasal washes without antiserum led to isolation of over 100 clones, which were all identical to the mdv (data not shown). when nasal washes were cloned in the presence of antiserum, only nine clones were isolated. genome composition analysis showed that all isolates were triple reassortants, which had inherited pb2 and na genes from mdv, pa gene from pr8, and ha gene from nibrg-23. the origin of the other gene segments (pb1, np, m, ns) in the genome of guinea pig-derived reassortants varied. reassuringly, all reassortants generated in vivo had the phenotype typical of the mdv. the severity of influenza outbreaks is partly determined by efficient spreading of the causative virus strain between human hosts. however, little is known about mechanisms underlying influenza virus transmission in humans. guinea pigs have been shown to be a suitable model for influenza transmission studies. 1 our in vivo study showed that influenza a viruses vary in their transmissibility. nib-rg-23 and indo ⁄ 5 viruses were able to transmit to naïve animals caged distantly from infected animals. in contrast, cold-adapted viruses, the same as those used for licensed laivs, showed no signs of transmission from one guinea pig to another. our study also provided evidence of a lower level of transmissibility of the novel pandemic h1n1 virus compared to the nibrg-23 and indo ⁄ 5 h5n1 strains evaluated. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 2 in our study, the mdv inoculated into guinea pigs appeared to interfere with and even offer protection from transmission of the highly transmissible nibrg-23 virus. the ability to immunize with the laiv and subsequently block the spread of a homologous h3n2 subtype and a heterologous h3n2 subtype influenza virus between guinea pigs has been shown. 3 interference between cold-adapted and wildtype influenza virus infection was the most likely explanation for the data observed in our study. the mdv inoculated into guinea pigs might in some way interfere with transmission of highly transmissible influenza viruses. it is believed by some that widespread use of laiv could increase the potential risk of reassortment of the vaccine strain with circulating influenza viruses immediately following vaccination. however, it was shown that any such potential reassortments would most likely lead to yet attenuated viruses. 4 our in vivo studies have shown that introduction of mdv genes into the genome of nib-rg-23 virus led to the generation of triple reassortants inherited pb2 and na genes of mdv and ha gene of h5n1 virus. all isolates possessed phenotypical markers associated with attenuation of mdv. our data suggest that even if a reassortment event of such rare occurrence between a laiv strain and a circulating virus were to occur, it would most likely lead to a reassortant that would retain highly attenuated phenotypic properties of the vaccine strain. our data strongly support the safety of laivs, especially those developed against highly transmissible h5n1 and h1n1 pandemic influenza viruses. this information builds upon databases that have clearly shown the low likelihood of transmitting an laiv, as well as the high likelihood of any field reassortment of laiv with a circulating influenza virus to retain important properties of the cold-adapted, temperature-sensitive vaccine master composition. very interestingly, we also present data that show the potential of a laiv to prevent the transmission of highly infectious influenza viruses, perhaps identifying a broader role for laiv in the overall scheme of influenza virus prophylactic use. background: schlieren imaging is a non-invasive, real-time airflow visualization technique that relies on differences in air temperatures (and the resulting changes in the refractive index) to allow exhaled human airflows to be seen clearly against the background of more-stationary, ambient air. recently, this technique, well-known to engineers, has been applied to better understand and characterize airflow behaviors associated with everyday, as well as healthcarerelated, human respiratory activities. materials and methods: as a surrogate marker for the behavior of airborne infectious agents, schlieren imaging was used to visualize the airflow patterns produced by adult human volunteers of different ages while coughing with and without the wearing of standard surgical and n95 masks. results: the cough plumes were generally similar in shape and range for all the adult volunteers used in this study. although both the surgical and n95 masks decelerated and blocked some of the forward momentum of the coughed airflows, much of the cough plume was redirected and escaped around the top, bottom, and side edges of the masks to merge with the volunteer's natural, verticallymoving thermal plume. conclusions: schlieren imaging is a safe technique for visualizing exhaled airflows from human volunteers without the need for potentially-irritant or toxic particle tracers. findings from these schlieren imaging experiments will assist the development of more effective aerosol infection control guidelines in healthcare premises where patients infected with potentially airborne infectious agents (e.g., influenza and tuberculosis) are present. these infectious agents may be transmitted to healthcare workers, other patients, and their visitors by way of exhaled airflows. with the recent influenza pandemic 1,2 and the ongoing concerns about human cases of avian influenza h5n1 infections, 3 there is now a very real concern about the potential for the aerosol transmission of respiratory pathogens. such concerns amongst staff and patients in healthcare environments have led to a greater emphasis on the understanding and control of infectious airflows. 4,5 previous visualization techniques have used potentially-toxic or irritant gas or particulate tracers with hazardous laser light sources that have precluded the use of human volunteers as subjects. instead, various forms of lung models that simulate human respiratory patterns with such particulate tracers have been used. 6, 7 schlieren imaging is a technique familiar to engineers and offers a non-invasive (i.e., no tracer required) airflow visualization method that depends only on differences in the refractive index of the warmer, human-exhaled air and the cooler ambient air. 8 the use of a simple incandescent or light-emitting diode (i.e., non-laser) light source is safe and allows human volunteers to be used as experimental subjects, where their exhaled airflows are then observed using a large, precise spherical or parabolic telescopic mirror and a camera, and are recorded for later analysis and presentation. [9] [10] [11] the analysis of these patterns of 'real-life' human airflows will be useful in optimizing aerosol infection control guidelines, which aim to reduce the transmission of airborne infectious agents to other healthcare personnel, patients, or their visitors. the images and analysis presented here have all been obtained from the large 1 m diameter parabolic mirror (figure 1 ) situated at the gas dynamics laboratory of penn state (directed by gary s. settles). this large schlie-ren imaging system has been in use for over 30 years to obtain high quality schlieren images for various engineering applications. it has only recently been applied to clinically-relevant imaging. the objective of this paper is to augment and expand upon the details of the methods and results presented in an earlier study using this same schlieren imaging system. 10 the aim of this series of studies is to visualize and capture a series of airflow images produced by coughing from adult human volunteers of different ages (24-80 years old). these included males (three of 26 years, one of 80 years of age) and females (one of 24 years, one of 30-40 years, and one of 40-50 years of age). each volunteer was tested with and without wearing either a standard surgical mask or n95 mask. more specifically, the aim was to visualize the extent and direction of leakage around the mask whilst each subject was coughing. penn state institutional approval for experiments involving human subjects was also obtained. each volunteer was asked to stand approximately 1 m in front of the schlieren mirror, facing across the surface of the mirror on one side, and to cough several times as the real-time, color image and video footage was recorded by the operator (using a nikon d90 camera; nikon inc. melville, ny, usa). this process was repeated whilst each volunteer was wearing a standard surgical mask then an n95 mask (supplied by 3mô, st paul, mn, usa). some of the schlieren images obtained from some of these volunteers have been published previously: for a 26-year old male, 9 the 24 year-old female and a 26-year old male, 10 and the 40-50 year-old female. 11 this article completes this series of schlieren images obtained from these experiments by including the images recorded for the older, 80 year-old man. generally, it was found that the shape of the cough plumes (shown in the figure as darker shadows emanating from the subject's mouth) produced by adult humans of different ages was relatively similar. cough plumes are roughly conical in shape and very turbulent, usually passing beyond the extent of the 1 m mirror (figure 2a) . a previous detailed study of one of these images measured a maximum airflow velocity of 8 m ⁄ second for an adult cough. 9 similarly, the effects of wearing surgical and n95 masks can be generalized across different ages. wearing a surgical mask allows leakage of the coughed air from the sides, top, and bottom of the mask ( figure 2b ). there is also some leakage through the mask, as indicated by the darker patches of air directly in front of the mask ( figure 2b, c) . the useful effect of the mask appears to be a deceleration and redirection of this coughed (and potentially infectious) air into the natural, upward-rising human thermal plume, which captures it and carries it upwards where it is diluted and less likely to transmit infection to others. the effects of the n95 mask are similar (i.e., deceleration and redirection), yet due to its tighter (mask-fitted) face seal, more of the coughed air appears to penetrate the front of the mask ( figure 2c ). this penetrating air is, however, also decelerated sufficiently to allow the wearer's natural thermal plume to carry it upwards. 10, 11 discussion from these series of schlieren images presented in this and other related studies, [9] [10] [11] it is clear that schlieren imaging offers a safe, non-invasive, real-time technique to visualize human exhaled airflows for all age groups. it is apparent that, at least where airflow patterns are an acceptable surrogate marker for airborne transmission risks, there are beneficial effects of wearing either type of mask, even when the mask fit is relatively poor. this is often the case when n95-style masks are purchased and used by the general public -in contrast to the situation with healthcare workers, who are often accurately fit-tested for this type of mask. the immediate significance of this can be seen when masks are bought by parents for their children. often, these will not be of pediatric size and the mask-fit will be loose. children are well-known to be major sources of infection in the community because of their relatively poor immunity to many types of infectious agents due to their young age and, therefore, limited past-exposure history. 12 these images allow infection control teams to literally see how far and how fast potentially-infectious human exhaled airflows can travel from an individual. this may have significant implications for guidance on the wearing of masks for infected staff and patients, on ward bed-spacing, as well as for the types of masks to be used in different situations. the important practical potential lies in the non-intrusive visualization of airflows associated with human volunteers, to assist in heightening the awareness amongst healthcare workers of the risks and potential for the airborne transmission of infectious agents, as well as the development of more effective aerosol infection control policies. schlieren images can be analysed more quantitatively, e.g., with the 'schlieren-piv' technique, 9, 13 though this additional quantitative data is probably more of research interest than being of immediate practical use to everyday hospital infection control teams. these are the subtypes that we have studied. clearly, the question arises as to whether the changes in antigenicity are coupled with changes in germicide susceptibility. we have employed a modified log-reduction method 3 in a cell culture system employing mdck cells 4 in serum-free ex-cellô 5 medium supplemented with trypsin. microscopic examination of cpe was the marker for infectivity together with plaque assay. we confirmed antiviral potency by using specific subtype influenza identification subtype technology, quidel quickvue ò influenza a + b test. the log inactivation and percent inactivation by bac after a 60 second contact time for the h1, h2, and h3 pandemic strains are as follows: a ⁄ swine ⁄ iowa ⁄ 12 ⁄ 30 h1n1, 3ae5 log ⁄ 99ae97%; a ⁄ swine ⁄ cal ⁄ 2009 h1n1, 4ae8 logs ⁄ 99ae998%; a ⁄ j305 ⁄ 57 ⁄ h2n2, 5 logs ⁄ 99ae999%; and a ⁄ hong kong 8 ⁄ 68 h3n2, 5ae0 logs ⁄ 99ae999% (table 1 ). comparable results of antiviral efficacy are obtained with the tcid 50 and plaque assays against all subtypes studied. when performing the plaque assay the sensitivity of virus recovery was better in the vessel with a larger surface area and overall recovery was in agreement with the potency determined by tcid 50 assay. in our plaque assay, we inoculated a ⁄ hong kong ⁄ 8 ⁄ 68 virus dilutions into two different vessels with 2 hours adsorption time: 6-well plate and t-25 flask, 9 ml inoculum per replicate. virus titers obtained were: 1ae4 · 10 6 pfu ⁄ ml from 6-well plate and 2ae2 · 10 6 pfu ⁄ ml from t-25 flask ( table 2 ). the discrepancy on virus potency can possibly be explained as: the binding of virus to host cell occurs only when virus gets a chance to interact with the cell on the monolayer during adsorption time. the percentage of virus population in the inoculum that has the opportunity to bind to the cell mainly depends on the surface area where this interaction takes place. therefore, in our experiment the plaque assay in the t-25 flask gave higher virus recovery 2ae2 10 6 versus 1ae4 · 10 6 pfu ⁄ ml. the increased virus recovery can translate into better sensitivity of the test system for disinfectant and antiviral agents. the potency of the virus used in this study was determined by tcid 50 was 5 · 10 6 tcid 50 ⁄ ml. rapid diagnostic testing for influenza (quickvue ò influenza a + b test, quidel) for aj305 versus bac was studied. the presence of influenza viral nucleoprotein a determined by quickvue kit correlated 100% with the viral infection based on by cpe in viral culture. interestingly, the inactivation of viral nucleoprotein was able to be revealed with diagnostic kit in the dilutions of virus ⁄ bac reaction mixture, which possessed prominent cytotoxic effect for the host cells in viral culture system. this type of molecular testing method is useful for interpreting antiviral efficacy against a background of cytotoxicity. these experiments are intended for the sponsor to substantiate to us fda that their antiviral substances are safe and effective. the data shows that the three hemagglutinin subtypes were highly susceptible to the quaternary ammonium compound in the short term in vitro experiment. the appearance of novel subtypes in the future can be met with the assurance that disinfectant and ⁄ or antiseptic resistance will be unlikely. certainly, from the above data, although genetic reassortment of human and swine viruses may modulate influenza pathogenesis and limit existing vaccine benefit, it is not likely be a factor in control of viruses on environmental surfaces by benzalkonium-type disinfectant ⁄ cleaning agents in community or health care environments. table 2 . comparison of viral titer obtained in different vessels using quantal tcid 50 and plaque assay methods plaque assay tcid 50 assay t-25 (25 cm 2 ) 6-well plate (9 cm 2 ) tcid 50 ⁄ ml tcid ⁄ ml 2ae2 · 10 6 pfu ⁄ ml 1ae4 · 10 6 pfu ⁄ ml 5 · 10 6 2ae5 · 10 6 options for the control of influenza vii outbreak influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. in this paper we share our experience of managing an outbreak of viral respiratory infection in an acf very early in the 2009 influenza pandemic and also describe some of the emerging issues relating to crossreacting antibodies to the pandemic (h1n1) 2009 influenza virus in the very elderly. the outbreak investigation was conducted as part of an urgent public health intervention initiated by the new south wales (nsw) department of health during the early stages of the first southern hemisphere wave of the 2009 pandemic. nose and throat swabs for nucleic acid testing (nat) plus acute and convalescent serum samples (6 weeks apart) were collected from all the residents of an acf where an influenza-like illness (ili) outbreak occurred. the investigation revealed dual outbreaks of pandemic (h1n1) 2009 influenza and rhinovirus infection. out of 28 residents, three had laboratory confirmed influenza [two with pandemic (h1n1) 2009], and 10 had rhinovirus infection on nat. testing of acute sera collected from every subject found elevated ( ‡1:40) pandemic (h1n1) 2009 hai antibody in 60% (9 ⁄ 15) subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1) compared with none of the 13 residents aged under 85 years (born after 1924 and median age 79 years; gmt 10ae1, p = 0ae01). the acf was closed to visi-tors for 7 days. the symptomatic residents received treatment-dose oseltamivir, and all other residents were given oseltamivir prophylaxis. more than one virus may be circulating in an acf with an ili outbreak at any one time in winter. a significant proportion of elderly residents had pre-existing cross reacting antibody to the pandemic (h1n1) 2009, which may explain the minimal clinical impact of pandemic (h1n1) 2009 in this elderly population. influenza is one of the leading causes of infectious death in elderly people, principally due to co-morbidities and declining immune competence with age. it is the most important agent in outbreaks of respiratory illness. 1 influenza in aged care facilities (acfs) is associated with an increased risk of poor health outcomes among residents, including death. 2 the clinical presentation of influenza in residents of acfs can be subtle, with a blunted febrile response and a non-specific decline in mental and functional status. 3 residents commonly have underlying diseases that can be exacerbated by influenza infection, and in addition, they are at higher risk of serious influenza-related complications than community dwelling elderly people. 4 people aged over 65 years are also at higher risk of influenza-related death, and more than 90% of annual influenza-related mortality is usually confined to this high risk group. 5 in australia, influenza and pneumonia have sub-stantial health impacts; recorded as being the underlying causes of death for 2623 persons in 2007. 6 since the world health organization declared an influenza pandemic in june 2009, australia has suffered one of the highest rates of confirmed infection during the first southern hemisphere wave. by late october 2009 there were 187 reported deaths due to pandemic influenza in australia, 7 and to date there have been about 18 449 deaths reported worldwide. 8 although disproportionately far fewer elderly people developed clinical influenza during the current pandemic than occurs with seasonal influenza, their case-fatality rate remained substantial. 9 early in the pandemic (june 2009), we investigated a suspected pandemic influenza outbreak in a rural acf in the state of nsw, australia. the epidemiology (including virulence and clinical outcome in the elderly) of the pandemic (h1n1) 2009 virus was mostly unknown at the time of investigation, and as time passed, this investigation provided clarity on some important issues of the influenza epidemiology in the elderly population. in this paper we share our experience of managing a dual outbreak of viral respiratory infections early in the pandemic, and also describe some of the emerging issues relating to the cross-reacting antibodies to pandemic influenza in the very elderly. the outbreak investigation was conducted as part of urgent public health intervention initiated by the nsw department of heath in conjunction with the local public health unit, the national centre for immunisation research and surveillance (ncirs), and the institute of clinical pathology and medical research (a who national influenza centre). to determine the extent and cause of the outbreak, a public health research doctor (gk) was dispatched from sydney over a weekend to assist with outbreak investigation and control. on june 12th 2009, the greater southern public health unit surveillance officer (bd) received a report of a possible pandemic (h1n1) 2009 outbreak in a local acf. on investigation, it was discovered that 3 days earlier a 77 year old female resident had become generally unwell, but without specific symptoms of influenza like illness (ili). soon after, nine of the 27 co-residents (but no staff) had developed symptoms suggestive of influenza. one other resident had returned from a melbourne (victoria) hospital (where pandemic (h1n1) 2009 was known to be circulating) the previous week after surgery, but did not have ili symptoms. on june 10th, the 10 symptomatic residents had nasal swabs taken by the local doctor for influenza [including pandemic (h1n1) 2009] nucleic acid testing (nat). there was rising concern due to reports of widespread pandemic (h1n1) 2009 influenza in a local army camp just over the border in nearby victoria, where pandemic (h1n1) 2009 influenza was known to be circulating widely. on june 12th, the 77 year old lady proved nat positive for pandemic (h1n1) 2009, but none of the other samples were pandemic (h1n1) 2009 nat positive. concern arose that there might be an outbreak of pandemic (h1n1) 2009 in the facility, and that some of the swabs from other residents might be false negatives. between 14 and 15 june, after consent was obtained, directly or through next of kin in 13 demented residents, all 28 submitted to venipuncture for serology, 27 successfully, and the other 18 as yet un-swabbed residents were swabbed. basic demographic data were collected from every resident with clinical information on co-morbidities and current medication use. convalescent blood samples were collected after 4 weeks on 16th july 2009 from 23 of the 28 residents. swabs were sent to icpmr where nat for influenza a [including pandemic (h1n1) 2009] and b was performed. the acute and convalescent serum samples were tested later (in december 2009), using haemagglutination inhibition assay (hai) to detect pandemic (h1n1) 2009 antibody. 10, 11 interventions the acf was closed to visitors from 12th until 18th june. treatment of the positive case and the nine symptomatic residents, with twice daily oseltamivir, was begun on saturday june 13th, and all other residents were started on once daily oseltamivir prophylaxis. the facility manager and local general practitioner (gp) monitored patient health on a daily basis, and none had to stop oseltamivir due to adverse events. one resident with ili who was known to have moderately impaired renal function was given once daily rather than twice daily oseltamivir treatment. the age range of the residents was 58-97 years with a median of 85 years. all residents had underlying medical conditions, e.g., chronic cardiac and respiratory diseases ( table 1) testing of acute sera collected from every subject found elevated ( ‡1:40) cross-reacting hai antibody to the pandemic (h1n1) 2009 in 60% (9 ⁄ 15) of subjects aged 85 years or more (born before 1925 and median age 88 years; geometric mean titre-gmt 48ae1). however, the hai titre was consistently <1:40 and significantly lower (gmt 10ae1, p = 0ae01) in the 13 residents aged under 85 years (range 58-83 years, median 79 years) (figure 1 ). the index case (nat positive) did not show a significant raise in hai level in convalescence (going from 20 to 40). the pandemic (h1n1) 2009 case that was determined by serology was pandemic (h1n1) 2009 nat negative. to our surprise, seven of the other 18 asymptomatic residents had rhinovirus detected on extended nat (reported on june 25th), despite being asymptomatic at time of swabbing and remaining so. the original nine influenza nat negative samples were then tested and three of these were also nat positive for rhinovirus; in total, ten proved nat positive for rhinovirus (35ae7%). the serologically confirmed pandemic (h1n1) 2009 case was also positive for rhinovirus infection. of interest was that only one resident had a documented fever. this investigation illustrates some of the difficulties in managing and investigating possible influenza outbreaks in real time in the context of an influenza pandemic. 12 finding a nat positive case of pandemic (h1n1) 2009 influenza among many other symptomatic cases raised the possibility (although not the probability) that pandemic (h1n1) 2009 was the cause of the outbreak. rhinovirus infection, however, was confirmed by nat in ten residents. this outbreak illustrates that more than one virus (in this case 2 and perhaps 3) may be circulating in an acf at any one time in winter. in ili outbreaks in acfs, broad laboratory testing is recommended; nat is the most sensitive method of detecting influenza or other viruses in respiratory tract samples. 13 studies have found that the pandemic (h1n1) 2009 haemagglutinin (ha) gene is more closely related phylogenetically to the 1918 h1n1 virus and classical swine influenza a ⁄ h1n1 viruses than more recent seasonal human influenza a ⁄ h1n1 viruses. 14 it is antigenically similar to the 1918 h1n1 pandemic virus in terms of the immunodominant antibody response to haemagglutinin. [15] [16] [17] it is likely that individuals alive during the emergence and initial persistence of the 1918 pandemic virus would have higher levels of cross-reacting hai antibodies to the pandemic (h1n1) 2009, which would contribute towards better clinical protection. 18 in our investigation, 60% of the residents born before 1925 (aged 85 years or above in 2009) had pre-existing cross-reacting hai antibody to the pandemic (h1n1) 2009. in elderly populations, severe illness may be associated with organisms typically considered to be mild, such as rhinovirus. however, studies have shown that nursing home residents may be susceptible to outbreaks of rhinovirus that may cause mild to severe respiratory illness, particularly in those with a history of lung disease. one rhinovirus outbreak in a nursing home in the usa caused 12 fatalities. 19 another outbreak showed residents with underlying lung disease are more likely to have longer infection, require antibiotics, develop bronchospasm, and have difficulty breathing; two residents with underlying lung disease required emergency treatment and one died. 20 a previous influenza outbreak in a nsw aged care facility in 2006 caused significant mortality and morbidity. that outbreak resulted in 14 hospital admissions and six deaths. 21 in our investigation we have found that 17% of the residents had chronic lung disease and 64% had chronic cardiac conditions both considered as high risk for severe complications of both rhinovirus and influenza infection. however, there were no hospitalisations or deaths in our outbreak investigation. indeed only one resident developed fever, indicating that non-specific signs of illness (such as in our index case) may be the only, or early, indication of an ili. our own experience with managing other ili outbreaks has also taught us that staff of acfs may not be vigilant enough to detect fevers. in this outbreak, the nursing home staff, local gp, public health unit and the outbreak investigation team and supporting laboratory staff acted quickly and in a coordinated way. pre-existing cross-reacting antibody in the very elderly (aged ‡85 years) probably helped to limit the spread of the pandemic virus (compared to the circulation of rhinovirus) within the acf. exposure to the 1918 pandemic (or a close variant occurring before 1925) appears to be responsible for a high hai titre in the very elderly, which contributed towards better clinical protection. however, wider testing early on would have alerted us more quickly to the main cause of the outbreak. treatment and prophylactic use of oseltamivir may also have contributed to halting the spread of pandemic (h1n1) 2009 and also to symptom relief. pandemic (h1n1) 2009 influenza virus (ah1pdm) has spread worldwide since march 2009. in a paper of ah1pdm, 25% of infected individuals have experienced gastrointestinal symptoms such as diarrhea and vomiting, which is higher than that of seasonal influenza. however, little is known whether viable virus shed from stool and replication of viruses are ongoing in the gastrointestinal tract. 1,2 viral load and isolation of ah1pdm in cell culture in stool samples has been reported. 3 stool specimens were collected from 35 patients suspected to have pandemic (h1n1) 2009 infection from november 2009 through may 2010. virus isolation was conducted in cell culture by using madin-darby canine kidney (mdck) cells and taqman based rt-pcr from 10% (w ⁄ v) stool suspension in phosphate-buffered saline. taqman based rt-pcr was conducted by using primers, probes, and positive controls provided by niid (national institute of infectious diseases of japan). to confirm presence of ah1pdm viral rna, lamp (loop-mediated isothermal amplification) was used as supplemental testing. of patients, one child (case 1) submitted one nasal swab and four stool samples, another one nasal swab and two stool samples, and the other one stool sample. informed consent was obtained. strand specific rt-nested pcr was performed for only case 1 by using only one primer at the rt reaction and also assayed neu5aca2-3gal and neu5aca2-6gal binding specificity about isolated strain derived from nasal swab and stool. receptor binding specificity was performed using a solid-phase binding assay with the sialylglycopolymers (poly a-l-glutamic acid backbones containing neu5aca2-3galb1-4glcnacb-pap or neu5aca2-6galb1-4glcnacb-pap bond as described. 4 ) nucleotide sequences of the ha gene of ah1pdm viruses isolated from stool sample and nasal swab were analysed. in order to exclude the possibility of contamination, the stool samples and nasal swabs were subjected to virus isolation separately. after getting the results on the nucleotide sequence, we also confirmed no strain harboring identical sequence was isolated in our laboratory before and after the day of sample collection. ah1pdm viral rna was detected in nine (25%) of the subjects from stool samples. among nine subjects, one case (case no. 1) was positive for viral isolation. case1, a healthy 9-year-old girl, experienced fever and abdominal pain, and the others had gastrointestinal symptoms without upper respiratory symptoms. in case 1, influenza a virus was diagnosed by rapid antigen test on the day of symptom onset. viable ah1pdm virus was isolated from the stool sample and nasal swab on the second day from onset using mdck cells (table 1 ). viral load decreased gradually after symptom onset. however, viral shedding was still present 8 days after symptom onset. positive stranded rna was detected 6 days after symptom onset from the stool specimen ( figure 1 ). above two ah1pdm strains (isolated from nasal swab and stool specimen) bound exclusively to human type receptor, neu5aca2-6gal. sequence analysis demonstrated that isolated virus from stool samples was identical with that from nasal swabs in comparison of ha gene (990 bp). ah1pdm influenza virus was isolated from the stool and nasal swab samples in the same patient simultaneously by using mdck cells. our results suggests the detection of viral rna and viable ah1pdm influenza virus from stool samples may serve as a potential mode of transmission and has important implications in understanding the context of ah1pdm influenza virus. strategies to prevent transmission of influenza include use of respirators. ffp2 and n95 respirators are certified to fil-ter at least 95% of particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. [1] [2] [3] we have developed a proprietary acid-polymer formulation to coat a standard ffp2 respirator with an antiviral layer. we aimed to test this coated respirator for antiviral efficacy against a range of influenza viruses. a series of tests compared the antiviral efficacy of coated and uncoated respirators in conditions designed to simulate real-life exposure to influenza by varying the route of inoculation, contact time, temperature, humidity, moisture, and contaminating substances. we also investigated whether infectious viruses could be transferred from contaminated respirator surfaces to gloves. we tested human, swine, and avian influenza viruses, including influenza a and b viruses. influenza a subtypes were the a ⁄ h1n1 2009 pandemic strain, seasonal h1n1, h5n1, h3n2, h5n9, and h2n2. in each test, suspensions of influenza viruses were prepared to 4-8 log 10 tcid 50 ⁄ ml in mem. in some tests, organic contaminants (yeast, bsa, and mucin) were added. one set of respirators was maintained at 40°c and 75% relative humidity for 24 hours before the viral challenge, and repeatedly sprayed with he-pes buffer to simulate respiratory secretions. for each test, three coated (glaxosmithkline actiprotect) and three uncoated (sperian willson easy fit) ffp2 respirator samples were inoculated with 0ae2 ml of a viral suspension, which was applied with a pipette, sprayed, or aerosolised to create airborne droplets. after 1 minute at room temperature (on a shaker), the respirator samples were assayed for the presence of infectious viruses using standard methods. 4 in one test, after a 1 minute contact time of the respirator with the virus, nitrile gloves were applied with light pressure to the outer surface of inoculated respirator samples and then assayed after 1 minute. samples were put into test medium (mem, supplemented with antibiotics [penicillin, gentamycin, or streptomycin] and amphotericin b or l-glutamine). the supernatants were vortexed, extracted, and used to prepare serial 10-fold dilutions in mem. each dilution was used to inoculate four wells of rmk cells in a multi-well plate, and these cultures were incubated and scored over 7 days for cytopathic effects, cytotoxicity, and viability. (some tests substituted mdck cells; others used inoculated embryonated chick eggs.) all tests included negative cell controls, cytotoxicity controls, and neutralisation controls. the spearman-karber formula was used to calculate viral loads as tcid 50 or eid 50 . 4 antiviral efficacy was calculated from the difference between the geometric mean loads of influenza virus on the coated and uncoated respirators after 1 minute of exposure. the viral loads applied to respirators in these experiments ranged from 5ae5 to 8ae1 log 10 tcid 50 , and were therefore high in comparison with respiratory secretions from infected patients at the peak of influenza symptoms (range 3-7 log 10 tcid 50 ). 1 tables 1-2 show that the average viral loads detected on uncoated ffp2 respirator samples remained high in all conditions tested, ranging from 3ae2 to 6ae9 log 10 tcid 50 (or 4ae5-5ae0 log 10 eid 50 ). in contrast, the average viral load on coated respirators after 1 minute of exposure ranged from below the limits of detection to £1ae5 log 10 tcid 50 (1ae0 log 10 eid 50 ). therefore, the relative antiviral efficacy of the coating ranged from ‡2ae7 to 6ae4 log 10 . table 1 shows that the relative antiviral efficacy of the coated mask remained high in simulated-use conditions such as organic contaminants and repeated saturation at high temperature and humidity. in the experiment to test transfer of viruses from respirators, the gloves applied to regular uncoated inoculated respirators had a viral load of 3ae5 log 10 eid 50 (table 1) . by contrast, no viruses were detected on either the coated respirators or the gloves applied to them. the relative reduction in contamination was therefore ‡2ae5 log 10 . ‡4ae8 log 10 viral load with organic contaminants* 6ae1 0 ae7 5 ae3 log 10 viral load after heat, moisture, and simulated secretions** 5ae1 0 ae8 4 ae3 log 10 viral load transferred to glove** 3ae5 £1ae0 ‡2ae5 log 10 eid 50 *influenza subtype was a ⁄ h5n1, and strain was vnh5n1-pr8 ⁄ cdc-rg. **influenza subtype was a ⁄ h3n2, and the strain was hong kong ⁄ 8 ⁄ 68. results are mean log 10 tcid 50 , unless specified otherwise. results are mean log 10 tcid 50 , unless specified otherwise, based on an infectivity assay in triplicate. limits of detection varied. *2009 pandemic strains. **results are mean log 10 eid 50 , based on a haemagglutinin assay in duplicate. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 301-327 strategies to prevent transmission of influenza include use of respirators, and many guidelines have recommended that healthcare workers wear respirators in certain healthcare settings for protection against pandemic influenza. 1-3 ffp3 respirators are certified in europe to filter at least 99% of nacl particles (0ae3 lm in diameter), and ffp2 and ffp1 respirators must filter at least 95% and 80% of these particles, respectively. influenza a viruses are typically 0ae1 lm, and can be carried in aerosolised droplets smaller than 1 lm in diameter, which can disperse widely, remain airborne for 8 hours, and be inhaled deeply into the respiratory tract. 4 we have developed an acid-polymer formulation to coat the outer layer of a standard ffp2 respirator, in order to provide antiviral activity on the outer surface. we compared this coated respirator against standard ffp1, ffp2, and ffp3 respirators for filtration of aerosolised influenza viruses. the aim was to simulate protection against infectious viruses in droplets released when infected people cough and sneeze, and during aerosol-generating procedures in healthcare settings. the first assay compared three samples of coated ffp2 respirators (glaxosmithkline actiprotect) with three ffp2 controls (sperian willson easy fit). for each test, suspensions of influenza a (h3n2) at 7ae1 log 10 tcid 50 ⁄ ml in 0ae1· minimum essential medium (mem) were aerosolised with a nebulizer. the airborne droplets were introduced into a sterile chamber upstream of a respirator sample for 2 minutes, at a flow rate of 28ae3 l ⁄ minute. constant airflow was maintained for another 2 minutes after exposure to the virus. then the collection dish in the downstream sieve sampler (anderson) was assayed for infectious viruses using standard techniques. 5 briefly, serial dilutions of the collection medium (mem with 1% fbs, 5% gelatine, and 2% hepes, supplemented with antibiotics and amphotericin b) in mem + trypsin were used to inoculate madin-darby canine kidney epithelial (mdck) cells in quadruplicate in a multi-well plate. these cultures were then incubated and scored over 4-6 days for cytopathic effects, cytotoxicity, and viability. negative cell controls and cytotoxicity and neutralisation controls were also performed. the spearman-karber formula was used to calculate tcid 50 . the second assay compared five samples of coated respirators with five ffp1 controls (3m 9310) and five ffp3 controls (3m 1863). a suspension of influenza a (h1n1), at 8ae3 tcid 50 ⁄ ml, was nebulized for 1 minute and 40 seconds into the aerosol chamber, at a flow rate of 28ae3 l ⁄ minute, followed by constant airflow for 5 minutes after exposure to the virus. then the collection medium in the downstream chamber (as before, with 1% nahco 3 ) was assayed as described above. initial viral loads in the first and second assays were 8ae1 and 7ae9 log 10 tcid 50 , respectively, and were therefore high in comparison with respiratory secretions from infected patients at the peak of their influenza symptoms (range 3-7 log 10 t-cid 50 ). table 1 shows that the average viral load that passed through the uncoated ffp2 respirators in the first assay was 3ae6 log 10 tcid 50 . the average viral load that passed through the coated respirators was 1ae4 log 10 tcid 50 . therefore, for active filtration of viruses, the relative efficacy of the respirator with antiviral coating was 2ae2 log 10 greater than the uncoated respirator. for surface inactivation, the relative antiviral efficacy of the coated respirator was 3ae9 log 10. in the second study, table 2 shows that the average viral load that passed through the uncoated ffp1 respirators was 5ae2 log 10 tcid 50 . in contrast, 3ae1 log 10 tcid 50 passed through the coated ffp2 respirators. by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp1 respirators reduced the viral load by 3ae7 log 10 , and the coated ffp2 by 5ae8 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load by 2ae1 log 10 more than the ffp1 respirators. in this second study, the average viral load that passed through the uncoated ffp3 respirators was also 5ae2 log 10 tcid 50 . by comparison with the viral load when no respirator was present (8ae9 log 10 tcid 50 ), the ffp3 respirators reduced the viral load by 3ae7 log 10 . therefore, for active filtration of viruses, the respirators with antiviral coating reduced the viral load passing through the mask by 2ae1 log 10 more than the ffp3 respirators. table 2 also shows that the coated respirators reduced the infectious viruses remaining on the mask surfaces by 3ae8 log 10 more than the ffp1 respirators, and 3ae6 log 10 more than the ffp3 respirators. even with a very high viral challenge, the coated respirators prevented passage of at least an additional 2ae1 log 10 infectious viruses, compared with uncoated respirators. large numbers of infectious virions passed through all uncoated respirators tested. ffp3 respirators were no more effective than ffp1 respirators at blocking airborne influenza viruses. based on these in-vitro results, respirators with the antiviral coating could be expected to provide more protection than standard respirators from the risk of inhaling influenza viruses. strategies to prevent transmission of influenza include use of respiratory protection. ffp2 and n95 respirators are certified to filter at least 95% of nacl particles (0ae3 lm in diameter), and many guidelines have recommended that healthcare workers wear these respirators in certain healthcare settings to protect against infection from patients with pandemic influenza. 1,2 we have developed a proprietary acid-polymer formulation, designed to coat a standard respirator and inactivate influenza viruses on contact. we tested this coated respirator for cytotoxicity, skin irritation, and sensitisation potential. the antiviral coating was also tested for stability and leaching under extreme environmental conditions, such as physical abrasion and simulated breathing at different temperatures, levels of humidity and co 2 , and saturation with contaminants. eight coated respirators were tested at standard relative humidity (60% rh) for 8 hours, and one at elevated humidity (80% rh) for 2 hours. four coated masks were treated with synthetic blood or oral secretions, and then tested at 30% rh for 1 hour. the sample respirators were sealed onto a mannequin head inside an airtight chamber, and air at 30°c and 5000 ppm co 2 was pumped through the masks by a cyclic breathing machine at 24 l ⁄ minute. a 37 mm glass-fibre filter was placed behind the respirator, over the mannequin's mouth opening. at the end of all tests, these filters were eluted and analysed using high-performance liquid chromatography (hplc). standard in vitro methods were used to assess the cytotoxicity of the coated polyester and uncoated polypropylene layers of the respirator (glaxosmithkline actiprotect). 3 samples were extracted in minimum essential medium (mem), supplemented with serum, penicillin, streptomycin, amphotericin b, and l-glutamine, at 37°c for 24 hours. triplicate monolayers of mouse fibroblast cells (l-929) were dosed with each extract (including a reagent control and negative and positive controls), and incubated at 37°c in 5% co 2 for 48 hours. after 48 hours of incubation with samples or controls, the monolayers of mouse fibroblast cells were examined microscopically for abnormal cell morphology or cellular degeneration. samples of the coated respirator (comprising four polypropylene layers bonded to the coated polyester outer layer) were applied under occlusive patch conditions to the skin of 51 adults. controls, including individual layers, were applied in the same way. in a separate patch test, samples of the coated polyester outer layer and controls were applied under the same conditions to 219 adults. after 24 hours, test patches and controls were removed. sites were then scored for itching, erythema, oedema, epidermal damage, and papular response after 48 and 72 hours. the patches were applied three times a week for 3 weeks. to evaluate sensitisation, test patches were applied 10-15 days later for 24 hours at different sites to the original samples. after this challenge, skin was assessed and graded for sensitisation potential after 48 and 72 hours. table 1 shows that no residues of the antiviral coating or degradation products were detected in the air that had passed through any of the eight respirators. cytotoxicity tests showed that the coated respirator material caused 10% cell lysis or toxicity, classified as slight reactivity (grade 1), and that uncoated material caused no cell lysis or toxicity (grade 0) ( table 2) . results for positive and negative controls were severe reactivity and no reaction, respectively. from the results of the two human repeat-insult patch tests, neither the coated or uncoated layers nor the fullthickness respirator fabric caused irritation (including itching, erythema, edema, vesiculation, epidermal damage, papules, or reactions beyond the patch site) or sensitisation in any of the adult volunteers at any of the time points. based on these results, in conjunction with published data on acute and repeat-dose toxicity, mutagenicity, local irritation, dermal sensitisation, and inhalation safety for all components of the antiviral coating, the potential topical or inhalation exposure to the coated antiviral respirator does not pose a safety risk. the antiviral coating is durable and stable, and stays on the outer surface of the respirator, even in extreme environmental conditions. the coated respirator is non-irritating and non-sensitising. therefore, this respirator is considered to be well-tolerated and safe for its intended use. ies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. knowing how influenza virus is transmitted at home and in school is the key to preventing its spread. at the previous two meetings of this conference, 1,3 we introduced our study of household transmission of seasonal influenza and reported our conclusion that protracted survival of the virus even after treatment increases household transmission, and is a major factor in the transmission of the virus to infants. on the other hand, during the recent pandemic, many schoolchildren developed serious respiratory tract disorders, which again highlights the significance of schoolbased transmission of the disease. in this study, we compared transmission of a new influenza strain at home and in school with that of seasonal influenza and proposed countermeasures. the for the analysis of school-based transmission, the epidemic status of seasonal influenza in 4237 children at six elementary schools over the past two seasons (2002-2003 and 2003-2004 seasons) was compared with that of pdmh1 in 1913 children at two primary schools. using observational data of school-based transmission, we also constructed a model for influenza transmission 3, 4 and evaluated the effects of factors that could affect influenza transmission (e.g., antibody prevalence, transmission rate, non-infectious latent period, infectious latent period, school closure) through the use of 1000 simulations. in this study, a diagnosis of influenza was confirmed by rapid influenza antigen detection kit. we previously reported the high sensitivity of the kits, 5-7 not only for seasonal influenza, but also for h1n1 pandemic 2009 compared to virus isolation and pcr. serum antibody was not investigated. most of the index patients were treated with oseltamivir or zanamivir, and 30 patients were treated with amatadine. no treatment was done for 16 patients. no nai therapy was done as prophylaxis within the family. the incidence of households with an initial case patient who subsequently infected another member of the household was 21ae6% (275 of 1271 households) for seasonal influenza or 18ae3% (112 of 612 households) for pdmh1. thus, the household incidence of pdmh1 was lower than that of seasonal influenza. in addition, the percentage of family members in households who were infected by initial case patients (household transmission rate) was 10ae5% (375 of 3573 individuals) for seasonal influenza or 7ae9% (141 of 1792 individuals) for pdmh1. thus, the household transmission rate was also lower for pdmh1 than that for seasonal influenza. effect of family size on household incidence and household transmission rate an analysis of the effect of family size on household incidence showed that, in families consisting of 2-7 individuals, the incidence of seasonal influenza in order of increasing family size was 3ae5%, 17ae4%, 24ae5%, 24ae2%, 32ae4%, and 42ae9%, respectively, and the incidence of pdmh1 was 0ae0%, 13ae5%, 17ae8%, 24ae7%, 42ae9%, and 33ae3%, respectively, indicating that household incidence tends to increase with increasing family size. in contrast, no definite relationship was noted between household transmission rate and family size. transmission rates for seasonal influenza in order of increasing family size were 3ae5%, 10ae6%, 11ae8%, 8ae0%, 10ae3%, and 7ae1%, respectively, or 0ae0%, 6ae7%, 7ae7%, 8ae0%, 15ae2%, and 5ae6%, respectively, for pdmh1 (shown in table 1 ). effect of age cohort of initial case patient in household on household incidence and household transmission rate an analysis of the effect of the age cohort of the initial case patient in the household on household incidence and transmission rate showed that the household incidence of seasonal influenza in c1, c2, c3, and c4 was 26ae7% (112 of 419 households), 21ae4% (66 of 308 households), 16ae3% (21 of 129 households), 9ae9% (9 of 91 households), and for m and f was 19ae6% (37 of 189 households) and 22ae5% (29 of 129 households), respectively. therefore, household incidence was the highest in c1, followed by the parents. when the initial case patient was a child, the household incidence increased with decreasing patient age. in contrast, the household incidence of pdmh1 in c1, c2, c3, and c4 was 23ae1% (36 of 156 households), 17ae3% (42 of 243 households), 8ae1% (9 of 111 households), 10ae0% (4 of 40 households), and for m and f was 30ae8% (12 of 39 households) and 39ae1% (9 of 23 households), respectively. therefore, household incidence was higher when the initial case patient was a parent, rather than a child. the household transmission rates for seasonal influenza from c1 to f were 13ae4%, 10ae1%, 6ae0%, 3ae9%, 10ae3%, and 12ae6%, respectively. therefore, as for household incidence, the highest rate (13ae4%) was observed in c1. the corresponding household transmission rates for pdmh1 were 9ae9%, 6ae9%, 4ae0%, 4ae3%, 12ae7%, and 22ae1%, respectively, with the highest transmission rates observed for infections from parents (shown in table 1 ). if the rate of individuals with a secondary infection transmitted from the initial case patient in a household is presented as a percentage of the total number of affected individuals, the rates for seasonal influenza and pdmh1 were 22ae8% (375 of 1646 individuals) and 18ae9% (142 of 753 individuals), respectively. therefore, the rate of individuals with a secondary infection was lower for pdmh1 than that for seasonal influenza. by age cohort, the corresponding rates of individuals for seasonal influenza in c1, c2, c3, and c4 were 22ae3% (120 of 539 individuals), 11ae5% (40 of 348 individuals), 12ae2% (18 of 147 individuals), 7ae1% (7 of 98 individuals), and for m and f was 42ae7% (141 of 330 individuals) and 27ae1% (48 of 177 individuals), respectively. for pdmh1, the corresponding rates in c1, c2, c3, and c4 were 24ae3% (50 of 206 individuals), 9ae3% (25 of 268 individuals), 9ae0% (11 of 122 individuals), 4ae8% (2 of 42 individuals), and for m and f was 51ae9% (42 of 81 individuals) and 35ae3% (12 of 34 individuals), respectively. these findings indicate that, especially in the case of pdmh1, most secondary infections in parents tend to be transmitted from another household member. the mean annual prevalence of seasonal influenza and the new influenza strain at the elementary schools for the two seasons was 19ae6% and 10ae3%, respectively, whereas the prevalence determined 7 days after appearance of the first case in school was 3ae9% and 4ae5%, respectively. in the recent season at the same elementary schools, however, the prevalence was a high 43ae5%. since the prevalence at 7 days after the appearance of the first case in school was already 17ae5%, these data show that the influenza virus spread quickly throughout the schools. at the schools with high transmission rates in the early period of the pandemic, new infections were confirmed even 4 days after the school closure action was taken. these findings indicate that pdmh1, the current influenza virus, has a long latent period during which it becomes infectious and spreads from infected individuals to numerous others in their vicinity. we constructed a model for influenza transmission in schools and estimated the time course of changes in the number of expected cases and the expected prevalence during the season. in this model, school children were divided into six groups depending on the stage of infection: uninfected period with no immunity, non-infectious latent period, infectious latent period, onset, post-onset infectious period, and immune period. it was assumed that schoolbased transmission occurred during the infectious latent period prior to onset and that no infections occurred during the post-onset infectious period because children were absent from school. due to the long latent period of pdmh1, the distribution of the non-infectious latent period of pdmh1 was established as (day 1, day 2, day 3, day 4) = (10%, 40%, 40%, 10%) and the distribution of the infectious period as (day 0, day 1, day 2) = (10%, 80%, 10%). when simulations were performed under these conditions using the model for school-based transmission of influenza in which children from classes with an outbreak were kept at home for 3 days, the time course of changes in the number of affected individuals actually observed and the time course of changes in the number of expected cases were determined. the expected prevalence under these conditions was 35%. to evaluate the effect of school closure, simulations were performed based on the assumption that children from affected classes were not kept at home for 3 days. it was shown that there was an increase in the expected number of cases during the 3 days corresponding to the period of actual school closure and that the expected prevalence increased to 52%. based on these findings, it was concluded that keeping children home from classes with an outbreak is an effective means of controlling the transmission of influenza in schools (shown in figure 1 ). if the transmissibility of pdmh1 virus at home is estimated based on the speed of transmission and the degree to which pdmh1 is prevalent in schools, it would be expected that the household transmission of pdmh1 is also higher than that of seasonal influenza. in fact, the opposite is the case. this paradox can be explained in two ways. 1. the number of children aged 19 or more and parents with pdmh1 influenza as a percentage of the total number of affected individuals is lower than those with seasonal influenza (20ae8% versus 37ae2%). further, although the number of parents with a secondary infection was high at home, the percentage of the total number of individuals with pdmh1 was a low 3ae8% (29 of 754 individuals), compared to that for seasonal influenza (5ae5% [91 of 1640 individuals]). in other words, adults are less susceptible to pdmh1 infections and there was a correspondingly small number of affected individuals. therefore, it was considered that the transmission rate at home was lower than that at school for this reason. 2. the percentage of households with more than one affected individual within the same family was higher for pdmh1 at 28ae5% (157 of 550 households) than for seasonal influenza at 24ae4% (309 of 1265 households). in the patients secondarily infected with pdmh1, 26ae8% of them showed symptoms of infection 10 days or more after the onset in the first patient, suggesting that they were not infected at home, and the actual household transmission was 20ae9% (115 of 550 households). therefore, although the prevalence was higher for pdmh1, it seems that household transmission was lower because households with an affected individual implemented satisfactory control measures against infection. seasonal influenza differs greatly from pdmh1 influenza in its transmissibility at home and in school. in the household transmission of pdmh1 influenza, both the household incidence and household transmission rate of pdmh1 were low compared to those for seasonal influenza. although transmission of seasonal influenza from infants to parents was marked, in the case of pdmh1, the reverse was true with transmission from parents to children being predomi-nant. it should be noted that household transmission in mothers was common in all eight seasons, suggesting the need to reconsider control measures against infection when nursing unwell family members. in the case of school-based transmission, pdmh1 was more prevalent than seasonal influenza, indicating that the virus spread quickly throughout the schools. this difference was attributed to the long infectious latent period when pdmh1 rapidly became rampant in the schools. an analysis of school-based transmission using a model for influenza transmission showed that, when 10% of the student population is infected, schools should be closed for five consecutive days in order to minimize the spread of the disease. the effectiveness of seasonal influenza vaccine in preventing pandemic and seasonal influenza infection: a randomized controlled trial introduction household transmission has been estimated to account for one-third of all influenza transmission, 1,2 and children are at high risk of spreading the disease. with reference to previous evidence, 3-15 some vaccine deployment strategies target children to prevent them from infection and transmitting influenza. 16 nevertheless, few studies evaluated the effectiveness of vaccinating children in reducing household transmission. 10, 11 during 2008-09, a pilot randomized controlled trial was conducted to investigate such effect by studying households with school age children randomized to receive trivalent inactivated seasonal influenza vaccine (tiv). 17 the monovalent vaccine against pandemic influenza a (h1n1) (ph1n1) had yet been available until the end of the first wave. various conclusions have been made as to whether seasonal influenza vaccine might possibly protect against ph1n1. [18] [19] [20] [21] [22] [23] [24] [25] we report findings on the effectiveness of tiv against ph1n1 observed in our cohort. households were screened if they expressed interest after receiving invitation letters distributed via their children's school or an existing pediatric cohort study. 26 to be eligible, the household had to include at least one child aged 6-15 years who was not allergic or hypersensitive to any of the tiv components. children known to have immunosuppressive conditions or other contraindications against tiv were also excluded. written consent and assent were obtained from participants aged above 18 years and those aged 8-17 years, respectively. proxy written consent was obtained from legal guardians or parents for participants younger than 18 years. ethical approval was obtained from the institutional review board of the university of hong kong. consented households were allocated to the tiv and placebo group (in ratio 3:2) according a code generated by block randomization with random block sizes of 5, 10, and 15. an independent nurse prepared 0. one child (study subjects) from each household in the tiv group received a single dose of tiv with one child from each household in the placebo group receiving a single dose of saline placebo. parents and legal guardians were asked to report any adverse reactions 4 days following vaccination. all participants, study nurses, and other research staff were blinded to the allocation and administration of vaccine or placebo. the vaccine allocation sequence was only disclosed to the investigators at completion of the study. serum specimens were collected from subjects shortly before (november-december 2008), one month after vaccination (december 2008-january 2009), and after the winter (april 2009) and summer influenza seasons (august-october 2009). serum specimens were obtained from household contacts at baseline and after the winter and summer influenza seasons. all household members recorded any fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, coryza, or myalgia daily on a symptom diary. they were also invited to report to the study hotline immediately if they experienced at least 2 of the above signs or symptoms. as a response, the study nurse would visit the households with any sick members and collect nose and throat swab from all household members. the households were also telephoned monthly or increased to fortnightly during influenza seasons to monitor for signs and symptoms and remind them to report to the hotline. supermarket or book vouchers (for children) were given to the households including us$13 for each serum specimen collected, us$6.5 for each home visit, and us$65 for completion of the study. serologically-indicated influenza infection was the primary outcome of this study. it was define as a ‡4 fold rise in antibody titer within each influenza season. other study outcomes included rt-pcr confirmed influenza virus infection, acute respiratory illness (ari) (two of any of the above listed signs or symptoms), and influenza-like illness (ili) (fever ‡37.8°c with cough or sore throat). antibody titers against the vaccine strains were obtained by testing each serum specimens by haemagluttination inhibition (hai). viral microneutralization (vn) using standard methods was found to be more sensitive than hai in detecting antibody response against a ⁄ california ⁄ 04 ⁄ 2009(h1n1) in another study conducted by our group 27 and was, therefore, used in this study. the sera was initially diluted at 1 ⁄ 10 and further tested in serial doubling dilutions. nose and throat swabs were tested by reverse transcription polymerase chain reaction (rt-pcr) for influenza a and b viruses. technical details of the laboratory methods have been reported elsewhere. 27, 28 fisher's exact test and chi-squared tests were used to compare count data including occurrence of side effects, laboratory confirmed, and clinically defined influenza infections. wilcoxon signed-rank test were used to compare the serum antibody titers between groups. exact binomial method or the wald approximation was used to estimate 95% confidence intervals where appropriate. all analyses were carried out in r version 2.8.1 (r development core team, vienna, austria). twenty-five primary and secondary schools in the district of the study clinic were invited to participate. to parents of three schools that agreed to take part and another study cohort, 3690 invitation letters were sent and 105 households were enrolled. personal referrals were made from these parents to enroll 14 additional households. among 119 enrolled households, 1 subject with history of epileptic seizure was assessed to be contra-indicated against receiving the vaccine. blood taking failed in another subject, and both of them withdrew from the study. eleven households did not complete the study. table 1 shows subject and household contacts of the tiv and placebo group were similar in demographics and prior influenza vaccination history. antibody titers before vaccination were comparable between groups (data not shown). most study subjects who received tiv showed antibody titer ‡40 against the vaccine strains 1 month after receiving tiv, and the proportion was significantly higher than those who received placebo (a ⁄ h1n1 93% in tiv versus 68% in placebo group, p < 0.01; a ⁄ h3n2 97% versus 61%, p < 0.01; b 99% versus 91%, p = 0.15). none of the study subjects had antibody titer ‡40 against ph1n1 following receipt of seasonal tiv. no serious adverse reactions were reported, and only pain at injection sites was slightly higher in tiv group (data not shown). subjects who received tiv had lower rates of serologically confirmed seasonal influenza a(h1n1) (8% versus 21%, p = 0.10), a(h3n2) (7% versus 12%, p = 0.49) and b infection (3% versus 8%, p = 0.36, although the differences were not statistically significant (table 2) . study subjects had higher rate of serologically confirmed ph1n1 infection (32% versus 17%, p = 0.09), yet it was not statistically significant. after adjusting for potential cross reactive antibody response, 31% of subjects in tiv versus 12% in placebo groups showed ph1n1 infection confirmed by either serology or rt-pcr (p = 0.04). little differences were observed for rt-pcr confirmed infection, ari, and ili in results combining the winter and summer influenza seasons. during winter season when seasonal influenza predominated, study subjects who had received tiv showed a lower tendency to develop ili (15% versus 23%, p = 0.43) or ari (46% versus 54%, p = 0.52). an opposite tendency was seen (ili 27% versus 19%, p = 0.43; ari 49% versus 44%, p = 0.68) during summer when ph1n1 predominated. however, these differences were not statistically significant. rates of ili in subjects infected with ph1n1 did not differ statistical significantly between subject who received tiv and placebo (40% versus 25%, p = 0.30). the study was not powered to detect indirect benefits to household contacts of vaccines resulting from reduced household transmission. attack rates were found to be similar between household contacts of subjects received tiv and placebo (data not shown). to examine potential factors that might affect risk of laboratory confirmed ph1n1 infection, a multivariable logistic regression model was fitted to study all subjects and their household contacts. younger participants aged below 16 years were found to have a higher risk (<16 years or = 6.60, 95% ci 2.17, 20.13; 16-45 years or = 2.53, 95% ci 0.80, 7.99, >45 or = 1.00). after adjusting for age, sex, and date of study completion, receipt of tiv for the 2008-9 influenza season was not found to affect risk of ph1n1 infection. however, participants who had laboratory confirmed seasonal influenza infection during the study period had 65% lower risk of ph1n1 infection (infected with seasonal influenza or = 0.35, 95% ci 0.14, 0.87; not infected with seasonal influenza or = 1.00). as (see table s1 for winter and summer results separately). influenza-like illness (ili) defined as temperature ‡37.8°c plus cough or sore throat; acute respiratory illness (ari) defined at least any two of fever ‡37.8°c, chills, headache, sore throat, cough, presence of phlegm, nasal congestion, runny nose, muscle or joint pain. limited by the sample size, we were not able to differentiate between the protective effect of seasonal a(h1n1) and a(h3n2) infection against ph1n1. other details of the results from the study were published elsewhere. 17 discussion a non-significantly higher rate of ph1n1 infection was observed in study subjects who received tiv compared to placebo. results from a multivariable logistic regression suggested that such a pattern might be explained by more common seasonal influenza infection in placebo group prior to the pandemic, protecting the placebo group against ph1n1. seasonal influenza infection within 3-6 months observed in our study might have conferred better cross protection than tiv against ph1n1. this resembles similar previous findings on cross protection between influenza infections in human and animal studies. [29] [30] [31] [32] [33] [34] [35] [36] however, the same phenomenon has not been observed in some studies on seasonal influenza vaccine against ph1n1. 18, 21, [23] [24] [25] apart from differences in study design and vaccine used, we speculate that a short time interval between ph1n1 and most recent seasonal influenza peak activities might be crucial for the phenomenon. hong kong is a subtropical area where the 2009 pandemic was preceded immediately by summer seasonal influenza circulation and a few months apart from the winter 2008-9 influenza peak. if cross protection from seasonal influenza lasts for only a short period, it might have waned below partial cross protection from tiv over time from last seasonal influenza infection. the current study is limited by a small sample size, and further studies are required to confirm our hypothesis. while tiv is only effective against matching strains, a universal influenza vaccine could provide better protection against the ever evolving influenza viruses. introduction immunisation of healthy, as well as high risk, children has been the focus of much recent attention both in prevention of seasonal influenza and during the 2009 h1n1 pandemic. detailed information on reactogenicity, particularly for newer vaccine formulations that include adjuvants, is limited. we recently reported results of a head-to-head comparison of two 2009 h1n1 pandemic influenza vaccines in children in the uk. 1 here we present new, detailed analyses of reactogenicity data from that study, which has important potential implications for future paediatric influenza vaccine development and use. we compared the safety, reactogenicity, and immunogenicity of two h1n1 influenza vaccines, one as03 b (tocopherol based oil in water emulsion) adjuvanted egg culture derived split virion, the other non-adjuvanted cell culture derived whole virion, given as two dose schedules 21 days apart, in a randomised, open label trial as previously reported. the study was age stratified (6 months to under 3 years & 3-12 years) to ensure adequate data in young children. age appropriate safety data (simplified for under 5 year olds) were collected for 7 days after each vaccine dose and serum was collected at enrolment & 21 days after the second dose. nine hundred-thirty seven children received vaccines as per-protocol. when comparing the two vaccines, grade 3 ( ‡50 mm) local reactions were seen more frequently following the adjuvanted than the non-adjuvanted vaccine in both age groups, after both vaccine doses. in children over 5 years old, 7ae2% versus 1ae1%, p < 0ae001, after dose one; 8ae5% versus 1ae1%, p = 0ae002, after dose two, in children under 5 years old, 1ae5% versus 0ae0%, p = 0ae06, after dose one (non significant, ns); 5ae9% versus 0ae0%, p < 0ae001 after dose two. fever ‡38°c (axillary measurement) was seen more frequently following the second dose of the adjuvanted vaccine compared to the non-adjuvanted vaccine in <5 year olds (22ae4% versus 12ae5%; p < 0ae05). looking specifically at the adjuvanted vaccine in under 5 year olds, comparing the second dose with the first, there were significantly higher rates of fever ‡38°c (axillary measurement) (22ae4% versus 8ae9%, p < 0ae001), local grade 3 ( ‡50 mm) reactions (5ae9% versus 1ae5%, p = 0ae02), pain (39ae4% versus 31ae5, p = 0ae02), use of analgesia or antipyretic medication (43ae7% versus 31ae5%, p < 0ae001), and decreased activity (31ae9% versus 20ae4%, p < 0ae001). the adjuvanted vaccine was significantly more immunogenic, most notably in the younger children. in <3 year olds, haemagglutination inhibition (hi) seroconversion rates were 98ae2% versus 80ae1%, p < 0ae001. among all general and local reactions measured, only the maximum temperature measured during the 7 days after the second dose of the adjuvanted vaccine showed a significant (positive) association with post vaccination hi titres. for each 1°c rise in temperature there was a 27% increase in titre (p < 0ae001). these reactogenicity data demonstrate a step towards the future possibility of one-dose influenza immunisation programmes for young children associated with low rates of fever and other reactions. the occurrence of fever following adjuvanted vaccine, seen particularly after a second dose in younger children, was quantitatively associated with enhanced antibody titres. this association was not seen with unadjuvanted vaccine. this apparent difference between the relatedness of the pyrogenic and immunogenic effects of the two vaccines merits further investigation. novel adjuvants appear to have the potential to overcome the relatively poor immunogenicity previously experienced with inactivated influenza vaccines in infants and young children. however, careful adjustment may be needed to optimise the balance between high protection and acceptable reaction rates. tries causing sporadic human infections. vaccination has been used as an effective public health tool for influenza prophylaxis. the goal of this study was to evaluate live attenuated influenza vaccine (laiv) vaccine candidates for subtypes h1 and h5. the attenuated phenotype of h1 and h5 laiv candidates has been proven in experiments in ovo and in vivo. in randomized clinical trials among adult volunteers, no significant adverse reactions attributable to the live vaccine occurred. our results indicate that pandemic laiv candidates were well tolerated and elicited serum, local, and cellular immune responses. the emergence and spread of highly pathogenic avian influenza h5n1 viruses in avian populations and concurrent infections in humans since 1997 has prompted efforts to develop vaccines for use in the event of an influenza pandemic. in 2009, the world faced a new h1n1 pandemic. immunization with inactivated or live vaccines is the primary measure for preventing influenza. laivs appear to be safe and efficacious, and might possibly provide broader immune responses than inactivated vaccines. our study evaluated laiv pandemic candidates as part of the global influenza pandemic preparation project outlined by the who. capacity of the viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) was evaluated by routine technique in embryonated hen eggs. laiv and placebo were supplied by microgen (irkutsk, russia). the monovalent laiv was produced from the pandemic vaccine candidates and formulated to contain 10 7 and 10 6ae9 eid 50 per dose (0ae5 ml) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 92, respectively. the vaccine or placebo was administered intranasally with a single-use dosing nasal sprayer. two doses were given at an interval of 21 days. one hundred-ninety healthy adults aged 18-60 years were randomly divided into groups to receive either pandemic vaccine candidates (204) or placebo (28) . subjects were informed about purposes and methods of the study and potential risks associated with participation. all participants had an hai antibody titer of £1:10 to a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) pandemic virus. in all there were 47 and 42 vaccines and 19 and 8 participants who received placebo, and were further tested for immune responses to h1n1 or h5n2 pandemic vaccine, respectively. another 29 participants vaccinated with h1n1 laiv were children between 12 to18 years old. before the children were vaccinated, their parents were advised about study and their consent was required before any child was enrolled. on the advice of the national ethics committee, we did not include a placebo group in this study. individuals were not enrolled if they had an acute illness or fever at the beginning of the study or a history of egg allergy. immune responses of subjects were assessed by routine hai test (evaluation of serum igg antibodies), elisa (evaluation of iga antibodies eluted from the nasal swabs into steril pbs), and cytokine flow cytometry assay (evaluation of virus-specific cd3 + cd4 + ifnc + and cd3 + cd8 + ifnc + peripheral blood mononuclear cells). the results of phenotypic analysis in ovo showed that pandemic vaccine candidates retained the cold adapted-temperature sensitive (ca ⁄ ts) phenotype, typical of the coldadapted parental mdv. in contrast and as expected, a ⁄ california ⁄ 07 ⁄ 2009 and a ⁄ duck ⁄ potsdam ⁄ 1406-86 parental strains had the non-ts ⁄ non-ca phenotype typical of wt viruses. the h5n2 pandemic vaccine candidate demonstrated an attenuated phenotype in mice and in java macaques and did not infect chickens. the vaccine attenuation study confirmed the attenuated phenotype of a a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 pandemic laiv candidate in mouse, ferret, and guinea pig models. the phase i ⁄ ii randomized, controlled, double-blind clinical study safety evaluation of pandemic vaccine candidates in adults clinical examination of subjects who received two doses of pandemic vaccine candidates indicated that both vaccines were well tolerated. no fever reactions were observed after the first or second vaccination. after the first vaccination, 33ae0% and 40ae0% of reactogenicity events consisting of catarrhal symptoms, such as pharyngeal irritation or hyperemia, were observed for h1n1 and h5n2 vaccine candidates, respectively. after revaccination, subjects did not report local or systemic reactions. to determine whether a serological response occurred in the cohort of immunologically naïve subjects vaccinated with pandemic vaccine candidates, hai and elisa tests were used (table 1) . post-vaccination geometrical mean titers (gmt) among 20 subjects who received two doses of h5n2 vaccine were significantly higher than pre-vaccination titers. the frequency of ‡4 fold antibody rises was significantly higher (47ae1%) after revaccination than after one dose (5ae9%). the percentage of subjects with post-vaccination serum hai titers to h5n2 ‡ 1:20 was 47ae1% and for titers ‡1:40, it was 29ae4%. no seroconversions in the placebo group were detected. the virus-specific nasal iga antibody response to vaccination after two doses of the h5n2 vaccine candidate demonstrated significant increases of ‡4 fold rise iga antibodies (65%) compared to one dose. cumulative data of h5n2 vaccination (all applied tests) showed 35% and 80% of conversions after the first and the second vaccination, respectively. increasing h5n2 vaccine virus infectivity from 10 6ae9 to 10 8ae3 eid 50 ⁄ dose lead to an enhancement of post-vaccination hai titers in vaccinees after the first vaccination to homologous h5n2 antigen from 5ae9% to 31ae0% of ‡4 fold antibody rises. values of post-vaccination serum hai antibody titers in subjects vaccinated with another pandemic vaccine candidate, a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38, also proved to be rather low. after the primary vaccination, the percentage of subjects with hai protective antibody titers ‡1:40 were 2ae1%. after revaccination, this parameter increased to 17ae0%. four-fold increases in serum hai antibody titres were four-fold conversions after the first and the second vaccination was 23ae4% and 34ae4%, respectively. elisa antibodies in nasal swabs showed had an advantage in detecting induction of local iga as compared to serum hai antibodies. after revaccination four-fold serum hai antibody conversions were 34ae4% vs. 63ae8% of iga conversions in nasal swabs, respectively. taking into account cumulative data of h1n1 vaccination (hai and elisa data), the obtained results were here and in the 42ae5% and 70ae2% of conversions after the first and the second vaccination, respectively. fourty-seven subjects were vaccinated with h1n1 laiv, and 19 who received a placebo were chosen for evaluation of cellular immune response by cytokine assays. after revaccination, the mean increases of both cd4 + and cd8 + memory cells were significantly higher in vaccinated subjects compared to the placebo group. interestingly, the same effect of vaccination was observed in vaccinees without detectable conversions of hai antibody titers. even after a single vaccination, the rate of subjects with significant increases of these cells in the blood was 37ae5% (cd8 + ) and 75% (cd4 + ). after the revaccination, the percentage of subjects with significant increases in cd8 + and in cd4 + cells was 68ae8%. immunogenicity of h1n1 pandemic vaccine candidate in children hai antibody results among children aged 12 to 18 years proved to be significantly higher when compared to adult subjects: after the first vaccination, 41ae4% of the children seroconverted; after revaccination, seroconversions reached 83ae3% ( table 2 ). the gmt rise to h1n1 vaccine with primary vaccination was 3:1; after revaccination it increased to 6:7. benefits of vaccination with laiv to aid in the control of influenza outbreaks are acknowledged by the who. 1 many years of laiv seasonal trials have shown excellent tolerability and low reactogenicity. [2] [3] [4] indeed, data showed that live influenza vaccines cause minimal systemic, local, and thermal reactions, generally from 0 to 3%. a different situation was observed in the cohort of immunologically naïve volunteers vaccinated with pandemic vaccines. the rate of local reactions to a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 and a ⁄ 17 ⁄ duck ⁄ potsdam ⁄ 86 ⁄ 9 vaccine candidates increased to 33ae0% and 40ae0%, respectively. after revaccination no significant local and systemic reactions were observed. this confirms, indirectly, the development of a sufficiently high level of protection after the first vaccination with pandemic laiv. the most important criterion for assessing the quality of vaccines is their estimated safety, epidemic effectiveness, and immunogenicity. however, current regulatory documentation 5 mandates that induction of serum antibodies, measured by hai, as the only criterion for a laiv immunogenicity evaluation. in addition to the standard hai assay, we determined serum (igg) and local (iga) antibodies in adult subjects vaccinated with an h1n1 pandemic vaccine candidate. evaluation of overall results obtained in these additional serological tests, as well as those from the hai assay, showed an immune response to the vaccine in the majority of subjects (42ae5% of ab seroconversions after the single vaccination and 70ae2% after revaccination, respectively). these data show that methods used to routinely measure laiv immunogenicity should be revised to include a number of additional immunological methods such as igg and iga elisa, and cytokine assays consistent with the recently updated who recommendations on laiv monitoring. these clinical studies clearly demonstrated that pandemic laiv candidates are effective at generating pandemic specific influenza immunity. a key finding from this study is that it may be practical to give the vaccine as a single dose to both children and adults. evaluation of our laiv pandemic vaccine candidates was performed as part of the global influenza pandemic preparation project outlined by the who. 6 it was considered that laiv could be produced in greater quantities and more rapidly than inactivated vaccines. together with the generation of herd immunity by laiv, this suggests that laiv implementation during the first wave of a pandemic may provide significant social, economic, and health benefits to the community. authors are thankful to path for the financial support of h1n1 pandemic vaccine study. we are grateful for the the main evolutionary mechanism of influenza viruses during inter-pandemic period is the antigenic drift, but the epidemiological picture of circulating viruses is complicated by a high level of heterogeneity of strains, even though drift does not occur, due to co-circulation of drifted and old strains or to co-circulation of viruses belonging to the same type ⁄ subtype but with different antigenic patterns. [1] [2] [3] [4] [5] [6] lack of data exists on the impact of the wide heterogeneity of circulating strains on the seroprotection and on-field effectiveness of influenza vaccine: in particular, little is known about the ability of influenza vaccine to elicit an effective immune response against isolates with few amino acid mutations with respect to vaccine strains that represent the majority of circulating viruses. mf59-adjuvanted vaccines, which are currently used for the prevention of seasonal influenza epidemics in elderly, are showed to confer higher seroprotection against homologous and drifted a(h3n2) strains than non-adjuvanted vaccines. [7] [8] [9] the broader immune response showed by mf59-adjuvanted vaccine was measured using hi and nt assays against egg-grown drifted strains representing vaccine composition changes during the following seasons, but its ability to elicit a broader immune response against circulating viruses belonging to vaccine cluster and presenting amino acid mutations onto antigenic sites or against on-field isolates not-antigenically distant from vaccine strains has not yet been investigated. showing amino acid changes onto antigenic sites in position 145 (n145k), 189 (n189k), and 227 (p227s) with respect to a ⁄ california ⁄ 7 ⁄ 04. in particular, a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04 presents n126d amino acid mutation detected in clade 2 a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses. 1 the ha sequences of a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 2 ⁄ 05, genoa ⁄ 11 ⁄ 05, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 fell within the clade represented by the ha of a ⁄ califor-nia ⁄ 7 ⁄ 04; among these isolates, a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 05 showed antigenic site sequences very close to that of the 2005 ⁄ 06 vaccine strain, whereas ha sequences of a ⁄ genoa ⁄ 62 ⁄ 05, a ⁄ genoa ⁄ ⁄ 47 ⁄ 05 and a ⁄ genoa ⁄ 59 ⁄ 05 posses amino changes onto antigenic site a(r142k), c(g50e) and d(r208k), respectively. the ha sequences of more recent isolates fell within the clade represented by the ha of a ⁄ brisbane ⁄ 10 ⁄ 07 and characterized by the amino acid changes, relative to the ha of a a ⁄ california ⁄ 7 ⁄ 04, g50e and k140i, with the exception of a ⁄ genoa ⁄ 3 ⁄ 07, showing r142g and l157s amino acid changes present in viruses belonging to a ⁄ nepal ⁄ 921 ⁄ 06 clade. measure of genetic distance between vaccine and circulating strains was calculated as previously described by gupta. 10 two blood samples were collected from each subject, just before and 22 ± 2 day post-vaccination. all sera were stored at )20°c. all samples were tested at the laboratory of health sciences department, university of genoa, by haemagglutination-inhibition (hi) and neutralization (nt) assays, performed following the who criteria and standardised method in our laboratory, respectively. [11] [12] [13] guinea pig red blood cells were used for hi assay. all samples were assayed twice for hi and for nt. the obtained antibody titre was expressed as the reciprocal of the last sera haemagglutinating or inhibiting virus dilution. immunogenicity was determined by: geometric mean titre (gmt); mean-fold increase (mfi; ratio of post-to pre-vaccination titre); seroprotection rate (the percentage of subjects achieving an hi and nt titre ‡40 iu); and seroconversion rate (percentage of subjects with a fourfold increase in hi or nt antibody titers, providing a minimal post vaccination titer of 1:40). post-vaccination gmt was reported as ratio, with the corresponding 95% confidence interval, of gmts after vaccination with mf59-adjuvanted vaccine and with non-adjuvanted subunit vaccine. seroprotection and seroconversion rate 95% confidence interval was calculated using modified wald method. comparisons of seroconversion and seroprotection rates between subunit and mf59-adjuvanted vaccine groups have been analyzed by fischer's exact test. the results were evaluated against the committee for medicinal products for human use (chmp) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria be met: mfi >2; seroprotection rate >60%, or seroconversion rate >30%. furthermore, hi titres were also transformed into binary logarithms, corrected for pre-vaccination status, as described by beyer et al. 14 and were expressed as median titres, with the corresponding 25°-75°i nter-quantile range. comparisons of corrected post-vaccination titers between subunit and mf59-adjuvanted vaccine groups were analyzed by wilcoxon test. difference in immunogenicity profile between vaccine groups, expressed by ratio of different parameters, was correlated with genetic and antigenic distance between vaccine and viruses used in the study using spearman test. pre-vaccination titres were not significantly different between vaccine groups, for all 15 strains (data not shown). post-vaccination gmt ratios between mf59-adjuvanted and non-adjuvanted vaccine groups determined using hi and nt assays, with the corresponding 95% confidence interval, according to viral strain are shown in figure 1 . both vaccines met chmp requirements for mfi (>2), seroconversion (>30%), and seroprotection rate (>60%) against a ⁄ wyoming ⁄ 3 ⁄ 03-like, with the exception of a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ california ⁄ 7 ⁄ 04-like circulating viruses and against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ 7 ⁄ 04, and a ⁄ wisconsin ⁄ 67 ⁄ 05 strains; the immune response against a ⁄ genoa ⁄ 13 ⁄ 04 met the requirements for mfi and seroprotection rate only in mf59-adjuvanted vaccine group. requirements for mfi, seroconversion, and seroprotection rate against the a ⁄ brisbane ⁄ 10 ⁄ 07-like virus a ⁄ genoa ⁄ 2 ⁄ 07 and the a ⁄ nepal ⁄ 921 ⁄ 06-like genoa ⁄ 3 ⁄ 07 viruses and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 strain were reached only in subjects vaccinated with the mf59adjuvanted vaccine. a similar pattern emerged from the analysis of mfi, seroconversion and seroprotection rates using nt assays. subjects vaccinated with the mf59-adjuvanted vaccine showed significantly higher post-vaccination hi gmts against a ⁄ wyoming ⁄ 3 ⁄ 03-like, a ⁄ california ⁄ 7 ⁄ 04-like, a ⁄ nepal ⁄ 921 ⁄ 06-like and a ⁄ brisbane ⁄ 10 ⁄ 07like viruses, with the exception of a ⁄ genoa ⁄ 3 ⁄ 06, and against egg-grown a ⁄ california ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07 strains, compared with individuals immunized with the non-adjuvanted vaccine ( figure 1 ). the mf59-adjuvanted vaccine also induced significantly higher seroconversion and seroprotection rates against following correction for pre-vaccination status, hi titres were significantly higher for the mf59-adjuvanted vaccine group when evaluated against a ⁄ wyoming ⁄ 3 ⁄ 03-like viruses, a ⁄ brisbane ⁄ 10 ⁄ 07-like a ⁄ genoa ⁄ 2 ⁄ 07, and a ⁄ nepal ⁄ 921 ⁄ 06-like a ⁄ genoa ⁄ 3 ⁄ 07 strain ( figure 2 ). pre-vaccination titre corrected response was higher in subjects vaccinated with mf59 adjuvanted vaccine also against egg-grown a ⁄ wyoming ⁄ 3 ⁄ 03, a ⁄ california ⁄ ⁄ 7 ⁄ 04, a ⁄ wisconsin ⁄ 67 ⁄ 05, and a ⁄ brisbane ⁄ 10 ⁄ 07. among viruses more closely related to a ⁄ california ⁄ 7 ⁄ 04, subjects immunized with mf59-adjuvanted vaccine showed a significantly higher corrected titres against a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05 strains compared with the non-adjuvanted vaccine ( figure 2) . spearman test showed a clear correlation between the distances and the advantage offered by mf59 expressed by ratio between mfi, post-vaccination gmts, corrected post-vaccination median, seroconversion, and seroprotection rates calculated using hi test in the two vaccine groups. similarly, ratio between mfi, seroconversion, and seroprotection rates calculated with nt test correlated with the genetic and antigenic distance between vaccine and viruses used for the study. the ability of mf59 to enhance the immunogenicity and to elicit a broader immune response against drifted strains than non-adjuvanted vaccine is consistent with other findings reported during the last decade. [7] [8] [9] 15 in subjects vaccinated with the mf59-adjuvanted vaccine containing a ⁄ california ⁄ 7 ⁄ 04, the immune response, expressed by a number of parameters, such as crude and corrected postvaccination titers, seroconversion, and seroprotection rates calculated using hi and nt assays, is higher than that observed in individuals immunized with subunit vaccine when it is evaluated against a drifted strains, such as a ⁄ brisbane ⁄ 10 ⁄ 07-like and a ⁄ nepal ⁄ 921 ⁄ 06-like strains, and against egg-grown a ⁄ brisbane ⁄ 10 ⁄ 07 virus. for the first time in this study, the impact of heterogeneity of circulating strains antigenically close to the vaccine on the antibody response elicited by mf59-and non-adiuvanted vaccines is evaluated. immune response against viruses isolated during the 2004 ⁄ 05 season, that appear more phylogenetically close to 2004 ⁄ 05 vaccine strain a ⁄ wyoming ⁄ 3 ⁄ 03, was higher in subjects vaccinated with mf59-adiuvanted vaccine as demonstrated by higher crude and corrected post-vaccination hi titres and higher postvaccination nt titres, with the exception of a ⁄ genoa ⁄ 27 ⁄ 04, against whom the nt post-vaccination gmt is identical in mf59 and subunit vaccine groups. furthermore, hi seroconversion and seroprotection rates were higher in mf59 vaccine group when evaluated against a ⁄ genoa ⁄ 13 ⁄ 04 and a ⁄ genoa ⁄ 27 ⁄ 04. as far as the immune response against a ⁄ california ⁄ 7 ⁄ 04-like viruses, the small number of enrolled subjects did not allow appreciating differences using qualitative response indicators, but crude post-vaccination hi titres were higher in mf59 vaccine group for all the strains. interestingly, a ⁄ california ⁄ 7 ⁄ 04-like viruses with at least one amino acid change onto antigenic sites, i.e. a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, showed a more marked difference in terms of response between the two vaccine groups. individuals immunized with mf59-adiuvanted vaccine showed higher corrected post-vaccination hi titres and post-vaccination nt titres in comparison with subjects vaccinated with plain vaccine. these response indicators were similar in the two vaccine groups when the response was evaluated against a ⁄ genoa ⁄ 2 ⁄ 05 and a ⁄ genoa ⁄ 11 ⁄ 04, which present no amino acid changes onto antigenic sites and identical hi titers respect with a ⁄ california ⁄ 7 ⁄ 04 at molecular and antigenic characterization, respectively. thus, the advantage offered by mf59 in terms of higher immunogenicity expressed by higher post-vaccination hi titres is observable also against viruses showing antigenic and molecular pattern undistinguishable from vaccine strain, but it became even more evident as the antigenic and molecular distance between vaccine and circulating strains grew. as emerged for a ⁄ genoa ⁄ 59 ⁄ 04, a ⁄ genoa ⁄ 47 ⁄ 05, and a ⁄ genoa ⁄ 62 ⁄ 05, one amino acid was a sufficient change in antigenic sites for 2-fold decrease of hi titre against homologous vaccine strain to observe 2-fold higher post-vaccination nt titers (mf59 ⁄ subunit postvaccination gmt ratio range between 1ae79 and 2ae45, figure 1) and one-dilution higher corrected post-vaccination hi titers in mf59 vaccine group ( figure 2) . finally, the correlation between the distance and the improvement offered by mf59 in terms of higher immunogenicity clearly emerged by spearman correlation analysis: it remains wellfounded both using a number of different response parameters obtained from hi and nt assays and calculating the distance by serological and genetic methods. outbreaks of h1n1pdm in pigs in commercial swine operations have been reported in several countries. in all incidents, epidemiological investigations have linked humans as the possible source of the infection to pigs. experimentally, it was established that the virus is pathogenic and transmits readily in pigs. 1 the natural outbreaks of h1n1pdm and laboratory studies underscore the threat that the virus poses to the swine industry and highlight the need for developing effective control strategies. in the united states, a trivalent live attenuated influenza vaccine (flumistò) has been licensed for use in humans since 2003. 2 in swine medicine, however, temperature-sensitive laivs are not available. currently, only inactivated vaccines are available for pigs, but they provide limited protection against antigenically diverse influenza viruses. additionally, the use of inactivated vaccines has been associated with enhanced pneumonia when immunized pigs were challenged with divergent viruses. 3 thus, the development of laivs has the potential to circumvent the drawbacks associated with commercial vaccines. with the aim of developing laiv temperature-sensitive influenza vaccines against the h1n1pdm virus, we have used reverse genetics to introduce attenuation markers in the polymerase genes of a swine-like tr h3n2 influenza virus, a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04). 4 we chose this isolate because it grows well in both eggs and cell culturebased substrates, displays a broad host range, and has internal genes similar to the h1n1pdm virus. safety and efficacy studies of the ty ⁄ 04 att vaccine candidates in pigs demonstrated that this vaccine backbone is attenuated in swine and conferred sterilizing immunity upon an aggressive intratracheal challenge of pigs with the 2009 h1n1 pandemic virus. thus, introduction of genetic signatures for att in the backbone of a swine-like tr influenza virus resulted in highly attenuated and efficacious live influenza vaccines with promising applications veterinary medicine. 293-t cells and mdck cells were maintained as previously described. 5 a ⁄ turkey ⁄ ohio ⁄ 313053 ⁄ 04 (h3n2) (ty ⁄ 04) has options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 been previously described and it was kindly provided by yehia saif, ohio state university. 4 a ⁄ california ⁄ 04 ⁄ 09 (h1n1) (ca ⁄ 04) was kindly provided by the centers for disease control and prevention (cdc). generation of recombinant viruses by reverse genetics (rg) was done using a previously described method. 6 the genetic signatures for attenuation 5 were introduced into the pb2 and pb1 genes of ty ⁄ 04. ny 2:6 ty ⁄ 04 att is a 2:6 reassortant with the surface genes from the a ⁄ new york ⁄ 18 ⁄ 2009 (h1n1) virus and the ty ⁄ 04 att internal genes. all viruses were amplified in mdck cells to produce viral stocks. twenty-five pigs were divided into five groups (n = 5) and intranasally inoculated with 10 5 tcid 50 ⁄ animal of either h3n2:6ty ⁄ 04 att or with ny(h1n1)2:6ty ⁄ 04 att vaccines diluted in 2 ml of mem. two other groups were similarly inoculated with h3n2:6ty ⁄ 04 wt and h3n2:6ty ⁄ 04 rg and served as controls, whereas a fifth group was mockvaccinated with pbs alone. clinical observations were performed as previously described. 1, 7 efficacy of h1n1 ty ⁄ 04 att vaccine in pigs fourty pigs were divided in four groups (n = 10)( table 1) . group 1 was vaccinated with 10 5 tcid 50 ⁄ animal of ny(h1n1)2:6ty ⁄ 04 att through intranasal route, whereas group 2 was vaccinated intramuscularly with 2 ml of an adjuvanted uv-inactivated ca ⁄ 04 vaccine (uvadj-ca ⁄ 04). 7 group 3, non-vaccinated and challenged (nv+ca ⁄ 04), and group 4, non-vaccinated, mock-challenged (nv+mock), were also included. pigs were boosted two weeks later. fourteen days post boost (dpb), pigs from groups 1-3 were challenged intratracheally with 2 ml of 1 · 10 5 tcid 50 of ca ⁄ 04. following challenge, pigs were monitored using methods as previously described. 7 all statistical analyses were performed using graphpad prism software version 5ae00 (graphpad software inc., san diego, ca). the differences were considered statistically significant at p < 0ae05. the ty ⁄ 04 att-based vaccines are attenuated in swine pigs inoculated with wt ty ⁄ 04 viruses developed fever (>40°c) that peaked 24 hpi ( figure 1a) and shed large amounts of in nasal secretions ( figure 1b) . similarly, viral titers in bronchoalveolar lavage fluid (balf) collected at 3 dpi ranged from 10 5 to 10 6 tcid 50 ⁄ ml ( figure 1c ). at necropsy, the lungs from animals inoculated with these viruses had severe pneumonia ( figure 1d ). in contrast, none of the animals inoculated with h3n2 or h1n1 ty ⁄ 04 att viruses developed clinical signs following vaccination, indicating that the ty ⁄ 04 att viruses were safe for administration to pigs ( figure 1a) . correspondingly, there was 100-1000 fold less virus shedding from the nose of pigs vaccinated with ty ⁄ 04 att viruses as compared to unmodified ty ⁄ 04 viruses. in general, ny(h1n1)2:6ty ⁄ 04 att -vaccinated pigs shed less virus than h3n2:6 ty ⁄ 04 att inoculated pigs ( figure 1b ). in addition, viral titers in balf were significantly reduced (p < 0ae01) in ty ⁄ 04 attvaccinated pigs as compared to ty ⁄ 04 wt-infected pigs ( figure 1c ). although both vaccines caused mild gross and microscopic lesions in the lungs, the percentage of lung 0ae2 ± 0ae1* 0 ± 0* 0 ± 0* 0 ± 0* balf, bronchoalveolar lavage fluid, uvadj-ca ⁄ 04, uv-inactivated ca ⁄ 04 vaccine; nv+ca ⁄ 04, non-vaccinated, challenged positive control group; nv+mock, non-vaccinated, non-challenged negative control group. *significantly different from nv+ca ⁄ 04 control group at p < 0ae05. geometric mean hi titer against ca ⁄ 04 at the day of challenge. à percentage of macroscopic lung lesions given as mean score ± sem. § average viral titer (log 10 ) measure as tcid 50 per ml. -average viral titer (log 10 ) in balf at 5 dpc. involvement was not significantly different from mock-vaccinated pigs, corroborating the clinical findings that these vaccines are sufficiently attenuated in pigs ( figure 1d, e) . histopathologically, nasal turbinates and trachea obtained from pigs immunized with either vaccine were similar to control animals, as opposed to the wt-inoculated pigs ( figure 1e ). vaccination with h1n1 ty ⁄ 04 att-based vaccines provides sterilizing immunity against h1n1pdm in pigs the clinical performance in pigs of the h1n1 vaccines is summarized in table 1 . nv+ca ⁄ 04 animals had macroscopic pneumonia, viral replication in balf and shedding in the nose. uvadj-ca ⁄ 04 vaccine provided satisfactory protection, but this protection was not sterilizing. remarkably, animals vaccinated with ny(h1n1)2:6ty ⁄ 04 att had sterilizing immunity. in both vaccine groups there was a significant reduction (p < 0ae001) in the percentage of macroscopic lung pathology compared to the nv+ca ⁄ 04 group. control pigs had neither significant macroscopic nor microscopic lesions in the lungs. hi antibody titers measured at the day of challenge in both vaccine groups were approximately the same (table 1 ). in the present study, we developed for the first time, temperature-sensitive laiv for use in pigs. data from our safety studies showed that both the h3n2 and h1n1 ty ⁄ 04 att vaccines were attenuated in pigs. although the ty ⁄ 04 att vaccines were detected in balf samples, the level of viral replication was significantly reduced in comparison to unmodified virus and, more importantly, caused no overt clinical signs. a minimal amount of replication is likely beneficial for eliciting t-cell responses to internal genes that may provide heterologous cross-protection. one of the most challenging tasks in producing effective live attenuated vaccines is to achieve an adequate balance between safety and efficacy. by introducing the att modifications into the polymerase genes of a swine-like tr strain, this desirable balance was achieved. the vaccines were histopathologic scores of nasal turbinates, trachea and lungs at 3 dpi. ny(h1n1)2:6ty ⁄ 04 att (a virus that carries the surface genes of a ⁄ new york ⁄ 18 ⁄ 09 (h1n1) and ty ⁄ 04 att internal genes). all h3n2 viruses have their surface genes derived from ty ⁄ 04. values are shown as the mean ± sem. * p < 0ae05; **p < 0ae01; *** p < 0ae001. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 328-394 attenuated in pigs and, more importantly, provided sterilizing immunity upon an aggressive challenge with pandemic h1n1 as opposed to an experimental ca ⁄ 04 inactivated vaccine, which elicited protective but not sterilizing immunity in all animals. in the face of influenza pandemics that have the ability to overcome the species barriers such as the 2009 h1n1, the supply of vaccines for use in agriculture could be jeopardized. our cell culture-based live att h1n1 vaccines could be an attractive alternative for this possible pandemic vaccine shortage. because the ty ⁄ 04 att live vaccines developed here are efficacious in swine, are easier to manufacture than inactivated vaccines, and do not require adjuvants, our study represents a major advance in vaccine development for the 2009 h1n1 pandemic. in conclusion, our second generation of live att influenza vaccines based on modifications of the pb2 and pb1 genes of ty ⁄ 04 retains its safety properties in vivo and can induce excellent protection against aggressive h1n1 challenges in the swine host. influenza virus is one of the most important respiratory pathogens worldwide. 1, 2 type a influenza causes an acute disease of the upper airways, and affects 20-40 million persons yearly. moreover, the threat of human influenza epidemic and pandemic has dramatically increased in recent years. 3 vaccination is one of the crucial interventions for reducing the spread and impact of influenza. the generally used parenteral inactivated influenza vaccines induce mainly systemic antibody responses and only weak cell-mediated immunity and low levels if any mucosal immunity. on the other hand, intranasal immunization with live virus can induces a broad spectrum of both systemic and mucosal antibodies, and the immune response localized in the mucosa blocks the virus even during the first phase of infection. unfortunately, the use of live vaccines is always associated with a certain risk. the development of a crossprotective vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. we induced intrasubtypic and intersubtypic cross-protection in balb ⁄ c mice by intratracheal (it) immunization with inactivated influenza viruses together with dead delipidated bacillus firmus (dbf) as an adjuvant. ten days after the 2nd immunization dose, the mice were infected with live influenza virus b ⁄ lee ⁄ 40 lethal for mice (total infection dose corresponded to 5 · ld 50 ) or a⁄ pr 8 ⁄ 34 (total infection dose corresponded to 0ae5-5 ld 50 ). dbf adjuvant markedly increased both systemic and mucosal anti-viral antibody formation when applied together with inactivated influenza a or b viruses. 4 protective significance was tested in vivo. mice were preimmunized with 1) pbs (controls), 2) dbf alone, 3) virus alone, and 4) vir-us+dbf. influenza b virus strains b ⁄ lee and b ⁄ yamanashi 166 ⁄ 98 (58 years phylogenetically distant and antigenically substantially different, especially in terms of the main protective antigen -surface haemagglutinin) or two different influenza a subtypes -a ⁄ pr 8 ⁄ 34 (h 1 n 1 ) and a ⁄ california 7 ⁄ 04 (h 3 n 2 ) -were used (figures 1 and 2) . the mice were challenged with 5 · ld 50 of either b ⁄ lee ⁄ 40 or a ⁄ pr 8 ⁄ 34 as appropriate. all controls died. the mice treated with dbf alone died with a delay or survived, which could be explained by stimulation of innate immunity. the animals immunized with virus alone were protected against homologous strains. adjuvant immunization was cross-protective: the mice immunized with a heterologous b strain (figure 2 ) fell ill (pronounced body mass loss), but almost all survived and recovered. 5 the mice immunized with a heterologous a subtype were excellently protected (negligible weight loss and zero mortality). 6 intratracheal dbf (500 lg per mouse) given to non-immunized mice 24 hour before influenza infection eliminated the lethal effect in 40-100% of infected animals depending on infection dose (0ae5-5 ld50); in mice infected with lower than lethal doses (0ae5 ld50), weight loss was minimized or did not occur. the current mode of vaccination-induced immunity is mostly effective against a homologous strain of the virus used for vaccination. the attention is therefore focused on vaccines that are able to induce cross-protection and could be effective also in case of sudden appearance of a new virus variant. inactivated influenza viruses are known to be often insufficiently effective when used for mucosal immunization and for induction of cross-protection against drifted influenza viruses or novel subtypes. the drawback of vaccination with dead virus can be overcome by using a suitable adjuvant. mouse models were successfully immunized with vaccine containing inactivated virus in combination with cholera toxin or the escheria coli heat-labile toxin (lt). [7] [8] [9] the use of cholera toxin in humans is precluded because of its high toxicity; a number of lt mutants that retain their adjuvant activity have been prepared; these mutants were likewise tested on the mouse model and should not cause any serious side effect in humans. for this reason, current studies aim at finding a suitable and safe mucosal and systemic immune response. dbf has been shown to be a very efficient adjuvant for mucosal immunization stimulating both innate and adaptive immunity. intratracheal immunization with inactivated influenza viruses and dbf as adjuvant induced efficient and even heterosubtypic cross-protection. dbf given 24 hour before infection provided partial protection probably because of its strong stimulatory effect on the innate immunity. temperature-sensitive and cold-adapted candidates for live attenuated influenza vaccine with genomic composition of 7:1 based on highly pathogenic influenza a ⁄ h5n1 viruses with pandemic potential were generated by the replacement of six internal genes from the influenza a ⁄ puerto rico ⁄ 8 ⁄ 34 (pr8) virus from pr8-based rg-candidates for inactivated vaccine with appropriate internal genes of influenza a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor virus (mdv) for russian laiv by methods of classical reassortment. all attempts to capture avian n1 neuraminidase into the genome of the mdv laiv production were ineffective. 6:2 reassortants were not generated. step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv in some cases was the only possibility to generate influenza a ⁄ h5n2 cold-adapted vaccine reassortants. difficulties in generating 6:2 reassortants could be explained by a substantial gene constellation in the genome of pr8based h5n1 reassortant viruses. strong coupling of pb2 ⁄ pr8 and avian n1 genes in a ⁄ h5n1-pr8-rg reassortants was revealed. annually updated laiv strains are generated by classical reassortment of circulating influenza viruses with well characterized, attenuated, ts ⁄ ca mdvs. resulting attenuated reassortants inherit the relevant ha and na of wild type parental virus and six internal genes of the mdv. 1 candidates for inactivated influenza vaccines based upon avian influenza viruses with pandemic potential are generally generated by reverse genetics methods. 2 in these cases, like with laiv, vaccine strains are 6:2 reassortants which possess the modified ha and na from potentially pandemic virus and six internal genes from the pr8 virus. the pr8 virus is considered to be of low virulence, i.e. attenuated, for humans, yet offers properties of high seed virus growth for influenza vaccine production. the ha of avian h5 influenza viruses with pandemic potential is engineered to remove four basic amino acid codons from the cleavage site of ha, resulting in a virus that is considered attenuated for natural hosts and safe for people. the objective of this study was to safely generate vaccine candidates for a laiv using highly pathogenic avian influenza viruses by the replacement of six internal pr8 genes in the genome of candidates for inactivated vaccine subtype h5n1 (a ⁄ h5n1-pr8-rg) with internal genes of the laiv mdv by methods of classical reassortment. len17-mdv and a ⁄ h5n1-pr8-rg virus were co-infected in embryonated chicken eggs. five rounds of selective propagation were performed, three of which were at low temperature (25°c). the production and selection of reassortants were carried out in the presence of rabbit antiserum to len17-mdv. cloning by endpoint dilution was performed in each of the last three passages. a virus sample in an open petri dish was rocked gently for 20 sec while being irradiated with a ge 15 watt germicidal lamp at a distance of 20 cm from the dish. the residual infection titer was measured by titration in embryonated chicken eggs. genome composition of reassortant viruses was monitored by rflp analysis. 3 in addition, capacity of reassortant viruses to grow at optimum, low, and elevated temperatures (ca ⁄ ts phenotype) for influenza viruses was determined by virus titration in chicken eggs. reassortment of the mdv with the vn-pr or indo-pr viruses either resulted in reassortants that contained six internal genes from len17-mdv. however, all generated clones contained the na from the mdv. of ten such 7:1 reassortants based on vn-pr three reassortants had the pa gene from pr8 and one had ns gene from pr8. 6:2 reassortants from the targeted h5n1 composition were not generated. after repeated attempts, 7:1 temperature sensitive and cold adapted reassortants based on vn-pr and indo-pr viruses were obtained, but again, none had inherited the avian n1 neuraminidase (table 1) . in contrast, nibrg-23 didn't reassort with the mdv at all. twelve unsucsessful attempts to develop 6:2 or 7:1 reassortants of nibrg-23 with mdv showed that the classical reassortment procedure (cloning by limited dilutions in the presence of anti-mdv serum, followed by co-infection of equal doses of two parental viruses in eggs and two selective passages at 25°c) did not work for this virus pair. to disharmonize the incredibly strong gene constellation of nibrg-23, various modifications of the co-infection step were studied, such as: altering the nibrg-23 to mdv ratio (from 1:1 to 1: tions of anti-mdv serum alone or together with anti-pr8 serum. it was noted that even if the h5n1 to mdv ratio was 1:10 6 , the clones obtained were presumably parental h5n1 viruses without the transfer of any mdv-genes into genome of nibrg-23. in all, 234 clones were isolated, and 209 of them were identical to nibrg-23 parental virus. in nine clones, only the pa gene from mdv was included, whereas in three clones only the 'cold' ns gene was included (data not shown). using uv inactivation of nibrg-23 prior co-infection was more encouraging. after the first round of co-infection of partially uv-inactivated nibrg-23 with mdv (at ratio 1:10 2 ), reassortants that inherited several internal genes of mdv were obtained in the context of the nibrg-23 background (b3, c2, c4, d1) ( table 2 ). some of them (c2, c4, d1) were chosen for the next round of co-infection. after the second round of co-infection, c2, c4, and d1 'intermediate' reassortants with mdv (at ratio 1:1 or 1:10) 7:1 vaccine reassortants finally were obtained. live attenuated influenza vaccine is considered as one of the most promising pandemic vaccines. according to the who there is evidence that laiv might be more effective than inactivated vaccines. 4 this study attempted the safe development of laiv for potential pandemic highly pathogenic avian a ⁄ h5n1 viruses on the base of rg-reassortants for inactivated vaccine with modified h5 hemagglutinin and mdv for laiv. replacement of pr8based internal genes into genome of vn-pr and indo-pr reassortants with appropriate genes of mdv was realized by the classical reassortment procedure. difficulties were encountered in obtaining 6:2 reassortants that contained both the ha and na from the wild type avian h5n1 parental virus. in attempts to reassort the nibrg-23 with mdv, the classical reassortment procedure was unsuccessful. the challenge faced was to break an incredibly strong gene constellation of the nibrg-23 virus. partial uv-inactivation of nibrg-23 was encouraged in replacement of some pr8 internal genes with mdv genes 5 in some cases avian-human reassortant viruses with gull h13n6 and human influenza h1n1 genes were difficult to generate, and reassortants with the desired genotype of six gull virus genes with human influenza a h1 and n1 genes were not isolated despite repeated attempts. the gull pb2, np, and ns genes were not present in any of the gull-human h1n1 reassortants generated. 6 it is difficult to fully understand potential reasons for observed difficulties to reassort some avian viruses with human strains. unsuccessful attempts to develop 6:2 vaccine reassortants may be caused by an observed strong connection of pb2 and na genes in the genome of a ⁄ h5n1-pr8-rg viruses. in our attempts, each reassortant that possessed avian n1 neuraminidase inheritied pb2 gene of pr8 as well. and vice versa, the 'cold' pb2 gene always appeared to be coupled with the n2 neuraminidase of the mdv. in some cases, step by step co-infection of triple reassortants (h5n1-h1n1-h2n2) with h2n2 mdv may be the only possibility to generate a cold-adapted vaccine reassortant. our studies demonstrate unique and significant challenges that are faced in the development of influenza vaccines for avian influenza viruses with pandemic potential. such challenges must be further studied to identify methodologies to allow for rapid development and response to emerging viruses in a crisis. it is imperative that these studies be continued and expanded to identify either mechanisms of such tight gene constellations in influenza viruses produced by rg-derived vaccine strains or inability some genes of human h2n2 and avian h5n1 viruses to cross. in addition, further studies to improve the efficiency of classical reassortment processes will be conducted. during the period from 1997 to 2009, avian influenza outbreaks among humans have been registered in 15 countries of asia, europe, and africa. morbidity and mortality of humans followed the global spread of avian influenza h5n1 among wild and domestic birds, which caused great economic loss to the poultry industry in many regions including some highly developed countries. the global threat from avian influenza forced scientists to develop technologies for the production of a ⁄ h5n1 human vaccine. the development of ai a ⁄ h5n1 vaccines using strains isolated in kazakhstan and the organization of local production and creation of strategic stockpiles of effective vaccines is the an important issue for public health protection in the republic of kazakhstan. to address this, a scientific program 'influenza a ⁄ h5n1 vaccine development for public health protection in kazakhstan' was approved and financed from 2008 to 2010. in this article we give basic results of the development of a recombinant ai a ⁄ h5n1 inactivated whole virion vaccine with aluminium hydroxide as adjuvant for public health protection in kazakhstan. [1] [2] [3] the development of vaccine technology was conducted with the use of a ⁄ astanarg ⁄ 6:2 ⁄ 2009(a ⁄ h5n1) recombinant strain made of a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05(h5n1) and a ⁄ pr ⁄ 8 ⁄ 34(h1n1) strains by the reverse genetics. inactivation of virus containing allantoic fluid was carried out with the use of formalin in different concentrations. complete-ness of the virus inactivation was tested by 3-fold virus passaging in embryos. 4, 5 purification and concentration of the inactivated viruscontaining allantoic fluid was conducted with the use of ultra filtration in tangential flow, which was followed by gel filtration. then we evaluated the content of total protein, hemagglutinin, and ovalbumin in purified and concentrated material. 6 vaccine was composed of clarified and inactivated virus concentrate with the known ha dose containment, and 0ae4% aluminum hydroxide was added in 1:1 proportions. composition components and quality control of finished vaccine was determined in the stages of semi-finished product and finished biopreparation. determination of quantitative ovalbumin content was conducted by elisa applying a strip test-system chicken egg ovalbumin elisa kit cat. n 6050 (alpha diagnostic international, usa). vaccine immunogenicity was evaluated by hai micro test in u-bottom 96-well plates produced by 'costar' (usa). 7 vaccine apyrogenicity was evaluated after intravenous injection of the studied preparation to rabbits. 8, 9 for confirmation of the results vaccine series were tested for bacterial endotoxins with the use of limulus amebocyte lysate produced by charles river laboratories, inc. usa. 8 the vaccine toxicity was evaluated in white mice with body weight 18-20 gm and in rats with body weight 180-210 g both males and females according to glp principles. 9 allergenic characteristics of the inactivated vaccine was determined in white outbred mice and guinea-pigs both males and females according to 'methodic guideline for evaluation of allergenic characteristics of pharmacological substances'. 10 in the first series of experiments, we conducted work for obtaining influenza a(h5n1) recombinant strain. bidirectional expression plasmid phw_b754 with full-length sequences of ha and na gene segments of the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) isolated in kazakhstan were synthesized in geneart ag, (regensburg, germany). ha gene was modified by deleting the region encoding multiple basic amino acid rrrk motif in ha cleavage site. moreover, to prevent recovery of repeating basic amino acids motif due to polymerase slide, we inserted replacements g fi t and k fi t. thus the ha cleavage site consists of the following sequence ntpqgerrrkkrglfgai ntpqtetrglfgai. the basic amino acid motif of highly pathogenic strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was replaced by the sequence tetr ⁄ glf, which is characteristic of low pathogenic strains of influenza h5n1. sequence of gene coding na in the strain a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) was cloned without modifications. the other segments pb1, pb2, pa, np, m and ns were obtained from influenza virus ivr-116 and synthesized and cloned in two-forked expression plasmid phw_b754 in geneart ag company, germany. the origin of genetic segments of vaccine strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) is presented in table 1 . vero cell culture (134 passage) (who) was received from european cell culture collection (salisbury, wiltshire sp4 0jg, great britain). the cell culture was grown in dmem ⁄ f12 medium with the addition of 10% of fetal bovine serum and 2 mm l-glutamine. to obtain reassortant virus a ⁄ astana ⁄ 6 ⁄ 05r-6:2, vero cells were infected with correlative plasmids by way of electroporation using nucleofector ii (amaxa) equipment. infected cells were placed in 6-well plates. after 6 hour, dmem ⁄ f12 medium was changed into 4 ml of opti-pro sfm (gibco) medium adding 2 mm l-glutamine and 1lg ⁄ ml trypsin. two days after cytopathic effect appearance supernatant was collected and used for infection of spf-eggs. the virus a ⁄ astanarg ⁄ 6 ⁄ 05-6:2 was grown in chicken embryos, and then virus titer was determined in chicken embryos and madine-darby canine kidney (mdck) cell culture. the titer of two final a ⁄ astanarg6 ⁄ 05-6:2 virus stocks was 9ae1 log 10 eid 50 ⁄ ml (chicken embryos); 1ae8 log 10 tcid 50 ⁄ ml (mdck cells); ha titer 1:512. a ⁄ chicken ⁄ astana ⁄ 6 ⁄ 05 (h5n1) virus contains motif of repeating basic amino acids in ha cleavage site. it is known that this sequence is the main determinant of ai virus pathogenicity. that is why this site was deleted in vaccine candidate strain. sequence results confirmed that influenza virus a ⁄ astanarg ⁄ 6 ⁄ 05r-6:2 strain ha gene sequence contains modified ha cleavage site and keeps mutations inserted for prevention of return to virus wild type. to confirm stability of modified ha gene sequence, five additional passages of recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 were conducted in chicken embryos. sequencing and following phylogenetic analysis of the recombinant strain a ⁄ astana rg ⁄ 6 ⁄ 05-6:2 ha gene sequence proved the presence of modification in ha cleavage site. deletion of pathogenicity site of the obtained virus was confirmed by lethality test for chicken embryos, intravenous pathogenicity test in chicken, and in plaque-forming test with trypsin. pathogenicity test in chicken embryos showed that recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 is capable of growing up to high titers without causing embryos' death. a ⁄ astanarg6 ⁄ 05-6:2 strain pathogenicity evaluation was conducted in 5-6 week-age white leghorns chicken, and this study proved that the strain a ⁄ astanarg6 ⁄ 05-6:2 (h5n1) is not virus pathogenicity inductor in chickens, which got intravenous injections of this virus (pathogenicity index is equal to 0). h5n1 strain ha cleavage site modification provides its cleavage capability only with tripsin-like proteases, which shows low level of pathogenicity. aiming at confirmation of ha cleavage site modification, we experimentally studied virus replication ability both with trypsin and without this enzyme. and we got the following results. in the plaque-forming test, a ⁄ astanarg6 ⁄ 05-6:2 strain produced plaques in mdck cells only with trypsin, proving the trypsin-dependent phenotype characteristic of low pathogenic avian influenza viruses. to prove the ha subtype antigenic analyses of a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain was conducted by means of serological methods in hemagglutinin inghibition test with the use of postinfection antisera of rabbits and rats (influenza research institute swd rams), standard serum received from cdc, atlanta, usa. hai test proved that a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain belongs to h5 subtype. furthermore, toxicity of vaccine candidate strain was evaluated by way of subcutaneous injection of viral material to balb mice. the strain appeared to be non-toxic for white mice getting subcutaneous injection of 0ae5 ml of the preparation. the conducted research showed that according to all tested characteristics, a ⁄ astana ⁄ 6 ⁄ 05r-6:2 strain can be used for influenza a ⁄ h5n1 inactivated vaccine production. according to its genetic characteristics, this strain belongs to the group of vaccine strains recommended by who for the development of influenza pre-pandemic inactivated vaccines. we determined basic cultivation parameters of the recombinant strain a ⁄ astanarg6 ⁄ 05-6:2 in 10-11 day chicken embryos. the determined parameters are the following: infection dose, 1000-10 000 eid 50 ; cultivation period, 72 hour; incubation temperature, 33°c. these cultivation parameters allow obtaining virus containing material with biological eid and hemagglutinating activity of 8ae5-9ae0 log 10 eid 50 ⁄ cm 3 and 1:512 ha titre and even higher. in the next series of experiments, we conducted research on the determination of optimal sequence of technological stages of virus clarification, concentration, and inactivation in the order of vaccine production. samples of viral material were subjected to inactivation before and after clarification and concentration. the regimen of virus inactivation by formaldehyde with final concentration of 0ae05%, period of inactivation of 3 days, temperature of inactivation medium of 4-6°c, ph of inactivation medium of 7-7ae5. on the basis of the conducted experiments we determined that the selected regimen of inactivation provides complete and irreversible inactivation of viral suspensions of the hpai strain irrespective of the kind of inactivated material. we did not observe reduction of ha activity in non-clarified viral suspensions. however, when we inactivated clarified and concentrated material, ha activity reduced by an order of magnitude. comparison of forms and sizes of virion structural elements in native (non-clarified) and formalin inactivated preparations did not reveal any significant differences. concentration of virus particles in the studied preparations was similar. the selected inactivation regimen provides obtaining completely avirulent viral suspension of the strain a ⁄ astanarg6 ⁄ 05-6:2, and it does not influence the structure of the virus. on the basis of the experiments results, we selected method of viral allantoic fluid inactivation without preliminary clarification. during further research, we tried to get highly clarified viral concentrate. this study resulted in the combined scheme, which includes clarification of inactivated viral allantoic fluid by low speed centrifugation at 4000 circulations per min for 30 minutes, filtration through membrane filters with pore diameter of 0ae45 lm, ultrafilatration ⁄ diafiltration, gel filtration in 6b sepharose, and sterilization of viral suspension through membrane filters with pore diameter of 0ae22 lm. the experiments resulted in the development of production technology of embryonic inactivated vaccine based on recombinant strain a ⁄ astanarg ⁄ 6:2 ⁄ 2009 (h5n1) contain-ing aluminium hydroxide as adjuvant. the developed influenza a ⁄ h5n1 human vaccine has the trade name kazfluvacò. its composition components are presented in table 2 . preclinical testing of the vaccine kazfluvacò was conducted according to the following parameters: general health condition of animals, change of body weight and temperature of immunised animals (for ferrets), presence of post vaccination antibodies response in sera, forming protective immune response against reassortant viruses of h5 subtype, study of acute and chronic toxicity of three experimental vaccine series in different doses and semi-finished vaccine product applying different ways of injection, study of allergic and immunotoxic characteristics of the vaccine, as well as study of pyrogenic reaction and analysis for bacterial endotoxins presence. [11] [12] [13] [14] preclinical tests of kazfluvacò vaccine safety showed that this vaccine does not have toxic effect on organisms of warm-blooded laboratory animals. double intramuscular injection of kazfluvacò vaccine in inoculative dose does not effect appearance, general health condition, behaviour of animals, their muscular strength and physical activity, does not have negative effect on biochemical parameters of blood and basic physical functions of animals organism, and does not cause pathomorphological changes. this shows the safety of the vaccine. local irritation action was not observed. the results of the vaccine allergic action study showed that the vaccine does not have allergic effect at the intravenous injection. the research also showed that the vaccine does not have negative effect on immune system of laboratory animals. research conducted on mice and ferrets showed high immunogenic activity of the vaccine at one-and two-dose regimen of injection. the research showed 100% of protective effect of kazfluvacò vaccine at two-dose injection regimen in ferrets infected by homological strain of influenza virus. the devised inactivated influenza a ⁄ h5n1 vaccine kaz-fluvacò is a safe and immunogenic biopreparation that is not worse than the overseas analogues in its immunobiological characteristics. [15] [16] [17] [18] to date the whole-virion inactivated influenza a ⁄ h5n1 vaccines of the producers such as omnivest (hungary), biken, denka seiken, kitasato institute, kaketsuken (japan), gsk biologicals (belgium), sinovac biotech (china) are registered. all of them are produced on the basis of chicken embryos and aluminum is used as an adjuvant. kazfluvacò differs from its analogues in the flowchart of the virus purification and concentration that makes possible to produce a safer preparation. 19, 20 the results of the conducted research and preclinical testing allow starting work towards implementation of phase i preclinical tests on volunteers. it is planned to conduct a randomized blind placebo-controlled phase i study on double application of kazfluvacò vaccine in increasing doses. the preparation will be administered to volunteers aged 18-60 years for assessment of its safety and immunogenicity in doses of 7ae5 and 15ae0 lg of ha. when the world health organization (who) announced the sixth phase of a ⁄ h1n1v influenza pandemic, scientists all over the world started investigation to develop technology for production of prophylactic means against the disease. having taken into consideration the threat of a pandemic for kazakhstan, the ministry of education and science of the republic of kazakhstan launched the program ''monitoring, study, and development of diagnostic, prophylactic, and therapeutic means for influenza a ⁄ h1n1.'' this paper presents the experimental data obtained at the ribsp in the course of the studies towards the development of technology for production of an inactivated a ⁄ h1n1 influenza vaccine, as well as the results of pre-clinical testing of the developed vaccine. the development of vaccine production technology was conducted with the use of who recommended vaccine strain nibrg-121xp constructed by the method of reverse genetics in the national institute for biological standards and control (nibsc, great britain). the virus was inactivated with formalin at different final concentrations, and the extent of inactivation was evaluated via threefold virus passages in developing chicken embryos. 1 the inactivated virus was purified and concentrated by the method of ultrafiltration in tangential flow followed by gel filtration. the purified and concentrated material was evaluated judging on the total protein, hemagglutinin (ha), and ovalbumin. the vaccine was prepared by pooling the purified and concentrated virus material with the certain weight content of ha and the work solution of aluminum hydroxide (0ae4%) in the ratio 1:1. the ovalbumin content was quantified in elisa with the use of the strip test system chicken egg ovalbumin elisa kit (cat. no. 6050 alpha diagnostic international, san antonio, texas, usa). weight content of the virus ha was determined according to sominina, burtseva. 2 the content of the residual formaldehyde, aluminum (al +3 ) ions, and thiomersal in the vaccine was measured according to the operating instructions. 3 the vaccine immunogenicity was assessed in the hemagglutination inhibition test, which was carried out as a microassay in 96-welled u-bottomed plates (''costar'', new york, usa). 3, 4 apyrogenicity of the vaccine was assessed post intravenous administration of the tested preparation to rabbits. 5, 6 to confirm the obtained results the vaccine batches were tested for bacterial endotoxins with use of the limulus amebocyte lysate (charles river laboratories, inc., wilmington, ma, usa). 7 the toxicity of the vaccine was assayed in white mice weighing 18-20 g and in rats weighing 180-210 g (male and female) in compliance with the principles of good laboratory practice. 8 allergenic properties of the inactivated vaccine were determined according to the ''operating instructions on assessment of allergenic properties of pharmaceutical substances'' 9 in white outbred laboratory mice and guinea-pigs of both sexes. the first step in the course of developing technology for vaccine production was to determine the major conditions for influenza virus cultivation: usage of 10-days embryonated chicken eggs at the infectious dose within 1000-10 000 eid 50 , incubation temperature (34 ± 0ae5)°c, and duration of the incubation period 72 hours. the established parameters for virus cultivation made it possible to produce virus-containing materials of infectious activity within 8ae5-9ae0 log eid 50 ⁄ cm 3 and hemagglutinating activity 1:256 and higher. in the subsequent experiments, an optimal method for virus inactivation was selected. on the basis of the experimental findings, the following conditions for inactivation of the native virus-containing material were elected: formalin of 0ae05% final concentration as an inactivating agent; inactivation period of 72 hours at temperature (4 ± 2)°c. these conditions provide the complete inactivation of the virus (nibrg-121xp strain) material, did not impact distinctly the structural organization of the virus, and did not reduce the antigenic activity. as it is well known, virus purification and concentration means very much in the development of technology for production of an inactivated whole-virion influenza vaccine. the investigation into optimization of the technological step of purification and concentration of the recombinant influenza virus nibrg-121xp strain resulted in selection of an optimal pattern including such steps as clarification of the virus suspension by filtration through membranes with pore size 0ae45 lm, virus concentration by ultrafiltration in a tangential flow, dialysis filtration in a tangential flow, gel filtration on sepharose 6b, and sterilization of the viral suspension through membrane filters with pore size 0ae22 lm. the studies conducted by the ribsp specialists resulted in the development of technology for production of the first domestic whole-virion inactivated a ⁄ h1n1 influenza vaccine with aluminum hydroxide as adjuvant and with the brand name refluvac ò . the key processing characteristics of the whole-virion inactivated a ⁄ h1n1 influenza vaccine vaccine refluvac ò are shown in table 1 . simultaneous with the performance of all process operations, the parameters such as sterility, inactivation extent, ph, vaccine specificity, total protein content, weight content of has, aluminum and formalin contents, content of thiomersal, and ovalbumin, pyrogenicity of the vaccine and its immunogenicity for mice, were optimized. the key qualitative characteristics of the designed influenza a ⁄ h1n1 vaccine refluvac ò are shown in table 2 . before implementation of phase i clinical trials on volunteers, preclinical testing of three experimental batches of refluvac for immunogenic activity and safety was carried out. it was conducted in three laboratory bases of research institutions: the toxicology institute ⁄ federal medicobiological agency, russia (st petersburg), the research institute for biological safety problems (republic of kazakhstan), and the influenza research institute ⁄ north-western branch of the russian academy of medical sciences (st petersburg), with use of different animal models (mice, rats, chinchilla rabbits, guinea-pigs, ferrets). the results of the preclinical testing are as follows: • electron microscopy of the preparation has shown that the viral particles are well dispersed and do not aggregate. the portion of whole (intact) particles is over 95%, which is evidence of virion integrity; • assessment of polypeptide composition of the vaccine refluvac by electrophoresis in 10% polyacrylamide gel with sodium dodecyl sulfate has shown the vaccine to contain both surface antigens (ha, na) and highly purified inner virion proteins (np, m1) that are typespecific antigens, so the vaccine is a preparation of full immunological value; • judging on the parameters of acute and chronic toxicity for white mice and rats of both sexes, the vaccine is a non-toxic and safe preparation; • under conditions of a chronic experiment on white mice and rats, it was found that refluvac does not produce changes in behavior, somatic, or vegetative responses; • assay of hematological and biochemical blood characteristics of white mice and rats following vaccine administration did not reveal any significant differences as compared to the animals of the control group; • refluvac does not cause allergenic and immunotoxic impact; • the vaccine refluvac does not cause local irritative effect; • refluvac is apyrogenic for laboratory animals; • the pathomorphological and hystopathological analysis did not reveal any changes due to immunization in animal organs; • testing of immunogenic characteristics of the vaccine on mice and ferrets has shown formation of hemagglutinating antibodies in animals after single administration; • refluvac induces 100% protection in immunized ferrets at their challenge with the wild-type influenza virus a ⁄ california ⁄ 07 ⁄ 2009 (h1n1v). the results of the performed preclinical testing have allowed concluding that refluvac, an inactivated whole-virion vaccine with aluminum hydroxide as adjuvant, is a safe and highly effective preparation against influenza a ⁄ h1n1v. the implemented study resulted in development of technology for production of the first domestic inactivated allantoic whole-virion influenza a ⁄ h1n1 vaccine with aluminum hydroxide as an adjuvant under the brand name refluvac ò based on the recombinant strain nibrg-121xp. the devised pandemic vaccine meets who requirements as well as requirements concerning safety and immunogenicity of the national pharmacopeias of the republic of kazakhstan and russian federation. [9] [10] [11] [12] the devised technology for vaccine production differs from the previous technologies for production of allantoic whole-virion influenza a ⁄ h1n1 vaccines in its processdependent parameters. presence of an adjuvant (aluminum hydroxide) increases significantly the vaccine immunogenicity and allows maximal reduction of the dose of the administered antigen that, in turn, results in diminished reactogenicity of the vaccine. aluminum hydroxide is an adjuvant that is most frequently used in clinical practice. 13 to date the results of the double-centered randomized study of the europe-licensed vaccine fluval p [monovalent inactivated whole-virion influenza vaccine with aluminum phosphate based on strain a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) nymc x-179a (omninvest, pilisborosjeno, hungary)] that is similar to the refluvac preparation are published. the data of this research are an evidence of safety and high immunological effectiveness of the vaccine in dose 6 lg ha at single administration both in adults and elderly persons. 14 the results of the pre-clinical tests allow recommending carrying out phase 1 clinical testing of the refluvac ò vaccine for safety and immunogenicity. single immunization of volunteers with refluvac ò in doses 3ae75, 7ae50, and 15ae00 lg of ha are planned. mid 50, respectively. the study results confirm that new h1n1 laiv and h7n3 laiv candidates are safe and immunogenic and confer protection from homologues influenza virus infection in mice. the recent emergence of a new pandemic h1n1 virus and the threat of transmission of avian viruses to humans had stimulated research and development of live attenuated cold-adapted influenza vaccines against newly appeared influenza viruses. formulations of live attenuated influenza a vaccine (laiv) against pandemic influenza strains, including h1n1, h5n1, h9n2, and h7n3 are currently being tested in preclinical and phase i clinical studies. 1 the following paper describes the preclinical study of new h1n1 and h7n3 laiv candidates in mice. the study addressed the following three objectives: (i) to demonstrate that cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates are indistinguishable from the parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) master donor strain (mds) virus with regard to replication efficiency in upper and lower respiratory tract of mice; (ii) to demonstrate the immunogenicity of different doses of cold-adapted (ca) reassortant influenza a(h1n1) and a(h7n3) vaccine candidates in mice; and (iii) to demonstrate the protective efficacy of cold-adapted (ca) reassortant influenza a(h5n1) and a(h7n3) vaccine candidates in mice against a homologous wild-type virus challenge. the a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) reassortant containing the ha and na genes from a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) and six other genes from mds, the a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) reassortant containing the ha and na genes from a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and six other genes from a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) were generated by classical genetic reassortment in embryonated chicken eggs (ec). viruses were propagated in 10days old eggs (34°c, 48 hours). fifty percent egg infectious dose (eid 50 ) titers were determined by serial titration of viruses in eggs. titers were calculated by the method of reed and muench. 2 female balb ⁄ c mice, 6-8 weeks of age were used in all experiments. mice were lightly anesthetized with ether and then inoculated intranasally (i.n.) with 50 ll of infectious virus diluted in phosphate-buffered saline (pbs). mice were inoculated with 100 mid 50 (50% mouse infectious dose) of a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1), a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), and a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) mds. viral loads were measured in respiratory and brain tissues collected at 3 and 6 days post-infection (dpi). tissue homogenates prepared using a disruptor and clarified supernatants were titrated on eggs at permissive temperature to determine infectious concentrations. groups of animals were inoculated with 1000 mid 50 or 100 mid 50 of either h1n1 laiv or h7n3 laiv intranasally after collecting a pre-immunization blood sample. a second blood sample was collected at 28 dpi. on the same day, the animals received a second intranasal inoculation with the same virus that was used for priming at 0 dpi. to assess protection, all animals were infected 42 dpi with either 100 mid 50 of a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) or 100 mid 50 a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) virus by the intranasal route. four animals from each group were euthanized at 45 dpi, and the respiratory and systemic organs were harvested for virus titration. a forth blood sample was collected at 56 dpi from the remaining animals. hi antibody titers were determined for individual serum samples collected on days 0, 28, 42, and 56. body weights were taken daily following challenge through day 14 postchallenge. sera were tested for hi against homologous h1n1 and h7n3 viruses. the h1n1 laiv, h7n3 laiv and h2n2 mds influenza viruses replicate in mice lungs at level 2ae1-2ae3 lgeid 50 ⁄ ml at 3 dpi (figure 1 ). at 6 dpi, replication of the viruses in the lungs decreased to 1ae6-2ae0 lgeid 50 ⁄ ml (data not shown). in contrast, the wild-type virus a ⁄ mallard ⁄ netherlands ⁄ 00 (h7n3) demonstrated high level replication in lungs -6ae4 lgeid 50 ⁄ ml. the levels of replication of studied viruses in nasal turbinates were 2ae5-3ae7 lg eid 50 ⁄ ml at 3 dpi (figure 1) , and 2ae0-2ae2 lgeid 50 ⁄ ml at 6 dpi (data not shown). there were no significant differences between the viruses in regard to replication in upper respiratory tract of mice. thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3) and a ⁄ 17 ⁄ california ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates was indistinguishable from parental a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in terms of replication in the lungs and noses of mice at 3 and 6 dpi. no virus was found in the brain tissue of immunized mice at 3 and 6 dpi (in undiluted samples tested). thus, it was shown that a ⁄ 17 ⁄ mallard ⁄ netherlands ⁄ 00 ⁄ 95 (h7n3), a ⁄ 17 ⁄ cali-fornia ⁄ 2009 ⁄ 38 (h1n1) vaccine candidates are identical to a ⁄ leningrad ⁄ 134 ⁄ 17 ⁄ 57 (h2n2) in lacking neuroivasive capacity, and all three viruses similarly fail to replicate in the brain. it was shown that all immunized animals survived after challenge with wild-type a ⁄ mallard ⁄ netherlands ⁄ 12 ⁄ 00 (h7n3) virus. the mice in vaccine groups showed no signs of morbidity. average weight changes were tracked from day 2 to day 6 in all study groups, but the changes did not exceed 5%. as shown in figure 2 , the challenge virus actively replicated in respiratory tissue taken from mock immunized animals (5ae7 lgeid 50 in the lung and 4ae2 lgeid 50 in the nose), but failed to infect the brain and spleen. on the other hand, in both h7n3 laiv vaccinated groups, all tested organs were free from presence of challenge virus. thus, immunization of mice with either 1000 mid 50 or 100 mid 50 h7n3 laiv protected the animals from the subsequent challenge infection with a homologous with wild-type h7n3 virus. both h1n1 and h7n3 laiv candidates were found to be immunogenic. after one dose of 100 mid 50 of h1n1 laiv, gmt of hi antibodies were 17ae6. one dose of 1000 mid 50 or 100 mid 50 h7n3 laiv elicited hi antibody level with gmt of 8ae7 and 6ae6, respectively. the second dose of h7n3 laiv further stimulated serum hi antibody levels to gmt 40ae0 and 23ae3, for 1000 mid 50 or 100 mid 50, respectively (data not shown). the mouse model is widely used to better understand the pathogenicity of avian influenza viruses for mammalian species, to be able to predict the pandemic potential of such viruses, and to develop improved methods for the prevention and control of the virus in a potential pandemic. 3 a subset of the h7 viruses was evaluated for the ability to replicate and cause disease in balb ⁄ c mice following intranasal administration. h7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. 4 there is limited preclinical information available for laiv. thus, live monovalent vaccine against pandemic influenza virus h1n1 (influvir) was tested for acute toxicity and its effect on the systems and organs of laboratory animals. according to toxicology and necroscopy results, the live monovalent influenza vaccine influvir, when applied intranasally, was safe and was well tolerated. 5 in our current study we demonstrate that a(h1n1) and a(h7n3) laiv are indistinguishable from the parental mds virus with regards to replication kinetics in the upper and lower respiratory tract of mice. both h1n1 and h7n3 laiv candidates were immunogenic and protect mice against subsequent a challenge with the wild-type virus. live attenuated cold-adapted (ca) influenza vaccines are an effective means for the control of influenza, most likely due to their ability to induce both humoral and cellular immune responses. in our study we confirm that new h1n1 laiv and h7n3 laiv candidates are safe, immunogenic, and confer protection from influenza infection in mice. health organization (who) declared a pandemic by raising the worldwide pandemic alert level to phase 6. therefore, h1n1 inactivated monovalent vaccine formulated with our proprietary oil-in-water emulsion based adjuvant was evaluated in ferrets for its potential to induce with low antigen dose efficient, robust, and rapid protective immunity against a wild type challenge virus (a ⁄ netherlands ⁄ 602 ⁄ 2009). this adjuvant was also tested in ferrets in a h5n1 avian influenza model for its ability to induce a cross-clade immunity and cross-protection. two independent studies (a&b) were carried out with male and female outbred ferrets (musleta putorius furo) in compliance with ''guide for the care and use of laboratory animals,'' ilar recommendations and aaalac standards. ferrets used in both studies were influenza seronegative by anti-nucleoprotein elisa and by hi assay against the pandemic and seasonal strains. in study a, four groups of seven ferrets aged approximately of 6 months received one or two im vaccinations 3 weeks apart of either af03-adjuvanted (3ae8 lg of ha with af03) or unadjuvanted ( body weight loss was monitored as an indicator of disease and a mean body weight loss of 20% was recorded in the control group at day of necropsy. body weight loss was reduced to £10% and £8% in animals that had received 1 and 2 doses of either unadjuvanted or af03-adjuvanted vaccine, respectively. viral lung titration showed high levels of virus replication ( ‡4ae7 tcid50 ⁄ g tissue) in the lungs of all control ferrets 4 days after challenge. one or two administrations of unadjuvanted vaccine reduced lung viral load by 2 and 3 log 10 , respectively. interestingly, ferrets that received either one or two doses of af03-adjuvanted h1n1 2009 vaccine, showed significantly greater reduction of lung viral loads (>4 log 10 ). no virus was detected in the lungs of 6 ⁄ 7 (86%) animals immunized with a single injection of the af03-adjuvanted vaccine and in 100% of ferrets vaccinated twice. assessment of viral shedding from the upper respiratory tract showed that the af03-adjuvanted a ⁄ h1n1 monovalent vaccine was able to reduce the viral load in the nose and in the throat by 3ae8 and 3ae1 log 10 , respectively, as compared to the control group. conversely, viral loads were only slightly reduced in the nose and mostly unchanged in the throat in ferrets immunized with either one or two doses of unadjuvanted a ⁄ h1n1 monovalent vaccine. gross pathology and histology examinations revealed lung lesions consistent with influenza a ⁄ h1n1 virus infechowever, a second dose of af03-adjuvanted vaccine strongly increased hi and mn titers, which persisted for 3 months (table 2 ). antibody responses cross-reactive to heterologous clade 2.2 strain were elicited ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine. hi antibody titers ‡40 crossreactive to clade 2.2 and persistent up to d110 were observed in vaccinated animals. an inter-clade low crossreactive hi response to a clade 1 strain was only detected in a few ferrets that had been vaccinated with the af03-adjuvanted clade 2.1. all af03-adjuvanted clade 2.1 antigen vaccinated animals survived challenge either with the homologous or heterologous virus until euthanized day 5. after challenge, mean body temperature and mean body weights were monitored as indicators of disease. in the control ferrets, mean body temperature increased by 2-3°c (depending on the challenge virus strain) 24 h post challenge, with an accompanying mean body weight loss ranging from 15ae4% to 20ae7%. ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine showed a lower and delayed fever compared to control ferrets that received the same viral challenge, whereas no significant differences were observed between vaccinated animals and their respective controls upon challenge with clade 2.2 or clade 0 viruses. body weight loss was reduced in all vaccinated animals when compared to controls after challenge with either the homologous clade 2.1 strain or with one of the heterologous strains. lung virus titration showed high levels of virus replication in all control animals 5 days after homologous challenge with the clade 2.1 virus. lung viral loads of all ferrets immunized with the af03-adjuvanted clade 2.1 vaccine were reduced more than 4 log 10 . vaccination resulted in complete viral clearance from the lungs of 80% of animals assessed 5 days after challenge. as compared to controls, a reduction of the mean viral load of about 2 log 10 was observed in ferrets vaccinated with the af03-adjuvanted clade 2.1 vaccine after heterologous challenge with either the clade 0 or clade 1 virus. conversely, vaccination with af03-adjuvanted clade 2.1 vaccine did not result in reduction of lung viral loads after challenge with the clade 2.2 heterologous virus strain. titration of pharyngeal swabs showed high levels of viral shedding in all control ferrets after challenge with clade 2.1 strain, whereas virus was not detected in any vaccinated animal. similarly, 5 log 10 reduction of viral shedding was seen in vaccinated versus control ferrets following clade 1 heterologous challenge. lower reductions in viral shedding were observed after clade 2.2 challenge (2ae6 log 10 ) and clade 0 challenge (1ae4 log 10 ). gross pathology and histology revealed lung lesions consistent with influenza a ⁄ h5n1 virus infection all control animals challenged with the clade 2.1, clade 2.2 or clade 0 strains. mild to moderate lung lesions were observed in control animals following challenge with clade 1 virus. macroscopic evaluation (percentage of affected lung parenchyma) and histopathological analysis (extent and severity of alveolitis, alveolar oedema and hemorrhage) showed that lung lesions were significantly reduced in af03-adjuvanted clade 2.1 vaccinated animals after challenge with the homologous clade 2.1 virus strain as compared to controls. similarly, a reduction of the macroscopic and microscopic lung lesions was observed in vaccinated animals upon heterologous challenge with clade 2.2 and clade 0 virus strains, whereas no differences were observed between control and vaccinated animals after challenge with clade 1 virus. the results of these ferret challenge studies demonstrated that low doses of pandemic influenza vaccines formulated with an oil-in-water emulsion adjuvant, af03, elicited strong antibody responses specific to the immunizing strain. importantly, these vaccines provided protection after homologous challenge with complete virus clearance in ferret lungs and reduced viral shedding from the upper respiratory tract suggesting an ability to reduce virus transmission. moreover, af03-adjuvanted h5n1 vaccine can provide cross-protection upon challenge with different h5n1clades by preventing mortality and reducing the viral burden in the lower and the upper respiratory tract. in conclusion, the results of these studies highlighted the ability of af03-adjuvanted influenza vaccines to induce potent immune responses and full protection in ferrets against homologous challenge and suggested that protection may be mediated, at least in part, by antigenspecific humoral immunity. since 1993, outbreaks of h9n2 influenza virus infection in poultry have occurred in eurasian countries. phylogenetic and antigenic analysis of h9n2 isolates revealed that there are three sublineages, consisting of g1, g9, and korean, among ha genes of the eurasian h9n2 viruses. h9n2 viruses do not cause severe disease in poultry, but co-infection of h9n2 viruses with bacteria such as staphylococcus aureus, haemophilus paragallinarum, or attenuated coronavirus vaccine may exacerbate the disease. 1,2 h9n2 viruses were isolated from domestic pigs in china and korea and from humans with febrile respiratory illness in hong kong in 1998 kong in , 1999 kong in , and 2003 it is, thus, postulated that in the present study, h9 virus strains were analyzed antigenically and phylogenetically to select a proper h9n2 vaccine strain. inactivated whole virus particle vaccine was prepared, and its potency against h9 virus challenge was assessed in mice. viral rnas were extracted from the allantoic fluid of chicken embryos infected with viruses by using a commercial kit (trizol ls reagent; invitrogen, california, usa) and reverse-transcribed with the uni12 primer 6 and m-mlv reverse transcriptase (invitrogen). the primers used for the ha gene amplification were h9-101f 7 and h9-1341r. for phylogenetic analysis, sequence data of the genes together with those from public database were analyzed by the neighbor-joining method. 8 h9 influenza viruses were analyzed by hemagglutinationinhibition (hi) test. 9 chicken hyperimmunized antisera against seven h9 viruses were prepared according to previous report. 10 virus replication and pathogenicity against embryonated chicken eggs viruses were inoculated into 10-day-old embryonated chicken eggs and incubated for 48 hours at 35°c. ha titers and 50% egg infectious dose (eid 50 ) were measured every 12 hours post-inoculation. pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was evaluated by mean death time (mdt) as described previously. 11 dk ⁄ hok ⁄ 49 ⁄ 98 was injected into the allantoic cavities of 10-day-old embryonated chicken eggs and propagated at 35°c for 48 hours. the virus in the allantoic fluids (512ha) was purified by differential centrifugation and sedimentation through a sucrose gradient according to previous report. 12 the concentration of protein was measured by od using ultrospec 3100 pro (amersham biosciences, tokyo, japan). the purified virus was inactivated with 0ae1% formalin at 4°c for 7 days. immunization of mice and challenge of immunized mice with hk ⁄ 1073 ⁄ 99 four-week-old female balb ⁄ c mice were purchased from japan slc, inc. (shizuoka, japan). the mice were injected subcutaneously with 10, 2, 0ae4, or 0ae08 lg proteins of inactivated dk ⁄ hok ⁄ 49 ⁄ 98 whole virus vaccine. two weeks later, the mice were boosted by subcutaneous injection with the same dose of the vaccine. control mice were injected with pbs. serum samples were tested by enzyme-linked immunosorbent assay (elisa) according to previous report. 10 one week after the second vaccination, 10 mice in each group were challenged intranasally with 30 ll of 10 6ae5 eid 50 of hk ⁄ 1073 ⁄ 99 under anesthesia. on 3 days postinfection, five mice in each group were sacrificed, and the lungs were separately homogenized to make a 10% (w ⁄ v) suspension with minimal essential medium (nissui, tokyo, japan). the virus titers of the supernatants of lung tissue homogenates were calculated in 10-day-old embryonated chicken eggs and expressed as the eid 50 ⁄ gram of tissue. the other five mice in each group were monitored for body weight for 14 days after challenge. the ha genes of 22 h9 viruses were sequenced and analyzed by the neighbor-joining method. all of the 22 h9 viruses were classified into the eurasian lineage ( figure 1) . eleven, seven, and four strains were classified in the korean, g9, and g1 sublineages, respectively. the h9 viruses of the korean and g9 sublineages were isolated from waterfowl, poultry, pigs, and humans in the east asian countries, and those of the g1 sublineage were isolated from poultry in the west asian countries. the cross-reactivity between these antisera and h9n2 viruses were analyzed by hi test. the antisera against h9 viruses belonging to the korean sublineage were broadly cross-reacted to h9 viruses belonging to the g9 and g1 sublineages. h9 viruses belonging to the korean lineage were reacted to the antisera against h9 viruses belonging to the g9 and g1 sublineage compared with h9 viruses belonging to the other sublineage (data not shown). thus, it was suggested that h9 vaccine strain should be selected from the viruses of korean sublineage to prepare for the vaccine strain of h9 viruses. dk ⁄ hok ⁄ 49 ⁄ 98 replicated efficiently in 10-day-old embryonated chicken eggs (data not shown). pathogenicity of dk ⁄ hok ⁄ 49 ⁄ 98 against embryonated chicken eggs was determined by mdt. dk ⁄ hok ⁄ 49 ⁄ 98 was low pathogenic against embryonated chicken eggs (data not shown) and was selected as an h9 vaccine strain. to assess the potency of the vaccine against h9 virus infection, mice vaccinated subcutaneously with inactivated dk ⁄ hok ⁄ 49 ⁄ 98 were challenged intra-nasally with hk ⁄ 1073 ⁄ 99. immunogenicity of the inactivated vaccine was assessed by measuring the igg antibodies in mouse sera by elisa. antibody was detected in the group of mice injected 10 lg protein after the first immunization and detected in the group of mice injected 2 lg protein after the second immunization. thus, potency of the present inactivated whole virus vaccine was demonstrated in mice. next, to assess the protective immunity of the inactivated vaccine in mice, viral titers in the lungs was determined. the virus titers in the lungs were 10 1ae5 -10 3ae7 eid 50 ⁄ g in the groups of mice injected 10, and 2 lg protein, and 10 5ae3 -10 6ae0 eid 50 ⁄ g in the other vaccinated groups. body weight reduction of mice were observed in the group of mice injected 0ae4, 0ae08 lg protein, and control groups from 3 dpi, and reached to 10% body weight loss from 4-to 6-day post-infection ( figure 2 ). this result correlates with antibody titer in mouse sera and viral titers in the lungs. these results suggest that the test h9 inactivated whole vaccine confers prevent of weight loss and reduction of virus replication against h9 influenza virus infection in mice. recently, h9n2 viruses of all of three sublineage have been isolated from wild birds and poultry in worldwide. h9n2 viruses were isolated from pigs and humans in china 3 and korea, suggesting that h9n2 virus would be a potential for a pandemic influenza virus in human population. h9n2 viruses were isolated from pigs in china and korea and were classified into the g9 and korean sublineage. in human cases, all h9n2 virus isolated from humans in china was classified into the g1 sublineage. it was suggested that h9n2 viruses isolated from pigs and humans vary in antigenicity of isolates between the korean, g9, and g1 sublineages. therefore, it is important for the preparedness of influenza pandemic to develop h9 influenza virus vaccine, which could broadly cross-react to antisera of all sublineage viruses. so, we selected the vaccine candidate strain, dk ⁄ hok ⁄ 49 ⁄ 98, which could broadly cross-react to antisera of all sublineage viruses, and which could replicate in this study, it was suggested that the test vaccine has potency to protect against challenge with h9 virus using mice for mammalian model. the challenge virus, hk ⁄ 1073 ⁄ 99, was isolated from human, replicates efficiently in mice, and shows pathogenicity in mice. the test vaccine inhibited viral replication and body weight loss in mice. whole inactivated vaccine produced protective immunity, supporting our approach of using whole virus particles for vaccine development. furthermore, whole particle virus vaccine could induce igg and mucosal iga levels after intranasal vaccination with whole particle vaccine. 13 the present results may facilitate the studies of the vaccine for future pandemic caused by h9 influenza virus in humans. tants to attempt to improve growth. to determine whether wild type h1n1pdm grew better in the novartis mdck suspension cell line (mdck33016pf) than in eggs, isolations from h1n1pdm positive clinical samples were attempted in both substrates. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . however the yields were lower than observed with seasonal viruses. little improvement in virus yield was seen with extra passaging or dilutions of h1n1pdm viruses isolated in mdck33016pf cells. with the emergence of the swine-origin pandemic h1n1 (h1n1pdm) influenza in april 2009, 1 the need for efficient production of a suitable vaccine was a high priority. 2 virus isolates were distributed by the who for the urgent development of suitable vaccine strains early in the pandemic. vaccine viruses can be grown in embryonated chicken eggs 2 or in certified mammalian cells. 3, 4 unfortunately wildtype h1n1pdm virus strains distributed by the who grew poorly in cell lines and eggs, requiring the generation of a series of 3 conventional and 8 reverse genetics 5 derived reassortants to attempt to improve growth. from these reassortants, only the conventional egg derived reassortants nymc-x-179a and nymc-x-181 (both based on one of the earliest known viruses a ⁄ california ⁄ 7 ⁄ 2009) showed high enough growth and yield in eggs and cell culture to make them suitable for vaccine manufacture. these reassortants, while acceptable, still only gave haemagglutinin (ha) yields of approximately 60% that of seasonal h1n1 reassortants. to determine if more recent wild type h1n1pdm viruses grew better in the novartis mdck suspension cell line (mdck33016pf), h1n1pdm positive clinical samples were cultured in mdck33016pf cells and also in embryonated hen's eggs. in addition, to improve virus yields from mdck33016pf isolates, extended passaging of three wild type h1n1pdm influenza viruses was performed using various virus dilutions at each passage level. the results were assessed using various serological and molecular biology techniques and compared to viruses isolated in eggs and conventional mdck cells. h1n1pdm viruses were received at the centre from who national influenza centres, who influenza collaborating centres and other regional laboratories and hospitals in australia, new zealand, and the asia ⁄ pacific region. viruses were received as original clinical specimens consisting of nasal swabs, throat swabs, nasopharyngeal aspirates, or nasal washes that had previously been shown to be h1n1pdm positive by real time rt-pcr. these specimens were then cultured in mdck33016pf cells with serum free medium containing trypzean (optaflu) 3 and also independently inoculated into the allantoic cavity of 11 day-old embryonated hen's eggs. virus cultures in mdck33016pf cells were sampled at 48 and 72 hour and evaluated by various means including ha titres. at 72 hour, virus cultures were further passaged at varying dilutions ranging from 10 )5 to 10 )8 up to a total of 10 passages. embryonated hen's eggs were incubated at 35°c for 3 days and allantoic fluid was harvested and ha titres performed to determine whether a further passage was required in order to improve growth. the conventional reassortants were produced by a mixed infection of eggs or mdck 33016pf cells with the wild type virus and a donor virus carrying the internal genes of the a ⁄ puerto rico ⁄ 8 ⁄ 34 virus. the reassortants were obtained by sequential passages using immuno-selective antisera against the surface antigen of the donor virus to remove virus populations carrying the ha and na protein of the donor strain. 6 the reverse genetics viruses were rescued in vero cells using the 12 plasmid system. 7 both types of reassortants were generated and supplied by who collaborating centres and essential regulatory laboratories except the nvd-c-07 strain, which was produced by novartis. in this small study with recent h1n1pdm viruses, the isolation rate was higher in mdck33016pf cells (89%) (31 ⁄ 35) compared to allantoically inoculated eggs (66%) (23 ⁄ 35) . assessment of ha titres, however, showed higher ha titres in egg-isolated viruses compared to viruses isolated in mdck33016pf cells after two passages. egg generated or cell generated reassortant viruses gave higher ha titres compared to the homologous wild type viruses (table 1) . no amino acid changes were observed in mdck33016pf isolated influenza viruses compared to original specimens or viruses isolated in conventional atcc derived mdck cells, unlike egg isolated viruses which showed a number of amino acid changes, many consistent with egg adaptation mutations (table 1) . 10 viruses isolated in mdck33016pf cells grouped phylogenetically with viruses isolated in conventional atcc derived mdck cells or viruses sequenced from original clinical samples, while egg isolated viruses grouped slightly differently (data not shown). as a result of the poor growth of h1n1pdm viruses in mdck33016pf cells, serial dilutions were performed over a number of passages ( figure 1 ). based on the results obtained from the virus isolates, a ⁄ victoria ⁄ 2081 ⁄ 2009, a ⁄ wellington ⁄ 188 ⁄ 2009, and a ⁄ darwin ⁄ 2131 ⁄ 2009, a supplemental protocol was developed and used in the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 (figure 1 ). only small differences in ha titer were seen between different dilutions, and copy number showed a similar trend to ha titer at each passage ( figure 1 ). following the supplemental protocol for the isolation of a ⁄ brisbane ⁄ 10 ⁄ 2010 results showed slightly higher ha titres with little variation between passages. the egg derived reassortants nymc-x-179a and nymc-x-181 were also assessed for growth in mdck33016pf cells and were found to be superior by ha titer to other conventional reassortants (egg or cell derived), reverse genetics derived reassortants, or wild type viruses (table 1) . two methods were used to determine the ratio of ha to other viral proteins: densiometric analysis using sds-page and reversed-phase hplc using a subtype specific standard. 11 ha content in different vaccine seeds of influenza a subtypes demonstrated that the ha content per total virus protein from the nymc h1n1pdm reassortants was significantly different to the seasonal influenza a subtypes. for the seasonal h1n1 the ratio of ha to p4 p5 p6 p7 p8 p9 p10 p3 p4 p5 p6 p7 p8 p9 p10 p3 n and m1 was ‡30%, for the h3n2 the ratio of ha to n and m1 was £30%, while for the pandemic a ⁄ h1n1, the ratio of ha to n and m1 was much lower at £20% (data not shown). the results of this study has observed the growth of a series of 2009-2010 h1n1pdm viruses in vaccine suitable mdck33016pf cells to be generally lower than what has been seen with other seasonal influenza viruses. 11 little improvement in virus yield was seen with extra passaging of h1n1pdm viruses isolated and passaged in mdck33016pf cells. passaging up to 10 times in mdck33016pf cells using dilutions ranging from 10 )5 to 10 )8 resulted in supernatants with viral ha titres ranging from 8 ha ⁄ 25 ll to 512 ha ⁄ 25 ll. the isolation rate of h1n1pdm viruses was higher in mdck33016pf cells (89%) compared to allantoically inoculated (and passaged) eggs (66%), a trend also seen in previous work with seasonal influenza viruses. 12 in contrast a study by hussain and colleagues 13 found similar rates of isolation and replication of seasonal influenza viruses in mdck cells and eggs. the virus load as determined by matrix gene copy number showed a similar trend to ha titers. two of the isolates exhibited small rises and falls in ha titer during passaging, while a third, a ⁄ victoria ⁄ 2081 ⁄ 2009 gave consistently higher titers. interestingly this virus was unable to be isolated in eggs. the ha sequences of all strains were assessed at p1, p2, p3, p4, p10 and when available compared to the original clinical sample ha sequence. mdck33016pf-isolated viruses had few if any changes in their ha amino acid sequence, while the majority of egg isolates showed 1-2 amino acid changes compared to the clinical sample, with an egg adaption change (l191i) evident in a number of them. the ha sequence of one of the better growing viruses, a ⁄ victoria ⁄ 2081 ⁄ 2009, was found to have a g155e change compared to the a ⁄ california ⁄ 7 ⁄ 2009 reference virus. this change was also seen in the virus isolated in conventional, adherent mdck cells. these viruses with g155e change when tested by hai have shown reduced reactivity with ferret antisera to a ⁄ california ⁄ 7 ⁄ 2009-like viruses, but normal reactivity with ferret antisera to h1n1pdm a ⁄ bayern ⁄ 69 ⁄ 2009-like viruses. despite this mutation all mdck33016pf derived viruses appeared to be a ⁄ california ⁄ 7 ⁄ 2009-like by hai. the h1n1pdm egg-derived reassortants (nymc x-179a and nymc x-181) when grown in mdck33016pf cells were superior to wild type h1n1pdm viruses, reverse genetics derived reassortants, and other egg-derived reassortants. the yields of haemagglutinin from the nymc h1n1pdm reassortants were still below those seen with sea-sonal h1n1 reassortants as was also seen in eggs. this trend has also been noted in other studies. 10 in summary, attempts to improve growth and yield of the h1n1pdm wild types for mdck 331016pf cells by extended passaging were not successful, and reassortants did not perform as well as seasonal h1n1 reassortants have in the past. however, using higher dilutions for the passaging of h1n1pdm viruses in mdck33016pf cells did result in higher ha titres (a ⁄ brisbane ⁄ 10 ⁄ 2010). further work is therefore required to generate pandemic h1n1 seed viruses that grow well in a variety of cell culture and egg based vaccine production systems. the aim of this study is to evaluate antibody response to influenza virus neuraminidase (na) following immunization with live attenuated influenza vaccine (laiv). we adjusted the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. (1990) to assay neuraminidase inhibition (ni) antibody in sera taken from immunized mice and from human subjects in a clinical trial. for the assay, we prepared the a(h7n1) reassortant virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009 (h1n1) and the hemagglutinin (ha) of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7). in addition, we used an na-specific igg elisa assay to test sera from immunized mice and volunteers. in mice, one dose of laiv induced ni antibody of a geometric mean titer (gmt) of 31ae7, compared to 10ae6 in the control group. gmt of ni from human subjects who received two doses of pandemic a(h1n1) were significantly higher than pre-vaccination titers. in unvaccinated human subjects, na-specific cross-reactive antibodies to pandemic a(h1n1) were detected more often than cross-reactive antibodies to ha. antibody response to influenza virus na contributes to the overall immune response to influenza and may provide partial protection against influenza infection and reduce severity of disease in the host. 1 a number of preclinical studies using purified or recombinant na have shown that various two-dose vaccine regimens in mice may significantly reduce pulmonary virus titers following viral challenge. [2] [3] [4] a plasmid dna-vaccine model demonstrated cross-reactive antibodies to human n1 in mice could provide partial protection against a lethal challenge against h5n1 or recombinant pr8 bearing the avian n1. 4 immunogenicity of current influenza vaccines, including laivs, is measured primarily as a level of strain-specific hemagglutination inhibition (hi) antibodies. 5 however, the who meeting on the role of na in inducing protective immunity against influenza infection (2008) specified a need to develop suitable assays for anti-na antibody detection to enhance influenza vaccine evaluation in preclinical and clinical studies. 6 the aim of the current study was to evaluate anti-na antibodies to pandemic a(h1n1) 2009 influenza virus following laiv immunization. the rn1 ⁄ 09-swine a(h7n1) reassortant influenza virus containing the na of a ⁄ california ⁄ 07 ⁄ 2009(h1n1) and the ha of a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) generated by classical genetic reassortment in embryonated chicken eggs (ce). parental a ⁄ equine ⁄ prague ⁄ 1 ⁄ 56(h7n7) influenza virus was obtained from the center for disease control and prevention, atlanta, ga, usa. viruses were propagated in 10 day old ce and purified by sedimentation out of the allantoic fluid, followed by ultracentrifugation on 30-60% sucrose step gradient. for the mouse studies, 10 week old cba mice were inoculated intranasally with one dose 10 7 eid 50 ⁄ 0ae05 ml of a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) vaccine strain or received 0ae05 ml pbs. blood samples were collected on day 15 post inoculation. healthy young adults were immunized twice, 10 or 21 days apart in the fall 2009 with a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) laiv manufactured by microgen, irkutsk, russia. for the human studies, peripheral blood specimens were collected from volunteers before vaccination, 21 days after the first vaccination, and 21 days after the second dose of vaccine. sera from five subjects diagnosed with influenza a(h1n1) were collected in december 2009, 3 to 4 weeks post infection and kindly provided by e. vo ıtsekhovskaia from biotechnology laboratory, institute of influenza, rams. also, sera obtained in 2005 from unvaccinated vol-unteers were tested for presence of cross-reactive antibodies to a ⁄ california ⁄ 07 ⁄ 2009 (h1n1). sera were treated with a receptor-destroying enzyme from vibrio cholera (denka-seiken, tokyo, japan) and then were tested in duplicates for hemagglutination-inhibition (hi) h1 specific antibodies by standard procedures 7 using a ⁄ 17 ⁄ california ⁄ 09 ⁄ 38(h1n1) test antigen. the peroxidase-linked lectin micro-procedure previously reported by lambre, et al. 8 was adjusted to assay ni antibody. briefly, 96-well plates (sarstedt, inc., nümbrecht, germany) were coated overnight with 150 ll of 50 lg ⁄ ml fetuin. the purified a(h7n1) reassortant virus was diluted in pbs with 1% bsa and 10 mm ca 2+ to give a four times higher optical density at 450 nm (od 450 ) compared to control wells not containing virus. fifty-microliter volumes of serially diluted serum samples were incubated with an equal volume of prediluted virus for 1 hour at 37°c. after incubation, the plates were washed and neuraminidase activity was measured by subsequently adding peroxidase-labeled lectin (2 lg ⁄ ml; sigma, st. louis, mo, usa), incubating for 1 hour at room temperature, washing the plates, and adding 100 ll of peroxidase substrate (tmb). the reaction was stopped after 5 minute by adding 100 ll of 1n sulfuric acid. od values were measured at 450 nm using the universal microplate reader (el x 800; bio-tek instruments, inc., winooski, vt, usa). the ni titers were expressed as the reciprocal dilution that gave 50% od of positive control (virus, no serum control). in addition we used an igg elisa assay 9 with 0ae5 lg ⁄ ml of purified na from a ⁄ california ⁄ 07 ⁄ 09(h1n1) to test sera from immunized mice and volunteers. data were analyzed with statistica software (version 6ae0) (statsoft, inc. tulsa, oklahoma, usa). geometric mean titers (gmt) were calculated and used to represent the antibody response. the comparisons were made within groups between pre-and postvaccinated titers (expressed as log 2 ) after first and second vaccination using wilcoxon matched pairs test. to compare multiple independent groups we used a kruskal-wallis anova with subsequent multiple pairwise comparison based on kruskal-wallis' sums of ranks. a p-value of <0ae05 was considered to be statistically significant. in mice, one dose of laiv induced antibody responses to both ha and na components of the a ⁄ california ⁄ 07 ⁄ 2009(h1n1) influenza virus vaccine (table 1) . geometric mean titers of ni antibody levels from vaccinated mice were 31ae7 and were significantly higher compared to those in unvaccinated control animals (p < 0ae02). elisa igg titers expressed as log 10 were 1ae7 compared to 1ae0 in control group. there was good correlation between antibody rises obtained using ni or elisa tests (r = 0ae7). in a study during the fall of 2009, 85% of 60 examined unvaccinated subjects were negative to pandemic a(h1n1) (hi titers £1:10). serum hi antibody titers to pandemic a(h1n1) ‡1:40 were considered to be protective against *the postvaccination gmts of hi antibodies after revaccination were higher than respective prevaccination titers (p = 0ae04) **the postvaccination gmts of ni antibodies after revaccination were higher than respective prevaccination titers (p = 0ae02) serum hi and ni antibodies to a ⁄ california ⁄ 07 ⁄ 09(h1n1) after one or two doses of pandemic laiv were evaluated in subjects who had pre-vaccination hi titers £1:10 ( table 2) . post-vaccination gmts of a(h1n1)-specific antibodies were significantly higher than pre-vaccination titers only among subjects who received two doses of laiv ( table 2 ). the frequency of subjects with ‡ fourfold rises in hi antibody titers was higher after two doses (32ae3%) compared to responses after one dose (7ae1%) although the differences were not statistically significant ( table 2 ). the highest antibody titers of hi and ni antibodies were achieved after natural infection (p < 0ae01 compared to all post-vaccination groups). all five subjects with confirmed influenza also had high levels of n1-specific igg measured by elisa using purified na as the coating antigen (data not shown). influenza ha and na surface proteins are primary targets of neutralizing antibodies that provide protection against influenza infection. the correlation of strain-specific hi antibody titers ‡1:40 to protection of 50% of the subjects against influenza infection is based on a number of reports published in 1980s. 10 serum antibodies against viral na as result of influenza infection or vaccination also can neutralize the virus from infecting cells; however, little is known about protective levels of such antibodies. to evaluate ni antibodies directed against pandemic a(h1n1) we used the reassortant a(h7n1) influenza virus with mismatched ha to avoid non-specific inhibition. we demonstrated laiv immunization effectively increased levels of ni antibody, although in smaller amounts compared to influenza infection. our data suggest that an antibody to neuraminidase, resulting from an earlier infection of the circulating seasonal influenza a(h1n1), evidently cross-reacted with the n1 of pandemic influenza virus, perhaps due to the previously reported 17% of conserved na epitopes in pandemic a(h1n1). 11 the peroxidase-linked lectin test using the reassortant a(h7n1) influenza virus was shown to be a sensitive and time effective means of revealing homologous and cross-reactive anti-na antibodies after laiv immunization or influenza infection. this could be a useful method for influenza vaccine evaluation. significant levels of anti-na antibodies detected in peripheral serum from subjects infected with wildtype h1n1 virus or with h1n1 laiv. and the cross-antibody response to ph1n1. for calculation of geometric mean titer (gmt), a titer of <10 was assigned a value of 5. statistical significance was determined by paired t-test. cross-reactive antibody response to ph1n1 in vaccinated populations of seasonal influenza virus table 1 shows the antibody response to seasonal influenza viruses and ph1n1 of participants. before vaccination, no or little antibody response to ph1n1 had been detected in all age groups. vaccination with seasonal influenza vaccines resulted in seroresponse in over 70% of subjects, except children aged 0-7 years (68%) and subjects aged of 18-59 years (69%) vaccinated with 2007-2008 season influenza vaccine and adults aged ‡60 years (59%) vaccinated with 2008-2009 season influenza vaccine. seroconversion was detected in over 40% of subjects of all ages. postvaccination to prevaccination gmt ratios for response to seasonal influenza viruses was more than 2ae5-fold. in contrast, seroresponse to a ⁄ california ⁄ 07 ⁄ 2009 after vaccination with 2007-2008 and 2008-2009 seasonal influenza vaccines were detected in only 3% and 4% of those aged 0-7 years, 3% of those aged 8-17 years, 7% and 3% of those aged 18-59 years, 7% and 5% of those aged ‡60 years, respectively. seroconversion in all participants ranged from 2% to 4%, and postvaccination to prevaccination gmt ratios were <2ae5-fold. preexisting antibody response to ph1n1 among subjects born before 1920s in china according to a recent report, people who were born from 1910 to 1929 had a preexisting immunity to ph1n1. 7 although only a very low level of cross-reactive antibody response to ph1n1 had been observed among older subjects aged more than 60 years old in china, we further analyzed these data by different age distribution of subjects, which can trace back to the previous infection that is genetically and antigenically more closely related to this new ph1n1 influenza virus. the proportion of seroresponse to ph1n1 with the titer of 40, 80, and 160 (highest titer detected from participants of all ages in this study) and the value of gmt were analyzed according to the birth decade of subjects from 1915. similarly, a peak of antibody response and the value of gmt occurred both in subjects born from 1915 to 1925 and sharply decreased afterward ( figure 1 ). the seroresponse of subjects born in and before 1925 is significantly higher than subjects born afterward (p < 0ae05). similar to recent studies in some asia countries (guangxi province of china and singapore), limited antibody response to ph1n1 had been detected in children and adults. 8, 9 but, some other studies from european countries (finland, germany, the united kingdom) and the united states reported a high proportion of older individuals aged >60 years with pre-existing cross-reactive antibodies to ph1n1, which may possibly ba a result of previous exposure to antigenically related h1n1 influenza viruses circulating in earlier decades or a lifetime of exposure to influenza a, which has resulted in broad heterosubtypic immunity among older individuals in those countries. previous infection and vaccination with a ⁄ new jersey ⁄ 76 may also contribute to the high level of cross-reactive antibody response to ph1n1 among adults older than 60 years in the us. 10, 11 the peak of the antibody response to ph1n1 in subjects born between 1915 and 1925, which is consistent with recent reports, may suggest the previous viral infections of 1918 spanish flu or closely related influenza viruses, which is before and little after the year of 1918. recent antigenic report of new ph1n1 viruses indicated that they are antigenically homogeneous among historical viruses, which are most similar to classical swine a(h1n1) viruses. a number of reviews [12] [13] [14] [15] confirmed that the 1918 virus is the likely ancestor of all four of the human and swine h1n1 and h3n2 lineages, as well as the 'extinct' h2n2 lineage. in 1930, a(h1n1) influenza viruses were first isolated from swine. they have been shown to be antigenically highly similar to the recently reconstructed human 1918 a(h1n1) virus. 16 the cellular responses may contribute to the sustaining and long term antibody response. probably, boosting by persisting antigenically related viruses in the early decades of the 20th century, may have contributed to the ability of these subjects to sustain memory b cells, 17 and it is well established that a subset of plasma cells is long-lived, and these cells contribute to durable humoral immune responses, 18 such as that observed after childhood smallpox vaccination. furthermore, t cells that recognize cross-reactive epitopes are preserved and might be enriched in the memory population; the course of each infection is influenced by the t-cell memory pool that has been laid down by a host's history of previous successive infections. 19 our study indicated that wide transmission of this new virus or any antigenically close related influenza a(h1n1) viruses may not have circulated among populations in china before the outbreak of ph1n1. our data also suggests the need for vaccination with ph1n1 vaccine in all age groups. hypo-and agammaglobulinemia patients have an impaired immune system and are particularly susceptible to bacterial infections that are normally defended against by antibodies. therefore, patients routinely receive replacement therapy with immunoglobulins isolated from healthy blood donors. 1 these patients are also prone to get viral infections, possibly due to defects in toll-like receptors 8 and 9. 2 because these patients lack an antigen specific humoral immune response, they are rarely vaccinated. the ability of hypogammaglobulinemic patients to produce a specific cell-mediated immune response upon vaccination has only been sparsely investigated. in contrast to local mucosal antibodies, vaccine-induced cell-mediated immunity is not believed to protect against pathogen entry per se, but may be sufficient to provide protection against severe disease and death following transmission of some microbes. 3, 4 the aim of this pilot study was to investigate if influenza vaccination of hypogammaglobulinemic patients can induce an influenza-specific cell-mediated immune response. we therefore vaccinated hypogammaglobulinemic patients and healthy controls with pandemic h1n1 virus vaccine and subsequently investigated the bcell and t-cell responses. the percentages of ifn-c, il-2, and tnf-a cytokine producing cd4+ th1-cells were determined, as these cytokines are important indicators of cell-mediated immunity. five a-or hypogammaglobulinemic patients were classified based on the freiburg classification 5 : patient #1 is diagnosed with x-linked agammaglobulinemia, patient #2 and #4 are in group ia, patient #3 is in group ib and patient #5 is in group ii. the monovalent egg grown split virus vaccine adjuvanted with as03 was manufactured by glaxosmithkline (gsk), belgium. the vaccine strain was produced by reassortment between influenza a ⁄ california ⁄ 7 ⁄ 2009 (h1n1) and a ⁄ pr ⁄ 8 ⁄ 34 (h1n1) to produce a ⁄ california ⁄ 7 ⁄ 2009-like virus (x179a). the vaccine was mixed with adjuvant to contain 7ae5 lg haemagglutinin (ha) of a ⁄ california ⁄ 7 ⁄ 2009-like virus (h1n1), squalene (21ae38 mg), dl-atocopherol (23ae72 mg), and polysorbate 8 (9ae72 mg) per ml. healthy controls and hypogammaglobulinemia patients were vaccinated by intramuscular (im) injection. hypogammaglobulinemia patients received one or two vaccine doses 21 days apart. the intention was to vaccinate the hypogammaglobulinemic patients with two doses of 3ae75 lg ha, but 7ae5 lg ha was inadvertently administered to the patients as the first dose. for patient #4 this was the second dose as he had received an initial dose of 3ae75 lg ha 3 months prior to the study. patient #1, #2, and #5 received a second dose of 3ae75 lg ha. four healthy controls were immunised with one dose of 3ae75 lg ha according to norwegian national guidelines. peripheral blood mononuclear cells (pbmcs) were harvested and washed in pbs with 10% fbs. the pbmcs were resuspended in lymphocyte medium (rpmi 1640 with l-glutamine, 0ae1 mm non-essential amino acids, 10 mm hepes ph 7ae4, 1 mm sodium pyruvate, 100 iu ⁄ ml penicillin, 100 lg ⁄ ml streptomycin, 0ae25 lg ⁄ ml fungizone and 10% fbs) prior to use in the enzyme-linked immunospot (elispot) and influenza-specific cd4+ t-cell assays. serum haemagglutination inhibition antibodies were tested by a standard method using 8 ha units and 0ae7% turkey erythrocytes. all samples were tested in duplicate and the test was repeated at least two times. titres <10 were assigned a value of 5 for calculation purposes. for numeration of antibody-secreting cells (asc), an eli-spot assay was conducted as previously described 6 with the following modifications. ninety-six well elispot plates were coated with 2 lg ⁄ ml of a ⁄ california ⁄ 7 ⁄ 2009like (x179a) h1n1 virus diluted in pbs overnight at 4°c. after blocking with rpmi (10% fbs), 10 5 pbmcs were added and incubated (37°c, 5% co 2 ) for 16 hour. secreted antibodies were detected with biotinylated goat anti-human igg, iga and igm specific antibody (southern biotech, birmingham, alabama, usa), incubated for 2 hour at room temperature and developed with extravidin peroxidase and aec substrate. the numbers of spots were counted using an elispot reader (immunoscanô) and immunospot ò software. the influenza-specific cd4+ th1-cell response was measured by intracellular cytokine production of ifn-c, il-2, and tnf-a. peripheral blood mononuclear cells (10 6 per well) were incubated for 16 hour (37°c, 5% co 2 ) in 200 ll lymphocyte medium containing 1 lg ⁄ ml anti-cd28, 1 lg ⁄ ml anti-cd49d, 0ae7 lg ⁄ ml monensin, 1 lg ⁄ ml brefeldin a, (bd biosciences, franklin lakes, new jersey, usa), and the h1n1 influenza split virus vaccine x179a (either 2ae5 lg ⁄ ml or 10 lg ⁄ ml ha). basal cytokine production was determined by incubating pbmcs in lymphocyte medium without influenza virus, and the percentage of cytokine positive cells without influenza stimulation were subtracted from influenza-stimulated cells. cells were stained for cd3, cd4, cd8, ifn-c, il-2, and tnf-a (bd biosciences) as previously described. 7 finally, cells were resuspended in pbs containing 5% fbs and 0ae1% sodium azide and analysed by bd facscanto flow cytometer (300 000-500 000 cells acquired). flowjo v8ae8ae6 (tree star, ashland, oregon, usa) was used for data analysis. five to six fold lower gmts were found in the patient group as compared to the healthy controls throughout the study ( figure 1a) . the lowest hi titres were obtained in patients #1, #2, and #3, whilst patients #4 and #5 and all healthy controls fulfilled two of three european medicines agency committee for medicinal products for human use (chmp) 8 seasonal influenza vaccine licensing criteria, by obtaining an hi titre >40 and a mean geometric increase of 2ae5 between pre-and post-vaccination. thus, the hi data indicate that two vaccine doses was sufficient to induce a protective hi antibody response in two out of five of the hypogammaglobulinemia patients tested in this study. the numbers of influenza-specific iga, igg, and igm asc were tested pre-vaccination and 7 days post-vaccination with the h x179a virus. few or no ascs were detected pre-vaccination (data not shown). at 7 days post-vaccination the patient's iga, igg, and igm asc levels were significantly lower (p < 0ae01) compared to the healthy controls ( figure 1b) . but, the post-vaccination asc numbers in the patients were generally higher than at pre-vaccination stage (0-2 ascs). patient #3 had the highest iga and igg asc numbers, followed by patients #2 and #5, whilst patient #4 and #1 had few or no asc's. these results confirm that the patients are indeed hypogammaglobulinemic and that some of the patients (#1 and #4) could be agammaglobulinemic in the context of producing influenza-specific antibodies. the asc levels of patients #2, #3, and #5 were lower than those of the healthy controls, but could possibly be adequate for reducing the severity of influenza disease. the influenza-specific th1-cell response was evaluated by stimulating pbmcs with the influenza x179a virus 21, 42, and 90 days post-vaccination. stimulation of healthy control pbmcs with x179a 21 days after vaccination, induced ifn-c, il-2, and tnf-a production by an average of 0ae10%, 0ae09%, and 0ae03% cd4+ t-cells, respectively. patient #1 and #2 had higher responses than the healthy controls and stimulation with x179a induced 0ae45%, 0ae34%, and 0ae05% of t-cells from patient #2 to produce ifn-c, il-2, and tnf-a, respectively (figure 2a) . the response of patient #2 was further boosted by a second vaccine dose, which resulted in 0ae73%, 0ae54%, and 0ae25% cd4 t-cells producing ifn-c, il-2, and tnf-a, respectively at day 42 ( figure 2b ). these results show that the hypogammaglobulinemia patients studied here did not have a common impaired influenza-specific cd4+ th1 cytokine response. rather, there was a tendency towards increased responses, suggesting that the diminished antigen specific b-cell responses could induce a compensatory antigen specific th1-cell response. the results from this pilot study suggest that some hypogammaglobulinemia patients may benefit from influenza vaccination. we found very different patient responses to influenza vaccination, but some of the patients (patient #2 and #3) did mount low influenza-specific asc responses. in addition, the vaccine-induced hi antibody titres above the protective level in patient #4 and #5. these results are in accordance with previous publications, which described that polypeptide vaccines induce humoral responses in subgroups of common variable immunodeficiency patients. [9] [10] [11] in this study, we also investigated cell-mediated immunity and found the percentages of homologous and cross-reactive influenza-specific cd4+ th1-cells to be in the same range (for patient #3, #4, and #5) or higher (for patient #1 and #2) in the a-or hypogammaglobulinemic patients compared to the healthy controls. the higher response is probably due to the patients having received a vaccine dose of 7ae5 lg ha, whilst the controls received 3ae75 lg ha. in addition, the patients received a second booster dose, which influences the day 42 and 3 months responses. nonetheless, these results are the first to demonstrate that proliferation of pandemic influenza antigen specific th1cells can be induced in hypogammaglobulinemic patients. in addition, vaccination induced influenza-specific asc's in some patients. the findings are promising and provide hope that hypogammaglobulineamic patients could be vaccinated against influenza and other diseases preventable by figure 2 . peripheral blood mononuclear cells s from patients and healthy controls were isolated at day 21 (a), 42 (b), and day 90 (c) and stimulated for 17 hour with x179a virus before staining and flow cytometric analysis. the figure shows the mean ± sd frequency of influenza-specific cd4+ cytokine producing cells (%) where the basal cytokine production from unstimulated cells has been subtracted. data for the hypogammaglobulinemia patients are additionally shown as a number for each patient. **significantly higher frequency of il-2 producing cd4+ t-cells in the patients compared to the healthy controls (students t-test p < 0ae05). titres are presented as the geometric mean titre ± 95% confidence interval. elispot data (b) are presented as the mean number of influenza-specific iga, igg, and igm ascs per 100 000 peripheral blood mononuclear cells ± sem. data for the hypogammaglobulinemia patients are additionally presented by a number for each patient. *significantly higher numbers of ascs were detected in the healthy controls as compared with the hypogammaglobulinemia group (students t-test, p < 0ae01). vaccination. however, this hypothesis should be tested in larger clinical studies. the influenza virus undergoes antigenic evolution under intense immune selection pressure from herd immunity in humans through the process called antigenic drift and shift. 1,2 because of antigenic drift, yearly updating of vaccine strain is needed. a mismatch between the circulating strains and the vaccine strain in the subsequent season is often encountered, resulting in reduction of vaccine effectiveness and lack of protection from the circulating strain. 3 in order to address this, a universal influenza vaccine based on a more conserved part of the influenza virus, which is not affected by antigenic change and that is conserved across all strains, remains the ultimate goal to afford cross-protection to drifted strains as well as to other subtypes of influenza which may arise from antigenic shift. 4, 5 previous studies have investigated the potential of the m2e. 6, 7 m2e has remained highly conserved since it was first isolated in 1933. 6 several studies have examined the use of m2e as a vaccine component, using various approaches including proteins, peptides, dna vectors, and attenuated viral vectors. 6, [8] [9] [10] [11] [12] [13] although m2e is a weak antigen, by linking the protein to a carrier hepatitis b virus core particle, protection against influenza has been achieved in mice particularly when administered with an adjuvant. 3 some articles found that vaccination with m2e coupled to hbc induces protective antibodies, whereas the contribution of t cells to protection was negligible. protection induced by vaccination with m2e-hbc was weak overall and failed to prevent weight loss in vaccinated infected animals, and mice succumbed to high dose infection. 14 we aimed to address the poor immunogenicity of m2e-hbc by using igv as adjuvant. igv domain is common and conserved in the tim family. ligand binding sites of t cell immunoglobulin mucin (tim) located at igv domain. [15] [16] [17] tim function is done by anti tim antibody which recognized the ligand binding sites of igv domain. 18 tim family members share a common motif, including an igv domain. they are differentially expressed on th1 cells and th2 cells with the ability to regulate the immune system. 19, 20 the igv domain of human b7-2 is sufficient to co-stimulate t lymphocytes and induce cytokine secretion. 21 soo hoo et al. vaccinated with tim-1 antibody and inactivated influenza and found enhanced vaccine-specific immune response. 22 we report here for the first time the use of igv recombinant protein as adjuvant to immunize mice with influenza m2e-hbc. results indicated that igv can induce the strong cellular immune response and cross reaction with different subtype influenza virus antigen. target igv may be used to develop the new method for vaccination strategies. expression and purification of recombinant igv protein rna was extracted from healthy human pbmc. one-step rt-pcr (qiagen, valencia, ca, usa) was done for the amplification igv gene. the pcr product was purified and cloned into pet32a (novagen, madison, germany). the resultant construct pet32a-igv has a histidine (his) tag (6his) at the n terminus. dna sequence of the insert was determined by sequencing. igv. recombinant protein was expressed in escherichia coli and was purified on a ni column (novagen). the purified protein was examined by sds-page and western blotting. six-eight weeks female balb ⁄ c mice (institute of zoology chinese academy of sciences, china) was used for the study. mice were immunized twice intradermally with 10 ug m2e-hbc (provided by cnic, china) combined with different doses of recombinant igv protein 5, 10, 50 ug, respectively, or without igv as control. the area proximal to the tibialis anterior muscle was sterilized with 70% ethanol and different groups of mice were injected bilaterally with 5, 10, 50 ug igv plus 10 ug m2e-hbc in 50 ul phosphate buffer saline per mouse using a 1 ml syringe with attached 1 ⁄ 2¢¢ 30g needle. the immunization was given at 3 weeks intervals. four blood samples were obtained from every mouse: before immunization, after the first and second immunization, and after virus challenge by retro-orbital plexus puncture. after clotting and centrifugation, serum samples were collected and stored at )80°c prior to use for assays. mouse-adapted a ⁄ pr ⁄ 8 ⁄ 34 (h1n1), a ⁄ brisbane ⁄ 1 ⁄ 10 (h3n2), a ⁄ xinjiang ⁄ 1 ⁄ 2006 (h5n1), and a ⁄ guangzhou ⁄ 333 ⁄ 1999 (h9n2) were provided by chinese national influenza centre. nine to eleven days old embroynated specific pathogen free (spf) chicken eggs were inoculated with virus, and the eggs were incubated at 35°c for 2-3 days. the allantoic fluid was collected and purified by sucrose density gradient centrifugation, and the virus was inactivated by formaldehyde at 4°c overnight. to identify igg, igg1, igg2a against m2e, elisa assays were used. in brief, 96-well (nunc, brunei, denmark) were coated with 100 ul ⁄ well of m2e recombinant protein (provided by gene lab of ivdc, xuanwu district, beijing, china) in carbonate buffer (ph 9ae6) overnight at 4°c. immediately before use, the coated plates were incubated with blocking solution (2% bsa in pbs) for 2 h at 37°c and washed four times with pbs containing 0ae05% tween 20 (pbs-t). the serum samples were serially diluted and added in the plates. the detection color was developed by adding hrp-labeled goat anti-mouse igg, igg1, or igg2a ( figure 1) . no cross-strain response was observed in the control group. the igv adjuvented groups show splenocytes stimulation with seasonal h1n1, h3n2, h5n1, and h9n2 antigens. m2e-hbc immunization without igv showed splenocyte stimulation, but the extent was lower than animals immunized in the presence of the igv adjuvent. these data suggested that igv had enhanced effect on priming against the conserved viral antigen matrix protein and generation cross-strain immune response. influenza is a respiratory disease causing epidemics every year. h5n1 viruses and swine-origin h1n1 have also infected humans in recent years. seasonal influenza vaccine cannot cope with significant antigenic drift or with the emergence of pandemic viruses of different subtypes not contained in the vaccine. the high extent of conservation of the m2e makes it a promising immunogen. a vaccine based on coupling of the m2e peptide to an appropriate carrier may provide a universal vaccine with effectiveness and safety. m2e based vaccination induces protective antibodies not only in mice, but also in ferrets and monkeys. the carrier hepatitis b core as carrier with m2e forms a virus like particle (vlp). vaccination with m2e coupled to hbc induces protective antibody, 11 whereas the contribution of t cell protection was negligible. protection induced by vaccination with m2 coupled to hbc was weak overall. in order to improve the vaccination effect of m2e-hbc, new adjuvant igv was evaluated in combination with the m2e-hbc. the tim molecules are a recently discovered class of proteins with the ability to regulate the immune system. crystal structures of the tim molecules has revealed a unique, conserved structure with ligand-binding sites in the igv domain. to determine the potential immunostimulatory molecular properties of igv, we have evaluated immune response of the igv in combination with m2e-hbc vlp. previous papers reported that vlp immunized mice can induce the th1 and th2 immune response. 23 different adjuvant combined vlp can produce biased immune response th1 ⁄ th2 mixed immune response, or th1-preferred th1 ⁄ th2 profile. 24 thus, the response following the use of igv as a new adjuvant combined with m2e-hbc vlp needs to be evaluated. results indicated that igv combined groups showed th1 biased immune response and enhanced cross reactive t cell immune responses. this may show that igv immunized the mice and antiigv antibody can cross link the igv on t cells and enhance the cell figure 1 . t cell proliferation assay. mice were immunized twice with 50, 10, 5, 0 ug ⁄ ml igv plus m2e-hbc, respectively, and naive group was immunized with pbs. three weeks after a boosting immunization, spleens were harvested from immunized and naive mice. different subtypes of inactivated virus antigen (a) h1n1, (b) h3n2, (c) h5n1, (d) h9n2 were added and cocultured with different group splenocytes for 96 h. quick cell proliferation assay kit was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. data were showed were shown as mean values. the difference between naive group and different doses igv plus m2e groups was determined using the student's t-test. all significance level is p < 0ae05. response. we also evaluated the cross-protection produced by igv combined m2e-hbc. we challenge with mouseadapted strain pr8 and prove the cross protection via reaction between the cells from the immunized animal and different subtypes of virus antigen. some subtypes of virus cannot infect the mice naturally, and therefore, virus challenge cannot be used to evaluate the effect. we co-cultured the t cells with inactivated antigen h1, h3, h5, and h9, and t cell proliferation was measured. results indicated that after immunization with igv plus m2e-hbc, the t cells show cross-protection with other subtypes. this provides evidence that igv can enhance the cross protection across subtypes. the results of this study demonstrated that recombinant igv can be useful as an adjuvant and polarize the m2e-hbc vlp immune response to a th1 profile. igv induced the m2e-hbc vlp to induce t cell proliferation and cross-reactive responses to different influenza virus subtypes. this finding represents a new direction for the promotion of cell mediated immunity in m2e based vaccine against influenza. a core european protocol, i-move, describing the methods to estimate influenza vaccine effectiveness (ive) was proposed by the european centre for disease prevention and control (ecdc) and epiconcept for the 2009-2010 season. it includes a case control method for pooled analysis based on a randomized ''systematic'' sample of swabs. 1, 2 collection of swabs using a non randomized, i.e., ''ad hoc,'' sampling strategy, left at the appreciation of sentinel practitioners, provides a greater number of cases and con-trols for ive estimation more easily than using a systematic randomized sampling strategy. the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using both sampling methods. 3, 4 during the circulation of pandemic influenza 2009 viruses in france, it gave an opportunity to compare ive estimates using systematic randomized versus non systematic ''ad hoc'' sampling. influenza vaccine effectiveness was estimated by a casecontrol methodology according to ecdc i-move protocol, using on the one hand a systematic random sampling, on the other hand ''ad hoc'' non random sampling. the study was proposed to 608 primary care practitioners of the grog network (493 general practitioners and 115 pediatricians) trained to collect data and swabs. the study population was patients from the community of all ages consulting a grog practitioner for an influenza like illness (ili) and having a nasal or throat swab taken within an interval of <5 days after symptom onset. ili was defined according to the european union (eu) case definition as sudden onset of symptoms with at least one of the following four systemic symptoms: fever or feverishness, malaise, headache, myalgia; and at least one of the following three respiratory symptoms: cough, sore throat, shortness of breath. swabs were performed through usual surveillance. no ethical approval was needed, but an oral informed consent was requested. cases were excluded if they refused to participate in the study or if they were unable to give informed consent or to follow the interview in native language because of aphasia, reduced consciousness, or other reasons. an individual was considered as vaccinated against pandemic influenza if he or she reported having received a pandemic influenza vaccination during the current season, and if at least one vaccine dose occurred more than 14 days before ili onset. the study period started with the initiation of active influenza surveillance by the grog network, i.e., 15 days after the beginning of the influenza vaccination campaign, and finished at the end of the influenza period defined as the last week with at least one swab positive for influenza within the grog network. ''ad hoc'' sampling patients from which swabs were taken were selected by the grog practitioners during the study period. systematic random sampling during the same period, patients were selected at random as follows. an age-group 0-4 years (gps and pediatricians); 5-14 years (gps and pediatricians); 15-64 years (gps); 65 years or more (gps) was assigned to each practitioner, who was requested to swab the first patient of the week presenting with an ili within the pre-assigned age-group. swabs were collected in appropriate transport medium (virocult ò , viralpack ò , utm copan ò ) and sent by post to the laboratory in triple packaging following the international guidelines for the transport of infectious substances (category b, classification un 3373). laboratory confirmation of influenza was by rt-pcr to detect currently circulating influenza a (subtypes h3, seasonal and pandemic h1) and b viruses. an influenza case was defined as an ili case with a respiratory sample positive for influenza during the study period. controls were cases of ili having a swab negative for influenza during the study period. the outcome of interest is laboratory confirmed influenza. confounding factors and effects modifiers identified during the i-move preliminary study 5 were registered: risk factors, chronic diseases, severity of underlying conditions, smoking history, former vaccinations, and functional status. data on cases and controls were collected by the practitioners using a standardized questionnaire adapted from the i-move study. questionnaires were sent by the practitioners with the swab to the virology laboratory, and sent to the grog national coordination. data entry and validation were ensured by open rome through the vircases computing tool. validation steps included control of exhaustiveness of centralization of questionnaires, comparison of data entered by the labs and the national grog coordination, coherence control, and identification of missing data. analysis was done for the two sampling groups (systematic and ad hoc) on cases ⁄ controls following the european method proposed by epiconcept, using excel 2007 ª (microsoft corp. redmond, washington, usa) and stata ª . baseline characteristics of cases and controls in unmatched studies were compared using the chi-square test, fisher's exact test, or the mann-whitney test (depending on the nature of the variable and the sample size). the association between vaccination status and baseline characteristics was assessed for both case and control groups. the vaccine effectiveness was computed as ive = 1)or (odds ratio). an exact 95% confidence interval (ci) was computed around the point estimate. analysis was stratified according to age groups, time (month of onset), presence or absence of chronic disease, and previous influenza vaccination. effect modification was assessed comparing the or across the strata of the baseline characteristics. confounding factors were assessed by comparing crude and adjusted or for each baseline characteristic. a multivariable logistic regression analysis was conducted to control for negative and positive confounding factors using a complete case analysis (with records with missing data dropped) and using multiple imputation with chained equations. the complete model included age group, number of gp visits, onset week, seasonal vaccination, previous seasonal influenza vaccination, presence of chronic disease and associated hospitalizations in the previous 12 months, gender, and smoking status. variables were tested for multi-colinearity. interactions were tested using the likelihood ratio test (or wald test) and included in the model if significant at 5% level. a model with fewer variables (age group, number of gp visit, onset week, and seasonal vaccination) was also tested. several models were applied to both the ''ad hoc'' and systematic sampling groups of cases and controls. as shown in table 1 , whatever the analysis method used, the ''ad hoc'' sampling strategy led to a slightly lower estimate of ive. the ci were extended when data were missing and reduced when using multiple imputations with chained equations. however, from a statistical point of view, comparison of ''ad hoc'' versus systematic strategies is not straightforward, because ''ad hoc'' sampling is not randomized and does not allow comparisons with statistical tests using statistical distribution laws. there are more missing data with the ad hoc sampling method. this is mainly due to our validation procedure: in the case of missing data in the systematic sampling group, as required by the i-move study protocol, queries were sent to sentinel practitioners using mail and phone calls. this specific heavy workload is not usually performed during routine surveillance and has not been achieved for the ''ad hoc'' sampling group given the great number of cases and controls (2690). within the framework of the i-move study, several items were added to the grog's usual clinical form accompanying swabs (hospitalizations, number of gp visits, smoking status, help needed for bathing or walking). in 2009-2010, gps explained that this added workload was not compatible with their daily additional workload due to the pandemic situation. therefore, many of them refused to fill these new items systematically and threatened to leave the network. we thus obtained that the ''i-move items'' would be filled in for the clinical forms linked to systematic sampling, but were not in a position to obtain that for ''ad hoc'' sampling. the weekly distribution of systematic swabbing is not similar to that of ad hoc swabbing. the percentage of ad hoc swabs was higher than systematic swabs during the pandemic wave (mid-november to end of december) during which time the percentage of swabs positive for influenza was also higher ( figure 1 ). this could explain the higher rate of positive swabs within the ''ad hoc'' samples. the vaccination campaign was launched by the ministry of health on october 20, 2009, 6 and vaccination coverage increased during the surveillance period. in february, the vaccination coverage was 10ae2% in patients swabbed in the systematic group (9ae6% on imputed data) and 11ae5% [4ae4-23ae4] in the ad hoc group (11ae9% on imputed data). at the national level, vaccine coverage is estimated at 7ae95%. 7 due to the over-mediatisation of pandemic vaccination and to rumors about its poor effectiveness, overconsultation of vaccinated patients and over-swabbing of vaccinated patients in the ad hoc group are not surprising. age distribution is significantly different between our two samples (p < 0ae0001): the rate of 5-14 years old is lower in the systematic sampling group (25ae5%) than in the ad hoc sampling group (35ae7%). this can be explained by the fact that for the systematic sampling procedure, each grog practitioner had to swab the first ili patient in his assigned age group, whereas for ''ad hoc'' sampling, every grog practitioner could swab any ili patient irrespective of age. given the emphasis by health authorities and media on the burden of pandemic influenza among children and teenagers, one can hypothesize that when they were able to, sentinel practitioners focused on these age groups. gps in the ad hoc sampling scheme seem to have been more likely to select cases and further, to select vaccinated cases. those patients may have consulted earlier with specific symptoms (strong headache being more prevalent among cases). over-swabbing of patients having these symptoms in the ad hoc group is likely. the 2009-2010 pandemic influenza season was markedly different from previous ones: vaccination rate increased during and mainly after the pandemic peak; behaviors were strongly modified by unusual media hype; clinical features and risk factors might be different. it will be necessary to see if similar results are observed during a regular influenza season during which the vaccination rate increases before the epidemic peak with usual messages about vaccination and usual clinical influenza features. influenza early warning networks can estimate ive, taking into account many covariates. from a stakeholders and patients point of view, during the 2009-2010 influenza pandemic wave, there were no major discrepancies between ive estimated with an ad hoc sampling strategy, based on sentinel practitioners instinct, and ive estimated with a systematic random sampling strategy whatever the multivariable analysis methodology. although from a statistical point of view, comparison of the two strategies is not readily feasible because of the non random nature of ad hoc sampling. this latter strategy seems to result in slightly lower ive estimates, which could potentially be attributed to sentinel practitioners swabbing behavior. the ability to avoid missing data is a key point to decide which sampling method must be adopted, because ci extent depends greatly on the proportion of missing data among covariates. to match ive evaluation to surveillance networks practicality, selection of only those data essential for the study endpoint and easily collected by sentinel practitioners is paramount. it will be necessary to determine if results similar to those observed during the 2009-2010 pandemic season are found during a regular influenza season. influenza a viruses are important pathogens which remain a major cause of morbidity and mortality worldwide, and large numbers of the human population are affected every year. the first influenza pandemic in this century broke out in humans in march 2009, and it was declared to be pandemic by mid-june. as of 1 august jul 2010, the pandemic virus had caused more than 18 449 deaths worldwide, according to the world health organization (http:// www.who.int/csr/don/2010_08_06/en/index.html). the infection and spread of the pandemic influenza was reduced in part due to the use of vaccines. however, the lack of h1n1pdm vaccine early in the pandemic illustrates the need to improve vaccine production and to generate vaccines that induce stronger cross-protection. inactivated split vaccines or live attenuated influenza virus vaccines (laivs) against h1n1pdm viruses were approved for human use by the united states food and drug administration. both the inactivated vaccines and laivs are produced by creating reassortant viruses that generally contain six vrnas (pb2, pb1, pa, np, m, and ns) from a master donor strain, plus the two glycoprotein vrnas (ha and na) from a virus that antigenically matches the strain predicted to circulate in upcoming influenza season (e.g. a ⁄ ca ⁄ 07 ⁄ 2009). the reference viruses containing inactivated split virus vaccines are produced in embryonated chicken eggs, and primarily result in the production of antibodies that recognize the viral glycoproteins. both of these vaccine approaches require significant lead time for vaccine production, and modern approaches to speed preparation of vaccines and improve their efficacy is a global priority. 1, 2 the ns1 protein of influenza a virus is a multifunctional protein that plays important roles in virus replication and as potent type i ifn antagonist. 3, 4 mutations and ⁄ or deletions in ns1 typically induce stronger ifn responses by the host; those in turn suppress the replication of influenza virus 3-7 and can enhance immune recognition. [8] [9] [10] [11] in this study, we created a panel of experimental h1n1pdm ns-laiv candidates that have different deletions in the ns vrna and analyzed the vaccine potential of each ns-laiv in mice and ferrets to identify the best candidate(s). wt h1n1pdm influenza a virus a ⁄ new york ⁄ 1682 ⁄ 2009 (ny1682) was created by reverse-genetics directly from a human swab specimen collected in new york state in april 2009. 12 deletions were introduced into the ny1682 ns plasmid to create three mutant ns segments: ns1-73, ns1-126, and nsd5. nucleotides 246-482 (cdna of ns segment) and 405-482 were replaced by stop codons to generate ns1-73 and ns1-126; nucleotides 612-626 were deleted to generate nsd5, whose open reading frames for ns1 and nep were maintained. recombinant viruses were generated by co-transfection of eight reverse-genetics plasmids carrying the cdna of each gene segment into 293t ⁄ mdck cocultured monolayer adapted from hoffmann et al. 12, 13 mouse studies experiments were performed in a biosafety level 3 laboratories approved by the u.s. centers for disease control and prevention and the u.s. department of agriculture, and were conducted under approved animal care and use protocols. groups of 6-week-old female balb ⁄ cj (jackson laboratory, bar harbor, me, usa) were anesthetized with isoflurane and inoculated intranasally with 10 5 tcid 50 of each recombinant virus in 20 ll of pbs diluent, or pbs as controls. body weights and clinical symptoms of the mice were monitored daily for 10 days. nine mice in each group were euthanized on 1, 2, and 5 days post inoculation (dpi), and nasal washes and lungs were collected for virus titration by tcid 50 assay in mdck cells. at 30 dpi, 12 mice per group were challenged intranasally with 5 · 10 4 tcid 50 (100 ld 50 in 6-week-old mice) of a mouse-adapted variant of ny1682 (a ⁄ ny ⁄ 1682 ⁄ 2009-ma7) (accepted, journal of virology). disease symptoms and weights of the vaccinated mice were monitored for 10 days, and four mice from each virus group were euthanized at 3 and 6 days post challenge. lungs were removed and homogenized for virus titration by tcid 50 assay. the mice that became moribund or lost >25% of their starting body weight were euthanized for humane reasons. male fitch ferrets (triple f farms, sayre, pa, usa), 7-10 months of age and serologically negative by hemagglutination inhibition (hi) assay for currently circulating influenza viruses were used in this study. groups of 3 or 4 ferrets were inoculated intranasally with 10 6ae5 tcid 50 of one of the viruses: ny1682 wt (n = 4), ns1-73 (n = 3), ns1-126 (n = 4), or nsd5 (n = 4). ferrets were monitored for clinical signs through 14 dpi as previously described. 14 nasal washes were collected on 1, 3, 5, and 7 dpi and were titrated in mdck cells by tcid 50 assay. serum was isolated from blood collected 6ae5 weeks after immunization and used for neutralization assays. the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 15 6ae5 weeks postimmunization and monitored for clinical signs of disease through 14 dpi. nasal washes were collected on 1, 3, 5, and 7 dpi, and were titrated in mdck cells by plaque assay. using reverse genetics, we created three laiv candidates weight loss of wt virus inoculated mice became evident at 3 dpi, and the mice did not recover until 8 dpi (figure 1a) . in contrast, mice inoculated with any one of the vaccine candidates had no clinical signs of disease and continued to gain weight at the same rate as did the mockinoculated mice ( figure 1a ). viral titers in the lungs of ns1-73, and ns1-126 infected mice were $100-fold lower than titers from wt virus-infected mice at all the time points analyzed (1, 2, and 5 dpi) ( figure 1b) . notably, the nsd5 laiv was cleared from the mouse lungs very rapidly, and the mean titers were $100-fold and 50 000-fold lower than the titers of the wt virus at 1 and 2 dpi, respectively ( figure 1b) . the vaccinated mice were challenged with a mouseadapted variant of ny1682 (accepted, journal of virology) on 30 dpi. no disease symptoms were observed in the mice immunized by any of the ns-laiv candidates or the wt control. in contrast, disease symptoms including ruffled fur, hunched posture, and weight loss were observed in the mock-immunized mice as early as 2 days post challenge (dpc); the symptoms progressed to severe disease, and the animals showed dramatic weight loss, became moribund, and succumbed to infection by 5 dpc (figure 1c ). high titers of virus ($10 7 tcid 50 ⁄ ml) were present in the mock-immunized mice at 3 dpc and at 6 dpc ( figure 1d ). in contrast, virus was not detected in the lungs of immunized mice ( figure 1d ). this challenge data demonstrates that all of the ns-laiv candidates, including the highly attenuated nsd5, induced sterilizing immunity that protected mice from a lethal ny1682 h1n1pdm variant. groups of ferrets were intranasally immunized with 10 6ae5 tcid 50 of each vaccine candidate or the wt virus. the titer of viruses recovered from nasal washes ranged from 10 6ae8 to 10 4ae5 tcid 50 ⁄ ml through day 5 in the wt virusinfected group, while the ns-laivs showed various degrees of attenuation (figure 2a) . the viral titer of all of the ns-laivs is at least 100-fold lower than that of wt in the nasal wash collected at 1 dpi. the ns1-73 laiv was the least attenuated in ferrets, and its replication was similar to that observed in mice. relative to the wt virus, the ns1-126 laiv showed 100-fold reduction in titer, and the nsd5 laiv was below the limit of detection (at least 1000fold reduction) at 3 dpi. sera from blood collected 6ae5 weeks after immunization was analyzed for the presence of neutralizing antibodies by micro-neutralization assays. the ns-laiv candidates all induced very strong neutralizing antibody responses (1280-5120) that were similar to the titer elicited by wt virus infection ( figure 2b ). the ferrets were challenged with 10 6 pfu of a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm) 6ae5 weeks post immunization. little disease or weight loss were observed in the naïve ferrets, and the ferrets immunized by infection with wt virus or the ns-laiv candidates didn't show any disease symptoms or weight loss. in contrast to the high titer of virus detected in the naïve ferrets through 5 dpc, the ns-laiv immunized ferrets had very low levels of a ⁄ mexico ⁄ 4482 ⁄ 2009 in their nasal washes at 1 dpc ( figure 2c ). the ferrets immunized with the ny1682 ns-laivs had $10 000-to 100 000-fold lower viral titers than did the naïve animals ( figure 2d ). in summary, the ns-laiv candidates dramatically inhibited initial replication of the h1n1pdm virus under stringent challenge conditions (10 6 pfu), and that the vaccinated animals rapidly cleared the infection (to below the limit of detection, by 3 dpc). our results demonstrate that all of the ns-laiv candidates are attenuated compared to the wt h1n1pdm virus, and the degree of attenuation is dependent on the specific ns mutation. ns1-73 was the least attenuated and does not represent a good vaccine candidate; whereas, nsd5 and ns1-126 were highly attenuated in both the mouse and ferret models. although they were markedly attenuated, they elicited strong neutralizing antibody responses and protected mice and ferrets from subsequent challenge. nsd5 has a subtle in-frame deletion (15 nt) that affects both the ns1 (residues 196-200) and nep (residues 39-43), and is analogous to a naturally attenuated variant of a normally highly pathogenic h5n1 virus (a ⁄ sw ⁄ fj ⁄ 03). 16 the analogous ns1 deletion in a ⁄ sw ⁄ fj ⁄ 03 (residues 191-195) was shown to reduce binding to host cleavage and polyadenylation specificity factor (cpsf), reduce ns1 protein stability, and enhance the type i ifn response of this h5n1 virus. 16 our study indicates that deletion of these 15nt in the ns vrna of the h1n1pdm also stimulates the host ifn response, specifically, ifn-ß, ifn-k1, ip10, and mxa (data not shown). the role of the deletion of residues 39-43 from nep has not been elucidated, but the induction of ifn and isgs by nsd5 was similar to, or slightly lower than, their induction by ns-126, suggesting that the nep mutation also has an attenuating effect that warrants future investigation. in summary, we have generated a panel of laivs directly from a swab specimen containing a new pandemic virus and analyzed their attenuation and immunogenicity in two animal models. our study demonstrates that nsd5 is a novel ns-laiv that could be used to create laivs for diverse influenza a viruses. this study also validates the use of ns-laiv candidates, which are not only highly attenuated, but they also elicit strong innate and adaptive immune responses, resulting in protection of mice from subsequent challenge with a lethal mouse-adapted variant of ny1682, and ferrets from challenge with a ⁄ mexico ⁄ 4482 ⁄ 2009 (h1n1pdm). currently, a total of approximately 300 million doses of inactivated influenza vaccine are being produced worldwide each year. one of the limitations in vaccine production is poor growth of human isolates in embryonated chicken eggs. this is essential to develop high yield seed viruses for large scale production of influenza vaccines. influenza a vaccine production utilizes high yield reassortants carrying ha and na genes from a wild type (wt) strain with generally 5-6 internal genes from the a ⁄ pr ⁄ 8 ⁄ 34 (pr8) strain, an highly egg adapted high growth donor strain. 1 influenza b vaccines, however, have been produced directly from wt strains, partly because no high yield donor analogous to pr8 has been identified. in recent years, reverse genetics has been used as an alternative means of developing high growth vaccine viruses. 2, 3 since in this plasmid-based technology, a 6:2 reassortant (six internal genes from a donor strain and two surface antigen genes from wild type strain) can be directly rescued, reverse genetics-derived reassortant viruses were expected to grow as efficiently as those derived from classical reassortment. however, reverse genetics reassortants have not produced the expected high growth for several reasons: (i) the 6:2 configuration is not always the best for virus yield, (ii) there is no process included for positive selection of adaptive mutants from quasispecies, and (iii) cell-derived viruses are not readily adapted to grow efficiently in eggs. our laboratory at new york medical college has been preparing b reassortants for several years by classical reassortment using b ⁄ lee ⁄ 40 as a donor. it has been possible to develop b reassortants, which produce higher virus yields than wt strains in eggs, and it was found that the np gene of b ⁄ lee ⁄ 40 was important in producing high yield b reassortants. 4 however, b ⁄ lee ⁄ 40 is inconsistent in providing high yield properties to b reassortants. in this study, in an attempt to find an alternative donor, we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 for developing high yield b reassortants. as a wt strain, b ⁄ brisbane ⁄ 60 ⁄ 2008 was used, which is one of the recommended influenza b virus vaccine strains for the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. we found that b ⁄ panama ⁄ 45 ⁄ 90 is a useful donor, and some of the resultant reassortants were considered as vaccine candidates. 5 b reassortant viruses were prepared by the classical reassortment method described by kilbourne. 1 the antiserum to b ⁄ panama ⁄ 45 ⁄ 90 hemagglutinin and neuraminidase (hana) was raised in this study by immunizing rabbits with hana isolated from b ⁄ panama ⁄ 45 ⁄ 90; purified igg was used for antibody selection. the yields of the reassortants and their corresponding parent viruses were assessed by hemagglutination assay. viral rna was extracted directly from the allantoic fluid and amplified by rt-pcr to produce cdna for analyzing the gene composition. restriction fragment length polymorphism (rflp) analyses were performed to determine the origin of each gene segment of the high yield reassortants. restriction enzyme sets for each gene segment are available upon request. in this study we investigated the usefulness of b ⁄ panama ⁄ 45 ⁄ 90 as a donor for transferring high yield phenotype. b ⁄ panama ⁄ 45 is a yamagata lineage strain with high growth phenotype (ha titer: 1024-2048). b ⁄ panama ⁄ 45 ⁄ 90 itself was a recommended b virus vaccine strain for 1989 ⁄ 90-1994 ⁄ 95 seasons. as a wt virus, a victoria lineage strain, b ⁄ brisbane ⁄ 60 ⁄ 2008, was used, which is a recommended b virus vaccine strain for use in the 2009 ⁄ 10 and 2010 ⁄ 11 seasons. reassortants were prepared according to classical reassortment protocol. after co-infection of b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008, progeny viruses carrying surface antigens (ha and na) of the vaccine strain were negatively selected by anti-b ⁄ panama hana antibodies, followed by passages without antibodies for positive selection of eggadapted viruses and finally limited dilution cloning. nymc bx-33, bx-33b, bx-33d, and r-3a are representative of resultant reassortants, which have significantly higher ha titers than the wt strain. the complete gene compositions of these reassortants were determined by rt-pcr ⁄ rflp analyses. as shown in table 1 , all of these reassortants contained the pb2 of b ⁄ panama ⁄ 45 ⁄ 90. other genes of b ⁄ panama ⁄ 45 ⁄ 90 (np of bx-33, m of bx-33b, and pb1 of bx-33d) may not be involved in the high virus yield, since no significant growth difference among these reassortants in eggs was found as assayed by hemagglutination test. accordingly, the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is considered to be the sole factor involved in the high yield phenotype donated to the vaccine strain. we previously found that the b ⁄ lee ⁄ 40 np gene was important in producing high yield b reassortants. it was of interest to examine whether b ⁄ lee ⁄ 40 np and b ⁄ panama pb2 could work together to produce even higher yields. to test this possibility, bx-33b (2:6 reassortant: pb2 and m genes from b ⁄ panama and the rest of the genes from b ⁄ brisbane) was selected and further reassorted with b ⁄ lee ⁄ 40. despite some difficulty in removing the na gene of b ⁄ lee ⁄ 40 (r-4c, 7b, 10b in table 2 ), by monitoring ha and na genes of resultant viruses after each antibody selection passage with anti b ⁄ lee ⁄ 40 hana antibodies, we were able to isolate and clone a triple reassortant, nymc bx-35, which contains the np gene from b ⁄ lee ⁄ 40 and pb2 and m genes from b ⁄ panama; the remaining genes are from b ⁄ brisbane ⁄ 60 ⁄ 2008 (table 2 ). in comparison with bx-33b, no significant growth enhancement (nor reduction) in eggs was found for bx-35 over that seen for bx-33b. nevertheless, bx-35 stably produces high virus yield and has been utilized as a seed virus for influenza b vaccine production for the 2010-2011 season by one or more vaccine manufacturers. there are contradictory reports 6-8 about the usefulness of reassortment for high yield influenza b viruses. however, we have been preparing b reassortants for several years by classical reassortment 1 using b ⁄ lee ⁄ 40 as a donor, and have been able to generate higher virus yield than wt strains. 4 in this study, we found that b ⁄ panama ⁄ 45 ⁄ 90 serves as an efficient donor in providing the high growth capacity to b ⁄ brisbane ⁄ 60 ⁄ 2008 (a recommended vaccine virus of victoria lineage for 2009 ⁄ 10-2010 ⁄ 11 seasons), and that the pb2 of b ⁄ panama ⁄ 45 ⁄ 90 is associated with the high yield phenotype. this particular strain from yamagata lineage might be useful to prepare high yield reassortants for other victoria lineage vaccine viruses. we noticed in this study that there may be segment incompatibilities between b ⁄ panama ⁄ 45 ⁄ 90 and b ⁄ brisbane ⁄ 60 ⁄ 2008. as shown in table 1 , the pa and ns genes of all the high yield reassortants examined are derived from wt, b ⁄ brisbane ⁄ 60 ⁄ 2008, not from the donor, b ⁄ panama ⁄ 45 ⁄ 90. this indicates that in this reassortment, the pa and ns genes are not replaceable with that of the donor to obtain high yield viruses. this degree of incompatibility might be common in b reassortment, resulting in low donor ⁄ wt reassortants, such as 2:6 and even 1:7 reassortants that we obtained in this study. if this is the case, reverse genetics based on 6:2 configuration may not result in generating high yield b reassortants unless a variety of donor ⁄ wt combinations are designed. one can speculate that in influenza b viruses, the surface glycoproteins (ha and na) and some of the internal proteins are functionally more closely related than in influenza a virus, as was seen in that pa and ns genes of b ⁄ brisbane ⁄ 60 ⁄ 2008 reassort together with the ha and na genes of the same parent (table 1 ). in our recent study on a reassortment between b ⁄ lee ⁄ 40 and b ⁄ panama ⁄ 45 ⁄ 90, it appeared that ha shapes overall gene constellations of the resultant reassortants, namely the reassortants tend to have more internal genes from the same parent of ha, no matter which parent's ha is selected by antibodies against the surface antigens of the other parent (data not shown). because of success in influenza a virus reassortment with pr8, it is generally believed that reassortant with 6:2 or 5:3 configuration is optimal for virus yield. this may be the case in most instances of influenza a reassortment, but is not necessarily so in b reassortment. as shown in this study, only a single donor gene is capable of improving the yield of vaccine strain by reassortment. influenza a ⁄ h1n1v has spread rapidly in all parts of the world in 2009 as a true pandemic. 1 epidemic events in russia occurred during the last week of september 2009 starting from far east region (yuzhno-sakhalinsk). kaliningrad (the western most russian city) was the second starting point of the epidemic. during october 2009 the epidemic spread over the whole russian territory. in a short period the new virus started to change genetically as it began to adapt to human populations during this pandemic (http://www.who.int; http://www.euroflu.org). in the period from may to december 2009, 1558 clinical samples (nasopharyngeal swabs and postmortem materials) of patients with influenza-like illness from different regions of russian federation were analyzed to confirm the diagnosis using real-time reverse transcription pcr (rrt-pcr). clinical nasopharyngeal swabs and bronchoalveolar lavage and post mortal fragments of trachea, lungs, bronchi, spleen from saint petersburg hospitals and 49 basic laboratories of federal influenza center were included in this study. all specimens were taken from patients with influenza-like illness or viral pneumonia. specimens were tested by rrt-pcr according to cdc protocols, i.e. using superscript iii platinum one-step qrt-pcr system (invitrogen) with primers and probes for infa, h1 seasonal, and h1sw (biosearch technologies). in addition, the test-systems 'amplisense influenza virus a ⁄ b-fl' and 'influenza virus a ⁄ h1-swine-fl' for pcr-detection, typing and subtyping of influenza viruses were also used. these test-systems are produced by central institute of epidemiology, moscow, russia and recommended by russian ministry of health as tests for influenza diagnosis. sequencing was carried out on an abi prism 3100-avant genetic analyzer (applied biosystems, usa) with bigdye terminator cycle sequencing kit. phylogenetic analysis was performed using programs vector nti 10.0 (invitrogen) and mega 4.1 (psu, usa) by maximum likelihood with the tim+i+g model for ha, and -hky+i+g model for na. evolutionary model was selected by akaike information criterion (aic) in model-test (posada, crandall, 1998). statistical reliability of tree branches was evaluated by bootstrap test (100 replications). immunohistochemical study was performed using novalink antibodies to ha and np with novocastra visualization system. influenza virus a ⁄ h1n1v rna was detected in 409 patients with severe form of influenza-like illness and 163 fatal cases. out of pcr-confirmed flu recovered cases 58% were patients under 14 years of age, 41% were aged 15-64 years, and 1% were older than 65 years. mean age of recovered patients was 15ae7 years (from 1 month to 77 years). viral rna in postmortem materials was detected mostly in lung tissue (86% of specimens) and trachea fragments (64%), and less commonly in spleen (17%). mean age of the deceased with confirmed flu (h1n1v) infection was 39ae3 years with age ranging from 7 months to 75 years. in 84% of fatal cases, influenza was complicated by viral or secondary bacterial pneumonia. median time from the onset of illness until death was 10 days. according to our data, 4% of patients died had diabetes, 4ae5% were obese, and 4% were pregnant women in the 2nd or 3rd trimester. ha and np were detected by immunohistochemical assay in lung tissue of dead patients with confirmed influenza virus a ⁄ h1n1v infection. ha and np was revealed in the endothelium of different sized blood vessels (capillaries and arterioles). these influenza virus proteins were also detected in some tissue macrophages apart from epithelium and endothelium. the localization of the two proteins was different: ha is mostly localized in cell membrane and cytoplasm, and np -mostly in the nucleus. here we present data on molecular genetic characteristics of 51 strains of pandemic virus, 40 strains obtained from clinical specimens, and 11 from post mortal ones isolated in the research institute of influenza. all the strains studied contain the s31n substitution in m2 protein, which indicates resistance to the adamantane antivirals, and have no h275y substitution in the neuraminidase, which indicates resistance to oseltamivir. the phylogenetic analysis showed that russian viruses were similar to influenza viruses a ⁄ texas ⁄ 05 ⁄ 2009 and a ⁄ california ⁄ 07 ⁄ 2009 (ha similarity 98ae9%). all russian viruses could be divided in two clusters: the first one includes viruses similar to the reference strain a ⁄ california ⁄ 07 ⁄ 2009, and the second one, which is the majority of viruses analyzed includes strains with substitutions ha s203t, na n248d, v106i, and ns i23v (figure 1 ). bootstrap support was 59. the isolates with ha s203t substitution can be classified in one of the five minor genome variants of a ⁄ h1n1v viruses found in the united states and mexico in 2009. 2 several viruses had strain-specific substitutions in antigenic sites sb and ca and the mutation d222g in ha receptor-binding site. the substitution of amino acid residue asp to gly at position 222 of ha was found in eight of eleven isolates (73%) from postmortem lung and trachea samples and two of forty isolates (5%) from nasopharyngeal swabs of patients with severe course of the disease. appearance of amino acid substitutions in the ha receptor-binding site (d187e and d222g ⁄ e) could be associated with influenza virus passaging on eggs. 3 five strains that contained g at position 222 of ha were isolated from post mortal specimens on mdck cells in this study, thereby excluding the possibility of substitution appearance hence to virus adaptation on eggs. in order to reveal genome changes in a(h1n1)v, strains isolated on the territory of russian federation during the 2009 pandemic, 67 full genome sequences from genbank, and research institute of influenza database were analyzed comparing two groups of viruses (isolated before and after 22 sept 2009). nine amino acid changes observed predominantly in late pandemic strains were found. five of them (s128p, s162n, d222g, v234i, v321i) reside in ha, two in na (i263v, n386k), two in pb2 (k340n, t588i), and one in pa (f35l). towards the end of the epidemic the viral population had demonstrated statistically certain rise in number of strains containing mutations in four genes. difference between groups was statistically significant (chisquare test, p = 0ae05). if v 2 > 3ae84, than difference between early and late strains is statistically significant. additionally fisher's test determined whether 'early strains' and 'late strains' differ significantly in the proportion of 'no mutation event' and 'mutation event' attributed to them in each particular position. all calculations were performed in fisher_tk freeware by vladimir belyaev similar to calcfisher (haseeb, 2003) fully described here (http://www.jstatsoft.org/v08/i21/paper). we have selected positions with statistically significant amino acid changes in late strains (p-value 0ae05). according to full genome analysis of influenza virus a ⁄ h1n1v 2009 strains, seven clades were distinguished, but the divergence between representatives of different clades remained small. 4 (figure 2 ). besides the strain a ⁄ perth ⁄ 12 ⁄ 2010 also contains substitution s181f in the same ha antigenic site. according to data obtained, the 2009 epidemic in russia was caused only by influenza virus a ⁄ h1n1v. unlike the previous epidemic periods when most severe influenza cases were registered among the children under 5 years and among elderly people aged over 65 years, the first wave of pandemic due to influenza virus a (h1n1)v resulted in increased level of mortality mainly among the people aged 18-53 years. though all pandemic viruses showed comparative genetic homogeneity, some evolutionary trends could be outlined. for clarification of the exact pathogenic role of mutation d222g in ha receptor binding site, further studies are necessary. full-genome analysis of influenza virus a ⁄ h1n1v strains circulating in the southern hemisphere in the new epidemic season 2010 revealed the phylogenetic subgroup distinguished by seven substitutions in inner proteins (pb2, pb1, np, ns1) and sa antigenic site of ha (n142d). the changes revealed could be caused by adaptation of the virus to an immunized human population. nasal and throat swabs (placed in 3 ml mem and frozen at )80°c until use for viral rna extraction and tissue culture inoculation) were collected from patients with febrile illness, i.e., >38ae0°c. samples were received from clinics in us embassies and us military laboratories located throughout the world since the initial who declaration of 2009 novel h1n1 outbreaks as a global pandemic on june 11, 2009. viral isolates were obtained from inoculating cultures of mdck cells with 0ae1-0ae2 ml viral suspensions collected in mem originated from patients after 2-7 days incubation. [10] [11] [12] [13] due to low viral titers in normal clinical samples, most of full viral genome sequences were derived from viral stocks obtained by tissue culturing passages (mdck, 1-2 times). viral rna was extracted from clarified supernatant fluid of nasal ⁄ throat swabs or mdck cultures using the 'charge-switch' rna extraction system based on the user manual protocol from the manufacturer (invitrogen inc., ca, usa). total rna was eluted into volume equal to original sample volume, i.e., 100 ll starting viral supernatant used to yield final 100 ll rna in molecular grade water (invotrogen inc.) and stored at )80°c until tested. generating ⁄ preparing overlapped cdnas for full genome coverage of 2009 novel h1n1 viruses by multiple rt-pcr amplifications the first step in the high-throughput sequencing pipeline for full influenza genome sequences was to establish a robust rt-pcr amplification scheme consisting 67 different rt-pcr primer pairs covering all 8 rna segments to ensure 100% amplification coverage of full viral genomes of all the incoming targeted viruses (houng, hs. 2011, submitted for publication). extracted viral rna (5 ll), derived from mostly mdck culturing stock or clinical sample containing sufficient viral load (>100 infectious units per ml) was added to primer-free rt-pcr total master mixture (10ae5 ll) for each virus followed by adding primer pair (2 ll, 3 pmole ⁄ ll per primer). rt-pcr was then performed: rt reaction through two hold-steps (50°c, 15 minutes and 94°c, 2 minutes); 35 cycling amplifications (94°c for 15 seconds, 53°c for 15 seconds, 72°c for 2ae5 minutes). specific cdna amplicons corresponding to each individual primer pair were routinely monitored and visualized by agarose gel electrophoresis. pooled 67 cdna products (2-5 lg) from each viral rt-pcr amplification run were used as sequencing substrates according to the roche 454 flx user manual and bulletins by incorporating adaptors containing individually multiplex identifier [mid]-key assigned to each individually pooled viral cdna. up to 12 different mid-keyed viral cdna were further pooled together to be clonally amplified on capture beads in water-in-oil emulsion micro-reactors (em amplifications), and pyrosequenced using one of two regions of a 40 · 75 mm picotiterplate. for each individual viral genome containing multiple assemblies (8 rna segments), we obtained sff file(s) containing raw sequencing reads from which nucleotide sequence data and phredlike quality scores were extracted. on average, 80ae0-90ae0% of 3-9 million mid-key specific nucleotides were extracted and mapped for consensus genome sequences. roche 454 gsmapper (v. 2.0 and 2.3) software was used to assemble all sequencing raw data and sff files into consensus sequences. new reference mapping projects were created to assemble each individually mid-keyed viral cdna into consensus viral sequences. one of the earliest 2009 h1n1 genomes of california origin, a ⁄ california ⁄ 04 ⁄ 2009(h1n1), deposited in genbank, was routinely employed as a reference genome sequence for most of gsmapper projects. the resultant consensus sequences obtained were further verified and validated through the ncbi annotation utility check and ultimately deposited to the ncbi influenza database, genbank. nucleotide sequences specific to each individual rna gene were aligned by the geneious pro 4.7.5 software (http://www.geneious.com). 14 trees were built based on the tamura-nei genetic distance model using the neighbor-joining method with no outgroup used via geneious pro 4.7.5. phylogenetic trees of the 2009 h1n1 genomes were constructed by importing fasta files containing specific concatenated target sequences of pb1, pb2, pa, ha, np, na, mp, and ns from each individual virus into the geneous pro software and going through the sequence assembling and tree building steps. high-throughput pyrosequencing of pooled 2009 novel h1n1 cdnas by roche 454 flx system up to 24 viral cdnas could be routinely sequenced to completion for 24 different full viral genomes from a single roche 454 flx picotiter plate by utilizing the combination of pico-titer plate's two distinct regions as well as 12 different mid-keyed adaptors. the 'shotgun' sequencing approach employed in this study is a feasible method to viral isolates (n) sequence multiple pooled 2009 h1n1 viral genomes. for each pyrosequencing experiment, approximately 800 000-1 000 000 passed key reads (single fragment per bead) were obtained that yielded readable nucleic acid sequences. among those close to a million passed key reads, only 500 000-600 000 passed key reads had an average sequencing read length of >220 bps, defined as 'long reads' (220 bps · 500 000 reads = total of 110 million bases of nucleic acid sequences) that were used to assemble into influenza genome sequences. mathematically, 110-130 million bases of raw sequencing data from each single roche 454 flx experiment would provide sufficient sequencing bases to cover 24 full genome sequences with approximate 3-400 · of sequencing depth coverage of influenza a with average genome size of 13 500 bps for the total of eight segmented rnas. so far, more than 100 full 2009 h1n1 genomes sampled worldwide have been successfully sequenced and deposited in the ncbi database by division of viral diseases, walter reed army institute of research (wrair). the bioinformatics derived from unique viral genome sequences generated from this study based on constant rt-pcr amplification scheme and identical roche 454 pyrosequencing protocols provide a reliable data set in predicting the evolutionary patterns of pandemic viruses. wrair received clinical samples from us embassies and military personnel throughout the world since the initial who announcement of 2009 novel h1n1 outbreaks. nearly equal distributions of sequenced viruses derived from three broadly categorized geographic regions, north america, central ⁄ south america, and asia ⁄ europe ⁄ africa (data not shown). besides the geographic distribution pattern of viral isolates, figure 1 displays the viral isolation time lines of all the sequenced viruses reflecting two peaks that coincided with two waves of 2009 pandemic infections, early-mid summer and fall of 2009. 15 phylogenetic trees of the eight influenza a segments of all sequenced viruses were tentatively generated. it was found that the substitution frequencies per site for the ha, na, and ns genes are at much higher rate than the other five genes, pb2, pb1, pa, np, and mp genes (data not shown). the observed higher genetic variations for ha and na genes of 2009 h1n1 are consistent with the historical genomic and epidemiological dynamics data of human influenza a revealing higher temporal fluctuations in ha and na genes. [16] [17] [18] [19] analysis of full influenza genomes containing concatenated eight complete rna segments revealed the existence of two distinctive genetic clades in circulation since the beginning of 2009 pandemic, as shown in fig-ure 2. it is noteworthy that all viruses of mexico and california origins (clade 1 shown at the top of figure 2 ) were isolated at the beginning of 2009 pandemic prior to the isolation of all other viruses belonging to the second genetic clade 2. 18, 20 discussion during the past decade, the advance of dna sequencing technology, such as development of ngs, in making full viral genome sequences readily available have enabled study of far broader and more detailed aspects of evolutionary change for any new emergent infectious pathogen. the massive sequencing capacity of roche 454 flx system allows simultaneously process and sequence millions of individual cdna molecules, in contrast to processing and sequencing individual cdna fragments by conventional sanger sequencing method. within a short period of few months since the beginning of the 2009 pandemics, wrair accomplished large number of representative 2009 h1n1 full genomes of worldwide origins via roche 454 flx system. sequencing data derived from this study illustrates a much higher genetic variation rate for ha and na genes of 2009 h1n1 that is compatible to the higher temporal fluctuation rate for ha and na genes of seasonal influenza a derived from decades of intensive monitoring and comparison studies and analyses. [16] [17] [18] [19] following the mexican and us reported cases, confirmed outbreaks of 2009 swine h1n1 rapidly proliferated and spread throughout europe, asia, africa, and south america, most probably via global airline travel. 5, 6 it seemed that new cases in the us and most cases throughout the world had been clinically mild relative to the initial reported cases in mexico. [21] [22] [23] [24] here we demonstrate through the phylogenetic relationship of sequenced 2009 h1n1 full genomes that the clinical isolates could be divided into two different clades of viruses, i.e., the clade 1 genetic group contains only viruses isolated at the beginning (march ⁄ april 2009, mexico and california) of 2009 pandemics and the rest of other viruses all belong to the 2nd genetic group, clade 2. thus, it's likely that the currently circulating 2009 h1n1 of clade 2 causing worldwide infections is genetically different from the initial 2009 h1n1 isolates that caused the early infections in mexico and california. 18, 20 introduction a pandemic influenza virus (2009 h1n1) was recently introduced into the human population. the hemagglutinin (ha) gene of 2009 h1n1 is derived from 'classical swine h1n1' virus, which likely shares a common progenitor strain with the human h1n1 virus that caused the pandemic in 1918. 1 since antigenic changes of influenza virus ha occur more slowly in swine than in humans, 2 we hypothesized that 2009 h1n1 might still retain an antigenic structure similar to that of 1918 h1n1 or the early isolates of its descendants. in this study, we compared ha antigenic structures of 2009 h1n1 and human h1n1 viruses by a molecular modeling approach to demonstrate the existence of shared epi-topes for neutralizing antibodies. we found that has of 2009 h1n1 and the 1918 h1n1 virus shared a significant number of amino acid residues in known antigenic regions. from this observation, we hypothesize that the 2009 h1n1 ha antigenic sites will be targeted by antibody-mediated selection pressure in humans in the near future. we further discuss possible directions of antigenic changes in the evolutionary process of 2009 h1n1. sequence data of ha genes modeller 9v6 was used for homology modeling of ha structures. after 100 models of the ha trimer were generated, the model was chosen by a combination of the mod-eller objective function value and the discrete optimized protein energy statistical potential score. after addition of hydrogen atoms, the model was refined by energy minimization with the minimization protocols in the accelrys discovery studio 2.1 software package using a charmm force field. steepest descent followed by conjugate gradient minimizations was carried out until the root mean square gradient was less than or equal to 0ae01 kcal ⁄ mol ⁄ a. the generalized born implicit solvent model was used to model the effects of solvation. the ha model was finally evaluated by using procheck, whatcheck, and verify-3d. custom-made programs were developed with the ruby language and used for investigating the numbers of potential n-glycosylation sites and candidate codons (cand1) in ha sequences. it is known that the h1 ha molecules have four distinct antigenic sites: sa, sb, ca, and cb. 3, 4 as a result, these sites consist of the most variable amino acids in the ha molecule of the seasonal human h1n1 viruses that have been subjected to antibody-mediated immune pressure since its emergence in 1918, although it was absent in humans from 1957 to 1976. to investigate the structures of these antigenic sites of 2009 h1n1, 3d structures of the ha molecules of sc1918, the recent seasonal human h1n1 virus (br2007), and 2009 h1n1 (ca2009) were constructed by a homology modeling approach, and compared by mapping all the amino acid residues that were distinct from those of sc1918 ha (data not shown). we found that most of these antigenic sites of br2007 ha predominantly contained altered amino acid residues if compared with sc1918. by contrast, amino acid residues at these positions were relatively conserved in ca2009 ha when compared with sc1918 ha. notably, the sa and sb sites, which contain many amino acids involved in neutralizing epitopes near the receptor binding pockets, remain almost intact ( table 1 ), suggesting that antibodies raised by natural infection with sc1918 or its antigenically related descendant viruses play a role in specific immunity against ca2009. these observations lead us to hypothesize that such antigenic sites involving the conserved amino acids will soon be targeted by antibody-mediated selection pressure in the human population. based on this hypothesis, we speculated that 2009 h1n1 would undergo patterns of amino acid substitutions in ha similar to those seen in seasonal human h1n1 viruses during its epidemic period (i.e. those that have been substituted since 1934) (figure 1) . we then predicted possible amino acid substitutions of 2009 h1n1 from the sequence similarity of the antigenic sites. for example, both sc1918 and ca2009 had an asn residue at position 142 in the sa site. for sc1918, the residue at this position has altered from asn to lys since 1942. combining these two facts, it seems reasonable to hypothesize that ca2009 will also undergo an amino acid substitution from asn to lys at position 142 in the future. interestingly, we found that some of the recent variants of the 2009 h1n1 virus have indeed undergone substitutions identical to those predicted in figure 1 . it is important to monitor whether such variants will be selected and survive in sustained circulation in humans. next, we analyzed the acquisition of potential n-glycosylation sites associated with antigenic changes. previously, we reported that cand1 sites, a set of three codons that require single nucleotide substitution to produce n-glycosylation sequons, were important motifs to rapidly acquire n-glycosylation sequons. 5 therefore, we investigated the number and location of potential n-glycosylation sites and cand1 sites in 2009 h1n1 ha. we found that ca2009 also had a single n-glycosylation sequon at the same position in the globular head region of ha, and lacked the multiple n-glycosylations that have been observed in the antigenic changes of the human h1n1 virus during the early epidemic of this virus. we also found that ca2009 ha possessed three cand1 sites that were present at the same position in sc1918 ha (positions of the first asn residue, 177, 179, and 184). of these, the cand1 sites with positions at 177 and 179 had actually become potential n-glycosylation sites in human h1n1 viruses. this result suggests the likelihood of additional n-glycosylation at these sites during future antigenic changes of 2009 h1n1 ha. notably, some of the recent 2009 h1n1 variants (as of march 24, 2010) have an additional n-glycosylation sequon at position 179, where the 1918 h1n1 virus readily acquired an n-glycosylation site during its circulation. the present study suggests that the antigenic structure of 2009 h1n1 ha is similar, at least in part, to that of the 1918 h1n1 ha. the 2009 and 1918 h1n1 has share unique three-codon motifs that are important to readily acquire n-glycosylation sequons in their globular head region. based on these similarities, we predicted possible amino acid substitutions that might be associated with future antigenic changes of 2009 h1n1, and confirmed that such substitutions occurred in some of the recent variants of this virus. the present study provides an insight into likely future antigenic changes in the evolutionary process of 2009 h1n1 in the human population. influenza viruses are classified into three types, a, b, and c, based upon the antigenic properties of nucleoproteins and matrix proteins. influenza a virus infects a wide range of hosts, including human, bird, swine, equine, and marine mammal species, while influenza b and c are less pathogenic than influenza a and are mainly found in humans, although there is evidence that they can also infect other species. influenza a has evolved in association with its various hosts on different continents for extended periods of time. 1 to survive as a successful pathogen, the influenza viruses have developed a number of mechanisms, including antigenic mutation and genome reassortment, to continuously evolve and evade the surveillance of the host immune systems. antigenic and genetic analyses have provided important insights into the molecular dynamics of influenza virus evolution. 2 however, a comprehensive understanding of influenza viral genetic divergence and diversity remains lacking. neuraminidase (na) is a major surface glycoprotein of influenza a and b, but is absent in influenza c. it plays a key role in virus replication through removing sialic acids from the surface of the host cell and releasing newly formed virions. influenza a viral na genes are classified into nine subtypes (na1-na9) based upon their antigenic properties, while na genes of influenza b are not classified into subtypes. furthermore, most na subtypes of influenza a have evolved into distinct lineages and sub-lineages, which correspond to specific hosts or geographical locations. in this study, we conducted large-scale analyses of influenza na sequences in order to infer their evolution and to identify lineages (or sub-lineages) of influenza a viruses. a total of 11 454 na sequences that excluded laboratory recombinant sequences were downloaded from genbank. sequences were aligned with muscle and mafft. the alignments were adjusted manually using translatorx, 3 based upon corresponding protein sequences. phylogenetic analyses were conducted using the maximum-likelihood (ml) method in raxml. 4 a set of perl scripts were written by us to facilitate this computational analysis. lineages and sub-lineages were determined based on the topology of the ml trees. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 5 but with the following modifications: a single digit is used to represent one of the nine subtypes and a letter is used to represent a lineage; a sub-lineage is also represented using a digit; a dot is used to separate a lineage and a sublineage. for example, 2a.2 means na2 subtype, lineage a, and sub-lineage 2. the time of most recent common ancestor (tmrca) was estimated using the bayesian mcmc method in beast. 6 in all cases, we employed the gtr + u4 nucleotide substitution model, in which the first and second codon positions are allowed different rates relative to the third codon position. all data sets were analyzed under a relaxed molecular clock and the bayesian skyline population coalescent prior. the maximum clade credibility (mcc) tree across all plausible trees was computed from the beast trees using the treeannotator program, with the first 10% trees removed as burn-in. phylogenetic analysis based upon na sequences revealed two large groups corresponding to influenza a and b, respectively ( figure 1a ). within influenza a, two subgroups were found, one consisting of na1, na4, na5, and na8 and the other consisting of the remaining five subtypes. subtype na9 was found to be a sister subtype of na6, na1 being a sister subtype of na4, and na8 a sister subtype of na5. finally, each na subtype forms a distinct cluster, indicating its genetic uniqueness. influenza a and b viral na were estimated to have diverged around 2641 years ago ( figure 1b ). however, it had large 95% hpd values which ranged from 1259 years to 4299 years ago. the na subtypes of influenza a diverged from more than 1000 to several hundred years ago. the time of most recent common ancestor (tmrca) of each subtype of influenza a virus was generally recent and ranged from the calendar years 1767 to 1928 (figure 1b ). in addition, the tmrca for influenza b viral na was dated back to 1936. a total of 23 lineages were identified in influenza a (table 1) . three lineages, 1a, 1b, and 1c, were identified for na1 based upon the tree topology. linage 1a originated from avian viruses and was further divided into 5 sub-lineages: 1a.0, 1a.1, 1a.2, 1a.3, and 1a.4. linage 1b consists of north american swine influenza viruses whereas 1c is a human lineage. two large lineages, 2a and 2b, were identified in na2. lineage 2a is a human-specific lineage. interestingly, five major swine clades were observed within this lineage. lineage 2b is an avian-specific lineage, and consists of 3 sub-lineages, 2b.0, 2b.1, and 2b.2. three lineages were found in na3. lineage 3a was found in north american avian, 3b in eurasian ⁄ oceanian avian, and 3c also in avian, but it does not show any geographical pattern. for na4, na5, and na6, each was classified into 2 lineages, one found in north american avian (4a, 5a, 6a) and the other in eurasian ⁄ oceanian avian (4b, 5b, 6b). three lineages identified respectively in na7 and na8 are north american avian (7a, 8a), equine (7b, 8b), and eurasian avian (7c, 8c). na9 was also found to have 3 lineages: north american avian (9a), eurasian ⁄ oceanian avian i (9b), and eurassian ⁄ oceanian avian ii (9c), respectively. in this study, we conducted large-scale phylogeny and evolutionary analyses using influenza viral na sequences. the results showed that divergence between influenza a and b viruses occurred earlier than between any influenza a subtypes. this observation was consistent with previous findings based upon phylogenetic analysis of the ha gene, one of the most important genes related to host infection. 7 within influenza a, two sub-groups were found, one consisting of na1, 4, 5, and 8 and the other consisting of the rest of five subtypes (na2, 3, 6, 7, 9) . this observation does not agree with the result described by liu et al., 8 where na subtypes 1, 3, 4, 5, and 8 formed one group and the remaining four subtypes (na2, 6, 7, and 9) formed the other group. this difference is apparently caused by the fact that an outgroup was not used in their phylogenetic analyses. in the present study, both influenza a and b viral na sequences were included in the analysis. high bootstrap values were obtained for major groups, indicating that the inferred evolutionary relationship should be highly reliable. classification and designation of the lineages and sublineages within the influenza a virus are essential for studies of viral evolution, ecology, and epidemiology. a total of 23 lineages were identified within nine influenza a viral na subtypes and with the majority of the identified lineages found to be host or geographic specific or both. our results demonstrated a comprehensive view for the evolution of na genes and provided a framework for the inference of evolutionary history of pandemic viruses and for further exploring of viral circulations in multiple hosts. for example, the global pandemics of human h1n1 in 1918, h2n2 in 1957, the pandemic of human h3n2 virus in 1968, 9 the crisis of h5n1 hpai in hong kong in 1997, 10 and swine-origin h1n1 influenza in 2009, 11 all can be mapped onto the lineages and sub-lineages identified in this study. such information will facilitate not only identification of known genetic origins but also early detection of novel influenza a viruses. influenza viruses constantly evolve to avoid the human immune pressure in the process of antigenic drift. 1 through sequencing of viral genomes, the rates and direction of virus evolution can be observed. moreover, comparison of protein sequences allows us to determine amino acid substitutions that are related to immune pressure and antigenic drift. the creation of global influenza genetic databases, along with concurrent development of analytical tools, allows the comparison of multiple influenza virus strains. 2 the main aim of this study was to perform antigenic and genetic comparison of pandemic influenza viruses (h1n1) isolated during the 2009-2010 pandemic in ukraine and in other countries. nasopharyngeal swabs and autopsy materials collected from infected patients were received from the areas of ukraine. in addition, field isolates of influenza viruses from the 2009 ⁄ 10 season and strain specific serum were used for identification by hemagglutinin inhibition assay. influenza viruses were identified and subtyped using real-time rt-pcr analyses using cdc primers and adopted protocols. 3 sequencing was performed in two world health organization (who) influenza collaboration centers (centers for disease control and prevention, atlanta and national institute for medical research, london). hemagglutinin inhibition assay was conducted using chicken and guinea pig red blood cells following standard who protocols. 4 the all 6 ukrainian isolates of influenza viruses, which were isolated in ukraine during august-november 2009, were identified as a ⁄ california the phylogenetic analyses confirmed the evolutionary relationship between ukrainian isolates and viruses from other countries, which were isolated during the first wave of the pandemic. high genetic and antigenic conservation of pandemic influenza viruses from ukraine and other countries also were demonstrated. considering that the emergence of the novel pandemic influenza strain occurred in countries of northern hemisphere during summer, it was very interesting and significant tracking the dynamics of genetic changes in influenza viruses, which were isolated at the beginning of epidemic and those isolated during the rise of the epidemic in ukraine. influenza a virus causes moderate to severe epidemics annually and catastrophic pandemics sporadically. due to the evasiveness of the influenza virus and the nature of its genome (eight single-stranded and negative-sense rna segments), it is essential to understand the evolution of this important pathogen. 1 influenza virus evolves by two major mechanisms: mutation and reassortment. antigenic and genetic analyses have revealed partially the molecular dynamics of influenza virus evolution. 2,3 however, important questions, such as how many genotypes in the influenza a virus, remain unanswered. one of the major issues pertaining to this genotyping problem is how many lineages or sub-lineages can be determined for a subtype and according to what criteria. because of the unique structure of the influenza a viral genome, the computational genotyping methods developed for other viruses cannot be applied to the influenza virus. constructing phylogenetic trees is a powerful technique for the identification of evolutionary groupings (i.e., lineages ⁄ clades). however, for large trees, it is hard to determine how many lineages and the boundaries for each lineage. in this regard, multivariate analysis methods, such as multidimensional scaling (mds) and model-based hierarchical clustering, both taking advantage of dimension reduction and visualization, can complement conventional phylogenetic methods. 4 hemagglutinin (ha), the fastest evolving segment, is recognized as the most important gene in the influenza virus that plays a key role in viral pathogenesis. however, we have only limited knowledge of lineages and sub-lineages occurring in the hemagglutinin (ha) gene of influenza a virus, 5 although much effort has been made in assigning clades or sub-clades in highly pathogenic avian influenza (hpai) virus ha. 6 in this study, both model-based hierarchical clustering and phylogenetic methods were used for sequence analysis. one objective for this study is to explore and develop a more accurate lineage approach for further comprehensive influenza lineage and genotype analyses. a total of 11 821 hemagglutinin (ha) sequences (approximately 1778 nucleotides long), excluding laboratory recombinant sequences, were downloaded from genbank as of march, 2010. 7 sequences were aligned with muscle 8 and mafft. 9 the genetic distance matrix of all pairwise sequences was computed using the k2p model under mega 5.0. we then used the distance matrix as input to the cmdscale module in r 2.5.1 for the mds analysis. the principle coordinates resulting from mds were used for the model-based hierarchical clustering analysis, again in r 2.5.1 (the r foundation. available at: http://www.r-project.org/). the bayesian information criterion (bic) values were computed based upon ten different statistical data models -eii, vii, eei, evi, vei, vvi, eee, eev, vev, and vvv. the highest bic value was used to determine the number of clusters in the given sequence data. phylogenetic analysis was conducted using maximumlikelihood (ml) in raxml. 10 raxml uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large-scale sequence analysis. all the analyses were conducted on the supercomputer cluster (holland computing center, http://hcc.unl.edu/main/index.php). the trees were visualized in figtree (version 1.3.1) . lineages and sub-lineages were determined based on both the topology of the ml trees and model-based clustering results. additional information such as hosts, geographical regions, and circulation years were also considered in the classification. we used the same lineage nomenclature as described in, 11 with the following modifications: lineage analysis was conducted for each ha subtype, which agrees with the convention of influenza virologists that 16 ha subtypes were identified in influenza a virus; ha lineages are represented with digits and letters, where the digit(s) represent one of the 16 subtypes and a letter represents a lineage; here, we present sub-lineages or sub-sub-lineages also in digits, with smaller numbers representing earlier lineages or sub-lineages within the same subtype (e.g., lineage 1 occurs earlier than lineage 2); the digit 0 is used to indicate inclusion of ancestral viruses in a lineage (or sub-lineage); a dot is used to separate lineages, sub-lineages, and sub-sub-lineages. for example, 1a.1ae2 means ha1 subtype, lineage a, and sub-lineage 1, and sub-sub-lineage 2. the sub-lineage level can be extended as necessary. the model-based clustering method corroborates commonly used phylogenetic methods in lineage and sub-lineage assignment. here we use the h12 subtype as an example to show the lineage and sub-lineage assignment. the bayesian information criterion (bic) reaches its maximum when the number of clusters for h12 equals 3, regardless of which mode we choose ( figure 1a ). therefore, based on bic, the optimal number of clusters for the h12 subtype is 3. as a result, a total of 3 clusters based upon the vvv model were identified ( figure 1b) . a significant correlation was found in lineage assignments by the phylogenetic method and the model-based hierarchical clustering method ( figure 1b,c) . lineages 12a and 12b were identified for h12, which correspond to north american avian and eurasian avian, respectively. lineage 12a was further divided into 2 sub-lineages, 12a.0, 12a.1, where 12a.0 is the ancestral sub-lineage in 12a. based on both model-based hierarchical clustering and phylogenetic analyses, a total of 41 distinct lineages were identified among 16 subtypes, averaging out to be 2ae56 lineages per subtype ( table 1 ). the majority of the identified lineages were found to be host or geographic specific or both. for example, three lineages, 1a, 1b, and 1c, were identified for ha1. lineage 1a was further divided into two sub-lineages, 1a.0 and 1a.1. the 1a.0 is swine-specific, whereas 1a.1 is a human pandemic 2009 h1n1 sub-line how to accurately identify an evolutionary lineage of influenza a viruses is challenging. one commonly used approach is molecular phylogeny, where phylogenetic trees are constructed, and the tree topology is used for lineage determination. here, we used a bayesian model-based clustering method, along with phylogenetic methods, to decide lineages and sub-lineages of influenza a viruses based upon sequence data. the results demonstrated that the modelbased clustering method corroborates phylogenetic methods and increases the accuracy of lineage assignment. one salient feature of this study is its large-scale analysis of all available influenza a hemagglutinin sequences. a total of 41 distinct lineages and 81 sub-lineages were classified; the majority of them were found to be host or geographic specific. this observation agrees largely with previous findings. 5 we are conducting further analyses of other influenza a segments 12 and expect to identify their lineages and create a comprehensive genotypes database for all influenza a viruses. such information will allow us to detect the genetic origin of newly found viruses, track their genetic changes, and identify potential genome reassortments. a hierarchical nomenclature system has been proposed and adopted for hpai ha clades and sub-clades by who influenza surveillance centers. 6 wan et al. also proposed a hierarchical approach for influenza a viral genotypes system. 13 the work presented here is one of the first steps towards the development of a nomenclature system for influenza a virus lineages (at the segment level) and genotypes (at the genome level). whether the naming system will be accepted and used by the influenza research community is more challenging than the lineage analysis itself. identification of the genetic origins of influenza a viruses will enhance our understanding the evolution and adaptation mechanisms of influenza viruses. the phylogenetic analysis is the traditional approach to identify the influenza progenitor. first, the nucleotide sequences are aligned using multiple sequence alignment methods, such as clustalw, 3 muscle, 4 and t-coffee. 5 second, phylogenetic analysis is performed on these aligned sequences to infer their evolutionary relationship using neighbor-joining (nj), 6 likelihood, or bayesian inference. 8 bootstrap analyses or computation of posterior probability are usually applied to estimate the phylogenetic uncertainty. however, this phylogenetic analysis is time consuming due to intensive computations in multiple sequence alignments and phylogenetic inferences. it is difficult to perform an analysis using this method on a large dataset, for instance, with more than 1000 taxa, as is the common case for influenza studies. alternatively, blast 9 is applied to identify the prototype genes in the database. blast determines a similarity by identifying initial short matches and starting local alignments. since influenza viral sequences have very high similarities, especially for most conserved regions, blast usually generates a large number of outputs, which will not be helpful for progenitor identification. since blast is a local sequence alignment, the results from blast may not reflect the global evolutionary information between the sequences. the blast scores cannot be used to define the evolutionary relations between viruses, especially in the context of the entire genetic pool. recently, we have developed a distance measurement method, complete composition vector (ccv), that can calculate genetic distance between influenza a viruses without performing multiple sequence alignments. 10, 11 we also adapted the minimum spanning tree (mst) clustering algorithm for influenza reassortment identification. 12 the application of this approach in the analyses of pb2 genes of influenza a virus showed that the integration of ccv and mst allows us to identify the potential progenitor genes rapidly and effectively. based on these results, here we develop a webserver called ipminer for influenza progenitor identification. ipminer can identify potential progenitors for a query sequence against all public influenza datasets within a few minutes. in order to improve the computing efficiency, 31 distance matrices were pre-computed by ccv, and they include 16 for ha (h1 to 16), 9 for na (n1 to n9), and one for each of the internal gene segments (pb2, pb1, pa, np, ns, and mp). these 31 pre-computed matrices will be updated weekly. ipminer just needs to compute the query matrices for a query sequence and sequences in the database. the standalone ccv program is also available at http://sysbio.cvm.msstate.edu/ipminer. in order to identify the influenza progenitor genes, ipminer first integrates the query matrix and a corresponding pre-computed matrix into a full distance matrix, which is then clustered by mst clustering algorithm. we adapted the threshold we measured previously in mst, u + nr, where u is the average distance and r is the standard deviation of a cluster. 12 as a result, mst will generate a hierarchical structure for the clusters. in each cluster, we will randomly select 20 viruses or 10% of the cluster size if this cluster has more than 200 viruses. ipminer will return the viruses with the smallest distances when the search reaches to the lowest level (the largest n) in this hierarchical structure. our analyses have shown that the level 5 has generally yielded good results for influenza a viruses. to visualize the overall mst structure, ipminer applies multi-dimensional scaling (mds) method to project all the viruses in the genetic pool onto a two dimensional graph, and the precursor viruses are marked in different shapes ( figure 1 ). the users can select other prototype viruses from the graph for further phylogenetic analyses. a single job with one query sequence takes <2 min. the genbank identifiers and associated genetic distances and sequence identities are displayed. the users can download the sequences for the identified precursor viruses as well as those from the prototypes viruses. in addition, for the users' convenience, ipminer generates a phylogenetic tree using nj method implemented in phylip 13 to illustrate the phylogenetic relationship among the query sequence(s), the identified progenitors, and the selected prototypes viruses. the programs in this solution package are written in java. the shell scripts are written in korn shell script in order to achieve high performance. cascading style sheets (css) are used for a consistent look across the pages. this also enables to change the overall design just by replacing the css definition file. php has been used as server side scripting and is written in java. in order to achieve high performance for computing in a genomic scale, we apply hash function or a binary tree, which enables that the precursor identification has a time complexity of o(n). for single queries, the users can visualize the results online. for batch queries of multiple sequences, the results will be sent to the users by e-mail. ipminer has been tested on microsoft internet explorer, mozilla firefox, and safari. the users need javascript to obtain full function of ipminer server. the webserver is available at http://sysbio.cvm.msstate.edu/ipminer. in summary, ipminer webserver has three major computational features for influenza progenitor identification: (i) it calculates the genetic distances through ccv and identifies the viruses with the shortest ccv distances against the query virus to be the progenitor genes; (ii) it projects influenza viruses onto a two dimensional map, which illustrates the global relationship between the progenitor genes and other viruses in the genetic pool; and (iii) it performs phylogenetic analyses between the query virus, the identified progenitor genes, and other selected prototype viruses. ipminer provides a user friendly web service for influenza progenitor identification in real time. the gisaid initiative offers an alternative to current public-domain database models in response to growing needs of the global influenza community for the sharing of genetic sequence and associated epidemiological and clinical data of all influenza strains. gisaid's publicly accessible epifluô database is governed by a unique sharing mechanism that protects the rights of the submitter, while permitting ongoing research as well as the development of medical interventions, such as drugs and vaccines. for the gisaid initiative, the max planck institute for informatics (mpii) saarbrücken, germany, has developed a web portal that is accessible at http://www.gisaid.org featuring the gisaid epifluô database that offers a unique collection of nucleotide sequence and other relevant data on influenza viruses. the database is based on software by oracle and the dante ò system by a3systems gmbh, germany. extensive metadata are also collected for most isolates. the database provides features for searching, filtering specific datasets for download, and user friendly upload functionality. to uphold gisaid's unique sharing mechanism, all users must positively identify themselves. while access is free of charge, all users agree that they will not attach any restrictions on the data, but will acknowledge both the originator of the specimen and the submitter of the data, and seek to undertake to collaborate with the submitter. all uploaded sequence data are submitted to rigorous curation by the friedrich-loeffler-institute for animal health (fli), germany. the database has been live since september 14, 2009. among its contributors are all five who collaborating centers for influenza who routinely contribute data in addition to using the epifluô database for their semiannual vaccine strain selection. to provide a complete picture of data, all data available in the public domain is routinely imported. as of october 8, 2010, the rapidly growing gisaid dataset comprises 166 989 nucleotide sequences (from 48 779 isolates) with 28 949 (from 11 857 isolates) uniquely submitted to this database. software development is underway to continually extend the spectrum of available data analysis tools. the intergovernmental process of the 62nd world health assembly specifically mentions gisaid as a publicly available database for depositing virus sequence data. starting in 2011, germany's federal ministry of food, agriculture and consumer protection will be the long-term host of the gisaid platform. the mpii will continue to develop the portal and database software and enable gisaid to act as a catalyst for the development of advanced bioinformatics software connected directly to the database. gisaid has become an indispensible resource for the international scientific community on influenza. the consortium will expand its activities and offers to catalyze research and development on a wide variety of issues pertaining to risk analysis, drug development, and therapy of influenza. options for the control of influenza vii ª 2011 blackwell publishing ltd, influenza and other respiratory viruses, 5 (suppl. 1), 416-424 the pandemic h1n1 virus emerged in 2009 and spread rapidly throughout the world, principally affecting children and young adults. as this virus is new to the human population, it is important to determine if these influenza infections are more commonly associated with other respiratory pathogens compared to previously circulating influenza strains. co-infecting respiratory viruses may cause increased morbidity in individuals with pandemic h1n1, and may also be unwanted contaminants in influenza vaccines if original clinical samples containing these adventitious viruses are used to directly inoculate certified cell lines for vaccine production. to examine this issue, stored rna from 300 original clinical samples (nasal swabs, nasal aspirates, throat swabs) from australian and new zealand subjects that were collected in 2009 that were positive for pandemic h1n1 and 300 samples collected in 2008 that were positive for seasonal influenza by real time pcr assay (using the cdc, usa kits), were subjected to a resplex ii -panel version 2.0 (qiagen) pathogen screen. the resplex ii assay detects 18 common respiratory viruses, such as respiratory syncytial viruses (rsv a, b), influenza a and b viruses, parainfluenza viruses (piv1-4), human metapneumo-viruses (hmpv), coxsackieviruses ⁄ echovirus (cvev), rhinoviruses (rhv), adenoviruses (adv b, e), coronaviruses (nl63, hku1, 229e, oc43), and bocaviruses. resplex ii uses a combination of multiplex rt-pcr, hybridization of pcr onto target specific beads followed by detection using luminex-xmap technology. original clinical samples were received at the center from who national influenza centers, who influenza collaborating centers, and other regional laboratories and hospitals from australia, new zealand, and the asia ⁄ pacific region. most samples were from australia and new zealand. these samples consisted of nasal swabs, nasopharyngeal swabs, nasal washes, throat washes, and throat swabs. all samples were stored at )80°c until rna was extracted. rna was extracted from 200 ll of clinical sample using either the magnapure extraction system (roche, australia) or the qiaxtractor system (qiagen, australia) according to the manufacturer's recommendations with an elution volume of 90 ll and stored at )80°c until used. a 5 ll aliquot of rna was used to amplify the selected influenza virus gene using specific primers and probes as supplied by cdc (atlanta, usa) 1 along with super-script iii platinum one-step rt-pcr reagents (invitrogen, australia). real time pcr detection was performed on a 7500 fast system with sds software (applied biosystems, ca, usa). a cut off of a cycle threshold (c t ) of 35 or below was considered positive. resplex ii panel ver2.0 detection the qiagen molecular differential detection (mdd) system was used, which combines qiaplex amplification (multiplex rt-pcr) with detection on the liquichip 200 workstation (luminex's xmap microsphere based multiplexing system) and qiaplex mdd software according to the manufacturer's instructions. a low level cutoff was used (150) to obtain maximum sensitivity. from the 300 clinical specimens that were positive for influenza from 2008 by real time pcr, there were 18 (6%) a(h1n1) seasonal influenza viruses, 106 (35ae3%) a(h3n2) viruses, 174 (58%) b viruses, and 2 (0ae7%) viruses which were influenza a positive, but could not be typed. clinical samples from 2009 selected to study were all influenza a(h1n1) pandemic 2009 positive by real time pcr. detection of influenza virus in respiratory samples was much lower with the resplex ii assay (using a low cut off of 150 units) for pandemic influenza a virus (160 ⁄ 320; sensitivity 53ae3%) and to a lesser extent for seasonal influenza a (116 ⁄ 126; sensitivity of 92ae1%) and b viruses (161 ⁄ 174; sensitivity of 92ae5%) when compared to real time pcr. there were relatively few co-infecting respiratory viruses with either pandemic h1 infections in 2009 (5ae7%) or seasonal influenza infections in 2008 (6ae3%) ( table 1 ). the most common dual infection seen with pandemic h1n1 2009 viruses and 2008 seasonal b viruses was with cvev (7 ⁄ 18; 2009 and 5 ⁄ 14; 2008, respectively) while for 2008 a(h3) viruses there were no dominant co-infecting viruses ( table 2 ). in 2009 one case was detected with three respiratory pathogens in the same sample, a 25 year old female who had pandemic h1n1, cvev, and rhv, and in a 2008 seasonal influenza sample, one case with a triple infection was detected (bocavirus, piv2 and influenza b). the median age of subjects with co-infections was younger for both pandemic h1n1 with a median age of 10 years (range: 1 months to 27 years), compared to the full 2009 sample set which had a median age of 29 years (range: 1 months to 85 years), while for the patients from 2008 with seasonal influenza viruses with co-infections they had a median age of 6ae5 years (range: 1 months to 26 years) compared to all 2008 samples which had a median age of 16 years (range: 1 months to 84 years). there was good concordance in detecting influenza a and b in respiratory samples collected in 2008 between real time rt-pcr and the resplex ii system (100% versus >92ae1% for seasonal influenza a and b respectively). this data compares well with other studies such as li et al. 2 who found that resplex ii had 82ae8% sensitivity and 100% specificity for seasonal influenza a viruses and 90ae0% sensitivity and 100% specificity for influenza b viruses. in contrast, the present study found only 53ae3% sensitivity for the resplex ii detection of influenza a with the 2009 samples that were positive for pandemic h1n1 by real time rt-pcr. a recent study by rebbapragada et al. 3 also showed lower sensitivity for pandemic h1n1 viruses in nasopharyngeal samples with the resplex ii system (55% sensitivity and 100% specificity) compared to other commercial platforms seeplex rvp (95% sensitivity and 100% specificity) and luminex rvp (100% sensitivity and 97% specificity). interestingly the latest version of the resplex system offered by qiagen the resplex ii plus panel ruo now has a separate target for the pandemic h1n1 virus (mexico 2009). 4 in terms of detection of other respiratory viruses such as piv-1, piv-3, rsv and hmpv, high sensitivities (87ae5%, 72ae2%, 73ae2%, and 80%, respectively) and specificities (99ae7-100%) compared to taqman rt-pcr have been reported from testing of nasal wash and nasopharyngeal clinical samples. 5 in both the seasonal influenza positive 2008 and the pandemic h1n1 positive (by real time rt-pcr) clinical specimens, few other respiratory viruses were detected. only 18 of the 2008 samples had another virus detectable and one had two other viruses, while in 2009 16 out had another virus and one had two other viruses detected from a total of 300 influenza virus positive samples collected in each year. enteroviruses, coronaviruses, and parainfluenza viruses were most often found with both seasonal and pandemic infections. younger age appeared to be associated with co-infections with those subjects in 2008 with dual infections having a median age of only 10 years compared to the study groups 29 years; and similarly for 2009, the median age for subjects with dual infections was only 6ae5 years compared to the study groups' median age of 16 years. a study by chong et al. 6 on 298 nasopharyngeal swabs collected during 2007-2009 using resplex ii and luminex xtag rvp fast, they found dual respiratory virus infections in 27 ⁄ 179 (15ae1%) of samples and only 2 (0ae7%) with triple respiratory viral infections; however, these were from cases with any combination of multiple respiratory viruses not necessarily influenza, although influenza positive cases were the most common respiratory virus detected (37ae4% of all positive samples). given the low level and variety of viral co-infections along with both seasonal and pandemic influenza seen in this study, it is unlikely that influenza infections predispose subjects to particular respiratory viruses, but may still allow bacterial colonization, such as has been seen with severe and fatal cases with pandemic h1n1 with various bacteria including streptococcus pneumoniae, streptococcus pyogenes, staphylococcus aureus, or haemophilus influenzae. 7, 8 low levels of other respiratory viruses along with the finding that certain cell lines (like the mdck 33016-cells used in this study) do not propagate a number of these viruses (e.g. rsv a and b, rhinoviruses, coronaviruses), but do propagate others (e.g. parainfluenza 3) 9 should make testing for unwanted viruses that might be co-isolated with influenza viruses more focused and hence easier to detect and eliminate this isolate for future vaccine production. global influenza surveillance is one of the most important approaches to combat spread of disease. current laboratory methods for characterizing influenza are time-consuming and labor-intensive, and few viral strains undergo full characterization. 1 even fewer strains from domestic poultry and swine or from wild aquatic birds are wellcharacterized. these strains are important for global surveillance since they are thought to be the precursors to pandemic influenza strains. 2 we have designed a highthroughput global bio laboratory to address these surveillance needs. the goal of this project was to develop highspeed and high-volume laboratory capabilities for extensive surveillance and rapid and accurate detection and analysis of influenza. the workflow consists of surveillance, sample transportation, laboratory testing, data management and analysis. five robotic systems have been designed for this laboratory: sample accessioning, biobanking, screening, viral culture, and sequencing. sample accessioning logs barcodes, centrifuges, and aliquots samples are then sent to biobanking. the robotic biobank stores samples at )80°c and reformats tubes for screening. the screening system extracts rna and confirms the presence and subtype of influenza. aliquots of positive samples are sent to the viral culturing system for scale-up. finally, cultured samples are extracted and sent to the sequencing system for full genome sequencing. the sample accessioning, sequencing, and biobanking systems have been built, delivered, and validation processes are currently being completed. robotic screening and culturing systems have been fully designed and are ready to be built. a biosafety level 3-enhanced containment laboratory was built to enable the flow of samples containing highly pathogenic avian influenza viruses. in full operation, this approach to surveillance is designed to enable the sequencing of up to10 000 full virus genomes per year, more than the total of all full influenza genomes sequenced to date. the design of a robotic laboratory for influenza surveillance presents unique challenges and opportunities. before a robotic system is built, each assay is worked out on the bench top, each movement of the plates and reagents is defined, and the laboratory information management system (lims) must be able to address each step of the process. alternate assays are conceived for processes that are not automation-friendly. waste streams, worker safety, and space constraints are considered. each possibility is taken to reduce processes that have the potential to aerosolize or cross-contaminate influenza samples. instruments must be found that fit the capabilities needed. detailed specifications for each of the robotic systems were written including all the parameters listed above. once the systems are built, a long validation process takes place where the processes and instruments in each system are adjusted to function together properly. finally, a validation study is performed to ensure that the system is able to produce useful data for influenza research. the entire process takes months from start to finish for each robotic system and requires complete cooperation from a diverse team of researchers. the accessioning system logs initial sample information with the lims system. samples arrive in barcoded cryotubes. the liquid handler brings all samples up to a common volume and clarifies samples by centrifugation. samples are then transferred from screw-cap sample vials into storage plates containing 96 individually punchable storage tubes. each tube (0ae3 ml) is individually identifiable with a 2d barcode on the bottom. six archive aliquots are made, and tubes are individually weld-sealed for storage. tips for aspiration are fixed and undergo a high-pressure plasma process between each use to sterilize tips and destroy nucleic acids. samples are stored at )80°c. each module has a capacity of 600 remp plates or $60 000 samples. the automated freezer system can assemble requested samples as 96-well plates while samples remain frozen. the screening system uses magnetic bead extraction chemistry, real-time pcr, and a liquid handling system to extract samples, confirm and quantify the presence of influenza, and reformat extracted samples for input into the sequencing system. serotype of human influenza samples will be performed by real-time pcr. many samples will not have enough material for further analysis and will need to be scaled up. the culturing system combines incubators, a liquid handling platform, plate reader, and real-time pcr to culture, monitor growth, harvest, and quantify influenza. when the system is not being used for culture and scale-up, it can be used to assay previously cultured influenza samples for drug resistance. a challenge to sequencing large numbers of influenza samples is the manpower required for sample preparation. the sequencing system has the capacity to prepare up to 10 000 samples for sequencing per year for sanger sequencing. sanger sequencing was chosen because it is well-established for influenza surveillance, and automation-friendly. the system is designed to work with multiple primer sets (48, 96, 192) . robotic systems all report to the lims. each process completion, plate movement, and data point are entered and checked by an online, web-based lims. status updates, notification, reporting, and data analysis can be achieved without entering the bsl3 containment facility. routine data analysis such as determining whether a cultured sample is ready to be harvested will be performed by the lims. complex data analysis, while still requiring significant human input, will be made easier by the data-acquisition functions of the lims. the implementation of a high-throughput influenza surveillance laboratory will provide an influenza research and response capacity that far exceeds what is available today. with the addition of each new system, we add a new capability to the influenza community and new opportunities to foster partnerships and collaborations with government, foundations, businesses, and academic institutions. this laboratory will not only enable cutting edge research, but will also enable a more effective response of near real-time surveillance during a pandemic outbreak. pandemics of 1957 and 1968 were believed to arise from avian influenza viruses. 1 the tropism of avian and human seasonal influenza viruses for the human lower respiratory tract deserves investigation. the target cell types that support replication of avian influenza a viruses in the human respiratory tract in the early stages of clinical infection have not well defined. in a previous autopsy studies of human h5n1 disease, influenza a virus were found to infect alveolar epithelial cells 2 and macrophages. 3 in this study, viral infectivity and replication competence of human and high and low pathogenic avian influenza viruses were systematically investigated in the human conducting and lower respiratory tract using ex vivo organ cultures. we compared the replication kinetics of human seasonal influenza viruses (h1n1 and h3n2), low pathogenic avian influenza viruses (h9n2, h5n8) with that of the highly pathogenic h5n1 viruses isolated from human h5n1 disease. a range of human seasonal influenza a viruses of subtypes h1n1 and h3n2 viruses were included in this study from 1975 to 2009. two isolates of low pathogenic avian influenza a (lpai) (h9n2) viruses from different virus lineages isolated from poultry in hong kong in 1997, a low pathogenic influenza a (h5n8) virus isolate from wild ducks in hong kong in 2005, and two virus isolates of highly pathogenic avian influenza (hpai) a subtype h5n1 were included. fragments of human bronchi and lung were cut into multiple 2-3 mm fragments within 2 hours of collection and infected in parallel with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml and as control cultures were infected with ultraviolet light inactivated virus. these tissues fragments were infected for 1 hours and washed twice with pbs and incubated for 1, 24, and 48h at 37°c. the bronchial tissue was cultured in an air-liquid interface using sponge. viral yield was assessed by titration in mdck cells. one part of the infected tissue were fixed in formalin and processed for immunohistochemistry for influenza antigen. other part of infected tissue was homogenized and underwent rna extraction, and the expression of influenza virus matrix gene was measured by quantitative rt-pcr. human bronchus ex vivo cultures supported human seasonal influenza virus to replicate efficiently. avian influenza h9n2 virus replicated, although less efficiently than that of seasonal influenza viruses, whereas hpai h5n1 did not productively replicate in ex vivo cultures of human bronchus. this is in agreement with our previous finding in the well-differentiated bronchial epithelial cells in vitro. 4 on the other hand, human lung ex vivo cultures supported prominent productive replication of human seasonal influenza h1n1 ( figure 1a ) and hpai h5n1 ( figure 1f ) viruses. lpai, such as h9n2 ( figure 1c -d) and h5n8 ( figure 1e ), also replicated productively, but with a lower viral yield. surprisingly, the replication of human influenza h3n2 viruses ( figure 1b ) across the last three decades was greatly inhibited. there are clear differences in viral tropism of human seasonal and avian influenza viruses for replication in the human bronchus and lung. hpai h5n1 virus can infect and productively replicate in the lower lung, which may account for the severity of human h5n1 disease, but not in the conducting airways. surprisingly, there are marked differences in the replication competence of seasonal influenza viruses in ex vivo lung tissues, with influenza h1n1 viruses being able to replicate efficiently while h3n2 viruses do not. this may be related to the more strict siaa2-6gal binding preference of h3n2 viruses. on the other hand, the efficient replication of influenza h1n1 viruses in the alveolar spaces indicates factors other than tissues tropism alone play a role in the differences in disease severity between human seasonal h1n1 and avian h5n1 virus infections. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells, suggesting that viral protein(s) other than m gene-translational products facilitates the splicing of viral mrnas. the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, raising the possibility that c ⁄ ns1 or c ⁄ ns2 enhances the splicing of viral mrnas. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, whereas it was inhibited by that of a ⁄ ns1. these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. pre-mrnas of the influenza a virus m and ns genes are poorly spliced in virus-infected cells. 1, 2 the inefficient splicing of viral pre-mrnas can be understood partly by the fact that influenza a virus ns1 protein is associated with spliceosomes and inhibits pre-mrna splicing. 3, 4 cis-acting sequences in the ns1 transcript also negatively regulate splicing. 5 by contrast, in influenza c virus-infected cells, the predominant transcript from the m gene is spliced mrna. 6 the present study was performed to investigate the mechanism by which influenza c virus m gene-specific mrna (m mrna) is readily spliced. 7 the yamagata ⁄ 1 ⁄ 88 strain of influenza c virus was grown in the amniotic cavity of 9-day-old embryonated hen's eggs. cos-1 and 293t cells were cultured in dulbecco's modified eagle's medium containing 10% fetal calf serum. subconfluent monolayers of cos-1 cells were transfected with pme18s containing influenza c virus m gene cdna using the lipofectamine procedure and then incubated at 37°c. total rna was extracted from both the transfected cells and cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 virus using the rneasy mini kit (qiagen). ribonuclease protection assay was performed using a ribonuclease protection assay kit rpa iii (ambion). 6 briefly, a [ 33 p]-labeled influenza c virus rna 6-specific rna probe (vrna sense) was synthesized by in vitro transcription and hybridized with the total rna at 42°c overnight. hybrids were digested with rnase a (0ae08 u) and rnase t1 (3 u) at 37°c for 30 minutes and then analyzed on a 4% polyacrylamide gel containing 4 m urea. hmv-ii cells infected with c ⁄ yamagata ⁄ 1 ⁄ 88 and cos-1 cells transfected with pme18s expressing influenza c virus ns1 were fixed with carbon tetrachloride at various times after infection and transfection, respectively. the cells were then stained by an indirect method using anti-gst ⁄ ns1 serum as the primary antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit igg (seikagaku kogyo) as the secondary antibody. the splicing efficiency of influenza c virus m gene-specific mrna (m mrna) in infected cells was higher than that in m gene-transfected cells the ratio of m1 encoded by a spliced m mrna to cm2 encoded by an unspliced m mrna in influenza c virusinfected cells was about 10 times larger than that in m gene-transfected cells. ribonuclease protection assays showed that the splicing of m mrna in infected cells was much higher than that in m gene-transfected cells (figure 1 ). these data suggest that viral protein(s) other than m gene-translational products facilitates viral mrna splicing. the influenza c virus ns gene translational product may up-regulate the splicing of viral mrnas the unspliced and spliced mrnas of the influenza c virus ns gene encode two nonstructural (ns) proteins, ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2), respectively. the introduction of translational premature termination into the ns gene, which blocked the synthesis of c ⁄ ns1 and c ⁄ ns2 proteins, drastically reduced the splicing of ns mrna, suggesting that c ⁄ ns1 or c ⁄ ns2 enhances viral mrna splicing. immunofluorescent staining showed that ns1 localized in the nucleus in the early phase of infection, and was distributed in both the nucleus and cytoplasm in the late phase of infection, raising the possibility that influenza c virus ns1 protein plays a role in viral mrna splicing that occurs in the nucleus. the splicing of influenza c virus m mrna was increased by co-expression of c ⁄ ns1, whereas it was reduced by co-expression of influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m mrna was also increased by co-expression of c ⁄ ns1, though it was inhibited by that of a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1, but not a ⁄ ns1, can up-regulate the splicing of viral mrnas. 7 in influenza a virus-infected cells, splicing is controlled so that the steady-state amount of spliced mrnas is only 5-10% of that of unspliced mrnas. 1, 2 the mechanisms by which influenza a virus ns pre-mrnas are poorly spliced have been investigated and the following confirmed. influenza a virus ns1 protein associates with spliceosomes and inhibits pre-mrna splicing. 3, 4 two cis-acting sequences in the ns1 transcript (positions 153-465 in the intron and positions 775-860 in the 3¢ exon region) inhibit splicing. 5 by contrast, influenza c virus m gene-specific mrna (m mrna) is efficiently spliced in influenza c virus-infected cells. 6 in this study, we examined the mechanism by which influenza c virus m mrna is efficiently spliced and the regulatory mechanism of the splicing of ns gene-specific mrna (ns mrna). the introduction of a translational pre-mature termination into the influenza c virus ns gene, thereby blocking the synthesis of influenza c virus ns1 (c ⁄ ns1) and ns2 (c ⁄ ns2) proteins, drastically reduced the splicing rate of ns mrna. we further examined whether c ⁄ ns1 potentially facilitates viral mrna splicing. the splicing rate of m mrna of influenza c virus was increased by co-expression with c ⁄ ns1, whereas it was reduced by co-expression with influenza a virus ns1 protein (a ⁄ ns1) (figure 2a ). the splicing of influenza a virus m gene-specific mrna was also increased by co-expression with c ⁄ ns1, though it was inhibited by co-expression with a ⁄ ns1 ( figure 2b ). these results suggest that influenza c virus ns1 can facilitate viral mrna splicing, but in no way inhibit it, which is in striking contrast to the inhibitory effect of influenza a virus ns1 on pre-mrna splicing. 3, 4 the mechanism for splicing enhancement by c ⁄ ns1 also remains to be determined. we speculate that c ⁄ ns1 may interact with some host proteins involved in splicing, thereby leading to an up-regulation in splicing, or that c ⁄ ns1 may bind to pre-mrna, increasing its accessibility to the spliceosome. the spliced mrna of the influenza c virus m gene encodes the m1 protein, which plays an important role in virus formation and determines virion morphology. 8, 9 therefore, it is speculated that the mechanism for efficient splicing of m mrna, which provides the m1 protein necessary for virus assembly in a redundant amount, has been maintained in the influenza c virus. by contrast, unspliced mrna from the influenza c virus m gene encodes the cm2 ion channel, which is permeable to chloride ions, 10 and also has ph-modulating activity. 11 although the role of the influenza c virus cm2 ion channel in virus replication remains to be determined, it is conceivable that the over-expression of the cm2 protein has a deleterious effect on virus replication since the fact that a high level of influenza a virus m2 protein expression inhibits the rate of intracellular transport of the influenza a virus ha protein and other integral membrane glycoproteins has been demonstrated. 12 if this is the case, efficient splicing of m mrna may control the amount of cm2 synthesized to optimize virus replication. therefore, we speculate that efficient splicing of m mrna leads to a high level of m1 expression and the reduced expression of cm2, thereby creating conditions that are optimal for virus replication. in this study, we provided evidence that c ⁄ ns1 facilitates the splicing of m mrna. furthermore, c ⁄ ns1 may regulate the splicing efficiency of its own ns mrna during infection, controlling the amount of c ⁄ ns1 and c ⁄ ns2 proteins in infected cells. c ⁄ ns2 plays an important role in the nuclear export of vrnp, and is also associated with vrnp in the later stages of infection in virus-infected cells and is incorporated into virions, 13 suggesting that c ⁄ ns2 is involved, not only in the sorting of vrnp into the assembly site, but also in virus assembly. therefore, it is likely that there is a mechanism by which an appropriate amount of c ⁄ ns2 is provided during infection to accomplish these functions. in conclusion, c ⁄ ns1, which enhances the splicing of viral mrna, may regulate both the expression level of m gene-derived m1 and cm2 proteins, and that of ns gene-derived ns1 and ns2 proteins, thereby leading to optimal virus replication. propagation of the human influenza viruses in embryonated hen's eggs always results in a selection of variants with amino acid substitutions in the hemagglutinin (ha) that affect viral receptor-binding characteristics (reviewed 1 ). brookes et al. 2 recently studied infection in pigs using the egg-grown virus that contained a mixture of the original a ⁄ california ⁄ 07 ⁄ 09 (h1n1pdm) and its two egg-adaptation mutants with single amino acid substitutions d222g and q223r (225 and 226 in h3 numbering system). only the original virus and the variant with 222g were detected in the directly inoculated animals, indicating that the variant with 223r failed to infect. only the original virus was detected in nasal secretions of contact infected pigs, suggesting that the d222g mutant failed to transmit. in contrast, there was an apparent selection of the d222g mutant in the lower respiratory tract samples from directly inoculated pigs. the d222g substitution is of a special interest as it can emerge during virus replication in humans and was associated with severe and fatal cases of pandemic influenza in 2009-2010 3-7 and 1918. 8 here we compared phenotypic properties of the original clinical isolate of h1n1pdm virus a ⁄ hamburg ⁄ 5 ⁄ 09 and its d222g and d223r mutants to explain observed effects of these mutations on virus replication in swine 2 and to predict their potential effects on virus replication in humans. a ⁄ hamburg ⁄ 5 ⁄ 09 (ham) was isolated from clinical material by two passages in mdck cells. the virus was passaged twice in 11-day-old embryonated hen's eggs and plaqued in mdck cells. the plaques were amplified in mdck cells and the sequences of the viral ha were determined. the variants with single mutation d222g and q223r were aliquoted and designated ham-e and ham-e1, respectively. the receptor-binding specificity of the viruses was assessed by assaying their binding to desialylated-resialylated peroxidase-labeled fetuin containing either a2-3-linked sialic acid (2-3-fet) or a2-6-linked sialic acid (2-6-fet). 9 in brief, viruses adsorbed in the wells of 96-well eia micro plates were incubated with serial dilutions of 2-3-fet or 2-6-fet, and the amount of bound fetuin probe was quantified by peroxidase activity. the binding data were converted to scatchard plots (a490 ⁄ c versus a490), and the association constants of the virus-fetuin complexes were determined from the slopes of these plots. viral cell tropism and replication efficiency in human airway epithelium were studied using fully differentiated cultures of human tracheo-bronchial epithelial cells (htbe). 10, 11 to determine cell tropism, cultures were infected at a moi 1, fixed 8 hours after infection, and double immuno-stained for virus antigen and cilia of ciliated cells. infected cells were counted under the microscope (100· objective with oil immersion) in the epithelial segment that included 15-30 consecutive microscopic fields containing between 5% and 20% ciliated cells relative to the total number of superficial cells. percentages of infected ciliated cells and infected non-ciliated cells relative to the total number of infected cells were calculated. ten segments per culture were analyzed and the results were averaged. to compare growth kinetics of ham and ham-e, replicate htbe cultures were infected with 200 plaque-forming units of the viruses followed by incubation at 35°c under airliquid interface conditions. at 24, 48, and 72 hours postinfection, we added dmem to the apical compartments of the cultures and incubated for 30 minutes at 35°c. the apical washes were harvested, stored at )80°c, and analyzed simultaneously for the presence of infectious virus by titration in mdck cells as described previously. 11 the non-egg-adapted h1n1pdm virus ham, similarly to the seasonal human virus a ⁄ memphis ⁄ 14 ⁄ 96 (h1n1), bound to 2-6-fet ( figure 1a ) and did not show any significant binding to 2-3-fet. this result contrasted with the binding of h1n1pdm viruses to several 2-3-specific probes in carbohydrate microarray analysis. 12 reduced avidity of virus interactions with soluble glycoprotein in solution as compared to its binding to the probe clustered on the microarray surface could account for these differences in the assay results. the d222g mutant ham-e differed from the parent virus by its ability to bind to 3-fet and by its reduced binding to 6-fet. the q223r mutant only bound to 2-3-fet, although less strongly than did the avian virus a ⁄ duck ⁄ alberta ⁄ 119 ⁄ 98 (h1n1). the viral cell tropism in htbe cultures ( figure 1b ) correlated with receptor specificity. ham and mem ⁄ 96 showed a typical human-virus-like tropism 10, 11 with preferential infection of non-ciliated cells (<5% of infected cells were ciliated). the mutant with 223r and control duck virus displayed a typical avian-virus-like tropism (preferential infection of ciliated cells). the d222g mutant displayed a cell tropism that was intermediate between those of human and avian viruses; in particular, this mutant infected significantly higher proportion of ciliated cells than ham and mem ⁄ 96. observed alteration of receptor specificity and cell tropism ( figure 1 ) suggested that egg-derived mutations can affect replication of the h1n1pdm virus in human airway epithelium. to test this, we first compared the capacity of the viruses to initiate infection in htbe cultures. replicate cultures were infected with identical doses of the viruses, fixed 8 hours post-infection, and immuno-stained for viral antigen. under these conditions, ham and ham-e infected comparable numbers of cells, whereas ham-e1 infected at least 10 times less cells (data not shown). this result indicated that the mutation q223r markedly impaired the ability of ham-e1 to infect human airway epithelial cultures. we next compared two other viruses ham and ham-e for their multi-cycle replication in htbe cultures and found that the original virus reached threefold higher peak titers 48 hours post infection than did the d222g mutant ( figure 2 ). the d222g mutation in h1n1pdm virus facilitates virus binding to 2-3-linked receptors and alters viral cell tropism in human airway epithelium. these changes could account for increased replication of the d222g mutant in the lower respiratory tract in humans 5-7 and pigs 2 and correlation of this mutation with severe pulmonary disease. [3] [4] [5] [6] [7] the d222g mutant replicates less efficiently in human airway cultures than the original virus. this finding correlates with an apparent lack of transmission of variants with 222g in humans 3 and pigs. 2 egg-derived mutation q223r abolishes virus binding to 2-6-linked receptors and strongly decreases infection in cultures of human airway epithelium. this result agrees with poor infectivity of the q223r mutant in pigs 2 and highlights potential pitfalls of using egg-adapted viruses with this mutation for the preparation of live influenza vaccines. nin-esterase-fusion (hef), nucleoprotein (np), matrix (m1) protein, cm2, and the non-structural proteins ns1 and ns2. 1,2 cm2 is the second membrane protein of the virus and is encoded by rna segment 6 (m gene). [3] [4] [5] [6] [7] [8] it is composed of three distinct domains: a 23-residue n-terminal extracellular domain, a 23-residue transmembrane domain, and a 69-residue cytoplasmic domain. 3, 9, 10 it is abundantly expressed at the plasma membranes of infected cells and is incorporated in a small amount into virions. 10,11 cm2 forms disulphide-linked dimers and tetramers, and is posttranslationally modified by n-glycosylation, palmitoylation, and phosphorylation. [10] [11] [12] analyses of a number of cm2 mutants revealed the positions of the amino acids involved in the posttranslational modifications. 10, 13 evidence was obtained that the n-glycosylation was not required for either the formation of disulfide-linked multimers or transport to the cell surface, 10 and that none of dimer-or tetramer-formation, palmitoylation or phosphorylation was essential to the transport of cm2 to the cell surface. 13 in the present study, in order to investigate the effect of cm2 palmitoylation on influenza c virus replication, we generated a cm2 palmitoylation-deficient influenza c virus, in which a cysteine at residue 65 of cm2 was mutated to alanine, and examined the viral growth and viral protein synthesis in infected cells. 293t and hmv-ii cells were maintained as described previously. 14,15 llc-mk 2 cells were maintained at 37°c in minimal essential medium with 5% foetal bovine serum and 5% calf serum. monoclonal antibodies (mabs) against the hef, np, and m1 proteins of c ⁄ ann arbor ⁄ 1 ⁄ 50 (aa ⁄ 50), and antisera against the aa ⁄ 50 virion and the cm2 protein were prepared as described previously. 3, [16] [17] [18] the seven pol i plasmids for the expression of viral rnas of aa ⁄ 50, and the nine plasmid dnas for the expression of the influenza c viral proteins were reported previously. 6, 14 plasmid dna, ppoli ⁄ cm2-acy(-), in which 995-tgt-997 of the m gene was replaced with 995-gct-997, was constructed based on ppoli ⁄ m. to generate a recombinant wild-type (rwt) virus, the above-mentioned 16 plasmids were transfected into 293t cells as described previously. 6 to rescue a mutant virus, rcm2-c65a, a recombinant influenza c virus lacking a cm2 palmitoylation site, the plasmid ppoli ⁄ cm2-acy(-), instead of ppoli ⁄ m, was transfected together with the other 15 plas-mids. at 90 hours posttransfection (p.t.), the respective culture medium of the transfected-293t cells was inoculated into the amniotic cavity of 9-day-old embryonated chicken eggs, and a stock of the recombinant virus was prepared. the infectious titres of the stocked recombinant viruses and the supernatants of recombinant-infected hmv-ii cells were determined according to the procedure reported previously. 19 radioimmunoprecipitation hmv-ii cells infected with recombinants were labeled with [ 35 s]methionine or [ 3 h]palmitic acid. cells were then disrupted and subjected to immunoprecipitation with the indicated antibodies. the immunoprecipitates obtained were then analysed by sds-page on 17ae5% gels containing 4 m urea, and processed for fluorography. 18 flotation analysis was performed according to the procedure described previously. 6 to examine whether the cm2 protein without palmitoylation is synthesized in rcm2-c65a-infected cells, hmv-ii cells infected with the recombinants were subjected to , and the lysates of the cells were immunoprecipitated with anti-cm2 serum and analysed by sds-page. as shown in figure 1 , the cm2 protein was synthesized both in the rwt-and rcm2-c65a-infected cells, but no incorporation of [ 3 h]palmitic acid into the cm2 proteins synthesized in the rcm2-c65ainfected cells was observed, indicating that cm2 in the rcm2-c65a-infected cells was not palmitoylated. the rwt or rcm2-c65a viruses were infected to hmv-ii cells at an m.o.i. of 5 and incubated at 33°c for up to 96 hours. the infectious titres (p.f.u. ⁄ ml) of rwt were approximately 5-to 10-fold higher than those of rcm2-c65a at 24-96 hours p.i. (data not shown), indicating that rwt grew more efficiently than did rcm2-c65a. thus palmitoylation of cm2 appears to have some effect on the generation of infectious virions in cultured cells. to investigate the reason(s) for the difference in growth kinetics between the two recombinants, we analysed viral proteins synthesized in the infected hmv-ii cells. pulsechase experiments of hmv-ii cells revealed no significant differences in the synthesis and maturation of the hef, np, m1, and cm2 proteins between the rwt-and rcm2-c65a-infected cells (data not shown). the infected cells pulse-labeled and chased were respectively immunoprecipitated with anti-cm2 serum in the presence of 50 mm iodoacetamide and analysed by sds-page in non-reducing condition. in both populations of infected cells, several bands corresponding to cm2a-monomer, -dimer, and -tetramer, as well as cm2b-dimer and -tetramer were detected (data not shown). these results demonstrate an absence of any significant differences between palmitoylation-deficient cm2 and authentic cm2 in terms of conformational maturation and transport in infected cells. membrane flotation analysis revealed that no significant differences in the kinetics of the hef, m1, and cm2 proteins were observed between rwt-and rcm2-c65ainfected cells (data not shown). in contrast, a slight difference in np kinetics was observed. the pulse-labeled np proteins were recovered in the bottom fractions in both rwt-and rcm2-c65a-infected cells. in the chase experiment, the amount of membrane-associated np proteins in fractions 3 and 4 was 30 % of the total np in the rwt-infected cells, which was higher than that (19%) in the rcm2-c65a-infected cells (data not shown). this finding may suggest that the affinity of the np protein, presumably representing the viral ribonucleoprotein (vrnp) complex, to the plasma membrane in the rcm2-c65ainfected cells is lower than that in rwt-infected cells, leading to the less efficient generation of infectious virions. since cm2 is structurally similar to m2, an influenza a virus membrane protein known to be involved in infectious virus production, [20] [21] [22] [23] [24] it is possible that the cytoplasmic tail of cm2 participates in the genome packaging through interaction with vrnp. in the present study, we showed that the affinity of np to the plasma membrane of rcm2-c65a-infected cells was slightly lower than that to the plasma membrane of rwt-infected cells. this observation may suggest that palmitoylation of cm2 is involved in the viral ribonucleoprotein (vrnp) incorporation, leading to efficient infectious virion generation. we hypothesize that palmitoylation contributes to proper regional structure formation in the cm2 cytoplasmic tail, which is competent to recruit vrnp efficiently into virions. alternatively, the cm2 cytoplasmic tail without palmitoylation is not likely to reach the proper conformation, resulting in reduced interaction with vrnp and less efficient generation of infectious progeny virions. the questions of if and how the m1 protein is involved in the interaction between the cm2 cytoplasmic tail and np remains to be clarified. we showed that cm2 synthesized in rcm2-c65ainfected cells was oligomerized and transported to the cell surface. this finding is consistent with the previous observation that palmitoylation is not required for the transport of cm2 to the cell surface in cm2-expressing cos-1 cells. 13 however, the use of reverse-genetics system has enabled us to conclude that the palmitoylation of cm2 is required for efficient infectious virus production. this suggests that the significance of the other posttranslational modifications of cm2 during virus replication can be clarified using recombinant viruses lacking the respective modification sites. sialic acid (sia) linked glycoproteins are the classical influenza receptors for influenza virus haemagglutinin to bind. the distribution of sia on cell surfaces is one of the determinants of host tropism, and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. previous research has shown the differences in apical versus basolateral infection and release of different influenza virus from polarized epithelial cells 1 and correlated this with sialic acid distribution in the human respiratory tract. moreover, mass spectrometric analysis was recently employed to elucidate the glycans present in the tissue in a higher resolution in human lung. 2 the objective of this study was to examine in detail the distribution of these sia-linked glycans at the cellular level by the use of confocal microscopy. human primary type i-like and type ii pneumocytes were isolated from human non-tumor lung tissue by tissue fragmentation, percoll density gradient centrifugation, and magnetic cell sorting. 1 the cells were seeded on coverslips and maintained in small airway growth medium. when confluence was reached, cell monolayers were fixed with 4% paraformaldehyde. we used the plant lectins, sambucus nigra glutinin (sna) from roche which binds to siaa2-6gal, maackia amurensis agglutinin (maa)i and maaii from vector lab, which bind the siaa2-3gal linked glycans using vector red as fluorescent chromogen. the cells were counter-stained with dapi or with fitc-conjugated antibody against endoplasmic recticulum (protein disulfideisomerase, pdi). the cells were imaged with multi-photon excitation laser scanning microscopy using zeiss 510 lsm. the optical cross-section pictures were reconstructed by zeiss lsm510 meta. we found that there was more binding of maai and ma-aii to type ii pneumocytes than type i-like pneumocytes and more overall binding of these lectins than binding of sna ( figure 1 ). in keeping with results from other polarized cells there was more binding to the apical than basolateral aspect, thus, explaining the previously published data on apical versus basolateral infection. 1 as sialic acid has been implicated in the targeting of proteins to the surface, the relative lack of sialic acid on the basolateral aspect can explain why there is little seasonal influenza virus dissemination to the systemic circulation in human infections. furthermore, though there was little binding of sna to the figure 1 . primary human type i-like and type ii pneumocytes stained with lecins (red), pdi (green), and dapi (blue) and imaged captured with confocal microscope. apical or basolateral aspects of the pneumocytes, the experimental findings of infection by influenza h3n2 virus that has a strict siaa2-6gal tropism 3 suggests that there are siaa2-6gal glycans present, which are not readily bound by the lectin sna. the in vitro model of primary human type i-like and type ii pneumocytes system formed a polarized epithelium that has a similar lectin distribution to human alveoli in vivo which demonstrated that it is a physiologically relevant model to study the tropism and pathogenesis of influenza a virus. human disease caused by highly pathogenic avian influenza (hpai) h5n1 virus is associated with fulminant viral pneumonia and mortality rates in excess of 60%. 1 cytokine dysregulation is thought to contribute to its pathogenesis. 2, 3 we previously found delayed onset of apoptosis in h5n1 infected human macrophages and, therefore, a longer survival time of the target cells for prolonged virus replication and cytokine and chemokine secretion, which may contribute to the pathogenesis of h5n1 disease in humans. 4 as bronchial and alveolar epithelial cells are target cells of influenza virus because of their proximal physiological location and interaction with macrophages, we further investigated if the differential onset of apoptosis could be found in influenza h5n1 and seasonal influenza h1n1 infected human respiratory epithelia. we dissected the apoptotic pathways triggered by influenza virus infection. seasonal influenza h1n1 virus (a ⁄ hk ⁄ 54 ⁄ 98), a low pathogenic avian influenza h9n2 lineage isolated from poultry (a ⁄ quail ⁄ hk ⁄ g1 ⁄ 97), and two virus isolates of hpai a subtype (a ⁄ hk ⁄ 483 ⁄ 97 and a ⁄ vn ⁄ 1203 ⁄ 04) were included. primary human bronchial and alveolar epithelial cells were infected with influenza viruses at moi of 2 and the cell monolayer was collected at 24, 30, and 48 hours post infection for tunel assay, and supernatant were collected for ldh assay. fragments of human lung tissues were cut into multiple 2-3 mm fragments within 2 hours of collection and infected with influenza a viruses at a titer of 10 6 tcid 50 ⁄ ml. these tissues fragments were infected for 1 hours and incubated for 48 hours at 37°c. one part of the infected tissue was fixed in formalin and processed for immunohistochemistry for influenza antigen, and the other part was homogenized and underwent rna extraction. apoptosis cdna superarray platform (sabioscience) was employed to conduct apoptosis pathway analysis. in bronchial epithelial cells, seasonal influenza h1n1 virus induced a high percentage of apoptotic cells by tunel assay at 24, 30, and 48 hours post infection with a peak of (figure 1) . a similar observation of delayed onset of apoptosis was found in influenza h5n1 and h9n2 infected alveolar epithelial cells. besides, cdna array data of ex vivo infected human lung showed that both influenza h5n1 and h1n1 virus induced trail expression compared with mock-infected tissue (approximately 15 folds) at 48 hours post infection, but influenza h3n2 virus infected lung induced significantly more trail (27 folds compared to mock infected cells), albeit with a limited viral replication ( figure 2 ). influenza h3n2 virus infected lung also elicited more tnf-alpha and fasr transcription than either h5n1 or h1n1. these observations can account for the greater apoptotic response in influenza h3n2 virus infected lung. as little impact on the expression of intrinsic pathway components was observed, it seems that the apoptotic response to influenza virus infection in lung was mainly through the extrinsic pathways. no significant changes in the expression of anti-apoptotic protein gene was found, except for a moderate induction of birc3 by influenza h3n2 virus, which may act to modulate the apoptotic response. the delayed onset of apoptosis by hpai h5n1 and low pathogenic avian influenza h9n2 virus infected respiratory epithelial cells may be a mechanism for the influenza viruses to have more prolonged replication within the human respiratory tract, and this may contribute to the pathogenesis of human disease. hemagglutination (ha) assay 10% crbc suspension was treated by 625 mu a2, 3-specific sialidase at 37°c for 15 minutes. complete elimination of a2, 3-receptor on sialidase-treated crbcs was confirmed by receptor staining and flow cytometry. ha assay of live viruses with 1% crbc or 1% sialidase-treated crbc were performed in bsl-3 facility. synthetic 3¢sln-paa-biotin(pa191), 6¢sln-paa-biotin(pa190), 6¢sln-ln-paa-biotin(pa343) was provided by the scripps research institute (tsri). as described elsewhere with some modifications, 3 generally, serial dilutions of sialyglycopolymers were coated in 96-well-flat-bottom polystryrene plates, and 32 hau live virus ⁄ well were added. alternatively, the plates were precoated with 5 lg ⁄ ml sialyglycopolymers, and then 8, 16, 32, 64, 128 hau live virus ⁄ well influenza viruses were added. rabbit antisera against a ⁄ ah ⁄ 1 ⁄ 05 diluted in pbs containing 1% bsa was added into the wells. bound antibody was detected by use of hrp-conjugated anti-rabbit igg antibody and tetramethylbenzidine substrate solution. each sample was determined in duplicates and the absorbance read at 450 nm. a total of 31 h5n1 virus strains were obtained from 2003 to 2009. the name and passage history of influenza viruses used in the study are listed in table 1 . as the same sequences of eight rna segments were detected in a ⁄ js ⁄ 1 ⁄ 07 and a ⁄ js ⁄ 2 ⁄ 07, only a ⁄ js ⁄ 2 ⁄ 07 was tested here. three amantadine-resistant variants with m2 mutation of screening of receptor-binding preference by ha assay representative results from three sets of independent experiments are shown in table 1 . complete ha with sialidasetreated crbcs, which were only with a2,6-receptors, was detected in human influenza virus (a ⁄ brisbane ⁄ 59 ⁄ 2007, h1n1) and two human h5n1 virus strains, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. high binding of a2,3 oligosaccharides to h5n1 viruses was detected ( figure 1a -c). and enhanced a2, 6-binding preference was also detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. the a2, 6-binding was dose dependent for sialyglycopolymers and virus titer. notably, as compared with a ⁄ gd ⁄ 1 ⁄ 06 of both short-and long-a2, 6 recognition, a ⁄ gx ⁄ 1 ⁄ 08 prefers to bind to long-a2, six oligosaccharides at low viral titer ( figure 1b,c) . however, both of them showed strong affinity to short-and long-a2, 6 oligosaccharides at high viral loads ( figure 1d ). sialoside-, galactoside-, mannoside-and sulfo-os-binding are the four types of carbohydrate-binding properties of influenza virus. 12 binding of influenza virus to the a2, 3-or a2, 6-linked sialylated glycans on cell surface is important for host range restriction, and the preference to a2, 3 of h5n1 virus limited its efficient infection in human. here, dual receptor-binding preferences were detected in a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08, which are of clade 2ae3ae4. although there is no direct evidence supporting the occurrence of human-to-human transmission in these infection events or the association between viral virulence and receptor-binding switching, viral systemic disseminations are found in the both fatal cases (data not shown). furthermore, with the introduction of clade 2ae3ae4 into the adjacent countries of china, 4 the finding of h5n1 virus with 2-6binding in human should be of concern. though h5n1 virus with human-type receptor-binding was isolated from one patient treated by oseltamivir and those viruses were with ha and ⁄ or na substitutions, 13 whether the substitutions responsible for receptor specificity switching is pre-existed or selected in human host remains unknown. our finding that three mutant viruses bearing m2 mutations of a30s, a30t, and s31n cloned from one isolate a ⁄ hb ⁄ 1 ⁄ 06 suggested it is likely that the resistant viruses emerged in the host environment. no variation was found in their ha and na sequence, and all of them show high affinity to a2-3-binding. our data suggest that the binding-specificity was not affected by the mutations on viral envelope protein m2. with the adaptation from wild aquatic birds to domestic poultry or even in human host environment, influenza virus may possess broader carbohydrate-binding spectrum or topology conformation. 11, 14 we demonstrated differential a2, 6-binding property of two human h5n1 viruses, a ⁄ gd ⁄ 1 ⁄ 06 and a ⁄ gx ⁄ 1 ⁄ 08. though minor effect of short-a2, 6-binding was detected in viruses a ⁄ gx ⁄ 1 ⁄ 08 at low virus titer, both were of high affinity to long-a2, 6 glycans, even at the low titer which are rich on apical side of human upper respiratory epithelia. 11 notably, no evident binding preference switching was detected in the viruses isolated from the sporadic human infection cases at the early of 2009 in china (table 1) . however, higher affinity to the long-a2, 6 glycans was observed in bj ⁄ 1 ⁄ 09, gz ⁄ 1 ⁄ 09, and xj ⁄ 1 ⁄ 09 (data not shown). the discrepancy from the findings obtained by sialidase-treated crbc maybe associated with a limited abundance of n-linked a2-6 with long branches on crbc, as demonstrated in a recent study. 11 therefore, glycan dose-dependent binding assay is valuable and should be applied in flu surveillance. the underlying cause of the tendency is unknown, and further research on receptor-binding specificity of h5n1 viruses is required. influenza a viruses of migrating wild aquatic birds in 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promotes influenza a virus replication through an annexin ii-dependent pathway in absence of neuraminidase costimulatory receptors in a teleost fish: typical cd28, elusive ctla4 trypsin increases pseudorabies virus production through activation of the erk signalling pathway characterization of hla-g1, -g2, -g3, and -g4 isoforms transfected in a human melanoma cell line immunosuppressive hla-g molecule is upregulated in alveolar epithelial cells after influenza a virus infection influenza virus propagation is impaired by inhibition of the raf ⁄ mek ⁄ erk signalling cascade influenza viruses and the nf-kappab signaling pathway -towards a novel concept of antiviral therapy the immunotolerance role of hla-g hla-g1 co-expression boosts the hla class i-mediated nk lysis inhibition hla-g inhibits the allogeneic proliferative response hla-g-mediated inhibition of antigen-specific cytotoxic t lymphocytes the world health organization. h1n1 influenza vaccine task force pandemic influenza: a 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induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? proinflammatory cytokine responses induced by influenza a (h5n1) viruses in primary human alveolar and bronchial epithelial cells differential onset of apoptosis in influenza a virus h5n1-and h1n1-infected human blood macrophages avian flu: influenza virus receptors in the human airway haemagglutinin mutations responsible for the binding of h5n1 influenza a viruses to humantype receptors an avian influenza h5n1 virus that binds to a human-type receptor evolution of highly pathogenic h5n1 avian influenza viruses in vietnam between evolutionary dynamics and emergence of panzootic h5n1 influenza viruses writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza a (h5n1) virus recent avian h5n1 viruses exhibit increased propensity for acquiring human receptor specificity a simple screening assay for receptor switching of avian influenza viruses glycan topology determines human adaptation of avian h5n1 virus hemagglutinin h5n1 chicken influenza viruses display a high binding affinity for neu5acalpha2-3galbeta1-4(6-hso3)glcnac-containing receptors a strain of human influenza a virus binds to extended but not short gangliosides as assayed by thin-layer chromatography overlay search for additional influenza virus to cell interactions avian flu: isolation of drug-resistant h5n1 virus the surface glycoproteins of h5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties this study was supported by the li ka shing foundation, the national institutes of health (niaid contract hhsn266200700005c), and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and we thank all french and vietnamese field staff involved in the data collection in viet nam for their enthusiasm and support and we are grateful to the pig farmers participating in the study for their cooperation and patience. this study was a part of the gripavi project and was funded by the french ministry of foreign affairs. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants commission (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we acknowledge the food and environmental hygiene department of hong kong for facilitating the study. this work was supported by the national institute of allergy and infectious diseases (niaid) contract hhsn266200700005c, the li ka shing foundation, and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. we gratefully acknowledge our colleagues from iiii, shantou university and skleid, hku for their excellent technical assistance. the study was supported by the rfcid commissioned study (lab#16) from research fund secretariat, food and health bureau, hong kong sar; area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06), hong kong sar; and by niaid contract (sjceirs, hhsn26620070005c), nih, usa.ferrets in all groups inoculated with a ⁄ turkey ⁄ 15 ⁄ 06 virus survived the infection and were observed once daily for 14 days. below lower limit of detection (<0ae75 log 10 eid 50 ⁄ ml).statistical cutoff of ic 50 values for nai susceptibility, determined by x 0ae75 + 3iqr. outliers with ic 50 above this cutoff and >10 times the mean ic 50 for each drug were characterized as extreme outliers; those with known drug-resistance mutations such as h275y were classified as resistant and analyzed separately. h275 wildtype, oseltamivir-susceptible isolates. h275y variants, oseltamivir-resistant virus isolates. iqr, interquartile ranges; nai, neuraminidase inhibitors. we wish to thank our collaborators in the who global influenza surveillance network and united states public health laboratories for the submission of virus isolates and clinical specimens. we also thank our colleagues from the virus reference team and the influenza sequence activity, influenza division, cdc, for their valuable technical assis-the findings and conclusions of this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention (cdc). we are indebted to yonas araya, theresa wolter, and ivan gomez-osorio for their excellent laboratory techniques and animal handling assistance. we would like to thank andrea ferrero for her laboratory managerial skills. this research was possible through funding by the cdc-hhs grant (1u01ci000355), niaid-nih grant, (r01ai052155), csrees-usda grant (1865-05523), and niaid-nih contract (hhsn266186700010c). we thank c bazzoli for advice. this work was supported by a grant from the european union fp7 project flu-modcont (no. 20160). we thank staff at seoul, incheon, daejeon, gwangju, gangwon, and jeonbuk provincial research institute of health and environments for their laboratory testing. additionally, we would like to acknowledge the contributions of participating sentinel doctors for evaluating the new rat kit. this study was supported by a grant from the korea cdc. we thank roche applied science for providing the materials and equipment for this evaluation. this research was supported in part by the national institute of allergy and infectious diseases (niaid) contract hhsn26600700005c and the area of excellence scheme of the university grants committee (grant aoe ⁄ m-12 ⁄ 06) of the hong kong sar government. the authors would like express their sincere thanks to cdc, usa for supporting the routine surveillance of ili in we would like to acknowledge the australian red cross blood service (the blood service) and the australian government, which fully fund the blood service for the provision of blood products and services to the australian community. we also wish to thank the donors and staff of the blood service, who have assisted in provision of specimens for testing in this protocol, as well as the staff at the who we are grateful to liping long for her assistance in map generation. this project was supported by nih niaid 1rc1ai086830. cz is supported partially by canadian nserc postdoc fellowship. the authors thank the national investigation team based at the national institute of health (istituto superiore di sanita'), italy (in particular antonino bella, maria cristina rota, stefania salmaso) for providing their support in data collection, and the european union this study was supported in part by a grant-in-aid (21249061) and the special coordination funds for promoting science and technology of ministry of education, science, sports and culture of japan. this study was supported in part by a grant-in-aid from the ministry of education, science, and culture of japan (21249061) and the special coordination funds for promoting science and technology of mext of japan. the work described here was supported by phs grant ai-66349 (jam) and alsac. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. py, po, dw and kb are employees of glaxosmithkline. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the studies were funded by gsk consumer healthcare, and gsk investigators were involved in all stages of the study conduct and analysis. this study was funded by glaxosmithkline. we thank all authors for their participation in data gathering and analysis, and in writing this manuscript. the stud authors are thankful to path for the financial support of this research. we would like to acknowledge jessica d'amico and dr. rick bright of path for their editorial review. this study was supported by path. the authors would like to thank rick bright, jessica d'amico, and vadim tsvetnitsky for editing assistance. the we thank dr. m. enami (kanazawa university) for generously providing plasmids containing cdnas to influenza a virus m and ns genes. we also gratefully thank dr. r. sho (department of public health, yamagata university faculty of medicine) for statistical analysis. some data shown in this study have also been presented in the reference paper. 7 this work was supported in part by a grant-in-aid for scientific research from the ministry of education, culture, sports, science, and technology, japan, takeda science foundation, terumo life science foundation, and a grant-in-aid from the global coe program of the japan society for the promotion of science. we thank markus eickmann for his help in isolation and initial characterization of a ⁄ hamburg ⁄ 5 ⁄ 09 and for providing antisera against h1n1pdm. this study was supported by the european union fp6 global a(h1n1)2009 genetic characterization, molecular evolution dynamics, antiviral susceptibility profiles, and inference of public health implications require nation and region wide systematic analysis of circulating virus. in this study we analysed the genetic and antiviral drug susceptibility profiles of pandemic a(h1n1)2009 influenza virus circulating in portugal. genetic profile analysis was performed in 37 isolates to the hemagglutinin (ha), neuraminidase (na) and mp genes, and in six of these isolates the pb1, pb2, pa, np and ns genes were also analysed. antiviral drug susceptibility profile was analysed for 96 isolates, phenotypically and genotypically to neuraminidase inhibitors (nai) and genotypically to amantadine. the point mutations identified in ha, na, and mp genes of different strains do not seem to evidence an evolutionary trend. this is in agreement with the genetic and antigenic homogeneity that has being described for a(h1n1) 2009 virus. all analysed strains were found to be resistant to amantadine, and five of these strains exhibited a reduced susceptibility profile to nai, three only for oseltamivir and two for both inhibitors. introduction: the dynamics of pandemic influenza a ⁄ h1n1 compared to seasonal strains of influenza is not clearly understood. it is important to understand the patterns of viral shedding and symptoms over time in community-based infections.materials and methods: household infections were followed-up in two large community-based studies. patterns of viral shedding, symptoms and signs, and tympanic temperature were plotted over time and grouped according to strain for analysis.results: the patterns of viral shedding, symptoms and signs, and tympanic temperature in three influenza a strains (pandemic a ⁄ h1n1, seasonal a ⁄ h1n1, and seasonal a ⁄ h3n2) were comparable. peak viral shedding occurred close to the onset of symptoms and resolved after 6-7 days. patterns of viral shedding in influenza b virus infections differed.discussion: the patterns of viral shedding and clinical course of pandemic influenza a ⁄ h1n1 infections were broadly similar to seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. only the clinical course of seasonal influenza b infections was similar to pandemic influenza a ⁄ h1n1. the dynamics of pandemic influenza a ⁄ h1n1 were observed to be largely alike to the dynamics of seasonal influenza a ⁄ h1n1 and a ⁄ h3n2. the coated respirators inactivated a broad range of influenza strains within 1 minute, including the 2009 pandemic strain and human, swine, and avian influenza viruses. antiviral effectiveness was not reduced by hot, humid conditions or repeated saturation, which might occur during prolonged use of respirators. in contrast, infectious virions were detected on the surfaces of all uncoated ffp2 respirators, and could be transferred to glove surfaces during handling of contaminated masks. growth of the viruses was monitored by ha titer using turkey red blood cells, by quantitative real time rt-pcr (qrt-pcr) to detect the influenza a matrix gene, and also by flow cytometry to detect virus positive cells using monoclonal antibodies (imagen influenza virus a and b). 8, 9 matrix gene copy number was determined using qrt-pcr and analysed using the sequence detection software on a 7500 fast system sds (applied biosystems, california, usa). further characterisation was performed through sequence analysis and the ha inhibition (hai) assay. 8 sequence analysis was performed using dnastar 8 and all sequences obtained were compared with the sequence of either the original clinical specimen if available or the conventional atcc derived mdck cell isolate. the hai assay was used to characterize the viruses against a panel of known standard reference viruses and their homologous ferret antiserum.options for the control of influenza vii abstract background: we measured the cross-reactive antibody response to pandemic h1n1 2009 in children and adults before and after vaccination with [2007] [2008] [2008] [2009] influenza season vaccines as part of the rapid public health response to the emergence of ph1n1 and to provide evidence for ph1n1 vaccination policy development in mainland china. materials and methods: archived serum specimens from previous vaccine studies were detected by hemagglutination inhibition assay. results: limited crossreactive antibody response to ph1n1 had been detected among participants of all age groups before and after they had been vaccinated with 2007-2008, 2008-2009 influenza seasonal vaccines. vaccination with seasonal influenza viruses resulted in limited seroconversion to ph1n1 in all age groups, compared with 59-94% of seroconversion to seasonal influenza viruses. but similar to recent studies, a peak of cross-reactive antibody response to ph1n1 was observed in 23% and 39% of participants born from 1915 to 1925 before and after vaccination. conclusions: in order to protect our populations in china, our study strongly suggests vaccination with ph1n1 is required in all age groups and that older populations born before 1925 may be associated with a lower infection rate of ph1n1. on april 15 and april 17, 2009, cases of ph1n1 were identified in specimens obtained from two epidemiologically unlinked patients in the united states and soon thereafter in texas and mexico. 1 since that time, the virus has spread across the globe. assessment of cross-reactive antibody response to the ph1n1 after vaccination with sea-sonal influenza vaccine was first reported from us centers of disease control and prevention (us cdc). according to their results, the seasonal influenza vaccines provided little or no protection against the ph1n1, but some degree of preexisting immunity to the virus existed, especially among adults aged ‡60 years. 2 in this study, using archived serum samples from previous vaccine studies, we measure the level of cross-reactive antibody response to ph1n1 in children and adults vaccinated intramuscularly with trivalent inactivated vaccine developed for the northern hemi serum specimens were collected and provided by provincial centers for disease control and prevention of china as a public health response to the emergence of ph1n1 exempt from human-subjects review. a total of 1308 serum samples were collected from xinjiang uygur autonomous region, yunnan, and shandong provinces. all the serum specimens were grouped by the age of subjects (0-7, 8-17, 18-59, ‡60 years) and by different influenza seasons.hemagglutination inhibition assay was performed according to standard procedures in this study. [3] [4] [5] as with h1n1 components of the vaccine, the seasonal influenza viruses used in this study were a ⁄ solomon islands ⁄ 3 ⁄ 2006 and a ⁄ brisbane ⁄ 59 ⁄ 2007. the ph1n1 influenza virus used in this study was a ⁄ california ⁄ 07 ⁄ 2009 provided by us cdc. all the viruses were propagated in specific pathogenfree embryonated chicken eggs and inactivated by 1& paraformaldehyde. the criteria 6 recommended by the european agency for the evaluation of medical product was applied for the assessment of seasonal influenza vaccine gmt, geometric mean titer; hi, hemagglutination inhibition. three weeks after boosting immunization, spleens were harvested from immunized and control mice. splenocytes were prepared by lymphocyte separation media (ez-sepô, shen zhen, china). the cells were washed and resuspended in complete rpmi-1640 containing fetal bovine serum (hyclone, logan, ut, usa), glutamax, 5 um b-me. splenocytes were cultured in vitro in the presence of inactivated h1n1, h3n2, h5n1, and h9n2 influenza virus antigen for 96 h. quick cell proliferation assay kit (biovision, san francisco, ca, usa) was used to detect the cell proliferation. the 420-480 nm absorbance was read on a plate reader. all experiments have been repeated at least three times.results are presented as mean standard error of the mean (sem). comparison of the data was performed using the student's t-test. significance was defined as a p value of <0ae05. to evaluate the adjuvant effect of recombinant igv, the anti m2e antibody subclasses was measured. igg1 and igg2a were detected after the first and second immunization ( table 1 ). the ratio of igg2a ⁄ igg1 was calculated. immunization with only m2e-hbc showed a lower igg2a ⁄ igg1 ratio <0ae5. igv combined with m2e-hbc led to a high igg2a ⁄ igg1 ratio of up to 5-15 after first and second immunization. these igg subclass distributions indicated that igv can induce a th1 immune response. to determine whether the splenocytes were stimulated in vitro with different subtypes of inactivated influenza antigen after the igv plus m2e-hbc antigen immunization, h1n1, h3n2, h5n1, h9n2 inactivated antigen was used table 1 . the serum igg, igg1, igg2a, and igg2a ⁄ igg1 ratio were measured by elisa after first and second immunization. m2e were coated on the 96 wells plate overnight, and serial dilution sera of day 7, 14, 21 after first and second immunization were added 0, 2ae5, 5, 10, 20, 40 ug ⁄ ml of igg, igg1, igg2a purified antibody were also added for obtaining the standard curve. hrp-labeled goat anti-mouse igg, igg1, or igg2a was then added, washed, and the optical density was read at 492 nm. the results were showed at mean ± sem. day after first immunization days after second immunizationoptions for the control of influenza vii the french grog (groupes régionaux d'observation de la grippe) early warning network collects more than 5000 specimens yearly from cases of acute respiratory illness (ari), using two sampling methods: systematic randomized and non systematic ''ad hoc'' sampling. although vaccines against influenza a virus are the most effective method by which to combat infection, it is clear that their production needs to be accelerated and their efficacy improved. a panel of recombinant live attenuated human influenza a vaccines (laivs), including ns1-73, ns1-126, nsd5, were generated by rationally engineering mutations directly into the genome of a pandemic-h1n1 virus. the vaccine potential of each laiv was determined through analysis of attenuation, immunogenicity, and their ability to protect mice and ferrets. the data indicate that the novel nsd5-laiv was ideally attenuated and elicited strong protective immunity. this study also shows that attenuating mutations can be rapidly engineered into the genomes of emerging ⁄ circulating influenza a viruses in order to produce laivs. the influenza virus exhibits complicated evolutionary dynamics due to multiple reasons, such as diverse hosts, high mutation rates, and rapid replications. in this study, large-scale analyses of 11 454 influenza neuraminidase (na) sequences revealed influenza a and b na genes diverged first around 2641 years ago, and subsequently the na subtypes of influenza a emerged around 1125 years ago. all nine na subtypes of influenza a were genetically distinct from each other, with a total of 23 lineages identified. in addition, five and three sub-lineages were further identified in lineage 1a of na1 and lineage 2b of na2, respectively. the majority of lineages and sub-lineages were found to be host or geographic specific. this study provides not only a better understanding of influenza na evolution, but also a database of lineages and sub-lineages that can be used for early detection of novel genetic changes for improved influenza surveillance. although phylogenetic approaches are commonly used and often found to be powerful, how to accurately identify lineages or sub-lineages of a gene segment of the influenza a virus remains a challenging issue. in this study, we address this issue by analyzing hemagglutinin (ha) sequences using a combination of statistical and phylogenetic methods. following a hierarchical nomenclature system that uses a letter to represent a lineage and a digit for a sub-lineage, we identified 41 distinct lineages and 81 sub-lineages in all 16 ha subtypes through large-scale analyses of 11 821 influenza a hemagglutinin sequences. the majority of the lineages or sub-lineages were host or geographic specific or both. further analysis of other segments will allow us to construct a comprehensive database for influenza a lineages and genotypes, facilitating early detection of new viral strains and genome reassortments and hence improve influenza surveillance. identification of the genetic origin of influenza a viruses will facilitate understanding of the genomic dynamics, evolutionary pathway, and viral fitness of influenza a viruses. the exponential increases of influenza sequences have expanded the coverage of influenza genetic pool, thus potentially reducing the biases for influenza progenitor identification. however, these large amounts of data generate a great challenge in progenitor identification. clinical (nasopharyngeal swabs) and post-mortem materials (fragments of trachea, bronchi, lungs, spleen) were obtained from clinics and ⁄ or out-patients from st. petersburg and from 19 base virological laboratories (bvls) of the research institute of influenza in different regions of the country, which cover approximately 3 ⁄ 4 of the territory of russia. the informed consent for the bio-materials collection and studies was obtained from research subjects or from their relatives in cases of post-mortem materials. isolation of viruses was carried out in the mdck cell culture (cdc, atlanta, ga, usa) and in 10-day-old chicken embryos (e). isolation was done according the standard internationally accepted methods. 3 the reaction of hemagglutination (ha) and the inhibition of hemagglutination (hai) were performed according the who recommended standard method. 3 for the identification of epidemic isolates, we used the hyperimmune diagnostic bovine or ovine antisera annually obtained from the who reference center (cdc). for a detailed antigenic analysis we used the hyperimmune rat antisera against epidemic and reference influenza strains during the period from july 20, 2009 up to april 30, 2010, we have obtained 772 swabs from clinics and out-patients in st. petersburg and 374 swabs from the bvls. in this period, rather high incidence of lethality from pneumonia was observed, which developed on the background of the pandemic flu h1n1v. thus, we received from bvls 173 postmortem materials from 91 deceased patients which manifested pcr+ influenza h1n1v-specific rna. all materials were tested for a possibility of isolation of influenza virus h1n1v both in eggs and in mdck cells. pcr-negative materials were discarded. we isolated 229 strains of pandemic influenza from the materials collected in st. petersburg and region, which comprised 29ae7% of the total number of analyzed samples. at the same time, we did not isolate any other sub-types of influenza in the season 2009-2010 except the pandemic flu. from the swabs purchased from bvls, 47 strains were isolated, which compose 37ae9% of the pcr+ samples, and 35 strains from the post-mortem materials (43ae2% of the pcr+ samples).altogether in the season 2009-2010, we isolated, retrieved, and analyzed in hai 453 influenza strains. 91ae2% of them were pandemic strains a(h1n1)v, and only 7ae9% influenza b viruses. these data together with the epidemiologic data and the results of pcr-diagnostic provide evidence in favor of nearly mono-etiological character of epidemic season 2009-2010 in russia for pandemic influenza a(h1n1)v.though the isolation of pandemic viruses was fulfilled in two traditional model systems, in the case of pandemic 2009 virus, we could observe the tendency of preferential multiplication in embryos compared to mdck, especially in cases of post-mortem material for which chicken embryos are the preferential system of isolation.h1n1v viruses, which were isolated and passaged in mdck, even with significant ha titers, quickly lost their ha activity provided they were kept at +4°c. moreover, some other tested cell lines proved to be practically nonsensitive to the pandemic viruses h1n1v. we used hai reaction for the typing and antigenic characterization of isolated viruses. in the course of isolation of viruses in the reported period, we produced rat polyclonal antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 (h1n1)v and the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 -the virus isolated during the epidemic 1976 in the united states and also of the swine origin -and to the 'swine' strains a ⁄ sw ⁄ 1976 ⁄ 31 and a ⁄ iowa ⁄ 15 ⁄ 30. the hai results of representative strains are given in figure 1 . table 1 shows that the isolated strains were homogenous in their antigenic properties and interacted with the diagnostic antiserum cdc for a(h1n1)v and also with the antisera to the strains a ⁄ california ⁄ 07 ⁄ 09 and a ⁄ st. petersburg ⁄ 56 ⁄ 09 up to 1-1 ⁄ 4 homologous titer. viruses that were isolated from post-mortem materials did not differ by their antigenic characteristics from those isolated from swabs of live patients. only two strains could be attributed to the drift-variants of the strain a ⁄ california ⁄ 07 ⁄ 09 because they reacted with the appropriate antiserum up to 1 ⁄ 8 homologous titer; these strains were a ⁄ pskov ⁄ 1 ⁄ 09 and a ⁄ belgorod ⁄ 6 ⁄ 09. it is interesting that the isolated strains reacted with the antisera to the strains a ⁄ new jersey ⁄ 8 ⁄ 76 and a ⁄ sw ⁄ 1976 ⁄ 31 to 1 ⁄ 4-1 ⁄ 8, and some particular strains even to 1 ⁄ 2 homologous titer. it is even more interesting that some pandemic isolates reacted with the antiserum to the strain iowa isolated in 1930 up to 1 ⁄ 4-1 ⁄ 8 homologous titer. despite of the fact that since the outbreak of 'swine flu' in the usa in new jersey 30 years had gone (and for the strain iowa this period is nearly 70 years) the ha of these viruses and of the pandemic influenza 2009 share some common antigenic determinants as was shown in hai.one more interesting feature of a considerable part of isolated strains is their capability to react with high titers with normal equine serum heated to 56 and to 80°c, while all the strains of swine origin isolated earlier were inhibitor-resistant ( figure 1 ). russian isolates of 2009 divided, in this respect, in two clear and approximately equal in number groups: one of them is similar to the reference strain amino acid substitutions, among them more than 30 were disclosed in antigenic sites, so the degree of similarity to this strain is 78%. a new site of glycosylation was also discovered in the position 276 of ha. 4 essential distinctions of the aminoacid sequence of ha and antigenic properties of the h1n1v strains as compared with actual circulating and vaccine strains is one of the factors that determine the pandemic potential of this new influenza virus.according to the literature, the mutation in the ha gene d222g could cause a broadening of the spectrum of receptor specificity of influenza virus by the acquisition of the capacity to bind both the residues a(2 fi 6) and a(2 fi 3) of the sialic acid of cellular receptors. 5 both types of receptors are present at the human respiratory tract, but in different parts of it, and they exist in different proportions. 6 according to the data of the european center of disease control and prevention (ecdc), the varieties g222 of the h1n1v virus were isolated in 20 countries from subjects deceased of influenza or who suffered a severe form of illness, as well as from those who sustained only a light course of influenza. 7 concerning the strains isolated in rii, this mutation was discovered in nine cases: four were isolated from live patients and five from post-mortem materials. thus, there are no convincing data at present that could prove a causal relationship of the given substitution and the aggravation of a disease course. this is in accordance with previous observations. 7 concerning the resistance of studied strains to the widely used antiviral preparations, it was shown that all tested strains possessed the substitution s31n in the m2 protein that determine the resistance to adamantanes. there was no substitution in the position 275 of neuraminidase (na), which determines the resistance to oseltamivir (h275y). these substitutions are the characteristic indices of the eurasian lineage of swine influenza viruses. 8 thus all studied russian h1n1v isolates were resistant to adamantanes (rimantadine) and sensitive to oseltamivir. respiratory clinical samples taken in 2008 and 2009 that tested positive by real time reverse transcription (rt)-pcr for seasonal influenza viruses (a and b) and pandemic h1n1 2009 respectively were assessed for other respiratory viruses using the resplex ii panel ver2.0 system distributed by qiagen. results showed that co-infections with another 16 respiratory viruses were relatively rare, with a small number of samples having another co-infecting virus present, very few samples having two other viruses detectable in their samples, and none with further viruses. this low number of co-infecting viruses and the ability of certain cell lines not to support infection with particular viruses may make primary isolation of influenza viruses in cell lines easier than might have been thought previously. cm2 is the second membrane protein of influenza c virus and is posttranslationally modified by phosphorylation, palmitoylation, n-glycosylation, and dimer ⁄ tetramer formation. in the present study, we generated rcm2-c65a, a recombinant influenza c virus lacking cm2 palmitoylation site, and examined viral growth and viral protein synthesis in the recombinant-infected cells. the rcm2-c65a virus grew less efficiently than did the wild-type virus. membrane flotation analysis of the infected cells revealed that less np was recovered in the plasma membrane fractions of the rcm2-c65a-infected cells than that in the wild-type virus-infected cells, suggesting that palmitoylation of cm2 is involved in the affinity of the ribonucleoprotein complex to the plasma membrane, leading to the efficient generation of infectious viruses. influenza c virus has seven single-stranded rna segments of negative polarity, encoding pb2, pb1, p3, haemagglutiboth the a2, 6 linkage and its topology on target cells were critical for human adaptation of influenza a viruses. the binding preference of avian flu virus h5n1 ha to the a2, 3-linked sialylated glycans is considered the major factor that limited its efficient infection in human. currently, the switch in binding-specificity of human h5n1 viruses from a2, 3 to a2, 6-glycans did naturally occur, and limited humanto-human transmission was found. to monitor their potential adaptation in the human population, receptor-binding specificity surveillance was made in china. here, the binding specificity of 32 human h5n1 virus strains isolated from 2003 to 2009 was demonstrated. dual binding preference to a2, 3 and a2, 6-glycans were found in a ⁄ guangdong ⁄ 1 ⁄ 06 and a ⁄ guangxi ⁄ 1 ⁄ 08. furthermore, both of them showed a high affinity to the long-branched a2, 6-glycans, which predominate on the upper respiratory epithelial in human. our data suggests that the existence of h5n1 virus with binding specificity to humans should be of concern.introduction via envelope glycoprotein hemagglutinin (ha), influenza viruses bind to cell-surface glycosylated oligosaccharides terminated by sialic acids (sa) where their linkage is celland species-specific. differential receptor binding preference is a host barrier for influenza virus transmission. although most h5n1 viruses have low affinity to neu5aca2, 6gal (human-type) receptor, recent findings suggested that the adaptation of h5n1 virus to human by mutations in the receptor-binding site (rbs) do indeed happen and resulted in enhanced affinity to human-type receptor. [1] [2] [3] in contrast to its putative precursor, a ⁄ gs ⁄ gd ⁄ 1 ⁄ 96, diverse genotypes were presented in currently circulating h5n1 virus, accelerating evolution and widespread occurrence. 4, 5 to date, 10 distinct phylogenetic clades (0-9) were identified based on h5n1 ha, and the confirmed human infections were caused by clade 0, 1, 2ae1, 2ae2, 2ae3, and 7. 6 in china, human h5n1 disease was mostly caused by clade2ae3ae4, which was identified in 28 isolates from 39 confirmed patients from 17 provinces since 2003. clade 7 and clade 2ae2 are responsible for the case in 2003 and 2006, respectively. two current cases of 2009 and 2010 were due to clade 2ae3ae2. now information on receptor property has been documented in some h5n1 viruses of clade 1, 2ae1, and 2ae2. [1] [2] [3] 7 little is known about h5n1 virus of clade 2ae3ae4, particularly from human.recently, a2, 3-specific sialidase-treated red blood cell (rbc) agglutination assay was developed and used for receptor specificity screening of h5n1 virus. 3, 8 the a2, 6 or a2, 3-binding preference can be distinguished by the change of hemagglutination titer reacted with rbcs and enzymatic rbcs. since fine receptor specificity existed in h5n1 viruses, 9,10 the glycan array including sulfated-, fucosylated-, linear sialosides, di-sialosides, or direct binding assay with synthetic polyacrylamide (paa)-based sialylglycopolymers was also recommended for the receptor-specificity surveillance on h5n1 viruses. furthermore, the long-branched a2, 6 sialylated glycans were currently identified to predominate on the upper respiratory epithelial in human and the recognition of this topology, 6¢sln-ln is the key determinant for the human-adaptation of influenza a virus. 11 here, we analyzed the receptor-binding specificity of human h5n1 viruses isolated in china from 2003 to 2009. since 2003, a total of 39 h5n1 infection cases were confirmed in china from 17 provinces. the pharyngeal swabs and lower airway aspirations from the patients were collected within 12 days after disease onset, maintained in viral-transport medium, and tested within 24 hours.options for the control of influenza vii key: cord-006089-08g206kf authors: stevens, james; blixt, ola; paulson, james c.; wilson, ian a. title: glycan microarray technologies: tools to survey host specificity of influenza viruses date: 2006-10-02 journal: nat rev microbiol doi: 10.1038/nrmicro1530 sha: doc_id: 6089 cord_uid: 08g206kf new technologies are urgently required for rapid surveillance of the current h5n1 avian influenza a outbreaks to gauge the potential for adaptation of the virus to the human population, a crucial step in the emergence of pandemic influenza virus strains. owing to the species-specific nature of the interaction between the virus and host glycans, attention has recently focused on novel glycan array technologies that can rapidly assess virus receptor specificity and the potential emergence of human-adapted h5n1 viruses. influenza, an acute viral disease of the respiratory tract, affects millions of people each year. as members of the orthomyxoviridae family, influenza viruses have an envelope and a negative sense, single-stranded and segmented rna genome. of the three groups of influenza virus (a, b and c), type a accounts for all known major epidemics and pandemics. influenza a virus subtypes are named according to their surface antigens: haemagglutinin (ha) and neuraminidase (na). currently, all sixteen ha (h1 to h16) and nine na (n1 to n9) serotypes of influenza a virus circulate in the avian population and, consequently, birds are believed to be an important reservoir for influenza a viruses. in the past century, influenza a viruses with only three ha (h1, h2 and h3) and two na (n1 and n2) serotypes have adapted sufficiently to humans to produce pandemic strains: h1n1 in 1918, h2n2 in 1957 and h3n2 in 1968 (refs 1, 2) . the antigenic shifts that occurred in 1957 and 1968 were a result of avian-human reassortment: in 1957, three of the eight avian gene segments, and in 1968, two avian gene segments were reassorted into existing, human-adapted viruses. this reassortment event is believed by some to occur in animals such as pigs, which can serve as a mixing vessel, as these animals are susceptible to both avian and human influenza virus 3 . the genetic origin of the 1918 pandemic h1n1 virus, which killed about 50 million people worldwide 4 is not known, and there is an ongoing debate as to whether or not the virus 'jumped' directly to the human population or adapted through reassortment 1, [5] [6] [7] . since late 2003, circulating h5n1 avian influenza virus strains have reached epizootic levels in both domestic and wild bird populations across asia. recently, the h5n1 avian influenza virus has spread into europe, the middle east and the african continent. an unprecedented level of global concern surrounds the circulating highly pathogenic h5n1 avian influenza a virus, with fears that the next human influenza pandemic could arise from these strains. so far, the spread of h5n1 avian influenza virus to the human population has been limited; as of 20 june 2006 there have been around 228 welldocumented human cases, but these cases have been associated with a high mortality rate of 130 deaths 8 , although the rate might be inflated by the lack of reporting of asymptomatic cases. the influenza a virus surface is coated with three antigenic proteins: ha, which is responsible for virus binding to receptors on the host cell, internalization and subsequent membrane fusion events in the endosomal pathway of the infected cell; na, which is a sialidase involved in the removal of sialic acid from the infected cell surface to allow the escape of progenitor viral particles after budding; and the m2 ion channel protein, which is involved in the acidification of the inside of the viral particle during internalization through the endosomal pathway. although all three coat proteins are targets for the host immune system, ha is by far the most abundant antigen on the viral surface and, therefore, harbours the primary epitopes for neutralizing antibodies. several factors are involved in hostrange restriction of influenza viruses, but these will not be discussed here, as they have been reviewed recently 2 . here, we focus on receptor specificity of influenza virus ha. influenza viruses use host glycans, which contain sialic acid (n-acetylneuraminic acid (neuac)) moieties as cell-surface receptors, which vary in structure from species to species. humanadapted influenza viruses have has with a binding preference for cell receptors that contain α2-6-linked sialic-acid moieties (neuacα2-6gal), which are found predominately on epithelial cells of the human upper respiratory tract, whereas avian viruses prefer receptors with α2-3-linked sialic acid moieties (neuacα2-3gal) [9] [10] [11] , which are found on epithelial cells in the intestines and respiratory tract of birds (fig. 1) . this subtle difference in the recognition of glycan structure is a key determinant of the species barrier that prevents avian influenza viruses from easily infecting humans. another barrier to infection of humans by avian viruses are mucins produced by specialized epithelial cells that lubricate and protect the otherwise exposed upper respiratory tract epithelium. these proteins are rich in α2-3-linked sialosides (sialylated glycans), and function to trap avian influenza viruses and prevent their attachment to epithelial cells 12 recent studies have revealed that some human epithelial cells in the lower respiratory tract contain α2-3-linked sialic acids and can be directly infected by avian influenza viruses 10, 11, 13 . although humans have been infected by the h5n1 avian influenza virus, most documented cases either lived or worked in close proximity to infected poultry and were, therefore, exposed to a sufficiently high viral load that would increase the chances of the virus escaping the protective mucin layer to infect cells in the lower respiratory tract. the restricted replication of h5n1 avian influenza viruses in the lower respiratory tract in humans, where the virus cannot be readily spread by sneezing and coughing 11 , is believed to account in part for the inefficient human-to-human transmission of h5n1 viruses so far. several other species have been implicated in the origin of new human influenza viruses. the respiratory epithelial cells of pigs have receptors that express both α2-3and α2-6-linked sialic acids and can be infected by human and avian viruses, which indicates that domesticated pigs could be a 'melting pot' for the emergence of new reassortant viruses 9 (fig. 2) . there is evidence that some terrestrial avian species, such as quail, also harbour both types of sialic-acid receptor in the trachea and intestine and, therefore, could also support the creation and spread of reassortments among avian and human influenza viruses 14, 15 . the current h5n1 strains show increased transmissibility to other mammals, including domestic cats 16 , captive wild cats (tigers and leopards) 17 and indonesian pigs, in which the virus causes asymptomatic infection 18 , although the report on indonesian pigs has yet to be confirmed. importantly, no change has been reported in the epidemiology of h5n1 virus transmission and it still remains firmly an avian virus. however, with the constantly increasing host range for h5n1 viruses, many believe that intermediate hosts might ultimately provide the vehicle for h5n1 influenza viruses to adapt to the human population 19 . furthermore, as described above, in the correct conditions, the h5n1 virus can infect humans directly 8 , and it could subsequently mutate to a strain that can effect human-to-human transmission. in view of the rapid geographic spread of the h5n1 avian influenza virus in the bird population and the increasing numbers of confirmed human cases, new technologies are urgently required for the surveillance of current influenza outbreaks to gauge the potential for adaptation to the human population, a crucial step in the emergence of a pandemic strain. this perspective article discusses existing methods that are used to study influenza virus receptor specificity and then introduces recently described glycan microarray technologies that are used to study the glycan-binding properties of several influenza virus has, including a current h5 ha. methods to assess ha receptor specificity one important aspect of surveillance is to determine whether a particular avian influenza virus strain is adapting to human receptors. for many years, techniques to study the interactions of the influenza virus with host cell receptors have used whole viruses. this includes haemagglutination assays, which measure the ability of intact influenza virus and other viruses to agglutinate red blood cells (rbcs), and haemagglutination inhibition assays, which detect and quantify antibodies and soluble receptor analogues that inhibit rbc agglutination by intact viruses [20] [21] [22] [23] [24] . with the identification and isolation of specific α2-3-sialidases and α2-6-sialidases, and advances in technologies for genetic manipulation of the influenza genome, a haemadsorption assay based on desialylation of endogenous sialic acid from rbcs, followed by selective linkage-specific resialylation of rbcs, and subsequent analysis of their binding to viruses or cells transfected with influenza ha, has become routine in many laboratories 21, 24 . although such assays potentially reflect the natural binding of a virus to host cell receptors, the outcome is subject to many factors that are often difficult to control. for example, the quality of cell preparations can vary owing to the relative instability of rbcs, and the extensive variability in transfection efficiencies. the activity of the sialyltransferase enzymes might differ substantially between batches and suppliers and lead to varying degrees of enzymatic resialylation, which can skew results and mask any important specificity differences among viral strains. another factor to consider when using whole-cell assays is the polyvalent cooperative effect of the virus interaction with multiple heterogeneous receptor ligands on the cell surface. therefore, these assays reflect only the overall 'global' affinity for α2-3or α2-6-linked sialic-acid receptors and cannot distinguish among the various classes and modifications of sialylated glycans. since the early 1990s, new technologies have emerged to overcome the problems of cell-based assays. one of the most successful methods reported in recent years uses an assay that is based on competition for binding to solid-phase immobilized virus between a horseradish-peroxidaseconjugated blood sialyl glycoprotein, called fetuin, and unlabelled α2-3 and α2-6 sialyloligosaccharides 25 . the α2-3 and α2-6 sialyloligosaccharides are coupled to polyacrylamide, providing increased valency to compensate for the low ha affinity (mm range) for receptor analogues [26] [27] [28] . such assays have increased our understanding of the diversity of the receptor specificities of influenza viruses from both birds and mammals 14,29-32 , including h5 influenza viruses 33, 34 . these assays have been used to evaluate details of influenza virus receptor specificity beyond the standard analysis for recognition of neuacα2-3gal and neuacα2-6gal linkages, demonstrating that influenza virus receptor specificity is more complex than previously recognized. however, major drawbacks of these assays are that they are relatively low throughput, and have been optimized for screening with whole viruses, thereby restricting the study of new pathogenic strains to specialized laboratories. in the united states, biosafety level 2 (bsl-2) facilities are recommended for working with contemporary, circulating human influenza strains, low pathogenicity avian influenza strains, and equine and swine influenza viruses 35 , whereas increased biosafety level 3 (bsl-3) practices are recommended for non-contemporary and highly pathogenic avian influenza (hpai) viruses. indeed, all of the reconstructed viruses that contain one or more gene segments from the 1918 influenza virus were generated and handled under bsl-3 enhanced (bsl-3+) laboratory conditions 36 , in accordance with the guidelines from the national institutes of health and the cdc 35 . any study that analyses potential humanization of the current h5 hpai strains by mutagenesis of the viral genome would require extreme care and a minimum of bsl-3 facilities (table 1) . since 2002, studies of the interactions of glycan-binding proteins with their glycan ligands have advanced significantly through the development of glycan microarray technologies [37] [38] [39] [40] [41] [42] [43] . such technologies allow investigation of glycan-binding protein interactions on a single chip to which hundreds of different glycan structures can be coupled 40 . arrays have already been used for , arose from genetic reassortment, in which gene segments from an avian virus were mixed with genetic material from a co-infecting human virus, probably through an intermediate host, such as a pig (the mixing vessel theory). the haemagglutinin (ha) of human influenza a viruses has a binding preference for cell receptors that contain α2-6-linked sialic-acid moieties, whereas avian viruses bind preferentially to α2-3-linked sialic acids moieties. the respiratory epithelial cells of pigs have receptors that express both α2-3and α2-6-linked sialic-acid moieties, and can be infected by both human and avian viruses. the resulting viral progeny will either be intact avian or human virus, which can only infect their respective hosts or, if reassortment yields functional virus (usually swapped ha, pb2 and/or neuraminidase (na)), a new pandemic strain might emerge with the ability to retain efficient human-to-human transfer, but be sufficiently different (for example, by a species change in ha) to reduce the ability of the host to mount an effective immune response. pb2, polymerase basic-2 protein. 37, 40, 47 , human glycan-binding proteins involved in both the innate and adaptive immune system 40, [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] , virus glycanbinding proteins 40, 58 and even the binding of whole cells 59 . a glycan microarray that is well suited for the analysis of influenza virus receptor specificity was developed by the consortium for functional glycomics (cfg), funded by the national institute of general medical sciences 40 . the microarray comprises a library of structurally defined sugars with amino-terminal linkers that are robotically printed on amino-reactive n-hydroxysuccinimide-activated glass slides to yield a covalent amide bond. the library of more than 260 glycans range in size from monosaccharides to decasaccharides, 61 of which contain neuac, the type of sialic-acid moiety found in humans. of these, 38 contain neuac in α2-3 linkages, 16 have α2-6 linkages and 7 have α2-8 linkages, and a further 16 contain sialic acids found in other species (for example, n-glycolylneuraminic acid), sialic-acid analogues or have β-linkages. therefore, the array offers an unprecedented opportunity to simultaneously assess the details of the specificity of influenza virus has towards numerous glycans that have not been previously studied. from studies conducted so far, the array seems to have value for analysis of both whole virus and recombinant ha (fig. 3) . simultaneous binding of recombinant has to the different glycans is detected by fluorescent antibodies that bind to a histidine (his) tag on the recombinant ha (fig. 3) . details of the general properties, use and design of this technology have been well described elsewhere 42, 43 , and a regularly updated database of over 300 published and unpublished examples of plant, microbial and mammalian glycan-binding proteins analysed on the cfg glycan array can be found online (see further information). here, we focus specifically on the applications of glycan microarrays to characterize influenza-host-receptor interactions. recombinant ha assays. while the cfg glycan microarray was being developed, a research programme was initiated to structurally characterize the ha from the 1918 influenza virus. the usual method for ha production is bromelain release from intact viruses, followed by sucrose density gradient centrifugation and anion-exchange chromatography 60 . however, owing to the increased biosafety requirements for making intact 1918 viruses, a new recombinant route was sought. ha is normally expressed on the influenza a virus as a homotrimeric, membrane-bound glycoprotein. each monomer is synthesized as a single polypeptide (ha0) that is cleaved by specific host proteases into two subunits, a membrane-proximal ha2 chain and a membrane-distal ha1 chain, that forms the receptor-binding pocket for binding to complex glycans with terminal sialic acids. to form functional trimers, ha was produced in a baculovirus expression system 61 with the ectodomain engineered as a fusion construct to a carboxy-terminal, trimerizing 'foldon' sequence from the bacteriophage t4 fibritin 62 . after removal of the trimerization domain by thrombin cleavage, the intact ha0 was crystallized and its structure was solved 61 . this important methodological advance for the production of the 1918 pandemic ha has recently been applied to determine the crystal structure of an avian influenza h5 ha 63 and a homotrimeric viral coat protein from the parainfluenza virus 64 , demonstrating its usefulness for the production of other homotrimeric proteins for structural and immunological analysis. for glycan microarray analysis, glycanbinding proteins have to be directly or indirectly labelled with a fluorescent tag 43 . the ha cloning strategy introduces a his tag at the carboxyl terminus of the ha2 chain to enable rapid purification from the culture media. with the commercial preliminary experiments 65 used his-tagged recombinant ha pre-complexed to a mouse fluorescent anti-penta-his antibody (six ha-binding sites per complex), but this failed to detect binding to the array owing to the weak ha affinity (mm range) for receptor analogues 26 . binding to a secondary fluorescent anti-mouse-igg1 antibody (resulting in twelve ha-binding sites per complex) allowed the detection of fluorescence. it was concluded that the additional multivalency enabled detection through avidity effects, comparable to the natural interaction of ha trimers on the viral envelope with the host cell. the intensity of the fluorescent signal determines whether the binding is recorded as strong, weak or absent. the ha produced by this method can also be used for crystallization and structural studies. whole-virus analysis involves glycan microarray analysis with intact virus. iex, ionexchange chromatography; na, neuraminidase; m2, m2 ion-channel protein; sec, size exclusion chromatography. availability of anti-his-tag antibodies, the ha can be pre-complexed with fluorescent primary and/or secondary antibodies (fig. 3) . because the affinity of ha for its glycan ligand is low (mm range) 26 , the use of such complexes has proved highly advantageous as the secondary antibodies provide increased valency to obtain detectable signals. another advantage of this technology is the small amount of protein that is required for analysis (~1.5-15 µg), so that the bulk of the expressed protein can be used for crystallization trials. so far, several viral has from human and avian h1, h3 and h5 serotypes have been produced and analysed on the array, including variants from the 1918 pandemic influenza virus, and from the highly pathogenic h5 avian influenza strain viet04 (a/vietnam/1203/2004) 63, 65 . striking differences in the receptor specificity of the different has were revealed not only for the type of glycan sialic-acid linkage, that is, either an α2-3 and/or an α2-6 sialic-acid linkage, but also in the fine specificity of has for other glycan modifications, such as fucosylation, sulphation and additional sialylation 63, 65 (fig. 4) . for example, a single amino-acid difference between the two variants of the 1918 pandemic influenza virus was sufficient to switch the ha from a classic human α2-6linked sialoside-binding pattern (asp 225 ), as seen with the a/south carolina/1/1918 (sc) ha, to mixed specificity with a significant reduction in binding to α2-6linked sialosides and an increased affinity for α2-3-linked sialosides, particularly for glycans with an additional negative charge, such as a sulphate or sialic acid (fig. 4a,b) for the a/new york/1/1918 (ny) strain ha (gly 225 ) 65 . from these results, it was proposed that the extra cavity arising from gly 225 in the 1918 ny strain ha might allow more flexibility in its glycan binding. this would enable n-acetylglucosamine (glcnac) to adopt an orientation within the ha receptor-binding site, that would allow lys 222 , which is maintained in nearly all has from avian and human h1, h2 and h5 serotypes, to interact preferentially with the 6-sulphated moieties. although the biological significance of binding to sialylated and sulphated glycans is not clear at present, human mucins have been shown to contain 6-sulphated glycans, demonstrating that the necessary enzymes are present in the human epithelium and, therefore, that sialylated sulphated structures might be of importance in virus-host interactions 33, 66 . whereas binding of both the sc and the h3 (a/moscow/10/1999) human viruses are highly restricted to α2-6-linked sialosides (fig. 4a,e) , the array data clearly showed that not all human has have identical ligand preferences. for example, the human h1 (a/texas/36/1991) ha has a strong preference for α2-6 human receptor analogues, but also binds weakly to a range of α2-3-linked sialosides 65 (fig. 4c) . it is of interest that this relatively recent h1 strain also has the asp 190 gly 225 combination, similar to the 1918 ny strain (fig. 4b) , yet results from the glycan microarray show that it is better adapted to human receptors with a higher preference for α2-6 sialosides, presumably owing to other as-yet-unidentified residues. both the avian h5 (a/vietnam/1203/2004) and h3 (a/duck/ukraine/1/1963) serotype has reveal a classic preference for α2-3 avian receptor analogues (fig. 4d,f) . comparison between these two avian subtypes shows the power of the array in differentiating ha binding to glycan ligands that contain additional fucose linkages to glcnac. ligands 25-29 on the array are all fucosylated glycans and it is clear that the human h3 ha does not bind to such ligands (fig. 4f) , whereas the h5 ha does bind them, albeit weakly (fig. 4d) . the use of such analyses allows the ready identification of a receptor footprint for each virus. therefore, it is clear that, although human and avian virus has have a primary specificity for either α2-6or α2-3-linked sialosides, each virus might use a different range of glycan receptors for cell entry, and the ability of a virus to infect a host cell might depend on features in the glycan structure, other than simply the type of sialic-acid linkage. it has been suggested that the use of recombinant has produced in baculovirus expression systems in these microarray studies might not reflect the situation of natural virus ha expression. like mammalian cells, insect cells can assemble, transfer and trim n-glycans to produce high mannose or paucimannose products 67 . but insect cells differ from mammalian cells in that they are unable to process these glycan products any further to produce complex glycans that contain terminal galactose and/or sialic acids. however, the presence of influenza na, a sialidase, ensures that complex glycans of influenza has usually terminate only in galactose and, therefore, the size of the n-glycans that are elaborated by insect cells approximate the size of the complex n-glycans in mammalian host cells. indeed, our studies using insect-cell-produced has, and cell-based assays with whole viruses show that glycans produced in both systems can affect receptor binding through steric hindrance if the n-glycan is positioned close enough to the receptor-binding site of the ha 65, 68 . in addition, results for both avian h3 (a/duck/ukraine/1/1963) and h1 (a/duck/alberta/35/1976) has 63,65 were in agreement with previous whole-viral studies 21, 23 . a key advantage of the baculovirus expression of recombinant ha is the ability to introduce specific mutations in the ha receptor-binding site to assess their effect on receptor specificity using the array. for example, different mutations known to switch the receptor specificity of pandemic h1, h2 and h3 viruses from an avian to a human preference were introduced into the h5n1 viral isolate, viet04, one of the most pathogenic viruses studied so far 69, 70 . in pandemic h1, h2 and h3 viruses, two different combinations of two mutations effectively switched receptor specificity from α2-6 to α2-3-linked glycans and viceversa. although no clear switch in receptor specificity was observed for the h5 ha, the mutations that switched the specificity of h3 viruses from avian to human (at residues 226 and 228) not only reduced overall avian specificity for the h5 ha, but also increased specificity/avidity towards biantennary sialosides 63 , which might function as receptors on human lung epithelial cells. these combined effects could allow an h5n1 virus to escape entrapment by mucins and increase binding to susceptible lung epithelial cells and so might provide a possible route by which h5n1 viruses could become established in the human population. current studies are ongoing to determine whether mutations of any other residues around the ha binding pocket can cause a more obvious switch in specificity. therefore, incorporation of mutations in and around the receptor-binding site into the recombinant ha can assess the effects on binding specificity without the need to generate potentially dangerous mutant viruses. although the glycan microarray technologies are still in their infancy, the use of this technology for the analysis of whole viruses is an important goal, and the preliminary results so far look promising. experiments with a/puerto rico/8/1934 virus reveal both α2-3 and α2-6 receptor specificity, which is in agreement with experiments from cell-based assays 23, 40 . studies that compare viruses with their equivalent recombinant has are ongoing but have to take into account the increased valency of the virus, which might enhance binding to weak ligands. furthermore, activity of the viral na can also influence the outcome by destroying glycans on the array; or the na itself could mediate binding of the virus, although the use of na inhibitors could provide a partial solution for both scenarios. categorizing influenza virus receptor specificity into two main types based on preferential recognition of α2-6 and α2-3 linkages continues to be useful for understanding the adaptation of influenza strains to the individual receptors that are present on the epithelial cells of different host species. most recently, staining of human airway epithelium by plant lectins documented for the first time that human upper airway cells have predominantly α2-6-linked glycans, whereas the lower airway surprisingly has increasing numbers of cells that express α2-3-linked glycans [11] [12] [13] 71 . as avian viruses have now been shown to infect human epithelial cells that have α2-3-linked glycans 10, 11, 13 , this observation has been proposed to account for the propensity of the h5n1 virus to localize to the lower lung during human infection and, as mentioned previously, the subsequent lack of human-to-human transmission by virus shedding 11, 13 . however, the ability to screen for the receptor specificity of influenza virus for a highly diverse set of sialic-acid-containing glycans has revealed that the situation is more complex, with different virus strains binding preferentially to novel structures (such as sulphated and sialylated glycans) and markedly different binding patterns to glycan structures in a given class 33, 63, 65 . the biological relevance of this new binding data is not yet fully understood, in part owing to a lack of knowledge about the range of glycan structures on the surface of human and avian epithelial cells and the protective mucin secretions 66 . additional information about the glycan structures produced by the natural hosts of influenza combined with the emerging data from glycan array analysis is certain to provide new biological insights relevant to modes of influenza adaptation across species barriers. a major goal for worldwide influenza surveillance is the continuing development of new technologies that can rapidly characterize the evolution of strains that emerge in humans and in avian populations. currently, viruses are collected, shipped to the who or other secure facilities, such as the cdc, where the viruses are serotyped and undergo partial sequence analysis, before informed guesses are made as to whether any identified mutations signify antigenic drift that is relevant for incorporation into the next influenza vaccine or, in the case of avian viruses, signal significant adaptation to the human population. this powerful glycan microarray technology can now facilitate mapping of the coarse and fine specificity of previous, current and emerging influenza viruses. a key advantage of using the baculovirus expression system described here is that we can now obtain specificity data directly from pathological specimens without the need for prior growth in eggs or other laboratory hosts. this issue is important because several earlier observations show that ha receptor variants of human viruses can be selected by growth in eggs, which could bias subsequent binding studies 72 . the use of such a recombinant system reduces the probability of unwanted mutations that can happen when maintaining whole viruses in a laboratory environment. the technology described here, however, is limited by its reliance on existing who surveillance methods to correctly identify the type and subtype of each virus before any analysis can be done. the recombinant assay requires knowledge of the dna sequence of the ha prior to cloning and expression studies, whereas the whole-cell assay requires a prior knowledge of the virus subtype to select a suitable reactive antibody. of interest are recent advances in dna array technologies for influenza a typing: current methods for identification require 3-7 days, but a recent report describes the development of a low-density oligonucleotide array technology (fluchip) for rapid identification of h1n1, h3n2 and h5n1 influenza a virus subtypes within 12 hours (with an accuracy of >72%) 73, 74 . commercial products are already available, such as an influenza microarray from combimatrix corporation that requires <5 hours for completion, and that is claimed to be able to identify all known strains of influenza a, including differentiating between highly pathogenic and less pathogenic strains 75 . by working together, future development of dna and glycan microarray technologies could enable rapid assessment of emerging viruses to detect virus subtypes and receptor specificities that increase the risk to the human population, and to eliminate guesswork from sequence analyses alone. a recent and exciting development is an agreement between the cdc and the cfg that will provide a customized influenza glycan microarray to allow the cdc to assess its usefulness for screening influenza field isolates. although such technological developments still require improvement in both their accuracy and sensitivity of detection, in the future, portable machines might be used to conduct such tests in the field, adding a new dimension to characterizing and assessing avian influenza outbreaks. such knowledge could aid decisions for deployment of emergency services to contain a potential outbreak. characterization of the 1918 influenza virus polymerase genes host range restriction and pathogenicity in the context of influenza pandemic the nucleoprotein as a possible major factor in determining host specificity of influenza h3n2 viruses updating the accounts: global mortality of the 1918-1920 'spanish' influenza pandemic molecular virology: was the 1918 flu avian in origin? molecular virology: was the 1918 pandemic caused by a bird flu molecular virology: was the 1918 pandemic caused by a bird flu? 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h3 influenza a and influenza b viruses share a common high binding affinity for 6′-sialyl(n-acetyllactosamine) avian influenza a viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the ha receptor-binding site early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals receptor specificity of influenza viruses from birds and mammals: new data on involvement of the inner fragments of the carbohydrate chain h5n1 chicken influenza viruses display a high binding affinity for neu5acα2-3galβ1-4(6-hso 3 )glcnac-containing receptors evolution of the receptor binding phenotype of influenza a (h5) viruses characterization of the reconstructed 1918 spanish influenza pandemic virus carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells the use of carbohydrate microarrays to study carbohydrate-cell interactions and to detect pathogens oligosaccharide microarrays to decipher the glyco code printed covalent glycan array for ligand profiling of diverse glycan binding proteins hydrogel glycan microarrays design of carbohydrate multiarrays sweet spots in functional glycomics carbohydrate array analysis of anti-tn antibodies and lectins reveals unexpected specificities: implications for diagnostic and vaccine development glycan array identifies specific signatures of anti-glycan autoantibodies in sera of breast cancer patients: diagnostic, prognostic and therapeutic opportunities cancer-associated carbohydrate identification in hodgkin's lymphoma by carbohydrate array profiling defining the binding specificity of commercially available plant lectins using a printed glycan array carbohydrate profiling reveals a distinctive role for the c-type lectin mgl in the recognition of helminth parasites and tumor antigens by dendritic cells mouse siglec-f and human siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6'-sulfo-sialyl lewis x as a preferred glycan ligand ligands for the β-glucan receptor, dectin-1, assigned using 'designer' microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides the carbohydrate-recognition domain of dectin-2 is a c-type lectin with specificity for high mannose selective binding of the scavenger receptor c-type lectin to lewisx trisaccharide and related glycan ligands glycan array screening reveals a candidate ligand for siglec-8 masking of cd22 by cis ligands does not prevent redistribution of cd22 to sites of cell contact carbohydrate profiling reveals a distinctive role for the c-type lectin mgl in the recognition of helminth parasites and tumor antigens by dendritic cells siglecs in innate immunity high and low affinity carbohydrate ligands revealed for murine sign-r1 by carbohydrate array and cell binding approaches, and differing specificities for sign-r3 and langerin identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation intact cell adhesion to glycan microarrays h5 avian and h9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes structure of the uncleaved human h1 hemagglutinin from the extinct 1918 influenza virus stabilization of short collagen-like triple helices by protein engineering structure and receptor specificity of the hemagglutinin from an h5n1 influenza virus structure of the parainfluenza virus 5 f protein in its metastable, prefusion conformation glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis baculovirus as versatile vectors for protein expression in insect and mammalian cells importance of hemagglutinin glycosylation for the biological functions of influenza virus lethality to ferrets of h5n1 influenza viruses isolated from humans and poultry in 2004 avian influenza (h5n1) viruses isolated from humans in asia in 2004 exhibit increased virulence in mammals human-specific regulation of α2-6-linked sialic acids sialic acid-α 2,6gal-specific clones of a/duck/ukraine/1/63 revert to sialic acid-α 2,3gal-specific wild type in ovo robust sequence selection method used to develop the fluchip diagnostic microarray for influenza virus experimental evaluation of the fluchip diagnostic microarray for influenza virus surveillance use of semiconductor-based oligonucleotide microarrays for influenza a virus subtype identification and sequencing the work was supported in part by grants from the national institute of allergy and infectious diseases, and the national institute of general medical sciences. the authors thank ruben donis for suggestions. this is publication 18250 from the scripps research institute. the authors declare no competing financial interests. the following term in this article is linked online to: